U.S. patent application number 14/849531 was filed with the patent office on 2016-08-04 for compounds and methods for diagnosis and immunotherapy of tuberculosis.
This patent application is currently assigned to CORIXA CORPOATION. The applicant listed for this patent is Antonio Campos-Neto, Shyian Jen, Michael Lodes, Pamela Ovendale, Yasir Skeiky. Invention is credited to Antonio Campos-Neto, Shyian Jen, Michael Lodes, Pamela Ovendale, Yasir Skeiky.
Application Number | 20160222070 14/849531 |
Document ID | / |
Family ID | 26880723 |
Filed Date | 2016-08-04 |
United States Patent
Application |
20160222070 |
Kind Code |
A1 |
Campos-Neto; Antonio ; et
al. |
August 4, 2016 |
COMPOUNDS AND METHODS FOR DIAGNOSIS AND IMMUNOTHERAPY OF
TUBERCULOSIS
Abstract
Compounds and methods for diagnosing tuberculosis or for
inducing protective immunity against tuberculosis are disclosed.
The compounds provided include polypeptides that contain at least
one immunogenic portion of one or more Mycobacterium proteins and
DNA molecules encoding such polypeptides. Diagnostic kits
containing such polypeptides or DNA sequences and a suitable
detection reagent may be used for the detection of Mycobacterium
infection in patients and biological samples. Antibodies directed
against such polypeptides are also provided. In addition, such
compounds may be formulated into vaccines and/or pharmaceutical
compositions for immunization against Mycobacterium infection.
Inventors: |
Campos-Neto; Antonio;
(Banbridge Island, WA) ; Skeiky; Yasir; (Seattle,
WA) ; Ovendale; Pamela; (Everett, WA) ; Jen;
Shyian; (Seattle, WA) ; Lodes; Michael;
(Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Campos-Neto; Antonio
Skeiky; Yasir
Ovendale; Pamela
Jen; Shyian
Lodes; Michael |
Banbridge Island
Seattle
Everett
Seattle
Seattle |
WA
WA
WA
WA
WA |
US
US
US
US
US |
|
|
Assignee: |
CORIXA CORPOATION
Seattle
WA
|
Family ID: |
26880723 |
Appl. No.: |
14/849531 |
Filed: |
September 9, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13560514 |
Jul 27, 2012 |
|
|
|
14849531 |
|
|
|
|
11928957 |
Oct 30, 2007 |
|
|
|
13560514 |
|
|
|
|
11476254 |
Jun 27, 2006 |
7935353 |
|
|
11928957 |
|
|
|
|
09793306 |
Feb 26, 2001 |
7087713 |
|
|
11476254 |
|
|
|
|
09073010 |
May 5, 1998 |
6613881 |
|
|
09793306 |
|
|
|
|
08859381 |
May 20, 1997 |
|
|
|
09073010 |
|
|
|
|
60223828 |
Aug 8, 2000 |
|
|
|
60185027 |
Feb 25, 2000 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/06 20180101;
A61K 48/00 20130101; G01N 2469/20 20130101; A61K 2039/55566
20130101; G01N 33/5695 20130101; C07K 14/35 20130101; C12Q 1/689
20130101; A61K 2039/53 20130101; A61K 39/04 20130101; C07K 2319/00
20130101; A61P 37/04 20180101; C12Q 2600/158 20130101; A61K 38/00
20130101; C12Q 1/6883 20130101 |
International
Class: |
C07K 14/35 20060101
C07K014/35; A61K 39/04 20060101 A61K039/04 |
Claims
1. An isolated nucleic acid comprising a nucleotide sequence
encoding a polypeptide sequence comprising the amino acid sequence
of SEQ ID NO: 138.
2. The isolated nucleic acid of claim 1, consisting of the nucleic
acid sequence encoding a polypeptide sequence comprising the amino
acid sequence of SEQ ID NO: 138.
3. An isolated nucleic acid comprising a nucleotide sequence
encoding a polypeptide sequence having at least 90% identity to the
sequence of SEQ ID NO: 138.
4. The isolated nucleic acid sequence of claim 3 comprising a
nucleotide sequence encoding a polypeptide sequence having at least
95% identity to the sequence of SEQ ID NO: 138.
5. A composition comprising a physiologically acceptable carrier
and the nucleic acid of claim 1.
6. An isolated nucleic acid comprising a nucleotide sequence
encoding a polypeptide sequence comprising an immunogenic fragment
of a polypeptide consisting of the amino acid sequence of SEQ ID
NO: 138, wherein the fragment is at least 9 amino acids in
length.
7. A pharmaceutical composition comprising the nucleic acid of
claim 6 and a physiologically acceptable carrier.
8. A composition comprising an isolated nucleic acid comprising a
nucleotide sequence encoding a polypeptide sequence comprising an
immunogenic fragment of a polypeptide consisting of the amino acid
sequence of SEQ ID NO: 138, wherein the fragment is at least 9
amino acids in length, and a pharmaceutically acceptable
carrier.
9. A nucleic acid comprising a nucleotide sequence encoding a
fusion protein comprising an isolated polypeptide comprising an
immunogenic fragment of the polypeptide consisting of the amino
acid sequence of SEQ ID NO: 138, wherein the fragment is at least 9
amino acids in length, and a second polypeptide.
10. The fusion protein of claim 9, wherein the second polypeptide
is a Mycobacterium tuberculosis antigen.
11. The composition of claim 16, wherein the non-specific immune
response enhancer is an adjuvant.
12. A composition comprising: a nucleic acid encoding a fusion
protein comprising an isolated polypeptide comprising an
immunogenic fragment of the polypeptide consisting of the amino
acid sequence of SEQ ID NO: 138, wherein the fragment is at least 9
amino acids in length and a second polypeptide; and a
pharmaceutically acceptable carrier.
13. An isolated nucleic acid comprising the sequence of SEQ ID NO:
137.
14. The isolated nucleic acid of claim 13 consisting of the
sequence of SEQ ID NO: 137.
15. The isolated nucleic acid sequence of claim 13 having at least
85% identity to the sequence of SEQ ID NO: 137.
16. The isolated nucleic acid sequence of claim 15 having at least
90% identity to the sequence of SEQ ID NO: 137.
17. The isolated nucleic acid sequence of claim 16 having at least
95% identity to the sequence of SEQ ID NO: 137.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. Ser. No.
13/560,514, filed 27 Jul. 2012 which is a continuation of U.S. Ser.
No. 11/928,957, filed 30 Oct. 2007; now abandoned; which is a
continuation of U.S. Ser. No. 11/476,254, filed 27 Jun. 2006, now
U.S. Pat. No. 7,935,353; which is a continuation of U.S. Ser. No.
09/793,306, filed 26 Feb. 2001, now U.S. Pat. No. 7,087,713; which
claims priority to U.S. Ser. No. 60/223,828, filed 8 Aug. 2000; and
U.S. Ser. No. 60/185,027, filed 25 Feb. 2000; and is a continuation
in part of U.S. Ser. No. 09/073,010, filed 5 May 1998, now U.S.
Pat. No. 6,613,881, which is a continuation in part of U.S. Ser.
No. 08/859,381, filed 20 May 1997, now abandoned.
BACKGROUND OF THE INVENTION
[0002] Tuberculosis is a chronic, infectious disease, that is
generally caused by infection with Mycobacterium tuberculosis. It
is a major disease in developing countries, as well as an
increasing problem in developed areas of the world, with about 8
million new cases and 3 million deaths each year. Although the
infection may be asymptomatic for a considerable period of time,
the disease is most commonly manifested as an acute inflammation of
the lungs, resulting in fever and nonproductive cough. If left
untreated, serious complications and death typically result.
[0003] Although tuberculosis can generally be controlled using
extended antibiotic therapy, such treatment is not sufficient to
prevent the spread of the disease. Infected individuals may be
asymptomatic, but contagious, for some time. In addition, although
compliance with the treatment regimen is critical, patient behavior
is difficult to monitor. Some patients do not complete the course
of treatment, which can lead to ineffective treatment and the
development of drug resistance.
[0004] Inhibiting the spread of tuberculosis will require effective
vaccination and accurate, early diagnosis of the disease.
Currently, vaccination with live bacteria is the most efficient
method for inducing protective immunity. The most common
Mycobacterium employed for this purpose is Bacillus Calmette-Guerin
(BCG), an avirulent strain of Mycobacterium bovis. However, the
safety and efficacy of BCG is a source of controversy and some
countries, such as the United States, do not vaccinate the general
public. Diagnosis is commonly achieved using a skin test, which
involves intradermal exposure to tuberculin PPD (protein-purified
derivative). Antigen-specific T cell responses result in measurable
induration at the injection site by 48-72 hours after injection,
which indicates exposure to Mycobacterial antigens. Sensitivity and
specificity have, however, been a problem with this test, and
individuals vaccinated with BCG cannot be distinguished from
infected individuals.
[0005] While macrophages have been shown to act as the principal
effectors of M. tuberculosis immunity, T cells are the predominant
inducers of such immunity. The essential role of T cells in
protection against M. tuberculosis infection is illustrated by the
frequent occurrence of M. tuberculosis in AIDS patients, due to the
depletion of CD4 T cells associated with human immunodeficiency
virus (HIV) infection. Mycobacterium-reactive CD4 T cells have been
shown to be potent producers of gamma-interferon (IFN-.gamma.),
which, in turn, has been shown to trigger the anti-mycobacterial
effects of macrophages in mice. While the role of IFN-.gamma. in
humans is less clear, studies have shown that
1.25-dihydroxy-vitamin D3, either alone or in combination with
IFN-.gamma. or tumor necrosis factor-alpha, activates human
macrophages to inhibit M. tuberculosis infection. Furthermore, it
is known that IFN-.gamma. stimulates human macrophages to make
1,25-dihydroxy-vitamin D3. Similarly, 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
and Kaufmann, in Tuberculosis: Pathogenesis, Protection and
Control, Bloom (ed.), ASM Press. Washington, D.C. (1994).
[0006] Accordingly, there is a need in the art for improved
diagnostic methods for detecting tuberculosis, as well as for
vaccines and methods for preventing the infection. The present
invention fulfills this need and further provides other related
advantages.
SUMMARY OF THE INVENTION
[0007] Briefly stated, the present invention provides compounds and
methods for preventing and diagnosing tuberculosis.
[0008] In one embodiment, polypeptides are provided that comprise
an immunogenic portion of a Mycobacterium antigen, preferably a
Mycobacterium tuberculosis antigen, or a variant of such an antigen
that differs only in conservative substitutions and/or
modifications, wherein the antigen comprises an amino acid sequence
encoded by a polynucleotide having the nucleotide sequence recited
in SEQ ID NO: 145, 149, 150, 151, 152, 153, 154, 155, 156, 157,
158, 159, 160, 162, and 164, the complements of said sequences, or
a nucleotide sequence that hybridizes to the sequence set forth in
SEQ ID NO:145, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,
159, 160, 162, and 164, or an immunogenic fragment thereof. In a
second embodiment, the present invention provides polypeptides
comprising an immunogenic portion of a Mycobacterium antigen,
preferably a Mycobacterium tuberculosis antigen, having the amino
acid sequence described in SEQ ID NO:146, 161, or 163 or variants
or immunogenic fragments thereof.
[0009] In related aspects, nucleotide sequences encoding the above
polypeptides, recombinant expression vectors comprising these
nucleotide sequences and host cells transformed or transfected with
such expression vectors are also provided. In particular, the
present invention provides an isolated polynucleotide that
specifically hybridizes under moderately stringent conditions to a
second polynucleotide comprising a nucleotide sequence selected
from the group consisting of SEQ ID NO:145, 149, 150, 151, 152,
153, 154, 155, 156, 157, 158, 159, 160, 162, and 164. In some
embodiments, the isolated polynucleotide specifically hybridizes to
the second polynucleotide under highly stringent conditions.
[0010] In another aspect, the present invention provides fusion
proteins comprising a first polypeptide encoded by a polynucleotide
having the sequence set forth in SEQ ID NO: 145, 149, 150, 151,
152, 153, 154, 155, 156, 157, 158, 159, 160, 162, and 164, or a
fragment thereof, and a second polypeptide. In one embodiment, the
first and second polypeptides are heterologous. Alternatively, the
fusion proteins of the invention may comprise a first polypeptide
encoded by a polynucleotide having a sequence selected from the
group consisting of SEQ ID NO:145, 149, 150, 151, 152, 153, 154,
155, 156, 157, 158, 159, 160, 162, and 164, or an immunogenic
fragment thereof, and a known Mycobacterium antigen, preferably a
M. tuberculosis antigen.
[0011] In further aspects of the subject invention, methods and
diagnostic kits are provided for detecting Mycobacterium infection
in a patient. The methods comprise contacting a biological sample
with at least one of the above polypeptides and detecting in the
sample the presence of antibodies that bind to the polypeptide or
polypeptides, thereby detecting Mycobacterium infection in the
biological sample. In a preferred embodiment, the Mycobacterium
infection is a M. tuberculosis infection.
[0012] Suitable biological samples include whole blood, sputum,
serum, plasma, saliva, cerebrospinal fluid and urine. The
diagnostic kits comprise one or more of the above polypeptides in
combination with a detection reagent.
[0013] The present invention also provides methods for detecting
Mycobacterium infection, comprising obtaining a biological sample
from a patient, contacting the sample with at least one
oligonucleotide primer in a polymerase chain reaction, the
oligonucleotide primer being specific for a nucleotide sequence
encoding the above polypeptides, and detecting in the sample a
nucleotide sequence that amplifies in the presence of the first and
second oligonucleotide primers. In one embodiment, the
oligonucleotide primer comprises at least about 10 contiguous
nucleotides of such a nucleotide sequence. In a preferred
embodiment, the Mycobacterium infection is a M. tuberculosis
infection.
[0014] In a further aspect, the present invention provides a method
for detecting Mycobacterium infection in a patient, comprising
obtaining a biological sample from the patient, contacting the
sample with an oligonucleotide probe specific for a nucleotide
sequence encoding the above polypeptides, and detecting in the
sample a nucleotide sequence that hybridizes to the oligonucleotide
probe. In one embodiment, the oligonucleotide probe comprises at
least about 15 contiguous nucleotides of such a nucleotide
sequence. In a preferred embodiment, the Mycobacterium infection is
a M. tuberculosis infection.
[0015] In yet another aspect, methods are provided for detecting
Mycobacterium infection in a patient, such methods comprising the
steps of contacting a biological sample with a polypeptide, wherein
the polypeptide comprises an amino acid sequence encoded by a
polynucleotide having a nucleotide sequence selected from the group
consisting of SEQ ID NO:145, 149, 150, 151, 152, 153, 154, 155,
156, 157, 158, 159, 160, 162, and 164, the complements of said
sequences, or a nucleotide sequence that hybridizes to a sequence
selected from the group consisting of SEQ ID NO:145, 149, 150, 151,
152, 153, 154, 155, 156, 157, 158, 159, 160, 162, and 164, or an
immunogenic fragment thereof, and detecting in the sample the
presence of antibodies that bind to the polypeptide, thereby
detecting Mycobacterium infection in the biological sample. In a
preferred embodiment, the Mycobacterium infection is a M.
tuberculosis infection. Diagnostic kits for use in such methods are
also provided.
[0016] In another aspect, the present invention provides
antibodies, both polyclonal and monoclonal, that bind to the
polypeptides described above, as well as methods for their use in
the detection of Mycobacterium infection.
[0017] Within other aspects, the present invention provides
pharmaceutical compositions that comprise one or more of the above
polypeptides, or a polynucleotide encoding such polypeptides, and a
physiologically acceptable carrier or an adjuvant, e.g., SBAS-2,
QS-21, ENHANZYN (Detox), MPL, 3D-MPL, CWS, GM-CSF, SAF, ISCOMS,
MF-59, RC-529, AS2, AS2', AS2'', AS4, AS6, TDM, AGP, CPG, Leif,
saponin, and saponin mimetics, and derivatives thereof or mixtures
thereof. In another aspect, the present invention provides
pharmaceutical compositions that comprise one or more of the above
polypeptides, or a polynucleotide encoding such polypeptides, and
an adjuvant such as BCG. In another aspect the present invention
provides methods in which one or more of the above polypeptides, or
a polynucleotide encoding such polypeptides is administered to a
subject who has been exposed to BCG. The invention also provides
vaccines comprising one or more of the polypeptides as described
above and a non-specific immune response enhancer, together with
vaccines comprising one or more polynucleotides encoding such
polypeptides and a non-specific immune response enhancer.
[0018] In yet another aspect, methods are provided for inducing
protective immunity in a patient, comprising administering to a
patient an effective amount of one or more of the above
polypeptides.
[0019] In further aspects of this invention, methods and diagnostic
kits are provided for detecting tuberculosis in a patient. The
methods comprise contacting dermal cells of a patient with one or
more of the above polypeptides and detecting an immune response on
the patient's skin. The diagnostic kits comprise one or more of the
above polypeptides in combination with an apparatus sufficient to
contact the polypeptide(s) with the dermal cells of a patient.
[0020] In yet another aspect, methods are provided for detecting
tuberculosis in a patient, such methods comprising contacting
dermal cells of a patient with one or more polypeptides encoded by
a nucleotide sequence selected from the group consisting of SEQ ID
NO:145, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,
162, and 164, the complements of said sequences, or nucleotide
sequences that hybridize to a sequence selected from the group
consisting of SEQ ID NO:145, 149, 150, 151, 152, 153, 154, 155,
156, 157, 158, 159, 160, 162, and 164, and detecting an immune
response on the patient's skin. Diagnostic kits for use in such
methods are also provided.
[0021] In additional aspects of the invention, methods are provided
for inhibiting the development of a Mycobacterium infection in a
patient. In one embodiment, inhibiting the development of a
Mycobacterium infection comprises administering to a patient an
effective amount of a pharmaceutical composition or a vaccine of
the invention. In another embodiment, inhibiting the development of
a Mycobacterium infection in the patient comprises administering to
a patient an effective amount of an antibody of the invention. In a
preferred embodiment, the Mycobacterium infection is a M.
tuberculosis infection.
[0022] These and other aspects of the present invention will become
apparent upon reference to the following detailed description and
attached drawings. All references disclosed herein are hereby
incorporated by reference in their entirety as if each was
incorporated individually.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1A and 1B illustrate the stimulation of proliferation
and interferon-.gamma. production, respectively, in T cells derived
from a first PPD-positive donor (referred to as D7) by recombinant
ORF-2 and synthetic peptides to ORF-2.
[0024] FIGS. 2A and 2B illustrate the stimulation of proliferation
and interferon-.gamma. production, respectively, in T cells derived
from a second PPD-positive donor (referred to as D160) by
recombinant ORF-2 and synthetic peptides to ORF-2.
[0025] FIG. 3 shows the nucleotide sequence of mTTC#3 (SEQ ID
NO:145).
[0026] FIG. 4 shows the amino acid sequence of mTCC#3 (SEQ ID
NO:146).
[0027] FIG. 5 shows the 5' nucleotide sequence of P1 (SEQ ID NO:
149).
[0028] FIG. 6 shows the nucleotide sequence of P2 (SEQ ID NO:
150).
[0029] FIG. 7 shows the 3' nucleotide sequence of P3 (SEQ ID NO:
151).
[0030] FIG. 8 shows the nucleotide sequence of P4 (SEQ ID
NO:152).
[0031] FIG. 9 shows the nucleotide sequence of P6 (SEQ ID NO:
153)
[0032] FIG. 10 shows the nucleotide sequence of P7 (SEQ ID
NO:154)
[0033] FIG. 11 shows the nucleotide sequence of P8 (SEQ ID
NO:155)
[0034] FIG. 12 shows the nucleotide sequence of P9 (SEQ ID
NO:156)
[0035] FIG. 13 shows the 5' nucleotide sequence of P10 (SEQ ID
NO:157)
[0036] FIG. 14 shows the 5' nucleotide sequence of P11 (SEQ ID NO:
158)
[0037] FIG. 15 shows the 3' nucleotide sequence of P12 (SEQ ID
NO:159)
[0038] FIG. 16 shows the full length nucleotide and amino acid
sequence of MO-1 (SEQ ID NO:160 (nucleotide) and SEQ ID NO: 161
(amino acid).
[0039] FIG. 17 shows the full length nucleotide and amino acid
sequence of MO-2 (SEQ ID NO:162 (nucleotide) and SEQ ID NO: 163
(amino acid).
[0040] FIG. 18 shows the full length nucleotide sequence of
TbH4/XP-1 (MTB48) (SEQ ID NO:164).
[0041] SEQ ID NO:1 is the cDNA sequence of Tb224 [0042] SEQ ID NO:2
is the cDNA sequence of Tb636 [0043] SEQ ID NO:3 is the cDNA
sequence of Tb424 [0044] SEQ ID NO:4 is the cDNA sequence of Tb436
[0045] SEQ ID NO:5 is the cDNA sequence of Tb398 [0046] SEQ ID NO:6
is the cDNA sequence of Tb508 [0047] SEQ ID NO:7 is the cDNA
sequence of Tb441 [0048] SEQ ID NO:8 is the cDNA sequence of Tb475
[0049] SEQ ID NO:9 is the cDNA sequence of Tb488 [0050] SEQ ID NO:
10 is the cDNA sequence of Tb465 [0051] SEQ ID NO:11 is the cDNA
sequence of Tb431 [0052] SEQ ID NO:12 is the cDNA sequence of Tb472
[0053] SEQ ID NO: 13 is the predicted amino acid sequence of Tb224
[0054] SEQ ID NO: 14 is the predicted amino acid sequence of Tb636
[0055] SEQ ID NO:15 is the predicted amino acid sequence of Tb431
[0056] SEQ ID NO:16 is the amino acid sequence of Tb424 ORF-1
[0057] SEQ ID NO: 17 is the amino acid sequence of Tb424 ORF-2
[0058] SEQ ID NO:18 is the amino acid sequence of Tb436 ORF-1
[0059] SEQ ID NO:19 is the amino acid sequence of Tb436 ORF-2
[0060] SEQ ID NO:20 is the amino acid sequence of Tb398 ORF-1
[0061] SEQ ID NO:21 is the amino acid sequence of Tb398 ORF-2
[0062] SEQ ID NO:22 is the amino acid sequence of Tb508 ORF-1
[0063] SEQ ID NO:23 is the amino acid sequence of Tb508 ORF-2
[0064] SEQ ID NO:24 is the amino acid sequence of Tb441 ORF-1
[0065] SEQ ID NO:25 is the amino acid sequence of Tb441 ORF-2
[0066] SEQ ID NO:26 is the amino acid sequence of Tb475 ORF-1
[0067] SEQ ID NO:27 is the amino acid sequence of Tb475 ORF-2
[0068] SEQ ID NO:28 is the amino acid sequence of Tb488 ORF-1
[0069] SEQ ID NO:29 is the amino acid sequence of Tb488 ORF-2
[0070] SEQ ID NO:30 is the amino acid sequence of Tb465 ORF-1
[0071] SEQ ID NO:31 is the amino acid sequence of Tb465 ORF-2
[0072] SEQ ID NO:32 is the amino acid sequence of Tb424 ORF-U
[0073] SEQ ID NO:33 is the amino acid sequence of Tb436 ORF-U
[0074] SEQ ID NO:34 is the amino acid sequence of ORF-1-1 [0075]
SEQ ID NO:35 is the amino acid sequence of ORF-1-2 [0076] SEQ ID
NO:36 is the amino acid sequence of ORF-1-3 [0077] SEQ ID NO:37 is
the amino acid sequence of ORF-1-4 [0078] SEQ ID NO:38 is the amino
acid sequence of ORF-1-5 [0079] SEQ ID NO:39 is the amino acid
sequence of ORF-1-6 [0080] SEQ ID NO:40 is the amino acid sequence
of ORF-1-7 [0081] SEQ ID NO:41 is the amino acid sequence of
ORF-1-8 [0082] SEQ ID NO:42 is the amino acid sequence of ORF-1-9
[0083] SEQ ID NO:43 is the amino acid sequence of ORF-1-10 [0084]
SEQ ID NO:44 is the amino acid sequence of ORF-1-11 [0085] SEQ ID
NO:45 is the amino acid sequence of ORF-1-12 [0086] SEQ ID NO:46 is
the amino acid sequence of ORF-1-13 [0087] SEQ ID NO:47 is the
amino acid sequence of ORF-1-14 [0088] SEQ ID NO:48 is the amino
acid sequence of ORF-1-15 [0089] SEQ ID NO:49 is the amino acid
sequence of ORF-1-16 [0090] SEQ ID NO:50 is the amino acid sequence
of ORF-1-17 [0091] SEQ ID NO:51 is the amino acid sequence of
ORF-2-1 [0092] SEQ ID NO:52 is the amino acid sequence of ORF-2-2
[0093] SEQ ID NO:53 is the amino acid sequence of ORF-2-3 [0094]
SEQ ID NO:54 is the amino acid sequence of ORF-2-4 [0095] SEQ ID
NO:55 is the amino acid sequence of ORF-2-5 [0096] SEQ ID NO:56 is
the amino acid sequence of ORF-2-6 [0097] SEQ ID NO:57 is the amino
acid sequence of ORF-2-7 [0098] SEQ ID NO:58 is the amino acid
sequence of ORF-2-8 [0099] SEQ ID NO:59 is the amino acid sequence
of ORF-2-9 [0100] SEQ ID NO:60 is the amino acid sequence of
ORF-2-10 [0101] SEQ ID NO:61 is the amino acid sequence of ORF-2-11
[0102] SEQ ID NO:62 is the amino acid sequence of ORF-2-12 [0103]
SEQ ID NO:63 is the amino acid sequence of ORF-2-13 [0104] SEQ ID
NO:64 is the amino acid sequence of ORF-2-14 [0105] SEQ ID NO:65 is
the amino acid sequence of ORF-2-15 [0106] SEQ ID NO:66 is the
amino acid sequence of ORF-2-16 [0107] SEQ ID NO:67 is the amino
acid sequence of ORF-2-17 [0108] SEQ ID NO:68 is the amino acid
sequence of ORF-2-18 [0109] SEQ ID NO:69 is the amino acid sequence
of ORF-2-19 [0110] SEQ ID NO:70 is the amino acid sequence of
ORF-2-20 [0111] SEQ ID NO:71 is the amino acid sequence of ORF-2-21
[0112] SEQ ID NO:72 is the amino acid sequence of ORF-2-22 [0113]
SEQ ID NO:73 is the amino acid sequence of ORF-2-23 [0114] SEQ ID
NO:74 is the amino acid sequence of ORF-2-24 [0115] SEQ ID NO:75 is
the amino acid sequence of ORF-2-25 [0116] SEQ ID NO:76 is the
amino acid sequence of ORF-2-26 [0117] SEQ ID NO:77 is the amino
acid sequence of ORF-2-27 [0118] SEQ ID NO:78 is the amino acid
sequence of ORF-2-28 [0119] SEQ ID NO:79 is the amino acid sequence
of ORF-2-29 [0120] SEQ ID NO:80 is the amino acid sequence of
ORF-2-30 [0121] SEQ ID NO:81-82 are the amino acid sequence of two
overlapping peptides to the open reading frame of Tb224 [0122] SEQ
ID NO:83 is the full-length cDNA sequence of Tb431 (which contains
an ORF encoding Mtb-40) [0123] SEQ ID NO:84 is the amino acid
sequence of MSF-1 [0124] SEQ ID NO:85 is the amino acid sequence of
MSF-2 [0125] SEQ ID NO:86 is the amino acid sequence of MSF-3
[0126] SEQ ID NO:87 is the amino acid sequence of MSF-4 [0127] SEQ
ID NO:88 is the amino acid sequence of MSF-5 [0128] SEQ ID NO:89 is
the amino acid sequence of MSF-6 [0129] SEQ ID NO:90 is the amino
acid sequence of MSF-7 [0130] SEQ ID NO:91 is the amino acid
sequence of MSF-8 [0131] SEQ ID NO:92 is the amino acid sequence of
MSF-9 [0132] SEQ ID NO:93 is the amino acid sequence of MSF-10
[0133] SEQ ID NO:94 is the amino acid sequence of MSF-1 [0134] SEQ
ID NO:95 is the amino acid sequence of MSF-12 [0135] SEQ ID NO:96
is the amino acid sequence of MSF-13 [0136] SEQ ID NO:97 is the
amino acid sequence of MSF-14 [0137] SEQ ID NO:98 is the amino acid
sequence of MSF-15 [0138] SEQ ID NO:99 is the amino acid sequence
of MSF-16 [0139] SEQ ID NO:100 is the amino acid sequence of MSF-17
[0140] SEQ ID NO: 101 is the amino acid sequence of MSF-18 [0141]
SEQ ID NO:102 is the cDNA sequence of Tb867 [0142] SEQ ID NO:103 is
the cDNA sequence of Tb391 [0143] SEQ ID NO:104 is the cDNA
sequence of Tb470 [0144] SEQ ID NO:105 is the cDNA sequence of
Tb838 [0145] SEQ ID NO:106-107 are the cDNA sequences of Tb962
[0146] SEQ ID NO:108 is the full-length cDNA sequence of Tb472
[0147] SEQ ID NO:109 is the predicted amino acid sequence of the
protein encoded by Tb472 (referred to as MSL) [0148] SEQ ID NO: 110
is the amino acid sequence of MSL-1 [0149] SEQ ID NO: 111 is the
amino acid sequence of MSL-2 [0150] SEQ ID NO: 112 is the amino
acid sequence of MSL-3 [0151] SEQ ID NO:113 is the amino acid
sequence of MSL-4 [0152] SEQ ID NO:114 is the amino acid sequence
of MSL-5 [0153] SEQ ID NO:115 is the amino acid sequence of MSL-6
[0154] SEQ ID NO: 116 is the amino acid sequence of MSL-7 [0155]
SEQ ID NO:117 is the amino acid sequence of MSL-8 [0156] SEQ ID
NO:118 is the amino acid sequence of MSL-9 [0157] SEQ ID NO:119 is
the amino acid sequence of MSL-10 [0158] SEQ ID NO:120 is the amino
acid sequence of MSL-11 [0159] SEQ ID NO:121 is the amino acid
sequence of MSL-12 [0160] SEQ ID NO:122 is the amino acid sequence
of MSL-13 [0161] SEQ ID NO: 123 is the amino acid sequence of
MSL-14 [0162] SEQ ID NO: 124 is the amino acid sequence of MSL-15
[0163] SEQ ID NO:125 is the DNA sequence of the full-length open
reading frame of Tb470 (which encodes Mtb-40) [0164] SEQ ID NO:126
is the determined amino acid sequence of Mtb-40 [0165] SEQ ID
NO:127 is the cDNA sequence of Tb366 [0166] SEQ ID NO:128 is the
cDNA sequence of Tb433 [0167] SEQ ID NO:129 is the cDNA sequence of
Tb439 [0168] SEQ ID NO: 130-131 are the cDNA sequences of Tb372
[0169] SEQ ID NO: 132 is the cDNA sequence of Tb390R5C6 [0170] SEQ
ID NO: 133-134 are the cDNA sequences of Tb390R2C11 [0171] SEQ ID
NO:135 is the 5' cDNA sequence of Y1-26C1 [0172] SEQ ID NO:136 is
the 5' cDNA sequence of Y1-86C11 [0173] SEQ ID NO: 137 is the
full-length cDNA sequence of hTcc#1 [0174] SEQ ID NO:138 is the
predicted amino acid sequence of hTcc#1 [0175] SEQ ID NO:139 is the
cDNA sequence of mTCC#1 [0176] SEQ ID NO: 140 is the cDNA sequence
of mTCC#2 [0177] SEQ ID NO: 141 is the predicted amino acid
sequence of mTCC#1 [0178] SEQ ID NO: 142 is the predicted amino
acid sequence of mTCC#2 [0179] SEQ ID NO: 143 is the amino acid
sequence of MTb9.8 [0180] SEQ ID NO:144 is the amino acid sequence
of Tb#470 [0181] SEQ ID NO: 145 is the full length nucleotide
sequence of mTTC#3 [0182] SEQ ID NO:146 is the predicted amino acid
sequence of mTTC#3 [0183] SEQ ID NO:147 and 148 are the sequences
of primers used to amplify the full-length coding sequence of
mTTC#3 [0184] SEQ ID NO:149 is the 5' nucleotide sequence of P1
[0185] SEQ ID NO:150 is the nucleotide sequence of P2 [0186] SEQ ID
NO:151 is the 3' nucleotide sequence of P3 [0187] SEQ ID NO:152 is
the nucleotide sequence of P4 [0188] SEQ ID NO:153 is the
nucleotide sequence of P6 [0189] SEQ ID NO: 154 is the nucleotide
sequence of P7 [0190] SEQ ID NO:155 is the nucleotide sequence of
P8 [0191] SEQ ID NO:156 is the nucleotide sequence of P9 [0192] SEQ
ID NO:157 is the 5' nucleotide sequence of P10 [0193] SEQ ID NO:158
is the 5' nucleotide sequence of P11 [0194] SEQ ID NO:159 is the 3'
nucleotide sequence of P12 [0195] SEQ ID NO:160 is the full length
nucleotide sequence of MO-1 [0196] SEQ ID NO:161 is the full length
amino acid sequence of MO-1. [0197] SEQ ID NO:162 is the full
length nucleotide sequence of MO-2 [0198] SEQ ID NO:163 is the full
length amino acid sequence of MO-2 [0199] SEQ ID NO:164 is the full
length nucleotide sequence of TbH4/XP-1 (MTB48).
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
I. Introduction
[0200] As noted above, the present invention is generally directed
to compositions and methods for preventing, treating and diagnosing
tuberculosis. In particular, the present invention relates to
Mycobacterium antigens, optionally from a species such as M.
tuberculosis, M. bovis, M. smegmatis, BCG, M. leprae, M.
scrofulaceum, M. avium-intracellulare, M. marinum, M. ulcerans, M.
kansasii, M. xenopi M. szulgai, M. fortuium, or M. chelonei. In
particular, the invention relates to Mycobacterium polypeptides and
immunogenic fragments thereof, polynucleotides that encode the
polypeptides and immunogenic fragments thereof, and methods of
using such compositions in the treatment, prevention and diagnosis
of Mycobacterium infection. In one embodiment of the invention, the
polypeptides of the invention are used to diagnose tuberculosis. In
another embodiment of the invention, the polypeptides of the
invention are used to induce an immune response in a patient in
order to prevent Mycobacterium infection, and in particular
tuberculosis, or to reduce the probability of pathological
responses typical of Mycobacterium infection, and in particular
tuberculosis, in a patient. In another embodiment of the invention,
the polynucleotides of the invention are used to produce DNA
vaccines, or for diagnostic purposes.
II. Definitions
[0201] "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). The term also
encompasses ribonucleotides including 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.
[0202] 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. 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 native
polypeptide. The effect on the immunogenicity of the encoded
polypeptide may generally be assessed as described herein. The term
"variants" also encompasses interspecies homologs. 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.
[0203] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms 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 polymers. As used herein, the terms encompass amino acid
chains of any length, including full length proteins (i.e.,
antigens), wherein the amino acid residues are linked by covalent
peptide bonds. Thus, for instance, a polypeptide comprising an
immunogenic portion of an antigen may consist entirely of the
immunogenic portion, or may contain additional sequences. The
additional sequences may be derived from the native Mycobacterium
antigen or may be heterologous, and such sequences may (but need
not) be immunogenic.
[0204] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function hi 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, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs 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.
[0205] 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.
[0206] The compositions and methods of this invention also
encompass variants of the above polypeptides. A polypeptide
"variant," as used herein, is a polypeptide that differs from the
recited polypeptide only in conservative substitutions and/or
modifications, such that the therapeutic and/or immunogenic
properties of the polypeptide are retained. Polypeptide variants
preferably exhibit at least about 70%, more preferably at least
about 90% and most preferably at least about 95% identity to the
identified polypeptides. For polypeptides with immunoreactive
properties, variants may, alternatively, be identified by modifying
the amino acid sequence of one of the above polypeptides, and
evaluating the immunoreactivity of the modified polypeptide. For
polypeptides useful for the generation of diagnostic binding
agents, a variant may be identified by evaluating a modified
polypeptide for the ability to generate antibodies that detect the
presence or absence of Mycobacterium infection, and in particular
tuberculosis. Alternatively, variants of the claimed antigens that
may be usefully employed in the inventive diagnostic methods may be
identified by evaluating modified polypeptides for their ability to
detect antibodies present in the sera of Mycobacterium-infected
patients. Such modified sequences may be prepared and tested using,
for example, the representative procedures described herein.
[0207] A "conservative substitution" applies to both amino acid and
nucleic acid sequences. With respect to particular nucleic acid
sequences, conservative substitutions refers to changes in the
nucleic acid sequence that result in nucleic acids encoding
identical or essentially identical amino acid sequences, or where
the nucleic acid does not encode an amino acid sequence, to
essentially identical sequences. Because of 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 are
"silent variations," 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 recognize that
each codon in a nucleic acid (except AUG, which is ordinarily the
only codon for methionine, and TOG, 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 which encodes a polypeptide is implicit in each described
sequence.
[0208] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservative substitution" where the
alteration results in the substitution of an amino acid with a
chemically similar amino acid and where the alteration has minimal
influence on the immunogenic properties, secondary structure and
hydropathic nature of the polypeptide. 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.
[0209] The following eight groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Glycine (G);
[0210] 2) Aspartic acid (D), Glutamic acid (B);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
[0211] (see, e.g., Creighton, Proteins (1984)).
[0212] "Immunogenic," as used herein, refers to the ability to
elicit an immune response (e.g., cellular or humoral) in a patient,
such as a human, and/or in a biological sample (in vitro). In
particular, antigens that are immunogenic (and immunogenic portions
or other variants of such antigens) are recognized by a B-cell
and/or a T-cell surface antigen receptor. Antigens that are
immunogenic (and immunogenic portions or other variants of such
antigens) are capable of stimulating cell proliferation,
interleukin-12 production and/or interferon-.gamma. production in
biological samples comprising one or more cells selected from the
group of T cells, NK cells, B cells and macrophages, where the
cells are derived from an Mycobacterium-immune individual.
Polypeptides comprising at least an immunogenic portion of one or
more Mycobacterium antigens may generally be used to detect
tuberculosis or to induce protective immunity against tuberculosis
in a patient.
[0213] "Fusion polypeptide" or "fusion protein" refers to a protein
having at least two heterologous polypeptides covalently linked,
preferably Mycobacterium sp. polypeptides, 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,
subsequences, 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). The complete
sequence of the Mycobacterium tuberculosis genome can be found at
http://www.sanger.ac.uk and at
http://www.pasteur.fr/mycdb/(MycDB).
[0214] 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-.gamma., IL-2, and IL-12) tend to favor 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 and TNF-.beta.)
tend to favor the induction of humoral immune responses.
[0215] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0216] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
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.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For stringent hybridization, a positive signal is at
least two times background, preferably 10 times background
hybridization. Exemplary stringent hybridization conditions
include: 50% formamide, 5.times.SSC and 1% SDS incubated at
42.degree. C. or 5.times.SSC and 1% SDS incubated at 65.degree. C.,
with a wash in 0.2.times.SSC and 0.1% SDS at 65.degree. C.
[0217] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides that 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
cased, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately"
stringent hybridization conditions include a hybridization in a
buffer of 40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a
wash in 1.times.SSC at 45.degree. C. A positive hybridization is at
least twice background. Those of ordinary skill will readily
recognize that alternative hybridization and wash conditions can be
utilized to provide conditions of similar stringency.
[0218] 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 subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 55%, 60%, 65%, 70%, 75%, or 80% identity,
preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity
over a specified window 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 complement of a test sequence.
Preferably, the identity exists over a region that is at least
about 25 amino acids or nucleotides in length, or more preferably
over a region that is 50-100 amino acids or nucleotides in
length.
[0219] 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 than calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0220] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 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.
[0221] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, A model of
evolutionary change in proteins--Matrices for detecting distant
relationships, In: Dayhoff (ed.) Atlas of Protein Sequence and
Structure, National Biomedical Research Foundation, Washington D.C.
Vol. 5, Suppl. 3, pp. 345-358 (1978); Hein, Unified Approach to
Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol.
183, Academic Press, Inc., San Diego, Calif. (1990); Higgins and
Sharp, CABIOS 5:151-153 (1989); Myers and Muller, CABIOS 4:11-17
(1988); Robinson, Comb. Theor 11:105 (1971); Santou and Nes, Mol.
Biol. Evol. 4:406-425 (1987); Sneath and Sokal, Numerical
Taxonomy--the Principles and Practice of Numerical Taxonomy,
Freeman Press, San Francisco, Calif. (1973); Wilbur and Lipman,
Proc. Natl. Acad. Sci. USA 80:726-730 (1983).
[0222] Alternatively, optimal alignment of sequences for comparison
can be conducted, e.g., by the local homology algorithm of Smith
and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology
alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443
(1970), by the search for similarity method of Pearson and Lipman,
Proc. Natl. 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)).
[0223] A preferred 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 Altachul
et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altachul et al., J.
Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0
are used, with the parameters described herein, to determine
percent sequence identity for the nucleic acids and proteins of the
invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
(http://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) of 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 and 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.
[0224] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul, Proc. Natl. 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.
[0225] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
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, Ig, IgM, IgA, IgD and IgB,
respectively.
[0226] 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
kD) and one "heavy" chain (about 50-70 kD). 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 (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0227] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized 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)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 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)'.sub.2 dimer into an Fab'
monomer. The Fab' monomer is essentially Fab with part of the hinge
region (see, e.g., 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 synthesized 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))
[0228] As used herein, an antibody, or antigen-binding fragment
thereof is said to "specifically bind" to a polypeptide of interest
if it reacts at a detectable level (within, for example, an ELISA)
with the polypeptide of interest, and does not react detectably
with unrelated proteins under similar conditions. As used herein,
"binding" refers to a noncovalent association between two separate
molecules such that a complex is formed. The ability to bind may be
evaluated by, for example, determining a binding constant for the
formation of the complex. The binding constant is the value
obtained when the concentration of the complex is divided by the
product of the component concentrations. In general, two compounds
are said to "bind," in the context of the present invention, when
the binding constant for complex formation exceeds about 10.sup.3
l/mol. The binding constant may be determined using methods well
known in the art.
[0229] As used herein, a "biological sample" is any
antibody-containing sample obtained from a patient. Preferably, the
sample is whole blood, sputum, serum, plasma, saliva, cerebrospinal
fluid or urine. More preferably, the sample is a blood, serum or
plasma sample obtained from a patient or a blood supply.
[0230] In the context of the present invention, a "patient" refers
to any warm-blooded animal, preferably a human. A patient may be
afflicted with a disease, or may be free of detectable disease
and/or infection.
III. Preparation of Mycobacterium Polypeptides and Nucleic
Acids
[0231] In general, Mycobacterium antigens and DNA sequences
encoding such antigens may be prepared using any of a variety of
procedures. Here and throughout the specification, the
Mycobacterium antigens are preferably M. tuberculosis antigens.
[0232] A. Polynucleotides of the Invention
[0233] DNA sequences encoding antigens may be identified, for
example, by screening an appropriate Mycobacterium genomic or cDNA
expression library with sera obtained from patients infected with
Mycobacterium. Alternatively, sera from mice immunized with
Mycobacterium antigens can be used. In some embodiments, sera is
obtained from mice immunized with blood or urine from syngencic
mice infected with Mycobacterium. Such screens may generally be
performed using techniques well known to those of ordinary skill in
the art, such as those described in Sambrook et al., Molecular
Cloning. A Laboratory Manual, Cold Spring Harbor Laboratories, Cold
Spring Harbor, N.Y. (1989).
[0234] DNA sequences encoding the antigens of the present invention
may also be obtained by screening an appropriate Mycobacterium cDNA
or genomic DNA library for DNA sequences that hybridize to
degenerate oligonucleotides derived from partial amino acid
sequences of isolated antigens. Degenerate oligonucleotide
sequences for use in such a screen may be designed and synthesized,
and the screen may be performed as described, for example, in
Sambrook et al., supra, and references cited therein.
[0235] For hybridization techniques, a partial sequence may be
labeled (e.g., by nick-translation or end-labeling with .sup.32P)
using well known techniques. A bacterial or bacteriophage library
is then screened by hybridizing filters containing denatured
bacterial colonies (or lawns containing phage plaques) with the
labeled probe (see Sambrook et al., supra). Hybridizing 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 are then
assembled into a single contiguous sequence. A full length cDNA
molecule can be generated by ligating suitable fragments, using
well known techniques.
[0236] Amplification techniques may also be employed, using the
above oligonucleotides in methods well known in the art, to isolate
a nucleic acid probe from a cDNA or genomic library. The library
screen for obtaining a full length coding sequence from a partial
cDNA sequence may then be performed using the isolated probe.
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.degree.
C. to 72.degree. C. The amplified region may be sequenced and
overlapping sequences assembled into a contiguous sequence.
[0237] 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 circularized 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 hybridizes to a polyA region or vector
sequence, to identify sequences that are 5' and 3' of a known
sequence. Optionally, capture PCR (Lagestrom et al., PCR Methods
Applic. 1:111-119 (1991)) and walking PCR (Parker et al, Nucl.
Acids. Res. 19:3055-60 (1991)) can also be used. Methods for
amplification further include the ligase chain reaction (LCR; see,
e.g., EP patent application publication 320, 308), the Qbeta
Replicase method (see, e.g., PCT/US87/00880), the isothermal
amplification method, the Strand Displacement Amplification (SDA),
the cyclic probe reaction (CPR), the transcription-based
amplification systems (TAS; see, e.g., PCT/US88/10315), as well as
other methods known to those of skill in the art (see, e.g., GB
patent application No. 2,202,328; PCT/US89/01025; and EP patent
application publication No. 329,822). Other methods employing
amplification may also be employed to obtain a full length cDNA
sequence.
[0238] 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.
[0239] Polynucleotide variants may generally be prepared by any
method known in the art, including chemical synthesis by, for
example, solid phase phosphoramidite chemical synthesis.
Modifications in a polynucleotide sequence may also be introduced
using standard mutagenesis techniques, such as
oligonucleotide-directed site-specific mutagenesis (see Adelman et
al., DNA 2:183 (1983)). Alternatively, RNA molecules may be
generated by in vitro or in vivo transcription of DNA sequences
encoding a Mycobacterium polypeptide, or portion thereof, provided
that the DNA is incorporated into a vector with a suitable RNA
polymerase promoter (such as T7 or SP6). Certain portions may be
used to prepare an encoded polypeptide, as described infra. In
addition, or alternatively, a portion may be administered to a
patient such that the encoded polypeptide is generated in vivo
(e.g., by transfecting antigen-presenting cells, such as dendritic
cells, with a cDNA construct encoding a Mycobacterium polypeptide,
and administering the transfected cells to the patient).
[0240] A portion of a sequence complementary to a coding sequence
(i.e., an antisense polynucleotide) may also be used as a probe or
to modulate gene expression. cDNA constructs that can be
transcribed into antisense RNA may also be introduced into cells or
tissues to facilitate the production of antisense RNA. An antisense
polynucleotide may be used, as described herein, to inhibit
expression of a Mycobacterium protein. Antisense technology can be
used to control gene expression through triple-helix formation,
which compromises the ability of the double helix to open
sufficiently for the binding of polymerases, transcription factors
or regulatory molecules (see Gee et al., In Huber and Carr,
Molecular and Immunologic Approaches, Futura Publishing Co., Mt.
Kisco, N.Y. (1994)). Alternatively, an antisense molecule may be
designed to hybridize with a control region of a gene (e.g.,
promoter, enhancer or transcription initiation site), and block
transcription of the gene; or to block translation by inhibiting
binding of a transcript to ribosomes.
[0241] A portion of a coding sequence or of a complementary
sequence may also be designed as a probe or primer to detect gene
expression. Probes may be labeled with a variety of reporter
groups, such as radionuclides and enzymes, and are preferably at
least 10 nucleotides in length, more preferably at least 20
nucleotides in length and still more preferably at least 30
nucleotides in length. Primers, as noted above, are preferably
22-30 nucleotides in length.
[0242] Any polynucleotide may be further modified to increase
stability in vivo. Possible modifications include, but are not
limited to, the addition of flanking sequences at the 5' and/or 3'
ends; the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages in the backbone; and/or the inclusion of
nontraditional bases such as inosine, queosine and wybutosine, as
well as acetyl-methyl-, thio- and other modified forms of adenine,
cytidine, guanine, thymine and uridine.
[0243] Nucleotide sequences as described herein may be joined to a
variety of other nucleotide sequences using established recombinant
DNA techniques. For example, a polynucleotide may be cloned into
any of a variety of cloning vectors, including plasmids, phagemids,
lambda phage derivatives and cosmids. Vectors of particular
interest include expression vectors, replication vectors, probe
generation vectors and sequencing vectors. In general, a vector
will contain an origin of replication functional in at least one
organism, convenient restriction endonuclease sites and one or more
selectable markers. Other elements will depend upon the desired
use, and will be apparent to those of ordinary skill in the
art.
[0244] Within certain embodiments, polynucleotides may be
formulated so as to permit entry into a cell of a mammal, and
expression therein. Such formulations are particularly useful for
therapeutic purposes, as described infra. Those of ordinary skill
in the art will appreciate that there are many ways to achieve
expression of a polynucleotide in a target cell, and any suitable
method may be employed. For example, a polynucleotide may be
incorporated into a viral vector such as, but not limited to,
adenovirus, adeno-associated virus, retrovirus, or vaccinia or
other pox virus (e.g., avian pox virus). The polynucleotides may
also be administered as naked plasmid vectors. Techniques for
incorporating DNA into such vectors are well known to those of
ordinary skill in the art. A retroviral vector may additionally
transfer or incorporate a gene for a selectable marker (to aid in
the identification or selection of transduced cells) and/or a
targeting moiety, such as a gene that encodes a ligand for a
receptor on a specific target cell, to render the vector target
specific. Targeting may also be accomplished using an antibody, by
methods known to those of ordinary skill in the art.
[0245] Other formulations for therapeutic purposes include
colloidal dispersion systems, such as macromolecule complexes,
nanocapsules, microspheres, beads, and lipid-based systems
including oil-in-water emulsions, micelles, mixed micelles, and
liposomes. A preferred colloidal system for use as a delivery
vehicle in vitro and in vivo is a liposome (i.e., an artificial
membrane vesicle). The preparation and use of such systems is well
known in the art.
[0246] B. Polypeptides of the invention
[0247] Within the context of the present invention, polypeptides
may comprise at least an immunogenic portion of a Mycobacterium
antigen, or a variant thereof, as described herein. As noted above,
a Mycobacterium antigen is a protein that is expressed by cells
infected with Mycobacterium. In a preferred embodiment the
Mycobacterium antigen is a Mycobacterium tuberculosis antigen.
Proteins that are Mycobacterium antigens also react detectably
within an immunoassay (such as an ELISA) with antisera from a
patient infected with Mycobacterium, and preferably with M.
tuberculosis. Polypeptides as described herein may be of any
length. Additional sequences derived from the native protein and/or
heterologous sequences may be present, and such sequences may (but
need not) possess further immunogenic or antigenic properties.
[0248] Genomic or cDNA libraries derived from Mycobacterium, and
preferably from M. tuberculosis, may be screened directly using
peripheral blood mononuclear cells (PBMCs) or T cell lines or
clones derived from one or more Mycobacterium-immune individuals.
In a preferred embodiment, the Mycobacterium-immune individuals are
M. tuberculosis-immune individuals. Direct library screens may
generally be performed by assaying pools of expressed recombinant
proteins for the ability to induce proliferation and/or
interferon-.gamma. production in T cells derived from a
Mycobacterium-immune individual. Potential T cell antigens may be
first selected based on antibody reactivity, as described above.
Purified antigens are then evaluated for their ability to elicit an
appropriate immune response (e.g., cellular) using, for example,
the representative methods described infra. Immunogenic antigens
may then be partially sequenced using techniques such as
traditional Edman chemistry (see Edman and Berg, Eur. J. Biochem.
80:116-132 (1967)).
[0249] Immunogenic antigens may also be produced recombinantly
using a DNA sequence that encodes the antigen, which has been
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, and expressed in an
appropriate host. 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., supra; and Ausubel et al., supra.
[0250] Polypeptides may comprise a signal (or leader) sequence at
the N-terminal end of the protein, which co-translationally or
post-translationally directs transfer of the protein. The
polypeptide may also be conjugated to a linker or other sequence
for ease of synthesis, purification or identification of the
polypeptide (e.g., poly-His), or to enhance binding of the
polypeptide to a solid support. For example, a polypeptide may be
conjugated to an immunoglobulin Fe region.
[0251] Portions and other variants of Mycobacterium antigens may be
generated by synthetic or recombinant means. Synthetic polypeptides
having fewer than about 100 amino acids, and generally fewer than
about 50 amino acids, may be generated using techniques well known
in the art. For example, such polypeptides may be synthesized using
any of the commercially available solid-phase techniques, such as
the Merrifield solid-phase synthesis method, where amino acids are
sequentially added to a growing amino acid chain (see Merrifield,
J. Am. Chem. Soc. 85:2149-2146 (1963)). Equipment. for automated
synthesis of polypeptides is commercially available from suppliers
such as Perkin Elmer/Applied BioSystems Division, Inc., Foster
City, Calif., and may be operated according to the manufacturer's
instructions. Variants of a native antigen may generally be
prepared using standard mutagenesis techniques, such as
oligonucleotide-directed site-specific mutagenesis. Sections of the
DNA sequence may also be removed using standard techniques to
permit preparation of truncated polypeptides.
[0252] Recombinant polypeptides containing portions and/or variants
of a native antigen may be readily prepared from a DNA sequence
encoding the polypeptide using a variety of techniques well known
to those of ordinary skill in the art. For example, supernatants
from suitable host/vector systems which secrete recombinant protein
into culture media may be first concentrated using a commercially
available filter. Following concentration, the concentrate may be
applied to a suitable purification matrix such as an affinity
matrix or an ion exchange resin. Finally, one or more reverse phase
HPLC steps can be employed to further purify a recombinant
protein.
[0253] Any of a variety of expression vectors known to those of
ordinary skill in the art may be employed to express recombinant
polypeptides of the present invention. Expression may be achieved
in any appropriate host cell (e.g., prokaryotic, yeast and higher
eukaryotic cell) that has been transformed or transfected with an
expression vector containing a DNA molecule that encodes a
recombinant polypeptide. Suitable expression vector/host systems
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. Examples of expression
vectors for use in bacterial systems include, e.g., multifunctional
E. coli cloning and expression vectors such as BLUESCRIPT
(Stratagene) and pIN vectors (see Van Heeke and Schuster, J. Biol.
Chem. 264:5503-5509 (1989)). 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 (see, e.g., Ausubel et al., supra; and Grant et al.,
Methods Enzymol. 153:516-544 (1987)). In cases where plant
expression vectors are used, the expression of sequences encoding
polypeptides may be driven by any of a number of promoters,
including, but not limited to, the 35S and 19S promoters of CaMV,
the omega leader sequence from TMV (Takamatsu, EMBO J. 6:307-311
(1987)), as well as plant promoters such as the small subunit of
RUBISCO or heat-shock promoters (Coruzzi et al., EMBO J.
3:1671-1680 (1984); Broglie at al., Science 224:838-843 (1984); and
Winter et al., Results Probl. Cell Differ. 17:85-105 (1991)). A
variety of expression vectors are also available for expression in
insect systems. For example, suitable vectors for expression in
Spodoptera frugiperda cells or in Trichoplusia include, but are not
limited to the Autographa californica nuclear polyhedrosis virus
(AcNPV). Furthermore, viral-based expression systems can also be
used to express the polypeptide(s) of interest in mammalian host
cells. Preferably, the host cells employed are E. coli, yeast or
mammalian cell lines, such as COS or CHO. The DNA sequences
expressed in this manner may encode naturally occurring antigens,
portions of naturally occurring antigens, or other variants
thereof.
[0254] In general, regardless of the method of preparation, the
polypeptides disclosed herein are prepared in substantially pure
form. Preferably, the polypeptides are at least about 80% pure,
more preferably at least about 90% pure and most preferably at
least about 99% pure. For use in the methods described herein,
however, such substantially pure polypeptides may be combined.
[0255] In one embodiment, the subject invention discloses
polypeptides comprising at least an immunogenic portion of a M.
tuberculosis antigen (or a variant of such an antigen) that
comprises the amino acid sequences encoded by (a) the DNA sequence
of SEQ ID NO:145, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,
159, 160, 162, and 164; (b) the complement of such DNA sequence, or
(c) a DNA sequence substantially homologous to the sequence of (a)
or (b). In a related embodiment, the present invention provides
polypeptides comprising at least an immunogenic portion of a M.
tuberculosis antigen having the amino acid sequence provided in SEQ
ID NO:146, 161, or 163, and variants thereof.
[0256] The Mycobacterium antigens provided herein include variants
that are encoded by DNA sequences which are substantially
homologous to one or more of DNA sequences specifically recited
herein.
[0257] C. Fusion Polypeptides
[0258] In one embodiment, the present invention provides fusion
proteins comprising multiple polypeptides of the invention or,
alternatively, a polypeptide of the present invention and a known
Mycobacterium antigen, preferably a M. tuberculosis antigen.
Examples of such known Mycobacterium antigens include, but are not
limited to, e.g., 38 kD antigen described in Andersen and Hansen,
Infect. Immun. 57:2481-2488 (1989) (Genbank Accession No. M30046)
and ESAT-6 previously identified in M. bovis (Accession No. U34848)
and in M. tuberculosis (Sorensen et al., Infec. Immun. 63:1710-1717
(1995). Examples of suitable Mycobacterium antigens are disclosed
in U.S. patent application Ser. Nos. 09/056,556, 09/223,040 and
09/287,849, and in U.S. provisional patent application Nos.
60/158,338 and 60/158,425, herein each incorporated by reference.
Variants of such fusion proteins are also provided.
[0259] The fusion proteins of the present invention may also
include a fusion partner which may, for example, assist in
providing T helper epitopes (an immunological fusion partner),
preferably T helper epitopes recognized by humans, or 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. Examples of such proteins include tetanus, tuberculosis
and hepatitis proteins (see, e.g., Stoute at al., New Engl. J. Med.
336:86-91, 1997). 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.
[0260] 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 are 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.
[0261] 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.
[0262] The fusion proteins of the present invention may also
include a linker peptide between the first and second polypeptides.
A peptide linker sequence may be employed to separate, for example,
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. Nat. Acad. Sci. USA 83:8258-8262 (1986); U.S. Pat. Nos.
4,935,233 and 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.
[0263] Fusion proteins may generally be prepared using standard
techniques, including chemical conjugation. Preferably, a fusion
protein is expressed as a recombinant 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.
[0264] The ligated DNA sequences are operably linked to suitable
transcriptional or translational regulatory elements. The
regulatory elements responsible for expression of DNA are located
only 5' to the DNA sequence encoding the first polypeptide.
Similarly, stop codons required to end translation and
transcription termination signals are only present 3' to the DNA
sequence encoding the second polypeptide.
[0265] In general, polypeptides (including fusion proteins) and
polynucleotides as described herein are isolated. An "isolated"
polypeptide or polynucleotide is one that is removed from its
original environment. 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.
D. Immunogenicity of the Polypeptides of the Invention
[0266] Regardless of the method of preparation, the antigens and
immunogenic portions thereof described herein have the ability to
`induce an immunogenic response. More specifically, the antigens
have the ability to react with sera obtained from a
Mycobacterium-infected individual and/or to induce proliferation
and/or cytokine production (i.e., interferon-y and/or
interleukin-12 production) in T cells, NK cells, B cells and/or
macrophages derived from a Mycobacterium-immune individual. Here
and throughout the specification, the Mycobacterium-immune
individual is preferably an M. tuberculosis-immune individual.
[0267] Reactivity with sera obtained from a Mycobacterium-infected
individual may be evaluated using, for example, the representative
ELISA assays described herein, where an absorbance reading with
sera from infected individuals that is at least three standard
deviations above the absorbance obtained with sera from uninfected
individuals is considered positive.
[0268] The selection of cell type for use in evaluating an
immunogenic response to a antigen will, of course, depend on the
desired response. For example, interleukin-12 production is most
readily evaluated using preparations containing B cells and/or
macrophages. A Mycobacterium-immune individual (e.g., an M.
tuberculosis-Immune individual) is one who is considered to be
resistant to the development of the disease (e.g., tuberculosis) by
virtue of having mounted an effective T cell response to
Mycobacterium (i.e., substantially free of disease symptoms). Such
individuals may be identified based on a strongly positive (i.e.,
greater than about 10 mm diameter induration) intradermal skin test
response to tuberculosis proteins (PPD) and an absence of any signs
or symptoms of, e.g., tuberculosis disease. T cells, NK cells, B
cells and macrophages derived from Mycobacterium-immune individuals
may be prepared using methods known to those of ordinary skill in
the art. For example, a preparation of PBMCs (i.e., peripheral
blood mononuclear cells) may be employed without further separation
of component cells. PBMCs may generally be prepared, for example,
using density centrifugation through FICOLL.TM. (Winthrop
Laboratories, NY).
[0269] T cells for use in the assays described herein may also be
purified directly from PBMCs. Alternatively, an enriched T cell
line reactive against mycobacterial proteins, or T cell clones
reactive to individual mycobacterial proteins, may be employed.
Such T cell clones may be generated by, for example, culturing
PBMCs from Mycobacterium immune individuals with mycobacterial
proteins for a period of 2-4 weeks. This allows expansion of only
the mycobacterial protein-specific T cells, resulting in a line
composed solely of such cells. These cells may then be cloned and
tested with individual proteins, using methods known to those of
ordinary skill in the art, to more accurately define individual T
cell specificity. In general, antigens that test positive in assays
for proliferation and/or cytokine production (i.e.,
interferon-.gamma. and/or interleukin-12 production) performed
using T cells, NK cells, B cells and/or macrophages derived from an
Mycobacterium-immune individual are considered immunogenic. Such
assays may be performed, for example, using the representative
procedures described infra. Immunogenic portions of such antigens
may be identified using similar assays, and may be present within
the polypeptides described herein.
[0270] The ability of a polypeptide (e.g., an immunogenic antigen,
or a portion or other variant thereof) to induce cell proliferation
is evaluated by contacting the cells (e.g., T cells and/or NK
cells) with the polypeptide and measuring the proliferation of the
cells. In general, the amount of polypeptide that is sufficient for
evaluation of about 10.sup.3 cells ranges from about 10 ng/ml to
about 100 .mu.g/ml and preferably is about 10 .mu.g/ml. The
incubation of a polypeptide with cells is typically performed at
37.degree. C. for about six days. Following incubation with the
polypeptide, the cells are assayed for a proliferative response,
which may be evaluated by methods known to those of ordinary skill
in the art, such as exposing the cells to a pulse of radiolabeled
thymidine and measuring the incorporation of label into cellular
DNA. In general, a polypeptide that results in at least a three
fold increase in proliferation above background (i.e., the
proliferation observed for cells cultured without polypeptide) is
considered to be able to induce proliferation.
[0271] The ability of a polypeptide to stimulate the production of
interferon-.gamma. and/or interleukin-12 in cells may be evaluated
by contacting the cells with the polypeptide and measuring the
level of interferon-.gamma. or interleukin-12 produced by the
cells. In general, the amount of polypeptide that is sufficient for
the evaluation of about 10.sup.5 cells ranges from about 10 ng/ml
to about 100 .mu.g/ml and preferably is about 10 .mu.g/ml. The
polypeptide may, but need not, be immobilized on a solid support,
such as a bead or a biodegradable microsphere, such as those
described in, e.g., U.S. Pat. Nos. 4,897,268 and 5,075,109. The
incubation of a polypeptide with the cells is typically performed
at 37.degree. C. for about six days. Following incubation with the
polypeptide, the cells are assayed for interferon-.gamma. and/or
interleukin-12 (or one or more subunits thereof) production, which
may be evaluated by methods known to those of ordinary skill in the
art, such as an enzyme-linked immunosorbent assay (ELISA) or, in
the case of the IL-12 P70 heterodimer, a bioassay such as an assay
measuring proliferation of T cells. In general, a polypeptide that
results in the production of at least 50 pg of interferon-.gamma.
per ml of cultured supernatant (containing 10.sup.4-10.sup.5 T
cells per ml) is considered able to stimulate the production of
interferon-.gamma.. A polypeptide that stimulates the production of
at least 10 pg/ml of IL-12 P70 subunit, and/or at least 100 pg/ml
of IL-12 P40 subunit, per 10.sup.5 macrophages or B cells (or per
3.times.10.sup.5 PBMC) is considered able to stimulate the
production of IL-12.
[0272] In general, immunogenic antigens are those antigens that
stimulate proliferation and/or cytokine production (i.e.,
interferon-.gamma. and/or interleukin-12 production) in T cells, NK
cells, B cells and/or macrophages derived from at least about 25%
of Mycobacterium-immune individuals. Among these immunogenic
antigens, polypeptides having superior therapeutic properties may
be distinguished based on the magnitude of the responses in the
above assays and based on the percentage of individuals for which a
response is observed. In addition, antigens having superior
therapeutic properties will not stimulate proliferation and/or
cytokine production in vitro in cells derived from more than about
25% of individuals that are not Mycobacterium-immune, thereby
eliminating responses that are not specifically due to
Mycobacterium-responsive cells. Those antigens that induce a
response in a high percentage of T cell, NK cell, B cell and/or
macrophage preparations from Mycobacterium-immune individuals (with
a low incidence of responses in cell preparations from other
individuals) have superior therapeutic properties.
[0273] Antigens with superior therapeutic properties may also be
identified based on their ability to diminish the severity of
Mycobacterium infection in experimental animals, when administered
as a vaccine. Suitable vaccine preparations for use on experimental
animals are described in detail below. Efficacy may be determined
based on the ability of the antigen to provide at least about a 50%
reduction in bacterial numbers and/or at least about a 40% decrease
in mortality following experimental infection. Suitable
experimental animals include, e.g., mice, guinea pigs and
primates.
[0274] Antigens having superior diagnostic properties may generally
be identified based on the ability to elicit a response in an
intradermal skin test performed on an individual with active
tuberculosis, but not in a test performed on an individual who is
not infected with Mycobacterium. Skin tests may generally be
performed as described below, with a response of at least 5 mm
induration considered positive.
[0275] Immunogenic portions of Mycobacterium antigens may be
prepared and identified using well known techniques, such as those
summarized in Paul, Fundamental Immunology, 3d ed., Raven Press,
pp. 243-247 (1993) and references cited therein. Such techniques
include screening polypeptide portions of the native antigen for
immunogenic properties and in particular, e.g., ability to react
with antigen-specific antibodies, antisera and/or T-cell lines or
clones. As used herein, antisera and antibodies are
"antigen-specific" if they specifically bind to an antigen (i.e.,
they react with the protein in an ELISA or other immunoassay, and
do not react detectably with unrelated proteins). Such antisera and
antibodies may be prepared as described herein, and using well
known techniques. The representative ELISAs as well as the
proliferation and cytokine production assays described herein may
generally be employed in these screens. An immunogenic portion of a
polypeptide is a portion that, within such representative assays,
generates a signal or an immune response (e.g., proliferation,
interferon-.gamma. production and/or interleukin-12 production)
that is not substantially less than that generated by the full
length polypeptide. In other words, an immunogenic portion of a
Mycobacterium antigen generates at least about 20%, and preferably
about 100%, of the signal and/or immune response induced by the
full length antigen in the model ELISA or proliferation assay
described herein, respectively. An immunogenic portion may also, or
alternatively, stimulate the production of at least about 20%, and
preferably about 100%, of the interferon-.gamma. and/or
interleukin-12 induced by the full length antigen in the model
assay described herein. Such immunogenic portions may also react
within such assays at a level that is greater than the reactivity
of the full length polypeptide. Such screens may generally be
performed using methods well known to those of ordinary skill in
the art, such as those described in Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory (1988). For use in
the methods described herein, substantially pure polypeptides may
be combined.
IV. Antibodies
[0276] The present invention further provides agents, such as
antibodies and antigen-binding fragments thereof, that specifically
bind to the polypeptides of the invention. Binding agents may be
capable of differentiating between patients infected or not with
Mycobacterium, and in particular with M. tuberculosis, using the
representative assays provided infra. In other words, antibodies or
other binding agents that bind to a Mycobacterium antigen will
generate a signal indicating the presence of tuberculosis in at
least about 20% of patients with the disease, and will generate a
negative signal indicating the absence of the disease in at least
about 90% of individuals without tuberculosis. To determine whether
a binding agent satisfies this requirement, biological samples
(e.g., blood, sera, urine, sputum, saliva, etc.) from patients with
and without tuberculosis (as determined using standard clinical
tests) may be assayed as described herein for the presence of
polypeptides that bind to the binding agent. It will be apparent
that a statistically significant number of samples with and without
the disease should be assayed. Each binding agent should satisfy
the above criteria; however, those of ordinary skill in the art
will recognize that binding agents may be used in combination to
improve sensitivity.
[0277] Any agent that satisfies the above requirements may be a
binding agent. For example, a binding agent may be a ribosome, with
or without a peptide component, an RNA molecule or a polypeptide.
In a preferred embodiment, a binding agent is an antibody or an
antigen-binding fragment thereof.
[0278] Antibodies may be prepared by any of a variety of techniques
known to those of ordinary skill in the art (see, e.g., Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory (1988)). In general, antibodies can be produced by cell
culture techniques, including the generation of monoclonal
antibodies as described herein, or via transfection of antibody
genes into suitable bacterial or mammalian cell hosts, in order to
allow for the production of recombinant antibodies. In one
technique, an immunogen comprising the immunogenic polypeptide is
initially injected into any of a wide variety of mammals (e.g.,
mice, rats, rabbits, sheep and goats). In this step, the
polypeptides of the invention may serve as the immunogen without
modification. Alternatively, particularly for relatively short
polypeptides, a superior immune response may be elicited if the
polypeptide is joined to a carrier protein, such as bovine serum
albumin or keyhole limpet hemocyanin. The immunogen is injected
into the animal host, preferably according to a predetermined
schedule incorporating one or more booster immunizations, and the
animals are bled periodically. Polyclonal antibodies specific for
the polypeptide may then be purified from such antisera by, for
example, affinity chromatography using the polypeptide coupled to a
suitable solid support.
[0279] Polyclonal antibodies raised to a fusion protein of the
invention can also be obtained by selecting only those polyclonal
antibodies that are specifically immunoreactive with the fusion
protein of interest and not with the individual polypeptide
components of the fusion protein. This selection may be achieved by
subtracting out antibodies that cross-react with the individual
polypeptide components of the fusion protein of interest.
[0280] Alternatively, antibodies that recognize each or all of the
individual polypeptide components of a fusion protein may be useful
in the context of the present invention.
[0281] Monoclonal antibodies specific for the immunogenic
polypeptide of interest may be prepared, for example, using the
technique of Kohier and Milstein, Eur. J. Immunol. 6:511-519
(1976), and improvements thereto. Briefly, these methods involve
the preparation of immortal cell lines capable of producing
antibodies having the desired specificity (i.e., reactivity with
the polypeptide of interest). Such cell lines may be produced, for
example, from spleen cells obtained from an animal immunized as
described above. The spleen cells are then immortalized by, for
example, fusion with a myeloma cell fusion partner, preferably one
that is syngeneic with the immunized animal. A variety of fusion
techniques may be employed. For example, the spleen cells and
myeloma cells may be combined with a nonionic detergent for a few
minutes and then plated at low density on a selective medium that
supports the growth of hybrid cells, but not myeloma cells. A
preferred selection technique uses HAT (hypoxanthine, aminopterin,
thymidine) selection. After a sufficient time, usually about 1 to 2
weeks, colonies of hybrids are observed. Single colonies are
selected and their culture supernatants tested for binding activity
against the polypeptide. Hybridomas having high reactivity and
specificity are preferred.
[0282] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the
hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host, such as, e.g., a mouse. Monoclonal antibodies may
then be harvested from the ascites fluid or the blood. Contaminants
may be removed from the antibodies by conventional techniques, such
as chromatography, gel filtration, precipitation, and extraction.
The polypeptides of this invention may be used in the purification
process in, for example, an affinity chromatography step.
[0283] Within certain embodiments, the use of antigen-binding
fragments of antibodies may be preferred. Such fragments include
Fab fragments, which may be prepared using standard techniques.
Briefly, immunoglobulins may be purified from rabbit serum by
affinity chromatography on Protein A bead columns (see Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory (1988)) and digested by papain to yield Fab and Fe
fragments. The Fab and Fc fragments may be separated by affinity
chromatography on protein A bead columns.
[0284] Antibodies may be used in diagnostic tests to detect the
presence of Mycobacterium antigens using assays similar to those
detailed infra and other techniques well known to those of skill in
the art, thereby providing a methods for detecting Mycobacterium
infection, and in particular tuberculosis, in a patient.
[0285] Monoclonal antibodies of the present invention may be
coupled to one or more therapeutic agents. Suitable agents in this
regard include, but are not limited to, drugs, toxins, and
derivatives thereof. Preferred drugs include, e.g., penicillin,
rifampin, isoniazid, pyrazinamide, ethambutol, streptomycin, etc.
These drugs can be obtained from a natural source or be
semisynthetic or synthetic compounds. Preferred toxins include
ricin, abrin, Diphtheria toxin, cholera toxin, goelonin,
Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral
protein.
[0286] A therapeutic agent may be coupled (e.g., covalently bonded)
to a suitable monoclonal antibody either directly or indirectly
(e.g., via a linker group). A direct reaction between an agent and
an antibody is possible when each possesses a substituent capable
of reacting with the other. For example, a nucleophilic group, such
as an amino or sulfhydryl group, on one may be capable of reacting
with a carbonyl-containing group, such as an anhydride or an acid
halide, or with an alkyl group containing a good leaving group
(e.g., a halide) on the other.
[0287] Alternatively, it may be desirable to couple a therapeutic
agent and an antibody via a linker group. A linker group can
function as a spacer to distance an antibody from an agent in order
to avoid interference with binding capabilities. A linker group can
also serve to increase the chemical reactivity of a substituent on
an agent or an antibody, and thus increase the coupling efficiency.
An increase in chemical reactivity may also facilitate the use of
agents, or functional groups on agents, which otherwise would not
be possible.
[0288] It will be evident to those skilled in the art that a
variety of bifunctional or polyfunctional reagents, both homo- and
hetero-functional (such as those described in the catalog of the
Pierce Chemical Co., Rockford, Ill.), may be employed as the linker
group. Coupling may be effected, for example, through amino groups,
carboxyl groups, sulfhydryl groups or oxidized carbohydrate
residues. There are numerous references describing such
methodology, including, e.g., U.S. Pat. No. 4,671,958.
[0289] Where a therapeutic agent is more potent when free from the
antibody portion of the immunoconjugates of the present invention,
it may be desirable to use a linker group which is cleavable during
or upon internalization into a cell. A number of different
cleavable linker groups have been described. The mechanisms for the
intracellular release of an agent from these linker groups include
cleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.
4,489,710), by irradiation of a photolabile bond (e.g., U.S. Pat.
No. 4,625,014), by hydrolysis of derivatized amino acid side chains
(e.g., U.S. Pat. No. 4,638,045), by serum complement-mediated
hydrolysis (e.g., U.S. Pat. No. 4,671,958), and acid-catalyzed
hydrolysis (e.g., U.S. Pat. No. 4,569,789).
[0290] It may be desirable to couple more than one agent to an
antibody. In one embodiment, multiple molecules of an agent are
coupled to one antibody molecule. In another embodiment, more than
one type of agent may be coupled to one antibody. Regardless of the
particular embodiment, immunoconjugates with more than one agent
may be prepared in a variety of ways. For example, more than one
agent may be coupled directly to an antibody molecule, or linkers
that provide multiple sites for attachment can be used.
Alternatively, a carrier can be used.
[0291] A carrier may bear the agents in a variety of ways,
including covalent bonding either directly or via a linker group.
Suitable carriers include proteins such as, e.g., albumins (e.g.,
U.S. Pat. No. 4,507,234), peptides and polysaccharides such as,
e.g., aminodextran (e.g., U.S. Pat. No. 4,699,784). A carrier may
also bear an agent by noncovalent bonding or by encapsulation, such
as within a liposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and
4,873,088).
[0292] A variety of routes of administration for the antibodies and
immunoconjugates may be used. Typically, administration will be,
e.g., intravenous, intramuscular, subcutaneous, intranasal, or
buccal. It will be evident that the precise dose of the
antibody/immunoconjugate will vary depending upon the antibody
used, the antigen density in the cells, and the rate of clearance
of the antibody.
V. T Cells
[0293] 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 CEPRATE.TM. system, available from CellPro
Inc., Bothell Wash. (see also U.S. Pat. Nos. 5,240,856 and
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.
[0294] T cells may be stimulated with a Mycobacterium polypeptide,
a polynucleotide encoding a Mycobacterium 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, a Mycobacterium polypeptide or
polynucleotide is present within a delivery vehicle, such as a
microsphere, to facilitate the generation of specific T cells.
[0295] T cells are considered to be specific for a Mycobacterium
polypeptide if the T cells 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-labeling cultures
of T cells with tritiated thymidine and measuring the amount of
tritiated thymidine incorporated into DNA). Contact with a
Mycobacterium polypeptide (100 ng/ml-100 .mu.g/ml, preferably 200
ng/ml-25 .mu.g/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-.gamma.) is indicative of T cell activation (see Coligan et
al., Current Protocols in Immunology, vol. 1, Wiley Interscience,
Greene (1998)). T cells that have been activated in response to a
Mycobacterium polypeptide, polynucleotide or polypeptide-expressing
APC may be CD4.sup.+ and/or CD8.sup.+. Mycobacterium
polypeptide-specific T cells may be expanded using standard
techniques. Within preferred embodiments, the T cells are derived
from a patient, or from a related or unrelated donor, and are
administered to the patient following stimulation and
expansion.
[0296] For therapeutic purposes, CD4.sup.+ or CD8.sup.+T cells that
proliferate in response to a Mycobacterium 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 Mycobacterium polypeptide (e.g., 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 synthesize a Mycobacterium polypeptide.
Alternatively, one or more T cells that proliferate in the presence
of a Mycobacterium polypeptide can be expanded in number by
cloning. Methods for cloning cells are well known in the art, and
include limiting dilution. Following expansion, the cells may be
administered back to the patient as described, for example, by
Chang et al., Crit. Rev. Oncol. Hematol. 22:213 (1996).
VI. Diagnostic Assays
[0297] A. Diagnostic Assays with Mycobacterium Polypeptides
[0298] In another aspect, the present invention provides methods
for using the polypeptides described above to diagnose
Mycobacterium infection, and in particular tuberculosis. In this
aspect, methods are provided for detecting Mycobacterium infection
in a biological sample, using one or more of the above
polypeptides, alone or in combination. In embodiments in which
multiple polypeptides are employed, polypeptides other than those
specifically described herein, such as the 38 kD antigen described
above, may be included. The polypeptide(s) are used in an assay, as
described infra, to determine the presence or absence of antibodies
to the polypeptide(s) in a biological sample (e.g., whole blood,
sputum, serum, plasma, saliva, cerebrospinal fluid, urine, etc.)
relative to a predetermined cut-off value. The presence of such
antibodies indicates previous sensitization to mycobacterial
antigens which may be indicative of Mycobacterium infection, and in
particular tuberculosis.
[0299] In embodiments in which more than one polypeptide is
employed, the polypeptides used are preferably complementary (i.e.,
one component polypeptide will tend to detect infection in samples
where the infection would not be detected by another component
polypeptide). Complementary polypeptides may generally be
identified by using each polypeptide individually to evaluate serum
samples obtained from a series of patients known to be infected
with Mycobacterium. After determining which samples test positive
(as described below) with each polypeptide, combinations of two or
more polypeptides may be formulated that are capable of detecting
infection in most, or all, of the samples tested. Such polypeptides
are complementary. For example, approximately 25-30% of sera from
tuberculosis-infected individuals are negative for antibodies to
any single protein, such as the above-mentioned 38 kD antigen.
Complementary polypeptides may, therefore, be used in combination
with the 38 kD antigen to improve sensitivity of a diagnostic
test.
[0300] There are a variety of assay formats known to those of
ordinary skill in the art for using one or more polypeptides to
detect antibodies in a sample. See, e.g., Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
(1988), which is incorporated herein by reference. In general, the
presence or absence of tuberculosis in a patient may be determined
by (a) contacting a biological sample obtained from a patient with
one or more polypeptides or fusion proteins of the invention; (b)
detecting in the sample a level of antibody that binds to the
polypeptide(s) or the fusion protein(s); and (c) comparing the
level of antibody with a predetermined cut-off value.
[0301] In a preferred embodiment, the assay involves the use of a
polypeptide immobilized on a solid support to bind to and remove
the antibody from the sample. The bound antibody may then be
detected using a detection reagent that contains a reporter group.
Suitable detection reagents include antibodies that bind to the
antibody/polypeptide complex and free polypeptide labeled with a
reporter group (e.g., in a semi-competitive assay). Alternatively,
a competitive assay may be utilized, in which an antibody that
binds to the polypeptide of interest is labeled with a reporter
group and allowed to bind to the immobilized antigen after
incubation of the antigen with the sample. The extent to which
components of the sample inhibit the binding of the labeled
antibody to the polypeptide is indicative of the reactivity of the
sample with the immobilized polypeptide.
[0302] The solid support may be any solid material known to those
of ordinary skill in the art to which the antigen may be attached.
For example, the solid support may be a test well in a microtiter
plate or a nitrocellulose or other suitable membrane.
Alternatively, the support may be a bead or disc, such as glass,
fiberglass, latex or a plastic material such as polystyrene or
polyvinylchloride. The support may also be a magnetic particle or a
fiber optic sensor, such as those disclosed, for example, in U.S.
Pat. No. 5,359,681.
[0303] The polypeptides may be bound to the solid support using a
variety of techniques known to those of ordinary skill in the art,
which are amply described in the patent and scientific literature.
In the context of the present invention, the term "bound" refers to
both noncovalent association, such as adsorption, and covalent
attachment (which may be a direct linkage between the antigen and
functional groups on the support or may be a linkage by way of a
cross-linking agent). Binding by adsorption to a well in a
microtiter plate or to a membrane is preferred. In such cases,
adsorption may be achieved by contacting the polypeptide, in a
suitable buffer, with the solid support for a suitable amount of
time. The contact time varies with temperature, but is typically
between about 1 hour and 1 day. In general, contacting a well of a
plastic microtiter plate (such as polystyrene or polyvinylchloride)
with an amount of polypeptide ranging from about 10 ng to about 1
.mu.g, and preferably about 100 ng, is sufficient to bind an
adequate amount of antigen.
[0304] Covalent attachment of the polypeptide of interest to a
solid support may generally be achieved by first reacting the
support with a bifunctional reagent that reacts with both the
support and a functional group, such as a hydroxyl or amino group,
on the polypeptide. For example, the polypeptide may be bound to
supports having an appropriate polymer coating using benzoquinone
or by condensation of an aldehyde group on the support with an
amine and an active hydrogen on the polypeptide (see, e.g., Pierce
Immunotechnology Catalog and Handbook, at A12-A13 (1991)).
[0305] In certain embodiments, the assay is an enzyme linked
immunosorbent assay (ELISA). This assay may be performed by first
contacting a polypeptide antigen that has been immobilized on a
solid support, commonly the well of a microtiter plate, with the
sample, such that antibodies present within the sample that
recognize the polypeptide of interest are allowed to bind to the
immobilized polypeptide. Unbound sample is then removed from the
immobilized polypeptide and a detection reagent capable of binding
to the immobilized antibody-polypeptide complex is added. The
amount of detection reagent that remains bound to the solid support
is then determined using a method appropriate for the specific
detection reagent.
[0306] More specifically, once the polypeptide is immobilized on
the support as described above, the remaining protein binding sites
on the support are typically blocked. Any suitable blocking agent
known to those of ordinary skill in the art, such as bovine serum
albumin or TWEEN 20.TM. (Sigma Chemical Co., St. Louis, Mo.), may
be employed. The immobilized polypeptide is then incubated with the
sample, and the antibody is allowed to bind to the antigen. The
sample may be diluted with a suitable diluent, such as
phosphate-buffered saline (PBS) prior to incubation. In general, an
appropriate contact time (i.e., incubation time) is a period of
time that is sufficient to detect the presence of antibody within a
Mycobacterium-infected sample. Preferably, the contact time is
sufficient to achieve a level of binding that is at least 95% of
that achieved at equilibrium between bound and unbound antibody.
Those of ordinary skill in the art will recognize that the time
necessary to achieve equilibrium may be readily determined by
assaying the level of binding that occurs over a period of time. At
room temperature, an incubation time of about 30 minutes is
generally sufficient.
[0307] Unbound sample may then be removed by washing the solid
support with an appropriate buffer, such as PBS containing 0.1%
TWEEN 20.TM.. Detection reagent may then be added to the solid
support. An appropriate detection reagent is any compound that
binds to the immobilized antibody-polypeptide complex and that can
be detected by any of a variety of means known to those in the art.
Preferably, the detection reagent contains a binding agent (such
as, for example, Protein A, Protein G, immunoglobulin, lectin or
free antigen) conjugated to a reporter group. Preferred reporter
groups include enzymes (such as horseradish peroxidase),
substrates, cofactors, inhibitors; dyes, radionuclides, luminescent
groups, fluorescent groups and biotin. The conjugation of a binding
agent to the reporter group may be achieved using standard methods
known to those of ordinary skill in the art. Common binding agents
may also be purchased conjugated to a variety of reporter groups
from many commercial sources (e.g., Zymed Laboratories, San
Francisco, Calif., and Pierce, Rockford, Ill.).
[0308] The detection reagent is then incubated with the immobilized
antibody-polypeptide complex for an amount of time sufficient to
detect the bound antibody. An appropriate amount of time may
generally be determined from the manufacturer's instructions or by
assaying the level of binding that occurs over a period of time.
Unbound detection reagent is then removed and bound detection
reagent is detected using the reporter group. The method employed
for detecting the reporter group depends upon the nature of the
reporter group. For radioactive groups, scintillation counting or
autoradiographic methods are generally appropriate. Spectroscopic
methods may be used to detect dyes, luminescent groups and
fluorescent groups. Biotin may be detected using avidin, coupled to
a different reporter group (commonly a radioactive or fluorescent
group or an enzyme). Enzyme reporter groups may generally be
detected by the addition of substrate (generally for a specific
period of time), followed by spectroscopic or other analysis of the
reaction products.
[0309] To determine the presence or absence of anti-Mycobacterium
antibodies in the sample, the signal detected from the reporter
group that remains bound to the solid support is generally compared
to a signal that corresponds to a predetermined cut-off value. In
one preferred embodiment, the cut-off value is the average mean
signal obtained when the immobilized antigen is incubated with
samples from an uninfected patient. In general, a sample generating
a signal that is three standard deviations above the predetermined
cut-off value is considered positive for Mycobacterium infection.
In another embodiment, the cut-off value is determined using a
Receiver Operator Curve, according to the method of Sackett et al.,
Clinical Epidemiology: A Basic Science for Clinical Medicine,
Little Brown and Co., pp. 106-107 (1985). Briefly, in this
embodiment, the cut-off value may be determined from a plot of
pairs of true positive rates (i.e., sensitivity) and false positive
rates (100% specificity) that correspond to each possible cut-off
value for the diagnostic test result. The cut-off value on the plot
that is the closest to the upper left-hand corner (i.e., the value
that encloses the largest area) is the most accurate cut-off value,
and a sample generating a signal that is higher than the cut-off
value determined by this method may be considered positive.
Alternatively, the cut-off value may be shifted to the left along
the plot, to minimize the false positive rate, or to the right, to
minimize the false negative rate. In general, a sample generating a
signal that is higher than the cut-off value determined by this
method is considered positive for tuberculosis.
[0310] In a related embodiment, the assay is performed in a rapid
flow-through or strip test format, wherein the antigen is
immobilized on a membrane, such as, e.g., nitrocellulose. In the
flow-through test, antibodies within the sample bind to the
immobilized polypeptide as the sample passes through the membrane.
A detection reagent (e.g., protein A-colloidal gold) then binds to
the antibody-polypeptide complex as the solution containing the
detection reagent flows through the membrane. The detection of
bound detection reagent may then be performed as described above.
In the strip test format, one end of the membrane to which the
polypeptide is bound is immersed in a solution containing the
sample. The sample migrates along the membrane through a region
containing the detection reagent and to the area of immobilized
polypeptide. The concentration of the detection reagent at the
polypeptide indicates the presence of anti-Mycobacterium antibodies
in the sample. Typically, the concentration of detection reagent at
that site generates a pattern, such as a line, that can be read
visually. The absence of such a pattern indicates a negative
result. In general, the amount of polypeptide immobilized on the
membrane is selected to generate a visually discernible pattern
when the biological sample contains a level of antibodies that
would be sufficient to generate a positive signal in an ELISA, as
discussed supra. Preferably, the amount of polypeptide immobilized
on the membrane ranges from about 25 ng to about 1 .mu.g, and more
preferably from about 50 ng to about 500 ng. Such tests can
typically be performed with a very small amount (e.g., one drop) of
patient serum or blood.
[0311] In another aspect, this invention provides methods for using
one or more of the polypeptides described above to diagnose
Mycobacterium infection, and in particular tuberculosis, using a
skin test. As used herein, a "skin test" is any assay performed
directly on a patient in which a delayed-type hypersensitivity
(DTH) reaction (such as swelling, reddening or dermatitis) is
measured following intradermal injection of one or more
polypeptides as described above. Such injection may be achieved
using any suitable device sufficient to contact the polypeptide or
polypeptides with dermal cells of the patient, such as a tuberculin
syringe or 1 ml syringe. Preferably, the reaction is measured at
least 48 hours after injection, more preferably 48-72 hours.
[0312] The DTH reaction is a cell-mediated immune response which is
greater in patients that have been exposed previously to the test
antigen (i.e., the immunogenic portion of the polypeptide employed,
or a variant thereof). The response may be measured visually, using
a ruler. In general, a response that is greater than about 0.5 cm
in diameter, preferably greater than about 1.0 cm in diameter, is a
positive response, indicative of Mycobacterium infection, which may
or may not be manifested as an active disease.
[0313] The polypeptides of this invention are preferably
formulated, for use in a skin test, as pharmaceutical compositions
containing a polypeptide and a physiologically acceptable carrier,
as described infra. Such compositions typically contain one or more
of the above polypeptides in an amount ranging from about 1 .mu.g
to about 100 .mu.g, preferably from about 10 .mu.g to about 50
.mu.g in a volume of 0.1 ml. Preferably, the carrier employed in
such pharmaceutical compositions is a saline solution with
appropriate preservatives, such as phenol and/or TWEEN 80.TM..
[0314] In a preferred embodiment, a polypeptide employed in a skin
test is of sufficient size such that it remains at the site of
injection for the duration of the reaction period. In general, a
polypeptide that is at least 9 amino acids in length is sufficient.
The polypeptide is also preferably broken down by macrophages
within hours of injection to allow presentation to T-cells. Such
polypeptides may contain repeats of one or more of the above
sequences and/or other immunogenic or non-immunogenic
sequences.
[0315] Of course. numerous other assay protocols exist that are
suitable for use with the polypeptides of the present invention.
The above descriptions are intended to be exemplary only.
[0316] B. Diagnostic Assays with Polynucleotides Encoding
Mycobacterium Polypeptides
[0317] Antibodies may be used in diagnostic tests to detect the
presence of Mycobacterium antigens using assays similar to those
detailed above and other techniques well known to those of skill in
the art, thereby providing a method for detecting Mycobacterium
infection, and in particular tuberculosis, in a patient.
[0318] Diagnostic reagents of the present invention may also
comprise DNA sequences encoding one or more of the above
polypeptides, or one or more portions thereof. Alternatively,
Mycobacterium infection can be detected based on the level of mRNA
encoding a Mycobacterium antigen in a biological sample. For
example, at least two oligonucleotide primers may be employed in a
polymerase chain reaction (PCR) based assay to amplify
Mycobacterium-specific cDNA derived from a biological sample,
wherein at least one of the oligonucleotide primers is specific for
(i.e., hybridizes to) a DNA molecule encoding a polypeptide of the
present invention. The presence of the amplified cDNA is then
detected using techniques well known in the art, such as gel
electrophoresis. Similarly, oligonucleotide probes specific for a
DNA molecule encoding a polypeptide of the present invention may be
used in a hybridization assay to detect the presence of a
polypeptide of the invention in a biological sample.
[0319] To permit hybridization under assay conditions,
oligonucleotide primers and probes should comprise an
oligonucleotide sequence that has at least about 60%, preferably at
least about 75% and more preferably at least about 90%, identity to
a portion of a polynucleotide encoding a Mycobacterium antigen that
is at least 10 nucleotides, and preferably at least 20 nucleotides,
in length. Preferably, oligonucleotide primers and/or probes
hybridize to a polynucleotide encoding a polypeptide described
herein under moderately stringent conditions, as defined above.
Oligonucleotide primers and/or probes which may be usefully
employed in the diagnostic methods described herein preferably are
at least 10-40 nucleotides in length. In a preferred embodiment,
the oligonucleotide primers comprise at least 10 contiguous
nucleotides, more preferably at least 15 contiguous nucleotides, of
a DNA molecule having the sequence of SEQ ID NO: 145, 149, 150,
151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, and 164.
Primers or probes may thus be used to detect Mycobacterium-specific
sequences in biological samples. DNA probes or primers comprising
oligonucleotide sequences described above may be used alone, in
combination with each other, or with previously identified
sequences, such as the 38 kD antigen discussed above.
[0320] Techniques for both PCR. based assays and hybridization
assays are well known in the art (see, for example, Mullis et al,
Cold Spring Harbor Symp. Quant. Biol., 51:263 (1987); Erlich ed.,
PCR Technology, Stockton Press, NY (1989)).
[0321] One preferred assay employs RT-PCR, in which PCR is applied
in conjunction with reverse transcription. Typically, RNA is
extracted from a biological sample and is reverse transcribed to
produce cDNA molecules. PCR amplification using at least one
specific primer generates a cDNA molecule, which may be separated
and visualized using, for example, gel electrophoresis.
Amplification may be performed on biological samples taken from a
test patient and from an individual who is not afflicted with
Mycobacterium infection. The amplification reaction may be
performed on several dilutions of cDNA spanning two orders of
magnitude. A two-fold or greater increase in expression in several
dilutions of the test patient sample as compared to the same
dilutions of the non-infected sample is typically considered
positive.
[0322] C. Diagnostic Assays Using the Detection of T Cells
[0323] A Mycobacterium infection may also, or alternatively, be
detected based on the presence of T cells that specifically react
with a Mycobacterium protein in a biological sample. Within certain
methods, a biological sample comprising CD4.sup.+ and/or CD8.sup.+T
cells isolated from a patient is incubated with a Mycobacterium
polypeptide, a polynucleotide encoding such a polypeptide and/or an
APC that expresses at least an immunogenic portion of such a
polypeptide, and the presence or absence of specific activation of
the T cells is detected. Suitable biological samples include, but
are not limited to, isolated T cells. For example, T cells may be
isolated from a patient by routine techniques (such as by
Ficoll/Hypaque density gradient centrifugation of peripheral blood
lymphocytes). T cells may be incubated in vitro for 2-9 days
(typically 4 days) at 37.degree. C. with a Mycobacterium
polypeptide of the invention (at a concentration of, e.g., 5-25
.mu.g/ml). It may be desirable to incubate another aliquot of a T
cell sample in the absence of the Mycobacterium polypeptide to
serve as a control. For CD4.sup.+T cells, activation is preferably
detected by evaluating proliferation of the T cells. For CD8.sup.+T
cells, activation is preferably detected by evaluating cytolytic
activity. A level of proliferation that is at least two fold
greater and/or a level of cytolytic activity that is at least 20%
greater than in disease-free patients indicates the presence of a
Mycobacterium infection in the patient.
[0324] D. Diagnostic Assays for Monitoring the Progression of the
Infection
[0325] In another embodiment, Mycobacterium proteins and
polynucleotides encoding such proteins may be used as markers for
monitoring the progression of a Mycobacterium infection. In this
embodiment, assays as described above for the diagnosis of a
Mycobacterium infection may be performed over time, and the change
in the level of reactive polypeptide(s) evaluated. For example, the
assays may be performed every 24-72 hours for a period of 1 month
to 6-12 months, and thereafter performed as needed. In general, the
Mycobacterium infection is progressing in those patients in whom
the level of polypeptide detected by the binding agent increases
over time. In contrast, the Mycobacterium infection is not
progressing when the level of reactive polypeptide either remains
constant or decreases with time.
[0326] As noted above, to improve sensitivity, multiple
Mycobacterium markers may be assayed within a given sample. It will
be apparent that binding agents specific for different proteins
provided herein may be combined within a single assay. Further,
multiple primers or probes may be used concurrently. The selection
of Mycobacterium protein markers may be based on routine
experiments to determine combinations that result in optimal
sensitivity.
VII. Therapeutic Applications
[0327] In another aspect, the present invention provides methods
for using one or more of the above polypeptides or fusion proteins
(or DNA molecules encoding such polypeptides) to induce protective
immunity against Mycobacterium infection in a patient to either
prevent or treat Mycobacterium infection, and in particular
tuberculosis.
[0328] A. Pharmaceutical Compositions
[0329] In additional embodiments, the present invention concerns
formulation of the polypeptides, fusion proteins or DNA molecules
disclosed herein in pharmaceutically-acceptable solutions for
administration to a cell or an animal, either alone, or in
combination with one or more other modalities of therapy. The
pharmaceutical compositions of the invention may comprise one or
more polypeptides, each of which may contain one or more of the
above sequences (or variants thereof), and a physiologically
acceptable carrier.
[0330] It will also be understood that, if desired, the
polypeptide, fusion protein and nucleic acid molecule compositions
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. In particular, such
pharmaceutical compositions may also contain other Mycobacterium
antigens, either incorporated into a combination polypeptide or
present within a separate polypeptide. 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 synthesized as described herein.
Likewise, such compositions may further comprise substituted or
derivatized RNA or DNA compositions.
[0331] 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.
[0332] 1. Oral Administration
[0333] 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.
[0334] 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 (see, e.g., Mathiowitz et al., Nature 386:410-414
(1997); Hwang et al., Crit Rev Ther Drug Carrier Syst. 15:243-84
(1998); U.S. Pat. Nos. 5,641,515; 5,580,579; and 5,792,451). 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
flavoring agent, such as peppermint, oil of wintergreen, or cherry
flavoring. 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 compound sucrose as a
sweetening agent methyl and propylparabens as preservatives, a dye
and flavoring, such as cherry or orange flavor, 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 compounds may be incorporated
into sustained-release preparation and formulations.
[0335] Typically, these formulations may contain at least about
0.1% of the active compound or more, although the percentage of the
active ingredient(s) may, of course, be varied and may conveniently
be between about 1 or 2% and about 60% or 70% or more of the weight
or volume of the total formulation. 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.
[0336] 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.
[0337] 2. Injectable Delivery
[0338] In certain circumstances it will be desirable to deliver the
pharmaceutical compositions disclosed herein parenterally,
intravenously, intramuscularly, or even intraperitoneally as
described in, e.g., U.S. Pat. Nos. 5,543,158; 5,641,515; and
5,399,363. 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.
[0339] 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). 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.
[0340] 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 Pharmaceutical
Sciences 15th Edition, pages 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.
[0341] 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.
[0342] 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.
[0343] 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.
[0344] 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.
[0345] 3. Nasal Delivery
[0346] In certain embodiments, the pharmaceutical compositions may
be delivered by intranasal sprays, inhalation; and/or other aerosol
delivery vehicles. Methods for delivering genes, nucleic acids, and
peptide compositions directly to the lungs via nasal aerosol sprays
has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212.
Likewise, the delivery of drugs using intranasal microparticle
resins (Takenaga et al., J Controlled Release 52:81-87 (1998)) and
lysophosphatidyl-glycerol compounds (see, e.g., U.S. Pat. No.
5,725,871) 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.
[0347] 4. Liposome-, Nanocapsule-, and Microparticle-Mediated
Delivery
[0348] 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.
[0349] Such formulations may be preferred for the introduction of
pharmaceutically-acceptable formulations of the polypeptides,
fusion proteins and nucleic acids disclosed herein. The formation
and use of liposomes is generally known to those of skill in the
art (see, e.g., Couvreur et al., FEBS Lett. 84(2):323-326 (1977);
Couvreur (1988); Lasic, Trends Biotechnol. 16(7):307-321 (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 and
Papahadjopoulos, Proc Natl Acad Sci USA. 85(18):6949-6953 (1988);
Allen and Choun (1987); U.S. Pat. No. 5,741,516). Further, various
methods of liposome and liposome like preparations as potential
drug carriers have been reviewed (Takakura, Nippon Rinsho
56(3):691-695 (1998); Chandran at al., Indian J Exp Biol.
35(8):801-809 (1997); Margalit, Crit Rev Ther Drug Carrier Syst.
12(2-3):233-261 (1995); U.S. Pat. Nos. 5,567,434; 5,552,157;
5,565,213; 5,738,868; and 5,795,587).
[0350] 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. J Biol Chem.
265(27):16337-16342 (1990); Muller et al, DNA Cell Biol.
9(3):221-229 (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 and Martin, Chem Phys Lipids 40(2-4):347-358 (1986);
Heath et al., Biochim Biophys Acta. 862(1):72-80 (1986);
Balazsovits et al., Cancer Chemother Pharmacol. 23(2):81-6, (1989);
Fresta and Puglisi, J. Drug Target 4(2):95-101 (1996)),
radiotherapeutic agents (Pikul et al., Arch Surg. 122(12):1417-1420
(1987)), enzymes (Imaizumi et al., Stroke 21(9): 1312-1317 (1990);
Imaizumi et al., Acta Neurochir Suppl (Wien) 51:236-238 (1990)),
viruses (Faller and Baltimore, J Virol. 49(1):269-272 (1984)),
transcription factors and allosteric effectors (Nicolau and
Gersonde, Naturwissenschaften 66(11):563-566 (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., J Infect
Dis. 151(4):704-710 (1985); Lopez-Berestein et al., Cancer Drug
Deliv. 2(3):183-189 (1985); Coune, Infection 16(3):141-147 (1988);
Sculier et al., Eur. J. Cancer Clin. Oncol. 24(3):527-38 (1988)).
Furthermore, several studies suggest that the use of liposomes is
not associated with autoimmune responses, toxicity or gonadal
localization after systemic delivery (Mori and Fukatsu, Epilepsia
33(6):994-1000 (1992)).
[0351] 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 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 .ANG.,
containing an aqueous solution in the core.
[0352] 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.
[0353] In addition to the teachings of Couvreur et al. (1977),
supra; Couvreur et al. (1988), supra), 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.
[0354] 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.
[0355] 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.
[0356] 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.
[0357] 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.
[0358] 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 hours or days,
depending on their composition, and half lives in the blood range
from minutes to several hours. 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.
[0359] 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.
[0360] 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., Pharm Res.
15(7):1056-1062 (1998); Douglas et al., Crit. Rev. Ther. Drug
Carrier Syst. 3(3):233-261 (1987)). To avoid side effects due to
intracellular polymeric overloading, such ultrafine particles
(sized around 0.1 .mu.m) 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., J. Pharm. Sci. 69(2):199-202 (1980);
Couvreur et al., (1988), supra; zur Muhlen et al., Eur. J. Pharm.
Biopharm. 45(2):149-155 (1998); Zambaux et al., J. Controlled
Release 50(1-3):31-40 (1998); Pinto-Alphandry et al. (1995); and
U.S. Pat. No. 5,145,684).
[0361] B. Vaccines
[0362] In certain preferred embodiments of the present invention,
vaccines are provided. The vaccines will generally comprise one or
more pharmaceutical compositions, such as those discussed above, in
combination with a non-specific immune response enhancer. A
non-specific immune response enhancer may be any substance that
enhances or potentiates an immune response (antibody and/or
cell-mediated) to an exogenous antigen. Examples of non-specific
immune response enhancers 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). Vaccine preparation is generally described in, for
example, Powell and Newman, eds., "Vaccine Design (the subunit and
adjuvant approach)," Plenum Press (NY, 1995). Vaccines may be
designed to generate antibody immunity and/or cellular immunity
such as that arising from CTL or CD4+T cells.
[0363] Pharmaceutical compositions and vaccines 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 Mycobacterium antigens may be
present, either incorporated into a fusion polypeptide or as a
separate compound, within the composition or vaccine. Polypeptides
may, but need not, be conjugated to other macromolecules as
described, for example, within U.S. Pat. Nos. 4,372,945 and
4,474,757. Pharmaceutical compositions and vaccines may generally
be used for prophylactic and therapeutic purposes.
[0364] Illustrative vaccines may contain DNA encoding one or more
of the polypeptides as described above, such that the polypeptide
is generated in situ. Such a polynucleotide may comprise DNA, RNA,
a modified nucleic acid or a DNA/RNA hybrid. As noted above, the
nucleic acid 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 (such as
Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of
the polypeptide on its cell surface or secretes such an epitope. 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 at 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 vaccine may comprise both a
polynucleotide and a polypeptide component. Such vaccines may
provide for an enhanced immune response.
[0365] In a related aspect, a DNA vaccine as described supra may be
administered simultaneously with or sequentially to either a
polypeptide of the present invention or a known Mycobacterium
antigen, such as the 38 kD antigen described above For example,
administration of DNA encoding a polypeptide of the present
invention, either "naked" or in a delivery system as described
supra, may be followed by administration of an antigen in order to
enhance the protective immune effect of the vaccine.
[0366] It will be apparent that a vaccine 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).
[0367] While any suitable carrier known to those of ordinary skill
in the art may be employed in the vaccine 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.
[0368] 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.
[0369] Any of a variety of immunostimulants may be employed in the
vaccines 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 aluminum 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. In another embodiment, BCG is used as
an adjuvant. In addition, the vaccine can be administered to a
subject previously exposed to BCG. 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.);
AS-2 and derivatives thereof (SmithKline Beecham, Philadelphia,
Pa.); CWS, TDM, Leif, aluminum salts such as aluminum 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 and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or
-12, may also be used as adjuvants.
[0370] Within the vaccines provided herein, the adjuvant
composition is preferably designed to induce an immune response
predominantly of the Th1 type. High levels of Th1-type cytokines
(e.g., IFN-.gamma., TNF.alpha., IL-2 and IL-12) tend to favor the
induction of cell mediated immune responses to an administered
antigen. In contrast, high levels of Th2-type cytokines (e.g.,
IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral
immune responses. Following application of a vaccine as provided
herein, a patient will 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 Mosmann & Coffman, Ann. Rev. Immunol. 7:145-173
(1989).
[0371] Preferred adjuvants for use in eliciting a predominantly
Th1-type response include, for example, a combination of
monophosphoryl lipid A, preferably 3-de-Oacylated monophosphoryl
lipid A (3D-MPL), together with an aluminum salt. MPL adjuvants are
available from Corixa Corporation (Seattle, Wash.; see U.S. Pat.
Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing
oligonucleotides (in which the CpG dinucleotide is unmethylated)
also induce a predominantly Th1 response. 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). Another preferred adjuvant
comprises a saponin, such as Quil A, or derivatives thereof,
including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,
Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa
saponins. Other preferred formulations include more than one
saponin in the adjuvant combinations of the present invention, for
example combinations of at least two of the following group
comprising QS21, QS7, Quil A, p-escin, or digitonin.
[0372] 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 ISCOMs. 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.RTM. to
increase viscosity, or may be formulated in a dry powder form with
a powder excipient such as lactose.
[0373] In one preferred 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.RTM.
adjuvant, 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. Another particularly
preferred adjuvant formulation employing QS21, 3D-MPL.RTM. adjuvant
and tocopherol in an oil-in-water emulsion is described in WO
95/17210.
[0374] 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. Preferably the formulation additionally comprises an oil
in water emulsion and tocopherol.
[0375] Other preferred adjuvants include Montanide ISA 720 (Seppic,
France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59
(Chiron), the SBAS series of adjuvants (e.g., SBAS-2, AS2', AS2,''
SBAS-4, or SBAS6, available from SmithKline Beecham, Rixensart,
Belgium), Detox (Corixa, Hamilton, Mont.), RC-529 (Corixa,
Hamilton, Mont.) 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.
[0376] Other preferred adjuvants include adjuvant molecules of the
general formula (I): HO(CH.sub.2CH.sub.2O).sub.n-A-R,
wherein, n is 1-50, A is a bond or --C(O)--, R is C.sub.1-50 alkyl
or Phenyl C.sub.1-50 alkyl.
[0377] One embodiment of the present invention consists of a
vaccine formulation 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 C.sub.1-50, preferably
C.sub.4-C.sub.20 alkyl and most preferably C.sub.12 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
(12.sup.th edition: entry 7717). These adjuvant molecules are
described in WO 99/52549.
[0378] The polyoxyethylene ether according to the general formula
(I) above may, if desired, be combined with another adjuvant. For
example, a preferred adjuvant combination is preferably with CpG as
described in the pending UK patent application GB 9820956.2.
[0379] Any vaccine 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.
[0380] 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 and the nature of the condition to be treated or
prevented.
[0381] C. Delivery Vehicles
[0382] Any of a variety of delivery vehicles may be employed within
pharmaceutical compositions and vaccines to facilitate production
of an antigen-specific immune response that targets infected cells.
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, e.g., 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.
[0383] Certain preferred embodiments of the present invention use
dendritic cells or progenitors thereof as antigen-presenting cells.
Dendritic cells are highly potent APCs (Banchereau and 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 and 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
a vaccine (see Zitvogel et al., Nature Med. 4:594-600 (1998)).
[0384] 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
TNF.alpha. 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, TNF.alpha., CD40 ligand, LPS, flt3 ligand and/or
other compound(s) that induce maturation and proliferation of
dendritic cells.
[0385] Dendritic cells are conveniently categorized as "immature"
and "mature" cells, which allows a simple way to discriminate
between two well characterized phenotypes. However, this
nomenclature should not be construed to exclude all possible
intermediate stages of differentiation. Immature dendritic cells
are characterized as APC with a high capacity for antigen uptake
and processing, which correlates with the high expression of
Fc.gamma. 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 4-1BB).
[0386] APCs may generally be transfected with a polynucleotide
encoding a Mycobacterium antigen (or portion or other variant
thereof) such that the Mycobacterium polypeptide, or an immunogenic
portion thereof is expressed on the cell surface. Such transfection
may take place er vivo, and a composition or vaccine comprising
such transfected cells may then be used for therapeutic purposes,
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, e.g., 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
Mycobacterium 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.
[0387] D. Therapeutic Applications of the Compositions of the
Invention
[0388] In further aspects of the present invention, the
compositions described supra may be used for immunotherapy of
Mycobacterium infection, and in particular tuberculosis. Within
such methods, pharmaceutical compositions and vaccines are
typically administered to a patient to either prevent the
development of Mycobacterium infection or to treat a patient
afflicted with Mycobacterium infection. Mycobacterium infection may
be diagnosed using criteria generally accepted in the art, such as,
e.g., in the case of tuberculosis, fever, acute inflammation of the
lung and/or non-productive cough. Pharmaceutical compositions and
vaccines may be administered either prior to or following a
treatment such as administration of conventional drugs.
Administration may be by any suitable route, including, e.g.,
intravenous, intraperitoneal, intramuscular, subcutaneous,
intranasal, intradermal, oral, etc.
[0389] Within certain embodiments, immunotherapy may be active
immunotherapy, in which treatment relies on the in vivo stimulation
of the endogenous host immune system to react against Mycobacterium
infection with the administration of immune response-modifying
agents (such as polypeptides and polynucleotides as provided
herein).
[0390] Within other embodiments, immunotherapy may be passive
immunotherapy, in which treatment involves the delivery of agents
with established Mycobacterium-immune reactivity (such as effector
cells or antibodies) that can directly or indirectly mediate
anti-Mycobacterium infection effects and do not necessarily depend
on an intact host immune system. Examples of effector cells include
T cells as discussed above, T lymphocytes (such as CD8.sup.+
cytotoxic T lymphocytes and CD4.sup.+T-helper tumor-infiltrating
lymphocytes), killer cells (such as Natural Killer cells and
lymphokine-activated killer cells), B cells and antigen-presenting
cells (such as dendritic cells and macrophages) expressing a
polypeptide of the invention. T cell receptors and antibody
receptors specific for the polypeptides recited herein may be
cloned, expressed and transferred into other vectors or effector
cells for adoptive immunotherapy. The polypeptides provided herein
may also be used to generate antibodies or anti-idiotypic
antibodies (as described above and in U.S. Pat. No. 4,918,164) for
passive immunotherapy.
[0391] Effector cells may generally be obtained in sufficient
quantities for adoptive immunotherapy by growth in vitro, as
described herein. Culture conditions for expanding single
antigen-specific effector cells to several billion in number with
retention of antigen recognition in vivo are well known in the art.
Such in vitro culture conditions typically use intermittent
stimulation with antigen, often in the presence of cytokines (such
as IL-2) and non-dividing feeder cells. As noted above,
immunoreactive polypeptides as provided herein may be used to
rapidly expand antigen-specific T cell cultures in order to
generate a sufficient number of cells for immunotherapy. In
particular, antigen-presenting cells, such as dendritic, macrophage
or B cells, may be pulsed with immunoreactive polypeptides or
transfected with one or more polynucleotides using standard
techniques well known in the art. For example, antigen-presenting
cells can be transfected with a polynucleotide having a promoter
appropriate for increasing expression in a recombinant virus or
other expression system. Cultured effector cells for use in therapy
must be able to grow and distribute widely, and to survive long
term in vivo. Studies have shown that cultured effector cells can
be induced to grow in vivo and to survive long term in substantial
numbers by repeated stimulation with antigen supplemented with IL-2
(see, for example, Cheever et al., Immunological Reviews 157:177,
(1997)).
[0392] Alternatively, a vector expressing a polypeptide recited
herein may be introduced into antigen presenting cells taken from a
patient and clonally propagated ex vivo for transplant back into
the same patient. Transfected cells may be reintroduced into the
patient using any means known in the art, preferably in sterile
form by, e.g., injection, intranasal or oral administration.
[0393] E. Formulation and Administration
[0394] Vaccines and pharmaceutical compositions may be presented in
unit-dose or multi-dose containers, such as scaled 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, a vaccine or
pharmaceutical composition may be stored in a freeze-dried
condition requiring only the addition of a sterile liquid carrier
immediately prior to use.
[0395] Routes and frequency of administration, as well as dosage,
may vary from individual to individual and may parallel those
currently being employed in immunization using BCG. In general, the
pharmaceutical compositions and vaccines may be administered, e.g.,
by injection (e.g., intracutaneous, intramuscular, intravenous or
subcutaneous), intranasally (e.g., by aspiration) or orally.
Between 1 and 3 doses may be administered for a 1-36 week period.
Preferably, 3 doses are administered, at intervals of 3-4 months,
and booster vaccinations may be given periodically thereafter.
Alternate protocols may be appropriate for individual patients. A
suitable dose is an amount of polypeptide or DNA that, when
administered as described supra, is capable of raising an immune
response in an immunized patient sufficient to protect the patient
from Mycobacterium infection for at least 1-2 years. When used for
a therapeutic purpose, a suitable dose is the amount that is
capable of raising and immune response in a patient that is
sufficient to obtain an improved clinical outcome (e.g., more
frequent cure) in treated patients as compared to non-treated
patients. Increases in preexisting immune responses to a
Mycobacterium protein generally correlate with an improved clinical
outcome. Such immune responses may generally be evaluated using
standard proliferation, cytotoxicity or cytokine assays, which may
be performed using samples obtained from a patient before and after
treatment.
[0396] In general, the amount of polypeptide present in a dose (or
produced in situ by the DNA in a dose) ranges from about 1 pg to
about 100 mg per kg of host, typically from about 10 pg to about 1
mg, and preferably from about 100 pg to about 1 .mu.g. Suitable
dose sizes will vary with the size of the patient, but will
typically range from about 0.1 ml to about 5 ml.
[0397] F. Diagnostic Kits
[0398] 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.
For example, one container within a kit may contain a monoclonal
antibody or fragment thereof that specifically binds to a
Mycobacterium antigen. 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.
[0399] Alternatively, a kit may be designed to detect the level of
mRNA encoding a Mycobacterium antigen in a biological sample. Such
kits generally comprise at least one oligonucleotide probe or
primer, as described above, that hybridizes to a polynucleotide
encoding a Mycobacterium antigen. 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
Mycobacterium antigen.
[0400] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0401] 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.
VII. Examples
Example 1
Purification and Characterization of M. Tuberculosis Polypeptides
Using CD4+T Cell Lines Generated from Human PBMC
[0402] M. tuberculosis antigens of the present invention were
isolated by expression cloning of cDNA libraries of M. tuberculosis
strains H37Rv and Erdman essentially as described by Sanderson et
al. (J. Exp. Med., 182:1751-1757 (1995)) and were shown to induce
PBMC proliferation and IFN-.gamma. in an immunoreactive T cell
line.
[0403] Two CD4+T cell lines, referred to as DC-4 and DC-5, were
generated against dendritic cells infected with M. tuberculosis.
Specifically, dendritic cells were prepared from adherent PBMC from
a single donor and subsequently infected with tuberculosis.
Lymphocytes from the same donor were cultured under limiting
dilution conditions with the infected dendritic cells to generate
the CD4+T cell lines DC-4 and DC-5. These cell lines were shown to
react with crude soluble proteins from M. tuberculosis but not with
Tb38-1. Limiting dilution conditions were employed to obtain a
third CD4+T cell line, referred to as DC-6, which was shown to
react with both crude soluble proteins and Tb38-1.
[0404] Genomic DNA was isolated from the M. tuberculosis strains
H37Rv and Erdman and used to construct expression libraries in the
vector pBSK(-) using the Lambda ZAP expression system (Stratagene,
La Jolla, Calif.). These libraries were transformed into E. coli,
pools of induced E. coli cultures were incubated with dendritic
cells, and the ability of the resulting incubated dendritic cells
to stimulate cell proliferation and IFN-.gamma. production in the
CD4+T cell line DC-6 was examined as described below in Example 2.
Positive pools were fractionated and re-tested until pure M.
tuberculosis clones were obtained.
[0405] Nineteen clones were isolated, of which nine were found to
contain the previously identified M. tuberculosis antigens TbH-9
and Tb38-1, disclosed in U.S. patent application Ser. No.
08/533,634. The determined cDNA sequences for the remaining ten
clones (hereinafter referred to as Tb224, Tb636, Tb424, Tb436,
Tb398, Tb508, Tb441, Tb475, Tb488 and Tb465) are provided in SEQ ID
NO: 1-10, respectively. The corresponding predicted amino acid
sequences for Tb224 and Tb636 are provided in SEQ ID NO:13 and 14,
respectively. The open reading frames for these two antigens were
found to show some homology to TbH-9. Tb224 and Tb636 were also
found to be overlapping clones.
[0406] Tb424, Th436, Tb398. Th508, Tb441, Tb475, Tb488 and Tb465
were each found to contain two small open reading frames (referred
to as ORF-1 and ORF-2) or truncated forms thereof, with minor
variations in ORF-1 and ORF-2 being found for each clone. The
predicted amino acid sequences of ORF-1 and ORF-2 for Tb424, Tb436,
Tb398, Tb508, Tb441, Tb475, Th488 and Tb465 are provided in SEQ ID
NO:16 and 17, 18 and 19, 20 and 21, 22 and 23, 24 and 25, 26 and
27, 28 and 29, and 30 and 31, respectively. In addition, clones
Tb424 and Tb436 were found to contain a third apparent open reading
frame, referred to as ORF-U. The predicted amino acid sequences of
ORF-U for Tb424 and Tb436 are provided in SEQ ID NO:32 and 33,
respectively. Tb424 and Tb436 were found to be either overlapping
clones or recently duplicated/transposed copies. Similarly Th398,
Tb508 and Tb465 were found to be either overlapping clones or
recently duplicated/transposed copies, as were Tb475 and Tb488.
[0407] These sequences were compared with known sequences in
publicly available sequence databases using the BLASTN system. No
homologies to the antigens Tb224 and Tb431 were found. Tb636 was
found to be 100% identical to a cosmid previously identified in M.
tuberculosis. Similarly, Tb508, Tb488, Tb398, Tb424, Tb436, Tb441,
Tb465 and Tb475 were found to show homology to known M.
tuberculosis cosmids. In addition, Tb488 was found to have 100%
homology to M. tuberculosis topoisomerase I.
[0408] Seventeen overlapping peptides to the open reading frames
ORF-1 (referred to as 1-1-1-17; SEQ ID NO:34-50, respectively) and
thirty overlapping peptides to the open reading frame ORF-2
(referred to as 2-1-2-30, SEQ ID NO:51-80, respectively) were
synthesized using the procedure described below in Example 4.
[0409] The ability of the synthetic peptides and of recombinant
ORF-1 and ORF-2 to induce T cell proliferation and IFN-.gamma.
production in PBMC from PPD-positive donors was assayed as
described below in Example 2. FIGS. 1A-B and 2A-B illustrate
stimulation of T cell proliferation and IFN-.gamma. by recombinant
ORF-2 and the synthetic peptides 2-1-2-16 for two donors, referred
to as D7 and D160, respectively. Recombinant ORF-2 (referred to as
MTI) stimulated T cell proliferation and IFN-.gamma. production in
PBMC from both donors. The amount of PBMC stimulation seen with the
individual synthetic peptides varied with each donor, indicating
that each donor recognizes different epitopes on ORF-2. The
proteins encoded by ORF-1, ORF-2 and ORF-U were subsequently named
MTS, MTI and MSF, respectively.
[0410] Eighteen overlapping peptides to the sequence of MSF
(referred to as MSF-1-MSF-18; SEQ ID) NO:84-101, respectively) were
synthesized and their ability to stimulate T cell proliferation and
IFN-.gamma. production in a CD4+T cell line generated against M.
tuberculosis culture filtrate was examined as described below. The
peptides referred to as MSF-12 and MSF-13 (SEQ ID NO:95 and 96,
respectively) were found to show the highest levels of
reactivity.
[0411] Two overlapping peptides (SEQ ID NO:81 and 82) to the open
reading frame of Tb224 were synthesized and shown to induce T cell
proliferation and IFN-.gamma. production in PBMC from PPD-positive
donors.
[0412] Two CD4+T cell lines from different donors were generated
against M. tuberculosis infected dendritic cells using the above
methodology. Screening of the M. tuberculosis cDNA expression
library described above using this cell line, resulted in the
isolation of two clones referred to as Tb867 and Tb391. The
determined cDNA sequence for Tb867 (SEQ ID NO:102) was found to be
identical to the previously isolated M. tuberculosis cosmid
SCY22G10, with the candidate reactive open reading frame encoding a
750 amino acid M. tuberculosis protein kinase. Comparison of the
determined cDNA sequence for Tb391 (SEQ ID NO: 103) with those in
publicly available sequence databases revealed no significant
homologies to known sequences.
[0413] In further studies, CD4+T cell lines were generated against
M. tuberculosis culture filtrate, essentially as outlined above,
and used to screen the M. tuberculosis Erdman cDNA expression
library described above. Five reactive clones, referred to as
Tb431, Tb472, Tb470, Tb838 and Tb962 were isolated. The determined
cDNA sequences for Tb431, Tb472, Tb470, and Tb838 are provided in
SEQ ID NO: 11, 12, 104 and 105, respectively, with the determined
cDNA sequences for Tb962 being provided in SEQ ID NO: 106 and 107.
The corresponding predicted amino acid sequence for Tb431 is
provided in SEQ ID NO: 15.
[0414] Subsequent studies led to the isolation of a full-length
cDNA sequence for Tb472 (SEQ ID NO: 108). Overlapping peptides were
synthesized and used to identify the reactive open reading frame.
The predicted amino acid sequence for the protein encoded by Tb472
(referred to as MSL) is provided in SEQ ID NO: 109. Comparison of
the sequences for Tb472 and MSL with those in publicly available
sequence databases as described above, revealed no homologies to
known sequences. Fifteen overlapping peptides to the sequence of
MSL (referred to as MSL-1-MSL-15; SEQ ID NO: 110-124, respectively)
were synthesized and their ability to stimulate T cell
proliferation and IFN-.gamma. production in a CD4+T cell line
generated against M. tuberculosis culture filtrate was examined as
described below. The peptides referred to as MSL-10 (SEQ ID NO:
119) and MSL-11 (SEQ ID NO: 120) were found to show the highest
level of reactivity. Comparison of the determined cDNA sequence for
Tb838 with those in publicly available sequence databases revealed
identity to the previously isolated M. tuberculosis cosmid SCY07H7.
Comparison of the determined cDNA sequences for the clone Tb962
with those in publicly available sequence databases revealed some
homology to two previously identified M. tuberculosis cosmids, one
encoding a portion of bactoferritin. However, recombinant
bactoferritin was not found to be reactive with the T cell line
used to isolate Tb962.
[0415] The clone Tb470, described above, was used to recover a
full-length open reading frame (SEQ ID NO: 125) that showed
homology with TbH9 and was found to encode a 40 kDa antigen,
referred to as Mtb40. The determined amino acid sequence for Mtb40
is provided in SEQ ID NO:126. Similarly, subsequent studies led to
the isolation of the full-length cDNA sequence for Tb431, provided
in SEQ ID NO:83, which was also determined to contain an open
reading frame encoding Mtb40. Tb470 and Tb431 were also found to
contain a potential open reading frame encoding a U-ORF-like
antigen.
[0416] Screening of an M. tuberculosis Erdman cDNA expression
library with multiple CD4+T cell lines generated against A.
tuberculosis culture filtrate, resulted in the isolation of three
clones, referred to as Tb366, Tb433 and Tb439. The determined cDNA
sequences for Tb366, Tb433 and Tb439 are provided in SEQ ID NO:127,
128 and 129, respectively. Comparison of these sequences with those
in publicly available sequence databases revealed no significant
homologies to Th366. Th433 was found to show some homology to the
previously identified M. tuberculosis antigen MPTS3. Tb439 was
found to show 100% identity to the previously isolated M.
tuberculosis cosmid SCY02B10.
[0417] A CD4+T cell line was generated against M. tuberculosis PPD,
essentially described above, and used to screen the above M.
tuberculosis Erdman cDNA expression library. One reactive clone
(referred to as Tb372) was isolated, with the determined cDNA
sequences being provided in SEQ ID NO:130 and 131. Comparison of
these sequences with those in publicly available sequence databases
revealed no significant homologies.
[0418] In further studies, screening of an M. tuberculosis cDNA
expression library with a CD4+T cell line generated against
dendritic cells that had been infected with tuberculosis for 8
days, as described above, led to the isolation of two clones
referred to as Th390R5C6 and Th390R2C11. The determined cDNA
sequence for Tb390R5C6 is provided in SEQ ID NO:132, with the
determined cDNA sequences for Th390R2C11 being provided in SEQ ID
NO:133 and 134. Th390R5C6 was found to show 100% identity to a
previously identified M. tuberculosis cosmid.
[0419] In subsequent studies, the methodology described above was
used to screen an M. tuberculosis genomic DNA library prepared as
follows. Genomic DNA from M. tuberculosis Erdman strain was
randomly sheared to an average size of 2 kb, and blunt ended with
Klenow polymerase, followed by the addition of EcoRI adaptors. The
insert was subsequently ligated into the Screen phage vector
(Novagen, Madison, Wis.) and packaged in vitro using the PhageMaker
extract (Novagen). The phage library (referred to as the Erd
.lamda.Screen library) was amplified and a portion was converted
into a plasmid expression library by an autosubcloning mechanism
using the E. coli strain BM25.8 (Novagen). Plasmid DNA was purified
from BM25.8 cultures containing the pSCREEN recombinants and used
to transform competent cells of the expressing host strain
BL21(DE3)pLysS. Transformed cells were aliquoted into 96 well
microtiter plates with each well containing a pool size of
approximately 50 colonies. Replica-plates of the 96 well plasmid
library format were induced with IPTG to allow recombinant protein
expression. Following induction, the plates were centrifuged to
pellet the E. coli which was used directly in T cell expression
cloning of a CD4+T cell line prepared from a PPD-positive donor
(donor 160) as described above. Pools containing E. coli expressing
M. tuberculosis T cell antigens were subsequently broken down into
individual colonies and reassayed in a similar fashion to identify
positive hits.
[0420] Screening of the T cell line from donor 160 with one 96 well
plate of the Erd .lamda.Screen library provided a total of nine
positive hits. Previous experiments on the screening of the pBSK
library described above with T cells from donor 160 suggested that
most or all of the positive clones would be TbH-9. Tb38-1 or MTI
(disclosed in U.S. patent application Ser. No. 08/533,634) or
variants thereof. However, Southern analysis revealed that only
three wells hybridized with a mixed probe of TbH-9, Tb38-1 and MTI.
Of the remaining six positive wells, two were found to be
identical. The determined 5' cDNA sequences for two of the isolated
clones (referred to as YI-26C1 and YI-86C11) are provided in SEQ ID
NO:135 and 136, respectively. The full length cDNA sequence for the
isolated clone referred to as hTcc#1 is provided in SEQ ID NO:137,
with the corresponding predicted amino acid sequence being provided
in SEQ ID NO:138. Comparison of the sequences of hTcc#1 to those in
publicly available sequence databases as described above, revealed
some homology to the previously isolated M. tuberculosis cosmid
MTCY07H7B.06.
Example 2
Induction of T Cell Proliferation and Interferon-.gamma. Production
by M. tuberculosis Antigens
[0421] The ability of recombinant M. tuberculosis antigens to
induce T-cell proliferation and interferon-.gamma. production may
be determined as follows.
[0422] Proteins may be induced by IPTG and purified by gel elution,
as described in Skeiky et al., J. Exp. Med. 181:1527-1537 (1995).
The purified polypeptides are then screened for the ability to
induce T-cell proliferation in PBMC preparations. The PBMCs from
donors known to be PPD skin test positive and whose T-cells are
known to proliferate in response to PPD are cultured in medium
comprising RPMI 1640 supplemented with 10% pooled human serum and
50 .mu.g/ml gentamicin. Purified polypeptides are added in
duplicate at concentrations of 0.5 to 10 .mu.g/ml. After six days
of culture in 96-well round-bottom plates in a volume of 200 .mu.l,
50 .mu.l of medium is removed from each well for determination of
IFN-.gamma. levels, as described below. The plates are then pulsed
with 1 .mu.Ci/well of tritiated thymidine for a further 18 hours,
harvested and tritium uptake determined using a gas scintillation
counter. Fractions that result in proliferation in both replicates
three fold greater than the proliferation observed in cells
cultured in medium alone are considered positive.
[0423] IFN-.gamma. is measured using an enzyme-linked immunosorbent
assay (ELISA). ELISA plates are coated with a mouse monoclonal
antibody directed to human IFN-.gamma. (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 washed 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-.gamma. serum
diluted 1:3000 in PBS/10% normal goat serum is 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.) is added 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 is stopped after 20 min with 1 N sulfuric acid.
Optical density is determined at 450 nm using 570 nm as a reference
wavelength. Fractions that result in both replicates giving an OD
two fold greater than the mean OD from cells cultured in medium
atone, plus 3 standard deviations, are considered positive.
Example 3
Purification and Characterization of M. tuberculosis Polypeptides
Using CD4+T Cell Lines Generated from a Mouse M. tuberculosis
Model
[0424] Infection of C57BL/6 mice with M. tuberculosis results in
the development of a progressive disease for approximately 2-3
weeks. The disease progression is then halted as a consequence of
the emergence of a strong protective T cell-mediated immune
response. This infection model was used to generate T cell lines
capable of recognizing protective M. tuberculosis antigens.
[0425] Specifically, spleen cells were obtained from C57BL/6 mice
infected with M. tuberculosis for 28 days and used to raise
specific anti-M. tuberculosis T cell lines as described above. The
resulting CD4+T cell lines, in conjunction with normal antigen
presenting (spleen) cells from C57BL/6 mice were used to screen the
M. tuberculosis Erd .lamda.Screen library described above. One of
the reactive library pools, which was found to be highly
stimulatory of the T cells, was selected and the corresponding
active clone (referred to as Y288C10) was isolated.
[0426] Sequencing of the clone Y2SSC10 revealed that it contains
two potential genes, in tandem. The determined cDNA sequences for
these two genes (referred to as mTCC#1 and mTCC#2) are provided in
SEQ ID NO:139 and 140, respectively, with the corresponding
predicted amino acid sequences being provided in SEQ ID NO: 141 and
142, respectively. Comparison of these sequences with those in
publicly available sequence databases revealed identity to unknown
sequences previously found within the M. tuberculosis cosmid
MTY21C12. The predicted amino acid sequences of mTCC#1 and mTCC#2
were found to show some homology to previously identified members
of the TbH9 protein family, discussed above.
Example 4
Synthesis of Synthetic Polypeptides
[0427] Polypeptides may be synthesized on a Millipore 9050 peptide
synthesizer using FMOC chemistry with HIPTU
(O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate)
activation. A Gly-Cys-Gly sequence may be attached to the amino
terminus of the peptide to provide a method of conjugation or
labeling of the peptide. Cleavage of the peptides from the solid
support may be carried out using the following cleavage mixture:
trifluoroacetic acid:ethanedithiol:thioanisole:water:phenol
(40:1:2:2:3). After cleaving for 2 hours, the peptides may be
precipitated in cold methyl-t-butyl-ether. The peptide pellets may
then be dissolved in water containing 0.1% trifluoroacetic acid
(TFA) and lyophilized prior to purification by C18 reverse phase
HPLC. A gradient of 0-60% acetonitrile (containing 0.1% TFA) in
water (containing 0.1% TFA) may be used to elute the peptides.
Following lyophilization of the pure fractions, the peptides may be
characterized using electrospray mass spectrometry and by amino
acid analysis.
Example 5
Use of Representative Antigens for Serodiagnosis of
Tuberculosis
[0428] The diagnostic properties of representative M. tuberculosis
antigens may be determined by examining the reactivity of antigens
with sera from tuberculosis infected patients and from normal
donors as described below.
[0429] Assays are performed in 96-well plates coated with 200 ng
antigen diluted to 50 .mu.l in carbonate coating buffer, pH 9.6.
The wells are coated overnight at 4.degree. C. (or 2 hours at
37.degree. C.). The plate contents are then removed and the wells
are blocked for 2 hours with 200 .mu.l of PBS/1% BSA. After the
blocking step, the wells are washed five times with PBS/0.1% TWEEN
20.TM.. 50 .mu.l sera, diluted 1:100 in PBS/0.1% TWEEN 20/0.1% BSA,
is then added to each well and incubated for 30 minutes at room
temperature. The plates are washed again five times with PBS/0.1%
TWEEN 20.TM..
[0430] The enzyme conjugate (horseradish peroxidase--Protein A,
Zymed, San 30 Francisco, Calif.) is then 1:10,000 in PBS/0.1% TWEEN
20.TM./0.1% BSA, and 50 .mu.l of the diluted conjugate is added to
each well and incubated for 30 minutes at room temperature.
Following incubation, the wells are washed five times with PBS/0.1%
TWEEN 20.TM. 100 .mu.l of tetramethylbenzidine peroxidase (TMB)
substrate (Kirkegaard and Perry Laboratories, Gaithersburg, Md.) is
added, undiluted, and incubated for about 15 minutes. The reaction
is stopped with the addition of 100 .mu.l of 1 NH.sub.2SO.sub.4 to
each well, and the plates are read at 450 nm.
Example 6
Murine T Cell Expression Cloning of an MTB Antigen Associated with
the Control of TB Infection
[0431] Genomic DNA form M. tuberculosis Erdman strain was randomly
sheared to an average size of 2 kb, blunt ended with Klenow
polymerase and followed by the addition of EcoRI adaptors. The
insert was subsequently ligated into the Screen phage vector
predigested with EcoRI (Novagen, Madison, Wis.) and packaged in
vitro using the PhageMaker extract (Novagen, Madison, Wis.). The
phage library (Erd Screen) was amplified and a portion converted
into a plasmid expression library (pScreen) by autosubcloning using
the E. coli host strain BM25.8 as suggested by the manufacturer
(Novagen, Madison, Wis.). Plasmid DNA was purified from BM25.8
cultures containing pScreen recombinants and used to transform
competent cells of the expressing host strain BL21(DB3)pLysS.
Transformed cells were aliquoted into 96 well micro titer plates
with each well containing a pool size of 50 colonies. Replica
plates of the 96 well plasmid library format were induced with IPTG
to allow recombinant protein expression. Following induction, the
plates were centrifuged to pellet the E. coli and the bacterial
pellet was resuspended in 200 td of IX PBS. The general principle
is based on the direct recognition by the T cells of the antigens
presented by antigen presenting cells that have internalized a
library of E. coli-containing expressed recombinant antigens. The
M. tuberculosis library was initially divided in pools containing
approximately 50-100 transformants/ml distributed in 96-well
microtiter plates and stored in a replica plate manner. Adherent
spleen cells were fed with the E. coli pools and incubated for
processing for 2 h. After washing the adherent cells were exposed
to specific T cell lines in the presence of gentamycin (50
.mu.g/ml) to inhibit the bacterial growth. T cell recognition of
pool containing M. tuberculosis antigens was then detected by
proliferation (3H thymidine incorporation). Wells that scored
positive were then broken down using the same protocol until a
single clone was detected. The gene was then sequenced, sub-cloned,
expressed and the recombinant protein evaluated. Nucleotide
sequence comparison of the 0.6 kb insert of clone mTTC#3 with the
GenBank database revealed that it is comprised of the amino
terminal portion of gene MTV014.03e (locus MTV014; accession
#e1248750) of the Mtb H37Rv strain. The full length nucleotide
sequence of mTTC#3 (SEQ ID NO:145) is a 1.86 kb fragment comprising
the entire ORF with a predicted molecular weight of .about.57 kDa
(SEQ ID NO: 146). Thus, to maintain consistency with our
nomenclature, mTTC#3 is referred to hereafter as MTB57. The full
length coding portion of mTTC#3 (MTB57) was PCR amplified using the
following primer pairs: 5'(5'-CAA TTA CAT ATG CAT CAC CAT CAC CAT
CAC ATG AAT TAT TCG GTG TTG CCG (SEQ ID NO: 147)) and 3' (5'-CAA
TTA AAG CTT TTA GGG CTG ACC GAA GAA GCC (SEQ ID NO:148))h3. The
full length nucleic acid coding sequence of mTTC#3 and the
corresponding predicted amino acid sequence are provided in FIGS. 3
and 4, respectively.
Example 7
Identification of Mycobacterium Tuberculosis Antigens Excreted in
Urine of Infected Mice
[0432] Antigen were prepared by infecting intravenously C57BL/6
mice with 4.10.sup.7 colony forming units (CFU) of M. tuberculosis.
14 days later the animals were bled and their urine was collected
in microfuge tubes. Sera were obtained at room temperature. Both
sera and urine were centrifuged at 10,000 g for 15 minutes followed
by filtration in 0.2 u sterile membranes.
[0433] Antibodies were produced against the antigens by immunizing
normal C57BL/6 mice with either the sera or the urine from the M.
tuberculosis infected C57BL/6 mice. The adjuvant used was
incomplete Freund's adjuvant (IFA). Immunization was carried out
according to the following protocol: on day 1, mice were injected
in the footpad or in the base of the tail with a mix containing 100
Id of either serum or urine and 100 .mu.l of IFA; on day 14, a mix
containing 100 .mu.l of either serum or urine and 100 .mu.l of IFA
was injected intraperitoneally to the mice; finally on day 28,
either 200 .mu.l of serum or 50 .mu.l of urine were injected to the
mice intraperitoneally. By using syngeneic mice for the antibody
production, only antibodies specific for foreign antigens present
in the blood circulation or urine of the C57BLU6 mice, i.e., M.
tuberculosis antigens, are generated. On day 35, 100 .mu.l of blood
were collected by eye-bleeding the immunized mice. ELISA assays
were performed with the obtained sera using a M. tuberculosis crude
lysate. The ELISA experiments revealed that all the mice immunized
with either sera or urine from infected donors produced anti-M.
tuberculosis antibodies in titers varying from 1/40 to 1/320. No
anti-M. tuberculosis antibodies were found in the sera obtained
from the mice before the immunizations.
[0434] The antiserum made against the proteins excreted in the
urine was used to screen a Mtb expression library prepared in the
lambda screen phage expression system. Positive clones were
purified and their corresponding inserts sequenced. These inserts
were named P1, 2, 3, 4, 6, 7, 8, 9, 10, 11 and 12 (SEQ ID
NO:149-159).
Example 8
Identification of Mycobacterium Tuberculosis Antigens Using CD4+T
Cell Expression Cloning
[0435] Expression screening using a number of T cell lines
generated from healthy PPD-positive individuals has been employed
to identify M. tuberculosis clones encoding reactive antigens.
Pools of M. tuberculosis recombinant clones (expressed in E. coli)
were fed to dendritic cells. Autologous T cell lines were incubated
with the dendritic cells and proliferation and INF-gamma production
was measured. Reactive pools were fractionated and re-tested until
pure M. tuberculosis clones were achieved. This approach allows for
direct screening for T cell antigens. A related approach has been
used to identify Listeria monocytogenes antigens (see J. Exp. Med.
182:1751-1757 (1995).
[0436] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for the purpose of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Sequence CWU 1
1
16411886DNAMycobacterium tuberculosismisc_feature(1)..(1886)n is a,
c, g or t 1cgctctggtg accaccaact tcttcggtgt caacaccatc ccgatcgccc
tcaacgaggc 60cgactacctg cgcatgtgga tccaggccgc caccgtcatg agccactatc
aagccgtcgc 120gcacgaaatc tggtgtctcc atgaatangc cagttcggga
aagccgtggg ccagtatcac 180cacgggtgcg ccgggctcac cggcctcgac
cactcgcagt cgcacgccgt tggtatcaac 240taaccgtncn gtangtgcgc
ccatcgtctc accaaatcac accgggcacc ggcctgagaa 300gggcttgggg
agcanccaga ggcgattgtc gcgggtgctg ccgcgcatca ttgatcggcc
360ggccggacca ntcgggcctc ccttgacgtc cggatcncac ttcctgtgca
gctggcatgg 420ctacagctca cagtgactgc cccacgattg ccggccaggt
ccagttcaaa ttccggtgaa 480ttcgcggaca aaagcagcag gtcaaccaac
cgcagtcagt cgagggtccc aaacgtgagc 540caatcggtga aatggcttgc
tgcagtgaca ccggtcacag gcttagccga cagcaccgga 600atagctcagg
cgggctatag agtcctatag aaacatttgc tgatagaatt aaccgctgtc
660ttggcgtgat cttgatacgg ctcgccgtgc gaccggttgg ctcagtagct
gaccaccatg 720taacccatcc tcggcaggtg tctactaagg cgagacaccg
cattggtggg gctgcatcgc 780aaatcggtcc gagcatgtag cactgccgtt
atcccgggat agcaaaccac ccggaaccag 840ggctatccca gtcgctctcc
gacggaggcc gtttcgcttt ccgttgcccg ataactcccg 900agtggatatc
ggcgttatca nattcaggct tttcttcgca aggtaccggt gttcgctata
960ttcggatatc tcggacggat aattactaaa acttcagtgg tttagataag
gccgccgcaa 1020tacttcgccg atcttgccga gcgcaacgga tttccatcgt
cggttttcgt cgccttatca 1080aacatgatcg gagataatga cagatcggcc
tagctaggtg tttagcggac gcgatttagg 1140acaaccgaga tttgctttgc
ctcgcaacca tgagagcgcc ccgcttcgac gccgaatcgg 1200gtgagtgatg
gtgggttagc acagccctga ttgcgccacc ggcgaggtga ttgtgcccgc
1260cacgaggccg ccgccggcta gccccatgag cacgntatat agactctcct
gcaacagatc 1320tcataccgat cgaaggcgaa gcgcaggcat cgacgtcgga
gacactgcct tgggatcgcg 1380ccgcctacac ggcggttggc gcattgtcgc
agcgcagttg caggagggca aatgtgcgca 1440gacgatgtag tcgacaacaa
gtgnacatgc cgtcttcacg aactcaaaac tgacgatctg 1500cttagcatga
aaaaaactgt tgacatcggc caagcatgac agccagactg taggcctacg
1560cgtgcaatgc agaaccaagg ntatgcatgg aatcgacgac cgttgagata
ggcggcaggc 1620atgagcagag cgttcatcat cgatccaacg atcagtgcca
ttgacggctt gtacgacctt 1680ctggggattg gaatacccaa ccaagggggt
atcctttact cctcactaga gtacttcgaa 1740aaagccctgg aggagctggc
agcagcgttt ccgggtgatg gctggttagg ttcggccgcg 1800gacaaatacg
ccggcaaaaa ccgcaaccac gtgaattttt tccaggaact ggcagacctc
1860gatcgtcagc tcatcagcct gatcca 188622305DNAMycobacterium
tuberculosismisc_feature(1)..(2305)n is a, c, g or t 2ggcacgcgct
ggccgcgcaa tacaccgaaa ttgcaacgga actcgcaagc gtgctcgctg 60cggtgcaggc
aagctcgtgg caggggccca gcgccgaccg gttcgtcgtc gcccatcaac
120cgttccggta ttggctaacc cacgctgcca cggtggccac cgcagcagcc
gccgcgcacn 180aaacggccgc cgccgggtat acgtccgcat tggggggcat
gcctacgcta gccgagttgg 240cggccaacca tgccatgcac ggcgctctgg
tgaccaccaa cttcttcggt gtcaacacca 300tcccgatcgc cctcaacgag
gccgactacc tgcgcatgtg gatccaggcc gccaccgtca 360tgagccacta
tcaagccgtc gcgcacgaaa gcgtggcggc gacccccagc acgccgccgg
420cgccgcagat agtgaccagt gcggccagct cggcggctag cagcagcttc
cccgacccga 480ccaaattgat cctgcagcta ctcaaggatt tcctggagct
gctgcgctat ctggctgttg 540agctgctgcc ggggccgctc ggcgacctca
tcgcccaggt gttggactgg ttcatctcgt 600tcgtgtccgg tccagtcttc
acgtttctcg cctacctggt gctggaccca ctgatctatt 660tcggaccgtt
cgccccgctg acgagtccgg tcctgttgcc tgctgtggag ttacgcaacc
720gcctcaaaac cgccaccgga ctgacgctgc cacctaccgt gattttcgat
catcccactc 780ccactgcggt cgccgagtat gtcgcccagc aaatgtctgg
cagccgccca acggaatccg 840gtgatccgac gtcgcaggtt gtcgaacccg
ctcgtgccga attcggcacg agtgctgttc 900atcaaatccc cccgagacct
gcggacaccc ggcgcgcttg ccgacatcga gatgatgtcc 960cgcgagatag
cagaattgcc caacatcgtg atggtgcggg gcttgacccg accgaacggg
1020gaacctctga aggagaccaa ggtctcgttt caggctggtg aagtgggcgg
caagctcgac 1080gaagcgacca ccctgctcga agagcacgga ggcgagctgg
accagctgac cggcggtgcg 1140caccagttgg ccgacgccct cgcccaaata
cgcaacgaaa tcaatggggc cgtggccagc 1200tcgagcggga tagtcaacac
cctgcaggcc atgatggacc tgatgggcgg tgacaagacc 1260atccgacaac
tggaaaatgc gtcccaatat gtcgggcgca tgcgggctct gggggacaat
1320ctgagcggga ccgtcaccga tgccgaacaa atcgccactt gggccagccc
tatggtcaac 1380gccctcaact ccagcccggt gtgtaacagc gatcccgcct
gtcggacgtc gcgcgcacag 1440ttggcggcga ttgtccaggc gcaggacgac
ggcctgctca ggtccatcag agcgctagcc 1500gtcaccctgc aacagacgca
ggaataccag acactcgccc ggacggtgag cacactggac 1560gggcaactga
agcaagtcgt cagcaccctc aaagcggtcg acggcctacc caccaaattg
1620gctcaaatgc agcaaggagc caacgctctc gccgacggca gcgcagcgct
ggcggcaggc 1680gtgcaggaat tggtcgatca ggtcaaaaag atgggctcag
ggctcaacga ggccgccgac 1740ttcctgttgg ggatcaagcg ggatgcggac
aagccgtcaa tggcgggctt caacattcca 1800ccgcagattt tttcgaggga
cgagttcaag aagggcgccc agattttcct gtcggccgat 1860ggtcatgcgg
cgcggtactt cgtgcagagc gcgctgaatc cggccaccac cgaggcgatg
1920gatcaggtca acgatatcct ccgtgttgcg gattccgcgc gaccgaatac
cgaactcgag 1980gatgccacga taggtctggc gggggttccg actgcgctgc
gggatatccg cgactactac 2040aacagcgata tgaaattcat cgtcattgcg
acgatcgtta tcgtattctt gattctcgtc 2100attctgntgc gcgcacttgt
ggntccgata tatctgatag gctcggtgct gatttcttac 2160ttgtcggccc
taggcatagg aactttcgtt ttccaattga tactgggcca ggaaatgcat
2220tggagcctgc cgggactgtc cttcatatta ttggttgcca tcggcgctga
ctacaacatg 2280ctgctcattt cacgcatccg cgacg
230531741DNAMycobacterium tuberculosismisc_feature(1)..(1741)n is
a, t, c or g 3ccgctctctt tcaacgtcat aagttcggtg ggccagtcgg
ccgcgcgtgc atatggcacc 60aataacgcgt gtcccatgga tacccggacc gcacgacggt
agagcggatc agcgcagccg 120gtgccgaaca ctaccgcgtc cacgctcagc
cctgccgcgt tgcggaagat cgagcccagg 180ttctcatggt cgttaacgcc
ttccaacact gcgacggtgc gcgccccggc gaccacctga 240gcaacgctcg
gctccggcac ccggcgcgcg gctgccaaca ccccacgatt gagatggaag
300ccgatcaccc gtgccatgac atcagccgac gctcgatagt acggcgcgcc
gacaccggcc 360agatcatcct tgagctcggc cagccggcgg tcggtgccga
acagcgccag cggcgtgaac 420cgtgaggcca gcatgcgctg caccaccagc
acaccctcgg cgatcaccaa cgccttgccg 480gtcggcagat cgggacnacn
gtcgatgctg ttcaggtcac ggaaatcgtc gagccgtggg 540tcgtcgggat
cgcagacgtc ctgaacatcg aggccgtcgg ggtgctgggc acaacggcct
600tcggtcacgg gctttcgtcg accagagcca gcatcagatc ggcggcgctg
cgcaggatgt 660cacgctcgct gcggttcagc gtcgcgagcc gctcagccag
ccactcttgc agagagccgt 720tgctgggatt aattgggaga ggaagacagc
atgtcgttcg tgaccacaca gcggaagccc 780tggcagctgc ggcggcgaac
ctacagggta ttggcacgac aatgaacgcc cagaacgcgg 840ccgcggctgc
tccaaccacc ggagtagtgc ccgcagccgc cgatgaagta tcagcgctga
900ccgcggctca gtttgctgcg cacgcgcaga tgtaccaaac ggtcagcgcc
caggccgcgg 960ccattcacga aatgttcgtg aacacgctgg tggccagttc
tggctcatac gcggccaccg 1020aggcggccaa cgcagccgct gccggctgaa
cgggctcgca cgaacctgct gaaggagagg 1080gggaacatcc ggagttctcg
ggtcaggggt tgcgccagcg cccagccgat tcagntatcg 1140gcgtccataa
cagcagacga tctaggcatt cagtactaag gagacaggca acatggcctc
1200acgttttatg acggatccgc atgcgatgcg ggacatggcg ggccgttttg
aggtgcacgc 1260ccagacggtg gaggacgagg ctcgccggat gtgggcgtcc
gcgcaaaaca tttccggtgc 1320gggctggagt ggcatggccg aggcgacctc
gctagacacc atgacctaga tgaatcaggc 1380gtttcgcaac atcgtgaaca
tgctgcacgg ggtgcgtgac gggctggttc gcgacgccaa 1440caantacgaa
cagcaagagc aggcctccca gcagatcctg agcagntagc gccgaaagcc
1500acagctgngt acgntttctc acattaggag aacaccaata tgacgattaa
ttaccagttc 1560ggggacgtcg acgctcatgg cgccatgatc cgcgctcagg
cggcgtcgct tgaggcggag 1620catcaggcca tcgttcgtga tgtgttggcc
gcgggtgact tttggggcgg cgccggttcg 1680gtggcttgcc aggagttcat
tacccagttg ggccgtaact tccaggtgat ctacgagcag 1740g
174142836DNAMycobacterium tuberculosismisc_feature(1)..(2836)n is
a, c, t or g 4gttgattccg ttcgcggcgc cgccgaagac caccaactcc
gctggggtgg tcgcacaggc 60ggttgcgtcg gtcagctggc cgaatcccaa tgattggtgg
ctcngtgcgg ttgctgggct 120cgattacccc cacggaaagg acgacgatcg
ttcgtttgct cggtcagtcg tacttggcga 180cgggcatggc gcggtttctt
acctcgatcg cacagcagct gaccttcggc ccagggggca 240caacggctgg
ctccggcgga gcctggtacc caacgccaca attcgccggc ctgggtgcag
300gcccggcggt gtcggcgagt ttggcgcggg cggagccggt cgggaggttg
tcggtgccgc 360caagttgggc cgtcgcggct ccggccttcg cggagaagcc
tgaggcgggc acgccgatgt 420ccgtcatcgg cgaagcgtcc agctgcggtc
agggaggcct gcttcgaggc ataccgctgg 480cgagagcggg gcggcgtaca
ggcgccttcg ctcaccgata cgggttccgc cacagcgtga 540ttacccggtc
tccgtcggcg ggatagcttt cgatccggtc tgcgcggccg ccggaaatgc
600tgcagatagc gatcgaccgc gccggtcggt aaacgccgca cacggcacta
tcaatgcgca 660cggcgggcgt tgatgccaaa ttgaccgtcc cgacggggct
ttatctgcgg caagatttca 720tccccagccc ggtcggtggg ccgataaata
cgctggtcag cgcgactctt ccggctgaat 780tcgatgctct gggcgcccgc
tcgacgccga gtatctcgag tgggccgcaa acccggtcaa 840acgctgttac
tgtggcgtta ccacaggtga atttgcggtg ccaactggtg aacacttgcg
900aacgggtggc atcgaaatca acttgttgcg ttgcagtgat ctactctctt
gcagagagcc 960gttgctggga ttaattggga gaggaagaca gcatgtcgtt
cgtgaccaca cagccggaag 1020ccctggcagc tgcggcggcg aacctacagg
gtattggcac gacaatgaac gcccagaacg 1080cggccgcggc tgctccaacc
accggagtag tgcccgcagc cgccgatgaa gtatcagcgc 1140tgaccgcggc
tcagtttgct gcgcacgcgc agatgtacca aacggtcagc gcccaggccg
1200cggccattca cgaaatgttc gtgaacacgc tggtggccag ttctggctca
tacgcggcca 1260ccgaggcggc caacgcagcc gctgccggct gaacgggctc
gcacgaacct gctgaaggag 1320agggggaaca tccggagttc tcgggtcagg
ggttgcgcca gcgcccagcc gattcagcta 1380tcggcgtcca taacagcaga
cgatctaggc attcagtact aaggagacag gcaacatggc 1440ctcacgtttt
atgacggatc cgcatgcgat gcgggacatg gcgggccgtt ttgaggtgca
1500cgcccagacg gtggaggacg aggctcgccg gatgtgggcg tccgcgcaaa
acatttccgg 1560tgcgggctgg agtggcatgg ccgaggcgac ctcgctagac
accatgacct agatgaatca 1620ggcgtttcgc aacatcgtga acatgctgca
cggggtgcgt gacgggctgg ttcgcgacgc 1680caacaactac gaacagcaag
agcaggcctc ccagcagatc ctgagcagct agcgccgaaa 1740gccacagctg
cgtacgcttt ctcacattag gagaacacca atatgacgat taattaccag
1800ttcggggacg tcgacgctca tggcgccatg atccgcgctc aggcggcgtc
gcttgaggcg 1860gagcatcagg ccatcgttcg tgatgtgttg gccgcgggtg
acttttgggg cggcgccggt 1920tcggtggctt gccaggagtt cattacccag
ttgggccgta acttccaggt gatctacgag 1980caggccaacg cccacgggca
gaaggtgcag gctgccggca acaacatggc gcaaaccgac 2040agcgccgtcg
gctccagctg ggcctaaaac tgaacttcag tcgcggcagc acaccaacca
2100gccggtgtgc tgctgtgtcc tgcagttaac tagcactcga ccgctgaggt
agcgatggat 2160caacagagta cccgcaccga catcaccgtc aacgtcgacg
gcttctggat gcttcaggcg 2220ctactggata tccgccacgt tgcgcctgag
ttacgttgcc ggccgtacgt ctccaccgat 2280tccaatgact ggctaaacga
gcacccgggg atggcggtca tgcgcgagca gggcattgtc 2340gtcaacgacg
cggtcaacga acaggtcgct gcccggatga aggtgcttgc cgcacctgat
2400cttgaagtcg tcgccctgct gtcacgcggc aagttgctgt acggggtcat
agacgacgag 2460aaccagccgc cgggttcgcg tgacatccct gacaatgagt
tccgggtggt gttggcccgg 2520cgaggccagc actgggtgtc ggcggtacgg
gttggcaatg acatcaccgt cgatgacgtg 2580acggtctcgg atagcgcctc
gatcgccgca ctggtaatgg acggtctgga gtcgattcac 2640cacgccgacc
cagccgcgat caacgcggtc aacgtgccaa tggaggagat ctcgtgccga
2700attcggcacg aggcacgagg cggtgtcggt gacgacggga tcgatcacga
tcatcgaccg 2760gccgggatcc ttggcgatct cgttgagcac gacccgggcc
cgcgggaagc tctgcgacat 2820ccatgggttc ttcccg
28365900DNAMycobacterium tuberculosismisc_feature(1)..(900)n is a,
t, c or g 5aacatgctgc acggggtgcg tgacgggctg gttcgcgacg ccaacaacta
cgagcagcaa 60gagcaggcct cccagcagat cctcagcagc taacgtcagc cgctgcagca
caatactttt 120acaagcgaag gagaacaggt tcgatgacca tcaactatca
gttcggtgat gtcgacgctc 180acggcgccat gatccgcgct caggccgggt
tgctggaggc cgaacatcag gccatcattc 240gtgatgtgtt gaccgcgagt
gacttttggg gcggcgccgg ttcggcggcc tgccaggggt 300tcattaccca
attgggccgt aacttccagg tgatctacga acaggccaac gcccacgggc
360agaaggtgca ggctgccggc aacaacatgg cgcaaaccga cagcgccgtc
ggctccagct 420gggcctgaca ccaggccaag gccagggacg tggtgtacga
gtgaaggttc ctcgcgtgat 480ccttcgggtg gcagtctagg tggtcagtgc
tggggtgttg gtggtttgct gcttggcggg 540ttcttcggtg ctggtcagtg
ctgctcgggc tcgggtgagg acctcgaggc ccaggtagcg 600ccgtccttcg
atccattcgt cgtgttgttc ggcgaggacg gctccgacga ggcggatgat
660cgaggcgcgg tcggggaaga tgcccacgac gtcggttcgg cgtcgtacct
ctcggttgag 720gcgttcctgg gggttgttgg accagatttg gcgccagatc
ttcttgggga aggcggtgaa 780cgccagcagg tcggtgcggg cggtgtcgan
gtgctcggcc accgcgggga gtttgtcggt 840cagagcgtcg agtacccgat
catattgggc aacaactgat tcggcgttgg gctggtcgta
90061905DNAMycobacterium tuberculosismisc_feature(1)..(1905)n is a,
t, c or g 6gctcgccgga tgtgggcgtc cgcgcaaaac atttccggtg cgggctggag
tggcatggcc 60gaggcgacct cgctagacac catggcccag atgaatcagg cgtttcgcaa
catcgtgaac 120atgctgcacg gggtgcgtga cgggctggtt cgcgacgcca
acaactacga gcagcaagag 180caggcctccc agcagatcct cagcagctaa
cgtcagccgc tgcagcacaa tacttttaca 240agcgaaggag aacaggttcg
atgaccatca actatcagtt cggtgatgtc gacgctcacg 300gcgccatgat
ccgcgctcag gccgggttgc tggaggccga gcatcaggcc atcattcgtg
360atgtgttgac cgcgagtgac ttttggggcg gcgccggttc ggcggcctgc
caggggttca 420ttacccagtt gggccgtaac ttccaggtga tctacgaaca
agccaacacc cacgggcaga 480aggtgcaagc tgccggcaac aacatggcgc
aaaccgacag cgccgtcngc tccagctggg 540cctgacacca ggccaaggcc
agggacgtgg tgtacnagtg aaggttcctc gcgtgatcct 600tcgggtggca
gtctaggtgg tcagtgctgg ggtgttggtg gtttgctgct tggcgggttc
660ttcggtgctg gtcagtgctg ctcgggctcg ggtgaggacc tcgaggccca
ggtagcgccg 720tccttcgatc cattcgtcgt gttgttcggc gaggacngct
ccgacgangc ggatgatcga 780ggcgcggtcg gggaagatgc ccacgacgtc
ggttcggcgt cgtacctctc ggttgaagcg 840ttcctggggg ccaccgcttg
gcgccnangc actccacgcc aattcgtcnc acctaacagc 900ggtggccaac
gactatgact acgacaccgt ttttgccagg gccctcnaaa ggatctgcgc
960gtcccggcga cacgcttttt gcgataagta cctccggcaa ttctatgagt
gtactgcggn 1020ccgcgaaaac cgcaagggag ttgggtgtga cggttnttgc
aaatgacggg cgaatccggc 1080ggccagctgg cagaattcgc agatttcttg
atcaacgtcc cgtcacgcga caccgggcga 1140atccaggaat ctcacatcgt
ttttattcat gcgatctccg aacatgtcga acacgcgctt 1200ttcgcgcctc
gccaatagga aagccgatcc ttacgcggcc attcgaaaga tggtcgcgga
1260acgtgcggga caccaatggt gtctcttcct cgatagagac ggggtcatca
atcgacaagt 1320ggtcggcgac tacgtacgga actggcggca gtttgaatgg
ttgcccgggg cggcgcgggc 1380gttgaagaag ctacgggcat gggctccgta
catcgttgtc gtgacaaacc agcagggcgt 1440gggtgccgga ttgatgagcg
ccgtcgacgt gatggtgata catcggcacc tccaaatgca 1500gcttgcatcc
gatggcgtgc tgatagatgg atttcaggtt tgcccgcacc accgttcgca
1560gcggtgtggc tgccgtaagc cgagaccggg tctggtcctc gactggctcg
gacgacaccc 1620cgacagtgag ccattgctga gcatcgtggt tggggacagc
ctcagcgatc ttgacattgg 1680cacacaacgt cgccgctgct gccggtgcat
gtgccagtgt ccagataggg ggcgccagtt 1740ctggcggtgt cgctgacgcg
tcatttgact cgctctggga gttcgctgtc gcagtcggac 1800atgcgcgggg
ggagcggggc taatggcgat cttgcgcggg cgagcgccgt ngcggntcgg
1860actnngcggt ggcgggacag acgtggaacc gtactcgagc cagtt
190572921DNAMycobacterium tuberculosismisc_feature(1)..(2921)n is
a, t, c or g 7cgggatgccg tggtggttgg tattgcccaa accctggcgc
tggtccccgg ggtatccagg 60tccgggtcga ccatcagcgc tggactgttt ctcggactcg
accgtgaact ggccgcccga 120ttcggattcc tgctggccat tccagcggtg
ttcgcctccg ggttgttctc gttgcccgac 180gcattccacc cggtaaccga
gggcatgagc gctactggcc cgcagttgct ggtggccacc 240ctgatcgcgt
tcgtcctcgg tctgaccgcg gtggcctggc tgctgcggtt tctggtgcga
300cacaacatgt actggttcgt cggctaccgg gtgctcgtcg ggacgggcat
gctcgtgctg 360ctggctaccg ggacggtagc cgcgacatga ccgtcatctt
gctacgccat gcccgttcca 420cctcgaacac cgcgggcgtg ctggccggcc
ggtccggcgt cgacctcgac gagaaggggc 480gcgagcaggc caccgggttg
atcgatcgaa ttggtgacct gccgatccgg gcggtcgcgt 540cttctccaat
gctgcggtgt caacgcaccg tcgaaccgct ggccgaggcg ctgtgcctgg
600agccgctcat cgatgaccgg ttctccgaag tcgactacgg cgaatggact
ggcagaaaaa 660tcggtgacct ggtcgacgag ccgttgtggc gggtagtcca
ggcccacccc agcgcggcgg 720tgtttcccgg cggtgagggt ttggcgcagg
tgcagacgtg gttgtcctga cggatttcca 780tgccggggaa caccaagacc
ggatcggcac tggcggtcgc cggcgaaaac ccggccgcca 840atagggcgac
cgtcgctgcg aatgcgcgtg gtaccaggcg gaccaccttg aactcccatc
900cgtcggggcc aagcgcatcg cccgccgccg gttacggcta aggcgtacca
aaacccgacg 960gtaatacttc ggcaatgtcg ggtcncgacg ttaccgagac
gtgaccagng aggcngcggc 1020attggattta tcgatggtgc gcggttccca
ncccggcggt ccgaanacgt agcccagccg 1080atcccgcaga cgtgttgccg
accgccagtc acgcacgatc gccacgtact cgcgggtctg 1140cagcttccag
atgttgaacg tgtcgacccg cttggtcagg ccataatgcg gtcggaatag
1200ctccggctga aagctaccga acaggcggtc ccagatgatg aggatgccgc
catagttctt 1260gtccanatac accgggtcca ttccgtggtg gacccggtgg
tgcgacgggg tattgaagac 1320gaattcgaac caccgcggca gcctgtcgat
ccgctcggtg tgcacccaga actggtagat 1380caagttcagc gaccaattgc
agaacaccat ccaaggggga agccccatca gtggcagcgg 1440aacccacatg
agaatctcgc cgctgttgtt ccantttctg gcgcagcgcg gtggcgaagt
1500tgaagtattc gctggagtga tgcgcctggt gggtagccca gatcagccga
actcggtggg 1560cgatgcggtg ataggagtag tacagcagat cgacaccaac
gatcgcgatc acccaggtgt 1620accaccggtg ggcggacagc tgccaggggg
caaggtaggc atagattgcg gcataaccga 1680gcagggcaag ggacttccag
ccggcggtgg tggctatcga aaccagcccc atcgagatgc 1740tggccaccga
gtcgcgggtg aggtaagcgc ccgaggcggg ccgtggctgc ccggtagcag
1800cggtctcgat gctttccagc ttgcgggccg ccgtccattc gagaatcagc
agcaatagaa 1860aacatggaat ggcgaacagt accgggtccc gcatttcctc
gggcagcgct gagaagaatc 1920cggcgacggc atggccgagg cgacctcgnt
agacaccatg acccagatga atcaggcgtt 1980tcgcaacatc gtgaacatgc
tgcacggggt gcgtgacggg ctggttcgcg acgccaacaa 2040ntacgaacag
caagagcagg cctcccagca gatcctcagc agctgacccg gcccgacgac
2100tcaggaggac acatgaccat caactatcaa ttcggggacg tcgacgctca
cggcgccatg 2160atccgcgctc aggccgggtc gctggaggcc gagcatcagg
ccatcatttc tgatgtgttg 2220accgcgagtg acttttgggg cggcgccggt
tcggcggcct gccaggggtt cattacccag 2280ctgggccgta acttccaggt
gatntacgag caggccaacg cccacgggca gaaggtgcag 2340gctgccggca
acaacatggc acaaaccgac agcgccgtcg gctccagctg ggcataaagn
2400tggcttaagg cccgcgccgt caattacaac gtggccgcac accggttggt
gtgtggccac 2460gttgttatct gaacgactaa ctacttcgac ctgctaaagt
cggcgcgttg atccccggtc 2520ggatggtgct gaactgggaa gatggcctca
atgcccttgt tgcggaaggg attgaggcca 2580tcgtgtttcg tactttaggc
gatcagtgct ggttgtggga gtcgctgctg cccgacgagg 2640tgcgccgact
gcccgaggaa ctggcccggg tggacgcatt gttggacgat ccggcgttct
2700tcgccccgtt cgtgccgttc ttcgacccgc gcaggggccg gccgtcgacg
ccgatggagg 2760tctatctgca gttgatgttt gtgaagttcc gctaccggct
gggctatgag tcgctgtgcc 2820gggaggtggc tgattcgatc acctgacggc
ggttttgccg cattgcgctg gacgggtcgg
2880tgccgcatcc gaccacattg atgaagctca ccacgcgttg c
292181704DNAMycobacterium tuberculosismisc_feature(1)..(1704)n is
a, t, c or g 8cgcgatcgtc gtcaacgang tcgaccgtca ccacggactg
atcaacaagt tcgcaggcga 60cgccgccctg gccatcttcg gagccccgaa ccgcctcgac
cgtcccgaag acgccgcgct 120ggccgccgcc cgggccatan ccgancggct
ggccnacgag atgcccgagg tccaagccgg 180catcggggtg gcggcaggcc
anatcgtcgc cggcaatgtc ggcgccaagc aaagattcna 240atacacagtg
gtcggcaagc cggtcaacca ngcggcccga ttgtgcgaac tggccaaatc
300acaccccgcg cgattgggtc tcgcccgctc ggctcatggt cacccaattc
aaggactact 360ttggcctggc gcacgacctg ccgaagtggg cgagtgaagg
cgccaaagcc gccggtgagg 420ccgccaaggc gttgccggcc gccgttccgg
ccattccgag tgctggcctg agcggcgttg 480cgggcgccgt cggtcaggcg
gcgtcggtcg ggggattgaa ggttccggcc gtttggaccg 540ccacgacccc
ggcggcgagc cccgcggtgc tggcggcgtc caacggcctc ggagccgcgg
600ccgccgctga aggttcgaca cacgcgtttg gcgggatgcc gctcatgggt
ancggtgccg 660gacgtgcgtt taacaacttc gctgcccctc gatacggatt
caagccgacc gtgatcgccc 720aaccgccggc tggcggatga ccaactacgt
tcgttgatcg aggatcgaat tcnacgattc 780aaagggagga attcatatga
cctcncgttt tatgacggat ccgcacgcna tncgggacat 840ggcgggccgt
tttgaggtgc acgcccagac ggtggaggac gaggctngcn ggatgtgggc
900gtccgcgcaa aacatttccg gtgcgggctg gagtggcatg gccgaggcga
cctcgntaga 960caccatggcc cagatgaatc aggcgtttcn caacatcgtg
aacatgctgc acggggtgng 1020tgacgggctg gttcgcgacg ccaacaacta
cgaacagcaa gagcaggcct cccagcagat 1080cctcagcagc tgacccggcc
cgacgactca ggaggacaca tgaccatcaa ctatcaattc 1140ggggacgtcg
acgctcatgg cgccatgatc cgcgctntgg ccgggttgct ggaggccgag
1200catcaggcca tcatttctga tgtgttgacc gcgagtgact tttggggcgg
cgccggttcg 1260gcggcctgcc aggggttcat tacccagttg ggccgtaact
tccaggtgat ttacgagcag 1320gccaacgccc acgggcagaa ggtgcaggct
gccggcaaca acatggcaca aaccgacagc 1380gccgtnggnt ccagctgggc
ctaacccggg tcntaagttg ggtccgcgca gggcgggccg 1440atcagcgtng
actttggcgc ccgatacacg ggcatnttnt ngtcgggaac actgcgcccg
1500cgtcagntgc ccgcttcccc ttgttnggcg acgtgctcgg tgatggcttt
gacgaccgct 1560tcgccggcgc ggccaatcaa ttggtcgcgc ttgcctntag
cccattcgtg cgacgcccgc 1620ggcgccgcga gttgtccctt gaaataagga
atcacagcac gggcgaacag ctcataggag 1680tgaaaggttg ccgtggcggg gccc
170492286DNAMycobacterium tuberculosismisc_feature(1)..(2286)n is
a, t, c or g 9ccgtcttggc gtctgggcgc attgtgatct gggccanttg
cccctccacc cagaccgcgc 60ccagcttgtc gatccagccc gcgacccgga ttgccaccgc
gcgaaccggg aacggattct 120ccgctgaatt ctgggtcact tcgcagtcgc
gcgggtgatc ctgttggcga ncagcgtctg 180gaacgggcgt cnaacgcgtg
ccgtaagccc agcgtgtacg ccgtcagccc gacgccgatg 240ccgaatgcct
tgccgcccaa gctgagccgc gcgggctcca ccaagagcgt cacggtgagc
300cagccaacca gatgcaaggc gacgatcacc gcgaagtgcc gaattcggca
cgagaggtgc 360tggaaatcca gcaatacgcc cgcgagccga tctcgttgga
ccagaccatc ggcgacgang 420gcgacagnca gcttggcgat ttcatcgaaa
acagcgaggc ggtggtggnc gtcgacgcgg 480tgtccttcac tttgctgcat
gatcaactgc antcggtgct ggacacgctc tccgagcgtg 540aggcgggcgt
ggtgcggcta cgcttcggcc ttaccgacgg ccagccgcgc acccttgacg
600agatcggcca ggtctacggc gtgacccggg aacgcatccg ccagatcgaa
tccaagacta 660tgtcgaagtt gcgccatccg agccgctcac aggtcctgcg
cgactatcgt gccgaattcg 720gcacgagccg ttttgaggtg cacgcccaga
cggtggagga cgaggctcgc cggatgtggg 780cgtccgcgca aaacatttcc
ggtgcgggct ggagtggcat ggccgangcg acctcgctag 840acaccatggc
ccagatgaat caggcgtttc gcaacatcgt gaacatgctg cacggggtgc
900gtgacgggct ggttcgcgac gccaacaact acgaacagca agagcaggcc
tcccagcaga 960tcctcagcag ctgacccggc ccgacgactc aggaggacac
atgaccatca actatcaatt 1020cggggacgtc gacgctcatg gcgccatgat
ccgcgctctg gccgggttgc tggaggccga 1080gcatcaggcc atcatttctg
atgtgttgac cgcgagtgac ttttggggcg gcgccggttc 1140ggcggcctgc
caggggttca ttacccagtt gggccgtaac ttccaggtga tctacgagca
1200ggccaacgcc cacgggcaga aggtgcaggc tgccggcaac aacatggcac
aaaccgacag 1260cgccgtcggc tccagctggg cctaacccgg gtcctaagtt
gggtccgcgc agggcgggcc 1320gatcagcgtc gactttggcg cccgatacac
gggcatgtng tngtcgggaa cactgcgccc 1380gcgtcagctg cccgcttccc
cttgttcggc gacgtgctcg gtgatggctt tgacgaccgc 1440ttcgccggcg
cggccaatca attggtcgcg cttgcctcta gcctcgtgcc gaattcggca
1500cgagggtgct ggtgccgcgc tatcggcagc acgtgagctc cacgacgaac
tcatcccagt 1560gctgggttcc gcggagttcg gcatcggcgt gtcggccgga
agggccatcg ccggccacat 1620cggcgctcaa gcccgcttcg agtacaccgt
catcggcgac ccggtcaacg aggccgcccg 1680gctcaccgaa ctggccaaag
tcgaggatgg ccacgttctg gcgtcggcga tcgcggtcag 1740tggcgccctg
gacgccgaag cattgtgttg ggatgttggc gaggtggttg agctccgcgg
1800acgtgctgca cccacccaac tagccaggcc aatgaatntg gcngcacccg
aagaggtttc 1860cagcgaagta cgcggctagt cgcgcttggc tgcnttcttc
gccggcacct tccgggcagc 1920tttcctggct ggccgttttg ccggaccccg
ggctcggcga tcggccaaca gctcggcggc 1980gcgctcgtcg gttatggaag
ccacgtngtc gcccttacgc aggctggcat tggtctcacc 2040gtcggtgacg
tacggcccga atcggccgtc cttgatgacc attggcttgc cagacgccgg
2100atntgntccc agctcgcgca gcggcggagc cgaagcgctt tgccggccac
gacntttcgg 2160ctctgngtag atnttcaggg cttcgtcgag cgngatggtg
aatatatggt cttcggtgac 2220cagtgatcga gaatcgttgc cgcgctttag
atacggtcng tagcgcccgt tctgcgcggt 2280gatntc
2286101136DNAMycobacterium tuberculosismisc_feature(1)..(1136)n is
a, t, c or g 10gggcatcttc cccgaccgcg cctcgatcat ccgcctcgtc
ggagccgtcc tcgccgaaca 60acacgacgaa tggatcgaag gacggcgcta cctgggcctc
gaggtcctca cccgagcccg 120agcagcactg accagcaccg aagaaccgcc
aagcagcaaa ccaccaacac cccagcactg 180accacctaga ctgccacccg
aaggatcacg cgaggaacct tcactcgtac accacgtccc 240tggccttggc
ctggtgtcag gcccagctgg agccgacggc gctgtcggtt tgcgccatgt
300tgttgccggc agcctgcacc ttctgcccgt gggcgttggc ctgctcgtag
atcacctgga 360agttacggcc caactgggta atgaacccct ggcaggccgc
cgaaccggcg ccgccccaaa 420agtcactcgc ggtcaacaca tcacgaatga
tggcctgatg ctcggcctcc agcaacccgg 480cctgagcgcg gatcatggcg
ccgtgagcgt cgacatcacc gaactgatag ttgatggtca 540tcgaacctgt
tctccttcgc ttgtaaaagt attgtgctgc agcggctgac gttagctgct
600gaggatctgc tgggaggcct gctcttgcct cgtgccgaat tcggcacgag
aggccgcctt 660cgaagaaatc ctttgagaat tcgccaaggc cgtcgaccca
gcatggggtc agctcgccag 720ccgcgccggc tggcaaccgt tcccgctcga
gaaagacctg gaggaatacc agtgacaaac 780gacctcccag acgtccgaga
gcgtgacggc ggtccacgtc ccgctcctcc tgctggcggg 840ccacgcttgt
cagacgtgtg ggtttacaac gggcgggcgt acgacctgag tgagtggatt
900tccaagcatc ccggcggcgc cttnttcatt gggcggacca agaaccgcga
catcaccgca 960atcgtcaagt cctaccatcg tgatccggcg attgtcgagc
gaatcctgca gcggaggtac 1020gcgttgggcc gcgacgcaac ccctagggac
atccacccca agcacaatgc accggcattt 1080ctgttcaaag acgacttcaa
cagctggcgg gacaccccga agtatcgatt ngacga 113611967DNAMycobacterium
tuberculosismisc_feature(1)..(967)n is a, t, c or g 11tgagcgccaa
ccctaccgtc ggttcgtcac acggaccgca tggcctgctc cgcggactgc 60cgctagggtc
gcggatcact cggcgtagcg gcgcctttgc ccaccgatat gggttccgtc
120acagtgtggt tgcccgcccg ccatcggccg gataacgcca tgacctcagc
tcggcagaaa 180tgacaatgct cccaaaggcg tgagcacccg aagacaacta
agcaggagat cgcatgccgt 240ttgtgactac ccaaccagaa gcactggcgg
cggcggccgg cagtctgcag ggaatcggct 300ccgcattgaa cgcccagaat
gcggctgcgg cgactcccac gacgggggtg gtccggcggc 360cgccgatgaa
ntgtcggcgc tgacggcggc tcagttcgcg gcacacgccc agatctatca
420ggccgtcagc gcccaggccg cggcgattca cgagatgttc gtcaacactc
tacagatgag 480ctcagggtcg tatgctgcta ccgaggccgc caacgcggcc
gcggccggnt agaggagtca 540ctgcgatgga ttttggggcg ttgccgccgg
aggtcaattc ggtgcggatg tatgccgttc 600ctggctcggc accaatggtc
gctgcggcgt cggcctggaa cgggttggcc gcggagctga 660gttcggcggc
caccggttat gagacggtga tcactcagct cagcagtgag gggtggctag
720gtccggcgtc agcggcgatg gccgaggcag ttgcgccgta tgtggcgtgg
atgagtgccg 780ctgcggcgca agccgagcag gcggccacac aggccagggc
cgccgcggcc gcttttgagg 840cggcgtttgc cgcgacggtg cctccgccgt
tgatcgcggc caaccgggct tcgttgatgc 900agctgatctc gacgaatgtc
tttggtcaga acacctcggc gatcgcggcc gccgaagctc 960agtacgg
96712585DNAMycobacterium tuberculosismisc_feature(1)..(585)n is a,
t, c or g 12tggattccga tagcggtttc ggcccctcga cgggcgacca cggcgcgcag
gcctccgaac 60ggggggccgg gacgctggga ttcgccggga ccgcaaccaa agaacgccgg
gtccgggcgg 120tcgggctgac cgcactggcc ggtgatgagt tcggcaacgg
cccccggatg ccgatggtgc 180cggggacctg ggagcagggc agcaacgagc
ccgaggcgcc cgacggatcg gggagagggg 240gaggcgacgg cttaccgcac
gacagcaagt aaccgaattc cgaatcacgt ggacccgtac 300gggtcgaaag
gagagatgtt atgagccttt tggatgctca tatcccacag ttggtggcct
360cccagtcggc gtttgccgcc aaggcggggc tgatgcggca cacgatcggt
caggccgagc 420aggcggcgat gtcggctcag gcgtttcacc agggggagtc
gtcggcggcg tttcaggccg 480cccatgcccg gtttgtggcg gcggccgcca
aagtcaacac cttgttggat gtcgcgcagg 540cgaatctggg tgaggccgcc
ggtacctatg tggccgccga tgctg 58513144PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(144)X = any amino acid 13Ala Leu Val
Thr Thr Asn Phe Phe Gly Val Asn Thr Ile Pro Ile Ala 1 5 10 15 Leu
Asn Glu Ala Asp Tyr Leu Arg Met Trp Ile Gln Ala Ala Thr Val 20 25
30 Met Ser His Tyr Gln Ala Val Ala His Glu Ile Trp Cys Leu His Glu
35 40 45 Xaa Ala Ser Ser Gly Lys Pro Trp Ala Ser Ile Thr Thr Gly
Ala Pro 50 55 60 Gly Ser Pro Ala Ser Thr Thr Arg Ser Arg Thr Pro
Leu Val Ser Thr 65 70 75 80 Asn Arg Xaa Val Xaa Ala Pro Ile Val Ser
Pro Asn His Thr Gly His 85 90 95 Arg Pro Glu Lys Gly Leu Gly Ser
Xaa Gln Arg Arg Leu Ser Arg Val 100 105 110 Leu Pro Arg Ile Ile Asp
Arg Pro Ala Gly Pro Xaa Gly Pro Pro Leu 115 120 125 Thr Ser Gly Ser
His Phe Leu Cys Ser Trp His Gly Tyr Ser Ser Gln 130 135 140
14352PRTMycobacterium tuberculosisMISC_FEATURE(1)..(352)X = any
amino acid 14His Ala Leu Ala Ala Gln Tyr Thr Glu Ile Ala Thr Glu
Leu Ala Ser 1 5 10 15 Val Leu Ala Ala Val Gln Ala Ser Ser Trp Gln
Gly Pro Ser Ala Asp 20 25 30 Arg Phe Val Val Ala His Gln Pro Phe
Arg Tyr Trp Leu Thr His Ala 35 40 45 Ala Thr Val Ala Thr Ala Ala
Ala Ala Ala His Xaa Thr Ala Ala Ala 50 55 60 Gly Tyr Thr Ser Ala
Leu Gly Gly Met Pro Thr Leu Ala Glu Leu Ala 65 70 75 80 Ala Asn His
Ala Met His Gly Ala Leu Val Thr Thr Asn Phe Phe Gly 85 90 95 Val
Asn Thr Ile Pro Ile Ala Leu Asn Glu Ala Asp Tyr Leu Arg Met 100 105
110 Trp Ile Gln Ala Ala Thr Val Met Ser His Tyr Gln Ala Val Ala His
115 120 125 Glu Ser Val Ala Ala Thr Pro Ser Thr Pro Pro Ala Pro Gln
Ile Val 130 135 140 Thr Ser Ala Ala Ser Ser Ala Ala Ser Ser Ser Phe
Pro Asp Pro Thr 145 150 155 160 Lys Leu Ile Leu Gln Leu Leu Lys Asp
Phe Leu Glu Leu Leu Arg Tyr 165 170 175 Leu Ala Val Glu Leu Leu Pro
Gly Pro Leu Gly Asp Leu Ile Ala Gln 180 185 190 Val Leu Asp Trp Phe
Ile Ser Phe Val Ser Gly Pro Val Phe Thr Phe 195 200 205 Leu Ala Tyr
Leu Val Leu Asp Pro Leu Ile Tyr Phe Gly Pro Phe Ala 210 215 220 Pro
Leu Thr Ser Pro Val Leu Leu Pro Ala Val Glu Leu Arg Asn Arg 225 230
235 240 Leu Lys Thr Ala Thr Gly Leu Thr Leu Pro Pro Thr Val Ile Phe
Asp 245 250 255 His Pro Thr Pro Thr Ala Val Ala Glu Tyr Val Ala Gln
Gln Met Ser 260 265 270 Gly Ser Arg Pro Thr Glu Ser Gly Asp Pro Thr
Ser Gln Val Val Glu 275 280 285 Pro Ala Arg Ala Glu Phe Gly Thr Ser
Ala Val His Gln Ile Pro Pro 290 295 300 Arg Pro Ala Asp Thr Arg Arg
Ala Cys Arg His Arg Asp Asp Val Pro 305 310 315 320 Arg Asp Ser Arg
Ile Ala Gln His Arg Asp Gly Ala Gly Leu Asp Pro 325 330 335 Thr Glu
Arg Gly Thr Ser Glu Gly Asp Gln Gly Leu Val Ser Gly Trp 340 345 350
15141PRTMycobacterium tuberculosisMISC_FEATURE(1)..(141)X = any
amino acid 15Met Asp Phe Gly Ala Leu Pro Pro Glu Val Asn Ser Val
Arg Met Tyr 1 5 10 15 Ala Val Pro Gly Ser Ala Pro Met Val Ala Ala
Ala Ser Ala Trp Asn 20 25 30 Gly Leu Ala Ala Glu Leu Ser Ser Ala
Ala Thr Gly Tyr Glu Thr Val 35 40 45 Ile Thr Gln Leu Ser Ser Glu
Gly Trp Leu Gly Pro Ala Ser Ala Ala 50 55 60 Met Ala Glu Ala Val
Ala Pro Tyr Val Ala Trp Met Ser Ala Ala Ala 65 70 75 80 Ala Gln Ala
Glu Gln Ala Ala Thr Gln Ala Arg Ala Ala Ala Ala Ala 85 90 95 Phe
Glu Ala Ala Phe Ala Ala Thr Val Pro Pro Pro Leu Ile Ala Ala 100 105
110 Asn Arg Ala Ser Leu Met Gln Leu Ile Ser Thr Asn Val Phe Gly Gln
115 120 125 Asn Thr Ser Ala Ile Ala Ala Ala Glu Ala Gln Tyr Gly 130
135 140 1658PRTMycobacterium tuberculosisMISC_FEATURE(1)..(58)X =
any amino acid 16Met Ala Ser Arg Phe Met Thr Asp Pro His Ala Met
Arg Asp Met Ala 1 5 10 15 Gly Arg Phe Glu Val His Ala Gln Thr Val
Glu Asp Glu Ala Arg Arg 20 25 30 Met Trp Ala Ser Ala Gln Asn Ile
Ser Gly Ala Gly Trp Ser Gly Met 35 40 45 Ala Glu Ala Thr Ser Leu
Asp Thr Met Thr 50 55 1767PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(67)X = any amino acid 17Met Thr Ile
Asn Tyr Gln Phe Gly Asp Val Asp Ala His Gly Ala Met 1 5 10 15 Ile
Arg Ala Gln Ala Ala Ser Leu Glu Ala Glu His Gln Ala Ile Val 20 25
30 Arg Asp Val Leu Ala Ala Gly Asp Phe Trp Gly Gly Ala Gly Ser Val
35 40 45 Ala Cys Gln Glu Phe Ile Thr Gln Leu Gly Arg Asn Phe Gln
Val Ile 50 55 60 Tyr Glu Gln 65 1858PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(58)X = any amino acid 18Met Ala Ser
Arg Phe Met Thr Asp Pro His Ala Met Arg Asp Met Ala 1 5 10 15 Gly
Arg Phe Glu Val His Ala Gln Thr Val Glu Asp Glu Ala Arg Arg 20 25
30 Met Trp Ala Ser Ala Gln Asn Ile Ser Gly Ala Gly Trp Ser Gly Met
35 40 45 Ala Glu Ala Thr Ser Leu Asp Thr Met Thr 50 55
1994PRTMycobacterium tuberculosisMISC_FEATURE(1)..(94)X = any amino
acid 19Met Thr Ile Asn Tyr Gln Phe Gly Asp Val Asp Ala His Gly Ala
Met 1 5 10 15 Ile Arg Ala Gln Ala Ala Ser Leu Glu Ala Glu His Gln
Ala Ile Val 20 25 30 Arg Asp Val Leu Ala Ala Gly Asp Phe Trp Gly
Gly Ala Gly Ser Val 35 40 45 Ala Cys Gln Glu Phe Ile Thr Gln Leu
Gly Arg Asn Phe Gln Val Ile 50 55 60 Tyr Glu Gln Ala Asn Ala His
Gly Gln Lys Val Gln Ala Ala Gly Asn 65 70 75 80 Asn Met Ala Gln Thr
Asp Ser Ala Val Gly Ser Ser Trp Ala 85 90 2030PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(30)X = any amino acid 20Asn Met Leu
His Gly Val Arg Asp Gly Leu Val Arg Asp Ala Asn Asn 1 5 10 15 Tyr
Glu Gln Gln Glu Gln Ala Ser Gln Gln Ile Leu Ser Ser 20 25 30
2194PRTMycobacterium tuberculosisMISC_FEATURE(1)..(94)X = any amino
acid 21Met Thr Ile Asn Tyr Gln Phe Gly Asp Val Asp Ala His Gly Ala
Met 1 5 10 15 Ile Arg Ala Gln Ala Gly Leu Leu Glu Ala Glu His Gln
Ala Ile Ile 20 25 30 Arg Asp Val Leu Thr Ala Ser Asp Phe Trp Gly
Gly Ala Gly Ser Ala 35 40 45 Ala Cys Gln Gly Phe Ile Thr Gln Leu
Gly Arg Asn Phe Gln Val Ile 50 55 60 Tyr Glu Gln Ala Asn Ala His
Gly Gln Lys Val Gln Ala Ala Gly Asn 65 70 75 80 Asn Met Ala Gln Thr
Asp Ser Ala Val Gly Ser Ser Trp Ala 85 90 2269PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(69)X = any amino acid 22Ala Arg Arg
Met Trp Ala Ser Ala Gln Asn Ile Ser Gly Ala Gly Trp 1 5 10 15 Ser
Gly Met Ala Glu Ala Thr Ser Leu Asp Thr Met Ala Gln Met Asn 20 25
30 Gln Ala Phe Arg Asn Ile Val Asn Met Leu His Gly Val Arg Asp Gly
35 40
45 Leu Val Arg Asp Ala Asn Asn Tyr Glu Gln Gln Glu Gln Ala Ser Gln
50 55 60 Gln Ile Leu Ser Ser 65 2394PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(94)X = any amino acid 23Met Thr Ile
Asn Tyr Gln Phe Gly Asp Val Asp Ala His Gly Ala Met 1 5 10 15 Ile
Arg Ala Gln Ala Gly Leu Leu Glu Ala Glu His Gln Ala Ile Ile 20 25
30 Arg Asp Val Leu Thr Ala Ser Asp Phe Trp Gly Gly Ala Gly Ser Ala
35 40 45 Ala Cys Gln Gly Phe Ile Thr Gln Leu Gly Arg Asn Phe Gln
Val Ile 50 55 60 Tyr Glu Gln Ala Asn Thr His Gly Gln Lys Val Gln
Ala Ala Gly Asn 65 70 75 80 Asn Met Ala Gln Thr Asp Ser Ala Val Xaa
Ser Ser Trp Ala 85 90 2452PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(52)X = any amino acid 24Gly Met Ala
Glu Ala Thr Ser Xaa Asp Thr Met Thr Gln Met Asn Gln 1 5 10 15 Ala
Phe Arg Asn Ile Val Asn Met Leu His Gly Val Arg Asp Gly Leu 20 25
30 Val Arg Asp Ala Asn Xaa Tyr Glu Gln Gln Glu Gln Ala Ser Gln Gln
35 40 45 Ile Leu Ser Ser 50 2594PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(94)X = any amino acid 25Met Thr Ile
Asn Tyr Gln Phe Gly Asp Val Asp Ala His Gly Ala Met 1 5 10 15 Ile
Arg Ala Gln Ala Gly Ser Leu Glu Ala Glu His Gln Ala Ile Ile 20 25
30 Ser Asp Val Leu Thr Ala Ser Asp Phe Trp Gly Gly Ala Gly Ser Ala
35 40 45 Ala Cys Gln Gly Phe Ile Thr Gln Leu Gly Arg Asn Phe Gln
Val Xaa 50 55 60 Tyr Glu Gln Ala Asn Ala His Gly Gln Lys Val Gln
Ala Ala Gly Asn 65 70 75 80 Asn Met Ala Gln Thr Asp Ser Ala Val Gly
Ser Ser Trp Ala 85 90 2696PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(96)X = any amino acid 26Met Thr Ser
Arg Phe Met Thr Asp Pro His Ala Met Arg Asp Met Ala 1 5 10 15 Gly
Arg Phe Glu Val His Ala Gln Thr Val Glu Asp Glu Ala Arg Arg 20 25
30 Met Trp Ala Ser Ala Gln Asn Ile Ser Gly Ala Gly Trp Ser Gly Met
35 40 45 Ala Glu Ala Thr Ser Leu Asp Thr Met Ala Gln Met Asn Gln
Ala Phe 50 55 60 Arg Asn Ile Val Asn Met Leu His Gly Val Arg Asp
Gly Leu Val Arg 65 70 75 80 Asp Ala Asn Asn Tyr Glu Gln Gln Glu Gln
Ala Ser Gln Gln Ile Leu 85 90 95 2794PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(94)X = any amino acid 27Met Thr Ile
Asn Tyr Gln Phe Gly Asp Val Asp Ala His Gly Ala Met 1 5 10 15 Ile
Arg Ala Xaa Ala Gly Leu Leu Glu Ala Glu His Gln Ala Ile Ile 20 25
30 Ser Asp Val Leu Thr Ala Ser Asp Phe Trp Gly Gly Ala Gly Ser Ala
35 40 45 Ala Cys Gln Gly Phe Ile Thr Gln Leu Gly Arg Asn Phe Gln
Val Ile 50 55 60 Tyr Glu Gln Ala Asn Ala His Gly Gln Lys Val Gln
Ala Ala Gly Asn 65 70 75 80 Asn Met Ala Gln Thr Asp Ser Ala Val Gly
Ser Ser Trp Ala 85 90 2881PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(81)X = any amino acid 28Arg Phe Glu
Val His Ala Gln Thr Val Glu Asp Glu Ala Arg Arg Met 1 5 10 15 Trp
Ala Ser Ala Gln Asn Ile Ser Gly Ala Gly Trp Ser Gly Met Ala 20 25
30 Xaa Ala Thr Ser Leu Asp Thr Met Ala Gln Met Asn Gln Ala Phe Arg
35 40 45 Asn Ile Val Asn Met Leu His Gly Val Arg Asp Gly Leu Val
Arg Asp 50 55 60 Ala Asn Asn Tyr Glu Gln Gln Glu Gln Ala Ser Gln
Gln Ile Leu Ser 65 70 75 80 Ser 2994PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(94)X = any amino acid 29Met Thr Ile
Asn Tyr Gln Phe Gly Asp Val Asp Ala His Gly Ala Met 1 5 10 15 Ile
Arg Ala Leu Ala Gly Leu Leu Glu Ala Glu His Gln Ala Ile Ile 20 25
30 Ser Asp Val Leu Thr Ala Ser Asp Phe Trp Gly Gly Ala Gly Ser Ala
35 40 45 Ala Cys Gln Gly Phe Ile Thr Gln Leu Gly Arg Asn Phe Gln
Val Ile 50 55 60 Tyr Glu Gln Ala Asn Ala His Gly Gln Lys Val Gln
Ala Ala Gly Asn 65 70 75 80 Asn Met Ala Gln Thr Asp Ser Ala Val Gly
Ser Ser Trp Ala 85 90 3011PRTMycobacterium tuberculosis 30Gln Glu
Gln Ala Ser Gln Gln Ile Leu Ser Ser 1 5 10 3194PRTMycobacterium
tuberculosis 31Met Thr Ile Asn Tyr Gln Phe Gly Asp Val Asp Ala His
Gly Ala Met 1 5 10 15 Ile Arg Ala Gln Ala Gly Leu Leu Glu Ala Glu
His Gln Ala Ile Ile 20 25 30 Arg Asp Val Leu Thr Ala Ser Asp Phe
Trp Gly Gly Ala Gly Ser Ala 35 40 45 Ala Cys Gln Gly Phe Ile Thr
Gln Leu Gly Arg Asn Phe Gln Val Ile 50 55 60 Tyr Glu Gln Ala Asn
Ala His Gly Gln Lys Val Gln Ala Ala Gly Asn 65 70 75 80 Asn Met Ala
Gln Thr Asp Ser Ala Val Gly Ser Ser Trp Ala 85 90
3299PRTMycobacterium tuberculosis 32Met Ser Phe Val Thr Thr Gln Pro
Glu Ala Leu Ala Ala Ala Ala Ala 1 5 10 15 Asn Leu Gln Gly Ile Gly
Thr Thr Met Asn Ala Gln Asn Ala Ala Ala 20 25 30 Ala Ala Pro Thr
Thr Gly Val Val Pro Ala Ala Ala Asp Glu Val Ser 35 40 45 Ala Leu
Thr Ala Ala Gln Phe Ala Ala His Ala Gln Met Tyr Gln Thr 50 55 60
Val Ser Ala Gln Ala Ala Ala Ile His Glu Met Phe Val Asn Thr Leu 65
70 75 80 Val Ala Ser Ser Gly Ser Tyr Ala Ala Thr Glu Ala Ala Asn
Ala Ala 85 90 95 Ala Ala Gly 3399PRTMycobacterium tuberculosis
33Met Ser Phe Val Thr Thr Gln Pro Glu Ala Leu Ala Ala Ala Ala Ala 1
5 10 15 Asn Leu Gln Gly Ile Gly Thr Thr Met Asn Ala Gln Asn Ala Ala
Ala 20 25 30 Ala Ala Pro Thr Thr Gly Val Val Pro Ala Ala Ala Asp
Glu Val Ser 35 40 45 Ala Leu Thr Ala Ala Gln Phe Ala Ala His Ala
Gln Met Tyr Gln Thr 50 55 60 Val Ser Ala Gln Ala Ala Ala Ile His
Glu Met Phe Val Asn Thr Leu 65 70 75 80 Val Ala Ser Ser Gly Ser Tyr
Ala Ala Thr Glu Ala Ala Asn Ala Ala 85 90 95 Ala Ala Gly
3415PRTArtificial SequencePeptide ORF-1-1 34Asp Pro His Ala Met Arg
Asp Met Ala Gly Arg Phe Glu Val His 1 5 10 15 3515PRTArtificial
SequencePeptide ORF-1-2 35Arg Asp Met Ala Gly Arg Phe Glu Val His
Ala Gln Thr Val Glu 1 5 10 15 3615PRTArtificial SequencePeptide
ORF-1-3 36Arg Phe Glu Val His Ala Gln Thr Val Glu Asp Glu Ala Arg
Arg 1 5 10 15 3715PRTArtificial SequencePeptide ORF-1-4 37Ala Gln
Thr Val Glu Asp Glu Ala Arg Arg Met Trp Ala Ser Ala 1 5 10 15
3815PRTArtificial SequencePeptide ORF-1-5 38Asp Glu Ala Arg Arg Met
Trp Ala Ser Ala Gln Asn Ile Ser Gly 1 5 10 15 3915PRTArtificial
SequencePeptide ORF-1-6 39Met Trp Ala Ser Ala Gln Asn Ile Ser Gly
Ala Gly Trp Ser Gly 1 5 10 15 4015PRTArtificial SequencePeptide
ORF-1-7 40Gln Asn Ile Ser Gly Ala Gly Trp Ser Gly Met Ala Glu Ala
Thr 1 5 10 15 4116PRTArtificial SequencePeptide ORF-1-8 41Ala Gly
Trp Ser Gly Met Ala Glu Ala Thr Ser Leu Asp Thr Met Thr 1 5 10 15
4215PRTArtificial SequencePeptide ORF-1-9 42Met Ala Glu Ala Thr Ser
Leu Asp Thr Met Ala Gln Met Asn Gln 1 5 10 15 4314PRTArtificial
SequencePeptide ORF-1-10 43Ser Leu Asp Thr Met Ala Gln Met Asn Gln
Ala Phe Arg Asn 1 5 10 4415PRTArtificial SequencePeptide ORF-1-11
44Ala Gln Met Asn Gln Ala Phe Arg Asn Ile Val Asn Met Leu His 1 5
10 15 4515PRTArtificial SequencePeptide ORF-1-12 45Ala Phe Arg Asn
Ile Val Asn Met Leu His Gly Val Arg Asp Gly 1 5 10 15
4615PRTArtificial SequencePeptide ORF-1-13 46Val Asn Met Leu His
Gly Val Arg Asp Gly Leu Val Arg Asp Ala 1 5 10 15 4715PRTArtificial
SequencePeptide ORF-1-14 47Gly Val Arg Asp Gly Leu Val Arg Asp Ala
Asn Asn Tyr Glu Gln 1 5 10 15 4815PRTArtificial SequencePeptide
ORF-1-15 48Leu Val Arg Asp Ala Asn Asn Tyr Glu Gln Gln Glu Gln Ala
Ser 1 5 10 15 4916PRTArtificial SequencePeptide ORF-1-16 49Asn Asn
Tyr Glu Gln Gln Glu Gln Ala Ser Gln Gln Ile Leu Ser Ser 1 5 10 15
5017PRTArtificial SequencePeptide ORF-1-17 50Met Ala Ser Arg Phe
Met Thr Asp Pro His Ala Met Arg Asp Met Ala 1 5 10 15 Gly
5115PRTArtificial SequencePeptide ORF-2-1 51Met Thr Ile Asn Tyr Gln
Phe Gly Asp Val Asp Ala His Gly Ala 1 5 10 15 5215PRTArtificial
SequencePeptide ORF-2-2 52Gln Phe Gly Asp Val Asp Ala His Gly Ala
Met Ile Arg Ala Gln 1 5 10 15 5315PRTArtificial SequencePeptide
ORF-2-23 53Asp Ala His Gly Ala Met Ile Arg Ala Gln Ala Ala Ser Leu
Glu 1 5 10 15 5415PRTArtificial SequencePeptide ORF-2-4 54Met Ile
Arg Ala Gln Ala Ala Ser Leu Glu Ala Glu His Gln Ala 1 5 10 15
5515PRTArtificial SequencePeptide ORF-2-5 55Ala Ala Ser Leu Glu Ala
Glu His Gln Ala Ile Val Arg Asp Val 1 5 10 15 5615PRTArtificial
SequencePeptide ORF-2-6 56Ala Glu His Gln Ala Ile Val Arg Asp Val
Leu Ala Ala Gly Asp 1 5 10 15 5715PRTArtificial SequencePeptide
ORF-2-7 57Ile Val Arg Asp Val Leu Ala Ala Gly Asp Phe Trp Gly Gly
Ala 1 5 10 15 5816PRTArtificial SequencePeptide ORF-2-8 58Leu Ala
Ala Gly Asp Phe Trp Gly Gly Ala Gly Ser Val Ala Cys Gln 1 5 10 15
5916PRTArtificial SequencePeptide ORF-2-9 59Leu Ala Ala Gly Asp Phe
Trp Gly Gly Ala Gly Ser Val Ala Cys Gln 1 5 10 15 6015PRTArtificial
SequencePeptide ORF-2-10 60Gly Ser Val Ala Cys Gln Glu Phe Ile Thr
Gln Leu Gly Arg Asn 1 5 10 15 6118PRTArtificial SequencePeptide
ORF-2-11 61Gln Glu Phe Ile Thr Gln Leu Gly Arg Asn Phe Gln Val Ile
Tyr Glu 1 5 10 15 Gln Ala 6215PRTArtificial SequencePeptide
ORF-2-12 62Arg Asn Phe Gln Val Ile Tyr Glu Gln Ala Asn Ala His Gly
Gln 1 5 10 15 6315PRTArtificial SequencePeptide ORF-2-13 63Ile Tyr
Glu Gln Ala Asn Ala His Gly Gln Lys Val Gln Ala Ala 1 5 10 15
6415PRTArtificial SequencePeptide ORF-2-14 64Asn Ala His Gly Gln
Lys Val Gln Ala Ala Gly Asn Asn Met Ala 1 5 10 15 6515PRTArtificial
SequencePeptide ORF-2-16 65Lys Val Gln Ala Ala Gly Asn Asn Met Ala
Gln Thr Asp Ser Ala 1 5 10 15 6616PRTArtificial SequencePeptide
ORF-2-16 66Gly Asn Asn Met Ala Gln Thr Asp Ser Ala Val Gly Ser Ser
Trp Ala 1 5 10 15 6715PRTArtificial SequencePeptide ORF-2-17 67Asp
Ala His Gly Ala Met Ile Arg Ala Leu Ala Gly Leu Leu Glu 1 5 10 15
6815PRTArtificial SequencePeptide ORF-2-18 68Asp Ala His Gly Ala
Met Ile Arg Ala Gln Ala Gly Leu Leu Glu 1 5 10 15 6915PRTArtificial
SequencePeptide ORF-2-19 69Met Ile Arg Ala Leu Ala Gly Leu Leu Glu
Ala Glu His Gln Ala 1 5 10 15 7015PRTArtificial SequencePeptide
ORF-2-20 70Met Ile Arg Ala Gln Ala Gly Leu Leu Glu Ala Glu His Gln
Ala 1 5 10 15 7115PRTArtificial SequencePeptide ORF-2-21 71Ala Gly
Leu Leu Glu Ala Glu His Gln Ala Ile Ile Ser Asp Val 1 5 10 15
7215PRTArtificial SequencePeptide ORF-2-22 72Ala Gly Leu Leu Glu
Ala Glu His Gln Ala Ile Ile Arg Asp Val 1 5 10 15 7315PRTArtificial
SequencePeptide ORF-2--23 73Ala Glu His Gln Ala Ile Ile Ser Asp Val
Leu Thr Ala Ser Asp 1 5 10 15 7415PRTArtificial SequencePeptide
ORF-2-24 74Ala Glu His Gln Ala Ile Ile Arg Asp Val Leu Thr Ala Ser
Asp 1 5 10 15 7515PRTArtificial SequencePeptide ORF-2-25 75Ile Ile
Ser Asp Val Leu Thr Ala Ser Asp Phe Trp Gly Gly Ala 1 5 10 15
7615PRTArtificial SequencePeptide ORF-2-26 76Ile Ile Arg Asp Val
Leu Thr Ala Ser Asp Phe Trp Gly Gly Ala 1 5 10 15 7716PRTArtificial
SequencePeptide ORF-2-27 77Leu Thr Ala Ser Asp Phe Trp Gly Gly Ala
Gly Ser Ala Ala Cys Gln 1 5 10 15 7815PRTArtificial SequencePeptide
ORF-2-28 78Phe Trp Gly Gly Ala Gly Ser Ala Ala Cys Gln Gly Phe Ile
Thr 1 5 10 15 7915PRTArtificial SequencePeptide ORF-2-29 79Gly Ser
Ala Ala Cys Gln Gly Phe Ile Thr Gln Leu Gly Arg Asn 1 5 10 15
8015PRTArtificial SequencePeptide ORF-2-30 80Gln Gly Phe Ile Thr
Gln Leu Gly Arg Asn Phe Gln Val Ile Tyr 1 5 10 15 8125PRTArtificial
SequenceOverlapping peptide of ORF Tb224 81Val Thr Thr Asn Phe Phe
Gly Val Asn Thr Ile Pro Ile Ala Leu Asn 1 5 10 15 Glu Ala Asp Tyr
Leu Arg Met Trp Ile 20 25 8225PRTArtificial SequenceOverlapping
peptide of ORF of Tb224 82Asn Glu Ala Asp Tyr Leu Arg Met Trp Ile
Gln Ala Ala Thr Val Met 1 5 10 15 Ser His Tyr Gln Ala Val Ala His
Glu 20 25 83967DNAMycobacterium tuberculosismisc_feature(1)..(967)n
is a, t, c or g 83tgagcgccaa ccctaccgtc ggttcgtcac acggaccgca
tggcctgctc cgcggactgc 60cgctagggtc gcggatcact cggcgtagcg gcgcctttgc
ccaccgatat gggttccgtc 120acagtgtggt tgcccgcccg ccatcggccg
gataacgcca tgacctcagc tcggcagaaa 180tgacaatgct cccaaaggcg
tgagcacccg aagacaacta agcaggagat cgcatgccgt 240ttgtgactac
ccaaccagaa gcactggcgg cggcggccgg cagtctgcag ggaatcggct
300ccgcattgaa cgcccagaat gcggctgcgg cgactcccac gacgggggtg
gtccggcggc 360cgccgatgaa ntgtcggcgc tgacggcggc tcagttcgcg
gcacacgccc agatctatca 420ggccgtcagc gcccaggccg cggcgattca
cgagatgttc gtcaacactc tacagatgag 480ctcagggtcg tatgctgcta
ccgaggccgc caacgcggcc gcggccggnt agaggagtca 540ctgcgatgga
ttttggggcg ttgccgccgg aggtcaattc ggtgcggatg tatgccgttc
600ctggctcggc accaatggtc gctgcggcgt cggcctggaa cgggttggcc
gcggagctga 660gttcggcggc caccggttat gagacggtga tcactcagct
cagcagtgag gggtggctag 720gtccggcgtc agcggcgatg gccgaggcag
ttgcgccgta tgtggcgtgg atgagtgccg 780ctgcggcgca agccgagcag
gcggccacac aggccagggc cgccgcggcc gcttttgagg 840cggcgtttgc
cgcgacggtg cctccgccgt tgatcgcggc caaccgggct tcgttgatgc
900agctgatctc gacgaatgtc tttggtcaga acacctcggc gatcgcggcc
gccgaagctc 960agtacgg 9678415PRTArtificial SequencePeptide MSF-1
84Met Ser Phe Val Thr Thr Gln Pro Glu Ala Leu Ala Ala Ala Ala 1 5
10 15 8515PRTArtificial SequencePeptide MSF-2 85Thr Gln Pro Glu Ala
Leu Ala Ala Ala Ala Ala Asn Leu Gln Gly 1 5 10 15 8615PRTArtificial
SequencePeptide MSF-3
86Leu Ala Ala Ala Ala Ala Asn Leu Gln Gly Ile Gly Thr Thr Met 1 5
10 15 8715PRTArtificial SequencePeptide MSF-4 87Ala Asn Leu Gln Gly
Ile Gly Thr Thr Met Asn Ala Gln Asn Ala 1 5 10 15 8815PRTArtificial
SequencePeptide MSF-5 88Ile Gly Thr Thr Met Asn Ala Gln Asn Ala Ala
Ala Ala Ala Pro 1 5 10 15 8915PRTArtificial SequencePeptide MSF-6
89Asn Ala Gln Asn Ala Ala Ala Ala Ala Pro Thr Thr Gly Val Val 1 5
10 15 9015PRTArtificial SequencePeptide MSF-7 90Ala Ala Ala Ala Pro
Thr Thr Gly Val Val Pro Ala Ala Ala Asp 1 5 10 15 9115PRTArtificial
SequencePeptide MSF-8 91Thr Thr Gly Val Val Pro Ala Ala Ala Asp Glu
Val Ser Ala Leu 1 5 10 15 9215PRTArtificial SequencePeptide MSF-9
92Pro Ala Ala Ala Asp Glu Val Ser Ala Leu Thr Ala Ala Gln Phe 1 5
10 15 9315PRTArtificial SequencePeptide MSF-10 93Glu Val Ser Ala
Leu Thr Ala Ala Gln Phe Ala Ala His Ala Gln 1 5 10 15
9415PRTArtificial SequencePeptide MSF-11 94Thr Ala Ala Gln Phe Ala
Ala His Ala Gln Met Tyr Gln Thr Val 1 5 10 15 9515PRTArtificial
SequencePeptide MSF-12 95Ala Ala His Ala Gln Met Tyr Gln Thr Val
Ser Ala Gln Ala Ala 1 5 10 15 9616PRTArtificial SequencePeptide
MSF-13 96Met Tyr Gln Thr Val Ser Ala Gln Ala Ala Ala Ile His Glu
Met Phe 1 5 10 15 9715PRTArtificial SequencePeptide MSF-14 97Ser
Ala Gln Ala Ala Ala Ile His Glu Met Phe Val Asn Thr Leu 1 5 10 15
9815PRTArtificial SequencePeptide MSF-15 98Ala Ile His Glu Met Phe
Val Asn Thr Leu Val Ala Ser Ser Gly 1 5 10 15 9915PRTArtificial
SequencePeptide MSF-16 99Phe Val Asn Thr Leu Val Ala Ser Ser Gly
Ser Tyr Ala Ala Thr 1 5 10 15 10015PRTArtificial SequencePeptide
MSF-17 100Val Ala Ser Ser Gly Ser Tyr Ala Ala Thr Glu Ala Ala Asn
Ala 1 5 10 15 10114PRTArtificial SequencePeptide MSF-18 101Ser Tyr
Ala Ala Thr Glu Ala Ala Asn Ala Ala Ala Ala Gly 1 5 10
1021784DNAMycobacterium tuberculosismisc_feature(1)..(1784)n is a,
t, c or g 102attcgttcct gccgcagcta aatcccgggg acatcgtcgc cggccagtac
gaggtcaaag 60gctgcatcgc gcacggcgga ctgggctgga tctacctcgc tctcgaccgc
aatgtcaacg 120gccgtccggt ggtgctcaag ggcctggtgc attccggtga
tgccgaagcg caggcaatgg 180cgatggccga acgccagttc ctggccgagg
tggtgcaccc gtcgatcgtg cagatcttca 240actttgtcga gcacaccgac
aggcacgggg atccggtcgg ctacatcgtg atggaatacg 300tcggcgggca
atcgctcaaa cgcagcaagg gtcanaaact gcccgtcgcg gaggccatcg
360cctacctgct ggagatcctg ccggcgctga gctacctgca ttccatcggc
ttggtctaca 420acgacctgaa gccggaaaac atcatgctga ccgaggaaca
gctcaagctg atcgacctgg 480gcgcggtatc gcggatcaac tcgttcggct
acctctacgg gaccccaggc ttccaggcgc 540ccgagatcgt gcggaccggt
ccgacggtgg ccaccgacat ctacaccgtg ggacgcacgc 600tcgcggcgct
cacgctggac ctgcccaccc gcaatggccg ttatgtggat gggctacccg
660aagacgaccc ggtgctgaaa acctacgact cttacggccg gttgctgcgc
agggccatcg 720accccgatcc gcggcaacgg ttcaccaccg ccgaagagat
gtccgcgcaa ttgacgggcg 780tgttgcggga ggtggtcgcc cagacaccgg
ggtgccgcgg ccaggctatc aacgatcttc 840agtcccagtc ggtcgacatt
tggagtggac tgctggtggc gcacaccgac gtgtatctgg 900acgggcaggt
gcacgcggag aagctgaccg ccaacgagat cgtgaccgcg ctgtcggtgc
960cgctggtcga tccgaccgac gtcgcagctt cggtcctgca ggccacggtg
ctctcccagc 1020cggtgcagac cctagactcg ntgcgcgcgg cccgccacgg
tgcgctggac gccgacggcg 1080tcgattntcc gagtcagtgg agctgccgct
aatggaagtc cgcgcgctgc tggatctcgg 1140cgatgtggcc aaggccaccc
gaaaactcga cgatctggcc gaacgcgttg gctggcgatg 1200gcgattggtc
tggtaccggg ccgtcgccga gctgctcacc ggcgactatg actcggccac
1260caaacatttc accgaggtgc tggatacctt tcccggcgag ctggcgccca
agctcgccct 1320ggccgccacc gccgaactag ccggcaacac cgacgaacac
aagttctatc agacggtgtg 1380gagcaccaac gacggcgtga tctcggcggc
tttcggactg gccagagccc ggtcggccga 1440aggtgatcgg gtcggcgccg
tgcgcacgct cgacgaggta ccgcccactt ctcggcattt 1500caccacggca
cggctgacca gcgcggtgac tctgttgtcc ggccggtcaa cgagtgaagt
1560caccgaggaa cagatccgcg acgccgcccg aagagtggag gcgctgcccc
cgaccgaacc 1620acgcgtgctg cagatccgcg ccctggtgct gggtggcgcg
ctggactggc tgaaggacaa 1680caaggccagc accaaccaca tcctcggttt
cccgttcacc agtcacgggc tgcggctggg 1740tgtcgaggcg tcactgcgca
gcctggcccg ggtagctccc actc 1784103766DNAMycobacterium
tuberculosismisc_feature(1)..(766)n is a, t, c or g 103acaanacact
cggnggcngc cgntccggcc tgatcgtcgg tgatcagcnt cgtgccaaan 60tcggcacaag
gtgcgcgctn cccaangagt tcttcgccgc ngtgcgngcn naactggcct
120atcntggttg ggtgccgtcc cgcanaaccc gcgaacttaa acccatttta
accgggcagg 180aagtttccta catntacccn ngsnanccaa ccgggccgcc
nanaantccg tcctggantc 240cgancggttc ccggtgttcg ccgcactgct
gaccggcacg gantatccgc aggcggcgtt 300ggccaacgcg tgggtgcaac
tggcctacgg tgcgcaccas gacgccatca ccggctcgga 360gtccgaccag
gtactcaatg ctggcgacca caccagccag cagaccaaac tggtgcacgc
420cgatctccag gcgcgccggc ccggtggcat acggattggt cgaaaccaat
ccgaaggaat 480tcatcacgga cggtcacgga aaacgatcgc cccaatgggn
ggacnacccn agccaggcgn 540attnaccgtt naacaagttg gngtaggttc
tttgatatcg ancaaccgat acggancggn 600ccgcggaatg gtagaccacc
accagtgccc ncangtngtg caccagtttg gtcatcgccc 660gcagatcggt
gaccccgcca agcgttccgg atgcggagat gasggtgacc agccnggttg
720acctgttgat caggttntcc cagtgccacg tcggcagctg gccggt
7661041231DNAMycobacterium tuberculosismisc_feature(1)..(1231)n is
a, t, c or g 104cggcacgaga atgtcgcctg tgcctcgata gccacttgcg
tgtggtcgcg ctgccagcgg 60gtcagccagg tcgcctggtc caggccatcg ggccggcgca
ggagcgcgat gttggccaga 120cccggtgtac gagaaccgga ctcgacnaag
tgtcggcgct gacggcggct cagttcgcgg 180cacacgccca gatctatcag
gccgtcagcg cccaggccgc ggcgattcac gagatgttcg 240tcaacactct
acagatnanc tcagggtcgt atgctgctac cgaggccgcc aacgcggccg
300cggccggcta gaggagtcac tgcgatggat tttggggcgt tgccgccgga
ggtcaattcg 360gtgcggatgt atgccggtcc tggctcggca ccaatggtcg
ctgcggcgtc ggcctggaac 420gggttggccg cggagctgag ttcggcggcc
accggttatg agacggtgat cactcagctc 480agcagtgagg ggtggctagg
tccggcgtca gcggcgatgg ccgaggcagt tgcgccgtat 540gtggcgtgga
tgagtgccgc tgcggcgcaa gccgagcagg cggccacaca ggccagggcc
600gccgcggccg cttttgaggc ggcgtttgcc gcgacggtgc ctccgccgtt
gatcgcggcc 660aaccgggctt cgttgatgca gctgatctcg acgaatgtct
ttggtcagaa cacctcggcg 720atcgcggccg ccgaagctca gtacggcgag
atgtgggccc aagactccgc ggcgatgtat 780gcctacgcgg gcagttcggc
gagcgcctcg gcggtcacgc cgtttagcac gccgccgcag 840attgccaacc
cgaccgctca gggtacgcag gccgcggccg tggccaccgc cgccggtacc
900gcccagtcga cgctgacgga gatgatcacc gggctaccca acgcgctgca
aagcctcacc 960tcacntctgt tgcagtcgtc taacggtccg ctgtcgtggc
tgtggcagat cttgttcggc 1020acgcccaatt tccccacctc aatttcggca
ctgctgaccg acctgcagcc ctacgcgagc 1080ttnttntata acaccgaggg
cctgccgtac ttcagcatcg gcatgggcaa caacttcatt 1140cagtcggcca
agaccctggg attgatcggc taggcggcac cggctgcggt cgcggntgct
1200ggggatnccg ccaagggctt gcctcgtgcc g 12311052041DNAMycobacterium
tuberculosismisc_feature(1)..(2041)n is a, t, c or g 105cggcacgagc
tcgtgccgat cagtgccatt gacggcttgt acgaccttct ggggattgga 60atacccaacc
aagggggtat cctttactcc tcactagagt acttcgaaaa agccctggag
120gagctggcag cagcgtttcc gggtgatggc tggttaggtt cggccgcgga
caaatacgcc 180ggcaaaaacc gcaaccacgt gaattttttc caggaactgg
cagacctcga tcgtcagctc 240atcagcctga tccacgacca ggccaacgcg
gtccagacga cccgcgacat cctggagggc 300gccaagaaag gtctcgagtt
cgtgcgcccg gtggctgtgg acctgaccta catcccggtc 360gtcgggcacg
ccctatcggc cgccttccan gcgccgtttt gcgcgggcgc gatggccgta
420gtgggcggcg cgcttgccta cttggtcgtg aaaacgctga tcaacgcgac
tcaactcctc 480aaattgcttg ccaaattggc ggagttggtc gcggccgcca
ttgcggacat catttcggat 540gtggcggaca tcatcaaggg catcctcgga
gaagtgtggg agttcatcac aaacgcgctc 600aacggcctga aagagctttg
ggacaagctc acggggtggg tgaccggact gttctctcga 660gggtggtcga
acctggagtc cttctttgcg ggcgtccccg gcttgaccgg cgcgaccagc
720ggcttgtcgc aagtgactgg cttgttcggt gcggccggtc tgtccgcatc
gtcgggcttg 780gctcacgcgg atagcctggc gagctcagcc agcttgcccg
ccctggccgg cattgggggc 840gggtccggtt ttgggggctt gccgagcctg
gctcaggtcc atgccgcctc aactcggcag 900gcgctacggc cccgagctga
tggcccggtc ggcgccgctg ccgagcaggt cggcgggcag 960tcgcagctgg
tctccgcgca gggttcccaa ggtatgggcg gacccgtagg catgggcggc
1020atgcacccct cttcgggggc gtcgaaaggg acgacgacga agaagtactc
ggaaggcgcg 1080gcggcgggca ctgaagacgc cgagcgcgcg ccagtcgaag
ctgacgcggg cggtgggcaa 1140aaggtgctgg tacgaaacgt cgtctaacgg
catggcgagc caaatccatt gctagccagc 1200gcctaacaac gcgcaatgct
aaacggaagg gacacgatca atgacggaaa acttgaccgt 1260ccagcccgag
cgtctcggtg tactggcgtc gcaccatgac aacgcggcgg tcgatgcntc
1320ctcgggcgtc gaagctgccg ctggcctagg cgaatctgtg gcgatcactc
acggtccgta 1380ctgctcacag ttcaacgaca cgttaaatgt gtacttgact
gcccacaatg ccctgggctc 1440gtccttgcat acggccggtg tcgatctcgc
caaaagtctt cgaattgcgg cgaagatata 1500tagcgaggcc gacgaagcgt
ggcgcaaggc tatcgacggg ttgtttacct gaccacgttt 1560gctgcccgca
gtgcaggcca cgacgtagcg caggtcgtgt ccctcgtagg cgtggatgcg
1620accggccagc accagcaccc ggtgcgcacc gatgggcacg gacagtagct
cgcccgcatg 1680cccggctgcg gttggcggca caaacccggg cagttcggcc
tgcggcagca cggtggtngg 1740ggagcccaac gccgcaacgg ccggtaacca
tcccgacccg agcacgaccg agacgtcatg 1800ttcgccgatc ccggtgcggt
cagcgatgac ctgcgccgcc cgccgggcca gtttgtcggg 1860atcggggcgc
gggtcagcca cactgggcga gcttaactga gccgctcgcc ggggagcggg
1920tgctngtcga tgagatactg cgagcatgcc agcagccagc gcatccgacc
gcgtcgagga 1980attggtgcgg cgccgtggtg gcgagctggt cgagctgtcc
catgccatcc acctcgtgcc 2040g 20411061202DNAMycobacterium
tuberculosismisc_feature(1)..(1202)n is a, t, c or g 106gagctcaccg
ctatcaacca atactttctg cactccaaga tgcaggacaa ctggggtttt 60accgagctgg
cggcccacac ccgcgcggag tcgttcgacg aaatgcggca cgccgaggaa
120atcaccgatc gcatcttgtt gctggatggt ttgccgaact accagcgcat
cggttcgttg 180cgtatcggcc agacgctccg cgagcaattt gaggccgatc
tggcgatcga atacgacgtg 240ttgaatcgtc tcaagccagg aatcgtcatg
tgccgggaga aacaggacac caccagcgcc 300gtactgctgg agaaaatcgt
tgccgacgag gaagaacaca tcgactactt ggaaacgcag 360ctggagctga
tggacaagct aggagaggag ctttactcgg cgcagtgcgt ctctcgccca
420ccgacctgat gcccgcttga ggattctccg ataccactcc gggcgccgct
gacaagctct 480agcatcgact cgaacagcga tgggagggcg gatatggcgg
gccccacagc accgaccact 540gcccccaccg caatccgagc cggtggcccg
ctgctcagtc cggtgcgacg caacattatt 600ttcaccgcac ttgtgttcgg
ggtgctggtc gctgcgaccg gccaaaccat cgttgtgccc 660gcattgccga
cgatcgtcgc cgagctgggc agcaccgttg accagtcgtg ggcggtcacc
720agctatctgc tggggggaac actskygkkk ktgkkgksks ksrmrmkctc
ggtgatctgc 780tcggccgcaa cagggtgctg ctaggctccg tcgtggtctt
cgtcgttggc tctgtgctgt 840gcgggttatc gcagacgatg accatgctgg
cgatctctcg cgcactgcag ggcgtcggtg 900ccggtgcgat ttccgtcacc
gcctacgcgc tggccgctga ggtggtccca ctgcgggacc 960gtggccgcta
ccagggcgtc ttangtgcgg tgttcggtgt caacacggtc accggtccgc
1020tgctgggggg ctggctcacc gactatctga gctggcggtg ggcgttccga
ccaccagccc 1080catcaccgac ccgatcgcgg tcatcgcggc gaacaccgcc
ctcgcggcgt tgcgggcagg 1140tcccttgggg aacgtggtcc cacagcgcca
gaacggtcgg aaatgcgatg gccgacccac 1200ac 1202107496DNAMycobacterium
tuberculosismisc_feature(1)..(496)n is a, t, c or g 107ggcggcggca
gttggccagc agttngggcg ggggagccgg ttcggngacc aagaaatcgg 60cctgggcaag
cagccgggac cgcgnaccgt gatcagttng gatcgccggg accgccgccg
120accaangcca ttccgccgnt gaggaagtcg gaantntgcg cagtgatgac
gccctgctgc 180aacgcntccc ggattgccga gcggatcgcc gccgaacggc
ggtgctcacc accggcgagc 240acccctacng acaggcccgc atagctgaat
gacgccgggt naccgccgtc ccntccaccg 300nganatcggc ccggangcaa
aagatccgtc ggcgctccgc ctcggcgacg acagccacgt 360tcacccgcgc
gttatcggtg gccgcgatcg cataccaggc gccgtcaagg tngccgtygc
420ggtagtcacg caccgacaag gtgatytggt ccatcgcctn gacggcgggg
gtgacgctgg 480gggcgatcam gtgcac 496108849DNAMycobacterium
tuberculosismisc_feature(1)..(849)n is a, t, c or g 108tggattccga
tagcggtttc ggcccctcga cgggcgacca cggcgcgcag gcctccgaac 60ggggggccgg
gacgctggga ttcgccggga ccgcaaccaa agaacgccgg gtccgggcgg
120tcgggctgac cgcactggcc ggtgatgagt tcggcaacgg cccccggatg
ccgatggtgc 180cggggacctg ggagcagggc agcaacgagc ccgaggcgcc
cgacggatcg gggagagggg 240gaggcgacgg cttaccgcac gacagcaagt
aaccgaattc cgaatcacgt ggacccgtac 300gggtcgaaag gagagatgtt
atgagccttt tggatgctca tatcccacag ttggtggcct 360cccagtcggc
gtttgccgcc aaggcggggc tgatgcggca cacgatcggt caggccgagc
420aggcggcgat gtcggctcag gcgtttcacc agggggagtc gtcggcggcg
tttcaggccg 480cccatgcccg gtttgtggcg gcggccgcca aagtcaacac
cttgttggat gtcgcgcagg 540cgaatctggg tgaggccgcc ggtacctatg
tggccgccga tgctgcggcc gcgtcgacct 600ataccgggtt ctgatcgaac
cctgctgacc gagaggactt gtgatgtcgc aaatcatgta 660caactacccc
gcgatgttgg gtcacgccgg ggatatggcc ggatatgccg gcacgctgca
720gagcttgggt gccgagatcg ccgtggagca ggccgcgttg cagagtgcgt
ggcagggcga 780taccgggatc acgtatcagg cgtggcaggc acantggtaa
ccangccang gaagatttgg 840tgcgggcct 84910997PRTMycobacterium
tuberculosis 109Met Ser Leu Leu Asp Ala His Ile Pro Gln Leu Val Ala
Ser Gln Ser 1 5 10 15 Ala Phe Ala Ala Lys Ala Gly Leu Met Arg His
Thr Ile Gly Gln Ala 20 25 30 Glu Gln Ala Ala Met Ser Ala Gln Ala
Phe His Gln Gly Glu Ser Ser 35 40 45 Ala Ala Phe Gln Ala Ala His
Ala Arg Phe Val Ala Ala Ala Ala Lys 50 55 60 Val Asn Thr Leu Leu
Asp Val Ala Gln Ala Asn Leu Gly Glu Ala Ala 65 70 75 80 Gly Thr Tyr
Val Ala Ala Asp Ala Ala Ala Ala Ser Thr Tyr Thr Gly 85 90 95 Phe
11015PRTArtificial SequencePeptide MSL-1 110Met Ser Leu Leu Asp Ala
His Ile Pro Gln Leu Val Ala Ser Gln 1 5 10 15 11115PRTArtificial
SequencePeptide MSL-2 111Ala His Ile Pro Gln Leu Val Ala Ser Gln
Ser Ala Phe Ala Ala 1 5 10 15 11215PRTArtificial SequencePeptide
MSL-3 112Leu Val Ala Ser Gln Ser Ala Phe Ala Ala Lys Ala Gly Leu
Met 1 5 10 15 11315PRTArtificial SequencePeptide MSL-4 113Ser Ala
Phe Ala Ala Lys Ala Gly Leu Met Arg His Thr Ile Gly 1 5 10 15
11415PRTArtificial SequencePeptide MSL-5 114Lys Ala Gly Leu Met Arg
His Thr Ile Gly Gln Ala Glu Gln Ala 1 5 10 15 11515PRTArtificial
SequencePeptide MSL-6 115Arg His Thr Ile Gly Gln Ala Glu Gln Ala
Ala Met Ser Ala Gln 1 5 10 15 11615PRTArtificial SequencePeptide
MSL-7 116Gln Ala Glu Gln Ala Ala Met Ser Ala Gln Ala Phe His Gln
Gly 1 5 10 15 11715PRTArtificial SequencePeptide MSL-8 117Ala Met
Ser Ala Gln Ala Phe His Gln Gly Glu Ser Ser Ala Ala 1 5 10 15
11815PRTArtificial SequencePeptide MSL-9 118Ala Phe His Gln Gly Glu
Ser Ser Ala Ala Phe Gln Ala Ala His 1 5 10 15 11915PRTArtificial
SequencePeptide MSL-10 119Glu Ser Ser Ala Ala Phe Gln Ala Ala His
Ala Arg Phe Val Ala 1 5 10 15 12015PRTArtificial SequencePeptide
MSL-11 120Phe Gln Ala Ala His Ala Arg Phe Val Ala Ala Ala Ala Lys
Val 1 5 10 15 12115PRTArtificial SequencePeptide MSL-12 121Ala Arg
Phe Val Ala Ala Ala Ala Lys Val Asn Thr Leu Leu Asp 1 5 10 15
12215PRTArtificial SequencePeptide MSL-13 122Ala Ala Ala Lys Val
Asn Thr Leu Leu Asp Val Ala Gln Ala Asn 1 5 10 15
12315PRTArtificial SequencePeptide MSL-14 123Asn Thr Leu Leu Asp
Val Ala Gln Ala Asn Leu Gly Glu Ala Ala 1 5 10 15
12416PRTArtificial SequencePeptide MSL-15 124Val Ala Gln Ala Asn
Leu Gly Glu Ala Ala Gly Thr Tyr Val Ala Ala 1 5 10 15
1251692DNAMycobacterium tuberculosismisc_feature(1)..(1692)n is a,
t, c or g 125cggcacgaga atgtcgcctg tgcctcgata gccacttgcg tgtggtcgcg
ctgccagcgg 60gtcagccagg tcgcctggtc caggccatcg ggccggcgca ggagcgcgat
gttggccaga 120cccggtgtac gagaaccgga ctcgacnaag tgtcggcgct
gacggcggct cagttcgcgg 180cacacgccca gatctatcag gccgtcagcg
cccaggccgc ggcgattcac gagatgttcg 240tcaacactct acagatnanc
tcagggtcgt atgctgctac cgaggccgcc aacgcggccg 300cggccggcta
gaggagtcac tgcgatggat tttggggcgt tgccgccgga ggtcaattcg
360gtgcggatgt atgccggtcc tggctcggca ccaatggtcg ctgcggcgtc
ggcctggaac 420gggttggccg cggagctgag ttcggcggcc accggttatg
agacggtgat cactcagctc 480agcagtgagg ggtggctagg tccggcgtca
gcggcgatgg ccgaggcagt tgcgccgtat 540gccgcggccg cttttgaggc
ggcgtttgcc gcgacggtgc ctccgccgtt gatcgcggcc 600aaccgggctt
cgttgatgca gctgatctcg acgaatgtct ttggtcagaa cacctcggcg
660atcgcggccg ccgaagctca gtacggcgag atgtgggccc aagactccgc
ggcgatgtat 720gcctacgcgg gcagttcggc gagcgcctcg gcggtcacgc
cgtttagcac gccgccgcag 780attgccaacc cgaccgctca gggtacgcag
gccgcggccg tggccaccgc cgccggtacc 840gcccagtcga cgctgacgga
gatgatcacc gggctaccca acgcgctgca aagcctcacc 900tcacntctgt
tgcagtcgtc taacggtccg ctgtcgtggc tgtggcagat cttgttcggc
960acgcccaatt
tccccacctc aatttcggca ctgctgaccg acctgcagcc ctacgcgagc
1020ttnttntata acaccgaggg cctgccgtac ttcagcatcg gcatgggcaa
caacttcatt 1080cagtcggcca agaccctggg attgatcggc taggcggcac
cggctgcggt cgcggctgct 1140ggggatgccg ccaagggctt gcctggactg
ggcgggatgc tcggtggcgg gccggtggcg 1200gcgggtctgg gcaatgcggc
ttcggttggc aagctgtcgg tgccgccggt gtggantgga 1260ccgttgcccg
ggtcggtgac tccgggggct gctccgctac cggtgagtac ggtcagtgcc
1320gccccggagg cggcgcccgg aagcctgttg ggcggcctgc cgctanctgg
tgcgggcggg 1380gccggcgcgg gtccacgcta cggattccrt cccaccgtca
tggctcgccc acccttcgmc 1440gggatagtcg ctgccgcaac gtattaacgc
gccggcctcg gctggtgtgg tccgctgcgg 1500gtggcaattg gtcngcgccg
aaatctcngt gggttatttn cggtgggatt ttttcccgaa 1560gccgggttca
ncaccggatt tcctaacggt cccgcnactc tcgtgccgaa ttcngcacta
1620agtgacgtcc ggcggaaacc cgttgggtnt gaaagcttca gaaaggcccg
ctcccagggg 1680ttcggcaaac gg 1692126400PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(400)X = any amino acid 126Met Asp Phe
Gly Ala Leu Pro Pro Glu Val Asn Ser Val Arg Met Tyr 1 5 10 15 Ala
Gly Pro Gly Ser Ala Pro Met Val Ala Ala Ala Ser Ala Trp Asn 20 25
30 Gly Leu Ala Ala Glu Leu Ser Ser Ala Ala Thr Gly Tyr Glu Thr Val
35 40 45 Ile Thr Gln Leu Ser Ser Glu Gly Trp Leu Gly Pro Ala Ser
Ala Ala 50 55 60 Met Ala Glu Ala Val Ala Pro Tyr Val Ala Trp Met
Ser Ala Ala Ala 65 70 75 80 Ala Gln Ala Glu Gln Ala Ala Thr Gln Ala
Arg Ala Ala Ala Ala Ala 85 90 95 Phe Glu Ala Ala Phe Ala Ala Thr
Val Pro Pro Pro Leu Ile Ala Ala 100 105 110 Asn Arg Ala Ser Leu Met
Gln Leu Ile Ser Thr Asn Val Phe Gly Gln 115 120 125 Asn Thr Ser Ala
Ile Ala Ala Ala Glu Ala Gln Tyr Gly Glu Met Trp 130 135 140 Ala Gln
Asp Ser Ala Ala Met Tyr Ala Tyr Ala Gly Ser Ser Ala Ser 145 150 155
160 Ala Ser Ala Val Thr Pro Phe Ser Thr Pro Pro Gln Ile Ala Asn Pro
165 170 175 Thr Ala Gln Gly Thr Gln Ala Ala Ala Val Ala Thr Ala Ala
Gly Thr 180 185 190 Ala Gln Ser Thr Leu Thr Glu Met Ile Thr Gly Leu
Pro Asn Ala Leu 195 200 205 Gln Ser Leu Thr Ser Xaa Leu Leu Gln Ser
Ser Asn Gly Pro Leu Ser 210 215 220 Trp Leu Trp Gln Ile Leu Phe Gly
Thr Pro Asn Phe Pro Thr Ser Ile 225 230 235 240 Ser Ala Leu Leu Thr
Asp Leu Gln Pro Tyr Ala Ser Xaa Xaa Tyr Asn 245 250 255 Thr Glu Gly
Leu Pro Tyr Phe Ser Ile Gly Met Gly Asn Asn Phe Ile 260 265 270 Gln
Ser Ala Lys Thr Leu Gly Leu Ile Gly Ser Ala Ala Pro Ala Ala 275 280
285 Val Ala Ala Ala Gly Asp Ala Ala Lys Gly Leu Pro Gly Leu Gly Gly
290 295 300 Met Leu Gly Gly Gly Pro Val Ala Ala Gly Leu Gly Asn Ala
Ala Ser 305 310 315 320 Val Gly Lys Leu Ser Val Pro Pro Val Trp Xaa
Gly Pro Leu Pro Gly 325 330 335 Ser Val Thr Pro Gly Ala Ala Pro Leu
Pro Val Ser Thr Val Ser Ala 340 345 350 Ala Pro Glu Ala Ala Pro Gly
Ser Leu Leu Gly Gly Leu Pro Leu Xaa 355 360 365 Gly Ala Gly Gly Ala
Gly Ala Gly Pro Arg Tyr Gly Phe Xaa Pro Thr 370 375 380 Val Met Ala
Arg Pro Pro Phe Xaa Gly Ile Val Ala Ala Ala Thr Tyr 385 390 395 400
127474DNAMycobacterium tuberculosis 127ggcacgagca ccagttgacc
cgcgaagaac ctgaccgcgc cacccagcgc cgcccgcatc 60accggccccg tcccacgaac
cttttcggta aacgagccac tccagcggag atcggtaccg 120cccgacgcat
ttggtgtaag gaccacctcg ccgaagtagt cctggacggg tgtcctcgcg
180ccaaccagct tgtagacgtg gcgacggtcc tgctcatact cgacggtctc
ttcctgcacg 240aacaccggcc acatgcctag tttgcggatg gccccgatgc
cgccgggcgc gggatcaccg 300cgtcgcgccc aactcgattg agcaacgatg
ggcttggccc aggtcgccca gttgccaccg 360tctgtcacga gccgaaacaa
ggttgcagcc ggcgcgctgc tggtcttggt gacctcgaac 420gaaaatttcc
gacccgacat gcgcgactcc cgaaacgaca actgaagctc gtgc
4741281431DNAMycobacterium tuberculosismisc_feature(1)..(1431)n is
a, t, c or g 128ctgcgcgccg gaaaaaanta ttactggcag gaccggcaga
atgcatggtg atattccggt 60gatgaggccg ccgaggaacc gactagtgcg agggtcaaca
catcggttat tcgttgccgt 120ttaggtcttg gatctgccgg gacggcaacg
agttggcagg accgctcacg cgagcgctgt 180tgacagagtc ggttcacgtc
gaactcgcca cccgtcagat gcgaatgata gccacatcgg 240ccacaccatc
gacggcgtcg aagtcgccgt cgtgggtcac gaccggcacc ccttgcgacg
300tggcaacggc agcggccctc accggacggg accgagatcg tcggtggtgt
cgccagtgag 360cgttgcgagg tcgcgggtgc aatcccgcat ctgcttgcgt
atgccgaagc cgccgcagca 420gctcgtctcg actcaaccat cggcgccgtg
cgggctgcct gcggtcagca gcgcaacggg 480tttgccgttg gcagtgatgg
tgatgtcttc gccggcctgc acgcgccgta gcagcccggc 540ggtgttgttg
cgcagttcgc gagacgcgac ttcagcaggc atgctgcggg gatcggcttg
600cgctgggcgc ggtgtcaccg tcatgcgctt gggatatcac gtgatctatc
ggcacgaagc 660cgccggatga gcgaggcaaa ccgcctacac gggctgcctc
gccttgaccg cgccgaacgt 720tactgtgccg ggggcatcag caccgtatcg
atcatgtaca ccgtcgcgtg ggcggtgtga 780ctccgccaca taccaaacgg
gcgttgttga ccatgagtcg tcgcgggcgc ctatcaccgt 840caggtcggca
ccttgcaggt ctgatgggtg ccgtcgatcc tgctcggact cgcctggccg
900gctatcacgt ggtaggtcag gatgctgctg agcagcttgg cgtcagtctt
gagttgatcg 960atagtggccg ccggcagctt gtcgaatgcg gcgttggtgg
gggcgaaaac ggtgtactcg 1020ccgccgttga gggtgtcgac cagattcaca
tccgggttca gcttgcccga cagagccgag 1080gtcagggtac tgagcatcgg
gttgttggaa gccgcggtag cgaccgggtc ttgcgccatt 1140ccggccaccg
atccgggacc ggtgggattt tgcgccgcgt attgcgcgca cccacgacca
1200atcaggtccg ctgcggtcag ccattgccgc cgtggtaacg ggcgccgccg
ggctggtcgc 1260cggtttcggg ctggtgtctt gcgacacggg tttggtgctc
gaacaacccg ctaagaacgc 1320aatcgcgatg gctgcgaggc tcgctgctgc
ggccggtttg gcctgaacgt tgatcatcgc 1380ttcgattcct ttgcttctgc
ggcggcgttg aacgccgtcc tcctgggtgg a 1431129279DNAMycobacterium
tuberculosismisc_feature(1)..(279)a is a, t, c or g 129gcacgagagt
cgtatctttg cacccagcgc ccgtaggaaa ccgctggcct ggctaactca 60gatgcgggcg
gccgtcgatt cgagaggtaa ccgatcgccc gccgacaatg ggttacccac
120cgagactgat tgccgcgcag ccgccttcga cgtgtaagcg ccggttcgtg
catgcccgga 180acggctgcac tcacggacct tctacgtagt acgtgacgga
cttttacgca ttatcgctga 240cgatctttgc ctcccaggac tccagaatct actcgtgcc
2791301470DNAMycobacterium tuberculosismisc_feature(1)..(1470)n is
a, t, c or g 130accgccaccc gcagcccgga atcaccgtcg gtaacctgcg
aatacaattt cttcatcgac 60gacttcgcga acagcgaacc cgagcccacc gcctgatagc
cttcttcctc gatgttccaa 120ccgccggcgg cgtcgaacga aacgatacga
cccgcgctct gcgggtcaga cgcatgaatg 180tcgtagcccg ccagcaacgg
caacgccagc agaccctgca tcgcggccgc cagattgcca 240cgcaccataa
tcgccagccg gttgattttg ccggcaaacg tcagcggcac accctcgagc
300ttctcgtagt gctcaagttc cacggcatac agccgggcaa actcaaccgc
gaccgcagcc 360gtgccagcga tgccggtagc ggtgtagtca tcggtgatat
acaccttgcg cacatcacgc 420ccagaaatca tgttgccctg cgtcgaacgc
cggtcacccg ccatgacaac accgccgggg 480tatttcagcg cgacaatggt
ggtgccgtgc ggcagttgcg catcgccgcc tgcgagtggc 540gcaccgccgc
tgatgcttgc cggcagcaac tccggcgcct ggcggcgcag gaagtcaagt
600gaaagaagat aggtctacag cgggtgttcc agagagtgaa ttaatggaca
ggcgatcggg 660caacggccag gtcactgtcc gcccttttgg acgtatgcgc
ggacgaagtc ctcggcgttc 720tcctcgagga cgtcgtcgat ttcgtcgagc
agatcgtcgg tctcctcggt cagcttttcg 780cgacgctcct ggcccgcggc
ggtgctgccg gcgatgtcgt catcatcgcc gccgccaccg 840ccacgcttgg
tctgctcttg cgccatcgcc gcctcctgct tcctcatggc ctttcaaaag
900gccgcgggtg cgcgtcacac gcccgctgtc tttctctcac ctaccggtca
acaccaacgt 960ttcccggcct aaccaggctt agcgaggctc agcggtcagt
tgctctacca gctccacggc 1020actgtccacc gaatccagca acgcaccaac
atgcgcctta ctaccccgca acggctccag 1080cgtcgggatg cgaaccagcg
agtcgccgcc aggtcgaaga tcaccgagtc ccagctagcc 1140gcggcgatat
cagccccgaa ccggcgcagg catttcgccg cggaaatacg cgcgggtgtc
1200ggtcggcggt tctccaccgc actcagcacc tggtgtttcg gtgactaaac
gctttatcga 1260gccgcgcgcg accagccggt tgtacaggcc cttgtccagc
cggacatcgg agtactgcag 1320gttgacgagg tgcagccggg gcgccgacca
gctcaggttc tcccgctgcc ggaaaccgtc 1380gagcagccgc agtttggccg
gccagtccag cagctccgcg caatccatcg ggtcacgctc 1440gagctgatcc
agcacgtgtg cccaggtttc 14701311059DNAMycobacterium
tuberculosismisc_feature(1)..(1059)n is a, t, c or g 131attcccatcg
ctccggcacc tatcaccagg tagtcggttt cgatggtttt cgccggccct 60tgcgttggcc
tgggccacgg gtcgttcatg ggccctcctg tgcggattgg aatttgtgac
120aacgaaatcg ggcgatcggt gagcaatcgt cgccgatgca agacacgctt
tcgctgccgc 180ggcgtcaggt ggagtttagg ccagcgtaac aacgtagacc
ggccactgac caaaccccaa 240acccacaaac cctggacgca tgcgggtctc
gggcgtcaaa ttccgggtag atatcgtata 300ccgatatcgg atgccgtagc
cttatcgagg catgagacgc ccgctagacc cacgcgatat 360tccagatgag
ctgcggcgac ggctggggct cttggatgcg gtggtgatcg ggcttgggtc
420catgatcggt gccggaatct ttgctcgtgc cgaattcggc acgagctcgt
gccgaattcg 480gcacgagatt ccaatcccca gaaggtcgta caagccgtca
atggcacttg atcgttggat 540cgatgatgaa cgctctgctc atgcctgccg
cctatctcaa cggtcgtcga ttccatgcat 600tagccttggt tctgcattgc
acgcgtaggg cctacagtct ggctgtcatg cttggccgat 660gtcaacagtt
tttttcatgc taagcagatc gtcagttttg agttcgtgaa gacggcatgt
720tcacttgttg tcgactacat cgtctgcgca catttgccct cctgcaactg
cgctgcgaca 780atgcgccaac cgccgtgtag ctcgtgccga attcggcacg
aggatccacc ggagatggcc 840gacgactacg acgaggcctg gatgctcaac
accgtgttcg actatcacaa cgagaacgca 900aaagaagagg tcatccatct
cgtgcccgac gtgaacaagg agagggggcc catcgaactc 960gtaaccaagg
tagacaaaga gggacatcag actcgtctac gatggggagc cacgttttca
1020tacaaggaac atcctaagtt ttgattcggg aacatccta
1059132153DNAMycobacterium tuberculosismisc_feature(1)..(153)n is
a, t, c or g 132gcacgaggca ttggcgggca tctgcataaa cggtgacgta
tcagcacaaa acagcggaga 60gaacaacatg cgatcagaac gtctccggtg gctggtagcc
gcagaaggtc cgttcgcctc 120ggtgtatttc gacgactcgc acgactcgtg ccg
153133387DNAMycobacterium tuberculosis 133ccgcgcggtc gatcagcgag
ccaggcaaaa actccgtcga gcccgagtcg atgatggtca 60cccggcgcag catctggcga
acgatcacct cgatgtgctt gtcgtggatc gacacacctt 120gggcgcggta
gacctcctgg acctcgcgaa ccaggtgtat ctgcacctcg cgggggccct
180gcacccgcag cacctcatgc gggtcggccg agccttccat cagctgctgg
cccacctcga 240cgtggtcgcc atcggagagc acccgttcgg aaccgtcttc
gtgcttgaac acccgcagcc 300gctgccgctt ggagatcttg tcgtagacca
cttcctcacc gccgtcgtca ggaacgatgg 360tgatcttgta gaaccgctcg ccgtcct
387134389DNAMycobacterium tuberculosis 134gttcagcacg gctatccgat
tgtgccgttc gcttcggtgg gtgctgaaca cggcatcgac 60atcgtgctcg acaacgaatc
cccactgctg gcaccggtcc agttcctcgc cgagaagctg 120ctcggcacca
aagacggtcc ggcgctggtc cgtggtgtcg gactgacacc ggtaccgcgc
180cccgaacggc agtattactg gttcggcgag ccaaccgaca ccacagagtt
tatggggcag 240caagccgacg ataacgccgc acgcagggtg cgcgagcgtg
ccgccgccgc tatcgaacac 300ggcatcgagc tgatgctggc cgagcgcgca
gccgatccaa atcgatccct ggtcggacgg 360ctcttgcgct cggacgccta aggcgcccc
389135480DNAMycobacterium tuberculosismisc_feature(1)..(480)n is a,
t, c or g 135cccgcggtcg gaatgatccc cgtctcgtcg cgcgcccatt tgatgctgtt
gatgagctgt 60ttggagaagc ccggttggcg taccggtgag ccggaatatc tgttggaagc
gtcaccggat 120gtncacatga antncnttgn cccngtngcg gtnttggntg
nggnaaacac gtgttgtnta 180agccttgntg gnctcgnaag ngccgtngac
gcctgtgtcg ccgaagataa tgagcacctg 240acggttggcg ggatcgccgt
tatcccaagg aattccgagg tcggtcccgg agatgccgaa 300gcgttccagg
gtcttgttgg ggctgtccgg tccggtcacc cactcggcga gggatgtggn
360agccccggcg agcgtggcac caggatccgg cgccgccgcc ggagcagggt
cggnngctgn 420nctgnnttcc tnnngccnaa ttnnactccn ncnacaanct
tgnnnccgac tcnnacccgn 480136587DNAMycobacterium
tuberculosismisc_feature(1)..(587)n is a, t, g or c 136gcacgaggct
accggcgcgt cgcccgccat gccctggatg cacgcgtagc cacccgtnca 60tncagcgggt
cagccgccgc gtccgggctt aacgctatag cagctgcaaa caacccagcg
120ccggcaatta ctttgatgtt gaaccgatga ccatngcctn cgngtncaat
ctcntctctt 180ngcgcgccnc tatttnngcc atanatttgg ttnnanncgn
aacgctagac gtatcgagtt 240ccttttcgac caccggctca attgtcagca
tcctatgggg aacatgagcc ccgccgcacc 300gggccgtttc caaatggtga
cgtcacaacg gtgtcacaag ccagcgcaat gtccgcggta 360gggacgcggc
ggctgggatc ggtggggtga gcgcccggct tctcaaagcg aggggagccc
420cgggactctt accggccgaa ggcggcgggt gtcactgatc taggctgacg
gccagtggtt 480gntnagccaa caaggatgac nacaaataan ccgagganag
acangngacg gnccganang 540ctnanccggn nttgnncnaa nnnnacncac
ttntaccgnn cttatgn 5871371200DNAMycobacterium tuberculosis
137caggcatgag cagagcgttc atcatcgatc caacgatcag tgccattgac
ggcttgtacg 60accttctggg gattggaata cccaaccaag ggggtatcct ttactcctca
ctagagtact 120tcgaaaaagc cctggaggag ctggcagcag cgtttccggg
tgatggctgg ttaggttcgg 180ccgcggacaa atacgccggc aaaaaccgca
accacgtgaa ttttttccag gaactggcag 240acctcgatcg tcagctcatc
agcctgatcc acgaccaggc caacgcggtc cagacgaccc 300gcgacatcct
ggagggcgcc aagaaaggtc tcgagttcgt gcgcccggtg gctgtggacc
360tgacctacat cccggtcgtc gggcacgccc tatcggccgc cttccaggcg
ccgttttgcg 420cgggcgcgat ggccgtagtg ggcggcgcgc ttgcctactt
ggtcgtgaaa acgctgatca 480acgcgactca actcctcaaa ttgcttgcca
aattggcgga gttggtcgcg gccgccattg 540cggacatcat ttcggatgtg
gcggacatca tcaagggcac cctcggagaa gtgtgggagt 600tcatcacaaa
cgcgctcaac ggcctgaaag agctttggga caagctcacg gggtgggtga
660ccggactgtt ctctcgaggg tggtcgaacc tggagtcctt ctttgcgggc
gtccccggct 720tgaccggcgc gaccagcggc ttgtcgcaag tgactggctt
gttcggtgcg gccggtctgt 780ccgcatcgtc gggcttggct cacgcggata
gcctggcgag ctcagccagc ttgcccgccc 840tggccggcat tgggggcggg
tccggttttg ggggcttgcc gagcctggct caggtccatg 900ccgcctcaac
tcggcaggcg ctacggcccc gagctgatgg cccggtcggc gccgctgccg
960agcaggtcgg cgggcagtcg cagctggtct ccgcgcaggg ttcccaaggt
atgggcggac 1020ccgtaggcat gggcggcatg cacccctctt cgggggcgtc
gaaagggacg acgacgaaga 1080agtactcgga aggcgcggcg gcgggcactg
aagacgccga gcgcgcgcca gtcgaagctg 1140acgcgggcgg tgggcaaaag
gtgctggtac gaaacgtcgt ctaacggcat ggcgagccaa
1200138392PRTMycobacterium tuberculosis 138Met Ser Arg Ala Phe Ile
Ile Asp Pro Thr Ile Ser Ala Ile Asp Gly 1 5 10 15 Leu Tyr Asp Leu
Leu Gly Ile Gly Ile Pro Asn Gln Gly Gly Ile Leu 20 25 30 Tyr Ser
Ser Leu Glu Tyr Phe Glu Lys Ala Leu Glu Glu Leu Ala Ala 35 40 45
Ala Phe Pro Gly Asp Gly Trp Leu Gly Ser Ala Ala Asp Lys Tyr Ala 50
55 60 Gly Lys Asn Arg Asn His Val Asn Phe Phe Gln Glu Leu Ala Asp
Leu 65 70 75 80 Asp Arg Gln Leu Ile Ser Leu Ile His Asp Gln Ala Asn
Ala Val Gln 85 90 95 Thr Thr Arg Asp Ile Leu Glu Gly Ala Lys Lys
Gly Leu Glu Phe Val 100 105 110 Arg Pro Val Ala Val Asp Leu Thr Tyr
Ile Pro Val Val Gly His Ala 115 120 125 Leu Ser Ala Ala Phe Gln Ala
Pro Phe Cys Ala Gly Ala Met Ala Val 130 135 140 Val Gly Gly Ala Leu
Ala Tyr Leu Val Val Lys Thr Leu Ile Asn Ala 145 150 155 160 Thr Gln
Leu Leu Lys Leu Leu Ala Lys Leu Ala Glu Leu Val Ala Ala 165 170 175
Ala Ile Ala Asp Ile Ile Ser Asp Val Ala Asp Ile Ile Lys Gly Thr 180
185 190 Leu Gly Glu Val Trp Glu Phe Ile Thr Asn Ala Leu Asn Gly Leu
Lys 195 200 205 Glu Leu Trp Asp Lys Leu Thr Gly Trp Val Thr Gly Leu
Phe Ser Arg 210 215 220 Gly Trp Ser Asn Leu Glu Ser Phe Phe Ala Gly
Val Pro Gly Leu Thr 225 230 235 240 Gly Ala Thr Ser Gly Leu Ser Gln
Val Thr Gly Leu Phe Gly Ala Ala 245 250 255 Gly Leu Ser Ala Ser Ser
Gly Leu Ala His Ala Asp Ser Leu Ala Ser 260 265 270 Ser Ala Ser Leu
Pro Ala Leu Ala Gly Ile Gly Gly Gly Ser Gly Phe 275 280 285 Gly Gly
Leu Pro Ser Leu Ala Gln Val His Ala Ala Ser Thr Arg Gln 290 295 300
Ala Leu Arg Pro Arg Ala Asp Gly Pro Val Gly Ala Ala Ala Glu Gln 305
310 315 320 Val Gly Gly Gln Ser Gln Leu Val Ser Ala Gln Gly Ser Gln
Gly Met 325 330 335 Gly Gly Pro Val Gly Met Gly Gly Met His Pro Ser
Ser Gly Ala Ser 340 345 350 Lys Gly Thr Thr Thr Lys Lys Tyr Ser Glu
Gly Ala Ala Ala Gly Thr 355 360 365 Glu Asp Ala Glu Arg Ala Pro Val
Glu Ala Asp Ala Gly Gly Gly Gln 370 375 380 Lys Val Leu Val Arg Asn
Val Val 385 390 139439DNAMycobacterium tuberculosis 139acgtttaccc
atgccgtcgg tgcagagcaa cgccagacaa cacaaagtag tctaattccg 60ttataaagca
gacatttccg tggttatgta gaagatgtcg accgatcaga tgaagcgatc
120cgcgtcaggt ggtatccgat gtcttttgtg
accatccagc cggtggtctt ggcagccgcg 180acgggggact tgccgacgat
cggtaccgcc gtgagtgctc ggaacacagc cgtctgtgcc 240ccgacgacgg
gggtgttacc ccctgctgcc aatgacgtgt cggtcctgac ggcggcccgg
300ttcaccgcgc acaccaagca ctaccgagtg gtgagtaagc cggccgcgct
ggtccatggc 360atgttcgtgg ccctcccggc ggccaccgcc gatgcgtatg
cgaccaccga ggccgtcaat 420gtggtcgcga ccggttaag
4391401441DNAMycobacterium tuberculosis 140gaggttgctg gcaatggatt
tcgggctttt acctccggaa gtgaattcaa gccgaatgta 60ttccggtccg gggccggagt
cgatgctagc cgccgcggcc gcctgggacg gtgtggccgc 120ggagttgact
tccgccgcgg tctcgtatgg atcggtggtg tcgacgctga tcgttgagcc
180gtggatgggg ccggcggcgg ccgcgatggc ggccgcggca acgccgtatg
tggggtggct 240ggccgccacg gcggcgctgg cgaaggagac ggccacacag
gcgagggcag cggcggaagc 300gtttgggacg gcgttcgcga tgacggtgcc
accatccctc gtcgcggcca accgcagccg 360gttgatgtcg ctggtcgcgg
cgaacattct ggggcaaaac agtgcggcga tcgcggctac 420ccaggccgag
tatgccgaaa tgtgggccca agacgctgcc gtgatgtaca gctatgaggg
480ggcatctgcg gccgcgtcgg cgttgccgcc gttcactcca cccgtgcaag
gcaccggccc 540ggccgggccc gcggccgcag ccgcggcgac ccaagccgcc
ggtgcgggcg ccgttgcgga 600tgcacaggcg acactggccc agctgccccc
ggggatcctg agcgacattc tgtccgcatt 660ggccgccaac gctgatccgc
tgacatcggg actgttgggg atcgcgtcga ccctcaaccc 720gcaagtcgga
tccgctcagc cgatagtgat ccccaccccg ataggggaat tggacgtgat
780cgcgctctac attgcatcca tcgcgaccgg cagcattgcg ctcgcgatca
cgaacacggc 840cagaccctgg cacatcggcc tatacgggaa cgccggcggg
ctgggaccga cgcagggcca 900tccactgagt tcggcgaccg acgagccgga
gccgcactgg ggccccttcg ggggcgcggc 960gccggtgtcc gcgggcgtcg
gccacgcagc attagtcgga gcgttgtcgg tgccgcacag 1020ctggaccacg
gccgccccgg agatccagct cgccgttcag gcaacaccca ccttcagctc
1080cagcgccggc gccgacccga cggccctaaa cgggatgccg gcaggcctgc
tcagcgggat 1140ggctttggcg agcctggccg cacgcggcac gacgggcggt
ggcggcaccc gtagcggcac 1200cagcactgac ggccaagagg acggccgcaa
acccccggta gttgtgatta gagagcagcc 1260gccgcccgga aaccccccgc
ggtaaaagtc cggcaaccgt tcgtcgccgc gcggaaaatg 1320cctggtgagc
gtggctatcc gacgggccgt tcacaccgct tgtagtagcg tacggctatg
1380gacgacggtg tctggattct cggcggctat cagagcgatt ttgctcgcaa
cctcagcaaa 1440g 144114199PRTMycobacterium tuberculosis 141Met Ser
Phe Val Thr Ile Gln Pro Val Val Leu Ala Ala Ala Thr Gly 1 5 10 15
Asp Leu Pro Thr Ile Gly Thr Ala Val Ser Ala Arg Asn Thr Ala Val 20
25 30 Cys Ala Pro Thr Thr Gly Val Leu Pro Pro Ala Ala Asn Asp Val
Ser 35 40 45 Val Leu Thr Ala Ala Arg Phe Thr Ala His Thr Lys His
Tyr Arg Val 50 55 60 Val Ser Lys Pro Ala Ala Leu Val His Gly Met
Phe Val Ala Leu Pro 65 70 75 80 Ala Ala Thr Ala Asp Ala Tyr Ala Thr
Thr Glu Ala Val Asn Val Val 85 90 95 Ala Thr Gly
142423PRTMycobacterium tuberculosis 142Met Asp Phe Gly Leu Leu Pro
Pro Glu Val Asn Ser Ser Arg Met Tyr 1 5 10 15 Ser Gly Pro Gly Pro
Glu Ser Met Leu Ala Ala Ala Ala Ala Trp Asp 20 25 30 Gly Val Ala
Ala Glu Leu Thr Ser Ala Ala Val Ser Tyr Gly Ser Val 35 40 45 Val
Ser Thr Leu Ile Val Glu Pro Trp Met Gly Pro Ala Ala Ala Ala 50 55
60 Met Ala Ala Ala Ala Thr Pro Tyr Val Gly Trp Leu Ala Ala Thr Ala
65 70 75 80 Ala Leu Ala Lys Glu Thr Ala Thr Gln Ala Arg Ala Ala Ala
Glu Ala 85 90 95 Phe Gly Thr Ala Phe Ala Met Thr Val Pro Pro Ser
Leu Val Ala Ala 100 105 110 Asn Arg Ser Arg Leu Met Ser Leu Val Ala
Ala Asn Ile Leu Gly Gln 115 120 125 Asn Ser Ala Ala Ile Ala Ala Thr
Gln Ala Glu Tyr Ala Glu Met Trp 130 135 140 Ala Gln Asp Ala Ala Val
Met Tyr Ser Tyr Glu Gly Ala Ser Ala Ala 145 150 155 160 Ala Ser Ala
Leu Pro Pro Phe Thr Pro Pro Val Gln Gly Thr Gly Pro 165 170 175 Ala
Gly Pro Ala Ala Ala Ala Ala Ala Thr Gln Ala Ala Gly Ala Gly 180 185
190 Ala Val Ala Asp Ala Gln Ala Thr Leu Ala Gln Leu Pro Pro Gly Ile
195 200 205 Leu Ser Asp Ile Leu Ser Ala Leu Ala Ala Asn Ala Asp Pro
Leu Thr 210 215 220 Ser Gly Leu Leu Gly Ile Ala Ser Thr Leu Asn Pro
Gln Val Gly Ser 225 230 235 240 Ala Gln Pro Ile Val Ile Pro Thr Pro
Ile Gly Glu Leu Asp Val Ile 245 250 255 Ala Leu Tyr Ile Ala Ser Ile
Ala Thr Gly Ser Ile Ala Leu Ala Ile 260 265 270 Thr Asn Thr Ala Arg
Pro Trp His Ile Gly Leu Tyr Gly Asn Ala Gly 275 280 285 Gly Leu Gly
Pro Thr Gln Gly His Pro Leu Ser Ser Ala Thr Asp Glu 290 295 300 Pro
Glu Pro His Trp Gly Pro Phe Gly Gly Ala Ala Pro Val Ser Ala 305 310
315 320 Gly Val Gly His Ala Ala Leu Val Gly Ala Leu Ser Val Pro His
Ser 325 330 335 Trp Thr Thr Ala Ala Pro Glu Ile Gln Leu Ala Val Gln
Ala Thr Pro 340 345 350 Thr Phe Ser Ser Ser Ala Gly Ala Asp Pro Thr
Ala Leu Asn Gly Met 355 360 365 Pro Ala Gly Leu Leu Ser Gly Met Ala
Leu Ala Ser Leu Ala Ala Arg 370 375 380 Gly Thr Thr Gly Gly Gly Gly
Thr Arg Ser Gly Thr Ser Thr Asp Gly 385 390 395 400 Gln Glu Asp Gly
Arg Lys Pro Pro Val Val Val Ile Arg Glu Gln Pro 405 410 415 Pro Pro
Gly Asn Pro Pro Arg 420 14397PRTMycobacterium tuberculosis 143Met
Ser Leu Leu Asp Ala His Ile Pro Gln Leu Val Ala Ser Gln Ser 1 5 10
15 Ala Phe Ala Ala Lys Ala Gly Leu Met Arg His Thr Ile Gly Gln Ala
20 25 30 Glu Gln Ala Ala Met Ser Ala Gln Ala Phe His Gln Gly Glu
Ser Ser 35 40 45 Ala Ala Phe Gln Ala Ala His Ala Arg Phe Val Ala
Ala Ala Ala Lys 50 55 60 Val Asn Thr Leu Leu Asp Val Ala Gln Ala
Asn Leu Gly Glu Ala Ala 65 70 75 80 Gly Thr Tyr Val Ala Ala Asp Ala
Ala Ala Ala Ser Thr Tyr Thr Gly 85 90 95 Phe 14499PRTMycobacterium
tuberculosisMISC_FEATURE(1)..(99)X = any amino acid 144Cys Arg Leu
Cys Leu Asp Ser His Leu Arg Val Val Ala Leu Pro Ala 1 5 10 15 Gly
Gln Pro Gly Arg Leu Val Gln Ala Ile Gly Pro Ala Gln Glu Arg 20 25
30 Asp Val Gly Gln Thr Arg Cys Thr Arg Thr Gly Leu Asp Xaa Val Ser
35 40 45 Ala Leu Thr Ala Ala Gln Phe Ala Ala His Ala Gln Ile Tyr
Gln Ala 50 55 60 Val Ser Ala Gln Ala Ala Ala Ile His Glu Met Phe
Val Asn Thr Leu 65 70 75 80 Gln Xaa Xaa Ser Gly Ser Tyr Ala Ala Thr
Glu Ala Ala Asn Ala Ala 85 90 95 Ala Ala Gly
1451794DNAMycobacterium tuberculosis 145atgcatcacc atcaccatca
catgaattat tcggtgttgc cgccggagat taattcgttg 60cggatgttta ccggtgcggg
gtctgcgccg atgcttgcgg catcggtggc ttgggatggt 120ttggccgcgg
agttggcggt ggcggcgtcc tcgtttgggt cggtgacttc ggggttggcg
180ggtcagtcct ggcagggtgc ggcggcggcg gcgatggccg cggcggcggc
gccgtatgcg 240gggtggttgg ctgctgcggc ggcgcgggcc gctggcgcgt
cggctcaggc caaggcggtg 300gccagtgcgt ttgaggcggc gcgggcggcg
acggtgcatc cgatgctggt ggcggccaac 360cgtaatgcgt ttgtgcagtt
ggtgttgtcg aatctgtttg ggcagaatgc gccggcgatc 420gcggccgctg
aggcgatgta tgaacagatg tgggccgccg atgtggccgc gatggtgggc
480tatcacggcg gggcatcggc ggccgcggcg cagctgtcgt cgtggtcaat
tggtctgcag 540caggcgttgc cagctgcgcc atcggcgctg gccgccgcga
tcggcctcgg caacatcggc 600gtcgggaacc tgggcggcgg gaacaccggt
gactacaatc tgggcagcgg aaattccggc 660aacgccaacg taggtagcgg
aaactccggc aacgccaatg tgggcagcgg aaatgacggt 720gccacgaatt
tgggcagcgg aaatatcggc aacaccaatc tcggcagcgg aaacgttggc
780aatgtcaatc tgggcagcgg aaaccgaggc tttggaaacc tcggcaacgg
aaactttggc 840agtgggaacc tgggcagtgg aaacaccgga agtaccaact
tcggcggcgg aaatctcggt 900tccttcaact tgggcagtgg aaacatcggc
tcctccaaca tcggtttcgg aaacaacggc 960gacaataacc tcggcctcgg
gaacaatggc aacaacaaca tcggttttgg gctcaccggc 1020gacaacttgg
tgggcattgg cgcgctgaac tcgggcatcg ggaatctagg tttcgggaac
1080tcgggtaaca acaacatcgg tttcttcaac tctggcaaca acaacgtggg
cttcttcaat 1140tcgggcaaca acaacttcgg ctttggaaac gcgggcgaca
tcaacacggg cttcggaaac 1200gccggcgaca ccaacacggg cttcggaaac
gccggcttct tcaatatggg catcgggaac 1260gcgggcaacg aagacatggg
cgtcgggaac ggcggttcct ttaacgtggg cgttggcaat 1320gcgggcaacc
aaagtgtggg ctttggcaac gcgggcaccc taaacgtggg cttcgcaaac
1380gcgggcagta tcaatacggg attcgcgaac tcgggcagca tcaatacggg
cggtttcgac 1440tcgggcgacc ggaacaccgg gtttggaagc tcggtcgacc
aatccgtttc gagctcgggc 1500ttcggcaaca ccggcatgaa ttcctcaggc
ttctttaaca cgggcaatgt ttcggctggc 1560tatgggaaca acggtgacgt
tcagtcgggc atcaataaca ccaactccgg cggcttcaac 1620gtcggcttct
ataactcggg tgccggcacc gtgggcatcg caaactctgg cctgcagacc
1680acaggcattg cgaactcggg caccctcaac acgggtgtgg cgaacacggg
tgaccacagc 1740tcggggggct tcaatcaggg cagtgaccag tcgggcttct
tcggtcagcc ctaa 1794146597PRTMycobacterium tuberculosis 146Met His
His His His His His Met Asn Tyr Ser Val Leu Pro Pro Glu 1 5 10 15
Ile Asn Ser Leu Arg Met Phe Thr Gly Ala Gly Ser Ala Pro Met Leu 20
25 30 Ala Ala Ser Val Ala Trp Asp Gly Leu Ala Ala Glu Leu Ala Val
Ala 35 40 45 Ala Ser Ser Phe Gly Ser Val Thr Ser Gly Leu Ala Gly
Gln Ser Trp 50 55 60 Gln Gly Ala Ala Ala Ala Ala Met Ala Ala Ala
Ala Ala Pro Tyr Ala 65 70 75 80 Gly Trp Leu Ala Ala Ala Ala Ala Arg
Ala Ala Gly Ala Ser Ala Gln 85 90 95 Ala Lys Ala Val Ala Ser Ala
Phe Glu Ala Ala Arg Ala Ala Thr Val 100 105 110 His Pro Met Leu Val
Ala Ala Asn Arg Asn Ala Phe Val Gln Leu Val 115 120 125 Leu Ser Asn
Leu Phe Gly Gln Asn Ala Pro Ala Ile Ala Ala Ala Glu 130 135 140 Ala
Met Tyr Glu Gln Met Trp Ala Ala Asp Val Ala Ala Met Val Gly 145 150
155 160 Tyr His Gly Gly Ala Ser Ala Ala Ala Ala Gln Leu Ser Ser Trp
Ser 165 170 175 Ile Gly Leu Gln Gln Ala Leu Pro Ala Ala Pro Ser Ala
Leu Ala Ala 180 185 190 Ala Ile Gly Leu Gly Asn Ile Gly Val Gly Asn
Leu Gly Gly Gly Asn 195 200 205 Thr Gly Asp Tyr Asn Leu Gly Ser Gly
Asn Ser Gly Asn Ala Asn Val 210 215 220 Gly Ser Gly Asn Ser Gly Asn
Ala Asn Val Gly Ser Gly Asn Asp Gly 225 230 235 240 Ala Thr Asn Leu
Gly Ser Gly Asn Ile Gly Asn Thr Asn Leu Gly Ser 245 250 255 Gly Asn
Val Gly Asn Val Asn Leu Gly Ser Gly Asn Arg Gly Phe Gly 260 265 270
Asn Leu Gly Asn Gly Asn Phe Gly Ser Gly Asn Leu Gly Ser Gly Asn 275
280 285 Thr Gly Ser Thr Asn Phe Gly Gly Gly Asn Leu Gly Ser Phe Asn
Leu 290 295 300 Gly Ser Gly Asn Ile Gly Ser Ser Asn Ile Gly Phe Gly
Asn Asn Gly 305 310 315 320 Asp Asn Asn Leu Gly Leu Gly Asn Asn Gly
Asn Asn Asn Ile Gly Phe 325 330 335 Gly Leu Thr Gly Asp Asn Leu Val
Gly Ile Gly Ala Leu Asn Ser Gly 340 345 350 Ile Gly Asn Leu Gly Phe
Gly Asn Ser Gly Asn Asn Asn Ile Gly Phe 355 360 365 Phe Asn Ser Gly
Asn Asn Asn Val Gly Phe Phe Asn Ser Gly Asn Asn 370 375 380 Asn Phe
Gly Phe Gly Asn Ala Gly Asp Ile Asn Thr Gly Phe Gly Asn 385 390 395
400 Ala Gly Asp Thr Asn Thr Gly Phe Gly Asn Ala Gly Phe Phe Asn Met
405 410 415 Gly Ile Gly Asn Ala Gly Asn Glu Asp Met Gly Val Gly Asn
Gly Gly 420 425 430 Ser Phe Asn Val Gly Val Gly Asn Ala Gly Asn Gln
Ser Val Gly Phe 435 440 445 Gly Asn Ala Gly Thr Leu Asn Val Gly Phe
Ala Asn Ala Gly Ser Ile 450 455 460 Asn Thr Gly Phe Ala Asn Ser Gly
Ser Ile Asn Thr Gly Gly Phe Asp 465 470 475 480 Ser Gly Asp Arg Asn
Thr Gly Phe Gly Ser Ser Val Asp Gln Ser Val 485 490 495 Ser Ser Ser
Gly Phe Gly Asn Thr Gly Met Asn Ser Ser Gly Phe Phe 500 505 510 Asn
Thr Gly Asn Val Ser Ala Gly Tyr Gly Asn Asn Gly Asp Val Gln 515 520
525 Ser Gly Ile Asn Asn Thr Asn Ser Gly Gly Phe Asn Val Gly Phe Tyr
530 535 540 Asn Ser Gly Ala Gly Thr Val Gly Ile Ala Asn Ser Gly Leu
Gln Thr 545 550 555 560 Thr Gly Ile Ala Asn Ser Gly Thr Leu Asn Thr
Gly Val Ala Asn Thr 565 570 575 Gly Asp His Ser Ser Gly Gly Phe Asn
Gln Gly Ser Asp Gln Ser Gly 580 585 590 Phe Phe Gly Gln Pro 595
14751DNAArtificial SequencemTTC#3 (MTB57) 5' PCR amplication primer
147caattacata tgcatcacca tcaccatcac atgaattatt cggtgttgcc g
5114833DNAArtificial SequencemTTC#3 (MTB57) 3' PCR amplification
primer 148caattaaagc ttttagggct gaccgaagaa gcc
33149421DNAArtificial Sequence5' P1 lambda phage insert
149ggatccgaat tctgcacgag ggnngacgac ganctttgca cacgagcgat
ggcaaccctc 60acgtccgcgc aaaccccgcg cgaggccgta gagcaattcg tcgagctgat
ggtcgacgat 120ccggtgcgcg ggcgcgtgct gttgctggcg ccggcggtag
aaccggccct gacccggtcg 180ggcgcggagt ggatgcccaa cttcatcgag
ttgctgcaac gcaagttgtc ccgcatcgtt 240gatccagttc tgcagaaact
ggtcgccacc agcttgatcg gcgctcttac cggtctgttc 300accgcatatc
tgaacggacg gctgggagcc acccgcaagc aattcatcga ctactgcgtc
360aacatgttgc tcagcaccgc cgcacctacg caccgcaccg cgagcgggga
gaatccgaac 420a 421150675DNAArtificial SequenceP2 lambda phage
insert 150gatccgaatt cggcacgagt cgaggccacc gcttccatgg ccaggcccac
atnttgatcg 60gcgtggtggc cacgcccggt gtgaagtgct gttggccgtg atgtcggatt
acagtctcgg 120cgtgcccgac gagacaggcc ttggtgctga cgcggcgcgc
gcgtgaagtg gcgctgacac 180agcacattgg ggtatccgcg gagaccgatc
gggccgtcgt ccccaagctg cgccaggcct 240atgacagcct ggtgtgcggt
cgccgccggc ttggcgccat tggagccgag atcgagaacg 300cggtggccca
tcagcgcgcg ctgggccttg acaccccggc cggtgcccgt aacttctccc
360ggtttctcgc caccaaagca cacgacatca cgcgagtgct ggcagcaacc
gccgcggaat 420cccaggccgg cgcggcgcgg ttgcgatccc tggcttcgtc
ctatcaggct gtgggatttg 480gccccaaacc ccaggagccg cctccggatc
cagtgccatt tccgccctac cagccgaagg 540tgtgggcggc gtgccgggcg
cgtggccaag acccggacaa ggtcgtcagg acgttccatc 600acgcgccgat
gagcgcgaga ttccgctcgc ttactcgtgc cgaattsgga tctgatatcg
660ccatggcctt gtcgt 675151276DNAArtificial Sequence3' P3 lambda
phage insert 151tgatcggtca atgcgcagta ctggtgacct agcgccgccg
cggtggccat catctcctcg 60atcggcgcgg acccgtccga ccagttcgaa tgcagatgca
gatccccgcg caatgcggca 120cggatcgccc ctccaccgag atcctcagcg
tcagcgcgta attcagccag caggtccggc 180tcgcggccag accaggcctg
ggcgatgact ttcgcggttt tgggaccgat acccgccagc 240gactgccagc
tgttggcctg gccgtgccgc tgccgc 276152456DNAArtificial SequenceP4
lambda phage insert 152ggatccgaat tctgcacgag gangaagtca tactgccgtc
atacacnttt gtctntaccg 60ccaacgcctt cgtgttgcgc ggtggtgtgc cagtctttgt
cgatattcgg cccgacacgc 120tcaacattga tgaaactcgc atcgtagacg
ccatcacccc gcgaaccaag gccatcgtcc 180ccgttcacta tgccggcgtg
gcctgcgaga tggacgcgat catgaagatc gccacgcacc 240acaacctggc
ggtggtcgaa gacgcggccc aaggcgcgat ggcgtcgtat cgtgggcggg
300cgctcggcag catcggcgac ctgggagcgc tctcatttca cgagaccaag
aatgtgattt 360ccggcgaagg cggcgccctg cttgtcaact cataagactt
cctgctccgg gcaganattc 420tcagggaaaa gggcaccaat nncagccngc ttcctt
456153341DNAArtificial SequenceP6 lambda phage insert 153gatatcggat
cggaattcgg cacgaggtgc ccntgggggg acaactggtg cacaagaggt
60tcgtccgtcc
cggtcctntc gtatagggac aggtttcctc aagtttctga cgcgcgcggc
120ggatagagac cgaactgtct cacgacgttc taaacccagc tcgcgtgccg
ctttaatggg 180cgaacagccc aacccttggg acctgctcca gccccaggat
gcgacgagcc gacatcgagg 240tgccaaacca tcccgtcgat atggactctt
ggggaagatc agcctgttat ccccggggta 300ccttttatcc gttgagcgac
accccttcca ctcgggggtg c 341154396DNAArtificial SequenceP7 lambda
phage insert 154gatccgaatt cagagcggcg acccgtgctc caagctcctt
cagcgtcgtc acgggctcat 60cctatccggc agatcagcag gcggttcctc cgcaaagtgc
ggctgcaacc taccgacttc 120gtgcgcggcg aggaacgcgc cccctggggg
tatccgcccg cgtcagacaa cagtgcctcg 180gtctgatcgg taataggcga
ccgcctcgag gtccacatcc gccacctgct cgaaacgtca 240ggtcttgggg
tgcggggtgt accggacggt atgcgcccag atcgtgccgt ctcggaatac
300gaaagtatcg actccgtcgt cgactcggct gaccgcggaa ttcgcggtcc
actccaggaa 360cagtatgtcg ccctcgaaga tttgggtctt taagtc
396155172DNAArtificial SequenceP8 lambda phage insert 155ggatccgaat
tcggcacgag gagtatcagc agaggtcgga gaaggtgctg accgaataca 60acaacaaggc
agccctggaa ccggtaaacc cgccgaagcc tccccccgcc atcaagatcg
120acccgccccc gcctccgcaa gagcagggat tgatccctgg cttcctgatg cc
17215693DNAArtificial SequenceP9 lambda phage insert 156ggatccgaat
tcggcacgag ccagaacctc gccngccccg ggcggcagng acaccaactg 60gacaccacgc
cgcggatcgg cnngagcagc gcc 93157609DNAArtificial Sequence5' P10
lambda phage insert 157gatccgaatt cggcacgaga agaatntgac ccnncnccng
tggctgatgc gagagcttnc 60ttntttcttc cccccantgg ttggacgggg tcgtcacagc
gggcattcta agtcccgcgg 120gccacaaaag gcagtgccgc ggaacttctt
ggcccaaacg ggcacccggc tacgtgcgca 180ccgcgaccgt cgacaactgg
tcggcgagcc ggtccgggga atccaccatc gagaacgtcc 240gtgctccctc
gattacctcg aaacgggcgc gcgggatggt cgcggcgagc cgttgaccgt
300tctcgagtgc gaagaacacg tcatccgccg accacgcgat gagcgccggc
ttgtcgaatt 360caggcagccg ggcggcgact gcggtggtga cttcggtgcg
cagcgatagc gagagctgac 420gcaggtcttc ggcgatggcc gggttggata
gcgccggacg aacccaggcc cgggtgagat 480ggtcgatgtt gtggtgcgac
aaaccggcat acgcgcggtt tacgcgcggc cggtgcccgc 540atcacctgga
tcgcggcccg gaacagggtg gccgatttcg cggncaggat cacctgnttt 600gaggatcgg
609158301DNAArtificial Sequence5' P11 lambda phage insert
158ggatccgaat tcggcacgag tgcggtgcct atctgcgttg gccagtacct
cgcggacctg 60gcgagtgcgg acgcgcaggc tatcgaagtg ggcctaaaga cggcggacgt
ggcgcccgtt 120gccgtacgac ctgcagcggc gccgccgttg cgtgagtctg
ccgcggtgcg accggaggcc 180aggctggtgt cggcggtggc gccagctccc
gcgggcacgt cggcgtcggt gctggcttcg 240gatcggggtg ccggcgtgtt
ggggtttgcc gggaccgctg gcaaggantc cnttgggcgt 300c
301159261DNAArtificial Sequence3' P12 lambda phage insert
159ggatccgaat cgcactcgcg ggtctgctgg acgagtggac gccggtgatc
gccggcgccg 60aactgggcga gcacccctac acgccgatca cgccggagtc gatccggcgg
gccgcgcagc 120tcggcgacga cctaccggtg gcgtggaagc accgcagcga
gcgctacacc gagaagctgg 180ccacccccga caccagcgtc gccgacctgg
tcggcgacgt cgacccgatc aaggttgccg 240agggccgcag cctcggggat c
2611601404DNAMycobacterium tuberculosismisc_feature(1)..(1404)n is
a, t, c or g 160tgagattggc agaccggtga gcaccggata cagccacgca
aagttcgtca ccacgagggc 60cacgtagcag cagacgacga tcagccccag tgtgcgtcgt
tcggagccct gaccggggtg 120atagaggata tcgccgagaa ccagcgaaat
gcccatcacc agaaatggcg ccatggtcgc 180tgcgtagaag aagtacatct
gccggtcgat gtcggcgaac cacggcagcc aaccggcgca 240gtagccgacc
aggaccaccg cataacgcca gtcccggcgc acaaacatac gccaccccgc
300gtatgccagg actggcaccg ccagccacca catcgcgggc gtgccgacca
gcatctcggc 360cttgacgcac gactgtgcgc cgcagcctgc aacgtcttgc
tggtcgatgg cgtacagcac 420cggccgcaac gacatgggcc aggtccacgg
tttggattcc caagggtggt agttgcctgc 480ggaattcgtc aggcccgcgt
ggaagtggaa cgctttggcg gtgtagtgcc agagcgagcg 540cacggcgtcg
ggcagcggaa caaccgagtt gcgaccgacc gcttgaccga ccgcatgccg
600atcgatcgcg gtctcggacg cgaaccacgg agcgtaggtg gccagataga
ccgcgaacgg 660gatcaacccc agcgcatacc cgctgggaag cacgtcacgc
cgcactgtcc ccagccacgg 720tctttgcact tggtactgac gtcgcgccgc
cacgtcgaac gccagcgcca tcgcgccgaa 780gaacagcacg aagtacacgc
cggaccactt ggtggcgcaa gccaatccca gcagcacccc 840ggcgccgaac
cgccaccagc gcacacccac ccgcggtccc cacacggtgg cggcgctgcg
900gccggccagc agagcgatgt gcatccgttc gcgaacctga tcgcggtcga
cgatgagcgc 960gccgaacgcc gcgacgacga agaacgtcag gaagccgtcc
agcagcgcgg tccgcgcggt 1020gacgaagctg accccgtcgc agatcagcag
caccccggcg atggcgccga ccaatgtcga 1080ccggctgatc cgccgcacga
tccgcaccac cagcgccacc aggaccacac ccagcagggc 1140gccggtgaac
cgccagccga atccgttgta accgaagatg gcctccccga tcgcgatcag
1200ctgcttaccg accggcgggt gaaccaccag gccgtacccg gggttgtctt
ccaccccatg 1260gttgttcagc acctgccagg cctggggtgc gtaatgcttc
tcgtcgaaga tgggggtgcc 1320ggcatcggtc agcgagccca ggttcaggaa
ccgggtcacc gtggccagca gcgtgatcag 1380gccggtcacg atccagccgc gtaa
1404161453PRTMycobacterium tuberculosis 161Val Arg His His Glu Gly
His Val Ala Ala Asp Asp Asp Gln Pro Gln 1 5 10 15 Cys Ala Ser Phe
Gly Ala Leu Thr Gly Val Ile Glu Asp Ile Ala Glu 20 25 30 Asn Gln
Arg Asn Ala His His Gln Lys Trp Arg His Gly Arg Cys Val 35 40 45
Glu Glu Val His Leu Pro Val Asp Val Gly Glu Pro Arg Gln Pro Thr 50
55 60 Gly Ala Val Ala Asp Gln Asp His Arg Ile Thr Pro Val Pro Ala
His 65 70 75 80 Lys His Thr Pro Pro Arg Val Cys Gln Asp Trp His Arg
Gln Pro Pro 85 90 95 His Arg Gly Arg Ala Asp Gln His Leu Gly Leu
Asp Ala Arg Leu Cys 100 105 110 Ala Ala Ala Cys Asn Val Leu Leu Val
Asp Gly Val Gln His Arg Pro 115 120 125 Gln Arg His Gly Pro Gly Pro
Arg Phe Gly Phe Pro Arg Val Val Val 130 135 140 Ala Cys Gly Ile Arg
Gln Ala Arg Val Glu Val Glu Arg Phe Gly Gly 145 150 155 160 Val Val
Pro Glu Arg Ala His Gly Val Gly Gln Arg Asn Asn Arg Val 165 170 175
Ala Thr Asp Arg Leu Thr Asp Arg Met Pro Ile Asp Arg Gly Leu Gly 180
185 190 Arg Glu Pro Arg Ser Val Gly Gly Gln Ile Asp Arg Glu Arg Asp
Gln 195 200 205 Pro Gln Arg Ile Pro Ala Gly Lys His Val Thr Pro His
Cys Pro Gln 210 215 220 Pro Arg Ser Leu His Leu Val Leu Thr Ser Arg
Arg His Val Glu Arg 225 230 235 240 Gln Arg His Arg Ala Glu Glu Gln
His Glu Val His Ala Gly Pro Leu 245 250 255 Gly Gly Ala Ser Gln Ser
Gln Gln His Pro Gly Ala Glu Pro Pro Pro 260 265 270 Ala His Thr His
Pro Arg Ser Pro His Gly Gly Gly Ala Ala Ala Gly 275 280 285 Gln Gln
Ser Asp Val His Pro Phe Ala Asn Leu Ile Ala Val Asp Asp 290 295 300
Glu Arg Ala Glu Arg Arg Asp Asp Glu Glu Arg Gln Glu Ala Val Gln 305
310 315 320 Gln Arg Gly Pro Arg Gly Asp Glu Ala Asp Pro Val Ala Asp
Gln Gln 325 330 335 His Pro Gly Asp Gly Ala Asp Gln Cys Arg Pro Ala
Asp Pro Pro His 340 345 350 Asp Pro His His Gln Arg His Gln Asp His
Thr Gln Gln Gly Ala Gly 355 360 365 Glu Pro Pro Ala Glu Ser Val Val
Thr Glu Asp Gly Leu Pro Asp Arg 370 375 380 Asp Gln Leu Leu Thr Asp
Arg Arg Val Asn His Gln Ala Val Pro Gly 385 390 395 400 Val Val Phe
His Pro Met Val Val Gln His Leu Pro Gly Leu Gly Cys 405 410 415 Val
Met Leu Leu Val Glu Asp Gly Gly Ala Gly Ile Gly Gln Arg Ala 420 425
430 Gln Val Gln Glu Pro Gly His Arg Gly Gln Gln Arg Asp Gln Ala Gly
435 440 445 His Asp Pro Ala Ala 450 1621863DNAMycobacterium
tuberculosismisc_feature(1)..(1863)n is a, t, c or g 162gtggcgctcg
tcgtgcagaa gtacggcgga tcctcggtgg ccgacgccga acggattcgc 60cgcgtcgccg
aacgcatcgt cgccaccaag aagcaaggca atgacgtcgt cgtcgtcgtc
120tctgccatgg gggataccac cgacgacctg ctggatctgg ctcagcaggt
gtgcccggcg 180ccgccgcctc gggagctgga catgctgctt accgccggtg
aacgcatctc gaatgcgttg 240gtggccatgg ccatcgagtc gctcggcgcg
catgcccggt cgttcaccgg ttcgcaggcc 300ggggtgatca ccaccggcac
ccacggcaac gccaagatca tcgacgtcac gccggggcgg 360ctgcaaaccg
cccttgagga ggggcgggtc gttttggtgg ccggattcca aggggtcagc
420caggacacca aggatgtcac gacgttgggc cgcggcggct cggacaccac
cgccgtcgcc 480atggccgccg cgctgggtgc cgatgtctgt gagatctaca
ccgacgtgga cggcatcttc 540agcgccgacc cgcgcatcgt gcgcaacgcc
cgaaagctcg acaccgtgac cttcgaggaa 600atgctcgaga tggcggcctg
cggcgccaag gtgctgatgc tgcgctgcgt ggaatacgct 660cgccgccata
atattccggt gcacgtccgg tcgtcgtact cggacagacc gggcaccgtc
720gttgtcggat cgatcaagga cgtacccatg gaagacccca tcctgaccgg
agtcgcgcac 780gaccgcagcg aggccaaggt gaccatcgtc gggctgcccg
acatccccgg gtatgcggcc 840aaggtgttta gggcggtggc cgacgccgac
gtcaacatcg acatggtgct gcagaacgtc 900tccaaggtcg aggacggcaa
gaccgacatc accttcacct gctcccgcga cgtcgggccc 960gccgccgtgg
aaaaactgga ctcgctcaga aacgagatcg gcttctcaca gctgctgtac
1020gacgaccaca tcggcaaggt atcgctgatc ggtgccggca tgcgcagcca
ccccggggtc 1080accgcgacgt tctgtgaggc gctggcggcg gtgggggtca
acatcgagct gatctccacc 1140tcggagatca ggatctcggt gttgtgccgc
gacaccgaac tggacaaggc cgtggtcgcg 1200ctgcatgaag cgttcgggct
cggcggcgac gaggaggcca cggtgtacgc ggggacggga 1260cggtagatgg
gcctgtcaat agggatcgtg ggggccaccg gtcaggtggg tcaggtcatg
1320cgcacgttgc tcgacgagcg ggatttcccg gcgagcgcgg tgcggttctt
cgcgtcggcc 1380cgatcgcagg gccgcaagct ggccttccgc ggccaggaga
tcgaagtgga agacgccgag 1440acggccgacc cgagcgggct ggatatcgcg
ttgttctccg ccggctcggc catgtcgaag 1500gtgcaggcgc cccgctttgc
ggcggccgga gtcacggtga tcgacaactc gtcggcgtgg 1560cgtaaggacc
ccgacgtgcc gttggtggtg tccgaggtga actttgaacg cgacgcgcac
1620cgccggccca aggctcgtgc cgctcgtgcc gaattcggca cgagccgacg
tggtcggcaa 1680cgtcctggat cgcgggcagc tggttgttga ggatgaatcc
gtccaccagg tggtaggagc 1740cgaacgaaga ttccaccgtc gtcgtcaacg
tggccgcatt gccgtacgaa tcgacgacgc 1800tgaggtggct ggtgccatgc
tcaggcactg gcggggcgac ggccgtcggt gcgccgaagt 1860ccc
1863163421PRTMycobacterium tuberculosisMISC_FEATURE(1)..(421)X =
any amino acid 163Val Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser
Val Ala Asp Ala 1 5 10 15 Glu Arg Ile Arg Arg Val Ala Glu Arg Ile
Val Ala Thr Lys Lys Gln 20 25 30 Gly Asn Asp Val Val Val Val Val
Ser Ala Met Gly Asp Thr Thr Asp 35 40 45 Asp Leu Leu Asp Leu Ala
Gln Gln Val Cys Pro Ala Pro Pro Pro Arg 50 55 60 Glu Leu Asp Met
Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu 65 70 75 80 Val Ala
Met Ala Ile Glu Ser Leu Gly Ala His Ala Arg Ser Phe Thr 85 90 95
Gly Ser Gln Ala Gly Val Ile Thr Thr Gly Thr His Gly Asn Ala Lys 100
105 110 Ile Ile Asp Val Thr Pro Gly Arg Leu Gln Thr Ala Leu Glu Glu
Gly 115 120 125 Arg Val Val Leu Val Ala Gly Phe Gln Gly Val Ser Gln
Asp Thr Lys 130 135 140 Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp
Thr Thr Ala Val Ala 145 150 155 160 Met Ala Ala Ala Leu Gly Ala Asp
Val Cys Glu Ile Tyr Thr Asp Val 165 170 175 Asp Gly Ile Phe Ser Ala
Asp Pro Arg Ile Val Arg Asn Ala Arg Lys 180 185 190 Leu Asp Thr Val
Thr Phe Glu Glu Met Leu Glu Met Ala Ala Cys Gly 195 200 205 Ala Lys
Val Leu Met Leu Arg Cys Val Glu Tyr Ala Arg Arg His Asn 210 215 220
Ile Pro Val His Val Arg Ser Ser Tyr Ser Asp Arg Pro Gly Thr Val 225
230 235 240 Val Val Gly Ser Ile Lys Asp Val Pro Met Glu Asp Pro Ile
Leu Thr 245 250 255 Gly Val Ala His Asp Arg Ser Glu Ala Lys Val Thr
Ile Val Gly Leu 260 265 270 Pro Asp Ile Pro Gly Tyr Ala Ala Lys Val
Phe Arg Ala Val Ala Asp 275 280 285 Ala Asp Val Asn Ile Asp Met Val
Leu Gln Asn Val Ser Lys Val Glu 290 295 300 Asp Gly Lys Thr Asp Ile
Thr Phe Thr Cys Ser Arg Asp Val Gly Pro 305 310 315 320 Ala Ala Val
Glu Lys Leu Asp Ser Leu Arg Asn Glu Ile Gly Phe Ser 325 330 335 Gln
Leu Leu Tyr Asp Asp His Ile Gly Lys Val Ser Leu Ile Gly Ala 340 345
350 Gly Met Arg Ser His Pro Gly Val Thr Ala Thr Phe Cys Glu Ala Leu
355 360 365 Ala Ala Val Gly Val Asn Ile Glu Leu Ile Ser Thr Ser Glu
Ile Arg 370 375 380 Ile Ser Val Leu Cys Arg Asp Thr Glu Leu Asp Lys
Ala Val Val Ala 385 390 395 400 Leu His Glu Ala Phe Gly Leu Gly Gly
Asp Glu Glu Ala Thr Val Tyr 405 410 415 Ala Gly Thr Gly Arg 420
1641379DNAMycobacterium tuberculosismisc_feature(1)..(1379)n is a,
t, c or g 164atgacgcagt cgcagaccgt gacggtggat cagcaagaga ttttgaacag
ggccaacgag 60gtggaggccc cgatggcgga cccaccgact gatgtcccca tcacaccgtg
cgaactcacg 120gcggctaaaa acgccgccca acagctggta ttgtccgccg
acaacatgcg ggaatacctg 180gcggccggtg ccaaagagcg gcagcgtctg
gcgacctcgc tgcgcaacgc ggccaaggcg 240tatggcgagg ttgatgagga
ggctgcgacc gcgctggaca acgacggcga aggaacgtgc 300aggcagaatc
ggccggggcc gtcggagggg acagttcggc cgaactaacc gatacgccga
360gggtggccac ggccggtgaa cccaacttca tggatctcaa agaagcggca
aggaagctcg 420aaacgggcga ccaaggcgca tcgctcgcgc actttgcgga
tgggtggaac actttcaacc 480tgacgctgca aggcgacgtc aagcggttcc
gggggtttga caactgggaa ggcgatgcgg 540ctaccgcttg cgaggcttcg
ctcgatcaac aacggcaatg gatactccac atggccaaat 600tgagcgctgc
gatggccaag caggctcaat atgtcgcgca gctgcacgtg tgggctaggc
660gggaacatcc gacttatgaa gacatagtcg ggctcgaacg gctttacgcg
gaaaaccctt 720cggcccgcga ccaaattctc ccggtgtacg cggagtatca
gcagaggtcg gagaaggtgc 780tgaccgaata caacaacaag gcagccctgg
aaccggtaaa cccgccgaag cctccccccg 840ccatcaagat cgacccgccc
ccgcctccgc aagagcaggg attgatccct ggcttcctga 900tgccgccgtc
tgacggctcc ggtgtgactc ccggtaccgg gatgccagcc gcaccgatgg
960ttccgcctac cggatcgccg ggtggtggcc tcccggctga cacggcggcg
cagctgacgt 1020cggctgggcg ggaagccgca gcgctgtcgg gcgacgtggc
ggtcaaagcg gcatcgctcg 1080gtggcggtgg aggcggcggg gtgccgtcgg
cgccgttggg atccgcgatc gggggcgccg 1140aatcggtgcg gcccgctggc
gctggtgaca ttgccggctt aggccaggga agggccggcg 1200gcggcgccgc
gctgggcggc ggtggcatgg gaatgccgat gggtgccgcg catcagggac
1260aagggggcgc caagtccaag ggttctcagc aggaagacga ggcgctctac
accgaggatc 1320gggcatggac cgaggccgtc attggtaacc gtcggcgcca
ggacagtaag gagtcgaag 1379
* * * * *
References