Note: Descriptions are shown in the official language in which they were submitted.
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Methods of diagnosis and treatment of M. tuberculosis infection
and reagents therefor V
Related application data
This application claims priority from Australian Patent Application No.
2006900452
filed on January 31 2006, the contents of which are incorporated herein in
their
entirety.
Field of the invention
io The present invention relates to novel diagnostic, prognostic and
therapeutic reagents
for infection of an animal subject such as a hun?an by llI tuberculosis, and
conditions
associated with such infections, such as, for example, tuberculosis. More
particularly,
the present invention provides the first enabling disclosure of the expression
in an
infected subject of a protein of M. tziber ctalosis designated "S9" (SEQ ID
NO: 1) and
immunogenic epitopes thereof suitable for the preparation of immunological
reagents,
such as, for example, antigenic proteins/peptides and/or antibodies, for the
diagnosis,
prognosis and therapy of infection, and vaccine development.
Background of the invention
1. Generallnforrn.ation
As used herein the term "derived from" shall be taken to indicate that a
specified
integer may be obtained from a particular source albeit not necessarily
directly from
that source.
Unless the context requires otherwise or specifically stated to the contrary,
integers,
steps, or elements of the invention recited herein as singular integers, steps
or elements
clearly encompass both singular and plural forms of the recited integers,
steps or
elements.
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The embodiments of the invention described herein with respect to any single
embodiment and, in particular, with respect to any protein or a use thereof in
the
diagnosis, prognosis or therapy of M. tuberculosis shall be taken to apply
inutatis
nautandis to any other embodiment of the invention described herein.
The diagnostic embodiments described here for individual subjects clearly
apply
mutatis mutandis to the epidemiology of a population, racial group or sub-
group or to
the diagnosis or prognosis of individuals having a particular MHC restriction.
All such
variations of the invention are readily derived by the skilled artisan based
upon the
1o subject matter described herein.
Throughout this specification, unless the context requires otherwise, the word
"comprise", or variations such as "comprises" or "comprising", will be
understood to
imply the inclusion of a stated step or element or integer or group of steps
or elements
or integers but not the exclusion of any other step or element or integer or
group of
elements or integers.
Those skilled in the art will appreciate that the invention described herein
is susceptible
to variations and modifications other than those specifically described. It is
to be
understood that the invention includes all such variations and modifications.
The
invention also includes all of the steps, features, compositions and compounds
referred
to or indicated in this specification, individually or collectively, and any
and all
combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific examples
described
herein. Functionally equivalent products, compositions and methods are clearly
within
the scope of the invention, as described herein.
The present invention is performed without undue experimentation using, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology,
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3
proteomics, virology, recombining DNA technology, peptide synthesis in
solution,
solid phase peptide synthesis, and immunology. Such procedures are described,
for
example, in the following texts that are incorporated by reference:
1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I,
II, and III;
2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed.,
1985),
IRL Press, Oxford, whole of text;
3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL
Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-
22;
Atkinson et al., pp35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151;
4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J.
Higgins, eds., 1985) IRL Press, Oxford, whole of text;
5. Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press,
Oxford, whole of text;
6. Perbal, B., A Practical Guide to Molecular Cloning (1984);
7. Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press,
Inc.), whole of series;
8. J.F. Ramalho Ortigao, "The Chemistry of Peptide Synthesis" In: Knowledge
database of Access to Virtual Laboratory website (Interactiva, Germany);
9. Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R.L. (1976).
Biochein.
Biophys. Res. Cornmun. 73 336-342
10. Merrifield, R.B. (1963). J. Arn. Chem. Soc. 85, 2149-2154.
11. Barany, G. and Merrifield, R.B. (1979) in Tize Peptides (Gross, E. and
Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York.
12. Wunsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der
Organischen Chernie (Muler, E., ed.), vol. 15, 4th edn., Parts 1 and 2,
Thieme,
Stuttgart.
13. Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag,
Heidelberg.
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14. Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis,
Springer- verlag, Heidelberg.
15. Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.
16. Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986, Blacl.-well Scientific Publications).
17. Wilkins M. R., Williams K. L., Appel R. D. and Hochstrasser (Eds) 1997
Proteorne Research: New Frontiers in Functional Genomics Springer, Berlin.
io 2. Description of the related art
Tuberculosis is a chronic, infectious disease that is generally caused by
infection with
Alycobacteritini tuberculosis. It is a major disease in developing countries,
as well as an
increasing problem in developed areas of the world, with about eight million
new cases
and three million deaths each year. Although the infection may be asymptomatic
for a
considerable period of time, the disease is most commonly manifested as an
acute
inflammation of the lungs, resulting in fever and a productive cough. If left
untreated,
RI tuberculosis infection may progress beyond the primary infection site in
the lungs to
any organ in the body and generally results in serious complications and
death.
2o The problems of the rapidly growing global incidence of tuberculosis and
microbial
resistance have been often described by many workers in the health care
industry and
are well known to skilled artisans in that field. In particular there is a
growing
recognition that new diagnostics, drugs and vaccines are urgently needed.
The immunological mechanisms by which M. tuberculosis maintains and multiplies
within the host are poorly understood. Consequently, any new information
regarding
the immunological relationship between tuberculosis and the host could clearly
be
used in many different ways to improve diagnosis, therapy and treatment of
that
disease.
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The incidence of tuberculosis is especially common in late-staging AIDS
patients, a
majority of whoni suffer from it. In fact, HIV infection is a most important
risk factor
for the development of active tuberculosis in purified protein derivative
(PPD)-
tuberculin-positive subjects, and the risk of acquisition of tuberculosis
infection in
5 HIV-infected immune-suppressed individuals may be markedly enhanced compared
to
those individuals that are not HIV-infected. It is also likely that co-
infections with
HIV-1, and M. tuberculosis mediate a shortened HIV symptom-free period and
shortened survival time in subjects, possibly by triggering increased viral
replication
and virus load that results in depletion of CD4+ T-cells and immune deficiency
or
i.o immune suppression (Corbett et al 2003; Ho, Mem. Inst. Oswaldo Cruz, 91,
385-387,
1996).
The sequencing of the Mycobacterium tuberculosis genome has facilitated an
enormous
research effort to identify potential M. tuberculosis proteins that
theoretically may be
expressed by the organism. However, sequence data alone are insufficient to
conclude
that any particular protein is expressed in vivo by the organism, let alone
during
infection of a human or other animal subject. Nor does the elucidation of open
reading
frames in the genome of M. tubef culosis indicate that any particular protein
encoded or
actually expressed by the bacterium comprises any immunodominant B-cell
epitopes or
2o T-cell epitopes that are required for the preparation of diagnostic,
prognostic and
therapeutic immunological reagents. For example, to conclude that a particular
protein
of A7 tuberculosis or a peptide fragment derived there from has efficacy as a
diagnostic
reagerit in an immunoassay format, or is suitable for use in a vaccine
preparation, it is
necessary to show that the protein is expressed during infectious cycle of the
bacterium,
and that the host organism mounts an immune response to the protein, and/or to
a
peptide fragment that comprises a B cell epitope or T-cell epitope (e.g., CD8+-
restricted
CTL epitope).
The ability to grow M. tuberculosis in culture has provided a convenient model
to
identify expressed tuberculosis proteins in vitro. However, the culture
envirorunent is
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markedly different to the environment of a human macrophage, lung, or
extrapulmonary site where M. tuberculosis is found in vivo. Recent evidence
indicates
that the protein expression profile of intracellular parasites, such as, for
example, M.
tuberculosis, varies markedly depending on environmental cues, such that the
expression profile of the organism in vitro may not accurately reflect the
expression
profile of the organism in situ.
Infection with M. tuberculosis bacilli, or reactivation of a latent infection,
induces a
host response comprising the recruitment of monocytes and macrophages to the
site of
io infection. As more immune cells accumulate a nodule of granulomata forms
comprising immune cells and host tissue that have been destroyed by the
cytotoxic
products of macrophages. As the disease progresses, macrophage enzymes cause
the
hydrolysis of protein, lipid and nucleic acids resulting in liquefaction of
surrounding
tissue and granuloma formation. Eventually the lesion ruptures and the bacilli
are
released into the surrounding lung, blood or lymph system.
During this infection cycle, the bacilli are exposed to four distinct host
environments,
being alveoli macrophage, caseous granuloma, extracellular lung and
extrapulmonary
sites, such as, for example the kidneys or peritoneal cavities, lymph, bone,
or spine.
It is thought that bacilli can replicate to varying degrees in all these
environments,
however, little is known about the environmental conditions at each site. All
four host
environments are distinct, suggesting that the expression profile of M.
tuberculosis in
each environnient will be different.
Accordingly, the identification of M, tuberculosis proteins from logarithmic
phase
cultures does not necessarily suggest which proteins are expressed or highly
immunogenic in each environment in vivo. Similarly, the identification of M.
tuberculosis proteins in a macrophage grown in vitro will not necessarily
emulate the
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protein expression profile of M. tuberculosis in caseous granuloma, highly
aerated
lung, or at an extrapulmonary site having a low oxygen content.
Furthermore, M. tuberculosis infection within the host can be seen as a
dynamic event
where the host immune system is continually trying to encapsulate and destroy
bacilli
through destruction of infected macrophages. Consequently, the M.
t.uberculosis bacilli
progress through cycles of intracellular growth, destruction (where both
intracellular
and secreted bacterial proteins are exposed and destroyed), and rapid
extracellular
multiplication. Host and pathogen interaction is a result of many factors,
which can not
be replicated in vitro.
Accordingly, until the present invention, it was not clear which M.
tuberculosis proteins
were the most highly expressed and/or highly immunologically active or
immunogenic
proteins of M. tuberculosis in any particular environment in vivo.
There clearly remains a need for rapid and cost-effective diagnostic and
prognostic
reagents for determining infection by M. tuberculosis and/or disease
conditions
associated therewith.
Summary of invention
In work leading up to the present invention, the inventors sought to elucidate
the range
of proteins expressed by A2 tuberculosis in a range of in vivo environn7ents,
to thereby
identify liighly expressed and/or highly immunogenic M. tuberculosis proteins.
The inventors used a proteomics approach to identify M. tuberculosis proteins
expressed in vivo and present in the body fluids of a cohort of diseased
patients,
including sputum, pleural fluid, plasma and serum. An M. tuberculosis protein
was
identified in vivo by 2-dimensional electrophoresis of immunoglobulin-
containing sera,
or alternatively, mixtures of sera and plasma, obtained previously from a
cohort of M.
tuberculosis-infected patients. The amino acid sequences of peptide fragments
were
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8
determined by mass spectrometry, and shown to align to the amino acid sequence
of the
30S ribosomal protein postulated to be encoded by the M. tuberculosis genome,
designated "S9" (SEQ ID NO: 1). In particular, matched peptides aligned to
amino
acid residues 71-78 of the putative S9 protein i.e., sequence APLVTVDR (SEQ ID
NO:
2); amino acid residues 64-70 of the putative S9 protein i.e., sequence
VHQQLIK
(SEQ ID NO: 3); amino acid residues 49-54 of the putative S9 protein i.e.,
sequence
FDLNGR (SEQ ID NO: 4); and amino acid residues 64-78 of the putative S9
protein
i.e., sequence VHQQLIKAPLVTVDR (SEQ ID NO: 5).
io The inventors have also made antibodies that bind to S9-derived peptides
for the
development of antigen-based diagnostic and prognostic assays. For example,
antibodies have been prepared against recombinant Sp protein by immunization
of
chickens and mice, and against a synthetic peptide comprising the N-terminal
21 amino
acid residues of the S9 protein (i.e., SEQ ID NO: 6) bound to keyhole limpet
hemocyanin (KLH). For determining quantitative titer of antibodies, an N-
terminal
sequence of S9 protein was produced with a C-terminal spacer sequence and
attached
to biotin (SEQ ID NO: 7). As exemplified herein, antibodies raised against the
N-
terminal peptide (SEQ ID NO: 6) were shown to bind to isolated S9 protein, the
peptide
immunogen in Western blots, endogenous S9 protein in clinical samples e.g.,
sputum,
2o and S9 protein in the cytosol and membrane fractions of Mycobacteriun
tuberculosis
strain H37Rv. Also exemplified herein antibodies prepared against full-length
recombinant S9 protein are useful in ELISA and sandwich ELISA assays for
detecting
expression of S9 protein in both clinical and laboratory M. tuberculosis
isolates, and for
detecting S9 protein at very low levels and in samples comprising serum or
sputum.
Antibodies against the full-length recombinant protein are high-affinity
antibodies
capable of detecting M. tuber-culosis S9 protein at sub-nanogram/ml or sub-
picogram/ml levels.
In antigen-based assays, 100% of TB-positive subjects in a cohort of 20 South
African
3o TB subjects were detected using an antibody that binds to the protein
(i.e., 100%
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9
sensitivity). In contrast, only 25% of TB-negative subjects were detected in a
cohort of
20 Australian non-TB respiratory disease subjects e.g., having bronchitis or
pneumonia,
were detected using an antibody that binds to the protein (i.e., 75%
specificity). These
data indicate that the presence of the S9 protein is correlated with a TB
diagnosis.
In multianalyte assays, e.g., using an antibody that binds to S9 protein and
antibodies
that bind to one or more M. tuber=culosis proteins e.g., Bsx protein and/or
glutamine
synthetase (GS) protein, high sensitivity and specificity are also achieved.
For
example, a multianalyte assay using an antibody that binds to S9 protein and
Bsx
protein to screen cohorts comprising about 14-20 samples, sensitivity is about
83% and
specificity is about 85%.
Antibodies that bind to the amino acid sequence set forth in SEQ ID NO: 1 or a
B-cell
epitope thereof have also been shown to be present in subjects during
extrapulmonary
infection by M. tuberculosis, in at least one population. The detection of
such
antibodies is a suitable assay readout for the diagnosis of tuberculosis. In
this respect,
the inventors determined that recombinant S9 protein comprising the sequence
set forth
in SEQ ID NO: 1 and peptides comprising the immunodominant B-cell epitope
within
SEQ ID NO: 2-7 are useful in antibody-based diagnostic tests for tuberculosis,
including multianalyte tests, by virtue of their high sensitivity and
specificity. Other
peptides derived from the full-length sequence of the S9 protein are also
useful for such
tests, e.g., as primary ligands or as secondary ligands in a multi-analyte
assay format,
by virtue of their high specificity.
These findings have provided the means for producing novel diagnostics for the
detection of M. tuberculosis infection in a subject, and novel prognostic
indicators for
the progression of infection or a disease state associated therewith.
Preferably, the S9
protein or a B-cell epitope thereof is useful for the early diagnosis of
infection or
disease. It will also be apparent to the skilled person that such prognostic
indicators as
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described herein may be used in conjunction with therapeutic treatments for
tuberculosis or an infection associated therewith.
Accordingly, the present invention provides the means for producing novel
diagnostics
5 for the detection of M. tubei-culosis infection in a subject, and novel
prognostic
indicators for the progression of infection or a disease state associated
therewith, either
by detecting S9 solus or as part of a multi-analyte test. Preferably, the S9
protein or a
B-cell epitope thereof is useful for the early diagnosis of infection or
disease. It will
also be apparent to the skilled person that such prognostic indicators as
described
io herein may be used in conjunction with therapeutic treatments for
tuberculosis or an
infection associated therewith.
For example, the present invention provides an isolated or recombinant
immunogenic
S9 protein of Mycobczcteriun2. tuberculosis or an immunogenic S9 peptide or
immunogenic S9 fragment or epitope thereof.
Preferably, the isolated or recombinant immunogenic S9 protein of M.
tuberculosis
comprises the amino acid sequence set forth in SEQ ID NO: 1 or having an amino
acid
sequence that is at least about 95% identical to SEQ ID NO: 1.
Preferably, the immunogenic S9 peptide is a synthetic peptide. Preferably the
S9
peptide, fragment or epitope comprises at least about 5 consecutive amino acid
residues
of the sequence set forth in SEQ ID NO: 1, more preferably at least about 10
consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1,
even more
preferably at least about 15 consecutive amino acid residues of the sequence
set forth in
SEQ ID NO: 1, and still more preferably at least about 5 consecutive amino
acid
residues of the sequence set forth in SEQ ID NO: 1 fused to about 1-5
additional amino
acid residues at the N-terminus and/or the C-terminus.
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In a particularly preferred embodiment, the S9 peptide, fragment or epitope
comprises
an amino acid sequence set forth in any one of SEQ ID Nos: 2-7 and preferably,
a
sequence selected from the group consisting of SEQ ID NOs: 6 and 7, and more
preferably SEQ ID NO: 6, or an immunologically cross-reactive variant of any
one of
said sequences that comprises an amino acid sequence that is at least about
95%
identical thereto.
It will be apparent from the disclosure that a preferred immunogenic S9
peptide,
fragment or epitope comprises an amino acid sequence of at least about 5
consecutive
io amino acid residues positioned between about residue I to about residue 50
of SEQ ID
NO: 1, more preferably at least about 5 consecutive amino acid residues
positioned
between about residue 1 to about residue 25 of SEQ ID NO: 1. Still more
preferably, a
preferred immunogenic S9 peptide, fragment or epitope comprises an amino acid
sequence of at least about 5 consecutive amino acid residues positioned
between
residue 1 to residue 20 of SEQ ID NO: 1, corresponding to at least 5
consecutive
residues of the sequence set forth in SEQ ID NO: 6. This includes any peptides
comprising an N-terminal ei:tension of up to about 5 amino acid residues in
length
and/or a C-terminal extension of up to about 5 amino acid residues in length.
It is clearly within the scope of the present invention for the isolated or
recombinant
immunogenic S9 protein of MUcobacterium tuberculosis or an immunogenic S9
peptide
or immunogenic S9 fragment or epitope thereof to comprise one or more labels
or
detectable moieties e.g., to facilitate detection or isolation or
immobilization. Preferred
labels include, for example, biotin, glutathione-S-transferase (GST), FLAG
epitope,
hexa-histidine, (3-galactosidase, horseradish peroxidase, streptavidin or
gold.
The present invention also provides a fusion protein comprising one or more
immunogenic S9 peptides, fragments or epitopes according to any embodiment
described herein. For example, the N-terminal and C-terminal portions of S9
protein
can be fused. The skilled artisan will be aware that it is preferred to
include an internal
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linking residue e.g., cysteine in such compositions of matter. Alternatively,
a preferred
fusion protein comprises a linker separating an immunogenic S9 peptide from
one or
more other peptide moieties, such as, for example, a single amino acid residue
(e.g.,
glycine, cysteine, lysine), a peptide linker (e.g., a non-immunogenic peptide
such as a
poly-lysine or poly-glycine), poly-carbon linker comprising up to about 6 or 8
or 10 or
12 carbon residues, or a chemical linker. Such linkers may facilitate antibody
production or vaccine formulation e.g., by permitting linkage to a lipid or
hapten, or to
permit cross-linking or binding to a ligand. The expression of proteins as
fusions may
also enhance their solubility.
Preferred fusion proteins will comprise the S9 protein, peptide, fragment or
epitope
fused to a carrier protein, detectable label or reporter molecule e.g.,
glutathione-S-
transferase (GST), FLAG epitope, hexa-histidine, P-galactosidase, thioredoxin
(TRX)
(La Vallie et al., Bio/Technologv 11, 187-193, 1993), maltose binding protein
(MBP),
Eschericlaia coli NusA protein (Fayard, E.M.S., Thesis, University of
Oklahonia, USA,
1999; Harrison, inNovations 11, 4-7, 2000), E. coli BFR (Harrison, inNovations
11, 4-
7, 2000) and E. coli. GrpE (Harrison, inNovations 11, 4-7, 2000).
The present invention also provides an isolated protein aggregate comprising
one or
2o more immunogenic S9 peptides, fragments or epitopes according to any
embodiment
described herein. Preferred protein aggregates will comprise the protein,
peptide,
fragment or epitope complexed to an immunoglobulin e.g., IgA, IgM or IgG, such
as,
for example as a circulating immune complex (CIC). Exemplary protein
aggregates
may be derived, for example, derived from an antibody-containing biological
sample of
a subject.
The present invention also encompasses the use of the isolated or recombinant
immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9
peptide
or immunogenic S9 fragment or epitope thereof according to any embodiment
3o described herein for detecting a past or present infection or latent
infection by. M.
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tuberculosis in a subject, wherein said infection is determined by the binding
of
antibodies in a sample obtained from the subject to said isolated or
recombinant
immunogenic S9 protein or an immunogenic S9 peptide or immunogenic S9 fragment
or epitope.
The present invention also encompasses the use of the isolated or recombinant
immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9
peptide
or immunogenic S9 fragment or epitope thereof according to any embodiment
described herein for eliciting the production of antibodies that bind to M.
tuberculosis
io S9.
The present invention also encompasses the use of the isolated or recombinant
immunogenic S9 protein of Mvcobacterium tuber=culosis or an immunogenic S9
peptide
or immunogenic S9 fragment or epitope thereof according to any embodiment
described herein in the preparation of a medicament for immunizing a subject
e.g., to
produce antibodies against the S9 protein and/or to protect against infection
by M.
tuber=culosis.
The present invention also provides a pharmaceutical composition comprising
the
isolated or recombinant immunogenic S9 protein of Mycobacteriuna tuberculosis
or an
immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according
to
any embodiment described herein in combination with a pharmaceutically
acceptable
diluent, e.g., an adjuvant.
The present invention also provides an isolated nucleic acid encoding the
isolated or
recombinant immunogenic S9 protein of Mvcobacteriurn tuberculosis or an
immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according
to
any embodiment described herein eg., for the preparation of nucleic acid based
vaccines or for otherwise expressing the immunogenic polypeptide, protein,
peptide,
fragment or epitope.
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The present invention also provides a cell expressing the isolated or
recombinant
inlmunogenic S9 protein of Mycobacteriurn tuberculosis or an immunogenic S9
peptide
or immunogenic S9 fragment or epitope thereof according to any embodiment
described herein. The cell may preferably consist of an antigen- presenting
cell (APC)
that expresses the isolated or recombinant immunogenic S9 protein of
Mycobacteriurn
tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or
epitope
thereof e.g., on its surface.
io The present invention- also provides an isolated ligand, e.g., a small
molecule, peptide,
antibody, or immune reactive fragment of an antibody, that binds specifically
to the
isolated or recombinant immunogenic S9 protein of Mycobacteriurra tuberculosis
or an
inimunogenic S9 peptide or immunogenic S9 fragment or epitope thereof
according to
any embodiment described herein, or to a fusion protein or protein aggregate
comprising said immunogenic S9 protein, peptide, fragment or epitope.
Preferred
ligands are peptides or antibodies. Preferred antibodies include, for example,
a
monoclonal or polyclonal antibody preparation. This extends to any isolated
antibody-
producing cell or antibody-producing cell population, e.g., a hybridoma or
plasmacytoma producing antibodies that bind to a S9 protein or immunogenic
fragment
of a S9 protein or other immunogenic peptide comprising a sequence derived
from the
sequence of a S9 protein.
The present invention also provides for the use of the isolated ligand
according to any
embodiment described herein, especially any peptide ligand, antibody or an
immune-
reactive fragment thereof in medicine.
The present invention also provides for the use of the isolated ligand
according to any
embodiment described herein, especially any peptide ligand, antibody or an
immune-
reactive fragment thereof for detecting a past or present (i.e., active)
infection or a
latent infection by M. tuber=culosis in a subject, wherein said infection is
determined by
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the binding of the ligand to M. tuberculosis S9 protein or an immunogenic
fragment or
epitope thereof present in a biological sample obtained from the subject.
The present invention also provides for the use of the isolated ligand
according to any
5 embodiment described herein, especially any peptide ligand, antibody or an
immune-
reactive fragment thereof for identifying the bacterium M. tuberculosis or
cells infected
by M. tuberculosis or for sorting or counting of said bacterium or said cells.
The isolated ligand according to any embodiment described herein, especially
any
io peptide ligand, antibody or an immune-reactive fragment thereof, is also
useful in
therapeutic, diagnostic and research applications for detecting a past or
present
infection, or a latent infection, by Al. tzeberculosis as determined by the
binding of the
ligand to an M. tuberculosis S9 protein or an immunogenic fragment or epitope
thereof
present in a biological sample from a subject (i.e., an antigen-based
immunoassay).
Other applications of the subject ligands include the purification and study
of the
diagnostic/prognostic S9 protein, identification of cells infected with M.
tuberculosis,
or for sorting or counting of such cells.
2o The ligands are also useful in therapy, including prophylaxis, diagnosis,
or prognosis,
and the use of such ligands for the manufacture of a medicament for use in
treatment of
infection by M. tuberculosis. For example, specific humanized antibodies or
other
ligands are produced that bind and neutralize a S9 protein or M. tuberculosis,
especially
in vivo. The humanized antibodies or other ligands are used as in the
preparation of a
medicament for treating TB-specific disease or M. tuberculosis infection in a
human
subject, such as, for example, in the treatment of an active or chronic 1b1.
tuber=culosis
infection.
The present invention also provides a composition comprising the isolated
ligand
3o according to any embodiment described herein, especially any peptide
ligand, antibody
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16
or an immune-reactive fragment thereof, and a pharmaceutically acceptable
carrier,
diluent or excipient.
The present invention also provides a method of diagnosing tuberculosis or an
infection
by M. tuberculosis in a subject comprising detecting in a biological sample
from said
subject antibodies that bind to an immunogenic S9 protein or an immunogenic S9
peptide or immunogenic S9 fragment or epitope thereof, the presence of said
antibodies
in the sample is indicative of infection. In a related embodiment, the
presence of said
antibodies in the sample is indicative of infection. The infection may be a
past or
1o active infection, or a latent infection, however this assay format is
particularly useful
for detecting active infection and/or recent infection.
For example, the method may be an immunoassay, e.g., comprising contacting a
biological sample derived from the subject with the isolated or recombinant
immunogenic S9 protein of Mvcobacteriuna tuberculosis or an immunogenic S9
peptide
or immunogenic S9 fragment or epitope thereof according to any embodiment
described herein (e.g., a peptide comprising an amino acid sequence set forth
in any
one of SEQ ID Nos: 2-7 and preferably, a sequence selected from the group
consisting
of SEQ ID NOs: 6 and 7, and still more preferably SEQ ID NO: 6, or an
immunologically cross-reactive variant of any one of said sequences that
comprises an
amino acid sequence that is at least about 95% identical tliereto) for a time
and under
conditions sufficient for an antigen-antibody complex to form and then
detecting the
formation of an antigen-antibody complex. The sample is an antibody-containing
sample e.g., a sample that comprises blood or serum or an immunoglobulin
fraction
obtained from the subject. The sample may contain circulating antibodies , in
the form
of complexes with S9 antigenic fragments. Generally, the antigen-antibody
complex
will be detected in such assay formats using antibodies capable of binding to
the
patient's immunoglobulin e.g., anti-human Ig antibodies.
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17
It is within the scope of the present invention to include a multi-analyte
test in this
assay format, wherein multiple antigenic epitopes are used to confirm a
diagnosis
obtained using a S9 peptide. For example, the patient sample may be contacted
with S9
or immunogenic S9 peptide or fragment or epitope and with aA~1. tuber=culosis
Bsx
protein (e.g., SwissProt Database Accession No. 053759) or immunogenic peptide
derived there from, e.g., a peptide derived from a Bsx protein, or comprising
a
sequence, selected from the group consisting of: MRQLAERSGVSNPYL (SEQ ID
NO: 8), ERGLRKPSADVLSQI (SEQ ID NO: 9), LRKPSADVLSQIAKA (SEQ ID
NO: 10), PSADVLSQIAKALRV (SEQ ID NO: 11), SQIAKALRVSAEVLY (SEQ ID
1o NO: 12), AKALRVSAEVLYVRA (SEQ ID NO: 13), VRAGILEPSETSQVR (SEQ
ID No: 14), TAITERQKQILLDIY (SEQ ID NO; 15), SQIAKALRVSAEVLYVRAC
(SEQ ID NO: 16), MSSEEKLCDPTPTDD (SEQ ID NO: 17) and
VRAGILEPSETSQVRC (SEQ ID NO: 18). Immunogenic M tuberculosis Bsx and
peptide derivatives for detecting tuberculosis or infection by M. tuberculosis
are also
described in detail in the instant applicant's co-pending International Patent
Application
No. PCT/AU2005/001254 filed August 19, 2005 the disclosure of which is
incorporated herein in its entirety.
Alternatively, or in addition, the patient sample may be contacted with S9 or
inimunogenic S9 peptide or fragment or epitope and with a M. tuberculosis
glutamine
synthetase (GS) protein (e.g., SwissProt Database Accession No. 033342) or
immunogenic peptide derived there from, e.g., a peptide derived from a surface-
exposed region of a GS protein, or comprising the sequence
RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO: 19) or
WASGYRGLTPASDYNIDYAI (SEQ ID NO: 20). Immunogenic M. tteberculosis GS
and peptide derivatives for detecting tuberculosis or infection by M.
tuberculosis are
also described in detail in the instant applicant's co-pending International
Patent
Application No. PCT/AU2005/000930 filed June 24 2005 the disclosure of which
is
incorporated herein in its entirety.
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Assays for one or more secondary analytes e.g., antibodies that bind to Bsx
and/or
glutamine synthetase, are conveniently performed in the same manner as for
detecting
antibodies that bind to S9 in serum or plasma or other body fluid. The assays
may be
performed simultaneously or at different times, and using the same or
different patient
samples. The assays may also be performed in the same reaction vessel,
provided that
different detection systems are used to detect the different antibodies, e.g.,
anti-human
Ig labelled using different reporter molecules such as different coloured
dyes,
fluorophores, radionucleotides or enzynzes.
io As used herein, the term "infection" shall be understood to mean invasion
and/or
colonisation by a microorganism and/or multiplication of a micro-organism, in
particular, a bacterium or a virus, in the respiratory tract of a subject.
Such an infection
may be unapparent or result in local cellular injury. The infection may be
localised,
subclinical and temporary or alternatively may spread by extension to become
an acute
or chronic clinical infection. The infection niay also be a past infection
wherein
residual S9 antigen, or alternatively, reactive host antibodies that bind to
isolated S9
protein or peptides, remain in the host. The infection may also be a latent
infection, in
which the microorganism is present in a subject, however the subject does not
exhibit
symptoms of disease associated with the organism. Preferably, the infection is
a
pulmonary or extra-pulmonary infection by M. tuberculosis, and more preferably
an
extra-pulmonary infection. By "pulmonary" infection is meant an infection of
the
airway of the lung, such as, for example, an infection of the lung tissue,
bronchi,
bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, or
alveoli. By "extra-
pulmonary" is meant outside the lung, encompassing, for example, kidneys,
lymph,
urinary tract, bone, skin, spinal fluid, intestine, peritoneal, pleural and
pericardial
cavities.
The antibodies of the present invention are also useful in the diagnosis of
tuberculosis
or infection by M. tuberculosis. For example, the present invention also
provides a
method of diagnosing tuberculosis or infection by M. tuberculosis in a subject
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19
comprising detecting in a biological sample from said subject an immunogenic
S9
protein or an immunogenic S9 peptide or immunogenic S9 fragment or epitope
thereof,
wherein the presence of said protein or immunogenic fragment or epitope in the
sample
is indicative of disease, disease progression or infection. In a related
embodiment, the
presence of said protein or immunogenic fragment or epitope in the sample is
indicative
of infection.
For example, the method may be an immunoassay, e.g., comprising contacting a
biological sample derived from the subject with an antibody that binds to the
io endogenous S9 protein of Mycobacterium tuberculosis or an inimunogenic S9
peptide
or immunogenic S9 fragment or epitope thereof according to any embodiment
described herein (e.g., comprising an amino acid sequence set forth in any one
of SEQ
ID Nos: 2-7 and preferably, comprising SEQ ID NO: 6 or 7, and still more
preferably
SEQ ID NO: 6, or an immunologically cross-reactive variant of any one of said
sequences that comprises an amino acid sequence that is at least about 95%
identical
thereto) for a time and under conditions sufficient for an antigen-antibody
complex to
form and then detecting the formation of an antigen-antibody complex.
Preferred
samples according to this embodiment are those samples in which M.
tuberculosis or
peptide fragments from bacterial debris are likely to be found, or
immunoglobulin-
containing fraction, e.g., an extract from brain, breast, ovary, lung, colon,
pancreas,
testes, liver, muscle, bone or mixtures thereof; body fluid(s) such as sputum,
serum,
plasma, whole blood, saliva, urine, pleural fluid or mixtures thereof or
derivatives
thereof e.g., sputum, serum, plasma, whole blood, saliva, urine, pleural
fluid, etc. The
sample may contain circulating antibodies complexed with S9 antigenic
fragments.
It is within the scope of the present invention to include a multi-analyte
test in this
assay format, wherein multiple antibodies are used to confirm a diagnosis
obtained
using antibodies that bind to the S9 protein or epitope. For example, the
patient sample
may be contacted with antibodies that bind to S9 or immunogenic S9 peptide or
fragment or epitope and with antibodies that bind to M. tuberculosis Bsx
protein (e.g.,
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SwissProt Database Accession No. 053759) or antibodies that bind to an
immunogenic
peptide derived there from, e.g., a peptide derived from a Bsx protein, or
comprising a
sequence selected from the group consisting of: MRQLAERSGVSNPYL (SEQ ID NO:
8), ERGLRKPSADVLSQI (SEQ ID NO: 9), LRKPSADVLSQIAKA (SEQ ID NO:
5 10), PSADVLSQIAKALRV (SEQ ID NO: 11), SQIAhALRVSAEVLY (SEQ ID NO:
12), AKALRVSAEVLYVRA (SEQ ID NO: 13), VRAGILEPSETSQVR (SEQ ID No:
14), TAITERQKQILLDIY (SEQ ID NO; 15), SQIAKALRVSAEVLYVRAC (SEQ
ID NO: 16), MSSEEKLCDPTPTDD (SEQ ID NO: 17) and VRAGILEPSETSQVRC
(SEQ ID NO: 18). Antibodies that bind to an immunogenic M tuberculosis Bsx
1o protein or peptide for detecting tuberculosis or infection by Rl.
tuberculosis are also
described in detail in the instant applicant's co-pending International Patent
Application
No. PCT/AU2005/001254 filed August 19, 2005 the disclosure of which is
incorporated herein in its entirety.
15 Alternatively, or in addition, the patient sample may be contacted with
antibodies that
bind to S9 protein or immunogenic S9 peptide or fragment or epitope and with
antibodies that bind to an immunogenic M. tuberculosis glutamine synthetase
(GS)
protein (e.g., SwissProt Database Accession No. 033342) or antibodies that
bind to an
immunogenic peptide derived from GS, e.g., a peptide derived from a surface-
exposed
2o region of a GS protein, or comprising the sequence
RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO: 19) or
WASGYRGLTPASDYNIDYAI (SEQ ID NO: 20). Antibodies that bind to an
immunogenic M. tuberculosis GS or peptide for detecting tuberculosis or
infection by
M. tuberculosis are also described in detail in the instant applicant's co-
pending
International Patent Application No. PCT/AU2005/000930 filed June 24 2005 the
disclosure of which is incorporated herein in its entirety.
Assays for one or more secondary analytes e.g., Bsx and/or glutamine
synthetase, are
conveniently performed in the same manner as for detecting S9 protein in the
sample.
3o The assays may be performed simultaneously or at different times, and using
the same
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21
or different patient samples. The assays may also be performed in the same
reaction
vessel, provided that different detection systems are used to detect the bound
antibodies, e.g., secondary antibodies that bind to the anti-S9 antibodies and
antibodies
that bind to the secondary analyte(s).
As with antibody-based assays, antigen-based assay systems can comprise an
immunoassay e.g.; contacting a biological sample derived from the subject with
one or
more isolated ligands according to any embodiment described herein, especially
any
peptide ligand, antibody or an immune-reactive fragment thereof capable of
binding to
io a S9 protein or an immunogenic fragment or epitope thereof, and detecting
the
formation of a complex e.g., an antigen-antibody complex. In a particularly
preferred
embodiment, the ligand is an antibody, preferably a polyclonal or monoclonal
antibody
or antibody fragment that binds specifically to the isolated or recombinant
immunogenic S9 protein of Mycobacteriutn tubef=culosis or an immunogenic S9
peptide
or immunogenic S9 fragment or epitope thereof according to any embodiment
described herein or to a fusion protein or protein aggregate comprising said
immunogenic S9 protein, peptide, fragment or epitope. Vdhilst useful for
subjects who
are not immune-compromized, e.g., HIV-negative subjects, the assay is also
particularly useful for detecting TB in a subject that is immune compromised
or
immune deficient, e.g., a subject that is infected with human immunodeficiency
virus
(i.e., "HIV+"). The samples used for conducting such assays include, for
exaniple, (i)
an extract from a tissue selected from the group consisting of brain, breast,
ovary, lung,
colon, pancreas, testes, liver, muscle, bone and mixtures thereof; (ii) body
fluid(s)
selected from the group consisting of sputum, serum, plasma, whole blood,
saliva,
urine, pleural fluid and mixtures thereof; and (iii) samples derived from body
fluid(s)
selected from the group consisting of sputum, serum, plasma, whole blood,
saliva,
urine, pleural fluid and mixtures thereof.
The present invention also provides a method for determining the response of a
subject
3o having tuberculosis or an infection by M. tuberculosis to treatment with a
therapeutic
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compound for said tuberculosis or infection, said method comprising detecting
a S9
protein or an immunogenic fragment or epitope thereof in a biological sample
from said
subject, wherein a level of the protein or fragment or epitope that is
enhanced, or not
decreased or decreasing, compared to the level of that protein or fragment or
epitope
detectable in a normal or healthy subject indicates that the subject is not
responding to
said treatment or has not been rendered free of disease or infection. For
example, the
method can comprise an immunoassay e.g., contacting a biological sample
derived
from the subject with one or more antibodies capable of binding to a S9
protein or an
immunogenic fragment or epitope thereof, and detecting the formation of an
antigen-
io antibody complex. In a particularly preferred embodiment, an antibody is an
isolated
or recombinant antibody or immune reactive fragment of an antibody that binds
specifically to the isolated or recombinant immunogenic S9 protein of
Mycobacteriuira
tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or
epitope
thereof according to any embodiment described herein or to a fusion protein or
protein
aggregate comprising said immunogenic S9 protein, peptide, fragment or
epitope.
Whilst useful for subjects who are not immune-compromized, e.g., HIV-negative
subjects, the diagnostic assay of the present invention is also particularly
useful for
detecting TB in a subject that is immune compromised or immune deficient,
e.g., a
subject that is HN+. The samples used for conducting such assays include, for
2o example, (i) an extract from a tissue selected from the group consisting of
brain, breast,
ovary, lung, colon, pancreas, testes, liver, muscle, bone and mixtures
thereof; (ii) body
fluid(s) selected from the group consisting of sputum, serum, plasma, whole
blood,
saliva, urine, pleural fluid and mixtures thereof; and (iii) samples derived
from body
fluid(s) selected from the group consisting of sputum, serum, plasma, whole
blood,
saliva, urine, pleural fluid and mixtures thereof.
The present invention also provides a method for determining the response of a
subject
having tuberculosis or an infection by M. tuberculosis to treatment with a
therapeutic
compound for said tuberculosis or infection, said method comprising detecting
a S9
protein or an immunogenic fragment or epitope thereof in a biological sample
from said
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23
subject, wherein a level of the protein or fragment or epitope that is lower
than the level
of the protein or fragment or epitope detectable in a subject suffering from
tuberculosis
or infection by 141 tuberculosis indicates that the subject is responding to
said treatment
or has been rendered free of disease or infection. For example, the method can
comprise an immunoassay e.g., contacting a biological sample derived from the
subject
with one or more antibodies capable of binding to a S9 protein or an
immunogenic
fragment or epitope thereof, and detecting the formation of an antigen-
antibody
complex. In a particularly preferred embodiment, an antibody is an isolated or
recombinant antibody or immune reactive fragment of an antibody that binds
1o specifically to the isolated or recombinant immunogenic S9 protein of
Mycobacteriuln
tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or
epitope
thereof according to any embodiment described herein or to a fusion protein or
protein
aggregate comprising said imniunogenic S9 protein, peptide, fragment or
epitope.
Whilst useful for subjects who are not immune-compromized, e.g., HIV-negative
subjects, the diagnostic assay of the present invention is also particularly
useful for
detecting TB in a subject that is immune compromised or immune deficient,
e.g., a
subject that is HIV+. The samples used for conducting such assays include, for
example, (i) an extract from a tissue selected from the group consisting of
brain, breast,
ovary, lung, colon, pancreas, testes, liver, muscle, bone and mixtures
thereof; (ii) body
fluid(s) selected from the group consisting of sputum, serum, plasma, whole
blood,
saliva, urine, pleural fluid and mixtures thereof; and (iii) samples derived
from body
fluid(s) selected from the group consisting of sputum, serum, plasma, whole
blood,
saliva, urine, pleural fluid and mixtures thereof.
The present invention also provides a.method of monitoring disease
progression,
responsiveness to therapy or infection status by M. tuberculosis in a subject
comprising
determining the level of a S9 protein or an immunogenic fragment or epitope
thereof in
a biological sample from said subject at different times, wherein a change in
the level
of the S9. protein, fragment or epitope indicates a change in disease
progression,
3o responsiveness to therapy or infection status of the subject. In a
preferred embodiment,
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24
the method further comprises administering a compound for the treatment of
tuberculosis or infection by M. tuberculosis when the level of S9 protein,
fragment or
epitope increases over time. For example, the method can comprise an
immunoassay
e.g., contacting a biological sample derived from the subject with one or more
antibodies capable of binding to a S9 protein or an immunogenic fragment or
epitope
thereof, and detecting the formation of an antigen-antibody complex. In a
particularly
preferred embodiment, an antibody is an isolated or recombinant antibody or
immune
reactive fragment of an antibody that binds specifically to the isolated or
recombinant
immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9
peptide
io or immunogenic S9 fragment or epitope thereof according to any embodiment
described herein or to a fusion protein or protein aggregate comprising said
immunogenic S9 protein, peptide, fragment or epitope. Whilst useful for
subjects who
are not iinmune-compromized, e.g., HIV-negative subjects, the diagnostic assay
of the
present invention is particularly useful for detecting TB in a subject that is
immune
compromised or immune deficient, e.g., a subject that is HIV+. The samples
used for
conducting such assays include, for example, (i) an extract from a tissue
selected from
the group consisting of brain, breast, ovary, lung, colon, pancreas, testes,
liver, muscle,
bone and mixtures thereof; (ii) body fluid(s) selected from the group
consisting of
sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures
thereof;
2o and (iii) samples derived from body fluid(s) selected from the group
consisting of
sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures
thereof.
In a particularly preferred embodiment, circulating immune complexes (CICs)
are
detected in an antigen-based assay platform or antibody-based assay platform.
For
antigen-based assay platforms, the detection of CICs may provide a signal
amplification over the detection of isolated antigen in circulation, by virtue
of
detecting the immunoglobulin moiety of the CIC. In accordance with this
embodiment, a capture reagent e.g., a capture antibody is used to capture the
S9
antigen (S9 polypeptide or an immune reactive fragment or epitope thereof)
complexed with the subject's immunoglobulin, in addition to isolated antigen
in the
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subject's circulation. Anti-Ig antibodies, optionally conjugated to a
detectable label,
are used to specifically bind the captured CIC thereby detecting CIC patient
samples.
Within the scope of this invention, the anti-Ig antibody binds preferentially
to IgM,
IgA or IgG in the sample. In a particularly preferred embodiment, the anti-Ig
antibody
5 binds to human Ig, e.g., human IgA, human IgG or human IgM. The anti-Ig
antibody
may be conjugated to any standard detectable label known in the art. This is
particularly useful for detecting infection by a pathogenic agent, e.g., a
bacterium or
virus, or for the diagnosis of any disease or disorder associated with CICs.
Accordingly, the diagnostic methods described according to any embodiment
herein
io are amenable to a modification wherein the sample derived from the subject
comprises
one or more circulating immune complexes comprising immunoglobulin (Ig) bound
to
S9 protein of A'Iycobacteriurn tuberculosis or one or more immunogenic S9
peptides,
fragments or epitopes thereof and wherein detecting the formation of an
antigen-
antibody complex comprises contacting an anti-Ig antibody with an
immunoglobulin
15 moiety of the circulating immune complex(es) for a time and under
conditions
sufficient for a complex to form than then detecting the bound anti-Ig
antibody.
It is also within the scope of the present invention to include a multi-
analyte test in one
or more of the preceding antigen-based assay formats, wherein multiple
antibodies of
2o different specificities are used to confirm a diagnosis obtained using anti-
S9 antibodies,
thereby enhancing specificity and/or selectivity. For example, the patient
sample may
be contacted with antibodies that bind to S9 or immunogenic S9 peptide or
fragment or
epitope and antibodies that bind to M. tuberculosis Bsx or glutamine
synthetase (GS)
proteins or immunogenic peptide derived there from, e.g., antibodies prepared
against a
25 peptide derived from a surface-exposed region of a Bsx or GS protein or
comprising a
sequence selected from the group consisting of SEQ ID Nos: 8-20. Antibodies
that
bind to immunogenic A?: tuberculosis Bsx peptides are also described in detail
in the
instant applicant's co-pending International Patent Application No. No.
PCT/AU2005/001254 filed August 19, 2005 the disclosure of which is
incorporated
3o herein in its entirety; and antibodies that bind to M. tuberculosis GS
peptides are also
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26
described in detail in the instant applicant's co-pending International Patent
Application
No. PCT/AU2005/000930 filed June 24 2005 the disclosure of which is also
incorporated herein in its entirety. The antigen-antibody complexes formed are
then
detected using antibodies capable of binding to each protein analyte, or in
the case of
CIC detections, antibodies capable of binding to human immunoglobulins. The
assays
may be performed simultaneously or at different times, and using the same or
different
patient samples. The assays may also be performed in the same reaction vessel,
provided that different detection systems are used to detect the different
antigens or
CICs comprising the different antigens, e.g., anti-human Ig labelled using
different
io reporter molecules such as different coloured dyes, fluorophores,
radionucleotides,
enzymes, or colloidal gold particles; or differentially-labelled anti-S9
antibodies, anti-
Bsx antibodies, and anti-GS antibodies. As with other immunoassays described
herein,
the secondary antibody is optionally conjugated to a suitable detectable label
e.g.,
horseradish peroxidase (HRP) or f3-galactosidase or 13-glucosidase, colloidal
gold
particles, amongst others. Standard methods for employing such labels in the
detection
of the complexes formed will be apparent to the skilled artisan.
The present invention also provides a method of treatment of tuberculosis or
infection
by M. tuber-culosis comprising:
(i) performing a diagnostic method accordiiig to any embodiment described
herein
thereby detecting the presence of M. tuberculosis infection in a biological
sample from a subject; and
(ii) administering a therapeutically effective amount of a pharmaceutical
composition to reduce the number of pathogenic bacilli in the lung, blood or
lymph system of the subject.
The present invention also provides a method of treatment of tuberculosis or
infection
by M. tubei-culosis comprising:
(i) performing a diagnostic method according to any embodiment described
herein
thereby detecting the presence of A7 tuber=culosis infection in a biological
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27
sample from a subject being treated with a first pharmaceutical composition;
and
(ii) administering a therapeutically effective amount of a second
pharmaceutical
composition to reduce the number of pathogenic bacilli in the lung, blood or
lymph system of the subject.
The present invention also provides a method of treatment of tuberculosis in a
subject
comprising performing a diagnostic method or prognostic method as described
herein.
In one embodiment, the present invention provides a method of prophylaxis
io comprising:
(i) detecting the presence of Al. tubet=culosis infection in a biological
sample from a
subject; and
(ii) administering a therapeutically effective amount of a pharmaceutical
composition to reduce the number of pathogenic bacilli in the lung, blood or
lymph system of the subject.
More particularly, an immunogenic S9 protein or one or more immunogenic S9
peptides, fragments or epitopes thereof induce(s) the specific production of a
high titer
antibody when administered to an animal subject.
Accordingly, the invention also provides a method of eliciting the production
of
antibody against M. tuberculosis comprising administering an immunogenic S9
protein
or one or more immunogenic S9 peptides or immunogenic S9 fragments or epitopes
thereof to said subject for a time and under conditions sufficient to elicit
the production
of antibodies, such as, for example, neutralizing antibodies that bind to hI.
tuberculosis.
The present invention clearly contemplates the use of an immunogenic S9
protein or
one or more immunogenic S9 peptides or immunogenic S9 fragments or epitopes
thereof in the preparation of a therapeutic or prophylactic subunit vaccine
against M.
tuberculosis infection in a human or other animal subject.
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Accordingly, this invention also provides a vaccine comprising an immunogenic
S9
protein or one or more immunogenic S9 peptides or immunogenic S9 fragments or
epitopes thereof in combination with a pharmaceutically acceptable diluent.
Preferably,
the protein or peptide(s) or fragment(s) or epitope(s) thereof is(are)
formulated with a
suitable adjuvant.
Alternatively, the peptide or derivative or variant is formulated as a
cellular vaccine via
the administration of an autologous or allogeneic antigen presenting cell
(APC) or a
io dendritic cell that has been treated in vitr-o so as to present the peptide
on its surface.
Nucleic acid-based vaccines that comprise nucleic acid, such as, for example,
DNA or
RNA, encoding an immunogenic S9 protein or one or more immunogenic S9 peptides
or immunogenic S9 fragments or epitopes thereof cloned into a suitable vector
(eg.
vaccinia, canary pox, adenovirus, or other eukaryotic virus vector) are also
contemplated. Preferably, DNA encoding an immunogenic S9 protein or an
immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof is
formulated
into a DNA vaccine, such as, for example, in combination witli the existing
Calmette-
Guerin (BCG) or an immune adjuvant such as vaccinia virus, Freund's adjuvant
or
2o another immune stimulant.
The present invention further provides for the use of an immunogenic S9
protein or one
or more immunogenic S9 peptides or one or more immunogenic S9 fragments or one
or
more epitopes thereof in the preparation of a composition for the prophylactic
or
therapeutic treatment or diagnosis of tuberculosis or infection by M.
tuberculosis in a
subject, such as, for example, a subject infected with HIV-1 and/or HIV-2,
including
the therapeutic treatment of a latent M. tuber=culosis infection in a human
subject.
In an alternative embodiment, the present invention provides for the use of an
immunogenic S9 protein or one or more immunogenic S9 peptides or one or more
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immunogenic S9 fragments or one or more epitopes thereof in the preparation of
a
composition for the prophylactic or therapeutic treatment or diagnosis of
tuberculosis
or infection by M. tuberculosis in a subject wherein the subject has been
subjected
previously to antiviral therapy against HN-1 and/or HN-2.
The present invention also provides a kit for detecting M. tuberculosis
infection in a
biological sample, said kit comprising:
(i) one or more isolated antibodies or immune reactive fragments thereof that
bind
specifically to the isolated or recombinant immunogenic S9 protein of
Mycobacteriuna tuberculosis or an immunogenic S9 peptide or immunogenic S9
fragment or epitope thereof according to any embodiment described herein or to
a fusion protein or protein aggregate comprising said immunogenic S9 protein,
peptide, fragment or epitope; and
(ii) means for detecting the formation of an antigen-antibody complex,
optionally packaged with instructions for use.
The present invention also provides a kit for detecting M. tuberculosis
infection in a
biological sample, said kit comprising:
(i) isolated or recombinant immunogenic S9 protein of Mycobacteriunz tuber
culosis
or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof
according to any embodiment described herein; and
(ii) means for detecting the formation of an antigen-antibody complex,
optionally packaged with instructions for use.
The assays described herein are amenable to any assay format, and particularly
to solid
phase ELISA, flow through immunoassay formats, lateral flow formats, capillary
formats, and for the purification or isolation of immunogenic proteins,
peptides,
fragments and epitopes and CICs.
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Accordingly, the present invention also provides a solid matrix having
adsorbed
thereto an isolated or recombinant S9 protein or an immunogenic S9 peptide or
immunogenic S9 fragment or epitope thereof according to any one embodiment
described herein or a fusion protein or protein aggregate comprising said
5 immunogenic S9 protein, peptide, fragment or epitope. For example, the solid
matrix
may comprise a membrane, e.g., nylon or nitrocellulose. Alternatively, the
solid
matrix may comprise a polystyrene or polycarbonate microwell plate or part
thereof
(e.g., one or more wells of a microtiter plate), a dipstick, a glass support,
or a
chromatography resin.
In an alternative embodiment, the invention also provides a solid matrix
having
adsorbed thereto an antibody that binds to an isolated or recombinant S9
protein or an
immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according
to
any embodiment described herein or to a fusion protein or protein aggregate
comprising said immunogenic S9 protein, peptide, fragment or epitope. For
example,
the solid matrix may comprise a membrane, e.g., nylon or nitrocellulose.
Alternatively, the solid matrix may comprise a polystyrene or polycarbonate
microwell plate or part thereof (e.g., one or more wells of a microtiter
plate), a
dipstick, a glass support, or a chromatography resin.
It is clearly within the scope of the present invention for such solid
matrices to
comprise additional antigens and/or antibodies as required to perform an assay
described herein, especially for multianalyte tests employing multiple
antigens or
multiple antibodies.
Brief description of the drawings
Figure I is a copy of a photographic representation showing a polyacrylamide
gel
within which proteins isolated from an immunoglobulin fraction isolated from a
TB
subject have been separated using two-dimensional gel electrophoresis. The
position of
M. tuberculosis ribosomal protein S9 is indicated.
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31
Figure 2 is a graphical representation showing the titration of polyclonal
antibody R9
its corresponding biotinylated peptide coated onto a 5 Rg/ml streptavidin
plate at 3
g/ml.
Figure 3 is a graphical representation showing the titration of the peptide
comprising
the amino acid sequence MTETT PAPQT PAAPA GPAQS FGSGL-Biotin from
20,480 pg/ml to 10 pg/ml against the rabbit sera raised against this peptide
linked to
KHL. Solid diamonds represent 40 g/ml of antibody. Solid squares represent 20
lo Rg/ml of antibody. Grey triangles represent 10 Rg/ml of antibody. Grey
squares
represent 0 g/ml of antibody.
Figure 4a is a copy of a photographic representation showing a Western blot to
detect
M. tuberculosis ribosomal protein S9 in samples from subjects suffering from
TB. The
position of a band corresponding to S9 is indicated by the arrow at the right-
hand side
of the figure. The sample number is indicated at the top of the figure and the
HIV
status of each patient is indicated at the base of the figure. The molecular
weight is
indicated at the left-hand side of the figure.
2o Figure 4b is a copy of a photographic representation showing a Western blot
to detect
M. tuberculosis ribosomal protein S9 in samples from control subjects, i.e.,
subjects
that do not suffer from TB. The position of a band corresponding to S9 is
indicated by
the arrow at the right-hand side of the figure. The sample number is indicated
at the
top of the figure and the molecular weight is indicated at the left-hand side
of the
figure.
Figure 5 is a graphical representation showing the binding affinities of
different
antibodies prepared against recombinant M. tuber=culosis ribosomal protein S9
for the
immunizing antigen, as determined by ELISA. Reconibinant S9 protein was
diluted
serially 1:2 (v/v) from 500 ng/ml starting concentration to 7.8 ng/ml, and 50
l aliquots
CA 02638761 2008-07-31
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32
of each dilution were used to coat the wells of an ELISA plate (x-axis).
Following
washing to remove unbound antigen, distinct antibodies prepared by
immunization of
chickens (i.e., a polyclonal antibody designated Ch27) or mice (i.e., an
antibody
designated Mo1025F) with recombinant full-length S9 protein were contacted
separately with the adsorbed antigen at a concentration of 5 g/ml. Following
incubation at room temperature for 1 hour, plates were washed, incubated with
50 l of
a 1:5000 (v/v) dilution of secondary antibody (i.e., sheep anti-chicken IgG
for detection
of bound Ch27 antibody; and donkey anti-mouse IgG for detection of bound
Mo1025F
antibody) conjugated to horseradish peroxidase (HRP), washed, incubated with
TMB
io for 30 mins, and absorbance at 595-600 nm was determined (y-axis). Data
show that
both antibodies detect recombinant S9 protein by ELISA.
Figure 6 is a graphical representation showing sandwich ELISA results using
antibody
Ch27 as capture antibody and antibody Mo1025F as detection antibody for
assaying
recombinant Al. tuberculosis ribosomal protein S9. An ELISA plate was coated
overnight with capture antibody Ch27 at 5 gg/ml and 2.5 gg/ml concentrations.
Following washing to remove unbound antibody, recombinant S9 protein was
diluted
serially 1:2 (v/v) from 500 ng/mi starting concentration to 7.8 ng/ml, and 50
l aliquots
of each dilution were added the wells of the antibody-coated ELISA plates (x-
axis).
2o Following incubation for 1 hour and washing to remove unbound antigen,
detection
antibody Mo1025F was contacted with the bound antigen-body complexes at
concentrations in the range of 1.25 g/ml to 5 g/ml. Following incubation at
room
temperature for 1 hour, plates were washed, incubated with 50 l of a 1:5000
(v/v)
dilution of secondary antibody (i.e., donkey anti-mouse IgG) conjugated to
horseradish
peroxidase (HRP), washed, incubated with TMB for 30 mins, and absorbance at
595-
600 nm was determined (y-axis). Data show no background signal with this
antibody
combination. Optimum signal was detected using capture antibody at a
concentration of
5 gg/ml with detection antibody in the concentration range of 1.25 g/ml to 5
g/ml,
which conditions provided a half-maximum detection of about 24 ng/ml M.
tuberculosis ribosomal protein S9.
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33
Figure 7 is a graphical representation showing sandwich ELISA results using
antibody
Mo1025F as capture antibody and antibody Ch27 as detection antibody for
assaying
recombinant M. tubef-culosis ribosomal protein S9. An ELISA plate was coated
overnight with capture antibody Mo1025F at 5 g/ml and 2.5 g/ml
concentrations.
Following washing to remove unbound antibody, recombinant S9 protein was
diluted
serially 1:2 (v/v) from 500 ng/mi starting concentration to 7.8 ng/ml, and 50
gl aliquots
of each dilution were added the wells of the antibody-coated ELISA plates (x-
axis).
Following incubation for 1 hour and washing to remove unbound antigen,
detection
io antibody Ch27 was contacted with the bound antigen-body complexes at
concentrations
in the range of 1.25 g/ml to 5 :g/ml. Following incubation at room
temperature for 1
hour, plates were washed, incubated with 50 gl of a 1:5000 (v/v) dilution of
secondary
antibody (i.e., sheep anti-chicken IgG) conjugated to horseradish peroxidase
(HRP),
washed, incubated with TMB for 30 mins, and absorbance at 595-600 nm was
determined (y-axis). Data show significant background cross-reactivity in the
absence
of added antigen using this antibody combination. Optimum signal was detected
using
capture antibody at a concentration of 2.5 g/ml or 5 g/ml with detection
antibody at a
concentration of 5 g/ml under the conditions tested.
2o Figure 8 is a graphical representation showing sandwich ELISA results using
antibody
Ch27 as capture antibody, antibody Mo1025F as detection antibody and an HRP-
conjugated secondary antibody, for assaying low concentrations of recombinant
11~I.
tuberculosis ribosomal protein S9. An ELISA plate was coated overnight with
capture
antibody Ch27 at 5 g/ml concentration. Following washing to remove unbound
antibody, recombinant S9 protein was diluted serially 1:2 (v/v) from 150 ng/ml
starting
concentration to 18.31 pg/ml, and 50 l aliquots of each dilution were added
the wells
of the antibody-coated ELISA plates (x-axis). Following incubation for 1 hour
and
washing to remove unbound antigen, detection antibody Mo1025F was contacted
with
the bound antigen-body complexes at 2.5 gg/ml concentration. Following
incubation at
room temperature for 1 hour, plates were washed, incubated with 50 l of a
1:5000
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34
(v/v) dilution of secondary antibody (i.e., donkey anti-mouse IgG) conjugated
to
horseradish peroxidase (HRP), washed, incubated with TMB for 30 mins, and
absorbance at 595-600 nm was determined (y-axis). Data show no background
signal
with this antibody combination, a detection limit of 996 pg/ml Af.
tuberculosis
ribosomal protein S9, and half-maximum detection of about 28 ng/ml AI tuber-
culosis
ribosomal protein S9 under the conditions tested. Error bars show one standard
deviation from the mean (n=3).
Figure 9 is a graphical representation showing sandwich ELISA results using
antibody
io Ch27 as capture antibody, antibody Mo1025F as detection antibody and a
biotinylated
secondary antibody for assaying low concentrations of recombinant M.
tuberculosis
ribosomal protein S9. ELISA was performed essentially as described in the
legend to
Figure 8 except that recombinant S9 protein was diluted serially 1:2 (v/v)
from 20
ng/ml starting concentration to 4.77 pg/ml concentration (x-axis); the
incubation with
secondary antibody was or 1 hour with a biotinylated donkey anti-mouse Ig
followed
by incubation with a modified streptavidin-HRP conjugate (poly-40) at 1:5000
(v/v)
dilution; and bound antibody- antigen- atitibody complexes were detected by
washing
plates, incubating with TMB for 10 mins, and measuring absorbance at 595-600
nm (y-
axis). Data show low background signal, a detection limit of about 150 pg/ml
M.
tuberculosis ribosomal protein S9, and half-maximum detection of about 6 ng/ml
M.
tuberculosis ribosomal protein S9 using the biotinylated secondary antibody.
Error
bars show one standard deviation from the mean (n=3).
Figure 10 is a graphical representation showing sandwich ELISA results using
antibody
Ch27 as capture antibody, antibody Mo1025F as detection antibody, a
biotinylated
secondary antibody and iterative antigen binding ( also termed herein
"replacement
amplification") for assaying low concentrations of recombinant M.
tuber=cT.slosis
ribosomal protein S9. ELISA was performed essentially as described in the
legend to
Figure 9 except that recombinant S9 protein was diluted serially 1:2 (v/v)
from 1.0
g/mi starting concentration to 0.238 fg/ml concentration (x-axis); and antigen
binding
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WO 2007/087679 PCT/AU2007/000093
was repeated 5 times i.e., an aliquot of antigen in blocking buffer was
incubated with
immobilized capture antibody for 1 hour, removed, another aliquot added, and
the
procedure repeated until five aliquots had been, added. Absorbance at 595-600
nm is
indicated on the y-axis. Data show low background signal, and a detection
limit of
5 about 84 pg/ml M. tubef=culosis ribosomal protein S9 using the biotinylated
secondary
antibody in combination with iterative antigen binding. Error bars show one
standard
deviation from the mean (n=3).
Figure 11 is a graphical representation of sandwich ELISA results showing lack
of
io significant cross-reactivity of antibodies against M. tuber=culosis
ribosomal protein S9
with Escherichia coli, Bacillus subtilis or Pseudot?zoraas aerugitiosa. Assay
conditions
were essentially as described in the legend to Figure 9 except that purified
recombinant
S9 protein was replaced with 500 ng/ml or 50 g/ml of a cellular extract as
indicated on
the x-axis. As a positive control, cellular extract from the M. tuberculosis
laboratory
15 strain H37Rv was used. As a negative control for each assay, buffer without
cellular
extract was used. Data show the change in absorbance at 595-600nni i.e.,
following
subtraction of background absorbance for each sample. Error bars show one
standard
deviation from the mean (n=3).
2o Figure 12 is a graphical representation of sandwich ELISA results showing
detection of
M. tuberculosis ribosomal protein S9 in the clinical M. tuber=culosis isolate
CSU93, and
lack of signal suppression in plasma. Assay conditions were essentially as
described in
the legend to Figure 11 except that cellular extracts were from M. tuber-
culosis
laboratory strain H37Rv and CSU93, as indicated on the x-axis. For
determination of
25 signal suppression by plasma, cellular extract at the concentration
indicated was diluted
into plasma, as indicated on the x-axis. As a negative control for each assay,
buffer or
plasma without cellular extract was used. The change in absorbance at 595-
600nm i.e.,
following subtraction of background absorbance for each sample is shown on the
y-
axis. Error bars show one standard deviation from the mean (n=3). Data show
that
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36
plasma does not suppress signal in this assay, and that the assay is capable
of detecting
both clinical and laboratory isolates of AI tuberculosis.
Figure 13a is a copy of a photographic representation showing a Western blot
to detect
Al. tubef-culosis BSX protein in samples from subjects suffering from TB. The
position
of a band corresponding to BSX is indicated by the arrow at the right-hand
side of the
figure. The sample number is indicated at the top of the figure and the HIV
status of
each patient is indicated at the base of the figure. The molecular weight is
indicated at
the left-hand side of the figure.
io.
Figure 13b is a copy of a photographic representation showing a Western blot
to detect
M. tubei=culosis BSX protein in samples from control subjects, i.e., subjects
that do not
suffer from TB. The position of a band corresponding to BSX is indicated by
the arrow
at the right-hand side of the figure. The sample number is indicated at the
top of the
figure and the molecular weight is indicated at the left-hand side of the
figure.
Figure 14 is a copy of a photographic representation showing a Western blot to
detect
A7 tuberculosis BSX protein in a fraction captured with Protein-G
(immunoglobulin
containing fraction) and the flow-through fraction from three different
subjects. The
fraction and patient number is indicated at the top of the figure. The
molecular weight
is indicated at the left-hand side of the figure and the size of the BSX
protein is
indicated at the right-hand side of the figure.
Figure 15 is a graphical representation showing a comparison of the
concentration of
recombinant BSX detected using a chicken anti-BSX polyclonal antibody
preincubated
with recombinant BSX (solid diamonds); a chicken anti-BSX antibody without
preincubation (grey squares); a rabbit anti-BSX polyclonal antibody (solid
triangles)
and a mouse anti-BSX monoclonal antibody (solid squares). The concentration of
the
recombinant protein is indicated on the X-axis and the optical density
indicated on the
Y-axis.
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37
Figure 16 is a graphical representation showing the detection of recombinant
BSX
using a sandwich ELISA in which monoclonal antibody 403B was used as a capture
reagent and polyclonal antibody C44 was used as a detection reagent. Titrating
amounts of recombinant. BSX from 50 ng/ml down to 0.39 ng/ml were screened.
Concentrations of detection and capture reagents are indicated. The
concentration of
BSX is shown on the X-axis and the mean OD is shown on the Y-axis.
Figure 17 is a graphical representation showing the detection of BSX in sputa
of TB
1o and control subjects using a Sandwich ELISA. The optical density is
indicated on the
Y-axis and the sample type and number is indicated on the X-axis.
Figure 18 is a graphical representation showing the detection of recombinant
BSX
using an amplified sandwich ELISA in which monoclonal antibody 403B was used
as a
capture reagent detection reagent (as indicated) and polyclonal antibody C44
was used
as a detection reagent or capture reagent (as indicated). Titrating amounts of
recombinant BSX from 50 ng/ml to 0.39 ng/ml were screened. Concentrations of
detection and capture reagents are indicated. The concentration of BSX is
shown on
the X-axis and the mean OD is shown on the Y-axis.
Figure 19 is a graphical representation showing the detection of recombinant
BSX
using an amplified ELISA in which C44 is used as a capture reagent. Purified
chicken
anti-BSX pAb C44 was immobilised onto an ELISA plate as a capture antibody at
a
concentration of 20 g/ml using 50 l per well. Titrating amounts of
recombinant BSX
from 10 ng/ml down to 0.078 ng/ml were then screened by sequential addition of
purified rabbit anti-BSX (Peptide 28) pAb at a concentration of 5 g/ml, and
then a
goat anti-rabbit IgG at a dilution of either 1/30000 or 1/60000, as a second
Detector.
Donkey anti-Goat IgG HRP at a dilution of 1/5000 and TMB were used for signal
detection.
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38
Figure 20 is a graphical representation showing the measurement of detection
limits of
standard sandwich ELISA versus biotin based Amplification System. Purified
rabbit
anti-BSX pAb R16 was immobilised onto an ELISA plate at a concentration of 20
g/ml. Titrating amounts of recombinant BSX were added at a concentration of 50
ng/ml down to 0.39 ng/ml for 1 hr unless specified otherwise (i.e 2 hr).
Antigen
detection was performed using either a standard sandwich system where chicken
anti-
BSX pAb C44 was added at a concentration of 5 g/ml followed by sheep anti-
chicken
IgG HRP conjugate at a dilution of 1/5000, or an amplifying system where
chicken
anti-BSX was first added at 5 g/ml followed by donkey anti-chicken IgG biotin
io conjugate at various dilutions as specified above, and finally streptavidin-
HRP at a
1/5000 dilution. Background (i.e. signal without BSX present) has been
subtracted
from the above curves.
Figure 21 is a graphical representation showing detection of titrating amounts
of
recombinant BSX using a Biotin -based amplified ELISA. Purified rabbit anti-
BSX
(anti-Peptide 28) pAb R16 was immobilised onto an ELISA plate as a capture
antibody
at a concentration of either 20 or 40 g/ml. Titrating amounts of recombinant
BSX
froni 10 ng/ml down to 4.9 pg/ml were then screened by sequential addition of
purified
chicken anti-BSX pAb C44 at a concentration of 5 g/ml, and then a donkey anti-
chicken IgG biotin conjugate at a dilution of 1:20,000 (v/v) as a second
detector.
Streptavidin HRP conjugate at a dilution of 1:5000 (v/v) and TMB were used for
signal
detection, Background OD intensity was obtained for both of the rabbit anti-
BSX.
capture concentrations where the recombinant BSX was not added.
Figure 22 is a graphical representation showing screening of sputum for
endogenous
BSX using sandwich ELISA with a' Biotin Amplification System. Sputum samples
(50
ul + 50 ul blocking buffer) from South African TB patients and control
patients with
non-TB respiratory disease from South Africa (prefix `M') and Australia
(prefix
`CGS') respectively were screened by sandwich ELISA for the presence of BSX
antigen. Purified rabbit anti-BSX (peptide 28) pAb was immobilised onto the
ELISA
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39
plate as a capture antibody at a concentration of 20 g/ml. Purified chicken
anti-BSX
pAb, C44, at a concentration of 5 g/ml, was used as the detector antibody.
Biotinylated donkey anti-chicken IgG at a dilution of 1/20000 was used as a
second
detector. Streptavidin HRP at a dilution of 1/5000 and TMB were used for
signal
detection. Sputum from control patient CGS25 was spiked with 5 ng/ml and 1
ng/ml
recombinant BSX as a positive control.
Figure 23 is a graphical representation showing the Effect of Multiple Sample
Loads on
Detection of BSX by Amplified Sandwich ELISA. Rabbit anti-BSX pAb R16 was
io immobilised onto an ELISA plate as the capture antibody at a concentration
of 20
g/ml using 50 ul per well. Sputum samples from TB patients and non-TB
respiratory
disease control patients were diluted at a 1:1 ratio with blocker solution.
The positive
control is recombinant BSX at ing/mi spiked in CGS23 sputum sample. Sputum
samples were either (i) incubated for 1 hr as per a standard ELISA; (ii)
incubated for 2
hr; or (iii) incubated for 2 hr, removed and fresh sputum added for an
additional 1 hr of
incubation. Endogenous BSX was detected using purified chicken anti-BSX pAb
C44
at 5 g/ml, followed by donkey anti-chicken IgG biotin conjugate at a dilution
of
1/20,000 and finally with streptavidin HRP conjugate at 1/5000 dilution.
2o Detailed description of the preferred embodiments
Isolated or recornbinant S9 protein and irnnzunogenic fragnaents and epitopes
thereof
One aspect of the present invention provides an isolated or recombinant S9
protein or
an immunogenic fragment or epitope thereof.
This aspect of the invention encompasses any synthetic or recombinant peptides
derived from a S9 protein referred to herein, including the full-length S9
protein, and/or
a derivative or analogue of a S9 protein or an immunogenic fragment or epitope
thereof.
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As used herein, the term "S9" shall be taken to mean any peptide, polypeptide,
or
protein having at least about 80% amino acid sequence identityto the amino
acid
sequence set forth in SEQ ID NO: 1. Preferably, the percentage identity of a
S9 protein
to SEQ ID NO: 1 is at least about 85%, more preferably at least about 90%,
even more
5 preferably at least about 95% and still more preferably at least about 99%.
The present
invention is not to be restricted to the use of the exemplified M.
tuberculosis S9 protein
because, as will be known to those skilled in the art, it is possible to
define a fragment
of a protein having sequence identity and immunological equivalence to a
region of the
exemplified M. tuberculosis S9 protein without undue experimentation.
In determining whether or not two amino acid sequences fall within the defined
percentage identity limits supra, those skilled in the art will be aware that
it is possible
to conduct a side-by-side comparison of the amino acid sequences. In such
comparisons or alignments, differences will arise in the positioning of non-
identical
residues depending upon the algorithm used to perform the alignment. In the
present
context, references to percentage identities and similarities between two or
more amino
acid sequences shall be taken to refer to the number of identical and similar
residues
respectively, between said sequences as detei-mined using any standard
algorithm
known to those skilled in the art. In particular, amino acid identities and
similarities are
calculated using software of the Computer Genetics Group, Inc., University
Research
Park, Maddison, Wisconsin, United States of America, eg., using the GAP
program of
Devereaux et al., Nucl. Acids Res. 12, 387-395, 1984, which utilizes the
algorithm of
Needleman and Wunsch, J. Mol. Biol. 48, 443-453, 1970. Alternatively, the
CLUSTAL W algorithm of Thompson et al., Nucl. Acids Res. 22, 4673-4680, 1994,
is
used to obtain an alignment of multiple sequences, wherein it is necessary or
desirable
to maxiniise the number of identical/similar residues and to minimise the
number
and/or length of sequence gaps in the alignment. Amino acid sequence
alignments can
also be performed using a variety of other commercially available sequence
analysis
programs, such as, for example, the BLAST program available at NCBI.
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41
Particularly preferred fragments include those that include an epitope, in
particular a B
cell epitope or T cell epitope.
A B-cell epitope is conveniently derived from the amino acid sequence of an
immunogenic S9 protein. Idiotypic and anti-idiotypic B cell epitopes against
which an
immune response is desired are specifically encompassed by the invention, as
are lipid-
modified B cell epitopes or a Group B protein. A preferred B-cell epitope will
be
capable of eliciting the production of antibodies when administered to a
mammal,
preferably neutralizing antibody against M. tacberculosis, and more
preferably, a high
io titer neutralizing antibody. Shorter B cell epitopes are preferred, to
facilitate peptide
synthesis. Preferably, the length of the B cell epitope will not exceed about
30 amino
acids in length. More preferably, the B cell epitope sequence consists of
about 25
amino acid residues or less, and more preferably less than 20 amino acid
residues, and
even more preferably about 5-20 amino acid residues in length derived from the
sequence of the full-length protein.
A CTL epitope is also conveniently derived from the full length amino acid
sequence
of a S9 protein and will generally consist of at least about 9 contiguous
amino acids of
said S9 protein and have an amino acid sequence that interacts at a
significant level
with a MHC Class I allele as determined using a predictive algorithm for
determining
MHC Class I-binding epitopes, such as, for example, the SYFPEITHI algorithm of
the
University of Tuebingen, Germany, or the algorithm of the HLA Peptide Binding
Predictions program of the Biolnformatics and Molecular Analysis Section
(BIMAS)
of the National Institutes of Health of the Government of the United States of
America.
More preferably, the CTL epitope will have an amino acid sequence that binds
to
and/or stabilizes a MHC Class I molecule on the surface of an antigen
presenting cell
(APC). Even more preferably, the CTL epitope will have a sequence that induces
a
memory CTL response or elicits IFN-y expression by a T cell, such as, for
example,
CD8+ T cell, cytotoxic T cell (CTL). Still even more preferably, the CTL will
have a
sequence that stimulates CTL activity in a standard cytotoxicity assay.
Particularly
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42
preferred CTL epitopes of a S9 protein are capable of eliciting a cellular
immune
response against M. tuberculosis in human cells or tissues, such as, for
example, by
recognizing and lysing human cells infected with M. tuberculosis, thereby
providing or
enhancing cellular immunity against A2 tuberculosis.
Suitable fragments will be at least about 5, e.g., 10, 12, 15 or 20 amino
acids in length.
They may also be less than 200, 100 or 50 amino acids in length.
Preferably, an immunogenic fragment or epitope of S9 comprises an amino acid
lo sequence set forth in any one of SEQ ID Nos: 2-7, and preferably an
immunogenic
fragment or epitope thereof comprising an amino acid sequence selected from
the
group consisting of: SEQ ID NO: 6 or SEQ ID NO: 7.
The amino acid sequence of a S9 protein or immunogenic fragment or epitope
thereof
may be modified for particular purposes according to methods well known to
those of
skill in the art without adversely affectiiig its immune function. For
example, particular
peptide residues may be derivatized or chemically modified in order to enhance
the
immune response or to permit coupling of the peptide to other agents,
particularly
lipids. It also is possible to change particular amino acids within the
peptides without
2o disturbing the overall structure or antigenicity of the peptide. Such
changes are
therefore termed "conservative" changes and tend to rely on the hydrophilicity
or
polarity of the residue. The size and/or charge of the side chains also are
relevant
factors in determining which substitutions are conservative.
The present invention clearly encompasses a covalent fusion between one or
more
immunogenic S9 peptides, including a homo-dimer, homo-trimer, homo-tetramer or
higlier order homo-multimer of a peptide, or a hetero-dimer, hetero-trimer,
hetero-
tetramer or higher order hetero-multimer comprising two or more different
immunogenic peptides.
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43
The present invention also encompasses a non-covalent aggregate between one or
more
immunogenic S9 peptides, e.g., held together by ionic, hydrostatic or other
interaction
known in the art or described herein.
It is well understood by the skilled artisan that, inherent in the definition
of a
biologically functional equivalent protein is the concept that there is a
limit to the
number of changes that may be made within a defined portion of the molecule
and still
result in a molecule with an acceptable level of equivalent biological
activity.
Biologically functional equivalent proteins are thus defined herein as those
proteins in
io which specific amino acids are substituted. Particular embodiments
encompass variants
that have one, two, three, four, five or more variations in the amino acid
sequence of
the peptide. Of course, a plurality of distinct proteins/peptides with
different
substitutions may easily be made and used in accordance with the invention.
Those skilled in the art are well aware that the following substitutions are
permissible
conservative substitutions (i) substitutions involving arginine, lysine and
histidine; (ii)
substitutions involving alanine, glycine and serine; and (iii) substitutions
involving
phenylalanine, tryptophan and tyrosine. Derivatives incorporating such
conservative
substitutions are defined herein as biologically or immunologically functional
2o equivalents.
The importance of the hydropathic amino acid index in conferring interactive
biological
function on a protein is generally understood in the art (Kyte & Doolittle, J.
Mol. Biol.
157, 105-132, 4982). It is known that certain amino acids may be substituted
for other
amino acids having a similar hydropathic index or score and still retain a
similar
biological activity. The hydropathic index of amino acids also may be
considered in
determining a conservative substitution that produces a functionally
equivalent
molecule. Each amino acid has been assigned a hydropathic index on the basis
of their
hydrophobicity and charge characteristics, as follows: isoleucine (+4.5);
valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine
(+1.9);
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44
alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-
0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-
3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In making changes
based
upon the hydropathic index, the substitution of amino acids whose hydropathic
indices
are within .+/- 0.2 is preferred. More preferably, the substitution will
involve amino
acids having hydropathic indices within .+/- 0.1, and more preferably within
about +/-
0.05.
It is also understood in the art that the substitution of like amino acids is
made
io effectively on the basis of hydrophilicity, particularly where the
biological functional
equivalent protein or peptide thereby created is intended for use in
immunological
embodiments, as in the present case (e.g. US Patent No. 4,554,101), In fact,
the greatest
local average hydrophilicity of a protein, as governed by the hydrophilicity
of its
adjacent amino acids, correlates with its immunogenicity and antigenicity. As
detailed
in US Patent No. 4,554,101, the following hydrophilicity values have been
assigned to
amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +/- 0.1);
glutamate
(+3.0 +/- 0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine
(0); threonine
(-0.4); proline (-0.5 +/- 0.1); alanine (-0.5); histidine (-0.5); cysteine (-
1.0); methionine
(-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-
2o 2.5); tryptophan (-3.4). In making changes based upon similar
hydrophilicity values, it
is preferred to substitute amino acids having hydrophilicity values within
about +/- 0.2
of each other, more preferably within about +/- 0.1, and even more preferably
within
about +/- 0.05
The S9 polypeptide or peptide fragment thereof comprising an epitope is
readily
synthesized using standard techniques, such as the Merrifield method of
synthesis
(Merrifield, J Ana Chein Soc, 85,:2149-2154, 1963) and the myriad of available
improvements on that technology (see e.g., Synthetic Peptides: A User's Guide,
Grant,
ed. (1992) W.H. Freeman & Co., New York, pp. 382; Jones (1994) The Chemical
Synthesis of Peptides, Clarendon Press, Oxford, pp. 230.); Barany, G. and
Merrifield,
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WO 2007/087679 PCT/AU2007/000093
R.B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-
284,
Academic Press, New York; Wunsch, E., ed. (1974) Synthese von Peptiden in
Houben-
Weyls,Metoden der Organischen Chemie (Muler, E., ed:), vol. 15, 4th edn.,
Parts 1 and
2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis,
Springer-
5 Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Pr=actice of
Peptide
Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide
Protein
Res. 25, 449-474.d/
As is known in the art, synthetic peptides can be produced with additional
hydrophilic
io N-terminal and/or C-terminal amino acids added to the sequence of a
fragment or B-
cell epitope derived from the full-length S9 protein, such as, for example, to
facilitate
synthesis or improve peptide solubility. Glycine and/or serine residues are
particularly
preferred for this purpose. Each of the peptides set forth in SEQ ID NO 2-6
may be
modified to include additional spacer sequences flanking the S9 fragments,
said spacers
15 comprising hetero-polymers (trimers or tetramers) comprising glycine and
serine e.g.,
as in SEQ ID NO: 7.
The peptides of the invention are readily modified for diagnostic purposes,
for
example, by addition of a natural or synthetic liapten, an antibiotic,
hormone, steroid,
2o nucleoside, nucleotide, nucleic acid, an enzyme, enzyme substrate, an
enzyme
inhibitor, biotin, avidin, streptavidin, polyhistidine tag, glutathione, GST,
polyethylene
glycol, a peptidic polypeptide moiety (e.g. tuftsin, poly-lysine), a
fluorescence marker
(e.g. FITC, RITC, dansyl, luminol or coumarin), a bioluminescence marker, a
spin
label, an alkaloid, biogenic amine, vitamin, toxin (e.g. digoxin, phalloidin,
amanitin,
25 tetrodotoxin), or a complex-forming agent. Biotinylated peptides are
especially
preferred.
In another embodiment, a S9 protein is produced as a recombinant protein.
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46
For expressing protein by recombinant means, a protein-encoding nucleotide
sequence
is placed in operable connection with a promoter or other regulatory sequence
capable
of regulating expression in a cell-free system or cellular system. In one
embodiment of
the invention, nucleic acid comprising a sequence that encodes a S9 protein or
an
epitope thereof in operable connection with a suitable promoter sequence, is
expressed
in a suitable cell for a time and under conditions sufficient for expression
to occur.
Nucleic acid encoding the S9 protein is readily derived from the publicly
available
amino acid sequence.
io In another embodiment, a S9 protein is produced as a recombinant fusion
protein, such
as for example, to aid in extraction and purification. To produce a fusion
polypeptide,
the open reading frames are covalently linked in the same reading frame, such
as, for
example, using standard cloning procedures as described by Ausubel et al.
(Current
Protocols in Molecular Biology, Wiley Interscience, ISBN 047150338, 1992), and
expressed under control of a promoter. Examples of fusion protein partners
include
glutathione-S-transferase (GST), FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), hexa-
histidine, GAL4 (DNA binding and/or transcriptional activation domains) and (3-
galactosidase. It may also be convenient to include a proteolytic cleavage
site between
the fusion protein partner and the protein sequence of interest to allow
removal of
fusion protein sequences. Preferably the fusion protein will not hinder the
immune
function of the S9 protein.
Reference herein to a "promoter" is to be taken in its broadest context and
includes the
transcriptional regulatory sequences of a classical genomic gene, including
the TATA
box which is required for accurate transcription initiation, with or without a
CCAAT
box sequence and additional regulatory elements (i.e., upstream activating
sequences,
enhancers and silencers) which alter gene expression in response to
developmental
and/or external stimuli, or in a tissue-specific manner. In the present
context, the term
"promoter" is also used to describe a recombinant, synthetic or fusion
molecule, or
3o derivative which confers, activates or enhances the expression of a nucleic
acid
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47
molecule to which it is operably connected, and which encodes the polypeptide
or
peptide fragment. Preferred promoters can contain additional copies of one or
more
specific regulatory elements to further enhance expression and/or to alter the
spatial
expression and/or temporal expression of the said nucleic acid molecule.
Placing a nucleic acid molecule under the regulatory control of, i.e., "in
operable
connection with", a promoter sequence means positioning said molecule such
that
expression is controlled by the promoter sequence. Promoters are generally
positioned
5' (upstream) to the coding sequence that they control.
The prerequisite for producing intact polypeptides and peptides in bacteria
such as E.
coli is the use.of a strong promoter with an effective ribosome binding site.
Typical
promoters suitable for expression in bacterial cells such as E. coli include,
but are not
limited to, the lacz promoter, temperature-sensitive XL or XR promoters, T7
promoter or
the IPTG-inducible tac promoter. A number of other vector systems for
expressing the
nucleic acid molecule of the invention in E. coli are well-known in the art
and are
described, for example, in Ausubel et al (In: Current Protocols in Molecular
Biology.
Wiley Interscience, ISBN 047150338, 1987) or Sambrook et al (In: Molecular
cloning,
A laboratory manual, second edition, Cold Spring Harbor Laboratory, Cold
Spring
2o Harbor, N.Y., 1989). Numerous plasmids with suitable promoter sequences for
expression in bacteria and efficient ribosome binding sites have been
described, such as
for example, phC30 (XL: Shimatake and Rosenberg, Nature 292, 128, 1981);
pKKl73-
3(tac: Amann and Brosius, Gerae 40, 183, 1985), pET-3 (T7: Studier and Moffat,
J.
Mol. Biol. 189, 113, 1986); the pBAD/TOPO or pBAD/Thio-TOPO series of vectors
containing an arabinose-inducible promoter (Invitrogen, Carlsbad, CA), the
latter of
which is designed to also produce fusion proteins with thioredoxin to enhance
solubility of the expressed protein; the pFLEX series of expression vectors
(Pfizer Inc.,
CT, USA); or the pQE series of expression vectors (Qiagen, CA), amongst
others.
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48
Typical promoters suitable for expression in viruses of eukaryotic cells and
eukaryotic
cells include the SV40 late promoter, SV40 early promoter and cytomegalovirus
(CMV) promoter, CMV IE (cytomegalovirus immediate early) promoter amongst
others. Preferred vectors for expression in mammalian cells (eg. 293, COS,
CHO, lOT
cells, 293T cells) include, but are not limited to, the pcDNA vector suite
supplied by
Invitrogen, in particular pcDNA 3.1 myc-His-tag comprising the CMV promoter
and
encoding a C-terminal 6xHis and MYC tag; and the retrovirus vector pSRatkneo
(Muller et al., Mol. Cell. Biol., 11, 1785, 1991). The vector pcDNA 3.1 inyc-
His
(Invitrogen) is particularly preferred for expressing a secreted form of a S9
protein or a
1o derivative thereof in 293T cells, wherein the expressed peptide or protein
can be
purified free of conspecific proteins, using standard affinity techniques that
employ a
Nickel column to bind the protein via the His tag.
A wide range of additional host/vector systems suitable for expressing the
diagnostic
protein of the present invention or an immunological derivative (eg., an
epitope or other
fragment) thereof are available publicly, and described, for example, in
Sambrook et al
(In: Molecular cloning, A laboratory manual, second edition, Cold Spring
Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989).
Means for introducing the isolated nucleic acid molecule or a gene construct
comprising same into a cell for expression are well-known to those skilled in
the art.
The technique used for a given organism depends on the known successful
techniques.
Means for introducing recombinant DNA into animal cells include
microinjection,
transfection mediated by DEAE-dextran, transfection mediated by liposomes such
as
by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA),
PEG-
mediated DNA uptake, electroporation and microparticle bombardrrient such as
by
using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst
others.
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49
Proteins of the invention can be produced in an isolated form, preferably
substantially
free of conspecific protein. Antibodies and other affinity ligands are
particularly
preferred for producing isolated protein. Preferably, the protein will be in a
preparation
wherein more than about 90% (e.g. 95%, 98% or 99%) of the protein in the
preparation
is a S9 protein or an epitope thereof.
Isolated or recornbinant secondaiy analyte pr-otein, peptides and epitopes
ther=eof
It is to be understood that methods described herein above for the production
of
isolated and recombinant S9 protein or immunogenic fragments thereof apply
nzutatis
io m.ictandis to the production of secondary analyte proteins, peptides and
fragments that
are to be used in an immunoassay format e.g., for the purposes of diagnosis or
prognosis of tuberculosis or infection by M. tuberculosis, antibody
production, analyte
purification, vaccine forrnulation, etc. As will be understood by the skilled
artisan,
such extrapolation is dependent on substituting the S9 immunogen for the
secoridary
analyte in question e.g., M. tuberculosis Bsx protein or GS protein or
immunogenic
fragment thereof according to any embodiment described herein. Such
substitution is
readily performed without undue experimentation fro the disclosure herein.
For convenience, preferred secondary analytes e.g., for use in multi-analyte
antigen-
2o based tests, will comprise an amino acid sequence selected from the group
set forth in
SEQ ID NOs: 8-19.
For example, the M. tuber-caclosis Bsx protein can be expressed and fragments
obtained
therefrom by standard means, or alternatively, synthetic peptides can be
synthesized
based on the sequence of the full-length protein (e.g., comprising the
sequence set forth
in SwissProt Database Accession No. 053759). Exemplary immunogenic peptides
from the full-length Bsx protein will comprise a sequence selected from the
group
consisting of: MRQLAERSGVSNPYL (SEQ ID NO: 8), ERGLRKPSADVLSQI (SEQ
ID NO: 9), LRKPSADVLSQIAKA (SEQ ID NO: 10), PSADVLSQIAKALRV (SEQ
ID NO: 11), SQIAKALRVSAEVLY (SEQ ID NO: 12), AKALRVSAEVLYVRA
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(SEQ ID NO: 13), VRAGILEPSETSQVR (SEQ ID No: 14), TAITERQKQILLDIY
(SEQ ID NO; 15), SQIAhALRVSAEVLYVRAC (SEQ ID NO: 16),
MSSEEKLCDPTPTDD (SEQ ID NO: 17) and VRAGILEPSETSQVRC (SEQ ID NO:
18). Methods for producing such fragments are described in detail in the
instant
5 applicant's co-pending International Patent Application No.
PCT/AU2005/001254 filed
August 19, 2005 the disclosure of which is incorporated herein in its
entirety.
Alternatively, or in addition, M. tuberctslosis glutamine synthetase (GS)
protein can be
expressed and fragments obtained therefrom by standard means, or
alternatively,
io s}mthetic peptides can be synthesized based on the sequence of the full-
length protein
(e.g., comprising the sequence set forth in SwissProt Database Accession No.
033342).
Exemplary immunogen fragments of the GS protein are derived from a surface-
exposed
region of a GS protein, or comprise the sequence
RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO: 19) or
15 WASGYRGLTPASDYNIDYAI (SEQ ID NO: 20). Methods for producing such
fragments are described in detail in the instant in the instant applicant's co-
pending
International Patent Application No. PCT/AU2005/000930 filed June 24 2005 the
disclosure of which is incorporated herein in its entirety.
2o Antibodies that bind to a S9 protein or an epitope tltereof
A second aspect of the present invention provides an antibody that binds
specifically to
a S9 protein or an immunogenic fragment or epitope thereof, such as, for
example, a
monoclonal or polyclonal antibody preparation suitable for use in the assays
described
herein.
Reference herein to antibody or antibodies includes whole polyclonal and
monoclonal
antibodies, and parts thereof, either alone or conjugated with other moieties.
Antibody
parts include Fab and F(ab)2 fragments and single chain antibodies. The
antibodies may
be made in uivo in suitable laboratory animals, or, in the case of engineered
antibodies
(Single Chain Antibodies or SCABS, etc) using recombinant DNA techniques in
vitro.
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51
In accordance with this aspect of the invention, the antibodies may be
produced for the
purposes of immunizing the subject, in which case high titer or neutralizing
antibodies
that bind to a B cell epitope will be especially preferred. Suitable subjects
for
immunization will, of course, depend upon the immunizing antigen or antigenic
B cell
epitope. It is contemplated that the present invention will be broadly
applicable to the
iinmunization of a wide range of animals, such as, for example, farm animals
(e.g.
horses, cattle, sheep, pigs, goats, chickens, ducks, turkeys, and the like),
laboratory
animals (e.g. rats, mice, guinea pigs, rabbits), domestic animals (cats, dogs,
birds and
io the like), feral or wild exotic animals (e.g. possums, cats, pigs, buffalo,
wild dogs and
the like) and humans.
Alternatively, the antibodies may be for commercial or diagnostic purposes, in
which
case the subject to whom the S9 protein or immunogenic fragment or epitope
thereof is
administered will most likely be a laboratory or farm animal. A wide range of
animal
species are used for the production of antisera. Typically the animal used for
production of antisera is a rabbit, mouse, rabbit, rat, hamster, guinea pig,
goat, sheep,
pig, dog, horse, or chicken. Because of the relatively large blood volumes of
rabbits
and sheep, these are preferred choice for production of polyclonal antibodies.
2o However, as will be known to those skilled in the art, larger amounts of
immunogen are
required to obtain high antibodies from large animals as opposed to smaller
animals
such as mice. In such cases, it will be desirable to isolate the antibody from
the
immunized animal.
Preferably, the antibody is a high titer antibody. By "high titer" means a
sufficiently
high titer to be suitable for use in diagnostic or therapeutic applications.
As will be
known in the art, there is some variation in what might be considered "high
titer". For
most applications a titer of at least about 103-104 is preferred. More
preferably, the
antibody titer will be in the range from about 104 to about 105 , even more
preferably in
the range from about 105 to about 106.
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52
More preferably, in the case of B cell epitopes from pathogens, viruses or
bacteria, the
antibody is a neutralizing antibody (i.e. it is capable of neutralizing the
infectivity of
the organism from which the B cell epitope is derived).
To generate antibodies, the S9 protein or immunogenic fragment or epitope
thereof,
optionally formulated with any suitable or desired carrier, adjuvant, BRM, or
pharmaceutically acceptable excipient, is conveniently administered in the
form of an
injectable composition. Injection may be intranasal, intramuscular, sub-
cutaneous,
1o intravenous, intradermal, intraperitoneal, or by other known route. For
intravenous
injection, it is desirable to include one or more fluid and nutrient
replenishers. Means
for preparing and characterizing antibodies are well known in the art. (See,
e.g.,
ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, 1988,
incorporated herein by reference).
Preferred immunogenic peptides for generating polyclonal or monoclonal
antibodies
are selected from the group set forth in the Sequence Listing. In one
embodiment, an
immunogenic peptide such as, for example, an immunogenic peptide comprising
the
amino acid sequence set forth in SEQ ID NO: 6 or an immunogenic fragment
thereof,
is covalently coupled to an immunogenic carrier protein, such as Diphtheria
toxoid
(DT), Keyhole Limpet Hemocyanin (KLH), tetanus toxoid (TT) or the nuclear
protein
of influenza virus (NP), using one of several conjugation chemistries known in
the art.
This enhances the immunogenicity of peptides that are otherwise not highly
immunogenic in animals e.g., mice, rats, chickens etc.
Methods of preparing carrier proteins for such coupling are well known in the
art. For
instance, DT is preferably produced by purification of the toxin from a
culture of
Coryraebacteriunz diphtheriae followed by chemical detoxification, but is
alternatively
made by purification of a recombinant, or genetically detoxified analogue of
the toxin
(for example, CRM197, or other mutants as described in U.S. Pat. Nos.
4,709,017,
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53
5,843,711, 5,601,827, and 5,917,017). Preferably, the toxoid is derivatized
using as a
spacer a bridge of up to 6 carbons, such as provided by use of the adipic acid
hydrazide
derivative of diphtheria toxoid (D-AH).
For coupling, peptides derived from the full-length S9 protein can be
synthesized
chemically or produced by recombinant expression means, treated with
hydroxylamine
to form free sulfhydryl groups, and cross-linked via the free sulfhydryl
groups to a
maleimide-modified diphtheria toxoid, tetanus toxoid or influenza NP protein
or other
carrier molecule. One of the most specific and reliable conjugation
chemistries uses a
io cysteine residue in the peptide and a'maleimide group added to the carrier
protein, to
form a stable thioether bond (Lee, A.C., et al., Mol. Lnrraisnol. 17, 749-756
1980). For
example, if no sulfhydryl groups are present in the peptide, the S9-derived
peptides can
be prior modified by the addition of a C-terminal cysteine residue e.g., SEQ
ID NO: 6
to facilitate this procedure. The immunogenic S9 peptides are preferably
produced
under non-denaturing conditions, treated with hydroxylamine, thiol reducing
agents or
by acid or base hydrolysis to generate free sulfhydryl groups and the free
sulfhydryl-
containing peptide is conjugated to a carrier by chemical bonding via the free
sulfhydryl groups. Such conjugation may be by use of a suitable bis-maleimide
compound. Alternatively, the conjugation of the HA protein may be to a
maleimide-
modified carrier protein, such as diphtheria toxoid, tetanus toxoid or
influenza (NP)
protein or to a carbohydrate, such as alginic acid, dextran or polyethylene
glycol. Such
maleimide-modified carrier molecules may be formed by reaction of the carrier
molecule with a hetero-bifunctional cross-linker of the maleimide-N-
hydroxysuccinimide ester type. Examples of such bifunctional esters include
maleimido-caproic-N-hydroxysuccinimide ester (MCS), maleimido-benzoyl-N-
hydroxysuccinimide ester (MBS), maleimido-benzoylsul-fosuccinimide ester
(sulfo-
MBS), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC),
succinimidyl-4-(p-maleimido-phenyl)butyrate (SMPP), sulfosuccinimidyl-4-(N-
maleimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC) and sulfosuccinimidyl-4-
(p-maleimidophenyl) butyrate (sulfo-SMPP). The N-hydroxy-succinimide ester
moiety
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54
reacts with the amine groups of the carrier protein leaving the maleimide
moiety free to
react with the sulfhydryl groups on the antigen to form the cross-linked
material.
The conjugate molecules so produced may be purified and employed in
immunogenic
compositions to elicit, upon administration to a host, an immune response to
the S9
peptide which is potentiated in comparison to S9 peptide alone.
Diphtheria toxoid is obtained commercially or prepared from Coiynebacterium
diphtheriae grown in submerged culture by standard methods. The production of
io Diphtheria Toxoid is divided into five stages, namely maintenance of the
working seed,
growth of Coi-jmebacterium diphtheriae, harvest of Diphtheria Toxin,
detoxification of
Diphtheria Toxin and concentration of Diphtheria Toxoid. The purified
diphtheria
toxoid (DT) used as carrier in the preparation is preferably a commercial
toxoid
modified (derivatized) by the attachment of a spacer molecule, such as adipic
acid
dihydrazide (ADH), using the water-soluble carbodiimide condensation method.
The
modified toxoid, typically the adipic hydrazide derivative D-AH, is then freed
from
unreacted ADH.
The efficacy of the S9 protein or immunogenic fragment or epitope thereof in
producing an antibody is established by injecting an animal, for example, a
mouse,
chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig, with a
formulation
comprising the S9 protein or immunogenic fragment or epitope thereof, and then
monitoring the immune response to the B cell epitope, as described in the
Examples.
Both primary and secondary immune responses are monitored. The antibody titer
is
determined using any conventional immunoassay, such as, for example, ELISA, or
radio immunoassay.
The production of polyclonal antibodies may be monitored by sampling blood of
the
immunized animal at various points following immunization. A second, booster
injection, may be given, if required to achieve a desired antibody titer. The
process of
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boosting and titering is repeated until a suitable titer is achieved. When a
desired level
of immunogenicity is obtained, the immunized animal is bled and the serum
isolated
and stored, and/or the animal is used to generate monoclonal antibodies
(Mabs).
5 Monoclonal antibodies are particularly preferred. For the production of
monoclonal
antibodies (Mabs) any one of a number of well-known techniques may be used,
such
as, for example, the procedure exemplified in US Patent No. 4,196,265,
incorporated
herein by reference.
1o For example, a suitable animal will be immunized with an effective amount
of the S9
protein or immunogenic fragment or epitope thereof under conditions sufficient
to
stimulate antibody producing cells. Rodents such as rabbits, mice and rats are
preferred
animals, however, the use of sheep or frog cells is also possible. The use of
rats may
provide certain advantages, but mice or rabbits are preferred, with the BALB/c
mouse
15 being most preferred as the most routinely used animal and one that
generally gives a
higher percentage of stable fusions. Rabbits are known to provide high
affinity
monoclonal antibodies.
Following immtinization, somatic cells with the potential for producing
antibodies,
20 specifically B lymphocytes (B cells), are selected for use in the MAb
generating
protocol. These cells may be obtained from biopsies of spleens, tonsils or
lymph nodes,
or from a peripheral blood sample. Spleen cells and peripheral blood cells are
preferred,
the former because they are a rich source of antibody-producing cells that are
in the
dividing plasmablast stage, and the latter because peripheral blood is easily
accessible.
25 Often, a panel of animals will have been immunized and the spleen of animal
with the
highest antibody titer removed. Spleen lymphocytes are obtained by
homogenizing the
spleen with a syringe. Typically, a spleen from an immunized mouse contains
approximately 5 x 107 to 2 x 108 lymphocytes.
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56
The B cells from the immunized animal are then fused with cells of an immortal
myeloma cell, generally derived from the same species as the animal that was
immunized with the S9 protein or immunogenic fragment or epitope thereof.
Myeloma
cell lines suited for use in hybridoma-producing fusion procedures preferably
are non-
antibody-producing, have high fusion efficiency and enzyme deficiencies that
render
them incapable of growing in certain selective media which support the growth
of only
the desired fused cells, or hybridomas. Any one of a number of myeloma cells
may be
used and these are known to those of skill in the art (e.g. murine P3-X63/Ag8,
X63-
AgS.653, NS1/1.Ag 4 1, .Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7
io and S 194/5XX0; or rat R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266,
GM1500-GRG2, LICR-LON-HMy2 and UC729-6). A preferred murine myeloma cell
is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily
available
from the NIGMS Human Genetic Mutant Cell Repository under Accession No.
GM3573. Alternatively, a murine myeloma SP2/0 non-producer cell line that is 8-
azaguanine-resistant is used.
To generate hybrids of antibody-producing spleen or lymph node cells and
myeloma
cells, somatic cells are mixed with myeloma cells in a proportion between
about 20:1 to
about 1:1, respectively, in the presence of an agent or agents (chemical or
electrical)
that promote the fusion of cell membranes. Fusion methods using Sendai virus
have
been described by Kohler and Milstein, Nature 256, 495-497, 1975; and Kohler
and
Milstein, Eu7-. J. Irnntunol. 6, 511-519, 1976. Methods using polyethylene
glycol
(PEG), such as 37% (v/v) PEG, are described in detail by Gefter et al.,
Somatic Cell
Genet. 3, 231-236, 1977. The use of electrically induced fusion methods is
also
appropriate.
Hybrids are amplified by culture in a selective medium comprising an agent
that blocks
the de novo synthesis of nucleotides in the tissue culture media. Exemplary
and
preferred agents are aminopterin, methotrexate and azaserine. Aminopterin and
methotrexate block de novo synthesis of both purines and pyrimidines, whereas
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57
azaserine blocks only purine synthesis. Where aminopterin or methotrexate is
used, the
media is supplemented with hypoxanthine and thymidine as a source of
nucleotides
(HAT medium). Where azaserine is used, the media is supplemented with
hypoxanthine.
The preferred selection medium is HAT, because only those hybridomas capable
of
operating nucleotide salvage pathways are able to survive in HAT medium,
whereas
myeloma cells are defective in key enzymes of the salvage pathway, (e.g.,
hypoxanthine phosphoribosyl transferase or HPRT), and they cannot survive. B
cells
io can operate this salvage pathway, but they have a limited life span in
culture and
generally die within about two weeks. Accordingly, the only cells that can
survive in
the selective media are those hybrids formed from myeloma and B cells.
The amplified hybridomas are subjected to a functional selection for antibody
specificity and/or titer, such as, for example, by immunoassay (e.g.
radioimmunoassay,
enzynle immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and
the
like).
The selected hybridomas are serially diluted and cloned into individual
antibody-
producing cell lines, which clones can then be propagated indefinitely to
provide
MAbs. The cell lines niay be exploited for MAb production in two basic ways. A
sample of the hybridoma is injected, usually in the peritoneal cavity, into a
histocompatible animal of the type that was used to provide the somatic and
myeloma
cells for the original fusion. The injected animal develops tumors secreting
the specific
monoclonal antibody produced by the fused cell hybrid. The body fluids of the
animal,
such as serum or ascites fluid, can then be tapped to provide MAbs in high
concentration. The individual cell lines could also be cultured in vitro,
where the MAbs
are naturally secreted into the culture medium from which they are readily
obtained in
high concentrations. MAbs produced by either means may be further purified, if
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58
desired, using filtration, centrifugation and various chromatographic methods
such as
HPLC or affinity chromatography.
Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to
produce cell lines secreting monoclonal antibodies (mAbs) against immunogenic
S9
peptide antigens. In this process, BALB/cByJ female mice are immunized with an
amount of the peptide antigen over a period of about 2 to about 3 months.
During this
time, test bleeds are taken from the immunized mice at regular intervals to
assess
antibody responses in a standard ELISA. The spleens of mice having antibody
titers of
io at least about 1,000 are used for subsequent ABL-MYC infection employing
replication-incompetent retrovirus comprising the oncogenes v-abl and c-ntyc.
Splenocytes are transplanted into naive mice which then develop ascites fluid
containing cell lines producing monoclonal antibodies (mAbs) against the S9
peptide
antigen. The mAbs are purified from ascites using protein G or protein A,
e.g., bound
to a solid matrix, depending on the isotype of the mAb. Because there is no
hybridoma
fusion, an advantage of the ABL-MYC process is that it is faster, more cost
effective,
and higher yielding than conventional mAb production methods. In addition, the
diploid plasmacytomas produced by this metliod are intrinsically more stable
than
polyploid hybridomas, because the ABL-MYC retrovirus infects only cells in the
spleen that have been stimulated by the immunizing antigen. ABL-MYC then
transforms those activated B-cells into immortal, mAb-producing plasma cells
called
plasmacytomas. A"plasmacytoma" is an immortalized plasma cell that is capable
of
uncontrolled cell division. Since a plasmacytoma begins with just one cell,
all of the
plasmacytomas produced from it are therefore identical, and moreover, produce
the
same desired "monoclonal" antibody. As a result, no sorting of undesirable
cell lines is
required. The ABL-MYC technology is described generically in detail in the
following
disclosures which are incorporated by reference herein:
1. Largaespada et al., Curr. Top. Microbiol. hnniunol., 166, 91-96. 1990;
2. Weissinger et al.,Proc. Natl. Acad. Sci. USA, 88, 8735-8739, 1991;
. go 3. Largaespada et al., Oncogene, 7, 811-819, 1992;
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59
4. Weissinger et al.,1. Immisnol. Methods 168, 123-130, 1994;
5. Largaespada et al., J. Innnzun.ol. Methods. 197(1-2), 85-95, 1996; and
6. Kumar et al., Irnmuno. Letters 65, 153-159, 1999.
Monoclonal antibodies of the present invention also include anti-idiotypic
antibodies
produced by methods well-known in the art. Monoclonal antibodies according to
the
present invention also may be monoclonal heteroconjugates, (i.e., hybrids of
two or
more antibody molecules). In another embodiment, monoclonal antibodies
according to
the invention are chimeric monoclonal antibodies. In one approach, the
chimeric
io monoclonal antibody is engineered by cloning recombinant DNA containing the
promoter, leader, and variable-region sequences from a mouse anti-PSA
producing cell
and the constant-region exons from a human antibody gene. The antibody encoded
by
such a recombinant gene is a mouse-human chimera. Its antibody specificity is
determined by the variable region derived from mouse sequences. Its isotype,
which is
determined by the constant region, is derived from human DNA.
In another embodiment, the monoclonal antibody according to the present
invention is
a "humanized" monoclonal antibody, produced by any one of a number of
techniques
well-known in the art. That is, mouse complementary determining regions
("CDRs")
2o are transferred from heavy and light V-chains of the mouse Ig into a human
V-domain,
followed by the replacement of some human residues in the framework regions of
their
murine counterparts. "Humanized" monoclonal antibodies in accordance with this
invention are especially suitable for use in vivo in diagnostic and
therapeutic methods.
As stated above, the monoclonal antibodies and fragments thereof according to
this
invention are multiplied according to in vitro and in vivo methods well-known
in the
art. Multiplication in vitro is carried out in suitable culture media such as
Dulbecco's
modified Eagle medium or RPMI 1640 medium, optionally replenished by a
mammalian serum such as fetal calf serum or trace elements and growth-
sustaining
supplements, e.g., feeder cells, such as normal mouse peritoneal exudate
cells, spleen
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cells, bone marrow macrophages or the like. In vitro production provides
relatively
pure antibody preparations and allows scale-up to give large amounts of the
desired
antibodies. Techniques for large scale hybridoma cultivation under tissue
culture
conditions are known in the art and include homogenous suspension culture,
(e.g., in an
5 airlift reactor or in a continuous stirrer reactor or immobilized or
entrapped cell
culture).
Large amounts of the monoclonal antibody of the present invention also may be
obtained by multiplying hybridoma cells in vivo. Cell clones are injected into
niammals
io which are histocompatible with the parent cells, (e.g., syngeneic mice, to
cause growth
of antibody-producing tumors. Optionally, the animals are primed with a
hydrocarbon,
especially oils such as Pristane (tetramethylpentadecane) prior to injection.
In accordance with the present invention, fragments of the monoclonal antibody
of the
15 invention are obtained from monoclonal antibodies produced as described
above, by
methods which include digestion with enzymes such as pepsin or papain and/or
cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal
antibody
fragments encompassed by the present invention are synthesized using an
automated
peptide synthesizer, or they may be produced manually using techniques well
known in
20 the art.
The monoclonal conjugates of the present invention are prepared by methods
known in
the art, e.g., by reacting a monoclonal antibody prepared as described above
with, for
instance, an enzyme in the presence of a coupling agent such as glutaraldehyde
or
25 periodate. Conjugates with fluorescein markers are prepared in the presence
of these
coupling agents, or by reaction with an isothiocyanate. Conjugates with metal
chelates
are similarly produced. Other moieties to which antibodies may be conjugated
include
radionuclides such as, for example, 3H, 125I, =32P, =35S, 14C, 51Cr, 36C1,
57Co, 53Co, 5917e,
7$Se, and 152Eu.
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61
i ne present invention clearly includes antibodies when coupled to any
detectable
ligand or reagent, including, for example, an enzyme such as horseradish
peroxidase or
alkaline phosphatase, or a fluorophore, radionuclide, coloured dye, gold
particle,
colloidal gold, etc.
Radioactively labelled monoclonal antibodies of the present invention are
produced
according to well-knowii methods in the art. For instance, monoclonal
antibodies are
iodinated by contact with sodium or potassium iodide and a chemical oxidizing
agent
such as sodium hypochlorite, or an enzymatic oxidizing agent, such as
lactoperoxidase.
io Monoclonal antibodies according to the invention may be labelled witli
technetium99 by
ligand exchange process, for example, by reducing pertechnate with stannous
solution,
chelating the reduced technetium onto a Sephadex column and applying the
antibody to
this column or by direct labelling techniques, (e.g., by incubating
pertechnate, a
reducing agent such as SNC12, a buffer solution such as sodium-potassium
phthalate
solution, and the antibody).
Any immunoassay may be used to monitor antibody production by the S9 protein
or
immunogenic fragment or epitope thereof . Immunoassays, in their most simple
and
direct sense, are binding assays. Certain preferred immunoassays are the
various types
of enzynie linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA)
known in the art. Immunohistochemical detection using tissue sections is also
particularly useful. However, it will be readily appreciated that detection is
not limited
to such techniques, and Western blotting, dot blotting, FACS analyses, and the
like may
also be used.
Most preferably, the assay will be capable of generating quantitative results.
For example, antibodies are tested in simple competition assays. A known
antibody
preparation that binds to the B cell epitope and the test antibody are
incubated with an
3o antigen composition comprising the B cell epitope, preferably in the
context of the
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62
native antigen. "Antigen composition" as used herein means any composition
that
contains some version of the B cell epitope in an accessible form. Antigen-
coated wells
of an ELISA plate are particularly preferred. In one embodiment, one would pre-
mix
the known antibodies with varying amounts of the test antibodies (e.g., 1:1,
1:10 and
1:100) for a period of time prior to applying to the antigen composition. If
one of the
known antibodies is labelled, direct detection of the label bound to the
antigen is
possible; comparison to an unmixed sample assay will determine competition by
the
test antibody and, hence, cross-reactivity. Alternatively, using secondary
antibodies
specific for either the known or test antibody, one will be able to determine
io competition.
An antibody that binds to the antigen composition will be able to effectively
compete
for binding of the known antibody and thus will significantly reduce binding
of the
latter. The reactivity of the known antibodies in the absence of any test
antibody is the
control. A significant reduction in reactivity in the presence of a test
antibody is
indicative of a test antibody that binds to the B cell epitope (i.e., it cross-
reacts with the
known antibody).
In one exemplary ELISA, the antibodies that bind to the S9 protein or
immunogenic
fragment or B cell epitope are immobilized onto a selected surface exhibiting
protein
affinity, such as a well in a polystyrene microtiter plate. Then, a
composition
containing a peptide comprising the B cell epitope is added to the wells.
After binding
and washing to remove non-specifically bound immune complexes, the bound
epitope
may be detected. Detection is generally achieved by the addition of a second
antibody
that is known to bind to the B cell epitope and is linked to a detectable
label. This type
of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the
addition of said second antibody, followed by the addition of a third antibody
that has
binding affinity for the second antibody, with the third antibody being linked
to a
detectable label.
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In an alternative embodiment (i.e., amplified ELISA), antibodies that bind to
the S9
protein or immunogenic fragment or B cell epitope are immobilized onto a
selected
surface exhibiting protein affinity, such as a well in a polystyrene
microtiter plate.
Then, a composition containing a peptide comprising the B cell epitope is
added to the
wells. After binding and washing to remove non-specifically bound immune
complexes, antibodies that bind to the B cell epitope are contacted with the
bound
peptide for a time and under conditions sufficient for a complex to form. The
signal is
then aniplified using secondary and preferably tertiary, antibodies that bind
to the
antibodies recognising the B cell epitope. Detection is then achieved by the
addition of
io a further antibody that is known to bind to the secondary or tertiary
antibodies, linked
to a detectable label.
In another exemplary immunoassay format applicable to both flow through and
solid
phase ELISA, antibodies that bind to the immunogenic S9 protein or immunogenic
S9
peptide or immunogenic S9 fragment or B cell epitope are immobilized onto a
selected
surface exhibitiiig protein affinity, such as a well in a polystyrene
microtiter plate or a
column. A sample comprising the immunogenic S9 protein or immunogenic peptide
or
immunogenic fragment comprising the B cell epitope to which the antibody binds
is
added for a time and under conditions sufficient for an antigen-antibody
complex to
form. In this case, the added S9 protein, peptide or fragment is complexed
with human
Ig. In the case of patient sera, for example, the peptide is preferably
complexed with
human Ig by virtue of an immune response of the patient to the M. tuberculosis
S9
protein. After binding and washing to remove non-specifically bound immune
complexes, the bound epitope is detected by the addition of a second antibody
that is
known to bind to human Ig in the immune complex and is linked to a detectable
label.
This is a modified "sandwich ELISA". Detection may also be achieved by the
addition
of said second antibody, followed by the addition of a third antibody that has
binding
affinity for the second antibody, with the third antibody being linked to a
detectable
label.
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64
tinuooaies oi me invention may be bound to a solid support and/or packaged
into kits
in a suitable container along with suitable reagents, controls, instructions
and.the like.
Antibodies that bind to a secondary analvte
It is to be understood that methods described herein above for the production
of
antibodies against the S9 protein or an immunogenic fragment thereof apply
inutatis
inutandis to the production of antibodies against a secondary analyte that is
to be used
in an inlmunoassay format e.g., for the purposes of diagnosis or prognosis of
tuberculosis or infection by M. tuberculosis. As will be understood by the
skilled
io artisan, such extrapolation is dependent on substituting the S9 immunogen
for the
secondary analyte in question e.g., Nl. tuberculosis Bsx protein or GS protein
or
immunogenic fragment thereof according to any embodiment described hereiii.
Such
substitution is readily performed without undue experimentation fro the
disclosure
herein.
For convenience, preferred immunizing peptides for the production of
antibodies
against secondary analytes e.g., for use in multi-analyte antigen-based tests,
will
comprise an amino acid sequence selected from the group set forth in SEQ ID
NOs: 8-
20.
For example, antibodies that bind to M. tuberculosis Bsx protein can be
prepared from
the full-length protein (e.g., comprising the sequence set forth in SwissProt
Database
Accession No. 053759) or from a peptide fragment thereof e.g., comprising a
sequence
selected from the group consisting of: MRQLAERSGVSNPYL (SEQ ID NO: 8),
ERGLRKPSADVLSQI (SEQ ID NO: 9), LRKPSADVLSQIAKA. (SEQ ID NO: 10),
PSADVLSQIAKALRV (SEQ ID NO: 11), SQIAKALRVSAEVLY (SEQ ID NO: 12),
AKALRVSAEVLYVRA (SEQ ID NO: 13), VRAGILEPSETSQVR (SEQ ID No: 14),
TAITERQKQILLDIY (SEQ ID NO; 15), SQIAKALRVSAEVLYVRAC (SEQ ID
NO: 16), MSSEEKLCDPTPTDD (SEQ ID NO: 17) and VRAGILEPSETSQVRC
(SEQ ID NO: 18). Antibodies that bind to an immunogenic M. tuberculosis Bsx
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protein or peptide for detecting tuberculosis or infection by M. tuberculosis
are also
described in detail in the instant applicant's co-pending International Patent
Application
No. PCT/AU2005/001254 filed August 19, 2005 the disclosure of which is
incorporated herein in its entirety.
5
Alternatively, or in addition, antibodies that bind to M. tuberculosis
glutamine
synthetase (GS) protein (e.g., comprising the sequence set forth in SwissProt
Database
Accession No. 033342) or from an immunogenic peptide derived thereof e.g.,
comprising a surface-exposed region of a GS protein, or comprising the
sequence
io RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO: 19) or
WASGYRGLTPASDYNIDYAI (SEQ ID NO: 20). Antibodies that bind to an
immunogenic M. tuberculosis GS or peptide for detecting tuberculosis or
infection by
M. tuberculosis are also described in detail in the instant applicant's co-
pending.
International Patent Application No. PCT/AU2005/000930 filed June 24 2005 the
15 disclosure of which is incorporated herein in its entirety.
The present invention clearly contemplates antibodies agaisnt secondary
analytes other
than Bsx or Gs or immunogenic fragnients thereof, the description of which is
provided
for the purposes of exemplification.
Diagnostic/prognostic rnetlaods for detecting tuberculosis or M. tuberculosis
infection
1. Antigen-based ass~s
This invention provides a method of diagnosing tuberculosis or an infection by
lll.
tuberculosis in a subject comprising detecting in a biological sample from
said subject
a S9 protein or 'an immunogenic fragment or epitope thereof, wherein the
presence of
said protein or immunogenic fragment or epitope in the sample is indicative of
infection.
One advantage of detecting M. tuberculosis antigen, as opposed to an antibody-
based
3o assay is that severely immune-compromized patients may not produce antibody
at
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66
detectable levels, and the level of the antibody in any patient does not
reflect bacilli
burden. On the other hand antigen levels should reflect bacilli burden and,
being a
product of the bacilli, are a direct method of detecting its presence.
In one embodiment of the diagnostic assays of the invention, there is provided
a
method for detecting M. tuberculosis infection in a subject, the method
comprising
contacting a biological sample derived from the subject with an antibody
capable of
binding to a S9 protein or an immunogenic fragment or epitope thereof, and
detecting
the formation of an antigen-antibody complex.
In another embodiment, the diagnostic assays of the invention are useful for
determining the progression of tuberculosis or an infection by M. tuber
culosis in a
subject. In accordance with these prognostic applications of the invention,
the level of
S9 protein or an immunogenic fragment or epitope thereof in a biological
sample is
positively correlated with the infectious state. For example, a. level of the
S9 protein or
an immunogenic fragment thereof that is less than the level of the S9 protein
or
fragment detectable in a subject suffering from the symptoms of tuberculosis
or an
infection indicates that the subject is recovering from the infection.
Similarly, a higher
level of the protein or fragment in a sample from the subject compared to a
healthy
individual indicates that the subject has not been rendered free of the
disease or
infection.
Accordingly, a further embodiment of the present invention provides a method
for
determining the response of a.subject having tuberculosis or an infection by
M.
tuberculosis to treatment with a therapeutic compound for said tuberculosis or
infection, said method comprising detecting a S9 protein or an immunogenic
fragment
or epitope thereof in a biological sample from said subject, wherein a level
of the
protein or fragment or epitope that is enhanced compared to the level of that
protein or
fragment or epitope detectable in a normal or healthy subject indicates that
the subject
is not responding to said treatment or has not been rendered free of disease
or infection.
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In an alternative embodiment, the present invention provides a method for
determining
the response of a subject having tuberculosis or an infection by M. tubet-
culosis to
treatment with a therapeutic compound for said tuberculosis or infection, said
method
comprising detecting a S9 protein or an immunogenic fragment or epitope
thereof in a
biological sample from said subject, wherein a level of the protein or
fragment or
epitope that is lower than the level of the protein or fragment or epitope
detectable in a
subject suffering from tuberculosis or infection by M. tuberculosis indicates
that the
subject is responding to said treatment or has been rendered free of disease
or infection.
io Clearly, if the level of the S9 protein or fragment or epitope thereof is
not detectable in
the subject, the subject has responded to treatment.
In a further embodiment, the amount of a S9 protein in a biological sample
derived
from a patient is compared to the amount of the same protein detected in a
biological
sample previously derived from the same patient. As will be apparent to a
person
skilled in the art, this method may be used to continually monitor a patient
with a latent
infection or a. patient that has developed tuberculosis. In this way a patient
may be
monitored for the onset or progression of an infection or disease, with the
goal of
commencing treatment before an infection is established, particularly in an
HIV+
2o individual.
Alternatively, or in addition, the amount of a protein detected in a
biological sample
derived from a subject with tuberculosis may be compared to a reference
sample,
wherein the reference sample is derived from one or more tuberculosis patients
that do
not suffer from an infection or disease or alternatively, one or more
tuberculosis
patients that have recently received successful treatment for infection and/or
one or
more subjects that do not have tuberculosis and that do not suffer from an
infection or
disease.
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In one embodiment, a S9 protein or imnlunogenic firagment thereof is not
detected in a
reference sample, however, said S9 protein or immunogenic fragment thereof is
detected in the patient sample, indicating that the patient from whom the
sample was
derived is suffering from tuberculosis or infection by M: tuberculosis or will
develop an
acute infection.
Alternatively, the amount of S9 protein or immunogenic fragment thereof may be
enhanced in the patient sample compared to the level detected in a reference
sample.
Again, this indicates that the patient from whom the biological sample was
isolated is
io suffering from tuberculosis or infection by M. tuberculosis or will develop
an acute
infection.
In one embodiment of the diagnostic/prognostic methods described herein, the
biological sample is obtained previously from the subject. In accordance with
such an
embodiment, the prognostic or diagnostic method is performed ex vivo.
In yet another embodiment, the subject diagnostic/prognostic methods further
comprise
processing the sample from the subject to produce a derivative or extract that
comprises
the analyte (eg., pleural fluid or sputum or serum).
Suitable samples include extracts from tissues such as brain, breast, ovary,
lung, colon,
pancreas, testes, liver, muscle and bone tissues, or body fluids such as
sputum, serum,
plasma, whole blood, sera or pleural fluid.
Preferably, the biological sample is a bodily fluid or tissue sample selected
from the
group consisting of: saliva, plasma, blood, serum, sputum, urine, and lung.
Other
samples are not excluded.
It will be apparent from the description herein that preferred samples may
comprise
circulating immune complexes comprising the S9 protein or fragments thereof
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69
complexed with human immunoglobulin. The detection of such immune complexes is
clearly within the scope of the present invention. In accordance with this
embodiment,
a capture reagent e.g., a capture antibody is used to capture the S9 antigen
(S9
polypeptide or an immunoactive fragment or epitope thereof) complexed with the
subject's immunoglobulin, in addition to isolated antigen in the subject's
circulation.
Anti-Ig antibodies, optionally conjugated to a detectable label, are used to
specifically
bind the captured CIC thereby detecting CIC patient samples. Within the scope
of this
invention, the anti-Ig antibody binds preferentially to IgM, IgA or IgG in the
sample.
In a particularly preferred embodiment, the anti-Ig antibody binds to human
Ig, e.g.,
io human IgA, human IgG or human IgM. The anti-Ig antibody may be conjugated
to any
standard detectable label known in the art. This is particularly useful for
detecting
infection by a pathogenic agent, e.g., a bacterium or virus, or for the
diagnosis of any
disease or disorder associated with CICs. Accordingly, the diagnostic inethods
described according to any embodiment herein are amenable to a modification
wherein
the sample derived from the subject comprises one or more circulating immune
complexes comprising immunoglobulin (Ig) bound to S9 protein of M)~cobacterium
tuberculosis or one or more immunogenic S9 peptides, fragments or epitopes
thereof
and wherein detecting the formation of an antigen-antibody complex comprises
contacting an anti-Ig antibody with an immunoglobulin moiety of the
circulating
immune complex(es) for a time and under conditions sufficient for a complex to
form
than then detecting the bound anti-Ig antibody.
The present invention clearly encompasses multianalyte tests for diagnosing
infection
by M. tuberculosis. For example, assays for detecting antibodies that bind to
M.
tuber=culosis S9 protein can be combined with assays for detecting M.
tuber=culosis Bsx
or glutamine synthetase (GS) protein. In this respect, the present inventors
have also
produced plasmacytomas producing monoclonal antibodies that bind to an
immunogenic fragment or peptide or epitope of Bsx or GS.
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2. Antibody-based assays
The present invention provides a method of diagnosing tuberculosis or an
infection by
M. tuberculosis in a subject comprising detecting in a biological sample from
said
subject antibodies that bind to a S9 protein or an immunogenic,fragment or
epitope
5 thereof, wherein the presence of said antibodies in the sample is indicative
of infection.
The infection may be a past or present infection, or a latent infection.
'Antibody-based assays are primarily used for detecting active infections by
M.
tuberculosis. Preferably, the clinical history of the subject is considered
due to residual
io antibody levels that may persist in recent past infections or chronic
infections by M.
tuberculosis.
The format is inexpensive and highly sensitive, however not as useful as an
antigen-
based assay format for detecting infection in immune-compromized individuals.
15 However, antibody-based assays are clearly useful for detecting M.
tubef=culosis
infections in HIV" or HIV} individuals who are not immune-compromized.
In one alternative embodiment, the present invention provides a method for
detecting
M. tubei=culosis infection in a subject, the method comprising contacting a
biological
20 sample derived from the subject with a S9 protein or an immunogenic
fragment or
epitope thereof and detecting the formation of an antigen-antibody complex.
In another embodiment, the diagnostic assays of the invention are useful for
determining the progression of tuberculosis or an infection by Af tuber-
culosis in a
25 subject. In accordance with these prognostic applications of the invention,
the amount
of antibodies that bind to a S9 protein or fragment or epitope in blood or
serum,
plasma, or an immunoglobulin fraction from the subject is positively
correlated with
the infectious state. For example, a level of the anti-S9 antibodies thereto
that is less
than the level of the anti-S9 antibodies detectable in a subject suffering
from the
30 symptoms of tuberculosis or an infection indicates that the subject is
recovering from
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71
the infection. Similarly, a higher level of the antibodies in a sample from
the subject
compared to a healthy individual indicates that the subject has not been
rendered free of
the disease or infection.
In a further embodiment of the present invention provides a method for
determining the
response of a subject having tuberculosis or an infection by M. tuberculosis
to
treatment with a therapeutic compound for said tuberculosis or infection, said
method
comprising detecting antibodies that bind to a S9 protein or an immunogenic
fragment
or epitope thereof in a biological sample from said subject, wherein a level
of the
io antibodies that is enhanced compared to the level of the antibodies
detectable in a
normal or healthy subject indicates that the subject is not responding to said
treatment
or has not been rendered free of disease or infection.
In an alternative embodiment, the present invention provides a method for
determining
the response of a subject having tuberculosis or an infection by M.
tuberculosis to
treatment with a therapeutic compound for said tuberculosis or infection, said
method
comprising detecting antibodies that bind to a S9 protein or an immunogenic
fragment
or epitope thereof in a biological sample from said subject, wherein a level
of the
antibodies that is lower than the level of the antibodies detectable in a
subject suffering
from tuberculosis or infection by M. iaiberculosis indicates that the subject
is
responding to said treatment or has been rendered free of disease or
infection.
The amount of an antibody against the S9 protein or fragment that is detected
in a
biological sample from a subject with tuberculosis may be compared to a
reference
sample, wherein the reference sample is derived from one or more healthy
subjects who
have not been previously infected with M. tuberculosi.s or not recently-
infected with M.
tuberculosis. Such negative control subjects will have a low circulating
antibody titer
making them suitable standards in antibody-based assay formats. For example,
antibodies that bind to a S9 protein or immunogenic fragment thereof are not
detected
in the reference sample and only in a patient sample, indicating that the
patient from
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72
whom the sample was derived is suffering from tuberculosis or infection by M.
tuberculosis or will develop an acute infection.
In one embodiment of the diagnostic/prognostic methods described herein, the
biological sample is obtained previously from the subject. In accordance with
such.an
embodiment, the prognostic or diagnostic method is performed ex vivo.
In yet another embodiment, the subject diagnostic/prognostic methods further
comprise
processing the sample from the subject to produce a derivative or extract that
comprises
1o the analyte (e.g., blood, serum, plasma, or any immunoglobulin-containing
sample).
Suitable sarriples include, for example, extracts from tissues comprising an
immunoglobulin such as blood, bone, or body fluids such as serum, plasma,
whole
blood, an immunoglobulin-containing fraction of serum, an immunoglobulin-
containing fraction of plasma, an immunoglobulin-containing fraction of blood.
3. Detection systems
Preferred detection systems contemplated herein include any known assay for
detecting
proteins or antibodies in a biological sample isolated from a human subject,
such as, for
2o example, SDS/PAGE, isoelectric focusing, 2-dimensional gel electrophoresis
comprising SDS/PAGE and isoelectric focusing, an immunoassay, a detection
based
system using an antibody or non-antibody ligand of the protein, such as, for
example, a
small molecule (e.g. a chemical compound, agonist, antagonist, allosteric
modulator,
competitive inhibitor, or non-competitive inhibitor, of the protein). In
accordance with
these embodiments, the antibody or small molecule may be used in any standard
solid
phase or solutioii phase assay format amenable to the detection of proteins.
Optical or
fluorescent detection, such as, for example, using mass spectrometry, MALDI-
TOF,
biosensor technology, evanescent fiber optics, or fluorescence resonance
energy
transfer, is clearly encompassed by the present invention. Assay systems
suitable for
use in high throughput screening of mass samples, particularly a high
throughput
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73
spectroscopy resonance method (e.g. MALDI-TOF, electrospray MS or nano-
electrospray MS), are particularly contemplated.
Immunoassay formats are particularly preferred, e.g., selected from the group
consisting of, an immunoblot, a Western blot, a dot blot, an enzyme linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay.
Modified immunoassays utilizing fluorescence resonance energy transfer (FRET),
isotope-coded affinity tags (ICAT), mass spectrometry, e.g., matrix-assisted
laser
desorption/ionization time of flight (MALDI-TOF), electrospray ionization
(ESI),
io biosensor technology, evanescent fiber-optics technology or protein chip
technology
are also useful.
Preferably, the assay is a semi-quantitative assay or quantitative assay.
Standard solid phase ELISA formats are particularly useful in determining the
concentration of a protein or antibody from a variety of patient samples.
In one form such as an assay involves immobilising a biological sample
comprising
anti-S9 antibodies, or alternatively S9 protein or an immunogenic fragment
thereof,
onto a solid matrix, such as, for example a polystyrene or polycarbonate
microwell or
dipstick, a membrane, or a glass support (e.g. a glass slide).
In the case of an antigen-based assay, an immobilised antibody that
specifically binds a
S9 protein is brought into direct contact with the biological sample, and
forms a direct
bond with any of its target protein present in said sample. For an antibody-
based assay,
an immobilised isolated or recombinant S9 protein or an immunogenic fragment
or
epitope thereof will be contacted with the biological sample. The added
antibody or
protein in solution is generally labelled with a detectable reporter molecule,
such as for
example, colloidal gold, a fluorescent label (e.g. FITC or Texas Red) or an
enzyme
(e.g. horseradish peroxidase (HRP)), alkaline phosphatase (AP) or P-
galactosidase.
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Alternatively, or in addition, a second labelled antibody can be used that
binds to the
first antibody or to the isolated/recombinant S9 antigen. Following washing to
remove
any unbound antibody or S9 antigen, the label may be detected either directly,
in the
case of a fluorescent label, or through the addition of a substrate, such as
for example
hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indol-beta-D-
galaotopyranoside (x-gal).
Such ELISA based systems are particularly suitable for quantification of the
amount of
a protein or antibody in a sample, such as, for example, by calibrating the
detection
io system against known amounts of a standard.
In another form, an ELISA consists of immobilizing an antibody that
specifically binds
a S9 protein on a solid matrix, such as, for example, a membrane, a
polystyrene or
polycarbonate microwell, a polystyrene or polycarbonate dipstick or a glass
support. A
patient sample is then brought into physical relation with said antibody, and
the antigen
in the sample is bound or `captured'. The bound protein can then be detected
using a
labelled antibody. For example if the protein is captured from a human sample,
an anti-
human antibody is used to detect the captured protein.
One example of this embodiment of the invention comprises:
(i) immobilizing an antibody that specifically binds an immunogenic S9 peptide
of
the invention to a solid matrix or support (e.g., a peptide comprising a
sequence
set forth in an), one or more of SEQ ID NOs: 2-7);
(ii) contacting the bound antibody with a sample obtained from a subject,
preferably
an antibody-containing sample such as blood, serum or Ig-containing fraction
thereof for a time and under conditions sufficient for the immobilized
antibody
to bind to an S9 protein or fragment thereof in the sample thereby forming an
antigen-antibody complex; and
(iii) detecting the antigen-antibody complex formed in a process comprising
contacting said complex with an antibody that recognizes human Ig, wherein the
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presence of said human Ig indicates the presence of M. tuberculosis in the
patient sample.
In accordance with this embodiment, specificity of the immobilized antibody
ensures
5 that only isolated or immunocomplexed S9 protein or fragments comprising the
epitope
that the antibody recognizes actually bind, whilst specificity of anti-human
Ig ensures
that only immunocomplexed S9 protein or fragment is detected. In this context,
the
term "immunocomplexed" shall be taken to mean that the S9 protein or fragments
thereof in the patient sample are complexed with human Ig such as human IgA or
1o human IgM or human IgG, etc. Accordingly, this embodiment is particularly
useful for
detecting the presence of M. tuberculosis or an infection by M. tuberculosis
that has
produced an imniune response in a subject. By appropriately selecting the
detection
antibody, e.g., anti-human IgA or anti-human IgG or anti-human IgM, it is
further
possible to isotype the immune response of the subject. Such detection
antibodies that
15 bind to human IgA, IgM and IgG are publicly available to the art.
Alternatively or in addition to the preceding embodiments, a third labelled
antibody can
be used that binds the second (detecting) antibody.
20 It will be apparent to the skilled person that the assay formats described
herein are
amenable to high throughput formats, such as, for example automation of
screening
processes, or a niicroarray format as described in Mendoza et al,
Biotechniaues 27(4):
778-788, 1999. Furthermore, variations of the above described assay will be
apparent to
those skilled in the art, such as, for example, a competitive ELISA.
Alternatively, the presence of anti-S9 antibodies, or alternatively a S9
protein or an
immunogenic fragment thereof, is detected using a radioimmunoassay (RIA). The
basic
principle of the assay is the use of a radiolabelled antibody or antigen to
detect antibody
antigen interactions. For example, an antibody that specifically binds to a S9
protein
can be bound to a solid support and a biological sample brought into direct
contact with
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76
said antibody. To detect the bound an.tigen, an isolated and/or recombinant
form of the
antigen is radiolabelled is brought into contact with the same antibody.
Following
washing the amount of bound radioactivity is detected. As any antigen in the
biological
sample inhibits binding of the radiolabelled antigen the amount of
radioactivity
detected is inversely proportional to the amount of antigen in the sample.
Such an assay
may be quantitated by using a standard curve using increasing known
concentrations of
the isolated antigen.
As will be apparent to the skilled artisan, such an assay may be modified to
use any
io reporter molecule, such as, for example, an enzyme or a fluorescent
molecule, in place
of a radioactive label.
Western blotting is also useful for detecting a S9 protein or an immunogenic
fragment
thereof. In such an assay, protein from a biological sample is separated using
sodium
dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) using
techniques well known in the art and described in, for example, Scopes (In:
Protein
Purification: Principles and Practice, Third Edition, Springer Verlag, 1994).
Separated
proteins are then transferred to a solid support, such as, for example, a
membrane or
more specifically, nitrocellulose membrane, nylon membrane or PVDF membrane,
using methods well known in the art, for example, electrotransfer. This
membrane may
then be blocked and probed with a labelled antibody or ligand that
specifically binds a
S9 protein. Alternatively,* a labelled secondary, or even tertiary, antibody
or ligand can
be used to detect the binding of a specific primary antibody.
High-throughput methods for detecting the presence or absence of anti-S9
antibodies,
or alternatively S9 protein or an immunogenic fragment thereof are
particularly
preferred.
In one embodiment, mass spectrometry, e.g., MALDI-TOF is used for the rapid
identification of a protein that has been separated by either one- or two-
dimensional gel
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77
electrophoresis. Accordingly, there is no need to detect the proteins of
interest using an
antibody or ligand that specifically binds to the protein of interest. Rather,
proteins
from a biological sample are separated using gel electrophoresis using methods
well
known in the art and those proteins at approximately the correct molecular
weight
and/or isoelectric point are analysed using MALDI-TOF to determine the
presence or
absence of a protein of interest.
Alternatively, mass spectrometry, e.g., MALDI or ESI, or a combination of
approaches
is used to determine the concentration of a particular protein in a biological
sample,
io such as, for example sputum. Such proteins are preferably well
characterised previously
with regard to parameters such as molecular weight and isoelectric point.
Biosensor devices generally employ an electrode surface in combination with
current or
impedance measuring elements to be integrated into a device in combination
with the
assay substrate (such as that described in U.S. Patent No. 5,567,301). An
antibody or
ligand that specifically binds to a protein of interest is preferably
incorporated onto the
surface of a biosensor device and a biological sample isolated from a patient
(for
example sputum that has been solubilised using the methods described herein)
contacted to said device. A change in the detected current or impedance by the
2o biosensor device indicates protein binding to said antibody or ligand. Some
forms of
biosensors known in the art also rely on surface plasmon resonance to detect
protein
interactions, whereby a change in the surface plasmon resonance surface of
reflection is
indicative of a protein binding to a ligand or antibody (U.S. Patent No.
5,485,277 and
5,492,840).
Biosensors are of particular use in high throughput analysis due to the ease
of adapting
such systems to micro- or nano-scales. Furthermore, such systems are
conveniently
adapted to incorporate several detection reagents, allowing for multiplexing
of
diagnostic reagents in a single biosensor unit. This permits the simultaneous
detection
of several epitopes in a small amount of body fluids.
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Evanescent biosensors are also preferred as they do not require the
pretreatment of a
biological sample prior to detection of a protein of interest. An evanescent
biosensor
generally relies upon light of a predetermined wavelength interacting with a
fluorescent
molecule, such as for example, a fluorescent antibody attached near the
probe's surface,
to emit fluorescence at a different wavelength upon binding of the diagnostic
protein to
the antibody or ligand.
To produce protein chips, the proteins, peptides, polypeptides, antibodies or
ligands
io that are able to bind specific antibodies or proteins of interest are bound
to a solid
support such as for example glass, polycarbonate, polytetrafluoroethylene,
polystyrene,
silicon oxide, metal or silicon nitride. This immobilization is either direct
(e.g. by
covalent linkage, such as, for example, Schiff s base formation, disulfide
linkage, or
amide or urea bond formation) or indirect. Methods of generating a protein
chip are
known in the art and are described in for example U.S. Patent Application No.
20020136821, 20020192654, 20020102617 and U.S. Patent No. 6,391,625. In order
to
bind a protein to a solid support it is often necessary to treat the solid
support so as to
create chemically reactive groups on the surface, such as, for example, with
an
aldehyde-containing silane reagent. Alternatively, an antibody or ligand may
be
captured on a microfabricated polyacrylamide gel pad and accelerated into the
gel
using microelectrophoresis as described in, Arenkov et al. Anal. Biochem.
278:123-
131, 2000.
A protein chip is preferably generated such that several proteins, ligands or
antibodies
are arrayed on said chip. This format permits the simultaneous screening for
the
presence of several proteins in a sample.
Alternatively, a protein chip may comprise only one protein, ligand or
antibody, and be
used to screen one or more patient samples for the presence of one polypeptide
of
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79
interest. Such a chip may also be used to simultaneously screen an array of
patient
samples for a polypeptide of interest.
Preferably, a sample to be analysed using a protein chip is attached to a
reporter
molecule, such as, for example, a fluorescent molecule, a radioactive
molecule, an
enzyme, or an antibody that is detectable using methods well known in the art.
Accordingly, by contacting a protein chip with a labelled sample and
subsequent
washing to remove any unbound proteins the presence of a bound protein is
detected
using methods well known in the art, such as, for example using a DNA
microarray
io reader.
Alternatively, biomolecular interaction analysis-mass spectrometry (BIA-MS) is
used
to rapidly detect and characterise a protein present in complex biological
samples at the
low- to sub-femptamole (fmol) level (Nelson et al. Electrophoresis .11: 1155-
1163,
2000). One technique useful in the analysis of a protein chip is surface
enhanced laser
desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS)
technology
to characterise a protein bound to the protein chip. Alternatively, the
protein chip is
analysed using ESI as described in U.S. Patent Application 20020139751.
2o As will be apparent to the skilled artisan, protein chips are particularly
amenable to
multiplexing of detection reagents. Accordingly, several antibodies or ligands
each
able to specifically bind a different peptide or protein may be bound to
different regions
of said protein chip. Analysis of a biological sample using said chip then
permits the
detecting of multiple proteins of interest, or multiple B cell epitopes of the
S9 protein.
Multiplexing of diagnostic and prognostic markers is particularly contemplated
in the
present invention.
In a further embodiment, the samples are analysed using ICAT or ITRAC,
essentially
as described in US Patent Application No. 20020076739. This system relies upon
the
labelling of a protein sample from one source (i.e. a healthy individual) with
a reagent
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and the labelling of a protein sample from another source (i.e. a tuberculosis
patient)
with a second reagent that is chemically identical to the first reagent, but
differs in mass
due to isotope composition. It is preferable that the first and second
reagents also
comprise a biotin molecule. Equal concentrations of the two samples are then
mixed,
5 and peptides recovered by avidin affinity chromatography. Samples are then
analysed
using mass spectrometry. Any difference in peak heights between the heaNry and
light
peptide ions directly correlates with a difference in protein abundance in a
biological
sample. The identity of such proteins may then be determined using a method
well
known in the art, such as, for example MALDI-TOF, or ESI.
In a particularly preferred embodiment, a biological sample comprising anti-S9
antibodies, or alternatively S9 protein or an immunogenic fragment thereof, is
subjected to 2-dimensional gel electroplioresis. In accordance with this
embodiment, it
is preferable to remove certain particulate matter from the sample prior to
electrophoresis, such as, for example, by centrifugation, filtering, or a
combination of
centrifugation and filtering. Proteins in the biological sample are then
separated. For
example, the proteins may be separated according to their charge using
isoelectric
focussing and/or according to their molecular weight. Two-dimensional
separations
allow various isoforms of proteins to be identified, as proteins with similar
molecular
weight are also separated by their charge. Using mass spectrometry, it is
possible to
determine whether or not a protein of interest is present in a patient sample.
As will be apparent to those skilled in the art a diagnostic or prognostic
assay described
herein may be a multiplexed assay. As used herein the. term "multiplex", shall
be
understood not only to mean the detection of two or more diagnostic or
prognostic
markers in a single sample simultaneously, but also to encompass consecutive
detection
of two or more diagnostic or prognostic markers in a single sample,
simultaneous
detection of two or more diagnostic or prognostic markers in distinct but
matched
samples, and consecutive detection of two or more diagnostic or prognostic
markers in
3o distinct but matched samples. As used lierein the term "matched samples"
shall be
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81
understood to mean t<vo or more samples derived from the same initial
biological
sample, or two or more biological samples isolated at the same point in time.
Accordingly, a multiplexed assay may comprise an assay that detects several
anti-S9
antibodies and/or S9 epitopes in the same reaction and simultaneously, or
alternatively,
it may detect other one or more antigens/antibodies in addition to one or more
anti-S9
antibodies and/or S9 epitopes.
The present invention clearly contemplates multiplexed assays for detecting S9
io antibodies and epitopes in addition to detecting CD4+ T-helper cells via
one or more
receptors on the cell surface and/or one or more HIV-1 and/or HIV-2 antigens.
Such
assays are particularly useful for simultaneously obtaining information on co-
infection
with M. tubef culosis and HIV-1 and/or HIV-2, and/or for determining whether
or not a
subject with M. tubef-culosis is immune-compromised. Clearly, such multiplexed
assay
formats are useful for monitoring the health of an HIV+/TB+ individual.
As will be apparent to the skilled artisan, if such an assay is antibody or
ligand based,
both of these antibodies must function under the same conditions.
2o 4. Biological samples and reference samples
Preferably, the biological sample in which a S9 protein or anti-S9 antibody is
detected
is a sample selected from the group consisting of lung, lymphoid tissue
associated with
the lung, paranasal sinuses, bronchi, a bronchiole, alveolus, ciliated mucosal
epithelia
of the respiratory tract, mucosal epithelia of the respiratory tract,
broncheoalveolar
lavage fluid (BAL), alveolar lining fluid, sputum, mucus, saliva, blood,
serum, plasma,
urine, peritoneal fluid, pericardial fluid, pleural fluid, squamous epithelial
cells of the
respiratory tract, a mast cell, a goblet cell, a pneumocyte (type 1 or type
2), an intra
epithelial dendritic cell, a PBMC, a neutrophil, a monocyte, or any
immunoglobulin-
containing fraction of any one or more of said tissues, fluids or cells.
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In one embodiment a biological sample is obtained previously from a patient.
In one embodiment a biological sample is obtained from a subject by a method
selected
from the group consisting of surgery or other excision method, aspiration of a
body
fluid such as hypertonic saline or propylene glycol, broncheoalveolar lavage,
bronchoscopy, saliva collection with a glass tube, salivette (Sarstedt AG,
Sevelen,
Switzerland), Ora-sure (Epitope Technologies Pty Ltd, Melbourne, Victoria,
Australia),
omni-sal (Saliva Diagnostic Systems, Brooklyn, NY, USA) and blood collection
using
any method well known in the art, such as, for example using a syringe.
It is particularly preferred that a biological sample is sputum, isolated from
lung of a
patient using, for example the method described in Gershman, N.H. et al,
JAllergy Clin
Innnunol, 10(4): 322-328, 1999. Preferably, the sputum is expectorated i.e.,
coughed
naturally.
In another preferred embodiment a biological sample is plasma that has been
isolated
from blood collected from a patient using a method well known in the art.
In one embodiment, a biological sample is treated to lyse a cell in said
sample. Such
methods include the use of detergents, enzymes, repeatedly freezing and
thawing said
cells, sonication or vortexing said cells in the presence of glass beads,
amongst others.
In another embodiment, a biological sample is treated to denature a protein
present in
said sample. Methods of denaturing a protein include heating a sample,
treating a
sample with 2-mercaptoethanol, dithiotreitol (DTT), N-acetylcysteine,
detergent or
other compound such as, for example, guanidinium or urea. For example, the use
of
DTT is preferred for liquefying sputum.
In yet another embodiment, a biological sample is treated to concentrate a
protein is
said sample. Methods of concentrating proteins include precipitation, freeze
drying, use
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83
of funnel tube gels (TerBush and Novick, Journal of Biomolecular Techniques,
10(3);
1999), ultrafiltration or dialysis.
As will be apparent, the diagnostic and prognostic methods provided by the
present
invention require a degree of quantification to determine either, the amount
of a protein
that is diagnostic or prognostic of an infection or disease. Such
quantification can be
determined by the inclusion of appropriate reference samples in the assays
described
herein, wherein said reference samples are derived from healthy or normal
individuals.
io In one embodiment, the reference sample comprises for example cells, fluids
or tissues
from a healthy subject who has not been previously or recently infected and is
not
suffering from an infection or disease. Conveniently, such reference samples
are from
fluids or tissues that do not require surgical resection or intervention to
obtain them.
Accordingly, bodily fluids and derivatives thereof are preferred. Highly
preferred
reference samples comprise sputum, mucus, saliva, blood, serum, plasma, urine,
BAL
fluid, peritoneal fluid, pericardial fluid, pleural fluid, a PBMC, a
neutrophil, a
monocyte; or aily immunoglobulin-containing fraction of any one or more of
said
tissues, fluids or cells.
2o A reference sample and a test (or patient) sample are processed, analysed
or assayed
and data obtained for a reference sample and a test sample are compared. In
one
embodinlent, a reference sample and a test sample are processed, analysed or
assayed at
the same time. In another embodiment, a reference sample and a test sample are
processed, analysed or assayed at a different time.
In an alternate embodiment, a reference sample is not included in an assay.
Instead, a
reference sample may be derived from an established data set that has been
previously
generated. Accordingly, in one embodiment, a reference sample comprises data
from a
sample population study of healthy individuals, such as, for example,
statistically
significant data for the healthy range of the integer being tested. Data
derived from
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processing, analysing or assaying a test sample is then compared to data
obtained for
the sample population.
Data obtained from a sufficiently large number of reference samples so as to
be
representative of a population allows the generation of a data set for
determining the
average level of a particular parameter. Accordingly, the amount of a protein
that is
diagnostic or prognostic of an infection or disease can be determined for any
population
of individuals, and for any sample derived from said individual, for
subsequent
comparison to levels of the expression product determined for a sample being
assayed.
io Where such normalized data sets are relied upon, internal controls are
preferably
included in each assay conducted to control for variation.
Diagnostic assay kits
The present invention provides a kit for detecting M. tuberculosis infection
in a
biological sample. In one embodiment, the kit comprises:
(i) one or more isolated antibodies that bind to a S9 protein or an
immunogenic
fragment or epitope thereof; and
(ii) means for detecting the formation of an antigen-antibody complex.
In an alternative embodiment, the kit comprises:
(i) an isolated or recombinant S9 protein or an immunogenic fragment or
epitope
thereof; and
(ii) means for detecting the formation of an antigen-antibody complex.
The antibodies, immunogenic S9 peptide, and detection means of the subject kit
are
preferably selected from the antibodies and immunogenic S9 peptides described
herein
above and those embodiments shall be taken to be incorporated by reference
herein
from the description. The selection of compatible kit components for any assay
format
will be readily apparent to the skilled artisan from the description.
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In a particularly preferred embodiment, the subject kit comprises:
(i) an antibody that binds to an isolated or recombinant or synthetic peptide
comprising an amino acid sequence selected from the group consisting of SEQ
IDNOs: 1, 2, 3, 4, 5, 6 and 7; and
5 (ii) anti-human Ig.
Preferably, the kit further comprises an amount of one or more peptides each
comprising an amino acid sequence selected from the group consisting of SEQ ID
NOs:
1-7, or a fusion between any two or more of said peptides.
Optionally, the kit further comprises means for the detection of the binding
of an
antibody, fragment thereof or a ligand to a S9 protein. Such means include a
reporter
molecule such as, for example, an enzyme (such as horseradish peroxidase or
alkaline
phosphatase), a substrate, a cofactor, an inhibitor, a dye, a radionucleotide,
a
luminescent group, a fluorescent group, biotin or a colloidal particle, such
as colloidal
gold or selenium. Preferably such a reporter molecule is directly linked to
the antibody
or ligand.
In yet another embodiment, a kit may additionally comprise a reference sample.
Such a
2o reference sample may for example, be a protein sample derived from a
biological
sample isolated from one or more tuberculosis subjects. Alternatively, a
reference
sample may comprise a biological sample isolated from one or more normal
healthy
individuals. Such a reference sample is optionally included in a kit for a
diagnostic or
prognostic assay.
In another embodiment, a reference sample comprises a peptide that is detected
by an
antibody or a ligand. Preferably, the peptide is of known concentration. Such
a peptide
is of particular use as a standard. Accordingly various known concentrations
of such a
peptide may be detected using a prognostic or diagnostic assay described
herein.
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In yet another embodiment, a kit optionally comprises means for sample
preparations,
such as, for example, a means for cell lysis. Preferably such means are means
of
solubilizing sputum, such as, for example, a detergent (e.g., tributyl
phosphine,
C7BZO, dextran sulfate, DTT, N-acetylcysteine, or polyoxyethylenesorbitan
monolaurate).
In yet another embodiment, a kit comprises means for protein isolation (Scopes
(In:
Protein Purification: Principles and Practice, Third Edition, Springer Verlag,
1994).
Pt=ophylactic and thef=apeutic method
The S9 protein or immunogenic fragment or epitope thereof can induce the
specific
production of a high titer antibody when administered to an animal subject.
Accordingly, the invention provides a method of eliciting the production of
antibody
against h~l. tuberculosis comprising administering an isolated S9 protein or
an
immunogenic fragment or epitope thereof to said subject for a time and under
conditions sufficient to elicit the production of antibodies, such as, for
example,
neutralizing antibodies that bind to M. tuber=culosis.
It is within the scope of the present invention to further administer one or
more second
antigens e.g., M. tuberculosis Bsx or GS or immunogenic fragment thereof for a
time
and under conditions sufficient to elicit the production of antibodies, such
as, for
example, neutralizing antibodies that bind to M. tuberculosis. Such
administration may
be at the same time as administering S9 protein or fragment (i.e., co-
administration) or
alternatively, before or after the S9 protein or fragment is administered to a
subject.
Preferably, the neutralizing antibodies according got any of the preceding
embodiments
are high titer neutralizing antibodies.
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The effective amount of S9 protein or other protein or epitope thereof to
produce
antibodies varies upon the nature of the immunogenic B cell epitope, the route
of
administration, the animal used for immunization, and the nature of the
antibody
sought. All such variables are empirically determined by art-recognized means.
In a preferred embodiment, the invention provides a method of inducing
immunity
against M. tuberculosis in a subject comprising administering to said subject
an isolated
or recombinant S9 protein or immunogenic fragment or epitope thereof for a
time and
under conditions sufficient to elicit a humoral immune response against said
an isolated
1o or recombinant S9 protein or immunogenic fragment or epitope.
It is also within the scope of the present invention. to further administer
one or more
second antigens e.g., M. tuberculosis Bsx or GS or immunogenic fragment
thereof for a
time and under conditions sufficient to elicit a humoral immune response
against that
antigen. Such administration may be at the same time as administering S9
protein or
fragment (i.e., co-administration) or alternatively, before or after the S9
protein or
fragment is administered to a subject.
The immunizing antigen may be administered in the form of any convenient
formulation as described herein.
By "humoral immune response" means that a secondary immune response is
generated
against the immunizing antigen sufficient to prevent infection by M.
tuberculosis.
Preferably, the humoral immunity generated includes eliciting in the subject a
sustained
level of antibodies that bind to a B cell epitope in the immunizing antigen.
By a
"sustained level of antibodies" is meant a sufficient level of circulating
antibodies that
bind to the B cell epitope to prevent infection by M. tuberculosis.
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Preferably, antibodies levels are sustained for at least about six months or 9
months or
12 months or 2 years.
In an alternative embodiment, the present invention provides a niethod of
enhancing the
immune system of a subject comprising administering an immunologically active
S9
protein or an epitope thereof or a vaccine composition comprising said S9
protein or
epitope for a time and under conditions sufficient to confer or enhance
resistance
against M. tuberculosis in said subject.
io It is also within the scope of the present invention to further administer
one or more
second antigens e.g.,1lL tuberculosis Bsx or GS or immunogenic fragment
thereof for a
time and under conditions sufficient to confer or enhance resistance against
M.
tzsbereulosis in said subject. Such administration may be at the same time as
administering S9 protein or fragment (i.e., co-administration) or
alternatively, before or
after the S9 protein or fragment is administered to a subject.
By "confer or enhance resistance" is meant that a M. tuberculosis-specific
immune
response occurs in said subject, said response being selected from the group
consisting
of:
(i) an antibody against a S9 protein ofM. tuberculosis or an epitope of said
protein
is produced in said subject;
(ii) neutralizing antibodies that bind to M. tuberculosis are produced in said
subject;
(iii) a cytotoxic T lymphocyte (CTL) and/or a CTL precursor that is specific
for a S9
protein of M. tuberculosis is activated in the subject; and
(iv) the subject has enhanced immunity to a subsequent M. tuberculosis
infection or
reactivation of a latent M. tuberculosis infection.
The invention will be understood to encompass a method of providing or
enhancing
immunity against M. t.uberculosis in an uninfected human subject comprising
3o administering to said subject an immunologically active S9 protein or an
epitope
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thereof or a vaccine composition comprising said S9 protein or epitope for a
time and
under conditions sufficient to provide immunological memory against a future
infection
by Rl. tuberculosis.
It is also within the scope of the present invention to further administer one
or more
second antigens e.g., M. tuberculosis Bsx or GS or immunogenic fragment
thereof for a
time and under conditions sufficient to provide iinmunological memory against
a future
infection by M. tuberculosis. Such administration may be at the same time as
administering S9 protein or fragment (i.e., co-administration) or
alternatively, before or
after the S9 protein or fragment is administered to a subject.
The present invention provides a method of treatment of tuberculosis in a
subject
comprising performing a diagnostic method or prognostic method as described
herein.
In one embodiment, the present invention provides a method of prophylaxis
comprising:
(i) detecting the presence of M. tuberculosis infection in a biological sample
from a
subject; and
(ii) administering a therapeutically effective ainount of a pharmaceutical
composition described herein to reduce the number of pathogenic bacilli in the
lung, blood or lymph system of the subject.
As will be apparent fro the disclosure herein, suitable compositions according
to this
embodiment comprise S9 protein or immunogenic fragment thereof optionally with
on
or more other immunogen M. tubet-culosis proteins or peptide fragments, in
combination with a pharmaceutically acceptable carrier or excipient. It is
clearly
within the scope of the present invention for such compositions to include S9
protein or
fragment thereof according to any embodiment described herein e.g., any one of
SEQ
ID NOs: 1-7, and one or more second antigens e.g., M. tuberculosis Bsx or GS
or
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immunogenic fragments thereof e.g., as set forth in any one of SEQ ID NOs: 8-
20 or a
subset thereof.
Preferably, the composition is administered to a subject harboring a latent or
active M.
5 tuberculosis infection.
Without being bound by any theory or mode of action, the therapeutic method
enhances
the ability of a T cell to recognize and lyse a cell harboring M.
tuber=culosis, or that the
ability of a T cell to recognize a T cell epitope of an antigen of M.
tuberculosis is
1o enhanced, either transiently or in a sustained manner. Similarly,
reactivation of a T cell
population may occur following activation of a latent M. tubercztilosis
infection, or
following re-infection with M tuberculosis, or following immunization of a
previously-
infected subject with a S9 protein or epitope or vaccine composition of the
invention.
Standard methods can be used to determine whether or not CTL activation has
occurred
15 in the subject, such as, for example, using cytotoxicity assays, ELISPOT,
or
determining IFN-y production in PBMC of the subject.
Preferably, the peptide or derivative or variant or vaccine composition is
administered
for a time and under conditions sufficient to elicit or enhance the expansion
of CD8+ T
20 cells. Still more preferably, the peptide or derivative or variant or
vaccine composition
is administered for a time and under conditions sufficient for M. tuberculosis
-specific
cell mediated immunity (CMI) to be enhanced in the subject.
By "Af. tuber=culosis -specific CMI" is meant that the activated and clonally
expanded
25 CTLs are MHC-restricted and specific for a CTL epitope of the invention.
CTLs are
classified based on antigen specificity and MHC restriction, (i.e., non-
specific CTLs
and antigen-specific, MHC-restricted CTLs). Non-specific CTLs are composed of
various cell types, including NK cells and antibody-dependent cytotoxicity,
and can
function very early in the immune response to decrease pathogen load, while
antigen-
30 specific responses are still being established. In contrast, MHC-restricted
CTLs achieve
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optimal activity later than non-specific CTL, generally before antibody
production.
Antigen-specific CTLs inhibit or reduce the spread of M. tuberculosis and
preferably
terminate infection.
CTL activation, clonal expansion, or CMI can be induced systemically or
compartmentally localized. In the case of compartmentally localized effects,
it is
preferred to utilize a vaccine composition suitably formulated for
administration to that
-compartment. On the other hand, there are no such stringent requirements for
inducing
CTL activation, expansion or CMI systemically in the subject.
The effective amount of S9 protein or epitope thereof, optionally in
combination with
one or more other proteins or epitopes e.g., derived from Bsx or GS proteins
of M.
tubef=culosis, to be administered solus or in a vaccine composition to elicit
CTL
activation, clonal expansion or CMI, varies upon the nature of the immunogenic
epitope, the route of administration, the weight, age, sex, or general health
of the
subject immunized, and the nature of the CTL response sought. All such
variables are
empirically determined by art-recognized means.
The S9 protein or epitope thereof, optionally in combination with one or more
other
proteins or epitopes e.g., derived from Bsx or GS proteins of M. tuberculosis,
and
optionally formulated with any suitable or desired carrier, adjuvant, BRIVI,
or
pharmaceutically acceptable excipient, is conveniently administered in the
form of an
injectable composition. Injection may be intranasal, intramuscular, sub-
cutaneous,
intravenous, intradermal, intraperitoneal, or by other known route. For
intravenous
injection, it is desirable to include one or more fluid and nutrient
replenishers.
The optimum dose to be administered and the preferred route for administration
are
established using animal models, such as, for example, by injecting a mouse,
rat, rabbit,
guinea pig, dog, horse, cow, goat or pig, with a formulation comprising the
peptide, and
then monitoring the CTL immune response to the epitope using any conventional
assay.
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Adoptive transfer techniques may also be used to confer or enhance resistance
against
M. tuberculosis infection or to prevent or reduce the severity of a
reactivated latent
infection. Accordingly, in a related embodiment, there is provided a method of
enhancing or conferring immunity against M. tubereulosis in an uninfected
human
subject comprising contacting ex vivo a T cell obtained from a human subject
with an
immunologically active S9 protein or an epitope thereof or a vaccine
composition
comprising said protein or epitope for a time and under conditions sufficient
to confer
M. tuberculosis activity on said T cells.
In a preferred embodiment, the invention provides a method of enhancing the M.
tuberculosis -specific cell mediated immunity of a human subject, said method
comprising:
(i) contacting ex vivo a T cell obtained from a human subject with an
immunologically active S9 protein or a CTL epitope thereof or a vaccine
composition comprising said protein or epitope for a time and under conditions
sufficient to confer A7 tuberculosis activity on said T cells; and
(ii) introducing the activated T cells autologously to the subject or
allogeneically to
another human subject.
As with other embodiments described herein, the present invention encompasses
the
administration of additional immunogenic proteins or epitopes e.g., derived
from Bsx
or GS proteins of M. tuberculosis.
The T cell may be a CTL or CTL precursor cell.
The human subject from whom the T cell is obtained may be the same subject or
a
different subject to the subject being treated. The subject being treated can
be any
human subject carrying a latent or active M. tuberculosis infection or at risk
of M.
tuberculosis infection or reactivation of 1l4. tuberculosis infection or a
person who is
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otherwise in need of obtaining vaccination against M. tuberculosis or desirous
of
obtaining vaccination against M tuberculosis.
Such adoptive transfer is preferably carried out and M. tuberculosis
reactivity assayed
essentially as described by Einsele et al., Blood 99, 3916-3922, 2002, which
procedures
are incorporated herein by reference.
By "M. tuber=culosis activity" is meant that the T cell is rendered capable of
being
activated as defined herein above (i.e. the T cell will recognize and lyse a
cell harboring
a.o M. tuberculosis or able to recognize a T cell epitope of an antigen of M.
tuberculosis,
either transiently or in a sustained manner). Accordingly, it is particularly
preferred for
the T cell to be a CTL precursor which by the process of the invention is
rendered able
to recognize and lyse a cell harboring M. tuberculosis or able to recognize a
T cell
epitope of an antigen of M. tuberculosis, either transiently or in a sustained
manner.
For such an ex vivo application, the T cell is preferably contained in a
biological sample
obtained from a human subject, such as, for example, a biopsy specimen
comprising a
primary or central lymphoid organ (eg. bone marrow or thyrnus) or a secondary
or
peripheral lymphoid organ (eg, blood, PBMC or a buffy coat fraction derived
there
from).
Preferably, the T cell or specimen comprising the T cell was obtained
previously from a
human subject, such as, for example, by a consulting physician who has
referred the
specimen to a pathology laboratory for analysis.
Preferably, the subject method further comprises obtaining a saniple
comprising the T
cell of the subject, and more preferably, obtaining said sample from said
subject.
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Formulatiofts
The present invention clearly contemplates the use of the S9 protein or an
immunogenic fragment or epitope thereof ih the preparation of a therapeutic or
prophylactic subunit vaccine against Al. tuberculosis infection in a human or
other
animal subject.
Accordingly, the invention provides a pharmaceutical composition or vaccine
comprising a S9 protein or an immunogenic fragment or epitope thereof in
combination
with a pharmaceutically acceptable diluent.
In a preferred enibodiment, the composition according to this embodiment
comprises
S9 protein or immunogenic fragment thereof optionally with on or more other
immunogenic M. tuberculosis proteins or peptide fragments, in combination with
a
pharmaceutically acceptable carrier or excipient. It is clearly within the
scope of the
present invention for such compositions to include S9 protein or fragment
thereof
according to any embodiment described herein e.g., any one of SEQ ID NOs: 1-7,
and
one or more second antigens e.g., M. tuberculosis Bsx or GS or immunogenic
fragments thereof e.g., as set forth in any one of SEQ ID NOs: 8-20 or a
subset thereof.
2o The S9 protein, and optional other protein, or immunogenic fragment or
epitope thereof
is conveniently formulated in a pharmaceutically acceptable excipient or
diluent, such
as, for example, an aqueous solvent, non-aqueous solvent, non-toxic excipient,
such as
a salt, preservative, buffer and the like. Examples of non-aqueous solvents
are
propylene glycol, polyethylene glycol, vegetable oil and injectable organic
esters such
as ethyloleate. Aqueous solvents include water, alcoholic/aqueous solutions,
saline
solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose,
etc.
Preservatives include antimicrobial, anti-oxidants, chelating agents and inert
gases. The
pH and exact concentration of the various components the pharmaceutical
composition
are adjusted according to routine skills in the art.
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In certain situations, it may also be desirable to formulate the S9 protein
and optional
other protein or an immunogenic fragnlent or epitope thereof, with an adjuvant
to
enhance the immune response to the B cell epitope. Again, this is strictly not
essential.
Such adjuvants include all acceptable immunostimulatory compounds such as, for
5 example, a cytokine, toxin, or synthetic composition. Exemplary adjuvants
include IL-
1, IL-2, BCG, aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine
(thur-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to
as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-
dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethyla.mine (CGP) 1983A,
referred
1o to as MTP-PE), lipid A, MPL and RIBI, which contains three components
extracted
from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall
skeleton
(MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.
Particularly preferred adjuvants for use in a vaccine against M. tuberculosis
are
15 described for example by Elhay and Andersen Inanaunol. Cell Biol. 75, 595-
603, 1997;
or Lindblad et al., Infect. Irnmun. 65, 1997.
It may be desirable to co-administer biologic response modifiers (BRM) with
the S9
protein or immunogenic fragment or epitope thereof, to down regulate
suppressor T cell
2o activity. Exemplary BRM's include, but are not limited to, Cimetidine (CIM;
1200
mg/d) (Smith/Kline, PA, USA); Indomethacin (IND; 150 mg/d) (Lederle, NJ, USA);
or
low-dose Cyclophosphamide (CYP; 75, 150 or 300 mg/m`) (Johnson/Mead, NJ, USA).
Preferred vehicles for administration of the S9 protein and optional other
protein, or
25 immunogenic fragment ' or epitope thereof, include liposomes. Liposomes are
microscopic vesicles that consist of one or more lipid bilayers surrounding
aqueous
compartments. (Bakker-Woudenberg et al., Eur. J. Clin. Microbiol. Infect. Dis.
12(Suppl. 1), S61 (1993); and Kim, Drugs 46, 618 (1993)). Liposomes are
similar in
composition to cellular membranes and as a result, liposomes generally are
3o administered safely and are biodegradable.
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Techniques for preparation of liposomes and the formulation (e.g.,
encapsulation) of
various molecules, including peptides and oligonucleotides, with liposomes are
well
known to the skilled artisan.
Depending on the method of preparation, liposomes may be unilamellar or
multilamellar, and can vary in size with diameters ranging from 0.02 gm to
greater than
m. A variety of agents are encapsulated in liposomes. Hydrophobic agents
partition
in the bilayers and hydrophilic agents partition within the inner aqueous
space(s)
io (Machy et al., LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John
Libbey 1987), and Ostro et al., Anterican J. Hosp. Pharrn. 46, 1576 (1989)).
Liposomes can also adsorb to virtually any type of cell and then release the
encapsulated agent. Alternatively, the liposome fuses with the target cell,
whereby the
contents of the liposome empty into the target cell. Alternatively, an
absorbed liposome
may be endocytosed by cells that are phagocytic. Endocytosis is followed by
intralysosomal degradation of liposomal lipids and release of the encapsulated
agents
(Scherphof et al., Ann. N.Y. Acad. Sci. 446, 368 (1985)). In the present
context, the S9
protein or immunogenic fragment or epitope thereof may be localized on the
surface of
the liposome, to facilitate antigen presentation without disruption of the
liposome or
endocytosis. Irrespective of the mechanism or delivery, however, the result is
the
intracellular disposition of the associated S9 protein or immunogenic fragment
or
epitope thereof.
Liposomal vectors may be anionic or cationic. Anionic liposomal vectors
include pH
sensitive liposomes which disrupt or fuse with the endosomal membrane
following
endocytosis and endosome acidification. Cationic liposomes are preferred for
mediating mammalian cell transfection in vitro, or general delivery of nucleic
acids, but
are used for delivery of other therapeutics, such as peptides or lipopeptides.
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Cationic liposome preparations are made by conventional methodologies (Feigner
et al,
Proc. Nat'l Acad. Sci USA 84, 7413 (1987); Schreier, Liposoine Res. 2, 145
(1992)).
Commercial preparations, such as Lipofectin (Life Technologies, Inc.,
Gaithersburg,
Md. USA), are readily available. The amount of liposomes to be administered
are
optimized based on a dose response curve. Feigner et al., supra.
Other suitable liposomes that are used in the methods of the invention include
multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar
vesicles
(UV), small unilamellar vesicles (SUV), medium-sized unilamellar vesicles
(MUV),
1o large unilamellar vesicles (LL1V), giant unilamellar vesicles (GUV),
multivesicular
vesicles (MVV), single or oligolamellar vesicles made by reverse-phase
evaporation
method (REV), multilamellar vesicles made by the reverse-phase evaporation
method
(MLV-REV), stable plurilamellar vesicles (SPLV), frozen and thawed MLV
(FATMLV), vesicles prepared by extrusion methods (VET), vesicles prepared by
French press (FPV), vesicles prepared by fusion (FUV), dehydration-rehydration
vesicles (DRV), and bubblesonies (BSV). The skilled artisan will recognize
that the
techniques for preparing these liposomes are well known in the art. (See
COLLOIDAL
DRUG DELIVERY SYSTEMS, vol. 66, J. Kreuter, ed., Marcel Dekl:er, Inc. 1994).
Other forms of delivery particle, for example, microspheres and the like, also
are
contemplated for delivery of the S9 protein and optional other protein, or
immunogenic
fragment or epitope thereof.
Guidance in preparing suitable formulations and pharmaceutically effective
vehicles,
are found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES,
chapters 83-92, pages 1519-1714. (Mack Publishing Company 1990) (Remington's),
which are hereby incorporated by reference.
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Alternatively, the peptide or derivative or variant is formulated as a
cellular vaccine via
the administration of an autologous or allogeneic antigen presenting cell
(APC) or a
dendritic cell that has been treated in vitro so as to present the peptide on
its surface.
Nucleic acid-based vaccines that comprise nucleic acid, such as, for example,
DNA or
RNA, encoding the immunologically active S9 protein and optional other
protein, or
epitope(s) thereof, and cloned into a suitable vector (eg. vaccinia, canary
pox,
adenovirus, or other eukaryotic virus vector) are also contemplated.
Preferably, DNA
encoding a S9 protein and optional other protein, is formulated into a DNA
vaccine,
io such as, for exaniple, in combination with the existing Calmette-Guerin
(BCG) or an
inimune adjuvant such as vaccinia virus, Freund's adjuvant or another immune
stimulant.
The present invention is further described with reference to the following non-
limiting
examples.
EXAMPLE I
Preparation of serum or plasma
Patient serum or plasma is applied to a column of protein G-SepharoseTM
(Amersham
Biosciences), previously equilibrated with 20mM phosphate buffer pH 7.0 and
incubated on ice with occasional inversion. The mixture is centrifuged at
6000g for 10
minutes at 4 C and the supernatant decanted. The SepharoseTM pellet is washed
with
20mM phosphate buffer. The IgG bound to the SepharoseTM is eluted by addition
of
50mM glycine pH 2.7 for 20 minutes. After centrifugation as above, the
supernatant is
discarded and the glycine step repeated. The supernatant is then precipitated
with cold
acetone at -20 C for 48h then centrifuged at 5000g for 20mins at 4 C. The
precipitate
is resolubilised in 1-2mis of sample buffer containing 5M urea, 2M thiourea,
2%
CHAPS, 2% SB3-10 and 40mM Tris, then simultaneously reduced with 5mM tributyl
phosphine and alkylated with 10mM acrylamide for 1 h.
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EXAMPLE 2
Analytical methods
The protein content of the samples is estimated using a Bradford assay.
Samples were
diluted with sample buffer as above replacing 40mM Tris with 5mM Tris.
Prior to rehydration of IPG strips, samples are centrifuged at 21000 x g for
10 minutes.
The supernatant is collected and 10 l of 1% Orange G(Sigma) per ml added as an
io indicator dye.
Two-dirnensional gel electrophoresis
First Dirnension
Dry 11 cm IPG strips (Amersham-Biosciences) are rehydrated for 16-24 hours
with
180 1 of protein sample. Rehydrated strips are focussed on a Protean IEF Cell
(Bio-
Rad, Hercules, CA) or Proteome System's IsoElectrIQ electrophoresis equipment
for
approx 140 kVhr at a niaximum of 10 kV. Focussed strips are then equilibrated
in
urea/SDS/Tris-HC1/bromophenol blue buffer.
Second Dinzensi.on
Equilibrated strips are inserted into loading wells of 6-15% (w/v) Tris-
acetate SDS-
PAGE pre-cast 10cm x 15cm GelChips (Proteome Systems, Sydney Australia).
Electrophoresis is performed at 50mA per gel for 1.5 hours, or until the
tracking dye
reached the bottom of the gel. Proteins are stained using SyproRuby (Molecular
Probes). Gel images are scanned after destaining using an Alphalmager System
(Alpha
Innotech Corp.). Gels are then stained with Coomassie G-250 to assist
visualisation of
protein spots in subsequent analyses. A representative gel showing the
position` of an
identified protein is shown in Figure 1.
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R1ass Spectrorraetry:
Prior to mass spectrometry protein samples are prepared by in-gel tryptic
digestion.
Protein gel pieces are excised, destained, digested and desalted using an
XciseT", an
excision/liquid handling robot (Proteome Systems, Sydney, Australia and
Shimadzu-
Biotech, Kyoto, Japan) in association' with the Montage In-Gel Digestion Kit
(developed by Proteome Systems and distributed by Millipore, Billerica, Ma,
01821,
USA). Prior to spot cutting, the 2-D gel is incubated in water to maintain a
constant
size and prevent drying. Subsequently, the 2-D gel is placed on the lciseTm, a
digital
io image was captured and the spots to be cut are selected. After automated
spot excision,
gel pieces are subjected to automated liquid handling and in-gel digestion.
Briefly, each
spot is destained with 100 l of 50% (v/v) acetonitrile in 50 mM ammonium
bicarbonate. The gel pieces are dried by adding 100% acetonitrile, the
acetonitrile is
removed after 5 seconds and the gels dried completely by evaporating the
residual
acetonitrile at 37 C. Proteolytic digestion is performed by rehydrating the
dried gel
pieces with 30 l of 20 mM ammonium bicarbonate (pH 7.8) containing 5 g/ml
modified porcine trypsin and incubated at 30 C overnight.
Ten gl of the tryptic peptide mixture is removed to a clean microtiter plate
in the event
that additional analysis by Liquid Chromatography (LC) - Electrospray
lonisation (ESI)
MS was required.
Automated desalting and concentration of tryptic peptides prior to MALDI-TOF
MS is
performed using C18 ZipTip (Millipore, Bedford, MA). Adsorbed peptides are
eluted
from the tips onto a 384-position MALDI-TOF sample target plate (Kratos,
Manchester, UK or Bruker Daltronics, Germany) using 2 1 of 2 mg/ml a-cyano-4-
hydroxycinnamic acid in 90% (v/v) acetonitrile and 0.085% (v/v) TFA.
Digests are analyzed using an Axima-CFR MALDI-TOF mass spectrometer (Kratos,
Manchester, UK) in positive ion reflectron mode. A nitrogen laser with a
wavelength of
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101
337 nm is used to irradiate the sample. The spectra are acquired in automatic
mode in
the mass range 600 Da to 4000 Da applying a 64-point raster to each sample
spot. Only
spectra passing certain criteria are saved. All spectra undergo an internal
two point
calibration using an autodigested trypsin peak mass, m/z 842.51 Da and spiked
adrenocorticotropic hormone (ACTH) peptide, m/z 2465.117 Da. Software designed
by
Proteome Systems, as contained in the web-based proteomic data management
system
BioinformatIQTM (Proteome Systems), is used to extract isotopic peaks from MS
spectra.
1o Protein identification is performed by matching the monoisotopic masses of
the tryptic
peptides (i.e. the peptide mass fingerprint) with the theoretical masses from
protein
databases using IonIQTM or MASCOTTM database search software (Proteome System
Limited, North Ryde, Sydney, Australia). Querying was done against the non-
redundant SwissProt (Release 40) and TrEMBL (Release 20) databases (June 2002
version), and protein identities are ranked through a modification of the
MOWSE
scoring system. Propionamide-cysteine (cys-PAM) or carboxyamidomethyl-cysteine
(cys-CAM) and oxidized methionine modifications are taken into account and a
mass
tolerance of 100 ppm was allowed.
Miscleavage sites are only considered after an initial search without
miscleavages had
been performed. The following criteria are used to evaluate the search
results: the
MOWSE score, the number and intensity of peptides matching the candidate
protein,
the coverage of the candidate protein's sequence by the matching peptides and
the gel
location.
In addition, or alternatively, proteins are analysed using LC-ESI-MS. Tryptic
digest
solutions of proteins (10 l) are analysed by nanoflow LC/MS using an LCQ Deca
Ion
Trap mass spectrometer (ThermoFinnigan, San Jose, CA) equipped with a Surveyor
LC
system composed of an autosampler and pump. Peptides are separated using a
3o PepFinder kit (Thermo-Finnigan) coupled to a C18 PicoFrit column (New
Objective).
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Gradient elution from water containing 0.1% (v/v) formic acid (mobile phase A)
to
90% (v/v) acetonitrile containing 0.1% (v/v) forinic acid (niobile phase B) is
performed
over a 30-60-minute period. The mass spectrometer is set up to acquire three
scan
events - one full scan (range from 400 to 2000 amu) followed by two data
dependant
MS/MS scans.
Bioinforrnatic Analysis:
Following automated collection of mass spectra peaks, data are processed as
follows.
All spectra were firstly checked for correct calibration of peptide masses.
Spectra are
io then processed to remove background noise including masses corresponding to
trypsin
peaks and matrix. The data are then searched against publicly-available
SwissProt and
TrEMBL databases using Proteome Systems search engine IonIQTM v69 and/or
MASCOTTM. PSD data is searched against the same databases using the in-house
search engine FragmentastIQTM. LC MS-MS data is also searched against the
databases
using the SEQUEST search engine software.
EXAMPLE 3
Identification of S9 protein as a diagnostic marker of M. tuber-culosis
infection
2o A protein having a molecular weight of about 30.2 kDa was recognized in the
immunoglobulin fraction of sera from TB+ samples. The sequences of four
peptides
from MALDI-TOF data (SEQ ID Nos: 2-5 inclusive) matched a protein having
SwissProt Accession No. P66638 (SEQ ID NO: 1). The percent coverage of P66638
by these 4 peptides (SEQ ID NOs: 2-5) was about 14-15%, suggesting that the
peptide
fragments were derived from this same protein marker. This conclusion was
supported
by there being only six theoretical tryptic peptides with zero miscleavages,
and fourteen
theoretical tryptic peptides having one miscleavage.
The identified protein having the amino acid sequence set forth in SEQ ID NO:
1 was
3o designated as "S9". Interestingly, the estimated molecular weight of the S9
protein is
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only about 16.4 kDa, and the estimated isoelectric point of S9 is about 10.7.
Since the
obsenied molecular weight of the S9 protein was about 14 kDa higher than the
estimated value, the protein is most likely post-translationally modified
e.g., by
glycosylation, or co-migrates with another molecular species such as nucleic
acid.
EXAMPLE 4
Antibodies against synthetic S9 peptides
Svnthesis of S9 Peptides
A synthetic peptide comprising the sequence H-MTETT PAPQT PAAPA GPAQS FC-
1o NH2 from 30S ribosomal protein S9 was synthesized to 78% purity as
determined by
liquid chromatography by Mimotopes using solid phase peptide synthesis
technology.
This peptide was coupled to keyhole limpet Hemocyanin (KHL) via a
Maleimidocaproyl-N-Hydroxysuccinimide linker.
To facilitate detection of antibodies raised against this epitope the peptide
was also
synthesized with a GSGL spacer and attached to biotin (PAPQT PAAPA GPAQS
FGSGL-Biotin) to 93% purity by liquid chromatography.
Antibody production
2o A rabbit was injected with 600 g per dose of the synthetic peptide
comprising the
amino acid sequence H-MTETT PAPQT PAAPA GPAQS FC-NH2 linked to KHL
according to the following injection protocol:
Prebleed Week 0
Primary Inoculation Week 0
lst Booster Week 3
2" Booster Week 6
Test Bleed Week 7.5
rd Booster Week 9
Bleedout Week 10.5
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After 10.5 weeks the rabbit was bled out. All blood was collected in sterile
containers
and incubated at 37 C to accelerate clotting. The containers were centrifuged
and the
serum removed and re-centrifuged to remove the remaining red cells.
Antibody titratioit
Streptavidin (Sigma Aldrich) was diluted to 5 g/ml in ddH2O and incubated in
a Nunc
plate overnight at 4 C. The solution was then flicked out and 250 L of
blocking buffer
(1 %(w/v) casein, 0.1 %(v/v) Tween 20, 0.1 %(w/v) sodium azide in PBS) added
to
io each well and incubated at room temperature for 1 hour. The blocking buffer
was
flicked out and biotinylated peptide (corresponding to the immunogen injected
into the
rabbit) was added in blocking buffer at 3 g/ml (50 1/well) and incubated for
one hour
at room temperature on a shaker. The plate was washed in an EIx405 Auto Plate
Washer (Bio-Tek Instruments Inc., Winooski, VT), with 0.5 x PBS / 0.05% (v/v)
Tween 20 solution and excess solution tapped out on a paper towel. The rabbit
sera
was diluted in blocking buffer 2 fold from 1: 500 to 1: 1,024,000 and
incubated from 1
hour at 50 ul/well at room temperature on a shaker. The plate was washed with
the
plate washer using 0.5 x PBS / 0.05% (v/v) Tween 20 solution and excess
solution
tapped out on a paper towel. Binding of the rabbit antibody to its
corresponding
2o epitope was detected using HRP-conjugated sheep anti-rabbit (Chemicon)
diluted 1 in
10,000 in conjugate diluent buffer. Fifty millilitres were added to each well
and
incubated for one hour at room temperature on a shaker. The plate was washed
with
the plate washer using 0.5 x PBS and excess solution tapped out on a paper
towel.,
Fifty millilitres of TMB (3,3',5',5-Tetramethylbenzidine; Sigma) was added to
each
well and the plate incubated in the dark for 30 minutes. Development was
stopped with
50 L/well of 0.5M sulphuric acid. The optical density of each well was read
with a
microtiter plate reader (PowerWavex 340 plate reader, Bio-Tek Instruments
Inc.,
Winooski, VT) using a wavelength of 450nm and an extinction at 620nm. The
titration
results are shown in Figure 2.
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Peptide titratiofi
To titrate the peptide, a protocol essentially as described supra was used.
However, the
biotinylated peptide was titrated from 20,480 pg/ml to 10 pg/ml and the rabbit
sera was
first purified down an affinity peptide column and added to the ELISA at 10
g/ml, 20
g/m1 and 40 g/ml. Results of this analysis are shown in Figure 3.
EXAMPLE 5
Antigen-based diagnosis of tuberculosis or infection by M. tuberculosis:
Detection of S9 in sputum from TB subjects
The R9 antibody described in Example 4 was used to detect S9 protein in
samples from
TB subjects and 20 subjects suffering from a non-TB subject. Briefly, sputum
(12 1) from TB or non-TB patients was loaded onto 4-12% ID gradient SDS
15 polyacrylamide gels and separated by electrophoresis. Proteins were then
electrotransferred onto PVDF membrane. Membranes were then blocked in solution
containing 1% casein in IX PBS, 0.1% Tween-20 (PBST) at room temperature (RT)
for 2 hours. Membranes were then incubated with 15 g/ml purified rabbit anti-
S9
polyclonal antibody solution (i.e., R9) at RT for 2 hr, following by 3 x 10min
washes
20 with PBST. Membranes were then incubated with 1:10,000 dilution of sheep
anti-rabbit
IgG-HRP conjugated antibody solution at RT for 1 hr, followed by 5 x 10 min
washes
with times PBST. Membranes were finally treated with `Femto' chemiluminescence
reagents (Pierce) for 5 min before exposure to x-ray films.
Ribosomal protein S9 was detected in 20/20 South African TB patients'
(Sensitivity =
100%) and 5/20 Australian non-TB respiratory disease patients (Specificity =
75%)
using the rabbit R9 polyclonal antibody (see Figures 4a and 4b).
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EXAMPLE 6
Antigen-based detection of M. tuberculosis:
Antibody R9 detects M. tuberculosis ribosomal protein S9 in cultured M.
tuberculosis
To confirm that the polyclonal antibody R9 was capable of detecting a protein
expressed by M. tuberculosis, Western blotting was performed using protein
extracted
from the M. tuber=culvsis laboratory train H37Rv. Cytosolic and membrane
proteins
were extracted and analysed using Western blotting essentially as described in
Example
4. Antibody R9 detected a protein of the correct molecular weight in reduced
cytosolic
1o samples, reduced membrane samples and non-reduced cytosolic/membrane
samples.
Accordingly, ribosomal protein S9 is expressed by Al. tuberculosis, e.g.,
strain H37Rv,
a fact that has been previously unrecognized.
EXAMPLE 7
Antibody-based diagnosis of tuberculosis or infection by M. tuberculosis:
Detection of antibodies against ribosomal protein S9
Peptides predicted to be exposed on the surface of the ribosomal S9 protein
were
conjugated to biotin. Streptavidin was immobilised onto an ELISA plate at 50
l per
well at a concentration of 5 g/ml. Wells were incubated with appropriate
peptides at 3
g/ml diluted in blocking buffer, followed by addition plasma from each of 44
TB and
,44 non-TB subjects, diluted 1/50. Bound human IgG were detected with sheep
anti-
Human IgG HRP conjugate diluted at 1/10,000, then colour development with TMB
substrate at 50 ul per well.. ROC curve analysis was used to determine (i)
sensitivity at
95% specificity; and (ii) optimum sensitivity and specificity.
Plasma and/or sputum antibodies from non-TB subjects were found to have
minimal
cross-reactivity to two peptides tested. In particular, one peptide had a
sensitivity at
95% specificity of 17.3%, an optimal sensitivity of 56.8% and an optimum
specificity
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of 79.6%. The other peptide had a sensitivity at 95% specificity of 8.3%, an
optimal
sensitivity of 56.8% and an optimum specificity of 79.6%.
EXAMPLE 8
Antibodies prepared against recombinant R2: tuberculosis S9 protein
Two additional antibody preparations were prepared against the full length
recombinantly-expressed M. tuberculosis protein (SEQ ID NO: 1), by
immunization of
chickens and mice using standard procedures. Two separate batches of chicken
io polyclonal antisera were raised against the S9 protein. Herein, the chicken
anti-S9
polyclonal sera are designated "Ch27", and mouse anti-S9 antibodies are
designated
"Mo 1025F". These antibody preparations were found to have the highest
sensitivity of
detection for the S9 protein in ELISA assays, compared to other antibodies
produced,
including bivalent F(ab)2 fragments produced by phage display of S9 peptides
and a
further polyclonal antibody raised against a purified S9 peptide (data not
shown).
Data presented in Figure 5 show that the antibodies Ch27 and Mo1025F prepared
against recombinant M. tuberculosis ribosomal protein S9 are capable of
detecting
recombinant S9 protein by standard ELISA, and suggest that the mouse antibody
Mo1025F may have particular utility as a diagnostic reagent due to its higher
titer (i.e.,
half-maximum detection of about 93 ng/ml S9 protein and detection limit of
about
8ng/ml under the conditions used) compared to antibody Ch27 (half-maximum
detection of greater than 125 ng/ml S9 protein and detection limit of about 32
ng/ml
under the conditions used).
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EXAMPLE 9
Sandwich ELISA for detecting Af tuberculosis S9 protein using
antibodies prepared against recombinant S9 protein
A sandwich ELISA was developed employing two antibodies prepared against
recombinant M. tubet-culosis ribosomal protein S9, in particular the chicken
polyclonal
antibody designated Ch27 and the mouse antibody Mo1025F (Example 8).
1. Preferred antibody orientation
io In a first set of experiments, sandwich ELISA was performed to determine
optimum
capture and detection antibodies, and appropriate antibody concentrations for
use.
Briefly, two ELISA plates were coated with either Ch27 or Mo 1025F antibodies
at 2.5
g/ml and 5 g/ml concentrations in blocking buffer. Following washing to
remove
unbound antibody, 50 l aliquots of recombinant S9 protein, diluted serially
in blocking
buffer 1:2 (v/v) from 500 ng/mi starting concentration to 7.8 ng/ml, were
added the
wells of the antibody-coated ELISA plates. Following incubation for 1 hour and
washing to remove unbound antigen, the alternate detection antibody (i.e.,
Mo1025F
for detection of Ch27-S9 complexes and Ch27 for detection of Mo1025F-S9
complexes) was contacted with the plates at concentrations in the range of
1.25 g/ml
to 5 g/ml. Following incubation at room temperature for 1 hour, plates were
washed
as before, incubated with 50 1 of a 1:5000 (v/v) dilution of donkey anti-mouse
IgG
conjugated to horseradish peroxidase (HRP), washed as before, incubated with
TMB
for 30 mins, and the absorbance at 595-600nm was determined.
Data presented in Figures 6 and 7 indicate that the preferred, albeit not
essential,
orientation of antibodies to achieve higher signal per unit of recombinant S9
protein in
sandwich ELISA is obtained using Ch27 as the capture antibody and Mo1025F as a
detection, antibody. Minimal cross-reactivity between antibodies is also
observed with
this antibody orientation, as indicated by the baseline value in Figure 6 when
no S9 is
present in the sample.
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2. Optimizing the limits of detection
To determine the limits of detection of the sandwich ELISA for recombinant M.
tuberculosis S9 protein, the assay was also performed using a serial dilution
of S9
protein, in the concentration range from 18.31 pg/ml to 150 ng/ml. Data
presented in
Figure 8 indicate that, under the assay conditions tested, there was no
background
signal with this antibody combination, and concentrations as low as about 996
pg/ml M.
tuberculosis ribosomal protein S9 could be detected, with half-maximum
detection of
1o about 28 ng/ml A7. tuberczilosis ribosomal protein S9. Such sensitivity of
detection
coupled with low background in sandwich ELISA is considered by the inventors
to be
within useful limits.
a) Use of biotinylated secondary antibody
To further enhance the detection limits of the sandwich ELISA assay, the
inventors
investigated whether or not a biotinylated secondary antibody could improve
sensitivity. As shown in Figure 9, the use of a biotinylated secondary
antibody and
streptavidin poly-40 horseradish peroxidase (HRP) conjugate provided some
increase
in sensitivity of detection, with a statistically significant limit of
detection as low as
2o about 150 pg/ml recombinant AI. tubei-culosis ribosomal protein S9. Under
these
conditions, the sandwich ELISA was also capable of detecting about 6 ng/ml M.
tuberculosis ribosomal protein S9 at half-maximal signal.
b) Replacement amplification
To further enhance sandwich ELISA sensitivity, the inventors further modified
the
basic assay by employing iterative antigen binding following coating of the
ELISA
plate with capture antibody. Essentially, this results in an increased amount
of antigen
being bound to the capture antibody notwithstanding the 50 l volume
limitations of a
96-well ELISA plate. Briefly, this iterative antigen loading involves
repeating the
3o antigen binding step in the sandwich ELISA several times, e.g., 2 or 3 or 4
or 5 times,
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etc. before washing and adding detection antibody. Naturally, each aliquot of
antigen
sample is removed following a standard incubation period before the next
aliquot is
added. The number of iterations can be modified to optimize the assay (e.g.,
parameters such as signal: noise ratio, detection limit and amount of antigen
detected at
half-maximum signal), depending upon the nature of the sample being tested
(e.g.,
sample type), without undue experimentation.
As shown in Figure 10, five iterations, of sample loading (i.e., a 5x
replacement
amplification) provided a low background signal, and a detection limit of
about 84
1o pg/ml M. tuberculosis ribosomal protein S9. As- the assay shown in Figure
10 was not
performed under conditions reaching signal saturation, no estimation of the
amount of
antigen detected at half-maximum signal was possible. Notwithstanding, an
approximate 2-fold increase in sensitivity of detection of recombinant M.
tuberculosis
ribosomal protein S9 was obtained by iterative antigen loading.
c) Sample dilution to reduce signal suppression from sample
To assess whether or not factors are present in biological samples that are to
be tested
using the sandwich ELISA of the present invention, e.g., in a point-of-care or
field test
format, different concentrations of recombinant M. tuberculosis ribosomal
protein S9
(i.e., 0.8-16 ng/ml) were mixed with serial dilutions of plasma or sputum and
tested in
an assay format utilizing biotinylated secondary antibody aiid streptavidin
poly-40
horseradish peroxidase (HRP) conjugate, as described herein above.
Under the conditions tested, no significant suppression of signal was observed
for
plasma-containing samples, and a minimum loss in signal strength could be
compensated by performing iterative sample loading for at least one round
(Figure 12).
In contrast to plasma, sputum did produce some signal suppression, especially
when
added essentially undiluted to assays. However, this signal suppression can be
overcome partially by diluting sputum samples, and compensating for the
reduced
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antigen in the sample, by performing iterative sample loading as described
herein
above. Preferably, a 1:2 (v/v) dilution or a 1:3 (v/v) dilution of sputum into
blocking
buffer and two or three iterations of sample loading permits sufficient
recovery of
signal strength to compensate for the signal suppression observed with sputum
samples
(data not shown).
In summary, the available data suggest that the sandwich ELISA for the
detection of S9
protein offers excellent sensitivity with low background signal. Further
enhancement
in the sensitivity of detection may be obtained by directly biotinylating the
detection
lo antibody Mo1025F to thereby permit amplification without the use of a
secondary
antibody.
EXAMPLE 10
Low cross-reactivity between antibodies against recombinant S9 protein and
Escherichia coli, Bacillus subtilis or Pseudornonas aeruginosa in sandwich
ELISA
To further assess the suitability of S9 as a diagnostic marker for the
presence of Al.
tuberculosis in biological samples, the inventors compared antibody cross-
reactivities
in sandwich ELISA performed as described in Example 9 between cellular
extracts of
11f. tuberculosis strain H37Rv (a laboratory strain), Escherichia coli,
Bacillus subtilis or
Pseudonzonas aeruginosa.
Briefly, an ELISA plate was coated overnight with capture antibody Ch27 at 5
g/ml
concentration. Following washing to remove unbound antibody, 500 ng/ml or 50
g/ml
of a cellular extract from each microorganism were added the wells of the
antibody-
coated ELISA plates. As a negative control for each assay, buffer without
cellular
extract was used. Following incubation for 1 hour and washing to remove
unbound
antigen, detection antibody Mo1025F was contacted with. the bound antigen-body
complexes at 2.5 g/ml concentration. Following incubation at room temperature
for 1
3o hour, plates were washed, incubated with 50 l of a 1:5000 (v/v) dilution
of secondary
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antibody (i.e., biotinylated donkey anti-mouse IgG and poly-40 streptavidin-
HRP
conjugate) for 1 hour, washed again, incubated with TMB for 10 mins, and the
absorbance at 595-600 nm was determined. No iterative sample loading was
performed
in this experiment.
Data presented in Figure 11 show low cross-reactivity of antibodies against M.
tuberculosis ribosomal protein S9 with Eschef ichia coli, Bacillus saibtilis
or
Pseudonionas aeruginosa cellular extracts under the conditions tested.
EXAMPLE 11
Reactivity between antibodies against recombinant S9 protein and laboratory
and
clinical isolates of M. tuberculosis in sandwich ELISA
To further assess the suitability of S9 as a diagnostic marker for the
presence of M.
tuberculosis in biological samples, the inventors compared antibody
reactivities in
sandwich ELISA performed as described in Example 9 between cellular extracts
of the
clinical If. tubei=culosis strain CSU93 and the laboratory M. tubej culosis
strain H37Rv.
2o Briefly, an ELISA plate was coated overnight with capture antibody Ch27 at
5 g/ml
concentration. Following washing to remove unbound antibody, 500 ng/ml or 50
g/ml
of a cellular extract from each isolate were added the wells of the antibody-
coated
ELISA plates. As a negative control for each assay, buffer without cellular
extract was
used. Following incubation for 1 hour and washing to remove unbound antigen,
detection antibody Mo1025F was contacted with the bound antigen-body complexes
at
2.5 g/ml concentration. Following incubation at room temperature for 1 hour,
plates
were washed, incubated with 50 l of a 1:5000 (v/v) dilution of secondary
antibody
(i.e., biotinylated donkey anti-mouse IgG and poly-40 streptavidin-HRP
conjugate) for
1 hour, washed again, incubated with TMB for 10 mins, and the absorbance at
595-600
nm was determined. No iterative sample loading was performed.
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Data presented in Figure 12 show that A7: tubercaslosis ribosomal protein S9
is present
in both the clinical Al. tuberculosis isolate CSU93 and the laboratory strain
H37Rv.
Additionally, since the signals obtained for both isolates are approximately
half the
maximum signal obtained under the assay conditions tested (e.g., see Figure
9), the
assay results suggest that endogenous S9 protein may be present at similar
levels in
both the clinical and laboratory isolates, or alternatively, that factors
suppressing signal
strength in one strain compensate for an over production of S9 protein by that
strain
relative to the other strain.
EXAMPLE 12
Antigen-based diagnosis of tuberculosis or infection by M. tuberculosis:
Western Blot analysis of BSX levels in sputum
Chicken polyclonal antibodies against full-length recombinant BSX protein or a
peptide from BSX were produced using standard methods. The antibody against
the
full-length protein The antibody was purified by affinity chromatography using
immobilised recombinant protein (without NUS).
Sputum (12u1) from TB and non-TB patients was loaded onto 4-12% 1D gradient
SDS
polyacrylamide gels and separated using electrophoresis. Proteins were then
electrotransferred onto PVDF membrane. All the membranes were blocked in
solution
containing 1% casein in IX PBS, 0.1% Tween-20 (PBST) at room temperature (RT)
for 2 hours. Membranes were then incubated with 10 g/ml purified chicken anti-
BSX
pAb solution at RT for 2 hr, following by 3 x 10min washes with PBST.
Membranes
were then incubated with 1:25,000 dilution of sheep anti-chicken IgG-HRP
conjugated
antibody solution at RT for I hr, followed by 5 x 10 min 'washes with times
PBST.
Membranes were finally treated with `Femto' chemiluminescence reagents
(Pierce) for
3o 5 min before exposure to x-ray films.
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Screening for BSX in sputum detected positive signal in 15/19 South African TB
patients (Sensitivity = 78.9%) and 4/18 Australian non-TB respiratory disease
patients
(Specificity = 77.8%) using a purified chicken antibody raised against a NUS-
conjugated recombinant protein (Figures 13a and 13b).
By combining the results of the Western blot for BSX and those of the Western
blot for
S9 (described in Example 5) the sensitivity of the multi-analyte assay was
increased to
83% (15/18) and the specificity to = 85% (2/14). In particular, S9 was
detected in 20
1o patients in the TB group and in 5 patients in the non-TB group. BSX was
detected in 15
patients, also at different levels, in the TB group and 5 patients in the non-
TB group.
There is an overlap of 18 TB and 14 non-TB patients that were screened for
both S9
and BSX. 15 out of 18 TB samples (83.3%) show positive signal for both
proteins. 10
out of 14 non-TB samples (71.4%) show negative results for both proteins. Only
2 non-
TB patients (14.3%) have positive signals for both proteins. It is important
to
appreciate that non-TB controls are those patients presenting with clinical
symptoms of
TB but have been diagnosed with other respiratory disease such as pneumonia or
bronchitis based on negative results for smear and culture testing for TB.
Given the
sensitivity level of these current diagnostic tests, there is -30% chance that
some of
these controls may indeed have undiagnosed TB. As a consequence, the
specificity for
the multi-analyte (or single analyte) assay may be higher than that actually
observed in
the current analysis.
Equivalent or better results will be obtained using optimized sandwich ELISA
for S9 as
described herein above, in combination with ELISA for detection of BSX.
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EXAMPLE 13
Antigen-based diagnosis of tuberculosis or infection by M. tuberculosis:
Detection of BSX in immunoglobulin fraction
The Immunoglobulin fraction of four different sputum samples was isolated
using
Protein-G SepharoseTM, and the flow through fraction were loaded onto a 4-12%
1D
gradient SDS polyacrylamide gel and separated by electrophoresis. Proteins
were then
electrotransferred onto PVDF membrane. All the membranes were blocked in
solution
containing 1% casein in 1 X PBS, 0.1% Tween-20 (PBST) at room temperature (RT)
io for 2 hours. Membranes were then incubated with 10 g/rnl purified chicken
anti-BSX
polyclonal antibody described in Example 12 at RT for 2 hr, following by 3x
10min
washes with PBST. Membranes were then incubated with 1:25,000 dilution of
sheep
anti-chicken IgG-HRP conjugated antibody solution at RT for 1 hr, followed by
5x 10
min washes with times PBST. Membranes were finally treated with `Femto'
chemiluminescence reagents (Pierce) for 5 min before exposure to x-ray films.
reagents
for 5 min before exposed on x-ray films.
As shown in Figure 14, BSX is detected in the flow through fraction (i.e., not
bound by
immunoglobulin) but not in the immunoglobulin fraction. These results indicate
that
2o BSX is not bound in circulating immune complexes in patient samples,
indicating that
this protein represents a good candidate for an antigen based assay for
diagnosing M.
tuberculosis infection and/or TB.
EXAMPLE 14
Antigen-based diagnosis of tuberculosis or infection by M. tuberculosis:
An ELISA to detect M. tuberculosis BSX
An ELISA assay was performed using one of three different anti-BSX antibodies,
3o namely rabbit polyclonal anti-BSX antibody (raised against a BSX peptide)
designated
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R16, a chicken anti-BSX polyclonal antibody designated C44 (raised against
recombinant protein) and a mouse anti-BSX monoclonal antibody designated 403B
(raised against the C-terminus of BSX). Briefly, the ELISA was performed as
follows:
The ELISA plate was coated with various anti-BSX proteins including chicken
(Ch)
anti-BSX pAb C44, rabbit (Ra) anti-BSX pAb R16, and mouse (Mo) anti-BSX mAb
403B all at 20 g/ml using 50 l per well. Titrating amounts of recombinant
BSX were
added at a concentration of 50 ng/ml down to 3 pg/ml. Antigen detection was
performed using either rabbit anti-BSX at 10 g/ml (with and without pre-
incubation
1o with the recombinant BSX protein) followed by detection using sheep anti-
rabbit Ig
HRP conjugate at a 1/5000 dilution (for chicken capture system), or chicken
anti-BSX
pAb C44 at 20 g/ml followed by sheep anti-chicken IgG HRP conjugate at 1/5000
dilution (for mouse and rabbit capture systems). Data are presented in Figure
15.
EXAMPLE 15
Antigen-based diagnosis of tuberculosis or infection by M. tuberculosis:
Detection of M. tuberculosis by sandwich ELISA
Deternzining the detection. liinit of the sandwich ELISA
Subsequent to determining detection limits of anti-BSX mAb 403B and pAb C44
for
detection of purified recombinant BSX our initial studies addressed
optimisation of a
sandwich ELISA using mAb 403B as a capture antibody and pAb C44 as a detector
antibody. Briefly, Anti-BSX mAb 403B was immobilised onto an ELISA plate as a
capture antibody at concentrations ranging from 10-40 g/ml as specified
above.
Titrating amounts of recombinant BSX from 50 ng/ml down to 0.39 ng/ml were
then
screened using a purified chicken anti-BSX pAb, C44, at concentrations of
either 10 or
20 g/ml as specified above as the detector antibody followed by incubations
with a
sheep anti-chicken IgG HRP at a dilution of 1/5000 and TMB for signal
detection. Data
are presented in Figure 16.
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Under these conditions, the limit of detection of recombinant BSX was - 2-3
ng/ml.
Detectirag BSX in patient sanZples
Sputum samples (50 ul + 50 ul blocking buffer) from South African TB patients
and
control patients with non-TB respiratory disease from South Africa (prefix
`M') and
Australia (prefix `CGS'), respectively, were screened by sandwich ELISA for
the
presence of BSX antigen. Purified rabbit anti-BSX (peptide 28) pAb, R16, was
immobilised onto the ELISA plate as a Capture antibody at a concentration of
20
g/ml. Purified chicken anti-BSX pAb, C44, at a concentration of 5 g/ml, was
used as
io the Detector antibody. Sheep anti-chicken IgG HRP at a dilution of 1/5000
and TMB
were used for signal detection. Sputum from control patient CGS25 was spiked
with 5
ng/ml recombinant BSX as a positive control (red). Results are shown in Figure
17.
EXAMPLE 16
Antigen-based diagnosis of tuberculosis or infection by M. tuberculosis:
Detection of M. tuberculosis BSX in sputum by amplified sandwich ELISA
ELISA plates were coated with either purified anti-BSX mAb 403B at a
concentration
of 40 g/ml or purified chicken anti-BSX pAb C44 at a concentration of 5 g/m1
using
2o 50 ul per well. Titrating amounts of purified recombinant BSX were added at
a
concentration of 50 ng/ml down to 0.39 ng/ml. Two amplification systems were
performed using either chicken anti-BSX at a concentration of 10 g/ml
followed by
donkey anti-chicken IgG biotin conjugate at various dilutions and finally
streptavidin-
HRP at a 1/5000 dilution, or anti-BSX mAb 403B at various concentrations
followed
by goat anti-mouse IgG at 1/30000 dilution and donkey anti-goat IgG HRP
conjugate at
a 1/5000 dilution. The amplified systems were used to compare to a basic
antigen
detecting system where chicken anti-BSX was used at a concentration of 10
g/ml
followed by sheep anti-chicken IgG HRP conjugate at a 1/5000 dilution.
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As shown in Figure 1 S, the amplified ELISA was approximately 10 fold more
sensitive
than the standard ELISA. Signal intensity is slightly higher when using the
rabbit pAb
as a capture and the chicken pAb as the first detector Ab in the amplified
system
(Figure 19).
We have also investigated an amplified ELISA system which, as shown in Figures
20
and 21, uses purified rabbit anti-BSX pAb R16 as a capture antibody and
purified
chicken anti-BSX pAb C44 as a detector antibody followed by amplification with
a
biotinylated secondary detector Ab. This system provided a further 2-fold
increase in
io sensitivity compared the amplification systems described earlier (Figure
20).
We have now also performed studies using the amplified biotin based ELISA to
screen
clinical sputum samples from TB and non-TB respiratory disease control
patients,
always keeping in mind in the non-TB respiratory disease group there may be up
to 30-
40% of the patients having TB co-infections due to the poor sensitivity of
smear
microscopy and culture assays (Figure 22).
To investigate if antibody sites were being saturated with endogenous BSX we
also
compared the effect of (i) incubation time; and (ii) sequential incubations
with a fresh
2o aliquot of a sputum sample from the same respective patient. The increase
from a 1 hr
to a 2 hr incubation did not have any effect on signal intensity. In contrast,
preliminary
data indicates that sequential incubations with 2 different sample loads of
sputum
increased signal intensity (Figure 23). Whilst the increase is not large,
these
preliminary observations warrant further investigation. Interestingly, the
increase in
signal intensity was most marked for detection of a recombinant protein as a
positive
control.
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EXAMPLE 17
Antibody-based diagnosis of tuberculosis or infection by M. tuberculosis:
ELISA using M. tuberculosis Bsx protein fragments to diagnose the presence of
antibodies against R7 tuberculosis
1. Sera and peptides
A total of 30 TB-positive samples and 52 TB-negative samples were screened
with the
io following peptides derived from the Bsx protein: MRQLAERSGVSNPYL (SEQ ID
NO: 8), ERGLRKPSADVLSQI (SEQ ID NO: 9), LRKPSADVLSQIAKA (SEQ ID
NO: 10), PSADVLSQIAKALRV (SEQ ID NO: 11), SQIAKALRVSAEVLY (SEQ ID
NO: 12), AKALRVSAEVLYVRA (SEQ ID NO: 13), VRAGILEPSETSQVR (SEQ
ID No: 14), TAITERQKQILLDIY (SEQ ID NO; 15), SQIAKALRVSAEVLYVRAC
15 (SEQ ID NO: 16), MSSEEKLCDPTPTDD (SEQ ID NO: 17) and
VRAGILEPSETSQVRC (SEQ ID NO: 18). In each case, the peptides were
biotinylated to facilitate their detection.
These samples included sera from South African (S.A.) Zulu TB-positive
individuals,
20 S.A. Zulu TB-negative individuals, S.A. Caucasian TB-negative individuals,
World
Health Organisation (WHO) TB-positive individuals of unknown race, 'WHO TB-
negative individuals of unknown race, and Australian Caucasian TB-negative
control
individuals and plasma from Chinese TB-positive individuals and Chinese TB-
negative
individuals.
Samples were screened for the presence of antibodies using an ELISA system
developed as described below.
2. ELISA Assav
3o Nunc-Immuno module maxisorp wells were coated overnight at room temperature
or at
4 C over the weekend with 100 l/well of 5 g/mi streptavidin diluted in milli-Q
water.
The streptavidin was flicked out of the wells and each well was blocked with
200 1
phosphate-buffered saline (PBS) containing 1.0% (w/v) casein, 0.1 %(v/v) Tween
20
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and 0.1 %(w/v) Azide (blocker) per well. After 1 hour, the blocker was
removed, and
each well was coated with 100 l of biotinylated peptide in blocker for 1 hour,
with
agitation of the plate. Subsequently, each well was washed 5 times with
PBS/0.1 %
Tween 20, allowed to dry on absorbent paper, and either stored at 4 C with
dessicant,
or used immediately. This was followed by incubation for 1 hour with agitation
in 50 l
of patient serum or plasma, diluted 1:50 in blocker. Following this
incubation, all wells
were washed 5 times, using PBS/0.1% Tween 20 in a laminar flow, and tapped
dry.
Then 100 1 sheep anti-human IgG horse radish peroxidase (HRP) conjugate was
added
to each well. The conjugate was diluted 1:10,000 (v/v) in PBS/0.1% (w/v)
casein/0.1%
io (v/v) Tween 20/0.1 %(w/v) thimerosal, and incubated for 1 hour with
agitation. Each
well was then washed 4 times using PBS/0.1% (v/v) Tween 20, and twice using
PBS.
Finally, 100 1 liquid TMB substrate based system (Sigma) was added to each
well, and
the wells incubated at room temperature in the dark for 20 mins. Reactions
were
stopped by addition of l00 10.5M sulfuric acid. Each peptide was assayed in
duplicate
and repeated if duplicates did not appear to be reproducible.
Alongside the patient samples, four control samples were also tested, as
follows:
1. Negative control: streptavidin/peptide'-14/no serum or plasma/conjugate;
2. Peptide Control: streptavidin/no peptide/patient serum or
plasma/conjugate;
3. Positive control: streptavidin/peptide 24/S.A. serum 7/conjugate; and
4. Serum background: no streptavidin/no peptide/patient serum or
plasma/conjugate.
S.A. serum 7 was used for the positive control, due to its consistent
reproducible
positive results found in preliminary ELISA experimentation.
3. Data analysis
Immunogenic peptides represent outliers in the distribution of peptide
absorbencies and
are detected following log transformation normalisation by calculation of a
normal
score statistic, with a mean and standard deviation estimated by a robust M-
Estimator.
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4. Results
Mass screening of the TB-positive and TB-negative samples for the presence of
antibodies to Bsx peptides demonstrate that about 47% of TB-positive samples
contain
anti-Bsx antibodies. A small number of TB-negative patients may test positive
for any
Bsx peptide. Differentiation of the total patient population to include HIV
status will
elucidate a TB/HIV correlation, where about 76% of the TB-positive samples
that
contain anti-Bsx antibodies are also HN+. In the S.A. group, about 80% of the
S.A.
TB-positive/HIV+ samples should contain antibodies to Bsx.
Conversely, in Chinese populations that are HIV- and categorised according to
their
pulmonary diagnosis, none of the extra-pulmonary or pulmonary TB-positive
plasma
should contain antibodies to Bsx, and only a small number of TB-negative
plasma
screened may contain anti-Bsx antibodies to one Bsx peptide.
In summary, ELISA analysis of TB positive and TB negative serum or plasma
reveals a
2o number of immunogenic Bsx peptides containing B cell epitopes of the full-
length Bsx
protein of A1. tuberculosis.
Several peptides are non-immunogenic in any control TB-negative serum or
control
plasma tested e.g., in sera from TB-negative S.A. Zulu subjects. These data
reinforce
the suitability of Bsx and/or any of its peptides as a diagnostic reagent, and
as an
immunogen for the preparation of monoclonal antibodies suitable for use in an
antigen-
based assay for A?: tubei-culosis infection.
Furthermore, the correlation between HIV status and TB status with respect to
serological reactivity of a Bsx peptide has many therapeutic advantages, such
as, for
example, the ability to detect TB and HIV status and/or monitoring the TB
status in
HIV+ individuals. To further emphasise the correlation between TB and HIV, it
is
important to note that all of the Chinese samples investigated were HIV-
negative.
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The absence of detectable antibodies that bind to Bsx in plasma from patients
in a
Chinese cohort may be associated with pulmonary TB being confined to the lung,
whereas in the South African patients HIV positive status is often associated
with
extrapulmonary disease, which is more systemic. Alternatively, Bsx may not be
as
highly expressed in Chinese compared to South African TB patients.
EXAMPLE 18
Antibody-based diagnosis of tuberculosis or infection by M. tuberculosis:
Screening of TB and non-TB sera against synthetic peptides
derived from the Bsx protein
1. Synthetic pe tp ides
Three peptides were synthesised from the amino acid sequence of the putative
transcriptional regulator Bsx (SwissProt entry number 053759) and evaluated as
capture agents for Human immunoglobulin G in TB-positive sera. One peptide,
designated Bsx (23-24) peptide (SEQ ID NO: 16) comprises the sequence of a
highly
immunogenic Bsx peptide with additional N-terminal and C-terminal sequences
flanking this sequence in the full-length protein and conjugated C-terminally
to a
cysteine residue. Another peptide, designated N-C terminal (SEQ ID NO: 17)
comprised the N-terminal seven residues of Bsx protein fused to the C-terminal
seven
residues of Bsx by an intervening cysteine residue. A third peptide,
designated peptide
28 (SEQ ID NO: 18) comprises another Bsx peptide conjugated C-terminally to a
cysteine residue.
For ELISA formats, the peptides set forth in SEQ ID NOs: 16-18 additionally
comprised an N-terminal linker (Ser-Gly-Ser-Gly) to the base peptide, to
facilitate
binding of the peptide to solid matrices.
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The C-terminal and internal cysteine residues were included to facilitate
cross-linking
of the peptides for subsequent antibody production.
2. Sera/plasma
Sera and plasma were a panel obtained from 41- 44 TB-positive patients (i.e.,
TB-
positive sera) in each experiment, and 51 healthy control subjects (i.e., non-
TB sera).
3. ELISA Assay
Peptides comprising SEQ ID NOs: 16-18 were coated on ELISA trays at 3 g/m1 on
a
io streptavidin base of 5 g/ml and then probed (after blocking) with Non-TB
control sera
and Known TB-positive sera and plasma. Sera and plasma were diluted 1/50 (v/v)
prior to use. Capture of human IgG was traced with enzyme-linked sheep anti-
human
IgG and tetramethylbenzidine (TMB) substrate.
4. Statistical 'analyses
The sensitivity and specificity were analysed by taking the average substrate
product
OD values (from the conjugated peroxidase/TMB reaction) and calculating the
cut-off
values for significance at two standard deviations above the average and three
standard
deviations above the mean (i.e., at the 95% and 99.7% significance levels,
respectively). For the control sera, one sample produced an outlier OD value
by Dixon's
outlier test (N = 30). The analyses were compared including or excluding this
outlier.
As used herein, term "sensitivity" in the context of a diagnostic/prognostic
assay is
understood to mean the proportion of TB-positive subjects that are diagnosed
using a
particular assay method (i.e., a "true" positive). Accordingly, an assay that
has
increased sensitivity is capable of detecting a greater proportion of TB-
infected subjects
than an assay with reduced or lower sensitivity.
As used herein, the term "specificity" in the context of a
diagnostic/prognostic assay is
understood to mean the proportion of non-TB subjects (i.e., non-infected
subjects) that
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do not return a positive result using a particular assay method (i.e., "true"
negatives).
Accordingly, an assay that has increased or enhanced specificity returns fewer
false
positive results or is capable of distinguishing between infected and non-
infected
subjects to a greater degree than an assay with a reduced specificity.
4. Results
a) Bsx (23-24) Peptide (SEQ ID NO: 16)
Bsx (23-24) peptide sequence showed a significant binding to confirmed TB-
positive
sera. Data indicate that a peptide comprising the sequence set forth in SEQ ID
NO: 16
io selectively identifies antibodies that bind to M. tuber=culosis in patient
sera. Data also
show that the sensitivity and specificity with these revised criteria are
relatively
unchanged irrespective of whether or not the outliers is omitted, however
there is a
marginal increase in sensitivity at the 3 standard deviation level.
b) N-C terniinal (SEQ ID NO: 17) czrad Peptide 28 (SEQ ID NO: 18)
These two peptides showed only weak interaction against a range of confirmed
TB-
positive sera. Assays using these peptides were not highly sensitive, albeit
specific in
so far as they omit false positive detection.
2o The data indicate that Bsx (23-24) peptide (SEQ ID NO: 16) has utility in
antibody-
based assays to detected tuberculosis in patient samples, especially sera. The
other two
peptides tested in this example (SEQ ID NOs: 17 and/or 18) also have utility
in
eliminating false positive detection e.g., as part of a multi-analyte test.
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EXAMPLE 19
Antibody-based diagnosis of tuberculosis or infection by M. tuberculosis:
Screening of TB and non-TB sera against recombinant full-length
Bsx protein
1. Sera/ lp asma
Sera and plasma were from 44 TB-positive (smear or culture) Chinese and South
Africaii patients (i.e., TB-positive sera), and 44 healthy control subjects
(i.e., non-TB
sera).
2. ELISA Assav
Recombinant Bsx protein was coated directly onto ELISA trays at 5 g/ml and
then
probed (after blocking) with Non-TB control sera, and known TB-positive sera
and
plasma diluted 1/100 (v/v) in buffer. Capture of human IgG was traced with
enzyme-
linked sheep anti-human IgG and tetramethylbenzidine (TMB) substrate.
3. Statistical analyses
The sensitivity and specificity were analysed by taking the average substrate
product
OD values (from the conjugated peroxidase/TMB reaction) and calculating the
cut-off
values for significance at two standard deviations above the average and three
standard
deviations above the mean (i.e., at the 95% and 99.7% significance levels,
respectively).
4. Results
Recombinant Bsx protein assayed under these conditions was highly specific in
detecting TB-positive sera. Sensitivity of the assay over the populations
tested was
intermediate between SEQ ID NO: 16 and SEQ ID NOs: 17-18.
On the other hand, the sensitivity of the assay in South African TB sera
smears or
culture positives is higher than the overall sensitivity (i.e., 35% compared
to 25% at
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three standard deviations cut-off value). Using Chinese smear or culture TB
sera, the
sensitivity of the assay is lower than the overall sensitivity (i.e., 11%
compared to 25%
at three standard deviatioiis cut-off value). In both Chinese and South
African
populations, the specificity of the assay is 100%, indicating robustness in
this
parameter.
EXAMPLE 20
Antibody-based diagnosis of tuberculosis or infection by M. tuberculosis:
Screening of TB and non-TB sera according to HIV status
1. Sera/plasma
Sera and plasma were obtained from the following subjects:
(i) Five (5) TB-positive and HIV-negative smear or culture South African
patients
(i.e., TB+ HIV` sera/plasma);
(ii) Twenty one (21) TB-positive and HIV-positive smear or culture South
African
patients (i.e., TB+ HIV+ sera/plasma); and
(iii) Twenty (20) TB-negative and HN-negative smear or culture subjects (i.e.,
healthy control sera/plasma).
2o 2. ELISA Assay
Recombinant Bsx protein or Bsx (23-24) peptide (SEQ ID NO: .16) was coated
directly
onto ELISA trays at 5 g/ml and then probed (after blocking) with Non-TB
control sera
and known TB-positive sera diluted 1/100 (v/v) in buffer. Alternatively, the
Bsx(23-
24) peptide was used as described in the preceding examples. Capture of human
IgG
was traced with enzyme-linked sheep anti-human IgG and tetramethylbenzidine
(TMB)
substrate.
3. Statistical analyses
The sensitivity and specificity were analysed by taking the average substrate
product
OD values (from the conjugated peroxidase/TMB reaction) and calculating the
cut-off
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values for significance at two standard deviations above the average and three
standard
deviations above the mean (i.e., at the 95% and 99.7% significance levels,
respectively).
4. Results
Recombinant Bsx protein assayed under these conditions was highly specific in
detecting TB-positive sera. Sensitivity of the assay over the populations
tested was
also quite high for HIV+ patients. Similar results were obtained using the
Bsx(23-24)
peptide. Thus, the full-length recombinant Bsx protein and Bsx(23-24) peptide
io separately detect about 40-45% of TB+ HIV+ subjects, and, in a multianalyte
test
format, detect about 65% to 70% of TB+HIV+ subjects, with only about 5% false-
positive detection.
On the other hand, the sensitivity of the assay in South African TB sera
and/or plasma
smears or culture positives is higher than the overall sensitivity (i.e., 35%
compared to
25% at three standard deviations cut-off value). Using Chinese smear or
culture TB
sera/plasma, the sensitivity of the assay is lower than the overall
sensitivity (i.e., 11%
compared to 25% at three standard deviations cut-off value). In both Chinese
and
South African populations, the specificity of the assay is absolute i.e., 100%
indicating
2o robustness in this parameter.
These data indicate that the full-length Bsx protein, e.g., expressed as a
recombinant
protein, can be used in combination with a synthetic peptide comprising the
dominant
B-cell epitope identified herein e.g., Bsx(23-24) peptide (SEQ ID NO: 16) , to
diagnose
the presence of an active infection or recent past infection by M.
tuberculosis.
For example, recombinant full-length Bsx and Bsx(23-24) peptide are both
biotinylated
and immobilized onto a streptavidin base (5 g/ml) that has been preadsorbed
onto
wells of a microtiter pl ate. Standard ELISA reactions are carried out wherein
(i)
patient sera and control sera, each diluted 1/100 (v/v) in buffer, are added
to separate
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wells, and (ii) capture of human IgG in the sera by the immobilized protein
and peptide
is traced using enzyme-linked sheep anti-HuIgG detected using
tetramethylbenzidine
(TMB) substrate.
EXAMPLE 21
Isolation of monoclonal antibodies that bind to M. tuberculosis GS peptide
1. Antigen selection
ELISA assays were performed to deterniine those GS peptide fragments against
which
1o an immune response was detected in sera from TB subjects and no immune
response
was detected in control subjects.
The amino acid sequence of the GS identified as being immunogenic in TB
subjects
(glutamine synthetase A4 or g1nA4) was aligned with the amino acid sequence of
other
known TB glutamine synthetases (g1nA, glnA2 and gInA3) and shown to have only
25% amino acid sequence identity with other known glutamine synthetase
homologs.
GS peptides were selected that are specifically immunoreactive with sera from
TB+
subjects and not comprise sequences not conserved with other glutamine
synthetases.
2o Finally, 3-dimensional protein modelling was used to determine a region of
the GS
protein of the invention that was likely to be on the surface of the protein
in vivo.
Based on all of the shtdies described supra two peptides were selected that
were
immunogenic in TB sera and not control sera, corresponded to a non-conserved
region
of GS and are likely to be on the surface of the GS protein in vivo. These
peptides
comprise the following sequences:
(i) RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO: 19); and
(ii) WASGYRGLTPASDYNIDYAI (SEQ ID NO: 20)
Antibodies that selectively bind to these peptides are unlikely to cross-react
with
3o another glutamine synthetase proteins.
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The two peptides were selected as antigens for antibody production,
synthesized and
attached to diphtheria toxoid.
2. Antibody production
Antigen
Approximately 6 mgs of peptide antigen consisting of the sequence
RGTDGSAVFADSNGPHGMSSMFRSF (set forth in SEQ ID NO: 19) conjugated to
diphtheria toxoid was provided to NeoClone, Madison, Wisconsin, USA for
generation
io of monoclonal antibodies according to their standard protocol. About 1 mg
of the
peptide was provided as biotinylated peptide for quality control.
Immunization
Five BALB/cByJ female mice were immunized with peptide conjugated to carrier
according to Neoclone's standard immunization process.
Test Bleeds
Test bleeds of the immunized mice were perfoimed at regular intervals for use
in the,
quality control sera ELISAs using biotinylated peptide. Polyclonal sera having
the
2o highest titer were determined using ELISA. Mice having polyclonal antibody
titers of
at least 1,000 were used for the ABL-MYC infection process.
Infection
The spleens of 3 mice having the highest titer of polyclonal antibodies cross-
reactive
with peptide antigen were used for the ABL-MYC infection, according to
NeoClone's
standard infection procedure.
Transplantation
The splenocytes of the ABL-MYC-infected mice were transplanted into
approximately
3o 20 naive mice.
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Ascites developrnent
Ascites fluid developed in the transplanted mice were isolated and screened
for cells
producing monoclonal antibodies (mAbs) that bind to the target peptide
antigen. A cell
line (i.e., plasmacytoma) producing a mAb designated 426C was isolated.
Binding
affinity and isotype specificity of the mAb 426C was confirmed using ELISA.
The mAb designated 426C was provided in 1 ml aliquots (approximately) in
ascites,
together with the associated cell line.
The mAb designated 426C is purified from ascites using protein G or protein A
columns.
3. Antibody titration
The monoclonal antibody designated 426C was coated on the bottom of an ELISA
plate at 20 1 and (i) an immunogenic glutamine synthetase (GS) peptide
biotinylated at
the N-terminus or (ii) a negative control peptide biotinylated at the N-
terminus, were
added at various concentrations to 10 pg/ml as indicated in Table 3a. The
biotinylated
GS peptide used had the sequence: SGSGRGTDGSAVFADSNGPHGMSSMFRSFC
(SEQ ID NO: 21). The peptide was detected by binding of streptavidin HRP
conjugate
under standard conditions. Absorbances were determined at 450nm and 620nm, and
tlie difference in absorbance at 450nm and 620nm determined. Average data for
duplicate samples were obtained. The data obtained show that the antibodies
capture
the immunogenic GS peptide antigen at concentrations of about 10 pg/ml or
greater, at
a signal:noise ratio of at least about 2Ø These data demonstrate efficacy of
the
antibodies as a capture reagent in immunoassays.
In a further assay to titer the monoclonal antibodies, the peptide (i.e., SEQ
ID NO: 21)
was coated onto the bottom of the ELISA plate at a concentration of about 3
gl.
3o Duplicate aliquots of the monoclonal antibody-producing plasmacytoma
designated
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426C, and duplicate aliquots of a negative control monoclonal antibody were
added at
various final concentrations to 10 pg/ml. Binding of the antibody was then
detected
using sheep anti-mouse HRP antibody conjugate under standard conditions.
Absorbances were determined at 450nm and 620nm, and the difference in
absorbance
at 450nm and 620nm determined. Average data were obtained. The data show that
the
antibody successfully detects GS above assay background at concentrations of
antibody
as low as 10 pg/ml, therefore demonstrating efficacy as a detection reagent in
immunoassays.
EXAMPLE 22
Antigen-based diagnosis of tuberculosis or infection by M. tuberculosis:
Solid phase ELISA using mAb 426 to detect circulating immune complexes
comprising
M. tubef culosis glutamine synthetase (GS) polypeptide or GS fragments
This example describes an ELISA for the detection of circulating immune
complexes
(CIC) bound to M. tubereulosis glutamine synthetase (GS) in patient samples
comprising circulating immune complexes or antibodies, such as a bodily fluid
selected
from the group consisting of blood, sera, sputa, plasma, pleural fluid,
saliva, urine etc.
Whilst the assay is described herein for the detection of CIC comprising R?:
tuberculosis GS using mAb 426C, the skilled artisan will be aware that the
assay is
broadly applicable to the detection of any CIC comprising an antigen against
which a
capture antibody has been produced. In general, the assay uses antibodies that
bind
specific epitopes on a target antigen found, for example, in sputa and/or sera
from a
subject that is infected with a pathogen (i.e., the subject has an active
infection). The
antibodies are used in a capture ELISA to bind CIC comprising the target
antigen and
the bound CIC are detected by contacting a secondary antibody that recognizes
human
Ig, e.g. anti-human IgA or anti-human IgG antibody, for a time and under
conditions
sufficient for binding to occur and then detecting the bound secondary
antibody. For
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example, the secondary antibody may be conjugated to a detectable label e.g.,
horseradish peroxidase (HRP).
Additionally, whilst exemplified herein for TB, it is to be understood that
the
immunoassay format described herein is useful for detecting any disease or
disorder
which is associated with the presence of CIC, including any infection, Johne's
disease,
Bovine TB, or Crohne's disease.
Additionally, whilst the assay is described herein for ELISA, it is to be
appreciated that
1o the generic assay is readily applicable to any immunoassay format e.g., a
rapid point-
of-care diagnostic format, flow-through format, etc.
An advantage of this assay format is that it directly shows an active vs.
latent infection.
This immunoassay format is particularly useful for discriminating between
active TB
infection and other, non-TB infections, and for monitoring a response of a TB
patient to
treatment.
ELISA based assay
Monoclonal antibody 426C that binds to M. tubei=culosis glutamine synthetase
at a
concentration of 20 g/ml in water, was coated onto the bottom of one or more
NUNC
plates. Plates were left to dry at 37 C overnight. The, plates were blocked
for 1 to 3
hours at room temperature in blocking buffer [ 1%(w/v) casein/0.1 % (v/v)
Tween-20 in
0.5M phosphate buffered saline (PBS)]. The wells were flicked or tapped to
remove
blocking solution, and patient sera diluted 1:50 (v/v) in blocking buffer
(50u1/well)
added. The plates were then incubated for 1 hour at room temperature e.g., on
a
rotating shaker. The plates were washed about 3-5 times with 0.1 %(v/v) Tween-
20 in
0.5M phosphate buffered saline (PBS) such as, for example, using an automated
,plate
washer. Sheep anti-hunlan IgG antibody or anti-human IgA antibody, diluted
1:5000
(v/v) in blocking buffer was added to wells. The plates were then incubated
for 1 hour
3o at room temperature e.g., on a rotating shaker. The plates were washed as
before, and
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TMB was added to the wells (50 ul/well). Plates were incubated for about 30
minutes,
and the reactions were then stopped by addition of 0.5M H2SO4 (50 ul/well).
Absorbances of eacli well was read at wavelengths of 450nm and 620nm, and the
differences in these wavelengths is determined (i.e.A4so-A62o).
The incubation periods and volumes of reagents specified in the preceding
paragraph
can be changed without affecting the parameters of the test. Preferably, the
concentrations of the patient sera, the capture antibody (e.g., mAb 426C) and
the
detecting antibodies (i.e., anti-human IgG antibody or anti-human IgA antibody
or anti-
1o human IgM antibody).
Results
Sera/plasma from 45 South African subjects with confirmed TB were screened and
compared with 19 (black) control sera/plasma and 14 (white) control
sera/plasma.
Three other South African sera/plasma were also included that had been
diagnosed with
diseases other than TB. A substantial number of the 45 TB sera tested detected
levels
of immune complexes comprising GS at greater than 3 standard deviations above
control average. Furthermore, of the 36 non-TB sera/plasma, one was greater
than 3
standard deviations above control average indicating that that the assay a
high level of
specificity.
When the limit was set at two standard deviations the true positive rate was
substantially increased while the false positive rate did not change
substantially.
Sera/plasma from 49 Chinese subjects with clinically-confirmed TB were also
screened
using the ELISA assay. Again this assay detected increased levels (greater
than 2 or 3
times standard deviation of the control average) of CIC comprising GS in TB
subjects.
Furthermore, or the 41 of non-TB subjects only 5 returned readings greater
than 2 or 3
standard deviations above control average indicating that that the assay a
high level of
specificity.
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These results clearly indicate that the monoclonal antibody 426C is specific
for GS of
M. tuberculosis and does not cross react with human proteins to a significant
degree.
EXAMPLE 23
Antigen-based diagnosis of tuberculosis or infection by M. tuberculosis:
Point-of-care test for diagnosing an active infection
by 111 tuberculosis using mAb 426
1o Monoclonal antibody 426C is striped onto a nitrocellulose membrane at a
concentration
of bet`veen about 0.5 and about 4 mg/ ml. The nitrocellulose membrane is
allowed to
dry at 40 C for 20 minutes. The nitrocellulose sheet is then cut into a 1 cm x
1 cm
squares and inserted into the base of the DiagnostIQ device (Proteome Systems
Ltd) on
top of a cellulose pad. The Pre-incubation frame is attached to the base and
the test
performed according to the procedure below.
1. About l 00ul to about 500u1 of patient 'or control seralplasma are added to
the
pre-incubation well of the DiagnostIQ format with 150ul of gold conjugated to
an anti-
human IgG and/or IgA antibody.
2. The sera/plasma are incubated with the nitrocellulose strip membrane for 30
seconds and the pre-incubation frame is pushed down onto the base of the test.
3. After about 1 minute, 2-4 drops of wash solution (0.5% Tween 20 in 0.1M
phosphate buffer) is added to the pre-incubation well and allowed to flow
through the
device.
4. The pre-incubation frame is removed and the signal read by visually
interpreted
or read in a Readrite optical reader.
In a modification of this example, additional antibodies targeted against
other specific
epitopes on the same or different M. tuberculosis antigen are employed
alongside mAb
426C. Additionally, the present invention clearly encompasses conjugation of
the anti-
IgG and/or anti-IgA antibody to the same gold particle to ensure the same
amount of
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label is applied in each test. The gold particles may also be dried onto the
pre-
incubation pads, to thereby avoid the later addition of conjugate. Sensitivity
of the
assay may also be improved by increasing the amount of sera tested in each
sample.
EXAMPLE 24
Isolation of monoclonal antibodies that bind to M. tuberculosis GS peptide
1. Antibody production
lo Antigen
Approximately 6 mgs of peptide antigen consisting of the sequence
WASGYRGLTPASDYNIDYAIC (set forth in SEQ ID NO: 22) conjugated to
diphtheria toxoid is provided to NeoClone, Madison, Wisconsin, USA for
generation of
monoclonal antibodies according to their standard protocol. About 1 mg of the
peptide
is also provided as biotinylated peptide for quality control.
Inamunizatiora
Five BALB/cByJ female mice are immunized with peptide conjugated to carrier
according to Neocione's standard immunization process.
Test Bleeds
Test bleeds of the immunized mice are performed at regular intervals for use
in the
quality control sera ELISAs using biotinylated peptide. Polyclonal sera having
the
highest titer are determined using ELISA. Mice having polyclonal antibody
titers of at
least 1,000 are used for the ABL-MYC infection process.
Infection
The spleens of 3 mice having the highest titer of polyclonal antibodies cross-
reactive
with peptide antigen are used for the ABL-MYC infection, according to
NeoClone's
standard infection procedure.
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Ti=ansplantation
The splenocytes of the ABL-MYC-infected mice are transplanted into
approximately
20 naive mice.
Ascites developnaent
Ascited fluid developed in the transplanted mice is isolated and screened for
cells
producing monoclonal antibodies (mAbs) that bind to the target peptide
antigen. Cell
lines (i.e., plasmacytoma) producing mAbs that bind to the peptide antigen are
isolated.
io Binding affinity and isotype specificity of the mAbs is confirmed using
ELISA.
A mAb that binds to the peptide antigen are is purified from ascites using
protein G or
protein A columns.
Antibody titration is performed essentially as described in the preceding
examples.
