WO2007087679A1 - Procédé de diagnostic et de traitement d'une infection à m. tuberculosis et réactifs correspondants - Google Patents

Procédé de diagnostic et de traitement d'une infection à m. tuberculosis et réactifs correspondants Download PDF

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Publication number
WO2007087679A1
WO2007087679A1 PCT/AU2007/000093 AU2007000093W WO2007087679A1 WO 2007087679 A1 WO2007087679 A1 WO 2007087679A1 AU 2007000093 W AU2007000093 W AU 2007000093W WO 2007087679 A1 WO2007087679 A1 WO 2007087679A1
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Prior art keywords
protein
immunogenic
tuberculosis
peptide
antibody
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PCT/AU2007/000093
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English (en)
Inventor
Andrew Sloane
Susanne Pedersen
Robert Cole
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Proteome Systems Limited
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Priority claimed from AU2006900452A external-priority patent/AU2006900452A0/en
Application filed by Proteome Systems Limited filed Critical Proteome Systems Limited
Priority to CA002638761A priority Critical patent/CA2638761A1/fr
Publication of WO2007087679A1 publication Critical patent/WO2007087679A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to novel diagnostic, prognostic and therapeutic reagents for infection of an animal subject such as a human by M. tuberculosis, and conditions associated with such infections, such as, for example, tuberculosis. More particularly, the present invention provides the first enabling disclosure of the expression in an infected subject of a protein of M. tuberculosis designated "S9" (SEQ ID NO: 1) and immunogenic epitopes thereof suitable for the preparation of immunological reagents, such as, for example, antigenic proteins/peptides an ⁇ Vo ⁇ antibodies, for the diagnosis, prognosis and therapy of infection, and vaccine development.
  • S9 protein of M. tuberculosis designated "S9" (SEQ ID NO: 1)
  • immunogenic epitopes thereof suitable for the preparation of immunological reagents, such as, for example, antigenic proteins/peptides an ⁇ Vo ⁇ antibodies, for the diagnosis, prognosis and therapy of infection, and vaccine development.
  • 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.
  • 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:
  • Tuberculosis is a chronic, infectious disease that is generally caused by infection with Mycobacterium tuberculosis. It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about eight million new cases and three million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as an acute inflammation of the lungs, resulting in fever and a productive cough. If left untreated, M. tuberculosis infection may progress beyond the primary infection site in the lungs to any organ in the body and generally results in serious complications and death.
  • tuberculosis maintains and multiplies within the host
  • 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.
  • 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.
  • PPD purified protein derivative
  • tuberculosis or a peptide fragment derived there from has efficacy as a diagnostic reagent in an immunoassay format, or is suitable for use in a vaccine preparation, it is necessary to show that the protein is expressed during infectious cycle of the bacterium, and that the host organism mounts an immune response to the protein, and/or to a peptide fragment that comprises a B cell epitope or T-cell epitope (e.g., CD8 + -restricted CTL epitope).
  • a B cell epitope or T-cell epitope e.g., CD8 + -restricted CTL epitope
  • M. tuberculosis The ability to grow M. tuberculosis in culture has provided a convenient model to identify expressed tuberculosis proteins in vitro.
  • 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.
  • a host response comprising the recruitment of monocytes and macrophages to the site of infection.
  • monocytes and macrophages 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.
  • 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.
  • 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.
  • M. tuberculosis proteins from logarithmic phase cultures does not necessarily suggest which proteins are expressed or highly immunogenic in each environment in vivo.
  • 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.
  • 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.
  • 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.
  • the inventors sought to elucidate the range of proteins expressed by M. tuberculosis in a range of in vivo environments, to thereby identify highly expressed and/or highly immunogenic M. tuberculosis proteins.
  • the inventors used a proteomics approach to identify M. tuberculosis proteins expressed in vivo and present in the body fluids of a cohort of diseased patients, including sputum, pleural fluid, plasma and serum.
  • An M. tuberculosis protein was identified in vivo by 2-dimensional electrophoresis of immunoglobulin-containing sera, or alternatively, mixtures of sera and plasma, obtained previously from a cohort of M. tuberculosis-infected patients.
  • the amino acid sequences of peptide fragments were determined by mass spectrometry, and shown to align to the amino acid sequence of the 3OS ribosomal protein postulated to be encoded by the M. tuberculosis genome, designated "S9" (SEQ ID NO: 1).
  • matched peptides aligned to amino acid residues 71-78 of the putative S9 protein i.e., sequence APLVTVDR (SEQ ID NO: 2); amino acid residues 64-70 of the putative S9 protein i.e., sequence VHQQLIK (SEQ ID NO: 3); amino acid residues 49-54 of the putative S9 protein i.e., sequence FDLNGR (SEQ ID NO: 4); and amino acid residues 64-78 of the putative S9 protein i.e., sequence VHQQLIKAPLVTVDR (SEQ ID NO: 5).
  • the inventors have also made antibodies that bind to S9-derived peptides for the development of antigen-based diagnostic and prognostic assays.
  • antibodies have been prepared against recombinant Sp protein by immunization of chickens and mice, and against a synthetic peptide comprising the N-terminal 21 amino acid residues of the S9 protein (i.e., SEQ ID NO: 6) bound to keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • an N-terminal sequence of S9 protein was produced with a C-terminal spacer sequence and attached to biotin (SEQ ED NO: 7).
  • antibodies raised against the N- terminal peptide were shown to bind to isolated S9 protein, the peptide immunogen in Western blots, endogenous S9 protein in clinical samples e.g., sputum, and S9 protein in the cytosol and membrane fractions of Mycobacterium tuberculosis strain H37Rv.
  • antibodies prepared against full-length recombinant S9 protein are useful in ELISA and sandwich ELISA assays for detecting expression of S9 protein in both clinical and laboratory M. tuberculosis isolates, and for detecting S9 protein at very low levels and in samples comprising serum or sputum.
  • Antibodies against the full-length recombinant protein are high-affinity antibodies capable of detecting M. tuberculosis S9 protein at sub-nanogram/ml or sub- picogram/ml levels.
  • multianalyte assays e.g., using an antibody that binds to S9 protein and antibodies that bind to one or more M. tuberculosis proteins e.g., Bsx protein and/or glutamine synthetase (GS) protein
  • high sensitivity and specificity are also achieved.
  • a multianalyte assay using an antibody that binds to S9 protein and Bsx protein to screen cohorts comprising about 14-20 samples sensitivity is about 83% and specificity is about 85%.
  • Antibodies that bind to the amino acid sequence set forth in SEQ ID NO: 1 or a B-cell epitope thereof have also been shown to be present in subjects during extrapulmonary infection by M. tuberculosis, in at least one population.
  • the detection of such antibodies is a suitable assay readout for the diagnosis of tuberculosis.
  • the inventors determined that recombinant S9 protein comprising the sequence set forth in SEQ ED NO: 1 and peptides comprising the immunodominant B-cell epitope within SEQ ID NO: 2-7 are useful in antibody-based diagnostic tests for tuberculosis, including multianalyte tests, by virtue of their high sensitivity and specificity.
  • peptides derived from the full-length sequence of the S9 protein are also useful for such tests, e.g., as primary ligands or as secondary ligands in a multi-analyte assay format, by virtue of their high specificity.
  • 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.
  • the S9 protein or a B-cell epitope thereof is useful for the early diagnosis of infection or disease. It will also be apparent to the skilled person that such prognostic indicators as described herein may be used in conjunction with therapeutic treatments for tuberculosis or an infection associated therewith.
  • the present invention provides the means for producing novel diagnostics for the detection of M. tuberculosis infection in a subject, and novel prognostic indicators for the progression of infection or a disease state associated therewith, either by detecting S9 solus or as part of a multi-analyte test.
  • 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.
  • the present invention provides an isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof.
  • the isolated or recombinant immunogenic S9 protein of M. tuberculosis comprises the amino acid sequence set forth in SEQ ID NO: 1 or having an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1.
  • the immunogenic S9 peptide is a synthetic peptide.
  • the S9 peptide, fragment or epitope comprises at least about 5 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1, more preferably at least about 10 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1, even more preferably at least about 15 consecutive amino acid residues of the sequence set forth in SEQ ED NO: 1, and still more preferably at least about 5 consecutive amino acid residues of the sequence set forth in SEQ ID NO: 1 fused to about 1-5 additional amino acid residues at the N-terminus and/or the C-terminus.
  • the S9 peptide, fragment or epitope comprises an amino acid sequence set forth in any one of SEQ ID Nos: 2-7 and preferably, a sequence selected from the group consisting of SEQ ID NOs: 6 and 7, and more preferably SEQ ID NO: 6, or an immunologically cross-reactive variant of any one of said sequences that comprises an amino acid sequence that is at least about 95% identical thereto.
  • a preferred immunogenic S9 peptide, fragment or epitope comprises an amino acid sequence of at least about 5 consecutive amino acid residues positioned between about residue 1 to about residue 50 of SEQ ID NO: 1, more preferably at least about 5 consecutive amino acid residues positioned between about residue 1 to about residue 25 of SEQ ID NO: 1. Still more preferably, a preferred immunogenic S9 peptide, fragment or epitope comprises an amino acid sequence of at least about 5 consecutive amino acid residues positioned between residue 1 to residue 20 of SEQ ID NO: 1, corresponding to at least 5 consecutive residues of the sequence set forth in SEQ ID NO: 6. This includes any peptides comprising an N-terminal extension of up to about 5 amino acid residues in length and/or a C-terminal extension of up to about 5 amino acid residues in length.
  • the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof to comprise one or more labels or detectable moieties e.g., to facilitate detection or isolation or immobilization.
  • Preferred labels include, for example, biotin, glutathione-S-transferase (GST), FLAG epitope, hexa-histidine, ⁇ -galactosidase, horseradish peroxidase, streptavidin or gold.
  • the present invention also provides a fusion protein comprising one or more immunogenic S9 peptides, fragments or epitopes according to any embodiment described herein.
  • a fusion protein comprising one or more immunogenic S9 peptides, fragments or epitopes according to any embodiment described herein.
  • the N-terminal and C-terminal portions of S9 protein can be fused.
  • an internal linking residue e.g., cysteine in such compositions of matter.
  • a preferred fusion protein comprises a linker separating an immunogenic S9 peptide from one or more other peptide moieties, such as, for example, a single amino acid residue (e.g., glycine, cysteine, lysine), a peptide linker (e.g., a non-immunogenic peptide such as a poly-lysine or poly-glycine), poly-carbon linker comprising up to about 6 or 8 or 10 or 12 carbon residues, or a chemical linker.
  • linkers may facilitate antibody production or vaccine formulation e.g., by permitting linkage to a lipid or hapten, or to permit cross-linking or binding to a ligand.
  • the expression of proteins as fusions may also enhance their solubility.
  • Preferred fusion proteins will comprise the S9 protein, peptide, fragment or epitope fused to a carrier protein, detectable label or reporter molecule e.g., glutathione-S- transferase (GST), FLAG epitope, hexa-histidine, ⁇ -galactosidase, thioredoxin (TRX) (La Vallie et ah, Bio/Technology 11, 187-193, 1993), maltose binding protein (MBP), Escherichia coli NusA protein (Fayard, E.M.S., Thesis, University of Oklahoma, USA, 1999; Harrison, inNovations 11, 4-7, 2000), E. coli BFR (Harrison, inNovations 11, A- 7, 2000) and E. coli GrpE (Harrison, inNovations 11, A-I, 2000).
  • GST glutathione-S- transferase
  • FLAG epitope hexa-histidine
  • the present invention also provides an isolated protein aggregate comprising one or more immunogenic S9 peptides, fragments or epitopes according to any embodiment described herein.
  • Preferred protein aggregates will comprise the protein, peptide, fragment or epitope complexed to an immunoglobulin e.g., IgA, IgM or IgG, such as, for example as a circulating immune complex (CIC).
  • ICC circulating immune complex
  • Exemplary protein aggregates may be derived, for example, derived from an antibody-containing biological sample of a subject.
  • the present invention also encompasses the use of the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein for detecting a past or present infection or latent infection by. M, tuberculosis in a subject, wherein said infection is determined by the binding of antibodies in a sample obtained from the subject to said isolated or recombinant immunogenic S9 protein or an immunogenic S9 peptide or immunogenic S9 fragment or epitope.
  • the present invention also encompasses the use of the isolated or recombinant immunogenic S 9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein for eliciting the production of antibodies that bind to M. tuberculosis S9.
  • the present invention also encompasses the use of the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein in the preparation of a medicament for immunizing a subject e.g., to produce antibodies against the S9 protein and/or to protect against infection by M. tuberculosis.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein in combination with a pharmaceutically acceptable diluent, e.g., an adjuvant.
  • the present invention also provides an isolated nucleic acid encoding the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein eg., for the preparation of nucleic acid based vaccines or for otherwise expressing the immunogenic polypeptide, protein, peptide, fragment or epitope.
  • the present invention also provides a cell expressing the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein.
  • the cell may preferably consist of an antigen- presenting cell (APC) that expresses the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof e.g., on its surface.
  • APC antigen-presenting cell
  • the present invention also provides an isolated ligand, e.g., a small molecule, peptide, antibody, or immune reactive fragment of an antibody, that binds specifically to the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein, or to a fusion protein or protein aggregate comprising said immunogenic S9 protein, peptide, fragment or epitope.
  • Preferred ligands are peptides or antibodies.
  • Preferred antibodies include, for example, a monoclonal or polyclonal antibody preparation.
  • any isolated antibody- producing cell or antibody-producing cell population e.g., a hybridoma or plasmacytoma producing antibodies that bind to a S9 protein or immunogenic fragment of a S9 protein or other immunogenic peptide comprising a sequence derived from the sequence of a S9 protein.
  • the present invention also provides for the use of the isolated ligand according to any embodiment described herein, especially any peptide ligand, antibody or an immune- reactive fragment thereof in medicine.
  • the present invention also provides for the use of the isolated ligand according to any embodiment described herein, especially any peptide ligand, antibody or an immune- reactive fragment thereof for detecting a past or present (i.e., active) infection or a latent infection by M, tuberculosis in a subject, wherein said infection is determined by the binding of the ligand to M. tuberculosis S9 protein or an immunogenic fragment or epitope thereof present in a biological sample obtained from the subject.
  • the present invention also provides for the use of the isolated ligand according to any embodiment described herein, especially any peptide ligand, antibody or an immune- reactive fragment thereof for identifying the bacterium M. tuberculosis or cells infected by M. tuberculosis or for sorting or counting of said bacterium or said cells.
  • the isolated ligand according to any embodiment described herein, especially any peptide ligand, antibody or an immune-reactive fragment thereof, is also useful in therapeutic, diagnostic and research applications for detecting a past or present infection, or a latent infection, by M. tuberculosis as determined by the binding of the ligand to an M. tuberculosis S9 protein or an immunogenic fragment or epitope thereof present in a biological sample from a subject (i.e., an antigen-based immunoassay).
  • Other applications of the subject ligands include the purification and study of the diagnostic/prognostic S9 protein, identification of cells infected with M. tuberculosis, or for sorting or counting of such cells.
  • the ligands are also useful in therapy, including prophylaxis, diagnosis, or prognosis, and the use of such ligands for the manufacture of a medicament for use in treatment of infection by M, tuberculosis.
  • specific humanized antibodies or other ligands are produced that bind and neutralize a S9 protein or M, tuberculosis, especially in vivo.
  • the humanized antibodies or other ligands are used as in the preparation of a medicament for treating TB-specific disease or M. tuberculosis infection in a human subject, such as, for example, in the treatment of an active or chronic M. tuberculosis infection.
  • the present invention also provides a composition
  • a composition comprising the isolated ligand according to any embodiment described herein, especially any peptide ligand, antibody or an immune-reactive fragment thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention also provides a method of diagnosing tuberculosis or an infection by M. tuberculosis in a subject comprising detecting in a biological sample from said subject antibodies that bind to an immunogenic S9 protein or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof, the presence of said antibodies in the sample is indicative of infection.
  • the presence of said antibodies in the sample is indicative of infection.
  • the infection may be a past or active infection, or a latent infection, however this assay format is particularly useful for detecting active infection and/or recent infection.
  • the method may be an immunoassay, e.g., comprising contacting a biological sample derived from the subject with the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein (e.g., a peptide comprising an amino acid sequence set forth in any one of SEQ ID Nos: 2-7 and preferably, a sequence selected from the group consisting of SEQ ID NOs: 6 and 7, and still more preferably SEQ ID NO: 6, or an immunologically cross-reactive variant of any one of said sequences that comprises an amino acid sequence that is at least about 95% identical thereto) for a time and under conditions sufficient for an antigen-antibody complex to form and then detecting the formation of an antigen-antibody complex.
  • an immunoassay e.g., comprising contacting a biological sample derived from the subject with the isolated or recombinant immunogenic S9 protein of
  • the sample is an antibody-containing sample e.g., a sample that comprises blood or serum or an immunoglobulin fraction obtained from the subject.
  • the sample may contain circulating antibodies in the form of complexes with S9 antigenic fragments.
  • the antigen-antibody complex will be detected in such assay formats using antibodies capable of binding to the patient's immunoglobulin e.g., anti-human Ig antibodies.
  • the patient sample may be contacted with S9 or immunogenic S9 peptide or fragment or epitope and with a M.
  • tuberculosis Bsx protein e.g., SwissProt Database Accession No. 053759
  • immunogenic peptide derived there from e.g., a peptide derived from a Bsx protein, or comprising a sequence selected from the group consisting of: MRQLAERSGVSNPYL (SEQ ID NO: 8), ERGLRKPSADVLSQI (SEQ ID NO: 9), LRKPSADVLSQIAKA (SEQ ID NO: 10), PSADVLSQIAKALRV (SEQ ID NO: 11), SQIAKALRVSAEVLY (SEQ ID NO: 12), AKALRVSAEVLYVRA (SEQ ID NO: 13), VRAGILEPSETSQVR (SEQ ID No: 14), TAITERQKQILLDIY (SEQ ID NO; 15), SQIAKALRVS AEVLYVRAC (SEQ ID NO: 16), MSSEEKLCDPTPTDD (SEQ ID NO: 17) and
  • the patient sample may be contacted with S9 or immunogenic S9 peptide or fragment or epitope and with a M. tuberculosis glutamine synthetase (GS) protein (e.g., SwissProt Database Accession No. 033342) or immunogenic peptide derived there from, e.g., a peptide derived from a surface- exposed region of a GS protein, or comprising the sequence RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO: 19) or WASGYRGLTPASDYNIDYAI (SEQ ID NO: 20).
  • GS M. tuberculosis glutamine synthetase
  • GS M. tuberculosis glutamine synthetase
  • immunogenic peptide derived there from e.g., a peptide derived from a surface- exposed region of a GS protein, or comprising the sequence RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO:
  • tuberculosis are also described in detail in the instant applicant's co-pending International Patent Application No. PCT/AU2005/000930 filed June 24 2005 the disclosure of which is incorporated herein in its entirety.
  • Assays for one or more secondary analytes e.g., antibodies that bind to Bsx and/or glutamine synthetase, are conveniently performed in the same manner as for detecting antibodies that bind to S9 in serum or plasma or other body fluid.
  • the assays may be performed simultaneously or at different times, and using the same or different patient samples.
  • the assays may also be performed in the same reaction vessel, provided that different detection systems are used to detect the different antibodies, e.g., anti-human Ig labelled using different reporter molecules such as different coloured dyes, fluorophores, radionucleotides or enzymes.
  • different detection systems are used to detect the different antibodies, e.g., anti-human Ig labelled using different reporter molecules such as different coloured dyes, fluorophores, radionucleotides or enzymes.
  • 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.
  • a micro-organism in particular, a bacterium or a virus
  • Such an infection may be unapparent or result in local cellular injury.
  • the infection may be localised, subclinical and temporaiy or alternatively may spread by extension to become an acute or chronic clinical infection.
  • the infection may also be a past infection wherein residual S9 antigen, or alternatively, reactive host antibodies that bind to isolated S9 protein or peptides, remain in the host.
  • the infection may also be a latent infection, in which the microorganism is present in a subject, however the subject does not exhibit symptoms of disease associated with the organism.
  • the infection is a pulmonary or extra-pulmonary infection by M. tuberculosis, and more preferably an extra-pulmonary infection.
  • 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.
  • extrapulmonary is meant outside the lung, encompassing, for example, kidneys, lymph, urinary tract, bone, skin, spinal fluid, intestine, peritoneal, pleural and pericardial cavities.
  • the antibodies of the present invention are also useful in the diagnosis of tuberculosis or infection by M. tuberculosis.
  • the present invention also provides a method of diagnosing tuberculosis or infection by M. tuberculosis in a subject comprising detecting in a biological sample from said subject an immunogenic S9 protein or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof, wherein the presence of said protein or immunogenic fragment or epitope in the sample is indicative of disease, disease progression or infection.
  • the presence of said protein or immunogenic fragment or epitope in the sample is indicative of infection.
  • the method may be an immunoassay, e.g., comprising contacting a biological sample derived from the subject with an antibody that binds to the endogenous S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein (e.g., comprising an amino acid sequence set forth in any one of SEQ ID Nos: 2-7 and preferably, comprising SEQ ID NO: 6 or 7, and still more preferably SEQ ID NO: 6, or an immunologically cross-reactive variant of any one of said sequences that comprises an amino acid sequence that is at least about 95% identical thereto) for a time and under conditions sufficient for an antigen-antibody complex to form and then detecting the formation of an antigen-antibody complex.
  • an immunoassay e.g., comprising contacting a biological sample derived from the subject with an antibody that binds to the endogenous S9 protein of Mycobacterium tuberculosis or an
  • Preferred samples according to this embodiment are those samples in which M. tuberculosis or peptide fragments from bacterial debris are likely to be found, or immunoglobulin- containing fraction, e.g., an extract from brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle, bone or mixtures thereof; body fluid(s) such as sputum, serum, plasma, whole blood, saliva, urine, pleural fluid or mixtures thereof or derivatives thereof e.g., sputum, serum, plasma, whole blood, saliva, urine, pleural fluid, etc.
  • the sample may contain circulating antibodies complexed with S9 antigenic fragments.
  • the patient sample may be contacted with antibodies that bind to S9 or immunogenic S9 peptide or fragment or epitope and with antibodies that bind to M. tuberculosis Bsx protein (e.g., SwissProt Database Accession No.
  • MRQLAERSGVSNPYL SEQ ID NO: 8
  • ERGLRKPSADVLSQI SEQ ID NO: 9
  • LRKPSADVLSQIAKA SEQ ID NO: 10
  • PSADVLSQIAKALRV SEQ ID NO: 11
  • SQIAKALRVSAEVLY SEQ ID NO: 12
  • AKALRVSAEVLYVRA SEQ ID NO: 13
  • VRAGILEPSETSQVR SEQ ID No: 14
  • TAITERQKQILLDIY SEQ ID NO; 15
  • SQIAKALRVSAEVLY VRAC SEQ ID NO: 16
  • MSSEEKLCDPTPTDD SEQ ID NO: 17
  • VRAGILEPSETSQVRC SEQ ID NO: 18
  • Antibodies that bind to an immunogenic M. tuberculosis Bsx protein or peptide for detecting tuberculosis or infection by M. tuberculosis are also described in detail in the instant applicant's co-pending International Patent Application No. PCT/AU2005/001254 filed August 19, 2005 the disclosure of which is incorporated herein in its entirety.
  • the patient sample may be contacted with antibodies that bind to S9 protein or immunogenic S9 peptide or fragment or epitope and with antibodies that bind to an immunogenic M. tuberculosis glutamine synthetase (GS) protein (e.g., SwissProt Database Accession No. 033342) or antibodies that bind to an immunogenic peptide derived from GS, e.g., a peptide derived from a surface-exposed region of a GS protein, or comprising the sequence RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO: 19) or WASGYRGLTPASDYNIDYAI (SEQ ID NO: 20). Antibodies that bind to an immunogenic M.
  • GS tuberculosis glutamine synthetase
  • an immunogenic peptide derived from GS e.g., a peptide derived from a surface-exposed region of a GS protein, or comprising the sequence RGTDGSAVFADS
  • tuberculosis GS or peptide for detecting tuberculosis or infection by M. tuberculosis are also described in detail in the instant applicant's co-pending International Patent Application No. PCT/AU2005/000930 filed June 24 2005 the disclosure of which is incorporated herein in its entirety.
  • Assays for one or more secondary analytes are conveniently performed in the same manner as for detecting S9 protein in the sample.
  • the assays may be performed simultaneously or at different times, and using the same or different patient samples.
  • the assays may also be performed in the same reaction vessel, provided that different detection systems are used to detect the bound antibodies, e.g., secondary antibodies that bind to the anti-S9 antibodies and antibodies that bind to the secondary analyte(s).
  • antigen-based assay systems can comprise an immunoassay e.g.; contacting a biological sample derived from the subject with one or more isolated ligands according to any embodiment described herein, especially any peptide ligand, antibody or an immune-reactive fragment thereof capable of binding to a S9 protein or an immunogenic fragment or epitope thereof, and detecting the formation of a complex e.g., an antigen-antibody complex.
  • an immunoassay e.g.; contacting a biological sample derived from the subject with one or more isolated ligands according to any embodiment described herein, especially any peptide ligand, antibody or an immune-reactive fragment thereof capable of binding to a S9 protein or an immunogenic fragment or epitope thereof, and detecting the formation of a complex e.g., an antigen-antibody complex.
  • the ligand is an antibody, preferably a polyclonal or monoclonal antibody or antibody fragment that binds specifically to the isolated or recombinant immunogenic S9 protein of Mycobacterium, tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein or to a fusion protein or protein aggregate comprising said immunogenic S9 protein, peptide, fragment or epitope.
  • the assay is also particularly useful for detecting TB in a subject that is immune compromised or immune deficient, e.g., a subject that is infected with human immunodeficiency virus (i.e., "HTV+").
  • a subject that is immune compromised or immune deficient e.g., a subject that is infected with human immunodeficiency virus (i.e., "HTV+”).
  • the samples used for conducting such assays include, for example, (i) an extract from a tissue selected from the group consisting of brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle, bone and mixtures thereof; (ii) body fluid(s) selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof; and (iii) samples derived from body fluid(s) selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.
  • the present invention also provides a method for determining the response of a subject having tuberculosis or an infection by M. tuberculosis to treatment with a therapeutic compound for said tuberculosis or infection, said method comprising detecting a S9 protein or an immunogenic fragment or epitope thereof in a biological sample from said subject, wherein a level of the protein or fragment or epitope that is enhanced, or not decreased or decreasing, compared to the level of that protein or fragment or epitope detectable in a normal or healthy subject indicates that the subject is not responding to said treatment or has not been rendered free of disease or infection.
  • the method can comprise an immunoassay e.g., contacting a biological sample derived from the subject with one or more antibodies capable of binding to a S9 protein or an immunogenic fragment or epitope thereof, and detecting the formation of an antigen- antibody complex.
  • an antibody is an isolated or recombinant antibody or immune reactive fragment of an antibody that binds specifically to the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein or to a fusion protein or protein aggregate comprising said immunogenic S9 protein, peptide, fragment or epitope.
  • the diagnostic assay of the present invention is also particularly useful for detecting TB in a subject that is immune compromised or immune deficient, e.g., a subject that is HIV+.
  • the samples used for conducting such assays include, for example, (i) an extract from a tissue selected from the group consisting of brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle, bone and mixtures thereof; (ii) body fluid(s) selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof; and (iii) samples derived from body fluid(s) selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.
  • the present invention also provides a method for determining the response of a subject having tuberculosis or an infection by M. tuberculosis to treatment with a therapeutic compound for said tuberculosis or infection, said method comprising detecting a S9 protein or an immunogenic fragment or epitope thereof in a biological sample from said subject, wherein a level of the protein or fragment or epitope that is lower than the level of the protein or fragment or epitope detectable in a subject suffering from tuberculosis or infection by M. tuberculosis indicates that the subject is responding to said treatment or has been rendered free of disease or infection.
  • the method can comprise an immunoassay e.g., contacting a biological sample derived from the subject with one or more antibodies capable of binding to a S9 protein or an immunogenic fragment or epitope thereof, and detecting the formation of an antigen-antibody complex.
  • an antibody is an isolated or recombinant antibody or immune reactive fragment of an antibody that binds specifically to the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein or to a fusion protein or protein aggregate comprising said immunogenic S9 protein, peptide, fragment or epitope.
  • the diagnostic assay of the present invention is also particularly useful for detecting TB in a subject that is immune compromised or immune deficient, e.g., a subject that is HIV+.
  • the samples used for conducting such assays include, for example, (i) an extract from a tissue selected from the group consisting of brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle, bone and mixtures thereof; (ii) body fluid(s) selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof; and (iii) samples derived from body fluid(s) selected from the group consisting of sputum, serum, plasma, whole blood, saliva, urine, pleural fluid and mixtures thereof.
  • the present invention also provides a method of monitoring disease progression, responsiveness to therapy or infection status by M. tuberculosis in a subject comprising determining the level of a S9 protein or an immunogenic fragment or epitope thereof in a biological sample from said subject at different times, wherein a change in the level of the S9. protein, fragment or epitope indicates a change in disease progression, responsiveness to therapy or infection status of the subject.
  • the method further comprises administering a compound for the treatment of tuberculosis or infection by M. tuberculosis when the level of S9 protein, fragment or epitope increases over time.
  • the method can comprise an immunoassay e.g., contacting a biological sample derived from the subject with one or more antibodies capable of binding to a S9 protein or an immunogenic fragment or epitope thereof, and detecting the formation of an antigen-antibody complex.
  • an antibody is an isolated or recombinant antibody or immune reactive fragment of an antibody that binds specifically to the isolated or recombinant immunogenic S9 protein of Mycobacterium tuberculosis or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein or to a fusion protein or protein aggregate comprising said immunogenic S9 protein, peptide, fragment or epitope.
  • 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.
  • circulating immune complexes are detected in an antigen-based assay platform or antibody-based assay platform.
  • the detection of CICs may provide a signal amplification over the detection of isolated antigen in circulation, by virtue of detecting the immunoglobulin moiety of the CIC.
  • a capture reagent e.g., a capture antibody is used to capture the S9 antigen (S9 polypeptide or an immune reactive fragment or epitope thereof) complexed with the subject's immunoglobulin, in addition to isolated antigen in the subject's circulation.
  • Anti-Ig antibodies are used to specifically bind the captured CIC thereby detecting CIC patient samples.
  • the anti-Ig antibody binds preferentially to IgM, IgA or IgG in the sample.
  • the anti-Ig antibody binds to human Ig, e.g., human IgA, human IgG or human IgM.
  • the anti-Ig antibody may be conjugated to any standard detectable label known in the art. This is particularly useful for detecting infection by a pathogenic agent, e.g., a bacterium or virus, or for the diagnosis of any disease or disorder associated with CICs.
  • the diagnostic methods described according to any embodiment herein are amenable to a modification wherein the sample derived from the subject comprises one or more circulating immune complexes comprising immunoglobulin (Ig) bound to S9 protein of Mycobacterium tuberculosis or one or more immunogenic S9 peptides, fragments or epitopes thereof and wherein detecting the formation of an antigen- antibody complex comprises contacting an anti-Ig antibody with an immunoglobulin moiety of the circulating immune complex(es) for a time and under conditions sufficient for a complex to form than then detecting the bound anti-Ig antibody.
  • immunoglobulin Ig
  • the patient sample may be contacted with antibodies that bind to S9 or immunogenic S9 peptide or fragment or epitope and antibodies that bind to M.
  • Antibodies that bind to immunogenic M. tuberculosis Bsx peptides are also described in detail in the instant applicant's co-pending International Patent Application No. No.
  • PCT/AU2005/001254 filed August 19, 2005 the disclosure of which is incorporated herein in its entirety; and antibodies that bind to M, tuberculosis GS peptides are also described in detail in the instant applicant's co-pending International Patent Application No. PCT/AU2005/000930 filed June 24 2005 the disclosure of which is also incorporated herein in its entirety.
  • the antigen-antibody complexes formed are then detected using antibodies capable of binding to each protein analyte, or in the case of CIC detections, antibodies capable of binding to human immunoglobulins.
  • the assays may be performed simultaneously or at different times, and using the same or different patient samples.
  • the assays may also be performed in the same reaction vessel, provided that different detection systems are used to detect the different antigens or CICs comprising the different antigens, e.g., anti-human Ig labelled using different reporter molecules such as different coloured dyes, fluorophores, radionucleotides, enzymes, or colloidal gold particles; or differentially-labelled anti-S9 antibodies, anti- Bsx antibodies, and anti-GS antibodies.
  • the secondary antibody is optionally conjugated to a suitable detectable label e.g., horseradish peroxidase (HRP) or ⁇ -galactosidase or ⁇ -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
  • the present invention also provides a method of treatment of tuberculosis or infection by M. tuberculosis comprising:
  • 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.
  • the present invention provides a method of prophylaxis comprising:
  • an immunogenic S9 protein or one or more immunogenic S9 peptides, fragments or epitopes thereof induce(s) the specific production of a high titer antibody when administered to an animal subject.
  • the invention also provides a method of eliciting the production of antibody against M. tuberculosis comprising administering an immunogenic S9 protein or one or more imm ⁇ nogenic S9 peptides or immunogenic S9 fragments or epitopes thereof to said subject for a time and under conditions sufficient to elicit the production of antibodies, such as, for example, neutralizing antibodies that bind to M. tuberculosis.
  • the present invention clearly contemplates the use of an immunogenic S9 protein or one or more immunogenic S9 peptides or immunogenic S9 fragments or epitopes thereof in the preparation of a therapeutic or prophylactic subunit vaccine against M. tuberculosis infection in a human or other animal subject.
  • this invention also provides a vaccine comprising an immunogenic S9 protein or one or more immunogenic S9 peptides or immunogenic S9 fragments or epitopes thereof in combination with a pharmaceutically acceptable diluent.
  • the protein or peptide(s) or fragment(s) or epitope(s) thereof is(are) formulated with a suitable adjuvant.
  • 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.
  • APC autologous or allogeneic antigen presenting cell
  • dendritic cell that has been treated in vitro so as to present the peptide on its surface.
  • Nucleic acid-based vaccines that comprise nucleic acid, such as, for example, DNA or RNA, encoding an immunogenic S9 protein or one or more immunogenic S9 peptides or immunogenic S9 fragments or epitopes thereof cloned into a suitable vector (eg. vaccinia, canary pox, adenovirus, or other eukaryotic virus vector) are also contemplated.
  • a suitable vector eg. vaccinia, canary pox, adenovirus, or other eukaryotic virus vector
  • DNA encoding an immunogenic S 9 protein or an immunogenic S9 peptide or immunogenic 89 fragment or epitope thereof is formulated into a DNA vaccine, such as, for example, in combination with the existing Calmette- Guerin (BCG) or an immune adjuvant such as vaccinia virus, Freund's adjuvant or another immune stimulant.
  • BCG Calmette- Guerin
  • an immune adjuvant such as vaccinia virus, Freund's adjuvant or another immune stimulant.
  • the present invention further provides for the use of an immunogenic S9 protein or one or more immunogenic S9 peptides or one or more immunogenic S9 fragments or one or more epitopes thereof in the preparation of a composition for the prophylactic or therapeutic treatment or diagnosis of tuberculosis or infection by M. tuberculosis in a subject, such as, for example, a subject infected with HIV-I and/or HIV-2, including the therapeutic treatment of a latent M. tuberculosis infection in a human subject.
  • the present invention provides for the use of an immunogenic S9 protein or one or more immunogenic S9 peptides or one or more immunogenic S9 fragments or one or more epitopes thereof in the preparation of a composition for the prophylactic or therapeutic treatment or diagnosis of tuberculosis or infection by M. tuberculosis in a subject wherein the subject has been subjected previously to antiviral therapy against HIV-I and/or HIV-2.
  • the present invention also provides a kit for detecting M. tuberculosis infection in a biological sample, said kit comprising:
  • the present invention also provides a kit for detecting M. tuberculosis infection in a biological sample, said kit comprising:
  • the assays described herein are amenable to any assay format, and particularly to solid phase ELISA, flow through immunoassay formats, lateral flow formats, capillary formats, and for the purification or isolation of immunogenic proteins, peptides, fragments and epitopes and CICs.
  • the present invention also provides a solid matrix having adsorbed thereto an isolated or recombinant S9 protein or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any one embodiment described herein or a fusion protein or protein aggregate comprising said immunogenic S9 protein, peptide, fragment or epitope.
  • the solid matrix may comprise a membrane, e.g., nylon or nitrocellulose.
  • 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.
  • the invention also provides a solid matrix having adsorbed thereto an antibody that binds to an isolated or recombinant S9 protein or an immunogenic S9 peptide or immunogenic S9 fragment or epitope thereof according to any embodiment described herein or to a fusion protein or protein aggregate comprising said immunogenic S9 protein, peptide, fragment or epitope.
  • the solid matrix may comprise a membrane, e.g., nylon or nitrocellulose.
  • 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.
  • solid matrices to comprise additional antigens and/or antibodies as required to perform an assay described herein, especially for multianalyte tests employing multiple antigens or multiple antibodies.
  • Figure 1 is a copy of a photographic representation showing a polyacrylamide gel within which proteins isolated from an immunoglobulin fraction isolated from a TB subject have been separated using two-dimensional gel electrophoresis. The position of M. tuberculosis ribosomal protein S9 is indicated.
  • Figure 2 is a graphical representation showing the titration of polyclonal antibody R9 its corresponding biotinylated peptide coated onto a 5 ⁇ g/ml streptavidin plate at 3 ⁇ g/ml.
  • Figure 3 is a graphical representation showing the titration of the peptide comprising the amino acid sequence MTETT PAPQT PAAPA GPAQS FGSGL-Biotin from 20,480 pg/ml to 10 pg/ml against the rabbit sera raised against this peptide linked to KHL.
  • Solid diamonds represent 40 ⁇ g/ml of antibody.
  • Solid squares represent 20 ⁇ g/ml of antibody.
  • Grey triangles represent 10 ⁇ g/ml of antibody.
  • Grey squares represent 0 ⁇ g/ml of antibody.
  • Figure 4a is a copy of a photographic representation showing a Western blot to detect M. tuberculosis ribosomal protein S9 in samples from subjects suffering from TB.
  • the position of a band corresponding to S9 is indicated by the arrow at the right-hand side of the figure.
  • the sample number is indicated at the top of the figure and the HIV status of each patient is indicated at the base of the figure.
  • the molecular weight is indicated at the left-hand side of the figure.
  • Figure 4b is a copy of a photographic representation showing a Western blot to detect M. tuberculosis ribosomal protein S9 in samples from control subjects, i.e., subjects that do not suffer from TB.
  • the position of a band corresponding to S9 is indicated by the arrow at the right-hand side of the figure.
  • the sample number is indicated at the top of the figure and the molecular weight is indicated at the left-hand side of the figure.
  • Figure 5 is a graphical representation showing the binding affinities of different antibodies prepared against recombinant M. tuberculosis ribosomal protein S9 for the immunizing antigen, as determined by ELISA.
  • Recombinant S9 protein was diluted serially 1:2 (v/v) from 500 ng/ml starting concentration to 7.8 ng/ml, and 50 ⁇ l aliquots of each dilution were used to coat the wells of an ELISA plate (x-axis).
  • Figure 6 is a graphical representation showing sandwich ELISA results using antibody Ch27 as capture antibody and antibody Mol025F as detection antibody for assaying recombinant M. tuberculosis ribosomal protein S9.
  • An ELISA plate was coated overnight with capture antibody Ch27 at 5 ⁇ g/ml and 2.5 ⁇ g/ml concentrations. Following washing to remove unbound antibody, recombinant S9 protein was diluted serially 1 :2 (v/v) from 500 ng/ml starting concentration to 7.8 ng/ml, and 50 ⁇ l aliquots of each dilution were added the wells of the antibody-coated ELISA plates (x-axis).
  • detection antibody Mol025F was contacted with the bound antigen-body complexes at concentrations in the range of 1.25 ⁇ g/ml to 5 ⁇ g/ml. Following incubation at room temperature for 1 hour, plates were washed, incubated with 50 ⁇ l of a 1 :5000 (v/v) dilution of secondary antibody (i.e., donkey anti-mouse IgG) conjugated to horseradish peroxidase (HRP), washed, incubated with TMB for 30 mins, and absorbance at 595- 600 nm was determined (y-axis). Data show no background signal with this antibody combination.
  • secondary antibody i.e., donkey anti-mouse IgG conjugated to horseradish peroxidase (HRP)
  • FIG. 7 is a graphical representation showing sandwich ELISA results using antibody Mol025F as capture antibody and antibody Ch27 as detection antibody for assaying recombinant M. tuberculosis ribosomal protein S9.
  • An ELISA plate was coated overnight with capture antibody Mol025F at 5 ⁇ g/ml and 2.5 ⁇ g/ml concentrations.
  • recombinant S9 protein was diluted serially 1 :2 (v/v) from 500 ng/ml starting concentration to 7.8 ng/ml, and 50 ⁇ l aliquots of each dilution were added the wells of the antibody-coated ELISA plates (x-axis).
  • detection antibody Ch27 was contacted with the bound antigen-body complexes at concentrations in the range of 1.25 ⁇ g/ml to 5 ⁇ g/ml.
  • Figure 8 is a graphical representation showing sandwich ELISA results using antibody Ch27 as capture antibody, antibody Mo 1025 F as detection antibody and an HRP- conjugated secondary antibody, for assaying low concentrations of recombinant M. tuberculosis ribosomal protein S9.
  • An ELISA plate was coated overnight with capture antibody Ch27 at 5 ⁇ g/ml concentration. Following washing to remove unbound antibody, recombinant S9 protein was diluted serially 1 :2 (v/v) from 150 ng/ml starting concentration to 18.31 pg/ml, and 50 ⁇ l aliquots of each dilution were added the wells of the antibody-coated ELISA plates (x-axis).
  • detection antibody Mol025F was contacted with the bound antigen-body complexes at 2.5 ⁇ g/ml concentration. Following incubation at room temperature for 1 hour, plates were washed, incubated with 50 ⁇ l of a 1 :5000 (v/v) dilution of secondary antibody (i.e., donkey anti-mouse IgG) conjugated to horseradish peroxidase (HRP), washed, incubated with TMB for 30 mins, and absorbance at 595-600 nm was determined (y-axis). Data show no background signal with this antibody combination, a detection limit of 996 pg/ml M.
  • secondary antibody i.e., donkey anti-mouse IgG conjugated to horseradish peroxidase
  • Figure 9 is a graphical representation showing sandwich ELISA results using antibody Ch27 as capture antibody, antibody Mol025F as detection antibody and a biotinylated secondary antibody for assaying low concentrations of recombinant M. tuberculosis ribosomal protein S9.
  • ELISA was performed essentially as described in the legend to
  • Figure 10 is a graphical representation showing sandwich ELISA results using antibody Ch27 as capture antibody, antibody Mol025F as detection antibody, a biotinylated secondary antibody and iterative antigen binding (also termed herein “replacement amplification") for assaying low concentrations of recombinant M. tuberculosis ribosomal protein S9.
  • ELISA was performed essentially as described in the legend to
  • Figure 1 1 is a graphical representation of sandwich ELISA results showing lack of significant cross-reactivity of antibodies against M, tuberculosis ribosomal protein S9 with Escherichia coli, Bacillus subtilis or Pseudomonas aeruginosa.
  • Assay conditions were essentially as described in the legend to Figure 9 except that purified recombinant S9 protein was replaced with 500 ng/ml or 50 ⁇ g/ml of a cellular extract as indicated on the x-axis.
  • As a positive control cellular extract from the M, tuberculosis laboratory strain H37R.V was used.
  • buffer without cellular extract was used.
  • Figure 12 is a graphical representation of sandwich ELISA results showing detection of M. tuberculosis ribosomal protein S9 in the clinical M. tuberculosis isolate CSU93, and lack of signal suppression in plasma.
  • Assay conditions were essentially as described in the legend to Figure 1 1 except that cellular extracts were from M. tuberculosis laboratory strain H37Rv and CSU93, as indicated on the x-axis.
  • cellular extract at the concentration indicated was diluted into plasma, as indicated on the x-axis.
  • buffer or plasma without cellular extract was used as a negative control for each assay.
  • Figure 13a is a copy of a photographic representation showing a Western blot to detect M. tuberculosis BSX protein in samples from subjects suffering from TB.
  • the position of a band corresponding to BSX is indicated by the arrow at the right-hand side of the figure.
  • the sample number is indicated at the top of the figure and the HFV status of each patient is indicated at the base of the figure.
  • the molecular weight is indicated at the left-hand side of the figure.
  • Figure 13b is a copy of a photographic representation showing a Western blot to detect M. tuberculosis BSX protein in samples from control subjects, i.e., subjects that do not suffer from TB.
  • the position of a band corresponding to BSX is indicated by the arrow at the right-hand side of the figure.
  • the sample number is indicated at the top of the figure and the molecular weight is indicated at the left-hand side of the figure.
  • Figure 14 is a copy of a photographic representation showing a Western blot to detect M. tuberculosis BSX protein in a fraction captured with Protein-G (immunoglobulin containing fraction) and the flow-through fraction from three different subjects.
  • the fraction and patient number is indicated at the top of the figure.
  • the molecular weight is indicated at the left-hand side of the figure and the size of the BSX protein is indicated at the right-hand side of the figure.
  • Figure 15 is a graphical representation showing a comparison of the concentration of recombinant BSX detected using a chicken anti-BSX polyclonal antibody preincubated with recombinant BSX (solid diamonds); a chicken anti-BSX antibody without preincubation (grey squares); a rabbit anti-BSX polyclonal antibody (solid triangles) and a mouse anti-BSX monoclonal antibody (solid squares).
  • concentration of the recombinant protein is indicated on the X-axis and the optical density indicated on the Y-axis.
  • Figure 16 is a graphical representation showing the detection of recombinant BSX using a sandwich ELISA in which monoclonal antibody 403B was used as a capture reagent and polyclonal antibody C44 was used as a detection reagent. Titrating amounts of recombinant BSX from 50 ng/ml down to 0.39 ng/ml were screened. Concentrations of detection and capture reagents are indicated. The concentration of BSX is shown on the X-axis and the mean OD is shown on the Y-axis.
  • Figure 17 is a graphical representation showing the detection of BSX in sputa of TB and control subjects using a Sandwich ELISA.
  • the optical density is indicated on the Y-axis and the sample type and number is indicated on the X-axis.
  • Figure 18 is a graphical representation showing the detection of recombinant BSX using an amplified sandwich ELISA in which monoclonal antibody 403 B was used as a capture reagent detection reagent (as indicated) and polyclonal antibody C44 was used as a detection reagent or capture reagent (as indicated). Titrating amounts of recombinant BSX from 50 ng/ml to 0.39 ng/ml were screened. Concentrations of detection and capture reagents are indicated. The concentration of BSX is shown on the X-axis and the mean OD is shown on the Y-axis.
  • Figure 19 is a graphical representation showing the detection of recombinant BSX using an amplified ELISA in which C44 is used as a capture reagent.
  • Purified chicken anti-BSX pAb C44 was immobilised onto an ELISA plate as a capture antibody at a concentration of 20 ⁇ g/ml using 50 ⁇ l per well. Titrating amounts of recombinant BSX from 10 ng/ml down to 0.078 ng/ml were then screened by sequential addition of purified rabbit anti-BSX (Peptide 28) pAb at a concentration of 5 ⁇ g/ml, and then a goat anti-rabbit IgG at a dilution of either 1/30000 or 1/60000, as a second Detector.
  • FIG. 20 is a graphical representation showing the measurement of detection limits of standard sandwich ELISA versus biotin based Amplification System.
  • Purified rabbit anti-BSX pAb Rl 6 was immobilised onto an ELISA plate at a concentration of 20 ⁇ g/ml. Titrating amounts of recombinant BSX were added at a concentration of 50 ng/ml down to 0.39 ng/ml for 1 hr unless specified otherwise (i.e 2 hr).
  • Antigen detection was performed using either a standard sandwich system where chicken anti- BSX p Ab C44 was added at a concentration of 5 ⁇ g/ml followed by sheep anti-chicken IgG HRP conjugate at a dilution of 1/5000, or an amplifying system where chicken anti-BSX was first added at 5 ⁇ g/ml followed by donkey anti-chicken IgG biotin conjugate at various dilutions as specified above, and finally streptavidin-HRP at a 1/5000 dilution. Background (i.e. signal without BSX present) has been subtracted from the above curves.
  • Figure 21 is a graphical representation showing detection of titrating amounts of recombinant BSX using a Biotin -based amplified ELISA.
  • Purified rabbit anti-BSX (anti-Peptide 28) pAb Rl 6 was immobilised onto an ELISA plate as a capture antibody at a concentration of either 20 or 40 ⁇ g/ml.
  • recombinant BSX Titrating amounts of recombinant BSX from 10 ng/ml down to 4.9 pg/ml were then screened by sequential addition of purified chicken anti-BSX pAb C44 at a concentration of 5 ⁇ g/ml, and then a donkey anti- chicken IgG biotin conjugate at a dilution of 1:20,000 (v/v) as a second detector. Streptavidin HRP conjugate at a dilution of 1 :5000 (v/v) and TMB were used for signal detection. Background OD intensity was obtained for both of the rabbit anti-BSX capture concentrations where the recombinant BSX was not added.
  • Figure 22 is a graphical representation showing screening of sputum for endogenous BSX using sandwich ELISA with a Biotin Amplification System.
  • Sputum samples 50 ul + 50 ul blocking buffer
  • Sputum samples from South African TB patients and control patients with non-TB respiratory disease from South Africa (prefix 'M') and Australia (prefix 'CGS') respectively were screened by sandwich ELISA for the presence of BSX antigen.
  • Purified rabbit anti-BSX (peptide 28) pAb was immobilised onto the ELISA plate as a capture antibody at a concentration of 20 ⁇ g/ml.
  • Figure 23 is a graphical representation showing the Effect of Multiple Sample Loads on Detection of BSX by Amplified Sandwich ELISA.
  • Rabbit anti-BSX pAb Rl 6 was immobilised onto an ELISA plate as the capture antibody at a concentration of 20 ⁇ g/ml using 50 ul per well.
  • Sputum samples from TB patients and non-TB respiratory disease control patients were diluted at a 1 : 1 ratio with blocker solution.
  • the positive control is recombinant BSX at 1 ng/ml spiked in CGS23 sputum sample.
  • Sputum samples were either (i) incubated for 1 hr as per a standard ELISA; (ii) incubated for 2 hr; or (iii) incubated for 2 hr, removed and fresh sputum added for an additional 1 hr of incubation.
  • Endogenous BSX was detected using purified chicken anti-BSX pAb C44 at 5 ⁇ g/ml, followed by donkey anti-chicken IgG biotin conjugate at a dilution of 1/20,000 and finally with streptavidin HRP conjugate at 1/5000 dilution.
  • Isolated or recombinant S9 protein and immunogenic fragments and epitopes thereof One aspect of the present invention provides an isolated or recombinant S9 protein or an immunogenic fragment or epitope thereof.
  • S9 encompasses any synthetic or recombinant peptides derived from a S9 protein referred to herein, including the full-length S9 protein, and/or a derivative or analogue of a S9 protein or an immunogenic fragment or epitope thereof.
  • S9 shall be taken to mean any peptide, polypeptide, or protein having at least about 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ED NO: 1.
  • the percentage identity of a S9 protein to SEQ ED NO: 1 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • the present invention is not to be restricted to the use of the exemplified M. tuberculosis S9 protein because, as will be known to those skilled in the art, it is possible to define a fragment of a protein having sequence identity and immunological equivalence to a region of the exemplified M. tuberculosis S9 protein without undue experimentation,
  • 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.
  • the CLUSTAL W algorithm of Thompson et al, Nucl. Acids Res. 22, 4673-4680, 1994 is used to obtain an alignment of multiple sequences, wherein it is necessary or desirable to maximise the number of identical/similar residues and to minimise the number and/or length of sequence gaps in the alignment.
  • Amino acid sequence alignments can also be performed using a variety of other commercially available sequence analysis programs, such as, for example, the BLAST program available at NCBI.
  • Particularly preferred fragments include those that include an epitope, in particular a B cell epitope or T cell epitope.
  • a B-cell epitope is conveniently derived from the amino acid sequence of an immunogenic S9 protein. Idiotypic and anti-idiotypic B cell epitopes against which an immune response is desired are specifically encompassed by the invention, as are lipid- modified B cell epitopes or a Group B protein.
  • a preferred B-cell epitope will be capable of eliciting the production of antibodies when administered to a mammal, preferably neutralizing antibody against M. tuberculosis, and more preferably, a high titer neutralizing antibody. Shorter B cell epitopes are preferred, to facilitate peptide synthesis.
  • the length of the B cell epitope will not exceed about 30 amino acids in length. More preferably, the B cell epitope sequence consists of about 25 amino acid residues or less, and more preferably less than 20 amino acid residues, and even more preferably about 5-20 amino acid residues in length derived from the sequence of the full-length protein.
  • a CTL epitope is also conveniently derived from the full length amino acid sequence of a S9 protein and will generally consist of at least about 9 contiguous amino acids of said S9 protein and have an amino acid sequence that interacts at a significant level with a MHC Class I allele as determined using a predictive algorithm for determining MHC Class I-binding epitopes, such as, for example, the SYFPEITHI algorithm of the University of Tuebingen, Germany, or the algorithm of the HLA Peptide Binding Predictions program of the Biolnformatics and Molecular Analysis Section (BIMAS) of the National Institutes of Health of the Government of the United States of America.
  • a predictive algorithm for determining MHC Class I-binding epitopes such as, for example, the SYFPEITHI algorithm of the University of Tuebingen, Germany, or the algorithm of the HLA Peptide Binding Predictions program of the Biolnformatics and Molecular Analysis Section (BIMAS) of the National Institutes of Health of the
  • 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).
  • the CTL epitope will have a sequence that induces a memory CTL response or elicits IFN- ⁇ expression by a T cell, such as, for example, CDS + T cell, cytotoxic T cell (CTL).
  • CTL cytotoxic T cell
  • the CTL will have a sequence that stimulates CTL activity in a standard cytotoxicity assay.
  • Particularly preferred CTL epitopes of a S9 protein are capable of eliciting a cellular immune response against M. tuberculosis in human cells or tissues, such as, for example, by recognizing and lysing human cells infected with M. tuberculosis, thereby providing or enhancing cellular immunity against M. tuberculosis.
  • Suitable fragments will be at least about 5, e.g., 10, 12, 15 or 20 amino acids in length. They may also be less than 200, 100 or 50 amino acids in length.
  • an immunogenic fragment or epitope of S9 comprises an amino acid sequence set forth in any one of SEQ ID Nos: 2-7, and preferably an immunogenic fragment or epitope thereof comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 6 or SEQ ID NO: 7.
  • amino acid sequence of a S9 protein or immunogenic fragment or epitope thereof may be modified for particular purposes according to methods well known to those of skill in the art without adversely affecting its immune function.
  • 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.
  • the size and/or charge of the side chains also are relevant factors in determining which substitutions are conservative.
  • the present invention clearly encompasses a covalent fusion between one or more immunogenic S9 peptides, including a homo-dimer, homo-trimer, homo-tetramer or higher order homo-multimer of a peptide, or a hetero-dimer, hetero-trimer, hetero- tetramer or higher order hetero-multimer comprising two or more different immunogenic peptides.
  • the present invention also encompasses a non-covalent aggregate between one or more immunogenic S9 peptides, e.g., held together by ionic, hydrostatic or other interaction known in the art or described herein.
  • 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.
  • a plurality of distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention.
  • 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.
  • 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).
  • 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.
  • 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).
  • the S9 polypeptide or peptide fragment thereof comprising an epitope is readily synthesized using standard techniques, such as the Merrifield method of synthesis (Merrifield, J Am Chem Soc, S5,:2149-2154, 1963) and the myriad of available improvements on that technology (see e.g., Synthetic Peptides: A User's Guide, Grant, ed. (1992) W.H. Freeman & Co., New York, pp. 382; Jones (1994) The Chemical Synthesis of Peptides, Clarendon Press, Oxford, pp. 230.); Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol.
  • synthetic peptides can be produced with " additional hydrophilic N-terminal and/or C-terminal amino acids added to the sequence of a fragment or B- cell epitope derived from the full-length S9 protein, such as, for example, to facilitate synthesis or improve peptide solubility. Glycine and/or serine residues are particularly preferred for this purpose.
  • Each of the peptides set forth in SEQ ID NO 2-6 may be modified to include additional spacer sequences flanking the S9 fragments, said spacers comprising hetero-polymers (trimers or tetramers) comprising glycine and serine e.g., as in SEQ ID NO: 7.
  • the peptides of the invention are readily modified for diagnostic purposes, for example, by addition of a natural or synthetic hapten, an antibiotic, hormone, steroid, nucleoside, nucleotide, nucleic acid, an enzyme, enzyme substrate, an enzyme inhibitor, biotin, avidin, streptavidin, polyhistidine tag, glutathione, GST, polyethylene glycol, a peptidic polypeptide moiety (e.g. tuftsin, poly-lysine), a fluorescence marker (e.g. FITC, RITC, dansyl, luminol or coumarin), a bioluminescence marker, a spin label, an alkaloid, biogenic amine, vitamin, toxin (e.g. digoxin, phalloidin, amanitin, tetrodotoxin), or a complex-forming agent. Biotinylated peptides are especially preferred.
  • a S9 protein is produced as a recombinant protein.
  • 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.
  • nucleic acid comprising a sequence that encodes a S9 protein or an epitope thereof in operable connection with a suitable promoter sequence, is expressed in a suitable cell for a time and under conditions sufficient for expression to occur.
  • Nucleic acid encoding the S9 protein is readily derived from the publicly available amino acid sequence.
  • a S9 protein is produced as a recombinant fusion protein, such as for example, to aid in extraction and purification.
  • 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.
  • fusion protein partners include glutathione-S-transferase (GST), FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Lys), hexa- histidine, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ - galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the immune function of the S9 protein.
  • promoter 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.
  • promoter is also used to describe a recombinant, synthetic or fusion molecule, or derivative which confers, activates or enhances the expression of a nucleic acid molecule to which it is operably connected, and which encodes the polypeptide or peptide fragment.
  • Preferred promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or to alter the spatial expression and/or temporal expression of the said nucleic acid molecule.
  • Placing a nucleic acid molecule under the regulatory control of, i.e., "in operable connection with”, a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the coding sequence that they control.
  • the prerequisite for producing intact polypeptides and peptides in bacteria such as E. coli is the use of a strong promoter with an effective ribosome binding site.
  • Typical promoters suitable for expression in bacterial cells such as E. coli include, but are not limited to, the lac ⁇ promoter, temperature-sensitive ⁇ L or ⁇ R promoters, T7 promoter or the EPTG-inducible tac promoter.
  • a number of other vector systems for expressing the nucleic acid molecule of the invention in E. coli are well-known in the art and are described, for example, in Ausubel et al (In: Current Protocols in Molecular Biology.
  • 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.
  • CMV cytomegalovirus
  • Preferred vectors for expression in mammalian cells eg.
  • 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 pSR ⁇ tkneo (Muller et al, MoI. Cell. Biol, 11, 17S5, 1991).
  • the vector pcDNA 3.1 myc-His (Invitrogen) is particularly preferred for expressing a secreted form of a S9 protein or a derivative thereof in 293T cells, wherein the expressed peptide or protein can be purified free of conspecific proteins, using standard affinity techniques that employ a Nickel column to bind the protein via the His tag.
  • a wide range of additional host/vector systems suitable for expressing the diagnostic protein of the present invention or an immunological derivative (eg., an epitope or other fragment) thereof are available publicly, and described, for example, in Sambrook et al (In: Molecular cloning, A laboratory manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
  • Means for introducing the isolated nucleic acid molecule or a gene construct comprising same into a cell for expression are well-known to those skilled in the art. The technique used for a given organism depends on the known successful techniques. Means for introducing recombinant DNA into animal cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG- mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.
  • Proteins of the invention can be produced in an isolated form, preferably substantially free of conspecific protein. Antibodies and other affinity ligands are particularly preferred for producing isolated protein. Preferably, the protein will be in a preparation wherein more than about 90% (e.g. 95%, 98% or 99%) of the protein in the preparation is a S9 protein or an epitope thereof.
  • preferred secondary analytes e.g., for use in multi-analyte antigen- based tests, will comprise an amino acid sequence selected from the group set forth in SEQ ID NOs: 8-19.
  • the M. tuberculosis Bsx protein can be expressed and fragments obtained therefrom by standard means, or alternatively, synthetic peptides can be synthesized based on the sequence of the full-length protein (e.g., comprising the sequence set forth in SwissProt Database Accession No. 053759).
  • Exemplary immunogenic peptides from the full-length Bsx protein will comprise a sequence selected from the group consisting of: MRQLAERSGVSNPYL (SEQ ID NO: 8), ERGLRKPSADVLSQI (SEQ ID NO: 9), LRKPSADVLSQIAKA (SEQ ID NO: 10), PSADVLSQIAKALRV (SEQ ID NO: 11), SQIAKALRVSAEVLY (SEQ ID NO: 12), AKALRVSAEVLYVRA (SEQ ID NO: 13), VRAGILEPSETSQVR (SEQ ID No: 14), TAITERQKQILLDIY (SEQ ID NO; 15), SQIAKALRVSAEVLYVRAC (SEQ ID NO: 16), MSSEEKLCDPTPTDD (SEQ ID NO: 17) and VRAGILEPSETSQVRC (SEQ ID NO: 18).
  • M. tuberculosis glutamine synthetase (GS) protein can be expressed and fragments obtained therefrom by standard means, or alternatively, synthetic peptides can be synthesized based on the sequence of the full-length protein (e.g., comprising the sequence set forth in SwissProt Database Accession No. 033342).
  • Exemplary immunogen fragments of the GS protein are derived from a surface-exposed region of a GS protein, or comprise the sequence RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO: 19) or WASGYRGLTPASDYNIDYAI (SEQ ID NO: 20). Methods for producing such fragments are described in detail in the instant in the instant applicant's co-pending International Patent Application No. PCT/AU2005/000930 filed June 24 2005 the disclosure of which is incorporated herein in its entirety.
  • a second aspect of the present invention provides an antibody that binds specifically to a S9 protein or an immunogenic fragment or epitope thereof, such as, for example, a monoclonal or polyclonal antibody preparation suitable for use in the assays described herein.
  • 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.
  • 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.
  • 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
  • the antibodies may be for commercial or diagnostic purposes, in which case the subject to whom the S9 protein or immunogenic fragment or epitope thereof is administered will most likely be a laboratory or farm animal.
  • a wide range of animal species are used for the production of antisera.
  • the animal used for production of antisera is a rabbit, mouse, rabbit, rat, hamster, guinea pig, goat, sheep, pig, dog, horse, or chicken. Because of the relatively large blood volumes of rabbits and sheep, these are preferred choice for production of polyclonal antibodies.
  • 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.
  • the antibody is a high titer antibody.
  • high titer means a sufficiently high titer to be suitable for use in diagnostic or therapeutic applications. As will be known in the art, there is some variation in what might be considered “high titer”. For most applications a titer of at least about 10 3 -10 4 is preferred. More preferably, the antibody titer will be in the range from about 10 4 to about 10 5 , even more preferably in the range from about 10 5 to about 10 6 . More preferably, in the case of B cell epitopes from pathogens, viruses or bacteria, the antibody is a neutralizing antibody (i.e. it is capable of neutralizing the infectivity of the organism from which the B cell epitope is derived).
  • the S9 protein or immunogenic fragment or epitope thereof 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.
  • 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).
  • an immunogenic peptide such as, for example, an immunogenic peptide comprising the amino acid sequence set forth in SEQ ID NO: 6 or an immunogenic fragment thereof, is covalently coupled to an immunogenic carrier protein, such as Diphtheria toxoid (DT), Keyhole Limpet Hemocyanin (KLH), tetanus toxoid (TT) or the nuclear protein of influenza virus (NP), using one of several conjugation chemistries known in the art.
  • DT Diphtheria toxoid
  • KLH Keyhole Limpet Hemocyanin
  • TT tetanus toxoid
  • NP nuclear protein of influenza virus
  • DT is preferably produced by purification of the toxin from a culture of Corynebacte ⁇ wn diphtheriae followed by chemical detoxification, but is alternatively made by purification of a recombinant, or genetically detoxified analogue of the toxin (for example, CRM197, or other mutants as described in U.S. Pat. Nos. 4,709,017, 5,843,711, 5,601,827, and 5,917,017).
  • the toxoid is derivatized using as a spacer a bridge of up to 6 carbons, such as provided by use of the adipic acid hydrazide derivative of diphtheria toxoid (D-AH).
  • D-AH diphtheria toxoid
  • peptides derived from the full-length S9 protein can be synthesized chemically or produced by recombinant expression means, treated with hydroxylamine to form free sulfhydryl groups, and cross-linked via the free sulfhydryl groups to a maleimide-modified diphtheria toxoid, tetanus toxoid or influenza NP protein or other carrier molecule.
  • One of the most specific and reliable conjugation chemistries uses a cysteine residue in the peptide and a maleimide group added to the carrier protein, to form a stable thioether bond (Lee, A.C., et al, MoI. Immunol 17, 749-756 1980).
  • the S9-derived peptides can be prior modified by the addition of a C-terminal cysteine residue e.g., SEQ ID NO: ' 6 to facilitate this procedure.
  • the immunogenic S9 peptides are preferably produced under non-denaturing conditions, treated with hydroxylamine, thiol reducing agents or by acid or base hydrolysis to generate free sulfhydryl groups and the free sulfhydryl- containing peptide is conjugated to a carrier by chemical bonding via the free sulfhydryl groups.
  • Such conjugation may be by use of a suitable bis-maleimide compound.
  • the conjugation of the HA protein may be to a maleimide- modified carrier protein, such as diphtheria toxoid, tetanus toxoid or influenza (NP) protein or to a carbohydrate, such as alginic acid, dextran or polyethylene glycol.
  • a maleimide-modified carrier molecules may be formed by reaction of the carrier molecule with a hetero-bifunctional cross-linker of the maleimide-N- hydroxysuccinimide ester type.
  • bifunctional esters examples include maleimido-caproic-N-hydroxysuccinimide ester (MCS), maleimido-benzoyl-N- hydroxysuccinimide ester (MBS), maleimido-benzoylsul-fosuccinimide ester (sulfo- MBS), succinimidyl ⁇ 4-(N-maleimidornethyl) cyclohexane-1-carboxylate (SMCC), succinimidyl-4-(p-maleimido-phenyl)butyrate (SMPP), sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC) and sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate (sulfo-SMPP).
  • MCS maleimido-caproic-N-hydroxysuccinimide ester
  • conjugate molecules so produced may be purified and employed in immunogenic compositions to elicit, upon administration to a host, an immune response to the S9 peptide which is potentiated in comparison to S9 peptide alone.
  • Diphtheria toxoid is obtained commercially or prepared from Corynebacterium diphtheriae grown in submerged culture by standard methods.
  • the production of Diphtheria Toxoid is divided into five stages, namely maintenance of the working seed, growth of Cojynebacterium diphtheriae, harvest of Diphtheria Toxin, detoxification of Diphtheria Toxin and concentration of Diphtheria Toxoid.
  • the purified diphtheria toxoid (DT) used as carrier in the preparation is preferably a commercial toxoid modified (derivatized) by the attachment of a spacer molecule, such as adipic acid dihydrazide (ADH), using the water-soluble carbodiimide condensation method.
  • ADH adipic acid dihydrazide
  • the modified toxoid typically the adipic hydrazide derivative D-AH, is then freed from unreacted ADH.
  • the efficacy of the S9 protein or immunogenic fragment or epitope thereof in producing an antibody is established by injecting an animal, for example, a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig, with a formulation comprising the S9 protein or immunogenic fragment or epitope thereof, and then monitoring the immune response to the B cell epitope, as described in the Examples.
  • an animal for example, a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig
  • the antibody titer is determined using any conventional immunoassay, such as, for example, ELISA, or radio immunoassay.
  • 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).
  • Mabs monoclonal antibodies
  • Monoclonal antibodies are particularly preferred.
  • 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.
  • a suitable animal will be immunized with an effective amount of the S9 protein or immunogenic fragment or epitope thereof under conditions sufficient to stimulate antibody producing cells.
  • Rodents such as rabbits, mice and rats are preferred animals, however, the use of sheep or frog cells is also possible.
  • the use of rats may provide certain advantages, but mice or rabbits are preferred, with the BALB/c mouse being most preferred as the most routinely used animal and one that generally gives a higher percentage of stable fusions. Rabbits are known to provide high affinity monoclonal antibodies.
  • somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol.
  • B cells B lymphocytes
  • These cells may be obtained from biopsies of spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer removed. Spleen lymphocytes are obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 x 10 7 to 2 x 10 8 lymphocytes.
  • the B cells from the immunized animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal that was immunized with the S9 protein or immunogenic fragment or epitope thereof.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non- antibody-producing, have high fusion efficiency and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells, or hybridomas. Any one of a number of myeloma cells may be used and these are known to those of skill in the art (e.g.
  • 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.
  • a murine myeloma SP2/0 non-producer cell line that is 8- azaguanine-resistant is used.
  • somatic cells are mixed with myeloma cells in a proportion between about 20: 1 to about 1 :1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus have been described by Kohler and Milstein, Nature 256, 495-497, 1975; and Kohler and Milstein, Eur. J. Immunol 6, 51 1-519, 1976.
  • Methods using polyethylene glycol (PEG), such as 37% (v/v) PEG are described in detail by Gefter et al, Somatic Cell Genet. 3, 231-236, 1977.
  • the use of electrically induced fusion methods is also appropriate.
  • Hybrids are amplified by culture in a selective medium comprising an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • exemplary and preferred agents are aminopterin, methotrexate and azaserine.
  • Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • aminopterin or methotrexate the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium hypoxanthine
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT, because only those hybridomas capable of operating nucleotide salvage pathways are able to survive in HAT medium, whereas myeloma cells are defective in key enzymes of the salvage pathway, (e.g., hypoxanthine phosphoribosyl transferase or HPRT), and they cannot survive.
  • B cells can operate this salvage pathway, but they have a limited life span in culture and generally die within about two weeks. Accordingly, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.
  • the amplified hybridomas are subjected to a functional selection for antibody specificity and/or titer, such as, for example, by immunoassay (e.g. radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like).
  • immunoassay e.g. radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like.
  • the selected hybridomas are serially diluted and cloned into individual antibody- producing cell lines, which clones can then be propagated indefinitely to provide MAbs.
  • the cell lines may be exploited for MAb production in two basic ways.
  • a sample of the hybridoma is injected, usually in the peritoneal cavity, into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they are readily obtained in high concentrations.
  • MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to produce cell lines secreting monoclonal antibodies (mAbs) against immunogenic S9 peptide antigens.
  • mAbs monoclonal antibodies
  • BALB/cByJ female mice are immunized with an amount of the peptide antigen over a period of about 2 to about 3 months.
  • test bleeds are taken from the immunized mice at regular intervals to assess antibody responses in a standard ELISA.
  • the spleens of mice having antibody titers of at least about 1,000 are used for subsequent ABL-MYC infection employing replication-incompetent retrovirus comprising the oncogenes v-abl and c-myc.
  • Splenocytes are transplanted into naive mice which then develop ascites fluid containing cell lines producing monoclonal antibodies (mAbs) against the S9 peptide antigen.
  • the mAbs are purified from ascites using protein G or protein A, e.g., bound to a solid matrix, depending on the isotype of the mAb. Because there is no hybridoma fusion, an advantage of the ABL-MYC process is that it is faster, more cost effective, and higher yielding than conventional mAb production methods.
  • diploid plasmacytomas produced by this method are intrinsically more stable than polyploid hybridomas, because the ABL-MYC retrovirus infects only cells in the spleen that have been stimulated by the immunizing antigen. ABL-MYC then transforms those activated B-cells into immortal, mAb-producing plasma cells called plasmacytomas.
  • a "plasmacytoma” is an immortalized plasma cell that is capable of uncontrolled cell division. Since a plasmacytoma begins with just one cell, all of the plasmacytomas produced from it are therefore identical, and moreover, produce the same desired "monoclonal" antibody. As a result, no sorting of undesirable cell lines is required.
  • the ABL-MYC technology is described generically in detail in the following disclosures which are incorporated by reference herein:
  • 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).
  • monoclonal antibodies according to the invention are chimeric monoclonal antibodies.
  • 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.
  • 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.
  • CDRs mouse complementary determining regions
  • “Humanized" monoclonal antibodies in accordance with this invention are especially suitable for use in vivo in diagnostic and therapeutic methods.
  • 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.
  • suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium
  • a mammalian serum such as fetal calf serum or trace elements
  • growth-sustaining supplements e.g., feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like.
  • feeder cells such as normal mouse peritoneal exudate cells, spleen cells
  • 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.
  • the animals are primed with a hydrocarbon, especially oils such as Pristane (tetramethylpentadecane) prior to injection.
  • 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.
  • monoclonal antibody fragments encompassed by the present invention are synthesized using an automated peptide synthesizer, or they may be produced manually using techniques well known in the art.
  • the monoclonal conjugates of the present invention are prepared by methods known in the art, e.g., by reacting a monoclonal antibody prepared as described above with, for instance, an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents, or by reaction with an isothiocyanate. Conjugates with metal chelates are similarly produced. Other moieties to which antibodies may be conjugated include radionuclides such as, for example, 3 H, 125 I, . 32 P, . 35 S, 14 C, 51 Cr, 36 Cl, 57 Co, 58 Co, 5Q Fe,
  • 75 Se, and 152 Eu. i ne present invention clearly includes antibodies when coupled to any detectable ligand or reagent, including, for example, an enzyme such as horseradish peroxidase or alkaline phosphatase, or a fluorophore, radionuclide, coloured dye, gold particle, colloidal gold, etc.
  • an enzyme such as horseradish peroxidase or alkaline phosphatase, or a fluorophore, radionuclide, coloured dye, gold particle, colloidal gold, etc.
  • Radioactively labelled monoclonal antibodies of the present invention are produced according to well-known methods in the art. For instance, monoclonal antibodies are iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • a chemical oxidizing agent such as sodium hypochlorite
  • an enzymatic oxidizing agent such as lactoperoxidase.
  • Monoclonal antibodies according to the invention may be labelled with technetium" by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labelling techniques, (e.g., by incubating pertechnate, a reducing agent such as SNCl 2 , a buffer solution such as sodium-potassium phthalate solution, and the antibody).
  • a reducing agent such as SNCl 2
  • a buffer solution such as sodium-potassium phthalate solution
  • 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.
  • the assay will be capable of generating quantitative results.
  • 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.
  • one of the known antibodies is labelled, direct detection of the label bound to the antigen is possible; comparison to an unmixed sample assay will determine competition by the test antibody and, hence, cross-reactivity. Alternatively, using secondary antibodies specific for either the known or test antibody, one will be able to determine competition.
  • An antibody that binds to the antigen composition will be able to effectively compete for binding of the known antibody and thus will significantly reduce binding of the latter.
  • the reactivity of the known antibodies in the absence of any test antibody is the control.
  • a significant reduction in reactivity in the presence of a test antibody is indicative of a test antibody that binds to the B cell epitope (i.e., it cross-reacts with the known antibody).
  • the antibodies that bind to the S9 protein or immunogenic fragment or B cell epitope are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a composition containing a peptide comprising the B cell epitope is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound epitope may be detected. Detection is generally achieved by the addition of a second antibody that is known to bind to the B cell epitope and is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA".
  • Detection may also be achieved by the addition of said second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • antibodies that bind to the S9 protein or immunogenic fragment or B cell epitope are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a composition containing a peptide comprising the B cell epitope is added to the wells.
  • antibodies that bind to the B cell epitope are contacted with the bound peptide for a time and under conditions sufficient for a complex to form.
  • the signal is then amplified using secondary and preferably tertiary, antibodies that bind to the antibodies recognising the B cell epitope. Detection is then achieved by the addition of a further antibody that is known to bind to the secondary or tertiary antibodies, linked to a detectable label.
  • antibodies that bind to the immunogenic S9 protein or immunogenic S9 peptide or immunogenic S9 fragment or B cell epitope are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate or a column.
  • a sample comprising the immunogenic S9 protein or immunogenic peptide or immunogenic fragment comprising the B cell epitope to which the antibody binds is added for a time and under conditions sufficient for an antigen-antibody complex to form.
  • the added S9 protein, peptide or fragment is complexed with human Ig.
  • the peptide is preferably complexed with human Ig by virtue of an immune response of the patient to the M. tuberculosis S9 protein.
  • 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.
  • a second antibody that is known to bind to human Ig in the immune complex and is linked to a detectable label.
  • 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.
  • mmoo ⁇ ies oi me invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
  • Antibodies that bind to a secondary analyte It is to be understood that methods described herein above for the production of antibodies against the S9 protein or an immunogenic fragment thereof apply mutatis mutandis to the production of antibodies against a secondary analyte that is to be used in an immunoassay format e.g., for the purposes of diagnosis or prognosis of tuberculosis or infection by M. tuberculosis. As will be understood by the skilled artisan, such extrapolation is dependent on substituting the S9 immunogen for the secondary analyte in question e.g., M. tuberculosis Bsx protein or GS protein or immunogenic fragment thereof according to any embodiment described herein. Such substitution is readily performed without undue experimentation fro the disclosure herein.
  • preferred immunizing peptides for the production of antibodies against secondary analytes e.g., for use in multi-analyte antigen-based tests, will comprise an amino acid sequence selected from the group set forth in SEQ ID NOs: S- 20.
  • antibodies that bind to M. tuberculosis Bsx protein can be prepared from the full-length protein (e.g., comprising the sequence set forth in SwissProt Database Accession No. 053759) or from a peptide fragment thereof e.g., comprising a sequence selected from the group consisting of: MRQLAERSGVSNPYL (SEQ ID NO: 8), ERGLRKPSADVLSQI (SEQ ID NO: 9), LRKPSAD VLSQIAKA (SEQ ID NO: 10), PSADVLSQIAKALRV (SEQ ID NO: 11), SQIAKALRVS AEVLY (SEQ ID NO: 12), AKALRVSAEVLYVRA (SEQ ID NO: 13), VRAGILEPSETSQVR (SEQ ID No: 14), TAITERQKQILLD IY (SEQ ID NO; 15), SQIAKALR VSAEVLYVRAC (SEQ ID NO: 16), MSSEEKLCDPTPTDD (SEQ ID
  • Antibodies that bind to an immunogenic M. tuberculosis Bsx protein or peptide for detecting tuberculosis or infection by M. tuberculosis are also described in detail in the instant applicant's co-pending International Patent Application No. PCT/AU2005/001254 filed August 19, 2005 the disclosure of which is incorporated herein in its entirety.
  • antibodies that bind to M. tuberculosis glutamine synthetase (GS) protein e.g., comprising the sequence set forth in SwissProt Database Accession No. 033342
  • an immunogenic peptide derived thereof e.g., comprising a surface-exposed region of a GS protein, or comprising the sequence RGTDGSAVFADSNGPHGMSSMFRSF (SEQ ID NO: 19) or WASGYRGLTPASDYNIDYAI (SEQ ID NO: 20).
  • Antibodies that bind to an immunogenic M. tuberculosis GS or peptide for detecting tuberculosis or infection by M. tuberculosis are also described in detail in the instant applicant's co-pending International Patent Application No. PCT/AU2005/000930 filed June 24 2005 the disclosure of which is incorporated herein in its entirety.
  • the present invention clearly contemplates antibodies agaisnt secondary analytes other than Bsx or Gs or immunogenic fragments thereof, the description of which is provided for the purposes of exemplification.
  • This invention provides a method of diagnosing tuberculosis or an infection by M. tuberculosis in a subject comprising detecting in a biological sample from said subject a S9 protein or an immunogenic fragment or epitope thereof, wherein the presence of said protein or immunogenic fragment or epitope in the sample is indicative of infection.
  • M. tuberculosis antigen as opposed to an antibody-based assay is that severely immune-compromized patients may not produce antibody at detectable levels, and the level of the antibody in any patient does not reflect bacilli burden.
  • antigen levels should reflect bacilli burden and, being a product of the bacilli, are a direct method of detecting its presence.
  • a method for detecting M. tuberculosis infection in a subject comprising contacting a biological sample derived from the subject with an antibody capable of binding to a S9 protein or an immunogenic fragment or epitope thereof, and detecting the formation of an antigen-antibody complex.
  • the diagnostic assays of the invention are useful for determining the progression of tuberculosis or an infection by M. tuberculosis in a subject.
  • the level of S9 protein or an immunogenic fragment or epitope thereof in a biological sample is positively correlated with the infectious state. For example, a level of the S9 protein or an immunogenic fragment thereof that is less than the level of the S9 protein or fragment detectable in a subject suffering from the symptoms of tuberculosis or an infection indicates that the subject is recovering from the infection. Similarly, a higher level of the protein or fragment in a sample from the subject compared to a healthy individual indicates that the subject has not been rendered free of the disease or infection.
  • a further embodiment of the present invention provides a method for determining the response of a .subject having tuberculosis or an infection by M. tuberculosis to treatment with a therapeutic compound for said tuberculosis or infection, said method comprising detecting a S9 protein or an immunogenic fragment or epitope thereof in a biological sample from said subject, wherein a level of the protein or fragment or epitope that is enhanced compared to the level of that protein or fragment or epitope detectable in a normal or healthy subject indicates that the subject is not responding to said treatment or has not been rendered free of disease or infection.
  • the present invention provides a method for determining the response of a subject having tuberculosis or an infection by M.
  • tuberculosis to treatment with a therapeutic compound for said tuberculosis or infection, said method comprising detecting a S9 protein or an immunogenic fragment or epitope thereof in a biological sample from said subject, wherein a level of the protein or fragment or epitope that is lower than the level of the protein or fragment or epitope detectable in a subject suffering from tuberculosis or infection by M. tuberculosis indicates that the subject is responding to said treatment or has been rendered free of disease or infection. Clearly, if the level of the S9 protein or fragment or epitope thereof is not detectable in the subject, the subject has responded to treatment.
  • the amount of a S9 protein in a biological sample derived from a patient is compared to the amount of the same protein detected in a biological sample previously derived from the same patient.
  • this method may be used to continually monitor a patient with a latent infection or a patient that has developed tuberculosis. In this way a patient may be monitored for the onset or progression of an infection or disease, with the goal of commencing treatment before an infection is established, particularly in an HFV+ individual.
  • 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.
  • a S9 protein or immunogenic fragment thereof is not detected in a reference sample, however, said S9 protein or immunogenic fragment thereof is detected in the patient sample, indicating that the patient from whom the sample was derived is suffering from tuberculosis or infection by M, tuberculosis or will develop an acute infection.
  • the amount of S9 protein or immunogenic fragment thereof may be enhanced in the patient sample compared to the level detected in a reference sample. Again, this indicates that the patient from whom the biological sample was isolated is suffering from tuberculosis or infection by M. tuberculosis or will develop an acute infection.
  • the biological sample is obtained previously from the subject.
  • the prognostic or diagnostic method is performed ex vivo.
  • the subject diagnostic/prognostic methods further comprise processing the sample from the subject to produce a derivative or extract that comprises the analyte (eg., pleural fluid or sputum or serum).
  • a derivative or extract that comprises the analyte (eg., pleural fluid or sputum or serum).
  • Suitable samples include extracts from tissues such as brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle and bone tissues, or body fluids such as sputum, serum, plasma, whole blood, sera or pleural fluid.
  • the biological sample is a bodily fluid or tissue sample selected from the group consisting of: saliva, plasma, blood, serum, sputum, urine, and lung. Other samples are not excluded.
  • preferred samples may comprise circulating immune complexes comprising the S9 protein or fragments thereof complexed with human immunoglobulin.
  • a capture reagent e.g., a capture antibody is used to capture the S9 antigen (S9 polypeptide or an immunoactive fragment or epitope thereof) complexed with the subject's immunoglobulin, in addition to isolated antigen in the subject's circulation.
  • Anti-Ig antibodies optionally conjugated to a detectable label, are used to specifically bind the captured CIC thereby detecting CIC patient samples.
  • the anti-Ig antibody binds preferentially to IgM, IgA or IgG in the sample.
  • the anti-Ig antibody binds to human Ig, e.g., human IgA, human IgG or human IgM.
  • the anti-Ig antibody may be conjugated to any standard detectable label known in the art. This is particularly useful for detecting infection by a pathogenic agent, e.g., a bacterium or virus, or for the diagnosis of any disease or disorder associated with CICs.
  • the diagnostic methods described according to any embodiment herein are amenable to a modification wherein the sample derived from the subject comprises one or more circulating immune complexes comprising immunoglobulin (Ig) bound to S9 protein of Mycobacterium tuberculosis or one or more immunogenic S9 peptides, fragments or epitopes thereof and wherein detecting the formation of an antigen-antibody complex comprises contacting an anti-Ig antibody with an immunoglobulin moiety of the circulating immune complex(es) for a time and under conditions sufficient for a complex to form than then detecting the bound anti-Ig antibody.
  • immunoglobulin Ig
  • the present invention clearly encompasses multianalyte tests for diagnosing infection by M. tuberculosis.
  • assays for detecting antibodies that bind to M. tuberculosis S9 protein can be combined with assays for detecting M. tuberculosis Bsx or glutamine synthetase (GS) protein.
  • the present inventors have also produced plasmacytomas producing monoclonal antibodies that bind to an immunogenic fragment or peptide or epitope of Bsx or GS. 2.
  • the present invention provides a method of diagnosing tuberculosis or an infection by M. tuberculosis in a subject comprising detecting in a biological sample from said subject antibodies that bind to a S9 protein or an immunogenic fragment or epitope thereof, wherein the presence of said antibodies in the sample is indicative of infection.
  • the infection may be a past or present infection, or a latent infection.
  • Antibody-based assays are primarily used for detecting active infections by M. tuberculosis.
  • the clinical history of the subject is considered due to residual antibody levels that may persist in recent past infections or chronic infections by M. tuberculosis.
  • the format is inexpensive and highly sensitive, however not as useful as an antigen- based assay format for detecting infection in immune-compromized individuals.
  • antibody-based assays are clearly useful for detecting M. tuberculosis infections in HFV " or HIV + individuals who are not immune-compromized.
  • the present invention provides a method for detecting M. tuberculosis infection in a subject, the method comprising contacting a biological sample derived from the subject with a S9 protein or an immunogenic fragment or epitope thereof and detecting the formation of an antigen-antibody complex.
  • the diagnostic assays of the invention are useful for determining the progression of tuberculosis or an infection by M. tuberculosis in a subject.
  • the amount of antibodies that bind to a S9 protein or fragment or epitope in blood or serum, plasma, or an immunoglobulin fraction from the subject is positively correlated with the infectious state. For example, a level of the anti-S9 antibodies thereto that is less than the level of the anti-S9 antibodies detectable in a subject suffering from the symptoms of tuberculosis or an infection indicates that the subject is recovering from the infection. Similarly, a higher level of the antibodies in a sample from the subject compared to a healthy individual indicates that the subject has not been rendered free of the disease or infection.
  • a further embodiment of the present invention provides a method for determining the response of a subject having tuberculosis or an infection by M. tuberculosis to treatment with a therapeutic compound for said tuberculosis or infection, said method comprising detecting antibodies that bind to a S9 protein or an immunogenic fragment or epitope thereof in a biological sample from said subject, wherein a level of the antibodies that is enhanced compared to the level of the antibodies detectable in a normal or healthy subject indicates that the subject is not responding to said treatment or has not been rendered free of disease or infection.
  • the present invention provides a method for determining the response of a subject having tuberculosis or an infection by M. tuberculosis to treatment with a therapeutic compound for said tuberculosis or infection, said method comprising detecting antibodies that bind to a S9 protein or an immunogenic fragment or epitope thereof in a biological sample from said subject, wherein a level of the antibodies that is lower than the level of the antibodies detectable in a subject suffering from tuberculosis or infection by M. tuberculosis indicates that the subject is responding to said treatment or has been rendered free of disease or infection.
  • the amount of an antibody against the S9 protein or fragment that is detected in a biological sample from a subject with tuberculosis may be compared to a reference sample, wherein the reference sample is derived from one or more healthy subjects who have not been previously infected with M. tuberculosis or not recently-infected with M. tuberculosis.
  • a reference sample is derived from one or more healthy subjects who have not been previously infected with M. tuberculosis or not recently-infected with M. tuberculosis.
  • Such negative control subjects will have a low circulating antibody titer making them suitable standards in antibody-based assay formats.
  • antibodies that bind to a S9 protein or immunogenic fragment thereof are not detected in the reference sample and only in a patient sample, indicating that the patient from whom the sample was derived is suffering from tuberculosis or infection by M. tuberculosis or will develop an acute infection.
  • the biological sample is obtained previously from the subject.
  • the prognostic or diagnostic method is performed ex vivo.
  • the subject diagnostic/prognostic methods further comprise processing the sample from the subject to produce a derivative or extract that comprises the analyte (e.g., blood, serum, plasma, or any immunoglobulin-containing sample).
  • a derivative or extract that comprises the analyte (e.g., blood, serum, plasma, or any immunoglobulin-containing sample).
  • Suitable samples include, for example, extracts from tissues comprising an immunoglobulin such as blood, bone, or body fluids such as serum, plasma, whole blood, an immunoglobulin-containing fraction of serum, an immunoglobulin- containing fraction of plasma, an immunoglobulin-containing fraction of blood.
  • an immunoglobulin such as blood, bone, or body fluids such as serum, plasma, whole blood, an immunoglobulin-containing fraction of serum, an immunoglobulin- containing fraction of plasma, an immunoglobulin-containing fraction of blood.
  • Preferred detection systems contemplated herein include any known assay for detecting proteins or antibodies in a biological sample isolated from a human subject, such as, for example, SDS/PAGE, isoelectric focusing, 2-dimensional gel electrophoresis comprising SDS/PAGE and isoelectric focusing, an immunoassay, a detection based system using an antibody or non-antibody ligand of the protein, such as, for example, a small molecule (e.g. a chemical compound, agonist, antagonist, allosteric modulator, competitive inhibitor, or non-competitive inhibitor, of the protein).
  • the antibody or small molecule may be used in any standard solid phase or solution phase assay format amenable to the detection of proteins.
  • Optical or fluorescent detection such as, for example, using mass spectrometry, MALDI-TOF, biosensor technology, evanescent fiber optics, or fluorescence resonance energy transfer, is clearly encompassed by the present invention.
  • Assay systems suitable for use in high throughput screening of mass samples, particularly a high throughput spectroscopy resonance method e.g. MALDI-TOF, electrospray MS or nano- electrospray MS, are particularly contemplated.
  • Immunoassay formats are particularly preferred, e.g., selected from the group consisting of, an immunoblot, a Western blot, a dot blot, an enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay.
  • an immunoblot e.g., selected from the group consisting of, an immunoblot, a Western blot, a dot blot, an enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • FRET fluorescence resonance energy transfer
  • ICAT isotope-coded affinity tags
  • mass spectrometry e.g., matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), biosensor technology, evanescent fiber-optics technology or protein chip technology are also useful.
  • FRET fluorescence resonance energy transfer
  • ICAT isotope-coded affinity tags
  • MALDI-TOF matrix-assisted laser desorption/ionization time of flight
  • ESI electrospray ionization
  • biosensor technology evanescent fiber-optics technology or protein chip technology
  • 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.
  • an assay involves immobilising a biological sample comprising anti-S9 antibodies, or alternatively S9 protein or an immunogenic fragment thereof, onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide).
  • 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).
  • an immobilised antibody that specifically binds a S9 protein is brought into direct contact with the biological sample, and forms a direct bond with any of its target protein present in said sample.
  • an immobilised isolated or recombinant S9 protein or an immunogenic fragment or epitope thereof will be contacted with the biological sample.
  • the added antibody or protein in solution is generally labelled with a detectable reporter molecule, such as for example, colloidal gold, a fluorescent label (e.g. FITC or Texas Red) or an enzyme (e.g. horseradish peroxidase (HRP)), alkaline phosphatase (AP) or ⁇ -galactosidase.
  • a detectable reporter molecule such as for example, colloidal gold, a fluorescent label (e.g. FITC or Texas Red) or an enzyme (e.g. horseradish peroxidase (HRP)), alkaline phosphatase (AP) or ⁇ -galactosidase.
  • a second labelled antibody can be used that binds to the first antibody or to the isolated/recombinant S9 antigen.
  • the label may be detected either directly, in the case of a fluorescent label, or through the addition of a substrate, such as for example hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indol-beta-D- galaotopyranoside (x-gal).
  • Such ELISA based systems are particularly suitable for quantification of the amount of a protein or antibody in a sample, such as, for example, by calibrating the detection system against known amounts of a standard.
  • an ELISA consists of immobilizing an antibody that specifically binds a S9 protein on a solid matrix, such as, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick or a glass support.
  • a 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.
  • specificity of the immobilized antibody ensures that only isolated or immunocomplexed S9 protein or fragments comprising the epitope that the antibody recognizes actually bind, whilst specificity of anti-human Ig ensures that only immunocomplexed S9 protein or fragment is detected.
  • the term "immunocomplexed” shall be taken to mean that the S9 protein or fragments thereof in the patient sample are complexed with human Ig such as human IgA or human IgM or human IgG, etc. Accordingly, this embodiment is particularly useful for detecting the presence of M. tuberculosis or an infection by M. tuberculosis that has produced an immune response in a subject.
  • detection antibody e.g., anti-human IgA or anti-human IgG or anti-human IgM
  • detection antibodies that bind to human IgA, IgM and IgG are publicly available to the art.
  • a third labelled antibody can be used that binds the second (detecting) antibody.
  • the presence of anti-S9 antibodies, or alternatively a S9 protein or an immunogenic fragment thereof, is detected using a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • the basic principle of the assay is the use of a radiolabeled antibody or antigen to detect antibody antigen interactions.
  • an antibody that specifically binds to a S9 protein can be bound to a solid support and a biological sample brought into direct contact with said antibody.
  • 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.
  • 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.
  • 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.
  • any reporter molecule such as, for example, an enzyme or a fluorescent molecule, in place of a radioactive label.
  • Western blotting is also useful for detecting a S9 protein or an immunogenic fragment thereof.
  • 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 ( ⁇ r Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994).
  • SDS-PAGE sodium dodecyl sulphate
  • Separated proteins are then transferred to a solid support, such as, for example, a membrane or more specifically, nitrocellulose membrane, nylon membrane or PVDF membrane, using methods well known in the art, for example, electrotransfer.
  • This membrane may then be blocked and probed with a labelled antibody or ligand that specifically binds a S9 protein.
  • a labelled secondary, or even tertiary, antibody or ligand can be used to detect the binding of a specific primary antibody.
  • High-throughput methods for detecting the presence or absence of anti-S9 antibodies, or alternatively S 9 protein or an immunogenic fragment thereof are particularly preferred.
  • mass spectrometry e.g., MALDI-TOF is used for the rapid identification of a protein that has been separated by either one- or two-dimensional gel electrophoresis. Accordingly, there is no need to detect the proteins of interest using an antibody or ligand that specifically binds to the protein of interest. Rather, proteins from a biological sample are separated using gel electrophoresis using methods well known in the art and those proteins at approximately the correct molecular weight and/or isoelectric point are analysed using MALDI-TOF to determine the presence or absence of a protein of interest.
  • mass spectrometry e.g., MALDI or ESI
  • a biological sample such as, for example sputum.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a protein chip 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.
  • biomolecular interaction analysis-mass spectrometry is used to rapidly detect and characterise a protein present in complex biological samples at the low- to sub-femptamole (fmol) level (Nelson et al Electrophoresis 21: 1155-1 163, 2000).
  • One technique useful in the analysis of a protein chip is surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technology to characterise a protein bound to the protein chip.
  • SELDI-TOF-MS surface enhanced laser desorption/ionization-time of flight-mass spectrometry
  • the protein chip is analysed using ESI as described in U.S. Patent Application 20020139751.
  • protein chips are particularly amenable to multiplexing of detection reagents. Accordingly, several antibodies or ligands each able to specifically bind a different peptide or protein may be bound to different regions of said protein chip. Analysis of a biological sample using said chip then permits the detecting of multiple proteins of interest, or multiple B cell epitopes of the S9 protein. Multiplexing of diagnostic and prognostic markers is particularly contemplated in the present invention.
  • the samples are analysed using ICAT or ITRAC, essentially as described in US Patent Application No. 20020076739.
  • This system relies upon the labelling of a protein sample from one source (i.e. a healthy individual) with a reagent and the labelling of a protein sample from another source (i.e. a tuberculosis patient) with a second reagent that is chemically identical to the first reagent, but differs in mass due to isotope composition.
  • 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.
  • a biological sample comprising anti-S9 antibodies, or alternatively S9 protein or an immunogenic fragment thereof is subjected to 2-dimensional gel electrophoresis.
  • Proteins in the biological sample are then separated.
  • 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.
  • mass spectrometry it is possible to determine whether or not a protein of interest is present in a patient sample.
  • a diagnostic or prognostic assay described herein may be a multiplexed assay.
  • 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.
  • 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.
  • a multiplexed assay may comprise an assay that detects several anti-S9 antibodies and/or S9 epitopes in the same reaction and simultaneously, or alternatively, it may detect other one or more antigens/antibodies in addition to one or more anti-S9 antibodies and/or S9 epitopes.
  • the present invention clearly contemplates multiplexed assays for detecting S9 antibodies and epitopes in addition to detecting CD4+ T-helper cells via one or more receptors on the cell surface and/or one or more HIV-I and/or HIV-2 antigens.
  • Such assays are particularly useful for simultaneously obtaining information on co-infection with M. tuberculosis and HIV-I and/or HIV-2, and/or for determining whether or not a subject with M. tuberculosis is immune-compromised.
  • multiplexed assay formats are useful for monitoring the health of an HIV+/TB+ individual.
  • the biological sample in which a S9 protein or anti-S9 antibody is detected is a sample selected from the group consisting of lung, lymphoid tissue associated with the lung, paranasal sinuses, bronchi, a bronchiole, alveolus, ciliated mucosal epithelia of the respiratory tract, mucosal epithelia of the respiratory tract, broncheoalveolar lavage fluid (BAL), alveolar lining fluid, sputum, mucus, saliva, blood, serum, plasma, urine, peritoneal fluid, pericardial fluid, pleural fluid, squamous epithelial cells of the respiratory tract, a mast cell, a goblet cell, a pneumocyte (type 1 or type 2), an intra epithelial dendritic cell, a PBMC, a neutrophil, a monocyte, or any immunoglobulin- containing fraction of any one or more of said tissues, fluids or cells.
  • a biological sample is obtained from the group consist
  • 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.
  • a biological sample is sputum, isolated from lung of a patient using, for example the method described in Gershman, N. H. et al, J Allergy CHn Immunol, 10(4): 322-328, 1999.
  • the sputum is expectorated i.e., coughed naturally.
  • a biological sample is plasma that has been isolated from blood collected from a patient using a method well known in the art.
  • 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.
  • a biological sample is treated to denature a protein present in said sample.
  • Methods of denaturing a protein include heating a sample, treating a sample with 2-mercaptoethanol, dithiotreitol (DTT), N-acetylcysteine, detergent or other compound such as, for example, guanidinium or urea.
  • DTT dithiotreitol
  • N-acetylcysteine detergent or other compound such as, for example, guanidinium or urea.
  • the use of DTT is preferred for liquefying sputum.
  • 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.
  • 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.
  • 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.
  • the reference sample comprises for example cells, fluids or tissues from a healthy subject who has not been previously or recently infected and is not suffering from an infection or disease.
  • such reference samples are from fluids or tissues that do not require surgical resection or intervention to obtain them.
  • bodily fluids and derivatives thereof are preferred.
  • Highly preferred reference samples comprise sputum, mucus, saliva, blood, serum, plasma, urine, BAL fluid, peritoneal fluid, pericardial fluid, pleural fluid, a PBMC 5 a neutrophil, a monocyte . , or any immunoglobulin-containing fraction of any one or more of said tissues, fluids or cells.
  • a reference sample and a test (or patient) sample are processed, analysed or assayed and data obtained for a reference sample and a test sample are compared.
  • a reference sample and a test sample are processed, analysed or assayed at the same time.
  • a reference sample and a test sample are processed, analysed or assayed at a different time.
  • 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.
  • the present invention provides a kit for detecting M. tuberculosis infection in a biological sample.
  • the kit comprises:
  • the kit comprises:
  • an isolated or recombinant S9 protein or an immunogenic fragment or epitope thereof (i) an isolated or recombinant S9 protein or an immunogenic fragment or epitope thereof; and (ii) means for detecting the formation of an antigen-antibody complex.
  • the antibodies, immunogenic S9 peptide, and detection means of the subject kit are preferably selected from the antibodies and immunogenic S9 peptides described herein above and those embodiments shall be taken to be incorporated by reference herein from the description.
  • the selection of compatible kit components for any assay format will be readily apparent to the skilled artisan from the description.
  • the subject kit comprises:
  • the kit further comprises an amount of one or more peptides each comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7, or a fusion between any two or more of said peptides.
  • the kit further comprises means for the detection of the binding of an antibody, fragment thereof or a ligand to a S9 protein.
  • 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.
  • 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.
  • a reporter molecule is directly linked to the antibody or ligand.
  • a kit may additionally comprise a reference sample.
  • a reference sample may for example, be a protein sample derived from a biological sample isolated from one or more tuberculosis subjects.
  • 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.
  • a reference sample comprises a peptide that is detected by an antibody or a ligand.
  • the peptide is of known concentration.
  • a kit optionally comprises means for sample preparations, such as, for example, a means for cell lysis.
  • Such means are means of solubilizing sputum, such as, for example, a detergent (e.g., tributyl phosphine, C7BZO, dextran sulfate, DTT, N-acetylcysteine, or polyoxyethylenesorbitan monolaurate).
  • a detergent e.g., tributyl phosphine, C7BZO, dextran sulfate, DTT, N-acetylcysteine, or polyoxyethylenesorbitan monolaurate.
  • a kit comprises means for protein isolation (Scopes (In: Protein Purification: Principles and Practice, Third Edition, Springer Verlag, 1994).
  • the S9 protein or immunogenic fragment or epitope thereof can induce the specific production of a high titer antibody when administered to an animal subject.
  • the invention provides a method of eliciting the production of antibody against M. tuberculosis comprising administering an isolated S9 protein or an immunogenic fragment or epitope thereof to said subject for a time and under conditions sufficient to elicit the production of antibodies, such as, for example, neutralizing antibodies that bind to M. tuberculosis.
  • M. tuberculosis Bsx or GS or immunogenic fragment thereof for a time and under conditions sufficient to elicit the production of antibodies, such as, for example, neutralizing antibodies that bind to M. tubercidosis .
  • Such administration may be at the same time as administering S9 protein or fragment (i.e., co-administration) or alternatively, before or after the S9 protein or fragment is administered to a subject.
  • the neutralizing antibodies according got any of the preceding embodiments are high titer neutralizing antibodies.
  • the effective amount of S9 protein or other protein or epitope thereof to produce antibodies varies upon the nature of the immunogenic B cell epitope, the route of administration, the animal used for immunization, and the nature of the antibody sought. All such variables are empirically determined by art-recognized means.
  • the invention provides a method of inducing immunity against M. tuberculosis in a subject comprising administering to said subject an isolated or recombinant S9 protein or immunogenic fragment or epitope thereof for a time and under conditions sufficient to elicit a humoral immune response against said an isolated or recombinant S9 protein or immunogenic fragment or epitope.
  • one or more second antigens e.g., M. tuberculosis Bsx or GS or immunogenic fragment thereof for a time and under conditions sufficient to elicit a humoral immune response against that antigen.
  • Such administration may be at the same time as administering S9 protein or fragment (i.e., co-administration) or alternatively, before or after the S9 protein or fragment is administered to a subject.
  • the immunizing antigen may be administered in the form of any convenient formulation as described herein.
  • humoral immune response means that a secondary immune response is generated against the immunizing antigen sufficient to prevent infection by M. tuberculosis.
  • the humoral immunity generated includes eliciting in the subject a sustained level of antibodies that bind to a B cell epitope in the immunizing antigen.
  • sustained level of antibodies is meant a sufficient level of circulating antibodies that bind to the B cell epitope to prevent infection by M. tuberculosis.
  • antibodies levels are sustained for at least about six months or 9 months or 12 months or 2 years.
  • the present invention provides a method of enhancing the immune system of a subject comprising administering an immunologically active S9 protein or an epitope thereof or a vaccine composition comprising said S 9 protein or epitope for a time and under conditions sufficient to confer or enhance resistance against M. tuberculosis in said subject.
  • M. tuberculosis Bsx or GS or immunogenic fragment thereof for a time and under conditions sufficient to confer or enhance resistance against M. tuberculosis in said subject.
  • Such administration may be at the same time as administering S9 protein or fragment (i.e., co-administration) or alternatively, before or after the S9 protein or fragment is administered to a subject.
  • confer or enhance resistance is meant that a M. tuberculosis -specific immune response occurs in said subject, said response being selected from the group consisting of: (i) an antibody against a S9 protein of M. tuberculosis or an epitope of said protein is produced in said subject;
  • the invention will be understood to encompass a method of providing or enhancing immunity against M. tuberculosis in an uninfected human subject comprising administering to said subject an immunologically active S9 protein or an epitope thereof or a vaccine composition comprising said S9 protein or epitope for a time and under conditions sufficient to provide immunological memory against a future infection by M. tuberculosis.
  • M. tuberculosis Bsx or GS or immunogenic fragment thereof for a time and under conditions sufficient to provide immunological memory against a future infection by M. tuberculosis.
  • Such administration may be at the same time as administering S9 protein or fragment (i.e., co-administration) or alternatively, before or after the S9 protein or fragment is administered to a subject.
  • the present invention provides a method of treatment of tuberculosis in a subject comprising performing a diagnostic method or prognostic method as described herein.
  • 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
  • compositions according to this embodiment comprise S9 protein or immunogenic fragment thereof optionally with on or more other immunogen M. tuberculosis proteins or peptide fragments, in combination with a pharmaceutically acceptable carrier or excipient. It is clearly within the scope of the present invention for such compositions to include S9 protein or fragment thereof according to any embodiment described herein e.g., any one of SEQ ID NOs: 1-7, and one or more second antigens e.g., M. tuberculosis Bsx or GS or immunogenic fragments thereof e.g., as set forth in any one of SEQ ID NOs: 8-20 or a subset thereof.
  • the composition is administered to a subject harboring a latent or active M. tuberculosis infection.
  • 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.
  • reactivation of a T cell population may occur following activation of a latent M. tuberculosis infection, or following re-infection with M. tuberculosis, or following immunization of a previously- infected subject with a S9 protein or epitope or vaccine composition of the invention.
  • Standard methods can be used to determine whether or not CTL activation has occurred in the subject, such as, for example, using cytotoxicity assays, ELISPOT, or determining IFN - ⁇ production in PBMC of the subject.
  • the peptide or derivative or variant or vaccine composition is administered for a time and under conditions sufficient to elicit or enhance the expansion of CDS + T cells. Still more preferably, the peptide or derivative or variant or vaccine composition is administered for a time and under conditions sufficient for M. tuberculosis -specific cell mediated immunity (CMI) to be enhanced in the subject.
  • CMI tuberculosis -specific cell mediated immunity
  • M. tuberculosis -specific CMI is meant that the activated and clonally expanded CTLs are MHC-restricted and specific for a CTL epitope of the invention.
  • CTLs are classified based on antigen specificity and MHC restriction, (i.e., non-specific CTLs and antigen-specific, MHC-restricted CTLs).
  • Non-specific CTLs are composed of various cell types, including NK cells and antibody-dependent cytotoxicity, and can function very early in the immune response to decrease pathogen load, while antigen- specific responses are still being established.
  • 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.
  • compartmentally localized effects it is preferred to utilize a vaccine composition suitably formulated for administration to that compartment.
  • a vaccine composition suitably formulated for administration to that compartment.
  • the effective amount of S9 protein or epitope thereof, optionally in combination with one or more other proteins or epitopes e.g., derived from Bsx or GS proteins of M. tuberculosis, to be administered solus or in a vaccine composition to elicit CTL activation, clonal expansion or CMI, varies upon the nature of the immunogenic epitope, the route of administration, the weight, age, sex, or general health of the subject immunized, and the nature of the CTL response sought. All such variables are empirically determined by art-recognized means.
  • the S9 protein or epitope thereof 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.
  • the optimum dose to be administered and the preferred route for administration are established using animal models, such as, for example, by injecting a mouse, rat, rabbit, guinea pig, dog, horse, cow, goat or pig, with a formulation comprising the peptide, and then monitoring the CTL immune response to the epitope using any conventional assay.
  • Adoptive transfer techniques may also be used to confer or enhance resistance against M. tuberculosis infection or to prevent or reduce the severity of a reactivated latent infection. Accordingly, in a related embodiment, there is provided a method of enhancing or conferring immunity against M.
  • tuberculosis in an uninfected human subject comprising contacting ex vivo a T cell obtained from a human subject with an immunologically active S9 protein or an epitope thereof or a vaccine composition comprising said protein or epitope for a time and under conditions sufficient to confer M. tuberculosis activity on said T cells.
  • the invention provides a method of enhancing the M. tuberculosis -specific cell mediated immunity of a human subject, said method comprising:
  • the present invention encompasses the administration of additional immunogenic proteins or epitopes e.g., derived from Bsx or GS proteins of M. tuberculosis.
  • the T cell may be a CTL or CTL precursor cell.
  • the human subject from whom the T cell is obtained may be the same subject or a different subject to the subject being treated.
  • the subject being treated can be any human subject carrying a latent or active M. tuberculosis infection or at risk of M. tuberculosis infection or reactivation of M. tuberculosis infection or a person who is otherwise in need of obtaining vaccination against M. tuberculosis or desirous of obtaining vaccination against M. tuberculosis.
  • Such adoptive transfer is preferably carried out and M. tuberculosis reactivity assayed essentially as described by Einsele et ai, Blood 99, 3916-3922, 2002, which procedures are incorporated herein by reference.
  • M. tuberculosis activity is meant that the T cell is rendered capable of being activated as defined herein above (i.e. the T cell will recognize and lyse a cell harboring M. tuberculosis or able to recognize a T cell epitope of an antigen of M. tuberculosis, either transiently or in a sustained manner). Accordingly, it is particularly preferred for the T cell to be a CTL precursor which by the process of the invention is rendered able to recognize and lyse a cell harboring M. tuberculosis or able to recognize a T cell epitope of an antigen of M. tuberculosis, either transiently or in a sustained manner.
  • the T cell is preferably contained in a biological sample obtained from a human subject, such as, for example, a biopsy specimen comprising a primary or central lymphoid organ (eg. bone marrow or thymus) or a secondary or peripheral lymphoid organ (eg. blood, PBMC or a buffy coat fraction derived there from).
  • a biological sample obtained from a human subject, such as, for example, a biopsy specimen comprising a primary or central lymphoid organ (eg. bone marrow or thymus) or a secondary or peripheral lymphoid organ (eg. blood, PBMC or a buffy coat fraction derived there from).
  • a primary or central lymphoid organ eg. bone marrow or thymus
  • PBMC peripheral lymphoid organ
  • 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.
  • the subject method further comprises obtaining a sample comprising the T cell of the subject, and more preferably, obtaining said sample from said subject.
  • the present invention clearly contemplates the use of the S9 protein or an immunogenic fragment or epitope thereof in the preparation of a therapeutic or prophylactic subunit vaccine against M. tuberculosis infection in a human or other animal subject.
  • the invention provides a pharmaceutical composition or vaccine comprising a S9 protein or an immunogenic fragment or epitope thereof in combination with a pharmaceutically acceptable diluent,
  • the composition according to this embodiment comprises S9 protein or immunogenic fragment thereof optionally with on or more other immunogenic M. tuberculosis proteins or peptide fragments, in combination with a pharmaceutically acceptable carrier or excipient. It is clearly within the scope of the present invention for such compositions to include S9 protein or fragment thereof according to any embodiment described herein e.g., any one of SEQ ID NOs: 1-7, and one or more second antigens e.g., M. tuberculosis Bsx or GS or immunogenic fragments thereof e.g., as set forth in any one of SEQ ID NOs: 8-20 or a subset thereof.
  • the S9 protein, and optional other protein, or immunogenic fragment or epitope thereof is conveniently formulated in a pharmaceutically acceptable excipient or diluent, such as, for example, an aqueous solvent, non-aqueous solvent, non-toxic excipient, such as a salt, preservative, buffer and the like.
  • 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.
  • 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.
  • adjuvants include all acceptable immunostimulatory compounds such as, for example, a cytokine, toxin, or synthetic composition.
  • Exemplary adjuvants include IL- 1, IL-2, BCG, aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thur-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 1 1637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'- dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP) 1983A, referred to as MTP-PE), lipid A, MPL and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emul
  • BRM biologic response modifiers
  • exemplary BRM's include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA, USA); Indomethacin (IND; 150 mg/d) (Lederle, NJ, USA); or low-dose Cyclophosphamide (CYP; 75, 150 or 300 mg/m 2 ) (Johnson/Mead, NJ, USA).
  • Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments.
  • 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.
  • liposomes may be unilamellar or multilamellar, and can vary in size with diameters ranging from 0.02 ⁇ m to greater than 10 ⁇ m.
  • 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.
  • the liposome fuses with the target cell, whereby the contents of the liposome empty into the target cell.
  • an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents (Scherphof et al., Ann. N. Y. Acad. ScL 446, 368 (1985)).
  • the S9 protein or immunogenic fragment or epitope thereof may be localized on the surface of the liposome, to facilitate antigen presentation without disruption of the liposome or endocytosis. Irrespective of the mechanism or delivery, however, the result is the intracellular disposition of the associated S9 protein or immunogenic fragment or epitope thereof.
  • Liposomal vectors may be anionic or cationic.
  • Anionic liposomal vectors include pH sensitive liposomes which disrupt or fuse with the endosomal membrane following endocytosis and endosome acidification.
  • Cationic liposomes are preferred for mediating mammalian cell transfection in vitro, or general delivery of nucleic acids, but are used for delivery of other therapeutics, such as peptides or lipopeptides.
  • Cationic liposome preparations are made by conventional methodologies (Feigner et at, Proc. Nat'l Acad. Sci USA 84, 7413 (19S7); Schreier, Liposome Res. 2, 145 (1992)). Commercial preparations, such as Lipofectin (Life Technologies, Inc., Gaithersburg, Md. USA), are readily available. The amount of liposomes to be administered are optimized based on a dose response curve. Feigner et al., supra.
  • Suitable liposomes that are used in the methods of the invention include multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUV), medium-sized unilamellar vesicles (MUV), large unilamellar vesicles (LUV), giant unilamellar vesicles (GUV), multivesicular vesicles (MW), single or oligolamellar vesicles made by reverse-phase evaporation method (REV), multilamellar vesicles made by the reverse-phase evaporation method (MLV-REV), stable plurilamellar vesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared by extrusion methods (VET), vesicles prepared by French press (FPV), vesicles prepared by fusion
  • delivery particle for example, microspheres and the like, also are contemplated for delivery of the S9 protein and optional other protein, or immunogenic fragment or epitope thereof.
  • 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.
  • APC autologous or allogeneic antigen presenting cell
  • Nucleic acid-based vaccines that comprise nucleic acid, such as, for example, DNA or RNA, encoding the immunologically active S9 protein and optional other protein, or epitope(s) thereof, and cloned into a suitable vector (eg. vaccinia, canary pox, adenovirus, or other eukaryotic virus vector) are also contemplated.
  • a suitable vector eg. vaccinia, canary pox, adenovirus, or other eukaryotic virus vector
  • DNA encoding a S9 protein and optional other protein is formulated into a DNA vaccine, such as, for example, in combination with the existing Calmette-Guerin (BCG) or an immune adjuvant such as vaccinia virus, Freund's adjuvant or another immune stimulant.
  • BCG Calmette-Guerin
  • an immune adjuvant such as vaccinia virus, Freund's adjuvant or another immune stimulant.
  • Patient serum or plasma is applied to a column of protein G-SepharoseTM (Amersham Biosciences), previously equilibrated with 2OmM phosphate buffer pH 7.0 and incubated on ice with occasional inversion.
  • the mixture is centrifuged at 600Og for 10 minutes at 4 0 C and the supernatant decanted.
  • the SepharoseTM pellet is washed with 2OmM phosphate buffer.
  • the IgG bound to the SepharoseTM is eluted by addition of 5OmM glycine pH 2.7 for 20 minutes. After centrifugation as above, the supernatant is discarded and the glycine step repeated.
  • the protein content of the samples is estimated using a Bradford assay. Samples were diluted with sample buffer as above replacing 4OmM Tris with 5mM Tris.
  • Dry 1 1cm EPG strips (Amersham-Biosciences) are rehydrated for 16-24 hours with 180 ⁇ l of protein sample. Rehydrated strips are focussed on a Protean IEF Cell (B io- Rad, Hercules, CA) or Proteome System's IsoElectrIQ electrophoresis equipment for approx 140 kVhr at a maximum of 10 kV. Focussed strips are then equilibrated in urea/SDS/Tris-HCl/bromophenol blue buffer.
  • Protein gel pieces Prior to mass spectrometry protein samples are prepared by in-gel tryptic digestion. Protein gel pieces are excised, destained, digested and desalted using an XciseTM, an excision/liquid handling robot (Proteome Systems, Sydney, Australia and Shimadzu- Biotech, Kyoto, Japan) in association with the Montage In-GeI Digestion Kit (developed by Proteome Systems and distributed by Millipore, Billerica, Ma, 01821, USA). Prior to spot cutting, the 2-D gel is incubated in water to maintain a constant size and prevent drying. Subsequently, the 2-D gel is placed on the XciseTM, a digital image was captured and the spots to be cut are selected.
  • gel pieces are subjected to automated liquid handling and in-gel digestion. Briefly, each spot is destained with 100 ⁇ l of 50% (v/v) acetonitrile in 50 mM ammonium bicarbonate. The gel pieces are dried by adding 100% acetonitrile, the acetonitrile is removed after 5 seconds and the gels dried completely by evaporating the residual acetonitrile at 37°C. Proteolytic digestion is performed by rehydrating the dried gel pieces with 30 ⁇ l of 20 mM ammonium bicarbonate (pH 7.8) containing 5 ⁇ g/ml modified porcine trypsin and incubated at 3O 0 C overnight.
  • 20 mM ammonium bicarbonate pH 7.8
  • Digests are analyzed using an Axima-CFR MALDI-TOF mass spectrometer (Kratos, Manchester, LIK) in positive ion reflectron mode.
  • a nitrogen laser with a wavelength of 337 nm is used to irradiate the sample.
  • the spectra are acquired in automatic mode in the mass range 600 Da to 4000 Da applying a 64-point raster to each sample spot. Only spectra passing certain criteria are saved. All spectra undergo an internal two point calibration using an autodigested trypsin peak mass, m/z 842.51 Da and spiked adrenocorticotropic hormone (ACTH) peptide, m/z 2465.117 Da.
  • Software designed by Proteome Systems as contained in the web-based proteomic data management system BioinfbrmatIQ T (Proteome Systems), is used to extract isotopic peaks from MS spectra.
  • Protein identification is performed by matching the monoisotopic masses of the tryptic peptides (i.e. the peptide mass fingerprint) with the theoretical masses from protein databases using IonlQTM or MASCOTTM database search software (Proteome System Limited, North Ryde, Sydney, Australia). Querying was done against the non- redundant SwissProt (Release 40) and TrEMBL (Release 20) databases (June 2002 version), and protein identities are ranked through a modification of the MOWSE scoring system. Propionamide-cysteine (cys-PAM) or carboxyamidomethyl-cysteine (cys-CAM) and oxidized methionine modifications are taken into account and a mass tolerance of 100 ppm was allowed.
  • cys-PAM Propionamide-cysteine
  • cys-CAM carboxyamidomethyl-cysteine
  • oxidized methionine modifications are taken into account and a mass tolerance of 100 ppm was allowed.
  • Miscleavage sites are only considered after an initial search without miscleavages had been performed.
  • the following criteria are used to evaluate the search results: the MOWSE score, the number and intensity of peptides matching the candidate protein, the coverage of the candidate protein's sequence by the matching peptides and the gel location.
  • proteins are analysed using LC-ESI-MS. Tryptic digest solutions of proteins (10 ⁇ l) are analysed by nanoflow LC/MS using an LCQ Deca Ion
  • Trap mass spectrometer (ThermoFinnigan, San Jose, CA) equipped with a Surveyor LC system composed of an autosampler and pump. Peptides are separated using a PepFinder kit (Thermo-Finnigan) coupled to a Cl 8 PicoFrit column (New Objective). Gradient elution from water containing 0.1% (v/v) formic acid (mobile phase A) to 90% (v/v) acetonitrile containing 0.1% (v/v) formic acid (mobile phase B) is performed over a 30-60-minute period. The mass spectrometer is set up to acquire three scan events - one full scan (range from 400 to 2000 amu) followed by two data dependant MS/MS scans.
  • spectra were firstly checked for correct calibration of peptide masses. Spectra are then processed to remove background noise including masses corresponding to trypsin peaks and matrix. The data are then searched against publicly-available SwissProt and TrEMBL databases using Proteome Systems search engine IonlQTM v69 and/or MASCOTTM. PSD data is searched against the same databases using the in-house search engine FragmentastIQTM. LC MS-MS data is also searched against the databases using the SEQUEST search engine software.
  • a protein having a molecular weight of about 30.2 kDa was recognized in the immunoglobulin fraction of sera from TB + samples.
  • the sequences of four peptides from MALDI-TOF data (SEQ ID Nos: 2-5 inclusive) matched a protein having SwissProt Accession No. P66638 (SEQ ID NO: 1).
  • the percent coverage of P66638 by these 4 peptides (SEQ ID NOs: 2-5) was about 14-15%, suggesting that the peptide fragments were derived from this same protein marker. This conclusion was supported by there being only six theoretical tryptic peptides with zero miscleavages, and fourteen theoretical tryptic peptides having one miscleavage.
  • the identified protein having the amino acid sequence set forth in SEQ ID NO: 1 was designated as "S9".
  • the estimated molecular weight of the S9 protein is only about 16.4 kDa, and the estimated isoelectric point of S9 is about 10.7. Since the observed molecular weight of the S9 protein was about 14 kDa higher than the estimated value, the protein is most likely post-translationally modified e.g., by glycosylation, or co-migrates with another molecular species such as nucleic acid.
  • a synthetic peptide comprising the sequence H-MTETT PAPQT PAAPA GPAQS FC- NH 2 from 3OS ribosomal protein S9 was synthesized to 78% purity as determined by liquid chromatography by Mimotopes using solid phase peptide synthesis technology. This peptide was coupled to keyhole limpet Hemocyanin (KHL) via a Maleimidocaproyl-N-Hydroxysuccinimide linker.
  • KHL keyhole limpet Hemocyanin
  • the peptide was also synthesized with a GSGL spacer and attached to biotin (PAPQT PAAPA GPAQS FGSGL-Biotin) to 93% purity by liquid chromatography.
  • the rabbit was bled out. All blood was collected in sterile containers and incubated at 37°C to accelerate clotting. The containers were centrifuged and the serum removed and re-centrifuged to remove the remaining red cells.
  • Streptavidin (Sigma Aldrich) was diluted to 5 ⁇ g/ml in ddH 2 O and incubated in a Nunc plate overnight at 4 0 C. The solution was then flicked out and 250 ⁇ L of blocking buffer (1% (w/v) casein, 0.1% (v/v) Tween 20, 0.1% (w/v) sodium azide in PBS) added to each well and incubated at room temperature for 1 hour. The blocking buffer was flicked out and biotinylated peptide (corresponding to the immunogen injected into the rabbit) was added in blocking buffer at 3 ⁇ g/ml (50 ⁇ l/well) and incubated for one hour at room temperature on a shaker.
  • blocking buffer 1% (w/v) casein, 0.1% (v/v) Tween 20, 0.1% (w/v) sodium azide in PBS
  • the plate was washed in an Elx405 Auto Plate Washer (Bio-Tek Instruments Inc., Winooski, VT), with 0.5 x PBS / 0.05% (v/v) Tween 20 solution and excess solution tapped out on a paper towel.
  • the rabbit sera was diluted in blocking buffer 2 fold from 1 : 500 to 1 : 1,024,000 and incubated from 1 hour at 50 ul/well at room temperature on a shaker.
  • the plate was washed with the plate washer using 0.5 x PBS / 0.05% (v/v) Tween 20 solution and excess solution tapped out on a paper towel.
  • Binding of the rabbit antibody to its corresponding epitope was detected using HRP-conjugated sheep anti-rabbit (Chemicon) diluted 1 in 10,000 in conjugate diluent buffer. Fifty millilitres were added to each well and incubated for one hour at room temperature on a shaker. The plate was washed with the plate washer using 0.5 x PBS and excess solution tapped out on a paper towel- Fifty millilitres of TMB (3,3',5',5-Tetramethylbenzidine; Sigma) was added to each well and the plate incubated in the dark for 30 minutes. Development was stopped with 50 ⁇ L/well of 0.5M sulphuric acid.
  • the R9 antibody described in Example 4 was used to detect S9 protein in samples from 20 TB subjects and 20 subjects suffering from a non-TB subject. Briefly, sputum (12 ⁇ l) from TB or non-TB patients was loaded onto 4-12% ID gradient SDS polyacrylamide gels and separated by electrophoresis. Proteins were then electrotransferred onto PVDF membrane. Membranes were then blocked in solution containing 1% casein in IX PBS, 0.1% Tween-20 (PBST) at room temperature (RT) for 2 hours. Membranes were then incubated with 15 ⁇ g/ml purified rabbit anti-S9 polyclonal antibody solution (i.e., R9) at RT for 2 hr, following by 3 x lOmin washes with PBST.
  • PBST room temperature
  • Membranes were then incubated with 1 :10,000 dilution of sheep anti-rabbit IgG-HRP conjugated antibody solution at RT for 1 hr, followed by 5 x 10 min washes with times PBST. Membranes were finally treated with 'Femto' chemiluminescence reagents (Pierce) for 5 min before exposure to x-ray films.
  • Antibody R9 detects M, tuberculosis ribosomal protein S9 in cultured M. tuberculosis
  • Peptides predicted to be exposed on the surface of the ribosomal S9 protein were conjugated to biotin.
  • Streptavidin was immobilised onto an ELISA plate at 50 ⁇ l per well at a concentration of 5 ⁇ g/ml.
  • Wells were incubated with appropriate peptides at 3 ⁇ g/ml diluted in blocking buffer, followed by addition plasma from each of 44 TB and 44 non-TB subjects, diluted 1/50.
  • Bound human IgG were detected with sheep anti- Human IgG HRP conjugate diluted at 1/10,000, then colour development with TMB substrate at 50 ul per well..
  • ROC curve analysis was used to determine (i) sensitivity at 95% specificity; and (ii) optimum sensitivity and specificity.
  • Plasma and/or sputum antibodies from non-TB subjects were found to have minimal cross-reactivity to two peptides tested.
  • one peptide had a sensitivity at 95% specificity of 17.3%, an optimal sensitivity of 56.8% and an optimum specificity of 79.6%.
  • the other peptide had a sensitivity at 95% specificity of 8.3%, an optimal sensitivity of 56.8% and an optimum specificity of 79.6%.
  • a sandwich ELISA was developed employing two antibodies prepared against recombinant M. tuberculosis ribosomal protein S9, in particular the chicken polyclonal antibody designated Ch27 and the mouse antibody Mol025F (Example 8),
  • sandwich ELISA was performed to determine optimum capture and detection antibodies, and appropriate antibody concentrations for use. Briefly, two ELISA plates were coated with either Ch27 or Mol025F antibodies at 2.5 ⁇ g/ml and 5 ⁇ g/ml concentrations in blocking buffer. Following washing to remove unbound antibody, 50 ⁇ l aliquots of recombinant S9 protein, diluted serially in blocking buffer 1 :2 (v/v) from 500 ng/ml starting concentration to 7.8 ng/ml, were added the wells of the antibody-coated ELISA plates.
  • the alternate detection antibody i.e., Mol025F for detection of Ch27-S9 complexes and Ch27 for detection of Mol025F-S9 complexes
  • the alternate detection antibody was contacted with the plates at concentrations in the range of 1.25 ⁇ g/ml to 5 ⁇ g/ml.
  • plates were washed as before, incubated with 50 ⁇ l of a 1 :5000 (v/v) dilution of donkey anti-mouse IgG conjugated to horseradish peroxidase (HRP), washed as before, incubated with TMB for 30 mins, and the absorbance at 595-600nm was determined.
  • HRP horseradish peroxidase
  • the assay was also performed using a serial dilution of S9 protein, in the concentration range from 18.31 pg/ml to 150 ng/ml. Data presented in
  • Figure 8 indicate that, under the assay conditions tested, there was no background signal with this antibody combination, and concentrations as low as about 996 pg/ml M. tuberculosis ribosomal protein S9 could be detected, with half-maximum detection of about 28 ng/ml M. tuberculosis ribosomal protein S9.
  • concentrations as low as about 996 pg/ml M. tuberculosis ribosomal protein S9 could be detected, with half-maximum detection of about 28 ng/ml M. tuberculosis ribosomal protein S9.
  • Such sensitivity of detection coupled with low background in sandwich ELISA is considered by the inventors to be within useful limits.
  • biotinylated secondary antibody a biotinylated secondary antibody and streptavidin poly-40 horseradish peroxidase (HRP) conjugate provided some increase in sensitivity of detection, with a statistically significant limit of detection as low as about 150 pg/ml recombinant M. tuberculosis ribosomal protein S9. Under these conditions, the sandwich ELISA was also capable of detecting about 6 ng/ml M. tuberculosis ribosomal protein S9 at half-maximal signal.
  • HRP horseradish peroxidase
  • the inventors further modified the basic assay by employing iterative antigen binding following coating of the ELISA plate with capture antibody. Essentially, this results in an increased amount of antigen being bound to the capture antibody notwithstanding the 50 ⁇ l volume limitations of a 96-well ELISA plate. Briefly, this iterative antigen loading involves repeating the antigen binding step in the sandwich ELISA several times, e.g., 2 or 3 or 4 or 5 times, etc. before washing and adding detection antibody. Naturally, each aliquot of antigen sample is removed following a standard incubation period before the next aliquot is added.
  • the number of iterations can be modified to optimize the assay (e.g., parameters such as signal: noise ratio, detection limit and amount of antigen detected at half-maximum signal), depending upon the nature of the sample being tested (e.g., sample type), without undue experimentation.
  • parameters such as signal: noise ratio, detection limit and amount of antigen detected at half-maximum signal
  • sample loading i.e., a 5x replacement amplification
  • a detection limit of about 84 pg/ml M. tuberculosis ribosomal protein S9.
  • the assay shown in Figure 10 was not performed under conditions reaching signal saturation, no estimation of the amount of antigen detected at half-maximum signal was possible. Notwithstanding, an approximate 2-fold increase in sensitivity of detection of recombinant M. tuberculosis ribosomal protein S9 was obtained by iterative antigen loading.
  • sputum did produce some signal suppression, especially when added essentially undiluted to assays.
  • this signal suppression can be overcome partially by diluting sputum samples, and compensating for the reduced antigen in the sample, by performing iterative sample loading as described herein above.
  • a 1 :2 (v/v) dilution or a 1:3 (v/v) dilution of sputum into blocking buffer and two or three iterations of sample loading permits sufficient recovery of signal strength to compensate for the signal suppression observed with sputum samples (data not shown).
  • the available data suggest that the sandwich ELISA for the detection of S9 protein offers excellent sensitivity with low background signal. Further enhancement in the sensitivity of detection may be obtained by directly biotinylating the detection antibody Mol025F to thereby permit amplification without the use of a secondary antibody.
  • M. tuberculosis strain H37Rv a laboratory strain
  • Escherichia coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis or Pseudomonas aeruginosa.
  • an ELISA plate was coated overnight with capture antibody Ch27 at 5 ⁇ g/ml concentration. Following washing to remove unbound antibody, 500 ng/ml or 50 ⁇ g/ml of a cellular extract from each microorganism were added the wells of the antibody- coated ELISA plates. As a negative control for each assay, buffer without cellular extract was used. Following incubation for 1 hour and washing to remove unbound antigen, detection antibody Mol025F was contacted with the bound antigen-body complexes at 2.5 ⁇ g/ml concentration.
  • an ELISA plate was coated overnight with capture antibody Ch27 at 5 ⁇ g/ml concentration. Following washing to remove unbound antibody, 500 ng/ml or 50 ⁇ g/ml of a cellular extract from each isolate were added the wells of the antibody-coated ELISA plates. As a negative control for each assay, buffer without cellular extract was used. Following incubation for 1 hour and washing to remove unbound antigen, detection antibody Mol025F was contacted with the bound antigen-body complexes at 2.5 ⁇ g/ml concentration.
  • the assay results suggest that endogenous S9 protein may be present at similar levels in both the clinical and laboratory isolates, or alternatively, that factors suppressing signal strength in one strain compensate for an over production of S9 protein by that strain relative to the other strain.
  • Chicken polyclonal antibodies against full-length recombinant BSX protein or a peptide from BSX were produced using standard methods.
  • the antibody against the full-length protein The antibody was purified by affinity chromatography using immobilised recombinant protein (without NUS).
  • the sensitivity of the multi-analyte assay was increased to 83% (15/18) and the specificity to - 85% (2/14).
  • S9 was detected in 20 patients in the TB group and in 5 patients in the non-TB group.
  • BSX was detected in 15 patients, also at different levels, in the TB group and 5 patients in the non-TB group.
  • non-TB controls are those patients presenting with clinical symptoms of TB but have been diagnosed with other respiratory disease such as pneumonia or bronchitis based on negative results for smear and culture testing for TB. Given the sensitivity level of these current diagnostic tests, there is ⁇ 30% chance that some of these controls may indeed have undiagnosed TB. As a consequence, the specificity for the multi-analyte (or single analyte) assay may be higher than that actually observed in the current analysis.
  • the Immunoglobulin fraction of four different sputum samples was isolated using Protein-G SepharoseTM, and the flow through fraction were loaded onto a 4-12% ID gradient SDS polyacrylamide gel and separated by electrophoresis. Proteins were then electrotransferred onto PVDF membrane. All the membranes were blocked in solution containing 1% casein in IX PBS, 0.1% Tween-20 (PBST) at room temperature (RT) for 2 hours. Membranes were then incubated with 10 ⁇ g/ml purified chicken anti-BSX polyclonal antibody described in Example 12 at RT for 2 hr, following by 3x lOmin washes with PBST.
  • PBST room temperature
  • Membranes were then incubated with 1:25,000 dilution of sheep anti-chicken IgG-HRP conjugated antibody solution at RT for 1 hr, followed by 5x 10 min washes with times PBST. Membranes were finally treated with 'Femto' chemiluminescence reagents (Pierce) for 5 min before exposure to x-ray films, reagents for 5 min before exposed on x-ray films.
  • BSX is detected in the flow through fraction (i.e., not bound by immunoglobulin) but not in the immunoglobulin fraction.
  • Antigen-based diagnosis of tuberculosis or infection by M. tuberculosis An ELISA to detect M, tuberculosis BSX
  • An ELISA assay was performed using one of three different anti-BSX antibodies, namely rabbit polyclonal anti-BSX antibody (raised against a BSX peptide) designated R 16, a chicken anti-BSX polyclonal antibody designated C44 (raised against recombinant protein) and a mouse anti-BSX monoclonal antibody designated 403B (raised against the C-terminus of BSX).
  • rabbit polyclonal anti-BSX antibody (raised against a BSX peptide) designated R 16
  • C44 chicken anti-BSX polyclonal antibody
  • 403B mouse anti-BSX monoclonal antibody
  • the ELISA plate was coated with various anti-BSX proteins including chicken (Ch) anti-BSX pAb C44, rabbit (Ra) anti-BSX pAb R 16, and mouse (Mo) anti-BSX mAb 403B all at 20 ⁇ g/ml using 50 ⁇ l per well. Titrating amounts of recombinant BSX were added at a concentration of 50 ng/ml down to 3 pg/ml.
  • Antigen detection was performed using either rabbit anti-BSX at 10 ⁇ g/ml (with and without pre-incubation with the recombinant BSX protein) followed by detection using sheep anti-rabbit Ig HRP conjugate at a 1/5000 dilution (for chicken capture system), or chicken anti-BSX pAb C44 at 20 ⁇ g/ml followed by sheep anti-chicken IgG HRP conjugate at 1/5000 dilution (for mouse and rabbit capture systems). Data are presented in Figure 15.
  • Antigen-based diagnosis of tuberculosis or infection by M. tuberculosis Detection of M. tuberculosis by sandwich ELISA
  • recombinant BSX Titrating amounts of recombinant BSX from 50 ng/ml down to 0.39 ng/ml were then screened using a purified chicken anti-BSX pAb, C44, at concentrations of either 10 or 20 ⁇ g/ml as specified above as the detector antibody followed by incubations with a sheep anti-chicken IgG HRP at a dilution of 1/5000 and TMB for signal detection. Data are presented in Figure 16. Under these conditions, the limit of detection of recombinant BSX was ⁇ 2-3 ng/ml.
  • ELISA plates were coated with either purified anti-BSX mAb 403B at a concentration of 40 ⁇ g/ml or purified chicken anti-BSX pAb C44 at a concentration of 5 ⁇ g/ml using 50 ul per well. Titrating amounts of purified recombinant BSX were added at a concentration of 50 ng/ml down to 0.39 ng/ml.
  • Two amplification systems were performed using either chicken anti-BSX at a concentration of 10 ⁇ g/ml followed by donkey anti-chicken IgG biotin conjugate at various dilutions and finally streptavidin- HRP at a 1/5000 dilution, or anti-BSX mAb 403B at various concentrations followed by goat anti-mouse IgG at 1/30000 dilution and donkey anti-goat IgG HRP conjugate at a 1/5000 dilution.
  • the amplified systems were used to compare to a basic antigen detecting system where chicken anti-BSX was used at a concentration of 10 ⁇ g/ml followed by sheep anti-chicken IgG HRP conjugate at a 1/5000 dilution. As shown in Figure 18, the amplified ELISA was approximately 10 fold more sensitive than the standard ELISA. Signal intensity is slightly higher when using the rabbit pAb as a capture and the chicken pAb as the first detector Ab in the amplified system ( Figure 19).
  • Antibody-based diagnosis of tuberculosis or infection by M. tuberculosis ELISA using M. tuberculosis Bsx protein fragments to diagnose the presence of antibodies against M. tuberculosis
  • TAITERQKQILLDIY SEQ ID NO; 15
  • SQIAKALRVSAEVLYVRAC SEQ ID NO: 16
  • MSSEEKLCDPTPTDD SEQ ID NO: 17
  • VRAGILEPSETSQVRC SEQ ID NO: 18
  • the peptides were biotinylated to facilitate their detection.
  • each well was washed 5 times with PBS/0.1% Tween 20, allowed to dry on absorbent paper, and either stored at 4°C with dessicant, or used immediately. This was followed by incubation for 1 hour with agitation in 50 ⁇ l of patient serum or plasma, diluted 1 :50 in blocker. Following this incubation, all wells were washed 5 times, using PBS/0.1% Tween 20 in a laminar flow, and tapped dry. Then lOO ⁇ l sheep anti-human IgG horse radish peroxidase (HRP) conjugate was added to each well.
  • HR horse radish peroxidase
  • 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, lOO ⁇ l 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 lOO ⁇ l 0.5M sulfuric acid. Each peptide was assayed in duplicate and repeated if duplicates did not appear to be reproducible.
  • Peptide Control streptavidin/no peptide/patient serum or plasma/conjugate
  • Serum background no streptavidin/no peptide/patient serum or plasma/conjugate.
  • Immunogenic peptides represent outliers in the distribution of peptide absorbencies and are detected following log transformation normalisation by calculation of a normal score statistic, with a mean and standard deviation estimated by a robust M-Estimator. 4. Results
  • Mass screening of the TB-positive and TB-negative samples for the presence of antibodies to Bsx peptides demonstrate that about 47% of TB-positive samples contain anti-Bsx antibodies.
  • a small number of TB-negative patients may test positive for any Bsx peptide.
  • Differentiation of the total patient population to include HIV status will elucidate a TB/HIV correlation, where about 76% of the TB-positive samples that contain anti-Bsx antibodies are also HFV + .
  • about 80% of the S.A. TB-positive/HIV + samples should contain antibodies to Bsx.
  • ELISA analysis of TB positive and TB negative serum or plasma reveals a number of immunogenic Bsx peptides containing B cell epitopes of the full-length Bsx protein of M. tuberculosis.
  • the correlation between HIV status and TB status with respect to serological reactivity of a Bsx peptide has many therapeutic advantages, such as, for example, the ability to detect TB and HFV status and/or monitoring the TB status in HFV + individuals.
  • HFV HFV " negative.
  • the absence of detectable antibodies that bind to Bsx in plasma from patients in a Chinese cohort may be associated with pulmonary TB being confined to the lung, whereas in the South African patients HIV positive status is often associated with extrapulmonary disease, which is more systemic.
  • Bsx may not be as highly expressed in Chinese compared to South African TB patients.
  • EXAMPLE 18 Antibody-based diagnosis of tuberculosis or infection by M. tuberculosis:
  • Bsx (23-24) peptide SEQ ID NO: 16
  • Bsx (23-24) peptide comprises the sequence of a highly immunogenic Bsx peptide with additional N-terminal and C-terminal sequences flanking this sequence in the full-length protein and conjugated C-terminally to a cysteine residue.
  • N-C terminal SEQ ID NO: 17
  • a third peptide designated peptide 28 (SEQ ID NO: 18) comprises another Bsx peptide conjugated C-terminally to a cysteine residue.
  • the peptides set forth in SEQ ID NOs: 16-18 additionally comprised an N-terminal linker (Ser-Gly-Ser-Gly) to the base peptide, to facilitate binding of the peptide to solid matrices.
  • the C-terminal and internal cysteine residues were included to facilitate cross-linking of the peptides for subsequent antibody production.
  • Sera/plasma Sera and plasma were a panel obtained from 41- 44 TB-positive patients (i.e., TB- positive sera) in each experiment, and 51 healthy control subjects (i.e., non-TB sera).
  • Peptides comprising SEQ ID NOs: 16-18 were coated on ELISA trays at 3 ⁇ g/ml on a streptavidin base of 5 ⁇ g/ml and then probed (after blocking) with Non-TB control sera and Known TB-positive sera and plasma. Sera and plasma were diluted 1/50 (v/v) prior to use. Capture of human IgG was traced with enzyme-linked sheep anti-human IgG and tetramethylbenzidine (TMB) substrate.
  • TMB tetramethylbenzidine
  • the sensitivity and specificity were analysed by taking the average substrate product OD values (from the conjugated peroxidase/TMB reaction) and calculating the cut-off values for significance at two standard deviations above the average and three standard deviations above the mean (i.e., at the 95% and 99.7% significance levels, respectively).
  • sensitivity in the context of a diagnostic/prognostic assay is understood to mean the proportion of TB-positive subjects that are diagnosed using a particular assay method (i.e., a "true” positive). Accordingly, an assay that has increased sensitivity is capable of detecting a greater proportion of TB-infected subjects than an assay with reduced or lower sensitivity.
  • the term "specificity" in the context of a diagnostic/prognostic assay is understood to mean the proportion of non-TB subjects (i.e., non-infected subjects) that do not return a positive result using a particular assay method (i.e., "true” negatives). Accordingly, an assay that has increased or enhanced specificity returns fewer false positive results or is capable of distinguishing between infected and non-infected subjects to a greater degree than an assay with a reduced specificity.
  • Bsx (23-24) peptide sequence showed a significant binding to confirmed TB-positive sera.
  • Data indicate that a peptide comprising the sequence set forth in SEQ ID NO: 16 selectively identifies antibodies that bind to M. tuberculosis in patient sera.
  • Data also show that the sensitivity and specificity with these revised criteria are relatively unchanged irrespective of whether or not the outliers is omitted, however there is a marginal increase in sensitivity at the 3 standard deviation level.
  • Bsx (23-24) peptide (SEQ ED NO: 16) has utility in antibody- based assays to detected tuberculosis in patient samples, especially sera.
  • the other two peptides tested in this example (SEQ ID NOs: 17 and/or 18) also have utility in eliminating false positive detection e.g., as part of a multi-analyte test.
  • Antibody-based diagnosis of tuberculosis or infection by M. tuberculosis Screening of TB and non-TB sera against recombinant full-length
  • Sera and plasma were from 44 TB-positive (smear or culture) Chinese and South African patients (i.e., TB-positive sera), and 44 healthy control subjects (i.e., non-TB sera).
  • Recombinant Bsx protein was coated directly onto ELISA trays at 5 ⁇ g/ml and then probed (after blocking) with Non-TB control sera, and known TB-positive sera and plasma diluted 1/100 (v/v) in buffer. Capture of human IgG was traced with enzyme- linked sheep anti-human IgG and tetramethylbenzidine (TMB) substrate.
  • TMB tetramethylbenzidine
  • the sensitivity and specificity were analysed by taking the average substrate product OD values (from the conjugated peroxidase/TMB reaction) and calculating the cut-off values for significance at two standard deviations above the average and three standard deviations above the mean (i.e., at the 95% and 99.7% significance levels, respectively).
  • the sensitivity of the assay in South African TB sera smears or culture positives is higher than the overall sensitivity (i.e., 35% compared to 25% at three standard deviations cut-off value).
  • the sensitivity of the assay is lower than the overall sensitivity (i.e., 1 1% compared to 25% at three standard deviations cut-off value).
  • the specificity of the assay is 100%, indicating robustness in this parameter.
  • Antibody-based diagnosis of tuberculosis or infection by M. tuberculosis Screening of TB and non-TB sera according to HIV status
  • Recombinant Bsx protein or Bsx (23-24) peptide (SEQ ED NO: 16) was coated directly onto ELISA trays at 5 ⁇ g/ml and then probed (after blocking) with Non-TB control sera and known TB-positive sera diluted 1/100 (v/v) in buffer.
  • Bsx(23- 24) peptide was used as described in the preceding examples. Capture of human IgG was traced with enzyme-linked sheep anti-human IgG and tetramethylbenzidine (TMB) substrate.
  • the sensitivity and specificity were analysed by taking the average substrate product OD values (from the conjugated peroxidase/TMB reaction) and calculating the cut-off values for significance at two standard deviations above the average and three standard deviations above the mean (i.e., at the 95% and 99.7% significance levels, respectively).
  • Recombinant Bsx protein assayed under these conditions was highly specific in detecting TB-positive sera. Sensitivity of the assay over the populations tested was also quite high for HIV + patients. Similar results were obtained using the Bsx(23-24) peptide.
  • the full-length recombinant Bsx protein and Bsx(23-24) peptide separately detect about 40-45% of TB + HIV + subjects, and, in a multianalyte test format, detect about 65% to 70% of TB + HIV + subjects, with only about 5% false- positive detection.
  • the sensitivity of the assay in South African TB sera and/or plasma smears or culture positives is higher than the overall sensitivity (i.e., 35% compared to
  • the specificity of the assay is absolute i.e., 100% indicating robustness in this parameter.
  • Bsx(23-24) peptide SEQ ID NO: 16
  • recombinant full-length Bsx and Bsx(23-24) peptide are both biotinylated and immobilized onto a streptavidin base (5 ⁇ g/ml) that has been preadsorbed onto wells of a microtiter pi ate.
  • Standard ELISA reactions are carried out wherein (i) patient sera and control sera, each diluted 1/100 (v/v) in buffer, are added to separate wells, and (ii) capture of human IgG in the sera by the immobilized protein and peptide is traced using enzyme-linked sheep anti-HulgG detected using tetramethylbenzidine (TMB) substrate.
  • TMB tetramethylbenzidine
  • ELISA assays were performed to determine those GS peptide fragments against which an immune response was detected in sera from TB subjects and no immune response was detected in control subjects.
  • the amino acid sequence of the GS identified as being immunogenic in TB subjects was aligned with the amino acid sequence of other known TB glutamine synthetases (glnA, glnA2 and glnA3) and shown to have only 25% amino acid sequence identity with other known glutamine synthetase homologs.
  • GS peptides were selected that are specifically immunoreactive with sera from TB subjects and not comprise sequences not conserved with other glutamine synthetases.
  • RGTDGSAVFADSNGPHGMSSMFRSF SEQ ID NO: 19
  • WASGYRGLTPASDYNIDYAI SEQ ID NO: 20
  • Antibodies that selectively bind to these peptides are unlikely to cross-react with another glutamine synthetase proteins.
  • the two peptides were selected as antigens for antibody production, synthesized and attached to diphtheria toxoid.
  • peptide antigen consisting of the sequence RGTDGSAVFADSNGPHGMSSMFRSF (set forth in SEQ ID NO: 19) conjugated to diphtheria toxoid was provided to NeoClone, Madison, Wisconsin, USA for generation of monoclonal antibodies according to their standard protocol. About 1 mg of the peptide was provided as biotinylated peptide for quality control.
  • mice Five BALB/cByJ female mice were immunized with peptide conjugated to carrier according to Neoclone's standard immunization process.
  • 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.
  • 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.
  • mice The splenocytes of the ABL-MYC-infected mice were transplanted into approximately 20 naive mice. Ascites development
  • mAbs monoclonal antibodies
  • a cell line i.e., plasmacytoma
  • 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.
  • the monoclonal antibody designated 426C was coated on the bottom of an ELISA plate at 20 ⁇ l and (i) an immunogenic glutamine synthetase (GS) peptide biotinylated at the N-terminus or (ii) a negative control peptide biotinylated at the N-terminus, were added at various concentrations to 10 pg/ml as indicated in Table 3a.
  • the biotinylated GS peptide used had the sequence: SGSGRGTDGSAVFADSNGPHGMSSMFRSFC (SEQ ID NO: 21). The peptide was detected by binding of streptavidin HRP conjugate under standard conditions.
  • Absorbances were determined at 450nm and 620nm, and the difference in absorbance at 450nm and 620nm determined. Average data for duplicate samples were obtained. The data obtained show that the antibodies capture the immunogenic GS peptide antigen at concentrations of about 10 pg/ml or greater, at a signahnoise ratio of at least about 2.0. These data demonstrate efficacy of the antibodies as a capture reagent in immunoassays.
  • the peptide i.e., SEQ ID NO: 21
  • the peptide was coated onto the bottom of the ELISA plate at a concentration of about 3 ⁇ l.
  • Duplicate aliquots of the monoclonal antibody-producing plasmacytoma designated 426C, and duplicate aliquots of a negative control monoclonal antibody were added at various final concentrations to 10 pg/ml.
  • Binding of the antibody was then detected using sheep anti-mouse HRP antibody conjugate under standard conditions. Absorbances were determined at 450nm and 620nm, and the difference in absorbance at 450nm and 620nm determined. Average data were obtained. The data show that the antibody successfully detects GS above assay background at concentrations of antibody as low as 10 pg/ml, therefore demonstrating efficacy as a detection reagent in immunoassays.
  • Solid phase ELISA using mAb 426 to detect circulating immune complexes comprising
  • GS tuberculosis glutamine synthetase
  • 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.
  • circulating immune complexes CIC
  • GS tuberculosis glutamine synthetase
  • 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.
  • 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.
  • the secondary antibody may be conjugated to a detectable label e.g., horseradish peroxidase (HRP).
  • HRP horseradish peroxidase
  • 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.
  • 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.
  • 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.
  • Monoclonal antibody 426C that binds to M, tuberculosis glutamine synthetase at a concentration of 20 ⁇ g/ml in water was coated onto the bottom of one or more NUNC plates. Plates were left to dry at 37 0 C overnight. The. plates were blocked for 1 to 3 hours at room temperature in blocking buffer [1% (w/v) casein/0.1% (v/v) Tween-20 in 0.5M phosphate buffered saline (PBS)]. The wells were flicked or tapped to remove blocking solution, and patient sera diluted 1 :50 (v/v) in blocking buffer (50ul/well) added. The plates were then incubated for 1 hour at room temperature e.g., on a rotating shaker.
  • the plates were washed about 3-5 times with 0.1% (v/v) Tween-20 in 0.5M phosphate buffered saline (PBS) such as, for example, using an automated plate washer. Sheep anti-human IgG antibody or anti-human IgA antibody, diluted 1 :5000 (v/v) in blocking buffer was added to wells. The plates were then incubated for 1 hour at room temperature e.g., on a rotating shaker. The plates were washed as before, and TMB was added to the wells (50 ul/well). Plates were incubated for about 30 minutes, and the reactions were then stopped by addition of 0.5M H 2 SO 4 (50 ul/well). Absorbances of each well was read at wavelengths of 450nm and 620nm, and the differences in these wavelengths is determined (i.e.A 45O -A ⁇ 5 2 o).
  • PBS phosphate buffered saline
  • 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.
  • Sera/plasma from 45 South African subjects with confirmed TB were screened and compared with 19 (black) control sera/plasma and 14 (white) control sera/plasma. Three other South African sera/plasma were also included that had been diagnosed with diseases other than TB. A substantial number of the 45 TB sera tested detected levels of immune complexes comprising GS at greater than 3 standard deviations above control average. Furthermore, of the 36 non-TB sera/plasma, one was greater than 3 standard deviations above control average indicating that that the assay a high level of specificity.
  • Sera/plasma from 49 Chinese subjects with clinically-confirmed TB were also screened using the ELISA assay. Again this assay detected increased levels (greater than 2 or 3 times standard deviation of the control average) of CIC comprising GS in TB subjects. Furthermore, or the 41 of non-TB subjects only 5 returned readings greater than 2 or 3 standard deviations above control average indicating that that the assay a high level of specificity.
  • Point-of-care test for diagnosing an active infection by M. tuberculosis using mAb 426
  • Monoclonal antibody 426C is striped onto a nitrocellulose membrane at a concentration of between about 0.5 and about 4 mg/ ml.
  • the nitrocellulose membrane is allowed to dry at 4O 0 C for 20 minutes.
  • the nitrocellulose sheet is then cut into a 1 cm x 1 cm squares and inserted into the base of the DiagnostIQ device (Proteome Systems Ltd) on top of a cellulose pad.
  • the Pre-incubation frame is attached to the base and the test performed according to the procedure below.
  • the sera/plasma are incubated with the nitrocellulose strip membrane for 30 seconds and the pre-incubation frame is pushed down onto the base of the test.
  • wash solution (0.5% Tween 20 in 0.1 M phosphate buffer) is added to the pre-incubation well and allowed to flow through the device.
  • the pre-incubation frame is removed and the signal read by visually interpreted or read in a Readrite optical reader.
  • the present invention clearly encompasses conjugation of the anti- IgG and/or anti-IgA antibody to the same gold particle to ensure the same amount of label is applied in each test.
  • the gold particles may also be dried onto the preincubation pads, to thereby avoid the later addition of conjugate. Sensitivity of the assay may also be improved by increasing the amount of sera tested in each sample.
  • peptide antigen consisting of the sequence WASGYRGLTPASDYNIDYAIC (set forth in SEQ ID NO: 22) conjugated to diphtheria toxoid is provided to NeoClone, Madison, Wisconsin, USA for generation of monoclonal antibodies according to their standard protocol. About 1 mg of the peptide is also provided as biotinylated peptide for quality control.
  • mice Five BALB/cByJ female mice are immunized with peptide conjugated to carrier according to Neoclone's standard immunization process.
  • Test bleeds of the immunized mice are performed at regular intervals for use in the quality control sera ELISAs using biotinylated peptide. Potyclonal sera having the highest titer are determined using ELISA. Mice having polyclonal antibody titers of at least 1,000 are used for the ABL-MYC infection process.
  • 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.
  • the splenocytes of the ABL-MYC-infected mice are transplanted into approximately
  • mice 20 naive mice.
  • mAbs monoclonal antibodies
  • a mAb that binds to the peptide antigen are is purified from ascites using protein G or protein A columns.
  • Antibody titration is performed essentially as described in the preceding examples.

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Abstract

La présente invention concerne une protéine S9 ribosomale de M. tuberculosis isolée (SEQ ID NO: 1) ainsi que des fragments peptidiques immunogènes de cette protéine (l'une quelconque des SEQ ID NO: 2 à 7), et des anticorps produits dirigés contre la protéine entière et des fragments peptidiques immunogènes pour le diagnostic d'une infection à M. tuberculosis chez un sujet humain, par exemple en utilisant un format ELISA sandwich à base d'antigènes. La présente invention concerne également des tests multi-analytes dans lesquels les tests de diagnostic à base de S9 de la présente invention sont multiplexés avec la détection d'épitopes immunogènes d'autres protéines issues de M. tuberculosis, par exemple des épitopes de la protéine BSX (l'une quelconque des SEQ ID NO: 8 à 18) et/ou de la protéine glutamine synthétase (l'une quelconque des SEQ ID NO: 19 à 22) et/ou la détection d'anticorps dirigés contre une ou plusieurs autres protéines de M. tuberculosis chez le sujet humain, y compris des anticorps dirigés contre la BSX et/ou la glutamine synthétase.
PCT/AU2007/000093 2006-01-31 2007-01-31 Procédé de diagnostic et de traitement d'une infection à m. tuberculosis et réactifs correspondants WO2007087679A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003033530A2 (fr) * 2001-10-12 2003-04-24 Health Protection Agency Antigenes mycobacteriens exprimes a haute tension en oxygene
US6573361B1 (en) * 1999-12-06 2003-06-03 Monsanto Technology Llc Antifungal proteins and methods for their use
US20040029129A1 (en) * 2001-10-25 2004-02-12 Liangsu Wang Identification of essential genes in microorganisms

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6573361B1 (en) * 1999-12-06 2003-06-03 Monsanto Technology Llc Antifungal proteins and methods for their use
WO2003033530A2 (fr) * 2001-10-12 2003-04-24 Health Protection Agency Antigenes mycobacteriens exprimes a haute tension en oxygene
US20040029129A1 (en) * 2001-10-25 2004-02-12 Liangsu Wang Identification of essential genes in microorganisms

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
COLE S.T. ET AL.: "Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence", NATURE, vol. 393, no. 6685, 1998, pages 537 - 544, XP002434927 *

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