COMPOUNDS AND METHODS FOR IMMUNOTHERAPY AND DIAGNOSIS OF TUBERCULOSIS

Abstract

Compounds and methods for inducing protective immunity against tuberculosis are disclosed. The compounds provided include polypeptides that contain at least one immunogenic portion of one or more M. tuberculosis proteins and DNA molecules encoding such polypeptides. Such compounds may be formulated into vaccines and/or pharmaceutical compositions for immunization against M. tuberculosis infection, or may be used for the diagnosis of tuberculosis.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. application Ser. No. 08/730,510, filed Oct. 11, 1996; which claims priority from PCT Application no. PCT/US 96/14674, filed Aug. 30, 1996; and is a continuation-in-part of U.S. application Ser. No. 08/680,574, filed Jul. 12, 1996; which is a continuation-in-part of U.S. application Ser. No. 08/659,683, filed Jun. 5, 1996; which is a continuation-in-part of U.S. application Ser. No. 08/620,874, filed Marcia 22, 1996; which is a continuation-in-part of U.S. application Ser. No. 08/533,634, filed Sep. 22, 1995; which is a continuation-in-part of U.S. application Ser. No. 08/523,436, filed Sep. 1, 1995, now abandoned.

TECHNICAL FIELD

[0002] The present invention relates generally to detecting, treating and preventing Mycobacterium tuberculosis infection. The invention is more particularly related to polypeptides comprising a Mycobacterium tuberculosis antigen, or a portion or other variant thereof, and the use of such polypeptides for diagnosing and vaccinating against Mycobacterium tuberculosis infection.

BACKGROUND OF THE INVENTION

[0003] Tuberculosis is a chronic, infectious disease, that is generally caused by infection with Mycobacterium tuberculosis. It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about 8 million new cases and 3 million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as an acute inflammation of the lungs, resulting in fever and a nonproductive cough. If left untreated, serious complications and death typically result.

[0004] Although tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behavior is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistance.

[0005] Inhibiting the spread of tuberculosis requires effective vaccination and accurate, early diagnosis of the disease. Currently, vaccination with live bacteria is the most efficient method for inducing protective immunity. The most common Mycobacterium employed for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of Mycobacterium bovis. However, the safety and efficacy of BCG is a source of controversy and some countries, such as the United States, do not vaccinate the general public. Diagnosis is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative). Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, which indicates exposure to Mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.

[0006] While macrophages have been shown to act as the principal effectors of M. tuberculosis immunity, T cells are the predominant inducers of such immunity. The essential role of T cells in protection against M. tuberculosis infection is illustrated by the frequent occurrence of M. tuberculosis in AIDS patients, due to the depletion of CD4 T cells associated with human immunodeficiency virus (HIV) infection. Mycobacterium-reactive CD4 T cells have been shown to be potent producers of gamma-interferon (IFN-γ), which, in turn, has been shown to trigger the anti-mycobacterial effects of macrophages in mice. While the role of IFN-γ in humans is less clear, studies have shown that 1,25-dihydroxy-vitamin D3, either alone or in combination with IFN-γ or tumor necrosis factor-alpha, activates human macrophages to inhibit M. tuberculosis infection. Furthermore, it is known that IFN-γ stimulates human macrophages to make 1,25-dihydroxy-vitamin D3. Similarly, TL-12 has been shown to play a role in stimulating resistance to M. tuberculosis infection. For a review of the immunology of M. tuberculosis infection see Chan and Kaufmann in Tuberculosis: Pathogenesis, Protection and Control, Bloom (ed.), ASM Press, Washington, D.C., 1994.

[0007] Accordingly, there is a need in the art for improved vaccines and methods for preventing, treating and detecting tuberculosis. The present invention fulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

[0008] Briefly stated, this invention provides compounds and methods for preventing and diagnosing tuberculosis. In one aspect, polypeptides are provided comprising an immunogenic portion of a soluble M. tuberculosis antigen, or a variant of such an antigen that differs only in conservative substitutions and/or modifications. In one embodiment of this aspect, the soluble antigen has one of the following N-terminal sequences:

TABLE-US-00001 (a) (SEQ ID No. 120) Asp-Pro-Val-Asp-Ala-Val-Ile-Asn-Thr-Thr-Cys-Asn- Tyr-Gly-Gln-Val-Val-Ala-Ala-Leu; (b) (SEQ ID No. 121) Ala-Val-Glu-Ser-Gly-Met-Leu-Ala-Leu-Gly-Thr-Pro- Ala-Pro-Ser; (c) (SEQ ID No. 122) Ala-Ala-Met-Lys-Pro-Arg-Thr-Gly-Asp-Gly-Pro-Leu- Glu-Ala-Ala-Lys-Glu-Gly-Arg; (d) (SEQ ID No. 123) Tyr-Tyr-Trp-Cys-Pro-Gly-Gln-Pro-Phe-Asp-Pro-Ala- Trp-Gly-Pro; (e) (SEQ ID No. 124) Asp-Ile-Gly-Ser-Glu-Ser-Thr-Glu-Asp-Gln-Gln-Xaa- Ala-Val; (f) (SEQ ID No. 125) Ala-Glu-Glu-Ser-Ile-Ser-Thr-Xaa-Glu-Xaa-Ile-Val- Pro; (g) (SEQ ID No. 126) Asp-Pro-Glu-Pro-Ala-Pro-Pro-Val-Pro-Thr-Thr-Ala- Ala-Ser-Pro-Pro-Ser, (h) (SEQ ID No. 127) Ala-Pro-Lys-Thr-Tyr-Xaa-Glu-Glu-Leu-Lys-Gly-Thr- Asp-Thr-Gly; (i) (SEQ ID No. 128) Asp-Pro-Ala-Ser-Ala-Pro-Asp-Val-Pro-Thr-Ala-Ala- Gln-Leu-Thr-Ser-Leu-Leu-Asn-Ser-Leu-Ala-Asp-Pro- Asn-Val-Ser-Phe-Ala-Asn; (j) (SEQ ID No. 134) Xaa-Asp-Ser-Glu-Lys-Ser-Ala-Thr-Ile-Lys-Val-Thr- Asp-Ala-Ser; (k) (SEQ ID No. 135) Ala-Gly-Asp-Thr-Xaa-Ile-Tyr-Ile-Val-Gly-Asn-Leu- Thr-Ala-Asp; or (l) (SEQ ID No. 136) Ala-Pro-Glu-Ser-Gly-Ala-Gly-Leu-Gly-Gly-Thr-Val- Gln-Ala-Gly;

wherein Xaa may be any amino acid.

[0009] In a related aspect, polypeptides are provided comprising an immunogenic portion of an M. tuberculosis antigen, or a variant of such an antigen that differs only in conservative substitutions and/or modifications, the antigen having one of the following N-terminal sequences:

TABLE-US-00002 (m) (SEQ ID No. 137) Xaa-Tyr-Ile-Ala-Tyr-Xaa-Thr-Thr-Ala-Gly-Ile-Val- Pro-Gly-Lys-Ile-Asn-Val-His-Leu-Val; or (n) (SEQ ID No. 129) Asp-Pro-Pro-Asp-Pro-His-Gln-Xaa-Asp-Met-Thr-Lys- Gly-Tyr-Tyr-Pro-Gly-Gly-Arg-Arg-Xaa-Phe;

wherein Xaa may be any amino acid.

[0010] In another embodiment, the antigen comprises an amino acid sequence encoded by a DNA sequence selected from the group consisting of the sequences recited in SEQ ID Nos.: 1, 2, 4-10, 13-25, 52, 99 and 101, the complements of said sequences, and DNA sequences that hybridize to a sequence recited in SEQ ID Nos.: 1, 2, 4-10, 13-25, 52, 99 and 101 or a complement thereof under moderately stringent conditions.

[0011] In a related aspect, the polypeptides comprise an immunogenic portion of a M. tuberculosis antigen, or a variant of such an antigen that differs only in conservative substitutions and/or modifications, wherein the antigen comprises an amino acid sequence encoded by a DNA sequence selected from the group consisting of the sequences recited in SEQ ID Nos.: 26-51, 138 and 139, the complements of said sequences, and DNA sequences that hybridize to a sequence recited in SEQ ID Nos.: 26-51, 133 and 139 or a complement thereof under moderately stringent conditions.

[0012] In related aspects, DNA sequences encoding the above polypeptides, expression vectors comprising these DNA sequences and host cells transformed or transfected with such expression vectors are also provided.

[0013] In another aspect, the present invention provides fission proteins comprising a first and a second inventive polypeptide or, alternatively, an inventive polypeptide and a known M. tuberculosis antigen.

[0014] Within other aspects, the present invention provides pharmaceutical compositions that comprise one or more of the above polypeptides, or a DNA molecule encoding such polypeptides, and a physiologically acceptable carrier. The invention also provides vaccines comprising one or more of the polypeptides as described above and a non-specific immune response enhancer, together with vaccines comprising one or more DNA sequences encoding such polypeptides and a non-specific immune response enhancer.

[0015] In yet another aspect, methods are provided for inducing protective immunity in a patient, comprising administering to a patient an effective amount of one or more of the above polypeptides.

[0016] In further aspects of this invention, methods and diagnostic kits are provided for detecting tuberculosis in a patient. The methods comprise contacting dermal cells of a patient with one or more of the above polypeptides and detecting an immune response on the patient's skin. The diagnostic kits comprise one or more of the above polypeptides in combination with an apparatus sufficient to contact the polypeptide with the dermal cells of a patient.

[0017] In yet other aspects, methods are provided for detecting tuberculosis in a patient, such methods comprising contacting dermal cells of a patient with one or more polypeptides encoded by a DNA sequence selected from the group consisting of SEQ ID Nos.: 3, 11, 12, 140 and 141, the complements of said sequences, and DNA sequences that hybridize to a sequence recited in SEQ ID Nos.: 3, 11, 12, 140 and 141; and detecting an immune response on the patient's skin. Diagnostic kits for use in such methods are also provided.

[0018] These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS

[0019] FIGS. 1A and B illustrate the stimulation of proliferation and interferon-γ production in T cells derived from a first and a second M. tuberculosis-immune donor, respectively, by the 14 Kd, 20 Kd and 26 Kd antigens described in Example 1.

[0020] FIG. 2 illustrates the stimulation of proliferation and interferon-γ production in T cells derived from an M. tuberculosis-immune individual by the two representative polypeptides TbRa3 and TbRa9.

[0021] FIGS. 3A-D illustrate the reactivity of antisera raised against secretory M. tuberculosis proteins, the known M. tuberculosis antigen 85b and the inventive antigens Tb38-1 and TbH-9, respectively, with M. tuberculosis lysate (lane 2), M. tuberculosis secretory proteins (lane 3), recombinant Tb38-1 (lane 4), recombinant TbH-9 (lane 5) and recombinant 85b (lane 5).

[0022] FIG. 4A illustrates the stimulation of proliferation in a TbH-9-specific T cell clone by secretory M. tuberculosis proteins, recombinant TbH-9 and a control antigen, TbRa11.

[0023] FIG. 4B illustrates the stimulation of interferon-γ production in a TbH-9-specific T cell clone by secretory M. tuberculosis proteins, PPD and recombinant TbH-9.

[0024] FIGS. 5A and B illustrate the stimulation of proliferation and interferon-γ production in TbH9-specific T cells by the fusion protein TbH9-Tb38-1.

[0025] FIGS. 6A and B illustrate the stimulation of proliferation and interferon-γ production in Tb38-1-specific T cells by the fusion protein TbH9-Tb38-1.

[0026] FIGS. 7A and B illustrate the stimulation of proliferation and interferon-γ production in T cells previously shown to respond to both TbH-9 and Tb38-1 by the fusion protein TbH9-Tb38-1.

[0027] SEQ. ID NO. 1 is the DNA sequence of TbRa1. [0028] SEQ. ID NO. 2 is the DNA sequence of TbRa10. [0029] SEQ. ID NO. 3 is the DNA sequence of TbRa11. [0030] SEQ. ID NO. 4 is the DNA sequence of TbRa12. [0031] SEQ. ID NO. 5 is the DNA sequence of TbRa13. [0032] SEQ. ID NO. 6 is the DNA sequence of TbRa16. [0033] SEQ. ID NO. 7 is the DNA sequence of TbRa17. [0034] SEQ. ID NO. 8 is the DNA sequence of TbRa18. [0035] SEQ. ID NO. 9 is the DNA sequence of TbRa19. [0036] SEQ. ID NO. 10 is the DNA sequence of TbRa24. [0037] SEQ. ID NO. 11 is the DNA sequence of TbRa26. [0038] SEQ. ID NO. 12 is the DNA sequence of TbRa28. [0039] SEQ. ID NO. 13 is the DNA sequence of TbRa29. [0040] SEQ. ID NO. 14 is the DNA sequence of TbRa2A. [0041] SEQ. ID NO. 15 is the DNA sequence of TbRa3. [0042] SEQ. ID NO. 16 is the DNA sequence of TbRa32. [0043] SEQ. ID NO. 17 is the DNA sequence of TbRa35. [0044] SEQ. ID NO. 18 is the DNA sequence of TbRa36. [0045] SEQ. ID NO. 19 is the DNA sequence of TbRa4. [0046] SEQ. ID NO. 20 is the DNA sequence of TbRa9. [0047] SEQ. ID NO. 21 is the DNA sequence of TbRaB. [0048] SEQ. ID NO. 22 is the DNA sequence of TbRaC. [0049] SEQ. ID NO. 23 is the DNA sequence of TbRaD. [0050] SEQ. ID NO. 24 is the DNA sequence of YYWCPG. [0051] SEQ. ID NO. 25 is the DNA sequence of AAMK. [0052] SEQ. ID NO. 26 is the DNA sequence of TbL-23. [0053] SEQ. ID NO. 27 is the DNA sequence of TbL-24. [0054] SEQ. ID NO. 28 is the DNA sequence of TbL-25. [0055] SEQ. ID NO. 29 is the DNA sequence of TbL-28. [0056] SEQ. ID NO. 30 is the DNA sequence of TbL-29. [0057] SEQ. ID NO. 31 is the DNA sequence of TbH-5. [0058] SEQ. ID NO. 32 is the DNA sequence of TbH-8. [0059] SEQ. ID NO. 33 is the DNA sequence of TbH-9. [0060] SEQ. ID NO. 34 is the DNA sequence of TbM-1. [0061] SEQ. ID NO. 35 is the DNA sequence of TbM-3. [0062] SEQ. ID NO. 36 is the DNA sequence of TbM-6. [0063] SEQ. ID NO. 37 is the DNA sequence of TbM-7. [0064] SEQ. ID NO. 38 is the DNA sequence of TbM-9. [0065] SEQ. ID NO. 39 is the DNA sequence of TbM-12. [0066] SEQ. ID NO. 40 is the DNA sequence of TbM-13. [0067] SEQ. ID NO. 41 is the DNA sequence of TbM-14. [0068] SEQ. ID NO. 42 is the DNA sequence of TbM-15. [0069] SEQ. ID NO. 43 is the DNA sequence of TbH-4. [0070] SEQ. ID NO. 44 is the DNA sequence of TbH-4-FWD. [0071] SEQ. ID NO. 45 is the DNA sequence of TbH-12. [0072] SEQ. ID NO. 46 is the DNA sequence of Tb38-1. [0073] SEQ. ID NO. 47 is the DNA sequence of Tb38-4. [0074] SEQ. ID NO. 48 is the DNA sequence of TbL-17. [0075] SEQ. ID NO. 49 is the DNA sequence of TbL-20. [0076] SEQ. ID NO. 50 is the DNA sequence of TbL-21. [0077] SEQ. ID NO. 51 is the DNA sequence of TbH-16. [0078] SEQ. ID NO. 52 is the DNA sequence of DPEP. [0079] SEQ. ID NO. 53 is the deduced amino acid sequence of DPEP. [0080] SEQ. ID NO. 54 is the protein sequence of DPV N-terminal Antigen. [0081] SEQ. ID NO. 55 is the protein sequence of AVGS N-terminal Antigen. [0082] SEQ. ID NO. 56 is the protein sequence of AAMK N-terminal Antigen. [0083] SEQ. ID NO. 57 is the protein sequence of YYWC N-terminal Antigen. [0084] SEQ. ID NO. 58 is the protein sequence of DIGS N-terminal Antigen. [0085] SEQ. ID NO. 59 is the protein sequence of AEES N-terminal Antigen. [0086] SEQ. ID NO. 60 is the protein sequence of DPEP N-terminal Antigen. [0087] SEQ. ID NO. 61 is the protein sequence of APKT N-terminal Antigen. [0088] SEQ. ID NO. 62 is the protein sequence of DPAS N-terminal Antigen. [0089] SEQ. ID NO. 63 is the deduced amino acid sequence of TbRa1. [0090] SEQ. ID NO. 64 is the deduced amino acid sequence of TbRa10. [0091] SEQ. ID NO. 65 is the deduced amino acid sequence of TbRa11. [0092] SEQ. ID NO. 66 is the deduced amino acid sequence of TbRa12. [0093] SEQ. ID NO. 67 is the deduced amino acid sequence of TbRa13. [0094] SEQ. ID NO. 68 is the deduced amino acid sequence of TbRa16. [0095] SEQ. ID NO. 69 is the deduced amino acid sequence of TbRa17. [0096] SEQ. ID NO. 70 is the deduced amino acid sequence of TbRa18. [0097] SEQ. ID NO. 71 is the deduced amino acid sequence of TbRa19. [0098] SEQ. ID NO. 72 is the deduced amino acid sequence of TbRa24. [0099] SEQ. ID NO. 73 is the deduced amino acid sequence of TbRa26. [0100] SEQ. ID NO. 74 is the deduced amino acid sequence of TbRa28. [0101] SEQ. ID NO. 75 is the deduced amino acid sequence of TbRa29. [0102] SEQ. ID NO. 76 is the deduced amino acid sequence of TbRa2A. [0103] SEQ. ID NO. 77 is the deduced amino acid sequence of TbRa3. [0104] SEQ. ID NO. 78 is the deduced amino acid sequence of TbRa32. [0105] SEQ. ID NO. 79 is the deduced amino acid sequence of TbRa35. [0106] SEQ. ID NO. 80 is the deduced amino acid sequence of TbRa36. [0107] SEQ. ID NO. 81 is the deduced amino acid sequence of TbRa4. [0108] SEQ. ID NO. 82 is the deduced amino acid sequence of TbRa9. [0109] SEQ. ID NO. 83 is the deduced amino acid sequence of TbRaB. [0110] SEQ. ID NO. 84 is the deduced amino acid sequence of TbRaC. [0111] SEQ. ID NO. 85 is the deduced amino acid sequence of TbRaD. [0112] SEQ. ID NO. 86 is the deduced amino acid sequence of YYWCPG. [0113] SEQ. ID NO. 87 is the deduced amino acid sequence of TbAAMK. [0114] SEQ. ID NO. 88 is the deduced amino acid sequence of Tb38-1. [0115] SEQ. ID NO. 89 is the deduced amino acid sequence of TbH-4. [0116] SEQ. ID NO. 90 is the deduced amino acid sequence of TbH-8. [0117] SEQ. ID NO. 91 is the deduced amino acid sequence of TbH-9. [0118] SEQ. ID NO: 92 is the deduced amino acid sequence of TbH-12. [0119] SEQ. ID NO. 93 is the amino acid sequence of Tb38-1 Peptide 1. [0120] SEQ. ID NO. 94 is the amino acid sequence of Tb38-1 Peptide 2. [0121] SEQ. ID NO. 95 is the amino acid sequence of Tb38-1 Peptide 3. [0122] SEQ. ID NO. 96 is the amino acid sequence of Tb38-1 Peptide 4. [0123] SEQ. ID NO. 97 is the amino acid sequence of Tb38-1 Peptide 5. [0124] SEQ. ID NO. 98 is the amino acid sequence of Tb38-1 Peptide 6. [0125] SEQ. ID NO. 99 is the DNA sequence of DPAS. [0126] SEQ. ID NO. 100 is the deduced amino acid sequence of DPAS. [0127] SEQ. ID NO. 101 is the DNA sequence of DPV. [0128] SEQ. ID NO. 102 is the deduced amino acid sequence of DPV. [0129] SEQ. ID NO. 103 is the DNA sequence of ESAT-6. [0130] SEQ. ID NO. 104 is the deduced amino acid sequence of ESAT-6. [0131] SEQ. ID NO. 105 is the DNA sequence of TbH-8-2. [0132] SEQ. ID NO. 106 is the DNA sequence of TbH-9FL. [0133] SEQ. ID NO. 107 is the deduced amino acid sequence of TbH-9 μL. [0134] SEQ. ID NO. 108 is the DNA sequence of TbH-9-1. [0135] SEQ. ID NO. 109 is the deduced amino acid sequence of TbH-9-1. [0136] SEQ. ID NO. 110 is the DNA sequence of TbH-9-4. [0137] SEQ. ID NO. 111 is the deduced amino acid sequence of TbH-9-4. [0138] SEQ. ID NO. 112 is the DNA sequence of Tb38-1F2 IN. [0139] SEQ. ID NO. 113 is the DNA sequence of Tb38-2F2 RP. [0140] SEQ. ID NO. 114 is the deduced amino acid sequence of Tb37-FL. [0141] SEQ. ID NO. 115 is the deduced amino acid sequence of Tb38-IN. [0142] SEQ. ID NO. 116 is the DNA sequence of Tb38-1F3. [0143] SEQ. ID NO. 117 is the deduced amino acid sequence of Tb38-1F3. [0144] SEQ. ID NO. 118 is the DNA sequence of Tb38-1F5. [0145] SEQ. ID NO. 119 is the DNA sequence of Tb38-1F6. [0146] SEQ. ID NO. 120 is the deduced N-terminal amino acid sequence of DPV. [0147] SEQ. ID NO. 121 is the deduced N-terminal amino acid sequence of AVGS. [0148] SEQ. ID NO. 122 is the deduced N-terminal amino acid sequence of AAMK. [0149] SEQ. ID NO. 123 is the deduced N-terminal amino acid sequence of YYWC. [0150] SEQ. ID NO. 124 is the deduced N-terminal amino acid sequence of DIGS. [0151] SEQ. ID NO. 125 is the deduced N-terminal amino acid sequence of AEES. [0152] SEQ. ID NO. 126 is the deduced N-terminal amino acid sequence of DPEP. [0153] SEQ. ID NO. 127 is the deduced N-terminal amino acid sequence of APKT. [0154] SEQ. ID NO. 128 is the deduced amino acid sequence of DPAS. [0155] SEQ.

ID NO. 129 is the protein sequence of DPPD N-terminal Antigen. [0156] SEQ ID NO. 130-133 are the protein sequences of four DPPD cyanogen bromide fragments. [0157] SEQ ID NO. 134 is the N-terminal protein sequence of XDS antigen. [0158] SEQ ID NO. 135 is the N-terminal protein sequence of AGD antigen. [0159] SEQ ID NO. 136 is the N-terminal protein sequence of APE antigen. [0160] SEQ ID NO. 137 is the N-terminal protein sequence of XYI antigen. [0161] SEQ ID NO. 138 is the DNA sequence of TbH-29. [0162] SEQ ID NO. 139 is the DNA sequence of TbH-30. [0163] SEQ ID NO. 140 is the DNA sequence of TbH-32. [0164] SEQ ID NO. 141 is the DNA sequence of TbH-33. [0165] SEQ ID NO. 142 is the predicted amino acid sequence of TbH-29. [0166] SEQ ID NO. 143 is the predicted amino acid sequence of TbH-30. [0167] SEQ ID NO. 144 is the predicted amino acid sequence of TbH-32. [0168] SEQ ID NO. 145 is the predicted amino acid sequence of TbH-33. [0169] SEQ ID NO: 146-151 are PCR primers used in the preparation of a fusion protein containing TbRa3, 38 kD and Tb38-1. [0170] SEQ ID NO: 152 is the DNA sequence of the fusion protein containing TbRa3, 38 kD and Tb38-1. [0171] SEQ ID NO: 153 is the amino acid sequence of the fusion protein containing TbRa3, 38 kD and Tb38-1. [0172] SEQ ID NO: 154 is the DNA sequence of the M. tuberculosis antigen 38 kD. [0173] SEQ ID NO: 155 is the amino acid sequence of the M. tuberculosis antigen 38

DETAILED DESCRIPTION OF THE INVENTION

[0174] As noted above, the present invention is generally directed to compositions and methods for preventing, treating and diagnosing tuberculosis. The compositions of the subject invention include polypeptides that comprise at least one immunogenic portion of a M. tuberculosis antigen, or a variant of such an antigen that differs only in conservative substitutions and/or modifications. Polypeptides within the scope of the present invention include, but are not limited to, immunogenic soluble M. tuberculosis antigens. A "soluble M. tuberculosis antigen" is a protein of M. tuberculosis origin that is present in M. tuberculosis culture filtrate. As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds. Thus, a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences. The additional sequences may be derived from the native M. tuberculosis antigen or may be heterologous, and such sequences may (but need not) be immunogenic.

[0175] "Immunogenic," as used herein, refers to the ability to elicit an immune response (e.g., cellular) in a patient, such as a human, and/or in a biological sample. In particular, antigens that are immunogenic (and immunogenic portions or other variants of such antigens) are capable of stimulating cell proliferation, interleukin-12 production and/or interferon-γ production in biological samples comprising one or more cells selected from the group of T cells, NK cells, B cells and macrophages, where the cells are derived from an M. tuberculosis-immune individual. Polypeptides comprising at least an immunogenic portion of one or more M. tuberculosis antigens may generally be used to detect tuberculosis or to induce protective immunity against tuberculosis in a patient.

[0176] The compositions and methods of this invention also encompass variants of the above polypeptides. A "variant," as used herein, is a polypeptide that differs from the native antigen only in conservative substitutions and/or modifications, such that the ability of the polypeptide to induce an immune response is retained. Such variants may generally be identified by modifying one of the above polypeptide sequences, and evaluating the immunogenic properties of the modified polypeptide using, for example, the representative procedures described herein.

[0177] A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

[0178] Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenic properties, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

[0179] In a related aspect, combination polypeptides are disclosed. A "combination polypeptide" is a polypeptide comprising at least one of the above immunogenic portions and one or more additional immunogenic M. tuberculosis sequences, which are joined via a peptide linkage into a single amino acid chain. The sequences may be joined directly (i.e., with no intervening amino acids) or may be joined by way of a linker sequence (e.g., Gly-Cys-Gly) that does not significantly diminish the immunogenic properties of the component polypeptides.

[0180] In general, M. tuberculosis antigens, and DNA sequences encoding such antigens, may be prepared using any of a variety of procedures. For example, soluble antigens may be isolated from M. tuberculosis culture filtrate by procedures known to those of ordinary skill in the art, including anion-exchange and reverse phase chromatography. Purified antigens are then evaluated for their ability to elicit an appropriate immune response (e.g., cellular) using, for example, the representative methods described herein. Immunogenic antigens may then be partially sequenced using techniques such as traditional Edman chemistry. See Edman and Berg, Eur. J. Biochem. 80:116-132, 1967.

[0181] Immunogenic antigens may also be produced recombinantly using a DNA sequence that encodes the antigen, which has been inserted into an expression vector and expressed in an appropriate host. DNA molecules encoding soluble antigens may be isolated by screening an appropriate M. tuberculosis expression library with anti-sera (e.g., rabbit) raised specifically against soluble M. tuberculosis antigens. DNA sequences encoding antigens that may or may not be soluble may be identified by screening an appropriate M. tuberculosis genomic or cDNA expression library with sera obtained from patients infected with M. tuberculosis. Such screens may generally be performed using techniques well known to those of ordinary skill in the art, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989.

[0182] DNA sequences encoding soluble antigens may also be obtained by screening an appropriate M. tuberculosis cDNA or genomic DNA library for DNA sequences that hybridize to degenerate oligonucleotides derived from partial amino acid sequences of isolated soluble antigens. Degenerate oligonucleotide sequences for use in such a screen may be designed and synthesized, and the screen may be performed, as described (for example) in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989 (and references cited therein). Polymerase chain reaction (PCR) may also be employed, using the above oligonucleotides in methods well known in the art, to isolate a nucleic acid probe from a cDNA or genomic library. The library screen may then be performed using the isolated probe.

[0183] Alternatively, genomic or cDNA libraries derived from M. tuberculosis may be screened directly using peripheral blood mononuclear cells (PBMCs) or T cell lines or clones derived from one or more M. tuberculosis-immune individuals. In general, PBMCs and/or T cells for use in such screens may be prepared as described below. Direct library screens may generally be performed by assaying pools of expressed recombinant proteins for the ability to induce proliferation and/or interferon-γ production in T cells derived from an M. tuberculosis-immune individual. Alternatively, potential T cell antigens may be first selected based on antibody reactivity, as described above.

[0184] Regardless of the method of preparation, the antigens (and immunogenic portions thereof) described herein (which may or may not be soluble) have the ability to induce an immunogenic response. More specifically, the antigens have the ability to induce proliferation and/or cytokine production (i.e., interferon-γ and/or interleukin-12 production) in T cells, NK cells, B cells and/or macrophages derived from an M. tuberculosis-immune individual. The selection of cell type for use in evaluating an immunogenic response to a antigen will, of course, depend on the desired response. For example, interleukin-12 production is most readily evaluated using preparations containing B cells and/or macrophages. An M. tuberculosis-immune individual is one who is considered to be resistant to the development of tuberculosis by virtue of having mounted an effective T cell response to M. tuberculosis (i.e., substantially free of disease symptoms). Such individuals may be identified based on a strongly positive (i.e., greater than about 10 mm diameter induration) intradermal skin test response to tuberculosis proteins (PPD) and an absence of any signs or symptoms of tuberculosis disease. T cells, NK cells, B cells and macrophages derived from M. tuberculosis-immune individuals may be prepared using methods known to those of ordinary skill in the art. For example, a preparation of PBMCs (i.e., peripheral blood mononuclear cells) may be employed without further separation of component cells. PBMCs may generally be prepared, for example, using density centrifugation through Ficoll® (Winthrop Laboratories, NY). T cells for use in the assays described herein may also be purified directly from PBMCs. Alternatively, an enriched T cell line reactive against mycobacterial proteins, or T cell clones reactive to individual mycobacterial proteins, may be employed. Such T cell clones may be generated by, for example, culturing PBMCs from M. tuberculosis-immune individuals with mycobacterial proteins for a period of 2-4 weeks. This allows expansion of only the mycobacterial protein-specific T cells, resulting in a line composed solely of such cells. These cells may then be cloned and tested with individual proteins, using methods known to those of ordinary skill in the art, to more accurately define individual T cell specificity. In general, antigens that test positive in assays for proliferation and/or cytokine production (i.e., interferon-γ and/or interleukin-12 production) performed using T cells, NK cells, B cells and/or macrophages derived from an M. tuberculosis-immune individual are considered immunogenic. Such assays may be performed, for example, using the representative procedures described below. Immunogenic portions of such antigens may be identified using similar assays, and may be present within the polypeptides described herein.

[0185] The ability of a polypeptide (e.g., an immunogenic antigen, or a portion or other variant thereof) to induce cell proliferation is evaluated by contacting the cells (e.g., T cells and/or NK cells) with the polypeptide and measuring the proliferation of the cells. In general, the amount of polypeptide that is sufficient for evaluation of about 10⁵ cells ranges from about 10 ng/mL to about 100 μg/mL and preferably is about 10 μg/mL. The incubation of polypeptide with cells is typically performed at 37° C. for about six days. Following incubation with polypeptide, the cells are assayed for a proliferative response, which may be evaluated by methods known to those of ordinary skill in the art, such as exposing cells to a pulse of radiolabeled thymidine and measuring the incorporation of label into cellular DNA. In general, a polypeptide that results in at least a three increase in proliferation above background (i.e., the proliferation observed for cells cultured without polypeptide) is considered to be able to induce proliferation.

[0186] The ability of a polypeptide to stimulate the production of interferon-γ and/or interleukin-12 in cells may be evaluated by contacting the cells with the polypeptide and measuring the level of interferon-γ or interleukin-12 produced by the cells. In general, the amount of polypeptide that is sufficient for the evaluation of about 10⁵ cells ranges from about 10 ng/mL to about 100.1 g/mL and preferably is about 10 μg/mL. The polypeptide may, but need not, be immobilized on a solid support, such as a bead or a biodegradable microsphere, such as those described in U.S. Pat. Nos. 4,897,268 and 5,075,109. The incubation of polypeptide with the cells is typically performed at 37° C. for about six days. Following incubation with polypeptide, the cells are assayed for interferon-γ and/or interleukin-12 (or one or more subunits thereof), which may be evaluated by methods known to those of ordinary skill in the art, such as an enzyme-linked immunosorbent assay (ELISA) or, in the case of IL-12 P70 subunit, a bioassay such as an assay measuring proliferation of T cells. In general, a polypeptide that results in the production of at least 50 pg of interferon-γ per mL of cultured supernatant (containing 10⁴-10⁵ T cells per mL) is considered able to stimulate the production of interferon-γ. A polypeptide that stimulates the production of at least 10 pg/mL of IL-12 P70 subunit, and/or at least 100 pg/mL of IL-12 P40 subunit, per 10⁵ macrophages or B cells (or per 3×10⁵ PBMC) is considered able to stimulate the production of IL-12.

[0187] In general, immunogenic antigens are those antigens that stimulate proliferation and/or cytokine production (i.e., interferon-γ and/or interleukin-12 production) in T cells, NK cells, B cells and/or macrophages derived from at least about 25% of M. tuberculosis-immune individuals. Among these immunogenic antigens, polypeptides having superior therapeutic properties may be distinguished based on the magnitude of the responses in the above assays and based on the percentage of individuals for which a response is observed. In addition, antigens having superior therapeutic properties will not stimulate proliferation and/or cytokine production in vitro in cells derived from more than about 25% of individuals that are not M. tuberculosis-immune, thereby eliminating responses that are not specifically due to M. tuberculosis-responsive cells. Those antigens that induce a response in a high percentage of T cell, NK cell, B cell and/or macrophage preparations from M. tuberculosis-immune individuals (with a low incidence of responses in cell preparations from other individuals) have superior therapeutic properties.

[0188] Antigens with superior therapeutic properties may also be identified based on their ability to diminish the severity of M. tuberculosis infection in experimental animals, when administered as a vaccine. Suitable vaccine preparations for use on experimental animals are described in detail below. Efficacy may be determined based on the ability of the antigen to provide at least about a 50% reduction in bacterial numbers and/or at least about a 40% decrease in mortality following experimental infection. Suitable experimental animals include mice, guinea pigs and primates.

[0189] Antigens having superior diagnostic properties may generally be identified based on the ability to elicit a response in an intradermal skin test performed on an individual with active tuberculosis, but not in a test performed on an individual who is not infected with M. tuberculosis. Skin tests may generally be performed as described below, with a response of at least 5 mm induration considered positive.

[0190] Immunogenic portions of the antigens described herein may be prepared and identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3d ed., Raven Press, 1993, pp. 243-247 and references cited therein. Such techniques include screening polypeptide portions of the native antigen for immunogenic properties. The representative proliferation and cytokine production assays described herein may generally be employed in these screens. An immunogenic portion of a polypeptide is a portion that, within such representative assays, generates an immune response (e.g., proliferation, interferon-γ production and/or interleukin-12 production) that is substantially similar to that generated by the full length antigen. In other words, an immunogenic portion of an antigen may generate at least about 20%, and preferably about 100%, of the proliferation induced by the full length antigen in the model proliferation assay described herein. An immunogenic portion may also, or alternatively, stimulate the production of at least about 20%, and preferably about 100%, of the interferon-γ and/or interleukin-12 induced by the full length antigen in the model assay described herein.

[0191] Portions and other variants of M. tuberculosis antigens may be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc., Foster City, Calif., and may be operated according to the manufacturer's instructions. Variants of a native antigen may generally be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis. Sections of the DNA sequence may also be removed using standard techniques to permit preparation of truncated polypeptides.

[0192] Recombinant polypeptides containing portions and/or variants of a native antigen may be readily prepared from a DNA sequence encoding the polypeptide using a variety of techniques well known to those of ordinary skill in the art. For example, supernatants from suitable host/vector systems which secrete recombinant protein into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant protein.

[0193] Any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant polypeptides of this invention. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this mariner may encode naturally occurring antigens, portions of naturally occurring antigens, or other variants thereof.

[0194] In general, regardless of the method of preparation, the polypeptides disclosed herein are prepared in substantially pure form. Preferably, the polypeptides are at least about 80% pure, more preferably at least about 90% pure and most preferably at least about 99% pure. In certain preferred embodiments, described in detail below, the substantially pure polypeptides are incorporated into pharmaceutical compositions or vaccines for use in one or more of the methods disclosed herein.

[0195] In certain specific embodiments, the subject invention discloses polypeptides comprising at least an immunogenic portion of a soluble M. tuberculosis antigen having one of the following N-terminal sequences, or a variant thereof that differs only in conservative substitutions and/or modifications:

TABLE-US-00003 (a) (SEQ ID No. 120) Asp-Pro-Val-Asp-Ala-Val-Ile-Asn-Thr-Thr-Cys-Asn- Tyr-Gly-Gln-Val-Val-Ala-Ala-Leu; (b) (SEQ ID No. 121) Ala-Val-Glu-Ser-Gly-Met-Leu-Ala-Leu-Gly-Thr-Pro- Ala-Pro-Ser; (c) (SEQ ID No. 122) Ala-Ala-Met-Lys-Pro-Arg-Thr-Gly-Asp-Gly-Pro-Leu- Glu-Ala-Ala-Lys-Glu-Gly-Arg; (d) (SEQ ID No. 123) Tyr-Tyr-Trp-Cys-Pro-Gly-Gln-Pro-Phe-Asp-Pro-Ala- Trp-Gly-Pro; (e) (SEQ ID No. 124) Asp-Ile-Gly-Ser-Glu-Ser-Thr-Glu-Asp-Gln-Gln-Xaa- Ala-Val; (f) (SEQ ID. No. 125) Ala-Glu-Glu-Ser-Ile-Ser-Thr-Xaa-Glu-Xaa-Ile-Val- Pro; (g) (SEQ ID No. 126) Asp-Pro-Glu-Pro-Ala-Pro-Pro-Val-Pro-Thr-Ala-Ala- Ala-Ser-Pro-Pro-Ser; (h) (SEQ ID No. 127) Ala-Pro-Lys-Thr-Tyr-Xaa-Glu-Glu-Leu-Lys-Gly-Thr- Asp-Thr-Gly; (i) (SEQ ID No. 128) Asp-Pro-Ala-Ser-Ala-Pro-Asp-Val-Pro-Thr-Ala-Ala- Gln-Leu-Thr-Ser-Leu-Leu-Asn-Ser-Leu-Ala-Asp-Pro- Asn-Val-Ser-Phe-Ala-Asn; (j) (SEQ ID No. 134) Xaa-Asp-Ser-Glu-Lys-Ser-Ala-Thr-Ile-Lys-Val-Thr- Asp-Ala-Ser; (k) (SEQ ID No. 135) Ala-Gly-Asp-Thr-Xaa-Ile-Tyr-Lle-Val-Gly-Asn-Leu- Thr-Ala-Asp; or (l) (SEQ ID No. 136) Ala-Pro-Glu-Ser-Gly-Ala-Gly-Leu-Gly-Gly-Thr-Val- Gln-Ala-Gly;

wherein Xaa may be any amino acid, preferably a cysteine residue. A DNA sequence encoding the antigen identified as (g) above is provided in SEQ ID No. 52, and the polypeptide encoded by SEQ ID No. 52 is provided in SEQ ID No. 53. A DNA sequence encoding the antigen defined as (a) above is provided in SEQ ID No. 101; its deduced amino acid sequence is provided in SEQ ID No. 102. A DNA sequence corresponding to antigen (d) above is provided in SEQ ID No. 24 a DNA sequence corresponding to antigen (c) is provided in SEQ ID No. 25 and a DNA sequence corresponding to antigen (i) is provided in SEQ ID No. 99; its deduced amino acid sequence is provided in SEQ ID No. 100.

[0196] In a further specific embodiment, the subject invention discloses polypeptides comprising at least an immunogenic portion of an M. tuberculosis antigen having one of the following N-terminal sequences, or a variant thereof that differs only in conservative substitutions and/or modifications:

TABLE-US-00004 (m) (SEQ ID No 137) Xaa-Tyr-Ile-Ala-Tyr-Xaa-Thr-Thr-Ala-Gly-Ile-Val- Pro-Gly-Lys-Ile-Asn-Val-His-Leu-Val; or (n) (SEQ ID No. 129) Asp-Pro-Pro-Asp-Pro-His-Gln-Xaa-Asp-Met-Thr-Lys- Gly-Tyr-Tyr-Pro-Gly-Gly-Arg-Arg-Xaa-Phe;

wherein Xaa may be any amino acid, preferably a cysteine residue.

[0197] In other specific embodiments, the subject invention discloses polypeptides comprising at least an immunogenic portion of a soluble M. tuberculosis antigen (or a variant of such an antigen) that comprises one or more of the amino acid sequences encoded by (a) the DNA sequences of SEQ ID Nos.: 1, 2, 4-10, 13-25 and 52; (b) the complements of such DNA sequences, or (c) DNA sequences substantially homologous to a sequence in (a) or (b).

[0198] In further specific embodiments, the subject invention discloses polypeptides comprising at least an immunogenic portion of a M. tuberculosis antigen (or a variant of such an antigen), which may or may not be soluble, that comprises one or more of the amino acid sequences encoded by (a) the DNA sequences of SEQ ID Nos.: 26-51, 138 and 139, (b) the complements of such DNA sequences or (c) DNA sequences substantially homologous to a sequence in (a) or (b).

[0199] In the specific embodiments discussed above, the XL tuberculosis antigens include variants that are encoded by DNA sequences which are substantially homologous to one or more of DNA sequences specifically recited herein. "Substantial homology," as used herein, refers to DNA sequences that are capable of hybridizing under moderately stringent conditions. Suitable moderately stringent conditions include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-65° C., 5×SSC, overnight or, in the case of cross-species homology at 45° C., 0.5×SSC; followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS). Such hybridizing DNA sequences are also within the scope of this invention, as are nucleotide sequences that, due to code degeneracy, encode an immunogenic polypeptide that is encoded by a hybridizing DNA sequence.

[0200] In a related aspect, the present invention provides fusion proteins comprising a first and a second inventive polypeptide or, alternatively, a polypeptide of the present invention and a known M. tuberculosis antigen, such as the 38 kD antigen described in Andersen and Hansen, Infect. Immun. 57:2481-2488, 1989, (Genbank Accession No. M30046) or ESAT-6 (SEQ ID Nos. 103 and 104), together with variants of such fusion proteins. The fusion proteins of the present invention may also include linker peptide between the first and second polypeptides.

[0201] A DNA sequence encoding a fusion protein of the present invention is constructed using known recombinant DNA techniques to assemble separate DNA sequences encoding the first and second polypeptides into an appropriate expression vector. The 3' end of a DNA sequence encoding the first polypeptide is ligated, with or without a peptide linker; to the 5' end of a DNA sequence encoding the second polypeptide so that the reading frames of the sequences are in phase to permit mRNA translation of the two DNA sequences into a single fusion protein that retains the biological activity of both the first and the second polypeptides.

[0202] A peptide linker sequence may be employed to separate the first and the second polypeptides by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may be from 1 to about 50 amino acids in length. Peptide sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

[0203] The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons require to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.

[0204] In another aspect, the present invention provides methods for using one or more of the above polypeptides or fusion proteins (or DNA molecules encoding such polypeptides) to induce protective immunity against tuberculosis in a patient. As used herein, a "patient" refers to any warm-blooded animal, preferably a human. A patient may be afflicted with a disease, or may be free of detectable disease and/or infection. In other words, protective immunity may be induced to prevent or treat tuberculosis.

[0205] In this aspect, the polypeptide, fusion protein or DNA molecule is generally present within a pharmaceutical composition and/or a vaccine. Pharmaceutical compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof), and a physiologically acceptable carrier. Vaccines may comprise one or more of the above polypeptides and a non-specific immune response enhancer, such as an adjuvant or a liposome (into which the polypeptide is incorporated). Such pharmaceutical compositions and vaccines may also contain other M. tuberculosis antigens, either incorporated into a combination polypeptide or present within a separate polypeptide.

[0206] Alternatively, a vaccine may contain DNA encoding one or more polypeptides as described above, such that the polypeptide is generated in situ. In such vaccines, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on, its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

[0207] In a related aspect, a DNA vaccine as described above may be administered simultaneously with or sequentially to either a polypeptide of the present invention or a known M. tuberculosis antigen, such as the 38 kD antigen described above. For example, administration of DNA encoding a polypeptide of the present invention, either "naked" or in a delivery system as described above, may be followed by administration of an antigen in order to enhance the protective immune effect of the vaccine.

[0208] Routes and frequency of administration, as well as dosage, will vary from individual to individual and may parallel those currently being used in immunization using BCG. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Between 1 and 3 doses may be administered for a 1-36 week period. Preferably, 3 doses are administered, at intervals of 3-4 months, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of polypeptide or DNA that, when administered as described above, is capable of raising an immune response in an immunized patient sufficient to protect the patient from M. tuberculosis infection for at least 1-2 years. In general, the amount of polypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 μg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

[0209] While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

[0210] Any of a variety of adjuvants may be employed in the vaccines of this invention to nonspecifically enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a nonspecific stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and quil A.

[0211] In another aspect, this invention provides methods for using one or more of the polypeptides described above to diagnose tuberculosis using a skin test. As used herein, a "skin test" is any assay performed directly on a patient in which a delayed-type hypersensitivity (DTH) reaction (such as swelling, reddening or dermatitis) is measured following intradermal injection of one or more polypeptides as described above. Such injection may be achieved using any suitable device sufficient to contact the polypeptide or polypeptides with dermal cells of the patient, such as a tuberculin syringe or 1 mL syringe. Preferably, the reaction is measured at least 48 hours after injection, more preferably 48-72 hours.

[0212] The DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to the test antigen (i.e., the immunogenic portion of the polypeptide employed, or a variant thereof). The response may be measured visually, using a ruler. In general, a response that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter, is a positive response, indicative of tuberculosis infection, which may or may not be manifested as an active disease.

[0213] The polypeptides of this invention are preferably formulated, for use in a skin test, as pharmaceutical compositions containing a polypeptide and a physiologically acceptable carrier, as described above. Such compositions typically contain one or more of the above polypeptides in an amount ranging from about 1 μg to about 100 μg, preferably from about 10 μg to about 50 μg in a volume of 0.1 mL. Preferably, the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween 80®.

[0214] In a preferred embodiment, a polypeptide employed in a skin test is of sufficient size such that it remains at the site of injection for the duration of the reaction period. In general, a polypeptide that is at least 9 amino acids in length is sufficient. The polypeptide is also preferably broken down by macrophages within hours of injection to allow presentation to T-cells. Such polypeptides may contain repeats of one or more of the above sequences and/or other immunogenic or nonimmunogenic sequences.

[0215] The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1

Purification and Characterization of Polypeptides from M. tuberculosis Culture Filtrate

[0216] This example illustrates the preparation of M. tuberculosis soluble polypeptides from culture filtrate. Unless otherwise noted, all percentages in the following example are weight per volume.

[0217] M. tuberculosis (either H37Ra, ATCC No. 25177, or H37Rv, ATCC No. 25618) was cultured in sterile GAS media at 37° C. for fourteen days. The media was then vacuum filtered (leaving the bulk of the cells) through a 0.45μ filter into a sterile 2.5 L bottle. The media was next filtered through a 0.2μ filter into a sterile 4 L bottle and NaN₃ was added to the culture filtrate to a concentration of 0.04%. The bottles were then placed in a 4° C. cold room.

[0218] The culture filtrate was concentrated by placing the filtrate in a 12 L reservoir that had been autoclaved and feeding the filtrate into a 400 ml Amicon stir cell which had been rinsed with ethanol and contained a 10,000 kDa MWCO membrane. The pressure was maintained at 60 psi using nitrogen gas. This procedure reduced the 12 L volume to approximately 50 ml.

[0219] The culture filtrate was dialyzed into 0.1% ammonium bicarbonate using a 8,000 kDa MWCO cellulose ester membrane, with two changes of ammonium bicarbonate solution. Protein concentration was then determined by a commercially available BCA assay (Pierce, Rockford, Ill.).

[0220] The dialyzed culture filtrate was then lyophilized, and the polypeptides resuspended in distilled water. The polypeptides were dialyzed against 0.01 mM 1,3 bis[tris(hydroxymethyl)-methylamino]propane, pH 7.5 (Bis-Tris propane buffer), the initial conditions for anion exchange chromatography. Fractionation was performed using gel profusion chromatography on a POROS 146 II Q/M anion exchange column 4.6 mm×100 mm (Perseptive BioSystems, Framingham, Mass.) equilibrated in 0.01 mM Bis-Tris propane buffer pH 7.5. Polypeptides were eluted with a linear 0-0.5 M NaCl gradient in the above buffer system. The column eluent was monitored at a wavelength of 220 nm.

[0221] The pools of polypeptides eluting from the ion exchange column were dialyzed against distilled water and lyophilized. The resulting material was dissolved in 0.1% trifluoroacetic acid (TFA) pH 1.9 in water, and the polypeptides were purified on a Delta-Pak C18 column (Waters, Milford, Mass.) 300 Angstrom pore size, 5 micron particle size (3.9×150 mm). The polypeptides were eluted from the column with a linear gradient from 0-60% dilution buffer (0.1% TFA in acetonitrile). The flow rate was 0.75 ml/minute and the HPLC eluent was monitored at 214 nm. Fractions containing the eluted polypeptides were collected to maximize the purity of the individual samples. Approximately 200 purified polypeptides were obtained.

[0222] The purified polypeptides were then screened for the ability to induce T-cell proliferation in PBMC preparations. The PBMCs from donors known to be PPD skin test positive and whose T-cells were shown to proliferate in response to PPD and crude soluble proteins from MTB were cultured in medium comprising RPM: 1640 supplemented with 10% pooled human serum and 50 μg/ml gentamicin. Purified polypeptides were added in duplicate at concentrations of 0.5 to 10 pg/mL. After six days of culture in 96-well round-bottom plates in a volume of 200 μl of medium was removed from each well for determination of IFN-γ levels, as described below. The plates were then pulsed with 1 μCi/well of tritiated thymidine for a further 18 hours, harvested and tritium uptake determined using a gas scintillation counter. Fractions that resulted in proliferation in both replicates three fold greater than the proliferation observed in cells cultured in medium alone were considered positive.

[0223] IFN-γ was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with a mouse monoclonal antibody directed to human IFN-γ (PharMingen, San Diego, Calif.) in PBS for four hours at room temperature. Wells were then blocked with PBS containing 5% (W/V) non-fat dried milk for 1 hour at room temperature. The plates were then washed six times in PBS/0.2% TWEEN-20 and samples diluted 1:2 in culture medium in the ELISA plates were incubated overnight at room temperature. The plates were again washed and a polyclonal rabbit anti-human IFN-γ serum diluted 1:3000 in PBS/10% normal goat serum was added to each well. The plates were then incubated for two hours at room temperature, washed and horseradish peroxidase-coupled anti-rabbit IgG (Sigma Chemical So., St. Louis, Mo.) was added at a 1:2000 dilution in PBS/5% non-fat dried milk. After a further two hour incubation at room temperature, the plates were washed and TMB substrate added. The reaction was stopped after 20 min with 1 N sulfuric acid. Optical density was determined at 450 nm using 570 nm as a reference wavelength. Fractions that resulted in both replicates giving an OD two fold greater than the mean OD from cells cultured in medium alone, plus 3 standard deviations, were considered positive.

[0224] For sequencing, the polypeptides were individually dried onto Biobrene® (Perkin Elmer/Applied BioSystems Division, Foster City, Calif.) treated glass fiber filters. The filters with polypeptide were loaded onto a Perkin Elmer/Applied BioSystems Division Procise 492 protein sequencer. The polypeptides were sequenced from the amino terminal and using traditional Edman chemistry. The amino acid sequence was determined for each polypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards.

[0225] Using the procedure described above, antigens having the following N-terminal sequences were isolated:

TABLE-US-00005 (a) (SEQ ID No. 54) Asp-Pro-Val-Asp-Ala-Val-Ile-Asn-Thr-Thr-Xaa-Asn- Tyr-Gly-Gln-Val-Val-Ala-Ala-Leu; (b) (SEQ ID No. 55) Ala-Val-Glu-Ser-Gly-Met-Leu-Ala-Leu-Gly-Thr-Pro- Ala-Pro-Ser; (c) (SEQ ID No. 56) Ala-Ala-Met-Lys-Pro-Arg-Thr-Gly-Asp-Gly-Pro-Leu- Glu-Ala-Ala-Lys-Glu-Gly-Arg; (d) (SEQ ID No. 57) Tyr-Tyr-Trp-Cys-Pro-Gly-Gln-Pro-Phe-Asp-Pro-Ala- Trp-Gly-Pro; (e) (SEQ ID No. 58) Asp-Ile-Gly-Ser-Glu-Ser-Thr-Glu-Asp-Gln-Gln-Xaa- Ala-Val; (f) (SEQ ID No. 59) Ala-Glu-Glu-Ser-Ile-Ser-Thr-Xaa-Glu-Xaa-Ile-Val- Pro; (g) (SEQ ID No. 60) Asp-Pro-Glu-Pro-Ala-Pro-Pro-Val-Pro-Thr-Ala-Ala- Ala-Ala-Pro-Pro-Ala; and (h) (SEQ ID No. 61) Ala-Pro-Lys-Thr-Tyr-Xaa-Glu-Glu-Leu-Lys-Gly-Thr- Asp-Thr-Gly;

wherein Xaa may be any amino acid.

[0226] An additional antigen was isolated employing a microbore HPLC purification step in addition to the procedure described above. Specifically, 20 μl of a fraction comprising a mixture of antigens from the chromatographic purification step previously described, was purified on an Aquapore C18 column (Perkin Elmer/Applied Biosystems Division, Foster City, Calif.) with a 7 micron pore size, column size 1 mm×100 mm, in a Perkin Elmer/Applied Biosystems Division Model 172 HPLC. Fractions were eluted from the column with a linear gradient of 1%/minute of acetonitrile (containing 0.05% TFA) in water (0.05% TFA) at a flow rate of 80 μl/minute. The eluent was monitored at 250 nm. The original fraction was separated into 4 major peaks plus other smaller components and a polypeptide was obtained which was shown to have a molecular weight of 12.054 Kd (by mass spectrometry) and the following N-terminal sequence:

TABLE-US-00006 (i) (SEQ ID No. 62) Asp-Pro-Ala-Ser-Ala-Pro-Asp-Val-Pro-Thr-Ala-Ala- Gln-Gln-Thr-Ser-Leu-Leu-Asn-Asn-Leu-Ala-Asp-Pro- Asp-Val-Ser-Phe-Ala-Asp.

This polypeptide was shown to induce proliferation and IFN-γ production in PBMC preparations using the assays described above.

[0227] Additional soluble antigens were isolated from M. tuberculosis culture filtrate as follows. M. tuberculosis culture filtrate was prepared as described above. Following dialysis against Bis-Tris propane buffer, at pH 5.5, fractionation was performed using anion exchange chromatography on a Poros QE column 4.6×100 mm (Perseptive Biosystems) equilibrated in Bis-Tris propane buffer pH 5.5. Polypeptides were eluted with a linear 0-1.5 M NaCl gradient in the above buffer system at a flow rate of 10 ml/min. The column eluent was monitored at a wavelength of 214 nm.

[0228] The fractions eluting from the ion exchange column were pooled and subjected to reverse phase chromatography using a Poros R2 column 4.6×100 mm (Perseptive Biosystems). Polypeptides were eluted from the column with a linear gradient from 0-100% acetonitrile (0.1% TFA) at a flow rate of 5 ml/min. The eluent was monitored at 214 nm.

[0229] Fractions containing the eluted polypeptides were lyophilized and resuspended in 80 μl of aqueous 0.1% TFA and further subjected to reverse phase chromatography on a Vydac C4 column 4 6×150 mm (Western Analytical, Temecula, Calif.) with a linear gradient of 0-100% acetonitrile (0.1% TFA) at a flow rate of 2 ml/min. Eluent was monitored at 214 nm.

[0230] The fraction with biological activity was separated into one major peak plus other smaller components. Western blot of this peak onto PVDF membrane revealed three major bands of molecular weights 14 Kd, 20 Kd and 26 Kd. These polypeptides were determined to have the following N-terminal sequences, respectively: [0231] (j) Xaa-Asp-Ser-Glu-Lys-Ser-Ala-Thr-Ile-Lys-Val-Thr-Asp-Ala-Ser; (SEQ ID No. 134) [0232] (k) Ala-Gly-Asp-Thr-Xaa-Ile-Tyr-Ile-Val-Gly-Asn-Leu-Thr-Ala-Asp; (SEQ ID No. 135) and [0233] (l) Ala-Pro-Glu-Ser-Gly-Ala-Gly-Leu-Gly-Gly-Thr-Val-Gln-Ala-Gly; (SEQ ID No. 136), wherein Xaa may be any amino acid. Using the assays described above, these polypeptides were shown to induce proliferation and IFN-γ production in PBMC preparations. FIGS. 1A and B show the results of such assays using PBMC preparations from a first and a second donor, respectively.

[0234] DNA sequences that encode the antigens designated as (a), (c), (d) and (g) above were obtained by screening a genomic M. tuberculosis library using ³²P end labeled degenerate oligonucleotides corresponding to the N-terminal sequence and containing M. tuberculosis codon bias. The screen performed using a probe corresponding to antigen (a) above identified a clone having the sequence provided in SEQ ID No. 101. The polypeptide encoded by SEQ ID No. 101 is provided in SEQ ID No. 102. The screen performed using a probe corresponding to antigen (g) above identified a clone having the sequence provided in SEQ ID No. 52. The polypeptide encoded by SEQ ID No. 52 is provided in SEQ ID No. 53. The screen performed using a probe corresponding to antigen (d) above identified a clone having the sequence provided in SEQ ID No. 24, and the screen performed with a probe corresponding to antigen (c) identified a clone having the sequence provided in SEQ ID No: 25.

[0235] The above amino acid sequences were compared to known amino acid sequences in the gene bank using the DNA STAR system. The database searched contains some 173,000 proteins and is a combination of the Swiss, PIR databases along with translated protein sequences (Version 87). No significant homologies to the amino acid sequences for antigens (a)-(h) and (l) were detected.

[0236] The amino acid sequence for antigen (i) was found to be homologous to a sequence from M. leprae. The full length M. leprae sequence was amplified from genomic DNA using the sequence obtained from GENBANK. This sequence was then used to screen the M. tuberculosis library described below in Example 2 and a full length copy of the M. tuberculosis homologue was obtained (SEQ-ID No. 99).

[0237] The amino acid sequence for antigen (j) was found to be homologous to a known M. tuberculosis protein translated from a DNA sequence. To the best of the inventors' knowledge, this protein has not been previously shown to possess T-cell stimulatory activity. The amino acid sequence for antigen (k) was found to be related to a sequence from M. leprae.

[0238] In the proliferation and IFN-γ assays described above, using three PPD positive donors, the results for representative antigens provided above are presented in Table 1:

TABLE-US-00007 TABLE 1 RESULTS OF PBMC PROLIFERATION AND IFN-γ ASSAYS Sequence Proliferation IFN-γ (a) + - (c) +++ +++ (d) ++ ++ (g) +++ +++ (h) +++ +++

[0239] In Table 1, responses that gave a stimulation index (SI) of between 2 and 4 (compared to cells cultured in medium alone) were scored as +, an SI of 4-8 or 2-4 at a concentration of 1 μg or less was scored as ++ and an SI of greater than 8 was scored as +++. The antigen of sequence (i) was found to have a high SI (+++) for one donor and lower SI (++ and +) for the two other donors in both proliferation and IFN-γ assays. These results indicate that these antigens are capable of inducing proliferation and/or interferon-γ production.

Example 2

Use of Patient Sera to Isolate M. tuberculosis Antigens

[0240] This example illustrates the isolation of antigens from M. tuberculosis lysate by screening with serum from M. tuberculosis-infected individuals.

[0241] Dessicated M. tuberculosis H37Ra (Difco Laboratories) was added to a 2% NP40 solution, and alternately homogenized and sonicated three times. The resulting suspension was centrifuged at 13,000 rpm in microfuge tubes and the supernatant put through a 0.2 micron syringe filter. The filtrate was bound to Macro Prep DEAE beads (BioRad, Hercules, Calif.). The beads were extensively washed with 20 mM Tris pH 7.5 and bound proteins eluted with 1M NaCl. The 1M NaCl elute was dialyzed overnight against 10 mM Tris, pH 7.5. Dialyzed solution was treated with DNase and RNase at 0.05 mg/ml for 30 min. at room temperature and then with α-D-mannosidase, 0.5 U/mg at pH 4.5 for 3-4 hours at room temperature. After returning to pH 7.5, the material was fractionated via FPLC over a Bio Scale-Q-20 column (BioRad). Fractions were combined into nine pools, concentrated in a Centriprep 10 (Amicon, Beverley, Mass.) and then screened by Western blot for serological activity using a serum pool from M. tuberculosis-infected patients which was not immunoreactive with other antigens of the present invention.

[0242] The most reactive fraction was run in SDS-PAGE and transferred to PVDF. A band at approximately 85 Kd was cut out yielding the sequence: [0243] (m) Xaa-Tyr-Ile-Ala-Tyr-Xaa-Thr-Thr-Ala-Gly-Ile-Val-Pro-Gly-Lys-Ile-Asn-Val-H- is-Leu-Val; (SEQ ID No. 137), wherein Xaa may be any amino acid.

[0244] Comparison of this sequence with those in the gene bank as described above, revealed no significant homologies to known sequences.

Example 3

Preparation of DNA Sequences Encoding M. tuberculosis Antigens

[0245] This example illustrates the preparation of DNA sequences encoding M. tuberculosis antigens by screening a M. tuberculosis expression library with sera obtained from patients infected with M. tuberculosis, or with anti-sera raised against soluble M. tuberculosis antigens.

A. Preparation of M. tuberculosis Soluble Antigens Using Rabbit Anti-Sera

[0246] Genomic DNA was isolated from the M. tuberculosis strain H37Ra. The DNA was randomly sheared and used to construct an expression library using the Lambda ZAP expression system (Stratagene, La Jolla, Calif.). Rabbit anti-sera was generated against secretory proteins of the M. tuberculosis strains H37Ra , H37Rv and Erdman by immunizing a rabbit with concentrated supernatant of the M. tuberculosis cultures. Specifically, the rabbit was first immunized subcutaneously with 200 μg of protein antigen in a total volume of 2 ml containing 10 μg muramyl dipeptide (Calbiochem, La Jolla, Calif.) and 1 ml of incomplete Freund's adjuvant. Four weeks later the rabbit was boosted subcutaneously with 100 pg antigen in incomplete Freund's adjuvant. Finally, the rabbit was immunized intravenously four weeks later with 50 μg protein antigen. The anti-sera were used to screen the expression library as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989. Bacteriophage plaques expressing immunoreactive antigens were purified. Phagemid from the plaques was rescued and the nucleotide sequences of the M. tuberculosis clones deduced.

[0247] Thirty two clones were purified. Of these, 25 represent sequences that have not been previously identified in human M. tuberculosis. Recombinant antigens were expressed and purified antigens used in the immunological analysis described in Example 1. Proteins were induced by IPTG and purified by gel elution, as described to Skeiky et al., J. Exp. Med. 181:1527-1537, 1995. Representative sequences of DNA molecules identified in this screen are provided in SEQ ID Nos.: 1-25. The corresponding predicted amino acid sequences are shown in SEQ ID Nos. 63-87.

[0248] On comparison of these sequences with known sequences in the gene bank using the databases described above, it was found that the clones referred to hereinafter as TbRA2A, TbRA16, TbRA18, and TbRA29 (SEQ ID Nos. 76, 68, 70, 75) show some homology to sequences previously identified in Mycobacterium leprae but not in M. tuberculosis. TbRA11, TbRA26, TbRA28 and TbDPEP (SEQ ID Nos.: 65, 73, 74, 53) have been previously identified in M. tuberculosis. No significant homologies were found to TbRA1, TbRA3, TbRA4, TbRA9, TbRA10, TbRA13, TbRA17, TbRa19, TbRA29, TbRA32, TbRA36 and the overlapping clones TbRA35 and TbRA12 (SEQ ID Nos. 63, 77, 81, 82, 64, 67, 69, 71, 75, 78, 80, 79, 66). The clone TbRa24 is overlapping with clone TbRa29.

[0249] The results of PBMC proliferation and interferon-γ assays performed on representative recombinant antigens, and using T-cell preparations from several different M. tuberculosis-immune patients, are presented in Tables 2 and 3, respectively.

TABLE-US-00008 TABLE 2 RESULTS OF PBMC PROLIFERATION TO REPRESENTATIVE SOLUBLE ANTIGENS Patient Antigen 1 2 3 4 5 6 7 8 9 10 11 12 13 TbRa1 - - ± ++ - - ± ± - - + ± - TbRa3 - ± ++ - ± - - ++ ± - - - - TbRa9 - - nt nt ++ ++ nt nt nt nt nt nt nt TbRa10 - - ± ± ± + nt ± - + ± ± - TbRa11 ± ± + ++ ++ + nt - ++ ++ ++ ± nt TbRa12 - - + + ± ++ + ± ± - + - - TbRa16 nt nt nt nt - + nt nt nt nt nt nt nt TbRa24 nt nt nt nt - - nt nt nt nt nt nt nt TbRa26 - + nt nt - - nt nt nt nt nt nt nt TbRa29 nt nt nt nt - - nt nt nt nt nt nt nt TbRa35 ++ nt ++ ++ ++ ++ nt ++ ++ ++ ++ ++ nt TbRaB nt nt nt nt - - nt nt nt nt nt nt nt TbRaC nt nt nt nt - - nt nt nt nt nt nt nt TbRaD nt nt nt nt - - nt nt nt nt nt nt nt AAMK - - ± - - - nt - - - nt ± nt YY - - - - - - nt - - - nt + nt DPEP - + - ++ - - nt ++ ± + ± ± nt Control - - - - - - - - - - - - - nt = not tested

TABLE-US-00009 TABLE 3 RESULTS OF PBMC INTERFERON-γ PRODUCTION TO REPRESENTATIVE SOLUBLE ANTIGENS Patient Antigen 1 2 3 4 5 6 7 8 9 10 11 12 13 TbRa1 + ++ +++ + - ± - - + ± - TbRa3 - ± ++ - ± - - ++ ± - - - - TbRa9 ++ + nt nt ++ - nt nt nt nt nt nt nt TbRa10 + + ± ± ± + nt ± - + ± ± - TbRa11 ± + ++ ++ + nt - ++ ++ ++ ± nt TbRa12 - - + + ± +++ + ± ± - + - - TbRa16 nt nt nt nt + + nt nt nt nt nt nt nt TbRa24 nt nt nt nt + - nt nt nt nt nt nt nt TbRa26 ++ ++ nt nt + + nt nt nt nt nt nt nt TbRa29 nt nt nt nt + - nt nt nt nt nt nt nt TbRa35 ++ nt ++ ++ +++ +++ nt ++ ++ +++ +++ ++ nt TbRaB nt nt nt nt ++ + nt nt nt nt nt nt nt TbRaC nt nt nt nt + + nt nt nt nt nt nt nt TbRaD nt nt nt nt + + nt nt nt nt nt nt nt AAMK - - ± - - - nt - - - nt ± nt YY - - - - - - nt - - - nt + nt DPEP + + + +++ + - nt +++ ± + ± ± nt Control - - - - - - - - - - - - -

[0250] In Tables 2 and 3, responses that gave a stimulation index (SI) of between 1.2 and 2 (compared to cells cultured in medium alone) were scored as ±, a SI of 2-4 was scored as +, as SI of 4-8 or 2-4 at a concentration of 1 μg or less was scored as ++ and an SI of greater than 8 was scored as +++. In addition, the effect of concentration on proliferation and interferon-γ production is shown for two of the above antigens in the attached Figure. For both proliferation and interferon-γ production, TbRa3 was scored as ++ and TbRa9 as +.

[0251] These results indicate that these soluble antigens can induce proliferation and/or interferon-γ production in T-cells derived from an M. tuberculosis-immune individual.

B. Use of Patient Sera to Identify DNA Sequences Encoding M. tuberculosis Antigens

[0252] The genomic DNA library described above, and an additional H37Rv library, were screened using pools of sera obtained from patients with active tuberculosis. To prepare the H37Rv library, M. tuberculosis strain H37Rv genomic DNA was isolated, subjected to partial Sau3A digestion and used to construct an expression library using the Lambda Zap expression system (Stratagene, La Jolla, Calif.). Three different pools of sera, each containing sera obtained from three individuals with active pulmonary or pleural disease, were used in the expression screening. The pools were designated TbL, TbM and TbH, referring to relative reactivity with H37Ra lysate (i.e., TbL=low reactivity, TbM=medium reactivity and TbH=high reactivity) in both ELISA and immunoblot format. A fourth pool of sera from seven patients with active pulmonary tuberculosis was also employed. All of the sera lacked increased reactivity with the recombinant 38 kD M. tuberculosis H37Ra phosphate-binding protein.

[0253] All pools were pre-adsorbed with E. coli lysate and used to screen the H37Ra and H37Rv expression libraries, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab Laboratories, Cold Spring Harbor, N.Y., 1989. Bacteriophage plaques expressing immunoreactive antigens were purified. Phagemid from the plaques was rescued and the nucleotide sequences of the M. tuberculosis clones deduced.

[0254] Thirty two clones were purified. Of these, 31 represented sequences that had not been previously identified in human M. tuberculosis. Representative sequences of the DNA molecules identified are provided in SEQ ID Nos.: 26-51 and 105. Of these, TbH-8-2 (SEQ. ID NO. 105) is a partial clone of TbH-8, and TbH-4 (SEQ. ID NO. 43) and TbH-4-FWD (SEQ. ID NO. 44) are non-contiguous sequences from the same clone. Amino acid sequences for the antigens hereinafter identified as Tb38-1, TbH-4, TbH-8, TbH-9, and TbH-12 are shown in SEQ ID Nos.: 88-92. Comparison of these sequences with known sequences in the gene bank using the databases identified above revealed no significant homologies to TbH-4, TbH-8, TbH-9 and TbM-3, although weak homologies were found to TbH-9. TbH-12 was found to be homologous to a 34 kD antigenic protein previously identified in M. paratuberculosis (Acc. No. S28515). Tb38-1 was found to be located 34 base pairs upstream of the open reading frame for the antigen ESAT-6 previously identified in M. bovis (Acc. No. U34848) and in M. tuberculosis (Sorensen et al., Infec. Immun. 63:1710-1717, 1995).

[0255] Probes derived from Tb38-1 and TbH-9, both isolated from an H37Ra library, were used to identify clones in an H37Rv library. Tb38-1 hybridized to Tb38-1F2, Tb38-1F3, Tb38-1F5 and Tb38-1F6 (SEQ. ID NOS. 112, 113, 116, 118, and 119). (SEQ ID NOS. 112 and 113 are non-contiguous sequences from clone Tb38-1F2.) Two open reading frames were deduced in Tb38-IF2; one corresponds to Tb37FL (SEQ. ID. NO. 114), the second, a partial sequence, may be the homologue of Tb38-1 and is called Tb38-IN (SEQ. ID NO. 115). The deduced amino acid sequence of Tb38-1F3 is presented in SEQ. ID. NO. 117. A TbH-9 probe identified three clones in the H37Rv library: TbH-9-FL (SEQ. ID NO. 106), which may be the homologue of TbH-9 (R37Ra ), TbH-9-1 (SEQ. ID NO. 108), and TbH-9-4 (SEQ. ID NO. 110), all of which are highly related sequences to TbH-9. The deduced amino acid sequences for these three clones are presented in SEQ ID NOS. 107, 109 and 111.

[0256] Further screening of the M. tuberculosis genomic DNA library, as described above, resulted in the recovery of ten additional reactive clones, representing seven different genes. One of these genes was identified as the 38 Kd antigen discussed above, one was determined to be identical to the 14 Kd alpha crystallin heat shock protein previously shown to be present in M. tuberculosis, and a third was determined to be identical to the antigen TbH-8 described above. The determined DNA sequences for the remaining five clones (hereinafter referred to as TbH-29, TbH-30, TbH-32 and TbH-33) are provided in SEQ ID NO: 138-141, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 142-145, respectively. The DNA and amino acid sequences for these antigens were compared with those in the gene bank as described above. No homologies were found to the 5' end of TbH-29 (which contains the reactive open reading frame), although the 3' end of TbH-29 was found to be identical to the M. tuberculosis cosmid Y227. TbH-32 and TbH-33 were found to be identical to the previously identified M. tuberculosis insertion element IS6110 and to the M. tuberculosis cosmid Y50, respectively. No significant homologies to TbH-30 were found.

[0257] Positive phagemid from this additional screening were used to infect E. coli XL-1 Blue MRF', as described in Sambrook et al., supra. Induction of recombinant protein was accomplished by the addition of IPTG. Induced and uninduced lysates were run in duplicate on SDS-PAGE and transferred to nitrocellulose filters. Filters were reacted with human M. tuberculosis sera (1:200 dilution) reactive with TbH and a rabbit sera (1:200 or 1:250 dilution) reactive with the N-terminal 4 Kd portion of lacZ. Sera incubations were performed for 2 hours at room temperature. Bound antibody was detected by addition of ¹²⁵I-labeled Protein A and subsequent exposure to film for variable times ranging from 16 hours to 11 days. The results of the immunoblots are summarized in Table 4.

TABLE-US-00010 TABLE 4 Human M. tb Anti-lacZ Antigen Sera Sera TbH-29 45 Kd 45 Kd TbH-30 No reactivity 29 Kd TbH-32 12 Kd 12 Kd TbH-33 16 Kd 16 Kd

[0258] Positive reaction of the recombinant human M. tuberculosis antigens with both the human M. tuberculosis sera and anti-lacZ sera indicate that reactivity of the human M. tuberculosis sera is directed towards the fusion protein. Antigens reactive with the anti-lacZ sera but not with the human M. tuberculosis sera may be the result of the human M. tuberculosis sera recognizing conformational epitopes, or the antigen-antibody binding kinetics may be such that the 2 hour sera exposure in the immunoblot is not sufficient.

[0259] The results of T-cell assays performed on Tb38-1, ESAT-6 and other representative recombinant antigens are presented in Tables 5A, B and 6, respectively, below:

TABLE-US-00011 TABLE 5A RESULTS OF PBMC PROLIFERATION TO REPRESENTATIVE ANTIGENS Donor Antigen 1 2 3 4 5 6 7 8 9 10 11 Tb38.1 +++ + - - - ++ - + - ++ +++ ESAT-6 +++ + + + - + - + + ++ +++ TbH-9 ++ ++ - ++ ± ± ++ ++ ++ ++ ++

TABLE-US-00012 TABLE 5B RESULTS OF PBMC INTERFERON-γ PRODUCTION TO REPRESENTATIVE ANTIGENS Donor Antigen 1 2 3 4 5 6 7 8 9 10 11 Tb38.1 +++ + - + + +++ - ++ - +++ +++ ESAT-6 +++ + + + +- + - + + +++ +++ TbH-9 ++ ++ - +++ ± ± +++ +++ ++ +++ ++

TABLE-US-00013 TABLE 6 SUMMARY OF T-CELL RESPONSES TO REPRESENTATIVE ANTIGENS Proliferation Interferon-y Antigen patient 4 patient 5 patient 6 patient 4 patient 5 patient 6 total TbH9 ++ ++ ++ +++ ++ ++ 13 TbM7 - + - ++ + - 4 TbH5 - + + ++ ++ ++ 8 TbL23 - + ± ++ ++ + 7.5 TbH4 - ++ ± ++ ++ ± 7 - control - - - - - - 0

[0260] These results indicate that both the inventive M. tuberculosis antigens and ESAT-6 can induce proliferation and/or interferon-γ production in T-cells derived from an M. tuberculosis-immune individual. To the best of the inventors' knowledge, ESAT-6 has not been previously shown to stimulate human immune responses

[0261] A set of six overlapping peptides covering the amino acid sequence of the antigen Tb38-1 was constructed using the method described in Example 6. The sequences of these peptides, hereinafter referred to as pep 1-6, are provided in SEQ ID Nos. 93-98, respectively. The results of T-cell assays using these peptides are shown in Tables 7 and 8. These results confirm the existence, and help to localize T-cell epitopes within Tb38-1 capable of inducing proliferation and interferon-γ production in T-cells derived from an M. tuberculosis immune individual.

TABLE-US-00014 TABLE 7 RESULTS OF PBMC PROLIFERATION TO TB38-1 PEPTIDES Patient Peptide 1 2 3 4 5 6 7 8 9 10 11 12 13 pep1 - - - - ± - - - - ± - - + pep2 ± - - - ± - - - ± ± - - + pep3 - - - - - - - - ± - - - ± pep4 ++ - - - - - + - ± ± - - + pep5 ++ ± - - - - + - ± - - - + pep6 - ++ - - - - ± - ± + - - + Control - - - - - - - - - - - - -

TABLE-US-00015 TABLE 8 RESULTS OF PBMC INTEFERON-γ PRODUCTION TO TB38-1 PEPTIDES Patient Peptide 1 2 3 4 5 6 7 8 9 10 11 12 13 pep1 + - - - ± - - - - ± - - + pep2 - - - ± - - - ± ± - - + pep3 - - - - - - - - ± - - - ± pep4 ++ - - - - - + - ± ± - - + pep5 ++ ± - - - - + - ± - - - + pep6 + ++ - - - - ± - ± + - - + Control - - - - - - - - - - - - -

[0262] Studies were undertaken to determine whether the antigens TbH-9 and Tb38-1 represent cellular proteins or are secreted into M. tuberculosis culture media. In the first study, rabbit sera were raised against A) secretory proteins of M. tuberculosis, B) the known secretory recombinant M. tuberculosis antigen 85b, C) recombinant Tb38-1 and D) recombinant TbH-9, using protocols substantially the same as that as described in Example 3A. Total M. tuberculosis lysate, concentrated supernatant of M. tuberculosis cultures and the recombinant antigens 85b, TbH-9 and Tb38-1 were resolved on denaturing gels, immobilized on nitrocellulose membranes and duplicate blots were probed using the rabbit sera described above.

[0263] The results of this analysis using control sera (panel I) and antisera (panel II) against secretory proteins, recombinant 85b, recombinant Tb38-1 and recombinant TbH-9 are shown in FIGS. 3A-D, respectively, wherein the lane designations are as follows: 1) molecular weight protein standards; 2) 5 μg of M. tuberculosis lysate; 3) 5 μg secretory proteins; 4) 50 ng recombinant Tb38-1; 5) 50 ng recombinant TbH-9; and 6) 50 ng recombinant 85b. The recombinant antigens were engineered with six terminal histidine residues and would therefore be expected to migrate with a mobility approximately 1 kD larger that the native protein. In FIG. 3D, recombinant TbH-9 is lacking approximately 10 kD of the full-length 42 kD antigen, hence the significant difference in the size of the immunoreactive native TbH-9 antigen in the lysate lane (indicated by an arrow). These results demonstrate that Tb38-1 and TbH-9 are intracellular antigens and are not actively secreted by M. tuberculosis.

[0264] The finding that TbH-9 is an intracellular antigen was confirmed by determining the reactivity of TbH-9-specific human T cell clones to recombinant TbH-9, secretory M. tuberculosis proteins and PPD. A TbH-9-specific T cell clone (designated 131TbH-9) was generated from PBMC of a healthy PPD-positive donor. The proliferative response of 131TbH-9 to secretory proteins, recombinant TbH-9 and a control M. tuberculosis antigen, TbRa11, was determined by measuring uptake of tritiated thymidine, as described in Example 1. As shown in FIG. 4A, the clone 131TbH-9 responds specifically to TbH-9, showing that TbH-9 is not a significant component of M. tuberculosis secretory proteins. FIG. 4B shows the production of IFN-γ by a second TbH-9-specific T cell clone (designated PPD 800-10) prepared from PBMC from a healthy PPD-positive donor, following stimulation of the T cell clone with secretory proteins, PPD or recombinant TbH-9. These results further confirm that TbH-9 is not secreted by M. tuberculosis.

Example 4

Purification and Characterization of a Polypeptide from Tuberculin Purified Protein Derivative

[0265] An M. tuberculosis polypeptide was isolated from tuberculin purified protein derivative (PPD) as follows.

[0266] PPD was prepared as published with some modification (Seibert, F. et al., Tuberculin purified protein derivative. Preparation and analyses of a large quantity for standard. The American Review of Tuberculosis 44:9-25, 1941).

[0267] M. tuberculosis Rv strain was grown for 6 weeks in synthetic medium in roller bottles at 37° C. Bottles containing the bacterial growth were then heated to 100° C. in water vapor for 3 hours. Cultures were sterile filtered using a 0.22μ filter and the liquid phase was concentrated 20 times using a 3 kD cut-off membrane. Proteins were precipitated once with 50% ammonium sulfate solution and eight times with 25% ammonium sulfate solution. The resulting proteins (PPD) were fractionated by reverse phase liquid chromatography (RP-HPLC) using a C18 column (7.8×300 mM; Waters, Milford, Mass.) in a Biocad HPLC system (Perseptive Biosystems, Framingham, Mass.). Fractions were eluted from the column with a linear gradient from 0-100% buffer (0.1% TFA in acetonitrile). The flow rate was 10 ml/minute and eluent was monitored at 214 nm and 280 nm.

[0268] Six fractions were collected, dried, suspended in PBS and tested individually in M. tuberculosis-infected guinea pigs for induction of delayed type hypersensitivity (DTH) reaction. One fraction was found to induce a strong DTH reaction and was subsequently fractionated further by RP-HPLC on a microbore Vydac C18 column (Cat. No. 218TP5115) in a Perkin Elmer/Applied Biosystems Division Model 172 HPLC. Fractions were eluted with a linear gradient from 5-100% buffer (0.05% TFA in acetonitrile) with a flow rate of 80 μl/minute. Eluent was monitored at 215 nm. Eight fractions were collected and tested for induction of DTH in M. tuberculosis-infected guinea pigs. One fraction was found to induce strong DTH of about 16 mm induration. The other fractions did not induce detectable DTH. The positive fraction was submitted to SDS-PAGE gel electrophoresis and found to contain a single protein band of approximately 12 kD molecular weight.

[0269] This polypeptide, herein after referred to as DPPD, was sequenced from the amino terminal using a Perkin Elmer/Applied Biosystems Division Procise 492 protein sequencer as described above and found to have the N-terminal sequence shown in SEQ ID No.: 129. Comparison of this sequence with known sequences in the gene bank as described above revealed no known homologies. Four cyanogen bromide fragments of DPPD were isolated and found to have the sequences shown in SEQ ID Nos.: 130-133.

[0270] The ability of the antigen DPPD to stimulate human PBMC to proliferate and to produce IFN-γ was assayed as described in Example 1. As shown in Table 9, DPPD was found to stimulate proliferation and elicit production of large quantities of IFN-γ; more than that elicited by commercial PPD.

TABLE-US-00016 TABLE 9 RESULTS OF PROLIFERATION AND INTERFERON-γ ASSAYS TO DPPD PBMC Donor Stimulator Proliferation (CPM) IFN-γ (OD₄₅₀) A Medium 1,089 0.17 PPD (commercial) 8,394 1.29 DPPD 13,451 2.21 B Medium 450 0.09 PPD (commercial) 3,929 1.26 DPPD 6,184 1.49 C Medium 541 0.11 PPD (commercial) 8,907 0.76 DPPD 23,024 >2.70

Example 5

Use of Representative Antigens for Diagnosis of Tuberculosis

[0271] This example illustrates the effectiveness of several representative polypeptides in skin tests for the diagnosis of M. tuberculosis infection.

[0272] Individuals were injected intradermally with 100 μl of either PBS or PBS plus Tween 20® containing either 0.1 μg of protein (for TbH-9 and TbRa35) or 1.0 μg of protein (for TbRa38-1). Induration was measured between 5-7 days after injection, with a response of 5 mm or greater being considered positive. Of the 20 individuals tested, 2 were PPD negative and 18 were PPD positive. Of the PPD positive individuals, 3 had active tuberculosis, 3 had been previously infected with tuberculosis and 9 were healthy. In a second study, 13 PPD positive individuals were tested with 0.1 μg TbRa11 in either PBS or PBS plus Tween 20® as described above. The results of both studies are shown in Table 10.

TABLE-US-00017 TABLE 10 RESULTS OF DTH TESTING WITH REPRESENTATIVE ANTIGENS TbH-9 Tb38-1 TbRa35 Cumulative TbRa11 Pos/Total Pos/Total Pos/Total Pos/Total Pos/Total PPD 0/2 0/2 0/2 0/2 negative PPD positive healthy 5/9 4/9 4/9 6/9 1/4 prior TB 3/5 2/5 2/5 4/5 3/5 active 3/4 3/4 0/4 4/4 1/4 TOTAL 11/18 9/18 6/18 14/18 5/13

Example 6

Synthesis of Synthetic Polypeptides

[0273] Polypeptides may be synthesized on a Millipore 9050 peptide synthesizer using FMOC chemistry with HPTU (O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) activation. A Gly-Cys-Gly sequence may be attached to the amino terminus of the peptide to provide a method of conjugation or labeling of the peptide. Cleavage of the peptides from the solid support may be carried out using the following cleavage mixture: trifluoroacetic acid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleaving for 2 hours, the peptides may be precipitated in cold methyl-t-butyl-ether. The peptide pellets may then be dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized prior to purification by C18 reverse phase HPLC. A gradient of 0%-60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) may be used to elute the peptides. Following lyophilization of the pure fractions, the peptides may be characterized using electrospray mass spectrometry and by amino acid analysis.

Example 7

Preparation and Characterization of M. tuberculosis Fusion Proteins

[0274] A fusion protein containing TbRa3, the 38 kD antigen and Tb38-1 was prepared as follows.

[0275] Each of the DNA constructs TbRa3, 38 kD and Tb38-1 were modified by PCR in order to facilitate their fusion and the subsequent expression of the fusion protein TbRa3-38 kD-Tb38-1. TbRa3, 38 kD and Tb38-1 DNA was used to perform PCR using the primers PDM-64 and PDM-65 (SEQ ID NO: 146 and 147), PDM-57 and PDM-58 (SEQ ID NO: 148 and 149), and PDM-69 and PDM-60 (SEQ ID NO: 150 and 151), respectively. In each case, the DNA amplification was performed using 10 μl 10×Pfu buffer, 2 μl 10 mM dNTPs, 2 μl each of the PCR primers at 10 μM concentration, 81.5 μl water, 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.) and 1 μl DNA at either 70 ng/μl (for TbRa3) or 50 ng/μl (for 38 kD and Tb38-1). For TbRa3, denaturation at 94° C. was performed for 2 min, followed by 40 cycles of 96° C. for 15 sec and 72° C. for 1 min, and lastly by 72° C. for 4 min. For 38 kD, denaturation at 96° C. was performed for 2 min, followed by 40 cycles of 96° C. for 30 sec, 68° C. for 15 sec and 72° C. for 3 min, and finally by 72° C. for 4 min. For Tb38-1 denaturation at 94° C. for 2 min was followed by 10 cycles of 96° C. for 15 sec, 68° C. for 15 sec and 72° C. for 1.5 min, 30 cycles of 96° C. for 15 sec, 64° C. for 15 sec and 72° C. for 1.5, and finally by 72° C. for 4 min.

[0276] The TbRa3PCR fragment was digested with NdeI and EcoRI and cloned directly into pT7 L2 IL 1 vector using NdeI and EcoRI sites. The 38 kD PCR fragment was digested with Sse83871, treated with T4 DNA polymerase to make blunt ends and then digested with EcoRI for direct cloning into the pT7 L2Ra3-1 vector which was digested with StuI and EcoRI. The 38-1 PCR fragment was digested with Eco47III and EcoRI and directly subcloned into pT7 L2Ra3/38 kD-17 digested with the same enzymes. The whole fusion was then transferred to pET28b NT LMEIF-1 using NdeI and EcoRI sites. The fusion construct was confirmed by DNA sequencing.

[0277] The expression construct was transformed to BLR pLys S E. coli (Novagen, Madison, Wis.) and grown overnight in LB broth with kanamycin (30 μg/ml) and chloramphenicol (34 μg/ml). This culture (12 ml) was used to inoculate 500 ml 2XYT with the same antibiotics and the culture was induced with IPTG at an OD560 of 0.44 to a final concentration of 1.2 mM. Four hours post-induction, the bacteria were harvested and sonicated in 20 mM Tris (8.0), 100 mM NaCl, 0.1% DOC, 20 μg/ml Leupeptin, 20 mM PMSF followed by centrifugation at 26,000×g. The resulting pellet was resuspended in 8 M urea, 20 mM Tris (8.0), 100 mM NaCl and bound to Pro-bond nickel resin (Invitrogen, Carlsbad, Calif.). The column was washed several times with the above buffer then eluted with an imidazole gradient (50 mM, 100 mM, 500 mM imidazole was added to 8 M urea, 20 mM Tris (8.0), 100 mM NaCl). The eluates containing the protein of interest were then dialzyed against 10 mM Tris (8.0).

[0278] The DNA and amino acid sequences for the resulting fusion protein (hereinafter referred to as TbRa3-38 kD-Tb38-1) are provided in SEQ ID NO: 152 and 153, respectively.

[0279] A fusion protein containing the two antigens TbH-9 and Tb38-1 (hereinafter referred to as TbH9-Tb38-1) without a hinge sequence, was prepared using a similar procedure to that described above. The DNA sequence for the TbH9-Tb38-1 fusion protein is provided in SEQ ID NO: 156.

[0280] The ability of the fusion protein TbH9-Tb38-1 to induce T cell proliferation and IFN-γ production in PBMC preparations was examined using the protocol described above in Example 1. PBMC from three donors were employed: one who had been previously shown to respond to TbH9 but not Tb38-1 (donor 131); one who had been shown to respond to Tb38-1 but not TbH9 (donor 184); and one who had been shown to respond to both antigens (donor 201). The results of these studies (FIGS. 5-7, respectively) demonstrate the functional activity of both the antigens in the fusion protein.

[0281] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

Sequence CWU 1

1531766DNAMycobacterium tuberculosismisc_feature(1)..(765)ANY NUCLEOTIDE 1cgaggcaccg gtagtttgaa ccaaacgcac aatcgacggg caaacgaacg gaagaacaca 60accatgaaga tggtgaaatc gatcgccgca ggtctgaccg ccgcggctgc aatcggcgcc 120gctgcggccg gtgtgacttc gatcatggct ggcggcccgg tcgtatacca gatgcagccg 180gtcgtcttcg gcgcgccact gccgttggac ccggcatccg cccctgacgt cccgaccgcc 240gcccagttga ccagcctgct caacagcctc gccgatccca acgtgtcgtt tgcgaacaag 300ggcagtctgg tcgagggcgg catcgggggc accgaggcgc gcatcgccga ccacaagctg 360aagaaggccg ccgagcacgg ggatctgccg ctgtcgttca gcgtgacgaa catccagccg 420gcggccgccg gttcggccac cgccgacgtt tccgtctcgg gtccgaagct ctcgtcgccg 480gtcacgcaga acgtcacgtt cgtgaatcaa ggcggctgga tgctgtcacg cgcatcggcg 540atggagttgc tgcaggccgc agggnaactg attggcgggc cggnttcagc ccgctgttca 600gctacgccgc ccgcctggtg acgcgtccat gtcgaacact cgcgcgtgta gcacggtgcg 660gtntgcgcag ggncgcacgc accgcccggt gcaagccgtc ctcgagatag gtggtgnctc 720gncaccagng ancacccccn nntcgncnnt tctcgntgnt gnatga 7662752DNAMycobacterium tuberculosismisc_feature(1)..(752)ANY NUCLEOTIDE 2atgcatcacc atcaccatca cgatgaagtc acggtagaga cgacctccgt cttccgcgca 60gacttcctca gcgagctgga cgctcctgcg caagcgggta cggagagcgc ggtctccggg 120gtggaagggc tcccgccggg ctcggcgttg ctggtagtca aacgaggccc caacgccggg 180tcccggttcc tactcgacca agccatcacg tcggctggtc ggcatcccga cagcgacata 240tttctcgacg acgtgaccgt gagccgtcgc catgctgaat tccggttgga aaacaacgaa 300ttcaatgtcg tcgatgtcgg gagtctcaac ggcacctacg tcaaccgcga gcccgtggat 360tcggcggtgc tggcgaacgg cgacgaggtc cagatcggca agctccggtt ggtgttcttg 420accggaccca agcaaggcga ggatgacggg agtaccgggg gcccgtgagc gcacccgata 480gccccgcgct ggccgggatg tcgatcgggg cggtcctccg acctgctacg accggatttt 540ccctgatgtc caccatctcc aagattcgat tcttgggagg cttgagggtc ngggtgaccc 600ccccgcgggc ctcattcngg ggtntcggcn ggtttcaccc cntaccnact gccncccggn 660ttgcnaattc nttcttcnct gcccnnaaag ggaccnttan cttgccgctn gaaanggtna 720tccngggccc ntcctngaan ccccntcccc ct 7523813DNAMycobacterium tuberculosismisc_feature(1)..(813)ANY NUCLEOTIDE 3catatgcatc accatcacca tcacacttct aaccgcccag cgcgtcgggg gcgtcgagca 60ccacgcgaca ccgggcccga tcgatctgct agcttgagtc tggtcaggca tcgtcgtcag 120cagcgcgatg ccctatgttt gtcgtcgact cagatatcgc ggcaatccaa tctcccgcct 180gcggccggcg gtgctgcaaa ctactcccgg aggaatttcg acgtgcgcat caagatcttc 240atgctggtca cggctgtcgt tttgctctgt tgttcgggtg tggccacggc cgcgcccaag 300acctactgcg aggagttgaa aggcaccgat accggccagg cgtgccagat tcaaatgtcc 360gacccggcct acaacatcaa catcagcctg cccagttact accccgacca gaagtcgctg 420gaaaattaca tcgcccagac gcgcgacaag ttcctcagcg cggccacatc gtccactcca 480cgcgaagccc cctacgaatt gaatatcacc tcggccacat accagtccgc gataccgccg 540cgtggtacgc aggccgtggt gctcamggtc taccacaacg ccggcggcac gcacccaacg 600accacgtaca aggccttcga ttgggaccag gcctatcgca agccaatcac ctatgacacg 660ctgtggcagg ctgacaccga tccgctgcca gtcgtcttcc ccattgttgc aaggtgaact 720gagcaacgca gaccgggaca acwggtatcg atagccgccn aatgccggct tggaacccng 780tgaaattatc acaacttcgc agtcacnaaa naa 8134447DNAMycobacterium tuberculosis 4cggtatgaac acggccgcgt ccgataactt ccagctgtcc cagggtgggc agggattcgc 60cattccgatc gggcaggcga tggcgatcgc gggccagatc cgatcgggtg gggggtcacc 120caccgttcat atcgggccta ccgccttcct cggcttgggt gttgtcgaca acaacggcaa 180cggcgcacga gtccaacgcg tggtcgggag cgctccggcg gcaagtctcg gcatctccac 240cggcgacgtg atcaccgcgg tcgacggcgc tccgatcaac tcggccaccg cgatggcgga 300cgcgcttaac gggcatcatc ccggtgacgt catctcggtg aactggcaaa ccaagtcggg 360cggcacgcgt acagggaacg tgacattggc cgagggaccc ccggcctgat ttcgtcgygg 420ataccacccg ccggccggcc aattgga 4475604DNAMycobacterium tuberculosismisc_feature(1)..(604)ANY NUCLEOTIDE 5gtcccactgc ggtcgccgag tatgtcgccc agcaaatgtc tggcagccgc ccaacggaat 60ccggtgatcc gacgtcgcag gttgtcgaac ccgccgccgc ggaagtatcg gtccatgcct 120agcccggcga cggcgagcgc cggaatggcg cgagtgagga ggcgggcaat ttggcggggc 180ccggcgacgg ngagcgccgg aatggcgcga gtgaggaggt ggncagtcat gcccagngng 240atccaatcaa cctgnattcg gnctgngggn ccatttgaca atcgaggtag tgagcgcaaa 300tgaatgatgg aaaacgggng gngacgtccg ntgttctggt ggtgntaggt gnctgnctgg 360ngtngnggnt atcaggatgt tcttcgncga aanctgatgn cgaggaacag ggtgtncccg 420nnannccnan ggngtccnan cccnnnntcc tcgncganat cananagncg nttgatgnga 480naaaagggtg gancagnnnn aantngnggn ccnaanaanc nnnanngnng nnagntngnt 540nnntnttnnc annnnnnntg nngnngnncn nnncaancnn ntnnnngnaa nnggnttntt 600naat 6046633DNAMycobacterium tuberculosismisc_feature(1)..(633)ANY NUCLEOTIDE 6ttgcangtcg aaccacctca ctaaagggaa caaaagctng agctccaccg cggtggcggc 60cgctctagaa ctagtgkatm yyyckggctg cagsaatycg gyacgagcat taggacagtc 120taacggtcct gttacggtga tcgaatgacc gacgacatcc tgctgatcga caccgacgaa 180cgggtgcgaa ccctcaccct caaccggccg cagtcccgya acgcgctctc ggcggcgcta 240cgggatcggt ttttcgcggy gttggycgac gccgaggycg acgacgacat cgacgtcgtc 300atcctcaccg gygccgatcc ggtgttctgc gccggactgg acctcaaggt agctggccgg 360gcagaccgcg ctgccggaca tctcaccgcg gtgggcggcc atgaccaagc cggtgatcgg 420cgcgatcaac ggcgccgcgg tcaccggcgg gctcgaactg gcgctgtact gcgacatcct 480gatcgcctcc gagcacgccc gcttcgncga cacccacgcc cgggtggggc tgctgcccac 540ctggggactc agtgtgtgct tgccgcaaaa ggtcggcatc ggnctgggcc ggtggatgag 600cctgaccggc gactacctgt ccgtgaccga cgc 63371362DNAMycobacterium tuberculosis 7cgacgacgac ggcgccggag agcgggcgcg aacggcgatc gacgcggccc tggccagagt 60cggcaccacc caggagggag tcgaatcatg aaatttgtca accatattga gcccgtcgcg 120ccccgccgag ccggcggcgc ggtcgccgag gtctatgccg aggcccgccg cgagttcggc 180cggctgcccg agccgctcgc catgctgtcc ccggacgagg gactgctcac cgccggctgg 240gcgacgttgc gcgagacact gctggtgggc caggtgccgc gtggccgcaa ggaagccgtc 300gccgccgccg tcgcggccag cctgcgctgc ccctggtgcg tcgacgcaca caccaccatg 360ctgtacgcgg caggccaaac cgacaccgcc gcggcgatct tggccggcac agcacctgcc 420gccggtgacc cgaacgcgcc gtatgtggcg tgggcggcag gaaccgggac accggcggga 480ccgccggcac cgttcggccc ggatgtcgcc gccgaatacc tgggcaccgc ggtgcaattc 540cacttcatcg cacgcctggt cctggtgctg ctggacgaaa ccttcctgcc ggggggcccg 600cgcgcccaac agctcatgcg ccgcgccggt ggactggtgt tcgcccgcaa ggtgcgcgcg 660gagcatcggc cgggccgctc cacccgccgg ctcgagccgc gaacgctgcc cgacgatctg 720gcatgggcaa caccgtccga gcccatagaa accgcgttcg ccgcgctcag ccaccacctg 780gacaccgcgc cgcacctgcc gccaccgact cgtcaggtgg tcaggcgggt cgtggggtcg 840tggcacggcg agccaatgcc gatgagcagt cgctggacga acgagcacac cgccgagctg 900cccgccgacc tgcacgcgcc cacccgtctt gccctgctga ccggcctggc cccgcatcag 960gtgaccgacg acgacgtcgc cgcggcccga tccctgctcg acaccgatgc ggcgctggtt 1020ggcgccctgg cctgggccgc cttcaccgcc gcgcggcgca tcggcacctg gatcggcgcc 1080gccgccgagg gccaggtgtc gcggcaaaac ccgactgggt gagtgtgcgc gccctgtcgg 1140tagggtgtca tcgctggccc gagggatctc gcggcggcga acggaggtgg cgacacaggt 1200ggaagctgcg cccactggct tgcgccccaa cgccgtcgtg ggcgttcggt tggccgcact 1260ggccgatcag gtcggcgccg gcccttggcc gaaggtccag ctcaacgtgc cgtcaccgaa 1320ggaccggacg gtcaccgggg gtcaccctgc gcgcccaagg aa 136281458DNAMycobacterium tuberculosis 8gcgacgaccc cgatatgccg ggcaccgtag cgaaagccgt cgccgacgca ctcgggcgcg 60gtatcgctcc cgttgaggac attcaggact gcgtggaggc ccggctgggg gaagccggtc 120tggatgacgt ggcccgtgtt tacatcatct accggcagcg gcgcgccgag ctgcggacgg 180ctaaggcctt gctcggcgtg cgggacgagt taaagctgag cttggcggcc gtgacggtac 240tgcgcgagcg ctatctgctg cacgacgagc agggccggcc ggccgagtcg accggcgagc 300tgatggaccg atcggcgcgc tgtgtcgcgg cggccgagga ccagtatgag ccgggctcgt 360cgaggcggtg ggccgagcgg ttcgccacgc tattacgcaa cctggaattc ctgccgaatt 420cgcccacgtt gatgaactct ggcaccgacc tgggactgct cgccggctgt tttgttctgc 480cgattgagga ttcgctgcaa tcgatctttg cgacgctggg acaggccgcc gagctgcagc 540gggctggagg cggcaccgga tatgcgttca gccacctgcg acccgccggg gatcgggtgg 600cctccacggg cggcacggcc agcggaccgg tgtcgtttct acggctgtat gacagtgccg 660cgggtgtggt ctccatgggc ggtcgccggc gtggcgcctg tatggctgtg cttgatgtgt 720cgcacccgga tatctgtgat ttcgtcaccg ccaaggccga atcccccagc gagctcccgc 780atttcaacct atcggttggt gtgaccgacg cgttcctgcg ggccgtcgaa cgcaacggcc 840tacaccggct ggtcaatccg cgaaccggca agatcgtcgc gcggatgccc gccgccgagc 900tgttcgacgc catctgcaaa gccgcgcacg ccggtggcga tcccgggctg gtgtttctcg 960acacgatcaa tagggcaaac ccggtgccgg ggagaggccg catcgaggcg accaacccgt 1020gcggggaggt cccactgctg ccttacgagt catgtaatct cggctcgatc aacctcgccc 1080ggatgctcgc cgacggtcgc gtcgactggg accggctcga ggaggtcgcc ggtgtggcgg 1140tgcggttcct tgatgacgtc atcgatgtca gccgctaccc cttccccgaa ctgggtgagg 1200cggcccgcgc cacccgcaag atcgggctgg gagtcatggg tttggcggaa ctgcttgccg 1260cactgggtat tccgtacgac agtgaagaag ccgtgcggtt agccacccgg ctcatgcgtc 1320gcatacagca ggcggcgcac acggcatcgc ggaggctggc cgaagagcgg ggcgcattcc 1380cggcgttcac cgatagccgg ttcgcgcggt cgggcccgag gcgcaacgca caggtcacct 1440ccgtcgctcc gacgggca 14589862DNAMycobacterium tuberculosis 9acggtgtaat cgtgctggat ctggaaccgc gtggcccgct acctaccgag atctactggc 60ggcgcagggg gctggccctg ggcatcgcgg tcgtcgtagt cgggatcgcg gtggccatcg 120tcatcgcctt cgtcgacagc agcgccggtg ccaaaccggt cagcgccgac aagccggcct 180ccgcccagag ccatccgggc tcgccggcac cccaagcacc ccagccggcc gggcaaaccg 240aaggtaacgc cgccgcggcc ccgccgcagg gccaaaaccc cgagacaccc acgcccaccg 300ccgcggtgca gccgccgccg gtgctcaagg aaggggacga ttgccccgat tcgacgctgg 360ccgtcaaagg tttgaccaac gcgccgcagt actacgtcgg cgaccagccg aagttcacca 420tggtggtcac caacatcggc ctggtgtcct gtaaacgcga cgttggggcc gcggtgttgg 480ccgcctacgt ttactcgctg gacaacaagc ggttgtggtc caacctggac tgcgcgccct 540cgaatgagac gctggtcaag acgttttccc ccggtgagca ggtaacgacc gcggtgacct 600ggaccgggat gggatcggcg ccgcgctgcc cattgccgcg gccggcgatc gggccgggca 660cctacaatct cgtggtacaa ctgggcaatc tgcgctcgct gccggttccg ttcatcctga 720atcagccgcc gccgccgccc gggccggtac ccgctccggg tccagcgcag gcgcctccgc 780cggagtctcc cgcgcaaggc ggataattat tgatcgctga tggtcgattc cgccagctgt 840gacaacccct cgcctcgtgc cg 86210622DNAMycobacterium tuberculosismisc_feature(1)..(622)ANY NUCLEOTIDE 10ttgatcagca ccggcaaggc gtcacatgcc tccctgggtg tgcaggtgac caatgacaaa 60gacaccccgg gcgccaagat cgtcgaagta gtggccggtg gtgctgccgc gaacgctgga 120gtgccgaagg gcgtcgttgt caccaaggtc gacgaccgcc cgatcaacag cgcggacgcg 180ttggttgccg ccgtgcggtc caaagcgccg ggcgccacgg tggcgctaac ctttcaggat 240ccctcgggcg gtagccgcac agtgcaagtc accctcggca aggcggagca gtgatgaagg 300tcgccgcgca gtgttcaaag ctcggatata cggtggcacc catggaacag cgtgcggagt 360tggtggttgg ccgggcactt gtcgtcgtcg ttgacgatcg cacggcgcac ggcgatgaag 420accacagcgg gccgcttgtc accgagctgc tcaccgaggc cgggtttgtt gtcgacggcg 480tggtggcggt gtcggccgac gaggtcgaga tccgaaatgc gctgaacaca gcggtgatcg 540gcggggtgga cctggtggtg tcggtcggcg ggaccggngt gacgnctcgc gatgtcaccc 600cggaagccac ccgngacatt ct 622111200DNAMycobacterium tuberculosis 11ggcgcagcgg taagcctgtt ggccgccggc acactggtgt tgacagcatg cggcggtggc 60accaacagct cgtcgtcagg cgcaggcgga acgtctgggt cggtgcactg cggcggcaag 120aaggagctcc actccagcgg ctcgaccgca caagaaaatg ccatggagca gttcgtctat 180gcctacgtgc gatcgtgccc gggctacacg ttggactaca acgccaacgg gtccggtgcc 240ggggtgaccc agtttctcaa caacgaaacc gatttcgccg gctcggatgt cccgttgaat 300ccgtcgaccg gtcaacctga ccggtcggcg gagcggtgcg gttccccggc atgggacctg 360ccgacggtgt tcggcccgat cgcgatcacc tacaatatca agggcgtgag cacgctgaat 420cttgacggac ccactaccgc caagattttc aacggcacca tcaccgtgtg gaatgatcca 480cagatccaag ccctcaactc cggcaccgac ctgccgccaa caccgattag cgttatcttc 540cgcagcgaca agtccggtac gtcggacaac ttccagaaat acctcgacgg tgtatccaac 600ggggcgtggg gcaaaggcgc cagcgaaacg ttcagcgggg gcgtcggcgt cggcgccagc 660gggaacaacg gaacgtcggc cctactgcag acgaccgacg ggtcgatcac ctacaacgag 720tggtcgtttg cggtgggtaa gcagttgaac atggcccaga tcatcacgtc ggcgggtccg 780gatccagtgg cgatcaccac cgagtcggtc ggtaagacaa tcgccggggc caagatcatg 840ggacaaggca acgacctggt attggacacg tcgtcgttct acagacccac ccagcctggc 900tcttacccga tcgtgctggc gacctatgag atcgtctgct cgaaataccc ggatgcgacg 960accggtactg cggtaagggc gtttatgcaa gccgcgattg gtccaggcca agaaggcctg 1020gaccaatacg gctccattcc gttgcccaaa tcgttccaag caaaattggc ggccgcggtg 1080aatgctattt cttgacctag tgaagggaat tcgacggtga gcgatgccgt tccgcaggta 1140gggtcgcaat ttgggccgta tcagctattg cggctgctgg gccgaggcgg gatgggcgag 1200121155DNAMycobacterium tuberculosis 12gcaagcagct gcaggtcgtg ctgttcgacg aactgggcat gccgaagacc aaacgcacca 60agaccggcta caccacggat gccgacgcgc tgcagtcgtt gttcgacaag accgggcatc 120cgtttctgca acatctgctc gcccaccgcg acgtcacccg gctcaaggtc accgtcgacg 180ggttgctcca agcggtggcc gccgacggcc gcatccacac cacgttcaac cagacgatcg 240ccgcgaccgg ccggctctcc tcgaccgaac ccaacctgca gaacatcccg atccgcaccg 300acgcgggccg gcggatccgg gacgcgttcg tggtcgggga cggttacgcc gagttgatga 360cggccgacta cagccagatc gagatgcgga tcatggggca cctgtccggg gacgagggcc 420tcatcgaggc gttcaacacc ggggaggacc tgtattcgtt cgtcgcgtcc cgggtgttcg 480gtgtgcccat cgacgaggtc accggcgagt tgcggcgccg ggtcaaggcg atgtcctacg 540ggctggttta cgggttgagc gcctacggcc tgtcgcagca gttgaaaatc tccaccgagg 600aagccaacga gcagatggac gcgtatttcg cccgattcgg cggggtgcgc gactacctgc 660gcgccgtagt cgagcgggcc cgcaaggacg gctacacctc gacggtgctg ggccgtcgcc 720gctacctgcc cgagctggac agcagcaacc gtcaagtgcg ggaggccgcc gagcgggcgg 780cgctgaacgc gccgatccag ggcagcgcgg ccgacatcat caaggtggcc atgatccagg 840tcgacaaggc gctcaacgag gcacagctgg cgtcgcgcat gctgctgcag gtccacgacg 900agctgctgtt cgaaatcgcc cccggtgaac gcgagcgggt cgaggccctg gtgcgcgaca 960agatgggcgg cgcttacccg ctcgacgtcc cgctggaggt gtcggtgggc tacggccgca 1020gctgggacgc ggcggcgcac tgagtgccga gcgtgcatct ggggcgggaa ttcggcgatt 1080tttccgccct gagttcacgc tcggcgcaat cgggaccgag tttgtccagc gtgtacccgt 1140cgagtagcct cgtca 1155131771DNAMycobacterium tuberculosis 13gagcgccgtc tggtgtttga acggttttac cggtcggcat cggcacgggc gttgccgggt 60tcgggcctcg ggttggcgat cgtcaaacag gtggtgctca accacggcgg attgctgcgc 120atcgaagaca ccgacccagg cggccagccc cctggaacgt cgatttacgt gctgctcccc 180ggccgtcgga tgccgattcc gcagcttccc ggtgcgacgg ctggcgctcg gagcacggac 240atcgagaact ctcggggttc ggcgaacgtt atctcagtgg aatctcagtc cacgcgcgca 300acctagttgt gcagttactg ttgaaagcca cacccatgcc agtccacgca tggccaagtt 360ggcccgagta gtgggcctag tacaggaaga gcaacctagc gacatgacga atcacccacg 420gtattcgcca ccgccgcagc agccgggaac cccaggttat gctcaggggc agcagcaaac 480gtacagccag cagttcgact ggcgttaccc accgtccccg cccccgcagc caacccagta 540ccgtcaaccc tacgaggcgt tgggtggtac ccggccgggt ctgatacctg gcgtgattcc 600gaccatgacg ccccctcctg ggatggttcg ccaacgccct cgtgcaggca tgttggccat 660cggcgcggtg acgatagcgg tggtgtccgc cggcatcggc ggcgcggccg catccctggt 720cgggttcaac cgggcacccg ccggccccag cggcggccca gtggctgcca gcgcggcgcc 780aagcatcccc gcagcaaaca tgccgccggg gtcggtcgaa caggtggcgg ccaaggtggt 840gcccagtgtc gtcatgttgg aaaccgatct gggccgccag tcggaggagg gctccggcat 900cattctgtct gccgaggggc tgatcttgac caacaaccac gtgatcgcgg cggccgccaa 960gcctcccctg ggcagtccgc cgccgaaaac gacggtaacc ttctctgacg ggcggaccgc 1020acccttcacg gtggtggggg ctgaccccac cagtgatatc gccgtcgtcc gtgttcaggg 1080cgtctccggg ctcaccccga tctccctggg ttcctcctcg gacctgaggg tcggtcagcc 1140ggtgctggcg atcgggtcgc cgctcggttt ggagggcacc gtgaccacgg ggatcgtcag 1200cgctctcaac cgtccagtgt cgacgaccgg cgaggccggc aaccagaaca ccgtgctgga 1260cgccattcag accgacgccg cgatcaaccc cggtaactcc gggggcgcgc tggtgaacat 1320gaacgctcaa ctcgtcggag tcaactcggc cattgccacg ctgggcgcgg actcagccga 1380tgcgcagagc ggctcgatcg gtctcggttt tgcgattcca gtcgaccagg ccaagcgcat 1440cgccgacgag ttgatcagca ccggcaaggc gtcacatgcc tccctgggtg tgcaggtgac 1500caatgacaaa gacaccccgg gcgccaagat cgtcgaagta gtggccggtg gtgctgccgc 1560gaacgctgga gtgccgaagg gcgtcgttgt caccaaggtc gacgaccgcc cgatcaacag 1620cgcggacgcg ttggttgccg ccgtgcggtc caaagcgccg ggcgccacgg tggcgctaac 1680ctttcaggat ccctcgggcg gtagccgcac agtgcaagtc accctcggca aggcggagca 1740gtgatgaagg tcgccgcgca gtgttcaaag c 1771141058DNAMycobacterium tuberculosismisc_feature(1)..(1058)ANY NUCLEOTIDE 14ctccaccgcg gtggcggccg ctctagaact agtggatccc ccgggctgca ggaattcggc 60acgaggatcc gacgtcgcag gttgtcgaac ccgccgccgc ggaagtatcg gtccatgcct 120agcccggcga cggcgagcgc cggaatggcg cgagtgagga ggcgggcaat ttggcggggc 180ccggcgacgg cgagcgccgg aatggcgcga gtgaggaggc gggcagtcat gcccagcgtg 240atccaatcaa cctgcattcg gcctgcgggc ccatttgaca atcgaggtag tgagcgcaaa 300tgaatgatgg aaaacgggcg gtgacgtccg ctgttctggt ggtgctaggt gcctgcctgg 360cgttgtggct atcaggatgt tcttcgccga aacctgatgc cgaggaacag ggtgttcccg 420tgagcccgac ggcgtccgac cccgcgctcc tcgccgagat caggcagtcg cttgatgcga 480caaaagggtt gaccagcgtg cacgtagcgg tccgaacaac cgggaaagtc gacagcttgc 540tgggtattac cagtgccgat gtcgacgtcc gggccaatcc gctcgcggca aagggcgtat 600gcacctacaa cgacgagcag ggtgtcccgt ttcgggtaca aggcgacaac atctcggtga 660aactgttcga cgactggagc aatctcggct cgatttctga actgtcaact tcacgcgtgc 720tcgatcctgc cgctggggtg acgcagctgc tgtccggtgt cacgaacctc caagcgcaag 780gtaccgaagt gatagacgga atttcgacca ccaaaatcac cgggaccatc cccgcgagct 840ctgtcaagat gcttgatcct ggcgccaaga gtgcaaggcc ggcgaccgtg tggattgccc 900aggacggctc gcaccacctc gtccgagcga gcatcgacct cggatccggg tcgattcagc 960tcacgcagtc gaaatggaac gaacccgtca acgtcgacta ggccgaagtt gcgtcgacgc 1020gttgntcgaa acgcccttgt gaacggtgtc aacggnac 105815542DNAMycobacterium tuberculosismisc_feature(1)..(542)ANY NUCLEOTIDE 15gaattcggca cgagaggtga tcgacatcat cgggaccagc cccacatcct gggaacaggc 60ggcggcggag gcggtccagc gggcgcggga tagcgtcgat gacatccgcg tcgctcgggt 120cattgagcag gacatggccg tggacagcgc cggcaagatc acctaccgca tcaagctcga 180agtgtcgttc aagatgaggc cggcgcaacc gcgctagcac gggccggcga gcaagacgca 240aaatcgcacg gtttgcggtt gattcgtgcg attttgtgtc tgctcgccga ggcctaccag 300gcgcggccca ggtccgcgtg ctgccgtatc caggcgtgca tcgcgattcc ggcggccacg 360ccggagttaa tgcttcgcgt

cgacccgaac tgggcgatcc gccggngagc tgatcgatga 420ccgtggccag cccgtcgatg cccgagttgc ccgaggaaac gtgctgccag gccggtagga 480agcgtccgta ggcggcggtg ctgaccggct ctgcctgcgc cctcagtgcg gccagcgagc 540gg 54216913DNAMycobacterium tuberculosismisc_feature(1)..(913)ANY NUCLEOTIDE 16cggtgccgcc cgcgcctccg ttgcccccat tgccgccgtc gccgatcagc tgcgcatcgc 60caccatcacc gcctttgccg ccggcaccgc cggtggcgcc ggggccgccg atgccaccgc 120ttgaccctgg ccgccggcgc cgccattgcc atacagcacc ccgccggggg caccgttacc 180gccgtcgcca ccgtcgccgc cgctgccgtt tcaggccggg gaggccgaat gaaccgccgc 240caagcccgcc gccggcaccg ttgccgcctt ttccgcccgc cccgccggcg ccgccaattg 300ccgaacagcc amgcaccgtt gccgccagcc ccgccgccgt taacggcgct gccgggcgcc 360gccgccggac ccgccattac cgccgttccc gttcggtgcc ccgccgttac cggcgccgcc 420gtttgccgcc aatattcggc gggcaccgcc agacccgccg gggccaccat tgccgccggg 480caccgaaaca acagcccaac ggtgccgccg gccccgccgt ttgccgccat caccggccat 540tcaccgccag caccgccgtt aatgtttatg aacccggtac cgccagcgcg gcccctattg 600ccgggcgccg gagngcgtgc ccgccggcgc cgccaacgcc caaaagcccg gggttgccac 660cggccccgcc ggacccaccg gtcccgccga tccccccgtt gccgccggtg ccgccgccat 720tggtgctgct gaagccgtta gcgccggttc cgcsggttcc ggcggtggcg ccntggccgc 780cggccccgcc gttgccgtac agccaccccc cggtggcgcc gttgccgcca ttgccgccat 840tgccgccgtt gccgccattg ccgccgttcc cgccgccacc gccggnttgg ccgccggcgc 900cgccggcggc cgc 913171872DNAMycobacterium tuberculosismisc_feature(1)..(1872)ANY NUCLEOTIDE 17gactacgttg gtgtagaaaa atcctgccgc ccggaccctt aaggctggga caatttctga 60tagctacccc gacacaggag gttacgggat gagcaattcg cgccgccgct cactcaggtg 120gtcatggttg ctgagcgtgc tggctgccgt cgggctgggc ctggccacgg cgccggccca 180ggcggccccg ccggccttgt cgcaggaccg gttcgccgac ttccccgcgc tgcccctcga 240cccgtccgcg atggtcgccc aagtggcgcc acaggtggtc aacatcaaca ccaaactggg 300ctacaacaac gccgtgggcg ccgggaccgg catcgtcatc gatcccaacg gtgtcgtgct 360gaccaacaac cacgtgatcg cgggcgccac cgacatcaat gcgttcagcg tcggctccgg 420ccaaacctac ggcgtcgatg tggtcgggta tgaccgcacc caggatgtcg cggtgctgca 480gctgcgcggt gccggtggcc tgccgtcggc ggcgatcggt ggcggcgtcg cggttggtga 540gcccgtcgtc gcgatgggca acagcggtgg gcagggcgga acgccccgtg cggtgcctgg 600cagggtggtc gcgctcggcc aaaccgtgca ggcgtcggat tcgctgaccg gtgccgaaga 660gacattgaac gggttgatcc agttcgatgc cgcaatccag cccggtgatt cgggcgggcc 720cgtcgtcaac ggcctaggac aggtggtcgg tatgaacacg gccgcgtccg ataacttcca 780gctgtcccag ggtgggcagg gattcgccat tccgatcggg caggcgatgg cgatcgcggg 840ccaaatccga tcgggtgggg ggtcacccac cgttcatatc gggcctaccg ccttcctcgg 900cttgggtgtt gtcgacaaca acggcaacgg cgcacgagtc caacgcgtgg tcggaagcgc 960tccggcggca agtctcggca tctccaccgg cgacgtgatc accgcggtcg acggcgctcc 1020gatcaactcg gccaccgcga tggcggacgc gcttaacggg catcatcccg gtgacgtcat 1080ctcggtgaac tggcaaacca agtcgggcgg cacgcgtaca gggaacgtga cattggccga 1140gggacccccg gcctgatttg tcgcggatac cacccgccgg ccggccaatt ggattggcgc 1200cagccgtgat tgccgcgtga gcccccgagt tccgtctccc gtgcgcgtgg cattgtggaa 1260gcaatgaacg aggcagaaca cagcgttgag caccctcccg tgcagggcag ttacgtcgaa 1320ggcggtgtgg tcgagcatcc ggatgccaag gacttcggca gcgccgccgc cctgcccgcc 1380gatccgacct ggtttaagca cgccgtcttc tacgaggtgc tggtccgggc gttcttcgac 1440gccagcgcgg acggttccgn cgatctgcgt ggactcatcg atcgcctcga ctacctgcag 1500tggcttggca tcgactgcat ctgttgccgc cgttcctacg actcaccgct gcgcgacggc 1560ggttacgaca ttcgcgactt ctacaaggtg ctgcccgaat tcggcaccgt cgacgatttc 1620gtcgccctgg tcgacaccgc tcaccggcga ggtatccgca tcatcaccga cctggtgatg 1680aatcacacct cggagtcgca cccctggttt caggagtccc gccgcgaccc agacggaccg 1740tacggtgact attacgtgtg gagcgacacc agcgagcgct acaccgacgc ccggatcatc 1800ttcgtcgaca ccgaagagtc gaactggtca ttcgatcctg tccgccgaca gttnctactg 1860gcaccgattc tt 1872181482DNAMycobacterium tuberculosis 18cttcgccgaa acctgatgcc gaggaacagg gtgttcccgt gagcccgacg gcgtccgacc 60ccgcgctcct cgccgagatc aggcagtcgc ttgatgcgac aaaagggttg accagcgtgc 120acgtagcggt ccgaacaacc gggaaagtcg acagcttgct gggtattacc agtgccgatg 180tcgacgtccg ggccaatccg ctcgcggcaa agggcgtatg cacctacaac gacgagcagg 240gtgtcccgtt tcgggtacaa ggcgacaaca tctcggtgaa actgttcgac gactggagca 300atctcggctc gatttctgaa ctgtcaactt cacgcgtgct cgatcctgcc gctggggtga 360cgcagctgct gtccggtgtc acgaacctcc aagcgcaagg taccgaagtg atagacggaa 420tttcgaccac caaaatcacc gggaccatcc ccgcgagctc tgtcaagatg cttgatcctg 480gcgccaagag tgcaaggccg gcgaccgtgt ggattgccca ggacggctcg caccacctcg 540tccgagcgag catcgacctc ggatccgggt cgattcagct cacgcagtcg aaatggaacg 600aacccgtcaa cgtcgactag gccgaagttg cgtcgacgcg ttgctcgaaa cgcccttgtg 660aacggtgtca acggcacccg aaaactgacc ccctgacggc atctgaaaat tgacccccta 720gaccgggcgg ttggtggtta ttcttcggtg gttccggctg gtgggacgcg gccgaggtcg 780cggtctttga gccggtagct gtcgcctttg agggcgacga cttcagcatg gtggacgagg 840cggtcgatca tggcggcagc aacgacgtcg tcgccgccga aaacctcgcc ccaccggccg 900aaggccttat tggacgtgac gatcaagctg gcccgctcat accgggagga caccagctgg 960aagaagaggt tggcggcctc gggctcaaac ggaatgtaac cgacttcgtc aaccaccagg 1020agcggatagc ggccaaaccg ggtgagttcg gcgtagatgc gcccggcgtg gtgagcctcg 1080gcgaaccgtg ctacccattc ggcggcggtg gcgaacagca cccgatgacc ggcctgacac 1140gcgcgtatcg ccaggccgac cgcaagatga gtcttcccgg tgccaggcgg ggcccaaaaa 1200cacgacgtta tcgcgggcgg tgatgaaatc cagggtgccc agatgtgcga tggtgtcgcg 1260tttgaggcca cgagcatgct caaagtcgaa ctcttccaac gacttccgaa ccgggaagcg 1320ggcggcgcgg atgcggccct caccaccatg ggactcccgg gctgacactt cccgctgcag 1380gcaggcggcc aggtattctt cgtggctcca gttctcggcg cgggcgcgat cggccagccg 1440ggacactgac tcacgcaggg tgggagcttt caatgctctt gt 148219876DNAMycobacterium tuberculosismisc_feature(1)..(876)ANY NUCLEOTIDE 19gaattcggca cgagccggcg atagcttctg ggccgcggcc gaccagatgg ctcgagggtt 60cgtgctcggg gccaccgccg ggcgcaccac cctgaccggt gagggcctgc aacacgccga 120cggtcactcg ttgctgctgg acgccaccaa cccggcggtg gttgcctacg acccggcctt 180cgcctacgaa atcggctaca tcgnggaaag cggactggcc aggatgtgcg gggagaaccc 240ggagaacatc ttcttctaca tcaccgtcta caacgagccg tacgtgcagc cgccggagcc 300ggagaacttc gatcccgagg gcgtgctggg gggtatctac cgntatcacg cggccaccga 360gcaacgcacc aacaaggngc agatcctggc ctccggggta gcgatgcccg cggcgctgcg 420ggcagcacag atgctggccg ccgagtggga tgtcgccgcc gacgtgtggt cggtgaccag 480ttggggcgag ctaaaccgcg acggggtggt catcgagacc gagaagctcc gccaccccga 540tcggccggcg ggcgtgccct acgtgacgag agcgctggag aatgctcggg gcccggtgat 600cgcggtgtcg gactggatgc gcgcggtccc cgagcagatc cgaccgtggg tgccgggcac 660atacctcacg ttgggcaccg acgggttcgg tttttccgac actcggcccg ccggtcgtcg 720ttacttcaac accgacgccg aatcccaggt tggtcgcggt tttgggaggg gttggccggg 780tcgacgggtg aatatcgacc cattcggtgc cggtcgtggg ccgcccgccc agttacccgg 840attcgacgaa ggtggggggt tgcgcccgan taagtt 876201021DNAMycobacterium tuberculosismisc_feature(1)..(1021)ANY NUCLEOTIDE 20atccccccgg gctgcaggaa ttcggcacga gagacaaaat tccacgcgtt aatgcaggaa 60cagattcata acgaattcac agcggcacaa caatatgtcg cgatcgcggt ttatttcgac 120agcgaagacc tgccgcagtt ggcgaagcat ttttacagcc aagcggtcga ggaacgaaac 180catgcaatga tgctcgtgca acacctgctc gaccgcgacc ttcgtgtcga aattcccggc 240gtagacacgg tgcgaaacca gttcgacaga ccccgcgagg cactggcgct ggcgctcgat 300caggaacgca cagtcaccga ccaggtcggt cggctgacag cggtggcccg cgacgagggc 360gatttcctcg gcgagcagtt catgcagtgg ttcttgcagg aacagatcga agaggtggcc 420ttgatggcaa ccctggtgcg ggttgccgat cgggccgggg ccaacctgtt cgagctagag 480aacttcgtcg cacgtgaagt ggatgtggcg ccggccgcat caggcgcccc gcacgctgcc 540gggggccgcc tctagatccc tgggggggat cagcgagtgg tcccgttcgc ccgcccgtct 600tccagccagg ccttggtgcg gccggggtgg tgagtaccaa tccaggccac cccgacctcc 660cggnaaaagt cgatgtcctc gtactcatcg acgttccagg agtacaccgc ccggccctga 720gctgccgagc ggtcaacgag ttgcggatat tcctttaacg caggcagtga gggtcccacg 780gcggttggcc cgaccgccgt ggccgcactg ctggtcaggt atcggggggt cttggcgagc 840aacaacgtcg gcaggagggg tggagcccgc cggatccgca gaccgggggg gcgaaaacga 900catcaacacc gcacgggatc gatctgcgga ggggggtgcg ggaataccga accggtgtag 960gagcgccagc agttgttttt ccaccagcga agcgttttcg ggtcatcggn ggcnnttaag 1020t 102121321DNAMycobacterium tuberculosismisc_feature(1)..(321)ANY NUCLEOTIDE 21cgtgccgacg aacggaagaa cacaaccatg aagatggtga aatcgatcgc cgcaggtctg 60accgccgcgg ctgcaatcgg cgccgctgcg gccggtgtga cttcgatcat ggctggcggn 120ccggtcgtat accagatgca gccggtcgtc ttcggcgcgc cactgccgtt ggacccggna 180tccgcccctg angtcccgac cgccgcccag tggaccagnc tgctcaacag nctcgncgat 240cccaacgtgt cgtttgngaa caagggnagt ctggtcgagg gnggnatcgg nggnancgag 300ggngngnatc gncgancaca a 32122373DNAMycobacterium tuberculosismisc_feature(1)..(373)ANY NUCLEOTIDE 22tcttatcggt tccggttggc gacgggtttt gggngcgggt ggttaacccg ctcggccagc 60cgatcgacgg gcgcggagac gtcgactccg atactcggcg cgcgctggag ctccaggcgc 120cctcggtggt gnaccggcaa ggcgtgaagg agccgttgna gaccgggatc aaggcgattg 180acgcgatgac cccgatcggc cgcgggcagc gccagctgat catcggggac cgcaagaccg 240gcaaaaaccg ccgtctgtgt cggacaccat cctcaaacca gcgggaagaa ctgggagtcc 300ggtggatccc aagaagcagg tgcgcttgtg tatacgttgg ccatcgggca agaaggggaa 360cttaccatcg ccg 37323352DNAMycobacterium tuberculosismisc_feature(1)..(352)ANY NUCLEOTIDE 23gtgacgccgt gatgggattc ctgggcgggg ccggtccgct ggcggtggtg gatcagcaac 60tggttacccg ggtgccgcaa ggctggtcgt ttgctcaggc agccgctgtg ccggtggtgt 120tcttgacggc ctggtacggg ttggccgatt tagccgagat caaggcgggc gaatcggtgc 180tgatccatgc cggtaccggc ggtgtgggca tggcggctgt gcagctggct cgccagtggg 240gcgtggaggt tttcgtcacc gccagccgtg gnaagtggga cacgctgcgc gccatngngt 300ttgacgacga nccatatcgg ngattcccnc acatncgaag ttccgangga ga 35224726DNAMycobacterium tuberculosis 24gaaatccgcg ttcattccgt tcgaccagcg gctggcgata atcgacgaag tgatcaagcc 60gcggttcgcg gcgctcatgg gtcacagcga gtaatcagca agttctctgg tatatcgcac 120ctagcgtcca gttgcttgcc agatcgcttt cgtaccgtca tcgcatgtac cggttcgcgt 180gccgcacgct catgctggcg gcgtgcatcc tggccacggg tgtggcgggt ctcggggtcg 240gcgcgcagtc cgcagcccaa accgcgccgg tgcccgacta ctactggtgc ccggggcagc 300ctttcgaccc cgcatggggg cccaactggg atccctacac ctgccatgac gacttccacc 360gcgacagcga cggccccgac cacagccgcg actaccccgg acccatcctc gaaggtcccg 420tgcttgacga tcccggtgct gcgccgccgc ccccggctgc cggtggcggc gcatagcgct 480cgttgaccgg gccgcatcag cgaatacgcg tataaacccg ggcgtgcccc cggcaagcta 540cgacccccgg cggggcagat ttacgctccc gtgccgatgg atcgcgccgt ccgatgacag 600aaaataggcg acggttttgg caaccgcttg gaggacgctt gaagggaacc tgtcatgaac 660ggcgacagcg cctccaccat cgacatcgac aaggttgtta cccgcacacc cgttcgccgg 720atcgtg 72625580DNAMycobacterium tuberculosis 25cgcgacgacg acgaacgtcg ggcccaccac cgcctatgcg ttgatgcagg cgaccgggat 60ggtcgccgac catatccaag catgctgggt gcccactgag cgaccttttg accagccggg 120ctgcccgatg gcggcccggt gaagtcattg cgccggggct tgtgcacctg atgaacccga 180atagggaaca ataggggggt gatttggcag ttcaatgtcg ggtatggctg gaaatccaat 240ggcggggcat gctcggcgcc gaccaggctc gcgcaggcgg gccagcccga atctggaggg 300agcactcaat ggcggcgatg aagccccgga ccggcgacgg tcctttggaa gcaactaagg 360aggggcgcgg cattgtgatg cgagtaccac ttgagggtgg cggtcgcctg gtcgtcgagc 420tgacacccga cgaagccgcc gcactgggtg acgaactcaa aggcgttact agctaagacc 480agcccaacgg cgaatggtcg gcgttacgcg cacaccttcc ggtagatgtc cagtgtctgc 540tcggcgatgt atgcccagga gaactcttgg atacagcgct 58026160DNAMycobacterium tuberculosis 26aacggaggcg ccgggggttt tggcggggcc ggggcggtcg gcggcaacgg cggggccggc 60ggtaccgccg ggttgttcgg tgtcggcggg gccggtgggg ccggaggcaa cggcatcgcc 120ggtgtcacgg gtacgtcggc cagcacaccg ggtggatccg 16027272DNAMycobacterium tuberculosis 27gacaccgata cgatggtgat gtacgccaac gttgtcgaca cgctcgaggc gttcacgatc 60cagcgcacac ccgacggcgt gaccatcggc gatgcggccc cgttcgcgga ggcggctgcc 120aaggcgatgg gaatcgacaa gctgcgggta attcataccg gaatggaccc cgtcgtcgct 180gaacgcgaac agtgggacga cggcaacaac acgttggcgt tggcgcccgg tgtcgttgtc 240gcctacgagc gcaacgtaca gaccaacgcc cg 27228317DNAMycobacterium tuberculosis 28gcagccggtg gttctcggac tatctgcgca cggtgacgca gcgcgacgtg cgcgagctga 60agcggatcga gcagacggat cgcctgccgc ggttcatgcg ctacctggcc gctatcaccg 120cgcaggagct gaacgtggcc gaagcggcgc gggtcatcgg ggtcgacgcg gggacgatcc 180gttcggatct ggcgtggttc gagacggtct atctggtaca tcgcctgccc gcctggtcgc 240ggaatctgac cgcgaagatc aagaagcggt caaagatcca cgtcgtcgac agtggcttcg 300cggcctggtt gcgcggg 31729182DNAMycobacterium tuberculosis 29gatcgtggag ctgtcgatga acagcgttgc cggacgcgcg gcggccagca cgtcggtgta 60gcagcgccgg accacctcgc cggtgggcag catggtgatg accacgtcgg cctcggccac 120cscttcgggc gcgctacgaa acaccgcgac accgtgcgcg gcggcgccgg acgccgccgt 180gg 18230308DNAMycobacterium tuberculosis 30gatcgcgaag tttggtgagc aggtggtcga cgcgaaagtc tgggcgcctg cgaagcgggt 60cggcgttcac gaggcgaaga cacgcctgtc cgagctgctg cggctcgtct acggcgggca 120gaggttgaga ttgcccgccg cggcgagccg gtagcaaagc ttgtgccgct gcatcctcat 180gagactcggc ggttaggcat tgaccatggc gtgtaccgcg tgcccgacga tttggacgct 240ccgttgtcag acgacgtgct cgaacgcttt caccggtgaa gcgctacctc atcgacaccc 300acgtttgg 30831267DNAMycobacterium tuberculosis 31ccgacgacga gcaactcacg tggatgatgg tcggcagcgg cattgaggac ggagagaatc 60cggccgaagc tgccgcgcgg caagtgctca tagtgaccgg ccgtagaggg ctcccccgat 120ggcaccggac tattctggtg tgccgctggc cggtaagagc gggtaaaaga atgtgagggg 180acacgatgag caatcacacc taccgagtga tcgagatcgt cgggacctcg cccgacggcg 240tcgacgcggc aatccagggc ggtctgg 267321539DNAMycobacterium tuberculosis 32ctcgtgccga aagaatgtga ggggacacga tgagcaatca cacctaccga gtgatcgaga 60tcgtcgggac ctcgcccgac ggcgtcgacg cggcaatcca gggcggtctg gcccgagctg 120cgcagaccat gcgcgcgctg gactggttcg aagtacagtc aattcgaggc cacctggtcg 180acggagcggt cgcgcacttc caggtgacta tgaaagtcgg cttccgctgg aggattcctg 240aaccttcaag cgcggccgat aactgaggtg catcattaag cgacttttcc agaacatcct 300gacgcgctcg aaacgcggtt cagccgacgg tggctccgcc gaggcgctgc ctccaaaatc 360cctgcgacaa ttcgtcggcg gcgcctacaa ggaagtcggt gctgaattcg tcgggtatct 420ggtcgacctg tgtgggctgc agccggacga agcggtgctc gacgtcggct gcggctcggg 480gcggatggcg ttgccgctca ccggctatct gaacagcgag ggacgctacg ccggcttcga 540tatctcgcag aaagccatcg cgtggtgcca ggagcacatc acctcggcgc accccaactt 600ccagttcgag gtctccgaca tctacaactc gctgtacaac ccgaaaggga aataccagtc 660actagacttt cgctttccat atccggatgc gtcgttcgat gtggtgtttc ttacctcggt 720gttcacccac atgtttccgc cggacgtgga gcactatctg gacgagatct cccgcgtgct 780gaagcccggc ggacgatgcc tgtgcacgta cttcttgctc aatgacgagt cgttagccca 840catcgcggaa ggaaagagtg cgcacaactt ccagcatgag ggaccgggtt atcggacaat 900ccacaagaag cggcccgaag aagcaatcgg cttgccggag accttcgtca gggatgtcta 960tggcaagttc ggcctcgccg tgcacgaacc attgcactac ggctcatgga gtggccggga 1020accacgccta agcttccagg acatcgtcat cgcgaccaaa accgcgagct aggtcggcat 1080ccgggaagca tcgcgacacc gtggcgccga gcgccgctgc cggcaggccg attaggcggg 1140cagattagcc cgccgcggct cccggctccg agtacggcgc cccgaatggc gtcaccggct 1200ggtaaccacg cttgcgcgcc tgggcggcgg cctgccggat caggtggtag atgccgacaa 1260agcctgcgtg atcggtcatc accaacggtg acagcagccg gttgtgcacc agcgcgaacg 1320ccaccccggt ctccgggtct gtccagccga tcgagccgcc caagcccaca tgaccaaacc 1380ccggcatcac gttgccgatc ggcataccgt gatagccaag atgaaaattt aagggcacca 1440atagatttcg atccggcaga acttgccgtc ggttgcgggt caggcccgtg accagctccc 1500gcgacaagaa ccgtatgccg tcgatctcgc ctcgtgccg 153933851DNAMycobacterium tuberculosismisc_feature(1)..(851)ANY NUCLEOTIDE 33ctgcagggtg gcgtggatga gcgtcaccgc ggggcaggcc gagctgaccg ccgcccaggt 60ccgggttgct gcggcggcct acgagacggc gtatgggctg acggtgcccc cgccggtgat 120cgccgagaac cgtgctgaac tgatgattct gatagcgacc aacctcttgg ggcaaaacac 180cccggcgatc gcggtcaacg aggccgaata cggcgagatg tgggcccaag acgccgccgc 240gatgtttggc tacgccgcgg cgacggcgac ggcgacggcg acgttgctgc cgttcgagga 300ggcgccggag atgaccagcg cgggtgggct cctcgagcag gccgccgcgg tcgaggaggc 360ctccgacacc gccgcggcga accagttgat gaacaatgtg ccccaggcgc tgaaacagtt 420ggcccagccc acgcagggca ccacgccttc ttccaagctg ggtggcctgt ggaagacggt 480ctcgccgcat cggtcgccga tcagcaacat ggtgtcgatg gccaacaacc acatgtcgat 540gaccaactcg ggtgtgtcga tgaccaacac cttgagctcg atgttgaagg gctttgctcc 600ggcggcggcc gcccaggccg tgcaaaccgc ggcgcaaaac ggggtccggg cgatgagctc 660gctgggcagc tcgctgggtt cttcgggtct gggcggtggg gtggccgcca acttgggtcg 720ggcggcctcg gtacggtatg gtcaccggga tggcggaaaa tatgcanagt ctggtcggcg 780gaacggtggt ccggcgtaag gtttaccccc gttttctgga tgcggtgaac ttcgtcaacg 840gaaacagtta c 85134254DNAMycobacterium tuberculosis 34gatcgatcgg gcggaaattt ggaccagatt cgcctccggc gataacccaa tcaatcgaac 60ctagatttat tccgtccagg ggcccgagta atggctcgca ggagaggaac cttactgctg 120cgggcacctg tcgtaggtcc tcgatacggc ggaaggcgtc gacattttcc accgacaccc 180ccatccaaac gttcgagggc cactccagct tgtgagcgag gcgacgcagt cgcaggctgc 240gcttggtcaa gatc 254351227DNAMycobacterium tuberculosis 35gatcctgacc gaagcggccg ccgccaaggc gaagtcgctg ttggaccagg agggacggga 60cgatctggcg ctgcggatcg cggttcagcc gggggggtgc gctggattgc gctataacct 120tttcttcgac gaccggacgc tggatggtga ccaaaccgcg gagttcggtg gtgtcaggtt 180gatcgtggac cggatgagcg cgccgtatgt ggaaggcgcg tcgatcgatt tcgtcgacac 240tattgagaag caaggttcac catcgacaat cccaacgcca ccggctcctg cgcgtgcggg 300gattcgttca actgataaaa cgctagtacg accccgcggt gcgcaacacg tacgagcaca 360ccaagacctg accgcgctgg aaaagcaact gagcgatgcc ttgcacctga ccgcgtggcg 420ggccgccggc ggcaggtgtc acctgcatgg tgaacagcac ctgggcctga tattgcgacc 480agtacacgat tttgtcgatc gaggtcactt cgacctggga gaactgcttg cggaacgcgt 540cgctgctcag

cttggccaag gcctgatcgg agcgcttgtc gcgcacgccg tcgtggatac 600cgcacagcgc attgcgaacg atggtgtcca catcgcggtt ctccagcgcg ttgaggtatc 660cctgaatcgc ggttttggcc ggtccctccg agaatgtgcc tgccgtgttg gctccgttgg 720tgcggacccc gtatatgatc gccgccgtca tagccgacac cagcgcgagg gctaccacaa 780tgccgatcag cagccgcttg tgccgtcgct tcgggtagga cacctgcggc ggcacgccgg 840gatatgcggc gggcggcagc gccgcgtcgt ctgccggtcc cggggcgaag gccggttcgg 900dcggcgccga ggtcgtgggg gtagtccagg gcttggggtt cgtgggatga ggctcggggt 960acggcgccgg tccgttggtg ccgacaccgg ggttcggcga gtggggaccg ggcattgtgg 1020ttctcctagg gtggtggacg ggaccagctg ctagggcgac aaccgcccgt cgcgtcagcc 1080ggcagcatcg gcaatcaggt gagctcccta ggcaggctag cgcaacagct gccgtcagct 1140ctcaacgcga cggggcgggc cgcggcgccg ataatgttga aagactaggc aaccttagga 1200acgaaggacg gagattttgt gacgatc 122736181DNAMycobacterium tuberculosismisc_feature(1)..(181)ANY NUCLEOTIDE 36gcggtgtcgg cggatccggc gggtggttga acggcaacgg cggggccggc ggggccggcg 60ggaccggcgc taacggtggt gccggcggca acgcctggtt gttcggggcc ggcgggtccg 120gcggngccgg caccaatggt ggngtcggcg ggtccggcgg atttgtctac ggcaacggcg 180g 18137290DNAMycobacterium tuberculosismisc_feature(1)..(290)ANY NUCLEOTIDE 37gcggtgtcgg cggatccggc gggtggttga acggcaacgg cggtgtcggc ggccggggcg 60gcgacggcgt ctttgccggt gccggcggcc agggcggcct cggtgggcag ggcggcaatg 120gcggcggctc caccggcggc aacggcggtc ttggcggcgc gggcggtggc ggaggcaacg 180ccccggacgg cggcttcggt ggcaacggcg gtaagggtgg ccagggcggn attggcggcg 240gcactcagag cgcgaccggc ctcggnggtg acggcggtga cggcggtgac 2903834DNAMycobacterium tuberculosismisc_feature(1)..(34)ANY NUCLEOTIDE 38gatccagtgg catggngggt gtcagtggaa gcat 3439155DNAMycobacterium tuberculosismisc_feature(1)..(155)ANY NUCLEOTIDE 39gatcgctgct cgtccccccc ttgccgccga cgccaccggt cccaccgtta ccgaacaagc 60tggcgtggtc gccagcaccc ccggcaccgc cgacgccgga gtcgaacaat ggcaccgtcg 120tatccccacc attgccgccg gncccaccgg caccg 1554053DNAMycobacterium tuberculosismisc_feature(1)..(53)ANY NUCLEOTIDE 40atggcgttca cggggcgccg gggaccgggc agcccggngg ggccgggggg tgg 5341132DNAMycobacterium tuberculosismisc_feature(1)..(132)ANY NUCLEOTIDE 41gatccaccgc gggtgcagac ggtgcccgcg gcgccacccc gaccagcggc ggcaacggcg 60gcaccggcgg caacggcgcg aacgccaccg tcgtcggngg ggccggcggg gccggcggca 120agggcggcaa cg 13242132DNAMycobacterium tuberculosismisc_feature(1)..(132)ANY NUCLEOTIDE 42gatcggcggc cggnacggnc ggggacggcg gcaagggcgg naacgggggc gccgnagcca 60ccngccaaga atcctccgng tccnccaatg gcgcgaatgg cggacagggc ggcaacggcg 120gcancggcgg ca 13243702DNAMycobacterium tuberculosismisc_feature(1)..(701)ANY NUCLEOTIDE 43cggcacgagg atcggtaccc cgcggcatcg gcagctgccg attcgccggg tttccccacc 60cgaggaaagc cgctaccaga tggcgctgcc gaagtagggc gatccgttcg cgatgccggc 120atgaacgggc ggcatcaaat tagtgcagga acctttcagt ttagcgacga taatggctat 180agcactaagg aggatgatcc gatatgacgc agtcgcagac cgtgacggtg gatcagcaag 240agatttrgaa cagggccaac gaggtggagg ccccgatggc ggacccaccg actgatgtcc 300ccatcacacc gtgcgaactc acggnggnta aaaacgccgc ccaacagntg gtnttgtccg 360ccgacaacat gcgggaatac ctggcggccg gtgccaaaga gcggcagcgt ctggcgacct 420cgctgcgcaa cgcggccaag gngtatggcg aggttgatga ggaggctgcg accgcgctgg 480acaacgacgg cgaaggaact gtgcaggcag aatcggccgg ggccgtcgga ggggacagtt 540cggccgaact aaccgatacg ccgagggtgg ccacggccgg tgaacccaac ttcatggatc 600tcaaagaagc ggcaaggaag ctcgaaacgg gcgaccaagg cgcatcgctc gcgcactgng 660gggatgggtg gaacacttnc accctgacgc tgcaaggcga cg 70244298DNAMycobacterium tuberculosis 44gaagccgcag cgctgtcggg cgacgtggcg gtcaaagcgg catcgctcgg tggcggtgga 60ggcggcgggg tgccgtcggc gccgttggga tccgcgatcg ggggcgccga atcggtgcgg 120cccgctggcg ctggtgacat tgccggctta ggccagggaa gggccggcgg cggcgccgcg 180ctgggcggcg gtggcatggg aatgccgatg ggtgccgcgc atcagggaca agggggcgcc 240aagtccaagg gttctcagca ggaagacgag gcgctctaca ccgaggatcc tcgtgccg 298451058DNAMycobacterium tuberculosis 45cggcacgagg atcgaatcgc gtcgccggga gcacagcgtc gcactgcacc agtggaggag 60ccatgaccta ctcgccgggt aaccccggat acccgcaagc gcagcccgca ggctcctacg 120gaggcgtcac accctcgttc gcccacgccg atgagggtgc gagcaagcta ccgatgtacc 180tgaacatcgc ggtggcagtg ctcggtctgg ctgcgtactt cgccagcttc ggcccaatgt 240tcaccctcag taccgaactc ggggggggtg atggcgcagt gtccggtgac actgggctgc 300cggtcggggt ggctctgctg gctgcgctgc ttgccggggt ggttctggtg cctaaggcca 360agagccatgt gacggtagtt gcggtgctcg gggtactcgg cgtatttctg atggtctcgg 420cgacgtttaa caagcccagc gcctattcga ccggttgggc attgtgggtt gtgttggctt 480tcatcgtgtt ccaggcggtt gcggcagtcc tggcgctctt ggtggagacc ggcgctatca 540ccgcgccggc gccgcggccc aagttcgacc cgtatggaca gtacgggcgg tacgggcagt 600acgggcagta cggggtgcag ccgggtgggt actacggtca gcagggtgct cagcaggccg 660cgggactgca gtcgcccggc ccgcagcagt ctccgcagcc tcccggatat gggtcgcagt 720acggcggcta ttcgtccagt ccgagccaat cgggcagtgg atacactgct cagcccccgg 780cccagccgcc ggcgcagtcc gggtcgcaac aatcgcacca gggcccatcc acgccaccta 840ccggctttcc gagcttcagc ccaccaccac cggtcagtgc cgggacgggg tcgcaggctg 900gttcggctcc agtcaactat tcaaacccca gcgggggcga gcagtcgtcg tcccccgggg 960gggcgccggt ctaaccgggc gttcccgcgt ccggtcgcgc gtgtgcgcga agagtgaaca 1020gggtgtcagc aagcgcggac gatcctcgtg ccgaattc 105846327DNAMycobacterium tuberculosis 46cggcacgaga gaccgatgcc gctaccctcg cgcaggaggc aggtaatttc gagcggatct 60ccggcgacct gaaaacccag atcgaccagg tggagtcgac ggcaggttcg ttgcagggcc 120agtggcgcgg cgcggcgggg acggccgccc aggccgcggt ggtgcgcttc caagaagcag 180ccaataagca gaagcaggaa ctcgacgaga tctcgacgaa tattcgtcag gccggcgtcc 240aatactcgag ggccgacgag gagcagcagc aggcgctgtc ctcgcaaatg ggcttctgac 300ccgctaatac gaaaagaaac ggagcaa 32747170DNAMycobacterium tuberculosismisc_feature(1)..(170)ANY NUCLEOTIDE 47cggtcgcgat gatggcgttg tcgaacgtga ccgattctgt accgccgtcg ttgagatcaa 60ccaacaacgt gttggcgtcg gcaaatgtgc cgnacccgtg gatctcggtg atcttgttct 120tcttcatcag gaagtgcaca ccggccaccc tgccctcggn tacctttcgg 17048127DNAMycobacterium tuberculosis 48gatccggcgg cacggggggt gccggcggca gcaccgctgg cgctggcggc aacggcgggg 60ccgggggtgg cggcggaacc ggtgggttgc tcttcggcaa cggcggtgcc ggcgggcacg 120gggccgt 1274981DNAMycobacterium tuberculosis 49cggcggcaag ggcggcaccg ccggcaacgg gagcggcgcg gccggcggca acggcggcaa 60cggcggctcc ggcctcaacg g 8150149DNAMycobacterium tuberculosismisc_feature(1)..(149)ANY NUCLEOTIDE 50gatcagggct ggccggctcc ggccagaagg gcggtaacgg aggagctgcc ggattgtttg 60gcaacggcgg ggccggnggt gccggcgcgt ccaaccaagc cggtaacggc ggngccggcg 120gaaacggtgg tgccggtggg ctgatctgg 14951355DNAMycobacterium tuberculosismisc_feature(1)..(355)ANY NUCLEOTIDE 51cggcacgaga tcacacctac cgagtgatcg agatcgtcgg gacctcgccc gacggtgtcg 60acgcggnaat ccagggcggt ctggcccgag ctgcgcagac catgcgcgcg ctggactggt 120tcgaagtaca gtcaattcga ggccacctgg tcgacggagc ggtcgcgcac ttccaggtga 180ctatgaaagt cggcttccgc ctggaggatt cctgaacctt caagcgcggc cgataactga 240ggtgcatcat taagcgactt ttccagaaca tcctgacgcg ctcgaaacgc ggttcagccg 300acggtggctc cgccgaggcg ctgcctccaa aatccctgcg acaattcgtc ggcgg 35552999DNAMycobacterium tuberculosis 52atgcatcacc atcaccatca catgcatcag gtggacccca acttgacacg tcgcaaggga 60cgattggcgg cactggctat cgcggcgatg gccagcgcca gcctggtgac cgttgcggtg 120cccgcgaccg ccaacgccga tccggagcca gcgcccccgg tacccacaac ggccgcctcg 180ccgccgtcga ccgctgcagc gccacccgca ccggcgacac ctgttgcccc cccaccaccg 240gccgccgcca acacgccgaa tgcccagccg ggcgatccca acgcagcacc tccgccggcc 300gacccgaacg caccgccgcc acctgtcatt gccccaaacg caccccaacc tgtccggatc 360gacaacccgg ttggaggatt cagcttcgcg ctgcctgctg gctgggtgga gtctgacgcc 420gcccacttcg actacggttc agcactcctc agcaaaacca ccggggaccc gccatttccc 480ggacagccgc cgccggtggc caatgacacc cgtatcgtgc tcggccggct agaccaaaag 540ctttacgcca gcgccgaagc caccgactcc aaggccgtgc cccggttggg ctcggacatg 600ggtgagttct atatgcccta cccgggcacc cggatcaacc aggaaaccgt ctcgctcgac 660gccaacgggg tgtctggaag cgcgtcgtat tacgaagtca agttcagcga tccgagtaag 720ccgaacggcc agatctggac gggcgtaatc ggctcgcccg cggcgaacgc accggacgcc 780gggccccctc agcgctggtt tgtggtatgg ctcgggaccg ccaacaaccc ggtggacaag 840ggcgcggcca aggcgctggc cgaatcgatc cggcctttgg tcgccccgcc gccggcgccg 900gcaccggctc ctgcagagcc cgctccggcg ccggcgccgg ccggggaagt cgctcctacc 960ccgacgacac cgacaccgca gcggacctta ccggcctga 99953332PRTMycobacterium tuberculosis 53Met His His His His His His Met His Gln Val Asp Pro Asn Leu Thr1 5 10 15Arg Arg Lys Gly Arg Leu Ala Ala Leu Ala Ile Ala Ala Met Ala Ser 20 25 30Ala Ser Leu Val Thr Val Ala Val Pro Ala Thr Ala Asn Ala Asp Pro 35 40 45Glu Pro Ala Pro Pro Val Pro Thr Thr Ala Ala Ser Pro Pro Ser Thr 50 55 60Ala Ala Ala Pro Pro Ala Pro Ala Thr Pro Val Ala Pro Pro Pro Pro65 70 75 80Ala Ala Ala Asn Thr Pro Asn Ala Gln Pro Gly Asp Pro Asn Ala Ala 85 90 95Pro Pro Pro Ala Asp Pro Asn Ala Pro Pro Pro Pro Val Ile Ala Pro 100 105 110Asn Ala Pro Gln Pro Val Arg Ile Asp Asn Pro Val Gly Gly Phe Ser 115 120 125Phe Ala Leu Pro Ala Gly Trp Val Glu Ser Asp Ala Ala His Phe Asp 130 135 140Tyr Gly Ser Ala Leu Leu Ser Lys Thr Thr Gly Asp Pro Pro Phe Pro145 150 155 160Gly Gln Pro Pro Pro Val Ala Asn Asp Thr Arg Ile Val Leu Gly Arg 165 170 175Leu Asp Gln Lys Leu Tyr Ala Ser Ala Glu Ala Thr Asp Ser Lys Ala 180 185 190Ala Ala Arg Leu Gly Ser Asp Met Gly Glu Phe Tyr Met Pro Tyr Pro 195 200 205Gly Thr Arg Ile Asn Gln Glu Thr Val Ser Leu Asp Ala Asn Gly Val 210 215 220Ser Gly Ser Ala Ser Tyr Tyr Glu Val Lys Phe Ser Asp Pro Ser Lys225 230 235 240Pro Asn Gly Gln Ile Trp Thr Gly Val Ile Gly Ser Pro Ala Ala Asn 245 250 255Ala Pro Asp Ala Gly Pro Pro Gln Arg Trp Phe Val Val Trp Leu Gly 260 265 270Thr Ala Asn Asn Pro Val Asp Lys Gly Ala Ala Lys Ala Leu Ala Glu 275 280 285Ser Ile Arg Pro Leu Val Ala Pro Pro Pro Ala Pro Ala Pro Ala Pro 290 295 300Ala Glu Pro Ala Pro Ala Pro Ala Pro Ala Gly Glu Val Ala Pro Thr305 310 315 320Pro Thr Thr Pro Thr Pro Gln Arg Thr Leu Pro Ala 325 3305420PRTMycobacterium tuberculosisMISC_FEATURE(1)..(20)ANY AMINO ACID 54Asp Pro Val Asp Ala Val Ile Asn Thr Thr Xaa Asn Tyr Gly Gln Val1 5 10 15Val Ala Ala Leu 205515PRTMycobacterium tuberculosis 55Ala Val Glu Ser Gly Met Leu Ala Leu Gly Thr Pro Ala Pro Ser1 5 10 155619PRTMycobacterium tuberculosis 56Ala Ala Met Lys Pro Arg Thr Gly Asp Gly Pro Leu Glu Ala Ala Lys1 5 10 15Glu Gly Arg5715PRTMycobacterium tuberculosis 57Tyr Tyr Trp Cys Pro Gly Gln Pro Phe Asp Pro Ala Trp Gly Pro1 5 10 155814PRTMycobacterium tuberculosisMISC_FEATURE(1)..(14)ANY AMINO ACID 58Asp Ile Gly Ser Glu Ser Thr Glu Asp Gln Gln Xaa Ala Val1 5 105913PRTMycobacterium tuberculosisMISC_FEATURE(1)..(13)ANY AMINO ACID 59Ala Glu Glu Ser Ile Ser Thr Xaa Glu Xaa Ile Val Pro1 5 106017PRTMycobacterium tuberculosis 60Asp Pro Glu Pro Ala Pro Pro Val Pro Thr Ala Ala Ala Ala Pro Pro1 5 10 15Ala6115PRTMycobacterium tuberculosisMISC_FEATURE(1)..(15)ANY AMINO ACID 61Ala Pro Lys Thr Tyr Xaa Glu Glu Leu Lys Gly Thr Asp Thr Gly1 5 10 156230PRTMycobacterium tuberculosis 62Asp Pro Ala Ser Ala Pro Asp Val Pro Thr Ala Ala Gln Gln Thr Ser1 5 10 15Leu Leu Asn Asn Leu Ala Asp Pro Asp Val Ser Phe Ala Asp 20 25 3063187PRTMycobacterium tuberculosisMISC_FEATURE(1)..(187)ANY AMINO ACID 63Thr Gly Ser Leu Asn Gln Thr His Asn Arg Arg Ala Asn Glu Arg Lys1 5 10 15Asn Thr Thr Met Lys Met Val Lys Ser Ile Ala Ala Gly Leu Thr Ala 20 25 30Ala Ala Ala Ile Gly Ala Ala Ala Ala Gly Val Thr Ser Ile Met Ala 35 40 45Gly Gly Pro Val Val Tyr Gln Met Gln Pro Val Val Phe Gly Ala Pro 50 55 60Leu Pro Leu Asp Pro Ala Ser Ala Pro Asp Val Pro Thr Ala Ala Gln65 70 75 80Leu Thr Ser Leu Leu Asn Ser Leu Ala Asp Pro Asn Val Ser Phe Ala 85 90 95Asn Lys Gly Ser Leu Val Glu Gly Gly Ile Gly Gly Thr Glu Ala Arg 100 105 110Ile Ala Asp His Lys Leu Lys Lys Ala Ala Glu His Gly Asp Leu Pro 115 120 125Leu Ser Phe Ser Val Thr Asn Ile Gln Pro Ala Ala Ala Gly Ser Ala 130 135 140Thr Ala Asp Val Ser Val Ser Gly Pro Lys Leu Ser Ser Pro Val Thr145 150 155 160Gln Asn Val Thr Phe Val Asn Gln Gly Gly Trp Met Leu Ser Arg Ala 165 170 175Ser Ala Met Glu Leu Leu Gln Ala Ala Gly Xaa 180 18564148PRTMycobacterium tuberculosis 64Asp Glu Val Thr Val Glu Thr Thr Ser Val Phe Arg Ala Asp Phe Leu1 5 10 15Ser Glu Leu Asp Ala Pro Ala Gln Ala Gly Thr Glu Ser Ala Val Ser 20 25 30Gly Val Glu Gly Leu Pro Pro Gly Ser Ala Leu Leu Val Val Lys Arg 35 40 45Gly Pro Asn Ala Gly Ser Arg Phe Leu Leu Asp Gln Ala Ile Thr Ser 50 55 60Ala Gly Arg His Pro Asp Ser Asp Ile Phe Leu Asp Asp Val Thr Val65 70 75 80Ser Arg Arg His Ala Glu Phe Arg Leu Glu Asn Asn Glu Phe Asn Val 85 90 95Val Asp Val Gly Ser Leu Asn Gly Thr Tyr Val Asn Arg Glu Pro Val 100 105 110Asp Ser Ala Val Leu Ala Asn Gly Asp Glu Val Gln Ile Gly Lys Leu 115 120 125Arg Leu Val Phe Leu Thr Gly Pro Lys Gln Gly Glu Asp Asp Gly Ser 130 135 140Thr Gly Gly Pro14565230PRTMycobacterium tuberculosisMISC_FEATURE(1)..(230)ANY AMINO ACID 65Thr Ser Asn Arg Pro Ala Arg Arg Gly Arg Arg Ala Pro Arg Asp Thr1 5 10 15Gly Pro Asp Arg Ser Ala Ser Leu Ser Leu Val Arg His Arg Arg Gln 20 25 30Gln Arg Asp Ala Leu Cys Leu Ser Ser Thr Gln Ile Ser Arg Gln Ser 35 40 45Asn Leu Pro Pro Ala Ala Gly Gly Ala Ala Asn Tyr Ser Arg Arg Asn 50 55 60Phe Asp Val Arg Ile Lys Ile Phe Met Leu Val Thr Ala Val Val Leu65 70 75 80Leu Cys Cys Ser Gly Val Ala Thr Ala Ala Pro Lys Thr Tyr Cys Glu 85 90 95Glu Leu Lys Gly Thr Asp Thr Gly Gln Ala Cys Gln Ile Gln Met Ser 100 105 110Asp Pro Ala Tyr Asn Ile Asn Ile Ser Leu Pro Ser Tyr Tyr Pro Asp 115 120 125Gln Lys Ser Leu Glu Asn Tyr Ile Ala Gln Thr Arg Asp Lys Phe Leu 130 135 140Ser Ala Ala Thr Ser Ser Thr Pro Arg Glu Ala Pro Tyr Glu Leu Asn145 150 155 160Ile Thr Ser Ala Thr Tyr Gln Ser Ala Ile Pro Pro Arg Gly Thr Gln 165 170 175Ala Val Val Leu Xaa Val Tyr His Asn Ala Gly Gly Thr His Pro Thr 180 185 190Thr Thr Tyr Lys Ala Phe Asp Trp Asp Gln Ala Tyr Arg Lys Pro Ile 195 200 205Thr Tyr Asp Thr Leu Trp Gln Ala Asp Thr Asp Pro Leu Pro Val Val 210 215 220Phe Pro Ile Val Ala Arg225 23066132PRTMycobacterium tuberculosis 66Thr Ala Ala Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly Gln Gly Phe1 5 10 15Ala Ile Pro Ile Gly Gln Ala Met Ala Ile Ala Gly Gln Ile Arg Ser 20 25 30Gly Gly Gly Ser Pro Thr Val His Ile Gly Pro Thr Ala Phe Leu Gly 35 40 45Leu Gly Val Val Asp Asn Asn Gly Asn Gly Ala Arg Val Gln Arg Val 50 55 60Val Gly Ser Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly Asp Val65 70 75 80Ile Thr Ala Val Asp Gly Ala Pro Ile Asn Ser Ala Thr Ala Met Ala 85 90 95Asp Ala Leu Asn Gly His His Pro Gly Asp Val Ile Ser Val Asn Trp 100 105

110Gln Thr Lys Ser Gly Gly Thr Arg Thr Gly Asn Val Thr Leu Ala Glu 115 120 125Gly Pro Pro Ala 13067100PRTMycobacterium tuberculosisMISC_FEATURE(1)..(100)ANY AMINO ACID 67Val Pro Leu Arg Ser Pro Ser Met Ser Pro Ser Lys Cys Leu Ala Ala1 5 10 15Ala Gln Arg Asn Pro Val Ile Arg Arg Arg Arg Leu Ser Asn Pro Pro 20 25 30Pro Arg Lys Tyr Arg Ser Met Pro Ser Pro Ala Thr Ala Ser Ala Gly 35 40 45Met Ala Arg Val Arg Arg Arg Ala Ile Trp Arg Gly Pro Ala Thr Xaa 50 55 60Ser Ala Gly Met Ala Arg Val Arg Arg Trp Xaa Val Met Pro Xaa Val65 70 75 80Ile Gln Ser Thr Xaa Ile Arg Xaa Xaa Gly Pro Phe Asp Asn Arg Gly 85 90 95Ser Glu Arg Lys 10068163PRTMycobacterium tuberculosisMISC_FEATURE(1)..(163)ANY AMINO ACID 68Met Thr Asp Asp Ile Leu Leu Ile Asp Thr Asp Glu Arg Val Arg Thr1 5 10 15Leu Thr Leu Asn Arg Pro Gln Ser Arg Asn Ala Leu Ser Ala Ala Leu 20 25 30Arg Asp Arg Phe Phe Ala Xaa Leu Xaa Asp Ala Glu Xaa Asp Asp Asp 35 40 45Ile Asp Val Val Ile Leu Thr Gly Ala Asp Pro Val Phe Cys Ala Gly 50 55 60Leu Asp Leu Lys Val Ala Gly Arg Ala Asp Arg Ala Ala Gly His Leu65 70 75 80Thr Ala Val Gly Gly His Asp Gln Ala Gly Asp Arg Arg Asp Gln Arg 85 90 95Arg Arg Gly His Arg Arg Ala Arg Thr Gly Ala Val Leu Arg His Pro 100 105 110Asp Arg Leu Arg Ala Arg Pro Leu Arg Arg His Pro Arg Pro Gly Gly 115 120 125Ala Ala Ala His Leu Gly Thr Gln Cys Val Leu Ala Ala Lys Gly Arg 130 135 140His Arg Xaa Gly Pro Val Asp Glu Pro Asp Arg Arg Leu Pro Val Arg145 150 155 160Asp Arg Arg69344PRTMycobacterium tuberculosis 69Met Lys Phe Val Asn His Ile Glu Pro Val Ala Pro Arg Arg Ala Gly1 5 10 15Gly Ala Val Ala Glu Val Tyr Ala Glu Ala Arg Arg Glu Phe Gly Arg 20 25 30Leu Pro Glu Pro Leu Ala Met Leu Ser Pro Asp Glu Gly Leu Leu Thr 35 40 45Ala Gly Trp Ala Thr Leu Arg Glu Thr Leu Leu Val Gly Gln Val Pro 50 55 60Arg Gly Arg Lys Glu Ala Val Ala Ala Ala Val Ala Ala Ser Leu Arg65 70 75 80Cys Pro Trp Cys Val Asp Ala His Thr Thr Met Leu Tyr Ala Ala Gly 85 90 95Gln Thr Asp Thr Ala Ala Ala Ile Leu Ala Gly Thr Ala Pro Ala Ala 100 105 110Gly Asp Pro Asn Ala Pro Tyr Val Ala Trp Ala Ala Gly Thr Gly Thr 115 120 125Pro Ala Gly Pro Pro Ala Pro Phe Gly Pro Asp Val Ala Ala Glu Tyr 130 135 140Leu Gly Thr Ala Val Gln Phe His Phe Ile Ala Arg Leu Val Leu Val145 150 155 160Leu Leu Asp Glu Thr Phe Leu Pro Gly Gly Pro Arg Ala Gln Gln Leu 165 170 175Met Arg Arg Ala Gly Gly Leu Val Phe Ala Arg Lys Val Arg Ala Glu 180 185 190His Arg Pro Gly Arg Ser Thr Arg Arg Leu Glu Pro Arg Thr Leu Pro 195 200 205Asp Asp Leu Ala Trp Ala Thr Pro Ser Glu Pro Ile Ala Thr Ala Phe 210 215 220Ala Ala Leu Ser His His Leu Asp Thr Ala Pro His Leu Pro Pro Pro225 230 235 240Thr Arg Gln Val Val Arg Arg Val Val Gly Ser Trp His Gly Glu Pro 245 250 255Met Pro Met Ser Ser Arg Trp Thr Asn Glu His Thr Ala Glu Leu Pro 260 265 270Ala Asp Leu His Ala Pro Thr Arg Leu Ala Leu Leu Thr Gly Leu Ala 275 280 285Pro His Gln Val Thr Asp Asp Asp Val Ala Ala Ala Arg Ser Leu Leu 290 295 300Asp Thr Asp Ala Ala Leu Val Gly Ala Leu Ala Trp Ala Ala Phe Thr305 310 315 320Ala Ala Arg Arg Ile Gly Thr Trp Ile Gly Ala Ala Ala Glu Gly Gln 325 330 335Val Ser Arg Gln Asn Pro Thr Gly 34070485PRTMycobacterium tuberculosis 70Asp Asp Pro Asp Met Pro Gly Thr Val Ala Lys Ala Val Ala Asp Ala1 5 10 15Leu Gly Arg Gly Ile Ala Pro Val Glu Asp Ile Gln Asp Cys Val Glu 20 25 30Ala Arg Leu Gly Glu Ala Gly Leu Asp Asp Val Ala Arg Val Tyr Ile 35 40 45Ile Tyr Arg Gln Arg Arg Ala Glu Leu Arg Thr Ala Lys Ala Leu Leu 50 55 60Gly Val Arg Asp Glu Leu Lys Leu Ser Leu Ala Ala Val Thr Val Leu65 70 75 80Arg Glu Arg Tyr Leu Leu His Asp Glu Gln Gly Arg Pro Ala Glu Ser 85 90 95Thr Gly Glu Leu Met Asp Arg Ser Ala Arg Cys Val Ala Ala Ala Glu 100 105 110Asp Gln Tyr Glu Pro Gly Ser Ser Arg Arg Trp Ala Glu Arg Phe Ala 115 120 125Thr Leu Leu Arg Asn Leu Glu Phe Leu Pro Asn Ser Pro Thr Leu Met 130 135 140Asn Ser Gly Thr Asp Leu Gly Leu Leu Ala Gly Cys Phe Val Leu Pro145 150 155 160Ile Glu Asp Ser Leu Gln Ser Ile Phe Ala Thr Leu Gly Gln Ala Ala 165 170 175Glu Leu Gln Arg Ala Gly Gly Gly Thr Gly Tyr Ala Phe Ser His Leu 180 185 190Arg Pro Ala Gly Asp Arg Val Ala Ser Thr Gly Gly Thr Ala Ser Gly 195 200 205Pro Val Ser Phe Leu Arg Leu Tyr Asp Ser Ala Ala Gly Val Val Ser 210 215 220Met Gly Gly Arg Arg Arg Gly Ala Cys Met Ala Val Leu Asp Val Ser225 230 235 240His Pro Asp Ile Cys Asp Phe Val Thr Ala Lys Ala Glu Ser Pro Ser 245 250 255Glu Leu Pro His Phe Asn Leu Ser Val Gly Val Thr Asp Ala Phe Leu 260 265 270Arg Ala Val Glu Arg Asn Gly Leu His Arg Leu Val Asn Pro Arg Thr 275 280 285Gly Lys Ile Val Ala Arg Met Pro Ala Ala Glu Leu Phe Asp Ala Ile 290 295 300Cys Lys Ala Ala His Ala Gly Gly Asp Pro Gly Leu Val Phe Leu Asp305 310 315 320Thr Ile Asn Arg Ala Asn Pro Val Pro Gly Arg Gly Arg Ile Glu Ala 325 330 335Thr Asn Pro Cys Gly Glu Val Pro Leu Leu Pro Tyr Glu Ser Cys Asn 340 345 350Leu Gly Ser Ile Asn Leu Ala Arg Met Leu Ala Asp Gly Arg Val Asp 355 360 365Trp Asp Arg Leu Glu Glu Val Ala Gly Val Ala Val Arg Phe Leu Asp 370 375 380Asp Val Ile Asp Val Ser Arg Tyr Pro Phe Pro Glu Leu Gly Glu Ala385 390 395 400Ala Arg Ala Thr Arg Lys Ile Gly Leu Gly Val Met Gly Leu Ala Glu 405 410 415Leu Leu Ala Ala Leu Gly Ile Pro Tyr Asp Ser Glu Glu Ala Val Arg 420 425 430Leu Ala Thr Arg Leu Met Arg Arg Ile Gln Gln Ala Ala His Thr Ala 435 440 445Ser Arg Arg Leu Ala Glu Glu Arg Gly Ala Phe Pro Ala Phe Thr Asp 450 455 460Ser Arg Phe Ala Arg Ser Gly Pro Arg Arg Asn Ala Gln Val Thr Ser465 470 475 480Val Ala Pro Thr Gly 48571267PRTMycobacterium tuberculosis 71Gly Val Ile Val Leu Asp Leu Glu Pro Arg Gly Pro Leu Pro Thr Glu1 5 10 15Ile Tyr Trp Arg Arg Arg Gly Leu Ala Leu Gly Ile Ala Val Val Val 20 25 30Val Gly Ile Ala Val Ala Ile Val Ile Ala Phe Val Asp Ser Ser Ala 35 40 45Gly Ala Lys Pro Val Ser Ala Asp Lys Pro Ala Ser Ala Gln Ser His 50 55 60Pro Gly Ser Pro Ala Pro Gln Ala Pro Gln Pro Ala Gly Gln Thr Glu65 70 75 80Gly Asn Ala Ala Ala Ala Pro Pro Gln Gly Gln Asn Pro Glu Thr Pro 85 90 95Thr Pro Thr Ala Ala Val Gln Pro Pro Pro Val Leu Lys Glu Gly Asp 100 105 110Asp Cys Pro Asp Ser Thr Leu Ala Val Lys Gly Leu Thr Asn Ala Pro 115 120 125Gln Tyr Tyr Val Gly Asp Gln Pro Lys Phe Thr Met Val Val Thr Asn 130 135 140Ile Gly Leu Val Ser Cys Lys Arg Asp Val Gly Ala Ala Val Leu Ala145 150 155 160Ala Tyr Val Tyr Ser Leu Asp Asn Lys Arg Leu Trp Ser Asn Leu Asp 165 170 175Cys Ala Pro Ser Asn Glu Thr Leu Val Lys Thr Phe Ser Pro Gly Glu 180 185 190Gln Val Thr Thr Ala Val Thr Trp Thr Gly Met Gly Ser Ala Pro Arg 195 200 205Cys Pro Leu Pro Arg Pro Ala Ile Gly Pro Gly Thr Tyr Asn Leu Val 210 215 220Val Gln Leu Gly Asn Leu Arg Ser Leu Pro Val Pro Phe Ile Leu Asn225 230 235 240Gln Pro Pro Pro Pro Pro Gly Pro Val Pro Ala Pro Gly Pro Ala Gln 245 250 255Ala Pro Pro Pro Glu Ser Pro Ala Gln Gly Gly 260 2657297PRTMycobacterium tuberculosis 72Leu Ile Ser Thr Gly Lys Ala Ser His Ala Ser Leu Gly Val Gln Val1 5 10 15Thr Asn Asp Lys Asp Thr Pro Gly Ala Lys Ile Val Glu Val Val Ala 20 25 30Gly Gly Ala Ala Ala Asn Ala Gly Val Pro Lys Gly Val Val Val Thr 35 40 45Lys Val Asp Asp Arg Pro Ile Asn Ser Ala Asp Ala Leu Val Ala Ala 50 55 60Val Arg Ser Lys Ala Pro Gly Ala Thr Val Ala Leu Thr Phe Gln Asp65 70 75 80Pro Ser Gly Gly Ser Arg Thr Val Gln Val Thr Leu Gly Lys Ala Glu 85 90 95Gln73364PRTMycobacterium tuberculosis 73Gly Ala Ala Val Ser Leu Leu Ala Ala Gly Thr Leu Val Leu Thr Ala1 5 10 15Cys Gly Gly Gly Thr Asn Ser Ser Ser Ser Gly Ala Gly Gly Thr Ser 20 25 30Gly Ser Val His Cys Gly Gly Lys Lys Glu Leu His Ser Ser Gly Ser 35 40 45Thr Ala Gln Glu Asn Ala Met Glu Gln Phe Val Tyr Ala Tyr Val Arg 50 55 60Ser Cys Pro Gly Tyr Thr Leu Asp Tyr Asn Ala Asn Gly Ser Gly Ala65 70 75 80Gly Val Thr Gln Phe Leu Asn Asn Glu Thr Asp Phe Ala Gly Ser Asp 85 90 95Val Pro Leu Asn Pro Ser Thr Gly Gln Pro Asp Arg Ser Ala Glu Arg 100 105 110Cys Gly Ser Pro Ala Trp Asp Leu Pro Thr Val Phe Gly Pro Ile Ala 115 120 125Ile Thr Tyr Asn Ile Lys Gly Val Ser Thr Leu Asn Leu Asp Gly Pro 130 135 140Thr Thr Ala Lys Ile Phe Asn Gly Thr Ile Thr Val Trp Asn Asp Pro145 150 155 160Gln Ile Gln Ala Leu Asn Ser Gly Thr Asp Leu Pro Pro Thr Pro Ile 165 170 175Ser Val Ile Phe Arg Ser Asp Lys Ser Gly Thr Ser Asp Asn Phe Gln 180 185 190Lys Tyr Leu Asp Gly Val Ser Asn Gly Ala Trp Gly Lys Gly Ala Ser 195 200 205Glu Thr Phe Ser Gly Gly Val Gly Val Gly Ala Ser Gly Asn Asn Gly 210 215 220Thr Ser Ala Leu Leu Gln Thr Thr Asp Gly Ser Ile Thr Tyr Asn Glu225 230 235 240Trp Ser Phe Ala Val Gly Lys Gln Leu Asn Met Ala Gln Ile Ile Thr 245 250 255Ser Ala Gly Pro Asp Pro Val Ala Ile Thr Thr Glu Ser Val Gly Lys 260 265 270Thr Ile Ala Gly Ala Lys Ile Met Gly Gln Gly Asn Asp Leu Val Leu 275 280 285Asp Thr Ser Ser Phe Tyr Arg Pro Thr Gln Pro Gly Ser Tyr Pro Ile 290 295 300Val Leu Ala Thr Tyr Glu Ile Val Cys Ser Lys Tyr Pro Asp Ala Thr305 310 315 320Thr Gly Thr Ala Val Arg Ala Phe Met Gln Ala Ala Ile Gly Pro Gly 325 330 335Gln Glu Gly Leu Asp Gln Tyr Gly Ser Ile Pro Leu Pro Lys Ser Phe 340 345 350Gln Ala Lys Leu Ala Ala Ala Val Asn Ala Ile Ser 355 36074309PRTMycobacterium tuberculosis 74Gln Ala Ala Ala Gly Arg Ala Val Arg Arg Thr Gly His Ala Glu Asp1 5 10 15Gln Thr His Gln Asp Arg Leu His His Gly Cys Arg Arg Ala Ala Val 20 25 30Val Val Arg Gln Asp Arg Ala Ser Val Ser Ala Thr Ser Ala Arg Pro 35 40 45Pro Arg Arg His Pro Ala Gln Gly His Arg Arg Arg Val Ala Pro Ser 50 55 60Gly Gly Arg Arg Arg Pro His Pro His His Val Gln Pro Asp Asp Arg65 70 75 80Arg Asp Arg Pro Ala Leu Leu Asp Arg Thr Gln Pro Ala Glu His Pro 85 90 95Asp Pro His Arg Arg Gly Pro Ala Asp Pro Gly Arg Val Arg Gly Arg 100 105 110Gly Arg Leu Arg Arg Val Asp Asp Gly Arg Leu Gln Pro Asp Arg Asp 115 120 125Ala Asp His Gly Ala Pro Val Arg Gly Arg Gly Pro His Arg Gly Val 130 135 140Gln His Arg Gly Gly Pro Val Phe Val Arg Arg Val Pro Gly Val Arg145 150 155 160Cys Ala His Arg Arg Gly His Arg Arg Val Ala Ala Pro Gly Gln Gly 165 170 175Asp Val Leu Arg Ala Gly Leu Arg Val Glu Arg Leu Arg Pro Val Ala 180 185 190Ala Val Glu Asn Leu His Arg Gly Ser Gln Arg Ala Asp Gly Arg Val 195 200 205Phe Arg Pro Ile Arg Arg Gly Ala Arg Leu Pro Ala Arg Arg Ser Arg 210 215 220Ala Gly Pro Gln Gly Arg Leu His Leu Asp Gly Ala Gly Pro Ser Pro225 230 235 240Leu Pro Ala Arg Ala Gly Gln Gln Gln Pro Ser Ser Ala Gly Gly Arg 245 250 255Arg Ala Gly Gly Ala Glu Arg Ala Asp Pro Gly Gln Arg Gly Arg His 260 265 270His Gln Gly Gly His Asp Pro Gly Arg Gln Gly Ala Gln Arg Gly Thr 275 280 285Ala Gly Val Ala His Ala Ala Ala Gly Pro Arg Arg Ala Ala Val Arg 290 295 300Asn Arg Pro Arg Arg30575580PRTMycobacterium tuberculosis 75Ser Ala Val Trp Cys Leu Asn Gly Phe Thr Gly Arg His Arg His Gly1 5 10 15Arg Cys Arg Val Arg Ala Ser Gly Trp Arg Ser Ser Asn Arg Trp Cys 20 25 30Ser Thr Thr Ala Asp Cys Cys Ala Ser Lys Thr Pro Thr Gln Ala Ala 35 40 45Ser Pro Leu Glu Arg Arg Phe Thr Cys Cys Ser Pro Ala Val Gly Cys 50 55 60Arg Phe Arg Ser Phe Pro Val Arg Arg Leu Ala Leu Gly Ala Arg Thr65 70 75 80Ser Arg Thr Leu Gly Val Arg Arg Thr Leu Ser Gln Trp Asn Leu Ser 85 90 95Pro Arg Ala Gln Pro Ser Cys Ala Val Thr Val Glu Ser His Thr His 100 105 110Ala Ser Pro Arg Met Ala Lys Leu Ala Arg Val Val Gly Leu Val Gln 115 120 125Glu Glu Gln Pro Ser Asp Met Thr Asn His Pro Arg Tyr Ser Pro Pro 130 135 140Pro Gln Gln Pro Gly Thr Pro Gly Tyr Ala Gln Gly Gln Gln Gln Thr145 150 155 160Tyr Ser Gln Gln Phe Asp Trp Arg Tyr Pro Pro Ser Pro Pro Pro Gln 165 170 175Pro Thr Gln Tyr Arg Gln Pro Tyr Glu Ala Leu Gly Gly Thr Arg Pro 180 185 190Gly Leu Ile Pro Gly Val Ile Pro Thr Met Thr Pro Pro Pro Gly Met 195 200 205Val Arg Gln Arg Pro Arg Ala Gly Met Leu Ala Ile Gly Ala Val Thr 210 215 220Ile Ala Val Val Ser Ala Gly Ile Gly Gly Ala Ala Ala Ser Leu Val225 230 235 240Gly Phe Asn Arg Ala Pro Ala Gly Pro Ser Gly Gly Pro Val Ala Ala 245 250 255Ser Ala Ala Pro Ser Ile Pro Ala Ala Asn Met Pro Pro Gly Ser Val 260 265 270Glu Gln Val Ala Ala Lys Val Val Pro Ser Val Val Met Leu

Glu Thr 275 280 285Asp Leu Gly Arg Gln Ser Glu Glu Gly Ser Gly Ile Ile Leu Ser Ala 290 295 300Glu Gly Leu Ile Leu Thr Asn Asn His Val Ile Ala Ala Ala Ala Lys305 310 315 320Pro Pro Leu Gly Ser Pro Pro Pro Lys Thr Thr Val Thr Phe Ser Asp 325 330 335Gly Arg Thr Ala Pro Phe Thr Val Val Gly Ala Asp Pro Thr Ser Asp 340 345 350Ile Ala Val Val Arg Val Gln Gly Val Ser Gly Leu Thr Pro Ile Ser 355 360 365Leu Gly Ser Ser Ser Asp Leu Arg Val Gly Gln Pro Val Leu Ala Ile 370 375 380Gly Ser Pro Leu Gly Leu Glu Gly Thr Val Thr Thr Gly Ile Val Ser385 390 395 400Ala Leu Asn Arg Pro Val Ser Thr Thr Gly Glu Ala Gly Asn Gln Asn 405 410 415Thr Val Leu Asp Ala Ile Gln Thr Asp Ala Ala Ile Asn Pro Gly Asn 420 425 430Ser Gly Gly Ala Leu Val Asn Met Asn Ala Gln Leu Val Gly Val Asn 435 440 445Ser Ala Ile Ala Thr Leu Gly Ala Asp Ser Ala Asp Ala Gln Ser Gly 450 455 460Ser Ile Gly Leu Gly Phe Ala Ile Pro Val Asp Gln Ala Lys Arg Ile465 470 475 480Ala Asp Glu Leu Ile Ser Thr Gly Lys Ala Ser His Ala Ser Leu Gly 485 490 495Val Gln Val Thr Asn Asp Lys Asp Thr Pro Gly Ala Lys Ile Val Glu 500 505 510Val Val Ala Gly Gly Ala Ala Ala Asn Ala Gly Val Pro Lys Gly Val 515 520 525Val Val Thr Lys Val Asp Asp Arg Pro Ile Asn Ser Ala Asp Ala Leu 530 535 540Val Ala Ala Val Arg Ser Lys Ala Pro Gly Ala Thr Val Ala Leu Thr545 550 555 560Phe Gln Asp Pro Ser Gly Gly Ser Arg Thr Val Gln Val Thr Leu Gly 565 570 575Lys Ala Glu Gln 58076233PRTMycobacterium tuberculosis 76Met Asn Asp Gly Lys Arg Ala Val Thr Ser Ala Val Leu Val Val Leu1 5 10 15Gly Ala Cys Leu Ala Leu Trp Leu Ser Gly Cys Ser Ser Pro Lys Pro 20 25 30Asp Ala Glu Glu Gln Gly Val Pro Val Ser Pro Thr Ala Ser Asp Pro 35 40 45Ala Leu Leu Ala Glu Ile Arg Gln Ser Leu Asp Ala Thr Lys Gly Leu 50 55 60Thr Ser Val His Val Ala Val Arg Thr Thr Gly Lys Val Asp Ser Leu65 70 75 80Leu Gly Ile Thr Ser Ala Asp Val Asp Val Arg Ala Asn Pro Leu Ala 85 90 95Ala Lys Gly Val Cys Thr Tyr Asn Asp Glu Gln Gly Val Pro Phe Arg 100 105 110Val Gln Gly Asp Asn Ile Ser Val Lys Leu Phe Asp Asp Trp Ser Asn 115 120 125Leu Gly Ser Ile Ser Glu Leu Ser Thr Ser Arg Val Leu Asp Pro Ala 130 135 140Ala Gly Val Thr Gln Leu Leu Ser Gly Val Thr Asn Leu Gln Ala Gln145 150 155 160Gly Thr Glu Val Ile Asp Gly Ile Ser Thr Thr Lys Ile Thr Gly Thr 165 170 175Ile Pro Ala Ser Ser Val Lys Met Leu Asp Pro Gly Ala Lys Ser Ala 180 185 190Arg Pro Ala Thr Val Trp Ile Ala Gln Asp Gly Ser His His Leu Val 195 200 205Arg Ala Ser Ile Asp Leu Gly Ser Gly Ser Ile Gln Leu Thr Gln Ser 210 215 220Lys Trp Asn Glu Pro Val Asn Val Asp225 2307766PRTMycobacterium tuberculosis 77Val Ile Asp Ile Ile Gly Thr Ser Pro Thr Ser Trp Glu Gln Ala Ala1 5 10 15Ala Glu Ala Val Gln Arg Ala Arg Asp Ser Val Asp Asp Ile Arg Val 20 25 30Ala Arg Val Ile Glu Gln Asp Met Ala Val Asp Ser Ala Gly Lys Ile 35 40 45Thr Tyr Arg Ile Lys Leu Glu Val Ser Phe Lys Met Arg Pro Ala Gln 50 55 60Pro Arg657869PRTMycobacterium tuberculosis 78Val Pro Pro Ala Pro Pro Leu Pro Pro Leu Pro Pro Ser Pro Ile Ser1 5 10 15Cys Ala Ser Pro Pro Ser Pro Pro Leu Pro Pro Ala Pro Pro Val Ala 20 25 30Pro Gly Pro Pro Met Pro Pro Leu Asp Pro Trp Pro Pro Ala Pro Pro 35 40 45Leu Pro Tyr Ser Thr Pro Pro Gly Ala Pro Leu Pro Pro Ser Pro Pro 50 55 60Ser Pro Pro Leu Pro6579355PRTMycobacterium tuberculosis 79Met Ser Asn Ser Arg Arg Arg Ser Leu Arg Trp Ser Trp Leu Leu Ser1 5 10 15Val Leu Ala Ala Val Gly Leu Gly Leu Ala Thr Ala Pro Ala Gln Ala 20 25 30Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro Ala Leu 35 40 45Pro Leu Asp Pro Ser Ala Met Val Ala Gln Val Ala Pro Gln Val Val 50 55 60Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn Ala Val Gly Ala Gly Thr65 70 75 80Gly Ile Val Ile Asp Pro Asn Gly Val Val Leu Thr Asn Asn His Val 85 90 95Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe Ser Val Gly Ser Gly Gln 100 105 110Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr Gln Asp Val Ala 115 120 125Val Leu Gln Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala Ala Ile Gly 130 135 140Gly Gly Val Ala Val Gly Glu Pro Val Val Ala Met Gly Asn Ser Gly145 150 155 160Gly Gln Gly Gly Thr Pro Arg Ala Val Pro Gly Arg Val Val Ala Leu 165 170 175Gly Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu Glu Thr 180 185 190Leu Asn Gly Leu Ile Gln Phe Asp Ala Ala Ile Gln Pro Gly Asp Ser 195 200 205Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val Gly Met Asn Thr 210 215 220Ala Ala Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly Gln Gly Phe Ala225 230 235 240Ile Pro Ile Gly Gln Ala Met Ala Ile Ala Gly Gln Ile Arg Ser Gly 245 250 255Gly Gly Ser Pro Thr Val His Ile Gly Pro Thr Ala Phe Leu Gly Leu 260 265 270Gly Val Val Asp Asn Asn Gly Asn Gly Ala Arg Val Gln Arg Val Val 275 280 285Gly Ser Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly Asp Val Ile 290 295 300Thr Ala Val Asp Gly Ala Pro Ile Asn Ser Ala Thr Ala Met Ala Asp305 310 315 320Ala Leu Asn Gly His His Pro Gly Asp Val Ile Ser Val Asn Trp Gln 325 330 335Thr Lys Ser Gly Gly Thr Arg Thr Gly Asn Val Thr Leu Ala Glu Gly 340 345 350Pro Pro Ala 35580205PRTMycobacterium tuberculosis 80Ser Pro Lys Pro Asp Ala Glu Glu Gln Gly Val Pro Val Ser Pro Thr1 5 10 15Ala Ser Asp Pro Ala Leu Leu Ala Glu Ile Arg Gln Ser Leu Asp Ala 20 25 30Thr Lys Gly Leu Thr Ser Val His Val Ala Val Arg Thr Thr Gly Lys 35 40 45Val Asp Ser Leu Leu Gly Ile Thr Ser Ala Asp Val Asp Val Arg Ala 50 55 60Asn Pro Leu Ala Ala Lys Gly Val Cys Thr Tyr Asn Asp Glu Gln Gly65 70 75 80Val Pro Phe Arg Val Gln Gly Asp Asn Ile Ser Val Lys Leu Phe Asp 85 90 95Asp Trp Ser Asn Leu Gly Ser Ile Ser Glu Leu Ser Thr Ser Arg Val 100 105 110Leu Asp Pro Ala Ala Gly Val Thr Gln Leu Leu Ser Gly Val Thr Asn 115 120 125Leu Gln Ala Gln Gly Thr Glu Val Ile Asp Gly Ile Ser Thr Thr Lys 130 135 140Ile Thr Gly Thr Ile Pro Ala Ser Ser Val Lys Met Leu Asp Pro Gly145 150 155 160Ala Lys Ser Ala Arg Pro Ala Thr Val Trp Ile Ala Gln Asp Gly Ser 165 170 175His His Leu Val Arg Ala Ser Ile Asp Leu Gly Ser Gly Ser Ile Gln 180 185 190Leu Thr Gln Ser Lys Trp Asn Glu Pro Val Asn Val Asp 195 200 20581286PRTMycobacterium tuberculosisMISC_FEATURE(1)..(285)ANY AMINO ACID 81Gly Asp Ser Phe Trp Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Val1 5 10 15Leu Gly Ala Thr Ala Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln 20 25 30His Ala Asp Gly His Ser Leu Leu Leu Asp Ala Thr Asn Pro Ala Val 35 40 45Val Ala Tyr Asp Pro Ala Phe Ala Tyr Glu Ile Gly Tyr Ile Xaa Glu 50 55 60Ser Gly Leu Ala Arg Met Cys Gly Glu Asn Pro Glu Asn Ile Phe Phe65 70 75 80Tyr Ile Thr Val Tyr Asn Glu Pro Tyr Val Gln Pro Pro Glu Pro Glu 85 90 95Asn Phe Asp Pro Glu Gly Val Leu Gly Gly Ile Tyr Arg Tyr His Ala 100 105 110Ala Thr Glu Gln Arg Thr Asn Lys Xaa Gln Ile Leu Ala Ser Gly Val 115 120 125Ala Met Pro Ala Ala Leu Arg Ala Ala Gln Met Leu Ala Ala Glu Trp 130 135 140Asp Val Ala Ala Asp Val Trp Ser Val Thr Ser Trp Gly Glu Leu Asn145 150 155 160Arg Asp Gly Val Val Ile Glu Thr Glu Lys Leu Arg His Pro Asp Arg 165 170 175Pro Ala Gly Val Pro Tyr Val Thr Arg Ala Leu Glu Asn Ala Arg Gly 180 185 190Pro Val Ile Ala Val Ser Asp Trp Met Arg Ala Val Pro Glu Gln Ile 195 200 205Arg Pro Trp Val Pro Gly Thr Tyr Leu Thr Leu Gly Thr Asp Gly Phe 210 215 220Gly Phe Ser Asp Thr Arg Pro Ala Gly Arg Arg Tyr Phe Asn Thr Asp225 230 235 240Ala Glu Ser Gln Val Gly Arg Gly Phe Gly Arg Gly Trp Pro Gly Arg 245 250 255Arg Val Asn Ile Asp Pro Phe Gly Ala Gly Arg Gly Pro Pro Ala Gln 260 265 270Leu Pro Gly Phe Asp Glu Gly Gly Gly Leu Arg Pro Xaa Lys 275 280 28582173PRTMycobacterium tuberculosis 82Thr Lys Phe His Ala Leu Met Gln Glu Gln Ile His Asn Glu Phe Thr1 5 10 15Ala Ala Gln Gln Tyr Val Ala Ile Ala Val Tyr Phe Asp Ser Glu Asp 20 25 30Leu Pro Gln Leu Ala Lys His Phe Tyr Ser Gln Ala Val Glu Glu Arg 35 40 45Asn His Ala Met Met Leu Val Gln His Leu Leu Asp Arg Asp Leu Arg 50 55 60Val Glu Ile Pro Gly Val Asp Thr Val Arg Asn Gln Phe Asp Arg Pro65 70 75 80Arg Glu Ala Leu Ala Leu Ala Leu Asp Gln Glu Arg Thr Val Thr Asp 85 90 95Gln Val Gly Arg Leu Thr Ala Val Ala Arg Asp Glu Gly Asp Phe Leu 100 105 110Gly Glu Gln Phe Met Gln Trp Phe Leu Gln Glu Gln Ile Glu Glu Val 115 120 125Ala Leu Met Ala Thr Leu Val Arg Val Ala Asp Arg Ala Gly Ala Asn 130 135 140Leu Phe Glu Leu Glu Asn Phe Val Ala Arg Glu Val Asp Val Ala Pro145 150 155 160Ala Ala Ser Gly Ala Pro His Ala Ala Gly Gly Arg Leu 165 17083107PRTMycobacterium tuberculosisMISC_FEATURE(1)..(107)ANY AMINO ACID 83Arg Ala Asp Glu Arg Leu Asn Thr Thr Met Lys Met Val Lys Ser Ile1 5 10 15Ala Ala Gly Leu Thr Ala Ala Ala Ala Ile Gly Ala Ala Ala Ala Gly 20 25 30Val Thr Ser Ile Met Ala Gly Gly Pro Val Val Tyr Gln Met Gln Pro 35 40 45Val Val Phe Gly Ala Pro Leu Pro Leu Asp Pro Xaa Ser Ala Pro Xaa 50 55 60Val Pro Thr Ala Ala Gln Trp Thr Xaa Leu Leu Asn Xaa Leu Xaa Asp65 70 75 80Pro Asn Val Ser Phe Xaa Asn Lys Gly Ser Leu Val Glu Gly Gly Ile 85 90 95Gly Gly Xaa Glu Gly Xaa Xaa Arg Arg Xaa Gln 100 10584125PRTMycobacterium tuberculosisMISC_FEATURE(1)..(125)ANY AMINO ACID 84Val Leu Ser Val Pro Val Gly Asp Gly Phe Trp Xaa Arg Val Val Asn1 5 10 15Pro Leu Gly Gln Pro Ile Asp Gly Arg Gly Asp Val Asp Ser Asp Thr 20 25 30Arg Arg Ala Leu Glu Leu Gln Ala Pro Ser Val Val Xaa Arg Gln Gly 35 40 45Val Lys Glu Pro Leu Xaa Thr Gly Ile Lys Ala Ile Asp Ala Met Thr 50 55 60Pro Ile Gly Arg Gly Gln Arg Gln Leu Ile Ile Gly Asp Arg Lys Thr65 70 75 80Gly Lys Asn Arg Arg Leu Cys Arg Thr Pro Ser Ser Asn Gln Arg Glu 85 90 95Glu Leu Gly Val Arg Trp Ile Pro Arg Ser Arg Cys Ala Cys Val Tyr 100 105 110Val Gly His Arg Ala Arg Arg Gly Thr Tyr His Arg Arg 115 120 12585117PRTMycobacterium tuberculosisMISC_FEATURE(1)..(117)ANY AMINO ACID 85Cys Asp Ala Val Met Gly Phe Leu Gly Gly Ala Gly Pro Leu Ala Val1 5 10 15Val Asp Gln Gln Leu Val Thr Arg Val Pro Gln Gly Trp Ser Phe Ala 20 25 30Gln Ala Ala Ala Val Pro Val Val Phe Leu Thr Ala Trp Tyr Gly Leu 35 40 45Ala Asp Leu Ala Glu Ile Lys Ala Gly Glu Ser Val Leu Ile His Ala 50 55 60Gly Thr Gly Gly Val Gly Met Ala Ala Val Gln Leu Ala Arg Gln Trp65 70 75 80Gly Val Glu Val Phe Val Thr Ala Ser Arg Gly Lys Trp Asp Thr Leu 85 90 95Arg Ala Xaa Xaa Phe Asp Asp Xaa Pro Tyr Arg Xaa Phe Pro His Xaa 100 105 110Arg Ser Ser Xaa Gly 11586103PRTMycobacterium tuberculosis 86Met Tyr Arg Phe Ala Cys Arg Thr Leu Met Leu Ala Ala Cys Ile Leu1 5 10 15Ala Thr Gly Val Ala Gly Leu Gly Val Gly Ala Gln Ser Ala Ala Gln 20 25 30Thr Ala Pro Val Pro Asp Tyr Tyr Trp Cys Pro Gly Gln Pro Phe Asp 35 40 45Pro Ala Trp Gly Pro Asn Trp Asp Pro Tyr Thr Cys His Asp Asp Phe 50 55 60His Arg Asp Ser Asp Gly Pro Asp His Ser Arg Asp Tyr Pro Gly Pro65 70 75 80Ile Leu Glu Gly Pro Val Leu Asp Asp Pro Gly Ala Ala Pro Pro Pro 85 90 95Pro Ala Ala Gly Gly Gly Ala 1008788PRTMycobacterium tuberculosis 87Val Gln Cys Arg Val Trp Leu Glu Ile Gln Trp Arg Gly Met Leu Gly1 5 10 15Ala Asp Gln Ala Arg Ala Gly Gly Pro Ala Arg Ile Trp Arg Glu His 20 25 30Ser Met Ala Ala Met Lys Pro Arg Thr Gly Asp Gly Pro Leu Glu Ala 35 40 45Thr Lys Glu Gly Arg Gly Ile Val Met Arg Val Pro Leu Glu Gly Gly 50 55 60Gly Arg Leu Val Val Glu Leu Thr Pro Asp Glu Ala Ala Ala Leu Gly65 70 75 80Asp Glu Leu Lys Gly Val Thr Ser 858895PRTMycobacterium tuberculosis 88Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly Asn Phe Glu Arg Ile1 5 10 15Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val Glu Ser Thr Ala Gly 20 25 30Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly Thr Ala Ala Gln Ala 35 40 45Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys Gln Lys Gln Glu Leu 50 55 60Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly Val Gln Tyr Ser Arg65 70 75 80Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser Gln Met Gly Phe 85 90 9589166PRTMycobacterium tuberculosisMISC_FEATURE(1)..(165)ANY AMINO ACID 89Met Thr Gln Ser Gln Thr Val Thr Val Asp Gln Gln Glu Ile Leu Asn1 5 10 15Arg Ala Asn Glu Val Glu Ala Pro Met Ala Asp Pro Pro Thr Asp Val 20 25 30Pro Ile Thr Pro Cys Glu Leu Thr Xaa Xaa Lys Asn Ala Ala Gln Gln 35 40 45Xaa Val Leu Ser Ala Asp Asn Met Arg Glu Tyr Leu Ala Ala Gly Ala 50 55

60Lys Glu Arg Gln Arg Leu Ala Thr Ser Leu Arg Asn Ala Ala Lys Xaa65 70 75 80Tyr Gly Glu Val Asp Glu Glu Ala Ala Thr Ala Leu Asp Asn Asp Gly 85 90 95Glu Gly Thr Val Gln Ala Glu Ser Ala Gly Ala Val Gly Gly Asp Ser 100 105 110Ser Ala Glu Leu Thr Asp Thr Pro Arg Val Ala Thr Ala Gly Glu Pro 115 120 125Asn Phe Met Asp Leu Lys Glu Ala Ala Arg Lys Leu Glu Thr Gly Asp 130 135 140Gln Gly Ala Ser Leu Ala His Xaa Gly Asp Gly Trp Asn Thr Xaa Thr145 150 155 160Leu Thr Leu Gln Gly Asp 165905PRTMycobacterium tuberculosis 90Arg Ala Glu Arg Met1 591263PRTMycobacterium tuberculosisMISC_FEATURE(1)..(263)ANY AMINO ACID 91Val Ala Trp Met Ser Val Thr Ala Gly Gln Ala Glu Leu Thr Ala Ala1 5 10 15Gln Val Arg Val Ala Ala Ala Ala Tyr Glu Thr Ala Tyr Gly Leu Thr 20 25 30Val Pro Pro Pro Val Ile Ala Glu Asn Arg Ala Glu Leu Met Ile Leu 35 40 45Ile Ala Thr Asn Leu Leu Gly Gln Asn Thr Pro Ala Ile Ala Val Asn 50 55 60Glu Ala Glu Tyr Gly Glu Met Trp Ala Gln Asp Ala Ala Ala Met Phe65 70 75 80Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr Ala Thr Leu Leu Pro Phe 85 90 95Glu Glu Ala Pro Glu Met Thr Ser Ala Gly Gly Leu Leu Glu Gln Ala 100 105 110Ala Ala Val Glu Glu Ala Ser Asp Thr Ala Ala Ala Asn Gln Leu Met 115 120 125Asn Asn Val Pro Gln Ala Leu Lys Gln Leu Ala Gln Pro Thr Gln Gly 130 135 140Thr Thr Pro Ser Ser Lys Leu Gly Gly Leu Trp Lys Thr Val Ser Pro145 150 155 160His Arg Ser Pro Ile Ser Asn Met Val Ser Met Ala Asn Asn His Met 165 170 175Ser Met Thr Asn Ser Gly Val Ser Met Thr Asn Thr Leu Ser Ser Met 180 185 190Leu Lys Gly Phe Ala Pro Ala Ala Ala Ala Gln Ala Val Gln Thr Ala 195 200 205Ala Gln Asn Gly Val Arg Ala Met Ser Ser Leu Gly Ser Ser Leu Gly 210 215 220Ser Ser Gly Leu Gly Gly Gly Val Ala Ala Asn Leu Gly Arg Ala Ala225 230 235 240Ser Val Arg Tyr Gly His Arg Asp Gly Gly Lys Tyr Ala Xaa Ser Gly 245 250 255Arg Arg Asn Gly Gly Pro Ala 26092303PRTMycobacterium tuberculosis 92Met Thr Tyr Ser Pro Gly Asn Pro Gly Tyr Pro Gln Ala Gln Pro Ala1 5 10 15Gly Ser Tyr Gly Gly Val Thr Pro Ser Phe Ala His Ala Asp Glu Gly 20 25 30Ala Ser Lys Leu Pro Met Tyr Leu Asn Ile Ala Val Ala Val Leu Gly 35 40 45Leu Ala Ala Tyr Phe Ala Ser Phe Gly Pro Met Phe Thr Leu Ser Thr 50 55 60Glu Leu Gly Gly Gly Asp Gly Ala Val Ser Gly Asp Thr Gly Leu Pro65 70 75 80Val Gly Val Ala Leu Leu Ala Ala Leu Leu Ala Gly Val Val Leu Val 85 90 95Pro Lys Ala Lys Ser His Val Thr Val Val Ala Val Leu Gly Val Leu 100 105 110Gly Val Phe Leu Met Val Ser Ala Thr Phe Asn Lys Pro Ser Ala Tyr 115 120 125Ser Thr Gly Trp Ala Leu Trp Val Val Leu Ala Phe Ile Val Phe Gln 130 135 140Ala Val Ala Ala Val Leu Ala Leu Leu Val Glu Thr Gly Ala Ile Thr145 150 155 160Ala Pro Ala Pro Arg Pro Lys Phe Asp Pro Tyr Gly Gln Tyr Gly Arg 165 170 175Tyr Gly Gln Tyr Gly Gln Tyr Gly Val Gln Pro Gly Gly Tyr Tyr Gly 180 185 190Gln Gln Gly Ala Gln Gln Ala Ala Gly Leu Gln Ser Pro Gly Pro Gln 195 200 205Gln Ser Pro Gln Pro Pro Gly Tyr Gly Ser Gln Tyr Gly Gly Tyr Ser 210 215 220Ser Ser Pro Ser Gln Ser Gly Ser Gly Tyr Thr Ala Gln Pro Pro Ala225 230 235 240Gln Pro Pro Ala Gln Ser Gly Ser Gln Gln Ser His Gln Gly Pro Ser 245 250 255Thr Pro Pro Thr Gly Phe Pro Ser Phe Ser Pro Pro Pro Pro Val Ser 260 265 270Ala Gly Thr Gly Ser Gln Ala Gly Ser Ala Pro Val Asn Tyr Ser Asn 275 280 285Pro Ser Gly Gly Glu Gln Ser Ser Ser Pro Gly Gly Ala Pro Val 290 295 3009328PRTMycobacterium tuberculosis 93Gly Cys Gly Glu Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly Asn1 5 10 15Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile 20 259416PRTMycobacterium tuberculosis 94Asp Gln Val Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly1 5 10 159527PRTMycobacterium tuberculosis 95Gly Cys Gly Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala1 5 10 15Ala Gly Thr Ala Ala Gln Ala Ala Val Val Arg 20 259627PRTMycobacterium tuberculosis 96Gly Cys Gly Gly Thr Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu1 5 10 15Ala Ala Asn Lys Gln Lys Gln Glu Leu Asp Glu 20 259727PRTMycobacterium tuberculosis 97Gly Cys Gly Ala Asn Lys Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr1 5 10 15Asn Ile Arg Gln Ala Gly Val Gln Tyr Ser Arg 20 259828PRTMycobacterium tuberculosis 98Gly Cys Gly Ile Arg Gln Ala Gly Val Gln Tyr Ser Arg Ala Asp Glu1 5 10 15Glu Gln Gln Gln Ala Leu Ser Ser Gln Met Gly Phe 20 2599507DNAMycobacterium tuberculosis 99atgaagatgg tgaaatcgat cgccgcaggt ctgaccgccg cggctgcaat cggcgccgct 60gcggccggtg tgacttcgat catggctggc ggcccggtcg tataccagat gcagccggtc 120gtcttcggcg cgccactgcc gttggacccg gcatccgccc ctgacgtccc gaccgccgcc 180cagttgacca gcctgctcaa cagcctcgcc gatcccaacg tgtcgtttgc gaacaagggc 240agtctggtcg agggcggcat cgggggcacc gaggcgcgca tcgccgacca caagctgaag 300aaggccgccg agcacgggga tctgccgctg tcgttcagcg tgacgaacat ccagccggcg 360gccgccggtt cggccaccgc cgacgtttcc gtctcgggtc cgaagctctc gtcgccggtc 420acgcagaacg tcacgttcgt gaatcaaggc ggctggatgc tgtcacgcgc atcggcgatg 480gagttgctgc aggccgcagg gaactga 507100168PRTMycobacterium tuberculosis 100Met Lys Met Val Lys Ser Ile Ala Ala Gly Leu Thr Ala Ala Ala Ala1 5 10 15Ile Gly Ala Ala Ala Ala Gly Val Thr Ser Ile Met Ala Gly Gly Pro 20 25 30Val Val Tyr Gln Met Gln Pro Val Val Phe Gly Ala Pro Leu Pro Leu 35 40 45Asp Pro Ala Ser Ala Pro Asp Val Pro Thr Ala Ala Gln Leu Thr Ser 50 55 60Leu Leu Asn Ser Leu Ala Asp Pro Asn Val Ser Phe Ala Asn Lys Gly65 70 75 80Ser Leu Val Glu Gly Gly Ile Gly Gly Thr Glu Ala Arg Ile Ala Asp 85 90 95His Lys Leu Lys Lys Ala Ala Glu His Gly Asp Leu Pro Leu Ser Phe 100 105 110Ser Val Thr Asn Ile Gln Pro Ala Ala Ala Gly Ser Ala Thr Ala Asp 115 120 125Val Ser Val Ser Gly Pro Lys Leu Ser Ser Pro Val Thr Gln Asn Val 130 135 140Thr Phe Val Asn Gln Gly Gly Trp Met Leu Ser Arg Ala Ser Ala Met145 150 155 160Glu Leu Leu Gln Ala Ala Gly Asn 165101500DNAMycobacterium tuberculosis 101cgtggcaatg tcgttgaccg tcggggccgg ggtcgcctcc gcagatcccg tggacgcggt 60cattaacacc acctgcaatt acgggcaggt agtagctgcg ctcaacgcga cggatccggg 120ggctgccgca cagttcaacg cctcaccggt ggcgcagtcc tatttgcgca atttcctcgc 180cgcaccgcca cctcagcgcg ctgccatggc cgcgcaattg caagctgtgc cgggggcggc 240acagtacatc ggccttgtcg agtcggttgc cggctcctgc aacaactatt aagcccatgc 300gggccccatc ccgcgacccg gcatcgtcgc cggggctagg ccagattgcc ccgctcctca 360acgggccgca tcccgcgacc cggcatcgtc gccggggcta ggccagattg ccccgctcct 420caacgggccg catctcgtgc cgaattcctg cagcccgggg gatccactag ttctagagcg 480gccgccaccg cggtggagct 50010296PRTMycobacterium tuberculosis 102Val Ala Met Ser Leu Thr Val Gly Ala Gly Val Ala Ser Ala Asp Pro1 5 10 15Val Asp Ala Val Ile Asn Thr Thr Cys Asn Tyr Gly Gln Val Val Ala 20 25 30Ala Leu Asn Ala Thr Asp Pro Gly Ala Ala Ala Gln Phe Asn Ala Ser 35 40 45Pro Val Ala Gln Ser Tyr Leu Arg Asn Phe Leu Ala Ala Pro Pro Pro 50 55 60Gln Arg Ala Ala Met Ala Ala Gln Leu Gln Ala Val Pro Gly Ala Ala65 70 75 80Gln Tyr Ile Gly Leu Val Glu Ser Val Ala Gly Ser Cys Asn Asn Tyr 85 90 95103154DNAMycobacterium tuberculosis 103atgacagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccaggga 60aatgtcacgt ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca 120gcggcctggg gcggtagcgg ttcggaagcg tacc 15410451PRTMycobacterium tuberculosis 104Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser1 5 10 15Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly 20 25 30Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser 35 40 45Glu Ala Tyr 50105282DNAMycobacterium tuberculosismisc_feature(1)..(282)ANY NUCLEOTIDE 105cggtcgcgca cttccaggtg actatgaaag tcggcttccg nctggaggat tcctgaacct 60tcaagcgcgg ccgataactg aggtgcatca ttaagcgact tttccagaac atcctgacgc 120gctcgaaacg cggcacagcc gacggtggct ccgncgaggc gctgnctcca aaatccctga 180gacaattcgn cgggggcgcc tacaaggaag tcggtgctga attcgncgng tatctggtcg 240acctgtgtgg tctgnagccg gacgaagcgg tgctcgacgt cg 2821063058DNAMycobacterium tuberculosis 106gatcgtaccc gtgcgagtgc tcgggccgtt tgaggatgga gtgcacgtgt ctttcgtgat 60ggcataccca gagatgttgg cggcggcggc tgacaccctg cagagcatcg gtgctaccac 120tgtggctagc aatgccgctg cggcggcccc gacgactggg gtggtgcccc ccgctgccga 180tgaggtgtcg gcgctgactg cggcgcactt cgccgcacat gcggcgatgt atcagtccgt 240gagcgctcgg gctgctgcga ttcatgacca gttcgtggcc acccttgcca gcagcgccag 300ctcgtatgcg gccactgaag tcgccaatgc ggcggcggcc agctaagcca ggaacagtcg 360gcacgagaaa ccacgagaaa tagggacacg taatggtgga tttcggggcg ttaccaccgg 420agatcaactc cgcgaggatg tacgccggcc cgggttcggc ctcgctggtg gccgcggctc 480agatgtggga cagcgtggcg agtgacctgt tttcggccgc gtcggcgttt cagtcggtgg 540tctggggtct gacggtgggg tcgtggatag gttcgtcggc gggtctgatg gtggcggcgg 600cctcgccgta tgtggcgtgg atgagcgtca ccgcggggca ggccgagctg accgccgccc 660aggtccgggt tgctgcggcg gcctacgaga cggcgtatgg gctgacggtg cccccgccgg 720tgatcgccga gaaccgtgct gaactgatga ttctgatagc gaccaacctc ttggggcaaa 780acaccccggc gatcgcggtc aacgaggccg aatacggcga gatgtgggcc caagacgccg 840ccgcgatgtt tggctacgcc gcggcgacgg cgacggcgac ggcgacgttg ctgccgttcg 900aggaggcgcc ggagatgacc agcgcgggtg ggctcctcga gcaggccgcc gcggtcgagg 960aggcctccga caccgccgcg gcgaaccagt tgatgaacaa tgtgccccag gcgctgcaac 1020agctggccca gcccacgcag ggcaccacgc cttcttccaa gctgggtggc ctgtggaaga 1080cggtctcgcc gcatcggtcg ccgatcagca acatggtgtc gatggccaac aaccacatgt 1140cgatgaccaa ctcgggtgtg tcgatgacca acaccttgag ctcgatgttg aagggctttg 1200ctccggcggc ggccgcccag gccgtgcaaa ccgcggcgca aaacggggtc cgggcgatga 1260gctcgctggg cagctcgctg ggttcttcgg gtctgggcgg tggggtggcc gccaacttgg 1320gtcgggcggc ctcggtcggt tcgttgtcgg tgccgcaggc ctgggccgcg gccaaccagg 1380cagtcacccc ggcggcgcgg gcgctgccgc tgaccagcct gaccagcgcc gcggaaagag 1440ggcccgggca gatgctgggc gggctgccgg tggggcagat gggcgccagg gccggtggtg 1500ggctcagtgg tgtgctgcgt gttccgccgc gaccctatgt gatgccgcat tctccggcgg 1560ccggctagga gagggggcgc agactgtcgt tatttgacca gtgatcggcg gtctcggtgt 1620ttccgcggcc ggctatgaca acagtcaatg tgcatgacaa gttacaggta ttaggtccag 1680gttcaacaag gagacaggca acatggcctc acgttttatg acggatccgc acgcgatgcg 1740ggacatggcg ggccgttttg aggtgcacgc ccagacggtg gaggacgagg ctcgccggat 1800gtgggcgtcc gcgcaaaaca tttccggtgc gggctggagt ggcatggccg aggcgacctc 1860gctagacacc atggcccaga tgaatcaggc gtttcgcaac atcgtgaaca tgctgcacgg 1920ggtgcgtgac gggctggttc gcgacgccaa caactacgag cagcaagagc aggcctccca 1980gcagatcctc agcagctaac gtcagccgct gcagcacaat acttttacaa gcgaaggaga 2040acaggttcga tgaccatcaa ctatcaattc ggggatgtcg acgctcacgg cgccatgatc 2100cgcgctcagg ccgggttgct ggaggccgag catcaggcca tcattcgtga tgtgttgacc 2160gcgagtgact tttggggcgg cgccggttcg gcggcctgcc aggggttcat tacccagttg 2220ggccgtaact tccaggtgat ctacgagcag gccaacgccc acgggcagaa ggtgcaggct 2280gccggcaaca acatggcgca aaccgacagc gccgtcggct ccagctgggc ctgacaccag 2340gccaaggcca gggacgtggt gtacgagtga agttcctcgc gtgatccttc gggtggcagt 2400ctaagtggtc agtgctgggg tgttggtggt ttgctgcttg gcgggttctt cggtgctggt 2460cagtgctgct cgggctcggg tgaggacctc gaggcccagg tagcgccgtc cttcgatcca 2520ttcgtcgtgt tgttcggcga ggacggctcc gacgaggcgg atgatcgagg cgcggtcggg 2580gaagatgccc acgacgtcgg ttcggcgtcg tacctctcgg ttgaggcgtt cctgggggtt 2640gttggaccag atttggcgcc agatctgctt ggggaaggcg gtgaacgcca gcaggtcggt 2700gcgggcggtg tcgaggtgct cggccaccgc ggggagtttg tcggtcagag cgtcgagtac 2760ccgatcatat tgggcaacaa ctgattcggc gtcgggctgg tcgtagatgg agtgcagcag 2820ggtgcgcacc cacggccagg agggcttcgg ggtggctgcc atcagattgg ctgcgtagtg 2880ggttctgcag cgctgccagg ccgctgcggg cagggtggcg ccgatcgcgg ccaccaggcc 2940ggcgtgggcg tcgctggtga ccagcgcgac cccggacagg ccgcgggcga ccaggtcgcg 3000gaagaacgcc agccagccgg ccccgtcctc ggcggaggtg acctggatgc ccaggatc 3058107391PRTMycobacterium tuberculosis 107Met Val Asp Phe Gly Ala Leu Pro Pro Glu Ile Asn Ser Ala Arg Met1 5 10 15Tyr Ala Gly Pro Gly Ser Ala Ser Leu Val Ala Ala Ala Gln Met Trp 20 25 30Asp Ser Val Ala Ser Asp Leu Phe Ser Ala Ala Ser Ala Phe Gln Ser 35 40 45Val Val Trp Gly Leu Thr Val Gly Ser Trp Ile Gly Ser Ser Ala Gly 50 55 60Leu Met Val Ala Ala Ala Ser Pro Tyr Val Ala Trp Met Ser Val Thr65 70 75 80Ala Gly Gln Ala Glu Leu Thr Ala Ala Gln Val Arg Val Ala Ala Ala 85 90 95Ala Tyr Glu Thr Ala Tyr Gly Leu Thr Val Pro Pro Pro Val Ile Ala 100 105 110Glu Asn Arg Ala Glu Leu Met Ile Leu Ile Ala Thr Asn Leu Leu Gly 115 120 125Gln Asn Thr Pro Ala Ile Ala Val Asn Glu Ala Glu Tyr Gly Glu Met 130 135 140Trp Ala Gln Asp Ala Ala Ala Met Phe Gly Tyr Ala Ala Ala Thr Ala145 150 155 160Thr Ala Thr Ala Thr Leu Leu Pro Phe Glu Glu Ala Pro Glu Met Thr 165 170 175Ser Ala Gly Gly Leu Leu Glu Gln Ala Ala Ala Val Glu Glu Ala Ser 180 185 190Asp Thr Ala Ala Ala Asn Gln Leu Met Asn Asn Val Pro Gln Ala Leu 195 200 205Gln Gln Leu Ala Gln Pro Thr Gln Gly Thr Thr Pro Ser Ser Lys Leu 210 215 220Gly Gly Leu Trp Lys Thr Val Ser Pro His Arg Ser Pro Ile Ser Asn225 230 235 240Met Val Ser Met Ala Asn Asn His Met Ser Met Thr Asn Ser Gly Val 245 250 255Ser Met Thr Asn Thr Leu Ser Ser Met Leu Lys Gly Phe Ala Pro Ala 260 265 270Ala Ala Ala Gln Ala Val Gln Thr Ala Ala Gln Asn Gly Val Arg Ala 275 280 285Met Ser Ser Leu Gly Ser Ser Leu Gly Ser Ser Gly Leu Gly Gly Gly 290 295 300Val Ala Ala Asn Leu Gly Arg Ala Ala Ser Val Gly Ser Leu Ser Val305 310 315 320Pro Gln Ala Trp Ala Ala Ala Asn Gln Ala Val Thr Pro Ala Ala Arg 325 330 335Ala Leu Pro Leu Thr Ser Leu Thr Ser Ala Ala Glu Arg Gly Pro Gly 340 345 350Gln Met Leu Gly Gly Leu Pro Val Gly Gln Met Gly Ala Arg Ala Gly 355 360 365Gly Gly Leu Ser Gly Val Leu Arg Val Pro Pro Arg Pro Tyr Val Met 370 375 380Pro His Ser Pro Ala Ala Gly385 3901081725DNAMycobacterium tuberculosis 108gacgtcagca cccgccgtgc agggctggag cgtggtcggt tttgatctgc ggtcaaggtg 60acgtccctcg gcgtgtcgcc ggcgtggatg cagactcgat gccgctcttt agtgcaacta 120atttcgttga agtgcctgcg aggtatagga cttcacgatt ggttaatgta gcgttcaccc 180cgtgttgggg tcgatttggc cggaccagtc gtcaccaacg cttggcgtgc gcgccaggcg 240ggcgatcaga tcgcttgact accaatcaat cttgagctcc cgggccgatg ctcgggctaa 300atgaggagga gcacgcgtgt ctttcactgc gcaaccggag atgttggcgg ccgcggctgg 360cgaacttcgt tccctggggg caacgctgaa ggctagcaat gccgccgcag ccgtgccgac 420gactggggtg gtgcccccgg

ctgccgacga ggtgtcgctg ctgcttgcca cacaattccg 480tacgcatgcg gcgacgtatc agacggccag cgccaaggcc gcggtgatcc atgagcagtt 540tgtgaccacg ctggccacca gcgctagttc atatgcggac accgaggccg ccaacgctgt 600ggtcaccggc tagctgacct gacggtattc gagcggaagg attatcgaag tggtggattt 660cggggcgtta ccaccggaga tcaactccgc gaggatgtac gccggcccgg gttcggcctc 720gctggtggcc gccgcgaaga tgtgggacag cgtggcgagt gacctgtttt cggccgcgtc 780ggcgtttcag tcggtggtct ggggtctgac ggtggggtcg tggataggtt cgtcggcggg 840tctgatggcg gcggcggcct cgccgtatgt ggcgtggatg agcgtcaccg cggggcaggc 900ccagctgacc gccgcccagg tccgggttgc tgcggcggcc tacgagacag cgtataggct 960gacggtgccc ccgccggtga tcgccgagaa ccgtaccgaa ctgatgacgc tgaccgcgac 1020caacctcttg gggcaaaaca cgccggcgat cgaggccaat caggccgcat acagccagat 1080gtggggccaa gacgcggagg cgatgtatgg ctacgccgcc acggcggcga cggcgaccga 1140ggcgttgctg ccgttcgagg acgccccact gatcaccaac cccggcgggc tccttgagca 1200ggccgtcgcg gtcgaggagg ccatcgacac cgccgcggcg aaccagttga tgaacaatgt 1260gccccaagcg ctgcaacagc tggcccagcc agcgcagggc gtcgtacctt cttccaagct 1320gggtgggctg tggacggcgg tctcgccgca tctgtcgccg ctcagcaacg tcagttcgat 1380agccaacaac cacatgtcga tgatgggcac gggtgtgtcg atgaccaaca ccttgcactc 1440gatgttgaag ggcttagctc cggcggcggc tcaggccgtg gaaaccgcgg cggaaaacgg 1500ggtctgggcg atgagctcgc tgggcagcca gctgggttcg tcgctgggtt cttcgggtct 1560gggcgctggg gtggccgcca acttgggtcg ggcggcctcg gtcggttcgt tgtcggtgcc 1620gccagcatgg gccgcggcca accaggcggt caccccggcg gcgcgggcgc tgccgctgac 1680cagcctgacc agcgccgccc aaaccgcccc cggacacatg ctggg 1725109359PRTMycobacterium tuberculosis 109Val Val Asp Phe Gly Ala Leu Pro Pro Glu Ile Asn Ser Ala Arg Met1 5 10 15Tyr Ala Gly Pro Gly Ser Ala Ser Leu Val Ala Ala Ala Lys Met Trp 20 25 30Asp Ser Val Ala Ser Asp Leu Phe Ser Ala Ala Ser Ala Phe Gln Ser 35 40 45Val Val Trp Gly Leu Thr Thr Gly Ser Trp Ile Gly Ser Ser Ala Gly 50 55 60Leu Met Val Ala Ala Ala Ser Pro Tyr Val Ala Trp Met Ser Val Thr65 70 75 80Ala Gly Gln Ala Glu Leu Thr Ala Ala Gln Val Arg Val Ala Ala Ala 85 90 95Ala Tyr Glu Thr Ala Tyr Gly Leu Thr Val Pro Pro Pro Val Ile Ala 100 105 110Glu Asn Arg Ala Glu Leu Met Ile Leu Ile Ala Thr Asn Leu Leu Gly 115 120 125Gln Asn Thr Pro Ala Ile Ala Val Asn Glu Ala Glu Tyr Gly Glu Met 130 135 140Trp Ala Gln Asp Ala Ala Ala Met Phe Gly Tyr Ala Ala Thr Ala Ala145 150 155 160Thr Ala Thr Glu Ala Leu Leu Pro Phe Glu Asp Ala Pro Leu Ile Thr 165 170 175Asn Pro Gly Gly Leu Leu Glu Gln Ala Val Ala Val Glu Glu Ala Ile 180 185 190Asp Thr Ala Ala Ala Asn Gln Leu Met Asn Asn Val Pro Gln Ala Leu 195 200 205Gln Gln Leu Ala Gln Pro Ala Gln Gly Val Val Pro Ser Ser Lys Leu 210 215 220Gly Gly Leu Trp Thr Ala Val Ser Pro His Leu Ser Pro Leu Ser Asn225 230 235 240Val Ser Ser Ile Ala Asn Asn His Met Ser Met Met Gly Thr Gly Val 245 250 255Ser Met Thr Asn Thr Leu His Ser Met Leu Lys Gly Leu Ala Pro Ala 260 265 270Ala Ala Gln Ala Val Glu Thr Ala Ala Gln Asn Gly Val Trp Ala Met 275 280 285Ser Ser Leu Gly Ser Gln Leu Gly Ser Ser Leu Gly Ser Ser Gly Leu 290 295 300Gly Ala Gly Val Ala Ala Asn Leu Gly Arg Ala Ala Ser Val Gly Ser305 310 315 320Leu Ser Val Pro Pro Ala Trp Ala Ala Ala Asn Gln Ala Val Thr Pro 325 330 335Ala Ala Arg Ala Leu Pro Leu Thr Ser Leu Thr Ser Ala Ala Gln Thr 340 345 350Ala Pro Gly His Met Leu Gly 3551103027DNAMycobacterium tuberculosis 110agttcagtcg agaatgatac tgacgggctg tatccacgat ggctgagaca accgaaccac 60cgtcggacgc ggggacatcg caagccgacg cgatggcgtt ggccgccgaa gccgaagccg 120ccgaagccga agcgctggcc gccgcggcgc gggcccgtgc ccgtgccgcc cggttgaagc 180gtgaggcgct ggcgatggcc ccagccgagg acgagaacgt ccccgaggat atgcagactg 240ggaagacgcc gaagactatg acgactatga cgactatgag gccgcagacc aggaggccgc 300acggtcggca tcctggcgac ggcggttgcg ggtgcggtta ccaagactgt ccacgattgc 360catggcggcc gcagtcgtca tcatctgcgg cttcaccggg ctcagcggat acattgtgtg 420gcaacaccat gaggccaccg aacgccagca gcgcgccgcg gcgttcgccg ccggagccaa 480gcaaggtgtc atcaacatga cctcgctgga cttcaacaag gccaaagaag acgtcgcgcg 540tgtgatcgac agctccaccg gcgaattcag ggatgacttc cagcagcggg cagccgattt 600caccaaggtt gtcgaacagt ccaaagtggt caccgaaggc acggtgaacg cgacagccgt 660cgaatccatg aacgagcatt ccgccgtggt gctcgtcgcg gcgacttcac gggtcaccaa 720ttccgctggg gcgaaagacg aaccacgtgc gtggcggctc aaagtgaccg tgaccgaaga 780ggggggacag tacaagatgt cgaaagttga gttcgtaccg tgaccgatga cgtacgcgac 840gtcaacaccg aaaccactga cgccaccgaa gtcgctgaga tcgactcagc cgcaggcgaa 900gccggtgatt cggcgaccga ggcatttgac accgactctg caacggaatc taccgcgcag 960aagggtcagc ggcaccgtga cctgtggcgc atgcaggtta ccttgaaacc cgttccggtg 1020attctcatcc tgctcatgtt gatctctggg ggcgcgacgg gatggctata ccttgagcaa 1080tacgacccga tcagcagacg gactccggcg ccgcccgtgc tgccgtcgcc gcggcgtctg 1140acgggacaat cgcgctgttg tgtattcacc cgacacgtcg accaagactt cgctaccgcc 1200aggtcgcacc tcgccggcga tttcctgtcc tatacgacca gttcacgcag cagatcgtgg 1260ctccggcggc caaacagaag tcactgaaaa ccaccgccaa ggtggtgcgc gcggccgtgt 1320cggagctaca tccggattcg gccgtcgttc tggtttttgt cgaccagagc actaccagta 1380aggacagccc caatccgtcg atggcggcca gcagcgtgat ggtgacccta gccaaggtcg 1440acggcaattg gctgatcacc aagttcaccc cggtttaggt tgccgtaggc ggtcgccaag 1500tctgacgggg gcgcgggtgg ctgctcgtgc gagataccgg ccgttctccg gacaatcacg 1560gcccgacctc aaacagatct cggccgctgt ctaatcggcc gggttattta agattagttg 1620ccactgtatt tacctgatgt tcagattgtt cagctggatt tagcttcgcg gcagggcggc 1680tggtgcactt tgcatctggg gttgtgacta cttgagagaa tttgacctgt tgccgacgtt 1740gtttgctgtc catcattggt gctagttatg gccgagcgga aggattatcg aagtggtgga 1800cttcggggcg ttaccaccgg agatcaactc cgcgaggatg tacgccggcc cgggttcggc 1860ctcgctggtg gccgccgcga agatgtggga cagcgtggcg agtgacctgt tttcggccgc 1920gtcggcgttt cagtcggtgg tctggggtct gacgacggga tcgtggatag gttcgtcggc 1980gggtctgatg gtggcggcgg cctcgccgta tgtggcgtgg atgagcgtca ccgcggggca 2040ggccgagctg accgccgccc aggtccgggt tgctgcggcg gcctacgaga cggcgtatgg 2100gctgacggtg cccccgccgg tgatcgccga gaaccgtgct gaactgatga ttctgatagc 2160gaccaacctc ttggggcaaa acaccccggc gatcgcggtc aacgaggccg aatacgggga 2220gatgtgggcc caagacgccg ccgcgatgtt tggctacgcc gccacggcgg cgacggcgac 2280cgaggcgttg ctgccgttcg aggacgcccc actgatcacc aaccccggcg ggctccttga 2340gcaggccgtc gcggtcgagg aggccatcga caccgccgcg gcgaaccagt tgatgaacaa 2400tgtgccccaa gcgctgcaac aactggccca gcccacgaaa agcatctggc cgttcgacca 2460actgagtgaa ctctggaaag ccatctcgcc gcatctgtcg ccgctcagca acatcgtgtc 2520gatgctcaac aaccacgtgt cgatgaccaa ctcgggtgtg tcgatggcca gcaccttgca 2580ctcaatgttg aagggctttg ctccggcggc ggctcaggcc gtggaaaccg cggcgcaaaa 2640cggggtccag gcgatgagct cgctgggcag ccagctgggt tcgtcgctgg gttcttcggg 2700tctgggcgct ggggtggccg ccaacttggg tcgggcggcc tcggtcggtt cgttgtcggt 2760gccgcaggcc tgggccgcgg ccaaccaggc ggtcaccccg gcggcgcggg cgctgccgct 2820gaccagcctg accagcgccg cccaaaccgc ccccggacac atgctgggcg ggctaccgct 2880ggggcaactg accaatagcg gcggcgggtt cggcggggtt agcaatgcgt tgcggatgcc 2940gccgcgggcg tacgtaatgc cccgtgtgcc cgccgccggg taacgccgat ccgcacgcaa 3000tgcgggccct ctatgcgggc agcgatc 3027111396PRTMycobacterium tuberculosis 111Val Val Asp Phe Gly Ala Leu Pro Pro Glu Ile Asn Ser Ala Arg Met1 5 10 15Tyr Ala Gly Pro Gly Ser Ala Ser Leu Val Ala Ala Ala Lys Met Trp 20 25 30Asp Ser Val Ala Ser Asp Leu Phe Ser Ala Ala Ser Ala Phe Gln Ser 35 40 45Val Val Trp Gly Leu Thr Thr Gly Ser Trp Ile Gly Ser Ser Ala Gly 50 55 60Leu Met Val Ala Ala Ala Ser Pro Tyr Val Ala Trp Met Ser Val Thr65 70 75 80Ala Gly Gln Ala Glu Leu Thr Ala Ala Gln Val Arg Val Ala Ala Ala 85 90 95Ala Tyr Glu Thr Ala Tyr Gly Leu Thr Val Pro Pro Pro Val Ile Ala 100 105 110Glu Asn Arg Ala Glu Leu Met Ile Leu Ile Ala Thr Asn Leu Leu Gly 115 120 125Gln Asn Thr Pro Ala Ile Ala Val Asn Glu Ala Glu Tyr Gly Glu Met 130 135 140Trp Ala Gln Asp Ala Ala Ala Met Phe Gly Tyr Ala Ala Thr Ala Ala145 150 155 160Thr Ala Thr Glu Ala Leu Leu Pro Phe Glu Asp Ala Pro Leu Ile Thr 165 170 175Asn Pro Gly Gly Leu Leu Glu Gln Ala Val Ala Val Glu Glu Ala Ile 180 185 190Asp Thr Ala Ala Ala Asn Gln Leu Met Asn Asn Val Pro Gln Ala Leu 195 200 205Gln Gln Leu Ala Gln Pro Thr Lys Ser Ile Trp Pro Phe Asp Gln Leu 210 215 220Ser Glu Leu Trp Lys Ala Ile Ser Pro His Leu Ser Pro Leu Ser Asn225 230 235 240Ile Val Ser Met Leu Asn Asn His Val Ser Met Thr Asn Ser Gly Val 245 250 255Ser Met Ala Ser Thr Leu His Ser Met Leu Lys Gly Phe Ala Pro Ala 260 265 270Ala Ala Gln Ala Val Glu Thr Ala Ala Gln Asn Gly Val Gln Ala Met 275 280 285Ser Ser Leu Gly Ser Gln Leu Gly Ser Ser Leu Gly Ser Ser Gly Leu 290 295 300Gly Ala Gly Val Ala Ala Asn Leu Gly Arg Ala Ala Ser Val Gly Ser305 310 315 320Leu Ser Val Pro Gln Ala Trp Ala Ala Ala Asn Gln Ala Val Thr Pro 325 330 335Ala Ala Arg Ala Leu Pro Leu Thr Ser Leu Thr Ser Ala Ala Gln Thr 340 345 350Ala Pro Gly His Met Leu Gly Gly Leu Pro Leu Gly Gln Leu Thr Asn 355 360 365Ser Gly Gly Gly Phe Gly Gly Val Ser Asn Ala Leu Arg Met Pro Pro 370 375 380Arg Ala Tyr Val Met Pro Arg Val Pro Ala Ala Gly385 390 3951121616DNAMycobacterium tuberculosis 112catcggaggg agtgatcacc atgctgtggc acgcaatgcc accggagtaa ataccgcacg 60gctgatggcc ggcgcgggtc cggctccaat gcttgcggcg gccgcgggat ggcagacgct 120ttcggcggct ctggacgctc aggccgtcga gttgaccgcg cgcctgaact ctctgggaga 180agcctggact ggaggtggca gcgacaaggc gcttgcggct gcaacgccga tggtggtctg 240gctacaaacc gcgtcaacac aggccaagac ccgtgcgatg caggcgacgg cgcaagccgc 300ggcatacacc caggccatgg ccacgacgcc gtcgctgccg gagatcgccg ccaaccacat 360cacccaggcc gtccttacgg ccaccaactt cttcggtatc aacacgatcc cgatcgcgtt 420gaccgagatg gattatttca tccgtatgtg gaaccaggca gccctggcaa tggaggtcta 480ccaggccgag accgcggtta acacgctttt cgagaagctc gagccgatgg cgtcgatcct 540tgatcccggc gcgagccaga gcacgacgaa cccgatcttc ggaatgccct cccctggcag 600ctcaacaccg gttggccagt tgccgccggc ggctacccag accctcggcc aactgggtga 660gatgagcbgc ccgatgcagc agctgaccca gccgctgcag caggtgacgt cgttgttcag 720ccaggtgggc ggcaccggcg gcggcaaccc agccgacgag gaagccgcgc agatgggcct 780gctcggcacc agtccgctgt cgaaccatcc gctggctggt ggatcaggcc ccagcgcggg 840cgcgggcctg ctgcgcgcgg agtcgctacc tggcgcaggt gggtcgttga cccgcacgcc 900gctgatgtct cagctgatcg aaaagccggt tgccccctcg gtgatgccgg cggctgctgc 960cggatcgtcg gcgacgggtg gcgccgctcc ggtgggtgcg ggagcgatgg gccagggtgc 1020gcaatccggc ggctccacca ggccgggtct ggtcgcgccg gcaccgctcg cgcaggagcg 1080tgaagaagac gacgaggacg actgggacga agaggacgac tggtgagctc ccgtaatgac 1140aacagacttc ccggccaccc gggccggaag acttgccaac attttggcga ggaaggtaaa 1200gagagaaagt agtccagcat ggcagagatg aagaccgatg ccgctaccct cgcgcaggag 1260gcaggtaatt tcgagcggat ctccggcgac ctgaaaaccc agatcgacca ggtggagtcg 1320acggcaggtt cgttgcaggg ccagtggcgc ggcgcggcgg ggacggccgc ccaggccgcg 1380gtggtgcgct tccaagaagc agccaataag cagaagcagg aactcgacga gatctcgacg 1440aatattcgtc aggccggcgt ccaatactcg agggccgacg aggagcagca gcaggcgctg 1500tcctcgcaaa tgggcttctg acccgctaat acgaaaagaa acggagcaaa aacatgacag 1560agcagcagtg gaatttcgcg ggtatcgagg ccgcggcaag cgcaatccag ggaaat 1616113432DNAMycobacterium tuberculosis 113ctagtggatg ggaccatggc cattttctgc agtctcactg ccttctgtgt tgacattttg 60gcacgccggc ggaaacgaag cactggggtc gaagaacggc tgcgctgcca tatcgtccgg 120agcttccata ccttcgtgcg gccggaagag cttgtcgtag tcggccgcca tgacaacctc 180tcagagtgcg ctcaaacgta taaacacgag aaagggcgag accgacggaa ggtcgaactc 240gcccgatccc gtgtttcgct attctacgcg aactcggcgt tgccctatgc gaacatccca 300gtgacgttgc cttcggtcga agccattgcc tgaccggctt cgctgatcgt ccgcgccagg 360ttctgcagcg cgttgttcag ctcggtagcc gtggcgtccc atttttgctg gacaccctgg 420tacgcctccg aa 432114368PRTMycobacterium tuberculosisMISC_FEATURE(1)..(368)ANY AMINO ACID 114Met Leu Trp His Ala Met Pro Pro Glu Xaa Asn Thr Ala Arg Leu Met1 5 10 15Ala Gly Ala Gly Pro Ala Pro Met Leu Ala Ala Ala Ala Gly Trp Gln 20 25 30Thr Leu Ser Ala Ala Leu Asp Ala Gln Ala Val Glu Leu Thr Ala Arg 35 40 45Leu Asn Ser Leu Gly Glu Ala Trp Thr Gly Gly Gly Ser Asp Lys Ala 50 55 60Leu Ala Ala Ala Thr Pro Met Val Val Trp Leu Gln Thr Ala Ser Thr65 70 75 80Gln Ala Lys Thr Arg Ala Met Gln Ala Thr Ala Gln Ala Ala Ala Tyr 85 90 95Thr Gln Ala Met Ala Thr Thr Pro Ser Leu Pro Glu Ile Ala Ala Asn 100 105 110His Ile Thr Gln Ala Val Leu Thr Ala Thr Asn Phe Phe Gly Ile Asn 115 120 125Thr Ile Pro Ile Ala Leu Thr Glu Met Asp Tyr Phe Ile Arg Met Trp 130 135 140Asn Gln Ala Ala Leu Ala Met Glu Val Tyr Gln Ala Glu Thr Ala Val145 150 155 160Asn Thr Leu Phe Glu Lys Leu Glu Pro Met Ala Ser Ile Leu Asp Pro 165 170 175Gly Ala Ser Gln Ser Thr Thr Asn Pro Ile Phe Gly Met Pro Ser Pro 180 185 190Gly Ser Ser Thr Pro Val Gly Gln Leu Pro Pro Ala Ala Thr Gln Thr 195 200 205Leu Gly Gln Leu Gly Glu Met Ser Gly Pro Met Gln Gln Leu Thr Gln 210 215 220Pro Leu Gln Gln Val Thr Ser Leu Phe Ser Gln Val Gly Gly Thr Gly225 230 235 240Gly Gly Asn Pro Ala Asp Glu Glu Ala Ala Gln Met Gly Leu Leu Gly 245 250 255Thr Ser Pro Leu Ser Asn His Pro Leu Ala Gly Gly Ser Gly Pro Ser 260 265 270Ala Gly Ala Gly Leu Leu Arg Ala Glu Ser Leu Pro Gly Ala Gly Gly 275 280 285Ser Leu Thr Arg Thr Pro Leu Met Ser Gln Leu Ile Glu Lys Pro Val 290 295 300Ala Pro Ser Val Met Pro Ala Ala Ala Ala Gly Ser Ser Ala Thr Gly305 310 315 320Gly Ala Ala Pro Val Gly Ala Gly Ala Met Gly Gln Gly Ala Gln Ser 325 330 335Gly Gly Ser Thr Arg Pro Gly Leu Val Ala Pro Ala Pro Leu Ala Gln 340 345 350Glu Arg Glu Glu Asp Asp Glu Asp Asp Trp Asp Glu Glu Asp Asp Trp 355 360 365115100PRTMycobacterium tuberculosis 115Met Ala Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly1 5 10 15Asn Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val 20 25 30Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly 35 40 45Thr Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys 50 55 60Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly65 70 75 80Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser 85 90 95Gln Met Gly Phe 100116396DNAMycobacterium tuberculosis 116gatctccggc gacctgaaaa cccagatcga ccaggtggag tcgacggcag gttcgttgca 60gggccagtgg cgcggcgcgg cggggacggc cgcccaggcc gcggtggtgc gcttccaaga 120agcagccaat aagcagaagc aggaactcga cgagatctcg acgaatattc gtcaggccgg 180cgtccaatac tcgagggccg acgaggagca gcagcaggcg ctgtcctcgc aaatgggctt 240ctgacccgct aatacgaaaa gaaacggagc aaaaacatga cagagcagca gtggaatttc 300gcgggtatcg aggccgcggc aagcgcaatc cagggaaatg tcacgtccat tcattccctc 360cttgacgagg ggaagcagtc cctgaccaag ctcgca 39611780PRTMycobacterium tuberculosis 117Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val Glu Ser Thr Ala1 5 10 15Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly Thr Ala Ala Gln 20 25 30Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys Gln Lys Gln Glu 35 40 45Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly Val Gln Tyr Ser 50 55 60Arg Ala Asp Glu Glu Gln Gln Gln Ala

Leu Ser Ser Gln Met Gly Phe65 70 75 80118387DNAMycobacterium tuberculosis 118gtggatcccg atcccgtgtt tcgctattct acgcgaactc ggcgttgccc tatgcgaaca 60tcccagtgac gttgccttcg gtcgaagcca ttgcctgacc ggcttcgctg atcgtccgcg 120ccaggttctg cagcgcgttg ttcagctcgg tagccgtggc gtcccatttt tgctggacac 180cctggtacgc ctccgaaccg ctaccgcccc aggccgctgc gagcttggtc agggactgct 240tcccctcgtc aaggagggaa tgaatggacg tgacatttcc ctggattgcg cttgccgcgg 300cctcgatacc cgcgaaattc cactgctgct ctgtcatgtt tttgctccgt ttcttttcgt 360attagcgggt cagaagccca tttgcga 387119272DNAMycobacterium tuberculosis 119cggcacgagg atctcggttg gcccaacggc gctggcgagg gctccgttcc gggggcgagc 60tgcgcgccgg atgcttcctc tgcccgcagc cgcgcctgga tggatggacc agttgctacc 120ttcccgacgt ttcgttcggt gtctgtgcga tagcggtgac cccggcgcgc acgtcgggag 180tgttgggggg caggccgggt cggtggttcg gccggggacg cagacggtct ggacggaacg 240ggcgggggtt cgccgattgg catctttgcc ca 27212020PRTMycobacterium tuberculosis 120Asp Pro Val Asp Ala Val Ile Asn Thr Thr Cys Asn Tyr Gly Gln Val1 5 10 15Val Ala Ala Leu 2012115PRTMycobacterium tuberculosis 121Ala Val Glu Ser Gly Met Leu Ala Leu Gly Thr Pro Ala Pro Ser1 5 10 1512219PRTMycobacterium tuberculosis 122Ala Ala Met Lys Pro Arg Thr Gly Asp Gly Pro Leu Glu Ala Ala Lys1 5 10 15Glu Gly Arg12315PRTMycobacterium tuberculosis 123Tyr Tyr Trp Cys Pro Gly Gln Pro Phe Asp Pro Ala Trp Gly Pro1 5 10 1512414PRTMycobacterium tuberculosisMISC_FEATURE(1)..(14)ANY AMINO ACID 124Asp Ile Gly Ser Glu Ser Thr Glu Asp Gln Gln Xaa Ala Val1 5 1012513PRTMycobacterium tuberculosisMISC_FEATURE(1)..(13)ANY AMINO ACID 125Ala Glu Glu Ser Ile Ser Thr Xaa Glu Xaa Ile Val Pro1 5 1012617PRTMycobacterium tuberculosis 126Asp Pro Glu Pro Ala Pro Pro Val Pro Thr Thr Ala Ala Ser Pro Pro1 5 10 15Ser12715PRTMycobacterium tuberculosisMISC_FEATURE(1)..(15)ANY AMINO ACID 127Ala Pro Lys Thr Tyr Xaa Glu Glu Leu Lys Gly Thr Asp Thr Gly1 5 10 1512830PRTMycobacterium tuberculosis 128Asp Pro Ala Ser Ala Pro Asp Val Pro Thr Ala Ala Gln Leu Thr Ser1 5 10 15Leu Leu Asn Ser Leu Ala Asp Pro Asn Val Ser Phe Ala Asn 20 25 3012922PRTMycobacterium tuberculosisMISC_FEATURE(1)..(22)ANY AMINO ACID 129Asp Pro Pro Asp Pro His Gln Xaa Asp Met Thr Lys Gly Tyr Tyr Pro1 5 10 15Gly Gly Arg Arg Xaa Phe 201307PRTMycobacterium tuberculosis 130Asp Pro Gly Tyr Thr Pro Gly1 513110PRTMycobacterium tuberculosisMISC_FEATURE(1)..(1)ANY AMINO ACID 131Xaa Xaa Gly Phe Thr Gly Pro Gln Phe Tyr1 5 101329PRTMycobacterium tuberculosisMISC_FEATURE(1)..(1)ANY AMINO ACID 132Xaa Pro Xaa Val Thr Ala Tyr Ala Gly1 51339PRTMycobacterium tuberculosisMISC_FEATURE(1)..(9)ANY AMINO ACID 133Xaa Xaa Xaa Glu Lys Pro Phe Leu Arg1 513415PRTMycobacterium tuberculosisMISC_FEATURE(1)..(15)ANY AMINO ACID 134Xaa Asp Ser Glu Lys Ser Ala Thr Ile Lys Val Thr Asp Ala Ser1 5 10 1513515PRTMycobacterium tuberculosisMISC_FEATURE(1)..(15)ANY AMINO ACID 135Ala Gly Asp Thr Xaa Ile Tyr Ile Val Gly Asn Leu Thr Ala Asp1 5 10 1513615PRTMycobacterium tuberculosis 136Ala Pro Glu Ser Gly Ala Gly Leu Gly Gly Thr Val Gln Ala Gly1 5 10 1513721PRTMycobacterium tuberculosisMISC_FEATURE(1)..(21)ANY AMINO ACID 137Xaa Tyr Ile Ala Tyr Xaa Thr Thr Ala Gly Ile Val Pro Gly Lys Ile1 5 10 15Asn Val His Leu Val 20138882DNAMycobacterium tuberculosis 138gcaacgctgt cgtggccttt gcggtgatcg gtttcgcctc gctggcggtg gcggtggcgg 60tcaccatccg accgaccgcg gcctcaaaac cggtagaggg acaccaaaac gcccagccag 120ggaagttcat gccgttgttg ccgacgcaac agcaggcgcc ggtcccgccg cctccgcccg 180atgatcccac cgctggattc cagggcggca ccattccggc tgtacagaac gtggtgccgc 240ggccgggtac ctcacccggg gtgggtggga cgccggcttc gcctgcgccg gaagcgccgg 300ccgtgcccgg tgttgtgcct gccccggtgc caatcccggt cccgatcatc attcccccgt 360tcccgggttg gcagcctgga atgccgacca tccccaccgc accgccgacg acgccggtga 420ccacgtcggc gacgacgtcg ccgaccacgc cgccgaccac gccggtgacc acgccgccaa 480cgacgccgcc gaccacgccg gtgaccacgc cgccaacgac gccgccgacc acgccggtga 540ccacgccacc aacgaccgtc gccccgacga ccgtcgcccc gacgacggtc gctccgacca 600ccgtcgcccc gaccacggtc gctccagcca ccgccacgcc gacgaccgtc gctccgcagc 660cgacgcagca gcccacgcaa caaccaaccc aacagatgcc aacccagcag cagaccgtgg 720ccccgcagac ggtggcgccg gctccgcagc cgccgtccgg tggcgcaacg gcagcggcgg 780ggggcgactt attcggcggg ttctgatcac ggtcgcggct tcactacggt cggaggacat 840ggccggtgat gcggtgacgg tggtgctgcc ctgtctcaac ga 882139815DNAMycobacterium tuberculosismisc_feature(1)..(815)ANY NUCLEOTIDE 139ccatcaacca accgctcgcg ccgcccgcgc cgccggatcc gccgtcgccg ccacgcccgc 60cggtgcctcc ggtgcccccg ttgccgccgt cgccgccgtc gccgccgacc ggctgggtgc 120ctagggcgct gttaccgccc tggttggcgg ggacgccgcc ggcaccaccg gtaccgccga 180tggcgccgtt gccgccggcg gcaccgttgc caccgttgcc accgttgcca ccgttgccga 240ccagccaccc gccgcgacca ccggcaccgc cggcgccgcc cgcaccgccg gcgtgcccgt 300tcgtgcccgt accgccggca ccgccgttgc cgccgtcacc gccgacggaa ctaccggcgg 360acgcggcctg cccgccggcg ccgcccgcac cgccattggc accgccgtca ccgccggctg 420ggagtgccgc gattagggca ctgaccggcg caaccagcgc aagtactctc ggtcaccgag 480cacttccaga cgacaccaca gcacggggtt gtcggcggac tgggtgaaat ggcagccgat 540agcggctagc tgtcggctgc ggtcaacctc gatcatgatg tcgaggtgac cgtgaccgcg 600ccccccgaag gaggcgctga actcggcgtt gagccgatcg gcgatcggtt ggggcagtgc 660ccaggccaat acggggatac cgggtgtcna agccgccgcg agcgcagctt cggttgcgcg 720acngtggtcg gggtggcctg ttacgccgtt gtcntcgaac acgagtagca ggtctgctcc 780ggcgagggca tccaccacgc gttgcgtcag ctcgt 8151401152DNAMycobacterium tuberculosis 140accagccgcc ggctgaggtc tcagatcaga gagtctccgg actcaccggg gcggttcagc 60cttctcccag aacaactgct gaagatcctc gcccgcgaaa caggcgctga tttgacgctc 120tatgaccggt tgaacgacga gatcatccgg cagattgata tggcaccgct gggctaacag 180gtgcgcaaga tggtgcagct gtatgtctcg gactccgtgt cgcggatcag ctttgccgac 240ggccgggtga tcgtgtggag cgaggagctc ggcgagagtc agtatccgat cgagacgctg 300gacggcatca cgctgtttgg gcggccgacg atgacaacgc ccttcatcgt tgagatgctc 360aagcgtgagc gcgacatcca gctcttcacg accgacggcc actaccaggg ccggatctca 420acacccgacg tgtcatacgc gccgcggctc cgtcagcaag ttcaccgcac cgacgatcct 480gcgttctgcc tgtcgttaag caagcggatc gtgtcgagga agatcctgaa tcagcaggcc 540ttgattcggg cacacacgtc ggggcaagac gttgctgaga gcatccgcac gatgaagcac 600tcgctggcct gggtcgatcg atcgggctcc ctggcggagt tgaacgggtt cgagggaaat 660gccgcaaagg catacttcac cgcgctgggg catctcgtcc cgcaggagtt cgcattccag 720ggccgctcga ctcggccgcc gttggacgcc ttcaactcga tggtcagcct cggctattcg 780ctgctgtaca agaacatcat aggggcgatc gagcgtcaca gcctgaacgc gtatatcggt 840ttcctacacc aggattcacg agggcacgca acgtctcgtg ccgaattcgg cacgagctcc 900gctgaaaccg ctggccggct gctcagtgcc cgtacgtaat ccgctgcgcc caggccggcc 960cgccggccga ataccagcag atcggacagc gaattgccgc ccagccggtt ggagccgtgc 1020ataccgccgg cacactcacc ggcagcgaac aggcctggca ccgtggcggc gccggtgtcc 1080gcgtctactt cgacaccgcc catcacgtag tgacacgtcg gcccgacttc cattgcctgc 1140gttcggcacg ag 1152141655DNAMycobacterium tuberculosismisc_feature(1)..(655)ANY NUCLEOTIDE 141ctcgtgccga ttcggcaggg tgtacttgcc ggtggtgtan gccgcatgag tgccgacgac 60cagcaatgcg gcaacagcac ggatcccggt caacgacgcc acccggtcca cgtgggcgat 120ccgctcgagt ccgccctggg cggctctttc cttgggcagg gtcatccgac gtgtttccgc 180cgtggtttgc cgccattatg ccggcgcgcc gcgtcgggcg gccggtatgg ccgaangtcg 240atcagcacac ccgagatacg ggtctgtgca agctttttga gcgtcgcgcg gggcagcttc 300gccggcaatt ctactagcga gaagtctggc ccgatacgga tctgaccgaa gtcgctgcgg 360tgcagcccac cctcattggc gatggcgccg acgatggcgc ctggaccgat cttgtgccgc 420ttgccgacgg cgacgcggta ggtggtcaag tccggtctac gcttgggcct ttgcggacgg 480tcccgacgct ggtcgcggtt gcgccgcgaa agcggcgggt cgggtgccat caggaatgcc 540tcaccgccgc ggcactgcac ggccagtgcc gcggcgatgt cagccatcgg gacatcatgc 600tcgcgttcat actcctcgac cagtcggcgg aacagctcga ttcccggacc gccca 655142267PRTMycobacterium tuberculosis 142Asn Ala Val Val Ala Phe Ala Val Ile Gly Phe Ala Ser Leu Ala Val1 5 10 15Ala Val Ala Val Thr Ile Arg Pro Thr Ala Ala Ser Lys Pro Val Glu 20 25 30Gly His Gln Asn Ala Gln Pro Gly Lys Phe Met Pro Leu Leu Pro Thr 35 40 45Gln Gln Gln Ala Pro Val Pro Pro Pro Pro Pro Asp Asp Pro Thr Ala 50 55 60Gly Phe Gln Gly Gly Thr Ile Pro Ala Val Gln Asn Val Val Pro Arg65 70 75 80Pro Gly Thr Ser Pro Gly Val Gly Gly Thr Pro Ala Ser Pro Ala Pro 85 90 95Glu Ala Pro Ala Val Pro Gly Val Val Pro Ala Pro Val Pro Ile Pro 100 105 110Val Pro Ile Ile Ile Pro Pro Phe Pro Gly Trp Gln Pro Gly Met Pro 115 120 125Thr Ile Pro Thr Ala Pro Pro Thr Thr Pro Val Thr Thr Ser Ala Thr 130 135 140Thr Pro Pro Thr Thr Pro Pro Thr Thr Pro Val Thr Thr Pro Pro Thr145 150 155 160Thr Pro Pro Thr Thr Pro Val Thr Thr Pro Pro Thr Thr Pro Pro Thr 165 170 175Thr Pro Val Thr Thr Pro Pro Thr Thr Val Ala Pro Thr Thr Val Ala 180 185 190Pro Thr Thr Val Ala Pro Thr Thr Val Ala Pro Thr Thr Val Ala Pro 195 200 205Ala Thr Ala Thr Pro Thr Thr Val Ala Pro Gln Pro Thr Gln Gln Pro 210 215 220Thr Gln Gln Pro Thr Gln Gln Met Pro Thr Gln Gln Gln Thr Val Ala225 230 235 240Pro Gln Thr Val Ala Pro Ala Pro Gln Pro Pro Ser Gly Gly Arg Asn 245 250 255Gly Ser Gly Gly Gly Asp Leu Phe Gly Gly Phe 260 265143174PRTMycobacterium tuberculosis 143Ile Asn Gln Pro Leu Ala Pro Pro Ala Pro Pro Asp Pro Pro Ser Pro1 5 10 15Pro Arg Pro Pro Val Pro Pro Val Pro Pro Leu Pro Pro Ser Pro Pro 20 25 30Ser Pro Pro Thr Gly Trp Val Pro Arg Ala Leu Leu Pro Pro Trp Leu 35 40 45Ala Gly Thr Pro Pro Ala Pro Pro Val Pro Pro Met Ala Pro Leu Pro 50 55 60Pro Ala Ala Pro Leu Pro Pro Leu Pro Pro Leu Pro Pro Leu Pro Thr65 70 75 80Ser His Pro Pro Arg Pro Pro Ala Pro Pro Ala Pro Pro Ala Pro Pro 85 90 95Ala Cys Pro Phe Val Pro Val Pro Pro Ala Pro Pro Leu Pro Pro Ser 100 105 110Pro Pro Thr Glu Leu Pro Ala Asp Ala Ala Cys Pro Pro Ala Pro Pro 115 120 125Ala Pro Pro Leu Ala Pro Pro Ser Pro Pro Ala Gly Ser Ala Ala Ile 130 135 140Arg Ala Leu Thr Gly Ala Thr Ser Ala Ser Thr Leu Gly His Arg Ala145 150 155 160Leu Pro Asp Asp Thr Thr Ala Arg Gly Cys Arg Arg Thr Gly 165 17014435PRTMycobacterium tuberculosis 144Gln Pro Pro Ala Glu Val Ser Asp Gln Arg Val Ser Gly Leu Thr Gly1 5 10 15Ala Val Gln Pro Ser Pro Arg Thr Thr Ala Glu Asp Pro Arg Pro Arg 20 25 30Asn Arg Arg 35145104PRTMycobacterium tuberculosisMISC_FEATURE(1)..(104)ANY AMINO ACID 145Arg Ala Asp Ser Ala Gly Cys Thr Cys Arg Trp Cys Xaa Pro His Glu1 5 10 15Cys Arg Arg Pro Ala Met Arg Gln Gln His Gly Ser Arg Ser Thr Thr 20 25 30Pro Pro Gly Pro Arg Gly Arg Ser Ala Arg Val Arg Pro Gly Arg Leu 35 40 45Phe Pro Trp Ala Gly Ser Ser Asp Val Phe Pro Pro Trp Phe Ala Ala 50 55 60Ile Met Pro Ala Arg Arg Val Gly Arg Pro Val Trp Pro Xaa Val Asp65 70 75 80Gln His Thr Arg Asp Thr Gly Leu Cys Lys Leu Phe Glu Arg Arg Ala 85 90 95Gly Gln Leu Arg Arg Gln Phe Tyr 10014653DNAMycobacterium tuberculosis 146ggatccatat gggccatcat catcatcatc acgtgatcga catcatcggg acc 5314742DNAMycobacterium tuberculosis 147cctgaattca ggcctcggtt gcgccggcct catcttgaac ga 4214831DNAMycobacterium tuberculosis 148ggatcctgca ggctcgaaac caccgagcgg t 3114931DNAMycobacterium tuberculosis 149ctctgaattc agcgctggaa atcgtcgcga t 3115033DNAMycobacterium tuberculosis 150ggatccagcg ctgagatgaa gaccgatgcc gct 3315133DNAMycobacterium tuberculosis 151gagagaattc tcagaagccc atttgcgagg aca 331521992DNAMycobacterium tuberculosis 152tgttcttcga cggcaggctg gtggaggaag ggcccaccga acagctgttc tcctcgccga 60agcatgcgga aaccgcccga tacgtcgccg gactgtcggg ggacgtcaag gacgccaagc 120gcggaaattg aagagcacag aaaggtatgg cgtgaaaatt cgtttgcata cgctgttggc 180cgtgttgacc gctgcgccgc tgctgctagc agcggcgggc tgtggctcga aaccaccgag 240cggttcgcct gaaacgggcg ccggcgccgg tactgtcgcg actacccccg cgtcgtcgcc 300ggtgacgttg gcggagaccg gtagcacgct gctctacccg ctgttcaacc tgtggggtcc 360ggcctttcac gagaggtatc cgaacgtcac gatcaccgct cagggcaccg gttctggtgc 420cgggatcgcg caggccgccg ccgggacggt caacattggg gcctccgacg cctatctgtc 480ggaaggtgat atggccgcgc acaaggggct gatgaacatc gcgctagcca tctccgctca 540gcaggtcaac tacaacctgc ccggagtgag cgagcacctc aagctgaacg gaaaagtcct 600ggcggccatg taccagggca ccatcaaaac ctgggacgac ccgcagatcg ctgcgctcaa 660ccccggcgtg aacctgcccg gcaccgcggt agttccgctg caccgctccg acgggtccgg 720tgacaccttc ttgttcaccc agtacctgtc caagcaagat cccgagggct ggggcaagtc 780gcccggcttc ggcaccaccg tcgacttccc ggcggtgccg ggtgcgctgg gtgagaacgg 840caacggcggc atggtgaccg gttgcgccga gacaccgggc tgcgtggcct atatcggcat 900cagcttcctc gaccaggcca gtcaacgggg actcggcgag gcccaactag gcaatagctc 960tggcaatttc ttgttgcccg acgcgcaaag cattcaggcc gcggcggctg gcttcgcatc 1020gaaaaccccg gcgaaccagg cgatttcgat gatcgacggg cccgccccgg acggctaccc 1080gatcatcaac tacgagtacg ccatcgtcaa caaccggcaa aaggacgccg ccaccgcgca 1140gaccttgcag gcatttctgc actgggcgat caccgacggc aacaaggcct cgttcctcga 1200ccaggtcatt tccagccgct gccgcccgcg gtggtgaagt tgtctgacgc gttgatcgcg 1260acgatttcca gctagcctcg ttgaccacca cgcgacagca acctccgtcg ggccatcggg 1320ctgctttgcg gagcatgctg gcccgtgccg gtgaagtcgg ccgcgctggc ccggccatcc 1380ggtggttggg tgggataggt gcggtgatcc cgctgcttgc gctggtcttg gtgctggtgg 1440tgctggtcat cgaggcgatg ggtgcgatca ggctcaacgg gttgcatttc ttcaccgcca 1500ccgaatggaa tccaggcaac acctacggcg aaaccgttgt caccgacgcg tcgcccatcc 1560ggtcggcgcc tactacgggg cgttgccgct gatcgtcggg acgctggcga cctcggcaat 1620cgccctgatc atcgcggtgc cggtctctgt aggagcggcg ctggtgatcg tggaacggct 1680gccgaaacgg ttggccgagg ctgtgggaat agtcctggaa ttgctcgccg gaatccccag 1740cgtggtcgtc ggtttgtggg gggcaatgac gttcgggccg ttcatcgctc atcacatcgc 1800tccggtgatc gctcacaacg ctcccgatgt gccggtgctg aactacttgc gcggcgaccc 1860gggcaacggg gagggcatgt tggtgtccgg tctggtgttg gcggtgatgg tcgttcccat 1920tatcgccacc accactcatg acctgttccg gcaggtgccg gtgttgcccc gggagggcgc 1980gatcgggaat tc 1992153374PRTMycobacterium tuberculosis 153Val Lys Ile Arg Leu His Thr Leu Leu Ala Val Leu Thr Ala Ala Pro1 5 10 15Leu Leu Leu Ala Ala Ala Gly Cys Gly Ser Lys Pro Pro Ser Gly Ser 20 25 30Pro Glu Thr Gly Ala Gly Ala Gly Thr Val Ala Thr Thr Pro Ala Ser 35 40 45Ser Pro Val Thr Leu Ala Glu Thr Gly Ser Thr Leu Leu Tyr Pro Leu 50 55 60Phe Asn Leu Trp Gly Pro Ala Phe His Glu Arg Tyr Pro Asn Val Thr65 70 75 80Ile Thr Ala Gln Gly Thr Gly Ser Gly Ala Gly Ile Ala Gln Ala Ala 85 90 95Ala Gly Thr Val Asn Ile Gly Ala Ser Asp Ala Tyr Leu Ser Glu Gly 100 105 110Asp Met Ala Ala His Lys Gly Leu Met Asn Ile Ala Leu Ala Ile Ser 115 120 125Ala Gln Gln Val Asn Tyr Asn Leu Pro Gly Val Ser Glu His Leu Lys 130 135 140Leu Ala Gly Lys Val Leu Ala Ala Met Tyr Gln Gly Thr Ile Leu Thr145 150 155 160Trp Asp Asp Pro Gln Ile Ala Ala Leu Asn Pro Gly Val Asn Leu Pro 165 170 175Gly Thr Ala Val Val Pro Leu His Arg Ser Asp Gly Ser Gly Asp Thr 180 185 190Phe Leu Phe Thr Gln Tyr Leu Ser Lys Gln Asp Pro Glu Gly Trp Gly 195 200 205Lys Ser Pro Gly Phe Gly Thr Thr Val Asp Phe Pro Ala Val

Pro Gly 210 215 220Ala Leu Gly Glu Asn Gly Asn Gly Gly Met Val Thr Gly Cys Ala Glu225 230 235 240Thr Pro Gly Cys Val Ala Tyr Ile Gly Ile Ser Phe Leu Asp Gln Ala 245 250 255Ser Gln Arg Gly Leu Gly Glu Ala Gln Leu Gly Asn Ser Ser Gly Asn 260 265 270Phe Leu Leu Pro Asp Ala Gln Ser Ile Gln Ala Ala Ala Ala Gly Phe 275 280 285Ala Ser Lys Thr Pro Ala Asn Gln Ala Ile Ser Met Ile Asp Gly Pro 290 295 300Ala Pro Ala Gly Tyr Pro Ile Ile Asn Tyr Glu Tyr Ala Ile Val Asn305 310 315 320Asn Arg Gln Lys Asp Ala Ala Thr Ala Gln Thr Leu Gln Ala Phe Leu 325 330 335His Trp Ala Ile Thr Asp Gly Asn Lys Ala Ser Phe Leu Asp Gln Val 340 345 350His Phe Gln Pro Leu Pro Pro Ala Val Val Lys Leu Ser Asp Ala Leu 355 360 365Ile Ala Thr Ile Ser Ser 370

Claims

1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 107.

2. A fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO:107 and a second polypeptide.

3. A composition comprising a polypeptide of claim 1 and a physiologically acceptable carrier.

4. The composition of claim 3, further comprising a non-specific immune response enhancer.

5. The composition of claim 4, wherein the non-specific immune response enhancer is an adjuvant.

6. An isolated polypeptide comprising an immunogenic fragment of SEQ ID NO:107, wherein the immunogenic fragment of SEQ ID NO:107 is at least 9 amino acids in length.

7. A fusion protein comprising a first polypeptide comprising an immunogenic fragment of SEQ ID NO:107, wherein the immunogenic fragment of SEQ ID NO:107 is at least 9 amino acids in length and a second polypeptide.

8. A composition comprising a polypeptide of claim 6 and a physiologically acceptable carrier.

9. The composition of claim 8, further comprising a non-specific immune response enhancer.

10. The composition of claim 9, wherein the non-specific immune response enhancer is an adjuvant.

11. A fusion protein according to claim 2, where said second polypeptide is an M. tuberculosis antigen.

12. A composition comprising a fusion protein of claim 2 and a physiologically acceptable carrier.

13. The composition of claim 12, further comprising a non-specific immune response enhancer.

14. The composition of claim 13, wherein the non-specific immune response enhancer is an adjuvant.

15. A fusion protein according to claim 7, where said second polypeptide is an M. tuberculosis antigen.

16. A composition comprising a fusion protein of claim 7 and a physiologically acceptable carrier.

17. The composition of claim 16, further comprising a non-specific immune response enhancer.

18. The composition of claim 17, where the non-specific immune response enhancer is an adjuvant.

19. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO:107.

20. A fusion protein comprising a first polypeptide consisting of the amino acid sequence of SEQ ID NO:107 and a second polypeptide.

21. A fusion protein according to claim 20, where said second polypeptide is an M. tuberculosis antigen.

22. A composition comprising a polypeptide of claim 19 and a physiologically acceptable carrier.

23. A composition comprising a fusion protein of claim 20 and a physiologically acceptable carrier.

24. The composition of claim 22, further comprising a non-specific immune response enhancer.

25. The composition of claim 24, wherein the non-specific immune response enhancer is an adjuvant.

26. The composition of claim 23, further comprising a non-specific immune response enhancer.

27. The composition of claim 26, wherein the non-specific immune response enhancer is an adjuvant.

28. An isolated polypeptide consisting of an immunogenic fragment of SEQ ID NO:107, wherein the immunogenic fragment of SEQ ID NO:107 is at least 9 amino acids in length.

29. A fusion protein comprising a first polypeptide consisting of an immunogenic fragment of SEQ ID NO:107, wherein the immunogenic fragment of SEQ ID NO:107 is at least 9 amino acids in length, and a second polypeptide.

30. A fusion protein according to claim 29, where said second polypeptide is an M. tuberculosis antigen.

31. A composition comprising a polypeptide of claim 28 and a physiologically acceptable carrier.

32. A composition comprising a fusion protein of claim 29 and physiologically acceptable carrier.

33. The composition of claim 31, further comprising a non-specific immune response enhancer.

34. The composition of claim 33, wherein the non-specific immune response enhancer is an adjuvant.

35. The composition of claim 32, further comprising a non-specific immune response enhancer.

36. The composition of claim 35, wherein the non-specific immune response enhancer is an adjuvant.

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