WO2006000045A1

WO2006000045A1 - Novel methods of diagnosis and treatment of m. tuberculosis infection and reagents therefor i

Info

Publication number: WO2006000045A1
Application number: PCT/AU2005/000930
Authority: WO
Inventors: Robert Alan Cole; Rebecca Louise Harcourt
Original assignee: Proteome Systems Intellectual Property Pty Ltd
Priority date: 2004-06-25
Filing date: 2005-06-24
Publication date: 2006-01-05

Abstract

The present invention provides immunogenic peptides of M. tuberculosis antigens, antibodies thereto, diagnostic kits and assays for determining infection with M. tuberculosis in patient samples, and responsiveness to therapy.

Description

Novel methods of diagnosis and treatment of M. tuberculosis infection and reagents therefor I

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 M. tuberculosis glutamine synthetase (GS) (SEQ ID NO: 1) and immunogenic epitopes thereof suitable for the preparation of immunological reagents, such as, for example, antigenic proteins/peptides and/or antibodies, for the diagnosis, prognosis and therapy of infection, and vaccine development.

Background of the invention 1. 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 ah, pp35-81; Sproat et at, pp 83-115; and Wu et ah, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hanies & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; 5. Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; 6. Perbal, B., A Practical Guide to Molecular Cloning (1984); 7. Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series; 8. J.F. Ramalho Ortigao, "The Chemistry of Peptide Synthesis" In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); 9. Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R.L. (1976). Biochem. Biophys. Res. Commun. 73 336-342 10. Merrifield, R.B. (1963). J. Am. Chem. Soc. 85, 2149-2154. 11. 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. Wύnsch, 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. 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 non-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 HTV-I and M. tuberculosis mediate a shortened HIV symptom-free period and shortened survival time in subjects, possibly by triggering increased viral replication and virus load that results in depletion of CD4+ T-cells and immune deficiency or 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 therefrom 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 in the body fluids of a cohort of diseased patients, including sputum, pleural fluid, plasma and serum. A highly-expressed M. tuberculosis protein was identified in vivo in samples of a cohort of M. tuberculosis-infected patients, having high amino acid sequence identity to a sequence postulated to be encoded by the M. tuberculosis genome. The protein identified by the inventors comprised the amino acid sequence set forth in SEQ ID NO: 1.

The amino acid sequence set forth in SEQ ID NO: 1 was designated "glutamine synthetase A4" (also referred to herein as "glutamine synthetase" or "GS"). Sequence analysis indicated that the sequence of the M. tuberculosis protein GS comprised a region that appears to be a glutamine synthetase catalytic domain in addition to a region that appears to be a GS β-Grasp domain. GS is a key enzyme in nitrogen metabolism having dual functions in two essential biochemical reactions, ammonia assimilation and glutamine biosynthesis. GS condenses ammonia with glutamate, with the aid of ATP, to form glutamine. Glutamine is in turn a source of nitrogen in the biosynthesis of numerous nitrogen-containing metabolites, including amino acids, nucleotides, and amino sugars. The inventors further produced a PEPSET comprising 90 synthetic overlapping peptides (i.e., SEQ ID Nos: 2-91 or 94) covering the amino acid sequence of SEQ ID NO: 1, to determine whether the identified protein was present across geographic and racial boundaries and identify B-cell epitopes for subsequent monoclonal and polyclonal antibody production. TB-negative sera, and sera from TB-positive subjects, including both HIVVTB⁺ and HIV⁺/TB⁺ subjects, were screened for the presence of antibodies to each peptide in the PEPSET. Peptides that are immunogenic in the TB- positive cohort are selected and used in the diagnostic assays and formulations described herein.

Plasmacytomas were also produced that express antibodies against selected peptides. These cytomas are used routinely to produce hybridomas expressing monoclonal antibodies that bind to a B-cell epitope of M tuberculosis glutamine synthase in patient samples, thereby detecting the bacterium.

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 glutamine synthetase solus or as part of a multi-analyte test. Preferably, the GS 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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof. Preferably, the isolated or recombinant immunogenic GS 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 GS peptide is a synthetic peptide. Preferably the GS peptide, fragment or epitope comprises at least about 5 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1, more preferably at least about 10 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1, even more preferably at least about 15 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1, and still more preferably at least about 5 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1 fused to about 1-5 additional amino acid residues at the N-terminus and/or the C-terminus.

In a particularly preferred embodiment, the GS peptide, fragment or epitope comprises an amino acid sequence set forth in any one of SEQ ID Nos: 2-91 or 94 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 GS peptide, fragment or epitope comprises an amino acid sequence of at least about 5 consecutive amino acid residues positioned between about residue 265 to about residue 300 of SEQ ID NO: 1, more preferably at least about 5 consecutive amino acid residues positioned between about residue 270 to about residue 295 of SEQ ID NO: 1, still more preferably at least about 5 consecutive amino acid residues positioned between residue 271 to residue 295 of SEQ ID NO: 1. This corresponds to at least 5 consecutive residues of the sequence set forth in SEQ ID NO: 92, preferably further 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.

Another preferred immunogenic GS peptide, fragment or epitope comprises an amino acid sequence of at least about 5 consecutive amino acid residues positioned between about residue 155 to about residue 185 of SEQ ID NO: 1, more preferably at least about 5 consecutive amino acid residues positioned between about residue 160 to residue 180 of SEQ ID NO: 1 and still more preferably at least about 5 consecutive amino acid residues positioned between about residue 161 to residue 180 of SEQ ID NO: 1. This corresponds to at least 5 consecutive residues of the sequence set forth in SEQ ID NO: 94, preferably further 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.

Alternatively, or in addition, a preferred immunogenic GS peptide or immunogenic GS fragment or epitope comprises an amino acid sequence set forth in SEQ ID NO: 92, 93 or 94 or an immunologically cross-reactive variant thereof comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO: 92, 93 or 94. The regions of GS that correspond a peptide comprising the sequence set forth in SEQ ID NO: 92, 93 or 94 are poorly conserved amongst glutamine synthetase polypeptides (as shown in Figure 1). Accordingly, these peptides are useful for producing an antibody that selectively binds to a GS polypeptide or fragment thereof or epitope thereof of the invention.

It is clearly within the scope of the present invention for the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS 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, hexahistidine, β-galactosidase, horseradish peroxidase, streptavidin or gold.

The present invention also provides a fusion protein comprising one or more immunogenic GS peptides, fragments or epitopes according to any embodiment described herein. Preferred fusion proteins will comprise the protein, peptide, fragment or epitope fused to glutathione-S-transferase (GST), FLAG epitope, hexahistidine, or β- galactosidase.

The present invention also provides an isolated protein aggregate comprising one or more immunogenic GS 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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS 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 GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope.

The present invention also encompasses the use of the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment described herein for eliciting the production of antibodies that bind to M. tuberculosis glutamine synthetase.

The present invention also encompasses the use of the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment described herein in the preparation of a medicament for immunizing a subject against infection by M. tuberculosis. The present invention also provides a pharmaceutical composition comprising the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS 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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS 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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS 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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof e.g., on its surface.

The present invention also provides an isolated or recombinant antibody or immune reactive fragment of an antibody that binds specifically to the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment described herein, or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope. 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 GS protein or immunogenic fragment of a GS protein or other immunogenic peptide comprising a sequence derived from the sequence of a GS protein.

The present invention also provides for the use of the isolated or recombinant antibody according to any embodiment described herein or an immune-reactive fragment thereof in medicine.

The present invention also provides for the use of the isolated or recombinant antibody according to any embodiment described herein or an immune-reactive fragment thereof for detecting a past or present infection or a latent infection by M. tuberculosis in a subject, wherein said infection is determined by the binding of the antibody or fragment to M. tuberculosis GS 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 or recombinant antibody according to any embodiment described herein 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 or recombinant antibodies, or immune-reactive fragments thereof, are 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 antibody to an M. tuberculosis GS 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 antibodies include the purification and study of the diagnostic/prognostic GS protein, identification of cells infected with M. tuberculosis, or for sorting or counting of such cells. The antibodies and fragments thereof are also useful in therapy, including prophylaxis, diagnosis, or prognosis, and the use of such antibodies or fragments for the manufacture of a medicament for use in treatment of infection by M. tuberculosis. For example, specific humanized antibodies or ligands are produced that bind and neutralize a GS protein or M. tuberculosis, especially in vivo. The humanized antibodies or 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 or recombinant antibody according to any embodiment described herein and a pharmaceutically acceptable carrier, diluent or excipient.

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 GS protein or an immunogenic GS peptide or immunogenic GS 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. 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 GS 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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment described herein or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope. 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.

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 GS 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. 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 GS protein or an immunogenic fragment or epitope thereof, and detecting the formation of an antigen-antibody complex. Li 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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment described herein or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope. 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.

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 GS 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 GS 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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment described herein or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope. 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 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 of monitoring disease progression, responsiveness to therapy or infection status by M. tuberculosis in a subject comprising determining the level of a GS 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 GS 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 GS 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 GS protein or an immunogenic fragment or epitope thereof, and detecting the formation of an antigen-antibody complex, hi 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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment described herein or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope. 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.

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 against an immunogenic GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof, wherein the presence of said antibodies 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 the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment described herein 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 may be any antibody-containing sample e.g., a sample that comprises blood or serum or an immunoglobulin fraction obtained from the subject.

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 an immunogenic GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof, wherein the presence of said protein or immunogenic fragment or epitope in the sample is indicative of infection.

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 against an immunogenic GS protein or an immunogenic GS peptide or immunogenic GS 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.

As used herein, the term "infection" shall be understood to mean invasion and/or colonisation by a microorganism and/or multiplication of a micro-organism, in particular, a bacterium or a virus, in the respiratory tract of a subject. Such an infection may be unapparent or result in local cellular injury. The infection may be localised, subclinical and temporary or alternatively may spread by extension to become an acute or chronic clinical infection. The infection may also be a past infection wherein residual GS antigen, or alternatively, reactive host antibodies that bind to isolated GS protein or peptides, remain in the host. The infection may also be a latent infection, in which the microorganism is present in a subject, however the subject does not exhibit symptoms of disease associated with the organism. Preferably, the infection is a pulmonary or extra-pulmonary infection by M. tuberculosis, and more preferably an extra-pulmonary infection. By "pulmonary" infection is meant an infection of the airway of the lung, such as, for example, an infection of the lung tissue, bronchi, bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, or alveoli. By "extra¬ pulmonary" is meant outside the lung, encompassing, for example, kidneys, lymph, urinary tract, bone, skin, spinal fluid, intestine, peritoneal, pleural and pericardial cavities.

In a particularly preferred embodiment described herein, circulating immune complexes (CICs) are detected. In accordance with this embodiment, a capture reagent e.g., a capture antibody is used to capture the GS antigen (GS 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. 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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or one or more immunogenic GS 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.

Standard detection systems are used in the assays described herein, e.g., to detect the antigen-antibody complex formed in an immunoassay format. For example, the antigen or antibody moiety of the antigen-antibody complex can be detected using a secondary antibody 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 GS protein or one or more immunogenic GS 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 GS protein or one or more immunogenic GS peptides or immunogenic GS 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 against M. tuberculosis.

The present invention clearly contemplates the use of an immunogenic GS protein or one or more immunogenic GS peptides or immunogenic GS 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 GS protein or one or more immunogenic GS peptides or immunogenic GS 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 GS protein or one or more immunogenic GS peptides or immunogenic GS 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 GS protein or an immunogenic GS peptide or immunogenic GS 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 GS protein or one or more immunogenic GS peptides or one or more immunogenic GS 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, 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 GS protein or one or more immunogenic GS peptides or one or more immunogenic GS 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.

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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment described herein or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) 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 glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16; and (ii) means for detecting the formation of an antigen-antibody complex, optionally packaged with instructions for use.

The present invention also provides a method for detecting an immune response against an antigen in a subject, said method comprising: (i) contacting an antibody-containing biological sample obtained from the subject with an isolated or recombinant antibody that binds to the antigen for a time and under conditions sufficient for an antigen-antibody complex to form; (ii) contacting the antigen-antibody complex with an anti-Ig antibody for a time and under conditions sufficient for the anti-Ig antibody to bind to an immunoglobulin in the sample; and (iii) detecting the bound anti-Ig antibody, wherein binding complex formation at (i) and (ii) is indicative of an immune response in the subject to the antigen..

In a preferred embodiment the antigen is a protein of a pathogen or an immunogenic peptide or immunogenic fragment or epitope of said protein, e.g., a virus or a bacterium. The antibody that binds to the antigen can be a polyclonal antibody or a monoclonal antibody. Preferably, the antibody-containing biological sample comprises: (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; and/or (ii) body fluid(s) selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof; and/or (iii) is derived from body fluid(s) selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof. 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.

The present invention also provides a process for diagnosing an infection in a subject comprising performing the method described in the preceding paragraph or corresponding description elsewhere herein to thereby determine an immune response in the subject to an antigen, wherein the antigen is an antigen of an infectious agent producing said infection.

The present invention also provides a process for diagnosing a disease or disorder associated with the presence of one or more circulating immune complexes (CIC) in a subject's immune system said process comprising the method described in the preceding paragraph or corresponding description elsewhere herein to thereby determine an immune response in the subject to an antigen, wherein the antigen is present in the subject's immune system as a CIC. For example, the process may be used to diagnose tuberculosis in humans, bovine tuberculosis, Johne's disease or Crohne's disease.

Without conceding the generality of the method, a particularly preferred embodiment provides for the bacterium to be Mycobacterium tuberculosis. Without conceding the generality of the method, it is also preferred for the antigen to comprise the isolated or recombinant GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment described herein or a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope. Also without conceding the general applicability of the method, it is particularly preferred for the isolated or recombinant antibody that binds to the antigen to be an isolated or recombinant antibody or immune reactive fragment of an antibody that binds specifically to the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment herein, or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope.

Because the assays described herein are amenable to any assay format, and particularly to solid phase ELISA, flow through immunoassay formats, capillary formats, and for the purification or isolation of immunogenic proteins, peptides, fragments and epitopes and CICs, the present invention also provides a solid matrix having adsorbed thereto an isolated or recombinant GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one embodiment described herein or a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) 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.

Similarly, because the assays described herein are amenable to any assay format, and particularly to solid phase ELISA, flow through immunoassay formats, capillary formats, and for the purification or isolation of antibodies against immunogenic proteins, peptides, fragments or epitopes, the invention also provides a solid matrix having adsorbed thereto an antibody that binds to an isolated or recombinant GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any embodiment described herein or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) 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. Brief description of the drawings Figure 1 is a graphical representation showing a CLUSTAL W 1.81 alignment of glutamine synthetase A (glnA) of M. tuberculosis (P0A590), glutamine synthetase A2 (P64245), glutamine synthetase A3 (007752) and glutamine synthetase A4 (033342; an example of the GS of the invention). Bold regions represent peptides synthesized for the production of monoclonal antibodies against GS. "*" indicates identical or conserved residue in all sequences in the alignment; ":" indicates conserved substitutions; and "." indicates semi-conserved substitutions.

Figure 2 is a graphical representation showing the capture of an immunogenic glutamine synthase (GS) peptide having the sequence set forth in SEQ ID NO: 93 using the monoclonal antibody 426C prepared against an immunogenic GS peptide comprising the amino acid sequence RGTDGSAVFADSNGPHGMSSMFRSFC (SEQ ID NO: 92).

Figure 3 is a graphical representation showing the binding of the monoclonal antibody 426C prepared against an immunogenic GS peptide comprising the amino acid sequence RGTDGSAVFADSNGPHGMSSMFRSFC (SEQ ID NO: 92) to a fixed concentration of an immunogenic glutamine synthase (GS) peptide having the sequence set forth in SEQ ID NO: 93.

Detailed description of the preferred embodiments Isolated or recombinant GS protein and immunogenic fragments and epitopes thereof The present invention provides an isolated or recombinant immunogenic GS protein or an immunogenic fragment or epitope thereof. ^x

This encompasses any synthetic or recombinant immunogenic peptides derived from a GS protein referred to herein including the full-length GS protein and/or a derivative or analogue of a GS protein or an immunogenic fragment or epitope thereof. As used herein, the term "glutamine synthetase" or "GS" 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 ID NO: 1. Preferably, the percentage identity of a GS protein to SEQ ID 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 GS 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 GS protein without undue experimentation.

hi 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. hi 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 which 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 GS 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 Group B protein.

A CTL epitope is also conveniently derived from the full length amino acid sequence of a GS protein and will generally consist of at least about 9 contiguous amino acids of said GS 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 (BEVI AS) 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, CD8⁺ T cell, cytotoxic T cell (CTL). Still even more preferably, the CTL will have a sequence that stimulates CTL activity in a standard cytotoxicity assay. Particularly preferred CTL epitopes of a GS protein are capable of eliciting a cellular immune response against M. tuberculosis in human cells or tissues, such as, for example, by recognizing and lyzing human cells infected with M. tuberculosis, thereby providing or enhancing cellular immunity against M. tuberculosis.

Suitable fragments will be at least about 5, eg 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 peptide or epitope of GS comprises an amino acid sequence set forth in any one of SEQ ID Nos: 2-91 or 94.

More preferably, an immunogenic fragment or immunogenic GS peptide or epitope comprises an amino acid sequence of at least about 5 consecutive amino acid residues positioned from about residue 265 to about residue 300 of SEQ ID NO: 1, more preferably from about residue 270 to about residue 295 of SEQ ID NO: 1 and still more preferably from residue 271 to residue 295 of SEQ ID NO: 1. It will be apparent from the disclosure herein that a highly immunogenic portion of the full-length GS polypeptide thus comprises at least 5 consecutive residues of the sequence RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO: 92).

Immunogenic GS peptides derived from this region of the full-length GS polypeptide may comprise 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 consecutive amino acid residues thereof.

It is also within the scope of the invention for such immunogenic GS peptides to further comprise N-terminal and/or C-terminal extensions, e.g., an N-terminal extension of up to about 5 amino acid residues in length including the tetramer SGSG; and/or a C- terminal extension of up to about 5 amino acid residues in length including the addition of a single cysteine residue.

As with other embodiments described herein, an immunogenic GS peptide can also comprise one or more labels or detectable moieties to facilitate detection or isolation or immobilization of the peptide, e.g., biotin, without adversely affecting its immunogenic properties.

For example, a particularly preferred immunogenic GS peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 54-60 or mixtures thereof or fusions derived there from. In a preferred embodiment, an immunogenic GS peptide will comprise the amino acid sequence set forth in SEQ ID NO: 54. In a preferred embodiment, an immunogenic GS peptide will comprise the amino acid sequence set forth in SEQ ID NO: 55. In a preferred embodiment, an immunogenic GS peptide will comprise the amino acid sequence set forth in SEQ ID NO: 56. In a preferred embodiment, an immunogenic GS peptide will comprise the amino acid sequence set forth in SEQ ID NO: 57. In a preferred embodiment, an immunogenic GS peptide will comprise the amino acid sequence set forth in SEQ ID NO: 58. In a preferred embodiment, an immunogenic GS peptide will comprise the amino acid sequence set forth in SEQ ID NO: 59. In a preferred embodiment, an immunogenic GS peptide will comprise the amino acid sequence set forth in SEQ TD NO: 60. In a preferred embodiment, an immunogenic GS peptide will comprise the amino acid sequence set forth in SEQ ID NO: 92. In a preferred embodiment, an immunogenic GS peptide will comprise the amino acid sequence set forth in SEQ ID NO: 93 (i.e., the sequence of SEQ ID NO: 93 further comprising N-terminal and C-terminal extensions not adversely affecting immunogenicity of the base peptide). In a preferred embodiment, an immunogenic GS peptide will comprise the amino acid sequence set forth in SEQ ED NO: 94 (i.e., the sequence of SEQ ID NO: 94 further comprising N-terminal and C- terminal extensions not adversely affecting immunogenicity of the base peptide).

The amino acid sequence of a GS 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 hydrophilicity or polarity of the residue. The size and/or charge of the side chains also are relevant factors in determining which substitutions are conservative.

The present invention clearly encompasses a covalent fusion between one or more immunogenic GS 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 GS 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, implicit 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 immunogenic GS polypeptide or immunogenic GS peptide 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 GS protein, such as, for example, to facilitate synthesis or improve peptide solubility. Glycine and/or serine residues are particularly preferred for this purpose. As exemplified herein, each of the peptides set forth in SEQ E) Nos: 2-91 or 94 includes additional spacer sequences flanking the GS fragments, said spacers comprising heteropolymers (trimers or tetramers) comprising glycine and serine.

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, polyethylene glycol, a peptidic polypeptide moiety (e.g. tuftsin, polylysine), a fluorescence marker (e.g. FITC, PJTC, 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.

In another embodiment, a GS 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 GS 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 GS protein is readily derived from the publicly available amino acid sequence.

hi another embodiment, a GS 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), hexahistidine, 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 GS 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 lacz promoter, temperature-sensitive λi or X_R promoters, T7 promoter or the IPTG-inducible tac promoter. A number of other vector systems for expressing the nucleic acid molecule of the invention in E. coli are well-known in the art and are described, for example, in Ausubel et al {In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047150338, 1987) or Sambrook et al {In: Molecular cloning, A laboratory manual, second edition, Cold Spring Harbor Laboratory, Cold Spring 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), ρΕT-3 (17: 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, 1785, 1991). The vector pcDNA 3.1 myc-His (Invitrogen) is particularly preferred for expressing a secreted form of a GS 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 a Group TB protein or an immunological derivative 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 GS protein or an epitope thereof.

Antibodies that bind to a GS protein or an epitope thereof This invention provides an antibody that binds specifically to a GS 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.

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 GS 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, a mouse, rat, hamster, guinea pig, goat, sheep, pig, dog, horse, or chicken. Because of the relatively large blood volume of rabbits, a rabbit is a 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 10³-10⁴ is preferred. More preferably, the antibody titer will be in the range from about 10⁴ to about 10⁵ , even more preferably in the range from about 10⁵ to about 10⁶.

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 GS 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).

The efficacy of the GS protein or immunogenic fragment or epitope thereof in producing an antibody is established by injecting an animal, for example, a mouse, rat, rabbit, guinea pig, dog, horse, cow, goat or pig, with a formulation comprising the GS 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).

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 GS protein or immunogenic fragment or epitope thereof under conditions sufficient to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible. The use of rats may provide certain advantages, but mice 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.

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 biopsied 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 10⁷ to 2 x 10⁸ 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 GS 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-Il, MPC11-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, 511-519, 1976. Methods using polyethylene glycol (PEG), such as 37% (v/v) PEG, are described in detail by Gefter et at, 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 immunobinding assay, 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 GS 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 replicaton-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 GS 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 palsmacytomas 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 genetically in detail in the following disclosures which are incorporated by reference herein: 1. Largaespada et al, Curr. Top. Microbiol. Immunol, 166, 91-96. 1990; 2. Weissinger et al.Proc. Natl. Acad. Set. 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, Immuno. 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, ³II, ¹²⁵I, .³²P, .³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, and ¹⁵²Eu. Radioactively labeled 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 labeled with technetium" by ligand exchange process, for example, by reducing pertechnetate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labeling techniques, (e.g., by incubating pertechnate, a reducing agent such as SNCl₂, a buffer solution such as sodium- potassium phthalate solution, and the antibody).

Any immunoassay may be used to monitor antibody production by the GS 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 labeled, 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 against the immunogenic GS protein or immunogenic GS peptide or immunogenic GS 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 another exemplary immunoassay format applicable to both flow through and solid phase ELISA, antibodies that bind to the immunogenic GS protein or immunogenic GS peptide or immunogenic GS 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 GS 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 GS 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 GS 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.

As exemplified herein, the present inventors have produced a plasmacytoma producing monoclonal antibodies that bind to an immunogenic fragment or peptide or epitope of GS that comprises an amino acid sequence of at least about 5 consecutive amino acid residues positioned from about residue 265 to about residue 300 of SEQ ID NO: 1, more preferably from about residue 270 to about residue 295 of SEQ ID NO: 1 and still more preferably from residue 271 to residue 295 of SEQ ID NO: 1, and especially to at least 5 consecutive residues within the amino acid sequence RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO: 92). Thus, the antibodies bind to 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 consecutive amino acid residues of SEQ ID NO: 92. The epitope to which the antibodies bind maps to one or more of SEQ ID NOs: 54-60 or fusions derived there from, including the amino acid sequence set forth in SEQ ID NO: 54 or 55 or 56 or 57 or 58 or 59 or 60 or any overlapping region between any two or more of said sequences that also occurs in a naturally occurring GS polypeptide. As will be known to the skilled artisan, the antibodies can thus bind to multiple distinct GS peptides or fragments or to full-length GS, the only requirement being that the peptides, fragments or full-length polypeptide comprises a linear or conformational B-cell epitope recognized by the antibody. It will also be apparent from the foregoing that the antibodies of the present invention also bind with high affinity to an immunogenic GS peptide comprising the amino acid sequence set forth in SEQ ID NO: 93 (i.e., the sequence of SEQ DD NO: 93 further comprising N-terminal and C-terminal extensions not adversely affecting immunogenicity of the base peptide). Antibodies of the 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. Diagnostic/prognostic methods for detecting tuberculosis or M. tuberculosis infection L 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 GS 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 immunocompromised patients may not produce antibody at detectable levels, and the level of the antibody in any patient may 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 GS protein or an immunogenic fragment or epitope thereof, and detecting the formation of an antigen-antibody complex.

Li 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 GS 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 GS protein or an immunogenic fragment thereof that is less than the level of the GS 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 GS 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.

Li 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 GS 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 GS 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 GS protein in a biological sample derived from a patient is compared to the amount of the same protein detected in a biological sample previously derived from the same patient. As will be apparent to a person skilled in the art, this method may be used to continually monitor a patient with a latent infection or a patient that has developed tuberculosis. In this way a patient may be monitored for the onset or progression of an infection or disease, with the goal of commencing treatment before an infection is established, particularly in an HIV+ 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 GS protein or immunogenic fragment thereof is not detected in a reference sample, however, said GS 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 GS 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).

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, saliva, urine or pleural fluid. Sputa, sera and fractions derived from sputa or sera are particularly preferred.

Preferably, the biological sample is a bodily fluid or tissue sample selected from the group consisting of: blood, serum, sputum, urine, and lung. Other samples are not excluded.

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 against a GS 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 immunocompromised individuals. However, antibody-based assays are clearly useful for detecting M. tuberculosis infections in HIV- individuals who are not immunocompromised.

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 GS 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 against a GS protein or fragment or epitope in blood or serum or an immunoglobulin fraction from the subject is positively correlated with the infectious state. For example, a level of the anti-GS antibodies thereto that is less than the level of the anti-GS 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 against a GS 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 against a GS 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.

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 (blood, serum or immunoglobulin-containing fraction).

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 focussing, 2-dimensional gel electrophoresis comprising SDS/PAGE and isoelectric focussing, 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, eg., 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), 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-GS antibodies, or alternatively GS 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 antibody that specifically binds a GS protein is brought into direct contact with the immobilised 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 GS protein or an immunogenic fragment or epitope thereof is contacted with the sample. The added antibody or protein in solution is generally labelled with a detectable reporter molecule, such as for example, 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 GS antigen. Following washing to remove any unbound antibody or GS antigen, the label is 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 GS 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.

In one example, the present invention comprises: (i) immobilizing an antibody that specifically binds an immunogenic GS 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: 54-60, 92, 93 or 94); (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 a GS 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 GS protein or fragments comprising the epitope that the antibody recognizes actually bind, whilst specificity of anti-human Ig ensures that only immunocomplexed GS protein or fragment is detected. In this context, the term "immunocomplexed" shall be taken to mean that the GS 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 against 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-GS antibodies, or alternatively a GS 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 GS 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 GS 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 (Ini 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 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 GS 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-GS antibodies, or alternatively GS protein or an immunogenic fragment thereof are particularly preferred.

In one embodiment, 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, 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 al. Anal. Biochem. 278:123- 131, 2000.

A protein chip is preferably generated such that several proteins, ligands or antibodies are arrayed on said chip. This format permits the simultaneous screening for the presence of several proteins in a sample.

Alternatively, a protein chip may comprise only one protein, ligand or antibody, and be used to screen one or more patient samples for the presence of one polypeptide of 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-fmole level (Nelson et al. Electrophoresis 21: 1155-1163, 2000). One technique useful in the analysis of a protein chip is surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technology to characterise a protein bound to the protein chip. Alternatively, the protein chip is analysed using ESI as described in U.S. Patent Application 20020139751.

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 GS protein. Multiplexing of diagnostic and prognostic markers is particularly contemplated in the present invention.

In a further embodiment, the samples are analysed using ICAT, 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-GS antibodies, or alternatively GS 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-GS antibodies and/or GS 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-GS antibodies and/or GS epitopes.

The present invention clearly contemplates multiplexed assays for detecting GS 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 antigens. Such assays are particularly useful for simultaneously obtaining information on co-infection with M. tuberculosis and HIV-I, 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 GS protein or anti-GS 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, squamous epithelial cells of the respiratory tract, a mast cell, a goblet cell, a pneumocyte (type 1 or type 2), broncheoalveolar lavage fluid (BAL), alveolar lining fluid, an intra epithelial dendritic cell, sputum, mucus, saliva, blood, serum, plasma, a PBMC, a neutrophil and a monocyte.

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 Clin Immunol, 10(4): 322-328, 1999.

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, treatment with 2-mercaptoethanol, or treatment with detergents and other compounds such as, for example, guanidinium or urea. 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, tissue, plasma, serum, whole blood, sputum, saliva, or BAL fluid derived from the same subject when the individual was not suffering from an infection or disease. In another embodiment, the reference sample comprises (cells, tissue, plasma, serum, whole blood, sputum, saliva, or BAL fluid) derived from a normal healthy individual.

Accordingly, a reference sample and a test (or patient) sample are both 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 GS protein or an immunogenic GS peptide or 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 GS protein or an immunogenic GS peptide or fragment or epitope thereof; and (ii) means for detecting the formation of an antigen-antibody complex.

In another embodiment, the kit comprises: (i) one or more isolated antibodies that bind to an immunogenic GS protein or an immunogenic GS peptide or fragment or epitope thereof; (ii) an isolated or recombinant GS protein or an immunogenic GS peptide or fragment or epitope thereof; and (iii) means for detecting the formation of an antigen-antibody complex.

The antibodies, immunogenic GS peptide, and detection means of the subject kit are preferably selected from the antibodies and immunogenic GS peptides described herein above and those embodiments shall be taken to be incorporated by reference herein from the preceding description. The selection of compatible kit components for any assay format will be readily apparent to the skilled artisan from the preceding 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: 54-60, 92, 93 or 94; and (ii) anti-human Ig. Preferably, the kit further comprises an amount of one or more control peptides each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 54-60, 92, 93 or 94 and fusions 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 GS 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 (eg tributyl phosphine, C7BZO, dextran sulfate, 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 As exemplified herein, the immunogenic GS protein or immunogenic GS peptide or 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 GS 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 against M. tuberculosis.

Preferably, the neutralizing antibodies are high titer neutralizing antibodies.

The effective amount of GS protein or epitope 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. AU 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 GS 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 GS protein or immunogenic fragment or epitope.

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 against a B cell epitope in the immunizing antigen. By a "sustained level of antibodies" is meant a sufficient level of circulating antibodies against 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 GS protein or an epitope thereof or a vaccine composition comprising said GS protein or epitope for a time and under conditions sufficient to confer or enhance resistance against M. tuberculosis in said 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 GS protein of M. tuberculosis or an epitope of said protein is produced in said subject; (ii) neutralizing antibodies against M. tuberculosis are produced in said subject; (iii) a cytotoxic T lymphocyte (CTL) and/or a CTL precursor that is specific for a GS 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 GS protein or an epitope thereof or a vaccine composition comprising said GS protein or epitope for a time and under conditions sufficient to provide immunological memory against a future infection by M. tuberculosis.

The 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 to reduce the number of pathogenic bacilli in the lung, blood or lymph system of the subject.

Preferably, the GS protein or epitope or vaccine 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 GS 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 CD8⁺ 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, (ie., 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 GS protein or epitope thereof to be administered, either 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 GS protein or an 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.

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 GS 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 GS 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.

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 al, 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 (ie. the T cell will recognize and lyze 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 lyze 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 therefrom). 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 GS protein or an immunogenic GS peptide or immunogenic GS 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 GS protein or one or more immunogenic GS peptides, fragments or epitopes thereof in combination with a pharmaceutically acceptable diluent.

The GS protein or immunogenic peptide or 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 GS protein or 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-I, IL-2, BCG, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thur-MDP), N- acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'-dipalmitoyl-sn-glycero- 3-hydroxyphosphoryloxy)-ethylamine (CGP) 1983 A, 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 GS protein or immunogenic fragment or epitope thereof, to down regulate suppressor T cell activity. Exemplary BRM's include, but are not limited to, Cimetidine (CBvI; 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.sup.2) (Johnson/Mead, NJ, USA).

Preferred vehicles for administration of the GS 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 ah, Eur. J. CHn. Microbiol. Infect. Dis. 12(Suppl. I), 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. K Y. Acad. Sci. 446, 368 (1985)). In the present context, the GS 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 GS 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 al, Proc. Nat'l Acad. Sci USA 84, 7413 (1987); 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)₅ 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 GS 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 GS protein or epitope(s) and cloned into a suitable vector (eg. vaccinia, canary pox, adenovirus, or other eukaryotic virus vector) are also contemplated. Preferably, DNA encoding a GS 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 1.5ml of patient serum stored at -80°C was thawed at room temperature then applied to a 2ml column of protein G-sepharose (Amersham Biosciences), previously equilibrated with 2OmM phosphate buffer pH7 and incubated on ice for 30 minutes with occasional inversion. The mixture was spun at 600Og for 10 minutes at 4⁰C and the supernatant decanted. The sepharose pellet was washed with 2OmM phosphate buffer. The IgG bound to the sepharose was eluted by addition of 5OmM glycine pH2.7 for 20 minutes. After centrifugation as above, the supernatant was discarded and the glycine step repeated. The supernatant was collected from this second glycine elution and stored at -80°C. The supernant was precipitated with 10 volumes of cold acetone at -20⁰C for 48h then centrifuged at 5000g for 20mins at 4⁰C. The precipitate was 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. Samples were aliquoted into 250μl aliquots and stored at -80°C. Example 2 Analytical methods The protein content of the samples was estimated using a Bradford assay. Samples were diluted to 2mg/ml with sample buffer as above replacing 4OmM Tris with 5mM Tris.

Prior to rehydration of IPG strips, samples were centrifuged at 21000 x g for lOminutes. The supernatant was collected and lOμl of 1% Orange G (Sigma) per ml added as an indicator dye.

First Dimension Dry 11cm IPG strips (Amersham-Biosciences) were rehydrated for 16-24 hours with 180μl of protein sample. Rehydrated strips were focussed on a Protean IEF Cell (Bio- Rad, Hercules, CA) or Proteome System's IsoElectrIQ electrophoresis equipment for approx 140 kVhr at a maximum of 10 kV. Focussed strips were then equilibrated in urea/SDS/Tris-HCl/bromophenol blue buffer.

Second Dimension Equilibrated strips were inserted into loading wells of 6-15% (w/v) tris-acetate SDS- PAGE pre-cast 10cm x 15cm GelChips (Proteome Systems, Sydney Australia). Electrophoresis was performed at 50mA per gel for 1.5 hours, or until the tracking dye reached the bottom of the gel. Proteins were stained using SyproRuby (Molecular Probes). Gel images were scanned after destaining using an Alphalmager System (Alpha Innotech Corp.). Gels were then stained with Coomassie G-250 to assist visualisation of protein spots in subsequent analyses.

Mass Spectrometry: Prior to mass spectrometry it was necessary to prepare protein samples by in-gel tryptic digestion. Protein gel pieces were 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 was incubated in water to maintain a constant size and prevent drying. Subsequently, the 2-D gel was placed on the Xcise, a digital image was captured and the spots to be cut were selected. After automated spot excision, gel pieces were subjected to automated liquid handling and in-gel digestion. Briefly, each spot was destained with 100 μl of 50% (v/v) acetonitrile in 50 mM ammonium bicarbonate. The gel pieces were dried by adding 100% acetonitrile, the acetonitrile was removed after 5 seconds and the gels were dried completely by evaporating the residual acetonitrile at 37⁰C. Proteolytic digestion was 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 30°C overnight.

Ten μl of the tryptic peptide mixture was removed to a clean microtitre 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 was performed using Cl 8 ZipTip (Millipore, Bedford, MA). Adsorbed peptides were 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 were analysed using an Axima-CFR MALDI-TOF mass spectrometer (Kratos, Manchester, UK) in positive ion reflectron mode. A nitrogen laser with a wavelength of 337 nm was used to irradiate the sample. The spectra were 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 were saved. All spectra underwent an internal two point calibration using an autodigested trypsin peak mass, m/z 842.51 Da and spiked adenocorticotropic hormone (ACTH) peptide, m/z 2465.117 Da. Software designed by Proteome Systems, as contained in the web-based proteomic data management system BioinformatIQ^Tm (Proteome Systems), was used to extract isotopic peaks from MS spectra.

Protein identification was 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 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 were ranked through a modification of the MOWSE scoring system. Propionamide-cysteine (cys-PAM) or carboxyamidomethyl-cysteine (cys-CAM) and oxidized methionine modifications were taken into account and a mass tolerance of 100 ppm was allowed.

Miscleavage sites were only considered after an initial search without miscleavages had been performed. The following criteria were 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 were analysed using LC-ESI-MS. Tryptic digest solutions of proteins (10 μl) were 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 were 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) was performed over a 30-60-minute period. The mass spectrometer was set up to acquire three scan events - one full scan (range from 400 to 2000 amu) followed by two data dependant MS/MS scans. Proteins were identified using TurboSequest (Thermo-Finnigan) software. Peptides were identified from MS/MS spectra in which more than half of the experimental fragment ions matched theoretical ion values, and gave cross-correlation (a raw correlation score of the top candidate peptide), delta correlation (difference in correlation between the top two candidate peptides) and preliminary score (raw score used to rank candidate peptides) values greater than 2.2, 0.2, and 400, respectively.

Using this method a 49.7kDa protein was identified in a TB subject. This protein was analysed using MALDI-TOF MS and fragments (SEQ ID NO: 1) shown in Table 1 identified. Table 1 Identification of a diagnostic marker of M. tuberculosis infection. Data extracted from Proteome Systems' IonlQ database. PeptideID=peptide identification number DB Mass=theoretical mass of peptide User Mass=observed mass of peptide PPM error=error associated with mass of peptide in parts per million MC=miscleavages Pep Start=Amino acid number at beginning of peptide Pep End= Amino acid number at end of peptide 10 Mods=Modifications Sequence= Amino acid sequence

Accession number: O33342 Sequence name: HYPOTHETICAL 49.7 kDa PROTEIN (Glutamine synthetase, putative) Species: Mycobacterium tuberculosis Molecular weight: 49717 Isoelectric point: 5.06 to 15 MTGPGSPPLA WTELERLVAA GDVDTVIVAF TDMQGRLAGK RISGRHFVDD IATRGVECCS YLLAVDVDLN TVPGYAMASW DTGYGDMVMT PDLSTLRLIP WLPGTALVIA DLVWADGSEV AVSPRSILRR QLDRLKARGL VADVATELEF IVFDQPYRQA WASGYRGLTP ASDYNIDYAI LASSRMEPLL RDIRLGMAGA GLRFEAVKGE CNMGQQEIGF RYDEALVTCD NHAIYKNGAK EIADQHGKSL TFMAKYDERE GNSCHIHVSL RGTDGSAVFA DSNGPHGMSS MFRSFVAGQL ATLREFTLCY APTINSYKRF ADSSFAPTAL AWGLDNRTCA LRWGHGQNI RVECRVPGGD VNQYLAVAAL IAGGLYGIER GLQLPEPCVG NAYQGADVER LPVTLADAAV LFEDSALVRE AFGEDWAHY LNNARVELAA FNAAVTDWER IRGFERL 20 Amino acid coverage: 90 Percentage coverage: 19.69%

Example 3 Identification of a diagnostic marker of M. tuberculosis infection A protein having an isoelectric point of about 5.06 and a molecular weight of about 49717 Daltons was identified in one TB⁺ZHIV⁺ sample (gel number srl l2, protein spot 103). 6 peptides matched this protein from the MALDI-TOF data, two with 1 missed cleavage and four with no missed cleavages. The percentage coverage of the protein by these 6 peptides was 19.69%. One peptide had a methionine sulfoxide modification and one peptide had a cysteine modification. This data is presented in Table 1 and is extracted from the IonlQ database used to analyse the PMF data. This protein has the amino acid sequence set forth in SEQ ID NO: 1 and was designated as "glutamine synthetase", based upon the presence of both a glutamine synthetase catalytic domain and a glutamine synthetase β-Grab domain.

The amino acid sequence set forth in SEQ ID NO: 1 corresponded to a hypothetical protein of M. tuberculosis identified from in silico translation of open reading frames of the M. tuberculosis genome. Accordingly, this provides the first disclosure that the GS protein of M. tuberculosis is expressed during the infectious cycle of the bacterium in vivo. These data suggest that the M. tuberculosis GS protein identified in subjects is a good candidate protein for preparation of diagnostic and therapeutic reagents for tuberculosis.

Example 4 B cell epitope mapping of the GS protein of M. tuberculosis 1. Peptides To identify immunogenic epitopes of the GS protein, a set of synthetic peptides (PEPSET) was produced from the primary amino acid sequence shown in SEQ ID NO: 1. The synthetic peptides comprised the amino acid sequences set forth in SEQ ID Nos: 2-91, with additional N-terminal or C-terminal sequence extensions added compared to the sequence of the corresponding fragment in SEQ ID NO: 1. In particular, a synthetic peptide (#1 in Table 2) was produced consisting of the N- terminal 15 residues of SEQ ID NO: 1 with a C-terminal extension Gly-Ser-Gly. The base peptide sequence for this peptide is set forth in SEQ ID NO; 2. The remaining synthetic peptides (i.e., #2 to #91 in Table 2) in the PEPSET contained the N-terminal extension Ser-Gly-Ser-Gly added to the base peptide sequences of GS set forth in SEQ ID Nos: 3-91, respectively. The structures of the peptides are set forth in Table 2.

The peptides were labeled as indicated in Table 2 to facilitate their use in ELISA assays.

2. Serum samples Sera isolated from subjects suffering from TB and control samples isolated from subjects that do not suffer from TB are screened with the peptides of the PEPSET. These include sera from South African (S.A.) TB positive individuals, S.A. TB negative individuals, Chinese TB positive individuals, Chinese TB negative individuals, and Australian Caucasian TB negative control individuals.

Sera are screened for the presence of antibodies using an ELISA system developed as described below. TABLE2 No . Peptide structure Hydro MoIWt SEQ ID NO: (base peptide) 1 H- MTGPGSPPLAWTELEGSG ^■■BiocytAM 0.33 2,140.48 2 2 Biotin- SGSGSPPLAWTELERLVAA-^■NH2 0.49 2,166.50 3 3 Biotin- SGSGWTELERLVAAGDVDT-^•NH2 0.32 2,188.42 4 4 Biotin- SGSGRLVAAGDVDTVIVAF-^■NH2 0.48 2,059.39 5 5 Biotin- SGSGGDVDTVIVAFTDMQG-^■NH2 0.39 2,081.33 6 6 Biotin- SGSGVIVAFTDMQGRLAGK-^■NH2 0.39 2,119.51 7 7 Biotin- SGSGTDMQGRLAGKRISGR- NH2 0.07 2,159.50 8 8 Biotin- SGSGRLAGKRISGRHFVDD-^■NH2 0.13 2,240.55 9 9 Biotin- SGSGRISGRHFVDDIATRG-^■NH2 0.19 2,213.48 10 10 Biotin- SGSGHFVDDIATRGVECCS-^■NH2 0.38 2,165.43 11 11 Biotin- SGSGIATRGVECCSYLLAV-^•NH2 0.58 2,111.51 12 12 Biotin- SGSGVECCSYLLAVDVDLN- NH2 0.55 2,169.50 13 13 Biotin- SGSGYLLAVDVDLNTVPGY-^■NH2 0.53 2,165.47 14 14 Biotin- SGSGDVDLNTVPGYAMASW-^■NH2 0.44 2,152.41 15 15 Biotin- SGSGTVPGYAMASWDTGYG- NH2 0.43 2,089.31 16 16 Biotin- SGSGAMASWDTGYGDMVMT- NH2 0.43 2,149.45 17 17 Biotin- ΞGSGDTGYGDMVMTPDLST- NH2 0.32 2,116.35 18 18 Biotin- SGSGDMVMTPDLSTLRLIP- NH2 0.54 2,215.66 19 19 Biotin- SGSGPDLSTLRLIPWLPGT-^■NH2 0.62* 2,192.58 20 20 Biotin- SGSGLRLIPWLPGTALVIA- NH2 0.79* 2,146.64 21 21 Biotin- SGSGWLPGTALVIADLVWA- NH2 0.78* 2,138.53 22 22 Biotin- SGSGALVIADLVWADGSEV- NH2 0.53 2,071.35 23 23 Biotin- SGSGDLVWADGSEVAVSPR- NH2 0.33 2,114.34 24 24 Biotin- SGSGDGSEVAVSPRSILRR- NH2 0.18 2,155.44 25 25 Biotin- SGSGAVSPRSILRRQLDRL- NH2 0.26 2,293.70 26 26 Biotin- SGSGΞILRRQLDRLKARGL-NH2 0.20 2,308.76 27 27 Biotin- SGSGQLDRLKARGLVADVA- NH2 0.24 2,138.49 28 28 Biotin- SGSGKARGLVADVATELEF-NH2 0.29 2,132.44 29 29 Biotin- SGSGVADVATELEFIVFDQ-NH2 0.47 2,209.48 30 30 Biotin- SGSGTELEFIVFDQPYRQA-NH2 0.40 2,369.66 31 31 Biotin- SGSGIVFDQPYRQAWASGY-NH2 0.45 2,314.58 32 32 Biotin- SGSGPYRQAWASGYRGLTP-NH2 0.34 2,236.51 33 33 Biotin- SGSGWAΞGYRGLTPASDYN-NH2 0.30 2,171.35 34 34 Biotin- SGΞGRGLTPASDYNIDYAI-NH2 0.33 2,182.41 35 35 Biotin- SGSGASDYNIDYAILAΞSR-NH2 0.30 2,172.38 36 36 Biotin- SGSGIDYAILASSRMEPLL-NH2 0.53 2,205.60 37 37 Biotin- SGSGLASSRMEPLLRDIRL-NH2 0.36 2,283.72 38 38 Biotin- SGSGMEPLLRDIRLGMAGA-NH2 0.41 2,156.60 39 39 Biotin- SGSGRDIRLGMAGAGLRFE-NH2 0.27 2,175.54 40 TABLE 2 continued No . Peptide structure [ydro MoIWt SEQ ID NO: (base peptide) 40 Biotin- SGSGGMAGAGLRFEAVKGE-NH2 0.23 2,006.31 41 41 Biotin- SGSGGLRFEAVKGECNMGQ-NH2 0.24 2,152.48 42 42 Biotin- SGSGAVKGECNMGQQEIGF-NH2 0.28 2,124.42 43 43 Biotin- SGSGCNMGQQEIGFRYDEA-NH2 0.23 2,274.52 44 44 Biotin- SGSGQEIGFRYDEALVTCD-NH2 0.33 2,272.52 45 45 Biotin- SGΞGRYDEALVTCDNHAIY-NH2 0.32 2,296.54 46 46 Biotin- SGSGLVTCDNHAIYKNGAK-NH2 0.27 2,160.48 47 47 Biotin- SGSGNHAIYKNGAKEIADQ-NH2 0.10 2,185.42 48 48 Biotin- SGSGKNGAKEIADQHGKSL-NH2 0.02 2,109.36 49 49 Biotin- SGSGEIADQHGKSLTFMAK-NH2 0.25 2,189.52 50 50 Biotin- ΞGΞGHGKSLTFMAKYDERE-NH2 0.12 2,325.63 51 51 Biotin- SGSGTFMAKYDEREGNSCH-NH2 0.14 2,301.54 52 52 Biotin- SGSGYDEREGNSCHIHVΞL-NH2 0.24 2,272.48 53 53 Biotin- ΞGSGGNΞCHIHVSLRGTDG-NH2 0.27 2,066.28 54 54 Biotin- SGSGIHVSLRGTDGSAVFA-NH2 0.39 2,043.30 55 55 Biotin- SGSGRGTDGSAVFADSNGP-NH2 0.13 1,964.07 56 56 Biotin- SGSGSAVFADSNGPHGMSS-NH2 0.26 1,977.14 57 57 Biotin- SGSGDSNGPHGMSSMFRSF-NH2 0.27 2,170.41 58 58 Biotin- SGSGHGMΞSMFRSFVAGQL-NH2 0.45 2,168.52 59 59 Biotin- SGSGMFRSFVAGQLATLRE-NH2 0.41 2,239.62 60 60 Biotin- SGSGVAGQLATLREFTLCY-NH2 0.53 2,198.57 61 61 Biotin- SGΞGATLREFTLCYAPTIN-NH2 0.51 2,226.58 62 62 Biotin- SGSGFTLCYAPTINSYKRF-NH2 0.51 2,337.73 63 63 Biotin- SGSGAPTINSYKRFADSΞF-NH2 0.27 2,217.46 64 64 Biotin- SGΞGSYKRFADSSFAPTAL-NH2 0.31 2,174.44 65 65 Biotin- SGSGADSSFAPTALAWGLD-NH2 0.45 2,035.24 66 66 Biotin- SGSGAPTALAWGLDNRTCA-NH2 0.41 2,073.36 67 67 Biotin- SGΞGAWGLDNRTCALRWG-NH2 0.40 2,144.48 68 68 Biotin- SGSGNRTCALRWGHGQNI-NH2 0.29 2,151.48 69 69 Biotin- SGSGLRWGHGQNIRVECR-NH2 0.27 2,249.62 70 70 Biotin- SGSGHGQNIRVECRVPGGD-NH2 0.18 2,150.40 71 71 Biotin- SGSGRVECRVPGGDVNQYL-NH2 0.27 2,218.52 72 72 Biotin- SGSGVPGGDVNQYLAVAAL-NH2 0.45 2,000.28 73 73 Biotin- SGSGVNQYLAVAALIAGGL-NH2 0.57 1,986.34 74 74 Biotin- SGSGAVAALIAGGLYGIER-NH2 0.48 1,987.32 75 75 Biotin- SGSGIAGGLYGIERGLQLP-NH2 0.48 2,070.42 76 76 Biotin- SGSGYGIERGLQLPEPCVG-NH2 0.44 2,144.47 77 77 Biotin- SGSGGLQLPEPCVGNAYQG-NH2 0.41 2,059.32 78 78 Biotin- SGSGEPCVGNAYQGADVER-NH2 0.18 2,121.31 79 TABLE 2 continued No. Peptide structure ydio MoIWt SEQ ID NO: (base peptide) 79 Biotin- SGSGNAYQGADVERLPVTL-NH2 0.31 2,159.42 80 80 Biotin- ΞGΞGADVERLPVTLADAAV-NH2 0.35 2,053.34 81 81 Biotin- SGSGLPVTLADAAVLFEDS-NH2 0.49 2,074.35 82 82 Biotin- SGSGADAAVLFEDSALVRE-NH2 0.30 2,119.35 83 83 Biotin- SGSGLFEDSALVREAFGED-NH2 0.27 2,211.41 84 84 Biotin- SGSGALVREAFGEDWAHY-NH2 0.35 2,189.45 85 85 Biotin- ΞGSGAFGEDWAHYLNNAR-NH2 0.27 2,189.41 86 86 Biotin- SGSGWAHYLNNARVELAA-NH2 0.37 2,153.46 87 87 Biotin- SGSGLNNARVELAAFNAAV-NH2 0.34 2,086.37 88 88 Biotin- SGSGVELAAFNAAVTDWER-NH2 0.35 2,205.45 89 89 Biotin- SGSGFNAAVTDWERIRGFE-NH2 0.30 2,324.58 90 90 Biotin- SGSGAAVTDWERIRGFERL-OH 0.27 2,333.63 91 3. ELISA Assay Nunc-Immuno module maxisorp wells were coated overnight at room temperature or at 4°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 200 μl 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 or overnight, 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, 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, 80μl 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.

The following controls were used in the ELISA screening: (i) negative control - streptavidin/peptide/no serum/conjugate; (ii) peptide control - streptavidin/no peptide/patient serum/conjugate; (iii) positive control - streptavidin/control peptide/serum containing antibodies against control peptide/conjugate (iv) serum background - non streptavidin/no peptide/patient serum/conjugate 4. 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 the mean and standard deviation estimated by a robust M- Estimator.

The immunogenic peptides identified were categorized into two groups, namely weakly positive (WP) or positive. The tool used to make this distinction was the Biomarker Identification Package (Emphron Informatics Pty Ltd).

Based on manual interpretation of results and the results obtained using the Biomarker Identification Package it was determined that a result greater than 6 standard deviations above background or 8 standard deviations above background designate the WP and positive categories, respectively.

ELISA results demonstrate that subjects suffering from tuberculosis raise antibodies against specific peptide or regions of GS that do not appear in control subjects. Approximately 50% of TB subjects had raised antibodies against GS. This was also observed when data were separated into specific racial groups (Chinese or South African) or HIV status.

In all cases the immune responses observed in subjects were across a range of peptides spanning the GS PEPSET suggesting that the immune response against GS in TB subjects may spread across the whole protein or against one or more conformational epitopes. Example 5 Isolation of monoclonal antibodies that bind to M. tuberculosis GS peptide 1. Antigen selection The results of the ELISA assays described in Example 4 were used to determine peptides 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). As shown in Figure 1, glnA4 shares only 25% amino acid sequence identity with other known glutamine synthetase homologs.

Peptides were selected that correspond to regions of GS-A4 that are not conserved with other glutamine synthetases. Two of these regions are highlighted in bold in Figure 1.

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) RGTDGSAVFADSNGPHGMSSMFRSFC (SEQ ID NO: 92); and (ii) WASGYRGLTPASDYNIDYAIC (SEQ ID NO: 94)

As shown in Figure 1, the region corresponding to the peptide comprising the amino aid sequence set forth in SEQ ID NO: 92 (and SEQ ID NO: 93) is not conserved between glutamine synthetase polypeptides. Furthermore, the region corresponding to the peptide comprising the amino aid sequence set forth in SEQ ID NO: 94 is not conserved between glutamine synthetase polypeptides. Accordingly, an antibody that selectively binds any of these peptides is unlikely to cross-react with another glutamine synthetase.

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 RGTDGSAVFADSNGPHGMSSMFRSFC (set forth in SEQ ID NO: 92) 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 Ascited 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 20ug/ml 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 3 a. The biotinylated GS peptide used had the sequence: SGSGRGTDGSAVFADSNGPHGMSSMFRSFC (SEQ ID NO: 93). The peptide was detected by binding of streptavidin HRP conjugate under standard conditions. Absorbances were determined at 450nm and 620nm (Tables 3b and 3c, respectively), and the difference in absorbance at 450nm and 620nm determined (Table 3d). Average data for duplicate samples are presented in Table 3e. Figure 2 shows the amount of peptide captured by mAb 426C. The data show that the antibodies capture the immunogenic GS peptide antigen at concentrations of about lOpg/ml or greater, at a signal:noise 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 titre the monoclonal antibodies, the peptide (i.e., SEQ ID NO: 93) was coated onto the bottom of the ELISA plate at a concentration of about 3ug/ml as shown in Table 4a. 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 lOpg/ml, as shown in Table 4b. Binding of the antibody was then detected using sheep anti-mouse HRP antibody conjugate under standard conditions. Absorbances were determined at 450nm and 620nm (Tables 4c and 4d, respectively), and the difference in absorbance at 450nm and 620nni determined (Table 4e). Average data are presented in Table 4f and Figure 3. The data show that the antibody successfully detects GS above assay background at concentrations of antibody as low as 10pg/ml, therefore demonstrating efficacy as a detection reagent in immunoassays.

Example 6 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 synthase (Example 5), at a concentration of 20ug/ml in water, was coated onto the bottom of one or more NUNC plates. Plates were left to dry at 37⁰C 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 H₂SO₄ (50 ul/well). Absorbances of each well was read at wavelengths of 450nm and 620nm, and the differences in these wavelengths is determined (i.6.A₄₅₀- A₆₂₀).

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 from 45 South African subjects with confirmed TB were screened and compared with 19 (black) control sera and 14 (white) control sera. Three other South African sera 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, 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 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.

The results of the ELISA assays are summarized in Tables 5 a and 5b.

Example 7 Point-of-care test for diagnosing an active infection by M. tuberculosis using rnAb 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 4O⁰C 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 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 are incubated with the nitrocellulose strip membrane for 30 seconds and the pre-incubation frame is pushed down onto the base of the test. 3. After about 1 minute, 2-4 drops of wash solution (0.5% Tween 20 in 0.1M phosphate buffer) is added to the pre-incubation well and allowed to flow through the device. 4. The pre-incubation frame is removed and the signal read by visually interpreted or read in a Readrite optical reader.

In a modification of this example, additional antibodies targeted against other specific epitopes on the same or different M. tuberculosis antigen are employed alongside mAb 426C. Additionally, the present invention clearly encompasses conjugation of the anti- IgG and/or anti-IgA antibody to the same gold particle to ensure the same amount of label is applied in each test. The gold particles may also be dried onto the pre- incubation pads, to thereby avoid the later addition of conjugate. Sensitivity of the assay may also be improved by increasing the amount of sera tested in each sample.

Example 8 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: 94) 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. Polyclonal sera having the highest titer are determined using ELISA. Mice having polyclonal antibody titers of at least 1,000 are used for the ABL-MYC infection process.

Infection The spleens of 3 mice having the highest titer of polyclonal antibodies cross-reactive with peptide antigen are used for the ABL-MYC infection, according to NeoClone's standard infection procedure.

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 ten performed essentially as described in Example 5 TABLE 3a Key to ELISA Plates for Determining Titre of mAb 426C

A, B: mAb 426C and GS peptide C, D: mAb 426C without peptide E, F: mAb 426C and negative control peptide G, H: mAb 426C without peptide TABLE 3b Absorbance at 4S0nm for ELISA late shown in Table 3a

A₅ B: mAb 426C and GS peptide C₃ D: mAb 426C without peptide E, F: mAb 426C and negative control peptide O O G₃ H: mAb 426C without peptide

A, B: mAb 426C and GS peptide C, D: mAb 426C without peptide E, F: mAb 426C and negative control peptide G, H: mAb 426C without peptide TABLE 3a Key to ELISA Plates for Determining Titre of mAb 426C showin jS ind ne ative control e tide concentrations /ml used in du licate sam les

</> C CD

A, B: mAb 426C and GS peptide m C, D: mAb 426C without peptide </> O X E, F: mAb 426C and negative control peptide K) m m G, H: mAb 426C without peptide Ti c ι- m σ> TABLE 3b Absorbance at 450nm for ELISA plate shown in Table 3a

</> C CD

A, B: mAb 426C and GS peptide C, D: mAb 426C without peptide m E, F: mAb 426C and negative control peptide </> O X G, H: mAb 426C without peptide m m TJ c ι- m σ> TABLE 3c Absorbance at 620nm for ELISA late shown in Table 3 a

</> C CD

A, B: mAb 426C and GS peptide m </> X C, D: mAb 426C without peptide m m E, F: mAb 426C and negative control peptide G, H: mAb 426C without peptide TJ c ι- m σ> TABLE 3d Difference between absorbance at 450nm and absorbance at 620nm for ELISA plate shown in Table 3 a

</> C CD

m A₃B: mAb426C and GS peptide </> o X C₃D: mAb426C without peptide m m E₃F: mAb426C and negative control peptide G₃H: mAb426C without peptide TJ c ι- m σ> TABLE 3e Average Difference in Absorbance at 450nm relative to Absorbance at 620nm for ELISA plate shown in Table 3 a

</> C

CD A, B: mAb 426C and GS peptide C, D: mAb 426C without peptide E, F: mAb 426C and negative control peptide G, H: mAb 426C without peptide m </> O X m m Ti c ι- m σ> TABLE 4a Ke to ELISA Plates for Determinin Titre of mAb 426C showin GS e tide concentration

</> C CD

m </> o TABLE 4b -4 X m m Key to ELISA Plates for Determining Titre of mAb 426C showing mAb concentration used in duplicate samples Ti c ι- m σ>

A₃ B₅ E₃F: mAb 426C C,D, G₅H: negative control mAb </> C CD

A₅ B: GS peptide and mAb 426C m C₃D: GS peptide and negative control mAb </> O X E₅F: No peptide andmAb 426C m m G,H: No peptide and negative control mAb Ti c ι- m σ> TABLE 4d Absorbance at 620nm for ELISA plate shown in Table 4a and Table 4b

</> C CD

A₃ B: GS peptide and mAb 426C C₅D: GS peptide and negative control mAb m E₃F: No peptide and mAb 426C </> O X G₃H: No peptide and negative control mAb m m Ti c ι- m σ> TABLE 4e Difference in Absorbance at 450nm relative to Absorbance at 620nm for ELISA plate shown in Table 4a and Table 4b

</> C CD

A, B: GS peptide and mAb 426C m C,D: GS peptide and negative control mAb </> X E₅F: No peptide and mAb 426C m m G₅H: No peptide and negative control mAb Ti c ι- m σ> TABLE 4f Average Difference in Absorbance at 450nm relative to Absorbance at 620nm for ELISA plate shown in Table 4a and Table 4b

</> C

CD A, B: GS peptide and mAb 426C C₅D: GS peptide and negative control mAb E₅F: No peptide and mAb 426C G₅H: No peptide and negative control mAb m </> X m m Ti c ι- m σ> TABLE 4a Specificity and sensitivity of ELISA assay that detects CIC comprising GS for diagnosing TB in South African and Chinese populations

Ti c TP = true positives ι- m FN = False negatives σ> FP = False positives TN = True negatives TABLE 4b Overall specificity and sensitivity of ELISA assay that detects CIC comprising GS for diagnosing TB

</> C CD

m </> M X m

m Ti TP = true positives c ι- m FN = False negatives σ> FP = False positives TN = True negatives

Claims

We Claim: 1. An isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof.

2. The isolated or recombinant immunogenic GS protein of M. tuberculosis according to claim 1 comprising 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.

3. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 1 wherein said peptide is a synthetic peptide.

4. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 1 wherein said peptide, fragment or epitope comprises at least about 5 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1.

5. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 1 wherein said peptide, fragment or epitope comprises at least about 10 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1.

6. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 1 wherein said peptide, fragment or epitope comprises at least about 15 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1.

7. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 1 wherein said peptide, fragment or epitope comprises 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-teπninus and/or the C- terminus.

8. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 7 wherein said peptide, fragment or epitope comprises an amino acid sequence set forth in any one of SEQ ID Nos: 2-91 or 94 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.

9. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 1 wherein said peptide, fragment or epitope comprises an amino acid sequence of at least about 5 consecutive amino acid residues positioned between about residue 265 to about residue 300 of SEQ ID NO: 1.

10. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 9 wherein said peptide, fragment or epitope comprises at least about 5 consecutive amino acid residues positioned between about residue 270 to about residue 295 of SEQ ID NO : 1.

11. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 9 wherein said peptide, fragment or epitope comprises at least about 5 consecutive amino acid residues positioned between residue 271 to residue 295 of SEQ ID NO: 1.

12. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 1 wherein said peptide, fragment or epitope comprises at least 5 consecutive residues of the sequence set forth in SEQ ID NO: 92.

13. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 9 further 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.

14. The immunogenic GS peptide or immunogenic GS fragment or epitope according to claim 1 comprising an amino acid sequence set forth in SEQ ID NO: 92, 93 or 94 or an immunologically cross-reactive variant thereof comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO: 92, 93 or 94.

15. The isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to claim 1 wherein said protein, peptide, fragment or epitope comprises one or more labels or detectable moieties to facilitate detection or isolation or immobilization.

16. The isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to claim 15 wherein the label is biotin, glutathione-S-transferase (GST), FLAG epitope, hexahistidine, or β-galactosidase.

17. A fusion protein comprising one or more immunogenic GS peptides, fragments or epitopes according to claim 1.

18. The fusion protein according to claim 17 comprising glutathione-S-transferase (GST), FLAG epitope, hexahistidine, or β-galactosidase.

19. An isolated protein aggregate comprising between one or more isolated or recombinant immunogenic glutamine synthetase (GS) proteins of Mycobacterium tuberculosis or one or more immunogenic GS peptides, fragments or epitopes thereof according to claim 1.

20. The isolated protein aggregate according to claim 19 comprising immunoglobulin.

21. Use of the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to claim 1 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 GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope.

22. Use of the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to claim 1 to elicit the production of antibodies that bind to M. tuberculosis glutamine synthetase.

23. Use of the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to claim 1 in the preparation of a medicament for immunizing a subject against infection by M. tuberculosis.

24. An isolated or recombinant antibody or immune reactive fragment of an antibody that binds specifically to the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16 or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope.

25. The isolated antibody of claim 24 wherein the antibody is a polyclonal antibody.

26. The isolated antibody of claim 24 wherein the antibody is a monoclonal antibody.

27. An isolated antibody-producing cell or antibody-producing cell population that produces the monoclonal antibody of claim 26.

28. Use of the isolated or recombinant antibody according to claim 24 or an 5 immune-reactive fragment thereof for detecting a past or present infection or a latent infection by M. tuberculosis in a subject, wherein said infection is determined by the binding of the antibody or fragment to M. tuberculosis GS protein or an immunogenic fragment or epitope thereof present in a biological sample obtained from the subject.

10 29. Use of the isolated or recombinant antibody according to claim 24 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.

15 30. Use of the isolated or recombinant antibody according to claim 24 or an immune-reactive fragment thereof in medicine.

31. A composition comprising the isolated or recombinant antibody according to claim 24 and a pharmaceutically acceptable carrier, diluent or excipient. 0

32. A method of diagnosing tuberculosis or infection by M. tuberculosis in a subject comprising detecting in a biological sample from said subject an immunogenic GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof, wherein the presence of said protein or immunogenic fragment or epitope in 5 the sample is indicative of disease, disease progression or infection.

33. The method of claim 32 wherein said method comprises contacting a biological sample derived from the subject with one or more antibodies capable of binding to a GS protein or an immunogenic fragment or epitope thereof, and detecting the 0 formation of an antigen-antibody complex.

34. The method of claim 33 wherein an antibody is an isolated or recombinant antibody or immune reactive fragment of an antibody that binds specifically to the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16 or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope.

35. The method of claim 34 wherein an antibody is a polyclonal antibody.

36. The method of claim 34 wherein an antibody is a monoclonal antibody.

37. The method of claim 32 wherein the subject is immune compromised or immune deficient.

38. The method of claim 32 wherein the subject is HIV+.

39. The method of claim 32 wherein the sample comprises an extract from a tissue selected from the group consisting of brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle, bone and mixtures thereof.

40. The method of claim 32 wherein the sample comprises body fluid selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.

41. The method of claim 32 wherein the sample is derived from body fluid selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.

42. 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 GS 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.

43. The method of claim 42 wherein said method comprises contacting a biological sample derived from the subject with one or more antibodies capable of binding to a GS protein or an immunogenic fragment or epitope thereof, and detecting the formation of an antigen-antibody complex.

44. The method of claim 43 wherein an antibody is an isolated or recombinant antibody or immune reactive fragment of an antibody that binds specifically to the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16 or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope.

45. The method of claim 43 wherein an antibody is a polyclonal antibody.

46. The method of claim 43 wherein an antibody is a monoclonal antibody.

47. The method of claim 42 wherein the subject is immune compromised or immune deficient.

48. The method of claim 42 wherein the subject is HTV+.

49. The method of claim 42 wherein the sample comprises an extract from a tissue selected from the group consisting of brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle, bone and mixtures thereof.

50. The method of claim 42 wherein the sample comprises body fluid selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.

51. The method of claim 42 wherein the sample is derived from body fluid selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.

52. 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 GS 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.

53. The method of claim 52 wherein said method comprises contacting a biological sample derived from the subject with one or more antibodies capable of binding to a GS protein or an immunogenic fragment or epitope thereof, and detecting the formation of an antigen-antibody complex.

54. The method of claim 53 wherein an antibody is an isolated or recombinant antibody or immune reactive fragment of an antibody that binds specifically to the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16 or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope.

55. The method of claim 54 wherein an antibody is a polyclonal antibody.

56. The method of claim 54 wherein an antibody is a monoclonal antibody.

57. The method of claim 52 wherein the subject is immune compromised or immune deficient.

58. The method of claim 52 wherein the subject is HIV+.

59. The method of claim 52 wherein the sample comprises an extract from a tissue selected from the group consisting of brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle, bone and mixtures thereof.

60. The method of claim 52 wherein the sample comprises body fluid selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.

61. The method of claim 52 wherein the sample is derived from body fluid selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.

62. A method of monitoring disease progression, responsiveness to therapy or infection status by M. tuberculosis in a subject comprising determining the level of a GS 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 GS protein, fragment or epitope indicates a change in disease progression, responsiveness to therapy or infection status of the subject.

63. The method of claim 62 further comprising administering a compound for the treatment of tuberculosis or infection by M. tuberculosis when the level of GS protein, fragment or epitope increases over time.

5 64. The method of claim 63 wherein said method comprises contacting a biological sample derived from the subject with one or more antibodies capable of binding to a GS protein or an immunogenic fragment or epitope thereof, and detecting the formation of an antigen-antibody complex.

10 65. The method of claim 64 wherein an antibody is an isolated or recombinant antibody or immune reactive fragment of an antibody that binds specifically to the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16 or to a fusion 15 protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope.

66. The method of claim 64 wherein an antibody is a polyclonal antibody.

0 67. The method of claim 64 wherein an antibody is a monoclonal antibody.

68. The method of claim 62 wherein the subject is immune compromised or immune deficient.

5 69. The method of claim 62 wherein the subject is HIV+.

70. The method of claim 62 wherein the sample comprises an extract from a tissue selected from the group consisting of brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle, bone and mixtures thereof. 0

71. The method of claim 62 wherein the sample comprises body fluid selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.

72. The method of claim 62 wherein the sample is derived from body fluid selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.

73. A method of diagnosing tuberculosis or an infection by M. tuberculosis in a subject comprising detecting in a biological sample from said subject antibodies against an immunogenic GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof, wherein the presence of said antibodies in the sample is indicative of infection.

74. The method of claim 73 comprising contacting a biological sample derived from the subject with the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to claim 1 for a time and under conditions sufficient for an antigen-antibody complex to form and then detecting the formation of an antigen-antibody complex.

75. The method of claim 74 wherein the sample comprises blood or serum or an immunoglobulin fraction obtained from the subject.

76. The method of claim 33 wherein the sample derived from the subject comprises one or more circulating immune complexes comprising immunoglobulin (Ig) bound to glutamine synthetase (GS) protein of Mycobacterium tuberculosis or one or more immunogenic GS 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.

77. The method of claim 43 wherein the sample derived from the subject comprises one or more circulating immune complexes comprising immunoglobulin (Ig) bound to glutamine synthetase (GS) protein of Mycobacterium tuberculosis or one or more immunogenic GS 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.

78. The method of claim 53 wherein the sample derived from the subject comprises one or more circulating immune complexes comprising immunoglobulin (Ig) bound to glutamine synthetase (GS) protein of Mycobacterium tuberculosis or one or more immunogenic GS 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.

79. The method of claim 64 wherein the sample derived from the subject comprises one or more circulating immune complexes comprising immunoglobulin (Ig) bound to glutamine synthetase (GS) protein of Mycobacterium tuberculosis or one or more immunogenic GS 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.

80. A method of treatment of tuberculosis or infection by M. tuberculosis comprising: (i) performing the method according to claim 32 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.

81. A method of treatment of tuberculosis or infection by M. tuberculosis comprising: (i) performing the method according to claim 42 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.

82. A pharmaceutical composition comprising the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to claim 1 in combination with a pharmaceutically acceptable diluent.

83. The pharmaceutical composition of claim 82 further comprising an adjuvant.

84. Isolated nucleic acid encoding the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to claim 1.

85. A cell expressing the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to claim 1.

86. The cell of claim 85 wherein said cell is an antigen- presenting cell (APC) that expresses the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to claim 1 on its surface.

87. A kit for detecting M. tuberculosis infection in a biological sample, said kit comprising: (iii) one or more isolated antibodies or immune reactive fragments thereof that bind specifically to the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16 or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope; and (iv) means for detecting the formation of an antigen-antibody complex, optionally packaged with instructions for use.

88. A kit for detecting M. tuberculosis infection in a biological sample, said kit comprising: (iii) isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16; and (iv) means for detecting the formation of an antigen-antibody complex, optionally packaged with instructions for use.

89. A method for detecting an immune response against an antigen in a subject, said method comprising: (i) contacting an antibody-containing biological sample obtained from the subject with an isolated or recombinant antibody that binds to the antigen for a time and under conditions sufficient for an antigen-antibody complex to form; (ii) contacting the antigen-antibody complex with an anti-Ig antibody for a time and under conditions sufficient for the anti-Ig antibody to bind to an immunoglobulin in the sample; and (iii) detecting the bound anti-Ig antibody, wherein binding complex formation at (i) and (ii) is indicative of an immune response in the subject to the antigen..

90. The method of claim 89 wherein the antigen is a protein of a pathogen or an immunogenic peptide or immunogenic fragment or epitope of said protein.

91. The method of claim 90 wherein the pathogen is a virus.

92. The method of claim 90 wherein the pathogen is a bacterium.

93. The method of claim 92 wherein the bacterium is Mycobacterium tuberculosis.

94. The method of claim 89 wherein the antigen comprises the isolated or recombinant GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16 or a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope.

95. The method of claim 89 wherein the isolated or recombinant antibody that binds to the antigen is a polyclonal antibody.

96. The method of claim 89 wherein the isolated or recombinant antibody that binds to the antigen is a monoclonal antibody.

97. The method of claim 89 wherein the isolated or recombinant antibody that binds to the antigen is an isolated or recombinant antibody or immune reactive fragment of an antibody that binds specifically to the isolated or recombinant immunogenic glutamine synthetase (GS) protein of Mycobacterium tuberculosis or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16 or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope. 5

98. The method of claim 89 wherein the antibody-containing biological sample comprises an extract from a tissue selected from the group consisting of brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle, bone and mixtures thereof.

10 99. The method of claim 89 wherein the antibody-containing biological sample comprises body fluid selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.

100. The method of claim 89 wherein the antibody-containing biological sample is 15 derived from body fluid selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.

101. The method according to claim 89 wherein the anti-Ig antibody binds to IgM.

0 102. The method according to claim 89 wherein the anti-Ig antibody binds to IgA.

103. The method according to claim 89 wherein the anti-Ig antibody binds to IgG.

104. The method according to claim 89 wherein the anti-Ig antibody binds to human 5 Ig.

105. The method according to claim 89 wherein the anti-Ig antibody is conjugated to a detectable label.

0 106. A process for diagnosing an infection in a subject comprising performing the method according to claim 89 to thereby determine an immune response in the subject to an antigen, wherein the antigen is an antigen of an infectious agent producing said infection.

107. A process for diagnosing a disease or disorder associated with the presence of one or more circulating immune complexes (CIC) in a subject's immune system said process comprising performing the method according to claim 89 to thereby determine an immune response in the subject to an antigen, wherein the antigen is present in the subject's immune system as a CIC.

108. The process according to claim 107 wherein the disease is tuberculosis.

109. The process according to claim 107 wherein the disease is bovine tuberculosis.

110. The process according to claim 107 wherein a disease is Johne's disease.

111. The process according to claim 107 wherein a disease is Crohne's disease.

112. A solid matrix having adsorbed thereto an isolated or recombinant GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16 or a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope.

113. The solid matrix according to claim 112 comprising a membrane.

114. The solid matrix according to claim 113 wherein the membrane comprises nylon or nitrocellulose.

115. The solid matrix according to claim 112 comprising a polystyrene or polycarbonate microwell plate.

116. The solid matrix according to claim 112 comprising a dipstick.

117. The solid matrix according to claim 112 comprising a glass support.

118. The solid matrix according to claim 112 comprising a chromatography resin.

119. A solid matrix having adsorbed thereto an antibody that binds to an isolated or recombinant GS protein or an immunogenic GS peptide or immunogenic GS fragment or epitope thereof according to any one of claims 1 to 16 or to a fusion protein or protein aggregate comprising said immunogenic glutamine synthetase (GS) protein, peptide, fragment or epitope.

120. The solid matrix according to claim 119 comprising a membrane.

121. The solid matrix according to claim 120 wherein the membrane comprises nylon or nitrocellulose.

122. The solid matrix according to claim 119 comprising a polystyrene or polycarbonate microwell plate.

123. The solid matrix according to claim 119 comprising a dipstick.

124. The solid matrix according to claim 119 comprising a glass support.

125. The solid matrix according to claim 119 comprising a chromatography resin.