U.S. patent application number 13/164962 was filed with the patent office on 2011-11-03 for tuberculosis antigen detection assays and vaccines.
Invention is credited to Antonio Campos-Neto, Suely S. Kashino.
Application Number | 20110268758 13/164962 |
Document ID | / |
Family ID | 37504020 |
Filed Date | 2011-11-03 |
United States Patent
Application |
20110268758 |
Kind Code |
A1 |
Campos-Neto; Antonio ; et
al. |
November 3, 2011 |
TUBERCULOSIS ANTIGEN DETECTION ASSAYS AND VACCINES
Abstract
The present invention relates to isolated Tuberculosis (TB)
antigens that are useful in therapeutic and vaccine compositions
for stimulating a TB specific immunological response. The
identified antigens are also useful in diagnostic assays to
determine the presence of active TB in an individual. Accordingly,
the present invention includes polypeptide molecules, nucleic acid
molecules, vaccine compositions, diagnostic assays, and methods of
diagnosis and monitoring treatment related to these TB
antigens.
Inventors: |
Campos-Neto; Antonio;
(Westborough, MA) ; Kashino; Suely S.; (Boston,
MA) |
Family ID: |
37504020 |
Appl. No.: |
13/164962 |
Filed: |
June 21, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11478366 |
Jun 29, 2006 |
7968694 |
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13164962 |
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60717062 |
Sep 14, 2005 |
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60696439 |
Jul 1, 2005 |
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Current U.S.
Class: |
424/190.1 ;
530/324; 530/326; 530/327; 530/328; 530/329; 530/350; 530/403 |
Current CPC
Class: |
C07K 14/35 20130101;
G01N 2800/12 20130101; C12Q 2600/158 20130101; C07K 2319/00
20130101; C12Q 1/6883 20130101; G01N 33/5695 20130101; A61K 39/00
20130101; A61K 2039/53 20130101; A61P 31/06 20180101; G01N 33/6893
20130101 |
Class at
Publication: |
424/190.1 ;
530/328; 530/350; 530/327; 530/329; 530/326; 530/403; 530/324 |
International
Class: |
A61K 39/04 20060101
A61K039/04; A61P 31/06 20060101 A61P031/06; C07K 7/08 20060101
C07K007/08; C07K 7/06 20060101 C07K007/06; C07K 14/35 20060101
C07K014/35 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by a
grant, No. TDA30469A from The World Health Organization, and No. NH
AI43529 from the National Institutes of Health. The Government has
certain rights in the invention.
Claims
1. An isolated polypeptide molecule that consists of an immunogenic
portion of a Mycobacterium tuberculosis antigen, wherein said
antigen consisting of: a) an amino acid sequence encoded by a
nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or
combination thereof; b) an amino acid sequence encoded by a coding
region of a nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, or combination thereof; c) an amino acid sequence encoded by a
complement of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combination
thereof; d) an amino acid sequence encoded by a nucleic acid
molecule that hybridizes to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or
combination thereof under high stringency conditions, wherein said
conditions comprise 1.times.SSC, 1% SDS and 0.1-2 mg/ml denatured
calf thymus DNA at 65.degree. C.; or e) an amino acid sequence set
forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,
28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or combination
thereof
2. The isolated polypeptide molecule of claim 1, wherein the
isolated polypeptide molecule stimulates an immunogenic specific
Tuberculosis Bacterium (TB) response in a host.
3. An isolated polypeptide molecule that consists of an immunogenic
portion of a M. tuberculosis antigen, wherein said antigen having
greater than or equal to about 70% similarity with a sequence
consisting of: a) an amino acid sequence encoded by a nucleic acid
molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combination
thereof; b) an amino acid sequence encoded by a coding region of a
nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or
combination thereof; c) an amino acid sequence encoded by a
complement of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combination
thereof; d) an amino acid sequence encoded by a nucleic acid
molecule that hybridizes to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or
combination thereof, under high stringency conditions, wherein said
conditions comprise 1.times.SSC, 1% SDS and 0.1-2 mg/ml denatured
calf thymus DNA at 65.degree. C.; or e) an amino acid sequence set
forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,
28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or combination
thereof
4. The isolated polypeptide molecule of claim 3, wherein the
isolated polypeptide molecule stimulates an immunogenic specific TB
response in a host.
5. The isolated polypeptide molecule of claim 3, wherein the
nucleic acid molecule has greater than or equal to about 80%
similarity with said sequences.
6. The isolated polypeptide molecule of claim 5, wherein the
nucleic acid molecule has greater than or equal to about 90%
similarity with said sequences.
7. A fusion protein comprising a polypeptide selected from the
group consisting of: a) at least one of the polypeptides of claim
1; b) at least two of the polypeptides of claim 1; c) at least one
polypeptide of claim 1 and a known M. tuberculosis antigen; and d)
at least one polypeptide of claim 1 and an M. tuberculosis antigen
presented on a MHC Class-2 molecule.
8. A composition that comprises the polypeptide sequence of claim 1
and a physiologically acceptable carrier.
9. The composition of claim 8, further including an immune response
enhancer.
10. The composition of claim 9, wherein the immune response
enhancer is an adjuvant or another TB antigen.
11. The composition of claim 10, wherein the composition is a
vaccine composition.
12. The composition of claim 8, wherein the adjuvant includes at
least one component selected from the group consisting of 3D-MPL
and QS21.
13. The composition of claim 8, wherein the composition is
formulated in an oil and water emulsion.
14. The composition of claim 9, wherein the immune response
enhancer is an immunostimulatory cytokine or chemokine
15. A kit for diagnosing the presence or absence of M. tuberculosis
infection in a person, the kit comprises one or more reagents for
detecting the polypeptide molecule of claim 1.
16. A pharmaceutical composition comprising the polypeptide of
claim 1, and a carrier.
Description
RELATED APPLICATION
[0001] This application is a divisional application of U.S. patent
application Ser. No. 11/478,366, filed Jun. 29, 2006, which claims
the benefit of U.S. Provisional Application No. 60/717,062, filed
Sep. 14, 2005, entitled "Tuberculosis Antigen Detection Assays and
Vaccines" and claims the benefit of U.S. Provisional Application
No. 60/696,439, filed Jul. 1, 2005, entitled "Tuberculosis Antigen
Detection Assays and Vaccines." The entire teachings of the above
applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] More than one-third of the world population is infected with
Mycobacterium tuberculosis, the bacterium that causes the
Tuberculosis (TB) disease. Each year, 8 million people become
infected with TB, and 2 million people die from the disease. TB
significantly affects developing countries and is also becoming an
increasing problem in developed areas of the world.
[0004] Persons infected with TB can be asymptomatic for a
considerable period of time, and can be in a latent stage of the
disease. In its active state, the disease is often manifested with
an acute inflammation of the lungs, resulting in fever and a
nonproductive cough. If untreated, serious complications and death
typically result. Present diagnostic assays are often inaccurate,
and are unable to distinguish between persons in the latent stage
of the disease and those in the active stage.
[0005] Currently, vaccination with live bacteria is one method for
immunizing persons against the disease. However, TB vaccination
with certain live bacteria has often been the source of controversy
in some countries, including the United States, and consequently
has not been put into widespread use in the U.S. Additionally,
current diagnostic tests are many times unable to distinguish
between persons who have been immunized, and persons infected with
TB.
[0006] Effective vaccination and accurate early diagnosis of the
disease are important to control the disease. Consequently, a need
exists for effective diagnostic assays that detect active infection
by the TB bacteria. A further need exits for a vaccine that does
not use a live bacteria and provides a protective immunogenic
response to the disease.
SUMMARY OF THE INVENTION
[0007] The present invention relates to isolated polypeptide
molecules that have an immunogenic portion of a Mycobacterium
tuberculosis antigen, or a variant of the antigen that differs only
in conservative substitutions and/or modifications. The antigen has
an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or
combination thereof; or an amino acid sequence encoded by a nucleic
acid molecule having a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, or combination thereof; the coding region of SEQ ID NO:1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, or combination thereof; a complement of SEQ ID
NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, 47, or combination thereof; or a sequence
that hybridizes (e.g., under high stringency conditions) to SEQ ID
NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, 47, or combination thereof. In one
embodiment, the isolated polypeptide molecule stimulates an
immunogenic specific Tuberculosis (TB) response in a host or an
animal (e.g., human, mouse, pig, goat, monkey). In another
embodiment, the present invention includes an isolated polypeptide
molecule of an immunogenic portion of a M. tuberculosis antigen,
wherein the antigen comprises an amino acid sequence encoded by a
nucleic acid molecule having greater than or equal to about 70%
identity (e.g., about 80% identity, 90% identity, about 95%
identity) with any one of the sequences recited above. The present
invention, in one embodiment, includes an isolated polypeptide
molecule, wherein an amino acid sequence has greater than or equal
to about 70% similarity (e.g., about 80% similarity, about 90%
similarity, about 95% similarity) to the sequences recited
herein.
[0008] The present invention further embodies isolated nucleic acid
molecules that encode polypeptide molecules that have an
immunogenic portion of a M. tuberculosis antigen, or a variant of
the antigen that differs only in conservative substitutions and/or
modifications. The antigen is encoded by a nucleic acid molecule
having a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or
combination thereof; the coding region of SEQ ID NO: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, or combination thereof; a complement of SEQ ID NO:1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45, 47, or combination thereof; that hybridizes (e.g.,
under high stringency conditions) to SEQ ID NO: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, or combination thereof; or that encodes SEQ ID NO: 2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, or combination thereof The isolated nucleic acid
molecule encodes a polypeptide molecule that stimulates an
immunogenic specific TB response. The present invention also
includes isolated nucleic acid molecules that encode a polypeptide
molecule that has an immunogenic portion of a M. tuberculosis
antigen, wherein the antigen is encoded by a nucleic acid molecule
having greater than or equal to about 70% identity (e.g., about 80%
identity, 90% identity, about 95% identity) of the recited
sequences. The present invention, in one embodiment, includes
isolated nucleic acid molecules that encode polypeptide molecules
whose sequence has greater than or equal to about 70% similarity
(e.g., about 80% similarity, about 90% similarity, about 95%
similarity) to the sequences recited herein.
[0009] Yet another embodiment of the invention includes vectors,
plasmids and host cells that contain the nucleic acid molecules or
encode the polypeptide molecules described herein. The host cell
can be any cell including, e.g., E. coli, yeast and mammalian
cells. The present invention additionally includes probes that
hybridize under stringency conditions (e.g., high or moderate) to a
nucleic acid molecules described herein.
[0010] The present invention includes antibodies that bind to one
or more of the polypeptide molecules described herein. The antibody
can be a monoclonal antibody or a polyclonal antibody. The
invention also pertains to fusion proteins that comprise one or
more the polypeptide molecules described herein (e.g., two
polypeptide molecules). In addition to including the polypeptide
molecules of the present invention, the fusion protein can also
include other M. tuberculosis antigens, including those presented
on a MHC Class-2 molecule (e.g., a CD4+ T-cell pathway TB
antigen).
[0011] The present invention includes methods for stimulating a
specific immunogenic TB response in an individual, preventing or
reducing the severity of the TB disease, by administering an amount
of one or more of the polypeptide molecules or nucleic acid
molecules described herein (e.g., in a carrier). In another aspect,
the present invention includes compositions (e.g., vaccine
compositions or pharmaceutical compositions) having the polypeptide
molecules or nucleic acid molecules described herein, in a
physiologically acceptable carrier. The composition can also
include or can be co-administered with an immune response enhancer
(e.g., an adjuvant, another TB antigen, immunostimulatory cytokine
or chemokine) Examples of adjuvants include 3D-MPL and QS21. The
composition can be formulated in an oil in water emulsion.
[0012] The present invention further embodies methods for
monitoring treatment of the TB disease in an individual. The
methods include detecting the level of one or more M. tuberculosis
antigenic polypeptides described herein in a sample from the
individual;
[0013] and comparing the level with a standard. A level of TB
antigenic peptides that is higher than the standard indicates
ineffective treatment, and a level that is less than or equal to
the standard indicates effective treatment. The method of
monitoring treatment can also include detecting levels of one or
more of the TB antigenic peptides described herein in a sample at
one or more time points (e.g., a first or baseline time point, and
second time point after commencement of treatment), and comparing
the levels at the time points. Increases in the levels of the
peptides indicates ineffective treatment, and decrease or no change
in the levels indicates effective treatment.
[0014] The invention includes methods of diagnosing TB disease in
an individual by detecting the presence, absence, or levels of one
or more of the polypeptide molecules described herein. The
diagnostic assays of the present invention also allow one to
distinguish between an individual having the active TB disease and
immunity to TB. Such a method includes detecting the presence,
absence or level of one or more of the polypeptide molecules
described herein, and measuring the stimulation of a TB specific
immune response in the individual. The absence of the polypeptide
molecules in a sample from the individual, and the presence of a TB
specific immune response in the individual indicates that the
individual has acquired some immunity to the TB disease and not the
disease itself Measuring the stimulation of a TB specific immune
response includes measuring cell proliferation, interleukin-12
production, interferon-.gamma. levels, or a combination thereof, in
a sample from the individual.
[0015] The present invention includes, in an additional embodiment,
methods for detecting M. tuberculosis infection in a biological
sample, by assessing the presence of one or more of the polypeptide
molecules described herein in the sample. The presence of one or
more of the molecules indicate the presence of M. tuberculosis
infection; and the absence of one or more of the molecules indicate
the absence of M. tuberculosis infection. In particular, methods
include contacting the sample with an antibody (e.g., a detectably
labeled antibody) that binds with the polypeptide molecule,
sufficiently to allow formation of a complex between the sample and
the antibody, to thereby form an antigen-antibody complex; and
detecting the antigen-antibody complex. The presence of the complex
indicates the presence of M. tuberculosis infection, and the
absence of a complex indicates the absence of M. tuberculosis
infection. In one aspect, the method further includes contacting
the sample with a second antibody specific to the antigen or the
antigen-antibody complex. The polypeptide or the antibody can be
bound to a solid support, and the biological sample can be urine,
blood, sputum, cerebrospinal fluid, or tissue sample.
[0016] Methods for detecting M. tuberculosis infection in a
biological sample also include contacting the sample with at least
two oligonucleotide primers in a polymerase chain reaction, wherein
at least one of the oligonucleotide primers (e.g., at least about
10 contiguous bases) is specific for one or more of the isolated
nucleic acid molecules described herein, sufficiently to allow
amplification of the primers; and detecting in the sample the
amplified nucleic acid sequence. The presence any one of the
amplified nucleic acid sequences indicates M. tuberculosis
infection, and the absence of any one of the amplified nucleic acid
sequences indicates an absence of M. tuberculosis infection.
Another method for detecting M. tuberculosis infection in a
biological sample includes contacting the sample with one or more
oligonucleotide probes (e.g., at least about 15 contiguous bases)
specific for the nucleic acid molecule described herein under high
stringency conditions, sufficiently to allow hybridization between
the sample and the probe; and detecting the nucleic acid molecule
that hybridizes to the oligonucleotide probe in the sample. The
presence of hybridization of the probe indicates M. tuberculosis
infection, and the absence of hybridization indicates an absence of
M. tuberculosis infection.
[0017] Furthermore, the present invention includes kits for
diagnosing the presence or absence of M. tuberculosis infection in
a person. The kit comprises one or more reagents for detecting one
or more of polypeptide molecules or nucleic acid molecules
described herein. The reagents can include those that are used for
carrying out an enzyme-linked immunosorbent assay, a rapid
immunochromatographic assay, a flow cytometric analysis, or a
radioimmunoassay. Such kits can comprise one or more nucleic acid
molecules having a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
or combination thereof; the complements of said sequences, and
nucleic acid sequences that hybridize to a sequence recited in SEQ
ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, or combination; and a detection
reagent. Kits can include other items such as solid supports, and
detection agents (e.g., a reporter group like radioisotopes,
fluorescent groups, luminescent groups, enzymes, biotin and dye
particles; conjugated to a binding agent such as
anti-immunoglobulins, Protein G, Protein A and lectins). Advantages
of the present invention include new methods for preventing or
reducing the severity of the TB disease by providing an effective
vaccine composition. The assays of the present invention allow
simple and easy to administer urine tests to quickly and
efficiently distinguish between patients having active TB and those
who do not. New vaccine compositions and more effective diagnostic
assays will assist in reducing the present worldwide TB
problem.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-1G are schematics showing the nucleic acid
sequences (in Bold) (SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, and 27) and corresponding M. tuberculosis polypeptide
sequences (in Bold) (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, and 28) eluted from MHC class 1 molecules from the
macrophages of mice infected with the M. tuberculosis bacteria.
FIG. 1H is a table showing M. tuberculosis peptide sequences, SEQ
ID Nos: 2, 6, 10, 14, 18, 22, and 26, eluted from MHC class 1
molecules from the macrophages of mice infected with the M.
tuberculosis bacteria. The figure also shows The Institute for
Genomic Research (TIGR) annotation, the Swiss-Prot designation, and
the protein name.
[0019] FIG. 2 is a schematic showing a peptide, SEQ ID NO: 30, in
bold, found in the urine of patients with pulmonary tuberculosis,
and its corresponding nucleic acid sequence, SEQ ID NO: 29. The
figure also shows a DNA sequence (SEQ ID NO: 31) and its
corresponding protein sequence (SEQ ID NO: 32) which is a putative
molybdopterin biosynthesis protein that is found in M. tuberculosis
and has 100% homology to SEQ ID NO: 30.
[0020] FIG. 3 is a schematic showing the protocol used to purify
and sequence MHC Class 1 associated peptides isolated from M.
tuberculosis-infected macrophages.
[0021] FIG. 4 is a drawing showing an example of a
immunochromatographic assay for the detection of TB antigens of the
present invention in a sample.
[0022] FIG. 5 is a table showing M. tuberculosis peptide sequences,
SEQ ID Nos: 34, 38, 42, and 46, found in the urine of patients with
pulmonary tuberculosis and the M. tuberculosis donor protein.
[0023] FIGS. 6A-B are schematics showing the nucleic acid sequence
(in Bold and Underline) (SEQ ID NO: 33) and corresponding M.
tuberculosis polypeptide sequence (in Bold and Underline) (SEQ ID
NO: 34) found in the urine of patients with pulmonary tuberculosis.
Also depicted are Homoserine O-acetyltransferase nucleic acid and
polypeptide sequences from M. tuberculosis having 100% homology to
the isolated sequences (SEQ ID NOs: 35 and 36, respectively), along
with a table providing Genomic Research (TIGR) Locus name, primary
locus name, the Swiss-Prot designation, putative identification,
Gene Symbol, TIGR cellular roles, coordinates, DNA molecule name,
gene length, protein length, molecular weight, pl, percent GC,
enzyme Commission #, Kingdom, and Family.
[0024] FIGS. 7A-C are schematics showing the nucleic acid sequence
(in Bold and Underline) (SEQ ID NO: 37) and corresponding M.
tuberculosis polypeptide sequence (in Bold and Underline) (SEQ ID
NO: 38) found in the urine of patients with pulmonary tuberculosis.
Also depicted are Chromosome partition protein smc nucleic acid and
polypeptide sequences from M. tuberculosis having 100% homology to
the isolated sequences (SEQ ID NOs: 39 and 40, respectively), along
with a table providing the TIGR Locus name, primary locus name, the
Swiss-Prot designation, putative identification, Gene Symbol, TIGR
cellular roles, coordinates, DNA molecule name, gene length,
protein length, molecular weight, pl, percent GC, enzyme Commission
#, Kingdom, and Family.
[0025] FIGS. 8A-B are schematics showing the nucleic acid sequence
(in Bold and Underline) (SEQ ID NO: 41) and corresponding M.
tuberculosis polypeptide sequence (in Bold and Underline) (SEQ ID
NO: 42) found in the urine of patients with pulmonary tuberculosis.
Also depicted are Ornithine carbamoyltransferase nucleic acid and
polypeptide sequences from M. tuberculosis having 100% homology to
the isolated sequences (SEQ ID NOs: 43 and 44, respectively), along
with a table providing the TIGR Locus name, primary locus name, the
Swiss-Prot designation, putative identification, Gene Symbol, TIGR
cellular roles, coordinates, DNA molecule name, gene length,
protein length, molecular weight, pl, percent GC, enzyme Commission
#, Kingdom, and Family.
[0026] FIGS. 9A-B are schematics showing the nucleic acid sequence
(in Bold and Underline) (SEQ ID NO: 45) and corresponding M.
tuberculosis polypeptide sequence (in Bold and Underline) (SEQ ID
NO: 46) found in the urine of patients with pulmonary tuberculosis.
Also depicted are phosphoadenosine phosphosulfate reductase nucleic
acid and polypeptide sequences from M. tuberculosis having 100%
homology to the isolated sequences (SEQ ID NOs: 47 and 48,
respectively), along with a table providing the TIGR Locus name,
primary locus name, the Swiss-Prot designation, putative
identification, Gene Symbol, TIGR cellular roles, coordinates, DNA
molecule name, gene length, protein length, molecular weight, pl,
percent GC, enzyme Commission #, Kingdom, and Family.
[0027] FIG. 10 is a representation of Western Blot showing
over-expression and purification of recombinants MT1694, MT2462 and
MT2990 from E. coli lysates from non-induced cultures (lanes 1); E.
coli lysates from isopropyl-beta-D-thiogalactopyranoside (IPTG)
induced cultures (lanes 2); and purified recombinant proteins
(lanes 3), and the molecular weight markers (MWM).
[0028] FIG. 11 is a representation of Western Blot showing
identification of native MTB1694 and MT2462 in crude whole M.
tuberculosis cell lysate (Lane 1); culture filtrate (CF) proteins
(Lane 2); purified recombinant antigen (Lane 3); and molecular
weight markers (MWM), wherein antigens were electrophoresed and
transferred to nitrocellulose membrane followed by probing with
either rabbit anti-MTB 1694 or anti-MT2462 antisera.
[0029] FIG. 12A is a bar graph showing the recognition of MT1694
and MT2452 by proliferative responses expressed as counts per
minute (CPM) from healthy PPD (an intradermal skin test response to
tuberculosis proteins using a Purified Protein Derivative) negative
(donor 1) and PPD positive (donors 2-5) individuals following
stimulation with recombinant antigens (5 ug/ml).
[0030] FIG. 12B is a bar graph showing the recognition of MT1694
and MT2452 by IFN-.gamma. production of peripheral blood
mononuclear cells (PBMC) from healthy PPD negative (donor 1) and
PPD positive (donors 2-5) individuals following stimulation with
recombinant antigens (5 ug/ml).
DETAILED DESCRIPTION OF THE INVENTION
[0031] A description of preferred embodiments of the invention
follows. The present invention relates to vaccine compositions and
diagnostic assays for the Tuberculosis (TB) disease. The present
invention is based, in part, on the discovery of certain TB
antigenic peptides presented on the MHC class 1 macrophage, when a
host is infected with the bacteria that causes TB, namely the M.
tuberculosis bacteria. MHC class-1 macrophages are cells that play
a role in the CD8+ T-Cell pathway of the immune response. A second
discovery that forms the basis of the present invention is the
identification of a TB peptide present in the urine of people
infected with active TB. The identity of the specific antigenic TB
peptides presented on MHC class 1 cell, and the identity of the TB
peptide found in urine of infected patients, provide the
polypeptide sequences needed to create effective vaccines and/or
diagnostic tests.
[0032] TB vaccine compositions of the present invention can include
polypeptide sequences or nucleic acid sequences, and, optionally,
additional immune enhancing molecules. Hence, the present
invention, in part, relates to the specific antigenic TB
polypeptide sequences discovered, which are shown in FIGS. 1, 2,
and 5-9 namely, SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and combinations
thereof. SEQ ID NOs: 2, 6, 10, 14, 18, 22, and 26 were isolated by
infecting mice with significant amounts of M. tuberculosis
bacteria. About two weeks after the mice were infected, the spleen
was removed, and MHC class 1 molecules, molecules that present
antigens to CD8+ T cells, were separated, in accordance with the
methods detailed in Example 1. A number of peptides that were found
on MHC class 1 molecules were identified as being of M.
tuberculosis origin. SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, and 27, the nucleic acid sequences that encode these
identified polypeptides, are also shown in FIG. 1A-H.
[0033] SEQ ID NO: 30, MVIIELMRR, is a peptide found the in urine of
persons with pulmonary TB, and this sequence has 100% homology with
a M. tuberculosis biosynthesis protein, SEQ ID NO: 32, shown in
FIG. 2. The nucleic acid sequence, SEQ ID NO: 31, that encodes this
protein is also shown. Additionally, FIGS. 5-9 show SEQ ID NOs: 34,
38, 42, and 46 that were also found in the urine of persons with
pulmonary TB, and have 100% homology with SEQ ID NOs: 36, 40, 44,
and 48, respectively. The methods used to identify these TB protein
in the urine of patients infected with active
[0034] TB are described in Example 2.
[0035] Accordingly, the present invention relates to these
sequences, SEQ ID NO: 1-48, that have been identified as being
useful in eliciting a protective immune response against TB, and
for diagnostic assays for identifying persons with active TB.
Polypeptides and their Function
[0036] The present invention relates to isolated polypeptide
molecules that have been isolated including antigenic portions of
TB sequences presented by MHC class 1 molecule in an infected host,
and TB sequences isolated from urine of infected patients. The
present invention includes polypeptide molecules that contain the
sequence of any one of the antigenic TB amino acid sequences (SEQ
ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, or combinations thereof). See FIGS.
1, 2 and 5-9. The present invention also pertains to polypeptide
molecules that are encoded by nucleic acid sequences, SEQ ID NO: 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, or combinations thereof).
[0037] As used herein, the term "polypeptide" encompasses amino
acid chains of any length, including full length proteins (i.e.,
antigens), wherein the amino acid residues are linked by covalent
peptide bonds. Thus, a polypeptide comprising an immunogenic
portion of a M. tuberculosis antigen can consist entirely of the
immunogenic portion, or can contain additional sequences. The
additional sequences can be derived from the native M. tuberculosis
antigen or can be heterologous, and such sequences can (but need
not) be immunogenic. In general, 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.
[0038] Antigenic TB polypeptides of the present invention referred
to herein as "isolated" are polypeptides that separated away from
other proteins and cellular material of their source of origin.
Isolated antigenic TB polypeptides, peptides derived by infection
with the M. tuberculosis bacteria, include essentially pure
protein, proteins produced by chemical synthesis, by combinations
of biological and chemical synthesis and by recombinant methods.
The proteins of the present invention have been isolated and
characterized as to its physical characteristics using the
procedures described in the Exemplification, and can be done using
laboratory techniques for protein purification. Such techniques
include, for example, salting out, immunoprecipation, column
chromatography, high pressure liquid chromatography or
electrophoresis.
[0039] The compositions and methods of the present invention also
encompass variants of the above polypeptides and DNA molecules. 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,
antigenic and/or immunogenic properties of the polypeptide are
retained. A variant of a specific M. tuberculosis antigen will
therefore stimulate cell proliferation and/or IFN-.gamma. in Thl
cells raised against that specific antigen. Polypeptide variants
preferably exhibit at least about 70%, more preferably at least
about 90% and most preferably at least about 95% homology to the
identified polypeptides. For polypeptides with immunoreactive
properties, variants can, alternatively, be identified by modifying
the amino acid sequence of one of the above polypeptides, and
evaluating the immunoreactivity of the modified polypeptide. Such
modified sequences can be prepared and tested using, for example,
the representative procedures described herein.
[0040] As used herein, a "conservative substitution" is one in
which an amino acid is substituted for another amino acid that has
similar properties, such that one skilled in the art of peptide
chemistry would expect the secondary structure and hydropathic
nature of the polypeptide to be substantially unchanged. In
general, the following groups of amino acids represent conservative
changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys,
ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his;
and (5) phe, tyr, trp, his.
[0041] Variants can also, or alternatively, contain other
modifications, including the deletion or addition of amino acids
that have minimal influence on the antigenic properties, secondary
structure and hydropathic nature of the polypeptide. For example, a
polypeptide can be conjugated to a signal (or leader) sequence at
the N-terminal end of the protein which co-translationally or
post-translationally directs transfer of the protein. The
polypeptide can 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 can be
conjugated to an immunoglobulin Fc region.
[0042] The present invention also encompasses TB proteins and
polypeptides, variants thereof, or those having amino acid
sequences analogous to the amino acid sequences of antigenic TB
polypeptides described herein. Such polypeptides are defined herein
as antigenic TB analogs (e.g., homologues), or mutants or
derivatives. "Analogous" or "homologous" amino acid sequences refer
to amino acid sequences with sufficient identity of any one of the
TB amino acid sequences so as to possess the biological activity
(e.g., the ability to elicit a protective immune response to TB
bacteria) of any one of the native TB polypeptides. For example, an
analog polypeptide can be produced with "silent" changes in the
amino acid sequence wherein one, or more, amino acid residues
differ from the amino acid residues of any one of the TB protein,
yet still possesses the function or biological activity of the TB.
Examples of such differences include additions, deletions or
substitutions of residues of the amino acid sequence of TB. Also
encompassed by the present invention are analogous polypeptides
that exhibit greater, or lesser, biological activity of any one of
the TB proteins of the present invention. Such polypeptides can be
made by mutating (e.g., substituting, deleting or adding) one or
more amino acid or nucleic acid residues to any of the isolated TB
molecules described herein. Such mutations can be performed using
methods described herein and those known in the art. In particular,
the present invention relates to homologous polypeptide molecules
having at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%)
identity or similarity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or
combination thereof. Percent "identity" refers to the amount of
identical nucleotides or amino acids between two nucleotides or
amino acid sequences, respectfully. As used herein, "percent
similarity" refers to the amount of similar or conservative amino
acids between two amino acid sequences.
[0043] The polypeptides of the present invention, including a full
length sequence, partial sequences, functional fragments and
homologues, that allow for or assist in stimulating an immunogenic
specific or protective immune response to TB. "Immunogenic," as
used herein, refers to the ability to elicit an immune response
(e.g., cellular) in a patient, such as a human, and/or in a
biological sample. In particular, antigens that are immunogenic
(and immunogenic portions thereof) stimulate cell proliferation,
interleukin-12 production and/or interferon-.gamma. production in
biological samples comprising one or more cells (e.g., T cells, NK
cells, B cells and macrophage). Such cells are derived from an M.
tuberculosis-immune individual. Immunogenic portions of the
antigens described herein can be prepared and identified using the
techniques described herein. Other techniques, such as those
summarized in Paul, Fundamental Immunology, 3d ed., Raven Press,
1993, pp. 243-247 and references cited therein, can be used. Such
techniques include screening polypeptide portions of the native
antigen for immunogenic properties. An immunogenic portion of a
polypeptide is a portion that, within such assays, generates an
immune response (e.g., proliferation, interferon-.gamma. production
and/or interleukin-12 production) that is substantially similar to
that generated by the full-length antigen. In other words, an
immunogenic portion of an antigen can generate at least about 20%,
and preferably about 100%, of the proliferation induced by the full
length antigen in the model proliferation assay described herein.
An immunogenic portion can 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. As used herein, "TB"
or "TB disease" refers to the disease cause by the infection of M.
tuberculosis.
[0044] Homologous polypeptides can be determined using methods
known to those of skill in the art. Initial homology searches can
be performed at NCBI against the GenBank, EMBL and SwissProt
databases using, for example, the BLAST network service.
Altschuler, S. F., et al., J. Mol. Biol., 215:403 (1990),
Altschuler, S. F., Nucleic Acids Res., 25:3389-3402 (1998).
Computer analysis of nucleotide sequences can be performed using
the MOTIFS and the FindPatterns subroutines of the Genetics
Computing Group (GCG, version 8.0) software. Protein and/or
nucleotide comparisons were performed according to Higgins and
Sharp (Higgins, D. G. and Sharp, P. M., Gene, 73:237-244 (1988)
e.g., using default parameters).
[0045] Additionally, the individual isolated polypeptides of the
present invention are biologically active or functional and play
various roles in bacteria as well. For example, isolated
polypeptide, such as SEQ ID NO: 6 is a glutamine-transport
transmembrane protein ABC transporter. Likewise, SEQ ID NO: 22 is a
cationic amino acid transport integral member protein, and SEQ ID
NO: 26 is a Cationic transporting P-type ATPase. SEQ ID NO: 32, is
a molybdopterin biosynthesis protein. The present invention
includes fragments of these isolated amino acid sequences, yet
possess the function or biological activity of the sequence. For
example, polypeptide fragments comprising deletion mutants of the
antigenic TB proteins can be designed and expressed by well-known
laboratory methods. Fragments, homologues, or analogous
polypeptides can be evaluated for biological activity, as described
herein.
[0046] The present invention also encompasses biologically active
derivatives or analogs of the above described antigenic TB
polypeptides, referred to herein as peptide mimetics. Mimetics can
be designed and produced by techniques known to those of skill in
the art. (see e.g., U.S. Pat. Nos. 4,612,132; 5,643,873 and
5,654,276). These mimetics can be based, for example, on a specific
TB amino acid sequence and maintain the relative position in space
of the corresponding amino acid sequence. These peptide mimetics
possess biological activity similar to the biological activity of
the corresponding peptide compound, but possess a "biological
advantage" over the corresponding antigenic TB amino acid sequence
with respect to one, or more, of the following properties:
solubility, stability and susceptibility to hydrolysis and
proteolysis.
[0047] Methods for preparing peptide mimetics include modifying the
N-terminal amino group, the C-terminal carboxyl group, and/or
changing one or more of the amino linkages in the peptide to a
non-amino linkage. Two or more such modifications can be coupled in
one peptide mimetic molecule. Modifications of peptides to produce
peptide mimetics are described in U.S. Pat. Nos. 5,643,873 and
5,654,276. Other forms of the antigenic TB polypeptides,
encompassed by the present invention, include those which are
"functionally equivalent." This term, as used herein, refers to any
nucleic acid sequence and its encoded amino acid, which mimics the
biological activity of the TB polypeptides and/or functional
domains thereof.
TB Nucleic Acid Sequences, Plasmids, Vectors and Host Cells
[0048] The present invention, in one embodiment, includes an
isolated nucleic acid molecule having a sequence of SEQ ID NO: 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, or combinations thereof. See FIGS. 1, 2 and
5-9. The present invention includes sequences as recited in FIGS.
1, 2, and 5-9, as well as the coding regions thereof
[0049] As used herein, the terms "DNA molecule" or "nucleic acid
molecule" include both sense and anti-sense strands, cDNA, genomic
DNA, recombinant DNA, RNA, and wholly or partially synthesized
nucleic acid molecules. A nucleotide "variant" is a sequence that
differs from the recited nucleotide sequence in having one or more
nucleotide deletions, substitutions or additions. Such
modifications can be readily introduced using standard mutagenesis
techniques, such as oligonucleotide-directed site-specific
mutagenesis as taught, for example, by Adelman et al. (DNA 2:183,
1983).
[0050] Nucleotide variants can be naturally occurring allelic
variants, or non-naturally occurring variants. Variant nucleotide
sequences preferably exhibit at least about 70%, more preferably at
least about 80% and most preferably at least about 90% homology to
the recited sequence. Such variant nucleotide sequences will
generally hybridize to the recited nucleotide sequence under
stringent conditions. In one embodiment, "stringent conditions"
refers to prewashing in a solution of 6.times.SSC, 0.2% SDS;
hybridizing at 65.degree. Celsius, 6.times.SSC, 0.2% SDS overnight;
followed by two washes of 30 minutes each in 1.times.SSC, 0.1% SDS
at 65.degree. C. and two washes of 30 minutes each in
0.2.times.SSC, 0.1% SDS at 65.degree. C.
[0051] The present invention also encompasses isolated nucleic acid
sequences that encode TB polypeptides, and in particular, those
which encode a polypeptide molecule having an amino acid sequence
of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or combinations thereof.
These TB nucleic acid sequences encode polypeptides that stimulate
a protective immunogenic response to the M. tuberculosis bacteria
and/or are involved the functions further described herein.
[0052] As used herein, an "isolated" gene or nucleotide sequence
which is not flanked by nucleotide sequences which normally (e.g.,
in nature) flank the gene or nucleotide sequence (e.g., as in
genomic sequences) and/or has been completely or partially purified
from other transcribed sequences (e.g., as in a cDNA or RNA
library). Thus, an isolated gene or nucleotide sequence can include
a gene or nucleotide sequence which is synthesized chemically or by
recombinant means. Nucleic acid constructs contained in a vector
are included in the definition of "isolated" as used herein. Also,
isolated nucleotide sequences include recombinant nucleic acid
molecules and heterologous host cells, as well as partially or
substantially or purified nucleic acid molecules in solution. In
vivo and in vitro RNA transcripts of the present invention are also
encompassed by "isolated" nucleotide sequences. Such isolated
nucleotide sequences are useful for the manufacture of the encoded
antigenic TB polypeptide, as probes for isolating homologues
sequences (e.g., from other mammalian species or other organisms),
for gene mapping (e.g., by in situ hybridization), or for detecting
the presence (e.g., by Southern blot analysis) or expression (e.g.,
by Northern blot analysis) of related genes in cells or tissue.
[0053] The antigenic TB nucleic acid sequences of the present
invention include homologues nucleic acid sequences. "Analogous" or
"homologous" nucleic acid sequences refer to nucleic acid sequences
with sufficient identity of any one of the TB nucleic acid
sequences, such that once encoded into polypeptides, they possess
the biological activity of any one of the antigenic TB polypeptides
described herein. For example, an analogous nucleic acid molecule
can be produced with "silent" changes in the sequence wherein one,
or more, nucleotides differ from the nucleotides of any one of the
TB polypeptides described herein, yet, once encoded into a
polypeptide, still possesses its function or biological activity.
Examples of such differences include additions, deletions or
substitutions. Also encompassed by the present invention are
nucleic acid sequences that encode analogous polypeptides that
exhibit greater, or lesser, biological activity of the TB proteins
of the present invention. In particular, the present invention is
directed to nucleic acid molecules having at least about 70% (e.g.,
75%, 80%, 85%, 90% or 95%) identity with SEQ ID NO: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, or combinations thereof.
[0054] The nucleic acid molecules of the present invention,
including the full length sequences, the partial sequences,
functional fragments and homologues, once encoded into
polypeptides, elicit an specific immunogenic TB response, or has
the function of the polypeptide, as further described herein. The
homologous nucleic acid sequences can be determined using methods
known to those of skill in the art, and by methods described herein
including those described for determining homologous polypeptide
sequences. Immunogenic antigens can then be sequenced using
techniques such as Edman chemistry. See Edman and Berg, Eur. J.
Biochem. 80:116-132, 1967.
[0055] Also encompassed by the present invention are nucleic acid
sequences, DNA or RNA, which are substantially complementary to the
DNA sequences encoding the antigenic TB polypeptides of the present
invention, and which specifically hybridize with their DNA
sequences under conditions of stringency known to those of skill in
the art. As defined herein, substantially complementary means that
the nucleic acid need not reflect the exact sequence of the TB
sequences, but must be sufficiently similar in sequence to permit
hybridization with TB nucleic acid sequence under high stringency
conditions. For example, non-complementary bases can be
interspersed in a nucleotide sequence, or the sequences can be
longer or shorter than the TB nucleic acid sequence, provided that
the sequence has a sufficient number of bases complementary to the
TB sequence to allow hybridization therewith. Conditions for
stringency are described in e.g., Ausubel, F. M., et al., Current
Protocols in Molecular Biology, (Current Protocol, 1994), and
Brown, et al., Nature, 366:575 (1993); and further defined in
conjunction with certain assays.
[0056] Also encompassed by the present invention are nucleic acid
sequences, genomic DNA, cDNA, RNA or a combination thereof, which
are substantially complementary to the DNA sequences of the present
invention and which specifically hybridize with the antigenic TB
nucleic acid sequences under conditions of sufficient stringency
(e.g., high stringency) to identify DNA sequences with substantial
nucleic acid identity.
[0057] The present invention also includes portions and other
variants of M. tuberculosis antigens that are generated by
synthetic or recombinant means. Synthetic polypeptides having fewer
than about 100 amino acids, and generally fewer than about 50 amino
acids, can be generated using techniques well known to those of
ordinary skill in the art. For example, such polypeptides can be
synthesized using any of the commercially available solid-phase
techniques, such as the Merrifield solid-phase synthesis method,
where amino acids are sequentially added to a growing amino acid
chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963.
Equipment for automated synthesis of polypeptides is commercially
available from suppliers such as Applied BioSystems, Inc., Foster
City, Calif., and can be operated according to the manufacturer's
instructions. Variants of a native antigen can generally be
prepared using standard mutagenesis techniques, such as
oligonucleotide-directed site-specific mutagenesis. Sections of the
DNA sequence can also be removed using standard techniques to
permit preparation of truncated polypeptides.
[0058] In another embodiment, the present invention includes
nucleic acid molecules (e.g., probes or primers) that hybridize to
the TB sequences, SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combinations
thereof under high or moderate stringency conditions. In one
aspect, the present invention includes molecules that are or
hybridize to at least about 20 contiguous nucleotides or longer in
length (e.g., 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,
2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100,
3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000). Such
molecules hybridize to one of the TB nucleic acid sequences under
high stringency conditions. The present invention includes such
molecules and those that encode a polypeptide that has the
functions or biological activity described herein.
[0059] Typically the nucleic acid probe comprises a nucleic acid
sequence (e.g. SEQ ID
[0060] NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 39, 41, 43, 45, 47, or combinations thereof) and is
of sufficient length and complementarity to specifically hybridize
to a nucleic acid sequence that encodes a TB antigenic polypeptide.
For example, a nucleic acid probe can be at least about 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% the length of the TB
nucleic acid sequence. The requirements of sufficient length and
complementarity can be easily determined by one of skill in the
art. Suitable hybridization conditions (e.g., high stringency
conditions) are also described herein. Additionally, the present
invention encompasses fragments of the polypeptides of the present
invention or nucleic acid sequences that encodes a polypeptide
wherein the polypeptide has the biologically activity of the TB
polypeptides recited herein.
[0061] Such fragments are useful as probes for assays described
herein, and as experimental tools, or in the case of nucleic acid
fragments, as primers. A preferred embodiment includes primers and
probes which selectively hybridize to the nucleic acid constructs
encoding any one of the recited TB polypeptides. For example,
nucleic acid fragments which encode any one of the domains
described herein are also implicated by the present invention.
[0062] Stringency conditions for hybridization refers to conditions
of temperature and buffer composition which permit hybridization of
a first nucleic acid sequence to a second nucleic acid sequence,
wherein the conditions determine the degree of identity between
those sequences which hybridize to each other. Therefore, "high
stringency conditions" are those conditions wherein only nucleic
acid sequences which are very similar to each other will hybridize.
The sequences can be less similar to each other if they hybridize
under moderate stringency conditions. Still less similarity is
needed for two sequences to hybridize under low stringency
conditions. By varying the hybridization conditions from a
stringency level at which no hybridization occurs, to a level at
which hybridization is first observed, conditions can be determined
at which a given sequence will hybridize to those sequences that
are most similar to it. The precise conditions determining the
stringency of a particular hybridization include not only the ionic
strength, temperature, and the concentration of destabilizing
agents such as formamide, but also factors such as the length of
the nucleic acid sequences, their base composition, the percent of
mismatched base pairs between the two sequences, and the frequency
of occurrence of subsets of the sequences (e.g., small stretches of
repeats) within other non-identical sequences. Washing is the step
in which conditions are set so as to determine a minimum level of
similarity between the sequences hybridizing with each other.
Generally, from the lowest temperature at which only homologous
hybridization occurs, a 1% mismatch between two sequences results
in a 1.degree. C. decrease in the melting temperature (T.sub.m) for
any chosen SSC concentration. Generally, a doubling of the
concentration of SSC results in an increase in the T.sub.m of about
17.degree. C. Using these guidelines, the washing temperature can
be determined empirically, depending on the level of mismatch
sought. Hybridization and wash conditions are explained in Current
Protocols in Molecular Biology (Ausubel, F. M. et al., eds., John
Wiley & Sons, Inc., 1995, with supplemental updates) on pages
2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.
[0063] High stringency conditions can employ hybridization at
either (1) 1.times.SSC (10.times.SSC=3 M NaCl, 0.3 M
Na.sub.3-citrate . . . 2H.sub.2O (88 g/liter), pH to 7.0 with 1 M
HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calf
thymus DNA at 65.degree. C., (2) 1.times.SSC, 50% formamide, 1%
SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42.degree. C., (3) 1%
bovine serum albumin (fraction V), 1 mM Na.sub.2 . . . EDTA, 0.5 M
NaHPO.sub.4 (pH 7.2) (1 M NaHPO.sub.4=134 g Na.sub.2HPO.sub.4 . . .
7H.sub.2O, 4 ml 85% H.sub.3PO.sub.4 per liter), 7% SDS, 0.1-2 mg/ml
denatured calf thymus DNA at 65.degree. C., (4) 50% formamide,
5.times.SSC, 0.02 M Tris-HCl (pH 7.6), 1.times. Denhardt's solution
(100X=10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum
albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1%
SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42.degree. C., (5)
5.times.SSC, 5.times. Denhardt's solution, 1% SDS, 100 .mu.g/ml
denatured calf thymus DNA at 65.degree. C., or (6) 5.times.SSC,
5.times. Denhardt's solution, 50% formamide, 1% SDS, 100 .mu.g/ml
denatured calf thymus DNA at 42.degree. C., with high stringency
washes of either (1) 0.3-0.1.times.SSC, 0.1% SDS at 65.degree. C.,
or (2) 1 mM Na.sub.2EDTA, 40 mM NaHPO.sub.4 (pH 7.2), 1% SDS at
65.degree. C. The above conditions are intended to be used for
DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is
believed to be less than 18 base pairs in length, the hybridization
and wash temperatures should be 5-10.degree. C. below that of the
calculated T.sub.m of the hybrid, where T.sub.m in .degree.
C.=(2.times. the number of A and T bases)+(4.times. the number of G
and C bases). For hybrids believed to be about 18 to about 49 base
pairs in length, the T.sub.m in .degree. C.=(81.5.degree.
C.+16.6(log.sub.10M)+0.41(% G+C)-0.61 (% formamide)-500/L), where
"M" is the molarity of monovalent cations (e.g., Na.sup.+), and "L"
is the length of the hybrid in base pairs.
[0064] Moderate stringency conditions can employ hybridization at
either (1) 4.times.SSC, (10.times.SSC=3 M NaCl, 0.3 M
Na.sub.3-citrate . . . 2H.sub.2O (88 g/liter), pH to 7.0 with 1 M
HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calf
thymus DNA at 65.degree. C., (2) 4.times.SSC, 50% formamide, 1%
SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42.degree. C., (3) 1%
bovine serum albumin (fraction V), 1 mM Na.sub.2 . . . EDTA, 0.5 M
NaHPO.sub.4 (pH 7.2) (1 M NaHPO.sub.4=134 g Na.sub.2HPO.sub.4. . .
7H.sub.2O, 4 ml 85% H.sub.3PO.sub.4 per liter), 7% SDS, 0.1-2 mg/ml
denatured calf thymus DNA at 65.degree. C., (4) 50% formamide,
5.times.SSC, 0.02 M Tris-HCl (pH 7.6), 1.times. Denhardt's solution
(100.times.=10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine
serum albumin (fraction V), water to 500 ml), 10% dextran sulfate,
1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42.degree. C., (5)
5.times.SSC, 5.times. Denhardt's solution, 1% SDS, 100 .mu.g/ml
denatured calf thymus DNA at 65.degree. C., or (6) 5.times.SSC,
5.times. Denhardt's solution, 50% formamide, 1% SDS, 100 .mu.g/ml
denatured calf thymus DNA at 42.degree. C., with moderate
stringency washes of 1.times.SSC, 0.1% SDS at 65.degree. C. The
above conditions are intended to be used for DNA-DNA hybrids of 50
base pairs or longer. Where the hybrid is believed to be less than
18 base pairs in length, the hybridization and wash temperatures
should be 5-10.degree. C. below that of the calculated T.sub.m of
the hybrid, where T.sub.m in .degree. C.=(2.times. the number of A
and T bases)+(4.times. the number of G and C bases). For hybrids
believed to be about 18 to about 49 base pairs in length, the
T.sub.m in .degree. C.=(81.5.degree. C.+16.6(log.sub.10M)+0.41(%
G+C)-0.61 (% formamide)-500/L), where "M" is the molarity of
monovalent cations (e.g., Na+), and "L" is the length of the hybrid
in base pairs.
[0065] Low stringency conditions can employ hybridization at either
(1) 4.times.SSC, (10.times.SSC=3 M NaCl, 0.3 M Na.sub.3-citrate . .
. 2H.sub.2O (88 g/liter), pH to 7.0 with 1 M HCl), 1% SDS (sodium
dodecyl sulfate), 0.1-2 mg/ml denatured calf thymus DNA at
50.degree. C., (2) 6.times.SSC, 50% formamide, 1% SDS, 0.1-2 mg/ml
denatured calf thymus DNA at 40.degree. C., (3) 1% bovine serum
albumin (fraction V), 1 mM Na.sub.2 . . . EDTA, 0.5 M NaHPO.sub.4
(pH 7.2) (1 M NaHPO.sub.4=134 g Na.sub.2HPO.sub.4 . . . 7H.sub.2O,
4 ml 85% H.sub.3PO.sub.4 per liter), 7% SDS, 0.1-2 mg/ml denatured
calf thymus DNA at 50.degree. C., (4) 50% formamide, 5.times.SSC,
0.02 M Tris-HCl (pH 7.6), 1.times. Denhardt's solution
(100.times.=10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine
serum albumin (fraction V), water to 500 ml), 10% dextran sulfate,
1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 40.degree. C., (5)
5.times.SSC, 5.times. Denhardt's solution, 1% SDS, 100 .mu.g/ml
denatured calf thymus DNA at 50.degree. C., or (6) 5.times.SSC,
5.times. Denhardt's solution, 50% formamide, 1% SDS, 100 .mu.g/ml
denatured calf thymus DNA at 40.degree. C., with low stringency
washes of either 2.times.SSC, 0.1% SDS at 50.degree. C., or (2)
0.5% bovine serum albumin (fraction V), 1 mM Na.sub.2EDTA, 40 mM
NaHPO.sub.4 (pH 7.2), 5% SDS. The above conditions are intended to
be used for DNA-DNA hybrids of 50 base pairs or longer. Where the
hybrid is believed to be less than 18 base pairs in length, the
hybridization and wash temperatures should be 5-10.degree. C. below
that of the calculated T.sub.m of the hybrid, where T.sub.m in
.degree. C.=(2.times. the number of A and T bases)+(4.times. the
number of G and C bases). For hybrids believed to be about 18 to
about 49 base pairs in length, the T.sub.m in .degree.
C.=(81.5.degree. C.+16.6(log.sub.10M)+0.41(% G+C)-0.61 (%
formamide)-500/L), where "M" is the molarity of monovalent cations
(e.g., Na.+), and "L" is the length of the hybrid in base
pairs.
[0066] The TB nucleic acid sequences of the present invention, or a
fragment thereof, can also be used to isolate additional homologs.
For example, a cDNA or genomic DNA library from the appropriate
organism can be screened with labeled TB nucleic acid sequence to
identify homologous genes as described in e.g., Ausebel, et al.,
Eds., Current Protocols In Molecular Biology, John Wiley &
Sons, New York (1997).
[0067] Immunogenic antigens can be produced recombinantly using a
DNA sequence that encodes the antigen, which has been inserted into
an expression vector and expressed in an appropriate host cell. DNA
sequences encoding M. tuberculosis antigens can, for example, be
identified by screening an appropriate M. tuberculosis genomic or
cDNA expression library with sera obtained from patients infected
with M. tuberculosis. Such screens can 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. Degenerate oligonucleotide sequences for use in
such a screen can be designed and synthesized, and the screen can
be performed. Polymerase chain reaction (PCR) can 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 can then be performed using the isolated probe. The
present method can optionally include a labeled TB antigenic
probe.
[0068] Alternatively, genomic or cDNA libraries derived from M.
tuberculosis can be screened directly using peripheral blood
mononuclear cells (PBMCs) or T cell lines or clones derived from
one or more M. tuberculosis-immune individuals. In general, PBMCs
and/or T cells for use in such screens can be prepared as described
below. Direct library screens can 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 an M. tuberculosis-immune individual.
[0069] The invention also provides vectors, plasmids or viruses
containing one or more of the TB nucleic acid molecules (e.g.,
having the sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or
combinations thereof). Suitable vectors for use in eukaryotic and
prokaryotic cells are known in the art and are commercially
available or readily prepared by a skilled artisan. Additional
vectors can also be found, for example, in Ausubel, F. M., et al.,
Current Protocols in Molecular Biology, (Current Protocol, 1994)
and Sambrook et al., "Molecular Cloning: A Laboratory Manual," 2nd
ED. (1989).
[0070] Recombinant polypeptides containing portions and/or variants
of a native antigen can 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 can be first concentrated using a commercially
available filter. Following concentration, the concentrate can 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.
[0071] Any of a variety of expression vectors known to those of
ordinary skill in the art can be employed to express recombinant
polypeptides of this invention. Expression can be achieved in any
appropriate host cell that has been transformed or transfected with
an expression vector containing a DNA molecule that encodes a
recombinant polypeptide. Suitable host cells include prokaryotes,
yeast and higher eukaryotic cells. Preferably, the host cells
employed are E. coli, yeast or a mammalian cell line such as COS or
CHO. The DNA sequences expressed in this manner can encode
naturally occurring antigens, portions of naturally occurring
antigens, or other variants thereof.
[0072] Uses of plasmids, vectors or viruses containing the cloned
TB receptors or receptor fragments include one or more of the
following; (1) generation of hybridization probes for detection and
measuring level of TB in tissue or isolation of TB homologs; (2)
generation of TB mRNA or protein in vitro or in vivo; and (3)
generation of transgenic non-human animals or recombinant host
cells.
[0073] In one embodiment, the present invention encompasses host
cells transformed with the plasmids, vectors or viruses described
above. Nucleic acid molecules can be inserted into a construct
which can, optionally, replicate and/or integrate into a
recombinant host cell, by known methods. The host cell can be a
eukaryote or prokaryote and includes, for example, yeast (such as
Pichia pastorius or Saccharomyces cerevisiae), bacteria (such as E.
coli, M. tuberculosis, or Bacillus subtilis), animal cells or
tissue, insect Sf9 cells (such as baculoviruses infected SF9 cells)
or mammalian cells (somatic or embryonic cells, Human Embryonic
Kidney (HEK) cells, Chinese hamster ovary cells, HeLa cells, human
293 cells and monkey COS-7 cells). Host cells suitable in the
present invention also include a mammalian cell, a bacterial cell,
a yeast cell, an insect cell, and a plant cell.
[0074] The nucleic acid molecule can be incorporated or inserted
into the host cell by known methods. Examples of suitable methods
of transfecting or transforming cells include calcium phosphate
precipitation, electroporation, microinjection, infection,
lipofection and direct uptake. "Transformation" or "transfection"
as used herein refers to the acquisition of new or altered genetic
features by incorporation of additional nucleic acids, e.g., DNA.
"Expression" of the genetic information of a host cell is a term of
art which refers to the directed transcription of DNA to generate
RNA which is translated into a polypeptide. Methods for preparing
such recombinant host cells and incorporating nucleic acids are
described in more detail in Sambrook et al., "Molecular Cloning: A
Laboratory Manual," Second Edition (1989) and Ausubel, et al.
"Current Protocols in Molecular Biology," (1992), for example.
[0075] The host cell is then maintained under suitable conditions
for expression and recovery of the antigenic TB polypeptide of the
present invention. Generally, the cells are maintained in a
suitable buffer and/or growth medium or nutrient source for growth
of the cells and expression of the gene product(s). The growth
media are not critical to the invention, are generally known in the
art and include sources of carbon, nitrogen and sulfur. Examples
include Luria broth, Superbroth, Dulbecco's Modified Eagles Media
(DMEM), RPMI-1640, M199 and Grace's insect media. The growth media
can contain a buffer, the selection of which is not critical to the
invention. The pH of the buffered media can be selected and is
generally one tolerated by or optimal for growth for the host
cell.
[0076] The host cell is maintained under a suitable temperature and
atmosphere. Alternatively, the host cell is aerobic and the host
cell is maintained under atmospheric conditions or other suitable
conditions for growth. The temperature should also be selected so
that the host cell tolerates the process and can be for example,
between about 13-40 degree Celsius.
Antibodies and Methods of Assessment
[0077] Method for assessing the presence or absence of the
antigenic TB polypeptides described herein, in a sample, are
encompassed by the present invention. Suitable assays include
immunological methods, such as radioimmunoassay, enzyme-linked
immunosorbent assays (ELISA), chemiluminescence assays, and rapid
immunochromatographic assays. Any method known now or developed
later can be used for measuring antigenic TB polypeptides.
[0078] Antibodies reactive with any one of the antigenic TB
polypeptides, namely, SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or
combination thereof, or portions thereof can be used. In a
preferred embodiment, the antibodies specifically bind with
antigenic TB polypeptides or a portion thereof. The antibodies can
be polyclonal or monoclonal, and the term antibody is intended to
encompass polyclonal and monoclonal antibodies, and functional
fragments thereof. The terms polyclonal and monoclonal refer to the
degree of homogeneity of an antibody preparation, and are not
intended to be limited to particular methods of production.
[0079] In several of the preferred embodiments, immunological
techniques detect the presence, absence of levels of antigenic TB
polypeptides described herein by means of an anti-TB antibody
(i.e., one or more antibodies). The term "anti-TB antibody"
includes monoclonal and/or polyclonal antibodies, and mixtures or
cocktails thereof, and refers to antibodies specific to
polypeptides having a sequence set forth in SEQ ID Nos: 2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, or combination thereof, or portions thereof.
[0080] Anti-TB antibodies can be raised against appropriate
immunogens, such as isolated and/or recombinant antigenic TB
polypeptides described herein, analogs or portion thereof
(including synthetic molecules, such as synthetic peptides). In one
embodiment, antibodies are raised against an isolated and/or
recombinant antigenic TB polypeptides described herein or portion
thereof (e.g., a peptide) or against a host cell which expresses
recombinant antigenic TB polypeptides. In addition, cells
expressing recombinant antigenic TB polypeptides described herein,
such as transfected cells, can be used as immunogens or in a screen
for antibody which binds receptor.
[0081] Any suitable technique can prepare the immunizing antigen
and produce polyclonal or monoclonal antibodies. The art contains a
variety of these methods (see e.g., Kohler et al., Nature, 256:
495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et
al., Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat. No.
4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory
Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);
Current Protocols In Molecular Biology, Vol. 2 (Supplement 27,
Summer `94), Ausubel, F. M. et al., Eds., (John Wiley & Sons:
New York, N.Y.), Chapter 11, (1991)). Generally, fusing a suitable
immortal or myeloma cell line, such as SP2/0, with antibody
producing cells can produce a hybridoma. Animals immunized with the
antigen of interest provide the antibody producing cell, preferably
cells from the spleen or lymph nodes. Selective culture conditions
isolate antibody producing hybridoma cells while limiting dilution
techniques produce them. Researchers can use suitable assays such
as ELISA to select antibody producing cells with the desired
specificity.
[0082] Other suitable methods can produce or isolate antibodies of
the requisite specificity. Examples of other methods include
selecting recombinant antibody from a library or relying upon
immunization of transgenic animals such as mice.
[0083] The present invention includes assays to determine if a
person is infected with active TB, as compared with person who does
not have active TB (e.g., has latent TB, no TB infection, or has
been immunized against TB infection). Latent TB occurs when a
person has been infected with M. tuberculosis, but the bacteria is
dormant or inactive. Active TB infection refers to a person
infected with M. tuberculosis and the bacteria is acutely affecting
portions of the bodying, including the lungs, and other tissues.
The present invention, based on the discovery that certain TB
antigens are found in the urine of patients with active TB,
includes assays for determining the absence or presence of active
TB infection.
[0084] According to the method, an assay can determine the
presence, absence or level of antigenic TB polypeptides in a
biological sample. Such an assay includes combining the sample to
be tested with an antibody having specificity for antigenic TB
polypeptides described herein, under conditions suitable for
formation of a complex between antibody and antigenic TB
polypeptides, and detecting or measuring (directly or indirectly)
the formation of a complex. The sample can be obtained directly or
indirectly (e.g., provided by a healthcare provider), and can be
prepared by a method suitable for the particular sample (e.g.,
urine, sputum, cerebral spinal fluid, whole blood, platelet rich
plasma, platelet poor plasma, serum) and assay format selected.
Methods of combining sample and antibody, and methods of detecting
complex formation are also selected to be compatible with the assay
format.
[0085] Suitable labels can be detected directly, such as
radioactive, fluorescent or chemiluminescent labels. They can also
be indirectly detected using labels such as enzyme labels and other
antigenic or specific binding partners like biotin. Examples of
such labels include fluorescent labels such as fluorescein,
rhodamine, chemiluminescent labels such as luciferase, radioisotope
labels such as .sup.32P, .sup.125I, .sup.131I, enzyme labels such
as horseradish peroxidase, and alkaline phosphatase, galactosidase,
biotin, avidin, spin labels and the like. The detection of
antibodies in a complex can also be done immunologically with a
second antibody, which is then detected (e.g., by means of a
label). Conventional methods or other suitable methods can directly
or indirectly label an antibody. Labeled primary and secondary
antibodies can be obtained commercially or prepared using methods
know to one of skill in the art (see Harlow, E. and D. Lane, 1988,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory:
Cold Spring Harbor, N.Y.).
[0086] In a preferred embodiment, the presence, absence, or level
of antigenic TB polypeptides in a sample is determined using an
ELISA assay, a sandwich ELISA assay, or immunochromatographic
assay. For detection of antigenic TB polypeptides in a suitable
sample, a sample (e.g., urine) is collected. Samples can be
processed as known in the art. The assay further includes combining
a suitable sample with a composition having an anti-TB polypeptide
antibody as detector (e.g., biotinylated anti-TB polypeptides MAb
and HRP-streptavidin, or HRP-conjugated anti-TB polypeptides Mab),
and a solid support, such as a microtiter plate or dipstick, having
an anti-TB polypeptide capture antibody bound (directly or
indirectly) thereto. The detector antibody binds to a different
antigenic TB polypeptide epitope from that recognized by the
capture antibody, under conditions suitable for the formation of
the complex. The assay then involves determining the formation of
complex in the samples. The presence of one or more of the
antigenic TB polypeptide in a sample of an individual indicates the
presence of active TB infection, whereas the absence of a TB
polypeptide indicates that the patient does not have active TB
infection.
[0087] The solid support, such as a microtiter plate, dipstick,
bead, pad, strip, or other suitable support, can be coated directly
or indirectly with an anti-TB polypeptide antibody or TB specific
antigen. For example, an anti-TB polypeptide antibody can coat a
microtiter well, or a biotinylated anti-TB polypeptide Mab can be
added to a streptavidin coated support. With respect to a
immunochromatographic assay, a pad or strip can be coated with an
antibody specific for the antigen, and when a sample having the one
or more of antigens described herein comes into contact with the
antibody, the complex can turn a color with aid of a detector, as
further described herein. See FIG. 4. A variety of immobilizing or
coating methods as well as a number of solid supports can be used,
and can be selected according to the desired format.
[0088] In one immunochromatographic assay, a sample having the TB
antigens of the present invention can be added to the pad like that
shown in FIG. 4. The sample diffuses across a gold labeled antibody
(mAb#1) that is specific to a portion of the antigen, and a complex
between the antigen in the sample and the antibody is formed. As
the complex further diffuses across the pad having a second
antibody (mAb #2), the antibody binds to a different portion of the
antigen. When it hits the line labeled "line 1", the labeled
complex turns a color, and the control line ("line 2") will also
change color. If the sample does not contain the TB antigens of the
present invention, then line 1 will not turn color because the
gold-labeled antibody will not bind to the sample and therefore
will not come into contact with line 1.
[0089] In another embodiment, the sample (or an antigenic TB
polypeptide standard) is combined with the solid support
simultaneously with the detector antibody, and optionally with a
one or more reagents by which detection is monitored. For example,
the sample can be combined with the solid support simultaneously
with (a) HRP-conjugated anti-TB polypeptide Mab, or (b) a
biotinylated anti-TB polypeptide Mab and HRP-streptavidin.
[0090] A known amount of an antigenic TB polypeptide standard can
be prepared and processed as described above for a suitable sample.
This antigenic TB polypeptide standard assists in quantifying the
amount of antigenic TB polypeptides detected by comparing the level
of antigenic TB polypeptides in the sample relative to that in the
standard.
[0091] A physician, technician, apparatus or a qualified person can
compare the amount of detected complex with a suitable control to
determine if the levels are elevated. For example, the level of
antigenic TB polypeptides following treatment can be compared with
a baseline level prior to treatment, or with levels in normal
individuals or suitable controls. A decrease in or maintenance of
the levels of one or more TB polypeptides in the urine, as compared
to baseline levels, indicates that the treatment is working,
whereas increases in levels indicates that is not effective.
[0092] Typical assays for antigenic TB polypeptides are sequential
assays in which a plate is coated with first antibody, sample is
added, the plate is washed, second tagged antibody is added, and
the plate is washed and bound second antibody is quantified. In
another embodiment, a format in which antibodies and the sample are
added simultaneously, in a competitive ELISA format, can achieve
greater sensitivity.
[0093] A variety of methods can determine the amount of antigenic
TB polypeptides in complexes. For example, when HRP is used as a
label, a suitable substrate such as OPD can be added to produce
color intensity directly proportional to the bound anti-TB
polypeptides Mab (assessed e.g., by optical density), and therefore
to the antigenic TB polypeptides in the sample.
[0094] A technician, physician, qualified person or apparatus can
compare the results to a suitable control such as a standard, or
baseline levels of antigenic TB polypeptides in a sample from the
same donor. For example, the assay can be performed using a known
amount of antigenic TB polypeptides standard in lieu of a sample,
and a standard curved established. One can relatively compare known
amounts of the antigenic TB polypeptides standard to the amount of
complex formed or detected.
[0095] The nucleic acid that encodes the antigenic TB polypeptides
can also be assayed by hybridization, e.g., by hybridizing one of
the TB sequences provided herein (e.g., SEQ ID NO: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, or combinations thereof) or an oligonucleotide derived from
one of the sequences, to a DNA or RNA-containing tissue sample from
a person. Such a hybridization sequence can have a detectable
label, e.g., radioactive, fluorescent, etc., attached to allow the
detection of hybridization product. Such methods include contacting
the sample with one or more oligonucleotide probes (e.g., at least
about 15 contiguous bases) specific for the nucleic acid molecule
described herein under high stringency conditions, sufficiently to
allow hybridization between the sample and the probe; and detecting
the nucleic acid molecule that hybridizes to the oligonucleotide
probe in the sample. The presence of hybridization of the probe
indicates M. tuberculosis infection, and the absence of
hybridization indicates an absence of M tuberculosis infection.
Methods for hybridization are well known, and such methods are
provided in U.S. Pat. No. 5,837,490, by Jacobs et al., the entire
teachings of which are herein incorporated by reference in their
entirety. The design of the oligonucleotide probe should preferably
follow these parameters: (a) it should be designed to an area of
the sequence which has the fewest ambiguous bases ("N's"), if any,
and (b) it should be designed to have a T.sub.m of approx.
80.degree. Celsius (assuming 2.degree. Celsius for each A or T and
4.degree. Cfor each G or C).
[0096] The present invention encompasses detection of TB
polypeptides of the present invention in a sample using with PCR
methods using primers disclosed or derived from sequences described
herein. For example, the sequences described herein can be detected
by PCR using SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or combinations
thereof. PCR is the selective amplification of a target sequence by
repeated rounds of nucleic acid replication utilizing
sequence-specific primers and a thermostable polymerase. PCR allows
recovery of entire sequences between two ends of known sequence.
Specifically, contacting the sample with at least two
oligonucleotide primers in a PCR, wherein at least one of the
oligonucleotide primers (e.g., at least about 10 contiguous bases)
is specific for one or more of the isolated nucleic acid molecules
described herein. The two are contacted sufficiently to allow
amplification of the primers. The amplified nucleic acid sequence
in the sample is detected. The presence any one of the amplified
nucleic acid sequences indicate M. tuberculosis infection, and the
absence of any one of the amplified nucleic acid sequences indicate
an absence of M. tuberculosis infection. Methods of PCR are
described herein and are known in the art.
[0097] Hence, the present invention includes kits for the detection
of SEQ ID Nos:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or combinations thereof, or
the quantification of these sequences, having either antibodies
specific for them or a portion thereof, or a nucleic acid sequence
that can hybridize to the nucleic acid of encoding these
sequences.
[0098] Additional immunological or nucleic acid assessments can be
performed using methods known in the art. Assays, known in the art
or those later developed can be used to assess the antigenic TB
polypeptides in a sample.
[0099] In addition to measuring the presence of antigenic TB
polypeptides in a sample, assays exist to determine the efficacy of
a TB vaccine (e.g., the extent to which the immune response is
stimulated). These types of assays can be used together to fully
assess a person's TB status. For examples, an individual who has a
TB-specific immunogenic response, but tests negative to the
presence of one or more the antigenic TB polypeptides in a sample,
is one who has a level of immunity to the disease. However, a
person who has a TB-specific immunogenic response and tests
positive to the presence of the antigenic TB polypeptides of the
present invention is someone who likely has TB.
[0100] The efficacy of a TB vaccine can be measured by determining
the immunogenic response of the person who received the vaccine.
The TB antigens of the present invention (and immunogenic portions
thereof) described herein have the ability to induce an immunogenic
response. More specifically, the antigens have the ability to
induce proliferation and/or cytokine production (i.e.,
interferon-.gamma. and/or interleukin-12 production) in T cells, NK
cells, B cells and/or macrophages derived from an M.
tuberculosis-immune individual. See Example 3.
[0101] The selection of cell type for use in evaluating an
immunogenic response to a antigen will, of course, depend on the
desired response. For example, interleukin-12 production is most
readily evaluated using preparations containing B-cells and/or
macrophages. An M. tuberculosis-immune individual is one who is
considered to be resistant to the development of tuberculosis by
virtue of having mounted an effective T cell response to M.
tuberculosis (i.e., substantially free of disease symptoms). Such
individuals can be identified based on a strongly positive (i.e.,
greater than about 10 mm diameter induration) intradermal skin test
response to tuberculosis proteins using a Purified Protein
Derivative (PPD) and an absence of any signs or symptoms of
tuberculosis disease. T cells, NK cells, B cells and macrophages
derived from M. tuberculosis-immune individuals can 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) can be employed without further separation of component
cells. PBMCs can generally be prepared, for example, using density
centrifugation through Ficoll (Winthrop Laboratories, NY). T cells
for use in the assays described herein can 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, can be employed. Such T cell
clones can be generated by, for example, culturing PBMCs from M.
tuberculosis-immune individuals with mycobacterial proteins for a
period of 2-4 weeks. This allows expansion of only the
mycobacterial protein-specific T cells, resulting in a line
composed solely of such cells. These cells can 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
M. tuberculosis-immune individual are considered immunogenic. Such
assays can be performed, for example, using the representative
procedures described below. Immunogenic portions of such antigens
can be identified using similar assays, and can be present within
the polypeptides described herein.
[0102] The ability of a polypeptide (e.g., an immunogenic antigen,
or a portion or other variant thereof) to induce cell proliferation
can be 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.5 cells ranges from about 10 ng/mL to
about 100 .mu.g/mL and preferably is about 10 .mu.g/mL. The
incubation of polypeptide with cells is typically performed at
37.degree. C. for about six days. Following incubation with
polypeptide, the cells are assayed for a proliferative response,
which can be evaluated by methods known to those of ordinary skill
in the art, such as exposing cells to a pulse of radiolabeled
thymidine and measuring the incorporation of label into cellular
DNA. In general, a polypeptide that results in at least a three
fold increase in proliferation above background (i.e., the
proliferation observed for cells cultured without polypeptide) is
considered to be able to induce proliferation.
[0103] The ability of a polypeptide to stimulate the production of
interferon-.gamma. and/or interleukin-12 in cells can be evaluated
by contacting the cells with the polypeptide and measuring the
level of interferon-.gamma. or interleukin-12 produced by the
cells, as demonstrated in Example 3. 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 can, but need not,
be immobilized on a solid support, such as a bead or a
biodegradable microsphere, such as those described in U.S. Pat.
Nos. 4,897,268 and 5,075,109. The incubation of polypeptide with
the cells is typically performed at 37.degree. C. or about six
days. Following incubation with polypeptide, the cells are assayed
for interferon-.gamma. and/or interleukin-12 (or one or more
subunits thereof), which can be evaluated by methods known to those
of ordinary skill in the art, such as an enzyme-linked
immunosorbent assay (ELISA) or, in the case of IL-12 P70 subunit, a
bioassay such as an assay measuring proliferation of T cells. In
general, a polypeptide that results in the production of at least
50 pg of interferon-.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 3X10.sup.5 PBMC) is
considered able to stimulate the production of IL-12.
[0104] 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 M. tuberculosis-immune individuals. Among these immunogenic
antigens, polypeptides having superior therapeutic properties can
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 who are not M. tuberculosis-immune, thereby
eliminating responses that are not specifically due to M.
tuberculosis-responsive cells. Those antigens that induce a
response in a high percentage of T cell, NK cell, B cell and/or
macrophage preparations from M. tuberculosis-immune individuals
(with a low incidence of responses in cell preparations from other
individuals) have superior therapeutic properties.
Fusion Proteins, Vaccine Compositions, Mode and Manner of
Administration
[0105] The TB polypeptides of the present invention can be in the
form of a conjugate or a fusion protein, which can be manufactured
by known methods. In particular, 2 or more of the sequences, SEQ ID
NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, or 48 can be fused to one another, or with
other proteins, to provide a more effective vaccine composition,
and stimulate an improved immunogenic response. Other proteins that
can be used to make such a fusion protein include TB antigens that
simulate the CD4+ T cell pathway of the immune response. Examples
of such antigens include Antigen 85b, ESAT-6, MtB41, Mtb39. The TB
polypeptides of the present invention were isolated from MHC class
1 molecules, molecules known for presenting antigens to CD8+ T
cells. Although it is possible for these polypeptides to be also
presented in the CD4+ pathway, fusing a CD4+ T-cell pathway antigen
with one of the polypeptides of the present invention can serve to
increase effectiveness of the TB vaccine. Fusion proteins can be
manufactured according to known methods of recombinant DNA
technology. For example, fusion proteins can be expressed from a
nucleic acid molecule comprising sequences which code for a
biologically active portion of the TB polypeptides or the entire TB
polypeptides set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or
combinations thereof, and its fusion partner, for example another
sequence of the present invention, a portion of an immunoglobulin
molecule, or another TB antigen from the CD4+ T cell pathway. For
example, some embodiments can be produced by the intersection of a
nucleic acid encoding immunoglobulin sequences into a suitable
expression vector, phage vector, or other commercially available
vectors. The resulting construct can be introduced into a suitable
host cell for expression. Upon expression, the fusion proteins can
be isolated or purified from a cell by means of an affinity matrix.
By measurement of the alternations in the functions of transfected
cells occurring as a result of expression of recombinant TB
proteins, either the cells themselves or TB proteins produced from
the cells can be utilized in a variety of screening assays.
[0106] As noted above, in certain aspects the inventive
compositions comprise fusion proteins or DNA fusion molecules. Each
fusion protein comprises a first and a second inventive polypeptide
or, alternatively, a polypeptide of the present invention and a
known M. tuberculosis antigen, together with variants of such
fusion proteins. The fusion proteins of the present invention can
also include a linker peptide between the first and second
polypeptides. The DNA fusion molecules of the present invention
comprise a first and a second isolated DNA molecule, each isolated
DNA molecule encoding either an inventive M. tuberculosis antigen
or a known M. tuberculosis antigen.
[0107] A DNA sequence encoding a fusion protein of the present
invention is constructed using known recombinant DNA techniques to
assemble separate DNA sequences encoding the first and second
polypeptides into an appropriate expression vector, as described in
detail below. The 3' end of a DNA sequence encoding the first
polypeptide is ligated, with or without a peptide linker, to the 5'
end of a DNA sequence encoding the second polypeptide so that the
reading frames of the sequences are in phase to permit mRNA
translation of the two DNA sequences into a single fusion protein
that retains the biological activity of both the first and the
second polypeptides.
[0108] A peptide linker sequence can be employed to separate the
first and the second polypeptides by a distance sufficient to
ensure that each polypeptide folds into its secondary and tertiary
structures. Such a peptide linker sequence is incorporated into the
fusion protein using standard techniques well known in the art.
Suitable peptide linker sequences can 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 can
also be used in the linker sequence. Amino acid sequences which can
be usefully employed as linkers include those disclosed in Maratea
et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci.
USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No.
4,751,180. The linker sequence can be from 1 to about 50 amino
acids in length. Peptide sequences are not required when the first
and second polypeptides have non-essential N-terminal amino acid
regions that can be used to separate the functional domains and
prevent steric interference.
[0109] The ligated DNA sequences are operably linked to suitable
transcriptional or translational regulatory elements. The
regulatory elements responsible for expression of DNA are located
only 5' to the DNA sequence encoding the first polypeptides.
Similarly, stop codons require to end translation and transcription
termination signals are only present 3' to the DNA sequence
encoding the second polypeptide.
[0110] Efficacy of a vaccine including the isolated sequences of
the present invention can be determined based on the ability of the
antigen to provide at least about a 50% (e.g., about a 60%, about a
70%, about a 80%, about a 90%, or about a 100%) reduction in
bacterial numbers and/or at least about a 40% (e.g., about a 50%,
about a 60%, about a 70%, about a 80%, about a 90%, or about a
100%) decrease in mortality following experimental infection in a
challenge experiment. Suitable experimental animals include mice,
guinea pigs and primates.
[0111] The compositions of the present invention are preferably
formulated as either pharmaceutical compositions or as vaccines for
in the induction of protective immunity against tuberculosis in a
patient. A patient can be afflicted with a disease, or can be free
of detectable disease and/or infection. In other words, protective
immunity can be induced to prevent, reduce the severity of, or
treat tuberculosis.
[0112] In one embodiment, pharmaceutical compositions of the
present invention comprise one or more of the above polypeptides,
either present as a mixture or in the form of a fusion protein, and
a physiologically acceptable carrier. Similarly, vaccines comprise
one or more the above polypeptides and a non-specific immune
response enhancer, such as an adjuvant or a liposome (into which
the polypeptide is incorporated).
[0113] In another embodiment, a pharmaceutical composition and/or
vaccine of the present invention can contain one or more of the DNA
molecules of the present invention, either present as a mixture or
in the form of a DNA fusion molecule, each DNA molecule encoding a
polypeptide as described above, such that the polypeptide is
generated in situ. In such vaccines, the DNA can be present within
any of a variety of delivery systems known to those of ordinary
skill in the art, including nucleic acid expression systems,
bacterial and viral expression systems. Appropriate nucleic acid
expression systems contain the necessary DNA sequences for
expression in the patient (such as a suitable promoter and
terminating signal). Bacterial delivery systems involve the
administration of a bacterium (such as Bacillus-Calmette-Guerrin)
that expresses an immunogenic portion of the polypeptide on its
cell surface. In a preferred embodiment, the DNA can be introduced
using a viral expression system (e.g., vaccinia or other pox virus,
retrovirus, or adenovirus), which can involve the use of a
non-pathogenic (defective), replication competent virus. Techniques
for incorporating DNA into such expression systems are well known
to those of ordinary skill in the art. The DNA can 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 can be increased by coating the DNA onto biodegradable
beads, which are efficiently transported into the cells.
[0114] The antigenic TB molecules of the present invention can be
administered with or without a carrier. The terms "pharmaceutically
acceptable carrier" or a "carrier" refer to any generally
acceptable excipient or drug delivery composition that is
relatively inert and non-toxic. Exemplary carriers include sterile
water, salt solutions (such as Ringer's solution), alcohols,
gelatin, talc, viscous paraffin, fatty acid esters,
hydroxymethylcellulose, polyvinyl pyrolidone, calcium carbonate,
carbohydrates (such as lactose, sucrose, dextrose, mannose,
albumin, starch, cellulose, silica gel, polyethylene glycol (PEG),
dried skim milk, rice flour, magnesium stearate, and the like.
Suitable formulations and additional carriers are described in
Remington's Pharmaceutical Sciences, (17.sup.th Ed., Mack Pub. Co.,
Easton, Pa.). Such preparations can be sterilized and, if desired,
mixed with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers, coloring, preservatives and/or aromatic
substances and the like which do not deleteriously react with the
active compounds. Typical preservatives can include, potassium
sorbate, sodium metabisulfite, methyl paraben, propyl paraben,
thimerosal, etc. The compositions can also be combined where
desired with other active substances, e.g., enzyme inhibitors, to
reduce metabolic degradation. A carrier (e.g., a pharmaceutically
acceptable carrier) is preferred, but not necessary to administer
the compound.
[0115] The composition can be a liquid solution, suspension,
emulsion, tablet, pill, capsule, sustained release formulation, or
powder. The method of administration can dictate how the
composition will be formulated. For example, the composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc.
[0116] The antigenic TB molecules used in the invention can be
administered intravenously, parenterally, intramuscular,
subcutaneously, orally, nasally, topically, by inhalation, by
implant, by injection, or by suppository. The composition can be
administered in a single dose or in more than one dose over a
period of time to confer the desired effect.
[0117] The actual effective amounts of compound or drug can vary
according to the specific composition being utilized, the mode of
administration and the age, weight and condition of the patient.
For example, as used herein, an effective amount of the drug is an
amount which reduces the number of bacteria. Dosages for a
particular individual patient can be determined by one of ordinary
skill in the art using conventional considerations, (e.g. by means
of an appropriate, conventional pharmacological protocol).
[0118] For enteral or mucosal application (including via oral and
nasal mucosa), particularly suitable are tablets, liquids, drops,
suppositories or capsules. A syrup, elixir or the like can be used
wherein a sweetened vehicle is employed. Liposomes, microspheres,
and microcapsules are available and can be used.
[0119] Pulmonary administration can be accomplished, for example,
using any of various delivery devices known in the art such as an
inhaler. See. e.g., S. P. Newman (1984) in Aerosols and the Lung,
Clarke and Davis (eds.), Butterworths, London, England, pp.
197-224; PCT Publication No. WO 92/16192; PCT Publication No. WO
91/08760.
[0120] For parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. In particular, carriers for parenteral
administration include aqueous solutions of dextrose, saline, pure
water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil,
polyoxyethylene-polyoxypropylene block polymers, and the like.
Ampules are convenient unit dosages.
[0121] Biodegradable microspheres (e.g., polylactic galactide) can
also be employed as carriers for the pharmaceutical compositions of
this invention. Suitable biodegradable microspheres are disclosed,
for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
[0122] Any of a variety of adjuvants can be employed in the
vaccines of this invention to enhance the immune response. Most
adjuvants contain a substance designed to protect the antigen from
rapid catabolism, such as aluminum hydroxide or mineral oil, and a
nonspecific stimulator of immune responses, such as lipid A,
Bortadella pertussis or Mycobacterium tuberculosis. Suitable
adjuvants are commercially available and include, for example,
Freund's Incomplete Adjuvant and Freund's Complete Adjuvant /(Difco
Laboratories) and Merck Adjuvant 65 (Merck and Company, Inc.,
Rahway, N.J.). Other suitable adjuvants include alum, biodegradable
microspheres, monophosphoryl lipid A and quil A.
[0123] In the inventive vaccines, it is preferred that the adjuvant
induces an immune response comprising Thl aspects. Suitable
adjuvant systems include, for example, a combination of
monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl
lipid A (3D-MLP) together with an aluminum salt. An enhanced system
involves the combination of a monophosphoryl lipid A and a saponin
derivative, particularly the combination of 3D-MLP and the saponin
QS21 as disclosed in WO 94/00153, or a less reactogenic composition
where the QS21 is quenched with cholesterol as disclosed in WO
96/33739. Previous experiments have demonstrated a clear
synergistic effect of combinations of 3D-MLP and QS21 in the
induction of both humoral and Thl type cellular immune responses. A
particularly potent adjuvant formation involving QS21, 3D-MLP and
tocopherol in an oil-in-water emulsion is described in WO 95/17210
and is a preferred formulation.
[0124] The administration of the antigenic TB polypeptide molecules
of the present invention and other compounds can occur
simultaneously or sequentially in time. A DNA vaccine and/or
pharmaceutical composition as described above can be administered
simultaneously with or sequentially to an additional polypeptide of
the present invention, a known M. tuberculosis antigen, an immune
enhancer, or other compound known in the art that would be
administered with such a vaccine. The compound can be administered
before, after or at the same time as the antigenic TB molecules.
Thus, the term "co-administration" is used herein to mean that the
antigenic TB molecules and the additional compound (e.g., immune
stimulating compound) will be administered at times to achieve a
specific TB immune response, as described herein. The methods of
the present invention are not limited to the sequence in which the
compounds are administered, so long as the compound is administered
close enough in time to produce the desired effect.
[0125] Routes and frequency of administration of the inventive
pharmaceutical compositions and vaccines, as well as dosage, will
vary from individual to individual and can parallel those currently
being used in immunization using BCG. In general, the
pharmaceutical compositions and vaccines can be administered by
injection (e.g., intracutaneous, intramuscular, intravenous or
subcutaneous), intranasally (e.g., by aspiration), intralung, or
orally. Between 1 and 3 doses can be administered for a 1-36 week
period. Preferably, 3 doses are administered, at intervals of 3-4
months, and booster vaccinations can be given periodically
thereafter. Alternate protocols can be appropriate for individual
patients. A suitable dose is an amount of polypeptide or DNA that,
when administered as described above, is capable of raising an
immune response in an immunized patient sufficient to protect the
patient from M. tuberculosis infection for at least 1-2 years. In
general, the amount of polypeptide present in a dose (or produced
in situ by the DNA in a dose) ranges from about 1 pg to about 100
mg per kg of host, typically from about 10 pg to about 1 mg, and
preferably from about 100 pg to about 1 .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. EXEMPLIFICATION
Example 1
M. tuberculosis Peptides Associated With MHC Class 1 Molecules
[0126] A strategy to detect M. tuberculosis peptides associated
with MHC Class I molecules of mouse macrophages infected in vivo
was applied to identify the microbial antigens.
[0127] The rationale to search for M. tuberculosis antigens or
peptides associated with the MHC class I molecules of infected
cells is based on the fact that these host cell molecules are
crucial to present antigens to CD8+ T cells, a subset of T cells
involved in resistance and against tuberculosis. Therefore, this
approach of antigen discovery has direct implication in vaccine
development strategies against this disease.
[0128] The MHC class I-associated M. tuberculosis peptides were
isolated from adherent spleen cells of C57BL/6 mice infected with
107 CFU of M. tuberculosis. C57BL/6 mice are considered to be
relatively resistant to M. tuberculosis infection, therefore ideal
for these studies. The infection with a high inoculum is important
to achieve high degree of intra cellular infection in the animals'
spleen cells. Between days 10 and 14 of infection the mice spleens
contain approximately five times more cells than non-infected
controls. A great percentage of the spleen cells are macrophages
infected with M. tuberculosis. The mice were sacrificed and their
splenic adherent cells were obtained, lysed with CHAPS detergent
(Boehringer Mannheim Corp., Indianapolis, Ind.), and the MHC class
I molecules isolated by affinity purification on a protein G
sepharose column linked to the anti-H-2 K/D.sup.b monoclonal
antibody (ATCC HB-11). The procedure is summarized in FIG. 3.
[0129] The eluted peptides comprising a mixture of molecules below
5 kDa were ultra filtrated over a Millipore Ultrafree-CL 5 kDa
cutoff were then fractionated by microbore reverse phase HPLC
(Delta-Pak C18 column, 300 .ANG. pore size, 5.mu. particle). The
HPLC profiles rendered several peaks, which were individually
analyzed by microcapillary liquid chromatography coupled with
electrospray ionization mass spectrometry (LC-MS) to determine the
molecular weight and abundance of all peptide species. The mass of
the candidate peptides was then obtained by collision activated
dissociation (CAD) tandem mass spectra (MS/MS).
[0130] The peptide sequences were searched for identity with known
proteins in both the M. tuberculosis and mouse genome databases
(The Institute for Genomic Research). Several peptides had strong
homology with mouse protein. However seven peptide and nucleic acid
sequences were identified to being of M. tuberculosis origin (FIG.
1). The corresponding genes encoding the protein donor of these
peptides are being cloned and will be expressed using standard
procedures. In addition, bacterial plasmid DNA containing these M.
tuberculosis genes will be prepared. Both, recombinant protein and
plasmid DNA will be used in immunization protocols aiming to
investigate the protection potential of these molecules to protect
mice against challenge with virulent M. tuberculosis. The protein
formulation will the used to test CD4+ response and the DNA
immunization will target CD8+ T cell response.
Example 2
A M. tuberculosis Antigen Found in the Urine of Infected Mice
[0131] A strategy to detect M. tuberculosis antigens in the urine
of infected mice was applied to identify the microbial antigens in
the urine of patients with active tuberculosis.
[0132] Antigen detection assays, in contrast to conventional
serology, detect disease status and not the host antibody response
to the disease etiological agent. It can therefore be used for both
diagnosis and treatment follow up. In the present project an
antigen discovery approach to identify M. tuberculosis antigens in
the urine of human patients was used. Additionally, recombinant
proteins will be produced and validated as markers of active
disease.
[0133] Individual urines (15 mL) were loaded onto a 15 mL capacity
Vivaspin 5K MWCO filtered and centrifuged at 3000G at 4.degree. C.
until the retentate volume was <2 mL. The concentrate was
removed from the membrane by rinsing with 6M urea 100 mM ammonium
bicarbonate. Final concentrates were brought up to 4 mL with 6M
Urea 100 mM ammonium bicarbonate pH 8.5. To each protein
concentrate sample was added 20 mL of 2M DTT. Samples were vortexed
and incubated at 37.degree. C. for 1 hour. Reduced cysteines were
alkylated by adding 200 mL of 0.5M iodoacetamide to each sample.
Samples were vortexed and allowed to react for 1 hour at room
temperature in the dark. Residual iodoacetamide was quenched with
15 mL of 2M DTT. From the 4 mL stock sample, 300 uL was removed for
gel analysis.
[0134] Gels were imaged with a Kodak DC280 Digital Camera fitted
with a +10 Macro lens. Images were processed using Adobe Photoshop
and printed out. Gels were cut with a clean razor blade into 33 gel
slices each while marking positions of the cuts for each slice.
Slices were then placed into 2 mL centrifuge tubes. All further
processing of the samplers was perfomed in a dual isolation
BioSafety Cabinet. The tubes with gel slices were filled to 2 mL
with 50% methanol, 5% acetic acid, and rinsed three times with
additional aliquots of 50% methanol, 5% acetic acid. The destain
solution was removed and the tubes filled with 2 mL of
acetonitrile. After 30 minutes with agitation, the acetonitrile was
removed and replaced with 100 mM ammonium bicarbonate. After 30
minutes the ammonium bicarbonate solution was removed and the tubes
were filled with acetonitrile. This acetonitrile, ammonium
bicarbonate rinsing step was repeated once. The final acetonitrile
wash was removed just prior to digestion and the gel slices dried
for 30 minutes in a speedvac. The tubes containing the dried gel
slices were placed on ice and allowed to cool. Promega Sequencing
grade trypsin was dissolved in ice cold 50 mM ammonium bicarbonate
to a concentration of 13.3 mg/mL. Using a repeating pipettor, 125
mL of the trypsin solution was added to the bottom of each tube.
The gel slices were allowed to swell for 15 minutes on ice, after
which an additional 125 mL of 50 mM ammonium bicarbonate was added
to each tube. The tubes were then capped and incubated for 16 hours
at 37.degree. C. After digestion 650 mL of 50 mM ammonium
bicarbonate was added to each tube. The samples were then incubated
for 1 hour at 37.degree. C. The first set of extracts were removed
and placed into labeled Axygen 1.5 mL tubes, a second extraction of
650 mL ammonium bicarbonate was carried out and pooled with the
first. These extracts were then lyophilized while two acidic
extractions of 650 mL of 50% acetonitrile 0.1% formic acid were
carried out. All extracts were frozen to -80.degree. C. and
lyophilized to dryness in a ThermoSavant SC280 speedvac <10
mTorr. The lyophilate was redissolved into 200 mL of 5%
acetonitrile 0.1% formic acid. Appropriate volumes of each extract
(1-6, 10 mL)(7-12, 20 mL) (13-18, 35 mL) (19-33, 50 mL) were added
to a 96 well plate to approximate protein molarity load and protein
from each MW region of the gel, the plate was frozen to -80.degree.
C. and lyophilized again. All samples were redissolved in 12 mL of
5% acetonitrile 0.1% formic acid.
[0135] Samples were then evaluated by Mass Spectrometry on a LCQ
DECA XP plus Proteome X workstation from Thermofinnigan. For each
run 10 mL of each reconstituted sample were injected with a Famos
Autosampler while the separation was done on a 75 mm
i.d..times.18cm column packed with C18 media running at a 235 nL a
minute flow rate provided from a Surveyor MS pump with a flow
splitter with a gradient of 5-60% water 0.1% formic acid,
acetonitrile 0.1% formic acid over the course of 90 min. (2.5 hour
run), 180 minutes (4 hour run), or 400 minutes(8 hour run). In
between each set of samples was run two standards of a 5 Angio mix
peptides (Michrom BioResources) to ascertain column performance,
and observe any potential carryover that might have occurred. The
LCQ was run in a top five configuration with one MS scans and five
MS/MS scans. Dynamic exclusion was set to 1 with a limit of 30
seconds. Peptide ID's were made using Sequest through the Bioworks
Browser 3.1. Sequential database searches was made using the NCBI
RefSeqHuman Database using differential carbamidomethyl modified
cysteines and oxidized methionines, followed by further searches
using differential modifications. Secondary searches were performed
using Sequest using RefSeqHuman Gnomon predicted protein database.
In this fashion known and theoretical protein hits can be found
without compromising the statistical relevance of all the data.
Peptide score cutoff values were chosen at Xcorr of 1.8 for singly
charged ions, 2.5 for doubly charged ions, and 3.0 for triply
charged ions, along with deltaCN values of 0.1, and RSP values of
1. The cross correlation values chosen for each peptide assure a
high confidence match for the different charge states, while the
deltaCN cutoff insures the uniqueness of the peptide hit. The RSP
value of 1 ensured that the peptide matched the top hit in the
preliminary scoring and that the peptide fragment file only matched
to one protein hit. Using this state of art approach several human
peptides were identified but most importantly we were able thus far
to identify at least five M. tuberculosis peptides in the urine of
three out of the six urine samples studied. One of the sequences of
the identified peptide (MVIIELMRR--SEQ ID NO: 30) has identical
homology with the deduced sequence of a M. tuberculosis protein
(FIG. 2) [The Institute for Genomic Research (TIGR), and
SWISS-PROT/TrEMBL AC #033183]. Four other sequences were
identified, SEQ ID NO: 34, 38, 42, 46, shown in FIGS. 5-9,
respectively. These peptide sequence was found in three out of six
urine samples from tuberculosis patients. PCR primers were designed
and synthesized and used to amplify the full-length gene encoding
this protein from M. tuberculosis genomic DNA. A DNA fragment
(.about.1 kb) containing at the 5' end an Nde I site and at the 3'
end a Bam HI site, was obtained and is currently been used to clone
and expressed the gene.
Example 3
Production, Purification and Characterization of M tuberculosis
Recombinant Proteins
[0136] Concentrated efforts have been made to produce, purify, and
characterize the proteins of M. tuberculosis found in the urine of
patients with pulmonary tuberculosis, as described herein. Three of
them (MTP1694 (SEQ ID NO: 42), MTP2462 (SEQ ID NO: 46)) and (MT2990
(SEQ ID NO: 38)) have been obtained and their biochemical and
biological properties characterized. The three proteins were
purified from inclusion bodies with yields ranging from 10 to 15 mg
of purified protein per liter of induced culture. FIG. 10
illustrates the over-expression of the recombinant molecules, MT
1694, MT2462, and MT2990, in E. coli and shows that they migrated
to positions in the gel that correspond to their expected Molecular
Weight (MW). Recombinant proteins were expressed in E. coli
BL-21(DE3)/pLysS host cells with six His-tag amino terminal
residues. Proteins were purified by affinity chromatography using
Ni-NTA agarose matrix. Purity was evaluated by SDS-PAGE (4-20%
gradient polyacrylamide gel) followed by Coomassie blue stain.
Because the MT2990 gene codes for a protein of 130.6 kDa MW, which
is a molecule size that is difficult to express as recombinant
protein, overlapping/truncated forms of this molecule were cloned
and expressed. The cloned and expressed protein illustrated in FIG.
10 represents a fragment of the full length gene that spans 100
amino acids, both upstream and downstream of the amino acid
sequence of the M. tuberculosis protein (MT2990) that has identical
homology with the peptide found in the urine of tuberculosis
patients. This fragment of the molecule was chosen because this
portion of the protein it is stable and resistant to the host
proteolytic enzyme machinery, therefore a vaccine target.
[0137] Rabbit antisera to MTP1694 and MTP2462 recombinant proteins
were produced and an antiserum to MT2990 is being and can be
generated. The antisera to MTP1694 and MTP2462 were used to
validate identified proteins as a genuine M. tuberculosis
molecules, which was carried out by Western blot analyses using a
crude antigenic preparations of M. tuberculosis antigen (whole
bacterial cell extract) as well as culture filtrate(CF) or CF
antigens (supplied by the Dept. Microbiol. Immunol. Pathol.,
Colorado State University, Fort Collins Colo.). Antigens were
electrophoresed under reducing conditions in a 4-20% gradient gel
and transferred to nitrocellulose membrane followed by probing with
either rabbit anti-MTB1694 or anti-MT2462 antisera. Reactivity was
detected with peroxidase labeled goat-anti-rabbit immunoglobulin
and developed using a chemiluminescent reagent (ECL). FIG. 11
indicates that the anti-MTP1694 antiserum recognizes a band of
.about.37 kDa in both the mycobacterial lysate and culture filtrate
preparations (lanes 1 and 2) as well as, as expected, the
recombinant protein (lane 3). In contrast, the anti-MTP2462
antiserum recognizes a band of .about.27 kDa in only in the
bacterial lysate and not in the culture filtrate. The MW bands of
both antigens match their predicted MW. In addition, the MW of the
recombinant molecules are slightly higher than the native
molecules, which is expected because the recombinant molecules
have, in addition to the sequences of the native molecules, a
stretch of 16 amino acids derived from the cloning vector (a tag of
six histidines to facilitate purification and a thrombin site
composed of 10 amino acids to allow cleavage of the his-tag). These
results therefore confirm that both MTP1694 and MTP2462 are genuine
M. tuberculosis proteins that are actively produced during the
bacterial growth. In addition, because the antigen MTP1694 is
present in the culture filtrate preparation ("M. tuberculosis
secrete protein") these results demonstrate this molecule to be
useful as a vaccine.
[0138] To evaluate the use of the identified antigens as vaccine
targets for humans, the response of peripheral blood mononuclear
cells (PBMC) obtained from healthy PPD (an intradermal skin test
response to tuberculosis proteins using a Purified Protein
Derivative) positive donors to these antigens was analyzed. Thus
far, four healthy PPD positive and one PPD negative donors have
been tested with the antigens MTP1694 and MTP2462. The recombinant
protein (MT299) mentioned above, had not been available at the time
that these assays were performed. To test for the antigen
recognition, both antigen induced proliferative response and
production of IFN-.gamma. were used, following stimulation with
recombinant antigens (5 ug/ml). Proliferation was measured by
[3H]TdR incorporation and results are expressed as counts per
minute (CPM). IFN-.gamma. was measured by sandwich ELISA in the
culture supernatants. The results are expressed in FIGS. 12A and B
and indicate that both antigens are readily recognized by PBMC of
three out the four PPD positive donors. No response was observed
with the PBMC obtained from the PPD negative donor. These results
suggest that these antigens will be recognized by a high percentage
of the healthy PPD positive individuals (presumably resistant). In
addition, because the proliferative response was accompanied by the
production of IFN-.gamma., MT1649 and MT2462 is associated with
protection against tuberculosis, therefore these molecules are
vaccine targets.
[0139] The relevant teachings of all the references, patents and/or
patent applications cited herein are incorporated herein by
reference in their entirety.
[0140] The teachings of the following patents are incorporated
herein by reference in their entirety: U.S. Pat. No. 6,627,198,
entitled, "Fusion proteins of Mycobacterium tuberculosis antigens
and their uses;"U.S. Pat. No. 6,613,881, entitled, "Compounds for
immunotherapy and diagnosis of tuberculosis and methods of their
use;" "U.S. Pat. No. 6,592,877, entitled, "Compounds and methods
for immunotherapy and diagnosis of tuberculosis;" "U.S. Pat. No.
6,555,653, entitled, "Compounds for diagnosis of tuberculosis and
methods of their uses;"U.S. Pat. No. 6,544,522, entitled, "Fusion
proteins of Mycobacterium tuberculosis antigens and their uses;
"U.S. Pat. No. 6,458,366, entitled, "Compounds and methods for
diagnosis of tuberculosis;" U.S. Pat. No. 6,338,852 entitled,
"Compounds and methods for diagnosis of tuberculosis;" and "U.S.
Pat. No. 6,290,969, entitled, "Compounds and methods for
immunotherapy and diagnosis of tuberculosis."
[0141] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details can be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
48127DNAMycobacterium tuberculosis 1gacggctatg tcggcgcgcc tgcccac
2729PRTMycobacterium tuberculosis 2Asp Gly Tyr Val Gly Ala Pro Ala
His1 531260DNAMycobacterium tuberculosis 3gtgatcgacg gctggacgga
agaacagcac gaacccaccg ttaggcatga gcgcccagca 60gctccccaag acgttcggcg
ggtgatgttg ctgggttcgg ccgaacccag ccgggagctg 120gcgatcgcgt
tgcagggctt gggcgcggag gtgatcgccg tcgacggcta tgtcggcgcg
180cctgcccacc ggatagccga ccagtcggtg gtggtcacca tgaccgatgc
tgaagagctg 240acggcggtga tccggcggct gcaaccggat ttcttggtga
cggtcaccgc cgcggtgtct 300gtggatgctc tcgatgccgt cgagcaagcc
gacggcgagt gcactgagct ggtgccgaac 360gcccgtgccg tccggtgcac
ggccgaccgg gagggcctgc gccggctggc cgccgatcag 420ctcggcctgc
ccacagcccc gttctggttc gtcggatccc ttggcgaact tcaagcggtg
480gccgtccatg ctgggtttcc gttgctggtg agcccggtgg caggggtggc
tggccagggt 540agctcggtgg tcgccgggcc caacgaggtc gagcccgcct
ggcagcgcgc ggcaggccat 600caagtacagc cgcagactgg gggagtgagc
cctcgggtgt gcgccgagtc ggtggtcgag 660atcgagtttt tggtcaccat
gatcgttgtg tgcagtcagg gcccgaacgg gccgctcatc 720gagttctgtg
cacctatcgg tcatcgcgac gccgatgccg gtgagttgga atcctggcaa
780ccgcagaagc tgagcacggc ggcgctggac gcggccaagt cgatcgccgc
gcgcatcgtc 840aaggcgctcg ggggacgcgg ggttttcggc gtcgaattga
tgatcaacgg cgatgaggtg 900tatttcgccg atgtcaccgt gtgtcctgcc
gggagtgcct gggtcaccgt gcgcagccag 960cggctttcgg tgttcgaact
gcaggcccgg gcgatcctgg gtctggcggt ggacaccctg 1020atgatctcgc
cgggtgccgc gcgggtgatc aacccggacc acacggcagg ccgggcagcg
1080gtcggcgccg caccacctgc cgatgcgctg accggtgcgc tcggtgtgcc
ggaaagcgac 1140gtcgtgatat tcggccgcgg gcttggggtg gcgctggcca
ccgcacccga ggtggcaatc 1200gcccgcgaac gcgcccgcga agttgcatct
cggctaaatg tgccagactc acgcgagtga 12604419PRTMycobacterium
tuberculosis 4Met Ile Asp Gly Trp Thr Glu Glu Gln His Glu Pro Thr
Val Arg His1 5 10 15Glu Arg Pro Ala Ala Pro Gln Asp Val Arg Arg Val
Met Leu Leu Gly 20 25 30Ser Ala Glu Pro Ser Arg Glu Leu Ala Ile Ala
Leu Gln Gly Leu Gly 35 40 45Ala Glu Val Ile Ala Val Asp Gly Tyr Val
Gly Ala Pro Ala His Arg 50 55 60Ile Ala Asp Gln Ser Val Val Val Thr
Met Thr Asp Ala Glu Glu Leu65 70 75 80Thr Ala Val Ile Arg Arg Leu
Gln Pro Asp Phe Leu Val Thr Val Thr 85 90 95Ala Ala Val Ser Val Asp
Ala Leu Asp Ala Val Glu Gln Ala Asp Gly 100 105 110Glu Cys Thr Glu
Leu Val Pro Asn Ala Arg Ala Val Arg Cys Thr Ala 115 120 125Asp Arg
Glu Gly Leu Arg Arg Leu Ala Ala Asp Gln Leu Gly Leu Pro 130 135
140Thr Ala Pro Phe Trp Phe Val Gly Ser Leu Gly Glu Leu Gln Ala
Val145 150 155 160Ala Val His Ala Gly Phe Pro Leu Leu Val Ser Pro
Val Ala Gly Val 165 170 175Ala Gly Gln Gly Ser Ser Val Val Ala Gly
Pro Asn Glu Val Glu Pro 180 185 190Ala Trp Gln Arg Ala Ala Gly His
Gln Val Gln Pro Gln Thr Gly Gly 195 200 205Val Ser Pro Arg Val Cys
Ala Glu Ser Val Val Glu Ile Glu Phe Leu 210 215 220Val Thr Met Ile
Val Val Cys Ser Gln Gly Pro Asn Gly Pro Leu Ile225 230 235 240Glu
Phe Cys Ala Pro Ile Gly His Arg Asp Ala Asp Ala Gly Glu Leu 245 250
255Glu Ser Trp Gln Pro Gln Lys Leu Ser Thr Ala Ala Leu Asp Ala Ala
260 265 270Lys Ser Ile Ala Ala Arg Ile Val Lys Ala Leu Gly Gly Arg
Gly Val 275 280 285Phe Gly Val Glu Leu Met Ile Asn Gly Asp Glu Val
Tyr Phe Ala Asp 290 295 300Val Thr Val Cys Pro Ala Gly Ser Ala Trp
Val Thr Val Arg Ser Gln305 310 315 320Arg Leu Ser Val Phe Glu Leu
Gln Ala Arg Ala Ile Leu Gly Leu Ala 325 330 335Val Asp Thr Leu Met
Ile Ser Pro Gly Ala Ala Arg Val Ile Asn Pro 340 345 350Asp His Thr
Ala Gly Arg Ala Ala Val Gly Ala Ala Pro Pro Ala Asp 355 360 365Ala
Leu Thr Gly Ala Leu Gly Val Pro Glu Ser Asp Val Val Ile Phe 370 375
380Gly Arg Gly Leu Gly Val Ala Leu Ala Thr Ala Pro Glu Val Ala
Ile385 390 395 400Ala Arg Glu Arg Ala Arg Glu Val Ala Ser Arg Leu
Asn Val Pro Asp 405 410 415Ser Arg Glu539DNAMycobacterium
tuberculosis 5cttgcggcgg tggtgggcgt cgtcctggcg caggtgttg
39613PRTMycobacterium tuberculosis 6Leu Ala Ala Val Val Gly Val Val
Leu Ala Gln Val Leu1 5 1071050DNAMycobacterium tuberculosis
7atgctcttcg cggccctgcg tgacatgcaa tggagaaagc gccgcctggt catcacgatc
60atcagcaccg ggctgatctt cgggatgacg cttgttttga ccggactcgc gaacggcttc
120cgggtggagg cccggcacac cgtcgattcc atgggtgtcg atgtattcgt
cgtcagatcc 180ggcgctgctg gaccttttct gggttcaata ccgtttcccg
atgttgacct ggcccgagtg 240gccgctgaac ccggtgtcat ggccgcggcc
ccgttgggca gcgtggggac gatcatgaaa 300gaaggcacgt cgacgcgaaa
cgtcacggtc ttcggcgcgc ccgagcacgg acctggcatg 360ccacgggtct
cagagggtcg gtcaccgtcg aaaccggacg aagtcgcggc atcgagcacg
420atgggccgac acctcggtga cactgtcgag gtcggcgcgc gcagattgcg
ggtcgttggc 480attgtgccga attccaccgc gctggccaag atccccaatg
tcttcctcac gaccgagggc 540ttacagaaat tggcgtacaa cgggcagccg
aatatcacgt ccatcgggat cataggtatg 600ccccgacagc tgccggaggg
ttaccagact ttcgatcggg tgggcgctgt caatgatttg 660gtgcgcccat
tgaaggtcgc agtgaattcg atctcgatcg tggctgtttt gctgtggatt
720gtggcggtgc tgatcgtcgg ctcggtggtg tacctttcgg ctcttgagcg
gctacgtgac 780ttcgcggtgt tcaaggcgat tggcacgcca acgcgctcga
ttatggccgg gctcgcatta 840caggcgctgg tcattgcgtt gcttgcggcg
gtggtgggcg tcgtcctggc gcaggtgttg 900gcaccactgt ttccgatgat
tgtcgcggta cccgtcggtg cttacctggc gctaccggtg 960gccgcgatcg
tcatcggtct gttcgctagt gttgccggat tgaagcgcgt ggtgacggtc
1020gatcccgcgc aggcgttcgg aggtccctag 10508349PRTMycobacterium
tuberculosis 8Met Leu Phe Ala Ala Leu Arg Asp Met Gln Trp Arg Lys
Arg Arg Leu1 5 10 15Val Ile Thr Ile Ile Ser Thr Gly Leu Ile Phe Gly
Met Thr Leu Val 20 25 30Leu Thr Gly Leu Ala Asn Gly Phe Arg Val Glu
Ala Arg His Thr Val 35 40 45Asp Ser Met Gly Val Asp Val Phe Val Val
Arg Ser Gly Ala Ala Gly 50 55 60Pro Phe Leu Gly Ser Ile Pro Phe Pro
Asp Val Asp Leu Ala Arg Val65 70 75 80Ala Ala Glu Pro Gly Val Met
Ala Ala Ala Pro Leu Gly Ser Val Gly 85 90 95Thr Ile Met Lys Glu Gly
Thr Ser Thr Arg Asn Val Thr Val Phe Gly 100 105 110Ala Pro Glu His
Gly Pro Gly Met Pro Arg Val Ser Glu Gly Arg Ser 115 120 125Pro Ser
Lys Pro Asp Glu Val Ala Ala Ser Ser Thr Met Gly Arg His 130 135
140Leu Gly Asp Thr Val Glu Val Gly Ala Arg Arg Leu Arg Val Val
Gly145 150 155 160Ile Val Pro Asn Ser Thr Ala Leu Ala Lys Ile Pro
Asn Val Phe Leu 165 170 175Thr Thr Glu Gly Leu Gln Lys Leu Ala Tyr
Asn Gly Gln Pro Asn Ile 180 185 190Thr Ser Ile Gly Ile Ile Gly Met
Pro Arg Gln Leu Pro Glu Gly Tyr 195 200 205Gln Thr Phe Asp Arg Val
Gly Ala Val Asn Asp Leu Val Arg Pro Leu 210 215 220Lys Val Ala Val
Asn Ser Ile Ser Ile Val Ala Val Leu Leu Trp Ile225 230 235 240Val
Ala Val Leu Ile Val Gly Ser Val Val Tyr Leu Ser Ala Leu Glu 245 250
255Arg Leu Arg Asp Phe Ala Val Phe Lys Ala Ile Gly Thr Pro Thr Arg
260 265 270Ser Ile Met Ala Gly Leu Ala Leu Gln Ala Leu Val Ile Ala
Leu Leu 275 280 285Ala Ala Val Val Gly Val Val Leu Ala Gln Val Leu
Ala Pro Leu Phe 290 295 300Pro Met Ile Val Ala Val Pro Val Gly Ala
Tyr Leu Ala Leu Pro Val305 310 315 320Ala Ala Ile Val Ile Gly Leu
Phe Ala Ser Val Ala Gly Leu Lys Arg 325 330 335Val Val Thr Val Asp
Pro Ala Gln Ala Phe Gly Gly Pro 340 345927DNAMycobacterium
tuberculosis 9cgctccggcg cggccacacc cgttcgc 27109PRTMycobacterium
tuberculosis 10Arg Ser Gly Ala Ala Thr Pro Val Arg1
511171DNAMycobacterium tuberculosis 11gtgccggccg ctccgtcgac
aagagagaag gactgcatgc tggttttgca cggcttctgg 60tccaactccg gcgggatgcg
gctgtgggcg gaggactccg atctgctggt gaagagcccg 120agtcaggcgc
tgcgctccgg cgcggccaca cccgttcgcg gcgcccgctg a
1711256PRTMycobacterium tuberculosis 12Met Pro Ala Ala Pro Ser Thr
Arg Glu Lys Asp Cys Met Leu Val Leu1 5 10 15His Gly Phe Trp Ser Asn
Ser Gly Gly Met Arg Leu Trp Ala Glu Asp 20 25 30Ser Asp Leu Leu Val
Lys Ser Pro Ser Gln Ala Leu Arg Ser Gly Ala 35 40 45Ala Thr Pro Val
Arg Gly Ala Arg 50 551321DNAMycobacterium tuberculosis 13acctgcctcc
cattcgcact a 21147PRTMycobacterium tuberculosis 14Thr Cys Leu Pro
Phe Ala Leu1 515267DNAMycobacterium tuberculosis 15atgtccgcgc
cacccgctca agcgccggta tgtggcgcct tggcggctag gccaaccgcc 60cccggcaacg
ccagctgcac acgcccagcg aagcgcgatt gtcggtacgg gtcgcgctgc
120gaaacctgcc tcccattcgc actagcaaaa gactgtcgac aagcgagcag
tcgacttcag 180gccgcgaccg aacccgacga gacgacaaca acatctgtca
tctcaatgcg ctcaccagga 240tcgctacaat atcagccagc tacatga
2671688PRTMycobacterium tuberculosis 16Met Ser Ala Pro Pro Ala Gln
Ala Pro Val Cys Gly Ala Leu Ala Ala1 5 10 15Arg Pro Thr Ala Pro Gly
Asn Ala Ser Cys Thr Arg Pro Ala Lys Arg 20 25 30Asp Cys Arg Tyr Gly
Ser Arg Cys Glu Thr Cys Leu Pro Phe Ala Leu 35 40 45Ala Lys Asp Cys
Arg Gln Ala Ser Ser Arg Leu Gln Ala Ala Thr Glu 50 55 60Pro Asp Glu
Thr Thr Thr Thr Ser Val Ile Ser Met Arg Ser Pro Gly65 70 75 80Ser
Leu Gln Tyr Gln Pro Ala Thr 851724DNAMycobacterium tuberculosis
17acgaccatgc cgctgttcgc cgac 24188PRTMycobacterium tuberculosis
18Thr Thr Met Pro Leu Phe Ala Asp1 519492DNAMycobacterium
tuberculosis 19gtgccagagg tcacccgtga agaaccggca atcgatggat
ggttcaccac cgataaggcc 60ggcaacccgc atctgctcgg cggcaagtgt ccccagtgcg
gcacgtacgt cttcccaccc 120cgggcggaca attgtccgaa tccggcttgc
ggcagcgaca cactagagtc ggtcggactg 180tcgacccgcg gaaagctttg
gagctacacc gaaaaccggt acgccccgcc accgccgtac 240ccggcacccg
acccctttga gccgtttgcc gtggccgcgg tggaactggc cgacgaggga
300ctgatcgtgc tgggcaaagt ggtcgatggc acgctggccg ccgatctgaa
ggtcggcatg 360gagatggagc tgacgaccat gccgctgttc gccgacgacg
acggtgtgca gcgcatcgtc 420tacgcgtggc ggatcccatc gcgcgccggc
gacgatgcag agcgcagcga tgctgaggag 480cggcgccgat ga
49220163PRTMycobacterium tuberculosis 20Met Pro Glu Val Thr Arg Glu
Glu Pro Ala Ile Asp Gly Trp Phe Thr1 5 10 15Thr Asp Lys Ala Gly Asn
Pro His Leu Leu Gly Gly Lys Cys Pro Gln 20 25 30Cys Gly Thr Tyr Val
Phe Pro Pro Arg Ala Asp Asn Cys Pro Asn Pro 35 40 45Ala Cys Gly Ser
Asp Thr Leu Glu Ser Val Gly Leu Ser Thr Arg Gly 50 55 60Lys Leu Trp
Ser Tyr Thr Glu Asn Arg Tyr Ala Pro Pro Pro Pro Tyr65 70 75 80Pro
Ala Pro Asp Pro Phe Glu Pro Phe Ala Val Ala Ala Val Glu Leu 85 90
95Ala Asp Glu Gly Leu Ile Val Leu Gly Lys Val Val Asp Gly Thr Leu
100 105 110Ala Ala Asp Leu Lys Val Gly Met Glu Met Glu Leu Thr Thr
Met Pro 115 120 125Leu Phe Ala Asp Asp Asp Gly Val Gln Arg Ile Val
Tyr Ala Trp Arg 130 135 140Ile Pro Ser Arg Ala Gly Asp Asp Ala Glu
Arg Ser Asp Ala Glu Glu145 150 155 160Arg Arg
Arg21140DNAMycobacterium tuberculosis 21gtgctggtcg cactggccgc
gctgggcacc caaccgtggc aggacttcgc agagcaggaa 60accgccgggc tggccatcat
cttggacaac gtcacgcatg gcgaatgggc cagcacgatt 120ctggccgcgg
tgcggtggtc 1402247PRTMycobacterium tuberculosis 22Val Leu Val Ala
Leu Ala Ala Leu Gly Thr Gln Pro Trp Gln Asp Phe1 5 10 15Ala Glu Gln
Glu Thr Ala Gly Leu Ala Ile Ile Leu Asp Asn Val Thr 20 25 30His Gly
Glu Trp Ala Ser Thr Ile Leu Ala Ala Gly Ala Val Val 35 40
45231430DNAMycobacterium tuberculosis 23ttgccgacaa cgtcgatgag
ccttcgagaa ctgatgctgc ggcgccgccc ggtgagcggc 60gccccggtcg catccggggc
atcggggaac ctcaagcgga gtttcggcac cttccagctg 120accatgttcg
gggttggcgc gacgataggt accggcatct ttttcgtgct tgcccaggca
180gttccagagg ccggcccggg cgtgattgtt tcgttcatca tcgccggcat
cgccgctggg 240ctcgcggcta tctgctacgc ggaactggct tccgccgtgc
cgatttccgg gtcggcgtac 300tcctacgcgt acacgacgct gggcgaggcg
gtcgcgatgg tggtggcggc ctgcctactg 360ctggaatacg gggtagccac
cgcagcggtc gcggtcggct ggagtggcta cgtgaacaag 420ctgctgagta
atctgttcgg atttcagatg ccgcacgtat tgtcggcggc gccgtgggac
480acccatcccg gttgggtgaa cctgcccgcc gtcatcctga tcgggctatg
cgcgctgctg 540ttgattcgag gggccagcga gtcggcgagg gtcaacgcga
tcatggtgct gatcaagctc 600ggcgtgctgg gcatgttcat gatcatcgcg
ttcagcgcgt acagcgccga ccacctcaag 660gatttcgtcc cattcggcgt
cgccggcatc ggctccgcgg cgggcacgat cttcttctca 720tacatcggcc
ttgacgcggt gtcgaccgcc ggcgacgagg tgaaggaccc gcagaagacc
780atgccgcgtg cgctgatcgc agcgctggtg gtcgtcaccg gtgtctacgt
gctggtcgca 840ctggccgcgc tgggcaccca accgtggcag gacttcgcag
agcaggaaac cgccgggctg 900gccatcatct tggacaacgt cacgcatggc
gaatgggcca gcacgattct ggccgccggt 960gcggtggctc gattttcacc
gtcacgctgg tcaccatgta cggccagacc cggatcctgt 1020tcgcgatggg
gcgcgacggg ctgctgccgg cgcggttcgc gaaggtgaat ccgcgcacca
1080tgacgccggt gcacaacacg gtgatcgtcg cgatcttcgc atcgacgctg
gccgccttca 1140taccgctgga tagcttggcg gacatggtgt ccatcggcac
gctcaccgcg ttcagcgtgg 1200tggctgtggg tgtgatcgtt ctacgggtgc
gcgagcccga cttaccccga gggttcaagg 1260tacccggtta ccctgtgacg
cctgttcttt cggtgctggc ctgcgggtat atcctggcca 1320gcttgcactg
gtacacctgg ctggcgttca gcggatgggt ggcggtggca gtgatctttt
1380acctgatgtg gggtcggcac cacagtgcgc tcaacgagga agtgccgtga
143024475PRTMycobacterium tuberculosis 24Met Pro Thr Thr Ser Met
Ser Leu Arg Glu Leu Met Leu Arg Arg Arg1 5 10 15Pro Val Ser Gly Ala
Pro Val Ala Ser Gly Ala Ser Gly Asn Leu Lys 20 25 30Arg Ser Phe Gly
Thr Phe Gln Leu Thr Met Phe Gly Val Gly Ala Thr 35 40 45Ile Gly Thr
Gly Ile Phe Phe Val Leu Ala Gln Ala Val Pro Glu Ala 50 55 60Gly Pro
Gly Val Ile Val Ser Phe Ile Ile Ala Gly Ile Ala Ala Gly65 70 75
80Leu Ala Ala Ile Cys Tyr Ala Glu Leu Ala Ser Ala Val Pro Ile Ser
85 90 95Gly Ser Ala Tyr Ser Tyr Ala Tyr Thr Thr Leu Gly Glu Ala Val
Ala 100 105 110Met Val Val Ala Ala Cys Leu Leu Leu Glu Tyr Gly Val
Ala Thr Ala 115 120 125Ala Val Ala Val Gly Trp Ser Gly Tyr Val Asn
Lys Leu Leu Ser Asn 130 135 140Leu Phe Gly Phe Gln Met Pro His Val
Leu Ser Ala Ala Pro Trp Asp145 150 155 160Thr His Pro Gly Trp Val
Asn Leu Pro Ala Val Ile Leu Ile Gly Leu 165 170 175Cys Ala Leu Leu
Leu Ile Arg Gly Ala Ser Glu Ser Ala Arg Val Asn 180 185 190Ala Ile
Met Val Leu Ile Lys Leu Gly Val Leu Gly Met Phe Met Ile 195 200
205Ile Ala Phe Ser Ala Tyr Ser Ala Asp His Leu Lys Asp Phe Val Pro
210 215 220Phe Gly Val Ala Gly Ile Gly Ser Ala Ala Gly Thr Ile Phe
Phe Ser225 230 235 240Tyr Ile Gly Leu Asp Ala Val Ser Thr Ala Gly
Asp Glu Val Lys Asp 245 250 255Pro Gln Lys Thr Met Pro Arg Ala Leu
Ile Ala Ala Leu Val Val Val 260 265 270Thr Gly Val Tyr Val Leu Val
Ala Leu Ala Ala Leu Gly Thr Gln Pro 275 280 285Trp Gln Asp Phe Ala
Glu Gln Glu Thr Ala Gly Leu Ala Ile Ile Leu 290 295 300Asp Asn Val
Thr His Gly Glu Trp Ala Ser Thr Ile Leu Ala Ala Gly305 310 315
320Ala Val Val Ser Ile Phe Thr Val Thr Leu Val Thr Met Tyr Gly
Gln
325 330 335Thr Arg Ile Leu Phe Ala Met Gly Arg Asp Gly Leu Leu Pro
Ala Arg 340 345 350Phe Ala Lys Val Asn Pro Arg Thr Met Thr Pro Val
His Asn Thr Ile 355 360 365Val Ala Ile Phe Ala Ser Thr Leu Ala Ala
Phe Ile Pro Leu Asp Ser 370 375 380Leu Ala Asp Met Val Ser Ile Gly
Thr Leu Thr Ala Phe Ser Val Val385 390 395 400Ala Val Gly Val Ile
Val Leu Arg Val Arg Glu Pro Asp Leu Pro Arg 405 410 415Gly Phe Lys
Val Pro Gly Tyr Pro Val Thr Pro Val Leu Ser Val Leu 420 425 430Ala
Cys Gly Tyr Ile Leu Ala Ser Leu His Trp Tyr Thr Trp Leu Ala 435 440
445Phe Ser Gly Trp Val Ala Val Ala Val Ile Phe Tyr Leu Met Trp Gly
450 455 460Arg His His Ser Ala Leu Asn Glu Glu Val Pro465 470
4752554DNAMycobacterium tuberculosis 25tgcgcggtgg tgctggccac
catgccgccg ctgctttcgg cgatcgccaa cgca 542618PRTMycobacterium
tuberculosis 26Cys Ala Val Val Leu Ala Thr Met Pro Pro Leu Leu Ser
Ala Ile Ala1 5 10 15Asn Ala271974DNAMycobacterium tuberculosis
27atgaccttga ccgcttgtga agtaactgcc gcggaggctc ctttcgaccg cgtttcaaag
60accattcccc acccattgag ctggggagcc gcgctgtggt cggtagtctc cgtgcgctgg
120gccaccgtgg cgctgctgct gtttctcgcc ggactagtgg cgcaactgaa
cggtgctccc 180gaggccatgt ggtggacgct ttacctggcc tgttatctgg
ccggcggctg gggctcggca 240tgggcgggcg cacaagcgtt gcggaacaag
gcacttgatg tggatctgct gatgattgcc 300gcggcggtcg gagcggtcgc
gattgggcag atcttcgacg gcgcgctgct gatcgtgatc 360ttcgccacgt
ccggtgcgct ggatgacatt gccaccagac acaccgcgga atcggtcaaa
420ggcctgctgg acctcgcgcc ggatcaggcg gtggtggtcc agggcgacgg
cagcgaacgg 480gtggtggcgg ccagcgagct ggtggtgggg gaccgggtgg
tggtgcggcc gggggaccgg 540atacccgcag acggtgcggt gctgtcgggg
gctagcgacg tcgaccaacg ctcgatcacc 600ggtgaatcga tgccggtggc
caaggcccgc ggtgacgagg tgttcgccgg caccgtgaac 660ggatcgggtg
tattgcatct ggtggtcacc cgtgacccga gccagaccgt ggtagcccgc
720atcgtcgaac tggtcgccga cgcttcggcg acgaaggcca aaacccaact
gttcattgag 780aaaatcgagc aacgctactc cctgggcatg gtcgcggcca
cccttgccct catcgttatt 840ccgctgatgt tcggcgccga cctgcggccg
gtgctgctgc gcgccatgac cttcatgatc 900gtggcatcgc catgcgcggt
ggtgctggcc accatgccgc cgctgctttc ggcgatcgcc 960aacgcaggcc
gtcatggggt gctggtcaaa tccgcggtgg tcgtcgaacg cctggccgat
1020accagcatcg tcgctttgga caagaccggt acgctgaccc gtggcatccc
gcgactggct 1080tccgtcgcac cgctggaccc caacgtggtc gatgcccggc
gattgttgca attggcagct 1140gccgcagaac aatccagcga gcacccgctt
ggccgggcga tcgtcgcgga agctcgtcgg 1200cgtggtatcg ccataccgcc
cgccaaggac ttccgcgcgg tcccgggctg cggggtccac 1260gccctggtgg
gcaacgattt cgtcgagatc gccagcccgc aaagctaccg cggtgcaccg
1320ctagcagagc tggcgccgct cctttctgcc ggcgccactg ccgccatcgt
cttgttggat 1380ggagttgcca tcggtgtgct cgggctcacc gatcagcttc
gtccggatgc cgtggagtcc 1440gtcgcggcga tggctgcatt gaccgccgca
ccaccggtgc tgctcacggg tgacaacggg 1500cgagcggctt ggcgggtcgc
tcggaacgcc gggatcaccg atgtgcgagc cgcattgctg 1560cccgagcaga
aggttgaagt cgtgcgcaac ctgcaggccg gtggtcacca ggtgctgctc
1620gtcggcgacg gcgtcaacga cgctcccgcc atggccgccg cccgcgccgc
tgtcgccatg 1680ggcgccggcg ccgatctgac cctacagacc gcagacgggg
tgaccatacg ggacgaactg 1740cacaccatcc cgacgatcat cgggttggca
cggcaggcgc gccgggtggt caccgtcaac 1800ctggccatcg cggccacctt
catcgccgtc ctggtgctgt gggacctttt tgggcagctg 1860ccgctgccac
tgggtgtggt gggtcacgaa gggtccactg tgctggtggc cctcaacggc
1920atgcggctat tgaccaaccg gtcgtggcgg gccgcggctt cggctgcgcg ttag
197428657PRTMycobacterium tuberculosis 28Met Thr Leu Thr Ala Cys
Glu Val Thr Ala Ala Glu Ala Pro Phe Asp1 5 10 15Arg Val Ser Lys Thr
Ile Pro His Pro Leu Ser Trp Gly Ala Ala Leu 20 25 30Trp Ser Val Val
Ser Val Arg Trp Ala Thr Val Ala Leu Leu Leu Phe 35 40 45Leu Ala Gly
Leu Val Ala Gln Leu Asn Gly Ala Pro Glu Ala Met Trp 50 55 60Trp Thr
Leu Tyr Leu Ala Cys Tyr Leu Ala Gly Gly Trp Gly Ser Ala65 70 75
80Trp Ala Gly Ala Gln Ala Leu Arg Asn Lys Ala Leu Asp Val Asp Leu
85 90 95Leu Met Ile Ala Ala Ala Val Gly Ala Val Ala Ile Gly Gln Ile
Phe 100 105 110Asp Gly Ala Leu Leu Ile Val Ile Phe Ala Thr Ser Gly
Ala Leu Asp 115 120 125Asp Ile Ala Thr Arg His Thr Ala Glu Ser Val
Lys Gly Leu Leu Asp 130 135 140Leu Ala Pro Asp Gln Ala Val Val Val
Gln Gly Asp Gly Ser Glu Arg145 150 155 160Val Val Ala Ala Ser Glu
Leu Val Val Gly Asp Arg Val Val Val Arg 165 170 175Pro Gly Asp Arg
Ile Pro Ala Asp Gly Ala Val Leu Ser Gly Ala Ser 180 185 190Asp Val
Asp Gln Arg Ser Ile Thr Gly Glu Ser Met Pro Val Ala Lys 195 200
205Ala Arg Gly Asp Glu Val Phe Ala Gly Thr Val Asn Gly Ser Gly Val
210 215 220Leu His Leu Val Val Thr Arg Asp Pro Ser Gln Thr Val Val
Ala Arg225 230 235 240Ile Val Glu Leu Val Ala Asp Ala Ser Ala Thr
Lys Ala Lys Thr Gln 245 250 255Leu Phe Ile Glu Lys Ile Glu Gln Arg
Tyr Ser Leu Gly Met Val Ala 260 265 270Ala Thr Leu Ala Leu Ile Val
Ile Pro Leu Met Phe Gly Ala Asp Leu 275 280 285Arg Pro Val Leu Leu
Arg Ala Met Thr Phe Met Ile Val Ala Ser Pro 290 295 300Cys Ala Val
Val Leu Ala Thr Met Pro Pro Leu Leu Ser Ala Ile Ala305 310 315
320Asn Ala Gly Arg His Gly Val Leu Val Lys Ser Ala Val Val Val Glu
325 330 335Arg Leu Ala Asp Thr Ser Ile Val Ala Leu Asp Lys Thr Gly
Thr Leu 340 345 350Thr Arg Gly Ile Pro Arg Leu Ala Ser Val Ala Pro
Leu Asp Pro Asn 355 360 365Val Val Asp Ala Arg Arg Leu Leu Gln Leu
Ala Ala Ala Ala Glu Gln 370 375 380Ser Ser Glu His Pro Leu Gly Arg
Ala Ile Val Ala Glu Ala Arg Arg385 390 395 400Arg Gly Ile Ala Ile
Pro Pro Ala Lys Asp Phe Arg Ala Val Pro Gly 405 410 415Cys Gly Val
His Ala Leu Val Gly Asn Asp Phe Val Glu Ile Ala Ser 420 425 430Pro
Gln Ser Tyr Arg Gly Ala Pro Leu Ala Glu Leu Ala Pro Leu Leu 435 440
445Ser Ala Gly Ala Thr Ala Ala Ile Val Leu Leu Asp Gly Val Ala Ile
450 455 460Gly Val Leu Gly Leu Thr Asp Gln Leu Arg Pro Asp Ala Val
Glu Ser465 470 475 480Val Ala Ala Met Ala Ala Leu Thr Ala Ala Pro
Pro Val Leu Leu Thr 485 490 495Gly Asp Asn Gly Arg Ala Ala Trp Arg
Val Ala Arg Asn Ala Gly Ile 500 505 510Thr Asp Val Arg Ala Ala Leu
Leu Pro Glu Gln Lys Val Glu Val Val 515 520 525Arg Asn Leu Gln Ala
Gly Gly His Gln Val Leu Leu Val Gly Asp Gly 530 535 540Val Asn Asp
Ala Pro Ala Met Ala Ala Ala Arg Ala Ala Val Ala Met545 550 555
560Gly Ala Gly Ala Asp Leu Thr Leu Gln Thr Ala Asp Gly Val Thr Ile
565 570 575Arg Asp Glu Leu His Thr Ile Pro Thr Ile Ile Gly Leu Ala
Arg Gln 580 585 590Ala Arg Arg Val Val Thr Val Asn Leu Ala Ile Ala
Ala Thr Phe Ile 595 600 605Ala Val Leu Val Leu Trp Asp Leu Phe Gly
Gln Leu Pro Leu Pro Leu 610 615 620Gly Val Val Gly His Glu Gly Ser
Thr Val Leu Val Ala Leu Asn Gly625 630 635 640Met Arg Leu Leu Thr
Asn Arg Ser Trp Arg Ala Ala Ala Ser Ala Ala 645 650
655Arg2927DNAMycobacterium tuberculosis 29atggtgatca tagagctgat
gcgccgg 27309PRTMycobacterium tuberculosis 30Met Val Ile Ile Glu
Leu Met Arg Arg1 531996DNAMycobacterium tuberculosis 31atggtgatca
tagagctgat gcgccgggtg gtaggtctcg cacagggagc taccgccgag 60gtcgccgtct
atggcgaccg agatcgtgat ctcgcggagc gatggtgcgc gaacaccgga
120aacaccctgg tgcgcgccga cgtggaccag accggcgtcg gcaccctggt
ggtgcgccgc 180ggccatccgc ctgacccggc aagcgtgttg ggccccgacc
ggctacccgg ggtccggttg 240tggctgtaca ccaacttcca ctgcaacctg
tgctgcgact actgctgcgt ctcgtcgtca 300ccaagcaccc cgcatcgcga
actgggggcg gagcggatcg gccgaatcgt cggtgaagcg 360gcgcgctggg
gagtgcgcga actgttcctc accggcggtg agccgttcct gctgcccgac
420atcgacacga tcatcgcgac ctgtgtgaag cagttgccca ccaccgtcct
caccaacggc 480atggtgttca aagggcgggg tcggcgcgcg ctggaatccc
tacctagagg gctcgccttg 540cagatcagcc tggactcggc caccccggag
ctgcacgatg cgcaccgcgg cgcggggacg 600tgggtcaagg cagtagctgg
tatccggttg gcgctctcac ttggcttccg ggtgcgggtg 660gccgcgacgg
ttgccagccc cgcacctggc gagctgacgg cgtttcacga cttcctcgac
720gggcttggca tcgcacccgg ggatcagctg gtccggccga tcgcgctgga
gggcgccgcg 780tcgcaagggg tggcgctcac ccgcgaatcg ctggttcccg
aggtgaccgt caccgccgac 840ggcgtgtact ggcacccagt ggccgccacc
gacgagcgcg ccctggtcac ccgtaccgtc 900gaacccttga ccccggcgct
ggacatggta agccggctat tcgccgaaca gtggacacga 960gccgccgaag
aggccgcgtt gttcccgtgt gcgtag 99632331PRTMycobacterium tuberculosis
32Met Val Ile Ile Glu Leu Met Arg Arg Val Val Gly Leu Ala Gln Gly1
5 10 15Ala Thr Ala Glu Val Ala Val Tyr Gly Asp Arg Asp Arg Asp Leu
Ala 20 25 30Glu Arg Trp Cys Ala Asn Thr Gly Asn Thr Leu Val Arg Ala
Asp Val 35 40 45Asp Gln Thr Gly Val Gly Thr Leu Val Val Arg Arg Gly
His Pro Pro 50 55 60Asp Pro Ala Ser Val Leu Gly Pro Asp Arg Leu Pro
Gly Val Arg Leu65 70 75 80Trp Leu Tyr Thr Asn Phe His Cys Asn Leu
Cys Cys Asp Tyr Cys Cys 85 90 95Val Ser Ser Ser Pro Ser Thr Pro His
Arg Glu Leu Gly Ala Glu Arg 100 105 110Ile Gly Arg Ile Val Gly Glu
Ala Ala Arg Trp Gly Val Arg Glu Leu 115 120 125Phe Leu Thr Gly Gly
Glu Pro Phe Leu Leu Pro Asp Ile Asp Thr Ile 130 135 140Ile Ala Thr
Cys Val Lys Gln Leu Pro Thr Thr Val Leu Thr Asn Gly145 150 155
160Met Val Phe Lys Gly Arg Gly Arg Arg Ala Leu Glu Ser Leu Pro Arg
165 170 175Gly Leu Ala Leu Gln Ile Ser Leu Asp Ser Ala Thr Pro Glu
Leu His 180 185 190Asp Ala His Arg Gly Ala Gly Thr Trp Val Lys Ala
Val Ala Gly Ile 195 200 205Arg Leu Ala Leu Ser Leu Gly Phe Arg Val
Arg Val Ala Ala Thr Val 210 215 220Ala Ser Pro Ala Pro Gly Glu Leu
Thr Ala Phe His Asp Phe Leu Asp225 230 235 240Gly Leu Gly Ile Ala
Pro Gly Asp Gln Leu Val Arg Pro Ile Ala Leu 245 250 255Glu Gly Ala
Ala Ser Gln Gly Val Ala Leu Thr Arg Glu Ser Leu Val 260 265 270Pro
Glu Val Thr Val Thr Ala Asp Gly Val Tyr Trp His Pro Val Ala 275 280
285Ala Thr Asp Glu Arg Ala Leu Val Thr Arg Thr Val Glu Pro Leu Thr
290 295 300Pro Ala Leu Asp Met Val Ser Arg Leu Phe Ala Glu Gln Trp
Thr Arg305 310 315 320Ala Ala Glu Glu Ala Ala Leu Phe Pro Cys Ala
325 3303357DNAMycobacterium tuberculosis 33cgtgccaccg cagaccagat
cggcacgcag acaacgcaaa tcgcggccat caaagcc 573419PRTMycobacterium
tuberculosis 34Arg Ala Thr Ala Asp Gln Ile Gly Thr Gln Thr Thr Gln
Ile Ala Ala1 5 10 15Ile Lys Ala351140DNAMycobacterium tuberculosis
35atgacgatct ccgatgtacc cacccagacg ctgcccgccg aaggcgaaat cggcctgata
60gacgtcggct cgctgcaact ggaaagcggg gcggtgatcg acgatgtctg tatcgccgtg
120caacgctggg gcaaattgtc gcccgcacgg gacaacgtgg tggtggtctt
gcacgcgctc 180accggcgact cgcacatcac tggacccgcc ggacccggcc
accccacccc cggctggtgg 240gacggggtgg ccgggccgag tgcgccgatt
gacaccaccc gctggtgcgc ggtagctacc 300aatgtgctcg gcggctgccg
cggctccacc gggcccagct cgcttgcccg cgacggaaag 360ccttggggct
caagatttcc gctgatctcg atacgtgacc aggtgcaggc ggacgtcgcg
420gcgctggccg cgctgggcat caccgaggtc gccgccgtcg tcggcggctc
catgggcggc 480gcccgggccc tggaatgggt ggtcggctac ccggatcggg
tccgagccgg attgctgctg 540gcggcggtcg gtcgtgccac cgcagaccag
atcggcacgc agacaacgca aatcgcggcc 600atcaaagccg acccggactg
gcagagcggc gactaccacg agacggggag ggcaccagac 660gccgggctgc
gactcgcccg ccgcttcgcg cacctcacct accgcggcga gatcgagctc
720gacacccggt tcgccaacca caaccagggc aacgaggatc cgacggccgg
cgggcgctac 780gcggtgcaaa gttatctgga acaccaagga gacaaactgt
tatcccggtt cgacgccggc 840agctacgtga ttctcaccga ggcgctcaac
agccacgacg tcggccgcgg ccgcggcggg 900gtctccgcgg ctctgcgcgc
ctgcccggtg ccggtggtgg tgggcggcat cacctccgac 960cggctctacc
cgctgcgcct gcagcaggag ctggccgacc tgctgccggg ctgcgccggg
1020ctgcgagtcg tcgagtcggt ctacggacac gacggcttcc tggtggaaac
cgaggccgtg 1080ggcgaattga tccgccagac actgggattg gctgatcgtg
aaggcgcgtg tcggcggtga 114036379PRTMycobacterium tuberculosis 36Met
Thr Ile Ser Asp Val Pro Thr Gln Thr Leu Pro Ala Glu Gly Glu1 5 10
15Ile Gly Leu Ile Asp Val Gly Ser Leu Gln Leu Glu Ser Gly Ala Val
20 25 30Ile Asp Asp Val Cys Ile Ala Val Gln Arg Trp Gly Lys Leu Ser
Pro 35 40 45Ala Arg Asp Asn Val Val Val Val Leu His Ala Leu Thr Gly
Asp Ser 50 55 60His Ile Thr Gly Pro Ala Gly Pro Gly His Pro Thr Pro
Gly Trp Trp65 70 75 80Asp Gly Val Ala Gly Pro Ser Ala Pro Ile Asp
Thr Thr Arg Trp Cys 85 90 95Ala Val Ala Thr Asn Val Leu Gly Gly Cys
Arg Gly Ser Thr Gly Pro 100 105 110Ser Ser Leu Ala Arg Asp Gly Lys
Pro Trp Gly Ser Arg Phe Pro Leu 115 120 125Ile Ser Ile Arg Asp Gln
Val Gln Ala Asp Val Ala Ala Leu Ala Ala 130 135 140Leu Gly Ile Thr
Glu Val Ala Ala Val Val Gly Gly Ser Met Gly Gly145 150 155 160Ala
Arg Ala Leu Glu Trp Val Val Gly Tyr Pro Asp Arg Val Arg Ala 165 170
175Gly Leu Leu Leu Ala Val Gly Ala Arg Ala Thr Ala Asp Gln Ile Gly
180 185 190Thr Gln Thr Thr Gln Ile Ala Ala Ile Lys Ala Asp Pro Asp
Trp Gln 195 200 205Ser Gly Asp Tyr His Glu Thr Gly Arg Ala Pro Asp
Ala Gly Leu Arg 210 215 220Leu Ala Arg Arg Phe Ala His Leu Thr Tyr
Arg Gly Glu Ile Glu Leu225 230 235 240Asp Thr Arg Phe Ala Asn His
Asn Gln Gly Asn Glu Asp Pro Thr Ala 245 250 255Gly Gly Arg Tyr Ala
Val Gln Ser Tyr Leu Glu His Gln Gly Asp Lys 260 265 270Leu Leu Ser
Arg Phe Asp Ala Gly Ser Tyr Val Ile Leu Thr Glu Ala 275 280 285Leu
Asn Ser His Asp Val Gly Arg Gly Arg Gly Gly Val Ser Ala Ala 290 295
300Leu Arg Ala Cys Pro Val Pro Val Val Val Gly Gly Ile Thr Ser
Asp305 310 315 320Arg Leu Tyr Pro Leu Arg Leu Gln Gln Glu Leu Ala
Asp Leu Leu Pro 325 330 335Gly Cys Ala Gly Leu Arg Val Val Glu Ser
Val Tyr Gly His Asp Gly 340 345 350Phe Leu Val Glu Thr Glu Ala Val
Gly Glu Leu Ile Arg Gln Thr Leu 355 360 365Gly Leu Ala Asp Arg Glu
Gly Ala Cys Arg Arg 370 3753757DNAMycobacterium tuberculosis
37cgcaccgccg aggaacgcgc caacgcggtt cgcgggcggg ccgattcgct gcgccgt
573819PRTMycobacterium tuberculosis 38Arg Thr Ala Glu Glu Arg Ala
Asn Ala Val Arg Gly Arg Ala Asp Ser1 5 10 15Leu Arg
Arg393618DNAMycobacterium tuberculosis 39gtgtacctca agagtctgac
gttgaagggc ttcaagtcct tcgccgcgcc gacgacttta 60cgcttcgagc cgggcattac
ggccgtcgtt gggcccaacg gctccggcaa atccaatgtg 120gtcgatgccc
tggcgtgggt gatgggggag cagggggcaa agacgctgcg cggcggcaag
180atggaagacg tcatcttcgc cggcacctcg tcgcgtgcgc cgctgggccg
cgccgaagtc 240accgttagca tcgacaactc cgacaacgca ctgcctatcg
aatacaccga ggtgtcgatc 300acccgaagaa tgtttcgcga cggtgccagc
gaatacgaaa tcaacggcag cagttgccgt 360ttgatggatg tgcaggagtt
gctgagcgac tccggcatcg gccgtgagat gcatgtgatt 420gttgggcaag
ggaagctcga ggagatcttg cagtcgcggc ctgaggatcg gcgggcgttc
480atcgaggaag ccgccggtgt gctcaagcat cgcaagcgca aggaaaaagc
tctgcgcaaa 540ctcgacacga tggcggcgaa cctggcccgg ctcaccgatc
tgaccaccga gctccggcgt 600caactcaaac cgctgggccg gcaggccgag
gcggcccagc gtgccgcggc catccaagcc 660gatctgcgcg acgcccggct
gcgcctggcg gccgacgact tggtaagccg cagagccgaa 720cgggaagcgg
tctttcaggc cgaggctgcg atgcgccgcg agcatgacga ggccgccgcc
780cggctggcgg tggcatccga ggagctggcc gcgcatgagt ccgcggtcgc
cgaactctcg 840acgcgggccg agtcgatcca gcacacttgg ttcgggctgt
ctgcgctggc cgaacgggtg 900gacgctacgg tgcgcatcgc cagcgaacgc
gcccatcatc tcgatatcga gccggtagcg 960gtcagcgaca ccgaccccag
aaagcccgag gagctagaag ccgaggccca gcaggtggcc 1020gtcgccgagc
aacaactgtt agcggagctg gacgcggcgc gtgcccgact cgatgctgcc
1080cgtgcagagc tggccgaccg ggagcgccgc gccgccgagg ccgaccgggc
acacctggcg 1140gcggtccggg aggaggcgga ccgccgtgag ggactggcgc
ggctggctgg ccaggtggag 1200accatgcggg cgcgtgtcga atcgatcgat
gagagcgtgg cacggttgtc cgagcggatc 1260gaggatgccg caatgcgcgc
ccagcagacc cgagccgagt tcgaaaccgt gcagggccgc 1320atcggtgaac
tggatcaagg cgaggtcggc ctggatgagc accacgagcg tactgtggcc
1380gcgttgcggt tggccgacga acgcgtcgcc gagctgcaat ccgccgaacg
cgccgccgaa 1440cgccaggtgg catcgctacg ggctcgcatc gatgcgctcg
cagtggggct acagcgcaag 1500gacggcgcgg cgtggctggc gcacaatcgc
agtggcgcag ggcttttcgg ttcgatcgcc 1560caattggtga aggtacgttc
cggctatgaa gcggcactgg ccgcggcgct cgggccggcg 1620gccgacgcac
ttgcggtgga cggcctgact gccgcgggta gtgccgtcag cgcactcaaa
1680caagccgacg gcggtcgcgc ggtcctcgtg ctgagtgact ggccggcccc
gcaagccccc 1740caatccgcct cgggggagat gctgcctagc ggcgcccagt
gggccctaga cctggtcgag 1800tctccaccgc agttggttgg cgcgatgatc
gccatgcttt cgggtgtcgc ggtggtcaac 1860gacctgactg aggcaatggg
cctggtcgag attcgtccgg agctacgcgc ggtcaccgtt 1920gacggtgatc
tggtgggcgc cggctgggtc agcggcggat cggaccgcaa gctgtccacc
1980ttggaggtca cctccgagat cgacaaggcc aggagtgagc tggccgctgc
cgaggcgctg 2040gcggcgcaat tgaatgcggc cctggccggt gcgctgaccg
agcagtccgc ccgccaggac 2100gcggccgagc aagccttggc cgcgcttaac
gaatccgaca cggccatctc ggcgatgtac 2160gagcagctgg gccgcctcgg
gcaggaggcc cgcgcggcgg aagaagagtg gaaccggttg 2220ctgcagcagc
gtacggaaca ggaagccgtg cgcacacaga ctctcgacga cgtcatacaa
2280cttgagaccc agctgcgtaa ggcccaggag acccaacggg tgcaggtggc
ccaaccgatc 2340gaccgccagg cgatcagtgc cgctgccgat cgcgcccgcg
gtgtcgaagt ggaagcccgg 2400ctggcggtgc gcaccgccga ggaacgcgcc
aacgcggttc gcgggcgggc cgattcgctg 2460cgccgtgcgg cagcggcgga
acgtgaggcg cgggtgcggg ctcagcaagc acgcgccgca 2520agactgcatg
cggccgcggt ggccgcagcg gtcgccgact gcggacggct gctggccggg
2580cggttgcacc gggcggtgga cggggcgtcg caactgcgcg acgcgtcggc
cgcgcaacgt 2640cagcagcggt tagcggcgat ggccgcggtg cgcgacgagg
tgaacacgct gagcgcccga 2700gtgggggaac tcaccgattc gctgcaccgc
gacgagctgg ctaacgcgca ggcggcgctg 2760cgtatcgagc agcttgagca
gatggtgcta gagcagttcg gaatggcgcc ggccgacttg 2820atcaccgaat
acggtccaca tgtggcgcta ccaccgaccg agctcgagat ggctgagttc
2880gagcaagccc gcgaacgcgg cgagcaggtg attgcgcccg cccccatgcc
gttcgaccgg 2940gttacccagg agcgccgggc caaacgcgcc gagcgtgcgc
ttgccgagtt gggcagggtc 3000aacccgctgg cgctcgaaga gtttgctgcc
ttggaggagc gctacaattt cctgtccacc 3060caactcgagg atgtcaaggc
tgcccgcaag gatctgctgg gcgtcgtcgc cgatgttgac 3120gcccgcatcc
tgcaggtgtt caatgacgcg ttcgtagacg tggaacgcga atttcgcggc
3180gtgttcaccg cattgttccc cggtggtgaa ggacggctgc ggctgaccga
gcccgacgac 3240atgctcacca ccggcatcga ggtcgaagcc cgcccgccgg
gcaagaagat tacccgactg 3300tctttgctct ccggtggcga gaaggcgctg
accgcggtgg cgatgctggt cgcgatcttt 3360cgtgcccgtc catcgccgtt
ctacatcatg gacgaggtgg aggccgccct cgacgacgtg 3420aacctgcgcc
gactgctcag cctgttcgaa cagctgcgag agcagtcgca gatcatcatc
3480atcacccacc agaagccgac gatggaggtc gcggacgcac tgtacggcgt
aaccatgcag 3540aacgacggca tcaccgcggt catctcgcag cgcatgcgcg
gtcagcaggt ggatcagctg 3600gttaccaatt cctcgtag
3618401205PRTMycobacterium tuberculosis 40Met Tyr Leu Lys Ser Leu
Thr Leu Lys Gly Phe Lys Ser Phe Ala Ala1 5 10 15Pro Thr Thr Leu Arg
Phe Glu Pro Gly Ile Thr Ala Val Val Gly Pro 20 25 30Asn Gly Ser Gly
Lys Ser Asn Val Val Asp Ala Leu Ala Trp Val Met 35 40 45Gly Glu Gln
Gly Ala Lys Thr Leu Arg Gly Gly Lys Met Glu Asp Val 50 55 60Ile Phe
Ala Gly Thr Ser Ser Arg Ala Pro Leu Gly Arg Ala Glu Val65 70 75
80Thr Val Ser Ile Asp Asn Ser Asp Asn Ala Leu Pro Ile Glu Tyr Thr
85 90 95Glu Val Ser Ile Thr Arg Arg Met Phe Arg Asp Gly Ala Ser Glu
Tyr 100 105 110Glu Ile Asn Gly Ser Ser Cys Arg Leu Met Asp Val Gln
Glu Leu Leu 115 120 125Ser Asp Ser Gly Ile Gly Arg Glu Met His Val
Ile Val Gly Gln Gly 130 135 140Lys Leu Glu Glu Ile Leu Gln Ser Arg
Pro Glu Asp Arg Arg Ala Phe145 150 155 160Ile Glu Glu Ala Ala Gly
Val Leu Lys His Arg Lys Arg Lys Glu Lys 165 170 175Ala Leu Arg Lys
Leu Asp Thr Met Ala Ala Asn Leu Ala Arg Leu Thr 180 185 190Asp Leu
Thr Thr Glu Leu Arg Arg Gln Leu Lys Pro Leu Gly Arg Gln 195 200
205Ala Glu Ala Ala Gln Arg Ala Ala Ala Ile Gln Ala Asp Leu Arg Asp
210 215 220Ala Arg Leu Arg Leu Ala Ala Asp Asp Leu Val Ser Arg Arg
Ala Glu225 230 235 240Arg Glu Ala Val Phe Gln Ala Glu Ala Ala Met
Arg Arg Glu His Asp 245 250 255Glu Ala Ala Ala Arg Leu Ala Val Ala
Ser Glu Glu Leu Ala Ala His 260 265 270Glu Ser Ala Val Ala Glu Leu
Ser Thr Arg Ala Glu Ser Ile Gln His 275 280 285Thr Trp Phe Gly Leu
Ser Ala Leu Ala Glu Arg Val Asp Ala Thr Val 290 295 300Arg Ile Ala
Ser Glu Arg Ala His His Leu Asp Ile Glu Pro Val Ala305 310 315
320Val Ser Asp Thr Asp Pro Arg Lys Pro Glu Glu Leu Glu Ala Glu Ala
325 330 335Gln Gln Val Ala Val Ala Glu Gln Gln Leu Leu Ala Glu Leu
Asp Ala 340 345 350Ala Arg Ala Arg Leu Asp Ala Ala Arg Ala Glu Leu
Ala Asp Arg Glu 355 360 365Arg Arg Ala Ala Glu Ala Asp Arg Ala His
Leu Ala Ala Val Arg Glu 370 375 380Glu Ala Asp Arg Arg Glu Gly Leu
Ala Arg Leu Ala Gly Gln Val Glu385 390 395 400Thr Met Arg Ala Arg
Val Glu Ser Ile Asp Glu Ser Val Ala Arg Leu 405 410 415Ser Glu Arg
Ile Glu Asp Ala Ala Met Arg Ala Gln Gln Thr Arg Ala 420 425 430Glu
Phe Glu Thr Val Gln Gly Arg Ile Gly Glu Leu Asp Gln Gly Glu 435 440
445Val Gly Leu Asp Glu His His Glu Arg Thr Val Ala Ala Leu Arg Leu
450 455 460Ala Asp Glu Arg Val Ala Glu Leu Gln Ser Ala Glu Arg Ala
Ala Glu465 470 475 480Arg Gln Val Ala Ser Leu Arg Ala Arg Ile Asp
Ala Leu Ala Val Gly 485 490 495Leu Gln Arg Lys Asp Gly Ala Ala Trp
Leu Ala His Asn Arg Ser Gly 500 505 510Ala Gly Leu Phe Gly Ser Ile
Ala Gln Leu Val Lys Val Arg Ser Gly 515 520 525Tyr Glu Ala Ala Leu
Ala Ala Ala Leu Gly Pro Ala Ala Asp Ala Leu 530 535 540Ala Val Asp
Gly Leu Thr Ala Ala Gly Ser Ala Val Ser Ala Leu Lys545 550 555
560Gln Ala Asp Gly Gly Arg Ala Val Leu Val Leu Ser Asp Trp Pro Ala
565 570 575Pro Gln Ala Pro Gln Ser Ala Ser Gly Glu Met Leu Pro Ser
Gly Ala 580 585 590Gln Trp Ala Leu Asp Leu Val Glu Ser Pro Pro Gln
Leu Val Gly Ala 595 600 605Met Ile Ala Met Leu Ser Gly Val Ala Val
Val Asn Asp Leu Thr Glu 610 615 620Ala Met Gly Leu Val Glu Ile Arg
Pro Glu Leu Arg Ala Val Thr Val625 630 635 640Asp Gly Asp Leu Val
Gly Ala Gly Trp Val Ser Gly Gly Ser Asp Arg 645 650 655Lys Leu Ser
Thr Leu Glu Val Thr Ser Glu Ile Asp Lys Ala Arg Ser 660 665 670Glu
Leu Ala Ala Ala Glu Ala Leu Ala Ala Gln Leu Asn Ala Ala Leu 675 680
685Ala Gly Ala Leu Thr Glu Gln Ser Ala Arg Gln Asp Ala Ala Glu Gln
690 695 700Ala Leu Ala Ala Leu Asn Glu Ser Asp Thr Ala Ile Ser Ala
Met Tyr705 710 715 720Glu Gln Leu Gly Arg Leu Gly Gln Glu Ala Arg
Ala Ala Glu Glu Glu 725 730 735Trp Asn Arg Leu Leu Gln Gln Arg Thr
Glu Gln Glu Ala Val Arg Thr 740 745 750Gln Thr Leu Asp Asp Val Ile
Gln Leu Glu Thr Gln Leu Arg Lys Ala 755 760 765Gln Glu Thr Gln Arg
Val Gln Val Ala Gln Pro Ile Asp Arg Gln Ala 770 775 780Ile Ser Ala
Ala Ala Asp Arg Ala Arg Gly Val Glu Val Glu Ala Arg785 790 795
800Leu Ala Val Arg Thr Ala Glu Glu Arg Ala Asn Ala Val Arg Gly Arg
805 810 815Ala Asp Ser Leu Arg Arg Ala Ala Ala Ala Glu Arg Glu Ala
Arg Val 820 825 830Arg Ala Gln Gln Ala Arg Ala Ala Arg Leu His Ala
Ala Ala Val Ala 835 840 845Ala Ala Val Ala Asp Cys Gly Arg Leu Leu
Ala Gly Arg Leu His Arg 850 855 860Ala Val Asp Gly Ala Ser Gln Leu
Arg Asp Ala Ser Ala Ala Gln Arg865 870 875 880Gln Gln Arg Leu Ala
Ala Met Ala Ala Val Arg Asp Glu Val Asn Thr 885 890 895Leu Ser Ala
Arg Val Gly Glu Leu Thr Asp Ser Leu His Arg Asp Glu 900 905 910Leu
Ala Asn Ala Gln Ala Ala Leu Arg Ile Glu Gln Leu Glu Gln Met 915 920
925Val Leu Glu Gln Phe Gly Met Ala Pro Ala Asp Leu Ile Thr Glu Tyr
930 935 940Gly Pro His Val Ala Leu Pro Pro Thr Glu Leu Glu Met Ala
Glu Phe945 950 955 960Glu Gln Ala Arg Glu Arg Gly Glu Gln Val Ile
Ala Pro Ala Pro Met 965 970 975Pro Phe Asp Arg Val Thr Gln Glu Arg
Arg Ala Lys Arg Ala Glu Arg 980 985 990Ala Leu Ala Glu Leu Gly Arg
Val Asn Pro Leu Ala Leu Glu Glu Phe 995 1000 1005Ala Ala Leu Glu
Glu Arg Tyr Asn Phe Leu Ser Thr Gln Leu Glu 1010 1015 1020Asp Val
Lys Ala Ala Arg Lys Asp Leu Leu Gly Val Val Ala Asp 1025 1030
1035Val Asp Ala Arg Ile Leu Gln Val Phe Asn Asp Ala Phe Val Asp
1040 1045 1050Val Glu Arg Glu Phe Arg Gly Val Phe Thr Ala Leu Phe
Pro Gly 1055 1060 1065Gly Glu Gly Arg Leu Arg Leu Thr Glu Pro Asp
Asp Met Leu Thr 1070 1075 1080Thr Gly Ile Glu Val Glu Ala Arg Pro
Pro Gly Lys Lys Ile Thr 1085 1090 1095Arg Leu Ser Leu Leu Ser Gly
Gly Glu Lys Ala Leu Thr Ala Val 1100 1105 1110Ala Met Leu Val Ala
Ile Phe Arg Ala Arg Pro Ser Pro Phe Tyr 1115 1120 1125Ile Met Asp
Glu Val Glu Ala Ala Leu Asp Asp Val Asn Leu Arg 1130 1135 1140Arg
Leu Leu Ser Leu Phe Glu Gln Leu Arg Glu Gln Ser Gln Ile 1145 1150
1155Ile Ile Ile Thr His Gln Lys Pro Thr Met Glu Val Ala Asp Ala
1160 1165 1170Leu Tyr Gly Val Thr Met Gln Asn Asp Gly Ile Thr Ala
Val Ile 1175 1180 1185Ser Gln Arg Met Arg Gly Gln Gln Val Asp Gln
Leu Val Thr Asn 1190 1195 1200Ser Ser 12054148DNAMycobacterium
tuberculosis 41ctgcacgcgc agaaggcgct gctggtgtgg ctgctggagc gctcatga
484216PRTMycobacterium tuberculosis 42Arg Leu His Ala Gln Lys Ala
Leu Leu Val Trp Leu Leu Glu Arg Ser1 5 10 1543924DNAMycobacterium
tuberculosis 43gtgatcaggc atttcctgcg cgacgacgat ctgtccccgg
ccgaacaggc cgaggtgctc 60gagctcgcgg ccgagctgaa gaaagacccg gttagccgtc
gtcccctgca agggccgcgc 120ggggtggcgg tcatcttcga caagaactcc
acccgcaccc ggttctcctt cgagctgggc 180atcgcgcagc tgggcgggca
tgccgtcgtc gtcgacagcg gcagcaccca gctgggccgc 240gacgaaaccc
tgcaggacac cgcaaaggtg ttgtcccgct acgtcgatgc catcgtctgg
300cgaaccttcg gccaagagcg gctggacgcc atggcgtcgg tcgcgacggt
gcccgtgatc 360aacgcgctct ccgatgagtt ccatccgtgt caggtgttgg
ccgacctgca gaccatcgcc 420gaacgcaagg gggcgctgcg cggcctgagg
ttgtcctact tcggcgacgg cgccaacaac 480atggcccact cgctgctgct
cggcggggtc accgcgggta tccacgtcac cgtcgcggct 540cccgagggct
tcctgcccga cccgtcggtg cgggccgcgg ccgagcgccg cgcccaggat
600accggcgcct cggtgactgt gaccgccgac gcccacgcgg ccgccgccgg
cgccgacgtt 660ctggtcaccg acacctggac gtcgatgggc caggaaaacg
acgggttgga ccgagtgaag 720ccgtttcggc cgtttcagct caactcgcga
cttctggcgc tggccgactc ggatgccatc 780gtgttgcatt gcctgccggc
ccatcgcggc gacgagatca ccgacgcggt gatggacggg 840ccggccagcg
cggtgtggga cgaggccgaa aaccggctgc acgcgcagaa ggcgctgctg
900gtgtggctgc tggagcgctc atga 92444307PRTMycobacterium tuberculosis
44Met Ile Arg His Phe Leu Arg Asp Asp Asp Leu Ser Pro Ala Glu Gln1
5 10 15Ala Glu Val Leu Glu Leu Ala Ala Glu Leu Lys Lys Asp Pro Val
Ser 20 25 30Arg Arg Pro Leu Gln Gly Pro Arg Gly Val Ala Val Ile Phe
Asp Lys 35 40 45Asn Ser Thr Arg Thr Arg Phe Ser Phe Glu Leu Gly Ile
Ala Gln Leu 50 55 60Gly Gly His Ala Val Val Val Asp Ser Gly Ser Thr
Gln Leu Gly Arg65 70 75 80Asp Glu Thr Leu Gln Asp Thr Ala Lys Val
Leu Ser Arg Tyr Val Asp 85 90 95Ala Ile Val Trp Arg Thr Phe Gly Gln
Glu Arg Leu Asp Ala Met Ala 100 105 110Ser Val Ala Thr Val Pro Val
Ile Asn Ala Leu Ser Asp Glu Phe His 115 120 125Pro Cys Gln Val Leu
Ala Asp Leu Gln Thr Ile Ala Glu Arg Lys Gly 130 135 140Ala Leu Arg
Gly Leu Arg Leu Ser Tyr Phe Gly Asp Gly Ala Asn Asn145 150 155
160Met Ala His Ser Leu Leu Leu Gly Gly Val Thr Ala Gly Ile His Val
165 170 175Thr Val Ala Ala Pro Glu Gly Phe Leu Pro Asp Pro Ser Val
Arg Ala 180 185 190Ala Ala Glu Arg Arg Ala Gln Asp Thr Gly Ala Ser
Val Thr Val Thr 195 200 205Ala Asp Ala His Ala Ala Ala Ala Gly Ala
Asp Val Leu Val Thr Asp 210 215 220Thr Trp Thr Ser Met Gly Gln Glu
Asn Asp Gly Leu Asp Arg Val Lys225 230 235 240Pro Phe Arg Pro Phe
Gln Leu Asn Ser Arg Leu Leu Ala Leu Ala Asp 245 250 255Ser Asp Ala
Ile Val Leu His Cys Leu Pro Ala His Arg Gly Asp Glu 260 265 270Ile
Thr Asp Ala Val Met Asp Gly Pro Ala Ser Ala Val Trp Asp Glu 275 280
285Ala Glu Asn Arg Leu His Ala Gln Lys Ala Leu Leu Val Trp Leu Leu
290 295 300Glu Arg Ser3054566DNAMycobacterium tuberculosis
45cgctggaccg acgaaacctt cggcgacatc ggcggcgccg gcggcggcgt gagcggacat
60cgcggg 664622PRTMycobacterium tuberculosis 46Arg Trp Thr Asp Glu
Thr Phe Gly Asp Ile Gly Gly Ala Gly Gly Gly1 5 10 15Val Ser Gly His
Arg Gly 2047765DNAMycobacterium tuberculosis 47atgagcggcg
agacaaccag gctgaccgaa ccgcaactac gtgagctggc cgcgcgcgga 60gctgccgaac
tcgacggcgc caccgccacc gacatgttgc gctggaccga cgaaaccttc
120ggcgacatcg gcggcgccgg cggcggcgtg agcggacatc gcgggtggac
aacgtgcaac 180tacgtagttg cttccaacat ggctgatgcg gtgctggtgg
atctggccgc caaggtgcga 240ccgggcgtac cggtcatctt tcttgatacc
ggctaccact tcgtcgaaac aatcggcacc 300agagatgcga tcgagtccgt
ctatgacgtc cgggtgctca atgtcactcc ggagcacaca 360gtggccgagc
aggacgaact gctgggcaag gacttgttcg cccgcaaccc ccatgaatgc
420tgccggttgc gcaaggtcgt tcccctgggc aagacgctgc gtggctactc
cgcgtgggtg 480accgggctac ggcgggtcga tgcaccgacc cgggccaatg
ccccgctggt cagcttcgat 540gagacgttca aactagtgaa ggtcaacccg
ctggcggcgt ggaccgacca agatgtgcag 600gaatacattg ccgacaacga
cgtgctggtt aatccgcttg tgcgggaagg ctatccgtcg 660atcggttgcg
ctccgtgcac agccaaaccc gccgaaggcg ccgacccgcg cagcggacgc
720tggcaggggc tggccaagac cgaatgcggg ttgcacgcct cgtga
76548254PRTMycobacterium tuberculosis 48Met Ser Gly Glu Thr Thr Arg
Leu Thr Glu Pro Gln Leu Arg Glu Leu1 5 10 15Ala Ala Arg Gly Ala Ala
Glu Leu Asp Gly Ala Thr Ala Thr Asp Met 20 25 30Leu Arg Trp Thr Asp
Glu Thr Phe Gly Asp Ile Gly Gly Ala Gly Gly 35 40 45Gly Val Ser Gly
His Arg Gly Trp Thr Thr Cys Asn Tyr Val Val Ala 50 55 60Ser Asn
Met Ala Asp Ala Val Leu Val Asp Leu Ala Ala Lys Val Arg65 70 75
80Pro Gly Val Pro Val Ile Phe Leu Asp Thr Gly Tyr His Phe Val Glu
85 90 95Thr Ile Gly Thr Arg Asp Ala Ile Glu Ser Val Tyr Asp Val Arg
Val 100 105 110Leu Asn Val Thr Pro Glu His Thr Val Ala Glu Gln Asp
Glu Leu Leu 115 120 125Gly Lys Asp Leu Phe Ala Arg Asn Pro His Glu
Cys Cys Arg Leu Arg 130 135 140Lys Val Val Pro Leu Gly Lys Thr Leu
Arg Gly Tyr Ser Ala Trp Val145 150 155 160Thr Gly Leu Arg Arg Val
Asp Ala Pro Thr Arg Ala Asn Ala Pro Leu 165 170 175Val Ser Phe Asp
Glu Thr Phe Lys Leu Val Lys Val Asn Pro Leu Ala 180 185 190Ala Trp
Thr Asp Gln Asp Val Gln Glu Tyr Ile Ala Asp Asn Asp Val 195 200
205Leu Val Asn Pro Leu Val Arg Glu Gly Tyr Pro Ser Ile Gly Cys Ala
210 215 220Pro Cys Thr Ala Lys Pro Ala Glu Gly Ala Asp Pro Arg Ser
Gly Arg225 230 235 240Trp Gln Gly Leu Ala Lys Thr Glu Cys Gly Leu
His Ala Ser 245 250
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