Note: Descriptions are shown in the official language in which they were submitted.
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1
TUBERCULOSIS VACCINE AND METHOD FOR MAKING SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
60/667,243 filed on April 1 S' , 2005, which is hereby incorporated by
reference in its
entirety.
FIELD OF THE INVENTION
The present invention relates to an improved tuberculosis (TB) vaccine
and includes a method for making this vaccine. Vaccination against
mycobacterial
infection occurs through peptides and proteins expressed in response to sigma
K
stimulation. The invention further includes a method for determining the
potency of
tuberculosis (TB) strains which is based on the detection of an anti-sigma K
cell
response.
BACKGROUND OF THE INVENTION
Mycobacterium bovis Bacille Calmette-Guerin (BCG) strains have been
given to billions of people as vaccines against tuberculosis (TB) as their
derivation at
the Pasteur Institute between 1908 and 1921. While BCG immunization reliably
provides protection in animal models, their protection in human clinical
trials has
been inconsistent, leading to a number of hypotheses to explain these variable
findings (Fine, 1995; Agger and Andersen, 2002). One theory that has been the
subject of recent investigation pertains to the heterogeneity of BCG
preparations
distinct from each other and different from the vaccines first provided in the
early 20th
century (Mostowy et al ., 2003). Moreover, analysis of the elements implicated
in
BCG evolution indicates that genes encoding regulatory elements and antigenic
proteins are over-represented in the genomic deletions incurred by BCG strains
(Behr, 2002).
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The importance of antigenic proteins in TB pathogenesis and vaccine
development has been well established through their use in generating immunity
to
TB and the demonstration that disruption of the ESAT-6 region contributed to
the
derivation of BCG (Andersen, 1994; Harboe et al., 1996; Mahairas et al., 1996;
Pym
et al., 2002; Lewis et al., 2003; Brodin et al., 2004; Doherty et al., 2004).
Therefore,
the observation that BCG strains have suffered loss of antigenic proteins
during in
vitro passage is consistent with a potential impairment in their capacity to
serve as
immunizing agents. Of the described antigenic proteins of the Mycobacterium
tuberculosis complex, the M. bovis antigens MPB70 and MPB83 (also known as
MPT70 and MPT83 when studied in M. tuberculosis) figure prominently as
candidates for vaccine development (Fifis et al., 1994; Mustafa et al., 1998;
Chambers et al., 2002; 2004). Although the genes encoding these proteins have
not
been deleted in BCG evolution, production of these proteins by BCG vaccines
during
in vitro growth varies considerably. In certain BCG strains, such as BCG
Tokyo,
MPB70 represents the most abundant protein in the culture filtrate (Nagai et
al.,
1981). In other BCG strains, such as BCG Pasteur, production of MPB70 is
markedly
reduced, leading to the division of BCG strains into high-producers or low-
producers
(Miura et al., 1983; Harboe and Nagai, 1984). The same pattern of antigen
production across BCG strains has also been observed for MPB83, although the
differences have generally not been as dichotomous (Wiker et al., 1996).
To explore the reasons underlying these differences in production,
sequence-based analysis has been performed, but no mutations in the encoding
genes or their upstream promoter regions have been detected (Hewinson et al.,
1996; Vosloo et a/., 1997). Complementation of BCG Pasteur with mpb70 from BCG
Tokyo did not restore levels of MPB70 to those observed with BCG Tokyo,
suggesting differences in expression inherent to the parent BCG strain
(Matsumoto
et al., 1995). By targeted expression analysis, using reverse transcription
polymerase
chain reaction (RT-PCR) and Northern blots, an obvious difference in mpb70
transcription was observed between BCG Tokyo (high-producer) and BCG Pasteur
(low-producer) (Matsuo et al., 1995). However, the reason for this difference
has
remained unknown.
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Identification of the cause or causes behind the observed differences in
antigen production across tuberculosis vaccines would be useful for a variety
of
reasons, including the determination of the relative potency of the vaccines
and the
development of new, more effective vaccines. The present invention seeks to
meet
these and related needs.
SUMMARY OF THE INVENTION
Mycobacterium bovis Bacille Calmette-Guerin (BCG) strains are
genetically and phenotypically heterogeneous. Expression of the antigenic
proteins
MPB70 and MPB83 is known to vary considerably across BCG strains; however, the
reason for this phenotypic difference has remained unknown. By immunoblot, BCG
strains were separated into high- and low-producing strains. By quantitative
reverse
transcription polymerase chain reaction (RT-PCR), it was determined that
transcription of the antigen-encoding genes, mpb70 and mpb83, follows the same
strain pattern with mRNA levels reduced over 50-fold in low-producing strains.
Transcriptome comparison of the same BCG strains by DNA microarray revealed
two
gene regions consistently downregulated in low-producing strains compared with
high-producing strains, one including mpb70 (Rv2875) and mpb83 (Rv2873) and a
second that includes the predicted sigma factor, sigK. DNA sequence analysis
revealed a point mutation in the start codon of sigK in all low-producing BCG
strains.
Complementation of a low-producing strain, BCG Pasteur, with wild-type sigK
fully
restored MPB70 and MPB83 production. Microarray-based analysis and
confirmatory
RT-PCR of the complemented strains revealed an upregulation in gene
transcription
limited to the sigK and the mpb83/mpb70 gene regions. These data demonstrate
that
a mutation of sigK is responsible for decreased expression of MPB70 and MPB83
in
low-producing BCG strains and provide clues into the role of Mycobacterium
tuberculosis Sig K.
In one embodiment, the present invention relates to an improved
tuberculosis (TB) vaccine and includes a method for making this vaccine. This
vaccine is based on the complementation of a defective sigma K protein with a
wild-
type sigma K protein. In a specific embodiment, the nucleotide sequence for
wild-
type sigma K protein is introduced via a nucleotide vector.
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Host organisms to make the tuberculosis vaccine include but are not
limited to the following: M. tuberculosis, M. bovis, M. caprae, M. microti, M.
africanum, M. canettii, M. pinnipedii.
Alternatively, the tuberculosis vaccine is BCG and wild-type sigma K is
introduced into a host cell to stimulate the production of the immunogens
mpb70 and
mpb83 (or mpt70 an mpt83). Possible BCG strains are chosen from the following
non-limited group: BCG Russia, BCG Moreau, BCG Japan, BCG Sweden, BCG
Birkhaug, BCG Prague, BCG Glaxo, BCG Denmark, BCG Tice, BCG Connaught,
BCG Frappier, BCG Phipps and BCG Pasteur.
The vaccines produced in accordance with the present invention are
useful for the immunization of mammals against tuberculosis. Mammals include
but
are not limited to man, sheep, goats, pigs, deer, elk, bison, cows, steers,
bulls
and oxen.
In another embodiment, the invention relates to a method for determining
the potency of tuberculosis (TB) strains. In one embodiment, this method is
based
on the detection of an anti-sigma K cell response reflected in the overall
production of
mpb70, mpb83 or both of these antigenic proteins. (Instead of mpb70 and mpb83,
the antigenic proteins may be analogous proteins, such as mpt70 and mpt83.) In
another embodiment, the method is based on microarray hybridization and
analysis.
This method relies on the use of the newly identified nucleotide sequence
described
in the present specification for a mutant form of sigma K wherein the G at
position 3
is replaced by A. The nucleotide sequence of this mutant form of sigma K, as
well as
its peptidic sequence, are part of the present invention, as are nucleotide
and amino
acid fragments comprising the point mutation described here.
Further scope and applicability will become apparent from the detailed
description given hereinafter. It should be understood, however, that this
detailed
description, while indicating preferred embodiments of the invention, is given
by way
of example only, since various changes and modifications will become apparent
to
those skilled in the art.
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BRIEF DESCRIPTION OF THE FIGURES
In the appended drawings:
Figure 1: SDS-PAGE and immunoblotting of culture filtrate proteins (A
and C) and cell extracts (B and D) from BCG strains, using monoclonal
antibodies 1-
5 5c for MPB70 (A and B) and MBS43 for MPB83 (C and D). The strains used were
as
follows: Ja, BCG Japan; Pa, BCG Pasteur; Ru, BCG Russia; Sw, BCG Sweden; Bi,
BCG Birkhaug; Ph, BCG Phipps; GI, BCG Glaxo; Mo, BCG Moreau; Pr, BCG
Prague; Fr, BCG Frappier; Co, BCG Connaught; Ti, BCG Tice; De, BCG Denmark.
Figure 2: Expression of mpb70 (white) and mpb83 (grey) in M. bovis
BCG strains. Ratio of expression is to that of BCG Pasteur. All values were
normalized to the levels of sigA mRNA.
Figure 3: A and B. Expression of sigK (A) and mpb70 (white) and mpb83
(grey) (B) upon complementation of BCG Pasteur with sigK from M. tuberculosis
H37Rv, BCG Russia, BCG Birkhaug and BCG Pasteur. Values are expressed as a
ratio of mRNA copies in complemented strains compared with BCG
Pasteur::pMV306. All values were normalized to the levels of sigA mRNA and
error
bars represent the standard error of the mean. C and D. SDS-PAGE and
immunoblotting of culture filtrate proteins (C) and cell extracts (D) from
complemented BCG Pasteur, using monoclonal antibodies 1-5c for MPB70 (C) and
MBS43 for MPB83 (D). The strains used were as follows: 1, BCG Pasteur::pH37Rv;
2, BCG Pasteur::pRUSS; 3, BCG Pasteur::pBIRK; 4, BCG Pasteur::pPAST; 5, BCG
Pasteur::pMV306.
Figure 4: A. Expression analysis of the genes Rv0441c to Rv0450c. B.
Expression analysis for the genes Rv2870c to Rv2881c. Levels presented
represent
the ratio of mRNA copies in sigK-complemented strain to the control strain BCG
Pasteur::pMV306 (empty vector). All values were normalized to the levels of
sigA
mRNA and the ratio presented represents the mean of results from different
clones,
specifically BCG Pasteur::pH37Rv, BCG Pasteur::pRUSS and BCG Pasteur::pBIRK.
Figure 5: Protective efficacy against M. tuberculosis low-dose aerosol
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6
challenge in a guinea pig model.
Figure 6: Protective efficacy against M. tuberculosis i.v. challenge in
C57BL/6 mouse model; and
Figure 7: Immunogenicity, as measured by MPB70-induced gamma-
interferon production by spienocytes in mice.
DEFINITIONS AND TERMS
The terminology used herein is for the purpose of describing particular
embodiments only and is not intended to limit the scope of the present
invention.
Use of the singular forms "a," "an," and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example, reference to
"a
target polynucleotide" includes a plurality of target polynucleotides.
As used in this specification and claim(s), the words "comprising" (and
any form of comprising, such as "comprise" and "comprises"), "having" (and any
form
of having, such as "have" and "has"), "including" (and any form of including,
such as
"include" and "includes") or "containing" (and any form of containing, such as
"contain" and "contains"), are inclusive or open-ended and do not exclude
additional,
unrecited elements or process steps.
The term "about" is used to indicate that a value includes an inherent
variation of error for the device or the method being employed to determine
the
value.
Terms such as "connected," "attached," and "linked" may be used
interchangeably herein and encompass direct as well as indirect connection,
attachment, linkage or conjugation unless the context clearly dictates
otherwise.
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Where a value is explicitly recited, it is to be understood that values which
are about the same quantity or amount as the recited value are also within the
scope
of the invention, as are ranges based thereon.
Unless defined otherwise or the context clearly dictates otherwise, all
technical and scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this invention
belongs.
Although any methods and materials similar or equivalent to those described
herein
can be used in the practice or testing of the invention, the preferred methods
and
materials are now described.
All publications mentioned herein are hereby incorporated by reference
for the purpose of disclosing and describing the particular materials and
methodologies for which the reference was cited. The publications discussed
herein
are provided solely for their disclosure prior to the filing date of the
present
application. Nothing herein is to be construed as an admission that the
invention is
not entitled to antedate such disclosure by virtue of prior invention.
The terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic
acid molecule" are used interchangeably herein to refer to a polymeric form of
nucleotides of any length, and may comprise ribonucleotides,
deoxyribonucleotides,
analogs thereof, or mixtures thereof. These terms refer only to the primary
structure
of the molecule. Thus, the terms include triple-, double- and single-stranded
deoxyribonucleic acid ("DNA"), as well as triple-, double- and single-stranded
ribonucleic acid ("RNA"). They also include modified (for example, by
alkylation
and/or by capping) and unmodified forms of the polynucleotide.
More particularly, the terms "polynucleotide," "oligonucleotide," "nucleic
acid" and "nucleic acid molecule" include polydeoxyribonucleotides (containing
2-
deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA,
rRNA,
hRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide
which is an N- or C-glycoside of a purine or pyrimidine base, and other
polymers
containing a phosphate or other polyanionic backbone, and other synthetic
sequence-specific nucleic acid polymers provided that the polymers contain
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8
nucleobases in a configuration which allows for base pairing and base
stacking, such
as is found in DNA and RNA. There is no intended distinction in length between
the
terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid
molecule,"
and these terms are used interchangeably herein. Thus, these terms include,
for
example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' P5'
phosphoramidates,
2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double-
and
single-stranded RNA, and hybrids thereof, including, for example, hybrids
between
DNA and RNA, and also include known types of modifications, for example,
labels,
alkylation, "caps," substitution of one or more of the nucleotides with an
analog,
internucleotide modifications such as, for example, those with negatively
charged
linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those
containing
pendant moieties, such as, for example, proteins (including enzymes (e.g.
nucleases), toxins, antibodies, signal peptides, poly-L-lysine, etc.), those
with
intercalators (e.g., acridine, psoralen, etc.), those containing chelates (of,
e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those containing
alkylators,
those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as
well as
unmodified forms of the polynucleotide or oligonucleotide.
Standard A-T and G-C base pairs form under conditions which allow the
formation of hydrogen bonds between the N3-H and C4-oxy of thymidine and the
N1
and C6-NH2, respectively, of adenosine and between the C2-oxy, N3 and C4-NH2,
of cytidine and the C2-NH2, N'--H and C6-oxy, respectively, of guanosine.
Thus, for
example, guanosine (2-amino-6-oxy-9-.beta.-D-ribofuranosyl-purine) may be
modified to form isoguanosine (2-oxy-6-amino-9-.beta.-D-ribofuranosyl-purine).
Such
modification results in a nucleoside base which will no longer effectively
form a
standard base pair with cytosine. However, modification of cytosine (1-.beta.-
D-
ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-.beta.-D-
ribofuranosyl-
2-amino-4-oxy-pyrimidine) results in a modified nucleotide which will not
effectively
base pair with guanosine but will form a base pair with isoguanosine.
Isocytosine is
available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine may be
prepared by
the method described by Switzer et al. (1993) Biochemistry 32:10489-10496 and
references cited therein; 2'-deoxy-5-methyl-isocytidine may be prepared by the
method of Tor et al. (1993) J. Am. Chem. Soc. 115:4461-4467 and references
cited
therein; and isoguanine nucleotides may be prepared using the method described
by
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9
Switzer et al. (1993), supra, and Mantsch et al. (1993) Biochem. 14:5593-5601,
or by
the method described in U.S. Pat. No. 5,780,610 to Collins et al. Other
normatural
base pairs may be synthesized by the method described in Piccirilli et al.
(1990)
Nature 343:33-37 for the synthesis of 2,6-diaminopyrimidine and its complement
(1-
methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione). Other such modified
nucleotidic
units which form unique base pairs are known, such as those described in Leach
et
al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al., supra.
Hybridization conditions will typically include salt concentrations of less
than about 1 M, more usually less than about 500 mM and preferably less than
about
200 mM. Hybridization temperatures can be as low as 5 C., but are typically
greater
than 22 C., more typically greater than about 30 C., and preferably in excess
of
about 37 C. Longer fragments may require higher hybridization temperatures for
specific hybridization. Other factors may affect the stringency of
hybridization,
including base composition and length of the complementary strands, presence
of
organic solvents and extent of base mismatching, and the combination of
parameters
used is more important than the absolute measure of any one alone. Suitable
hybridization conditions for a given assay format can be determined by one of
skill in
the art; nonlimiting parameters which may be adjusted include concentrations
of
assay components, salts used and their concentration, ionic strength,
temperature,
buffer type and concentration, solution pH, presence and concentration of
blocking
reagents to decrease background binding such as repeat sequences or blocking
protein solutions, detergent type(s) and concentrations, molecules such as
polymers
which increase the relative concentration of the polynucleotides, metal ion(s)
and
their concentration(s), chelator(s) and their concentrations, and other
conditions
known in the art.
The target polynucleotide can be single-stranded, double-stranded, or
higher order, and can be linear or circular. Exemplary single-stranded target
polynucleotides include MRNA, rRNA, tRNA, hnRNA, ssRNA or ssDNA viral
genomes, although these polynucleotides may contain internally complementary
sequences and significant secondary structure. Exemplary double-stranded
target
polynucleotides include genomic DNA, mitochondrial DNA, chloroplast DNA, dsRNA
or dsDNA viral genomes, plasmids, phage, and viroids. The target
polynucleotide can
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be prepared synthetically or purified from a biological source. The target
polynucleotide may be purified to remove or diminish one or more undesired
components of the sample or to concentrate the target polynucleotide.
Conversely,
where the target polynucleotide is too concentrated for the particular assay,
the
5 target polynucleotide may be diluted.
Oligonucleotide probes or primers of the present invention may be of any
suitable length, depending on the particular assay format and the particular
needs
and targeted genomes employed. In general, the oligonucleotide probes or
primers
are at least 12 nucleotides in length, preferably between 15 and 24 molecules,
and
10 they may be adapted to be especially suited to a chosen nucleic acid
amplification
system. As commonly known in the art, the oligonucleotide probes and primers
can
be designed by taking into consideration the melting point of hybridization
thereof
with its targeted sequence (see below and in Sambrook et al., 1989, Molecular
Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al.,
1989,
in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
Probes of the invention can be utilized with naturally occurring
sugar-phosphate backbones as well as modified backbones including
phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the
like.
Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann.
Reports Med. Chem. 23:295 and Moran et al., 1987, Nucleic Acids Res., 14:5019.
Probes of the invention can be constructed of either ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA), and preferably of DNA.
The types of detection methods in which probes can be used include
Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern
blots
(RNA detection). Although less preferred, labeled proteins could also be used
to
detect a particular nucleic acid sequence to which it binds. Other detection
methods
include kits containing probes on a dipstick setup and the like.
Although the present invention is not specifically dependent on the use of
a label for the detection of a particular nucleic acid sequence, such a label
might be
beneficial, by increasing the sensitivity of the detection. Furthermore, it
enables
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11
automation. Probes can be labeled according to numerous well known methods
(Sambrook et al., 1989, supra). Non-limiting examples of labels include 3H,
14C, 32P,
and 35S. Non-limiting examples of detectable markers include ligands,
fluorophores,
chemiluminescent agents, enzymes, and antibodies. Other detectable markers for
use with probes, which can enable an increase in sensitivity of the method of
the
invention, include biotin and radionucleotides. It will become evident to the
person of
ordinary skill that the choice of a particular label dictates the manner in
which it is
bound to the probe.
As commonly known, radioactive nucleotides can be incorporated into
probes of the invention by several methods. Non-limiting examples thereof
include
kinasing the 5' ends of the probes using gamma 32P ATP and polynucleotide
kinase,
using the Klenow fragment of Pol I of E. coli in the presence of radioactive
dNTP
(e.g. uniformly labeled DNA probe using random oligonucleotide primers in low-
melt
gels), using the SP6/T7 system to transcribe a DNA segment in the presence of
one
or more radioactive NTP, and the like.
As used herein, "oligonucleotides" or "oligos" define a molecule having
two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo
will be
dictated by the particular situation and ultimately on the particular use
thereof and
adapted accordingly by the person of ordinary skill. An oligonucleotide can be
synthesized chemically or derived by cloning according to well known methods.
While they are usually in a single-stranded form, they can be in a double-
stranded
form and even contain a"regulatory region".
As used herein, a "primer" defines an oligonucleotide which is capable of
annealing to a target sequence, thereby creating a double stranded region
which can
serve as an initiation point for DNA synthesis under suitable conditions.
Primers can
be, for example, designed to be specific for certain alleles so as to be used
in an
allele-specific amplification system.
Amplification of a selected, or target, nucleic acid sequence may be
carried out by a number of suitable methods. See generally Kwoh et al., 1990,
Am.
Biotechnol. Lab. 8:14-25. Numerous amplification techniques have been
described
and can be readily adapted to suit particular needs of a person of ordinary
skill. Non-
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limiting examples of amplification techniques include polymerase chain
reaction
(PCR), ligase chain reaction (LCR), strand displacement amplification (SDA),
transcription-based amplification, the Q(3 replicase system and NASBA (Kwoh et
al.,
1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988,
BioTechnology
6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook
et
al., 1989, supra). Preferably, amplification will be carried out using PCR.
Polymerase chain reaction (PCR) is carried out in accordance with known
techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and
4,965,188 (the disclosures of all three U.S. Patent are incorporated herein by
reference). In general, PCR involves, a treatment of a nucleic acid sample
(e.g., in
the presence of a heat stable DNA polymerase) under hybridizing conditions,
with
one oligonucleotide primer for each strand of the specific sequence to be
detected.
An extension product of each primer which is synthesized is complementary to
each
of the two nucleic acid strands, with the primers sufficiently complementary
to each
strand of the specific sequence to hybridize therewith. The extension product
synthesized from each primer can also serve as a template for further
synthesis of
extension products using the same primers. Following a sufficient number of
rounds
of synthesis of extension products, the sample is analyzed to assess whether
the
sequence or sequences to be detected are present. Detection of the amplified
sequence may be carried out by visualization following EtBr staining of the
DNA
following gel electrophores, or using a detectable label in accordance with
known
techniques, and the like. For a review on PCR techniques (see PCR Protocols, A
Guide to Methods and Amplifications, Michael et al. Eds, Acad. Press, 1990).
As used herein, the term "gene" is well known in the art and relates to a
nucleic acid sequence defining a single protein or polypeptide. A "structural
gene"
defines a DNA sequence which is transcribed into RNA and translated into a
protein
having a specific amino acid sequence thereby giving rise to a specific
polypeptide or
protein. It will be readily recognized by the person of ordinary skill, that
the nucleic
acid sequence of the present invention can be incorporated into anyone of
numerous
established kit formats which are well known in the art.
A "heterologous" (e.g. a heterologous gene) region of a DNA molecule is
a subsegment of DNA within a larger segment that is not found in association
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13
therewith in nature. The term "heterologous" can be similarly used to define
two
polypeptidic segments not joined together in nature. Non-limiting examples of
heterologous genes include reporter genes such as luciferase, chloramphenicol
acetyl transferase, P-galactosidase, and the like which can be juxtaposed or
joined to
heterologous control regions or to heterologous polypeptides.
The term "vector" is commonly known in the art and defines a plasmid
DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into
which DNA of the present invention can be cloned. Numerous types of vectors
exist
and are well known in the art.
The term "expression" defines the process by which a gene is transcribed
into mRNA (transcription), the mRNA is then being translated (translation)
into one
polypeptide (or protein) or more.
The terminology "expression vector" defines a vector or vehicle as
described above but designed to enable the expression of an inserted sequence
following transformation into a host. The cloned gene (inserted sequence) is
usually
placed under the control of control element sequences such as promoter
sequences.
The placing of a cloned gene under such control sequences is often referred to
as
being operably linked to control elements or sequences.
Operably linked sequences may also include two segments that are
transcribed onto the same RNA transcript. Thus, two sequences, such as a
promoter
and a "reporter sequence" are operably linked if transcription commencing in
the
promoter will produce an RNA transcript of the reporter sequence. In order to
be
"operably linked" it is not necessary that two sequences be immediately
adjacent to
one another.
Expression control sequences will vary depending on whether the vector
is designed to express the operably linked gene in a prokaryotic or eukaryotic
host or
both (shuttle vectors) and can additionally contain transcriptional elements
such as
enhancer elements, termination sequences, tissue-specificity elements, and/or
translational initiation and termination sites.
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Prokaryotic expressions are useful for the preparation of large quantities
of the protein encoded by the DNA sequence of interest. This protein can be
purified
according to standard protocols that take advantage of the intrinsic
properties
thereof, such as size and charge (e.g. SDS gel electrophoresis, gel
filtration,
centrifugation, ion exchange chromatography...). In addition, the protein of
interest
can be purified via affinity chromatography using polyclonal or monoclonal
antibodies. The purified protein can be used for therapeutic applications.
The DNA construct can be a vector comprising a promoter that is
operably linked to an oligonucleotide sequence of the present invention, which
is in
turn, operably linked to a heterologous gene, such as the gene for the
luciferase
reporter molecule. "Promoter" refers to a DNA regulatory region capable of
binding
directly or indirectly to RNA polymerase in a cell and initiating
transcription of a
downstream (3' direction) coding sequence. For purposes of the present
invention,
the promoter is bound at its 3' terminus by the transcription initiation site
and extends
upstream (5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above background.
Within the
promoter will be found a transcription initiation site (conveniently defined
by mapping
with S1 nuclease), as well as protein binding domains (consensus sequences)
responsible for the binding of RNA polymerase. Eukaryotic promoters will
often, but
not always, contain "TATA" boxes and "CCAT" boxes. Prokaryotic promoters
contain
-10 and -35 consensus sequences, which serve to initiate transcription and the
transcript products contain Shine-Dalgarno sequences, which serve as ribosome
binding sequences during translation initiation.
As used herein, the designation "functional derivative" denotes, in the
context of a functional derivative of a sequence whether a nucleic acid or
amino acid
sequence, a molecule that retains a biological activity (either function or
structural)
that is substantially similar to that of the original sequence. This
functional derivative
or equivalent may be a natural derivative or may be prepared synthetically.
Such
derivatives include amino acid sequences having substitutions, deletions, or
additions of one or more amino acids, provided that the biological activity of
the
protein is conserved. The same applies to derivatives of nucleic acid
sequences
which can have substitutions, deletions, or additions of one or more
nucleotides,
provided that the biological activity of the sequence is generally maintained.
When
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relating to a protein sequence, the substituting amino acid generally has
chemico-
physical properties which are similar to that of the substituted amino acid.
The similar
chemico-physical properties include, similarities in charge, bulkiness,
hydrophobicity,
hydrophylicity and the like. The term "functional derivatives" is intended to
include
5 "fragments", "segments", "variants", "analogs" or "chemical derivatives" of
the subject
matter of the present invention.
Thus, the term "variant" refers herein to a protein or nucleic acid molecule
which is substantially similar in structure and biological activity to the
protein or
10 nucleic acid of the present invention.
The functional derivatives of the present invention can be synthesized
chemically or produced through recombinant DNA technology. All these methods
are
well known in the art.
The term "allele" defines an alternative form of a gene which occupies a
given locus on a chromosome.
As commonly known, a "mutation" is a detectable change in the genetic
material which can be transmitted to a daughter cell. As well known, a
mutation can
be, for example, a detectable change in one or more deoxyribonucleotide. For
example, nucleotides can be added, deleted, substituted for, inverted, or
transposed
to a new position. Spontaneous mutations and experimentally induced mutations
exist. A mutant polypeptide can be encoded from this mutant nucleic acid
molecule.
As used herein, the term "purified" refers to a molecule having been
separated from a cellular component. Thus, for example, a "purified protein"
has
been purified to a level not found in nature. A "substantially pure" molecule
is a
molecule that is lacking in most other cellular components.
As used herein, the terms "molecule", "compound", "agent" or "ligand" are
used interchangeably and broadly to refer to natural, synthetic or semi-
synthetic
molecules or compounds. The term "molecule" therefore denotes for example
chemicals, macromolecules, cell or tissue extracts (from plants or animals)
and the
like. Non limiting examples of molecules include nucleic acid molecules,
peptides,
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16
antibodies, carbohydrates and pharmaceutical agents. The agents can be
selected
and screened by a variety of means including random screening, rational
selection
and by rational design using for example protein or ligand modeling methods
such as
computer modeling. The terms "rationally selected" or "rationally designed"
are
meant to define compounds which have been chosen based on the configuration of
interacting domains of the present invention. As will be understood by the
person of
ordinary skill, macromolecules having non-naturally occurring modifications
are also
within the scope of the term "molecule". For example, peptidomimetics, well
known
in the pharmaceutical industry and generally referred to as peptide analogs
can be
generated by modeling as mentioned above. Similarly, in a preferred
embodiment,
the polypeptides of the present invention are modified to enhance their
stability. It
should be understood that in most cases this modification should not alter the
biological activity of the interaction domain.
For certainty, the sequences and polypeptides useful to practice the
invention include without being limited thereto mutants, homologs, subtypes,
alleles
and the like. It shall be understood that generally, the sequences of the
present
invention should encode a functional (albeit defective) interaction domain. It
will be
clear to the person of ordinary skill that whether an interaction domain of
the present
invention, variant, derivative, or fragment thereof retains its function in
binding to its
partner can be readily determined by using the teachings and assays of the
present
invention and the general teachings of the art.
A host cell or indicator cell has been "transfected" by exogenous or
heterologous DNA (e.g. a DNA construct) when such DNA has been introduced
inside the cell. The transfecting DNA may or may not be integrated (covalently
linked) into chromosomal DNA making up the genome of the cell. In prokaryotes,
yeast, and mammalian cells for example, the transfecting DNA may be maintained
on
a episomal element such as a plasmid. With respect to eukaryotic cells, a
stably
transfected cell is one in which the transfecting DNA has become integrated
into a
chromosome so that it is inherited by daughter cells through chromosome
replication.
This stability is demonstrated by the ability of the eukaryotic cell to
establish cell lines
or clones comprised of a population of daughter cells containing the
transfecting
DNA. Transfection methods are well known in the art (Sambrook et al., 1989,
supra;
Ausubel et al., 1994 supra).
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In general, techniques for preparing antibodies (including monoclonal
antibodies and hybridomas) and for detecting antigens using antibodies are
well
known in the art (Campbell, 1984, In "Monoclonal Antibody Technology:
Laboratory
Techniques in Biochemistry and Molecular Biology", Elsevier Science Publisher,
Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody- A
Laboratory
Manual, CSH Laboratories). The present invention also provides polyclonal,
monoclonal antibodies, or humanized versions thereof, chimeric antibodies and
the
like which inhibit or neutralize their respective interaction domains and/or
are specific
thereto.
From the specification and appended claims, the term therapeutic agent
should be taken in a broad sense so as to also include a combination of at
least two
such therapeutic agents. Further, the DNA segments or proteins according to
the
present invention can be introduced into individuals in a number of ways. For
example, erythropoietic cells can be isolated from the afflicted individual,
transformed
with a DNA construct according to the invention and reintroduced to the
afflicted
individual in a number of ways, including intravenous injection.
Alternatively, the
DNA construct can be administered directly to the afflicted individual, for
example, by
injection in the bone marrow. The DNA construct can also be delivered through
a
vehicle such as a liposome, which can be designed to be targeted to a specific
cell
type, and engineered to be administered through different routes.
For administration to humans, the prescribing medical professional will
ultimately determine the appropriate form and dosage for a given patient, and
this
can be expected to vary according to the chosen therapeutic regimen (e.g. DNA
construct, protein, cells), the response and condition of the patient as well
as the
severity of the disease.
Composition within the scope of the present invention should contain the
active agent (e.g. fusion protein, nucleic acid, and molecule) in an amount
effective to
achieve the desired therapeutic effect while avoiding adverse side effects.
Typically,
the nucleic acids in accordance with the present invention can be administered
to
mammals (e.g. humans) in doses ranging from 0.005 to 1 mg per kg of body
weight
per day of the mammal which is treated. Pharmaceutically acceptable
preparations
and salts of the active agent are within the scope of the present invention
and are
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well known in the art (Remington's Pharmaceutical Science, 16th Ed., Mack
Ed.).
For the administration of polypeptides, antagonists, agonists and the like,
the amount
administered should be chosen so as to avoid adverse side effects. The dosage
will
be adapted by the clinician in accordance with conventional factors such as
the
extent of the disease and different parameters from the patient. Typically,
0.001 to
50 mg/kg/day will be administered to the mammal.
The present invention may be included within a kit for determining the
characteristics (such as the potency) of a tuberculosis vaccine, comprising a
nucleic
acid, a protein or a ligand in accordance with the present invention. For
example, a
compartmentalized kit in accordance with the present invention includes any
kit in
which reagents are contained in separate containers. Such containers include
small
glass containers, plastic containers or strips of plastic or paper. Such
containers
allow the efficient transfer of reagents from one compartment to another
compartment such that the samples and reagents are not cross-contaminated and
the agents or solutions of each container can be added in a quantitative
fashion from
one compartment to another. Such containers will include a container which
will
accept the test sample (DNA protein or cells), a container which contains the
primers
used in the assay, containers which contain enzymes, containers which contain
wash
reagents, and containers which contain the reagents used to detect the
extension
products.
EXPERIMENTAL PROCEDURES
Bacterial cultures
Unless otherwise stated, BCG strains were grown at 37 C in Middlebrook
7H9 medium (Difco Laboratories, Detroit, MI) containing 0.05% Tween 80 (Sigma-
Aldrich, St. Louis, MO) and 10% albumin-dextrose-catalase (Becton Dickinson
and
Co., Sparks, MD) supplement on a rotating platform (Wheaton). Transformed BCG
strains were resuspended in 7H9 containing 15% glycerol and frozen in 1 ml
aliquots
at -80 C until needed. Frozen bacteria were thawed and diluted in fresh 7H9
medium
containing 10% albumin-dextrose-catalase and grown with rotation at 37 C.
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PCR amplification and sequencing across Rv0445c (sigK)
The sequence of Rv0445c (sigK) was determined by amplifying the gene
and flanking regions from isolates of M. canettii, M. tuberculosis H37Rv, M.
tuberculosis H37Ra, Mycobacterium africanum (n = 2), M. microti (n = 2), M.
caprae
(n = 2), M. bovis (n = 2) and 13 members of the BCG family - BCG Russia (ATCC
35740), BCG Moreau, BCG Japan, BCG Sweden, BCG Birkhaug (ATCC 35731),
BCG Prague, BCG Glaxo (ATCC 35741), BCG Denmark 1331 (ATCC 35733), BCG
Tice (ATCC 35743), BCG Frappier (ATCC 35735), BCG Connaught, BCG Phipps
(ATCC 35744) and BCG Pasteur 1173. The sequence was amplified using Primers
were left primer: 5'- agctcgagcagctcaaaatc-3'; and right primer: 5'-
acgcgtcaccccaactact-3' and amplicons were sequenced by di-deoxy terminal
sequencing at the McGill University and Genome Quebec Innovation Center. To
look
for differences between the amplified sequence and the prototype genome
sequences, results were compared by BLAST analysis to M. tuberculosis H37Rv
using Tuberculist (http://genolist.pasteur.fr/TubercuList/), M. tuberculosis
210 and
CDC1551 using the sequences provided at NCBI
(http://www.ncbi.nlm.nih.gov/sutils/genom-table.cgi), M. bovis AF2122/97 using
Bovilist (http://qenolist.pasteur.fr/BoviList/), and the assembly sequence of
BCG
Pasteur (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/m bovis).
RNA extraction
BCG strains grown to an OD600 of 0.3-0.5 were pelleted by
centrifugation, resuspended in 1 ml of wash buffer (0.5% Tween 80, 0.8% sodium
chloride) and transferred to 1.5 mi screw-cap cryovials. RNA was extracted by
a
modified phenol-chloroform extraction protocol as previously described (Belley
et al.,
2004). Genomic DNA contamination was removed by RNAeasy on-column digestion,
following the manufacturer's protocol (Qiagen, Mississauga, Canada). The
quality of
RNA was confirmed by denaturing gel electrophoresis (formaldehyde).
Real-time quantitative RT-PCR
Targeted gene expression levels were determined using RTPCR with molecular
beacons or sybr green, according to established protocols (Manganelli et al.,
1999;
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Mostowy et al., 2004). To provide a normalization standard for mRNA
expression,
expression of sigA was also determined, and the level of expression of a gene
of
interest divided by that of sigA to normalize for differences in total mRNA
extracted
(Manganelli et al., 1999). Sequences of the primers used for molecular beacon
and
5 sybr green analysis and the sequences of the molecular beacons used are as
listed
in Table 4.
Microarray analysis
10 Microarray hybridization and analysis were performed as previously
described (Mostowy et al., 2004). In brief, mRNA from BCG strains and
complemented strains was extracted during log-phase in vitro growth and
labelled
with Cy3 or Cy5 dUTP by reverse-transcriptase (Amersham Biosciences). Labelled
cDNA was hybridized to microarrays composed of oligonucleotide probes from the
15 TB Array-Ready Oligo SetTM (Operon) that had been printed onto
SigmascreenTM
microarray slides (Sigma). Initial comparisons were BCG Russia versus BCG
Pasteur, and BCG Birkhaug versus Denmark. After complenting BCG Pasteur with
wild-type sigK, comparisons of Pasteur::sigK versus Pasteur::pMV306 (empty
vector)
were also performed. In each case, duplicate hybridizations were performed for
each
20 dye combination (Cy3/Cy5 and Cy5/Cy3), resulting in four hybridizations per
comparison. Hybridized arrays were scanned with Scanarray 5000XL (PerkinElmer,
Freemont, CA) and hybridization results were quantified with Scanalyze
software
(http://rana.Stanford.EDU/software/).
Array analysis was performed as previously described (Mostowy et al.,
2004) in order to determine a z-score, indicative of how many standard
deviations a
data point lies from the population mean, for each gene. z-scores for each
gene were
averaged across replicates within each experiment to minimize the probability
of
observing such variation by chance alone and genes with average z-scores of 2
or
greater are presented.
Complementation of sigK
To complement BCG strain Pasteur, the sigK region (including the
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21
complete gene and 288 bp upstream) was amplified via PCR from M. tuberculosis
H37Rv, and BCG strains Russia and Birkhaug. As a control, the sigK from BCG
Pasteur was also complemented to determine the effect of having a second copy
of
the mutated gene. To complement, PCR was performed using the following primers
Rv0445cL and sigKR (left primer: 5'-agctcgagcagctcaaaatc-3'; right primer: 5'-
acgcgtcaccccaactact-3') and amplified products were cloned into the T-vector,
pDRIVE (Qiagen). The sigK region was then removed by digestion with Hindlll
and
Kpnl and ligated to the integrative mycobacterial vector pMV306 (de Stover et
al.,
1991) cut with the same restriction endonucleases. Integrity of the cloned
genes was
confirmed by DNA sequencing, then the resulting plasmids (pH37Rv, pRUSS, pBIRK
and pPAST) were electroporated into M. bovis BCG Pasteur cells, using
previously
described methods (Belley et al., 2004). The empty pMV306 vector was also
included as a control. Transformants were grown at 37 C on Middlebrook 7H10
agar
supplemented with 10% ADC [albumin (bovine fraction V), dextrose and catalase;
BD/BBL media] and kanamycin (25 mg ml-1). Complementation was PCR-confirmed
by amplifying the sigK gene with primers specific for the regions of pMV306
flanking
the sigK insert and these amplicons were sequence-confirmed for all
transformants.
Protein preparation and immunoblot analysis
BCG strains
BCG strains were cultivated as surface pellicles on liquid synthetic Sauton
medium
for 3 weeks at 37 C. The bacteria were washed and disrupted by a bead beater
to
yield a cellular extract and the culture medium was filtered to remove
residual
bacteria and concentrated by ammonium sulphate precipitation at 80%
saturation.
The antigens were separated under reducing conditions by horizontal SDSPAGE in
precast 8-18% gradient Excel gel using a Multiphor II unit 2117 (Amersham
Pharmacia). After separation, the proteins were transferred to a
nitrocellulose
membrane (pore size, 0.2 mm) by diffusion blotting (Olsen and Wiker, 1998) and
the
gel was stained with CBB. The membranes were blocked with PBS containing 2%
bovine serum albumin (BSA) and 1% gelatin and incubated with antibodies
overnight.
Bound antibodies were recognized by horseradish peroxidase (HRP)-labelled anti-
rabbit or anti-mouse Ig. As substrate, 3,3-diaminobenzidine was added to
visualize
the bound antibodies.
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BCG pasteur complemented strains
Cultures were grown at 37 C in 7H9 with 10% ADC, supplemented with
kanamycin (25 mg ml-1) for 7 days. The cultures were then centrifuged and the
supernatant was filtered with a 0.22 mm membrane filter and concentrated with
an
Amicon Ultra-15 Centrifugal Filter Unit, 10 000 MWCO. Cell pellets were frozen
and
whole-cell lysates were prepared by resuspending the cell pellet in 100 ml of
PBS
and boiling for 20 min. The culture filtrate proteins (CFP) were precipitated
by the
following protocol: 1 volume of sample was mixed with 3 volumes of methanol, 1
volume of chloroform, 4 volumes of water. Samples were centrifuged at max
speed
(-13 200 rpm) for 1 min. The upper phase was removed and replaced with 4
volumes of methanol and mixed briefly. Protein was pelleted at max speed for
15 min
(Wessel and Flugge, 1984). Protein was resuspended in 100 ml of PBS and the
final
concentration was determined using Coomassie Plus Protein Determination Kit
(Pierce) following standard protocol.
A total of 10 mg of CFP for each sample or 2 ml of cell lysate was loaded
in each lane. Samples were added to SDSloading buffer and heated to 80 C for 5
min. SDS-PAGE was performed under reducing conditions using the Mini-PROTEAN
3 electrophoresis system (Bio-Rad) with 12% polyacrylamide gels. Proteins were
transferred to a polyvinylidene difluoride membrane. Membranes were blocked in
PBS containing 2% BSA and 0.05% Tween 20, then probed with primary antibodies
for 1 h at room temperature. Bound antibodies were recognized by HRP-labelled
anti-mouse Ig. All antibodies were diluted in PBST with 1% BSA. Mouse
monoclonal
antibodies 1-5C (anti-MPB70) and MBS43 (anti-MPB83) (Wiker et al., 1998) were
used at a dilution of 1/500 and the HRP-conjugated anti-mouse antibody was
used at
a dilution of 1/10 000. Protein bands were detected using ECL PIusTMWestern
Blotting Detection Reagents (Amersham).
DETAILED DESCRIPTION
lmmunoblotting M. bovis BCG culture filtrate proteins and whole-cell extracts
To determine the production of MPB70 and MPB83 across BCG strains,
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culture filtrate proteins and whole-cell extracts blinded to strain identity
were
analysed. Upon decoding the samples, MPB70 was detected in substantial amounts
in the culture filtrates of BCG Russia, Birkhaug, Sweden, Japan and Moreau and
not
in the remaining strains (Figure 1A). In the whole-cell extracts, MPB70 was
not
detectable in any strain (Figure 1 B). MPB83 was detected in both the culture
filtrate
proteins and the wholecell extracts of BCG Russia, Birkhaug, Sweden, Japan and
Moreau (Figure 1 C). In the remaining strains of BCG, MPB83 could be detected
in
low amounts in the wholecell extracts (Figure 1D). These results are in
agreement
with previous results from a subset of these strains (Miura et al., 1983;
Harboe and
Nagai, 1984; Wiker et al., 1996) and indicate a clear delineation between
strains
obtained from the Pasteur Institute until 1927 (high-producers) versus strains
obtained, either directly or indirectly, in 1931 or later (low-producers).
Transcription of mpb70 and mpb83 in BCG strains
To determine whether transcriptional differences might correlate with
variations in protein production, quantitative RT-PCR was employed with
molecular
beacons to estimate relative mRNA levels for mpb70 and mpb83. As
immunoblotting
results pointed to distinctions between strains obtained before or after the
interval
1927-1931, the first and last strain obtained from each group were selected
(BCG
Russia and BCG Birkhaug for the earlier/high-producing group and BCG Danish
and
BCG Pasteur for the later/low-producing group). Consistent with previous
reports
indicating different mRNA expression (Matsuo et al., 1995), measured levels of
mpb70 and mpb83 mRNA were profoundly lower in BCG Danish and BCG Pasteur
as compared with BCG Birkhaug and BCG Russia with a calculated difference
greater than 50-fold (Figure 2).
Microarray analysis of BCG strains
To look for other differences in gene expression that might coincide with
transcription of mpb70 and mpb83, the same four BCG strains were studied by
whole
genome microarray, directly comparing BCG Russia with BCG Pasteur and BCG
Birkhaug with BCG Danish (Table 1). Consistent with the RT-PCR data, levels of
mpb70 and mpb83 were significantly lower in BCG Pasteur and BCG Danish, as
compared with BCG Russia and BCG Birkhaug. Also in this region, Rv2876 and
Rv2878c showed decreased expression in late/low-producing strains compared
with
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early/high-producing strains. A second region that showed consistent
downregulation
in the low-producing strains was Rv0445c-Rv0449c. Of these genes, the only
putative transcriptional regulator is Rv0445c (sigK), which is predicted to
encode an
alternate sigma factor, prompting further analysis.
Sequence analysis of sigK
Comparison of sigK across sequenced genomes indicated two
polymorphisms in BCG Pasteur compared with M. tuberculosis H37Rv. First,
located
at nucleotide -31 upstream from the sigK start codon, the adenine residue is
replaced
by a thymidine residue in BCG Pasteur. Sequencing this region across members
of
the M. tuberculosis complex and 13 BCG strains revealed that this mutation
represents an M. tuberculosis polymorphism; Mycobacterium canettii,
Mycobacterium
microti, Mycobacterium caprae, M. bovis and all BCG strains have the thymidine
residue at the -31 upstream position while sequenced M. tuberculosis strains
(H37Rv, 210 and CDC1551) have the adenine residue. Second, in M. tuberculosis
H37Rv, the start codon of sigK is the predominant start codon sequence AUG,
while
in BCG Pasteur there is a G-> A mutation at the third nucleotide, resulting in
an
altered AUA start codon. The AUG was observed in all members of the M.
tuberculosis complex except for the eight low-producing BCG strains obtained
after
1927 in which the altered AUA start codon was observed (Table 2). While the
codon
AUA has been identified as a functional start codon in Escherichia coli,
Bacillus
subtilis and Salmonella spp., levels of translation are substantially reduced
with this
codon compared with the conventional start codon AUG (Romero and Garcia, 1991;
Sussman et al., 1996). Because the start codon mutation correlated precisely
with
the BCG strains having decreased sigK and mpb83/mpb70 expression, the
functional
consequence of the sigK mutation was examined.
Effect of sigK complementation on transcription in BCG Pasteur
First, the effect of sigK complementation on its own expression by
quantitative RT-PCR was determined. Complementation with the empty vector or
with the mutant sigK resulted in no change of sigK expression, as seen with
BCG
Pasteur::pMV306 (empty vector) and BCG Pasteur::pPAST. This latter result
indicated that a second copy of the mutant gene did not alter levels of
transcription.
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In contrast, complementation with wild-type sigK, demonstrated with BCG
Pasteur::pH37Rv, BCG Pasteur::pRUSS and BCG Pasteur::pBIRK, showed a
marked increase in transcription of sigK (Figure 3A). Similar results were
obtained
with a second clone of each of the same strains (data not shown). As the
mutation in
5 sigK is predicted to impair translation, not transcription, and
complementation of wild-
type, but not mutant-type, sigK served to markedly increase sigK expression,
these
results signify that expression of this gene appears to be autoregulated, as
has been
described for other M. tuberculosis sigma factors (Helmann, 2002; Manganelli
et al.,
2004a).
Next, the effect of sigK complementation on mpb70 and mpb83 levels
was determined, using the same clones and mRNA preparations. In BCG
Pasteur::pPAST and BCG Pasteur::pMV306, levels of mRNA were comparable to
those previously demonstrated in low-producing strains of BCG. The same
strains in
which increased sigK expression was observed, BCG Pasteur::pH37Rv, BCG
Pasteur::pRUSS and BCG Pasteur::pBIRK, manifested highly increased
transcription
levels for mpb70 and mpb83, comparable to the levels observed with high-
producing
strains of BCG (Figure 3B). The same results were
obtained with a second clone of the same strains (data not shown).
Effect of sigK complementation on MPB70 and MPB83 production
To determine the effect of expression of wild-type sigK in BCG Pasteur
on protein synthesis, culture filtrate proteins and whole-cell lysates from
the sigK-
complemented strains of BCG Pasteur were analysed by sodium dodecyl sulphate-
polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. MPB70 was
detected in the culture filtrate proteins in BCG Pasteur::pH37Rv, BCG
Pasteur::pRUSS and BCG Pasteur::pBIRK, but could not be detected in
Pasteur::pPAST or BCG Pasteur::pMV306 (Figure 3C). Similarly, MPB83 was
detected in the whole-cell lysates of the same clones where MPB70 was
abundantly
detected in the culture filtrates (Figure 3D). Upon complementation of wild-
type sigK,
BCG Pasteur was able to produce MPB70 and MPB83 in a pattern consistent with
high-producing strains.
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Further expression analysis of genes neighbouring mpb70/mpb83 and sigK
Based on observed differences in expression of the genes neighbouring
sigK and mpb70/mpb83 by microarraybased analysis of four BCG strains,
quantitative RT-PCR was used to determine the effect of wild-type sigK
complementation on transcription in these regions. As shown in Figure 4A,
expression of Rv0443c-Rv0449c was increased over twofold with sigK
complementation. Transcription of Rv2874, Rv2876 and Rv2877c was increased
over 10-fold in the sigK-complemented strains compared with the control
strain.
Rv2878c (mpt53) was also increased, although to a lesser extent, with fourfold
increase seen in the sigKcomplemented strains. Contrary to expectations from
previous study indicating that Rv2871-Rv2874 are cotranscribed (Juarez et al.,
2001), no increase in expression of either Rv2871 or Rv2872 was detected,
suggesting that these two genes are under separate transcriptional control.
Based on
the differences in transcription, this sigK-regulated gene region includes
Rv2873
through Rv2878, but not Rv2872 or Rv2879 (Figure 4B).
Microarray-based analysis of sigK complementation
To further examine the role that sigK plays in global gene expression,
global transcription was analysed in BCG Pasteur::pH37Rv, BCG Pasteur::pRUSS
and BCG Pasteur::pBIRK compared with BCG Pasteur::pMV306 by DNA microarray.
Results revealed increased expression of the majority of genes presented in
Figure
4, with significant changes in expression as measured by both induction ratios
and z-
scores. Because of the stringency of the analysis performed, genes whose
expression was induced in three of four arrays, but whose expression could not
be
optimally quantified on the fourth, were not included in the table (e.g.
mpb83). Only
four genes were repressed with introduction of wild-type sigK; genes whose
expression was increased were restricted to the sigK and the mpb70/83 regions
(Table 3).
Comparative genomic studies have demonstrated the substantial genome
decay experienced by BCG strains during a half-century of in vitro passage
(Mostowy
et al., 2003). While DNA microarray-based comparisons have efficiently
uncovered
genomic deletions as the most evident form of evolution, these tools overlook
other
forms of genomic variability, such as duplications and single nucleotide
CA 02603298 2007-10-01
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27
polymorphisms (SNPs). With targeted study of specific genes has come the
recognition of numerous loss of function SNPs during BCG evolution, including
a
SNP that impairs synthesis of methoxymycolic acids (Behr et al., 2000), a SNP
predicted to decrease the DNA binding ability of a cAMP receptor protein (CRP)
homologue (Spreadbury et al., 2005) and the start codon mutation described in
this
report. Remarkably, along with the loss of the RD2 region (Mahairas et al.,
1996),
these three SNPs all coincide with the interval 1927-1931, suggesting either a
period
of considerable in vitro evolution or the replacement of one BCG stock with
another
at the Pasteur Institute in the late 1920s.
The data presented here indicate that the sigK SNP occurred between
1927 and 1931 at the Pasteur Institute and resulted, either directly or
indirectly, in a
major drop in production of the antigenic proteins MPB70 and MPB83. These two
proteins exhibit striking amino acid sequence homology (Hewinson et al., 1996)
and
both are exported, but localize differently. The single form of MPB70 is
secreted into
culture media while MPB83 is present in two forms, a 26 kDa lipoprotein which
remains associated with the mycobacterial cell wall and a 23 kDa form which is
found
in the culture media (Harboe et al., 1998). The structure of MPB70 has
recently been
solved and superimposition of MPB83 on the MPB70 structure confirmed the
overall
homology of the antigens (Carr et al., 2003). From immunologic studies, it is
known
that both proteins induce cellular and humoral responses in experimental
infection of
model hosts and natural infection of humans (Miura et al., 1983; Haslov et
al., 1987;
Fifis et al., 1994; Roche et al., 1994; Harboe et al., 1995; Wiker et al.,
1996;
Vordermeier et al., 2000; Lyashchenko et al., 2001). Based on these
observations,
both MBP70 and MPB83 have been developed as candidates for novel TB vaccine
development (Chambers et al., 2000; Morris et al., 2000; Al Attiyah et al.,
2003;
Tollefsen et al., 2003; Xue et al., 2004). As extracellular antigens have
consistently
been implicated in the induction of a protective immune response against M.
tuberculosis, it is remarkable that all BCG strains are unable to produce the
antigenic
proteins ESAT-6 and CFP-10 (lost with the RD1 deletion of 1908-1921), while
strains
obtained after 1927-1931 are also deficient (through the deletion of RD2) in
the
proteins MPB64 (Harboe et al., 1986; Li et al., 1993) and CFP-21 (Mahairas et
al.,
1996; Weldingh et al., 1998; Weldingh and Andersen, 1999) and functionally
deficient
in production of MPB70 and MPB83 via the sigK mutation described here.
CA 02603298 2007-10-01
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28
SigK is one of 10 extracytoplasmic function (ECF) sigma factors
annotated in the M. tuberculosis H37Rv genome. As the name suggests, these
regulatory elements mediate responses to changing external conditions
(Manganelli
et al., 2001; 2004b; Ando et al., 2003; Hu et al., 2004), with a common
feature being
their control over relatively small regulons (Bashyam and Hasnain, 2004). For
instance, by microarray analysis, the regulon of sigC has been estimated to
contain
13, 14 and 18 genes, in exponential, early and late stationary phase growth,
respectively (Sun et al., 2004), consistent with present observations of two
regions,
comprising 13 genes, being consistently upregulated in wild-type sigK-
complemented
strains. Mutants of sigC, sigD, sigE and sigH all exhibit reduced virulence in
animal
models (Kaushal et al., 2002; Calamita et al., 2004; Raman et al., 2004; Sun
et al.,
2004), but to date, there have been no published papers looking specifically
at M.
tuberculosis sigK. In transposon site hybridization studies, neither sigK nor
any of
the other genes in the regulon identified here was observed to be essential
for in vivo
growth in a murine model (Sassetti and Rubin, 2003). However, these
experiments
averaged the results for M. tuberculosis H37Rv and BCG Pasteur, and BCG
Pasteur
is now shown to be functionally deficient in this regulon, therefore the
impact of
mutations in these genes may have been minimized. An epidemiologic study of M.
tuberculosis isolates in San Francisco used genomic hybridization studies to
determine deletions in strains that had successfully caused TB, thereby
generating a
list of genes that are apparently nonessential for disease (Tsolaki et al.,
2004). Of
224 genes disrupted in at least one clinical isolate, none of the genes
implicated in
the sigK nor the mpb83/70 regions is featured, suggesting that loss
of these genes may be detrimental to disease causation.
The relevance of sigK-regulated genes is supported by transcriptome
analysis of M. tuberculosis during intracellular conditions, where mpb70 and
mpb83
figure among the most highly induced genes, across time points and in both
activated
and non-activated macrophages (Schnappinger et al., 2003). Additionally, in a
microarray-based study of M. tuberculosis expression during murine infection,
sigK
and mpt53 were among those genes noted as significantly dysregulated in vivo
(Talaat et al., 2004). Together, these results point to a potential role of
the sigK
regulon in the pathogenesis of TB that merits further attention.
The data presented here explain the difference in expression between
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29
high-producing and low-producing BCG strains. However, MPB70 and MPB83 are
also differentially produced by M. tuberculosis and M. bovis. Although these
organisms have identical, wild-type AUG sigK start codons, in vitro expression
is low
(although inducible) in M. tuberculosis and constitutively high in M. bovis
(Wiker et
a/., 1996). The constitutive in vitro production of MBP70 and MPB83 observed
in M.
bovis may therefore stem from unregulated activity of SigK. Activity of an ECF
sigma
can be mediated by a second protein, the anti-sigma factor, that functions
post-
translationally as a negative regulator to prevent constitutive expression of
the target
regulon (Helmann, 2002; Manganelli et al., 2004a). The sigma/anti-sigma pair
are
usually adjacent and co-transcribed genes; for instance, in M. tuberculosis,
RshA
(Rv3221A) is the anti-sigma factor for SigH (Rv3223c) while UsfX (Rv3287c) is
the
anti-sigma factor for SigF (Rv3286c) (Beaucher et al., 2002; Song et al.,
2003).
Consistent with this pattern, Rv0444c may encode the anti-sigma factor for
sigK
(Rv0445c), and by extension, mutations in Rv0444c might result in unregulated
expression of sigK. Ongoing investigations are pursuing this possibility,
based on the
presence of two non-synomyous SNPs in Rv0444c restricted to M. tuberculosis
complex species presenting constitutively high MPB70 production (data not
shown).
As BCG vaccines are given to an estimated 2 million infants per week,
there are important practical implications of these findings. The sigK
mutation
described here impairs the production of two immunodominant antigens, MPB70
and
MPB83, as well as transcription of mpb53. The importance of this deficit for
TB
immunization is unknown because strains of BCG that produce these antigens
have
never been utilized in a randomized clinical trial, although results from some
observational studies have suggested a greater potency to some of the
highproducer
strains (Kroger et al., 1994; Vitkova et a/., 1995). Based on the number of
documented differences between BCG strains obtained before 1927 and those
obtained after 1931, there is compelling rationale to perform a human trial
comparing
BCG strains from these two groups. Furthermore, these results are also
applicable
towards efforts to develop improved vaccines against TB.
CA 02603298 2007-10-01
WO 2006/102767 PCT/CA2006/000503
Recent advances have demonstrated that recombinant strains of BCG
expressing M. tuberculosis antigens provide an important avenue towards more
effective vaccines (Horwitz et al., 2000; Pym et al., 2003). These constructs
may
benefit from the inherent MPB70 and MPB83 expression of the high-producing
5 strains obtained before 1931, or alternatively, by correcting the sigK
mutation in later
strains to achieve the same result.
Example 1: IMMUNIZATION EXPERIMENTS WITH GUINEA PIGS
Recombinant BCG Pasteur expressing sigK from BCG Russia were used
10 in the experiments.
Hartley guinea pigs were vaccinated with 103 CFU of recombinant BCG.
Guinea pigs were rested for 10 weeks and then infected with a low dose aerosol
of
M. tuberculosis H37Rv. Viable count was performed at day 30 post challenge on
5
15 guinea pigs per group (Figure 5).
The results are shown in Table 5. Preliminary real-time PCR analysis of
blood samples taken from guinea pigs post-vaccination, pre-challenge: IFN-y -
negative for all guinea pigs.
Example 2: IMMUNIZATION EXPERIMENTS WITH MICE
The protocol used for mice was similar to that used for the guinea pigs
described in Example 1. Vaccination was with 106 bacteria injected sub-
cutaneously. The mice were challenged with 104 of Mycobacterium tuberculosis
10
weeks later. Results appeared 4 and 8 weeks after challenge (Figure 6).
For immunogenicity, the vaccine was once again 106 bacteria injected
sub-cutaneously. Spleens were harvested 28 days later, splenocytes were
isolated
and plated in tissue culture plates, then stimulated with either nothing
(control), PHA,
MPB 70 (2 doses) with gamma interferon being read in the supernatant by ELISA
(Figure 7).
CA 02603298 2007-10-01
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31
Although the present invention has been described hereinabove by way
of preferred embodiments thereof, it can be modified without departing from
the spirit,
scope and the nature of the subject invention, as defined in the appended
claims.
CA 02603298 2007-10-01
WO 2006/102767 PCT/CA2006/000503
32
~
~
~
C_ T C ~ C C
~ fn C_ C C_ ~
O :222 2O 2
'n. o 2 2 0_ a Q~
4) p fl. C. n a). '~ V N a)
c L
a - ~ ~o c o c 2 c
c o
~ (o 0 S
~ U L~ L L L d ~ 7
a aE 0 0E E E 2 2
tn U d fn U U
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w +z o O
Z Q) N Z O
ra/) N C C C N () ' y~ C
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CA 02603298 2007-10-01
WO 2006/102767 PCT/CA2006/000503
33
Table 2 Sequence analysis of sigK across M. tuberculosis complex
members.
Strains 31 bp prior to start 3bp of start codon
codon
M. canettii T G
M.tuberculosis H37Rv A G
M.tuberculosis H37Ra A G
M. africanum T G
M. microti T G
M.caprae T G
M.bovis
T G
BCG Russia T G
BCG Moreau T G
BCG Japan T G
BCG Sweden T G
BCG Birkhaug T G
BCG Prague T A
BCG Glaxo T A
BCG Denmark T A
BCG Tice T A
BCG Connaught T A
BCG Frappier T A
BCG Phipps T A
BCG Pasteur T A
CA 02603298 2007-10-01
WO 2006/102767 PCT/CA2006/000503
34
~
~
co U
~
C 0 c: C
a~ cn c c c
m O ~
p 2 N N~ OO ~ p 2
a Q o o m m a a a c o
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.r
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E
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0, U
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(a
a.
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U > 0 d: N O CD N M M LA N N M O
w y N Lf) 4 iI) lC) C'") N CV O) r-~ 6 M N
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N
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C7 c
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m V N CD r M N N
co CNO M
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O U
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L
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~ 4) Q OL
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c
!On
v . . ~.~O' r~ OJ' Oi v co eV- tt lp CO
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14- v'r v rr ao w oao mCO W co
o 0 0 0 0> ~>~~~>>
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H
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WO 2006/102767 PCT/CA2006/000503
Table 4 Sequences of primers and molecular beacons for quantitative RT-PCR
Gene Left Primer (5' to 3') Right Primer (5' to 3') Beacon
SigA tgcagtcggtgctggaca cgcgcaggacctgtgagc cctcgcgtcgaagttgcgccatccgagcgagg
SigK agtttgactccgccaaaggt gcaccatagcgcacttcc gcagcctgtcgaccgagtccgttggctgc
mpb70 ctcgaacaatccggagttgaca acaccgtgtactgaccgctgtt
gcagccagctcaatccgcaagtaaacctgggctgc
mpb83 atcaactcaagactgacgccaaac caccttgcagggtctgatgg
gcagccagcatcctgacctaccacgtgaggctgc
Dxr gggacttgaggtcatcgaaa tatggaaatcacaggcagca
Rv2871 gtatcgatgacgagctgtacc ttggacgatagatcgacacc
Rv2872 gactatcggggtttgcttga tcaacatcggacgctaactg
DipZ tcggttggtatcaggcctac ggtccaagtggcgtagttgt
Rv2876 agtgggagttcgacgtcagt acgtgatcaggaaccagtcc
Rv2877c ggttccatgtatggctacgg agccagatagatcgctacgc
mpb53 gttcggtctggccaatacac cgtccagaaccacaacacc
Rv2879c gcgacgggtgtctattgagt taccgtgcaggaaactcctt
Rv2880c aacacggtctgcatttcctc cccatacccatgaacaccac
CdsA tggtcgttgtctgcatgatt gagccattttccgggtagac
Rv0441 c tgggtgccaaaaaggtagac ccacaacagggtgacttcg
PPE10 caattcggcactgatgtttg caggctaggtactgggttgc
Rv0443 cgggtgcaggatatacaggt tagtaccccgacagcaggtc
Rv0444c ggccgagcaagttctgac gcagccacatctgatacacg
Rv0446c tggcaactgtgggtattcaa cagcaggtaggtcatcagca
ufaA1 ccgacctttcgacctagttg tgaacattgcgcacgaatac
Rv0448c cttctacgtttcgccgtttc tcatcgcgatctgtcttgtc
Rv0449c cccacacccactatctggac acatcgacatttccgactcc
MmpL4 ctaaattcgcgaacgactcc cttccagtgacgggacaaat
MmpS4 cct aaaaca caaaccat ccacgatatttcccatcacc
CA 02603298 2007-10-01
WO 2006/102767 PCT/CA2006/000503
36
~
~~ ~ ~'Cl- J -0 ~
N -
00 1- Lf7 O
> Lf? CV 7r O
J O O O O
(a ~
cn LL
U)
u CV O O
or- o Lf) N O
c+ ) Cq N C1l
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E
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=~ ~ U uc) r- cq
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) = aa cy o 0
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<D (D
Q aD~ U~
U U U
=~
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37
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