US20040057963A1 - Therapeutic TB vaccine - Google Patents

Therapeutic TB vaccine Download PDF

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US20040057963A1
US20040057963A1 US10/617,038 US61703803A US2004057963A1 US 20040057963 A1 US20040057963 A1 US 20040057963A1 US 61703803 A US61703803 A US 61703803A US 2004057963 A1 US2004057963 A1 US 2004057963A1
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Peter Andersen
Ida Rosenkrands
Anette Stryhn
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Statens Serum Institut SSI
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Statens Serum Institut SSI
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Priority to US10/617,038 priority Critical patent/US20040057963A1/en
Assigned to STATENS SERUM INSTITUTE reassignment STATENS SERUM INSTITUTE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: STRYHN, ANETTE, ANDERSEN, PETER, ROSENKRANDS, IDA
Publication of US20040057963A1 publication Critical patent/US20040057963A1/en
Priority to US12/785,053 priority patent/US8142797B2/en
Priority to US13/369,411 priority patent/US20130149324A1/en
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    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A

Definitions

  • the present invention discloses a therapeutic vaccine against latent or active tuberculosis infection caused by the tuberculosis complex microorganisms ( Mycobacterium tuberculosis., M.bovis, M.africanum ).
  • the invention furthermore discloses a multi-phase vaccine that can be administered either prophylactically or therapeutically as well as a diagnostic reagent for the detection of latent stages of tuberculosis.
  • Organisms of the tuberculosis complex can cause a variety of diseases, but the commonest route of invasion is by inhalation of bacteria. This initiates an infection in the lung, which can ultimately spread to other parts of the body. Normally, this infection is restricted in growth by the immune system, so that the majority of infected individuals show few signs apart from cough and fever, which eventually abates. Approximately 30% of individuals are unable to contain the infection and they will develop primary disease, which in many cases will eventually prove fatal. However, it is believed that even those individuals who apparently control the infection remain infected, probably for the rest of their life.
  • tuberculosis and other organisms of the TB complex are unique in that the mycobacteria can evade the immune response and survive for long periods in a refractory non-replicating or slowly-replicating stage. This is referred to as latent TB and is at present a very significant global health problem that is estimated to affect approximately 1 ⁇ 3 of the world's population (Anon., 2001).
  • M. tuberculosis The course of a M. tuberculosis infection runs essentially through 3 phases, as illustrated in FIG. 1.
  • the bacteria proliferate in the organs, until the immune response increases to the point at which it can control the infection, whereupon the bacterial load peaks and starts declining.
  • a latent phase is established where the bacterial load is kept stable at a low level.
  • M. tuberculosis goes from active multiplication to dormancy, essentially becoming non-replicating and remaining inside the granuloma.
  • the infection goes to the reactivation phase, where the dormant bacteria start replicating again.
  • BCG The only vaccine presently available for clinical use is BCG, a vaccine whose efficacy remains a matter of controversy. Although BCG consistently performs well in animal models of primary infection, it has clearly failed to control the TB epidemic. Consistent with that, BCG vaccination appears to provide protection against pediatric TB (which is due to primary infection), while offering little or no protection against adult disease (which is often reactivation of latent infection acquired in childhood). It has also been shown that vaccination of individuals who are currently sensitized to mycobacteria or latently infected is ineffective. Thus, current vaccination strategies, while effective against primary disease, fail to activate immune responses that efficiently control surviving dormant bacteria.
  • the present invention provides therapeutic vaccines based on molecules that are induced or upregulated under the conditions of low oxygen transmission and restricted nutrients found in the granuloma (i.e., the location of latent TB infection). These vaccines are therapeutic and contrast with prior art vaccines which are designed to elicit protective immune responses prior to infection (prophylactic vaccination) that are only effective against primary infection.
  • the immune responses elicited are powerless against the latent stage of the disease, because the bacteria have changed the antigens that they produce so that in essence they have altered their appearance and the immune system can no longer recognize them.
  • latency is a dynamic process, maintained by the immune response, as indicated by the dramatic increase in the risk of reactivation of TB after HIV infection or other events that compromise immunity. Therefore, an effective vaccination strategy to protect infected individuals (therapeutic vaccination) is possible, but only if it is directed against those antigens expressed in the latent stage.
  • the present invention provides a multiphase vaccine that combines components with prophylactic and therapeutic activity.
  • existing TB vaccines do not result in sterilizing immunity but rather control the infection at a subclinical level (thereby resulting in the subsequent establishment of latent infection.
  • the evasion of the primary immune response and the subsequent development of latent disease are probably at least in part due to the change in the antigenic profile of the invading bacteria.
  • vaccinating with antigens associated with latent TB prevents or reduces the establishment of latent infection and therefore, a vaccine incorporating antigens expressed by the bacteria both in the first logarithmic growth phase and during latent disease improve long-term immunity when used as a prophylactic vaccine.
  • a multiphase vaccine of the invention will also be efficient as a therapeutic vaccine thereby addressing the problem that the majority of the population in the third world who would receive a future TB vaccine could be already latently infected.
  • the immunodominant antigens identified in this invention may be used as diagnostic reagents.
  • We has abundantly demonstrated that antigens expressed by mycobacteria during the early stages of the infection, such as ESAT-6 (Early Secretory Antigen Target-6) are recognized in individuals who are in the process of developing primary TB, even though they are healthy at the time of diagnosis (Doherty 2002).
  • ESAT-6 Early Secretory Antigen Target-6
  • Doherty 2002 the large numbers of contacts who are exposed, and almost certainly infected, remain negative to this antigen. Since those individuals latently infected remain healthy by making an immune response against the latent bacteria, they must be making an immune response to those antigens expressed by the latent bacteria.
  • the antigens of the invention may also be used to diagnose latent infection and differentiate it from primary acute TB.
  • FIGS. 1A and 1B illustrate the results of testing in TB vaccination models.
  • Each square on the time axis represents one week.
  • Three prophylactic vaccinations two weeks apart are given 6 weeks prior to an aerosol infection.
  • the protective effect of the vaccines is measured 6 weeks after infection, in the acute phase of the infection.
  • a reactivation model is established, where aerosol infected mice are treated with anti- M tuberculosis drugs for 8 weeks from the peak of infection (6 weeks after infection). This induces a latent infection phase with a low bacterial load.
  • Four to five weeks into the latency phase three therapeutic vaccinations are given two weeks apart and the protective effect of the vaccines is measured as bacterial load in the reactivation phase, seven weeks after the last immunization.
  • FIGS. 2A and 2B illustrate prophylactic and therapeutic vaccine induced protection.
  • C57BI/6j mice were immunized 3 times with a 2-week interval with recombinant ESAT6, BCG or recombinant Rv2031c.
  • the immunization was given as a prophylactic vaccine 6 weeks before the mice were given a M. tuberculosis infection (approx. 250 bacilli) through the aerosol route with.
  • Bacterial numbers in the lung was enumerated 6 weeks post infection.
  • FIG. 2B the immunization was given as a therapeutic vaccine after a latent infection had been established.
  • Bacterial numbers in the lung was enumerated 8 weeks after the last immunization.
  • the data represents the mean of 5 individual mice.
  • FIG. 3 illustrates Rv2031c specific IFN- ⁇ responses.
  • Latent infected C57BI/6j mice were either not immunized or immunized with 3 ⁇ g recombinant Rv2031 3 times with a two-week interval.
  • One and two weeks post immunization mice were bleed and PBMCs isolated.
  • the frequency of IFN- ⁇ producing cells specific for either ESAT6 or Rv2031c was determined for both the rRv2031c immunized and the unimmunized group.
  • FIGS. 4A and 4B illustrate the results of epitope screening of RV2031c.
  • PBMCs from rRV2031c immunized latently infected C57BI/6j mice were analyzed for recognition of 20′mer overlapping peptides scanning through Rv2031c.
  • FIG. 4A the peptides were analyzed in pools of 3-4 peptides.
  • PBMCs (2 ⁇ 10 5 ) were incubated for 72 h with the peptide pools at 5 ⁇ g/ml per peptide. Supernatant was harvested and secreted IFN- ⁇ was quantitated by ELISA.
  • FIG. 4B individual peptides of positive pools were reanalyzed.
  • PBMCs (2 ⁇ 10 5 ) were incubated for 72 h with 1 ⁇ g/ml of each peptide. Secreted IFN- ⁇ in the supernatant was quantitated.
  • FIGS. 5A and 5B illustrate protection against reactivation conferred by therapeutic vaccine given during latent infection.
  • Latent infected C57BI/6j mice were immunized 3 times with or without rRv2031c.
  • Bacterial numbers in lung (FIG. 5A) and spleen (FIG. 5B) was enumerated 8 weeks after the last immunization. The data represents the mean of 8 individual mice.
  • FIGS. 6A and 6B illustrate Rv0569 specific IFN- ⁇ responses.
  • Latent infected C57BI/6j mice were vaccinated with 3 ⁇ g of either recombinant Rv0569 or recombinant ESAT6 in a DDA/MPL adjuvant.
  • the vaccines were given as 3 s.c. injections with a two-week interval and the induced immune response were evaluated 7 weeks after the last vaccination.
  • Isolated splenocytes (2 ⁇ 10 5 ) were incubated for 72 h with antigen at 1 ⁇ g/ml.
  • FIG. 6A Rv0569 specific response is measured in Rv0569-vaccinated and un-vaccinated latently infected mice; in FIG. 6B, the ESAT6 specific response is measured in ESAT6-vaccinated and un-vaccinated latently infected mice
  • FIGS. 7A and 7B illustrate therapeutic vaccine induced protection against reactivation.
  • Latently infected C57BI/6j mice were vaccinated once with BCG or 3 times with a 2-week interval with either recombinant Rv0569 or recombinant ESAT6.
  • the bacterial numbers was enumerated in FIG. 7A, the lung and in FIG. 7B, the spleen of vaccinated and un-vaccinated mice.
  • the data represents the mean of Log CFU per organ of 6-8 individual mice.
  • the invention is related to preventing, treating and detecting infections caused by species of the tuberculosis complex ( Mycobacterium tuberculosis, M. bovis, M. africanum ) by the use of a polypeptide comprising a M. tuberculosis antigen or an immunogenic portion or other variant thereof, or by the use of a DNA sequence encoding a M. tuberculosis antigen or an immunogenic portion or other variant thereof.
  • the invention discloses a new therapeutic vaccine against tuberculosis comprising antigens induced during the latent stage of TB-infection. It also discloses a multiphase vaccine incorporating a combination of prophylactic and therapeutic antigens as well as diagnostic reagents for the detection of the latent stage of M. tuberculosis infection.
  • the present invention discloses the use of one or more polypeptides, nucleic acids encoding these polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, for a therapeutic vaccine against tuberculosis.
  • polypeptides comprises one or more amino acid sequences selected from
  • the immunogenic portions are selected from the group consisting of the sequences presented in Table 1 and the nucleic acid sequences are selected from the sequences presented in Table 2.
  • the vaccine is a multiphase vaccine, where the polypeptides or fragments hereof are fused to other antigens with efficacy as prophylactic vaccines, where the fusion partner is selected from e.g. the group consisting of ESAT-6, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32.
  • the fusion partner is selected from e.g. the group consisting of ESAT-6, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32.
  • the invention further discloses a therapeutic vaccine against tuberculosis comprising one or more polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, or nucleic acids encoding these polypeptides.
  • the therapeutic and multiphase vaccine comprises an additional delivery system selected from among, live recombinant vaccines, that is gene-modified organisms such as bacteria or viruses expressing mycobacteria genes, or immunogenic delivery systems such as, DNA vaccines, that is plasmids expressing genes or gene fragments for the proteins described above, or protein vaccines, that is the proteins themselves or synthetic peptides derived from the proteins themselves delivered in a delivery system such as an adjuvant.
  • live recombinant vaccines that is gene-modified organisms such as bacteria or viruses expressing mycobacteria genes
  • immunogenic delivery systems such as, DNA vaccines, that is plasmids expressing genes or gene fragments for the proteins described above, or protein vaccines, that is the proteins themselves or synthetic peptides derived from the proteins themselves delivered in a delivery system such as an adjuvant.
  • the invention further discloses a therapeutic vaccine in which the amino acid sequence is lipidated so as to allow a self-adjuvanting effect of the polypeptide.
  • the invention also discloses a method for treating an animal, including a human being, with tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , comprising administering to the animal the above-mentioned vaccine.
  • virulent mycobacteria e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis
  • the invention also discloses a method for immunizing an animal, including a human being, against tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , comprising administering to the animal the above mentioned vaccine.
  • the invention discloses an immunogenic composition
  • an immunogenic composition comprising a polypeptide as defined above, preferably in the form of a vaccine or in the form of a diagnostic reagent.
  • the diagnostic reagent can be in the form of a skin test reagent (administered by the transcutaneous, subcutaneous or intradermal routes), a serological reagent or a reagent for stimulating a cell-mediated reaction.
  • the invention discloses a nucleic acid fragment in isolated form which
  • (a) comprises a nucleic acid sequence which encodes a polypeptide as defined above, or comprises a nucleic acid sequence complementary thereto;
  • (b) has a length of at least 10 nucleotides and hybridizes readily under stringent hybridization conditions with a nucleotide sequence selected from the nucleotide sequences presented in Table 2 or a sequence complementary thereto, or with a nucleotide sequence selected from a sequence in (a)
  • the nucleic acid fragment is preferably a DNA fragment.
  • the fragment can be used as a pharmaceutical.
  • the invention discloses a vaccine comprising a nucleic acid fragment according to the invention, optionally inserted in a vector, the vaccine effecting in vivo expression of antigen by a human being or other mammal or animal, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , in an animal, including a human being.
  • virulent mycobacteria e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis
  • the invention discloses the use of a nucleic acid fragment according to the invention for the preparation of a composition for the diagnosis of tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , and the use of a nucleic acid fragment according to the invention for the preparation of a pharmaceutical composition for the vaccination against tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis.
  • the invention discloses a vaccine for immunizing an human being or other mammal or animal, against tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , comprising as the effective component a non-pathogenic microorganism, wherein at least one copy of a DNA fragment comprising a DNA sequence encoding a polypeptide as defined above has been incorporated into the microorganism (e.g., placed on a plasmid or in the genome) in a manner allowing the microorganism to express and optionally secrete the polypeptide.
  • the invention discloses a replicable expression vector, which comprises a nucleic acid fragment according to the invention, and a transformed cell harboring at least one such vector.
  • the invention discloses a method for producing a polypeptide as defined above, comprising
  • the invention also discloses a method of diagnosing tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , in an animal, including a human being, comprising intradermally injecting, in the animal, a polypeptide as defined above or an immunogenic composition as defined above, a positive skin response at the location of injection being indicative of the animal having tuberculosis, and a negative skin response at the location of injection being indicative of the animal not having tuberculosis.
  • virulent mycobacteria e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis
  • the invention discloses a method for immunizing an animal, including a human being, against tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis , comprising administering to the animal the polypeptide as defined above, the immunogenic composition according to the invention, or the vaccine according to the invention.
  • virulent mycobacteria e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis
  • Another embodiment of the invention discloses a monoclonal or polyclonal antibody, which is specifically reacting with a polypeptide as defined above in an immuno assay, or a specific binding fragment of said antibody.
  • said antibody is for use as a diagnostic reagent, e.g. for detection of mycobacteria antigens in sputum, urine or other body fluids of an infected animal, including a human being.
  • the invention discloses a pharmaceutical composition that comprises an immunologically responsive amount of at least one member selected from the group consisting of:
  • a fusion polypeptide comprising at least one polypeptide or amino acid sequence according to (a) or (b) and at least one fusion partner;
  • nucleic acid sequence which has a length of at least 10 nucleotides and which hybridizes under stringent conditions with a nucleic acid sequence according to (d) or (e);
  • the invention discloses a method for stimulating an immunogenic response in an animal which comprises administering to said animal an immunologically stimulating amount of at least one member selected from the group consisting of:
  • a fusion polypeptide comprising at least one polypeptide or amino acid sequence according to (a) or (b) and at least one fusion partner;
  • nucleic acid sequence which has a length of at least 10 nucleotides and which hybridizes under stringent conditions with a nucleic acid sequence according to (d) or (e);
  • the vaccine, immunogenic composition and pharmaceutical composition according to the invention can be used therapeutically in a subject infected with a virulent mycobacterium combined with a prophylactic composition in a subject to prevent further infection with a virulent mycobacterium.
  • the invention also discloses a method for diagnosing previous or ongoing infection with a virulent mycobacterium, said method comprising
  • a sample e.g. a blood sample
  • a composition comprising an antibody according to the invention, a nucleic acid fragment according to the invention and/or a polypeptide as defined above, or
  • a sample e.g. a blood sample comprising mononuclear cells (e.g. T-lymphocytes), with a composition comprising one or more polypeptides as defined above in order to detect a positive reaction, e.g. proliferation of the cells or release of cytokines such as IFN- ⁇ .
  • a positive reaction e.g. proliferation of the cells or release of cytokines such as IFN- ⁇ .
  • the invention discloses a method of diagnosing Mycobacterium tuberculosis infection in a subject comprising:
  • polypeptide in the present invention should have its usual meaning. That is an amino acid chain of any length, including a full-length protein, oligopeptides, short peptides and fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds.
  • the polypeptide may be chemically modified by being glycosylated, by being lipidated (e.g. by chemical lipidation with palmitoyloxy succinimide as described by Mowat et al. 1991 or with dodecanoyl chloride as described by Lustig et al. 1976), by comprising prosthetic groups, or by containing additional amino acids such as e.g. a his-tag or a signal peptide.
  • Each polypeptide may thus be characterized by specific amino acids and be encoded by specific nucleic acid sequences. It will be understood that such sequences include analogues and variants produced by recombinant or synthetic methods wherein such polypeptide sequences have been modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide and still be immunogenic in any of the biological assays described herein. Substitutions are preferably “conservative”. These are defined according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. The amino acids in the third column are indicated in one-letter code. ALIPHATIC Non-polar GAP ILV Polar-uncharged CSTM NQ Polar-charged DE KR AROMATIC HFWY
  • a preferred polypeptide within the present invention is an immunogenic antigen from M. tuberculosis produced when the organism is subjected to the stresses associated with latent infection.
  • antigen can for example also be derived from the M. tuberculosis cell and/or M. tuberculosis culture filtrate.
  • a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences.
  • the additional sequences may be derived from the native M. tuberculosis antigen or be heterologous and such sequences may, but need not, be immunogenic.
  • Each polypeptide is encoded by a specific nucleic acid sequence. It will be understood that such sequences include analogues and variants hereof wherein such nucleic acid sequences have been modified by substitution, insertion, addition or deletion of one or more nucleic acids. Substitutions are preferably silent substitutions in the codon usage that will not lead to any change in the amino acid sequence, but may be introduced to enhance the expression of the protein.
  • substantially pure polypeptide fragment means a polypeptide preparation which contains at most 5% by weight of other polypeptide material with which it is natively associated (lower percentages of other polypeptide material are preferred, e.g. at most 4%, at most 3%, at most 2%, at most 1%, and at most 1 ⁇ 2%). It is preferred that the substantially pure polypeptide is at least 96% pure, i.e. that the polypeptide constitutes at least 96% by weight of total polypeptide material present in the preparation, and higher percentages are preferred, such as at least 97%, at least 98%, at least 99%, at least 99.25%, at least 99.5%, and at least 99.75%.
  • the polypeptide fragment is in “essentially pure form”, i.e. that the polypeptide fragment is essentially free of any other antigen with which it is natively associated, i.e. free of any other antigen from bacteria belonging to the tuberculosis complex or a virulent mycobacterium.
  • This can be accomplished by preparing the polypeptide fragment by means of recombinant methods in a nonmycobacterial host cell as will be described in detail below, or by synthesizing the polypeptide fragment by the well-known methods of solid or liquid phase peptide synthesis, e.g. by the method described by Merrifield or variations thereof.
  • virulent mycobacterium is understood a bacterium capable of causing the tuberculosis disease in an animal or in a human being.
  • virulent mycobacteria include but are not limited to M. tuberculosis, M. africanum , and M. bovis .
  • relevant animals are cattle, possums, badgers and kangaroos.
  • a TB patient an individual with culture or microscopically proven infection with virulent mycobacteria, and/or an individual clinically diagnosed with TB and who is responsive to anti-TB chemotherapy. Culture, microscopy and clinical diagnosis of TB are well known by any person skilled in the art.
  • PPD-positive individual an individual with a positive Mantoux test or an individual where PPD induces a positive in vitro recall response determined by release of IFN- ⁇ .
  • a latently infected individual an individual, who has been infected by a virulent mycobacterium, e.g. M. tuberculosis , but shows no sign of active tuberculosis. It is likely that individuals who have been vaccinated, e.g. by BCG, or treated for TB may still retain the mycobacteria within their bodies, although this is currently impossible to prove since such individuals would be expected to be positive if tested for PPD reactivity. Nonetheless, in its most accurate sense, “latently-infected” may be used to describe any individual who has M. tuberculosis residing in their tissues but who is not clinically ill.
  • DTH delayed type hypersensitivity reaction
  • IFN- ⁇ interferon-gamma.
  • the measurement of IFN- ⁇ is used as an indication of an immunological response.
  • nucleic acid fragment and “nucleic acid sequence” are understood any nucleic acid molecule including DNA, RNA, LNA (locked nucleic acids), PNA, RNA, dsRNA and RNA-DNA-hybrids. Also included are nucleic acid molecules comprising non-naturally occurring nucleosides. The term includes nucleic acid molecules of any length e.g. from 10 to 10000 nucleotides, depending on the use. When the nucleic acid molecule is for use as a pharmaceutical, e.g.
  • a molecule encoding at least one epitope is preferably used, having a length from about 18 to about 1000 nucleotides, the molecule being optionally inserted into a vector.
  • a molecule having a length of 10-100 is preferably used.
  • molecule lengths can be used, for instance a molecule having at least 12, 15, 21, 24, 27, 30, 33, 36, 39, 42, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 nucleotides (or nucleotide derivatives), or a molecule having at most 10000, 5000, 4000, 3000, 2000, 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30 or 20 nucleotides (or nucleotide derivatives).
  • stringent when used in conjunction with hybridization conditions is as defined in the art, i.e. the hybridization is performed at a temperature not more than 15-20° C. under the melting point T m , cf. Sambrook et al, 1989, pages 11.45-11.49.
  • the conditions are “highly stringent”, i.e. 5-10° C. under the melting point T m .
  • sequence identity indicates a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length.
  • the two sequences to be compared must be aligned to best possible fit allowing the insertion of gaps or alternatively, truncation at the ends of the protein sequences.
  • the sequence identity can be calculated as (N ref ⁇ N dif ) 100 /N ref , wherein N dif is the total number of non-identical residues in the two sequences when aligned and wherein N ref is the number of residues in one of the sequences.
  • Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (Pearson, 1988, or www.ncbi.nlm.nih.gov/cgi-bin/BLAST).
  • alignment is performed with the sequence alignment method ClustalW with default parameters as described by Thompson J., et al 1994, available at http://www2.ebi.ac.uk/clustalw/.
  • a preferred minimum percentage of sequence identity is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.
  • the polypeptide comprises an immunogenic portion of the polypeptide, such as an epitope for a B-cell or T-cell.
  • the immunogenic portion of a polypeptide is a part of the polypeptide, which elicits an immune response in an animal or a human being, and/or in a biological sample determined by any of the biological assays described herein.
  • the immunogenic portion of a polypeptide may be a T-cell epitope or a B-cell epitope.
  • Immunogenic portions can be related to one or a few relatively small parts of the polypeptide, they can be scattered throughout the polypeptide sequence or be situated in specific parts of the polypeptide.
  • epitopes have even been demonstrated to be scattered throughout the polypeptide covering the full sequence (Ravn et al 1999).
  • overlapping oligopeptides for the detection of MHC class II epitopes, preferably synthetic, having a length of e.g. 20 amino acid residues derived from the polypeptide.
  • These peptides can be tested in biological assays (e.g. the IFN- ⁇ assay as described herein) and some of these will give a positive response (and thereby be immunogenic) as evidence for the presence of a T cell epitope in the peptide.
  • B-cell epitopes can be determined by analyzing the B cell recognition to overlapping peptides covering the polypeptide of interest as, e.g., described in Harboe et al 1998.
  • the minimum length of a T-cell epitope has been shown to be at least 6 amino acids, it is normal that such epitopes are constituted of longer stretches of amino acids.
  • the polypeptide fragment of the invention has a length of at least 7 amino acid residues, such as at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, and at least 30 amino acid residues.
  • the polypeptide fragment has a length of at most 50 amino acid residues, such as at most 40, 35, 30, 25, and 20 amino acid residues.
  • the peptides having a length of from 10 to 20 amino acid residues will prove to be most efficient as MHC class II epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 18, such as 15, 14, 13, 12 and even 11 amino acid residues. It is expected that the peptides having a length of from 7 to 12 amino acid residues will prove to be most efficient as MHC class I epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 11, such as 10, 9, 8 and even 7 amino acid residues.
  • Immunogenic portions of polypeptides may be recognized by a broad part (high frequency) or by a minor part (low frequency) of the genetically heterogeneous human population.
  • high frequency some immunogenic portions induce high immunological responses (dominant), whereas others induce lower, but still significant, responses (subdominant).
  • High frequency> ⁇ low frequency can be related to the immunogenic portion binding to widely distributed MHC molecules (HLA type) or even by multiple MHC molecules (Sinigaglia, 1988, Kilgus, 1991).
  • the subdominant epitopes are however as relevant as are the dominant epitopes since it has been shown (Olsen, 2000) that such epitopes can induce protection regardless of the fact that they are not as strongly or broadly recognized.
  • a common feature of the polypeptides of the invention is their capability to induce an immunological response as illustrated in the examples. It is understood that a variant of a polypeptide of the invention produced by substitution, insertion, addition or deletion may also be immunogenic as determined by any of the assays described herein.
  • An immune individual is defined as a person or an animal, which has cleared or controlled an infection with virulent mycobacteria or has received a vaccination with M.bovis BCG.
  • the immune response may be monitored by one of the following methods:
  • An in vitro cellular response is determined by induction of the release of a relevant cytokine such as IFN- ⁇ from, or the induction of proliferation in lymphocytes withdrawn from an animal or human being currently or previously infected with virulent mycobacteria or immunized with the relevant polypeptide.
  • the induction being performed by the addition of the polypeptide or the immunogenic portion of the polypeptide to a suspension comprising from 2 ⁇ 10 5 cells to 4 ⁇ 10 5 cells per well.
  • the cells being isolated from either the blood, the spleen, the liver or the lung and the addition of the polypeptide or the immunogenic portion resulting in a concentration of not more than 20 ⁇ g per ml suspension and the stimulation being performed from two to five days.
  • a positive response is defined as being a response more than background plus two standard deviations.
  • the release of IFN- ⁇ can be determined by the ELISA method, which is well known to a person skilled in the art.
  • a positive response being a response more than background plus two standard deviations.
  • Other cytokines than IFN- ⁇ could be relevant when monitoring the immunological response to the polypeptide, such as IL-12, TNF- ⁇ , IL-4, IL-5, IL-10, IL-6, TGF- ⁇ .
  • Another and more sensitive method for detecting the immune response is the ELISpot method, in which the frequency of IFN- ⁇ producing cells is determined.
  • ELISpot method in which the frequency of IFN- ⁇ producing cells is determined.
  • an ELIspot plate MAHA, Millipore precoated with anti-murine IFN- ⁇ antibodies (PharMingen) graded numbers of cells isolated from either blood, spleen, or lung (typically between 1 to 4 ⁇ 10 5 cells/well) are incubated for 24-32 hrs in the presence of the polypeptide or the immunogenic portion resulting in a concentration of not more than 20 ⁇ g per ml.
  • the plates are subsequently incubated with biotinylated anti-IFN- ⁇ antibodies followed by a streptavidin-alkaline phosphatase incubation.
  • the IFN- ⁇ producing cells are identified by adding BCIP/NBT (Sigma), the relevant substrate giving rise to spots. These spots can be enumerated using a dissection microscope. It is also a possibility to determine the presence of mRNA coding for the relevant cytokine by the use of the PCR technique. Usually one or more cytokines will be measured utilizing for example PCR, ELISPOT or ELISA. It will be appreciated by a person skilled in the art that a significant increase or decrease in the amount of any of these cytokines induced by a specific polypeptide can be used in evaluation of the immunological activity of the polypeptide.
  • An in vitro cellular response may also be determined by the use of T cell lines derived from an immune individual or an M. tuberculosis -infected person where the T cell lines have been driven with either live mycobacteria, extracts from the bacterial cell or culture filtrate for 10 to 20 days with the addition of IL-2.
  • the induction being performed by addition of not more than 20 ⁇ g polypeptide per ml suspension to the T cell lines containing from 1 ⁇ 10 5 cells to 3 ⁇ 10 5 cells per well and incubation being performed from two to six days.
  • the induction of IFN- ⁇ or release of another relevant cytokine is detected by ELISA.
  • the stimulation of T cells can also be monitored by detecting cell proliferation using radioactively labeled Thymidine as described above. For both assays a positive response being a response more than background plus two standard deviations.
  • An in vivo cellular response may be determined as a positive DTH response after intradermal injection or local application patch of at most 100 ⁇ g of the polypeptide or the immunogenic portion to an individual who is clinically or subclinically infected with a virulent mycobacterium, a positive response having a diameter of at least 5 mm 72-96 hours after the injection or application.
  • An in vitro humoral response is determined by a specific antibody response in an immune or infected individual.
  • the presence of antibodies may be determined by an ELISA technique or a Western blot where the polypeptide or the immunogenic portion is absorbed to either a nitrocellulose membrane or a polystyrene surface.
  • the serum is preferably diluted in PBS from 1:10 to 1:100 and added to the absorbed polypeptide and the incubation being performed from 1 to 12 hours.
  • labeled secondary antibodies the presence of specific antibodies can be determined by measuring the OD e.g. by ELISA where a positive response is a response of more than background plus two standard deviations or alternatively a visual response in a Western blot.
  • Another relevant parameter is measurement of the protection in animal models induced after vaccination with the polypeptide in an adjuvant or after DNA vaccination.
  • Suitable animal models include primates, guinea pigs or mice, which are challenged with an infection of a virulent Mycobacterium. Readout for induced protection could be decrease of the bacterial load in target organs compared to non-vaccinated animals, prolonged survival times compared to non-vaccinated animals and diminished weight loss compared to non-vaccinated animals.
  • M. tuberculosis antigens and DNA sequences encoding such antigens, may be prepared using any one of a variety of procedures.
  • Immunogenic antigens may also be produced recombinantly using a DNA sequence encoding the antigen, which has been inserted into an expression vector and expressed in an appropriate host. Examples of host cells are E. coli .
  • the polypeptides or immunogenic portion hereof can also be produced synthetically having fewer than about 100 amino acids, and generally fewer than 50 amino acids and may be generated using techniques well known to those ordinarily skilled in the art, such as commercially available solid-phase techniques where amino acids are sequentially added to a growing amino acid chain.
  • Plasmid DNA can then be prepared from cultures of the host strain carrying the plasmid of interest, and purified using e.g. the Qiagen Giga-Plasmid column kit (Qiagen, Santa Clarita, Calif., USA) including an endotoxin removal step. It is preferred that plasmid DNA used for DNA vaccination is endotoxin free.
  • the immunogenic polypeptides may also be produced as fusion proteins, by which methods superior characteristics of the polypeptide of the invention can be achieved. For instance, fusion partners that facilitate export of the polypeptide when produced recombinantly, fusion partners that facilitate purification of the polypeptide, and fusion partners which enhance the immunogenicity of the polypeptide fragment of the invention are all interesting possibilities. Therefore, the invention also pertains to a fusion polypeptide comprising at least one polypeptide or immunogenic portion defined above and at least one fusion partner.
  • the fusion partner can, in order to enhance immunogenicity, be another polypeptide derived from M.
  • tuberculosis such as of a polypeptide fragment derived from a bacterium belonging to the tuberculosis complex, such as ESAT-6, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32 and alpha-crystalline, or at least one T-cell epitope of any of the above mentioned antigens (Skj ⁇ t et al 2000; Danish Patent application PA 2000 00666; Danish Patent application PA 1999 01020; U.S. patent application Ser. No.
  • the invention also pertains to a fusion polypeptide comprising mutual fusions of two or more of the polypeptides (or immunogenic portions thereof) of the invention.
  • fusion partners which could enhance the immunogenicity of the product, are lymphokines such as IFN- ⁇ , IL-2 and IL-12.
  • the fusion partner can e.g. be a bacterial fimbrial protein, e.g. the pilus components pilin and papA; protein A; the ZZ-peptide (ZZ-fusions are marketed by Pharmacia in Sweden); the maltose binding protein; glutathione S-transferase; ⁇ -galactosidase; or poly-histidine. Fusion proteins can be produced recombinantly in a host cell, which could be E. coli , and it is a possibility to induce a linker region between the different fusion partners.
  • polypeptides which are lipidated so that the immunogenic polypeptide is presented in a suitable manner to the immune system.
  • This effect is e.g. known from vaccines based on the Borrelia burgdorferi OspA polypeptide as described in e.g. WO 96/40718 A or vaccines based on the Pseudomonas aeruginosa OprI lipoprotein (Cote-Sierra J 1998).
  • Another possibility is N-terminal fusion of a known signal sequence and an N-terminal cystein to the immunogenic polypeptide. Such a fusion results in lipidation of the immunogenic polypeptide at the N-terminal cystein, when produced in a suitable production host.
  • Another part of the invention pertains to a vaccine composition
  • a vaccine composition comprising a polypeptide (or at least one immunogenic portion thereof) or fusion polypeptide according to the invention.
  • a vaccine composition comprises an immunologically and pharmaceutically acceptable carrier, vehicle or adjuvant.
  • An effective vaccine wherein a polypeptide of the invention is recognized by the animal, will in an animal model be able to decrease bacterial load in target organs, prolong survival times and/or diminish weight loss after challenge with a virulent Mycobacterium, compared to non-vaccinated animals
  • Suitable carriers are selected from the group consisting of a polymer to which the polypeptide(s) is/are bound by hydrophobic non-covalent interaction, such as a plastic, e.g. polystyrene, or a polymer to which the polypeptide(s) is/are covalently bound, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin or keyhole limpet haemocyanin.
  • Suitable vehicles are selected from the group consisting of a diluent and a suspending agent.
  • the adjuvant is preferably selected from the group consisting of dimethyldioctadecylammonium bromide (DDA), Quil A, poly I:C, aluminum hydroxide, Freund's incomplete adjuvant, IFN- ⁇ , IL-2, IL-12, monophosphoryl lipid A (MPL), Trehalose Dimycolate (TDM), Trehalose Dibehenate and muramyl dipeptide (MDP).
  • DDA dimethyldioctadecylammonium bromide
  • Quil A Quil A
  • poly I:C aluminum hydroxide
  • Freund's incomplete adjuvant IFN- ⁇
  • IL-2 poly I:C
  • MPL monophosphoryl lipid A
  • TDM Trehalose Dimycolate
  • Trehalose Dibehenate and muramyl dipeptide
  • agents such as aluminum hydroxide or phosphate (alum), synthetic polymers of sugars (Carbopol), aggregation of the protein in the vaccine by heat treatment, aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gramnegative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
  • Other possibilities involve the use of immune modulating substances such as cytokines or synthetic IFN- ⁇ inducers such as poly I:C in combination with the above-mentioned adjuvants.
  • a relevant antigen such as an antigen of the present invention can be conjugated to an antibody (or antigen binding antibody fragment) against the Fc ⁇ receptors on monocytes/macrophages.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired.
  • Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 ⁇ g to 1000 ⁇ g, such as in the range from about 1 ⁇ g to 300 ⁇ g, and especially in the range from about 10 ⁇ g to 50 ⁇ g.
  • Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • the manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and, to a lesser degree, the size of the person to be vaccinated.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and advantageously contain 10-95% of active ingredient, preferably 25-70%.
  • vaccines can be administered to prevent an infection with virulent mycobacteria, a prophylactic vaccine, and/or to treat established mycobacterial infection, a therapeutic vaccine.
  • the vaccine When administered to prevent an infection, the vaccine is given prophylactically, before definitive clinical signs, diagnosis or identification of an infection TB are present. Since the current vaccine BCG appears to induce an effective, but short-lived immune response, prophylactic vaccines may also be designed to be used as booster vaccines. Such booster vaccines are given to individuals who have previously received a vaccination, with the intention of prolonging the period of protection.
  • the previous vaccination may have provided sufficient immunity to prevent primary disease, but as discussed previously, boosting this immune response will not help against the latent infection.
  • the vaccine will necessarily have to be a therapeutic vaccine designed for efficacy against the latent stage of infection.
  • the vaccine according to the invention may comprise several different polypeptides in order to increase the immune response.
  • the vaccine may comprise two or more polypeptides or immunogenic portions, where all of the polypeptides are as defined above, or some but not all of the peptides may be derived from virulent mycobacteria.
  • the polypeptides not necessarily fulfilling the criteria set forth above for polypeptides may either act due to their own immunogenicity or merely act as adjuvants.
  • the vaccine may comprise 1-20, such as 2-20 or even 3-20 different polypeptides or fusion polypeptides, such as 3-10 different polypeptides or fusion polypeptides.
  • the invention also pertains to a method for immunizing an animal, including a human being, against TB caused by virulent mycobacteria, comprising administering to the animal the polypeptide of the invention, or a vaccine composition of the invention as described above, or a living vaccine described above.
  • the invention also pertains to a method for producing an immunologic composition according to the invention, the method comprising preparing, synthesizing or isolating a polypeptide according to the invention, and solubilizing or dispersing the polypeptide in a medium for a vaccine, and optionally adding other M. tuberculosis antigens and/or a carrier, vehicle and/or adjuvant substance.
  • the invention also relates to a vaccine comprising a nucleic acid fragment according to the invention, the vaccine effecting in vivo expression of antigen by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to infections caused by virulent mycobacteria in an animal, including a human being.
  • the efficacy of such a DNA vaccine can possibly be enhanced by administering the gene encoding the expression product together with a DNA fragment encoding a polypeptide that has the capability of modulating an immune response.
  • One possibility for effectively activating a cellular immune response for a vaccine can be achieved by expressing the relevant antigen in a vaccine in a non-pathogenic microorganism or virus.
  • a non-pathogenic microorganism or virus are Mycobacterium bovis BCG, Salmonella and Pseudomona and examples of viruses are Vaccinia Virus and Adenovirus.
  • Another important aspect of the present invention is an improvement of the living BCG vaccine presently available, wherein one or more copies of a DNA sequence encoding one or more polypeptide as defined above has been incorporated into the genome of the micro-organism in a manner allowing the micro-organism to express and secrete the polypeptide.
  • the incorporation of more than one copy of a nucleotide sequence of the invention is contemplated to enhance the immune response.
  • Another possibility is to integrate the DNA encoding the polypeptide according to the invention in an attenuated virus such as the vaccinia virus or Adenovirus (Rolph et al 1997).
  • the recombinant vaccinia virus is able to replicate within the cytoplasma of the infected host cell and the polypeptide of interest can therefore induce an immune response, which is envisioned to induce protection against TB.
  • the invention also relates to the use of a polypeptide or nucleic acid of the invention for use as therapeutic vaccines as have been described by D. Lowrie (Lowrie, 1999) using DNA vaccine encoding HSP65 from M. leprae .
  • Antigens with therapeutic properties may be identified based on their ability to diminish the severity of M. tuberculosis infection in experimental animals or prevent reactivation of previous infection, when administered as a vaccine.
  • the composition used for therapeutic vaccines can be prepared as described above for vaccines.
  • the invention also relates to a method of diagnosing latent TB caused by a virulent mycobacterium in an animal, including a human being, comprising intradermally injecting, in the animal, a polypeptide according to the invention, a positive skin response at the location of injection being indicative of the animal having TB, and a negative skin response at the location of injection being indicative of the animal not having TB.
  • a blood sample comprising mononuclear cells (i.e. T-lymphocytes) from a patient is contacted with a sample of one or more polypeptides of the invention.
  • This contacting can be performed in vitro and a positive reaction could e.g. be proliferation of the T-cells or release of cytokines such as IFN- ⁇ into the extracellular phase.
  • a positive reaction could e.g. be proliferation of the T-cells or release of cytokines such as IFN- ⁇ into the extracellular phase.
  • cytokines such as IFN- ⁇ into the extracellular phase.
  • the invention therefore also relates to an in vitro method for diagnosing latent infection in an animal or a human being with a virulent mycobacterium, the method comprising providing a blood sample from the animal or human being, and contacting the sample from the animal with the polypeptide of the invention, a significant release into the extracellular phase of at least one cytokine by mononuclear cells in the blood sample being indicative of the animal being sensitized.
  • a positive response being a response more than release from a blood sample derived from a patient without the TB diagnosis plus two standard deviations.
  • the invention also relates to the in vitro method for diagnosing ongoing or previous sensitization in an animal or a human being with a virulent mycobacterium, the method comprising providing a blood sample from the animal or human being, and by contacting the sample from the animal with the polypeptide of the invention demonstrating the presence of antibodies recognizing the polypeptide of the invention in the serum sample.
  • the immunogenic composition used for diagnosing may comprise 1-20, such as 2-20 or even 3-20 different polypeptides or fusion polypeptides, such as 3-10 different polypeptides or fusion polypeptides.
  • nucleic acid probes encoding the polypeptide of the invention can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample.
  • a method of determining the presence of mycobacterial nucleic acids in an animal, including a human being, or in a sample, comprising administering a nucleic acid fragment of the invention to the animal or incubating the sample with the nucleic acid fragment of the invention or a nucleic acid fragment complementary thereto, and detecting the presence of hybridized nucleic acids resulting from the incubation (by using the hybridization assays which are well-known in the art), is also included in the invention.
  • Such a method of diagnosing TB might involve the use of a composition comprising at least a part of a nucleotide sequence as defined above and detecting the presence of nucleotide sequences in a sample from the animal or human being to be tested which hybridize with the nucleic acid fragment (or a complementary fragment) by the use of PCR technique.
  • a monoclonal or polyclonal antibody which is specifically reacting with a polypeptide of the invention in an immunoassay, or a specific binding fragment of said antibody, is also a part of the invention.
  • the antibodies can be produced by methods known to a person skilled in the art. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of a polypeptide according to the present invention and, if desired, an adjuvant.
  • the monoclonal antibodies according to the present invention may, for example, be produced by the hybridoma method first described by Kohler and Milstein (Kohler and Milstein, 1975), or may be produced by recombinant DNA methods such as described in U.S. Pat. No.
  • the monoclonal anti-bodies may also be isolated from phage libraries generated using the techniques described by McCafferty et al (McCafferty, 1990), for example. Methods for producing antibodies are described in the literature, e.g. in U.S. Pat. No. 6,136,958.
  • a sample of a potentially infected organ or body fluid from an infected individual may be contacted with such an antibody recognizing a polypeptide of the invention.
  • the demonstration of the reaction by means of methods well known in the art between the sample and the antibody will be indicative of an ongoing infection. It is of course also a possibility to demonstrate the presence of anti-mycobacterial antibodies in serum or other body fluids by contacting a serum sample from a subject with at least one of the polypeptide fragments of the invention and using well-known methods for visualizing the reaction between the antibody and antigen.
  • an antibody, a nucleic acid fragment and/or a polypeptide of the invention can be used either alone, or as a constituent in a composition.
  • Such compositions are known in the art, and comprise compositions in which the antibody, the nucleic acid fragment or the polypeptide of the invention is coupled, preferably covalently, to at least one other molecule, e.g. a label (e.g. radioactive or fluorescent) or a carrier molecule.
  • a number of M tuberculosis genes are induced under low oxygen conditions.
  • the upregulation of the genes listed in table 2 has been determined at either the mRNA (Sherman, 2001) or protein (Boon, 2001, Rosenkrands, 2002) level
  • the coding region of these selected antigens is amplified by PCR using the primer sets listed in table 3.
  • TABLE 3 Prim r sequences for PCR amplification of selected low oxygen induced antigens SEQ ID Rv no.
  • PCR reactions were carried out using Platinum Tag DNA Polymerase (GIBCO BRL) in a 50 ⁇ l reaction volume containing 60 mM Tris-SO 4 (pH 8.9), 18 mM Ammonium Sulfate, 0.2 mM of each of the four nucleotides, 0.2 ⁇ M of each primer and 10 ng of M. tuberculosis H37Rv chromosomal DNA.
  • the reaction mixtures were initially heated to 95° C. for 5 min., followed by 35 cycles of: 95° C. for 45 sec, 60° C. for 45 sec and 72° C. for 2 min.
  • the amplification products were precipitated by PEG/MgCl 2 , and dissolved in 50 ⁇ L TE buffer.
  • DNA fragments were cloned and expressed in Gateway Cloning system (Life Technology).
  • 5 ⁇ L of DNA fragment was mixed with 1 ⁇ L of pDONR201, 2 ⁇ L of BP CLONASE enzyme mix and 2 ⁇ L of BP reaction buffer. The recombination reactions were carried out at 25° C. for 60 min. After Proteinase K treatment at 37° C. for 10 min., 5 ⁇ L of each sample was used to transform E. coli DH5 ⁇ competent cells. Transformants were selected on LB plates containing 50 ⁇ g/mL kanamycin. One bacterial clone from each transformation was grown in 3 mL LB medium containing 50 ⁇ g/mL kanamycin and plasmid DNA was isolated (Qiagen).
  • lysozyme 0.1 mg/mL
  • DNase I 2.5 ⁇ g/mL
  • the recombinant protein forms inclusion bodies and can be pelleted by centrifugation at 27.000 ⁇ g for 15 min.
  • Protein pellets were solubilized by adding 20 ml of sonication buffer (8 M urea, 50 mM Na 2 HPO 4 , 100 mM Tris-HCl, pH 8.0) and sonicating 5 ⁇ 30 sec pulses interrupted by a 30 sec pause. After another centrifugation at 27.000 ⁇ g for 15 min., supernatants were applied to 10 mL TALON columns (Clontech). The columns were washed with 50 mL sonication buffer.
  • Bound proteins were eluted by lowering pH (8 M urea, 50 mM Na 2 HPO 4 , 100 mM Tris-HCl, pH 4.5). 5 mL fractions were collected and analyzed by SDS-PAGE. Fractions containing recombinant combinant protein were pooled. Further purification was achieved by anion- or cation-exchange chromatography on Hitrap columns (Pharmacia). Bound protein was eluted using a NaCl gradient from 0-500 mM in 3 M urea, 10 mM Tris-HCl, pH 8.0. All fractions were collected and analyzed on SDS-PAGE using Coomassie staining. Fractions containing recombinant protein were pooled. Final protein concentrations were determined by micro BCA (Pierce).
  • a prophylactic vaccine given prior to infection should induce an immune response sufficiently strong to prevent or dampen the initial proliferation of the bacteria in the acute phase and thereby reduce the ensuing disease.
  • na ⁇ ve mice are immunized 3 times, 2 weeks apart with recombinant antigens.
  • the mice are given an aerosol infection with approximately 250 M. tuberculosis bacilli.
  • the protective capacity of the vaccine is evaluated by enumeration of the bacteria in spleen and lung 6 weeks post-infection.
  • FIG. 1B To define the optimal components for a therapeutic vaccine, a murine reactivation model of latent TB has been established (van Pinxteren, 2000) (FIG. 1B).
  • An aerosol infection with approximately 250 M. tuberculosis bacilli is given and at the peak of infection 6 weeks later, the mice receive an 8-week course of anti-mycobacterial drug treatment of isoniazid and rifabutin given in the drinking water. This reduces the bacterial load in spleen and lung to a low level (about 500 bacteria per organ).
  • This latent phase of low chronic infection is stable for 9-10 weeks after which a slow spontaneous reactivation occurs.
  • the therapeutic vaccine is given as 3 subcutaneous (s.c.) immunizations about 5 weeks after cessation of drug treatment.
  • the effect of the therapeutic vaccine is evaluated as protection against reactivation determined by enumeration of bacteria in spleen and lung 7 weeks after the last immunization.
  • BCG, ESAT6, and Rv2031c one of the most prominent proteins induced under low oxygen conditions (Rosenkrands, 2002), were analyzed for their prophylactic and therapeutic vaccine potential.
  • Na ⁇ ve or latently infected C57BI mice were immunized with one s.c. injection of 2.5 ⁇ 10 5 BCG, or 3 s.c. immunizations of 10 ⁇ g of either recombinant ESAT6 or recombinant Rv2031c in a DDA/MPL adjuvant.
  • the vaccinations were done in groups of 5 mice and protective capacity of the vaccines was evaluated as described above.
  • ESAT6 offers protection against acute phase infection at the level of BCG (FIG. 2A). However, neither of the two shows any protective effect against reactivation of the infection when given during latent infection (FIG. 2B).
  • Rv2031c the low oxygen induced antigen, offers no protection against the acute phase of the infection when given as a prophylactic vaccine, but gives some protection against reactivation when given as a therapeutic vaccine. That is, some antigens, here exemplified by ESAT6, though potent as prophylactic vaccines have no effect as therapeutic vaccines. In contrast, other antigens, here exemplified by Rv2031c, can be efficient therapeutic vaccines although they have no effect or only negligible effect as prophylactic vaccines.
  • the frequency of Rv2031c-specific and ESAT6-specific IFN- ⁇ -producing cells were determined in both the rRv2031c immunized and the unimmunized group (FIG. 3).
  • the rRv2031c immunization has increased the frequency of Rv2031c-specific IFN- ⁇ producing cells by a factor of 43 as compared to the unimmunized group.
  • the frequency of ESAT6-specific IFN- ⁇ producing cells is significantly higher in the unimmunized group.
  • ESAT6 is an antigen produced in high amounts by the actively-growing M. tuberculosis bacteria.
  • the level of the ESAT6 specific immune response in infected mice could therefore be indicative the degree of actively-growing infection in the animals.
  • Recent reports have in fact demonstrated such a correlation between the level of ESAT6 response and degree of disease in both M. tuberculosis -infected humans and M. bovis -infected cattle (Doherty, 2002, Vordermeier, 2002). Therefore, the higher ESAT6 response in the unimmunized group of latently-infected mice could be indicative of a transition into the reactivation phase, where the bacteria are again beginning to multiply.
  • FIG. 5 shows the bacterial load in the lung (A) and the spleen (B) of both rRv2031c-immunized and unimmunized mice.
  • Rv0569 is also a low oxygen induced antigen described in WO0179274 and illustrates very well the potential as a therapeutic vaccine.
  • Rv0569 which is highly up regulated under low oxygen growth conditions [Rosenkrands et al, 2002, 184(13): 3485-91], was analyzed for its ability to protect against reactivation given as a therapeutic vaccine in the latent phase of TB infection.
  • Latent infected C57BI mice were vaccinated with 3 s.c. injections of 3 ⁇ g recombinant Rv0569 and for comparison with 3 s.c. injections of 3 ⁇ g recombinant ESAT6 or one s.c. injection of BCG. The effect of the vaccine is evaluated 7 weeks after the last immunization.
  • the induced immune responses were analyzed for Rv0569 or ESAT6 specific responses in an in vitro recall assay.
  • Isolated splenocytes were incubated with 1 ⁇ g/ml of Rv0569 or 1 ⁇ g/ml of ESAT6 for 72 h.
  • the produced IFN ⁇ in the culture supernatant was quantitated in a standard sandwich ELISA.
  • FIG. 6 shows a nice Rv0569 specific IFN ⁇ response induced in the Rv0569 vaccinated group with practically no response in the un-vaccinated group.
  • the ESAT6 vaccination enhanced the ESAT6 specific response though a significant ESAT6 response persisted in the un-vaccinated latent infected group.
  • FIG. 7 shows the bacterial load in the lung and the spleen of both Rv0569-vaccinated, ESAT6-vaccinated, BCG vaccinated and un-vaccinated latently infected mice.
  • Patent application U.S. Ser. No. 09/0505,739 “Nucleic acid fragments and polypeptide fragments derived from M. tuberculosis”

Abstract

Therapeutic vaccines comprising polypeptides expressed during the latent stage of mycobacteria infection are provided, as are multiphase vaccines, and methods for treating and preventing tuberculosis.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a non-provisional of U.S. Patent Application No. 60/401,725, filed Aug. 7, 2002, and claims the benefit of the priority thereof.[0001]
    • BACKGROUND OF THE INVENTION
  • The present invention discloses a therapeutic vaccine against latent or active tuberculosis infection caused by the tuberculosis complex microorganisms ([0002] Mycobacterium tuberculosis., M.bovis, M.africanum). The invention furthermore discloses a multi-phase vaccine that can be administered either prophylactically or therapeutically as well as a diagnostic reagent for the detection of latent stages of tuberculosis.
  • Human tuberculosis caused by [0003] Mycobacterium tuberculosis (M. tuberculosis) is a severe global health problem, responsible for approx. 3 million deaths annually, according to the WHO. The worldwide incidence of new tuberculosis (TB) cases had been falling during the 1960s and 1970s but during recent decades this trend has markedly changed in part due to the advent of AIDS and the appearance of multidrug resistant strains of M. tuberculosis.
  • Organisms of the tuberculosis complex can cause a variety of diseases, but the commonest route of invasion is by inhalation of bacteria. This initiates an infection in the lung, which can ultimately spread to other parts of the body. Normally, this infection is restricted in growth by the immune system, so that the majority of infected individuals show few signs apart from cough and fever, which eventually abates. Approximately 30% of individuals are unable to contain the infection and they will develop primary disease, which in many cases will eventually prove fatal. However, it is believed that even those individuals who apparently control the infection remain infected, probably for the rest of their life. Certainly, individuals who have been healthy for years or even decades can suddenly develop tuberculosis, which has proven to be caused by the same organism they were infected with many years previously. [0004] M. tuberculosis and other organisms of the TB complex are unique in that the mycobacteria can evade the immune response and survive for long periods in a refractory non-replicating or slowly-replicating stage. This is referred to as latent TB and is at present a very significant global health problem that is estimated to affect approximately ⅓ of the world's population (Anon., 2001).
  • The course of a [0005] M. tuberculosis infection runs essentially through 3 phases, as illustrated in FIG. 1. During the acute phase, the bacteria proliferate in the organs, until the immune response increases to the point at which it can control the infection, whereupon the bacterial load peaks and starts declining. After this, a latent phase is established where the bacterial load is kept stable at a low level. In this phase M. tuberculosis goes from active multiplication to dormancy, essentially becoming non-replicating and remaining inside the granuloma. In some cases, the infection goes to the reactivation phase, where the dormant bacteria start replicating again. The full nature of the immune response that controls latent infection and the factors that lead to reactivation are largely unknown. However, there is some evidence for a shift in the dominant cell types responsible. While CD4 T cells are essential and sufficient for control of infection during the acute phase, studies suggest that CD8 T cell responses are more important in the latent phase. It is also likely that changes in the antigen-specificity of the response occur, as the bacterium modulates gene expression during its transition from active replication to dormancy.
  • The only vaccine presently available for clinical use is BCG, a vaccine whose efficacy remains a matter of controversy. Although BCG consistently performs well in animal models of primary infection, it has clearly failed to control the TB epidemic. Consistent with that, BCG vaccination appears to provide protection against pediatric TB (which is due to primary infection), while offering little or no protection against adult disease (which is often reactivation of latent infection acquired in childhood). It has also been shown that vaccination of individuals who are currently sensitized to mycobacteria or latently infected is ineffective. Thus, current vaccination strategies, while effective against primary disease, fail to activate immune responses that efficiently control surviving dormant bacteria. [0006]
  • At this point no vaccine has been developed that confers protection against reactivation whether given as a prophylactic vaccine prior to infection or as a therapeutic vaccine given to already latently infected individuals. [0007]
  • This makes the development of a new and improved vaccine against TB an urgent matter, which has been given a very high priority by the WHO. Many attempts to define protective mycobacterial substances have been made, and different investigators have reported increased resistance after experimental vaccination. However, these efforts have almost exclusively focused on the development of prophylactic vaccines for the prevention of disease (Doherty, 2002), and such vaccines have not been demonstrated to work if given in an immunotherapeutic fashion (Turner, 2000). [0008]
  • It has been suggested that the transition of [0009] M. tuberculosis from primary infection to latency is accompanied by changes in gene expression (see, for example, Honer zu Bentrup, 2001, which is incorporated herein by reference). In vitro hypoxic culture conditions, which mimic the conditions of low oxygen tension and restricted nutrients found in the granuloma (the location of the latent infection), have been used to analyze changes in gene expression and a number of antigens have been found that are induced or markedly upregulated under these conditions e.g. the 16 kDa antigen α-crystalline (Boon, 2001, Monahan, 2001, Florczyk 2001, Sherman 2001, Manganelli, 2001, all of which are incorporated herein by reference) and Rv0569 as described in Rosenkrands, 2002, and which is described in WO0179274.
  • As noted in the references cited above, it is already known that some genes are upregulated under conditions that mimic latency. However, these are a limited subset of the total gene expression during latent infection. Moreover, as one skilled in the art will readily appreciate, expression of a gene is not sufficient to make it a good vaccine candidate. The only way to determine if a protein is recognized by the immune system during latent infection with [0010] M. tuberculosis is to produce the given protein and test it in an appropriate assay as described herein. Of the more than 200 hundred antigens known to be expressed during primary infection, and tested as vaccines, less than a half dozen have demonstrated significant potential. So far only one antigen has been shown to have any potential as a therapeutic vaccine (Lowrie, 1999). However this vaccine only worked if given as a DNA vaccine, an experimental technique so far not approved for use in humans. Moreover, the technique has proved controversial, with other groups claiming that vaccination using this protocol induces either non-specific protection or even worsens disease (Turner, 2000).
  • What are needed are therapeutic vaccines that treat latent TB infection. [0011]
    • SUMMARY OF THE INVENTION
  • The present invention provides therapeutic vaccines based on molecules that are induced or upregulated under the conditions of low oxygen transmission and restricted nutrients found in the granuloma (i.e., the location of latent TB infection). These vaccines are therapeutic and contrast with prior art vaccines which are designed to elicit protective immune responses prior to infection (prophylactic vaccination) that are only effective against primary infection. The immune responses elicited are powerless against the latent stage of the disease, because the bacteria have changed the antigens that they produce so that in essence they have altered their appearance and the immune system can no longer recognize them. However, latency is a dynamic process, maintained by the immune response, as indicated by the dramatic increase in the risk of reactivation of TB after HIV infection or other events that compromise immunity. Therefore, an effective vaccination strategy to protect infected individuals (therapeutic vaccination) is possible, but only if it is directed against those antigens expressed in the latent stage. [0012]
  • Further, the present invention provides a multiphase vaccine that combines components with prophylactic and therapeutic activity. In contrast, existing TB vaccines do not result in sterilizing immunity but rather control the infection at a subclinical level (thereby resulting in the subsequent establishment of latent infection. After conventional prophylactic vaccination, the evasion of the primary immune response and the subsequent development of latent disease are probably at least in part due to the change in the antigenic profile of the invading bacteria. Thus, vaccinating with antigens associated with latent TB prevents or reduces the establishment of latent infection and therefore, a vaccine incorporating antigens expressed by the bacteria both in the first logarithmic growth phase and during latent disease improve long-term immunity when used as a prophylactic vaccine. A multiphase vaccine of the invention will also be efficient as a therapeutic vaccine thereby addressing the problem that the majority of the population in the third world who would receive a future TB vaccine could be already latently infected. [0013]
  • For a number of years, a major effort has been put into the identification of protective antigens for the development of novel prophylactic vaccines against TB and today a few antigens with demonstrated protective activity in prophylactic vaccines have been identified (e.g. ESAT-6, the 38 kDa antigen, Ag85A and Ag85B). Such molecules are useful components, which in combination with latency associated antigens such as α-crystalline, form a multiphase vaccine of the invention. Advantageously and in contrast to antigens in the art, the antigens described in the invention are incorporated in vaccines through the use of well-recognized vaccination technology, as demonstrated in provided examples. [0014]
  • Finally, the immunodominant antigens identified in this invention may be used as diagnostic reagents. Our group has abundantly demonstrated that antigens expressed by mycobacteria during the early stages of the infection, such as ESAT-6 (Early Secretory Antigen Target-6) are recognized in individuals who are in the process of developing primary TB, even though they are healthy at the time of diagnosis (Doherty 2002). However, the large numbers of contacts who are exposed, and almost certainly infected, remain negative to this antigen (Doherty 2002). Since those individuals latently infected remain healthy by making an immune response against the latent bacteria, they must be making an immune response to those antigens expressed by the latent bacteria. Thus, the antigens of the invention may also be used to diagnose latent infection and differentiate it from primary acute TB. [0015]
  • Other aspects and advantages of the invention will be readily apparent to one of skill in the art.[0016]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGS. 1A and 1B illustrate the results of testing in TB vaccination models. A schematic time schedule of the models for FIG. 1A, prophylactic vaccination and FIG. 1B, therapeutic vaccination. Each square on the time axis represents one week. Three prophylactic vaccinations two weeks apart are given 6 weeks prior to an aerosol infection. The protective effect of the vaccines is measured 6 weeks after infection, in the acute phase of the infection. For analysis of therapeutic vaccinations a reactivation model is established, where aerosol infected mice are treated with anti-[0017] M tuberculosis drugs for 8 weeks from the peak of infection (6 weeks after infection). This induces a latent infection phase with a low bacterial load. Four to five weeks into the latency phase three therapeutic vaccinations are given two weeks apart and the protective effect of the vaccines is measured as bacterial load in the reactivation phase, seven weeks after the last immunization.
  • FIGS. 2A and 2B illustrate prophylactic and therapeutic vaccine induced protection. C57BI/6j mice were immunized 3 times with a 2-week interval with recombinant ESAT6, BCG or recombinant Rv2031c. In FIG. 2A, the immunization was given as a [0018] prophylactic vaccine 6 weeks before the mice were given a M. tuberculosis infection (approx. 250 bacilli) through the aerosol route with. Bacterial numbers in the lung was enumerated 6 weeks post infection. In FIG. 2B, the immunization was given as a therapeutic vaccine after a latent infection had been established. Bacterial numbers in the lung was enumerated 8 weeks after the last immunization. The data represents the mean of 5 individual mice.
  • FIG. 3 illustrates Rv2031c specific IFN-γ responses. Latent infected C57BI/6j mice were either not immunized or immunized with 3 μg recombinant Rv2031 3 times with a two-week interval. One and two weeks post immunization mice were bleed and PBMCs isolated. The frequency of IFN-γ producing cells specific for either ESAT6 or Rv2031c was determined for both the rRv2031c immunized and the unimmunized group. In an ELIspot plate precoated with anti-IFN-γ antibodies graded numbers of PBMCs were incubated with either 2 μg/ml rRv2031c or 2 μg/ml rESAT6. After 32 h the plate was washed and incubated with biotinylated anti-INF-γ antibodies followed by a streptavidin-alkalinephosphatase incubation. The INFγ spots, representing individual IFN-γ producing cells were visualized using BCIP/NBT substrate. The results are shown as number RV2031c specific IFN-γ producing cell (black bars) and number of ESAT6 specific IFN-γ producing cell (hatch bars) per 10[0019] 6 PBMCs.
  • FIGS. 4A and 4B illustrate the results of epitope screening of RV2031c. PBMCs from rRV2031c immunized latently infected C57BI/6j mice were analyzed for recognition of 20′mer overlapping peptides scanning through Rv2031c. In FIG. 4A, the peptides were analyzed in pools of 3-4 peptides. PBMCs (2×10[0020] 5) were incubated for 72 h with the peptide pools at 5 μg/ml per peptide. Supernatant was harvested and secreted IFN-γ was quantitated by ELISA. In FIG. 4B, individual peptides of positive pools were reanalyzed. PBMCs (2×105) were incubated for 72 h with 1 μg/ml of each peptide. Secreted IFN-γ in the supernatant was quantitated.
  • FIGS. 5A and 5B illustrate protection against reactivation conferred by therapeutic vaccine given during latent infection. Latent infected C57BI/6j mice were immunized 3 times with or without rRv2031c. Bacterial numbers in lung (FIG. 5A) and spleen (FIG. 5B) was enumerated 8 weeks after the last immunization. The data represents the mean of 8 individual mice. [0021]
  • FIGS. 6A and 6B illustrate Rv0569 specific IFN-γ responses. Latent infected C57BI/6j mice were vaccinated with 3 μg of either recombinant Rv0569 or recombinant ESAT6 in a DDA/MPL adjuvant. The vaccines were given as 3 s.c. injections with a two-week interval and the induced immune response were evaluated 7 weeks after the last vaccination. Isolated splenocytes (2×10[0022] 5) were incubated for 72 h with antigen at 1 μg/ml. Supernatant was harvested and secreted IFN-γ was quantitated by ELISA using paired anti-murine IFN-γ antibodies (PharMingen) and recombinant IFN-γ (PharMingen) as standard. In FIG. 6A, Rv0569 specific response is measured in Rv0569-vaccinated and un-vaccinated latently infected mice; in FIG. 6B, the ESAT6 specific response is measured in ESAT6-vaccinated and un-vaccinated latently infected mice
  • FIGS. 7A and 7B illustrate therapeutic vaccine induced protection against reactivation. Latently infected C57BI/6j mice were vaccinated once with BCG or 3 times with a 2-week interval with either recombinant Rv0569 or recombinant ESAT6. Seven weeks after the last vaccination the bacterial numbers was enumerated in FIG. 7A, the lung and in FIG. 7B, the spleen of vaccinated and un-vaccinated mice. The data represents the mean of Log CFU per organ of 6-8 individual mice.[0023]
    • DETAILED DESCRIPTION OF THE INVENTION
  • The invention is related to preventing, treating and detecting infections caused by species of the tuberculosis complex ([0024] Mycobacterium tuberculosis, M. bovis, M. africanum) by the use of a polypeptide comprising a M. tuberculosis antigen or an immunogenic portion or other variant thereof, or by the use of a DNA sequence encoding a M. tuberculosis antigen or an immunogenic portion or other variant thereof. The invention discloses a new therapeutic vaccine against tuberculosis comprising antigens induced during the latent stage of TB-infection. It also discloses a multiphase vaccine incorporating a combination of prophylactic and therapeutic antigens as well as diagnostic reagents for the detection of the latent stage of M. tuberculosis infection.
  • The present invention discloses the use of one or more polypeptides, nucleic acids encoding these polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, for a therapeutic vaccine against tuberculosis. [0025]
  • The polypeptides comprises one or more amino acid sequences selected from [0026]
  • (a) The sequences presented in Table 1. [0027]
  • (b) an immunogenic portion, e.g. a T-cell epitope, of any one of the sequences in (a); and/or [0028]
  • (c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic. [0029]
    TABLE 1
    Amino acid sequences of selected low oxygen induced antigens
    SEQ ID
    Rv no. NO: Sequence
    Rv0569 91 MKAKVGDWLVIKGATIDQPDHRGLIIEVRSSDGSPPYVVRWLETDHVATVIPGPDA
    VVVTAEEQNAADERAQHRFGAVQSAILHARGT
    Rv0079 1 VEPKRSRLVVCAPEPSHAREFPDVAVFSGGRANASQAERLARAVGRVLADRGVTGG
    ARVRLTMANCADGPTLVQINLQVGDTPLRAQAATAGIDDLRPALIRLDRQIVRASA
    QWCPRPWPDRPRRRLTTPAEALVTRRKPVVLRRATPLQAIAAMDAMDYDVHLFTDA
    ETGEDAVVYRAGPSGLRLARQHHVFPPGWSRCRAPAGPPVPLIVNSRPTPVLTEAA
    AVDRAREHGLPFLFFTDQATGRGQLLYSRYDGNLGLITPTGDGVADGLA
    Rv0080 2 MSPGSRRASPQSAREVVELDRDEAMRLLASVDHGRVVFTRAALPAIRPVNHLVVDG
    RVIGRTRLTAKVSVAVRSSADAGVVVAYEADDLDPRRRTGWSVVVTGLATEVSDPE
    QVARYQRLLHPWVNMAMDTVVAIEPEIVTGIRIVADSRTP
    Rv0081 3 VESEPLYKLKAEFFKTLAHPARIRILELLVERDRSVGELLSSDVGLESSNLSQQLG
    VLRRAGVVAARRDGNAMIYSIAAPDIAELLAVARKVLARVLSDRVAVLEDLRAGG-
    SAT
    Rv0363c 4 MPIATPEVYAEMLGQAKQNSYAFPAINCTSSETVNAAIKGFADAGSDGIIQF
    STGGAEFGSGLGVKDMVTGAVALAEFTHVIAAKYPVNVALHTDHCPKDKLDS
    YVRPLLAISAQRVSKGGNPLFQSHMWDGSAVPIDENLAIAQELLKAAAAAKI
    ILEIEIGVVGGEEDGVANEINEKLYTSPEDFEKTIEALGAGEHGKYLLAATF
    GNVHGVYKPGNVKLRPDILAQGQQVAAAKLGLPADAKPFDFVFHGGSGSLKS
    EIEEALRYGVVKMNVDTDTQYAFTRPIAGHMFTNYDGVLKVDEVGVKKVYD
    PRSYLKKAEASMSQRVVQACNDLHCAGKSLTH
    Rv0572 5 MGEHAIKRHMRQRKPTKHPLAQKRGARILVFTDDPRRSVLIVPGCHLDSMRR
    EKNAYYFQDGNALVGMVVSGGTEYDADDRTYVVQLTDGRHTTESSFEH-
    SSPSRSPQSDDL
    Rv0574c 6 VAGNPDVVTVLLGGDVMLGRGVDQILPHPGKPQLRERYMRDATGYVRLAERV
    NGRIPLPVDWRWPWGEALAVLENTATDVCLINLETTITADGEFADRKPVCYR
    MHPDNVPALTALRPHVCALANNHILDFGYQGLTDTVALLAGAGIQSVGAGAD
    LLAARRSALVTVGHERRVIVGSVAAESSGVPESWAARRDRPGVWLIRDPAQR
    DVADDVAAQVLADKRPGDIAIVSMHWGSNWGYATAPGDVAFAHRLIDAGIDM
    VHGHSSHHPRPIEIYRGKPILYGCGDVVDDYEGIGGHESFRSELRLLYLTVT
    PDASGNLISLQMLPLRVSRMRLQRASQTDTEWLRNTIERISRRFGIRVVTRP
    DNLLEVVPAANLTSKE
    Rv1264 7 VTDHVREADDANIDDLLGDLGGTARAERAKLVEWLLEQGITPDEIRATNPPL
    LLATRHLVGDDGTYVSAREISENYGVDLELLQRVQRAVGLARVDDPDAVVHM
    RADGEAAARAQRFVELGLNPDQVVLVVRVLAEGLSHAAEAMRYTALEAIMRP
    GATELDIAKGSQALVSQIVPLLGPMIQDMLFMQLRHMMETEAVNAGERAAGK
    PLPGARQVTVAFADLVGFTQLGEVVSAEELGHLAGRLAGLARDLTAPPVWFI
    KTIGDAVMLVCPDPAPLLDTVLKLVEVVDTDNNFPRLRAGVASGMAVSRAGD
    WFGSPVNVASRVTGVARPGAVLVADSVREALGDAPEADGFQWSFAGPRRLRG
    IRGDVRLFRVRRGATRTGSGGAAQDDDLAGSSP
    Rv1592c 8 MVEPGNLAGATGAEWIGRPPHEELQRKVRPLLPSDDPFYFPPAGYQHAVPGT
    VLRSRDVELAFMGLIPQPVTATQLLYRTTNMYGNPEATVTTVIVPAELAPGQ
    TCPLLSYQCAIDAMSSRCFPSYALRRRAKALGSLTQMELLMISAALAEGWAV
    SVPDHEGPKGLWGSPYEPGYRVLDGIRAALNSERVGLSPATPIGLWGYSGGG
    LASAWAAEACGEYAPDLDIVGAVLGSPVGDLGHTFRRLNGTLLAGLPALVVA
    ALQHSYPGLARVIKEHANDEGRQLLEQLTEMTTVDAVIRMAGRDMGDFLDEP
    LEDILSTPEISHVFGDTKLGSAVPTPPVLIVQAVHDYLIDVSDIDALADSYT
    AGGANVTYHRDLFSEHVSLHPLSAPMTLRWLTDRFAGKPLTDHRVRTTWPTI
    FNPMTYAGMARLAVIAAKVITGRKLSRRPL
    Rv1733c 9 MIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTV
    SLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATS
    APPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAP
    PARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFC-
    TQR
    Rv1734c 10 MTNVGDQGVDAVFGVIYPPQVALVSFGKPAQRVCAVDGAIHVMTTVLATLPA
    DHGCSDDHRGALFFLSINELTRCAAVTG
    Rv1736c 11 VTVTPRTGSRIEELLARSGREFIPGEISADLRTVTRRGGRDGDVFYRDRWSHDK
    VVRSTHGVNCTGSCSWKIYVKDDIITWETQETDYPSVGPDRPEYEPRGCPRGAA
    FSWYTYSPTRVRHPYARGVLVEMYREAKARLGDPVAAWADIQADPRRRRRYQRA
    RGKGGLVRVSWAEATEMIAAAHVHTISTYGPDRVAGFSPIPAMSMVSHAAGSRF
    VELIGGVMTSFYDWYADLPVASPQVFGDQTDVPESGDWWDVVWQCASVLLTYPN
    SRQLGTAEELLAHIDGPAADLLGRTVSELRRADPLTAATRYVDTFDLRGPATLY
    LTYWTAGDTRNRGREMLAFAQTYRSTDVAPPRGETPDFLPVVLEFAATVDPEAG
    RRLLSGYRVPIAALCNALTEAALPYAHTVAAVCRTGDMMGELEFWTVVPYVTMTI
    VAVGSWWRYRYDKFGWTTRSSQLYESRLLRIASPMFHFGILVVIVGHGIGLVIP
    QSWTQAAGLSEGAYHVQAVVLGSIAGITTLAGVTLLIYRRRTRGPVFMATTVND
    KVMYLVLVAAIVAGLGATALGSGVVGEAYNYRETVSVWFRSVWVLQPRGDLMAE
    APLYYQIHVLIGLALFALWPFTRLVHAFSAPIGYLFRPYIIYRSREELVLTR-
    PRRRGW
    Rv1737c 12 MRGQAANLVLATWISVVNFWAWNLIGPLSTSYARDMSLSSAEASLLVATPILVG
    ALGRIVTGPLTDRFGGRAMLIAVTLASILPVLAVGVAATMGSYALLVFFGLFLG
    VAGTTFAVGIPFANNWYQPARRGFSTGVFGMGMVGTALSAFFTPRFVRWFGLFT
    THAIVAAALASTAVVAMVVLRDAPYFRPNADPVLPRLKAIAAARLPVTWEMSFLYA
    IVFGGFVAFSNYLPTYITTIYGFSTVDAGARTAGFALAIXVLARPVGGWLSDRIA
    PRHVVLASLAGTALLAFAAALQPPPEVWSAATFITTLAVCLGVGTGGVFAWVARR
    APAASVGSVTGTVAAAGGLGGYFPPLVMGATYDPVDNDYTVGLLLLVATALVAC-
    TYTALHAREPVSEEASR
    Rv1738c 13 MCGDQSDHVLQHWTVDISTDEHEGLTRAKARLRWREKELVGVGLARLNPADRNV
    PEIGDELSVARALSDLGKRMLKVSTHDTEAVTHQPARLLY
    Rv1739c 14 MIPTMTSAGWAPGVVQFREYQRRWLRGDVLAGLTVAAYLIPQAMAYATVAGLPP
    AAGLWASTAPLAIYALLGSSRQLSIGPESATALMTAAVLAPMAAGDLRRYAVLA
    ATLGLLVGLICLLAGTARLGFLASLRSRPVLVGYMAGIALVMISSQLGTITGTS
    VEGNEFFSEVHSFATSVTRVHWPTFVLAMSVLALLTMLTRWAPRAPGPITAVLA
    ATMLVAVMSLDAKGTAIVGRTPSGLPTPGVPPVSVEDLPALIIPAAGIAIVTFT
    DGVLTARAFAARRGQEVNANAELRAVGACNIAAGLTHGFPVSSSSSRTALADVV
    GGRTQLYSLIALGLVVIVMVFASGLLAMFPIAALGALVVYAALRLIDLSEFRRL
    ARFRRSELMLALATTAAVLGLGVFYGVLAAVALSILELLRRVAHPHDSVLGFVP
    GIAGMHDIDDYPQAKRVPGLVVYRYDAPLCFANAEDFRRRALTVVQDPGQVEW
    FVLNAESNVEVDLTALDALDQLRTELLRRGIVFAMARVKQDLRESLRAASLLDK
    IGEDHIFMTLPTAVQAFRRR
    Rv1813c 15 MITNLRRRTAMAAAGLGAALGLGILLVPTVDAHLANGSMSEVMMSEIAG
    LPIPPIIHYGATAYAPSGASGKAWHQRTPARAEQVALEKCGDKTCKVVS
    RFTRCGAVAYNGSKYQGGTGLTRRAAEDDAVNRLEGGRIVNWACN
    Rv1997c 16 LSASVSATTAHHGLPAHEVVLLLESDPYHGLSDGEAAQRLERFGPNTLAVVTPASL
    LARILRQFHHPLTYVLLVAGTTTAGLKEFVDAAVIFGVVVTNAIVGFIQESKAEAA
    LQGLRSMVHTHAKVVREGHEHTMPSEELVPGDLVLLAAGDKVPADLRLVRQTGLSV
    NESALTOESTPVHKDEVALPEGTPVADRRNIAYSGTLVTAGHGAGIVVATGAETEL
    GEIHRLVGAAEVVATPLTAKLAWFSKFLTIAILGLAALTFGVGLLRRQDAVETFTA
    AIALAVGAIPEGLPTAVTITLAIGMARMAKRPJANIRRLPAVETLGSTTVICADKTG
    TLTENQMTVQSIWTPHGEIRATGTGYAPDVLLCDTDDAPVPVNANAALRWSLLAGA
    CSNDAALVRDGTRWQIVGDPTEGAMLVVAAKAGFNPERLATTLPQVAAIPFSSERQ
    YMATLHRDGTDHVVLAKGAVERMLDLCGTEMGADGALRPLDRATVLPATEMLTSRG
    LRVLATGMGAGAGTPDDFDENVIPGSLALTGLQANSDPPPAAAASAVAACHSAGIA
    VKMITGDHAGTATAIATEVGLLDNTEPAAGSVLTGAELAALSADQYPEAVDTASVF
    ARVSPEQKLRLVQALQARGHVVAMTGDGVNDAPALRQANIGVAMGRGGTEVAKDAA
    DMVLTDDDFATIEAAVEEGRGVFDNLTKFITWTLPTNLGEGLVTLAAIAVGVALPI
    LPTQILWINMTTAIALGLMLAFEPKEAGIMTRPPRDPDQPLLTGWLVRRTLLVSTL
    LVASAWWLFAWELDNGAGLHEARTAALNLFVVVEAFYLFSCRSLTRSAWRLGMKAN
    RWIILGVSAQAIAQFAITYLPAMNMVFDTAPIDIGVWVRIFAVATAITIVVATDTL
    LPRIRAQPP
    Rv1998c 17 MSFHDLHHQGVPFVLPNAWDVPSALAYLAEGFTAIGTTSFGVSSSGGHPDGHPATR
    GANIALAAALAPLQCYVSVDIEDGYSDEPDATADYVAQLSTAGINIEDSSAEKLID
    PALAAAKIVAIKQRNPEVFVNARVDTYWLRQHADTTSTIQRALRYVDAGADGVFVP
    LANDPDELAELTRNIPCPVNTLPVPGLTIADLGELGVARVSTGSVPYSAGLYAAAH
    AARAVSDGEQLPRSVPYAELQARLVDYENRTSTT
    Rv2003c 18 VVKRSRATRLSPSIWSGWESPQCRSIRARLLLPRGRSRPPNADCCWNQLAVTPDTR
    MPASSAAGRDAAAYDAWYDSPTGRPILATEVAALRPLIEVFAQPRLEIGVGTGRFA
    DLLGVRFGLDPSRDALMFARRRGVLVANAVGEAVPFVSRHFGAVLMIWTLCFVTDP
    AAIFRETRRLLADGGGLVIGFLPRGTPWADLYALRAARGQPGYRDARFYTAAELEQ
    LLADSGFRVIARRCTLHQPPGLARYDIEAAHDGTQAGAGFVAISAVDQAHEPKD-
    DHPLESE
    Rv2005c 19 MSKPRKQHGVVVGVDGSLESDAAACWGATDAAMRNIPLTVVHVVNADVATWPPMPY
    PETWGVWQEDEGRQIVANAVKLAKEAVGADRKLSVKSELVFSTPVPTMVEISNEAE
    MVVLGSSGRGALARGLLGSVSSSLVRRAGCPVAVIHSDDAVIPDPQHAPVLVGIDC
    SPVSELATAVAFDEASRRGVELIAVHAWSDVEVVELPGLDFSAVQQEAELSLAERL
    AGWQERYPDVPVSRVVVCDRPARKLVQKSASAQLVVVGSHGRGGLTGMLLGSVSNA
    VLHAARVPVTVARQS
    Rv2007c 20 VTYVIGSECVDVMDKSCVQECPVDCIYEGARMLYINPDECVDCGACKPACRVEAIY
    WEGDLPDDQHQHLGDNAAFFHQVLPGRVAPLGSPGGAAAVGPIGVDTPLVAAIP-
    VECP
    Rv2028c 21 DANQSHKPPSIVVGIDGSKPAVQALWAVDEAASRDIPLRLLYAIEPDDPGYAAHGA
    AARKLAAAENAVRYAFTAVEAADRPVKVEVEITQERPVTSLIRASAAAALVCVGAI
    GVHHFRPERVGSTAAALALSAQCPVAIVRPHRVPIGRDAAWIVVEADGSSDIGVLL
    GAVMAEARLRDSPVRVVTCRQSGVGDTGDDVRASLDRWLARWQPRYPDVRVQSAAV
    HGELLDYLAGLGRSVHMVVLSASDQEHVEQLVGAPGNAVLQEAGCTLLVVGQQYL
    Rv2029c 22 MTEPAAWDEGKPRIITLTMNPALDITTSVDVVRPTEKMRCGAPRYDPGGGGINVAR
    IVHVLGGCSTALFPAGGSTGSLLMALLGDAGVPFRVIPIAASTRESFTVNESRTAK
    QYRFVLPGPSLTVAEQEQCLDELRGAAASAAFVVASGSLPPGVAADYYQRVADICR
    RSSTPLILDTSGGGLQHISSGVFLLKASVRELRECVGSELLTEPEQLAAAHELIDR
    GRAEVVVVSLGSQGALLATRHASHRFSSIPMTAVSGVGAGDAMVAAITVGLSRGWS
    LIKSVRLGNAAGAAMLLTPGTAACNRDDVERFFELAAEPTEVGQDQYVWHPIVN-
    PEASP
    Rv2030c 23 VLMTAAADVTRRSPRRVFRDRREAGRVLAELLAAYRDQPDVIVLGLARGGLPVAWE
    VAAALHAPLDAFVVRKLGAPGHDEFAVGALASGGRVVVNDDVVRGLRITPQQLRDI
    AEREGRELLRRESAYRGERPPTDTTGKTVTVVDDGLATGASMFAAVQALRDAQPAQ
    IVIAVPAAPESTCREFAGLVDDVVCATMPTPFLAVGESFWDFRQVTDEEVRRLLAT
    PTAGPSLRRPAASTAADVLRRVAIDAPGGVPTHEVLAELVGDARIVLIGESSHGTH
    EFYQARAAMTQWLIEEKGFGAVAAEADWPDAYRVNRYVRGLGEDTNADEALSGFER
    FPAWMWRNTVVRDFVEWLRTRNQRYESGALRQAGFYGLDLYSLHRSIQEVISYLDK
    VDPRAAARARARYACFDHACADDGQAYGFAAAEGAGPSCEREAVEQLVDVQRNALA
    YARQDGLLAEDELFYAQQNAQTVRDAEVYYRAMFSGRVTSWNLRDQHMAQTLGSLL
    THLDRHLDAPPARIVVWAHNSHVGDARATEVWADGQLTLGQIVRERYGDESRSIGE
    STYTGTVTAASEWGGIAQRKAVRPALHGSVEELFHQTADSFLVSARLSRDAEAPLD
    VVRLGRAIGVVYLPATERQSHYLHVRPADQFDAMIHIDQTRALEPLEVTSRWIAGE
    NPETYPTGL
    Rv2031c 24 MATTLPVQRHPRSLFPEFSELFAAEPSFAGLRPTFDTRLMRLEDEMKEGRYEVPAE
    LPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGA-
    DEDDIKATYDKGILTVSVAVSEGKPTEKHIQIRSTN
    Rv2032 25 MPDTMVTTDVIKSAVQLACRAPSLHNSQPWRWIAEDHTVALFLDKDRVLYATDHSG
    REALLGCGAVLDHFRVAMAAAGTTANVERFPNPNDPLHLASIDESPADFVTEGHRL
    RADAILLRRTDRLPFAEPPDWDLVESQLRTTVTADTVRIDVTADDMRPELAAASKL
    TESLRLYDSSYHAELFWWTGAFETSEGIPHSSLVSAAESDRVTFGRDFPVVANTDR
    RPEFGHDRSKVLVLSTYDNERASLLRCGEMLSAVLLDATMAGLATCTLTHITELHA
    SRDLVAALIGQPATPQALVRVGLAPEMEEPPPATPRRPIDEVFHVRAKDHR
    Rv2428 26 MPLLTIGDQFPAYQLTALIGGDLSKVDAKQPGDYFTTITSDEHPGKWRVVFEWPKD
    ETFVCPTEIAAFSKLNDEFEDRDAQILGVSIDSEFAHFQWRAQHNDLKTLPFPMLS
    DIKRELSQAAGVLNADGVADRVTFIVDPNNEIQFV-
    SATAGSVGRNVDEVLRVLDALQSDELCACNWRKGDPTLDAGELLKASA
    Rv2624c 27 MSGRGEPTMKTIIVGIDGSHAAITAALWGVDEAISRAVPLRLVSVIKPTHPSPDDY
    DRDLAHAERSLREAQSAVEAAGKLVKIETDIPRGPAGPVLVEASRDAEMICVGSVG
    IGRYASSILGSTATELAEKAHCPVAVMRSKVDQPASDINWIVVRMTDAPDNEAVLE
    YAAREAKLRQAPILALGGRPEELREIPDGEFERRVQDWHHRHPDVRVYPITTHTGI
    ARELADHDERVQLAVIGGGEAGQLARLVGPSGHPVFRHAECSVLVVRR
    Rv2625c 28 MRDAIPLGRIAGEVVNVHWSVLVILWLFTWSLATMLPGTVGGYPAVVYWLLGAGGA
    VMLLASLLAHELAHAVVARRAGVSVESVTLWLFGGVTALGGEAKTPKAAFRIAEAG
    PATSLALSATFGALAITLAGVRTPAIVISVAWWLATVNLLLGLFNLLPGAPLDGGR
    LVRAYLWRRHGDSVRAGIGAARAGRVVALVLIALGLAEFVAGGLVGGVWLAFIGWE
    IFAAAREEETRISTQQLEAGVRVADAMTAQPHTAPGWINVEDFIQRYVLGERHSAY
    PVADRDGSITGLVALRQLRDVAPSRRSTTSVGDIALPLHSVPTARPQEPLTALLER
    MAPLGPRSRALVTEGSAVVGIVTPSDVARLIDVYRLAQPEPTFTTSPQDADRFS-
    DAG
    Rv2727c 29 MASSASDGTHERSAFRLSPPVLSGAMGPFMHTGLYVAQSWRDYLGQQPDKLPIARP
    TIALAAQAFRDEIVLLGLKARRPVSNHRVFERISQEVAAGLEFYGNRRWLEKPSGF
    FAQPPPLTEVAVRKVKDRRRSFYRIFFDSGFTPHPGEPGSQRWLSYTANNREYALL
    LRHPEPRPWLVCVHGTEMGRAPLDLAVFRAWKLHDELGLNIVMPVLPMIIGPRGQGL
    PKGAVFPGEDVLDDVHGTAQAVWDIRRLLSWIRSQEEESLIGLNGLSLGGYIASLV
    ASLEEGLACAILGVPVADLIELLGRHCGLRHKDPRRHTVKMAEPIGRMISPLSLTP
    LVPMPGRFIYAGIADRLVHPREQVTRLWEHWGKPEIVWYPGGHTGFFQSRPVRREV
    QAALEQSGLLDAPRTQRDRSA
    Rv2628 30 MSTQRPRHSGIRAVGPYAWAGRCGRIGRWGVHQEAMMNLAIWHPRKVQSATIYQVT
    DRSHDGRTARVPGDEITSTVSGWLSELGTQSPLADELARAVRTGDWPAAYAIGEHL
    SVETAVAV
    Rv2629 31 MRSERLRWLVAAEGPFASVYFDDSHDTLDAVERREATWRDVRKHLESRDAKQELID
    SLEEAVRDSRPAVGQRGRALTATGEQVLVNEHLIGPPPATVIRLSDYPYVVPLIDL
    EMRRPTYVFAAVDHTGADVKLYQGATTSSTKIDGVGYPVHKPVTAGWNGYGDFQHT
    TEEAIRMNCRAVADHLTRLVDAADPEVVFVSGEVRSRTDLLSTLPQRVAVRVSQLH
    AGPRKSALDEEEIWDLTSAEPTRRRYAEITNVAQQFEAEIGRGSGLAAQGLAEVCA
    ALRDGDVDTLIVGELGEATVVTGKARTTVARDADMLSELGEPVDRVARADEALPFA
    AIAVGAALVRDDNRIAPLDGVGALLRYAATNRLGSHRS
    Rv2630 32 MLHRDDHINPPRPRGLDVPCARLRATNPLRALARCVQAGKPGTSSGHRSVPHTADL
    RTEAWAPTRDGCIRQAVLGTVESFLDLESAHAVHTRLRRLTADRDDDLLVAVLEEV
    IYLLDTVGETPVDLRLRDVDGGVDVTEATTDASTLVQVGAVPKAVSLNELRESQGR
    HGWRCAVTLDV
    Rv2659c 33 VTQTGKRQRRKFGRIRQFNSGRWQASYTGPDGRVYIAPKTFNAKIDAEAWLTDRRR
    EIDRQLWSPASGQEDRPGAPFGEYAEGWLKQRGIKDRTRAHYRKLLDNHILATFAD
    TDLRDITPAAVRRWYATTAVGTPTMRAHSYSLLRAIMQTALADDLIDSNPCRISGA
    STARRVHKIRPATLDELETITKAMPDPYQAFVLMAAWLAMRYGELTELRRKDIDLH
    GEVARVRRAVVRVGEGFKVTTPKSDAGVRDISIPPHLIPAIEDHLHKHVNPGRESL
    LFPSVNDPNRHLAPSALYRMFYKARKAAGRPDLRVHDLRHSGAVLAASTGATLAEL
    MQRLGHSTAGAALRYQHAAKGRDREIAALLSKLAENQEM
    Rv2780 34 MRVGIPTETKNNEFRVAITPAGVAELTRRGHEVLIQAGAGEGSAITDADPKAAGAQ
    LVGTADQVWADADLLLKVKEPIAAEYGRLRHGQILFTFLHLAASRACTDALLDSGT
    TSIAYETVQTADGALPLLAPMSEVAGRLAAQVGAYHLMRTQGGRGVLMGGVPGVEP
    ADVVVIGAGTAGYNAARIANGMGATVTVLDINIDKLRQLDAEPCGRIHTRYSSAYE
    LEGAVKRADLVIGAVLVPGAKAPKLVSNSLVAHMKPGAVLVDIAIDQGGCFEGSRP
    TTYDHPTFAVHDTLFYCVANMPASVPKTSTYALTNATMPYVLELADHGWRAACRSN
    PALAKGLSTHEGALLSERVATDLGVPFTEPASVLA
    Rv3126c 35 MVIRFDQTGSLVLSMKSLASLSFQRCLRENSSLVAALDRLDAAVDELSALSFDALT
    TPERDRARRDRDHHPWSRSRSQLSPRMAHGAVHQCQWPKAVWAVIDNP
    Rv3127 36 VLKNAVLLACRAPSVHNSQPWRWVAESGSEHTTVHLFVNRHRTVPATDHSGRQAII
    SCGAVLDHLRIAMTAAHWQANITRPPQPNQPDQLATVEFSPIDHVTAGQRNRAQAT
    LQRRTDRLPFDSPMYWHLFEPALRDAVDKDVAMLDVVSDDQRTRLVVASQLSEVLR
    RDDPYYHAELEWWTSPFVLAHGVPPDTLASDAERLRVDLGRDFPVRSYQNRRAELA
    DDRSKVLVLSTPSDTRADALRCGEVLSTILLECTMAGMATCTLTHLIESSDSRDTV
    RGLTRQRGEPQALIRVGIAPPLAAVPAPTPRRPLDSVLQIRQTPEKGRNASDRNAR
    ETGWFSPP
    Rv3128c 37 VWSASGGQCGKYLAASMVLQLDGLERHGVLEFGRDRYGPEVREELLAMSAASIDRY
    LKTAKAKDQISGVSTTKPSPLLRNSIKVRRAGDEVEAEPGFFEGDTVAHCGPTLKG
    EFAHTLNLTDVHIGWVFTRTVRNNARTHILAGLKASVTEIPHGITGLDFDNGTVFL
    NKPVISWAGDNGI-
    YFTRFRPYKKNH*ATIESKNNHLVRKYAFYYRYDTAEERAVLNRMWKLVNDRLNYL
    TPTIKPIGYASSADGRRRRLYDAPQTPLDRPLAARVLSAAQQADLITYRDSLNPAQ
    IGRKIADLQNRLLILAKEKTEQLYLANIPTALPDIHKGILIKAG
    Rv3129 38 VVQGRTVLFRTAEGAKLFSAVAKCAVAFEADDHNVAEGWSVIVKVRAQVLTTDAGV
    REAERAQLLPWTATLKRHCVRVIPWEITGRHFRFGPEPDRSQTFACEASSHNQR
    Rv3130c 39 MNHLTTLDAGFLKAEDVDRHVSLAIGALAVIEGPAPDQEAFLSSLAQRLRPCTRFG
    QRLRLRPFDLGAPKWVDDPDFDLGRHVWRIALPRPGNEDQLFELIADLMARRLDRG
    RPLWEVWVIEGLADSKWAILTKLHHCMADGIAATHLLAGLSDESMSDSFASNIHTT
    MQSQSASVRRGGFRVNPSEALTASTAVMAGIVRAAKGASEIAAGVLSPAASSLNGP
    ISDLRRYSAAKVPLADVEQVCRKFDVTINDVALAAITESYRNVLIQRGERPRFDSL
    RTLVPVSTRSNSALSKTDNRVSLMLPNLPVDQENPLQRLRIVHSRLTRAKAGGQRQ
    FGNTLMAIANRLPFPMTAWAVGLLMRLPQRGVVTVATNVPGPRRPLQIMGRRVLDL
    YPVSPIANQLRTSVAMLSYADDLYFGILADYDVVADAGQLARGIEDAVARLVAISK
    RRKVTRRRGALSLVV
    Rv3131 40 MNTHFPDAETVRTVLTLAVRAPSIHNTQPWRWRVCPTSLELFSRPDMQLRSTDPDG
    RELILSCGVALHHCVVALASLGWQAKVNRFPJDPKDRCHLATIGVQPLVPDQADVAL
    AAATPRRRTDRRAYSCWPVPGGDIALMAARAARGGVMLRQVSALDRMKAIVAQAVL
    DHVTDEEYLRELTIWSGRYGSVAGVPARNEPPSDPSAPIPGRLFAGPGLSQPSDVL
    PADDGAAILALGTETDDRLARLRAGEAASIVLLTATAMGLACCPITEPLEIAKTRD
    AVRAEVFGAGGYPQMLLRVGWAPINADPLPPTPRRELSQVVEWPEELLRQRC
    Rv3132 41 MTTGGLVDENDGAANRPLRHTLSQLRLHELLVEVQDRVEQIVEGRDRLDGLVEAML
    VVTAGLDLEATLRAIVHSATSLVDARYGAMEVHDRQHRVLHFVYEGIDEETVRRIG
    HLPKGLGVIGLLIEDPKPLRLDDVSAHPASIGFPPYHPPMRTFLGVPVRVRDESFG
    TLYLTDKTNGQPFSDDDEVLVQALAAAAGIAVANARLYQQAKARQSWIEATRDIAT
    ELLSGTEPATVFRLVAAEALKLTAADAALVAVPVDEDMPAADVGELLVIETVGSAV
    ASIVORTIPVAGAVLREVFVNGIPRRVDRVDLEGLDELADAGPALLLPLRARGTVA
    GVVVVLSQGGPGAFTDEQLEMMAAFADQAALAWQLATSQRRMRELDVLTDRDRIAR
    DLHDHVIQRLFAIGLALQGAVPHERNPEVQQRLSDVVDDLQDVIQEIRTTIYDLHG
    ASQGITRLRQRIDAAVAQFADSGLRTSVQFVGPLSVVDSALADQAEAVVREAVSNA
    VRHAKASTLTVRVKVDDDLCIEVTDNGRGLPDEFTGSGLTNLRQRAEQAGGEFTLA
    SVPGASGTVLRWSAPLSQ
    Rv3134c 42 MSDPRPARAVVVGIDGSRAATHAALWAVDEAVNRDIPLRLVYVIDPSQLSAAGEGG
    GQSAARAALHDASRKVEATGQPVKIETEVLCGRPLTKLMQESRSAAMLCVGSVGLD
    HVRGRRGSVAATLAGSALCPVAVIHPSPAEPATTSQVSAVVAEVDNGVVLRHAEEE
    ARLRGVPLRAVAVHAAETPDDVEQGSRLAHVHLSRRLAHWTRLYPEVRVDRAIAGG
    SACRHLAANAKPGQLFVADSHSAHELCGAYQPGCAVLTVRSANL
    Rv3841 43 MTEYECPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEER
    NHAMMLVQHLLDRDLRVEIPGVDTVRNQFDRPREALALALDQERTVTDQVGRLTAV
    ARDEGDFLGEQFMQWFLQEQIEEVALMATLVRVADPAGANLFELENFVAREVDVAP
    AASGAPHAAGGRL
    Rv3842c 44 MTWADEVLAGHPFVVAHRGASAARPEHTLAAYDLALKEGADGVECDVRLTRDGHL
    VCVHDRRLDRTSTGAGLVSTMTLAQLRELEYGAWHDSWRPDGSHGDTSLLTLDAL
    VSLVLDWHRPVKIFVETKHPVRYGSLVENKLLALLHRFGIAAPASADRSPAVVMS
    FSAAAVWRIRRAAPLLPTVLLGKTPRYLTSSAATAVGATAVGPSLPALKEYPQLV
    DRSAAQGRAVYCWNVDEYEDIDFCREVGVAWIGTHHPGRTKAWLEDGRANGTTR
    Rv3908 45 VSDGEQAKSRRRRGRRRGRRAAATAENHMDAQPAGDATPTPATAKRSRSRSPRRG
    STRMRTVHETSAGGLVIDGIDGPRDAQVAALIGRVDRRGRLLWSLPKGHTELGET
    AEQTAIREVAEETGTRGSVLAALGRIDYWFVTDGRRVHKTVHHYLMRFLGGELSD
    EDLEVAEVAWVPIRELPSRLAYADERRLAEVADE-
    LIDKLQSDGPAALPPLPPSSPRRRPQTHSRARHADDSAPGQHNGPGPGP
  • [0030]
    TABLE 2
    DNA sequences of selected low oxygen induced antigens
    SEQ ID
    Rv no. NO: Sequence
    Rv0079 46 Gtggaaccgaaacgcagtcgcctcgtcgtatgtgcacccgagccatcgcacgcgcg
    ggaattcccggatgtcgccgtattctccggcggccgggctaacgcatcccaggccg
    aacggttggctcgtgccgtgggtcgcgtgttggccgatcggggcgtcaccgggggt
    gctcgggtgcggctgaccatggcgaactgcgccgatgggccgacgctggtgcagat
    aaacctgcaggtaggtgacaccccattaagggcgcaggccgccaccgcgggcatcg
    atgatctgcgacccgcactgatcagactggatcgacagatcgtgcgggcgtcggca
    cagtggtgcccccggccttggccggatcggccccgccggcgattgaccacgccggc
    cgaggcgctagtcacccgccgcaaaccggtcgtgctaaggcgcgcaaccccgttgc
    aggcgattgccgctatggacgccatggactacgacgtgcatttgttcaccgacgcc
    gagacgggggaggacgctgtggtctatcgggctggaccgtcggggctgcggctggc
    ccgccagcaccacgtatttcccccaggatggtcacgttgtcgcgccccagccgggc
    cgccggtgccgctgattgtgaattcgcgtccgacaccggttctcacggaggccgcc
    gcggtggaccgggcgcgcgaacatggactgccattcctgtttttcaccgaccaggc
    caccggccgcggccagctgctctactcccgctacgacggcaacctcgggttgatca
    ccccgaccggtgacggcgttgccgacggtctggca
    Rv0080 47 Atgagcccgggctcgcggcgcgccagcccgcaaagcgcccgggaggtggtcgagct
    cgaccgtgacgaggcgatgcggttgctggccagcgttgaccatgggcgtgtggtgt
    tcacccgcgcggcgctgccggcgatccgtccagtcaatcacctcgtggtcgacggt
    cgggtgatcgggcgcacccgcctgacggccaaggtgtccgttgcggtgcgatcgag
    cgccgatgccggtgtcgtggtcgcctacgaagccgacgaccttgatccgcggcgtc
    ggacggggtggagtgtggtggtgacgggactggcgaccgaggtcagcgatcccgag
    caggttgcccgctaccagcggctgctacacccgtgggtgaacatggcgatggacac
    cgtggtcgcgatcgaacccgagatcgtcaccggcatccgcatcgttgctgactcgc
    gtacgccg
    Rv0081 48 gtggagtccgaaccgctgtacaagctcaaggcggagttcttcaaaacccttgcgca
    tccggcgcggatcaggattttggagctgctggtcgagcgggaccgttcggtcggtg
    agttgctgtcctcggacgtcggcctggagtcgtcgaacctgtcccagcagctgggt
    gtgctacgccgggcgggtgttgtcgcggcacgtcgtgacggcaacgcgatgatcta
    ttcgattgccgcacccgatatcgccgagctgctggcggtggcacgcaaggtgctgg
    ccagggtgctcagcgaccgggtggcggtgctagaggacctccgcgccggcggc-
    tcggccacg
    Rv0363c 49 Atgcctatcgcaacgcccgaggtctacgcggagatgctcggtcaggccaaacaaaa
    ctcgtacgctttcccggctatcaactgcacctcctcggaaaccgtcaacgccgcga
    tcaaaggtttcgccgacgccggcagtgacggaatcatccagttctcgaccggtggc
    gcagaattcggctccggcctcggggtcaaagacatggtgaccggtgcggtcgcctt
    ggcggagttcacccacgttatcgcggccaagtacccggtcaacgtggcgctgcaca
    ccgaccactgccccaaggacaagttggacagctatgtccggcccttgctggcgatc
    tcggcgcaacgcgtgagcaaaggtggcaatcctttgttccagtcgcacatgtggga
    cggctcggcagtgccaatcgatgagaacctggccatcgcccaggagctgctcaagg
    cggcggcggccgccaagatcattctggagatcgagatcggcgtcgtcggcggcgaa
    gaggacggcgtggcgaacgagatcaacgagaagctgtacaccagcccggaggactt
    cgagaaaaccatcgaggcgctgggcgccggtgagcacggcaaatacctgctggccg
    cgacgttcggcaacgtgcatggcgtctacaagcccggcaacgtcaagcttcgcccc
    gacatccttgcgcaagggcaacaggtggcggcggccaagctcggactgccggccga
    cgccaagccgttcgacttcgtgttccacggcggctcgggttcgcttaagtcggaga
    tcgaggaggcgctgcgctacggcgtggtgaagatgaacgtcgacaccgacacccag
    tacgcgttcacccgcccgatcgccggtcacatgttcaccaactacgacggagtgct
    caaggtcgatggcgaggtgggtgtcaagaaggtctacgacccgcgcagctacctca
    agaaggccgaagcttcgatgagccagcgggtcgttcaggcgtgcaatgacctgcac
    tgcgccggaaagtccctaacccac
    Rv0572c 50 atgggtgagcacgccatcaagcggcacatgcggcaacggaagcctacgaagcatcc
    cctagcccagaaacggggcgcgcggattctggtcttcaccgacgatccccgcagga
    gcgtcctcatagtgcccggttgccacctggattccatgcgccgagaaaagaacgcg
    tactacttccaggacggcaatgcgttggttgggatggttgtctcgggcggcacggt
    tgagtacgacgccgacgaccgcacatatgtcgtgcagctcaccgacggaaggcaca
    ccactgagtcatctttcgaacactcatcgccgagtcgatcacctcaatccgat-
    gaccta
    Rv0574c 51 gtggctggcaatcctgatgtggtgacggtgctgctgggcggtgacgtcatgctcgg
    ccgtggcgtcgatcagatcctgcctcatcccggcaaaccgcaattgcgcgaacggt
    atatgcgggatgcgaccggctatgttcgcctggccgagcgggtgaacgggcgcatt
    ccgctccccgtggattggcgctggccctggggcgaggcgttggcggtccttgagaa
    caccgcgaccgacgtctgtttgatcaatctggagacgacgatcaccgccgacggtg
    aattcgccgaccgcaaaccggtctgctaccggatgcacccggataacgtgccggcg
    ctgacggcattgcggccgcacgtgtgcgcgctggccaacaaccacattctcgattt
    cggctaccaggggctgaccgatacggtcgcggctctcgccggtgcggggatccaga
    gtgtcggggcgggagccgatttgctcgccgctcgccgctcggcgctagtcacggtt
    ggccatgaacgccgggtgatcgtcggctcggtagcggcggaatccagcggcgtccc
    cgaatcctgggccgcccgccgcgaccggcccggagtgtggttgatccgggatccgg
    cgcaacgcgacgtcgccgacgatgtggcggcacaggtgctggcggacaaacgcccc
    ggcgatatcgccatagtctcgatgcattggggatccaattggggctatgcgaccgc
    acccggcgacgtcgcgttcgcgcaccgactgatcgacgccggcatcgacatggtcc
    acggacattcctcgcaccatccgcggccaatcgagatatatcgcggtaaaccgatc
    ctgtacggatgcggtgacgtcgttgacgactacgaaggcatcggcgggcacgagtc
    gttccgcagtgaactgcgactgctgtatctgaccgtcaccgatcccgccagcggga
    acctgatctcgctgcagatgcttccactgcgagtgtcgcggatgcgcctacagcgt
    gcctcccagaccgacaccgaatggctccgcaacaccattgagcgcatcagccgccg
    gttcgggattcgagtcgtgactcgacccgacaacctgctggaggtcgttcccgctg
    ccaacctaacgagcaaggag
    Rv1264 52 gtgacagaccacgtgcgcgaggcggacgacgcgaacatcgacgatctgttgggcga
    cctgggcggtaccgcgcgcgccgagcgtgcgaagcttgtcgagtggttgctcgagc
    agggcatcacccccgacgagattcgggcgaccaacccgccgttgctgctggccacc
    cgccacctcgtcggcgacgacggcacctacgtatccgcaagggagattagcgagaa
    ctatggcgttgacctcgagctgctgcagcgggtgcagcgcgctgtcggtctggcca
    gagtggatgatcctgacgcggtggtgcacatgcgtgccgacggtgaggcggccgca
    cgcgcacagcggttcgttgagctggggctgaatcccgaccaagtcgtgctggtcgt
    gcgtgtgctcgccgagggcttgtcacacgccgccgaggccatgcgctacaccgcgc
    tggaggccattatgcggccgggggctaccgagttggacatcgcgaaggggtcgcag
    gcgctggtgagccagatcgtgccgctgctggggccgatgatccaggacatgctgtt
    catgcagctgcggcacatgatggagacggaggccgtcaacgccggagagcgtgcgg
    ccggcaagccgctaccgggagcgcgacaggtcaccgttgccttcgccgacctggtc
    ggtttcacccagctaggcgaagtggtgtcggccgaagagctagggcacctcgccgg
    gcggctggccggcctcgcgcgtgacctgaccgctccgccggtgtggttcattaaga
    cgatcggcgacgcggtcatgttggtctgtcctgatccggcgccattgctggacacc
    gtgctgaagctggtcgaggtcgtcgacaccgacaacaactttccccggctgcgagc
    cggcgtcgcctccgggatggcggttagccgggccggcgactggttcggcagcccgg
    tcaacgtggcaagccgggtgaccggggtggcgcgcccgggtgccgtgctggtcgcg
    gattcggtgcgggaggcccttggtgatgcccccgaagccgacggatttcagtggtc
    cttcgccggcccccgtcgcctcaggggaatccggggtgacgtcaggctttttcgag
    tccggcgaggggccactcgcaccggctccggcggcgcggcccaagacgacgatttg
    gccggctcgtcaccg
    Rv1592c 53 atggtagagcccggcaatttggcaggcgcgaccggcgccgaatggatcggccggcc
    accgcacgaggaattgcagcgcaaagtgcgcccgctgctgccatccgacgatccgt
    tctacttcccacctgccggctaccagcatgccgtgcccggaacggtgttgcgctcg
    cgcgatgtcgaactggcgtttatgggcttgattccgcagcccgtcaccgctaccca
    gctgctgtaccggaccacgaacatgtacggcaaccccgaggcgacggtgaccacgg
    tgatcgtcccagcggagcttgccccgggtcagacctgccccttgctgtcgtaccag
    tgtgcgatcgatgccatgtcgtcgcgctgttttccgtcatatgccctgcgacgacg
    ggccaaggccctggggtcactgacccaaatggagctgttgatgatcagcgccgcac
    ttgccgaaggatgggcggtatcagtacccgaccatgaagggccgaaagggctgtgg
    gggtcgccgtatgaacccggttaccgagtcctcgacggaatccgggctgccttgaa
    ttccgagcgtgtcgggttgtccccggcaacgccgatcgggctgtggggctactccg
    gcggcgggctggccagcgcgtgggccgccgaagcatgcggcgagtacgcaccggac
    ctagacatcgtcggcgccgtgctgggatcacccgtcggtgaccttggtcacacgtt
    ccgccggctcaatggcactcttcttgccggtctgcccgcgttggtggtggccgcgc
    tgcaacacagctaccccggcctggcccgggtgatcaaggagcacgccaacgacgaa
    ggacgtcagctgctggagcaactgacggagatgacaacggtagacgcagtgatccg
    gatggccggcagggacatgggtgacttcctcgacgaaccccttgaggacattctgt
    cgacgccggaaatttcccatgtcttcggcgacaccaagctgggtagcgcggtgccc
    accccgccggtattgatcgtgcaggccgtgcatgactacctcatcgacgtctctga
    catcgacgcgctcgctgacagctatacagccggcggcgccaacgtcacctaccacc
    gcgacctgttcagcgaacatgtgtccctgcacccgctgtcggccccaatgacgctt
    cgctggctcaccgaccggttcgccggcaagccactgaccgaccaccgcgtccggac
    cacgtggccgaccatcttcaacccgatgacctacgccggcatggcgagactggccg
    tgatcgcggccaaggtgatcaccggcaggaagttgagccgccgtccgctc
    Rv1733c 54 atgatcgccacaacccgcgatcgtgaaggagccaccatgatcacgtttaggctgcg
    cttgccgtgccggacgatactgcgggtgttcagccgcaatccgctggtgcgtggga
    cggatcgactcgaggcggtcgtcatgctgctggccgtcacggtctcgctgctgact
    atcccgttcgccgccgcggccggcaccgcagtccaggattcccgcagccacgtcta
    tgcccaccaggcccagacccgccatcccgcaaccgcgaccgtgatcgatcacgagg
    gggtgatcgacagcaacacgaccgccacgtcagcgccgccgcgcacgaagatcacc
    gtgcctgcccgatgggtcgtgaacggaatagaacgcagcggtgaggtcaacgcgaa
    gccgggaaccaaatccggtgaccgcgtcggcatttgggtcgacagtgccggtcagc
    tggtcgatgaaccagctccgccggcccgtgccattgcggatgcggccctggccgcc
    ttgggactctggttgagcgtcgccgcggttgcgggcgccctgctggcgctcactcg
    ggcgattctgatccgcgttcgcaacgccagttggcaacacgacatcgacagcctgt
    tctgcacgcagcgg
    Rv1734c 55 atgaccaacgtcggtgaccagggggttgacgcggtcttcggggtgatctacccacc
    tcaggtcgcgctggtcagtttcggcaagccggcacaacgagtttgcgccgtcgacg
    gcgcgatccacgtcatgacgaccgtgctggctacgctgcccgctgaccacggctgc
    agcgatgaccatcgcggcgcgctgttcttcctgtcgatcaacgagctgacgcggtg
    cgccgcagtaacagga
    Rv1736c 56 gtgacggtgacaccacggaccggcagccgcatcgaggagctgcttgcacgcagcgg
    ccggttcttcatcccgggtgagatctcggcggatctgcgtaccgtgacccgccgcg
    gcggccgcgacggcgacgtgttctatcgagaccggtggagccacgacaaggtggtc
    cgctccacacacggggtgaattgcaccgggtcgtgttcttggaagatctacgtcaa
    agacgacatcatcacctgggagacgcaggagaccgactatccgtcggtgggcccgg
    accggcccgagtatgagccccgcggctgcccgcgcggcgcggcgttttcctggtac
    acgtattcgccgacgcgggtgcgccatccgtacgcccgcggcgtgcttgtcgagat
    gtatcgggaggcgaaggcacgtttgggtgatccggtggcggcctgggccgacatcc
    aggccgacccgcggcggcgccgccgctaccagcgcgcccgcggcaagggcgggctg
    gtccgggtcagctgggccgaggccaccgagatgatcgccgccgcccacgtgcacac
    catctccacatacggcccggaccgggttgccggcttctcccccatcccggcgatgt
    ccatggtgagccacgccgcggggtcgcggttcgtggagctaatcggcggggtgatg
    acgtcgttctacgactggtacgccgacctgccggtggcctccccgcaggtgttcgg
    cgaccagaccgacgtgccggagtccggagattggtgggacgtggtgtggcaatgcg
    cctcggtgctgctgacctacccgaactcacggcaactcggcaccgcagaggaattg
    ctggcccacatcgacggtccggccgcggatctgttggggcgcacggtctctgagct
    gcgccgtgccgatccgctgaccgcggcgacccgctacgtcgacaccttcgacctgc
    gaggccgcgccaccctgtacctgacctactggaccgccggcgacacccgcaaccgc
    ggccgggagatgctggccttcgcccagacctaccgcagcaccgacgtcgcaccacc
    gcgcggcgagaccccggacttcctgccggtggtgctcgaattcgccgcgaccgtcg
    accccgaggcggggcgacggttgctgagcgggtaccgggtgcccatcgccgcgctg
    tgcaatgccctgaccgaggccgcattgccatacgcacacacggtggccgcggtatg
    ccggacgggtgacatgatgggcgaactcttctggaccgtcgtgccgtatgtgacga
    tgacgatcgtcgcggtcggctcctggtggcgctaccgctatgacaaattcggctgg
    accacccgctcgtcccagctgtacgagtcgcggctgctgcggatcgccagcccgat
    gtttcatttcggcatcctggtggtcatcgtcggccacggtatcgggctcgtgatcc
    cgcagtcgtggactcaggccgccggtttgagcgagggcgcatatcacgtgcaggcc
    gtcgtgctggggtcgatcgccggcatcaccaccttggccggcgttaccctgctgat
    ctaccggcggcgcacccgcgggccggtgttcatggctaccaccgtcaacgacaagg
    tgatgtacctcgtgctggtggcggcgatcgtcgcgggactgggtgcgacggcgttg
    ggctccggcgttgtcggcgaggcgtacaactaccgcgagacggtgtcggtgtggtt
    ccgctcggtgtgggtactgcaaccgcgcggggacctgatggccgaggctccgctgt
    attaccagatccatgtgctgatcgggttggcgttgttcgcgttgtggccgttcacc
    cggctggtacacgcgttcagcgccccgatcggctatctgttccgcccgtacatcat
    ctaccgcagccgcgaggagctggtgctaacgcggccgcggcggcgcgggtgg
    Rv1737c 57 atgagagggcaagcggccaatctcgtgctggccacctggatctcggtggtcaactt
    ctgggcgtggaacctgatcggcccgctgtcgaccagctacgcgcgtgacatgtcac
    tgtccagcgccgaggcgtcgctgctcgtcgccaccccgatcctggtgggtgccctt
    ggccgcatcgtcaccgggccgctcaccgaccgcttcggcgggcgcgccatgctcat
    cgcggtgacgctggcgtcgatcctcccggtgctcgcggtcggggtcgcggcaacca
    tgggctcctacgcgttgctggtgtttttcgggctcttcctgggcgttgccggcacg
    atcttcgccgtcggcatcccgttcgccaacaactggtaccagccggcgcggcgcgg
    tttctccaccggcgtgttcggtatgggcatggtcggcaccgcgctctcggcgttct
    tcaccccgcggtttgtacggtggttcggcctgttcaccacccacgccatcgtcgcg
    gccgcgctcgcgtcgaccgccgtggtggccatggtcgtgcttcgtgatgcacccta
    ctttcggcccaacgccgacccggtgctgcccaggctcaaggccgcggcacggttgc
    cggtgacctgggagatgtcgtttctgtacgcgatcgtgttcggcgggttcgtggcg
    ttcagcaactacctgcccacctacatcaccacgatctacgggttctccacggtcga
    cgcgggcgctcgcaccgccgggttcgccctggcggcggtgctggcccggccggtgg
    gcgggtggctctccgaccggatcgcaccgaggcacgtggtgctggcctcgctcgcc
    gggaccgcgctgctggcgttcgccgcggcgttgcagccgccgccggaggtgtggtc
    ggcggccaccttcatcaccctggcggtctgcctcggcgtgggcaccggcggcgtgt
    tcgcgtgggtggcccgccgcgccccggccgcatcggtcggctcggtcaccggaatc
    gtcgccgcggcaggcggattgggcggttacttcccgccgctggtgatgggcgcgac
    ctacgacccggtcgacaacgactacacggtcgggttgctgctgctggtggcgaccg
    cgctggtcgcgtgtacctacaccgcgctgcacgcgcgggagccggtgagtgaggag
    gcgtccagg
    Rv1738 58 atgtgcggcgaccagtcggatcacgtgctgcagcactggaccgtcgacatatcgat
    cgacgaacacgaaggattgactcgggcgaaggcacggctgcgttggcgggaaaagg
    aattggtgggtgttggcctggcaaggctcaatccggccgaccgcaacgtccccgag
    atcggcgatgaactctcggtcgcccgagccttgtccgacttggggaagcgaatgtt
    gaaggtgtcgacccacgacatcgaagctgttacccatcagccggcgcgattgttg-
    tat
    Rv1739c 59 atgattcccacgatgacatcggccggctgggcaccaggggtggtgcagttccgcga
    ataccaacggcgttggctgcgcggcgatgtcctcgccggcctgaccgtggccgcct
    atctgatcccgcaagcgatggcgtatgcgaccgtggcgggcctaccgccggcagcc
    gggctgtgggcgtcgatcgcgccgcttgccatttacgcactgctcggatcgtcccg
    gcagctttcaatcggcccggaatccgccaccgccttgatgacggcggccgtgctcg
    ctccgatggccgccggggatcttcgacgctatgccgttctggcggcaaccctcgga
    ttgctagtcggccttatctgcctactcgctggcacggcgcgactaggtttcctcgc
    cagcctgcgatcgcggccggtgctcgtcggatacatggccggcatcgcgcttgtca
    tgatctccagccaactcggcactatcaccggcacctcggtcgaaggcaacgaattc
    ttcagcgaagtacactctttcgcgactagcgtcacgcgagttcactggccgacttt
    tgtgttagccatgtctgtcctagcgctgctaactatgctcacgcggtgggcgccgc
    gcgcccccggaccgatcatcgcggttcttgcggccacgatgctagtggccgttatg
    tccttggatgccaaaggtattgcgattgtgggtcggataccttccggtctgccgac
    gccgggtgtgccgcccgtttcggtggaagacttgcgggcactgatcattccggctg
    ccgggatcgcgattgttaccttcaccgacggtgtgttgaccgcacgcgccttcgcc
    gctcgtcgaggtcaggaagtcaatgccaacgccgagctgcgcgcggtcggggcctg
    caacatcgccgccgggctgacacacggttttccggtgagttccagcagcagccgta
    ccgccctcgccgacgtcgtcggtggccgcacccagctgtactcgctgatcgcgttg
    gggcttgttgtcatcgtgatggttttcgcgagtgggctgctggccatgtttccgat
    cgccgctctgggcgctttggtggtatatgccgcgctacgcttgatcgacttgtcag
    aattccggcgactggcgcggtttcggcgcagcgaactcatgctggcactagccacc
    acagcagccgtgttaggcctaggagtgttctatggagtcctcgccgcggttgccct
    gtccatcctcgaactgcttcgtcgggtcgcacatccgcatgacagcgttctcgggt
    tcgtgccgggcattgccggcatgcacgacatcgatgactatccgcaggccaagcgc
    gtgcccgggctggtggtgtatcgctatgacgcgccgttgtgcttcgccaatgccga
    agacttccgcaggcgagcactgaccgtggtcgatcaggatccggggcaagtcgagt
    ggttcgtactcaacgccgaatccaatgtggaggtcgacctgactgcgctggatgcg
    ctcgaccaactccgcaccgagctgctgcgtcggggaatagtgttcgccatggcccg
    ggtcaaacaagacttgcgtgaatcactcagggccgccagtcttctcgataagattg
    gcgaagaccatatctttatgacattgcctaccgcagtgcaggcgttccgtcggcgc
    Rv1813c 60 atgatcacaaacctccgacgccgaaccgcgatggcagccgccggcctaggggctgc
    tctcgggctgggcatcctgctggttccgacggtggacgcccatctcgccaacggtt
    cgatgtcggaagtcatgatgtcggaaattgccgggttgcctatccctccgattatc
    cattacggggcgattgcctatgcccccagcggcgcgtcgggcaaagcgtggcacca
    gcgcacaccggcgcgagcagagcaagtcgcactagaaaagtgcggtgacaagactt
    gcaaagtggttagtcgcttcaccaggtgcggcgcggtcgcctacaacggctcgaaa
    taccaaggcggaaccggactcacgcgccgcgcggcagaagacgacgccgtgaaccg
    actcgaaggcgggcggatcgtcaactgggcgtgcaac
    Rv1997 61 ttgtcggcgtcagtgtctgccacgacggctcatcatggcttgccagcacatgaagt
    ggtgctgctgctggagagcgatccatatcacgggctgtccgacggcgaggccgccc
    aacgactagaacgcttcgggcccaacaccttggcggtggtaacgcgcgctagcttg
    ctggcccgcatcctgcggcagtttcatcacccgctgatctacgttctgctcgttgc
    cgggacgatcaccgccggtcttaaggaattcgttgacgccgcagtgatcttcggtg
    tggtggtgatcaatgcgatcgtgggtttcattcaagaatccaaggcagaggccgca
    ctgcagggcctgcgctccatggtgcacacccacgccaaggtggtgcgcgagggtca
    cgagcacacaatgccatccgaagagctggttcccggtgaccttgtgctgttagcgg
    ccggtgacaaggttcccgccgatttgcggctggtgcgacagaccggattgagcgtg
    aacgagtcagcacttaccggcgagtcgacgccggttcacaaggacgaggtggcgtt
    gccggagggcacaccggtcgctgatcgtcgcaatatcgcgtattccggcacattgg
    taaccgcgggccatggcgccgggatcgtcgtcgcgaccggcgccgaaaccgaactc
    ggtgagattcatcggctcgttggggccgccgaggttgtcgccacaccgctgaccgc
    gaagctggcgtggttcagcaagtttctgaccatcgccatcctgggtctggcagcgc
    tcacgttcggcgtgggtttgctgcgccggcaagatgccgtcgaaacgttcaccgct
    gcgatcgcgctggcggtcggggcaattcccgaaggtctgcccaccgccgtgaccat
    caccttggccatcggcatggcccggatggccaagcgccgcgcggtcattcgacgtc
    tacccgcggtggaaacgctgggcagcaccacggtcatctgcgccgacaagaccgga
    acgctgaccgagaatcagatgacggtccagtcgatctggacaccccacggtgagat
    ccgggcgaccggaacgggctatgcacccgacgtcctcctgtgcgacaccgacgacg
    cgccggttccggtgaatgccaatgcggcccttcgctggtcgctgctggccggtgcc
    tgcagcaacgacgccgcactggttcgcgacggcacacgctggcagatcgtcggcga
    tcccaccgagggcgcgatgctcgtcgtggccgccaaggccggcttcaacccggagc
    ggctggcgacaactctgccgcaagtggcagccataccgttcagttccgagcggcaa
    tacatggccaccctgcatcgcgacgggacggatcatgtggtgctggccaagggtgc
    tgtggagcgcatgctcgacctgtgcggcaccgagatgggcgccgacggcgcattgc
    ggccgctggaccgcgccaccgtgttgcgtgccaccgaaatgttgacttcccggggg
    ttgcgggtgctggcaaccgggatgggtgccggcgccggcactcccgacgacttcga
    cgaaaacgtgataccaggttcgctggcgctgaccggcctgcaagcgatgagcgatc
    caccacgagcggccgcggcatcggcggtggcggcctgccacagtgccggcattgcg
    gtaaaaatgattaccggtgaccacgcgggcaccgccacggcgatcgcaaccgaggt
    ggggttgctcgacaacactgaaccggcggcaggctcggtcctgacgggtgccgagc
    tggccgcgctgagcgcagaccagtacccggaggccgtggatacagccagcgtgttt
    gccagggtctctcccgagcagaagctgcggttggtgcaagcattgcaggccagggg
    gcacgtcgtcgcgatgaccggcgacggcgtcaacgacgccccggccttgcgtcagg
    ccaacattggcgtcgcgatgggccgcggtggcaccgaggtcgccaaggatgccgcc
    gacatggtgttgaccgacgacgacttcgccaccatcgaagccgcggtcgaggaagg
    ccgcggcgtattcgacaatctgaccaagttcatcacctggacgctgcccaccaacc
    tcggtgagggcctagtgatcttggccgccatcgctgttggcgtcgccttgccgatt
    ctgcccacccaaattctgtggatcaacatgaccacagcgatcgcgctcggactcat
    gctcgcgttcgagcccaaggaggccggaatcatgacccggccaccgcgcgaccccg
    accaaccgctgctgaccggctggcttgtcaggcggactcttctggtttccaccttg
    ctcgtcgccagcgcgtggtggctgtttgcatgggagctcgacaatggcgcgggcct
    gcatgaggcgcgcacggcggcgctgaacctgttcgtcgtcgtcgaggcgttctatc
    tgttcagctgccggtcgctgacccgatcggcctggcggctcggcatgttcgccaac
    cgctggatcatcctcggcgtcagtgcgcaggccatcgcgcaattcgcgatcacata
    tctacccgcgatgaatatggtgttcgacaccgcgccaatcgatatcggggtgtggg
    tgcgcatattcgctgtcgcgaccgcaatcacgattgtggtggccaccgacacgctg
    ctgccgagaatacgggcgcaaccgcca
    Rv1998c 62 atgagtttccacgatcttcatcaccaaggtgttccgttcgtgttgcccaacgcctg
    ggatgtgccgtcggccctggcctacctcgcggagggcttcacggctatcggcacaa
    ccagtttcggggtctcgtccagcggcgggcacccggacgggcaccgcgccactcgc
    ggcgccaacatcgcactggcggccgccctggcaccgctgcaatgctacgtcagcgt
    cgacatcgaggacggatacagcgacgaacccgacgccattgctgactacgtcgcac
    aactgtcgacagccggaatcaatatcgaggacagtagcgccgaaaagctcatcgac
    cccgccctggcagccgctaaaatcgttgcgatcaaacaacgtaaccccgaggtgtt
    cgtcaacgcccgcgtcgacacctattggttgcgccagcacgccgataccaccagca
    cgatccagcgcgcacttcgctacgtcgatgccggcgccgacggcgtctttgtccca
    ctggccaacgatcccgacgaacttgctgagctcactcgcaacattccgtgcccggt
    taacacgttgcccgtgcccggcttgacgatcgccgaccttggtgagctcggggtgg
    cccgggtgtcaaccggttcagtgccctacagcgcggggttgtatgcagcggcccac
    gcggctcgggccgtgagcgacggagagcagctgccacggtccgtaccgtacgccga
    actgcaggcacgcttggttgactacgagaaccgcacgagtacaacg
    Rv2003c 63 gtggtcaagcgctctcgggcaacccgactttcgccgagcatctggtccggatggga
    atcacctcagtgtcggtccattcgggcgcgattgctgctaccccggggtcggtcgc
    ggccgccgaacgccgattgttgctggaatcagctcgcggtgacgcctgacacccgg
    atgccggcatcgtcggccgccgggcgcgacgcggcggcctacgacgcctggtatga
    ctcacccaccgggcggccgatcctggcgaccgaggtcgccgcgttgcggccgctca
    tcgaggtctttgcccagccacgcttggaaatcggtgtcggtacaggacgtttcgcc
    gacctgctcggcgtgcggttcggactcgatccatcccgtgatgcgctgatgttcgc
    acgccggcgcggcgtcctggtcgccaatgccgtcggcgaggcggtccctttcgtca
    gccggcacttcggggcggtcctcatggcattcacgctctgtttcgtcaccgacccg
    gccgccatattccgggaaacgcggcgtctgctcgccgacggcggcggccttgttat
    cgggttcttgcctcgcgggacaccgtgggccgacctgtacgctctgcgcgcggccc
    gcggacagccaggctaccgcgacgcccgcttctacaccgcggccgaactcgaacaa
    ctgctcgcagactcgggattccgggtcatcgcccgccgctgcacgctgcaccaacc
    gccgggactcgcccggtacgacatcgaagccgcccatgacggtatccaagccggcg
    ccggcttcgttgctatctcggcggtcgaccaagcgcacgagcctaaggatgatcac
    ccactcgagtcggaa
    Rv2005c 64 atgtctaaaccccgcaagcagcacggagttgtcgtcggggtagatggttcgctcga
    atcggatgccgccgcctgttggggtgccaccgatgcggcgatgaggaacattccgc
    tgaccgtggtccacgtggtgaacgccgatgtagcgacgtggccgccgatgccgtat
    ccggagacctggggggtttggcaggaggacgagggtcgccagatcgtcgccaacgc
    cgtcaagctcgccaaagaggcggttggagcggatcgaaagctcagcgtaaagagcg
    agctcgtattttccacgccggtacctaccatggttgaaatctccaacgaggcagag
    atggtggtgttgggcagctcgggccggggagcgctggcccgaggcttgctcggttc
    ggtcagctcgagcctggtgcgacgcgccgggtgcccggtcgcggtcatccacagcg
    atgatgcggtgatccctgatccgcagcacgctcccgtgctggtgggaatcgacggt
    tcgccggtttcggagcttgcgacggcggtggcatttgacgaggcgtcgcgccgcgg
    cgtcgaactgatcgccgtgcacgcgtggagtgacgtcgaagtggtggaacttccgg
    gtttggacttctcggctgtacagcaggaagcggagcttagtctcgccgaacgcttg
    gcaggttggcaagaacgctatcccgatgtgccggtgagccgggttgtcgtttgcga
    tcgcccggcgcggaagctggtgcaaaagtcggcgtccgcccagcttgtcgtcgttg
    gcagtcatggccgaggtggcttgaccggcatgcttctggggtcggtcagtaacgcg
    gtcttacacgccgcgcgggtgccagtgatcgtggcacggcagtcg
    Rv2007c 65 gtgacctatgtgatcggtagtgagtgcgtggatgtgatggacaagtcctgtgtgca
    ggagtgtccggtcgactgtatctatgagggcgcccgaatgctctacatcaaccccg
    acgagtgcgtggattgtggtgcgtgcaaaccggcctgccgcgtcgaggcgatctac
    tgggaaggcgatctacccgacgatcaacaccagcatctgggggacaacgccgcctt
    tttccaccaagtcctgccgggccgagtggctccgctgggttcgccgggtggtgccg
    cagcggtgggcccgatcggagtcgacacgcctctggtcgcggctatcccggtg-
    gagtgccct
    Rv2028c 66 atgaaccaatcacacaaacccccatcgatcgtcgtcggtattgatggctcgaagcc
    ggccgtgcaagccgcactgtgggcggtcgacgaggcagccagccgtgacatcccgc
    tgcgtctgctgtacgcgatcgaacccgacgatcccgggtacgccgcacacggcgcg
    gcggctcgcaaactcgccgccgccgagaacgcggtgcgctacgcgttcacagcggt
    cgaggcggcggaccggccggtcaaggtcgaggtggagatcacccaggagcggccgg
    tcacctcgttgatccgcgcttcggcggctgctgccctggtgtgcgttggcgctatc
    ggcgtgcaccacttccgaccggagcgggtgggatctaccgcagcggccctggcgtt
    atcggcgcagtgcccagtggcgatcgtgcgaccccaccgggtccccatcggacgcg
    acgccgcatggatcgtcgtcgaggcggacgggtcgtccgatatcggtgttttgctg
    ggggcggtgatggccgaagcacggctgcgcgactcgccggttcgggtggtcacctg
    ccggcaatccggagtgggcgataccggggacgacgtccgtgccagcctggaccgct
    ggcttgcccgttggcaaccacggtatcccgatgtgcgggtgcaatcggcggcagtg
    cacggcgagctgctggattatctggctgggctgggtcgatcggtacacatggtggt
    gctcagcgcgagcgaccaggagcatgtggagcaacttgtgggagcgccgggcaacg
    ccgtgttgcaggaggccggctgcaccctgctggtcgtcggtcagcagtatctg
    Rv2029c 67 atgacggagccagcggcgtgggacgaaggcaagccgcgaatcatcactttgaccat
    gaaccccgccttggacatcacgacgagcgtcgacgtggtgcgcccgaccgagaaaa
    tgcgttgtggcgcacctcgctacgatcccggcggcggcggtatcaatgtcgcccgc
    attgtgcatgtcctcggcggttgctcgacagcactgttcccggccggcgggtcgac
    cgggagcctgctgatggcgctgctcggtgatgcgggagtgccatttcgcgtcattc
    cgatcgcggcctcgacgcgggagagcttcacggtcaacgagtccaggaccgccaag
    cagtatcgtttcgtgcttccggggccgtcgctgaccgtcgcggagcaggagcaatg
    cctcgacgaactgcgcggtgcggcggcttcggccgcctttgtggtggccagtggca
    gcctgccgccaggtgtggctgccgactactatcagcgggttgccgacatctgccgc
    cgatcgagcactccgctgatcctggatacatctggtggcgggttgcagcacatttc
    gtccggggtgtttcttctcaaggcgagcgtgcgggaactgcgcgagtgcgtcggat
    ccgaactgctgaccgagcccgaacaactggccgccgcacacgaactcattgaccgt
    gggcgcgccgaggtcgtggtggtctcgcttggatctcagggcgcgctattggccac
    acgacatgcgagccatcgattttcgtcgattccgatgaccgcggttagcggtgtcg
    gcgccggcgacgcgatggtggccgcgattaccgtgggcctcagccgtggctggtcg
    ctcatcaagtccgttcgcttgggaaacgcggcaggtgcagccatgctgctgacgcc
    aggcaccgcggcctgcaatcgcgacgatgtggagaggttcttcgagctggcggccg
    aacccaccgaagtcgggcaggatcaatacgtttggcacccgatcgttaacccggaa
    gcctcgcca
    Rv2030c 68 gtgctgatgaccgcagcggctgatgtcacccggcgctcgccgcggcgcgtgttccg
    tgaccgccgcgaggccggccgggtgctggcggaattactcgccgcctatcgggacc
    agccggacgtgattgtgctcggcttggcccggggtggcctcccggtcgcatgggag
    gttgccgcggcactgcatgccccgctagacgccttcgtcgtgcgcaaacttggtgc
    cccggggcatgacgagttcgccgttggtgcactggccagcggcggccgcgtcgtgg
    tcaatgacgacgtcgtgcggggcctgcggatcacaccgcagcaactgcgcgacatc
    gccgaacgtgagggtcgggaactgcttcggcgcgagtccgcctaccgcggcgagcg
    cccgcccaccgatatcaccggcaagacggtcattgtcgtcgatgacggtttggcca
    ccggcgcaagcatgttcgcggcggtacaggcattgcgcgatgcgcaaccagcgcag
    atcgtgattgccgtgccggcggcgccggagtccacgtgccgggagttcgccggcct
    cgtcgacgacgttgtgtgcgcgaccatgccgaccccgttcctggccgtcggtgagt
    cgttttgggacttccggcaggtcaccgacgaggaggtccgccggctcctggccacc
    ccgaccgctgggccgtcgctgcgccggcccgcggcgtcaacggcggccgatgttct
    gcgcagagtcgcgatcgacgcccccgggggtgttccgacgcacgaggtgttggcgg
    agctggtcggcgatgcacgaatcgtgttgatcggcgaaagctcgcacggcacacac
    gagttctaccaggcccgggccgccatgacacagtggctgatcgaggagaagggctt
    tggtgcggtagccgccgaggcggactggcccgacgcctaccgggtcaatcggtacg
    ttcgcggcctcggcgaggacaccaacgctgacgaggcgcttagcggattcgagcgg
    tttcccgcctggatgtggcgcaacaccgtggtccgagattttgtggaatggctgcg
    cacacgcaaccagcgctacgagtcgggcgcgctgcggcaagccggcttctacggtc
    tggatctttacagcctgcatcggtcgatccaagaggtgatcagctatctcgacaag
    gtcgacccgcgtgcggcggcacgggcgcgggcccggtatgcgtgcttcgaccatgc
    ctgcgccgatgacggtcaggcgtacggattcgcggccgcattcggcgccggtccgt
    cgtgcgaacgtgaagccgtcgagcaactggtcgacgttcagcgcaatgccctggcg
    tatgcgcgccaagacgggctgcttgccgaggacgaactgttctacgcccagcaaaa
    cgcgcagacggtgcgcgacgcagaggtgtattaccgggccatgttcagtggacgcg
    ttacctcgtggaacctgcgcgaccagcacatggcgcagacccttggcagtttgctg
    acgcatttggaccgacacctcgatgcgccgccggcgcgaatagtggtgtgggctca
    taactcccacgtgggtgacgcacgcgctaccgaggtgtgggccgacgggcagctca
    ccctcggccagatagtccgtgagcgatacggtgacgagtcgcgcagcatcggattc
    agcacgtacacgggcaccgtcaccgcggccagcgaatggggtggtatcgcccaacg
    caaagcggttcggccggcactgcacggcagtgtcgaggagctcttccaccagactg
    cagacagtttcctggtgtcagcgcggctaagccgcgacgccgaagccccgctggac
    gttgtccggttgggacgtgccatcggcgtcgtttatctaccggcaacggaacggca
    aagtcactacttgcacgtgcggcccgccgaccagttcgacgccatgatccacatcg
    atcagacccgtgccctggaacctctcgaggtgacgagccggtggatcgccggcgag
    aacccggaaacctacccgaccggtctg
    Rv2031c 69 Atggccaccacccttcccgttcagcgccacccgcggtccctcttccccgagttttc
    tgagctgttcgcggccttcccgtcattcgccggactccggcccaccttcgacaccc
    ggttgatgcggctggaagacgagatgaaagaggggcgctacgaggtacgcgcggag
    cttcccggggtcgaccccgacaaggacgtcgacattatggtccgcgatggtcagct
    gaccatcaaggccgagcgcaccgagcagaaggacttcgacggtcgctcggaattcg
    cgtacggttccttcgttcgcacggtgtcgctgccggtaggtgctgacgaggacgac
    attaaggccacctacgacaagggcattcttactgtgtcggtggcggtttcggaagg
    gaagccaaccgaaaagcacattcagatccggtccaccaac
    Rv2032 70 atgccggacaccatggtgaccaccgatgtcatcaagagcgcggtgcagttggcctg
    ccgcgcaccgtcgctccacaacagccagccctggcgctggatagccgaggaccaca
    cggttgcgctgttcctcgacaaggatcgggtgctttacgcgaccgaccactccggc
    cgggaagcgctgctggggtgcggcgccgtactcgaccactttcgggtggcgatggc
    ggccgcgggtaccaccgccaatgtggaacggtttcccaaccccaacgatcctttgc
    atctggcgtcaattgacttcagcccggccgatttcgtcaccgagggccaccgtcta
    agggcggatgcgatcctactgcgccgtaccgaccggctgcctttcgccgagccgcc
    ggattgggacttggtggagtcgcagttgcgcacgaccgtcaccgccgacacggtgc
    gcatcgacgtcatcgccgacgatatgcgtcccgaactggcggcggcgtccaaactc
    accgaatcgctgcggctctacgattcgtcgtatcatgccgaactcttttggtggac
    aggggcttttgagacttctgagggcataccgcacagttcattggtatcggcggccg
    aaagtgaccgggtcaccttcggacgcgacttcccggtcgtcgccaacaccgatagg
    cgcccggagtttggccacgaccgctctaaggtcctggtgctctccacctacgacaa
    cgaacgcgccagcctactgcgctgcggcgagatgctttccgccgtattgcttgacg
    ccaccatggctgggcttgccacctgcacgctgacccacatcaccgaactgcacgcc
    agccgagacctggtcgcagcgctgattgggcagcccgcaactccgcaagccttggt
    tcgcgtcggtctggccccggagatggaagagccgccaccggcaacgcctcggcgac
    caatcgatgaagtgtttcacgttcgggctaaggatcaccgg
    Rv2428 71 atgccactgctaaccattggcgatcaattccccgcctaccagctcaccgctctcat
    cggcggtgacctgtccaaggtcgacgccaagcagcccggcgactacttcaccacta
    tcaccagtgacgaacacccaggcaagtggcgggtggtgttcttttggccgaaagac
    ttcacgttcgtgtgccctaccgagatcgcggcgttcagcaagctcaatgacgagtt
    cgaggaccgcgacgcccagatcctgggggtttcgattgacagcgaattcgcgcatt
    tccagtggcgtgcacagcacaacgacctcaaaacgttacccttcccgatgctctcc
    gacatcaagcgcgaactcagccaagccgcaggtgtcctcaacgccgacggtgtggc
    cgaccgcgtgacctttatcgtcgaccccaacaacgagatccagttcgtctcggcca
    ccgccggttcggtgggacgcaacgtcgatgaggtactgcgagtgctcgacgccctc
    cagtccgacgagctgtgcgcatgcaactggcgcaagggcgacccgacgctagacgc
    tggcgaactcctcaaggcttcggcc
    Rv2624c 72 atgtctgggagaggagagccgacgatgaaaacaatcattgttggtatcgatggttc
    gcacgcggcgattacggccgcattgtggggggttgacgaggccatcagccgagcgg
    tgccgctgcgactggtctcagtgatcaagccgacacatccgtccccggacgactac
    gaccgcgaccttgcgcatgctgaaagatcgcttcgggaagcgcagtccgctgttga
    ggccgcgggcaagctcgtcaagatcgaaaccgacatcccccgcgggccagccggcc
    cggtgcttgtggaggcatcgcgcgacgccgagatgatctgcgtcggctccgtggga
    atcgggcgctacgccagctcgatcttgggttcgacggcaaccgagctggccgaaaa
    ggcgcattgcccggtcgccgtcatgcgctcaaaagtggaccagccagcgtctgaca
    tcaactggatcgtggtgcgcatgaccgacgcaccggataacgaggccgtgctggaa
    tacgctgcccgggaagcgaagttgcggcaagcgcccatactggcactcggcgggcg
    accggaggagctccgggagattccggacggcgaattcgaacgtcgcgtgcaggatt
    ggcaccaccgtcatcccgatgtgcgcgtctacccgatcaccactcacacgggtatt
    gcccggttcctggccgaccacgacgagcgcgtacagctggcagtgatcggcggtgg
    tgaggccggtcagctagcgcggctggtcgggccatccggacatccggtgttccgtc
    acgccgagtgttcggtgcttgtcgttcgccgc
    Rv2625c 73 atgcgtgatgcgatcccgcttgggcggatcgccgggtttgtggtgaacgtccactg
    gagcgtgttggtgatcctgtggttgttcacctggagtctggcgaccatgttgccgg
    gtaccgtcggaggctacccggccgtggtctattggcttctcggcgcaggtggcgcg
    gtcatgttgctggcgtcgctgttggctcatgagctcgcgcacgccgtcgtcgctcg
    tcgcgccggggtatccgttgagagcgtgacgttgtggctgttcggcggggtgaccg
    cgcttggcggcgaggcaaagacgcccaaagccgctttccggatcgcgttcgcgggt
    ccggctaccagcctggcgctgtcggcgacattcggtgcgttggccatcacgctcgc
    cggcgtgcggaccccggccatcgtgatcagcgttgcttggtggttggctactgtca
    acctgctgctggggctgttcaatctgctgcctggcgcgccgttggacggtgggcgg
    ttggtccgggcctatctgtggcgccgccacggcgatagtgtgcgcgccgggatcgg
    tgcggcgcgggccggacgggtggttgcgctggtcttgatcgcgttgggattggccg
    agtttgtggctggtggcctcgtcggtggggtctggttagccttcattggctggttt
    atcttcgctgccgctcgcgaggaggagacccggatttcgacccagcagctgtttgc
    cggggtgcgtgtggccgatgcgatgaccgcccaaccgcatacggctcccggatgga
    tcaatgtcgaggatttcatccagcgttacgtgcttggtgaacggcactcggcatat
    ccggttgccgatcgggacggatcgatcacgggcctggtggcattgcggcagctgcg
    cgatgttgcgcctagccggcgcagcactaccagcgtaggtgacattgcgctgccgc
    tgcacagcgtgccgaccgcccgaccacaagagccgctgaccgcgctcctagagcgg
    atggcaccgctcggcccgcgcagccgtgcgctggtcaccgaagggagcgcggtggt
    cggcatcgtcactcccagcgatgtcgcgcggctgattgacgtctaccggttggccc
    agccggaaccgacctttaccacgagtccccaagatgcggacaggttttccgatg-
    cgggg
    Rv2627c 74 atggcaagttctgcgagcgacggcacccacgaacgctcggcttttcgcctgagtcc
    accggtcttgagcggcgccatgggaccgttcatgcacaccggtctgtacgtcgctc
    aatcgtggcgcgactatctgggtcaacagcccgataaactgccgatcgcacggccc
    actattgccttagcggcgcaagcctttcgagacgaaatcgtcctgctgggcctcaa
    ggcacgacgtccggtcagcaatcatcgagtgttcgagcgcatcagccaagaagtgg
    ccgctggactggagttctatgggaatcgcagatggctggagaagcctagcggattt
    tttgcccagcccccaccgctcaccgaggtcgcggtccgaaaggtcaaggaccgcag
    acgctccttttatcgcatcttcttcgacagtgggtttacgccgcatccgggtgaac
    cgggcagccaacggtggctctcatacactgcgaacaatcgcgagtacgccctgtta
    ctgcggcacccagagccgcgtccctggctggtttgtgtacacggcaccgagatggg
    cagggccccgttggatctcgcggtgttccgcgcctggaagctgcatgacgaactcg
    gcctgaacattgtcatgccggttcttccgatgcatggtccccgcgggcaaggtctg
    ccgaagggcgccgtttttcccggagaagatgttctcgacgatgtgcatgggacggc
    tcaagcggtgtgggatatccggcggctgttgtcctggatacgatcgcaggaggagg
    agtcgctgatcgggttgaacggtctctcgctgggcggctacatcgcgtcattggtc
    gccagcctcgaagaaggtctcgcctgcgcgattctcggtgtcccagtggctgatct
    gatcgagttgttgggccgccactgcggtcttcggcacaaagacccccgccgccaca
    ccgtcaagatggccgaaccgatcggccgaatgatctcgccgctctcacttacgcca
    ctggtgcccatgccgggccgctttatctacgcgggcattgccgaccgactcgtgca
    tccacgcgaacaggtgactcgcctctgggagcactggggcaaacccgaaatcgtgt
    ggtatccaggcggtcacactggcttcttccagtcgcggccggtacgacggtttgtc
    caggctgcgctggagcagtcgggcctgttggacgcgccacggacacagcgcgac-
    cgttccgcc
    Rv2628 75 Atgtccacgcaacgaccgaggcactccggtattcgggctgttggcccctacgcatg
    ggccggccgatgtggtcggataggcaggtggggggtgcaccaggaggcgatgatga
    atctagcgatatggcacccgcgcaaggtgcaatccgccaccatctatcaggtgacc
    gatcgctcgcacgacgggcgcacagcacgggtgcctggtgacgagatcactagcac
    cgtgtccggttggttgtcggagttgggcacccaaagcccgttggccgatgagcttg
    cgcgtgcggtgcggatcggcgactggcccgctgcgtacgcaatcggtgagcacctg
    tccgttgagattgccgttgcggtc
    Rv2629 76 atgcgatcagaacgtctccggtggctggtagccgcagaaggtccgttcgcctcggt
    gtatttcgacgactcgcacgacactcttgatgccgtcgagcgccgggaagcgacgt
    ggcgcgatgtccggaagcatctcgaaagccgcgacgcgaagcaggagctcatcgac
    agcctcgaagaggcggtgcgggattctcgaccggccgtcggccagcgtggccgcgc
    gctgatcgcgaccggcgagcaagtactggtcaacgagcatctgatcggcccaccac
    cggctacggtgattcggctgtcggattatccgtacgtcgtgccattgatagacctt
    gagatgcggcgaccgacgtatgtatttgccgcggttgatcacaccggcgccgacgt
    caagctgtatcagggggccaccatcagttccacgaaaatcgatggggtcggctacc
    cggtgcacaagccggtcaccgccggctggaacggctacggcgacttccagcacacc
    accgaagaagccatccgaatgaactgccgcgcggtcgccgaccatctcacccgact
    ggtagacgctgccgaccccgaggtggtgttcgtgtccggcgaggtgcggtcacgca
    cagacctgctttccacattgccgcagcgggtggcggtccgggtgtcgcagctgcat
    gccggaccgcgcaaaagcgccttagacgaggaagagatctgggacctgacatccgc
    ggagttcacccggcggcggtacgccgaaatcaccaatgtcgcacaacaatttgagg
    cggagatcggacgcggatcggggctggcggcccaagggttggcggaggtgtgtgcg
    gctctgcgtgacggcgacgtcgacacgctgatcgtcggagagctaggcgaggccac
    cgtggtcaccggtaaagcgcgtactacggtcgcgcgggatgccgacatgttgtccg
    aactcggcgaaccggtagatcgcgtggcaagggccgatgaggcgttgccattcgcc
    gcgatcgcggtaggtgccgcattggtccgtgacgacaaccggatcgcgccactaga
    tggggtgggcgcattgctgcgttatgccgccaccaaccgactcggcagccata-
    gatcc
    Rv2630 77 Atgctgcaccgcgacgatcacatcaatccgccgcggccccgcgggttggatgttcc
    ttgcgcccgcctacgagcgacaaatcccctgcgcgccttggcgcgttgcgttcagg
    cgggcaagccgggcaccagttcagggcatcggtccgtgccgcatacggcggacttg
    cgaatcgaagcctgggcaccgacccgtgacggctgtatccggcaggcggtgctggg
    taccgtcgagagcttcctcgacctggaatccgcgcacgcggtccatacccggctgc
    gccggctgaccgcggatcgcgacgacgatctactggtcgcggtgctcgaggaggtc
    atttatttgctggacaccgtcggtgaaacgcctgtcgatctcaggctgcgcgacgt
    tgacgggggtgtcgacgtcacattcgcaacgaccgatgcgagtacgctagttcagg
    tgggtgccgtgccgaaggcggtgtcactcaacgaacttcggttctcgcagggtcgc
    cacggctggcgatgtgcggtaacgctcgatgtg
    Rv2659c 78 Gtgacgcaaaccggcaagcgtcagagacgcaaattcggtcgcatccgacagttcaa
    ctccggccgctggcaagccagctacaccggccccgacggccgcgtgtacatcgccc
    ccaaaaccttcaacgccaagatcgacgccgaagcatggctcaccgaccgccgccgc
    gaaatcgaccgacaactatggtccccggcatcgggtcaggaagaccgccccggagc
    cccattcggtgagtacgccgaaggatggctgaagcagcgtggaatcaaggaccgca
    cccgcgcccactatcgcaaactgctggacaaccacatcctggccaccttcgctgac
    accgacctacgcgacatcaccccggccgccgtgcgccgctggtacgccaccaccgc
    cgtgggcacaccgaccatgcgggcacactcctacagcttgctgcgcgcaatcatgc
    agaccgccttggccgacgacctgatcgactccaacccctgccgcatctcaggcgcg
    tccaccgcccgccgcgtccacaagatcaggcccgccaccctcgacgagctggaaac
    catcaccaaagccatgcccgacccctaccaggcgttcgtgctgatggcggcatggc
    tggccatgcgctacggcgagctgaccgaattacgccgcaaagacatcgacctgcac
    ggcgaggttgcgcgggtgcggcgggctgtcgttcgggtgggcgaaggcttcaaggt
    gacgacaccgaaaagcgatgcgggagtgcgcgacataagtatcccgccacatctga
    tacccgccatcgaagaccaccttcacaaacacgtcaaccccggccgggagtccctg
    ctgttcccatcggtcaacgaccccaaccgtcacctagcaccctcggcgctgtaccg
    catgttctacaaggcccgaaaagccgccggccgaccagacttacgggtgcacgacc
    ttcgacactccggcgccgtgttggctgcatccaccggcgccacactggccgaactg
    atgcagcggctaggacacagcacagccggcgccgcactccgctaccagcacgccgc
    caagggccgggaccgcgaaatcgccgcactgttaagcaaactggccgagaaccag-
    gagatg
    Rv2780 79 Atgcgcgtcggtattccgaccgagaccaaaaacaacgaattccgggtggccatcac
    cccggccggcgtcgcggaactaacccgtcgtggccatgaggtgctcatccaggcag
    gtgccggagagggctcggctatcaccgacgcggatttcaaggcggcaggcgcgcaa
    ctggtcggcaccgccgaccaggtgtgggccgacgctgatttattgctcaaggtcaa
    agaaccgatagcggcggaatacggccgcctgcgacacgggcagatcttgttcacgt
    tcttgcatttggccgcgtcacgtgcttgcaccgatgcgttgttggattccggcacc
    acgtcaattgcctacgagaccgtccagaccgccgacggcgcactacccctgcttgc
    cccgatgagcgaagtcgccggtcgactcgccgcccaggttggcgcttaccacctga
    tgcgaacccaagggggccgcggtgtgctgatgggcggggtgcccggcgtcgaaccg
    gccgacgtcgtggtgatcggcgccggcaccgccggctacaacgcagcccgcatcgc
    caacggcatgggcgcgaccgttacggttctagacatcaacatcgacaaacttcggc
    aactcgacgccgagttctgcggccggatccacactcgctactcatcggcctacgag
    ctcgagggtgccgtcaaacgtgccgacctggtgattggggccgtcctggtgccagg
    cgccaaggcacccaaattagtctcgaattcacttgtcgcgcatatgaaaccaggtg
    cggtactggtggatatagccatcgaccagggcggctgtttcgaaggctcacgaccg
    accacctacgaccacccgacgttcgccgtgcacgacacgctgttttactgcgtggc
    gaacatgcccgcctcggtgccgaagacgtcgacctacgcgctgaccaacgcgacga
    tgccgtatgtgctcgagcttgccgaccatggctggcgggcggcgtgccggtcgaat
    ccggcactagccaaaggtctttcgacgcacgaaggggcgttactgtccgaacgggt
    ggccaccgacctgggggtgccgttcaccgagcccgccagcgtgctggcc
    Rv3126c 80 Atggtcatccggtttgatcaaatagggtcattggtcctctcaatgaaatcccttgc
    gtcactgtcgtttcagcggtgtctgcgcgagaattctagtttggtcgcggcgctgg
    accggctcgatgctgcggtcgatgagctgagcgctttgtcgtttgatgcgttgacc
    actccggagcgggatcgcgcccgtcgcgaccgggaccatcatccttggtcccgctc
    ccgctcgcagttgtcgccacgaatggcgcacggtgcagtgcaccaatgccagtggc
    cgaaggcggtttgggctgtcattgacaatcca
    Rv3127 81 Gtgctcaagaacgcagtcttgctggcatgccgggcgccgtcggtgcacaacagcca
    gccctggcgttgggtggccgaaagcggctccgagcacactactgtgcacctgttcg
    tcaaccgccaccgaacggtgccggccaccgaccattccggccggcaagcgatcatc
    agttgcggtgccgtactcgatcaccttcgcatcgccatgacggccgcgcactggca
    ggcgaatatcactcgctttccccagccgaaccaacctgaccagttggccaccgtcg
    aattcagtcccatcgatcacgtcacggcgggacagcgaaaccgcgcccaggcgatt
    ctgcagcgccgaaccgatcggcttccgtttgacagcccgatgtactggcacctgtt
    tgagcccgcgctgcgcgacgccgtcgacaaagacgttgcgatgcttgatgtggtat
    ccgacgaccagcgaacacgactggtggtagcgtcacaactcagcgaagtcctgcgg
    cgggacgatccgtactatcacgccgaactcgaatggtggacttcaccgttcgtgct
    ggcccatggtgtgccgccggatacgctggcatcagacgccgaacgcttgcgggttg
    acctgggccgtgacttcccggtccggagctaccagaatcgccgtgccgagctagct
    gatgaccgatcgaaagtccttgtgctgtcgacccctagcgacacgcgagccgacgc
    actgaggtgtggcgaagtgctgtcgaccatcctactcgagtgcaccatggccggca
    tggctacctgcacgttgacccatctgatcgaatccagtgacagtcgtgacatcgtg
    cggggcctgacgaggcagcgaggcgagccgcaagccttgatccgggtagggatagc
    cccgccgttggcagcagttcccgcccccacaccacggcggccgctggacagcgtct
    tgcagattcgccagacgcccgagaaagggcgtaatgcctcagatagaaatgcccgt
    gaaacgggttggttcagcccgcct
    Rv3128c 82 gtgtggtccgcctcgggtgggcagtgcgggaagtatcttgccgcctcgatggtgct
    gcagcttgatgggttggaacgtcacggtgtgttggagtttgggcgtgaccgctatg
    gccccgaggtgcgtgaggagctgttggcgatgagtgcggccagcatcgatcgttat
    ctgaagaccgcgaaggccaaagaccagatatcgggtgtgtcgacgacgaaaccctc
    accactgctgcgtaattcgatcaaggttcgcagggccggcgatgaggtcgaggcgg
    agccggggttcttcgagggcgacaccgtcgcccattgcggtccgacgctcaaaggc
    gagttcgcccacaccctgaacttgaccgacgtgcacatcggatgggtgttcacccg
    caccgtccgcaacaacgcccgtacccacatcctcgccgggctcaaagcttctgtca
    ccgagatcccgcatgggataacgggtttagatttcgacaacggcaccgtgtttctc
    aacaagccggtcatcagctgggccggcgacaacggtatctacttcacccgctttcg
    cccgtacaagaaaaaccactaggccaccatcgagtccaagaacaaccacctggtcc
    gcaagtacgcgttctactaccgctatgacaccgccgaggaacgcgccgtgctcaac
    cggatgtggaagctggtcaacgaccgcctcaactacctcaccccgaccatcaaacc
    gatcgggtatgccagcagcgccgacggccgccgccgacgcctctacgatgccccac
    agacgccgctggaccggccactggccgcaagggtgctctccgcggcccagcaggcc
    gacctgatcacctaccgagacagcctcaaccccgcccagatcggccgcaaaatcgc
    cgacctgcagaaccgactcctcatcttggccaaggagaaaaccgagcagctctacc
    tcgctaacatccccaccgccctacccgacatccacaaaggcatcctgatcaagg-
    cgggc
    Rv3129 83 Gtggtgcaaggccgcaccgtgctgtttcgtaccgcggagggcgccaaattattttc
    agccgtcgcgaagtgcgcggtggctttcgaggcggacgaccacaacgttgccgagg
    gctggagcgtgatcgtcaaggttcgcgcccaggtgctgacgaccgacgcgggggtc
    cgcgaagccgaacgcgcccagttactaccgtggaccgcgacgctgaaacgtcactg
    tgtgcgggtgatcccgtgggagatcaccggccgccacttcaggttcggtccggaac
    cggaccgcagccagacctttgcctgcgaggcctcgtcacacaaccagcga
    Rv3130c 84 atgaatcacctaacgacacttgacgccgggtttctcaaggcagaagacgtggatcg
    gcacgtgagtctggcaatcggcgctctggcggtcatcgaggggccggctcccgatc
    aggaagccttcttatcgtcgctcgctcaacgcctacgtccctgtacccggttcggg
    cagcggttacgcctgcgcccgttcgacctcggtgcacccaaatgggtggacgatcc
    cgacttcgatcttggccgtcatgtgtggcgcatcgccttgccgcggcctggcaacg
    aagaccagttattcgagctgatcgccgatctgatggcgcgtcgtttggaccggggt
    cgaccgctgtgggaggtctgggtcatcgaaggcctggcggacagcaagtgggcgat
    cctgaccaaactgcaccactgcatggccgacggaatcgcggcgactcacctgctag
    ctgggctctccgatgaaagtatgagcgacagcttcgcgagcaacatccacacgacc
    atgcagtcgcaatccgcatctgtgcggcggggtggattccgtgtcaatccaagcga
    ggcgttgaccgcgtcgaccgccgtgatggcaggcatcgttcgcgcggccaagggtg
    ccagtgagatcgcggccggcgtgctaagtcccgccgcgtcgtcgttgaacgggccg
    atcagtgatttgcgtcgctacagcgcagcaaaggtccctctcgccgacgtcgaaca
    ggtgtgccggaaattcgacgtcaccatcaatgatgttgcgcttgccgcgattacgg
    aaagctaccgcaacgtcctcatccagcggggtgagcggcctaggtttgattcgctg
    cgtacgctagtgccggtctcgacgcgttccaacagcgctttgagcaagaccgataa
    ccgtgtttcgttaatgctgcccaacctgccggtggatcaagagaacccgctgcagc
    ggctgcggatcgtgcactcgcggctgactcgggccaaggcggggggacagagacaa
    ttcggaaatactttgatggcgattgccaaccgccttccgttccccatgaccgcatg
    ggcggtcgggctgttgatgcggctgccgcagcgtggtgttgtcaccgtggcgacaa
    atgtgccgggtccacgacggccgctgcagattatgggcagacgggtgcttgaccta
    tacccggtttcgccgatcgcgatgcaactgcgcaccagtgtcgcgatgctcagcta
    cgccgacgacctgtacttcgggatcctggccgactacgacgtggtagcagatgccg
    gccagctggcgcgaggaattgaagacgccgtcgcacggctggtggcgatcagtaag
    cggcgcaaggtgactcgcaggcgcggagcgctatcgctggttgtg
    Rv3131 85 atgaacacccatttcccggacgccgaaaccgtgcgaacggttctcaccctggccgt
    ccgggccccctccatccacaacacgcagccgtggcggtggcgggtatgcccgacga
    gtctggagctgttctctagacccgatatgcagctgcgtagcaccgatccggacggg
    cgtgagttgatcctcagctgtggtgtggcattgcaccactgcgtcgtcgctttggc
    gtcgctgggctggcaggccaaggtaaaccgtttccccgatcccaaggaccgctgcc
    atctggccaccatcggggtacaaccgcttgttcccgatcaggccgatgtcgccttg
    gcggcggccataccgcggcgacgcaccgatcggcgcgcctacagttgctggccggt
    gccaggaggtgacatcgcgttgatggccgcaagagcagcccgtggcggggtcatgc
    tgcggcaggtcagtgccctagaccgaatgaaagccattgtggcgcaggctgtcttg
    gaccacgtgaccgacgaggaatatctgcgcgagctcaccatttggagtgggcgcta
    cggttcagtggccggggttcccgcccgcaacgagccgccatcagaccccagtgccc
    cgatccccggtcgcctgttcgccgggcccggtctgtctcagccgtccgacgtctta
    cccgctgacgacggcgccgcgatcctggcactaggcaccgagacagacgaccggtt
    ggcccggctgcgcgccggcgaggccgccagcatcgtcttgttgaccgcgacggcaa
    tggggctggcgtgctgcccgatcaccgaaccgctggagatcgccaagacccgcgac
    gcggtccgtgccgaggtgttcggcgccggcggctacccccagatgctgctgcgagt
    gggttgggcaccgatcaatgccgacccgttgccaccgacgccacggcgcgaactgt
    cccaggtcgttgagtggccggaagagctactgcgacaacggtgc
    Rv3132c 86 atgacaacagggggcctcgtcgacgaaaacgacggcgccgcaatgcgtccactgcg
    tcacacgctctcccaactacgcctgcacgagctgctggtcgaggtgcaggaccggg
    tcgagcagatcgtcgagggccgggaccgcctcgatggtctggtggaggccatgctc
    gtggtcacagcgggcctggacctggaggcaaccctacgcgctatcgtgcattcagc
    gaccagccttgtcgatgcgcgctatggcgctatggaggtgcacgaccggcagcatc
    gggtattgcactttgtctatgaaggcatcgacgaggagaccgttcggcggatcggc
    cacctaccgaaaggcctaggcgtcatcgggctgctcatcgaagatcccaaaccgtt
    acggctggacgatgtttctgcgcacccggcctcgattggttttccgccgtatcatc
    cgccgatgcgtaccttcctcggggtaccggttcgggtgcgcgatgaatcgttcggc
    actctgtacctgactgacaagaccaacgggcaaccgttcagcgacgacgacgaggt
    tctggtccaggcgctggcggccgccgcgggtatcgcagtcgcgaatgcccggctct
    accagcaggctaaggcgcgtcagtcgtggatcgaggccacccgtgacatcgccacc
    gagttgttgtccggcaccgaacccgcgacggtgttccggcttgtcgccgcggaggc
    gctcaagctgacggcggctgacgctgccctggtagccgttcccgtcgacgaggaca
    tgcctgccgctgacgtgggggagctgctggtgattgaaacagtcggcagcgctgtg
    gcttccattgttgggcgaacgattccggtggcgggcgcggtgctgcgggaggtctt
    cgtcaacggcattccgcgacgggtcgaccgggtcgatttggaaggcctggacgaac
    tggccgacgcaggtccggcgctgctgttgccgctgcgggccagaggtaccgtagcg
    ggtgtcgttgttgtgctgagtcaaggcggtccaggggctttcaccgacgaacaact
    cgagatgatggccgcgttcgccgaccaggccgcgctggcttggcaattggccactt
    cgcaacgtcggatgcgcgaactcgacgtactgaccgaccgggatcgtatcgcccgt
    gacctccatgaccatgtcatccagcggctcttcgcgattggcctggctttgcaggg
    tgctgtcccgcacgaacgtaatcctgaagtgcagcaacgactctcggacgtggtag
    acgatctgcaagacgttatacaggaaatccggaccaccatttatgacctgcacgga
    gcatcgcagggtatcactcggctccggcagcgaatcgatgcggccgtagcccaatt
    tgccgactcggggttgcgcaccagcgttcaattcgtgggtccattgtcggtggtcg
    acagcgcgctcgccgatcaggccgaggcggtggttcgggaagcggtcagcaacgcg
    gttcgccatgcgaaggccagcacgttgaccgtccgggtcaaagtcgacgacgactt
    gtgcatcgaggtgaccgacaacggccgcgggctgcccgacgagttcaccggaagcg
    gcttaacgaacctgcggcagcgggcagagcaggccggcggcgaattcaccctcgcg
    agcgtaccgggcgcgagcggaacagtgctgcgatggtcagcaccgttgtcgcag
    Rv3134c 87 atgagcgatcctcggccagctcgggcagtggtcgttggtatcgacgggtcaagggc
    ggcaacgcatgcggcgttgtgggcggtcgatgaggcggtgaaccgagacattccgc
    tgcgactggtgtacgtcatcgatccgtcccaactgtccgccgccggcgagggcggt
    gggcaatcagcggcccgagcggcgctgcacgacgcctctcggaaggtcgaggccac
    cgggcaaccggtcaagatcgaaacggaggttctgtgcggcaggccgctcaccaagc
    tgatgcaggagtccaggtccgcggcgatgctgtgcgtcggttcggtggggcttgat
    catgtccgcggtcgccggggttcggtcgcggcgaccctggctgggtcggccttatg
    ccccgtggcggtgattcacccgtcgccggccgagccagcgacaacctcccaggtca
    gcgcggttgtcgcggaggtggacaatggtgtggtgctgcggcacgcattcgaggag
    gccaggctgcgcggagttccgctgcgggccgtggctgtccacgctgctgaaacacc
    cgatgacgtcgaacagggcagccggttggcgcatgtacacctgagccgtcggctcg
    cccactggacccggctctaccccgaggtgcgggtggatcgggccatcgccggcggc
    agtgcgtgccgtcatctggccgccaacgcaaagccgggtcagctgttcgtcgcgga
    ctcacactccgcgcacgaattgtgcggtgcataccagcccggatgcgccgtactta
    cggtacgcagtgccaacttg
    Rv3841 88 atgacagaatacgaagggcctaagacaaaattccacgcgttaatgcaggaacagat
    tcataacgaattcacagcggcacaacaatatgtcgcgatcgcggtttatttcgaca
    gcgaagacctgccgcagttggcgaagcatttttacagccaagcggtcgaggaacga
    aaccatgcaatgatgctcgtgcaacacctgctcgaccgcgaccttcgtgtcgaaat
    tcccggcgtagacacggtgcgaaaccagttcgacagaccccgcgaggcactggcgc
    tggcgctcgatcaggaacgcacagtcaccgaccaggtcggtcggctgacagcggtg
    gcccgcgacgagggcgatttcctcggcgagcagttcatgcagtggttcttgcagga
    acagatcgaagaggtggccttgatggcaaccctggtgcgggttgccgatcgggccg
    gggccaacctgttcgagctagagaacttcgtcgcacgtgaagtggatgtggcgccg
    gccgcatcaggcgccccgcacgctgccgggggccgcctc
    Rv3842c 89 atgacatgggccgacgaggtgctcgccggacatccctttgtggttgctcaccgtgg
    tgcgtcggcggctcggccggagcatacccttgccgcctacgacctggcgctcaaag
    agggcgccgacggcgtggaatgtgatgtgcggttgacccgggacgggcatctggtc
    tgtgtgcatgaccgccgcctggaccgaacctcgacgggagccggcttggtcagcac
    gatgacgctggcccagctacgcgagctggagtacggcgcgtggcacgacagctggc
    gccccgacggttcgcacggcgacaccagtctgctgaccctggacgcgcttgtttcg
    ctggttttggactggcaccggccggtgaagatcttcgtcgagaccaagcatcccgt
    ccgatacggctcgctggtggaaaacaagctgctggcgctgctacaccggttcggta
    ttgccgcacccgcctccgcagatcgatcccgtgcggtggtgatgtcgttttcggcc
    gccgcggtctggcggatccggcgggctgcaccgctgctgccgacggtgttgctcgg
    caagaccccccgatacctgaccagcagtgcggccacggcggtcggggcaaccgccg
    tgggaccctcactgcctgcgttaaaggaatatccgcaactcgttgaccgctcggca
    gctcagggccgggcggtgtactgctggaacgtcgatgagtacgaggacatcgactt
    ttgccgggaggtcggggtggcctggattggtactcaccaccccggccgcaccaagg
    cctggctggaagacgggcgggcgaacgggaccactcgc
    Rv3908 90 gtgtccgacggcgaacaagccaaatcacgtcgacgccgggggcggcgccgcgggcg
    gcgcgctgcggctacagccgagaatcacatggacgcccaaccggccggcgacgcca
    ccccgaccccggcaacggcgaagcggtcccggtcccgctcacctcgtcgcgggtcg
    actcggatgcgcaccgtgcacgaaacatcggctggagggttggtcattgacggtat
    cgacggtccacgagacgcgcaggtcgcggctctgatcggccgcgtcgaccggcgcg
    gccggctgctgtggtcgctacccaaggggcacatcgagttgggcgagaccgccgag
    cagaccgccatccgcgaggtcgccgaggagaccggcatccgcggcagtgtgctcgc
    cgcgctggggcgcatcgactactggttcgtcaccgacggccggcgggtgcacaaga
    ccgtccaccattatttgatgcggtttttaggcggagagctgtccgacgaagacctc
    gaggtagccgaggtagcctgggtgccgatccgggaactgccgtctcgactggccta
    cgccgacgaacgtcgactagccgaggtggccgacgaactgatcgacaagctgcaga
    gcgacggccccgccgcgcttccgccgctaccacccagctcgcctcgtcgacggccg
    caaacgcattcacgcgctcgtcatgccgatgactcagcaccgggtcagcacaacgg
    tcccgggccggggccg
  • Preferably the immunogenic portions are selected from the group consisting of the sequences presented in Table 1 and the nucleic acid sequences are selected from the sequences presented in Table 2. [0031]
  • In another embodiment, the vaccine is a multiphase vaccine, where the polypeptides or fragments hereof are fused to other antigens with efficacy as prophylactic vaccines, where the fusion partner is selected from e.g. the group consisting of ESAT-6, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32. [0032]
  • The invention further discloses a therapeutic vaccine against tuberculosis comprising one or more polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, or nucleic acids encoding these polypeptides. [0033]
  • Preferably, the therapeutic and multiphase vaccine comprises an additional delivery system selected from among, live recombinant vaccines, that is gene-modified organisms such as bacteria or viruses expressing mycobacteria genes, or immunogenic delivery systems such as, DNA vaccines, that is plasmids expressing genes or gene fragments for the proteins described above, or protein vaccines, that is the proteins themselves or synthetic peptides derived from the proteins themselves delivered in a delivery system such as an adjuvant. [0034]
  • The invention further discloses a therapeutic vaccine in which the amino acid sequence is lipidated so as to allow a self-adjuvanting effect of the polypeptide. [0035]
  • The invention also discloses a method for treating an animal, including a human being, with tuberculosis caused by virulent mycobacteria, e.g., by [0036] Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, comprising administering to the animal the above-mentioned vaccine.
  • The invention also discloses a method for immunizing an animal, including a human being, against tuberculosis caused by virulent mycobacteria, e.g., by [0037] Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, comprising administering to the animal the above mentioned vaccine.
  • In a still further embodiment, the invention discloses an immunogenic composition comprising a polypeptide as defined above, preferably in the form of a vaccine or in the form of a diagnostic reagent. The diagnostic reagent can be in the form of a skin test reagent (administered by the transcutaneous, subcutaneous or intradermal routes), a serological reagent or a reagent for stimulating a cell-mediated reaction. [0038]
  • In another embodiment, the invention discloses a nucleic acid fragment in isolated form which [0039]
  • (a) comprises a nucleic acid sequence which encodes a polypeptide as defined above, or comprises a nucleic acid sequence complementary thereto; or [0040]
  • (b) has a length of at least 10 nucleotides and hybridizes readily under stringent hybridization conditions with a nucleotide sequence selected from the nucleotide sequences presented in Table 2 or a sequence complementary thereto, or with a nucleotide sequence selected from a sequence in (a) [0041]
  • The nucleic acid fragment is preferably a DNA fragment. The fragment can be used as a pharmaceutical. [0042]
  • In another embodiment, the invention discloses a vaccine comprising a nucleic acid fragment according to the invention, optionally inserted in a vector, the vaccine effecting in vivo expression of antigen by a human being or other mammal or animal, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to tuberculosis caused by virulent mycobacteria, e.g. by [0043] Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, in an animal, including a human being.
  • In a further embodiment, the invention discloses the use of a nucleic acid fragment according to the invention for the preparation of a composition for the diagnosis of tuberculosis caused by virulent mycobacteria, e.g., by [0044] Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, and the use of a nucleic acid fragment according to the invention for the preparation of a pharmaceutical composition for the vaccination against tuberculosis caused by virulent mycobacteria, e.g., by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis.
  • In a still further embodiment, the invention discloses a vaccine for immunizing an human being or other mammal or animal, against tuberculosis caused by virulent mycobacteria, e.g. by [0045] Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, comprising as the effective component a non-pathogenic microorganism, wherein at least one copy of a DNA fragment comprising a DNA sequence encoding a polypeptide as defined above has been incorporated into the microorganism (e.g., placed on a plasmid or in the genome) in a manner allowing the microorganism to express and optionally secrete the polypeptide.
  • In another embodiment, the invention discloses a replicable expression vector, which comprises a nucleic acid fragment according to the invention, and a transformed cell harboring at least one such vector. [0046]
  • In another embodiment, the invention discloses a method for producing a polypeptide as defined above, comprising [0047]
  • (a) inserting a nucleic acid fragment according to the invention into a vector that is able to replicate in a host cell, introducing the resulting recombinant vector into the host cell, culturing the host cell in a culture medium under conditions sufficient to effect expression of the polypeptide, and recovering the polypeptide from the host cell or culture medium; [0048]
  • (b) isolating the polypeptide from a whole mycobacterium, e.g. [0049] Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, from culture filtrate or from lysates or fractions thereof; or
  • (c) synthesizing the polypeptide e.g. by solid or liquid phase peptide synthesis. [0050]
  • The invention also discloses a method of diagnosing tuberculosis caused by virulent mycobacteria, e.g. by [0051] Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, in an animal, including a human being, comprising intradermally injecting, in the animal, a polypeptide as defined above or an immunogenic composition as defined above, a positive skin response at the location of injection being indicative of the animal having tuberculosis, and a negative skin response at the location of injection being indicative of the animal not having tuberculosis.
  • In another embodiment, the invention discloses a method for immunizing an animal, including a human being, against tuberculosis caused by virulent mycobacteria, e.g. by [0052] Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, comprising administering to the animal the polypeptide as defined above, the immunogenic composition according to the invention, or the vaccine according to the invention.
  • Another embodiment of the invention discloses a monoclonal or polyclonal antibody, which is specifically reacting with a polypeptide as defined above in an immuno assay, or a specific binding fragment of said antibody. Preferably, said antibody is for use as a diagnostic reagent, e.g. for detection of mycobacteria antigens in sputum, urine or other body fluids of an infected animal, including a human being. [0053]
  • In a further embodiment the invention discloses a pharmaceutical composition that comprises an immunologically responsive amount of at least one member selected from the group consisting of: [0054]
  • (a) a polypeptide selected from the sequences presented in Table 1, or an immunogenic portion thereof; [0055]
  • (b) an amino acid sequence which has a sequence identity of at least 70% to any one of said polypeptides in (a) and is immunogenic; [0056]
  • (c) a fusion polypeptide comprising at least one polypeptide or amino acid sequence according to (a) or (b) and at least one fusion partner; [0057]
  • (d) a nucleic acid sequence which encodes a polypeptide or amino acid sequence according to (a), (b) or (c); [0058]
  • (e) a nucleic acid sequence which is complementary to a sequence according to (d); [0059]
  • (f) a nucleic acid sequence which has a length of at least 10 nucleotides and which hybridizes under stringent conditions with a nucleic acid sequence according to (d) or (e); and [0060]
  • (g) a non-pathogenic micro-organism which has incorporated (e.g. placed on a plasmid or in the genome) therein a nucleic acid sequence according to (d), (e) or (f) in a manner to permit expression of a polypeptide encoded thereby. [0061]
  • In a still further embodiment the invention discloses a method for stimulating an immunogenic response in an animal which comprises administering to said animal an immunologically stimulating amount of at least one member selected from the group consisting of: [0062]
  • (a) a polypeptide selected from the sequences presented in Table 1, or an immunogenic portion thereof; [0063]
  • (b) an amino acid sequence which has a sequence identity of at least 70% to any one of said polypeptides in (a) and is immunogenic; [0064]
  • (c) a fusion polypeptide comprising at least one polypeptide or amino acid sequence according to (a) or (b) and at least one fusion partner; [0065]
  • (d) a nucleic acid sequence which encodes a polypeptide or amino acid sequence according to (a), (b) or (c); [0066]
  • (e) a nucleic acid sequence that is complementary to a sequence according to (d); [0067]
  • (f) a nucleic acid sequence which has a length of at least 10 nucleotides and which hybridizes under stringent conditions with a nucleic acid sequence according to (d) or (e); and [0068]
  • (g) a non-pathogenic micro-organism which has incorporated therein (e.g. placed on a plasmid or in the genome) a nucleic acid sequence according to (d), (e) or (f) in a manner to permit expression of a polypeptide encoded thereby. [0069]
  • The vaccine, immunogenic composition and pharmaceutical composition according to the invention can be used therapeutically in a subject infected with a virulent mycobacterium combined with a prophylactic composition in a subject to prevent further infection with a virulent mycobacterium. [0070]
  • The invention also discloses a method for diagnosing previous or ongoing infection with a virulent mycobacterium, said method comprising [0071]
  • (a) contacting a sample, e.g. a blood sample, with a composition comprising an antibody according to the invention, a nucleic acid fragment according to the invention and/or a polypeptide as defined above, or [0072]
  • (b) contacting a sample, e.g. a blood sample comprising mononuclear cells (e.g. T-lymphocytes), with a composition comprising one or more polypeptides as defined above in order to detect a positive reaction, e.g. proliferation of the cells or release of cytokines such as IFN-γ. [0073]
  • Finally, the invention discloses a method of diagnosing [0074] Mycobacterium tuberculosisinfection in a subject comprising:
  • (a) contacting a polypeptide as defined above with a bodily fluid of the subject; [0075]
  • (b) detecting binding of a antibody to said polypeptide, said binding being an indication that said subject is infected by [0076] Mycobacterium tuberculosis or is susceptible to Mycobacterium tuberculosis infection.
  • Definitions [0077]
  • Polypeptides [0078]
  • The word “polypeptide” in the present invention should have its usual meaning. That is an amino acid chain of any length, including a full-length protein, oligopeptides, short peptides and fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds. [0079]
  • The polypeptide may be chemically modified by being glycosylated, by being lipidated (e.g. by chemical lipidation with palmitoyloxy succinimide as described by Mowat et al. 1991 or with dodecanoyl chloride as described by Lustig et al. 1976), by comprising prosthetic groups, or by containing additional amino acids such as e.g. a his-tag or a signal peptide. [0080]
  • Each polypeptide may thus be characterized by specific amino acids and be encoded by specific nucleic acid sequences. It will be understood that such sequences include analogues and variants produced by recombinant or synthetic methods wherein such polypeptide sequences have been modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide and still be immunogenic in any of the biological assays described herein. Substitutions are preferably “conservative”. These are defined according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. The amino acids in the third column are indicated in one-letter code. [0081]
    ALIPHATIC Non-polar GAP
    ILV
    Polar-uncharged CSTM
    NQ
    Polar-charged DE
    KR
    AROMATIC HFWY
  • A preferred polypeptide within the present invention is an immunogenic antigen from [0082] M. tuberculosis produced when the organism is subjected to the stresses associated with latent infection. Such antigen can for example also be derived from the M. tuberculosis cell and/or M. tuberculosis culture filtrate. Thus, a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences. The additional sequences may be derived from the native M. tuberculosis antigen or be heterologous and such sequences may, but need not, be immunogenic.
  • Each polypeptide is encoded by a specific nucleic acid sequence. It will be understood that such sequences include analogues and variants hereof wherein such nucleic acid sequences have been modified by substitution, insertion, addition or deletion of one or more nucleic acids. Substitutions are preferably silent substitutions in the codon usage that will not lead to any change in the amino acid sequence, but may be introduced to enhance the expression of the protein. [0083]
  • In the present context the term “substantially pure polypeptide fragment” means a polypeptide preparation which contains at most 5% by weight of other polypeptide material with which it is natively associated (lower percentages of other polypeptide material are preferred, e.g. at most 4%, at most 3%, at most 2%, at most 1%, and at most ½%). It is preferred that the substantially pure polypeptide is at least 96% pure, i.e. that the polypeptide constitutes at least 96% by weight of total polypeptide material present in the preparation, and higher percentages are preferred, such as at least 97%, at least 98%, at least 99%, at least 99.25%, at least 99.5%, and at least 99.75%. It is especially preferred that the polypeptide fragment is in “essentially pure form”, i.e. that the polypeptide fragment is essentially free of any other antigen with which it is natively associated, i.e. free of any other antigen from bacteria belonging to the tuberculosis complex or a virulent mycobacterium. This can be accomplished by preparing the polypeptide fragment by means of recombinant methods in a nonmycobacterial host cell as will be described in detail below, or by synthesizing the polypeptide fragment by the well-known methods of solid or liquid phase peptide synthesis, e.g. by the method described by Merrifield or variations thereof. [0084]
  • By the term “virulent mycobacterium” is understood a bacterium capable of causing the tuberculosis disease in an animal or in a human being. Examples of virulent mycobacteria include but are not limited to [0085] M. tuberculosis, M. africanum, and M. bovis. Examples of relevant animals are cattle, possums, badgers and kangaroos.
  • By “a TB patient” is understood an individual with culture or microscopically proven infection with virulent mycobacteria, and/or an individual clinically diagnosed with TB and who is responsive to anti-TB chemotherapy. Culture, microscopy and clinical diagnosis of TB are well known by any person skilled in the art. [0086]
  • By the term “PPD-positive individual” is understood an individual with a positive Mantoux test or an individual where PPD induces a positive in vitro recall response determined by release of IFN-γ. [0087]
  • By “a latently infected individual” is understood an individual, who has been infected by a virulent mycobacterium, e.g. [0088] M. tuberculosis, but shows no sign of active tuberculosis. It is likely that individuals who have been vaccinated, e.g. by BCG, or treated for TB may still retain the mycobacteria within their bodies, although this is currently impossible to prove since such individuals would be expected to be positive if tested for PPD reactivity. Nonetheless, in its most accurate sense, “latently-infected” may be used to describe any individual who has M. tuberculosis residing in their tissues but who is not clinically ill.
  • By the term “delayed type hypersensitivity reaction” (DTH) is understood a T-cell mediated inflammatory response elicited after the injection of a polypeptide into, or application to, the skin, said inflammatory response appearing 72-96 hours after the polypeptide injection or application. [0089]
  • By the term “IFN-γ” is understood interferon-gamma. The measurement of IFN-γ is used as an indication of an immunological response. [0090]
  • By the terms “nucleic acid fragment” and “nucleic acid sequence” are understood any nucleic acid molecule including DNA, RNA, LNA (locked nucleic acids), PNA, RNA, dsRNA and RNA-DNA-hybrids. Also included are nucleic acid molecules comprising non-naturally occurring nucleosides. The term includes nucleic acid molecules of any length e.g. from 10 to 10000 nucleotides, depending on the use. When the nucleic acid molecule is for use as a pharmaceutical, e.g. in DNA therapy, or for use in a method for producing a polypeptide according to the invention, a molecule encoding at least one epitope is preferably used, having a length from about 18 to about 1000 nucleotides, the molecule being optionally inserted into a vector. When the nucleic acid molecule is used as a probe, as a primer or in antisense therapy, a molecule having a length of 10-100 is preferably used. According to the invention, other molecule lengths can be used, for instance a molecule having at least 12, 15, 21, 24, 27, 30, 33, 36, 39, 42, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 nucleotides (or nucleotide derivatives), or a molecule having at most 10000, 5000, 4000, 3000, 2000, 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30 or 20 nucleotides (or nucleotide derivatives). [0091]
  • The term “stringent” when used in conjunction with hybridization conditions is as defined in the art, i.e. the hybridization is performed at a temperature not more than 15-20° C. under the melting point T[0092] m, cf. Sambrook et al, 1989, pages 11.45-11.49. Preferably, the conditions are “highly stringent”, i.e. 5-10° C. under the melting point Tm.
  • Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations thereof such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. [0093]
  • Sequence Identity [0094]
  • The term “sequence identity” indicates a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. The two sequences to be compared must be aligned to best possible fit allowing the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as (N[0095] ref−Ndif)100/Nref, wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC [SEQ ID NO: 184] will have a sequence identity of 75% with the sequence AATCAATC, SEQ ID NO: 185 (Ndif=2 and Nref=8). A gap is counted as non-identity of the specific residue(s), i.e. the DNA sequence AGTGTC [SEQ ID NO: 186] will have a sequence identity of 75% with the DNA sequence AGTCAGTC, SEQ ID NO: 187, (Ndif=2 and Nref=8). Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (Pearson, 1988, or www.ncbi.nlm.nih.gov/cgi-bin/BLAST). In one aspect of the invention, alignment is performed with the sequence alignment method ClustalW with default parameters as described by Thompson J., et al 1994, available at http://www2.ebi.ac.uk/clustalw/.
  • A preferred minimum percentage of sequence identity is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%. [0096]
  • Immunogenic Portion [0097]
  • In a preferred embodiment of the invention, the polypeptide comprises an immunogenic portion of the polypeptide, such as an epitope for a B-cell or T-cell. The immunogenic portion of a polypeptide is a part of the polypeptide, which elicits an immune response in an animal or a human being, and/or in a biological sample determined by any of the biological assays described herein. The immunogenic portion of a polypeptide may be a T-cell epitope or a B-cell epitope. Immunogenic portions can be related to one or a few relatively small parts of the polypeptide, they can be scattered throughout the polypeptide sequence or be situated in specific parts of the polypeptide. For a few polypeptides, epitopes have even been demonstrated to be scattered throughout the polypeptide covering the full sequence (Ravn et al 1999). In order to identify relevant T-cell epitopes which are recognized during an immune response, it is possible to use overlapping oligopeptides for the detection of MHC class II epitopes, preferably synthetic, having a length of e.g. 20 amino acid residues derived from the polypeptide. These peptides can be tested in biological assays (e.g. the IFN-γ assay as described herein) and some of these will give a positive response (and thereby be immunogenic) as evidence for the presence of a T cell epitope in the peptide. For the detection of MHC class I epitopes it is possible to predict peptides that will bind (Stryhn et al. 1996) and hereafter produce these peptides synthetic and test them in relevant biological assays, e.g. the IFN-γ assay as described herein. The peptides preferably having a length of, e.g., 8 to 11 amino acid residues derived from the polypeptide. B-cell epitopes can be determined by analyzing the B cell recognition to overlapping peptides covering the polypeptide of interest as, e.g., described in Harboe et al 1998. [0098]
  • Although the minimum length of a T-cell epitope has been shown to be at least 6 amino acids, it is normal that such epitopes are constituted of longer stretches of amino acids. Hence, it is preferred that the polypeptide fragment of the invention has a length of at least 7 amino acid residues, such as at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, and at least 30 amino acid residues. Hence, in important embodiments of the inventive method, it is preferred that the polypeptide fragment has a length of at most 50 amino acid residues, such as at most 40, 35, 30, 25, and 20 amino acid residues. It is expected that the peptides having a length of from 10 to 20 amino acid residues will prove to be most efficient as MHC class II epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 18, such as 15, 14, 13, 12 and even 11 amino acid residues. It is expected that the peptides having a length of from 7 to 12 amino acid residues will prove to be most efficient as MHC class I epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 11, such as 10, 9, 8 and even 7 amino acid residues. [0099]
  • Immunogenic portions of polypeptides may be recognized by a broad part (high frequency) or by a minor part (low frequency) of the genetically heterogeneous human population. In addition some immunogenic portions induce high immunological responses (dominant), whereas others induce lower, but still significant, responses (subdominant). High frequency><low frequency can be related to the immunogenic portion binding to widely distributed MHC molecules (HLA type) or even by multiple MHC molecules (Sinigaglia, 1988, Kilgus, 1991). [0100]
  • In the context of providing candidate molecules for a new vaccine against tuberculosis, the subdominant epitopes are however as relevant as are the dominant epitopes since it has been shown (Olsen, 2000) that such epitopes can induce protection regardless of the fact that they are not as strongly or broadly recognized. [0101]
  • Variants [0102]
  • A common feature of the polypeptides of the invention is their capability to induce an immunological response as illustrated in the examples. It is understood that a variant of a polypeptide of the invention produced by substitution, insertion, addition or deletion may also be immunogenic as determined by any of the assays described herein. [0103]
  • Immune Individual [0104]
  • An immune individual is defined as a person or an animal, which has cleared or controlled an infection with virulent mycobacteria or has received a vaccination with [0105] M.bovis BCG.
  • Immune Response [0106]
  • The immune response may be monitored by one of the following methods: [0107]
  • An in vitro cellular response is determined by induction of the release of a relevant cytokine such as IFN-γ from, or the induction of proliferation in lymphocytes withdrawn from an animal or human being currently or previously infected with virulent mycobacteria or immunized with the relevant polypeptide. The induction being performed by the addition of the polypeptide or the immunogenic portion of the polypeptide to a suspension comprising from 2×10[0108] 5 cells to 4×105 cells per well. The cells being isolated from either the blood, the spleen, the liver or the lung and the addition of the polypeptide or the immunogenic portion resulting in a concentration of not more than 20 μg per ml suspension and the stimulation being performed from two to five days. For monitoring cell proliferation the cells are pulsed with radioactive labeled Thymidine and after 16-22 hours of incubation detecting the proliferation by liquid scintillation counting. A positive response is defined as being a response more than background plus two standard deviations. The release of IFN-γ can be determined by the ELISA method, which is well known to a person skilled in the art. A positive response being a response more than background plus two standard deviations. Other cytokines than IFN-γ could be relevant when monitoring the immunological response to the polypeptide, such as IL-12, TNF-α, IL-4, IL-5, IL-10, IL-6, TGF-β. Another and more sensitive method for detecting the immune response is the ELISpot method, in which the frequency of IFN-γ producing cells is determined. In an ELIspot plate (MAHA, Millipore) precoated with anti-murine IFN-γ antibodies (PharMingen) graded numbers of cells isolated from either blood, spleen, or lung (typically between 1 to 4×105 cells/well) are incubated for 24-32 hrs in the presence of the polypeptide or the immunogenic portion resulting in a concentration of not more than 20 μg per ml. The plates are subsequently incubated with biotinylated anti-IFN-γ antibodies followed by a streptavidin-alkaline phosphatase incubation. The IFN-γ producing cells are identified by adding BCIP/NBT (Sigma), the relevant substrate giving rise to spots. These spots can be enumerated using a dissection microscope. It is also a possibility to determine the presence of mRNA coding for the relevant cytokine by the use of the PCR technique. Usually one or more cytokines will be measured utilizing for example PCR, ELISPOT or ELISA. It will be appreciated by a person skilled in the art that a significant increase or decrease in the amount of any of these cytokines induced by a specific polypeptide can be used in evaluation of the immunological activity of the polypeptide.
  • An in vitro cellular response may also be determined by the use of T cell lines derived from an immune individual or an [0109] M. tuberculosis-infected person where the T cell lines have been driven with either live mycobacteria, extracts from the bacterial cell or culture filtrate for 10 to 20 days with the addition of IL-2. The induction being performed by addition of not more than 20 μg polypeptide per ml suspension to the T cell lines containing from 1×105 cells to 3×105 cells per well and incubation being performed from two to six days. The induction of IFN-γ or release of another relevant cytokine is detected by ELISA. The stimulation of T cells can also be monitored by detecting cell proliferation using radioactively labeled Thymidine as described above. For both assays a positive response being a response more than background plus two standard deviations.
  • An in vivo cellular response may be determined as a positive DTH response after intradermal injection or local application patch of at most 100 μg of the polypeptide or the immunogenic portion to an individual who is clinically or subclinically infected with a virulent mycobacterium, a positive response having a diameter of at least 5 mm 72-96 hours after the injection or application. [0110]
  • An in vitro humoral response is determined by a specific antibody response in an immune or infected individual. The presence of antibodies may be determined by an ELISA technique or a Western blot where the polypeptide or the immunogenic portion is absorbed to either a nitrocellulose membrane or a polystyrene surface. The serum is preferably diluted in PBS from 1:10 to 1:100 and added to the absorbed polypeptide and the incubation being performed from 1 to 12 hours. By the use of labeled secondary antibodies the presence of specific antibodies can be determined by measuring the OD e.g. by ELISA where a positive response is a response of more than background plus two standard deviations or alternatively a visual response in a Western blot. [0111]
  • Another relevant parameter is measurement of the protection in animal models induced after vaccination with the polypeptide in an adjuvant or after DNA vaccination. Suitable animal models include primates, guinea pigs or mice, which are challenged with an infection of a virulent Mycobacterium. Readout for induced protection could be decrease of the bacterial load in target organs compared to non-vaccinated animals, prolonged survival times compared to non-vaccinated animals and diminished weight loss compared to non-vaccinated animals. [0112]
  • Preparation Methods [0113]
  • In general, [0114] M. tuberculosis antigens, and DNA sequences encoding such antigens, may be prepared using any one of a variety of procedures.
  • They may be purified as native proteins from the [0115] M. tuberculosis cell or culture filtrate by procedures such as those described above. Immunogenic antigens may also be produced recombinantly using a DNA sequence encoding the antigen, which has been inserted into an expression vector and expressed in an appropriate host. Examples of host cells are E. coli. The polypeptides or immunogenic portion hereof can also be produced synthetically having fewer than about 100 amino acids, and generally fewer than 50 amino acids and may be generated using techniques well known to those ordinarily skilled in the art, such as commercially available solid-phase techniques where amino acids are sequentially added to a growing amino acid chain.
  • In the construction and preparation of plasmid DNA encoding the polypeptide as defined for DNA vaccination a host strain such as [0116] E. coli can be used. Plasmid DNA can then be prepared from cultures of the host strain carrying the plasmid of interest, and purified using e.g. the Qiagen Giga-Plasmid column kit (Qiagen, Santa Clarita, Calif., USA) including an endotoxin removal step. It is preferred that plasmid DNA used for DNA vaccination is endotoxin free.
  • Fusion Proteins [0117]
  • The immunogenic polypeptides may also be produced as fusion proteins, by which methods superior characteristics of the polypeptide of the invention can be achieved. For instance, fusion partners that facilitate export of the polypeptide when produced recombinantly, fusion partners that facilitate purification of the polypeptide, and fusion partners which enhance the immunogenicity of the polypeptide fragment of the invention are all interesting possibilities. Therefore, the invention also pertains to a fusion polypeptide comprising at least one polypeptide or immunogenic portion defined above and at least one fusion partner. The fusion partner can, in order to enhance immunogenicity, be another polypeptide derived from [0118] M. tuberculosis, such as of a polypeptide fragment derived from a bacterium belonging to the tuberculosis complex, such as ESAT-6, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32 and alpha-crystalline, or at least one T-cell epitope of any of the above mentioned antigens (Skjøt et al 2000; Danish Patent application PA 2000 00666; Danish Patent application PA 1999 01020; U.S. patent application Ser. No. 09/0505,739; Rosenkrands et al 1998; Nagai et al 1991). The invention also pertains to a fusion polypeptide comprising mutual fusions of two or more of the polypeptides (or immunogenic portions thereof) of the invention.
  • Other fusion partners, which could enhance the immunogenicity of the product, are lymphokines such as IFN-γ, IL-2 and IL-12. In order to facilitate expression and/or purification, the fusion partner can e.g. be a bacterial fimbrial protein, e.g. the pilus components pilin and papA; protein A; the ZZ-peptide (ZZ-fusions are marketed by Pharmacia in Sweden); the maltose binding protein; glutathione S-transferase; β-galactosidase; or poly-histidine. Fusion proteins can be produced recombinantly in a host cell, which could be [0119] E. coli, and it is a possibility to induce a linker region between the different fusion partners.
  • Other interesting fusion partners are polypeptides, which are lipidated so that the immunogenic polypeptide is presented in a suitable manner to the immune system. This effect is e.g. known from vaccines based on the [0120] Borrelia burgdorferi OspA polypeptide as described in e.g. WO 96/40718 A or vaccines based on the Pseudomonas aeruginosa OprI lipoprotein (Cote-Sierra J 1998). Another possibility is N-terminal fusion of a known signal sequence and an N-terminal cystein to the immunogenic polypeptide. Such a fusion results in lipidation of the immunogenic polypeptide at the N-terminal cystein, when produced in a suitable production host.
  • Uses [0121]
  • Protein Vaccine [0122]
  • Another part of the invention pertains to a vaccine composition comprising a polypeptide (or at least one immunogenic portion thereof) or fusion polypeptide according to the invention. In order to ensure optimum performance of such a vaccine composition it is preferred that it comprises an immunologically and pharmaceutically acceptable carrier, vehicle or adjuvant. [0123]
  • An effective vaccine, wherein a polypeptide of the invention is recognized by the animal, will in an animal model be able to decrease bacterial load in target organs, prolong survival times and/or diminish weight loss after challenge with a virulent Mycobacterium, compared to non-vaccinated animals [0124]
  • Suitable carriers are selected from the group consisting of a polymer to which the polypeptide(s) is/are bound by hydrophobic non-covalent interaction, such as a plastic, e.g. polystyrene, or a polymer to which the polypeptide(s) is/are covalently bound, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin or keyhole limpet haemocyanin. Suitable vehicles are selected from the group consisting of a diluent and a suspending agent. The adjuvant is preferably selected from the group consisting of dimethyldioctadecylammonium bromide (DDA), Quil A, poly I:C, aluminum hydroxide, Freund's incomplete adjuvant, IFN-γ, IL-2, IL-12, monophosphoryl lipid A (MPL), Trehalose Dimycolate (TDM), Trehalose Dibehenate and muramyl dipeptide (MDP). [0125]
  • Preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231 and 4,599,230, all incorporated herein by reference. [0126]
  • Other methods of achieving adjuvant effect for the vaccine include use of agents such as aluminum hydroxide or phosphate (alum), synthetic polymers of sugars (Carbopol), aggregation of the protein in the vaccine by heat treatment, aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as [0127] C. parvum or endotoxins or lipopolysaccharide components of gramnegative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. Other possibilities involve the use of immune modulating substances such as cytokines or synthetic IFN-γ inducers such as poly I:C in combination with the above-mentioned adjuvants.
  • Another interesting possibility for achieving adjuvant effect is to employ the technique described in Gosselin et al., 1992 (which is hereby incorporated by reference herein). In brief, a relevant antigen such as an antigen of the present invention can be conjugated to an antibody (or antigen binding antibody fragment) against the Fcγ receptors on monocytes/macrophages. [0128]
  • The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired. Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 μg to 1000 μg, such as in the range from about 1 μg to 300 μg, and especially in the range from about 10 μg to 50 μg. Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations. [0129]
  • The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and, to a lesser degree, the size of the person to be vaccinated. [0130]
  • The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and advantageously contain 10-95% of active ingredient, preferably 25-70%. [0131]
  • In many instances, it will be necessary to have multiple administrations of the vaccine. Especially, vaccines can be administered to prevent an infection with virulent mycobacteria, a prophylactic vaccine, and/or to treat established mycobacterial infection, a therapeutic vaccine. When administered to prevent an infection, the vaccine is given prophylactically, before definitive clinical signs, diagnosis or identification of an infection TB are present. Since the current vaccine BCG appears to induce an effective, but short-lived immune response, prophylactic vaccines may also be designed to be used as booster vaccines. Such booster vaccines are given to individuals who have previously received a vaccination, with the intention of prolonging the period of protection. In instances where the individual has already become infected or is suspected to have become infected, the previous vaccination may have provided sufficient immunity to prevent primary disease, but as discussed previously, boosting this immune response will not help against the latent infection. In such a situation, the vaccine will necessarily have to be a therapeutic vaccine designed for efficacy against the latent stage of infection. A combination of a prophylactic vaccine and a therapeutic vaccine, which is active against both primary and latent infection, constitutes a multiphase vaccine. [0132]
  • Due to genetic variation, different individuals may react with immune responses of varying strength to the same polypeptide. Therefore, the vaccine according to the invention may comprise several different polypeptides in order to increase the immune response. The vaccine may comprise two or more polypeptides or immunogenic portions, where all of the polypeptides are as defined above, or some but not all of the peptides may be derived from virulent mycobacteria. In the latter example, the polypeptides not necessarily fulfilling the criteria set forth above for polypeptides may either act due to their own immunogenicity or merely act as adjuvants. [0133]
  • The vaccine may comprise 1-20, such as 2-20 or even 3-20 different polypeptides or fusion polypeptides, such as 3-10 different polypeptides or fusion polypeptides. [0134]
  • The invention also pertains to a method for immunizing an animal, including a human being, against TB caused by virulent mycobacteria, comprising administering to the animal the polypeptide of the invention, or a vaccine composition of the invention as described above, or a living vaccine described above. [0135]
  • The invention also pertains to a method for producing an immunologic composition according to the invention, the method comprising preparing, synthesizing or isolating a polypeptide according to the invention, and solubilizing or dispersing the polypeptide in a medium for a vaccine, and optionally adding other [0136] M. tuberculosis antigens and/or a carrier, vehicle and/or adjuvant substance.
  • DNA Vaccine. [0137]
  • The nucleic acid fragments of the invention may be used for effecting in vivo expression of antigens, i.e. the nucleic acid fragments may be used in so-called DNA vaccines as reviewed in Ulmer et al 1993, which is included by reference. [0138]
  • Hence, the invention also relates to a vaccine comprising a nucleic acid fragment according to the invention, the vaccine effecting in vivo expression of antigen by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to infections caused by virulent mycobacteria in an animal, including a human being. [0139]
  • The above mentioned definitions and distinctions of prophylactic-, booster-, therapeutic- and multiphase vaccines also applies for DNA vaccines [0140]
  • The efficacy of such a DNA vaccine can possibly be enhanced by administering the gene encoding the expression product together with a DNA fragment encoding a polypeptide that has the capability of modulating an immune response. [0141]
  • Live Recombinant Vaccines [0142]
  • One possibility for effectively activating a cellular immune response for a vaccine can be achieved by expressing the relevant antigen in a vaccine in a non-pathogenic microorganism or virus. Well-known examples of such microorganisms are [0143] Mycobacterium bovis BCG, Salmonella and Pseudomona and examples of viruses are Vaccinia Virus and Adenovirus.
  • Therefore, another important aspect of the present invention is an improvement of the living BCG vaccine presently available, wherein one or more copies of a DNA sequence encoding one or more polypeptide as defined above has been incorporated into the genome of the micro-organism in a manner allowing the micro-organism to express and secrete the polypeptide. The incorporation of more than one copy of a nucleotide sequence of the invention is contemplated to enhance the immune response. [0144]
  • Another possibility is to integrate the DNA encoding the polypeptide according to the invention in an attenuated virus such as the vaccinia virus or Adenovirus (Rolph et al 1997). The recombinant vaccinia virus is able to replicate within the cytoplasma of the infected host cell and the polypeptide of interest can therefore induce an immune response, which is envisioned to induce protection against TB. [0145]
  • Therapeutic Vaccine. [0146]
  • The invention also relates to the use of a polypeptide or nucleic acid of the invention for use as therapeutic vaccines as have been described by D. Lowrie (Lowrie, 1999) using DNA vaccine encoding HSP65 from [0147] M. leprae. Antigens with therapeutic properties may be identified based on their ability to diminish the severity of M. tuberculosis infection in experimental animals or prevent reactivation of previous infection, when administered as a vaccine. The composition used for therapeutic vaccines can be prepared as described above for vaccines.
  • Diagnostic Protein [0148]
  • The invention also relates to a method of diagnosing latent TB caused by a virulent mycobacterium in an animal, including a human being, comprising intradermally injecting, in the animal, a polypeptide according to the invention, a positive skin response at the location of injection being indicative of the animal having TB, and a negative skin response at the location of injection being indicative of the animal not having TB. [0149]
  • When diagnosis of latent infection with virulent mycobacteria is the aim, a blood sample comprising mononuclear cells (i.e. T-lymphocytes) from a patient is contacted with a sample of one or more polypeptides of the invention. This contacting can be performed in vitro and a positive reaction could e.g. be proliferation of the T-cells or release of cytokines such as IFN-γ into the extracellular phase. It is also conceivable to contact a serum sample from a subject with a polypeptide of the invention, the demonstration of a binding between antibodies in the serum sample and the polypeptide being indicative of previous or ongoing infection. [0150]
  • The invention therefore also relates to an in vitro method for diagnosing latent infection in an animal or a human being with a virulent mycobacterium, the method comprising providing a blood sample from the animal or human being, and contacting the sample from the animal with the polypeptide of the invention, a significant release into the extracellular phase of at least one cytokine by mononuclear cells in the blood sample being indicative of the animal being sensitized. A positive response being a response more than release from a blood sample derived from a patient without the TB diagnosis plus two standard deviations. The invention also relates to the in vitro method for diagnosing ongoing or previous sensitization in an animal or a human being with a virulent mycobacterium, the method comprising providing a blood sample from the animal or human being, and by contacting the sample from the animal with the polypeptide of the invention demonstrating the presence of antibodies recognizing the polypeptide of the invention in the serum sample. [0151]
  • The immunogenic composition used for diagnosing may comprise 1-20, such as 2-20 or even 3-20 different polypeptides or fusion polypeptides, such as 3-10 different polypeptides or fusion polypeptides. [0152]
  • Diagnostic DNA [0153]
  • The nucleic acid probes encoding the polypeptide of the invention can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample. [0154]
  • A method of determining the presence of mycobacterial nucleic acids in an animal, including a human being, or in a sample, comprising administering a nucleic acid fragment of the invention to the animal or incubating the sample with the nucleic acid fragment of the invention or a nucleic acid fragment complementary thereto, and detecting the presence of hybridized nucleic acids resulting from the incubation (by using the hybridization assays which are well-known in the art), is also included in the invention. Such a method of diagnosing TB might involve the use of a composition comprising at least a part of a nucleotide sequence as defined above and detecting the presence of nucleotide sequences in a sample from the animal or human being to be tested which hybridize with the nucleic acid fragment (or a complementary fragment) by the use of PCR technique. [0155]
  • Antibodies [0156]
  • A monoclonal or polyclonal antibody, which is specifically reacting with a polypeptide of the invention in an immunoassay, or a specific binding fragment of said antibody, is also a part of the invention. The antibodies can be produced by methods known to a person skilled in the art. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of a polypeptide according to the present invention and, if desired, an adjuvant. The monoclonal antibodies according to the present invention may, for example, be produced by the hybridoma method first described by Kohler and Milstein (Kohler and Milstein, 1975), or may be produced by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal anti-bodies may also be isolated from phage libraries generated using the techniques described by McCafferty et al (McCafferty, 1990), for example. Methods for producing antibodies are described in the literature, e.g. in U.S. Pat. No. 6,136,958. [0157]
  • A sample of a potentially infected organ or body fluid from an infected individual may be contacted with such an antibody recognizing a polypeptide of the invention. The demonstration of the reaction by means of methods well known in the art between the sample and the antibody will be indicative of an ongoing infection. It is of course also a possibility to demonstrate the presence of anti-mycobacterial antibodies in serum or other body fluids by contacting a serum sample from a subject with at least one of the polypeptide fragments of the invention and using well-known methods for visualizing the reaction between the antibody and antigen. [0158]
  • In diagnostics, an antibody, a nucleic acid fragment and/or a polypeptide of the invention can be used either alone, or as a constituent in a composition. Such compositions are known in the art, and comprise compositions in which the antibody, the nucleic acid fragment or the polypeptide of the invention is coupled, preferably covalently, to at least one other molecule, e.g. a label (e.g. radioactive or fluorescent) or a carrier molecule. [0159]
  • It will be understood that the following examples are illustrative of the present invention and are not a limitation thereof. A number of variations on the techniques, reagents, and conditions described in the following examples will be readily apparent to one of skill in the art. [0160]
    • EXAMPLES Example 1
  • Cloning and Expression of Low Oxygen Induced [0161] M. tuberculosis Antigens in E. coli.
  • A number of [0162] M tuberculosis genes are induced under low oxygen conditions. The upregulation of the genes listed in table 2 has been determined at either the mRNA (Sherman, 2001) or protein (Boon, 2001, Rosenkrands, 2002) level The coding region of these selected antigens is amplified by PCR using the primer sets listed in table 3.
    TABLE 3
    Prim r sequences for PCR amplification of selected low oxygen
    induced antigens
    SEQ
    ID
    Rv no. NO: Primer sequence
    Rv0079 Fwd  92 CACCGTGGAACCGAAACGCAGTCG
    Rvs  93 TTATGCCAGACCGTCGGCA
    Rv0080 Fwd  94 CACCATGAGCCCGGGCTCG
    Rvs  95 TTACGGCGTACGCGAGTCAG
    Rv0081 Fwd  96 CACCGTGGAGTCCGAACCGCTGTA
    Rvs  97 TTACGTGGCCGAGCCGC
    Rv0363c Fwd  98 CACCATGCCTATCGCAACGCCC
    (fba) Rvs  99 TTAGTGGGTTAGGGACTTTCCGG
    Rv0569 Fwd 100 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAAGGCAAAGGTCGGGGAC
    Rvs 101 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACGTTCCCCTGGCATGGA
    Rv0572c Fwd 102 CACCATGGGTGAGCACGCCATC
    Rvs 103 TTATAGGTCATCGGATTGAGGTGATC
    Rv0574c Fwd 104 CACCGTGGCTGGCAATCCTGATGT
    Rvs 105 TTACTCCTTGCTCGTTAGGTTGGC
    Rv1264 Fwd 106 CACCGTGACAGACCACGTGCGC
    Rvs 107 TTACGGTGACGAGCCGGC
    Rv1592c Fwd 108 CACCATGGTAGAGCCCGGCAATTTG
    Rvs 109 TTAGAGCGGACGGCGGCT
    Rv1733c Fwd 110 CACCATGATCGCCACAACCCGC
    Rvs 111 TTACCGCTGCGTGCAGAACA
    Rv1734c Fwd 112 CACCATGACCAACGTCGGTGACCA
    Rvs 113 TTATCCTGTTACTGCGGCGCA
    Rv1736c Fwd 114 CACCGTGACGGTGACACCACGGAC
    (narX) Rvs 115 TTACCACCCGCGCCGC
    Rv1737c Fwd 116 CACCATGAGAGGGCAAGCGGC
    (narK2) Rvs 117 TTACCTGGACGCCTCCTCACTC
    Rv1738 Fwd 118 CACCATGTGCGGCGACCAGTC
    Rvs 119 TTAATACAACAATCGCGCCGG
    Rv1739c Fwd 120 CACCATGATTCCCACGATGACATCG
    Rvs 121 TTAGCGCCGACGGAACG
    Rv1813c Fwd 122 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATCACAAACCTCCGACGC
    Rvs 123 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGTTGCACGCCCAGTTGAC
    Rv1997 Fwd 124 CACCTTGTCGGCGTCAGTGTCTGC
    (ctpF) Rvs 125 TTATGGCGGTTGCGCCC
    Rv1998c Fwd 126 CACCATGAGTTTCCACGATCTTCATCACC
    Rvs 127 TTACGTTGTACTCGTGCGGTTCTC
    Rv2003c Fwd 128 CACCGTGGTCAAGCGCTCTCGG
    Rvs 129 TTATTCCGACTCGAGTGGGTGA
    Rv2005c Fwd 130 CACCATGTCTAAACCCCGCAAGCA
    Rvs 131 TTACGACTGCCGTGCCACG
    Rv2007c Fwd 132 CACCGTGACCTATGTGATCGGTAGTGAGTG
    (fdxA) Rvs 133 TTAAGGGCACTCCACCGGGA
    Rv2028c Fwd 134 CACCATGAACCAATCACACAAACCCC
    Rvs 135 TTACAGATACTGCTGACCGACGACC
    Rv2029c Fwd 136 CACCATGACGGAGCCAGCGG
    (pfkB) Rvs 137 TTATGGCGAGGCTTCCGG
    Rv2030c Fwd 138 GGGGACAAGTTTGTACAAAAAAGCAGGCTTACTGATGACCGCAGCGGCT
    Rvs 139 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACAGACCGGTCGGG-
    TAGGTTT
    Rv2031c Fwd 140 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGCCACCACCCTTCCCGT
    (hspX) Rvs 141 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGTTGGTGGACCGGATCT-
    GAAT
    Rv2032 Fwd 142 CACCATGCCGGACACCATGGTG
    Rvs 143 TTAGTGATCCTTAGCCCGAACGTG
    Rv2428 Fwd 144 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCCACTGCTAACCATTGGC
    (ahpC) Rvs 145 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGGCCGAAGCCTTGAGGAGT
    Rv2624c Fwd 146 CACCATGTCTGGGAGAGGAGAGCCG
    Rvs 147 TTAGCGAACGACAAGCACCGA
    Rv2625c Fwd 148 GGGGACAAGTTTGTACAAAAAAGCAGGCTTACGTGATGCGATCCCGCT
    Rvs 149 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACCCCGCATCGGAAAACC
    Rv2627c Fwd 150 GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGCAAGTTCTGCGAGCGA
    Rvs 151 GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAGGAACGGTCGCGCTGTGT
    Rv2628 Fwd 152 CACCATGTCCACGCAACGACCG
    Rvs 153 TTAACCGCAACGGCAATCTCA
    Rv2629 Fwd 154, CACCATGCGATCAGAACGTCTCCG
    Rvs 155, TTAGGATCTATGGCTGCCGAGTC
    Rv2630 Fwd 156, CACCATGCTGCACCGCGACGA
    Rvs 157, TTACACATCGAGCGTTACCGCAC
    Rv2659c Fwd 158, GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGTGACGCAAACCGGCAA
    Rvs 159, GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACATCTCCTGGTTCTCGGCC
    Rv2780 Fwd 160, GGGGACAAGTTTGTACAAAAAAGCAGGCTTACGCGTCGGTATTCCGACC
    Rvs 161, GGGGACCACTTTGTACAAGAAAGCTGGGTCCTACACGCTGGCGGGCTC
    Rv3126c Fwd 162, CACCATGGTCATCCGGTTTGATCAAATA
    Rvs 163, TTATGGATTGTCAATGACAGCCCA
    Rv3127 Fwd 164, CACCGTGCTCAAGAACGCAGTCTTGC
    Rvs 165, TTAAGGCGGGCTGAACCAACC
    Rv3128c Fwd 166, CACCGTGTGGTCCGCCTCGG
    Rvs 167, TTAGCCCGCCTTGATCAGGA
    Rv3129 Fwd 168, CACCGTGGTGCAAGGCCGCA
    Rvs 169, TTATCGCTGGTTGTGTGACGAG
    Rv3130c Fwd 170, CACCATGAATCACCTAACGACACTTGACG
    Rvs 171, TTACACAACCAGCGATAGCGCTC
    Rv3131 Fwd 172, CACCATGAACACCCATTTCCCGG
    Rvs 173, TTAGCACCGTTGTCGCAGTAGCT
    Rv3132c Fwd 174, CACCATGACAACAGGGGGCCTCG
    Rvs 175, TTACTGCGACAACGGTGCTGAC
    Rv3134c Fwd 176, CACCATGAGCGATCCTCGGCCA
    Rvs 177, TTACAAGTTGGCACTGCGTACCG
    Rv3841 Fwd 178, CCGGCTGAGATCTATGACAGAATACGAAGGGC
    (bfrB) Rvs 179, CCCCGCCAGGGAACTAGAGGCGGC
    Rv3842c Fwd 180, CACCATGACATGGGCCGACGAG
    (glpQ1) Rvs 181, TTAGCGAGTGGTCCCGTTCG
    Rv3908 Fwd 182, CACCGTGTCCGACGGCGAACAA
    Rvs 183, TTACGGCCCCGGCCC
  • PCR reactions were carried out using Platinum Tag DNA Polymerase (GIBCO BRL) in a 50 μl reaction volume containing 60 mM Tris-SO[0163] 4 (pH 8.9), 18 mM Ammonium Sulfate, 0.2 mM of each of the four nucleotides, 0.2 μM of each primer and 10 ng of M. tuberculosis H37Rv chromosomal DNA. The reaction mixtures were initially heated to 95° C. for 5 min., followed by 35 cycles of: 95° C. for 45 sec, 60° C. for 45 sec and 72° C. for 2 min. The amplification products were precipitated by PEG/MgCl2, and dissolved in 50 μL TE buffer.
  • DNA fragments were cloned and expressed in Gateway Cloning system (Life Technology). First, to create Entry Clones, 5 μL of DNA fragment was mixed with 1 μL of pDONR201, 2 μL of BP CLONASE enzyme mix and 2 μL of BP reaction buffer. The recombination reactions were carried out at 25° C. for 60 min. After Proteinase K treatment at 37° C. for 10 min., 5 μL of each sample was used to transform [0164] E. coli DH5α competent cells. Transformants were selected on LB plates containing 50 μg/mL kanamycin. One bacterial clone from each transformation was grown in 3 mL LB medium containing 50 μg/mL kanamycin and plasmid DNA was isolated (Qiagen).
  • Second, to create expression clones, 2 μL of each entry clone DNA was mixed with 1 μL of His-tagged expression vector (pDest17), 2 μL LR reaction buffer, 2 μL LR CLONASE enzyme mix and 3 μL TE. After recombination at 25° C. for 60 min. and Proteinase K treatment at 37° C. for 10 min., 5 μL of each sample was used to transform [0165] E. coli BL21-SI competent cells. Transformants were selected on LBON (LB without NaCl) plates containing 100 μg/mL ampicillin. The resulting E. coli clones express recombinant proteins carrying a 6-histine tag at the N-terminal. All clones were confirmed by DNA sequencing.
  • Recombinant proteins were purified from transformed [0166] E. coli BL21-SI cells cultured in 900 mL LBON medium containing 100 μg/mL at 30° C. until OD600 =0.4-0.6. At this point 100 mL 3 M NaCl was added and 3 hours later bacteria were harvested by centrifugation. Bacteria pellets were resuspended in 20 mL bacterial protein extraction reagent (Pierce) incubated for 10 min. at room temperature and pelleted by centrifugation. Bacteria were lysed and their DNA digested by treating with lysozyme (0.1 mg/mL) and DNase I (2.5 μg/mL) at room temperature for 30 minutes, with gentle agitation. The recombinant protein forms inclusion bodies and can be pelleted by centrifugation at 27.000×g for 15 min. Protein pellets were solubilized by adding 20 ml of sonication buffer (8 M urea, 50 mM Na2HPO4, 100 mM Tris-HCl, pH 8.0) and sonicating 5×30 sec pulses interrupted by a 30 sec pause. After another centrifugation at 27.000×g for 15 min., supernatants were applied to 10 mL TALON columns (Clontech). The columns were washed with 50 mL sonication buffer. Bound proteins were eluted by lowering pH (8 M urea, 50 mM Na2HPO4, 100 mM Tris-HCl, pH 4.5). 5 mL fractions were collected and analyzed by SDS-PAGE. Fractions containing recombinant combinant protein were pooled. Further purification was achieved by anion- or cation-exchange chromatography on Hitrap columns (Pharmacia). Bound protein was eluted using a NaCl gradient from 0-500 mM in 3 M urea, 10 mM Tris-HCl, pH 8.0. All fractions were collected and analyzed on SDS-PAGE using Coomassie staining. Fractions containing recombinant protein were pooled. Final protein concentrations were determined by micro BCA (Pierce).
    • Example 2
  • Prophylactic Versus Therapeutic Vaccine. [0167]
  • Murine Vaccination Models. [0168]
  • A prophylactic vaccine given prior to infection should induce an immune response sufficiently strong to prevent or dampen the initial proliferation of the bacteria in the acute phase and thereby reduce the ensuing disease. In the murine prophylactic vaccine model outlined in FIG. 1A, naïve mice are immunized 3 times, 2 weeks apart with recombinant antigens. Six weeks after the last immunization, the mice are given an aerosol infection with approximately 250 [0169] M. tuberculosis bacilli. The protective capacity of the vaccine is evaluated by enumeration of the bacteria in spleen and lung 6 weeks post-infection.
  • To define the optimal components for a therapeutic vaccine, a murine reactivation model of latent TB has been established (van Pinxteren, 2000) (FIG. 1B). An aerosol infection with approximately 250 [0170] M. tuberculosis bacilli is given and at the peak of infection 6 weeks later, the mice receive an 8-week course of anti-mycobacterial drug treatment of isoniazid and rifabutin given in the drinking water. This reduces the bacterial load in spleen and lung to a low level (about 500 bacteria per organ). This latent phase of low chronic infection is stable for 9-10 weeks after which a slow spontaneous reactivation occurs. The therapeutic vaccine is given as 3 subcutaneous (s.c.) immunizations about 5 weeks after cessation of drug treatment. The effect of the therapeutic vaccine is evaluated as protection against reactivation determined by enumeration of bacteria in spleen and lung 7 weeks after the last immunization.
  • The Effect of the Antigens in a Prophylactic or a Therapeutic Vaccine. [0171]
  • BCG, ESAT6, and Rv2031c, one of the most prominent proteins induced under low oxygen conditions (Rosenkrands, 2002), were analyzed for their prophylactic and therapeutic vaccine potential. Naïve or latently infected C57BI mice were immunized with one s.c. injection of 2.5×10[0172] 5 BCG, or 3 s.c. immunizations of 10 μg of either recombinant ESAT6 or recombinant Rv2031c in a DDA/MPL adjuvant. The vaccinations were done in groups of 5 mice and protective capacity of the vaccines was evaluated as described above. FIG. 2 shows the bacterial load in the lung in the acute phase (A) and in the reactivation phase (B), after prophylactic and therapeutic vaccination respectively. ESAT6 (as previously described by Brandt, 2000) offers protection against acute phase infection at the level of BCG (FIG. 2A). However, neither of the two shows any protective effect against reactivation of the infection when given during latent infection (FIG. 2B). In contrast, Rv2031c, the low oxygen induced antigen, offers no protection against the acute phase of the infection when given as a prophylactic vaccine, but gives some protection against reactivation when given as a therapeutic vaccine. That is, some antigens, here exemplified by ESAT6, though potent as prophylactic vaccines have no effect as therapeutic vaccines. In contrast, other antigens, here exemplified by Rv2031c, can be efficient therapeutic vaccines although they have no effect or only negligible effect as prophylactic vaccines.
    • Example 3
  • Low Oxygen Induced Antigens, Rv2031c, as Therapeutic Vaccines: [0173]
  • There is a high variability in bacterial load intrinsic to the reactivation model in the latent and reactivation phase. The analysis of Rv2031c as a therapeutic vaccine was therefore repeated in groups of eight mice. As in the previous experiments the mice were given 3 s.c. immunizations of 10 μg rRv2031c in DDA/MPL. The induced immune responses were analyzed one week post immunization. The mice were partially bled and the PBMC from the blood purified and analyzed for Rv2031c- and ESAT6 specific recall responses. Using ELIspot technique, the frequency of Rv2031c-specific and ESAT6-specific IFN-γ-producing cells were determined in both the rRv2031c immunized and the unimmunized group (FIG. 3). The rRv2031c immunization has increased the frequency of Rv2031c-specific IFN-γ producing cells by a factor of 43 as compared to the unimmunized group. In contrast, the frequency of ESAT6-specific IFN-γ producing cells is significantly higher in the unimmunized group. ESAT6 is an antigen produced in high amounts by the actively-growing [0174] M. tuberculosis bacteria. The level of the ESAT6 specific immune response in infected mice could therefore be indicative the degree of actively-growing infection in the animals. Recent reports have in fact demonstrated such a correlation between the level of ESAT6 response and degree of disease in both M. tuberculosis-infected humans and M. bovis-infected cattle (Doherty, 2002, Vordermeier, 2002). Therefore, the higher ESAT6 response in the unimmunized group of latently-infected mice could be indicative of a transition into the reactivation phase, where the bacteria are again beginning to multiply.
  • To analyze the epitope recognition pattern of Rv2031c, fourteen overlapping peptides (each 20 amino acids long) covering the whole Rv2031c protein were synthesized. Initially the peptides were analyzed in 4 pools of 3-4 peptides. PBMCs from rRv2031c immunized latently-infected mice were incubated with the peptide pools (5 μg/ml per peptide) for 72 h. The specific peptide-induced IFN-γ production was quantitated in the supernatant in a standard sandwich ELISA using paired anti-murine IFN-γ antibodies (PharMingen) and recombinant IFN-γ (PharMingen) as standard. Both peptide pool 1-4 and 8-10 stimulated a significant IFN-γ response (FIG. 4A). The individual peptides of these two pools were re-analyzed (FIG. 4B). This clearly shows that the response to Rv2031c contains a dominant epitope, peptide 2 (PRSLFPEFSELFMFPSFAG, aa 11-30 of SEQ ID NO:24), and a subdominant epitope, peptide 9 (RTEQKDFDGRSEFAYGSFVR, aa 81-100 of SEQ ID NO:24). [0175]
  • The therapeutic effect of the rRv2031c immunizations was analyzed 7 weeks after the last immunization. FIG. 5 shows the bacterial load in the lung (A) and the spleen (B) of both rRv2031c-immunized and unimmunized mice. There is a clear reduction in the level of bacteria in both organs in the rRv2031c-immunized group. That is, the induction of Rv2031c T cell responses can participate in keeping the latent infection in check. [0176]
    • Example 4
  • Low Oxygen Induced Antigens, Rv0569, as Therapeutic Vaccines [0177]
  • Rv0569 is also a low oxygen induced antigen described in WO0179274 and illustrates very well the potential as a therapeutic vaccine. [0178]
  • We have established a murine reactivation model of latent TB [van Pinxteren et al, 2000, 30:3689-98], which is a variant of the so-called Cornell model. An aerosol infection is allowed to be established and at the peak of [0179] infection 6 weeks after, the mice receive an 8-week course of anti-mycobacterial drug treatment of isoniazid and rifabutin given in the drinking water. This reduces the bacterial load in spleen and lung to a low level. This latent phase of low chronic infection is stable for 9-10 weeks after which a slow spontaneous reactivation can be detected. This model is used to analyze the protective effect of a post exposure vaccine on reactivation.
  • Rv0569, which is highly up regulated under low oxygen growth conditions [Rosenkrands et al, 2002, 184(13): 3485-91], was analyzed for its ability to protect against reactivation given as a therapeutic vaccine in the latent phase of TB infection. Latent infected C57BI mice were vaccinated with 3 s.c. injections of 3 μg recombinant Rv0569 and for comparison with 3 s.c. injections of 3 μg recombinant ESAT6 or one s.c. injection of BCG. The effect of the vaccine is evaluated 7 weeks after the last immunization. The induced immune responses were analyzed for Rv0569 or ESAT6 specific responses in an in vitro recall assay. Isolated splenocytes were incubated with 1 μg/ml of Rv0569 or 1 μg/ml of ESAT6 for 72 h. The produced IFNγ in the culture supernatant was quantitated in a standard sandwich ELISA. FIG. 6 shows a nice Rv0569 specific IFNγ response induced in the Rv0569 vaccinated group with practically no response in the un-vaccinated group. The ESAT6 vaccination enhanced the ESAT6 specific response though a significant ESAT6 response persisted in the un-vaccinated latent infected group. [0180]
  • The Rv0569 induced protection against reactivation was determined by enumeration of bacteria in spleen and lung 7 weeks after the last immunization. FIG. 7 shows the bacterial load in the lung and the spleen of both Rv0569-vaccinated, ESAT6-vaccinated, BCG vaccinated and un-vaccinated latently infected mice. There is a clear reduction in the level of viable bacteria in both spleen and lungs of the Rv0569 vaccinated mice, whereas neither ESAT6 nor BCG are able to inhibit the growth of the [0181] M. tuberculosis bacteria when given as a vaccine during latent infection. That is, the induction of Rv0569 T cell responses can participate in keeping the latent infection in check.
    • References
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  • Boon, C., etal. 2001. J. Bacteriol, 183, 2677-2676. [0183]
  • Brandt, L., et al. 2000 Infect.Immun. 68:2; 791-795. [0184]
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  • All documents cited above are incorporated by reference herein. A variety of modifications and variations on the processes, conditions, reagents and compositions described herein will be readily apparent to one of skill in the art given the teachings of the present invention. Such modifications and variations are within the scope of the invention as set forth in the following claims. [0219]
  • 1 187 1 273 PRT Mycobacterium tuberculosis 1 Val Glu Pro Lys Arg Ser Arg Leu Val Val Cys Ala Pro Glu Pro Ser 1 5 10 15 His Ala Arg Glu Phe Pro Asp Val Ala Val Phe Ser Gly Gly Arg Ala 20 25 30 Asn Ala Ser Gln Ala Glu Arg Leu Ala Arg Ala Val Gly Arg Val Leu 35 40 45 Ala Asp Arg Gly Val Thr Gly Gly Ala Arg Val Arg Leu Thr Met Ala 50 55 60 Asn Cys Ala Asp Gly Pro Thr Leu Val Gln Ile Asn Leu Gln Val Gly 65 70 75 80 Asp Thr Pro Leu Arg Ala Gln Ala Ala Thr Ala Gly Ile Asp Asp Leu 85 90 95 Arg Pro Ala Leu Ile Arg Leu Asp Arg Gln Ile Val Arg Ala Ser Ala 100 105 110 Gln Trp Cys Pro Arg Pro Trp Pro Asp Arg Pro Arg Arg Arg Leu Thr 115 120 125 Thr Pro Ala Glu Ala Leu Val Thr Arg Arg Lys Pro Val Val Leu Arg 130 135 140 Arg Ala Thr Pro Leu Gln Ala Ile Ala Ala Met Asp Ala Met Asp Tyr 145 150 155 160 Asp Val His Leu Phe Thr Asp Ala Glu Thr Gly Glu Asp Ala Val Val 165 170 175 Tyr Arg Ala Gly Pro Ser Gly Leu Arg Leu Ala Arg Gln His His Val 180 185 190 Phe Pro Pro Gly Trp Ser Arg Cys Arg Ala Pro Ala Gly Pro Pro Val 195 200 205 Pro Leu Ile Val Asn Ser Arg Pro Thr Pro Val Leu Thr Glu Ala Ala 210 215 220 Ala Val Asp Arg Ala Arg Glu His Gly Leu Pro Phe Leu Phe Phe Thr 225 230 235 240 Asp Gln Ala Thr Gly Arg Gly Gln Leu Leu Tyr Ser Arg Tyr Asp Gly 245 250 255 Asn Leu Gly Leu Ile Thr Pro Thr Gly Asp Gly Val Ala Asp Gly Leu 260 265 270 Ala 2 152 PRT Mycobacterium tuberculosis 2 Met Ser Pro Gly Ser Arg Arg Ala Ser Pro Gln Ser Ala Arg Glu Val 1 5 10 15 Val Glu Leu Asp Arg Asp Glu Ala Met Arg Leu Leu Ala Ser Val Asp 20 25 30 His Gly Arg Val Val Phe Thr Arg Ala Ala Leu Pro Ala Ile Arg Pro 35 40 45 Val Asn His Leu Val Val Asp Gly Arg Val Ile Gly Arg Thr Arg Leu 50 55 60 Thr Ala Lys Val Ser Val Ala Val Arg Ser Ser Ala Asp Ala Gly Val 65 70 75 80 Val Val Ala Tyr Glu Ala Asp Asp Leu Asp Pro Arg Arg Arg Thr Gly 85 90 95 Trp Ser Val Val Val Thr Gly Leu Ala Thr Glu Val Ser Asp Pro Glu 100 105 110 Gln Val Ala Arg Tyr Gln Arg Leu Leu His Pro Trp Val Asn Met Ala 115 120 125 Met Asp Thr Val Val Ala Ile Glu Pro Glu Ile Val Thr Gly Ile Arg 130 135 140 Ile Val Ala Asp Ser Arg Thr Pro 145 150 3 114 PRT Mycobacterium tuberculosis 3 Val Glu Ser Glu Pro Leu Tyr Lys Leu Lys Ala Glu Phe Phe Lys Thr 1 5 10 15 Leu Ala His Pro Ala Arg Ile Arg Ile Leu Glu Leu Leu Val Glu Arg 20 25 30 Asp Arg Ser Val Gly Glu Leu Leu Ser Ser Asp Val Gly Leu Glu Ser 35 40 45 Ser Asn Leu Ser Gln Gln Leu Gly Val Leu Arg Arg Ala Gly Val Val 50 55 60 Ala Ala Arg Arg Asp Gly Asn Ala Met Ile Tyr Ser Ile Ala Ala Pro 65 70 75 80 Asp Ile Ala Glu Leu Leu Ala Val Ala Arg Lys Val Leu Ala Arg Val 85 90 95 Leu Ser Asp Arg Val Ala Val Leu Glu Asp Leu Arg Ala Gly Gly Ser 100 105 110 Ala Thr 4 344 PRT Mycobacterium tuberculosis 4 Met Pro Ile Ala Thr Pro Glu Val Tyr Ala Glu Met Leu Gly Gln Ala 1 5 10 15 Lys Gln Asn Ser Tyr Ala Phe Pro Ala Ile Asn Cys Thr Ser Ser Glu 20 25 30 Thr Val Asn Ala Ala Ile Lys Gly Phe Ala Asp Ala Gly Ser Asp Gly 35 40 45 Ile Ile Gln Phe Ser Thr Gly Gly Ala Glu Phe Gly Ser Gly Leu Gly 50 55 60 Val Lys Asp Met Val Thr Gly Ala Val Ala Leu Ala Glu Phe Thr His 65 70 75 80 Val Ile Ala Ala Lys Tyr Pro Val Asn Val Ala Leu His Thr Asp His 85 90 95 Cys Pro Lys Asp Lys Leu Asp Ser Tyr Val Arg Pro Leu Leu Ala Ile 100 105 110 Ser Ala Gln Arg Val Ser Lys Gly Gly Asn Pro Leu Phe Gln Ser His 115 120 125 Met Trp Asp Gly Ser Ala Val Pro Ile Asp Glu Asn Leu Ala Ile Ala 130 135 140 Gln Glu Leu Leu Lys Ala Ala Ala Ala Ala Lys Ile Ile Leu Glu Ile 145 150 155 160 Glu Ile Gly Val Val Gly Gly Glu Glu Asp Gly Val Ala Asn Glu Ile 165 170 175 Asn Glu Lys Leu Tyr Thr Ser Pro Glu Asp Phe Glu Lys Thr Ile Glu 180 185 190 Ala Leu Gly Ala Gly Glu His Gly Lys Tyr Leu Leu Ala Ala Thr Phe 195 200 205 Gly Asn Val His Gly Val Tyr Lys Pro Gly Asn Val Lys Leu Arg Pro 210 215 220 Asp Ile Leu Ala Gln Gly Gln Gln Val Ala Ala Ala Lys Leu Gly Leu 225 230 235 240 Pro Ala Asp Ala Lys Pro Phe Asp Phe Val Phe His Gly Gly Ser Gly 245 250 255 Ser Leu Lys Ser Glu Ile Glu Glu Ala Leu Arg Tyr Gly Val Val Lys 260 265 270 Met Asn Val Asp Thr Asp Thr Gln Tyr Ala Phe Thr Arg Pro Ile Ala 275 280 285 Gly His Met Phe Thr Asn Tyr Asp Gly Val Leu Lys Val Asp Gly Glu 290 295 300 Val Gly Val Lys Lys Val Tyr Asp Pro Arg Ser Tyr Leu Lys Lys Ala 305 310 315 320 Glu Ala Ser Met Ser Gln Arg Val Val Gln Ala Cys Asn Asp Leu His 325 330 335 Cys Ala Gly Lys Ser Leu Thr His 340 5 113 PRT Mycobacterium tuberculosis 5 Met Gly Glu His Ala Ile Lys Arg His Met Arg Gln Arg Lys Pro Thr 1 5 10 15 Lys His Pro Leu Ala Gln Lys Arg Gly Ala Arg Ile Leu Val Phe Thr 20 25 30 Asp Asp Pro Arg Arg Ser Val Leu Ile Val Pro Gly Cys His Leu Asp 35 40 45 Ser Met Arg Arg Glu Lys Asn Ala Tyr Tyr Phe Gln Asp Gly Asn Ala 50 55 60 Leu Val Gly Met Val Val Ser Gly Gly Thr Val Glu Tyr Asp Ala Asp 65 70 75 80 Asp Arg Thr Tyr Val Val Gln Leu Thr Asp Gly Arg His Thr Thr Glu 85 90 95 Ser Ser Phe Glu His Ser Ser Pro Ser Arg Ser Pro Gln Ser Asp Asp 100 105 110 Leu 6 380 PRT Mycobacterium tuberculosis 6 Val Ala Gly Asn Pro Asp Val Val Thr Val Leu Leu Gly Gly Asp Val 1 5 10 15 Met Leu Gly Arg Gly Val Asp Gln Ile Leu Pro His Pro Gly Lys Pro 20 25 30 Gln Leu Arg Glu Arg Tyr Met Arg Asp Ala Thr Gly Tyr Val Arg Leu 35 40 45 Ala Glu Arg Val Asn Gly Arg Ile Pro Leu Pro Val Asp Trp Arg Trp 50 55 60 Pro Trp Gly Glu Ala Leu Ala Val Leu Glu Asn Thr Ala Thr Asp Val 65 70 75 80 Cys Leu Ile Asn Leu Glu Thr Thr Ile Thr Ala Asp Gly Glu Phe Ala 85 90 95 Asp Arg Lys Pro Val Cys Tyr Arg Met His Pro Asp Asn Val Pro Ala 100 105 110 Leu Thr Ala Leu Arg Pro His Val Cys Ala Leu Ala Asn Asn His Ile 115 120 125 Leu Asp Phe Gly Tyr Gln Gly Leu Thr Asp Thr Val Ala Ala Leu Ala 130 135 140 Gly Ala Gly Ile Gln Ser Val Gly Ala Gly Ala Asp Leu Leu Ala Ala 145 150 155 160 Arg Arg Ser Ala Leu Val Thr Val Gly His Glu Arg Arg Val Ile Val 165 170 175 Gly Ser Val Ala Ala Glu Ser Ser Gly Val Pro Glu Ser Trp Ala Ala 180 185 190 Arg Arg Asp Arg Pro Gly Val Trp Leu Ile Arg Asp Pro Ala Gln Arg 195 200 205 Asp Val Ala Asp Asp Val Ala Ala Gln Val Leu Ala Asp Lys Arg Pro 210 215 220 Gly Asp Ile Ala Ile Val Ser Met His Trp Gly Ser Asn Trp Gly Tyr 225 230 235 240 Ala Thr Ala Pro Gly Asp Val Ala Phe Ala His Arg Leu Ile Asp Ala 245 250 255 Gly Ile Asp Met Val His Gly His Ser Ser His His Pro Arg Pro Ile 260 265 270 Glu Ile Tyr Arg Gly Lys Pro Ile Leu Tyr Gly Cys Gly Asp Val Val 275 280 285 Asp Asp Tyr Glu Gly Ile Gly Gly His Glu Ser Phe Arg Ser Glu Leu 290 295 300 Arg Leu Leu Tyr Leu Thr Val Thr Asp Pro Ala Ser Gly Asn Leu Ile 305 310 315 320 Ser Leu Gln Met Leu Pro Leu Arg Val Ser Arg Met Arg Leu Gln Arg 325 330 335 Ala Ser Gln Thr Asp Thr Glu Trp Leu Arg Asn Thr Ile Glu Arg Ile 340 345 350 Ser Arg Arg Phe Gly Ile Arg Val Val Thr Arg Pro Asp Asn Leu Leu 355 360 365 Glu Val Val Pro Ala Ala Asn Leu Thr Ser Lys Glu 370 375 380 7 397 PRT Mycobacterium tuberculosis 7 Val Thr Asp His Val Arg Glu Ala Asp Asp Ala Asn Ile Asp Asp Leu 1 5 10 15 Leu Gly Asp Leu Gly Gly Thr Ala Arg Ala Glu Arg Ala Lys Leu Val 20 25 30 Glu Trp Leu Leu Glu Gln Gly Ile Thr Pro Asp Glu Ile Arg Ala Thr 35 40 45 Asn Pro Pro Leu Leu Leu Ala Thr Arg His Leu Val Gly Asp Asp Gly 50 55 60 Thr Tyr Val Ser Ala Arg Glu Ile Ser Glu Asn Tyr Gly Val Asp Leu 65 70 75 80 Glu Leu Leu Gln Arg Val Gln Arg Ala Val Gly Leu Ala Arg Val Asp 85 90 95 Asp Pro Asp Ala Val Val His Met Arg Ala Asp Gly Glu Ala Ala Ala 100 105 110 Arg Ala Gln Arg Phe Val Glu Leu Gly Leu Asn Pro Asp Gln Val Val 115 120 125 Leu Val Val Arg Val Leu Ala Glu Gly Leu Ser His Ala Ala Glu Ala 130 135 140 Met Arg Tyr Thr Ala Leu Glu Ala Ile Met Arg Pro Gly Ala Thr Glu 145 150 155 160 Leu Asp Ile Ala Lys Gly Ser Gln Ala Leu Val Ser Gln Ile Val Pro 165 170 175 Leu Leu Gly Pro Met Ile Gln Asp Met Leu Phe Met Gln Leu Arg His 180 185 190 Met Met Glu Thr Glu Ala Val Asn Ala Gly Glu Arg Ala Ala Gly Lys 195 200 205 Pro Leu Pro Gly Ala Arg Gln Val Thr Val Ala Phe Ala Asp Leu Val 210 215 220 Gly Phe Thr Gln Leu Gly Glu Val Val Ser Ala Glu Glu Leu Gly His 225 230 235 240 Leu Ala Gly Arg Leu Ala Gly Leu Ala Arg Asp Leu Thr Ala Pro Pro 245 250 255 Val Trp Phe Ile Lys Thr Ile Gly Asp Ala Val Met Leu Val Cys Pro 260 265 270 Asp Pro Ala Pro Leu Leu Asp Thr Val Leu Lys Leu Val Glu Val Val 275 280 285 Asp Thr Asp Asn Asn Phe Pro Arg Leu Arg Ala Gly Val Ala Ser Gly 290 295 300 Met Ala Val Ser Arg Ala Gly Asp Trp Phe Gly Ser Pro Val Asn Val 305 310 315 320 Ala Ser Arg Val Thr Gly Val Ala Arg Pro Gly Ala Val Leu Val Ala 325 330 335 Asp Ser Val Arg Glu Ala Leu Gly Asp Ala Pro Glu Ala Asp Gly Phe 340 345 350 Gln Trp Ser Phe Ala Gly Pro Arg Arg Leu Arg Gly Ile Arg Gly Asp 355 360 365 Val Arg Leu Phe Arg Val Arg Arg Gly Ala Thr Arg Thr Gly Ser Gly 370 375 380 Gly Ala Ala Gln Asp Asp Asp Leu Ala Gly Ser Ser Pro 385 390 395 8 446 PRT Mycobacterium tuberculosis 8 Met Val Glu Pro Gly Asn Leu Ala Gly Ala Thr Gly Ala Glu Trp Ile 1 5 10 15 Gly Arg Pro Pro His Glu Glu Leu Gln Arg Lys Val Arg Pro Leu Leu 20 25 30 Pro Ser Asp Asp Pro Phe Tyr Phe Pro Pro Ala Gly Tyr Gln His Ala 35 40 45 Val Pro Gly Thr Val Leu Arg Ser Arg Asp Val Glu Leu Ala Phe Met 50 55 60 Gly Leu Ile Pro Gln Pro Val Thr Ala Thr Gln Leu Leu Tyr Arg Thr 65 70 75 80 Thr Asn Met Tyr Gly Asn Pro Glu Ala Thr Val Thr Thr Val Ile Val 85 90 95 Pro Ala Glu Leu Ala Pro Gly Gln Thr Cys Pro Leu Leu Ser Tyr Gln 100 105 110 Cys Ala Ile Asp Ala Met Ser Ser Arg Cys Phe Pro Ser Tyr Ala Leu 115 120 125 Arg Arg Arg Ala Lys Ala Leu Gly Ser Leu Thr Gln Met Glu Leu Leu 130 135 140 Met Ile Ser Ala Ala Leu Ala Glu Gly Trp Ala Val Ser Val Pro Asp 145 150 155 160 His Glu Gly Pro Lys Gly Leu Trp Gly Ser Pro Tyr Glu Pro Gly Tyr 165 170 175 Arg Val Leu Asp Gly Ile Arg Ala Ala Leu Asn Ser Glu Arg Val Gly 180 185 190 Leu Ser Pro Ala Thr Pro Ile Gly Leu Trp Gly Tyr Ser Gly Gly Gly 195 200 205 Leu Ala Ser Ala Trp Ala Ala Glu Ala Cys Gly Glu Tyr Ala Pro Asp 210 215 220 Leu Asp Ile Val Gly Ala Val Leu Gly Ser Pro Val Gly Asp Leu Gly 225 230 235 240 His Thr Phe Arg Arg Leu Asn Gly Thr Leu Leu Ala Gly Leu Pro Ala 245 250 255 Leu Val Val Ala Ala Leu Gln His Ser Tyr Pro Gly Leu Ala Arg Val 260 265 270 Ile Lys Glu His Ala Asn Asp Glu Gly Arg Gln Leu Leu Glu Gln Leu 275 280 285 Thr Glu Met Thr Thr Val Asp Ala Val Ile Arg Met Ala Gly Arg Asp 290 295 300 Met Gly Asp Phe Leu Asp Glu Pro Leu Glu Asp Ile Leu Ser Thr Pro 305 310 315 320 Glu Ile Ser His Val Phe Gly Asp Thr Lys Leu Gly Ser Ala Val Pro 325 330 335 Thr Pro Pro Val Leu Ile Val Gln Ala Val His Asp Tyr Leu Ile Asp 340 345 350 Val Ser Asp Ile Asp Ala Leu Ala Asp Ser Tyr Thr Ala Gly Gly Ala 355 360 365 Asn Val Thr Tyr His Arg Asp Leu Phe Ser Glu His Val Ser Leu His 370 375 380 Pro Leu Ser Ala Pro Met Thr Leu Arg Trp Leu Thr Asp Arg Phe Ala 385 390 395 400 Gly Lys Pro Leu Thr Asp His Arg Val Arg Thr Thr Trp Pro Thr Ile 405 410 415 Phe Asn Pro Met Thr Tyr Ala Gly Met Ala Arg Leu Ala Val Ile Ala 420 425 430 Ala Lys Val Ile Thr Gly Arg Lys Leu Ser Arg Arg Pro Leu 435 440 445 9 210 PRT Mycobacterium tuberculosis 9 Met Ile Ala Thr Thr Arg Asp Arg Glu Gly Ala Thr Met Ile Thr Phe 1 5 10 15 Arg Leu Arg Leu Pro Cys Arg Thr Ile Leu Arg Val Phe Ser Arg Asn 20 25 30 Pro Leu Val Arg Gly Thr Asp Arg Leu Glu Ala Val Val Met Leu Leu 35 40 45 Ala Val Thr Val Ser Leu Leu Thr Ile Pro Phe Ala Ala Ala Ala Gly 50 55 60 Thr Ala Val Gln Asp Ser Arg Ser His Val Tyr Ala His Gln Ala Gln 65 70 75 80 Thr Arg His Pro Ala Thr Ala Thr Val Ile Asp His Glu Gly Val Ile 85 90 95 Asp Ser Asn Thr Thr Ala Thr Ser Ala Pro Pro Arg Thr Lys Ile Thr 100 105 110 Val Pro Ala Arg Trp Val Val Asn Gly Ile Glu Arg Ser Gly Glu Val 115 120 125 Asn Ala Lys Pro Gly Thr Lys Ser Gly Asp Arg Val Gly Ile Trp Val 130 135 140 Asp Ser Ala Gly Gln Leu Val Asp Glu Pro Ala Pro Pro Ala Arg Ala 145 150 155 160 Ile Ala Asp Ala Ala Leu Ala Ala Leu Gly Leu Trp Leu Ser Val Ala 165 170 175 Ala Val Ala Gly Ala Leu Leu Ala Leu Thr Arg Ala Ile Leu Ile Arg 180 185 190 Val Arg Asn Ala Ser Trp Gln His Asp Ile Asp Ser Leu Phe Cys Thr 195 200 205 Gln Arg 210 10 80 PRT Mycobacterium tuberculosis 10 Met Thr Asn Val Gly Asp Gln Gly Val Asp Ala Val Phe Gly Val Ile 1 5 10 15 Tyr Pro Pro Gln Val Ala Leu Val Ser Phe Gly Lys Pro Ala Gln Arg 20 25 30 Val Cys Ala Val Asp Gly Ala Ile His Val Met Thr Thr Val Leu Ala 35 40 45 Thr Leu Pro Ala Asp His Gly Cys Ser Asp Asp His Arg Gly Ala Leu 50 55 60 Phe Phe Leu Ser Ile Asn Glu Leu Thr Arg Cys Ala Ala Val Thr Gly 65 70 75 80 11 652 PRT Mycobacterium tuberculosis 11 Val Thr Val Thr Pro Arg Thr Gly Ser Arg Ile Glu Glu Leu Leu Ala 1 5 10 15 Arg Ser Gly Arg Phe Phe Ile Pro Gly Glu Ile Ser Ala Asp Leu Arg 20 25 30 Thr Val Thr Arg Arg Gly Gly Arg Asp Gly Asp Val Phe Tyr Arg Asp 35 40 45 Arg Trp Ser His Asp Lys Val Val Arg Ser Thr His Gly Val Asn Cys 50 55 60 Thr Gly Ser Cys Ser Trp Lys Ile Tyr Val Lys Asp Asp Ile Ile Thr 65 70 75 80 Trp Glu Thr Gln Glu Thr Asp Tyr Pro Ser Val Gly Pro Asp Arg Pro 85 90 95 Glu Tyr Glu Pro Arg Gly Cys Pro Arg Gly Ala Ala Phe Ser Trp Tyr 100 105 110 Thr Tyr Ser Pro Thr Arg Val Arg His Pro Tyr Ala Arg Gly Val Leu 115 120 125 Val Glu Met Tyr Arg Glu Ala Lys Ala Arg Leu Gly Asp Pro Val Ala 130 135 140 Ala Trp Ala Asp Ile Gln Ala Asp Pro Arg Arg Arg Arg Arg Tyr Gln 145 150 155 160 Arg Ala Arg Gly Lys Gly Gly Leu Val Arg Val Ser Trp Ala Glu Ala 165 170 175 Thr Glu Met Ile Ala Ala Ala His Val His Thr Ile Ser Thr Tyr Gly 180 185 190 Pro Asp Arg Val Ala Gly Phe Ser Pro Ile Pro Ala Met Ser Met Val 195 200 205 Ser His Ala Ala Gly Ser Arg Phe Val Glu Leu Ile Gly Gly Val Met 210 215 220 Thr Ser Phe Tyr Asp Trp Tyr Ala Asp Leu Pro Val Ala Ser Pro Gln 225 230 235 240 Val Phe Gly Asp Gln Thr Asp Val Pro Glu Ser Gly Asp Trp Trp Asp 245 250 255 Val Val Trp Gln Cys Ala Ser Val Leu Leu Thr Tyr Pro Asn Ser Arg 260 265 270 Gln Leu Gly Thr Ala Glu Glu Leu Leu Ala His Ile Asp Gly Pro Ala 275 280 285 Ala Asp Leu Leu Gly Arg Thr Val Ser Glu Leu Arg Arg Ala Asp Pro 290 295 300 Leu Thr Ala Ala Thr Arg Tyr Val Asp Thr Phe Asp Leu Arg Gly Arg 305 310 315 320 Ala Thr Leu Tyr Leu Thr Tyr Trp Thr Ala Gly Asp Thr Arg Asn Arg 325 330 335 Gly Arg Glu Met Leu Ala Phe Ala Gln Thr Tyr Arg Ser Thr Asp Val 340 345 350 Ala Pro Pro Arg Gly Glu Thr Pro Asp Phe Leu Pro Val Val Leu Glu 355 360 365 Phe Ala Ala Thr Val Asp Pro Glu Ala Gly Arg Arg Leu Leu Ser Gly 370 375 380 Tyr Arg Val Pro Ile Ala Ala Leu Cys Asn Ala Leu Thr Glu Ala Ala 385 390 395 400 Leu Pro Tyr Ala His Thr Val Ala Ala Val Cys Arg Thr Gly Asp Met 405 410 415 Met Gly Glu Leu Phe Trp Thr Val Val Pro Tyr Val Thr Met Thr Ile 420 425 430 Val Ala Val Gly Ser Trp Trp Arg Tyr Arg Tyr Asp Lys Phe Gly Trp 435 440 445 Thr Thr Arg Ser Ser Gln Leu Tyr Glu Ser Arg Leu Leu Arg Ile Ala 450 455 460 Ser Pro Met Phe His Phe Gly Ile Leu Val Val Ile Val Gly His Gly 465 470 475 480 Ile Gly Leu Val Ile Pro Gln Ser Trp Thr Gln Ala Ala Gly Leu Ser 485 490 495 Glu Gly Ala Tyr His Val Gln Ala Val Val Leu Gly Ser Ile Ala Gly 500 505 510 Ile Thr Thr Leu Ala Gly Val Thr Leu Leu Ile Tyr Arg Arg Arg Thr 515 520 525 Arg Gly Pro Val Phe Met Ala Thr Thr Val Asn Asp Lys Val Met Tyr 530 535 540 Leu Val Leu Val Ala Ala Ile Val Ala Gly Leu Gly Ala Thr Ala Leu 545 550 555 560 Gly Ser Gly Val Val Gly Glu Ala Tyr Asn Tyr Arg Glu Thr Val Ser 565 570 575 Val Trp Phe Arg Ser Val Trp Val Leu Gln Pro Arg Gly Asp Leu Met 580 585 590 Ala Glu Ala Pro Leu Tyr Tyr Gln Ile His Val Leu Ile Gly Leu Ala 595 600 605 Leu Phe Ala Leu Trp Pro Phe Thr Arg Leu Val His Ala Phe Ser Ala 610 615 620 Pro Ile Gly Tyr Leu Phe Arg Pro Tyr Ile Ile Tyr Arg Ser Arg Glu 625 630 635 640 Glu Leu Val Leu Thr Arg Pro Arg Arg Arg Gly Trp 645 650 12 395 PRT Mycobacterium tuberculosis 12 Met Arg Gly Gln Ala Ala Asn Leu Val Leu Ala Thr Trp Ile Ser Val 1 5 10 15 Val Asn Phe Trp Ala Trp Asn Leu Ile Gly Pro Leu Ser Thr Ser Tyr 20 25 30 Ala Arg Asp Met Ser Leu Ser Ser Ala Glu Ala Ser Leu Leu Val Ala 35 40 45 Thr Pro Ile Leu Val Gly Ala Leu Gly Arg Ile Val Thr Gly Pro Leu 50 55 60 Thr Asp Arg Phe Gly Gly Arg Ala Met Leu Ile Ala Val Thr Leu Ala 65 70 75 80 Ser Ile Leu Pro Val Leu Ala Val Gly Val Ala Ala Thr Met Gly Ser 85 90 95 Tyr Ala Leu Leu Val Phe Phe Gly Leu Phe Leu Gly Val Ala Gly Thr 100 105 110 Ile Phe Ala Val Gly Ile Pro Phe Ala Asn Asn Trp Tyr Gln Pro Ala 115 120 125 Arg Arg Gly Phe Ser Thr Gly Val Phe Gly Met Gly Met Val Gly Thr 130 135 140 Ala Leu Ser Ala Phe Phe Thr Pro Arg Phe Val Arg Trp Phe Gly Leu 145 150 155 160 Phe Thr Thr His Ala Ile Val Ala Ala Ala Leu Ala Ser Thr Ala Val 165 170 175 Val Ala Met Val Val Leu Arg Asp Ala Pro Tyr Phe Arg Pro Asn Ala 180 185 190 Asp Pro Val Leu Pro Arg Leu Lys Ala Ala Ala Arg Leu Pro Val Thr 195 200 205 Trp Glu Met Ser Phe Leu Tyr Ala Ile Val Phe Gly Gly Phe Val Ala 210 215 220 Phe Ser Asn Tyr Leu Pro Thr Tyr Ile Thr Thr Ile Tyr Gly Phe Ser 225 230 235 240 Thr Val Asp Ala Gly Ala Arg Thr Ala Gly Phe Ala Leu Ala Ala Val 245 250 255 Leu Ala Arg Pro Val Gly Gly Trp Leu Ser Asp Arg Ile Ala Pro Arg 260 265 270 His Val Val Leu Ala Ser Leu Ala Gly Thr Ala Leu Leu Ala Phe Ala 275 280 285 Ala Ala Leu Gln Pro Pro Pro Glu Val Trp Ser Ala Ala Thr Phe Ile 290 295 300 Thr Leu Ala Val Cys Leu Gly Val Gly Thr Gly Gly Val Phe Ala Trp 305 310 315 320 Val Ala Arg Arg Ala Pro Ala Ala Ser Val Gly Ser Val Thr Gly Ile 325 330 335 Val Ala Ala Ala Gly Gly Leu Gly Gly Tyr Phe Pro Pro Leu Val Met 340 345 350 Gly Ala Thr Tyr Asp Pro Val Asp Asn Asp Tyr Thr Val Gly Leu Leu 355 360 365 Leu Leu Val Ala Thr Ala Leu Val Ala Cys Thr Tyr Thr Ala Leu His 370 375 380 Ala Arg Glu Pro Val Ser Glu Glu Ala Ser Arg 385 390 395 13 94 PRT Mycobacterium tuberculosis 13 Met Cys Gly Asp Gln Ser Asp His Val Leu Gln His Trp Thr Val Asp 1 5 10 15 Ile Ser Ile Asp Glu His Glu Gly Leu Thr Arg Ala Lys Ala Arg Leu 20 25 30 Arg Trp Arg Glu Lys Glu Leu Val Gly Val Gly Leu Ala Arg Leu Asn 35 40 45 Pro Ala Asp Arg Asn Val Pro Glu Ile Gly Asp Glu Leu Ser Val Ala 50 55 60 Arg Ala Leu Ser Asp Leu Gly Lys Arg Met Leu Lys Val Ser Thr His 65 70 75 80 Asp Ile Glu Ala Val Thr His Gln Pro Ala Arg Leu Leu Tyr 85 90 14 560 PRT Mycobacterium tuberculosis 14 Met Ile Pro Thr Met Thr Ser Ala Gly Trp Ala Pro Gly Val Val Gln 1 5 10 15 Phe Arg Glu Tyr Gln Arg Arg Trp Leu Arg Gly Asp Val Leu Ala Gly 20 25 30 Leu Thr Val Ala Ala Tyr Leu Ile Pro Gln Ala Met Ala Tyr Ala Thr 35 40 45 Val Ala Gly Leu Pro Pro Ala Ala Gly Leu Trp Ala Ser Ile Ala Pro 50 55 60 Leu Ala Ile Tyr Ala Leu Leu Gly Ser Ser Arg Gln Leu Ser Ile Gly 65 70 75 80 Pro Glu Ser Ala Thr Ala Leu Met Thr Ala Ala Val Leu Ala Pro Met 85 90 95 Ala Ala Gly Asp Leu Arg Arg Tyr Ala Val Leu Ala Ala Thr Leu Gly 100 105 110 Leu Leu Val Gly Leu Ile Cys Leu Leu Ala Gly Thr Ala Arg Leu Gly 115 120 125 Phe Leu Ala Ser Leu Arg Ser Arg Pro Val Leu Val Gly Tyr Met Ala 130 135 140 Gly Ile Ala Leu Val Met Ile Ser Ser Gln Leu Gly Thr Ile Thr Gly 145 150 155 160 Thr Ser Val Glu Gly Asn Glu Phe Phe Ser Glu Val His Ser Phe Ala 165 170 175 Thr Ser Val Thr Arg Val His Trp Pro Thr Phe Val Leu Ala Met Ser 180 185 190 Val Leu Ala Leu Leu Thr Met Leu Thr Arg Trp Ala Pro Arg Ala Pro 195 200 205 Gly Pro Ile Ile Ala Val Leu Ala Ala Thr Met Leu Val Ala Val Met 210 215 220 Ser Leu Asp Ala Lys Gly Ile Ala Ile Val Gly Arg Ile Pro Ser Gly 225 230 235 240 Leu Pro Thr Pro Gly Val Pro Pro Val Ser Val Glu Asp Leu Arg Ala 245 250 255 Leu Ile Ile Pro Ala Ala Gly Ile Ala Ile Val Thr Phe Thr Asp Gly 260 265 270 Val Leu Thr Ala Arg Ala Phe Ala Ala Arg Arg Gly Gln Glu Val Asn 275 280 285 Ala Asn Ala Glu Leu Arg Ala Val Gly Ala Cys Asn Ile Ala Ala Gly 290 295 300 Leu Thr His Gly Phe Pro Val Ser Ser Ser Ser Ser Arg Thr Ala Leu 305 310 315 320 Ala Asp Val Val Gly Gly Arg Thr Gln Leu Tyr Ser Leu Ile Ala Leu 325 330 335 Gly Leu Val Val Ile Val Met Val Phe Ala Ser Gly Leu Leu Ala Met 340 345 350 Phe Pro Ile Ala Ala Leu Gly Ala Leu Val Val Tyr Ala Ala Leu Arg 355 360 365 Leu Ile Asp Leu Ser Glu Phe Arg Arg Leu Ala Arg Phe Arg Arg Ser 370 375 380 Glu Leu Met Leu Ala Leu Ala Thr Thr Ala Ala Val Leu Gly Leu Gly 385 390 395 400 Val Phe Tyr Gly Val Leu Ala Ala Val Ala Leu Ser Ile Leu Glu Leu 405 410 415 Leu Arg Arg Val Ala His Pro His Asp Ser Val Leu Gly Phe Val Pro 420 425 430 Gly Ile Ala Gly Met His Asp Ile Asp Asp Tyr Pro Gln Ala Lys Arg 435 440 445 Val Pro Gly Leu Val Val Tyr Arg Tyr Asp Ala Pro Leu Cys Phe Ala 450 455 460 Asn Ala Glu Asp Phe Arg Arg Arg Ala Leu Thr Val Val Asp Gln Asp 465 470 475 480 Pro Gly Gln Val Glu Trp Phe Val Leu Asn Ala Glu Ser Asn Val Glu 485 490 495 Val Asp Leu Thr Ala Leu Asp Ala Leu Asp Gln Leu Arg Thr Glu Leu 500 505 510 Leu Arg Arg Gly Ile Val Phe Ala Met Ala Arg Val Lys Gln Asp Leu 515 520 525 Arg Glu Ser Leu Arg Ala Ala Ser Leu Leu Asp Lys Ile Gly Glu Asp 530 535 540 His Ile Phe Met Thr Leu Pro Thr Ala Val Gln Ala Phe Arg Arg Arg 545 550 555 560 15 143 PRT Mycobacterium tuberculosis 15 Met Ile Thr Asn Leu Arg Arg Arg Thr Ala Met Ala Ala Ala Gly Leu 1 5 10 15 Gly Ala Ala Leu Gly Leu Gly Ile Leu Leu Val Pro Thr Val Asp Ala 20 25 30 His Leu Ala Asn Gly Ser Met Ser Glu Val Met Met Ser Glu Ile Ala 35 40 45 Gly Leu Pro Ile Pro Pro Ile Ile His Tyr Gly Ala Ile Ala Tyr Ala 50 55 60 Pro Ser Gly Ala Ser Gly Lys Ala Trp His Gln Arg Thr Pro Ala Arg 65 70 75 80 Ala Glu Gln Val Ala Leu Glu Lys Cys Gly Asp Lys Thr Cys Lys Val 85 90 95 Val Ser Arg Phe Thr Arg Cys Gly Ala Val Ala Tyr Asn Gly Ser Lys 100 105 110 Tyr Gln Gly Gly Thr Gly Leu Thr Arg Arg Ala Ala Glu Asp Asp Ala 115 120 125 Val Asn Arg Leu Glu Gly Gly Arg Ile Val Asn Trp Ala Cys Asn 130 135 140 16 905 PRT Mycobacterium tuberculosis 16 Leu Ser Ala Ser Val Ser Ala Thr Thr Ala His His Gly Leu Pro Ala 1 5 10 15 His Glu Val Val Leu Leu Leu Glu Ser Asp Pro Tyr His Gly Leu Ser 20 25 30 Asp Gly Glu Ala Ala Gln Arg Leu Glu Arg Phe Gly Pro Asn Thr Leu 35 40 45 Ala Val Val Thr Arg Ala Ser Leu Leu Ala Arg Ile Leu Arg Gln Phe 50 55 60 His His Pro Leu Ile Tyr Val Leu Leu Val Ala Gly Thr Ile Thr Ala 65 70 75 80 Gly Leu Lys Glu Phe Val Asp Ala Ala Val Ile Phe Gly Val Val Val 85 90 95 Ile Asn Ala Ile Val Gly Phe Ile Gln Glu Ser Lys Ala Glu Ala Ala 100 105 110 Leu Gln Gly Leu Arg Ser Met Val His Thr His Ala Lys Val Val Arg 115 120 125 Glu Gly His Glu His Thr Met Pro Ser Glu Glu Leu Val Pro Gly Asp 130 135 140 Leu Val Leu Leu Ala Ala Gly Asp Lys Val Pro Ala Asp Leu Arg Leu 145 150 155 160 Val Arg Gln Thr Gly Leu Ser Val Asn Glu Ser Ala Leu Thr Gly Glu 165 170 175 Ser Thr Pro Val His Lys Asp Glu Val Ala Leu Pro Glu Gly Thr Pro 180 185 190 Val Ala Asp Arg Arg Asn Ile Ala Tyr Ser Gly Thr Leu Val Thr Ala 195 200 205 Gly His Gly Ala Gly Ile Val Val Ala Thr Gly Ala Glu Thr Glu Leu 210 215 220 Gly Glu Ile His Arg Leu Val Gly Ala Ala Glu Val Val Ala Thr Pro 225 230 235 240 Leu Thr Ala Lys Leu Ala Trp Phe Ser Lys Phe Leu Thr Ile Ala Ile 245 250 255 Leu Gly Leu Ala Ala Leu Thr Phe Gly Val Gly Leu Leu Arg Arg Gln 260 265 270 Asp Ala Val Glu Thr Phe Thr Ala Ala Ile Ala Leu Ala Val Gly Ala 275 280 285 Ile Pro Glu Gly Leu Pro Thr Ala Val Thr Ile Thr Leu Ala Ile Gly 290 295 300 Met Ala Arg Met Ala Lys Arg Arg Ala Val Ile Arg Arg Leu Pro Ala 305 310 315 320 Val Glu Thr Leu Gly Ser Thr Thr Val Ile Cys Ala Asp Lys Thr Gly 325 330 335 Thr Leu Thr Glu Asn Gln Met Thr Val Gln Ser Ile Trp Thr Pro His 340 345 350 Gly Glu Ile Arg Ala Thr Gly Thr Gly Tyr Ala Pro Asp Val Leu Leu 355 360 365 Cys Asp Thr Asp Asp Ala Pro Val Pro Val Asn Ala Asn Ala Ala Leu 370 375 380 Arg Trp Ser Leu Leu Ala Gly Ala Cys Ser Asn Asp Ala Ala Leu Val 385 390 395 400 Arg Asp Gly Thr Arg Trp Gln Ile Val Gly Asp Pro Thr Glu Gly Ala 405 410 415 Met Leu Val Val Ala Ala Lys Ala Gly Phe Asn Pro Glu Arg Leu Ala 420 425 430 Thr Thr Leu Pro Gln Val Ala Ala Ile Pro Phe Ser Ser Glu Arg Gln 435 440 445 Tyr Met Ala Thr Leu His Arg Asp Gly Thr Asp His Val Val Leu Ala 450 455 460 Lys Gly Ala Val Glu Arg Met Leu Asp Leu Cys Gly Thr Glu Met Gly 465 470 475 480 Ala Asp Gly Ala Leu Arg Pro Leu Asp Arg Ala Thr Val Leu Arg Ala 485 490 495 Thr Glu Met Leu Thr Ser Arg Gly Leu Arg Val Leu Ala Thr Gly Met 500 505 510 Gly Ala Gly Ala Gly Thr Pro Asp Asp Phe Asp Glu Asn Val Ile Pro 515 520 525 Gly Ser Leu Ala Leu Thr Gly Leu Gln Ala Met Ser Asp Pro Pro Arg 530 535 540 Ala Ala Ala Ala Ser Ala Val Ala Ala Cys His Ser Ala Gly Ile Ala 545 550 555 560 Val Lys Met Ile Thr Gly Asp His Ala Gly Thr Ala Thr Ala Ile Ala 565 570 575 Thr Glu Val Gly Leu Leu Asp Asn Thr Glu Pro Ala Ala Gly Ser Val 580 585 590 Leu Thr Gly Ala Glu Leu Ala Ala Leu Ser Ala Asp Gln Tyr Pro Glu 595 600 605 Ala Val Asp Thr Ala Ser Val Phe Ala Arg Val Ser Pro Glu Gln Lys 610 615 620 Leu Arg Leu Val Gln Ala Leu Gln Ala Arg Gly His Val Val Ala Met 625 630 635 640 Thr Gly Asp Gly Val Asn Asp Ala Pro Ala Leu Arg Gln Ala Asn Ile 645 650 655 Gly Val Ala Met Gly Arg Gly Gly Thr Glu Val Ala Lys Asp Ala Ala 660 665 670 Asp Met Val Leu Thr Asp Asp Asp Phe Ala Thr Ile Glu Ala Ala Val 675 680 685 Glu Glu Gly Arg Gly Val Phe Asp Asn Leu Thr Lys Phe Ile Thr Trp 690 695 700 Thr Leu Pro Thr Asn Leu Gly Glu Gly Leu Val Ile Leu Ala Ala Ile 705 710 715 720 Ala Val Gly Val Ala Leu Pro Ile Leu Pro Thr Gln Ile Leu Trp Ile 725 730 735 Asn Met Thr Thr Ala Ile Ala Leu Gly Leu Met Leu Ala Phe Glu Pro 740 745 750 Lys Glu Ala Gly Ile Met Thr Arg Pro Pro Arg Asp Pro Asp Gln Pro 755 760 765 Leu Leu Thr Gly Trp Leu Val Arg Arg Thr Leu Leu Val Ser Thr Leu 770 775 780 Leu Val Ala Ser Ala Trp Trp Leu Phe Ala Trp Glu Leu Asp Asn Gly 785 790 795 800 Ala Gly Leu His Glu Ala Arg Thr Ala Ala Leu Asn Leu Phe Val Val 805 810 815 Val Glu Ala Phe Tyr Leu Phe Ser Cys Arg Ser Leu Thr Arg Ser Ala 820 825 830 Trp Arg Leu Gly Met Phe Ala Asn Arg Trp Ile Ile Leu Gly Val Ser 835 840 845 Ala Gln Ala Ile Ala Gln Phe Ala Ile Thr Tyr Leu Pro Ala Met Asn 850 855 860 Met Val Phe Asp Thr Ala Pro Ile Asp Ile Gly Val Trp Val Arg Ile 865 870 875 880 Phe Ala Val Ala Thr Ala Ile Thr Ile Val Val Ala Thr Asp Thr Leu 885 890 895 Leu Pro Arg Ile Arg Ala Gln Pro Pro 900 905 17 258 PRT Mycobacterium tuberculosis 17 Met Ser Phe His Asp Leu His His Gln Gly Val Pro Phe Val Leu Pro 1 5 10 15 Asn Ala Trp Asp Val Pro Ser Ala Leu Ala Tyr Leu Ala Glu Gly Phe 20 25 30 Thr Ala Ile Gly Thr Thr Ser Phe Gly Val Ser Ser Ser Gly Gly His 35 40 45 Pro Asp Gly His Arg Ala Thr Arg Gly Ala Asn Ile Ala Leu Ala Ala 50 55 60 Ala Leu Ala Pro Leu Gln Cys Tyr Val Ser Val Asp Ile Glu Asp Gly 65 70 75 80 Tyr Ser Asp Glu Pro Asp Ala Ile Ala Asp Tyr Val Ala Gln Leu Ser 85 90 95 Thr Ala Gly Ile Asn Ile Glu Asp Ser Ser Ala Glu Lys Leu Ile Asp 100 105 110 Pro Ala Leu Ala Ala Ala Lys Ile Val Ala Ile Lys Gln Arg Asn Pro 115 120 125 Glu Val Phe Val Asn Ala Arg Val Asp Thr Tyr Trp Leu Arg Gln His 130 135 140 Ala Asp Thr Thr Ser Thr Ile Gln Arg Ala Leu Arg Tyr Val Asp Ala 145 150 155 160 Gly Ala Asp Gly Val Phe Val Pro Leu Ala Asn Asp Pro Asp Glu Leu 165 170 175 Ala Glu Leu Thr Arg Asn Ile Pro Cys Pro Val Asn Thr Leu Pro Val 180 185 190 Pro Gly Leu Thr Ile Ala Asp Leu Gly Glu Leu Gly Val Ala Arg Val 195 200 205 Ser Thr Gly Ser Val Pro Tyr Ser Ala Gly Leu Tyr Ala Ala Ala His 210 215 220 Ala Ala Arg Ala Val Ser Asp Gly Glu Gln Leu Pro Arg Ser Val Pro 225 230 235 240 Tyr Ala Glu Leu Gln Ala Arg Leu Val Asp Tyr Glu Asn Arg Thr Ser 245 250 255 Thr Thr 18 285 PRT Mycobacterium tuberculosis 18 Val Val Lys Arg Ser Arg Ala Thr Arg Leu Ser Pro Ser Ile Trp Ser 1 5 10 15 Gly Trp Glu Ser Pro Gln Cys Arg Ser Ile Arg Ala Arg Leu Leu Leu 20 25 30 Pro Arg Gly Arg Ser Arg Pro Pro Asn Ala Asp Cys Cys Trp Asn Gln 35 40 45 Leu Ala Val Thr Pro Asp Thr Arg Met Pro Ala Ser Ser Ala Ala Gly 50 55 60 Arg Asp Ala Ala Ala Tyr Asp Ala Trp Tyr Asp Ser Pro Thr Gly Arg 65 70 75 80 Pro Ile Leu Ala Thr Glu Val Ala Ala Leu Arg Pro Leu Ile Glu Val 85 90 95 Phe Ala Gln Pro Arg Leu Glu Ile Gly Val Gly Thr Gly Arg Phe Ala 100 105 110 Asp Leu Leu Gly Val Arg Phe Gly Leu Asp Pro Ser Arg Asp Ala Leu 115 120 125 Met Phe Ala Arg Arg Arg Gly Val Leu Val Ala Asn Ala Val Gly Glu 130 135 140 Ala Val Pro Phe Val Ser Arg His Phe Gly Ala Val Leu Met Ala Phe 145 150 155 160 Thr Leu Cys Phe Val Thr Asp Pro Ala Ala Ile Phe Arg Glu Thr Arg 165 170 175 Arg Leu Leu Ala Asp Gly Gly Gly Leu Val Ile Gly Phe Leu Pro Arg 180 185 190 Gly Thr Pro Trp Ala Asp Leu Tyr Ala Leu Arg Ala Ala Arg Gly Gln 195 200 205 Pro Gly Tyr Arg Asp Ala Arg Phe Tyr Thr Ala Ala Glu Leu Glu Gln 210 215 220 Leu Leu Ala Asp Ser Gly Phe Arg Val Ile Ala Arg Arg Cys Thr Leu 225 230 235 240 His Gln Pro Pro Gly Leu Ala Arg Tyr Asp Ile Glu Ala Ala His Asp 245 250 255 Gly Ile Gln Ala Gly Ala Gly Phe Val Ala Ile Ser Ala Val Asp Gln 260 265 270 Ala His Glu Pro Lys Asp Asp His Pro Leu Glu Ser Glu 275 280 285 19 285 PRT Mycobacterium tuberculosis 19 Val Val Lys Arg Ser Arg Ala Thr Arg Leu Ser Pro Ser Ile Trp Ser 1 5 10 15 Gly Trp Glu Ser Pro Gln Cys Arg Ser Ile Arg Ala Arg Leu Leu Leu 20 25 30 Pro Arg Gly Arg Ser Arg Pro Pro Asn Ala Asp Cys Cys Trp Asn Gln 35 40 45 Leu Ala Val Thr Pro Asp Thr Arg Met Pro Ala Ser Ser Ala Ala Gly 50 55 60 Arg Asp Ala Ala Ala Tyr Asp Ala Trp Tyr Asp Ser Pro Thr Gly Arg 65 70 75 80 Pro Ile Leu Ala Thr Glu Val Ala Ala Leu Arg Pro Leu Ile Glu Val 85 90 95 Phe Ala Gln Pro Arg Leu Glu Ile Gly Val Gly Thr Gly Arg Phe Ala 100 105 110 Asp Leu Leu Gly Val Arg Phe Gly Leu Asp Pro Ser Arg Asp Ala Leu 115 120 125 Met Phe Ala Arg Arg Arg Gly Val Leu Val Ala Asn Ala Val Gly Glu 130 135 140 Ala Val Pro Phe Val Ser Arg His Phe Gly Ala Val Leu Met Ala Phe 145 150 155 160 Thr Leu Cys Phe Val Thr Asp Pro Ala Ala Ile Phe Arg Glu Thr Arg 165 170 175 Arg Leu Leu Ala Asp Gly Gly Gly Leu Val Ile Gly Phe Leu Pro Arg 180 185 190 Gly Thr Pro Trp Ala Asp Leu Tyr Ala Leu Arg Ala Ala Arg Gly Gln 195 200 205 Pro Gly Tyr Arg Asp Ala Arg Phe Tyr Thr Ala Ala Glu Leu Glu Gln 210 215 220 Leu Leu Ala Asp Ser Gly Phe Arg Val Ile Ala Arg Arg Cys Thr Leu 225 230 235 240 His Gln Pro Pro Gly Leu Ala Arg Tyr Asp Ile Glu Ala Ala His Asp 245 250 255 Gly Ile Gln Ala Gly Ala Gly Phe Val Ala Ile Ser Ala Val Asp Gln 260 265 270 Ala His Glu Pro Lys Asp Asp His Pro Leu Glu Ser Glu 275 280 285 20 114 PRT Mycobacterium tuberculosis 20 Val Thr Tyr Val Ile Gly Ser Glu Cys Val Asp Val Met Asp Lys Ser 1 5 10 15 Cys Val Gln Glu Cys Pro Val Asp Cys Ile Tyr Glu Gly Ala Arg Met 20 25 30 Leu Tyr Ile Asn Pro Asp Glu Cys Val Asp Cys Gly Ala Cys Lys Pro 35 40 45 Ala Cys Arg Val Glu Ala Ile Tyr Trp Glu Gly Asp Leu Pro Asp Asp 50 55 60 Gln His Gln His Leu Gly Asp Asn Ala Ala Phe Phe His Gln Val Leu 65 70 75 80 Pro Gly Arg Val Ala Pro Leu Gly Ser Pro Gly Gly Ala Ala Ala Val 85 90 95 Gly Pro Ile Gly Val Asp Thr Pro Leu Val Ala Ala Ile Pro Val Glu 100 105 110 Cys Pro 21 279 PRT Mycobacterium tuberculosis 21 Met Asn Gln Ser His Lys Pro Pro Ser Ile Val Val Gly Ile Asp Gly 1 5 10 15 Ser Lys Pro Ala Val Gln Ala Ala Leu Trp Ala Val Asp Glu Ala Ala 20 25 30 Ser Arg Asp Ile Pro Leu Arg Leu Leu Tyr Ala Ile Glu Pro Asp Asp 35 40 45 Pro Gly Tyr Ala Ala His Gly Ala Ala Ala Arg Lys Leu Ala Ala Ala 50 55 60 Glu Asn Ala Val Arg Tyr Ala Phe Thr Ala Val Glu Ala Ala Asp Arg 65 70 75 80 Pro Val Lys Val Glu Val Glu Ile Thr Gln Glu Arg Pro Val Thr Ser 85 90 95 Leu Ile Arg Ala Ser Ala Ala Ala Ala Leu Val Cys Val Gly Ala Ile 100 105 110 Gly Val His His Phe Arg Pro Glu Arg Val Gly Ser Thr Ala Ala Ala 115 120 125 Leu Ala Leu Ser Ala Gln Cys Pro Val Ala Ile Val Arg Pro His Arg 130 135 140 Val Pro Ile Gly Arg Asp Ala Ala Trp Ile Val Val Glu Ala Asp Gly 145 150 155 160 Ser Ser Asp Ile Gly Val Leu Leu Gly Ala Val Met Ala Glu Ala Arg 165 170 175 Leu Arg Asp Ser Pro Val Arg Val Val Thr Cys Arg Gln Ser Gly Val 180 185 190 Gly Asp Thr Gly Asp Asp Val Arg Ala Ser Leu Asp Arg Trp Leu Ala 195 200 205 Arg Trp Gln Pro Arg Tyr Pro Asp Val Arg Val Gln Ser Ala Ala Val 210 215 220 His Gly Glu Leu Leu Asp Tyr Leu Ala Gly Leu Gly Arg Ser Val His 225 230 235 240 Met Val Val Leu Ser Ala Ser Asp Gln Glu His Val Glu Gln Leu Val 245 250 255 Gly Ala Pro Gly Asn Ala Val Leu Gln Glu Ala Gly Cys Thr Leu Leu 260 265 270 Val Val Gly Gln Gln Tyr Leu 275 22 339 PRT Mycobacterium tuberculosis 22 Met Thr Glu Pro Ala Ala Trp Asp Glu Gly Lys Pro Arg Ile Ile Thr 1 5 10 15 Leu Thr Met Asn Pro Ala Leu Asp Ile Thr Thr Ser Val Asp Val Val 20 25 30 Arg Pro Thr Glu Lys Met Arg Cys Gly Ala Pro Arg Tyr Asp Pro Gly 35 40 45 Gly Gly Gly Ile Asn Val Ala Arg Ile Val His Val Leu Gly Gly Cys 50 55 60 Ser Thr Ala Leu Phe Pro Ala Gly Gly Ser Thr Gly Ser Leu Leu Met 65 70 75 80 Ala Leu Leu Gly Asp Ala Gly Val Pro Phe Arg Val Ile Pro Ile Ala 85 90 95 Ala Ser Thr Arg Glu Ser Phe Thr Val Asn Glu Ser Arg Thr Ala Lys 100 105 110 Gln Tyr Arg Phe Val Leu Pro Gly Pro Ser Leu Thr Val Ala Glu Gln 115 120 125 Glu Gln Cys Leu Asp Glu Leu Arg Gly Ala Ala Ala Ser Ala Ala Phe 130 135 140 Val Val Ala Ser Gly Ser Leu Pro Pro Gly Val Ala Ala Asp Tyr Tyr 145 150 155 160 Gln Arg Val Ala Asp Ile Cys Arg Arg Ser Ser Thr Pro Leu Ile Leu 165 170 175 Asp Thr Ser Gly Gly Gly Leu Gln His Ile Ser Ser Gly Val Phe Leu 180 185 190 Leu Lys Ala Ser Val Arg Glu Leu Arg Glu Cys Val Gly Ser Glu Leu 195 200 205 Leu Thr Glu Pro Glu Gln Leu Ala Ala Ala His Glu Leu Ile Asp Arg 210 215 220 Gly Arg Ala Glu Val Val Val Val Ser Leu Gly Ser Gln Gly Ala Leu 225 230 235 240 Leu Ala Thr Arg His Ala Ser His Arg Phe Ser Ser Ile Pro Met Thr 245 250 255 Ala Val Ser Gly Val Gly Ala Gly Asp Ala Met Val Ala Ala Ile Thr 260 265 270 Val Gly Leu Ser Arg Gly Trp Ser Leu Ile Lys Ser Val Arg Leu Gly 275 280 285 Asn Ala Ala Gly Ala Ala Met Leu Leu Thr Pro Gly Thr Ala Ala Cys 290 295 300 Asn Arg Asp Asp Val Glu Arg Phe Phe Glu Leu Ala Ala Glu Pro Thr 305 310 315 320 Glu Val Gly Gln Asp Gln Tyr Val Trp His Pro Ile Val Asn Pro Glu 325 330 335 Ala Ser Pro 23 681 PRT Mycobacterium tuberculosis 23 Val Leu Met Thr Ala Ala Ala Asp Val Thr Arg Arg Ser Pro Arg Arg 1 5 10 15 Val Phe Arg Asp Arg Arg Glu Ala Gly Arg Val Leu Ala Glu Leu Leu 20 25 30 Ala Ala Tyr Arg Asp Gln Pro Asp Val Ile Val Leu Gly Leu Ala Arg 35 40 45 Gly Gly Leu Pro Val Ala Trp Glu Val Ala Ala Ala Leu His Ala Pro 50 55 60 Leu Asp Ala Phe Val Val Arg Lys Leu Gly Ala Pro Gly His Asp Glu 65 70 75 80 Phe Ala Val Gly Ala Leu Ala Ser Gly Gly Arg Val Val Val Asn Asp 85 90 95 Asp Val Val Arg Gly Leu Arg Ile Thr Pro Gln Gln Leu Arg Asp Ile 100 105 110 Ala Glu Arg Glu Gly Arg Glu Leu Leu Arg Arg Glu Ser Ala Tyr Arg 115 120 125 Gly Glu Arg Pro Pro Thr Asp Ile Thr Gly Lys Thr Val Ile Val Val 130 135 140 Asp Asp Gly Leu Ala Thr Gly Ala Ser Met Phe Ala Ala Val Gln Ala 145 150 155 160 Leu Arg Asp Ala Gln Pro Ala Gln Ile Val Ile Ala Val Pro Ala Ala 165 170 175 Pro Glu Ser Thr Cys Arg Glu Phe Ala Gly Leu Val Asp Asp Val Val 180 185 190 Cys Ala Thr Met Pro Thr Pro Phe Leu Ala Val Gly Glu Ser Phe Trp 195 200 205 Asp Phe Arg Gln Val Thr Asp Glu Glu Val Arg Arg Leu Leu Ala Thr 210 215 220 Pro Thr Ala Gly Pro Ser Leu Arg Arg Pro Ala Ala Ser Thr Ala Ala 225 230 235 240 Asp Val Leu Arg Arg Val Ala Ile Asp Ala Pro Gly Gly Val Pro Thr 245 250 255 His Glu Val Leu Ala Glu Leu Val Gly Asp Ala Arg Ile Val Leu Ile 260 265 270 Gly Glu Ser Ser His Gly Thr His Glu Phe Tyr Gln Ala Arg Ala Ala 275 280 285 Met Thr Gln Trp Leu Ile Glu Glu Lys Gly Phe Gly Ala Val Ala Ala 290 295 300 Glu Ala Asp Trp Pro Asp Ala Tyr Arg Val Asn Arg Tyr Val Arg Gly 305 310 315 320 Leu Gly Glu Asp Thr Asn Ala Asp Glu Ala Leu Ser Gly Phe Glu Arg 325 330 335 Phe Pro Ala Trp Met Trp Arg Asn Thr Val Val Arg Asp Phe Val Glu 340 345 350 Trp Leu Arg Thr Arg Asn Gln Arg Tyr Glu Ser Gly Ala Leu Arg Gln 355 360 365 Ala Gly Phe Tyr Gly Leu Asp Leu Tyr Ser Leu His Arg Ser Ile Gln 370 375 380 Glu Val Ile Ser Tyr Leu Asp Lys Val Asp Pro Arg Ala Ala Ala Arg 385 390 395 400 Ala Arg Ala Arg Tyr Ala Cys Phe Asp His Ala Cys Ala Asp Asp Gly 405 410 415 Gln Ala Tyr Gly Phe Ala Ala Ala Phe Gly Ala Gly Pro Ser Cys Glu 420 425 430 Arg Glu Ala Val Glu Gln Leu Val Asp Val Gln Arg Asn Ala Leu Ala 435 440 445 Tyr Ala Arg Gln Asp Gly Leu Leu Ala Glu Asp Glu Leu Phe Tyr Ala 450 455 460 Gln Gln Asn Ala Gln Thr Val Arg Asp Ala Glu Val Tyr Tyr Arg Ala 465 470 475 480 Met Phe Ser Gly Arg Val Thr Ser Trp Asn Leu Arg Asp Gln His Met 485 490 495 Ala Gln Thr Leu Gly Ser Leu Leu Thr His Leu Asp Arg His Leu Asp 500 505 510 Ala Pro Pro Ala Arg Ile Val Val Trp Ala His Asn Ser His Val Gly 515 520 525 Asp Ala Arg Ala Thr Glu Val Trp Ala Asp Gly Gln Leu Thr Leu Gly 530 535 540 Gln Ile Val Arg Glu Arg Tyr Gly Asp Glu Ser Arg Ser Ile Gly Phe 545 550 555 560 Ser Thr Tyr Thr Gly Thr Val Thr Ala Ala Ser Glu Trp Gly Gly Ile 565 570 575 Ala Gln Arg Lys Ala Val Arg Pro Ala Leu His Gly Ser Val Glu Glu 580 585 590 Leu Phe His Gln Thr Ala Asp Ser Phe Leu Val Ser Ala Arg Leu Ser 595 600 605 Arg Asp Ala Glu Ala Pro Leu Asp Val Val Arg Leu Gly Arg Ala Ile 610 615 620 Gly Val Val Tyr Leu Pro Ala Thr Glu Arg Gln Ser His Tyr Leu His 625 630 635 640 Val Arg Pro Ala Asp Gln Phe Asp Ala Met Ile His Ile Asp Gln Thr 645 650 655 Arg Ala Leu Glu Pro Leu Glu Val Thr Ser Arg Trp Ile Ala Gly Glu 660 665 670 Asn Pro Glu Thr Tyr Pro Thr Gly Leu 675 680 24 144 PRT Mycobacterium tuberculosis 24 Met Ala Thr Thr Leu Pro Val Gln Arg His Pro Arg Ser Leu Phe Pro 1 5 10 15 Glu Phe Ser Glu Leu Phe Ala Ala Phe Pro Ser Phe Ala Gly Leu Arg 20 25 30 Pro Thr Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu Met Lys Glu 35 40 45 Gly Arg Tyr Glu Val Arg Ala Glu Leu Pro Gly Val Asp Pro Asp Lys 50 55 60 Asp Val Asp Ile Met Val Arg Asp Gly Gln Leu Thr Ile Lys Ala Glu 65 70 75 80 Arg Thr Glu Gln Lys Asp Phe Asp Gly Arg Ser Glu Phe Ala Tyr Gly 85 90 95 Ser Phe Val Arg Thr Val Ser Leu Pro Val Gly Ala Asp Glu Asp Asp 100 105 110 Ile Lys Ala Thr Tyr Asp Lys Gly Ile Leu Thr Val Ser Val Ala Val 115 120 125 Ser Glu Gly Lys Pro Thr Glu Lys His Ile Gln Ile Arg Ser Thr Asn 130 135 140 25 331 PRT Mycobacterium tuberculosis 25 Met Pro Asp Thr Met Val Thr Thr Asp Val Ile Lys Ser Ala Val Gln 1 5 10 15 Leu Ala Cys Arg Ala Pro Ser Leu His Asn Ser Gln Pro Trp Arg Trp 20 25 30 Ile Ala Glu Asp His Thr Val Ala Leu Phe Leu Asp Lys Asp Arg Val 35 40 45 Leu Tyr Ala Thr Asp His Ser Gly Arg Glu Ala Leu Leu Gly Cys Gly 50 55 60 Ala Val Leu Asp His Phe Arg Val Ala Met Ala Ala Ala Gly Thr Thr 65 70 75 80 Ala Asn Val Glu Arg Phe Pro Asn Pro Asn Asp Pro Leu His Leu Ala 85 90 95 Ser Ile Asp Phe Ser Pro Ala Asp Phe Val Thr Glu Gly His Arg Leu 100 105 110 Arg Ala Asp Ala Ile Leu Leu Arg Arg Thr Asp Arg Leu Pro Phe Ala 115 120 125 Glu Pro Pro Asp Trp Asp Leu Val Glu Ser Gln Leu Arg Thr Thr Val 130 135 140 Thr Ala Asp Thr Val Arg Ile Asp Val Ile Ala Asp Asp Met Arg Pro 145 150 155 160 Glu Leu Ala Ala Ala Ser Lys Leu Thr Glu Ser Leu Arg Leu Tyr Asp 165 170 175 Ser Ser Tyr His Ala Glu Leu Phe Trp Trp Thr Gly Ala Phe Glu Thr 180 185 190 Ser Glu Gly Ile Pro His Ser Ser Leu Val Ser Ala Ala Glu Ser Asp 195 200 205 Arg Val Thr Phe Gly Arg Asp Phe Pro Val Val Ala Asn Thr Asp Arg 210 215 220 Arg Pro Glu Phe Gly His Asp Arg Ser Lys Val Leu Val Leu Ser Thr 225 230 235 240 Tyr Asp Asn Glu Arg Ala Ser Leu Leu Arg Cys Gly Glu Met Leu Ser 245 250 255 Ala Val Leu Leu Asp Ala Thr Met Ala Gly Leu Ala Thr Cys Thr Leu 260 265 270 Thr His Ile Thr Glu Leu His Ala Ser Arg Asp Leu Val Ala Ala Leu 275 280 285 Ile Gly Gln Pro Ala Thr Pro Gln Ala Leu Val Arg Val Gly Leu Ala 290 295 300 Pro Glu Met Glu Glu Pro Pro Pro Ala Thr Pro Arg Arg Pro Ile Asp 305 310 315 320 Glu Val Phe His Val Arg Ala Lys Asp His Arg 325 330 26 195 PRT Mycobacterium tuberculosis 26 Met Pro Leu Leu Thr Ile Gly Asp Gln Phe Pro Ala Tyr Gln Leu Thr 1 5 10 15 Ala Leu Ile Gly Gly Asp Leu Ser Lys Val Asp Ala Lys Gln Pro Gly 20 25 30 Asp Tyr Phe Thr Thr Ile Thr Ser Asp Glu His Pro Gly Lys Trp Arg 35 40 45 Val Val Phe Phe Trp Pro Lys Asp Phe Thr Phe Val Cys Pro Thr Glu 50 55 60 Ile Ala Ala Phe Ser Lys Leu Asn Asp Glu Phe Glu Asp Arg Asp Ala 65 70 75 80 Gln Ile Leu Gly Val Ser Ile Asp Ser Glu Phe Ala His Phe Gln Trp 85 90 95 Arg Ala Gln His Asn Asp Leu Lys Thr Leu Pro Phe Pro Met Leu Ser 100 105 110 Asp Ile Lys Arg Glu Leu Ser Gln Ala Ala Gly Val Leu Asn Ala Asp 115 120 125 Gly Val Ala Asp Arg Val Thr Phe Ile Val Asp Pro Asn Asn Glu Ile 130 135 140 Gln Phe Val Ser Ala Thr Ala Gly Ser Val Gly Arg Asn Val Asp Glu 145 150 155 160 Val Leu Arg Val Leu Asp Ala Leu Gln Ser Asp Glu Leu Cys Ala Cys 165 170 175 Asn Trp Arg Lys Gly Asp Pro Thr Leu Asp Ala Gly Glu Leu Leu Lys 180 185 190 Ala Ser Ala 195 27 272 PRT Mycobacterium tuberculosis 27 Met Ser Gly Arg Gly Glu Pro Thr Met Lys Thr Ile Ile Val Gly Ile 1 5 10 15 Asp Gly Ser His Ala Ala Ile Thr Ala Ala Leu Trp Gly Val Asp Glu 20 25 30 Ala Ile Ser Arg Ala Val Pro Leu Arg Leu Val Ser Val Ile Lys Pro 35 40 45 Thr His Pro Ser Pro Asp Asp Tyr Asp Arg Asp Leu Ala His Ala Glu 50 55 60 Arg Ser Leu Arg Glu Ala Gln Ser Ala Val Glu Ala Ala Gly Lys Leu 65 70 75 80 Val Lys Ile Glu Thr Asp Ile Pro Arg Gly Pro Ala Gly Pro Val Leu 85 90 95 Val Glu Ala Ser Arg Asp Ala Glu Met Ile Cys Val Gly Ser Val Gly 100 105 110 Ile Gly Arg Tyr Ala Ser Ser Ile Leu Gly Ser Thr Ala Thr Glu Leu 115 120 125 Ala Glu Lys Ala His Cys Pro Val Ala Val Met Arg Ser Lys Val Asp 130 135 140 Gln Pro Ala Ser Asp Ile Asn Trp Ile Val Val Arg Met Thr Asp Ala 145 150 155 160 Pro Asp Asn Glu Ala Val Leu Glu Tyr Ala Ala Arg Glu Ala Lys Leu 165 170 175 Arg Gln Ala Pro Ile Leu Ala Leu Gly Gly Arg Pro Glu Glu Leu Arg 180 185 190 Glu Ile Pro Asp Gly Glu Phe Glu Arg Arg Val Gln Asp Trp His His 195 200 205 Arg His Pro Asp Val Arg Val Tyr Pro Ile Thr Thr His Thr Gly Ile 210 215 220 Ala Arg Phe Leu Ala Asp His Asp Glu Arg Val Gln Leu Ala Val Ile 225 230 235 240 Gly Gly Gly Glu Ala Gly Gln Leu Ala Arg Leu Val Gly Pro Ser Gly 245 250 255 His Pro Val Phe Arg His Ala Glu Cys Ser Val Leu Val Val Arg Arg 260 265 270 28 393 PRT Mycobacterium tuberculosis 28 Met Arg Asp Ala Ile Pro Leu Gly Arg Ile Ala Gly Phe Val Val Asn 1 5 10 15 Val His Trp Ser Val Leu Val Ile Leu Trp Leu Phe Thr Trp Ser Leu 20 25 30 Ala Thr Met Leu Pro Gly Thr Val Gly Gly Tyr Pro Ala Val Val Tyr 35 40 45 Trp Leu Leu Gly Ala Gly Gly Ala Val Met Leu Leu Ala Ser Leu Leu 50 55 60 Ala His Glu Leu Ala His Ala Val Val Ala Arg Arg Ala Gly Val Ser 65 70 75 80 Val Glu Ser Val Thr Leu Trp Leu Phe Gly Gly Val Thr Ala Leu Gly 85 90 95 Gly Glu Ala Lys Thr Pro Lys Ala Ala Phe Arg Ile Ala Phe Ala Gly 100 105 110 Pro Ala Thr Ser Leu Ala Leu Ser Ala Thr Phe Gly Ala Leu Ala Ile 115 120 125 Thr Leu Ala Gly Val Arg Thr Pro Ala Ile Val Ile Ser Val Ala Trp 130 135 140 Trp Leu Ala Thr Val Asn Leu Leu Leu Gly Leu Phe Asn Leu Leu Pro 145 150 155 160 Gly Ala Pro Leu Asp Gly Gly Arg Leu Val Arg Ala Tyr Leu Trp Arg 165 170 175 Arg His Gly Asp Ser Val Arg Ala Gly Ile Gly Ala Ala Arg Ala Gly 180 185 190 Arg Val Val Ala Leu Val Leu Ile Ala Leu Gly Leu Ala Glu Phe Val 195 200 205 Ala Gly Gly Leu Val Gly Gly Val Trp Leu Ala Phe Ile Gly Trp Phe 210 215 220 Ile Phe Ala Ala Ala Arg Glu Glu Glu Thr Arg Ile Ser Thr Gln Gln 225 230 235 240 Leu Phe Ala Gly Val Arg Val Ala Asp Ala Met Thr Ala Gln Pro His 245 250 255 Thr Ala Pro Gly Trp Ile Asn Val Glu Asp Phe Ile Gln Arg Tyr Val 260 265 270 Leu Gly Glu Arg His Ser Ala Tyr Pro Val Ala Asp Arg Asp Gly Ser 275 280 285 Ile Thr Gly Leu Val Ala Leu Arg Gln Leu Arg Asp Val Ala Pro Ser 290 295 300 Arg Arg Ser Thr Thr Ser Val Gly Asp Ile Ala Leu Pro Leu His Ser 305 310 315 320 Val Pro Thr Ala Arg Pro Gln Glu Pro Leu Thr Ala Leu Leu Glu Arg 325 330 335 Met Ala Pro Leu Gly Pro Arg Ser Arg Ala Leu Val Thr Glu Gly Ser 340 345 350 Ala Val Val Gly Ile Val Thr Pro Ser Asp Val Ala Arg Leu Ile Asp 355 360 365 Val Tyr Arg Leu Ala Gln Pro Glu Pro Thr Phe Thr Thr Ser Pro Gln 370 375 380 Asp Ala Asp Arg Phe Ser Asp Ala Gly 385 390 29 413 PRT Mycobacterium tuberculosis 29 Met Ala Ser Ser Ala Ser Asp Gly Thr His Glu Arg Ser Ala Phe Arg 1 5 10 15 Leu Ser Pro Pro Val Leu Ser Gly Ala Met Gly Pro Phe Met His Thr 20 25 30 Gly Leu Tyr Val Ala Gln Ser Trp Arg Asp Tyr Leu Gly Gln Gln Pro 35 40 45 Asp Lys Leu Pro Ile Ala Arg Pro Thr Ile Ala Leu Ala Ala Gln Ala 50 55 60 Phe Arg Asp Glu Ile Val Leu Leu Gly Leu Lys Ala Arg Arg Pro Val 65 70 75 80 Ser Asn His Arg Val Phe Glu Arg Ile Ser Gln Glu Val Ala Ala Gly 85 90 95 Leu Glu Phe Tyr Gly Asn Arg Arg Trp Leu Glu Lys Pro Ser Gly Phe 100 105 110 Phe Ala Gln Pro Pro Pro Leu Thr Glu Val Ala Val Arg Lys Val Lys 115 120 125 Asp Arg Arg Arg Ser Phe Tyr Arg Ile Phe Phe Asp Ser Gly Phe Thr 130 135 140 Pro His Pro Gly Glu Pro Gly Ser Gln Arg Trp Leu Ser Tyr Thr Ala 145 150 155 160 Asn Asn Arg Glu Tyr Ala Leu Leu Leu Arg His Pro Glu Pro Arg Pro 165 170 175 Trp Leu Val Cys Val His Gly Thr Glu Met Gly Arg Ala Pro Leu Asp 180 185 190 Leu Ala Val Phe Arg Ala Trp Lys Leu His Asp Glu Leu Gly Leu Asn 195 200 205 Ile Val Met Pro Val Leu Pro Met His Gly Pro Arg Gly Gln Gly Leu 210 215 220 Pro Lys Gly Ala Val Phe Pro Gly Glu Asp Val Leu Asp Asp Val His 225 230 235 240 Gly Thr Ala Gln Ala Val Trp Asp Ile Arg Arg Leu Leu Ser Trp Ile 245 250 255 Arg Ser Gln Glu Glu Glu Ser Leu Ile Gly Leu Asn Gly Leu Ser Leu 260 265 270 Gly Gly Tyr Ile Ala Ser Leu Val Ala Ser Leu Glu Glu Gly Leu Ala 275 280 285 Cys Ala Ile Leu Gly Val Pro Val Ala Asp Leu Ile Glu Leu Leu Gly 290 295 300 Arg His Cys Gly Leu Arg His Lys Asp Pro Arg Arg His Thr Val Lys 305 310 315 320 Met Ala Glu Pro Ile Gly Arg Met Ile Ser Pro Leu Ser Leu Thr Pro 325 330 335 Leu Val Pro Met Pro Gly Arg Phe Ile Tyr Ala Gly Ile Ala Asp Arg 340 345 350 Leu Val His Pro Arg Glu Gln Val Thr Arg Leu Trp Glu His Trp Gly 355 360 365 Lys Pro Glu Ile Val Trp Tyr Pro Gly Gly His Thr Gly Phe Phe Gln 370 375 380 Ser Arg Pro Val Arg Arg Phe Val Gln Ala Ala Leu Glu Gln Ser Gly 385 390 395 400 Leu Leu Asp Ala Pro Arg Thr Gln Arg Asp Arg Ser Ala 405 410 30 120 PRT Mycobacterium tuberculosis 30 Met Ser Thr Gln Arg Pro Arg His Ser Gly Ile Arg Ala Val Gly Pro 1 5 10 15 Tyr Ala Trp Ala Gly Arg Cys Gly Arg Ile Gly Arg Trp Gly Val His 20 25 30 Gln Glu Ala Met Met Asn Leu Ala Ile Trp His Pro Arg Lys Val Gln 35 40 45 Ser Ala Thr Ile Tyr Gln Val Thr Asp Arg Ser His Asp Gly Arg Thr 50 55 60 Ala Arg Val Pro Gly Asp Glu Ile Thr Ser Thr Val Ser Gly Trp Leu 65 70 75 80 Ser Glu Leu Gly Thr Gln Ser Pro Leu Ala Asp Glu Leu Ala Arg Ala 85 90 95 Val Arg Ile Gly Asp Trp Pro Ala Ala Tyr Ala Ile Gly Glu His Leu 100 105 110 Ser Val Glu Ile Ala Val Ala Val 115 120 31 374 PRT Mycobacterium tuberculosis 31 Met Arg Ser Glu Arg Leu Arg Trp Leu Val Ala Ala Glu Gly Pro Phe 1 5 10 15 Ala Ser Val Tyr Phe Asp Asp Ser His Asp Thr Leu Asp Ala Val Glu 20 25 30 Arg Arg Glu Ala Thr Trp Arg Asp Val Arg Lys His Leu Glu Ser Arg 35 40 45 Asp Ala Lys Gln Glu Leu Ile Asp Ser Leu Glu Glu Ala Val Arg Asp 50 55 60 Ser Arg Pro Ala Val Gly Gln Arg Gly Arg Ala Leu Ile Ala Thr Gly 65 70 75 80 Glu Gln Val Leu Val Asn Glu His Leu Ile Gly Pro Pro Pro Ala Thr 85 90 95 Val Ile Arg Leu Ser Asp Tyr Pro Tyr Val Val Pro Leu Ile Asp Leu 100 105 110 Glu Met Arg Arg Pro Thr Tyr Val Phe Ala Ala Val Asp His Thr Gly 115 120 125 Ala Asp Val Lys Leu Tyr Gln Gly Ala Thr Ile Ser Ser Thr Lys Ile 130 135 140 Asp Gly Val Gly Tyr Pro Val His Lys Pro Val Thr Ala Gly Trp Asn 145 150 155 160 Gly Tyr Gly Asp Phe Gln His Thr Thr Glu Glu Ala Ile Arg Met Asn 165 170 175 Cys Arg Ala Val Ala Asp His Leu Thr Arg Leu Val Asp Ala Ala Asp 180 185 190 Pro Glu Val Val Phe Val Ser Gly Glu Val Arg Ser Arg Thr Asp Leu 195 200 205 Leu Ser Thr Leu Pro Gln Arg Val Ala Val Arg Val Ser Gln Leu His 210 215 220 Ala Gly Pro Arg Lys Ser Ala Leu Asp Glu Glu Glu Ile Trp Asp Leu 225 230 235 240 Thr Ser Ala Glu Phe Thr Arg Arg Arg Tyr Ala Glu Ile Thr Asn Val 245 250 255 Ala Gln Gln Phe Glu Ala Glu Ile Gly Arg Gly Ser Gly Leu Ala Ala 260 265 270 Gln Gly Leu Ala Glu Val Cys Ala Ala Leu Arg Asp Gly Asp Val Asp 275 280 285 Thr Leu Ile Val Gly Glu Leu Gly Glu Ala Thr Val Val Thr Gly Lys 290 295 300 Ala Arg Thr Thr Val Ala Arg Asp Ala Asp Met Leu Ser Glu Leu Gly 305 310 315 320 Glu Pro Val Asp Arg Val Ala Arg Ala Asp Glu Ala Leu Pro Phe Ala 325 330 335 Ala Ile Ala Val Gly Ala Ala Leu Val Arg Asp Asp Asn Arg Ile Ala 340 345 350 Pro Leu Asp Gly Val Gly Ala Leu Leu Arg Tyr Ala Ala Thr Asn Arg 355 360 365 Leu Gly Ser His Arg Ser 370 32 179 PRT Mycobacterium tuberculosis 32 Met Leu His Arg Asp Asp His Ile Asn Pro Pro Arg Pro Arg Gly Leu 1 5 10 15 Asp Val Pro Cys Ala Arg Leu Arg Ala Thr Asn Pro Leu Arg Ala Leu 20 25 30 Ala Arg Cys Val Gln Ala Gly Lys Pro Gly Thr Ser Ser Gly His Arg 35 40 45 Ser Val Pro His Thr Ala Asp Leu Arg Ile Glu Ala Trp Ala Pro Thr 50 55 60 Arg Asp Gly Cys Ile Arg Gln Ala Val Leu Gly Thr Val Glu Ser Phe 65 70 75 80 Leu Asp Leu Glu Ser Ala His Ala Val His Thr Arg Leu Arg Arg Leu 85 90 95 Thr Ala Asp Arg Asp Asp Asp Leu Leu Val Ala Val Leu Glu Glu Val 100 105 110 Ile Tyr Leu Leu Asp Thr Val Gly Glu Thr Pro Val Asp Leu Arg Leu 115 120 125 Arg Asp Val Asp Gly Gly Val Asp Val Thr Phe Ala Thr Thr Asp Ala 130 135 140 Ser Thr Leu Val Gln Val Gly Ala Val Pro Lys Ala Val Ser Leu Asn 145 150 155 160 Glu Leu Arg Phe Ser Gln Gly Arg His Gly Trp Arg Cys Ala Val Thr 165 170 175 Leu Asp Val 33 375 PRT Mycobacterium tuberculosis 33 Val Thr Gln Thr Gly Lys Arg Gln Arg Arg Lys Phe Gly Arg Ile Arg 1 5 10 15 Gln Phe Asn Ser Gly Arg Trp Gln Ala Ser Tyr Thr Gly Pro Asp Gly 20 25 30 Arg Val Tyr Ile Ala Pro Lys Thr Phe Asn Ala Lys Ile Asp Ala Glu 35 40 45 Ala Trp Leu Thr Asp Arg Arg Arg Glu Ile Asp Arg Gln Leu Trp Ser 50 55 60 Pro Ala Ser Gly Gln Glu Asp Arg Pro Gly Ala Pro Phe Gly Glu Tyr 65 70 75 80 Ala Glu Gly Trp Leu Lys Gln Arg Gly Ile Lys Asp Arg Thr Arg Ala 85 90 95 His Tyr Arg Lys Leu Leu Asp Asn His Ile Leu Ala Thr Phe Ala Asp 100 105 110 Thr Asp Leu Arg Asp Ile Thr Pro Ala Ala Val Arg Arg Trp Tyr Ala 115 120 125 Thr Thr Ala Val Gly Thr Pro Thr Met Arg Ala His Ser Tyr Ser Leu 130 135 140 Leu Arg Ala Ile Met Gln Thr Ala Leu Ala Asp Asp Leu Ile Asp Ser 145 150 155 160 Asn Pro Cys Arg Ile Ser Gly Ala Ser Thr Ala Arg Arg Val His Lys 165 170 175 Ile Arg Pro Ala Thr Leu Asp Glu Leu Glu Thr Ile Thr Lys Ala Met 180 185 190 Pro Asp Pro Tyr Gln Ala Phe Val Leu Met Ala Ala Trp Leu Ala Met 195 200 205 Arg Tyr Gly Glu Leu Thr Glu Leu Arg Arg Lys Asp Ile Asp Leu His 210 215 220 Gly Glu Val Ala Arg Val Arg Arg Ala Val Val Arg Val Gly Glu Gly 225 230 235 240 Phe Lys Val Thr Thr Pro Lys Ser Asp Ala Gly Val Arg Asp Ile Ser 245 250 255 Ile Pro Pro His Leu Ile Pro Ala Ile Glu Asp His Leu His Lys His 260 265 270 Val Asn Pro Gly Arg Glu Ser Leu Leu Phe Pro Ser Val Asn Asp Pro 275 280 285 Asn Arg His Leu Ala Pro Ser Ala Leu Tyr Arg Met Phe Tyr Lys Ala 290 295 300 Arg Lys Ala Ala Gly Arg Pro Asp Leu Arg Val His Asp Leu Arg His 305 310 315 320 Ser Gly Ala Val Leu Ala Ala Ser Thr Gly Ala Thr Leu Ala Glu Leu 325 330 335 Met Gln Arg Leu Gly His Ser Thr Ala Gly Ala Ala Leu Arg Tyr Gln 340 345 350 His Ala Ala Lys Gly Arg Asp Arg Glu Ile Ala Ala Leu Leu Ser Lys 355 360 365 Leu Ala Glu Asn Gln Glu Met 370 375 34 371 PRT Mycobacterium tuberculosis 34 Met Arg Val Gly Ile Pro Thr Glu Thr Lys Asn Asn Glu Phe Arg Val 1 5 10 15 Ala Ile Thr Pro Ala Gly Val Ala Glu Leu Thr Arg Arg Gly His Glu 20 25 30 Val Leu Ile Gln Ala Gly Ala Gly Glu Gly Ser Ala Ile Thr Asp Ala 35 40 45 Asp Phe Lys Ala Ala Gly Ala Gln Leu Val Gly Thr Ala Asp Gln Val 50 55 60 Trp Ala Asp Ala Asp Leu Leu Leu Lys Val Lys Glu Pro Ile Ala Ala 65 70 75 80 Glu Tyr Gly Arg Leu Arg His Gly Gln Ile Leu Phe Thr Phe Leu His 85 90 95 Leu Ala Ala Ser Arg Ala Cys Thr Asp Ala Leu Leu Asp Ser Gly Thr 100 105 110 Thr Ser Ile Ala Tyr Glu Thr Val Gln Thr Ala Asp Gly Ala Leu Pro 115 120 125 Leu Leu Ala Pro Met Ser Glu Val Ala Gly Arg Leu Ala Ala Gln Val 130 135 140 Gly Ala Tyr His Leu Met Arg Thr Gln Gly Gly Arg Gly Val Leu Met 145 150 155 160 Gly Gly Val Pro Gly Val Glu Pro Ala Asp Val Val Val Ile Gly Ala 165 170 175 Gly Thr Ala Gly Tyr Asn Ala Ala Arg Ile Ala Asn Gly Met Gly Ala 180 185 190 Thr Val Thr Val Leu Asp Ile Asn Ile Asp Lys Leu Arg Gln Leu Asp 195 200 205 Ala Glu Phe Cys Gly Arg Ile His Thr Arg Tyr Ser Ser Ala Tyr Glu 210 215 220 Leu Glu Gly Ala Val Lys Arg Ala Asp Leu Val Ile Gly Ala Val Leu 225 230 235 240 Val Pro Gly Ala Lys Ala Pro Lys Leu Val Ser Asn Ser Leu Val Ala 245 250 255 His Met Lys Pro Gly Ala Val Leu Val Asp Ile Ala Ile Asp Gln Gly 260 265 270 Gly Cys Phe Glu Gly Ser Arg Pro Thr Thr Tyr Asp His Pro Thr Phe 275 280 285 Ala Val His Asp Thr Leu Phe Tyr Cys Val Ala Asn Met Pro Ala Ser 290 295 300 Val Pro Lys Thr Ser Thr Tyr Ala Leu Thr Asn Ala Thr Met Pro Tyr 305 310 315 320 Val Leu Glu Leu Ala Asp His Gly Trp Arg Ala Ala Cys Arg Ser Asn 325 330 335 Pro Ala Leu Ala Lys Gly Leu Ser Thr His Glu Gly Ala Leu Leu Ser 340 345 350 Glu Arg Val Ala Thr Asp Leu Gly Val Pro Phe Thr Glu Pro Ala Ser 355 360 365 Val Leu Ala 370 35 104 PRT Mycobacterium tuberculosis 35 Met Val Ile Arg Phe Asp Gln Ile Gly Ser Leu Val Leu Ser Met Lys 1 5 10 15 Ser Leu Ala Ser Leu Ser Phe Gln Arg Cys Leu Arg Glu Asn Ser Ser 20 25 30 Leu Val Ala Ala Leu Asp Arg Leu Asp Ala Ala Val Asp Glu Leu Ser 35 40 45 Ala Leu Ser Phe Asp Ala Leu Thr Thr Pro Glu Arg Asp Arg Ala Arg 50 55 60 Arg Asp Arg Asp His His Pro Trp Ser Arg Ser Arg Ser Gln Leu Ser 65 70 75 80 Pro Arg Met Ala His Gly Ala Val His Gln Cys Gln Trp Pro Lys Ala 85 90 95 Val Trp Ala Val Ile Asp Asn Pro 100 36 344 PRT Mycobacterium tuberculosis 36 Val Leu Lys Asn Ala Val Leu Leu Ala Cys Arg Ala Pro Ser Val His 1 5 10 15 Asn Ser Gln Pro Trp Arg Trp Val Ala Glu Ser Gly Ser Glu His Thr 20 25 30 Thr Val His Leu Phe Val Asn Arg His Arg Thr Val Pro Ala Thr Asp 35 40 45 His Ser Gly Arg Gln Ala Ile Ile Ser Cys Gly Ala Val Leu Asp His 50 55 60 Leu Arg Ile Ala Met Thr Ala Ala His Trp Gln Ala Asn Ile Thr Arg 65 70 75 80 Phe Pro Gln Pro Asn Gln Pro Asp Gln Leu Ala Thr Val Glu Phe Ser 85 90 95 Pro Ile Asp His Val Thr Ala Gly Gln Arg Asn Arg Ala Gln Ala Ile 100 105 110 Leu Gln Arg Arg Thr Asp Arg Leu Pro Phe Asp Ser Pro Met Tyr Trp 115 120 125 His Leu Phe Glu Pro Ala Leu Arg Asp Ala Val Asp Lys Asp Val Ala 130 135 140 Met Leu Asp Val Val Ser Asp Asp Gln Arg Thr Arg Leu Val Val Ala 145 150 155 160 Ser Gln Leu Ser Glu Val Leu Arg Arg Asp Asp Pro Tyr Tyr His Ala 165 170 175 Glu Leu Glu Trp Trp Thr Ser Pro Phe Val Leu Ala His Gly Val Pro 180 185 190 Pro Asp Thr Leu Ala Ser Asp Ala Glu Arg Leu Arg Val Asp Leu Gly 195 200 205 Arg Asp Phe Pro Val Arg Ser Tyr Gln Asn Arg Arg Ala Glu Leu Ala 210 215 220 Asp Asp Arg Ser Lys Val Leu Val Leu Ser Thr Pro Ser Asp Thr Arg 225 230 235 240 Ala Asp Ala Leu Arg Cys Gly Glu Val Leu Ser Thr Ile Leu Leu Glu 245 250 255 Cys Thr Met Ala Gly Met Ala Thr Cys Thr Leu Thr His Leu Ile Glu 260 265 270 Ser Ser Asp Ser Arg Asp Ile Val Arg Gly Leu Thr Arg Gln Arg Gly 275 280 285 Glu Pro Gln Ala Leu Ile Arg Val Gly Ile Ala Pro Pro Leu Ala Ala 290 295 300 Val Pro Ala Pro Thr Pro Arg Arg Pro Leu Asp Ser Val Leu Gln Ile 305 310 315 320 Arg Gln Thr Pro Glu Lys Gly Arg Asn Ala Ser Asp Arg Asn Ala Arg 325 330 335 Glu Thr Gly Trp Phe Ser Pro Pro 340 37 336 PRT Mycobacterium tuberculosis 37 Val Trp Ser Ala Ser Gly Gly Gln Cys Gly Lys Tyr Leu Ala Ala Ser 1 5 10 15 Met Val Leu Gln Leu Asp Gly Leu Glu Arg His Gly Val Leu Glu Phe 20 25 30 Gly Arg Asp Arg Tyr Gly Pro Glu Val Arg Glu Glu Leu Leu Ala Met 35 40 45 Ser Ala Ala Ser Ile Asp Arg Tyr Leu Lys Thr Ala Lys Ala Lys Asp 50 55 60 Gln Ile Ser Gly Val Ser Thr Thr Lys Pro Ser Pro Leu Leu Arg Asn 65 70 75 80 Ser Ile Lys Val Arg Arg Ala Gly Asp Glu Val Glu Ala Glu Pro Gly 85 90 95 Phe Phe Glu Gly Asp Thr Val Ala His Cys Gly Pro Thr Leu Lys Gly 100 105 110 Glu Phe Ala His Thr Leu Asn Leu Thr Asp Val His Ile Gly Trp Val 115 120 125 Phe Thr Arg Thr Val Arg Asn Asn Ala Arg Thr His Ile Leu Ala Gly 130 135 140 Leu Lys Ala Ser Val Thr Glu Ile Pro His Gly Ile Thr Gly Leu Asp 145 150 155 160 Phe Asp Asn Gly Thr Val Phe Leu Asn Lys Pro Val Ile Ser Trp Ala 165 170 175 Gly Asp Asn Gly Ile Tyr Phe Thr Arg Phe Arg Pro Tyr Lys Lys Asn 180 185 190 His Ala Thr Ile Glu Ser Lys Asn Asn His Leu Val Arg Lys Tyr Ala 195 200 205 Phe Tyr Tyr Arg Tyr Asp Thr Ala Glu Glu Arg Ala Val Leu Asn Arg 210 215 220 Met Trp Lys Leu Val Asn Asp Arg Leu Asn Tyr Leu Thr Pro Thr Ile 225 230 235 240 Lys Pro Ile Gly Tyr Ala Ser Ser Ala Asp Gly Arg Arg Arg Arg Leu 245 250 255 Tyr Asp Ala Pro Gln Thr Pro Leu Asp Arg Pro Leu Ala Ala Arg Val 260 265 270 Leu Ser Ala Ala Gln Gln Ala Asp Leu Ile Thr Tyr Arg Asp Ser Leu 275 280 285 Asn Pro Ala Gln Ile Gly Arg Lys Ile Ala Asp Leu Gln Asn Arg Leu 290 295 300 Leu Ile Leu Ala Lys Glu Lys Thr Glu Gln Leu Tyr Leu Ala Asn Ile 305 310 315 320 Pro Thr Ala Leu Pro Asp Ile His Lys Gly Ile Leu Ile Lys Ala Gly 325 330 335 38 110 PRT Mycobacterium tuberculosis 38 Val Val Gln Gly Arg Thr Val Leu Phe Arg Thr Ala Glu Gly Ala Lys 1 5 10 15 Leu Phe Ser Ala Val Ala Lys Cys Ala Val Ala Phe Glu Ala Asp Asp 20 25 30 His Asn Val Ala Glu Gly Trp Ser Val Ile Val Lys Val Arg Ala Gln 35 40 45 Val Leu Thr Thr Asp Ala Gly Val Arg Glu Ala Glu Arg Ala Gln Leu 50 55 60 Leu Pro Trp Thr Ala Thr Leu Lys Arg His Cys Val Arg Val Ile Pro 65 70 75 80 Trp Glu Ile Thr Gly Arg His Phe Arg Phe Gly Pro Glu Pro Asp Arg 85 90 95 Ser Gln Thr Phe Ala Cys Glu Ala Ser Ser His Asn Gln Arg 100 105 110 39 463 PRT Mycobacterium tuberculosis 39 Met Asn His Leu Thr Thr Leu Asp Ala Gly Phe Leu Lys Ala Glu Asp 1 5 10 15 Val Asp Arg His Val Ser Leu Ala Ile Gly Ala Leu Ala Val Ile Glu 20 25 30 Gly Pro Ala Pro Asp Gln Glu Ala Phe Leu Ser Ser Leu Ala Gln Arg 35 40 45 Leu Arg Pro Cys Thr Arg Phe Gly Gln Arg Leu Arg Leu Arg Pro Phe 50 55 60 Asp Leu Gly Ala Pro Lys Trp Val Asp Asp Pro Asp Phe Asp Leu Gly 65 70 75 80 Arg His Val Trp Arg Ile Ala Leu Pro Arg Pro Gly Asn Glu Asp Gln 85 90 95 Leu Phe Glu Leu Ile Ala Asp Leu Met Ala Arg Arg Leu Asp Arg Gly 100 105 110 Arg Pro Leu Trp Glu Val Trp Val Ile Glu Gly Leu Ala Asp Ser Lys 115 120 125 Trp Ala Ile Leu Thr Lys Leu His His Cys Met Ala Asp Gly Ile Ala 130 135 140 Ala Thr His Leu Leu Ala Gly Leu Ser Asp Glu Ser Met Ser Asp Ser 145 150 155 160 Phe Ala Ser Asn Ile His Thr Thr Met Gln Ser Gln Ser Ala Ser Val 165 170 175 Arg Arg Gly Gly Phe Arg Val Asn Pro Ser Glu Ala Leu Thr Ala Ser 180 185 190 Thr Ala Val Met Ala Gly Ile Val Arg Ala Ala Lys Gly Ala Ser Glu 195 200 205 Ile Ala Ala Gly Val Leu Ser Pro Ala Ala Ser Ser Leu Asn Gly Pro 210 215 220 Ile Ser Asp Leu Arg Arg Tyr Ser Ala Ala Lys Val Pro Leu Ala Asp 225 230 235 240 Val Glu Gln Val Cys Arg Lys Phe Asp Val Thr Ile Asn Asp Val Ala 245 250 255 Leu Ala Ala Ile Thr Glu Ser Tyr Arg Asn Val Leu Ile Gln Arg Gly 260 265 270 Glu Arg Pro Arg Phe Asp Ser Leu Arg Thr Leu Val Pro Val Ser Thr 275 280 285 Arg Ser Asn Ser Ala Leu Ser Lys Thr Asp Asn Arg Val Ser Leu Met 290 295 300 Leu Pro Asn Leu Pro Val Asp Gln Glu Asn Pro Leu Gln Arg Leu Arg 305 310 315 320 Ile Val His Ser Arg Leu Thr Arg Ala Lys Ala Gly Gly Gln Arg Gln 325 330 335 Phe Gly Asn Thr Leu Met Ala Ile Ala Asn Arg Leu Pro Phe Pro Met 340 345 350 Thr Ala Trp Ala Val Gly Leu Leu Met Arg Leu Pro Gln Arg Gly Val 355 360 365 Val Thr Val Ala Thr Asn Val Pro Gly Pro Arg Arg Pro Leu Gln Ile 370 375 380 Met Gly Arg Arg Val Leu Asp Leu Tyr Pro Val Ser Pro Ile Ala Met 385 390 395 400 Gln Leu Arg Thr Ser Val Ala Met Leu Ser Tyr Ala Asp Asp Leu Tyr 405 410 415 Phe Gly Ile Leu Ala Asp Tyr Asp Val Val Ala Asp Ala Gly Gln Leu 420 425 430 Ala Arg Gly Ile Glu Asp Ala Val Ala Arg Leu Val Ala Ile Ser Lys 435 440 445 Arg Arg Lys Val Thr Arg Arg Arg Gly Ala Leu Ser Leu Val Val 450 455 460 40 332 PRT Mycobacterium tuberculosis 40 Met Asn Thr His Phe Pro Asp Ala Glu Thr Val Arg Thr Val Leu Thr 1 5 10 15 Leu Ala Val Arg Ala Pro Ser Ile His Asn Thr Gln Pro Trp Arg Trp 20 25 30 Arg Val Cys Pro Thr Ser Leu Glu Leu Phe Ser Arg Pro Asp Met Gln 35 40 45 Leu Arg Ser Thr Asp Pro Asp Gly Arg Glu Leu Ile Leu Ser Cys Gly 50 55 60 Val Ala Leu His His Cys Val Val Ala Leu Ala Ser Leu Gly Trp Gln 65 70 75 80 Ala Lys Val Asn Arg Phe Pro Asp Pro Lys Asp Arg Cys His Leu Ala 85 90 95 Thr Ile Gly Val Gln Pro Leu Val Pro Asp Gln Ala Asp Val Ala Leu 100 105 110 Ala Ala Ala Ile Pro Arg Arg Arg Thr Asp Arg Arg Ala Tyr Ser Cys 115 120 125 Trp Pro Val Pro Gly Gly Asp Ile Ala Leu Met Ala Ala Arg Ala Ala 130 135 140 Arg Gly Gly Val Met Leu Arg Gln Val Ser Ala Leu Asp Arg Met Lys 145 150 155 160 Ala Ile Val Ala Gln Ala Val Leu Asp His Val Thr Asp Glu Glu Tyr 165 170 175 Leu Arg Glu Leu Thr Ile Trp Ser Gly Arg Tyr Gly Ser Val Ala Gly 180 185 190 Val Pro Ala Arg Asn Glu Pro Pro Ser Asp Pro Ser Ala Pro Ile Pro 195 200 205 Gly Arg Leu Phe Ala Gly Pro Gly Leu Ser Gln Pro Ser Asp Val Leu 210 215 220 Pro Ala Asp Asp Gly Ala Ala Ile Leu Ala Leu Gly Thr Glu Thr Asp 225 230 235 240 Asp Arg Leu Ala Arg Leu Arg Ala Gly Glu Ala Ala Ser Ile Val Leu 245 250 255 Leu Thr Ala Thr Ala Met Gly Leu Ala Cys Cys Pro Ile Thr Glu Pro 260 265 270 Leu Glu Ile Ala Lys Thr Arg Asp Ala Val Arg Ala Glu Val Phe Gly 275 280 285 Ala Gly Gly Tyr Pro Gln Met Leu Leu Arg Val Gly Trp Ala Pro Ile 290 295 300 Asn Ala Asp Pro Leu Pro Pro Thr Pro Arg Arg Glu Leu Ser Gln Val 305 310 315 320 Val Glu Trp Pro Glu Glu Leu Leu Arg Gln Arg Cys 325 330 41 578 PRT Mycobacterium tuberculosis 41 Met Thr Thr Gly Gly Leu Val Asp Glu Asn Asp Gly Ala Ala Met Arg 1 5 10 15 Pro Leu Arg His Thr Leu Ser Gln Leu Arg Leu His Glu Leu Leu Val 20 25 30 Glu Val Gln Asp Arg Val Glu Gln Ile Val Glu Gly Arg Asp Arg Leu 35 40 45 Asp Gly Leu Val Glu Ala Met Leu Val Val Thr Ala Gly Leu Asp Leu 50 55 60 Glu Ala Thr Leu Arg Ala Ile Val His Ser Ala Thr Ser Leu Val Asp 65 70 75 80 Ala Arg Tyr Gly Ala Met Glu Val His Asp Arg Gln His Arg Val Leu 85 90 95 His Phe Val Tyr Glu Gly Ile Asp Glu Glu Thr Val Arg Arg Ile Gly 100 105 110 His Leu Pro Lys Gly Leu Gly Val Ile Gly Leu Leu Ile Glu Asp Pro 115 120 125 Lys Pro Leu Arg Leu Asp Asp Val Ser Ala His Pro Ala Ser Ile Gly 130 135 140 Phe Pro Pro Tyr His Pro Pro Met Arg Thr Phe Leu Gly Val Pro Val 145 150 155 160 Arg Val Arg Asp Glu Ser Phe Gly Thr Leu Tyr Leu Thr Asp Lys Thr 165 170 175 Asn Gly Gln Pro Phe Ser Asp Asp Asp Glu Val Leu Val Gln Ala Leu 180 185 190 Ala Ala Ala Ala Gly Ile Ala Val Ala Asn Ala Arg Leu Tyr Gln Gln 195 200 205 Ala Lys Ala Arg Gln Ser Trp Ile Glu Ala Thr Arg Asp Ile Ala Thr 210 215 220 Glu Leu Leu Ser Gly Thr Glu Pro Ala Thr Val Phe Arg Leu Val Ala 225 230 235 240 Ala Glu Ala Leu Lys Leu Thr Ala Ala Asp Ala Ala Leu Val Ala Val 245 250 255 Pro Val Asp Glu Asp Met Pro Ala Ala Asp Val Gly Glu Leu Leu Val 260 265 270 Ile Glu Thr Val Gly Ser Ala Val Ala Ser Ile Val Gly Arg Thr Ile 275 280 285 Pro Val Ala Gly Ala Val Leu Arg Glu Val Phe Val Asn Gly Ile Pro 290 295 300 Arg Arg Val Asp Arg Val Asp Leu Glu Gly Leu Asp Glu Leu Ala Asp 305 310 315 320 Ala Gly Pro Ala Leu Leu Leu Pro Leu Arg Ala Arg Gly Thr Val Ala 325 330 335 Gly Val Val Val Val Leu Ser Gln Gly Gly Pro Gly Ala Phe Thr Asp 340 345 350 Glu Gln Leu Glu Met Met Ala Ala Phe Ala Asp Gln Ala Ala Leu Ala 355 360 365 Trp Gln Leu Ala Thr Ser Gln Arg Arg Met Arg Glu Leu Asp Val Leu 370 375 380 Thr Asp Arg Asp Arg Ile Ala Arg Asp Leu His Asp His Val Ile Gln 385 390 395 400 Arg Leu Phe Ala Ile Gly Leu Ala Leu Gln Gly Ala Val Pro His Glu 405 410 415 Arg Asn Pro Glu Val Gln Gln Arg Leu Ser Asp Val Val Asp Asp Leu 420 425 430 Gln Asp Val Ile Gln Glu Ile Arg Thr Thr Ile Tyr Asp Leu His Gly 435 440 445 Ala Ser Gln Gly Ile Thr Arg Leu Arg Gln Arg Ile Asp Ala Ala Val 450 455 460 Ala Gln Phe Ala Asp Ser Gly Leu Arg Thr Ser Val Gln Phe Val Gly 465 470 475 480 Pro Leu Ser Val Val Asp Ser Ala Leu Ala Asp Gln Ala Glu Ala Val 485 490 495 Val Arg Glu Ala Val Ser Asn Ala Val Arg His Ala Lys Ala Ser Thr 500 505 510 Leu Thr Val Arg Val Lys Val Asp Asp Asp Leu Cys Ile Glu Val Thr 515 520 525 Asp Asn Gly Arg Gly Leu Pro Asp Glu Phe Thr Gly Ser Gly Leu Thr 530 535 540 Asn Leu Arg Gln Arg Ala Glu Gln Ala Gly Gly Glu Phe Thr Leu Ala 545 550 555 560 Ser Val Pro Gly Ala Ser Gly Thr Val Leu Arg Trp Ser Ala Pro Leu 565 570 575 Ser Gln 42 268 PRT Mycobacterium tuberculosis 42 Met Ser Asp Pro Arg Pro Ala Arg Ala Val Val Val Gly Ile Asp Gly 1 5 10 15 Ser Arg Ala Ala Thr His Ala Ala Leu Trp Ala Val Asp Glu Ala Val 20 25 30 Asn Arg Asp Ile Pro Leu Arg Leu Val Tyr Val Ile Asp Pro Ser Gln 35 40 45 Leu Ser Ala Ala Gly Glu Gly Gly Gly Gln Ser Ala Ala Arg Ala Ala 50 55 60 Leu His Asp Ala Ser Arg Lys Val Glu Ala Thr Gly Gln Pro Val Lys 65 70 75 80 Ile Glu Thr Glu Val Leu Cys Gly Arg Pro Leu Thr Lys Leu Met Gln 85 90 95 Glu Ser Arg Ser Ala Ala Met Leu Cys Val Gly Ser Val Gly Leu Asp 100 105 110 His Val Arg Gly Arg Arg Gly Ser Val Ala Ala Thr Leu Ala Gly Ser 115 120 125 Ala Leu Cys Pro Val Ala Val Ile His Pro Ser Pro Ala Glu Pro Ala 130 135 140 Thr Thr Ser Gln Val Ser Ala Val Val Ala Glu Val Asp Asn Gly Val 145 150 155 160 Val Leu Arg His Ala Phe Glu Glu Ala Arg Leu Arg Gly Val Pro Leu 165 170 175 Arg Ala Val Ala Val His Ala Ala Glu Thr Pro Asp Asp Val Glu Gln 180 185 190 Gly Ser Arg Leu Ala His Val His Leu Ser Arg Arg Leu Ala His Trp 195 200 205 Thr Arg Leu Tyr Pro Glu Val Arg Val Asp Arg Ala Ile Ala Gly Gly 210 215 220 Ser Ala Cys Arg His Leu Ala Ala Asn Ala Lys Pro Gly Gln Leu Phe 225 230 235 240 Val Ala Asp Ser His Ser Ala His Glu Leu Cys Gly Ala Tyr Gln Pro 245 250 255 Gly Cys Ala Val Leu Thr Val Arg Ser Ala Asn Leu 260 265 43 181 PRT Mycobacterium tuberculosis 43 Met Thr Glu Tyr Glu Gly Pro Lys Thr Lys Phe His Ala Leu Met Gln 1 5 10 15 Glu Gln Ile His Asn Glu Phe Thr Ala Ala Gln Gln Tyr Val Ala Ile 20 25 30 Ala Val Tyr Phe Asp Ser Glu Asp Leu Pro Gln Leu Ala Lys His Phe 35 40 45 Tyr Ser Gln Ala Val Glu Glu Arg Asn His Ala Met Met Leu Val Gln 50 55 60 His Leu Leu Asp Arg Asp Leu Arg Val Glu Ile Pro Gly Val Asp Thr 65 70 75 80 Val Arg Asn Gln Phe Asp Arg Pro Arg Glu Ala Leu Ala Leu Ala Leu 85 90 95 Asp Gln Glu Arg Thr Val Thr Asp Gln Val Gly Arg Leu Thr Ala Val 100 105 110 Ala Arg Asp Glu Gly Asp Phe Leu Gly Glu Gln Phe Met Gln Trp Phe 115 120 125 Leu Gln Glu Gln Ile Glu Glu Val Ala Leu Met Ala Thr Leu Val Arg 130 135 140 Val Ala Asp Arg Ala Gly Ala Asn Leu Phe Glu Leu Glu Asn Phe Val 145 150 155 160 Ala Arg Glu Val Asp Val Ala Pro Ala Ala Ser Gly Ala Pro His Ala 165 170 175 Ala Gly Gly Arg Leu 180 44 274 PRT Mycobacterium tuberculosis 44 Met Thr Trp Ala Asp Glu Val Leu Ala Gly His Pro Phe Val Val Ala 1 5 10 15 His Arg Gly Ala Ser Ala Ala Arg Pro Glu His Thr Leu Ala Ala Tyr 20 25 30 Asp Leu Ala Leu Lys Glu Gly Ala Asp Gly Val Glu Cys Asp Val Arg 35 40 45 Leu Thr Arg Asp Gly His Leu Val Cys Val His Asp Arg Arg Leu Asp 50 55 60 Arg Thr Ser Thr Gly Ala Gly Leu Val Ser Thr Met Thr Leu Ala Gln 65 70 75 80 Leu Arg Glu Leu Glu Tyr Gly Ala Trp His Asp Ser Trp Arg Pro Asp 85 90 95 Gly Ser His Gly Asp Thr Ser Leu Leu Thr Leu Asp Ala Leu Val Ser 100 105 110 Leu Val Leu Asp Trp His Arg Pro Val Lys Ile Phe Val Glu Thr Lys 115 120 125 His Pro Val Arg Tyr Gly Ser Leu Val Glu Asn Lys Leu Leu Ala Leu 130 135 140 Leu His Arg Phe Gly Ile Ala Ala Pro Ala Ser Ala Asp Arg Ser Arg 145 150 155 160 Ala Val Val Met Ser Phe Ser Ala Ala Ala Val Trp Arg Ile Arg Arg 165 170 175 Ala Ala Pro Leu Leu Pro Thr Val Leu Leu Gly Lys Thr Pro Arg Tyr 180 185 190 Leu Thr Ser Ser Ala Ala Thr Ala Val Gly Ala Thr Ala Val Gly Pro 195 200 205 Ser Leu Pro Ala Leu Lys Glu Tyr Pro Gln Leu Val Asp Arg Ser Ala 210 215 220 Ala Gln Gly Arg Ala Val Tyr Cys Trp Asn Val Asp Glu Tyr Glu Asp 225 230 235 240 Ile Asp Phe Cys Arg Glu Val Gly Val Ala Trp Ile Gly Thr His His 245 250 255 Pro Gly Arg Thr Lys Ala Trp Leu Glu Asp Gly Arg Ala Asn Gly Thr 260 265 270 Thr Arg 45 248 PRT Mycobacterium tuberculosis 45 Val Ser Asp Gly Glu Gln Ala Lys Ser Arg Arg Arg Arg Gly Arg Arg 1 5 10 15 Arg Gly Arg Arg Ala Ala Ala Thr Ala Glu Asn His Met Asp Ala Gln 20 25 30 Pro Ala Gly Asp Ala Thr Pro Thr Pro Ala Thr Ala Lys Arg Ser Arg 35 40 45 Ser Arg Ser Pro Arg Arg Gly Ser Thr Arg Met Arg Thr Val His Glu 50 55 60 Thr Ser Ala Gly Gly Leu Val Ile Asp Gly Ile Asp Gly Pro Arg Asp 65 70 75 80 Ala Gln Val Ala Ala Leu Ile Gly Arg Val Asp Arg Arg Gly Arg Leu 85 90 95 Leu Trp Ser Leu Pro Lys Gly His Ile Glu Leu Gly Glu Thr Ala Glu 100 105 110 Gln Thr Ala Ile Arg Glu Val Ala Glu Glu Thr Gly Ile Arg Gly Ser 115 120 125 Val Leu Ala Ala Leu Gly Arg Ile Asp Tyr Trp Phe Val Thr Asp Gly 130 135 140 Arg Arg Val His Lys Thr Val His His Tyr Leu Met Arg Phe Leu Gly 145 150 155 160 Gly Glu Leu Ser Asp Glu Asp Leu Glu Val Ala Glu Val Ala Trp Val 165 170 175 Pro Ile Arg Glu Leu Pro Ser Arg Leu Ala Tyr Ala Asp Glu Arg Arg 180 185 190 Leu Ala Glu Val Ala Asp Glu Leu Ile Asp Lys Leu Gln Ser Asp Gly 195 200 205 Pro Ala Ala Leu Pro Pro Leu Pro Pro Ser Ser Pro Arg Arg Arg Pro 210 215 220 Gln Thr His Ser Arg Ala Arg His Ala Asp Asp Ser Ala Pro Gly Gln 225 230 235 240 His Asn Gly Pro Gly Pro Gly Pro 245 46 819 DNA Mycobacterium tuberculosis 46 gtggaaccga aacgcagtcg cctcgtcgta tgtgcacccg agccatcgca cgcgcgggaa 60 ttcccggatg tcgccgtatt ctccggcggc cgggctaacg catcccaggc cgaacggttg 120 gctcgtgccg tgggtcgcgt gttggccgat cggggcgtca ccgggggtgc tcgggtgcgg 180 ctgaccatgg cgaactgcgc cgatgggccg acgctggtgc agataaacct gcaggtaggt 240 gacaccccat taagggcgca ggccgccacc gcgggcatcg atgatctgcg acccgcactg 300 atcagactgg atcgacagat cgtgcgggcg tcggcacagt ggtgcccccg gccttggccg 360 gatcggcccc gccggcgatt gaccacgccg gccgaggcgc tagtcacccg ccgcaaaccg 420 gtcgtgctaa ggcgcgcaac cccgttgcag gcgattgccg ctatggacgc catggactac 480 gacgtgcatt tgttcaccga cgccgagacg ggggaggacg ctgtggtcta tcgggctgga 540 ccgtcggggc tgcggctggc ccgccagcac cacgtatttc ccccaggatg gtcacgttgt 600 cgcgccccag ccgggccgcc ggtgccgctg attgtgaatt cgcgtccgac accggttctc 660 acggaggccg ccgcggtgga ccgggcgcgc gaacatggac tgccattcct gtttttcacc 720 gaccaggcca ccggccgcgg ccagctgctc tactcccgct acgacggcaa cctcgggttg 780 atcaccccga ccggtgacgg cgttgccgac ggtctggca 819 47 819 DNA Mycobacterium tuberculosis 47 gtggaaccga aacgcagtcg cctcgtcgta tgtgcacccg agccatcgca cgcgcgggaa 60 ttcccggatg tcgccgtatt ctccggcggc cgggctaacg catcccaggc cgaacggttg 120 gctcgtgccg tgggtcgcgt gttggccgat cggggcgtca ccgggggtgc tcgggtgcgg 180 ctgaccatgg cgaactgcgc cgatgggccg acgctggtgc agataaacct gcaggtaggt 240 gacaccccat taagggcgca ggccgccacc gcgggcatcg atgatctgcg acccgcactg 300 atcagactgg atcgacagat cgtgcgggcg tcggcacagt ggtgcccccg gccttggccg 360 gatcggcccc gccggcgatt gaccacgccg gccgaggcgc tagtcacccg ccgcaaaccg 420 gtcgtgctaa ggcgcgcaac cccgttgcag gcgattgccg ctatggacgc catggactac 480 gacgtgcatt tgttcaccga cgccgagacg ggggaggacg ctgtggtcta tcgggctgga 540 ccgtcggggc tgcggctggc ccgccagcac cacgtatttc ccccaggatg gtcacgttgt 600 cgcgccccag ccgggccgcc ggtgccgctg attgtgaatt cgcgtccgac accggttctc 660 acggaggccg ccgcggtgga ccgggcgcgc gaacatggac tgccattcct gtttttcacc 720 gaccaggcca ccggccgcgg ccagctgctc tactcccgct acgacggcaa cctcgggttg 780 atcaccccga ccggtgacgg cgttgccgac ggtctggca 819 48 342 DNA Mycobacterium tuberculosis 48 gtggagtccg aaccgctgta caagctcaag gcggagttct tcaaaaccct tgcgcatccg 60 gcgcggatca ggattttgga gctgctggtc gagcgggacc gttcggtcgg tgagttgctg 120 tcctcggacg tcggcctgga gtcgtcgaac ctgtcccagc agctgggtgt gctacgccgg 180 gcgggtgttg tcgcggcacg tcgtgacggc aacgcgatga tctattcgat tgccgcaccc 240 gatatcgccg agctgctggc ggtggcacgc aaggtgctgg ccagggtgct cagcgaccgg 300 gtggcggtgc tagaggacct ccgcgccggc ggctcggcca cg 342 49 1032 DNA Mycobacterium tuberculosis 49 atgcctatcg caacgcccga ggtctacgcg gagatgctcg gtcaggccaa acaaaactcg 60 tacgctttcc cggctatcaa ctgcacctcc tcggaaaccg tcaacgccgc gatcaaaggt 120 ttcgccgacg ccggcagtga cggaatcatc cagttctcga ccggtggcgc agaattcggc 180 tccggcctcg gggtcaaaga catggtgacc ggtgcggtcg ccttggcgga gttcacccac 240 gttatcgcgg ccaagtaccc ggtcaacgtg gcgctgcaca ccgaccactg ccccaaggac 300 aagttggaca gctatgtccg gcccttgctg gcgatctcgg cgcaacgcgt gagcaaaggt 360 ggcaatcctt tgttccagtc gcacatgtgg gacggctcgg cagtgccaat cgatgagaac 420 ctggccatcg cccaggagct gctcaaggcg gcggcggccg ccaagatcat tctggagatc 480 gagatcggcg tcgtcggcgg cgaagaggac ggcgtggcga acgagatcaa cgagaagctg 540 tacaccagcc cggaggactt cgagaaaacc atcgaggcgc tgggcgccgg tgagcacggc 600 aaatacctgc tggccgcgac gttcggcaac gtgcatggcg tctacaagcc cggcaacgtc 660 aagcttcgcc ccgacatcct tgcgcaaggg caacaggtgg cggcggccaa gctcggactg 720 ccggccgacg ccaagccgtt cgacttcgtg ttccacggcg gctcgggttc gcttaagtcg 780 gagatcgagg aggcgctgcg ctacggcgtg gtgaagatga acgtcgacac cgacacccag 840 tacgcgttca cccgcccgat cgccggtcac atgttcacca actacgacgg agtgctcaag 900 gtcgatggcg aggtgggtgt caagaaggtc tacgacccgc gcagctacct caagaaggcc 960 gaagcttcga tgagccagcg ggtcgttcag gcgtgcaatg acctgcactg cgccggaaag 1020 tccctaaccc ac 1032 50 339 DNA Mycobacterium tuberculosis 50 atgggtgagc acgccatcaa gcggcacatg cggcaacgga agcctacgaa gcatccccta 60 gcccagaaac ggggcgcgcg gattctggtc ttcaccgacg atccccgcag gagcgtcctc 120 atagtgcccg gttgccacct ggattccatg cgccgagaaa agaacgcgta ctacttccag 180 gacggcaatg cgttggttgg gatggttgtc tcgggcggca cggttgagta cgacgccgac 240 gaccgcacat atgtcgtgca gctcaccgac ggaaggcaca ccactgagtc atctttcgaa 300 cactcatcgc cgagtcgatc acctcaatcc gatgaccta 339 51 1140 DNA Mycobacterium tuberculosis 51 gtggctggca atcctgatgt ggtgacggtg ctgctgggcg gtgacgtcat gctcggccgt 60 ggcgtcgatc agatcctgcc tcatcccggc aaaccgcaat tgcgcgaacg gtatatgcgg 120 gatgcgaccg gctatgttcg cctggccgag cgggtgaacg ggcgcattcc gctccccgtg 180 gattggcgct ggccctgggg cgaggcgttg gcggtccttg agaacaccgc gaccgacgtc 240 tgtttgatca atctggagac gacgatcacc gccgacggtg aattcgccga ccgcaaaccg 300 gtctgctacc ggatgcaccc ggataacgtg ccggcgctga cggcattgcg gccgcacgtg 360 tgcgcgctgg ccaacaacca cattctcgat ttcggctacc aggggctgac cgatacggtc 420 gcggctctcg ccggtgcggg gatccagagt gtcggggcgg gagccgattt gctcgccgct 480 cgccgctcgg cgctagtcac ggttggccat gaacgccggg tgatcgtcgg ctcggtagcg 540 gcggaatcca gcggcgtccc cgaatcctgg gccgcccgcc gcgaccggcc cggagtgtgg 600 ttgatccggg atccggcgca acgcgacgtc gccgacgatg tggcggcaca ggtgctggcg 660 gacaaacgcc ccggcgatat cgccatagtc tcgatgcatt ggggatccaa ttggggctat 720 gcgaccgcac ccggcgacgt cgcgttcgcg caccgactga tcgacgccgg catcgacatg 780 gtccacggac attcctcgca ccatccgcgg ccaatcgaga tatatcgcgg taaaccgatc 840 ctgtacggat gcggtgacgt cgttgacgac tacgaaggca tcggcgggca cgagtcgttc 900 cgcagtgaac tgcgactgct gtatctgacc gtcaccgatc ccgccagcgg gaacctgatc 960 tcgctgcaga tgcttccact gcgagtgtcg cggatgcgcc tacagcgtgc ctcccagacc 1020 gacaccgaat ggctccgcaa caccattgag cgcatcagcc gccggttcgg gattcgagtc 1080 gtgactcgac ccgacaacct gctggaggtc gttcccgctg ccaacctaac gagcaaggag 1140 52 1191 DNA Mycobacterium tuberculosis 52 gtgacagacc acgtgcgcga ggcggacgac gcgaacatcg acgatctgtt gggcgacctg 60 ggcggtaccg cgcgcgccga gcgtgcgaag cttgtcgagt ggttgctcga gcagggcatc 120 acccccgacg agattcgggc gaccaacccg ccgttgctgc tggccacccg ccacctcgtc 180 ggcgacgacg gcacctacgt atccgcaagg gagattagcg agaactatgg cgttgacctc 240 gagctgctgc agcgggtgca gcgcgctgtc ggtctggcca gagtggatga tcctgacgcg 300 gtggtgcaca tgcgtgccga cggtgaggcg gccgcacgcg cacagcggtt cgttgagctg 360 gggctgaatc ccgaccaagt cgtgctggtc gtgcgtgtgc tcgccgaggg cttgtcacac 420 gccgccgagg ccatgcgcta caccgcgctg gaggccatta tgcggccggg ggctaccgag 480 ttggacatcg cgaaggggtc gcaggcgctg gtgagccaga tcgtgccgct gctggggccg 540 atgatccagg acatgctgtt catgcagctg cggcacatga tggagacgga ggccgtcaac 600 gccggagagc gtgcggccgg caagccgcta ccgggagcgc gacaggtcac cgttgccttc 660 gccgacctgg tcggtttcac ccagctaggc gaagtggtgt cggccgaaga gctagggcac 720 ctcgccgggc ggctggccgg cctcgcgcgt gacctgaccg ctccgccggt gtggttcatt 780 aagacgatcg gcgacgcggt catgttggtc tgtcctgatc cggcgccatt gctggacacc 840 gtgctgaagc tggtcgaggt cgtcgacacc gacaacaact ttccccggct gcgagccggc 900 gtcgcctccg ggatggcggt tagccgggcc ggcgactggt tcggcagccc ggtcaacgtg 960 gcaagccggg tgaccggggt ggcgcgcccg ggtgccgtgc tggtcgcgga ttcggtgcgg 1020 gaggcccttg gtgatgcccc cgaagccgac ggatttcagt ggtccttcgc cggcccccgt 1080 cgcctcaggg gaatccgggg tgacgtcagg ctttttcgag tccggcgagg ggccactcgc 1140 accggctccg gcggcgcggc ccaagacgac gatttggccg gctcgtcacc g 1191 53 1338 DNA Mycobacterium tuberculosis 53 atggtagagc ccggcaattt ggcaggcgcg accggcgccg aatggatcgg ccggccaccg 60 cacgaggaat tgcagcgcaa agtgcgcccg ctgctgccat ccgacgatcc gttctacttc 120 ccacctgccg gctaccagca tgccgtgccc ggaacggtgt tgcgctcgcg cgatgtcgaa 180 ctggcgttta tgggcttgat tccgcagccc gtcaccgcta cccagctgct gtaccggacc 240 acgaacatgt acggcaaccc cgaggcgacg gtgaccacgg tgatcgtccc agcggagctt 300 gccccgggtc agacctgccc cttgctgtcg taccagtgtg cgatcgatgc catgtcgtcg 360 cgctgttttc cgtcatatgc cctgcgacga cgggccaagg ccctggggtc actgacccaa 420 atggagctgt tgatgatcag cgccgcactt gccgaaggat gggcggtatc agtacccgac 480 catgaagggc cgaaagggct gtgggggtcg ccgtatgaac ccggttaccg agtcctcgac 540 ggaatccggg ctgccttgaa ttccgagcgt gtcgggttgt ccccggcaac gccgatcggg 600 ctgtggggct actccggcgg cgggctggcc agcgcgtggg ccgccgaagc atgcggcgag 660 tacgcaccgg acctagacat cgtcggcgcc gtgctgggat cacccgtcgg tgaccttggt 720 cacacgttcc gccggctcaa tggcactctt cttgccggtc tgcccgcgtt ggtggtggcc 780 gcgctgcaac acagctaccc cggcctggcc cgggtgatca aggagcacgc caacgacgaa 840 ggacgtcagc tgctggagca actgacggag atgacaacgg tagacgcagt gatccggatg 900 gccggcaggg acatgggtga cttcctcgac gaaccccttg aggacattct gtcgacgccg 960 gaaatttccc atgtcttcgg cgacaccaag ctgggtagcg cggtgcccac cccgccggta 1020 ttgatcgtgc aggccgtgca tgactacctc atcgacgtct ctgacatcga cgcgctcgct 1080 gacagctata cagccggcgg cgccaacgtc acctaccacc gcgacctgtt cagcgaacat 1140 gtgtccctgc acccgctgtc ggccccaatg acgcttcgct ggctcaccga ccggttcgcc 1200 ggcaagccac tgaccgacca ccgcgtccgg accacgtggc cgaccatctt caacccgatg 1260 acctacgccg gcatggcgag actggccgtg atcgcggcca aggtgatcac cggcaggaag 1320 ttgagccgcc gtccgctc 1338 54 630 DNA Mycobacterium tuberculosis 54 atgatcgcca caacccgcga tcgtgaagga gccaccatga tcacgtttag gctgcgcttg 60 ccgtgccgga cgatactgcg ggtgttcagc cgcaatccgc tggtgcgtgg gacggatcga 120 ctcgaggcgg tcgtcatgct gctggccgtc acggtctcgc tgctgactat cccgttcgcc 180 gccgcggccg gcaccgcagt ccaggattcc cgcagccacg tctatgccca ccaggcccag 240 acccgccatc ccgcaaccgc gaccgtgatc gatcacgagg gggtgatcga cagcaacacg 300 accgccacgt cagcgccgcc gcgcacgaag atcaccgtgc ctgcccgatg ggtcgtgaac 360 ggaatagaac gcagcggtga ggtcaacgcg aagccgggaa ccaaatccgg tgaccgcgtc 420 ggcatttggg tcgacagtgc cggtcagctg gtcgatgaac cagctccgcc ggcccgtgcc 480 attgcggatg cggccctggc cgccttggga ctctggttga gcgtcgccgc ggttgcgggc 540 gccctgctgg cgctcactcg ggcgattctg atccgcgttc gcaacgccag ttggcaacac 600 gacatcgaca gcctgttctg cacgcagcgg 630 55 240 DNA Mycobacterium tuberculosis 55 atgaccaacg tcggtgacca gggggttgac gcggtcttcg gggtgatcta cccacctcag 60 gtcgcgctgg tcagtttcgg caagccggca caacgagttt gcgccgtcga cggcgcgatc 120 cacgtcatga cgaccgtgct ggctacgctg cccgctgacc acggctgcag cgatgaccat 180 cgcggcgcgc tgttcttcct gtcgatcaac gagctgacgc ggtgcgccgc agtaacagga 240 56 1956 DNA Mycobacterium tuberculosis 56 gtgacggtga caccacggac cggcagccgc atcgaggagc tgcttgcacg cagcggccgg 60 ttcttcatcc cgggtgagat ctcggcggat ctgcgtaccg tgacccgccg cggcggccgc 120 gacggcgacg tgttctatcg agaccggtgg agccacgaca aggtggtccg ctccacacac 180 ggggtgaatt gcaccgggtc gtgttcttgg aagatctacg tcaaagacga catcatcacc 240 tgggagacgc aggagaccga ctatccgtcg gtgggcccgg accggcccga gtatgagccc 300 cgcggctgcc cgcgcggcgc ggcgttttcc tggtacacgt attcgccgac gcgggtgcgc 360 catccgtacg cccgcggcgt gcttgtcgag atgtatcggg aggcgaaggc acgtttgggt 420 gatccggtgg cggcctgggc cgacatccag gccgacccgc ggcggcgccg ccgctaccag 480 cgcgcccgcg gcaagggcgg gctggtccgg gtcagctggg ccgaggccac cgagatgatc 540 gccgccgccc acgtgcacac catctccaca tacggcccgg accgggttgc cggcttctcc 600 cccatcccgg cgatgtccat ggtgagccac gccgcggggt cgcggttcgt ggagctaatc 660 ggcggggtga tgacgtcgtt ctacgactgg tacgccgacc tgccggtggc ctccccgcag 720 gtgttcggcg accagaccga cgtgccggag tccggagatt ggtgggacgt ggtgtggcaa 780 tgcgcctcgg tgctgctgac ctacccgaac tcacggcaac tcggcaccgc agaggaattg 840 ctggcccaca tcgacggtcc ggccgcggat ctgttggggc gcacggtctc tgagctgcgc 900 cgtgccgatc cgctgaccgc ggcgacccgc tacgtcgaca ccttcgacct gcgaggccgc 960 gccaccctgt acctgaccta ctggaccgcc ggcgacaccc gcaaccgcgg ccgggagatg 1020 ctggccttcg cccagaccta ccgcagcacc gacgtcgcac caccgcgcgg cgagaccccg 1080 gacttcctgc cggtggtgct cgaattcgcc gcgaccgtcg accccgaggc ggggcgacgg 1140 ttgctgagcg ggtaccgggt gcccatcgcc gcgctgtgca atgccctgac cgaggccgca 1200 ttgccatacg cacacacggt ggccgcggta tgccggacgg gtgacatgat gggcgaactc 1260 ttctggaccg tcgtgccgta tgtgacgatg acgatcgtcg cggtcggctc ctggtggcgc 1320 taccgctatg acaaattcgg ctggaccacc cgctcgtccc agctgtacga gtcgcggctg 1380 ctgcggatcg ccagcccgat gtttcatttc ggcatcctgg tggtcatcgt cggccacggt 1440 atcgggctcg tgatcccgca gtcgtggact caggccgccg gtttgagcga gggcgcatat 1500 cacgtgcagg ccgtcgtgct ggggtcgatc gccggcatca ccaccttggc cggcgttacc 1560 ctgctgatct accggcggcg cacccgcggg ccggtgttca tggctaccac cgtcaacgac 1620 aaggtgatgt acctcgtgct ggtggcggcg atcgtcgcgg gactgggtgc gacggcgttg 1680 ggctccggcg ttgtcggcga ggcgtacaac taccgcgaga cggtgtcggt gtggttccgc 1740 tcggtgtggg tactgcaacc gcgcggggac ctgatggccg aggctccgct gtattaccag 1800 atccatgtgc tgatcgggtt ggcgttgttc gcgttgtggc cgttcacccg gctggtacac 1860 gcgttcagcg ccccgatcgg ctatctgttc cgcccgtaca tcatctaccg cagccgcgag 1920 gagctggtgc taacgcggcc gcggcggcgc gggtgg 1956 57 1185 DNA Mycobacterium tuberculosis 57 atgagagggc aagcggccaa tctcgtgctg gccacctgga tctcggtggt caacttctgg 60 gcgtggaacc tgatcggccc gctgtcgacc agctacgcgc gtgacatgtc actgtccagc 120 gccgaggcgt cgctgctcgt cgccaccccg atcctggtgg gtgcccttgg ccgcatcgtc 180 accgggccgc tcaccgaccg cttcggcggg cgcgccatgc tcatcgcggt gacgctggcg 240 tcgatcctcc cggtgctcgc ggtcggggtc gcggcaacca tgggctccta cgcgttgctg 300 gtgtttttcg ggctcttcct gggcgttgcc ggcacgatct tcgccgtcgg catcccgttc 360 gccaacaact ggtaccagcc ggcgcggcgc ggtttctcca ccggcgtgtt cggtatgggc 420 atggtcggca ccgcgctctc ggcgttcttc accccgcggt ttgtacggtg gttcggcctg 480 ttcaccaccc acgccatcgt cgcggccgcg ctcgcgtcga ccgccgtggt ggccatggtc 540 gtgcttcgtg atgcacccta ctttcggccc aacgccgacc cggtgctgcc caggctcaag 600 gccgcggcac ggttgccggt gacctgggag atgtcgtttc tgtacgcgat cgtgttcggc 660 gggttcgtgg cgttcagcaa ctacctgccc acctacatca ccacgatcta cgggttctcc 720 acggtcgacg cgggcgctcg caccgccggg ttcgccctgg cggcggtgct ggcccggccg 780 gtgggcgggt ggctctccga ccggatcgca ccgaggcacg tggtgctggc ctcgctcgcc 840 gggaccgcgc tgctggcgtt cgccgcggcg ttgcagccgc cgccggaggt gtggtcggcg 900 gccaccttca tcaccctggc ggtctgcctc ggcgtgggca ccggcggcgt gttcgcgtgg 960 gtggcccgcc gcgccccggc cgcatcggtc ggctcggtca ccggaatcgt cgccgcggca 1020 ggcggattgg gcggttactt cccgccgctg gtgatgggcg cgacctacga cccggtcgac 1080 aacgactaca cggtcgggtt gctgctgctg gtggcgaccg cgctggtcgc gtgtacctac 1140 accgcgctgc acgcgcggga gccggtgagt gaggaggcgt ccagg 1185 58 282 DNA Mycobacterium tuberculosis 58 atgtgcggcg accagtcgga tcacgtgctg cagcactgga ccgtcgacat atcgatcgac 60 gaacacgaag gattgactcg ggcgaaggca cggctgcgtt ggcgggaaaa ggaattggtg 120 ggtgttggcc tggcaaggct caatccggcc gaccgcaacg tccccgagat cggcgatgaa 180 ctctcggtcg cccgagcctt gtccgacttg gggaagcgaa tgttgaaggt gtcgacccac 240 gacatcgaag ctgttaccca tcagccggcg cgattgttgt at 282 59 1680 DNA Mycobacterium tuberculosis 59 atgattccca cgatgacatc ggccggctgg gcaccagggg tggtgcagtt ccgcgaatac 60 caacggcgtt ggctgcgcgg cgatgtcctc gccggcctga ccgtggccgc ctatctgatc 120 ccgcaagcga tggcgtatgc gaccgtggcg ggcctaccgc cggcagccgg gctgtgggcg 180 tcgatcgcgc cgcttgccat ttacgcactg ctcggatcgt cccggcagct ttcaatcggc 240 ccggaatccg ccaccgcctt gatgacggcg gccgtgctcg ctccgatggc cgccggggat 300 cttcgacgct atgccgttct ggcggcaacc ctcggattgc tagtcggcct tatctgccta 360 ctcgctggca cggcgcgact aggtttcctc gccagcctgc gatcgcggcc ggtgctcgtc 420 ggatacatgg ccggcatcgc gcttgtcatg atctccagcc aactcggcac tatcaccggc 480 acctcggtcg aaggcaacga attcttcagc gaagtacact ctttcgcgac tagcgtcacg 540 cgagttcact ggccgacttt tgtgttagcc atgtctgtcc tagcgctgct aactatgctc 600 acgcggtggg cgccgcgcgc ccccggaccg atcatcgcgg ttcttgcggc cacgatgcta 660 gtggccgtta tgtccttgga tgccaaaggt attgcgattg tgggtcggat accttccggt 720 ctgccgacgc cgggtgtgcc gcccgtttcg gtggaagact tgcgggcact gatcattccg 780 gctgccggga tcgcgattgt taccttcacc gacggtgtgt tgaccgcacg cgccttcgcc 840 gctcgtcgag gtcaggaagt caatgccaac gccgagctgc gcgcggtcgg ggcctgcaac 900 atcgccgccg ggctgacaca cggttttccg gtgagttcca gcagcagccg taccgccctc 960 gccgacgtcg tcggtggccg cacccagctg tactcgctga tcgcgttggg gcttgttgtc 1020 atcgtgatgg ttttcgcgag tgggctgctg gccatgtttc cgatcgccgc tctgggcgct 1080 ttggtggtat atgccgcgct acgcttgatc gacttgtcag aattccggcg actggcgcgg 1140 tttcggcgca gcgaactcat gctggcacta gccaccacag cagccgtgtt aggcctagga 1200 gtgttctatg gagtcctcgc cgcggttgcc ctgtccatcc tcgaactgct tcgtcgggtc 1260 gcacatccgc atgacagcgt tctcgggttc gtgccgggca ttgccggcat gcacgacatc 1320 gatgactatc cgcaggccaa gcgcgtgccc gggctggtgg tgtatcgcta tgacgcgccg 1380 ttgtgcttcg ccaatgccga agacttccgc aggcgagcac tgaccgtggt cgatcaggat 1440 ccggggcaag tcgagtggtt cgtactcaac gccgaatcca atgtggaggt cgacctgact 1500 gcgctggatg cgctcgacca actccgcacc gagctgctgc gtcggggaat agtgttcgcc 1560 atggcccggg tcaaacaaga cttgcgtgaa tcactcaggg ccgccagtct tctcgataag 1620 attggcgaag accatatctt tatgacattg cctaccgcag tgcaggcgtt ccgtcggcgc 1680 60 429 DNA Mycobacterium tuberculosis 60 atgatcacaa acctccgacg ccgaaccgcg atggcagccg ccggcctagg ggctgctctc 60 gggctgggca tcctgctggt tccgacggtg gacgcccatc tcgccaacgg ttcgatgtcg 120 gaagtcatga tgtcggaaat tgccgggttg cctatccctc cgattatcca ttacggggcg 180 attgcctatg cccccagcgg cgcgtcgggc aaagcgtggc accagcgcac accggcgcga 240 gcagagcaag tcgcactaga aaagtgcggt gacaagactt gcaaagtggt tagtcgcttc 300 accaggtgcg gcgcggtcgc ctacaacggc tcgaaatacc aaggcggaac cggactcacg 360 cgccgcgcgg cagaagacga cgccgtgaac cgactcgaag gcgggcggat cgtcaactgg 420 gcgtgcaac 429 61 2715 DNA Mycobacterium tuberculosis 61 ttgtcggcgt cagtgtctgc cacgacggct catcatggct tgccagcaca tgaagtggtg 60 ctgctgctgg agagcgatcc atatcacggg ctgtccgacg gcgaggccgc ccaacgacta 120 gaacgcttcg ggcccaacac cttggcggtg gtaacgcgcg ctagcttgct ggcccgcatc 180 ctgcggcagt ttcatcaccc gctgatctac gttctgctcg ttgccgggac gatcaccgcc 240 ggtcttaagg aattcgttga cgccgcagtg atcttcggtg tggtggtgat caatgcgatc 300 gtgggtttca ttcaagaatc caaggcagag gccgcactgc agggcctgcg ctccatggtg 360 cacacccacg ccaaggtggt gcgcgagggt cacgagcaca caatgccatc cgaagagctg 420 gttcccggtg accttgtgct gttagcggcc ggtgacaagg ttcccgccga tttgcggctg 480 gtgcgacaga ccggattgag cgtgaacgag tcagcactta ccggcgagtc gacgccggtt 540 cacaaggacg aggtggcgtt gccggagggc acaccggtcg ctgatcgtcg caatatcgcg 600 tattccggca cattggtaac cgcgggccat ggcgccggga tcgtcgtcgc gaccggcgcc 660 gaaaccgaac tcggtgagat tcatcggctc gttggggccg ccgaggttgt cgccacaccg 720 ctgaccgcga agctggcgtg gttcagcaag tttctgacca tcgccatcct gggtctggca 780 gcgctcacgt tcggcgtggg tttgctgcgc cggcaagatg ccgtcgaaac gttcaccgct 840 gcgatcgcgc tggcggtcgg ggcaattccc gaaggtctgc ccaccgccgt gaccatcacc 900 ttggccatcg gcatggcccg gatggccaag cgccgcgcgg tcattcgacg tctacccgcg 960 gtggaaacgc tgggcagcac cacggtcatc tgcgccgaca agaccggaac gctgaccgag 1020 aatcagatga cggtccagtc gatctggaca ccccacggtg agatccgggc gaccggaacg 1080 ggctatgcac ccgacgtcct cctgtgcgac accgacgacg cgccggttcc ggtgaatgcc 1140 aatgcggccc ttcgctggtc gctgctggcc ggtgcctgca gcaacgacgc cgcactggtt 1200 cgcgacggca cacgctggca gatcgtcggc gatcccaccg agggcgcgat gctcgtcgtg 1260 gccgccaagg ccggcttcaa cccggagcgg ctggcgacaa ctctgccgca agtggcagcc 1320 ataccgttca gttccgagcg gcaatacatg gccaccctgc atcgcgacgg gacggatcat 1380 gtggtgctgg ccaagggtgc tgtggagcgc atgctcgacc tgtgcggcac cgagatgggc 1440 gccgacggcg cattgcggcc gctggaccgc gccaccgtgt tgcgtgccac cgaaatgttg 1500 acttcccggg ggttgcgggt gctggcaacc gggatgggtg ccggcgccgg cactcccgac 1560 gacttcgacg aaaacgtgat accaggttcg ctggcgctga ccggcctgca agcgatgagc 1620 gatccaccac gagcggccgc ggcatcggcg gtggcggcct gccacagtgc cggcattgcg 1680 gtaaaaatga ttaccggtga ccacgcgggc accgccacgg cgatcgcaac cgaggtgggg 1740 ttgctcgaca acactgaacc ggcggcaggc tcggtcctga cgggtgccga gctggccgcg 1800 ctgagcgcag accagtaccc ggaggccgtg gatacagcca gcgtgtttgc cagggtctct 1860 cccgagcaga agctgcggtt ggtgcaagca ttgcaggcca gggggcacgt cgtcgcgatg 1920 accggcgacg gcgtcaacga cgccccggcc ttgcgtcagg ccaacattgg cgtcgcgatg 1980 ggccgcggtg gcaccgaggt cgccaaggat gccgccgaca tggtgttgac cgacgacgac 2040 ttcgccacca tcgaagccgc ggtcgaggaa ggccgcggcg tattcgacaa tctgaccaag 2100 ttcatcacct ggacgctgcc caccaacctc ggtgagggcc tagtgatctt ggccgccatc 2160 gctgttggcg tcgccttgcc gattctgccc acccaaattc tgtggatcaa catgaccaca 2220 gcgatcgcgc tcggactcat gctcgcgttc gagcccaagg aggccggaat catgacccgg 2280 ccaccgcgcg accccgacca accgctgctg accggctggc ttgtcaggcg gactcttctg 2340 gtttccacct tgctcgtcgc cagcgcgtgg tggctgtttg catgggagct cgacaatggc 2400 gcgggcctgc atgaggcgcg cacggcggcg ctgaacctgt tcgtcgtcgt cgaggcgttc 2460 tatctgttca gctgccggtc gctgacccga tcggcctggc ggctcggcat gttcgccaac 2520 cgctggatca tcctcggcgt cagtgcgcag gccatcgcgc aattcgcgat cacatatcta 2580 cccgcgatga atatggtgtt cgacaccgcg ccaatcgata tcggggtgtg ggtgcgcata 2640 ttcgctgtcg cgaccgcaat cacgattgtg gtggccaccg acacgctgct gccgagaata 2700 cgggcgcaac cgcca 2715 62 774 DNA Mycobacterium tuberculosis 62 atgagtttcc acgatcttca tcaccaaggt gttccgttcg tgttgcccaa cgcctgggat 60 gtgccgtcgg ccctggccta cctcgcggag ggcttcacgg ctatcggcac aaccagtttc 120 ggggtctcgt ccagcggcgg gcacccggac gggcaccgcg ccactcgcgg cgccaacatc 180 gcactggcgg ccgccctggc accgctgcaa tgctacgtca gcgtcgacat cgaggacgga 240 tacagcgacg aacccgacgc cattgctgac tacgtcgcac aactgtcgac agccggaatc 300 aatatcgagg acagtagcgc cgaaaagctc atcgaccccg ccctggcagc cgctaaaatc 360 gttgcgatca aacaacgtaa ccccgaggtg ttcgtcaacg cccgcgtcga cacctattgg 420 ttgcgccagc acgccgatac caccagcacg atccagcgcg cacttcgcta cgtcgatgcc 480 ggcgccgacg gcgtctttgt cccactggcc aacgatcccg acgaacttgc tgagctcact 540 cgcaacattc cgtgcccggt taacacgttg cccgtgcccg gcttgacgat cgccgacctt 600 ggtgagctcg gggtggcccg ggtgtcaacc ggttcagtgc cctacagcgc ggggttgtat 660 gcagcggccc acgcggctcg ggccgtgagc gacggagagc agctgccacg gtccgtaccg 720 tacgccgaac tgcaggcacg cttggttgac tacgagaacc gcacgagtac aacg 774 63 855 DNA Mycobacterium tuberculosis 63 gtggtcaagc gctctcgggc aacccgactt tcgccgagca tctggtccgg atgggaatca 60 cctcagtgtc ggtccattcg ggcgcgattg ctgctacccc ggggtcggtc gcggccgccg 120 aacgccgatt gttgctggaa tcagctcgcg gtgacgcctg acacccggat gccggcatcg 180 tcggccgccg ggcgcgacgc ggcggcctac gacgcctggt atgactcacc caccgggcgg 240 ccgatcctgg cgaccgaggt cgccgcgttg cggccgctca tcgaggtctt tgcccagcca 300 cgcttggaaa tcggtgtcgg tacaggacgt ttcgccgacc tgctcggcgt gcggttcgga 360 ctcgatccat cccgtgatgc gctgatgttc gcacgccggc gcggcgtcct ggtcgccaat 420 gccgtcggcg aggcggtccc tttcgtcagc cggcacttcg gggcggtcct catggcattc 480 acgctctgtt tcgtcaccga cccggccgcc atattccggg aaacgcggcg tctgctcgcc 540 gacggcggcg gccttgttat cgggttcttg cctcgcggga caccgtgggc cgacctgtac 600 gctctgcgcg cggcccgcgg acagccaggc taccgcgacg cccgcttcta caccgcggcc 660 gaactcgaac aactgctcgc agactcggga ttccgggtca tcgcccgccg ctgcacgctg 720 caccaaccgc cgggactcgc ccggtacgac atcgaagccg cccatgacgg tatccaagcc 780 ggcgccggct tcgttgctat ctcggcggtc gaccaagcgc acgagcctaa ggatgatcac 840 ccactcgagt cggaa 855 64 885 DNA Mycobacterium tuberculosis 64 atgtctaaac cccgcaagca gcacggagtt gtcgtcgggg tagatggttc gctcgaatcg 60 gatgccgccg cctgttgggg tgccaccgat gcggcgatga ggaacattcc gctgaccgtg 120 gtccacgtgg tgaacgccga tgtagcgacg tggccgccga tgccgtatcc ggagacctgg 180 ggggtttggc aggaggacga gggtcgccag atcgtcgcca acgccgtcaa gctcgccaaa 240 gaggcggttg gagcggatcg aaagctcagc gtaaagagcg agctcgtatt ttccacgccg 300 gtacctacca tggttgaaat ctccaacgag gcagagatgg tggtgttggg cagctcgggc 360 cggggagcgc tggcccgagg cttgctcggt tcggtcagct cgagcctggt gcgacgcgcc 420 gggtgcccgg tcgcggtcat ccacagcgat gatgcggtga tccctgatcc gcagcacgct 480 cccgtgctgg tgggaatcga cggttcgccg gtttcggagc ttgcgacggc ggtggcattt 540 gacgaggcgt cgcgccgcgg cgtcgaactg atcgccgtgc acgcgtggag tgacgtcgaa 600 gtggtggaac ttccgggttt ggacttctcg gctgtacagc aggaagcgga gcttagtctc 660 gccgaacgct tggcaggttg gcaagaacgc tatcccgatg tgccggtgag ccgggttgtc 720 gtttgcgatc gcccggcgcg gaagctggtg caaaagtcgg cgtccgccca gcttgtcgtc 780 gttggcagtc atggccgagg tggcttgacc ggcatgcttc tggggtcggt cagtaacgcg 840 gtcttacacg ccgcgcgggt gccagtgatc gtggcacggc agtcg 885 65 342 DNA Mycobacterium tuberculosis 65 gtgacctatg tgatcggtag tgagtgcgtg gatgtgatgg acaagtcctg tgtgcaggag 60 tgtccggtcg actgtatcta tgagggcgcc cgaatgctct acatcaaccc cgacgagtgc 120 gtggattgtg gtgcgtgcaa accggcctgc cgcgtcgagg cgatctactg ggaaggcgat 180 ctacccgacg atcaacacca gcatctgggg gacaacgccg cctttttcca ccaagtcctg 240 ccgggccgag tggctccgct gggttcgccg ggtggtgccg cagcggtggg cccgatcgga 300 gtcgacacgc ctctggtcgc ggctatcccg gtggagtgcc ct 342 66 837 DNA Mycobacterium tuberculosis 66 atgaaccaat cacacaaacc cccatcgatc gtcgtcggta ttgatggctc gaagccggcc 60 gtgcaagccg cactgtgggc ggtcgacgag gcagccagcc gtgacatccc gctgcgtctg 120 ctgtacgcga tcgaacccga cgatcccggg tacgccgcac acggcgcggc ggctcgcaaa 180 ctcgccgccg ccgagaacgc ggtgcgctac gcgttcacag cggtcgaggc ggcggaccgg 240 ccggtcaagg tcgaggtgga gatcacccag gagcggccgg tcacctcgtt gatccgcgct 300 tcggcggctg ctgccctggt gtgcgttggc gctatcggcg tgcaccactt ccgaccggag 360 cgggtgggat ctaccgcagc ggccctggcg ttatcggcgc agtgcccagt ggcgatcgtg 420 cgaccccacc gggtccccat cggacgcgac gccgcatgga tcgtcgtcga ggcggacggg 480 tcgtccgata tcggtgtttt gctgggggcg gtgatggccg aagcacggct gcgcgactcg 540 ccggttcggg tggtcacctg ccggcaatcc ggagtgggcg ataccgggga cgacgtccgt 600 gccagcctgg accgctggct tgcccgttgg caaccacggt atcccgatgt gcgggtgcaa 660 tcggcggcag tgcacggcga gctgctggat tatctggctg ggctgggtcg atcggtacac 720 atggtggtgc tcagcgcgag cgaccaggag catgtggagc aacttgtggg agcgccgggc 780 aacgccgtgt tgcaggaggc cggctgcacc ctgctggtcg tcggtcagca gtatctg 837 67 1017 DNA Mycobacterium tuberculosis 67 atgacggagc cagcggcgtg ggacgaaggc aagccgcgaa tcatcacttt gaccatgaac 60 cccgccttgg acatcacgac gagcgtcgac gtggtgcgcc cgaccgagaa aatgcgttgt 120 ggcgcacctc gctacgatcc cggcggcggc ggtatcaatg tcgcccgcat tgtgcatgtc 180 ctcggcggtt gctcgacagc actgttcccg gccggcgggt cgaccgggag cctgctgatg 240 gcgctgctcg gtgatgcggg agtgccattt cgcgtcattc cgatcgcggc ctcgacgcgg 300 gagagcttca cggtcaacga gtccaggacc gccaagcagt atcgtttcgt gcttccgggg 360 ccgtcgctga ccgtcgcgga gcaggagcaa tgcctcgacg aactgcgcgg tgcggcggct 420 tcggccgcct ttgtggtggc cagtggcagc ctgccgccag gtgtggctgc cgactactat 480 cagcgggttg ccgacatctg ccgccgatcg agcactccgc tgatcctgga tacatctggt 540 ggcgggttgc agcacatttc gtccggggtg tttcttctca aggcgagcgt gcgggaactg 600 cgcgagtgcg tcggatccga actgctgacc gagcccgaac aactggccgc cgcacacgaa 660 ctcattgacc gtgggcgcgc cgaggtcgtg gtggtctcgc ttggatctca gggcgcgcta 720 ttggccacac gacatgcgag ccatcgattt tcgtcgattc cgatgaccgc ggttagcggt 780 gtcggcgccg gcgacgcgat ggtggccgcg attaccgtgg gcctcagccg tggctggtcg 840 ctcatcaagt ccgttcgctt gggaaacgcg gcaggtgcag ccatgctgct gacgccaggc 900 accgcggcct gcaatcgcga cgatgtggag aggttcttcg agctggcggc cgaacccacc 960 gaagtcgggc aggatcaata cgtttggcac ccgatcgtta acccggaagc ctcgcca 1017 68 2043 DNA Mycobacterium tuberculosis 68 gtgctgatga ccgcagcggc tgatgtcacc cggcgctcgc cgcggcgcgt gttccgtgac 60 cgccgcgagg ccggccgggt gctggcggaa ttactcgccg cctatcggga ccagccggac 120 gtgattgtgc tcggcttggc ccggggtggc ctcccggtcg catgggaggt tgccgcggca 180 ctgcatgccc cgctagacgc cttcgtcgtg cgcaaacttg gtgccccggg gcatgacgag 240 ttcgccgttg gtgcactggc cagcggcggc cgcgtcgtgg tcaatgacga cgtcgtgcgg 300 ggcctgcgga tcacaccgca gcaactgcgc gacatcgccg aacgtgaggg tcgggaactg 360 cttcggcgcg agtccgccta ccgcggcgag cgcccgccca ccgatatcac cggcaagacg 420 gtcattgtcg tcgatgacgg tttggccacc ggcgcaagca tgttcgcggc ggtacaggca 480 ttgcgcgatg cgcaaccagc gcagatcgtg attgccgtgc cggcggcgcc ggagtccacg 540 tgccgggagt tcgccggcct cgtcgacgac gttgtgtgcg cgaccatgcc gaccccgttc 600 ctggccgtcg gtgagtcgtt ttgggacttc cggcaggtca ccgacgagga ggtccgccgg 660 ctcctggcca ccccgaccgc tgggccgtcg ctgcgccggc ccgcggcgtc aacggcggcc 720 gatgttctgc gcagagtcgc gatcgacgcc cccgggggtg ttccgacgca cgaggtgttg 780 gcggagctgg tcggcgatgc acgaatcgtg ttgatcggcg aaagctcgca cggcacacac 840 gagttctacc aggcccgggc cgccatgaca cagtggctga tcgaggagaa gggctttggt 900 gcggtagccg ccgaggcgga ctggcccgac gcctaccggg tcaatcggta cgttcgcggc 960 ctcggcgagg acaccaacgc tgacgaggcg cttagcggat tcgagcggtt tcccgcctgg 1020 atgtggcgca acaccgtggt ccgagatttt gtggaatggc tgcgcacacg caaccagcgc 1080 tacgagtcgg gcgcgctgcg gcaagccggc ttctacggtc tggatcttta cagcctgcat 1140 cggtcgatcc aagaggtgat cagctatctc gacaaggtcg acccgcgtgc ggcggcacgg 1200 gcgcgggccc ggtatgcgtg cttcgaccat gcctgcgccg atgacggtca ggcgtacgga 1260 ttcgcggccg cattcggcgc cggtccgtcg tgcgaacgtg aagccgtcga gcaactggtc 1320 gacgttcagc gcaatgccct ggcgtatgcg cgccaagacg ggctgcttgc cgaggacgaa 1380 ctgttctacg cccagcaaaa cgcgcagacg gtgcgcgacg cagaggtgta ttaccgggcc 1440 atgttcagtg gacgcgttac ctcgtggaac ctgcgcgacc agcacatggc gcagaccctt 1500 ggcagtttgc tgacgcattt ggaccgacac ctcgatgcgc cgccggcgcg aatagtggtg 1560 tgggctcata actcccacgt gggtgacgca cgcgctaccg aggtgtgggc cgacgggcag 1620 ctcaccctcg gccagatagt ccgtgagcga tacggtgacg agtcgcgcag catcggattc 1680 agcacgtaca cgggcaccgt caccgcggcc agcgaatggg gtggtatcgc ccaacgcaaa 1740 gcggttcggc cggcactgca cggcagtgtc gaggagctct tccaccagac tgcagacagt 1800 ttcctggtgt cagcgcggct aagccgcgac gccgaagccc cgctggacgt tgtccggttg 1860 ggacgtgcca tcggcgtcgt ttatctaccg gcaacggaac ggcaaagtca ctacttgcac 1920 gtgcggcccg ccgaccagtt cgacgccatg atccacatcg atcagacccg tgccctggaa 1980 cctctcgagg tgacgagccg gtggatcgcc ggcgagaacc cggaaaccta cccgaccggt 2040 ctg 2043 69 432 DNA Mycobacterium tuberculosis 69 atggccacca cccttcccgt tcagcgccac ccgcggtccc tcttccccga gttttctgag 60 ctgttcgcgg ccttcccgtc attcgccgga ctccggccca ccttcgacac ccggttgatg 120 cggctggaag acgagatgaa agaggggcgc tacgaggtac gcgcggagct tcccggggtc 180 gaccccgaca aggacgtcga cattatggtc cgcgatggtc agctgaccat caaggccgag 240 cgcaccgagc agaaggactt cgacggtcgc tcggaattcg cgtacggttc cttcgttcgc 300 acggtgtcgc tgccggtagg tgctgacgag gacgacatta aggccaccta cgacaagggc 360 attcttactg tgtcggtggc ggtttcggaa gggaagccaa ccgaaaagca cattcagatc 420 cggtccacca ac 432 70 993 DNA Mycobacterium tuberculosis 70 atgccggaca ccatggtgac caccgatgtc atcaagagcg cggtgcagtt ggcctgccgc 60 gcaccgtcgc tccacaacag ccagccctgg cgctggatag ccgaggacca cacggttgcg 120 ctgttcctcg acaaggatcg ggtgctttac gcgaccgacc actccggccg ggaagcgctg 180 ctggggtgcg gcgccgtact cgaccacttt cgggtggcga tggcggccgc gggtaccacc 240 gccaatgtgg aacggtttcc caaccccaac gatcctttgc atctggcgtc aattgacttc 300 agcccggccg atttcgtcac cgagggccac cgtctaaggg cggatgcgat cctactgcgc 360 cgtaccgacc ggctgccttt cgccgagccg ccggattggg acttggtgga gtcgcagttg 420 cgcacgaccg tcaccgccga cacggtgcgc atcgacgtca tcgccgacga tatgcgtccc 480 gaactggcgg cggcgtccaa actcaccgaa tcgctgcggc tctacgattc gtcgtatcat 540 gccgaactct tttggtggac aggggctttt gagacttctg agggcatacc gcacagttca 600 ttggtatcgg cggccgaaag tgaccgggtc accttcggac gcgacttccc ggtcgtcgcc 660 aacaccgata ggcgcccgga gtttggccac gaccgctcta aggtcctggt gctctccacc 720 tacgacaacg aacgcgccag cctactgcgc tgcggcgaga tgctttccgc cgtattgctt 780 gacgccacca tggctgggct tgccacctgc acgctgaccc acatcaccga actgcacgcc 840 agccgagacc tggtcgcagc gctgattggg cagcccgcaa ctccgcaagc cttggttcgc 900 gtcggtctgg ccccggagat ggaagagccg ccaccggcaa cgcctcggcg accaatcgat 960 gaagtgtttc acgttcgggc taaggatcac cgg 993 71 585 DNA Mycobacterium tuberculosis 71 atgccactgc taaccattgg cgatcaattc cccgcctacc agctcaccgc tctcatcggc 60 ggtgacctgt ccaaggtcga cgccaagcag cccggcgact acttcaccac tatcaccagt 120 gacgaacacc caggcaagtg gcgggtggtg ttcttttggc cgaaagactt cacgttcgtg 180 tgccctaccg agatcgcggc gttcagcaag ctcaatgacg agttcgagga ccgcgacgcc 240 cagatcctgg gggtttcgat tgacagcgaa ttcgcgcatt tccagtggcg tgcacagcac 300 aacgacctca aaacgttacc cttcccgatg ctctccgaca tcaagcgcga actcagccaa 360 gccgcaggtg tcctcaacgc cgacggtgtg gccgaccgcg tgacctttat cgtcgacccc 420 aacaacgaga tccagttcgt ctcggccacc gccggttcgg tgggacgcaa cgtcgatgag 480 gtactgcgag tgctcgacgc cctccagtcc gacgagctgt gcgcatgcaa ctggcgcaag 540 ggcgacccga cgctagacgc tggcgaactc ctcaaggctt cggcc 585 72 816 DNA Mycobacterium tuberculosis 72 atgtctggga gaggagagcc gacgatgaaa acaatcattg ttggtatcga tggttcgcac 60 gcggcgatta cggccgcatt gtggggggtt gacgaggcca tcagccgagc ggtgccgctg 120 cgactggtct cagtgatcaa gccgacacat ccgtccccgg acgactacga ccgcgacctt 180 gcgcatgctg aaagatcgct tcgggaagcg cagtccgctg ttgaggccgc gggcaagctc 240 gtcaagatcg aaaccgacat cccccgcggg ccagccggcc cggtgcttgt ggaggcatcg 300 cgcgacgccg agatgatctg cgtcggctcc gtgggaatcg ggcgctacgc cagctcgatc 360 ttgggttcga cggcaaccga gctggccgaa aaggcgcatt gcccggtcgc cgtcatgcgc 420 tcaaaagtgg accagccagc gtctgacatc aactggatcg tggtgcgcat gaccgacgca 480 ccggataacg aggccgtgct ggaatacgct gcccgggaag cgaagttgcg gcaagcgccc 540 atactggcac tcggcgggcg accggaggag ctccgggaga ttccggacgg cgaattcgaa 600 cgtcgcgtgc aggattggca ccaccgtcat cccgatgtgc gcgtctaccc gatcaccact 660 cacacgggta ttgcccggtt cctggccgac cacgacgagc gcgtacagct ggcagtgatc 720 ggcggtggtg aggccggtca gctagcgcgg ctggtcgggc catccggaca tccggtgttc 780 cgtcacgccg agtgttcggt gcttgtcgtt cgccgc 816 73 1179 DNA Mycobacterium tuberculosis 73 atgcgtgatg cgatcccgct tgggcggatc gccgggtttg tggtgaacgt ccactggagc 60 gtgttggtga tcctgtggtt gttcacctgg agtctggcga ccatgttgcc gggtaccgtc 120 ggaggctacc cggccgtggt ctattggctt ctcggcgcag gtggcgcggt catgttgctg 180 gcgtcgctgt tggctcatga gctcgcgcac gccgtcgtcg ctcgtcgcgc cggggtatcc 240 gttgagagcg tgacgttgtg gctgttcggc ggggtgaccg cgcttggcgg cgaggcaaag 300 acgcccaaag ccgctttccg gatcgcgttc gcgggtccgg ctaccagcct ggcgctgtcg 360 gcgacattcg gtgcgttggc catcacgctc gccggcgtgc ggaccccggc catcgtgatc 420 agcgttgctt ggtggttggc tactgtcaac ctgctgctgg ggctgttcaa tctgctgcct 480 ggcgcgccgt tggacggtgg gcggttggtc cgggcctatc tgtggcgccg ccacggcgat 540 agtgtgcgcg ccgggatcgg tgcggcgcgg gccggacggg tggttgcgct ggtcttgatc 600 gcgttgggat tggccgagtt tgtggctggt ggcctcgtcg gtggggtctg gttagccttc 660 attggctggt ttatcttcgc tgccgctcgc gaggaggaga cccggatttc gacccagcag 720 ctgtttgccg gggtgcgtgt ggccgatgcg atgaccgccc aaccgcatac ggctcccgga 780 tggatcaatg tcgaggattt catccagcgt tacgtgcttg gtgaacggca ctcggcatat 840 ccggttgccg atcgggacgg atcgatcacg ggcctggtgg cattgcggca gctgcgcgat 900 gttgcgccta gccggcgcag cactaccagc gtaggtgaca ttgcgctgcc gctgcacagc 960 gtgccgaccg cccgaccaca agagccgctg accgcgctcc tagagcggat ggcaccgctc 1020 ggcccgcgca gccgtgcgct ggtcaccgaa gggagcgcgg tggtcggcat cgtcactccc 1080 agcgatgtcg cgcggctgat tgacgtctac cggttggccc agccggaacc gacctttacc 1140 acgagtcccc aagatgcgga caggttttcc gatgcgggg 1179 74 1239 DNA Mycobacterium tuberculosis 74 atggcaagtt ctgcgagcga cggcacccac gaacgctcgg cttttcgcct gagtccaccg 60 gtcttgagcg gcgccatggg accgttcatg cacaccggtc tgtacgtcgc tcaatcgtgg 120 cgcgactatc tgggtcaaca gcccgataaa ctgccgatcg cacggcccac tattgcctta 180 gcggcgcaag cctttcgaga cgaaatcgtc ctgctgggcc tcaaggcacg acgtccggtc 240 agcaatcatc gagtgttcga gcgcatcagc caagaagtgg ccgctggact ggagttctat 300 gggaatcgca gatggctgga gaagcctagc ggattttttg cccagccccc accgctcacc 360 gaggtcgcgg tccgaaaggt caaggaccgc agacgctcct tttatcgcat cttcttcgac 420 agtgggttta cgccgcatcc gggtgaaccg ggcagccaac ggtggctctc atacactgcg 480 aacaatcgcg agtacgccct gttactgcgg cacccagagc cgcgtccctg gctggtttgt 540 gtacacggca ccgagatggg cagggccccg ttggatctcg cggtgttccg cgcctggaag 600 ctgcatgacg aactcggcct gaacattgtc atgccggttc ttccgatgca tggtccccgc 660 gggcaaggtc tgccgaaggg cgccgttttt cccggagaag atgttctcga cgatgtgcat 720 gggacggctc aagcggtgtg ggatatccgg cggctgttgt cctggatacg atcgcaggag 780 gaggagtcgc tgatcgggtt gaacggtctc tcgctgggcg gctacatcgc gtcattggtc 840 gccagcctcg aagaaggtct cgcctgcgcg attctcggtg tcccagtggc tgatctgatc 900 gagttgttgg gccgccactg cggtcttcgg cacaaagacc cccgccgcca caccgtcaag 960 atggccgaac cgatcggccg aatgatctcg ccgctctcac ttacgccact ggtgcccatg 1020 ccgggccgct ttatctacgc gggcattgcc gaccgactcg tgcatccacg cgaacaggtg 1080 actcgcctct gggagcactg gggcaaaccc gaaatcgtgt ggtatccagg cggtcacact 1140 ggcttcttcc agtcgcggcc ggtacgacgg tttgtccagg ctgcgctgga gcagtcgggc 1200 ctgttggacg cgccacggac acagcgcgac cgttccgcc 1239 75 360 DNA Mycobacterium tuberculosis 75 atgtccacgc aacgaccgag gcactccggt attcgggctg ttggccccta cgcatgggcc 60 ggccgatgtg gtcggatagg caggtggggg gtgcaccagg aggcgatgat gaatctagcg 120 atatggcacc cgcgcaaggt gcaatccgcc accatctatc aggtgaccga tcgctcgcac 180 gacgggcgca cagcacgggt gcctggtgac gagatcacta gcaccgtgtc cggttggttg 240 tcggagttgg gcacccaaag cccgttggcc gatgagcttg cgcgtgcggt gcggatcggc 300 gactggcccg ctgcgtacgc aatcggtgag cacctgtccg ttgagattgc cgttgcggtc 360 76 1122 DNA Mycobacterium tuberculosis 76 atgcgatcag aacgtctccg gtggctggta gccgcagaag gtccgttcgc ctcggtgtat 60 ttcgacgact cgcacgacac tcttgatgcc gtcgagcgcc gggaagcgac gtggcgcgat 120 gtccggaagc atctcgaaag ccgcgacgcg aagcaggagc tcatcgacag cctcgaagag 180 gcggtgcggg attctcgacc ggccgtcggc cagcgtggcc gcgcgctgat cgcgaccggc 240 gagcaagtac tggtcaacga gcatctgatc ggcccaccac cggctacggt gattcggctg 300 tcggattatc cgtacgtcgt gccattgata gaccttgaga tgcggcgacc gacgtatgta 360 tttgccgcgg ttgatcacac cggcgccgac gtcaagctgt atcagggggc caccatcagt 420 tccacgaaaa tcgatggggt cggctacccg gtgcacaagc cggtcaccgc cggctggaac 480 ggctacggcg acttccagca caccaccgaa gaagccatcc gaatgaactg ccgcgcggtc 540 gccgaccatc tcacccgact ggtagacgct gccgaccccg aggtggtgtt cgtgtccggc 600 gaggtgcggt cacgcacaga cctgctttcc acattgccgc agcgggtggc ggtccgggtg 660 tcgcagctgc atgccggacc gcgcaaaagc gccttagacg aggaagagat ctgggacctg 720 acatccgcgg agttcacccg gcggcggtac gccgaaatca ccaatgtcgc acaacaattt 780 gaggcggaga tcggacgcgg atcggggctg gcggcccaag ggttggcgga ggtgtgtgcg 840 gctctgcgtg acggcgacgt cgacacgctg atcgtcggag agctaggcga ggccaccgtg 900 gtcaccggta aagcgcgtac tacggtcgcg cgggatgccg acatgttgtc cgaactcggc 960 gaaccggtag atcgcgtggc aagggccgat gaggcgttgc cattcgccgc gatcgcggta 1020 ggtgccgcat tggtccgtga cgacaaccgg atcgcgccac tagatggggt gggcgcattg 1080 ctgcgttatg ccgccaccaa ccgactcggc agccatagat cc 1122 77 537 DNA Mycobacterium tuberculosis 77 atgctgcacc gcgacgatca catcaatccg ccgcggcccc gcgggttgga tgttccttgc 60 gcccgcctac gagcgacaaa tcccctgcgc gccttggcgc gttgcgttca ggcgggcaag 120 ccgggcacca gttcagggca tcggtccgtg ccgcatacgg cggacttgcg aatcgaagcc 180 tgggcaccga cccgtgacgg ctgtatccgg caggcggtgc tgggtaccgt cgagagcttc 240 ctcgacctgg aatccgcgca cgcggtccat acccggctgc gccggctgac cgcggatcgc 300 gacgacgatc tactggtcgc ggtgctcgag gaggtcattt atttgctgga caccgtcggt 360 gaaacgcctg tcgatctcag gctgcgcgac gttgacgggg gtgtcgacgt cacattcgca 420 acgaccgatg cgagtacgct agttcaggtg ggtgccgtgc cgaaggcggt gtcactcaac 480 gaacttcggt tctcgcaggg tcgccacggc tggcgatgtg cggtaacgct cgatgtg 537 78 1125 DNA Mycobacterium tuberculosis 78 gtgacgcaaa ccggcaagcg tcagagacgc aaattcggtc gcatccgaca gttcaactcc 60 ggccgctggc aagccagcta caccggcccc gacggccgcg tgtacatcgc ccccaaaacc 120 ttcaacgcca agatcgacgc cgaagcatgg ctcaccgacc gccgccgcga aatcgaccga 180 caactatggt ccccggcatc gggtcaggaa gaccgccccg gagccccatt cggtgagtac 240 gccgaaggat ggctgaagca gcgtggaatc aaggaccgca cccgcgccca ctatcgcaaa 300 ctgctggaca accacatcct ggccaccttc gctgacaccg acctacgcga catcaccccg 360 gccgccgtgc gccgctggta cgccaccacc gccgtgggca caccgaccat gcgggcacac 420 tcctacagct tgctgcgcgc aatcatgcag accgccttgg ccgacgacct gatcgactcc 480 aacccctgcc gcatctcagg cgcgtccacc gcccgccgcg tccacaagat caggcccgcc 540 accctcgacg agctggaaac catcaccaaa gccatgcccg acccctacca ggcgttcgtg 600 ctgatggcgg catggctggc catgcgctac ggcgagctga ccgaattacg ccgcaaagac 660 atcgacctgc acggcgaggt tgcgcgggtg cggcgggctg tcgttcgggt gggcgaaggc 720 ttcaaggtga cgacaccgaa aagcgatgcg ggagtgcgcg acataagtat cccgccacat 780 ctgatacccg ccatcgaaga ccaccttcac aaacacgtca accccggccg ggagtccctg 840 ctgttcccat cggtcaacga ccccaaccgt cacctagcac cctcggcgct gtaccgcatg 900 ttctacaagg cccgaaaagc cgccggccga ccagacttac gggtgcacga ccttcgacac 960 tccggcgccg tgttggctgc atccaccggc gccacactgg ccgaactgat gcagcggcta 1020 ggacacagca cagccggcgc cgcactccgc taccagcacg ccgccaaggg ccgggaccgc 1080 gaaatcgccg cactgttaag caaactggcc gagaaccagg agatg 1125 79 1113 DNA Mycobacterium tuberculosis 79 atgcgcgtcg gtattccgac cgagaccaaa aacaacgaat tccgggtggc catcaccccg 60 gccggcgtcg cggaactaac ccgtcgtggc catgaggtgc tcatccaggc aggtgccgga 120 gagggctcgg ctatcaccga cgcggatttc aaggcggcag gcgcgcaact ggtcggcacc 180 gccgaccagg tgtgggccga cgctgattta ttgctcaagg tcaaagaacc gatagcggcg 240 gaatacggcc gcctgcgaca cgggcagatc ttgttcacgt tcttgcattt ggccgcgtca 300 cgtgcttgca ccgatgcgtt gttggattcc ggcaccacgt caattgccta cgagaccgtc 360 cagaccgccg acggcgcact acccctgctt gccccgatga gcgaagtcgc cggtcgactc 420 gccgcccagg ttggcgctta ccacctgatg cgaacccaag ggggccgcgg tgtgctgatg 480 ggcggggtgc ccggcgtcga accggccgac gtcgtggtga tcggcgccgg caccgccggc 540 tacaacgcag cccgcatcgc caacggcatg ggcgcgaccg ttacggttct agacatcaac 600 atcgacaaac ttcggcaact cgacgccgag ttctgcggcc ggatccacac tcgctactca 660 tcggcctacg agctcgaggg tgccgtcaaa cgtgccgacc tggtgattgg ggccgtcctg 720 gtgccaggcg ccaaggcacc caaattagtc tcgaattcac ttgtcgcgca tatgaaacca 780 ggtgcggtac tggtggatat agccatcgac cagggcggct gtttcgaagg ctcacgaccg 840 accacctacg accacccgac gttcgccgtg cacgacacgc tgttttactg cgtggcgaac 900 atgcccgcct cggtgccgaa gacgtcgacc tacgcgctga ccaacgcgac gatgccgtat 960 gtgctcgagc ttgccgacca tggctggcgg gcggcgtgcc ggtcgaatcc ggcactagcc 1020 aaaggtcttt cgacgcacga aggggcgtta ctgtccgaac gggtggccac cgacctgggg 1080 gtgccgttca ccgagcccgc cagcgtgctg gcc 1113 80 312 DNA Mycobacterium tuberculosis 80 atggtcatcc ggtttgatca aatagggtca ttggtcctct caatgaaatc ccttgcgtca 60 ctgtcgtttc agcggtgtct gcgcgagaat tctagtttgg tcgcggcgct ggaccggctc 120 gatgctgcgg tcgatgagct gagcgctttg tcgtttgatg cgttgaccac tccggagcgg 180 gatcgcgccc gtcgcgaccg ggaccatcat ccttggtccc gctcccgctc gcagttgtcg 240 ccacgaatgg cgcacggtgc agtgcaccaa tgccagtggc cgaaggcggt ttgggctgtc 300 attgacaatc ca 312 81 1032 DNA Mycobacterium tuberculosis 81 gtgctcaaga acgcagtctt gctggcatgc cgggcgccgt cggtgcacaa cagccagccc 60 tggcgttggg tggccgaaag cggctccgag cacactactg tgcacctgtt cgtcaaccgc 120 caccgaacgg tgccggccac cgaccattcc ggccggcaag cgatcatcag ttgcggtgcc 180 gtactcgatc accttcgcat cgccatgacg gccgcgcact ggcaggcgaa tatcactcgc 240 tttccccagc cgaaccaacc tgaccagttg gccaccgtcg aattcagtcc catcgatcac 300 gtcacggcgg gacagcgaaa ccgcgcccag gcgattctgc agcgccgaac cgatcggctt 360 ccgtttgaca gcccgatgta ctggcacctg tttgagcccg cgctgcgcga cgccgtcgac 420 aaagacgttg cgatgcttga tgtggtatcc gacgaccagc gaacacgact ggtggtagcg 480 tcacaactca gcgaagtcct gcggcgggac gatccgtact atcacgccga actcgaatgg 540 tggacttcac cgttcgtgct ggcccatggt gtgccgccgg atacgctggc atcagacgcc 600 gaacgcttgc gggttgacct gggccgtgac ttcccggtcc ggagctacca gaatcgccgt 660 gccgagctag ctgatgaccg atcgaaagtc cttgtgctgt cgacccctag cgacacgcga 720 gccgacgcac tgaggtgtgg cgaagtgctg tcgaccatcc tactcgagtg caccatggcc 780 ggcatggcta cctgcacgtt gacccatctg atcgaatcca gtgacagtcg tgacatcgtg 840 cggggcctga cgaggcagcg aggcgagccg caagccttga tccgggtagg gatagccccg 900 ccgttggcag cagttcccgc ccccacacca cggcggccgc tggacagcgt cttgcagatt 960 cgccagacgc ccgagaaagg gcgtaatgcc tcagatagaa atgcccgtga aacgggttgg 1020 ttcagcccgc ct 1032 82 1011 DNA Mycobacterium tuberculosis 82 gtgtggtccg cctcgggtgg gcagtgcggg aagtatcttg ccgcctcgat ggtgctgcag 60 cttgatgggt tggaacgtca cggtgtgttg gagtttgggc gtgaccgcta tggccccgag 120 gtgcgtgagg agctgttggc gatgagtgcg gccagcatcg atcgttatct gaagaccgcg 180 aaggccaaag accagatatc gggtgtgtcg acgacgaaac cctcaccact gctgcgtaat 240 tcgatcaagg ttcgcagggc cggcgatgag gtcgaggcgg agccggggtt cttcgagggc 300 gacaccgtcg cccattgcgg tccgacgctc aaaggcgagt tcgcccacac cctgaacttg 360 accgacgtgc acatcggatg ggtgttcacc cgcaccgtcc gcaacaacgc ccgtacccac 420 atcctcgccg ggctcaaagc ttctgtcacc gagatcccgc atgggataac gggtttagat 480 ttcgacaacg gcaccgtgtt tctcaacaag ccggtcatca gctgggccgg cgacaacggt 540 atctacttca cccgctttcg cccgtacaag aaaaaccact aggccaccat cgagtccaag 600 aacaaccacc tggtccgcaa gtacgcgttc tactaccgct atgacaccgc cgaggaacgc 660 gccgtgctca accggatgtg gaagctggtc aacgaccgcc tcaactacct caccccgacc 720 atcaaaccga tcgggtatgc cagcagcgcc gacggccgcc gccgacgcct ctacgatgcc 780 ccacagacgc cgctggaccg gccactggcc gcaagggtgc tctccgcggc ccagcaggcc 840 gacctgatca cctaccgaga cagcctcaac cccgcccaga tcggccgcaa aatcgccgac 900 ctgcagaacc gactcctcat cttggccaag gagaaaaccg agcagctcta cctcgctaac 960 atccccaccg ccctacccga catccacaaa ggcatcctga tcaaggcggg c 1011 83 330 DNA Mycobacterium tuberculosis 83 gtggtgcaag gccgcaccgt gctgtttcgt accgcggagg gcgccaaatt attttcagcc 60 gtcgcgaagt gcgcggtggc tttcgaggcg gacgaccaca acgttgccga gggctggagc 120 gtgatcgtca aggttcgcgc ccaggtgctg acgaccgacg cgggggtccg cgaagccgaa 180 cgcgcccagt tactaccgtg gaccgcgacg ctgaaacgtc actgtgtgcg ggtgatcccg 240 tgggagatca ccggccgcca cttcaggttc ggtccggaac cggaccgcag ccagaccttt 300 gcctgcgagg cctcgtcaca caaccagcga 330 84 1389 DNA Mycobacterium tuberculosis 84 atgaatcacc taacgacact tgacgccggg tttctcaagg cagaagacgt ggatcggcac 60 gtgagtctgg caatcggcgc tctggcggtc atcgaggggc cggctcccga tcaggaagcc 120 ttcttatcgt cgctcgctca acgcctacgt ccctgtaccc ggttcgggca gcggttacgc 180 ctgcgcccgt tcgacctcgg tgcacccaaa tgggtggacg atcccgactt cgatcttggc 240 cgtcatgtgt ggcgcatcgc cttgccgcgg cctggcaacg aagaccagtt attcgagctg 300 atcgccgatc tgatggcgcg tcgtttggac cggggtcgac cgctgtggga ggtctgggtc 360 atcgaaggcc tggcggacag caagtgggcg atcctgacca aactgcacca ctgcatggcc 420 gacggaatcg cggcgactca cctgctagct gggctctccg atgaaagtat gagcgacagc 480 ttcgcgagca acatccacac gaccatgcag tcgcaatccg catctgtgcg gcggggtgga 540 ttccgtgtca atccaagcga ggcgttgacc gcgtcgaccg ccgtgatggc aggcatcgtt 600 cgcgcggcca agggtgccag tgagatcgcg gccggcgtgc taagtcccgc cgcgtcgtcg 660 ttgaacgggc cgatcagtga tttgcgtcgc tacagcgcag caaaggtccc tctcgccgac 720 gtcgaacagg tgtgccggaa attcgacgtc accatcaatg atgttgcgct tgccgcgatt 780 acggaaagct accgcaacgt cctcatccag cggggtgagc ggcctaggtt tgattcgctg 840 cgtacgctag tgccggtctc gacgcgttcc aacagcgctt tgagcaagac cgataaccgt 900 gtttcgttaa tgctgcccaa cctgccggtg gatcaagaga acccgctgca gcggctgcgg 960 atcgtgcact cgcggctgac tcgggccaag gcggggggac agagacaatt cggaaatact 1020 ttgatggcga ttgccaaccg ccttccgttc cccatgaccg catgggcggt cgggctgttg 1080 atgcggctgc cgcagcgtgg tgttgtcacc gtggcgacaa atgtgccggg tccacgacgg 1140 ccgctgcaga ttatgggcag acgggtgctt gacctatacc cggtttcgcc gatcgcgatg 1200 caactgcgca ccagtgtcgc gatgctcagc tacgccgacg acctgtactt cgggatcctg 1260 gccgactacg acgtggtagc agatgccggc cagctggcgc gaggaattga agacgccgtc 1320 gcacggctgg tggcgatcag taagcggcgc aaggtgactc gcaggcgcgg agcgctatcg 1380 ctggttgtg 1389 85 996 DNA Mycobacterium tuberculosis 85 atgaacaccc atttcccgga cgccgaaacc gtgcgaacgg ttctcaccct ggccgtccgg 60 gccccctcca tccacaacac gcagccgtgg cggtggcggg tatgcccgac gagtctggag 120 ctgttctcta gacccgatat gcagctgcgt agcaccgatc cggacgggcg tgagttgatc 180 ctcagctgtg gtgtggcatt gcaccactgc gtcgtcgctt tggcgtcgct gggctggcag 240 gccaaggtaa accgtttccc cgatcccaag gaccgctgcc atctggccac catcggggta 300 caaccgcttg ttcccgatca ggccgatgtc gccttggcgg cggccatacc gcggcgacgc 360 accgatcggc gcgcctacag ttgctggccg gtgccaggag gtgacatcgc gttgatggcc 420 gcaagagcag cccgtggcgg ggtcatgctg cggcaggtca gtgccctaga ccgaatgaaa 480 gccattgtgg cgcaggctgt cttggaccac gtgaccgacg aggaatatct gcgcgagctc 540 accatttgga gtgggcgcta cggttcagtg gccggggttc ccgcccgcaa cgagccgcca 600 tcagacccca gtgccccgat ccccggtcgc ctgttcgccg ggcccggtct gtctcagccg 660 tccgacgtct tacccgctga cgacggcgcc gcgatcctgg cactaggcac cgagacagac 720 gaccggttgg cccggctgcg cgccggcgag gccgccagca tcgtcttgtt gaccgcgacg 780 gcaatggggc tggcgtgctg cccgatcacc gaaccgctgg agatcgccaa gacccgcgac 840 gcggtccgtg ccgaggtgtt cggcgccggc ggctaccccc agatgctgct gcgagtgggt 900 tgggcaccga tcaatgccga cccgttgcca ccgacgccac ggcgcgaact gtcccaggtc 960 gttgagtggc cggaagagct actgcgacaa cggtgc 996 86 1734 DNA Mycobacterium tuberculosis 86 atgacaacag ggggcctcgt cgacgaaaac gacggcgccg caatgcgtcc actgcgtcac 60 acgctctccc aactacgcct gcacgagctg ctggtcgagg tgcaggaccg ggtcgagcag 120 atcgtcgagg gccgggaccg cctcgatggt ctggtggagg ccatgctcgt ggtcacagcg 180 ggcctggacc tggaggcaac cctacgcgct atcgtgcatt cagcgaccag ccttgtcgat 240 gcgcgctatg gcgctatgga ggtgcacgac cggcagcatc gggtattgca ctttgtctat 300 gaaggcatcg acgaggagac cgttcggcgg atcggccacc taccgaaagg cctaggcgtc 360 atcgggctgc tcatcgaaga tcccaaaccg ttacggctgg acgatgtttc tgcgcacccg 420 gcctcgattg gttttccgcc gtatcatccg ccgatgcgta ccttcctcgg ggtaccggtt 480 cgggtgcgcg atgaatcgtt cggcactctg tacctgactg acaagaccaa cgggcaaccg 540 ttcagcgacg acgacgaggt tctggtccag gcgctggcgg ccgccgcggg tatcgcagtc 600 gcgaatgccc ggctctacca gcaggctaag gcgcgtcagt cgtggatcga ggccacccgt 660 gacatcgcca ccgagttgtt gtccggcacc gaacccgcga cggtgttccg gcttgtcgcc 720 gcggaggcgc tcaagctgac ggcggctgac gctgccctgg tagccgttcc cgtcgacgag 780 gacatgcctg ccgctgacgt gggggagctg ctggtgattg aaacagtcgg cagcgctgtg 840 gcttccattg ttgggcgaac gattccggtg gcgggcgcgg tgctgcggga ggtcttcgtc 900 aacggcattc cgcgacgggt cgaccgggtc gatttggaag gcctggacga actggccgac 960 gcaggtccgg cgctgctgtt gccgctgcgg gccagaggta ccgtagcggg tgtcgttgtt 1020 gtgctgagtc aaggcggtcc aggggctttc accgacgaac aactcgagat gatggccgcg 1080 ttcgccgacc aggccgcgct ggcttggcaa ttggccactt cgcaacgtcg gatgcgcgaa 1140 ctcgacgtac tgaccgaccg ggatcgtatc gcccgtgacc tccatgacca tgtcatccag 1200 cggctcttcg cgattggcct ggctttgcag ggtgctgtcc cgcacgaacg taatcctgaa 1260 gtgcagcaac gactctcgga cgtggtagac gatctgcaag acgttataca ggaaatccgg 1320 accaccattt atgacctgca cggagcatcg cagggtatca ctcggctccg gcagcgaatc 1380 gatgcggccg tagcccaatt tgccgactcg gggttgcgca ccagcgttca attcgtgggt 1440 ccattgtcgg tggtcgacag cgcgctcgcc gatcaggccg aggcggtggt tcgggaagcg 1500 gtcagcaacg cggttcgcca tgcgaaggcc agcacgttga ccgtccgggt caaagtcgac 1560 gacgacttgt gcatcgaggt gaccgacaac ggccgcgggc tgcccgacga gttcaccgga 1620 agcggcttaa cgaacctgcg gcagcgggca gagcaggccg gcggcgaatt caccctcgcg 1680 agcgtaccgg gcgcgagcgg aacagtgctg cgatggtcag caccgttgtc gcag 1734 87 804 DNA Mycobacterium tuberculosis 87 atgagcgatc ctcggccagc tcgggcagtg gtcgttggta tcgacgggtc aagggcggca 60 acgcatgcgg cgttgtgggc ggtcgatgag gcggtgaacc gagacattcc gctgcgactg 120 gtgtacgtca tcgatccgtc ccaactgtcc gccgccggcg agggcggtgg gcaatcagcg 180 gcccgagcgg cgctgcacga cgcctctcgg aaggtcgagg ccaccgggca accggtcaag 240 atcgaaacgg aggttctgtg cggcaggccg ctcaccaagc tgatgcagga gtccaggtcc 300 gcggcgatgc tgtgcgtcgg ttcggtgggg cttgatcatg tccgcggtcg ccggggttcg 360 gtcgcggcga ccctggctgg gtcggcctta tgccccgtgg cggtgattca cccgtcgccg 420 gccgagccag cgacaacctc ccaggtcagc gcggttgtcg cggaggtgga caatggtgtg 480 gtgctgcggc acgcattcga ggaggccagg ctgcgcggag ttccgctgcg ggccgtggct 540 gtccacgctg ctgaaacacc cgatgacgtc gaacagggca gccggttggc gcatgtacac 600 ctgagccgtc ggctcgccca ctggacccgg ctctaccccg aggtgcgggt ggatcgggcc 660 atcgccggcg gcagtgcgtg ccgtcatctg gccgccaacg caaagccggg tcagctgttc 720 gtcgcggact cacactccgc gcacgaattg tgcggtgcat accagcccgg atgcgccgta 780 cttacggtac gcagtgccaa cttg 804 88 543 DNA Mycobacterium tuberculosis 88 atgacagaat acgaagggcc taagacaaaa ttccacgcgt taatgcagga acagattcat 60 aacgaattca cagcggcaca acaatatgtc gcgatcgcgg tttatttcga cagcgaagac 120 ctgccgcagt tggcgaagca tttttacagc caagcggtcg aggaacgaaa ccatgcaatg 180 atgctcgtgc aacacctgct cgaccgcgac cttcgtgtcg aaattcccgg cgtagacacg 240 gtgcgaaacc agttcgacag accccgcgag gcactggcgc tggcgctcga tcaggaacgc 300 acagtcaccg accaggtcgg tcggctgaca gcggtggccc gcgacgaggg cgatttcctc 360 ggcgagcagt tcatgcagtg gttcttgcag gaacagatcg aagaggtggc cttgatggca 420 accctggtgc gggttgccga tcgggccggg gccaacctgt tcgagctaga gaacttcgtc 480 gcacgtgaag tggatgtggc gccggccgca tcaggcgccc cgcacgctgc cgggggccgc 540 ctc 543 89 822 DNA Mycobacterium tuberculosis 89 atgacatggg ccgacgaggt gctcgccgga catccctttg tggttgctca ccgtggtgcg 60 tcggcggctc ggccggagca tacccttgcc gcctacgacc tggcgctcaa agagggcgcc 120 gacggcgtgg aatgtgatgt gcggttgacc cgggacgggc atctggtctg tgtgcatgac 180 cgccgcctgg accgaacctc gacgggagcc ggcttggtca gcacgatgac gctggcccag 240 ctacgcgagc tggagtacgg cgcgtggcac gacagctggc gccccgacgg ttcgcacggc 300 gacaccagtc tgctgaccct ggacgcgctt gtttcgctgg ttttggactg gcaccggccg 360 gtgaagatct tcgtcgagac caagcatccc gtccgatacg gctcgctggt ggaaaacaag 420 ctgctggcgc tgctacaccg gttcggtatt gccgcacccg cctccgcaga tcgatcccgt 480 gcggtggtga tgtcgttttc ggccgccgcg gtctggcgga tccggcgggc tgcaccgctg 540 ctgccgacgg tgttgctcgg caagaccccc cgatacctga ccagcagtgc ggccacggcg 600 gtcggggcaa ccgccgtggg accctcactg cctgcgttaa aggaatatcc gcaactcgtt 660 gaccgctcgg cagctcaggg ccgggcggtg tactgctgga acgtcgatga gtacgaggac 720 atcgactttt gccgggaggt cggggtggcc tggattggta ctcaccaccc cggccgcacc 780 aaggcctggc tggaagacgg gcgggcgaac gggaccactc gc 822 90 744 DNA Mycobacterium tuberculosis 90 gtgtccgacg gcgaacaagc caaatcacgt cgacgccggg ggcggcgccg cgggcggcgc 60 gctgcggcta cagccgagaa tcacatggac gcccaaccgg ccggcgacgc caccccgacc 120 ccggcaacgg cgaagcggtc ccggtcccgc tcacctcgtc gcgggtcgac tcggatgcgc 180 accgtgcacg aaacatcggc tggagggttg gtcattgacg gtatcgacgg tccacgagac 240 gcgcaggtcg cggctctgat cggccgcgtc gaccggcgcg gccggctgct gtggtcgcta 300 cccaaggggc acatcgagtt gggcgagacc gccgagcaga ccgccatccg cgaggtcgcc 360 gaggagaccg gcatccgcgg cagtgtgctc gccgcgctgg ggcgcatcga ctactggttc 420 gtcaccgacg gccggcgggt gcacaagacc gtccaccatt atttgatgcg gtttttaggc 480 ggagagctgt ccgacgaaga cctcgaggta gccgaggtag cctgggtgcc gatccgggaa 540 ctgccgtctc gactggccta cgccgacgaa cgtcgactag ccgaggtggc cgacgaactg 600 atcgacaagc tgcagagcga cggccccgcc gcgcttccgc cgctaccacc cagctcgcct 660 cgtcgacggc cgcaaacgca ttcacgcgct cgtcatgccg atgactcagc accgggtcag 720 cacaacggtc ccgggccggg gccg 744 91 88 PRT Mycobacterium tuberculosis 91 Met Lys Ala Lys Val Gly Asp Trp Leu Val Ile Lys Gly Ala Thr Ile 1 5 10 15 Asp Gln Pro Asp His Arg Gly Leu Ile Ile Glu Val Arg Ser Ser Asp 20 25 30 Gly Ser Pro Pro Tyr Val Val Arg Trp Leu Glu Thr Asp His Val Ala 35 40 45 Thr Val Ile Pro Gly Pro Asp Ala Val Val Val Thr Ala Glu Glu Gln 50 55 60 Asn Ala Ala Asp Glu Arg Ala Gln His Arg Phe Gly Ala Val Gln Ser 65 70 75 80 Ala Ile Leu His Ala Arg Gly Thr 85 92 24 DNA Artificial primer sequence 92 caccgtggaa ccgaaacgca gtcg 24 93 19 DNA Artificial primer sequence 93 ttatgccaga ccgtcggca 19 94 19 DNA Artificial primer sequence 94 caccatgagc ccgggctcg 19 95 20 DNA Artificial primer sequence 95 ttacggcgta cgcgagtcag 20 96 24 DNA Artificial primer sequence 96 caccgtggag tccgaaccgc tgta 24 97 17 DNA Artificial primer sequence 97 ttacgtggcc gagccgc 17 98 22 DNA Artificial primer sequence 98 caccatgcct atcgcaacgc cc 22 99 23 DNA Artificial primer sequence 99 ttagtgggtt agggactttc cgg 23 100 49 DNA Artificial primer sequence 100 ggggacaagt ttgtacaaaa aagcaggctt aaaggcaaag gtcggggac 49 101 50 DNA Artificial primer sequence 101 ggggaccact ttgtacaaga aagctgggtc ctacgttccc ctggcatgga 50 102 22 DNA Artificial primer sequence 102 caccatgggt gagcacgcca tc 22 103 26 DNA Artificial primer sequence 103 ttataggtca tcggattgag gtgatc 26 104 24 DNA Artificial primer sequence 104 caccgtggct ggcaatcctg atgt 24 105 24 DNA Artificial primer sequence 105 ttactccttg ctcgttaggt tggc 24 106 22 DNA Artificial primer sequence 106 caccgtgaca gaccacgtgc gc 22 107 18 DNA Artificial primer sequence 107 ttacggtgac gagccggc 18 108 25 DNA Artificial primer sequence 108 caccatggta gagcccggca atttg 25 109 18 DNA Artificial primer sequence 109 ttagagcgga cggcggct 18 110 22 DNA Artificial primer sequence 110 caccatgatc gccacaaccc gc 22 111 20 DNA Artificial primer sequence 111 ttaccgctgc gtgcagaaca 20 112 24 DNA Artificial primer sequence 112 caccatgacc aacgtcggtg acca 24 113 21 DNA Artificial primer sequence 113 ttatcctgtt actgcggcgc a 21 114 24 DNA Artificial primer sequence 114 caccgtgacg gtgacaccac ggac 24 115 16 DNA Artificial primer sequence 115 ttaccacccg cgccgc 16 116 21 DNA Artificial primer sequence 116 caccatgaga gggcaagcgg c 21 117 22 DNA Artificial primer sequence 117 ttacctggac gcctcctcac tc 22 118 21 DNA Artificial primer sequence 118 caccatgtgc ggcgaccagt c 21 119 21 DNA Artificial primer sequence 119 ttaatacaac aatcgcgccg g 21 120 25 DNA Artificial primer sequence 120 caccatgatt cccacgatga catcg 25 121 17 DNA Artificial primer sequence 121 ttagcgccga cggaacg 17 122 49 DNA Artificial primer sequence 122 ggggacaagt ttgtacaaaa aagcaggctt aatcacaaac ctccgacgc 49 123 51 DNA Artificial primer sequence 123 ggggaccact ttgtacaaga aagctgggtc ctagttgcac gcccagttga c 51 124 24 DNA Artificial primer sequence 124 caccttgtcg gcgtcagtgt ctgc 24 125 17 DNA Artificial primer sequence 125 ttatggcggt tgcgccc 17 126 29 DNA Artificial primer sequence 126 caccatgagt ttccacgatc ttcatcacc 29 127 24 DNA Artificial primer sequence 127 ttacgttgta ctcgtgcggt tctc 24 128 22 DNA Artificial primer sequence 128 caccgtggtc aagcgctctc gg 22 129 22 DNA Artificial primer sequence 129 ttattccgac tcgagtgggt ga 22 130 24 DNA Artificial primer sequence 130 caccatgtct aaaccccgca agca 24 131 19 DNA Artificial primer sequence 131 ttacgactgc cgtgccacg 19 132 30 DNA Artificial primer sequence 132 caccgtgacc tatgtgatcg gtagtgagtg 30 133 20 DNA Artificial primer sequence 133 ttaagggcac tccaccggga 20 134 26 DNA Artificial primer sequence 134 caccatgaac caatcacaca aacccc 26 135 25 DNA Artificial primer sequence 135 ttacagatac tgctgaccga cgacc 25 136 20 DNA Artificial primer sequence 136 caccatgacg gagccagcgg 20 137 18 DNA Artificial primer sequence 137 ttatggcgag gcttccgg 18 138 49 DNA Artificial primer sequence 138 ggggacaagt ttgtacaaaa aagcaggctt actgatgacc gcagcggct 49 139 53 DNA Artificial primer sequence 139 ggggaccact ttgtacaaga aagctgggtc ctacagaccg gtcgggtagg ttt 53 140 48 DNA Artificial primer sequence 140 ggggacaagt ttgtacaaaa aagcaggctt agccaccacc cttcccgt 48 141 54 DNA Artificial primer sequence 141 ggggaccact ttgtacaaga aagctgggtc ctagttggtg gaccggatct gaat 54 142 22 DNA Artificial primer sequence 142 caccatgccg gacaccatgg tg 22 143 24 DNA Artificial primer sequence 143 ttagtgatcc ttagcccgaa cgtg 24 144 52 DNA Artificial primer sequence 144 ggggacaagt ttgtacaaaa aagcaggctt aatgccactg ctaaccattg gc 52 145 52 DNA Artificial primer sequence 145 ggggaccact ttgtacaaga aagctgggtc ctaggccgaa gccttgagga gt 52 146 25 DNA Artificial primer sequence 146 caccatgtct gggagaggag agccg 25 147 21 DNA Artificial primer sequence 147 ttagcgaacg acaagcaccg a 21 148 48 DNA Artificial primer sequence 148 ggggacaagt ttgtacaaaa aagcaggctt acgtgatgcg atcccgct 48 149 50 DNA Artificial primer sequence 149 ggggaccact ttgtacaaga aagctgggtc ctaccccgca tcggaaaacc 50 150 51 DNA Artificial primer sequence 150 ggggacaagt ttgtacaaaa aagcaggctt aatggcaagt tctgcgagcg a 51 151 51 DNA Artificial primer sequence 151 ggggaccact ttgtacaaga aagctgggtc ctaggaacgg tcgcgctgtg t 51 152 22 DNA Artificial primer sequence 152 caccatgtcc acgcaacgac cg 22 153 21 DNA Artificial primer sequence 153 ttaaccgcaa cggcaatctc a 21 154 24 DNA Artificial primer sequence 154 caccatgcga tcagaacgtc tccg 24 155 23 DNA Artificial primer sequence 155 ttaggatcta tggctgccga gtc 23 156 21 DNA Artificial primer sequence 156 caccatgctg caccgcgacg a 21 157 23 DNA Artificial primer sequence 157 ttacacatcg agcgttaccg cac 23 158 48 DNA Artificial primer sequence 158 ggggacaagt ttgtacaaaa aagcaggctt agtgacgcaa accggcaa 48 159 52 DNA Artificial primer sequence 159 ggggaccact ttgtacaaga aagctgggtc ctacatctcc tggttctcgg cc 52 160 49 DNA Artificial primer sequence 160 ggggacaagt ttgtacaaaa aagcaggctt acgcgtcggt attccgacc 49 161 48 DNA Artificial primer sequence 161 ggggaccact ttgtacaaga aagctgggtc ctacacgctg gcgggctc 48 162 28 DNA Artificial primer sequence 162 caccatggtc atccggtttg atcaaata 28 163 24 DNA Artificial primer sequence 163 ttatggattg tcaatgacag ccca 24 164 26 DNA Artificial primer sequence 164 caccgtgctc aagaacgcag tcttgc 26 165 21 DNA Artificial primer sequence 165 ttaaggcggg ctgaaccaac c 21 166 20 DNA Artificial primer sequence 166 caccgtgtgg tccgcctcgg 20 167 20 DNA Artificial primer sequence 167 ttagcccgcc ttgatcagga 20 168 20 DNA Artificial primer sequence 168 caccgtggtg caaggccgca 20 169 22 DNA Artificial primer sequence 169 ttatcgctgg ttgtgtgacg ag 22 170 29 DNA Artificial primer sequence 170 caccatgaat cacctaacga cacttgacg 29 171 23 DNA Artificial primer sequence 171 ttacacaacc agcgatagcg ctc 23 172 23 DNA Artificial primer sequence 172 caccatgaac acccatttcc cgg 23 173 23 DNA Artificial primer sequence 173 ttagcaccgt tgtcgcagta gct 23 174 23 DNA Artificial primer sequence 174 caccatgaca acagggggcc tcg 23 175 22 DNA Artificial primer sequence 175 ttactgcgac aacggtgctg ac 22 176 22 DNA Artificial primer sequence 176 caccatgagc gatcctcggc ca 22 177 23 DNA Artificial primer sequence 177 ttacaagttg gcactgcgta ccg 23 178 32 DNA Artificial primer sequence 178 ccggctgaga tctatgacag aatacgaagg gc 32 179 24 DNA Artificial primer sequence 179 ccccgccagg gaactagagg cggc 24 180 22 DNA Artificial primer sequence 180 caccatgaca tgggccgacg ag 22 181 20 DNA Artificial primer sequence 181 ttagcgagtg gtcccgttcg 20 182 22 DNA Artificial primer sequence 182 caccgtgtcc gacggcgaac aa 22 183 15 DNA Artificial primer sequence 183 ttacggcccc ggccc 15 184 8 DNA Artificial reference sequence 184 agtcagtc 8 185 8 DNA Artificial reference sequence 185 aatcaatc 8 186 6 DNA Artificial reference sequence 186 agtgtc 6 187 8 DNA Artificial reference sequence 187 agtcagtc 8

Claims (22)

1. A method for inducing an immune response to latent tuberculosis in an individual, said method comprising the step of delivering a composition comprising one or more polypeptides or fragments thereof, which polypeptides are upregulated or expressed during the latent stage of the mycobacteria infection, and/or nucleic acids encoding these polypeptides.
2. The method according to claim 1, wherein said individual is infected by a virulent mycobacterium, e.g. M. tuberculosis, and is not vaccinated with BCG against tuberculosis.
3. The method according to claim 1, where the polypeptides upregulated during the latent stage of the mycobacteria infection, comprises one or more an amino acid sequences selected from
(a) SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45
(b) an immunogenic portion, e.g. a T-cell epitope, of any one of the sequences in (a); and/or
(c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic.
4. The method according to claim 3, where the immunogenic portions are selected from the group consisting of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45.
5. The method according to claim 1, where the nucleic acid sequences are selected from SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90.
6. A therapeutic vaccine against tuberculosis comprising one or more polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, and/or nucleic acids encoding these polypeptides.
7. A therapeutic vaccine according to claim 6 where the polypeptides upregulated during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, comprises one or more amino acid sequences selected from
(a) SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45
(b) an immunogenic portion, e.g. a T-cell epitope, of any one of the sequences in (a); and/or
(c) an amino acid sequence analogue having at least 70% sequence identity to any one of the sequences in (a) or (b) and at the same time being immunogenic.
8. A therapeutic vaccine according to claim 7, where the immunogenic portions are selected from the group consisting of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45.
9. A therapeutic vaccine according to claim 6, where the polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, are fused to other antigens expressed by bacteria within the mycobacteria family.
10. A therapeutic vaccine according to claim 9 where the fusion partners is selected from the group consisting of ESAT-6, ESAT-6-Ag85B, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32.
11. A therapeutic vaccine according to claims 6, where the nucleic acid sequence are selected from SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90.
12. A multiphase vaccine comprising antigen components with therapeutic activity according to claim 6 combined with antigen components with prophylactic activity.
13. A multiphase vaccine according to claim 12 where the antigen components with prophylactic activity comprises ESAT-6, ESAT-6-Ag85B, TB10.4, CFP10, RD1-ORF5, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein or MPT32.
14. A vaccine according to claim 6, comprising as the effective component a non-pathogenic microorganism, wherein at least one copy of a DNA fragment comprising an antigen component with therapeutic activity selected from SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90 or/and an antigen component with prophylactic activity has been incorporated into the genome of the microorganism in a manner allowing the microorganism to express and optionally secrete the polypeptide.
15. A vaccine according to claim 14 where the non-pathogenic microorganism is selected among bacteria or virus.
16. A vaccine according to claim 6, where the antigen components are recombinant polypeptides or synthetic peptides delivered in a delivery system such as an adjuvant.
17. A vaccine according to claims 6 in which the amino acid sequence is lipidated so as to allow a self-adjuvanting effect of the polypeptide.
18. A method for treating an animal, including a human being, with tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, comprising administering to the animal the vaccine according to claim 6.
19. A method for immunizing an animal, including a human being, against tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, comprising administering to the animal the vaccine according to claim 12.
20. A method of diagnosing tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, in an animal, including a human being, comprising application or intradermally injecting, in the animal, polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, and/or nucleic acids encoding these polypeptides, a positive skin response at the location of injection or application being indicative of the animal having tuberculosis, and a negative skin response at the location of injection or application being indicative of the animal not having tuberculosis.
21. A method for diagnosing previous or ongoing infection with a virulent mycobacterium, said method comprising contacting a sample, e.g. a blood sample, comprising mononuclear cells (e.g. T-lymphocytes), with a polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, in order to detect a positive reaction, e.g. proliferation of the cells or release of cytokines such as IFN-γ.
22. A method of diagnosing Mycobacterium tuberculosis infection in a subject comprising:
(a) contacting a polypeptides or fragments hereof, which polypeptides are expressed during the latent stage of the mycobacteria infection, which stage is characterized by low-oxygen tension in the microenvironment of the mycobacteria, with a bodily fluid of the subject;
(b) detecting binding of an antibody to said polypeptide, said binding being an indication that said subject is infected by Mycobacterium tuberculosis or is susceptible to Mycobacterium tuberculosis infection.
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