NOVEL LIVE RECOMBINANT BOOSTER VACCINE AGAINST TUBERCULOSIS

Abstract

Embodiments of the invention comprise an improved vaccine for generating an immune response and preventing or treating mycobacterial diseases such as tuberculosis in humans and animals. Embodiments of the invention also comprise a method for using the vaccine against such mycobacterial diseases.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional patent application No. 61/355,052, filed Jun. 15, 2010, the entire contents of which are incorporated herein by reference.

1. FIELD OF THE INVENTION

[0003] The present invention relates to methods and compositions of matter that are useful for preventing or reducing the possibility of infection caused by Mycobacterium tuberculosis, the agent of tuberculosis, and infection by other pathogenic strains of mycobacteria in humans and/or animals including Mycobacterium bovis and Mycobacterium leprae.

2. BACKGROUND

[0004] Around the world, intracellular bacteria are responsible for millions of deaths each year and untold suffering. Tuberculosis, caused by Mycobacterium tuberculosis, is a leading worldwide cause of death from an infectious disease, with millions of new cases and deaths reported each year. Initial infections of M. tuberculosis almost always occur through the inhalation of aerosolized particles as the pathogen can remain viable for weeks or months in moist or dry sputum. Although the primary site of the infection is in the lungs, the organism can also cause infection of the bones, spleen, meninges, and skin. Depending on the virulence of the particular strain and the resistance of the host, the infection and corresponding damage to the tissue may be minor or extensive.

[0005] While M. tuberculosis is a significant pathogen, other species of the genus Mycobacterium also cause disease in humans and animals and are clearly within the scope of the present invention. For example, M. bovis is closely related to M. tuberculosis and is responsible for tubercular infections in domestic animals such as cattle, pigs, sheep, horses, dogs and cats. Further, M. bovis may infect humans via the intestinal tract, typically from the ingestion of raw milk. The localized intestinal infection eventually spreads to the respiratory tract and is followed shortly by the classic symptoms of tuberculosis. Another important pathogenic vector of the genus Mycobacterium is M. leprae which causes millions of cases of the ancient disease leprosy. Currently, there is no effective vaccine to prevent it. A vaccine to prevent leprosy would potentially have widespread use in endemic areas such as India and Brazil. Other species of this genus which cause disease in animals and humans include M. kansasii, M. avium intracellulare, M. fortuitum, M. marinum, M. chelonei, M. africanum, M. ulcerans, M. microti, and M. scrofulaceum. The pathogenic mycobacterial species frequently exhibit a high degree of homology in their respective DNA and corresponding protein sequences and some species, such as M. tuberculosis and M. bovis are highly related.

[0006] With regard to alveolar or pulmonary infections by M. tuberculosis, the guinea pig model closely resembles the human pathology of the disease in many respects. Accordingly, it is well understood by those skilled in the art that it is appropriate to extrapolate the guinea pig model of this disease to humans and other mammals. As with humans, guinea pigs are susceptible to tubercular infection with low doses of the aerosolized human pathogen M. tuberculosis. Unlike humans where the initial infection is usually controlled, guinea pigs consistently develop disseminated disease upon exposure to the aerosolized pathogen, facilitating subsequent analysis. Further, both guinea pigs and humans display cutaneous delayed-type hypersensitivity reactions characterized by the development of a dense mononuclear cell induration or rigid area at the skin test site. Finally, the characteristic tubercular lesions of humans and guinea pigs exhibit similar morphology including the presence of Langhans giant cells. As guinea pigs are more susceptible to initial infection and progression of the disease than humans, any protection conferred in experiments using this animal model provides a strong indication that the same protective immunity may be generated in man or other less susceptible mammals. Accordingly, for purposes of explanation only and not for purposes of limitation, the present invention will be primarily demonstrated in the exemplary context of guinea pigs as the mammalian host. Those skilled in the art will appreciate that the present invention may be practiced with other mammalian hosts including humans, mice and domesticated animals.

[0007] The only currently available vaccine, Mycobacterium bovis strain Bacille Calmette-Guerin (BCG), is of variable efficacy. Many studies have failed to demonstrate significant protection (see, e.g. Fine (1989). "The BCG story: lessons from the past and implications for the future." Rev Infect Dis 11 Suppl 2: S353-9). A large carefully conducted meta-analysis has estimated the potency of BCG to be approximately 50% (see, e.g. Colditz, et al. (1994). "Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature." JAMA 271(9): 698-702). Despite its variable efficacy, several hundred million doses of the BCG vaccine are still administered to humans each year. Hence, a better vaccine or a vaccine that improves the potency of BCG by even a small amount could have a tremendous impact on the disease incidence.

[0008] A previous study examined the use of M. tuberculosis major extracellular proteins for immunizing against tuberculosis (see, e.g. Horwitz, et al. (1995). "Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis." Proc Natl Acad Sci USA 92(5): 1530-4). Another study examined a prime-boost vaccination strategy for boosting the level of protective immunity conferred by a prime vaccine such as BCG or recombinant BCG (see, e.g. Horwitz, et al. (2005). "Enhancing the protective efficacy of Mycobacterium bovis BCG vaccination against tuberculosis by boosting with the Mycobacterium tuberculosis major secretory protein." Infect Immun 73(8): 4676-83). In this study, the prime vaccine consisted of BCG or a recombinant BCG and the booster vaccine consisted of an M. tuberculosis major extracellular protein in adjuvant. Boosting BCG with such a vaccine enhanced the level of protective immunity conferred by BCG alone.

[0009] Other investigators have used various heterologous vectors to deliver M. tuberculosis proteins as booster vaccines for BCG, for example, adenovirus and modified vaccinia Ankara (see, e.g. McShane, et al. (2004). "Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans." Nat Med 10(11): 1240-4; Williams, et al. (2005). "Boosting with poxviruses enhances Mycobacterium bovis BCG efficacy against tuberculosis in guinea pigs." Infect Immun 73(6): 3814-6; Santosuosso, et al. (2006). "Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis." Infect Immun 74(8): 4634-43; Vordermeier, et al. (2009). "Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis." Infect Immun 77(8): 3364-73; Xing, et al. (2009). "Intranasal mucosal boosting with an adenovirus-vectored vaccine markedly enhances the protection of BCG-primed guinea pigs against pulmonary tuberculosis." PLoS One 4(6): e5856). However, such vectors are difficult to produce because the viruses have to be grown in cell culture. Moreover, the efficacy of these virus-vectored vaccines as booster vaccines for BCG has not been high, especially with routes of administration other than intranasal. These vaccines have given little or no protection in the more challenging guinea pig model.

[0010] Therefore, a safe and effective vaccine against M. tuberculosis or other species of the genus Mycobacterium that is more potent than the currently available vaccines is sorely needed. There is also a need for a booster vaccine or a vaccine that can improve the potency of the currently available vaccines by even a small amount. The disclosure provided herein meets this need.

SUMMARY OF THE INVENTION

[0011] Current commercially available vaccines are of limited efficacy against pulmonary tuberculosis. The present disclosure provides a vaccine and method for preventing, reducing the possibility of or treating tuberculosis in humans and animals that is more potent than the current commercially available vaccines and methods in protecting against pulmonary tuberculosis and dissemination of bacteria to the spleen and other organs. The present disclosure also provides a vaccine and method for preventing, reducing the possibility of or treating leprosy and other mycobacterial diseases. Moreover, the present invention provides a booster vaccine that is surprisingly and unexpectedly more potent than a protein-in-adjuvant vaccine or an adenovirus-based vaccine. In addition, the present disclosure provides a vaccine that is easier and cheaper to manufacture than both virus-vectored vaccines, which must be grown in tissue culture cells and then purified, and protein-in-adjuvant vaccines, where the protein needs to be purified. The vaccine described in the present disclosure can simply be grown in broth culture--no purification is necessary.

[0012] The invention disclosed herein has a number of embodiments. A typical embodiment comprises a composition of matter that includes attenuated Listeria monocytogenes that expresses a Mycobacterium tuberculosis polypeptide, for example the 30 kDa antigen 85B protein (SEQ ID NO: 4). In such compositions, the Listeria monocytogenes is attenuated by inactivation (e.g. via deletion) of one or more genes so that it does not express a functional protein such as a ActA protein (SEQ ID NO: 1) and/or a InlB protein (SEQ ID NO: 2). In certain embodiments of the invention, attenuated Listeria monocytogenes expresses prfA protein having a G155S substitution mutation (SEQ ID NO: 3). In some embodiments of the invention, the composition comprises one or more agents commonly used in vaccines such as a pharmaceutically acceptable carrier.

[0013] While the 30 kDa antigen 85B protein (SEQ ID NO: 4) is a commonly discussed embodiment of the invention, other proteins, either alone, or in combination can be expressed in the attenuated Listeria monocytogenes. Such proteins include: Mycobacterium tuberculosis 12 kDa fragment of 16 kDa membrane protein (SEQ ID NO:5); Mycobacterium tuberculosis 14 kDa MPT53 protein (SEQ ID NO: 6); Mycobacterium tuberculosis 16 kDa MPT63 protein (SEQ ID NO: 7); Mycobacterium tuberculosis 23 kDa SOD protein (SEQ ID NO: 8); Mycobacterium tuberculosis 23.5 kDa MPT64 protein (SEQ ID NO: 9); Mycobacterium tuberculosis 24 kDa MPT51 protein (SEQ ID NO: 10); Mycobacterium tuberculosis 32 kDa antigen 85A protein (SEQ ID NO: 11); Mycobacterium tuberculosis 32 kDa antigen 85C protein (SEQ ID NO: 12); Mycobacterium tuberculosis 45 kDa MPT32 protein (SEQ ID NO: 13); Mycobacterium tuberculosis 58 kDa glutamine synthetase protein (SEQ ID NO: 14); Mycobacterium tuberculosis 71 kDa HSP 70 protein (SEQ ID NO: 15); Mycobacterium tuberculosis 10.4 kDa EsxH protein (SEQ ID NO: 16); Mycobacterium tuberculosis 14 kDa alpha crystalline homolog protein (SEQ ID NO: 17); Mycobacterium tuberculosis 47 kDa isocitrate lysate protein (SEQ ID NO: 18); Mycobacterium tuberculosis 7.6 kDa hypothetical protein (SEQ ID NO: 19); Mycobacterium tuberculosis 80 kDa glcB protein (SEQ ID NO: 20) Mycobacterium tuberculosis 110 kDa can protein (SEQ ID NO: 21); and Mycobacterium tuberculosis 9.9 kDa ESAT-6 protein (SEQ ID NO: 22). A wide variety of combinations of proteins can be expressed in various embodiments of the invention. For example, in certain embodiments of the invention, one or more latency associated proteins (e.g. SEQ NOS: 17-19) are expressed in combination with one or more of the other proteins disclosed herein (e.g. SEQ NOS: 4, 6-16 and 20-22). In certain embodiments of the invention, the Mycobacterium tuberculosis 30 kDa antigen 85B protein is fused in frame with a heterologous protein sequence. Optionally, for example, the Mycobacterium tuberculosis 30 kDa antigen 85B protein is coupled to a heterologous protein sequence comprising the N-terminal 100 amino acids of the ActA protein. The protein expression can further be controlled by constructing expression cassettes to include certain regulatory sequences. In one illustrative embodiment of the invention, the expression of the Mycobacterium tuberculosis 30 kDa antigen 85B protein is controlled by an ActA promoter.

[0014] Another embodiment of the invention is a method of generating an immune response to a specific polypeptide, for example a Mycobacterium tuberculosis polypeptide such as the 30 kDa antigen 85B protein (SEQ ID NO: 4). Such methods include immunizing a mammal with a composition of matter disclosed herein, for example one comprising attenuated Listeria monocytogenes constructed to express Mycobacterium tuberculosis 30 kDa antigen 85B protein so that an antibody and/or a cellular immune response to Mycobacterium tuberculosis 30 kDa antigen 85B protein is generated. In an illustrative embodiment of invention, the Listeria monocytogenes does not express a functional ActA protein (SEQ ID NO: 1); does not express a functional InlB polypeptide (SEQ ID NO: 2); expresses prfA protein having a G155S substitution mutation (SEQ ID NO: 3); and expresses Mycobacterium tuberculosis 30 kDa antigen 85B protein (SEQ ID NO: 4). While the 30 kDa antigen 85B protein (SEQ ID NO: 4) is a commonly discussed embodiment of the invention, other proteins, either alone, or in combination can be expressed in the attenuated Listeria monocytogenes to generate an immune response.

[0015] Those of skill in this art understand that the immunization methods disclosed herein can be combined with other methodological steps. For example, certain embodiments of the invention include the step of further comprising immunizing the mammal with Mycobacterium bovis strain Bacille Calmette-Guerin (BCG). Typically in these embodiments, the BCG is used in a primary immunization and the attenuated Listeria monocytogenes is used in a booster immunization. In such embodiments of the invention, the mammal can be immunized intradermally, intranasally, orally, subcutaneously, percutaneouly, intramuscularly, intravenously, or by another conventional route of vaccine delivery.

[0016] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Referring now to the drawings and figures in which like reference numbers represent corresponding parts throughout:

[0018] FIG. 1 is a graph illustrating measured diameters of induration (mm±SE) of guinea pigs after being injected intradermally with 10 μg of purified recombinant M. tuberculosis 30 kDa major extracellular protein (Antigen 85B; r30). As described herein under Experiment 1, guinea pigs were first immunized with various vaccines and then tested for cutaneous delayed-type hypersensitivity (c-DTH) to purified recombinant M. tuberculosis 30 kDa major extracellular protein (r30).

[0019] FIG. 2 is a graph illustrating splenic lymphocyte proliferation to r30, PPD, and media alone (RPMI). As described herein under Experiment 1, guinea pigs were first immunized with various vaccines and then assayed for splenic lymphocyte proliferation to purified recombinant M. tuberculosis 30 kDa major extracellular protein.

[0020] FIG. 3 is a graph illustrating measured reciprocal antibody titer for r30. As described herein under Experiment 1, guinea pigs were first immunized with various vaccines and then assayed for antibody responses to purified recombinant M. tuberculosis 30 kDa major extracellular protein.

[0021] FIG. 4 is a graph illustrating net weight gains or losses of guinea pigs after being challenged with an aerosol generated from a 7.5 ml single-cell suspension containing 6.3×10⁴ colony-forming units (CFU) of M. tuberculosis. As described herein under Experiment 2, the guinea pigs were first immunized with various vaccines and then subsequently challenged with the aerosol containing M. tuberculosis.

[0022] FIG. 5 is a graph illustrating assay measurements (mean log CFU±SE) of colony forming units (CFU) of M. tuberculosis in the lungs and spleens of guinea pigs. As described herein under Experiment 2, the guinea pigs were first immunized with various vaccinations and then subsequently challenged with an aerosol generated from a 7.5 ml single-cell suspension containing 6.3×10⁴ colony-forming units (CFU) of M. tuberculosis.

[0023] FIG. 6 illustrates cellular and humoral immune responses induced by boosting BCG-immunized animals with rLm/Mtb30 vaccines. Mice in groups of 4 were immunized intradermally with PBS (Sham) or BCG at week 0 and groups of BCG-immunized animals were boosted intradermally with one of various rLm/Mtb30 vaccines, with the rAd30 vaccine, or with r30 in adjuvant at weeks 3 and 6. At week 7, mice were anesthetized, bled and euthanized. Splenocytes were assayed for lymphocyte proliferation (A) or intracellular cytokine (INFgamma) expression in response to r30 (C and D) stratified as to CD4+ or CD8+ cells. Serum was assayed for immunoglobulin-G level in response to the r30 protein (B). Values represent mean±SE.

[0024] FIG. 7 illustrates assay measurements of colony forming units (CFU) of M. tuberculosis in the lungs and spleens of mice. As described herein under Experiment 4, the mice were first immunized with various vaccinations and then subsequently challenged with an aerosol generated from a 7.5 ml single-cell suspension containing 6.3×10⁴ colony-forming units (CFU) of M. tuberculosis.

[0025] FIG. 8 is a graph illustrating interferon-γ production in mice in response to M. tuberculosis antigens. As described herein under Experiment 5, mice were immunized with various vaccines and the splenocytes were stimulated with medium alone, the recombinant 30 kD protein (r30) or M. tuberculosis Purified Protein Derivative (PPD) for three days. The splenocyte supernatant fluid was collected and assayed for the level of IFNγ by ELISA.

[0026] FIG. 9 illustrates the calculated the integrated MFI (iMFI) for cytokine-secreting CD4+ T-cells. As described herein under Experiment 5, mice were immunized with various vaccines and the splenocytes were stimulated with the mature recombinant 30 kD protein (r30) or a pool of three peptides of r30 (30p). The cells were then stained for the cytokines IFNγ, IL-2, and TNFα, and analyzed by multi-parameter flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

[0028] In the following description of the typical embodiment, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

[0029] In one aspect of the present disclosure, a novel vaccine vector comprising attenuated Listeria monocytogenes is described. In one embodiment, a vector comprising attenuated Listeria monocytogenes is used by itself as a primary vaccine or vaccinating agent. In another embodiment, a vector comprising attenuated Listeria monocytogenes is used for delivering M. tuberculosis proteins as a booster vaccine for another tuberculosis (TB) vaccine, such as BCG or recombinant BCG. In a further embodiment, a vector comprising attenuated Listeria monocytogenes is used for delivering mycobacteria proteins, such as proteins from Mycobacterium bovis and Mycobacterium leprae, as a primary or booster vaccine or vaccinating agent. Unexpectedly, utilizing a vector comprising attenuated Listeria monocytogenes for delivering mycobacteria proteins as a booster vaccine induces greater protective immunity than boosting with just the purified proteins in adjuvant or boosting with a recombinant adenovirus encoding the same proteins.

[0030] Attenuated Listeria monocytogenes can be used as vectors to deliver M. tuberculosis major extracellular proteins, which include but are not limited to the 30 kDa major secretory protein (Antigen 85B), 32A major secretory protein (Antigen 85A), 32B major secretory protein (Antigen 85C), the 23.5 kDa major secretory protein (a.k.a. MPT64), the 16 kDa major secretory protein, the 23 kDa subunit mass superoxide dismutase, the 58 kDa subunit mass glutamine synthetase, the 71 kDa subunit mass heat shock protein, the 12 kDa subunit mass exported fragment of the 16 kDa alpha-crystallin protein, and the 14 kDa secreted protein, etc. Such extracellular proteins have been shown to be immunoprotective against M. tuberculosis (see, e.g. Horwitz, et al. (1995). "Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis." Proc Natl Acad Sci USA 92(5): 1530-4).

[0031] In one exemplary implementation of the present disclosure, BCG is utilized as a first vaccine with a booster vaccine being a recombinant attenuated Listeria monocytogenes expressing the M. tuberculosis 30 kDa major secretory protein, a.k.a. Antigen 85B or r30 (rLm/Mtb30). In the Examples described herein, boosting with rLm/Mtb30 has been compared with boosting with purified M. tuberculosis 30 kDa major secretory protein in adjuvant or boosting with a recombinant Adenovirus expressing the M. tuberculosis 30 kDa major secretory protein. Surprisingly, this method of immunization is found to induce significantly greater protective immunity than boosting with just the purified M. tuberculosis protein in adjuvant or boosting with a recombinant adenovirus encoding the same protein.

[0032] In another aspect of the present disclosure, a composition of matter is described that is useful for preventing or reducing the possibility of infection caused by Mycobacterium tuberculosis, the agent of tuberculosis, or infection by other pathogenic strains of mycobacteria in humans and/or animals including Mycobacterium bovis and Mycobacterium leprae. Administration of the composition of matter comprising attenuated Listeria monocytogenes expressing a major extracellular protein induces a cell-mediated immune response to the recombinant major extracellular protein. This subsequently protects against infection by M. tuberculosis or other mycobacterial diseases. In yet another aspect of the present disclosure, a vaccination strategy is described wherein an attenuated Listeria monocytogenes expressing M. tuberculosis proteins is administered by itself. In one embodiment, rLm/Mtb30 is administered by itself.

[0033] In a further aspect of the present disclosure, a vaccination strategy is described wherein an attenuated Listeria monocytogenes expressing M. tuberculosis proteins is administered as a booster vaccine following immunization with another TB vaccine. In one embodiment, rLm/Mtb30 is administered as a heterologous booster vaccine following immunization with another TB vaccine, such as BCG or a recombinant BCG expressing the same protein. For example, BCG or recombinant BCG is administered first, and after a period of time, the rLm/Mtb30 vaccine is administered one or more times. The initial vaccination may be with BCG or any recombinant strain of BCG overexpressing and secreting one or more M. tuberculosis major extracellular proteins, including but not limited to the 30 kDa major secretory protein (Antigen 85B), 32A major secretory protein (Antigen 85A), 32B major secretory protein (Antigen 85C), the 23.5 kDa major secretory protein (a.k.a. MPT64), the 16 kDa major secretory protein, the 23 kDa subunit mass superoxide dismutase, the 58 kDa subunit mass glutamine synthetase, the 71 kDa subunit mass heat shock protein, the 12 kDa subunit mass exported fragment of the 16 kDa alpha-crystallin protein, the 14 kDa secreted protein, etc. The subsequent vaccination would be with recombinant attenuated Listeria monocytogenes expressing the same M. tuberculosis protein. The attenuated Listeria monocytogenes can be administered intradermally or by another route, e.g. intranasally, subcutaneously, percutaneously, intramuscularly, or even orally to a mammalian host. The vaccines or immunotherapeutic agents and methods for their use disclosed herein may be used to impart acquired immunity in a mammalian host against various intracellular pathogens, including but not limited to M. tuberculosis, M. bovis, M. kansasii, M. avium-intracellulare, M. fortuitum, M. chelonei, M. marinum, M. scrofulaceum, M. leprae, M. africanum, M. ulcerans, and M. microti.

[0034] As illustrated above, the invention disclosed herein has a number of embodiments. A typical embodiment comprises a composition of matter that includes live attenuated Listeria monocytogenes that expresses a Mycobacterium tuberculosis polypeptide, for example the 30 kDa antigen 85B protein (SEQ ID NO: 4). In such compositions, the Listeria monocytogenes is attenuated by inactivation (e.g. via deletion) of one or more genes so that it does not express a functional protein such as a ActA protein (SEQ ID NO: 1) and/or a InlB protein (SEQ ID NO: 2). In certain embodiments of the invention, attenuated Listeria monocytogenes expresses prfA protein having a G155S substitution mutation (SEQ ID NO: 3).

[0035] While the 30 kDa antigen 85B protein (also called herein the M. tuberculosis 30 kDa major secretory protein r30) is a commonly discussed embodiment of the invention, other proteins, either alone, or in combination can be expressed in the attenuated Listeria monocytogenes (e.g. an attenuated Listeria monocytogenes of strain 10403S). Such proteins include: Mycobacterium tuberculosis 12 kDa fragment of 16 kDa membrane protein (SEQ ID NO:5); Mycobacterium tuberculosis 14 kDa MPT53 protein (SEQ ID NO: 6); Mycobacterium tuberculosis 16 kDa MPT63 protein (SEQ ID NO: 7); Mycobacterium tuberculosis 23 kDa SOD protein (SEQ ID NO: 8); Mycobacterium tuberculosis 23.5 kDa MPT64 protein (SEQ ID NO: 9); Mycobacterium tuberculosis 24 kDa MPT51 protein (SEQ ID NO: 10); Mycobacterium tuberculosis 32 kDa antigen 85A protein (SEQ ID NO: 11); Mycobacterium tuberculosis 32 kDa antigen 85C protein (SEQ ID NO: 12); Mycobacterium tuberculosis 45 kDa MPT32 protein (SEQ ID NO: 13); Mycobacterium tuberculosis 58 kDa glutamine synthetase protein (SEQ ID NO: 14); Mycobacterium tuberculosis 71 kDa HSP 70 protein (SEQ ID NO: 15); Mycobacterium tuberculosis 10.4 kDa EsxH protein (SEQ ID NO: 16); Mycobacterium tuberculosis 14 kDa alpha crystalline homolog protein (SEQ ID NO: 17); Mycobacterium tuberculosis 47 kDa isocitrate lysate protein (SEQ ID NO: 18); Mycobacterium tuberculosis 7.6 kDa hypothetical protein (SEQ ID NO: 19); Mycobacterium tuberculosis 80 kDa glcB protein (SEQ ID NO: 20) Mycobacterium tuberculosis 110 kDa can protein (SEQ ID NO: 21); and Mycobacterium tuberculosis 9.9 kDa ESAT-6 protein (SEQ ID NO: 22). A wide variety of combinations of proteins can be expressed in various embodiments of the invention. For example, in certain embodiments of the invention, one or more latency associated proteins (e.g. SEQ NOS: 17-19) are expressed in combination with one or more of the other proteins disclosed herein (e.g. SEQ NOS: 4, 6-16 and 20-22).

[0036] In some embodiments of the invention, the composition comprises one or more agents used in vaccines such as a pharmaceutically acceptable carrier. Methods for formulating compositions of the invention for pharmaceutical administration are known to those of skill in the art. See, for example, Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro (ed.) 1995, Mack Publishing Company, Easton, Pa. Typically the immunogenic agents used in the methods of the invention combined with at pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" is used according to its art accepted meaning and is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Certain compositions of the invention can include an adjuvant. Immunologic adjuvants are commonly added to vaccines to stimulate the immune system's response to the target antigen, but do not in themselves confer immunity. Adjuvants can act in various ways in presenting an antigen to the immune system. A wide variety of adjuvants are known in the art, see e.g. Handbook of Pharmaceutical Additives: An International Guide to More Than 6000 Products by Trade Name, Chemical, Function, and Manufacturer by Michael Ash and Irene Ash (1996). In addition, certain embodiments of the invention further comprising a buffer system, for example phosphate buffered saline.

[0037] In certain embodiments of the invention, a protein such as the Mycobacterium tuberculosis 30 kDa antigen 85B protein is fused in frame with a heterologous protein sequence. Optionally, for example, the Mycobacterium tuberculosis 30 kDa antigen 85B protein is coupled to a heterologous protein sequence comprising the N-terminal 100 amino acids of the ActA protein. The protein expression can further be controlled by constructing expression cassettes to include certain regulatory sequences. In one illustrative embodiment of the invention, the expression of the Mycobacterium tuberculosis 30 kDa antigen 85B protein is controlled by an ActA promoter.

[0038] Another embodiment of the invention is a method of generating an antibody to a Mycobacterium tuberculosis polypeptide such as the 30 kDa antigen 85B protein (SEQ ID NO: 4) by immunizing a mammal with a composition of matter disclosed herein, for example one comprising attenuated Listeria monocytogenes constructed to express this protein so that an antibody to a Mycobacterium tuberculosis 30 kDa antigen 85B protein is generated. The term "mammal" as used herein refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human. In an illustrative embodiment of invention, the Listeria monocytogenes does not express a functional ActA protein (SEQ ID NO: 1); does not express a functional InlB polypeptide (SEQ ID NO: 2); expresses prfA protein having a G155S substitution mutation (SEQ ID NO: 3); and expresses Mycobacterium tuberculosis 30 kDa antigen 85B protein (SEQ ID NO: 4). While the 30 kDa antigen 85B protein (SEQ ID NO: 4) is a commonly discussed embodiment of the invention, other proteins, either alone, or in combination can be expressed in the attenuated Listeria monocytogenes to generate an immune response.

[0039] Those of skill in this art understand that the immunization methods disclosed herein can be combined with other methodological steps. For example, certain embodiments of the invention include the step of further comprising immunizing the mammal with Mycobacterium bovis strain Bacille Calmette-Guerin (BCG). Typically in these embodiments, the BCG is used in a primary immunization and the attenuated Listeria monocytogenes is used in a booster immunization. In such embodiments of the invention, the mammal can be immunized intranasally, orally, subcutaneously, percutaneouly, intramuscularly, or by anther conventional route of vaccine delivery.

[0040] Antibodies generated by methodological embodiments of the invention have a number of uses. In one exemplary use in vivo, antibodies to an antigen such as a Mycobacterium tuberculosis protein are useful to prevent and/or diminish the severity of a disease such as tuberculosis. In yet another exemplary use, antibodies to a Mycobacterium tuberculosis protein such as 30 kDa antigen 85B protein (SEQ ID NO: 4) can function as an essential element in a diagnostic assay. In particular, those of skill in this art understand that a wide variety of diagnostic assays use antigen specific antibodies to provide substantial beneficial information to medical personnel including ELISA assay, Western Assays, radioimmunoassays and the like. Such assays use antibodies specific for an antigen such as a protein expressed by a pathogenic organism to, for example, identify the presence of that organism in a sample. Examples of tuberculosis diagnosis using antibodies in ELISA systems are described in publications such as Radhakrishnan et al., J Infect Dis, 1991 163(3): 650-652 and Kashyap et al., Clin Diag Lab Immunol. 2005 12(6): 752-758, the contents of which are incorporated by reference herein. In addition, a number of commercially available kits are known in the art such as the Clearview TB ELISA system. As is known in the art, antibodies useful in such assays can be obtained, for example, from the supernatants of hybridomas generated by conventional methods and/or affinity purified from human sera (see, e.g., Groen et al., J Virol Methods. 1989 Feb; 23(2):195-203, the contents of which are incorporated by reference). In this context, antibodies generated by the methods of the invention can be readily adapted for use in such assays.

[0041] Those skilled in the art will appreciate that the exemplary discussions of M. tuberculosis that are provided herein are in no way intended to limit the scope of the present invention to the treatment of M. tuberculosis. Similarly, the teachings herein are not limited in any way to the treatment of tubercular infections. On the contrary, this invention may be used to advantageously provide safe and effective vaccines and immunotherapeutic agents against the immunogenic determinants of any pathogenic agent expressing extracellular products and thereby inhibit the infectious transmission of those organisms.

EXAMPLES

Materials and Methods

[0042] A. BCG Strain (Wild-type M. bovis BCG Tice)

[0043] This strain was maintained in 7H9 medium pH 6.7 (Difco) at 37° C. in a 5% CO₂-95% air atmosphere as unshaken cultures. Cultures were sonicated once or twice weekly for 5 min in a sonicating water bath to reduce bacterial clumping, as described (see, e.g. Horwitz, et al. (2000). "Recombinant bacillus calmette-guerin (BCG) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model." Proc Natl Acad Sci USA 97(25): 13853-8).

B. Recombinant Attenuated Listeria monocytogenes Vaccines 1. rLm/Mtb30(01)

[0044] a. Construction of rLm/Mtb30(01)

[0045] rLm/Mtb30(01), an attenuated recombinant Listeria monocytogenes expressing the M. tuberculosis 30 kDa major secretory protein (Mtb30), was constructed using the attenuated L. monocytogenes host strain, LmΔactA, an L. monocytogenes strain 10403S (serotype 1/2a) with a deletion of actA encoding one of virulence factors, ActA. The coding sequence for the mature peptide of the 30 kDa major secretory protein was PCR amplified from the genomic DNA of the M. tuberculosis 37HRv strain. The DNA fragments were cloned into pZErO (Invitrogen) vector. The identity of the inserted 30 kDa coding sequences was confirmed by nucleotide sequencing, and subcloned into the BamHI and Pad sites of the cloning vector pKB199, in such a way that the 30 kDa coding sequence was fused to the listeriolysin 0 signal sequence (LLO) downstream of the hemolysin (hly) promoter of L. monocytogenes. The expression cassette of the LLO-30 kDa fusion protein driven by the hly promoter was excised from the resultant vector and subsequently cloned into a site-specific integration vector pDP4189. The integration vector was transformed into SM10, the E. coli conjugation donor strain. Through conjugation, the plasmid was mobilized and transferred from its E. coli SM10 host into the recipient LmΔactA strain. The conjugation mixture was selected on plates containing streptomycin and chloramphenicol. The LmΔactA strain is insensitive to streptomycin since it is derived from the L. monocytogenes strain 10403S, a spontaneous mutant resistant to streptomycin. E. coli SM10 is sensitive to streptomycin, therefore was not able to grow on the selective plate. The plasmid pDP4189 carries the chloramphenicol resistance gene and is unable to replicate in Listeria. Under the selection pressure from chloramphenicol, the plasmid pDP4189/Mtb30 integrated in the 3' end of an arginine tRNA gene on the chromosome of LmΔactA strain. The resultant recombinant LmΔactA strain, rLm/Mtb30(01), carries a single copy of the 30 kDa expression cassette and is stable in the absence of antibiotic selection.

[0046] b. Protein Expression of Mtb30 in Broth.

[0047] The expression of 30 kDa major secretory protein by rLm/Mtb30 was tested in broth. A single colony of rLm/Mtb30(01) was inoculated into 3 ml Brain Heart Infusion (BHI) medium containing streptomycin and chloramphenicol and the bacteria were grown overnight at 37° C. with shaking. The overnight culture was inoculated into 40 ml fresh MOPS-buffered BHI medium containing streptomycin and chloramphenicol at an initial optical density (OD) of 0.05 at 540 nm. The culture was grown until late logarithmic phase at 37° C. with vigorous shaking before being harvested by centrifugation. The supernate was passed through a 0.2 μm filter membrane, and proteins in the culture filtrate were precipitated by trichloroacetic acid. The expression of the 30 kDa major secretory protein was analyzed by Western blotting using rabbit polyclonal antibody against the 30 kDa major secretory protein. It was found that the antibody reacted specifically with a protein band of 30 kDa, which was absent from the LmΔactA parental strain. This confirmed that the recombinant rLm/Mtb30 expresses the 30 kDa protein.

[0048] c. Protein Expression of Mtb 30 by rLm/Mtb30(01) in Human Macrophages

[0049] THP-1 cells were differentiated into a monolayer on a 24-well plate (2×10⁵ cells/well) in the presence of 100 nM PMA (phorbol 12-myristate 13-acetate) and in the absence of antibiotics. The rLm/Mtb30(01) culture was grown to late logarithmic phase (OD of 1.0 at 540 nm) in BHI broth and used to infect the THP-1 cell monolayer at a multiplicity of infection (MOI) of 50:1. After infection for 1 h at 37° C., the monolayer was washed twice with RPMI to remove extracellular bacteria and then treated with 1 ml medium containing gentamicin at a final concentration of 10 μg ml^-1 to kill any remaining extracellular bacteria. At 24 h post-infection, cells were harvested, washed once with PBS, and boiled for 7 min in Laemmli buffer before analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. It shows that bands of ˜30 kDa in size were detected in samples of macrophages infected by two clones of rLm/Mtb30(01), but not by the parental rLmΔactA strain. These two clones (#8 and #10) were amplified in BHI broth and used for animal experiments.

2. rLm/Mtb30(02) (Codon Optimized)

[0050] rLm/Mtb30(02), rLmΔactA expressing the 30 kDa protein codon-optimized for higher level of protein expression in L. monocytogenes (30 kDa(C-O)), was constructed using a method similar to that described above for rLm/Mtb30(01). The DNA sequence for a codon-optimized version of the 30 kDa major secretory protein (30 kDa(C-O)) was synthesized by DNA2.0 (Menlo Park, Calif.).

3. rLmΔactAΔinlB/Mtb30 (rLm/Mtb30(11))

[0051] To further reduce the toxicity of the LmΔactA vector, a second generation of rLm/Mtb30 vaccines were constructed using a L. monocytogenes with deletions in actA and inlB (LmΔactAΔinlB) as a vector. Deletions in actA and inlB retain the potency and diminish the toxicity of the Lm vector in vivo (see, e.g. Brockstedt, et al. (2004). "Listeria-based cancer vaccines that segregate immunogenicity from toxicity." Proc Natl Acad Sci USA 101(38): 13832-7). It was shown that the 30 kDa protein was readily detected by Coumassie blue staining in broth cultures of the vaccine and in extracts of macrophages infected with rLm/Mtb30(11).

4. rLmΔactAΔinlB/ActAN100-Mtb30 (rLm/Mtb30(03)(04))

[0052] It has been reported that the actA promoter has a higher activity than hly promoter in vivo and proteins fused with 100 amino acids of the N-terminus of ActA are highly immunogenic. Therefore to enhance the in vivo expression of 30 kDa protein by the rLm/Mtb30 vaccines, the 30 kDa major secretory protein was fused with the N-terminal 100 amino acids of ActA (ActAN100) under the control of the actA promoter and introduced the fusion protein expression cassette into the second generation of the rLmΔactAΔinlB vector. The following 2 additional second generation of rLm/Mtb30 vaccines have been constructed:

[0053] a. rLmΔactAΔinlB/ActAN100-Mtb30 (03)

[0054] b. rLmΔactAΔinlB/ActAN100-Mtb30-SL8 (04)

[0055] It has been shown that these second generation rLm/Mtb30 vaccines express the kDa major secretory protein in broth culture and in human macrophages at levels similar to that of the first generation rLm/Mtb30 vaccines. The safety of these vaccines in animals are further being tested.

5. rLmΔactAΔinlBΔuvrABprfA*/Mtb30 (12)(07)(08)

[0056] To further enhance the immunogenicity of the rLm/Mtb30 vaccines, the following third generation rLm/Mtb30 vaccines expressing high amounts of the 30 kDa protein have been constructed and characterized.

[0057] a. rLmactAΔinlBΔuvrABprfA*/Mtb30 (12)

[0058] b. rLmactAΔinlBΔuvrABprfA*/ActAN100-Mtb30 (07)

[0059] c. LmactAΔinlBΔuvrABprfA*/ActAN100-Mtb30-SL8 (08)

[0060] The third generation rLm/Mtb30 vaccines were constructed using as a vector an LmΔactAΔinlB with an additional deletion in uvrAB and a single mutation (G155S) in pfrA (LmΔactAΔinlBΔuvrABprfA*, or Lm/prfA*) provided by Aduro BioTech. The deletion in uvrAB was designed to facilitate inactivation of the rLm/prfA* vaccines by UV light (Brockstedt et al. 2005) and the prfA* mutation resulted in constitutive expression of the downstream genes, including actA and hly (see, e.g. Lauer, et al. (2008). "Constitutive Activation of the PrfA regulon enhances the potency of vaccines based on live-attenuated and killed but metabolically active Listeria monocytogenes strains." Infect Immun 76(8): 3742-53; Yan, et al. (2008). "Selected prfA* mutations in recombinant attenuated Listeria monocytogenes strains augment expression of foreign immunogens and enhance vaccine-elicited humoral and cellular immune responses." Infect Immun 76(8): 3439-50). It was shown that all three rLm/pfrA*/Mtb30 vaccines expressed the 30 kDa fusion proteins at significantly higher levels in broth than the corresponding vaccines derived from the first and second generation Lm vectors (rLmΔactA and rLmΔactAΔinlB)

[0061] C. Purified M. tuberculosis 30 kDa Major Secretory Protein

[0062] This protein was purified from recombinant M. smegmatis as described in Horwitz et al. 1995 (see, e.g. Horwitz, et al. (1995). "Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis." Proc Natl Acad Sci USA 92(5): 1530-4) and Harth et al. 1997 (see, e.g. Harth, et al. (1997). "High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets." Infect Immun 65(6): 2321-8).

D. Replication-Deficient Adenovirus Expressing the 30 kDa Major Secretory Protein: Generation of Strains and Recombinant Protein Expression

[0063] The replication-deficient recombinant adenovirus strain that expresses the 30 kDa major secretory protein of M. tuberculosis (Mtb30) was constructed using an AdenoVator system (Q Biogen). The Mtb30 coding sequence was first cloned into a transfer vector of pAdenoVator-CMV5 downstream of a modified immediate-early promoter of cytomegalovirus (CMV5), which allows for the production of high levels of heterologous proteins in mammalian cells. The transfer vector contains a kanamycin-resistance gene that allows for selection of recombinant adenoviral DNAs. The transfer vector containing the Mtb30 coding sequence was then co-transformed into E. coli together with an adenoviral plasmid DNA with deletions in viral early genes E1 and E3, pAdenoVatorΔE1/E3. The E1 but not the E3 gene is essential for adenovirus growth in mammalian cells. Thus the recombinant adenoviruses with E1 and E3 deletions are replication-deficient and can grow only on cells that express the E1 gene. Through homologous recombination between the transfer vector and the adenoviral plasmid DNA in E. coli, the Mtb30 gene driven by the CMV5 promoter was introduced into the deleted E1 region of the adenoviral plasmid DNA. Recombinant adenoviral DNAs were selected with kanamycin and confirmed by restriction enzyme analysis. A positive recombinant adenoviral DNA that was confirmed to contain the appropriate insert, pAdvΔE1E3/Mtb30, was linearized with restriction enzyme (Pad) and transfected into mammalian cells (293A) that express the E1 protein. The resultant replication-deficient recombinant adenoviruses were plaque purified and amplified in 293A cells for up to 4 passages. The expression of Mtb30 by the recombinant adenovirus, AdvΔE1E3/Mtb30, was confirmed by Western blotting using a rabbit polyclonal antibody to Mtb30. The replication-deficient recombinant adenovirus stock was prepared from 6×10⁸ 293A cells and purified by two rounds of ultracentrifugation on CsCl gradients. The amount of virus particles in the virus stock was measured by assessing the DNA content of lysed virus in solution and utilizing the extinction coefficient of 1.1×10¹² virus particles per OD₂₆₀ unit. The amount of infectious virus in the virus stock was measured by determining the tissue culture infectious dose 50 (TCID₅₀) in 293A cells. The purified viral particles were stored at -80° C. in 20 mM Tris, pH 8.0, 25 mM NaCl, 2.5% glycerol.

E. Animal Models

[0064] The studies of the efficacy of the vaccines utilized guinea pigs because the guinea pig model is especially relevant to human tuberculosis clinically, immunologically, and pathologically. In contrast to the mouse and rat, but like the human, the guinea pig a) is susceptible to low doses of aerosolized M. tuberculosis; b) exhibits strong cutaneous delayed-type hypersensitivity (DTH) to tuberculin; and c) displays Langhans giant cells and caseation in pulmonary lesions. However, whereas only about 10% of immunocompetent humans who are infected with M. tuberculosis develop active disease over their lifetime (half early after exposure and half after a period of latency), infected guinea pigs always develop early active disease. While guinea pigs differ from humans in this respect, the consistency with which they develop active disease after infection with M. tuberculosis is an advantage in trials of vaccine efficacy.

[0065] Additional immunology studies including studies requiring special immunology reagents were conducted in C57BL/6 mice.

F. Preparation of Primary Vaccination

[0066] Aliquots were removed from logarithmically growing wild-type or recombinant BCG cultures, and the bacteria were pelleted by centrifugation at 3,500×g for 15 min. The bacteria were then washed with 1× phosphate buffered saline (1×PBS, 50 mM sodium phosphate pH 7, 150 mM sodium chloride) and resuspended at a final concentration of 1×10⁴ colony forming units per ml in 1×PBS. The immunization inoculum contained 1,000 viable wild-type or recombinant BCG bacteria in a total volume of 100 μA.

G. Preparation of Booster Vaccination

[0067] 1. rLm/Mtb30.

[0068] The various rLm/Mtb30 vaccines were grown in broth. Aliquots were removed from late logarithmically growing rLm/Mtb30 cultures and the bacteria were pelleted by centrifugation at 3,500×g for 15 min. The bacteria were then washed once with 1× phosphate buffered saline (1×PBS, 50 mM sodium phosphate pH 7, 150 mM sodium chloride) and re-suspended at a final concentration of 2×10⁹ colony forming units per ml in 1× sterile PBS. The stock was stored at -80° C., and the titer was checked periodically by plating serial dilutions of the stock on BHI plates. No significant titer loss was found over the period of storage. Before each use in animals, one vial of rLm/Mtb30 was thawed immediately at 37° C., diluted in sterile saline to a final concentration of 2×10⁷ colony forming units per ml in 1× sterile PBS and kept on ice until use. The rLm/Mtb30 was administered intradermally at a dose of 1×10⁶ bacteria in a total of 50 μl per animal.

2. r30 in Adjuvant

[0069] 100 μg of the r30 protein was mixed with Syntex Adjuvant Formulation (SAF) as described in Horwitz et al. 1995 (see, e.g. Horwitz, et al. (1995). "Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis." Proc Natl Acad Sci USA 92(5): 1530-4).

3. rAd/Mtb30

[0070] The rAd/Mtb30 stock was thawed on ice, diluted in sterile PBS to a final concentration of 1×10¹¹ viral particles per mL and administered intradermally at a dose of 1×10¹¹ viral particles in a total 50 μL per animal.

EXPERIMENTS

Experiment 1

Immunogenicity of rLm/Mtb30 in Prime-Boost Vaccination Regimen

[0071] Specific-pathogen free 250-300 g outbred male Hartley strain guinea pigs from Charles River Breeding Laboratories, in groups of 6, were immunized intradermally as follows:

[0072] Group A: Sham

[0073] Group B: BCG Tice Parental Control (10³ CFU) at Week 0

[0074] Group C: BCG Tice Parental Control (10³ CFU) at Week 0 and 100 μg of r30 in SAF adjuvant at Week 4

[0075] Group D: BCG Tice Parental Control (10³ CFU) at Week 0 and rAd/Mtb30 at Week 4

[0076] Group E: BCG Tice Parental Control (10³ CFU) at Week 0 and Lm Empty Vector at Week 4

[0077] Group F: BCG Tice Parental Control (10³ CFU) at Week 0 and rLm/Mtb30 at Week 4

[0078] Group G: BCG Tice Parental Control (10³ CFU) at Week 0 and rLm/Mtb30 C-0 at Week 4

[0079] At week 8, animals were tested for cutaneous delayed-type hypersensitivity (c-DTH) to r30, and after the skin test was assessed, the animals were euthanized for assay of splenic lymphocyte proliferation and antibody responses to r30.

A. Cutaneous Delayed-type Hypersensitivity (DTH) to Purified Recombinant M. tuberculosis 30 kDa Major Extracellular Protein (r30)

[0080] Guinea pigs were shaved over the back and injected intradermally with 10 μg of purified recombinant M. tuberculosis 30 kDa major extracellular protein (r30) in 100 μl phosphate buffered saline. After 24 h, the diameter of erythema and induration was measured. Induration is most reflective of a c-DTH response. The results are summarized in Table 1 and FIG. 1.

TABLE-US-00001 TABLE 1 Cutaneous DTH - Experiment 1 Test Erythema Induration Group Vaccination Antigen (mm ± SE) (mm ± SE) A Sham r30 0 ± 0 0 ± 0 B BCG r30 0 ± 0 0 ± 0 C BCG + r30 r30 19.6 ± 1.9 19.6 ± 1.9 D BCG + rAd/Mtb30 r30 19.7 ± 1.4 19.7 ± 1.4 E BCG + Lm Vector r30 1.2 ± 0.7 0 ± 0 F BCG + rLm/Mtb30 r30 13.3 ± 1.1 13.3 ± 1.1 G BCG + rLm/Mtb30 C-O r30 12.3 ± 1.0 6.7 ± 3.1

[0081] These results showed that the animals immunized with the parental BCG Tice strain (Group F) and the sham-immunized animals (Group J) had no erythema and induration upon testing with the M. tuberculosis 30 kDa major secretory protein r30. In contrast, animals immunized with BCG and boosted with purified r30 in adjuvant or with recombinant vaccines expressing r30, including both recombinant adenovirus and recombinant L. monocytogenes all developed significantly increased levels of both erythema and, more importantly, induration in response to skin-testing with r30. Boosting with the empty L. monocytogenes vector did not induce a significant c-DTH response (0 induration). Both rLm/Mtb30 and rLm/Mtb30C-0 induced a significant c-DTH response.

B. Splenic Lymphocyte Proliferation to Purified Recombinant M. tuberculosis 30 kDa Major Extracellular Protein (r30)

[0082] Immediately after skin-testing, the animals were euthanized, their spleens removed, and splenic lymphocyte proliferation to r30 (100 μg/well) and PPD (10 μg/well) assayed. The results are shown in Table 2 and FIG. 2.

[0083] As expected, all but the sham-immunized animals reacted to the positive control antigen PPD. The sham and BCG-immunized animals had low lymphocyte proliferative activity. Animals immunized with BCG and boosted with either rAd/Mtb30 or the Lm vector had modest lymphocyte proliferative activity. Boosting BCG-immunized animals with r30 markedly increased lymphocyte proliferative activity approximately 4-fold vs. BCG-immunized animals. Boosting BCG-immunized animals with rLm/Mtb30 or rLm/Mtb 30 C-0 also markedly increased splenic lymphocyte proliferative activity by ˜7-fold and 4-fold, respectively vs. BCG immunized animals.

[0084] Thus, boosting with recombinant L. monocytogenes expressing the M. tuberculosis 30 kDa major secretory protein markedly increased splenic lymphocyte proliferation to r30 in guinea pigs.

TABLE-US-00002 TABLE 2 Splenic Lymphocyte Proliferation - Experiment 1 Test r30 (Mean PPD (Mean Group Vaccination Antigen CPM ± SE) CPM ± SE) A Sham r30 328 ± 102 259 ± 111 B BCG r30 194 ± 52 3032 ± 682 C BCG + r30 r30 774 ± 188 5483 ± 1164 D BCG + rAd/Mtb30 r30 527 ± 137 3620 ± 609 E BCG + Lm Vector r30 544 ± 94 2582 ± 454 F BCG + rLm/Mtb30 r30 1331 ± 249 2800 ± 505 G BCG + rLm/Mtb30 r30 851 ± 155 1732 ± 251 C-O

C. Antibody Responses to Purified Recombinant M. tuberculosis 30 kDa Major Extracellular Protein (r30)

[0085] Immediately after skin-testing, the animals were euthanized and their serum obtained. The serum was tested for antibody to r30 using an ELISA assay. The results are shown in Table 3 and FIG. 3.

TABLE-US-00003 TABLE 3 Antibody Titers - Experiment 1 Reciprocal Titer Group Vaccination Test Antigen (Geometric mean) A Sham r30 157 B BCG r30 ≦125 C BCG + r30 r30 35,919 D BCG + rAd/Mtb30 r30 14,254 E BCG + Lm Vector r30 140 F BCG + rLm/Mtb30 r30 223 G BCG + rLm/Mtb30C-O r30 ≦125

[0086] The results showed that sham-immunized animals and animals immunized only with BCG had negligible antibody titers. In contrast, animals immunized first with BCG and later boosted with r30 in adjuvant or rAd/Mtb30 had high antibody titers. Animals immunized first with BCG and later boosted with rLm/Mtb30 or rLm/Mtb30C-O had low antibody titers, perhaps reflecting the fact that the rLm produces r30 in the cytoplasm of host cells where it is likely processed such that the protein is not available for presentation to B cells.

Experiment 2

Protective Efficacy of rLm/Mtb30 in a Prime-Boost Vaccination Regimen in Guinea Pigs

[0087] Specific-pathogen free 250-300 g outbred male Hartley strain guinea pigs from Charles River Breeding Laboratories, in groups of 15 (except for the sham group, which had 9 animals), were immunized intradermally as follows:

[0088] Group A: Sham

[0089] Group B: BCG Tice Parental Control (10⁵ CFU) at Week 0

[0090] Group C: BCG Tice Parental Control (10⁵ CFU) at Week 0 and 100 μg of r30 in SAF adjuvant at Week 4

[0091] Group D: BCG Tice Parental Control (10⁵ CFU) at Week 0 and 100 μg of r30 in SAF adjuvant at Weeks 4 and 8

[0092] Group E: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rAd/Mtb30 at Week 4

[0093] Group F: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rAd/Mtb30 at Weeks 4 and 8

[0094] Group G: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rLm/Mtb30 at Week 4

[0095] Group H: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rLm/Mtb30 at Weeks 4 and 8

[0096] Twenty weeks after immunization, all animals were challenged with an aerosol generated from a 7.5 ml single-cell suspension containing 6.3×10⁴ colony-forming units (CFU) of M. tuberculosis. (Prior to challenge, the challenge strain, M. tuberculosis Erdman strain (ATCC 35801), had been passaged through outbred guinea pigs to maintain virulence, cultured on 7H11 agar, subjected to gentle sonication to obtain a single cell suspension, and frozen at -70° C.). This relatively high dose aerosol dose delivered ˜75 live bacilli to the lungs of each animal. The airborne route of infection was used because this is the natural route of infection for pulmonary tuberculosis. A large dose was used so as to induce measurable clinical illness in 100% of control animals within a relatively short time frame (10 weeks). Afterwards, guinea pigs were individually housed in stainless steel cages contained within a laminar flow biohazard safety enclosure and allowed free access to standard laboratory chow and water. The animals were observed for illness and weighed weekly for 10 weeks and then euthanized. The right lung and spleen of each animal was removed and cultured for CFU of M. tuberculosis on Middlebrook 7H11 agar for two weeks at 37° C., 5% CO₂-95% air atmosphere. The results were as follows:

A. Deaths

[0097] Four of the 9 sham-immunized animals died before the end of the experiment between weeks four and nine after challenge. Two of 15 animals in the group immunized first with BCG and then twice boosted with protein in adjuvant died in the last week of the experiment. All of the animals in the remaining groups survived until the end of the experiment.

B. Net Weight Gain (Loss) after Challenge

[0098] Animals in the sham-immunized group lost weight over the course of the experiment, with a mean net weight loss by the end of the experiment of 173 grams for the surviving animals (FIG. 4). Animals in the remaining groups maintained their weights but did not gain an appreciable amount of weight.

C. Organ Burden

[0099] The results of the assay for CFU in the lungs and spleens are shown in Table 4 and FIG. 5.

TABLE-US-00004 TABLE 4 CFU in Lungs and Spleens - Experiment 2 High Dose Challenge; 20 week immunization-Challenge Interval Lung Spleen (Mean Log (Mean Log Group Strain CFU ± SE) CFU ± SE) A Sham 7.28 ± .22 5.59 ± 0.41 B BCG 6.13 ± .20 4.13 ± 0.31 C BCG + r30 x1 6.09 ± .22 4.39 ± 0.37 D BCG + r30 x2 6.31 ± .13 4.52 ± 0.36 E BCG + rAd/Mtb30 x 1 5.93 ± .22 4.34 ± 0.28 F BCG + rAd/Mtb30 x 2 6.18 ± .20 4.13 ± 0.32 G BCG + rLm/Mtb30 x 1 5.86 ± .18 3.98 ± 0.24 H BCG + rLm/Mtb30 x 2 5.97 ± .21 3.73 ± 0.41

[0100] These results showed that animals immunized with BCG had much lower CFU in the lungs and spleens than the sham immunized animals. Animals immunized first with BCG and then boosted with r30 in adjuvant or rAd/Mtb30 once or twice had similar CFU to BCG immunized animals in this high dose challenge experiment with a 20-week immunization--challenge interval. Animals immunized first with BCG and then boosted with rLm/Mtb30 once or twice had fewer CFU in the lung and spleen than BCG immunized animals in all cases. Animals immunized with BCG and boosted once with rLm/Mtb30 had 0.3 log fewer CFU in the lungs and 0.15 log fewer CFU in the spleen than animals immunized with only BCG. Animals immunized with BCG and boosted twice with rLm/Mtb30 had 0.2 log fewer CFU in the lungs and 0.4 log fewer CFU in the spleen than animals immunized with only BCG.

[0101] Thus, in an experiment in which animals were challenged with a relatively high dose of aerosolized M. tuberculosis, such that nearly half of the sham-immunized animals died before the conclusion of the experiment, boosting BCG-immunized animals with rLm/Mtb30 improved protection against M. tuberculosis aerosol challenge.

Experiment 3

Immunogenicity of rLm/Mtb30 in the Mouse Model

[0102] Specific-pathogen free 6-8 week male C57BL/6 mice from Charles River Breeding Laboratories, in groups of 4, were immunized intradermally as follows:

[0103] Group A: Sham

[0104] Group B: BCG Tice Parental Control (10⁶ CFU) at Week 0

[0105] Group C: BCG Tice Parental Control (10⁶ CFU) at Week 0 and 100 μg of r30 in SAF adjuvant at Weeks 3 and 6

[0106] Group D: BCG Tice Parental Control (10⁶ CFU) at Week 0 and rLm/30(01) (rLmΔactA/LLO-Mtb30) at Weeks 3 and 6

[0107] Group E: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rLm/30(03) (rLmΔactAΔinlB/ActA-Mtb30) at Weeks 3 and 6

[0108] Group F: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rLm/30(04) (rLmΔactAΔinlB/ActA-Mtb30-5L8) at Weeks 3 and 6

[0109] Group G: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rLm/30(07) (rLmΔactAΔinlBΔuvrABprfA*/ActA-Mtb30) at Weeks 3 and 6

[0110] Group H: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rLm/Mtb30(08) (rLmΔactAΔinlBΔuvrABprfA*/ActA-Mtb30-5L8) at Weeks 3 and 6

[0111] Group I: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rLm/Mtb30(11) (rLmΔactAΔinlB/Mtb30) at Weeks 3 and 6

[0112] Group J: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rLm/Mtb30(12) (rLmΔactAΔinlB ΔuvrABprfA*/Mtb30) at Weeks 3 and 6

[0113] Group K: BCG Tice Parental Control (105 CFU) at Week 0 and rAd/Mtb30 at Weeks 3 and 6

[0114] One week after the last immunization (Week 7), animals were anesthetized with Ketamine/Xylazine, bled and euthanized. Serum was isolated and used to assay for levels of antibodies specific to r30 (see below). In addition, a single cell suspension of splenocytes was prepared for assay of lymphocyte proliferation and intracellular interferon-gamma in response to r30. Red cells were lysed with 1× PharmLyse (BD Biosciences).

Antibody Assay

[0115] Serum was analyzed for IgG level by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well high-binding capacity plates (Corning, N.Y.) were coated or not coated with 0.1 ml of the r30 protein diluted in carbonate/bi-carbonate buffer (50 mM NaHCO₃, 50 mM Na₂CO₃) to a final concentration of 10 μg/ml. Excess antigen was removed by washing three times with PBS. Sera at a starting dilution of 1:12.5 were diluted further through a two-fold series with PBS. The diluted sera were incubated with r30 coated on 96-well plates at ambient temperature for 3 h. The plates were subsequently incubated for 90 min at ambient temperature with alkaline phosphatase-conjugated goat anti-mouse IgA (Sigma, St. Louis, Mo.) at a dilution of 1:1000. The plates then were washed three times with PBS and 0.05% Tween-20. One hundred μl of p-nitrophenylphosphate substrate in diethanolamine buffer (Phosphatase Substrate kit, BioRad, Hercules, Calif.) was added to each well. The yellow color that developed was read at 414 nm for absorbance using a multiscan microplate reader (TiterTek, Huntsville, Ala.). The endpoint antibody titer was calculated as the reciprocal of the highest serum dilution that was 2 fold or above optical density units in r30 coated wells versus non-coated control wells.

Splenic Lymphocyte Proliferation and Intracellular Cytokine Staining

[0116] Splenocytes were incubated with or without r30 antigen and tested for lymphocyte proliferation and intracellular interferon gamma (IFNγ) expression. For the lymphocyte proliferation assay, splenocytes were allowed to proliferate for 48 hours. The amount of lymphocyte proliferation was detected by adding radioactive ³H (tritiated) thymidine for 2 hours, which was incorporated into the newly synthesized DNA of the dividing cells. The amount of radioactivity incorporated into DNA was measured in a scintillation counter and is proportional to the number of proliferating cells, which in turn is a function of the number of lymphocytes that were stimulated by a given antigen to proliferate.

[0117] To assay intracellular expression of IFNγ, splenocytes were incubated with or without r30 antigen in the presence of interleukin 2 (IL-2) for 24 hours. Intracellular cytokine staining was performed at day 6 after the last vaccination as described previously (see, e.g. Lee, et al. (2006). "Identification, recombinant expression, immunolocalization in macrophages, and T-cell responsiveness of the major extracellular proteins of Francisella tularensis." Infect Immun 74(7): 4002-13; Jia, et al. (2009). "Recombinant attenuated Listeria monocytogenes vaccine expressing Francisella tularensis Ig1C induces protection in mice against aerosolized Type A F. tularensis." Vaccine 27(8): 1216-29), using antibodies purchased from BD Biosciences Pharmingen. Briefly, after a 24 h incubation, Golgi-Plug (BD Pharmingen) was added and cells were incubated for an additional 11 h at 37° C. in a 5% CO₂ atmosphere. Cells were pelleted at 250×g for 5 min and resuspended in staining buffer (BD Pharmingen) containing Fc-Block (BD Pharmingen). After incubation for 15 min, cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-CD4 or PE-Cy5-labeled anti-CD8 antibody at a 1:100 dilution for 30 min, washed twice in staining buffer, fixed with Cytofix solution for 20 min, and washed twice with Perm/Wash solution. Cells were then stained for intracellular interferon gamma (IFNγ) with PE-labeled rat anti-mouse IFNγ or a PE-labeled isotypic control immunoglobulin G at a dilution of 1:100. All the incubations were performed on ice in the dark. Stained cells were washed, resuspended in staining buffer, and analyzed on a FACSCalibur flow cytometer using CellQuest software.

Results of Experiment 3 (Mouse Expt. TB01)

A. Lymphocyte Proliferation

[0118] As shown in FIG. 6A, mice primed with BCG and boosted with rLm/Mtb30 induced higher lymphocyte proliferative response to stimulation by r30 than sham-immunized mice and mice immunized with BCG alone. Among seven rLm/Mtb30 vaccines tested, vaccines derived from the rLmΔactAΔinlBΔuvrABprfA* vector (rLm/Mtb30(12), rLm/Mtb30(07) and rLm/Mtb30(08)) induced stronger immune responses than the corresponding vaccines derived from the rLmΔactAΔinlB vector (rLm/Mtb30(11), rLm/Mtb30(03) and rLm/Mtb30(04)). Mice primed with BCG and boosted with r30 or rAd30 also induced strong lymphocyte proliferation to r30.

B. Serum Antibody Level

[0119] As shown in FIG. 6 B, mice primed with BCG and boosted with r30+ SAF had significantly higher antibody levels than sham-immunized mice and mice primed with BCG and boosted with rLm/Mtb30.

C. Intracellular Expression of IFNγ

[0120] Consistent with the lymphocyte proliferation assay results, mice primed with BCG and boosted with rLm/Mtb30 vaccines derived from rLmΔactAΔinlBΔuvrABprfA* vector (rLm/Mtb30(12), rLm/Mtb30(07) and rLm/Mtb30(08)) had stronger CD4+ mediated immune responses than the corresponding vaccines derived from the rLmΔactAΔinlB vector (rLm/Mtb30(11), rLm/Mtb30(03) and rLm/Mtb30(04)), although the difference did not reach statistical significance (FIG. 6C). There was no significant difference in CD8+ mediated immune responses among mice immunized with BCG alone or primed with BCG and boosted with either rLm/Mtb30 or rAd30 vaccines (FIG. 6D).

Experiment 4

Protective Efficacy of Recombinant Listeria Monocytogenes Vaccines Secreting the M. Tuberculosis 30 kDa Major Secretory Protein (Antigen 85B) in the Mouse Model of Pulmonary Tuberculosis

[0121] Specific-pathogen free 6-8 week male C57BL/6 mice from Charles River Breeding Laboratories, in groups of 8, were immunized intradermally as follows:

[0122] Group A: Sham

[0123] Group B: BCG Tice Parental Control (10⁶ CFU) at Week 0

[0124] Group C: BCG Tice Parental Control (10⁶ CFU) at Week 0 and 100 μg of r30 in SAF adjuvant at Weeks 3 and 6

[0125] Group D: BCG Tice Parental Control (10⁶ CFU) at Week 0 and LmΔactA (Vector control) at Weeks 3 and 6

[0126] Group E: BCG Tice Parental Control (10⁶ CFU) at Week 0 and rLm/30(01) (rLmΔactA/LLO-Mtb30) at Weeks 3 and 6

[0127] Group F: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rLm/Mtb30(12) (rLmΔactAΔinlB ΔuvrABprfA*/Mtb30) at Weeks 3 and 6

[0128] Group G: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rLm/30(07) (rLmΔactAΔinlBΔuvrABprfA*/ActA-Mtb30) at Weeks 3 and 6

[0129] Group H: BCG Tice Parental Control (10⁵ CFU) at Week 0 and rAd/Mtb30 at Weeks 3 and 6

[0130] At Week 12, all animals were challenged with an aerosol generated from a 7.5 ml single-cell suspension containing 6.3×10⁴ colony-forming units (CFU) of M. tuberculosis. (Prior to challenge, the challenge strain, M. tuberculosis Erdman strain (ATCC 35801), had been passaged through outbred guinea pigs to maintain virulence, cultured on 7H11 agar, subjected to gentle sonication to obtain a single cell suspension, and frozen at -70° C.). This aerosol dose delivered ˜100 live bacilli to the lungs of each animal. The airborne route of infection was used because this is the natural route of infection for pulmonary tuberculosis.

[0131] At 6, 10, and 15 weeks after challenge, animals were euthanized. The lung and spleen of each animal was removed and cultured for CFU of M. tuberculosis on Middlebrook 7H11 agar for two weeks at 37° C., 5% CO₂-95% air atmosphere. The results are shown in FIG. 7.

[0132] These results show that animals immunized with BCG had lower CFU in the spleen and somewhat lower CFU in the lungs than the sham immunized animals. Animals immunized first with BCG and then boosted twice with r30 in adjuvant had slightly lower CFU in the spleen than BCG and significantly lower CFU in the lungs than BCG at all time points. Animals immunized first with BCG and then boosted twice with rAd/Mtb30 had lower CFU than BCG in the spleen only at the 6 week time point; at 10 and 15 weeks, these animals had similar CFU in the spleen. In the lung, these mice had lower CFU than BCG at all time points. Animals immunized first with BCG and then boosted twice with the Listeria vector control had CFU counts similar to BCG in the spleen at the 6 and 10 week timepoints and slightly greater but not significantly greater CFU counts in the spleen at 15 weeks. In the lungs, CFU counts were lower than BCG at 6 and 10 weeks, but similar to BCG at 15 weeks.

[0133] Results for the listeria vectored vaccines varied somewhat between early and late timepoints. At 6 weeks after challenge, rLm30(01) had significantly fewer CFU counts than BCG in both the spleen and lung. rLm30(12) and rLm30(07) did not have fewer CFU counts than BCG in the spleen but did have fewer CFU counts in the lung. At 10 weeks, all Listeria vectored vaccines were better than BCG in both the lungs and spleen. rLm30(12) was comparable to rLm30(01) in the spleen but had lower CFU counts in the lungs. rLm30(07) gave the lowest counts of all vaccines in the spleen and CFU counts comparable to rLm30(12) in the lungs. At 15 weeks after challenge, all Listeria vectored vaccines had lower CFU counts than the vector control in the lung and spleen; they also had slightly lower CFU counts than BCG in the spleen and moderately lower CFU counts than BCG in the lungs.

Experiment 5

Mouse Immunogenicity of Recombinant Listeria Vaccines

[0134] Mice (4 per group) were sham-immunized, immunized with BCG at Week 0, or primed with BCG at Week 0 and then boosted twice at Week 3 and 6 with a) the recombinant 30 kD protein in adjuvant; b) the Lm ΔactA vector, or c) one of five different rLm vaccines expressing the 30 kD protein --rLm30(01), rLm30(03), rLm30(07), rLm30(11), rLm30(12). At Week 10, the mice were euthanized, the spleen removed, a single cell suspension of lymphocytes prepared and used for studies as follows.

A. Interferon-γ Production in Response to M. Tuberculosis Antigens

[0135] The splenocytes were stimulated with medium alone, the recombinant 30 kD protein (r30) or M. tuberculosis Purified Protein Derivative (PPD) for three days. The splenocyte supernatant fluid was collected and assayed for the level of IFNγ by ELISA. The results are shown in FIG. 8. Data are the mean±S.E.

[0136] In the absence of antigen, essentially no IFNγ was secreted by the splenocytes. In the presence of the purified 30 kDa protein or PPD, splenocytes from sham-immunized mice, mice immunized with only BCG, and mice immunized with BCG and boosted with the Listeria vector control secreted little or no IFNγ. In contrast, splenocytes from mice primed with BCG and boosted with the M. tuberculosis 30 kDA protein or the recombinant Listeria vaccines expressing the M. tuberculosis 30 kDa protein generally produced large amounts of IFNγ.

B. Intracellular Cytokine Secretion

[0137] The splenocytes were stimulated with the mature recombinant 30 kD protein (r30) or a pool of three peptides of r30 (30p) for a total of 6 hours (the last 4 hours in the presence of Golgi-Plug), and stained first for CD4 and CD8 and subsequently for the cytokines IFNγ, IL-2, and TNFα, and analyzed by multi-parameter flow cytometry. The total frequency and mean fluorescence intensity (MFI) of each cytokine-secreting CD4+ T-cell was determined and the integrated MFI (iMFI) was calculated. The results are shown in FIG. 9. Data are the mean±S.E. after background subtraction of the identically gated population of cells from the same sample stimulated without antigen.

[0138] In the presence of the purified 30 kDa protein (r30) or the 30 kDa protein peptide pool (30p), splenocytes from sham-immunized mice, mice immunized only with BCG, and mice immunized with BCG and boosted with the Listeria vector control displayed low or negligible iMFI. In contrast, splenocytes from mice primed with BCG and boosted with the M. tuberculosis 30 kDA protein or the recombinant Listeria vaccines expressing the M. tuberculosis 30 kDa protein generally displayed moderate to large iMFI. Splenocytes from mice immunized with BCG and boosted with rLm30 (01), rLm30 (03), rLm30 (07), and rLm30 (12) had particularly large iMFI for each of the cytokines

[0139] This concludes the description of embodiments of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.

[0140] Those of skill in this art understand that aspects of this technology can be adapted to form a wide variety of embodiments of the invention. All literature and other references are incorporated herein by reference (e.g. WO 01/46473 and WO 02/094848). Literature describing methods and materials that relate to embodiments of the invention includes Brockstedt et al., (2004) Proc Natl Acad Sci USA 101(38): 13832-7. PMID 15365184; Brockstedt et al., (2005) Nat Med 11(8): 853-60. PMID 16041382; Colditz et al., (1994)." JAMA 271(9): 698-702. PMID 8309034; Fine, P. E. (1989) Rev Infect Dis 11 Suppl 2: S353-9. PMID 2652252; Harth et al., (1997) Infect Immun 65(6): 2321-8. PMID 9169770; Horwitz et al., (1995) Proc Natl Acad Sci USA 92(5): 1530-4. PMID 7878014; Horwitz et al., (2000) Proc Natl Acad Sci USA 97(25): 13853-8. PMID 11095745; Horwitz et al., (2005) Infect Immun 73(8): 4676-83. PMID 16040980; Jia et al., (2009) Vaccine 27(8): 1216-29. PMID 19126421; Lauer et al., (2008) Infect Immun 76(8): 3742-53. PMID 18541651; Lee et al., (2006) Infect Immun 74(7): 4002-13. PMID 16790773; McShane et al., (2004) Nat Med 10(11): 1240-4. PMID 15502839; Santosuosso et al., (2006) Infect Immun 74(8): 4634-43. PMID 16861651; Vordermeier et al., (2009) Infect Immun 77(8): 3364-73. PMID 19487476; Williams et al., (2005) Infect Immun 73(6): 3814-6. PMID 15908420; Xing et al., (2009) PLoS One 4(6): e5856. PMID 19516906; and Yan et al., (2008) Infect Immun 76(8): 3439-50. PMID 18474644

[0141] It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto. The above specification, examples and data provide a complete description of the manufacture and use of the apparatus and method of the invention. Since many embodiments of the invention can be made without departing from the scope of the invention, the invention resides in the claims hereinafter appended and the equivalents thereto.

M. Tuberculosis Polypeptide and Polynucleotide Sequences

TABLE-US-00005

[0142] TABLE 5 Sequences of M. tuberculosis Extracellular Protein Genes Rv numbers/ Protein (kDa), Initial Illustrative # (Alternate Names) sequences Lab/Author References 1 12 (fragment of 16 Rv2031c B.-Y. Lee et al. Infect. Immun. kDa Membrane FDTRL . . . (Sequenced the 60: 2066, 1992 Protein, alpha 16 kDa Membrane J. Bact. 174: 1352, crystallian family) Protein) 1992 MMP Verbon et al. (Sequenced the 16 kDa Membrane Protein gene) 2 14 (MPT53) Rv2878c Horwitz Lab (Tuberculist: 18 kDa) ADERL . . . Gen Bank 3 16 (MPT63/Mpb63) Rv1926c Horwitz Lab Infect. Immun. Harth et al. 65: 2321, 1997 Manca et al. Infect. Immun. 65: 16, 1997 4 23 (SOD/SodA) Rv3846 Zhang et al. Molec. Harth & Micro. 5: 381, 1991 Horwitz J. Biol. Chem. 274: 4281, 1999 5 23.5 (MPT64) Rv1980c Horwitz Lab Infect. Immun. Oettinger & 62: 2058, 1994 Anderson Infect. Immun. Harth et al. 65: 2321, 1997 6 24 (MPT51) Rv3803c Horwitz Lab Scand. J. Immunol. APYEN . . . (O'Hara et 41: 433, 1995 al. published identical homolog in M. bovis) 7 30 (Antigen Rv1886c Horwitz Lab J. DNA Seq. & 85B/FbpB) DeWit et al. Map. 4: 267, 1994 Harth et al. Infect. Immun. 65: 2321, 1997 8 32A (Antigen Rv3804c Horwitz Lab Infect. Immun. 85A/FbpA) Borremans 57: 3123, 1989 Harth et al. Infect. Immun. 65: 2321, 1997 9 32B (Antigen Rv0983 Content et al. Infect. Immun. 85C/pepD) 59: 3205, 1991 10 45 (MPT32/apa Rv1860 Laqueyerie et Infect. Immun. gene, modD) DPEPA . . . al. 63: 4003, 1995 11 58 (Glutamine Rv2220 Horwitz Lab J. Biol. Chem. Synthetase/GSI/ Harth & 272: 22728, 1997 glnA) Horwitz 12 71 (hsp/DnaK/ Rv0350 R. Young & D. Hsp70) ARAVG . . . Young Horwitz Lab 13 10.4 (EsxH/ Rv0288 ESAT-6 homolog) 14 14 (alpha Rv2031c crystalline homolog/heat shock protein HSPX) (Tuberculist: 16.2 kDa) 15 47 (isocitrate Rv0467 Mckinney J. D., et lyase/ICL/aceA) al, Nature, 406: 735, 2000 16 7.6 (Hypothetical Rv2660c Aaggard C, et al, protein) Nature Medicine, 17: 189, 2011 17 80 (glcB) Rv1837c Horwitz Lab TDRVSVGN . . . 18 110 (can) Rv1475c Horwitz Lab (Tuberculist: 102 kDa) NSKSVNSFGA . . . 19 9.9 (ESAT-6/esxA) Rv3875 Note 1: Proteins 14, 15, and 16 are latency-associated proteins Note 2: All sequences now available on-line as a result of the M. tuberculosis genome project. See: Cole et al. Nature. Nature 393: 537-544, 1998. Note 3: See also U.S. Pat. Nos. 7,622,107; 7,300,660; 7,002,002; 6,924,118; 6,818,223; 6,761,894; 6,752,993; 6,599,510; 6,471,967; 6,054,133; 6,013,660; and 5,108,745; and U.S. patent application Nos. 20110129492; 20100284963; 20100183547; and 20100092518, the contents of which are incorporated by reference. M. tuberculosis Protein and coding sequences noted in Table 5 above are provided below. Note: All sequences can be found for example by an online search using the terms genolist.pasteur.fr/TubercuList/.

1. 12 kDa (fragment of 16 kDa membrane protein, alpha crystalline homolog, heat shock protein HSPX, and number 14 in the protein list), 110 aa

TABLE-US-00006 (SEQ ID NO: 5) FDTRLMRLEDEMKEGRYEVRAELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVS L PVGADEDDIKATYDKGILTVSVAVSEGKPTEKHIQIRSTN 12 kDa fragment of 16 kDa membrane protein, MMP: 333 bp (SEQ ID NO: 23) ttcgacacccggttgatgcggctggaagacgagatgaaagaggggcgctacgaggtacgcgcggagctt cccggggtcgaccccgacaaggacgtcgacattatggtccgcgatggtcagctgaccatcaaggccgag cgcaccgagcagaaggacttcgacggtcgctcggaattcgcgtacggttccttcgttcgcacggtgtcg ctgccggtaggtgctgacgaggacgacattaaggccacctacgacaagggcattcttactgtgtcggtg gcggtttcggaagggaagccaaccgaaaagcacattcagatccggtccaccaactga

2. M. tuberculosis H37Rv|Rv2878c|Mpt53: 173 aa--Soluble Secreted Antigen Mpt53 Precursor. First Identified Protein Sequence Starts from ADERL . . . :

TABLE-US-00007 (SEQ ID NO: 6) MSLRLVSPIKAFADGIVAVAIAVVLMFGLANTPRAVAADERLQFTATTLSGAPFDGASLQGKPAVLWFW TPWCPFCNAEAPSLSQVAAANPAVTFVGIATRADVGAMQSFVSKYNLNFTNLNDADGVIWARYNVPWQP AFVFYRADGTSTFVNNPTAAMSQDELSGRVAALTS M. tuberculosis H37Rv|Rv2878c|mpt53: 522 bp - SOLUBLE SECRETED ANTIGEN MPT53 PRECURSOR (SEQ ID NO: 24) atgagtcttcgcctggtgtccccgatcaaggcgtttgcggacggcattgtggccgttgctatcgcggtt gtcctgatgttcggtctggccaatacaccgcgagcggtggcagccgatgaacgtctgcagttcaccgca accacgctcagcggtgctcccttcgatggcgcaagcctgcaaggcaagccggcggtgttgtggttctgg acgccgtggtgcccgttctgcaacgcagaagcccccagcctcagccaggtagcggccgctaatccggcg gtcaccttcgtcggaatcgccacccgcgccgacgtcggggcgatgcagagctttgtctcgaagtacaac ctgaatttcaccaacctcaatgacgccgatggtgtgatctgggcccgctacaacgtgccttggcaaccg gcatttgtgttctatcgcgcggacggcacatcgacgttcgtcaacaaccccaccgcggccatgtctcag gacgagctgtccggccgggtggctgcgctgacgtcctgacccggtgaacgaggcgctgatcggtttggc gttcgccgccgggttggtggctgcgctgaacccatgcgggtttgccatgttgccggcctacctgctgtt ggtggtgtatgggcaggattcggcgggccggacggggccgcttagcgcagtgggccgagcggcagccgc cacggtcgggatggcgctgggcttcttgacgg

3. M. tuberculosis H37Rv|Rv1926c|Mpt63: 159 aa--Immunogenic Protein Mpt63 (Antigen MPT63/MPB63) (16 kDa Immunoprotective Extracellular Protein)

TABLE-US-00008 (SEQ ID NO: 7) MKLTTMIKTAVAVVAMAAIATFAAPVALAAYPITGKLGSELTMTDTVGQVVLGWKVSDLKSSTAVIPGY PVAGQVWEATATVNAIRGSVTPAVSQFNARTADGINYRVLWQAAGPDTISGATIPQGEQSTGKIYFDVT GPSPTIVAMNNGMEDLLIWEP M. tuberculosis H37Rv|Rv1926c|mpt63: 480 bp - IMMUNOGENIC PROTEIN MPT63 (ANTIGEN MPT63/MPB63) (16 kDa IMMUNOPROTECTIVE EXTRACELLULAR PROTEIN) (SEQ ID NO: 25) atgaagctcaccacaatgatcaagacggcagtagcggtcgtggccatggcggccatcgcg acctttgcggcaccggtcgcgttggctgcctatcccatcaccggaaaacttggcagtgag ctaacgatgaccgacaccgttggccaagtcgtgctcggctggaaggtcagtgatctcaaa tccagcacggcagtcatccccggctatccggtggccggccaggtctgggaggccactgcc acggtcaatgcgattcgcggcagcgtcacgcccgcggtctcgcagttcaatgcccgcacc gccgacggcatcaactaccgggtgctgtggcaagccgcgggccccgacaccattagcgga gccactatcccccaaggcgaacaatcgaccggcaaaatctacttcgatgtcaccggccca tcgccaaccatcgtcgcgatgaacaacggcatggaggatctgctgatttgggagccgtag

4. M. tuberculosis H37Rv|Rv3846|SodA: 207 aa--Superoxide Dismutase (FE) SodA

TABLE-US-00009 (SEQ ID NO: 8) VAEYTLPDLDWDYGALEPHISGQINELHHSKHHATYVKGANDAVAKLEEARAKEDHSAILLNEKNLAFN LAGHVNHTIWWKNLSPNGGDKPTGELAAAIADAFGSFDKFRAQFHAAATTVQGSGWAALGWDTLGNKLL IFQVYDHQTNFPLGIVPLLLLDMWEHAFYLQYKNVKVDFAKAFWNVVNWADVQSRYAAATSQTKGLIFG M. tuberculosis H37Rv|Rv3846|sodA: 624 bp - SUPEROXIDE DISMUTASE(FE) SODA (SEQ ID NO: 26) gtggccgaatacaccttgccagacctggactgggactacggagcactggaaccgcacatc tcgggtcagatcaacgagcttcaccacagcaagcaccacgccacctacgtaaagggcgcc aatgacgccgtcgccaaactcgaagaggcgcgcgccaaggaagatcactcagcgatcttg ctgaacgaaaagaatctagctttcaacctcgccggccacgtcaatcacaccatctggtgg aagaacctgtcgcctaacggtggtgacaagcccaccggcgaactcgccgcagccatcgcc gacgcgttcggttcgttcgacaagttccgtgcgcagttccacgcggccgctaccaccgtg caggggtcgggctgggcggcactgggctgggacacactcggcaacaagctgctgatattc caggtttacgaccaccagacgaacttcccgctaggcattgttccgctgctgctgctcgac atgtgggaacacgccttctacctgcagtacaagaacgtcaaagtcgactttgccaaggcg ttttggaacgtcgtgaactgggccgatgtgcagtcacggtatgcggccgcgacctcgcag accaaggggttgatattcggctga

5. M. tuberculosis H37Rv|Rv1980c|Mpt64: 228 aa--Immunogenic Protein Mpt64 (Antigen MPT64/MPB64)

TABLE-US-00010 (SEQ ID NO: 9) VRIKIFMLVTAVVLLCCSGVATAAPKTYCEELKGTDTGQACQIQMSDPAYNINISLPSYYPDQKSLENY IAQTRDKFLSAATSSTPREAPYELNITSATYQSAIPPRGTQAVVLKVYQNAGGTHPTTTYKAFDWDQAY RKPITYDTLWQADTDPLPVVFPIVQGELSKQTGQQVSIAPNAGLDPVNYQNFAVTNDGVIFFFNPGELL PEAAGPTQVLVPRSAIDSMLA M. tuberculosis H37Rv|Rv1980c|mpt64: 687 bp - IMMUNOGENIC PROTEIN MPT64 (ANTIGEN MPT64/MPB64) (SEQ ID NO: 27) gtgcgcatcaagatcttcatgctggtcacggctgtcgttttgctctgttgttcgggtgtggccacggcc gcgcccaagacctactgcgaggagttgaaaggcaccgataccggccaggcgtgccagattcaaatgtcc gacccggcctacaacatcaacatcagcctgcccagttactaccccgaccagaagtcgctggaaaattac atcgcccagacgcgcgacaagttcctcagcgcggccacatcgtccactccacgcgaagccccctacgaa ttgaatatcacctcggccacataccagtccgcgataccgccgcgtggtacgcaggccgtggtgctcaag gtctaccagaacgccggcggcacgcacccaacgaccacgtacaaggccttcgattgggaccaggcctat cgcaagccaatcacctatgacacgctgtggcaggctgacaccgatccgctgccagtcgtcttccccatt gtgcaaggtgaactgagcaagcagaccggacaacaggtatcgatagcgccgaatgccggcttggacccg gtgaattatcagaacttcgcagtcacgaacgacggggtgattttcttcttcaacccgggggagttgctg cccgaagcagccggcccaacccaggtattggtcccacgttccgcgatcgactcgatgctggcctag

6. M. tuberculosis H37Rv|Rv3803c|FbpD: 299 aa--Secreted MPT51/MPB51 Antigen Protein FbpD (MPT51/MPB51 Antigen 85 Complex C) (AG58C) (Mycolyl Transferase 85C) (Fibronectin-Binding Protein C) (85C)

TABLE-US-00011 (SEQ ID NO: 10) MKGRSALLRALWIAALSFGLGGVAVAAEPTAKAAPYENLMVPSPSMGRDIPVAFLAGGPHAVYLLDAFN AGPDVSNWVTAGNAMNTLAGKGISVVAPAGGAYSMYTNWEQDGSKQWDTFLSAELPDWLAANRGLAPGG HAAVGAAQGGYGAMALAAFHPDRFGFAGSMSGFLYPSNTTTNGAIAAGMQQFGGVDTNGMWGAPQLGRW KWHDPWVHASLLAQNNTRVWVWSPTNPGASDPAAMIGQAAEAMGNSRMFYNQYRSVGGHNGHFDFPASG DNGWGSWAPQLGAMSGDIVGAIR M. tuberculosis H37Rv|Rv3803c|fbpD: 900 bp - SECRETED MPT51/MPB51 ANTIGEN PROTEIN FBPD (MPT51/MPB51 ANTIGEN 85 COMPLEX C) (AG58C) (MYCOLYL TRANSFERASE 85C) (FIBRONECTIN-BINDING PROTEIN C) (85C) (SEQ ID NO: 28) atgaagggtcggtcggcgctgctgcgggcgctctggattgccgcactgtcattcgggttg ggcggtgtcgcggtagccgcggaacccaccgccaaggccgccccatacgagaacctgatg gtgccgtcgccctcgatgggccgggacatcccggtggccttcctagccggtgggccgcac gcggtgtatctgctggacgccttcaacgccggcccggatgtcagtaactgggtcaccgcg ggtaacgcgatgaacacgttggcgggcaaggggatttcggtggtggcaccggccggtggt gcgtacagcatgtacaccaactgggagcaggatggcagcaagcagtgggacaccttcttg tccgctgagctgcccgactggctggccgctaaccggggcttggcccccggtggccatgcg gccgttggcgccgctcagggcggttacggggcgatggcgctggcggccttccaccccgac cgcttcggcttcgctggctcgatgtcgggctttttgtacccgtcgaacaccaccaccaac ggtgcgatcgcggcgggcatgcagcaattcggcggtgtggacaccaacggaatgtgggga gcaccacagctgggtcggtggaagtggcacgacccgtgggtgcatgccagcctgctggcg caaaacaacacccgggtgtgggtgtggagcccgaccaacccgggagccagcgatcccgcc gccatgatcggccaagccgccgaggcgatgggtaacagccgcatgttctacaaccagtat cgcagcgtcggcgggcacaacggacacttcgacttcccagccagcggtgacaacggctgg ggctcgtgggcgccccagctgggcgctatgtcgggcgatatcgtcggtgcgatccgctaa

7. M. tuberculosis H37Rv|Rv1886c|FbpB: 325 aa--Secreted Antigen 85-B FbpB (85B) (Antigen 85 Complex B) (Mycolyl Transferase 85B) (Fibronectin-Binding Protein B) (Extracellular Alpha-Antigen)

TABLE-US-00012 (SEQ ID NO: 4) MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSG GNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWE TFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLA MGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSS NLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG M. tuberculosis H37Rv|Rv1886c|fbpB: 978 bp - SECRETED ANTIGEN 85-B FBPB (85B) (ANTIGEN 85 COMPLEX B) (MYCOLYL TRANSFERASE 85B) (FIBRONECTIN-BINDING PROTEIN B) (EXTRACELLULAR ALPHA-ANTIGEN) (SEQ ID NO: 29) atgacagacgtgagccgaaagattcgagcttggggacgccgattgatgatcggcacggcagcggctgta gtccttccgggcctggtggggcttgccggcggagcggcaaccgcgggcgcgttctcccggccggggctg ccggtcgagtacctgcaggtgccgtcgccgtcgatgggccgcgacatcaaggttcagttccagagcggt gggaacaactcacctgcggtttatctgctcgacggcctgcgcgcccaagacgactacaacggctgggat atcaacaccccggcgttcgagtggtactaccagtcgggactgtcgatagtcatgccggtcggcgggcag tccagcttctacagcgactggtacagcccggcctgcggtaaggctggctgccagacttacaagtgggaa accttcctgaccagcgagctgccgcaatggttgtccgccaacagggccgtgaagcccaccggcagcgct gcaatcggcttgtcgatggccggctcgtcggcaatgatcttggccgcctaccacccccagcagttcatc tacgccggctcgctgtcggccctgctggacccctctcaggggatggggcctagcctgatcggcctcgcg atgggtgacgccggcggttacaaggccgcagacatgtggggtccctcgagtgacccggcatgggagcgc aacgaccctacgcagcagatccccaagctggtcgcaaacaacacccggctatgggtttattgcgggaac ggcaccccgaacgagttgggcggtgccaacatacccgccgagttcttggagaacttcgttcgtagcagc aacctgaagttccaggatgcgtacaacgccgcgggcgggcacaacgccgtgttcaacttcccgcccaac ggcacgcacagctgggagtactggggcgctcagctcaacgccatgaagggtgacctgcagagttcgtta ggcgccggctga

8. M. tuberculosis H37Rv|Rv3804c|FbpA: 338 aa--Secreted Antigen 85-A FbpA (Mycolyl Transferase 85A) (Fibronectin-Binding Protein A) (Antigen 85 Complex A)

TABLE-US-00013 (SEQ ID NO: 11) MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQF QSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTY KWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLI GLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFV RTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA M. tuberculosis H37Rv|Rv3804c|fbpA: 1017 bp - SECRETED ANTIGEN 85-A FBPA (MYCOLYL TRANSFERASE 85A) (FIBRONECTIN-BINDING PROTEIN A) (ANTIGEN 85 COMPLEX A) (SEQ ID NO: 30) atgcagcttgttgacagggttcgtggcgccgtcacgggtatgtcgcgtcgactcgtggtc ggggccgtcggcgcggccctagtgtcgggtctggtcggcgccgtcggtggcacggcgacc gcgggggcattttcccggccgggcttgccggtggagtacctgcaggtgccgtcgccgtcg atgggccgtgacatcaaggtccaattccaaagtggtggtgccaactcgcccgccctgtac ctgctcgacggcctgcgcgcgcaggacgacttcagcggctgggacatcaacaccccggcg ttcgagtggtacgaccagtcgggcctgtcggtggtcatgccggtgggtggccagtcaagc ttctactccgactggtaccagcccgcctgcggcaaggccggttgccagacttacaagtgg gagaccttcctgaccagcgagctgccggggtggctgcaggccaacaggcacgtcaagccc accggaagcgccgtcgtcggtctttcgatggctgcttcttcggcgctgacgctggcgatc tatcacccccagcagttcgtctacgcgggagcgatgtcgggcctgttggacccctcccag gcgatgggtcccaccctgatcggcctggcgatgggtgacgctggcggctacaaggcctcc gacatgtggggcccgaaggaggacccggcgtggcagcgcaacgacccgctgttgaacgtc gggaagctgatcgccaacaacacccgcgtctgggtgtactgcggcaacggcaagccgtcg gatctgggtggcaacaacctgccggccaagttcctcgagggcttcgtgcggaccagcaac atcaagttccaagacgcctacaacgccggtggcggccacaacggcgtgttcgacttcccg gacagcggtacgcacagctgggagtactggggcgcgcagctcaacgctatgaagcccgac ctgcaacgggcactgggtgccacgcccaacaccgggcccgcgccccagggcgcctag

9. M. tuberculosis H37Rv|Rv0983|PepD: 464 aa--Probable Serine Protease PepD (SERINE PROTEINASE) (MTB32B)

TABLE-US-00014 (SEQ ID NO: 12) MAKLARVVGLVQEEQPSDMTNHPRYSPPPQQPGTPGYAQGQQQTYSQQFDWRYPPSPPPQPTQYRQPYE ALGGTRPGLIPGVIPTMTPPPGMVRQRPRAGMLAIGAVTIAVVSAGIGGAAASLVGFNRAPAGPSGGPV AASAAPSIPAANMPPGSVEQVAAKVVPSVVMLETDLGRQSEEGSGIILSAEGLILTNNHVIAAAAKPPL GSPPPKTTVTFSDGRTAPFTVVGADPTSDIAVVRVQGVSGLTPISLGSSSDLRVGQPVLAIGSPLGLEG TVTTGIVSALNRPVSTTGEAGNQNTVLDAIQTDAAINPGNSGGALVNMNAQLVGVNSAIATLGADSADA QSGSIGLGFAIPVDQAKRIADELISTGKASHASLGVQVTNDKDTLGAKIVEVVAGGAAANAGVPKGVVV TKVDDRPINSADALVAAVRSKAPGATVALTFQDPSGGSRTVQVTLGKAEQ M. tuberculosis H37Rv|Rv0983|pepD: 1395 bp - PROBABLE SERINE PROTEASE PEPD (SERINE PROTEINASE) (MTB32B) (SEQ ID NO: 31) atggccaagttggcccgagtagtgggcctagtacaggaagagcaacctagcgacatgacg aatcacccacggtattcgccaccgccgcagcagccgggaaccccaggttatgctcagggg cagcagcaaacgtacagccagcagttcgactggcgttacccaccgtccccgcccccgcag ccaacccagtaccgtcaaccctacgaggcgttgggtggtacccggccgggtctgatacct ggcgtgattccgaccatgacgccccctcctgggatggttcgccaacgccctcgtgcaggc atgttggccatcggcgcggtgacgatagcggtggtgtccgccggcatcggcggcgcggcc gcatccctggtcgggttcaaccgggcacccgccggccccagcggcggcccagtggctgcc agcgcggcgccaagcatccccgcagcaaacatgccgccggggtcggtcgaacaggtggcg gccaaggtggtgcccagtgtcgtcatgttggaaaccgatctgggccgccagtcggaggag ggctccggcatcattctgtctgccgaggggctgatcttgaccaacaaccacgtgatcgcg gcggccgccaagcctcccctgggcagtccgccgccgaaaacgacggtaaccttctctgac gggcggaccgcacccttcacggtggtgggggctgaccccaccagtgatatcgccgtcgtc cgtgttcagggcgtctcc

10. M. tuberculosis H37Rv|Rv1860|Apa: 325 aa--Alanine and Proline Rich Secreted Protein Apa (Fibronectin Attachment Protein) (Immunogenic protein MPT32) (Antigen MPT-32) (45-kDa Glycoprotein) (45/47 kDa Antigen)

TABLE-US-00015 (SEQ ID NO: 13) MHQVDPNLTRRKGRLAALAIAAMASASLVTVAVPATANADPEPAPPVPTTAASPPSTAAAPPAPATPVA PPPPAAANTPNAQPGDPNAAPPPADPNAPPPPVIAPNAPQPVRIDNPVGGFSFALPAGWVESDAAHFDY GSALLSKTTGDPPFPGQPPPVANDTRIVLGRLDQKLYASAEATDSKAAARLGSDMGEFYMPYPGTRINQ ETVSLDANGVSGSASYYEVKFSDPSKPNGQIWTGVIGSPAANAPDAGPPQRWFVVWLGTANNPVDKGAA KALAESIRPLVAPPPAPAPAPAEPAPAPAPAGEVAPTPTTPTPQRTLPA M. tuberculosis H37Rv|Rv1860|apa: 978 bp - ALANINE AND PROLINE RICH SECRETED PROTIEN APA (FIBRONECTIN ATTACHMENT PROTIEN) (Immunogenic protein MPT32) (Antigen MPT-32) (45-kDa glycoprotein) (45/47 kDa antigen) (SEQ ID NO: 32) atgcatcaggtggaccccaacttgacacgtcgcaagggacgattggcggcactggctatcgcggcgatg gccagcgccagcctggtgaccgttgcggtgcccgcgaccgccaacgccgatccggagccagcgcccccg gtacccacaacggccgcctcgccgccgtcgaccgctgcagcgccacccgcaccggcgacacctgttgcc cccccaccaccggccgccgccaacacgccgaatgcccagccgggcgatcccaacgcagcacctccgccg gccgacccgaacgcaccgccgccacctgtcattgccccaaacgcaccccaacctgtccggatcgacaac ccggttggaggattcagcttcgcgctgcctgctggctgggtggagtctgacgccgcccacttcgactac ggttcagcactcctcagcaaaaccaccggggacccgccatttcccggacagccgccgccggtggccaat gacacccgtatcgtgctcggccggctagaccaaaagctttacgccagcgccgaagccaccgactccaag gccgcggcccggttgggctcggacatgggtgagttctatatgccctacccgggcacccggatcaaccag gaaaccgtctcgctcgacgccaacggggtgtctggaagcgcgtcgtattacgaagtcaagttcagcgat ccgagtaagccgaacggccagatctggacgggcgtaatcggctcgcccgcggcgaacgcaccggacgcc gggccccctcagcgctggtttgtggtatggctcgggaccgccaacaacccggtggacaagggcgcggcc aaggcgctggccgaatcgatccggcctttggtcgccccgccgccggcgccggcaccggctcctgcagag cccgctccggcgccggcgccggccggggaagtcgctcctaccccgacgacaccgacaccgcagcggacc ttaccggcctga

11. M. tuberculosis H37Rv|Rv2220|GlnA1: 478 aa--Glutamine Synthetase GlnA1 (Glutamine Synthase) (GS-I)

TABLE-US-00016 (SEQ ID NO: 14) VTEKTPDDVFKLAKDEKVEYVDVRFCDLPGIMQHFTIPASAFDKSVFDDGLAFDGSSIRGFQSIHESDM LLLPDPETARIDPFRAAKTLNINFFVHDPFTLEPYSRDPRNIARKAENYLISTGIADTAYFGAEAEFYI FDSVSFDSRANGSFYEVDAISGWWNTGAATEADGSPNRGYKVRHKGGYFPVAPNDQYVDLRDKMLTNLI NSGFILEKGHHEVGSGGQAEINYQFNSLLHAADDMQLYKYIIKNTAWQNGKTVTFMPKPLFGDNGSGMH CHQSLWKDGAPLMYDETGYAGLSDTARHYIGGLLHHAPSLLAFTNPTVNSYKRLVPGYEAPINLVYSQR NRSACVRIPITGSNPKAKRLEFRSPDSSGNPYLAFSAMLMAGLDGIKNKIEPQAPVDKDLYELPPEEAA SIPQTPTQLSDVIDRLEADHEYLTEGGVFTNDLIETWISFKRENEIEPVNIRPHPYEFALYYDV M. tuberculosis H37Rv|Rv2220|glnA1: 1437 bp - GLUTAMINE SYNTHETASE GLNA1 (GLUTAMINE SYNTHASE) (GS-I) (SEQ ID NO: 33) gtgacggaaaagacgcccgacgacgtcttcaaacttgccaaggacgagaaggtcgaatat gtcgacgtccggttctgtgacctgcctggcatcatgcagcacttcacgattccggcttcg gcctttgacaagagcgtgtttgacgacggcttggcctttgacggctcgtcgattcgcggg ttccagtcgatccacgaatccgacatgttgcttcttcccgatcccgagacggcgcgcatc gacccgttccgcgcggccaagacgctgaatatcaacttctttgtgcacgacccgttcacc ctggagccgtactcccgcgacccgcgcaacatcgcccgcaaggccgagaactacctgatc agcactggcatcgccgacaccgcatacttcggcgccgaggccgagttctacattttcgat tcggtgagcttcgactcgcgcgccaacggctccttctacgaggtggacgccatctcgggg tggtggaacaccggcgcggcgaccgaggccgacggcagtcccaaccggggctacaaggtc cgccacaagggcgggtatttcccagtggcccccaacgaccaatacgtcgacctgcgcgac aagatgctgaccaacctgatcaactccggcttcatcctggagaagggccaccacgaggtg ggcagcggcggacaggccgagatcaactaccagttcaattcgctgctgcacgccgccgac gacatgcagttgtacaagtacatcatcaagaacaccgcctggcagaacggcaaaacggtc acgttcatgcccaagccgctgttcggcgacaacgggtccggcatgcactgtcatcagtcg ctgtggaaggacggggccccgctgatgtacgacgagacgggttatgccggtctgtcggac acggcccgtcattacatcggcggcctgttacaccacgcgccgtcgctgctggccttcacc aacccgacggtgaactcctacaagcggctggttcccggttacgaggccccgatcaacctg gtctatagccagcgcaaccggtcggcatgcgtgcgcatcccgatcaccggcagcaacccg gtctatagccagcgcaaccggtcggcatgcgtgcgcatcccgatcaccggcagcaacccg tcggccatgctgatggcaggcctggacggtatcaagaacaagatcgagccgcaggcgccc gtcgacaaggatctctacgagctgccgccggaagaggccgcgagtatcccgcagactccg acccagctgtcagatgtgatcgaccgtctcgaggccgaccacgaatacctcaccgaagga ggggtgttcacaaacgacctgatcgagacgtggatcagtttcaagcgcgaaaacgagatc gagccggtcaacatccggccgcatccctacgaattcgcgctgtactacgacgtttaa

12. M. tuberculosis H37Rv|Rv0350|DnaK: 625 aa--Probable Chaperone Protein DnaK (Heat Shock Protein 70) (Heat Shock 70 KDA Protein) (HSP70)

TABLE-US-00017 (SEQ ID NO: 15) MARAVGIDLGTTNSVVSVLEGGDPVVVANSEGSRTTPSIVAFARNGEVLVGQPAKNQAVTNVDRTVRSV KRHMGSDWSIEIDGKKYTAPEISARILMKLKRDAEAYLGEDITDAVITTPAYFNDAQRQATKDAGQIAG LNVLRIVNEPTAAALAYGLDKGEKEQRILVFDLGGGTFDVSLLEIGEGVVEVRATSGDNHLGGDDWDQR VVDWLVDKFKGTSGIDLTKDKMAMQRLREAAEKAKIELSSSQSTSINLPYITVDADKNPLFLDEQLTRA EFQRITQDLLDRTRKPFQSVIADTGISVSEIDHVVLVGGSTRMPAVTDLVKELTGGKEPNKGVNPDEVV AVGAALQAGVLKGEVKDVLLLDVTPLSLGIETKGGVMTRLIERNTTIPTKRSETFTTADDNQPSVQIQV YQGEREIAAHNKLLGSFELTGIPPAPRGIPQIEVTFDIDANGIVHVTAKDKGTGKENTIRIQEGSGLSK EDIDRMIKDAEAHAEEDRKRREEADVRNQAETLVYQTEKFVKEQREAEGGSKVPEDTLNKVDAAVAEAK AALGGSDISAIKSAMEKLGQESQALGQAIYEAAQAASQATGAAHPGGEPGGAHPGSADDVVDAEVVDDG REAK M. tuberculosis H37Rv|Rv0350|dnaK: 1878 bp - PROBABLE CHAPERONE PROTEIN DNAK (HEAT SHOCK PROTEIN 70) (HEAT SHOCK 70 KDA PROTEIN) (HSP70) (SEQ ID NO: 34) atggctcgtgcggtcgggatcgacctcgggaccaccaactccgtcgtcctggttctggaa ggtggcgacccggtcgtcgtcgccaactccgagggctccaggaccaccccgtcaattgtc gcgttcgcccgcaacggtgaggtgctggtcggccagcccgccaagaaccaggcagtgacc aacgtcgatcgcaccgtgcgctcggtcaagcgacacatgggcagcgactggtccatagag attgacggcaagaaatacaccgcgccggagatcagcgcccgcattctgatgaagctgaag cgcgacgccgaggcctacctcggtgaggacattaccgacgcggttatcacgacgcccgcc tacttcaatgacgcccagcgtcaggccaccaaggacgccggccagatcgccggcctcaac gtgctgcggatcgtcaacgagccgaccgcggccgcgctggcctacggcctcgacaagggc gagaaggagcagcgaatcctggtcttcgacttgggtggtggcactttcgacgtttccctg ctggagatcggcgagggtgtggttgaggtccgtgccacttcgggtgacaaccacctcggc ggcgacgactgggaccagcgggtcgtcgattggctggtggacaagttcaagggcaccagc ggcatcgatctgaccaaggacaagatggcgatgcagcggctgcgggaagccgccgagaag gcaaagatcgagctgagttcgagtcagtccacctcgatcaacctgccctacatcaccgtc gacgccgacaagaacccgttgttcttagacgagcagctgacccgcgcggagttccaacgg atcactcaggacctgctggaccgcactcgcaagccgttccagtcggtgatcgctgacacc ggcatttcggtgtcggagatcgatcacgttgtgctcgtgggtggttcgacccggatgccc gcggtgaccgatctggtcaaggaactcaccggcggcaaggaacccaacaagggcgtcaac cccgatgaggttgtcgcggtgggagccgctctgcaggccggcgtcctcaagggcgaggtg aaagacgttctgctgcttgatgttaccccgctgagcctgggtatcgagaccaagggcggg gtgatgaccaggctcatcgagcgcaacaccacgatccccaccaagcggtcggagactttc accaccgccgacgacaaccaaccgtcggtgcagatccaggtctatcagggggagcgtgag atcgccgcgcacaacaagttgctcgggtccttcgagctgaccggcatcccgccggcgccg cgggggattccgcagatcgaggtcactttcgacatcgacgccaacggcattgtgcacgtc accgccaaggacaagggcaccggcaaggagaacacgatccgaatccaggaaggctcgggc ctgtccaaggaagacattgaccgcatgatcaaggacgccgaagcgcacgccgaggaggat cgcaagcgtcgcgaggaggccgatgttcgtaatcaagccgagacattggtctaccagacg gagaagttcgtcaaagaacagcgtgaggccgagggtggttcgaaggtacctgaagacacg ctgaacaaggttgatgccgcggtggcggaagcgaaggcggcacttggcggatcggatatt tcggccatcaagtcggcgatggagaagctgggccaggagtcgcaggctctggggcaagcg atctacgaagcagctcaggctgcgtcacaggccactggcgctgcccaccccggcggcgag ccgggcggtgcccaccccggctcggctgatgacgttgtggacgcggaggtggtcgacgac ggccgggaggccaagtga

13. M. tuberculosis H37Rv|Rv0288|EsxH: 96 aa--Low Molecular Weight Protein Antigen 7 EsxH (10 kDa Antigen) (CFP-7) (Protein TB10.4)

TABLE-US-00018 (SEQ ID NO: 16) MSQIMYNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAWQGDTGITYQAWQAQWNQAMEDLVRAY HAMSSTHEANTMAMMARDTAEAAKWGG M. tuberculosis H37Rv|Rv0288|esxH: 291 bp - LOW MOLECULAR WEIGHT PROTEIN ANTIGEN 7 ESXH (10 kDa ANTIGEN) (CFP-7) (PROTEIN TB10.4) (SEQ ID NO: 35) atgtcgcaaatcatgtacaactaccccgcgatgttgggtcacgccggggatatggccgga tatgccggcacgctgcagagcttgggtgccgagatcgccgtggagcaggccgcgttgcag agtgcgtggcagggcgataccgggatcacgtatcaggcgtggcaggcacagtggaaccag gccatggaagatttggtgcgggcctatcatgcgatgtccagcacccatgaagccaacacc atggcgatgatggcccgcgacacggccgaagccgccaaatggggcggctag

14. M. tuberculosis H37Rv|Rv2031c|HspX: 144 aa--Heat Shock Protein HspX (Alpha-Crystallin Homolog) (14 kDa Antigen) (HSP16.3)

TABLE-US-00019 (SEQ ID NO: 17) MATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRAELPGVDPDKDVDIM VRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADEDDIKATYDKGILTVSVAVSEGKPTEKHI QIRSTN M. tuberculosis H37Rv|Rv2031c|hspX: 435 bp - HEAT SHOCK PROTEIN HSPX (ALPHA-CRYSTALLIN HOMOLOG) (14 kDa ANTIGEN) (HSP16.3) (SEQ ID NO: 36) atggccaccacccttcccgttcagcgccacccgcggtccctcttccccgagttttctgag ctgttcgcggccttcccgtcattcgccggactccggcccaccttcgacacccggttgatg cggctggaagacgagatgaaagaggggcgctacgaggtacgcgcggagcttcccggggtc gaccccgacaaggacgtcgacattatggtccgcgatggtcagctgaccatcaaggccgag cgcaccgagcagaaggacttcgacggtcgctcggaattcgcgtacggttccttcgttcgc acggtgtcgctgccggtaggtgctgacgaggacgacattaaggccacctacgacaagggc attcttactgtgtcggtggcggtttcggaagggaagccaaccgaaaagcacattcagatc cggtccaccaactga

15. M. tuberculosis H37Rv|Rv0467|Icl: 428 aa--Isocitrate Lyase Icl (Isocitrase) (Isocitratase)

TABLE-US-00020 (SEQ ID NO: 18) MSVVGTPKSAEQIQQEWDTNPRWKDVTRTYSAEDVVALQGSVVEEHTLARRGAEVLWEQLHDLEWVNAL GALTGNMAVQQVRAGLKAIYLSGWQVAGDANLSGHTYPDQSLYPANSVPQVVRRINNALQRADQIAKIE GDTSVENWLAPIVADGEAGFGGALNVYELQKALIAAGVAGSHWEDQLASEKKCGHLGGKVLIPTQQHIR TLTSARLAADVADVPTVVIARTDAEAATLITSDVDERDQPFITGERTREGFYRTKNGIEPCIARAKAYA PFADLIWMETGTPDLEAARQFSEAVKAEYPDQMLAYNCSPSFNWKKHLDDATIAKFQKELAAMGFKFQF ITLAGFHALNYSMFDLAYGYAQNQMSAYVELQEREFAAEERGYTATKHQREVGAGYFDRIATTVDPNSS TTALTGSTEEGQFH M. tuberculosis H37Rv|Rv0467|icl: 1287 bp - ISOCITRATE LYASE ICL (ISOCITRASE) (ISOCITRATASE) (SEQ ID NO: 37) atgtctgtcgtcggcaccccgaagagcgcggagcagatccagcaggaatgggacacgaacccgcgctgg aaggacgtcacccgcacctactccgccgaggacgtcgtcgccctccagggcagcgtggtcgaggagcac acgctggcccgccgcggtgcggaggtgctgtgggagcagctgcacgacctcgagtgggtcaacgcgctg ggcgcgctgaccggcaacatggccgtccagcaggtgcgcgccggcctgaaggccatctacctgtcgggc tggcaggtcgccggcgatgccaacctgtccgggcacacctaccccgaccagagcctgtatcccgccaac tcggtgccgcaggtggtccgccggatcaacaacgcactgcagcgcgccgaccagatcgccaagatcgag ggcgatacttcggtggagaactggctggcgccgattgtcgccgacggcgaggccggctttggcggcgcg ctcaacgtctacgagctgcagaaagccctgatcgccgcgggcgttgcgggttcgcactgggaggaccag ttggcctctgagaagaagtgcggccacctgggcggcaaggtgttgatcccgacccagcagcacatccgc actttgacgtctgctcggctcgcggccgatgtggctgatgttcccacggtggtgatcgcccgtaccgac gccgaggcggccacgctgatcacctccgacgtcgacgagcgcgaccagccgttcatcaccggcgagcgc acccgggaaggcttctaccgcaccaagaacggcatcgagccttgcatcgctcgggcgaaggcctacgcc ccgttcgccgacttgatctggatggagaccggtaccccggacctcgaggccgcccggcagttctccgag gcggtcaaggcggagtacccggaccagatgctggcctacaactgctcgccatcgttcaactggaaaaag cacctcgacgacgccaccatcgccaagttccagaaggagctggcagccatgggcttcaagttccagttc atcacgctggccggcttccatgcgctgaactactcgatgttcgatctggcctacggctacgcccagaac cagatgagcgcgtatgtcgaactgcaggaacgcgagttcgccgccgaagaacggggctacaccgcgacc aagcaccagcgcgaggtcggcgccggctacttcgaccggattgccaccaccgtggacccgaattcgtcg accaccgcgttgaccggttccaccgaagagggccagttccactag

16. M. tuberculosis H37Rv|Rv2660c|Rv2660c: 75 aa--Hypothetical Protein

TABLE-US-00021 (SEQ ID NO: 19) VIAGVDQALAATGQASQRAAGASGGVTVGVGVGTEQRNLSVVAPSQFTFSSRSPDFVDETAGQSWCAIL GLNQFH M. tuberculosis H37Rv|Rv2660c|Rv2660c: 228 by - HYPOTHETICAL PROTEIN (SEQ ID NO: 38) gtgatagcgggcgtcgaccaggcgcttgcagcaacaggccaggctagccagcgggcggca ggcgcatctggtggggtcaccgtcggtgtcggcgtgggcacggaacagaggaacctttcg gtggttgcaccgagtcagttcacatttagttcacgcagcccagattttgtggatgaaacc gcaggtcaatcgtggtgcgcgatactgggattgaaccagtttcactag

17. M. tuberculosis H37Rv|Rv1837c|GlcB: 741 aa--Probable Malate Synthase G GlcB

TABLE-US-00022 (SEQ ID NO: 20) MTDRVSVGNLRIARVLYDFVNNEALPGTDIDPDSFWAGVDKVVADLTPQNQALLNARDELQAQIDKWHR RRVIEPIDMDAYRQFLTEIGYLLPEPDDFTITTSGVDAEITTTAGPQLVVPVLNARFALNAANARWGSL YDALYGTDVIPETDGAEKGPTYNKVRGDKVIAYARKFLDDSVPLSSGSFGDATGFTVQDGQLVVALPDK STGLANPGQFAGYTGAAESPTSVLLINHGLHIEILIDPESQVGTTDRAGVKDVILESAITTIMDFEDSV AAVDAADKVLGYRNWLGLNKGDLAAAVDKDGTAFLRVLNRDRNYTAPGGGQFTLPGRSLMFVRNVGHLM TNDAIVDTDGSEVFEGIMDALFTGLIAIHGLKASDVNGPLINSRTGSIYIVKPKMHGPAEVAFTCELFS RVEDVLGLPQNTMKIGIMDEERRTTVNLKACIKAAADRVVFINTGFLDRTGDEIHTSMEAGPMVRKGTM KSQPWILAYEDHNVDAGLAAGFSGRAQVGKGMWTMTELMADMVETKIAQPRAGASTAWVPSPTAATLHA LHYHQVDVAAVQQGLAGKRRATIEQLLTIPLAKELAWAPDEIREEVDNNCQSILGYVVRWVDQGVGCSK VPDIHDVALMEDRATLRISSQLLANWLRHGVITSADVRASLERMAPLVDRQNAGDVAYRPMAPNFDDSI AFLAAQELILSGAQQPNGYTEPILHRRRREFKARAAEKPAPSDRAGDDAAR M. tuberculosis H37Rv|Rv1837c|glcB: 2226 bp - PROBABLE MALATE SYNTHASE G GLCB (SEQ ID NO: 39) atgacagatcgcgtgtcggtgggcaacttgcgcatcgctcgggtgctctacgacttcgtg aacaatgaagccctgcctggcaccgatatcgacccggacagcttctgggcgggcgtcgac aaggtcgtcgccgacctgaccccgcagaaccaagctctgttgaacgcccgcgacgagctg caggcgcagatcgacaagtggcaccggcgtcgggtgatcgagcccatcgacatggatgcc taccgccagttcctcaccgagatcggctacctgcttcccgaacctgatgacttcaccatc accacgtccggtgtcgacgctgagatcaccacgaccgccggcccccagctggtggtgccg gtgctcaacgcgcggtttgctctgaacgcggccaacgctcgctggggctccctctacgac gccttgtatggcaacgatgtcatccccgagaccgacggcgccgaaaaaggccccacgtac aacaaggttcgtggcgacaaggtgatcgcgtatgcccgcaagttcctcgacgacagtgtt ccgctgtcgtcgggttcctttggcgacgccaccggtttcacagtgcaggatggccagctc gtggttgccttgccggataagtccaccggcctggccaaccccggccagttcgccggctac accggcgcagccgagtcgccgacatcggtgctgctaatcaatcacggtttgcacatcgag atcctgatcgatccggagtcgcaggtcggcaccaccgaccgggccggcgtcaaggacgtg atcctggaatccgcgatcaccacgatcatggacttcgaggactcggtggccgccgtggac gccgccgacaaggtgctgggttatcggaactggctcggcctgaacaagggcgacctggca gcagcggtagacaaggacggcaccgctttcctgcgggtgctcaatagggaccggaactac accgcacccggcggtggccagttcacgctgcctggacgcagcctcatgttcgtccgcaac gtcggtcacttgatgacgaatgacgccatcgtcgacactgacggcagcgaggtgttcgaa ggcatcatggatgccctattcaccggcctgatcgccatccacgggctaaaggccagcgac gtcaacgggccgctgatcaacagccgcaccggctccatctacatcgtcaagccgaagatg cacggtccggccgaggtggcgtttacctgcgaactgttcagccgggttgaagatgtgctg gggttgccgcaaaacaccatgaagatcggcatcatggacgaggaacgccggaccacggtc aacctcaaggcgtgcatcaaagctgccgcggaccgcgtggtgttcatcaacaccgggttc ctggaccgcaccggcgatgaaatccacacctcgatggaggccggcccgatggtgcgcaag ggcaccatgaagagccagccgtggatcttggcctacgaggaccacaacgtcgatgccggc ctggccgccgggttcagcggccgagcccaggtcggcaagggcatgtggacaatgaccgag ctgatggccgacatggtcgagacaaaaatcgcccagccgcgcgccggggccagcaccgcc tgggttccctctcccactgcggccaccctgcatgcgctgcactaccaccaggtcgacgtc gccgcggtgcaacaaggactggcggggaagcgtcgcgccaccatcgaacaattgctgacc attccgctggccaaggaattggcctgggctcccgacgagatccgcgaagaggtcgacaac aactgtcaatccatcctcggctacgtggttcgctgggttgatcaaggtgtcggctgctcg aaggtgcccgacatccacgacgtcgcgctcatggaggaccgggccacgctgcgaatctcc agccaattgttggccaactggctgcgccacggtgtgatcaccagcgcggatgtgcgggcc agcttggagcggatggcgccgttggtcgatcgacaaaacgcgggcgacgtggcataccga ccgatggcacccaacttcgacgacagtatcgccttcctggccgcgcaggagctgatcttg tccggggcccagcagcccaacggctacaccgagccgatcctgcaccgacgtcgtcgggag tttaaggcccgggccgctgagaagccggccccatcggacagggccggtgacgatgcggcc cgctag

18. M. tuberculosis H37Rv|Rv1475c|Acn: 943 aa--Probable Iron-Regulated Aconitate Hydratase Acn (Citrate hydro-lyase) (Aconitase)

TABLE-US-00023 (SEQ ID NO: 21) VTSKSVNSFGAHDTLKVGEKSYQIYRLDAVPNTAKLPYSLKVLAENLLRNEDGSNITKDHIEAIANWDP KAEPSIEIQYTPARVVMQDFTGVPCIVDLATMREAIADLGGNPDKVNPLAPADLVIDHSVIADLFGRAD AFERNVEIEYQRNGERYQFLRWGQGAFDDFKVVPPGTGIVHQVNIEYLASVVMTRDGVAYPDTCVGTDS HTTMVNGLGVLGWGVGGIEAEAAMLGQPVSMLIPRVVGFRLTGEIQPGVTATDVVLTVTEMLRQHGVVG KFVEFYGEGVAEVPLANRATLGNMSPEFGSTAAIFPIDEETIKYLRFTGRTPEQVALVEAYAKAQGMWH DPKHEPEFSEYLELNLSDVVPSIAGPKRPQDRIALAQAKSTFREQIYHYVGNGSPDSPHDPHSKLDEVV EETFPASDPGQLTFANDDVATDETVHSAAAHADGRVSNPVRVKSDELGEFVLDHGAVVIAAITSCTNTS NPEVMLGAALLARNAVEKGLTSKPWVKTTIAPGSQVVNDYYDRSGLWPYLEKLGFYLVGYGCTTCIGNS GPLPEEISKAVNDNDLSVTAVLSGNRNFEGRINPDVKMNYLASPPLVIAYALAGTMDFDFQTQPLGQDK DGKNVFLRDIWPSQQDVSDTIAAAINQEMFTRNYADVFKGDDRWRNLPTPSGNTFEWDPNSTYVRKPPY FEGMTAKPEPVGNISGARVLALLGDSVTTDHISPAGAIKPGTPAARYLDEHGVDRKDTNSFGSRRGNHE VMIRGTFANIRLRNQLLDDVSGGYTRDFTQPGGPQAFIYDAAQNYAAQHIPLVVFGGKEYGSGSSRDWA AKGTLLLGVRAVIAESFERIHRSNLIGMGVIPLQFPEGKSASSLGLDGTEVFDITGIDVLNDGKTPKTV CVQATKGDGATIEFDAVVRIDTPGEADYYRNGGILQYVLRNILKSG M. tuberculosis H37Rv|Rv1475c|acn: 2832 bp - PROBABLE IRON-REGULATED ACONITATE HYDRATASE ACN (Citrate hydro-lase) (Aconitase) (SEQ ID NO: 40) gtgactagcaaatctgtgaactcattcggagcccacgacaccctgaaggtcggcgaaaag agttaccagatctatcgtctcgacgccgtccccaataccgcgaaactcccctacagcctc aaagtgctcgccgagaacctgttgcgcaacgaggacggcagcaacatcaccaaggaccac atcgaggccatcgccaactgggaccctaaggccgagcccagcatcgagatccagtacacg cccgcccgggtggtgatgcaggacttcaccggcgtaccgtgcatcgtcgacttggccacc atgcgcgaggcgatcgccgatctgggcggcaacccggacaaggtcaacccgctggcgccc gcagacttggtgatcgaccactcggtgatcgccgatttgttcggccgcgccgacgcattc gagcgcaacgtcgaaatcgaataccagcgcaacggtgagcgttaccaattcctgcgctgg ggccaaggcgctttcgacgacttcaaagtggtgccgccgggcaccggcatcgtgcaccag gtcaatatcgagtacctggccagcgtggtgatgactcgcgacggagtggcctaccccgac acctgcgtgggcaccgactcacacaccaccatggtcaacggcctccctgtgctcgggtgg ggtgtcggcggcatcgaggcggaggccgcgatgctgggtcagccggtatcgatgctgatc ccgcgggtcgtgggtttcaggttgaccggcgagatccagccgggagtcaccgccaccgac gtggtgttgaccgtcaccgagatgctgcgccagcacggcgtcgtcggcaaattcgtcgag ttctacggcgagggcgtggccgaggtgccgctggccaaccgcgccaccctgggcaacatg agtcccgaattcggttccaccgcagcgattttcccgatcgacgaagaaaccatcaagtat ctgcggtttaccggtcgcacgccggagcaggtcgcactggtcgaggcctacgccaaggcg cagggcatgtggcacgatcccaagcacgagccggagttctcggaatacctcgaactcaac ctatccgacgtggtgccgtcgatcgccggaccaaagcgtccacaggaccgaatcgcgttg gcgcaagccaaatcaacattccgcgagcagatttaccactatgtcggcaatggttccccg gattccccccacgacccgcactcgaagctggacgaggtagtcgaggagacattcccggcc agcgacccggggcagctgacgttcgccaacgacgacgtcgccactgacgaaaccgtgcac tcggctgccgcgcatgccgatggccgggtgagcaacccagtgcgggtgaagtcggacgaa ctcggcgaattcgtgctcgaccacggcgcggtggtgattgccgcgatcacgtcctgcacc aacacctccaaccccgaagtaatgctgggcgcggcgctgctggcccgcaacgccgtcgaa aagggactgacctcgaagccgtgggtgaagaccacgattgccccgggctcgcaagtggtc aacgactactacgacagatccggcctgtggccgtatctggagaagctcggcttctatctg gtcggctacggctgcaccacctgcatcggcaactccgggccgctgcccgaggaaatctca aaagcggttaacgacaacgacctttcggtgaccgcggtactgtccggcaaccggaacttc gagggccgtatcaacccagacgtgaagatgaactacctggcatcgccgccgctggtcatc gcctacgcgctggccgggaccatggacttcgacttccaaacccagccgctcggtcaagac aaagacggtaagaacgtttttctccgcgatatctggccatcgcagcaggatgtctccgac accatcgccgcggcgatcaaccaggagatgttcacccgcaactacgccgacgtgttcaag ggcgacgaccgctggcgcaacctgccaaccccgagcggcaacacctttgagtgggacccg aattcgacctacgtgcgcaagccgccgtatttcgaggggatgacagccaaacccgaaccg gtcggcaacatcagcggtgcccgggtgctggcgctgctcggtgattcggtgaccaccgac cacatctcccccgccggcgccatcaagcccggaactcccgcggcgcgctacctcgacgaa cacggtgtcgaccgcaaggactacaactccttcggttctcgccgcggcaaccacgaggtg atgattcgtggcaccttcgccaacatccggctgcgtaaccaactgctagacgacgtgtcc ggcggttatacccgcgacttcacccagccgggcggtccgcaggcgttcatctacgacgcc gcgcagaactatgcggcgcaacacattccgctggttgtgttcggcggcaaagagtacggg tcgggttcgtcacgggactgggcggccaaaggcacattgctactgggcgtgcgggcggtg atcgccgagtcattcgagcggatccaccggtccaacctgatcggcatgggcgtgatcccg ctgcagttccccgaaggaaagtcagcgtcgtcgttgggactcgacggtaccgaggtcttc gacatcaccggtatcgacgtgcttaacgacggcaagacacccaagacggtgtgcgtccag gccaccaagggcgacggcgccacgatcgagttcgacgccgtggtgcgcatcgacaccccc ggtgaggcggactactaccgcaacggcggcatcctgcagtacgtgctgcgcaacatactg aagtcaggctga

19. M. tuberculosis H37Rv|Rv38751EsxA: 95 aa--6 KDA Early Secretory Antigenic Target EsxA (ESAT-6)

TABLE-US-00024 (SEQ ID NO: 22) MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNAL QNLARTISEAGQAMASTEGNVTGMFA M. tuberculosis H37Rv|Rv3875|esxA: 288 bp - 6 KDA EARLY SECRETORY ANTIGENIC TARGET ESXA (ESAT-6) (SEQ ID NO: 41) atgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccaggga aatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgca gcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgcc acggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggt caggcaatggcttcgaccgaaggcaacgtcactgggatgttcgcatag

Polynucleotide and Polypeptide Sequences from: Listeria Monocytogenes (EGD STRAIN)

ActA

TABLE-US-00025

[0143] actin-assembly inducing protein precursor (Listeria monocytogenes EGD-e) (SEQ ID NO: 1) MGLNRFMRAMMVVFITANCITINPDIIFAATDSEDSSLNTDEWEEEKTEEQPSEVNTGPR YETAREVSSRDIKELEKSNKVRNTNKADLIAMLKEKAEKGPNINNNNSEQTENAAINEEA SGADRPAIQVERRHPGLPSDSAAEIKKRRKAIASSDSELESLTYPDKPTKVNKKKVAKES VADASESDLDSSMQSADESSPQPLKANQQPFFPKVFKKIKDAGKWVRDKIDENPEVKKAI VDKSAGLIDQLLTKKKSEEVNASDFPPPPTDEELRLALPETPMLLGFNAPATSEPSSFEF PPPPTDEELRLALPETPMLLGFNAPATSEPSSFEFPPPPTEDELEIIRETASSLDSSFTR GDLASLRNAINRHSQNFSDFPPIPTEEELNGRGGRPTSEEFSSLNSGDFTDDENSETTEE EIDRLADLRDRGTGKHSRNAGFLPLNPFASSPVPSLSPKVSKISAPALISDITKKTPFKN PSQPLNVFNKKTTTKTVTKKPTPVKTAPKLAELPATKPQETVLRENKTPFIEKQAETNKQ SINMPSLPVIQKEATESDKEEMKPQTEEKMVEESESANNANGKNRSAGIEEGKLIAKSAE DEKAKEEPGNHTTLILAMLAIGVFSLGAFIKIIQLRKNN gene = "actA" (Listeria monocytogenes EGD-e) (SEQ ID NO: 42) GTGGGATTAAACAGATTTATGCGTGCGATGATGGTGGTTTTCATTACTGCCAATTGCATT ACGATTAACCCCGACATAATATTTGCAGCGACAGATAGCGAAGATTCTAGTCTAAACACA GATGAATGGGAAGAAGAAAAAACAGAAGAGCAACCAAGCGAGGTAAATACGGGACCAAGA TACGAAACTGCACGTGAAGTAAGTTCACGTGATATTAAAGAACTAGAAAAATCGAATAAA GTGAGAAATACGAACAAAGCAGACCTAATAGCAATGTTGAAAGAAAAAGCAGAAAAAGGT CCAAATATCAATAATAACAACAGTGAACAAACTGAGAATGCGGCTATAAATGAAGAGGCT TCAGGAGCCGACCGACCAGCTATACAAGTGGAGCGTCGTCATCCAGGATTGCCATCGGAT AGCGCAGCGGAAATTAAAAAAAGAAGGAAAGCCATAGCATCATCGGATAGTGAGCTTGAA AGCCTTACTTATCCGGATAAACCAACAAAAGTAAATAAGAAAAAAGTGGCGAAAGAGTCA GTTGCGGATGCTTCTGAAAGTGACTTAGATTCTAGCATGCAGTCAGCAGATGAGTCTTCA CCACAACCTTTAAAAGCAAACCAACAACCATTTTTCCCTAAAGTATTTAAAAAAATAAAA GATGCGGGGAAATGGGTACGTGATAAAATCGACGAAAATCCTGAAGTAAAGAAAGCGATT GTTGATAAAAGTGCAGGGTTAATTGACCAATTATTAACCAAAAAGAAAAGTGAAGAGGTA AATGCTTCGGACTTCCCGCCACCACCTACGGATGAAGAGTTAAGACTTGCTTTGCCAGAG ACACCAATGCTTCTTGGTTTTAATGCTCCTGCTACATCAGAACCGAGCTCATTCGAATTT CCACCACCACCTACGGATGAAGAGTTAAGACTTGCTTTGCCAGAGACGCCAATGCTTCTT GGTTTTAATGCTCCTGCTACATCGGAACCGAGCTCGTTCGAATTTCCACCGCCTCCAACA GAAGATGAACTAGAAATCATCCGGGAAACAGCATCCTCGCTAGATTCTAGTTTTACAAGA GGGGATTTAGCTAGTTTGAGAAATGCTATTAATCGCCATAGTCAAAATTTCTCTGATTTC CCACCAATCCCAACAGAAGAAGAGTTGAACGGGAGAGGCGGTAGACCAACATCTGAAGAA TTTAGTTCGCTGAATAGTGGTGATTTTACAGATGACGAAAACAGCGAGACAACAGAAGAA GAAATTGATCGCCTAGCTGATTTAAGAGATAGAGGAACAGGAAAACACTCAAGAAATGCG GGTTTTTTACCATTAAATCCGTTTGCTAGCAGCCCGGTTCCTTCGTTAAGTCCAAAGGTA TCGAAAATAAGCGCACCGGCTCTGATAAGTGACATAACTAAAAAAACGCCATTTAAGAAT CCATCACAGCCATTAAATGTGTTTAATAAAAAAACTACAACGAAAACAGTGACTAAAAAA CCAACCCCTGTAAAGACCGCACCAAAGCTAGCAGAACTTCCTGCCACAAAACCACAAGAA ACCGTACTTAGGGAAAATAAAACACCCTTTATAGAAAAACAAGCAGAAACAAACAAGCAG TCAATTAATATGCCGAGCCTACCAGTAATCCAAAAAGAAGCTACAGAGAGCGATAAAGAG GAAATGAAACCACAAACCGAGGAAAAAATGGTAGAGGAAAGCGAATCAGCTAATAACGCA AACGGAAAAAATCGTTCTGCTGGCATTGAAGAAGGAAAACTAATTGCTAAAAGTGCAGAA GACGAAAAAGCGAAGGAAGAACCAGGGAACCATACGACGTTAATTCTTGCAATGTTAGCT ATTGGCGTGTTCTCTTTAGGGGCGTTTATCAAAATTATTCAATTAAGAAAAAATAATTAA

LLO

TABLE-US-00026

[0144] listeriolysin O precursor (Listeria monocytogenes EGD-e) (SEQ ID NO: 43) MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADE IDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNA ISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNN AVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAIS EGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGR QVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIID GNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKIN IDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVY AKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIE gen = "hly" (Listeria monocytogenes EGD-e) (SEQ ID NO: 44) ATGAAAAAAATAATGCTAGTTTTTATTACACTTATATTAGTTAGTCTACCAATTGCGCAA CAAACTGAAGCAAAGGATGCATCTGCATTCAATAAAGAAAATTCAATTTCATCCATGGCA CCACCAGCATCTCCGCCTGCAAGTCCTAAGACGCCAATCGAAAAGAAACACGCGGATGAA ATCGATAAGTATATACAAGGATTGGATTACAATAAAAACAATGTATTAGTATACCACGGA GATGCAGTGACAAATGTGCCGCCAAGAAAAGGTTACAAAGATGGAAATGAATATATTGTT GTGGAGAAAAAGAAGAAATCCATCAATCAAAATAATGCAGACATTCAAGTTGTGAATGCA ATTTCGAGCCTAACCTATCCAGGTGCTCTCGTAAAAGCGAATTCGGAATTAGTAGAAAAT CAACCAGATGTTCTCCCTGTAAAACGTGATTCATTAACACTCAGCATTGATTTGCCAGGT ATGACTAATCAAGACAATAAAATCGTTGTAAAAAATGCCACTAAATCAAACGTTAACAAC GCAGTAAATACATTAGTGGAAAGATGGAATGAAAAATATGCTCAAGCTTATCCAAATGTA AGTGCAAAAATTGATTATGATGACGAAATGGCTTACAGTGAATCACAATTAATTGCGAAA TTTGGTACAGCATTTAAAGCTGTAAATAATAGCTTGAATGTAAACTTCGGCGCAATCAGT GAAGGGAAAATGCAAGAAGAAGTCATTAGTTTTAAACAAATTTACTATAACGTGAATGTT AATGAACCTACAAGACCTTCCAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAA GCGCTTGGAGTGAATGCAGAAAATCCTCCTGCATATATCTCAAGTGTGGCGTATGGCCGT CAAGTTTATTTGAAATTATCAACTAATTCCCATAGTACTAAAGTAAAAGCTGCTTTTGAT GCTGCCGTAAGCGGAAAATCTGTCTCAGGTGATGTAGAACTAACAAATATCATCAAAAAT TCTTCCTTCAAAGCCGTAATTTACGGAGGTTCCGCAAAAGATGAAGTTCAAATCATCGAC GGCAACCTCGGAGACTTACGCGATATTTTGAAAAAAGGCGCTACTTTTAATCGAGAAACA CCAGGAGTTCCCATTGCTTATACAACAAACTTCCTAAAAGACAATGAATTAGCTGTTATT AAAAACAACTCAGAATATATTGAAACAACTTCAAAAGCTTATACAGATGGAAAAATTAAC ATCGATCACTCTGGAGGATACGTTGCTCAATTCAACATTTCTTGGGATGAAGTAAATTAT GATCCTGAAGGTAACGAAATTGTTCAACATAAAAACTGGAGCGAAAACAATAAAAGCAAG CTAGCTCATTTCACATCGTCCATCTATTTGCCAGGTAACGCGAGAAATATTAATGTTTAC GCTAAAGAATGCACTGGTTTAGCTTGGGAATGGTGGAGAACGGTAATTGATGACCGGAAC TTACCACTTGTGAAAAATAGAAATATCTCCATCTGGGGCACCACGCTTTATCCGAAATAT AGTAATAAAGTAGATAATCCAATCGAATAA

Internalin B

TABLE-US-00027

[0145] internalin B (Listeria monocytogenes EGD-e) (SEQ ID NO: 2) MKEKHNPRRKYCLISGLAIIFSLWIIIGNGAKVQAETITVPTPIKQIFSDDAFAETIKDN LKKKSVTDAVTQNELNSIDQIIANNSDIKSVQGIQYLPNVTKLFLNGNKLTDIKPLANLK NLGWLFLDENKVKDLSSLKDLKKLKSLSLEHNGISDINGLVHLPQLESLYLGNNKITDIT VLSRLTKLDTLSLEDNQISDIVPLAGLTKLQNLYLSKNHISDLRALAGLKNLDVLELFSQ ECLNKPINHQSNLVVPNTVKNTDGSLVTPEIISDDGDYEKPNVKWHLPEFTNEVSFIFYQ PVTIGKAKARFHGRVTQPLKEVYTVSYDVDGTVIKTKVEAGTRITAPKPPTKQGYVFKGW YTEKNGGHEWNFNTDYMSGNDFTLYAVFKAETTEKAVNLTRYVKYIRGNAGIYKLPREDN SLKQGTLASHRCKALTVDREARNGGKLWYRLKNIGWTKAENLSLDRYDKMEYDKGVTAYA RVRNASGNSVWTKPYNTAGAKHVNKLSVYQGKNMRILREAKTPITTWYQFSIGGKVIGWV DTRALNTFYKQSMEKPTRLTRYVSANKAGESYYKVPVADNPVKRGTLAKYKNQKLIVDCQ ATIEGQLWYRIRTSSTFIGWTKAANLRAQK gene = "inlB" (Listeria monocytogenes EGD-e) (SEQ ID NO: 45) GTGAAAGAAAAGCACAACCCAAGAAGGAAGTATTGTTTAATCTCAGGTTTAGCTATTATT TTTAGTTTATGGATAATTATTGGAAACGGGGCGAAAGTACAAGCGGAGACTATCACCGTG CCAACGCCAATCAAGCAAATTTTTTCAGATGATGCTTTTGCAGAAACAATCAAAGACAAT TTAAAGAAAAAAAGTGTGACAGATGCAGTGACACAAAATGAATTAAATAGTATAGATCAA ATCATTGCGAATAATAGTGATATTAAATCCGTTCAAGGAATTCAGTATTTACCCAATGTG ACAAAGTTATTTTTAAACGGGAATAAACTAACAGATATAAAGCCCTTAGCAAACTTGAAA AATTTAGGATGGCTTTTTTTAGACGAAAATAAAGTTAAGGACCTAAGTTCGCTCAAGGAT TTAAAAAAATTAAAATCACTTTCTTTGGAGCATAATGGTATAAGTGATATAAACGGACTT GTTCATTTACCACAGCTGGAAAGTTTGTATTTGGGAAATAATAAAATAACGGATATAACG GTTCTTTCACGTTTAACTAAACTGGATACTTTGTCTCTCGAAGATAACCAAATTAGTGAT ATTGTGCCACTTGCAGGTTTAACTAAATTGCAGAACCTATATTTAAGTAAAAATCACATA AGCGATTTAAGAGCATTAGCAGGACTTAAAAATCTAGATGTTTTAGAATTATTTAGCCAA GAATGTCTTAATAAGCCTATTAATCATCAATCTAATTTGGTTGTTCCGAATACAGTGAAA AACACTGATGGGTCGTTAGTGACTCCAGAAATAATAAGTGATGATGGCGATTATGAAAAA CCTAATGTTAAATGGCATTTACCAGAATTTACAAATGAAGTGAGTTTTATTTTCTATCAG CCAGTCACTATTGGAAAAGCAAAAGCAAGATTTCATGGGAGAGTAACCCAACCACTGAAA GAGGTTTACACAGTAAGTTATGATGTTGATGGAACGGTAATAAAAACAAAAGTAGAAGCA GGGACGCGGATAACTGCACCTAAACCTCCGACTAAACAAGGCTATGTTTTTAAAGGATGG TATACTGAAAAAAATGGTGGGCATGAGTGGAATTTTAATACGGATTATATGTCCGGAAAT GATTTTACTTTGTACGCAGTATTTAAAGCGGAAACGACCGAAAAAGCAGTCAACTTAACC CGCTATGTCAAATATATTCGCGGGAATGCAGGCATCTACAAACTTCCACGAGAAGATAAC TCGCTTAAACAAGGAACTCTAGCCTCGCACCGCTGTAAAGCTCTAACTGTTGATAGAGAA GCCCGAAATGGCGGAAAATTATGGTACAGGTTAAAAAATATTGGCTGGACTAAAGCGGAA AACCTTTCCTTAGACCGATACGATAAAATGGAATATGACAAAGGGGTTACCGCTTATGCA AGAGTGAGAAATGCGTCTGGAAATTCGGTTTGGACAAAACCCTACAACACAGCCGGCGCT AAACACGTGAATAAGCTATCGGTCTACCAAGGTAAAAATATGCGTATCTTGCGCGAAGCC AAAACACCAATTACTACATGGTATCAATTTAGCATTGGTGGTAAAGTAATTGGTTGGGTC GATACCCGAGCACTTAACACATTCTACAAACAAAGCATGGAAAAGCCAACCCGTTTAACT CGTTATGTCAGCGCCAATAAAGCTGGCGAATCGTACTATAAAGTCCCGGTAGCAGATAAT CCAGTCAAAAGGGGTACTTTAGCCAAGTATAAAAATCAAAAGTTAATTGTTGATTGTCAA GCAACCATCGAAGGTCAACTTTGGTACCGAATAAGGACTAGTTCCACTTTCATTGGTTGG ACGAAAGCAGCTAATTTAAGGGCACAGAAATAA

PrfA

TABLE-US-00028

[0146] listeriolysin positive regulatory protein (Listeria monocytogenes EGD-e) (SEQ ID NO: 3) MNAQAEEFKKYLETNGIKPKQFHKKELIFNQWDPQEYCIFLYDGITKLTSISENGTIMNL QYYKGAFVIMSGFIDTETSVGYYNLEVISEQATAYVIKINELKELLSKNLTHFFYVFQTL QKQVSYSLAKFNDFSINGKLGSICGQLLILTYVYGKETPDGIKITLDNLTMQELGYSSGI AHSSAVSRIISKLKQEKVIVYKNSCFYVQNLDYLKRYAPKLDEWFYLACPATWGKLN gene = "prfA" complement(1 . . . 714)prfA (Listeria monocytogenes EGD-e) (SEQ ID NO: 46) TTAATTTAATTTTCCCCAAGTAGCAGGACATGCTAAATAAAACCATTCATCTAATTTAGG GGCATATCTTTTGAGATAATCAAGATTTTGTACATAAAAGCATGAATTTTTATACACGAT AACTTTCTCTTGCTTTAATTTGGAAATAATTCTGCTAACAGCTGAGCTATGTGCGATGCC ACTTGAATATCCTAACTCCTGCATTGTTAAATTATCCAGTGTAATCTTGATGCCATCAGG AGTTTCTTTACCATACACATAGGTCAGGATTAAAAGTTGACCGCAAATAGAGCCAAGCTT CCCGTTAATCGAAAAATCATTAAATTTAGCTAGACTGTATGAAACTTGTTTTTGTAGGGT TTGGAAAACATAGAAAAAGTGCGTAAGATTTTTGCTCAGTAGTTCTTTTAGTTCGTTTAT TTTGATAACGTATGCGGTAGCCTGCTCGCTAATGACTTCTAAATTATAATAGCCAACCGA TGTTTCTGTATCAATAAAGCCAGACATTATAACGAAAGCCCCTTTGTAGTATTGTAAATT CATGATGGTCCCGTTCTCGCTAATACTCGTGAGCTTTGTGATACCATCATATAGAAAAAT ACAATATTCTTGTGGATCCCATTGGTTAAAAATAAGTTCTTTTTTATGAAATTGTTTTGG TTTTATCCCGTTAGTTTCTAAATATTTTTTGAATTCTTCTGCTTGAGCGTTCAT

Sequence CWU 1

1

461639PRTListeria monocytogenes 1Met Gly Leu Asn Arg Phe Met Arg Ala Met Met Val Val Phe Ile Thr1 5 10 15 Ala Asn Cys Ile Thr Ile Asn Pro Asp Ile Ile Phe Ala Ala Thr Asp 20 25 30 Ser Glu Asp Ser Ser Leu Asn Thr Asp Glu Trp Glu Glu Glu Lys Thr 35 40 45 Glu Glu Gln Pro Ser Glu Val Asn Thr Gly Pro Arg Tyr Glu Thr Ala 50 55 60 Arg Glu Val Ser Ser Arg Asp Ile Lys Glu Leu Glu Lys Ser Asn Lys65 70 75 80 Val Arg Asn Thr Asn Lys Ala Asp Leu Ile Ala Met Leu Lys Glu Lys 85 90 95 Ala Glu Lys Gly Pro Asn Ile Asn Asn Asn Asn Ser Glu Gln Thr Glu 100 105 110 Asn Ala Ala Ile Asn Glu Glu Ala Ser Gly Ala Asp Arg Pro Ala Ile 115 120 125 Gln Val Glu Arg Arg His Pro Gly Leu Pro Ser Asp Ser Ala Ala Glu 130 135 140 Ile Lys Lys Arg Arg Lys Ala Ile Ala Ser Ser Asp Ser Glu Leu Glu145 150 155 160 Ser Leu Thr Tyr Pro Asp Lys Pro Thr Lys Val Asn Lys Lys Lys Val 165 170 175 Ala Lys Glu Ser Val Ala Asp Ala Ser Glu Ser Asp Leu Asp Ser Ser 180 185 190 Met Gln Ser Ala Asp Glu Ser Ser Pro Gln Pro Leu Lys Ala Asn Gln 195 200 205 Gln Pro Phe Phe Pro Lys Val Phe Lys Lys Ile Lys Asp Ala Gly Lys 210 215 220 Trp Val Arg Asp Lys Ile Asp Glu Asn Pro Glu Val Lys Lys Ala Ile225 230 235 240 Val Asp Lys Ser Ala Gly Leu Ile Asp Gln Leu Leu Thr Lys Lys Lys 245 250 255 Ser Glu Glu Val Asn Ala Ser Asp Phe Pro Pro Pro Pro Thr Asp Glu 260 265 270 Glu Leu Arg Leu Ala Leu Pro Glu Thr Pro Met Leu Leu Gly Phe Asn 275 280 285 Ala Pro Ala Thr Ser Glu Pro Ser Ser Phe Glu Phe Pro Pro Pro Pro 290 295 300 Thr Asp Glu Glu Leu Arg Leu Ala Leu Pro Glu Thr Pro Met Leu Leu305 310 315 320 Gly Phe Asn Ala Pro Ala Thr Ser Glu Pro Ser Ser Phe Glu Phe Pro 325 330 335 Pro Pro Pro Thr Glu Asp Glu Leu Glu Ile Ile Arg Glu Thr Ala Ser 340 345 350 Ser Leu Asp Ser Ser Phe Thr Arg Gly Asp Leu Ala Ser Leu Arg Asn 355 360 365 Ala Ile Asn Arg His Ser Gln Asn Phe Ser Asp Phe Pro Pro Ile Pro 370 375 380 Thr Glu Glu Glu Leu Asn Gly Arg Gly Gly Arg Pro Thr Ser Glu Glu385 390 395 400 Phe Ser Ser Leu Asn Ser Gly Asp Phe Thr Asp Asp Glu Asn Ser Glu 405 410 415 Thr Thr Glu Glu Glu Ile Asp Arg Leu Ala Asp Leu Arg Asp Arg Gly 420 425 430 Thr Gly Lys His Ser Arg Asn Ala Gly Phe Leu Pro Leu Asn Pro Phe 435 440 445 Ala Ser Ser Pro Val Pro Ser Leu Ser Pro Lys Val Ser Lys Ile Ser 450 455 460 Ala Pro Ala Leu Ile Ser Asp Ile Thr Lys Lys Thr Pro Phe Lys Asn465 470 475 480 Pro Ser Gln Pro Leu Asn Val Phe Asn Lys Lys Thr Thr Thr Lys Thr 485 490 495 Val Thr Lys Lys Pro Thr Pro Val Lys Thr Ala Pro Lys Leu Ala Glu 500 505 510 Leu Pro Ala Thr Lys Pro Gln Glu Thr Val Leu Arg Glu Asn Lys Thr 515 520 525 Pro Phe Ile Glu Lys Gln Ala Glu Thr Asn Lys Gln Ser Ile Asn Met 530 535 540 Pro Ser Leu Pro Val Ile Gln Lys Glu Ala Thr Glu Ser Asp Lys Glu545 550 555 560 Glu Met Lys Pro Gln Thr Glu Glu Lys Met Val Glu Glu Ser Glu Ser 565 570 575 Ala Asn Asn Ala Asn Gly Lys Asn Arg Ser Ala Gly Ile Glu Glu Gly 580 585 590 Lys Leu Ile Ala Lys Ser Ala Glu Asp Glu Lys Ala Lys Glu Glu Pro 595 600 605 Gly Asn His Thr Thr Leu Ile Leu Ala Met Leu Ala Ile Gly Val Phe 610 615 620 Ser Leu Gly Ala Phe Ile Lys Ile Ile Gln Leu Arg Lys Asn Asn625 630 635 2630PRTListeria monocytogenes 2Met Lys Glu Lys His Asn Pro Arg Arg Lys Tyr Cys Leu Ile Ser Gly1 5 10 15 Leu Ala Ile Ile Phe Ser Leu Trp Ile Ile Ile Gly Asn Gly Ala Lys 20 25 30 Val Gln Ala Glu Thr Ile Thr Val Pro Thr Pro Ile Lys Gln Ile Phe 35 40 45 Ser Asp Asp Ala Phe Ala Glu Thr Ile Lys Asp Asn Leu Lys Lys Lys 50 55 60 Ser Val Thr Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln65 70 75 80 Ile Ile Ala Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr 85 90 95 Leu Pro Asn Val Thr Lys Leu Phe Leu Asn Gly Asn Lys Leu Thr Asp 100 105 110 Ile Lys Pro Leu Ala Asn Leu Lys Asn Leu Gly Trp Leu Phe Leu Asp 115 120 125 Glu Asn Lys Val Lys Asp Leu Ser Ser Leu Lys Asp Leu Lys Lys Leu 130 135 140 Lys Ser Leu Ser Leu Glu His Asn Gly Ile Ser Asp Ile Asn Gly Leu145 150 155 160 Val His Leu Pro Gln Leu Glu Ser Leu Tyr Leu Gly Asn Asn Lys Ile 165 170 175 Thr Asp Ile Thr Val Leu Ser Arg Leu Thr Lys Leu Asp Thr Leu Ser 180 185 190 Leu Glu Asp Asn Gln Ile Ser Asp Ile Val Pro Leu Ala Gly Leu Thr 195 200 205 Lys Leu Gln Asn Leu Tyr Leu Ser Lys Asn His Ile Ser Asp Leu Arg 210 215 220 Ala Leu Ala Gly Leu Lys Asn Leu Asp Val Leu Glu Leu Phe Ser Gln225 230 235 240 Glu Cys Leu Asn Lys Pro Ile Asn His Gln Ser Asn Leu Val Val Pro 245 250 255 Asn Thr Val Lys Asn Thr Asp Gly Ser Leu Val Thr Pro Glu Ile Ile 260 265 270 Ser Asp Asp Gly Asp Tyr Glu Lys Pro Asn Val Lys Trp His Leu Pro 275 280 285 Glu Phe Thr Asn Glu Val Ser Phe Ile Phe Tyr Gln Pro Val Thr Ile 290 295 300 Gly Lys Ala Lys Ala Arg Phe His Gly Arg Val Thr Gln Pro Leu Lys305 310 315 320 Glu Val Tyr Thr Val Ser Tyr Asp Val Asp Gly Thr Val Ile Lys Thr 325 330 335 Lys Val Glu Ala Gly Thr Arg Ile Thr Ala Pro Lys Pro Pro Thr Lys 340 345 350 Gln Gly Tyr Val Phe Lys Gly Trp Tyr Thr Glu Lys Asn Gly Gly His 355 360 365 Glu Trp Asn Phe Asn Thr Asp Tyr Met Ser Gly Asn Asp Phe Thr Leu 370 375 380 Tyr Ala Val Phe Lys Ala Glu Thr Thr Glu Lys Ala Val Asn Leu Thr385 390 395 400 Arg Tyr Val Lys Tyr Ile Arg Gly Asn Ala Gly Ile Tyr Lys Leu Pro 405 410 415 Arg Glu Asp Asn Ser Leu Lys Gln Gly Thr Leu Ala Ser His Arg Cys 420 425 430 Lys Ala Leu Thr Val Asp Arg Glu Ala Arg Asn Gly Gly Lys Leu Trp 435 440 445 Tyr Arg Leu Lys Asn Ile Gly Trp Thr Lys Ala Glu Asn Leu Ser Leu 450 455 460 Asp Arg Tyr Asp Lys Met Glu Tyr Asp Lys Gly Val Thr Ala Tyr Ala465 470 475 480 Arg Val Arg Asn Ala Ser Gly Asn Ser Val Trp Thr Lys Pro Tyr Asn 485 490 495 Thr Ala Gly Ala Lys His Val Asn Lys Leu Ser Val Tyr Gln Gly Lys 500 505 510 Asn Met Arg Ile Leu Arg Glu Ala Lys Thr Pro Ile Thr Thr Trp Tyr 515 520 525 Gln Phe Ser Ile Gly Gly Lys Val Ile Gly Trp Val Asp Thr Arg Ala 530 535 540 Leu Asn Thr Phe Tyr Lys Gln Ser Met Glu Lys Pro Thr Arg Leu Thr545 550 555 560 Arg Tyr Val Ser Ala Asn Lys Ala Gly Glu Ser Tyr Tyr Lys Val Pro 565 570 575 Val Ala Asp Asn Pro Val Lys Arg Gly Thr Leu Ala Lys Tyr Lys Asn 580 585 590 Gln Lys Leu Ile Val Asp Cys Gln Ala Thr Ile Glu Gly Gln Leu Trp 595 600 605 Tyr Arg Ile Arg Thr Ser Ser Thr Phe Ile Gly Trp Thr Lys Ala Ala 610 615 620 Asn Leu Arg Ala Gln Lys625 630 3237PRTListeria monocytogenes 3Met Asn Ala Gln Ala Glu Glu Phe Lys Lys Tyr Leu Glu Thr Asn Gly1 5 10 15 Ile Lys Pro Lys Gln Phe His Lys Lys Glu Leu Ile Phe Asn Gln Trp 20 25 30 Asp Pro Gln Glu Tyr Cys Ile Phe Leu Tyr Asp Gly Ile Thr Lys Leu 35 40 45 Thr Ser Ile Ser Glu Asn Gly Thr Ile Met Asn Leu Gln Tyr Tyr Lys 50 55 60 Gly Ala Phe Val Ile Met Ser Gly Phe Ile Asp Thr Glu Thr Ser Val65 70 75 80 Gly Tyr Tyr Asn Leu Glu Val Ile Ser Glu Gln Ala Thr Ala Tyr Val 85 90 95 Ile Lys Ile Asn Glu Leu Lys Glu Leu Leu Ser Lys Asn Leu Thr His 100 105 110 Phe Phe Tyr Val Phe Gln Thr Leu Gln Lys Gln Val Ser Tyr Ser Leu 115 120 125 Ala Lys Phe Asn Asp Phe Ser Ile Asn Gly Lys Leu Gly Ser Ile Cys 130 135 140 Gly Gln Leu Leu Ile Leu Thr Tyr Val Tyr Gly Lys Glu Thr Pro Asp145 150 155 160 Gly Ile Lys Ile Thr Leu Asp Asn Leu Thr Met Gln Glu Leu Gly Tyr 165 170 175 Ser Ser Gly Ile Ala His Ser Ser Ala Val Ser Arg Ile Ile Ser Lys 180 185 190 Leu Lys Gln Glu Lys Val Ile Val Tyr Lys Asn Ser Cys Phe Tyr Val 195 200 205 Gln Asn Leu Asp Tyr Leu Lys Arg Tyr Ala Pro Lys Leu Asp Glu Trp 210 215 220 Phe Tyr Leu Ala Cys Pro Ala Thr Trp Gly Lys Leu Asn225 230 235 4325PRTMycobacterium tuberculosis 4Met Thr Asp Val Ser Arg Lys Ile Arg Ala Trp Gly Arg Arg Leu Met1 5 10 15 Ile Gly Thr Ala Ala Ala Val Val Leu Pro Gly Leu Val Gly Leu Ala 20 25 30 Gly Gly Ala Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val 35 40 45 Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val 50 55 60 Gln Phe Gln Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp65 70 75 80 Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn Thr Pro 85 90 95 Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Ile Val Met Pro Val 100 105 110 Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly 115 120 125 Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu 130 135 140 Leu Pro Gln Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser145 150 155 160 Ala Ala Ile Gly Leu Ser Met Ala Gly Ser Ser Ala Met Ile Leu Ala 165 170 175 Ala Tyr His Pro Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu 180 185 190 Leu Asp Pro Ser Gln Gly Met Gly Pro Ser Leu Ile Gly Leu Ala Met 195 200 205 Gly Asp Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Ser Ser 210 215 220 Asp Pro Ala Trp Glu Arg Asn Asp Pro Thr Gln Gln Ile Pro Lys Leu225 230 235 240 Val Ala Asn Asn Thr Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro 245 250 255 Asn Glu Leu Gly Gly Ala Asn Ile Pro Ala Glu Phe Leu Glu Asn Phe 260 265 270 Val Arg Ser Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Ala Ala Gly 275 280 285 Gly His Asn Ala Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp 290 295 300 Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu Gln Ser305 310 315 320 Ser Leu Gly Ala Gly 325 5110PRTMycobacterium tuberculosis 5Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu Met Lys Glu Gly Arg1 5 10 15 Tyr Glu Val Arg Ala Glu Leu Pro Gly Val Asp Pro Asp Lys Asp Val 20 25 30 Asp Ile Met Val Arg Asp Gly Gln Leu Thr Ile Lys Ala Glu Arg Thr 35 40 45 Glu Gln Lys Asp Phe Asp Gly Arg Ser Glu Phe Ala Tyr Gly Ser Phe 50 55 60 Val Arg Thr Val Ser Leu Pro Val Gly Ala Asp Glu Asp Asp Ile Lys65 70 75 80 Ala Thr Tyr Asp Lys Gly Ile Leu Thr Val Ser Val Ala Val Ser Glu 85 90 95 Gly Lys Pro Thr Glu Lys His Ile Gln Ile Arg Ser Thr Asn 100 105 110 6173PRTMycobacterium tuberculosis 6Met Ser Leu Arg Leu Val Ser Pro Ile Lys Ala Phe Ala Asp Gly Ile1 5 10 15 Val Ala Val Ala Ile Ala Val Val Leu Met Phe Gly Leu Ala Asn Thr 20 25 30 Pro Arg Ala Val Ala Ala Asp Glu Arg Leu Gln Phe Thr Ala Thr Thr 35 40 45 Leu Ser Gly Ala Pro Phe Asp Gly Ala Ser Leu Gln Gly Lys Pro Ala 50 55 60 Val Leu Trp Phe Trp Thr Pro Trp Cys Pro Phe Cys Asn Ala Glu Ala65 70 75 80 Pro Ser Leu Ser Gln Val Ala Ala Ala Asn Pro Ala Val Thr Phe Val 85 90 95 Gly Ile Ala Thr Arg Ala Asp Val Gly Ala Met Gln Ser Phe Val Ser 100 105 110 Lys Tyr Asn Leu Asn Phe Thr Asn Leu Asn Asp Ala Asp Gly Val Ile 115 120 125 Trp Ala Arg Tyr Asn Val Pro Trp Gln Pro Ala Phe Val Phe Tyr Arg 130 135 140 Ala Asp Gly Thr Ser Thr Phe Val Asn Asn Pro Thr Ala Ala Met Ser145 150 155 160 Gln Asp Glu Leu Ser Gly Arg Val Ala Ala Leu Thr Ser 165 170 7159PRTMycobacterium tuberculosis 7Met Lys Leu Thr Thr Met Ile Lys Thr Ala Val Ala Val Val Ala Met1 5 10 15 Ala Ala Ile Ala Thr Phe Ala Ala Pro Val Ala Leu Ala Ala Tyr Pro 20 25 30 Ile Thr Gly Lys Leu Gly Ser Glu Leu Thr Met Thr Asp Thr Val Gly 35 40 45 Gln Val Val Leu Gly Trp Lys Val Ser Asp Leu Lys Ser Ser Thr Ala 50 55 60 Val Ile Pro Gly Tyr Pro Val Ala Gly Gln Val Trp Glu Ala Thr Ala65 70 75 80 Thr Val Asn Ala Ile Arg Gly Ser Val Thr Pro Ala Val Ser Gln Phe 85 90 95 Asn Ala Arg Thr Ala Asp Gly Ile Asn Tyr Arg Val Leu Trp Gln Ala 100 105 110 Ala Gly Pro Asp Thr Ile Ser Gly Ala Thr Ile Pro Gln Gly Glu Gln 115 120 125 Ser Thr Gly Lys Ile Tyr Phe Asp Val Thr Gly Pro Ser Pro Thr Ile 130 135 140 Val Ala Met Asn Asn Gly Met Glu Asp Leu Leu Ile Trp Glu Pro145 150 155

8207PRTMycobacterium tuberculosis 8Val Ala Glu Tyr Thr Leu Pro Asp Leu Asp Trp Asp Tyr Gly Ala Leu1 5 10 15 Glu Pro His Ile Ser Gly Gln Ile Asn Glu Leu His His Ser Lys His 20 25 30 His Ala Thr Tyr Val Lys Gly Ala Asn Asp Ala Val Ala Lys Leu Glu 35 40 45 Glu Ala Arg Ala Lys Glu Asp His Ser Ala Ile Leu Leu Asn Glu Lys 50 55 60 Asn Leu Ala Phe Asn Leu Ala Gly His Val Asn His Thr Ile Trp Trp65 70 75 80 Lys Asn Leu Ser Pro Asn Gly Gly Asp Lys Pro Thr Gly Glu Leu Ala 85 90 95 Ala Ala Ile Ala Asp Ala Phe Gly Ser Phe Asp Lys Phe Arg Ala Gln 100 105 110 Phe His Ala Ala Ala Thr Thr Val Gln Gly Ser Gly Trp Ala Ala Leu 115 120 125 Gly Trp Asp Thr Leu Gly Asn Lys Leu Leu Ile Phe Gln Val Tyr Asp 130 135 140 His Gln Thr Asn Phe Pro Leu Gly Ile Val Pro Leu Leu Leu Leu Asp145 150 155 160 Met Trp Glu His Ala Phe Tyr Leu Gln Tyr Lys Asn Val Lys Val Asp 165 170 175 Phe Ala Lys Ala Phe Trp Asn Val Val Asn Trp Ala Asp Val Gln Ser 180 185 190 Arg Tyr Ala Ala Ala Thr Ser Gln Thr Lys Gly Leu Ile Phe Gly 195 200 205 9228PRTMycobacterium tuberculosis 9Val Arg Ile Lys Ile Phe Met Leu Val Thr Ala Val Val Leu Leu Cys1 5 10 15 Cys Ser Gly Val Ala Thr Ala Ala Pro Lys Thr Tyr Cys Glu Glu Leu 20 25 30 Lys Gly Thr Asp Thr Gly Gln Ala Cys Gln Ile Gln Met Ser Asp Pro 35 40 45 Ala Tyr Asn Ile Asn Ile Ser Leu Pro Ser Tyr Tyr Pro Asp Gln Lys 50 55 60 Ser Leu Glu Asn Tyr Ile Ala Gln Thr Arg Asp Lys Phe Leu Ser Ala65 70 75 80 Ala Thr Ser Ser Thr Pro Arg Glu Ala Pro Tyr Glu Leu Asn Ile Thr 85 90 95 Ser Ala Thr Tyr Gln Ser Ala Ile Pro Pro Arg Gly Thr Gln Ala Val 100 105 110 Val Leu Lys Val Tyr Gln Asn Ala Gly Gly Thr His Pro Thr Thr Thr 115 120 125 Tyr Lys Ala Phe Asp Trp Asp Gln Ala Tyr Arg Lys Pro Ile Thr Tyr 130 135 140 Asp Thr Leu Trp Gln Ala Asp Thr Asp Pro Leu Pro Val Val Phe Pro145 150 155 160 Ile Val Gln Gly Glu Leu Ser Lys Gln Thr Gly Gln Gln Val Ser Ile 165 170 175 Ala Pro Asn Ala Gly Leu Asp Pro Val Asn Tyr Gln Asn Phe Ala Val 180 185 190 Thr Asn Asp Gly Val Ile Phe Phe Phe Asn Pro Gly Glu Leu Leu Pro 195 200 205 Glu Ala Ala Gly Pro Thr Gln Val Leu Val Pro Arg Ser Ala Ile Asp 210 215 220 Ser Met Leu Ala225 10299PRTMycobacterium tuberculosis 10Met Lys Gly Arg Ser Ala Leu Leu Arg Ala Leu Trp Ile Ala Ala Leu1 5 10 15 Ser Phe Gly Leu Gly Gly Val Ala Val Ala Ala Glu Pro Thr Ala Lys 20 25 30 Ala Ala Pro Tyr Glu Asn Leu Met Val Pro Ser Pro Ser Met Gly Arg 35 40 45 Asp Ile Pro Val Ala Phe Leu Ala Gly Gly Pro His Ala Val Tyr Leu 50 55 60 Leu Asp Ala Phe Asn Ala Gly Pro Asp Val Ser Asn Trp Val Thr Ala65 70 75 80 Gly Asn Ala Met Asn Thr Leu Ala Gly Lys Gly Ile Ser Val Val Ala 85 90 95 Pro Ala Gly Gly Ala Tyr Ser Met Tyr Thr Asn Trp Glu Gln Asp Gly 100 105 110 Ser Lys Gln Trp Asp Thr Phe Leu Ser Ala Glu Leu Pro Asp Trp Leu 115 120 125 Ala Ala Asn Arg Gly Leu Ala Pro Gly Gly His Ala Ala Val Gly Ala 130 135 140 Ala Gln Gly Gly Tyr Gly Ala Met Ala Leu Ala Ala Phe His Pro Asp145 150 155 160 Arg Phe Gly Phe Ala Gly Ser Met Ser Gly Phe Leu Tyr Pro Ser Asn 165 170 175 Thr Thr Thr Asn Gly Ala Ile Ala Ala Gly Met Gln Gln Phe Gly Gly 180 185 190 Val Asp Thr Asn Gly Met Trp Gly Ala Pro Gln Leu Gly Arg Trp Lys 195 200 205 Trp His Asp Pro Trp Val His Ala Ser Leu Leu Ala Gln Asn Asn Thr 210 215 220 Arg Val Trp Val Trp Ser Pro Thr Asn Pro Gly Ala Ser Asp Pro Ala220 5 230 235 240 Ala Met Ile Gly Gln Ala Ala Glu Ala Met Gly Asn Ser Arg Met Phe 245 250 255 Tyr Asn Gln Tyr Arg Ser Val Gly Gly His Asn Gly His Phe Asp Phe 260 265 270 Pro Ala Ser Gly Asp Asn Gly Trp Gly Ser Trp Ala Pro Gln Leu Gly 275 280 285 Ala Met Ser Gly Asp Ile Val Gly Ala Ile Arg 290 295 11338PRTMycobacterium tuberculosis 11Met Gln Leu Val Asp Arg Val Arg Gly Ala Val Thr Gly Met Ser Arg1 5 10 15 Arg Leu Val Val Gly Ala Val Gly Ala Ala Leu Val Ser Gly Leu Val 20 25 30 Gly Ala Val Gly Gly Thr Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly 35 40 45 Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp 50 55 60 Ile Lys Val Gln Phe Gln Ser Gly Gly Ala Asn Ser Pro Ala Leu Tyr65 70 75 80 Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Phe Ser Gly Trp Asp Ile 85 90 95 Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gln Ser Gly Leu Ser Val Val 100 105 110 Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Gln Pro 115 120 125 Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu 130 135 140 Thr Ser Glu Leu Pro Gly Trp Leu Gln Ala Asn Arg His Val Lys Pro145 150 155 160 Thr Gly Ser Ala Val Val Gly Leu Ser Met Ala Ala Ser Ser Ala Leu 165 170 175 Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Val Tyr Ala Gly Ala Met 180 185 190 Ser Gly Leu Leu Asp Pro Ser Gln Ala Met Gly Pro Thr Leu Ile Gly 195 200 205 Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ser Asp Met Trp Gly 210 215 220 Pro Lys Glu Asp Pro Ala Trp Gln Arg Asn Asp Pro Leu Leu Asn Val225 230 235 240 Gly Lys Leu Ile Ala Asn Asn Thr Arg Val Trp Val Tyr Cys Gly Asn 245 250 255 Gly Lys Pro Ser Asp Leu Gly Gly Asn Asn Leu Pro Ala Lys Phe Leu 260 265 270 Glu Gly Phe Val Arg Thr Ser Asn Ile Lys Phe Gln Asp Ala Tyr Asn 275 280 285 Ala Gly Gly Gly His Asn Gly Val Phe Asp Phe Pro Asp Ser Gly Thr 290 295 300 His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Pro Asp305 310 315 320 Leu Gln Arg Ala Leu Gly Ala Thr Pro Asn Thr Gly Pro Ala Pro Gln 325 330 335 Gly Ala12464PRTMycobacterium tuberculosis 12Met Ala Lys Leu Ala Arg Val Val Gly Leu Val Gln Glu Glu Gln Pro1 5 10 15 Ser Asp Met Thr Asn His Pro Arg Tyr Ser Pro Pro Pro Gln Gln Pro 20 25 30 Gly Thr Pro Gly Tyr Ala Gln Gly Gln Gln Gln Thr Tyr Ser Gln Gln 35 40 45 Phe Asp Trp Arg Tyr Pro Pro Ser Pro Pro Pro Gln Pro Thr Gln Tyr 50 55 60 Arg Gln Pro Tyr Glu Ala Leu Gly Gly Thr Arg Pro Gly Leu Ile Pro65 70 75 80 Gly Val Ile Pro Thr Met Thr Pro Pro Pro Gly Met Val Arg Gln Arg 85 90 95 Pro Arg Ala Gly Met Leu Ala Ile Gly Ala Val Thr Ile Ala Val Val 100 105 110 Ser Ala Gly Ile Gly Gly Ala Ala Ala Ser Leu Val Gly Phe Asn Arg 115 120 125 Ala Pro Ala Gly Pro Ser Gly Gly Pro Val Ala Ala Ser Ala Ala Pro 130 135 140 Ser Ile Pro Ala Ala Asn Met Pro Pro Gly Ser Val Glu Gln Val Ala145 150 155 160 Ala Lys Val Val Pro Ser Val Val Met Leu Glu Thr Asp Leu Gly Arg 165 170 175 Gln Ser Glu Glu Gly Ser Gly Ile Ile Leu Ser Ala Glu Gly Leu Ile 180 185 190 Leu Thr Asn Asn His Val Ile Ala Ala Ala Ala Lys Pro Pro Leu Gly 195 200 205 Ser Pro Pro Pro Lys Thr Thr Val Thr Phe Ser Asp Gly Arg Thr Ala 210 215 220 Pro Phe Thr Val Val Gly Ala Asp Pro Thr Ser Asp Ile Ala Val Val225 230 235 240 Arg Val Gln Gly Val Ser Gly Leu Thr Pro Ile Ser Leu Gly Ser Ser 245 250 255 Ser Asp Leu Arg Val Gly Gln Pro Val Leu Ala Ile Gly Ser Pro Leu 260 265 270 Gly Leu Glu Gly Thr Val Thr Thr Gly Ile Val Ser Ala Leu Asn Arg 275 280 285 Pro Val Ser Thr Thr Gly Glu Ala Gly Asn Gln Asn Thr Val Leu Asp 290 295 300 Ala Ile Gln Thr Asp Ala Ala Ile Asn Pro Gly Asn Ser Gly Gly Ala305 310 315 320 Leu Val Asn Met Asn Ala Gln Leu Val Gly Val Asn Ser Ala Ile Ala 325 330 335 Thr Leu Gly Ala Asp Ser Ala Asp Ala Gln Ser Gly Ser Ile Gly Leu 340 345 350 Gly Phe Ala Ile Pro Val Asp Gln Ala Lys Arg Ile Ala Asp Glu Leu 355 360 365 Ile Ser Thr Gly Lys Ala Ser His Ala Ser Leu Gly Val Gln Val Thr 370 375 380 Asn Asp Lys Asp Thr Leu Gly Ala Lys Ile Val Glu Val Val Ala Gly385 390 395 400 Gly Ala Ala Ala Asn Ala Gly Val Pro Lys Gly Val Val Val Thr Lys 405 410 415 Val Asp Asp Arg Pro Ile Asn Ser Ala Asp Ala Leu Val Ala Ala Val 420 425 430 Arg Ser Lys Ala Pro Gly Ala Thr Val Ala Leu Thr Phe Gln Asp Pro 435 440 445 Ser Gly Gly Ser Arg Thr Val Gln Val Thr Leu Gly Lys Ala Glu Gln 450 455 460 13325PRTMycobacterium tuberculosis 13Met His Gln Val Asp Pro Asn Leu Thr Arg Arg Lys Gly Arg Leu Ala1 5 10 15 Ala Leu Ala Ile Ala Ala Met Ala Ser Ala Ser Leu Val Thr Val Ala 20 25 30 Val Pro Ala Thr Ala Asn Ala Asp Pro Glu Pro Ala Pro Pro Val Pro 35 40 45 Thr Thr Ala Ala Ser Pro Pro Ser Thr Ala Ala Ala Pro Pro Ala Pro 50 55 60 Ala Thr Pro Val Ala Pro Pro Pro Pro Ala Ala Ala Asn Thr Pro Asn65 70 75 80 Ala Gln Pro Gly Asp Pro Asn Ala Ala Pro Pro Pro Ala Asp Pro Asn 85 90 95 Ala Pro Pro Pro Pro Val Ile Ala Pro Asn Ala Pro Gln Pro Val Arg 100 105 110 Ile Asp Asn Pro Val Gly Gly Phe Ser Phe Ala Leu Pro Ala Gly Trp 115 120 125 Val Glu Ser Asp Ala Ala His Phe Asp Tyr Gly Ser Ala Leu Leu Ser 130 135 140 Lys Thr Thr Gly Asp Pro Pro Phe Pro Gly Gln Pro Pro Pro Val Ala145 150 155 160 Asn Asp Thr Arg Ile Val Leu Gly Arg Leu Asp Gln Lys Leu Tyr Ala 165 170 175 Ser Ala Glu Ala Thr Asp Ser Lys Ala Ala Ala Arg Leu Gly Ser Asp 180 185 190 Met Gly Glu Phe Tyr Met Pro Tyr Pro Gly Thr Arg Ile Asn Gln Glu 195 200 205 Thr Val Ser Leu Asp Ala Asn Gly Val Ser Gly Ser Ala Ser Tyr Tyr 210 215 220 Glu Val Lys Phe Ser Asp Pro Ser Lys Pro Asn Gly Gln Ile Trp Thr225 230 235 240 Gly Val Ile Gly Ser Pro Ala Ala Asn Ala Pro Asp Ala Gly Pro Pro 245 250 255 Gln Arg Trp Phe Val Val Trp Leu Gly Thr Ala Asn Asn Pro Val Asp 260 265 270 Lys Gly Ala Ala Lys Ala Leu Ala Glu Ser Ile Arg Pro Leu Val Ala 275 280 285 Pro Pro Pro Ala Pro Ala Pro Ala Pro Ala Glu Pro Ala Pro Ala Pro 290 295 300 Ala Pro Ala Gly Glu Val Ala Pro Thr Pro Thr Thr Pro Thr Pro Gln305 310 315 320 Arg Thr Leu Pro Ala 325 14478PRTMycobacterium tuberculosis 14Val Thr Glu Lys Thr Pro Asp Asp Val Phe Lys Leu Ala Lys Asp Glu1 5 10 15 Lys Val Glu Tyr Val Asp Val Arg Phe Cys Asp Leu Pro Gly Ile Met 20 25 30 Gln His Phe Thr Ile Pro Ala Ser Ala Phe Asp Lys Ser Val Phe Asp 35 40 45 Asp Gly Leu Ala Phe Asp Gly Ser Ser Ile Arg Gly Phe Gln Ser Ile 50 55 60 His Glu Ser Asp Met Leu Leu Leu Pro Asp Pro Glu Thr Ala Arg Ile65 70 75 80 Asp Pro Phe Arg Ala Ala Lys Thr Leu Asn Ile Asn Phe Phe Val His 85 90 95 Asp Pro Phe Thr Leu Glu Pro Tyr Ser Arg Asp Pro Arg Asn Ile Ala 100 105 110 Arg Lys Ala Glu Asn Tyr Leu Ile Ser Thr Gly Ile Ala Asp Thr Ala 115 120 125 Tyr Phe Gly Ala Glu Ala Glu Phe Tyr Ile Phe Asp Ser Val Ser Phe 130 135 140 Asp Ser Arg Ala Asn Gly Ser Phe Tyr Glu Val Asp Ala Ile Ser Gly145 150 155 160 Trp Trp Asn Thr Gly Ala Ala Thr Glu Ala Asp Gly Ser Pro Asn Arg 165 170 175 Gly Tyr Lys Val Arg His Lys Gly Gly Tyr Phe Pro Val Ala Pro Asn 180 185 190 Asp Gln Tyr Val Asp Leu Arg Asp Lys Met Leu Thr Asn Leu Ile Asn 195 200 205 Ser Gly Phe Ile Leu Glu Lys Gly His His Glu Val Gly Ser Gly Gly 210 215 220 Gln Ala Glu Ile Asn Tyr Gln Phe Asn Ser Leu Leu His Ala Ala Asp225 230 235 240 Asp Met Gln Leu Tyr Lys Tyr Ile Ile Lys Asn Thr Ala Trp Gln Asn 245 250 255 Gly Lys Thr Val Thr Phe Met Pro Lys Pro Leu Phe Gly Asp Asn Gly 260 265 270 Ser Gly Met His Cys His Gln Ser Leu Trp Lys Asp Gly Ala Pro Leu 275 280 285 Met Tyr Asp Glu Thr Gly Tyr Ala Gly Leu Ser Asp Thr Ala Arg His 290 295 300 Tyr Ile Gly Gly Leu Leu His His Ala Pro Ser Leu Leu Ala Phe Thr305 310 315 320 Asn Pro Thr Val Asn Ser Tyr Lys Arg Leu Val Pro Gly Tyr Glu Ala 325 330 335 Pro Ile Asn Leu Val Tyr Ser Gln Arg Asn Arg Ser Ala Cys Val Arg 340 345 350 Ile Pro Ile Thr Gly Ser Asn Pro Lys Ala Lys Arg Leu Glu Phe Arg 355 360 365 Ser Pro Asp Ser Ser Gly Asn Pro Tyr Leu Ala Phe Ser Ala Met Leu 370 375 380 Met Ala Gly Leu Asp Gly Ile Lys Asn Lys Ile Glu Pro Gln Ala Pro385 390 395 400 Val Asp Lys Asp Leu Tyr Glu Leu Pro Pro Glu Glu Ala Ala Ser Ile 405

410 415 Pro Gln Thr Pro Thr Gln Leu Ser Asp Val Ile Asp Arg Leu Glu Ala 420 425 430 Asp His Glu Tyr Leu Thr Glu Gly Gly Val Phe Thr Asn Asp Leu Ile 435 440 445 Glu Thr Trp Ile Ser Phe Lys Arg Glu Asn Glu Ile Glu Pro Val Asn 450 455 460 Ile Arg Pro His Pro Tyr Glu Phe Ala Leu Tyr Tyr Asp Val465 470 475 15625PRTMycobacterium tuberculosis 15Met Ala Arg Ala Val Gly Ile Asp Leu Gly Thr Thr Asn Ser Val Val1 5 10 15 Ser Val Leu Glu Gly Gly Asp Pro Val Val Val Ala Asn Ser Glu Gly 20 25 30 Ser Arg Thr Thr Pro Ser Ile Val Ala Phe Ala Arg Asn Gly Glu Val 35 40 45 Leu Val Gly Gln Pro Ala Lys Asn Gln Ala Val Thr Asn Val Asp Arg 50 55 60 Thr Val Arg Ser Val Lys Arg His Met Gly Ser Asp Trp Ser Ile Glu65 70 75 80 Ile Asp Gly Lys Lys Tyr Thr Ala Pro Glu Ile Ser Ala Arg Ile Leu 85 90 95 Met Lys Leu Lys Arg Asp Ala Glu Ala Tyr Leu Gly Glu Asp Ile Thr 100 105 110 Asp Ala Val Ile Thr Thr Pro Ala Tyr Phe Asn Asp Ala Gln Arg Gln 115 120 125 Ala Thr Lys Asp Ala Gly Gln Ile Ala Gly Leu Asn Val Leu Arg Ile 130 135 140 Val Asn Glu Pro Thr Ala Ala Ala Leu Ala Tyr Gly Leu Asp Lys Gly145 150 155 160 Glu Lys Glu Gln Arg Ile Leu Val Phe Asp Leu Gly Gly Gly Thr Phe 165 170 175 Asp Val Ser Leu Leu Glu Ile Gly Glu Gly Val Val Glu Val Arg Ala 180 185 190 Thr Ser Gly Asp Asn His Leu Gly Gly Asp Asp Trp Asp Gln Arg Val 195 200 205 Val Asp Trp Leu Val Asp Lys Phe Lys Gly Thr Ser Gly Ile Asp Leu 210 215 220 Thr Lys Asp Lys Met Ala Met Gln Arg Leu Arg Glu Ala Ala Glu Lys225 230 235 240 Ala Lys Ile Glu Leu Ser Ser Ser Gln Ser Thr Ser Ile Asn Leu Pro 245 250 255 Tyr Ile Thr Val Asp Ala Asp Lys Asn Pro Leu Phe Leu Asp Glu Gln 260 265 270 Leu Thr Arg Ala Glu Phe Gln Arg Ile Thr Gln Asp Leu Leu Asp Arg 275 280 285 Thr Arg Lys Pro Phe Gln Ser Val Ile Ala Asp Thr Gly Ile Ser Val 290 295 300 Ser Glu Ile Asp His Val Val Leu Val Gly Gly Ser Thr Arg Met Pro305 310 315 320 Ala Val Thr Asp Leu Val Lys Glu Leu Thr Gly Gly Lys Glu Pro Asn 325 330 335 Lys Gly Val Asn Pro Asp Glu Val Val Ala Val Gly Ala Ala Leu Gln 340 345 350 Ala Gly Val Leu Lys Gly Glu Val Lys Asp Val Leu Leu Leu Asp Val 355 360 365 Thr Pro Leu Ser Leu Gly Ile Glu Thr Lys Gly Gly Val Met Thr Arg 370 375 380 Leu Ile Glu Arg Asn Thr Thr Ile Pro Thr Lys Arg Ser Glu Thr Phe385 390 395 400 Thr Thr Ala Asp Asp Asn Gln Pro Ser Val Gln Ile Gln Val Tyr Gln 405 410 415 Gly Glu Arg Glu Ile Ala Ala His Asn Lys Leu Leu Gly Ser Phe Glu 420 425 430 Leu Thr Gly Ile Pro Pro Ala Pro Arg Gly Ile Pro Gln Ile Glu Val 435 440 445 Thr Phe Asp Ile Asp Ala Asn Gly Ile Val His Val Thr Ala Lys Asp 450 455 460 Lys Gly Thr Gly Lys Glu Asn Thr Ile Arg Ile Gln Glu Gly Ser Gly465 470 475 480 Leu Ser Lys Glu Asp Ile Asp Arg Met Ile Lys Asp Ala Glu Ala His 485 490 495 Ala Glu Glu Asp Arg Lys Arg Arg Glu Glu Ala Asp Val Arg Asn Gln 500 505 510 Ala Glu Thr Leu Val Tyr Gln Thr Glu Lys Phe Val Lys Glu Gln Arg 515 520 525 Glu Ala Glu Gly Gly Ser Lys Val Pro Glu Asp Thr Leu Asn Lys Val 530 535 540 Asp Ala Ala Val Ala Glu Ala Lys Ala Ala Leu Gly Gly Ser Asp Ile545 550 555 560 Ser Ala Ile Lys Ser Ala Met Glu Lys Leu Gly Gln Glu Ser Gln Ala 565 570 575 Leu Gly Gln Ala Ile Tyr Glu Ala Ala Gln Ala Ala Ser Gln Ala Thr 580 585 590 Gly Ala Ala His Pro Gly Gly Glu Pro Gly Gly Ala His Pro Gly Ser 595 600 605 Ala Asp Asp Val Val Asp Ala Glu Val Val Asp Asp Gly Arg Glu Ala 610 615 620 Lys625 1696PRTMycobacterium tuberculosis 16Met Ser Gln Ile Met Tyr Asn Tyr Pro Ala Met Leu Gly His Ala Gly1 5 10 15 Asp Met Ala Gly Tyr Ala Gly Thr Leu Gln Ser Leu Gly Ala Glu Ile 20 25 30 Ala Val Glu Gln Ala Ala Leu Gln Ser Ala Trp Gln Gly Asp Thr Gly 35 40 45 Ile Thr Tyr Gln Ala Trp Gln Ala Gln Trp Asn Gln Ala Met Glu Asp 50 55 60 Leu Val Arg Ala Tyr His Ala Met Ser Ser Thr His Glu Ala Asn Thr65 70 75 80 Met Ala Met Met Ala Arg Asp Thr Ala Glu Ala Ala Lys Trp Gly Gly 85 90 95 17144PRTMycobacterium tuberculosis 17Met Ala Thr Thr Leu Pro Val Gln Arg His Pro Arg Ser Leu Phe Pro1 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 Glu65 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 18428PRTMycobacterium tuberculosis 18Met Ser Val Val Gly Thr Pro Lys Ser Ala Glu Gln Ile Gln Gln Glu1 5 10 15 Trp Asp Thr Asn Pro Arg Trp Lys Asp Val Thr Arg Thr Tyr Ser Ala 20 25 30 Glu Asp Val Val Ala Leu Gln Gly Ser Val Val Glu Glu His Thr Leu 35 40 45 Ala Arg Arg Gly Ala Glu Val Leu Trp Glu Gln Leu His Asp Leu Glu 50 55 60 Trp Val Asn Ala Leu Gly Ala Leu Thr Gly Asn Met Ala Val Gln Gln65 70 75 80 Val Arg Ala Gly Leu Lys Ala Ile Tyr Leu Ser Gly Trp Gln Val Ala 85 90 95 Gly Asp Ala Asn Leu Ser Gly His Thr Tyr Pro Asp Gln Ser Leu Tyr 100 105 110 Pro Ala Asn Ser Val Pro Gln Val Val Arg Arg Ile Asn Asn Ala Leu 115 120 125 Gln Arg Ala Asp Gln Ile Ala Lys Ile Glu Gly Asp Thr Ser Val Glu 130 135 140 Asn Trp Leu Ala Pro Ile Val Ala Asp Gly Glu Ala Gly Phe Gly Gly145 150 155 160 Ala Leu Asn Val Tyr Glu Leu Gln Lys Ala Leu Ile Ala Ala Gly Val 165 170 175 Ala Gly Ser His Trp Glu Asp Gln Leu Ala Ser Glu Lys Lys Cys Gly 180 185 190 His Leu Gly Gly Lys Val Leu Ile Pro Thr Gln Gln His Ile Arg Thr 195 200 205 Leu Thr Ser Ala Arg Leu Ala Ala Asp Val Ala Asp Val Pro Thr Val 210 215 220 Val Ile Ala Arg Thr Asp Ala Glu Ala Ala Thr Leu Ile Thr Ser Asp225 230 235 240 Val Asp Glu Arg Asp Gln Pro Phe Ile Thr Gly Glu Arg Thr Arg Glu 245 250 255 Gly Phe Tyr Arg Thr Lys Asn Gly Ile Glu Pro Cys Ile Ala Arg Ala 260 265 270 Lys Ala Tyr Ala Pro Phe Ala Asp Leu Ile Trp Met Glu Thr Gly Thr 275 280 285 Pro Asp Leu Glu Ala Ala Arg Gln Phe Ser Glu Ala Val Lys Ala Glu 290 295 300 Tyr Pro Asp Gln Met Leu Ala Tyr Asn Cys Ser Pro Ser Phe Asn Trp305 310 315 320 Lys Lys His Leu Asp Asp Ala Thr Ile Ala Lys Phe Gln Lys Glu Leu 325 330 335 Ala Ala Met Gly Phe Lys Phe Gln Phe Ile Thr Leu Ala Gly Phe His 340 345 350 Ala Leu Asn Tyr Ser Met Phe Asp Leu Ala Tyr Gly Tyr Ala Gln Asn 355 360 365 Gln Met Ser Ala Tyr Val Glu Leu Gln Glu Arg Glu Phe Ala Ala Glu 370 375 380 Glu Arg Gly Tyr Thr Ala Thr Lys His Gln Arg Glu Val Gly Ala Gly385 390 395 400 Tyr Phe Asp Arg Ile Ala Thr Thr Val Asp Pro Asn Ser Ser Thr Thr 405 410 415 Ala Leu Thr Gly Ser Thr Glu Glu Gly Gln Phe His 420 425 1975PRTMycobacterium tuberculosis 19Val Ile Ala Gly Val Asp Gln Ala Leu Ala Ala Thr Gly Gln Ala Ser1 5 10 15 Gln Arg Ala Ala Gly Ala Ser Gly Gly Val Thr Val Gly Val Gly Val 20 25 30 Gly Thr Glu Gln Arg Asn Leu Ser Val Val Ala Pro Ser Gln Phe Thr 35 40 45 Phe Ser Ser Arg Ser Pro Asp Phe Val Asp Glu Thr Ala Gly Gln Ser 50 55 60 Trp Cys Ala Ile Leu Gly Leu Asn Gln Phe His65 70 75 20741PRTMycobacterium tuberculosis 20Met Thr Asp Arg Val Ser Val Gly Asn Leu Arg Ile Ala Arg Val Leu1 5 10 15 Tyr Asp Phe Val Asn Asn Glu Ala Leu Pro Gly Thr Asp Ile Asp Pro 20 25 30 Asp Ser Phe Trp Ala Gly Val Asp Lys Val Val Ala Asp Leu Thr Pro 35 40 45 Gln Asn Gln Ala Leu Leu Asn Ala Arg Asp Glu Leu Gln Ala Gln Ile 50 55 60 Asp Lys Trp His Arg Arg Arg Val Ile Glu Pro Ile Asp Met Asp Ala65 70 75 80 Tyr Arg Gln Phe Leu Thr Glu Ile Gly Tyr Leu Leu Pro Glu Pro Asp 85 90 95 Asp Phe Thr Ile Thr Thr Ser Gly Val Asp Ala Glu Ile Thr Thr Thr 100 105 110 Ala Gly Pro Gln Leu Val Val Pro Val Leu Asn Ala Arg Phe Ala Leu 115 120 125 Asn Ala Ala Asn Ala Arg Trp Gly Ser Leu Tyr Asp Ala Leu Tyr Gly 130 135 140 Thr Asp Val Ile Pro Glu Thr Asp Gly Ala Glu Lys Gly Pro Thr Tyr145 150 155 160 Asn Lys Val Arg Gly Asp Lys Val Ile Ala Tyr Ala Arg Lys Phe Leu 165 170 175 Asp Asp Ser Val Pro Leu Ser Ser Gly Ser Phe Gly Asp Ala Thr Gly 180 185 190 Phe Thr Val Gln Asp Gly Gln Leu Val Val Ala Leu Pro Asp Lys Ser 195 200 205 Thr Gly Leu Ala Asn Pro Gly Gln Phe Ala Gly Tyr Thr Gly Ala Ala 210 215 220 Glu Ser Pro Thr Ser Val Leu Leu Ile Asn His Gly Leu His Ile Glu225 230 235 240 Ile Leu Ile Asp Pro Glu Ser Gln Val Gly Thr Thr Asp Arg Ala Gly 245 250 255 Val Lys Asp Val Ile Leu Glu Ser Ala Ile Thr Thr Ile Met Asp Phe 260 265 270 Glu Asp Ser Val Ala Ala Val Asp Ala Ala Asp Lys Val Leu Gly Tyr 275 280 285 Arg Asn Trp Leu Gly Leu Asn Lys Gly Asp Leu Ala Ala Ala Val Asp 290 295 300 Lys Asp Gly Thr Ala Phe Leu Arg Val Leu Asn Arg Asp Arg Asn Tyr305 310 315 320 Thr Ala Pro Gly Gly Gly Gln Phe Thr Leu Pro Gly Arg Ser Leu Met 325 330 335 Phe Val Arg Asn Val Gly His Leu Met Thr Asn Asp Ala Ile Val Asp 340 345 350 Thr Asp Gly Ser Glu Val Phe Glu Gly Ile Met Asp Ala Leu Phe Thr 355 360 365 Gly Leu Ile Ala Ile His Gly Leu Lys Ala Ser Asp Val Asn Gly Pro 370 375 380 Leu Ile Asn Ser Arg Thr Gly Ser Ile Tyr Ile Val Lys Pro Lys Met385 390 395 400 His Gly Pro Ala Glu Val Ala Phe Thr Cys Glu Leu Phe Ser Arg Val 405 410 415 Glu Asp Val Leu Gly Leu Pro Gln Asn Thr Met Lys Ile Gly Ile Met 420 425 430 Asp Glu Glu Arg Arg Thr Thr Val Asn Leu Lys Ala Cys Ile Lys Ala 435 440 445 Ala Ala Asp Arg Val Val Phe Ile Asn Thr Gly Phe Leu Asp Arg Thr 450 455 460 Gly Asp Glu Ile His Thr Ser Met Glu Ala Gly Pro Met Val Arg Lys465 470 475 480 Gly Thr Met Lys Ser Gln Pro Trp Ile Leu Ala Tyr Glu Asp His Asn 485 490 495 Val Asp Ala Gly Leu Ala Ala Gly Phe Ser Gly Arg Ala Gln Val Gly 500 505 510 Lys Gly Met Trp Thr Met Thr Glu Leu Met Ala Asp Met Val Glu Thr 515 520 525 Lys Ile Ala Gln Pro Arg Ala Gly Ala Ser Thr Ala Trp Val Pro Ser 530 535 540 Pro Thr Ala Ala Thr Leu His Ala Leu His Tyr His Gln Val Asp Val545 550 555 560 Ala Ala Val Gln Gln Gly Leu Ala Gly Lys Arg Arg Ala Thr Ile Glu 565 570 575 Gln Leu Leu Thr Ile Pro Leu Ala Lys Glu Leu Ala Trp Ala Pro Asp 580 585 590 Glu Ile Arg Glu Glu Val Asp Asn Asn Cys Gln Ser Ile Leu Gly Tyr 595 600 605 Val Val Arg Trp Val Asp Gln Gly Val Gly Cys Ser Lys Val Pro Asp 610 615 620 Ile His Asp Val Ala Leu Met Glu Asp Arg Ala Thr Leu Arg Ile Ser625 630 635 640 Ser Gln Leu Leu Ala Asn Trp Leu Arg His Gly Val Ile Thr Ser Ala 645 650 655 Asp Val Arg Ala Ser Leu Glu Arg Met Ala Pro Leu Val Asp Arg Gln 660 665 670 Asn Ala Gly Asp Val Ala Tyr Arg Pro Met Ala Pro Asn Phe Asp Asp 675 680 685 Ser Ile Ala Phe Leu Ala Ala Gln Glu Leu Ile Leu Ser Gly Ala Gln 690 695 700 Gln Pro Asn Gly Tyr Thr Glu Pro Ile Leu His Arg Arg Arg Arg Glu705 710 715 720 Phe Lys Ala Arg Ala Ala Glu Lys Pro Ala Pro Ser Asp Arg Ala Gly 725 730 735 Asp Asp Ala Ala Arg 740 21943PRTMycobacterium tuberculosis 21Val Thr Ser Lys Ser Val Asn Ser Phe Gly Ala His Asp Thr Leu Lys1 5 10 15 Val Gly Glu Lys Ser Tyr Gln Ile Tyr Arg Leu Asp Ala Val Pro Asn 20 25 30 Thr Ala Lys Leu Pro Tyr Ser Leu Lys Val Leu Ala Glu Asn Leu Leu 35 40 45 Arg Asn Glu Asp Gly Ser Asn Ile Thr Lys Asp His Ile Glu Ala Ile 50 55 60 Ala Asn Trp Asp Pro Lys Ala Glu Pro Ser Ile Glu Ile Gln Tyr Thr65 70 75 80 Pro Ala Arg Val Val Met Gln Asp Phe Thr Gly Val Pro Cys Ile Val 85 90 95 Asp Leu Ala Thr Met Arg Glu Ala Ile Ala Asp Leu Gly Gly Asn Pro

100 105 110 Asp Lys Val Asn Pro Leu Ala Pro Ala Asp Leu Val Ile Asp His Ser 115 120 125 Val Ile Ala Asp Leu Phe Gly Arg Ala Asp Ala Phe Glu Arg Asn Val 130 135 140 Glu Ile Glu Tyr Gln Arg Asn Gly Glu Arg Tyr Gln Phe Leu Arg Trp145 150 155 160 Gly Gln Gly Ala Phe Asp Asp Phe Lys Val Val Pro Pro Gly Thr Gly 165 170 175 Ile Val His Gln Val Asn Ile Glu Tyr Leu Ala Ser Val Val Met Thr 180 185 190 Arg Asp Gly Val Ala Tyr Pro Asp Thr Cys Val Gly Thr Asp Ser His 195 200 205 Thr Thr Met Val Asn Gly Leu Gly Val Leu Gly Trp Gly Val Gly Gly 210 215 220 Ile Glu Ala Glu Ala Ala Met Leu Gly Gln Pro Val Ser Met Leu Ile225 230 235 240 Pro Arg Val Val Gly Phe Arg Leu Thr Gly Glu Ile Gln Pro Gly Val 245 250 255 Thr Ala Thr Asp Val Val Leu Thr Val Thr Glu Met Leu Arg Gln His 260 265 270 Gly Val Val Gly Lys Phe Val Glu Phe Tyr Gly Glu Gly Val Ala Glu 275 280 285 Val Pro Leu Ala Asn Arg Ala Thr Leu Gly Asn Met Ser Pro Glu Phe 290 295 300 Gly Ser Thr Ala Ala Ile Phe Pro Ile Asp Glu Glu Thr Ile Lys Tyr305 310 315 320 Leu Arg Phe Thr Gly Arg Thr Pro Glu Gln Val Ala Leu Val Glu Ala 325 330 335 Tyr Ala Lys Ala Gln Gly Met Trp His Asp Pro Lys His Glu Pro Glu 340 345 350 Phe Ser Glu Tyr Leu Glu Leu Asn Leu Ser Asp Val Val Pro Ser Ile 355 360 365 Ala Gly Pro Lys Arg Pro Gln Asp Arg Ile Ala Leu Ala Gln Ala Lys 370 375 380 Ser Thr Phe Arg Glu Gln Ile Tyr His Tyr Val Gly Asn Gly Ser Pro385 390 395 400 Asp Ser Pro His Asp Pro His Ser Lys Leu Asp Glu Val Val Glu Glu 405 410 415 Thr Phe Pro Ala Ser Asp Pro Gly Gln Leu Thr Phe Ala Asn Asp Asp 420 425 430 Val Ala Thr Asp Glu Thr Val His Ser Ala Ala Ala His Ala Asp Gly 435 440 445 Arg Val Ser Asn Pro Val Arg Val Lys Ser Asp Glu Leu Gly Glu Phe 450 455 460 Val Leu Asp His Gly Ala Val Val Ile Ala Ala Ile Thr Ser Cys Thr465 470 475 480 Asn Thr Ser Asn Pro Glu Val Met Leu Gly Ala Ala Leu Leu Ala Arg 485 490 495 Asn Ala Val Glu Lys Gly Leu Thr Ser Lys Pro Trp Val Lys Thr Thr 500 505 510 Ile Ala Pro Gly Ser Gln Val Val Asn Asp Tyr Tyr Asp Arg Ser Gly 515 520 525 Leu Trp Pro Tyr Leu Glu Lys Leu Gly Phe Tyr Leu Val Gly Tyr Gly 530 535 540 Cys Thr Thr Cys Ile Gly Asn Ser Gly Pro Leu Pro Glu Glu Ile Ser545 550 555 560 Lys Ala Val Asn Asp Asn Asp Leu Ser Val Thr Ala Val Leu Ser Gly 565 570 575 Asn Arg Asn Phe Glu Gly Arg Ile Asn Pro Asp Val Lys Met Asn Tyr 580 585 590 Leu Ala Ser Pro Pro Leu Val Ile Ala Tyr Ala Leu Ala Gly Thr Met 595 600 605 Asp Phe Asp Phe Gln Thr Gln Pro Leu Gly Gln Asp Lys Asp Gly Lys 610 615 620 Asn Val Phe Leu Arg Asp Ile Trp Pro Ser Gln Gln Asp Val Ser Asp625 630 635 640 Thr Ile Ala Ala Ala Ile Asn Gln Glu Met Phe Thr Arg Asn Tyr Ala 645 650 655 Asp Val Phe Lys Gly Asp Asp Arg Trp Arg Asn Leu Pro Thr Pro Ser 660 665 670 Gly Asn Thr Phe Glu Trp Asp Pro Asn Ser Thr Tyr Val Arg Lys Pro 675 680 685 Pro Tyr Phe Glu Gly Met Thr Ala Lys Pro Glu Pro Val Gly Asn Ile 690 695 700 Ser Gly Ala Arg Val Leu Ala Leu Leu Gly Asp Ser Val Thr Thr Asp705 710 715 720 His Ile Ser Pro Ala Gly Ala Ile Lys Pro Gly Thr Pro Ala Ala Arg 725 730 735 Tyr Leu Asp Glu His Gly Val Asp Arg Lys Asp Tyr Asn Ser Phe Gly 740 745 750 Ser Arg Arg Gly Asn His Glu Val Met Ile Arg Gly Thr Phe Ala Asn 755 760 765 Ile Arg Leu Arg Asn Gln Leu Leu Asp Asp Val Ser Gly Gly Tyr Thr 770 775 780 Arg Asp Phe Thr Gln Pro Gly Gly Pro Gln Ala Phe Ile Tyr Asp Ala785 790 795 800 Ala Gln Asn Tyr Ala Ala Gln His Ile Pro Leu Val Val Phe Gly Gly 805 810 815 Lys Glu Tyr Gly Ser Gly Ser Ser Arg Asp Trp Ala Ala Lys Gly Thr 820 825 830 Leu Leu Leu Gly Val Arg Ala Val Ile Ala Glu Ser Phe Glu Arg Ile 835 840 845 His Arg Ser Asn Leu Ile Gly Met Gly Val Ile Pro Leu Gln Phe Pro 850 855 860 Glu Gly Lys Ser Ala Ser Ser Leu Gly Leu Asp Gly Thr Glu Val Phe865 870 875 880 Asp Ile Thr Gly Ile Asp Val Leu Asn Asp Gly Lys Thr Pro Lys Thr 885 890 895 Val Cys Val Gln Ala Thr Lys Gly Asp Gly Ala Thr Ile Glu Phe Asp 900 905 910 Ala Val Val Arg Ile Asp Thr Pro Gly Glu Ala Asp Tyr Tyr Arg Asn 915 920 925 Gly Gly Ile Leu Gln Tyr Val Leu Arg Asn Ile Leu Lys Ser Gly 930 935 940 2295PRTMycobacterium tuberculosis 22Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser1 5 10 15 Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly 20 25 30 Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser 35 40 45 Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu 50 55 60 Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly65 70 75 80 Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala 85 90 95 23714DNAListeria monocytogenes 23ttaatttaat tttccccaag tagcaggaca tgctaaataa aaccattcat ctaatttagg 60ggcatatctt ttgagataat caagattttg tacataaaag catgaatttt tatacacgat 120aactttctct tgctttaatt tggaaataat tctgctaaca gctgagctat gtgcgatgcc 180acttgaatat cctaactcct gcattgttaa attatccagt gtaatcttga tgccatcagg 240agtttcttta ccatacacat aggtcaggat taaaagttga ccgcaaatag agccaagctt 300cccgttaatc gaaaaatcat taaatttagc tagactgtat gaaacttgtt tttgtagggt 360ttggaaaaca tagaaaaagt gcgtaagatt tttgctcagt agttctttta gttcgtttat 420tttgataacg tatgcggtag cctgctcgct aatgacttct aaattataat agccaaccga 480tgtttctgta tcaataaagc cagacattat aacgaaagcc cctttgtagt attgtaaatt 540catgatggtc ccgttctcgc taatactcgt gagctttgtg ataccatcat atagaaaaat 600acaatattct tgtggatccc attggttaaa aataagttct tttttatgaa attgttttgg 660ttttatcccg ttagtttcta aatatttttt gaattcttct gcttgagcgt tcat 71424333DNAMycobacterium tuberculosis 24ttcgacaccc ggttgatgcg gctggaagac gagatgaaag aggggcgcta cgaggtacgc 60gcggagcttc ccggggtcga ccccgacaag gacgtcgaca ttatggtccg cgatggtcag 120ctgaccatca aggccgagcg caccgagcag aaggacttcg acggtcgctc ggaattcgcg 180tacggttcct tcgttcgcac ggtgtcgctg ccggtaggtg ctgacgagga cgacattaag 240gccacctacg acaagggcat tcttactgtg tcggtggcgg tttcggaagg gaagccaacc 300gaaaagcaca ttcagatccg gtccaccaac tga 33325722DNAMycobacterium tuberculosis 25atgagtcttc gcctggtgtc cccgatcaag gcgtttgcgg acggcattgt ggccgttgct 60atcgcggttg tcctgatgtt cggtctggcc aatacaccgc gagcggtggc agccgatgaa 120cgtctgcagt tcaccgcaac cacgctcagc ggtgctccct tcgatggcgc aagcctgcaa 180ggcaagccgg cggtgttgtg gttctggacg ccgtggtgcc cgttctgcaa cgcagaagcc 240cccagcctca gccaggtagc ggccgctaat ccggcggtca ccttcgtcgg aatcgccacc 300cgcgccgacg tcggggcgat gcagagcttt gtctcgaagt acaacctgaa tttcaccaac 360ctcaatgacg ccgatggtgt gatctgggcc cgctacaacg tgccttggca accggcattt 420gtgttctatc gcgcggacgg cacatcgacg ttcgtcaaca accccaccgc ggccatgtct 480caggacgagc tgtccggccg ggtggctgcg ctgacgtcct gacccggtga acgaggcgct 540gatcggtttg gcgttcgccg ccgggttggt ggctgcgctg aacccatgcg ggtttgccat 600gttgccggcc tacctgctgt tggtggtgta tgggcaggat tcggcgggcc ggacggggcc 660gcttagcgca gtgggccgag cggcagccgc cacggtcggg atggcgctgg gcttcttgac 720gg 72226480DNAMycobacterium tuberculosis 26atgaagctca ccacaatgat caagacggca gtagcggtcg tggccatggc ggccatcgcg 60acctttgcgg caccggtcgc gttggctgcc tatcccatca ccggaaaact tggcagtgag 120ctaacgatga ccgacaccgt tggccaagtc gtgctcggct ggaaggtcag tgatctcaaa 180tccagcacgg cagtcatccc cggctatccg gtggccggcc aggtctggga ggccactgcc 240acggtcaatg cgattcgcgg cagcgtcacg cccgcggtct cgcagttcaa tgcccgcacc 300gccgacggca tcaactaccg ggtgctgtgg caagccgcgg gccccgacac cattagcgga 360gccactatcc cccaaggcga acaatcgacc ggcaaaatct acttcgatgt caccggccca 420tcgccaacca tcgtcgcgat gaacaacggc atggaggatc tgctgatttg ggagccgtag 48027624DNAMycobacterium tuberculosis 27gtggccgaat acaccttgcc agacctggac tgggactacg gagcactgga accgcacatc 60tcgggtcaga tcaacgagct tcaccacagc aagcaccacg ccacctacgt aaagggcgcc 120aatgacgccg tcgccaaact cgaagaggcg cgcgccaagg aagatcactc agcgatcttg 180ctgaacgaaa agaatctagc tttcaacctc gccggccacg tcaatcacac catctggtgg 240aagaacctgt cgcctaacgg tggtgacaag cccaccggcg aactcgccgc agccatcgcc 300gacgcgttcg gttcgttcga caagttccgt gcgcagttcc acgcggccgc taccaccgtg 360caggggtcgg gctgggcggc actgggctgg gacacactcg gcaacaagct gctgatattc 420caggtttacg accaccagac gaacttcccg ctaggcattg ttccgctgct gctgctcgac 480atgtgggaac acgccttcta cctgcagtac aagaacgtca aagtcgactt tgccaaggcg 540ttttggaacg tcgtgaactg ggccgatgtg cagtcacggt atgcggccgc gacctcgcag 600accaaggggt tgatattcgg ctga 62428687DNAMycobacterium tuberculosis 28gtgcgcatca agatcttcat gctggtcacg gctgtcgttt tgctctgttg ttcgggtgtg 60gccacggccg cgcccaagac ctactgcgag gagttgaaag gcaccgatac cggccaggcg 120tgccagattc aaatgtccga cccggcctac aacatcaaca tcagcctgcc cagttactac 180cccgaccaga agtcgctgga aaattacatc gcccagacgc gcgacaagtt cctcagcgcg 240gccacatcgt ccactccacg cgaagccccc tacgaattga atatcacctc ggccacatac 300cagtccgcga taccgccgcg tggtacgcag gccgtggtgc tcaaggtcta ccagaacgcc 360ggcggcacgc acccaacgac cacgtacaag gccttcgatt gggaccaggc ctatcgcaag 420ccaatcacct atgacacgct gtggcaggct gacaccgatc cgctgccagt cgtcttcccc 480attgtgcaag gtgaactgag caagcagacc ggacaacagg tatcgatagc gccgaatgcc 540ggcttggacc cggtgaatta tcagaacttc gcagtcacga acgacggggt gattttcttc 600ttcaacccgg gggagttgct gcccgaagca gccggcccaa cccaggtatt ggtcccacgt 660tccgcgatcg actcgatgct ggcctag 68729900DNAMycobacterium tuberculosis 29atgaagggtc ggtcggcgct gctgcgggcg ctctggattg ccgcactgtc attcgggttg 60ggcggtgtcg cggtagccgc ggaacccacc gccaaggccg ccccatacga gaacctgatg 120gtgccgtcgc cctcgatggg ccgggacatc ccggtggcct tcctagccgg tgggccgcac 180gcggtgtatc tgctggacgc cttcaacgcc ggcccggatg tcagtaactg ggtcaccgcg 240ggtaacgcga tgaacacgtt ggcgggcaag gggatttcgg tggtggcacc ggccggtggt 300gcgtacagca tgtacaccaa ctgggagcag gatggcagca agcagtggga caccttcttg 360tccgctgagc tgcccgactg gctggccgct aaccggggct tggcccccgg tggccatgcg 420gccgttggcg ccgctcaggg cggttacggg gcgatggcgc tggcggcctt ccaccccgac 480cgcttcggct tcgctggctc gatgtcgggc tttttgtacc cgtcgaacac caccaccaac 540ggtgcgatcg cggcgggcat gcagcaattc ggcggtgtgg acaccaacgg aatgtgggga 600gcaccacagc tgggtcggtg gaagtggcac gacccgtggg tgcatgccag cctgctggcg 660caaaacaaca cccgggtgtg ggtgtggagc ccgaccaacc cgggagccag cgatcccgcc 720gccatgatcg gccaagccgc cgaggcgatg ggtaacagcc gcatgttcta caaccagtat 780cgcagcgtcg gcgggcacaa cggacacttc gacttcccag ccagcggtga caacggctgg 840ggctcgtggg cgccccagct gggcgctatg tcgggcgata tcgtcggtgc gatccgctaa 90030978DNAMycobacterium tuberculosis 30atgacagacg tgagccgaaa gattcgagct tggggacgcc gattgatgat cggcacggca 60gcggctgtag tccttccggg cctggtgggg cttgccggcg gagcggcaac cgcgggcgcg 120ttctcccggc cggggctgcc ggtcgagtac ctgcaggtgc cgtcgccgtc gatgggccgc 180gacatcaagg ttcagttcca gagcggtggg aacaactcac ctgcggttta tctgctcgac 240ggcctgcgcg cccaagacga ctacaacggc tgggatatca acaccccggc gttcgagtgg 300tactaccagt cgggactgtc gatagtcatg ccggtcggcg ggcagtccag cttctacagc 360gactggtaca gcccggcctg cggtaaggct ggctgccaga cttacaagtg ggaaaccttc 420ctgaccagcg agctgccgca atggttgtcc gccaacaggg ccgtgaagcc caccggcagc 480gctgcaatcg gcttgtcgat ggccggctcg tcggcaatga tcttggccgc ctaccacccc 540cagcagttca tctacgccgg ctcgctgtcg gccctgctgg acccctctca ggggatgggg 600cctagcctga tcggcctcgc gatgggtgac gccggcggtt acaaggccgc agacatgtgg 660ggtccctcga gtgacccggc atgggagcgc aacgacccta cgcagcagat ccccaagctg 720gtcgcaaaca acacccggct atgggtttat tgcgggaacg gcaccccgaa cgagttgggc 780ggtgccaaca tacccgccga gttcttggag aacttcgttc gtagcagcaa cctgaagttc 840caggatgcgt acaacgccgc gggcgggcac aacgccgtgt tcaacttccc gcccaacggc 900acgcacagct gggagtactg gggcgctcag ctcaacgcca tgaagggtga cctgcagagt 960tcgttaggcg ccggctga 978311017DNAMycobacterium tuberculosis 31atgcagcttg ttgacagggt tcgtggcgcc gtcacgggta tgtcgcgtcg actcgtggtc 60ggggccgtcg gcgcggccct agtgtcgggt ctggtcggcg ccgtcggtgg cacggcgacc 120gcgggggcat tttcccggcc gggcttgccg gtggagtacc tgcaggtgcc gtcgccgtcg 180atgggccgtg acatcaaggt ccaattccaa agtggtggtg ccaactcgcc cgccctgtac 240ctgctcgacg gcctgcgcgc gcaggacgac ttcagcggct gggacatcaa caccccggcg 300ttcgagtggt acgaccagtc gggcctgtcg gtggtcatgc cggtgggtgg ccagtcaagc 360ttctactccg actggtacca gcccgcctgc ggcaaggccg gttgccagac ttacaagtgg 420gagaccttcc tgaccagcga gctgccgggg tggctgcagg ccaacaggca cgtcaagccc 480accggaagcg ccgtcgtcgg tctttcgatg gctgcttctt cggcgctgac gctggcgatc 540tatcaccccc agcagttcgt ctacgcggga gcgatgtcgg gcctgttgga cccctcccag 600gcgatgggtc ccaccctgat cggcctggcg atgggtgacg ctggcggcta caaggcctcc 660gacatgtggg gcccgaagga ggacccggcg tggcagcgca acgacccgct gttgaacgtc 720gggaagctga tcgccaacaa cacccgcgtc tgggtgtact gcggcaacgg caagccgtcg 780gatctgggtg gcaacaacct gccggccaag ttcctcgagg gcttcgtgcg gaccagcaac 840atcaagttcc aagacgccta caacgccggt ggcggccaca acggcgtgtt cgacttcccg 900gacagcggta cgcacagctg ggagtactgg ggcgcgcagc tcaacgctat gaagcccgac 960ctgcaacggg cactgggtgc cacgcccaac accgggcccg cgccccaggg cgcctag 101732738DNAMycobacterium tuberculosis 32atggccaagt tggcccgagt agtgggccta gtacaggaag agcaacctag cgacatgacg 60aatcacccac ggtattcgcc accgccgcag cagccgggaa ccccaggtta tgctcagggg 120cagcagcaaa cgtacagcca gcagttcgac tggcgttacc caccgtcccc gcccccgcag 180ccaacccagt accgtcaacc ctacgaggcg ttgggtggta cccggccggg tctgatacct 240ggcgtgattc cgaccatgac gccccctcct gggatggttc gccaacgccc tcgtgcaggc 300atgttggcca tcggcgcggt gacgatagcg gtggtgtccg ccggcatcgg cggcgcggcc 360gcatccctgg tcgggttcaa ccgggcaccc gccggcccca gcggcggccc agtggctgcc 420agcgcggcgc caagcatccc cgcagcaaac atgccgccgg ggtcggtcga acaggtggcg 480gccaaggtgg tgcccagtgt cgtcatgttg gaaaccgatc tgggccgcca gtcggaggag 540ggctccggca tcattctgtc tgccgagggg ctgatcttga ccaacaacca cgtgatcgcg 600gcggccgcca agcctcccct gggcagtccg ccgccgaaaa cgacggtaac cttctctgac 660gggcggaccg cacccttcac ggtggtgggg gctgacccca ccagtgatat cgccgtcgtc 720cgtgttcagg gcgtctcc 73833978DNAMycobacterium tuberculosis 33atgcatcagg tggaccccaa cttgacacgt cgcaagggac gattggcggc actggctatc 60gcggcgatgg ccagcgccag cctggtgacc gttgcggtgc ccgcgaccgc caacgccgat 120ccggagccag cgcccccggt acccacaacg gccgcctcgc cgccgtcgac cgctgcagcg 180ccacccgcac cggcgacacc tgttgccccc ccaccaccgg ccgccgccaa cacgccgaat 240gcccagccgg gcgatcccaa cgcagcacct ccgccggccg acccgaacgc accgccgcca 300cctgtcattg ccccaaacgc accccaacct gtccggatcg acaacccggt tggaggattc 360agcttcgcgc tgcctgctgg ctgggtggag tctgacgccg cccacttcga ctacggttca 420gcactcctca gcaaaaccac cggggacccg ccatttcccg gacagccgcc gccggtggcc 480aatgacaccc gtatcgtgct cggccggcta gaccaaaagc tttacgccag cgccgaagcc 540accgactcca aggccgcggc ccggttgggc tcggacatgg gtgagttcta tatgccctac 600ccgggcaccc ggatcaacca ggaaaccgtc tcgctcgacg ccaacggggt gtctggaagc 660gcgtcgtatt acgaagtcaa gttcagcgat ccgagtaagc cgaacggcca gatctggacg 720ggcgtaatcg gctcgcccgc ggcgaacgca ccggacgccg ggccccctca gcgctggttt 780gtggtatggc tcgggaccgc caacaacccg gtggacaagg gcgcggccaa ggcgctggcc 840gaatcgatcc ggcctttggt cgccccgccg ccggcgccgg caccggctcc tgcagagccc 900gctccggcgc cggcgccggc cggggaagtc gctcctaccc cgacgacacc gacaccgcag 960cggaccttac cggcctga 978341437DNAMycobacterium tuberculosis 34gtgacggaaa agacgcccga cgacgtcttc aaacttgcca aggacgagaa ggtcgaatat 60gtcgacgtcc ggttctgtga cctgcctggc atcatgcagc acttcacgat tccggcttcg

120gcctttgaca agagcgtgtt tgacgacggc ttggcctttg acggctcgtc gattcgcggg 180ttccagtcga tccacgaatc cgacatgttg cttcttcccg atcccgagac ggcgcgcatc 240gacccgttcc gcgcggccaa gacgctgaat atcaacttct ttgtgcacga cccgttcacc 300ctggagccgt actcccgcga cccgcgcaac atcgcccgca aggccgagaa ctacctgatc 360agcactggca tcgccgacac cgcatacttc ggcgccgagg ccgagttcta cattttcgat 420tcggtgagct tcgactcgcg cgccaacggc tccttctacg aggtggacgc catctcgggg 480tggtggaaca ccggcgcggc gaccgaggcc gacggcagtc ccaaccgggg ctacaaggtc 540cgccacaagg gcgggtattt cccagtggcc cccaacgacc aatacgtcga cctgcgcgac 600aagatgctga ccaacctgat caactccggc ttcatcctgg agaagggcca ccacgaggtg 660ggcagcggcg gacaggccga gatcaactac cagttcaatt cgctgctgca cgccgccgac 720gacatgcagt tgtacaagta catcatcaag aacaccgcct ggcagaacgg caaaacggtc 780acgttcatgc ccaagccgct gttcggcgac aacgggtccg gcatgcactg tcatcagtcg 840ctgtggaagg acggggcccc gctgatgtac gacgagacgg gttatgccgg tctgtcggac 900acggcccgtc attacatcgg cggcctgtta caccacgcgc cgtcgctgct ggccttcacc 960aacccgacgg tgaactccta caagcggctg gttcccggtt acgaggcccc gatcaacctg 1020gtctatagcc agcgcaaccg gtcggcatgc gtgcgcatcc cgatcaccgg cagcaacccg 1080aaggccaagc ggctggagtt ccgaagcccc gactcgtcgg gcaacccgta tctggcgttc 1140tcggccatgc tgatggcagg cctggacggt atcaagaaca agatcgagcc gcaggcgccc 1200gtcgacaagg atctctacga gctgccgccg gaagaggccg cgagtatccc gcagactccg 1260acccagctgt cagatgtgat cgaccgtctc gaggccgacc acgaatacct caccgaagga 1320ggggtgttca caaacgacct gatcgagacg tggatcagtt tcaagcgcga aaacgagatc 1380gagccggtca acatccggcc gcatccctac gaattcgcgc tgtactacga cgtttaa 1437351878DNAMycobacterium tuberculosis 35atggctcgtg cggtcgggat cgacctcggg accaccaact ccgtcgtctc ggttctggaa 60ggtggcgacc cggtcgtcgt cgccaactcc gagggctcca ggaccacccc gtcaattgtc 120gcgttcgccc gcaacggtga ggtgctggtc ggccagcccg ccaagaacca ggcagtgacc 180aacgtcgatc gcaccgtgcg ctcggtcaag cgacacatgg gcagcgactg gtccatagag 240attgacggca agaaatacac cgcgccggag atcagcgccc gcattctgat gaagctgaag 300cgcgacgccg aggcctacct cggtgaggac attaccgacg cggttatcac gacgcccgcc 360tacttcaatg acgcccagcg tcaggccacc aaggacgccg gccagatcgc cggcctcaac 420gtgctgcgga tcgtcaacga gccgaccgcg gccgcgctgg cctacggcct cgacaagggc 480gagaaggagc agcgaatcct ggtcttcgac ttgggtggtg gcactttcga cgtttccctg 540ctggagatcg gcgagggtgt ggttgaggtc cgtgccactt cgggtgacaa ccacctcggc 600ggcgacgact gggaccagcg ggtcgtcgat tggctggtgg acaagttcaa gggcaccagc 660ggcatcgatc tgaccaagga caagatggcg atgcagcggc tgcgggaagc cgccgagaag 720gcaaagatcg agctgagttc gagtcagtcc acctcgatca acctgcccta catcaccgtc 780gacgccgaca agaacccgtt gttcttagac gagcagctga cccgcgcgga gttccaacgg 840atcactcagg acctgctgga ccgcactcgc aagccgttcc agtcggtgat cgctgacacc 900ggcatttcgg tgtcggagat cgatcacgtt gtgctcgtgg gtggttcgac ccggatgccc 960gcggtgaccg atctggtcaa ggaactcacc ggcggcaagg aacccaacaa gggcgtcaac 1020cccgatgagg ttgtcgcggt gggagccgct ctgcaggccg gcgtcctcaa gggcgaggtg 1080aaagacgttc tgctgcttga tgttaccccg ctgagcctgg gtatcgagac caagggcggg 1140gtgatgacca ggctcatcga gcgcaacacc acgatcccca ccaagcggtc ggagactttc 1200accaccgccg acgacaacca accgtcggtg cagatccagg tctatcaggg ggagcgtgag 1260atcgccgcgc acaacaagtt gctcgggtcc ttcgagctga ccggcatccc gccggcgccg 1320cgggggattc cgcagatcga ggtcactttc gacatcgacg ccaacggcat tgtgcacgtc 1380accgccaagg acaagggcac cggcaaggag aacacgatcc gaatccagga aggctcgggc 1440ctgtccaagg aagacattga ccgcatgatc aaggacgccg aagcgcacgc cgaggaggat 1500cgcaagcgtc gcgaggaggc cgatgttcgt aatcaagccg agacattggt ctaccagacg 1560gagaagttcg tcaaagaaca gcgtgaggcc gagggtggtt cgaaggtacc tgaagacacg 1620ctgaacaagg ttgatgccgc ggtggcggaa gcgaaggcgg cacttggcgg atcggatatt 1680tcggccatca agtcggcgat ggagaagctg ggccaggagt cgcaggctct ggggcaagcg 1740atctacgaag cagctcaggc tgcgtcacag gccactggcg ctgcccaccc cggcggcgag 1800ccgggcggtg cccaccccgg ctcggctgat gacgttgtgg acgcggaggt ggtcgacgac 1860ggccgggagg ccaagtga 187836291DNAMycobacterium tuberculosis 36atgtcgcaaa tcatgtacaa ctaccccgcg atgttgggtc acgccgggga tatggccgga 60tatgccggca cgctgcagag cttgggtgcc gagatcgccg tggagcaggc cgcgttgcag 120agtgcgtggc agggcgatac cgggatcacg tatcaggcgt ggcaggcaca gtggaaccag 180gccatggaag atttggtgcg ggcctatcat gcgatgtcca gcacccatga agccaacacc 240atggcgatga tggcccgcga cacggccgaa gccgccaaat ggggcggcta g 29137435DNAMycobacterium tuberculosis 37atggccacca cccttcccgt tcagcgccac ccgcggtccc tcttccccga gttttctgag 60ctgttcgcgg ccttcccgtc attcgccgga ctccggccca ccttcgacac ccggttgatg 120cggctggaag acgagatgaa agaggggcgc tacgaggtac gcgcggagct tcccggggtc 180gaccccgaca aggacgtcga cattatggtc cgcgatggtc agctgaccat caaggccgag 240cgcaccgagc agaaggactt cgacggtcgc tcggaattcg cgtacggttc cttcgttcgc 300acggtgtcgc tgccggtagg tgctgacgag gacgacatta aggccaccta cgacaagggc 360attcttactg tgtcggtggc ggtttcggaa gggaagccaa ccgaaaagca cattcagatc 420cggtccacca actga 435381287DNAMycobacterium tuberculosis 38atgtctgtcg tcggcacccc gaagagcgcg gagcagatcc agcaggaatg ggacacgaac 60ccgcgctgga aggacgtcac ccgcacctac tccgccgagg acgtcgtcgc cctccagggc 120agcgtggtcg aggagcacac gctggcccgc cgcggtgcgg aggtgctgtg ggagcagctg 180cacgacctcg agtgggtcaa cgcgctgggc gcgctgaccg gcaacatggc cgtccagcag 240gtgcgcgccg gcctgaaggc catctacctg tcgggctggc aggtcgccgg cgatgccaac 300ctgtccgggc acacctaccc cgaccagagc ctgtatcccg ccaactcggt gccgcaggtg 360gtccgccgga tcaacaacgc actgcagcgc gccgaccaga tcgccaagat cgagggcgat 420acttcggtgg agaactggct ggcgccgatt gtcgccgacg gcgaggccgg ctttggcggc 480gcgctcaacg tctacgagct gcagaaagcc ctgatcgccg cgggcgttgc gggttcgcac 540tgggaggacc agttggcctc tgagaagaag tgcggccacc tgggcggcaa ggtgttgatc 600ccgacccagc agcacatccg cactttgacg tctgctcggc tcgcggccga tgtggctgat 660gttcccacgg tggtgatcgc ccgtaccgac gccgaggcgg ccacgctgat cacctccgac 720gtcgacgagc gcgaccagcc gttcatcacc ggcgagcgca cccgggaagg cttctaccgc 780accaagaacg gcatcgagcc ttgcatcgct cgggcgaagg cctacgcccc gttcgccgac 840ttgatctgga tggagaccgg taccccggac ctcgaggccg cccggcagtt ctccgaggcg 900gtcaaggcgg agtacccgga ccagatgctg gcctacaact gctcgccatc gttcaactgg 960aaaaagcacc tcgacgacgc caccatcgcc aagttccaga aggagctggc agccatgggc 1020ttcaagttcc agttcatcac gctggccggc ttccatgcgc tgaactactc gatgttcgat 1080ctggcctacg gctacgccca gaaccagatg agcgcgtatg tcgaactgca ggaacgcgag 1140ttcgccgccg aagaacgggg ctacaccgcg accaagcacc agcgcgaggt cggcgccggc 1200tacttcgacc ggattgccac caccgtggac ccgaattcgt cgaccaccgc gttgaccggt 1260tccaccgaag agggccagtt ccactag 128739228DNAMycobacterium tuberculosis 39gtgatagcgg gcgtcgacca ggcgcttgca gcaacaggcc aggctagcca gcgggcggca 60ggcgcatctg gtggggtcac cgtcggtgtc ggcgtgggca cggaacagag gaacctttcg 120gtggttgcac cgagtcagtt cacatttagt tcacgcagcc cagattttgt ggatgaaacc 180gcaggtcaat cgtggtgcgc gatactggga ttgaaccagt ttcactag 228402226DNAMycobacterium tuberculosis 40atgacagatc gcgtgtcggt gggcaacttg cgcatcgctc gggtgctcta cgacttcgtg 60aacaatgaag ccctgcctgg caccgatatc gacccggaca gcttctgggc gggcgtcgac 120aaggtcgtcg ccgacctgac cccgcagaac caagctctgt tgaacgcccg cgacgagctg 180caggcgcaga tcgacaagtg gcaccggcgt cgggtgatcg agcccatcga catggatgcc 240taccgccagt tcctcaccga gatcggctac ctgcttcccg aacctgatga cttcaccatc 300accacgtccg gtgtcgacgc tgagatcacc acgaccgccg gcccccagct ggtggtgccg 360gtgctcaacg cgcggtttgc tctgaacgcg gccaacgctc gctggggctc cctctacgac 420gccttgtatg gcaccgatgt catccccgag accgacggcg ccgaaaaagg ccccacgtac 480aacaaggttc gtggcgacaa ggtgatcgcg tatgcccgca agttcctcga cgacagtgtt 540ccgctgtcgt cgggttcctt tggcgacgcc accggtttca cagtgcagga tggccagctc 600gtggttgcct tgccggataa gtccaccggc ctggccaacc ccggccagtt cgccggctac 660accggcgcag ccgagtcgcc gacatcggtg ctgctaatca atcacggttt gcacatcgag 720atcctgatcg atccggagtc gcaggtcggc accaccgacc gggccggcgt caaggacgtg 780atcctggaat ccgcgatcac cacgatcatg gacttcgagg actcggtggc cgccgtggac 840gccgccgaca aggtgctggg ttatcggaac tggctcggcc tgaacaaggg cgacctggca 900gcagcggtag acaaggacgg caccgctttc ctgcgggtgc tcaataggga ccggaactac 960accgcacccg gcggtggcca gttcacgctg cctggacgca gcctcatgtt cgtccgcaac 1020gtcggtcact tgatgacgaa tgacgccatc gtcgacactg acggcagcga ggtgttcgaa 1080ggcatcatgg atgccctatt caccggcctg atcgccatcc acgggctaaa ggccagcgac 1140gtcaacgggc cgctgatcaa cagccgcacc ggctccatct acatcgtcaa gccgaagatg 1200cacggtccgg ccgaggtggc gtttacctgc gaactgttca gccgggttga agatgtgctg 1260gggttgccgc aaaacaccat gaagatcggc atcatggacg aggaacgccg gaccacggtc 1320aacctcaagg cgtgcatcaa agctgccgcg gaccgcgtgg tgttcatcaa caccgggttc 1380ctggaccgca ccggcgatga aatccacacc tcgatggagg ccggcccgat ggtgcgcaag 1440ggcaccatga agagccagcc gtggatcttg gcctacgagg accacaacgt cgatgccggc 1500ctggccgccg ggttcagcgg ccgagcccag gtcggcaagg gcatgtggac aatgaccgag 1560ctgatggccg acatggtcga gacaaaaatc gcccagccgc gcgccggggc cagcaccgcc 1620tgggttccct ctcccactgc ggccaccctg catgcgctgc actaccacca ggtcgacgtc 1680gccgcggtgc aacaaggact ggcggggaag cgtcgcgcca ccatcgaaca attgctgacc 1740attccgctgg ccaaggaatt ggcctgggct cccgacgaga tccgcgaaga ggtcgacaac 1800aactgtcaat ccatcctcgg ctacgtggtt cgctgggttg atcaaggtgt cggctgctcg 1860aaggtgcccg acatccacga cgtcgcgctc atggaggacc gggccacgct gcgaatctcc 1920agccaattgt tggccaactg gctgcgccac ggtgtgatca ccagcgcgga tgtgcgggcc 1980agcttggagc ggatggcgcc gttggtcgat cgacaaaacg cgggcgacgt ggcataccga 2040ccgatggcac ccaacttcga cgacagtatc gccttcctgg ccgcgcagga gctgatcttg 2100tccggggccc agcagcccaa cggctacacc gagccgatcc tgcaccgacg tcgtcgggag 2160tttaaggccc gggccgctga gaagccggcc ccatcggaca gggccggtga cgatgcggcc 2220cgctag 2226412832DNAMycobacterium tuberculosis 41gtgactagca aatctgtgaa ctcattcgga gcccacgaca ccctgaaggt cggcgaaaag 60agttaccaga tctatcgtct cgacgccgtc cccaataccg cgaaactccc ctacagcctc 120aaagtgctcg ccgagaacct gttgcgcaac gaggacggca gcaacatcac caaggaccac 180atcgaggcca tcgccaactg ggaccctaag gccgagccca gcatcgagat ccagtacacg 240cccgcccggg tggtgatgca ggacttcacc ggcgtaccgt gcatcgtcga cttggccacc 300atgcgcgagg cgatcgccga tctgggcggc aacccggaca aggtcaaccc gctggcgccc 360gcagacttgg tgatcgacca ctcggtgatc gccgatttgt tcggccgcgc cgacgcattc 420gagcgcaacg tcgaaatcga ataccagcgc aacggtgagc gttaccaatt cctgcgctgg 480ggccaaggcg ctttcgacga cttcaaagtg gtgccgccgg gcaccggcat cgtgcaccag 540gtcaatatcg agtacctggc cagcgtggtg atgactcgcg acggagtggc ctaccccgac 600acctgcgtgg gcaccgactc acacaccacc atggtcaacg gcctgggtgt gctcgggtgg 660ggtgtcggcg gcatcgaggc ggaggccgcg atgctgggtc agccggtatc gatgctgatc 720ccgcgggtcg tgggtttcag gttgaccggc gagatccagc cgggagtcac cgccaccgac 780gtggtgttga ccgtcaccga gatgctgcgc cagcacggcg tcgtcggcaa attcgtcgag 840ttctacggcg agggcgtggc cgaggtgccg ctggccaacc gcgccaccct gggcaacatg 900agtcccgaat tcggttccac cgcagcgatt ttcccgatcg acgaagaaac catcaagtat 960ctgcggttta ccggtcgcac gccggagcag gtcgcactgg tcgaggccta cgccaaggcg 1020cagggcatgt ggcacgatcc caagcacgag ccggagttct cggaatacct cgaactcaac 1080ctatccgacg tggtgccgtc gatcgccgga ccaaagcgtc cacaggaccg aatcgcgttg 1140gcgcaagcca aatcaacatt ccgcgagcag atttaccact atgtcggcaa tggttccccg 1200gattcccccc acgacccgca ctcgaagctg gacgaggtag tcgaggagac attcccggcc 1260agcgacccgg ggcagctgac gttcgccaac gacgacgtcg ccactgacga aaccgtgcac 1320tcggctgccg cgcatgccga tggccgggtg agcaacccag tgcgggtgaa gtcggacgaa 1380ctcggcgaat tcgtgctcga ccacggcgcg gtggtgattg ccgcgatcac gtcctgcacc 1440aacacctcca accccgaagt aatgctgggc gcggcgctgc tggcccgcaa cgccgtcgaa 1500aagggactga cctcgaagcc gtgggtgaag accacgattg ccccgggctc gcaagtggtc 1560aacgactact acgacagatc cggcctgtgg ccgtatctgg agaagctcgg cttctatctg 1620gtcggctacg gctgcaccac ctgcatcggc aactccgggc cgctgcccga ggaaatctca 1680aaagcggtta acgacaacga cctttcggtg accgcggtac tgtccggcaa ccggaacttc 1740gagggccgta tcaacccaga cgtgaagatg aactacctgg catcgccgcc gctggtcatc 1800gcctacgcgc tggccgggac catggacttc gacttccaaa cccagccgct cggtcaagac 1860aaagacggta agaacgtttt tctccgcgat atctggccat cgcagcagga tgtctccgac 1920accatcgccg cggcgatcaa ccaggagatg ttcacccgca actacgccga cgtgttcaag 1980ggcgacgacc gctggcgcaa cctgccaacc ccgagcggca acacctttga gtgggacccg 2040aattcgacct acgtgcgcaa gccgccgtat ttcgagggga tgacagccaa acccgaaccg 2100gtcggcaaca tcagcggtgc ccgggtgctg gcgctgctcg gtgattcggt gaccaccgac 2160cacatctccc ccgccggcgc catcaagccc ggaactcccg cggcgcgcta cctcgacgaa 2220cacggtgtcg accgcaagga ctacaactcc ttcggttctc gccgcggcaa ccacgaggtg 2280atgattcgtg gcaccttcgc caacatccgg ctgcgtaacc aactgctaga cgacgtgtcc 2340ggcggttata cccgcgactt cacccagccg ggcggtccgc aggcgttcat ctacgacgcc 2400gcgcagaact atgcggcgca acacattccg ctggttgtgt tcggcggcaa agagtacggg 2460tcgggttcgt cacgggactg ggcggccaaa ggcacattgc tactgggcgt gcgggcggtg 2520atcgccgagt cattcgagcg gatccaccgg tccaacctga tcggcatggg cgtgatcccg 2580ctgcagttcc ccgaaggaaa gtcagcgtcg tcgttgggac tcgacggtac cgaggtcttc 2640gacatcaccg gtatcgacgt gcttaacgac ggcaagacac ccaagacggt gtgcgtccag 2700gccaccaagg gcgacggcgc cacgatcgag ttcgacgccg tggtgcgcat cgacaccccc 2760ggtgaggcgg actactaccg caacggcggc atcctgcagt acgtgctgcg caacatactg 2820aagtcaggct ga 283242288DNAMycobacterium tuberculosis 42atgacagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga 60aatgtcacgt ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca 120gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc 180acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt 240caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcatag 288431920DNAListeria monocytogenes 43gtgggattaa acagatttat gcgtgcgatg atggtggttt tcattactgc caattgcatt 60acgattaacc ccgacataat atttgcagcg acagatagcg aagattctag tctaaacaca 120gatgaatggg aagaagaaaa aacagaagag caaccaagcg aggtaaatac gggaccaaga 180tacgaaactg cacgtgaagt aagttcacgt gatattaaag aactagaaaa atcgaataaa 240gtgagaaata cgaacaaagc agacctaata gcaatgttga aagaaaaagc agaaaaaggt 300ccaaatatca ataataacaa cagtgaacaa actgagaatg cggctataaa tgaagaggct 360tcaggagccg accgaccagc tatacaagtg gagcgtcgtc atccaggatt gccatcggat 420agcgcagcgg aaattaaaaa aagaaggaaa gccatagcat catcggatag tgagcttgaa 480agccttactt atccggataa accaacaaaa gtaaataaga aaaaagtggc gaaagagtca 540gttgcggatg cttctgaaag tgacttagat tctagcatgc agtcagcaga tgagtcttca 600ccacaacctt taaaagcaaa ccaacaacca tttttcccta aagtatttaa aaaaataaaa 660gatgcgggga aatgggtacg tgataaaatc gacgaaaatc ctgaagtaaa gaaagcgatt 720gttgataaaa gtgcagggtt aattgaccaa ttattaacca aaaagaaaag tgaagaggta 780aatgcttcgg acttcccgcc accacctacg gatgaagagt taagacttgc tttgccagag 840acaccaatgc ttcttggttt taatgctcct gctacatcag aaccgagctc attcgaattt 900ccaccaccac ctacggatga agagttaaga cttgctttgc cagagacgcc aatgcttctt 960ggttttaatg ctcctgctac atcggaaccg agctcgttcg aatttccacc gcctccaaca 1020gaagatgaac tagaaatcat ccgggaaaca gcatcctcgc tagattctag ttttacaaga 1080ggggatttag ctagtttgag aaatgctatt aatcgccata gtcaaaattt ctctgatttc 1140ccaccaatcc caacagaaga agagttgaac gggagaggcg gtagaccaac atctgaagaa 1200tttagttcgc tgaatagtgg tgattttaca gatgacgaaa acagcgagac aacagaagaa 1260gaaattgatc gcctagctga tttaagagat agaggaacag gaaaacactc aagaaatgcg 1320ggttttttac cattaaatcc gtttgctagc agcccggttc cttcgttaag tccaaaggta 1380tcgaaaataa gcgcaccggc tctgataagt gacataacta aaaaaacgcc atttaagaat 1440ccatcacagc cattaaatgt gtttaataaa aaaactacaa cgaaaacagt gactaaaaaa 1500ccaacccctg taaagaccgc accaaagcta gcagaacttc ctgccacaaa accacaagaa 1560accgtactta gggaaaataa aacacccttt atagaaaaac aagcagaaac aaacaagcag 1620tcaattaata tgccgagcct accagtaatc caaaaagaag ctacagagag cgataaagag 1680gaaatgaaac cacaaaccga ggaaaaaatg gtagaggaaa gcgaatcagc taataacgca 1740aacggaaaaa atcgttctgc tggcattgaa gaaggaaaac taattgctaa aagtgcagaa 1800gacgaaaaag cgaaggaaga accagggaac catacgacgt taattcttgc aatgttagct 1860attggcgtgt tctctttagg ggcgtttatc aaaattattc aattaagaaa aaataattaa 192044529PRTListeria monocytogenes 44Met Lys Lys Ile Met Leu Val Phe Ile Thr Leu Ile Leu Val Ser Leu1 5 10 15 Pro Ile Ala Gln Gln Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30 Glu Asn Ser Ile Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser 35 40 45 Pro Lys Thr Pro Ile Glu Lys Lys His Ala Asp Glu Ile Asp Lys Tyr 50 55 60 Ile Gln Gly Leu Asp Tyr Asn Lys Asn Asn Val Leu Val Tyr His Gly65 70 75 80 Asp Ala Val Thr Asn Val Pro Pro Arg Lys Gly Tyr Lys Asp Gly Asn 85 90 95 Glu Tyr Ile Val Val Glu Lys Lys Lys Lys Ser Ile Asn Gln Asn Asn 100 105 110 Ala Asp Ile Gln Val Val Asn Ala Ile Ser Ser Leu Thr Tyr Pro Gly 115 120 125 Ala Leu Val Lys Ala Asn Ser Glu Leu Val Glu Asn Gln Pro Asp Val 130 135 140 Leu Pro Val Lys Arg Asp Ser Leu Thr Leu Ser Ile Asp Leu Pro Gly145 150 155 160 Met Thr Asn Gln Asp Asn Lys Ile Val Val Lys Asn Ala Thr Lys Ser 165 170 175 Asn Val Asn Asn Ala Val Asn Thr Leu Val Glu Arg Trp Asn Glu Lys 180 185 190 Tyr Ala Gln Ala Tyr Pro Asn Val Ser Ala Lys Ile Asp Tyr Asp Asp 195 200 205 Glu Met Ala Tyr Ser Glu Ser Gln Leu Ile Ala Lys Phe Gly Thr Ala 210 215 220 Phe Lys Ala Val Asn Asn Ser Leu Asn Val Asn Phe Gly Ala Ile Ser225 230 235 240 Glu Gly Lys Met Gln Glu Glu Val Ile Ser Phe Lys Gln Ile Tyr Tyr 245 250 255 Asn Val Asn Val Asn Glu Pro Thr Arg Pro Ser Arg Phe Phe Gly Lys 260 265

270 Ala Val Thr Lys Glu Gln Leu Gln Ala Leu Gly Val Asn Ala Glu Asn 275 280 285 Pro Pro Ala Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu 290 295 300 Lys Leu Ser Thr Asn Ser His Ser Thr Lys Val Lys Ala Ala Phe Asp305 310 315 320 Ala Ala Val Ser Gly Lys Ser Val Ser Gly Asp Val Glu Leu Thr Asn 325 330 335 Ile Ile Lys Asn Ser Ser Phe Lys Ala Val Ile Tyr Gly Gly Ser Ala 340 345 350 Lys Asp Glu Val Gln Ile Ile Asp Gly Asn Leu Gly Asp Leu Arg Asp 355 360 365 Ile Leu Lys Lys Gly Ala Thr Phe Asn Arg Glu Thr Pro Gly Val Pro 370 375 380 Ile Ala Tyr Thr Thr Asn Phe Leu Lys Asp Asn Glu Leu Ala Val Ile385 390 395 400 Lys Asn Asn Ser Glu Tyr Ile Glu Thr Thr Ser Lys Ala Tyr Thr Asp 405 410 415 Gly Lys Ile Asn Ile Asp His Ser Gly Gly Tyr Val Ala Gln Phe Asn 420 425 430 Ile Ser Trp Asp Glu Val Asn Tyr Asp Pro Glu Gly Asn Glu Ile Val 435 440 445 Gln His Lys Asn Trp Ser Glu Asn Asn Lys Ser Lys Leu Ala His Phe 450 455 460 Thr Ser Ser Ile Tyr Leu Pro Gly Asn Ala Arg Asn Ile Asn Val Tyr465 470 475 480 Ala Lys Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp Arg Thr Val Ile 485 490 495 Asp Asp Arg Asn Leu Pro Leu Val Lys Asn Arg Asn Ile Ser Ile Trp 500 505 510 Gly Thr Thr Leu Tyr Pro Lys Tyr Ser Asn Lys Val Asp Asn Pro Ile 515 520 525 Glu 451589DNAListeria monocytogenes 45atgaaaaaaa taatgctagt ttttattaca cttatattag ttagtctacc aattgcgcaa 60caaactgaag caaaggatgc atctgcattc aataaagaaa attcaatttc atccatggca 120ccaccagcat ctccgcctgc aagtcctaag acgccaatcg aaaagaaaca cgcggatgaa 180atcgataagt atatacaagg attggattac aataaaaaca atgtattagt ataccacgga 240gatgcagtga caaatgtgcc gccaagaaaa ggttacaaag atggaaatga atatattgtt 300gtggagaaaa agaagaaatc catcaatcaa aataatgcag acattcaagt tgtgaatgca 360atttcgagcc taacctatcc aggtgctctc gtaaaagcga attcggaatt agtagaaaat 420caaccagatg ttctccctgt aaaacgtgat tcattaacac tcagcattga tttgccaggt 480atgactaatc aagacaataa aatcgttgta aaaaatgcca ctaaatcaaa cgttaacaac 540gcagtaaata cattagtgga aagatggaat gaaaaatatg ctcaagctta tccaaatgta 600gtgcaaaaat tgattatgat gacgaaatgg cttacagtga atcacaatta attgcgaaat 660ttggtacagc atttaaagct gtaaataata gcttgaatgt aaacttcggc gcaatcagtg 720aagggaaaat gcaagaagaa gtcattagtt ttaaacaaat ttactataac gtgaatgtta 780atgaacctac aagaccttcc agatttttcg gcaaagctgt tactaaagag cagttgcaag 840cgcttggagt gaatgcagaa aatcctcctg catatatctc aagtgtggcg tatggccgtc 900aagtttattt gaaattatca actaattccc atagtactaa agtaaaagct gcttttgatg 960ctgccgtaag cggaaaatct gtctcaggtg atgtagaact aacaaatatc atcaaaaatt 1020cttccttcaa agccgtaatt tacggaggtt ccgcaaaaga tgaagttcaa atcatcgacg 1080gcaacctcgg agacttacgc gatattttga aaaaaggcgc tacttttaat cgagaaacac 1140caggagttcc cattgcttat acaacaaact tcctaaaaga caatgaatta gctgttatta 1200aaaacaactc agaatatatt gaaacaactt caaaagctta tacagatgga aaaattaaca 1260tcgatcactc tggaggatac gttgctcaat tcaacatttc ttgggatgaa gtaaattatg 1320atcctgaagg taacgaaatt gttcaacata aaaactggag cgaaaacaat aaaagcaagc 1380tagctcattt cacatcgtcc atctatttgc caggtaacgc gagaaatatt aatgtttacg 1440ctaaagaatg cactggttta gcttgggaat ggtggagaac ggtaattgat gaccggaact 1500taccacttgt gaaaaataga aatatctcca tctggggcac cacgctttat ccgaaatata 1560gtaataaagt agataatcca atcgaataa 1589461893DNAListeria monocytogenes 46gtgaaagaaa agcacaaccc aagaaggaag tattgtttaa tctcaggttt agctattatt 60tttagtttat ggataattat tggaaacggg gcgaaagtac aagcggagac tatcaccgtg 120ccaacgccaa tcaagcaaat tttttcagat gatgcttttg cagaaacaat caaagacaat 180ttaaagaaaa aaagtgtgac agatgcagtg acacaaaatg aattaaatag tatagatcaa 240atcattgcga ataatagtga tattaaatcc gttcaaggaa ttcagtattt acccaatgtg 300acaaagttat ttttaaacgg gaataaacta acagatataa agcccttagc aaacttgaaa 360aatttaggat ggcttttttt agacgaaaat aaagttaagg acctaagttc gctcaaggat 420ttaaaaaaat taaaatcact ttctttggag cataatggta taagtgatat aaacggactt 480gttcatttac cacagctgga aagtttgtat ttgggaaata ataaaataac ggatataacg 540gttctttcac gtttaactaa actggatact ttgtctctcg aagataacca aattagtgat 600attgtgccac ttgcaggttt aactaaattg cagaacctat atttaagtaa aaatcacata 660agcgatttaa gagcattagc aggacttaaa aatctagatg ttttagaatt atttagccaa 720gaatgtctta ataagcctat taatcatcaa tctaatttgg ttgttccgaa tacagtgaaa 780aacactgatg ggtcgttagt gactccagaa ataataagtg atgatggcga ttatgaaaaa 840cctaatgtta aatggcattt accagaattt acaaatgaag tgagttttat tttctatcag 900ccagtcacta ttggaaaagc aaaagcaaga tttcatggga gagtaaccca accactgaaa 960gaggtttaca cagtaagtta tgatgttgat ggaacggtaa taaaaacaaa agtagaagca 1020gggacgcgga taactgcacc taaacctccg actaaacaag gctatgtttt taaaggatgg 1080tatactgaaa aaaatggtgg gcatgagtgg aattttaata cggattatat gtccggaaat 1140gattttactt tgtacgcagt atttaaagcg gaaacgaccg aaaaagcagt caacttaacc 1200cgctatgtca aatatattcg cgggaatgca ggcatctaca aacttccacg agaagataac 1260tcgcttaaac aaggaactct agcctcgcac cgctgtaaag ctctaactgt tgatagagaa 1320gcccgaaatg gcggaaaatt atggtacagg ttaaaaaata ttggctggac taaagcggaa 1380aacctttcct tagaccgata cgataaaatg gaatatgaca aaggggttac cgcttatgca 1440agagtgagaa atgcgtctgg aaattcggtt tggacaaaac cctacaacac agccggcgct 1500aaacacgtga ataagctatc ggtctaccaa ggtaaaaata tgcgtatctt gcgcgaagcc 1560aaaacaccaa ttactacatg gtatcaattt agcattggtg gtaaagtaat tggttgggtc 1620gatacccgag cacttaacac attctacaaa caaagcatgg aaaagccaac ccgtttaact 1680cgttatgtca gcgccaataa agctggcgaa tcgtactata aagtcccggt agcagataat 1740ccagtcaaaa ggggtacttt agccaagtat aaaaatcaaa agttaattgt tgattgtcaa 1800gcaaccatcg aaggtcaact ttggtaccga ataaggacta gttccacttt cattggttgg 1860acgaaagcag ctaatttaag ggcacagaaa taa 1893

Claims

1. A composition of matter comprising attenuated Listeria monocytogenes, wherein the Listeria monocytogenes: does not express a functional ActA protein (SEQ ID NO: 1); and expresses Mycobacterium tuberculosis 30 kDa antigen 85B protein (SEQ ID NO: 4).

2. The composition of claim 1, wherein the Listeria monocytogenes further expresses at least one protein from the following group: Mycobacterium tuberculosis 12 kDa fragment of 16 kDa membrane protein (SEQ ID NO:5); Mycobacterium tuberculosis 14 kDa MPT53 protein (SEQ ID NO: 6); Mycobacterium tuberculosis 16 kDa MPT63 protein (SEQ ID NO: 7); Mycobacterium tuberculosis 23 kDa SOD protein (SEQ ID NO: 8); Mycobacterium tuberculosis 23.5 kDa MPT64 protein (SEQ ID NO: 9); Mycobacterium tuberculosis 24 kDa MPT51 protein (SEQ ID NO: 10); Mycobacterium tuberculosis 32 kDa antigen 85A protein (SEQ ID NO: 11); Mycobacterium tuberculosis 32 kDa antigen 85C protein (SEQ ID NO: 12); Mycobacterium tuberculosis 45 kDa MPT32 protein (SEQ ID NO: 13); Mycobacterium tuberculosis 58 kDa glutamine synthetase protein (SEQ ID NO: 14); Mycobacterium tuberculosis 71 kDa HSP 70 protein (SEQ ID NO: 15); Mycobacterium tuberculosis 10.4 kDa EsxH protein (SEQ ID NO: 16); Mycobacterium tuberculosis 14 kDa alpha crystalline homolog protein (SEQ ID NO: 17); Mycobacterium tuberculosis 47 kDa isocitrate lysate protein (SEQ ID NO: 18); Mycobacterium tuberculosis 7.6 kDa hypothetical protein (SEQ ID NO: 19); Mycobacterium tuberculosis 80 kDa glcB protein (SEQ ID NO: 20); Mycobacterium tuberculosis 110 kDa can protein (SEQ ID NO: 21); or Mycobacterium tuberculosis 9.9 kDa ESAT-6 protein (SEQ ID NO: 22).

3. The composition of claim 1, wherein the Listeria monocytogenes further expresses: at least one protein from group A: Mycobacterium tuberculosis 12 kDa fragment of 16 kDa membrane protein (SEQ ID NO:5); Mycobacterium tuberculosis 14 kDa MPT53 protein (SEQ ID NO: 6); Mycobacterium tuberculosis 16 kDa MPT63 protein (SEQ ID NO: 7); Mycobacterium tuberculosis 23 kDa SOD protein (SEQ ID NO: 8); Mycobacterium tuberculosis 23.5 kDa MPT64 protein (SEQ ID NO: 9); Mycobacterium tuberculosis 24 kDa MPT51 protein (SEQ ID NO: 10); Mycobacterium tuberculosis 32 kDa antigen 85A protein (SEQ ID NO: 11); Mycobacterium tuberculosis 32 kDa antigen 85C protein (SEQ ID NO: 12); Mycobacterium tuberculosis 45 kDa MPT32 protein (SEQ ID NO: 13); Mycobacterium tuberculosis 58 kDa glutamine synthetase protein (SEQ ID NO: 14); Mycobacterium tuberculosis 71 kDa HSP 70 protein (SEQ ID NO: 15); Mycobacterium tuberculosis 10.4 kDa EsxH protein (SEQ ID NO: 16); Mycobacterium tuberculosis 80 kDa glcB protein (SEQ ID NO: 20); Mycobacterium tuberculosis 110 kDa can protein (SEQ ID NO: 21); or Mycobacterium tuberculosis 9.9 kDa ESAT-6 protein (SEQ ID NO: 22); at least one protein from group B: Mycobacterium tuberculosis 14 kDa alpha crystalline homolog protein (SEQ ID NO: 17); Mycobacterium tuberculosis 47 kDa isocitrate lysate protein (SEQ ID NO: 18); or Mycobacterium tuberculosis 7.6 kDa hypothetical protein (SEQ ID NO: 19).

4. The composition of claim 1, wherein the Listeria monocytogenes: does not express a functional InlB protein (SEQ ID NO: 2); and/or expresses prfA protein having a G155S substitution mutation (SEQ ID NO: 3).

5. The composition of claim 1, further comprising at least one of a pharmaceutically acceptable carrier or an adjuvant.

6. The composition of claim 1, further comprising a buffer system.

7. The composition of claim 1, wherein the Mycobacterium tuberculosis 30 kDa antigen 85B protein is fused in frame with a heterologous protein sequence.

8. The composition of claim 1, wherein the Mycobacterium tuberculosis 30 kDa antigen 85B protein is coupled to a heterologous protein sequence comprising the N-terminal 100 amino acids of the ActA protein.

9. The composition of claim 4, wherein the expression of the Mycobacterium tuberculosis 30 kDa antigen 85B protein is controlled by an actA promoter.

10. A method of generating an antibody to a Mycobacterium tuberculosis 30 kDa antigen 85B protein (SEQ ID NO: 4) comprising the steps of: immunizing a mammal with a composition of matter comprising attenuated Listeria monocytogenes, wherein the Listeria monocytogenes: does not express a functional ActA protein (SEQ ID NO: 1); and expresses Mycobacterium tuberculosis 30 kDa antigen 85B protein (SEQ ID NO: 4); such that an antibody to a Mycobacterium tuberculosis 30 kDa antigen 85B protein is generated.

11. The method of claim 10, further comprising immunizing the mammal with Mycobacterium bovis strain Bacille Calmette-Guerin (BCG).

12. The method of claim 11, wherein the BCG is used in a primary immunization and the attenuated Listeria monocytogenes is used in a booster immunization.

13. The method of claim 10, wherein the Listeria monocytogenes: does not express a functional InlB protein (SEQ ID NO: 2); and/or expresses prfA protein having a G155S substitution mutation (SEQ ID NO: 3).

14. The method of claim 10, wherein the mammal is immunized intranasally, subcutaneously, intradermally, intramuscularly or orally.

15. The method of claim 10, wherein the mammal is a guinea pig or a mouse.

16. The method of claim 10, wherein the mammal is a human.

17. The method of claim 10, wherein the Listeria monocytogenes further expresses at least one protein from the following group: Mycobacterium tuberculosis 12 kDa fragment of 16 kDa membrane protein (SEQ ID NO:5); Mycobacterium tuberculosis 14 kDa MPT53 protein (SEQ ID NO: 6); Mycobacterium tuberculosis 16 kDa MPT63 protein (SEQ ID NO: 7); Mycobacterium tuberculosis 23 kDa SOD protein (SEQ ID NO: 8); Mycobacterium tuberculosis 23.5 kDa MPT64 protein (SEQ ID NO: 9); Mycobacterium tuberculosis 24 kDa MPT51 protein (SEQ ID NO: 10); Mycobacterium tuberculosis 32 kDa antigen 85A protein (SEQ ID NO: 11); Mycobacterium tuberculosis 32 kDa antigen 85C protein (SEQ ID NO: 12); Mycobacterium tuberculosis 45 kDa MPT32 protein (SEQ ID NO: 13); Mycobacterium tuberculosis 58 kDa glutamine synthetase protein (SEQ ID NO: 14); Mycobacterium tuberculosis 71 kDa HSP 70 protein (SEQ ID NO: 15); Mycobacterium tuberculosis 10.4 kDa EsxH protein (SEQ ID NO: 16); Mycobacterium tuberculosis 14 kDa alpha crystalline homolog protein (SEQ ID NO: 17); Mycobacterium tuberculosis 47 kDa isocytrate lysate protein (SEQ ID NO: 18); Mycobacterium tuberculosis 7.6 kDa hypothetical protein (SEQ ID NO: 19); Mycobacterium tuberculosis 80 kDa glcB protein (SEQ ID NO: 20); Mycobacterium tuberculosis 110 kDa can protein (SEQ ID NO: 21); or Mycobacterium tuberculosis 9.9 kDa ESAT-6 protein (SEQ ID NO: 22).

18. The method of claim 10, wherein the Listeria monocytogenes further expresses at least one protein from group A: Mycobacterium tuberculosis 12 kDa fragment of 16 kDa membrane protein (SEQ ID NO:5); Mycobacterium tuberculosis 14 kDa MPT53 protein (SEQ ID NO: 6); Mycobacterium tuberculosis 16 kDa MPT63 protein (SEQ ID NO: 7); Mycobacterium tuberculosis 23 kDa SOD protein (SEQ ID NO: 8); Mycobacterium tuberculosis 23.5 kDa MPT64 protein (SEQ ID NO: 9); Mycobacterium tuberculosis 24 kDa MPT51 protein (SEQ ID NO: 10); Mycobacterium tuberculosis 32 kDa antigen 85A protein (SEQ ID NO: 11); Mycobacterium tuberculosis 32 kDa antigen 85C protein (SEQ ID NO: 12); Mycobacterium tuberculosis 45 kDa MPT32 protein (SEQ ID NO: 13); Mycobacterium tuberculosis 58 kDa glutamine synthetase protein (SEQ ID NO: 14); Mycobacterium tuberculosis 71 kDa HSP 70 protein (SEQ ID NO: 15); Mycobacterium tuberculosis 10.4 kDa EsxH protein (SEQ ID NO: 16); Mycobacterium tuberculosis 80 kDa glcB protein (SEQ ID NO: 20); Mycobacterium tuberculosis 110 kDa can protein (SEQ ID NO: 21); or Mycobacterium tuberculosis 9.9 kDa ESAT-6 protein (SEQ ID NO: 22); at least one protein from group B: Mycobacterium tuberculosis 14 kDa alpha crystalline homolog protein (SEQ ID NO: 17); Mycobacterium tuberculosis 47 kDa isocytrate lysate protein (SEQ ID NO: 18); or Mycobacterium tuberculosis 7.6 kDa hypothetical protein (SEQ ID NO: 19).

19. The method of claim 10, wherein the Mycobacterium tuberculosis 30 kDa antigen 85B protein is coupled to a heterologous protein sequence comprising the N-terminal 100 amino acids of the ActA protein.

20. The method of claim 19, wherein the expression of the Mycobacterium tuberculosis 30 kDa antigen 85B protein is controlled by an ActA promoter.

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