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 The present invention relates to novel diagnostic, prognostic and therapeutic reagents for infection of an animal subject such as a human by M. 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. tuberculosis designated "S9" (SEQ ID NO: 1) and immunogenic epitopes thereof suitable for the preparation of immunological reagents, such as, for example, antigenic proteins/peptides anαVoτ antibodies, for the diagnosis, prognosis and therapy of infection, and vaccine development.

Background of the invention /. General Information

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.

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 mutatis mutandis 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 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.

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 VoIs I,

II, and III;

2. DNA Cloning: A Practical Approach, VoIs. 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 α/., 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) ERJL 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). Biochem. Biophys. Res. Commun. 73 336-342

10. Merrifield, R.B. (1963). J. Am. Chem. Soc. 85, 2149-2154.

1 1. Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York.

12. Wϋnsch, E., ed. (1974) Synthese von Peptiden in Honben-Weyls Metoden der Organischen Chemie (Muler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart.

13. Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer- Verlag, Heidelberg.

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, VoIs. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications).

17. Wilkins M. R., Williams K. L., Appel R. D. and Hochstrasser (Eds) 1997 Proteome Research: New Frontiers in Functional Genomics Springer, Berlin.

2. Description of the related art

Tuberculosis is a chronic, infectious disease that is generally caused by infection with Mycobacterium 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, M. 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.

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.

The incidence of tuberculosis is especially common in late-staging AIDS patients, a majority of whom 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 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-I, and M. tuberculosis mediate a shortened HF/ 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 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. tuberculosis indicate that any particular protein encoded or actually expressed by the bacterium comprises any immunodominant B-cell epitopes or 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 M. tuberculosis or a peptide fragment derived there from has efficacy as a diagnostic reagent 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 environment is

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 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 environment 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

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. tuberculosis 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 M. tuberculosis in a range of in vivo environments, to thereby identify highly 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

determined by mass spectrometry, and shown to align to the amino acid sequence of the 3OS 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).

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 ED 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, and S9 protein in the cytosol and membrane fractions of Mycobacterium 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. tuberculosis 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 TB subjects were detected using an antibody that binds to the protein (i.e., 100%

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. tuberculosis 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 ED 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

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 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, 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 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 Mycobacterium 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 ED 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.

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 amino acid residues positioned between about residue 1 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 extension 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 Mycobacterium 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, β-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

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, β-galactosidase, thioredoxin (TRX) (La Vallie et ah, Bio/Technology 11, 187-193, 1993), maltose binding protein (MBP), Escherichia coli NusA protein (Fayard, E.M.S., Thesis, University of Oklahoma, USA, 1999; Harrison, inNovations 11, 4-7, 2000), E. coli BFR (Harrison, inNovations 11, A- 7, 2000) and E. coli GrpE (Harrison, inNovations 11, A-I, 2000).

The present invention also provides an isolated protein aggregate comprising one or 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 described herein for detecting a past or present infection or latent infection by. M,

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 S 9 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 S9.

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 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. tuberculosis.

The present invention also provides a pharmaceutical composition comprising 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 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 Mycobacterium 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.

The present invention also provides a cell expressing 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. The cell may preferably consist of an antigen- presenting cell (APC) that expresses the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof e.g., on its surface.

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 Mycobacterium 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. 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, tuberculosis in a subject, wherein said infection is determined by

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 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 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 M. tuberculosis 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.

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 M. tuberculosis infection.

The present invention also provides a composition comprising the isolated ligand according to any embodiment described herein, especially any peptide ligand, antibody

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 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 Mycobacterium 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 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. 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.

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 a M. tuberculosis 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 NO: 12), AKALRVSAEVLYVRA (SEQ ID NO: 13), VRAGILEPSETSQVR (SEQ ID No: 14), TAITERQKQILLDIY (SEQ ID NO; 15), SQIAKALRVS AEVLYVRAC (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 immunogenic 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. tuberculosis 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.

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 enzymes.

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 temporaiy or alternatively may spread by extension to become an acute or chronic clinical infection. The infection may 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 "extrapulmonary" 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

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 endogenous S9 protein of Mycobacterium tuberculosis or an immunogenic 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.,

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: 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), SQIAKALRVSAEVLY VRAC (SEQ ID NO: 16), MSSEEKLCDPTPTDD (SEQ ID NO: 17) and VRAGILEPSETSQVRC (SEQ ID NO: 18). Antibodies that bind to an immunogenic M. tuberculosis Bsx 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.

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 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. 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 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 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 Mycobacterium, 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., HF/-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., "HTV+"). 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 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, 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- 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 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., HFV-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 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 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. 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 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., HFV-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, responsiveness to therapy or infection status of the subject. In a preferred embodiment,

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 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., HlV-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; 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

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., 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 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 Mycobacterium 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.

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 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 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 M. 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 herein in its entirety; and antibodies that bind to M, tuberculosis GS peptides 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 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 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 β-galactosidase or β-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. tuberculosis comprising: (i) performing a diagnostic method according 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. tuberculosis comprising:

(i) performing a diagnostic method according to any embodiment described herein thereby detecting the presence of M. tuberculosis infection in a biological

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 comprising:

(i) 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.

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 immμnogenic 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 M. 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.

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 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 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 S 9 protein or an immunogenic S9 peptide or immunogenic 89 fragment or epitope thereof is formulated into a DNA vaccine, such as, for example, in combination with the existing Calmette- Guerin (BCG) or an immune adjuvant such as vaccinia virus, Freund's adjuvant or 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-I and/or HIV-2, including the therapeutic treatment of a latent M. tuberculosis 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

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 HIV-I and/or HIV-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 Mycobacterium 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 Mycobacterium tuberculosis 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.

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 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 1 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.

Figure 2 is a graphical representation showing the titration of polyclonal antibody R9 its corresponding biotinylated peptide coated onto a 5 μg/ml streptavidin plate at 3 μg/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 μg/ml of antibody. Solid squares represent 20 μg/ml of antibody. Grey triangles represent 10 μg/ml of antibody. Grey squares represent 0 μg/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.

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. tuberculosis ribosomal protein S9 for the immunizing antigen, as determined by ELISA. Recombinant S9 protein was diluted serially 1:2 (v/v) from 500 ng/ml starting concentration to 7.8 ng/ml, and 50 μl aliquots

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 Mol025F) with recombinant full-length S9 protein were contacted separately with the adsorbed antigen at a concentration of 5 μg/ml. Following incubation at room temperature for 1 hour, plates were washed, incubated with 50 μl 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 Mol025F antibody) conjugated to horseradish peroxidase (HRP), washed, incubated with TMB for 30 mins, and absorbance at 595-600 nm was deteπnined (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 Mol025F as detection antibody for assaying recombinant M. tuberculosis ribosomal protein S9. An ELISA plate was coated overnight with capture antibody Ch27 at 5 μg/ml and 2.5 μg/ml concentrations. Following washing to remove unbound antibody, recombinant S9 protein was diluted serially 1 :2 (v/v) from 500 ng/ml starting concentration to 7.8 ng/ml, and 50 μl 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 Mol025F was contacted with the bound antigen-body complexes at concentrations in the range of 1.25 μg/ml to 5 μg/ml. Following incubation at room temperature for 1 hour, plates were washed, incubated with 50 μl 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 μg/ml with detection antibody in the concentration range of 1.25 μg/ml to 5 μg/ml, which conditions provided a half-maximum detection of about 24 ng/ml M. tuberculosis ribosomal protein S9.

Figure 7 is a graphical representation showing sandwich ELISA results using antibody Mol025F as capture antibody and antibody Ch27 as detection antibody for assaying recombinant M. tuberculosis ribosomal protein S9. An ELISA plate was coated overnight with capture antibody Mol025F at 5 μg/ml and 2.5 μg/ml concentrations. Following washing to remove unbound antibody, recombinant S9 protein was diluted serially 1 :2 (v/v) from 500 ng/ml starting concentration to 7.8 ng/ml, and 50 μl 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 Ch27 was contacted with the bound antigen-body complexes at concentrations in the range of 1.25 μg/ml to 5 μg/ml. Following incubation at room temperature for 1 hour, plates were washed, incubated with 50 μl 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 μg/ml or 5 μg/ml with detection antibody at a concentration of 5 μg/ml under the conditions tested.

Figure 8 is a graphical representation showing sandwich ELISA results using antibody Ch27 as capture antibody, antibody Mo 1025 F as detection antibody and an HRP- conjugated secondary antibody, for assaying low concentrations of recombinant M. tuberculosis ribosomal protein S9. An ELISA plate was coated overnight with capture antibody Ch27 at 5 μg/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 μl 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 Mol025F was contacted with the bound antigen-body complexes at 2.5 μg/ml concentration. Following incubation at room temperature for 1 hour, plates were washed, incubated with 50 μl 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, a detection limit of 996 pg/ml M. tuberculosis ribosomal protein S9, and half-maximum detection of about 28 ng/ml M. tuberculosis 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 Ch27 as capture antibody, antibody Mol025F 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-antibody 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 Mol025F as detection antibody, a biotinylated secondary antibody and iterative antigen binding ( also termed herein "replacement amplification") for assaying low concentrations of recombinant M. tuberculosis 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 μg/ml starting concentration to 0.238 fg/ml concentration (x-axis); and antigen binding

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 about 84 pg/ml M. tuberculosis 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 1 1 is a graphical representation of sandwich ELISA results showing lack of significant cross-reactivity of antibodies against M, tuberculosis ribosomal protein S9 with Escherichia coli, Bacillus subtilis or Pseudomonas aeruginosa. 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 μg/ml of a cellular extract as indicated on the x-axis. As a positive control, cellular extract from the M, tuberculosis laboratory strain H37R.V was used. As a negative control for each assay, buffer without cellular extract was used. Data show the change in absorbance at 595-600nm i.e., following subtraction of background absorbance for each sample. Error bars show one standard deviation from the mean (n=3).

Figure 12 is a graphical representation of sandwich ELISA results showing detection of M. tuberculosis ribosomal protein S9 in the clinical M. tuberculosis isolate CSU93, and lack of signal suppression in plasma. Assay conditions were essentially as described in the legend to Figure 1 1 except that cellular extracts were from M. tuberculosis laboratory strain H37Rv and CSU93, as indicated on the x-axis. For determination of 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

plasma does not suppress signal in this assay, and that the assay is capable of detecting both clinical and laboratory isolates of M. tuberculosis.

Figure 13a is a copy of a photographic representation showing a Western blot to detect M. tuberculosis 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 HFV 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.

Figure 13b is a copy of a photographic representation showing a Western blot to detect M. tuberculosis 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 M. 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.

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 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 403 B 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 μg/ml using 50 μl 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 μg/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.

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 Rl 6 was immobilised onto an ELISA plate at a concentration of 20 μg/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 p Ab C44 was added at a concentration of 5 μg/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 μg/ml followed by donkey anti-chicken IgG biotin 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 Rl 6 was immobilised onto an ELISA plate as a capture antibody at a concentration of either 20 or 40 μg/ml. Titrating amounts of recombinant BSX from 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 μg/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

plate as a capture antibody at a concentration of 20 μg/ml. Purified chicken anti-BSX pAb, C44, at a concentration of 5 μg/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 Rl 6 was immobilised onto an ELISA plate as the capture antibody at a concentration of 20 μg/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 1 ng/ml 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 μg/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.

Detailed description of the preferred embodiments

Isolated or recombinant S9 protein and immunogenic fragments 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.

As used herein, the term "S9" shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ED NO: 1. Preferably, the percentage identity of a S9 protein to SEQ ED NO: 1 is at least about 85%, more preferably at least about 90%, even more 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 determined 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 ah, Nucl. Acids Res. 12, 387-395, 1984, which utilizes the algorithm of Needleman and Wunsch, J. MoI. 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 maximise 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.

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. tuberculosis, and more preferably, a high 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-γ expression by a T cell, such as, for example, CDS ⁺ 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

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 M. 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 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 affecting 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 disturbing the overall structure or antigenicity of the peptide. Such changes are therefore termed "conservative" changes and tend to rely on the hydrophiliciry 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 higher 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.

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 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 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. MoI. Biol. 157, 105-132, 1982). 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);

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 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 (- 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 Am Chem Soc, S5,: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,

R.B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York; Wϋnsch, E., ed. (1974) Synthese von Peptiden in Houben- Weyls Metoden der Organischen Chemie (Mϋler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer- Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice 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 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 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 hapten, an antibiotic, hormone, steroid, 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, tetrodotoxin), or a complex-forming agent. Biotinylated peptides are especially preferred.

In another embodiment, a S9 protein is produced as a recombinant protein.

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.

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 β- 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 derivative which confers, activates or enhances the expression of a nucleic acid

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 lac∑ promoter, temperature-sensitive λ _L or λR promoters, T7 promoter or the EPTG-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 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, pKC30 (λ _L : Shimatake and Rosenberg, Nature 292, 128, 1981); pKK173- 3 (tac: Amann and Brosius, Gene 40, 183, 1985), pET-3 (T7: Studier and Moffat, J. MoI. 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.

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, 1OT 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 pSRαtkneo (Muller et al, MoI. Cell. Biol, 11, 17S5, 1991). The vector pcDNA 3.1 myc-His (Invitrogen) is particularly preferred for expressing a secreted form of a S9 protein or a 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 bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.

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 recombinant secondary anatyte protein, peptides and epitopes thereof

It is to be understood that methods described herein above for the production of isolated and recombinant S9 protein or immunogenic fragments thereof apply mutatis mutandis 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 formulation, etc. As will be understood by the skilled artisan, such extrapolation is dependent on substituting the S9 immunogen for the secondary 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- based tests, will comprise an amino acid sequence selected from the group set forth in SEQ ID NOs: 8-19.

For example, the M. tuberculosis 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

(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). Methods for producing such fragments are 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, M. tuberculosis glutamine synthetase (GS) 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. 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 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.

Antibodies that bind to a S9 protein or an epitope thereof

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 vivo in suitable laboratory animals, or, in the case of engineered antibodies (Single Chain Antibodies or SCABS, etc) using recombinant DNA techniques in vitro.

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 immunization 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 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. 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 10 ³ -10 ⁴ is preferred. More preferably, the antibody titer will be in the range from about 10 ⁴ to about 10 ⁵ , even more preferably in the range from about 10 ⁵ to about 10 ⁶ .

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, 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 Corynebacteήwn 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,

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 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, MoI. Immunol 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-maleimidornethyl) cyclohexane-1-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

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 Corynebacterium diphtheriae grown in submerged culture by standard methods. The production of Diphtheria Toxoid is divided into five stages, namely maintenance of the working seed, growth of Cojynebacterium 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

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).

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.

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 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 immunization, somatic cells with the potential for producing antibodies, 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. 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 10 ⁷ to 2 x 10 ⁸ lymphocytes.

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- Ag8.653, NSl/l .Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-I l, MPCl 1-X45-GTG 1.7 and S194/5XX0; 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-I myeloma cell line (also termed P3-NS-l-Ag4-l), 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, Eur. J. Immunol 6, 51 1-519, 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

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 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, enzyme 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 may 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

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 at least about 1,000 are used for subsequent ABL-MYC infection employing replication-incompetent retrovirus comprising the oncogenes v-abl and c-myc. 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 method 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, Ciirr. Top. Microbiol. Immunol., 166, 91-96. 1990;

2. Weissinger et ai.Proc. Natl. Acad. ScL USA, 88, 8735-8739, 1991; 3. Largaespada et al, Oncogene, 7, 811-819, 1992;

4. Weissinger et al, J. Immunol. Methods 168, 123-130, 1994;

5. Largaespada et al, J. Immunol. Methods. 197(1-2), 85-95, 1996; and

6. Kumar et al, Immiino. 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 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") 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

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 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 mammals 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 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 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 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, ³ H, ¹²⁵ I, . ³² P, . ³⁵ S, ¹⁴ C, ⁵¹ Cr, ³⁶ Cl, ⁵⁷ Co, ⁵⁸ Co, ^5Q Fe,

⁷⁵ Se, and ¹⁵² Eu.

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-known 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. Monoclonal antibodies according to the invention may be labelled with technetium" 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 SNCl ₂ , 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 enzyme 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 antigen composition comprising the B cell epitope, preferably in the context of the

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 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.

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-specifϊcally 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 amplified using secondary and preferably tertiary, antibodies that bind to the antibodies recognising the B cell epitope. Detection is then achieved by the addition of 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 exhibiting 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.

mmooαies 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 analyte 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 mutatis mutandis to the production of antibodies against a secondary analyte that is to be used in an immunoassay format e.g., for the purposes of diagnosis or prognosis of tuberculosis or infection by M. tuberculosis. As will be understood by the skilled artisan, such extrapolation is dependent on substituting the S9 immunogen for the secondary 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 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: S- 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), LRKPSAD VLSQIAKA (SEQ ID NO: 10), PSADVLSQIAKALRV (SEQ ID NO: 11), SQIAKALRVS AEVLY (SEQ ID NO: 12), AKALRVSAEVLYVRA (SEQ ID NO: 13), VRAGILEPSETSQVR (SEQ ID No: 14), TAITERQKQILLD IY (SEQ ID NO; 15), SQIAKALR VSAEVLYVRAC (SEQ ID NO: 16), MSSEEKLCDPTPTDD (SEQ ID NO: 17) and VRAGILEPSETSQVRC (SEQ ED NO: 18). Antibodies that bind to an immunogenic M. tuberculosis Bsx

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.

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 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.

The present invention clearly contemplates antibodies agaisnt secondary analytes other than Bsx or Gs or immunogenic fragments thereof, the description of which is provided for the purposes of exemplification.

Diagnostic/prognostic methods for detecting tuberculosis or M. tuberculosis infection

1. Antigen-based assays

This 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 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 assay is that severely immune-compromized patients may not produce antibody at

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. tuberculosis 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.

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 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. 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 HFV+ 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.

In one embodiment, a S9 protein or immunogenic fragment 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 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

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., 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 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 Mycobacterium 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. tuberculosis S9 protein can be combined with assays for detecting M. tuberculosis 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.

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 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 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. However, antibody-based assays are clearly useful for detecting M. tuberculosis infections in HFV ^" or HIV ⁺ individuals who are not immune-compromized.

In one alternative embodiment, the present invention provides a method for detecting M. tuberculosis infection in a subject, the method comprising contacting a biological 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 M. tuberculosis in a 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 symptoms of tuberculosis or an infection indicates that the subject is recovering from

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 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. tuberculosis 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. tuberculosis 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

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 the analyte (e.g., blood, serum, plasma, or any immunoglobulin-containing sample).

Suitable samples 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 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 solution 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

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), 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 β-galactosidase.

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 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 any 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

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 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 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 immune 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 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.

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 microarray format as described in Mendoza et al, Biotechniques 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 radiolabeled 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

said antibody. To detect the bound antigen, an isolated and/or recombinant form of the antigen is radiolabeled 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 radiolabeled 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 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 (λr 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 S 9 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

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, 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 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.

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 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 a 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

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 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 21: 1155-1 163, 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.

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

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, and peptides recovered by avidin affinity chromatography. Samples are then analysed using mass spectrometry. Any difference in peak heights between the heavy 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 electrophoresis. 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 distinct but matched samples. As used herein the term "matched samples" shall be

understood to mean two 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 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-I and/or HIV-2 antigens. Such assays are particularly useful for simultaneously obtaining information on co-infection with M. tuberculosis and HIV-I and/or HIV-2, and/or for determining whether or not a subject with M. tuberculosis 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.

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.

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, J Allergy CHn Immunol, 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

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.

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 ₅ a neutrophil, a monocyte _. , or any immunoglobulin-containing fraction of any one or more of said tissues, fluids or cells.

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 embodiment, 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

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. 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.

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

ID NOs: 1, 2, 3, 4, 5, 6 and 7; and (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 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.

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).

Prophylactic and therapeutic 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 M. 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. tuberculosis.

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. tubercidosis . 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.

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 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.

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 method 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 S 9 protein or epitope for a time and under conditions sufficient to confer or enhance resistance against M. tuberculosis in said subject.

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 confer or enhance resistance against M. tuberculosis 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 of M. 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. tuberculosis in an uninfected human subject comprising administering to said subject 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 provide immunological memory against a future infection by M. 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 immunological 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 amount 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. 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.

Preferably, the composition is administered to a subject harboring a latent or active M. 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. tuberculosis, or that the ability of a T cell to recognize a T cell epitope of an antigen of M. tuberculosis is enhanced, either transiently or in a sustained manner. Similarly, reactivation of a T cell population may occur following activation of a latent M. tuberculosis 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 in the subject, such as, for example, using cytotoxicity assays, ELISPOT, or determining IFN -γ 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 CDS ⁺ T 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 "M. tuberculosis -specific CMI" is meant that the activated and clonally expanded 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- specific responses are still being established. In contrast, MHC-restricted CTLs achieve

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. tuberculosis, 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, BRJvI, 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.

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. tuberculosis 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 M. 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 M. tuberculosis infection or a person who is

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 ai, Blood 99, 3916-3922, 2002, which procedures are incorporated herein by reference.

By "M. tuberculosis 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 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 thymus) 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 sample comprising the T cell of the subject, and more preferably, obtaining said sample from said subject.

Formulations

The present invention clearly contemplates the use of the S9 protein or an immunogenic fragment or epitope thereof in the preparation of a therapeutic or prophylactic subunit vaccine against M. 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 embodiment, 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.

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.

In certain situations, it may also be desirable to formulate the S9 protein and optional other protein or an immunogenic fragment 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 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 1 1637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'- dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP) 1983A, referred 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 described for example by Elhay and Andersen Immunol. Cell Biol. 75, 595-603, 1997; or Lindblad et ah, Infect. Immun. 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 activity. Exemplar}' 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 ² ) (Johnson/Mead, NJ, USA).

Preferred vehicles for administration of the S9 protein and optional other protein, or 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. CHn. 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 administered safely and are biodegradable.

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 μm to greater than 10 μm. A variety of agents are encapsulated in liposomes. Hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s) (Machy et al., LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John Libbey 1987), and Ostro et al, American J. Hosp. Pharm. 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. ScL 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.

Cationic liposome preparations are made by conventional methodologies (Feigner et at, Proc. Nat'l Acad. Sci USA 84, 7413 (19S7); Schreier, Liposome 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), large unilamellar vesicles (LUV), giant unilamellar vesicles (GUV), multivesicular vesicles (MW), 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 bubblesomes (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 Dekker, 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.

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, such as, for example, in combination with the existing Calmette-Guerin (BCG) or an immune 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 1 Preparation of serum or plasma

Patient serum or plasma is applied to a column of protein G-Sepharose™ (Amersham Biosciences), previously equilibrated with 2OmM phosphate buffer pH 7.0 and incubated on ice with occasional inversion. The mixture is centrifuged at 600Og for 10 minutes at 4 ⁰ C and the supernatant decanted. The Sepharose™ pellet is washed with 2OmM phosphate buffer. The IgG bound to the Sepharose™ is eluted by addition of 5OmM 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 -2O ⁰ C for 48h then centrifuged at 5000g for 20mins at 4 ⁰ C. The precipitate is resolubilised in l-2mls of sample buffer containing 5M urea, 2M thiourea, 2% CHAPS, 2% SB3-10 and 4OmM Tris, then simultaneously reduced with 5mM tributyl phosphine and alkylated with 1OmM acrylamide for Ih.

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 4OmM Tris with 5mM Tris.

Prior to rehydration of EPG strips, samples are centrifuged at 21000 x g for 10 minutes. The supernatant is collected and lOμl of 1% Orange G (Sigma) per ml added as an indicator dye.

Two-dimensional gel electrophoresis First Dimension

Dry 1 1cm EPG strips (Amersham-Biosciences) are rehydrated for 16-24 hours with 180μl of protein sample. Rehydrated strips are focussed on a Protean IEF Cell (B io- Rad, Hercules, CA) or Proteome System's IsoElectrIQ electrophoresis equipment for approx 140 kVhr at a maximum of 10 kV. Focussed strips are then equilibrated in urea/SDS/Tris-HCl/bromophenol blue buffer.

Second Dimension

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.

Mass Spectrometry:

Prior to mass spectrometry protein samples are prepared by in-gel tryptic digestion. Protein gel pieces are excised, destained, digested and desalted using an Xcise™, an excision/liquid handling robot (Proteome Systems, Sydney, Australia and Shimadzu- Biotech, Kyoto, Japan) in association with the Montage In-GeI 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 Xcise™, a digital 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 μl 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 μl of 20 mM ammonium bicarbonate (pH 7.8) containing 5 μg/ml modified porcine trypsin and incubated at 3O ⁰ C overnight.

Ten μl of the tryptic peptide mixture is removed to a clean microtiter plate in the event that additional analysis by Liquid Chromatography (LC) - Electrospray Ionisation (ESI) MS was required.

Automated desalting and concentration of tryptic peptides prior to MALDI-TOF MS is performed using Cl 8 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 μl of 2 mg/ml α-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, LIK) in positive ion reflectron mode. A nitrogen laser with a wavelength of

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 BioinfbrmatIQ ^T (Proteome Systems), is used to extract isotopic peaks from MS spectra.

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 IonlQ™ or MASCOT™ 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 μl) 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 PepFinder kit (Thermo-Finnigan) coupled to a Cl 8 PicoFrit column (New Objective).

Gradient elution from water containing 0.1% (v/v) formic acid (mobile phase A) to 90% (v/v) acetonitrile containing 0.1% (v/v) formic acid (mobile 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.

Bioinformatic 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 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 IonlQ™ v69 and/or MASCOT™. PSD data is searched against the same databases using the in-house search engine FragmentastIQ™. 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. tuberculosis infection

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 designated as "S9". Interestingly, the estimated molecular weight of the S9 protein is

only about 16.4 kDa, and the estimated isoelectric point of S9 is about 10.7. Since the observed 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 Synthesis ofS9 Peptides

A synthetic peptide comprising the sequence H-MTETT PAPQT PAAPA GPAQS FC- NH ₂ from 3OS 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 A rabbit was injected with 600μg per dose of the synthetic peptide comprising the amino acid sequence H-MTETT PAPQT PAAPA GPAQS FC-NH ₂ linked to KHL according to the following injection protocol:

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 titration

Streptavidin (Sigma Aldrich) was diluted to 5 μg/ml in ddH ₂ O and incubated in a Nunc plate overnight at 4 ⁰ C. The solution was then flicked out and 250μL of blocking buffer (1% (w/v) casein, 0.1% (v/v) Tween 20, 0.1% (w/v) sodium azide in PBS) added to 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 μg/ml (50 μl/well) and incubated for one hour at room temperature on a shaker. The plate was washed in an Elx405 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 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 50μL/well of 0.5M sulphuric acid. The optical density of each well was read with a micrόtiter plate reader (PowerWaveχ 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.

Peptide titration

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 μg/ml, 20 μg/ml and 40 μg/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 20 TB subjects and 20 subjects suffering from a non-TB subject. Briefly, sputum (12μl) from TB or non-TB patients was loaded onto 4-12% ID gradient SDS 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 μg/ml purified rabbit anti-S9 polyclonal antibody solution (i.e., R9) at RT for 2 hr, following by 3 x lOmin washes 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).

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. tuberculosis 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 samples, reduced membrane samples and non-reduced cytosolic/membrane samples. Accordingly, ribosomal protein S9 is expressed by M. 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 μl per well at a concentration of 5 μg/ml. Wells were incubated with appropriate peptides at 3 μg/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

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 M. 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 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 "Mol025F". 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 Mol025F prepared against recombinant M. tuberculosis ribosomal protein S9 are capable of detecting recombinant S9 protein by standard ELISA, and suggest that the mouse antibody Mol025F 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).

EXAMPLE 9

Sandwich ELISA for detecting M. tuberculosis S9 protein using antibodies prepared against recombinant S9 protein

A sandwich ELISA was developed employing two antibodies prepared against recombinant M. tuberculosis ribosomal protein S9, in particular the chicken polyclonal antibody designated Ch27 and the mouse antibody Mol025F (Example 8),

1. Preferred antibody orientation 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 Mol025F antibodies at 2.5 μg/ml and 5 μg/ml concentrations in blocking buffer. Following washing to remove unbound antibody, 50 μl aliquots of recombinant S9 protein, diluted serially in blocking buffer 1 :2 (v/v) from 500 ng/ml 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., Mol025F for detection of Ch27-S9 complexes and Ch27 for detection of Mol025F-S9 complexes) was contacted with the plates at concentrations in the range of 1.25 μg/ml to 5 μg/ml. Following incubation at room temperature for 1 hour, plates were washed as before, incubated with 50μl 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 Mol025F 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.

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 about 28 ng/ml M. tuberculosis 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 about 150 pg/ml recombinant M. tuberculosis 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 μl volume limitations of a 96-well ELISA plate. Briefly, this iterative antigen loading involves repeating the antigen binding step in the sandwich ELISA several times, e.g., 2 or 3 or 4 or 5 times,

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 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 and streptavidin poly-40 horseradish peroxidase (HRJP) 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

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 antibody Mol025F 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 Pseudomonas aeruginosa 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 cross-reactivities in sandwich ELISA performed as described in Example 9 between cellular extracts of M. tuberculosis strain H37Rv (a laboratory strain), Escherichia coli, Bacillus subtilis or Pseudomonas aeruginosa.

Briefly, an ELISA plate was coated overnight with capture antibody Ch27 at 5 μg/ml concentration. Following washing to remove unbound antibody, 500 ng/ml or 50 μg/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 Mol025F was contacted with the bound antigen-body complexes at 2.5 μg/ml concentration. Following incubation at room temperature for 1 hour, plates were washed, incubated with 50 μl 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 in this experiment.

Data presented in Figure 11 show low cross-reactivity of antibodies against M. tuberculosis ribosomal protein S9 with Escherichia coli, Bacillus subtilis or Pseudomonas 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 M. tuberculosis strain CSU93 and the laboratory M. tuberculosis strain H37Rv.

Briefly, an ELISA plate was coated overnight with capture antibody Ch27 at 5 μg/ml concentration. Following washing to remove unbound antibody, 500 ng/ml or 50 μg/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 Mol025F was contacted with the bound antigen-body complexes at 2.5 μg/ml concentration. Following incubation at room temperature for 1 hour, plates were washed, incubated with 50 μl 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.

Data presented in Figure 12 show that M. tuberculosis ribosomal protein S9 is present in both the clinical M. 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 (12ul) from TB and non-TB patients was loaded onto 4-12% ID 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 μg/ml purified chicken anti-BSX pAb solution at RT for 2 hr, following by 3 x lOmin 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 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.

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 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.

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 Sepharose™, and the flow through fraction were loaded onto a 4-12% ID 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 IX PBS, 0.1% Tween-20 (PBST) at room temperature (RT) for 2 hours. Membranes were then incubated with 10 μg/ml purified chicken anti-BSX polyclonal antibody described in Example 12 at RT for 2 hr, following by 3x lOmin 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 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, namely rabbit polyclonal anti-BSX antibody (raised against a BSX peptide) designated

R 16, 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 R 16, and mouse (Mo) anti-BSX mAb 403B all at 20 μg/ml using 50 μl 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 μg/ml (with and without pre-incubation 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 μg/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

Determining the detection limit 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 μg/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 μg/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.

Under these conditions, the limit of detection of recombinant BSX was ~ 2-3 ng/ml.

Detecting BSX in patient samples

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, Rl 6, was immobilised onto the ELISA plate as a Capture antibody at a concentration of 20 μg/ml. Purified chicken anti-BSX pAb, C44, at a concentration of 5 μg/ml, was used as 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 μg/ml or purified chicken anti-BSX pAb C44 at a concentration of 5 μg/ml using 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 μg/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 μg/ml followed by sheep anti-chicken IgG HRP conjugate at a 1/5000 dilution.

As shown in Figure 18, 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 Rl 6 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 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 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.

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 M. tuberculosis

1. Sera and peptides

A total of 30 TB-positive samples and 52 TB-negative samples were screened with the 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: 1 1), SQIAKALRVSAEVLY (SEQ ID

NO: 12), AKALRVSAEVLYYRA (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). In each case, the peptides were biotinylated to facilitate their detection.

These samples included sera from South African (S. A.) Zulu TB-positive individuals, 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 Assay Nunc-Immuno module maxisorp wells were coated overnight at room temperature or at 4 ⁰ C over the weekend with lOOμl/well of 5μg/ml streptavidin diluted in milli-Q water. The streptavidin was flicked out of the wells and each well was blocked with 200μl phosphate-buffered saline (PBS) containing 1.0% (w/v) casein, 0.1% (v/v) Tween 20

and 0.1% (w/v) Azide (blocker) per well. After 1 hour, the blocker was removed, and each well was coated with lOOμl 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 50μl 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 lOOμl 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% (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, lOOμl 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 lOOμl 0.5M 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 24/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.

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 HFV ⁺ . 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 HFV ^" 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 number of immunogenic Bsx peptides containing B cell epitopes of the full-length Bsx protein of M. 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 M. tuberculosis 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 HFV status and/or monitoring the TB status in HFV ⁺ individuals. To further emphasise the correlation between TB and HFV, it is important to note that all of the Chinese samples investigated were HFV ^" negative.

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 peptides 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.

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. ELlSA Assay

Peptides comprising SEQ ID NOs: 16-18 were coated on ELISA trays at 3 μg/ml on a streptavidin base of 5 μg/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

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 selectively identifies antibodies that bind to M. tuberculosis 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 terminal (SEQ ID NO: 17) and 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.

The data indicate that Bsx (23-24) peptide (SEQ ED 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.

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/plasma

Sera and plasma were from 44 TB-positive (smear or culture) Chinese and South African patients (i.e., TB-positive sera), and 44 healthy control subjects (i.e., non-TB sera).

2. ELISA Assay

Recombinant Bsx protein was coated directly onto ELISA trays at 5 μg/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

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., 1 1% compared to 25% at three standard deviations 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 HIV-negative smear or culture subjects (i.e., healthy control sera/plasma).

2. ELISA Assay

Recombinant Bsx protein or Bsx (23-24) peptide (SEQ ED NO: 16) was coated directly onto ELISA trays at 5 μg/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

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 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 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 μg/ml) that has been preadsorbed onto wells of a microtiter pi 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

wells, and (ii) capture of human IgG in the sera by the immobilized protein and peptide is traced using enzyme-linked sheep anti-HulgG 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 determine those GS peptide fragments against which 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 glnA4) was aligned with the amino acid sequence of other known TB glutamine synthetases (glnA, glnA2 and glnA3) 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.

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 studies 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 another glutamine synthetase proteins.

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 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 performed at regular intervals for use in the . quality control sera ELISAs using biotinylated peptide. Polyclonal sera having the 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 20 naive mice.

Ascites development

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 20μl 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 the 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 signahnoise ratio of at least about 2.0. 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 μl. Duplicate aliquots of the monoclonal antibody-producing plasmacytoma designated

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. tuberculosis glutamine synthetase (GS) polypeptide or GS fragments

This example describes an ELISA for the detection of circulating immune complexes (CIC) bound to M. tuberculosis 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 M. 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

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 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, tuberculosis glutamine synthetase at a concentration of 20 μg/ml in water, was coated onto the bottom of one or more NUNC plates. Plates were left to dry at 37 ⁰ 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 (50ul/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-human 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 at room temperature e.g., on a rotating shaker. The plates were washed as before, and

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 H ₂ SO ₄ (50 ul/well). Absorbances of each well was read at wavelengths of 450nm and 620nm, and the differences in these wavelengths is determined (i.e.A _45O -A _< 5 ₂ o).

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- 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.

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 M. tuberculosis using mAb 426

Monoclonal antibody 426C is striped onto a nitrocellulose membrane at a concentration of between about 0.5 and about 4 mg/ ml. The nitrocellulose membrane is allowed to dry at 4O ⁰ 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 lOOul to about 500ul of patient or control sera/plasma 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 0.1 M 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

label is applied in each test. The gold particles may also be dried onto the preincubation 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 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.

Immunization

Five BALB/cByJ female mice are immunized with peptide conjugated to carrier according to Neoclone'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. Potyclonal 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.

Transplantation

The splenocytes of the ABL-MYC-infected mice are transplanted into approximately

20 naive mice.

Ascites development

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, 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.