U.S. patent application number 09/791171 was filed with the patent office on 2002-07-18 for nucleic acid fragments and polypeptide fragments derived from m. tuberculosis.
This patent application is currently assigned to STATENS SERUM INSTITUT. Invention is credited to Andersen, Peter, Florio, Walter, Nielsen, Rikke, Oettinger, Thomas, Rasmussen, Peter Birk, Rosenkrands, Ida, Weldingh, Karin.
Application Number | 20020094336 09/791171 |
Document ID | / |
Family ID | 29273299 |
Filed Date | 2002-07-18 |
United States Patent
Application |
20020094336 |
Kind Code |
A1 |
Andersen, Peter ; et
al. |
July 18, 2002 |
Nucleic acid fragments and polypeptide fragments derived from M.
tuberculosis
Abstract
The present invention is based on the identification and
characterization of a number of M. tuberculosis derived novel
proteins and protein fragments (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,
16, 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, and
168-171). The invention is directed to the polypeptides and
immunologically active fragments thereof, the genes encoding them,
immunological compositions such as vaccines and skin test reagents
containing the polypeptides. Another part of the invention is based
on the surprising discovery that fusions between ESAT-6 and MPT59
are superior immunogens compared to each of the unfused proteins,
respectively.
Inventors: |
Andersen, Peter; (Bronshoj,
DK) ; Nielsen, Rikke; (Frederiksberg C, DK) ;
Oettinger, Thomas; (Hellerup, DK) ; Rasmussen, Peter
Birk; (Kobenhaven O, DK) ; Rosenkrands, Ida;
(Kobenhaven O, DK) ; Weldingh, Karin; (Kobenhaven
N, DK) ; Florio, Walter; (Frederiksberg C,
DK) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG LLP
745 FIFTH AVENUE
NEW YORK
NY
10151
US
|
Assignee: |
STATENS SERUM INSTITUT
|
Family ID: |
29273299 |
Appl. No.: |
09/791171 |
Filed: |
February 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09791171 |
Feb 20, 2001 |
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09050739 |
Mar 30, 1998 |
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60044624 |
Apr 18, 1997 |
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60070488 |
Jan 5, 1998 |
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Current U.S.
Class: |
424/190.1 ;
435/183 |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 39/00 20130101; A61K 2039/51 20130101; C07K 2319/00 20130101;
C07K 14/35 20130101 |
Class at
Publication: |
424/190.1 ;
435/183 |
International
Class: |
A61K 039/02; C12N
009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 2, 1997 |
DK |
0376/97 |
Nov 10, 1997 |
DK |
1277/97 |
Claims
1. A substantially pure polypeptide fragment which a) comprises an
amino acid sequence selected from the sequences shown in SEQ ID NO:
2, 4, 6, 8, 10, 12, 14, 16, 17-23, 42, 48, 50, 52, 54, 56, 58, 60,
62, 64, 66, 68, 70, 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149,
151, 153, and 168-171, b) comprises a subsequence of the
polypeptide fragment defined in a) which has a length of at least 6
amino acid residues, said subsequence being immunologically
equivalent to the polypeptide defined in a) with respect to the
ability of evoking a protective immune response against infections
with mycobacteria belonging to the tuberculosis complex or with
respect to the ability of eliciting a diagnostically significant
immune response indicating previous or ongoing sensitization with
antigens derived from mycobacteria belonging to the tuberculosis
complex, or c) comprises an amino acid sequence having a sequence
identity with the polypeptide defined in a) or the subsequence
defined in b) of at least 70% and at the same time being
immunologically equivalent to the polypeptide defined in a) with
respect to the ability of evoking a protective immune response
against infections with mycobacteria belonging to the tuberculosis
complex or with respect to the ability of eliciting a
diagnostically significant immune response indicating previous or
ongoing sensitization with antigens derived from mycobacteria
belonging to the tuberculosis complex, with the proviso that i) the
polypeptide fragment is in essentially pure form when consisting of
the amino acid sequence 1-96 of SEQ ID NO: 2 or when consisting of
the amino acid sequence 87-108 of SEQ ID NO: 4 fused to
.beta.-galactosidase, ii) the degree of sequence identity in c) is
at least 95% when the polypeptide comprises a homologue of a
polypeptide which has the amino acid sequence SEQ ID NO: 12 or a
subsequence thereof as defined in b), and iii) the polypeptide
fragment contains a threonine residue corresponding to position 213
in SEQ ID NO: 42 when comprising an amino acid sequence of at least
6 amino acids in SEQ ID NO: 42.
2. The polypeptide fragment according to claim 1 in essentially
pure form.
3. The polypeptide fragment according to claim 1 or 2, which
comprises an epitope for a T-helper cell.
4. The polypeptide fragment according to any of the preceding
claims, which has a length of at least 7 amino acid residues, such
as at least 8, at least 9, at least 10, at least 12, at least 14,
at least 16, at least 18, at least 20, at least 22, at least 24,
and at least 30 amino acid residues.
5. The polypeptide fragment according to any of the preceding
claims, which is free from amino acid residues -30 to -1 in SEQ ID
NO: 6 and/or -32 to -1 in SEQ ID NO: 10 and/or -8 to -1 in SEQ ID
NO: 12 and/or -32 to -1 in SEQ ID NO: 14 and/or -33 to -1 in SEQ ID
NO: 42 and/or -38 to -1 in SEQ ID NO: 52 and/or -33 to -1 in SEQ ID
NO: 56 and/or -56 to -1 in SEQ ID NO: 58 and/or -28 to -1 in SEQ ID
NO: 151.
6. The polypeptide fragment according to any of the preceding
claims which is free from any signal sequence.
7. The polypeptide fragment according to any of the preceding
claims which 1) induces a release of IFN-.gamma. from primed memory
T-lymphocytes withdrawn from a mouse within 2 weeks of primary
infection or within 4 days after the mouse has been rechallenge
infected with mycobacteria belonging to the tuberculosis complex,
the induction performed by the addition of the polypeptide to a
suspension comprising about 200.000 spleen cells per ml, the
addition of the polypeptide resulting in a concentration of 1-4
.mu.g polypeptide per ml suspension, the release of IFN-.gamma.
being assessable by determination of IFN-.gamma. in supernatant
harvested 2 days after the addition of the polypeptide to the
suspension, and/or 2) induces a release of IFN-.gamma. of at least
300 pg above background level from about 1000,000 human PBMC
(peripheral blood mononuclear cells) per ml isolated from TB
patients in the first phase of infection, or from healthy BCG
vaccinated donors, or from healthy contacts to TB patients, the
induction being performed by the addition of the polypeptide to a
suspension comprising the about 1,000,000 PBMC per ml, the addition
of the polypeptide resulting in a concentration of 1-4 .mu.g
polypeptide per ml suspension, the release of IFN-.gamma. being
assessable by determination of IFN-.gamma. in supernatant harvested
2 days after the addition of the polypeptide to the suspension;
and/or 3) induces an IFN-.gamma. release from bovine PBMC derived
from animals previously sensitized with mycobacteria belonging to
the tuberculosis complex, said release being at least two times the
release observed from bovine PBMC derived from animals not
previously sensitized with mycobacteria belonging to the
tuberculosis complex.
8. A polypeptide fragment according to any of the preceding claims,
wherein the sequence identity in c) is at least 80%, such as at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, and at least 99.5%.
9. A fusion polypeptide comprising at least one polypeptide
fragment according to any of the preceding claims and at least one
fusion partner.
10. A fusion polypeptide according to claim 56, wherein the fusion
partner is selected from the group consisting of a polypeptide
fragment as defined in any of claims 1-8, and an other polypeptide
fragment derived from a bacterium belonging to the tuberculosis
complex, such as ESAT-6 or at least one T-cell epitope thereof,
MPB64 or at least one T-cell epitope thereof, MPT64 or at least one
T-cell epitope thereof, and MPB59 or at least one T-cell epitope
thereof.
11. A fusion polypeptide fragment which comprises 1) a first amino
acid sequence including at least one stretch of amino acids
constituting a T-cell epitope derived from the M. tuberculosis
protein ESAT-6, and a second amino acid sequence including at least
one T-cell epitope derived from a M. tuberculosis protein different
from ESAT-6 and/or including a stretch of amino acids which
protects the first amino acid sequence from in vivo degradation or
post-translational processing; or 2) a first amino acid sequence
including at least one stretch of amino acids constituting a T-cell
epitope derived from the M. tuberculosis protein MPT59, and a
second amino acid sequence including at least one T-cell epitope
derived from a M. tuberculosis protein different from MPT59 and/or
including a stretch of amino acids which protects the first amino
acid sequence from in vivo degradation or post-translational
processing.
12. A fusion polypeptide fragment according to claim 11, wherein
the first amino acid sequence is situated C-terminally to the
second amino acid sequence.
13. A fusion polypeptide fragment according to claim 11, wherein
the first amino acid sequence is situated N-terminally to the
second amino acid sequence.
14. A fusion polypeptide fragment according to any of claims 11-13,
wherein the at least one T-cell epitope included in the second
amino acid sequence is derived from a M. tuberculosis polypeptide
selected from the group consisting of a polypeptide fragment
according to any of claims 1-55, DnaK, GroEL, urease, glutamine
synthetase, the proline rich complex, L-alanine dehydrogenase,
phosphate binding protein, Ag 85 complex, HBHA (heparin binding
hemagglutinin), MPT51, MPT64, superoxide dismutase, 19 kDa
lipoprotein, .alpha.-crystallin, GroES, MPT59 when the first T-cell
epitope is derived from ESAT-6, and ESAT-6 when the first T-cell
epitope is derived from MPT59.
15. A fusion polypeptide fragment according to any of claims 11-14,
wherein the first and second T-cell epitopes each have a sequence
identity of at least 70% with the natively occurring sequence in
the proteins from which they are derived.
16. A fusion polypeptide according to any of claims 11-15, wherein
the first and/or second amino acid sequence have a sequence
identity of at least 70% with the protein from which they are
derived.
17. A fusion polypeptide fragment according to any of claims 11-16,
wherein the first amino acid sequence is the amino acid sequence of
ESAT-6 or of MPT59 and/or the second amino acid sequence is the
amino acid sequence of a M. tuberculosis polypeptide selected from
the group consisting of a polypeptide fragment according to any of
claims 1-8, DnaK, GroEL, urease, glutamine synthetase, the proline
rich complex, L-alanine dehydrogenase, phosphate binding protein,
Ag 85 complex, HBHA (heparin binding hemagglutinin), MPT51, MPT64,
superoxide dismutase, 19 kDa lipoprotein, .alpha.-crystallin,
GroES, ESAT-6 when the first amino acid sequence is that of MPT59,
and MPT59 when the first amino acid sequence is that of ESAT-6.
18. A fusion polypeptide fragment according to any of claims 11-17,
which comprises ESAT-6 fused to MPT59.
19. A fusion polypeptide fragment according to claim 18, wherein no
linkers are introduced between the two amino acid sequences.
20. A polypeptide according to any of the preceding claims which is
lipidated so as to allow a self-adjuvating effect of the
polypeptide.
21. A substantially pure polypeptide according to any of claims
1-20 for use as a pharmaceutical.
22. The use of a substantially pure polypeptide according to any of
claims 1-20 in the preparation of a pharmaceutical composition for
the diagnosis of or vaccination against tuberculosis caused by
Mycobacterium tuberculosis, Mycobacterium africanum or
Mycobacterium bovis.
23. A nucleic acid fragment in isolated form which 1) comprises a
nucleic acid sequence which encodes a polypeptide as defined in any
of claims 1-20, or comprises a nucleic acid sequence complementary
thereto, 2) has a length of at least 10 nucleotides and hybridizes
readily under stringent hybridization conditions with a nucleic
acid fragment which has a nucleotide sequence selected from
35 SEQ ID NO: 1 or a sequence complementary thereto, SEQ ID NO: 3
or a sequence complementary thereto, SEQ ID NO: 5 or a sequence
complementary thereto, SEQ ID NO: 7 or a sequence complementary
thereto, SEQ ID NO: 9 or a sequence complementary thereto, SEQ ID
NO: 11 or a sequence complementary thereto, SEQ ID NO: 13 or a
sequence complementary thereto, SEQ ID NO: 15 or a sequence
complementary thereto, SEQ ID NO: 41 or a sequence complementary
thereto, SEQ ID NO: 47 or a sequence complementary thereto, SEQ ID
NO: 49 or a sequence complementary thereto, SEQ ID NO: 51 or a
sequence complementary thereto, SEQ ID NO: 53 or a sequence
complementary thereto, SEQ ID NO: 55 or a sequence complementary
thereto, SEQ ID NO: 57 or a sequence complementary thereto, SEQ ID
NO: 59 or a sequence complementary thereto, SEQ ID NO: 61 or a
sequence complementary thereto, SEQ ID NO: 63 or a sequence
complementary thereto, SEQ ID NO: 65 or a sequence complementary
thereto, SEQ ID NO: 67 or a sequence complementary thereto, SEQ ID
NO: 69 or a sequence complementary thereto, SEQ ID NO: 71 or a
sequence complementary thereto, SEQ ID NO: 87 or a sequence
complementary thereto, SEQ ID NO: 89 or a sequence complementary
thereto, SEQ ID NO: 91 or a sequence complementary thereto, SEQ ID
NO: 93 or a sequence complementary thereto, SEQ ID NO: 140 or a
sequence complementary thereto, SEQ ID NO: 142 or a sequence
complementary thereto, SEQ ID NO: 144 or a sequence complementary
thereto, SEQ ID NO: 146 or a sequence complementary thereto, SEQ ID
NO: 148 or a sequence complementary thereto, SEQ ID NO: 150 or a
sequence complementary thereto, and SEQ ID NO: 152 or a sequence
complementary thereto,
with the proviso that when the nucleic acid fragment comprises a
subsequence of SEQ ID: 41, then the nucleic acid fragment contains
an A corresponding to position 781 in SEQ ID NO: 41 and when the
nucleic acid fragment comprises a subsequence of a nucleotide
sequence exactly complementary to SEQ ID NO: 41, then the nucleic
acid fragment comprises a T corresponding to position 781 in SEQ ID
NO: 41.
24. A nucleic acid fragment according to claim 23, which is a DNA
fragment.
25. A vaccine comprising a nucleic acid fragment according to claim
23 or 24, the vaccine effecting in vivo expression of antigen by an
animal, including a human being, to whom the vaccine has been
administered, the amount of expressed antigen being effective to
confer substantially increased resistance to infections with
mycobacteria of the tuberculosis complex in an animal, including a
human being.
26. A nucleic acid fragment according to claim 23 or 24 for use as
a pharmaceutical.
27. The use of a nucleic acid fragment according to claim 23 or 24
in the preparation of a pharmaceutical composition for the
diagnosis of or vaccination against tuberculosis caused by
Mycobacterium tuberculosis, Mycobacterium africanum or
Mycobacterium bovis.
28. An immunologic composition comprising a polypeptide according
to any of claims 1-20.
29. An immunologic composition according to claim 28, which further
comprises an immunologically and pharmaceutically acceptable
carrier, vehicle or adjuvant.
30. An immunologic composition according to claim 29, wherein the
carrier is selected from the group consisting of a polymer to which
the polypeptide(s) is/are bound by hydrophobic non-covalent
interaction, such as a plastic, e.g. polystyrene, a polymer to
which the polypeptide (s) is/are covalently bound, such as a
polysaccharide, and a polypeptide, e.g. bovine serum albumin,
ovalbumin or keyhole limpet hemocyanin; the vehicle is selected
from the group consisting of a diluent and a suspending agent; and
the adjuvant is selected from the group consisting of
dimethyldioctadecylammonium bromide (DDA), Quil A, poly I:C,
Freund's incomplete adjuvant, IFN-.gamma. IL-2, IL-12,
monophosphoryl lipid A (MPL), and muramyl dipeptide (MDP).
31. An immunologic composition according to any of claims 28 to 30,
comprising at least two different polypeptide fragments, each
different polypeptide fragment being a polypeptide according to any
of claims 1-67.
32. An immunologic composition according to claim 78, comprising
3-20 different polypeptide fragments, each different polypeptide
fragment being according to any of claims 1-20.
33. An immunologic composition according to any of claims 28-32,
which is in the form of a vaccine.
34. An immunologic composition according to any of claims 28-32,
which is in the form of a skin test reagent.
35. A vaccine for immunizing an animal, including a human being,
against tuberculosis caused by mycobacteria belonging to the
tuberculosis complex, comprising as the effective component a
non-pathogenic microorganism, wherein at least one copy of a DNA
fragment comprising a DNA sequence encoding a polypeptide according
to any of claims 1-20 has been incorporated into the genome of the
microorganism in a manner allowing the microorganism to express and
optionally secrete the polypeptide.
36. A vaccine according to claim 35, wherein the microorganism is a
bacterium.
37. A vaccine according to claim 36, wherein the bacterium is
selected from the group consisting of the genera Mycobacterium,
Salmonella, Pseudomonas and Eschericia.
38. A vaccine according to claim 37, wherein the microorganism is
Mycobacterium bovis BCG, such as Mycobacterium bovis BCG strain:
Danish 1331.
39. A vaccine according to any of claims 35-38, wherein at least 2
copies of a DNA fragment encoding a polypeptide according to any of
claims 1-20 are incorporated into the genome of the
microorganism.
40. A vaccine according to claim 39, wherein the number of copies
is at least 5.
41. A replicable expression vector which comprises a nucleic acid
fragment according to claim 23 or 24.
42. A vector according to claim 41, which is selected from the
group consisting of a virus, a bacteriophage, a plasmid, a cosmid,
and a microchromosome.
43. A transformed cell harbouring at least one vector according to
claim 41 or 42.
44. A transformed cell according to claim 43, which is a bacterium
belonging to the tuberculosis complex, such as a M. tuberculosis
bovis BCG cell.
45. A transformed cell according to claim 43 or 44, which expresses
a polypeptide according to any of claims 1-20.
46. A method for producing a polypeptide according to any of claims
1-20, comprising inserting a nucleic acid fragment according to
claim 23 or 24 into a vector which is able to replicate in a host
cell, introducing the resulting recombinant vector into the host
cell, culturing the host cell in a culture medium under conditions
sufficient to effect expression of the polypeptide, and recovering
the polypeptide from the host cell or culture medium; or isolating
the polypeptide from a short-term culture filtrate as defined in
claim 1; or isolating the polypeptide from whole mycobacteria of
the tuberculosis complex or from lysates or fractions thereof, e.g.
cell wall containing fractions; or synthesizing the polypeptide by
solid or liquid phase peptide synthesis.
47. A method for producing an immunologic composition according to
any of claims 28-32 comprising preparing, synthesizing or isolating
a polypeptide according to any of claims 1-20, and solubilizing or
dispersing the polypeptide in a medium for a vaccine, and
optionally adding other M. tuberculosis antigens and/or a carrier,
vehicle and/or adjuvant substance, or cultivating a cell according
to any of claims 37-45, and transferring the cells to a medium for
a vaccine, and optionally adding a carrier, vehicle and/or adjuvant
substance.
48. A method of diagnosing tuberculosis caused by Mycobacterium
tuberculosis, Mycobacterium africanum or Mycobacterium bovis in an
animal, including a human being, comprising intradermally
injecting, in the animal, a polypeptide according to any of claims
1-20 or an immunologic composition according to claim 34, a
positive skin response at the location of injection being
indicative of the animal having tuberculosis, and a negative skin
response at the location of injection being indicative of the
animal not having tuberculosis.
49. A method for immunising an animal, including a human being,
against tuberculosis caused by mycobacteria belonging to the
tuberculosis complex, comprising administering to the animal the
polypeptide according to any of claims 1-20, the immunologic
composition according to claim 33, or the vaccine according to any
of claims 35-40.
50. A method according to claim 49, wherein the polypeptide,
immunologic composition, or vaccine is administered by the
parenteral (such as intravenous and intraarterially),
intraperitoneal, intramuscular, subcutaneous, intradermal, oral,
buccal, sublingual, nasal, rectal or transdermal route.
51. A method for diagnosing ongoing or previous sensitization in an
animal or a human being with bacteria belonging to the tuberculosis
complex, the method comprising providing a blood sample from the
animal or human being, and contacting the sample from the animal
with the polypeptide according to any of claims 1-20, a significant
release into the extracellular phase of at least one cytokine by
mononuclear cells in the blood sample being indicative of the
animal being sensitized.
52. A composition for diagnosing tuberculosis in an animal,
including a human being, comprising a polypeptide according to any
of claims 1-20, or a nucleic acid fragment according to claim 23 or
24, optionally in combination with a means for detection.
53. A monoclonal or polyclonal antibody, which is specifically
reacting with a polypeptide according to any of claims 1-20 in an
immuno assay, or a specific binding fragment of said antibody.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a number of immunologically
active, novel polypeptide fragments derived from the Mycobacterium
tuberculosis, vaccines and other immunologic compositions
containing the fragments as immunogenic components, and methods of
production and use of the polypeptides. The invention also relates
to novel nucleic acid fragments derived from M. tuberculosis which
are useful in the preparation of the polypeptide fragments of the
invention or in the diagnosis of infection with M. tuberculosis.
The invention further relates to certain fusion polypeptides,
notably fusions between ESAT-6 and MPT59.
BACKGROUND OF THE INVENTION
[0002] Human tuberculosis (hereinafter designated "TB") caused by
Mycobacterium tuberculosis is a severe global health problem
responsible for approximately 3 million deaths annually, according
to the WHO. The worldwide incidence of new TB cases has been
progressively falling for the last decade but the recent years has
markedly changed this trend due to the advent of AIDS and the
appearance of multidrug resistant strains of M. tuberculosis.
[0003] The only vaccine presently available for clinical use is
BCG, a vaccine which efficacy remains a matter of controversy. BCG
generally induces a high level of acquired resistance in animal
models of TB, but several human trials in developing countries have
failed to demonstrate significant protection. Notably, BCG is not
approved by the FDA for use in the United States.
[0004] This makes the development of a new and improved vaccine
against TB an urgent matter which has been given a very high
priority by the WHO. Many attempts to define protective
mycobacterial substances have been made, and from 1950 to 1970
several investigators reported an increased resistance after
experimental vaccination. However, the demonstration of a specific
long-term protective immune response with the potency of BCG has
not yet been achieved by administration of soluble proteins or cell
wall fragments, although progress is currently being made by
relying on polypeptides derived from short term-culture filtrate,
cf. the discussion below.
[0005] Immunity to M. tuberculosis is characterized by three basic
features; i) Living bacilli efficiently induces a protective immune
response in contrast to killed preparations; ii) Specifically
sensitized T lymphocytes mediate this protection; iii) The most
important mediator molecule seems to be interferon gamma
(INF-.gamma.).
[0006] Short term-culture filtrate (ST-CF) is a complex mixture of
proteins released from M. tuberculosis during the first few days of
growth in a liquid medium (Andersen et al., 1991). Culture
filtrates has been suggested to hold protective antigens recognized
by the host in the first phase of TB infection (Andersen et al.
1991, Orme et al. 1993). Recent data from several laboratories have
demonstrated that experimental subunit vaccines based on culture
filtrate antigens can provide high levels of acquired resistance to
TB (Pal and Horwitz, 1992; Roberts et al., 1995; Andersen, 1994;
Lindblad et al., 1997). Culture filtrates are, however, complex
protein mixtures and until now very limited information has been
available on the molecules responsible for this protective immune
response. In this regard, only two culture filtrate antigens have
been described as involved in protective immunity, the low mass
antigen ESAT-6 (Andersen et al., 1995 and EP-A-0 706 571) and the
31 kDa molecule Ag85B (EP-0 432 203).
[0007] There is therefore a need for the identification of further
antigens involved in the induction of protective immunity against
TB in order to eventually produce an effective sub-unit
vaccine.
OBJECT OF THE INVENTION
[0008] It is an object of the invention to provide novel antigens
which are effective as components in a subunit vaccine against TB
or which are useful as components in diagnostic compositions for
the detection of infection with mycobacteria, especially
virulence-associated mycobacteria. The novel antigens may also be
important drug targets.
SUMMARY OF THE INVENTION
[0009] The present invention is i.a. based on the identification
and characterization of a number of previously uncharacterized
culture filtrate antigens from M. tuberculosis. In animal models of
TB, T cells mediating immunity are focused predominantly to
antigens in the regions 6-12 and 17-30 kDa of ST-CF. In the present
invention 8 antigens in the low molecular weight region (CFP7,
CFP7A, CFP7B, CFP8A, CFP8B, CFP9, CFP10A, and CFP11) and 18
antigens (CFP16, CFP17, CFP19, CFP19B, CFP20, CFP21, CFP22, CFP22A,
CFP23, CFP23A, CFP23B, CFP25, CFP26, CFP27, CFP28, CFP29, CFP30A,
and CFP30B) in the 17-30 kDa region have been identified. Of these,
CFP19A and CFP23 have been selected because they exhibit relatively
high homologies with CFP21 and CFP25, respectively, in so far that
a nucleotide homology sequence search in the Sanger Database (cf.
below) with the genes encoding CFP21 and CFP25, (cfp25 and
cfp21respectively), shows homology to two M. tuberculosis DNA
sequences, orf19A and orf23. The two sequences, orf19a and orf23,
encode to putative proteins CFP19A and CFP23 with the molecular
weights of approx. 19 and 23 kDa respectively. The identity, at
amino acid level, to CFP21 and CFP25 is 46% and 50% , respectively,
for both proteins. CFP21 and CFP25 have been shown to be dominant
T-cell antigens, and it is therefore believed that CFP19A and CFP23
are possible new T-cell antigens.
[0010] Furthermore, a 50 kDa antigen (CFP50) has been isolated from
culture filtrate and so has also an antigen (CWP32) isolated from
the cell wall in the 30 kDa region.
[0011] The present invention is also based on the identification of
a number of putative antigens from M. tuberculosis which are not
present in Mycobacterium bovis BCG strains. The nucleotide
sequences encoding these putative antigens are: rd1-orf2, rd1-orf3,
rd1-orf4, rd1-orf5, rd1-orf5, rd1-orf9a, and rd1-orf9b.
[0012] Finally, the invention is based on the surprising discovery
that fusions between ESAT-6 and MPT59 are superior immunogens
compared to the unfused proteins, respectively.
[0013] The encoding genes for 33 of the antigens have been
determined, the distribution of a number of the antigens in various
mycobacterial strains investigated and the biological activity of
the products characterized. The panel hold antigens with potential
for vaccine purposes as well as for diagnostic purposes, since the
antigens are all secreted by metabolizing mycobacteria.
[0014] The following table lists the antigens of the invention by
the names used herein as well as by reference to relevant SEQ ID
NOs of N-terminal sequences, full amino acid sequences and
sequences of DNA encoding the antigens:
1 N-terminal Nucleotide Amino acid sequence sequence sequence
Antigen SEQ ID NO: SEQ ID NO: SEQ ID NO: CFP7 1 2 CFP7A 81 47 48
CFP7B 168 146 147 CFP8A 73 148 149 CFP8B 74 150 151 CFP9 3 4 CFP10A
169 140 141 CFP11 170 142 143 CFP16 79 63 64 CFP17 17 5 6 CFP19 82
49 50 CFP19A 51 52 CFP19B 80 CFP20 18 7 8 CFP21 19 9 10 CFP22 20 11
12 CFP22A 83 53 54 CFP23 55 56 CFP23A 76 CFP23B 75 CFP25 22 13 14
CFP25A 78 65 66 CFP27 84 57 58 CFP28 22 CFP29 23 15 16 CFP30A 85 59
60 CFP30B 171 144 145 CFP50 86 61 62 MPT51 41 42 CWP32 77 152 153
RD1-ORF8 67 68 RD1-ORF2 71 72 RD1-ORF9B 69 70 RD1-ORF3 87 88
RD1-ORF9A 93 94 RD1-ORF4 89 90 RD2-ORF5 91 92 MPT59- 172 ESAT6
ESAT6- 173 MPT59
[0015] It is well-known in the art that T-cell epitopes are
responsible for the elicitation of the acquired immunity against
TB, whereas B-cell epitopes are without any significant influence
on acquired immunity and recognition of mycobacteria in vivo. Since
such T-cell epitopes are linear and are known to have a minimum
length of 6 amino acid residues, the present invention is
especially concerned with the identification and utilisation of
such T-cell epitopes.
[0016] Hence, in its broadest aspect the invention relates to a
substantially pure polypeptide fragment which
2 a) comprises an amino acid sequence selected from the sequences
shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23,
42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of
72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, and any
one of 168- 171,
[0017] b) comprises a subsequence of the polypeptide fragment
defined in a) which has a length of at least 6 amino acid residues,
said subsequence being immunologically equivalent to the
polypeptide defined in a) with respect to the ability of evoking a
protective immune response against infections with mycobacteria
belonging to the tuberculosis complex or with respect to the
ability of eliciting a diagnostically significant immune response
indicating previous or ongoing sensitization with antigens derived
from mycobacteria belonging to the tuberculosis complex, or
[0018] c) comprises an amino acid sequence having a sequence
identity with the polypeptide defined in a) or the subsequence
defined in b) of at least 70% and at the same time being
immunologically equivalent to the polypeptide defined in a) with
respect to the ability of evoking a protective immune response
against infections with mycobacteria belonging to the tuberculosis
complex or with respect to the ability of eliciting a
diagnostically significant immune response indicating previous or
ongoing sensitization with antigens derived from mycobacteria
belonging to the tuberculosis complex,
[0019] with the proviso that
[0020] i) the polypeptide fragment is in essentially pure form when
consisting of the amino acid sequence 1-96 of SEQ ID NO: 2 or when
consisting of the amino acid sequence 87-108 of SEQ ID NO: 4 fused
to .beta.-galactosidase,
[0021] ii) the degree of sequence identity in c) is at least 95%
when the polypeptide comprises a homologue of a polypeptide which
has the amino acid sequence SEQ ID NO: 12 or a subsequence thereof
as defined in b), and
[0022] iii) the polypeptide fragment contains a threonine residue
corresponding to position 213 in SEQ ID NO: 42 when comprising an
amino acid sequence of at least 6 amino acids in SEQ ID NO: 42.
[0023] Other parts of the invention pertains to the DNA fragments
encoding a polypeptide with the above definition as well as to DNA
fragments useful for determining the presence of DNA encoding such
polypeptides.
DETAILED DISCLOSURE OF THE INVENTION
[0024] In the present specification and claims, the term
"polypeptide fragment" denotes both short peptides with a length of
at least two amino acid residues and at most 10 amino acid
residues, oligopeptides (11-100 amino acid residues), and longer
peptides (the usual interpretation of "polypeptide", i.e. more than
100 amino acid residues in length) as well as proteins (the
functional entity comprising at least one peptide, oligopeptide, or
polypeptide which may be chemically modified by being glycosylated,
by being lipidated, or by comprising prosthetic groups). The
definition of polypeptides also comprises native forms of
peptides/proteins in mycobacteria as well as recombinant proteins
or peptides in any type of expression vectors transforming any kind
of host, and also chemically synthesized peptides.
[0025] In the present context the term "substantially pure
polypeptide fragment" means a polypeptide preparation which
contains at most 5% by weight of other polypeptide material with
which it is natively associated (lower percentages of other
polypeptide material are preferred, e.g. at most 4%, at most 3% at
most 2%, at most 1%, and at most 1/2% ). It is preferred that the
substantially pure polypeptide is at least 96% pure, i.e. that the
polypeptide constitutes at least 96% by weight of total polypeptide
material present in the preparation, and higher percentages are
preferred, such as at least 97%, at least 98%, at least 99%, at
least 99,25%, at least 99,5%, and at least 99,75%. It is especially
preferred that the polypeptide fragment is in "essentially pure
form", i.e. that the polypeptide fragment is essentially free of
any other antigen with which it is natively associated, i.e. free
of any other antigen from bacteria belonging to the tuberculosis
complex. This can be accomplished by preparing the polypeptide
fragment by means of recombinant methods in a non-mycobacterial
host cell as will be described in detail below, or by synthesizing
the polypeptide fragment by the well-known methods of solid or
liquid phase peptide synthesis, e.g. by the method described by
Merrifield or variations thereof.
[0026] The term "subsequence" when used in connection with a
polypeptide of the invention having a SEQ ID NO selected from 2, 4,
6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58,
60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143,
145, 147, 149, 151, 153, and any one of 168-171 denotes any
continuous stretch of at least 6 amino acid residues taken from the
M. tuberculosis derived polypeptides in SEQ ID NO: 2, 4, 6, 8, 10,
12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145,
147, 149, 151, 153, or any one of 168-171 and being immunological
equivalent thereto with respect to the ability of conferring
increased resistance to infections with bacteria belonging to the
tuberculosis complex. Thus, included is also a polypeptide from
different sources, such as other bacteria or even from eukaryotic
cells.
[0027] When referring to an "immunologically equivalent"
polypeptide is herein meant that the polypeptide, when formulated
in a vaccine or a diagnostic agent (i.e. together with a
pharmaceutically acceptable carrier or vehicle and optionally an
adjuvant), will
[0028] I) confer, upon administration (either alone or as an
immunologically active constituent together with other antigens),
an acquired increased specific resistance in a mouse and/or in a
guinea pig and/or in a primate such as a human being against
infections with bacteria belonging to the tuberculosis complex
which is at least 20% of the acquired increased resistance
conferred by Mycobacterium bovis BCG and also at least 20% of the
acquired increased resistance conferred by the parent polypeptide
comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23,
42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of
72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, or any
one of 168-171 (said parent polypeptide having substantially the
same relative location and pattern in a 2DE gel prepared as the 2DE
gel shown in FIG. 6, cf. the examples), the acquired increased
resistance being assessed by the observed reduction in
mycobacterial counts from spleen, lung or other organ homogenates
isolated from the mouse or guinea pig receiving a challenge
infection with a virulent strain of M. tuberculosis, or, in a
primate such as a human being, being assessed by determining the
protection against development of clinical tuberculosis in a
vaccinated group versus that observed in a control group receiving
a placebo or BCG (preferably the increased resistance is higher and
corresponds to at least 50% of the protective immune response
elicited by M. bovis BCG, such as at least 60%, or even more
preferred to at least 80% of the protective immune response
elicited by M. bovis BCG, such as at least 90%; in some cases it is
expected that the increased resistance will supersede that
conferred by M. bovis BCG, and hence it is preferred that the
resistance will be at least 100t, such as at least 110% of said
increased resistance); and/or
[0029] II) elicit a diagnostically significant immune response in a
mammal indicating previous or ongoing sensitization with antigens
derived from mycobacteria belonging to the tuberculosis complex;
this diagnostically significant immune response can be in the form
of a delayed type hypersensitivity reaction which can e.g. be
determined by a skin test, or can be in the form of IFN-.gamma.
release determined e.g. by an IFN-.gamma. assay as described in
detail below. A diagnostically significant response in a skin test
setup will be a reaction which gives rise to a skin reaction which
is at least 5 mm in diameter and which is at least 65% (preferably
at least 75% such as at the least 85%) of the skin reaction
(assessed as the skin reaction diameter) elicited by the parent
polypeptide comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any
one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149, 151,
153, or any one of 168-171.
[0030] The ability of the polypeptide fragment to confer increased
immunity may thus be assessed by measuring in an experimental
animal, e.g. a mouse or a guinea pig, the reduction in
mycobacterial counts from the spleen, lung or other organ
homogenates isolated from the experimental animal which have
received a challenge infection with a virulent strain of
mycobacteria belonging to the tuberculosis complex after previously
having been immunized with the polypeptide, as compared to the
mycobacterial counts in a control group of experimental animals
infected with the same virulent strain, which experimental animals
have not previously been immunized against tuberculosis. The
comparison of the mycobacterial counts may also be carried out with
mycobacterial counts from a group of experimental animals receiving
a challenge infection with the same virulent strain after having
been immunized with Mycobacterium bovis BCG.
[0031] The mycobacterial counts in homogenates from the
experimental animals immunized with a polypeptide fragment
according to the present invention must at the most be 5 times the
counts in the mice or guinea pigs immunized with Mycobacterium
bovis BCG, such as at the most 3 times the counts, and preferrably
at the most 2 times the counts.
[0032] A more relevant assessment of the ability of the polypeptide
fragment of the invention to confer increased resistance is to
compare the incidence of clinical tuberculosis in two groups of
individuals (e.g. humans or other primates) where one group
receives a vaccine as described herein which contains an antigen of
the invention and the other group receives either a placebo or an
other known TB vaccine (e.g. BCG). In such a setup, the antigen of
the invention should give rise to a protective immunity which is
significantly higher than the one provided by the administration of
the placebo (as determined by statistical methods known to the
skilled artisan).
[0033] The "tuberculosis-complex" has its usual meaning, i.e. the
complex of mycobacteria causing TB which are Mycobacterium
tuberculosis, Mycobacterium bovis, Mycobacterium bovis BCG, and
Mycobacterium africanum.
[0034] In the present context the term "metabolizing mycobacteria"
means live mycobacteria that are multiplying logarithmically and
releasing polypeptides into the culture medium wherein they are
cultured.
[0035] The term "sequence identity" indicates a quantitative
measure of the degree of homology between two amino acid sequences
or between two nucleotide sequences of equal length: The sequence
identity can be calculated as 1 ( N ref - N dif ) 100 N ref ,
[0036] wherein N.sub.dif is the total number of non-identical
residues in the two sequences when aligned and wherein N.sub.ref is
the nunber of residues in one of the sequences. Hence, the DNA
sequence AGTCAGTC will have a sequence identity of 75% with the
sequence AATCAATC (N.sub.dif=2 and N.sub.ref=8).
[0037] The sequence identity is used here to illustrate the degree
of identity between the amino acid sequence of a given polypeptide
and the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12,
14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64,
66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147,
149, 151, 153, or any one of 168-171. The amino acid sequence to be
compared with the amino acid sequence shown in SEQ ID NO: 2, 4, 6,
8, 10, 12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58,
60, 62, 64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143,
145, 147, 149, 151, 153, or any one of 168-171 may be deduced from
a DNA sequence, e.g. obtained by hybridization as defined below, or
may be obtained by conventional amino acid sequencing methods. The
sequence identity is preferably determined on the amino acid
sequence of a mature polypeptide, i.e. without taking any leader
sequence into consideration.
[0038] As appears from the above disclosure, polypeptides which are
not identical to the polypeptides having SEQ ID NO: 2, 4, 6, 8, 10,
12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145,
147, 149, 151, 153, or any one of 168-171 are embraced by the
present invention. The invention allows for minor variations which
do not have an adverse effect on immunogenicity compared to the
parent sequences and which may give interesting and useful novel
binding properties or biological functions and immunogenicities
etc.
[0039] Each polypeptide fragment may thus be characterized by
specific amino acid and nucleic acid sequences. It will be
understood that such sequences include analogues and variants
produced by recombinant methods wherein such nucleic acid and
polypeptide sequences have been modified by substitution,
insertion, addition and/or deletion of one or more nucleotides in
said nucleic acid sequences to cause the substitution, insertion,
addition or deletion of one or more amino acid residues in the
recombinant polypeptide. When the term DNA is used in the
following, it should be understood that for the number of purposes
where DNA can be substituted with RNA, the term DNA should be read
to include RNA embodiments which will be apparent for the man
skilled in the art. For the purposes of hybridization, PNA may be
used instead of DNA, as PNA has been shown to exhibit a very
dynamic hybridization profile (PNA is described in Nielsen P E et
al., 1991, Science 254: 1497-1500).
[0040] In both immunodiagnostics and vaccine preparation, it is
often possible and practical to prepare antigens from segments of a
known immunogenic protein or polypeptide. Certain epitopic regions
may be used to produce responses similar to those produced by the
entire antigenic polypeptide. Potential antigenic or immunogenic
regions may be identified by any of a number of approaches, e.g.,
Jameson-Wolf or Kyte-Doolittle antigenicity analyses or Hopp and
Woods (1981) hydrophobicity analysis (see, e.g., Jameson and Wolf,
1988; Kyte and Doo-little, 1982; or U.S. Pat. No. 4,554,101).
Hydrophobicity analysis assigns average hydrophilicity values to
each amino acid residue from these values average hydrophilicities
can be calculated and regions of greatest hydrophilicity
determined. Using one or more of these methods, regions of
predicted antigenicity may be derived from the amino acid sequence
assigned to the polypeptides of the invention.
[0041] Alternatively, in order to identify relevant T-cell epitopes
which are recognized during an immune response, it is also possible
to use a "brute force" method: Since T-cell epitopes are linear,
deletion mutants of polypeptides having SEQ ID NO: 2, 4, 6, 8, 10,
12, 14, 16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145,
147, 149, 151, 153, or any one of 168-171 will, if constructed
systematically, reveal what regions of the polypeptides are
essential in immune recognition, e.g. by subjecting these deletion
mutants to the IFN-.gamma. assay described herein. Another method
utilises overlapping oligomers (preferably synthetic having a
length of e.g. 20 amino -acid residues) derived from polypeptides
having SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42,
48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86,
88, 90, 92, 94, 141, 143, 145, 147, 149, 151, 153, or any one of
168-171. Some of these will give a positive response in the
IFN-.gamma. assay whereas others will not.
[0042] In a preferred embodiment of the invention, the polypeptide
fragment of the invention comprises an epitope for a T-helper
cell.
[0043] Although the minimum length of a T-cell epitope has been
shown to be at least 6 amino acids, it is normal that such epitopes
are constituted of longer stretches of amino acids. Hence it is
preferred that the polypeptide fragment of the invention has a
length of at least 7 amino acid residues, such as at least 8, at
least 9, at least 10, at least 12, at least 14, at least 16, at
least 18, at least 20, at least 22, at least 24, and at least 30
amino acid residues.
[0044] As will appear from the examples, a number of the
polypeptides of the invention are natively translation products
which include a leader sequence (or other short peptide sequences),
whereas the product which can be isolated from short-term culture
filtrates from bacteria belonging to the tuberculosis complex are
free of these sequences. Although it may in some applications be
advantageous to produce these polypeptides recombinantly and in
this connection facilitate export of the polypeptides from the host
cell by including information encoding the leader sequence in the
gene for the polypeptide, it is more often preferred to either
substitute the leader sequence with one which has been shown to be
superior in the host system for effecting export, or to totally
omit the leader sequence (e.g. when producing the polypeptide by
peptide synthesis. Hence, a preferred embodiment of the invention
is a polypeptide which is free from amino acid residues -30 to -1
in SEQ ID NO: 6 and/or -32 to -1 in SEQ ID NO: 10 and/or -8 to -1
in SEQ ID NO: 12 and/or -32 to -1 in SEQ ID NO: 14 and/or -33 to -1
in SEQ ID NO: 42 and/or -38 to -1 in SEQ ID NO: 52 and/or -33 to -1
in SEQ ID NO: 56 and/or -56 to -1 in SEQ ID NO: 58 and/or -28 to -1
in SEQ ID NO: 151.
[0045] In another preferred embodiment, the polypeptide fragment of
the invention is free from any signal sequence; this is especially
interesting when the polypeptide fragment is produced synthetically
but even when the polypeptide fragments are produced recombinantly
it is normally acceptable that they are not exported by the host
cell to the periplasm or the extracellular space; the polypeptide
fragments can be recovered by traditional methods (cf. the
discussion below) from the cytoplasm after disruption of the host
cells, and if there is need for refolding of the polypeptide
fragments, general refolding schemes can be employed, cf. e.g. the
disclosure in WO 94/18227 where such a general applicable refolding
method is described.
[0046] A suitable assay for the potential utility of a given
olypeptide fragment derived from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,
16, any one of 17-23, 42, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,
68, 70, any one of 72-86, 88, 90, 92, 94, 141, 143, 145, 147, 149,
151, 153, or any one of 168-171 is to assess the ability of the
polypeptide fragment to effect IFN-.gamma. release from primed
memory T-lymphocytes. Polypeptide fragments which have this
capability are according to the invention especially interesting
embodiments of the invention: It is contemplated that polypeptide
fragments which stimulate T lymphocyte immune response shortly
after the onset of the infection are important in the control of
the mycobacteria causing the infection before the mycobacteria have
succeeded in multiplying up to the number of bacteria that would
have resulted in fulminant infection.
[0047] Thus, an important embodiment of the invention is a
polypeptide fragment defined above which
[0048] 1) induces a release of IFN-.gamma. from primed memory
T-lymphocytes withdrawn from a mouse within 2 weeks of primary
infection or within 4 days after the mouse has been rechallenge
infected with mycobacteria belonging to the tuberculosis complex,
the induction performed by the addition of the polypeptide to a
suspension comprising about 200,000 spleen cells per ml, the
addition of the polypeptide resulting in a concentration of 1-4
.mu.g polypeptide per ml suspension, the release of IFN-.gamma.
being assessable by determination of IFN-.gamma. in supernatant
harvested 2 days after the addition of the polypeptide to the
suspension, and/or
[0049] 2) induces a release of IFN-.gamma. of at least 1,500 pg/ml
above background level from about 1,000,000 human PBMC (peripheral
blood mononuclear cells) per ml isolated from TB patients in the
first phase of infection, or from healthy BCG vaccinated donors, or
from healthy contacts to TB patients, the induction being performed
by the addition of the polypeptide to a suspension comprising the
about 1,000,000 PBMC per ml, the addition of the polypeptide
resulting in a concentration of 1-4 .mu.g polypeptide per ml
suspension, the release of IFN-.gamma. being assessable by
determination of IFN-.gamma. in supernatant harvested 2 days after
the addition of the polypeptide to the suspension; and/or
[0050] 3) induces an IFN-.gamma. release from bovine PBMC derived
from animals previously sensitized with mycobacteria belonging to
the tuberculosis complex, said release being at least two times the
release observed from bovine PBMC derived from animals not
previously sensitized with mycobacteria belonging to the
tuberculosis complex.
[0051] Preferably, in alternatives 1 and 2, the release effected by
the polypeptide fragment gives rise to at least 1,500 pg/ml
IFN-.gamma. in the supernatant but higher concentrations are
preferred, e.g. at least 2,000 pg/ml and even at least 3,000 pg/ml
IFN-.gamma. in the supernatant. The IFN-.gamma. release from bovine
PBMC can e.g. be measured as the optical density (OD) index over
background in a standard cytokine ELISA and should thus be at least
two, but higher numbers such as at least 3, 5, 8, and 10 are
preferred.
[0052] The polypeptide fragments of the invention preferably
comprises an amino acid sequence of at least 6 amino acid residues
in length which has a higher sequence identity than 70 percent with
SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, any one of 17-23, 42, 48,
50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, any one of 72-86, 88,
90, 92, 94, 141, 143, 145, 147, 149, 151, 153, or any one of
168-171. A preferred minimum percentage of sequence identity is at
least 80%, such as at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, and at least 99.5%.
[0053] As mentioned above, it will normally be interesting to omit
the leader sequences from the polypeptide fragments of the
invention. However, by producing fusion polypeptides, superior
characteristics of the polypeptide fragments of the invention can
be achieved. For instance, fusion partners which facilitate export
of the polypeptide when produced recombinantly, fusion partners
which facilitate purification of the polypeptide, and fusion
partners which enhance the immunogenicity of the polypeptide
fragment of the invention are all interesting possibilities.
Therefore, the invention also pertains to a fusion polypeptide
comprising at least one polypeptide fragment defined above and at
least one fusion partner. The fusion partner can, in order to
enhance immunogenicity, e.g. be selected from the group consisting
of another polypeptide fragment as defined above (so as to allow
for multiple expression of relevant epitopes), and an other
polypeptide derived from a bacterium belonging to the tuberculosis
complex, such as ESAT-6, MPB64, MPT64, and MPB59 or at least one
T-cell epitope of any of these antigens. Other immunogenicity
enhancing polypeptides which could serve as fusion partners are
T-cell epitopes (e.g. derived from the polypeptides ESAT-6, MPB64,
MPT64, or MPB59) or other immunogenic epitopes enhancing the
immunogenicity of the target gene product, e.g. lymphokines such as
INF-.gamma., IL-2 and IL-12. In order to facilitate expression
and/or purification the fusion partner can e.g. be a bacterial
fimbrial protein, e.g. the pilus components pilin and papA; protein
A; the ZZ-peptide (ZZ-fusions are marketed by Pharmacia in Sweden);
the maltose binding protein; gluthatione S-transferase;
.beta.-galactosidase; or poly-histidine.
[0054] Other interesting fusion partners are polypeptides which are
lipidated and thereby effect that the immunogenic polypeptide is
presented in a suitable manner to the immune system. This effect is
e.g. known from vaccines based on the Borrelia burgdorferi OspA
polypeptide, wherein the lipidated membrane anchor in the
polypeptide confers a self-adjuvating effect to the polypeptide
(which is natively lipidated) when isolated from cells producing
it. In contrast, the OspA polypeptide is relatively silent
immunologically when prepared without the lipidation anchor.
[0055] As evidenced in Example 6A, the fusion polypeptide
consisting of MPT59 fused directly N-terminally to ESAT-6 enhances
the immunogenicity of ESAT-6 beyond what would be expected from the
immunogenicities of MPT59 and ESAT-6 alone. The precise reason for
this surprising finding is not yet known, but it is expected that
either the presence of both antigens lead to a synergistic effect
with respect to immunogenicity or the presence of a sequence
N-terminally to the ESAT-6 sequence protects this immune dominant
protein from loss of important epitopes known to be present in the
N-terminus. A third, alternative, possibility is that the presence
of a sequence C-terminally to the MPT59 sequence enhances the
immunologic properties of this antigen.
[0056] Hence, one part of the invention pertains to a fusion
polypeptide fragment which comprises a first amino acid sequence
including at least one stretch of amino acids constituting a S-cell
epitope derived from the M. tuberculosis protein ESAT-6 or MPT59,
and a second amino acid sequence including at least one T-cell
epitope derived from a M. tuberculosis protein different from
ESAT-6 (if the first stretch of amino acids are derived from
ESAT-6) or MPT59 (if the first stretch of amino acids are derived
from MPT59) and/or including a stretch of amino acids which
protects the first amino acid sequence from in vivo degradation or
post-translational processing. The first amino acid sequence may be
situated N- or C-terminally to the second amino acid sequence, but
in line with the above considerations regarding protection of the
ESAT-6 N-terminus it is preferred that the first amino acid
sequence is C-terminal to the second when the first amino acid
sequence is derived from ESAT-6.
[0057] Although only the effect of fusion between MPT59 and ESAT6
has been investigated at present, it is believed that ESAT6 and
MPT59 or epitopes derived therefrom could be advantageously be
fused to other fusion partners having substantially the same effect
on overall immunogenicity of the fusion construct. Hence, it is
preferred that such a fusion polypeptide fragment according of the
invention is one, wherein the at least one T-cell epitope included
in the second amino acid sequence is derived from a M. tuberculosis
polypeptide (the "parent" polypeptide) selected from the group
consisting of a polypeptide fragment according to the present
invention and described in detail above and in the examples, or the
amino acid sequence could be derived from any one of the M.
tuberculosis proteins DnaK, GroEL, urease, glutamine synthetase,
the proline rich complex, L-alanine dehydrogenase, phosphate
binding protein, Ag 85 complex, HBHA (heparin binding
hemagglutinin), MPT51, MPT64, superoxide dismutase, 19 kDa
lipoprotein, .alpha.-crystallin, GroES, MPT59 (when the first amino
acid sequence is derived from ESAT-6), and ESAT-6 (when the first
amino acid sequence is derived from MPT59). It is preferred that
the first and second T-cell epitopes each have a sequence identity
of at least 70% with the natively occurring sequence in the
proteins from which they are derived and it is even further
preferred that the first and/or second amino acid sequence has a
sequence identity of at least 70% with the protein from which they
are derived. A most preferred embodiment of this fusion polypeptide
is one wherein the first amino acid sequence is the amino acid
sequence of ESAT-6 or MPT59 and/or the second amino acid sequence
is the full-length amino acid sequence of the possible "parent"
polypeptides listed above.
[0058] In the most preferred embodiment, the fusion polypeptide
fragment comprises ESAT-6 fused to MPT59 (advantageously, ESAT-6 is
fused to the C-terminus of MPT59) and in one special embodiment,
there are no linkers introduced between the two amino acid
sequences constituting the two parent polypeptide fragments.
[0059] Another part of the invention pertains to a nucleic acid
fragment in isolated form which
[0060] 1) comprises a nucleic acid sequence which encodes a
polypeptide or fusion polypeptide as defined above, or comprises a
nucleic acid sequence complementary thereto, and/or
[0061] 2) has a length of at least 10 nucleotides and hybridizes
readily under stringent hybridization conditions (as defined in the
art, i.e. 5-10.degree. C. under the melting point T.sub.m, cf.
Sambrook et al, 1989, pages 11.45-11.49) with a nucleic acid
fragment which has a nucleotide sequence selected from
3 SEQ ID NO: 1 or a sequence complementary thereto, SEQ ID NO: 3 or
a sequence complementary thereto, SEQ ID NO: 5 or a sequence
complementary thereto, SEQ ID NO: 7 or a sequence complementary
thereto, SEQ ID NO: 9 or a sequence complementary thereto, SEQ ID
NO: 11 or a sequence complementary thereto, SEQ ID NO: 13 or a
sequence complementary thereto, SEQ ID NO: 15 or a sequence
complementary thereto, SEQ ID NO: 41 or a sequence complementary
thereto, SEQ ID NO: 47 or a sequence complementary thereto, SEQ ID
NO: 49 or a sequence complementary thereto, SEQ ID NO: 51 or a
seguence complementary thereto, SEQ ID NO: 53 or a sequence
complementary thereto, SEQ ID NO: 55 or a sequence complementary
thereto, SEQ ID NO: 57 or a sequence complementary thereto, SEQ ID
NO: 59 or a sequence complementary thereto, SEQ ID NO: 61 or a
sequence complementary thereto, SEQ ID NO: 63 or a sequence
complementary thereto, SEQ ID NO: 65 or a sequence complementary
thereto, SEQ ID NO: 67 or a sequence complementary thereto, SEQ ID
NO: 69 or a sequence complementary thereto, SEQ ID NO: 71 or a
sequence complementary thereto, SEQ ID NO: 87 or a sequence
complementary thereto, SEQ ID NO: 89 or a sequence complementary
thereto, SEQ ID NO: 91 or a sequence complementary thereto, SEQ ID
NO: 93 or a sequence complementary thereto, SEQ ID NO: 140 or a
sequence complementary thereto, SEQ ID NO: 142 or a sequence
complementary thereto, SEQ ID NO: 144 or a sequence complementary
thereto, SEQ ID NO: 146 or a sequence complementary thereto, SEQ ID
NO: 148 or a sequence complementary thereto, SEQ ID NO: 150 or a
sequence complementary thereto, and SEQ ID NO: 152 or a sequence
complementary thereto,
[0062] with the proviso that when the nucleic acid fragment
comprises a subsequence of SEQ ID NO: 41, then the nucleic acid
fragment contains an A corresponding to position 781 in SEQ ID NO:
41 and when the nucleic acid fragment comprises a subsequence of a
nucleotide sequence exactly complementary to SEQ ID NO: 41, then
the nucleic acid fragment comprises a T corresponding to position
781 in SEQ ID NO: 41.
[0063] It is preferred that the nucleic acid fragment is a DNA
fragment.
[0064] To provide certainty of the advantages in accordance with
the invention, the preferred nucleic acid sequence When employed
for hybridization studies or assays includes sequences that are
complementary to at least a 10 to 40, or so, nucleotide stretch of
the selected sequence. A size of at least 10 nucleotides in length
helps to ensure that the fragment will be of sufficient length to
form a duplex molecule that is both stable and selective. Molecules
having complementary sequences over stretches greater than 10 bases
in length are generally preferred, though, in order to increase
stability and selectivity of the hybrid, and thereby improve the
quality and degree of specific hybrid molecules obtained. Hence,
the term "subsequence" when used in connection with the nucleic
acid fragments of the invention is intended to indicate a
continuous stretch of at least 10 nucleotides exhibits the above
hybridization pattern. Normally this will require a minimum
sequence identity of at least 70% with a subsequence of the
hybridization partner having SEQ ID NO: 1, 3, 5, 7, 9, 11, 12, 15,
21, 41, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 87, 89,
91, 93, 140, 142, 144, 146, 148, 150, or 152. It is preferred that
the nucleic acid fragment is longer than 10 nucleotides, such as at
least 15, at least 20, at least 25, at least 30, at least 35, at
least 40, at least 45, at least 50, at least 55, at least 60, at
least 65, at least 70, and at least 80 nucleotides long, and the
sequence identity should preferable also be higher than 70%, such
as at least 75%, at least 80%, at least 85%, at least 90%, at least
92%, at least 94%, at least 96%, and at least 98%. It is most
preferred that the sequence identity is 100%. Such fragments may be
readily prepared by, for example, directly synthesizing the
fragment by chemical means, by application of nucleic acid
reproduction technology, such as the PCR technology of U.S. Pat.
No. 4,603,102, or by introducing selected sequences into
recombinant vectors for recombinant production.
[0065] It is well known that the same amino acid may be encoded by
various codons, the codon usage being related, inter alia, to the
preference of the organisms in question expressing the nucleotide
sequence. Thus, at least one nucleotide or codon of a nucleic acid
fragment of the invention may be exchanged by others which, when
expressed, result in a polypeptide identical or substantially
identical to the polypeptide encoded by the nucleic acid fragment
in question. The invention thus allows for variations in the
sequence such as substitution, insertion (including introns),
addition, deletion and rearrangement of one or more nucleotides,
which variations do not have any substantial effect on the
polypeptide encoded by the nucleic acid fragment or a subsequence
thereof. The term "substitution" is intended to mean the
replacement of one or more nucleotides in the full nucleotide
sequence with one or more different nucleotides, "addition" is
understood to mean the addition of one or more nucleotides at
either end of the full nucleotide sequence, "insertion" is intended
to mean the introduction of one or more nucleotides within the full
nucleotide sequence, "deletion" is intended to indicate that one or
more nucleotides have been deleted from the full nucleotide
sequence whether at either end of the sequence or at any suitable
point within it, and "rearrangement" is intended to mean that two
or more nucleotide residues have been exchanged with each
other.
[0066] The nucleotide sequence to be modified may be of CDNA or
genomic origin as discussed above, but may also be of synthetic
origin. Furthermore, the sequence may be of mixed cDNA and genomic,
mixed cDNA and synthetic or genomic and synthetic origin as
discussed above. The sequence may have been modified, e.g. by
site-directed mutagenesis, to result in the desired nucleic acid
fragment encoding the desired polypeptide. The following discussion
focused on modifications of nucleic acid encoding the polypeptide
should be understood to encompass also such possibilities, as well
as the possibility of building up the nucleic acid by ligation of
two or more DNA fragments to obtain the desired nucleic acid
fragment, and combinations of the above-mentioned principles.
[0067] The nucleotide sequence may be modified using any suitable
technique which results in the production of a nucleic acid
fragment encoding a polypeptide of the invention. The modification
of the nucleotide sequence encoding the amino acid sequence of the
polypeptide of the invention should be one which does not impair
the immunological function of the resulting polypeptide.
[0068] A preferred method of preparing variants of the antigens
disclosed herein is site-directed mutagenesis. This technique is
useful in the preparation of individual peptides, or biologically
functional equivalent proteins or peptides, derived from the
antigen sequences, through specific mutagenesis of the underlying
nucleic acid. The technique further provides a ready ability to
prepare and test sequence variants, for example, incorporating one
or more of the foregoing considerations, by introducing one or more
nucleotide sequence changes into the nucleic acid. Site-specific
mutagenesis allows the production of mutants through the use of
specific oligonucleotide sequences which encode the nucleotide
sequence of the desired mutation, as well as a sufficient number of
adjacent nucleotides, to provide a primer sequence of sufficient
size and sequence complexity to form a stable duplex on both sides
of the deletion junction being traversed. Typically, a primer of
about 17 to 25 nucleotides in length is preferred, with about 5 to
10 residues on both sides of the junction of the sequence being
altered. In general, the technique of site-specific mutagenesis is
well known in the art as exemplified by publications (Adelman et
al., 1983). As will be appreciated, the technique typically employs
a phage vector which exists in both a single stranded and double
stranded form. Typical vectors useful in site-directed mutagenesis
include vectors such as the M13 phage (Messing et al., 1981). These
phage are readily commercially available and their use is generally
well known to those skilled in the art.
[0069] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector which
includes within its sequence a nucleic acid sequence which encodes
the polypeptides of the invention. An oligonucleotide primer
bearing the desired mutated sequence is prepared, generally
synthetically, for example by the method of Crea et al. (1978).
This primer is then annealed with the single-stranded vector, and
subjected to DNA polymerizing enzymes such as E. coli polymerase I
Klenow fragment, in order to complete the synthesis of the
mutation-bearing strand. Thus, a heteroduplex is formed wherein one
strand encodes the original non-mutated sequence and the second
strand bears the desired mutation. This heteroduplex vector is then
used to transform appropriate cells, such as E. coli cells, and
clones are selected which include recombinant vectors bearing the
mutated sequence arrangement.
[0070] The preparation of sequence variants of the selected nucleic
acid fragments of the invention using site-directed mutagenesis is
provided as a means of producing potentially useful species of the
genes and is not meant to be limiting as there are other ways in
which sequence variants of the nucleic acid fragments of the
invention may be obtained. For example, recombinant vectors
encoding the desired genes may be treated with mutagenic agents to
obtain sequence variants (see, e.g., a method described by
Eichenlaub, 1979) for the mutagenesis of plasmid DNA using
hydroxylamine.
[0071] The invention also relates to a replicable expression vector
which comprises a nucleic acid fragment defined above, especially a
vector which comprises a nucleic acid fragment encoding a
polypeptide fragment of the invention.
[0072] The vector may be any vector which may conveniently be
subjected to recombinant DNA procedures, and the choice of vector
will often depend on the host cell into which it is to be
introduced. Thus, the vector may be an autonomously replicating
vector, i.e. a vector which exists as an extrachromosomal entity,
the replication of which is independent of chromosomal replication;
examples of such a vector are a plasmid, phage, cosmid,
mini-chromosome or virus. Alternatively, the vector may be one
which, when introduced in a host cell, is integrated in the host
cell genome and replicated together with the chromosome(s) into
which it has been integrated.
[0073] Expression vectors may be constructed to include any of the
DNA segments disclosed herein. Such DNA might encode an antigenic
protein specific for virulent strains of mycobacteria or even
hybridization probes for detecting mycobacteria nucleic acids in
samples. Longer or shorter DNA segments could be used, depending on
the antigenic protein desired. Epitopic regions of the proteins
expressed or encoded by the disclosed DNA could be included as
relatively short segments of DNA. A wide variety of expression
vectors is possible including, for example, DNA segments encoding
reporter gene products useful for identification of heterologous
gene products and/or resistance genes such as antibiotic resistance
genes which may be useful in identifying transformed cells.
[0074] The vector of the invention may be used to transform cells
so as to allow propagation of the nucleic acid fragments of the
invention or so as to allow expression of the polypeptide fragments
of the invention. Hence, the invention also pertains to a
transformed cell harbouring at least one such vector according to
the invention, said cell being one which does not natively harbour
the vector and/or the nucleic acid fragment of the invention
contained therein. Such a transformed cell (which is also a part of
the invention) may be any suitable bacterial host cell or any other
type of cell such as a unicellular eukaryotic organism, a fungus or
yeast, or a cell derived from a multicellular organism, e.g. an
animal or a plant. It is especially in cases where glycosylation is
desired that a mammalian cell is used, although glycosylation of
proteins is a rare event in prokaryotes. Normally, however, a
prokaryotic cell is preferred such as a bacterium belonging to the
genera Mycobacterium, Salmonella, Pseudomonas, Bacillus and
Eschericia. It is preferred that the transformed cell is an E.
coli, B. subtilis, or M. bovis BCG cell, and it is especially
preferred that the transformed cell expresses a polypeptide
according of the invention. The latter opens for the possibility to
produce the polypeptide of the invention by simply recovering it
from the culture containing the transformed cell. In the most
preferred embodiment of this part of the invention the transformed
cell is Mycobacterium bovis BCG strain: Danish 1331, which is the
Mycobacterium bovis strain Copenhagen from the Copenhagen BCG
Laboratory, Statens Seruminstitut, Denmark.
[0075] The nucleic acid fragments of the invention allow for the
recombinant production of the polypeptides fragments of the
invention. However, also isolation from the natural source is a way
of providing the polypeptide fragments as is peptide synthesis.
[0076] Therefore, the invention also pertains to a method for the
preparation of a polypeptide fragment of the invention, said method
comprising inserting a nucleic acid fragment as defined above into
a vector which is able to replicate in a host cell, introducing the
resulting recombinant vector into the host cell (transformed cells
may be selected using various techniques, including screening by
differential hybridization, identification of fused reporter gene
products, resistance markers, anti-antigen antibodies and the
like), culturing the host cell in a culture medium under conditions
sufficient to effect expression of the polypeptide (of course the
cell may be cultivated under conditions appropriate to the
circumstances, and if DNA is desired, replication conditions are
used), and recovering the polypeptide from the host cell or culture
medium; or
[0077] isolating the polypeptide from a short-term culture filtrate
as defined in claim 1; or
[0078] isolating the polypeptide from whole mycobacteria of the
tuberculosis complex or from lysates or fractions thereof, e.g.
cell wall containing fractions, or
[0079] synthesizing the polypeptide by solid or liquid phase
peptide synthesis.
[0080] The medium used to grow the transformed cells may be any
conventional medium suitable for the purpose. A suitable vector may
be any of the vectors described above, and an appropriate host cell
may be any of the cell types listed above. The methods employed to
construct the vector and effect introduction thereof into the host
cell may be any methods known for such purposes within the field of
recombinant DNA. In the following a more detailed description of
the possibilities will be given:
[0081] In general, of course, prokaryotes are preferred for the
initial cloning of nucleic sequences of the invention and
constructing the vectors useful in the invention. For example, in
addition to the particular strains mentioned in the more specific
disclosure below, one may mention by way of example, strains such
as E. coli K12 strain 294 (ATCC No. 31446), E. coli B, and E. coli
X 1776 (ATCC No. 31537). These examples are, of course, intended to
be illustrative rather than limiting.
[0082] Prokaryotes are also preferred for expression. The
aforementioned strains, as well as E. coli W3110 (F-, lambda-,
prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis,
or other enterobacteriaceae such as Salmonella typhimurium or
Serratia marcesans, and various Pseudomonas species may be used.
Especially interesting are rapid-growing mycobacteria, e.g. M.
smegmatis, as these bacteria have a high degree of resemblance with
mycobacteria of the tuberculosis complex and therefore stand a good
chance of reducing the need of performing post-translational
modifications of the expression product.
[0083] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species (see, e.g., Bolivar et al., 1977,
Gene 2: 95). The pBR322 plasmid contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR plasmid, or other microbial
plasmid or phage must also contain, or be modified to contain,
promoters which can be used by the microorganism for
expression.
[0084] Those promoters most commonly used in recombinant DNA
construction include the B-lactamase (penicillinase) and lactose
promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel
et al., 1979) and a tryptophan (trp) promoter system (Goeddel et
al., 1979; EPO Appl. Publ. No. 0036776). While these are the most
commonly used, other microbial promoters have been discovered and
utilized, and details concerning their nucleotide sequences have
been published, enabling a skilled worker to ligate them
functionally with plasmid vectors (Siebwenlist et al., 1980).
Certain genes from prokaryotes may be expressed efficiently in E.
coli from their own promoter sequences, precluding the need for
addition of another promoter by artificial means.
[0085] After the recombinant preparation of the polypeptide
according to the invention, the isolation of the polypeptide may
for instance be carried out by affinity chromatography (or other
conventional biochemical procedures based on chromatography), using
a monoclonal antibody which substantially specifically binds the
polypeptide according to the invention. Another possibility is to
employ the simultaneous electroelution technique described by
Andersen et al. in J. Immunol. Methods 161: 29-39.
[0086] According to the invention the post-translational
modifications involves lipidation, glycosylation, cleavage, or
elongation of the polypeptide.
[0087] In certain aspects, the DNA sequence information provided by
this invention allows for the preparation of relatively short DNA
(or RNA or PNA) sequences having the ability to specifically
hybridize to mycobacterial gene sequences. In these aspects,
nucleic acid probes of an appropriate length are prepared based on
a consideration of the relevant sequence. The ability of such
nucleic acid probes to specifically hybridize to the mycobacterial
gene sequences lend them particular utility in a variety of
embodiments. Most importantly, the probes can be used in a variety
of diagnostic assays for detecting the presence of pathogenic
organisms in a given sample. However, either uses are envisioned,
including the use of the sequence information for the preparation
of mutant species primers, or primers for use in preparing other
genetic constructs.
[0088] Apart from their use as starting points for the synthesis of
polypeptides of the invention and for hybridization probes (useful
for direct hybridization assays or as primers in e.g. PCR or other
molecular amplification methods) the nucleic acid fragments of the
invention may be used for effecting in vivo expression of antigens,
i.e. the nucleic acid fragments may be used in so-called DNA
vaccines. Recent research have revealed that a DNA fragment cloned
in a vector which is non-replicative in eukaryotic cells may be
introduced into an animal (including a human being) by e.g.
intramuscular injection or percutaneous administration (the
so-called "gene gun" approach). The DNA is taken up by e.g. muscle
cells and the gene of interest is expressed by a promoter which is
functioning in eukaryotes, e.g. a viral promoter, and the gene
product thereafter stimulates the immune system. These newly
discovered methods are reviewed in Ulmer et al., 1993, which hereby
is included by reference.
[0089] Hence, the invention also relates to a vaccine comprising a
nucleic acid fragment according to the invention, the vaccine
effecting in vivo expression of antigen by an animal, including a
human being, to whom the vaccine has been administered, the amount
of expressed antigen being effective to confer substantially
increased resistance to infections with mycobacteria of the
tuberculosis complex in an animal, including a human being.
[0090] The efficacy of such a "DNA vaccines" can possibly be
enhanced by administering the gene encoding the expression product
together with a DNA fragment encoding a polypeptide which has the
capability of modulating an immune response. For instance, a gene
encoding lymphokine precursors or lymphokines (e.g. IFN-.gamma.,
IL-2, or IL-12) could be administered together with the gene
encoding the immunogenic protein, either by administering two
separate DNA fragments or by administering both DNA fragments
included in the same vector. It also is a possibility to administer
DNA fragments comprising a multitude of nucleotide sequences which
each encode relevant epitopes of the polypeptides disclosed herein
so as to effect a continuous sensitization of the immune system
with a broad spectrum of these epitopes.
[0091] As explained above, the polypeptide fragments of the
invention are excellent candidates for vaccine constituents or for
constituents in an immune diagnostic agent due to their
extracellular presence in culture media containing metabolizing
virulent mycobacteria belonging to the tuberculosis complex, or
because of their high homologies with such extracellular antigens,
or because of their absence in M. bovis BCG.
[0092] Thus, another part of the invention pertains to an
immunologic composition comprising a polypeptide or fusion
polypeptide according to the invention. In order to ensure optimum
performance of such an immunologic composition it is preferred that
it comprises an immunologically and pharmaceutically acceptable
carrier, vehicle or adjuvant.
[0093] Suitable carriers are selected from the group consisting of
a polymer to which the polypeptide(s) is/are bound by hydrophobic
non-covalent interaction, such as a plastic, e.g. polystyrene, or a
polymer to which the polypeptide(s) is/are covalently bound, such
as a polysaccharide, or a polypeptide, e.g. bovine serum albumin,
ovalbumin or keyhole limpet haemocyanin. Suitable vehicles are
selected from the group consisting of a diluent and a suspending
agent. The adjuvant is preferably selected from the group
consisting of dimethyldioctadecylammon- ium bromide (DDA), Quil A,
poly I:C, Freund's incomplete adjuvant, IFN-.gamma., IL-2, IL-12,
monophosphoryl lipid A (MPL), and muramyl dipeptide (MDP).
[0094] A preferred immunologic composition according to the present
invention comprising at least two different polypeptide fragments,
each different polypeptide fragment being a polypeptide or a fusion
polypeptide defined above. It is preferred that the immunologic
composition comprises between 3-20 different polypeptide fragments
or fusion polypeptides.
[0095] Such an immunologic composition may preferably be in the
form of a vaccine or in the form of a skin test reagent.
[0096] In line with the above, the invention therefore also pertain
to a method for producing an immunologic composition according to
the invention, the method comprising preparing, synthesizing or
isolating a polypeptide according to the invention, and
solubilizing or dispersing the polypeptide in a medium for a
vaccine, and optionally adding other M. tuberculosis antigens
and/or a carrier, vehicle and/or adjuvant substance.
[0097] Preparation of vaccines which contain peptide sequences as
active ingredients is generally well understood in the art, as
exemplified by U.S. Pat. Nos.4,608,251; 4,601,903; 4,599,231;
4,599,230; 4,596,792; and 4,578,770, all incorporated herein by
reference. Typically, such vaccines are prepared as injectables
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. The preparation may also be emulsified. The active
immunogenic ingredient is often mixed with excipients which are
pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients are, for example, water, saline,
dextrose, glycerol, ethanol, or the like, and combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, or adjuvants which enhance the effectiveness of
the vaccines.
[0098] The vaccines are conventionally administered parenterally,
by injection, for example, either subcutaneously or
intramuscularly. Additional formulations which are suitable for
other modes of administration include suppositories and, in some
cases, oral formulations. For suppositories, traditional binders
and carriers may include, for example, polyalkalene glycols or
triglycerides; such suppositories may be formed from mixtures
containing the active ingredient in the range of 0.5% to 10%,
preferably 1-2%. Oral formulations include such normally employed
excipients as, for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate, and the like. These compositions take the form
of solutions, suspensions, tablets, pills, capsules, sustained
release formulations or powders and contain 10-95% of active
ingredient, preferably 25-70%.
[0099] The proteins may be formulated into the vaccine as neutral
or salt forms. Pharmaceutically acceptable salts include acid
addition salts (formed with the free amino groups of the peptide)
and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic
oxalic, tartaric, mandelic, and the like. Salts formed with the
free carboxyl groups may also be derived from inorganic bases such
as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the
like.
[0100] The vaccines are administered in a manner compatible with
the dosage formulation, and in such amount as will be
therapeutically effective and immunogenic. The quantity to be
administered depends on the subject to be treated, including, e.g.,
the capacity of the individual's immune system to mount an immune
response, and the degree of protection desired. Suitable dosage
ranges are of the order of several hundred micrograms active
ingredient per vaccination with a preferred range from about 0.1
.mu.g to 1000 .mu.g, such as in the range from about 1 .mu.g to 300
.mu.g, and especially in the range from about 10 .mu.g to 50 .mu.g.
Suitable regimens for initial administration and booster shots are
also variable but are typified by an initial administration
followed by subsequent inoculations or other administrations.
[0101] The manner of application may be varied widely. Any of the
conventional methods for administration of a vaccine are
applicable. These are believed to include oral application on a
solid physiologically acceptable base or in a physiologically
acceptable dispersion, parenterally, by injection or the like. The
dosage of the vaccine will depend on the route of administration
and will vary according to the age of the person to be vaccinated
and, to a lesser degree, the size of the person to be
vaccinated.
[0102] Some of the polypeptides of the vaccine are sufficiently
immunogenic in a vaccine, but for some of the others the immune
response will be enhanced if the vaccine further comprises an
adjuvant substance.
[0103] Various methods of achieving adjuvant effect for the vaccine
include use of agents such as aluminum hydroxide or phosphate
(alum), commonly used as 0.05 to 0.1 percent solution in phosphate
buffered saline, admixture with synthetic polymers of sugars
(Carbopol) used as 0.25 percent solution, aggregation of the
protein in the vaccine by heat treatment with temperatures ranging
between 700 to 101.degree. C. for 30 second to 2 minute periods
respectively. Aggregation by reactivating with pepsin treated (Fab)
antibodies to albumin, mixture with bacterial cells such as C.
parvum or endotoxins or lipopolysaccharide components of
gram-negative bacteria, emulsion in physiologically acceptable oil
vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20
percent solution of a perfluorocarbon (Fluosol-DA) used as a block
substitute may also be employed. According to the invention DDA
(dimethyldioctadecylammonium bromide) is an interesting candidate
for an adjuvant, but also Freund's complete and incomplete
adjuvants as well as QuilA and RIBI are interesting possibilities.
Further possibilities are monophosphoryl lipid A (MPL), and muramyl
dipeptide (MDP).
[0104] Another highly interesting (and thus, preferred) possibility
of achieving adjuvant effect is to employ the technique described
in Gosselin et al., 1992 (which is hereby incorporated by reference
herein). In brief, the presentation of a relevant antigen such as
an antigen of the present invention can be enhanced by conjugating
the antigen to antibodies (or antigen binding antibody fragments)
against the Fc.gamma. receptors on monocytes/macrophages.
Especially conjugates between antigen and anti-Fc.gamma.RI have
been demonstrated to enhance immunogenicity for the purposes of
vaccination.
[0105] Other possibilities involve the use of immune modulating
substances such as lymphokines (e.g. IFN-.gamma., IL-2 and IL-12)
or synthetic IFN-.gamma.inducers such as poly I:C in combination
with the above-mentioned adjuvants. As discussed in example 3, it
is contemplated that such mixtures of antigen and adjuvant will
lead to superior vaccine formulations.
[0106] In many instances, it will be necessary to have multiple
administrations of the vaccine, usually not exceeding six
vaccinations, more usually not exceeding four vaccinations and
preferably one or more, usually at least about three vaccinations.
The vaccinations will normally be at from two to twelve week
intervals, more usually from three to five week intervals. Periodic
boosters at intervals of 1-5 years, usually three years, will be
desirable to maintain the desired levels of protective immunity.
The course of the immunization may be followed by in vitro
proliferation assays of PBL (peripheral blood lymphocytes)
co-cultured with ESAT-6 or ST-CF, and especially by measuring the
levels of IFN-.gamma. released form the primed lymphocytes. The
assays may be performed using conventional labels, such as
radionuclides, enzymes, fluorescers, and the like. These techniques
are well known and may be found in a wide variety of patents, such
as U.S. Pat. Nos. 3,791,932; 4,174,384 and 3,949,064, as
illustrative of these types of assays.
[0107] Due to genetic variation, different individuals may react
with immune responses of varying strength to the same polypeptide.
Therefore, the vaccine according to the invention may comprise
several different polypeptides in order to increase the immune
response. The vaccine may comprise two or more polypeptides, where
all of the polypeptides are as defined above, or some but not all
of the peptides may be derived from a bacterium belonging to the M.
tuberculosis complex. In the latter example the polypeptides not
necessarily fulfilling the criteria set forth above for
polypeptides may either act due to their own immunogenicity or
merely act as adjuvants. Examples of such interesting polypeptides
are MPB64, MPT64, and MPB59, but any other substance which can be
isolated from mycobacteria are possible candidates.
[0108] The vaccine may comprise 3-20 different polypeptides, such
as 3-10 different polypeptides.
[0109] One reason for admixing the polypeptides of the invention
with an adjuvant is to effectively activate a cellular immune
response. However, this effect can also be achieved in other ways,
for instance by expressing the effective antigen in a vaccine in a
non-pathogenic microorganism. A well-known example of such a
microorganism is Mycobacterium bovis BCG.
[0110] Therefore, another important aspect of the present invention
is an improvement of the living BCG vaccine presently available,
which is a vaccine for immunizing an animal, including a human
being, against TB caused by mycobacteria belonging to the
tuberculosis-complex, comprising as the effective component a
microorganism, wherein one or more copies of a DNA sequence
encoding a polypeptide as defined above has been incorporated into
the genome of the microorganism in a manner allowing the
microorganism to express and secrete the polypeptide.
[0111] In the present context the term "genome" refers to the
chromosome of the microorganisms as well as extrachromosomally DNA
or RNA, such as plasmids. It is, however, preferred that the DNA
sequence of the present invention has been introduced into the
chromosome of the non-pathogenic microorganism, since this will
prevent loss of the genetic material introduced.
[0112] It is preferred that the non-pathogenic microorganism is a
bacterium, e.g. selected from the group consisting of the genera
Mycobacterium, Salmonella, Pseudomonas and Eschericia.
[0113] It is especially preferred that the non-pathogenic
microorganism is Mycobacterium bovis BCG, such as Mycobacterium
bovis BCG strain: Danish 1331.
[0114] The incorporation of one or more copies of a nucleotide
sequence encoding the polypeptide according to the invention in a
mycobacterium from a M. bovis BCG strain will enhance the
immunogenic effect of the BCG strain. The incorporation of more
than one copy of a nucleotide sequence of the invention is
contemplated to enhance the immune response even more, and
consequently an aspect of the invention is a vaccine wherein at
least 2 copies of a DNA sequence encoding a polypeptide is
incorporated in the genome of the microorganism, such as at least 5
copies. The copies of DNA sequences may either be identical
encoding identical polypeptides or be variants of the same DNA
sequence encoding identical or homologues of a polypeptide, or in
another embodiment be different DNA sequences encoding different
polypeptides where at least one of the polypeptides is according to
the present invention.
[0115] The living vaccine of the invention can be prepared by
cultivating a transformed non-pathogenic cell according to the
invention, and transferring these cells to a medium for a vaccine,
and optionally adding a carrier, vehicle and/or adjuvant
substance.
[0116] The invention also relates to a method of diagnosing TB
caused by Mycobacterium tuberculosis, Mycobacterium africanum or
Mycobacterium bovis in an animal, including a human being,
comprising intradermally injecting, in the animal, a polypeptide
according to the invention or a skin test reagent described above,
a positive skin response at the location of injection being
indicative of the animal having TB, and a negative skin response at
the location of injection being indicative of the animal not having
TB. A positive response is a skin reaction having a diameter of at
least 5 mm, but larger reactions are preferred, such as at least 1
cm, 1.5 cm, and at least 2 cm in diameter. The composition used as
the skin test reagent can be prepared in the same manner as
described for the vaccines above.
[0117] In line with the disclosure above pertaining to vaccine
preparation and use, the invention also pertains to a method for
immunising an animal, including a human being, against TB caused by
mycobacteria belonging to the tuberculosis complex, comprising
administering to the animal the polypeptide of the invention, or a
vaccine composition of the invention as described above, or a
living vaccine described above. Preferred routes of administration
are the parenteral (such as intravenous and intraarterially),
intraperitoneal, intramuscular, subcutaneous, intradermal, oral,
buccal, sublingual, nasal, rectal or transdermal route.
[0118] The protein ESAT-6 which is present in short-term culture
filtrates from mycobacteria as well as the esat-6 gene in the
mycobacterial genome has been demonstrated to have a very limited
distribution in other mycobacterial strains that M. tuberculosis,
e.g. esat-6 is absent in both BCG and the majority of mycobacterial
species isolated from the environment, such as M. avium and M.
terrae. It is believed that this is also the case for at least one
of the antigens of the present invention and their genes and
therefore, the diagnostic embodiments of the invention are
especially well-suited for performing the diagnosis of on-going or
previous infection with virulent mycobacterial strains of the
tuberculosis complex, and it is contemplated that it will be
possible to distinguish between 1) subjects (animal or human) which
have been previously vaccinated with e.g. BCG vaccines or subjected
to antigens from non-virulent mycobacteria and 2) subjects which
have or have had active infection with virulent mycobacteria.
[0119] A number of possible diagnostic assays and methods can be
envisaged:
[0120] When diagnosis of previous or ongoing infection with
virulent mycobacteria is the aim, a blood sample comprising
mononuclear cells (i.a. T-lymphocytes) from a patient could be
contacted with a sample of one or more polypeptides of the
invention. This contacting can be performed in vitro and a positive
reaction could e.g. be proliferation of the T-cells or release
cytokines such as .gamma.-interferon into the extracellular phase
(e.g. into a culture supernatant); a suitable in vivo test would be
a skin test as described above. It is also conceivable to contact a
serum sample from a subject to contact with a polypeptide of the
invention, the demonstration of a binding between antibodies in the
serum sample and the polypeptide being indicative of previous or
ongoing infection.
[0121] The invention therefore also relates to an in vitro method
for diagnosing ongoing or previous sensitization in an animal or a
human being with bacteria belonging to the tuberculosis complex,
the method comprising providing a blood sample from the animal or
human being, and contacting the sample from the animal with the
polypeptide of the invention, a significant release into the
extracellular phase of at least one cytokine by mononuclear cells
in the blood sample being indicative of the animal being
sensitized. By the term "significant release" is herein meant that
the release of the cytokine is significantly higher than the
cytokine release from a blood sample derived from a non-tuberculous
subject (e.g. a subject which does not react in a traditional skin
test for TB). Normally, a significant release is at least two times
the release observed from such a sample.
[0122] Alternatively, a sample of a possibly infected organ may be
contacted with an antibody raised against a polypeptide of the
invention. The demonstration of the reaction by means of methods
well-known in the art between the sample and the antibody will be
indicative of ongoing infection. It is of course also a possibility
to demonstrate the presence of anti-mycobacterial antibodies in
serum by contacting a serum sample from a subject with at least one
of the polypeptide fragments of the invention and using well-known
methods for visualizing the reaction between the antibody and
antigen.
[0123] Also a method of determining the presence of mycobacterial
nucleic acids in an animal, including a human being, or in a
sample, comprising administering a nucleic acid fragment of the
invention to the animal or incubating the sample with the nucleic
acid fragment of the invention or a nucleic acid fragment
complementary thereto, and detecting the presence of hybridized
nucleic acids resulting from the incubation (by using the
hybridization assays which are well-known in the art), is also
included in the invention. Such a method of diagnosing TB might
involve the use of a composition comprising at least a part of a
nucleotide sequence as defined above and detecting the presence of
nucleotide sequences in a sample from the animal or human being to
be tested which hybridize with the nucleic acid fragment (or a
complementary fragment) by the use of PCR technique.
[0124] The fact that certain of the disclosed antigens are not
present in M. bovis BCG but are present in virulent mycobacteria
point them out as interesting drug targets; the antigens may
constitute receptor molecules or toxins which facilitate the
infection by the mycobacterium, and if such functionalities are
blocked the infectivity of the mycobacterium will be diminshed.
[0125] To determine particularly suitable drug targets among the
antigens of the invention, the gene encoding at least one of the
polypeptides of the invention and the necessary control sequences
can be introduced into avirulent strains of mycobacteria (e.g. BCG)
so as to determine which of the polypeptides are critical for
virulence. Once particular proteins are identified as critical
for/contributory to virulence, anti-mycobacterial agents can be
designed rationally to inhibit expression of the critical genes or
to attack the critical gene products. For instance, antibodies or
fragments thereof (such as Fab and (Fab').sub.2 fragments can be
prepared against such critical polypeptides by methods known in the
art and thereafter used as prophylactic or therapeutic agents.
Alternatively, small molecules can be screened for their ability to
selectively inhibit expression of the critical gene products, e.g.
using recombinant expression systems which include the gene's
endogenous promoter, or for their ability to directly interfere
with the action of the target. These small molecules are then used
as therapeutics or as prophylactic agents to inhibit mycobacterial
virulence.
[0126] Alternatively, anti-mycobacterial agents which render a
virulent mycobacterium avirulent can be operably linked to
expression control sequences and used to transform a virulent
mycobacterium. Such anti-mycobacterial agents inhibit the
replication of a specified mycobacterium upon transcription or
translation of the agent in the mycobacterium. Such a "newly
avirulent" mycobacterium would constitute a superb alternative to
the above described modified BCG for vaccine purposes since it
would be immunologically very similar to a virulent mycobacterium
compared to e.g. BCG.
[0127] Finally, a monoclonal or polyclonal antibody, which is
specifically reacting with a polypeptide of the invention in an
immuno assay, or a specific binding fragment of said antibody, is
also a part of the invention. The production of such polyclonal
antibodies requires that a suitable animal be immunized with the
polypeptide and that these antibodies are subsequently isolated,
suitably by immune affinity chromatography. The production of
monoclonals can be effected by methods well-known in the art, since
the present invention provides for adequate amounts of antigen for
both immunization and screening of positive hybridomas.
LEGENDS TO THE FIGURES
[0128] FIG. 1: Long term memory immune mice are very efficiently
protected towards an infection with M. tuberculosis. Mice were
given a challenge of M. tuberculosis and spleens were isolated at
different time points. Spleen lymphocytes were stimulated in vitro
with ST-CF and the release of IFN-.gamma. investigated (panel A).
The counts of CFU in the spleens of the two groups of mice are
indicated in panel B. The memory immune mice control infection
within the first week and produce large quantities of IFN-.gamma.
in response to antigens in ST-CF.
[0129] FIG. 2: T cells involved in protective immunity are
predominantly directed to molecules from 6-12 and 17-38 kDa.
Splenic T cells were isolated four days after the challenge with M.
tuberculosis and stimulated in vitro with narrow molecular mass
fractions of ST-CF. The release of IFN-.gamma.was investigated
[0130] FIG. 3: Nucleotide sequence (SEQ ID NO: 1) of cfp7. The
deduced amino acid sequence (SEQ ID NO: 2) of CFP7 is given in
conventional one-letter code below the nucleotide sequence. The
putative ribosome-binding site is written in underlined italics as
are the putative -10 and -35 regions. Nucleotides written in bold
are those encoding CFP7.
[0131] FIG. 4. Nucleotide sequence (SEQ ID NO: 3) of cfp9. The
deduced amino acid sequence (SEQ ID NO: 4) of CFP9 is given in
conventional one-letter code below the nucleotide sequence. The
putative ribosome-binding site Shine Delgarno sequence is written
in underlined italics as are the putative -10 and -35 regions.
Nucleotides in bold writing are those encoding CFP9. The nucleotide
sequence obtained from the lambda 226phage is double
underlined.
[0132] FIG. 5: Nucleotide sequence of mpt5l. The deduced amino acid
sequence of MPT51 is given in a one-letter code below the
nucleotide sequence. The signal is indicated in italics. the
putative potential ribosome-binding site is underlined. The
nucleotide difference and amino acid difference compared to the
nucleotide sequence of MPB51 (Ohara et al., 1995) are underlined at
position 780. The nucleotides given in italics are not present in
M. tuberculosis H37Rv.
[0133] FIG. 6: the position of the purified antigens in the 2DE
system have been determined and mapped in a reference gel. The
newly purified antigens are encircled and the position of
well-known proteins are also indicated.
EXAMPLE 1
[0134] Identification of single culture filtrate antigens involved
in protective immunity
[0135] A group of efficiently protected mice was generated by
infecting 8-12 weeks old female C57Bl/6j mice with 5.times.10.sup.4
M. tuberculosis i.v. After 30 days of infection the mice were
subjected to 60 days of antibiotic treatment with isoniazid and
were then left for 200-240 days to ensure the establishment of
resting long-term memory immunity. Such memory immune mice are very
efficiently protected against a secondary infection (FIG. 1). Long
lasting immunity in this model is mediated by a population of
highly reactive CD4 cells recruited to the site of infection and
triggered to produce large amounts of IFN-.gamma. in response to
ST-CF (FIG. 1) (Andersen et al. 1995).
[0136] We have used this model to identify single antigens
recognized by protective T cells. Memory immune mice were
reinfected with 1.times.10.sup.6 M. tuberculosis i.v. and splenic
lymphocytes were harvested at day 4-6 of reinfection, a time point
where this population is highly reactive to ST-CF. The antigens
recognized by these T cells were mapped by the multi-elution
technique (Andersen and Heron, 1993). This technique divides
complex protein mixtures separated in SDS-PAGE into narrow
fractions in a physiological buffer. These fractions were used to
stimulate spleen lymphocytes in vitro and the release of
IFN-.gamma. was monitored (FIG. 2). Long-term memory immune mice
did not recognize these fractions before TB infection, but splenic
lymphocytes obtained during the recall of protective immunity
recognized a range of culture filtrate antigens and peak production
of IFN-.gamma. was found in response to proteins of apparent
molecular weight 6-12 and 17-30 kDa (FIG. 2). It is therefore
concluded that culture filtrate antigens within these regions are
the major targets recognized by memory effector T-cells triggered
to release IFN-.gamma. during the first phase of a protective
immune response.
EXAMPLE 2
[0137] Cloning of genes expressing low mass culture filtrate
antigens
[0138] In example 1 it was demonstrated that antigens in the low
molecular mass fraction are recognized strongly by cells isolated
from memory immune mice. Monoclonal antibodies (mAbs) to these
antigens were therefore generated by immunizing with the low mass
fraction in RIBI adjuvant (first and second immunization) followed
by two injections with the fractions in aluminium hydroxide. Fusion
and cloning of the reactive cell lines were done according to
standard procedures (Kohler and Milstein 1975). The procedure
resulted in the provision of two mAbs: ST-3 directed to a 9 kDa
culture filtrate antigen (CFP9) and PV-2 directed to a 7 kDa
antigen (CFP7), when the molecular weight is estimated from
migration of the antigens in an SDS-PAGE.
[0139] In order to identify the antigens binding to the Mab's, the
following experiments were carried out:
[0140] The recombinant .lambda.gt11 M. tuberculosis DNA library
constructed by R. Young (Young, R. A. et al. 1985) and obtained
through the World Health Organization IMMTUB programme
(WHO.0032.wibr) was screened for phages expressing gene products
which would bind the monoclonal antibodies ST-3 and PV-2.
[0141] Approximately 1.times.10.sup.5 pfu of the gene library
(containing approximately 25% recombinant phages) were plated on
Eschericia coli Y1090 (DlacU169, proA.sup.+, Dlon, araD139, supF,
trpC22::tn100 [pMC9] ATCC#37197) in soft agar and incubated for 2,5
hours at 42.degree. C.
[0142] The plates were overlaid with sheets of nitrocellulose
saturated with isopropyl-.beta.-D-thiogalactopyranoside and
incubation was continued for 2,5 hours at 37.degree. C. The
nitrocellulose was removed and incubated with samples of the
monoclonal antibodies in PBS with Tween 20 added to a final
concentration of 0.05%. Bound monoclonal antibodies were visualized
by horseradish peroxidase-conjugated rabbit anti-mouse
immunoglobulins (P260, Dako, Glostrup, DK) and a staining reaction
involving 5,5',3,3'-tetramethylbenzidine and H.sub.2O.sub.2.
[0143] Positive plaques were recloned and the phages originating
from a single plaque were used to lysogenize E. coli Y1089
(DlacU169, proA.sup.+, Dlon, araD139, strA, hfl150 [chr::tn10]
[pMC9] ATCC nr. 37196). The resultant lysogenic strains were used
to propagate phage particles for DNA extraction. These lysogenic E.
coli strains have been named:
[0144] AA226 (expressing ST-3 reactive polypeptide CFP9) which has
been deposited 28 June 1993 with the collection of Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the
accession number DSM 8377 and in accordance with the provisions of
the Budapest Treaty, and AA242 (expressing PV-2 reactive
polypeptide CFP7) which has
[0145] been deposited 28 June 1993 with the collection of Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the
accession number DSM 8379 and in accordance with the provisions of
the Budapest Treaty.
[0146] These two lysogenic E. coli strains are disclosed in WO
95/01441 as are the mycobacterial polypeptide products expressed
thereby. However, no information concerning the amino acid
sequences of these polypeptides or their genetic origin are given,
and therefore only the direct expression products of AA226 and
AA242 are made available to the public.
[0147] The st-3 binding protein is expressed as a protein fused to
.beta.-galactosidase, whereas the pv-2 binding protein appears to
be expressed in an unfused version.
[0148] Sequencing of the nucleotide sequence encoding the PV-2 and
ST-3 binding protein
[0149] In order to obtain the nucleotide sequence of the gene
encoding the pv-2 binding protein, the approximately 3 kb M.
tuberculosis derived EcoRI--EcoRI fragment from AA242 was subcloned
in the EcoRI site in the pBluescriptSK+(Stratagene) and used to
transform E. coli XL-lBlue (Stratagene).
[0150] Similarly, to obtain the nucleotide sequence of the gene
encoding the st-3 binding protein, the approximately 5 kb M.
tuberculosis derived EcoRI--EcoRI fragment from AA226 was subcloned
in the EcoRI site in the pBluescriptSK+(Stratagene) and used to
transform E. coli XL-lBlue (Stratagene).
[0151] The complete DNA sequence of both genes were obtained by the
dideoxy chain termination method adapted for supercoiled DNA by use
of the Sequenase DNA sequencing kit version 1.0 (United States
Biochemical Corp., Cleveland, Ohio) and by cycle sequencing using
the Dye Terminator system in combination with an automated gel
reader (model 373A; Applied Biosystems) according to the
instructions provided. The sequences DNA are shown in SEQ ID NO: 1
(CFP7) and in SEQ ID NO: 3 (CFP9) as well as in FIGS. 3 and 4,
respectively. Both strands of the DNA were sequenced.
[0152] CFP7
[0153] An open reading frame (ORF) encoding a sequence of 96 amino
acid residues was identified from an ATG start codon at position
91-93 extending to a TAG stop codon at position 379-381. The
deduced amino acid sequence is shown in SEQ ID NO: 2 (and in FIG. 3
where conventional one-letter amino acid codes are used).
[0154] CFP7 appear to be expressed in E. coli as an unfused
version. The nucleotide sequence at position 78-84 is expected to
be the Shine Delgarno sequence and the sequences from position
47-50 and 14-19 are expected to be the -10 and -35 regions,
respectively:
[0155] CFP9
[0156] The protein recognised by ST-3 was produced as a
.beta.-galactosi-dase fusion protein, when expressed from the AA226
lambda phage. The fusion protein had an approx. size of 116-117 kDa
(Mw for .beta.-galactosidase 116.25 kDa) which may suggest that
only part of the CFP9 gene was included in the lambda clone
(AA226).
[0157] Based on the 90 bp nucleotide sequence obtained on the
insert from lambda phage AA226, a search of homology to the
nucleotide sequence of the M. tuberculosis genome was performed in
the Sanger database (Sanger Mycobacterium tuberculosis
database):
[0158] http://www.sanger.ac.uk/pathogens/TB-blast-server.html;
[0159] Williams, 1996). 100% identity to the cloned sequence was
found on the MTCY48 cosmid. An open reading frame (ORF) encoding a
sequence of 109 amino acid residues was identified from a GTG start
codon at position 141-143 extending to a TGA stop codon at position
465-467. The deduced amino acid sequence is shown in FIG. 4 using
conventional one letter code.
[0160] The nucleotide sequence at position 123-130 is expected to
be the Shine Delgarno sequence and the sequences from position
73-78 and 4-9 are expected to be the -10 and -35 region
respectively (FIG. 4). The ORF overlapping with the 5'-end of the
sequence of AA229 is shown in FIG. 4 by double underlining.
[0161] Subcloning CFP7 and CFP9 in expression vectors
[0162] The two ORFs encoding CFP7 and CFP9 were PCR cloned into the
pMST24 (Theisen et al., 1995) expression vector pRVN01 or the
pQE-32 (QIAGEN) expression vector pRVN02, respectively.
[0163] The PCR amplification was carried out in a thermal reactor
(Rapid cycler, Idaho Technology, Idaho) by mixing 10 ng plasmid DNA
with the mastermix (0.5 .mu.M of each oligonucleotide tide primer,
0.25 .mu.M BSA (Stratagene), Low sall buffer (20 mM Tris-HCl, pH
8.8, 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 2 mM MgSO.sub.4 and
0,1% Triton X-100) (Stratagene), 0.25 mM of each deoxynucleoside
triphosphate and 0.5 U Taq Plus Long DNA polymerase (Stratagene)).
Final volume was 10 .mu.l (all concentrations given are
concentrations in the final volume). Predenaturation was carried
out at 94.degree. C. for 30 s. 30 cycles of the following was
performed; Denaturation at 94.degree. C. for 30 s, annealing at
55.degree. C. for 30 s and elongation at 72.degree. C. for 1
min.
[0164] The oligonucleotide primers were synthesised automatically
on a DNA synthesizer (Applied Biosystems, Forster City, Calif.,
ABI-391, PCR-mode), deblocked, and purified by ethanol
precipitation.
[0165] The cfp7 oligonucleotides (TABLE 1) were synthesised on the
basis of the nucleotide sequence from the CFP7 sequence (FIG. 3).
The oligonucleotides were engineered to include an SmaI restriction
enzyme site at the 5' end and a BamHI restriction enzyme site at
the 3' end for directed subcloning.
[0166] The cfp9 oligonucleotides (TABLE 1) were synthesized partly
on the basis of the nucleotide sequence from the sequence of the
AA229 clone and partly from the identical sequence found in the
Sanger database cosmid MTCY48 (FIG. 4). The oligonucleotides were
engineered to include a SmaI restriction enzyme site at the 5' end
and a HindIII restriction enzyme site at the 3' end for directed
subcloning.
[0167] CFP7
[0168] By the use of PCR a SmaI site was engineered immediately 5'
of the first codon of the ORF of 291 bp, encoding the cfp7 gene, so
that only the coding region would be expressed, and a BamHI site
was incorporated right after the stop codon at the 3' end. The 291
bp PCR fragment was cleaved by SmaI and BamHI, purified from an
agarose gel and subcloned into the SmaI--BamHI sites of the pMST24
expression vector. Vector DNA containing the gene fusion was used
to transform the E. coli XL1-Blue (pRVN01).
[0169] CFP9
[0170] By the use of PCR a SmaI site was engineered immediately 5'
of the first codon of an ORF of 327 bp, encoding the cfp9 gene, so
that only the coding region would be expressed, and a HindIII site
was incorporated after the stop codon at the 3' end. The 327 bp PCR
fragment was cleaved by SmaI and HindIII, purified from an agarose
gel, and subcloned into the SmaI--HindIII sites of the pQE-32
(QIAGEN) expression vector. Vector DNA containing the gene fusion
was used to transform the E. coli XL1-Blue (pRVN02).
[0171] Purification of recombinant CFP7 and CFP9
[0172] The ORFs were fused N-terminally to the (His).sub.6-tag (cf.
EP-A-0 282 242). Recombinant antigen was prepared as follows:
Briefly, a single colony of E. coli harbouring either the pRVN01 or
the pRVN02 plasmid, was inoculated into Luria-Bertani broth
containing 100 .mu.g/ml ampicillin and 12.5 .mu.g/ml tetracycline
and grown at 37.degree. C. to OD.sub.60onm=0.5. IPTG
(isopropyl-.beta.-D-thiogalactoside) was then added to a final
concentration of 2 mM (expression was regulated either by the
strong IPTG inducible Ptac or the T5 promoter) and growth was
continued for further 2 hours. The cells were harvested by
centrifugation at 4,200.times.g at 4.degree. C. for 8 min. The
pelleted bacteria were stored overnight at -20.degree. C. The
pellet was resuspended in BC 40/100 buffer (20 rrM Tris-HCl pH 7.9,
20% glycerol, 100 mM KCl, 40 mM Imidazole) and cells were broken by
sonication (5 times for 30 s with intervals of 30 s) at 4.degree.
C. followed by centrifugation at 12,000.times.g for 30 min at
4.degree. C., the supernatant (crude extract) was used for
purification of the recombinant antigens.
[0173] The two Histidine fusion proteins (His-rCFP7 and His-rCFP9)
were purified from the crude extract by affinity chromatography on
a Ni.sup.2+-NTA column from QIAGEN with a volume of 100 ml.
His-rCFP7 and His-rCFP9 binds to Ni.sup.2+. After extensive washes
of the column in BC 40/100 buffer, the fusion protein was eluted
with a BC 1000/100 buffer containing 100 mM imidazole, 20 mM Tris
pH 7.9, 20% glycerol and 1 M KCl. subsequently, the purified
products were dialysed extensively against 10 mM Tris pH 8.0.
His-rCFP7 and His-rCFP9 were then separated from contaminants by
fast protein liquid chromatography (FPLC) over an anion-exchange
column (Mono Q, Pharmacia, Sweden). in 10 mM Tris pH 8.0 with a
linear gradient of NaCl from 0 to 1 M. Aliquots of the fractions
were analyzed by 10%-20% gradient sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE). Fractions containing
purified either purified His-rCFP7 or His-rCFP9 were pooled.
4TABLE 1 Sequence of the cfp7 and cfp9 oligonucleotides.sup.a.
Orientation and Position.sup.b oligonucleotide Sequences (5'
.fwdarw. 3') (nucleotide) Sense pvR3 GCAACACCCGGGATGTCGCAAATCATG
91-105 (SEQ ID NO: 43) (SEQ ID NO: 1) stR2
GTAACACCCGGGGTGGCCGCCGACCCG 141-155 (SEQ ID NO: 44) (SEQ ID NO: 3)
Antisense pvF4 CTACTAAGCTTGGATCCCTAGCCGCCCCATTTGGCGG 381-362 (SEQ
ID NO: 45) (SEQ ID NO: 1) stF2
CTACTAAGCTTCCATGGTCAGGTCTTTTCGATGCTTAC 467-447 (SEQ ID NO: 46) (SEQ
ID NO: 3) .sup.aThe cfp7 oligonucleotides were based on the
nucleotide sequence shown in FIG. 3 (SEQ ID NO: 1). The cfp9
oligonucleotides were based on the nucleotide sequence shown in
FIG. 4 (SEQ ID NO: 3). Nucleotides underlined are not contained in
the nucleotide sequence of cfp7 and cfp9. .sup.bThe positions
referred to are of the non-underlined part of the primers and
correspond to the nucleotide sequence shown in FIG. 3 and FIG. 4,
respectively.
EXAMPLE 2A
[0174] Identification of antigens which are not expressed in BCG
strains.
[0175] In an effort to control the treat of TB, attenuated bacillus
Calmette-Gurin (BCG) has been used as a live attenuated vaccine.
BCG is an attenuated derivative of a virulent Mycobacterium bovis.
The original BCG from the Pasteur Institute in Paris, France was
developed from 1908 to 1921 by 231 passages in liquid culture and
has never been shown to revert to virulence in animals, indicating
that the attenuating mutation(s) in BCG are stable deletions and/or
multiple mutations which do not readily revert. While physiological
differences between BCG and M. tuberculosis and M. bovis has been
noted, the attenuating mutations which arose during serial passage
of the original BCG strain has been unknown until recently. The
first mutations described are the loss of the gene encoding MPB64
in some BCG strains (Li et al., 1993, Oettinger and Andersen, 1994)
and the gene encoding ESAT-6 in all BCG strain tested (Harboe et
al., 1996), later 3 large deletions in BCG have been identified
(Mahairas et al., 1996). The region named RD1 includes the gene
encoding ESAT-6 and an other (RD2) the gene encoding MPT64. Both
antigens have been shown to have diagnostic potential and ESAT-6
has been shown to have properties as a vaccine candidate (cf.
PCT/DK94/00273 and PCT/DK/00270). In order to find new M.
tuberculosis specific diagnostic antigens as well as antigens for a
new vaccine against TB, the RD1 region (17.499 bp) of M.
tuberculosis H37Rv has been analyzed for Open Reading Frames (ORF).
ORFs with a minimum length of 96 bp have been predicted using the
algorithm described by Borodovsky and McIninch (1993), in total 27
ORFs have been predicted, 20 of these have possible diagnostic
and/or vaccine potential, as they are deleted from all known BCG
strains. The predicted ORFs include ESAT-6 (RD1-ORF7) and CFP10
(RD1-ORF6) described previously (Srensen et al., 1995), as a
positive control for the ability of the algorithm. In the present
is described the potential of 7 of the predicted antigens for
diagnosis of TB as well as potential as candidates for a new
vaccine against TB.
[0176] Seven open reading frames (ORF) from the 17,499 kb RD1
region (Accession no. U34848) with possible diagnostic and vaccine
potential have been identified and cloned.
[0177] Identification of the ORF's rd1-orf2, rd1-orf3, rd1-orf4,
rd1-orf5, rd1-orf2, rd1-orf9a, and rd1-orf9b.
[0178] The nucleotide sequence of rd1-orf2 from M. tuberculosis
H37Rv is set forth in SEQ ID NO: 71. The deduced amino acid
sequence of RDl-ORF2 is set forth in SEQ ID NO: 72.
[0179] The nucleotide sequence of rd1-orf3 from M. tuberculosis
H37Rv is set forth in SEQ ID NO: 87. The deduced amino acid
sequence of RD1-ORF2 is set forth in SEQ ID NO: 88.
[0180] The nucleotide sequence of rd1-orf4 from M. tuberculosis
H37Rv is set forth in SEQ ID NO: 89. The deduced amino acid
sequence of RD1-ORF2 is set forth in SEQ ID NO: 90.
[0181] The nucleotide sequence of rd1-orf5 from M. tuberculosis
H37Rv is set forth in SEQ ID NO: 91. The deduced amino acid
sequence of RD1-ORF2 is set forth in SEQ ID NO: 92.
[0182] The nucleotide sequence of rd1-orf8 from M. tuberculosis
H37Rv is set forth in SEQ ID NO: 67. The deduced amino acid
sequence of RD1-ORF2 is set forth in SEQ ID NO: 68.
[0183] The nucleotide sequence of rd1-orf9a from M. tuberculosis
H37Rv is set forth in SEQ ID NO: 93. The deduced amino acid
sequence of RD1-ORF2 is set forth in SEQ ID NO: 94.
[0184] The nucleotide sequence of rd1-orf9b from M. tuberculosis
H37Rv is set forth in SEQ ID NO: 69. The deduced amino acid
sequence of RD1-ORF2 is set forth in SEQ ID NO: 70.
[0185] The DNA sequence rd1-orf2 (SEQ ID NO: 71) contained an open
reading frame starting with an ATG codon at position 889-891 and
ending with a termination codon (TAA) at position 2662-2664
(position numbers referring to the location in RD1). The deduced
amino acid sequence (SEQ ID NO: 72) contains 591 residues
corresponding to a molecular weight of 64,525.
[0186] The DNA sequence rd1-orf3 (SEQ ID NO: 87) contained an open
reading frame starting with an ATG codon at position 2807-2809 and
ending with a termination codon (TAA) at position 3101-3103
(position numbers referring to the location in RD1). The deduced
amino acid sequence (SEQ ID NO: 88) contains 98 residues
corresponding to a molecular weight of 9,799.
[0187] The DNA sequence rd1-orf4 (SEQ ID NO: 89) contained an open
reading frame starting with a GTG codon at position 4014-4012 and
ending with a termination codon (TAG) at position 3597-3595
(position numbers referring to the location in RD1). The deduced
amino acid sequence (SEQ ID NO: 90) contains 139 residues
corresponding to a molecular weight of 14,210.
[0188] The DNA sequence rd1-orf5 (SEQ ID NO: 91) contained an open
reading frame starting with a GTG codon at position 3128-3130 and
ending with a termination codon (TGA) at position 4241-4243
(position numbers referring to the location in RD1). The deduced
amino acid sequence (SEQ ID NO: 92) contains 371 residues
corresponding to a molecular weight of 37,647.
[0189] The DNA sequence rd1-orf8 (SEQ ID NO: 67) contained an open
reading frame starting with a GTG codon at position 5502-5500 and
ending with a termination codon (TAG) at position 5084-5082
(position numbers referring to the location in RD1), and the
deduced amino acid sequence (SEQ ID NO: 68) contains 139 residues
with a molecular weight of 11,737.
[0190] The DNA sequence rd1-orf9a (SEQ ID NO: 93) contained an open
reading frame starting with a GTG codon at position 6146-6148 and
ending with a termination codon (TAA) at position 7070-7072
(position numbers referring to the location in RD1). The deduced
amino acid sequence (SEQ ID NO: 94) contains 308 residues
corresponding to a molecular weight of 33,453.
[0191] The DNA sequence rd1-orf9b (SEQ ID NO: 69) contained an open
reading frame starting with an ATG codon at position 5072-5074 and
ending with a termination codon (TAA) at position 7070-7072
(position numbers referring to the location in RD1). The deduced
amino acid sequence (SEQ ID NO: 70) contains 666 residues
corresponding to a molecular weight of 70,650.
[0192] Cloning of the ORF's rd1-orf2, rd1-orf3, rd1-orf4, rd1-orf5,
rd1-orf8, rd1-orf9a, and rd1-orf9b.
[0193] The ORF's rd1-orf2, rd1-orf3, rd1-orf4, rd1-orf5, rd1-orf8,
rd1-orf9a and rd1-orf9b were PCR cloned in the pMST24 (Theisen et
al., 1995) (rd1-orf3) or the pQE32 (QIAGEN) (rd1-orf2, rd1-orf4,
rd1-orf5, rd1-orf8, rd1-orf9a and rd1-orf9b) expression vector.
Preparation of oligonucleotides and PCR amplification of the
rd1-orf encoding genes, was carried out as described in example 2.
Chromosomal DNA from M. tuberculosis H37Rv was used as template in
the PCR reactions. Oligonucleotides were synthesized on the basis
of the nucleotide sequence from the RD1 region (Accession no.
U34848). The oligonucleotide primers were engineered to include an
restriction enzyme site at the 5' end and at the 3' end by which a
later subcloning was possible. Primers are listed in TABLE 2.
[0194] rd1-orf2.
[0195] A BamHI site was engineered immediately 5' of the first
codon of rd1-orf2, and a HindIII site was incorporated right after
the stop codon at the 3' end. The gene rd1-orf2 was subcloned in
pQE32, giving pTO96.
[0196] rd1-orf3.
[0197] A SmaI site was engineered immediately 5' of the first codon
of rd1-orf3, and a NcoI site was incorporated right after the stop
codon at the 3' end. The gene rd1-orf3 was subcloned in pMST24,
giving pTO87.
[0198] rd1-orf34.
[0199] A BamHI site was engineered immediately 5' of the first
codon of rd1-orf4, and a HindIII site was incorporated right after
the stop codon at the 3' end. The gene rd1-orf4 was subcloned in
pQE32, giving pTO89.
[0200] rd1-orf5.
[0201] A BamHI site was engineered immediately 5' of the first
codon of rd1-orf5, and a HindIII site was incorporated right after
the stop codon at the 3' end. The gene rd1-orf5 was subcloned in
pQE32, giving pTO88.
[0202] rd1-orf8.
[0203] A BamHI site was engineered immediately 5' of the first
codon of rd1-orf8, and a NcoI site was incorporated right after the
stop codon at the 3' end. The gene rd1-orf8 was subcloned in
pMST24, giving pTO98.
[0204] rd1-orf9a.
[0205] A BamHI site was engineered immediately 5' of the first
codon of rd1-orf9a, and a HindIII site was incorporated right after
the stop codon at the 3' end. The gene rd1-orf9a was subcloned in
pQE32, giving pTO91.
[0206] rd1-orf9b.
[0207] A ScaI site was engineered immediately 5' of the first codon
of rd1-orf9b, and a Hind III site was incorporated right after the
stop codon at the 3' end. The gene rd1-orf9b was subcloned in
pQE32, giving pTO90.
[0208] The PCR fragments were digested with the suitable
restriction enzymes, purified from an agarose gel and cloned into
either pMST24 or pQE-32. The seven constructs were used to
transform the E. coli XL1-Blue. Endpoints of the gene fusions were
determined by the dideoxy chain termination method. Both strands of
the DNA were sequenced.
[0209] Purification of recombinant RD1-ORF2, RD1-ORF3, RD1-ORF4,
RD1-ORF5, RD1-ORF8, RD1-ORF9a and RD1-ORF9b.
[0210] The rRD1-ORFs were fused N-terminally to the
(His).sub.6-tag. Recombinant antigen was prepared as described in
example 2 (with the exception that pTO91 was expressed at
30.degree. C. and not at 37.degree. C.), using a single colony of
E. coli harbouring either the pTO87, pTO88, pTO89, pTO90, pTO91,
pTO96 or pTO98 for inoculation. Purification of recombinant antigen
by Ni.sup.2+ affinity chromatography was also carried out as
described in example 2. Fractions containing purified
His-rRD1-ORF2, His-rRD1-ORF3 His-rRD1-ORF4, His-rRD1-ORF5,
His-rRD1-ORF8, His-rRD1-ORF9a or His-rRD1-ORF9b were pooled. The
His-rRD1-ORF's were extensively dialysed against 10 mM Tris/HCl, pH
8.5, 3 M urea followed by an additional purification step performed
on an anion exchange column (Mono Q) using fast protein liquid
chromatography (FPLC) (Pharmacia, Uppsala, Sweden). The
purification was carried out in 10 mM Tris/HCl, pH 8.5, 3 M urea
and protein was eluted by a linear gradient of NaCl from 0 to 1 M.
Fractions containing the His-rRD1-ORF's were pooled and
subsequently dialysed extensively against 25 mM Hepes, pH 8.0
before use.
5TABLE 2 Sequence of the rd1-orf's oligonucleotides.sup.a.
Orientation and oligonucleotide Sequences (5' .fwdarw. 3') Position
(nt) Sense PD1-ORF2f CTGGGGATCCGCATGACTGCTGAACCG 886-903 PD1-ORF3f
CTTCCCGGGATGGAAAAAATGTCAC 2807-2822 RD1-ORF4f
GTAGGATCCTAGGAGACATCAGCGGC 4028-4015 RD1-ORF5f
CTGGGGATCCGCGTGATCACCATGCTGTGG 3028-3045 RD1-ORF8f
CTCGGATCCTGTGGGTGCAGGTCCGGCGATGGGC 5502-5479 RD1-ORF9af
GTGATGTGAGCTCAGGTGAAGAAGGTGAAG 6144-6160 RD1-ORFF9bf
GTGATGTGAGCTCCTATGGCGGCCGACTACGAC 5072-5089 Antisense PD1-ORF2r
TGCAAGCTTTTAACCGGCGCTTGGGGGTGC 2664-2644 RD1-ORF3r
GATGCCATGGTTAGGCGAAGACGCCGGC 3103-3086 RD1-ORF4r
CGATCTAAGCTTGGCAATGGAGGTCTA 3582-3597 RD1-ORF5r
TGCAAGCTTTCACCAGTCGTCCTCTTCGTC 4243-4223 RD1-ORF8r
CTCCCATGGCTACGACAAGCTCTTCCGGCCGC 5083-5105 PDI-ORF9a/br
CGATCTAAGCTTTCAACGACGTCCAGCC 7073-7056 .sup.aThe oligonucleotides
were constructed from the Accession number U34484 nucleotide
sequence (Mahairas et al., 1996). Nucleotides (nt) underlined are
not contained in the nucleotide sequence of PD1-ORF's. The
positions correspond to the nucleotide sequence of Accession number
U34484.
[0211] The nucleotide sequences of rd1-orf2, rd1-orf3, rd1-orf4,
rd1-orf5, rd1-orf8, rd1-orf9a, and rd1-orf9b from M. tuberculosis
H37Rv are set forth in SEQ ID NO: 71, 87, 89, 91, 67, 93, and 69,
respectively. The deduced amino acid sequences of rd1-orf2,
rd1-orf3, rd1-orf4 rd1-orf5, rd1-orf8, rd1-orf9a, and rd1-orf9b are
set forth in SEQ ID NO: 72, 88, 90, 92, 68, 94, and 70,
respectively.
EXAMPLE 3
[0212] Cloning of the genes expressing 17-30 kDa antigens from
ST-CF
[0213] Isolation of CFP17, CFP20. CFP21. CFP22, CFP25, and
CFP28
[0214] ST-CF was precipitated with ammonium sulphate at 80%
saturation. The precipitated proteins were removed by
centrifugation and after resuspension washed with 8 M urea. CHAPS
and glycerol were added to a final concentration of 0.5% (w/v) and
5% (v/v) respectively and the protein solution was applied to a
Rotofor isoelectrical Cell (BioRad). The Rotofor Cell had been
equilibrated with an 8 M urea buffer containing 0.5% (w/v) CHAPS,
5% (v/v) glycerol, 3% (v/v) Biolyt 3/5 and 1% (v/v) Biolyt 4/6
(BioRad). Isoelectric focusing was performed in a pH gradient from
3-6. The fractions were analyzed on silver-stained 10-20% SDS-PAGE.
Fractions with similar band patterns were pooled and washed three
times with PBS on a Centriprep concentrator (Amicon) with a 3 kDa
cut off membrane to a final volume of 1-3 ml. An equal volume of
SDS containing sample buffer was added and the protein solution
boiled for 5 min before further separation on a Prep Cell (BioRad)
in a matrix of 16% polyacrylamide under an electrical gradient.
Fractions containing pure proteins with an molecular mass from
17-30 kDa were collected.
[0215] Isolation of CFP29
[0216] Anti-CFP29, reacting with CFP29 was generated by
immunization of BALB/c mice with crushed gel pieces in RIBI
adjuvant (first and second immunization) or aluminium hydroxide
(third immunization and boosting) with two week intervals. SDS-PAGE
gel pieces containing 2-5 .mu.g of CFP29 were used for each
immunization. Mice were boosted with antigen 3 days before removal
of the spleen. Generation of a monoclonal cell line producing
antibodies against CFP29 was obtained essentially as decribed by
Kohler and Milstein (1975). Screening of supernatants from growing
clones was carried out by immunoblotting of nitrocellulose strips
containing ST-CF separated by SDS-PAGE. Each strip contained
approximately 50 .mu.g of ST-CF. The antibody class of anti-CFP29
was identified as IgM by the mouse monoclonal antibody isotyping
kit, RPN29 (Amersham) according to the manufacturer's
instructions.
[0217] CFP29 was purified by the following method: ST-CF was
concentrated 10 fold by ultrafiltration, and ammonium sulphate
precipitation in the 45 to 55% saturation range was performed. The
pellet was redissolved in 50 mM sodium phosphate, 1.5 M ammonium
sulphate, pH 8.5, and subjected to thiophilic adsorption
chromatography (Porath et al., 1985) on an Affi-T gel column
(Kem-En-Tec). Protein was eluted by a linear 1.5 to 0 M gradient of
ammonium sulphate and fractions collected in the range 0.44 to 0.31
M ammonium sulphate were identified as CFP29 containing fractions
in Western blot experiments with mAb Anti-CFP29. These fractions
were pooled and anion exchange chromatography was performed on a
Mono Q HR 5/5 column connected to an FPLC system (Pharmacia). The
column was equilibrated with 10 mM Tris-HCl, pH 8.5 and the elution
was performed with a linear gradient from 0 to 500 mM NaCl. From
400 to 500 mM sodium chloride, rather pure CFP29 was eluted. As a
final purification step the Mono Q fractions containing CFP29 were
loaded on a 12.5% SDS-PAGE gel and pure CFP29 was obtained by the
multi-elution technique (Andersen and Heron, 1993).
[0218] N-terminal sequencing and amino acid analysis
[0219] CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28 were washed
with water on a Centricon concentrator (Amicon) with cutoff at 10
kDa and then applied to a ProSpin concentrator (Applied Biosystems)
where the proteins were collected on a PVDF membrane. The membrane
was washed 5 times with 20% methanol before sequencing on a Procise
sequencer (Applied Biosystems).
[0220] CFP29 containing fractions were blotted to PVDF membrane
after tricine SDS-PAGE (Ploug et al., 1989). The relevant bands
were excised and subjected to amino acid analysis (Barkholt and
Jensen, 1989) and N-terminal sequence analysis on a Procise
sequencer (Applied Biosystems).
[0221] The following N-terminal sequences were obtained:
6 For CFP17: A/S E L D A P A Q A G T E X A V (SEQ ID NO: 17) For
CFP20: A Q I T L R G N A I N T V G E (SEQ ID NO: 18) For CFP21: D P
X S D I A V V F A R G T H (SEQ ID NO: 19) For CFP22: T N S P L A T
A T A T L H T N (SEQ ID NO: 20) For CFP25: A X P D A E V V F A R G
R F E (SEQ ID NO: 21) For CFP28: X I/V Q K S L E L I V/T V/F T A
D/Q E (SEQ ID NO: 22) For CFP29: M N N L Y R D L A P V T E A A W A
E I (SEQ ID NO: 23)
[0222] "X" denotes an amino acid which could not be determined by
the sequencing method used, whereas a "/" between two amino acids
denotes that the sequencing method could not determine which of the
two amino acids is the one actually present.
[0223] Cloning the gene encoding CFP29
[0224] The N-terminal sequence of CFP29 was used for a homology
search in the EMBL database using the TFASTA program of the
Genetics Computer Group sequence analysis software package. The
search identified a protein, Linocin M18, from Brevibacterium
linens that shares 74% identity with the 19 N-terminal amino acids
of CFP29.
[0225] Based on this identity between the N-terminal sequence of
CFP29 and the sequence of the Linocin M18 protein from
Brevibacterium linens, a set of degenerated primers were
constructed for PCR cloning of the M. tuberculosis gene encoding
CFP29. PCR reactions were containing 10 ng of M. tuberculosis
chromosomal DNA in 1.times.low salt Taq+ buffer from Stratagene
supplemented with 250 .mu.M of each of the four nucleotides
(Boehringer Mannheim), 0,5 mg/ml BSA (IgG technology), 1% DMSO
(Merck), 5 pmoles of each priner and 0.5 unit Tag+ DNA polymerase
(Stratagene) in 10 .mu.l reaction volume. Reactions were initially
heated to 94.degree. C. for 25 sec. and run for 30 cycles of the
program; 94.degree. C. for 15 sec., 55.degree. C. for 15 sec. and
72.degree. C. for 90 sec, using thermocycler equipment from Idaho
Technology.
[0226] An approx. 300 bp fragment was obtained using primers with
the sequences:
7 1: 5'-CCCGGCTCGAGAACCTSTACCGCGACCTSGCSCC (SEQ ID NO: 24) 2:
5'-GGGCCGGATCCGASGCSGCGTCCTTSACSGGYTGCCA (SEQ ID NO: 25)
[0227] where S=G/C and Y=T/C
[0228] The fragment was excised from a 1% agarose gel, purified by
Spin-X spinn columns (Costar), cloned into pBluescript SK II+-T
vector (Stratagene) and finally sequenced with the Sequenase kit
from United States Biochemical.
[0229] The first 150 bp of this sequence was used for a homology
search using the Blast program of the Sanger Mycobacterium
tuberculosis database:
[0230]
(http//www.sanger.ac.uk/projects/M-tuberculosis/blast_server).
[0231] This program identified a Mycobacterium tuberculosis
sequence on cosmid cy444 in the database that is nearly 100%
identical to the 150 bp sequence of the CFP29 protein. The sequence
is contained within a 795 bp open reading frame of which the 5' end
translates into a sequence that is 100% identical to the
N-terminally sequenced 19 amino acids of the purified CFP29
protein.
[0232] Finally, the 795 bp open reading frame was PCR cloned under
the same PCR conditions as described above using the primers:
8 3: 5'-GGAAGCCCCATATGAACAATCTCTACCG (SEQ ID NO: 26) 4:
5'-CGGGCTCAGCCCTTAGTGACTGAGCGCGACCG (SEQ ID NO: 27)
[0233] The resulting DNA fragments were purified from agarose gels
as described above sequenced with primer 3 and 4 in addition to the
following primers:
9 5: 5'-GGACGTTCAAGCGACACATCGCCG-3' (SEQ ID NO: 115) 6:
5'-CAGCACGAACGCGCCGTCGATGGC-3' (SEQ ID NO: 116)
[0234] Three independent cloned were sequenced. All three clones
were in 100% agreement with the sequence on cosmid cy444.
[0235] All other DNA manipulations were done according to Maniatis
et al. (1989).
[0236] All enzymes other than Taq polymerase were from New England
Biolabs.
[0237] Homology searches in the Sanger database
[0238] For CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28 the
N-terimnal amino acid sequence from each of the proteins were used
for a homology search using the blast program of the Sanger
Mycobacterium tuberculosis database:
[0239] http://www.sanger.ac.uk/pathogens/TB-blast-server.html.
[0240] For CFP29 the first 150 bp of the DNA sequence was used for
the search. Furthermore, the EMBL database was searched for
proteins with homology to CFP29.
[0241] Thereby, the following information were obtained:
[0242] CFP17
[0243] Of the 14 determined amino acids in CFP17 a 93% identical
sequence was found with MTCY1A11.16c. The difference between the
two sequences is in the first amino acid: It is an A or an S in the
N-terminal determined sequenced and a S in MTCY1A11. From the
N-terminal sequencing it was not possible to determine amino acid
number 13.
[0244] Within the open reading frame the translated protein is 162
amino acids long. The N-terminal of the protein purified from
culture filtrate starts at amino acid 31 in agreement with the
presence of a signal sequence that has been cleaved off. This gives
a length of the mature protein of 132 amino acids, which
corresponds to a theoretical molecular mass of 13833 Da and a
theoretical pI of 4.4. The observed mass in SDS-PAGE is 17 kDa.
[0245] CFP20
[0246] A sequence 100% identical to the 15 determined amino acids
of CFP20 was found on the translated cosmid cscy09F9. A stop codon
is found at amino acid 166 from the amino acid M at position 1.
This gives a predicted length of 165 amino acids, which corresponds
to a theoretical molecular mass of 16897 Da and a pI of 4.2. The
observed molecular weight in a SDS-PAGE is 20 kDa.
[0247] Searching the GenEMBL database using the TFASTA algorithm
(Pearson and Lipman, 1988) revealed a number of proteins with
homology to the predicted 164 amino acids long translated
protein.
[0248] The highest homology, 51.5% identity in a 163 amino acid
overlap, was found to a Haemophilus influenza Rd toxR reg.
(HIHI0751).
[0249] CFP21
[0250] A sequence 1000% identical to the 14 determined amino acids
of CFP21 was found at MTCY39. From the N-terminal sequencing it was
not possible to determine amino acid number 3; this amino acid is a
C in MTCY39. The amino acid C can not be detected on a Sequencer
which is probably the explanation of this difference.
[0251] Within the open reading frame the translated protein is 217
amino acids long. The N-terminally determined sequence from the
protein purified from culture filtrate starts at amino acid 33 in
agreement with the presence of a signal sequence that has been
cleaved off. This gives a length of the mature protein of 185 amino
acids, which corresponds to a theoretical molecular weigh at 18657
Da, and a theoretical pI at 4,6. The observed weight in a SDS-PAGE
is 21 kDa.
[0252] In a 193 amino acids overlap the protein has 32,6% identity
to a cutinase precursor with a length of 209 amino acids
(CUTI_ALTBR P41744).
[0253] A comparison of the 14 N-terminal determined amino acids
with the translated region (RD2) deleted in M. bovis BCG revealed a
100% identical sequence (mb3484) (Mahairas et al. (1996)).
[0254] CFP22
[0255] A sequence 100% identical to the 15 determined amino acids
of CFP22 was found at MTCY10H4. Within the open reading frame the
translated protein is 182 amino acids long. The N-terminal sequence
of the protein purified from culture filtrate starts at amino acid
8 and therefore the length of the protein occurring in M.
tuberculosis culture filtrate is 175 amino acids. This gives a
theoretical molecular weigh at 18517 Da and a pI at 6.8. The
observed weight in a SDS-PAGE is 22 kDa.
[0256] In an 182 amino acids overlap the translated protein has
90,1% identity with E235739; a peptidyl-prolyl cis-trans
isomerase.
[0257] CFP25
[0258] A sequence 93% identical to the 15 determined amino acids
was found on the cosmid MTCY339.08c. The one amino acid that
differs between the two sequences is a C in MTCY339.08c and a X
from the N-terminal sequence data. On a Sequencer a C can not be
detected which is a probable explanation for this difference.
[0259] The N-terminally determined sequence from the protein
purified from culture filtrate begins at amino acid 33 in agreement
with the presence of a signal sequence that has been cleaved off.
This gives a length of the mature protein of 187 amino acids, which
corresponds to a theoretical molecular weigh at 19665 Da, and a
theoretical pI at 4.9. The observed weight in a SDS-PAGE is 25
kDa.
[0260] In a 217 amino acids overlap the protein has 42.9% identity
to CFP21 (MTCY39.35).
[0261] CFP28
[0262] No homology was found when using the 10 determined amino
acid residues 2-8, 11, 12, and 14 of SEQ ID NO: 22 in the database
search.
[0263] CFP29
[0264] Sanger database searching: A sequence nearly 100% identical
to the 150 bp sequence of the CFP29 protein was found on cosmid
cy444. The sequence is contained within a 795 bp open reading frame
of which the 5' end translates into a sequence that is 100%
identical to the N-terminally sequenced 19 amino acids of the
purified CFP29 protein. The open reading frame encodes a 265 amino
acid protein.
[0265] The amino acid analysis performed on the purified protein
further confirmed the 4dentity of CFP29 with the protein encoded in
open reading frame on cosmid 444.
[0266] EMBL database searching: The open reading frame encodes a
265 amino acid protein that is 58% identical and 74% similar to the
Linocin M18 protein (61% identity on DNA level). This is a 28.6 kDa
protein with bacteriocin activity (Valds-Stauber and Scherer, 1994;
Valds-Stauber and Scherer, 1996). The two proteins have the same
length (except for 1 amino acid) and share the same theoretical
physicochemical properties. We therefore suggest that CFP29 is a
mycobacterial homolog to the Brevibacterium linens Linocin M18
protein.
[0267] The amino acid sequences of the purified antigens as picked
from the Sanger database are shown in the following list. The amino
acids determined by N-terminal sequencing are marked with bold.
10 CFP17 (SEQ ID NO: 6): 1 MTDMNPDIEK DQTSDEVTVE TTSVFRADFL
SELDAPAQAG TESAVSGVEG 51 LPPGSALLVV KRGPNAGSRF LLDQAITSAG
RHPDSDIFLD DVTVSRRHAE 101 FRLENNEFNV VDVGSLNGTY VNREPVDSAV
LANGDEVQIG KFRLVFLTGP 151 KQGEDDGSTG GP CFP20 (SEQ ID NO: 8): 1
MAQITLRGNA INTVGELPAV GSPAPAFTLT GGDLGVISSD QFRGKSVLLN 51
IFPSVDTPVC ATSVRTFDER AAASGATVLC VSKDLPFAQK RFCGAEGTEN 101
VMPASAFRDS FGEDYGVTIA DGPMAGLLAR AIVVIGADGN VAYTELVPEI 151
AQEPNYEAAL AALGA CFP21 (SEQ ID NO: 10): 1 MTPRSLVRIV GVVVATTLAL
VSAPAGGRAA HADPCSDIAV 41 VFARGTHQAS GLGDVGEAFV DSLTSQVGGR
SIGVYAVNYP ASDDYRASAS 91 NGSDDASAHI QRTVASCPNT RIVLGGYSQG
ATVIDLSTSA MPPAVADHVA 141 AVALFGEPSS GFSSMLWGGG SLPTIGPLYS
SKTINLCAPD DPICTGGGNI 191 MAHVSYVQSG MTSQAATFAA NRLDHAG CFP22 (SEQ
ID NO: 12): 1 MADCDSVTNS PLATATATLH TNRGDIKIAL FGNHAPKTVA
NFVGLAQGTK 51 DYSTQNASGG PSGPFYDGAV FHRVIQGFMI QGGDPTGTGR
GGPGYKFADE 101 FHPELQFDKP YLLAMANAGP GTNGSQFFIT VGKTPHLNRR
ETIFGEVIDA 151 ESQRVVEAIS KTATDGNDRP TDPVVIESIT IS CFP25 (SEQ ID
NO: 14): 1 MGAAAAMLAA VLLLTPITVP AGYPOAVAPA TAACPDAEVV FARGRFEPPG
51 IGTVGNAFVS ALRSKVNKNV GVYAVKYPAD NQIDVGANDM SAHIQSMANS 101
CPNTRLVPGG YSLGAAVTDV VLAVPTQMWG FTNPLPPGSD EHIAAVALFG 151
NGSQWVGPIT NFSPAYNDRT IELCHGDDPV CHPADPNTWE ANWPQHLAGA 201
YVSSGMVNQA ADFVAGKLQ CFP29 (SEQ ID NO: 16): 1 MNNLYRDLAP VTEAAWAEIE
LEAARTFKRH IAGRRVVDVS DPGGPVTAAV 51 STGRLIDVKA PTNGVIAHLR
ASKPLVRLRV PFTLSRNEID DVERGSKDSD 101 WEPVKEAAKK LAFVEDRTIF
EGYSAASIEG IRSASSNPAL TLPEDPREIP 151 DVISQALSEL RLAGVDGPYS
VLLSADVYTK VSETSDHGYP IREHLNRLVD 201 GDIIWAPAID GAFVLTTRGG
DFDLQLGTDV AIGYASHDTD TVRLYLQETL 251 TFLCYTAEAS VALSH
[0268] For all six proteins the molecular weights predicted from
the sequences are in agreement with the molecular weights observed
on SDS-PAGE.
[0269] Cloning of the genes encoding CFP17. CFP20. CFP21, CFP22 and
CFP25.
[0270] The genes encoding CFP17, CFP20, CFP21, CFP22 and CFP25 were
all cloned into the expression vector pMCT6, by PCR amplification
with gene specific primers, for recombinant expression in E. coli
of the proteins. PCR reactions contained 10 ng of M. tuberculosis
chromosomal DNA in 1.times.low salt Taq+ buffer from Stratagene
supplemented with 250 mM of each of the four nucleotides
(Boehringer Mannheim), 0,5 mg/ml BSA (IgG technology), 1% DMSO
(Merck), 5 pmoles of each primer and 0.5 unit Tag+ DNA polymerase
(Stratagene) in 10 .mu.l reaction volume. Reactions were initially
heated to 94.degree. C. for 25 sec. and run for 30 cycles according
to the following program; 94.degree. C. for 10 sec., 55.degree. C.
for 10 sec. and 72.degree. C. for 90 sec, using thermocycler
equipment from Idaho Technology.
[0271] The DNA fragments were subsequently run on 1% agarose gels,
the bands were excised and purified by Spin-X spin columns (Costar)
and cloned into pBluescript SK II+-T vector (Stratagene). Plasmid
DNA was thereafter prepared from clones harbouring the desired
fragments, digested with suitable restriction enzymes and subcloned
into the expression vector pMCT6 in frame with 8 histidine residues
which are added to the N-terminal of the expressed proteins. The
resulting clones were hereafter sequenced by use of the dideoxy
chain termination method adapted for supercoiled DNA using the
Sequenase DNA sequencing kit version 1.0 (United States Biochemical
Corp., USA) and by cycle sequencing using the Dye Terminator system
in combination with an automated gel reader (model 373A; Applied
Biosystems) according to the instructions provided. Both strands of
the DNA were sequenced.
[0272] For cloning of the individual antigens, the following gene
specific primers were used:
11 CFP17: Primers used for cloning of cfp17: OPBR-51:
ACAGATCTGTGACGGACATGAACCCG (SEQ ID NO: 117) OPBR-52:
TTTTCCATGGTCACGGGCCCCCGGTACT (SEQ ID NO: 118) OPBR-51 and OPBR-52
create BglII and NcoI sites, respectively, used for the cloning in
pMCT6. CFP20: Primers used for cloning of cfp20: OPBR-53:
ACAGATCTGTGCCCATGGCACAGATA (SEQ ID NO: 119) OPBR-54:
TTTAAGCTTCTAGGCGCCCAGCGCGGC (SEQ ID NO: 120) OPBR-53 and OPBR-54
create BglII and HinDIII sites, respectively, used for the cloning
in pMCT6. CFP21: Primers used for cloning of cfp21: OPBR-55:
ACAGATCTGCGCATGCGGATCCGTGT (SEQ ID NO: 121) OPBR-56:
TTTTCCATGGTCATCCGGCGTGATCGAG (SEQ ID NO: 122) OPBR-55 and OPBR-56
create BglII and NcoI sites, respectively, used for the cloning in
pMCT6. CFP22: Primers used for cloning of cfp22: OPBR-57:
ACAGATCTGTAATGGCAGACTGTGAT (SEQ ID NO: 123) OPBR-58:
TTTTCCATGGTCAGGA~ATGGTGATCGA (SEQ ID NO: 124) OPBR-57 and OPBR-58
create BglII and NcoI sites, respectively, used for the cloning in
pMCT6. CPP25: Primers used for cloning of cfp25: OPBR-59:
ACAGATCTGCCGGCTACCCCGGTGCC (SEQ ID NO: 125) OPBR-60:
TTTTCCATGGCTATTGCAGCTTTCCGGC (SEQ ID NO: 126)
[0273] OPBR-59 and OPBR-60 create Bg1II and NcoI sites,
respectively, used for the cloning in pMCT6.
[0274] Expression/purification of recombinant CFP17. CFP20. CFP21,
CFP22 and CFP25 proteins.
[0275] Expression and metal affinity purification of recombinant
proteins was undertaken essentially as described by the
manufacturers. For each protein, 1 1 LB-media containing 100
.mu.g/ml ampicillin, was inoculated with 10 ml of an overnight
culture of XL1-Blue cells harbouring recombinant pMCT6 plasmids.
Cultures were shaken at 37.degree. C. until they reached a density
of OD.sub.600=0.4-0.6. IPTG was hereafter added to a final
concentration of 1 mM and the cultures were further incubated 4-16
hours. Cells were harvested, resuspended in 1.times. sonication
buffer+8 M urea and sonicated 5.times.30 sec. with 30 sec. pausing
between the pulses.
[0276] After centrifugation, the lysate was applied to a column
containing 25 ml of resuspended Talon resin (Clontech, Palo Alto,
USA). The column was washed and eluted as described by the
manufacturers.
[0277] After elution, all fractions (1.5 ml each) were subjected to
analysis by SDS-PAGE using the Mighty Small (Hoefer Scientific
Instruments, USA) system and the protein concentrations were
estimated at 280 nm. Fractions containing recombinant protein were
pooled and dialysed against 3 M urea in 10 mM Tris-HCl, pH 8.5. The
dialysed protein was further purified by FPLC (Pharmacia, Sweden)
using a 6 ml Resource-Q column, eluted with a linear 0-1 M gradient
of NaCl. Fractions were analyzed by SDS-PAGE and protein
concentrations were estimated at OD.sub.280. Fractions containing
protein were pooled and dialysed against 25 mM Hepes buffer, pH
8.5.
[0278] Finally the protein concentration and the LPS content were
determined by the BCA (Pierce, Holland) and LAL (Endosafe,
Charleston, USA) tests, respectively.
EXAMPLE 3A
[0279] Identification of CFP7A, CFP8A, CFP8B, CFP16, CFP19, CFP19B,
CFP22A, CFP23A, CFP23B, CFP25A, CFP27, CFP3OA, CWP32 and CFP50.
[0280] Identification of CFP16 and CFP19B.
[0281] ST-CF was precipitated with ammonium sulphate at 80%
saturation. The precipitated proteins were removed by
centrifugation and after resuspension washed with 8 M urea. CHAPS
and glycerol were added to a final concentration of 0.5% (w/v) and
5% (v/v) respectively and the protein solution was applied to a
Rotofor isoelectrical Cell (BioRad). The Rotofor Cell had been
equilibrated with a 8M urea buffer containing 0.5% (w/v) CHAPS, 5%
(v/v) glycerol, 3% (v/v) Biolyt 3/5 and 1% (v/v) Biolyt 4/6
(BioRad). Isoelectric focusing was performed in a pH gradient from
3-6. The fractions were analyzed on silver-stained 10-20% SDS-PAGE.
Fractions with similar band patterns were pooled and washed three
times with PBS on a Centriprep concentrator (Amicon) with a 3 kDa
cut off membrane to a final volume of 1-3 ml. An equal volume of
SDS containing sample buffer was added and the protein solution
boiled for 5 min before further separation on a Prep Cell (BioRad)
in a matrix of 16% polyacrylamide under an electrical gradient.
Fractions containing well separated bands in SDS-PAGE were selected
for N-terminal sequencing after transfer to PVDF membrane.
[0282] Isolation of CFP8A, CFP8B. CFPl9, CFP23A. and CFP23B.
[0283] ST-CF was precipitated with ammonium sulphate at 80%
saturation and redissolved in PBS, pH 7.4, and dialysed 3 times
against 25 mM Piperazin-HCl, pH 5.5, and subjected to
chromatofocusing on a matrix of PBE 94 (Pharmacia) in a column
connected to an FPLC system (Pharmacia). The column was
equilibrated with 25 mM Piperazin-HCl, pH 5.5, and the elution was
performed with 10% PB74-HCl, pH 4.0 (Pharmacia). Fractions with
similar band patterns were pooled and washed three times with PBS
on a Centriprep concentrator (Amicon) with a 3 kDa cut off membrane
to a final volume of 1-3 ml and separated on a Prepcell as
described above.
[0284] Identification of CFP22A
[0285] ST-CF was concentrated approximately 10 fold by
ultrafiltration and proteins were precipitated at 80% saturation,
redissolved in PBS, pH 7.4, and dialysed 3 times against PBS, pH
7.4. 5.1 ml of the dialysed ST-CF was treated with RNase (0.2
mg/ml, QUIAGEN) and DNase (0.2 mg/ml, Boehringer Mannheim) for 6 h
and placed on top of 6.4 ml of 48% (w/v) sucrose in PBS, pH 7.4, in
Sorvall tubes (Ultracrimp 03987, DuPont Medical Products) and
ultracentrifuged for 20 h at 257,300.times.g.sub.max, 10.degree. C.
The pellet was redissolved in 200 .mu.l of 25 mM Tris-192 mM
glycine, 0.1% SDS, pH 8.3.
[0286] Identification of CFP7A, CFP25A, CFP27, CFP30A and CFP50
[0287] For CFP27, CFP30A and CFP50 ST-CF was concentrated
approximately 10 fold by ultrafiltration and ammonium sulphate
precipitation in the 45 to 55% saturation range was performed.
Proteins were redissolved in 50 mM sodium phosphate, 1.5 M ammonium
sulphate, pH 8.5, and subjected to thiophilic adsorption
chromatography on an Affi-T gel column (Kem-En-Tec). Proteins were
eluted by a 1.5 to 0 M decreasing gradient of ammonium sulphate.
Fractions with similar band patterns in SDS-PAGE were pooled and
anion exchange chromatography was performed on a Mono Q HR 5/5
column connected to an FPLC system (Pharmacia). The column was
equilibrated with 10 mM Tris-HCl, pH 8.5, and the elution was
performed with a gradient of NaCl from 0 to 1 M. Fractions
containing well separated bands in SDS-PAGE were selected.
[0288] CFP7A and CFP25A were obtained as described above except for
the following modification: ST-CF was concentrated approximately 10
fold by ultrafiltration and proteins were precipitated at 80%
saturation, redissolved in PBS, pH 7.4, and dialysed 3 times
against PBS, pH 7.4. Ammonium sulphate was added to a concentration
of 1.5 M, and ST-CF proteins were loaded on an Affi T-gel column.
Elution from the Affi T-gel column and anion exchange were
performed as described above.
[0289] Isolation of CWP32
[0290] Heat treated H37Rv was subfractionated into subcellular
fractions as described in Sorensen et al 1995. The Cell wall
fraction was resuspended in 8 M urea, 0.2% (w/v) N-octyl
.beta.-.sub.D glucopyranoside (Sigma) and 5% (v/v) glycerol and the
protein solution was applied to a Rotofor isoelectrical Cell
(BioRad) which was equilibrated with the same buffer. Isoelectric
focusing was performed in a pH gradient from 3-6. The fractions
were analyzed by SDS-PAGE and fractions containing well separated
bands were polled and subjected to N-terminal sequencing after
transfer to PVDF membrane.
[0291] N-terminal sequencing
[0292] Fractions containing CFP7A, CFP8A, CFP8B, CFP16, CFP19,
CFP19B, CFP22A, CFP23A, CFP23B, CFP27, CFP30A, CWP32, and CFP50A
were blotted to PVDF membrane after Tricine SDS-PAGE (Ploug et al,
1989). The relevant bands were excised and subjected to N-terminal
amino acid sequence analysis on a Procise 494 sequencer (Applied
Biosystems). The fraction containing CFP25A was blotted to PVDF
membrane after 2-DE PAGE (isoelectric focusing in the first
dimension and Tricin SDS-PAGE in the second dimension). The
relevant spot was excised and sequenced as described above.
[0293] The following N-terminal sequences were obtained:
12 CFP7A: AEDVRAEIVA SVLEVVVNEG DQIDKGDVVV LLESMYMEIP (SEQ ID NO:
81) VLAEAAGTVS CFP8A: DPVDDAFIAKLNTAG (SEQ ID NO: 73) CFP8B:
DPVDAIINLDNYGX (SEQ ID NO: 74) CFP1G: AKLSTDELLDAFKEM (SEQ ID NO:
79) CFP19: TTSPDPYAALPKLPS (SEQ ID NO: 82) CFP19B: DPAXAPDVPTAAQLT
(SEQ ID NO: 80) CFP22A: TEYEGPKTKF HALMQ (SEQ ID NO: 83) CFP23A:
VIQ/AGMVT/GHIHXVAG (SEQ ID NO: 76) CFP23B: AEMKXFKNAIVQEID (SEQ ID
NO: 75) CFP2SA: AIEVSVLRVF TDSDG (SEQ ID NO: 78) CWP32:
TNIVVLIKQVPDTWS (SEQ ID NO: 77) CFP27: TTIVALKYPG GVVMA (SEQ ID NO:
84) CFP30A: SFPYFISPEX AMRE (SEQ ID NO: 85) CFP50: THYDVVVLGA GPGGY
(SEQ ID NO: 86)
[0294] N-terminal homology searching in the Sanger database and
identification of the corresponding genes.
[0295] The N-terminal amino acid sequence from each of the proteins
was used for a homology search using the blast program of the
Sanger Mycobacterium tuberculosis database:
[0296] http:
//www.sanger.ac.uk/projects/m-tuberculosis/TB-blast-server.
[0297] For CFP23B, CFP23A, and CFP19B no similarities were found in
the Sanger database. This could be due to the fact that only
approximately 70% of the M. tuberculosis genome had been sequenced
when the searches were performed. The genes encoding these proteins
could be contained in the remaining 30% of the genome for which no
sequence data is yet available.
[0298] For CFP7A, CFP8A, CFP8B, CFP16, CFP19, CFP19B , CFP22A,
CFP25A, CFP27, CFP30A, CWP32, and CFP50, the following information
was obtained:
[0299] CFP7A: Of the 50 determined amino acids in CFP7A a 98%
identical sequence was found in cosmid csCY07D1 (contig 256):
Score=226 (100.4 bits), Expect=1.4e-24, P=1.4e-24 Identities=49/50
(98%), Positives=49/50 (98%), Frame=-1
13 Query: 1 AEDVRAEIVASVLEVVVNEGDQIDKGDVVVLLESMYMEIPVLAEAAGTVS 50
AEDVRAEIVASVLEVVVNEGDQIDKGDVVVLLESM MEIPVLAEAAGTVS Sbjct: 257679
AEDVRAEIVASVLEVVVNEGDQIDKGDVVVLLESMKMEIPVLAEAAGTV- S 257530 (SEQ ID
NOs: 127, 128, and 129)
[0300] The identity is found within an open reading frame of 71
amino acids length corresponding to a theoretical MW of CFP7A of
7305.9 Da and a pI of 3.762. The observed molecular weight in an
SDS-PAGE gel is 7 kDa.
[0301] CFP8A: A sequence 80% identical to the 15 N-terminal amino
acids was found on contig TB.sub.--1884. The N-terminally
determined sequence from the protein purified from culture filtrate
starts at amino acid 32. This gives a length of the mature protein
of 98 amino acids corresponding to a theoretical MW of 9700 Da and
a pI of 3.72 This is in good agreement with the observed MW on
SDS-PAGE at approximately 8 kDa. The full length protein has a
theoretical MW of 1289 Da and a pI of 4.38.
[0302] CFP8B: A sequence 71% identical to the 14 N-terminal amino
acids was found on contig TB.sub.--653. However, careful
re-evaluation of the original N-terminal sequence data confirmed
the identification of the protein. The N-terminally determined
sequence from the protein purified from culture filtrate starts at
amino acid 29. This gives a length of the mature protein of 82
amino acids corresponding to a theoretical MW of 8337 Da and a pI
of 4.23. This is in good agreement with the observed MW on SDS-PAGE
at approximately 8 kDa. Analysis of the amino acid sequence
predicts the presence of a signal peptide which has been cleaved of
the mature protein found in culture filtrate.
[0303] CFP16: The 15 aa N-terminal sequence was found to be 100%
identical to a sequence found on cosmid MTCY20H1.
[0304] The identity is found within an open reading frame of 130
amino acids length corresponding to a theoretical MW of CFP16 of
13440.4 Da and a pI of 4.59. The observed molecular weight in an
SDS-PAGE gel is 16 kDa.
[0305] CFP19: The 15 aa N-terminal sequence was found to be 100%
identical to a sequence found on cosmid MTCY270.
[0306] The identity is found within an open reading frame of 176
amino acids length corresponding to a theoretical MW of CFP19 of
18633.9 Da and a pI of 5.41. The observed molecular weight in an
SDS-PAGE gel is 19 kDa.
[0307] CFP22A: The 15 aa N-terminal sequence was found to be 100%
identical to a sequence found on cosmid MTCY1A6.
[0308] The identity is found within an open reading frame of 181
amino acids length corresponding to a theoretical MW of CFP22A of
20441.9 Da and a pI of 4.73. The observed molecular weight in an
SDS-PAGE gel is 22 kDa.
[0309] CFP25A: The 15 aa N-terminal sequence was found to be 100%
identical to a sequence found on contig 255.
[0310] The identity is found within an open reading frame of 228
amino acids length corresponding to a theoretical MW of CFP25A of
24574.3 Da and a pI of 4.95. The observed molecular weight in an
SDS-PAGE gel is 25 kDa.
[0311] CFP27: The 15 aa N-terminal sequence was found to be 100%
identical to a sequence found on cosmid MTCY261.
[0312] The identity is found within an open reading frame of 291
amino acids length. The N-terminally determined sequence from the
protein purified from culture filtrate starts at amino acid 58.
This gives a length of the mature protein of 233 amino acids, which
corresponds to a theoretical molecular weigh at 24422.4 Da, and a
theoretical pI at 4.64. The observed weight in an SDS-PAGE gel is
27 kDa.
[0313] CFP30A: Of the 13 determined amino acids in CFP30A, a 1000%
identical sequence was found on cosmid MTCY261.
[0314] The identity is found within an open reading frame of 248
amino acids length corresponding to a theoretical MW of CFP30A of
26881.0 Da and a pI of 5.41. The observed molecular weight in an
SDS-PAGE gel is 30 kDa.
[0315] CWP32: The 15 amino acid N-terminal sequence was found to be
100% identical to a sequence found on contig 281. The identity was
found within an open reading frame of 266 amino acids length,
corresponding to a theoretical MW of CWP32 of 28083 Da and a pI of
4.563. The observed molecular weight in an SDS-PAGE gel is 32
kDa.
[0316] CFP50: The 15 aa N-terminal sequence was found to be 100%
identical to a sequence found in MTV038.06. The identity is found
within an open reading frame of 464 amino acids length
corresponding to a theoretical MW of CFP50 of 49244 Da and a pI of
5.66. The observed molecular weight in an SDS-PAGE gel is 50
kDa.
[0317] Use of homology searching in the EMBL database for
identification of CFP19A and CFP23.
[0318] Homology searching in the EMBL database (using the GCG
package of the Biobase, .ANG.rhus-DK) with the amino acid sequences
of two earlier identified highly immunoreactive ST-CF proteins,
using the TFASTA algorithm, revealed that these proteins (CFP21 and
CFP25, EXAMPLE 3) belong to a family of fungal cutinase homologs.
Among the most homologous sequences were also two Mycobacterium
tuberculosis sequences found on cosmid MTCY13E12. The first,
MTCY13El2.04 has 46% and 50% identity to CFP25 and CFP21
respectively. The second, MTCY13E12.05, has also 46% and 50%
identity to CFP25 and
[0319] CFP21. The two proteins share 62.5% aa identity in a 184
residues overlap. On the basis of the high homology to the strong
T-cell antigens CFP21 and CFP25, respectively, it is believed that
CFP19A and CFP23 are possible new T-cell antigens.
[0320] The first reading frame encodes a 254 amino acid protein of
which the first 26 aa constitute a putative leader peptide that
strongly indicates an extracellular location of the protein. The
mature protein is thus 228 aa in length corresponding to a
theoretical MW of 23149.0 Da and a Pi of 5.80. The protein is named
CFP23.
[0321] The second reading frame encodes an 231 aa protein of which
the first 44 aa constitute a putative leader peptide that strongly
indicates an extracellular location of the protein. The mature
protein is thus 187 aa in length corresponding to a theoretical MW
of 19020.3 Da and a Pi of 7.03. The protein is named CFP19A.
[0322] The presence of putative leader peptides in both proteins
(and thereby their presence in the ST-CF) is confirmed by
theoretical sequence analysis using the signalp program at the
Expasy molecular Biology server
[0323] (http://expasy.hcuge.ch/www/tools.html).
[0324] Searching for homologies to CFP7A, CFP16, CFP19, CFP19A,
CFPl9B, CFP22A, CFP23, CFP25A, CFP27, CFP30A, CWP32 and CFP50 in
the EMBL database.
[0325] The amino acid sequences derived from the translated genes
of the individual antigens were used for homology searching in the
EMBL and Genbank databases using the TFASTA algorithm, in order to
find homologous proteins and to address eventual functional roles
of the antigens.
[0326] CFP7A: CFP7A has 44% identity and 70% similarity to
hypothhetical Methanococcus jannaschii protein (M. janraschii from
base 1162199-1175341), as well as 43% and 38% identity and 68 and
64% similarity to the C-terminal part of B. stearothermophilus
pyruvate carboxylase and Streptococcus mutans biotin carboxyl
carrier protein.
[0327] CFP7A contains a consensus sequence EAMKM for a biotin
binding site motif which in this case was slightly modified (ESMKM
in amino acid residues 34 to 38). By incubation with alkaline
phosphatase conjugated streptavidin after SDS-PAGE and transfer to
nitrocellulose it was demonstrated that native CFP7A was
biotinylated.
[0328] CFP16: RplL gene, 130 aa. Identical to the M. bovis 50s
ribosomal protein L7/L12 (acc. No P37381).
[0329] CFP19: CFP19 has 47% identity and 55% similarity to E.coli
pectinesterase homolog (ybhc gene) in a 150 aa overlap.
[0330] CFP19A: CFP19A has between 38% and 45% identity to several
cutinases from different fungal sp.
[0331] In addition CFP19A has 46% identity and 61% similarity to
CFP25 as well as 50% identity and 64t similarity to CFP21 (both
proteins are earlier isolated from the ST-CF).
[0332] CFP19B: No apparent homology
[0333] CFP22A: No apparent homology
[0334] CFP23: CFP23 has between 38% and 46% identity to several
cutinases from different fungal sp.
[0335] In addition CFP23 has 46% identity and 61% similarity to
CFP25 as well as 50% identity and 63% similarity to CFP21 (both
proteins are earlier isolated from the ST-CF).
[0336] CFP25A: CFP25A has 95% identity in a 241 aa overlap to a
putative M. tuberculosis thymidylate synthase 450 aa accession No
p28176).
[0337] CFP27: CFP27 has 81% identity to a hypothetical M. leprae
protein and 64% identity and 78% similarity to Rhodococcus sp.
proteasome beta-type subunit 2 (prcB(2) gene).
[0338] CFP30A: CFP30A has 67% identity to Rhodococcus proteasome
alfa-type 1 subunit.
[0339] CWP32: The CWP32 N-terminal sequence is 100% identical to
the Mycobacterium leprae sequence MLCB637.03.
[0340] CFP50: The CFP50 N-terminal sequence is 100% identical to a
putative lipoamide dehydrogenase from M. leprae (Accession
415183)
[0341] Cloning of the genes encoding CFP7A, CFP8A, CFP8B, CFP16,
CFPl9, CFP19A, CFP22A, CFP23, CFP25A, CFP27, CFP30A, CWP32, and
CFP50.
[0342] The genes encoding CFP7A, CFP8A, CFP8B, CFP16, CFP19,
CFP19A, CFP22A, CFP23, CFP25A, CFP27, CFP30A, CWP32 and CFP50 were
all cloned into the expression vector pMCT6, by PCR amplification
with gene specific primers, for recombinant expression in E. coli
of the proteins.
[0343] PCR reactions contained 10 ng of M. tuberculosis chromosomal
DNA in 1.times. low salt Taq+ buffer from Stratagene supplemented
with 250 mM of each of the four nucleotides (Boehringer Mannheim),
0,5 mg/ml BSA (IgG technology), 1% DMSO (Merck), 5 pmoles of each
primer and 0.5 unit Tag+ DNA polymerase (Stratagene) in 10 ml
reaction volume. Reactions were initially heated to 94.degree. C.
for 25 sec. and run for 30 cycles of the program; 94.degree. C. for
10 sec., 55.degree. C. for 10 sec. and 72.degree. C. for 90 sec.
using thermocycler equipment from Idaho Technology.
[0344] The DNA fragments were subsequently run on 1% agarose gels,
the bands were excised and purified by Spin-X spin columns (Costar)
and cloned into pBluescript SK II+-T vector (Stratagene). Plasmid
DNA was hereafter prepared from clones harbouring the desired
fragments, digested with suitable restriction enzymes and subcloned
into the expression vector pMCT6 in frame with 8 histidines which
are added to the N-terminal of the expressed proteins. The
resulting clones were hereafter sequenced by use of the dideoxy
chain termination method adapted for supercoiled DNA using the
Sequenase DNA sequencing kit version 1.0 (United States Biochemical
Corp., USA) and by cycle sequencing using the Dye Terminator system
in combination with an automated gel reader (model 373A; Applied
Biosystems) according to the instructions provided. Both strands of
the DNA were sequenced.
[0345] For cloning of the individual antigens, the following gene
specific primers were used:
14 CFP7A: Primers used for cloning of cfp7A: OPBR-79:
AAGAGTAGATCTATGATGGCCGAGGATGTTCGCG (SEQ ID NO: 95) OPBR-80:
CGGCGACGACGGATCCTACCGCGTCGG (SEQ ID NO: 96) OPBR-79 and OPBR-80
create BglII and BamHI sites, respectively, used for the cloning in
pMCT6. CFP8A: Primers used for cloning of cfp8A: CFP8A-F:
CTGAGATCTATGAACCTACGGCGCC (SEQ ID NO: 154) CFP8A-R:
CTCCCATGGTACCCTAGGACCCGGGCAGCCCCGGC (SEQ ID NO: 155) CFP8A-F and
CFP8A-R create BglII and NcoI sites, respectively, used for the
cloning in pMCT6. CFP8B: Primers used for cloning of cfp8B:
CFP8B-F: CTGAGATCTATGAGGCTGTCGTTGACCGC (SEQ ID NO: 156) CFP8B-R:
CTCCCCGGGCTTAATAGTTGTTGCAGGAGC (SEQ ID NO: 157) CFP8B-F and CFP8B-R
create BglII and SmaI sites, respectively, used for the cloning in
pMCT6. CFPl6: Primers used for cloning of Cfpl6: OPBR-104:
CCGGGAGATCTATGGCAAAGCTCTCCACCGACG (SEQ ID NOs: 111 and 130)
OPBR-105: CGCTGGGCAGAGCTACTTGACGGTGACGGTGG (SEQ ID NOs: 112 and
131) OPBR-l04 and OPBR-105 create BglII and NcoI sites,
respectively, used for the cloning in pMCT6. CFPl9: Primers used
for cloning of Cfpl9: OPBR-96: GAGGAAGATCTATGACAACTTCACCCGACCCG
(SEQ ID NO: 107) OPBR-97. CATGAAGCCATGGCCCGCAGGCTGCATG (SEQ ID NO:
108) OPBR-96 and OPBR-97 create BglII and NcoI sites, respectively,
used for the cloning in pMCT6. CFPl9A: Primers used for cloning of
Cfpl9A: OPBR-88: CCCCCCAGATCTGCACCACCGGCATCGGC- GGGC (SEQ ID NO:
99) OPBR-89. GCGGCGGATCCGTTGCTTAGCCGG (SEQ ID NO: 100) OPBR-88 and
OPBR-89 create BglII and BamHI sites, respectively, used for the
cloning in pMCT6. CFP22A: Primers used for cloning of Cfp22A:
OPBR-90: CCGGCTGAGATCTATGACAGAATACGAAGGGC (SEQ ID NO: 101) OPBR-91:
CCCCGCCAGGGAACTAGAGGCGGC (SEQ ID NO: 102) OPBR-90 and OPBR-91
create BglII and NcoI sites, respectively, used for the cloning in
pMCT6. CFP23: Primers used for cloning of Cfp23: OPBR-86:
CCTTGGGAGATCTTTGGACCCCGGTTGC (SEQ ID NO: 97) OPBR-87:
GACGAGATCTTATGGGCTTACTGAC (SEQ ID NO: 98) OPBR-86 and OPBR-87 both
create a BglII site used for the cloning in pMCT6. CFP25A: Primers
used for cloning of Cfp25A: OPBR-106:
GGCCCAGATCTATGGCCATTGAGGTTTCGGTGFTGC (SEQ ID NO: 113) OPBR-107:
CGCCGTGTTGCATGGCAGCGCTGAGC (SEQ ID NO: 114) OPBR-106 and OPBR-107
create BglII and NcoI sites, respectively, used for the cloning in
pMCT6. CFP27: Primers used for cloning of Cfp27: OPBR-92:
CTGCCGAGATCTACCACCATTGTCGCGCTGAAATACCC (SEQ ID NO: 103) OPBR-93:
CGCCATGGCCTTACGCGCCAACTCG (SEQ ID NO: 104) OPBR-92 and OPBR-93
create BglII and NcoI sites, respectively, used for the cloning in
pMCT6. CFP30A: Primers used for cloning of Cfp30A: OPBR-94:
GGCGGAGATCTGTGAGTTTTCCGTATTTCATC (SEQ ID NO: 105) OPBR-95:
CGCGTCGAGCCATGGTTAGGCGCAG (SEQ ID NO: 106) OPBR-94 and OPBR-95
create BglII and NcoI sites, respectively, used for the cloning in
pMCT6. CWP32: Primers used for cloning of cwp32: CWP32-F:
GCTTAGATCTATGATTTTCTGGGCAA- CCAGGTA (SEQ ID NO: 158) CWP32-R:
GCTTCCATGGGCGAGGCACAGGCGTGGGAA (SEQ ID NO: 159) CWP32-F and CWP32-R
create BglII and NcoI sites, respectively, used for the cloning in
pMCT6. CFP50: Primers used for cloning of cfp50: OPBR-100:
GGCCGAGATCTGTGACCCACTATGACGTCGTCG (SEQ ID NO: 109) OPBR-101:
GGCGCCCATGGTCAGAAATTGATCATGTGGCCAA (SEQ ID NO: 110)
[0346] OPBR-100 and OPBR-101 create Bg1II and NcoI sites,
respectively, used for the cloning in pMCT6.
[0347] Expression/purification of recombinant CFP7A, CFP8A, CFP8B,
CFP16, CFPl9, CFP19A, CFP22A, CFP23, CFP25A, CFP27, CFP30A, CWP32,
and CFP50 proteins.
[0348] Expression and metal affinity purification of recombinant
proteins was undertaken essentially as described by the
manufacturers. For each protein, 1 l LB-media containing 100
.mu.g/ml ampicillin, was inoculated with 10 ml of an overnight
culture of XL1-Blue cells harbouring recombinant pMCT6 plasmids.
Cultures were shaken at 37.degree. C. until they reached a density
of OD.sub.600=0.4-0.6. IPTG was hereafter added to a final
concentration of 1 mM and the cultures were further incubated 4-16
hours. Cells were harvested, resuspended in 1.times. sonication
buffer+8 M urea and sonicated 5.times.30 sec. with 30 sec. pausing
between the pulses.
[0349] After centrifugation, the lysate was applied to a column
containing 25 ml of resuspended Talon resin (Clontech, Palo Alto,
USA). The column was washed and eluted as described by the
manufacturers.
[0350] After elution, all fractions (1.5 ml each) were subjected to
analysis by SDS-PAGE using the Mighty Small (Hoefer Scientific
Instruments, USA) system and the protein concentrations were
estimated at 280 nm. Fractions containing recombinant protein were
pooled and dialysed against 3 M urea in 10 mM Tris-HC1, pH 8.5. The
dialysed protein was further purified by FPLC (Pharmacia, Sweden)
using a 6 ml Resource-Q column, eluted with a linear 0-1 M gradient
of NaCl. Fractions were analyzed by SDS-PAGE and protein
concentrations were estimated at OD.sub.280. Fractions containing
protein were pooled and dialysed against 25 mM Hepes buffer,
p8.5.
[0351] Finally the protein concentration and the LPS content were
determined by the BCA (Pierce, Holland) and LAL (Endosafe,
Charleston, USA) tests, respectively.
EXAMPLE 3B
[0352] Identification of CFP7B, CFP10A, CFP11 and CFP30B.
[0353] Isolation of CFP7B
[0354] ST-CF was precipitated with ammonium sulphate at 80%
saturation and redissolved in PBS, pH 7.4, and dialyzed 3 times
against 25 mM Piperazin-HCl, pH 5.5, and subjected to
cromatofocusing on a matrix of PBE 94 (Pharmacia) in a column
connected to an FPLC system (Pharmacia). The column was
equilibrated with 25 mM Piperazin-HCl, pH 5.5, and the elution was
performed with 10% PB74-HCl, pH 4.0 (Pharmacia). Fractions with
similar band patterns were pooled and washed three times with PBS
on a Centriprep concentrator (Am-con) with a 3 kDa cut off membrane
to a final volume of 1-3 ml. An equal volume of SDS containing
sample buffer was added and the protein solution boiled for 5 min
before further separation on a MultiEluter (BioRad) in a matrix of
10-20% polyacrylamid (Andersen,P. & Heron,I., 1993). The
fraction containing a well separated band below 10 kDa was selected
for N-terminal sequencing after transfer to a PVDF membrane.
[0355] Isolation of CFPll
[0356] ST-CF was precipitated with ammonium sulphate at 80%
saturation. The precipitated proteins were removed by
centrifugation and after resuspension washed with 8 M urea. CHAPS
and glycerol were added to a final concentration of 0.5% (w/v) and
5% (v/v) respectively and the protein solution was applied to a
Rotofor isoelectrical Cell (BioRad). The Rotofor Cell had been
equilibrated with an 8M urea buffer containing 0.5% (t/w) CHAPS, 5%
(v/v) glycerol, 3% (v/v) Biolyt 3/5 and 1% (v/v) Biolyt 4/6
(BioRad). Isoelectric focusing was performed in a pH gradient from
3-6. The fractions were analyzed on silver-stained 10-20% SDS-PAGE.
The fractions in the pH gradient 5.5 to 6 were pooled and washed
three times with PBS on a Centriprep concentrator (Amicon) with a 3
kDa cut off membrane to a final volume of 1 ml. 300 mg of the
protein preparation was separated on a 10-20% Tricine SDS-PAGE
(Ploug et al 1989) and transferred to a PVDF membrane and Coomassie
stained. The lowest band occurring on the membrane was excised and
submitted for N-terminal sequencing.
[0357] Isolation of CFP1OA and CFP30B
[0358] ST-CF was concentrated approximately 10-fold by
ultrafiltration and ammonium sulphate precipitation at 80%
saturation. Proteins were redissolved in 50 mM sodium phosphate,
1.5 M ammonium sulphate, pH 8.5, and subjected to thiophilic
adsorption chromatography on an Affi-T gel column (Kem-En-Tec).
Proteins were eluted by a 1.5 to 0 M decreasing gradient of
ammonium sulphate. Fractions with similar band patterns in SDS-PAGE
were pooled and anion exchange chromatography was performed on a
Mono Q HR 5/5 column connected to an FPLC system (Pharmacia). The
column was equilibrated with 10 mM Tris-HCl, pH 8.5, and the
elution was performed with a gradient of NaCl from 0 to 1 M.
Fractions containing well separated bands in SDS-PAGE were
selected.
[0359] Fractions containing CFP10A and CFP30B were blotted to PVDF
membrane after 2-DE-PAGE (Ploug et al, 1989). The relevant spots
were excised and subjected to N-terminal amino acid sequence
analysis.
[0360] N-terminal sequencing
[0361] N-terminal amino acid sequence analysis was performed on a
Procise 494 sequencer (applied Biosystems).
[0362] The following N-terminal sequences were obtained:
15 CFP7B: PQGTVKWFNAEKGFG (SEQ ID NO: 168) CFP10A: NVTVSIPTILRPXXX
(SEQ ID NO: 169) CFP11: TRFMTDPHAMRDMAG (SEQ ID NO: 170) CFP30B:
PKRSEYRQGTPNWVD (SEQ ID NO: 171)
[0363] "X" denotes an amino acid which could not be determined by
the sequencing method used.
[0364] N-terminal homology searching in the Sanger database and
identification of the corresponding genes.
[0365] The N-terminal amino acid sequence from each of the proteins
was used for a homology search using the blast program of the
Sanger Mycobacterium tuberculosis genome database:
[0366]
http//www.sanger.ac.uk/projects/m-tuberculosis/TB-blast-server.
[0367] For CFP11 a sequence 100% identical to 15 N-terminal amino
acids was found on contig TB.sub.--1314. The identity was found
within an open reading frame of 98 amino acids length corresponding
to a theoretical MW of 10977 Da and a pI of 5.14.
[0368] Amino acid number one can also be an Ala (insted of a Thr)
as this sequence was also obtained (results not shown), and a 100%
identical sequence to this N-terminal is found on contig
TB.sub.--671 and on locus MTCI364.09.
[0369] For CFP7B a sequence 100% identical to 15 N-terminal amino
acids was found on contig TB.sub.--2044 and on locus MTY15C10.04
with EMBL accession number: z95436. The identity was found within
an open reading frame of 67 amino acids length corresponding to a
theoretical MW of 7240 Da and a pI of 5.18.
[0370] For CFP10A a sequence 100% identical to 12 N-terminal amino
acids was found on contig TB.sub.--752 and on locus CY130.20 with
EMBL accession number: Q10646 and Z73902. The identity was found
within an open reading frame of 93 amino acids length corresponding
to a theoretical MW of 9557 Da and a pI of 4.78.
[0371] For CFP30B a sequence 100% identical to 15 N-terminal amino
acids was found on contig TB.sub.--335. The identity was found
within an open reading frame of 261 amino acids length
corresponding to a theoretical MW of 27345 Da and a pI of 4.24.
[0372] The amino acid sequences of the purified antigens as picked
from the Sanger database are shown in the following list.
16 CFP7B (SEQ ID NO: 147) 1 MPQGTVKWFN AEKGFGFIAP EDGSADVFVH
YTEIQGTGFR TLEENQKVEF 51 EIGHSPKGPQ ATGVRSL CFP10A (SEQ ID NO: 141)
1 MNVTVSIPTI LRPHTGGQKS VSASGDTLGA VISDLEANYS GISERLMDPS 51
SPGKLHRFVN IYVNDEDVRF SGGLATAIAD GDSVTILPAV AGG CFP11 protein
sequence (SEQ ID NO: 143) 1 MATRFMTDPH ANRDMAGRFE VHAQTVEDEA
RRMWASAQNI SGAGWSGMAE 51 ATSLDTMAQM NQAFRNIVNM LHGVRDGLVR
DANNYEQQEQ ASQQILSS CFP30B (SEQ ID NO: 145) 1 MPKRSEYRQG TPNWVDLQTT
DQSAAKKFYT SLFGWGYDDN PVPGGGGVYS 51 MATLNGEAVA AIAPMPPGAP
EGMPPIWNTY IAVDDVDAVV DKVVPGGGQV 101 MMPAFDIGDA GRMSFITDPT
GAAVGLWQAN RHIGATLVNE TGTLIWNELL 151 TDKPDLALAF YEAVVGLTHS
SMEIAAGQNY RVLKAGDAEV GGCMEPPMPG 201 VPNHWHVYFA VDDADATAAK
AAAAGGQVIA EPADIPSVGR FAVLSDPQGA 251 IFSVLKPAPQ Q
[0373] Cloning of the genes encoding CFP7B, CFP10A, CFP11, and
CFP30B.
[0374] PCR reactions contained 10 ng of M. tuberculosis chromosomal
DNA in 1.times. low salt Taq+ buffer from Stratagene supplemented
with 250 mM of each of the four nucleotides (Boehringer Mannheim),
0,5 mg/ml BSA (IgG technology), 1% DMSO (Merck), 5 pmoles of each
primer and 0.5 unit Tag+ DNA polymerase (Stratagene) in 10 ml
reaction volume. Reactions were initially heated to 94.degree. C.
for 25 sec. and run for 30 cycles of the program; 94.degree. C. for
10 sec., 55.degree. C. for 10 sec. and 72.degree. C. for 90 sec.,
using thermocycler equipment from Idaho Technology.
[0375] The DNA fragments were subsequently run on 1% agarose gels,
the bands were excised and purified by Spin-X spin columns (Costar)
and cloned into pBluscript SK II+-T vector (Stratagene). Plasmid
DNA was hereafter prepared from clones harbouring the desired
fragments, digested with suitable restriction enzymes and subcloned
into the expression vector pMCT6 in frame with 8 histidines which
are added to the N-terminal of the expressed proteins. The
resulting clones were hereafter sequenced by use of the dideoxy
chain termination method adapted for supercoiled DNA using the
Sequenase DNA sequencing kit version 1.0 (United States Biochemical
Corp., USA) and by cycle sequencing using the Dye Terminator system
in combination with an automated gel reader (model 373A; Applied
Biosystems) according to the instructions provided. Both strands of
the DNA were sequenced.
[0376] For cloning of the individual antigens, the following gene
specific primers were used:
17 CFP7B: Primers used for cloning of cfp7B: CFP7B-F:
CTGAGATCTAGAATGCCACAGGGAACTGTG (SEQ ID NO: 160) CFP7B-R:
TCTCCCGGGGGTAACTCAGAGCGAGCGGAC (SEQ ID NO: 161) CFP7B-F and CFP7B-R
create BglII and SmaI sites, respectively, used for the cloning in
pMCT6. CFP10A: Primers used for cloning of cfp10A: CFP10A-F:
CTGAGATCTATGAACGTCACCGTATCC (SEQ ID NO: 162) CFP10A-R:
TCTCCCGGGGCTCACCCACCGGCCACG (SEQ ID NO: 163) CFP10A-F and CFP10A-R
create BglII and SmaI sites, respectively, used for the cloning in
pMCT6. CFP11: Primers used for cloning of cfp11: CFP11-F:
CTGAGATCTATGGCAACACGTTTTATGACG (SEQ ID NO: 164) CFP11-R:
CTCCCCGGGTTAGCTGCTGAGGATCTGCTH (SEQ ID NO: 165) CFP11-F and CFP11-R
create BglII and SinaI sites, respectively, used for the cloning in
pMCTG. CFP30B: Primers used for cloning of cfp30B: CFP30B-F:
CTGAAGATCTATGCCCAAGAGAAGCGAATAC (SEQ ID NO: 166) CFP30B-R:
CGGCAGCTGCTAGCATTCTCCGAATCTGCCG (SEQ ID NO: 167) CFP30B-F and
CFP30B-R create BglII and PvuII sites, respectively, used for the
cloning in pMCT6.
[0377] Expression/purification of recombinant CFP7B, CFP10A, CFP11
and CFP30B protein.
[0378] Expression and metal affinity purification of recombinant
protein was undertaken essentially as described by the
manufacturers. 1 l LB-media containing 100 .mu.g/ml ampicillin, was
inoculated with 10 ml of an overnight culture of XL1-Blue cells
harbouring recombinant pMCT6 plasmid. The culture was shaken at
37.degree. C. until it reached a density of OD.sub.600=0.5. IPTG
was hereafter added to a final concentration of 1 mM and the
culture was further incubated 4 hours. Cells were harvested,
resuspended in 1.times. sonication buffer+8 M urea and sonicated
5.times.30 sec. with 30 sec. pausing between the pulses.
[0379] After centrifugation, the lysate was applied to a column
containing 25 ml of resuspended Talon resin (Clontech, Palo Alto,
USA). The column was washed and eluted as described by the
manufacturers.
[0380] After elution, all fractions (1.5 ml each) were subjected to
analysis by SDS-PAGE using the Mighty Small (Hoefer Scientific
Instruments, USA) system and the protein concentrations were
estimated at 280 nm. Fractions containing recombinant protein were
pooled and dialysed against 3 M urea in 10 mM Tris-HCl, pH 8.5. The
dialysed protein was further purified by FPLC (Pharmacia, Sweden)
using a 6 ml Resource-Q column, eluted with a linear 0-1 M gradient
of NaCl. Fractions were analysed by SDS-PAGE and protein
concentrations were estimated at OD.sub.280. Fractions containing
protein were pooled and dialysed against 25 mM Hepes buffer, pH
8.5.
[0381] Finally the protein concentration and the LPS content was
determined by the BCA (Pierce, Holland) and LAL (Endosafe,
Charleston, USA) tests, respectively.
EXAMPLE 4
[0382] Cloning of the gene expressing CFP26 (MPT51)
[0383] Synthesis and design of probes
[0384] Oligonucleotide primers were synthesized automatically on a
DNA synthesizer (Applied Biosystems, Forster City, Calif., ABI-391,
PCR-mode) deblocked and purified by ethanol precipitation.
[0385] Three oligonucleotides were synthesized (TABLE 3) on the
basis of the nucleotide sequence from mpb51 described by Ohara et
al. (1995). The oligonucleotides were engineered to include an
EcoRI restriction enzyme site at the 5' end and at the 3' end by
which a later subcloning was possible.
[0386] Additional four oligonucleotides were synthesized on the
basis of the nucleotide sequence from MPT51 (FIG. 5 and SEQ ID NO:
41). The four combinations of the primers were used for the PCR
studies.
[0387] DNA cloning and PCR technology
[0388] Standard procedures were used for the preparation and
handling of DNA (Sambrook et al., 1989). The gene mpt5l was cloned
from M. tuberculosis H37Rv chromosomal DNA by the use of the
polymerase chain reactions (PCR) technology as described previously
(Oettinger and Andersen, 1994). The PCR product was cloned in the
pBluescriptSK+(Stratagene).
[0389] Cloning of mpt51
[0390] The gene, the signal sequence and the Shine Delgarno region
of MPT51 was cloned by use of the PCR technology as two fragments
of 952 bp and 815 bp in pBluescript SK+, designated pTO52 and
pTO53.
[0391] DNA Sequencing
[0392] The nucleotide sequence of the cloned 952 bp M. tuberculosis
H37Rv PCR fragment, pTO52, containing the Shine Dalgarno sequence,
the signal peptide sequence and the structural gene of MPT51, and
the nucleotide sequence of the cloned 815 bp PCR fragment
containing the structural gene of MPT51, pTO53, were determined by
the dideoxy chain termination method adapted for supercoiled DNA by
use of the Sequenase DNA sequencing kit version 1.0 (United States
Biochemical Corp., Cleveland, Ohio) and by cycle sequencing using
the Dye Terminator system in combination with an automated gel
reader (model 373A; Applied Biosystems) according to the
instructions provided. Both strands of the DNA were sequenced.
[0393] The nucleotide sequences of pTO52 and pTO53 and the deduced
amino acid sequence are shown in FIG. 5. The DNA sequence contained
an open reading frame starting with a ATG codon at position 45-47
and ending with a termination codon (TAA) at position 942-944. The
nucleotide sequence of the first 33 codons was expected to encode
the signal sequence. On the basis of the known N-terminal amino
acid sequence (Ala-Pro-Tyr-Glu-Asn) of the purified MPT51 (Nagai et
al., 1991) and the features of the signal peptide, it is presumed
that the signal peptidase recognition sequence (Ala-X-Ala) (von
Heijne, 1984) is located in front of the N-terminal region of the
mature protein at position 144. Therefore, a structural gene
encoding MPT51, mpt51, derived from M. tuberculosis H37Rv was found
to be located at position 144-945 of the sequence shown in FIG. 5.
The nucleotide sequence of mpt51 differed with one nucleotide
compared to the nucleotide sequence of MPB51 described by Ohara et
al. (1995) (FIG. 5). In mpt51 at position 780 was found a
substitution of a guanine to an adenine. From the deduced amino
acid sequence this change occurs at a first position of the codon
giving a amino acid change from alanine to threonine. Thus it is
concluded, that mpt51consists of 801 bp and that the deduced amino
acid sequence contains 266 residues with a molecular weight of
27,842, and MPT51 show 99,8% identity to MPB51.
[0394] Subcloning of mpt51
[0395] An EcoRI site was engineered immediately 5' of the first
codon of mpt51so that only the coding region of the gene encoding
MPT51 would be expressed, and an EcoRI site was incorporated right
after the stop codon at the 3' end.
[0396] DNA of the recombinant plasmid pTO53 was cleaved at the
EcoRI sites. The 815 bp fragment was purified from an agarose gel
and subcloned into the EcoRI site of the pMAL-cR1 expression vector
(New England Biolabs), pTO54. Vector DNA containing the gene fusion
was used to transform the E. coli XL1-Blue by the standard
procedures for DNA manipulation.
[0397] The endpoints of the gene fusion were determined by the
dideoxy chain termination method as described under section DNA
sequencing. Both strands of the DNA were sequenced.
[0398] Preparation and purification of rMPT51
[0399] Recombinant antigen was prepared in accordance with
instructions provided by New England Biolabs. Briefly, single
colonies of E. coli harbouring the pTO54 plasmid were inoculated
into Luria-Bertani broth containing 50 .mu.g/ml ampicillin and 12.5
.mu.g/ml tetracycline and grown at 37.degree. C. to
2.times.10.sup.8 cells/ml. Isopropyl-.beta.-D-thiogalactoside
(IPTG) was then added to a final concentration of 0.3 mM and growth
was continued for further 2 hours. The pelleted bacteria were
stored overnight at -20.degree. C. in new column buffer (20 mM
Tris/HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol
(DTT))and thawed at 4.degree. C. followed by incubation with 1
mg/ml lysozyme on ice for 30 min and sonication (20 times for 10
sec with intervals of 20 sec). After centrifugation at
9,000.times.g for 30 min at 4.degree. C., the maltose binding
protein -MPT51fusion protein (MBP-rMPT51) was purified from the
crude extract by affinity chromatography on amylose resin column.
MBP-rMPT51 binds to amylose. After extensive washes of the column,
the fusion protein was eluted with 10 mM maltose. Aliquots of the
fractions were analyzed on 10% SDS-PAGE. Fractions containing the
fusion protein of interest were pooled and was dialysed extensively
against physiological saline.
[0400] Protein concentration was determined by the BCA method
supplied by Pierce (Pierce Chemical Company, Rockford, Ill.).
18TABLE 3 Sequence of the mpt51 oligonucleotides.sup.a. Orientation
and position.sup.b oligonucleotide.sup.a Sequences (5' .fwdarw. 3')
(nucleotide) Sense MPT51-1 CTCGAATTCGCCGGGTGCACACAG 6-21 (SEQ ID
NO: 28) (SEQ ID NO: 41) MPT51-3 CTCGAATTCGCCCCATACGAGAAC 143-158
(SEQ ID NO: 29) (SEQ ID NO: 41) MPT51-5 GTGTATCTGCTGGAC 228-242
(SEQ ID NO: 30) (SEQ ID NO: 41) MPT51-7 CCGACTGGCTGGCCG 418-432
(SEQ ID NO: 31) (SEQ ID NO: 41) Antisense MPT51-2
GAGGAATTCGCTTAGCGGATCGCA 946-932 (SEQ ID NO: 32) (SEQ ID NO: 41)
MPT51-4 CCCACATTCCGTTGG 642-628 (SEQ ID NO: 33) (SEQ ID NO: 41)
MPT51-6 GTCCAGCAGATACAC 242-228 (SEQ ID NO: 34) (SEQ ID NO: 41)
.sup.aThe oligonucleotides MPT51-1 and MPT51-2 were constructed
from the MPB51 nucleotide sequence (Ohara et al., 1995). The other
oligonucleotides constructions were based on the nucleotide
sequence obtained from mpt52 reported in this work Nucleotides (nt)
underlined are not contained in the nucleotide sequence of MPB/T51.
.sup.bThe positions referred to are of the non-underlined parts of
the primers and correspond to the nucleotide sequence shown in SEQ
ID NO: 41.
[0401] Cloning of mpt51 in the expression vector pMST24.
[0402] A PCR fragment was produced from pTO52 using the primer
combination MPT51-F and MPT51-R (TABLE 4). A BamHI site was
engineered immediately 5' of the first codon of mpt51 so that only
the coding region of the gene encoding MPT51 would be expressed,
and an NcoI site was incorporated right after the stop codon at the
3' end.
[0403] The PCR product was cleaved at the BamHI and the NcoI site.
The 811 bp fragment was purified from an agarose gel and subcloned
into the BamHI and the NcoI site of the pMST24 expression vector,
pTO86. Vector DNA containing the gene fusion was used to transform
the E. coli XL1-Blue by the standard procedures for DNA
manipulation.
[0404] The nucleotide sequence of complete gene fusion was
determined by the dideoxy chain termination method as described
under section DNA sequencing. Both strands of the DNA were
sequenced.
[0405] Preparation and purification of rMPT51.
[0406] Recombinant antigen was prepared from single colonies of E.
coli harbouring the pTO86 plasmid inoculated into Luria-Bertani
broth containing 50 .mu.g/ml ampicillin and 12.5 .mu.g/ml
tetracycline and grown at 37.degree. C. to 2.times.10.sup.8
cells/ml. Isopropyl-.beta.-D-thiogalactoside (IPTG) was then added
to a final concentration of 1 mM and growth was continued for
further 2 hours. The pelleted bacteria were resuspended in BC
100/20 buffer (100 mM KCl, 20 mM Imidazole, 20 mM Tris/HCl, pH 7.9,
20% glycerol). Cells were broken by sonication (20 times for 10 sec
with intervals of 20 sec). After centrifugation at 9,000.times.g
for 30 min. at 4.degree. C. the insoluble matter was resuspended in
BC 100/20 buffer with 8 M urea followed by sonication and
centrifugation as above. The 6.times.His tag-MPT51 fusion protein
(His-rMPT51) was purified by affinity chromatography on Ni-NTA
resin column (Qiagen, Hilden, Germany). His-rMPT51 binds to Ni-NTA.
After extensive washes of the column, the fusion protein was eluted
with BC 100/40 buffer (100 mM KCl, 40 mM Imidazole, 20 mM Tris/HCl,
pH 7.9, 20% glycerol) with 8 M urea and BC 1000/40 buffer (1000 mM
KCl, 40 mM Imidazole, 20 mM Tris/HCl, pH 7.9, 20% glycerol) with 8
M urea. His-rMPT51 was extensive dialysed against 10 mM Tris/HC1,
pH 8.5, 3 M urea followed by purification using fast protein liquid
chromatography (FPLC) (Pharmacia, Uppsala, Sweden), over an anion
exchange column (Mono Q) using 10 mM Tris/HCl, pH 8.5, 3 M urea
with a 0-1 M NaCl linear gradient. Fractions containing rMPT51 were
pooled and subsequently dialysed extensively against 25 mM Hepes,
pH 8.0 before use.
[0407] Protein concentration was determined by the BCA method
supplied by Pierce (Pierce Chemical Company, Rockford, Ill.). The
lipopolysaccharide (LPS) content was determined by the limulus
amoebocyte lysate test (LAL) to be less than 0.004 ng/.mu.g rMPT5,
and this concentration had no influence on cellular activity.
19TABLE 4 Sequence of the mpt51 oligonucleotides. Orientation and
oligo- Position nucleotide Sequences (5' .fwdarw. 3') (nt) Sense
MPT51-F CTCGGATCCTGCCCCATACGAGAACCTG 139-156 Antisense MPT51-R
CTCCCATGGTTAGCGGATCGCACCQ 939-924
EXAMPLE 4A
[0408] Cloning of the ESAT6-MPT59 and the MPT59-ESAT6 hybrides.
[0409] Background for ESAT-MPT59 and MPT59-ESAT6 fusion
[0410] Several studies have demonstrated that ESAT-6 is a an
immunogen which is relatively difficult to adjuvate in order to
obtain consistent results when immunizing therewith. To detect an
in vitro recognition of ESAT-6 after immunization with the antigen
is very difficult compared to the strong recognition of the antigen
that has been found during the recall of memory immunity to M.
tuberculosis. ESAT-6 has been found in ST-CF in a truncated version
were amino acids 1-15 have been deleted. The deletion includes the
main T-cell epitopes recognized by C57BL/6j mice (Brandt et al.,
1996). This result indicates that ESAT-6 either is N-terminally
processed or proteolytically degraded in STCF. In order to optimize
ESAT-6 as an immunogen, a gene fusion between ESAT-6 and another
major T cell antigen MPT59 has been constructed. Two different
construct have been made: MPT59-ESAT-6 (SEQ ID NO: 172) and
ESAT-6-MPT59 (SEQ ID NO: 173). In the first hybrid ESAT-6 is
N-terminally protected by MPT59 and in the latter it is expected
that the fusion of two dominant T-cell antigens can have a
synergistic effect.
[0411] The genes encoding the ESAT6-MPT59 and the MPT59-ESAT6
hybrides were cloned into the expression vector pMCT6, by PCR
amplification with gene specific primers, for recombinant
expression in E. coli of the hybrid proteins.
[0412] Construction of the hybrid MPT59-ESAT6.
[0413] The cloning was carried out in three steps. First the genes
encoding the two components of the hybrid, ESAT6 and MPT59, were
PCR amplified using the following primer constructions:
20 ESAT6: OPBR-4: GGCGCCGGCAAGCTTGCCATGACAGAGCAGCAG- TGG (SEQ ID
NO: 132) OPBR-28: CGAACTCGCCGGATCCCGTGTTTCGC (SEQ ID NO: 133)
OPBR-4 and OPBR-28 create HinDIII and BamHI sites, respectively.
MPT59: OPBR-48: GGCAACCGCGAGATCTTTCTCCCGGCCGGGGC (SEQ ID NO: 134)
OPBR-3: GGCAAGCTTGCCGGCGCCTAACGAACT (SEQ ID NO: 135)
[0414] OPBR-48 and OPBR-3 create Bg1II and HinDIII, respectively.
Additionally OPBR-3 deletes the stop codon of MPT59.
[0415] PCR reactions contained 10 ng of M. tuberculosis chromosomal
DNA in 1.times. low salt Taq+ buffer from Stratagene supplemented
with 250 mM of each of the four nucleotides (Boehringer Mannheim),
0,5 mg/ml BSA (IgG technology), 1% DMSO (Merck), 5 pmoles of each
primer and 0.5 unit Tag+ DNA polymerase (Stratagene) in 10 .mu.l
reaction volume. Reactions were initially heated to 94.degree. C.
for 25 sec. and run for 30 cycles of the program; 94.degree. C. for
10 sec., 55.degree. C. for 10 sec. and 72.degree. C. for 90 sec,
using thermocycler equipment from Idaho Technology.
[0416] The DNA fragments were subsequently run on 1% agarose gels,
the bands were excised and purified by Spin-X spin columns
(Costar). The two PCR fragments were digested with HinDIII and
ligated. A PCR amplification of the ligated PCR fragments encoding
MPT59-ESAT6 was carried out using the primers OPBR-48 and OPBR-28.
PCR reaction was initially heated to 94.degree. C. for 25 sec. and
run for 30 cycles of the program; 94.degree. C. for 30 sec.,
55.degree. C. for 30 sec. and 72.degree. C. for 90 sec. The
resulting PCR fragment was digested with Bg1II and BamHI and cloned
into the expression vector pMCT6 in frame with 8 histidines which
are added to the N-terminal of the expressed protein hybrid. The
resulting clones were hereafter sequenced by use of the dideoxy
chain termination method adapted for supercoiled DNA using the
Sequenase DNA sequencing kit version 1.0 (United States Biochemical
Corp., USA) and by cycle sequencing using the Dye Terminator system
in combination with an automated gel reader (model 373A; Applied
Biosystems) according to the instructions provided. Both strands of
the DNA were sequenced.
[0417] Construction of the hybrid ESAT6-MPT59.
[0418] Construction of the hybrid ESAT6-MPT59 was carried out as
described for the hybrid MPT59-ESAT6. The primers used for the
construction and cloning were:
21 ESAT6: OPBR-75: GGACCCAGATCTATGACAGAGCAGCAGTGG (SEQ ID NO: 136)
OPBR-76: CCGGCAGCCCCGGCCGGGAGAAAAGCTTTGCGAACATCC- CAGTGACG (SEQ ID
NO: 137) OPBR-75 and OPBR-76 create BglII and HinDIII sites,
respectively. Additionally OPBR-76 deletes the stop codon of ESAT6.
MPT59: OPBR-77: GTTCGCAAAGCTTTTCTCCCGGCCGGGGCTGCCGGTCGAGTACC (SEQ
ID NO: 128) OPBR-18: CCTTCGGTGGATCCCGTCAG (SEQ ID NO: 139) OPBR-77
and OPBR-l8 create HinDIII and BamHI sites, respectively.
[0419] Expression/purification of MPT59-ESAT6 and ESAT6-MPT59
hybrid proteins.
[0420] Expression and metal affinity purification of recombinant
proteins was undertaken essentially as described by the
manufacturers. For each protein, 1 1 LB-media containing 100
.mu.g/ml ampicillin, was inoculated with 10 ml of an overnight
culture of XLl-Blue cells harbouring recombinant pMCT6 plasmids.
Cultures were shaken at 37.degree. C. until they reached a density
of OD.sub.600=0.4-0.6. IPTG was hereafter added to a final
concentration of 1 mM and the cultures were further incubated 4-16
hours. Cells were harvested, resuspended in 1.times. sonication
buffer+8 M urea and sonicated 5.times.30 sec. with 30 sec. pausing
between the pulses.
[0421] After centrifugation, the lysate was applied to a column
containing 25 ml of resuspended Talon resin (Clontech, Palo Alto,
USA). The column was washed and eluted as described by the
manufacturers.
[0422] After elution, all fractions (1.5 ml each) were subjected to
analysis by SDS-PAGE using the Mighty Small (Hoefer Scientific
Instruments, USA) system and the protein concentrations were
estimated at 280 nm. Fractions containing recombinant protein were
pooled and dialysed against 3 M urea in 10 mM Tris-HCl, pH 8.5. The
dialysed protein was further purified by FPLC (Pharmacia, Sweden)
using a 6 ml Resource-Q column, eluted with a linear 0-1 M gradient
of NaCl. Fractions were analyzed by SDS-PAGE and protein
concentrations were estimated at OD.sub.280. Fractions containing
protein were pooled and dialysed against 25 mM Hepes buffer, pH
8.5.
[0423] Finally the protein concentration and the LPS content were
determined by the BCA (Pierce, Holland) and LAL (Endosafe,
Charleston, USA) tests, respectively.
[0424] The biological activity of the MPT59-ESAT6 fusion protein is
described in Example 6A.
EXAMPLE 5
[0425] Mapping of the purified antigens in a 2DE system.
[0426] In order to characterize the purified antigens they were
mapped in a 2-dimensional electrophoresis (2DE) reference system.
This consists of a silver stained gel containing ST-CF proteins
separated by isoelectrical focusing followed by a separation
according to size in a polyacrylamide gel electro-phoresis. The 2DE
was performed according to Hochstrasser et al. (1988). 85 .mu.g of
ST-CF was applied to the isoelectrical focusing tubes where BioRad
ampholytes BioLyt 4-6 (2 parts) and BioLyt 5-7 (3 parts) were
included. The first dimension was performed in acrylamide/piperazin
diacrylamide tube gels in the presence of urea, the detergent CHAPS
and the reducing agent DTT at 400 V for 18 hours and 800 V for 2
hours. The second dimension 10-20% SDS-PAGE was performed at 100 V
for 18 hours and silver stained. The identification of CFP7, CFP7A,
CFP7B, CFP8A, CFP8B, CFP9, CFP11, CFP16, CFP17, CFP19, CFP20,
CFP21, CFP22, CFP25, CFP27, CFP28, CFP29, CFP30A, CFP50, and MPT51
in the 2DE reference gel were done by comparing the spot pattern of
the purified antigen with ST-CF with and without the purified
antigen. By the assistance of an analytical 2DE software system
(Phoretix International, UK) the spots have been identified in FIG.
6. The position of MPT51 and CFP29 were confirmed by a Western blot
of the 2DE gel using the Mab's anti-CFP29 and HBT 4.
EXAMPLE 6
[0427] Biological activity of the purified antigens.
[0428] IFN-.gamma. induction in the mouse model of TB infection
[0429] The recognition of the purified antigens in the mouse model
of memory immunity to TB (described in example 1) was investigated.
The results shown in TABLE 5 are representative for three
experiments.
[0430] A very high IFN-.gamma. response was induced by two of the
antigens CFP17 and CFP21 at almost the same high level as
ST-CF.
22TABLE 5 IFN-.gamma. release from splenic memory effector cells
from C57BL/6J mice isolated after reinfection with M. tuberculosis
after stimulation with native antigens. Antigen.sup.a IFN-.gamma.
(pg/ml).sup.b ST-CF 12564 CFP7 ND.sup.d CFP9 ND CFP17 9251 CFP20
2388 CFP21 10732 CFP22 + CFP25.sup.c 5342 CFP26 (MPT51) ND CFP28
2818 CFP29 3700 The data is derived from a representative
experiment out of three. .sup.aSTCF was tested in a concentration
of 5 pg/ml and the individual antigens in a concentration of 2
.mu.g/ml. .sup.bFour days after rechallenge a pool of cells from
three mice were tested. The results are expressed as mean of
duplicate values and the difference between duplicate cultures are
<15% of mean. The IFN-.gamma. release of cultures incubated
without antigen was 390 pg/ml. .sup.cA pool of CFP22 and CFP25 was
tested. .sup.dND, not determined.
[0431] Skin test reaction in TB infected guinea pigs
[0432] The skin test activity of the purified proteins was tested
in M. tuberculosis infected guinea pigs.
[0433] 1 group of guinea pigs was infected via an ear vein with
1.times.10.sup.4 CFU of M. tuberculosis H37Rv in 0,2 ml PBS. After
4 weeks skin tests were performed and 24 hours after injection
erythema diameter was measured.
[0434] As seen in TABLES 6 and 6a all of the antigens induced a
significant Delayed Type Hypersensitivity (DTH) reaction.
23TABLE 6 DTH erythema diameter in guinea pigs infected with 1
.times. 10.sup.4 CFU of M. tuberculosis, after stimulation with
native antigens. Antigen.sup.a Skin reaction (mm).sup.b Control
2.00 PPD.sup.c 15.40 (0.53) CFP7 ND.sup.e CFP9 ND CFP17 11.25
(0.84) CFP20 8.88 (0.13) CFP21 12.44 (0.79) CFP22 + CFP25.sup.d
9.19 (3.10) CFP26 (MPT51) ND CFP28 2.90 (1.28) CFP29 6.63 (0.88)
The values presented are the mean of erythema diameter of four
animals and the SEM's are indicated in the brackets. For PPD and
CFP29 the values are mean of erythema diameter of ten animals.
.sup.aThe antigens were tested in a concentration of 0.1 .mu.g
except for CFP29 which was tested in a concentration of 0.8 .mu.g.
.sup.bThe skin reactions are measured in mm erythema 24 h after
intradermal injection. .sup.c10 TU of PPD was used. .sup.dA pool of
CFP22 and CFP25 was tested. .sup.eND, not determined.
[0435] Together these analyses indicate that most of the antigens
identified were highly biologically active and recognized during TB
infection in different animal models.
24TABLE 6a DTH erythema diameter of recombinant antigens in outbred
guinea pigs infected with 1 .times. 10.sup.4 CFU of M.
Tuberculosis. Antigen.sup.a Skin reaction (mm).sup.b Control 2.9
(0.3) PPD.sup.a 14.5 (1.0) CFP 7a 13.6 (1.4) CFP 17 6.8 (1.9) CFP
20 6.4 (1.4) CFP 21 5.3 (0.7) CFP 25 10.8 (0.8) CFP 29 7.4 (2.2)
MPT 51 4.9 (1.1) The values presented are the mean of erythema
diameter of four animals and the SEM's are indicated in the
brackets. For Control, PPD, and CFP 20 the values are mean of
erythema diameter of eight animals. .sup.aThe antigens were tested
in a concentration of 1.0 .mu.g. .sup.bThe skin test reactions are
measured in mm erythema 24 h after intradermal infection. .sup.c10
TU of PPD was used.
[0436] Biological activity of the purified recombinant
antigens.
[0437] Interferon-.gamma. induction in the mouse model of TB
infection.
[0438] Primary infections
[0439] 8 to 12 weeks old female C57BL/6j(H-2.sup.b),
CBA/J(H-2.sup.k), DBA.2(H-2.sup.d) and A.SW(H-2.sup.s) mice
(Bomholtegaard, Ry) were given intravenous infections via the
lateral tail vein with an inoculum of 5.times.10.sup.4 M.
tuberculosis suspended in PBS in a vol. of 0.1 ml. 14 days
postinfection the animals were sacrificed and spleen cells were
isolated and tested for the recognition of recombinant antigen.
[0440] As seen in TABLE 7 the recombinant antigens rCFP7A, rCFP17,
rCFP21, rCFP25, and rCFP29 were all recognized in at least two
strains of mice at a level comparable to ST-CF. rMPT51 and rCFP7
were only recognized in one or two strains respectively, at a level
corresponding to no more than 1/3 of the response detected after
ST-CF stimulation. Neither of the antigens rCFP20 and rCFP22 were
recognized by any of the four mouse strains.
[0441] Memory responses
[0442] 8-12 weeks old female C57BL/6j(H-2.sup.b) mice
(Bomholtegaard, Ry) were given intravenous infections via the
lateral tail vein with an inoculum of 5.times.10.sup.4 M.
tuberculosis suspended in PBS in a vol. of 0.1 ml. After 1 month of
infection the mice were treated with isoniazid (Merck and Co.,
Rahway, N.J.) and rifabutin (Farmatalia Carlo Erba, Milano, Italy)
in the drinking water, for two months. The mice were rested for 4-6
months before being used in experiments. For the study of the
recall of memory immunity, animals were infected with an inoculum
of 1.times.10.sup.6 bacteria i.v. and sacrificed at day 4
postinfection. Spleen cells were isolated and tested for the
recognition of recombinant antigen. As seen from TABLE 8,
IFN-.gamma. release after stimulation with rCFP17, rCFP21 and
rCFP25 was at the same level as seen from spleen cells stimulated
with ST-CF. Stimulation with rCFP7, rCFP7A and rCFP29 all resulted
in an IFN-.gamma. no higher than 1/3 of the response seen with
ST-CF. rCFP22 was not recognized by IFN-.gamma. producing cells.
None of the antigens stimulated IFN-.gamma. release in naive mice.
Additionally non of the antigens were toxic to the cell
cultures.
25TABLE 7 T cell responses in primary TB infection. Name c57BL/6J
(H2.sup.b) DBA.2 (H2.sup.d) CBA/J (H2.sup.k) A.SW (H2.sup.s) rCFP7
+ + - - rCFP7A +++ +++ +++ + rCFP17 +++ + +++ + rCFP20 - - - -
rCFP21 +++ +++ +++ + rCFP22 - - - - rCFP25 +++ ++ +++ + rCFP29 +++
+++ +++ ++ rMPT51 + - - - Mouse IFN-.gamma. release during recall
of memory immunity to M. tuberculosis. -: no response; +: 1/3 of
ST-CF; ++: 2/3 of ST-CF; +++: level of ST-CF.
[0443]
26TABLE 8 T cell responses in memory immune animals. Name Memory
response rCFP7 + rCFP7A ++ rCFP17 +++ rCFP21 +++ rCFP22 - rCFP29 +
rCFP25 +++ rMPT51 + Mouse IFN-.gamma. release 14 days after primary
infection with M. tuberculosis. -: no response; +: 1/3 of ST-CF;
++: 2/3 of ST-CF; +++: level of ST-CF.
[0444] Interferon-.gamma. induction in human TB patients and BCG
vaccinated people.
[0445] Human donors
[0446] PBMC were obtained from healthy BCG vaccinated donors with
no known exposure to patients with TB and from patients with
culture or microscopy proven infection with Mycobacterium
tuberculosis. Blood samples were drawn from the TB patients 1-4
months after diagnosis.
[0447] Lymphocyte preparations and cell culture
[0448] PBMC were freshly isolated by gradient centrifugatin of
heparinized blood on Lymphoprep (Nycomed, Oslo, Norway). The cells
were resuspended in complete medium: RPMI 1640 (Gibco, Grand
Island, N.Y.) supplemented with 40 .mu./ml -streptomycin, 40 U/ml
penicillin, and 0.04 mM/ml glutamine, (all from Gibco Laboratories,
Paisley, Scotland) and 10% normal human ABO serum (NHS) from the
local blood bank. The number and the viability of the cells were
determined by trypan blue staining. Cultures were established with
2,5.times.10.sup.5 PBMC in 200 .mu.l in microtitre plates (Nunc,
Roskilde, Denmark) and stimulated with no antigen, ST-CF, PPD
(2.5.mu.g/ml); rCFP7, rCFP7A, rCFP17, rCFP20, rCFP21, rCFP22,
rCFP25, rCFP26, rCFP29, in a final concentration of 5 .mu.g/ml.
Phytohaemagglutinin, 1 .mu.g/ml (PHA, Difco laboratories, Detroit,
Mich. was used as a positive control. Supernatants for the
detection of cytokines were harvested after 5 days of culture,
pooled and stored at -80.degree. C. until use.
[0449] Cytokine analysis
[0450] Interferon-.gamma. (IFN-.gamma.) was measured with a
standard ELISA technique using a commercially available pair of
mAb's from Endogen and used according to the instructions for use.
Recombinant IFN-.gamma. (Gibco laboratories) was used as a
standard. The detection level for the assay was 50 pg/ml. The
variation between the duplicate wells did not exceed 10% of the
mean. Responses of 9 individual donors are shown in TABLE 9.
[0451] A seen in TABLE 9 high levels of IFN-.gamma. release are
obtained after stimulation with several of the recombinant
antigens. rCFP7a and rCFP17 gives rise to responses comparable to
STCF in almost all donors. rCFP7 seems to be most strongly
recognized by BCG vaccinated healthy donors. rCFP21, rCFP25,
rCFP26, and rCFP29 gives rise to a mixed picture with intermediate
responses in each group, whereas low responses are obtained by
rCFP20 and rCFP22.
27TABLE 9 Mean values of results from the stimulation of human
blood cells from 7 BCG vaccinated and 7 TB patients with
recombinant antigens. SE values are given for each antigen. ST-CF
and M. avium culture filtrate are shown for the comparison. donor:
no ag PHA PPD STCF CFP7 CFP17 CFP7A CFP20 CFP21 CFP22 CFP25 CFP26
CFP29 Controls, Healthy, BCG vaccinated, no known TB exposure 1 6
9564 6774 3966 7034 69 1799 58 152 73 182 946 86 2 48 12486 6603
8067 3146 10044 5267 29 6149 51 1937 526 2065 3 190 11929 10000
8299 8015 11563 8641 437 3194 669 2531 8076 6098 4 10 21029 4106
3537 1323 1939 5211 1 284 1 1344 20 125 5 1 18750 14209 13027 17725
8038 19002 1 3008 1 2103 974 8181 TB patients, 1-4 month after
diagnosis 6 9 8973 5096 6145 852 4250 4019 284 1131 48 2400 1078
4584 7 1 12413 6281 3393 168 6375 4505 11 4335 16 3082 1370 5115 8
4 11915 7671 7375 104 2753 3356 119 407 437 2069 712 5284 9 32
22130 16417 17213 8450 9783 16319 91 5957 67 10043 13313 9953
EXAMPLE 6A
[0452] Four groups of 6-8 weeks old, female C57Bl/6J mice
(Bomholtegard, Denmark) were immunized subcutaneously at the base
of the tail with vaccines of the following compositions:
[0453] Group 1: 10 .mu.g ESAT-6/DDA (250 .mu.g)
[0454] Group 2: 10 .mu.g MPT59/DDA (250 .mu.g)
[0455] Group 3: 10 .mu.g MPT59-ESAT-6/DDA (250 .mu.g)
[0456] Group 4: Adjuvant control group: DDA (250 .mu.g) in NaCl
[0457] The animals were injected with a volume of 0.2 ml. Two weeks
after the first injection and 3 weeks after the second injection
the mice were boosted a little further up the back. One week after
the last immunization the mice were bled and the blood cells were
isolated. The immune response induced was monitored by release of
IFN-.gamma. into the culture supernatants when stimulated in vitro
with relevant antigens (see the following table).
28 Immunogen For restimulation.sup.a): Ag in vitro 10 .mu.g/dose no
antigen ST-CF ESAT-6 MPT59 ESAT-6 219 .+-. 219 569 .+-. 569 835
.+-. 633 -- MPT59 0 802 .+-. 182 -- 5647 .+-. 159 Hybrid: 127 .+-.
127 7453 .+-. 581 15133 .+-. 861 16363 .+-. 1002 MPT59- ESAT-6
.sup.a)Blood cells were isolated 1 week after the last immunization
and the release of IFN-.gamma. (pg/ml) after 72 h of antigen
stimulation (5 .mu.g/ml) was measured. The values shown are mean of
triplicates performed on cells pooled from three mice .+-. SEM
.sup.b)-- not determined
[0458] The experiment demonstrates that immunization with the
hybrid stimulates T cells which recognize ESAT-6 and MPT59 stronger
than after single antigen immunization. Especially the recognition
of ESAT-6 was enhanced by immunization with the MPT59-ESAT-6
hybrid. IFN-y release in control mice immunized with DDA never
exceeded 1000 pg/ml.
EXAMPLE 6B
[0459] The recombinant antigens were tested individually as subunit
vaccines in mice. Eleven groups of 6-8 weeks old, female C57Bl/6j
mice (Bomholteg{dot over (a)}rd, Denmark) were immunized
subcutaneously at the base of the tail with vaccines of the
following composition:
[0460] Group 1: 10 .mu.g CFP7
[0461] Group 2: 10 .mu.g CFP17
[0462] Group 3: 10 .mu.g CFP21
[0463] Group 4: 10 .mu.g CFP22
[0464] Group 5: 10 .mu.g CFP25
[0465] Group 6: 10 .mu.g CFP29
[0466] Group 7: 10 .mu.g MPT51
[0467] Group 8: 50 .mu.g ST-CF
[0468] Group 9: Adjuvant control group
[0469] Group 10: BCG 2,5.times.10.sup.5/ml, 0,2 ml
[0470] Group 11: Control group: Untreated
[0471] All the subunit vaccines were given with DDA as adjuvant.
The animals were vaccinated with a volume of 0.2 ml. Two weeks
after the first injection and three weeks after the second
injection group 1-9 were boosted a little further up the back. One
week after the last injection the mice were bled and the blood
cells were isolated. The immune response induced was monitored by
release of IFN-.gamma. into the culture supernatant when stimulated
in vitro with the homologous protein.
[0472] 6 weeks after the last immunization the mice were aerosol
challenged with 5.times.10.sup.6 viable Mycobacterium tuberculosis
/ml. After 6 weeks of infection the mice were killed and the number
of viable bacteria in lung and spleen of infected mice was
determined by plating serial 3-fold dilutions of organ homogenates
on 7Hll plates. Colonies were counted after 2-3 weeks of
incubation. The protective efficacy is expressed as the difference
between log.sub.10 values of the geometric mean of counts obtained
from five mice of the relevant group and the geometric mean of
counts obtained from five mouse of the relevant control group.
[0473] The results from the experiments are presented in the
following table.
[0474] Immunogenicity and protective efficacy in mice, of ST-CF and
7 subunit vaccines
29 Immunogenicity and protective efficacy in mice, of ST-CF and 7
subunit vaccines Subunit Vaccine Immunogenicity Protective efficacy
ST-CF +++ +++ CFP7 ++ - CFP17 +++ +++ CFP21 +++ ++ CFP22 - - CFP25
+++ +++ CFP29 +++ +++ MPT51 +++ ++ +++ Strong immunogen/high
protection (level of BCG) ++ Medium immunogen/medium protection -
No recognition/no protection
[0475] In conclusion, we have identified a number of proteins
inducing high levels of protection. Three of these CFP17, CFP25 and
CFP29 giving rise to similar levels of protection as ST-CF and BCG
while two proteins CFP21 and MPT51 induces protections around 2/3
the level of BCG and ST-CF. Two of the proteins CFP7 and CFP22 did
not induce protection in the mouse model.
EXAMPLE 7
[0476] Species distribution of cfp7, cfp9, mpt51, rd1-orf2,
rd1-orf3, rd1-orf4, rd1-orf5, rd1-orf8, rd1-orf9a and rd1-orf9b as
well as of cfp7a, cfp7b, cfp10a, cfpl7, cfp21, cfp21, cfp22,
cfp22a, cfp23, cfp25 and cfp25a.
[0477] Presence of cfg7, cfp9, mpt51, rd1-orf2, rd1-orf3, rd1-orf4,
rd1-orf5, rd1-orf8, rd1-orf9a and rd1-orf9b in different
mycobacterial species.
[0478] In order to determine the distribution of the cfp7, cfp9,
mpt51, rd1-orf2, rd1-orf3, rd1-orf4, rd1-orf5, rd1-orf8, rd1-orf9a
and rd1-orf9b genes in species belonging to the M.
tuberculosis-complex and in other mycobacteria PCR and/or Southern
blotting was used. The bacterial strains used are listed in TABLE
10. Genomic DNA was prepared from mycobacterial cells as described
previously (Andersen et al. 1992).
[0479] PCR analyses were used in order to determine the
distribution of the cfp7, cfp9 and mpt5l gene in species belonging
to the tuberculosis-complex and in other mycobacteria. The
bacterial strains used are listed in TABLE 10. PCR was performed on
genomic DNA prepared from mycobacterial cells as described
previously (Andersen et al., 1992).
[0480] The oligonucleotide primers used were synthesised
automatically on a DNA synthesizer (Applied Biosystems, Forster
City, Calif., ABI-391, PCR-mode), deblocked, and purified by
ethanol precipitation. The primers used for the analyses are shown
in TABLE 11.
[0481] The PCR amplification was carried out in a thermal reactor
(Rapid cycler, Idaho Technology, Idaho) by mixing 20 ng chromosomal
with the mastermix (contained 0.5 .mu.M of each oligonucleotide
primer, 0.25 .mu.M BSA (Stratagene), low salt buffer (20 mM
Tris-HCl, pH8.8, 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 2 mM
MgSO.sub.4 and 0,1% Triton X-100) (Stratagene), 0.25 mM of each
deoxynucleoside triphosphate and 0.5 U Taq Plus Long DNA polymerase
(Stratagene)). Final volume was 10 .mu.l (all concentrations given
are concentrations in the final volume). Predenaturation was
carried out at 94.degree. C. for 30 s. 30 cycles of the following
was performed: Denaturation at 94.degree. C. for 30 s, annealing at
55.degree. C. for 30 s and elongation at 72.degree. C. for 1
min.
[0482] The following primer combinations were used (the length of
the amplified products are given in parentheses):
[0483] mpt51: MPT51-3 and MPT51-2 (820 bp), MPT51-3 and MPT51-6
(108 bp), MPT51-5 and MPT51-4 (415 bp), MPT51-7 and MPT51-4 (325
bp).
[0484] cfp7: pVF1 and PVR1 (274 bp), pVF1 and PVR2 (197 bp), pVF3
and PVR1 (302 bp), pVF3 and PVR2 (125 bp). cfp9: stR3 and stF1 (351
bp).
30TABLE 10 Mycobacterial strains used in this Example. Species and
strain(s) Source 1. M. tuberculosis H 3 7 R vATCC.sup.a (ATCC
27294) 2. H 3 7 R aATCC (ATCC 25177) 3. Erdman Obtained from A.
Lazlo, Ottawa, Canada 4. M. bovis BCG substrain: SSI.sup.b Danish
1331 5. Chinese SSI.sup.c 6. Canadian SSI.sup.c 7. Glaxo SSI.sup.c
8. Russia SSI.sup.c 9. Pasteur SSI.sup.c 10. Japan WHO.sup.e 11. M.
bovis MNC 27 SSI.sup.c 12. M. africanum Isolated from a Danish
patient 13. M. leprae (armadillo-derived) Obtained from J. M.
Colston, London, UK 14. M. avium (ATCC 15769) ATCC 15. M. kansasii
(ATCC 12478) ATCC 16. M. marinum (ATCC 927) ATCC 17. M.
scrofulaceum (ATCC 19275) ATCC 18. M. intercellulare (ATCC 15985)
ATCC 19. M. fortuitum (ATCC 6841) ATCC 20. M. xenopi Isolated from
a Danish patient 21. M. flavescens Isolated from a Danish patient
22. M. szulgai Isolated from a Danish patient 23. M. terrae
SSI.sup.c 24. E. coli SSI.sup.d 25. S. aureus SSI.sup.d
.sup.aAmerican Type Culture Collection, USA. .sup.bStatens Serum
Institut, Copenhagen, Denmark. .sup.cOur collection Department of
Mycobacteriology, Statens Serum Institut, Copenhagen, Denmark.
.sup.dDepartment of Clinical Microbiology, Statens Serum Institut,
Denmark. .sup.eWHO International Laboratory for Biological
Standards, Statens Serum Institut, Copenhagen, Denmark.
[0485]
31TABLE 11 Sequence of the mpt51, cfp7 and cfp9 oligonucleotides.
Orien- tation and oligo- nucleo- Position.sup.b tide Sequences (5'
.fwdarw. 3').sup.a (nucleotides) Sense MPT51-
CTCGAATTCGCCGGGTGCACACAG 6-21 1 (SEQ ID NO: 28) (SEQ ID NO: 41)
MPT51- CTCGAATTCGCCCCATACGAGAAC 143-158 3 (SEQ ID NO: 29) (SEQ ID
NO: 41) MPT51- GTGTATCTGCTGGAC 228-242 5 (SEQ ID NO: 30) (SEQ ID
NO: 41) MPT51- CCGACTGGCTGGCCG 418-432 7 (SEQ ID NO: 31) (SEQ ID
NO: 41) pvR1 GTACGAGAATTCATGTCGCAAATCATG 91-105 (SEQ ID NO: 35)
(SEQ ID NO: 1) pvR2 GTACGAGAATTCGAGCTTGGGGTGCCG 168-181 (SEQ ID NO:
36) (SEQ ID NO: 1) stR3 CGATTCCAAGCTTGTGGCCGCCGACCCG 141-155 (SEQ
ID NO: 37) (SEQ ID NO: 3) Antisense MPT51- GAGGAATTCGCTTAGCGGATCGCA
946-932 2 (SEQ ID NO: 32) (SEQ ID NO: 41) MPT51- CCCACATTCCGTTGG
642-628 4 (SEQ ID NO: 33) (SEQ ID NO: 41) MPT51- GTCCAGCAGATACAC
242-228 6 (SEQ ID NO: 34) (SEQ ID NO: 41) pvF1
CGTTAGGGATCCTCATCGCCATGGTGTTGG 340-323 (SEQ ID NO: 38) (SEQ ID NO:
1) pvF3 CGTTAGGGATCCGGTTCCACTGTGCC 268-255 (SEQ ID NO: 39) (SEQ ID
NO: 1) stF1 CGTTAGGGATCCTCAGGTCTTTTCGATG 467-452 (SEQ ID NO: 40)
(SEQ ID NO: 3) .sup.aNucleotides underlined are not contained in
the nucleotide sequences of mpt51, cfp7, and cfp9. .sup.bThe
positions referred to are of the non-underlined parts of the
primers and correspond to the nucleotide sequence shown in SEQ ID
NOs: 41, 1, and 3 for mpt51, cfp7, and cfp9, respectively.
[0486] The Southern blotting was carried out as described
previously (Oettinger and Andersen, 1994) with the following
modifications: 2 .mu.g of genomic DNA was digested with PvuII,
electrophoresed in an 0.8% agarose gel, and transferred onto a
nylon membrane (Hybond N-plus; Amersham International plc, Little
Chalfont, United Kingdom) with a vacuum transfer device (Milliblot,
TM-v; Millipore Corp., Bedford, Mass.). The cfp7, cfp9, mpt51,
rd1-orf2, rd1-orf3, rd1-orf4, rd1-orf5, rd1-orf8, rd1-orf9a and
rd1-orf9b gene fragments were amplified by PCR from the plasmids
pRVN01, pRVN02, pT052, pT087, pT088, pT089, pT090, pT091, pT096 or
pT098 by using the primers shown in TABLE 11 and TABLE 2 (in
Example 2a). The probes were labelled non-radioactively with an
enhanced chemiluminescence kit (ECL; Amersham International plc,
Little Chalfont, United Kingdom). Hybridization and detection was
performed according to the instructions provided by the
manufacturer. The results are summarized in TABLES 12 and 13.
32TABLE 12 Interspecies analysis of the cfp7, cfp9 and mpt51 genes
by PCR and/or Southern blotting and of MPT51 protein by Western
blotting. Western PCR Southern blot blot Species and strain cfp7
cfp9 mpt51 cfp7 cfp9 mpt51 MPT51 1. M. tub. H37Rv + + + + + + + 2.
M. tub. H37Ra + + + N.D. N.D. + + 3. M. tub. Erdmann + + + + + + +
4. M. bovis + + + + + 5. M. bovis BCG + + + + + + + Danish 1331 6.
M. bovis BCG + + N.D. + + + N.D. Japan 7. M. bovis BCG + + N.D. + +
N.D. N.D. Chinese 8. M. bovis BCG + + N.D. + + N.D. N.D. Canadian
9. M. bovis BCG + + N.D. + + N.D. N.D. Glaxo 10. M. bovis BCG + +
N.D. + + N.D. N.D. Russia 11. M. bovis BCG + + N.D. + + N.D. N.D.
Pasteur 12. M. africanum + + + + + + + 13. M. leprae - - - - - - -
14. M. avium + + - + + + - 15. M. kansasii + - - + + + - 16. M.
marinum - (+) - + + + - 17. M. scrofulaceum - - - - - - - 18. M.
intercellulare + (+) - + + + - 19. M. fortuitum - - - - - - - 20.
M. flavescens + (+) - + + + N.D. 21. M. xenopi - - - N.D. N.D. + -
22. M. szulgai (+) (+) - - + - - 23. M. terrae - - N.D. N.D. N.D.
N.D. N.D. +, positive reaction; -, no reaction, N.D. not
determined
[0487] cfp7, cfp9 and mpt51 were found in the M. tuberculosis
coinplex including BCG and the environmental mycobacteria; M.
avium, M. kansasii, M. marinum, M. intracellular and M. flavescens.
cfp9 was additionally found in M. szulgai and mpt5l in M.
xenopi.
[0488] Furthermore the presence of native MPT51 in culture
filtrates from different mycobacterial strains was investigated
with western blots developed with Mab HBT4.
[0489] There is a strong band at around 26 kDa in M. tuberculosis
H37Rv, Ra, Erdman, M. bovis AN5, M. bovis BCG substrain Danish 1331
and M. africanum. No band was seen in the region in any other
tested mycobacterial strains.
33TABLE 13a Interspecies analysis of the rd1-orf2, rd1-orf3,
rd1-orf4, rd1-orf5, rd1-orf8, rd1- orf9a and rd1-orf9b genes by
Southern blotting. Species and strain rd1-orf2 rd1-orf3 rd1-orf4
rd1-orf5 rd1-orf8 rd1-orf9a rd1-orf9b 1. M. tub. H37Rv + + + + + +
+ 2. M. bovis + + + + N.D. + + 3. M. bovis BCG + - - - N.D. - -
Danish 1331 4. M. bovis + - - - N.D. - - BCG Japan 5. M. avium - -
- - N.D. - - 6. M. kansasii - - - - N.D. - - 7. M. marinum + - + -
N.D. - - 8. M. scrofulaceum + - - - N.D. - - 9. M. intercellulare -
- - - N.D. - - 10. M. fortuitum - - - - N.D. - - 11. M. xenopi - -
- - N.D. - - 12. M. szulgai + - - - N.D. - - +, positive reaction;
-, no reaction, N.D. not determined.
[0490] Positive results for rd1-orf2, rd1-orf3, rd1-orf4, rd1-orf5,
rd1-orf8, rd1-orf9a and rd1-orf9b were only obtained when using
genomic DNA from M. tuberculosis and M. bovis, and not from M.
bovis BCG or other mycobacteria analyzed except rd1-orf4 which also
was found in M. marinum.
[0491] Presence of cfpv7a cfp7b, cfp10a, cfp17, cfp20, cfp21,
cfp22, cfp22a, cfp23, cfp25 and cfp25a in different mycobacterial
species.
[0492] Southern blotting was carried out as described for rd1-orf2,
rd1-orf3, rd1-orf4, rd1-orf5, rd1-orf8, rd1-orf9a and rd1-orf9b.
The cfp7a, cfp7b, cfploa, cfp17, cfp20, cfp21, cfp22, cfp22a,
cfp23, cfp25 and cfp25a gene fragments were amplified by PCR from
the recombinant pMCT6 plasmids encoding the individual genes. The
primers used (same as the primers used for cloning) are described
in example 3, 3A and 3B. The results are summarized in Table
13b.
34TABLE 13b Interspecies analysis of the cfp7a, cfp7b, cfp10a,
cfp17, cfp20, cfp21, cfp22, cfp22a, cfp23, cfp25, and cfp25a genes
by Southern blotting. Species and strain cfp7a cfp7b cfp10a cfp17
cfp20 cfp21 cfp22 cfp22a cfp23 cfp25 cfp25a 1. M. tub. H37Rv + + +
+ + + + + + + + 2. M. bovis + + + + + + + + + + + 3. M. bovis BCG +
+ + + + N.D. + + + + + Danish 1331 4. M. bovis + + + + + + + + + +
+ BCG Japan 5. M. avium + N.D. - + - + + + + + - 6. M. kansasii -
N.D. + - - - + - + - - 7. M. marinum + + - + + + + + + + + 8. M.
scrofulaceum - - + - + + - + + + - 9. M. intercellulare + + - + - +
+ - + + - 10. M. fortuitum - N.D. - - - - - - + - - 11. M. xenopi +
+ + + + + + + + + + 12. M. szulgai + + - + + + + + + + + +,
positive reaction; -, no reaction, N.D. not determined.
[0493] LIST OF REFERENCES
[0494] Andersen, P. and Heron, I, 1993, J. Immunol. Methods 161:
29-39.
[0495] Andersen, {dot over (A)}. B. et al., 1992, Infect. Immun.
60: 2317-2323.
[0496] Andersen P., 1994, Infect. Immun. 62: 2536-44.
[0497] Andersen P. et al., 1995, J. Immunol. 154: 3359-72
[0498] Barkholt, V. and Jensen, A. L., 1989, Anal. Biochem. 177:
318-322.
[0499] Borodovsky, M., and J. McIninch. 1993, Computers Chem. 17:
123-133.
[0500] van Dyke M. W. et al., 1992. Gene pp. 99-104.
[0501] Gosselin et al., 1992, J. Immunol. 149: 3477-3481.
[0502] Harboe, M. et al., 1996, Infect. Immun. 64: 16-22.
[0503] von Heijne, G., 1984, J. Mol. Biol. 173: 243-251.
[0504] Hochstrasser, D.F. et al., 1988, Anal.Biochem. 173:
424-435
[0505] Kohler, G. and Milstein, C., 1975, Nature 256: 495-497.
[0506] Li, H. et al., 1993, Infect. Immun. 61: 1730-1734.
[0507] Lindblad E.B. et al., 1997, Infect. Immun. 65: 623-629.
[0508] Mahairas, G. G. et al., 1996, J. Bacteriol 178:
1274-1282.
[0509] Maniatis T. et al., 1989, "Molecular cloning: a laboratory
manual", 2nd ed., Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.
[0510] Nagai, S. et al., 1991, Infect. Immun. 59: 372-382.
[0511] Oettinger, T. and Andersen, {dot over (A)}. B., 1994,
Infect. Immun. 62: 2058-2064.
[0512] Ohara, N. et al., 1995, Scand. J. immunol. 41: 233-442.
[0513] Pal P. G. and Horwitz M. A., 1992, Infect. Immun. 60:
47B1-92.
[0514] Pearson, W. R. and Lipman D. J., 1988. Proc. Natl. Acad.
Sci. USA 85: 2444-2448.
[0515] Ploug, M. et al., 1989, Anal. Biochem. 181: 33-39.
[0516] Porath, J. et al., 1985, FEBS Lett. 185: 306-310.
[0517] Roberts, A.D. et al., 1995, Immunol. 85: 502-508.
[0518] Srensen, A.L. et al., 1995, Infect. Immun. 63:
1710-1717.
[0519] Theisen, M. et al., 1995, Clinical and Diagnostic Laboratory
Immunology, 2: 30-34.
[0520] Valds-Stauber, N. and Scherer, S., 1994, Appl. Environ.
Microbiol. 60: 3809-3814.
[0521] Valds-Stauber, N. and Scherer, S., 1996, Appl. Environ.
Microbiol. 62: 1283-1286.
[0522] Williams, N., 1996, Science 272: 27.
[0523] Young, R. A. et al., 1985, Proc. Natl. Acad. Sci. USA 82:
2583-2587.
Sequence CWU 1
1
173 1 381 DNA Mycobacterium tuberculosis 1 ggccgccggt acctatgtgg
ccgccgatgc tgcggacgcg tcgacctata ccgggttctg 60 atcgaaccct
gctgaccgag aggacttgtg atgtcgcaaa tcatgtacaa ctaccccgcg 120
atgttgggtc acgccgggga tatggccgga tatgccggca cgctgcagag cttgggtgcc
180 gagatcgccg tggagcaggc cgcgttgcag agtgcgtggc agggcgatac
cgggatcacg 240 tatcaggcgt ggcaggcaca gtggaaccag gccatggaag
atttggtgcg ggcctatcat 300 gcgatgtcca gcacccatga agccaacacc
atggcgatga tggcccgcga caccgccgaa 360 gccgccaaat ggggcggcta g 381 2
96 PRT Mycobacterium tuberculosis 2 Met Ser Gln Ile Met Tyr Asn Tyr
Pro Ala Met Leu Gly His Ala Gly 1 5 10 15 Asp Met Ala Gly Tyr Ala
Gly Thr Leu Gln Ser Leu Gly Ala Glu Ile 20 25 30 Ala Val Glu Gln
Ala Ala Leu Gln Ser Ala Trp Gln Gly Asp Thr Gly 35 40 45 Ile Thr
Tyr Gln Ala Trp Gln Ala Gln Trp Asn Gln Ala Met Glu Asp 50 55 60
Leu Val Arg Ala Tyr His Ala Met Ser Ser Thr His Glu Ala Asn Thr 65
70 75 80 Met Ala Met Met Ala Arg Asp Thr Ala Glu Ala Ala Lys Trp
Gly Gly 85 90 95 3 467 DNA Mycobacterium tuberculosis 3 gggtagccgg
accacggctg ggcaaagatg tgcaggccgc catcaaggcg gtcaaggccg 60
gcgacggcgt cataaacccg gacggcacct tgttggcggg ccccgcggtg ctgacgcccg
120 acgagtacaa ctcccggctg gtggccgccg acccggagtc caccgcggcg
ttgcccgacg 180 gcgccgggct ggtcgttctg gatggcaccg tcactgccga
actcgaagcc gagggctggg 240 ccaaagatcg catccgcgaa ctgcaagagc
tgcgtaagtc gaccgggctg gacgtttccg 300 accgcatccg ggtggtgatg
tcggtgcctg cggaacgcga agactgggcg cgcacccatc 360 gcgacctcat
tgccggagaa atcttggcta ccgacttcga attcgccgac ctcgccgatg 420
gtgtggccat cggcgacggc gtgcgggtaa gcatcgaaaa gacctga 467 4 108 PRT
Mycobacterium tuberculosis 4 Met Ala Ala Asp Pro Glu Ser Thr Ala
Ala Leu Pro Asp Gly Ala Gly 1 5 10 15 Leu Val Val Leu Asp Gly Thr
Val Thr Ala Glu Leu Glu Ala Glu Gly 20 25 30 Trp Ala Lys Asp Arg
Ile Arg Glu Leu Gln Glu Leu Arg Lys Ser Thr 35 40 45 Gly Leu Asp
Val Ser Asp Arg Ile Arg Val Val Met Ser Val Pro Ala 50 55 60 Glu
Arg Glu Asp Trp Ala Arg Thr His Arg Asp Leu Ile Ala Gly Glu 65 70
75 80 Ile Leu Ala Thr Asp Phe Glu Phe Ala Asp Leu Ala Asp Gly Val
Ala 85 90 95 Ile Gly Asp Gly Val Arg Val Ser Ile Glu Lys Thr 100
105 5 889 DNA Mycobacterium tuberculosis 5 cgggtctgca cggatccggg
ccgggcaggg caatcgagcc tgggatccgc tggggtgcgc 60 acatcgcgga
cccgtgcgcg gtacggtcga gacagcggca cgagaaagta gtaagggcga 120
taataggcgg taaagagtag cgggaagccg gccgaacgac tcggtcagac aacgccacag
180 cggccagtga ggagcagcgg gtgacggaca tgaacccgga tattgagaag
gaccagacct 240 ccgatgaagt cacggtagag acgacctccg tcttccgcgc
agacttcctc agcgagctgg 300 acgctcctgc gcaagcgggt acggagagcg
cggtctccgg ggtggaaggg ctcccgccgg 360 gctcggcgtt gctggtagtc
aaacgaggcc ccaacgccgg gtcccggttc ctactcgacc 420 aagccatcac
gtcggctggt cggcatcccg acagcgacat atttctcgac gacgtgaccg 480
tgagccgtcg ccatgctgaa ttccggttgg aaaacaacga attcaatgtc gtcgatgtcg
540 ggagtctcaa cggcacctac gtcaaccgcg agcccgtgga ttcggcggtg
ctggcgaacg 600 gcgacgaggt ccagatcggc aagttccggt tggtgttctt
gaccggaccc aagcaaggcg 660 aggatgacgg gagtaccggg ggcccgtgag
cgcacccgat agccccgcgc tggccgggat 720 gtcgatcggg gcggtcctcg
acctgctacg accggatttt cctgatgtca ccatctccaa 780 gattcgattc
ttggaggctg agggtctggt gacgccccgg cgggcctcat cggggtatcg 840
gcggttcacc gcatacgact gcgcacggct gcgattcatt ctcactgcc 889 6 162 PRT
Mycobacterium tuberculosis 6 Met Thr Asp Met Asn Pro Asp Ile Glu
Lys Asp Gln Thr Ser Asp Glu 1 5 10 15 Val Thr Val Glu Thr Thr Ser
Val Phe Arg Ala Asp Phe Leu Ser Glu 20 25 30 Leu Asp Ala Pro Ala
Gln Ala Gly Thr Glu Ser Ala Val Ser Gly Val 35 40 45 Glu Gly Leu
Pro Pro Gly Ser Ala Leu Leu Val Val Lys Arg Gly Pro 50 55 60 Asn
Ala Gly Ser Arg Phe Leu Leu Asp Gln Ala Ile Thr Ser Ala Gly 65 70
75 80 Arg His Pro Asp Ser Asp Ile Phe Leu Asp Asp Val Thr Val Ser
Arg 85 90 95 Arg His Ala Glu Phe Arg Leu Glu Asn Asn Glu Phe Asn
Val Val Asp 100 105 110 Val Gly Ser Leu Asn Gly Thr Tyr Val Asn Arg
Glu Pro Val Asp Ser 115 120 125 Ala Val Leu Ala Asn Gly Asp Glu Val
Gln Ile Gly Lys Phe Arg Leu 130 135 140 Val Phe Leu Thr Gly Pro Lys
Gln Gly Glu Asp Asp Gly Ser Thr Gly 145 150 155 160 Gly Pro 7 898
DNA Mycobacterium tuberculosis 7 tcgactccgg cgccaccggg caggatcacg
gtgtcgacgg ggtcgccggg gaatcccacg 60 ataaccactc ttcgcgccat
gaatgccagt gttggccagg cgctggcctg gcgtccacgc 120 cacacaccgc
acagattagg acacgccggc ggcgcagccc tgcccgaaag accgtgcacc 180
ggtcttggca gactgtgccc atggcacaga taaccctgcg aggaaacgcg atcaataccg
240 tcggtgagct acctgctgtc ggatccccgg ccccggcctt caccctgacc
gggggcgatc 300 tgggggtgat cagcagcgac cagttccggg gtaagtccgt
gttgctgaac atctttccat 360 ccgtggacac accggtgtgc gcgacgagtg
tgcgaacctt cgacgagcgt gcggcggcaa 420 gtggcgctac cgtgctgtgt
gtctcgaagg atctgccgtt cgcccagaag cgcttctgcg 480 gcgccgaggg
caccgaaaac gtcatgcccg cgtcggcatt ccgggacagc ttcggcgagg 540
attacggcgt gaccatcgcc gacgggccga tggccgggct gctcgcccgc gcaatcgtgg
600 tgatcggcgc ggacggcaac gtcgcctaca cggaattggt gccggaaatc
gcgcaagaac 660 ccaactacga agcggcgctg gccgcgctgg gcgcctaggc
tttcacaagc cccgcgcgtt 720 cggcgagcag cgcacgattt cgagcgctgc
tcccgaaaag cgcctcggtg gtcttggccc 780 ggcggtaata caggtgcagg
tcgtgctccc acgtgaaggc gatggcaccg tggatctgaa 840 gagcggagcc
ggcgcataac acaaaggttt ccgcggtctg cgccttcgcc agcggcgc 898 8 165 PRT
Mycobacterium tuberculosis 8 Met Ala Gln Ile Thr Leu Arg Gly Asn
Ala Ile Asn Thr Val Gly Glu 1 5 10 15 Leu Pro Ala Val Gly Ser Pro
Ala Pro Ala Phe Thr Leu Thr Gly Gly 20 25 30 Asp Leu Gly Val Ile
Ser Ser Asp Gln Phe Arg Gly Lys Ser Val Leu 35 40 45 Leu Asn Ile
Phe Pro Ser Val Asp Thr Pro Val Cys Ala Thr Ser Val 50 55 60 Arg
Thr Phe Asp Glu Arg Ala Ala Ala Ser Gly Ala Thr Val Leu Cys 65 70
75 80 Val Ser Lys Asp Leu Pro Phe Ala Gln Lys Arg Phe Cys Gly Ala
Glu 85 90 95 Gly Thr Glu Asn Val Met Pro Ala Ser Ala Phe Arg Asp
Ser Phe Gly 100 105 110 Glu Asp Tyr Gly Val Thr Ile Ala Asp Gly Pro
Met Ala Gly Leu Leu 115 120 125 Ala Arg Ala Ile Val Val Ile Gly Ala
Asp Gly Asn Val Ala Tyr Thr 130 135 140 Glu Leu Val Pro Glu Ile Ala
Gln Glu Pro Asn Tyr Glu Ala Ala Leu 145 150 155 160 Ala Ala Leu Gly
Ala 165 9 1054 DNA Mycobacterium tuberculosis 9 ataatcagct
caccgttggg accgacctcg accaggggtc ctttgtgact gccgggcttg 60
acgcggacga ccacagagtc ggtcatcgcc taaggctacc gttctgacct ggggctgcgt
120 gggcgccgac gacgtgaggc acgtcatgtc tcagcggccc accgccacct
cggtcgccgg 180 cagtatgtca gcatgtgcag atgactccac gcagccttgt
tcgcatcgtt ggtgtcgtgg 240 ttgcgacgac cttggcgctg gtgagcgcac
ccgccggcgg tcgtgccgcg catgcggatc 300 cgtgttcgga catcgcggtc
gttttcgctc gcggcacgca tcaggcttct ggtcttggcg 360 acgtcggtga
ggcgttcgtc gactcgctta cctcgcaagt tggcgggcgg tcgattgggg 420
tctacgcggt gaactaccca gcaagcgacg actaccgcgc gagcgcgtca aacggttccg
480 atgatgcgag cgcccacatc cagcgcaccg tcgccagctg cccgaacacc
aggattgtgc 540 ttggtggcta ttcgcagggt gcgacggtca tcgatttgtc
cacctcggcg atgccgcccg 600 cggtggcaga tcatgtcgcc gctgtcgccc
ttttcggcga gccatccagt ggtttctcca 660 gcatgttgtg gggcggcggg
tcgttgccga caatcggtcc gctgtatagc tctaagacca 720 taaacttgtg
tgctcccgac gatccaatat gcaccggagg cggcaatatt atggcgcatg 780
tttcgtatgt tcagtcgggg atgacaagcc aggcggcgac attcgcggcg aacaggctcg
840 atcacgccgg atgatcaaag actgttgtcc ctataccgct ggggctgtag
tcgatgtaca 900 ccggctggaa tctgaagggc aagaacccgg tattcatcag
gccggatgaa atgacggtcg 960 ggcggtaatc gtttgtgttg aacgcgtaga
gccgatcacc gccggggctg gtgtagacct 1020 caatgtttgt gttcgccggc
agggttccgg atcc 1054 10 217 PRT Mycobacterium tuberculosis 10 Met
Thr Pro Arg Ser Leu Val Arg Ile Val Gly Val Val Val Ala Thr 1 5 10
15 Thr Leu Ala Leu Val Ser Ala Pro Ala Gly Gly Arg Ala Ala His Ala
20 25 30 Asp Pro Cys Ser Asp Ile Ala Val Val Phe Ala Arg Gly Thr
His Gln 35 40 45 Ala Ser Gly Leu Gly Asp Val Gly Glu Ala Phe Val
Asp Ser Leu Thr 50 55 60 Ser Gln Val Gly Gly Arg Ser Ile Gly Val
Tyr Ala Val Asn Tyr Pro 65 70 75 80 Ala Ser Asp Asp Tyr Arg Ala Ser
Ala Ser Asn Gly Ser Asp Asp Ala 85 90 95 Ser Ala His Ile Gln Arg
Thr Val Ala Ser Cys Pro Asn Thr Arg Ile 100 105 110 Val Leu Gly Gly
Tyr Ser Gln Gly Ala Thr Val Ile Asp Leu Ser Thr 115 120 125 Ser Ala
Met Pro Pro Ala Val Ala Asp His Val Ala Ala Val Ala Leu 130 135 140
Phe Gly Glu Pro Ser Ser Gly Phe Ser Ser Met Leu Trp Gly Gly Gly 145
150 155 160 Ser Leu Pro Thr Ile Gly Pro Leu Tyr Ser Ser Lys Thr Ile
Asn Leu 165 170 175 Cys Ala Pro Asp Asp Pro Ile Cys Thr Gly Gly Gly
Asn Ile Met Ala 180 185 190 His Val Ser Tyr Val Gln Ser Gly Met Thr
Ser Gln Ala Ala Thr Phe 195 200 205 Ala Ala Asn Arg Leu Asp His Ala
Gly 210 215 11 949 DNA Mycobacterium tuberculosis 11 agccgctcgc
gtggggtcaa ccgggtttcc acctgctcac tcattttgcc gcctttctgt 60
gtccgggccg aggcttgcgc tcaataactc ggtcaagttc cttcacagac tgccatcact
120 ggcccgtcgg cgggctcgtt gcgggtgcgc cgcgtgcggg tttgtgttcc
gggcaccggg 180 tgggggcccg cccgggcgta atggcagact gtgattccgt
gactaacagc ccccttgcga 240 ccgctaccgc cacgctgcac actaaccgcg
gcgacatcaa gatcgccctg ttcggaaacc 300 atgcgcccaa gaccgtcgcc
aattttgtgg gccttgcgca gggcaccaag gactattcga 360 cccaaaacgc
atcaggtggc ccgtccggcc cgttctacga cggcgcggtc tttcaccggg 420
tgatccaggg cttcatgatc cagggtggcg atccaaccgg gacgggtcgc ggcggacccg
480 gctacaagtt cgccgacgag ttccaccccg agctgcaatt cgacaagccc
tatctgctcg 540 cgatggccaa cgccggtccg ggcaccaacg gctcacagtt
tttcatcacc gtcggcaaga 600 ctccgcacct gaaccggcgc cacaccattt
tcggtgaagt gatcgacgcg gagtcacagc 660 gggttgtgga ggcgatctcc
aagacggcca ccgacggcaa cgatcggccg acggacccgg 720 tggtgatcga
gtcgatcacc atctcctgac ccgaagctac gtcggctcgt cgctcgaata 780
caccttgtgg acccgccagg gcacgtggcg gtacaccgac acgccgttgg ggccgttcaa
840 ccggacgccc tcacgccaag tccgctcacc tttggccgcg accggcgtaa
ccggcagcgg 900 taagcgcatc gagcacctcc actgggtcgg tgccgagatc
ccagcggga 949 12 182 PRT Mycobacterium tuberculosis 12 Met Ala Asp
Cys Asp Ser Val Thr Asn Ser Pro Leu Ala Thr Ala Thr 1 5 10 15 Ala
Thr Leu His Thr Asn Arg Gly Asp Ile Lys Ile Ala Leu Phe Gly 20 25
30 Asn His Ala Pro Lys Thr Val Ala Asn Phe Val Gly Leu Ala Gln Gly
35 40 45 Thr Lys Asp Tyr Ser Thr Gln Asn Ala Ser Gly Gly Pro Ser
Gly Pro 50 55 60 Phe Tyr Asp Gly Ala Val Phe His Arg Val Ile Gln
Gly Phe Met Ile 65 70 75 80 Gln Gly Gly Asp Pro Thr Gly Thr Gly Arg
Gly Gly Pro Gly Tyr Lys 85 90 95 Phe Ala Asp Glu Phe His Pro Glu
Leu Gln Phe Asp Lys Pro Tyr Leu 100 105 110 Leu Ala Met Ala Asn Ala
Gly Pro Gly Thr Asn Gly Ser Gln Phe Phe 115 120 125 Ile Thr Val Gly
Lys Thr Pro His Leu Asn Arg Arg His Thr Ile Phe 130 135 140 Gly Glu
Val Ile Asp Ala Glu Ser Gln Arg Val Val Glu Ala Ile Ser 145 150 155
160 Lys Thr Ala Thr Asp Gly Asn Asp Arg Pro Thr Asp Pro Val Val Ile
165 170 175 Glu Ser Ile Thr Ile Ser 180 13 1060 DNA Mycobacterium
tuberculosis 13 tggaccttca ccggcggtcc cttcgcttcg ggggcgacac
ctaacatact ggtcgtcaac 60 ctaccgcgac accgctggga ctttgtgcca
ttgccggcca ctcggggccg ctgcggcctg 120 gaaaaattgg tcgggcacgg
gcggccgcgg gtcgctacca tcccactgtg aatgatttac 180 tgacccgccg
actgctcacc atgggcgcgg ccgccgcaat gctggccgcg gtgcttctgc 240
ttactcccat caccgttccc gccggctacc ccggtgccgt tgcaccggcc actgcagcct
300 gccccgacgc cgaagtggtg ttcgcccgcg gccgcttcga accgcccggg
attggcacgg 360 tcggcaacgc attcgtcagc gcgctgcgct cgaaggtcaa
caagaatgtc ggggtctacg 420 cggtgaaata ccccgccgac aatcagatcg
atgtgggcgc caacgacatg agcgcccaca 480 ttcagagcat ggccaacagc
tgtccgaata cccgcctggt gcccggcggt tactcgctgg 540 gcgcggccgt
caccgacgtg gtactcgcgg tgcccaccca gatgtggggc ttcaccaatc 600
ccctgcctcc cggcagtgat gagcacatcg ccgcggtcgc gctgttcggc aatggcagtc
660 agtgggtcgg ccccatcacc aacttcagcc ccgcctacaa cgatcggacc
atcgagttgt 720 gtcacggcga cgaccccgtc tgccaccctg ccgaccccaa
cacctgggag gccaactggc 780 cccagcacct cgccggggcc tatgtctcgt
cgggcatggt caaccaggcg gctgacttcg 840 ttgccggaaa gctgcaatag
ccacctagcc cgtgcgcgag tctttgcttc acgctttcgc 900 taaccgacca
acgcgcgcac gatggagggg tccgtggtca tatcaagaca agaagggagt 960
aggcgatgca cgcaaaagtc ggcgactacc tcgtggtgaa gggcacaacc acggaacggc
1020 atgatcaaca tgctgagatc atcgaggtgc gctccgcaga 1060 14 219 PRT
Mycobacterium tuberculosis 14 Met Gly Ala Ala Ala Ala Met Leu Ala
Ala Val Leu Leu Leu Thr Pro 1 5 10 15 Ile Thr Val Pro Ala Gly Tyr
Pro Gly Ala Val Ala Pro Ala Thr Ala 20 25 30 Ala Cys Pro Asp Ala
Glu Val Val Phe Ala Arg Gly Arg Phe Glu Pro 35 40 45 Pro Gly Ile
Gly Thr Val Gly Asn Ala Phe Val Ser Ala Leu Arg Ser 50 55 60 Lys
Val Asn Lys Asn Val Gly Val Tyr Ala Val Lys Tyr Pro Ala Asp 65 70
75 80 Asn Gln Ile Asp Val Gly Ala Asn Asp Met Ser Ala His Ile Gln
Ser 85 90 95 Met Ala Asn Ser Cys Pro Asn Thr Arg Leu Val Pro Gly
Gly Tyr Ser 100 105 110 Leu Gly Ala Ala Val Thr Asp Val Val Leu Ala
Val Pro Thr Gln Met 115 120 125 Trp Gly Phe Thr Asn Pro Leu Pro Pro
Gly Ser Asp Glu His Ile Ala 130 135 140 Ala Val Ala Leu Phe Gly Asn
Gly Ser Gln Trp Val Gly Pro Ile Thr 145 150 155 160 Asn Phe Ser Pro
Ala Tyr Asn Asp Arg Thr Ile Glu Leu Cys His Gly 165 170 175 Asp Asp
Pro Val Cys His Pro Ala Asp Pro Asn Thr Trp Glu Ala Asn 180 185 190
Trp Pro Gln His Leu Ala Gly Ala Tyr Val Ser Ser Gly Met Val Asn 195
200 205 Gln Ala Ala Asp Phe Val Ala Gly Lys Leu Gln 210 215 15 1198
DNA Mycobacterium tuberculosis 15 cagatgctgc gcaacatgtt tctcggcgat
ccggcaggca acaccgatcg agtgcttgac 60 ttttccaccg cggtgaccgg
cggactgttc ttctcaccca ccatcgactt tctcgaccat 120 ccaccgcccc
taccgcaggc ggcgacgcca actctggcag ccgggtcgct atcgatcggc 180
agcttgaaag gaagcccccg atgaacaatc tctaccgcga tttggcaccg gtcaccgaag
240 ccgcttgggc ggaaatcgaa ttggaggcgg cgcggacgtt caagcgacac
atcgccgggc 300 gccgggtggt cgatgtcagt gatcccgggg ggcccgtcac
cgcggcggtc agcaccggcc 360 ggctgatcga tgttaaggca ccaaccaacg
gcgtgatcgc ccacctgcgg gccagcaaac 420 cccttgtccg gctacgggtt
ccgtttaccc tgtcgcgcaa cgagatcgac gacgtggaac 480 gtggctctaa
ggactccgat tgggaaccgg taaaggaggc ggccaagaag ctggccttcg 540
tcgaggaccg cacaatattc gaaggctaca gcgccgcatc aatcgaaggg atccgcagcg
600 cgagttcgaa cccggcgctg acgttgcccg aggatccccg tgaaatccct
gatgtcatct 660 cccaggcatt gtccgaactg cggttggccg gtgtggacgg
accgtattcg gtgttgctct 720 ctgctgacgt ctacaccaag gttagcgaga
cttccgatca cggctatccc atccgtgagc 780 atctgaaccg gctggtggac
ggggacatca tttgggcccc ggccatcgac ggcgcgttcg 840 tgctgaccac
tcgaggcggc gacttcgacc tacagctggg caccgacgtt gcaatcgggt 900
acgccagcca cgacacggac accgagcgcc tctacctgca ggagacgctg acgttccttt
960 gctacaccgc cgaggcgtcg gtcgcgctca gccactaagg cacgagcgcg
agcaatagct 1020 cctatggcaa gcggccgcgg gttgggtgtg ttcggagctg
ggctggtgga cggtgcgcag 1080 ggcctggaag acggtgcggg ctaggcggcg
tttgaggcag cgtagtgctg cgcgtttggt 1140 tttcccggcg tcttgcagcc
tttggtagta ggcctggccc cggctgtcgg tcatccgg 1198 16 265 PRT
Mycobacterium tuberculosis 16 Met Asn Asn Leu Tyr Arg Asp Leu Ala
Pro Val Thr Glu Ala Ala Trp 1 5 10 15 Ala Glu Ile Glu Leu Glu Ala
Ala Arg Thr Phe Lys Arg His Ile Ala 20 25 30 Gly Arg Arg Val Val
Asp Val Ser Asp Pro Gly Gly Pro Val Thr Ala 35 40 45 Ala Val Ser
Thr Gly Arg Leu Ile Asp Val Lys Ala Pro Thr Asn Gly 50 55
60 Val Ile Ala His Leu Arg Ala Ser Lys Pro Leu Val Arg Leu Arg Val
65 70 75 80 Pro Phe Thr Leu Ser Arg Asn Glu Ile Asp Asp Val Glu Arg
Gly Ser 85 90 95 Lys Asp Ser Asp Trp Glu Pro Val Lys Glu Ala Ala
Lys Lys Leu Ala 100 105 110 Phe Val Glu Asp Arg Thr Ile Phe Glu Gly
Tyr Ser Ala Ala Ser Ile 115 120 125 Glu Gly Ile Arg Ser Ala Ser Ser
Asn Pro Ala Leu Thr Leu Pro Glu 130 135 140 Asp Pro Arg Glu Ile Pro
Asp Val Ile Ser Gln Ala Leu Ser Glu Leu 145 150 155 160 Arg Leu Ala
Gly Val Asp Gly Pro Tyr Ser Val Leu Leu Ser Ala Asp 165 170 175 Val
Tyr Thr Lys Val Ser Glu Thr Ser Asp His Gly Tyr Pro Ile Arg 180 185
190 Glu His Leu Asn Arg Leu Val Asp Gly Asp Ile Ile Trp Ala Pro Ala
195 200 205 Ile Asp Gly Ala Phe Val Leu Thr Thr Arg Gly Gly Asp Phe
Asp Leu 210 215 220 Gln Leu Gly Thr Asp Val Ala Ile Gly Tyr Ala Ser
His Asp Thr Asp 225 230 235 240 Thr Glu Arg Leu Tyr Leu Gln Glu Thr
Leu Thr Phe Leu Cys Tyr Thr 245 250 255 Ala Glu Ala Ser Val Ala Leu
Ser His 260 265 17 15 PRT Mycobacterium tuberculosis VARIANT (1)
Ala is Ala or Ser 17 Ala Glu Leu Asp Ala Pro Ala Gln Ala Gly Thr
Glu Xaa Ala Val 1 5 10 15 18 15 PRT Mycobacterium tuberculosis 18
Ala Gln Ile Thr Leu Arg Gly Asn Ala Ile Asn Thr Val Gly Glu 1 5 10
15 19 15 PRT Mycobacterium tuberculosis UNSURE (3) Xaa is unknown
19 Asp Pro Xaa Ser Asp Ile Ala Val Val Phe Ala Arg Gly Thr His 1 5
10 15 20 15 PRT Mycobacterium tuberculosis 20 Thr Asn Ser Pro Leu
Ala Thr Ala Thr Ala Thr Leu His Thr Asn 1 5 10 15 21 15 PRT
Mycobacterium tuberculosis UNSURE (2) Xaa is unknown 21 Ala Xaa Pro
Asp Ala Glu Val Val Phe Ala Arg Gly Arg Phe Glu 1 5 10 15 22 15 PRT
Mycobacterium tuberculosis UNSURE (1) Xaa is unknown 22 Xaa Ile Gln
Lys Ser Leu Glu Leu Ile Val Val Thr Ala Asp Glu 1 5 10 15 23 19 PRT
Mycobacterium tuberculosis 23 Met Asn Asn Leu Tyr Arg Asp Leu Ala
Pro Val Thr Glu Ala Ala Trp 1 5 10 15 Ala Glu Ile 24 34 DNA
Mycobacterium tuberculosis 24 cccggctcga gaacctstac cgcgacctsg cscc
34 25 37 DNA Mycobacterium tuberculosis 25 gggccggatc cgasgcsgcg
tccttsacsg gytgcca 37 26 28 DNA Mycobacterium tuberculosis 26
ggaagcccca tatgaacaat ctctaccg 28 27 32 DNA Mycobacterium
tuberculosis 27 cgcgctcagc ccttagtgac tgagcgcgac cg 32 28 24 DNA
Mycobacterium tuberculosis 28 ctcgaattcg ccgggtgcac acag 24 29 25
DNA Mycobacterium tuberculosis 29 ctcgaattcg cccccatacg agaac 25 30
15 DNA Mycobacterium tuberculosis 30 gtgtatctgc tggac 15 31 15 DNA
Mycobacterium tuberculosis 31 ccgactggct ggccg 15 32 24 DNA
Mycobacterium tuberculosis 32 gaggaattcg cttagcggat cgca 24 33 15
DNA Mycobacterium tuberculosis 33 cccacattcc gttgg 15 34 15 DNA
Mycobacterium tuberculosis 34 gtccagcaga tacac 15 35 27 DNA
Mycobacterium tuberculosis 35 gtacgagaat tcatgtcgca aatcatg 27 36
27 DNA Mycobacterium tuberculosis 36 gtacgagaat tcgagcttgg ggtgccg
27 37 28 DNA Mycobacterium tuberculosis 37 cgattccaag cttgtggccg
ccgacccg 28 38 30 DNA Mycobacterium tuberculosis 38 cgttagggat
cctcatcgcc atggtgttgg 30 39 26 DNA Mycobacterium tuberculosis 39
cgttagggat ccggttccac tgtgcc 26 40 28 DNA Mycobacterium
tuberculosis 40 cgttagggat cctcaggtct tttcgatg 28 41 952 DNA
Mycobacterium tuberculosis 41 gaattcgccg ggtgcacaca gccttacacg
acggaggtgg acacatgaag ggtcggtcgg 60 cgctgctgcg ggcgctctgg
attgccgcac tgtcattcgg gttgggcggt gtcgcggtag 120 ccgcggaacc
caccgccaag gccgccccat acgagaacct gatggtgccg tcgccctcga 180
tgggccggga catcccggtg gccttcctag ccggtgggcc gcacgcggtg tatctgctgg
240 acgccttcaa cgccggcccg gatgtcagta actgggtcac cgcgggtaac
gcgatgaaca 300 cgttggcggg caaggggatt tcggtggtgg caccggccgg
tggtgcgtac agcatgtaca 360 ccaactggga gcaggatggc agcaagcagt
gggacacctt cttgtccgct gagctgcccg 420 actggctggc cgctaaccgg
ggcttggccc ccggtggcca tgcggccgtt ggcgccgctc 480 agggcggtta
cggggcgatg gcgctggcgg ccttccaccc cgaccgcttc ggcttcgctg 540
gctcgatgtc gggctttttg tacccgtcga acaccaccac caacggtgcg atcgcggcgg
600 gcatgcagca attcggcggt gtggacacca acggaatgtg gggagcacca
cagctgggtc 660 ggtggaagtg gcacgacccg tgggtgcatg ccagcctgct
ggcgcaaaac aacacccggg 720 tgtgggtgtg gagcccgacc aacccgggag
ccagcgatcc cgccgccatg atcggccaaa 780 ccgccgaggc gatgggtaac
agccgcatgt tctacaacca gtatcgcagc gtcggcgggc 840 acaacggaca
cttcgacttc ccagccagcg gtgacaacgg ctggggctcg tgggcgcccc 900
agctgggcgc tatgtcgggc gatatcgtcg gtgcgatccg ctaagcgaat tc 952 42
299 PRT Mycobacterium tuberculosis 42 Met Lys Gly Arg Ser Ala Leu
Leu Arg Ala Leu Trp Ile Ala Ala Leu 1 5 10 15 Ser Phe Gly Leu Gly
Gly Val Ala Val Ala Ala Glu Pro Thr Ala Lys 20 25 30 Ala Ala Pro
Tyr Glu Asn Leu Met Val Pro Ser Pro Ser Met Gly Arg 35 40 45 Asp
Ile Pro Val Ala Phe Leu Ala Gly Gly Pro His Ala Val Tyr Leu 50 55
60 Leu Asp Ala Phe Asn Ala Gly Pro Asp Val Ser Asn Trp Val Thr Ala
65 70 75 80 Gly Asn Ala Met Asn Thr Leu Ala Gly Lys Gly Ile Ser Val
Val Ala 85 90 95 Pro Ala Gly Gly Ala Tyr Ser Met Tyr Thr Asn Trp
Glu Gln Asp Gly 100 105 110 Ser Lys Gln Trp Asp Thr Phe Leu Ser Ala
Glu Leu Pro Asp Trp Leu 115 120 125 Ala Ala Asn Arg Gly Leu Ala Pro
Gly Gly His Ala Ala Val Gly Ala 130 135 140 Ala Gln Gly Gly Tyr Gly
Ala Met Ala Leu Ala Ala Phe His Pro Asp 145 150 155 160 Arg Phe Gly
Phe Ala Gly Ser Met Ser Gly Phe Leu Tyr Pro Ser Asn 165 170 175 Thr
Thr Thr Asn Gly Ala Ile Ala Ala Gly Met Gln Gln Phe Gly Gly 180 185
190 Val Asp Thr Asn Gly Met Trp Gly Ala Pro Gln Leu Gly Arg Trp Lys
195 200 205 Trp His Asp Pro Trp Val His Ala Ser Leu Leu Ala Gln Asn
Asn Thr 210 215 220 Arg Val Trp Val Trp Ser Pro Thr Asn Pro Gly Ala
Ser Asp Pro Ala 225 230 235 240 Ala Met Ile Gly Gln Thr Ala Glu Ala
Met Gly Asn Ser Arg Met Phe 245 250 255 Tyr Asn Gln Tyr Arg Ser Val
Gly Gly His Asn Gly His Phe Asp Phe 260 265 270 Pro Ala Ser Gly Asp
Asn Gly Trp Gly Ser Trp Ala Pro Gln Leu Gly 275 280 285 Ala Met Ser
Gly Asp Ile Val Gly Ala Ile Arg 290 295 43 27 DNA Mycobacterium
tuberculosis 43 gcaacacccg ggatgtcgca aatcatg 27 44 27 DNA
Mycobacterium tuberculosis 44 gtaacacccg gggtggccgc cgacccg 27 45
37 DNA Mycobacterium tuberculosis 45 ctactaagct tggatcccta
gccgccccat ttggcgg 37 46 38 DNA Mycobacterium tuberculosis 46
ctactaagct tccatggtca ggtcttttcg atgcttac 38 47 450 DNA
Mycobacterium tuberculosis 47 gtgccgcgct ccccagggtt cttatggttc
gatatacctg agtttgatgg aagtccgatg 60 accagcagtc agcatacggc
atggccgaaa agagtggggt gatgatggcc gaggatgttc 120 gcgccgagat
cgtggccagc gttctcgaag tcgttgtcaa cgaaggcgat cagatcgaca 180
agggcgacgt cgtggtgctg ctggagtcga tgaagatgga gatccccgtc ctggccgaag
240 ctgccggaac ggtcagcaag gtggcggtat cggtgggcga tgtcattcag
gccggcgacc 300 ttatcgcggt gatcagctag tcgttgatag tcactcatgt
ccacactcgg tgatctgctc 360 gccgaacaca cggtgctgcc gggcagcgcg
gtggaccacc tgcatgcggt ggtcggggag 420 tggcagctcc ttgccgactt
gtcgtttgcc 450 48 71 PRT Mycobacterium tuberculosis 48 Met Ala Glu
Asp Val Arg Ala Glu Ile Val Ala Ser Val Leu Glu Val 1 5 10 15 Val
Val Asn Glu Gly Asp Gln Ile Asp Lys Gly Asp Val Val Val Leu 20 25
30 Leu Glu Ser Met Lys Met Glu Ile Pro Val Leu Ala Glu Ala Ala Gly
35 40 45 Thr Val Ser Lys Val Ala Val Ser Val Gly Asp Val Ile Gln
Ala Gly 50 55 60 Asp Leu Ile Ala Val Ile Ser 65 70 49 750 DNA
Mycobacterium tuberculosis 49 gggtacccat cgatgggttg cggttcggca
ccgaggtgct aacgcacttg ctgacacact 60 gctagtcgaa aacgaggcta
gtcgcaacgt cgatcacacg agaggactga ccatgacaac 120 ttcacccgac
ccgtatgccg cgctgcccaa gctgccgtcc ttcagcctga cgtcaacctc 180
gatcaccgat gggcagccgc tggctacacc ccaggtcagc gggatcatgg gtgcgggcgg
240 ggcggatgcc agtccgcagc tgaggtggtc gggatttccc agcgagaccc
gcagcttcgc 300 ggtaaccgtc tacgaccctg atgcccccac cctgtccggg
ttctggcact gggcggtggc 360 caacctgcct gccaacgtca ccgagttgcc
cgagggtgtc ggcgatggcc gcgaactgcc 420 gggcggggca ctgacattgg
tcaacgacgc cggtatgcgc cggtatgtgg gtgcggcgcc 480 gcctcccggt
catggggtgc atcgctacta cgtcgcggta cacgcggtga aggtcgaaaa 540
gctcgacctc cccgaggacg cgagtcctgc atatctggga ttcaacctgt tccagcacgc
600 gattgcacga gcggtcatct tcggcaccta cgagcagcgt tagcgcttta
gctgggttgc 660 cgacgtcttg ccgagccgac cgcttcgtgc agcgagccga
acccgccgtc atgcagcctg 720 cgggcaatgc cttcatggat gtccttggcc 750 50
176 PRT Mycobacterium tuberculosis 50 Met Thr Thr Ser Pro Asp Pro
Tyr Ala Ala Leu Pro Lys Leu Pro Ser 1 5 10 15 Phe Ser Leu Thr Ser
Thr Ser Ile Thr Asp Gly Gln Pro Leu Ala Thr 20 25 30 Pro Gln Val
Ser Gly Ile Met Gly Ala Gly Gly Ala Asp Ala Ser Pro 35 40 45 Gln
Leu Arg Trp Ser Gly Phe Pro Ser Glu Thr Arg Ser Phe Ala Val 50 55
60 Thr Val Tyr Asp Pro Asp Ala Pro Thr Leu Ser Gly Phe Trp His Trp
65 70 75 80 Ala Val Ala Asn Leu Pro Ala Asn Val Thr Glu Leu Pro Glu
Gly Val 85 90 95 Gly Asp Gly Arg Glu Leu Pro Gly Gly Ala Leu Thr
Leu Val Asn Asp 100 105 110 Ala Gly Met Arg Arg Tyr Val Gly Ala Ala
Pro Pro Pro Gly His Gly 115 120 125 Val His Arg Tyr Tyr Val Ala Val
His Ala Val Lys Val Glu Lys Leu 130 135 140 Asp Leu Pro Glu Asp Ala
Ser Pro Ala Tyr Leu Gly Phe Asn Leu Phe 145 150 155 160 Gln His Ala
Ile Ala Arg Ala Val Ile Phe Gly Thr Tyr Glu Gln Arg 165 170 175 51
800 DNA Mycobacterium tuberculosis 51 tcatgaggtt catcggggtg
atcccacgcc cgcagccgca ttcgggccgc tggcgagccg 60 gtgccgcacg
ccgcctcacc agcctggtgg ccgccgcctt tgcggcggcc acactgttgc 120
ttacccccgc gctggcacca ccggcatcgg cgggctgccc ggatgccgag gtggtgttcg
180 cccgcggaac cggcgaacca cctggcctcg gtcgggtagg ccaagctttc
gtcagttcat 240 tgcgccagca gaccaacaag agcatcggga catacggagt
caactacccg gccaacggtg 300 atttcttggc cgccgctgac ggcgcgaacg
acgccagcga ccacattcag cagatggcca 360 gcgcgtgccg ggccacgagg
ttggtgctcg gcggctactc ccagggtgcg gccgtgatcg 420 acatcgtcac
cgccgcacca ctgcccggcc tcgggttcac gcagccgttg ccgcccgcag 480
cggacgatca catcgccgcg atcgccctgt tcgggaatcc ctcgggccgc gctggcgggc
540 tgatgagcgc cctgacccct caattcgggt ccaagaccat caacctctgc
aacaacggcg 600 acccgatttg ttcggacggc aaccggtggc gagcgcacct
aggctacgtg cccgggatga 660 ccaaccaggc ggcgcgtttc gtcgcgagca
ggatctaacg cgagccgccc catagattcc 720 ggctaagcaa cggctgcgcc
gccgcccggc cacgagtgac cgccgccgac tggcacaccg 780 cttaccacgg
ccttatgctg 800 52 226 PRT Mycobacterium tuberculosis 52 Met Ile Pro
Arg Pro Gln Pro His Ser Gly Arg Trp Arg Ala Gly Ala 1 5 10 15 Ala
Arg Arg Leu Thr Ser Leu Val Ala Ala Ala Phe Ala Ala Ala Thr 20 25
30 Leu Leu Leu Thr Pro Ala Leu Ala Pro Pro Ala Ser Ala Gly Cys Pro
35 40 45 Asp Ala Glu Val Val Phe Ala Arg Gly Thr Gly Glu Pro Pro
Gly Leu 50 55 60 Gly Arg Val Gly Gln Ala Phe Val Ser Ser Leu Arg
Gln Gln Thr Asn 65 70 75 80 Lys Ser Ile Gly Thr Tyr Gly Val Asn Tyr
Pro Ala Asn Gly Asp Phe 85 90 95 Leu Ala Ala Ala Asp Gly Ala Asn
Asp Ala Ser Asp His Ile Gln Gln 100 105 110 Met Ala Ser Ala Cys Arg
Ala Thr Arg Leu Val Leu Gly Gly Tyr Ser 115 120 125 Gln Gly Ala Ala
Val Ile Asp Ile Val Thr Ala Ala Pro Leu Pro Gly 130 135 140 Leu Gly
Phe Thr Gln Pro Leu Pro Pro Ala Ala Asp Asp His Ile Ala 145 150 155
160 Ala Ile Ala Leu Phe Gly Asn Pro Ser Gly Arg Ala Gly Gly Leu Met
165 170 175 Ser Ala Leu Thr Pro Gln Phe Gly Ser Lys Thr Ile Asn Leu
Cys Asn 180 185 190 Asn Gly Asp Pro Ile Cys Ser Asp Gly Asn Arg Trp
Arg Ala His Leu 195 200 205 Gly Tyr Val Pro Gly Met Thr Asn Gln Ala
Ala Arg Phe Val Ala Ser 210 215 220 Arg Ile 225 53 700 DNA
Mycobacterium tuberculosis 53 ctaggaaagc ctttcctgag taagtattgc
cttcgttgca taccgccctt tacctgcgtt 60 aatctgcatt ttatgacaga
atacgaaggg cctaagacaa aattccacgc gttaatgcag 120 gaacagattc
ataacgaatt cacagcggca caacaatatg tcgcgatcgc ggtttatttc 180
gacagcgaag acctgccgca gttggcgaag catttttaca gccaagcggt cgaggaacga
240 aaccatgcaa tgatgctcgt gcaacacctg ctcgaccgcg accttcgtgt
cgaaattccc 300 ggcgtagaca cggtgcgaaa ccagttcgac agaccccgcg
aggcactggc gctggcgctc 360 gatcaggaac gcacagtcac cgaccaggtc
ggtcggctga cagcggtggc ccgcgacgag 420 ggcgatttcc tcggcgagca
gttcatgcag tggttcttgc aggaacagat cgaagaggtg 480 gccttgatgg
caaccctggt gcgggttgcc gatcgggccg gggccaacct gttcgagcta 540
gagaacttcg tcgcacgtga agtggatgtg gcgccggccg catcaggcgc cccgcacgct
600 gccgggggcc gcctctagat ccctggcggg gatcagcgag tggtcccgtt
cgcccgcccg 660 tcttccagcc aggccttggt gcggccgggg tggtgagtac 700 54
181 PRT Mycobacterium tuberculosis 54 Met Thr Glu Tyr Glu Gly Pro
Lys Thr Lys Phe His Ala Leu Met Gln 1 5 10 15 Glu Gln Ile His Asn
Glu Phe Thr Ala Ala Gln Gln Tyr Val Ala Ile 20 25 30 Ala Val Tyr
Phe Asp Ser Glu Asp Leu Pro Gln Leu Ala Lys His Phe 35 40 45 Tyr
Ser Gln Ala Val Glu Glu Arg Asn His Ala Met Met Leu Val Gln 50 55
60 His Leu Leu Asp Arg Asp Leu Arg Val Glu Ile Pro Gly Val Asp Thr
65 70 75 80 Val Arg Asn Gln Phe Asp Arg Pro Arg Glu Ala Leu Ala Leu
Ala Leu 85 90 95 Asp Gln Glu Arg Thr Val Thr Asp Gln Val Gly Arg
Leu Thr Ala Val 100 105 110 Ala Arg Asp Glu Gly Asp Phe Leu Gly Glu
Gln Phe Met Gln Trp Phe 115 120 125 Leu Gln Glu Gln Ile Glu Glu Val
Ala Leu Met Ala Thr Leu Val Arg 130 135 140 Val Ala Asp Arg Ala Gly
Ala Asn Leu Phe Glu Leu Glu Asn Phe Val 145 150 155 160 Ala Arg Glu
Val Asp Val Ala Pro Ala Ala Ser Gly Ala Pro His Ala 165 170 175 Ala
Gly Gly Arg Leu 180 55 950 DNA Mycobacterium tuberculosis 55
tgggctcggc actggctctc ccacggtggc gcgctgattt ctccccacgg taggcgttgc
60 gacgcatgtt cttcaccgtc tatccacagc taccgacatt tgctccggct
ggatcgcggg 120 taaaattccg tcgtgaacaa tcgacccatc cgcctgctga
catccggcag ggctggtttg 180 ggtgcgggcg cattgatcac cgccgtcgtc
ctgctcatcg ccttgggcgc tgtttggacc 240 ccggttgcct tcgccgatgg
atgcccggac gccgaagtca cgttcgcccg cggcaccggc 300 gagccgcccg
gaatcgggcg cgttggccag gcgttcgtcg actcgctgcg ccagcagact 360
ggcatggaga tcggagtata cccggtgaat tacgccgcca gccgcctaca gctgcacggg
420 ggagacggcg ccaacgacgc catatcgcac attaagtcca tggcctcgtc
atgcccgaac 480 accaagctgg tcttgggcgg ctattcgcag ggcgcaaccg
tgatcgatat cgtggccggg 540 gttccgttgg gcagcatcag ctttggcagt
ccgctacctg cggcatacgc agacaacgtc 600 gcagcggtcg cggtcttcgg
caatccgtcc aaccgcgccg gcggatcgct gtcgagcctg 660 agcccgctat
tcggttccaa ggcgattgac ctgtgcaatc ccaccgatcc gatctgccat 720
gtgggccccg gcaacgaatt cagcggacac atcgacggct acatacccac ctacaccacc
780 caggcggcta gtttcgtcgt gcagaggctc cgcgccgggt cggtgccaca
tctgcctgga 840 tccgtcccgc agctgcccgg gtctgtcctt cagatgcccg
gcactgccgc accggctccc 900 gaatcgctgc acggtcgctg acgctttgtc
agtaagccca taaaatcgcg 950 56 262 PRT Mycobacterium tuberculosis 56
Met Asn Asn Arg Pro Ile Arg Leu Leu Thr Ser Gly Arg Ala Gly Leu 1 5
10 15 Gly Ala Gly Ala Leu Ile Thr Ala Val Val Leu Leu Ile Ala Leu
Gly 20 25 30 Ala Val Trp Thr Pro Val Ala Phe Ala Asp Gly Cys Pro
Asp Ala Glu 35 40 45 Val Thr Phe Ala Arg Gly Thr Gly Glu Pro Pro
Gly Ile Gly Arg Val 50 55 60 Gly Gln Ala Phe Val Asp Ser Leu Arg
Gln Gln Thr Gly Met Glu Ile 65 70 75 80 Gly Val Tyr Pro Val Asn Tyr
Ala Ala Ser Arg Leu Gln Leu His Gly 85 90 95 Gly Asp Gly Ala Asn
Asp Ala Ile Ser His Ile Lys Ser Met Ala Ser 100 105 110 Ser Cys Pro
Asn Thr Lys Leu Val Leu Gly Gly Tyr Ser Gln Gly Ala 115 120 125 Thr
Val Ile Asp Ile Val Ala Gly Val Pro Leu Gly Ser Ile Ser Phe 130 135
140 Gly Ser Pro Leu Pro Ala Ala Tyr Ala Asp Asn Val Ala Ala Val Ala
145 150 155 160 Val Phe Gly Asn Pro Ser Asn Arg Ala Gly Gly Ser Leu
Ser Ser Leu 165 170 175 Ser Pro Leu Phe Gly Ser Lys Ala Ile Asp Leu
Cys Asn Pro Thr Asp 180 185 190 Pro Ile Cys His Val Gly Pro Gly Asn
Glu Phe Ser Gly His Ile Asp 195 200 205 Gly Tyr Ile Pro Thr Tyr Thr
Thr Gln Ala Ala Ser Phe Val Val Gln 210 215 220 Arg Leu Arg Ala Gly
Ser Val Pro His Leu Pro Gly Ser Val Pro Gln 225 230 235 240 Leu Pro
Gly Ser Val Leu Gln Met Pro Gly Thr Ala Ala Pro Ala Pro 245 250 255
Glu Ser Leu His Gly Arg 260 57 1000 DNA Mycobacterium tuberculosis
57 cgaggagacc gacgatctgc tcgacgaaat cgacgacgtc ctcgaggaga
acgccgagga 60 cttcgtccgc gcatacgtcc aaaagggcgg acagtgacct
ggccgttgcc cgatcgcctg 120 tccattaatt cactctctgg aacacccgct
gtagacctat cttctttcac tgacttcctg 180 cgccgccagg cgccggagtt
gctgccggca agcatcagcg gcggtgcgcc actcgcaggc 240 ggcgatgcgc
aactgccgca cggcaccacc attgtcgcgc tgaaataccc cggcggtgtt 300
gtcatggcgg gtgaccggcg ttcgacgcag ggcaacatga tttctgggcg tgatgtgcgc
360 aaggtgtata tcaccgatga ctacaccgct accggcatcg ctggcacggc
tgcggtcgcg 420 gttgagtttg cccggctgta tgccgtggaa cttgagcact
acgagaagct cgagggtgtg 480 ccgctgacgt ttgccggcaa aatcaaccgg
ctggcgatta tggtgcgtgg caatctggcg 540 gccgcgatgc agggtctgct
ggcgttgccg ttgctggcgg gctacgacat tcatgcgtct 600 gacccgcaga
gcgcgggtcg tatcgtttcg ttcgacgccg ccggcggttg gaacatcgag 660
gaagagggct atcaggcggt gggctcgggt tcgctgttcg cgaagtcgtc gatgaagaag
720 ttgtattcgc aggttaccga cggtgattcg gggctgcggg tggcggtcga
ggcgctctac 780 gacgccgccg acgacgactc cgccaccggc ggtccggacc
tggtgcgggg catctttccg 840 acggcggtga tcatcgacgc cgacggggcg
gttgacgtgc cggagagccg gattgccgaa 900 ttggcccgcg cgatcatcga
aagccgttcg ggtgcggata ctttcggctc cgatggcggt 960 gagaagtgag
ttttccgtat ttcatctcgc ctgagcaggc 1000 58 291 PRT Mycobacterium
tuberculosis 58 Met Thr Trp Pro Leu Pro Asp Arg Leu Ser Ile Asn Ser
Leu Ser Gly 1 5 10 15 Thr Pro Ala Val Asp Leu Ser Ser Phe Thr Asp
Phe Leu Arg Arg Gln 20 25 30 Ala Pro Glu Leu Leu Pro Ala Ser Ile
Ser Gly Gly Ala Pro Leu Ala 35 40 45 Gly Gly Asp Ala Gln Leu Pro
His Gly Thr Thr Ile Val Ala Leu Lys 50 55 60 Tyr Pro Gly Gly Val
Val Met Ala Gly Asp Arg Arg Ser Thr Gln Gly 65 70 75 80 Asn Met Ile
Ser Gly Arg Asp Val Arg Lys Val Tyr Ile Thr Asp Asp 85 90 95 Tyr
Thr Ala Thr Gly Ile Ala Gly Thr Ala Ala Val Ala Val Glu Phe 100 105
110 Ala Arg Leu Tyr Ala Val Glu Leu Glu His Tyr Glu Lys Leu Glu Gly
115 120 125 Val Pro Leu Thr Phe Ala Gly Lys Ile Asn Arg Leu Ala Ile
Met Val 130 135 140 Arg Gly Asn Leu Ala Ala Ala Met Gln Gly Leu Leu
Ala Leu Pro Leu 145 150 155 160 Leu Ala Gly Tyr Asp Ile His Ala Ser
Asp Pro Gln Ser Ala Gly Arg 165 170 175 Ile Val Ser Phe Asp Ala Ala
Gly Gly Trp Asn Ile Glu Glu Glu Gly 180 185 190 Tyr Gln Ala Val Gly
Ser Gly Ser Leu Phe Ala Lys Ser Ser Met Lys 195 200 205 Lys Leu Tyr
Ser Gln Val Thr Asp Gly Asp Ser Gly Leu Arg Val Ala 210 215 220 Val
Glu Ala Leu Tyr Asp Ala Ala Asp Asp Asp Ser Ala Thr Gly Gly 225 230
235 240 Pro Asp Leu Val Arg Gly Ile Phe Pro Thr Ala Val Ile Ile Asp
Ala 245 250 255 Asp Gly Ala Val Asp Val Pro Glu Ser Arg Ile Ala Glu
Leu Ala Arg 260 265 270 Ala Ile Ile Glu Ser Arg Ser Gly Ala Asp Thr
Phe Gly Ser Asp Gly 275 280 285 Gly Glu Lys 290 59 900 DNA
Mycobacterium tuberculosis 59 ttggcccgcg cgatcatcga aagccgttcg
ggtgcggata ctttcggctc cgatggcggt 60 gagaagtgag ttttccgtat
ttcatctcgc ctgagcaggc gatgcgcgag cgcagcgagt 120 tggcgcgtaa
gggcattgcg cgggccaaaa gcgtggtggc gctggcctat gccggtggtg 180
tgctgttcgt cgcggagaat ccgtcgcggt cgctgcagaa gatcagtgag ctctacgatc
240 gggtgggttt tgcggctgcg ggcaagttca acgagttcga caatttgcgc
cgcggcggga 300 tccagttcgc cgacacccgc ggttacgcct atgaccgtcg
tgacgtcacg ggtcggcagt 360 tggccaatgt ctacgcgcag actctaggca
ccatcttcac cgaacaggcc aagccctacg 420 aggttgagtt gtgtgtggcc
gaggtggcgc attacggcga gacgaaacgc cctgagttgt 480 atcgtattac
ctacgacggg tcgatcgccg acgagccgca tttcgtggtg atgggcggca 540
ccacggagcc gatcgccaac gcgctcaaag agtcgtatgc cgagaacgcc agcctgaccg
600 acgccctgcg tatcgcggtc gctgcattgc gggccggcag tgccgacacc
tcgggtggtg 660 atcaacccac ccttggcgtg gccagcttag aggtggccgt
tctcgatgcc aaccggccac 720 ggcgcgcgtt ccggcgcatc accggctccg
ccctgcaagc gttgctggta gaccaggaaa 780 gcccgcagtc tgacggcgaa
tcgtcgggct gagtccgaaa gtccgacgcg tgtctgggac 840 cccgctgcga
cgttaactgc gcctaacccc ggctcgacgc gtcgccggcc gtcctgactt 900 60 248
PRT Mycobacterium tuberculosis 60 Met Ser Phe Pro Tyr Phe Ile Ser
Pro Glu Gln Ala Met Arg Glu Arg 1 5 10 15 Ser Glu Leu Ala Arg Lys
Gly Ile Ala Arg Ala Lys Ser Val Val Ala 20 25 30 Leu Ala Tyr Ala
Gly Gly Val Leu Phe Val Ala Glu Asn Pro Ser Arg 35 40 45 Ser Leu
Gln Lys Ile Ser Glu Leu Tyr Asp Arg Val Gly Phe Ala Ala 50 55 60
Ala Gly Lys Phe Asn Glu Phe Asp Asn Leu Arg Arg Gly Gly Ile Gln 65
70 75 80 Phe Ala Asp Thr Arg Gly Tyr Ala Tyr Asp Arg Arg Asp Val
Thr Gly 85 90 95 Arg Gln Leu Ala Asn Val Tyr Ala Gln Thr Leu Gly
Thr Ile Phe Thr 100 105 110 Glu Gln Ala Lys Pro Tyr Glu Val Glu Leu
Cys Val Ala Glu Val Ala 115 120 125 His Tyr Gly Glu Thr Lys Arg Pro
Glu Leu Tyr Arg Ile Thr Tyr Asp 130 135 140 Gly Ser Ile Ala Asp Glu
Pro His Phe Val Val Met Gly Gly Thr Thr 145 150 155 160 Glu Pro Ile
Ala Asn Ala Leu Lys Glu Ser Tyr Ala Glu Asn Ala Ser 165 170 175 Leu
Thr Asp Ala Leu Arg Ile Ala Val Ala Ala Leu Arg Ala Gly Ser 180 185
190 Ala Asp Thr Ser Gly Gly Asp Gln Pro Thr Leu Gly Val Ala Ser Leu
195 200 205 Glu Val Ala Val Leu Asp Ala Asn Arg Pro Arg Arg Ala Phe
Arg Arg 210 215 220 Ile Thr Gly Ser Ala Leu Gln Ala Leu Leu Val Asp
Gln Glu Ser Pro 225 230 235 240 Gln Ser Asp Gly Glu Ser Ser Gly 245
61 1560 DNA Mycobacterium tuberculosis 61 gagtcattgc ctggtcggcg
tcattccgta ctagtcggtt gtcggacttg acctactggg 60 tcaggccgac
gagcactcga ccattagggt aggggccgtg acccactatg acgtcgtcgt 120
tctcggagcc ggtcccggcg ggtatgtcgc ggcgattcgc gccgcacagc tcggcctgag
180 cactgcaatc gtcgaaccca agtactgggg cggagtatgc ctcaatgtcg
gctgtatccc 240 atccaaggcg ctgttgcgca acgccgaact ggtccacatc
ttcaccaagg acgccaaagc 300 atttggcatc agcggcgagg tgaccttcga
ctacggcatc gcctatgacc gcagccgaaa 360 ggtagccgag ggcagggtgg
ccggtgtgca cttcctgatg aagaagaaca agatcaccga 420 gatccacggg
tacggcacat ttgccgacgc caacacgttg ttggttgatc tcaacgacgg 480
cggtacagaa tcggtcacgt tcgacaacgc catcatcgcg accggcagta gcacccggct
540 ggttcccggc acctcactgt cggccaacgt agtcacctac gaggaacaga
tcctgtcccg 600 agagctgccg aaatcgatca ttattgccgg agctggtgcc
attggcatgg agttcggcta 660 cgtgctgaag aactacggcg ttgacgtgac
catcgtggaa ttccttccgc gggcgctgcc 720 caacgaggac gccgatgtgt
ccaaggagat cgagaagcag ttcaaaaagc tgggtgtcac 780 gatcctgacc
gccacgaagg tcgagtccat cgccgatggc gggtcgcagg tcaccgtgac 840
cgtcaccaag gacggcgtgg cgcaagagct taaggcggaa aaggtgttgc aggccatcgg
900 atttgcgccc aacgtcgaag ggtacgggct ggacaaggca ggcgtcgcgc
tgaccgaccg 960 caaggctatc ggtgtcgacg actacatgcg taccaacgtg
ggccacatct acgctatcgg 1020 cgatgtcaat ggattactgc agctggcgca
cgtcgccgag gcacaaggcg tggtagccgc 1080 cgaaaccatt gccggtgcag
agactttgac gctgggcgac catcggatgt tgccgcgcgc 1140 gacgttctgt
cagccaaacg ttgccagctt cgggctcacc gagcagcaag cccgcaacga 1200
aggttacgac gtggtggtgg ccaagttccc gttcacggcc aacgccaagg cgcacggcgt
1260 gggtgacccc agtgggttcg tcaagctggt ggccgacgcc aagcacggcg
agctactggg 1320 tgggcacctg gtcggccacg acgtggccga gctgctgccg
gagctcacgc tggcgcagag 1380 gtgggacctg accgccagcg agctggctcg
caacgtccac acccacccaa cgatgtctga 1440 ggcgctgcag gagtgcttcc
acggcctggt tggccacatg atcaatttct gagcggctca 1500 tgacgaggcg
cgcgagcact gacacccccc agatcatcat gggtgccatc ggtggtgtgg 1560 62 464
PRT Mycobacterium tuberculosis 62 Met Thr His Tyr Asp Val Val Val
Leu Gly Ala Gly Pro Gly Gly Tyr 1 5 10 15 Val Ala Ala Ile Arg Ala
Ala Gln Leu Gly Leu Ser Thr Ala Ile Val 20 25 30 Glu Pro Lys Tyr
Trp Gly Gly Val Cys Leu Asn Val Gly Cys Ile Pro 35 40 45 Ser Lys
Ala Leu Leu Arg Asn Ala Glu Leu Val His Ile Phe Thr Lys 50 55 60
Asp Ala Lys Ala Phe Gly Ile Ser Gly Glu Val Thr Phe Asp Tyr Gly 65
70 75 80 Ile Ala Tyr Asp Arg Ser Arg Lys Val Ala Glu Gly Arg Val
Ala Gly 85 90 95 Val His Phe Leu Met Lys Lys Asn Lys Ile Thr Glu
Ile His Gly Tyr 100 105 110 Gly Thr Phe Ala Asp Ala Asn Thr Leu Leu
Val Asp Leu Asn Asp Gly 115 120 125 Gly Thr Glu Ser Val Thr Phe Asp
Asn Ala Ile Ile Ala Thr Gly Ser 130 135 140 Ser Thr Arg Leu Val Pro
Gly Thr Ser Leu Ser Ala Asn Val Val Thr 145 150 155 160 Tyr Glu Glu
Gln Ile Leu Ser Arg Glu Leu Pro Lys Ser Ile Ile Ile 165 170 175 Ala
Gly Ala Gly Ala Ile Gly Met Glu Phe Gly Tyr Val Leu Lys Asn 180 185
190 Tyr Gly Val Asp Val Thr Ile Val Glu Phe Leu Pro Arg Ala Leu Pro
195 200 205 Asn Glu Asp Ala Asp Val Ser Lys Glu Ile Glu Lys Gln Phe
Lys Lys 210 215 220 Leu Gly Val Thr Ile Leu Thr Ala Thr Lys Val Glu
Ser Ile Ala Asp 225 230 235 240 Gly Gly Ser Gln Val Thr Val Thr Val
Thr Lys Asp Gly Val Ala Gln 245 250 255 Glu Leu Lys Ala Glu Lys Val
Leu Gln Ala Ile Gly Phe Ala Pro Asn 260 265 270 Val Glu Gly Tyr Gly
Leu Asp Lys Ala Gly Val Ala Leu Thr Asp Arg 275 280 285 Lys Ala Ile
Gly Val Asp Asp Tyr Met Arg Thr Asn Val Gly His Ile 290 295 300 Tyr
Ala Ile Gly Asp Val Asn Gly Leu Leu Gln Leu Ala His Val Ala 305 310
315 320 Glu Ala Gln Gly Val Val Ala Ala Glu Thr Ile Ala Gly Ala Glu
Thr 325 330 335 Leu Thr Leu Gly Asp His Arg Met Leu Pro Arg Ala Thr
Phe Cys Gln 340 345 350 Pro Asn Val Ala Ser Phe Gly Leu Thr Glu Gln
Gln Ala Arg Asn Glu 355 360 365 Gly Tyr Asp Val Val Val Ala Lys Phe
Pro Phe Thr Ala Asn Ala Lys 370 375 380 Ala His Gly Val Gly Asp Pro
Ser Gly Phe Val Lys Leu Val Ala Asp 385 390 395 400 Ala Lys His Gly
Glu Leu Leu Gly Gly His Leu Val Gly His Asp Val 405 410 415 Ala Glu
Leu Leu Pro Glu Leu Thr Leu Ala Gln Arg Trp Asp Leu Thr 420 425 430
Ala Ser Glu Leu Ala Arg Asn Val His Thr His Pro Thr Met Ser Glu 435
440 445 Ala Leu Gln Glu Cys Phe His Gly Leu Val Gly His Met Ile Asn
Phe 450 455 460 63 550 DNA Mycobacterium tuberculosis 63 ggcccggctc
gcggccgccc tgcaggaaaa gaaggcctgc ccaggcccag actcagccga 60
gtagtcaccc agtaccccac accaggaagg accgcccatc atggcaaagc tctccaccga
120 cgaactgctg gacgcgttca aggaaatgac cctgttggag ctctccgact
tcgtcaagaa 180 gttcgaggag accttcgagg tcaccgccgc cgctccagtc
gccgtcgccg ccgccggtgc 240 cgccccggcc ggtgccgccg tcgaggctgc
cgaggagcag tccgagttcg acgtgatcct 300 tgaggccgcc ggcgacaaga
agatcggcgt catcaaggtg gtccgggaga tcgtttccgg 360 cctgggcctc
aaggaggcca aggacctggt cgacggcgcg cccaagccgc tgctggagaa 420
ggtcgccaag gaggccgccg acgaggccaa ggccaagctg gaggccgccg gcgccaccgt
480 caccgtcaag tagctctgcc cagcgtgttc ttttgcgtct gctcggcccg
tagcgaacac 540 tgcgcccgct 550 64 130 PRT Mycobacterium tuberculosis
64 Met Ala Lys Leu Ser Thr Asp Glu Leu Leu Asp Ala Phe Lys Glu Met
1 5 10 15 Thr Leu Leu Glu Leu Ser Asp Phe Val Lys Lys Phe Glu Glu
Thr Phe 20 25 30 Glu Val Thr Ala Ala Ala Pro Val Ala Val Ala Ala
Ala Gly Ala Ala 35 40 45 Pro Ala Gly Ala Ala Val Glu Ala Ala Glu
Glu Gln Ser Glu Phe Asp 50 55 60 Val Ile Leu Glu Ala Ala Gly Asp
Lys Lys Ile Gly Val Ile Lys Val 65 70 75 80 Val Arg Glu Ile Val Ser
Gly Leu Gly Leu Lys Glu Ala Lys Asp Leu 85 90 95 Val Asp Gly Ala
Pro Lys Pro Leu Leu Glu Lys Val Ala Lys Glu Ala 100 105 110 Ala Asp
Glu Ala Lys Ala Lys Leu Glu Ala Ala Gly Ala Thr Val Thr 115 120 125
Val Lys 130 65 900 DNA Mycobacterium tuberculosis 65 tgaacgccat
cgggtccaac gaacgcagcg ctacctgatc accaccgggt ctgttagggc 60
tcttccccag gtcgtacagt cgggccatgg ccattgaggt ttcggtgttg cgggttttca
120 ccgattcaga cgggaatttc ggtaatccgc tgggggtgat caacgccagc
aaggtcgaac 180 accgcgacag gcagcagctg gcagcccaat cgggctacag
cgaaaccata ttcgtcgatc 240 ttcccagccc cggctcaacc accgcacacg
ccaccatcca tactccccgc accgaaattc 300 cgttcgccgg acacccgacc
gtgggagcgt cctggtggct gcgcgagagg gggacgccaa 360 ttaacacgct
gcaggtgccg gccggcatcg tccaggtgag ctaccacggt gatctcaccg 420
ccatcagcgc ccgctcggaa tgggcacccg agttcgccat ccacgacctg gattcacttg
480 atgcgcttgc cgccgccgac cccgccgact ttccggacga catcgcgcac
tacctctgga 540 cctggaccga ccgctccgct ggctcgctgc gcgcccgcat
gtttgccgcc aacttgggcg 600 tcaccgaaga cgaagcgacc ggtgccgcgg
ccatccggat taccgattac ctcagccgtg 660 acctcaccat cacccagggc
aaaggatcgt tgatccacac cacctggagt cccgagggct 720 gggttcgggt
agccggccga gttgtcagcg acggtgtggc acaactcgac tgacgtagag 780
ctcagcgctg ccgatgcaac acggcggcaa ggtgatcctg caggggttgc ccgaccgcgc
840 gcatctgcaa cgagtacgaa agctcgtcgc cgtcgatgcg gtaggaacgg
tcaagggcgg 900 66 228 PRT Mycobacterium tuberculosis 66 Met Ala Ile
Glu Val Ser Val Leu Arg Val Phe Thr Asp Ser Asp Gly 1 5 10 15 Asn
Phe Gly Asn Pro Leu Gly Val Ile Asn Ala Ser Lys Val Glu His 20 25
30 Arg Asp Arg Gln Gln Leu Ala Ala Gln Ser Gly Tyr Ser Glu Thr Ile
35 40 45 Phe Val Asp Leu Pro Ser Pro Gly Ser Thr Thr Ala His Ala
Thr Ile 50 55 60 His Thr Pro Arg Thr Glu Ile Pro Phe Ala Gly His
Pro Thr Val Gly 65 70 75 80 Ala Ser Trp Trp Leu Arg Glu Arg Gly Thr
Pro Ile Asn Thr Leu Gln 85 90 95 Val Pro Ala Gly Ile Val Gln Val
Ser Tyr His Gly Asp Leu Thr Ala 100 105 110 Ile Ser Ala Arg Ser Glu
Trp Ala Pro Glu Phe Ala Ile His Asp Leu 115 120 125 Asp Ser Leu Asp
Ala Leu Ala Ala Ala Asp Pro Ala Asp Phe Pro Asp 130
135 140 Asp Ile Ala His Tyr Leu Trp Thr Trp Thr Asp Arg Ser Ala Gly
Ser 145 150 155 160 Leu Arg Ala Arg Met Phe Ala Ala Asn Leu Gly Val
Thr Glu Asp Glu 165 170 175 Ala Thr Gly Ala Ala Ala Ile Arg Ile Thr
Asp Tyr Leu Ser Arg Asp 180 185 190 Leu Thr Ile Thr Gln Gly Lys Gly
Ser Leu Ile His Thr Thr Trp Ser 195 200 205 Pro Glu Gly Trp Val Arg
Val Ala Gly Arg Val Val Ser Asp Gly Val 210 215 220 Ala Gln Leu Asp
225 67 500 DNA Mycobacterium tuberculosis 67 gtttgtggtg tcggtggtct
ggggggcgcc aactgggatt cggttggggt gggtgcaggt 60 ccggcgatgg
gcatcggagg tgtgggtggt ttgggtgggg ccggttcggg tccggcgatg 120
ggcatggggg gtgtgggtgg tttgggtggg gccggttcgg gtccggcgat gggcatgggg
180 ggtgtgggtg gtttagatgc ggccggttcc ggcgagggcg gctctcctgc
ggcgatcggc 240 atcggagttg gcggaggcgg aggtgggggt gggggtggcg
gcggcggggc cgacacgaac 300 cgctccgaca ggtcgtcgga cgtcgggggc
ggagtctggc cgttgggctt cggtaggttt 360 gccgatgcgg gcgccggcgg
aaacgaagca ctggggtcga agaacggctg cgctgccata 420 tcgtccggag
cttccatacc ttcgtgcggc cggaagagct tgtcgtagtc ggccgccatg 480
acaacctctc agagtgcgct 500 68 139 PRT Mycobacterium tuberculosis 68
Met Gly Ala Gly Pro Ala Met Gly Ile Gly Gly Val Gly Gly Leu Gly 1 5
10 15 Gly Ala Gly Ser Gly Pro Ala Met Gly Met Gly Gly Val Gly Gly
Leu 20 25 30 Gly Gly Ala Gly Ser Gly Pro Ala Met Gly Met Gly Gly
Val Gly Gly 35 40 45 Leu Asp Ala Ala Gly Ser Gly Glu Gly Gly Ser
Pro Ala Ala Ile Gly 50 55 60 Ile Gly Val Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly 65 70 75 80 Ala Asp Thr Asn Arg Ser Asp
Arg Ser Ser Asp Val Gly Gly Gly Val 85 90 95 Trp Pro Leu Gly Phe
Gly Arg Phe Ala Asp Ala Gly Ala Gly Gly Asn 100 105 110 Glu Ala Leu
Gly Ser Lys Asn Gly Cys Ala Ala Ile Ser Ser Gly Ala 115 120 125 Ser
Ile Pro Ser Cys Gly Arg Lys Ser Leu Ser 130 135 69 2050 DNA
Mycobacterium tuberculosis 69 agcgcactct gagaggttgt catggcggcc
gactacgaca agctcttccg gccgcacgaa 60 ggtatggaag ctccggacga
tatggcagcg cagccgttct tcgaccccag tgcttcgttt 120 ccgccggcgc
ccgcatcggc aaacctaccg aagcccaacg gccagactcc gcccccgacg 180
tccgacgacc tgtcggagcg gttcgtgtcg gccccgccgc cgccaccccc acccccacct
240 ccgcctccgc caactccgat gccgatcgcc gcaggagagc cgccctcgcc
ggaaccggcc 300 gcatctaaac cacccacacc ccccatgccc atcgccggac
ccgaaccggc cccacccaaa 360 ccacccacac cccccatgcc catcgccgga
cccgaaccgg ccccacccaa accacccaca 420 cctccgatgc ccatcgccgg
acctgcaccc accccaaccg aatcccagtt ggcgcccccc 480 agaccaccga
caccacaaac gccaaccgga gcgccgcagc aaccggaatc accggcgccc 540
cacgtaccct cgcacgggcc acatcaaccc cggcgcaccg caccagcacc gccctgggca
600 aagatgccaa tcggcgaacc cccgcccgct ccgtccagac cgtctgcgtc
cccggccgaa 660 ccaccgaccc ggcctgcccc ccaacactcc cgacgtgcgc
gccggggtca ccgctatcgc 720 acagacaccg aacgaaacgt cgggaaggta
gcaactggtc catccatcca ggcgcggctg 780 cgggcagagg aagcatccgg
cgcgcagctc gcccccggaa cggagccctc gccagcgccg 840 ttgggccaac
cgagatcgta tctggctccg cccacccgcc ccgcgccgac agaacctccc 900
cccagcccct cgccgcagcg caactccggt cggcgtgccg agcgacgcgt ccaccccgat
960 ttagccgccc aacatgccgc ggcgcaacct gattcaatta cggccgcaac
cactggcggt 1020 cgtcgccgca agcgtgcagc gccggatctc gacgcgacac
agaaatcctt aaggccggcg 1080 gccaaggggc cgaaggtgaa gaaggtgaag
ccccagaaac cgaaggccac gaagccgccc 1140 aaagtggtgt cgcagcgcgg
ctggcgacat tgggtgcatg cgttgacgcg aatcaacctg 1200 ggcctgtcac
ccgacgagaa gtacgagctg gacctgcacg ctcgagtccg ccgcaatccc 1260
cgcgggtcgt atcagatcgc cgtcgtcggt ctcaaaggtg gggctggcaa aaccacgctg
1320 acagcagcgt tggggtcgac gttggctcag gtgcgggccg accggatcct
ggctctagac 1380 gcggatccag gcgccggaaa cctcgccgat cgggtagggc
gacaatcggg cgcgaccatc 1440 gctgatgtgc ttgcagaaaa agagctgtcg
cactacaacg acatccgcgc acacactagc 1500 gtcaatgcgg tcaatctgga
agtgctgccg gcaccggaat acagctcggc gcagcgcgcg 1560 ctcagcgacg
ccgactggca tttcatcgcc gatcctgcgt cgaggtttta caacctcgtc 1620
ttggctgatt gtggggccgg cttcttcgac ccgctgaccc gcggcgtgct gtccacggtg
1680 tccggtgtcg tggtcgtggc aagtgtctca atcgacggcg cacaacaggc
gtcggtcgcg 1740 ttggactggt tgcgcaacaa cggttaccaa gatttggcga
gccgcgcatg cgtggtcatc 1800 aatcacatca tgccgggaga acccaatgtc
gcagttaaag acctggtgcg gcatttcgaa 1860 cagcaagttc aacccggccg
ggtcgtggtc atgccgtggg acaggcacat tgcggccgga 1920 accgagattt
cactcgactt gctcgaccct atctacaagc gcaaggtcct cgaattggcc 1980
gcagcgctat ccgacgattt cgagagggct ggacgtcgtt gagcgcacct gctgttgctg
2040 ctggtcctac 2050 70 666 PRT Mycobacterium tuberculosis 70 Met
Ala Ala Asp Tyr Asp Lys Leu Phe Arg Pro His Glu Gly Met Glu 1 5 10
15 Ala Pro Asp Asp Met Ala Ala Gln Pro Phe Phe Asp Pro Ser Ala Ser
20 25 30 Phe Pro Pro Ala Pro Ala Ser Ala Asn Leu Pro Lys Pro Asn
Gly Gln 35 40 45 Thr Pro Pro Pro Thr Ser Asp Asp Leu Ser Glu Arg
Phe Val Ser Ala 50 55 60 Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro
Pro Pro Pro Thr Pro Met 65 70 75 80 Pro Ile Ala Ala Gly Glu Pro Pro
Ser Pro Glu Pro Ala Ala Ser Lys 85 90 95 Pro Pro Thr Pro Pro Met
Pro Ile Ala Gly Pro Glu Pro Ala Pro Pro 100 105 110 Lys Pro Pro Thr
Pro Pro Met Pro Ile Ala Gly Pro Glu Pro Ala Pro 115 120 125 Pro Lys
Pro Pro Thr Pro Pro Met Pro Ile Ala Gly Pro Ala Pro Thr 130 135 140
Pro Thr Glu Ser Gln Leu Ala Pro Pro Arg Pro Pro Thr Pro Gln Thr 145
150 155 160 Pro Thr Gly Ala Pro Gln Gln Pro Glu Ser Pro Ala Pro His
Val Pro 165 170 175 Ser His Gly Pro His Gln Pro Arg Arg Thr Ala Pro
Ala Pro Pro Trp 180 185 190 Ala Lys Met Pro Ile Gly Glu Pro Pro Pro
Ala Pro Ser Arg Pro Ser 195 200 205 Ala Ser Pro Ala Glu Pro Pro Thr
Arg Pro Ala Pro Gln His Ser Arg 210 215 220 Arg Ala Arg Arg Gly His
Arg Tyr Arg Thr Asp Thr Glu Arg Asn Val 225 230 235 240 Gly Lys Val
Ala Thr Gly Pro Ser Ile Gln Ala Arg Leu Arg Ala Glu 245 250 255 Glu
Ala Ser Gly Ala Gln Leu Ala Pro Gly Thr Glu Pro Ser Pro Ala 260 265
270 Pro Leu Gly Gln Pro Arg Ser Tyr Leu Ala Pro Pro Thr Arg Pro Ala
275 280 285 Pro Thr Glu Pro Pro Pro Ser Pro Ser Pro Gln Arg Asn Ser
Gly Arg 290 295 300 Arg Ala Glu Arg Arg Val His Pro Asp Leu Ala Ala
Gln His Ala Ala 305 310 315 320 Ala Gln Pro Asp Ser Ile Thr Ala Ala
Thr Thr Gly Gly Arg Arg Arg 325 330 335 Lys Arg Ala Ala Pro Asp Leu
Asp Ala Thr Gln Lys Ser Leu Arg Pro 340 345 350 Ala Ala Lys Gly Pro
Lys Val Lys Lys Val Lys Pro Gln Lys Pro Lys 355 360 365 Ala Thr Lys
Pro Pro Lys Val Val Ser Gln Arg Gly Trp Arg His Trp 370 375 380 Val
His Ala Leu Thr Arg Ile Asn Leu Gly Leu Ser Pro Asp Glu Lys 385 390
395 400 Tyr Glu Leu Asp Leu His Ala Arg Val Arg Arg Asn Pro Arg Gly
Ser 405 410 415 Tyr Gln Ile Ala Val Val Gly Leu Lys Gly Gly Ala Gly
Lys Thr Thr 420 425 430 Leu Thr Ala Ala Leu Gly Ser Thr Leu Ala Gln
Val Arg Ala Asp Arg 435 440 445 Ile Leu Ala Leu Asp Ala Asp Pro Gly
Ala Gly Asn Leu Ala Asp Arg 450 455 460 Val Gly Arg Gln Ser Gly Ala
Thr Ile Ala Asp Val Leu Ala Glu Lys 465 470 475 480 Glu Leu Ser His
Tyr Asn Asp Ile Arg Ala His Thr Ser Val Asn Ala 485 490 495 Val Asn
Leu Glu Val Leu Pro Ala Pro Glu Tyr Ser Ser Ala Gln Arg 500 505 510
Ala Leu Ser Asp Ala Asp Trp His Phe Ile Ala Asp Pro Ala Ser Arg 515
520 525 Phe Tyr Asn Leu Val Leu Ala Asp Cys Gly Ala Gly Phe Phe Asp
Pro 530 535 540 Leu Thr Arg Gly Val Leu Ser Thr Val Ser Gly Val Val
Val Val Ala 545 550 555 560 Ser Val Ser Ile Asp Gly Ala Gln Gln Ala
Ser Val Ala Leu Asp Trp 565 570 575 Leu Arg Asn Asn Gly Tyr Gln Asp
Leu Ala Ser Arg Ala Cys Val Val 580 585 590 Ile Asn His Ile Met Pro
Gly Glu Pro Asn Val Ala Val Lys Asp Leu 595 600 605 Val Arg His Phe
Glu Gln Gln Val Gln Pro Gly Arg Val Val Val Met 610 615 620 Pro Trp
Asp Arg His Ile Ala Ala Gly Thr Glu Ile Ser Leu Asp Leu 625 630 635
640 Leu Asp Pro Ile Tyr Lys Arg Lys Val Leu Glu Leu Ala Ala Ala Leu
645 650 655 Ser Asp Asp Phe Glu Arg Ala Gly Arg Arg 660 665 71 1890
DNA Mycobacterium tuberculosis 71 gcagcgatga ggaggagcgg cgccaacggc
ccgcgccggc gacgatgcaa agcgcagcga 60 tgaggaggag cggcgcgcat
gactgctgaa ccggaagtac ggacgctgcg cgaggttgtg 120 ctggaccagc
tcggcactgc tgaatcgcgt gcgtacaaga tgtggctgcc gccgttgacc 180
aatccggtcc cgctcaacga gctcatcgcc cgtgatcggc gacaacccct gcgatttgcc
240 ctggggatca tggatgaacc gcgccgccat ctacaggatg tgtggggcgt
agacgtttcc 300 ggggccggcg gcaacatcgg tattgggggc gcacctcaaa
ccgggaagtc gacgctactg 360 cagacgatgg tgatgtcggc cgccgccaca
cactcaccgc gcaacgttca gttctattgc 420 atcgacctag gtggcggcgg
gctgatctat ctcgaaaacc ttccacacgt cggtggggta 480 gccaatcggt
ccgagcccga caaggtcaac cgggtggtcg cagagatgca agccgtcatg 540
cggcaacggg aaaccacctt caaggaacac cgagtgggct cgatcgggat gtaccggcag
600 ctgcgtgacg atccaagtca acccgttgcg tccgatccat acggcgacgt
ctttctgatc 660 atcgacggat ggcccggttt tgtcggcgag ttccccgacc
ttgaggggca ggttcaagat 720 ctggccgccc aggggctggg gttcggcgtc
cacgtcatca tctccacgcc acgctggaca 780 gagctgaagt cgcgtgttcg
cgactacctc ggcaccaaga tcgagttccg gcttggtgac 840 gtcaatgaaa
cccagatcga ccggattacc cgcgagatcc cggcgaatcg tccgggtcgg 900
gcagtgtcga tggaaaagca ccatctgatg atcggcgtgc ccaggttcga cggcgtgcac
960 agcgccgata acctggtgga ggcgatcacc gcgggggtga cgcagatcgc
ttcccagcac 1020 accgaacagg cacctccggt gcgggtcctg ccggagcgta
tccacctgca cgaactcgac 1080 ccgaacccgc cgggaccaga gtccgactac
cgcactcgct gggagattcc gatcggcttg 1140 cgcgagacgg acctgacgcc
ggctcactgc cacatgcaca cgaacccgca cctactgatc 1200 ttcggtgcgg
ccaaatcggg caagacgacc attgcccacg cgatcgcgcg cgccatttgt 1260
gcccgaaaca gtccccagca ggtgcggttc atgctcgcgg actaccgctc gggcctgctg
1320 gacgcggtgc cggacaccca tctgctgggc gccggcgcga tcaaccgcaa
cagcgcgtcg 1380 ctagacgagg ccgctcaagc actggcggtc aacctgaaga
agcggttgcc gccgaccgac 1440 ctgacgacgg cgcagctacg ctcgcgttcg
tggtggagcg gatttgacgt cgtgcttctg 1500 gtcgacgatt ggcacatgat
cgtgggtgcc gccgggggga tgccgccgat ggcaccgctg 1560 gccccgttat
tgccggcggc ggcagatatc gggttgcaca tcattgtcac ctgtcagatg 1620
agccaggctt acaaggcaac catggacaag ttcgtcggcg ccgcattcgg gtcgggcgct
1680 ccgacaatgt tcctttcggg cgagaagcag gaattcccat ccagtgagtt
caaggtcaag 1740 cggcgccccc ctggccaggc atttctcgtc tcgccagacg
gcaaagaggt catccaggcc 1800 ccctacatcg agcctccaga agaagtgttc
gcagcacccc caagcgccgg ttaagattat 1860 ttcattgccg gtgtagcagg
acccgagctc 1890 72 591 PRT Mycobacterium tuberculosis 72 Met Thr
Ala Glu Pro Glu Val Arg Thr Leu Arg Glu Val Val Leu Asp 1 5 10 15
Gln Leu Gly Thr Ala Glu Ser Arg Ala Tyr Lys Met Trp Leu Pro Pro 20
25 30 Leu Thr Asn Pro Val Pro Leu Asn Glu Leu Ile Ala Arg Asp Arg
Arg 35 40 45 Gln Pro Leu Arg Phe Ala Leu Gly Ile Met Asp Glu Pro
Arg Arg His 50 55 60 Leu Gln Asp Val Trp Gly Val Asp Val Ser Gly
Ala Gly Gly Asn Ile 65 70 75 80 Gly Ile Gly Gly Ala Pro Gln Thr Gly
Lys Ser Thr Leu Leu Gln Thr 85 90 95 Met Val Met Ser Ala Ala Ala
Thr His Ser Pro Arg Asn Val Gln Phe 100 105 110 Tyr Cys Ile Asp Leu
Gly Gly Gly Gly Leu Ile Tyr Leu Glu Asn Leu 115 120 125 Pro His Val
Gly Gly Val Ala Asn Arg Ser Glu Pro Asp Lys Val Asn 130 135 140 Arg
Val Val Ala Glu Met Gln Ala Val Met Arg Gln Arg Glu Thr Thr 145 150
155 160 Phe Lys Glu His Arg Val Gly Ser Ile Gly Met Tyr Arg Gln Leu
Arg 165 170 175 Asp Asp Pro Ser Gln Pro Val Ala Ser Asp Pro Tyr Gly
Asp Val Phe 180 185 190 Leu Ile Ile Asp Gly Trp Pro Gly Phe Val Gly
Glu Phe Pro Asp Leu 195 200 205 Glu Gly Gln Val Gln Asp Leu Ala Ala
Gln Gly Leu Gly Phe Gly Val 210 215 220 His Val Ile Ile Ser Thr Pro
Arg Trp Thr Glu Leu Lys Ser Arg Val 225 230 235 240 Arg Asp Tyr Leu
Gly Thr Lys Ile Glu Phe Arg Leu Gly Asp Val Asn 245 250 255 Glu Thr
Gln Ile Asp Arg Ile Thr Arg Glu Ile Pro Ala Asn Arg Pro 260 265 270
Gly Arg Ala Val Ser Met Glu Lys His His Leu Met Ile Gly Val Pro 275
280 285 Arg Phe Asp Gly Val His Ser Ala Asp Asn Leu Val Glu Ala Ile
Thr 290 295 300 Ala Gly Val Thr Gln Ile Ala Ser Gln His Thr Glu Gln
Ala Pro Pro 305 310 315 320 Val Arg Val Leu Pro Glu Arg Ile His Leu
His Glu Leu Asp Pro Asn 325 330 335 Pro Pro Gly Pro Glu Ser Asp Tyr
Arg Thr Arg Trp Glu Ile Pro Ile 340 345 350 Gly Leu Arg Glu Thr Asp
Leu Thr Pro Ala His Cys His Met His Thr 355 360 365 Asn Pro His Leu
Leu Ile Phe Gly Ala Ala Lys Ser Gly Lys Thr Thr 370 375 380 Ile Ala
His Ala Ile Ala Arg Ala Ile Cys Ala Arg Asn Ser Pro Gln 385 390 395
400 Gln Val Arg Phe Met Leu Ala Asp Tyr Arg Ser Gly Leu Leu Asp Ala
405 410 415 Val Pro Asp Thr His Leu Leu Gly Ala Gly Ala Ile Asn Arg
Asn Ser 420 425 430 Ala Ser Leu Asp Glu Ala Ala Gln Ala Leu Ala Val
Asn Leu Lys Lys 435 440 445 Arg Leu Pro Pro Thr Asp Leu Thr Thr Ala
Gln Leu Arg Ser Arg Ser 450 455 460 Trp Trp Ser Gly Phe Asp Val Val
Leu Leu Val Asp Asp Trp His Met 465 470 475 480 Ile Val Gly Ala Ala
Gly Gly Met Pro Pro Met Ala Pro Leu Ala Pro 485 490 495 Leu Leu Pro
Ala Ala Ala Asp Ile Gly Leu His Ile Ile Val Thr Cys 500 505 510 Gln
Met Ser Gln Ala Tyr Lys Ala Thr Met Asp Lys Phe Val Gly Ala 515 520
525 Ala Phe Gly Ser Gly Ala Pro Thr Met Phe Leu Ser Gly Glu Lys Gln
530 535 540 Glu Phe Pro Ser Ser Glu Phe Lys Val Lys Arg Arg Pro Pro
Gly Gln 545 550 555 560 Ala Phe Leu Val Ser Pro Asp Gly Lys Glu Val
Ile Gln Ala Pro Tyr 565 570 575 Ile Glu Pro Pro Glu Glu Val Phe Ala
Ala Pro Pro Ser Ala Gly 580 585 590 73 15 PRT Mycobacterium
tuberculosis 73 Asp Pro Val Asp Asp Ala Phe Ile Ala Lys Leu Asn Thr
Ala Gly 1 5 10 15 74 14 PRT Mycobacterium tuberculosis UNSURE (14)
Xaa is unknown 74 Asp Pro Val Asp Ala Ile Ile Asn Leu Asp Asn Tyr
Gly Xaa 1 5 10 75 15 PRT Mycobacterium tuberculosis UNSURE (5) Xaa
is unknown 75 Ala Glu Met Lys Xaa Phe Lys Asn Ala Ile Val Gln Glu
Ile Asp 1 5 10 15 76 14 PRT Mycobacterium tuberculosis VARIANT (3)
Ala is Ala or Gln 76 Val Ile Ala Gly Met Val Thr His Ile His Xaa
Val Ala Gly 1 5 10 77 15 PRT Mycobacterium tuberculosis 77 Thr Asn
Ile Val Val Leu Ile Lys Gln Val Pro Asp Thr Trp Ser 1 5 10 15 78 15
PRT Mycobacterium tuberculosis 78 Ala Ile Glu Val Ser Val Leu Arg
Val Phe Thr Asp Ser Asp Gly 1 5 10 15 79 15 PRT Mycobacterium
tuberculosis 79 Ala Lys Leu Ser Thr Asp Glu Leu Leu Asp Ala Phe Lys
Glu Met 1 5 10 15 80 15 PRT Mycobacterium tuberculosis VARIANT (4)
Asp is Asp or Glu 80 Asp Pro Ala Asp Ala Pro Asp Val Pro Thr Ala
Ala Gln Leu Thr 1 5 10 15 81 50 PRT Mycobacterium tuberculosis
81 Ala Glu Asp Val Arg Ala Glu Ile Val Ala Ser Val Leu Glu Val Val
1 5 10 15 Val Asn Glu Gly Asp Gln Ile Asp Lys Gly Asp Val Val Val
Leu Leu 20 25 30 Glu Ser Met Tyr Met Glu Ile Pro Val Leu Ala Glu
Ala Ala Gly Thr 35 40 45 Val Ser 50 82 15 PRT Mycobacterium
tuberculosis 82 Thr Thr Ser Pro Asp Pro Tyr Ala Ala Leu Pro Lys Leu
Pro Ser 1 5 10 15 83 15 PRT Mycobacterium tuberculosis 83 Thr Glu
Tyr Glu Gly Pro Lys Thr Lys Phe His Ala Leu Met Gln 1 5 10 15 84 15
PRT Mycobacterium tuberculosis 84 Thr Thr Ile Val Ala Leu Lys Tyr
Pro Gly Gly Val Val Met Ala 1 5 10 15 85 15 PRT Mycobacterium
tuberculosis UNSURE (10) Xaa is unknown 85 Ser Phe Pro Tyr Phe Ile
Ser Pro Glu Xaa Ala Met Arg Glu Xaa 1 5 10 15 86 15 PRT
Mycobacterium tuberculosis 86 Thr His Tyr Asp Val Val Val Leu Gly
Ala Gly Pro Gly Gly Tyr 1 5 10 15 87 450 DNA Mycobacterium
tuberculosis 87 agcccggtaa tcgagttcgg gcaatgctga ccatcgggtt
tgtttccggc tataaccgaa 60 cggtttgtgt acgggataca aatacaggga
gggaagaagt aggcaaatgg aaaaaatgtc 120 acatgatccg atcgctgccg
acattggcac gcaagtgagc gacaacgctc tgcacggcgt 180 gacggccggc
tcgacggcgc tgacgtcggt gaccgggctg gttcccgcgg gggccgatga 240
ggtctccgcc caagcggcga cggcgttcac atcggagggc atccaattgc tggcttccaa
300 tgcatcggcc caagaccagc tccaccgtgc gggcgaagcg gtccaggacg
tcgcccgcac 360 ctattcgcaa atcgacgacg gcgccgccgg cgtcttcgcc
taataggccc ccaacacatc 420 ggagggagtg atcaccatgc tgtggcacgc 450 88
98 PRT Mycobacterium tuberculosis 88 Met Glu Lys Met Ser His Asp
Pro Ile Ala Ala Asp Ile Gly Thr Gln 1 5 10 15 Val Ser Asp Asn Ala
Leu His Gly Val Thr Ala Gly Ser Thr Ala Leu 20 25 30 Thr Ser Val
Thr Gly Leu Val Pro Ala Gly Ala Asp Glu Val Ser Ala 35 40 45 Gln
Ala Ala Thr Ala Phe Thr Ser Glu Gly Ile Gln Leu Leu Ala Ser 50 55
60 Asn Ala Ser Ala Gln Asp Gln Leu His Arg Ala Gly Glu Ala Val Gln
65 70 75 80 Asp Val Ala Arg Thr Tyr Ser Gln Ile Asp Asp Gly Ala Ala
Gly Val 85 90 95 Phe Ala 89 460 DNA Mycobacterium tuberculosis 89
gcaaccggct tttcgatcag ctgagacatc agcggcgtgc gggtcaacga cccacctgcg
60 ccaggtagcg actccgcgcg cagcaggccc gcgcccgcgc tggggcctga
tccaccagcc 120 agcggatggt tcgacagcgg actggtgccg agcaggccca
tctgcgcggc ttcctcgtcg 180 gctgggttgc cgccgccggt gccgcccacc
tggctgaaca acgacgtcac ctgctgcagc 240 ggctgggtca gctgctgcat
cgggccgctc atctcaccca gttggccgag ggtctgggta 300 gccgccggcg
gcaactggcc aaccggtgtt gagctgccag gggagggcat tccgaagatc 360
gggttcgtcg tgctctggct cgcgccggga tcaaggatcg acgccatcgg ctcgagcttc
420 tcgaaaagcg tgttaaccgc ggtctcggcc tggtagacct 460 90 139 PRT
Mycobacterium tuberculosis 90 Met Arg Val Asn Asp Pro Pro Ala Pro
Gly Ser Asp Ser Ala Arg Ser 1 5 10 15 Arg Pro Ala Pro Ala Leu Gly
Pro Asp Pro Pro Ala Ser Gly Trp Phe 20 25 30 Asp Ser Gly Leu Val
Pro Ser Arg Pro Ile Cys Ala Ala Ser Ser Ser 35 40 45 Ala Gly Leu
Pro Pro Pro Val Pro Pro Thr Trp Leu Asn Asn Asp Val 50 55 60 Thr
Cys Cys Ser Gly Trp Val Ser Cys Cys Ile Gly Pro Leu Ile Ser 65 70
75 80 Pro Ser Trp Pro Arg Val Trp Val Ala Ala Gly Gly Asn Trp Pro
Thr 85 90 95 Gly Val Glu Leu Pro Gly Glu Gly Ile Pro Lys Ile Gly
Phe Val Val 100 105 110 Leu Trp Leu Ala Pro Gly Ser Arg Ile Asp Ala
Ile Gly Ser Ser Phe 115 120 125 Ser Lys Ser Val Leu Thr Ala Val Ser
Ala Trp 130 135 91 1200 DNA Mycobacterium tuberculosis 91
taataggccc ccaacacatc ggagggagtg atcaccatgc tgtggcacgc aatgccaccg
60 gagctaaata ccgcacggct gatggccggc gcgggtccgg ctccaatgct
tgcggcggcc 120 gcgggatggc agacgctttc ggcggctctg gacgctcagg
ccgtcgagtt gaccgcgcgc 180 ctgaactctc tgggagaagc ctggactgga
ggtggcagcg acaaggcgct tgcggctgca 240 acgccgatgg tggtctggct
acaaaccgcg tcaacacagg ccaagacccg tgcgatgcag 300 gcgacggcgc
aagccgcggc atacacccag gccatggcca cgacgccgtc gctgccggag 360
atcgccgcca accacatcac ccaggccgtc cttacggcca ccaacttctt cggtatcaac
420 acgatcccga tcgcgttgac cgagatggat tatttcatcc gtatgtggaa
ccaggcagcc 480 ctggcaatgg aggtctacca ggccgagacc gcggttaaca
cgcttttcga gaagctcgag 540 ccgatggcgt cgatccttga tcccggcgcg
agccagagca cgacgaaccc gatcttcgga 600 atgccctccc ctggcagctc
aacaccggtt ggccagttgc cgccggcggc tacccagacc 660 ctcggccaac
tgggtgagat gagcggcccg atgcagcagc tgacccagcc gctgcagcag 720
gtgacgtcgt tgttcagcca ggtgggcggc accggcggcg gcaacccagc cgacgaggaa
780 gccgcgcaga tgggcctgct cggcaccagt ccgctgtcga accatccgct
ggctggtgga 840 tcaggcccca gcgcgggcgc gggcctgctg cgcgcggagt
cgctacctgg cgcaggtggg 900 tcgttgaccc gcacgccgct gatgtctcag
ctgatcgaaa agccggttgc cccctcggtg 960 atgccggcgg ctgctgccgg
atcgtcggcg acgggtggcg ccgctccggt gggtgcggga 1020 gcgatgggcc
agggtgcgca atccggcggc tccaccaggc cgggtctggt cgcgccggca 1080
ccgctcgcgc aggagcgtga agaagacgac gaggacgact gggacgaaga ggacgactgg
1140 tgagctcccg taatgacaac agacttcccg gccacccggg ccggaagact
tgccaacatt 1200 92 371 PRT Mycobacterium tuberculosis 92 Met Ile
Thr Met Leu Trp His Ala Met Pro Pro Glu Leu Asn Thr Ala 1 5 10 15
Arg Leu Met Ala Gly Ala Gly Pro Ala Pro Met Leu Ala Ala Ala Ala 20
25 30 Gly Trp Gln Thr Leu Ser Ala Ala Leu Asp Ala Gln Ala Val Glu
Leu 35 40 45 Thr Ala Arg Leu Asn Ser Leu Gly Glu Ala Trp Thr Gly
Gly Gly Ser 50 55 60 Asp Lys Ala Leu Ala Ala Ala Thr Pro Met Val
Val Trp Leu Gln Thr 65 70 75 80 Ala Ser Thr Gln Ala Lys Thr Arg Ala
Met Gln Ala Thr Ala Gln Ala 85 90 95 Ala Ala Tyr Thr Gln Ala Met
Ala Thr Thr Pro Ser Leu Pro Glu Ile 100 105 110 Ala Ala Asn His Ile
Thr Gln Ala Val Leu Thr Ala Thr Asn Phe Phe 115 120 125 Gly Ile Asn
Thr Ile Pro Ile Ala Leu Thr Glu Met Asp Tyr Phe Ile 130 135 140 Arg
Met Trp Asn Gln Ala Ala Leu Ala Met Glu Val Tyr Gln Ala Glu 145 150
155 160 Thr Ala Val Asn Thr Leu Phe Glu Lys Leu Glu Pro Met Ala Ser
Ile 165 170 175 Leu Asp Pro Gly Ala Ser Gln Ser Thr Thr Asn Pro Ile
Phe Gly Met 180 185 190 Pro Ser Pro Gly Ser Ser Thr Pro Val Gly Gln
Leu Pro Pro Ala Ala 195 200 205 Thr Gln Thr Leu Gly Gln Leu Gly Glu
Met Ser Gly Pro Met Gln Gln 210 215 220 Leu Thr Gln Pro Leu Gln Gln
Val Thr Ser Leu Phe Ser Gln Val Gly 225 230 235 240 Gly Thr Gly Gly
Gly Asn Pro Ala Asp Glu Glu Ala Ala Gln Met Gly 245 250 255 Leu Leu
Gly Thr Ser Pro Leu Ser Asn His Pro Leu Ala Gly Gly Ser 260 265 270
Gly Pro Ser Ala Gly Ala Gly Leu Leu Arg Ala Glu Ser Leu Pro Gly 275
280 285 Ala Gly Gly Ser Leu Thr Arg Thr Pro Leu Met Ser Gln Leu Ile
Glu 290 295 300 Lys Pro Val Ala Pro Ser Val Met Pro Ala Ala Ala Ala
Gly Ser Ser 305 310 315 320 Ala Thr Gly Gly Ala Ala Pro Val Gly Ala
Gly Ala Met Gly Gln Gly 325 330 335 Ala Gln Ser Gly Gly Ser Thr Arg
Pro Gly Leu Val Ala Pro Ala Pro 340 345 350 Leu Ala Gln Glu Arg Glu
Glu Asp Asp Glu Asp Asp Trp Asp Glu Glu 355 360 365 Asp Asp Trp 370
93 1000 DNA Mycobacterium tuberculosis 93 gacgcgacac agaaatcctt
aaggccggcg gccaaggggc cgaaggtgaa gaaggtgaag 60 ccccagaaac
cgaaggccac gaagccgccc aaagtggtgt cgcagcgcgg ctggcgacat 120
tgggtgcatg cgttgacgcg aatcaacctg ggcctgtcac ccgacgagaa gtacgagctg
180 gacctgcacg ctcgagtccg ccgcaatccc cgcgggtcgt atcagatcgc
cgtcgtcggt 240 ctcaaaggtg gggctggcaa aaccacgctg acagcagcgt
tggggtcgac gttggctcag 300 gtgcgggccg accggatcct ggctctagac
gcggatccag gcgccggaaa cctcgccgat 360 cgggtagggc gacaatcggg
cgcgaccatc gctgatgtgc ttgcagaaaa agagctgtcg 420 cactacaacg
acatccgcgc acacactagc gtcaatgcgg tcaatctgga agtgctgccg 480
gcaccggaat acagctcggc gcagcgcgcg ctcagcgacg ccgactggca tttcatcgcc
540 gatcctgcgt cgaggtttta caacctcgtc ttggctgatt gtggggccgg
cttcttcgac 600 ccgctgaccc gcggcgtgct gtccacggtg tccggtgtcg
tggtcgtggc aagtgtctca 660 atcgacggcg cacaacaggc gtcggtcgcg
ttggactggt tgcgcaacaa cggttaccaa 720 gatttggcga gccgcgcatg
cgtggtcatc aatcacatca tgccgggaga acccaatgtc 780 gcagttaaag
acctggtgcg gcatttcgaa cagcaagttc aacccggccg ggtcgtggtc 840
atgccgtggg acaggcacat tgcggccgga accgagattt cactcgactt gctcgaccct
900 atctacaagc gcaaggtcct cgaattggcc gcagcgctat ccgacgattt
cgagagggct 960 ggacgtcgtt gagcgcacct gctgttgctg ctggtcctac 1000 94
308 PRT Mycobacterium tuberculosis 94 Met Lys Lys Val Lys Pro Gln
Lys Pro Lys Ala Thr Lys Pro Pro Lys 1 5 10 15 Val Val Ser Gln Arg
Gly Trp Arg His Trp Val His Ala Leu Thr Arg 20 25 30 Ile Asn Leu
Gly Leu Ser Pro Asp Glu Lys Tyr Glu Leu Asp Leu His 35 40 45 Ala
Arg Val Arg Arg Asn Pro Arg Gly Ser Tyr Gln Ile Ala Val Val 50 55
60 Gly Leu Lys Gly Gly Ala Gly Lys Thr Thr Leu Thr Ala Ala Leu Gly
65 70 75 80 Ser Thr Leu Ala Gln Val Arg Ala Asp Arg Ile Leu Ala Leu
Asp Ala 85 90 95 Asp Pro Gly Ala Gly Asn Leu Ala Asp Arg Val Gly
Arg Gln Ser Gly 100 105 110 Ala Thr Ile Ala Asp Val Leu Ala Glu Lys
Glu Leu Ser His Tyr Asn 115 120 125 Asp Ile Arg Ala His Thr Ser Val
Asn Ala Val Asn Leu Glu Val Leu 130 135 140 Pro Ala Pro Glu Tyr Ser
Ser Ala Gln Arg Ala Leu Ser Asp Ala Asp 145 150 155 160 Trp His Phe
Ile Ala Asp Pro Ala Ser Arg Phe Tyr Asn Leu Val Leu 165 170 175 Ala
Asp Cys Gly Ala Gly Phe Phe Asp Pro Leu Thr Arg Gly Val Leu 180 185
190 Ser Thr Val Ser Gly Val Val Val Val Ala Ser Val Ser Ile Asp Gly
195 200 205 Ala Gln Gln Ala Ser Val Ala Leu Asp Trp Leu Arg Asn Asn
Gly Tyr 210 215 220 Gln Asp Leu Ala Ser Arg Ala Cys Val Val Ile Asn
His Ile Met Pro 225 230 235 240 Gly Glu Pro Asn Val Ala Val Lys Asp
Leu Val Arg His Phe Glu Gln 245 250 255 Gln Val Gln Pro Gly Arg Val
Val Val Met Pro Trp Asp Arg His Ile 260 265 270 Ala Ala Gly Thr Glu
Ile Ser Leu Asp Leu Leu Asp Pro Ile Tyr Lys 275 280 285 Arg Lys Val
Leu Glu Leu Ala Ala Ala Leu Ser Asp Asp Phe Glu Arg 290 295 300 Ala
Gly Arg Arg 305 95 34 DNA Mycobacterium tuberculosis 95 aagagtagat
ctatgatggc cgaggatgtt cgcg 34 96 27 DNA Mycobacterium tuberculosis
96 cggcgacgac ggatcctacc gcgtcgg 27 97 28 DNA Mycobacterium
tuberculosis 97 ccttgggaga tctttggacc ccggttgc 28 98 25 DNA
Mycobacterium tuberculosis 98 gacgagatct tatgggctta ctgac 25 99 33
DNA Mycobacterium tuberculosis 99 ccccccagat ctgcaccacc ggcatcggcg
ggc 33 100 24 DNA Mycobacterium tuberculosis 100 gcggcggatc
cgttgcttag ccgg 24 101 32 DNA Mycobacterium tuberculosis 101
ccggctgaga tctatgacag aatacgaagg gc 32 102 24 DNA Mycobacterium
tuberculosis 102 ccccgccagg gaactagagg cggc 24 103 38 DNA
Mycobacterium tuberculosis 103 ctgccgagat ctaccaccat tgtcgcgctg
aaataccc 38 104 25 DNA Mycobacterium tuberculosis 104 cgccatggcc
ttacgcgcca actcg 25 105 32 DNA Mycobacterium tuberculosis 105
ggcggagatc tgtgagtttt ccgtatttca tc 32 106 25 DNA Mycobacterium
tuberculosis 106 cgcgtcgagc catggttagg cgcag 25 107 32 DNA
Mycobacterium tuberculosis 107 gaggaagatc tatgacaact tcacccgacc cg
32 108 28 DNA Mycobacterium tuberculosis 108 catgaagcca tggcccgcag
gctgcatg 28 109 33 DNA Mycobacterium tuberculosis 109 ggccgagatc
tgtgacccac tatgacgtcg tcg 33 110 36 DNA Mycobacterium tuberculosis
110 ggcgcccatg gtcagaaatt gatcatgtgg ccaacc 36 111 33 DNA
Mycobacterium tuberculosis 111 ccgggagatc tatggcaaag ctctccaccg acg
33 112 32 DNA Mycobacterium tuberculosis 112 cgctgggcag agctacttga
cggtgacggt gg 32 113 36 DNA Mycobacterium tuberculosis 113
ggcccagatc tatggccatt gaggtttcgg tgttgc 36 114 26 DNA Mycobacterium
tuberculosis 114 cgccgtgttg catggcagcg ctgagc 26 115 24 DNA
Mycobacterium tuberculosis 115 ggacgttcaa gcgacacatc gccg 24 116 24
DNA Mycobacterium tuberculosis 116 cagcacgaac gcgccgtcga tggc 24
117 26 DNA Mycobacterium tuberculosis 117 acagatctgt gacggacatg
aacccg 26 118 28 DNA Mycobacterium tuberculosis 118 ttttccatgg
tcacgggccc ccggtact 28 119 26 DNA Mycobacterium tuberculosis 119
acagatctgt gcccatggca cagata 26 120 27 DNA Mycobacterium
tuberculosis 120 tttaagcttc taggcgccca gcgcggc 27 121 26 DNA
Mycobacterium tuberculosis 121 acagatctgc gcatgcggat ccgtgt 26 122
28 DNA Mycobacterium tuberculosis 122 ttttccatgg tcatccggcg
tgatcgag 28 123 26 DNA Mycobacterium tuberculosis 123 acagatctgt
aatggcagac tgtgat 26 124 28 DNA Mycobacterium tuberculosis 124
ttttccatgg tcaggagatg gtgatcga 28 125 26 DNA Mycobacterium
tuberculosis 125 acagatctgc cggctacccc ggtgcc 26 126 28 DNA
Mycobacterium tuberculosis 126 ttttccatgg ctattgcagc tttccggc 28
127 50 PRT Mycobacterium tuberculosis 127 Ala Glu Asp Val Arg Ala
Glu Ile Val Ala Ser Val Leu Glu Val Val 1 5 10 15 Val Asn Glu Gly
Asp Gln Ile Asp Lys Gly Asp Val Val Val Leu Leu 20 25 30 Glu Ser
Met Tyr Met Glu Ile Pro Val Leu Ala Glu Ala Ala Gly Thr 35 40 45
Val Ser 50 128 49 PRT Mycobacterium tuberculosis 128 Ala Glu Asp
Val Arg Ala Glu Ile Val Ala Ser Val Leu Glu Val Val 1 5 10 15 Val
Asn Glu Gly Asp Gln Ile Asp Lys Gly Asp Val Val Val Leu Leu 20 25
30 Glu Ser Met Met Glu Ile Pro Val Leu Ala Glu Ala Ala Gly Thr Val
35 40 45 Ser 129 50 PRT Mycobacterium tuberculosis 129 Ala Glu Asp
Val Arg Ala Glu Ile Val Ala Ser Val Leu Glu Val Val 1 5 10 15 Val
Asn Glu Gly Asp Gln Ile Asp Lys Gly Asp Val Val Val Leu Leu 20 25
30 Glu Ser Met Lys Met Glu Ile Pro Val Leu Ala Glu Ala Ala Gly Thr
35 40 45 Val Ser 50 130 33 DNA Mycobacterium tuberculosis 130
ccgggagatc tatggcaaag ctctccaccg acg 33 131 32 DNA Mycobacterium
tuberculosis 131 cgctgggcag agctacttga cggtgacggt gg 32 132 36 DNA
Mycobacterium tuberculosis 132 ggcgccggca agcttgccat gacagagcag
cagtgg 36 133 26 DNA Mycobacterium tuberculosis 133 cgaactcgcc
ggatcccgtg tttcgc 26 134 32 DNA Mycobacterium tuberculosis 134
ggcaaccgcg agatctttct cccggccggg gc 32 135 27 DNA Mycobacterium
tuberculosis 135 ggcaagcttg ccggcgccta acgaact 27 136 30 DNA
Mycobacterium tuberculosis 136 ggacccagat ctatgacaga gcagcagtgg 30
137 47 DNA Mycobacterium tuberculosis 137 ccggcagccc cggccgggag
aaaagctttg cgaacatccc agtgacg 47 138 44 DNA Mycobacterium
tuberculosis 138 gttcgcaaag cttttctccc ggccggggct
gccggtcgag tacc 44 139 20 DNA Mycobacterium tuberculosis 139
ccttcggtgg atcccgtcag 20 140 450 DNA Mycobacterium tuberculosis 140
tggcgctgtc accgaggaac ctgtcaatgt cgtcgagcag tactgaaccg ttccgagaaa
60 ggccagcatg aacgtcaccg tatccattcc gaccatcctg cggccccaca
ccggcggcca 120 gaagagtgtc tcggccagcg gcgatacctt gggtgccgtc
atcagcgacc tggaggccaa 180 ctattcgggc atttccgagc gcctgatgga
cccgtcttcc ccaggtaagt tgcaccgctt 240 cgtgaacatc tacgtcaacg
acgaggacgt gcggttctcc ggcggcttgg ccaccgcgat 300 cgctgacggt
gactcggtca ccatcctccc cgccgtggcc ggtgggtgag cggagcacat 360
gacacgatac gactcgctgt tgcaggcctt gggcaacacg ccgctggttg gcctgcagcg
420 attgtcgcca cgctgggatg acgggcgaga 450 141 93 PRT Mycobacterium
tuberculosis 141 Met Asn Val Thr Val Ser Ile Pro Thr Ile Leu Arg
Pro His Thr Gly 1 5 10 15 Gly Gln Lys Ser Val Ser Ala Ser Gly Asp
Thr Leu Gly Ala Val Ile 20 25 30 Ser Asp Leu Glu Ala Asn Tyr Ser
Gly Ile Ser Glu Arg Leu Met Asp 35 40 45 Pro Ser Ser Pro Gly Lys
Leu His Arg Phe Val Asn Ile Tyr Val Asn 50 55 60 Asp Glu Asp Val
Arg Phe Ser Gly Gly Leu Ala Thr Ala Ile Ala Asp 65 70 75 80 Gly Asp
Ser Val Thr Ile Leu Pro Ala Val Ala Gly Gly 85 90 142 480 DNA
Mycobacterium tuberculosis 142 ggtgttcccg cggccggcta tgacaacagt
caatgtgcat gacaagttac aggtattagg 60 tccaggttca acaaggagac
aggcaacatg gcaacacgtt ttatgacgga tccgcacgcg 120 atgcgggaca
tggcgggccg ttttgaggtg cacgcccaga cggtggagga cgaggctcgc 180
cggatgtggg cgtccgcgca aaacatctcg ggcgcgggct ggagtggcat ggccgaggcg
240 acctcgctag acaccatggc ccagatgaat caggcgtttc gcaacatcgt
gaacatgctg 300 cacggggtgc gtgacgggct ggttcgcgac gccaacaact
acgagcagca agagcaggcc 360 tcccagcaga tcctcagcag ctaacgtcag
ccgctgcagc acaatacttt tacaagcgaa 420 ggagaacagg ttcgatgacc
atcaactatc agttcggtga tgtcgacgct catggcgcca 480 143 98 PRT
Mycobacterium tuberculosis 143 Met Ala Thr Arg Phe Met Thr Asp Pro
His Ala Met Arg Asp Met Ala 1 5 10 15 Gly Arg Phe Glu Val His Ala
Gln Thr Val Glu Asp Glu Ala Arg Arg 20 25 30 Met Trp Ala Ser Ala
Gln Asn Ile Ser Gly Ala Gly Trp Ser Gly Met 35 40 45 Ala Glu Ala
Thr Ser Leu Asp Thr Met Ala Gln Met Asn Gln Ala Phe 50 55 60 Arg
Asn Ile Val Asn Met Leu His Gly Val Arg Asp Gly Leu Val Arg 65 70
75 80 Asp Ala Asn Asn Tyr Glu Gln Gln Glu Gln Ala Ser Gln Gln Ile
Leu 85 90 95 Ser Ser 144 940 DNA Mycobacterium tuberculosis 144
gccccagtcc tcgatcgcct catcgccttc accggccgcc agccgaccgc aggccacgtg
60 tccgccacct aacgaaagga tgatcatgcc caagagaagc gaatacaggc
aaggcacgcc 120 gaactgggtc gaccttcaga ccaccgatca gtccgccgcc
aaaaagttct acacatcgtt 180 gttcggctgg ggttacgacg acaacccggt
ccccggaggc ggtggggtct attccatggc 240 cacgctgaac ggcgaagccg
tggccgccat cgcaccgatg cccccgggtg caccggaggg 300 gatgccgccg
atctggaaca cctatatcgc ggtggacgac gtcgatgcgg tggtggacaa 360
ggtggtgccc gggggcgggc aggtgatgat gccggccttc gacatcggcg atgccggccg
420 gatgtcgttc atcaccgatc cgaccggcgc tgccgtgggc ctatggcagg
ccaatcggca 480 catcggagcg acgttggtca acgagacggg cacgctcatc
tggaacgaac tgctcacgga 540 caagccggat ttggcgctag cgttctacga
ggctgtggtt ggcctcaccc actcgagcat 600 ggagatagct gcgggccaga
actatcgggt gctcaaggcc ggcgacgcgg aagtcggcgg 660 ctgtatggaa
ccgccgatgc ccggcgtgcc gaatcattgg cacgtctact ttgcggtgga 720
tgacgccgac gccacggcgg ccaaagccgc cgcagcgggc ggccaggtca ttgcggaacc
780 ggctgacatt ccgtcggtgg gccggttcgc cgtgttgtcc gatccgcagg
gcgcgatctt 840 cagtgtgttg aagcccgcac cgcagcaata gggagcatcc
cgggcaggcc cgccggccgg 900 cagattcgga gaatgctaga agctgccgcc
ggcgccgccg 940 145 261 PRT Mycobacterium tuberculosis 145 Met Pro
Lys Arg Ser Glu Tyr Arg Gln Gly Thr Pro Asn Trp Val Asp 1 5 10 15
Leu Gln Thr Thr Asp Gln Ser Ala Ala Lys Lys Phe Tyr Thr Ser Leu 20
25 30 Phe Gly Trp Gly Tyr Asp Asp Asn Pro Val Pro Gly Gly Gly Gly
Val 35 40 45 Tyr Ser Met Ala Thr Leu Asn Gly Glu Ala Val Ala Ala
Ile Ala Pro 50 55 60 Met Pro Pro Gly Ala Pro Glu Gly Met Pro Pro
Ile Trp Asn Thr Tyr 65 70 75 80 Ile Ala Val Asp Asp Val Asp Ala Val
Val Asp Lys Val Val Pro Gly 85 90 95 Gly Gly Gln Val Met Met Pro
Ala Phe Asp Ile Gly Asp Ala Gly Arg 100 105 110 Met Ser Phe Ile Thr
Asp Pro Thr Gly Ala Ala Val Gly Leu Trp Gln 115 120 125 Ala Asn Arg
His Ile Gly Ala Thr Leu Val Asn Glu Thr Gly Thr Leu 130 135 140 Ile
Trp Asn Glu Leu Leu Thr Asp Lys Pro Asp Leu Ala Leu Ala Phe 145 150
155 160 Tyr Glu Ala Val Val Gly Leu Thr His Ser Ser Met Glu Ile Ala
Ala 165 170 175 Gly Gln Asn Tyr Arg Val Leu Lys Ala Gly Asp Ala Glu
Val Gly Gly 180 185 190 Cys Met Glu Pro Pro Met Pro Gly Val Pro Asn
His Trp His Val Tyr 195 200 205 Phe Ala Val Asp Asp Ala Asp Ala Thr
Ala Ala Lys Ala Ala Ala Ala 210 215 220 Gly Gly Gln Val Ile Ala Glu
Pro Ala Asp Ile Pro Ser Val Gly Arg 225 230 235 240 Phe Ala Val Leu
Ser Asp Pro Gln Gly Ala Ile Phe Ser Val Leu Lys 245 250 255 Pro Ala
Pro Gln Gln 260 146 280 DNA Mycobacterium tuberculosis 146
ccgaaaggcg gtgcaccgca cccagaagaa aaggaaagat cgagaaatgc cacagggaac
60 tgtgaagtgg ttcaacgcgg agaaggggtt cggctttatc gcccccgaag
acggttccgc 120 ggatgtattt gtccactaca cggagatcca gggaacgggc
ttccgcaccc ttgaagaaaa 180 ccagaaggtc gagttcgaga tcggccacag
ccctaagggc ccccaggcca ccggagtccg 240 ctcgctctga gttacccccg
cgagcagacg caaaaagccc 280 147 67 PRT Mycobacterium tuberculosis 147
Met Pro Gln Gly Thr Val Lys Trp Phe Asn Ala Glu Lys Gly Phe Gly 1 5
10 15 Phe Ile Ala Pro Glu Asp Gly Ser Ala Asp Val Phe Val His Tyr
Thr 20 25 30 Glu Ile Gln Gly Thr Gly Phe Arg Thr Leu Glu Glu Asn
Gln Lys Val 35 40 45 Glu Phe Glu Ile Gly His Ser Pro Lys Gly Pro
Gln Ala Thr Gly Val 50 55 60 Arg Ser Leu 65 148 540 DNA
Mycobacterium tuberculosis 148 atcgtgtcgt atcgagaacc ccggccggta
tcagaacgcg ccagagcgca aacctttata 60 acttcgtgtc ccaaatgtga
cgaccatgga ccaaggttcc tgagatgaac ctacggcgcc 120 atcagaccct
gacgctgcga ctgctggcgg catccgcggg cattctcagc gccgcggcct 180
tcgccgcgcc agcacaggca aaccccgtcg acgacgcgtt catcgccgcg ctgaacaatg
240 ccggcgtcaa ctacggcgat ccggtcgacg ccaaagcgct gggtcagtcc
gtctgcccga 300 tcctggccga gcccggcggg tcgtttaaca ccgcggtagc
cagcgttgtg gcgcgcgccc 360 aaggcatgtc ccaggacatg gcgcaaacct
tcaccagtat cgcgatttcg atgtactgcc 420 cctcggtgat ggcagacgtc
gccagcggca acctgccggc cctgccagac atgccggggc 480 tgcccgggtc
ctaggcgtgc gcggctccta gccggtccct aacggatcga tcgtggatgc 540 149 129
PRT Mycobacterium tuberculosis 149 Met Asn Leu Arg Arg His Gln Thr
Leu Thr Leu Arg Leu Leu Ala Ala 1 5 10 15 Ser Ala Gly Ile Leu Ser
Ala Ala Ala Phe Ala Ala Pro Ala Gln Ala 20 25 30 Asn Pro Val Asp
Asp Ala Phe Ile Ala Ala Leu Asn Asn Ala Gly Val 35 40 45 Asn Tyr
Gly Asp Pro Val Asp Ala Lys Ala Leu Gly Gln Ser Val Cys 50 55 60
Pro Ile Leu Ala Glu Pro Gly Gly Ser Phe Asn Thr Ala Val Ala Ser 65
70 75 80 Val Val Ala Arg Ala Gln Gly Met Ser Gln Asp Met Ala Gln
Thr Phe 85 90 95 Thr Ser Ile Ala Ile Ser Met Tyr Cys Pro Ser Val
Met Ala Asp Val 100 105 110 Ala Ser Gly Asn Leu Pro Ala Leu Pro Asp
Met Pro Gly Leu Pro Gly 115 120 125 Ser 150 400 DNA Mycobacterium
tuberculosis 150 atagtttggg gaaggtgtcc ataaatgagg ctgtcgttga
ccgcattgag cgccggtgta 60 ggcgccgtgg caatgtcgtt gaccgtcggg
gccggggtcg cctccgcaga tcccgtggac 120 gcggtcatta acaccacctg
caattacggg caggtagtag ctgcgctcaa cgcgacggat 180 ccgggggctg
ccgcacagtt caacgcctca ccggtggcgc agtcctattt gcgcaatttc 240
ctcgccgcac cgccacctca gcgcgctgcc atggccgcgc aattgcaagc tgtgccgggg
300 gcggcacagt acatcggcct tgtcgagtcg gttgccggct cctgcaacaa
ctattaagcc 360 catgcgggcc ccatcccgcg acccggcatc gtcgccgggg 400 151
110 PRT Mycobacterium tuberculosis 151 Met Arg Leu Ser Leu Thr Ala
Leu Ser Ala Gly Val Gly Ala Val Ala 1 5 10 15 Met Ser Leu Thr Val
Gly Ala Gly Val Ala Ser Ala Asp Pro Val Asp 20 25 30 Ala Val Ile
Asn Thr Thr Cys Asn Tyr Gly Gln Val Val Ala Ala Leu 35 40 45 Asn
Ala Thr Asp Pro Gly Ala Ala Ala Gln Phe Asn Ala Ser Pro Val 50 55
60 Ala Gln Ser Tyr Leu Arg Asn Phe Leu Ala Ala Pro Pro Pro Gln Arg
65 70 75 80 Ala Ala Met Ala Ala Gln Leu Gln Ala Val Pro Gly Ala Ala
Gln Tyr 85 90 95 Ile Gly Leu Val Glu Ser Val Ala Gly Ser Cys Asn
Asn Tyr 100 105 110 152 990 DNA Mycobacterium tuberculosis 152
aatagtaata tcgctgtgcg gttgcaaaac gtgtgaccga ggttccgcag tcgagcgctg
60 cgggccgcct tcgaggagga cgaaccacag tcatgacgaa catcgtggtc
ctgatcaagc 120 aggtcccaga tacctggtcg gagcgcaagc tgaccgacgg
cgatttcacg ctggaccgcg 180 aggccgccga cgcggtgctg gacgagatca
acgagcgcgc cgtggaggaa gcgctacaga 240 ttcgggagaa agaggccgcc
gacggcatcg aagggtcggt aaccgtgctg acggcgggcc 300 ccgagcgcgc
caccgaggcg atccgcaagg cgctgtcgat gggtgccgac aaggccgtcc 360
acctaaagga cgacggcatg cacggctcgg acgtcatcca aaccgggtgg gctttggcgc
420 gcgcgttggg caccatcgag ggcaccgagc tggtgatcgc aggcaacgaa
tcgaccgacg 480 gggtgggcgg tgcggtgccg gccatcatcg ccgagtacct
gggcctgccg cagctcaccc 540 acctgcgcaa agtgtcgatc gagggcggca
agatcaccgg cgagcgtgag accgatgagg 600 gcgtattcac cctcgaggcc
acgctgcccg cggtgatcag cgtgaacgag aagatcaacg 660 agccgcgctt
cccgtccttc aaaggcatca tggccgccaa gaagaaggaa gttaccgtgc 720
tgaccctggc cgagatcggt gtcgagagcg acgaggtggg gctggccaac gccggatcca
780 ccgtgctggc gtcgacgccc aaaccggcca agactgccgg ggagaaggtc
accgacgagg 840 gtgaaggcgg caaccagatc gtgcagtacc tggttgccca
gaaaatcatc taagacatac 900 gcacctccca aagacgagag cgatataacc
catggctgaa gtactggtgc tcgttgagca 960 cgctgaaggc gcgttaaaga
aggtcagcgc 990 153 266 PRT Mycobacterium tuberculosis 153 Met Thr
Asn Ile Val Val Leu Ile Lys Gln Val Pro Asp Thr Trp Ser 1 5 10 15
Glu Arg Lys Leu Thr Asp Gly Asp Phe Thr Leu Asp Arg Glu Ala Ala 20
25 30 Asp Ala Val Leu Asp Glu Ile Asn Glu Arg Ala Val Glu Glu Ala
Leu 35 40 45 Gln Ile Arg Glu Lys Glu Ala Ala Asp Gly Ile Glu Gly
Ser Val Thr 50 55 60 Val Leu Thr Ala Gly Pro Glu Arg Ala Thr Glu
Ala Ile Arg Lys Ala 65 70 75 80 Leu Ser Met Gly Ala Asp Lys Ala Val
His Leu Lys Asp Asp Gly Met 85 90 95 His Gly Ser Asp Val Ile Gln
Thr Gly Trp Ala Leu Ala Arg Ala Leu 100 105 110 Gly Thr Ile Glu Gly
Thr Glu Leu Val Ile Ala Gly Asn Glu Ser Thr 115 120 125 Asp Gly Val
Gly Gly Ala Val Pro Ala Ile Ile Ala Glu Tyr Leu Gly 130 135 140 Leu
Pro Gln Leu Thr His Leu Arg Lys Val Ser Ile Glu Gly Gly Lys 145 150
155 160 Ile Thr Gly Glu Arg Glu Thr Asp Glu Gly Val Phe Thr Leu Glu
Ala 165 170 175 Thr Leu Pro Ala Val Ile Ser Val Asn Glu Lys Ile Asn
Glu Pro Arg 180 185 190 Phe Pro Ser Phe Lys Gly Ile Met Ala Ala Lys
Lys Lys Glu Val Thr 195 200 205 Val Leu Thr Leu Ala Glu Ile Gly Val
Glu Ser Asp Glu Val Gly Leu 210 215 220 Ala Asn Ala Gly Ser Thr Val
Leu Ala Ser Thr Pro Lys Pro Ala Lys 225 230 235 240 Thr Ala Gly Glu
Lys Val Thr Asp Glu Gly Glu Gly Gly Asn Gln Ile 245 250 255 Val Gln
Tyr Leu Val Ala Gln Lys Ile Ile 260 265 154 25 DNA Mycobacterium
tuberculosis 154 ctgagatcta tgaacctacg gcgcc 25 155 35 DNA
Mycobacterium tuberculosis 155 ctcccatggt accctaggac ccgggcagcc
ccggc 35 156 29 DNA Mycobacterium tuberculosis 156 ctgagatcta
tgaggctgtc gttgaccgc 29 157 30 DNA Mycobacterium tuberculosis 157
ctccccgggc ttaatagttg ttgcaggagc 30 158 33 DNA Mycobacterium
tuberculosis 158 gcttagatct atgattttct gggcaaccag gta 33 159 30 DNA
Mycobacterium tuberculosis 159 gcttccatgg gcgaggcaca ggcgtgggaa 30
160 30 DNA Mycobacterium tuberculosis 160 ctgagatcta gaatgccaca
gggaactgtg 30 161 30 DNA Mycobacterium tuberculosis 161 tctcccgggg
gtaactcaga gcgagcggac 30 162 27 DNA Mycobacterium tuberculosis 162
ctgagatcta tgaacgtcac cgtatcc 27 163 27 DNA Mycobacterium
tuberculosis 163 tctcccgggg ctcacccacc ggccacg 27 164 30 DNA
Mycobacterium tuberculosis 164 ctgagatcta tggcaacacg ttttatgacg 30
165 30 DNA Mycobacterium tuberculosis 165 ctccccgggt tagctgctga
ggatctgcth 30 166 31 DNA Mycobacterium tuberculosis 166 ctgaagatct
atgcccaaga gaagcgaata c 31 167 31 DNA Mycobacterium tuberculosis
167 cggcagctgc tagcattctc cgaatctgcc g 31 168 15 PRT Mycobacterium
tuberculosis 168 Pro Gln Gly Thr Val Lys Trp Phe Asn Ala Glu Lys
Gly Phe Gly 1 5 10 15 169 15 PRT Mycobacterium tuberculosis UNSURE
(15) Xaa is unknown 169 Asn Val Thr Val Ser Ile Pro Thr Ile Leu Arg
Pro Xaa Xaa Xaa 1 5 10 15 170 15 PRT Mycobacterium tuberculosis
VARIANT (1) Thr could also be Ala 170 Thr Arg Phe Met Thr Asp Pro
His Ala Met Arg Asp Met Ala Gly 1 5 10 15 171 15 PRT Mycobacterium
tuberculosis 171 Pro Lys Arg Ser Glu Tyr Arg Gln Gly Thr Pro Asn
Trp Val Asp 1 5 10 15 172 404 PRT Mycobacterium tuberculosis 172
Met Ala Thr Val Asn Arg Ser Arg His His His His His His His His 1 5
10 15 Ile Glu Gly Arg Ser Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr
Leu 20 25 30 Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val
Gln Phe Gln 35 40 45 Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu
Leu Asp Gly Leu Arg 50 55 60 Ala Gln Asp Asp Tyr Asn Gly Trp Asp
Ile Asn Thr Pro Ala Phe Glu 65 70 75 80 Trp Tyr Tyr Gln Ser Gly Leu
Ser Ile Val Met Pro Val Gly Gly Gln 85 90 95 Ser Ser Phe Tyr Ser
Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly 100 105 110 Cys Gln Thr
Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gln 115 120 125 Trp
Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser Ala Ala Ile 130 135
140 Gly Leu Ser Met Ala Gly Ser Ser Ala Met Ile Leu Ala Ala Tyr His
145 150 155 160 Pro Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu
Leu Asp Pro 165 170 175 Ser Gln Gly Met Gly Pro Ser Leu Ile Gly Leu
Ala Met Gly Asp Ala 180 185 190 Gly Gly Tyr Lys Ala Ala Asp Met Trp
Gly Pro Ser Ser Asp Pro Ala 195 200 205 Trp Glu Arg Asn Asp Pro Thr
Gln Gln Ile Pro Lys Leu Val Ala Asn 210 215 220 Asn Thr Arg Leu Trp
Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu 225 230 235 240 Gly Gly
Ala Asn Ile Pro Ala Glu Phe Leu Glu Asn Phe Val Arg Ser 245 250 255
Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn 260
265 270 Ala Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp Glu Tyr
Trp 275 280 285 Gly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu Gln Ser
Ser Leu Gly 290 295 300 Ala Gly Lys Leu Ala Met Thr Glu Gln
Gln Trp Asn Phe Ala Gly Ile 305 310 315 320 Glu Ala Ala Ala Ser Ala
Ile Gln Gly Asn Val Thr Ser Ile His Ser 325 330 335 Leu Leu Asp Glu
Gly Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp 340 345 350 Gly Gly
Ser Gly Ser Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp 355 360 365
Ala Thr Ala Thr Glu Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr 370
375 380 Ile Ser Glu Ala Gly Gln Ala Met Ala Ser Thr Glu Gly Asn Val
Thr 385 390 395 400 Gly Met Phe Ala 173 403 PRT Mycobacterium
tuberculosis 173 Met Ala Thr Val Asn Arg Ser Arg His His His His
His His His His 1 5 10 15 Ile Glu Gly Arg Ser Met Thr Glu Gln Gln
Trp Asn Phe Ala Gly Ile 20 25 30 Glu Ala Ala Ala Ser Ala Ile Gln
Gly Asn Val Thr Ser Ile His Ser 35 40 45 Leu Leu Asp Glu Gly Lys
Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp 50 55 60 Gly Gly Ser Gly
Ser Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp 65 70 75 80 Ala Thr
Ala Thr Glu Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr 85 90 95
Ile Ser Glu Ala Gly Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr 100
105 110 Gly Met Phe Ala Lys Leu Phe Ser Arg Pro Gly Leu Pro Val Glu
Tyr 115 120 125 Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys
Val Gln Phe 130 135 140 Gln Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr
Leu Leu Asp Gly Leu 145 150 155 160 Arg Ala Gln Asp Asp Tyr Asn Gly
Trp Asp Ile Asn Thr Pro Ala Phe 165 170 175 Glu Trp Tyr Tyr Gln Ser
Gly Leu Ser Ile Val Met Pro Val Gly Gly 180 185 190 Gln Ser Ser Phe
Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala 195 200 205 Gly Cys
Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro 210 215 220
Gln Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser Ala Ala 225
230 235 240 Ile Gly Leu Ser Met Ala Gly Ser Ser Ala Met Ile Leu Ala
Ala Tyr 245 250 255 His Pro Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser
Ala Leu Leu Asp 260 265 270 Pro Ser Gln Gly Met Gly Pro Ser Leu Ile
Gly Leu Ala Met Gly Asp 275 280 285 Ala Gly Gly Tyr Lys Ala Ala Asp
Met Trp Gly Pro Ser Ser Asp Pro 290 295 300 Ala Trp Glu Arg Asn Asp
Pro Thr Gln Gln Ile Pro Lys Leu Val Ala 305 310 315 320 Asn Asn Thr
Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu 325 330 335 Leu
Gly Gly Ala Asn Ile Pro Ala Glu Phe Leu Glu Asn Phe Val Arg 340 345
350 Ser Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Ala Ala Gly Gly His
355 360 365 Asn Ala Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp
Glu Tyr 370 375 380 Trp Gly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu
Gln Ser Ser Leu 385 390 395 400 Gly Ala Gly
* * * * *
References