WO2023281263A1

WO2023281263A1 - Diagnosis of latent tuberculosis

Info

Publication number: WO2023281263A1
Application number: PCT/GB2022/051751
Authority: WO
Inventors: Danai PAPAKONSTANTINOU; Matt O'Shea; Alan Mcnally; Adam Cunningham
Original assignee: The University Of Birmingham
Priority date: 2021-07-08
Filing date: 2022-07-07
Publication date: 2023-01-12
Also published as: GB202109846D0

Abstract

The invention relates to methods for determining whether a patient suspected of being infected with Mycobacterium tuberculosis has an active or latent tuberculosis infection; to the use of certain Mycobacterium tuberculosis proteins as biomarkers for diagnosing latent tuberculosis infections; to a kit of parts comprising the Mycobacterium tuberculosis proteins; and to methods of treating latent tuberculosis infections.

Description

DIAGNOSIS OF LATENT TUBERCULOSIS FIELD OF THE INVENTION

[0001] The present invention relates to methods for determining whether a patient suspected of being infected with Mycobacterium tuberculosis has active tuberculosis infection or latent tuberculosis infection; to the use of certain Mycobacterium tuberculosis proteins as biomarkers for diagnosing latent tuberculosis infection; to a kit of parts comprising the Mycobacterium tuberculosis proteins; and to methods of treating latent tuberculosis infection.

BACKGROUND [0002] Tuberculosis (TB) is responsible for approximately 1.4 million deaths worldwide annually. Globally TB is one of the top 10 causes of death and it is the leading cause from a single infectious agent. In 2018 the United Nations confirmed Sustainable Development Goals to end the TB epidemic by 2030 and the WHO End TB strategy defines targets including a 90% reduction in TB deaths and an 80% reduction in the TB incidence rate by 2030; one arm of this strategy involves increased preventative treatment for people with latent TB infection (LTBI) [https://wwwwho.int/news-room/fact-sheets/detail/tuberculosis].

[0003] TB is caused by Mycobacterium tuberculosis (M tuberculosis or M.tb ) and approximately 5-10% of people go on to develop active tuberculosis infection (ATBI) within the first 2-5 years following infection [Anderson et a!., Globa! Tuberculosis Report (2018) Geneva: WHO]. The remainder of the infected individuals will either eliminate the infection, or will develop a persistent immune response to M.tb antigens without displaying symptoms of the active disease - these latter individuals may be classed as having LTBI, which at some future date could develop into ATBI Since it is estimated that up to 25% of the world’s population (-1.7 billion people) has LTBI, this clearly represents a huge reservoir of potential disease and transmission [Global Tuberculosis Report (2020) Geneva: WHO].

[0004] Tuberculin skin testing (TST) and Interferon gamma release assays (using ESAT6/CFP10 or ESAT6/CFP10 and TB7.7 antigens) are commonly used as screening tests for TB, but cannot differentiate between active and latent infection.

[0005] The only current laboratory method that is capable of differentiating between active and latent TB infection is the detection of TB bacilli in sputum or other bodily fluid by microscopic examination (however this method has low sensitivity), culture, or more advanced molecular techniques (such as PCR and whole-genome sequencing). Those methods are only used if the patients are symptomatic (e.g. coughing or have a fever) and are either time-consuming or expensive. When an individual is asymptomatic, an Interferon gamma release assay (IGRA) test is initially offered in order to detect if the individual had previous immunological exposure to TB. If the IGRA is positive, it is normally followed by a detailed clinical assessment and further diagnostic tests (such as X-ray, CT scan, blood tests, or in some cases bronchoscopy) in order for a diagnosis of latent TB to be made. These additional tests provide indications that the individual may have latent TB, but cannot completely differentiate between active and latent TB either Once a diagnosis of LTBI is strongly indicated, then the patient is typically offered a 3-month LTBI treatment according to the relevant national guidelines [e.g NICE guidelines 2016] This treatment reduces their chance of TB reactivation by more than 80%.

[0006] Therefore, there is an urgent need for a simple diagnostic test for latent TB which is easy to use, less time consuming, less invasive and/or cheaper than the current testing methods. The present invention was devised with the foregoing in mind.

BRIEF SUMMARY OF THE DISCLOSURE

[0007] Surprisingly, the inventors have identified certain biomarkers which are capable of distinguishing between active and latent TB. These biomarkers are antigen proteins expressed by Mycobacterium tuberculosis, namely SugC and Mpt83. It has been unexpectedly discovered that in subjects having latent TB, these antigens trigger a stronger immune response than in subjects having active TB, thereby facilitating easier diagnosis of LTBI.

[0008] In a first aspect of the invention there is provided a method for determining whether a patient suspected of being infected with Mycobacterium tuberculosis has active tuberculosis infection (ATBI) or latent tuberculosis infection (LTBI), the method comprising testing a biological sample from the patient for the presence of: a. immune cells responsive to a M tuberculosis antigen selected from SugC and/or Mpt83; or b. a product of immune cells against SugC or Mpt83.

[0009] In a convenient embodiment the immune cells responsive to a M tuberculosis antigen selected from SugC and/or Mpt83 are T cells Conveniently the T cells are stimulated by the SugC and/or Mpt83 antigens to express one or more cytokines which may be detected by an appropriate method. [0010] In another embodiment the product of the immune cells against SugC or Mpt83 comprise one or more antibodies against SugC or Mpt83. Conveniently, the antibodies are detected by an appropriate method.

[0011] In a second aspect of the invention there is provided a method for assessing a patient for tuberculosis infection comprising: testing a biological sample from the patient in an interferon gamma release assay (IGRA) against each of the following M tuberculosis antigens:

(i) at least one of SugC and Mpt83; and

(ii) at least one of CFP-10, ESAT6 and TB7.7 (Rv2654c); and characterising the patient as non-immunologically exposed to M.tb, having active tuberculosis, or having latent tuberculosis based on the assay results. Such method may also be able to identify individuals that have received Bacillus Calmette-Guerin (BCG) vaccine.

[0012] In a third aspect of the invention there is provided a method for determining whether a patient with a Mycobacterium tuberculosis infection has active tuberculosis infection (ATBI) or latent tuberculosis infection (LTBI), comprising: contacting a biological sample from the patient with a M tuberculosis SugC and/or Mpt83 antigen; and detecting for the presence of T cells that have been stimulated by the antigen to produce a cytokine, such as interferon gamma, wherein the number or amount of antigen stimulated cells in the biological sample that produce this cytokine indicates whether the patient has ATBI or LTBI.

[0013] In a fourth aspect of the invention there is provided the use of Mycobacterium tuberculosis SugC and/or Mpt83 protein as a biomarker for distinguishing between latent tuberculosis and active tuberculosis.

[0014] In a fifth aspect of the invention there is provided the use of recombinant or synthetic Mycobacterium tuberculosis SugC and/or Mpt83 antigen as a tool for distinguishing between latent tuberculosis and active tuberculosis, wherein the antigen is full-length protein or one or more peptides thereof.

[0015] In a sixth aspect of the invention there is provided a kit of parts comprising one or more M tuberculosis antigens selected from: (i) SugC protein, (ii) one or more peptides of SugC protein, (iii) Mpt83 protein, and (iii) one or more peptides of Mpt83 protein; and instructions for use (a) in detecting M tuberculosis SugC or pt83 activated T cells in a biological sample, and/or (b) in determining a patient’s tuberculosis infection status, in particular whether they have LTBI. [0016] In a seventh aspect of the invention there is provided a drug selected from isoniazid, rifampicin, or rifapentine, or a combination thereof, for use in the treatment of LTBI comprising:

(i) determining whether a patient has LTBI according to a method as described herein; and (ii) if the patient is identified in step (i) as having LTBI, administering the isoniazid, rifampicin, rifapentine, or a combination thereof, to the patient.

[0017] In an eighth aspect of the invention there is provided a method of treating a patient with latent tuberculosis (LTB), comprising: (i) determining whether the patient has LTB according to a method as described herein; and

(ii) if the patient is identified in step (i) as having LTB, administering to the patient drugs selected from: isoniazid, or rifampicin, or isoniazid and rifampicin, or isoniazid and rifapentine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order that the invention may be more clearly understood, one or more embodiments thereof will now be described, by way of example only, in relation to an experimental study and with reference to the accompanying drawings, of which:

Figure 1 : Demographic Characteristics of Example 2 Study Participants: (A) Ethnic background (^* any other ethnic background); (B) gender.

Figure 2: ELI-Spot Assay Results - T cell IFN-y secretion in response to PPD presented as spot forming units per 300000 PBMCs for the 4 blood donor groups: healthy control, BCG unvaccinated (HC-BCG(-)); healthy control, BCG vaccinated (HC-BCG(+)); latent TBI (latent) and active TB (active). The results are represented on a log 10 scale for better visualisation.

Figure 3: ELI-Spot Assay Results -T cell I FN-y secretion in response to SugC peptide pools 1-4 presented as spot forming units per 300000 PBMCs for the 4 blood donor groups: healthy control, BCG unvaccinated (HC-BCG(-)); healthy control, BCG vaccinated (HC- BCG(+)); latent TBI (latent) and active TB (active). Figure 4: ELI-Spot Assay Results - T cell IFN-y secretion in response to Mpt83 peptide pools 1-3 presented as spot forming units per 300000 PBMCs for the 4 blood donor groups: healthy control, BCG unvaccinated (HC-BCG(-)); healthy control, BCG vaccinated (HC- BCG(+)); latent TBI (latent) and active TB (active).

DETAILED DESCRIPTION [0019] The practice of particular embodiments of the invention will employ, unless indicated specifically to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, preferred embodiments of compositions, methods and materials are described herein.

Definitions

[0021] The articles "a,” “an,” and “the” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.

[0022] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and/or” should be understood to mean either one, or both of the alternatives.

[0023] As used herein, the term “about" or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. [0024] The term “active TB” (ATB) or “active TB infection” (ATBI) refers to an

Mycobacterium tuberculosis infection wherein the subject has either signs (e.g. crepitations, bronchial breathing, lymphadenopathy), or symptoms (e.g cough, fever, fatigue, chest pain) of active disease, or positive confirmatory test results (e.g. abnormal chest x-ray, microbiological confirmation of the infection), or all of the above. ATB and ATBI are used interchangeably herein.

[0025] The term “latent TB” (LTB) or “latent TB infection” (LTBI) refers to an Mycobacterium tuberculosis infection wherein the subject does not have any symptoms, or signs of TB disease, or positive confirmatory test results (e.g. chest x-ray, abnormal blood results), but has an immunological exposure to the Mycobacterium tuberculosis (e.g. they test positive to a tuberculin skin test or TB blood test). LTB and LTBI are used interchangeably herein.

[0026] The term “biological sample” is used herein to refer to a sample which may be obtained, taken or extracted from the patient suspected of being infected with Mycobacterium tuberculosis. Examples include whole blood, peripheral blood mononuclear cells (PBMCs), plasma, serum, saliva, sputum, urine, bronchoalveolar lavage (BAL), any cavity fluid, and samples from bone marrow, spleen, lymph node, pleural or any other tissue amenable to biopsy.

[0027] The term “immune cells” refers to leukocytes and lymphocytes. In particular, it refers to T lymphocytes (T cells) and B lymphocytes (B cells).

[0028] SugC is a trehalose import ATP-binding protein having the following FASTA amino acid sequence:

>sp|P9WQI3|SUGC_MYCTU OS=Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) OX=83332 GN=sugC PE=1 SV=1 (SEQ ID NO: 123)

MAEIVLDHVNKSYPDGHTAVRDLNLTIADGEFLILVGPSGCGKTTTLNMIAGLEDISSGE LRIAGERVNEKAPKDRDIAMVFQSYALYPHMTVRQNIAFPLTLAKMRKADIAQKVSETAK ILDLTNLLDRKPSQLSGGQRQRVAMGRAIVRHPKAFLMDEPLSNLDAKLRVQMRGEIAQL QRRLGTTTVYVTHDQTEAMTLGDRWVMYGGIAQQIGTPEELYERPANLFVAGFIGSPAM NFFPARLTAIGLTLPFGEVTLAPEVQGVIAAHPKPENVIVGVRPEHIQDAALIDAYQRIR ALTFQVKVNLVESLGADKYLYFTTESPAVHSVQLDELAEVEGESALHENQFVARVPAESK VAIGQSVELAFDTARLAVFDADSGANLTIPHRA.

[0029] Mpt83 is a cell surface glycolipoprotein having the following FASTA amino acid sequence: >sp| P9WN F3| M P83_M YCTU OS=Mycobacterium tuberculosis (strain ATCC 25618 /

H37Rv) OX=83332 GN=mpt83 PE=1 SV=1 (SEQ ID NO: 124)

MINVQAKPAAAASLAAIAIAFLAGCSSTKPVSQDTSPKPATSPAAPVTTAAMADPAADLI GRGCAQYAAQNPTGPGSVAGMAQDPVATAASNNPMLSTLTSALSGKLNPDVNLVDTLN GGEYTVFAPTNAAFDKLPAATIDQLKTDAKLLSSILTYHVIAGQASPSRIDGTHQTLQGADL TVIGARDDLMVNNAGLVCGGVHTANATVYMIDTVLMPPAQ

[0030] CFP-10 protein has the following amino acid sequence (UniProt P6WNK5): > ycobacterium tuberculosis H37Rv|Rv3874|esxB (SEQ ID NO: 125) MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAWRF QEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF.

[0031] ESAT6 protein has the following amino acid sequence (UniProt P9WNK7): > ycobacterium tuberculosis H37Rv|Rv3875|esxA (SEQ ID NO: 126)

MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKUWWVGGSGSEAYQGVQQKW

DATATELNNALQNLARTISEAGQAMASTEGNVTGMFA.

[0032] TB7.7 protein (Rv2654c) has the following FASTA amino acid sequence: >sp|P9WJ11 |Y2654_MYCTU Antitoxin Rv2654c OS=Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) OX=83332 GN=Rv2654c PE=1 SV=1 (SEQ ID NO: 127)

MSGHALAARTLLAAADELVGGPPVEASAAALAGDAAGAWRTAAVELARALVRAVAESHG

VAAVLFAATAAAAAAVDRGDPP. [0033] Full length protein refers to the complete protein as translated. The full-length protein sequence for SugC is disclosed in SEQ ID NO: 123. It is 393 amino acids in length. The full-length protein sequence for Mpt83 is disclosed in SEQ ID NO: 124. It is 220 amino acids in length and the first 24 amino acids represent the signal sequence.

[0034] A fragment of the protein could be an N- or C-terminally truncated fragment, e.g. one lacking signal peptide, which could comprise at least 80% of the length of the full-length protein or it could be a much smaller peptide fragment, such as one 10-30 amino acids in length.

[0035] A peptide is a short chain of two or more amino acids that are connected to one another by peptide bonds. Nominally a protein is made up of multiple peptide units joined together. However, the cut-off number for defining a peptide and protein is often arbitrary. For the purposes of this application a peptide is a chain of two or more connected amino acids that is shorter than the full-length protein. Accordingly, it may also be referred to as a fragment of the protein.

[0036] Peptides useful in the present invention will typically be at least 10 amino acids long, such as at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60 amino acids long and typically will be of a contiguous sequence when compared to the full-length protein. Suitably, if the intention is to elicit an immune response or to use the peptide in an IGRA assay, the antigen will be full-length protein, a single peptide or a pool of peptides (e.g. between about 10 and 30 amino acids long) and which derive from different (sometimes overlapping) regions of the full-length protein. The Examples herein have used peptides 15 amino acids in length, but peptides of differing lengths can be used. A pool of peptides comprises at least 2 distinct peptides. By distinct we mean they are not identical. Any 2 distinct peptides could overlap when compared to the full-length protein, or they could be from completely different (non-overlapping) regions of the protein. A pool of peptides could comprise any number of distinct peptides. Suitably, the pool of peptides comprises at least 2, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30 or more distinct peptides. [0037] As used herein, the term “in vitro” means performed or taking place in a test tube, culture dish, or elsewhere outside a living organism. As used herein the term also includes ex vivo because the analysis takes place outside an organism.

[0038] As used herein, the term “isolated” means material that is substantially or essentially free from components that normally accompany it in its native state. In particular embodiments, the term “obtained” or “derived” is used synonymously with isolated.

[0039] A “subject,” “individual,” or “patient” as used herein, includes any animal that exhibits a symptom of a condition that can be detected or identified with compositions contemplated herein. Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals (such as horses, cows, sheep, pigs), and domestic animals or pets (such as a cat or dog). In particular embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human primate and, in a particular embodiment, the subject is a human.

[0040] Any embodiment described herein can be applied to any aspect of the invention unless indicated otherwise or it is apparent to the person of skill in the art that such embodiment cannot apply.

Methods of Detecting LTBI

[0041] The inventors hypothesised that highly conserved M.tb proteins with essential roles in M.tb biology might generate immune responses in infected subjects and therefore could constitute novel TB diagnostic biomarkers. Having recently performed an in-depth bioinformatics analysis of 8535 M.tb genomes [Papakonstantinou et al., mSphere (2021 Apr 28); 6(2)], the inventors decided to investigate possible biomarker candidates. This investigation led to the surprising discovery that two antigens are suitable as biomarkers capable of producing a differential immune response between subjects having LTBI and those having ATBI.

[0042] The present invention therefore relies on looking for evidence that the body has encountered a M. tuberculosis infection and effected an immune reaction against SugC/Mpt83 antigens, such as detection of T cells that have previously been exposed to these antigens or antibodies that have been raised against these antigens by B-cells. [0043] Accordingly, in a first aspect of the invention there is provided a method for determining whether a patient suspected of being infected with Mycobacterium tuberculosis has active tuberculosis infection (ATBI) or latent tuberculosis infection (LTBI), the method comprising testing a biological sample from the patient for the presence of: a. immune cells responsive to a M tuberculosis antigen selected from SugC and/or Mpt83; or b. a product of immune cells against SugC or Mpt83.

[0044] Over the last decades evidence suggests that latent TB is a dynamic process. It is proposed that most of the M.tb bacilli exist inside the macrophages in a dormant, nonreplicating state. However, evidence suggests that during latent TB there are also bacilli which are actively replicating and they are in a constant ‘battle’ with the host’s immune response. When this balance gets disrupted, uncontrolled bacterial replication will promote reactivation and active TB disease. The findings of the present invention indicate that SugC and Mpt83 most likely are expressed during latent TB infection.

[0045] In an embodiment, the biological sample is selected from the group consisting of: whole blood, peripheral blood mononuclear cells (PBMCs), plasma, serum, saliva, sputum, urine, any cavity fluid (such as pleural effusion), bone marrow, spleen, lymph node, pleural or any other tissue amenable to biopsy in the human body that has been affected by TB. In a convenient embodiment, the biological sample is selected from the group consisting of whole blood, peripheral blood mononuclear cells (PBMCs), plasma and serum. In a more convenient embodiment, the biological sample is selected from the group consisting of peripheral blood mononuclear cells (PBMCs), and serum. In a most convenient embodiment, the biological sample is peripheral blood mononuclear cells (PBMCs).

[0046] In an embodiment the immune cells responsive to a M. tuberculosis antigen selected from SugC and/or Mpt83 are T cells. Conveniently, the T cells are stimulated by the SugC and/or Mpt83 antigen to express one or more cytokines which may be detected by an appropriate method. The T cells may have been previously exposed to the SugC and/or Mpt83 antigen.

[0047] Suitable cytokines expressed by T cells in response to antigenic stimulation comprise interferon gamma (IFN-g), tumour necrosis factor alpha (TNF-a), interleukin 2 (IL2), interleukin 4 (IL4), interleukin 17 (IL17), and any other human cytokine or chemokine of relevance to TB immunology. In a convenient embodiment, the expressed cytokine is interferon gamma (IFN-g), tumour necrosis factor alpha (TNF-a), interleukin 2 (IL2), interleukin 4 (IL4) or interleukin 17 (IL17). In a most convenient embodiment, the expressed cytokine is IFN-g. [0048] Therefore, in an embodiment, the presence of cells (e.g. T cells) responsive to

SugC and/or Mpt83 in the biological sample is tested by a suitable detection method. Suitable detection methods will be apparent to those skilled in the art. Methods for analysing for a biomarker in a biological sample are well known in the art. Such methods may include western blots, enzyme linked immunosorbent assay (ELISA), immunoprecipitation, immunofluorescence, flow cytometry, immunochemistry, or bead-based immunochemistry. Particularly suitable methods may include ELISA, multiplex strategies, ELISPOT, immunochromatography techniques, Western blotting, immunofluorescence, FACS, or radioimmunoassays.

[0049] In an embodiment, the presence of T cells responsive to SugC and/or Mpt83 in the biological sample is detected by a cytokine release assay. In a convenient embodiment, the T cell-expressed cytokine is interferon gamma and the interferon gamma is detected using an interferon gamma release assay (IGRA).

[0050] IGRA testing is used as a standard testing technique for the diagnosis of TB [Pai et at., Clin Microbiol Rev. (2014); 27(1):3-20] Two commonly used IGRAs are available on the market: the QuantiFERON®-TB Gold In-Tube (QFT) assay and the T-SPOT®.TB assay. For example, the commercially available T-SPOT®.TB assay uses the M.tb antigens ESAT-6 and CFP-10 and is based on the ELISPOT technique, which quantifies the number of IFN- g-producing T cells (spot-forming units). Similarly, the QFT assay is another IGRA commonly used for TB screening. It is an ELISA-based, whole blood assay, that uses the same M.tb peptides as the T-SPOT (i.e. ESAT-6 and CFP-10), as well as peptides from the TB7.7 (Rv2654c) protein in an in-tube format. The result is reported as quantification of IFN- g in international units (IU) per millilitre. Such ESAT-6/CFP-10/TB7.7 antigen assays are able to distinguish between M.tb infection and BCG-vaccination, since T cells in blood samples from BCG-vaccinated individuals do not respond to the ESAT-6 or CFP-10 antigens. These assays are unable, however, to distinguish between LTBI and ATB.

[0051] Applying the present invention, a SugC/Mpt83 IGRA test may comprise the following steps:

(i) prepare plates coated with anti-IFN-g antibody;

(ii) incubate biological sample (e.g. PB Cs) and SugC and/or Mpt83 antigen in plates in (i);

(iii) wash and add a primary antibody (e.g. biotinylated anti IFN-g antibody);

(iv) wash and add a second antibody (e.g. alkaline phosphatase-labelled streptavidin);

(v) wash and add a developer (e.g. BCIP);

(vi) wash and analyse the number of spot-forming cells [0052] The person of skill in the art is able to devise and carry out a suitable assay to detect the expression of the cytokine produced by the cells following contact with a SugC and/or Mpt83 antigen.

[0053] Suitable assay methods are described in more detail below in the Examples section. ll

[0054] In an alternative embodiment, the biological sample from the patient is tested for the presence of a product of immune cells against SugC or Mpt83. This product may be secreted, released or expressed by immune cells. In an embodiment, the immune cells are B ceils. In an embodiment, the product of the immune cells comprises one or more antibodies against SugC or Mpt83.

[0055] In a convenient embodiment, the presence of the antibodies is detected by using an enzyme linked immunosorbent assay (ELISA), an immunochromatographic test, or any exploratory immunological test capable of detecting antibodies. In a convenient embodiment, the presence of the antibodies is detected by using an enzyme linked immunosorbent assay (ELISA). Suitable methods for carrying out an ELISA assay for the detection of SugC or Mpt83 antibodies can be readily determined by one skilled in the art. The enzyme linked immunosorbent assay (ELISA) was originally described by Engvall and Perlmann [“Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G” !mmunochemistry (1971) 8(9):871-874] and has been shown to be a highly sensitive technique capable of detecting specific proteins (e.g. antibodies) in complex mixtures of proteins. The ELISA technique is well established and typically involves (i) coating/capture of antigens in the test sample to the surface of microtitre plate (e.g. by directly adsorption to the plate or by fist attaching a capture antibody to the plate surface); (ii) plate blocking by addition of irrelevant proteins to cover all unsaturated surface-binding sites if the plate/well; (iii) probing - by incubating with antigen-specific antibodies that bind to the antigens; and (i) signal detection - detection of a signal produced by the specific antibody directly (e.g. using an enzyme-conjugated primary antibody) or indirectly (e.g. using a matches set of unlabelled primary antibody and conjugated secondary antibody) . Various ELISA formats exist including direct, indirect and capture “sandwich”. [0056] An ELISA test could be used to detect the presence of M. tuberculosis SugC or

Mpt83 antigen in the sample, or the presence of antibodies raised by the body against the SugC or Mpt83 antigens.

[0057] In an embodiment, the biological sample is serum and the serum sample is tested for antibodies against SugC or Mpt83. [0058] One or both of the SugC and Mpt83 antigens may be used in the method according to the first aspect.

[0059] Therefore, in an embodiment, the presence of immune cells responsive to:

(i) SugC and Mpt83; or

(ii) SugC; or (iii) Mpt83; is determined. [0060] In an embodiment, the presence of antibodies against:

(i) SugC and Mpt83; or

(ii) SugC; or

(iii) Mpt83; is determined.

[0061] As mentioned previously, in subjects having latent TB, these antigens trigger a stronger immune response than in subjects having active TB, thereby facilitating diagnosis of LTBI.

[0062] Accordingly, in an embodiment, in the method according to the first aspect: a. the presence of immune cells responsive to the M.tb SugC and/or Mpt83 antigen; or b. the presence of the product of immune cells against SugC or Mpt83; is indicative that the patient has LTBI. Suitably, for option b. the product of immune cells against SugC or Mpt83 are antibodies against SugC or Mpt83. Such antibodies will have been produced by B-ceils.

[0063] In a particular embodiment, the methods of the present invention further comprise a step consisting of comparing the levels or number of immune cells or products detected with a reference value indicative of whether said subject has ATBI or LTBI.

[0064] A reference value can be a threshold value or a cut-off value. Typically, a “threshold value” or “cut-off value” can be determined experimentally or empirically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value reflects the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). [0065] For example, when employing the antigens of the invention in an IGRA test, the person skilled in the art may compare the cytokine production levels (e.g. spots or scores) obtained according to the method of the invention with one or more defined threshold values. In one embodiment of the present invention, the threshold value is or has been derived from the cytokine production level (e.g. spots or scores) determined in a control sample derived from one or more subjects who are substantially healthy (i.e. having no latent tuberculosis infection). In one embodiment of the present invention, the threshold value is or has been derived from the cytokine production level (e.g. spots or scores) determined in a control sample derived from one or more subjects who suffers from latent tuberculosis infection. In one embodiment of the present invention, the threshold value is or has been derived from the cytokine production level (e.g. spots or scores) determined in a control sample derived from one or more subjects who suffers from active tuberculosis infection. [0066] In an embodiment, a patient having LTBI will have a higher level in the biological sample of either immune cells (e.g. T cells) responsive to the M.tb SugC and/or pt83 antigen, or the product of immune cells (e.g. antibodies) against the M.tb SugC or Mpt83 antigen, than a patient having ATBI or a control sample. In an embodiment, the higher level is a greater than 1 fold difference relative to the ATBI/control sample, such as a fold difference of 1 5, 2.0, 2.5, 3.0, 35, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 105, 11 , 11.5, 12, 12.5, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any ranges therebetween. In one embodiment, the higher level is between 1 and 75 fold difference relative to the ATB/control sample, such as between 1 .5 and 10, in particular between 1.5 and 5. [0067] In an embodiment, a PBMC sample from a patient having LTBI will have on average greater than 25 spot-forming units (SFU) per 3 x 10⁵ PBMCs, when assayed in an IGRA test using SugC antigen. In a convenient embodiment, a PBMC sample from a patient having LTBI will have on average greater than 40 (such as greater than 50) SFU per 3x 10⁵ PBMCs, when assayed in an IGRA test using SugC antigen. In a convenient embodiment, a PBMC sample from a patient having LTBI will have on average greater than 5 (such as greater than 10) SFU per 3 x 10⁵ PBMCs, when assayed in an IGRA test using Mpt83 antigen.

[0068] In an embodiment of the first aspect of the invention, there is provided a method for determining whether a patient suspected of being infected with M tuberculosis has active tuberculosis (ATB) infection or latent tuberculosis (LTBI) infection, the method comprising testing a biological sample from the patient for the presence of T cells responsive to a M tuberculosis antigen selected from SugC and/or Mpt83 using an interferon gamma release assay (IGRA).

[0069] In an embodiment of the first aspect of the invention, there is provided a method for determining whether a patient suspected of being infected with M tuberculosis has active tuberculosis (ATB) infection or latent tuberculosis (LTBI) infection, the method comprising testing a biological sample from the patient for the presence of T cells responsive to a M tuberculosis antigen selected from SugC and/or Mpt83 using an interferon gamma release assay (IGRA); wherein the biological sample comprises PBMCs.

[0070] In an embodiment of the first aspect of the invention, there is provided a method for determining whether a patient suspected of being infected with M tuberculosis has active tuberculosis (ATB) infection or latent tuberculosis (LTBI) infection, the method comprising testing a biological sample from the patient for the presence of T cells responsive to a M tuberculosis antigen selected from SugC and/or Mpt83 using an interferon gamma release assay (IGRA); wherein the biological sample comprises PBMCs; and wherein the presence of T cells responsive to the M. tuberculosis antigen is indicative that the patient has LTBI. [0071] It is envisaged that a SugC/Mpt83 test according to the present invention could be used in conjunction with the existing methodologies for diagnosing and assessing TB. For example, it could be used in conjunction with the standard CFP10/ESAT6 T-spot IGRA test (orthe QuantiFERON®-TB using ESAT-6, CFP10, and TB77); either in one combined IGRA test, or subsequent to a standard (CFP10/ESAT6 or ESAT-6/CFP10/TB7.7) IGRA test.

[0072] In a two-step testing protocol, the first IGRA test (using CFP10/ESAT6 or ESAT- 6/CFP10/TB7.7) could be used to see if a patient has had immunological exposure to Mycobacterium tuberculosis. Although these tests can differentiate between TB-infected and BCG-vaccinated individuals, they cannot, however, differentiate between active and latent TB disease. Accordingly, a SugC/Mpt83 test according to the present invention could then be carried out as a second test on those patients identified from the first test as having a TB infection, to readily determine if their disease is active or latent.

[0073] In a second aspect of the invention there is provided a method for assessing a patient for tuberculosis infection comprising: testing a biological sample from the patient in an interferon gamma release assay (IGRA) against each of the following M tuberculosis antigens:

(i) at least one of SugC and Mpt83; and

(ii) at least one of CFP-10, ESAT6 and TB7.7(Rv2654c); and characterising the patient as non-immunologically exposed to M tuberculosis , having active tuberculosis, or having latent tuberculosis based on the assay results.

[0074] As mentioned previously, a CFP-10/ESAT6 IGRA test can distinguish between BCG-vaccinated, non-immunologically exposed to M tuberculosis and TB-infected patients. The present invention can distinguish between LTBI and ATBI. Therefore, although the method of the present invention may not be able to distinguish between BCG-vaccinated and LTBI, when used in conjunction with CFP-10/ESAT6 IGRA testing it is possible to further categorise the patients between BCG-vaccinated, non-immunologically exposed to M tuberculosis, ATBI and LTBI.

[0075] In a convenient embodiment of the second aspect, there is provided a method wherein: (i) an IGRA test using CFP-10, ESAT6 and/or TB7.7 is carried out first and an IGRA test using SUGC and/or MPT83 is carried out second; or

(ii) an IGRA test using SugC and/or Mpt83 is carried out first and an IGRA test using CFP-10, ESAT6 and/or TB7.7 is carried out second; or

(iii) an IGRA test using CFP-10, ESAT6 and/or TB7.7, and using SugC and/or Mpt83 is carried simultaneously. [0076] In a third aspect of the invention there is provided a method for determining whether a patient with a Mycobacterium tuberculosis infection has active tuberculosis infection (ATBI) or latent tuberculosis infection (LTBI), comprising: contacting a biological sample from the patient with a M tuberculosis SugC and/or Mpt83 antigen; and detecting for the presence of T cells that have been stimulated by the antigen to produce a cytokine, such as interferon gamma, wherein the number or amount of antigen stimulated cells in the biological sample that produce this cytokine indicates whether the patient has ATBI or LTBI.

[0077] Suitably the cells in the sample that are stimulated to produce the cytokine when contacted with M. tuberculosis SugC and/or Mpt83 antigen comprise T cells. [0078] In an embodiment, a biological sample from the patient is also contacted with a M.tb antigen selected from: CFP-10, ESAT6 and TB7.7. In an embodiment, the biological sample from the patient is also contacted with M.tb CFP-10 and ESAT6, or M.tb CFP-10, ESAT6 and TB7.7

[0079] In an embodiment of the third aspect, the patient has been previously identified as having a M.tb infection by a standard IGRA TB test, such as one utilising antigens against CFP-10, ESAT6 and/or TB7.7.

[0080] In an embodiment of the first or third aspects of the invention, the patient has not been vaccinated with a BCG vaccine.

[0081] In a convenient embodiment of the methods according to the first, second or third aspects of the invention, the M.tb antigen may be the full-length protein ora peptide fragment or fragments thereof, wherein the full-length amino acid sequences of the SugC and Mpt83 proteins are listed hereinabove Therefore, in an embodiment, the M tb SugC and/or Mpt83 antigen is selected from:

(i) recombinant or synthetic full-length SugC protein, or one or more peptides of SugC protein; and/or

(ii) recombinant or synthetic full-length Mpt83 protein, or one or more peptides of Mpt83 protein

[0082] In a convenient embodiment, the antigen is selected from full-length SugC protein, and full-length Mpt83 protein, or a mixture thereof. In a convenient embodiment, the antigen is full-length SugC protein. In a convenient embodiment, the antigen is full-length Mpt83 protein. In a convenient embodiment, the antigen is a mixture of full-length SugC protein, and full-length Mpt83 protein.

[0083] In a convenient embodiment, the antigen comprises one or more peptides of the full-length SugC protein (SEQ ID 123). Conveniently, these may be, or may comprise, one or more of the peptides as listed in Table 1 below. Table 1. SugC 15-mer peptide sequences overlapping by 10 amino acids

[0084] Table 1 lists 15-mer amino acid fragments of the full-length protein, which may be used in the methods of the present invention. The skilled person will appreciate that other length fragments including peptides may also be used to potentially stimulate cells to express cytokine when contacted with the antigen. Therefore, in an embodiment, the antigen comprises a plurality of peptides of the full-length SugC protein, wherein the peptides may be of the same length or different lengths to each other In an embodiment, the antigen comprises a pool (such as 3-15, or 4-10) of distinct peptides derived from the full-length SugC protein. [0085] In an embodiment, the antigen comprises one or more of the SugC peptides as listed in Table 3 in the Examples below In an embodiment, the antigen comprises one or more SugC peptides comprising a sequence selected from the following sequences:

SEQ ID 1 MAEIVLDHVNKSYPD

SEQ ID 4 GHTAVRDLNLTIADG

SEQ ID 7 EFLILVGPSGCGKTT

SEQ ID 78 TTLNMIAGLEDISSG

SEQ ID 13 LRIAGERVNEKAPKD

SEQ ID 15 KAPKDRDIAMVFQSY

SEQ ID 16 RDIAMVFQSYALYPH

SEQ ID 17 VFQSYALYPHMTVRQ.

[0086] In an embodiment, the antigen comprises one or more of the SugC peptides comprising a sequence selected from the following sequences:

SEQ ID 48 GSPAMNFFPARLTAI SEQ ID 54 QGVIAAHPKPENVIV SEQ ID 56 ENVIVGVRPEHIQDA SEQ ID 57 GVRPEHIQDAALIDA SEQ ID 58 HIQDAALIDAYQRIR SEQ ID 59 ALI DAYQRIRALTFQ SEQ ID 60 YQRIRALTFQVKVNL SEQ ID 61 ALTFQVKVNLVESLG SEQ ID 62 VKVN LVESLGADKYL.

[0087] In an embodiment, the antigen comprises a SugC peptide comprising one of the listed sequences having SEQ ID NO 1 to 78. For the avoidance of doubt, for example, the SugC peptide could be a 30-mer peptide that comprises one of the 15-mer SugC peptide sequences disclosed hereinabove.

[0088] In a convenient embodiment, the antigen comprises one or more peptides of the full-length Mpt83 protein (SEQ ID 124). Conveniently, these may be, or may comprise, one or more of the peptides as listed in Table 2 below. Table 2. Mpt83 15-mer peptide sequences overlapping by 10 amino acids

[0089] Table 2 lists 15-mer amino acid fragments of the full-length protein, which may be used in the methods of the present invention. The skilled person will appreciate that other length fragments including peptides may also be used to potentially stimulate cells to express cytokine when contacted with the antigen. Therefore, in an embodiment, the antigen comprises a plurality of peptides of the full-length Mpt83 protein, wherein the peptides may be of the same length or different lengths to each other. In an embodiment, the antigen comprises a pool (such as 3-15, or 4-10) of distinct peptides derived from the full-length Mpt83 protein.

[0090] In an embodiment, the antigen comprises one or more of the Mpt83 peptides as listed in Table 4 in the Examples below In an embodiment, the antigen comprises one or more Mpt83 peptides comprising a sequence selected from the following sequences:

SEQ ID 79 MINVQAKPAAAASLA SEQ ID 121 AIAIAFLAGCSSTK SEQ ID 90 AADLIGRGCAQYAAQ SEQ ID 122 AG AQDPVATAASN SEQ ID 97 SNNPMLSTLTSALSG.

[0091] In an embodiment, the antigen comprises a Mpt83 peptide comprising one of the listed sequences having SEQ ID NO 79 to 122. For the avoidance of doubt, for example, the Mpt83 peptide could be a 30-mer peptide that comprises one of the 15-mer Mpt83 peptide sequences disclosed hereinabove.

[0092] In a convenient embodiment of the methods according to the first, second or third aspects of the invention, the biological sample is selected from the group consisting of: whole blood, peripheral blood mononuclear cells (PBMCs), plasma, serum, saliva, sputum, urine, bronchoalveolar lavage (BAL), any cavity fluid (such as pleural effusion), bone marrow, spleen, lymph node, pleural and any other tissue amenable to biopsy in the human body that has been affected by TB. In a convenient embodiment, the biological sample is selected from the group consisting of whole blood, peripheral blood mononuclear cells (PBMCs), plasma and serum, such as PBMCs and serum. In a most convenient embodiment, the biological sample is peripheral blood mononuclear cells (PBMCs). [0093] In particular embodiments of the methods according to the first, second or third aspects of the invention, the biological sample has previously been taken from the patient.

[0094] In a convenient embodiment of the methods according to the first, second or third aspects of the invention, the patient is a human.

Use of Biomarkers to Detect LTBI [0095] In a fourth aspect of the invention there is provided the use of Mycobacterium tuberculosis SugC and/or Mpt83 protein as a biomarker for distinguishing between latent tuberculosis and active tuberculosis.

[0096] Suitably a user relies on detection of Mycobacterium tuberculosis SugC and/or Mpt83 protein, or an antibody against either or both of these proteins, in a method or test on a sample from a patient with, or suspected of having, tuberculosis infection to distinguishing between latent tuberculosis and active tuberculosis.

[0097] In a fifth aspect of the invention there is provided the use of recombinant or synthetic Mycobacterium tuberculosis SugC and/or Mpt83 antigen as a tool for distinguishing between latent tuberculosis and active tuberculosis, wherein the antigen is (a) full-length SugC protein or one or more peptides thereof; or (b) full-length Mpt83 protein or one or more peptides thereof.

[0098] The uses according to the fourth or fifth aspects of the invention may be carried out according to the methods of the first to third aspects of the present invention. Therefore, all the various embodiments, and combinations thereof, described herein in relation the first to third aspects may similarly apply to the fourth and fifth aspects.

Kit of Parts for Detecting LTBI

[0099] In a sixth aspect of the invention there is provided a kit of parts comprising one or more M tuberculosis antigens selected from:

(i) SugC protein, (ii) one or more peptides of SugC protein, (iii) Mpt83 protein, and (iii) one or more peptides of Mpt83 protein; and instructions for use:

(a) in detecting M tuberculosis SugC or Mpt83 activated T cells in a biological sample; and/or (b) in determining a patient’s tuberculosis infection status, in particular whether they have LTBI.

[00100] In a convenient embodiment the kit of parts comprises SugC protein and Mpt83 protein. In an alternative embodiment the kit of parts comprises one or more peptides derived from SugC protein. Conveniently, these may be one or more of the peptides as listed in Table 1 or Table 3 herein. In an alternative embodiment the kit of parts comprises one or more peptides derived from Mpt83 protein. Conveniently, these may be one or more of the peptides as listed in Table 2 or Table 4 herein. In an alternative embodiment the kit of parts comprises one or more peptides derived from Sug C protein and Mpt83 protein Conveniently, these may comprise one or more of the peptides as listed in Table 1 or 3 and one or more of the peptides as listed in Table 2 or Table 4 herein.

[00101] In an embodiment, the instructions for use describe (a) suitable assay procedures for the detection of a cytokine expressed by M.tb SugC or Mpt83 activated T cells in a biological sample (such as a blood sample from a patient to be tested with the kit for LTBI). In a convenient embodiment the cytokine is interferon gamma and the instructions describes suitable procedure for carrying out an interferon gamma release assay (IGRA). Further details may be provided in the instructions according to the methods and uses described herein in relation to the present invention.

[00102] In an embodiment, the instructions for use describe (b) how a patient’s tuberculosis infection status may be determined (in particular whether they have LTBI), by reference to the detection procedures described in part (a) above. In a convenient embodiment, the expression of a cytokine by M.tb SugC or Mpt83 activated T cells in the biological sample and its subsequent detection in a suitable assay, may be used to determine that the patient has latent TB and not active TB. Further details may be provided in the instructions according to the methods and uses described herein in relation to the present invention.

[00103] Suitably, the instructions for use may include suitable threshold levels that are used to discriminate between LTBI and ATBI and optionally also the statistical significance associated with making a diagnosis of LTBI based on the test data. For example, if the threshold level is detection of 20 spots in a set volume of sample and the sample comprises 50 spots the patient could be identified as LTBI with, for example, a statistical significance or accuracy of 99.5%; whereas if the patient had 21 spots the statistical significance or accuracy might be 96%. The threshold values and statistical significance values can be determined using conventional analyses on suitable numbers of test subjects. Typically, a “threshold value” or “cut-off value” can be determined experimentally or empirically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).

[00104] For example, when employing the antigens of the invention in an IGRA test, the person skilled in the art may compare the cytokine production levels (e.g. spots or scores) obtained according to the method of the invention with one or more defined threshold values. In one embodiment of the present invention, the threshold value is or has been derived from the cytokine production level (e.g. spots or scores) determined in a control sample derived from one or more subjects who are substantially healthy (i.e. having no latent tuberculosis infection). In one embodiment of the present invention, the threshold value is or has been derived from the cytokine production level (e.g. spots or scores) determined in a control sample derived from one or more subjects who suffers from latent tuberculosis infection. In one embodiment of the present invention, the threshold value is or has been derived from the cytokine production level (e.g. spots or scores) determined in a control sample derived from one or more subjects who suffers from active tuberculosis infection.

[00105] In an embodiment, the kit of parts also comprises one or more of the following:

(i) CFP-10 protein, (ii) one or more peptides of CFP-10 protein, (iii) ESAT6 protein, (iii) one or more peptides of ESAT6 protein, (v) TB 7.7 protein, and (vi) one or more peptides of TB7.7 protein. [00106] Conveniently, in this embodiment, the kit of parts further comprises instructions for use:

(c) in detecting M tuberculosis CFP-10, ESAT6 or TB7.7 activated T cells in a biological sample; and/or

(d) in determining a patient’s tuberculosis infection status, in particular whether they are non-immunologically exposed to M tuberculosis, have active tuberculosis, or have latent tuberculosis.

Treatment of LTBI

[00107] Current recommended current treatments options for latent TBI include (i) 6- or 9- month isoniazid monotherapy; (ii) 3-months of once-weekly isoniazid plus rifapentine; (iii) 3 months of daily isoniazid plus rifampicin; or (iv) 4 months of daily rifampicin. Clinicians will choose the most appropriate treatment on a case by case basis, taking into consideration any coexisting medical conditions that a patient may have, the potential for drug-drug interactions and importantly, whether is it likely that the patient has contracted a drug- resistant strain of M tuberculosis. [00108] According to a seventh aspect of the invention there is provided a drug selected from isoniazid, rifampicin or rifapentine, or a combination thereof, for use in the treatment of latent tuberculosis infection (LTBI) comprising:

(i) determining whether a patient has LTBI according to a method as described herein; and

(ii) if the patient is identified in step (i) as having LTBI administering the isoniazid, rifampicin or rifapentine, or a combination thereof, to the patient.

[00109] In an embodiment, step (i) is carried out to detect the presence of immune cells responsive to a M tuberculosis antigen selected from SugC and/or Mpt83, according to a method as described herein.

[00110] In an embodiment, in step (ii) either:

(a) isoniazid;

(b) rifampicin;

(c) isoniazid and rifampicin in combination; or (d) isoniazid and rifapentine in combination; are administered to the patient with LTBI.

[00111] In an eighth aspect of the invention there is provided a method of treating a patient with latent tuberculosis, comprising:

(i) determining whether the patient has latent TB according to a method as described herein; and

(ii) if the patient is identified in step (i) as having latent TB, administering to the patient drugs selected from: isoniazid, or rifampicin, or isoniazid & rifampicin or isoniazid & rifapentine.

[00112] In an embodiment, step (i) is carried out to detect the presence of immune cells responsive to a M tuberculosis antigen selected from SugC and/or Mpt83, or a product of immune cells against SugC or Mpt83, such as anti-SugC or anti-Mpt83 antibodies, according to a method as described herein.

[00113] In an embodiment, after the patient has completed step (ii) of either the seventh or eighth aspects of the invention, it may be desirable to perform additional tests to confirm whether the drug treatment has been successful. These tests may comprise tests to determine if the patient still is characterised as having LTBI Therefore, in an embodiment, there is provided the drug for use according to the seventh aspect, or the method of treatment according to eighth aspect, wherein following an appropriate period after step (ii), the effectiveness of the treatment is assessed by repeating step (i). [00114] Suitably an appropriate period after step (ii) is at least 3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48, or 60 months.

[00115] The following numbered statements 1-34 are not claims, but instead describe certain aspects and embodiments of the invention: 1. A method for determining whether a patient suspected of being infected with

Mycobacterium tuberculosis has active tuberculosis infection (ATBI) or latent tuberculosis infection (LTBI) comprising testing a biological sample from the patient for the presence of: a. immune cells responsive to a M tuberculosis antigen selected from SugC and/or Mpt83; or b. a product of immune cells against SugC or Mpt83.

2. The method according to statement 1 , wherein the immune cells responsive to a M tuberculosis antigen are T cells.

3. The method according to statement 2, wherein the T cells express one or more cytokines when contacted with the M tuberculosis antigen. 4. The method according to any one of statements 1 to 3, wherein the presence of T cells responsive to M tuberculosis antigen are detected by contacting the biological sample with the M tuberculosis antigen and detecting for cells that are stimulated to express one or more cytokines.

5. The method according to statement 3 or 4, wherein the cytokine is selected from the group consisting of: interferon gamma, tumour necrosis factor alpha, interleukin 2, interleukin 4, interleukin 17, and any other human cytokine or chemokine of relevance to TB immunity.

6. The method according to statement 4 or 5, wherein the expressed cytokine is detected using a cytokine release assay (CRA). 7. The method according to any one of statements 4 to 6, wherein the expressed cytokine is interferon gamma.

8. The method according to statement 7, wherein the interferon gamma is detected using an interferon gamma release assay (IGRA)

9. The method according to statement 1, wherein the product of the immune cells against SugC or pt83 comprise one or more antibodies against SugC or Mpt83

10 The method according to statement 9, wherein the presence of the one or more antibodies is detected using enzyme linked immunosorbent assay (ELISA), an immunochromatographic test, or any exploratory immunological test capable of detecting antibodies. 11 The method according to any one of the preceding statements, wherein the biological sample is selected from the group consisting of: whole blood, peripheral blood mononuclear cells (PBMCs), plasma, serum, saliva, sputum, bronchoalveolar lavage (BAL), and urine, any cavity fluid (such as pleural effusion), bone marrow, spleen, lymph node, pleural or any other tissue amenable to biopsy in the human body that has been affected by TB.

12 The method according to any one of the preceding statements, wherein the patient is a human.

13 The method according to any one of the preceding statements, wherein the antigen is selected from SugC protein and Mpt83 protein or a mixture thereof.

14 The method according to any one of the preceding statements, wherein the presence of immune cells responsive to: (i) SugC and Mpt83; or

(ii) SugC; or

(iii) Mpt83, is determined.

15 The method according to any one of the preceding statements, wherein the antigen is full-length protein or a fragment thereof. 16 The method according to any one of statements 1 to 14, wherein the antigen comprises one or more peptides of the full-length SugC and/or Mpt83 protein.

17 The method according to any one of the preceding statements, wherein the presence of immune cells responsive to the M tuberculosis antigen or a product of immune ceils against SugC or Mpt83 is indicative that the patient has LTBI 18. A method for assessing a patient for tuberculosis infection comprising: testing a biological sample from the patient in an interferon gamma release assay (IGRA) against each of the following M tuberculosis antigens:

(i) at least one of SugC and Mpt83; and

(ii) at least one of CFP-10, ESAT6 and TB7 7(Rv2654c); and characterising the patient as non-immunologically exposed to M.tb, having active tuberculosis or having latent tuberculosis based on the assay results.

19 The method according to statement 18, wherein:

(i) an IGRA test using CFP-10, ESAT6 and/or TB7.7 is carried out first and an IGRA test using SugC and/or Mpt83 is carried out second; or (ii) an IGRA test using SUGC and/or MPT83 is carried out first and an IGRA test using CFP-10, ESAT6 and TB7.7 is carried out second; or

(iii) an IGRA test using CFP-10, ESAT6 and TB7.7 and using SugC and/or pt83 is carried simultaneously.

20 A method for determining whether a patient with a Mycobacterium tuberculosis infection has active tuberculosis infection (ATBI) or latent tuberculosis infection (LTBI), comprising: contacting a biological sample from the patient with a M tuberculosis SugC and/or Mpt83 antigen; and detecting for the presence of T cells that have been stimulated by the antigen to produce a cytokine, such as interferon gamma, wherein the number or amount of antigen stimulated T cells in the biological sample that produce this cytokine indicates whether the patient has ATBI or LTBI.

21 The method according to statement 20, wherein the M tuberculosis SugC and/or Mpt83 antigen is selected from:

(i) recombinant or synthetic full length SugC protein or one or more peptides of SugC protein; and/or

(ii) recombinant or synthetic full length Mpt83 protein or one or more peptides of Mpt83 protein 22 The method according to statement 21, wherein the one or more peptides of SugC protein comprise one or more peptides as in Table 1 or Table 3; and/or the one or more peptides of MPT83 protein comprise one or more peptides as in Table 2 or Table 4.

23 The method according to any one of statements 20-22, wherein a biological sample from the patient is also contacted with a M tuberculosis antigen selected from: CFP-10, ESAT6 and TB7.7

24 The method of statement 23, wherein the biological sample from the patient is also contacted with M tuberculosis CFP-10 and ESAT6 or M tuberculosis CFP-10, ESAT6 and TB7.7

25 The method according to any one of statements 20-22, wherein the patient has been identified as having a Mycobacterium tuberculosis infection by standard IGRA TB test, such as one utilising antigens against CFP-10, ESAT6 and/or TB77.

26 Use of Mycobacterium tuberculosis SugC and/or Mpt83 protein as a biomarker for distinguishing between latent tuberculosis and active tuberculosis.

27 Use of recombinant or synthetic Mycobacterium tuberculosis SugC and/or Mpt83 antigen as a tool for distinguishing between latent tuberculosis and active tuberculosis, wherein the antigen is full-length protein or one or more peptides thereof.

28 A kit of parts comprising one or more M tuberculosis antigens selected from:

(i) SugC protein, (ii) one or more peptides of SugC protein, (iii) Mpt83 protein, and (iii) one or more peptides of pt83 protein, and instructions for use (a) in detecting M tuberculosis SugC or Mpt83 activated T cells in a biological sample, and/or (b) in determining a patient’s tuberculosis infection status, in particular whether they have LTBI.

29 The kit of parts according to statement 28, which also comprises one or more of the following: (i) CFP-10 protein, (ii) one or more peptides of CFP-10 protein, (iii) ESAT6 protein, (iii) one or more peptides of ESAT6 protein, (v) TB 7.7 protein, and (vi) one or more peptides of TB7.7 protein. 30 A drug selected from isoniazid, rifampicin, rifapentine, or a combination thereof, for use in the treatment of LTBI comprising:

(iii) determining whether a patient has LTBI according to the method of any one of statements 1 - 25, and (iv) administering the isoniazid, rifampicin, rifapentine or a combination thereof, to the patient.

31 The drug for use according to statement 30, wherein in step (ii) the patient administering (a) isoniazid or (b) rifampicin or (c) isoniazid and rifampicin in combination, or (d) isoniazid and rifapentine in combination. 32 A method of treating a patient with LTBI, comprising:

(iii) determining whether the patient has LTBI according to the method of any one of statements 1 - 25, and

(iv) if the patient is identified in step (i) as having LTBI, administering to the patient drugs selected from: isoniazid, or rifampicin, or isoniazid & rifampicin or isoniazid & rifapentine.

33 The drug for use according to statements 30 or 31 or the method of treatment according to statement 32, wherein step (i) is carried out to detect the presence of immune cells responsive to a M tuberculosis antigen selected from SugC and/or Mpt83, according to the method of any one of statements 1 - 25. 34 The drug for use according to statements 30, 31 or 33, or the method of treatment according to statements 32 or 33, wherein following an appropriate period after step (ii), the effectiveness of the treatment is assessed by repeating step (i).

EXAMPLES

Materials · Peptides were ordered from GL Biochem (Shanghai) Ltd

• Lymphoprep density gradient medium (Stemcell Technologies)

• R0 (RPMI 1640 with L- glutamine (Gibco), penicillin/streptomycin (Gibco))

• R10 (RPMI 1640 with L- glutamine (Gibco), penicillin/streptomycin (Gibco), Sodium pyruvate (Gibco), (FBS) - heat inactivated (Gibco)) · Fetal bovine serum (FBS) - heat inactivated (Gibco)

• Benzonase® Nuclease (Merck - 99%purity)

• Carbonate-bicarbonate buffer (Sigma Aldrich)

• anti-IFN-y capture antibody (Ab) (Mabtech- monoclonal antibody DIK- 1mg/ml)

• 0.45 mm hydrophobic plate; IP sterile (Merck) · Purified protein derivative (PPD BOVIGAM-ThermoFisher Scientific) Equipment

• Leucocep tubes (Greiner-bio-one)

• Elispot reader (Autoimmun Diagnostika GmbH - Al D)

Example 1 - Preparation of SugC and Mpt83 peptide pools [00116] Peptide sequences of the SugC and Mpt83 proteins were chosen based on MHC class ll-binding prediction analysis of the respective proteins. A combination of T cell epitope prediction algorithms for identifying sites which most frequently bind with MHC class II were used. The approach consisted of combining the results from different T cell epitope prediction algorithms and choosing epitopes with moderate to high prediction scores. The initial prediction was based on Sturniolo model [Sturniolo et al., Nat Biotechnol. (1999); 17(6):555-61] and all the moderate and high scorers identified by this model were then checked against seven additional algorithms in the IEDB database [Vita ef al., Nucleic Acids Res. (2018) doi: 10 1093/nar/gky1006] Synthetic peptides were reconstituted in DMSO. Peptide pools were then created using these peptides as shown in Tables 3 and 4 at a final concentration of 50pg/ml.

Table 3. SugC peptide pools

SugC - Pool 1

MAEIVLDHVNKSYPD (SEQ ID NO: 1)

GHTAVRDLNLTIADG (SEQ ID NO: 4)

EFLILVGPSGCGKTT (SEQ ID NO: 7)

TTLNMIAGLEDISSG (SEQ ID NO: 78)

LRIAGERVNEKAPKD (SEQ ID NO: 13)

KAPKDRDIAMVFQSY (SEQ ID NO: 15)

RDIAMVFQSYALYPH (SEQ ID NO: 16)

VFQSYALYPHMTVRQ (SEQ ID NO: 17)

SugC - Pool 2

RDLNLTIADGEFLIL (SEQ ID NO: 5)

TIADGEFLILVGPSG (SEQ ID NO:6)

EFLILVGPSGCGKTT (SEQ ID NO: 7)

TTLNMIAGLEDISSG (SEQ ID NO: 78)

LRIAGERVNEKAPKD (SEQ ID NO: 13)

KAPKDRDIAMVFQSY (SEQ ID NO: 15)

RDIAMVFQSYALYPH (SEQ ID NO: 16)

VFQSYALYPHMTVRQ (SEQ ID NO: 17)

SugC - Pool 3

GSPAMNFFPARLTAI (SEQ ID NO: 48)

QGVIAAHPKPENVIV (SEQ ID NO: 54)

ENVIVGVRPEHIQDA (SEQ ID NO: 56)

GVRPEHIQDAALIDA (SEQ ID NO: 57)

HIQDAALIDAYQRIR (SEQ ID NO: 58) ALI DAYQRI RALTFQ (SEQ ID NO: 59) YQRIRALTFQVKVNL (SEQ ID NO: 60) ALTFQVKVNLVESLG (SEQ ID NO: 61) VKVNLVESLGADKYL (SEQ ID NO: 62)

SugC - Pool 4

DKYLYFTTESPAVHS (SEQ ID NO: 128) SVQLDELAEVEGESA (SEQ ID NO: 67) LHENQFVARVPAESK (SEQ ID NO: 70) PAESKVAIGQSVELA (SEQ ID NO: 72) SVELAFDTARLAVFD (SEQ ID NO: 74)

Table 4. Mpt83 peptide pools

Mpt83 - Pool 1

MINVQAKPAAAASLA (SEQ ID NO: 79) AIAIAFLAGCSSTK (SEQ ID NO: 121) AADLIGRGCAQYAAQ (SEQ ID NO: 90) AGMAQDPVATAASN (SEQ ID NO: 122) SNNPMLSTLTSALSG (SEQ ID NO: 97) Mpt83 - Pool 2

KLNPDVNLVDTLNGG (SEQ ID NO: 100) VNLVDTLNGGEYTVF (SEQ ID NO: 101) GEYTVFAPTNAAFD (SEQ ID NO: 129) AAFDKLPAATIDQL(SEQ ID NO: 130) DQLKTDAKLLSSILT (SEQ ID NO: 107) Mpt83 - Pool 3

DAKLLSSILTYHVIA (SEQ ID NO: 108) SSILTYHVIAGQASP (SEQ ID NO: 109) YHVI AGQASPSRI DG (SEQ ID NO: 110) GQASPSRIDGTHQTL (SEQ ID NO: 111) TVIGARDDLMVNNAG (SEQ ID NO: 115) RDDLMVNNAGLVCGG (SEQ ID NO: 116) AGLVCGGVHTANA (SEQ ID NO: 131) ATVYMIDTVLMPPAQ (SEQ ID NO: 120)

Example 2 - Isolation of PBMCs from study participant blood samples [00117] 62 participants were recruited to this study. Adults with a new diagnosis of latent

TB infection (LTBI) or active TB (ATB) disease (both pulmonary and extra-pulmonary) were identified locally at Birmingham Chest Clinic (part of the University Hospital of Birmingham) through TB nurse specialists, respiratory and infectious diseases physicians.

[00118] Individuals with ATB were confirmed microbiologically (sputum or BAL TB culture positive, tissue culture positive or preliminary TB PCR positive). Individuals with LTBI had a positive IGRA test and subsequent relevant tests, imaging and clinical examination that pointed to latent TB diagnosis. The donors who participated in the study, were asked to provide basic demographic information including age, ethnic origin and country of birth. They were also asked questions pertinent to their diagnosis and immune profile, which included: history of BCG vaccination, travel history, previous TB infection, history of conditions causing immunosuppression, anti-tuberculosis medication and other medications. Healthy controls (BCG vaccinated and non BCG vaccinated) were identified either at the screening clinic (in Birmingham Chest Clinic) or at the University of Birmingham (IBR, Institute of Immunology & Immunotherapy). A similar process was followed, prior to proceeding to blood withdrawal. [00119] Each participant donated blood samples and the donor classification is shown in Table 5.

Table 5. Study donor classification

Donors Number Percentage

Healthy control - BCG vaccinated (HC-BCG (+)) 24 39

Healthy control - BCG non-vaccinated (HC-BCG (-)) 10 16

Latent TB infection (LTBI) 21 34

Active TB infection (ATBI) 7 il

Total 62 100

[00120] The demographic characteristics of the study participants are demonstrated in Fig 1 . The majority of participants recruited were white (any background) and Asian (mainly

Indian-Pakistani). There was an equal number of males and females recruited. The median age of the donors was 31.5.

PBMC isolation

[00121] Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood samples as follows.

[00122] 50 ml of blood was drawn from study participants. 15 ml of Lymphoprep density gradient medium was added to each 50 ml Leucocep tube and centrifuged at 700 x g for 1 min in order to get the Lymphoprep below the porous membrane in the Leucocep tube. Following that, 50ml of heparinized whole blood was divided and loaded into two of the Leucocep tubes (i.e. approximately 25 ml in each tube) and diluted 1:1 with R0 medium. The tubes were centrifuged at 1000 x g for 14 min at room temperature with the brake turned off. At this point, the cool cells containers were placed in the fridge and the metal ring placed at room temperature, in preparation for the cryopreservation stage. [00123] Following the main separation stage, the plasma was removed from the interface using a serological pipette and the PBMC layer was collected with a sterile Pasteur pipette and loaded into a 50 ml Falcon tube. The PBMCs were washed by topping up with 45 ml of R0 and then span at 700 x g at room temperature with the brake set at highest setting. Supernatants were discarded and the cells’ pellets were reconstituted. Cells from each donor were subsequently transferred into one 50ml Falcon tube and washed again by topping up with 45 ml of R0. After the second wash, supernatants were discarded and cell pellets were reconstituted with 10 ml of R10 medium, ready for counting. Counting was performed after diluting the cells 1:1 with trypan blue and visualized using a haemocytometer under a microscope. The mean of two counts was used to calculate the concentration of the cells.

PBMC freezing

[00124] For cryopreservation of PBMCs, cells were centrifuged at 700 x g for 5 min at room temperature, supernatants were discarded and cells reconstituted with cold FCS (0.5 ml of FCS per vial to be frozen). Subsequently the cells were left to rest in the fridge for approximately 20 min. An equal amount of ice cold FCS with 20% Dimethyl sulfoxide (D SO) was added to the cell mix exactly before the cells were transferred into cryovials. Cells were aliquoted (1 ml) into cryovials at a concentration between 5-7 x 10⁶/ml, placed into the cool cell containers with metal ring replaced and immediately transferred into -80°C freezer. After 1-2 days, the vials were transferred to liquid nitrogen (FIBRC facility) for long term storage. PBMC samples were thawed using standard protocols, in order to be used in subsequent immunological assays.

PBMC thawing

[00125] Cryopreserved PBMCs were retrieved from -80°C or liquid nitrogen and placed on dry ice. Cryovials were thawed one at the time by hand until a small amount of ice remained. Using a Pasteur pipette, approximately 0.5 ml of warm (37°C) R10 medium was added into each cryovial and transferred into a 15ml Falcon tube containing 9 ml of warm (37°C) R10 medium for a 1:10 dilution. Subsequently, the tube was centrifuged at 700 x g for 5 min at room temperature. The supernatant was discarded and the cell pellet was reconstituted in 2 ml of warm (37°C) R10 medium. For each 1x10⁶ PBMC in the cryovial 1 pi of Benzonase® Nuclease (approx 25 Units) was added and then incubated at 37°C for 1 hour minimum.

Example 3 - Ex-vivo T cell ELIspot assay protocols [00126] Carbonate-bicarbonate buffer (0 05M) was created by dissolving 1 capsule in

100ml of sterile water. Following that, the buffer was filter-sterilised with a 0.22 micron filter. The coating solution was made by adding anti-IFN-g capture antibody (Ab) to the above buffer for a final concentration of 15 m9/hhI. 50 mI of coating solution was added to each well of a 0.45 mΐh hydrophobic IP sterile plate and gently tapped to ensure that the entire surface of the well is covered. The plate was incubated at 4°C for 1-4 days. Subsequently, the plate was washed 5 times with sterile PBS (120 mI/well), and a 100 mI of R10 was added in each well to block the plate. The plate was subsequently incubated for 2-5 h at 37°C.

[00127] Whilst the plate was incubated, the cryopreserved PBMCs needed for the experiment were taken out of the liquid nitrogen or -80°C freezer and were thawed as described in Example 2. The PBMCs were rested in Benzonase® Nuclease for 1 h minimum and following that were washed once at 700 x g for 5 min at room temperature. The supernatant was discarded and the cell pellet was resuspended in R10 in order to achieve a concentration of 3.75x10⁶/ml (i.e. 0.3 x 10⁶ PBMC/80 mI/well). The appropriate concentration of PBMCs was defined after adequate optimisation and testing of 25 healthy control (BCG vaccinated) donors. Different dilutions of PBMCs were prepared in each donor vial as necessary to achieve the above concentration. The blocking solution was flicked (without any washing step) and 80 mI of the cell suspension in R10 was added to each well.

[00128] Antigens were prepared at the concentrations indicated in Table 6 and 20 mI of each antigen was added to the plate in duplicate. Phytohemagglutinin-L (PHA-L), a lectin from Phaseolus vulgaris was used as a general positive control, since it is a very potent lymphocyte mitogen Purified protein derivative from M.bovis culture was used as a more specific positive control for TB. The plate was then incubated for 18h at 37°C.

Table 6. Antigen concentrations

Antigen _ Stock Concentration/mass _ Final concentration

PPD 30000 lU/ l 150 pg/ml

PHA 5 mg 50 m /hiI

Peptide pools_ 10 g_ 50 mg/ml

Antigens were diluted in PBS to reach the desired working concentration. All the antigens were further diluted by 1 :5 when were added on the plate (i.e. 20 mI of antigen added in 80 pi of cell suspension).

[00129] After the overnight incubation, the plates were washed 5 times with sterile PBS containing 0.05% Tween 20 and 50mI of biotin anti- IFN-g Ab (biotinylated monoclonal antibody 7B6-1 - diluted 1 : 1000 in PBS) was added in duplicate wells. The plates were then incubated for 2h at room temperature. The plates were washed 5 times with PBS 0.05% Tween 20, flicked and blotted and 50 mI streptavidin-alkaline phosphatase (diluted 1/1000 in PBS) was added in each duplicate well and incubated at room temperature for 1 h. The plates were washed 5 times with PBS containing 0.05% Tween 20, flicked and blotted and 50 mI BCIP developer (Sigma Aldrich) was added in each well for approximately 3-15 min. The substrate reaction was terminated by washing the plate in tap water 3-4 times. The plate was dried on the bench overnight and then read the next day using an Elispot reader using AID 7S software. All the ELISpot plates were kept long-term in a dry and dark place.

Example 4 -ELIspot Assay Results

[00130] The IFN-g responses to PPD were as expected, with the highest responses being observed in those donors with ATB, LTBI and historic BCG vaccination (Fig. 2). Responses of the ATB and LTB groups were higher than those observed in donors with historic BCG vaccination. There was statistically significant difference between the healthy control BCG negative group and all the other groups (Kruskal Wallis - 28.25, p < 0.001).

IFN-v responses to SuqC [00131] Figure 3 shows the results of the ELIspot assays carried out with the SugC peptide pools 1-4.

[00132] The highest concentrations of spot-forming units (SFU) were observed for the latent TB and HC-BCG(+) donor cohorts in SugC pools 1, 2 and 3. There was a clear distinction between active TB and latent TB groups with these peptide pools (p<0.05 in SugC Pool 1 & 3).

IFN-v responses to Mpt83

[00133] Figure 4 shows the results of the ELIspot assays carried out with the Mpt83 peptide pools 1-3.

[00134] The majority of the IFN-y responses to Mpt83 peptide pools 2 and 3 were similar to the non-stimulated controls, whereas more than half the donors in the latent TB group had high IFN-g responses to Mpt83 pool 1. There was a clear distinction between the active TB and latent TB groups in pool 1 ( p<0.05 in Mpt83 Pool 1).

Conclusions

[00135] The ELIspot assay results indicate that both SugC and Mpt83 peptide pools are capable of stimulating an IFN-g response in participants that have latent TB, whereas this response was surprisingly absent from the participants having active TB. Thus, Mpt83 and SugC may represent immunogenic proteins that would elicit this differential response between active and latent TB and therefore could be used as potential diagnostic biomarkers. [00136] The SugC peptide pools had the greatest IFN- y responses overall SugC is a relatively large protein, part of an ABC sugar transporter that specifically is responsible for the uptake of trehalose, a carbohydrate that is not found in mammals. Studies indicate that this transporter system is essential for the growth of M.tb within the macrophages during the acute phase and thus it's essential for virulence [Kalscheuer etal., Proc Natl Acad Sci USA (2010) 107 (50):21761-6]

[00137] Mpt83 pool 1 peptides also gave very good IFN-g responses in LTBI donors compared to the ATB group. Mpt83, a cell surface glycoprotein, has been synthesised before and has shown relatively good responses as a TB vaccine candidate in mice [Ahmad et al ., J Genet Eng Biotechnol. (2018) 16(2):335-40; Chen et al., J Immunol. (2012) 188(2):668- 77]

[00138] The unexpected observation that active TB patients do not have adequate IFN-g responses to these proteins (Mpt83, SugC) may be a reflection that these antigens may be under-recognised by the immune system during the active phase of the infection. One possible reason for that, may be that bacilli are predominantly within the macrophages whilst granulomas are constructed during acute pulmonary TB. Another possible explanation of why active TB patients do not have an adequate response to these antigens, may be the relatively localised nature of pulmonary TB infection. Interestingly, active TB patients show substantial responses to PPD, which is used as a positive control for TB, but do not show adequate responses to the specific peptide pools of these proteins. This makes the significance of the present invention more substantial, as it provides a means for readily differentiating between active and latent TB infections An IGRA test using the SugC and/or Mpt83 proteins, or a mixture of peptides from these proteins, could potentially lead to significant reductions in the resources (trained staff and necessity for additional tests) required to identify patients who have latent TB and therefore may require treatment, when compared to the processes currently employed to evaluate latent TB.

[00139] It is envisaged that a SugC/Mpt83 test according to the present invention could be used in conjunction with the existing methodologies for diagnosing and assessing TB. For example, it could be used in conjunction with the standard CFP10/ESAT6 T-spot IGRA test (orthe QuantiFERON®-TB using ESAT-6, CFP10, and TB77); either in one combined IGRA test, or subsequent to a standard (CFP10/ESAT6 or ESAT-6/CFP10/TB7.7) IGRA test.

[00140] In a two-step testing protocol, the first IGRA test (using CFP10/ESAT6 or ESAT- 6/CFP10/TB7.7) would be used to see if a patient has had immunological exposure to Mycobacterium tuberculosis. Although these tests can differentiate between TB-infected and BCG-vaccinated individuals, they cannot, however, differentiate between active and latent TB disease. Accordingly, a SugC/Mpt83 test according to the present invention would then be carried out as a second test on those patients identified from the first test as having a TB infection, to readily determine if their disease is active or latent.

Claims

1. A method for determining whether a patient suspected of being infected with Mycobacterium tuberculosis (M.tb) has active tuberculosis infection (ATBI) or latent tuberculosis infection (LTBI) comprising testing a biological sample from the patient for the presence of: a. immune cells responsive to a M.tb antigen selected from SugC and/or Mpt83; or b. a product of immune cells against SugC or Mpt83.

2. The method according to claim 1, wherein the immune cells responsive to a M.tb antigen are T cells.

3. The method according to claim 2, wherein the T cells express one or more cytokines when contacted with the M.tb antigen.

4. The method according to any one of claims 1 to 3, wherein the presence of T cells responsive to M.tb antigen are detected by contacting the biological sample with the M.tb antigen and detecting for cells that are stimulated to express one or more cytokines.

5. The method according to claim 3 or 4, wherein the cytokine is selected from the group consisting of: interferon gamma, tumour necrosis factor alpha, interleukin 2, interleukin 4, interleukin 17, and any other human cytokine or chemokine of relevance to TB immunity.

6. The method according to claim 4 or 5, wherein the expressed cytokine is detected using a cytokine release assay (CRA).

7. The method according to any one of claims 4 to 6, wherein the expressed cytokine is interferon gamma; and optionally wherein the interferon gamma is detected using an interferon gamma release assay (IGRA)

8. The method according to claim 1 , wherein the product of the immune cells against SugC or Mpt83 comprises one or more antibodies against SugC or Mpt83.

9. The method according to claim 8, wherein the presence of the one or more antibodies is detected using an enzyme linked immunosorbent assay (ELISA), an immunochromatographic test, or any exploratory immunological test capable of detecting antibodies. 10 The method according to any one of the preceding claims, wherein the presence of immune cells responsive to:

(i) SugC and Mpt83; or

(ii) SugC; or (iii) Mpt83; is determined.

11 The method according to any one of the preceding claims, wherein: a. the presence of immune cells responsive to the M.tb SugC and/or Mpi83 antigen; or b. the presence of the product of immune cells against SugC or Mpt83; is indicative that the patient has LTBI.

12 A method for determining whether a patient with a M.tb infection has active tuberculosis infection (ATBI) or latent tuberculosis infection (LTBI), comprising: contacting a biological sample from the patient with a M.tb SugC and/or Mpt83 antigen; and detecting for the presence of cells that have been stimulated by the antigen to produce a cytokine, such as interferon gamma, wherein the number or amount of antigen stimulated cells in the biological sample that produce this cytokine indicates whether the patient has ATBI or LTBI.

13 The method according to claim 12, wherein a biological sample from the patient is also contacted with a M.tb antigen selected from: CFP-10, ESAT6 and TB7.7. 14 The method according to claim 12, wherein the patient has been previously identified as having a M.tb infection by a standard IGRA TB test, such as one utilising antigens against CFP-10, ESAT6 and/or TB7.7.

15 The method according to any one of the preceding claims, wherein the M.tb SugC and/or Mpt83 antigen is selected from: (i) recombinant or synthetic full-length SugC protein, or one or more peptides of

SugC protein; and/or

16 The method according to claim 15, wherein the antigen is selected from full-length SugC protein, and full-length Mpt83 protein, or a mixture thereof.

17 The method according to claim 15, wherein the one or more peptides of SugC protein comprise one or more peptides as listed in Table 1 or Table 3; and/or the one or more peptides of Mpt83 protein comprise one or more peptides as listed in Table 2 or Table 4. 18 The method according to any one of the preceding claims, wherein the biological sample is selected from the group consisting of: whole blood, peripheral blood mononuclear cells (PBMCs), plasma, serum, saliva, sputum, urine, bronchoalveolar lavage (BAL), any cavity fluid (such as pleural effusion), bone marrow, spleen, lymph node, pleural and any other tissue amenable to biopsy in the human body that has been affected by TB.

19 The method according to any one of the preceding claims, wherein the patient is a human.

20 A kit of parts comprising one or more M.tb antigens selected from:

(i) SugC protein, (ii) one or more peptides of SugC protein, (iii) Mpt83 protein, and (iii) one or more peptides of Mpt83 protein, and instructions for use (a) in detecting M.tb SugC or Mpt83 activated T cells in a biological sample, and/or (b) in determining a patient’s tuberculosis infection status, in particular whether they have LTBI.

21 The kit of parts according to claim 20, which also comprises one or more of the following:

(i) CFP-10 protein, (ii) one or more peptides of CFP-10 protein, (iii) ESAT6 protein, (iii) one or more peptides of ESAT6 protein, (v) TB 7.7 protein, and (vi) one or more peptides of TB7.7 protein.

22 A drug selected from isoniazid, rifampicin, or rifapentine, or a combination thereof, for use in the treatment of LTBI comprising:

(i) determining whether a patient has LTBI according to the method of any one of claims 1 - 19; and

(ii) if the patient is identified in step (i) as having LTBI, administering the isoniazid, rifampicin, or rifapentine, or a combination thereof, to the patient. 23 The drug for the use according to claim 22, wherein in step (ii) either:

(a) isoniazid;

(b) rifampicin;

(c) isoniazid and rifampicin in combination; or

(d) isoniazid and rifapentine in combination; are administered to the patient with LTBI.

24 The drug for the use according to claims 22 or 23, wherein step (i) is carried out to detect the presence of immune cells responsive to a M.tb antigen selected from SugC and/or Mpt83, according to the method of any one of claims 1 - 19. 25 The drug for the use according to any one of claims 22 to 24, wherein following an appropriate period after step (ii), the effectiveness of the treatment is assessed by repeating step (i).