Note: Descriptions are shown in the official language in which they were submitted.
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NUCLEIC ACID FRAGMENTS AND POLYPEPTIDE FRAGMENTS DERIVED FROM
M. TUBERCULOSIS
FIELD OF THE INVENTION
The present invention relates to a number of immunologically active,-novel
polypeptide
fragments derived from the Mycobacterium tuberculosis, vaccines and other
immuno-
logic 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
prepara-
tion of the polypeptide fragments of the invention or in the diagnosis of
infection with
M. tuberculosis .
BACKGROUND OF THE INVENTION
Human tuberculosis (hereinafter designated "TB") caused by Mycobacterium
tubercu-
losis 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.
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 resis-
tance 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.
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
de-
fine protective mycobacterial substances have been made, and from 1950 to 7
970
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
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cell wall fragments, although progress is currently being made by relying on
polypep-
tides derived from short term-culture filtrate, cf. the discussion below.
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-y).
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 resis-
tance 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 pro-
tective 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 fEP-0 432
203).
There is therefore a need for the identification of further antigens involved
in the in-
duction of protective immunity against TB in order to eventually produce an
effective
subunit vaccine.
OBJECT OF THE INVENTION
It is an object of the invention to provide novel antigens which are effective
as com-
ponents in a subunit vaccine against TB or which are useful as components in
diag-
nostic compositions for the detection of infection with mycobacteria,
especially viru-
lence-associated mycobacteria. The novel antigens may also be important drug
tar-
gets.
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SUMMARY OF THE INVENTION
The present invention is i.a. based on the identification and characterization
of a num-
ber of previously uncharacterized culture filtrate antigens from M.
tuberculosis. In
animal models of TB, T cells mediating immunity are focused predominantly to
anti-
gens in the regions 6-12 and 17-30 kDa of ST-CF. In the present invention 6
antigens
in the low molecular weight region (ORF7-1, ORF7-2, ORF11-1, ORF11-2, ORF11-3,
ORF1 1-4? have been identified.
Furthermore immunological and biological data on several important antigens
are
presented.
The encoding genes for 8 antigens have been determined. The panel hold
antigens
with potential for vaccine purposes as well as for diagnostic purposes, since
the
antigens are all secreted by metabolizing mycobacteria.
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:
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Antigen N-terminal sequenceNucleotide sequenceAmino acid sequence
SEQ ID NO: SEQ ID NO: SEQ ID NO:
CFP7 1 2
CFP7A 81 47 48
CFP7B 168 i46 147
CFPBA 73 748 149
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
CFP2 7 19 9 10
CFP22 20 71 12
CFP22A 83 53 54
CFP23 55 56
CFP23A 76
CFP23B 75
CFP25 21 i3 14
CFP25A 78 65 66
CFP27 84 57 58
CFP28 22
CFP29 23 15 16
CFP30A 85 59 60
CFP34B 171 144 145
CFP50 86 61 . 62
MPT51 41 42
CWP32 77 152 153
RD 1-ORF8 67 68
RD1-ORF2 71 72
RDi-ORF9B 69 70
RD1-ORF3 87 88
RD 1-ORF9A 93 94
RD 1-ORF4 89 90
RD1-ORF5 91 92
MPT59- 172
ESAT6
ESAT6- 173
MPT59
ORF7-1 174 175
ORF7-2 176 177
ORF71-1 178 179
ORF 1 180 181
1-2
ORF 1 182 183
1-3
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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 in-
fluence on acquired immunity and recognition of mycobacteria in vivo. Since
such T-
5 cell epitopes are linear and are known to have a minimum length of 6 amino
acid resi-
dues, the present invention is especially concerned with the identification
and utifisa-
Lion of such T-cell epitopes.
Hence, in its broadest aspect the invention relates to 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, 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, any one of 168-171, 175, 177, 179, 181, 183, and 185
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 pro-
tective immune response against infections with mycobacteria belonging to the
tuber-
culosis 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 poly-
peptide 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
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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 ~3-galactosidase,
ii) the degree of sequence identity in c) is at least 95% when the polypeptide
com-
prises 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.
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 pre
sence of DNA encoding such polypeptides.
DETAILED DISCLOSURE OF THE INVENTION
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 (1 1-100 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,
oiigopeptide,
or polypeptide which may be chemically modified by being glycosylated, by
being lipi-
dated, or by comprising prosthetic groups). The definition of polypeptides
also com-
prises native forms of peptides/proteins in mycobacteria as well as
recombinant pro-
teins or peptides in any type of expression vectors transforming any kind of
host, and
also chemically synthesized peptides.
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
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most %z %). 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 mate-
rial 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
poiypeptide
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
de-
scribed by Merrifield or variations thereof.
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, any one of 168-171, 175, 177, 179, 181,
183,
and 185 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, any one of 168-171,
175, 177, 179, 181, 183, and 185 and being immunological equivalent thereto
with
respect to the ability of conferring increased resistance to infections with
bacteria be-
longing to the tuberculosis complex. Thus, included is also a polypeptide from
diffe-
rent sources, such as other bacteria or even from eukaryotic cells.
When referring to an "irnmunologically 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
1) confer, upon administration /either atone or as an immunologically active
con-
stituent 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
infec-
tions with bacteria belonging to the tuberculosis complex which is at least
20% of the
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acquired increased resistance conferred by Mycobacterium bouts BCG and also at
least
20% of the acquired increased resistance conferred by the parent polypeptide
com-
prising 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, any one of 168-171, 175, 177, 179, 181, 183, or 185 (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
re-
ceiving 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
develop-
ment 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.
bouts
BCG, such as at least 60%, or even more preferred to at least 80% of the
protective
immune response elicited by M. bouts BCG, such as at least 90%; in some cases
it is
expected that the increased resistance will supersede that conferred by M.
bouts BCG,
and hence it is preferred that the resistance will be at least 100%, such as
at least
1 10% of said increased resistance); and/or
II) elicit a diagnostically significant immune response in a mammal indicating
pre-
vious 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-y release determined e.g. by an IFN-
y 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
polypep- -
tide 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, any one of 168-171, 175, 177, 179, 181, 183, or
185.
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The ability of the polypeptide fragment to confer increased immunity may thus
be as-
sessed by measuring in an experimental animal, e.g. a mouse or a guinea pig,
the re-
duction 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
tuber-
culosis. The comparison of the mycobacterial counts may also be carried out
with my-
cobacterial counts from a group of experimental animals receiving a challenge
infec-
tion with the same virulent strain after having been immunized with
Mycobacterium
bovis BCG.
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 preferably at the most 2
times the
counts.
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 le.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 de-
termined by statistical methods known to the skilled artisan).
In the context of the present application, the term "wide genetically" should
be under-
stood in a meaning of at least two strains. That is, if a polypeptide is
recognised by at
least two different strains, it is considered to have a wide genetically
recognition.
A subunit vaccine component is defined as a reagent which stimulates
protective im-
munity in an animal model of infection with an organism of the M. tuberculosis
com-
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plex, when given prior to infection and which also generates a significant
immune re-
sponses in human volunteers.
The "tuberculosis-complex" has its usual meaning, i.e. the complex of
mycobacteria
5 causing TB which are Mycobacterium tuberculosis, Mycobacterium bovis,
Mycobacte
rium bovis BCG, and Mycobacterium africanum. -
In the present context the term "metabolizing mycobacteria" means live
mycobacteria
that are multiplying logarithmically and releasing polypeptides into the
culture medium
10 wherein they are cultured.
The term "sequence identity" indicates a quantitative measure of the degree of
ho-
mology between two amino acid sequences or between two nucleotide sequences of
equal length, of if not of equal length aligned to best possible fit: The
sequence
identity can be calculated as ("~' ""')I°" 1, wherein Nd;, is the total
number of non-
h., l
identical residues in the two sequences when aligned and wherein N,e~ is the
number
of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will
have
a sequence identity of 75 % with the sequence AATCAATC (Nd;, = 2 and N,e, =
8). A
gap is counted as non-identity of the specific residue(s), i.e. the DNA
sequence
AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC
(Nd;, = 2 and N,e, = 8). Sequence identity can alternatively be calculated by
the BLASTP
program ((Pearson W.R and D.J. Lipman (1988) PNAS USA 85:2444-2448) in the
EMBL database (www.ncbi.nlm.govlcgi-bin/BLAST). Generally, the default
settings
with respect to e.g. "scoring matrix" and "gap penalty" will be used for
alignment.
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, any one of 168-171, 175, 177, 179, 181, 183, or 185. The amino
acid sequence to be compared with the amino acid sequence shown in SEQ ID NO:
2,
4, 6, 8, 10, 12, 14, 7 6, 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,
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any one of 168-171, 175, 177, 179, 181, 183, or 185 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 de-
termined on the amino acid sequence of a mature polypeptide, i.e. without
taking any
leader sequence into consideration.
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, any one of 168-171, 175, 177, 179, 181, 7
83,
or 185 are embraced by the present invention. The invention allows for minor
varia-
tions 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 bio-
logical functions and immunogenicities etc.
Each polypeptide fragment may thus be characterized by specific amino acid and
nu-
cleic acid sequences. It will be understood that such sequences include
analogues and
variants produced by recombinant methods wherein such nucleic acid and
poiypeptide
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, in-
section, 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 or LNA may be used
instead
of DNA. 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?. LNA (Locked
Nucleic
Acids) is a recently introduced oligonucleotide analogue containing-bicyclo
nucleoside
monomers (Koshkin et al., 1998, 54, 3607-3630;Nielsen, N.K. et al.
J.Am.Chem.Soc
1998, 120, 5458-5463).
In both immunodiagnostics and vaccine preparation, it is often possible and
practical
to prepare antigens from segments of a known immunogenic protein or
poiypeptide.
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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 an-
tigenicity analyses or Hopp and Woods (1981 ) hydrophobicity analysis (see,
e.g., Ja-
meson and Wolf, 1988; Kyte and Doolittle, 1982; or U.S. Patent No. 4,554,101).
Hy-
drophobicity 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
polypep-
tides of the invention.
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 poiypeptides having SEQ !D 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, any
one
of 168-171, 175, 177, 179, 181, 183, or 185 will, if constructed
systematically, re-
veal what regions of the polypeptides are essential in immune recognition,
e.g. by
subjecting these deletion mutants to the IFN-y 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, any one of 168-
171,
175, 177, 179, 181, 183, or 185. Some of these will give a positive response
in the
IFN-Y assay whereas others will not.
In a preferred embodiment of the invention, the polypeptide fragment of the
invention
comprises an epitope for a T-helper cell.
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
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13
feast 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.
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
fil-
trates from bacteria belonging to the tuberculosis complex are free of these
sequen-
ces. Although it may in some applications be advantageous to produce these
polypep-
tides recombinantly and in this connection facilitate export of the
poiypeptides 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
SEC2 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 andlor -38 to -1 in SEQ ID NO: 52 andlor -33 to -1
in SEQ
ID NO: 56 and/or -56 to -1 in SEQ ID NO: 58 andlor -28 to -1 in SEQ ID NO:
151.
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
re-
combinantly 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. -
A suitable assay for the potential utility of a given polypeptide fragment
derived from
SEQ ID NO: 2, 4, 6, 8, 1 O, 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, any one of 168-171, 175, 177, 179, 181, 183, or 185 is to
assess
the ability of the polypeptide fragment to effect IFN-y release from primed
memory T-
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14
lymphocytes. Poiypeptide 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. _' '
It is presently contemplated that when this application refers to IFN-y
release as a
measure of immunogenicity, other cytokines could be relevant, such as IL-12,
TNF-a.,
IL-4, IL-5, IL-10, IL-6, TGF-~3. Usually one or more cytokines will be
measured utilising
for example the PCR technique or ELISA. It will be appreciated by the person
skilled in
the art, that a significant increase or decrease in any~of these cytokines
will be indica-
tive of an immunological effective polypeptide or polypeptide fragment.
Thus, an important embodiment of the invention is a polypeptide fragment
defined
above which
1 ) induces a release of IFN-y from primed memory T-lymphocytes withdrawn from
a mouse within 2 weeks of primary infection or within 4 days after the mouse
has
been re-challenge 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 con-
centration of 1-4 ~g potypeptide per mi suspension, the release of IFN-y being
assess-
able by determination of IFN-'y in supernatant harvested 2 days after the
addition of
the polypeptide to the suspension, and/or
2) induces a release of 1FN-y of at least 1,500 pglml 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 do-
nors, 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 ~g
polypep-
tide per ml suspension, the release of IFN-y being assessable by determination
of IFN-y
CA 02319380 2000-08-O1
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in supernatant harvested 2 days after the addition of the polypeptide to the
suspen-
sion; and/or
3) induces an IFN-y release from bovine PBMC derived from animals previously
5 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.
Preferably, in alternatives 1 and 2, the release effected by the polypeptide
fragment
10 gives rise to at least 1,500 pg/ml IFN-y in the supernatant but higher
concentrations
are preferred, e.g. at least 2,000 pglml and even at least 3,000 pg/ml IFN-y
in the su-
pernatant. The IFN-y 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.
The poiypeptide fragments of the invention preferably comprises an amino acid
se-
quence of at least 6 amino acid residues in length which has a higher sequence
iden-
tity 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, any one of 168-171, 175, 177, 179, 181,
183, or 185. A preferred minimum percentage of sequence identity is at least
80%,
such as at least 85%, at least 90%, at feast 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%.
As mentioned above, it will normally be interesting to omit the leader
sequences from
the polypeptide fragments of the invention. However, by producing fusion
polypep-
tides, superior characteristics of the polypeptide fragments of the invention
can be
achieved. For instance, fusion partners which facilitate export of the
polypeptide when
3O produced recombinantiy, fusion partners which facilitate purification of
the polypep-
tide. and fusion partners which enhance the immunogenicity of the potypeptide
frag-
ment of the invention are all interesting possibilities. Therefore, the
invention also per-
tains 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 irn-
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16
munogenicity, e.g. be selected from the group consisting of another
polypeptide frag-
ment 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 com-
plex, such as ESAT-6, CFP7, CFP10, CFP17, CFP21, CFP25, CFP29, MPB59, MPT59,
MPB64, and MPT64 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-y, 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; ~i-galactosidase; or poly-histidine.
Other interesting fusion partners are polypeptides which are lipidated and
thereby ef-
fect that the immunogenic polypeptide is presented in a suitable manner to the
im-
mune system. This effect is e.g. known from vaccines based on the Borrelia
burgdor-
feri OspA polypeptide, wherein the lipidated membrane anchor in the
polypeptide con-
fers a self-adjuvating effect to the polypeptide (which is natively lipidated)
when iso-
lated from cells producing it. In contrast, the OspA pofypeptide is relatively
silent im-
munologically when prepared without the lipidation anchor.
As evidenced in Example 6A, the fusion polypeptide consisting of MPT59 fused
di
rectly 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 pre-
cise 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
immuno-
genicity 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 immunoiogic properties of this
anti-
gen.
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17
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 T-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
de-
rived from a M. tuberculosis protein different from ESAT-6 (if the first
stretch of
amino acids are derived from ESAT-6) or MPT59 (it the first stretch o~ 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 maybe 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.
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 ef-
fect 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, a-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 se-
quence 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
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18
MPT59 and/or the second amino acid sequence is the full-length amino acid
sequence
of the possible "parent" polypeptides listed above.
In the mast 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.
Another part of the invention pertains to a nucleic acid fragment in isolated
form
which
1 ) comprises a nucleic acid sequence which encodes a polypeptide or fusion
poly-
peptide as defined above, or comprises a nucleic acid sequence complementary
there-
to, and/or
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°C under the
melting point Tm,
cf. Sambrook et al, 1989, pages 1 1.45-11.49) with a nucleic acid fragment
which has
a nucleotide sequence selected from
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,
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19
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,
SEQ ID NO: 152 or a sequence complementary thereto,
SEQ ID NO: 174 or a sequence complementary thereto,
SEQ ID NO: 176 or a sequence complementary thereto,
SEQ ID NO: 178 or a sequence complementary thereto,
SEQ ID NO: 180 or a sequence complementary thereto,
SEQ ID NO: 182 or a sequence complementary thereto, and
SEQ ID NO: 184 or a sequence complementary thereto
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
posi-
tion 781 in SEQ ID NO: 41 and when the nucleic acid fragment comprises a subse-
quence 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.
It is preferred that the nucleic acid fragment is a DNA fragment.
CA 02319380 2000-08-O1
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To provide certainty of the advantages in accordance with the invention, the
preferred
nucleic acid sequence when employed for hybridization studies or assays
includes se-
quences 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 heaps to
ensure that
5 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
frag-
ments of the invention is intended to indicate a continuous stretch of at
feast 10 nu-
cleotides 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, 1 1, 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, 152, 174,
176,
178, 180, 182, or 184. tt 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 feast 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. Patent 4,603,102,
or by
introducing selected sequences into recombinant vectors for recombinant
production.
it is well known that the same amino acid may be encoded by various codons,
the co
don usage being related, inter alia, to the preference of the organisms in
question ex
pressing 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
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21
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.
The nucleotide sequence to be modified may be of cDNA or genomic origin as
discus-
sed 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
mu-
tagenesis, to result in the desired nucleic acid fragment encoding the desired
polypep-
tide. 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.
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.
A preferred method of preparing variants of the antigens disclosed herein is
site-di-
rected mutagenesis. This technique is useful in the preparation of individual
peptides,
or biologically functional equivalent proteins or peptides, derived from the
antigen se-
quences, through specific mutagenesis of the underlying nucleic acid. The
technique
CA 02319380 2000-08-O1
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22
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 su#icient 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 pre-
ferred, with about 5 to 10 residues on both sides of the junction of the
sequence be-
ing altered.
In general, the technique of site-specific mutagenesis is well known in the
art as ex-
emplified 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 M 13 phage (Messing et al., 1981 ). These phage are readily
commercially
available and their use is generally well known to those skilled in the art.
fn general, site-directed mutagenesis in accordance herewith is performed by
first ob-
taining a single-stranded vector which includes within its sequence a nucleic
acid se-
quence 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
polymer-
ase 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 hetero-
duplex 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.
The preparation of sequence variants of the selected nucleic acid fragments of
the in-
vention using site-directed mutagenesis is provided as a means of producing
poten-
tially useful species of the genes and is not meant to be limiting as there
are other
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23
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
de-
scribed by Eichenlaub, 1979y for the mutagenesis of plasmid DNA using hydroxyl-
s amine.
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 frag-
ment encoding a polypeptide fragment of the invention.
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 chromosomes) into which it has been integrated.
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
expres-
sion vectors is possible including, for example, DNA segments encoding
reporter gene
products useful for identification of heterologous gene products andlor
resistance
genes such as antibiotic resistance genes which may be useful in identifying
trans-
formed cells.
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.poly-
peptide fragments of the invention. Hence, the invention also pertains to a
transform-
ed cell harbouring at least one such vector according to the invention, said
cell being
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24
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
multiceilular
organism, e.g. an animal or a plant. It is especially in cases where
glycosylation is de-
sired 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
Es-
chericia. 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
polypep-
tide 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
My-
cobacterium bovis strain Copenhagen from the Copenhagen BCG Laboratory,
Statens
Seruminstitut, Denmark.
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.
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 ltransformed cells may be
selected us-
ing 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
isolating the polypeptide from a short-term culture filtrate as defined in
claim 1; or
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isolating the poiypeptide 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.
5
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
10 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:
In general, of course, prokaryotes are preferred for the initial cloning of
nucleic se-
quences of the invention and constructing the vectors useful in the invention.
For ex-
1 5 ample, 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.
20 Prokaryotes are also preferred for expression. The aforementioned strains,
as well as
E. coli W31 10 (F-, lambda-, prototrophic, ATCC No. 2733251, bacilli such as
Bacillus
subtilis, or other enterobacteriaceae such as Salmonella typhimurium or
Serratia mar-
cesans, and various Pseudomonas species may be used. Especially interesting
are
rapid-growing mycobacteria, e.g. M. smegmatis, as these bacteria have a high
degree
25 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.
In general, plasmid vectors containing replicon and control sequences which
are de-
rived 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
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26
ampiciliin 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
microorgan-
ism for expression.
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; Goeddef 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
con-
cerning 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 se-
quences, precluding the need for addition of another promoter by artificial
means.
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 tech-
pique described by Andersen et a!. in J. lmmunol. Methods 7 6'! : 29-39.
According to the invention the post-translationai modifications involves
lipidation, gly-
cosylation, cleavage, or elongation of the polypeptide.
In certain aspects, the DNA sequence information provided by this invention
allows for
the preparation of relatively short DNA for 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 rele-
want 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
detect-
ing the presence of pathogenic organisms in a given sample. However, either
uses are
CA 02319380 2000-08-O1
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27
envisioned, including the use of the sequence information for the preparation
of mu-
tant species primers, or primers for use in preparing other genetic
constructs.
Apart from their use as starting points for the synthesis of polypeptides of
the inven
tion 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
UI-
mer et al., 1993, which hereby is included by reference.
Hence, the invention also relates to a vaccine comprising a nucleic acid
fragment ac-
cording to the invention, the vaccine effecting in vivo expression of antigen
by an ani-
mal, including a human being, to whom the vaccine has been administered, the
amount of expressed antigen being effective to confer substantially increased
resis-
tance to infections with mycobacteria of the tuberculosis complex in an
animal, inclu-
ding a human being.
The efficacy of such a "DNA vaccine" can possibly be enhanced by administering
the
gene encoding the expression product together with a DNA fragment encoding a
poly-
peptide which has the capability of modulating an immune response. For
instance, a
gene encoding lymphokine precursors or lymphokines (e.g. IFN-y, IL-2, or IL-
12) could
be administered together with the gene encoding the immunogerlic 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 com-
prising a multitude of nucleotide sequences which each encode relevant
epitopes of
the polypeptides disclosed herein so as to effect a continuous sensitization
of the im-
mune system with a broad spectrum of these epitopes.
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28
As explained above, the polypeptide fragments of the invention are excellent
candi-
dates for vaccine constituents or for constituents in an immune diagnostic
agent due
to their extracellular presence in culture media containing metabolizing
virulent myco-
bacteria belonging to the tuberculosis complex, or because of their high
homologies
with such extracellular antigens, or because of their absence in M. bovis BCG.
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.
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 difuent and a suspending agent. The adjuvant is preferably selected from
the
group consisting of dimethyldioctadecylammonium bromide (DDA), Quil A, poly
I:C,
Freund's incomplete adjuvant, IFN-y, IL-2, IL-12, monophosphoryl lipid A
(MPL), and
muramyi dipeptide (MDP).
A preferred immunologic composition according to the present invention
comprising at
least two different polypeptide fragments, each different polypeptide fragment
being a
poiypeptide or a fusion polypeptide defined above. It is preferred that the
immunologic
composition comprises between 3-20 different polypeptide fragments or fusion
poly-
peptides.
Such an immunologic composition may preferably be in the form of a vaccine or
in the
form of a skin test reagent.
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
pre-
paring, synthesizing or isolating a polypeptide according to the invention,
and solubi-
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29
lining or dispersing the potypeptide in a medium for a vaccine, and optionally
adding
other M. tuberculosis antigens and/or a carrier, vehicle and/or adjuvant
substance.
Preparation of vaccines which contain peptide sequences as active ingredients
is gen-
erally well understood in the art, as exemplified by U.S. Patents 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
pharma-
ceutically acceptable and compatible with the active ingredient. Suitable
excipients
are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and
combina-
tions thereof. In addition, if desired, the vaccine may contain minor amounts
of auxil-
iary substances such as wetting or emulsifying agents, pH buffering agents, or
adju-
vants which enhance the effectiveness of the vaccines.
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 for-
mutations. 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 1.0%, preferably 1-
2%. Oral
formulations include such normally employed excipients as, for example,
pharmaceu-
tical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cel-
lulose, magnesium carbonate, and the like. These compositions take the form of
solu-
tions, suspensions, tablets, pills, capsules, sustained release formulations
or powders
and contain 10-95% of active ingredient, preferably 25-70%.
The proteins may be formulated into the vaccine as neutral or salt forms.
Pharmaceu-
tically 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, hy-
drochloric or phosphoric acids, or such organic acids as acetic oxalic,
tartaric, man-
delic, 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,
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or ferric hydroxides, and such organic bases as isopropylamine,
trimethytamine, 2-eth-
ylamino ethanol, histidine, procaine, and the like.
The vaccines are administered in a manner compatible with the dosage
formulation,
5 and in such amount as will be therapeutically effective and immunogenic. The
quantity
to be administered depends on the subject to be treated, including, a 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
micro-
grams active ingredient per vaccination with a preferred range from about 0.1
ug to
10 1000 gg, such as in the range from about 1 ~g to 300 ug, and especially in
the range
from about 10 ug to 50 pg. Suitable regimens for initial administration and
booster
shots are also variable but are typified by an initial administration followed
by subse-
quent inoculations or other administrations.
15 The manner of application may be varied widely. Any of the conventional
methods far
administration of a vaccine are applicable. These are believed to include oral
applica-
tion on a solid physiologically acceptable base or in a physiologically
acceptable dis-
persion, 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 vac-
20 cinated and, to a lesser degree, the size of the person to be vaccinated.
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
fur-
ther comprises an adjuvant substance.
Various methods of achieving adjuvant effect for the vaccine include use of
agents
such as aluminum hydroxide or phosphate lalum), commonly used as 0.05 to 0.1
per-
cent solution in phosphate buffered saline, admixture with synthetic polymers
of sug-
ars (Carbopol) used as 0.25 percent solution, aggregation of the protein in
the vaccine
by heat treatment with temperatures ranging between 70° to 101
°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
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31
percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute
may also
be employed. According to the invention DDA (dimethyldioctadecylammonium bro-
mide) is an interesting candidate for an adjuvant, but also Freund's complete
and in-
complete adjuvants as well as QuilA and RIBI are interesting possibilities.
Further pos-
y sibilities are monophosphoryl lipid A (MPL), and muramyl dipeptide (MDP).
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
incorpo-
rated 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
anti-
bodies (or antigen binding antibody fragments) against the Fcy receptors on
mono-
cyteslmacrophages. Especially conjugates between antigen and anti-FcyRl have
been
demonstrated to enhance immunogenicity for the purposes of vaccination.
Other possibilities involve the use of immune modulating substances such as
lympho-
kines (e.g. IFN-y, IL-2 and IL-12) or synthetic IFN-y inducers such as poly
I:C in combi-
nation with the above-mentioned adjuvants. As discussed in example 3, it is
contem-
plated that such mixtures of antigen and adjuvant will lead to superior
vaccine formu-
lations.
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 im-
munization 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-y 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. Patent Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of
these
types of assays.
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32
Due to genetic variation, different individuals may react with immune
responses of
varying strength to the same polypeptide. Therefore, the vaccine according to
the in-
vention may comprise several different polypeptides in order to increase the
immune
response. The vaccine may comprise two or more polypeptides, where all of the
poly-
peptides 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 sub-
stance which can be isolated from mycobacteria are possible candidates.
The vaccine may comprise 3-20 different polypeptides, such as 3-10 different
poly-
peptides.
One reason for admixing the polypeptides of the invention with an adjuvant is
to ef-
fectively 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 bouts BCG.
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
tuber-
culosis-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 poiypeptide.
In the present context the term "genome" refers to the chromosome of the
microor- -
ganisms as well as extrachromosomally DNA or RNA, such as plasmids. It is, how-
ever, 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.
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33
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. It is especially preferred that the non-pathogenic
microorganism is My-
cobacterium bouts BCG, such as Mycobacterium bouts BCG strain: Danish 1331.
The incorporation of one or more copies of a nucleotide sequence encoding the
poly-
peptide according to the invention in a mycobacterium from a M. bouts 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
se-
quences 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.
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.
The invention also relates to a method of diagnosing TB caused by
Mycobacterium
tuberculosis, Mycobacterium afiicanum or Mycobacterium bouts in an animal,
includ-
ing a human being, comprising intradermaNy injecting, in the animal, a
polypeptide ac-
cording to the invention or a skin test reagent described above, a positive
skin re-
sponse 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.
In line with the disclosure above pertaining to vaccine preparation and use,
the inven-
lion also pertains to a method for immunising an animal, including a human
being,
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34
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), in-
s traperitoneal, intramuscular, subcutaneous, intradermal, oral, buccal,
sublingual, nasal,
rectal or transdermal route.
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 fn 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
vacci-
nated 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.
A number of possible diagnostic assays and methods can be envisaged:
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
con-
tacting can be performed in vitro and a positive reaction could e.g, be
proliferation of
the T-cells or release cytokines such as y-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
infec-
Lion.
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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
ani-
mal or human being, and contacting the sample from the animal with the
polypeptide
5 of the invention, a significant release into the extracelluiar phase of at
least one cyto-
kine 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
10 skin test for TB). Normally, a significant release is at least two times
the release ob-
served from such a sample.
Alternatively, a sample of a possibly infected organ may be contacted with an
anti-
body raised against a polypeptide of the invention. The demonstration of the
reaction
15 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.
Also a method of determining the presence of mycobacterial nucleic acids in an
ani-
mal, 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 de-
tecting 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 inven-
tion. Such a method of diagnosing TB might involve the use of a composition
compri-
sing at least a part of a nucleotide sequence as defined above and-detecting
the pre-
sence 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.
The fact that certain of the disclosed antigens are not present in M. bouts
BCG but are
present in virulent mycobacteria point them out as interesting drug targets;
the anti-
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36
gens may constitute receptor molecules or toxins which facilitate the
infection by the
mycobacterium, and if such functionalities are blocked the infectivity of the
mycobac-
terium will be diminshed.
To determine particularly suitable drug targets among the antigens of the
invention,
the gene encoding at least one of the polypeptides of the invention arid 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')2 fragments can be prepared against such critical polypeptides by
methods
known in the art and thereafter used as prophylactic or therapeutic agents.
Alterna-
tively, small molecules can be screened for their ability to selectively
inhibit expression
of the critical gene products, e.g. using recombinant expression systems which
in-
clude 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 pro-
phylactic agents to inhibit mycobacterial virulence.
Alternatively, anti-mycobacterial agents which render a virulent mycobacterium
aviru-
lent 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
myco-
bacterium. Such a "newly avirulent" mycobacterium would constitute a superb
alter-
native 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.
Finally, a monoclonal or polyclonal antibody, which is specifically reacting
with a poly-
peptide 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 anti- -
bodies are subsequently isolated, suitably by immune affinity chromatography.
The
production of monoclonals can be effected by methods well-known in the art,
since
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the present invention provides for adequate amounts of antigen for both
immunization
and screening of positive hybridomas.
LEGENDS TO THE FIGURES
Fig. 1: Long term memory immune mice are very efficiently protected towards an
in-
fection 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-y 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-y in
response to antigens in ST-CF.
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-'y was investigated
Fig. 3: Nucleotide sequence (SEQ ID NO: 1 ) of cfp7. The deduced amino acid se-
quence (SEQ ID NO: 2) of CFP7 is given in conventional one-letter code below
the nu-
cleotide 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.
Fig. 4. Nucleotide sequence (SEQ ID NO: 3) of cfp9. The deduced amino acid se-
quence (SEQ ID NO: 4) of CFP9 is given in conventional one-letter code below
the nu-
cleotide 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 226 phage is double underlined.
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
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38
italics. The putative potential ribosome-binding site is underlined. The
nucleotide differ-
ence 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.
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 posi-
tion of well-known proteins are also indicated.
EXAMPLE 1
Identification of single culture filtrate antigens involved in p~oteciive
immunity
A group of efficiently protected mice was generated by infecting 8-12 weeks
old fe-
male C57B1/6j mice with 5 x 10° 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
immu-
nity. 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-y in response to ST-CF (Fig. 1 ) /Andersen et al. 1995).
We have used this model to identify single antigens recognized by protective T
cells.
Memory immune mice were reinfected with 1 x 106 M. tuberculosis i.v. and
splenic
lymphocytes were harvested at day 4-6 of reinfection, a time point where this
popula-
tion is highly reactive to ST-CF. The antigens recognized by these T cells
were
mapped by the multi-elution technique IAndersen 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 vi-
tro and the release of IFN-y was monitored (Fig. 2). Long-term memory immune
mice
did not recognize these fractions before TB infection, but sptenic lymphocytes
ob-
tained during the recall of protective immunity recognized a range of culture
filtrate
antigens and peak production of IFN-y was found in response to proteins of
apparent
molecular weight 6-12 and 17-30 kDa (Fig. 2). It is therefore concluded that
culture
CA 02319380 2000-08-O1
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39
filtrate antigens within these regions are the major targets recognized by
memory ef-
fector T-cells triggered to release IFN-y during the first phase of a
protective immune
response.
EXAMPLE 2
Cloning of genes expressing low mass culture filtrate antigens
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 anti-
bodies (mAbs) to these antigens were therefore generated by immunizing with
the low
mass fraction in RIBI adjuvant (first and second immunization) followed by two
injec-
lions with the fractions in aluminium hydroxide. Fusion and cloning of the
reactive cell
lines were done according to standard procedures (Kohler and Milstein i 975).
The
procedure resulted in the provision of two mAbs: ST-3 directed to a 9 kDa
culture fil-
trate 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.
In order to identify the antigens binding to the Mab's, the following
experiments were
carried out:
The recombinant ~.gt1 1 M. tuberculosis DNA library constructed by R. Young
(Young,
R.A. et al. 1985? and obtained through the World Health Organization IMMTUB
pro-
gramme (WH0.0032.wibr) was screened for phages expressing gene products which
would bind the monoclonal antibodies ST-3 and PV-2.
Approximately 1 x 105 pfu of the gene library (containing approximately 25 %
recom-
binant phages) were plated on Eschericia colt Y 1090 (DIacU 169,-proA+, Dlon,
araD139, supF, trpC22::tn10 (pMC9) ATCC#37197) in soft agar and incubated for
2,5 hours at 42°C.
The plates were overlaid with sheets of nitrocellulose saturated with
isopropyl-~3-D-
thiogalactopyranoside and incubation was continued for 2,5 hours at
37°C. The nitro-
cellulose was removed and incubated with samples of the monoclonal antibodies
in
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PBS with Tween 20 added to a final concentration of 0.05%. Bound monoclonal
anti-
bodies were visualized by horseradish peroxidase-conjugated rabbit anti-mouse
immu-
noglobulins (P260, Dako, Glostrup, DK) and a staining reaction involving
5,5',3,3'-
tetramethylbenzidine and HZOz.
5
Positive plaques were recfoned and the phages originating from a single plaque
were
used to lysogenize E. coli Y1089 (DIacU169, proA+, Dlon, araD139, strA, hf1150
[chr::tn 10] [pMC9] ATCC nr. 37196). The resultant lysogenic strains were used
to
propagate phage particles for DNA extraction. These lysogenic E. coli strains
have
10 been named:
AA226 (expressing ST-3 reactive polypeptide CFP9) which has been deposited 28
June 1993 with the collection of Deutsche Sammlung von Mikroorganismen and
Zell-
kulturen GmbH (DSM) under the accession number DSM 8377 and in accordance with
15 the provisions of the Budapest Treaty, and
AA242 (expressing PV-2 reactive polypeptide CFP7) which has been deposited 28
June 1993 with the collection of Deutsche Sammiung von Mikroorganismen and ZeN-
kulturen GmbH (DSM) under the accession number DSM 8379 and in accordance with
20 the provisions of the Budapest Treaty.
These two lysogenic E. coli strains are disclosed in WO 95101441 as are the
myco-
bacterial polypeptide products expressed thereby. However, no information
concerning
the amino acid sequences of these polypeptides or their genetic origin are
given, and
25 therefore only the direct expression products of AA226 and AA242 are made
available to the public.
The st-3 binding protein is expressed as a protein fused to G3-galactosidase,
whereas
the pv-2 binding protein appears to be expressed in an unfused version.
Seauencing of the nucleotide se4uence encoding the PV-2 and ST-3 binding
protein
In order to obtain the nucleotide sequence of the gene encoding the pv-2
binding pro-
tein, the approximately 3 kb M. tuberculosis derived EcoRl - EcoRl fragment
from
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41
AA242 was subcloned in the EcoRl site in the pBluescriptSK + (Stratagene) and
used
to transform E. coli XL-1 Blue (Stratagene).
Similarly, to obtain the nucleotide sequence of the gene encoding the st-3
binding pro-
tein, the approximately 5 kb M. tuberculosis derived EcoRl - EcoRl fragment
from
AA226 was subcloned in the EcoRl site in the pBluescriptSK + (Stratagene) and
used
to transform E. coli XL-1 Blue (Stratagene).
The complete DNA sequence of both genes were obtained by the dideoxy chain
termi-
nation method adapted for supercoiled DNA by use of the Sequenase DNA
sequencing
kit version 1.0 (United States Biochemical Corp., Cleveland, OH) and by cycle
se-
quencing 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.
CFP7
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).
CFP7 appear to be expressed in E. coli as an unfused version. The nucleotide
se-
quence at position 78-84 is expected to be the Shine Delgarno sequence and the
se-
quences from position 47-50 and 14-19 are expected to be the -10 and -35
regions,
respectively:
CFP9
The protein recognised by ST-3 was produced as a G3-galactosidase fusion
protein,
when expressed from the AA226 lambda phage. The fusion protein had an approx.
size of 1 16 - 1 l7kDa (Mw for (3-galactosidase 116.25 kDa) which may suggest
that
only part of the CFP9 gene was included in the lambda clone (AA226).
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Based on the 90 by 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):
http:/lwww.sanger.ac.uk/pathogenslTB-blast-server.html;
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 resi-
dues 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.
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.
Subcloning CFP7 and CFP9 in expression vectors
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.
The PCR amplification was carried out in a thermal reactor (Rapid cycler,
Idaho Tech-
nology, Idaho) by mixing 10 ng plasmid DNA with the mastermix (0.5 NM of each
oli-
gonucleotide primer, 0.25 uM BSA (Stratagene), low salt buffer (20 mM Tris-
HCI, pH
8.8, 10 mM KCI, 10 mM (NH4)ZSO4, 2 mM MgSO, and 0,1 % Triton X-100) (Strata- ~
gene), 0.25 mM of each deoxynucleoside triphosphate and 0.5 U Taq Plus Long
DNA
polymerase (Stratagene)). Final volume was 10 NI (all concentrations given are
con-
centrations in the final volume). ~Predenaturation was carried out at
94°C for 30 s. 30
cycles of the following was performed; Denaturation at 94°C for 30 s,
annealing at
55°C for 30 s and elongation at 72°C for 1 min.
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The oligonucleotide primers were synthesised automatically on a DNA
synthesizer
(Applied Biosystems, Forster City, Ca, ABI-391, PCR-mode?, deblocked, and
purified
by ethanol precipitation.
The cfp7 oligonucleotides (TABLE 1 ) were synthesised on the basis ef the
nucleotide
sequence from the CFP7 sequence (Fig. 31. The oligonucleotides were engineered
to
include an Smal restriction enzyme site at the 5' end and a BamHl restriction
enzyme
site at the 3' end for directed subcloning.
The cfp9 oligonucleotides (TABLE 1 ) were synthesized partly on the basis of
the nu-
cleotide 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 Smal restriction enzyme site at the 5' end and a
Hindlll
restriction enzyme site at the 3' end for directed subcloning.
CFP7
By the use of PCR a Smal 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
ex-
pressed, and a BamHl site was incorporated right after the stop codon at the
3' end.
The 291 by PCR fragment was cleaved by Smal and BamHl, purified from an
agarose
gel and subcloned into the Smal - BamHl sites of the pMST24 expression vector.
Vec-
tor DNA containing the gene fusion was used to transform the E. coli XL1-Blue
(pRVN01 ).
CFP9
By the use of PCR a Smal 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
ex-
pressed, and a Hindlll site was incorporated after the stop codon at the 3'
end. The
327 by PCR fragment was cleaved by Smal and Hindlll, purified from an agarose
gel,
and subcloned into the Smai - Hindlll sites of the pQE-32 (QIAGEN) expression
vector.
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Vector DNA containing the gene fusion was used to transform the E. coli XL1-
Blue
(pRVN02).
Purification of recombinant CFP7 and CFP9
The ORFs were fused N-terminally to the (His)6-tag (cf. EP-A-0 282- 242).
Recombi-
nant antigen was prepared as fotlows: Briefly, a single colony of E. coli
harbouring
either the pRVN01 or the pRVN02 plasmid, was inoculated into Luria-Bertani
broth
containing 100 Ng/ml ampicillin and 12.5 ug/ml tetracycline and grown at
37°C to
OD6~°m = 0.5. IPTG (isopropyl-[3-D-thiogalactoside) was then added to a
final concen-
tration of 2 mM (expression was regulated either by the strong IPTG inducible
Pta~ or
the T5 promoter) and growth was continued for further 2 hours. The cells were
har-
vested by centrifugation at 4,200 x g at 4°C for 8 min. The pelleted
bacteria were
stored overnight at -20°C. The pellet was resuspended in BC 40/100
buffer (20 mM
Tris-HCI pH 7.9, 20% glycerol, 100 mM KCI, 40 mM lmidazole) and cells were
broken
by sonication (5 times for 30 s with intervals of 30 s) at 4°C.
followed by cenirifuga-
tion at 12,000 x g for 30 min at 4°C, the supernatant (crude extract)
was used for
purification of the recombinant antigens.
The two Histidine fusion proteins (His-rCFP7 and His-rCFP9) were purified from
the
crude extract by affinity chromatography on a Ni2r-NTA column from QIAGEN with
a
volume of 100 ml. His-rCFP7 and His-rCFP9 binds to Ni2+. After extensive
washes of
the column in BC 401100 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
KCI.
subsequently, the purified products were dialysed extensively against 10 mM
Tris pH
8Ø 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 NaCI from 0 to 1 M.
Aliquots
of the fractions were analyzed by 10%-20% gradient sodium dodecyl sulphate
poly-
acrylamide gel electrophoresis (SDS-PAGE). Fractions containing purified
either
purified His-rCFP7 or His-rCFP9 were pooled.
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TABLE 1. Sequence of the cfp7 and cfp9 oligonucleotidesa.
Orientation and Sequences (5' -~ 3') Position° (nucleo-
oligonucleotide tide)
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 CTACTAAGCTTGGATCCCTAGCCG- 381-362
CCCCATTTGGCGG (SEQ ID NO: 1 )
(SEQ ID NO: 45)
stF2 CTACTAAGCTTCCATGGTCAGGTC- 467 - 447
TTTTCGATGCTTAC (SEQ ID NO: 3)
(SEQ 1D NO: 46)
a The cfp7 oligonucfeotides were based on the nucleotide sequence shown in
Fig. 3
(SEQ ID NO: 1 ). The cfp9 oligonucleotides were based on the nucleotide
sequence
5 shown in Fig. 4 (SEQ ID NO: 3).
Nucleotides underlined are not contained in the nucleotide sequence of cfp7
and cfp9.
The positions referred to are of the non-underlined part of the primers and
corre-
spond to the nucleotide sequence shown in Fig. 3 and Fig. 4, respectively.
10 EXAMPLE 2A
Identification of antigens which are not expressed in BCG strains.
In an effort to control the treat of TB, attenuated bacillus Calmette-Guerin
(BCG) has
15 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
mutations) in
BCG are stable deletions and/or multiple mutations which do not readily
revert. While
20 physiological differences between BCG and M. tuberculosis and M, bovis has
been
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46
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
Ander-
sen, 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 RD 1 includes the gene encoding ESAT-6 and an otheF (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/DK94100273 and PCT/DK100270). In order to find new M. tuberculosis
specific
diagnostic antigens as well as antigens for a new vaccine against TB, the RD 1
region
(17.499 bp) of M. tuberculosis H37Rv has been analyzed for Open Reading Frames
(ORF). ORFs with a minimum length of 96 by have been predicted using the
algorithm
described by Borodovsky and Mclninch (1993), in total 27 ORFs have been
predicted,
of these have possible diagnostic and/or vaccine potential, as they are
deleted
15 from all known BCG strains. The predicted ORFs include ESAT-6 (RD1-ORF7)
and
CFP10 (RD1-ORF6) described previously (Srarensen 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.
Seven open reading frames (ORF) from the 17,499kb RD1 region (Accession no.
034848) with possible diagnostic and vaccine potential. have been identified
and
cloned.
Identification of the ORF's rdl-orf2, rd 1-orf3 rd 1-orf4 rd 1-orf5 rd 1-orf8
rd 1-orf9a,
and rdl-orf9b.
The nucleotide sequence of rd 1-orf2 from M. tuberculosis H37Rv is set forth
in SEQ
ID NO: 71. The deduced amino acid sequence of RD1-ORF2 is set forth in SEQ ID
NO:
72.
The nucleotide sequence of rd 1-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 1D
NO:
88.
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The nucleotide sequence of rd 1-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.
The nucleotide sequence of rd 1-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.
The nucleotide sequence of rd 1-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.
The nucleotide sequence of rd 1-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.
The nucleotide sequence of rd 1-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.
The DNA sequence rd 1-orf2 (SEQ ID NO: 71 ) contained an open reading frame
start-
ing 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
RD 1 ). The
deduced amino acid sequence (SEQ ID NO: 72) contains 591 residues
corresponding
to a molecular weight of 64,525.
The DNA sequence rdl-orf3 (SEQ 1D NO: 87) contained an open reading frame
start-
ing 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.
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The DNA sequence rdl-orf4 (SEQ ID NO: 89) contained an open reading frame
start-
ing with a GTG codon at position 4014 - 4012 and ending with a termination
codon
(TAG) at position 3597 - 3595 lposition numbers referring to the location in
RD 1 ). The
deduced amino acid sequence (SEQ ID NO: 90) contains 139 residues
corresponding -
to a molecular weight of 14,210.
The DNA sequence rdl-orf5 (SEQ ID NO: 91) contained an open reading frame
start-
ing 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 N0: 92) contains 371 residues
corresponding
to a molecular weight of 37,647.
The DNA sequence rdl-orf8 (SEQ ID NO: 67) contained an open reading frame
start-
ing with a GTG codon at positron 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
mo-
lecular weight of 1 1,737.
The DNA sequence rdl-orf9a (SEQ ID NO: 93) contained an open reading frame
starting with a GTG codon at position 6146 - 6148 and ending with a
termination co-
don (TAA) at position 7070 - 7072 (position numbers referring to the location
in RD1 ).
The deduced amino acid sequence (SEQ 1D NO: 94) contains 308 residues corre-
sponding to a molecular weight of 33,453.
The DNA sequence rdl-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
cor-
responding to a molecular weight of 70,650.
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Cloning of the ORF's rd1-orf2, rdl-orf3. rdl-orf4, rdl-orf5. rd1-orf8, rdl-
orf9a, and
rd 1-orf96.
The O R F's rd 1-orf2, rd 1-orf3, rd 1-orf4, rd 1-orf5, rd 1-orf8, rd 1-orf9a
and rd 1-orf9b
were PCR cloned in the pMST24 (Theisen et al., 1995) (rdl-orf3) or the pQE32
(QIAGEN) (rd l -orf2, rd l-orf4, rd l-orf5, rd l-orf8, rd 1-orf9a and rd l
orf96) 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.
tuber
culosis H37Rv was used as template in the PCR reactions. Oligonucleotides were
syn-
thesized on the basis of the nucleotide sequence from the RD1 region
(Accession no.
U34848). The oligonucleotide primers were engineered to include an restriction
en-
zyme site at the 5' end and at the 3' end by which a lafer subcloning was
possible.
Primers are listed in TABLE 2.
rdl-orf2. A BamHl site was engineered immediately 5' of the first codon of rdl-
orf2,
and a Hindlll site was incorporated right after the stop codon at the 3' end.
The gene
rdl-orf2 was subcloned in pQE32, giving pT096.
rd 1-orf3. A Smal site was engineered immediately 5' of the first codon of rd
1-orf3,
and a Ncol site was incorporated right after the stop codon at the 3' end. The
gene
rd1-orf3 was subcloned in pMST24, giving pT087.
rd 1-orf4. A BamHl site was engineered immediately 5' of the first codon of rd
1-orf4,
and a Hindlll site was incorporated right after the stop codon at the 3' end.
The gene
rd 1-orf4 was subcloned in pQE32, giving pT089.
rd1-orf5. A BamHl site was engineered immediately 5' of the first codon of rdl-
orf5,
and a Hindllt site was incorporated right after the stop codon at the 3' end.
The gene
rd 1-orf5 was subcloned in pQE32, giving pT088.
_ rdl-orf8. A BamHl site was engineered immediately 5' of the first codon of
rd1-orf8,
and a Ncol site was incorporated right after the stop codon at the 3' end. The
gene
rd1-orf8 was subcloned in pMST24, giving pT098.
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rdl-orf9a. A BamHl site was engineered immediately 5' of the first codon of
rd1-
orf9a, and a Hindlll site was incorporated right after the stop codon at the
3' end. The
gene rd 1-orf9a was subcloned in pQE32, giving pT091.
5 rd1-orf96. A Scal site was engineered immediately 5' of the first codon of
rdl-orf9b,
and a Hind llt site was incorporated right after the stop codon at the 3' end.
The gene
rdl-orf96 was subcloned in pQE32, giving pT090.
The PCR fragments were digested with the suitable restriction enzymes,
purified from
10 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.
Purification of recombinant RD1-ORF2, RD1-ORF3, RD1-ORF4, RD1-ORF5, RD1-ORFB,
15 RD1-ORF9a and RD1-ORF9b.
The rRD1-ORFs were fused N-terminally to the (His)6 -tag. Recombinant antigen
was
prepared as described in example 2 (with the exception that pT091 was
expressed at
30°C and not at 37°C?, using a single colony of E. coli
harbouring either the pT087,
20 pT088, pT089, pT090, pT091, pT096 or pT098 for inoculation. Purification of
re-
combinant antigen by Niz+ 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-ORFS, His-rRD1-ORFB, His-rRD1-ORF9a or His-rRD1-ORF9b were
pooled. The His-rRD1-ORF's were extensively dialysed against 10 mM Tris/HCI,
pH
25 8.5, 3 M urea followed by an additional purification step performed on an
anion ex-
change column (Mono Q) using fast protein liquid chromatography (FPLC)
(Pharmacia,
Uppsala, Sweden). The purification was carried out in 10 mM Tris/HCI, pH 8.5,
3 M
urea and protein was eluted by a linear gradient of NaCI from 0 to 1 M.
Fractions con-
taining the His-rRD1-ORF's were pooled and subsequently dialysed extensively
against
30 25 mM Hepes, pH 8.0 before use.
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Table 2. Sequence of the rd l-orf's oiigonucteotidesa.
Orientation and Sequences (5'--> 3') Position (nt)
oligo-
nucleotide
Sense
RD1-ORF2f CTGGGGATCCGCATGACTGCTGAACCG -886 - 903
RD1-ORF3f CTTCCCGGGATGGAAAAAATGTCAC 2807 - 2822
RD1-ORF4f GTAGGATCCTAGGAGACATCAGCGGC 4028 - 4015
RD1-ORFSf CTGGGGATCCGCGTGATCACCAT- 3028 - 3045
GCTGTGG
RD 1-ORF8f CTCGGATCCTGTGGGTGCAGGTCCGGC 5502 - 5479
GATGGGC
RD1-ORF9af GTGATGTGAGCTCAGGTGAAGAA- 6144 - 6160
GGTGAAG
RD1-ORF9bf GTGATGTGAGCTCCTATGGCGGCCGAC- 5072 - 5089
TACGAC
Antisense
RD1-ORF2r TGCAAGCTTTTAACCGGCGCTTGGGGGT 2664 - 2644
GC
RD1-ORF3r GATGCCATGGTTAGGCGAAGACGC- 3103 - 3086
CGGC
RD1-ORF4r CGATCTAAGCTTGGCAATGGAGGTCTA 3582 - 3597
RD1-ORFSr TGCAAGCTTTCACCAGTCGTCCT- 4243 - 4223
CTTCGTC
RD1-ORF8r CTCCCATGGCTACGACAAGCTCTTC- 5083 - 5105
CGGCCGC
RD1-ORF9a/br CGATCTAAGCTTTCAACGACGTCCAGCC 7073 - 7056
a The oligonucteotides were constructed from the Accession number U34484
nucleo-
tide sequence (Mahairas et al., 1996). Nucleotides (nt) underlined are not
contained in
the nucleotide sequence of RD1-ORF's. The positions correspond to the
nucleotide se-
quence of Accession number U34484.
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The nucleotide sequences of rd 1-orf2, rd 1-orf3, rd 1-orf4, rd 1-orf5, rd 1-
orf8, rd 1-
orf9a, and rdl-orf96 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 rd 1-
orf2,
rd 1-orf3, rd 1-orf4 rd 1-orf5, rd 1-orf8, rd 1-orf9a, and rd 1-orf96 are set
forth in SEQ ID
NO: 72, 88, 90, 92, 68, 94, and 70, respectively.
EXAMPLE 3
Cloning of the genes expressing 17-30 kDa antigens from ST CF
Isolation of CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28
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
pat-
terns 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 i-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.
Isolation of CFP29
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 Ng of CFP29 were used for each immunization. Mice were
boosted with antigen 3 days before removal of the spleen. Generation of a
monoclonal
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cell line producing antibodies against CFP29 was obtained essentially as
described by
Kohler and Milstein (1975). Screening of supernatants from growing clones was
car-
ried out by immunoblotting of nitrocellulose strips containing ST-CF separated
by SDS-
PAGE. Each strip contained approximately 50 Ng of ST-CF. The antibody class of
anti-
s CFP29 was identified as IgM by the mouse monoclonal antibody isotyping kit,
RPN29
(Amersham) according to the manufacturer's instructions. -
CFP29 was purified by the following method: ST-CF was concentrated 10 fold by
ul-
trafiltration, 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 am-
monium sulphate, pH 8.5, and subjected to thiophilic adsorption chromatography
(Po-
rath 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
West-
ern 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-HCI,
pH
8.5 and the elution was performed with a linear gradient from 0 to 500 mM
NaCI.
From 400 to 500 mM sodium chloride, rather pure CFP29 was eluted. As a final
puri-
fication 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).
N-terminal seguencing and amino acid analysis
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
mem-
brane. The membrane was washed 5 times with 20% methanol before sequencing on
a Procise sequencer (Applied Biosystems).
CFP29 containing fractions were blotted to PVDF membrane after tricine SDS-
PAGE
(Ploug et al., 19891. The i-elevant bands were excised and subjected to amino
acid
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analysis (Barkholt and Jensen, 1989) and N-terminal sequence analysis on a
Procise
sequencer (Applied Biosystems).
The following N-terminal sequences were obtained:
ForCFPI7:A/SELDAPAQAGTEXAV (SEQIDN0:17)
ForCFP20:AQITLRGNAINTVGE (SEQIDN0:18)
ForCFP2I:DPXSDIAVVFARGTH (SEQIDN0:19)
ForCFP22:TNSPLATATATLHTN (SEQIDN0:20)
ForCFP25:AXPDAEV VFARGRFE (SEQIDN0:21)
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)
ForCFP29:MN NLYRDLAPVTEA AWAEI (SEQIDN0:23)
"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.
Cloning the gene encoding CFP29
The N-terminal sequence of CFP29 was used for a homology search in the EMBL da-
tabase using the TFASTA program of the Genetics Computer Group sequence
analysis
software package. The search identified a protein, Linocin M 18, from
Brevibacterium
linens that shares 74% identity with the 19 N-terminal amino acids of CFP29.
Based on this identity between the N-terminal sequence of CFP29 and the
sequence
of the Linocin M 18 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 x low
salt
Taq + buffer from Stratagene supplemented with 250 ~M of each of the four
nucleo- -
tides (Boehringer Mannheim), 0,5 mg/ml BSA (IgG technologyl. 1 % DMSO (Merck),
5
pmoles of each primer and 0.5 unit Tag+ DNA polymerase (Stratagene) in 10 ~I
reac- '
tion volume. Reactions were initially heated to 94°C for 25 sec. and
run for 30 cycles
of the program; 94°C for 15 sec., 55°C for 15 sec. and
72°C for 90 sec, using ther-
mocycler equipment from Idaho Technology.
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An approx. 300 by fragment was obtained using primers with the sequences:
1: 5'-CCCGGCTCGAGAACCTSTACCGCGACCTSGCSCC (SEQ ID NO: 24)
5 2:5'-GGGCCGGATCCGASGCSGCGTCCTTSACSGGYTGCCA
(SEQ-ID NO: 25)
-where S = GlC and Y = T/C
The fragment was excised from a 1 % agarose gel, purified by Spin-X spine
columns
10 (Costar), cloned into pBluescript SK II + - T vector (Stratagene) and
finally sequenced
with the Sequenase kit from United States Biochemical.
The first 150 by of this sequence was used for a homology search using the
Blast
program of the Sanger Mycobacterium tuberculosis database:
(http//www.sanger.ac.uk/projects/M-tuberculosis/blast_server).
This program identified a Mycobacterium tuberculosis sequence on cosmid cy444
in
the database that is nearly 100% identical to the 150 by sequence of the CFP29
pro-
tein. The sequence is contained within a 795 by 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.
Finally, the 795 by open reading frame was PCR cloned under the same PCR condi-
tions as described above using the primers:
3: 5'-GGAAGCCCCATATGAACAATCTCTACCG (SEQ ID NO: 26)
4: 5'-CGCGCTCAGCCCTTAGTGACTGAGCGCGACCG (SEQ ID NO: 27)
The resulting DNA fragments were purified from agarose gels as described above
se-
quenced with primer 3 and 4 in addition to the following primers:
5: 5'-GGACGTTCAAGCGACACATCGCCG-3' (SEQ ID NO: 115)
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6: 5'-CAGCACGAACGCGCCGTCGATGGC-3' (SEQ ID NO: 1 16)
Three independent cloned were sequenced. All three clones were in 100%
agreement
with the sequence on cosmid cy444.
All other DNA manipulations were done according to Maniatis et al. E7 989).
All enzymes other than Taq polymerase were from New England Biolabs.
Homology searches in the Sanger database
For CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28 the N-terminal amino acid se-
quence from each of the proteins were used for a homology search using the
blast
program of the Sanger Mycobacterium tuberculosis database:
http://www.sanger.ac.uk/pathogenslTB-blast-server.html.
For CFP29 the first 150 by of the DNA sequence was used for the search.
Further-
more, the EMBL database was searched for proteins with homology to CFP29.
Thereby, the following information were obtained:
CFP17
Of the 14 determined amino acids in CFP17 a 93% identical sequence was found
with
MTCY 1 A 1 1.16c. 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 MTCY1A1
1.
From the N-terminal sequencing it was not possible to determine amino acid
number
13.
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 agree-
meat 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
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molecular mass of 13833 Da and a theoretical pl of 4.4. The observed mass in
SDS-
PAGE is 17 kDa.
CFP20
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 pl of 4.2. The
ob-
served molecular weight in a SDS-PAGE is 20 kDa.
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
tong translated protein.
The highest homology, 51.5% identity in a 163 amino acid overlap, was found to
a
Haemophilus influenza Rd toxR reg. (HIH10751 ).
CFP21
A sequence 100% 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.
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 cor-
responds to a theoretical molecular weigh at 18657 Da, and a theoretical pl at
4,6.
The observed weight in a SDS-PAGE is 21 kDa.
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).
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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)
(Mahai-
ras et al. (1996)).
CFP22
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 cul-
ture filtrate is 175 amino acids. This gives a theoretical molecular weigh at
18517 Da
and a pl at 6.8. The observed weight in a SDS-PAGE is 22 kDa.
In an 182 amino acids overlap the translated protein has 90,1 % identity with
E235739; a peptidyl-prolyl cis-trans isomerase.
CFP25
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.
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 pl
at 4.9.
The observed weight in a SDS-PAGE is 25 kDa.
In a 217 amino acids overlap the protein has 42.9% identity to CFP21
(MTCY39.35).
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CFP28
No homology was found when using the 10 determined amino acid residues 2-8, 1
1,
12, and 14 of SEQ ID NO: 22 in the database search.
CFP29
Sanger database searching: A sequence nearly 100% identical to the 150 by
sequen-
ce of the CFP29 protein was found on cosmid cy444. The sequence is contained
within a 795 by 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.
The amino acid analysis performed on the purified protein further confirmed
the iden-
tity of CFP29 with the protein encoded in open reading frame on cosmid 444.
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 levely. This is a 28.6 kDa protein with bacteriocin activity (Valdes-
Stauber and
Scherer, 1994; Valdes-Stauber and Scherer, 1996). The two proteins have the
same
length (except far 1 amino acid) and share the same theoretical
physicochemical pro-
perties. We therefore suggest that CFP29 is a mycobacterial homolog to the
Brevibac-
terium linens Linocin M 18 protein.
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.
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
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CFP20 (SEQ ID NO: 8):
1 MAQITLRGNA INTVGELPAV GSPAPAFTLT GGDLGVISSD QFRGKSVLLN
5 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 MTSOAATFAA NRLDHAG
CFP22 (SEQ ID NO: 12):
1 MADCDSVTNS PLATATATLH TNRGDIKiAL FGNHAPKTVA NFVGLAQGTK
51 DYSTQNASGG PSGPFYDGAV FHRVIQGFMI QGGDPTGTGR GGPGYKFADE
101 FHPELQFDKP YLLAMANAGP GTNGSQFFIT VGKTPHLNRR HTIFGEVIDA
151 ESQRVVEAIS KTATDGNDRP TDPVVIESIT IS
CFP25 (SEQ ID NO: 14):
1 MGAAAAMLAA VLLLTPITVP AGYPGAVAPA 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
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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
For all six proteins the molecular weights predicted from the sequences are in
agree-
ment with the molecular weights observed on SDS-PAGE.
Cloning of the genes encoding CFP17, CFP20 CFP21 CFP22 and CFP25
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 x low
salt
Taq + buffer from Stratagene supplemented with 250 mM of each of the four
nucleo-
tides (Boehringer Mannheim), 0,5 mg/ml BSA (IgG technology), 1 % DMSO (Merck),
5
pmoles of each primer and 0.5 unit Tag+ DNA polymerise (Stratagene) in 10 pl
reac-
Lion volume. Reactions were initially heated to 94°C for 25 sec. and
run for 30 cycles
according to the following program; 94°C for 10 sec., 55°C for
10 sec. and 72°C for
90 sec, using thermocycler equipment from Idaho Technology.
The DNA fragments were subsequently run on 1 % agarose gels, the bands were ex-
cised and purified by Spin-X spin columns (Costar) and cloned into pBluescript
SK 11 +
- 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
sequen-
ced by use of the dideoxy chain termination method adapted for supercoiled DNA
us-
ing 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
instruc-
tions provided. Both strands of the DNA were sequenced.
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s2
For cloning of the individual antigens, the following gene specific primers
were used:
CFP17: Primers used for cloning of cfp17:
OPBR-51: ACAGATCTGTGACGGACATGAACCCG (SEQ ID NO: 1 17)
OPBR-52: TTTTCCATGGTCACGGGCCCCCGGTACT (SEQ ID NO: 118)
OPBR-51 and OPBR-52 create Bglll and Ncol 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 Bglll and HinDlll 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 Bglll and Ncol sites, respectively, used for the
cloning
in pMCT6.
CFP22: Primers used for cloning of cfp22:
OPBR-57. ACAGATCTGTAATGGCAGACTGTGAT (SEQ ID NO: 123)
OPBR-58: TTTTCCATGGTCAGGAGATGGTGATCGA (SEQ ID NO: 124) -
OPBR-57 and OPBR-58 create Bglll and Ncol sites, respectively, used for the
cloning
in pMCT6.
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CFP25: Primers used for cloning of cfp25:
OPBR-59: ACAGATCTGCCGGCTACCCCGGTGCC (SEQ iD NO: 125)
OPBR-60: TTTTCCATGGCTATTGCAGCTTTCCGGC (SEQ ID NO: 126)
OPBR-59 and OPBR-60 create Bglll and Ncol sites, respectively, used for the
cloning
in pMCT6.
Expression/purification of recombinant CFP17, CFP20, CFP21, CFP22 and CFP25
pro-
teins.
Expression and metal affinity purification of recombinant proteins was
undertaken es-
sentially as described by the manufacturers. For each protein, 1 I LB-media
containing
100 Ng/ml ampicillin, was inoculated with 10 ml of an overnight culture of XL1-
Blue
cells harbouring recombinant pMCT6 plasmids. Cultures were shaken at 37
°C until
they reached a density of ODsoo = 0.4 - 0.6. IPTG was hereafter added to a
final con-
centration of 1 mM and the cultures were further incubated 4 - 16 hours. Cells
were
harvested, resuspended in 1 X sonication buffer + 8 M urea and sonicated 5 X
30
sec. with 30 sec. pausing between the pulses.
After centrifugation, the lysate was applied to a column containing 25 ml of
resus-
pended Talon resin (Clontech, Palo Alto, USA). The column was washed and
eluted as
described by the manufacturers.
After elution, all fractions (1.5 ml each) were subjected to analysis by SDS-
PAGE us-
ing the Mighty Small (Hoefer Scientific Instruments, USA) system and the
protein con-
centrations were estimated at 280 nm. Fractions containing recombinant protein
were
pooled and dialysed against 3 M urea in 10 mM Tris-HCI, 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 NaCI. Fractions were analyzed by SDS-
PAGE
and protein concentrations were estimated at ODZ8o. Fractions containing
protein
were pooled and dialysed against 25 mM Hepes buffer, pH 8.5.
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Finally the protein concentration and the LPS content were determined by the
BCA
(Pierce, Holland) and LAL (Endosafe, Charleston, USA) tests, respectively.
EXAMPLE 3A
Identification of CFP7A, CFPBA, CFPBB, CFP 16, CFP 19, CFP 19B, CFP22A,
CFP23A,
CFP23B, CFP25A, CFP27, CFP30A, CWP32 and CFP50.
Identification of CFP16 and CFP19B.
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 % (vlv) Biolyt 4/6
(Bio-
Rad). 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
pat-
terns 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 mi. 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.
Isolation of CFP8A, CFPBB, CFP19, CFP23A, and CFP23B.
ST-CF was precipitated with ammonium sulphate at 80% saturation and
redissolved in
PBS, pH 7.4, and dialysed 3 times against 25mM Piperazin-HCI, 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-HCI,
pH
5.5, and the elution was performed with 10% PB74-HCI, pH 4.0 (Pharmacia). Frac-
tions with similar band patterns were pooled and washed three times with PBS
on a
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Centriprep concentrator (Amicon) with a 3 kDa cut oft membrane to a final
volume of
1-3 ml and separated on a Prepcell as described above.
Identification of CFP22A
5
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
10 on top of 6.4 ml of 48 % (wlv) sucrose in PBS, pH 7.4, in Sorvall tubes
(Ultracrimp
03987, DuPont Medical Products) and ultracentrifuged for 20 h at 257,300 x
gmax.
10°C. The pellet was redissolved in 200 NI of 25 mM Tris-192 mM
glycine, 0.1
SDS, pH 8.3.
15 Identification of CFP7A, CFP25A, CFP27, CFP30A and CFP50
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 ammo-
20 nium 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 gra-
dient of ammonium sulphate. Fractions with simitar band patterns in SDS-PAGE
were
pooled and anion exchange chromatography was performed on a Mono Q HR 5/5 col-
umn connected to an FPLC system (Pharmacia). The column was equilibrated with
10
25 mM Tris-HCI, pH 8.5, and the elution was performed with a gradient of NaCI
from 0
to 1 M. Fractions containing well separated bands in SDS-PAGE were selected.
CFP7A and CFP25A were obtained as described above except far the following
modi-
fication: ST-CF was concentrated approximately 10 fold by ultrafiltration and
proteins
30 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.
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Isolation of CWP32
Heat treated H37Rv was subfractionated into subcellular fractions as described
in Se-
rensen et al 1995. The Cell wall fraction was resuspended in 8 M urea, 0.2 %
(w/v)
N-octyl (3-p gfucopyranoside (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. Isoetectric focusing was performed in a pH gradient from 3-6. The
frac-
tions were analyzed by SDS-PAGE and fractions containing well separated bands
were
polled and subjected to N-terminal sequencing after transfer to PVDF membrane.
N-terminal seguencing
Fractions containing CFP7A, CFPBA, CFPBB, CFP16, CFP19, CFP19B, CFP22A,
CFP23A, CFP23B, CFP27, CFP30A, CWP32, and CFP50A were blotted to PVDF
membrane after Tricine SDS-PAGE IPloug et al, 1989). The relevant bands were
ex-
cised and subjected to N-terminal amino acid sequence analysis on a Procise
494 se-
quencer (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
de-
scribed above.
The following N-terminal sequences were obtained:
CFP7A: AEDVRAEIVA SVLEVVVNEG DQIDKGDVVV
LLESMYMEIP
VLAEAAGTVS (SEQ ID NO: 81 )
CFPBA: DPVDDAFtAKLNTAG (SEQ ID NO: 73)
CFPBB: DPVDAtINLDNYGX (SEQ ID NO: 74)
CFP16: AKLSTDELLDAFKEM (~EQ ID NO: 79)
CFP19: TTSPDPYAALPKLPS (SEQ ID NO: 82) -
CFP19B: DPAXAPDVPTAAQLT (SEQ ID NO: 80)
CFP22A: TEYEGPKTKF HALMQ (SEQ tD NO: 83)
CFP23A: VIQ/AGMVT/GHIHXVAG (SEQ ID NO: 76)
CFP23B: AEMKXFKNAIVQEtD (SEQ tD NO: 75)
CFP25A: AIEVSVLRVF TDSDG (SEQ ID NO: 78)
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CWP32: TNIVVLIKQVPDTWS (SEQ ID NO: 77)
CFP27: TTIVALKYPG GVVMA (SEQ ID NO: 84)
CFP30A: SFPYFISPEX AMRE (SEO ID NO: 85)
CFP50: THYDVVVLGA GPGGY (SEQ ID NO: 86)
N-terminal homology searching in the Sanger database and identification of the
corre-
sponding genes.
The N-terminal amino acid sequence from each of the proteins was used for a
homo-
logy search using the blast program of the Sanger Mycobacterium tuberculosis
data-
base:
http://www.Banger.ac.uk/projects/m-tuberculosis/TB-blast-server.
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 ge-
nome 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.
For CFP7A, CFPBA, CFPBB, CFP16, CFP19, CFP19B , CFP22A, CFP25A, CFP27,
CFP30A, CWP32, and CFP50, the following information was obtained:
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 = 49150 (98%), Positives = 49/50 (98%), Frame = -i
Query: 1 AEDVRAEIVASVLEVVVNEGDQIDKGDVVVLLESMYMEIPVLAEAAGTVS
30 AEDVRAEIVASVLEVVVNEGDQIDKGDVVVLLESM MEIPVLAEAAGTVS
Sbjct: 257679 AEDVRAEIVASVLEVVVNEGDOIDKGDVVVLLESMKMEIPVLAEAAGTVS
257530
(SEQ ID NOs: 127, 128, and 129)
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The identity is found within an open reading frame of 71 amino acids length
corre-
sponding to a theoretical MW of CFP7A of 7305.9 Da and a pl of 3.762. The ob-
served molecular weight in an SDS-PAGE gel is 7 kDa.
CFPBA: A sequence 80% identical to the 15 N-terminal amino acids-was found on
coniig TB-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 pl 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 12989 Da and a pl of 4.38.
CFPBB: A sequence 71 % identical to the 14 N-terminal amino acids was found on
contig TB_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 pl 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
pres-
ence of a signal peptide which has been cleaved of the mature protein found in
culture
filtrate.
CFP16: The 15 as N-terminal sequence was found to be 100% identical to a
sequence found on cosmid MTCY20H1.
The identity is found within an open reading frame of 130 amino acids length
corre-
sponding to a theoretical MW of CFP16 of 13440.4 Da and a pl of 4.59. The ob-
served molecular weight in an SDS-PAGE gel is 16 kDa.
CFP19: The 15 as N-terminal sequence was found to be 100% identical to a
sequence found on cosmid MTCY270.
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The identity is found within an open reading frame of 176 amino acids length
corre-
sponding to a theoretical MW of CFP19 of 18633.9 Da and a pl of 5.41 . The ob-
served molecular weight in an SDS-PAGE gel is 19 kDa.
CFP22A: The 15 as N-terminal sequence was found to be 100% identical to a se-
quence found on cosmid MTCY 1 A6.
The identity is found within an open reading frame of 181 amino acids length
corre-
sponding to a theoretical MW of CFP22A of 20441.9 Da and a pl of 4.73. The ob-
served molecular weight in an SDS-PAGE gel is 22 kDa.
CFP25A: The 15 as N-terminal sequence was found to be 100% identical to a se-
quence found on contig 255.
The identity is found within an open reading frame of 228 amino acids length
corre-
sponding to a theoretical MW of CFP25A of 24574.3 Da and a pl of 4.95. The ob-
served molecular weight in an SDS-PAGE gel is 25 kDa.
CFP27: The 15 as N-terminal sequence was found to be 100% identical to a
sequence found on cosmid MTCY261.
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
pl at
4.64. The observed weight in an SDS-PAGE gel is 27 kDa.
CFP30A: Of the 13 determined amino acids in CFP30A, a 100% identical sequence
was found on cosmid MTCY261.
The identity is found within an open reading frame of 248 amino acids length
corre-
sponding to a theoretical MW of CFP30A of 26881.0 Da and a pl of 5.41. The ob-
served molecular weight in an SDS-PAGE gel is 30 kDa.
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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 pl of 4.563. The observed molecular weight in an SDS-PAGE gel is 32 kDa.
5
CFP50: The 15 as 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 pl of 5.66. The observed molecular weight in an SDS-PAGE gel is 50 kDa.
Use of homology searchinca in the EMBL database for identification of CFP19A
and
CFP23.
Homology searching in the EMBL database (using the GCG package of the Biobase,
0
Arhus-DK) with the amino acid sequences of two earlier identified highly
immunoreac-
tive ST-CF proteins, using the TFASTA algorithm, revealed that these proteins
tCFP21
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, MTCY13E12.04 has 46% and 50% identity
to CFP25 and CFP21 respectively. The second, MTCY13E12.05, has also 46% and
50% identity to CFP25 and CFP21. The two proteins share 62.5% as 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.
The first reading frame encodes a 254 amino acid protein of which the first 26
as
constitute a putative leader peptide that strongly indicates an extracellular
location of
the protein. The mature protein is thus 228 as in length corresponding to a
theoretical
MW of 23149.0 Da and a Pi of 5.80. The protein is named CFP23.
The second reading frame encodes an 231 as protein of which the first 44 as
consti-
tute a putative leader peptide that strongly indicates an extracellular
location of the
protein. The mature protein is thus 187 as in length corresponding to a
theoretical
MW of 19020.3 Da and a Pi of 7.03. The protein is named CFP19A.
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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
(http:/lexpasy.hcuge.chlwwwltools.html). -
Searching for homologies to CFP7A, CFP16, CFP19, CFP19A, CFP19B, CFP22A,
CFP23, CFP25A, CFP27, CFP30A, CWP32 and CFP50 in the EMBL database.
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
func-
tional roles of the antigens.
CFP7A: CFP7A has 44% identity and 70% similarity to hypothetical Methanococcus
jannaschii protein (M. jannaschii from base 1 162199-1 175341 ), as well as
43% and
38% identity and 68 and 64% similarity to the C-terminal part of B.
stearothermophi-
lus pyruvate carboxylase and Streptococcus mutans biotin carboxyl carrier
protein.
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
incuba-
tion with alkaline phosphatase conjugated streptavidin after SDS-PAGE and
transfer to
nitrocellulose it was demonstrated that native CFP7A was biotinylated.
CFP16: RAIL gene, 130 aa. Identical to the M. bovis 50s ribosomal protein
L7/L12
(acc. No P37381 ).
CFP19: CFP19 has 47% identity and 55% similarity to E.coli pectinesterase
homolog
(ybhC gene) in a 150 as overlap.
CFP19A: CFP19A has between 38% and 45% identity to several cutinases from dif-
ferent fungal sp.
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In addition CFP19A has 46% identity and 61 % similarity to CFP25 as well as
50%
identity and 64% similarity to CFP21 (both proteins are earlier isolated from
the ST-
CF).
CFP19B: No apparent homology
CFP22A: No apparent homology
CFP23: CFP23 has between 38% and 46% identity to several cutinases from
different
fungal sp.
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).
CFP25A: CFP25A has 95% identity in a 241 as overlap to a putative M.
tuberculosis
thymidylate synthase (450 as accession No p28176).
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).
CFP30A: CFP30A has 67% identity to Rhodococcus proteasome alfa-type 1 subunit.
CWP32: The CWP32 N-terminal sequence is 100% identical to the Mycobacterium
leprae sequence MLCB637.03.
CFP50: The CFP50 N-terminal sequence is 100% identical to a putative lipoamide
de-
hydrogenase from M. leprae (Accession 415183)
Cloning of the genes encoding CFP7A, CFPBA, CFPBB, CFP16 CFP19 CFP19A,
CFP22A, CFP23, CFP25A, CFP27, CFP30A, CWP32, and CFP50.
The genes encoding CFP7A, CFPBA, CFP8B, CFP16, CFP19, CFP19A, CFP22A,
CFP23, CFP25A, CFP27, CFP30A, CWP32 and CFP50 were all cloned into the ex-
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pression vector pMCT6, by PCR amplification with gene specific primers, for
recombi-
nant expression in E. coli of the proteins.
PCR reactions contained 10 ng of M. tuberculosis chromosomal DNA in 1 X low
salt
Taq+ buffer from Stratagene supplemented with 250 mM of each of the four
nucleo-
tides (Boehringer Mannheim), 0,5 mg/ml BSA (IgG technology), 1 % DMSO (Merck),
5
pmoles of each primer and 0.5 unit Tag + DNA poiymerase (Stratagene) in 10 ml
reac-
tion volume. Reactions were initially heated to 94°C for 25 sec. and
run for 30 cycles
of the program; 94°C for 10 sec., 55°C for 10 sec. and
72°C for 90 sec, using ther-
mocycler equipment from Idaho Technology.
The DNA fragments were subsequently run on 1 % agarose gels, the bands were ex-
cised 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 Oye 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.
For cloning of the individual antigens, the following gene specific primers
were used:
CFP7A: Primers used for cloning of cfp7A:
OPBR-79: AAGAGTAGATCTATGATGGCCGAGGATGTTCGCCz. (SEQ ID NO:
95)
OPBR-80: CGGCGACGACGGATCCTACCGCGTCGG (SEQ ID NO: 96)
OPBR-79 and OPBR-80 create Bglll and BamHl sites, respectively, used for the
cloning
in pMCT6.
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CFPBA: Primers used for cloning of cfpBA:
CFPBA-F: CTGAGATCTATGAACCTACGGCGCC (SEa ID NO: 154)
CFPBA-R: CTCCCATGGTACCCTAGGACCCGGGCAGCCCCGGC (SEQ ID NO:
155)
CFPBA-F and CFPBA-R create Bglll and Ncol sites, respectively, used for the
cloning in
pMCT6.
CFPBB: Primers used for cloning of cfp88:
CFPBB-F: CTGAGATCTATGAGGCTGTCGTTGACCGC . (SEQ ID NO: 156)
CFPBB-R: CTCCCCGGGCTTAATAGTTGTTGCAGGAGC (SEQ ID NO. 157)
CFPBB-F and CFPBB-R create Bglll and Smal sites, respectively, used for the
cloning in
pMCT6.
CFP16: Primers used for cloning of cfpl6:
OPBR-104: CCGGGAGATCTATGGCAAAGCTCTCCACCGACG
(SEQ ID NOs: 1 1 1 and 130)
OPBR-105: CGCTGGGCAGAGCTACTTGACGGTGACGGTGG
(SEQ ID NOs: 1 12 and 131 )
OPBR-104 and OPBR-105 create Bglll and Ncol sites, respectively, used for the
clon-
ing in pMCT6.
CFP19: Primers used for cloning of cfpl9:
OPBR-96: GAGGAAGATCTATGACAACTTCACCCGACCCG
(SEQ ID NO: 107)
OPBR-97. CATGAAGCCATGGCCCGCAGGCTGCATG
(SEQ ID NO: 108?
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OPBR-96 and OPBR-97 create Bglll and Ncol sites, respectively, used for the
cloning
in pMCT6.
CFP19A: Primers used for cloning of cfp19A:
5
OPBR-88: CCCCCCAGATCTGCACCACCGGCATCGGCGGGC -
(SEQ ID NO: 99)
OPBR-89. GCGGCGGATCCGTTGCTTAGCCGG (SEQ ID NO: 100)
10 OPBR-88 and OPBR-89 create Bglll and BamHl sites, respectively, used for
the cloning
in pMCT6.
CFP22A: Primers used for cloning of cfp22A:
15 OPBR-90: CCGGCTGAGATCTATGACAGAATACGAAGGGC
(SEQ ID NO: 101 )
OPBR-91: CCCCGCCAGGGAACTAGAGGCGGC (SEQ ID NO: 102)
OPBR-90 and OPBR-91 create Bglll and Ncol sites, respectively, used for the
cloning
20 in pMCT6.
CFP23: Primers used for cloning of cfp23:
OPBR-86: CCTTGGGAGATCTTTGGACCCCGGTTGC
25 (SEQ ID NO: 97)
OPBR-87: GACGAGATCTTATGGGCTTACTGAC (SEQ 1D NO: 98)
OPBR-86 and OPBR-87 both create a Bglll site used for the cloning in pMCT6.
30 CFP25A: Primers used for cloning of cfp25A:
OPBR-106: GGCCCAGATCTATGGCCATTGAGGTTTCGGTGTTGC
(SEQ ID NO: 113)
OPBR-107: CGCCGTGTTGCATGGCAGCGCTGAGC (SEQ ID NO: 1 14)
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OPBR-106 and OPBR-107 create BgRI and Ncol sites, respectively, used for the
clon-
ing 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 Bglll and Ncol sites, respectively, used for the
cloning in
pMCT6.
CFP30A: Primers used for cloning of cfp30A:
OPBR-94: GGCGGAGATCTGTGAGTTTTCCGTATTTCATC
ISEQ ID NO: 105)
OPBR-95: CGCGTCGAGCCATGGTTAGGCGCAG (SEQ ID NO: 106)
OPBR-94 and OPBR-95 create Bglll and Ncol sites, respectively, used for the
cloning in
pMCT6.
CWP32: Primers used for cloning of cwp32:
CWP32-F: GCTTAGATCTATGATTTTCTGGGCAACCAGGTA
(SEQ ID NO: 158)
CWP32-R: GCTTCCATGGGCGAGGCACAGGCGTGGGAA (SEQ 1D NO: 1591
CWP32-F and CWP32-R create Bg/11 and Ncol sites, respectively, used for the
cloning
in pMCT6.
CFP50: Primers used for cloning of cfp50:
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OPBR-100: GGCCGAGATCTGTGACCCACTATGACGTCGTCG
(SEQ ID NO: 109)
OPBR-101: GGCGCCCATGGTCAGAAATTGATCATGTGGCCAA
(SEQ ID NO: 110)
OPBR-100 and OPBR-101 create Bglll and Ncol sites, respectively, used for the
clon-
ing in pMCT6.
Expression/purification of recombinant CFP7A, CFPBA, CFP8B CFP16 CFP19
CFP19A. CFP22A, CFP23, CFP25A, CFP27, CFP30A, CWP32 and CFP50 proteins
Expression and metal affinity purification of recombinant proteins was
undertaken es-
sentially as described by the manufacturers. For each protein, 1 I LB-media
containing
100 ug/ml ampicillin, was inoculated with 10 ml of an overnight culture of XL1-
Blue
cells harbouring recombinant pMCT6 plasmids. Cultures were shaken at
37°C until
they reached a density of OD6~ = 0.4 - 0.6. IPTG was hereafter added to a
final con-
centration of 1 mM and the cultures were further incubated 4-16 hours. Cells
were
harvested, resuspended in 1 X sonication buffer + 8 M urea and sonicated 5 X
30 sec.
with 30 sec. pausing between the pulses.
After centrifugation, the iysate was applied to a column containing 25 ml of
resus-
pended Talon resin (Clontech, Palo Alto, USA). The column was washed and
eluted as
described by the manufacturers.
After elution, all fractions (1.5 ml each) were subjected to analysis by SDS-
PAGE us-
ing the Mighty Small (Hoefer Scientific Instruments, USA) system and the
protein con-
centrations were estimated at 280 nm. Fractions containing recombinant protein
were
pooled and dialysed against 3 M urea in 10 mM Tris-HCI, pH 8.5. The dialysed
protein
was further purified by FPLC (Pharmacia, Sweden) using a 6 ml Resource-Q
column,
eluted with a linear O-1 M gradient of NaCI. Fractions were analyzed by SDS-
PAGE
and protein concentrations were estimated at ODZeo. Fractions containing
protein were
pooled and dialysed against 25 mM Hepes buffer, pH 8.5.
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Finally the protein concentration and the LPS content were determined by the
BCA
(Pierce, Holland) and LAL (Endosafe, Charleston, USA) tests, respectively.
EXAMPLE 3B
Identification of CFP7B, CFP10A, CFPI 1 and CFP30B.
Isolation of CFP7B
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-HCI, pH 5.5, and
subject-
ed 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-HCI,
pH
5.5, and the elution was performed with 10% PB74-HCI, pH 4.0 (Pharmacia). Frac-
tions 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
'!-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,l., 1993). The fraction
containing a
well separated band below 10 kDa was selected for N-terminal sequencing after
trans-
fer to a PVDF membrane.
Isolation of CFP1 1
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 % (wlv)
and 5%
(v/v) respectively and the protein solution was applied to a Rotofor
isoelectrical Csll
(BioRadl. The Rotofor Cell had been equilibrated w'tth an 8M urea buffer
containing
0.5 % (w/v) CHAPS, 5% (viv) glycerol, 3% (v!v) Biolyt 3/5 and 1 % (vlv) 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
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(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
occur-
ring on the membrane was excised and submitted for N-terminal sequencing.
Isolation of CFP10A and CFP30B
ST-CF was concentrated approximately 10-fold by ultrafiltration and ammonium
sul-
phate 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
pat-
terns 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-HCI, pH 8.5, and the elution was performed
with a
gradient of NaCI from 0 to 1 M. Fractions containing well separated bands in
SDS-
PAGE were selected.
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-
termi-
nal amino acid sequence analysis.
N-terminal seauencing
N-terminal amino acid sequence analysis was performed on a Procise 494
sequencer
(applied Biosystems).
The following N-terminal sequences were obtained:
CFP7B: PQGTVKWFNAEKGFG (SEQ ID NO: 168)
CFP10A: NVTVSIPTILRPXXX (SEQ ID NO: 169)
CFP1 1: TRFMTDPHAMRDMAG (SEQ lD NO: 170)
CFP30B: PKRSEYRQGTPNWVD (SEQ ID NO: 171 )
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"X" denotes an amino acid which could not be determined by the sequencing
method
used.
N-terminal homolo4y searching in the Sanger database and identification of the
corre- '
5 s~ondinc,~genes.
The N-terminal amino acid sequence from each of the proteins was used for a
homo-
logy search using the blast program of the Sanger Mycobacterium tuberculosis
ge-
nome database:
http//www.sanger.ac.uklprojectslm-tuberculosis/TB-blast-server.
For CFP1 1 a sequence 100% identical to 15 N-terminal amino acids was found on
contig TB_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 pl of 5.14.
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_671 and on locus MTC1364.09.
For CFP7B a sequence 100% identical to 15 N-terminal amino acids was found on
contig TB-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
corre-
sponding to a theoretical MW of 7240 Da and a pl of 5.18.
For CFP10A a sequence 100% identical to 12 N-terminal amino acids was found on
contig TB 752 and on locus CY130.20 with EMBL accession number: Q10646 and
273902. The identity was found within an open reading frame of 93 amino acids
length corresponding to a theoretical MW of 9557 Da and a pl of 4.78. -
For CFP30B a sequence 100% identical to 15 N-terminal amino acids was found on
.
contig TB 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 pl of 4.24.
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The amino acid sequences of the purified antigens as picked from the Sanger
database
are shown in the following list.
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
CFP1 1 protein sequence (SEQ ID NO: 143)
1 MATRFMTDPH AMRDMAGRFE VHAQTVEDEA RRMWASAQNI SGAGWSGMAE
51 ATSLDTMAOM 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
20i VPNHWHVYFA VDDADATAAK AAAAGGQV1A EPADIPSVGR FAVLSDPQGA
251 IFSVLKPAPQ Q
Cloning of the 4enes encoding CFP7B, CFP10A, CFP11. and CFP30B.
PCR reactions contained 10 ng of M. tuberculosis chromosomal DNA in 1 X low
salt
Taq + buffer from Stratagene supplemented with 250 mM of each of the four
nucleo-
tides (Boehringer Mannheim!. 0,5 mg/ml BSA (IgG technology), 1 % DMSO (Merck),
5
pmoies of each primer and 0.5 unit Tag + DNA polymerase (Stratagene) in 10 ml
reac-
tion volume. Reactions were initially heated to 94°C for 25 sec. and
run for 30 cycles
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of the program; 94°C for 10 sec., 55°C for 10 sec. and
72°C for 90 sec., using ther-
mocycler equipment from Idaho Technology.
The DNA fragments were subsequently run on 1 % agarose geis, the bands were ex-
cised and purified by Spin-X spin columns (Costar) and cloned into pBluscript
SK ll + -
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.
For cloning of the individual antigens, the following gene specific primers
were used:
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 Bglfl and Smal sites, respectively, used for the
cloning in
pMCT6.
CFP10A: Primers used for cloning of cfplOA:
CFP10A-F: CTGAGATCTATGAACGTCACCGTATCC (SEQ ID NO: 162)
CFP10A-R: TCTCCCGGGGCTCACCCACCGGCCACG ISEQ ID NO: 163)
CFP10A -F and CFP10A -R create Bglll and Smal sites, respectively, used for
the
cloning in pMCT6.
CFP1 1: Primers used for cloning of cfpll
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CFP11-F: CTGAGATCTATGGCAACACGTTTTATGACG (SEQ ID NO: 164)
CFP11-R: CTCCCCGGGTTAGCTGCTGAGGATCTGCTH (SEQ ID NO: 165)
CFP1 1-F and CFP1 1-R create Bglll and Smal sites, respectively, used for the
cloning in
pMCT6.
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 BgAI and Pvull sites, respectively, used for the
cloning
in pMCT6.
Expression/purification of recombinant CFP7B. CFP10A, CFP11 and CFP30B
protein.
Expression and metal affinity purification of recombinant protein was
undertaken es-
sentially as described by the manufacturers. 1 I LB-media containing 100 Nglml
ampi-
cillin, was inoculated with 10 ml of an overnight culture of XL1-Blue cells
harbouring
recombinant pMCT6 plasmid. The culture was shaken at 37 °C until it
reached a den-
sity of OD6~ = 0.5. IPTG was hereafter added to a final concentration of i mM
and
the culture was further incubated 4 hours. Cells were harvested, resuspended
in 1 X
sonication buffer + 8 M urea and sonicated 5 X 30 sec. with 30 sec. pausing be-
tween the pulses.
After centrifugation, the lysate was applied to a column containing 25 ml of
resus-
pended Talon resin (Clontech, Palo Alto, USA). The column was washed and
eluted as
described by the manufacturers.
After elution, all fractions (1.5 ml each) were subjected to analysis by SDS-
PAGE us-
ing the Mighty Small (Hoefer Scientific Instruments, USA) system and the
protein con-
centrations were estimated at 280 nm. Fractions containing recombinant protein
were
pooled and dialysed against 3 M urea in 10 mM Tris-HCI, pH 8.5. The dialysed
protein
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was further purified by FPLC (Pharmacia, Sweden) using a 6 ml Resource-Q
column,
eluted with a linear 0-1 M gradient of NaCI. Fractions were analysed by SDS-
PAGE
and protein concentrations were estimated at ODZeo. Fractions containing
protein
were pooled and dialysed against 25 mM Hepes buffer, pH 8.5. '
Finally the protein concentration and the LPS content was determined by the
BCA
(Pierce, Holland) and LAL (Endosafe, Charleston, USA) tests, respectively.
EXAMPLE 3C
Using homology searching for identification of ORF11-1, ORF1 1-2, ORF11-3 and
ORFI 1-4.
A search of the Mycobacterium tuberculosis Sanger sequence database with the
amino acid sequences of CFP1 1, a previously identified ST-CF protein,
identified 4
new very homologous proteins. All 4 proteins were at least 96% homologous to
CFP1 1.
On the basis of the strong homology to CFP1 1, it is belived that ORF1 1-1,
ORF11-2,
ORF11-3 and ORF1 1-4 are potential new T-cell antigens.
The first open reading frame, MTCY10G2.1 1, homologous to CFP11, encodes a pro-
tein of 98 amino acids corresponding to a theoretical molecular mass of
10994Da and
a pl of 5.14. The protein was named ORF11-1.
The second open reading frame, MTC1364.09, homologous to CFP11, encodes a pro-
tein of 98 amino acids corresponding to a theoretical molecular mass of
10964Da and
a pl of 5.14. The protein was named ORF11-2.
The third open reading frame, MTV049.14, has an in frame stop codon. Because
of
the very conserved DNA sequence in this position amongst the 4 open reading
frames
it is however suggested that this is due to a sequence mistake.
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The "T" in position 175 of the DNA sequence is therefor suggested to be a "C"
as in
the four other ORF's. The Q in position 59 in the amino acid sequence would
have
been a "stop" if the T in position 175 in the DNA sequence had not been
substituted.
The open reading frame encodes a protein of 98 amino acids corresponding to a
theo-
5 retical molecular mass of 10994Da and a pl of 5.14. The protein was named
ORF11-
3.
The fourth open reading frame, MTCY15C10.32, homologous to CFP1 1, encodes a
protein of 98 amino acids corresponding to a theoretical molecular mass of 1
1024Da
10 and a pl of 5.14. The protein was named ORF1 1-4.
Using homology searching for identification of ORF7-1 and ORF7-2.
A search of the Mycobacterium tuberculosis Sanger sequence database with the
15 amino acid sequences of a previously identified immunoreactive ST-CF
protein, CFP7,
identified 2 new very homologous proteins. The protein ORF7-1 (MTV012.33) was
84% identical to CFP7, with a primary structure of the same size as CFP7, and
the
protein ORF7-2 (MTV012.31? was 68% identical to CFP7 in a 69 amino acid
overlap.
On the basis of the strong homology to the potent human T-cell antigen CFP7,
ORF7-1
20 and ORF7-2 are belived to be potential new T-cell antigens.
The first open reading frame homologous to CFP7, encodes a protein of 96 amino
ac-
ids corresponding to a theoretical molecular mass of 10313Da and a pl of
4.186. The
protein was named ORF7-1.
The second open reading frame homologous to CFP7, encodes a protein of 120
amino
acids corresponding to a theoretical molecular mass of 1 2923.00 Da and a pl
of
7.889. The protein was named ORF7-2.
Clonin4 of the homoloctous orf7-1 and orf7-2.
Since ORF7-l and ORF7-2 are nearly identical to CFP7 it was nessesary to use
the
flanking DNA regions in the cloning procedure, to ensure the cloning of the
correct
ORF. Two PCR reactions were carried out with two different primer sets. PCR
reaction
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1 was carried out using M. tuberculosis chromosomal DNA and a primerset corre-
sponding to the flanking DNA. PCR reaction 2 was carried out directly on the
first PCR
product using ORF specific primers which introduced restriction sites for use
in the
later cloning procedure. '
The sequences of the primers used are given below;
Orf7-1:
Primers used for the initial PCR reaction (1) using M. tuberculosis
chromosomal DNA
as template;
Sence: MTV012.33-R1: 5'- GGAATGAAAAGGGGTTtGTG -~3' (SEQ ID NO: 186)
Antisence:MTV012.33-F1: 5'- GACCACGCCCGCGCCGTGTG - 3'(SEQ ID N0:187)
Primers used for the second round of PCR (2) using PCR product 1 as template;
Sence: MTV012.33-R2: 5' - GCAACACCCGGGATGTCGCAGATTATG - 3'
(SEQ ID NO: 188)
(introduces a Smal upstream of the orf7-1 start codon)
Antisence:MTV012.33-F2: 5' - CTAAGCTTGGATCCCTAGCCGCCCCACTTG - 3'
((SEQ ID NO: 189)
(introduces a BamHl downstream of the orf7-1 stop codon).
Orf7-2:
Primers used for the initial PCR reaction (1 ) using M. tuberculosis
chromosomal DNA
as template;
Sence: MTV012.31-R1: 5'- GAATATTTGAAAGGGATTCGTG - 3' (SEQ 1D NO: 190) .
Antisence:MTV012.31-F1: 5'- CTACTAAGCTTGGATCCTTAGTCTCCGGCG - 3'
(SEQ ID NO: 191 ) .
(introduces a BamHl downstream of the orf7-2 stop codon)
Primers used for the second round of PCR (2) using PCR product 1 as template;
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Sence: MTV012.31-R2: 5' -GCAACACCCGGGGTGTCGCAGAGTATG- 3'
(SEQ ID NO: 192)
(introduces a Smal upstream of the orf7-2 start codon)
Antisence:MTV012.31-F1: 5'- CTACTAAGCTTGGATCCTTAGTCTCCGGCG - 3'
(SEQ ID NO: 193)
(introduces a BamHl downstream of the orf7-2 stop codon)
The genes encoding ORF7-1 and ORF7-2 were cloned into the expression vector
pMST24, by PCR amplification with gene specific primers, for recombinant
expression
in E. coli of the proteins.
The first PCR reactions contained either 10 ng of M. tuberculosis chromosomal
DNA
(PCR reaction 1 ) or 1 Ong PCR product 1 (PCR reaction 2) in 1 x low salt Taq
+ buffer
from Stratagene supplemented with 250 mM of each of the four nucleotides
(Boehrin-
ger 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°C for 25 sec. and run for
30 cycles of
the program; 94°C for 10 sec., 55°C for 10 sec. and 72°C
for 90 sec, using thermo-
cycler equipment from Idaho Technology.
The DNA fragments were subsequently run on 1 % agarose gels, the bands were ex-
cised 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 pMST24 in frame with 6 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.
Expression/purification of recombinant ORF7-1 and ORF7-2 protein.
Expression and metal affinity purification of recombinant protein was
undertaken es-
sentially as described by the manufacturers. 1 I LB-media containing 100 Ng/ml
ampi-
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cillin, was inoculated with 10 ml of an overnight culture of XL1-Blue cells
harbouring
recombinant pMCT6 plasmid. The culture was shaken at 37 °C until it
reached a den-
sity of OD600 = 0.5. IPTG was hereafter added to a final concentration of 1 mM
and
the culture was further incubated 2 hours. Cells were harvested, resuspended
in 1 X
sonication buffer + 8 M urea and sonicated 5 X 30 sec. with 30 sec. pausing be-
tween the pulses.
After centrifugation, the lysate was applied to a column containing 25 ml of
resus-
pended Talon resin (Clontech, Palo Alto, USA). The column was washed and
eluted as
described by the manufacturers.
After elution, all fractions (1.5 ml each) were subjected to analysis by SDS-
PAGE us-
ing the Mighty Small (Hoefer Scientific Instruments, USA) system. Fractions
contain-
ing recombinant protein were pooled and dialysed against 3 M urea in 10 mM
Tris-
HCI, pH 8.5. The dialysed protein was further purified by FPLC fPharmacia,
Sweden)
using a 6 ml Resource-Q column, eluted with a linear 0-1 M gradient of NaCI.
Frac-
tions were analysed by SDS-PAGE. Fractions containing protein were pooted and
dia-
lysed against 25 mM Hepes buffer, pH 8.5.
Finally the protein concentration and the LPS content was determined by the
BCA
(Pierce, Holland) and LAL (Endosafe, Charleston, USA) tests, respectively.
EXAMPLE 4
Cloning of the gene expressing CFP26 (MPT51 J
Svnthesis and desi4n of probes
Oligonucleotide primers were synthesized automatically on a DNA synthesizer
(Applied
Biosystems, Forster City, Ca, ABI-391, PCR-mode) deblocked and purified by
ethanol
precipitation.
Three oligonucleotides were synthesized (TABLE 3) on the basis of the
nucleotide se-
quence from mpb51 described by Ohara et al. (1995). The oligonucleotides were
en-
gineered to include an EcoRl restriction enzyme site at the 5' end and at the
3' end by
which a later subcloning was possibte.
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Additional four oligonucleotides were synthesized on the basis of the
nucleotide se-
quence from MPT51 (Fig. 5 and SEQ ID NO: 41 ). The four combinations of the
primers were used for the PCR studies.
DNA cloning and PCR technology '
Standard procedures were used for the preparation and handling of DNA
(Sambrook et
al., 1989). The gene mpt51 was cloned from M. tuberculosis H37Rv chromosomal
DNA by the use of the polymerase chain reactions (PCR) technology as described
pre-
viously (Oettinger and Andersen, 1994). The PCR product was cloned in the
pBluescriptSK + (Stratagene).
Cloning of mpt51
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 by and 815 by in
pBluescript
SK + , designated pT052 and pT053.
DNA Seguencing
The nucleotide sequence of the cloned 952 by M. tuberculosis H37Rv PCR
fragment,
pT052, containing the Shine Dalgarno sequence, the signal peptide sequence and
the
structural gene of MPT51, and the nucleotide sequence of the cloned 815 by PCR
fragment containing the structural gene of MPT51, pT053, were determined by
the
dideoxy chain termination method adapted for supercoiled DNA by use of the
Seque-
nase DNA sequencing kit version 1.0 (United States Biochemical Corp.,
Cleveland,
OH) 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.
The nucleotide sequences of pT052 and pT053 and the deduced amino acid
sequence are shown in Figure 5. The DNA sequence contained an open reading
frame
starting with a ATG colon at position 45 - 47 and ending with a termination
colon
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(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 - Giu - Asn) of the purified MPT51 (Nagai et
al., 1991 )
and the features of the signal peptide, it is presumed that the signal
peptidase
5 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, mpt5l, 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
10 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 mpt51 consists of
801 by
and that the deduced amino acid sequence contains 266 residues with a
molecular
15 weight of 27,842, and MPT51 show 99,8% identity to MPB51.
Subcloninq of mpt51
An EcoRl site was engineered immediately 5' of the first codon of mpt51 so
that only
20 the coding region of the gene encoding MPT51 would be expressed, and an
EcoRl site
was incorporated right after the stop codon at the 3' end.
DNA of the recombinant plasmid pT053 was cleaved at the EcoRl sites. The 815
by
fragment was purified from an agarose gel and subcloned into the EcoRl site of
the
25 pMAL-cRl expression vector (New England Biolabs), pT054. Vector DNA
containing
the gene fusion was used to transform the E. coli XL1-Blue by the standard
proce-
dunes for DNA manipulation.
The endpoints of the gene fusion were determined by the dideoxy chain
termination
30 method as described under section DNA sequencing. Both strands of the DNA
were
sequenced.
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Preearation and purification of rMPT51
Recombinant antigen was prepared in accordance with instructions provided by
New
England Biolabs. Briefly, single colonies of E. colt harbouring the pT054
plasmid were
inoculated into Luria-Bertani broth containing 50 Ng/mf ampiciliin and 12.5
~rg/ml tetra-
cycline and grown at 37°C to 2 x 10$ cells/ml. Isopropyl-~i-D-
thiogalaCtoside (IPTG)
was then added to a final concentration of 0.3 mM and growth was continued for
fur-
ther 2 hours. The pelleted bacteria were stored overnight at -20°C in
new column
buffer (20 mM Tris/HCI, pH 7.4, 200 mM NaCI, 1 mM EDTA, 1 mM dithiothreitol
(DTT))and thawed at 4 °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). Atter
centrifugation
at 9,000 x g for 30 min at 4°C, the maltose binding protein -MPT51
fusion protein
(MBP-rMPT51 ) was purified from the crude extract by affinity chromatography
on
amyfose 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.
Protein concentration was determined by the BCA method supplied by Pierce
(Pierce
Chemical Company, Rockford, IL).
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TABLE 3.
Sequence of the mpt51 oligonucleotidesa.
Orientation Sequences (5'-a 3') Positionb
and oli- (nucleotide)
gonucleotidea
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)
a The oligonucieotides MPT51-1 and MPT51-2 were constructed from the MPB51 nu-
cleotide sequence (Ohara et al., 1995). The other oligonucleotides
constructions were
based on the nucleotide sequence obtained from mpt51 reported in this work.
Nucleo-
tides (nt) underlined are not contained in the nucleotide sequence of MPB/T51.
The positions referred to are of the non-underlined parts of the primers and
corre- -
spond to the nucleotide sequence shown in SEQ 1D NO: 41.
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Cloning of mpt51 in the expression vector pMST24
A PCR fragment was produced from pT052 using the primer combination MPT51-F
and MPT51-R (TABLE 4). A BamHl site was engineered immediately 5' of the first
co-
don of mpt51 so that only the coding region of the gene encoding MPT51 would
be
expressed, and an Ncol site was incorporated right after the stop codon at the
3' end.
The PCR product was cleaved at the BamHl and the Ncol site. The 81 1 by
fragment
was purified from an agarose gel and subcloned into the BamHl and the Ncol
site of
the pMST24 expression vector, pT086. Vector DNA containing the gene fusion was
used to transform the E. colt XL1-Blue by the standard procedures for DNA
manipula-
Lion.
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.
Preparation and purification of rMPT51.
Recombinant antigen was prepared from single colonies of E. colt harbouring
the
pT086 pfasmid inoculated into Luria-Bertani broth containing 50 Ng/ml
ampicillin and
12.5 Ng/ml tetracycline and grown at 37°C to 2 x 1 O$ cellslml.
Isopropyl-(3-D-thioga-
lactoside (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
100120
buffer (100 mM KCI, 20 mM Imidazole, 20 mM Tris/HCI, pH 7.9, 20 % glycerol).
Cells
were broken by sonication (20 times for 10 sec with intervals of 20 sect'.
After cen-
trifugation at 9,000 x g for 30 min. at 4°C the insoluble matter was
resuspended in
BC 100/20 buffer with 8 M urea followed by sonication and centrifugation as
above.
The 6 x His tag-MPTSi fusion protein (His-rMPT51 ) was purified by affinity
chroma-
tography 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 KCI, 40 mM Imidazole, 20 mM Tris/HCI, pH 7.9, 20 % gly-
cerol) with 8 M urea and BC 1000/40 buffer (1000 mM KCI, 40 mM Imidazole, 20
mM Tris/HCI, pH 7.9, 20 % glycerol) with 8 M urea. His-rMPT51 was extensive
dia-
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lysed against 10 mM TrisiHCl, 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 TrisiHCl, pH 8.5, 3 M urea with a 0 - 1 M
NaCI linear gradient. Fractions containing rMPT51 were pooled and subsequently
dia-
fysed extensively against 25 mM Hepes, pH 8.0 before use.
Protein concentration was determined by the BCA method supplied by Pierce
(Pierce
Chemical Company, Rockford, IL).
The iipopoiysaccharide (LPS) content was determined by the limulus amoebocyte
ly-
sate test (LAL) to be less than 0.004 nglug rMPT5l; and this concentration had
no
influence on cellular activity.
TABLE 4. Sequence of the mpt51 oligonucleotides.
Orientation and Sequences (5' --~ 3') Position
oligonucleotide (nt)
Sense
MPT51-F CTCGGATCCTGCCCCATACGAGAACCTG 139 - 156
Antisense
MPT51-R CTCCCATGGTTAGCGGATCGCACCG 939 - 924
EXAMPLE 4A
Cloning of the ESA T6-MPT59 and the MPT59-ESA T6 hybrides.
Background for ESAT-MPT59 and MPT59-ESAT6 fusion -
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
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a truncated version were amino acids 1-15 have been deleted. The deletion
includes
the main T-cell epitopes recognized by C57BLi6j 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
5 and another major T cell antigen MPT59 has been constructed. Two different
con-
struct have been made: MPT59-ESAT-6 (SEQ ID NO: 172) and ESAT-6-MPT59 iSEQ
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 syner-
gistic effect.
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.
Construction of the hybrid MPT59-ESAT6.
The cloning was carried out in three steps. First the genes encoding the two
cornpo-
nests of the hybrid, ESAT6 and MPT59, were PCR amplified using the following
primer constructions:
ESAT6:
OPBR-4: GGCGCCGGCAAGCTTGCCATGACAGAGCAGCAGTGG
(SEQ ID NO: 132)
OPBR-28: CGAACTCGCCGGATCCCGTGTTTCGC (SEQ ID NO: 133)
OPBR-4 and OPBR-28 create HinDlll and BamHl sites, respectively.
MPT59:
OPBR-48: GGCAACCGCGAGATCTTTCTCGCGGCCGGGGC (SEQ tD NO: 134)
OPBR-3: GGCAAGCTTGCCGGCGCCTAACGAACT (SEQ ID NO: 135)
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OPBR-48 and OPBR-3 create Bglll and HinDlll, respectively. Additionally OPBR-3
de-
letes the stop codon of MPT59.
PCR reactions contained 10 ng of M. tuberculosis chromosomal DNA in 1 x low
salt
Taq+ buffer from Stratagene supplemented with 250 mM of each of the four
nucleo-
tides (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 u!
reac-
tion volume. Reactions were initially heated to 94°C for 25 sec. and
run for 30 cycles
of the program; 94°C for 10 sec., 55°C for 10 sec. and
72°C for 90 sec, using ther-
mocycler equipment from Idaho Technology.
The DNA fragments were subsequently run on 1 % agarose gels, the bands were ex-
cised and purified by Spin-X spin columns (Costar). The two PCR fragments were
di-
gested with HinDlll and ligated. A PCR amplification of the iigated PCR
fragments en-
coding MPT59-ESAT6 was carried out using the primers OPBR-48 and OPBR-28. PCR
reaction was initially heated to 94°C for 25 sec. and run for 30 cycles
of the pro-
gram; 94°C for 30 sec., 55°C for 30 sec. and 72°C for 90
sec. The resulting PCR
fragment was digested with Bglil and BamHl 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
se-
quencing kit version 1.0 (United States Biochemical Corp., USA) and by cycle
se-
quencing 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.
Construction of the hybrid ESAT6-MPT59.
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:
ESAT6:
OPBR-75: GGACCCAGATCTATGACAGAGCAGCAGTGG (SEQ ID NO: 136)
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OPBR-76: CCGGCAGCCCCGGCCGGGAGAAAAGCTTTGCGAACATCCCAGTGACG
(SEQ ID NO: 137)
OPBR-75 and OPBR-76 create Bglll and HinDlll sites, respectively. Additionally
OPBR-
76 deletes the stop colon of ESAT6.
MPT59:
OPBR-77: GTTCGCAAAGCTTTTCTCCCGGCCGGGGCTGCCGGTCGAGTACC
(SEQ 1D NO: 138)
OPBR-18: CCTTCGGTGGATCCCGTCAG (SEQ iD NO: 139)
OPBR-77 and OPBR-18 create HinDlll and BamHl sites, respectively.
Expression/purification of MPT59-ESAT6 and ESAT6-MPT59 hybrid proteins.
Expression and metal affinity purification of recombinant proteins was
undertaken es-
sentially as described by the manufacturers. For each protein, 1 I LB-media
containing
100 ~uglmt ampicillin, was inoculated with 10 ml of an overnight culture of
XL1-Blue
cells harbouring recombinant pMCT6 plasmids. Cultures were shaken at 37
°C until
they reached a density of ODsoo = 0.4 - 0.6. IPTG was hereafter added to a
final con-
centration of 1 mM and the cultures were further incubated 4 - 16 hours. Cells
were
harvested, resuspended in 1 X sonication buffer + 8 M urea and sonicated 5 X
30
sec. with 30 sec. pausing between the pulses.
After centrifugation, the lysate was applied to a column containing 25 ml of
resus-
pended Talon resin (Clontech, Palo Alto, USA). The column was washed and
eluted as
described by the manufacturers.
After elution, all fractions (1.5 ml each) were subjected to analysis by SDS-
PAGE us-
ing the Mighty Small (Hoefer Scientific Instruments, USA) system and the
protein con-
centrations were estimated at 280 nm. Fractions containing recombinant protein
were
pooled and dialysed against 3 M urea in 10 mM Tris-HCI, pH 8.5. The dialysed
protein
was further purified by FPLC (Pharmacia, Sweden) using a 6 ml Resource-Q
column,
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eluted with a linear O-1 M gradient of NaCI. Fractions were analyzed by SDS-
PAGE
and protein concentrations were estimated at oD28o. Fractions containing
protein
were pooled and dialysed against 25 mM Hepes buffer, pH 8.5.
Finally the protein concentration and the LPS content were determined by the
BCA
(Pierce, Holland) and LAL (Endosafe, Charleston, USA) tests, respectively.
The biological activity of the MPT59-ESAT6 fusion protein is described in
Example 6A.
EXAMPLE 5
Mapping of the purified antigens in a 2DE system.
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 electrophoresis. The 2DE was performed
according to
Hochstrasser et al. (1988). 85 ug 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 in-
ciuded. 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, CFPBA, CFPBB, 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 f?een
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.
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EXAMPLE 6
Biological activity of the purified antigens.
IFN-y induction in the mouse model of TB infection
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 re-
presentative for three experiments.
A very high IFN-y response was induced by two of the antigens CFP17 and CFP21
at
almost the same high level as ST-CF.
TABLE 5
IFN-y release from splenic memory effector cells from C57BL16J mice isolated
after
reinfection with M. tuberculosis after stimulation with native antigens.
Antigena IFN-y (pg/ml)b
ST-CF 12564
CFP7 NDd
CFP9 ND
CFP17 9251
CFP20 2388
CFP21 10732
CFP22 + CFP25~ 5342
CFP26 (MPT51) ND
CFP28 2818
CFP29 3700
The data is derived from a representative experiment out of three.
a ST-CF was tested in a concentration of 5 pg/ml and the individual antigens
in a con-
centration of 2 wg/ml.
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° Four 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
cul-
tures are < 15% of mean. The IFN-y release of cultures incubated without
antigen
was 390 pg/ml.
' A pool of CFP22 and CFP25 was tested.
d ND, not determined. -
Skin test reaction in TB infected guinea pigs
The skin test activity of the purified proteins was tested in M. tuberculosis
infected
guinea pigs.
1 group of guinea pigs was infected via an ear vein with 1 x 10' CFU of M.
tubercu-
losis H37Rv in 0,2 ml PBS. After 4 weeks skin tests were performed and 24
hours
after injection erythema diameter was measured.
As seen in TABLES 6 and 6a all of the antigens induced a significant Delayed
Type
Hypersensitivity (DTH) reaction.
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TABLE 6
DTH erythema diameter in guinea pigs infected with 1 x 10° CFU of M.
tuberculosis,
after stimulation with native antigens.
Antigens Skin reaction (mm)b
Control 2.00
PPD~ 15.40 (0.53)
CFP 7 NDe
CFP9 ND
CFP17 11.25 (0.84)
CFP20 8.88 (0..13)
CFP21 12.44 (0.79)
CFP22 + CFP25d 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
ery-
thema diameter of ten animals.
a The antigens were tested in a concentration of 0,1 ~g~ except for CFP29
which was
tested in a concentration of 0,8 fig.
b The skin reactions are measured in mm erythema 24 h after intradermat
injection.
' i 0 TU of PPD was used.
A pool of CFP22 and CFP25 was tested.
a ND, not determined.
Together these analyses indicate that most of the antigens identified were
highly bio-
logically active and recognized during TB infection in different animal
models.
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TABLE 6a
DTH erythema diameter of recombinant antigens in outbred guinea pigs infected
with
1 x 10° CFU of M. Tuberculosis.
Antigens Skin reaction (mm}b
Control 2.9 (0.3)
PPD 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.
a The antigens were tested in a concentration of 1,0 ug.
The skin test reactions are measured in mm erythema 24 h after intradermal
infec-
tion.
10 TU of PPD was used.
Table 6B.
DTH erythema diameter in guinea pigs i.v. infected with 1 x i 04 CFU M.
tuberculo-
sis, after stimulation with 10 g antigen.
Antigen Mean (mm) SEM
PBS 3,25 0,48
PPD (2TU) 10,88 1
nCFP7B 7,0 0,46
nCFPl9 6,5 0,74
MPT59-ESAT6 14,75 I,5
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The values presented are the mean of erythema diameter of four animals.
The results in Table 6B indicates biological activity of nCFP7B, nCFP19 and
MPT59-
ESAT-6. MPT59-ESAT-6 resulting in a DTH response at the level of PPD.
Biological activity of the purified recombinant antigens.
Interferon-Y induction in the mouse model of TB infection.
Primary infections. 8 to 12 weeks old female C57BU6jtH-2b), CBAIJ(H-2k),
DBA.2iH-
2d) and A.SWiH-25) mice (Bomholtegaard, Ry) were given intravenous infections
via
the lateral tail vein with an inoculum of 5 x 104 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.
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
stimula-
tion. Neither of the antigens rCFP20 and rCFP22 were recognized by any of the
four
mouse strains.
As shown in TABLE 7A, the recombinant antigens rCFP27, RD1-ORF2, MPT59-
ESAT6, rCFPIOA, rCFPl9, and rCFP25A were all recognized in at least two
strains of
mice at a level comparable to ST-CF, whereas ESAT6-MPT59, rCFP23, and rCFP30B
only were recognized in one strain at this level. rCFP30A , RD1-ORFS, rCFP16
gave
rise to an IFN-y release in two mice strains at a level corresponding to 2/3
of the re-
sponse after stimulation with ST-CF. RD1-ORF3 was recognized in two strains at
a
level of 1/3 of ST-CF.
The native CFP7B was recognized in two strains at a level of 1/3 of the
response seen
after stimulation with ST-CF.
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Memory responses. 8-12 weeks old female C57BL/6j(H-2b) mice (Bomholtegaard,
Ry)
were given intravenous infections via the lateral tail vein with an inoculum
of 5 x 10°
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, NJ) and rifabutin
(Farma-
talia 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 x 106 bacteria
i.v. and
sacrificed at day 4 postinfection. Spleen cells were isolated and tested for
the recogni-
tion of recombinant antigen.
As seen from TABLE 8, IFN-y 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-y no higher
than 1 /3
of the response seen with ST-CF. rCFP22 was not recognized by IFN-y producing
cells. None of the antigens stimulated iFN-y release in naive mice.
Additionally non of
the antigens were toxic to the cell cultures.
As shown in TABLE 8A, IFN-y release after stimulation with RD1-ORF2, MPT59-
ESAT6, ESAT6-MPT59, and rCFP19 was at the same level as seen from spleen cells
stimulated with ST-CF. Stimulation with rCFPIOA and rCFP30A gave rise to an
IFN-y
release of 213 of the response after stimulation with ST-CF, whereas rCFP27,
RD1-
ORFS, rCFP23, rCFP25A and rCFP30B all resulted in an IFN-Y release no higher
than
1/3 of the response seen with ST-CF. RD1-ORF3 and rCFPl6 were not recognized
by
IFN-y producing memory cells.
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TABLE 7. T cell responses in primary TB infection.
Name c57BL/6J (H2b) DBA. 2 (H2d) CBA/J (H2k) A. SW (H2S
rCFP7 + + -
rCFP7A +++ +++ +++ +
rCFPl7 +++ + +++ -t
rCFP20 - - - -
rCFP21 +++ +++ +++ +
rCFP22 - - - -
rCFP25 +++ ++ +++ +
rCFP29 +++ +++ ++_ . ++
rMPT51 + - - -
Mouse IFN-y release 14 days after primary infectiowrvith M. tuberculosis.
-:no response; + : 1 I3 of ST-CF; + + : 2/3 of ST-CF; + + + : level of ST-CF.
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TABLE 7A. T cell responses in primary TB infection.
Name C57B1 6j (H2 DBA. 2 (H2) CBA/J (H2") A. SW (H2
) )
rCFP27 ++ ++ +++ +++ '
rCFP30A - + ++ ++
RDl-ORF2 +++ +++ +++ _ ++
RDl-ORF3 - - + +
RD1-ORFS + + ++ ++
MPT59-ESAT6 +++ +++ +++ ++
ESAT6-MPT59 +++ - + -
rCFPIOA +++ n.d. +++ n.d.
rCFPl6 ++ n.d. -~+ n.d.
rCFPl9 +++ n.d. +++ n.d.
rCFP23 ++ n.d. +++ n.d.
rCFP25A +++ n.d. +++ n.d.
rCFP30B + n.d. +++ n.d.
CFP7B(native) + n.d. + n.d.
Mouse IFN-y release 14 days after primary infection with M. tuberculosis.
no response; + : 113 of ST-CF; + + : 213 of ST-CF; + + + : level of ST-CF.
n.d. = not determined.
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TABLE 8. T cell responses in memory immune animals.
Name Memory response
rCFP7 +
rCFP7A ++
rCFPl7 +++
rCFP21 +++
rCFP22 -
rCFP29 +
rCFP25 +++
rMPT51 +
Mouse IFN-y 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.
TABLE 8A. T cell responses in memory immune animals.
Name Memory response
rCFP27 +
rCFP30A ++
RD1-ORF2 +++
RD1-ORF3 -
RD1-ORF5 +
MPT59-ESAT6 +++
ESAT6-MPT59 +++
rCFPIOA ++
rCFPl6 -
rCFPl9 +++
rCFP23 +
rCFP25A +
rCFP30B +
Mouse IFN-y release during recall of memory immunity to M. tuberculosis.
-: no response; + : 1 i3 of ST-CF; + + : 2/3 of ST-CF; + + + : level of ST-CF.
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Interferon-y induction in human TB patients and BCG vaccinated people.
Human donors: PBMC were obtained from healthy BCG vaccinated donors with no
v
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.
Lymphocyte preparations and cell culture: PBMC were freshly isolated by
gradient cen-
trifugation of heparinized blood on Lymphoprep (Nycomed, Oslo, Norway). The
cells
were resuspended in complete medium: RPMI 1640 (Gibco, Grand Island, N.Y.) sup-
plemented with 40 ~rglml 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 de-
termined by trypan blue staining. Cultures were established with 2,5 x 105
PBMC in
200 ~ul in microtitre plates (Nunc, Roskilde, Denmark) and stimulated with no
antigen,
ST-CF, PPD (2.5Ng1m1); rCFP7, rCFP7A, rCFP17, rCFP20, rCFP21, rCFP22, rCFP25,
rCFP26, rCFP29, in a final concentration of 5 Ng/ml. Phytohaemagglutinin, 1
Ng/ml
(PHA, Difco laboratories, Detroit, MI. was used as a positive control.
Supernatants for
the detection of cytokines were harvested after 5 days of culture, pooled and
stored
at -80°C until use.
Cytokine analysis: Interferon-y (IFN-y ) was measured with a standard ELISA
tech-
nique using a commercially available pair of mAb's from Endogen and used
according
to the instructions for use. Recombinant lFN-y (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.
A seen in TABLE 9 high levels of IFN-y release are obtained after stimulation
with sev-
eral of the recombinant antigens. rCFP7a and rCFPl7 gives rise to responses
compa-
rable 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.
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As is seen from Table 9A RD1-ORF2 and RD1-ORES give rise to IFN-y responses
close
to the level of ST-CF. Between 60% and 90% of the donors show high IFN-y
respon-
ses (> 1000 pglml). rCFP30A gives rise to a mixed response with 40-50% high re-
sponders, whilst low responses are obtained with RD1-ORF3.
As seen from Table 9B MPT59-ESAT6 and ESAT6-MPT59 both give rise to IFN-y re-
sponses at the level of ST-CF and 67-89 % show high responses ( > 1000 pg/ml).
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Table 9A_
Results from the stimulation of human blood cells from 10 healthy BCG
vaccinated or
non vaccinated ST-CF responsive healthy donors and 10 Tb patients with
recombinant
antigen are shown. ST-CF, PPD and PHA are included for comparison. Results are
given in pg
IFN-y/ml and negative values below 300 pg/ml are shown as " < ". nd = not
done.
Controls, Healthy BCG vaccinated, or non vaccinated ST-CF positive
Donor no ag PHA PPD STCF RDl-ORF2 RD1-ORF3 RDl-ORFS rCFP30A
< nd 3500 4200 1250 < 690 nd
11 < nd 5890 4040 5650 880 9030 nd
12 < nd 6480 3330 2310 < 3320 nd
13 < nd 7440 4570 920 < 1230 nd
14 < 8310 nd 2990 1870 < 4880 <
< 10820 nd 4160 5690 < 810 3380
16 < 8710 nd 5690 1630 < 5600 <
17 < 7020 4480 5340 2030 nd 670 <
18 < 8370 6250 4780 3850 nd 370 1730
19 < 8520 1600 310 5110 nd 2330 1800
10 Tb patients,
1-4 month after
diagnosis
Donor no ag PHA PPD STCF RD1-ORF2 RD1-ORF3 RD1-ORF5 rCFP30A
- -
c nd 10670 12 2020 c 9670 nd
680
21 < nd 3010 1420 850 < 350 nd
22 < nd 8450 7850 430 < 1950 nd
23 < 10060 nd 3730 < < 350 <
24 < 10830 nd 6180 2090 < 320 730
< 9000 nd 3200 4760 < 4960 2820
26 < 10740 nd 7650 4710 < 1170 2280
27 < 7550 6430 6220 2030 nd 3390 3069
28 < 8090 5790 4850 1100 nd 2095 550
29 < 7790 4800 4260 2800 nd 1210 420
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Table 9B.
Results from the stimulation of human blood cells from 9 Healthy BCG
vaccinated, or -
non vaccinated ST-CF positive and 8 Tb patients with recombinant MPT59-ESAT6
and
ESAT6-MPT59 are shown. ST-CF, PPD and PHA are included for comparison. Results
are given in pg IFN-ylml and negative values below 300 pg/ml are shown as " <
". nd
= not done. -
Controls, Healthy BCG vaccinated, or non vaccinated ST-CF positive.
Donor no ag PHA PPD STCF MPT59- ESAT6-
ESAT6 MPT59
1 < 9560 6770 3970 2030 <
2 < 12490 6600 8070 5660 5800
4 < 21030 4100 3540 < <
5 < 18750 14200 13030 8540 <
11 < nd 5890 4040 4930 8870
12 < nd 6470 3330 2070 6450
14 < 8310 nd 2990 10270 11030
< 10830 nd 4160 3880 4540
16 < 8710 nd 5690 2240 5820
10 Tb patients, 1-4 month after diagnosis
Donor no ag PHA PPD STCF MPT59- ESAT6-
ESAT6 MPT59
6 < 8970 5100 6150 4150 4120
7 < 12410 6280 3390 5050 2040
8 < 11920 7670 7370 800 1350
9 < 22130 16420 17210 13660 5630
23 < 10070 nd 3730 1740 2390
24 < 10820 nd 6180 1270 1570
< 9010 nd 3200 3680 5340
26 < 10740 nd 7650 2070 620
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EXAMPLE 6A
Four groups of 6-8 weeks old, female C578116J mice (Bomholtegard, Denmark)
were
immunized subcutaneously at the base of the tail with vaccines of the
following com-
positions:
Group 1: 10 Ng ESAT-fi/DDA (250,ug)
Group 2: 10 ~g MPT59lDDA (250Ng)
Group 3: 10 Ng MPT59-ESAT-6 /DDA (250 Ng)
Group 4: Adjuvant control group: DDA (250 fig) in NaCI
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-y into
the cul-
ture supernatants when stimulated in vitro with relevant antigens (see the
following
table).
Immunogen For restimulation Ag vitro
. in
10 pg/dose
no antigen ST-CF ESAT-6 MPT59
ESAT-6 - 219 219 569 569 835 633 -
MPT59 0 802 1B2 - 5647 159
Hybrid: 127 127 7453 581 15133 861 16363
MPT59-ESAT-6 1002
a' Blood cells were isolated 1 week after the last immunization and the
release of
IFN-y (pglml) after 72h of antigen stimulation (5 ~rg/ml) was measured.
The values shown are mean of triplicates performed on cells pooled from three
mice ~ SEM
°' - not determined
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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/mf.
EXAMPLE 6B
The recombinant antigens were tested individually as subunit vaccines in mice.
Eleven
groups of 6-8 weeks old, female C57B116j mice (Bomholtegard, Denmark) were
immu-
nized subcutaneously at the base of the tail with vaccines of the following
composi-
tion:
Group 1: 10 Ng CFP7
Group 10 ~g CFP17
2:
Group 3: 10 Ng CFP21
Group 4: 10 Ng CFP22
Group 5: l0lrg CFP25
Group 6: 10 Ng CFP29
Group 10 Ng MPT51
7:
Group 8: 50 dug ST-CF
Group 9: Adjuvant control
group
Group 10: BCG 2,5 x 1051m1,
0,2 ml
Group 1 Control group: Untreated
1:
All the subunit vaccines were given with DDA as adjuvant. The animals were
vacci-
nated 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-y into the culture
super-
natant when stimulated in vitro with the homologous protein.
fi weeks after the last immunization the mice were aerosol challenged with 5 x
106
viable Mycobacterium tuberculosislml. After 6 weeks of infection the mice were
killed
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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 7H11 plates.
Colonies were
counted after 2-3 weeks of incubation. The protective efficacy is expressed as
the dif-
ference between 1 og,o 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.
The results from the experiments are presented in the following table.
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
In conclusion, we have identified a number of proteins inducing high levels of
protec-
tion. Three of these CFP17, CFP25 and CFP29 giving rise to similar levels of
protec-
tion 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.
As is described for rCFP7, rCFP17, rCFP21, rCFP22, rCFP25, rCFP29 and rMPT51
the
two antigens rCFP7A and rCFP30A were tested individually as subunit vaccines
in
mice. C57BI/6j mice were immunized as described for rCFP7, rCFP17, rCFP21,
rCFP22, rCFP25, rCFP29 and rMPT51 using either 10~g rCFP7A or 10~g rCFP30A.
Controls were the same as in the experiment including rCFP7, rCFP17, rCFP21,
rCFP22, rCFP25, rCFP29 and rMPT5l.
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Immunogenicity and protective efficacy in mice, of ST-CF and
2 subunit vaccines.
Subunit vaccine Immunogenicity Protective efficacy
ST-CF +++ +++
rCFP7A +++ +++
rCFP30A +++ -
+ + + Strong immunogen/high protection (level of BCG)
+ + Medium immunogen/medium protection
- No recognition/no protection
In conclusion we have identified two strong immunogens of~which one, rCFP7A,
in-
duces protection at the level of ST-CF.
EXAMPLE 7
Species distribution of cfp7, cfp9, mpt5l, rd 1-orf2, rd 1-orf3, rd 1-orf4, rd
1-orf5, rd 1-
orf8, rd 1-orf9a and rd 1-orf9b as well as of cfp7a, cfp7b, cfp lOa, cfp 17,
cfp20,
cfp2l, cfp22, cfp22a, cfp23, cfp25 and cfp25a.
Presence of cfp7 cfp9 mpt51 rd 1-orf2 rd 1-orf3 rd 1-orf4 rd 1-orf5 rd 1-orf8,
rd 1-
orf9a and rdl-orf96 in different mycobacterial species.
In order to determine the distribution of the cfp7, cfp9, mpt51, rdl-orf2, rdl-
orf3,
rd 1-orf4, rd 1-orf5, rd 1-orf8, rd 1-orf9a and rd 1-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). "
,
PCR analyses were used in order to determine the distribution of the cfp7,
cfp9 and
mpt51 gene in species belonging to the tuberculosis-complex and in other
mycobacte- ,
ria. 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).
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The oligonucleotide primers used were synthesised automatically on a DNA
synthe-
sizer (Applied Biosystems, Forster City, Ca, ABI-391, PCR-mode), deblocked,
and puri-
' fied by ethanol precipitation. The primers used for the analyses are shown
in TABLE
11.
The PCR amplification was carried out in a thermal reactor (Rapid cycler,
Idaho Tech-
nology, Idaho) by mixing 20 ng chromosomal with the mastermix (contained 0.5
NM
of each oligonucleotide primer, 0.25 NM BSA (Stratagene), low salt buffer (20
mM
Tris-HCI, pH8.8 , 10 mM KCI, 10 mM (NHQ)ZS04, 2 mM MgS04 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 girl (all
concentrations given
are concentrations in the final volume). Predenaturation was carried out at
94°C for
30 s. 30 cycles of the following was performed: Denaturation at 94°C
for 30 s,
annealing at 55°C for 30 s and elongation at 72°C for 1 min.
The following primer combinations were used (the length of the amplified
products are
given in parentheses):
mpt5l: 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).
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).
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TABLE 10_
Mycobacterial strains used in this Example.
Species and strains) Source
1. M. tuberculosis H37Rv ATCC°
(ATCC 27294) .
2_ H37Ra ATCC _
(ATCC 25177}
3, Erdman Obtained from A. Lazlo,
Ottawa, Canada
4. M. bovis BCG substrain:Danish SSIb
1331
5. Chinese SSI'
6_ Canadian SSI'
7, Glaxo SSI'
g_ Russia SSI'
9. Pasteur SSI'
10. Japan WHOe
11. M. bovis MNC 27 SSI'
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. interce11u1are (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'
24. E. cola SSI°
25. S.aureus SSId
a American Type Culture Collection, USA.
b Statens Serum institut, Copenhagen, Denmark.
' Our collection Department of Mycobacteriology, Statens Serum Institut,
Copenha-
gen, Denmark.
Department of Clinical Microbiology, Statens Serum Institut, Denmark.
a WHO International Laboratory for Biological Standards, Statens Serum
Institut, Co-
penhagen, Denmark.
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TABLE 1 1.
Sequence of the mpt5l , cfp7 and cfp9 oligonucleotides.
Orientation and Sequences (5'--~3')a Position°
oligonucleotide (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
(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 lD NO: 36) (SEQ ID NO:
1 )
stR3 CGATTCCAAGCTTGTGGCCGCCGACCCG 141 - 155
(SEO 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)
5
a Nucleotides underlined are not contained in the nucleotide sequences of
mpt51,
cfp7, and cfp9.
b The positions referred to are of the non-underlined parts of the primers and
corre-
spond to the nucleotide sequence shown in SEQ ID NOs: 41, 1, and 3 for mpt51,
cfp7, and cfp9, respectively.
The Southern blotting was carried out as described previously (Oettinger and
Ander-
sen, 1994) with the following modifications: 2 erg of genomic DNA was digested
with
Pvull, electrophoresed in an 0.8% agarose gel, and transferred onto a nylon
membrane
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(Hybond N-plus; Amersham International plc, Little Chalfont, United Kingdom)
with a
vacuum transfer device (Milliblot, TM-v; Millipore Corp., Bedford, MA). The
cfp7,
cfp9, m pt 51, rd 1-orf2, rd 1-orf3, rd 1-orf4, rd 1-orf5, rd 1-orf8, rd 1-
orf9a and rd 1-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 1 1 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.
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TABLE 12. Interspecies analysis of the cfp7, cfp9 and mpt51 genes by PCR
and/or
Southern blotting and of MPT51 protein by Western blotting.
,PCR ,Southern ,Western
blot
' ' ;blot
Species and strain , cfp7cfp mpt51 cfp9 mpt5 , MPT51
,
cfp7
' 9 ' 1
1. M. tub. H37Rv ~+ + + ,+ + + ~+
2. M. tub. H37Ra + + + N.D. N.D. + +
3. M. tub. Erdmann+ + + + + + +
4. M. bovis + + + + +
S. M. bovis BCG :+ + + .+ + + :+
Danish 331 : '
1
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 . - - . - -
I4. M. avium ;+ + - ;+ + + ,
15. M. kansasii ;+ - - ;+ + + ;-
16 . marinum - ( - + + + -
M. +
)
17. M. scrofulaceum- - - - - - -
18. M. intercellul-+ (+) - + ~ + + -
are
19. M. fortui tum : - - : - -
20. M. flavescens ;+ (+} - ;+ + + ;N.D.
2I. 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.
cfp7, cfp9 and mpt51 were found in the M. tuberculosis complex_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 mpt51 in M.
xenopi.
Furthermore the presence of native MPT51 in culture filtrates from different
mycobac-
terial strains was investigated with western blots developed with Mab HBT4.
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There is a strong band at around 26 kDa in M, tuberculosis H37Rv, Ra, Erdman,
M.
bovis ANS, M. bovis BCG substrain Danish 1331 and M. africanum. No band was
seen in the region in any other tested mycobacteriai strains.
TA8LE 13a. Interspecies analysis of the rd 1-orf2, rd 1-orf3, rd 1-orf4, rd 1-
orf5, rd 1-
orf8, rdl-orf9a and rdl-orf9b genes by Southern blotting. -
Species and rd1- rd1- rd1- rd1- rd1- rd1- rd1-
strain orf2 orf3 orfg orf5 orf8 orf9a orf9b
1. M. tub. + + + + + + +
H37Rv
2. M. bovis + + + + N.D. + +
3. M. bovis + - - - N.D. - -
BCG
Danish . .
1331
4. M. bovis + - - - N.D. - -
BCG Japan
5. M. aviu.m - - - - N.D. - -
6. M. - _ _ _ N.D. - _
kansasii
7. M. marinum+ - + - N.D. - -
8. M. scrofu-+ - - - N.D. - -
laceum
9. M. - - - - N.D. - -
intercellu-
lare
10. M. - - - - N.D. - -
fortui tum
11. M. xenopi- - - - N.D. - -
12_ M. + - - - . N.D. - -
szulgai
+, positive reaction; -, no reaction, N.D. not determined.
Positive results for rd 1-orf2, rd 1-orf3, rd 1-orf4, rd 1-orf5, rd 1-orf8, rd
1-orf9a and rd 1-
orf96 were only obtained when using genomic DNA from M. tuberculosis and M. bo-
vis, and not from M. bovis BCG or other mycobacteria analyzed except rd 1-orf4
which
also was found in M. marinum.
Presence of cfA7a cfv7b cfp lOa cfp 17 cfp20 cfp21 cfp22 cfp22a cfp23 cfp25
and cfp25a in different mycobacterial species.
Southern blotting was carried out as described for rd l-orf2, rd l-orf3, rd 1-
orf4, rd l-
orf5, rd 1-orf8, rd l-orf9a and rd 1-orf9b. The cfp7a, cfp7b, cfp lOa, cfp 17,
cfp20,
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cfp2l, 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.
TABLE 13b. Interspecies analysis of the cfp7a, cfp7b, cfp 10a, cfp 17, cfp20,
cfp2l,
cfp22, cfp22a, cfp23, cfp25, and cfp25a genes by Southern blotting.
Species and cfp7acfp7 cfp- cfp cfp cfp2cfp2 cfp-cfp cfp cfp-
strain b l0a 17 20 1 2 22a 23 25 25a
1. M. tub. + + + + + + + + + + +
H37Rv
2. M. bovis + + + + + + + + + + +
3. M. bovis + + + + + N.D.+ + + + +
BCG
Danish 1331
4. M. bovis + + + + + + + + + + +
BCG Japan
5. M. avium + N.D. - + - + + + + + -
6. M. kansasii- N.D. + - - - + - + - -
7. M. marinum+ + - + + + + + + + +
8. M. scrofu-- - + - + + - + + + -
laceum
9. M. intercel-+ + - + - + + - + + -
lulare
10. M. fortui-- N.D. - - - - - - + - -
tum
11. M. xenopi+ + + + + + + + + + +
12. M. szulgai+ + - + + + + + + + +
-
+, positive reaction; -, no reaction, N.D. not determined.
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