Patent application title: NOVEL COMPOSITIONS AND METHODS
Inventors:
Pascal Mettens (Rixensart, BE)
Pascal Mettens (Rixensart, BE)
James Brown (Collegeville, PA, US)
James Brown (Collegeville, PA, US)
Dennis Murphy (Collegeville, PA, US)
Dennis Murphy (Collegeville, PA, US)
IPC8 Class: AA61K3904FI
USPC Class:
4241901
Class name: Antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same disclosed amino acid sequence derived from bacterium (e.g., mycoplasma, anaplasma, etc.)
Publication date: 2011-05-19
Patent application number: 20110117119
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Patent application title: NOVEL COMPOSITIONS AND METHODS
Inventors:
Pascal Mettens
James Brown
Dennis Murphy
Agents:
Assignees:
Origin: ,
IPC8 Class: AA61K3904FI
USPC Class:
Publication date: 05/19/2011
Patent application number: 20110117119
Abstract:
The present invention is directed to a polypeptide which comprises: (i)
an Rv3616c protein sequence; (ii) a variant of an Rv3616c protein
sequence; or (iii) an immunogenic fragment of an Rv3616c protein sequence
for use in the treatment or prevention of latent TB. In other aspects the
invention is directed to associated polynucleotides, fusion proteins and
methods for the treatment or prevention of latent tuberculosis.Claims:
1-45. (canceled)
46. A method for the treatment or prevention of latent tuberculosis comprising the administration of a polypeptide comprising: (i) an Rv3616c protein sequence; (ii) a variant of an Rv3616c protein sequence; or (iii) an immunogenic fragment of an Rv3616c protein sequence; to a subject in need thereof, wherein said polypeptide induces an immune response.
47. A method for the treatment or prevention of latent tuberculosis comprising the administration of a polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising: (i) an Rv3616c protein sequence; (ii) a variant of an Rv3616c protein sequence; or (iii) an immunogenic fragment of an Rv3616c protein sequence; to a subject in need thereof, wherein said polynucleotide induces an immune response.
48-51. (canceled)
52. The method according to claim 46, wherein the subject has active tuberculosis.
53. The method according to claim 46, wherein the subject has latent tuberculosis.
54. The method according to claim 46, wherein the subject does not have tuberculosis.
55-66. (canceled)
67. The method according to claim 46, wherein the treatment or prevention of latent tuberculosis refers to the treatment of latent tuberculosis.
68. The method according to claim 46, wherein the treatment or prevention of latent tuberculosis refers to the prevention of latent tuberculosis.
69. The method according to claim 46, wherein the treatment or prevention of latent tuberculosis refers to the prevention of reactivation of tuberculosis.
70. The method according to claim 46, wherein the treatment or prevention of latent tuberculosis refers to the delay of reactivation of tuberculosis.
71-86. (canceled)
87. The method according to claim 47, wherein the subject has active tuberculosis.
88. The method according to claim 47, wherein the subject has latent tuberculosis.
89. The method according to claim 47, wherein the subject does not have tuberculosis.
90. The method according to claim 47, wherein the treatment or prevention of latent tuberculosis refers to the treatment of latent tuberculosis.
91. The method according to claim 47, wherein the treatment or prevention of latent tuberculosis refers to the prevention of latent tuberculosis.
92. The method according to claim 47, wherein the treatment or prevention of latent tuberculosis refers to the prevention of reactivation of tuberculosis.
93. The method according to claim 47, wherein the treatment or prevention of latent tuberculosis refers to the delay of reactivation of tuberculosis.
94. The method according to claim 46, wherein the polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO:1.
95. The method according to claim 47, wherein the polynucleotide comprises a nucleic acid sequence at least 90% identical to SEQ ID NO:2.
Description:
FIELD OF THE INVENTION
[0001] The present invention relates to polypeptides and polynucleotides for use in the treatment or prevention of tuberculosis, in particular for use in the treatment or prevention of latent tuberculosis and in the prevention or delay of reactivation of tuberculosis (and also to related methods). The present invention further relates to pharmaceutical and immunogenic compositions comprising said polypeptides and polynucleotides, and to methods for the diagnosis of tuberculosis (in particular latent tuberculosis).
BACKGROUND OF THE INVENTION
[0002] Tuberculosis (TB) is a chronic infectious disease caused by infection with Mycobacterium tuberculosis and other Mycobacterium species. It is a major disease in developing countries, as well as an increasing problem in developed areas of the world. More than 2 billion people are believed to be infected with TB bacilli, with about 9.2 million new cases of TB and 1.7 million deaths each year. 10% of those infected with TB bacilli will develop active TB, each person with active TB infecting an average of 10 to 15 others per year. While annual incidence rates have peaked globally, the number of deaths and cases is still rising due to population growth (World Health Organisation Tuberculosis Facts 2008).
[0003] Mycobacterium tuberculosis infects individuals through the respiratory route. Alveolar macrophages engulf the bacterium, but it is able to survive and proliferate by inhibiting phagosome fusion with acidic lysosomes. A complex immune response involving CD4+ and CD8+ T cells ensues, ultimately resulting in the formation of a granuloma. Central to the success of Mycobacterium tuberculosis as a pathogen is the fact that the isolated, but not eradicated, bacterium may persist for long periods, leaving an individual vulnerable to the later development of active TB.
[0004] Fewer than 5% of infected individuals develop active TB in the first years after infection. The granuloma can persist for decades and is believed to contain live Mycobacterium tuberculosis in a state of dormancy, deprived of oxygen and nutrients. However, recently it has been suggested that the majority of the bacteria in the dormancy state are located in non-macrophage cell types spread throughout the body (Locht et al, Expert Opin. Biol. Ther. 2007 7(11):1665-1677). The development of active TB occurs when the balance between the host's natural immunity and the pathogen changes, for example as a result of an immunosuppressive event (Anderson P Trends in Microbiology 2007 15(1):7-13; Ehlers S Infection 2009 37(2):87-95).
[0005] A dynamic hypothesis describing the balance between latent TB and active TB has also been proposed (Cardana P-J Inflammation & Allergy--Drug Targets 2006 6:27-39; Cardana P-J Infection 2009 37(2):80-86).
[0006] Although an infection may be asymptomatic for a considerable period of time, the active disease is most commonly manifested as an acute inflammation of the lungs, resulting in tiredness, weight loss, fever and a persistent cough. If untreated, serious complications and death typically result.
[0007] Tuberculosis can generally be controlled using extended antibiotic therapy, although 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 behaviour 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.
[0008] Multidrug-resistant TB (MDR-TB) is a form which fails to respond to first line medications. 5% of all TB cases are MDR-TB, with an estimated 490,000 new MDR-TB cases occurring each year. Extensively drug-resistant TB (XDR-TB) occurs when resistance to second line medications develops on top of MDR-TB. It is estimated that 40,000 new cases of the virtually untreatable XDR-TB arise annually (World Health Organisation Tuberculosis Facts 2008).
[0009] Even if a full course of antibiotic treatment is completed, infection with M. tuberculosis may not be eradicated from the infected individual and may remain as a latent infection that can be reactivated.
[0010] In order to control the spread of tuberculosis, effective vaccination and accurate early diagnosis of the disease are of utmost importance.
[0011] Diagnosis of latent TB infection is commonly achieved using the tuberculin skin test, which involves intradermal exposure to tuberculin protein-purified derivative (PPD). 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 always be easily distinguished from infected individuals (this is particularly important in light of the fact that BCG does not protect against latent infection). In general, individuals who have received BCG but are not infected by M. tuberculosis show a PPD reaction below 10 mm in diameter whereas people who have a PPD reaction above 10 mm in diameter are considered to have been infected by M. tuberculosis. However, this rule is not applicable to individuals with immunosuppression due to HIV infection, which may result in a PPD reaction below 10 mm in diameter); or in endemic countries, where people infected by non-tuberculosis mycobacteria can show a PPD reaction above 10 mm in diameter.
[0012] Progress over recent years has seen the development of in vitro T cell based assays, based on interferon-gamma release and using antigens which are more specific to M. tuberculosis than PPD, namely ESAT-6 and CFP-10. These high specificity tests appear to be at least as sensitive as the tuberculin skin test and also demonstrate less cross-reactivity due to BCG vaccination. See Pai M et al Expert Rev. Mol. Diagn. 2006 6(3):413-422 for a recent review of latent TB diagnosis. However, since ESAT-6/CFP-10 are early stage antigens, assays based on ESAT-6/CFP-10 may only perform optimally in recently infected people. Consequently, the identification of new antigens specifically associated with latent tuberculosis may aid the development of more sensitive assays that could detect longer-term latent infections.
[0013] There remains a need for effective strategies for the treatment and prevention of tuberculosis, in particular the treatment and prevention of latent TB and the prevention of reactivation of TB.
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention relates generally to the identification of Rv3616c as a latent TB antigen and to related methods and uses in the prevention and treatment of latent TB, and the prevention or delay of TB reactivation.
[0015] The present invention provides a polypeptide which comprises: [0016] (i) an Rv3616c protein sequence; [0017] (ii) a variant of an Rv3616c protein sequence; or [0018] (iii) an immunogenic fragment of an Rv3616c protein sequence; for use as a medicament in the treatment or prevention of latent TB.
[0019] A further aspect of the invention relates to a method for the treatment or prevention of latent TB comprising the administration of a safe and effective amount of a polypeptide comprising: [0020] (i) an Rv3616c protein sequence; [0021] (ii) a variant of an Rv3616c protein sequence; or [0022] (iii) an immunogenic fragment of an Rv3616c protein sequence; to a subject in need thereof, wherein said polypeptide induces an immune response, in particular an immune response against Mycobacterium tuberculosis.
[0023] The use of a polypeptide comprising: [0024] (i) an Rv3616c protein sequence; [0025] (ii) a variant of an Rv3616c protein sequence; or [0026] (iii) an immunogenic fragment of an Rv3616c protein sequence; in the manufacture of a medicament for the treatment or prevention of latent TB, represents another aspect of the invention.
[0027] Also provided is a polynucleotide comprising a nucleic acid sequence encoding a polypeptide which comprises: [0028] (i) an Rv3616c protein sequence; [0029] (ii) a variant of an Rv3616c protein sequence; or [0030] (iii) an immunogenic fragment of an Rv3616c protein sequence; for use as a medicament in the treatment or prevention of latent TB.
[0031] A further aspect of the invention relates to a method for the treatment or prevention of latent TB comprising the administration of a safe and effective amount of a polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising: [0032] (i) an Rv3616c protein sequence; [0033] (ii) a variant of an Rv3616c protein sequence; or [0034] (iii) an immunogenic fragment of an Rv3616c protein sequence; to a subject in need thereof, wherein said polynucleotide induces an immune response, in particular an immune response against Mycobacterium tuberculosis.
[0035] Use of a polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising: [0036] (i) an Rv3616c protein sequence; [0037] (ii) a variant of an Rv3616c protein sequence; or [0038] (iii) an immunogenic fragment of an Rv3616c protein sequence; in the manufacture of a medicament for the treatment or prevention of latent TB, represents another aspect of the invention.
[0039] Additionally, there is provided a pharmaceutical composition comprising: [0040] (a) a polypeptide which comprises: [0041] (i) an Rv3616c protein sequence; [0042] (ii) a variant of an Rv3616c protein sequence; or [0043] (iii) an immunogenic fragment of an Rv3616c protein sequence; or [0044] (b) a polynucleotide comprising a nucleic acid sequence encoding the polypeptide of (a); [0045] and [0046] (c) a pharmaceutically acceptable carrier or excipient, for use as a medicament in the treatment or prevention of latent TB.
[0047] Further, there is provided an immunogenic composition comprising: [0048] (a) a polypeptide which comprises: [0049] (i) an Rv3616c protein sequence; [0050] (ii) a variant of an Rv3616c protein sequence; or [0051] (iii) an immunogenic fragment of an Rv3616c protein sequence; or [0052] (b) a polynucleotide comprising a nucleic acid sequence encoding the polypeptide of (a); [0053] and [0054] (c) a non-specific immune response enhancer, for use as a medicament in the treatment or prevention of latent TB.
[0055] Additionally provided is an antibody or fragment thereof which specifically binds to a polypeptide comprising: [0056] (i) an Rv3616c protein sequence; [0057] (ii) a variant of an Rv3616c protein sequence; or [0058] (iii) an immunogenic fragment of an Rv3616c protein sequence, for use in diagnosis of latent TB (such as methods for the diagnosis of latent tuberculosis comprising determining the presence of an antibody or fragment thereof which specifically binds to the polypeptides of the invention in a biological sample from a test subject).
[0059] There is also provided a method for the treatment of latent TB comprising the steps: [0060] (i) Identifying a subject as having a latent TB infection (e.g. by PPD or T cell based assays); and [0061] (ii) administering to said subject a safe and effective amount of a polypeptide or polynucleotide as described herein (such as in the form of a pharmaceutical composition or immunogenic composition).
[0062] Also provided is the use of a polypeptide of the present invention in the manufacture of a diagnostic kit for the identification of latent TB in a test subject.
[0063] In one embodiment the subject receiving a polypeptide, polynucleotide or composition may have active tuberculosis (e.g. active infection by M. tuberculosis). In a second embodiment the subject may have latent tuberculosis (e.g. dormant infection by M. tuberculosis). In a third embodiment the subject may be free from tuberculosis (e.g. free from infection by M. tuberculosis).
[0064] A subject receiving a polypeptide, polynucleotide or composition may have previously been vaccinated for tuberculosis (e.g. vaccinated against infection by M. tuberculosis), such as having been vaccinated with Bacillus Calmette-Guerin (BCG). Alternatively, a subject receiving a polypeptide, polynucleotide or composition of the invention may not have previously been vaccinated for tuberculosis (e.g. not vaccinated against infection by M. tuberculosis), such as not having been vaccinated with Bacillus Calmette-Guerin (BCG).
DESCRIPTION OF THE FIGURES
[0065] FIG. 1: Rv3616c peptide alignment with full length sequence.
[0066] FIG. 2: PBMC responses to Rv3616c peptides.
[0067] FIG. 3: Percentage of CD4 and CD8 cells from immunised CB6F1 mice expressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at day 21 (i.e. 7 days post second immunisation).
[0068] FIG. 4: Cytokine profile at day 21 (i.e. 7 days post second immunisation) of the antigen specific CD4 response in immunised CB6F1 mice.
[0069] FIG. 5: Cytokine profile at day 21 (i.e. 7 days post second immunisation) of the antigen specific CD8 response in immunised CB6F1 mice.
[0070] FIG. 6: Percentage of CD4 and CD8 cells from immunised CB6F1 mice expressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at day 35 (i.e. 7 days post third immunisation).
[0071] FIG. 7: Cytokine profile at day 35 (i.e. 7 days post third immunisation) of the antigen specific CD4 response in immunised CB6F1 mice.
[0072] FIG. 8: Cytokine profile at day 35 (i.e. 7 days post third immunisation) of the antigen specific CD8 response in immunised CB6F1 mice.
[0073] FIG. 9: Percentage of CD4 and CD8 cells from immunised C57BL/6 mice expressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at day 21 (i.e. 7 days post second immunisation).
[0074] FIG. 10: Cytokine profile at day 21 (i.e. 7 days post second immunisation) of the antigen specific CD4 response in immunised C57BL/6 mice.
[0075] FIG. 11: Percentage of CD4 and CD8 cells from immunised C57BL/6 mice expressing IFN-gamma and/or IL-2 and/or TNF-alpha cytokines at day 35 (i.e. 7 days post third immunisation).
[0076] FIG. 12: Cytokine profile at day 35 (i.e. 7 days post third immunisation) of the antigen specific CD4 response in immunised C57BL/6 mice.
[0077] FIG. 13: Cytokine profile at day 35 (i.e. 7 days post third immunisation) of the antigen specific CD8 response in immunised C57BL/6 mice.
[0078] FIG. 14: Antigen-specific CD4 T cell responses in naive and latently infected humans.
DESCRIPTION OF THE LISTED SEQUENCES
[0079] SEQ ID No: 1: polypeptide sequence of Rv3616c from M. tuberculosis H37Rv strain.
[0080] SEQ ID No: 2: polynucleotide sequence of Rv3616c from M. tuberculosis H37Rv strain.
[0081] SEQ ID No: 3: polypeptide sequence of Rv3616c from M. tuberculosis CDC1551 strain.
[0082] SEQ ID No: 4: polypeptide sequence of Rv3616c from M. tuberculosis F11 strain.
[0083] SEQ ID No: 5: polypeptide sequence of Rv3616c from M. tuberculosis Haarlem A strain.
[0084] SEQ ID No: 6: polypeptide sequence of Rv3616c from M. tuberculosis C strain.
[0085] SEQ ID No: 7: polypeptide sequence of Rv3616c from BCG.
[0086] SEQ ID No: 8: polypeptide sequence of Mtb8.4.
[0087] SEQ ID No: 9: polypeptide sequence of Mtb9.8.
[0088] SEQ ID No: 10: polypeptide sequence of Mtb9.9.
[0089] SEQ ID No: 11: polypeptide sequence of Ra12.
[0090] SEQ ID No: 12: polypeptide sequence of Ra35.
[0091] SEQ ID No: 13: polypeptide sequence of TbH9.
[0092] SEQ ID No: 14: polypeptide sequence of Mtb41.
[0093] SEQ ID No: 15: polypeptide sequence of ESAT-6.
[0094] SEQ ID No: 16: polypeptide sequence of Ag85A.
[0095] SEQ ID No: 17: polypeptide sequence of Ag85B.
[0096] SEQ ID No: 18: polypeptide sequence of alpha-crystallin.
[0097] SEQ ID No: 20: polypeptide sequence of Mtb32A.
[0098] SEQ ID No: 21: polypeptide sequence of Ser/Ala mutated mature Mtb32A.
[0099] SEQ ID No: 22: polypeptide sequence of TB10.4.
[0100] SEQ ID No: 23: polypeptide sequence of Mtb72f.
[0101] SEQ ID No: 24: polypeptide sequence of M72.
[0102] SEQ ID No: 25: polypeptide sequence of Mtb71f.
[0103] SEQ ID No: 26: polypeptide sequence of M92 fusion.
[0104] SEQ ID No: 27: polypeptide sequence of M103 fusion.
[0105] SEQ ID No: 28: polypeptide sequence of M114 fusion.
[0106] SEQ ID No: 29: putative human CD4 cell epitope 1.
[0107] SEQ ID No: 30: putative human CD4 cell epitope 2.
[0108] SEQ ID No: 31: putative human CD4 cell epitope 3.
[0109] SEQ ID No: 32: putative human CD4 cell epitope 4.
[0110] SEQ ID No: 33: putative human CD4 cell epitope 5.
[0111] SEQ ID No: 34: putative human CD4 cell epitope 6.
[0112] SEQ ID No: 35: putative human CD4 cell epitope 7.
[0113] SEQ ID No: 36: putative human CD4 cell epitope 8.
[0114] SEQ ID No: 37: putative human CD4 cell epitope 9.
[0115] SEQ ID No: 38: putative human CD4 cell epitope 10.
[0116] SEQ ID No: 39: putative human CD4 cell epitope 11.
[0117] SEQ ID No: 40: putative human CD4 cell epitope 12.
[0118] SEQ ID No: 41: putative human CD4 cell epitope 13.
[0119] SEQ ID No: 42: putative human CD4 cell epitope 14.
[0120] SEQ ID No: 43: putative human CD4 cell epitope 15.
[0121] SEQ ID No: 44: putative human CD4 cell epitope 16.
[0122] SEQ ID No: 45: putative human CD4 cell epitope 17.
[0123] SEQ ID No: 46: putative human CD4 cell epitope 18.
[0124] SEQ ID No: 47: putative human CD4 cell epitope 19.
[0125] SEQ ID No: 48: putative human CD8 cell epitope 1.
[0126] SEQ ID No: 49: putative human CD8 cell epitope 2.
[0127] SEQ ID No: 50: putative human CD8 cell epitope 3.
[0128] SEQ ID No: 51: putative human CD8 cell epitope 4.
[0129] SEQ ID No: 52: putative human CD8 cell epitope 5.
[0130] SEQ ID No: 53: putative human CD8 cell epitope 6.
[0131] SEQ ID No: 54: putative human CD8 cell epitope 7.
[0132] SEQ ID No: 55: putative human CD8 cell epitope 8.
[0133] SEQ ID No: 57: putative human CD8 cell epitope 10.
[0134] SEQ ID No: 58: putative human CD8 cell epitope 11.
[0135] SEQ ID No: 59: putative human CD8 cell epitope 12.
[0136] SEQ ID No: 60: putative human CD8 cell epitope 13.
[0137] SEQ ID No: 61: putative human CD8 cell epitope 14.
[0138] SEQ ID No: 62: putative human CD8 cell epitope 15.
[0139] SEQ ID No: 63: putative human CD8 cell epitope 16.
[0140] SEQ ID No: 64: putative human CD8 cell epitope 17.
[0141] SEQ ID No: 65: putative human CD8 cell epitope 18.
[0142] SEQ ID No: 66: putative human CD8 cell epitope 19.
[0143] SEQ ID No: 67: putative human CD8 cell epitope 20.
[0144] SEQ ID No: 68: putative human CD8 cell epitope 21.
[0145] SEQ ID No: 69: putative human CD8 cell epitope 22.
[0146] SEQ ID No: 70: putative human CD8 cell epitope 23.
[0147] SEQ ID No: 71: putative human CD8 cell epitope 24.
[0148] SEQ ID No: 72: putative human CD8 cell epitope 25.
[0149] SEQ ID No: 73: putative human CD8 cell epitope 26.
[0150] SEQ ID No: 74: putative human CD8 cell epitope 27.
[0151] SEQ ID No: 75: putative human CD8 cell epitope 28.
[0152] SEQ ID No: 76: putative human CD8 cell epitope 29.
[0153] SEQ ID No: 77: putative human CD8 cell epitope 30.
[0154] SEQ ID No: 78: putative human CD8 cell epitope 31.
[0155] SEQ ID No: 79: putative human CD8 cell epitope 32.
[0156] SEQ ID No: 80: putative human CD8 cell epitope 33.
[0157] SEQ ID No: 81: putative human CD8 cell epitope 34.
[0158] SEQ ID No: 82: putative human CD8 cell epitope 35.
[0159] SEQ ID No: 83: putative human CD8 cell epitope 36.
[0160] SEQ ID No: 84: putative human CD8 cell epitope 37.
[0161] SEQ ID No: 85: putative human CD8 cell epitope 38.
[0162] SEQ ID No: 86: putative human CD8 cell epitope 39.
[0163] SEQ ID No: 87: putative human CD8 cell epitope 40.
[0164] SEQ ID No: 88: putative human CD8 cell epitope 41.
[0165] SEQ ID No: 89: putative human CD8 cell epitope 42.
[0166] SEQ ID No: 90: putative human CD8 cell epitope 43.
[0167] SEQ ID No: 91: putative human CD8 cell epitope 44.
[0168] SEQ ID No: 92: putative human CD8 cell epitope 45.
[0169] SEQ ID No: 94: putative human CD8 cell epitope 47.
[0170] SEQ ID No: 95: putative human CD8 cell epitope 48.
[0171] SEQ ID No: 96: putative human CD8 cell epitope 49.
[0172] SEQ ID No: 97: putative human CD8 cell epitope 50.
[0173] SEQ ID No: 98: putative human CD8 cell epitope 51.
[0174] SEQ ID No: 99: putative human CD8 cell epitope 52.
[0175] SEQ ID No: 100: putative human CD8 cell epitope 53.
[0176] SEQ ID No: 101: putative human CD8 cell epitope 54.
[0177] SEQ ID No: 102: putative human CD8 cell epitope 55.
[0178] SEQ ID No: 103: putative human CD8 cell epitope 56.
[0179] SEQ ID No: 104: putative human CD8 cell epitope 57.
[0180] SEQ ID No: 105: putative human CD8 cell epitope 58.
[0181] SEQ ID No: 106: putative human CD8 cell epitope 59.
[0182] SEQ ID No: 107: putative human CD8 cell epitope 60.
[0183] SEQ ID No: 108: putative human CD8 cell epitope 61.
[0184] SEQ ID No: 109: putative human CD8 cell epitope 62.
[0185] SEQ ID No: 110: putative human CD8 cell epitope 63.
[0186] SEQ ID No: 111: putative human CD8 cell epitope 64.
[0187] SEQ ID No: 112: putative human CD8 cell epitope 65.
[0188] SEQ ID No: 113: putative human CD8 cell epitope 66.
[0189] SEQ ID No: 114: putative human CD8 cell epitope 67.
[0190] SEQ ID No: 115: putative human CD8 cell epitope 68.
[0191] SEQ ID No: 116: putative human CD8 cell epitope 69.
[0192] SEQ ID No: 117: putative human CD8 cell epitope 70.
[0193] SEQ ID No: 118: putative human CD8 cell epitope 71.
[0194] SEQ ID No: 119: putative human CD8 cell epitope 72.
[0195] SEQ ID No: 120: putative human CD8 cell epitope 73.
[0196] SEQ ID No: 121: putative human CD8 cell epitope 74.
[0197] SEQ ID No: 122: putative human CD8 cell epitope 75.
[0198] SEQ ID No: 123: putative human CD8 cell epitope 76.
[0199] SEQ ID No: 124: putative human CD8 cell epitope 77.
[0200] SEQ ID No: 125: putative human CD8 cell epitope 78.
[0201] SEQ ID No: 126: putative human CD8 cell epitope 79.
[0202] SEQ ID No: 127: peptide 1.
[0203] SEQ ID No: 128: peptide 2.
[0204] SEQ ID No: 129: peptide 3.
[0205] SEQ ID No: 131: peptide 5.
[0206] SEQ ID No: 132: peptide 6.
[0207] SEQ ID No: 133: peptide 7.
[0208] SEQ ID No: 134: peptide 8.
[0209] SEQ ID No: 135: peptide 9.
[0210] SEQ ID No: 136: peptide 10.
[0211] SEQ ID No: 137: peptide 11.
[0212] SEQ ID No: 138: peptide 12.
[0213] SEQ ID No: 139: peptide 13.
[0214] SEQ ID No: 140: peptide 14.
[0215] SEQ ID No: 141: peptide 15.
[0216] SEQ ID No: 142: peptide 16.
[0217] SEQ ID No: 143: peptide 17.
[0218] SEQ ID No: 144: peptide 18.
[0219] SEQ ID No: 145: peptide 19.
[0220] SEQ ID No: 146: peptide 20.
[0221] SEQ ID No: 147: peptide 21.
[0222] SEQ ID No: 148: peptide 22.
[0223] SEQ ID No: 149: peptide 23.
[0224] SEQ ID No: 150: peptide 24.
[0225] SEQ ID No: 151: peptide 25.
[0226] SEQ ID No: 152: peptide 26.
[0227] SEQ ID No: 153: peptide 27.
[0228] SEQ ID No: 154: peptide 28.
[0229] SEQ ID No: 155: peptide 29.
[0230] SEQ ID No: 156: peptide 30.
[0231] SEQ ID No: 157: polypeptide sequence of Rv1753c from M. tuberculosis H37Rv strain.
[0232] SEQ ID No: 158: polypeptide sequence of Rv2386c from M. tuberculosis H37Rv strain.
[0233] SEQ ID No: 159: polypeptide sequence of Rv2707c from M. tuberculosis H37Rv strain.
DETAILED DESCRIPTION
[0234] 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 M. bovis which was developed over 60 years ago. However, the safety and efficacy of BCG is a source of controversy--while protecting against severe disease manifestation in children, BCG does not prevent the establishment of latent TB or reactivation of pulmonary disease in adult life. Additionally, some countries, such as the United States, do not vaccinate the general public with this agent.
[0235] Almost all new generation TB vaccines which are currently in clinical development have been designed as pre-exposure vaccines. These include subunit vaccines, which have been particularly effective in boosting immunity induced by prior BCG vaccination, and advanced live mycobacterial vaccines which aim to replace BCG with more efficient and/or safer strains. Although these vaccines aim to improve resistance to infection, they are likely to be less effective as post-exposure or therapeutic vaccines in latent TB cases (Lin M Y et al Endocrine, Metabolic & Immune Disorders--Drug Targets 2008 8:15-29).
[0236] Several of the proteins which are strongly expressed during the early stages of Mycobacterium infection have been shown to provide strong protective efficacy in animal vaccination models. However, vaccination with antigens which are highly expressed during the early stages of infection may not provide an optimal immune response for dealing with later stages of infection. Adequate control during the later stages of infection may require T cells which are specific for the particular antigens which are expressed at that time.
[0237] Post-exposure vaccines which directly target the dormant persistent bacteria may aid in protecting against TB reactivation, thereby enhancing TB control, or even enabling clearance of the infection. A vaccine targeting latent TB could therefore significantly and economically reduce global TB infection rates.
[0238] Subunit vaccines based on late stage antigens could also be utilised in combination with early stage antigens to provide a multiphase vaccine. Alternatively, late stage antigens could be used to complement and improve BCG vaccination (either by boosting the BCG response or through the development of advanced recombinant BCG strains).
[0239] Rv3616c, also known as Mtb40 or HTCC1, has previously been implicated in the immune responses associated with tuberculosis (see, for example, WO98/53075). Al-Attiyah et al. Clin. Exp. Immunol. 2004 138:139-144 have shown that Rv3616c is well recognised (through PMBC proliferation and IFN-gamma production) by pulmonary tuberculosis patients. Mustafa et al. Infect. Immun. 2006 74(8):4566-4572 have investigated the recognition of Rv3616c by M. bovis infected and BCG vaccinated cattle.
[0240] Recently, a range of M. tuberculosis vaccine candidates have been proposed based on a bioinformatics analysis of the whole genome M. tuberculosis genome (Zvi et al. BMC Medical Genetics 2008 1:18) and on the testing of differentially expressed proteins in actively and latently infected individuals (Schuck S D et al. PLoS ONE 2009 4(5):e5590).
[0241] While macrophages have been shown to act as the principal effectors of Mycobacterium immunity, T cells are the predominant inducers of such immunity. The essential role of T cells in protection against tuberculosis is illustrated by the increased rates of TB reactivation in human immunodeficiency virus infected individuals, due to the associated depletion of CD4+ T cells. Furthermore, adoptive transfer of CD4+ T cells taken at the height of the primary immune response to M. tuberculosis has been shown to confer protection against M. tuberculosis in T cell deficient mice (Orme et al J. Exp. Med. 1983 158:74-83).
[0242] Mycobacterium-reactive CD4+ T cells have been shown to be potent producers of γ-interferon (IFN-γ), which, in turn, has been shown to trigger the anti-mycobacterial effects of macrophages in mice (Flynn et al. J. Exp. Med. 1993 178:2249-2254). 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, interleukin-12 (IL-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 & Kaufmann, Tuberculosis: Pathogenesis, Protection and Control (Bloom ed., 1994), Tuberculosis (2nd ed., Rom and Garay, eds., 2003), and Harrison's Principles of Internal Medicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds., 2005).
[0243] The present invention relates generally to the identification of Rv3616c as a TB antigen which is associated with latent TB and to related methods and uses in the prevention and treatment of latent TB, and the prevention or delay of TB reactivation.
[0244] The invention therefore provides an Rv3616c protein, variant thereof or immunogenic fragment thereof, or a polynucleotide encoding said protein, variant or fragment, for use in the treatment or prevention of latent TB. Suitably, the use may be specifically in the treatment of latent TB. Alternatively, the use may be in the prevention or delay of TB reactivation (especially the delay of TB reactivation, for example by a period of months, years or even indefinitely).
[0245] The term "Mycobacterium species of the tuberculosis complex" includes those species traditionally considered as causing the disease tuberculosis, as well as Mycobacterium environmental and opportunistic species that cause tuberculosis and lung disease in immune compromised patients, such as patients with AIDS, e.g., M. tuberculosis, M. bovis, or M. africanum, BCG, M. avium, M. intracellulare, M. celatum, M. genavense, M. haemophilum, M. kansasii, M. simiae, M. vaccae, M. fortuitum, and M. scrofulaceum (see, e.g., Harrison's Principles of Internal Medicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds., 2005). The present invention is particularly directed to infection with M. tuberculosis.
[0246] The term "active infection" refers to an infection (e.g. infection by M. tuberculosis) with manifested disease symptoms and/or lesions (suitably with manifested disease symptoms).
[0247] The terms "inactive infection", "dormant infection" or "latent infection" refer to an infection (e.g. infection by M. tuberculosis) without manifested disease symptoms and/or lesions (suitably without manifested disease symptoms).
[0248] The term "primary tuberculosis" refers to clinical illness (manifestation of disease symptoms) directly following infection (e.g. infection by M. tuberculosis). See, Harrison's Principles of Internal Medicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds., 2005).
[0249] The terms "secondary tuberculosis" or "postprimary tuberculosis" refer to the reactivation of a dormant, inactive or latent infection (e.g. infection by M. tuberculosis). See, Harrison's Principles of Internal Medicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al., eds., 2005).
[0250] The term "tuberculosis reactivation" refers to the later manifestation of disease symptoms in an individual that tests positive for infection (e.g. in a tuberculin skin test, suitably in an in vitro T cell based assay) test but does not have apparent disease symptoms. The positive diagnostic test indicates that the individual is infected, however, the individual may or may not have previously manifested active disease symptoms that had been treated sufficiently to bring the tuberculosis into an inactive or latent state. It will be recognised that methods for the prevention, delay or treatment of tuberculosis reactivation can be initiated in an individual manifesting active symptoms of disease.
[0251] The term "drug resistant" tuberculosis refers to an infection (e.g. infection by M. tuberculosis) wherein the infecting strain is not held static or killed (i.e. is resistant to) one or more of so-called "front-line" chemotherapeutic agents effective in treating tuberculosis (e.g., isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide).
[0252] The term "multi-drug resistant" tuberculosis refers to an infection (e.g. infection by M. tuberculosis) wherein the infecting strain is resistant to two or more of "front-line" chemotherapeutic agents effective in treating tuberculosis.
[0253] A "chemotherapeutic agent" refers to a pharmacological agent known and used in the art to treat tuberculosis (e.g. infection by M. tuberculosis). Exemplified pharmacological agents used to treat tuberculosis include, but are not limited to amikacin, aminosalicylic acid, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, kanamycin, pyrazinamide, rifamycins (i.e., rifampin, rifapentine and rifabutin), streptomycin, ofloxacin, ciprofloxacin, clarithromycin, azithromycin and fluoroquinolones. "First-line" or "Front-line" chemotherapeutic agents used to treat tuberculosis that is not drug resistant include isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide. "Second-line" chemotherapeutic agents used to treat tuberculosis that has demonstrated drug resistance to one or more "first-line" drugs include ofloxacin, ciprofloxacin, ethionamide, aminosalicylic acid, cycloserine, amikacin, kanamycin and capreomycin. Such pharmacological agents are reviewed in Chapter 48 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, Hardman and Limbird eds., 2001.
[0254] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Suitably a polypeptide according to the present invention will consist only of naturally occurring amino acid residues, especially those amino acids encoded by the genetic code.
[0255] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogues refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
[0256] Suitably an amino acid is a naturally occurring amino acid or an amino acid analogue, especially a naturally occurring amino acid and in particular those amino acids encoded by the genetic code.
[0257] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Suitably the term "nucleic acid" refers to naturally occurring deoxyribonucleotides or ribonucleotides and polymers thereof.
[0258] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated (suitably it refers to the sequence explicitly indicated). Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
[0259] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
[0260] By the term `Rv3616c protein sequence` as used herein is meant the polypeptide sequence provided in SEQ ID No: 1 or a homologue thereof from a Mycobacterium species of the tuberculosis complex, e.g., a species such as M. tuberculosis, M. bovis, or M. africanum, or a Mycobacterium species that is environmental or opportunistic and that causes opportunistic infections such as lung infections in immune compromised hosts (e.g., patients with AIDS), e.g., BCG, M. avium, M. intracellulare, M. celatum, M. genavense, M. haemophilum, M. kansasii, M. simiae, M. vaccae, M. fortuitum, and M. scrofulaceum (see, e.g., Harrison's Principles of Internal Medicine, Chapter 150, pp. 953-966, 16th ed., Braunwald, et al., eds., 2005).
[0261] To ensure a high efficacy rate among vaccinated hosts, the components of a vaccine should be well conserved among the stains of clinical significance. Suitably, the Rv3616c protein is derived from M. tuberculosis H37Rv (i.e. the polypeptide sequence provided in SEQ ID No: 1) or a homologue thereof from another M. tuberculosis strain (such as CDC1551, F11, Haarlem A and C strains). Strains of M. tuberculosis which are associated with drug resistance (e.g. MDR or especially XDR) are a particularly valuable basis for the Rv3616c protein sequence.
Strains of interest include: [0262] CDC1551--transmissible and virulent strain [0263] Haarlem family (such as Haarlem A)--Drug resistant strains found in crowded human populations. Members of the Haarlem family of M. tuberculosis strains have been found in many parts of the world. The first representative of the family was discovered in Haarlem, The Netherlands. [0264] KZN4207-- Drug sensitive isolate from patients in KwaZulu-Natal, South Africa [0265] KZN1435--Multiple drug resistant (MDR) isolate from patients in KwaZulu-Natal, South Africa [0266] KZN605-- Extensively drug resistant (XDR) isolate from patients in KwaZulu-Natal, South Africa [0267] C--Highly transmitted in New York City. In one study this strain was found to be more common among injection drug users and resistant to reactive nitrogen intermediates (Friedman et al. J. Infect. Dis. 1997 176(2):478-84) [0268] 94_M4241A--Isolated in San Francisco in 1994 from a patient born in China. This strain was previously analysed by genomic deletion analysis (Gagneux et al., PNAS 2006 103(8):2869-2873). [0269] 02--1987--Isolated in San Francisco in 2002 from a patient born in South Korea. This strain was previously analyzed by genomic deletion analysis (Gagneux et al., PNAS 2006 103(8):2869-2873). [0270] T92--Isolated in San Francisco in 1999 from a patient born in The Philippines. This strain was published in Hirsh et al. PNAS 2004 101:4871-4876). [0271] T85--Isolated in San Francisco in 1998 from a patient born in China. This strain was published in Hirsh et al. PNAS 2004 101:4871-4876). [0272] EAS054--Isolated in San Francisco in 1993 from a patient born in India. This strain was previously analyzed by genomic deletion analysis (Gagneux et al., PNAS 2006 103(8):2869-2873).
[0273] Gagneux et al., PNAS 2006 103(8):2869-2873 and Herbert et al. Infect. Immun. 2007 75(12):5798-5805 provide valuable background on the range of M. tuberculosis strains which are known to exist.
[0274] Most suitably, the Rv3616c protein is selected from the polypeptide sequences provided in SEQ ID Nos: 1 and 3-7, in particular SEQ ID Nos: 1 and 3-6, such as SEQ ID No: 1.
[0275] Polynucleotides of particular interest are those comprising (such as consisting of) a sequence encoding: [0276] (i) an Rv3616c protein sequence; [0277] (ii) a variant of an Rv3616c protein sequence; or [0278] (iii) an immunogenic fragment of an Rv3616c protein sequence. Polynucleotides will suitably comprise (such as consisting of) a variant of SEQ ID NO: 2 or fragment of SEQ ID NO: 2 which encodes an immunogenic fragment of an Rv3616c protein.
Combinations
[0279] The Rv3616c related polypeptides of use in the present invention can further comprise other components designed to enhance their immunogenicity or to improve these antigens in other respects. For example, improved isolation of the polypeptide antigens may be facilitated through the addition of a stretch of histidine residues (commonly known as a his-tag) towards one end of the antigen.
[0280] The term "his-tag" refers to a string of histidine residues, typically six residues, that are inserted within the reference sequence. To minimise disruption of the activity associated with the reference sequence, a his-tag is typically inserted at the N-terminus, usually immediately after the initiating methionine residue, or else at the C-terminus. They are usually heterologous to the native sequence but are incorporated since they facilitate isolation by improving the protein binding to immobilised metal affinity chromatography resins (IMAC). Generally speaking the presence or absence of a his-tag is not of significance from the point of view of eliciting a desirable immune response against the reference protein. However, to avoid the risk of an adverse reaction against the his-tag itself, it is considered best to minimise the length of the his-tag e.g. to four or fewer residues, in particular two residues (or to exclude the use of a his-tag entirely).
[0281] To improve the magnitude and/or breadth of the elicited immune response the compositions, polypeptides and nucleic acids can comprise multiple copies of the inventive antigen and/or additional heterologous polypeptides (or polynucleotides encoding them) from Mycobacterium species (in particular M. tuberculosis).
[0282] One skilled in the art will recognise that when a number of components are utilised in combination, the precise presentation can be varied. For example, a Rv3616c component and an additional copy of the antigen or an additional heterologous antigen component could be presented: [0283] (1) as two individual polypeptide components; [0284] (2) as a fusion protein comprising both polypeptide components; [0285] (3) as one polypeptide and one polynucleotide component; [0286] (4) as two individual polynucleotide components; [0287] (5) as a single polynucleotide encoding two individual polypeptide components; or [0288] (6) as a single polynucleotide encoding a fusion protein comprising both polypeptide components.
[0289] This flexibility applies equally to situations where three or more components are used in combination. However, for convenience, it is often desirable that when a number of components are present they are contained within a single fusion protein or a polynucleotide encoding a single fusion protein. In one embodiment of the invention all antigen components are provided as polypeptides (e.g. within a single fusion protein). In an alternative embodiment of the invention all antigen components are provided as polynucleotides (e.g. a single polynucleotide, such as one encoding a single fusion protein).
[0290] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
[0291] "Fusion polypeptide" or "fusion protein" refers to a protein having at least two heterologous polypeptides (e.g. at least two Mycobacterium sp. polypeptides) covalently linked, either directly or via an amino acid linker. The polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein can be in any order. This term also refers to conservatively modified variants, polymorphic variants, alleles, mutants, immunogenic fragments, and interspecies homologs of the antigens that make up the fusion protein. Mycobacterium tuberculosis antigens are described in Cole et al., Nature 393:537 (1998), which discloses the entire Mycobacterium tuberculosis genome. Antigens from other Mycobacterium species that correspond to M. tuberculosis antigens can be identified, e.g., using sequence comparison algorithms, as described herein, or other methods known to those of skill in the art, e.g., hybridisation assays and antibody binding assays.
[0292] The term "fused" refers to the covalent linkage between two polypeptides in a fusion protein. The polypeptides are typically joined via a peptide bond, either directly to each other or via an amino acid linker. Optionally, the peptides can be joined via non-peptide covalent linkages known to those of skill in the art.
[0293] Exemplary M. tuberculosis antigens which may be combined with Rv3616c include one or more of (e.g. 1 to 5, such as 1 to 3, in particular 1) the following (such as one or more of (i) to (xi)): [0294] (i) Mtb8.4 (also known as DPV and Rv1174c), the polypeptide sequence of which is described in SEQ ID No: 102 of WO97/09428 (cDNA in SEQ ID No: 101) and in Coler et al Journal of Immunology 1998 161:2356-2364. Of particular interest is the mature Mtb8.4 sequence which is absent the leading signal peptide (i.e. amino acid residues 15-96 from SEQ ID No: 102 of WO97/09428). The full-length polypeptide sequence of Mtb8.4 is shown in SEQ ID No: 8; [0295] (ii) Mtb9.8 (also known as MSL and Rv0287), the polypeptide sequence of which is described in SEQ ID No: 109 of WO98/53075 (fragments of MSL are disclosed in SEQ ID Nos: 110-124 of WO98/53075, SEQ ID Nos: 119 and 120 being of particular interest) and also in Coler et al Vaccine 2009 27:223-233 (in particular the reactive fragments shown in FIG. 2 therein). The full-length polypeptide sequence for Mtb9.8 is shown in SEQ ID No: 9; [0296] (iii) Mtb9.9 (also known as Mtb9.9A, MTI, MTI-A and Rv1793) the polypeptide sequence of which is described in SEQ ID No: 19 of WO98/53075 and in Alderson et al Journal of Experimental Medicine 2000 7:551-559 (fragments of MTI are disclosed in SEQ ID Nos: 17 and 51-66 of WO98/53075, SEQ ID Nos: 17, 51, 52, 53, 56 and 62-65 being of particular interest). A number of polypeptide variants of MTI are described in SEQ ID Nos: 21, 23, 25, 27, 29 and 31 of WO98/53075 and in Alderson et al Journal of Experimental Medicine 2000 7:551-559. The full-length polypeptide sequence for Mtb9.9 is shown in SEQ ID No: 10; [0297] (iv) Ra12 (also known as Mtb32A C-terminal antigen) the polypeptide sequence of which is described in SEQ ID No: 10 of WO01/98460 and in Skeiky et al Journal of Immunology 2004 172:7618-7682. The full-length polypeptide sequence for Ra12 is shown in SEQ ID No: 11; [0298] (v) Ra35 (also known as Mtb32A N-terminal antigen) the polypeptide sequence of which is described in SEQ ID No: 8 of WO01/98460 and in Skeiky et al Journal of Immunology 2004 172:7618-7682. The full-length polypeptide sequence for Ra35 is shown in SEQ ID No: 12; [0299] (vi) TbH9 (also known as Mtb39, Mtb39A, TbH9FL and Rv1196) the polypeptide sequence of which is described in SEQ ID No: 107 of WO97/09428, and also in Dillon et al Infection and Immunity 1999 67(6):2941-2950 and Skeiky et al Journal of Immunology 2004 172:7618-7682. The full-length polypeptide sequence for TbH9 is shown in SEQ ID No: 13; [0300] (vii) Mtb41 (also known as MTCC2 and Rv0915c) the polypeptide sequence of which is described in SEQ ID No: 142 of WO98/53075 (cDNA in SEQ ID No: 140) and in Skeiky et al Journal of Immunology 2000 165:7140-7149. The full-length polypeptide sequence for Mtb41 is shown in SEQ ID No: 14; [0301] (viii) ESAT-6 (also known as esxA and Rv3875) the polypeptide sequence of which is described in SEQ ID No: 103 of WO97/09428 (cDNA in SEQ ID No: 104) and in Sorensen et al Infection and Immunity 1995 63(5):1710-1717. The full-length polypeptide sequence for ESAT-6 is shown in SEQ ID No: 15; [0302] (ix) Ag85 complex antigens (e.g. Ag85A, also known as fbpA and Rv3804c; or Ag85B, also known as fbpB and Rv1886c) which are discussed, for example, in Content et al Infection and Immunity 1991 59:3205-3212 and in Huygen et al Nature Medicine 1996 2(8):893-898. The full-length polypeptide sequence for Ag85A is shown in SEQ ID No: 16 (the mature protein of residues 43-338, i.e. lacking the signal peptide, being of particular interest). The full-length polypeptide sequence for Ag85B is shown in SEQ ID No: 17 (the mature protein of residues 41-325, i.e. lacking the signal peptide, being of particular interest); [0303] (x) Alpha-crystallin (also known as hspX and Rv2031 c) which is described in Verbon et al Journal of Bacteriology 1992 174:1352-1359 and Friscia et al Clinical and Experimental Immunology 1995 102:53-57 (of particular interest are the fragments corresponding to residues 71-91, 21-40, 91-110 and 111-130)). The full-length polypeptide sequence for alpha-crystallin is shown in SEQ ID No: 18; [0304] (xi) Mpt64 (also known as Rv1980c) which is described in Roche et al Scandinavian Journal of Immunology 1996 43:662-670. The full-length polypeptide sequence for MPT64 is shown in SEQ ID No: 19 (the mature protein of residues 24-228, i.e. lacking the signal peptide, being of particular interest): [0305] (xii) Mtb32A, the polypeptide sequence of which is described in SEQ ID No: 2 (full-length) and residues 8-330 of SEQ ID No: 4 (mature) of WO01/98460, especially variants having at least one of the catalytic triad mutated (e.g. the catalytic serine residue, which may for example be mutated to alanine). The full-length polypeptide sequence for Mtb32A is shown in SEQ ID No: 20. The mature form of Mtb32A having a Ser/Ala mutation is shown in SEQ ID No: 21; [0306] (xiii) TB10.4, the full-length polypeptide sequence for TB10.4 is shown in SEQ ID No: 22; [0307] (xiv) Rv1753c, the full-length polypeptide sequence for Rv1753c from Mycobacterium tuberculosis H37Rv is shown in SEQ ID No: 157; [0308] (xv) Rv2386c, the full-length polypeptide sequence for Rv2386c from Mycobacterium tuberculosis H37Rv is shown in SEQ ID No: 158; and/or [0309] (xvi) Rv2707c, the full-length polypeptide sequence for Rv2707c from Mycobacterium tuberculosis H37Rv is shown in SEQ ID No: 159. or combinations thereof, such as (for example combinations such as (a) to (g)): [0310] (a) a combination of Ra12, TbH9 and Ra35 components, for example in the form of a fusion protein, such as Mtb72f. The polypeptide sequence of Mtb72f is described in SEQ ID No: 6 of WO2006/117240 (cDNA in SEQ ID No: 5) and in Skeiky et al Journal of Immunology 2004 172:7618-7682 (where it incorporates an optional His-tag to aid purification, when utilised in the present invention suitably Mtb72f is absent the optional histidine residues). The polypeptide sequence for Mtb72f is shown in SEQ ID No: 23; [0311] (b) a combination of Ra12, TbH9 and Ser/Ala mutated Ra35 (i.e. where the catalytic serine residue has been replaced with alanine) components, for example in the form of a fusion protein, such as M72. The polypeptide sequence of M72 is described in SEQ ID No: 4 of WO2006/117240 (cDNA in SEQ ID No: 3) where it incorporates an optional double histidine to aid manufacture, when utilised in the present invention M72 may also incorporate a double histidine though suitably M72 is absent the optional double histidine (i.e. residues 4-725 from SEQ ID No: 4 of WO2006/117240 are of particular interest). The polypeptide sequence for M72 is shown in SEQ ID No: 24; [0312] (c) a combination of Mtb8.4, Mtb9.8, Mtb9.9 and Mtb41 components, for example in the form of a fusion protein, such as Mtb71f. The polypeptide sequence of Mtb71f is described in SEQ ID No: 16 of WO99/051748 (cDNA in SEQ ID No: 15), where it incorporates an optional His-tag to aid purification, when utilised in the present invention suitably Mtb71f corresponds to amino acid residues 9-710 of SEQ ID NO: 16 from WO99/051748. The polypeptide sequence for Mtb71f is shown in SEQ ID No: 25; [0313] (d) a combination of Mtb72f or M72 (suitably without optional histidine residues to aid expression) with Mtb9.8 and Mtb9.9, for example in a fusion protein. The polypeptide sequence for an M72-Mtb9.9-Mtb9.8 fusion is shown in SEQ ID No: 26 (M92 fusion), when used in the present invention, the M72-Mtb9.9-Mtb9.8 fusion may optionally incorporate a double histidine following the initiating methionine residue to aid manufacture; [0314] (e) a combination of Mtb72f or M72 (suitably without optional histidine residues to aid expression) with Ag85B, for example in a fusion protein, such Mtb103f. The polypeptide sequence of Mtb103f is described in SEQ ID No: 18 of WO03/070187 (cDNA in SEQ ID No: 10), where it incorporates an optional His-tag to aid purification, when utilised in the present invention suitably Mtb103f corresponds to amino acid residues 8-1016 of SEQ ID No: 18 from WO03/070187. Also of particular interest is M103, i.e. Mtb103f incorporating a Ser/Ala mutation in the Ra35 component, when utilised in the present invention suitably M103 corresponds to amino acid residues 8-1016 of SEQ ID No: 18 from WO03/070187 wherein the Ser residue at position 710 has been replaced with Ala. The polypeptide sequence for M103 is shown in SEQ ID No: 27, when used in the present invention, the M72-Mtb9.9-Mtb9.8 fusion may optionally incorporate a double histidine following the initiating methionine residue to aid manufacture; [0315] (f) a combination of Mtb72f or M72 (suitably without optional histidine residues to aid expression) with Mtb41, for example in a fusion protein, such Mtb114f. The polypeptide sequence of Mtb114f is described in SEQ ID No: 16 of WO03/070187 (cDNA in SEQ ID No: 9), where it incorporates an optional His-tag to aid purification, when utilised in the present invention suitably Mtb114f corresponds to amino acid residues 8-1154 of SEQ ID No: 16 from WO03/070187. Also of particular interest is M114, i.e. Mtb114f incorporating a Ser/Ala mutation in the Ra35 component, when utilised in the present invention suitably M114 corresponds to amino acid residues 8-1154 of SEQ ID No: 16 from WO03/070187 wherein the Ser residue at position 710 has been replaced with Ala. The polypeptide sequence for M114 is shown in SEQ ID No: 28, when used in the present invention, the M72-Mtb9.9-Mtb9.8 fusion may optionally incorporate a double histidine following the initiating methionine residue to aid manufacture; [0316] (g) a combination of Ag85B and ESAT-6 components, such as in a fusion described in Doherty et al Journal of Infectious Diseases 2004 190:2146-2153; and/or [0317] (h) a combination of Ag85B and TB10.4 components, such as in a fusion described in Dietrich et al Journal of Immunology 2005 174(10):6332-6339 190:2146-2153.
[0318] Combinations of an Rv3616c component and an Rv1753c component are of particular interest. Obviously such combinations could optionally contain other additional antigen components (e.g. an M72 component).
[0319] Another combination of interest comprises an Rv3616c component and an M72 component.
[0320] A further combination of interest comprises an Rv3616c component and an Rv2386c component.
[0321] Other combinations of interest include those comprising an Rv3616c component and an Rv2707c component.
[0322] An additional combination of interest comprises an Rv3616c component and an alpha-crystallin component.
[0323] The skilled person will recognise that combinations need not rely upon the specific sequences described in above in (i)-(xvi) and (a)-(h), and that conservatively modified variants (e.g. having at least 70% identity, such as at least 80% identity, in particular at least 90% identity and especially at least 95% identity) or immunogenic fragments (e.g. at least 20% of the full length antigen, such as at least 50% of the antigen, in particular at least 70% and especially at least 80%) of the described sequences can be used to achieve the same practical effect.
[0324] Each of the above individual antigen sequences is also disclosed in Cole et al Nature 1998 393:537-544 and Camus Microbiology 2002 148:2967-2973. The genome of M. tuberculosis H37Rv is publicly available, for example at the Welcome Trust Sanger Institute website (www.sanger.ac.uk/Projects/M_tuberculosis/) and elsewhere.
[0325] Many of the above antigens are also disclosed in U.S. patent application Ser. Nos. 08/523,435, 08/523,436, 08/658,800, 08/659,683, 08/818,111, 08/818,112, 08/942,341, 08/942,578, 08/858,998, 08/859,381, 09/056,556, 09/072,596, 09/072,967, 09/073,009, 09/073,010, 09/223,040, 09/287,849 and in PCT patent applications PCT/US98/10407, PCT/US98/10514, PCT/US99/03265, PCT/US99/03268, PCT/US99/07717, WO97/09428 and WO97/09429, WO98/16645, WO98/16646, each of which is herein incorporated by reference.
[0326] The compositions, polypeptides, and nucleic acids of the invention can also comprise additional polypeptides from other sources. For example, the compositions and fusion proteins of the invention can include polypeptides or nucleic acids encoding polypeptides, wherein the polypeptide enhances expression of the antigen, e.g., NS1, an influenza virus protein (see, e.g. WO99/40188 and WO93/04175). The nucleic acids of the invention can be engineered based on codon preference in a species of choice, e.g., humans (in the case of in vivo expression) or a particular bacterium (in the case of polypeptide production).
[0327] The Rv3616c component may also be administered with one or more chemotherapeutic agents effective against tuberculosis (e.g. M. tuberculosis infection). Examples of such chemotherapeutic agents include, but are not limited to, amikacin, aminosalicylic acid, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, kanamycin, pyrazinamide, rifamycins (i.e., rifampin, rifapentine and rifabutin), streptomycin, ofloxacin, ciprofloxacin, clarithromycin, azithromycin and fluoroquinolones. Such chemotherapy is determined by the judgment of the treating physician using preferred drug combinations. "First-line" chemotherapeutic agents used to treat tuberculosis (e.g. M. tuberculosis infection) that is not drug resistant include isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide.
[0328] "Second-line" chemotherapeutic agents used to treat tuberculosis (e.g. M. tuberculosis infection) that has demonstrated drug resistance to one or more "first-line" drugs include ofloxacin, ciprofloxacin, ethionamide, aminosalicylic acid, cycloserine, amikacin, kanamycin and capreomycin.
[0329] Conventional chemotherapeutic agents are generally administered over a relatively long period (ca. 9 months). Combination of conventional chemotherapeutic agents with the administration of a Rv3616c component according to the present invention may enable the chemotherapeutic treatment period to be reduced (e.g. to 8 months, 7 months, 6 months, 5 months, 4 months, 3 months or less) without a decrease in efficacy.
[0330] Of particular interest is the use of an Rv3616c component in conjunction with Bacillus Calmette-Guerin (BCG). For example, in the form of a modified BCG which recombinantly expresses Rv3616c (or a variant or fragment thereof as described herein). Alternatively, the Rv3616c component may be used to enhance the response of a subject to BCG vaccination, either by co-administration or by boosting a previous BCG vaccination. When used to enhance the response of a subject to BCG vaccination, the Rv3616c component may obviously be provided in the form of a polypeptide or a polynucleotide (optionally in conjunction with additional antigenic components as described above).
[0331] The skilled person will recognise that combinations of components need not be administered together and may be applied: separately or in combination; at the same time, sequentially or within a short period; though the same or through different routes. Nevertheless, for convenience it is generally desirable (where administration regimes are compatible) to administer a combination of components as a single composition.
[0332] The polypeptides, polynucleotides and compositions of the present invention will usually be administered to humans, but are effective in other mammals including domestic mammals (e.g., dogs, cats, rabbits, rats, mice, guinea pigs, hamsters, chinchillas) and agricultural mammals (e.g., cows, pigs, sheep, goats, horses).
Immunogenic Fragments
[0333] T cell epitopes are short contiguous stretches of amino acids which are recognised by T cells (e.g. CD4+ or CD8+ T cells). Identification of T cell epitopes may be achieved through epitope mapping experiments which are well known to the person skilled in the art (see, for example, Paul, Fundamental Immunology, 3rd ed., 243-247 (1993); Beiβbarth et al Bioinformatics 2005 21(Suppl. 1):i29-i37).
[0334] Alternatively, epitopes may be predicted using the approaches discussed in the Examples.
[0335] As a result of the crucial involvement of the T cell response in tuberculosis, it is readily apparent that fragments of the full length Rv3616c polypeptide which contain at least one T cell epitope will be immunogenic and may contribute to immunoprotection. Such fragments are referred to herein as immunogenic fragments.
[0336] Immunogenic fragments according to the present invention will typically comprise at least 9 contiguous amino acids from the full length polypeptide sequence (e.g. at least 10), such as at least 12 contiguous amino acids (e.g. at least 15 or at least 20 contiguous amino acids), in particular at least 50 contiguous amino acids, such as at least 100 contiguous amino acids (for example at least 200 contiguous amino acids). Suitably the immunogenic fragments will be at least 20%, such as at least 50%, at least 70% or at least 80% of the length of the full length polypeptide sequence.
[0337] It will be understood that in a diverse out-bred population, such as humans, different HLA types mean that specific epitopes may not be recognised by all members of the population. Consequently, to maximise the level of recognition and scale of immune response to a polypeptide, it is generally desirable that an immunogenic fragment contains a plurality of the epitopes from the full length sequence (suitably all epitopes).
[0338] Particular fragments of the Rv3616c protein which may be of use include those containing at least one CD4+ epitope, suitably at least two CD4+ epitopes and especially all CD4+ epitopes (such as those epitopes described in the Examples and in SEQ ID Nos: 29-47, particularly those associated with a plurality of HLA alleles, e.g. those associated with 2, 3, 4, 5 or more alleles).
[0339] Other fragments of the Rv3616c protein which may be of use include those containing at least one CD8 epitope, suitably at least two CD8 epitopes and especially all CD8 epitopes (such as those epitopes described in the Examples and in SEQ ID Nos: 48-126, particularly those associated with a plurality of HLA alleles, e.g. those associated with 2, 3, 4, 5 or more alleles).
[0340] Where an individual fragment of the full length polypeptide is used, such a fragment is considered to be immunogenic where it elicits a response which is at least 20%, suitably at least 50% and especially at least 75% (such as at least 90%) of the activity of the reference sequence in an in vitro restimulation assay of PBMC or whole blood with specific antigens (e.g. restimulation for a period of between several hours to up to two weeks, such as up to one day, 1 day to 1 week or 1 to 2 weeks) that measures the activation of the cells via lymphoproliferation, production of cytokines in the supernatant of culture (measured by ELISA, CBA etc) or characterisation of T and B cell responses by intra and extracellular staining (e.g. using antibodies specific to immune markers, such as CD3, CD4, CD8, IL2, TNFa, IFNg, CD40L, CD69 etc) followed by analysis with a flowcytometer. Suitably, a fragment is considered to be immunogenic where it elicits a response which is at least 20%, suitably at least 50% and especially at least 75% (such as at least 90%) of the activity of the reference sequence in a T cell proliferation and/or IFN-gamma production assay.
[0341] In some circumstances a plurality of fragments of the full length polypeptide (which may or may not be overlapping and may or may not cover the entirety of the full length sequence) may be used to obtain an equivalent biological response to the full length sequence itself. For example, at least two immunogenic fragments (such as three, four or five) as described above, which in combination provide at least 50%, suitably at least 75% and especially at least 90% activity of the reference sequence in an in vitro restimulation assay of PBMC or whole blood (e.g. a T cell proliferation and/or IFN-gamma production assay).
[0342] Peptides of SEQ ID Nos: 127-156 are fragments of particular interest (especially those in SEQ ID Nos: 127-133 and 143-156).
Variants
[0343] "Variants" or "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
[0344] Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations lead to "silent" or "degenerate" variants, which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognise that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
[0345] A polynucleotide of the invention may contain a number of silent variations (for example, 1-50, such as 1-25, in particular 1-5, and especially 1 codon(s) may be altered) when compared to the reference sequence. A polynucleotide of the invention may contain a number of non-silent conservative variations (for example, 1-50, such as 1-25, in particular 1-5, and especially 1 codon(s) may be altered) when compared to the reference sequence. Non-silent variations are those which result in a change in the encoded amino acid sequence (either though the substitution, deletion or addition of amino acid residues). Those skilled in the art will recognise that a particular polynucleotide sequence may contain both silent and non-silent conservative variations.
[0346] In respect of variants of a protein sequence, the skilled person will recognise that individual substitutions, deletions or additions to polypeptide, which alters, adds or deletes a single amino acid or a small percentage of amino acids is a "conservatively modified variant" where the alteration(s) results in the substitution of an amino acid with a functionally similar amino acid or the substitution/deletion/addition of residues which do not substantially impact the biological function of the variant.
[0347] Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
[0348] A polypeptide of the invention may contain a number of conservative substitutions (for example, 1-50, such as 1-25, in particular 1-10, and especially 1 amino acid residue(s) may be altered) when compared to the reference sequence. In general, such conservative substitutions will fall within one of the amino-acid groupings specified below, though in some circumstances other substitutions may be possible without substantially affecting the immunogenic properties of the antigen. The following eight groups each contain amino acids that are typically conservative substitutions for one another: [0349] 1) Alanine (A), Glycine (G); [0350] 2) Aspartic acid (D), Glutamic acid (E); [0351] 3) Asparagine (N), Glutamine (Q); [0352] 4) Arginine (R), Lysine (K); [0353] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); [0354] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); [0355] 7) Serine (S), Threonine (T); and [0356] 8) Cysteine (C), Methionine (M) [0357] (see, e.g., Creighton, Proteins 1984).
[0358] Suitably such substitutions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen.
[0359] Protein variants may also include those wherein additional amino acids are inserted compared to the reference sequence, for example, such insertions may occur at 1-10 locations (such as 1-5 locations, suitably 1 or 2 locations, in particular 1 location) and may, for example, involve the addition of 50 or fewer amino acids at each location (such as 20 or fewer, in particular 10 or fewer, especially 5 or fewer). Suitably such insertions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen. One example of insertions includes a short stretch of histidine residues (e.g. 2-6 residues) to aid expression and/or purification of the antigen in question.
[0360] Protein variants include those wherein amino acids have been deleted compared to the reference sequence, for example, such deletions may occur at 1-10 locations (such as 1-5 locations, suitably 1 or 2 locations, in particular 1 location) and may, for example, involve the deletion of 50 or fewer amino acids at each location (such as 20 or fewer, in particular 10 or fewer, especially 5 or fewer). Suitably such deletions do not occur in the region of an epitope, and do not therefore have a significant impact on the immunogenic properties of the antigen.
[0361] The skilled person will recognise that a particular protein variant may comprise substitutions, deletions and additions (or any combination thereof).
[0362] Methods of determining the epitope regions of an antigen are described and exemplified in the Examples.
[0363] Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity (such as at least about 95%, at least about 98% or at least about 99%) to the associated reference sequence.
[0364] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or sub-sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 70% identity, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the compliment of a test sequence. Optionally, the identity exists over a region that is at least about 25 to about 50 amino acids or nucleotides in length, or optionally over a region that is 75-100 amino acids or nucleotides in length. Suitably, the comparison is performed over a window corresponding to the entire length of the reference sequence.
[0365] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
[0366] A "comparison window", as used herein, references to a segment in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0367] One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395 (1984).
[0368] Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (website at www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
[0369] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
[0370] The present invention also extends to polynucleotides comprising a first nucleotide sequence which selectively hybridises under moderately stringent conditions (such as under highly stringent conditions) to the complement of a second nucleotide sequence which encodes a polypeptide comprising: [0371] (i) an Rv3616c protein sequence; [0372] (ii) a variant of an Rv3616c protein sequence; or [0373] (iii) an immunogenic fragment of an Rv3616c protein sequence, for the treatment or prevention of latent TB.
[0374] The phrase "highly stringent hybridisation conditions" refers to conditions under which a probe will hybridise to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Highly stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. An extensive guide to the hybridisation of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridisation and the strategy of nucleic acid assays" (1993). Generally, highly stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridise to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Highly stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Highly stringent conditions may also be achieved with the addition of destabilising agents such as formamide. For selective or specific hybridisation, a positive signal is at least two times background, optionally 10 times background hybridisation.
[0375] Exemplary highly stringent hybridisation conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.
[0376] Nucleic acids that do not hybridise to each other under highly stringent conditions are still functionally equivalent if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridise under moderately stringent hybridisation conditions.
[0377] Exemplary "moderately stringent hybridisation conditions" include a hybridisation in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridisation is at least twice background. Those of ordinary skill will readily recognise that alternative hybridisation and wash conditions can be utilised to provide conditions of similar stringency.
[0378] The phrase "selectively (or specifically) hybridises to" refers to the binding, duplexing, or hybridising of a molecule only to a particular nucleotide sequence under stringent hybridisation conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
[0379] In any event, variants of a polypeptide sequence will have essentially the same activity as the reference sequence (in the case of polynucleotides, variant polynucleotide sequences will encode a polypeptide which has essentially the same activity as the reference sequence). By essentially the same activity is meant at least 50%, suitably at least 75% and especially at least 90% activity of the reference sequence in an in vitro restimulation assay of PBMC or whole blood with specific antigens (e.g. restimulation for a period of between several hours to up to two weeks, such as up to one day, 1 day to 1 week or 1 to 2 weeks) that measures the activation of the cells via lymphoproliferation, production of cytokines in the supernatant of culture (measured by ELISA, CBA etc) or characterisation of T and B cell responses by intra and extracellular staining (e.g. using antibodies specific to immune markers, such as CD3, CD4, CD8, IL2, TNFa, IFNg, CD40L, CD69 etc) followed by analysis with a flowcytometer. Suitably, by essentially the same activity is meant at least 50%, suitably at least 75% and especially at least 90% activity of the reference sequence in a T cell proliferation and/or IFN-gamma production assay.
Polynucleotide Compositions
[0380] As used herein, the term "polynucleotide" refers to a molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a polynucleotide encoding a polypeptide refers to a polynucleotide segment that contains one or more coding sequences yet is substantially isolated away from, or purified free from, total genomic DNA of the species from which the polynucleotide is obtained.
[0381] As will be understood by those skilled in the art, the polynucleotides of use in this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.
[0382] "Isolated," as used herein, means that a polynucleotide is substantially away from other coding sequences, and that the polynucleotide does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. An isolated nucleic acid is separated from other open reading frames that flank the gene and encode proteins other than the gene. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
[0383] As will be recognised by the skilled artisan, polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
[0384] Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a Mycobacterium antigen or a portion thereof) or may comprise a variant, or a biological or functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as further described below, preferably such that the immunogenicity of the encoded polypeptide is not diminished, relative to the reference protein. The effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein.
[0385] In additional embodiments, the present invention provides isolated polynucleotides and polypeptides comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein. For example, polynucleotides are provided by this invention that comprise at least about 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of the reference sequence disclosed herein as well as all intermediate lengths there between. It will be readily understood that "intermediate lengths", in this context, means any length between the quoted values, such as 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like.
[0386] Moreover, it will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear relatively low identity to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention, for example polynucleotides that are optimized for human and/or primate codon selection. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridisation, amplification and/or database sequence comparison).
Polynucleotide Identification and Characterisation
[0387] Polynucleotides may be identified, prepared and/or manipulated using any of a variety of well established techniques. For example, a polynucleotide may be identified, as described in more detail below, by screening a microarray of cDNAs. Such screens may be performed, for example, using a Synteni microarray (Palo Alto, Calif.) according to the manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA 93:10614-10619 (1996) and Heller et al., Proc. Natl. Acad. Sci. USA 94:2150-2155 (1997)). Alternatively, polynucleotides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as M. tuberculosis cells. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequences provided herein, and may be purchased or synthesised.
[0388] An amplified portion of a polynucleotide may be used to isolate a full length gene from a suitable library (e.g., a M. tuberculosis cDNA library) using well known techniques. Within such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5' and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5' sequences.
[0389] For hybridisation techniques, a partial sequence may be labeled (e.g., by nick-translation or end-labeling with 32P) using well known techniques. A bacterial or bacteriophage library is then generally screened by hybridising filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual (2000)). Hybridising colonies or plaques are selected and expanded, and the DNA is isolated for further analysis. cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences may be generated to identify one or more overlapping clones. The complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones. The resulting overlapping sequences can then be assembled into a single contiguous sequence. A full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.
[0390] Alternatively, there are numerous amplification techniques for obtaining a full length coding sequence from a partial cDNA sequence. Within such techniques, amplification is generally performed via PCR. Any of a variety of commercially available kits may be used to perform the amplification step. Primers may be designed using, for example, software well known in the art. Primers are preferably 22-30 nucleotides in length, have a GC content of at least 50% and anneal to the target sequence at temperatures of about 68° C. to 72° C. The amplified region may be sequenced as described above, and overlapping sequences assembled into a contiguous sequence.
[0391] One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 16:8186 (1988)), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularised by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. Within an alternative approach, sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region. A variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591. Another such technique is known as "rapid amplification of cDNA ends" or RACE. This technique involves the use of an internal primer and an external primer, which hybridises to a polyA region or vector sequence, to identify sequences that are 5' and 3' of a known sequence. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic. 1:111-19 (1991)) and walking PCR (Parker et al., Nucl. Acids. Res. 19:3055-60 (1991)). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.
[0392] In certain instances, it is possible to obtain a full length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank. Searches for overlapping ESTs may generally be performed using well known programs (e.g., NCBI BLAST searches), and such ESTs may be used to generate a contiguous full length sequence. Full length DNA sequences may also be obtained by analysis of genomic fragments.
Polynucleotide Expression in Host Cells
[0393] Polynucleotide sequences or fragments thereof which encode polypeptides, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.
[0394] As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
[0395] Moreover, the polynucleotide sequences can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product. For example, DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. In addition, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.
[0396] Natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of polypeptide activity, it may be useful to encode a chimeric protein that can be recognised by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence, so that the polypeptide may be cleaved and purified away from the heterologous moiety.
[0397] Sequences encoding a desired polypeptide may be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H. et al., Nucl. Acids Res. Symp. Ser. pp. 215-223 (1980), Horn et al., Nucl. Acids Res. Symp. Ser. pp. 225-232 (1980)). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or a portion thereof. For example, peptide synthesis can be performed using various solid-phase techniques (Roberge et al., Science 269:202-204 (1995)) and automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer, Palo Alto, Calif.).
[0398] A newly synthesised peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, Proteins, Structures and Molecular Principles (1983)) or other comparable techniques available in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of a polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
[0399] In order to express a desired polypeptide, the nucleotide sequences encoding the polypeptide, or functional equivalents, may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (2000), and Ausubel et al., Current Protocols in Molecular Biology (updated annually).
[0400] A variety of expression vector/host systems may be utilised to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
[0401] The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector--enhancers, promoters, 5' and 3' untranslated regions--which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.
[0402] In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are needed, for example for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke &Schuster, J. Biol. Chem. 264:5503-5509 (1989)); and the like. pGEX Vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
[0403] In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. Other vectors containing constitutive or inducible promoters include GAP, PGK, GAL and ADH. For reviews, see Ausubel et al. (supra) and Grant et al., Methods Enzymol. 153:516-544 (1987) and Romas et al. Yeast 8 423-88 (1992).
[0404] In cases where plant expression vectors are used, the expression of sequences encoding polypeptides may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680 (1984); Broglie et al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, e.g., Hobbs in McGraw Hill Yearbook of Science and Technology pp. 191-196 (1992)).
[0405] An insect system may also be used to express a polypeptide of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding the polypeptide may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which the polypeptide of interest may be expressed (Engelhard et al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)).
[0406] In mammalian host cells, a number of viral-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. Methods and protocols for working with adenovirus vectors are reviewed in Wold, Adenovirus Methods and Protocols, 1998. Additional references regarding use of adenovirus vectors can be found in Adenovirus: A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References, 2004.
[0407] Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf. et al., Results Probl. Cell Differ. 20:125-162 (1994)).
[0408] In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, HEK293, and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.
[0409] For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines which stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
[0410] Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-32 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-23 (1990)) genes which can be employed in tk--or aprt--cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70 (1980)); npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilise indole in place of tryptophan, or hisD, which allows cells to utilise histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. U.S.A. 85:8047-51 (1988)). Recently, the use of visible markers has gained popularity with such markers as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).
[0411] Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding a polypeptide is inserted within a marker gene sequence, recombinant cells containing sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a polypeptide-encoding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
[0412] Alternatively, host cells which contain and express a desired polynucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridisations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
[0413] A variety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilising monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be employed. These and other assays are described, among other places, in Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).
[0414] A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labelled hybridisation or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labelling or PCR amplification using a labelled nucleotide. Alternatively, the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits. Suitable reporter molecules or labels, which may be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
[0415] Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides may be designed to contain signal sequences which direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen. San Diego, Calif.) between the purification domain and the encoded polypeptide may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilised metal ion affinity chromatography) as described in Porath et al., Prot. Exp. Purif. 3:263-281 (1992) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll et al., DNA Cell Biol. 12:441-453 (1993)).
In Vivo Polynucleotide Delivery Techniques
[0416] In additional embodiments, genetic constructs comprising one or more of the polynucleotides of the invention are introduced into cells in vivo. This may be achieved using any of a variety or well known approaches, several of which are outlined below for the purpose of illustration.
[0417] 1. Adenovirus
[0418] One of the preferred methods for in vivo delivery of one or more nucleic acid sequences involves the use of an adenovirus expression vector. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express a polynucleotide that has been cloned therein in a sense or antisense orientation. Of course, in the context of an antisense construct, expression does not require that the gene product be synthesised.
[0419] The expression vector comprises a genetically engineered form of an adenovirus. Knowledge of the genetic organisation of adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus & Horwitz, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
[0420] Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titre, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 6-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.
[0421] In a current system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
[0422] Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E1 proteins (Graham et al., 1977). Since the E3 region is dispensable from the adenovirus genome (Jones & Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, the D3 or both regions (Graham & Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra kB of DNA. Combined with the approximately 5.5 kB of DNA that is replaceable in the E1 and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kB, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the E1-deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).
[0423] Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the currently preferred helper cell line is 293.
[0424] Racher et al. (1995) have disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into 1 litre siliconised spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.
[0425] Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain a conditional replication-defective adenovirus vector for use in the present invention, since Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
[0426] As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus E1 region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the E1-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al. (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.
[0427] Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titres, e.g., 109-1011 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet & Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz & Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993).
[0428] Adenovirus vectors may originate from human adenovirus. Alternatively they may originate from adenovirus of other species e.g. chimpanzee which may have the advantage that the viral vectors are not neutralised by antibodies against human adenovirus circulating in many human subjects (see e.g.: Tatsis N et al Gene Therapy 2006 13:421-429).
[0429] Adenovirus type 35, which is relatively uncommon and therefore there are low levels of pre-existing immunity to the vector itself, has been used as a delivery system in certain tuberculosis vaccines which are being developed (see for example, Radosevic et al Infection and Immunity 2007 75(8):4105-4115). Adenovirus type 35 may also be of particular value in the present invention as a delivery vector.
[0430] 2. Retroviruses
[0431] The retroviruses are a group of single-stranded RNA viruses characterised by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
[0432] In order to construct a retroviral vector, a nucleic acid encoding one or more oligonucleotide or polynucleotide sequences of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas & Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).
[0433] A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors.
[0434] A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989).
[0435] 3. Adeno Associated Viruses
[0436] AAV (Ridgeway, 1988; Hermonat & Muzycska, 1984) is a parovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) that has not been linked to any disease. It is also classified as a dependovirus, because its replication is dependent on the presence of a helper virus, such as adenovirus. Five serotypes have been isolated, of which AAV-2 is the best characterised. AAV has a single-stranded linear DNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to form an icosahedral virion of 20 to 24 nm in diameter (Muzyczka & McLaughlin, 1988).
[0437] The AAV DNA is approximately 4.7 kilobases long. It contains two open reading frames and is flanked by two ITRs. There are two major genes in the AAV genome: rep and cap. The rep gene codes for proteins responsible for viral replications, whereas cap codes for capsid protein VP1-3. Each ITR forms a T-shaped hairpin structure. These terminal repeats are the only essential cis components of the AAV for chromosomal integration. Therefore, the AAV can be used as a vector with all viral coding sequences removed and replaced by the cassette of genes for delivery. Three viral promoters have been identified and named p5, p19, and p40, according to their map position. Transcription from p5 and p19 results in production of rep proteins, and transcription from p40 produces the capsid proteins (Hermonat & Muzyczka, 1984).
[0438] There are several factors that prompted researchers to study the possibility of using rAAV as an expression vector. One is that the requirements for delivering a gene to integrate into the host chromosome are surprisingly few. It is necessary to have the 145-bp ITRs, which are only 6% of the AAV genome. This leaves room in the vector to assemble a 4.5-kb DNA insertion. While this carrying capacity may prevent the AAV from delivering large genes, it is amply suited for delivering antisense constructs.
[0439] AAV is also a good choice of delivery vehicles due to its safety. There is a relatively complicated rescue mechanism: not only wild type adenovirus but also AAV genes are required to mobilise rAAV. Likewise, AAV is not pathogenic and not associated with any disease. The removal of viral coding sequences minimises immune reactions to viral gene expression, and therefore, rAAV does not evoke an inflammatory response.
[0440] 4. Other Viral Vectors as Expression Constructs
[0441] Other viral vectors may be employed as expression constructs in the present invention for the delivery of oligonucleotide or polynucleotide sequences to a host cell. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Coupar et al., 1988), lentiviruses, polio viruses and herpes viruses may be employed. Other poxvirus derived vectors, such as fowl-pox derived vectors, may also be expected to be of use. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Coupar et al., 1988; Horwich et al., 1990).
[0442] With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al., 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. The hepatotropism and persistence (integration) were particularly attractive properties for liver-directed gene transfer. Chang et al. (1991) introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titres of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al., 1991).
[0443] Additional `viral` vectors include virus like particles (VLPs) and phages.
[0444] 5. Non-Viral Vectors
[0445] In order to effect expression of the oligonucleotide or polynucleotide sequences of the present invention, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. As described above, one preferred mechanism for delivery is via viral infection where the expression construct is encapsulated in an infectious viral particle.
[0446] Once the expression construct has been delivered into the cell the nucleic acid encoding the desired oligonucleotide or polynucleotide sequences may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the construct may be stably integrated into the genome of the cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronisation with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
[0447] In certain embodiments of the invention, the expression construct comprising one or more oligonucleotide or polynucleotide sequences may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed, for example, by any method which physically or chemically permeabilises the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty & Reshef (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.
[0448] Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
[0449] Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated.
[0450] Bacteria may also be utilised as a delivery method (e.g. listeria, see WO2004/11048) and in particular BCG.
Polypeptide Compositions
[0451] Generally, a polypeptide of use in the invention will be an isolated polypeptide (i.e. separated from those components with which it may usually be found in nature).
[0452] For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.
[0453] Polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by DNA sequences as described above may be readily prepared from the DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. 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, such as mammalian cells and plant cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO. Supernatants from suitable host/vector systems which secrete recombinant protein or polypeptide 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 polypeptide.
[0454] Polypeptides of the invention, immunogenic fragments thereof, and other variants having less than about 100 amino acids, and generally less than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesised 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 Perkin Elmer/Applied BioSystems Division (Foster City, Calif.), and may be operated according to the manufacturer's instructions.
[0455] Within certain specific embodiments, a polypeptide may be a fusion protein that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, e.g., Stoute et al., New Engl. J. Med. 336:86-91 (1997)). A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognised by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the protein.
[0456] Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.
[0457] A peptide linker sequence may be employed to separate the first and second polypeptide components 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 generally be from 1 to about 50 amino acids in length. Linker 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.
[0458] Within preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenzae virus, NS1 (hemaglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.
[0459] In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292 (1986)). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10:795-798 (1992)). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.
T Cells
[0460] Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for a Mycobacterium antigen. Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a patient, using a commercially available cell separation system, such as the Isolex® System, available from Nexell Therapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No. 5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.
[0461] T cells may be stimulated with a polypeptide of the invention, polynucleotide encoding such a polypeptide, and/or an antigen presenting cell (APC) that expresses such a polypeptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide. Preferably, the polypeptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.
[0462] T cells are considered to be specific for a polypeptide of the invention if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide. T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res. 54:1065-1070 (1994)). Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labelling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA). Contact with a polypeptide of the invention (100 ng/ml-100 μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days should result in at least a two fold increase in proliferation of the T cells. Contact as described above for 2-3 hours should result in activation of the T cells, as measured using standard cytokine assays in which a two fold increase in the level of cytokine release (e.g., TNF or IFN-γ) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. 1 (1998)). T cells that have been activated in response to a polypeptide, polynucleotide or polypeptide-expressing APC may be CD4.sup.+ and/or CD8.sup.+. Protein-specific T cells may be expanded using standard techniques. Within preferred embodiments, the T cells are derived from a patient, a related donor or an unrelated donor, and are administered to the patient following stimulation and expansion.
[0463] For therapeutic purposes, CD4.sup.+ or CD8.sup.+ T cells that proliferate in response to a polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways. For example, the T cells can be re-exposed to a polypeptide, or a short peptide corresponding to an immunogenic portion of such a polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesise a polypeptide. Alternatively, one or more T cells that proliferate in the presence of the protein can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution.
Pharmaceutical Compositions
[0464] In additional embodiments, the polynucleotide, polypeptide, T-cell and/or antibody compositions disclosed herein will be formulated in pharmaceutically-acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
[0465] It will also be understood that, if desired, the nucleic acid segment (e.g., RNA or DNA) that expresses a polypeptide as disclosed herein may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents, including chemotherapeutic agents effective against a M. tuberculosis infection. In fact, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The compositions may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesised as described herein. Likewise, such compositions may further comprise substituted or derivatised RNA or DNA compositions.
[0466] Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation. Other routes of administration include via the mucosal surfaces.
[0467] Typically, formulations comprising a therapeutically effective amount deliver about 0.1 ug to about 1000 ug of polypeptide per administration, more typically about 2.5 ug to about 100 ug of polypeptide per administration. In respect of polynucleotide compositions, these typically deliver about 10 ug to about 20 mg of the inventive polynucleotide per administration, more typically about 0.1 mg to about 10 mg of the inventive polynucleotide per administration
[0468] Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
[0469] 1. Oral Delivery
[0470] In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to an animal. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
[0471] The active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. No. 5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavouring agent, such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. A syrup of elixir may contain the active component, sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavouring, such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active components may be incorporated into sustained-release preparation and formulations.
[0472] For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
[0473] 2. Injectable Delivery
[0474] In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, intradermally, or even intraperitoneally as described in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363 (each specifically incorporated herein by reference in its entirety). Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0475] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[0476] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologics standards.
[0477] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0478] The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
[0479] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
[0480] The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.
[0481] 3. Nasal and Buccal Delivery
[0482] In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, buccal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs eg via nasal and buccal aerosol sprays has been described e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).
[0483] 4. Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery
[0484] In certain embodiments, the inventors contemplate the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the compositions of the present invention into suitable host cells. In particular, the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
[0485] Such formulations may be preferred for the introduction of pharmaceutically-acceptable formulations of the nucleic acids or constructs disclosed herein. The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy for intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon & Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No. 5,741,516, specifically incorporated herein by reference in its entirety). Further, various methods of liposome and liposome like preparations as potential drug carriers have been reviewed (Takakura, 1998; Chandran et al., 1997; Margalit, 1995; U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporated herein by reference in its entirety).
[0486] Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al., 1990; Muller et al., 1990). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs (Heath & Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989; Fresta & Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987), enzymes (Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses (Faller & Baltimore, 1984), transcription factors and allosteric effectors (Nicolau & Gersonde, 1979) into a variety of cultured cell lines and animals. In addition, several successful clinical trails examining the effectiveness of liposome-mediated drug delivery have been completed (Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier et al., 1988). Furthermore, several studies suggest that the use of liposomes is not associated with autoimmune responses, toxicity or gonadal localization after systemic delivery (Mori & Fukatsu, 1992).
[0487] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 Å, containing an aqueous solution in the core.
[0488] Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the peptide compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, i.e. in the aqueous spaces and within the bilayer itself, respectively. It is possible that the drug-bearing liposomes may even be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation.
[0489] In addition to the teachings of Couvreur et al. (1977; 1988), the following information may be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
[0490] In addition to temperature, exposure to proteins can alter the permeability of liposomes. Certain soluble proteins, such as cytochrome c, bind, deform and penetrate the bilayer, thereby causing changes in permeability. Cholesterol inhibits this penetration of proteins, apparently by packing the phospholipids more tightly. It is contemplated that the most useful liposome formations for antibiotic and inhibitor delivery will contain cholesterol.
[0491] The ability to trap solutes varies between different types of liposomes. For example, MLVs are moderately efficient at trapping solutes, but SUVs are extremely inefficient. SUVs offer the advantage of homogeneity and reproducibility in size distribution, however, and a compromise between size and trapping efficiency is offered by large unilamellar vesicles (LUVs). These are prepared by ether evaporation and are three to four times more efficient at solute entrapment than MLVs.
[0492] In addition to liposome characteristics, an important determinant in entrapping compounds is the physicochemical properties of the compound itself. Polar compounds are trapped in the aqueous spaces and nonpolar compounds bind to the lipid bilayer of the vesicle. Polar compounds are released through permeation or when the bilayer is broken, but nonpolar compounds remain affiliated with the bilayer unless it is disrupted by temperature or exposure to lipoproteins. Both types show maximum efflux rates at the phase transition temperature.
[0493] Liposomes interact with cells via four different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
[0494] The fate and disposition of intravenously injected liposomes depend on their physical properties, such as size, fluidity, and surface charge. They may persist in tissues for h or days, depending on their composition, and half lives in the blood range from min to several h. Larger liposomes, such as MLVs and LUVs, are taken up rapidly by phagocytic cells of the reticuloendothelial system, but physiology of the circulatory system restrains the exit of such large species at most sites. They can exit only in places where large openings or pores exist in the capillary endothelium, such as the sinusoids of the liver or spleen. Thus, these organs are the predominate site of uptake. On the other hand, SUVs show a broader tissue distribution but still are sequestered highly in the liver and spleen. In general, this in vivo behavior limits the potential targeting of liposomes to only those organs and tissues accessible to their large size. These include the blood, liver, spleen, bone marrow, and lymphoid organs.
[0495] Targeting is generally not a limitation in terms of the present invention. However, should specific targeting be desired, methods are available for this to be accomplished. Antibodies may be used to bind to the liposome surface and to direct the antibody and its drug contents to specific antigenic receptors located on a particular cell-type surface. Carbohydrate determinants (glycoprotein or glycolipid cell-surface components that play a role in cell-cell recognition, interaction and adhesion) may also be used as recognition sites as they have potential in directing liposomes to particular cell types. Mostly, it is contemplated that intravenous injection of liposomal preparations would be used, but other routes of administration are also conceivable.
[0496] Alternatively, the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention. Such particles may be are easily made, as described (Couvreur et al., 1980; 1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry et al., 1995 and U.S. Pat. No. 5,145,684, specifically incorporated herein by reference in its entirety).
[0497] Skin patches may also be utilised for transcutaneous delivery.
Immunogenic Compositions
[0498] In certain preferred embodiments of the present invention, immunogenic compositions are provided. The immunogenic compositions will generally comprise one or more polypeptides or polynucleotides, such as those discussed above, in combination with an immunostimulant. An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is incorporated; see, e.g., Fullerton, U.S. Pat. No. 4,235,877).
[0499] Preparation of immunogenic compositions is generally described in, for example, Powell & Newman, eds., Vaccine Design (the subunit and adjuvant approach) (1995). Pharmaceutical compositions and immunogenic compositions within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other M. tuberculosis antigens may be present, either incorporated into a fusion polypeptide or as a separate compound, within the pharmaceutical or immunogenic composition.
[0500] Illustrative immunogenic compositions may contain a polynucleotide (e.g. DNA) encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ (thereby eliciting an immune response). As noted above, 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, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198 (1998), and references cited therein. 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 host cell (for example, a Mycobacterium, Bacillus or Lactobacillus strain, including Bacillus-Calmette-Guerrin or Lactococcus lactis) that expresses the polypeptide (e.g. on its cell surface or secretes the polypeptide) (see, for example, Ferreira, et al., An Acad Bras Cienc (2005) 77:113-124; and Raha, et al., Appl Microbiol Biotechnol (2005) PubMedID 15635459). 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. Suitable systems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86:317-321 (1989); Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103 (1989); Flexner et al., Vaccine 8:17-21 (1990); U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627 (1988); Rosenfeld et al., Science 252:431-434 (1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994); Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502 (1993); Guzman et al., Circulation 88:2838-2848 (1993); and Guzman et al., Cir. Res. 73:1202-1207 (1993). 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. It will be apparent that a immunogenic composition may comprise both a polynucleotide and a polypeptide component. Such immunogenic composition may provide for an enhanced immune response.
[0501] It will be apparent that an immunogenic composition may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides provided herein. Such salts may be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).
[0502] While any suitable carrier known to those of ordinary skill in the art may be employed in the immunogenic compositions of this invention, the type of carrier will vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular 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., polylactate polyglycolate) 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; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252. One may also employ a carrier comprising the particulate-protein complexes described in U.S. Pat. No. 5,928,647, which are capable of inducing a class I-restricted cytotoxic T lymphocyte responses in a host.
[0503] Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using well known technology.
[0504] Any of a variety of immunostimulants may be employed in the immunogenic compositions of this invention. For example, an adjuvant may be included. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminium hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium species or Mycobacterium derived proteins. For example, delipidated, deglycolipidated M. vaccae ("pVac") can be used. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS01B, AS02A, AS15, AS-2 and derivatives thereof (GlaxoSmithKline, Philadelphia, Pa.); CWS (cell wall skeleton from a tubercule bacillus), TDM (trehalose dicorynomycolate), Leif (Leishmania elongation initiation factor), aluminium salts such as aluminium hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A (MPL®); and quil A (e.g. QS-21). Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
[0505] An adjuvant refers to the components in a vaccine or therapeutic composition that increase the specific immune response to the antigen (see, e.g., Edelman, AIDS Res. Hum Retroviruses 8:1409-1411 (1992)). Adjuvants induce immune responses of the Th1-type and Th-2 type response. Th1-type cytokines (e.g., IFN-γ, IL-2, and IL-12) tend to favour the induction of cell-mediated immune response to an administered antigen, while Th-2 type cytokines (e.g., IL-4, IL-5, 11-6, IL-10) tend to favour the induction of humoral immune responses. Adjuvants capable of preferential stimulation of a Th-1 cell-mediated immune response are described in WO 94/00153 and WO 95/17209.
[0506] Within the immunogenic compositions provided herein, the adjuvant composition is preferably designed to induce an immune response predominantly of the Th1 type. Following application of a immunogenic composition as provided herein, a patient will typically support an immune response that includes Th1- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Janeway, et al., Immunobiology, 5th Edition, 2001.
[0507] The Rv3616c compositions usually comprise one or more adjuvants, e.g., AS01B (3-de-O-acylated monophosphoryl lipid A (3D-MPL®) and QS21 in a liposome formulation; see, U.S. Patent Publication No. 2003/0143240); AS02 A (3D-MPL® and QS21 and an oil in water emulsion; see, Bojang, et al., Lancet (2001) 358:1927); ENHANZYN® (Detox); 3D-MPL®; saponins including Quil A and its components e.g. QS21 and saponin mimetics; CWS (cell wall skeleton from a tubercule bacillus); TDM (trehalose dicorynomycolate); aminoalkyl glucosaminide 4-phosphates (AGPs); immunostimulatory oligonucleoptides e.g. CPG; Leif (Leishmania elongation initiation factor); and derivatives thereof. In a preferred embodiment, an Rv3616c polypeptide is administered with one or more adjuvants selected from the group consisting of 3D-MPL® and QS21 in a liposome formulation e.g. AS01B and 3D-MPL® and QS21 and an oil in water emulsion (e.g. AS02A). Adjuvant systems AS01B and AS02A are further described in Pichyangkul, et al., Vaccine (2004) 22:3831-40.
[0508] When delivering the Rv3616c antigen as a nucleic acid, it can be delivered, for example, in a viral vector (i.e., an adenovirus vector), or in a mutant bacterium host cell (i.e., a mutant, avirulent Mycobacterium, Lactobacillus or Bacillus host cell including Bacillus Calmette-Guerin (BCG) and Lactococcus lactis).
[0509] Preferred adjuvants for use in eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A (MPL®) preferably 3-O-deacylated monophosphoryl lipid A (3D-MPL®), optionally with an aluminium salt (see, for example, Ribi, et al., 1986, Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY, pp. 407-419; GB 2122204B; GB 2220211; and U.S. Pat. No. 4,912,094). A preferred form of 3D-MPL® is in the form of an emulsion having a small particle size less than 0.2 mm in diameter, and its method of manufacture is disclosed in WO 94/21292. Aqueous formulations comprising monophosphoryl lipid A and a surfactant have been described in WO 98/43670. Exemplified preferred adjuvants include AS01B (MPL® and QS21 in a liposome formulation), 3D-MPL® and QS21 in a liposome formulation, AS02A (MPL® and QS21 and an oil in water emulsion), 3D-MPL® and QS21 and an oil in water emulsion, and AS15, available from GlaxoSmithKline. MPL® adjuvants are available from GlaxoSmithKline (see U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).
[0510] CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Th1 response. CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352 (1996). CpG when formulated into immunogenic compositions, is generally administered in free solution together with free antigen (WO 96/02555; McCluskie and Davis, supra) or covalently conjugated to an antigen (WO 98/16247), or formulated with a carrier such as aluminium hydroxide ((Hepatitis surface antigen) Davis et al. supra; Brazolot-Millan et al., Proc. Natl. Acad. Sci., USA, 1998, 95(26), 15553-8). CpG is known in the art as being an adjuvant that can be administered by both systemic and mucosal routes (WO 96/02555, EP 468520, Davis et al., J. Immunol, 1998, 160(2):870-876; McCluskie and Davis, J. Immunol., 1998, 161(9):4463-6).
[0511] Another preferred adjuvant is a saponin or saponin mimetics or derivatives, such as Quil A, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A (MPL®) and saponin derivative, such as the combination of QS21 and 3D-MPL® as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL® and tocopherol in an oil-in-water emulsion is described in WO 95/17210. Additional saponin adjuvants of use in the present invention include QS7 (described in WO 96/33739 and WO 96/11711) and QS17 (described in U.S. Pat. No. 5,057,540 and EP 0 362 279 B1).
[0512] Alternatively the saponin formulations may be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. The saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOM® s. Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM®. The saponins may also be formulated with excipients such as CARBOPOL® to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.
[0513] In one embodiment, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D-MPL® adjuvant, as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol containing liposomes, as described in WO 96/33739. Other suitable formulations comprise an oil-in-water emulsion and tocopherol. Another suitable adjuvant formulation employing QS21, 3D-MPL® adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
[0514] Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 as disclosed in WO 00/09159. Suitably the formulation additionally comprises an oil in water emulsion and tocopherol.
[0515] Other suitable adjuvants include MONTANIDE® ISA 720 (Seppic, France), SAF (Chiron, Calif., United States), ISCOMS® (CSL), MF-59 (Chiron), the SBAS series of adjuvants (SmithKline Beecham, Rixensart, Belgium), Detox (Corixa), RC-529 (Corixa) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. patent application Ser. Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1. SmithKline Beecham and Corixa Corporation are now part of GlaxoSmithKline.
[0516] Other suitable adjuvants include adjuvant molecules of the general formula (I):
HO(CH2CH2O)n-A-R
wherein, n is 1-50, A is a bond or--C(O)--, R is C1-50 alkyl or Phenyl C1-50 alkyl.
[0517] A further adjuvant of interest is shiga toxin b chain, used for example as described in WO2005/112991.
[0518] One embodiment of the present invention consists of an immunogenic composition comprising a polyoxyethylene ether of general formula (I), wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is C1-50, preferably C4-C20 alkyl and most preferably C1-2 alkyl, and A is a bond. The concentration of the polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12th edition: entry 7717). These adjuvant molecules are described in WO 99/52549.
[0519] Any immunogenic composition provided herein may be prepared using well known methods that result in a combination of antigen, immune response enhancer and a suitable carrier or excipient. The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel (composed of polysaccharides, for example) that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology (see, e.g., Coombes et al., Vaccine 14:1429-1438 (1996)) and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.
[0520] Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. Such carriers include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see, e.g., U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release.
[0521] Any of a variety of delivery vehicles may be employed within pharmaceutical compositions and immunogenic compositions to facilitate production of an antigen-specific immune response. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
[0522] Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau& Steinman, Nature 392:245-251 (1998)) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic immunity (see Timmerman & Levy, Ann. Rev. Med. 50:507-529 (1999)). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within an immunogenic composition (see Zitvogel et al., Nature Med. 4:594-600 (1998)).
[0523] Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.
[0524] Dendritic cells are conveniently categorised as "immature" and "mature" cells, which allows a simple way to discriminate between two well characterised phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterised as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcγ receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1 BB).
[0525] APCs may generally be transfected with a polynucleotide encoding a protein (or portion or other variant thereof) such that the polypeptide, is expressed on the cell surface. Such transfection may take place ex vivo, and a pharmaceutical composition or immunogenic composition comprising such transfected cells may then be used, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and Cell Biology 75:456-460 (1997). Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
[0526] Immunogenic compositions and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, an immunogenic composition or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
[0527] In some embodiments, a "priming" or first administration of an Rv3616c polypeptide (including variants, immunogenic fragments or fusion proteins), or polynucleotide encoding said polypeptide, is followed by one or more "boosting" or subsequent administrations of an Rv3616c polypeptide (including variants, immunogenic fragments or fusion proteins) or polynucleotide encoding said polypeptide ("prime and boost" method). For instance, a first administration with an Rv3616c polypeptide (including variants, immunogenic fragments or fusion proteins) or polynucleotide encoding said polypeptide is followed by one or more subsequent administrations of an Rv3616c polypeptide (including variants, immunogenic fragments or fusion proteins) or polynucleotide encoding said polypeptide.
[0528] In one embodiment, a first administration with an Rv3616c polypeptide or polynucleotide is followed by one or more subsequent administrations of an Rv3616c polypeptide. In one embodiment, a first administration with an Rv3616c polypeptide or polynucleotide is followed by one or more subsequent administrations of an Rv3616c polynucleotide. Usually the first or "priming" administration and the second or "boosting" administration are given about 2-12 weeks apart, or up to 4-6 months apart. Subsequent "booster" administrations are given about 6 months apart, or as long as 1, 2, 3, 4 or 5 years apart. Conventional booster treatment (e.g., a protein priming administration followed by a protein boosting administration) may also useful be in preventing or treating tuberculosis (e.g. preventing or treating latent tuberculosis, in particular preventing or delay tuberculosis reactivation).
Antibodies
[0529] "Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognises an antigen. The recognised immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
[0530] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
[0531] Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterised fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH--CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesised de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).
[0532] For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)). Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanised antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).
[0533] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to fusion proteins can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with fusion protein and not with individual components of the fusion proteins. This selection may be achieved by subtracting out antibodies that cross-react with the individual antigens. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) and Using Antibodies: A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10, 20 or 100 times background (e.g. binding to other Mycobacterium proteins, such as other Mycobacterium tuberculosis proteins).
Diagnostics
[0534] In another aspect, this invention provides methods for using one or more of the polypeptides described above to diagnose latent tuberculosis (for example using T cell response based assays or antibody based assays of conventional format).
[0535] For example, there is provided a method for determining latent M. tuberculosis infection in an individual comprising: [0536] (a) obtaining a sample from the individual; [0537] (b) contacting said sample with an isolated polypeptide which comprises: [0538] (i) an Rv3616c protein sequence; [0539] (ii) a variant of an Rv3616c protein sequence; or [0540] (iii) an immunogenic fragment of an Rv3616c protein sequence; [0541] (c) quantifying the sample response.
[0542] The sample may for example be whole blood or purified cells. Suitably the sample will contain peripheral blood mononucleated cells (PBMC). In one embodiment of the invention the individual will be seropositive. In a second embodiment of the invention the individual will be seronegative.
[0543] Suitably the individual will not previously have been vaccinated against M. tuberculosis infection (e.g. suitably the individual will not previously have been vaccinated with BCG).
[0544] The sample response may be quantified by a range of means known to those skilled in the art, including the monitoring of lymphocyte proliferation or the production of specific cytokines or antibodies. For example, T-cell ELISPOT may be used to monitor cytokines such as interferon gamma (IFNγ), interleukin 2 (IL2) and interleukin 5 (IL5). B-cell ELLISPOT may be used to monitor the stimulation of M. tuberculosis specific antigens. The cellular response may also be characterised by the use of by intra- and extra-cellular staining and analysis by a flow cytometer.
[0545] Methods of quantifying a sample proliferation response include: [0546] (i) pulsing cultured cells with a radiolabel (e.g. tritiated thymidine) and monitoring tritium uptake (e.g. gas scintillation); [0547] (ii) carboxyfluorsecein diacetate succinimidyl ester (CFSE) labelling and fluorescence monitoring of cell division using flow cytometry.
[0548] Quantifying a sample cytokine response includes in particular the monitoring of interferon gamma production.
[0549] When using such quantification methods, a positive response to an antigen may be defined by a signal to noise ratio (S/N ratio) of at least 2:1 (for example, at least 3:1 or at least 5:1).
[0550] In a further aspect of the present invention methods are provided to diagnose latent M. tuberculosis infection 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 an Rv153c polypeptide as described above (or variant, immunogenic fragments thereof or nucleotides encoding them). Such injection may be achieved using any suitable device sufficient to contact the antigen combinations with dermal cells of the patient, such as a tuberculin syringe or 1 mL syringe. The reaction is measured after a period of time, for example at least 48 hours after injection, especially 48-72 hours.
[0551] The DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to the test antigen. The response may be measured visually, using a ruler. In general, a response that is greater than about 0.5 cm in diameter, especially greater than about 1.0 cm in diameter, is a positive response, indicative of prior M. tuberculosis infection, which may or may not be manifested as an active disease.
[0552] For use in a skin test, the Rv3616c component is suitably formulated as a pharmaceutical composition containing a physiologically acceptable carrier. Suitably, the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween 80®.
[0553] The present invention further provides kits for use within any of the above diagnostic methods. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment.
[0554] For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a protein. Such antibodies or fragments may be provided attached to a support material, as described above. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.
[0555] Alternatively, a kit may be designed to detect the level of mRNA encoding a protein in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer, as described above, that hybridises to a polynucleotide encoding a protein. Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a protein of the invention.
[0556] Other diagnostics kits include those designed for the detection of cell mediated responses
[0557] (which may, for example, be of use in the diagnostic methods of the present invention). Such kits will typically comprise: [0558] (i) apparatus for obtaining an appropriate cell sample from a subject; [0559] (ii) means for stimulating said cell sample with an Rv3616c polypeptide (or variant thereof, immunogenic fragments thereof, or DNA encoding such polypeptides); [0560] (iii) means for detecting or quantifying the cellular response to stimulation.
[0561] Suitable means for quantifying the cellular response include a B-cell ELISPOT kit or alternatively a T-cell ELISPOT kit, which are known to those skilled in the art.
[0562] One possible kit comprises: [0563] (a) a polypeptide of the invention; and [0564] (b) a detection reagent suitable for direct or indirect detection of antibody binding.
[0565] Of particular interest are diagnostic kits tailored for quantifying T cell responses: [0566] A diagnostic kit comprising: [0567] (a) a polypeptide of the invention; and [0568] (b) apparatus sufficient to contact said polypeptide with the dermal cells of an individual. [0569] A diagnostic kit comprising: [0570] (a) a polypeptide of the invention; [0571] (b) apparatus sufficient to contact said polypeptide with a sample (e.g. whole blood or more suitably PBMC) from an individual; and [0572] (c) means to quantify the T cell response (e.g. proliferation or IFN-gamma production).
EXAMPLES
[0573] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.
Example 1
Identification of Rv3616c as a latent TB Vaccine Target
[0574] The gene Rv3616c encodes for a conserved hypothetical alanine and glycine rich protein. Rv3616c was selected based on a genome-wide analysis of Mycobacterium tuberculosis genes associated with dormancy phase maintenance and infectivity as in Murphy and Brown BMC. Infect. Dis. 2007 7:84-99. Potential dormancy phase gene targets in Mycobacterium tuberculosis were prioritised through a bioinformatics meta-analysis of published genome-wide DNA microarray datasets of bacterial gene expression under simulated dormancy conditions. Subcellular localisation of M. tuberculosis proteins encoded by genes, was subsequently carried out on the entire genome to identify vaccine targets.
[0575] Briefly, experimental conditions in the dormancy models were quite varied so a zero to five scoring system was developed to normalise these data based upon two criteria: 1) the relevance of the experimental conditions to the dormant state and 2) the rank order of expression. The maximum score for a particular experimental dataset was adjusted based on potential relevance to the clinical occurrence of dormancy phase M. tuberculosis infections. Table 1 shows the data sets collected for Step 1 together with the adjusted maximum scores for each dataset. Additional datasets on gene essentiality for growth were obtained from published studies using transposon-based knockout experiments (TraSH). Genes which had no effect on growth received a score of zero.
TABLE-US-00001 TABLE 1 Sources, experimental models, and scoring criteria for M. tuberculosis DNA microarray gene expression and genome-wide gene knock-out (growth phase essentiality). Timepoint: Reference Experimental model Maximum scorea Betts JC et al. Starvation under controlled O2 96 h: 3 Mol. Microbiol. 2002 43: 717-731 24 h: 2 4 h: 1 Hampshire T et al. Nutrient depletion under 62 and 75 d: 5 Tuberculosis. (Edinb.) 2004 84: 228-238 controlled O2 49 d: 4 18 d: 2 Muttucumaru DG et al. Wayne model of hypoxia# 14 d (NRP-2): 4 Tuberculosis. (Edinb.) 2004 84: 239-246 7 d (NRP-1): 2 Voskuil MI et al. Wayne model of hypoxia# 30 and 80 d: 5 Tuberculosis. (Edinb.) 2004 84: 218-227 14 and 20 d: 4 10 and 12 d: 3 6 and 8 d: 2 Schnappinger D et al. Infection of mouse macrophages, 24 and 48 h: 5 J. Exp. Med. 2003 198: 693-704 +/- γ-INF Karakousis PC et al. Hollow fiber subcutaneous 10 d: 3 J. Exp. Med. 2004 200: 647-657 implant in mice Talaat AM et al. Infection of mice. MTB harvested 28 d: 3 Proc. Natl. Acad. Sci. U.S.A 2004, from lungb 101: 4602-4607 Sassetti CM et al. TraSH mutated libraries grown on 14 d: 5 Mol. Microbiol. 2003 48: 77-84 solid media Rengarajan J et al. Infection of mouse macrophages, 7 d: 5 Proc. Natl. Acad. Sci. U.S.A 2005, +/- γ-INF with TraSH mutated 102: 8327-8332 libraries of M. tuberculosis Sassetti CM et al. C57BL/6J mice infected with 7, 14, 28 and Proc. Natl. Acad. Sci. U.S.A 2003 TraSH mutated libraries of 56 d: 5 100: 12989-12994 M. tuberculosis aMaximum score based on relevance as a dormancy model; h = hour; d = day.. bRatio of M. tuberculosis from Balb/c lung to MTB in aerated culture for 28 d. #Wayne LG and Hayes LG Infect. Immun. 1996 64: 2062-2069
[0576] Step 2--In applying the second criterion, the rank order of gene expression, gene scores from each dataset were ordered from highest to lowest based on expression ratio (fold expression in the experimental condition versus cells in log-phase liquid culture). The highest scoring gene received the maximum score for that particular dataset (listed in column 3 of Table 1. (e.g. 5, 4 . . . , 1 point)). The score was decreased by 0.005 points for each gene in order until zero, or the end of the data set was reached. Thus when the maximum score was 4 points, the 100th ranked gene would receive a score of 3.500. For a maximum score of 5 points, 1000 genes or 25% of the M. tuberculosis genome received a score. For experiments where data from multiple time points were collected, the maximum score across all time points was used as the final score.
[0577] In Step 3 scores for each gene in each of the experimental conditions were collected into a Microsoft Access database. Reference fields were added to facilitate prioritisation, such as the Refseq ID, Genbank function, Genbank note, Tuberculist classification, and KEGG and Sanger Center links. By combining the data from different studies and sources, a consensus view was reached about the particular genes and pathways most critical for survival in the dormant state.
[0578] In Step 4, a prioritised list of therapeutic targets was derived utilising the top 400 scoring genes (˜10% of the genome) supplemented by expert computational and manual analysis of biochemical pathways, enzymology, drug tractability, homology to human genes and other prior knowledge. The great majority of the high scoring genes come from the subset where two or three of the groups intersect.
[0579] In Step 5, the identification of subcellular localisation of M. tuberculosis proteins encoded by genes, was carried out on the entire genome. The heuristic used for membrane protein prediction is described in Chalker et al. J. Bacteriol. 2001 183:1259-1268. Average hydropathy profiles (H) (von Heijne G J. Mol. Biol. 1992 225:487-494) were generated using GES hydropathy values (Engelman D M et al. Annu. Rev. Biophys. Biophys. Chem. 1986 15:321-353) weighted using a trapezoid window. Using a process similar to the initial steps of the TopPred II algorithm (Claros M G et al. Comput. Appl. Biosci. 1994 10:685-686), helical transmembrane segments (TMS) were predicted for each peptide sequence by selecting 19 amino acids centered on the highest H value (MaxH), masking these from further consideration, and repeating the process until no peaks with a H of >0.5 remained. Subcellular locations were assigned based on the peak MaxH value, number of segments with a H of >1.0, and distribution and peak H values of the putative TMS. A MaxH cutoff of 1.15 was chosen to maximize the discrimination between two SwissProtein release 34 test datasets containing transmembrane and cytoplasmic proteins, respectively (Boyd D et al. Protein Sci. 1998 7:201-205). Proteins with a MaxH of <1.15 were classified as cytoplasmic, while those with a MaxH of >1.15 and at least three possible TMS were classified as membrane proteins. Anchored proteins were defined as having exactly two TMS, one starting before amino acid (aa) 35 and one having a H of >1.15 with the other having a H not lower than 0.5. SignalP with Gram positive settings was specifically used for M. bacterium to identify secreted proteins amongst those classified as either cytoplasmic or "unknown" in the heuristic analysis (Nielsen H et al. Protein Eng. 1997 10:1-6).
[0580] Rv3616c ranked very high as a vaccine antigen according to several criteria: [0581] (i) Rv3616c is consistently up-regulated across all models of dormancy. Among the entire suite of 3999 genes scored in the meta-analysis, Rv3616c was ranked in the top quartile of overexpressed genes across all dormancy models. The up-regulated score for Rv3616c was 6.52 which favourably compared with the top gene score of 22.28. [0582] (ii) Rv3616c ranked as being highly essential for survival in the mouse spleen infection model (scoring 4.945, out of a possible scoring of 5). [0583] (iii) Subcellular localisation predicted that Rv3616c protein is a membrane bound protein and thus has significant extracellular exposure, indicating suitability as a vaccine target. [0584] (iv) Rv3616c can a elicit protective response against initial tuberculosis challenge. [0585] (v) Rv3616c is broadly recognised as an antigen.
Example 2
Rv3616c Epitope Prediction
Method
[0586] T cell epitope prediction was based on the following approaches:
TABLE-US-00002 Prediction Name URL/References CD4 and Multipred website: antigen.i2r.a-star.edu.sg/multipred/ CD8 Zhang, G. L., Khan, A. M., Srinivasan, K. N., August, J. T. and Brusic, V. (2005) "MULTIPRE d: a computational system for prediction of promiscuous HLA binding peptides" Nucleic Acids Res. 33, W172-W179. SVMHC website: www-bs.informatik.uni-tuebingen.de/SVMHC "Prediction of MHC class I binding peptides, using SVMHC." Pierre Donnes and Arne Elofsson in: BMC Bioinformatics 2002 3: 25 CD4 ProPred website: www.imtech.res.in/raghava/propred/ Singh, H. and Raghava, G. P. S.(2001) "ProPred: Prediction of HLA-DR binding sites." Bioinformatics, 17(12), 1236-37. Tepitope2 In house program based on: H. Bian, J. Hammer (2004) "Discovery of promiscuous HLA-II- restricted T cell epitopes with TEPITOPE." Methods 34: 468-75 CD8 nHLA website: www.imtech.res.in/raghava/nhlapred/ Bhasin M. and Raghava G P S (2006) "A hybrid approach for predicting promiscuous MHC class I restricted T cell epitopes"; J. Biosci. 32: 31-42 NetCTL website: www.cbs.dtu.dk/services/NetCTL/ "An integrative approach to CTL epitope prediction. A combined algorithm integrating MHC-I binding, TAP transport efficiency, and proteasomal cleavage predictions." Larsen M. V., Lundegaard C., Kasper Lamberth, Buus S,. Brunak S., Lund O., and Nielsen M. European Journal of Immunology. 35(8): 2295-303. 2005 Epijen website: www.jenner.ac.uk/EpiJen/ Doytchinova, I. A., P. Guan, D. R. Flower. "EpiJen: a server for multi-step T cell epitope prediction." BMC Bioinformatics, 2006, 7, 131. Syfpeithi website: www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm Hans-Georg Rammensee, Jutta Bachmann, Niels Nikolaus Emmerich, Oskar Alexander Bachor, Stefan Stevanovic: "SYFPEITHI: database for MHC ligands and peptide motifs." Immunogenetics (1999) 50: 213-219 PredTAP website: antigen.i2r.a-star.edu.sg/predTAP/ Zhang, G. L., Petrovsky, N., Kwoh, C. K., August, J. T. and Brusic, V. (2006) "PREDTAP: a system for prediction of peptide binding to the human transporter associated with antigen processing." Immunome Res. 2(1), 3. PAPROC http://www.paproc2.de/paproc1/paproc1.html C. Kuttler, A. K. Nussbaum, T. P. Dick, H.-G. Rammensee, H. Schild, K. P. Hadeler, "An algorithm for the prediction of proteasomal cleavages", J. Mol. Biol. 298 (2000), 417-429 A. K. Nussbaum, C. Kuttler, K. P. Hadeler, H.-G. Rammensee, H. Schild, "PAProC: A Prediction Algorithm for Proteasomal Cleavages available on the WWW", Immunogenetics 53 (2001), 87-94
Results
TABLE-US-00003 [0587] TABLE 2 Putative Rv3616c human CD4+ T cell epitopes Putative CD4 Amino epitope acid Epitope number position sequence SEQ ID No: HLA allele 1 5 FIIDPTISA SEQ ID No: 29 DRB1_0301, DRB1_0401, DRB1_1101 2 31 ILYSSLEYF SEQ ID No: 30 DRB1_0301 3 36 LEYFEKALE SEQ ID No: 31 DRB1_1301 4 63 YAGKNRNHV SEQ ID No: 32 DRB1_0801 5 87 LIHDQANAV SEQ ID No: 33 DRB1_0301, DRB1_0401 6 111 FVRPVAVDL SEQ ID No: 34 DRB1_0101 7 119 LTYIPVVGH SEQ ID No: 35 DRB1_0401 8 121 YIPVVGHAL SEQ ID No: 36 DRB1_0101 9 151 YLVVKTLIN SEQ ID No: 37 DRB1_0401 10 152 LVVKTLINA SEQ ID No: 38 DRB1_1301 11 154 VKTLINATQ SEQ ID No: 39 DRB1_0401 12 164 LKLLAKLAE SEQ ID No: 40 DRB1_0301, DRB1_0801, DRB1_1101, DRB1_1301 13 173 LVAAAIADI SEQ ID No: 41 DRB1_0301, DRB1_1101, DRB1_1301 14 181 IISDVADII SEQ ID No: 42 DRB1_0301 15 197 WEFITNALN SEQ ID No: 43 DRB1_0401 16 252 LFGAAGLSA SEQ ID No: 44 DRB1_1501 17 264 LAHADSLAS SEQ ID No: 45 DRB1_0401 18 270 LASSASLPA SEQ ID No: 46 DRB1_0401 19 288 FGGLPSLAQ SEQ ID No: 47 DRB1_0401
TABLE-US-00004 TABLE 3 Putative Rv3616c human CD8+ T cell epitopes Putative CD8 Amino epitope acid Epitope number position sequence SEQ ID No: HLA allele 1 5 FIIDPTISA SEQ ID No: 48 A2 2 6 IIDPTISAI SEQ ID No: 49 A_0101, A2 3 9 PTISAIDGL SEQ ID No: 50 A2, A_0201, B7, B8 4 10 TISAIDGLY SEQ ID No: 51 A1, A_0101, A3, A_0301 5 12 SAIDGLYDL SEQ ID No: 52 A2, B_3501 6 13 AIDGLYDLL SEQ ID No: 53 A_0101, A_0201, B44 7 17 LYDLLGIGI SEQ ID No: 54 A24 8 25 IPNQGGILY SEQ ID No: 55 B7, A_0101, B_3501, B51 9 30 GILYSSLEY SEQ ID No: 56 A1, A_0101, A3, A_0301 10 33 YSSLEYFEK SEQ ID No: 57 A1, A_0301 11 35 SLEYFEKAL SEQ ID No: 58 A_0201, B7, Cw_0401, Cw_0602 12 38 YFEKALEEL SEQ ID No: 59 A24, A_2402, B8, Cw_0401, Cw_0602 13 39 FEKALEELA SEQ ID No: 60 B44, B_4403 14 69 NHVNFFQEL SEQ ID No: 61 A24, Cw_0602 15 76 ELADLDRQL SEQ ID No: 62 A_0201 16 77 LADLDRQLI SEQ ID No: 63 A_0101, B51 17 79 DLDRQLISL SEQ ID No: 64 A_0101, A_0201 18 80 LDRQLISLI SEQ ID No: 65 A24, B7, B51 19 94 AVQTTRDIL SEQ ID No: 66 B7 20 103 EGAKKGLEF SEQ ID No: 67 A24, B7 21 107 KGLEFVRPV SEQ ID No: 68 A_0201, B51 22 108 GLEFVRPVA SEQ ID No: 69 A_0101, A_0301 23 109 LEFVRPVAV SEQ ID No: 70 B44 24 111 FVRPVAVDL SEQ ID No: 71 B7, B8, B_3501 25 113 RPVAVDLTY SEQ ID No: 72 B7, A_0101, B_3501, B51 26 116 AVDLTYIPV SEQ ID No: 73 A2, A_0201 27 120 TYIPVVGHA SEQ ID No: 74 A24 28 121 YIPVVGHAL SEQ ID No: 75 A_0101, A2, A_0201, B7, B8 29 129 LSAAFQAPF SEQ ID No: 76 A1, B7, B_3501 30 130 SAAFQAPFC SEQ ID No: 77 A_0201 31 131 AAFQAPFCA SEQ ID No: 78 A_0301, B_3501 32 133 FQAPFCAGA SEQ ID No: 79 A2, A_0201 33 135 APFCAGAMA SEQ ID No: 80 B7, B_3501 34 136 PFCAGAMAV SEQ ID No: 81 A3 35 141 AMAVVGGAL SEQ ID No: 82 A2, A_0201, A24, B7 36 143 AVVGGALAY SEQ ID No: 83 A1, A3, A_0301, B7 37 147 GALAYLVVK SEQ ID No: 84 A3, A_0301 38 149 LAYLVVKTL SEQ ID No: 85 B8, B44, B51 39 150 AYLVVKTLI SEQ ID No: 86 A24 40 155 KTLINATQL SEQ ID No: 87 A_0201, A2, A_0301, A24 41 156 TLINATQLL SEQ ID No: 88 A2, A_0201, A3, A_0101, Cw_0401 42 158 INATQLLKL SEQ ID No: 89 B7, B8, Cw_0602 43 159 NATQLLKLL SEQ ID No: 90 A_2402, B7, B_3501, B44, Cw_0401, Cw_0602 44 162 QLLKLLAKL SEQ ID No: 91 A2, A_0201, A_0301, A_2402, B8, Cw_0401, Cw_0602 45 165 KLLAKLAEL SEQ ID No: 92 A2, A_0201, A_0301, B7, B8, Cw_0602 46 166 LLAKLAELV SEQ ID No: 93 A2, A_0201, A_0101, B8 47 169 KLAELVAAA SEQ ID No: 94 A2 48 170 LAELVAAAI SEQ ID No: 95 A1, A24, B51 49 173 LVAAAIADI SEQ ID No: 96 B7, B51 50 177 AIADIISDV SEQ ID No: 97 A2, A_0201, Cw_0602 51 178 IADIISDVA SEQ ID No: 98 A_0101, B_3501 52 182 ISDVADIIK SEQ ID No: 99 A1, A_0301 53 192 TLGEVWEFI SEQ ID No: 100 A2, A_0201 54 199 FITNALNGL SEQ ID No: 101 A2 55 202 NALNGLKEL SEQ ID No: 102 B51, A_2402, B_3501, Cw_0602 56 213 KLTGWVTGL SEQ ID No: 103 A2, A_0201 57 214 LTGWVTGLF SEQ ID No: 104 Al, A_0101, A24 58 225 GWSNLESFF SEQ ID No: 105 A24 59 228 NLESFFAGV SEQ ID No: 106 A2, A_0201 60 231 SFFAGVPGL SEQ ID No: 107 A2, A_0201, A24, Cw_0401 61 238 GLTGATSGL SEQ ID No: 108 A2, A_0201 62 246 LSQVTGLFG SEQ ID No: 109 A1, B8 63 247 SQVTGLFGA SEQ ID No: 110 A2 64 258 LSASSGLAH SEQ ID No: 111 Al, A3, B7, B8 65 260 ASSGLAHAD SEQ ID No: 112 Al, A3, A_0301 66 262 SGLAHADSL SEQ ID No: 113 A_0201 67 263 GLAHADSLA SEQ ID No: 114 A_0101, A_0201, A_0301 68 269 SLASSASLP SEQ ID No: 115 A_0201, A_0301 69 271 ASSASLPAL SEQ ID No: 116 B7 70 286 SGFGGLPSL SEQ ID No: 117 A2, A_0201, B51 71 291 LPSLAQVHA SEQ ID No: 118 B7, B_3501, B51 72 298 HAASTRQAL SEQ ID No: 119 B7, B8, B_3501 73 301 STRQALRPR SEQ ID No: 120 A3, A_0301 74 307 RPRADGPVG SEQ ID No: 121 B7, B_0702, B8, B51 75 319 EQVGGQSQL SEQ ID No: 122 B7, B44 76 350 GASKGTTTK SEQ ID No: 123 A3, A_0301 77 351 ASKGTTTKK SEQ ID No: 124 A3, A_0301 78 353 KGTTTKKYS SEQ ID No: 125 A_0301, B8 79 368 TEDAERAPV SEQ ID No: 126 B44
[0588] As can be seen from Tables 2 and 3, Rv3616c contains a number of predicted CD4+ and CD8 T cell epitopes. Furthermore, this information suggests that the protein carries epitopes that can be recognised by HLAs which occur worldwide (that is HLAs from Caucasian, African, Asian or Latin-American individuals--see website at www.allelefrequencies.net).
Example 3
Rv3616c Epitope Identification
[0589] A range of 30 overlapping peptides covering the full length of Rv3616c were prepared (see FIG. 1 for details and SEQ ID Nos: 127-156) and tested for their ability to stimulate PBMC from four PPD+ donors.
[0590] The data, shown in FIG. 2, reveals that peptides 1-7 and 17-30 were immunogenic for these individuals.
[0591] It should be noted that peptides 8-16 (amino acid residues 92-215) may be immunogenic in other individuals of differing HLA type.
Example 4
H37Rv Homologues
[0592] Rv3616c sequences from a number of M. tuberculosis strains and BCG were identified using BLASTP searches of GenBank (H37Rv reference sequence accession number NP--218133.1):
TABLE-US-00005 Strain Accession Number % identity CDC1551 NP_338263.1 99 F11 YP_001289574.1 99 Haarlem ZP_02248979.1 99 C ZP_00877472.1 99 BCG YP_979759.1 99
[0593] Alignment of the homologue sequences indicates a high level of identity.
Biological Assays
Quantification of T Cell Responses to Rv3616c
[0594] Polypeptides may be screened for their ability to activate T-cells (induction of proliferation and/or production of cytokines) in peripheral blood mononuclear cell (PBMC) or in whole blood preparations from infected (such as latently infected) individuals.
[0595] Latently infected individuals are usually identified by a skin test that has a diameter above 10 mm and without symptoms, with no Mtb positive culture, with a negative sputum negative and with no lesion (as detected by chest X-Ray).
[0596] A range of in vitro assays can be used based on PBMC samples or whole blood: after restimulation in presence of the antigen (or variant/immunogenic fragment thereof as appropriate) the proliferation of the cells may be determined (as measured by CFSE/flow cytometry) or the production of cytokines quantified (present in the supernatant of cultured cells and measured by ELISA, or, after intracellular staining of CD4 and CD8 T cells and analysis by flow cytometry).
[0597] For example, PBMC samples may be obtained from heparinised whole blood by Ficoll-Hypaque density gradient centrifugation following standard procedures. The cells may then be washed and cryopreserved in liquid nitrogen until testing (for further details see Lalvani A et al. J. Infect. Dis. 1999 180:1656-1664).
T Cell Proliferation
[0598] The specific immune response may be characterised by performing lymphocyte proliferation analysis using the tritiated thymidine. This technique assesses the cellular expansion upon in vitro stimulation to an antigen. In practice, cell proliferation is determined by estimating incorporation of tritiated-thymidine into DNA, a process closely related to underlying changes in cell number.
[0599] More suitably, lymphocyte proliferation may be performed using the succinimidyl ester of carboxyfluorsecein diacetate (CFSE). CFSE spontaneously and irreversibly couples to both intracellular and cell surface proteins by reaction with lysine side chains and other available amine groups. When lymphocyte cells divide, CFSE labelling is distributed equally between the daughter cells, which are therefore half as fluorescent as the parents. As a result, halving of cellular fluorescence intensity marks each successive generation in a population of proliferating cells and is readily followed by flow cytometry (for further details see Hodgkins, P D et al J. Exp. Med. 1996 184:277-281).
[0600] Practically, after thawing, PMBC may be washed and stained with CFSE before being cultivated (2×105 cells) for 72 hrs with 10 ug/ml of antigen in culture media (RPMI-1640 supplemented with glutamine, non essential amino acid, pyruvate and heat inactivated human AB serum). Cells may then harvested and their phenotype characterised using surface staining to identify memory CD8 and CD4+ T-Cells. Subsequently, flow cytometry analysis can be used to indicate the extent of lymphocyte proliferation in response to each antigen (proportion of cells with decreased CFSE intensity upon in vitro stimulation).
Cytokine Production
[0601] IFN-γ production (or the production of other cytokines such as e.g. IL2, TNF-alpha, IL5, IL12 etc) may be measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates may be coated with a mouse monoclonal antibody directed to human IFN-γ (PharMingen, San Diego, Calif.) in PBS for four hours at room temperature. Wells are then blocked with PBS containing 5% (W/V) non-fat dried milk for 1 hour at room temperature. The plates are then washed, for example, six times in PBS/0.2% TWEEN-20 and samples diluted 1:2 in culture medium in the ELISA plates are incubated overnight at room temperature. The plates are again washed and a polyclonal rabbit anti-human IFN-γ serum, for example, diluted 1:3000 in PBS/10% normal goat serum may be added to each well. The plates are then incubated for two hours at room temperature, washed and horseradish peroxidase-coupled anti-rabbit IgG (Sigma Chemical So., St. Louis, Mo.) may be added, for example, at a 1:2000 dilution in PBS/5% non-fat dried milk. After a further two hour incubation at room temperature, the plates are washed and TMB substrate added. The reaction may be stopped after 20 min with 1 N sulfuric acid. Optical density can then be determined at 450 nm using 570 nm as a reference wavelength. Typically, fractions that result in both replicates giving an OD two fold greater than the mean OD from cells cultured in medium alone may be considered positive.
Example 5
Immunogenicity in CB6F1 Mice
[0602] The immunogenicity of the antigen was evaluated in CB6F1 mice (first generation cross of BALB/c and C57BL/6 mice).
[0603] CB6F1 mice were immunised intramuscularly three times (on day 0, day 14 and day 28) with 0.5 ug of protein antigen in combination with the Adjuvant System AS01E (a liposomal adjuvant formulation comprising 3D-MPL and QS21).
[0604] The experimental design was as follows:
TABLE-US-00006 Group Day 0 Day 14 Day 28 1 0.5 ug Rv3616c/AS01E 0.5 ug 0.5 ug Rv3616c/AS01E Rv3616c/AS01E
[0605] A total of 24 mice were used in the protocol group.
[0606] Peripheral blood lymphocytes (PBL) were collected and pooled on day 21 (i.e. 7 days post second immunisation) and day 35 (i.e. 7 days post third immunisation) and the antigen-specific CD4 & CD8 T cell responses (as determined by CD4 or CD8 T cells producing IL-2 and/or IFN-gamma and/or TNF-alpha) were measured by flow cytometry after overnight in vitro restimulation with pools of 15mer peptides covering the sequences of interest. The detection of mouse T cells that express IL-2 and/or IFN-gamma and/or TNF-alpha was done by using short-term antigen-driven in vitro amplification of cytokine expression.
[0607] Briefly, PharmLyse solution (BD-Pharmingen) was added to heparinised mouse peripheral blood in order to lyse the red blood cells. The PBLs (Peripheral Blood Lymphocytes) obtained were washed and then incubated in the presence of a pool of 15-mer peptides--overlapping by 11 amino acids--covering the sequence of the antigen of interest and of 1 ug/ml of antibodies to CD28 and CD49d (BD-Pharmingen). Each 15-mer peptide was used at a final concentration of 1 ug/ml. Medium controls were also stimulated with antibodies to CD28 and CD49d.
[0608] The cytokine secretion blocking compound brefeldin-A (BD-Pharmingen) was added 2 h after the onset of the cultures at 37° C., 5% CO2 and the cells maintained at 37° C., 5% CO2 for 4 additional hours followed by overnight incubation at +4° C.
[0609] Cells were then harvested and stained with Pacific Blue-coupled anti-CD4 (BD--clone RM4-5, BD-Pharmingen) and peridinin chlorophyll A protein (PerCp) cyanin5.5 (Cy5.5)-coupled anti-CD8 alpha (clone 53-6.7, BD-Pharmingen) antibodies.
[0610] Cells were then washed, fixed, permeabilised (Cytofix-cytoperm kit, BD-Pharmingen) and stained with allophycocyanin-coupled anti-IFN-g antibodies (clone XMG1.2, BDPharmingen), fluorescein isothiocyanate (FITC)-coupled anti-IL-2 antibodies (clone JES 6-5H4, Beckman Coulter) and phycoerythrin (PE)-coupled anti-TNF alpha antibodies (clone MP6-XT22, BDPharmingen). After final washes, stained cells were analysed on a LSR II flow cytometer (Beckton-Dickinson). A minimum number of 10,000 cells were acquired in the CD8+ subset.
[0611] For further background see Walzer T et al Cell Immunol. 2000 206(1):16-25 and Maecker H T et al J. Immunol. Methods 2001 255(1-2):27-40.
[0612] As negative controls, some cells were also cultured overnight in vitro in culture medium (unstimulated). The antigen-specific responses were calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide-stimulated cells.
[0613] At each timepoint and for each group, the data was collected from 4 pools of 6 mice each. The data below is presented as the % of CD4 or CD8 T cells producing IL-2 and/or IFN-gamma and/or TNF-alpha. Each individual pool of mice is plotted (triangles) as well as the average value of the group (bar).
[0614] FIG. 3 shows that on day 21 (i.e. 7 days post second immunisation), Rv3616c-specific CD4 and CD8 T cell responses are detected in mice immunised with 0.5 ug of Rv3616c/AS01E.
[0615] FIG. 4 shows the cytokine profile of CD4 T cell response from the Rv3616c peptide pool-stimulated PBL (not medium removed) on day 21 (i.e. 7 days post second immunisation).
[0616] FIG. 5 shows the cytokine profile of CD8 T cell response from the Rv3616c peptide pool-stimulated PBL (not medium removed) on day 21 (i.e. 7 days post second immunisation).
[0617] FIG. 6 shows that on day 35 (i.e. 7 days post third immunisation), Rv3616c-specific CD4 and CD8 T cell responses are detected in mice immunised with 0.5 ug of Rv3616c/AS01E. The third dose increases the CD4 T cell response but not the CD8 T cell response. Due to technical difficulties, data was only available for a single pool.
[0618] FIG. 7 shows the cytokine profile of CD4 T cell response from the Rv3616c peptide pool-stimulated PBL (not medium removed) on day 35 (i.e. 7 days post third immunisation). Due to technical difficulties, data was only available for a single pool.
[0619] FIG. 8 shows the cytokine profile of CD8 T cell response from the Rv3616c peptide pool-stimulated PBL (not medium removed) on day 35 (i.e. 7 days post third immunisation). Due to technical difficulties, data was only available for a single pool.
Example 6
Immunogenicity in C57BL/6 Mice
[0620] The immunogenicity of the antigen was also evaluated in C57BL/6 mice. C57BL/6 mice were immunised intramuscularly three times (on day 0, day 14 and day 28) with 1 ug protein antigen in combination with a the Adjuvant System AS01E (a liposomal adjuvant formulation comprising 3D-MPL and QS21).
[0621] The experimental design was the following:
TABLE-US-00007 Group Day 0 Day 14 Day 28 1 1 ug Rv3616c/AS01E 1 ug 1 ug Rv3616c/AS01E Rv3616c/AS01E
[0622] Peripheral blood lymphocytes (PBL) were collected and pooled on day 21 (i.e. 7 days post second immunisation) and day 35 (i.e. 7 days post third immunisation) and the antigen-specific CD4 & CD8 T cell responses (as determined by CD4 or CD8 T cells producing IL-2 and/or IFN-gamma and/or TNF-alpha) were measured by flow cytometry after overnight in vitro restimulation with pools of 15mer peptides covering the sequences of interest. The procedure followed was as described previously.
[0623] As negative controls, some cells were also cultured overnight in vitro in culture medium (unstimulated). The antigen-specific responses were calculated by subtracting the average cytokine response produced by unstimulated cells from the average cytokine response produced by the peptide-stimulated cells.
[0624] At each timepoint and for each group, the data was collected from 4 pools of 6 mice each. The data below is presented as the % of CD4 or CD8 T cells producing IL-2 and/or IFN-gamma and/or TNF-alpha. Each individual pool of mice is plotted (triangles) as well as the average value of the group (bar).
[0625] FIG. 9 shows that on day 21 (i.e. 7 days post second immunisation), Rv3616c-specific CD4 and CD8 T cell responses are detected in mice immunised with 1 ug of Rv3616c/AS01E, although the antigen-specific CD8 T cell response is very low (cytokine profile data is therefore not shown).
[0626] FIG. 10 shows the cytokine profile of CD4 T cell response from the Rv3616c peptide pool-stimulated PBL (not medium removed) on day 21 (i.e. 7 days post second immunisation).
[0627] FIG. 11 shows that on day 35 (i.e. 7 days post third immunisation), Rv3616c-specific CD4 and CD8 T cell responses are detected in mice immunised with 1 ug of Rv3616c/AS01E. A third immunisation dose increases the CD4 T cell responses but only slightly the CD8 T cell response.
[0628] FIG. 12 shows the cytokine profile of CD4 T cell response from the Rv3616c peptide pool-stimulated PBL (not medium removed) on day 35 (i.e. 7 days post third immunisation).
[0629] FIG. 13 shows the cytokine profile of CD8 T cell response from the Rv3616c peptide pool-stimulated PBL (not medium removed) on day 35 (i.e. 7 days post third immunisation).
Example 7
In Vitro Recognition by PBMC from Humans with Latent TB
[0630] Experiments were performed in order to assess the peripheral T cell response specific to the inventive antigen in 4 TB naive healthy adults (PPD skin test=0 mm) and 8 TB latently Infected healthy adults (PPD skin test=15 mm or above) from South Africa
TABLE-US-00008 PPD Skin Test Data Induration Individual ID diameter Number (mm) 4 0 5 0 33 0 38 0 36 15 46 15 13 15 7 16 58 25 74 26 8 53 60 55
[0631] The cell-mediated immune (CMI) response was assessed by measuring cytokines on isolated peripheral blood mononuclear cells (PBMCs) by intracellular cytokine staining (ICS) assay.
[0632] ICS carried out was an adaptation of previously described methodology (see Von Eschen et al, Hum. Vaccin. 2009 5(7)). PBMCs were stimulated in vitro by one pool of 15-mer peptides--overlapping by 11 amino acids--covering the entire sequence of the antigen of interest. Cells were stimulated with peptides for 2 hours, further cultured overnight in the presence of Brefeldin A, processed for ICS and analysed using flow cytometry. The frequencies of the antigen-specific CD3+CD4+ or CD3+CD8+ T cells expressing IFN-gamma and/or TNF-alpha and/or IL-17 were measured. Medium-stimulated cell responses were subtracted from the responses obtained in peptide pools stimulated cells.
ICS: Antibodies
[0633] Anti-CD3 PO (Invitrogen--cat CD0330) [0634] Anti-CD4 PB (BD--cat 558116) [0635] Anti-CD8 APC--H7 (BD--cat 641400) [0636] Anti-IFNg AF700 (BD-Pharmingen--cat 557995) [0637] Anti-TNF PE-Cy7 (BD-Pharmingen--cat 557647) [0638] Anti-IL17 AF647 (BD-Pharmingen--cat 51-7178-71)
[0639] The results are presented as number of antigen-specific CD3+CD4+ T cells expressing TNF-alpha and IFN-gamma, per million of CD3+CD4+ T cells since these cells represent the main population of the antigen-specific CD4 T cells (the background response level due to the medium is removed). No antigen-specific CD3+CD8+ T cells were detected. FIG. 14 shows that an antigen-specific CD4 T cell response is measured in 6 out of 8 latently infected individuals (not in individuals number 7 and 74) when compared to the non-specific CD4 T cell response measured in the naive individuals.
[0640] In conclusion it may be noted that the Rv3616c antigen is capable of eliciting an immune response in both CB6F1 and C57BL/6 mice. Furthermore, the profile of cytokine production indicates that a large proportion of antigen-specific T-cells express a plurality of Th1 associated cytokines (i.e. a polyfunctional T-cell response is elicited). Importantly both CD4 and CD8 antigen-specific T-cells are present after immunisation, CD8 cells may be particularly important in a latent TB scenario. The relevance if Rv3616c to human infection is confirmed by the high level of recognition in latently infected individuals from South Africa and the absence of responses in naive subjects. Rv3616c may therefore be expected to be of substantial value in the prevention, treatment and diagnosis of latent tuberculosis infection.
[0641] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
[0642] All references referred to in this application, including patents and patent applications, are incorporated herein by reference to the fullest extent possible as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
[0643] Throughout the specification and the claims which follow, unless the context requires otherwise, the word `comprise`, and variations such as `comprises` and `comprising`, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.
Sequence CWU
1
1591392PRTMycobacterium tuberculosisMISC_FEATUREStrain H37Rv 1Met Ser Arg
Ala Phe Ile Ile Asp Pro Thr Ile Ser Ala Ile Asp Gly1 5
10 15Leu Tyr Asp Leu Leu Gly Ile Gly Ile
Pro Asn Gln Gly Gly Ile Leu 20 25
30Tyr Ser Ser Leu Glu Tyr Phe Glu Lys Ala Leu Glu Glu Leu Ala Ala
35 40 45Ala Phe Pro Gly Asp Gly Trp
Leu Gly Ser Ala Ala Asp Lys Tyr Ala 50 55
60Gly Lys Asn Arg Asn His Val Asn Phe Phe Gln Glu Leu Ala Asp Leu65
70 75 80Asp Arg Gln Leu
Ile Ser Leu Ile His Asp Gln Ala Asn Ala Val Gln 85
90 95Thr Thr Arg Asp Ile Leu Glu Gly Ala Lys
Lys Gly Leu Glu Phe Val 100 105
110Arg Pro Val Ala Val Asp Leu Thr Tyr Ile Pro Val Val Gly His Ala
115 120 125Leu Ser Ala Ala Phe Gln Ala
Pro Phe Cys Ala Gly Ala Met Ala Val 130 135
140Val Gly Gly Ala Leu Ala Tyr Leu Val Val Lys Thr Leu Ile Asn
Ala145 150 155 160Thr Gln
Leu Leu Lys Leu Leu Ala Lys Leu Ala Glu Leu Val Ala Ala
165 170 175Ala Ile Ala Asp Ile Ile Ser
Asp Val Ala Asp Ile Ile Lys Gly Thr 180 185
190Leu Gly Glu Val Trp Glu Phe Ile Thr Asn Ala Leu Asn Gly
Leu Lys 195 200 205Glu Leu Trp Asp
Lys Leu Thr Gly Trp Val Thr Gly Leu Phe Ser Arg 210
215 220Gly Trp Ser Asn Leu Glu Ser Phe Phe Ala Gly Val
Pro Gly Leu Thr225 230 235
240Gly Ala Thr Ser Gly Leu Ser Gln Val Thr Gly Leu Phe Gly Ala Ala
245 250 255Gly Leu Ser Ala Ser
Ser Gly Leu Ala His Ala Asp Ser Leu Ala Ser 260
265 270Ser Ala Ser Leu Pro Ala Leu Ala Gly Ile Gly Gly
Gly Ser Gly Phe 275 280 285Gly Gly
Leu Pro Ser Leu Ala Gln Val His Ala Ala Ser Thr Arg Gln 290
295 300Ala Leu Arg Pro Arg Ala Asp Gly Pro Val Gly
Ala Ala Ala Glu Gln305 310 315
320Val Gly Gly Gln Ser Gln Leu Val Ser Ala Gln Gly Ser Gln Gly Met
325 330 335Gly Gly Pro Val
Gly Met Gly Gly Met His Pro Ser Ser Gly Ala Ser 340
345 350Lys Gly Thr Thr Thr Lys Lys Tyr Ser Glu Gly
Ala Ala Ala Gly Thr 355 360 365Glu
Asp Ala Glu Arg Ala Pro Val Glu Ala Asp Ala Gly Gly Gly Gln 370
375 380Lys Val Leu Val Arg Asn Val Val385
39021179DNAMycobacterium tuberculosismisc_featureStrain H37Rv
2atgagcagag cgttcatcat cgatccaacg atcagtgcca ttgacggctt gtacgacctt
60ctggggattg gaatacccaa ccaagggggt atcctttact cctcactaga gtacttcgaa
120aaagccctgg aggagctggc agcagcgttt ccgggtgatg gctggttagg ttcggccgcg
180gacaaatacg ccggcaaaaa ccgcaaccac gtgaattttt tccaggaact ggcagacctc
240gatcgtcagc tcatcagcct gatccacgac caggccaacg cggtccagac gacccgcgac
300atcctggagg gcgccaagaa aggtctcgag ttcgtgcgcc cggtggctgt ggacctgacc
360tacatcccgg tcgtcgggca cgccctatcg gccgccttcc aggcgccgtt ttgcgcgggc
420gcgatggccg tagtgggcgg cgcgcttgcc tacttggtcg tgaaaacgct gatcaacgcg
480actcaactcc tcaaattgct tgccaaattg gcggagttgg tcgcggccgc cattgcggac
540atcatttcgg atgtggcgga catcatcaag ggcaccctcg gagaagtgtg ggagttcatc
600acaaacgcgc tcaacggcct gaaagagctt tgggacaagc tcacggggtg ggtgaccgga
660ctgttctctc gagggtggtc gaacctggag tccttctttg cgggcgtccc cggcttgacc
720ggcgcgacca gcggcttgtc gcaagtgact ggcttgttcg gtgcggccgg tctgtccgca
780tcgtcgggct tggctcacgc ggatagcctg gcgagctcag ccagcttgcc cgccctggcc
840ggcattgggg gcgggtccgg ttttgggggc ttgccgagcc tggctcaggt ccatgccgcc
900tcaactcggc aggcgctacg gccccgagct gatggcccgg tcggcgccgc tgccgagcag
960gtcggcgggc agtcgcagct ggtctccgcg cagggttccc aaggtatggg cggacccgta
1020ggcatgggcg gcatgcaccc ctcttcgggg gcgtcgaaag ggacgacgac gaagaagtac
1080tcggaaggcg cggcggcggg cactgaagac gccgagcgcg cgccagtcga agctgacgcg
1140ggcggtgggc aaaaggtgct ggtacgaaac gtcgtctaa
11793392PRTMycobacterium tuberculosisMISC_FEATUREStrain CDC1551 3Met Ser
Arg Ala Phe Ile Ile Asp Pro Thr Ile Ser Ala Ile Asp Gly1 5
10 15Leu Tyr Asp Leu Leu Gly Ile Gly
Ile Pro Asn Gln Gly Gly Ile Leu 20 25
30Tyr Ser Ser Leu Glu Tyr Phe Glu Lys Ala Leu Glu Glu Leu Ala
Ala 35 40 45Ala Phe Pro Gly Asp
Gly Trp Leu Gly Ser Ala Ala Asp Lys Tyr Ala 50 55
60Gly Lys Asn Arg Asn His Val Asn Phe Phe Gln Glu Leu Ala
Asp Leu65 70 75 80Asp
Arg Gln Leu Ile Ser Leu Ile His Asp Gln Ala Asn Ala Val Gln
85 90 95Thr Thr Arg Asp Ile Leu Glu
Gly Ala Lys Lys Gly Leu Glu Phe Val 100 105
110Arg Pro Val Ala Val Asp Leu Thr Tyr Ile Pro Val Val Gly
His Ala 115 120 125Leu Ser Ala Ala
Phe Gln Ala Pro Phe Cys Ala Gly Ala Met Ala Val 130
135 140Val Gly Gly Ala Leu Ala Tyr Leu Val Val Lys Thr
Leu Ile Asn Ala145 150 155
160Thr Gln Leu Leu Lys Leu Leu Ala Lys Leu Ala Glu Leu Val Ala Ala
165 170 175Ala Ile Ala Asp Ile
Ile Ser Asp Val Ala Asp Ile Ile Lys Gly Ile 180
185 190Leu Gly Glu Val Trp Glu Phe Ile Thr Asn Ala Leu
Asn Gly Leu Lys 195 200 205Glu Leu
Trp Asp Lys Leu Thr Gly Trp Val Thr Gly Leu Phe Ser Arg 210
215 220Gly Trp Ser Asn Leu Glu Ser Phe Phe Ala Gly
Val Pro Gly Leu Thr225 230 235
240Gly Ala Thr Ser Gly Leu Ser Gln Val Thr Gly Leu Phe Gly Ala Ala
245 250 255Gly Leu Ser Ala
Ser Ser Gly Leu Ala His Ala Asp Ser Leu Ala Ser 260
265 270Ser Ala Ser Leu Pro Ala Leu Ala Gly Ile Gly
Gly Gly Ser Gly Phe 275 280 285Gly
Gly Leu Pro Ser Leu Ala Gln Val His Ala Ala Ser Thr Arg Gln 290
295 300Ala Leu Arg Pro Arg Ala Asp Gly Pro Val
Gly Ala Ala Ala Glu Gln305 310 315
320Val Gly Gly Gln Ser Gln Leu Val Ser Ala Gln Gly Ser Gln Gly
Met 325 330 335Gly Gly Pro
Val Gly Met Gly Gly Met His Pro Ser Ser Gly Ala Ser 340
345 350Lys Gly Thr Thr Thr Lys Lys Tyr Ser Glu
Gly Ala Ala Ala Gly Thr 355 360
365Glu Asp Ala Glu Arg Ala Pro Val Glu Ala Asp Ala Gly Gly Gly Gln 370
375 380Lys Val Leu Val Arg Asn Val Val385
3904392PRTMycobacterium tuberculosisMISC_FEATUREStrain
F11 4Met Ser Arg Ala Phe Ile Ile Asp Pro Thr Ile Ser Ala Ile Asp Gly1
5 10 15Leu Tyr Asp Leu Leu
Gly Ile Gly Ile Pro Asn Gln Gly Gly Ile Leu 20
25 30Tyr Ser Ser Leu Glu Tyr Phe Glu Lys Ala Leu Glu
Glu Leu Ala Ala 35 40 45Ala Phe
Pro Gly Asp Gly Trp Leu Gly Ser Ala Ala Asp Lys Tyr Ala 50
55 60Gly Lys Asn Arg Asn His Val Asn Phe Phe Gln
Glu Leu Ala Asp Leu65 70 75
80Asp Arg Gln Leu Ile Ser Leu Ile His Asp Gln Ala Asn Ala Val Gln
85 90 95Thr Thr Arg Asp Ile
Leu Glu Gly Ala Lys Lys Gly Leu Glu Phe Val 100
105 110Arg Pro Val Ala Val Asp Leu Thr Tyr Ile Pro Val
Val Gly His Ala 115 120 125Leu Ser
Ala Ala Phe Gln Ala Pro Phe Cys Ala Gly Ala Met Ala Val 130
135 140Val Gly Gly Ala Leu Ala Tyr Leu Val Val Lys
Thr Leu Ile Asn Ala145 150 155
160Thr Gln Leu Leu Lys Leu Leu Ala Lys Leu Ala Glu Leu Val Ala Ala
165 170 175Ala Ile Ala Asp
Ile Ile Ser Asp Val Ala Asp Ile Ile Lys Gly Ile 180
185 190Leu Gly Glu Val Trp Glu Phe Ile Thr Asn Ala
Leu Asn Gly Leu Lys 195 200 205Glu
Leu Trp Asp Lys Leu Thr Gly Trp Val Thr Gly Leu Phe Ser Arg 210
215 220Gly Trp Ser Asn Leu Glu Ser Phe Phe Ala
Gly Val Pro Gly Leu Thr225 230 235
240Gly Ala Thr Ser Gly Leu Ser Gln Val Thr Gly Leu Phe Gly Ala
Ala 245 250 255Gly Leu Ser
Ala Ser Ser Gly Leu Ala His Ala Asp Ser Leu Ala Ser 260
265 270Ser Ala Ser Leu Pro Ala Leu Ala Gly Ile
Gly Gly Gly Ser Gly Phe 275 280
285Gly Gly Leu Pro Ser Leu Ala Gln Val His Ala Ala Ser Thr Arg Gln 290
295 300Ala Leu Arg Pro Arg Ala Asp Gly
Pro Val Gly Ala Ala Ala Glu Gln305 310
315 320Val Gly Gly Gln Ser Gln Leu Val Ser Ala Gln Gly
Ser Gln Gly Met 325 330
335Gly Gly Pro Val Gly Met Gly Gly Met His Pro Ser Ser Gly Ala Ser
340 345 350Lys Gly Thr Thr Thr Lys
Lys Tyr Ser Glu Gly Ala Ala Ala Gly Thr 355 360
365Glu Asp Ala Glu Arg Ala Pro Val Glu Ala Asp Ala Gly Gly
Gly Gln 370 375 380Lys Val Leu Val Arg
Asn Val Val385 3905392PRTMycobacterium
tuberculosisMISC_FEATUREStrain Haarlem A 5Met Ser Arg Ala Phe Ile Ile Asp
Pro Thr Ile Ser Ala Ile Asp Gly1 5 10
15Leu Tyr Asp Leu Leu Gly Ile Gly Ile Pro Asn Gln Gly Gly
Ile Leu 20 25 30Tyr Ser Ser
Leu Glu Tyr Phe Glu Lys Ala Leu Glu Glu Leu Ala Ala 35
40 45Ala Phe Pro Gly Asp Gly Trp Leu Gly Ser Ala
Ala Asp Lys Tyr Ala 50 55 60Gly Lys
Asn Arg Asn His Val Asn Phe Phe Gln Glu Leu Ala Asp Leu65
70 75 80Asp Arg Gln Leu Ile Ser Leu
Ile His Asp Gln Ala Asn Ala Val Gln 85 90
95Thr Thr Arg Asp Ile Leu Glu Gly Ala Lys Lys Gly Leu
Glu Phe Val 100 105 110Arg Pro
Val Ala Val Asp Leu Thr Tyr Ile Pro Val Val Gly His Ala 115
120 125Leu Ser Ala Ala Phe Gln Ala Pro Phe Cys
Ala Gly Ala Met Ala Val 130 135 140Val
Gly Gly Ala Leu Ala Tyr Leu Val Val Lys Thr Leu Ile Asn Ala145
150 155 160Thr Gln Leu Leu Lys Leu
Leu Ala Lys Leu Ala Glu Leu Val Ala Ala 165
170 175Ala Ile Ala Asp Ile Ile Ser Asp Val Ala Asp Ile
Ile Lys Gly Ile 180 185 190Leu
Gly Glu Val Trp Glu Phe Ile Thr Asn Ala Leu Asn Gly Leu Lys 195
200 205Glu Leu Trp Asp Lys Leu Thr Gly Trp
Val Thr Gly Leu Phe Ser Arg 210 215
220Gly Trp Ser Asn Leu Glu Ser Phe Phe Ala Gly Val Pro Gly Leu Thr225
230 235 240Gly Ala Thr Ser
Gly Leu Ser Gln Val Thr Gly Leu Phe Gly Ala Ala 245
250 255Gly Leu Ser Ala Ser Ser Gly Leu Ala His
Ala Asp Ser Leu Ala Ser 260 265
270Ser Ala Ser Leu Pro Ala Leu Ala Gly Ile Gly Gly Gly Ser Gly Phe
275 280 285Gly Gly Leu Pro Ser Leu Ala
Gln Val His Ala Ala Ser Thr Arg Gln 290 295
300Ala Leu Arg Pro Arg Ala Asp Gly Pro Val Gly Ala Ala Ala Glu
Gln305 310 315 320Val Gly
Gly Gln Ser Gln Leu Val Ser Ala Gln Gly Ser Gln Gly Met
325 330 335Gly Gly Pro Val Gly Met Gly
Gly Met His Pro Ser Ser Gly Ala Ser 340 345
350Lys Gly Thr Thr Thr Lys Lys Tyr Ser Glu Gly Ala Ala Ala
Gly Thr 355 360 365Glu Asp Ala Glu
Arg Ala Pro Val Glu Ala Asp Ala Gly Gly Gly Gln 370
375 380Lys Val Leu Val Arg Asn Val Val385
3906392PRTMycobacterium tuberculosisMISC_FEATUREStrain C 6Met Ser Arg Ala
Phe Ile Ile Asp Pro Thr Ile Ser Ala Ile Asp Gly1 5
10 15Leu Tyr Asp Leu Leu Gly Ile Gly Ile Pro
Asn Gln Gly Gly Ile Leu 20 25
30Tyr Ser Ser Leu Glu Tyr Phe Glu Lys Ala Leu Glu Glu Leu Ala Ala
35 40 45Ala Phe Pro Gly Asp Gly Trp Leu
Gly Ser Ala Ala Asp Lys Tyr Ala 50 55
60Gly Lys Asn Arg Asn His Val Asn Phe Phe Gln Glu Leu Ala Asp Leu65
70 75 80Asp Arg Gln Leu Ile
Ser Leu Ile His Asp Gln Ala Asn Ala Val Gln 85
90 95Thr Thr Arg Asp Ile Leu Glu Gly Ala Lys Lys
Gly Leu Glu Phe Val 100 105
110Arg Pro Val Ala Val Asp Leu Thr Tyr Ile Pro Val Val Gly His Ala
115 120 125Leu Ser Ala Ala Phe Gln Ala
Pro Phe Cys Ala Gly Ala Met Ala Val 130 135
140Val Gly Gly Ala Leu Ala Tyr Leu Val Val Lys Thr Leu Ile Asn
Ala145 150 155 160Thr Gln
Leu Leu Lys Leu Leu Ala Lys Leu Ala Glu Leu Val Ala Ala
165 170 175Ala Ile Ala Asp Ile Ile Ser
Asp Val Ala Asp Ile Ile Lys Gly Ile 180 185
190Leu Gly Glu Val Trp Glu Phe Ile Thr Asn Ala Leu Asn Gly
Leu Lys 195 200 205Glu Leu Trp Asp
Lys Leu Thr Gly Trp Val Thr Gly Leu Phe Ser Arg 210
215 220Gly Trp Ser Asn Leu Glu Ser Phe Phe Ala Gly Val
Pro Gly Leu Thr225 230 235
240Gly Ala Thr Ser Gly Leu Ser Gln Val Thr Gly Leu Phe Gly Ala Ala
245 250 255Gly Leu Ser Ala Ser
Ser Gly Leu Ala His Ala Asp Ser Leu Ala Ser 260
265 270Ser Ala Ser Leu Pro Ala Leu Ala Gly Ile Gly Gly
Gly Ser Gly Phe 275 280 285Gly Gly
Leu Pro Ser Leu Ala Gln Val His Ala Ala Ser Thr Arg Gln 290
295 300Ala Leu Arg Pro Arg Ala Asp Gly Pro Val Gly
Ala Ala Ala Glu Gln305 310 315
320Val Gly Gly Gln Ser Gln Leu Val Ser Ala Gln Gly Ser Gln Gly Met
325 330 335Gly Gly Pro Val
Gly Met Gly Gly Met His Pro Ser Ser Gly Ala Ser 340
345 350Lys Gly Thr Thr Thr Lys Lys Tyr Ser Glu Gly
Ala Ala Ala Gly Thr 355 360 365Glu
Asp Ala Glu Arg Ala Pro Val Glu Ala Asp Ala Gly Gly Gly Gln 370
375 380Lys Val Leu Val Arg Asn Val Val385
3907392PRTMycobacterium bovisMISC_FEATUREStrain BCG 7Met Ser Arg
Val Phe Ile Ile Asp Pro Thr Ile Ser Ala Ile Asp Gly1 5
10 15Leu Tyr Asp Leu Leu Gly Ile Gly Ile
Pro Asn Gln Gly Gly Ile Leu 20 25
30Tyr Ser Ser Leu Glu Tyr Phe Glu Lys Ala Leu Glu Glu Leu Ala Ala
35 40 45Ala Phe Pro Gly Asp Gly Trp
Leu Gly Ser Ala Ala Asp Lys Tyr Ala 50 55
60Gly Lys Asn Arg Asn His Val Asn Phe Phe Gln Glu Leu Ala Asp Leu65
70 75 80Asp Arg Gln Leu
Ile Ser Leu Ile His Asp Gln Ala Asn Ala Val Gln 85
90 95Thr Thr Arg Asp Ile Leu Glu Gly Ala Lys
Lys Gly Leu Glu Phe Val 100 105
110Arg Pro Val Ala Val Asp Leu Thr Tyr Ile Pro Val Val Gly His Ala
115 120 125Leu Ser Ala Ala Phe Gln Ala
Pro Phe Cys Ala Gly Ala Met Ala Val 130 135
140Val Gly Gly Ala Leu Ala Tyr Leu Val Val Lys Thr Leu Ile Asn
Ala145 150 155 160Thr Gln
Leu Leu Lys Leu Leu Ala Lys Leu Ala Glu Leu Val Ala Ala
165 170 175Ala Ile Ala Asp Ile Ile Ser
Asp Val Ala Asp Ile Ile Lys Gly Ile 180 185
190Leu Gly Glu Val Trp Glu Phe Ile Thr Asn Ala Leu Asn Gly
Leu Lys 195 200 205Glu Leu Trp Asp
Lys Leu Thr Gly Trp Val Thr Gly Leu Phe Ser Arg 210
215 220Gly Trp Ser Asn Leu Glu Ser Phe Phe Ala Gly Val
Pro Gly Leu Thr225 230 235
240Gly Ala Thr Ser Gly Leu Ser Gln Val Thr Gly Leu Phe Gly Ala Ala
245 250 255Gly Leu Ser Ala Ser
Ser Gly Leu Ala His Ala Asp Ser Leu Ala Ser 260
265 270Ser Ala Ser Leu Pro Ala Leu Ala Gly Ile Gly Gly
Gly Ser Gly Phe 275 280 285Gly Gly
Leu Pro Ser Leu Ala Gln Val His Ala Ala Ser Thr Arg Gln 290
295 300Ala Leu Arg Pro Arg Ala Asp Gly Pro Val Gly
Ala Ala Ala Glu Gln305 310 315
320Val Gly Gly Gln Ser Gln Leu Val Ser Ala Gln Gly Ser Gln Gly Met
325 330 335Gly Gly Pro Val
Gly Met Gly Gly Met His Pro Ser Ser Gly Ala Ser 340
345 350Lys Gly Thr Thr Thr Lys Lys Tyr Ser Glu Gly
Ala Ala Ala Gly Thr 355 360 365Glu
Asp Ala Glu Arg Ala Pro Val Glu Ala Asp Ala Gly Gly Gly Gln 370
375 380Lys Val Leu Val Arg Asn Val Val385
3908110PRTMycobacterium tuberculosismat_peptide(29)..(110) 8Met
Arg Leu Ser Leu Thr Ala Leu Ser Ala Gly Val Gly Ala Val Ala
-25 -20 -15Met Ser Leu Thr Val Gly Ala
Gly Val Ala Ser Ala Asp Pro Val Asp -10 -5
-1 1Ala Val Ile Asn Thr Thr Cys Asn Tyr Gly Gln Val Val Ala Ala
Leu5 10 15 20Asn Ala
Thr Asp Pro Gly Ala Ala Ala Gln Phe Asn Ala Ser Pro Val 25
30 35Ala Gln Ser Tyr Leu Arg Asn Phe
Leu Ala Ala Pro Pro Pro Gln Arg 40 45
50Ala Ala Met Ala Ala Gln Leu Gln Ala Val Pro Gly Ala Ala Gln
Tyr 55 60 65Ile Gly Leu Val Glu
Ser Val Ala Gly Ser Cys Asn Asn Tyr 70 75
80997PRTMycobacterium tuberculosis 9Met Ser Leu Leu Asp Ala His Ile
Pro Gln Leu Val Ala Ser Gln Ser1 5 10
15Ala Phe Ala Ala Lys Ala Gly Leu Met Arg His Thr Ile Gly
Gln Ala 20 25 30Glu Gln Ala
Ala Met Ser Ala Gln Ala Phe His Gln Gly Glu Ser Ser 35
40 45Ala Ala Phe Gln Ala Ala His Ala Arg Phe Val
Ala Ala Ala Ala Lys 50 55 60Val Asn
Thr Leu Leu Asp Val Ala Gln Ala Asn Leu Gly Glu Ala Ala65
70 75 80Gly Thr Tyr Val Ala Ala Asp
Ala Ala Ala Ala Ser Thr Tyr Thr Gly 85 90
95Phe1094PRTMycobacterium tuberculosis 10Met Thr Ile Asn
Tyr Gln Phe Gly Asp Val Asp Ala His Gly Ala Met1 5
10 15Ile Arg Ala Gln Ala Ala Ser Leu Glu Ala
Glu His Gln Ala Ile Val 20 25
30Arg Asp Val Leu Ala Ala Gly Asp Phe Trp Gly Gly Ala Gly Ser Val
35 40 45Ala Cys Gln Glu Phe Ile Thr Gln
Leu Gly Arg Asn Phe Gln Val Ile 50 55
60Tyr Glu Gln Ala Asn Ala His Gly Gln Lys Val Gln Ala Ala Gly Asn65
70 75 80Asn Met Ala Gln Thr
Asp Ser Ala Val Gly Ser Ser Trp Ala 85
9011132PRTMycobacterium tuberculosis 11Thr 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 Thr 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
13012195PRTMycobacterium tuberculosis 12Ala Pro Pro Ala Leu Ser Gln Asp
Arg Phe Ala Asp Phe Pro Ala Leu1 5 10
15Pro Leu Asp Pro Ser Ala Met Val Ala Gln Val Gly Pro Gln
Val Val 20 25 30Asn Ile Asn
Thr Lys Leu Gly Tyr Asn Asn Ala Val Gly Ala Gly Thr 35
40 45Gly Ile Val Ile Asp Pro Asn Gly Val Val Leu
Thr Asn Asn His Val 50 55 60Ile Ala
Gly Ala Thr Asp Ile Asn Ala Phe Ser Val Gly Ser Gly Gln65
70 75 80Thr Tyr Gly Val Asp Val Val
Gly Tyr Asp Arg Thr Gln Asp Val Ala 85 90
95Val Leu Gln Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala
Ala Ile Gly 100 105 110Gly Gly
Val Ala Val Gly Glu Pro Val Val Ala Met Gly Asn Ser Gly 115
120 125Gly Gln Gly Gly Thr Pro Arg Ala Val Pro
Gly Arg Val Val Ala Leu 130 135 140Gly
Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu Glu Thr145
150 155 160Leu Asn Gly Leu Ile Gln
Phe Asp Ala Ala Ile Gln Pro Gly Asp Ser 165
170 175Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val
Gly Met Asn Thr 180 185 190Ala
Ala Ser 19513391PRTMycobacterium tuberculosis 13Met 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
39014423PRTMycobacterium tuberculosis 14Met Asp Phe Gly Leu Leu Pro
Pro Glu Val Asn Ser Ser Arg Met Tyr1 5 10
15Ser Gly Pro Gly Pro Glu Ser Met Leu Ala Ala Ala Ala
Ala Trp Asp 20 25 30Gly Val
Ala Ala Glu Leu Thr Ser Ala Ala Val Ser Tyr Gly Ser Val 35
40 45Val Ser Thr Leu Ile Val Glu Pro Trp Met
Gly Pro Ala Ala Ala Ala 50 55 60Met
Ala Ala Ala Ala Thr Pro Tyr Val Gly Trp Leu Ala Ala Thr Ala65
70 75 80Ala Leu Ala Lys Glu Thr
Ala Thr Gln Ala Arg Ala Ala Ala Glu Ala 85
90 95Phe Gly Thr Ala Phe Ala Met Thr Val Pro Pro Ser
Leu Val Ala Ala 100 105 110Asn
Arg Ser Arg Leu Met Ser Leu Val Ala Ala Asn Ile Leu Gly Gln 115
120 125Asn Ser Ala Ala Ile Ala Ala Thr Gln
Ala Glu Tyr Ala Glu Met Trp 130 135
140Ala Gln Asp Ala Ala Val Met Tyr Ser Tyr Glu Gly Ala Ser Ala Ala145
150 155 160Ala Ser Ala Leu
Pro Pro Phe Thr Pro Pro Val Gln Gly Thr Gly Pro 165
170 175Ala Gly Pro Ala Ala Ala Ala Ala Ala Thr
Gln Ala Ala Gly Ala Gly 180 185
190Ala Val Ala Asp Ala Gln Ala Thr Leu Ala Gln Leu Pro Pro Gly Ile
195 200 205Leu Ser Asp Ile Leu Ser Ala
Leu Ala Ala Asn Ala Asp Pro Leu Thr 210 215
220Ser Gly Leu Leu Gly Ile Ala Ser Thr Leu Asn Pro Gln Val Gly
Ser225 230 235 240Ala Gln
Pro Ile Val Ile Pro Thr Pro Ile Gly Glu Leu Asp Val Ile
245 250 255Ala Leu Tyr Ile Ala Ser Ile
Ala Thr Gly Ser Ile Ala Leu Ala Ile 260 265
270Thr Asn Thr Ala Arg Pro Trp His Ile Gly Leu Tyr Gly Asn
Ala Gly 275 280 285Gly Leu Gly Pro
Thr Gln Gly His Pro Leu Ser Ser Ala Thr Asp Glu 290
295 300Pro Glu Pro His Trp Gly Pro Phe Gly Gly Ala Ala
Pro Val Ser Ala305 310 315
320Gly Val Gly His Ala Ala Leu Val Gly Ala Leu Ser Val Pro His Ser
325 330 335Trp Thr Thr Ala Ala
Pro Glu Ile Gln Leu Ala Val Gln Ala Thr Pro 340
345 350Thr Phe Ser Ser Ser Ala Gly Ala Asp Pro Thr Ala
Leu Asn Gly Met 355 360 365Pro Ala
Gly Leu Leu Ser Gly Met Ala Leu Ala Ser Leu Ala Ala Arg 370
375 380Gly Thr Thr Gly Gly Gly Gly Thr Arg Ser Gly
Thr Ser Thr Asp Gly385 390 395
400Gln Glu Asp Gly Arg Lys Pro Pro Val Val Val Ile Arg Glu Gln Pro
405 410 415Pro Pro Gly Asn
Pro Pro Arg 4201595PRTMycobacterium
tuberculosisINIT_MET(1)..(1)mat_peptide(2)..(95) 15Met Thr Glu Gln Gln
Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser-1 1 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 Gln Gly Val
Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu 50 55
60Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu
Ala Gly 65 70 75Gln Ala Met Ala Ser
Thr Glu Gly Asn Val Thr Gly Met Phe Ala80 85
9016338PRTMycobacterium tuberculosismat_peptide(43)..(338) 16Met Gln
Leu Val Asp Arg Val Arg Gly Ala Val Thr Gly Met Ser Arg -40
-35 -30Arg Leu Val Val Gly Ala Val Gly Ala Ala
Leu Val Ser Gly Leu Val -25 -20 -15Gly
Ala Val Gly Gly Thr Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly-10
-5 -1 1 5Leu Pro Val Glu Tyr Leu Gln Val
Pro Ser Pro Ser Met Gly Arg Asp 10 15
20Ile Lys Val Gln Phe Gln Ser Gly Gly Ala Asn Ser Pro Ala Leu
Tyr 25 30 35Leu Leu Asp Gly Leu
Arg Ala Gln Asp Asp Phe Ser Gly Trp Asp Ile 40 45
50Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gln Ser Gly Leu Ser
Val Val55 60 65 70Met
Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Gln Pro
75 80 85Ala Cys Gly Lys Ala Gly Cys
Gln Thr Tyr Lys Trp Glu Thr Phe Leu 90 95
100Thr Ser Glu Leu Pro Gly Trp Leu Gln Ala Asn Arg His Val
Lys Pro 105 110 115Thr Gly Ser Ala
Val Val Gly Leu Ser Met Ala Ala Ser Ser Ala Leu 120
125 130Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Val Tyr
Ala Gly Ala Met135 140 145
150Ser Gly Leu Leu Asp Pro Ser Gln Ala Met Gly Pro Thr Leu Ile Gly
155 160 165Leu Ala Met Gly Asp
Ala Gly Gly Tyr Lys Ala Ser Asp Met Trp Gly 170
175 180Pro Lys Glu Asp Pro Ala Trp Gln Arg Asn Asp Pro
Leu Leu Asn Val 185 190 195Gly Lys
Leu Ile Ala Asn Asn Thr Arg Val Trp Val Tyr Cys Gly Asn 200
205 210Gly Lys Pro Ser Asp Leu Gly Gly Asn Asn Leu
Pro Ala Lys Phe Leu215 220 225
230Glu Gly Phe Val Arg Thr Ser Asn Ile Lys Phe Gln Asp Ala Tyr Asn
235 240 245Ala Gly Gly Gly
His Asn Gly Val Phe Asp Phe Pro Asp Ser Gly Thr 250
255 260His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn
Ala Met Lys Pro Asp 265 270 275Leu
Gln Arg Ala Leu Gly Ala Thr Pro Asn Thr Gly Pro Ala Pro Gln 280
285 290Gly Ala29517325PRTMycobacterium
tuberculosismat_peptide(41)..(325) 17Met Thr Asp Val Ser Arg Lys Ile Arg
Ala Trp Gly Arg Arg Leu Met-40 -35 -30
-25Ile Gly Thr Ala Ala Ala Val Val Leu Pro Gly Leu Val Gly
Leu Ala -20 -15 -10Gly Gly
Ala Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val -5
-1 1 5Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly
Arg Asp Ile Lys Val 10 15 20Gln Phe
Gln Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp25
30 35 40Gly Leu Arg Ala Gln Asp Asp
Tyr Asn Gly Trp Asp Ile Asn Thr Pro 45 50
55Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Ile Val
Met Pro Val 60 65 70Gly Gly
Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly 75
80 85Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu
Thr Phe Leu Thr Ser Glu 90 95 100Leu
Pro Gln Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser105
110 115 120Ala Ala Ile Gly Leu Ser
Met Ala Gly Ser Ser Ala Met Ile Leu Ala 125
130 135Ala Tyr His Pro Gln Gln Phe Ile Tyr Ala Gly Ser
Leu Ser Ala Leu 140 145 150Leu
Asp Pro Ser Gln Gly Met Gly Pro Ser Leu Ile Gly Leu Ala Met 155
160 165Gly Asp Ala Gly Gly Tyr Lys Ala Ala
Asp Met Trp Gly Pro Ser Ser 170 175
180Asp Pro Ala Trp Glu Arg Asn Asp Pro Thr Gln Gln Ile Pro Lys Leu185
190 195 200Val Ala Asn Asn
Thr Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro 205
210 215Asn Glu Leu Gly Gly Ala Asn Ile Pro Ala
Glu Phe Leu Glu Asn Phe 220 225
230Val Arg Ser Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Ala Ala Gly
235 240 245Gly His Asn Ala Val Phe Asn
Phe Pro Pro Asn Gly Thr His Ser Trp 250 255
260Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu Gln
Ser265 270 275 280Ser Leu
Gly Ala Gly 28518144PRTMycobacterium
tuberculosisINIT_MET(1)..(1)mat_peptide(2)..(144) 18Met Ala Thr Thr Leu
Pro Val Gln Arg His Pro Arg Ser Leu Phe Pro-1 1 5
10 15Glu Phe Ser Glu Leu Phe Ala Ala Phe Pro
Ser Phe Ala Gly Leu Arg 20 25
30Pro Thr Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu Met Lys Glu
35 40 45Gly Arg Tyr Glu Val Arg
Ala Glu Leu Pro Gly Val Asp Pro Asp Lys 50 55
60Asp Val Asp Ile Met Val Arg Asp Gly Gln Leu Thr Ile Lys
Ala Glu 65 70 75Arg Thr Glu Gln Lys
Asp Phe Asp Gly Arg Ser Glu Phe Ala Tyr Gly80 85
90 95Ser Phe Val Arg Thr Val Ser Leu Pro Val
Gly Ala Asp Glu Asp Asp 100 105
110Ile Lys Ala Thr Tyr Asp Lys Gly Ile Leu Thr Val Ser Val Ala Val
115 120 125Ser Glu Gly Lys Pro
Thr Glu Lys His Ile Gln Ile Arg Ser Thr Asn 130
135 14019228PRTMycobacterium
tuberculosismat_peptide(24)..(228) 19Met Arg Ile Lys Ile Phe Met Leu Val
Thr Ala Val Val Leu Leu Cys -20 -15
-10Cys Ser Gly Val Ala Thr Ala Ala Pro Lys Thr Tyr Cys Glu Glu Leu
-5 -1 1 5Lys Gly Thr Asp Thr Gly Gln Ala
Cys Gln Ile Gln Met Ser Asp Pro10 15 20
25Ala Tyr Asn Ile Asn Ile Ser Leu Pro Ser Tyr Tyr Pro
Asp Gln Lys 30 35 40Ser
Leu Glu Asn Tyr Ile Ala Gln Thr Arg Asp Lys Phe Leu Ser Ala 45
50 55Ala Thr Ser Ser Thr Pro Arg Glu
Ala Pro Tyr Glu Leu Asn Ile Thr 60 65
70Ser Ala Thr Tyr Gln Ser Ala Ile Pro Pro Arg Gly Thr Gln Ala Val
75 80 85Val Leu Lys Val Tyr Gln Asn Ala
Gly Gly Thr His Pro Thr Thr Thr90 95
100 105Tyr Lys Ala Phe Asp Trp Asp Gln Ala Tyr Arg Lys
Pro Ile Thr Tyr 110 115
120Asp Thr Leu Trp Gln Ala Asp Thr Asp Pro Leu Pro Val Val Phe Pro
125 130 135Ile Val Gln Gly Glu Leu
Ser Lys Gln Thr Gly Gln Gln Val Ser Ile 140 145
150Ala Pro Asn Ala Gly Leu Asp Pro Val Asn Tyr Gln Asn Phe
Ala Val 155 160 165Thr Asn Asp Gly Val
Ile Phe Phe Phe Asn Pro Gly Glu Leu Leu Pro170 175
180 185Glu Ala Ala Gly Pro Thr Gln Val Leu Val
Pro Arg Ser Ala Ile Asp 190 195
200Ser Met Leu Ala 20520355PRTMycobacterium
tuberculosismat_peptide(33)..(355) 20Met Ser Asn Ser Arg Arg Arg Ser Leu
Arg Trp Ser Trp Leu Leu Ser -30 -25
-20Val Leu Ala Ala Val Gly Leu Gly Leu Ala Thr Ala Pro Ala Gln Ala
-15 -10 -5 -1Ala Pro Pro Ala
Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro Ala Leu1 5
10 15Pro Leu Asp Pro Ser Ala Met Val Ala Gln
Val Gly Pro Gln Val Val 20 25
30Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn Ala Val Gly Ala Gly Thr
35 40 45Gly Ile Val Ile Asp Pro Asn Gly
Val Val Leu Thr Asn Asn His Val 50 55
60Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe Ser Val Gly Ser Gly Gln65
70 75 80Thr Tyr Gly Val Asp
Val Val Gly Tyr Asp Arg Thr Gln Asp Val Ala 85
90 95Val Leu Gln Leu Arg Gly Ala Gly Gly Leu Pro
Ser Ala Ala Ile Gly 100 105
110Gly Gly Val Ala Val Gly Glu Pro Val Val Ala Met Gly Asn Ser Gly
115 120 125Gly Gln Gly Gly Thr Pro Arg
Ala Val Pro Gly Arg Val Val Ala Leu 130 135
140Gly Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu Glu
Thr145 150 155 160Leu Asn
Gly Leu Ile Gln Phe Asp Ala Ala Ile Gln Pro Gly Asp Ser
165 170 175Gly Gly Pro Val Val Asn Gly
Leu Gly Gln Val Val Gly Met Asn Thr 180 185
190Ala Ala Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly Gln Gly
Phe Ala 195 200 205Ile Pro Ile Gly
Gln Ala Met Ala Ile Ala Gly Gln Ile Arg Ser Gly 210
215 220Gly Gly Ser Pro Thr Val His Ile Gly Pro Thr Ala
Phe Leu Gly Leu225 230 235
240Gly Val Val Asp Asn Asn Gly Asn Gly Ala Arg Val Gln Arg Val Val
245 250 255Gly Ser Ala Pro Ala
Ala Ser Leu Gly Ile Ser Thr Gly Asp Val Ile 260
265 270Thr Ala Val Asp Gly Ala Pro Ile Asn Ser Ala Thr
Ala Met Ala Asp 275 280 285Ala Leu
Asn Gly His His Pro Gly Asp Val Ile Ser Val Thr Trp Gln 290
295 300Thr Lys Ser Gly Gly Thr Arg Thr Gly Asn Val
Thr Leu Ala Glu Gly305 310 315
320Pro Pro Ala21323PRTArtificial SequenceSer/Ala mutant of mature
Mtb32A 21Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro Ala Leu1
5 10 15Pro Leu Asp Pro
Ser Ala Met Val Ala Gln Val Gly Pro Gln Val Val 20
25 30Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn Ala
Val Gly Ala Gly Thr 35 40 45Gly
Ile Val Ile Asp Pro Asn Gly Val Val Leu Thr Asn Asn His Val 50
55 60Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe
Ser Val Gly Ser Gly Gln65 70 75
80Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr Gln Asp Val
Ala 85 90 95Val Leu Gln
Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala Ala Ile Gly 100
105 110Gly Gly Val Ala Val Gly Glu Pro Val Val
Ala Met Gly Asn Ser Gly 115 120
125Gly Gln Gly Gly Thr Pro Arg Ala Val Pro Gly Arg Val Val Ala Leu 130
135 140Gly Gln Thr Val Gln Ala Ser Asp
Ser Leu Thr Gly Ala Glu Glu Thr145 150
155 160Leu Asn Gly Leu Ile Gln Phe Asp Ala Ala Ile Gln
Pro Gly Asp Ala 165 170
175Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val Gly Met Asn Thr
180 185 190Ala Ala Ser Asp Asn Phe
Gln Leu Ser Gln Gly Gly Gln Gly Phe Ala 195 200
205Ile Pro Ile Gly Gln Ala Met Ala Ile Ala Gly Gln Ile Arg
Ser Gly 210 215 220Gly Gly Ser Pro Thr
Val His Ile Gly Pro Thr Ala Phe Leu Gly Leu225 230
235 240Gly Val Val Asp Asn Asn Gly Asn Gly Ala
Arg Val Gln Arg Val Val 245 250
255Gly Ser Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly Asp Val Ile
260 265 270Thr Ala Val Asp Gly
Ala Pro Ile Asn Ser Ala Thr Ala Met Ala Asp 275
280 285Ala Leu Asn Gly His His Pro Gly Asp Val Ile Ser
Val Thr Trp Gln 290 295 300Thr Lys Ser
Gly Gly Thr Arg Thr Gly Asn Val Thr Leu Ala Glu Gly305
310 315 320Pro Pro
Ala2296PRTMycobacterium tuberculosis 22Met Ser Gln Ile Met Tyr Asn Tyr
Pro Ala Met Leu Gly His Ala Gly1 5 10
15Asp Met Ala Gly Tyr Ala Gly Thr Leu Gln Ser Leu Gly Ala
Glu Ile 20 25 30Ala Val Glu
Gln Ala Ala Leu Gln Ser Ala Trp Gln Gly Asp Thr Gly 35
40 45Ile Thr Tyr Gln Ala Trp Gln Ala Gln Trp Asn
Gln Ala Met Glu Asp 50 55 60Leu Val
Arg Ala Tyr His Ala Met Ser Ser Thr His Glu Ala Asn Thr65
70 75 80Met Ala Met Met Ala Arg Asp
Thr Ala Glu Ala Ala Lys Trp Gly Gly 85 90
9523723PRTArtificial SequenceMtb72f 23Met Thr Ala Ala
Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly Gln Gly1 5
10 15Phe Ala Ile Pro Ile Gly Gln Ala Met Ala
Ile Ala Gly Gln Ile Arg 20 25
30Ser Gly Gly Gly Ser Pro Thr Val His Ile Gly Pro Thr Ala Phe Leu
35 40 45Gly Leu Gly Val Val Asp Asn Asn
Gly Asn Gly Ala Arg Val Gln Arg 50 55
60Val Val Gly Ser Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly Asp65
70 75 80Val Ile Thr Ala Val
Asp Gly Ala Pro Ile Asn Ser Ala Thr Ala Met 85
90 95Ala Asp Ala Leu Asn Gly His His Pro Gly Asp
Val Ile Ser Val Thr 100 105
110Trp Gln Thr Lys Ser Gly Gly Thr Arg Thr Gly Asn Val Thr Leu Ala
115 120 125Glu Gly Pro Pro Ala Glu Phe
Met Val Asp Phe Gly Ala Leu Pro Pro 130 135
140Glu Ile Asn Ser Ala Arg Met Tyr Ala Gly Pro Gly Ser Ala Ser
Leu145 150 155 160Val Ala
Ala Ala Gln Met Trp Asp Ser Val Ala Ser Asp Leu Phe Ser
165 170 175Ala Ala Ser Ala Phe Gln Ser
Val Val Trp Gly Leu Thr Val Gly Ser 180 185
190Trp Ile Gly Ser Ser Ala Gly Leu Met Val Ala Ala Ala Ser
Pro Tyr 195 200 205Val Ala Trp Met
Ser Val Thr Ala Gly Gln Ala Glu Leu Thr Ala Ala 210
215 220Gln Val Arg Val Ala Ala Ala Ala Tyr Glu Thr Ala
Tyr Gly Leu Thr225 230 235
240Val Pro Pro Pro Val Ile Ala Glu Asn Arg Ala Glu Leu Met Ile Leu
245 250 255Ile Ala Thr Asn Leu
Leu Gly Gln Asn Thr Pro Ala Ile Ala Val Asn 260
265 270Glu Ala Glu Tyr Gly Glu Met Trp Ala Gln Asp Ala
Ala Ala Met Phe 275 280 285Gly Tyr
Ala Ala Ala Thr Ala Thr Ala Thr Ala Thr Leu Leu Pro Phe 290
295 300Glu Glu Ala Pro Glu Met Thr Ser Ala Gly Gly
Leu Leu Glu Gln Ala305 310 315
320Ala Ala Val Glu Glu Ala Ser Asp Thr Ala Ala Ala Asn Gln Leu Met
325 330 335Asn Asn Val Pro
Gln Ala Leu Gln Gln Leu Ala Gln Pro Thr Gln Gly 340
345 350Thr Thr Pro Ser Ser Lys Leu Gly Gly Leu Trp
Lys Thr Val Ser Pro 355 360 365His
Arg Ser Pro Ile Ser Asn Met Val Ser Met Ala Asn Asn His Met 370
375 380Ser Met Thr Asn Ser Gly Val Ser Met Thr
Asn Thr Leu Ser Ser Met385 390 395
400Leu Lys Gly Phe Ala Pro Ala Ala Ala Ala Gln Ala Val Gln Thr
Ala 405 410 415Ala Gln Asn
Gly Val Arg Ala Met Ser Ser Leu Gly Ser Ser Leu Gly 420
425 430Ser Ser Gly Leu Gly Gly Gly Val Ala Ala
Asn Leu Gly Arg Ala Ala 435 440
445Ser Val Gly Ser Leu Ser Val Pro Gln Ala Trp Ala Ala Ala Asn Gln 450
455 460Ala Val Thr Pro Ala Ala Arg Ala
Leu Pro Leu Thr Ser Leu Thr Ser465 470
475 480Ala Ala Glu Arg Gly Pro Gly Gln Met Leu Gly Gly
Leu Pro Val Gly 485 490
495Gln Met Gly Ala Arg Ala Gly Gly Gly Leu Ser Gly Val Leu Arg Val
500 505 510Pro Pro Arg Pro Tyr Val
Met Pro His Ser Pro Ala Ala Gly Asp Ile 515 520
525Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro
Ala Leu 530 535 540Pro Leu Asp Pro Ser
Ala Met Val Ala Gln Val Gly Pro Gln Val Val545 550
555 560Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn
Ala Val Gly Ala Gly Thr 565 570
575Gly Ile Val Ile Asp Pro Asn Gly Val Val Leu Thr Asn Asn His Val
580 585 590Ile Ala Gly Ala Thr
Asp Ile Asn Ala Phe Ser Val Gly Ser Gly Gln 595
600 605Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr
Gln Asp Val Ala 610 615 620Val Leu Gln
Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala Ala Ile Gly625
630 635 640Gly Gly Val Ala Val Gly Glu
Pro Val Val Ala Met Gly Asn Ser Gly 645
650 655Gly Gln Gly Gly Thr Pro Arg Ala Val Pro Gly Arg
Val Val Ala Leu 660 665 670Gly
Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu Glu Thr 675
680 685Leu Asn Gly Leu Ile Gln Phe Asp Ala
Ala Ile Gln Pro Gly Asp Ser 690 695
700Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val Gly Met Asn Thr705
710 715 720Ala Ala
Ser24723PRTArtificial SequenceM72 24Met Thr Ala Ala Ser Asp Asn Phe Gln
Leu Ser Gln Gly Gly Gln Gly1 5 10
15Phe Ala Ile Pro Ile Gly Gln Ala Met Ala Ile Ala Gly Gln Ile
Arg 20 25 30Ser Gly Gly Gly
Ser Pro Thr Val His Ile Gly Pro Thr Ala Phe Leu 35
40 45Gly Leu Gly Val Val Asp Asn Asn Gly Asn Gly Ala
Arg Val Gln Arg 50 55 60Val Val Gly
Ser Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly Asp65 70
75 80Val Ile Thr Ala Val Asp Gly Ala
Pro Ile Asn Ser Ala Thr Ala Met 85 90
95Ala Asp Ala Leu Asn Gly His His Pro Gly Asp Val Ile Ser
Val Thr 100 105 110Trp Gln Thr
Lys Ser Gly Gly Thr Arg Thr Gly Asn Val Thr Leu Ala 115
120 125Glu Gly Pro Pro Ala Glu Phe Met Val Asp Phe
Gly Ala Leu Pro Pro 130 135 140Glu Ile
Asn Ser Ala Arg Met Tyr Ala Gly Pro Gly Ser Ala Ser Leu145
150 155 160Val Ala Ala Ala Gln Met Trp
Asp Ser Val Ala Ser Asp Leu Phe Ser 165
170 175Ala Ala Ser Ala Phe Gln Ser Val Val Trp Gly Leu
Thr Val Gly Ser 180 185 190Trp
Ile Gly Ser Ser Ala Gly Leu Met Val Ala Ala Ala Ser Pro Tyr 195
200 205Val Ala Trp Met Ser Val Thr Ala Gly
Gln Ala Glu Leu Thr Ala Ala 210 215
220Gln Val Arg Val Ala Ala Ala Ala Tyr Glu Thr Ala Tyr Gly Leu Thr225
230 235 240Val Pro Pro Pro
Val Ile Ala Glu Asn Arg Ala Glu Leu Met Ile Leu 245
250 255Ile Ala Thr Asn Leu Leu Gly Gln Asn Thr
Pro Ala Ile Ala Val Asn 260 265
270Glu Ala Glu Tyr Gly Glu Met Trp Ala Gln Asp Ala Ala Ala Met Phe
275 280 285Gly Tyr Ala Ala Ala Thr Ala
Thr Ala Thr Ala Thr Leu Leu Pro Phe 290 295
300Glu Glu Ala Pro Glu Met Thr Ser Ala Gly Gly Leu Leu Glu Gln
Ala305 310 315 320Ala Ala
Val Glu Glu Ala Ser Asp Thr Ala Ala Ala Asn Gln Leu Met
325 330 335Asn Asn Val Pro Gln Ala Leu
Gln Gln Leu Ala Gln Pro Thr Gln Gly 340 345
350Thr Thr Pro Ser Ser Lys Leu Gly Gly Leu Trp Lys Thr Val
Ser Pro 355 360 365His Arg Ser Pro
Ile Ser Asn Met Val Ser Met Ala Asn Asn His Met 370
375 380Ser Met Thr Asn Ser Gly Val Ser Met Thr Asn Thr
Leu Ser Ser Met385 390 395
400Leu Lys Gly Phe Ala Pro Ala Ala Ala Ala Gln Ala Val Gln Thr Ala
405 410 415Ala Gln Asn Gly Val
Arg Ala Met Ser Ser Leu Gly Ser Ser Leu Gly 420
425 430Ser Ser Gly Leu Gly Gly Gly Val Ala Ala Asn Leu
Gly Arg Ala Ala 435 440 445Ser Val
Gly Ser Leu Ser Val Pro Gln Ala Trp Ala Ala Ala Asn Gln 450
455 460Ala Val Thr Pro Ala Ala Arg Ala Leu Pro Leu
Thr Ser Leu Thr Ser465 470 475
480Ala Ala Glu Arg Gly Pro Gly Gln Met Leu Gly Gly Leu Pro Val Gly
485 490 495Gln Met Gly Ala
Arg Ala Gly Gly Gly Leu Ser Gly Val Leu Arg Val 500
505 510Pro Pro Arg Pro Tyr Val Met Pro His Ser Pro
Ala Ala Gly Asp Ile 515 520 525Ala
Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro Ala Leu 530
535 540Pro Leu Asp Pro Ser Ala Met Val Ala Gln
Val Gly Pro Gln Val Val545 550 555
560Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn Ala Val Gly Ala Gly
Thr 565 570 575Gly Ile Val
Ile Asp Pro Asn Gly Val Val Leu Thr Asn Asn His Val 580
585 590Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe
Ser Val Gly Ser Gly Gln 595 600
605Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr Gln Asp Val Ala 610
615 620Val Leu Gln Leu Arg Gly Ala Gly
Gly Leu Pro Ser Ala Ala Ile Gly625 630
635 640Gly Gly Val Ala Val Gly Glu Pro Val Val Ala Met
Gly Asn Ser Gly 645 650
655Gly Gln Gly Gly Thr Pro Arg Ala Val Pro Gly Arg Val Val Ala Leu
660 665 670Gly Gln Thr Val Gln Ala
Ser Asp Ser Leu Thr Gly Ala Glu Glu Thr 675 680
685Leu Asn Gly Leu Ile Gln Phe Asp Ala Ala Ile Gln Pro Gly
Asp Ala 690 695 700Gly Gly Pro Val Val
Asn Gly Leu Gly Gln Val Val Gly Met Asn Thr705 710
715 720Ala Ala Ser25702PRTArtificial
SequenceMtb71f 25Asp Pro Val Asp Ala Val Ile Asn Thr Thr Cys Asn Tyr Gly
Gln Val1 5 10 15Val Ala
Ala Leu Asn Ala Thr Asp Pro Gly Ala Ala Ala Gln Phe Asn 20
25 30Ala Ser Pro Val Ala Gln Ser Tyr Leu
Arg Asn Phe Leu Ala Ala Pro 35 40
45Pro Pro Gln Arg Ala Ala Met Ala Ala Gln Leu Gln Ala Val Pro Gly 50
55 60Ala Ala Gln Tyr Ile Gly Leu Val Glu
Ser Val Ala Gly Ser Cys Asn65 70 75
80Asn Tyr Glu Leu Met Thr Ile Asn Tyr Gln Phe Gly Asp Val
Asp Ala 85 90 95His Gly
Ala Met Ile Arg Ala Gln Ala Ala Ser Leu Glu Ala Glu His 100
105 110Gln Ala Ile Val Arg Asp Val Leu Ala
Ala Gly Asp Phe Trp Gly Gly 115 120
125Ala Gly Ser Val Ala Cys Gln Glu Phe Ile Thr Gln Leu Gly Arg Asn
130 135 140Phe Gln Val Ile Tyr Glu Gln
Ala Asn Ala His Gly Gln Lys Val Gln145 150
155 160Ala Ala Gly Asn Asn Met Ala Gln Thr Asp Ser Ala
Val Gly Ser Ser 165 170
175Trp Ala Thr Ser Met Ser Leu Leu Asp Ala His Ile Pro Gln Leu Val
180 185 190Ala Ser Gln Ser Ala Phe
Ala Ala Lys Ala Gly Leu Met Arg His Thr 195 200
205Ile Gly Gln Ala Glu Gln Ala Ala Met Ser Ala Gln Ala Phe
His Gln 210 215 220Gly Glu Ser Ser Ala
Ala Phe Gln Ala Ala His Ala Arg Phe Val Ala225 230
235 240Ala Ala Ala Lys Val Asn Thr Leu Leu Asp
Val Ala Gln Ala Asn Leu 245 250
255Gly Glu Ala Ala Gly Thr Tyr Val Ala Ala Asp Ala Ala Ala Ala Ser
260 265 270Thr Tyr Thr Gly Phe
Asp Ile Met Asp Phe Gly Leu Leu Pro Pro Glu 275
280 285Val Asn Ser Ser Arg Met Tyr Ser Gly Pro Gly Pro
Glu Ser Met Leu 290 295 300Ala Ala Ala
Ala Ala Trp Asp Gly Val Ala Ala Glu Leu Thr Ser Ala305
310 315 320Ala Val Ser Tyr Gly Ser Val
Val Ser Thr Leu Ile Val Glu Pro Trp 325
330 335Met Gly Pro Ala Ala Ala Ala Met Ala Ala Ala Ala
Thr Pro Tyr Val 340 345 350Gly
Trp Leu Ala Ala Thr Ala Ala Leu Ala Lys Glu Thr Ala Thr Gln 355
360 365Ala Arg Ala Ala Ala Glu Ala Phe Gly
Thr Ala Phe Ala Met Thr Val 370 375
380Pro Pro Ser Leu Val Ala Ala Asn Arg Ser Arg Leu Met Ser Leu Val385
390 395 400Ala Ala Asn Ile
Leu Gly Gln Asn Ser Ala Ala Ile Ala Ala Thr Gln 405
410 415Ala Glu Tyr Ala Glu Met Trp Ala Gln Asp
Ala Ala Val Met Tyr Ser 420 425
430Tyr Glu Gly Ala Ser Ala Ala Ala Ser Ala Leu Pro Pro Phe Thr Pro
435 440 445Pro Val Gln Gly Thr Gly Pro
Ala Gly Pro Ala Ala Ala Ala Ala Ala 450 455
460Thr Gln Ala Ala Gly Ala Gly Ala Val Ala Asp Ala Gln Ala Thr
Leu465 470 475 480Ala Gln
Leu Pro Pro Gly Ile Leu Ser Asp Ile Leu Ser Ala Leu Ala
485 490 495Ala Asn Ala Asp Pro Leu Thr
Ser Gly Leu Leu Gly Ile Ala Ser Thr 500 505
510Leu Asn Pro Gln Val Gly Ser Ala Gln Pro Ile Val Ile Pro
Thr Pro 515 520 525Ile Gly Glu Leu
Asp Val Ile Ala Leu Tyr Ile Ala Ser Ile Ala Thr 530
535 540Gly Ser Ile Ala Leu Ala Ile Thr Asn Thr Ala Arg
Pro Trp His Ile545 550 555
560Gly Leu Tyr Gly Asn Ala Gly Gly Leu Gly Pro Thr Gln Gly His Pro
565 570 575Leu Ser Ser Ala Thr
Asp Glu Pro Glu Pro His Trp Gly Pro Phe Gly 580
585 590Gly Ala Ala Pro Val Ser Ala Gly Val Gly His Ala
Ala Leu Val Gly 595 600 605Ala Leu
Ser Val Pro His Ser Trp Thr Thr Ala Ala Pro Glu Ile Gln 610
615 620Leu Ala Val Gln Ala Thr Pro Thr Phe Ser Ser
Ser Ala Gly Ala Asp625 630 635
640Pro Thr Ala Leu Asn Gly Met Pro Ala Gly Leu Leu Ser Gly Met Ala
645 650 655Leu Ala Ser Leu
Ala Ala Arg Gly Thr Thr Gly Gly Gly Gly Thr Arg 660
665 670Ser Gly Thr Ser Thr Asp Gly Gln Glu Asp Gly
Arg Lys Pro Pro Val 675 680 685Val
Val Ile Arg Glu Gln Pro Pro Pro Gly Asn Pro Pro Arg 690
695 70026920PRTArtificial SequenceM72-Mtb9.9-Mtb9.8
26Met Thr Ala Ala Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly Gln Gly1
5 10 15Phe Ala Ile Pro Ile Gly
Gln Ala Met Ala Ile Ala Gly Gln Ile Arg 20 25
30Ser Gly Gly Gly Ser Pro Thr Val His Ile Gly Pro Thr
Ala Phe Leu 35 40 45Gly Leu Gly
Val Val Asp Asn Asn Gly Asn Gly Ala Arg Val Gln Arg 50
55 60Val Val Gly Ser Ala Pro Ala Ala Ser Leu Gly Ile
Ser Thr Gly Asp65 70 75
80Val Ile Thr Ala Val Asp Gly Ala Pro Ile Asn Ser Ala Thr Ala Met
85 90 95Ala Asp Ala Leu Asn Gly
His His Pro Gly Asp Val Ile Ser Val Thr 100
105 110Trp Gln Thr Lys Ser Gly Gly Thr Arg Thr Gly Asn
Val Thr Leu Ala 115 120 125Glu Gly
Pro Pro Ala Glu Phe Met Val Asp Phe Gly Ala Leu Pro Pro 130
135 140Glu Ile Asn Ser Ala Arg Met Tyr Ala Gly Pro
Gly Ser Ala Ser Leu145 150 155
160Val Ala Ala Ala Gln Met Trp Asp Ser Val Ala Ser Asp Leu Phe Ser
165 170 175Ala Ala Ser Ala
Phe Gln Ser Val Val Trp Gly Leu Thr Val Gly Ser 180
185 190Trp Ile Gly Ser Ser Ala Gly Leu Met Val Ala
Ala Ala Ser Pro Tyr 195 200 205Val
Ala Trp Met Ser Val Thr Ala Gly Gln Ala Glu Leu Thr Ala Ala 210
215 220Gln Val Arg Val Ala Ala Ala Ala Tyr Glu
Thr Ala Tyr Gly Leu Thr225 230 235
240Val Pro Pro Pro Val Ile Ala Glu Asn Arg Ala Glu Leu Met Ile
Leu 245 250 255Ile Ala Thr
Asn Leu Leu Gly Gln Asn Thr Pro Ala Ile Ala Val Asn 260
265 270Glu Ala Glu Tyr Gly Glu Met Trp Ala Gln
Asp Ala Ala Ala Met Phe 275 280
285Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr Ala Thr Leu Leu Pro Phe 290
295 300Glu Glu Ala Pro Glu Met Thr Ser
Ala Gly Gly Leu Leu Glu Gln Ala305 310
315 320Ala Ala Val Glu Glu Ala Ser Asp Thr Ala Ala Ala
Asn Gln Leu Met 325 330
335Asn Asn Val Pro Gln Ala Leu Gln Gln Leu Ala Gln Pro Thr Gln Gly
340 345 350Thr Thr Pro Ser Ser Lys
Leu Gly Gly Leu Trp Lys Thr Val Ser Pro 355 360
365His Arg Ser Pro Ile Ser Asn Met Val Ser Met Ala Asn Asn
His Met 370 375 380Ser Met Thr Asn Ser
Gly Val Ser Met Thr Asn Thr Leu Ser Ser Met385 390
395 400Leu Lys Gly Phe Ala Pro Ala Ala Ala Ala
Gln Ala Val Gln Thr Ala 405 410
415Ala Gln Asn Gly Val Arg Ala Met Ser Ser Leu Gly Ser Ser Leu Gly
420 425 430Ser Ser Gly Leu Gly
Gly Gly Val Ala Ala Asn Leu Gly Arg Ala Ala 435
440 445Ser Val Gly Ser Leu Ser Val Pro Gln Ala Trp Ala
Ala Ala Asn Gln 450 455 460Ala Val Thr
Pro Ala Ala Arg Ala Leu Pro Leu Thr Ser Leu Thr Ser465
470 475 480Ala Ala Glu Arg Gly Pro Gly
Gln Met Leu Gly Gly Leu Pro Val Gly 485
490 495Gln Met Gly Ala Arg Ala Gly Gly Gly Leu Ser Gly
Val Leu Arg Val 500 505 510Pro
Pro Arg Pro Tyr Val Met Pro His Ser Pro Ala Ala Gly Asp Ile 515
520 525Ala Pro Pro Ala Leu Ser Gln Asp Arg
Phe Ala Asp Phe Pro Ala Leu 530 535
540Pro Leu Asp Pro Ser Ala Met Val Ala Gln Val Gly Pro Gln Val Val545
550 555 560Asn Ile Asn Thr
Lys Leu Gly Tyr Asn Asn Ala Val Gly Ala Gly Thr 565
570 575Gly Ile Val Ile Asp Pro Asn Gly Val Val
Leu Thr Asn Asn His Val 580 585
590Ile Ala Gly Ala Thr Asp Ile Asn Ala Phe Ser Val Gly Ser Gly Gln
595 600 605Thr Tyr Gly Val Asp Val Val
Gly Tyr Asp Arg Thr Gln Asp Val Ala 610 615
620Val Leu Gln Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala Ala Ile
Gly625 630 635 640Gly Gly
Val Ala Val Gly Glu Pro Val Val Ala Met Gly Asn Ser Gly
645 650 655Gly Gln Gly Gly Thr Pro Arg
Ala Val Pro Gly Arg Val Val Ala Leu 660 665
670Gly Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu
Glu Thr 675 680 685Leu Asn Gly Leu
Ile Gln Phe Asp Ala Ala Ile Gln Pro Gly Asp Ala 690
695 700Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val
Gly Met Asn Thr705 710 715
720Ala Ala Ser Ser Thr Met Thr Ile Asn Tyr Gln Phe Gly Asp Val Asp
725 730 735Ala His Gly Ala Met
Ile Arg Ala Gln Ala Ala Ser Leu Glu Ala Glu 740
745 750His Gln Ala Ile Val Arg Asp Val Leu Ala Ala Gly
Asp Phe Trp Gly 755 760 765Gly Ala
Gly Ser Val Ala Cys Gln Glu Phe Ile Thr Gln Leu Gly Arg 770
775 780Asn Phe Gln Val Ile Tyr Glu Gln Ala Asn Ala
His Gly Gln Lys Val785 790 795
800Gln Ala Ala Gly Asn Asn Met Ala Gln Thr Asp Ser Ala Val Gly Ser
805 810 815Ser Trp Ala Thr
Ser Met Ser Leu Leu Asp Ala His Ile Pro Gln Leu 820
825 830Val Ala Ser Gln Ser Ala Phe Ala Ala Lys Ala
Gly Leu Met Arg His 835 840 845Thr
Ile Gly Gln Ala Glu Gln Ala Ala Met Ser Ala Gln Ala Phe His 850
855 860Gln Gly Glu Ser Ser Ala Ala Phe Gln Ala
Ala His Ala Arg Phe Val865 870 875
880Ala Ala Ala Ala Lys Val Asn Thr Leu Leu Asp Val Ala Gln Ala
Asn 885 890 895Leu Gly Glu
Ala Ala Gly Thr Tyr Val Ala Ala Asp Ala Ala Ala Ala 900
905 910Ser Thr Tyr Thr Gly Phe Pro Trp
915 920271010PRTArtificial SequenceM103 27Met Thr Ala Ala
Ser Asp Asn Phe Gln Leu Ser Gln Gly Gly Gln Gly1 5
10 15Phe Ala Ile Pro Ile Gly Gln Ala Met Ala
Ile Ala Gly Gln Ile Arg 20 25
30Ser Gly Gly Gly Ser Pro Thr Val His Ile Gly Pro Thr Ala Phe Leu
35 40 45Gly Leu Gly Val Val Asp Asn Asn
Gly Asn Gly Ala Arg Val Gln Arg 50 55
60Val Val Gly Ser Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly Asp65
70 75 80Val Ile Thr Ala Val
Asp Gly Ala Pro Ile Asn Ser Ala Thr Ala Met 85
90 95Ala Asp Ala Leu Asn Gly His His Pro Gly Asp
Val Ile Ser Val Thr 100 105
110Trp Gln Thr Lys Ser Gly Gly Thr Arg Thr Gly Asn Val Thr Leu Ala
115 120 125Glu Gly Pro Pro Ala Glu Phe
Met Val Asp Phe Gly Ala Leu Pro Pro 130 135
140Glu Ile Asn Ser Ala Arg Met Tyr Ala Gly Pro Gly Ser Ala Ser
Leu145 150 155 160Val Ala
Ala Ala Gln Met Trp Asp Ser Val Ala Ser Asp Leu Phe Ser
165 170 175Ala Ala Ser Ala Phe Gln Ser
Val Val Trp Gly Leu Thr Val Gly Ser 180 185
190Trp Ile Gly Ser Ser Ala Gly Leu Met Val Ala Ala Ala Ser
Pro Tyr 195 200 205Val Ala Trp Met
Ser Val Thr Ala Gly Gln Ala Glu Leu Thr Ala Ala 210
215 220Gln Val Arg Val Ala Ala Ala Ala Tyr Glu Thr Ala
Tyr Gly Leu Thr225 230 235
240Val Pro Pro Pro Val Ile Ala Glu Asn Arg Ala Glu Leu Met Ile Leu
245 250 255Ile Ala Thr Asn Leu
Leu Gly Gln Asn Thr Pro Ala Ile Ala Val Asn 260
265 270Glu Ala Glu Tyr Gly Glu Met Trp Ala Gln Asp Ala
Ala Ala Met Phe 275 280 285Gly Tyr
Ala Ala Ala Thr Ala Thr Ala Thr Ala Thr Leu Leu Pro Phe 290
295 300Glu Glu Ala Pro Glu Met Thr Ser Ala Gly Gly
Leu Leu Glu Gln Ala305 310 315
320Ala Ala Val Glu Glu Ala Ser Asp Thr Ala Ala Ala Asn Gln Leu Met
325 330 335Asn Asn Val Pro
Gln Ala Leu Gln Gln Leu Ala Gln Pro Thr Gln Gly 340
345 350Thr Thr Pro Ser Ser Lys Leu Gly Gly Leu Trp
Lys Thr Val Ser Pro 355 360 365His
Arg Ser Pro Ile Ser Asn Met Val Ser Met Ala Asn Asn His Met 370
375 380Ser Met Thr Asn Ser Gly Val Ser Met Thr
Asn Thr Leu Ser Ser Met385 390 395
400Leu Lys Gly Phe Ala Pro Ala Ala Ala Ala Gln Ala Val Gln Thr
Ala 405 410 415Ala Gln Asn
Gly Val Arg Ala Met Ser Ser Leu Gly Ser Ser Leu Gly 420
425 430Ser Ser Gly Leu Gly Gly Gly Val Ala Ala
Asn Leu Gly Arg Ala Ala 435 440
445Ser Val Gly Ser Leu Ser Val Pro Gln Ala Trp Ala Ala Ala Asn Gln 450
455 460Ala Val Thr Pro Ala Ala Arg Ala
Leu Pro Leu Thr Ser Leu Thr Ser465 470
475 480Ala Ala Glu Arg Gly Pro Gly Gln Met Leu Gly Gly
Leu Pro Val Gly 485 490
495Gln Met Gly Ala Arg Ala Gly Gly Gly Leu Ser Gly Val Leu Arg Val
500 505 510Pro Pro Arg Pro Tyr Val
Met Pro His Ser Pro Ala Ala Gly Asp Ile 515 520
525Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro
Ala Leu 530 535 540Pro Leu Asp Pro Ser
Ala Met Val Ala Gln Val Gly Pro Gln Val Val545 550
555 560Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn
Ala Val Gly Ala Gly Thr 565 570
575Gly Ile Val Ile Asp Pro Asn Gly Val Val Leu Thr Asn Asn His Val
580 585 590Ile Ala Gly Ala Thr
Asp Ile Asn Ala Phe Ser Val Gly Ser Gly Gln 595
600 605Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr
Gln Asp Val Ala 610 615 620Val Leu Gln
Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala Ala Ile Gly625
630 635 640Gly Gly Val Ala Val Gly Glu
Pro Val Val Ala Met Gly Asn Ser Gly 645
650 655Gly Gln Gly Gly Thr Pro Arg Ala Val Pro Gly Arg
Val Val Ala Leu 660 665 670Gly
Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu Glu Thr 675
680 685Leu Asn Gly Leu Ile Gln Phe Asp Ala
Ala Ile Gln Pro Gly Asp Ala 690 695
700Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val Gly Met Asn Thr705
710 715 720Ala Ala Ser Ser
Gly Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu 725
730 735Gln Val Pro Ser Pro Ser Met Gly Arg Asp
Ile Lys Val Gln Phe Gln 740 745
750Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp Gly Leu Arg
755 760 765Ala Gln Asp Asp Tyr Asn Gly
Trp Asp Ile Asn Thr Pro Ala Phe Glu 770 775
780Trp Tyr Tyr Gln Ser Gly Leu Ser Ile Val Met Pro Val Gly Gly
Gln785 790 795 800Ser Ser
Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly
805 810 815Cys Gln Thr Tyr Lys Trp Glu
Thr Phe Leu Thr Ser Glu Leu Pro Gln 820 825
830Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser Ala
Ala Ile 835 840 845Gly Leu Ser Met
Ala Gly Ser Ser Ala Met Ile Leu Ala Ala Tyr His 850
855 860Pro Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala
Leu Leu Asp Pro865 870 875
880Ser Gln Gly Met Gly Pro Ser Leu Ile Gly Leu Ala Met Gly Asp Ala
885 890 895Gly Gly Tyr Lys Ala
Ala Asp Met Trp Gly Pro Ser Ser Asp Pro Ala 900
905 910Trp Glu Arg Asn Asp Pro Thr Gln Gln Ile Pro Lys
Leu Val Ala Asn 915 920 925Asn Thr
Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu 930
935 940Gly Gly Ala Asn Ile Pro Ala Glu Phe Leu Glu
Asn Phe Val Arg Ser945 950 955
960Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn
965 970 975Ala Val Phe Asn
Phe Pro Pro Asn Gly Thr His Ser Trp Glu Tyr Trp 980
985 990Gly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu
Gln Ser Ser Leu Gly 995 1000
1005Ala Gly 1010281148PRTArtificial SequenceM114 28Met Thr Ala Ala Ser
Asp Asn Phe Gln Leu Ser Gln Gly Gly Gln Gly1 5
10 15Phe Ala Ile Pro Ile Gly Gln Ala Met Ala Ile
Ala Gly Gln Ile Arg 20 25
30Ser Gly Gly Gly Ser Pro Thr Val His Ile Gly Pro Thr Ala Phe Leu
35 40 45Gly Leu Gly Val Val Asp Asn Asn
Gly Asn Gly Ala Arg Val Gln Arg 50 55
60Val Val Gly Ser Ala Pro Ala Ala Ser Leu Gly Ile Ser Thr Gly Asp65
70 75 80Val Ile Thr Ala Val
Asp Gly Ala Pro Ile Asn Ser Ala Thr Ala Met 85
90 95Ala Asp Ala Leu Asn Gly His His Pro Gly Asp
Val Ile Ser Val Thr 100 105
110Trp Gln Thr Lys Ser Gly Gly Thr Arg Thr Gly Asn Val Thr Leu Ala
115 120 125Glu Gly Pro Pro Ala Glu Phe
Met Val Asp Phe Gly Ala Leu Pro Pro 130 135
140Glu Ile Asn Ser Ala Arg Met Tyr Ala Gly Pro Gly Ser Ala Ser
Leu145 150 155 160Val Ala
Ala Ala Gln Met Trp Asp Ser Val Ala Ser Asp Leu Phe Ser
165 170 175Ala Ala Ser Ala Phe Gln Ser
Val Val Trp Gly Leu Thr Val Gly Ser 180 185
190Trp Ile Gly Ser Ser Ala Gly Leu Met Val Ala Ala Ala Ser
Pro Tyr 195 200 205Val Ala Trp Met
Ser Val Thr Ala Gly Gln Ala Glu Leu Thr Ala Ala 210
215 220Gln Val Arg Val Ala Ala Ala Ala Tyr Glu Thr Ala
Tyr Gly Leu Thr225 230 235
240Val Pro Pro Pro Val Ile Ala Glu Asn Arg Ala Glu Leu Met Ile Leu
245 250 255Ile Ala Thr Asn Leu
Leu Gly Gln Asn Thr Pro Ala Ile Ala Val Asn 260
265 270Glu Ala Glu Tyr Gly Glu Met Trp Ala Gln Asp Ala
Ala Ala Met Phe 275 280 285Gly Tyr
Ala Ala Ala Thr Ala Thr Ala Thr Ala Thr Leu Leu Pro Phe 290
295 300Glu Glu Ala Pro Glu Met Thr Ser Ala Gly Gly
Leu Leu Glu Gln Ala305 310 315
320Ala Ala Val Glu Glu Ala Ser Asp Thr Ala Ala Ala Asn Gln Leu Met
325 330 335Asn Asn Val Pro
Gln Ala Leu Gln Gln Leu Ala Gln Pro Thr Gln Gly 340
345 350Thr Thr Pro Ser Ser Lys Leu Gly Gly Leu Trp
Lys Thr Val Ser Pro 355 360 365His
Arg Ser Pro Ile Ser Asn Met Val Ser Met Ala Asn Asn His Met 370
375 380Ser Met Thr Asn Ser Gly Val Ser Met Thr
Asn Thr Leu Ser Ser Met385 390 395
400Leu Lys Gly Phe Ala Pro Ala Ala Ala Ala Gln Ala Val Gln Thr
Ala 405 410 415Ala Gln Asn
Gly Val Arg Ala Met Ser Ser Leu Gly Ser Ser Leu Gly 420
425 430Ser Ser Gly Leu Gly Gly Gly Val Ala Ala
Asn Leu Gly Arg Ala Ala 435 440
445Ser Val Gly Ser Leu Ser Val Pro Gln Ala Trp Ala Ala Ala Asn Gln 450
455 460Ala Val Thr Pro Ala Ala Arg Ala
Leu Pro Leu Thr Ser Leu Thr Ser465 470
475 480Ala Ala Glu Arg Gly Pro Gly Gln Met Leu Gly Gly
Leu Pro Val Gly 485 490
495Gln Met Gly Ala Arg Ala Gly Gly Gly Leu Ser Gly Val Leu Arg Val
500 505 510Pro Pro Arg Pro Tyr Val
Met Pro His Ser Pro Ala Ala Gly Asp Ile 515 520
525Ala Pro Pro Ala Leu Ser Gln Asp Arg Phe Ala Asp Phe Pro
Ala Leu 530 535 540Pro Leu Asp Pro Ser
Ala Met Val Ala Gln Val Gly Pro Gln Val Val545 550
555 560Asn Ile Asn Thr Lys Leu Gly Tyr Asn Asn
Ala Val Gly Ala Gly Thr 565 570
575Gly Ile Val Ile Asp Pro Asn Gly Val Val Leu Thr Asn Asn His Val
580 585 590Ile Ala Gly Ala Thr
Asp Ile Asn Ala Phe Ser Val Gly Ser Gly Gln 595
600 605Thr Tyr Gly Val Asp Val Val Gly Tyr Asp Arg Thr
Gln Asp Val Ala 610 615 620Val Leu Gln
Leu Arg Gly Ala Gly Gly Leu Pro Ser Ala Ala Ile Gly625
630 635 640Gly Gly Val Ala Val Gly Glu
Pro Val Val Ala Met Gly Asn Ser Gly 645
650 655Gly Gln Gly Gly Thr Pro Arg Ala Val Pro Gly Arg
Val Val Ala Leu 660 665 670Gly
Gln Thr Val Gln Ala Ser Asp Ser Leu Thr Gly Ala Glu Glu Thr 675
680 685Leu Asn Gly Leu Ile Gln Phe Asp Ala
Ala Ile Gln Pro Gly Asp Ala 690 695
700Gly Gly Pro Val Val Asn Gly Leu Gly Gln Val Val Gly Met Asn Thr705
710 715 720Ala Ala Ser Ser
Thr Met Asp Phe Gly Leu Leu Pro Pro Glu Val Asn 725
730 735Ser Ser Arg Met Tyr Ser Gly Pro Gly Pro
Glu Ser Met Leu Ala Ala 740 745
750Ala Ala Ala Trp Asp Gly Val Ala Ala Glu Leu Thr Ser Ala Ala Val
755 760 765Ser Tyr Gly Ser Val Val Ser
Thr Leu Ile Val Glu Pro Trp Met Gly 770 775
780Pro Ala Ala Ala Ala Met Ala Ala Ala Ala Thr Pro Tyr Val Gly
Trp785 790 795 800Leu Ala
Ala Thr Ala Ala Leu Ala Lys Glu Thr Ala Thr Gln Ala Arg
805 810 815Ala Ala Ala Glu Ala Phe Gly
Thr Ala Phe Ala Met Thr Val Pro Pro 820 825
830Ser Leu Val Ala Ala Asn Arg Ser Arg Leu Met Ser Leu Val
Ala Ala 835 840 845Asn Ile Leu Gly
Gln Asn Ser Ala Ala Ile Ala Ala Thr Gln Ala Glu 850
855 860Tyr Ala Glu Met Trp Ala Gln Asp Ala Ala Val Met
Tyr Ser Tyr Glu865 870 875
880Gly Ala Ser Ala Ala Ala Ser Ala Leu Pro Pro Phe Thr Pro Pro Val
885 890 895Gln Gly Thr Gly Pro
Ala Gly Pro Ala Ala Ala Ala Ala Ala Thr Gln 900
905 910Ala Ala Gly Ala Gly Ala Val Ala Asp Ala Gln Ala
Thr Leu Ala Gln 915 920 925Leu Pro
Pro Gly Ile Leu Ser Asp Ile Leu Ser Ala Leu Ala Ala Asn 930
935 940Ala Asp Pro Leu Thr Ser Gly Leu Leu Gly Ile
Ala Ser Thr Leu Asn945 950 955
960Pro Gln Val Gly Ser Ala Gln Pro Ile Val Ile Pro Thr Pro Ile Gly
965 970 975Glu Leu Asp Val
Ile Ala Leu Tyr Ile Ala Ser Ile Ala Thr Gly Ser 980
985 990Ile Ala Leu Ala Ile Thr Asn Thr Ala Arg Pro
Trp His Ile Gly Leu 995 1000
1005Tyr Gly Asn Ala Gly Gly Leu Gly Pro Thr Gln Gly His Pro Leu
1010 1015 1020Ser Ser Ala Thr Asp Glu
Pro Glu Pro His Trp Gly Pro Phe Gly 1025 1030
1035Gly Ala Ala Pro Val Ser Ala Gly Val Gly His Ala Ala Leu
Val 1040 1045 1050Gly Ala Leu Ser Val
Pro His Ser Trp Thr Thr Ala Ala Pro Glu 1055 1060
1065Ile Gln Leu Ala Val Gln Ala Thr Pro Thr Phe Ser Ser
Ser Ala 1070 1075 1080Gly Ala Asp Pro
Thr Ala Leu Asn Gly Met Pro Ala Gly Leu Leu 1085
1090 1095Ser Gly Met Ala Leu Ala Ser Leu Ala Ala Arg
Gly Thr Thr Gly 1100 1105 1110Gly Gly
Gly Thr Arg Ser Gly Thr Ser Thr Asp Gly Gln Glu Asp 1115
1120 1125Gly Arg Lys Pro Pro Val Val Val Ile Arg
Glu Gln Pro Pro Pro 1130 1135 1140Gly
Asn Pro Pro Arg 1145299PRTMycobacterium tuberculosis 29Phe Ile Ile
Asp Pro Thr Ile Ser Ala1 5309PRTMycobacterium tuberculosis
30Ile Leu Tyr Ser Ser Leu Glu Tyr Phe1 5319PRTMycobacterium
tuberculosis 31Leu Glu Tyr Phe Glu Lys Ala Leu Glu1
5329PRTMycobacterium tuberculosis 32Tyr Ala Gly Lys Asn Arg Asn His Val1
5339PRTMycobacterium tuberculosis 33Leu Ile His Asp Gln Ala
Asn Ala Val1 5349PRTMycobacterium tuberculosis 34Phe Val
Arg Pro Val Ala Val Asp Leu1 5359PRTMycobacterium
tuberculosis 35Leu Thr Tyr Ile Pro Val Val Gly His1
5369PRTMycobacterium tuberculosis 36Tyr Ile Pro Val Val Gly His Ala Leu1
5379PRTMycobacterium tuberculosis 37Tyr Leu Val Val Lys Thr
Leu Ile Asn1 5389PRTMycobacterium tuberculosis 38Leu Val
Val Lys Thr Leu Ile Asn Ala1 5399PRTMycobacterium
tuberculosis 39Val Lys Thr Leu Ile Asn Ala Thr Gln1
5409PRTMycobacterium tuberculosis 40Leu Lys Leu Leu Ala Lys Leu Ala Glu1
5419PRTMycobacterium tuberculosis 41Leu Val Ala Ala Ala Ile
Ala Asp Ile1 5429PRTMycobacterium tuberculosis 42Ile Ile
Ser Asp Val Ala Asp Ile Ile1 5439PRTMycobacterium
tuberculosis 43Trp Glu Phe Ile Thr Asn Ala Leu Asn1
5449PRTMycobacterium tuberculosis 44Leu Phe Gly Ala Ala Gly Leu Ser Ala1
5459PRTMycobacterium tuberculosis 45Leu Ala His Ala Asp Ser
Leu Ala Ser1 5469PRTMycobacterium tuberculosis 46Leu Ala
Ser Ser Ala Ser Leu Pro Ala1 5479PRTMycobacterium
tuberculosis 47Phe Gly Gly Leu Pro Ser Leu Ala Gln1
5489PRTMycobacterium tuberculosis 48Phe Ile Ile Asp Pro Thr Ile Ser Ala1
5499PRTMycobacterium tuberculosis 49Ile Ile Asp Pro Thr Ile
Ser Ala Ile1 5509PRTMycobacterium tuberculosis 50Pro Thr
Ile Ser Ala Ile Asp Gly Leu1 5519PRTMycobacterium
tuberculosis 51Thr Ile Ser Ala Ile Asp Gly Leu Tyr1
5529PRTMycobacterium tuberculosis 52Ser Ala Ile Asp Gly Leu Tyr Asp Leu1
5539PRTMycobacterium tuberculosis 53Ala Ile Asp Gly Leu Tyr
Asp Leu Leu1 5549PRTMycobacterium tuberculosis 54Leu Tyr
Asp Leu Leu Gly Ile Gly Ile1 5559PRTMycobacterium
tuberculosis 55Ile Pro Asn Gln Gly Gly Ile Leu Tyr1
5569PRTMycobacterium tuberculosis 56Gly Ile Leu Tyr Ser Ser Leu Glu Tyr1
5579PRTMycobacterium tuberculosis 57Tyr Ser Ser Leu Glu Tyr
Phe Glu Lys1 5589PRTMycobacterium tuberculosis 58Ser Leu
Glu Tyr Phe Glu Lys Ala Leu1 5599PRTMycobacterium
tuberculosis 59Tyr Phe Glu Lys Ala Leu Glu Glu Leu1
5609PRTMycobacterium tuberculosis 60Phe Glu Lys Ala Leu Glu Glu Leu Ala1
5619PRTMycobacterium tuberculosis 61Asn His Val Asn Phe Phe
Gln Glu Leu1 5629PRTMycobacterium tuberculosis 62Glu Leu
Ala Asp Leu Asp Arg Gln Leu1 5639PRTMycobacterium
tuberculosis 63Leu Ala Asp Leu Asp Arg Gln Leu Ile1
5649PRTMycobacterium tuberculosis 64Asp Leu Asp Arg Gln Leu Ile Ser Leu1
5659PRTMycobacterium tuberculosis 65Leu Asp Arg Gln Leu Ile
Ser Leu Ile1 5669PRTMycobacterium tuberculosis 66Ala Val
Gln Thr Thr Arg Asp Ile Leu1 5679PRTMycobacterium
tuberculosis 67Glu Gly Ala Lys Lys Gly Leu Glu Phe1
5689PRTMycobacterium tuberculosis 68Lys Gly Leu Glu Phe Val Arg Pro Val1
5699PRTMycobacterium tuberculosis 69Gly Leu Glu Phe Val Arg
Pro Val Ala1 5709PRTMycobacterium tuberculosis 70Leu Glu
Phe Val Arg Pro Val Ala Val1 5719PRTMycobacterium
tuberculosis 71Phe Val Arg Pro Val Ala Val Asp Leu1
5729PRTMycobacterium tuberculosis 72Arg Pro Val Ala Val Asp Leu Thr Tyr1
5739PRTMycobacterium tuberculosis 73Ala Val Asp Leu Thr Tyr
Ile Pro Val1 5749PRTMycobacterium tuberculosis 74Thr Tyr
Ile Pro Val Val Gly His Ala1 5759PRTMycobacterium
tuberculosis 75Tyr Ile Pro Val Val Gly His Ala Leu1
5769PRTMycobacterium tuberculosis 76Leu Ser Ala Ala Phe Gln Ala Pro Phe1
5779PRTMycobacterium tuberculosis 77Ser Ala Ala Phe Gln Ala
Pro Phe Cys1 5789PRTMycobacterium tuberculosis 78Ala Ala
Phe Gln Ala Pro Phe Cys Ala1 5799PRTMycobacterium
tuberculosis 79Phe Gln Ala Pro Phe Cys Ala Gly Ala1
5809PRTMycobacterium tuberculosis 80Ala Pro Phe Cys Ala Gly Ala Met Ala1
5819PRTMycobacterium tuberculosis 81Pro Phe Cys Ala Gly Ala
Met Ala Val1 5829PRTMycobacterium tuberculosis 82Ala Met
Ala Val Val Gly Gly Ala Leu1 5839PRTMycobacterium
tuberculosis 83Ala Val Val Gly Gly Ala Leu Ala Tyr1
5849PRTMycobacterium tuberculosis 84Gly Ala Leu Ala Tyr Leu Val Val Lys1
5859PRTMycobacterium tuberculosis 85Leu Ala Tyr Leu Val Val
Lys Thr Leu1 5869PRTMycobacterium tuberculosis 86Ala Tyr
Leu Val Val Lys Thr Leu Ile1 5879PRTMycobacterium
tuberculosis 87Lys Thr Leu Ile Asn Ala Thr Gln Leu1
5889PRTMycobacterium tuberculosis 88Thr Leu Ile Asn Ala Thr Gln Leu Leu1
5899PRTMycobacterium tuberculosis 89Ile Asn Ala Thr Gln Leu
Leu Lys Leu1 5909PRTMycobacterium tuberculosis 90Asn Ala
Thr Gln Leu Leu Lys Leu Leu1 5919PRTMycobacterium
tuberculosis 91Gln Leu Leu Lys Leu Leu Ala Lys Leu1
5929PRTMycobacterium tuberculosis 92Lys Leu Leu Ala Lys Leu Ala Glu Leu1
5939PRTMycobacterium tuberculosis 93Leu Leu Ala Lys Leu Ala
Glu Leu Val1 5949PRTMycobacterium tuberculosis 94Lys Leu
Ala Glu Leu Val Ala Ala Ala1 5959PRTMycobacterium
tuberculosis 95Leu Ala Glu Leu Val Ala Ala Ala Ile1
5969PRTMycobacterium tuberculosis 96Leu Val Ala Ala Ala Ile Ala Asp Ile1
5979PRTMycobacterium tuberculosis 97Ala Ile Ala Asp Ile Ile
Ser Asp Val1 5989PRTMycobacterium tuberculosis 98Ile Ala
Asp Ile Ile Ser Asp Val Ala1 5999PRTMycobacterium
tuberculosis 99Ile Ser Asp Val Ala Asp Ile Ile Lys1
51009PRTMycobacterium tuberculosis 100Thr Leu Gly Glu Val Trp Glu Phe
Ile1 51019PRTMycobacterium tuberculosis 101Phe Ile Thr Asn
Ala Leu Asn Gly Leu1 51029PRTMycobacterium tuberculosis
102Asn Ala Leu Asn Gly Leu Lys Glu Leu1
51039PRTMycobacterium tuberculosis 103Lys Leu Thr Gly Trp Val Thr Gly
Leu1 51049PRTMycobacterium tuberculosis 104Leu Thr Gly Trp
Val Thr Gly Leu Phe1 51059PRTMycobacterium tuberculosis
105Gly Trp Ser Asn Leu Glu Ser Phe Phe1
51069PRTMycobacterium tuberculosis 106Asn Leu Glu Ser Phe Phe Ala Gly
Val1 51079PRTMycobacterium tuberculosis 107Ser Phe Phe Ala
Gly Val Pro Gly Leu1 51089PRTMycobacterium tuberculosis
108Gly Leu Thr Gly Ala Thr Ser Gly Leu1
51099PRTMycobacterium tuberculosis 109Leu Ser Gln Val Thr Gly Leu Phe
Gly1 51109PRTMycobacterium tuberculosis 110Ser Gln Val Thr
Gly Leu Phe Gly Ala1 51119PRTMycobacterium tuberculosis
111Leu Ser Ala Ser Ser Gly Leu Ala His1
51129PRTMycobacterium tuberculosis 112Ala Ser Ser Gly Leu Ala His Ala
Asp1 51139PRTMycobacterium tuberculosis 113Ser Gly Leu Ala
His Ala Asp Ser Leu1 51149PRTMycobacterium tuberculosis
114Gly Leu Ala His Ala Asp Ser Leu Ala1
51159PRTMycobacterium tuberculosis 115Ser Leu Ala Ser Ser Ala Ser Leu
Pro1 51169PRTMycobacterium tuberculosis 116Ala Ser Ser Ala
Ser Leu Pro Ala Leu1 51179PRTMycobacterium tuberculosis
117Ser Gly Phe Gly Gly Leu Pro Ser Leu1
51189PRTMycobacterium tuberculosis 118Leu Pro Ser Leu Ala Gln Val His
Ala1 51199PRTMycobacterium tuberculosis 119His Ala Ala Ser
Thr Arg Gln Ala Leu1 51209PRTMycobacterium tuberculosis
120Ser Thr Arg Gln Ala Leu Arg Pro Arg1
51219PRTMycobacterium tuberculosis 121Arg Pro Arg Ala Asp Gly Pro Val
Gly1 51229PRTMycobacterium tuberculosis 122Glu Gln Val Gly
Gly Gln Ser Gln Leu1 51239PRTMycobacterium tuberculosis
123Gly Ala Ser Lys Gly Thr Thr Thr Lys1
51249PRTMycobacterium tuberculosis 124Ala Ser Lys Gly Thr Thr Thr Lys
Lys1 51259PRTMycobacterium tuberculosis 125Lys Gly Thr Thr
Thr Lys Lys Tyr Ser1 51269PRTMycobacterium tuberculosis
126Thr Glu Asp Ala Glu Arg Ala Pro Val1
512720PRTMycobacterium tuberculosis 127Met Ser Arg Ala Phe Ile Ile Asp
Pro Thr Ile Ser Ala Ile Asp Gly1 5 10
15Leu Tyr Asp Leu 2012820PRTMycobacterium
tuberculosis 128Ile Asp Gly Leu Tyr Asp Leu Leu Gly Ile Gly Ile Pro Asn
Gln Gly1 5 10 15Gly Ile
Leu Tyr 2012920PRTMycobacterium tuberculosis 129Asn Gln Gly
Gly Ile Leu Tyr Ser Ser Leu Glu Tyr Phe Glu Lys Ala1 5
10 15Leu Glu Glu Leu
2013020PRTMycobacterium tuberculosis 130Glu Lys Ala Leu Glu Glu Leu Ala
Ala Ala Phe Pro Gly Asp Gly Trp1 5 10
15Leu Gly Ser Ala 2013120PRTMycobacterium
tuberculosis 131Asp Gly Trp Leu Gly Ser Ala Ala Asp Lys Tyr Ala Gly Lys
Asn Arg1 5 10 15Asn His
Val Asn 2013220PRTMycobacterium tuberculosis 132Lys Asn Arg
Asn His Val Asn Phe Phe Gln Glu Leu Ala Asp Leu Asp1 5
10 15Arg Gln Leu Ile
2013320PRTMycobacterium tuberculosis 133Asp Leu Asp Arg Gln Leu Ile Ser
Leu Ile His Asp Gln Ala Asn Ala1 5 10
15Val Gln Thr Thr 2013420PRTMycobacterium
tuberculosis 134Ala Asn Ala Val Gln Thr Thr Arg Asp Ile Leu Glu Gly Ala
Lys Lys1 5 10 15Gly Leu
Glu Phe 2013520PRTMycobacterium tuberculosis 135Ala Lys Lys
Gly Leu Glu Phe Val Arg Pro Val Ala Val Asp Leu Thr1 5
10 15Tyr Ile Pro Val
2013627PRTMycobacterium tuberculosis 136Phe Val Arg Pro Val Ala Val Asp
Leu Thr Tyr Ile Pro Val Val Gly1 5 10
15His Ala Leu Ser Ala Ala Phe Gln Ala Pro Phe 20
2513720PRTMycobacterium tuberculosis 137Ala Ala Phe Gln
Ala Pro Phe Cys Ala Gly Ala Met Ala Val Val Gly1 5
10 15Gly Ala Leu Ala
2013820PRTMycobacterium tuberculosis 138Val Val Gly Gly Ala Leu Ala Tyr
Leu Val Val Lys Thr Leu Ile Asn1 5 10
15Ala Thr Gln Leu 2013920PRTMycobacterium
tuberculosis 139Leu Ile Asn Ala Thr Gln Leu Leu Lys Leu Leu Ala Lys Leu
Ala Glu1 5 10 15Leu Val
Ala Ala 2014020PRTMycobacterium tuberculosis 140Leu Ala Glu
Leu Val Ala Ala Ala Ile Ala Asp Ile Ile Ser Asp Val1 5
10 15Ala Asp Ile Ile
2014120PRTMycobacterium tuberculosis 141Ser Asp Val Ala Asp Ile Ile Lys
Gly Thr Leu Gly Glu Val Trp Glu1 5 10
15Phe Ile Thr Asn 2014220PRTMycobacterium
tuberculosis 142Val Trp Glu Phe Ile Thr Asn Ala Leu Asn Gly Leu Lys Glu
Leu Trp1 5 10 15Asp Lys
Leu Thr 2014320PRTMycobacterium tuberculosis 143Glu Leu Trp
Asp Lys Leu Thr Gly Trp Val Thr Gly Leu Phe Ser Arg1 5
10 15Gly Trp Ser Asn
2014420PRTMycobacterium tuberculosis 144Phe Ser Arg Gly Trp Ser Asn Leu
Glu Ser Phe Phe Ala Gly Val Pro1 5 10
15Gly Leu Thr Gly 2014520PRTMycobacterium
tuberculosis 145Gly Val Pro Gly Leu Thr Gly Ala Thr Ser Gly Leu Ser Gln
Val Thr1 5 10 15Gly Leu
Phe Gly 2014620PRTMycobacterium tuberculosis 146Gln Val Thr
Gly Leu Phe Gly Ala Ala Gly Leu Ser Ala Ser Ser Gly1 5
10 15Leu Ala His Ala
2014720PRTMycobacterium tuberculosis 147Ser Ser Gly Leu Ala His Ala Asp
Ser Leu Ala Ser Ser Ala Ser Leu1 5 10
15Pro Ala Leu Ala 2014820PRTMycobacterium
tuberculosis 148Ala Ser Leu Pro Ala Leu Ala Gly Ile Gly Gly Gly Ser Gly
Phe Gly1 5 10 15Gly Leu
Pro Ser 2014920PRTMycobacterium tuberculosis 149Gly Phe Gly
Gly Leu Pro Ser Leu Ala Gln Val His Ala Ala Ser Thr1 5
10 15Arg Gln Ala Leu
2015020PRTMycobacterium tuberculosis 150Ala Ser Thr Arg Gln Ala Leu Arg
Pro Arg Ala Asp Gly Pro Val Gly1 5 10
15Ala Ala Ala Glu 2015120PRTMycobacterium
tuberculosis 151Pro Val Gly Ala Ala Ala Glu Gln Val Gly Gly Gln Ser Gln
Leu Val1 5 10 15Ser Ala
Gln Gly 2015219PRTMycobacterium tuberculosis 152Gln Leu Val
Ser Ala Gln Gly Ser Gln Gly Met Gly Gly Pro Val Gly1 5
10 15Met Gly Gly 15320PRTMycobacterium
tuberculosis 153Pro Val Gly Met Gly Gly Met His Pro Ser Ser Gly Ala Ser
Lys Gly1 5 10 15Thr Thr
Thr Lys 2015420PRTMycobacterium tuberculosis 154Ser Lys Gly
Thr Thr Thr Lys Lys Tyr Ser Glu Gly Ala Ala Ala Gly1 5
10 15Thr Glu Asp Ala
2015520PRTMycobacterium tuberculosis 155Ala Ala Gly Thr Glu Asp Ala Glu
Arg Ala Pro Val Glu Ala Asp Ala1 5 10
15Gly Gly Gly Gln 2015620PRTMycobacterium
tuberculosis 156Arg Ala Pro Val Glu Ala Asp Ala Gly Gly Gly Gln Lys Val
Leu Val1 5 10 15Arg Asn
Val Val 201571053PRTMycobacterium tuberculosis 157Met Asn Phe
Ser Val Leu Pro Pro Glu Ile Asn Ser Ala Leu Ile Phe1 5
10 15Ala Gly Ala Gly Pro Glu Pro Met Ala
Ala Ala Ala Thr Ala Trp Asp 20 25
30Gly Leu Ala Met Glu Leu Ala Ser Ala Ala Ala Ser Phe Gly Ser Val
35 40 45Thr Ser Gly Leu Val Gly Gly
Ala Trp Gln Gly Ala Ser Ser Ser Ala 50 55
60Met Ala Ala Ala Ala Ala Pro Tyr Ala Ala Trp Leu Ala Ala Ala Ala65
70 75 80Val Gln Ala Glu
Gln Thr Ala Ala Gln Ala Ala Ala Met Ile Ala Glu 85
90 95Phe Glu Ala Val Lys Thr Ala Val Val Gln
Pro Met Leu Val Ala Ala 100 105
110Asn Arg Ala Asp Leu Val Ser Leu Val Met Ser Asn Leu Phe Gly Gln
115 120 125Asn Ala Pro Ala Ile Ala Ala
Ile Glu Ala Thr Tyr Glu Gln Met Trp 130 135
140Ala Ala Asp Val Ser Ala Met Ser Ala Tyr His Ala Gly Ala Ser
Ala145 150 155 160Ile Ala
Ser Ala Leu Ser Pro Phe Ser Lys Pro Leu Gln Asn Leu Ala
165 170 175Gly Leu Pro Ala Trp Leu Ala
Ser Gly Ala Pro Ala Ala Ala Met Thr 180 185
190Ala Ala Ala Gly Ile Pro Ala Leu Ala Gly Gly Pro Thr Ala
Ile Asn 195 200 205Leu Gly Ile Ala
Asn Val Gly Gly Gly Asn Val Gly Asn Ala Asn Asn 210
215 220Gly Leu Ala Asn Ile Gly Asn Ala Asn Leu Gly Asn
Tyr Asn Phe Gly225 230 235
240Ser Gly Asn Phe Gly Asn Ser Asn Ile Gly Ser Ala Ser Leu Gly Asn
245 250 255Asn Asn Ile Gly Phe
Gly Asn Leu Gly Ser Asn Asn Val Gly Val Gly 260
265 270Asn Leu Gly Asn Leu Asn Thr Gly Phe Ala Asn Thr
Gly Leu Gly Asn 275 280 285Phe Gly
Phe Gly Asn Thr Gly Asn Asn Asn Ile Gly Ile Gly Leu Thr 290
295 300Gly Asn Asn Gln Ile Gly Ile Gly Gly Leu Asn
Ser Gly Thr Gly Asn305 310 315
320Phe Gly Leu Phe Asn Ser Gly Ser Gly Asn Val Gly Phe Phe Asn Ser
325 330 335Gly Asn Gly Asn
Phe Gly Ile Gly Asn Ser Gly Asn Phe Asn Thr Gly 340
345 350Gly Trp Asn Ser Gly His Gly Asn Thr Gly Phe
Phe Asn Ala Gly Ser 355 360 365Phe
Asn Thr Gly Met Leu Asp Val Gly Asn Ala Asn Thr Gly Ser Leu 370
375 380Asn Thr Gly Ser Tyr Asn Met Gly Asp Phe
Asn Pro Gly Ser Ser Asn385 390 395
400Thr Gly Thr Phe Asn Thr Gly Asn Ala Asn Thr Gly Phe Leu Asn
Ala 405 410 415Gly Asn Ile
Asn Thr Gly Val Phe Asn Ile Gly His Met Asn Asn Gly 420
425 430Leu Phe Asn Thr Gly Asp Met Asn Asn Gly
Val Phe Tyr Arg Gly Val 435 440
445Gly Gln Gly Ser Leu Gln Phe Ser Ile Thr Thr Pro Asp Leu Thr Leu 450
455 460Pro Pro Leu Gln Ile Pro Gly Ile
Ser Val Pro Ala Phe Ser Leu Pro465 470
475 480Ala Ile Thr Leu Pro Ser Leu Asn Ile Pro Ala Ala
Thr Thr Pro Ala 485 490
495Asn Ile Thr Val Gly Ala Phe Ser Leu Pro Gly Leu Thr Leu Pro Ser
500 505 510Leu Asn Ile Pro Ala Ala
Thr Thr Pro Ala Asn Ile Thr Val Gly Ala 515 520
525Phe Ser Leu Pro Gly Leu Thr Leu Pro Ser Leu Asn Ile Pro
Ala Ala 530 535 540Thr Thr Pro Ala Asn
Ile Thr Val Gly Ala Phe Ser Leu Pro Gly Leu545 550
555 560Thr Leu Pro Ser Leu Asn Ile Pro Ala Ala
Thr Thr Pro Ala Asn Ile 565 570
575Thr Val Gly Ala Phe Ser Leu Pro Gly Leu Thr Leu Pro Ser Leu Asn
580 585 590Ile Pro Ala Ala Thr
Thr Pro Ala Asn Ile Thr Val Gly Ala Phe Ser 595
600 605Leu Pro Gly Leu Thr Leu Pro Ser Leu Asn Ile Pro
Ala Ala Thr Thr 610 615 620Pro Ala Asn
Ile Thr Val Ser Gly Phe Gln Leu Pro Pro Leu Ser Ile625
630 635 640Pro Ser Val Ala Ile Pro Pro
Val Thr Val Pro Pro Ile Thr Val Gly 645
650 655Ala Phe Asn Leu Pro Pro Leu Gln Ile Pro Glu Val
Thr Ile Pro Gln 660 665 670Leu
Thr Ile Pro Ala Gly Ile Thr Ile Gly Gly Phe Ser Leu Pro Ala 675
680 685Ile His Thr Gln Pro Ile Thr Val Gly
Gln Ile Gly Val Gly Gln Phe 690 695
700Gly Leu Pro Ser Ile Gly Trp Asp Val Phe Leu Ser Thr Pro Arg Ile705
710 715 720Thr Val Pro Ala
Phe Gly Ile Pro Phe Thr Leu Gln Phe Gln Thr Asn 725
730 735Val Pro Ala Leu Gln Pro Pro Gly Gly Gly
Leu Ser Thr Phe Thr Asn 740 745
750Gly Ala Leu Ile Phe Gly Glu Phe Asp Leu Pro Gln Leu Val Val His
755 760 765Pro Tyr Thr Leu Thr Gly Pro
Ile Val Ile Gly Ser Phe Phe Leu Pro 770 775
780Ala Phe Asn Ile Pro Gly Ile Asp Val Pro Ala Ile Asn Val Asp
Gly785 790 795 800Phe Thr
Leu Pro Gln Ile Thr Thr Pro Ala Ile Thr Thr Pro Glu Phe
805 810 815Ala Ile Pro Pro Ile Gly Val
Gly Gly Phe Thr Leu Pro Gln Ile Thr 820 825
830Thr Gln Glu Ile Ile Thr Pro Glu Leu Thr Ile Asn Ser Ile
Gly Val 835 840 845Gly Gly Phe Thr
Leu Pro Gln Ile Thr Thr Pro Pro Ile Thr Thr Pro 850
855 860Pro Leu Thr Ile Asp Pro Ile Asn Leu Thr Gly Phe
Thr Leu Pro Gln865 870 875
880Ile Thr Thr Pro Pro Ile Thr Thr Pro Pro Leu Thr Ile Asp Pro Ile
885 890 895Asn Leu Thr Gly Phe
Thr Leu Pro Gln Ile Thr Thr Pro Pro Ile Thr 900
905 910Thr Pro Pro Leu Thr Ile Glu Pro Ile Gly Val Gly
Gly Phe Thr Thr 915 920 925Pro Pro
Leu Thr Val Pro Gly Ile His Leu Pro Ser Thr Thr Ile Gly 930
935 940Ala Phe Ala Ile Pro Gly Gly Pro Gly Tyr Phe
Asn Ser Ser Thr Ala945 950 955
960Pro Ser Ser Gly Phe Phe Asn Ser Gly Ala Gly Gly Asn Ser Gly Phe
965 970 975Gly Asn Asn Gly
Ser Gly Leu Ser Gly Trp Phe Asn Thr Asn Pro Ala 980
985 990Gly Leu Leu Gly Gly Ser Gly Tyr Gln Asn Phe
Gly Gly Leu Ser Ser 995 1000
1005Gly Phe Ser Asn Leu Gly Ser Gly Val Ser Gly Phe Ala Asn Arg
1010 1015 1020Gly Ile Leu Pro Phe Ser
Val Ala Ser Val Val Ser Gly Phe Ala 1025 1030
1035Asn Ile Gly Thr Asn Leu Ala Gly Phe Phe Gln Gly Thr Thr
Ser 1040 1045
1050158450PRTMycobacterium tuberculosis 158Met Ser Glu Leu Ser Val Ala
Thr Gly Ala Val Ser Thr Ala Ser Ser1 5 10
15Ser Ile Pro Met Pro Ala Gly Val Asn Pro Ala Asp Leu
Ala Ala Glu 20 25 30Leu Ala
Ala Val Val Thr Glu Ser Val Asp Glu Asp Tyr Leu Leu Tyr 35
40 45Glu Cys Asp Gly Gln Trp Val Leu Ala Ala
Gly Val Gln Ala Met Val 50 55 60Glu
Leu Asp Ser Asp Glu Leu Arg Val Ile Arg Asp Gly Val Thr Arg65
70 75 80Arg Gln Gln Trp Ser Gly
Arg Pro Gly Ala Ala Leu Gly Glu Ala Val 85
90 95Asp Arg Leu Leu Leu Glu Thr Asp Gln Ala Phe Gly
Trp Val Ala Phe 100 105 110Glu
Phe Gly Val His Arg Tyr Gly Leu Gln Gln Arg Leu Ala Pro His 115
120 125Thr Pro Leu Ala Arg Val Phe Ser Pro
Arg Thr Arg Ile Met Val Ser 130 135
140Glu Lys Glu Ile Arg Leu Phe Asp Ala Gly Ile Arg His Arg Glu Ala145
150 155 160Ile Asp Arg Leu
Leu Ala Thr Gly Val Arg Glu Val Pro Gln Ser Arg 165
170 175Ser Val Asp Val Ser Asp Asp Pro Ser Gly
Phe Arg Arg Arg Val Ala 180 185
190Val Ala Val Asp Glu Ile Ala Ala Gly Arg Tyr His Lys Val Ile Leu
195 200 205Ser Arg Cys Val Glu Val Pro
Phe Ala Ile Asp Phe Pro Leu Thr Tyr 210 215
220Arg Leu Gly Arg Arg His Asn Thr Pro Val Arg Ser Phe Leu Leu
Gln225 230 235 240Leu Gly
Gly Ile Arg Ala Leu Gly Tyr Ser Pro Glu Leu Val Thr Ala
245 250 255Val Arg Ala Asp Gly Val Val
Ile Thr Glu Pro Leu Ala Gly Thr Arg 260 265
270Ala Leu Gly Arg Gly Pro Ala Ile Asp Arg Leu Ala Arg Asp
Asp Leu 275 280 285Glu Ser Asn Ser
Lys Glu Ile Val Glu His Ala Ile Ser Val Arg Ser 290
295 300Ser Leu Glu Glu Ile Thr Asp Ile Ala Glu Pro Gly
Ser Ala Ala Val305 310 315
320Ile Asp Phe Met Thr Val Arg Glu Arg Gly Ser Val Gln His Leu Gly
325 330 335Ser Thr Ile Arg Ala
Arg Leu Asp Pro Ser Ser Asp Arg Met Ala Ala 340
345 350Leu Glu Ala Leu Phe Pro Ala Val Thr Ala Ser Gly
Ile Pro Lys Ala 355 360 365Ala Gly
Val Glu Ala Ile Phe Arg Leu Asp Glu Cys Pro Arg Gly Leu 370
375 380Tyr Ser Gly Ala Val Val Met Leu Ser Ala Asp
Gly Gly Leu Asp Ala385 390 395
400Ala Leu Thr Leu Arg Ala Ala Tyr Gln Val Gly Gly Arg Thr Trp Leu
405 410 415Arg Ala Gly Ala
Gly Ile Ile Glu Glu Ser Glu Pro Glu Arg Glu Phe 420
425 430Glu Glu Thr Cys Glu Lys Leu Ser Thr Leu Thr
Pro Tyr Leu Val Ala 435 440 445Arg
Gln 450159324PRTMycobacterium tuberculosis 159Met Ser Asp Gln Val Pro
Lys Pro His Arg His His Ile Trp Arg Ile1 5
10 15Thr Arg Arg Thr Leu Ser Lys Ser Trp Asp Asp Ser
Ile Phe Ser Glu 20 25 30Ser
Ala Gln Ala Ala Phe Trp Ser Ala Leu Ser Leu Pro Pro Leu Leu 35
40 45Leu Gly Met Leu Gly Ser Leu Ala Tyr
Val Ala Pro Leu Phe Gly Pro 50 55
60Asp Thr Leu Pro Ala Ile Glu Lys Ser Ala Leu Ser Thr Ala His Ser65
70 75 80Phe Phe Ser Pro Ser
Val Val Asn Glu Ile Ile Glu Pro Thr Ile Gly 85
90 95Asp Ile Thr Asn Asn Ala Arg Gly Glu Val Ala
Ser Leu Gly Phe Leu 100 105
110Ile Ser Leu Trp Ala Gly Ser Ser Ala Ile Ser Ala Phe Val Asp Ala
115 120 125Val Val Glu Ala His Asp Gln
Thr Pro Leu Arg His Pro Val Arg Gln 130 135
140Arg Phe Phe Ala Leu Phe Leu Tyr Val Val Met Leu Val Phe Leu
Val145 150 155 160Ala Thr
Ala Pro Val Met Val Val Gly Pro Arg Lys Val Ser Glu His
165 170 175Ile Pro Glu Ser Leu Ala Asn
Leu Leu Arg Tyr Gly Tyr Tyr Pro Ala 180 185
190Leu Ile Leu Gly Leu Thr Val Gly Val Ile Leu Leu Tyr Arg
Val Ala 195 200 205Leu Pro Val Pro
Leu Pro Thr His Arg Leu Val Leu Gly Ala Val Leu 210
215 220Ala Ile Ala Val Phe Leu Ile Ala Thr Leu Gly Leu
Arg Val Tyr Leu225 230 235
240Ala Trp Ile Thr Arg Thr Gly Tyr Thr Tyr Gly Ala Leu Ala Thr Pro
245 250 255Ile Ala Phe Leu Leu
Phe Ala Phe Phe Gly Gly Phe Ala Ile Met Leu 260
265 270Gly Ala Glu Leu Asn Ala Ala Val Gln Glu Glu Trp
Pro Ala Pro Ala 275 280 285Thr His
Ala His Arg Leu Gly Asn Trp Leu Lys Ala Arg Ile Gly Val 290
295 300Gly Thr Thr Thr Tyr Ser Ser Thr Ala Gln His
Ser Ala Val Ala Ala305 310 315
320Glu Pro Pro Ser
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