WO2012167307A1

WO2012167307A1 - Diagnosis of mycobacterial infection

Info

Publication number: WO2012167307A1
Application number: PCT/AU2012/000644
Authority: WO
Inventors: Marc TEBRUEGGE; Richard Nigel CURTIS
Original assignee: The University Of Melbourne; Murdoch Childrens Research Institute
Priority date: 2011-06-06
Filing date: 2012-06-06
Publication date: 2012-12-13

Abstract

The present disclosure relates generally to the field of immunological-based diagnostic assays to detect infection and stage of infection by pathogens. Taught herein is an assay to detect mycobacterial infection and to distinguish between active and latent forms of mycobacterial infection. Kits and therapeutic protocols are also enabled by the present disclosure.

Description

DIAGNOSIS OF MYCOBACTERIAL INFECTION

FILING DATA [0001] This application is associated with and claims priority from Australian Provisional Patent Application No. 201 1902230 filed on 6 June, 201 1 entitled "Method of Treatment" and Australian Provisional Patent Application No. 201 1904523 filed on 31 October, 201 1 entitled "Diagnosis of Mycobacterial Infection" the entire contents of which, are incorporated herein by reference.

FIELD

[0002] The present disclosure relates generally to the field of immunological-based diagnostic assays to detect infection and stage of infection by pathogens. Taught herein is an assay to detect mycobacterial infection and to distinguish between active and latent forms of mycobacterial infection. Kits and therapeutic protocols are also enabled by the present disclosure.

BACKGROUND

[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0005] Immunological-based diagnostic assays are important tools in detecting a variety of disease conditions such as infection by pathogens. The effectiveness of these types of assays lies in part in the specificity of the components within the immune system. One form of immunological-based diagnostic assay involves the stimulation of immune cells (e.g. lymphocytes, macrophages, etc.) by antigens in fluid samples containing these types of cells followed by the detection of effector molecules such as cytokines produced by stimulated or activated immune cells. Effector molecules can be detected by techniques such as enzyme immunoassays, multiplex bead analysis, ELISpot and flow cytometry. An example of a T-cell assay is QuantiFERON-TB Gold In-Tube (QFT-GIT) [Cellestis Limited, Melbourne, Australia].

[0006] However, whilst the detection of an effector molecule, for example, can evince prior exposure of a subject to a pathogen or prior vaccination, on its own it will not necessarily distinguish between different stages of infection such as whether the infection is active or latent.

[0007] This is particularly problematic for the treatment of infection by mycobacteria, such as Mycobacterium tuberculosis, the causative agent of tuberculosis (TB).

[0008] Effective treatment and management of patients infected with M. tuberculosis requires sensitive and rapid laboratory diagnosis. Current diagnostic protocols are based on microscopic, culture and molecular procedures. With respect to microscopic methods, high mycobacterial concentrations are required in order to record a positive result (approximately from about 5 CFU/ml to 10,000 CFU/ml). Also, microscopy does not allow the distinction between M. tuberculosis and non-tuberculous mycobacteria. Culture tests incur unacceptable delays from a clinical management perspective with M tuberculosis colonies not being visible on solid media until after 10-24 days. Molecular diagnostics offers a highly sensitive option compared to microscopic and culture procedures when mycobacterial numbers have reached a threshold level. However, detection of mycobacterial DNA, for example, does not distinguish between active or latent infection. Traditionally, the tuberculin skin test (TST) has been widely used for decades as the primary tool to diagnose latent TB infection (LTBI) and as a supportive tool in the diagnosis of active TB. More recently, the QFT-GIT assay has been licensed for use as a diagnostic test. The latter assay is based on the use of M. tuberculosis preparations or antigens therefrom to stimulate immune cells to produce IFN-γ. Again, these tests do not discriminate between active and latent infection by mycobacteria or subjects who have previously been exposed to, or vaccinated against, infection by a pathogenic Mycobacterium.

[0009] TB is a significant threat worldwide but particularly in developing countries. With increasing immigration and relocation of peoples from high endemic regions to regions around the globe, the risk of the spread of TB is of ongoing concern. Many challenges still exist in managing TB including distinguishing between uninfected and infected individuals; distinguishing between individuals with an active infection and those with a latent infection (or who have had prior exposure to the pathogen) and assessing the impact of prior vaccination (i.e. BCG vaccination) on any immune-based diagnostic assays. Similarly, infection of cattle by Mycobacterium bovis can induce spontaneous abortions of fetal calves resulting in extensive loss to the industry. Mycobacterial infection is of significant concern to human and animal health.

[0010] It is apparent that whilst the tuberculin skin test (TST) and an assay for IFNy in an antigen-stimulated sample (e.g. the QFT-GIT assay) can on their own and with a certain level of sensitivity, distinguish between individuals who have or have not been exposed to ^' M. tuberculosis or who may have an infection, neither test can stratify an individual with respect to having an active or latent infection with any degree of certainty. Hence, there is a need to improve the sensitivity of detecting infection as well as classifying it as active or latent, not only in respect of M. tuberculosis but infection by a wide range of mycobacteria. SUMMARY

[0011 J Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO). The SEQ ID NOs correspond numerically to the sequence identifiers <400>1 (SEQ ID NO: l), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims. Abbreviations used herein are defined in Table 2.

[0012] The present disclosure is instructional for a method of stratifying a human or non- human subject with respect to prior or current exposure to a species of Mycobacterium, such as but not limited to Mycobacterium tuberculosis, or an immunological relative thereof. In an embodiment, the assay taiight herein characterizes the subject as:

(i) being currently infected by the Mycobacterium or an immunological relative thereof;

(ii) if infected, whether the infection is active or latent; and/or

(iii) if the subject has immunological memory of mycobacterial exposure or vaccination, whether the subject can exhibit immunoresponsiveness to challenge by a mycobacterial antigen. Hence, taught herein as a method of stratifying a subject with respect to exposure to a species of Mycobacterium, the method comprising selecting one or more antigens from the Mycobacterium species or an immunological relative thereof based on its or their capacity to stimulate production of effector molecules from immune cells in a fluid sample comprising immune cells from the subject in vitro, wherein the profile of the effector molecules produced characterizes the subject as:

(i) being currently infected by the Mycobacterium species or an immunological relative thereof;

(ii) if infected, whether the infection is active or latent; and/or

(iii) _ if the subject has immunological memory of Mycobacterium exposure or vaccination, whether the subject can exhibit immunoresponsiveness to challenge by the Mycobacterium or an antigen therefrom;

and determining the profile of effector molecules to thereby stratify the subject.

/ [0013] The assay enabled herein is based on selecting a mycobacterial antigen on the basis of which species or strain of Mycobacterium is to be screened and then executing an analysis of body fluid to determine the profile of effector molecules, including cytokines, stimulated by the antigen which distinguishes between infection and non-infection, active or latent infection and/or immunoresponsive or non-immunoresponsive to subsequent challenge. In this regard, enabled herein as a method of stratifying a subject with respect to exposure to a species of Mycobacterium, the method comprising selecting one or more antigens from the Mycobacterium species or an immunological relative thereof based on its or their capacity to stimulate production of effector molecules from immune cells in a fluid sample comprising immune cells from the subject in vitro, wherein the profile of the effector molecules produced characterizes the subject as: '

(ii) if infected, whether the infection is active or latent; and/or

(iii) if the subject has immunological memory of Mycobacterium exposure or vaccination, whether the subject can exhibit immunoresponsiveness to challenge by the Mycobacterium or an antigen therefrom;

and executing an analysis of the sample to determine the determine the profile of effector molecules to thereby stratify the subject.

[0014] Conveniently, the mycobacterial antigens are used in an antigen-stimulation assay conducted in a sample comprising immune cells such as lymphocytes, including T-cells, as well as macrophages. Whole blood is a particularly convenient source of fluid. In the case of M. tuberculosis, the mycobacterial antigens are selected from the list consisting of early secretory antigenic target-6 (ESAT-6), culture filtrate protein- 10 (CFP-10), tuberculosis protein 7.7 (TB7.7), purified protein derivative (PPD) and a killed M. tuberculosis preparation (MTBk). A single antigen may be employed or pools of antigens such as peptides SEQ ID NOs: l through 7 for ESAT-6, peptides SEQ ID NOs:8 through 13 for CFP-10 or peptides SEQ ID NOs:14 through 19 for TB7.7. Furthermore, combinations of different antigens may be used. [0015] ESAT-6 and CFP-10 or their equivalents are also useful antigens for virulent strains of Mycobacterium bovis as well as Mycobaterium leprae. Hence, the assay enabled herein is useful for screening for mycobacterial infection in human and non-human animals such as cattle.

[0016] As taught herein, a subject is diagnosed as having an infection by Mycobacterium, such as M. tuberculosis, or an immunologically related microorganism, when a mycobacterial antigen stimulates an elevation in one or more of IFN-γ, TNFa, IL-2, IL-13, IP- 10, IL-lra, GM-CSF, ΜΙΡ-Ιβ, IL-6, IL-8, MCP-1, MCP-3, IL-10 and/or IL-12. This is referred to herein as a "profile" of effector molecules. The profile may constitute a single or multiple effector molecules in terms of presence and/or level.

[0017] Particularly, infection is diagnosed with an elevation in IFN-γ, TNFa, IL-2, IL-13, IP- 10, IL- 1 ra, GM-CSF and/or MIP- 1 β.

[0018] Particularly, infection is diagnosed with an elevation in IFN-γ, TNFa, IL-2 and/or IL-13. [0019] Particularly, infection is diagnosed with an elevation in TNFa, especially in response to ESAT-6 or CFP- 10.

[0020] An infection is characterized as being active rather than latent if there is an elevation in IFN-γ, TNFa, IL-6, IL-lra, MCP-1, IL-10, IL-13, IP-10 and/or ΜΙΡ-1 β.

[0021] Particularly, active infection is associated with an elevation in TNFa, IL-lra, IL-6, IL-10 and/or MCP-1.

[0022] Particularly, active infection is associated with an elevation in TNFa.

[0023] Hence, a subject can be diagnosed directly with an active infection by M. tuberculosis or an immunologically-related microorganism if a mycobacterial antigen such as ESAT-6 or CFP-10 stimulates an elevation in TNFa. This applies to human and non- human subjects infected with other mycobacteria. An example of the latter is cattle infected by M. bovis.

[0024] In an embodiment, a subject is stratified as having an active M. tuberculosis infection when a Mycobacterium antigen listed in parenthesis stimulates an elevation in production of an effector molecule selected from the list consisting of TNFa (ESAT-6), TNFa (CFP-10), TNFa (MTBk), IL-lra (ESAT-6), IL-lra (PPD), IL-lra (MTBk), IL- 10 (PPD) and IL-10 (MTBk).

[0025] The ability to detect active versus latent infection and to accurately identify the causative agent, is important in protocols for the therapeutic management of subjects with an active or latent mycobacterial infection. Furthermore, a subject who has had prior exposure to Mycobacterium such as an earlier infection or by way of vaccination may or may not be immunoresponsive to a subsequent challenge to M. tuberculosis. Subjects are deemed immunoresponsive if, following exposure to ESAT-6, CFP-10, TB7.7, a peptide comprising an amino acid sequence having at lest 80% similarity to ESAT-6, CFP-10 or TB7.7, PPD or MTBk, there is an elevation in one or more of IFNa, TNFa, IL-2, IL-13, IP-10, IL-lra, GM-CSF, ΜΙΡ-Ι β, IL-6, IL-8, MCP-1 , MCP-3, IL-10 and/or IL-12, particularly IFNy, TNFa, IL-2, IL-13, IP-10, IL-lra, GM-CSF and/or ΜΙΡ-Ι β, particularly IFNy, TNFa, IL-2 and/or IL-13 and particularly, TNFa.

[0026] The assays enabled herein are useful in human and animal health maintenance. Furthermore, the assays may be conducted alone or in combination with more conventional assays such as the TST, the QFT-GIT assay and/or microscopic, culture or molecular procedures.

[0027] The effector molecules can be detected using in tube procedures such as at a point- of-care location or the assayed fluid can be screened for effector molecules at a diagnostic testing facility. 028] Kits for conducting the assays are further taught herein.

TABLE 1

Summary of sequence identifiers

SEQUENCE ID

DESCRIPTION NO:

1 Amino acid sequence - ESAT-6, PI

2 Amino acid sequence - ESAT-6, P2

3 Amino acid sequence - ESAT-6, P3

. 4 Amino acid sequence - ESAT-6, P4

5 Amino acid sequence - ESAT-6, P5

6 Amino acid sequence - ESAT-6, P6

7 Amino acid sequence - ESAT-6, P7

8 Amino acid sequence - CFP-10, PI

9 Amino acid sequence - CFP-10, P2

10 Amino acid sequence - CFP-10, P3

1 1 Amino acid sequence - CFP-10, P4

12 Amino acid sequence - CFP-10, P5

13 Amino acid sequence - CFP-10, P6

14 Amino acid sequence - TB7.7, PI

15 Amino acid sequence - TB7.7, P2

16 Amino acid sequence - TB7.7, P3

17 Amino acid sequence - TB7.7, P4

18 Amino acid sequence - TB7.7, P5

19 Amino acid sequence - TB7.7, P6 TABLE 2

Abbreviations

Abbreviation Definition

AuC Area under curve

BCG Bacille Calmette Guerin

BfA Brefeldin A

CFP-10 Culture filtrate protein 10 kD

ESAT-6 Early secretory antigenic target

IGRA Interferon gamma concentration in antigen-stimulated sample

IQR Interquartile range

LTBI Latent tuberculosis infection

MTBk Killed Mycobacterium tuberculosis

PPD Purified protein derivative

QFT-GIT QuantiFERON-TB Gold In-Tube [Cellesits Limited, Melbourne

Australia]; interferon gamma concentration in antigen-stimulated sample

ROC Receiver operating characteristic

SEB Staphylococcal enterotoxin B

TB Tuberculosis

TB7.7 Tuberculosis protein 7.7

TST Tuberculin skin test

BRIEF DESCRIPTION OF THE FIGURES

[0029] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0030] Figure 1 is a graphical representation of the distribution of TST results in the population of study subjects (children without palpable induration at the injection site excluded).

[0031] Figure 2 is a graphical representation of the distribution of TST results in children in the common discordance (TST+/IGRA-) group.

[0032] Figures 3A and 3B are graphical representations of correlation between TST result and background corrected IGRA [QFT-GIT assay]. (A) Data from the entire study population; (B) only the data from patients with a negative QFT-GIT assay result. Values exceeding 10 IU/ml are censored by the QFT-GIT ELISA system (read-out ">10 IU/ml'); these data are shown as 10 IU/ml in the figure. Not all results are visible due to overlap of identical values in multiple participants (e.g. TST induration 0 mm, interferon-gamma concentration 0.0 IU/ml).

[0033] Figures 4A through J are graphical representations of cytokine concentrations in the supernatants from whole blood assays measured by Luminex-based analysis in the seven diagnostic groups of study participants. All data are background corrected. The stimulant used is indicated on the figures. The results of the seven-group comparison by ruskal Wallis test is provided below the name of each cytokine; significant p-values (p<0.05) are highlighted in red.

[0034] Figures 5A and B are graphical representations showing statistical comparison of the background corrected cytokine responses in PPD (A) and killed MTB [MTBk] (B) stimulated samples in BCG-vaccinated (n=23) and BCG-nonvaccinated (n=49) children categorized as uninfected (shown on the left side of each graph). Significant p-values (i.e. p<0.05) are highlighted in red. Cytokine responses in the LTBI and the active TB group are shown on the right of each graph for comparison. [00351 Figures 6A and B are graphical representations of ROC curves for seven cytokines with the potential ability to discriminate TB-uninfected from TB-infected individuals. The corresponding stimulants are shown at the top of the figure. Area under curve (AUC) values are shown at the bottom left corner of each graph. [0036] Figure 7 is a graph representation showing performance of the calculated cut-off values for discriminatory cytokine/stimulant combinations in patients with active TB. Circles represent the background corrected responses in ESAT-6 stimulated samples, boxes those in CFP-10 stimulated samples, and triangles those in PPD stimulated samples. The black lines represent the medians. All y-axes are logio transformed (note that the vast majority of cytokine responses were far higher than the respective cut-offs, which is less obvious due to log transformation of the data). The cut-offs for IL- lra/ESAT-6 and IL- lra/CFP-10 cannot be shown on a log-transformed scale, as these are negative values. One cytokine response (IL-lra/CFP-10: -22.9 pg/ml) can also not be shown, but was above the respective cut-off (-23.6 pg/ml). The only response below any of the cut-offs was observed with IL-13 in a PPD stimulated sample.

[0037] Figure 8 is a graphical representation showing comparison of the TNF-a, IL-lra, IL-6, IL-10 and MCP-1 responses in children with LTBI and children with active TB in response to stimulation with ESAT-6, CFP-10, PPD or killed MTB. All data are background corrected. The black lines represent the medians. Red dotted lines represent the optimal cut-offs for the distinction between responses in patients with LTBI and responses in those with active TB (only in cytokine/stimulant combinations with comparatively high discriminatory ability). [0038] Figure 9 is a . graphical representation showing a comparison of the magnitude of cytokine responses in relation to the antigen used as stimulant in children in the LTBI and active TB groups. All data are background corrected and log transformed. The medians and interquartile range (IQR) are represented by red bars. Negative values cannot be shown on a log scale (i.e. these values are not displayed) but were included in the calculations of medians and IQR. Values related to TB7.7 were frequently negative; consequently the IQR for TB7.7 are not displayed. Medians that had negative values (which occurred with IL-lra and IL-13) are also not displayed. The blue dotted line in the graph related to interferon-gamma indicates the cut-off for a positive result in the QFT- GIT assay.

DETAILED DESCRIPTION

[0039] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any element or integer or method step or group of elements or integers or method steps. .

(0040] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells; reference to "an antigen" or "an effector molecule" includes a single antigen or effector molecule, as well as two or more antigens or effector molecules; reference to "the disclosure" includes a single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All such aspects are enabled within the width of the present invention.

[0041] Reference to an "agent", "reagent", "molecule" and "compound" includes single entities and combinations of two or more of such entities. A "combination" also includes a multi-part such as a two- or greater-part composition where the agents are provided separately and used or dispensed separately or admixed together prior to dispenzation. For example, a multi-component assay kit may comprise different mycobacterial peptide antigens for use in stimulating immune cells in a fluid sample or a cellular fraction thereof. A kit may further comprise a container adapted to receive a fluid sample containing immune cells.

[0042] The present disclosure teaches the use of peptide antigens from a species of Mycobacterium or an immunological relative thereof or a homolog or derivative or chemical analog of the antigen in the execution of an antigen-stimulation assay of fluid from a subject comprising immune cells or a cellular fraction thereof to stimulate production of effector molecules from the immune cells wherein the profile of type and/or level of the immune effector molecules is instructional as to whether:

(i) the subject is currently infected by the Mycobacterium or an immunological relative thereof;

(ii) if infected, whether the infection is active or latent; and/or

(iii) if the subject has immunological memory with respect to Mycobacterium exposure or vaccination, whether the subject is immunoresponsive to further challenge by the microorganism, its immunological relative or an antigen therefrom.

[0043] Hence, enabled herein is a method of stratifying a subject with respect to exposure to a species of Mycobacterium or an immunological relative thereof, the method comprising selecting one or more antigens from the Mycobacterium species or an immunological relative thereof or a homolog, derivative or chemical analog of the antigen or an immunological relative thereof based on its or their capacity to stimulate production of effector molecules from immune cells in a fluid sample from the subject in vitro, the profile of the effector molecules produced characterizing the subject as:

(i) being currently infected by the Mycobacterium species or an immunological relative thereof; . .

(ii) if infected, whether the infection is active or latent; and/or

(iii) if the subject has immunological memory of Mycobacterium exposure or vaccination, whether the subject can exhibit immunoresponsiveness to challenge by the

Mycobacterium antigen;

and determining the profile of effector molecules to thereby stratify the subject. By "determining the profile" includes executing a step of analyzing a body fluid sample to ascertain the level of one or more effector molecules in the sample.

(0044] A related embodiment teaches a method of stratifying a subject with respect to exposure to a species of Mycobacterium or an immunological relative thereof, the method comprising conducting a Mycobacterium antigen-stimulation assay in vitro in a fluid sample from the subject comprising immune cells or a cellular fraction thereof, the antigen selected on the basis of its capacity to stimulate production of effector molecules from cells in the fluid sample or its fraction which provides a profile of one or more effector molecules which stratifies the subject as:

(i) being currently infected by a Mycobacterium or an immunological relative thereof;

(ii) if infected, whether the infection is active or latent; and/or

(iii) if the subject has immunological memory of Mycobacterium exposure or vaccination, whether the subject can exhibit immunoresponsiveness to further challenge by the microorganism, its immunological relative or an antigen therefrom;

and then determining the profile of effector molecules to thereby stratify the subject.

[0045] The assay taught herein does not require all three aspects to be determined and the result may only be determined in respect of one or two of whether the subject is infected, if the infection is active or latent and/or if the subject is immunoresponsive. [0046] Reference to a "species" of Mycobacterium includes variants, strains and subspecies thereof. Particular species include Mycobacterium tuberculosis, Mycobacterium bovis (virulent), Mycobacterium bovis BCG, Mycobacterium africanum, Mycobacterium canetti, Mycobacterium caprae, Mycobacterium microti, Mycobacterium pinnipedii, Mycobacterium avium, Mycobacterium avium paratuberculosis, Mycobacterium avium silvaticum, Mycobacterium avium "hominissuis", Mycobacterium colombiense, Mycobacterium asiaticum, Mycobacterium gordonae, Mycobacterium gastri, Mycobacterium kansasii, Mycobacterium hiberniae, Mycobacterium nonchromogenicum, Mycobacterium terrae, Mycobacterium triviale, Mycobacterium ulcerans, Mycobacterium pseudoshottsii, Mycobacterium shottsii, Mycobacterium triplex, Mycobacterium genavense, Mycobacterium florentinum, Mycobacterium lentiflavum, Mycobacterium palustre, Mycobacterium kubicae, Mycobacterium parascrofulaceum, Mycobacterium heidelbergense, Mycobacterium interjectum, Mycobacterium simiae, Mycobact erium branderi, Mycobacterium coohii, Mycobacterium celatum, Mycobacterium bohemicum, Mycobacterium haemophilum, Mycobacterium malmoense, Mycobacterium szulgai, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium lepromatosis, Mycobacterium africanum, Mycobacterium botniense, Mycobacterium chimaera, Mycobacterium conspicuum, Mycobact erium doricum, Mycobacterium farcinogenes, Mycobacterium heckeshornense, Mycobacterium intracellular, Mycobacterium lacus, Mycobacterium marinum, Mycobacterium monacense, Mycobacterium montefwrense, Mycobacterium murale, Mycobacterium nebraskense, Mycobacterium saskatchewanense, Mycobacterium scrofulaceum, Mycobacterium shimoidei, Mycobacterium tusciae, Mycobacterium xenopi, Mycobacterium intermedium, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium bolletii, Mycobacterium fortuitum, Mycobacterium fortuitum subsp. acetamidolyticum, Mycobacterium boenickei, Mycobacterium peregrinum, Mycobacterium porcinum, Mycobacterium senegalense, Mycobacterium septicum, Mycobacterium neworleansense, Mycobacterium houstonense, Mycobacterium mucogenicum, Mycobacterium mageritense, Mycobacterium brisbanense, Mycobacterium cosmeticum, Mycobacterium parafortuitum, Mycobacterium austroafricanum, Mycobacterium diernhoferi, Mycobacterium hodleri, Mycobacterium neoaurum, Mycobacterium frederihbergense, Mycobacterium aurum, Mycobacterium vaccae, Mycobacterium chitae, Mycobacterium fallax, Mycobacterium confluentis, Mycobacterium flavescens, Mycobacterium madagascariense, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium goodii, Mycobacterium wolinskyi, Mycobacterium thermoresistibile, Mycobacterium gadium, My cobacterium komossense, Mycobacterium obuense, Mycobacterium sphagni, Mycobacterium agri, Mycobacterium aichiense, Mycobacterium alvei, Mycobacterium arupense, Mycobacterium brumae, Mycobacterium canariasense, Mycobacterium chubuense, Mycobacterium conceptionense, Mycobacterium duvalii, Mycobacterium elephantis, Mycobacterium gilvum, Mycobacterium hassiacum, Mycobacterium holsaticum, Mycobacterium immunogenum, Mycobacterium massiliense, Mycobacterium moriokaense, Mycobacterium psychrotolerans, Mycobacterium pyrenivorans, Mycobacterium vanbaalenii, Mycobacterium pulveris, Mycobacterium arosiense, Mycobacterium aubagnense, Mycobacterium caprae, Mycobacterium chlorophenolicum, Mycobacterium fluoroanthenivorans, Mycobacterium kumamotonense, Mycobacterium novocastrense, Mycobacterium parmense, Mycobacterium phocaicum, Mycobacterium poriferae, Mycobacterium rhodesiae, Mycobacterium seoulense and Mycobacterium tokaiense. This group include mycobacteria of the tuberculosis complex and non-tuberculous mycobacteria.

[0047] In an embodiment, the Mycobacterium is M. tuberculosis or an immunological relative thereof. The Mycobacterium antigen can be readily selected based on conserved or unique amino acid sequences amongst a group of Mycobacterium species. Generally, the antigen comprises a T-cell epitope. In an embodiment, the Mycobacterium antigen is ESAT-6, CFP-10, TB7.7 or purified protein derivative (PPD) from M. tuberculosis or is a killed M. tuberculosis preparation (MTBk) or an equivalent preparation from another species of Mycobacterium. For example, the antigen may have an amino acid sequence having at least about 80% similarity to one of ESAT-6, CFP-10 or TB7.7 after optimal alignment and carries a T-cell epitope. By "at least 80%" means 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%. Other mycobacteria which at least produce ESAT-6 and CFP-10 homologs include M. africanum, M. bovis (virulent strains), M. flavescens, M. gastri, M, kansasii, M. leprae, M. marinum, M. smegmatis and M. sulgai. The antigen may also be a homolog, derivative or chemical analog of a mycobacterial antigen such as ESAT-6, CFP-10 or TB7.7.

[0048] Enabled herein is a method for stratifying a subject with respect to exposure to a species of Mycobacterium, the method comprising selecting one or more mycobacterial antigens from the list consisting of ESAT-6, CFP-10, TB7.7, a peptide comprising an amino acid sequence having at lest 80% similarity to ESAT-6, CFP-10 or TB7.7, PPD and MTBk or other killed mycobacterial preparation based on its or their capacity to stimulate production of effector molecules from immune cells in a fluid sample from the subject in vitro, the profile of the effector molecules produced characterizing the subject as:

(i) being currently infected by a Mycobacterium or an immunological relative ^~ thereof;

(ii) if infected, whether the infection is active or latent; and/or

and determining the profile of effector molecules to thereby stratify the subject. [0049] Further, taught herein is a method for stratifying a subject with respect to exposure to M. tuberculosis or an immunological relative thereof, the method comprising conducting a M. tuberculosis antigen-stimulation assay in vitro in a fluid sample from the subject comprising immune cells or a cellular fraction thereof, the antigen selected from the list consisting of ESAT-6, CFP-10, TB7.7, a peptide comprising an amino acid sequence having at lest 80% similarity to ESAT-6, CFP-10 or TB7.7, PPD and MTBk and measuring a profile of levels of effector molecules, which profile stratifies the subject on the basis of:

(i) the subject is currently infected by M. tuberculosis or an immunological relative thereof;

(ii) if infected, whether the infection is active or latent; and/or

(iii) if the subject has immunological memory with respect to Mycobacterium exposure, whether the subject is immunoresponsive to further challenge by the microorganism or its immunological relative.

Taught herein is a method of stratifying a subject with respect to exposure to a species of Mycobacterium, the method comprising conducting an antigen-stimulation assay with immune cells from the subject, the antigen selected from early secretory antigenic target-6 (ESAT-6), culture filtrate protein- 10 (CFP-10), tuberculosis protein 7.7 (TB7.7), a peptide comprising an amino acid sequence having at least about 80% similarity to ESAT-6, CFP- 10 or TB7.7 after optimal alignment and carrying a T-cell epitope, purified protein derivative (PPD) and a killed M. tuberculosis preparation (MTBk) or other killed mycobacterial preparation, identifying effector molecules generated by the immune cells in response to the antigen, wherein:

(i) a subject is stratified as being infected with a species of Mycobacterium when there is an elevation in production of an effector molecule selected from the list consisting of IFN-γ, TNFa, IL-2, IL-13, IP-10, IL-lra, GM-CSF, MIP-1 B, IL-6, IL-8, MCP-1, MCP-3, IL-10 and IL-12;

(ii) a subject is stratified as having an active Mycobacterium infection when there is an elevation in production of an effector molecule selected from the list consisting of T Fa, IL-lra, IL-6, IL-10 and MCP-1 ;

(iii) a subject is stratified as being immunoresponsive to challenge by Mycobacterium when there is an elevation in production of an effector molecule selected from the list consisting of IFN-γ, TNFa, 1L-2, IL-13, IP-10, IL-lra, GM-CSF, MIP-1 B, IL- 6, IL-8, MCP-1, MCP-3, IL-10 and IL-12.

[0050] The assay of the present disclosure has both human and non-human application. In relation to non-human subjects, the diagnostic assays enabled herein have application in the livestock and poultry industries as well as the animal racing industries and in the maintenance of populations of companion animals and captured wild animals. For human applications, the assay enabled herein is useful in monitoring young children, adolescents, teenagers, adults and elderly individuals.

[0051] Reference to a "sample" includes whole blood, sputum and other respiratory fluid, tissue fluid, urine, cerebral spinal fluid and any other fluid containing immune cells (e.g. T-cells, B-cells, macrophages/monocytes). Reference to "T-cells" includes T-cells which produce effector molecules (e.g. cytokines) as well as regulatory T-cells. As indicated above, the assay involves executing a step of analyzing the content and level of effector molecules in the sample.

[0052] As indicated above, the effector molecule is generally a cytokine such as IFNy, TNFa, IL-2, IL-lra, IP-10, IL-13, GM-CSF, ΜΙΡ-Ιβ, IL-6, IL-8, MCP-1 , MCP-3, IL-10 and IL-12. [0053] As taught therein, an infected and non-infected subject can be distinguished by elevated levels of IFN-γ, TNFa, IL-2, IL-13, IP-10, IL-lra, GM-CSF, ΜΙΡ-Ιβ, IL-6, IL-8, MCP-1, MCP-3, IL-10 and/or IL-12. In an embodiment, infection is diagnosed with an elevation in IFN-γ, TNFa, IL-2, IL-13, IP-10, IL-lra, GM-CSF and/or MIP-Ι β. In a particular embodiment, the elevated effector molecules are IFN-γ, IP-10, TNFa and/or IL-13. Particularly, infection is diagnosed with an elevation in TNFa, especially in response to ESAT-6 or CFP- 10. [0054] Subjects with active or latent infection can be distinguished by elevated levels of IFN-γ, TNFa, IL-6, IL-lra, MCP-1, IL-2, IL-10, IL-13, IP-10 and/or ΜΙΡ-Ιβ. Particularly, active infection is associated with an elevation in TNFa, IL-lra, IL-6, IL-10 and/or MCP- 1. Particularly, active infection is associated with an elevation in TNFa.

[0055] In an embodiment, a subject is stratified as having an active M. tuberculosis infection when a Mycobacterium antigen listed in parenthesis stimulates an elevation in production of an^' effector molecule selected from the list consisting of TNFa (ESAT-6), TNFa (CFP- 10), TNFa (MTBk), IL- 1 ra (ESAT-6), IL- 1 ra (PPD), IL- 1 ra (MTBk), IL-10 (PPD) and IL-10 (MTBk).

[0056] Furthermore, a subject who has had prior exposure to Mycobacterium such as an earlier infection or by way of vaccination may or may not be immunoresponsive to a subsequent challenge to M. tuberculosis. Subjects are deemed immunoresponsive if following exposure to ESAT-6, CFP- 10, TB7.7, a peptide comprising an amino acid sequence having at lest 80% similarity to ESAT-6, CFP- 10 or TB7.7, PPD or MTBk, there is an elevation in one or more of IFNa, TNFa, IL-2, IL-13, IP-10, IL-lra, GM-CSF, MIP- 1β, IL-6, IL-8, MCP-1, MCP-3, IL-10 and/or IL-12, particularly IFNy, TNFa, IL-2, IL-13, IP-10, IL-lra, GM-CSF and/or ΜΙΡ-Ι β, particularly IFNy, TNFa, IL-2 and/or IL-13 and particularly, TNFa. A similar profile applies to infection by other mycobacteria such as M. bovis.

[0057] In conducting the assay, one or more of the effector molecules may be determined. The one or more effector molecules constitutes a profile in terms of which effector molecule is produced and/or the level of the molecule.

[0058] The present disclosure is therefore instructional for a method for stratifying a subject with respect to exposure to M. tuberculosis, the method comprising conducting a M. tuberculosis antigen-stimulation assay in vitro in a fluid sample from the subject comprising immune cells or a cellular fraction thereof, the antigen selected from the list consisting of ESAT-6, CFP-10, TB7.7, a peptide having an amino acid sequence with at least 80% similarity to ESAT-6, CFP-10 or TB7.7 after optimal alignment and carrying a T-cell epitope, PPD and MTBk and measuring a profile of levels of effector molecules, which profile stratifies the subject on the basis of:

(i) infected and non-infected subjects can be distinguished by elevated levels of IFN-γ, TNFa, IL-2, IL-lra, IL-6, IL-8, MCP-3, MIP-1 β and IL-10 in infected patients;

(ii) latent and active infections can be distinguished by elevated levels of TFNa, IL-lra, IL-6, IL-10 and MCP-Ι β and a decrease in IP- 10 in subjects with an active infection; and

(iii) subjects with a prior exposure to M. tuberculosis or an immunological relative thereof or who have been vaccinated are immunoresponsive to subsequent challenge when IFN-γ, IP- 10, IL-2, IL- lra, TNFa and MIP-1 β are elevated.

[0059] An embodiment of the assay taught herein includes a method for stratifying a subject with respect to infection by tuberculosis, the method comprising conducting an M. tuberculosis antigen-stimulation assay in vitro in fluid blood sample from the subject comprising immune cells or a cellular fraction thereof, the antigen selected from the list consisting of ESAT-6, CFP-10, TB7.7, a peptide having ah amino acid sequence with at least 80% similarity to ESAT-6, CFP-10 or TB7.7 after optimal alignment and carrying a T-cell epitope, PPD and MTBk and measuring an- elevation in the production of effector molecules wherein:

(i) an elevation in one or more of IFN-γ, TNFa, IL-2, IL-lra, IP- 10, IL- 13, GM-CSF, ΜΙΡ-Ιβ, MCP-3, IL-10 and/or IL-12 is indicative of infection by M. tuberculosis; and

(ii) an elevation in one or more of TNFa, IL-lra, IL-6, IL-10, MCP-Ιβ and/or

IP- 10 is indicative of active infection by M. tuberculosis.

[0060] In relation to this aspect, a subject may be regarded as not having an infection by M. tuberculosis when one or more of IFN-γ, TNFa, IL-2, IL-lra, IP-10, IL-13, GM-CSF, ΜΙΡ-Ιβ, IL-6, IL-8, MCP-1, MCP-3, IL-10 and/or IL-12 are not elevated. Similarly, a subject is diagnosed with a latent infection if one or more of TNFa, IL-lra, IL-6, IL-10, MCP- 1 β and/or IP- 10 are not elevated.

[0061] TNFci is a particularly useful marker for active infection by a mycobacterium. [0062] Further taught herein is a method for identifying a subject who is infected with tuberculosis, the method comprising conducting an M. tuberculosis antigen-stimulation assay in vitro in a fluid sample from the subject comprising immune cells or a cellular fraction thereof, the antigen selected from the list consisting of ESAT-6, CFP-10, TB7.7, a peptide having an amino acid sequence with at least 80% similarity to ESAT-6, CFP-10, TB7.7 after optimal alignment and carrying a T-cell epitope, PPD and MTBk and measuring the elevation in effector molecule production wherein an elevation in one or more of IFN-γ, TNFa, IL-2, IL-lra, IP-10, IL-13, GM-CSF, ΜΙΡ-Ιβ, IL-6, IL-8, MCP-1 , MCP-3, IL-10 and/or IL-12 is indicative of the subject having an M. tuberculosis infection. 10063] Within this profile, an elevation in TNFa, IL-lra, IL-6, IL-10, MCP-Ι β and/or IP- 10 is further indicative of an active infection. Particularly, an elevation in TNFa is indicative of active infection.

[0064] Hence, the present disclosure enables a method for identifying an individual with an active M. tuberculosis infection, the method comprising conducting an M. tuberculosis antigen-stimulation assay in vitro in a fluid sample from the subject comprising immune cell or a cellular fraction thereof, the antigen selected from the list consisting of ESAT-6, CFP-10, TB7.7, a peptide having an amino acid sequence with at least 80% similarity to ESAT-6, CFP-10 or TB7.7 after optimal alignment and carrying a T-cell epitope, PPD and MTBk and measuring the elevation in effector molecule production wherein an elevation in one or more of TNFa, IL-lra, IL-6, IL-10 and MCP-1 β and a decrease in IP-10 is indicative of the subject having an active M. tuberculosis infection. In an embodiment, TNFa is detected, such as after exposure to ESAT-6 or CFP-10.

^■ r

[0065] These aspects enabled herein equally apply to other mycobacteria such as M. bovis. [0066] The antigen- stimulation assay comprises incubating the fluid sample or a cellular fraction thereof containing lymphocytes in the presence of the Mycobacterial antigen for a time and under conditions sufficient for effector molecules to be produced. Additional agents may be included in the assay mixture including co-stimulants antibodies, heparin and carbohydrates. Regulatory T-cells which may dampen down an immune response may also be inhibited by the introduction of inhibitors. The assay may be conducted in any container such as a tube. In an embodiment the sample is whole blood or a cellular fraction thereof. [0067] The subject includes any human or non-human animal who is sensitive to infection by a particular species of Mycobacterium. For example, the subject may be a human who is infected by M. tuberculosis or M. leprae or the subject is a cow infected with M. bovis or other livestock animal, companion animal or captured wild animal infected by a mycobacterial organism.

[0068] Laboratory test animals may also be employed.

[0069] Examples of subjects include human and non-human primates, livestock animals (e.g. cattle, sheep, horses, goats, pigs), captive wild animals, companion animals (e.g. dogs, cats) and laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits). Animals include poultry birds.

[0070] Whilst whole blood is the most convenient sample, any sample comprising lymphocytes and other immune cells may be used such as lymph fluid, cerebral fluid, tissue fluid, respiratory fluid including nasal and pulmonary fluid and urine.

[0071] Blood or fluid volumes may be from 0.1 μΐ to 300 ml including from 0.5 μΐ to 20 ml. The present assay may also use acoustic microstreaming to improve mixing of components in the assay. Acoustic microstreaming is disclosed in International Patent Application No. PCT/AU01/00420 and in Petkovic-Duran et al. (2009) Biotechniques 47:827-834 and is applicable for samples comprising a small number of cells. (0072] The use of blood collection tubes compatible with standard automated laboratory systems render the assay amenable to analysis in large-scale sampling. Blood collection tubes also minimize handling costs and reduce laboratory exposure to whole blood and plasma and, hence, reduce the risk of laboratory personnel from contracting a pathogenic agent such as HIV or Hepatitis B virus (HBV) or Hepatitis C virus (HCV). In addition, depending on the assay to detect the type and/or level of effector molecule(s), the result may be obtained at a point-of-care site or in a diagnostic testing facility. [0073] The incubation step can also be combined with the collection tube.

[0074] Immune cells can lose the capacity to mount an immune response in whole blood after extended periods following blood draw from the subject. This can be obviated by conducting the antigen-stimulation assay at a point-of-care location such as in a physician's office, clinic, outpatient facility and veterinary clinic or on farms. Once antigen stimulation is complete, the requirement for fresh and active cells no longer exist. Effector molecules are stable in plasma or serum and, thus, the sample can be stored, or shipped without special conditions or rapid time requirements. [0075] The incubation step may be from 1 to 200 hours, including as 1 to 144 hours (6 days) or a time period in between including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 11, 1 12, 113, 114, 1 15, 1 16, 1 17, 118, 1 19, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 hours. A period of 16 to 24 hours is particularly convenient. [0076] The detection of the effector molecules may be measured at the protein or nucleic acid levels. Consequently, reference to "presence or level" of the immune effector molecule includes direct and indirect data. For example, high levels of cytokine mRN A are indirect data showing increased levels of the cytokine.

[0077] Ligands to the effector molecules are particularly useful in detecting and/or quantitating these molecules. Antibodies to the immune effectors are particularly useful. Techniques for the assays contemplated herein are known in the art and include, for example, radioimmunoassay, sandwich assays, ELISA and ELISpot. Reference to "antibodies" includes parts of antibodies, mammalianized (e.g. humanized) antibodies, deimmunized antibodies, recombinant or synthetic antibodies and hybrid and single chain antibodies. For skin tests, in humans, humanized or deimmunized antibodies are particularly contemplated herein to detect effector molecules.

[0078] Both polyclonal and monoclonal antibodies are obtainable by immunization with the effector molecules or antigenic fragments thereof and either type is utilizable for immunoassays. Methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the immune effector, or antigenic part thereof, collecting serum from the , animal and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

[0079] The use of monoclonal antibodies in an immunoassay is particularly useful because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. [0080] Another aspect enabled herein, therefore, is a method for detecting an immune effector molecule in supernatant fluid of a sample comprising lymphocytes from a subject which was subject to a mycobacterial antigen-stimulation assay, the method comprising contacting the supernatant fluid or an aliquot thereof with an antibody specific for the effector molecule or an antigenic fragment thereof for a time and under conditions sufficient for an antibody-effector complex to form, and then detecting the complex.

[0081] By "detecting" means quantitating or semi-quantitating. A "sample" includes whole blood or a fraction thereof or other fluid comprising lymphocytes and other immune cells which is used to conduct the antigen-stimulation assay. Generally, the supernatant fluid is screened for effector molecules. This method includes micro-arrays, macro-arrays and nano-arrays on planar or spherical solid supports. A micro- or macro-array is useful. A "sample" also includes a small volume sample of from about 0.5μ1 to ΙΟΟΟμΙ including 5μ1, Ι ΟμΙ, 20μ1, 50μ1 and ΙΟΟμΙ as well as larger volumes such as from 1 ml to about 200 ml such as 1 ml, 2 ml, 5 ml, 10 ml or 20 ml.

[0082] A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. [0083] The following is a description of one type of assay. An unlabeled antibody is immobilized on a solid substrate and the sample to be tested for the profile of effector molecules brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-effector molecule complex, a second antibody specific to the effector molecule, labeled with a reporter molecule capable of producing a detectable signal, is then added and incubated, allowing time sufficient for the formation of another complex of antibody-effector-labeled antibody. Any unreacted material is washed away, and the presence of the effector molecule is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of antigen. This generalized technique is well known to those skilled in the art as would be any of a number of variations.

[0084] In these assays, a first antibody having specificity for the instant effector molecule is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, spheres, discs of microplates, or any other surface suitable for conducting an immunoassay. Beads are particularly useful, especially in the context of a multiplex assay. The binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-120 minutes or where more convenient, overnight) and under suitable conditions (e.g. for about 20°C to about 40°C) to allow binding of effector molecule to the antibody. Following the incubation period, the antibody solid phase is washed and dried and incubated with a second antibody specific for a portion of the effector molecule. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the effector molecule. [0085] There are many variations to this assay. One particularly useful variation is a simultaneous assay where all or many of the components are admixed substantially simultaneously. Furthermore, binding of an antibody to a cytokine may be determined by binding of a labeled antibody directed to the first mentioned antibody. [0086] By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative/ The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules. Examples of suitable fluorophores are provided in Table 3. In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody-antigen complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen- antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample. Again, the present disclosure extends to a substantially simultaneous assay.

[0087] Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.

The fluorescent labeled antibody is allowed to bind to the first antibody-antigen complex.

After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the antigen of interest. Immunofluorescene and enzyme immunoassay techniques are both very well established in the art and are particularly preferred for the present method.

However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed. [0088] There is a range of other detection systems which may be employed including colloidal gold and all such detection systems are encompassed by the present disclosure. [0089] The present disclosure also contemplates genetic assays such as involving PCR analysis to detect RNA expression products of a genetic sequence encoding an immune effector. -

[0090] In one embodiment, PCR is conducted using pairs of primers, one or both of which are generally labeled with the same or a different reporter molecule capable of giving a distinguishable signal. The use of fluorophores is particularly useful in the practice of the present disclosure. Examples of suitable fluorophores may be selected from the list given in Table 3. Other labels include luminescence and phosphorescence as well as infrared dyes. These dyes or fluorophores may also be used as reporter molecules for antibodies.

TABLE 3

Ex: Peak excitation wavelength (nm) Em: Peak emission wavelength (nm) [00911 Any suitable method of analyzing fluorescence emission is encompassed herein. In this regard, techniques taught herein include but are not restricted to 2-photon and 3- photon time resolved fluorescence spectroscopy as, for example, disclosed by Lakowicz et al. (1997) Biophys. J. 72:567, fluorescence lifetime imaging as, for example, disclosed by Eriksson et al. (1993) Biophys. J. 2:64 and fluorescence resonance energy transfer as, for example, disclosed by Youvan et al. (1997) Biotechnology et elia J: 1-18.

[0092] Luminescence and phosphorescence may result respectively from a suitable luminescent or phosphorescent label as is known in the art. Any optical means of identifying such label may be used in this regard.

[0093| Infrared radiation may result from a suitable infrared dye. Exemplary infrared dyes that may be employed in the present disclosure include but are not limited to those disclosed in Lewis et al. (1999) Dyes Pigm. 42(2) Λ9Ί, Tawa et al. Mater. Res. Soc. Symp. Proc.488 [Electrical, Optical and Magnetic Properties of Organic Solid-State Materials IV], 885-890, Daneshvar et al (1999) J. Immunol. Methods 226(1-2): 1 19-128, Rapaport et al. (1999) Appl. Phys. Lett. 74(3):329-33 \ and Durig et al. (1993) J. Raman Spectrosc. 24(5).281-285. Any suitable infrared spectroscopic method may be employed to interrogate the infrared dye. For instance, fourier transform infrared spectroscopy as, for example, described by Rahman et al, J. Org. Chem. 63:6196, 1998 may be used in this regard.

[0094) Suitably, electromagnetic scattering may result from diffraction, reflection, polarization or refraction of the incident electromagnetic radiation including light and X-rays. Such scattering can be used to quantitate the level of mRNA or level of protein.

[0095] Flow cytometry is particularly useful in analyzing fluorophore emission. [0096] As is known in the art, flow cytometry is a high throughput technique which involves rapidly analyzing the physical and chemical characteristics of particles (e.g. labeled mRNA, DNA or proteins) as they pass through the path of one or more laser beams while suspended in a fluid stream. As each particle intercepts the laser beam, the scattered light and fluorescent light emitted by each cell or particle is detected and recorded using any suitable tracking algorithm.

[0097] A modern flow cytometer is able to perform these tasks up to 100,000 cells/particles s^"1. Through the use of an optical array of filters and dichroic mirrors, different wavelengths of fluorescent light can be separated and simultaneously detected. In addition, a number of lasers with different excitation wavelengths may be used. Hence, a variety of fluorophores can be used to target and examine, for example, different immune effectors within a sample or immune effectors from multiple subjects.

[0098J Suitable flow cytometers which may be used in the methods of the present disclosure include those which measure five to nine optical parameters (see Table 4) using a single excitation laser, commonly an argon ion air-cooled laser operating at 15 mW on its 488 nm spectral line. More advanced flow cytometers are capable of using multiple excitation lasers such as a HeNe laser (633 nm) or a HeCd laser (325 nm) in addition to the argon ion laser (488 or 514 nm).

TABLE 4

Exemplary optical parameters which may be measured by a flow cytometer.

using a 488 nm excitation laser

† width of bandpass filter

# longpass filter [0099] The assay enabled herein may be automated or semi-automated for high throughput screening or for screening for a number of immune effectors from the one subject. The automation is conveniently controlled by computer software. [0100] The present disclosure further contemplates therefore web-based and non-web- based systems where data on the profile of effector molecules produced in response to mycobacterial antigen stimulation of a sample from a subject are provided by a client server or other architecture platform to a central processor which analyses and compares to a control and optionally considers other information such as patient age, sex, weight and other medical conditions and then provides a report, such as, for example, a risk factor for infection severity or progression or status or an index of probability of infection development including whether the infection is active or latent. A business method is therefore also provided whereby blood or other fluid is collected in transportable tubes which is then analyzed for effector molecule profile after mycobacterial antigen stimulation at a defined location and the results then sent in the form of an electronic report via a client server or other architecture platform to a clinical care provider.

[0101] Hence, knowledge-based computer software and hardware also form part of the present disclosure. This facilitates clinical care to ascertain whether a mycobacterial disease condition including stage of infection.

[0102] In particular, the assays enabled by the instant disclosure may be used in existing or newly developed knowledge-based architecture or platforms associated with pathology services. For example, results from the assays are transmitted via a communications network (e.g. the internet) or telephone connection to a processing system in which an algorithm is stored and used to generate a predicted posterior probability value which translates to the correlation between effector molecule profile and stage of infection which is then forwarded to an end user in the form of a diagnostic or predictive report. This report may also form the basis of clinical care management and personalized medicine.

[0103] In accordance with this embodiment, levels of the immune effector molecule may be screened alone or in combination with other biomarkers or disease indicators such as the TST or a QFT-GIT assay as well as microscopic, cell culture and/or molecular assays.

[0104] As indicated above, the term "sample" as used herein means any sample containing one or more lymphocytes including, but not limited to, whole blood, a whole blood fraction, tissue extracts and freshly harvested cells as well as other fluid sample.

[0105] Contacting the chosen biological sample with the antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primar immune complexes) is generally a matter of adding the composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e. to bind to, any effector molecules present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, ELISpot, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[0106] In a further embodiment, the present disclosure enables kits for use with the methods described above. In one embodiment, an immunodetection kit for effector molecules is contemplated. In another embodiment, a kit for analysis of a sample from a subject having or suspected of developing TB or other mycobacterial disease condition. In a more particular embodiment, a kit for analysis of a sample from a subject having or suspected of developing a mycobacterial disease is contemplated. In an embodiment, a kit is for assessing whether a subject has an active or latent mycobacterial infection.

[0107] The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody or antigen, and detectable labels that are associated with or attached to a secondary binding ligand. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody or antigen, and secondary antibodies that have binding affinity for a human antibody. (0108] Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody or antigen, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label.

[0109] The kits may further comprise a suitably aliquoted composition of antigen or effector molecule, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay.

[0110] The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.

[0111] The container means of any of the kits generally includes at least one vial, test tube, flask, bottle, syringe or other container means, into which the testing agent, the antibody or antigen may be placed, and generally, suitably aliquoted. Where a second or third binding ligand or additional component is provided, the kit will also generally contain a second, third or other additional container into which this ligand or component may be placed. The kits taught by the present disclosure also typically include a means for containing the antibody, peptides derived from an antigen and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[0112] The present disclosure further teaches a method of treatment of a subject having a mycobacterial infection or suspected of having such an infection or is at risk of developing mycobacterial infection, the method comprising contacting a source of lymphocytes from the subject with a mycobacterial antigen and measuring the presence or elevation in the level of an immune effector molecule from immune cells wherein the presence or level of the immune effector molecule is indicative of active or latent infection and then selecting a suitable treatment regime. The assay enabled herein is also useful for monitoring the effectiveness of a treatment regime. The subject may be a human or non-human subject.

[0113] The instant disclosure also teaches the use of an effector molecule profile generated in response to stimulation of immune cells by a mycobacterial antigen in the manufacture of a diagnostic assay to distinguish between active and latent infection by a species of Mycobacterium. The Mycobacterium may be any of the species listed herein include M. tuberculosis and M. bovis.

EXAMPLES

[0114] Aspects disclosed herein are further described by the following non-limiting Examples. The Examples determine cytokine concentrations in supematants from whole blood assays following execution of an analysis.

MATERIALS AND METHODS

Tubes and plates

· 7.5 ml sodium-heparin blood collection tubes (Sarstedt, Lot 6299002)

• 1 ml sterile microtubes (Sarstedt, 72.693.005)

• 1.5 ml microtubes (Eppendorf , 0030 120.086)

• 2 ml sterile glass vials with caps (MP Biomedials, 096026099)

• 24-well plate, flat bottom (Nunclon surface, 142475)

· 96-well filter plate (Millipore, MX-plate)

Equipment and instruments

• Biological safety cabinet class II (Clyde-Apac, BH2000)

• Centrifuge (Eppendorf, Model5424)

· C0₂-incubator (Forma Scientific)

• Multipipette (Eppendorf Mulitpipette plus)

• Pipette aid (Integra Bioscience Pipetboy acu)

• Pipettes (Gilson: 21, 201, 100 1, 10001)

• Pipettes (Eppendorf: 2001, 1000 1)

· Refrigerator (Kelvinator, Model390)

• - 20 °C freezer (Westinghouse, Model Silhouette 393)

• -80 °C freezer (Thermo Fisher Scientific, Model905)

• Sonicating water bath (Unisonic, Model FXP4)

• Vacuum (Laboport Mini Laboratory Pump, N 816.1.2.KN.18)

· Vacuum Manifold (Millipore, Multiscreen HTS) • Vortex Mixer (Ratek Instruments, Model VM1)

• Vortex Mixer (Hwashin Technologies, Model250 VM)

• Water bath (Ratek Instruments, Model WB4)

• Multiplex Bio-Analyser (Luminex, Model Luminex 200)

REAGENTS Standard reagents

• Deionized sterile water (Milli-Q water; purified with Millipore system)

· Phosphate buffered saline (PBS) solution (Ambion, 9625)

• Dimethyl-sulfoxide (Calbiochem, 317275)

• Roswell Park Memorial Institute (RPMI) + L-glutamine medium (GIBCO, 1 1875-085)

• Ethylenediaminetetraacetic acid 0.5M (EDTA) (Sigma, E-7889)

• Bovine serum albumin (BSA) (Sigma, A-4503, Lots 079 1453 and 1 10M1310V) · Sodium azide (NaN3) (BDH Chemicals Ltd, 301 11)

Reagents for stimulation assays

• anti-CD28 (BD, 340975)

• anti-CD49d (BD, 340976)

· Brefeldin A (BfA) (Sigma Aldrich, B-7651)

Antigens for in vitro stimulation

• Staphylococcal enterotoxin B(SEB) (Sigma Aldrich, S-4881)

• Early secretory antigenic target (ESAT-6) peptide pool (JPT Peptide

Technologies, Berlin, Germany)

• Culture filtrate protein θ (CFP-10) peptide pool (JPT Peptide Technologies, Berlin, Germany)

• Rv2654 (TB7.7) peptide pool (JPT Peptide Technologies, Berlin, Germany)

• Purified protein derivative (PPD) (Statens Serum Institute, Copenhagen, Denmark, batch RT 50, Lot 219, Cat. no. 2390) • Killed Mycobacterium tuberculosis, (Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Australia)

[0115] The M. tuberculosis-specific peptide pools are defined in Tables 5 to 7.

TABLE 5

ESA T-6 Peptide Pools

Amino acid sequence SEQ ID NO:

ESAT-6 P1 MTEQQWNFAGIEAAASAIQG 1

P2 GIEAAASAIQGNVTSI 2

P3 SAIQGNVTSIHSLLDEGKQSLTKLA 3

P4 EG QSLTKLAAAWGGSGSEAYQGVQ 4

P5 SGSEAYQGVQQKWDATATELNNALQ 5

P6 TATELN ALQNLARTISEAGQAMAS 6

P7 NLARTISEAGQAMASTEGNVTGMFA 7

TABLE 6

CFP-10 Peptide Pools

Amino acid sequence SEQ ID NO:

CFP-10 PI MAEMKTDAATLAQEAGNFERISGDL 8

P2 GNFERISGDLKTQIDQVESTAGSLQ 9

P3 DQVESTAGSLQGQWRGAAGTMQAAV 10

P4 AAGTAAQAA WRFQEAANKQKQELD 11

P5 AANKQKQELDEISTNIRQAGVQYSR 12

P6 IRQAGVQYSRAD E EQQQALSSQMG F 13

TABLE 7

TB 7.7 Peptide Pools

Amino acid sequence SEQ ID NO:

TB7.7 P1 MSGHALAARTLLAAADEL 14

P2 AADEL VGGPPVEASAAAL 15

P3 ASAAALAGDAAGAWRTAA 16

P4 AWRTAA VELARALVRA VA 17

P5 LVRA VAESHG VAA VLFAA 18

P6 VLFAA TAAAAAA VDRGDPP 19

Luminex xMAP bead-based cytokine analysis

[0116] For the detection of cytokines in supernatants Millipore human cytokine/chemokine kits (MPXHCYTO -60K) were used. The Luminex xMAP technology is based on the use of microspheres that are color-coded with two 'internal' fluorescent dyes. This method allows the detection of up to one hundred analytes in a single multiplexed assay.

[0117] The microspheres (or beads) are coated with antibodies to a specific analyte (e.g. antihuman interferon-y antibody). If the analyte is present in the sample being assayed it is captured by the microsphere/antibody complex. In the next step a biotinylated detection antibody is added. The reaction mixture is then incubated with streptavidin-PE conjugate (reporter molecule), completing the reaction on the surface of each microsphere. Following a washing step the reaction mixture is analyzed in the Luminex 200 Bio-Analyser, which incorporates two lasers emitting light of different wavelengths. The first laser excites the internal dyes, allowing the determination of the bead identification number (e.g. the identification number corresponding to interferon-y). The second laser excites PE (i.e. the fluorescent dye on the reporter molecule), thereby allowing the determination of the amount of analyte bound to the micro sphere.

[0118] This Milliplex human cytokine/chemokine kits contain the following reagents: · Human cytokine/chemokine standard (MXH8060)

• Human cytokine quality controls 1 and 2(HXH6060)

• Serum matrix (MXHSM)

• Assay buffer (L-AB)

• 10 x wash buffer (L-WB)

· Human cytokine detection antibodies (MXH1060-1, 2, 3 or 4)

• Streptavidin-Phycoerythrin (L-SAPE3, 9, 10 or 1 1)

• Bead diluents (LBD)

• Cytokine/chemokine antibody-conjugated beads (Table 10) TABLE 8

Antibody-conjugated beads used in the Examples

Software

· Word 2008 for Mac (Microsoft, Redmond, Washington, USA)

• EndNote X2 (Thomson Corp., Stamford, Connecticut, USA)

• Excel2008 for Mac (Microsoft, Redmond, Washington, USA)

• PowerPoint 2008 for Mac (Microsoft, Redmond, Washington, USA)

• Photoshop CS4 Extended, version 11.0 (Adobe, San Jose, California, USA) · xPonent, version 3.1 (Luminex , Austin, Texas, USA)

■ · Stata 1 1 (StataCorp, College Station, Texas, USA)

• Prism 5, version 5.0c (GraphPad Software Inc., La Jolla, California, USA) STIMULATION ASSAYS

Whole blood intracellular cytokine assays Reagent and antigen preparation

Co-stimulatory antibodies

[0119] The co-stimulatory antibodies anti-CD28 and anti-CD49d were provided in 200 μΐ vials at a concentration of 1 mg/ml by the manufacturer. The vials were stored in a fridge at 2-6°C. On the day of the assay each co-stimulatory antibody was diluted 1 :20 with IxPBS (5 μΐ of each antibody added to 90 μΐ of IxPBS). For the stimulation assay 10 μΐ of the diluted antibody mix was added to each sample, resulting in a final concentration of 1 μg/ml of each antibody in the sample.

Staphylococcal enterotoxin B

[0120] Lyophilized staphylococcal enterotoxin B(SEB) was provided in 1 mg vials by the manufacturer. The contents was reconstituted with 1 ml of IxPBS to achieve a stock solution with a concentration of 1 mg/ml. This solution was divided into 25 μΐ aliquots and stored at - 20 °C. For use in the assay the aliquot was thawed and diluted with 225 μΐ of IxPBS. Of this solution 25 μΐ was added to the positive control sample, resulting in a final concentration of 5 μg/ml.

Mycobacterium tuberculosis-specific antigens

[0121] The custom-synthesized peptide pools of ESAT-6, CFP-10 and TB7.7 were provided freeze-dried in light-protected glass vials containing 25 μg of each peptide (ESAT-6: seven peptides; CFP-10 and TB7.7: six peptides) by the manufacturer. The vials were stored in a fridge at 2- 6°C. On the day of the assay the peptides were reconstituted with 20 μΐ of DMSO followed by vortexing to aid dissolution. Then 230 μΐ of I PBS was added, followed by further vortexing. Immediately prior to use the vials were sonicated for 30 seconds with 80 mHz. In the stimulation assays 50 μΐ of the reconstituted peptide pool was added to the sample, resulting in a final concentration of 10 μg/ml. Purified protein derivative

[0122] PPD was provided in 10 ml vials at a concentration of 1 mg/ml by the manufacturer. The contents was aliquoted into sterile glass vials (200 μΐ aliquot per vial) and stored at 2-6°C until use. In the stimulation assays 10 μΐ of PPD was added to the sample, resulting in a final concentration of 20 μ^πιΐ.

Killed Mycobacterium tuberculosis (MTBk)

[0123] Dissolved killed M. tuberculosis MTBk) was provided as 50 ml stock solutions at a concentration of 1 x 10⁸ CFU/ml. The contents were divided into 1 ml aliquots and stored at -80°C. Aliquots were thawed when needed and sonicated for 30 seconds with 80 mHz. Thawed aliquots were stored at 2-6°C for up to one month. Immediately prior to use in the assay the aliquot was once again sonicated for 30 seconds with 80 mHz. In the stimulation assays 16 μΐ of the solution was added to the sample. Ethylenediaminetetraacetic acid

[0124] EDTA was provided as a 500 mM stock solution and stored at room temperature (RT). A working solution was made by diluting the stock solution 1 :25 with l xPBS, resulting in a 20 mM solution, which was stored at 2-6°C. In the assays 40 μΐ of EDTA working solution was added to each sample, resulting in a 2 mM final concentration.

Initial incubation of whole blood samples

[0125] Subjects' blood was collected into sodium-heparin tubes and processed within 4 hours. All steps of the assay were performed in a laminar flow biological safety cabinet to avoid potential contamination. First the blood was transferred into nine labeled, sterile Sarstedt microtubes (500 μΐ per tube), followed by the addition of the co-stimulatory antibodies anti-CD28 (1 g ml) and anti-CD49d (1 μg ml) to each tube. Three samples were left unstimulated (one negative control, two samples later only partially stained, 'fluorescence-minus-one [FMO]' samples). The remaining samples were stimulated with SEB (positive control; stimulant added after initial incubation) or with mycobacterial antigens (ESAT-6, CFP-10, TB7.7, PPD or MTBk). This was followed by incubation in a water bath at 37°C for 20 - 24 hours. The following concentrations were used in the assays: SEB (5 μ^ταΐ) (ESAT-6, CFP-10 and TB7.7 (each at 10 μ πύ), PPD (20 μg/ml), MTBk (3.2 x 10^s CFU/ml).

Collection of supernatant and second incubation period

[0126] After the initial 20-24 hours of incubation, the microtubes were removed from the water bath. At this stage cells had settled at the base of the microtubes and a clearly separated layer of supernatant was visible at the top. Part of the supernatant (100 μΐ per sample) was removed and transferred into a 1.5 ml Eppendorf tube, followed by cryopreservation at -80°C.

Analysis of Supernatants

[0127] Cryopreserved supernatants were analyzed for the presence and levels of cytokines. Table 9 provides an overview of the properties of the cytokines included in the panel.

Preparation of supernatants

[0128] Cryopreserved supernatants were removed from the -80°C freezer and thawed at RT. Due to the fact that pilot experiments indicated that several cytokines were frequently present in concentrations exceeding the upper limit of detection (or dynamic range) of the Milliplex assays all supernatants were tested undiluted (neat) and in 1 :20 dilution. The following cytokines were analyzed undiluted in a 10-plex: IFN-γ, TNF-a, IL-lra IL-2, IL- 10, IL-12 (p40), IL-13, IL-15, IL-17, GM-CSF. The following cytokines were diluted 1 :20 with assay buffer and analyzed in a 7-plex: IP- 10, IL-6, IL-8, MCP-1, MCP-3, MIP 1-β, RANTES. Despite this approach in rare instances cytokine concentrations exceeded the upper limit of detection, which almost exclusively occurred with IP- 10 and RANTES. In these instances the respective sample was re-analyzed after 1 :50 dilution with assay buffer.

Preparation of immunoassay reagents

[0129] The vials containing the antibody -conjugated beads were removed from the kit, sonicated for 30 seconds in a waterbath, and vortexed at high speed while inverting the vials repeatedly for another 60 seconds. From each vial 80 μΐ was added to the mixing bottle from the kit, and brought to a final volume of 4 ml using the bead diluent. Quality controls (QC 1 and QC2) were reconstituted with 250 μΐ deionized water. The lOx assay wash buffer was diluted 1 :10 by adding 30 ml of wash buffer to 270 ml deionized water. The serum matrix was reconstituted by adding 1 ml of deionized water to the bottle containing lyophilized serum matrix.

Preparation of human cytokine standards

[0130] The cytokine standard was reconstituted with 250 μΐ of deionized water to give a concentration of 10,000 pg/ml. Then serial dilutions were prepared. Five Eppendorf tubes were labeled and 200 μΐ of assay buffer was added to each tube. Serial dilutions were achieved by adding 50 μΐ of the reconstituted standard to the 2,000 pg/ml tube. After vortexing, 50 μΐ of the 2,000 pg/ml standard was added to the 400 pg/ml tube. The same sequence of steps was performed until the last serial dilution (3.2 pg/ml) was reached. Immunoassay procedure

[0131] The assay filter plate was pre-wetted by adding 200 μΐ of assay buffer to each well. The plate was sealed, followed by mixing on a plate shaker for 10 minutes. Then the assay buffer was removed by vacuum, before adding 25 μΐ of each standard and the controls to their respective wells. Then 25 μΐ of assay buffer was added to 'background' and sample wells. Then 25 μΐ of supernatant was added to the respective sample wells, followed by adding 25 μΐ of serum matrix to background, standards and control wells. The mixing bottle with premixed beads was vortexed again for 60 seconds, and 25 μΐ of bead solution was added to each well. Then the assay plate was sealed, covered with a lid and allowed to incubate on a plate shaker for 60 minutes at RT.

EXA PLE 1

Methodology

Study participants

(0132) Children and adolescents up to 18 years of age were recruited at the Royal Children's Hospital (RCH), Melbourne, Australia. Eligible for study participation were all children undergoing screening for suspected LTBI or active TB. This comprised the following: (i) children with symptoms and signs suggestive of active TB (for example, but not limited to, persistent fever, persistent cough, weight loss, or night sweats), (ii) children with known TB contact, (iii) children who had recently migrated from countries with high TB prevalence. The exclusion criteria comprised the following: known immunodeficiency (congenital or acquired), current treatment with immunosuppressive medication (including oral steroids), or undergone TST between 6 to 52 weeks prior to presentation. (0133] Children were recruited from the inpatient setting (mainly those with suspected active TB), as well as the outpatient setting. The majority of patients were children with recent contact to an infectious case of active tuberculosis.

[0134] Demographic information, history and clinical findings were recorded on a standardized data collection sheet.

Diagnostic tests and samples for analysis

[0135] All participants underwent a tuberculin skin test (TST). For this test 0.1 ml of Tubersol (Sanofi Pasteur, Toronto, Ontario, Canada; bioequivalent to 5 Tuberculin Units PPD-S) was injected intradermally into the volar surface of the lower arm, and the result , (the diameter of induration at the injection site) was read after 48 to 72 hours by a healthcare professional specifically trained for this task. In addition, blood was obtained for the QFT-GIT (QuantiFERON-TB Gold In-Tube [Cellestis Limited, Melbourne, Australia]) assay, and an additional 10 ml for research tests were obtained into blood collection tubes containing sodium heparin. The blood collection tubes was processed within 4 hours. Categorization of participants

[0136] For the purpose of the study, a positive TST was defined as an induration of equal to or greater than 10 mm at 48 to 72 hours. The QFT-GIT assay results were interpreted according to manufacturer's guidelines (QFT-GIT package insert, Doc. No. CA05990301 A; Cellestis, Melbourne, Australia).

[0137] Participants were categorized according to their TST and IGRA results, as outlined in Table 10. Active TB was defined as either: (i) microbiological confirmation of infection with M. tuberculosis by culture or polymerase chain reaction (PCR); or (ii) a symptomatic patient fulfilling at least three of the following four criteria: (a) symptoms and signs consistent with active TB (chronic cough, persistent fever, night sweats, unexplained weight loss), (b) radiological findings suggestive of active TB, (c) presence of risk factors for TB infection (known TB contact, birth or previous residence in a country with high TB prevalence), and (d) response to treatment with anti-tuberculosis medication.

[0138] These stringent criteria for categorization were used to create unambiguous diagnostic groups, in order to reduce contamination of data by inclusion of patients with borderline result (e.g. children with a TST induration between 1 and 4 mm in diameter into the uninfected group).

TABLE 10

Diagnostic categorization of participants used in the Examples

Diagnostic category : TST | QFT-GIT Other criteria

induration . assay result j

Uninfected ; 0 mm ; Negative

Probable uninfected ' 1 - 4 mm Negative

Possible discordance ; 5 - 9 mm Negative Common discordance !≥ 10 mm Negative LTBI ≥ 10 mm Positive

Active TB : Microbiological confirmation and/or

presence of 3 of 4 criteria (irrespective of

I TST and IGRA result; see text above)

Reverse discordance ≤ 10 mm : Positive

Whole blood assays

[0139] The whole blood assays were performed as described in detail above. Briefly, following incubation of whole blood samples with mycobacterial antigens (ESAT-6, CFP- 10, TB7.7, PPD, and killed MTB), or staphylococcal enterotoxin B (positive control), or without stimulant (nil control) in the presence of co-stimulatory antibodies for 16 to 20 hours, 100 μΐ supernatant was harvested from each of the seven samples. Supernatants were then frozen at -80°C for batched analysis, and thawed immediately prior to analysis in the Luminex bead-based illiplex human cytokine assays.

Luminex cytokine analysis

[0140] The Milliplex assay protocol used to measure cytokine concentrations in supernatants is described in detail above. Two separate assay kits were used: (i) a 10-plex for IFN-γ, IL-lra, IL-2, IL-10, IL12(p40), IL-13, IL-15, IL-17, GM-CSF, and TNF-a for neat supernatants; and (ii) a 7-plex for IL-6, IL-8, IP-10, MCP-1 , MCP-3, ΜΙΡ-Ι β, and RANTES for supernatants diluted 1 :40 with assay buffer. Concentrations in diluted samples were automatically adjusted (by multiplying measured concentrations by a factor of 40) by the xPonent analytical software of the Luminex 200 Bio-Analyser used for acquisition.

Statistical analysis

|0141] Comparisons of multiple groups were done by using non-parametric ruskal Wallis tests using the statistical packages Stata and Prism. Categorical data were compared by using two-tailed chi square tests. A p-value < 0.05 was considered significant. Since the groups 'probable uninfected', 'possible discordance' and 'reverse discordance' were too small to allow meaningful statistical comparisons they were only included in the initial statistical analysis. The remaining four groups ('uninfected', 'common discordance', 'LTBI' and 'active TB') were analyzed in two-group comparisons using non-parametric Mann Whitney U tests, in all instances where the p-value calculated by the Kriiskal Wallis test was < 0.05, indicating that there was a difference between the groups. All figures were constructed with Prism and Excel.

EXAMPLE 2

Demographic and historical studies

[0142] A total of 140 patients were used in the study.

[0143] The number of participants in each diagnostic category, the demographic data and details related to history are shown in Table 1 1.

^T¾(clu¾eTrn¾rante from ew 2eaiani^" Aibrevitio^'nsfno^' : numfter; IQR^" interquartiTe range

[0144] As shown in Table 1 1, the median age differed significantly between the diagnostic groups. The median age was highest in the active TB group (15.0 years), followed by the discordant group and the LTBI group (12.1 and 1 1.6 years, respectively).

[0145] Regarding ethnic background the commonest countries of the families' origin were Somalia (n=33), India (n=14), the Philippines (n=12), Ethiopia (n=10) and New Zealand (n=10). Among patients with discordance (TST+/IGRA-; n=28) Asians represented the largest group (n=1 ; 46.4%); among patients with LTBI (n=16) Africans represented the largest group (n= 10; 62.5%); among cases of active TB (n=6) Africans represented the clear majority (n=5 ; 83.3%).

[0146] Almost half (42.1%) of the study participants were born in Australia or New Zealand. The second most common region of birth was Africa (29.3%), followed by Asia (20.7%). While there was no significant difference between the diagnostic groups regarding ethnic background (p=0.1812), there was a significant difference regarding the region of birth (pO.OOOl). The most notable difference was that the majority of LTBI cases and active TB cases were born in Africa (62.5% and 83.3%, respectively), _. while participants born in Africa only represented a minority in the uninfected group (18.7%).

[0147] A total of 57.9% of the study participants had a migration background. The proportion of participants with migration background was highest in the LTBI and the active TB group (93.8% and 100%, respectively), and lowest in the uninfected group (37.3%).

[0148] In the entire study population 53.6% of participants had a history of prior BCG vaccination. In the groups with common discordance, LTBI and active TB the majority of children had a history of BCG vaccination (75.0%, 87.5%, and 83.3%, respectively), and/or had a BCG scar in the deltoid region on examination (64.3%, 75.0%, and 83.3%, respectively). [0149] Almost two thirds (63.6%) of the study participants had a history of recent TB contact. The most common type of TB contact was a parent with active TB (24.3%), followed by a household contact (23.6%). Contact with a case of active TB outside the household was less common (15.7%).

[0150] Twelve (8.6%) children had clinical signs or symptoms compatible with active TB. Six of these were diagnosed with active TB; six had an alternative final diagnosis. All children with an alternative diagnosis had been born in Australia, but had different ethnic backgrounds, as shown in Table 12. None of these six children received anti-tuberculous therapy. The three patients with presumed bacterial infections recovered on treatment with 'conventional' antibiotics; the patient with presumed viral pneumonia recovered without specific treatment. In both patients diagnosed with malignancies (Langerhans cell histiocytosis and Hodgkin lymphoma) the diagnosis was based on lymph node histology.

- 58 - TABLE 12

Details of patients with signs or symptoms compatible with active TB in whom an alternative final diagnosis was made

[0151] Of the six patients diagnosed with active TB, only one had a known recent contact with a case of active TB, as shown in Table 13. The index case in this instance was a parent with sputum-smear positive pulmonary tuberculosis. All six patients were born in countries with high TB prevalence (i.e. a prevalence exceeding 40/100,000 inhabitants). Two patients had pulmonary TB, two had lymph node TB, one had TB of the cervical spine, and one presented with a chest wall tumor. The latter is a very unusual case, but confirmation was achieved by culture and PCR of bioptic material. In this case, and the two cases with cervical lymphadenopathy, histology showed caseating granulomata. All six patients were started on triple (rifampicin, isoniazid, pyrazinamide) or quadruple (the former three drugs plus ethambutol) anti-tuberculous treatment, and all made a full recovery. TABLE 13

Details of patient diagnosed with active TB

EXAMPLE 3

Tuberculin skin test (TST) and QFT-GIT test in study subjects

[0152] A total of 77 patients showed no TST induration at 48 to 72 hours, comprising the 75 participants in the uninfected group, and two in the reverse discordance group. The TST results of the remaining 63 study participants are shown in Figure 1. A total of 50 patients had positive TST results (defined as induration > 10 mm); of these, 31 patients had indurations > 15 mm (a criterion used by some other studies). The 50 TST-positive patients were distributed among the diagnostic groups as follows: 28 in the discordant group, 16 in the LTBI group and 6 in the active TB group. Figure 2 shows the TST results in the discordant group.

[0153) Figure 2 shows that among the 28 participants in the discordant group, 15 (53.6%) patients had a TST induration > 15 mm, while six (21.4%) had an induration exceeding 20 mm. This illustrates that a large proportion of patients in this group had strongly positive TST results, which are unlikely to be due to prior BCG vaccination alone. However, 22 of the patients in the discordant group had either a history of BCG vaccination or a scar suggestive of previous BCG vaccination on examination. In the remaining six there was neither a history of BCG vaccination, nor a visible scar in the deltoid area.

[0154] The correlation between TST induration and the background corrected interferon- gamma concentration in the antigen-stimulated sample of the QFT-GIT assay (i.e. interferon-gamma concentration in antigen-stimulated sample less the concentration in nil control sample) in the entire study population is shown in Figure 3A. A positive QFT-GIT assay result is defined by the manufacturer as a background corrected interferon-gamma concentration > 0.35 IU/ml; Figure 3B shows the results in the patients with a negative QFT-GIT assay result only.

[0155] As shown in Figure 3A there was a statistically significant positive correlation between the diameter of the TST induration and the interferon-gamma response in the GIT assay. However, the corresponding Spearman's correlation co-efficient (r) indicates that there was only moderate correlation between the two.

[0156] Figure 3B highlights that among participants with a QFT-GIT assay result classified as negative according to manufacturer's guidelines, several cases had background corrected interferon-gamma concentrations > 0.1 IU/ml (n=15; not all data points visible due to overlap). Seven of these participants had no palpable TST induration, one had an induration of 4 mm. The remaining seven participants had a positive TST result (i.e. > 10 mm induration; all in the common discordance group by definition). All seven had at least one risk factor for TB. Three had recent contact with adults with sputum-smear positive (i.e. highly infectious) pulmonary tuberculosis. One patient with a TST induration of 16 mm had a negative QFT-GIT result, as the background corrected interferon-gamma concentration (0.34 IU/ml) was just below the cut-off defining a positive assay result. [0157] It is probable that the seven patients with discordance had LTBI, which implies that the TST result represented the true-positive result, while the QFT-GIT assay produced a false-negative result. Notably, only three patients had evidence of prior BCG vaccination, which is frequently cited as the main reason for discordance. Two of these had TST indurations exceeding 20 mm, which is very rarely the result of previous BCG vaccination. In the absence of evidence of prior BCG vaccination or a history of previous non- tuberculoses mycobacterial (NTM) infection, there was no potential explanation for the positive TST result in the remaining four, indicating that they likely had LTBI. Of ten individuals with borderline results, only three had consistent results when tested serially over a four-week-period, while seven had inconsistent results (i.e. the latter converted from QFT- to QFT+, or reverted from QFT+ to QFT-; by "QFT" meaning "QFT-GIT"). This poor reproducibility of categorical assay results in cases with borderline interferon-gamma responses is concerning. Importantly, this test property may partly explain some of the discordance in the present study, as there is a distinct possibility that some of the participants ^' with borderline results would have had a positive QFT-GIT result (and thereby being concordantly TST+/QFT+) if the assay had been performed more than once. EXAMPLE 4

Cytokine concentrations in whole blood assays

[01S8] The results of the Luminex-based cytokine analysis of supematants from whole blood assays are shown in Figures 4A through J.

[0159J Three of the diagnostics groups only contained only a small number of individuals: the probable uninfected group (n=6), the possible discordance group (n=5) and the reverse discordance group (n=4). These groups were consequently excluded from further statistical comparisons, as these numbers are too small to provide meaningful data.

[0160] The statistical comparison of the cytokine responses in the four major groups - the uninfected, the common discordance, the LTBI and the active TB group - is shown in Tables 14 and 15.

[0161] Table 14 shows a summary of the statistical comparisons of the four major diagnostic groups regarding background corrected cytokine concentrations in supematants from whole blood assays following stimulation with MTB-specific peptides (EST-6, CFP- 10, and TB7.7). The third column shows the results of the Kruskal Wallis tests (i.e. four- group comparisons); all remaining values were calculated by Mann Whitney U tests (i.e. two-group comparisons). The latter were only performed in instances where the Kruskal Wallis test indicated a significant difference between the groups (i.e. p < 0.05).

TABLE 14

Statistical comparisons of the four major diagnostic groups regarding background corrected cytokine concentrations in supernatants from whole blood assays following stimulation with MTB-specific peptides (EST-6, CFP-10, and TB7.7)

[0162] Table 15 is a summary of the statistical comparisons of the four major diagnostic groups regarding background corrected cytokine concentrations in supernatants from whole blood assays following stimulation with PPD or killed MTB. The third column shows the results of the Kruskal Wallis tests (i.e. four-group comparisons); all remaining values were calculated by Mann Whitney U tests (i.e. two-group comparisons). The latter were only performed in instances where the Kruskal Wallis test indicated a significant difference between the groups (i.e. p < 0.05). TABLE 15

Statistical comparisons of the four major diagnostic groups regarding background corrected cytokine concentrations in supernatants from whole blood assays following stimulation with PPD or killed MTB

EXAMPLE 5

Cytokines which distinguish between TB-uninfected and TB-infected individuals [0163] There was a statistically significant difference between TB-uninfected and TB- infected participants (ie comparisons uninfected versus LTBI and uninfected vs. active TB) for a number of cytokines, indicating that these have a potential to be used for diagnostic purposes. The cytokines that potentially allow a distinction between TB-uninfected and TB-infected individuals differed between different antigens used for stimulation, as summarized in Table 16.

[0164] Summary illustrating the potential ability of different cytokines to distinguish between TB-uninfected and TB-infected (i.e. LTBI or active TB) individuals. Statistically significant differences (p < 0.05 in Mann Whitney U test) between groups are represented by dots (the corresponding numerical values are shown in Tables 14 and 15)

- 66 - TABLE 16

Summary illustrating the potential ability of different cytokines to distinguish between

TB-uninP _I10f- ected and TB-infected ( e. LTBI or active TB) individuals

[0165J As illustrated by Table 16 above, certain cytokines were lacking the ability to distinguish between TB-uninfected and TB-infected individuals, which included IL-15, IL- 17 and RANTES. For a number of other cytokines only a small number of comparisons reached statistical significance, indicating that their ability to distinguish between TB- uninfected and TB-infected individuals is likely to be relatively limited. This included IL- 6, IL-8, MCP-1 and MCP-3. To a lesser extent, this also applied to IL-10 and IL-12(p40), with only five and four of the comparisons reaching statistical significance, respectively.

[0166] According to the analyses presented in this Example, the most promising cytokines for the distinction between TB-uninfected and TB-infected individuals are IFN-γ, IP-10, TNF-a, IL-lra, IL-2, IL-13, GM-CSF and MIP-Ιβ. With the exception of comparisons related to TB7.7 (which produced very limited responses overall), IFN-γ, TNF-a, IL-2, and IL-13 reached statistical significance in every comparison shown in Table 16 (i.e. irrespective of the stimulant used). However, for a cytokine to be potentially useful as a diagnostic marker it is also important that there is no or only limited overlap between the responses in individuals in the TB-uninfected group and the TB-infected group. Figures 4A-D show that little or no overlap existed in ESAT-6 and CFP-10 stimulated samples between the uninfected group and the groups classified as LTBI and active TB regarding IFN-γ, IP- 10, TNF-a, IL-lra, IL-2, IL-13, and MIP-Ιβ. For all seven cytokines measured concentrations were highest in the active TB group. [0167] As shown in Figures 4G through J, there was overall more overlap in the concentrations of these seven cytokines between the uninfected group and the TB-infected groups (LTBI and active TB) when PPD or killed MTB were used for stimulation. This could potentially be explained by the immune system of some children in the uninfected group having been primed by prior BCG vaccination, resulting in the presence of T-cells reactive to certain antigens present in PPD and killed MTB. This would explain the general absence of cytokine responses in this group when peptides that are absent from all BCG vaccine strains (i.e. ESAT-6, CFP-10 and TB7.7) were used for stimulation. This hypothesis is tested in Example 6.

EXAMPLE 6

The influence of BCG vaccination on cytokine responses

[0168] To test whether prior BCG vaccination impacts on cytokine responses when heterogeneous antigens (i.e. PPD or killed MTB) are used for stimulation, participants in the uninfected group were divided into two subgroups: (i) children with a history of prior BCG vaccination (irrespective of the presence of a BCG scar); and (ii) children with a history of prior BCG vaccination. The cytokine responses in both groups were compared by use of Mann Whitney U tests, the results of which are shown in Figures 5 A and B.

[0169] As shown in Figure 5, there was a statistically significant difference between the cytokine responses in BCG-vaccinated and BCG-nonvaccinated children in the uninfected group with IFN-γ, IP- 10, IL-2, and IL-13. These differences were observed with both stimulants (i.e. PPD and killed MTB). With each of these four cytokines, responses were higher in the BCG-vaccinated subgroup compared to the BCG-nonvaccinated subgroup. In most instances there was a significant overlap between the results of the BCG-vaccinated subgroup and result of the LTBI group (in PPD stimulated samples: IFN-γ, IP- 10, and IL- 13; in killed MTB stimulated samples: IFN-γ, IP- 10, IL-2, and IL-13). These observations indicate that these cytokines would have very limited ability to distinguish between uninfected, BCG-vaccinated children and TB-infected children when PPD or MTBk are used as the stimulant (despite their ability to distinguish between uninfected, BCG- nonvaccinated children and TB-infected children when these heterogeneous antigens are used as stimulant). [0170] In contrast, with the remaining three cytokines, TNF-a, IL-lra, and ΜΙΡ-Ιβ, no significant differences between the BCG-vaccinated and BCG-nonvaccinated subgroups were observed, indicating that prior BCG vaccination had no impact on TNF-a, IL- 1 ra, and MIP-Ι β production in response to stimulation with PPD or killed MTB. This is an important observation, as the absence of confounding by prior BCG vaccination is a highly desirable property for any potential biomarker of TB infection. EXAMPLE 7

Performance characteristics of cytokines with the potential to distinguish between TB- uninfected and TB-infected individuals . (0171] To assess in more detail whether the seven cytokines (IFN-γ, IP-10, TNF-a, IL-lra, IL-2, IL-13, and MIP-Ι β) whose concentrations differed significantly between TB-uninfected and TB-infected individuals can potentially be used for diagnostic purposes, receiver operating characteristic (ROC) analyses were performed, the results of which are summarized in Table 17. For these analyses the data from children in the LTBI and the active TB group was grouped together (i.e. case values); data from the children in the uninfected group were used as control values. In addition, ROC curves were constructed for each cytokine/stimulant combination, which is shown in Figure 6.

[0172] Table 17 shows the results of the ROC analysis of data related to the seven cytokines with the potential ability to discriminate TB-uninfected from TB-infected individuals. For each cytokine/stimulant combination the cut-off was chosen at the background corrected cytokine concentration that achieved the highest values for both sensitivity and specificity.

TABLE 17

Results of the ROC analysis of data related to the seven cytokines with the potential ability to discriminate TB-uninfected from TB-infected individuals

[0173] Table 18 and Figure 6 show that overall IFN-γ, IP- 10, TNF-a, and IL-2 performed better than IL-lra, IL-13, and MIP-Ιβ. The former four cytokines achieved sensitivities and specificities universally exceeding 80% - and often exceeding 90% - when ESAT-6, CFP-10 or PPD were used as the stimulant. The highest sensitivity (100%) was achieved with the combination IL-2/PPD, with a corresponding specificity of 96.0%. The cut-offs were universally lower with CFP-10 as a stimulant, compared with ESAT-6, indicating that responses elicited by the latter are overall of greater magnitude. Also, sensitivity and specificity values for each cytokine were generally lower with CFP-10 than with ESAT-6. All cytokines performed least well when killed MTB was used as the stimulant.

[0174] The very high AUC values for IFN-γ, IP- 10, TNF-a, and IL-2 regarding ESAT-6, CFP-10 and PPD stimulated samples strongly indicate the ability of these cytokines to discriminate between TB-uninfected and TB-infected individuals. However, there is no gold standard for LTBI, and it is likely that IFN-γ performed comparatively well partly due to the fact that presence of a positive QFT-GIT assay result (i.e. presence of IFN-γ responses) formed part of the definition of LTBI. Nevertheless, application of the optimal cut-off values (shown in Table 17) to the data from patients with confirmed or highly probable active TB revealed that the cytokine responses in these children almost universally considerably exceeded all cut-offs, as shown in Figure 7, lending further support to the notion that these cytokines have discriminatory ability.

EXAMPLE 8

Cytokines that distinguish between latent TB infection and active tuberculosis

[0175] The statistical comparisons shown in Tables 16 and 17 indicate that the median concentrations of certain cytokines produced in response to stimulation with ESAT-6, CFP-10, PPD and killed MTB differed significantly between children with LTBI and those with active TB, suggesting that these cytokines may have some ability to discriminate between these two infection states. An overview of these data is shown in Table 18. [0176] Table 18 is a summary illustrating the potential ability of different cytokines to discriminate between individuals with LTBI and active TB. Statistically significant differences (p < 0.05 in Mann Whitney U test) between groups are represented by black dots; those approaching statistical significance (p = 0.05 - 0.1 in Mann Whitney U test) are represented by grey dots (the corresponding numerical values are sho₇wn in Tables 16 and 17).

TABLE 18

Summary illustrating the potential ability of different cytokines to discriminate between individuals with LTBI and active TB

(0177] Table 18 indicates that IFN-γ lacks the ability to discriminate between individuals with LTBI and those with active TB. TNF-a, IL-lra, IL-6, IL-10, and MCP-1 have some discriminatory ability. Due to the fact that the number of children with active TB was small, meaningful ROC analyses are not possible. However, a comparison of the cytokine responses in the LTBI and the active TB groups is shown in Figure 8.

[0178] As shown in Figure 8, the median TNF-a, IL-lra, IL-6, IL-10, and MCP-1 concentrations were almost universally higher in the active TB group, compared with the LTBI group, irrespective of the stimulant used. However, for most cytokine/stimulant combinations there was considerable overlap between the cytokine responses in both groups, indicating that the ability of these combinations to discriminate between the two disease states is limited. [0179] The best distinction between patients with LTBI and those with active TB was achieved with the combinations TNF-a/ESAT-6, TNF-a/CFP-10, IL-lra/PPD, IL- lra/MTBk, IL-10/PPD, and IL-10/MTBk, as shown in Figure 8 and summarized in Table 19.

TABLE 19

Performance of cytokine/stimulant combinations that can discriminate LTBlfrom active

Cytokine Stimulant Cut-off ! Number of cases Number of cases Total correctly

j I (pg/ml) correctly classified correctly classified classified

i as LTBI as active TB

TNF-a ^'. ESAT-6 80 13/16 5/6 j 81.8%

TNF-a j CFP-10 40 J 14/16 5/6 ; 86.4%

IL-1ra PPD 450 : 14/16 6/6 I 90.9%

IL-1ra \ MTBk I 500 ' 14/16 5/6 I 86.4%

IL-10 PPD I 100 j 16/16 6/6 ; 100%

IL-10 MTBk 1000 ! 15/16 5/6 ! 90.9% [0180] The data shown in Table 19 suggest that IL-10 (in samples stimulated with PPD or killed MTB) is best suited for the distinction between LTBI and active TB. However, as shown in Figure 4, there was considerable overlap between IL-10 responses in participants in the uninfected group and the responses in participants in the LTBI and active TB group. This indicates that this cytokine could not be used in isolation to diagnose infection and distinguish between disease states simultaneously. However, in a diagnostic test IL-10 could potentially be combined with another cytokine that can establish whether TB infection is present, using the IL-10 purely for the distinction between disease states. The same applies to IL-lra, for which an equally significant overlap between the responses in the uninfected and the groups with LTBI and active TB was observed (see Figure 4).

[0181] In contrast to IL-10 and IL-lra, TNF-a not only has the ability to discriminate between LTBI and active TB, but also achieves high levels of sensitivity and specificity regarding the distinction between TB-infected and TB-uninfected individuals. This suggests that diagnostic tests based on the detection of TNF-a responses could potentially simultaneously overcome two major challenges in the diagnosis of TB: (i) the distinction between uninfected and infected individuals; and (ii) the distinction between LTBI and active TB. Importantly, the latter distinction can not be made based on TST or IGRA results, and therefore any test that can achieve this would represent a major improvement. EXAMPLE 9

The magnitude of cytokine responses in relation to stimulatory antigens

[0182] As shown in Figure 4, the magnitude of cytokine responses differed between the different antigens used for stimulation. Overall, responses in children with LTBI or active TB were of greater magnitude when- heterogeneous antigens (i.e. PPD or killed MTB) were used for stimulation, compared to stimulation with single peptide antigens (i.e. ESAT-6, CFP-10 or TB7.7), as summarized in Figure 9. (0183) As shown in Figure 9, in children with LTBI or active TB stimulation with PPD or killed MTB produced stronger cytokine responses than stimulation with single peptide antigens. The concentrations of key cytokines in response to stimulation with PPD or killed MTB frequently exceeded those observed with stimulation with single peptide antigens several-fold. The only exception was IP- 10, where responses of similar magnitude were observed with ESAT-6, CFP-10, PPD and killed MTB (but not TB7.7).

[0184] The overall pattern regarding the magnitude of cytokine responses with single peptide antigens was identical in all seven key cytokines. Stimulation with ESAT-6 invariably produced higher median responses than stimulation with CFP-10. The lowest median responses were invariably observed with TB7.7. However, this does not imply that TB.7.7 is not useful as a stimulant. As apparent from Figure 9, only some patients responded to stimulation with TB7.7. However, some of these patients had responses of considerable magnitude. The graph related to interferon-gamma shows that in eleven cases interferon- gamma production occurred in response to stimulation with TB7.7. In four of these cases interferon-gamma responses even exceeded the cut-off value (a background corrected interferon-gamma concentration > 0.35 IU/ml; equivalent to 14 pg/ml) used for a positive result in the QFT-GIT assay (which uses a combination of all three peptide antigens).

[0185] The results outlined herein provide a method of stratifying a subject with respect to exposure to a species of Mycobacterium, said method comprising selecting one or more antigens from the Mycobacterium species or an immunological relative thereof based on its or their capacity to stimulate production of effector molecules from immune cells in a fluid sample comprising immune cells from the subject in vitro, wherein the profile of the effector molecules produced characterizes the subject as:

(ii) if infected, whether the infection is active or latent; and/or

[0186] Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure contemplates all such variations and modifications. The disclosure also enables all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively^ and any and all combinations of any two or more of the steps or features or compositions or compounds.

BIBLIOGRAPHY

Daneshvar et al. (1999) J. Immunol. Methods 226(1 -2) . 1 19-128 Durig et al. ( 1993) J. Raman Spectrosc. 24(5):2 1-285 Eriksson et al. (1993) Biophys. J. 2:64 Lakowicz et al. (1997) Biophys. J. 72:567 Lewis et al. ( 1999) Dyes Pigm. 42(2): 197 Petkovic-Duran et al. (2009) Biotechniques 47:827-834 Rapaport <?f a/. (1999) Appl. Phys. Lett. 74(3):329-3 \

Tawa et al., Mater. Res. Soc. Symp. Proc.488 [Electrical, Optical and Magnetic Properties of Organic Solid-State Materials IV], 885-890

Youvan et al. ( 1997) Biotechnology et elia 5: 1-18

Claims

CLAIMS:

1. A method of stratifying a subject with respect- to exposure to a species of Mycobacterium, said method comprising conducting an antigen-stimulation assay with immune cells from the subject, the antigen selected from early secretory antigenic target-6 (ESAT-6), culture filtrate protein-10 (CFP- 10), tuberculosis protein 7.7 (TB7.7), a peptide comprising an amino acid sequence having at least about 80% similarity to ESAT-6, CFP- 10 or TB7.7 after optimal alignment and carrying a T-cell epitope, purified protein derivative (PPD) and a killed M. tuberculosis preparation (MTBk) or other killed mycobacterial preparation, identifying effector molecules generated by the immune cells in response to the antigen, wherein:

(i) a subject is stratified as being infected with a species of Mycobacterium when there is an elevation in production of an effector molecule selected from the list consisting of IFN-γ, TNFa, IL-2, IL-13, IP-10, IL-lra, GM-CSF, MIP-1 B, IL-6, IL-8, MCP-1, CP-3, IL-10 and IL-12;

(ii) a subject is stratified as having an active Mycobacterium infection when there is an elevation in production of an effector molecule selected from the list consisting of TNFa, IL-lra, IL-6, IL- 10 and MCP- 1 ;

(iii) a subject is stratified as being immunoresponsive to challenge by Mycobacterium when there is an elevation in production of an effector molecule selected from the list consisting of IFN-γ, TNFa, IL-2, IL-13, IP-10, IL-lra, GM-CSF, MIP-1B, IL- 6, IL-8, MCP-1 , MCP-3, IL-10 and IL-12.

2. The method of Claim 1 wherein the Mycobacterium species is selected from the list consisting of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis (virulent), Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium smegmatis and Mycobacterium szulgai.

3. The method of Claim 1 wherein the subject is a human.

4. The method of Claim 1 wherein the subject is a livestock animal.

5. The method of Claim 3 wherein the Mycobacterium species is M. tuberculosis.

6. The method of Claim 1 wherein the ESAT-6 antigen is a pool of 7 peptides, each comprising an amino acid sequence selected from the group consisting of SEQ ID NO: l through 7, respectively.

7. The method of Claim 1 wherein the CFP-10 antigen is a pool of 6 peptides, each comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8 through 13, respectively.

8. The method of Claim 1 wherein the TB7.7 antigen is a pool of 6 antigens each comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14 through 19, respectively.

9. The method of Claim 1 wherein a subject is stratified as being infected with M. tuberculosis when there is an elevation in production of an effector molecule selected from the group consisting of IFN-γ, TNFa, IL-2, IL-13, IP- 10, IL-lra, GM-CSF and MIP- i p.

10. The method of Claim 9 wherein the subject is stratified as being infected with M. tuberculosis when there is an elevation in production of an effector molecule selected from IFN-γ, TNFa, IL-2 and IL-13.

1 1. The method of Claim 10 wherein the subject is stratified as being infected with M. tuberculosis when there is an elevation in production of TNFa.

12. The method of Claim 1 wherein a subject is stratified as having an active M. tuberculosis infection when there is an elevation in production of an effector molecule selected from the list consisting of TNFa, IL- 1 ra, IL-6, IL- 10 and MCP- 1.

13. The method of Claim 12 wherein a subject is stratified as having an active M. tuberculosis infection when there is an elevation in production of TNFa.

14. The method of Claim 1 wherein a subject is stratified as having an active M. tuberculosis infection when the antigen listed in parenthesis stimulates an elevation in production of an effector molecule selected from the list consisting of TNFa (ESAT-6), TNFa (CFRP- 10), IL- 1 ra (PPD), IL- 1 ra (MTBk), IL- 10 (PPD) and IL- 10 (MTBk).

15. The method of Claim 14 wherein the subject is stratified as having an active M. tuberculosis infection when ESAT-6 or CFP-10 stimulates an elevation in production of TNFa.

16. The method of Claim 1 wherein a subject is stratified as being immunoresponsive to challenge by Mycobacterium when there is an elevation in production of an effector molecule selected from the list consisting of IFN-γ, TNFa, IL-2, IL-13, IP- 10, IL-lra, GM-CSF and MIP-Ι β.

17. The method of Claim 16 wherein a subject is stratified as being immunoresponsive to M. tuberculosis challenge when there is an elevation in production of an effector molecule selected from the list consisting of IFN-γ, TNFa, IL-2 and IL-13.

18. The method of Claim 16 wherein a subject is stratified as being immunoresponsive to M. tuberculosis challenge when there is an elevation in production of an effector molecule selected from the list consisting of TNFa.

19. The method of any one of Claims 1 to 18 wherein the fluid sample is whole blood or a cellular fraction thereof. 1

20. Use of an effector molecule profile generated in response to stimulation of immune cells by a mycobacterial antigen in the manufacture of a diagnostic assay to distinguish between active and latent infection by a species of Mycobacterium.

21. Use of Claim 20 wherein the Mycobacterium species is is selected from the list consisting of M. tuberculosis, M. africanum, M. bovis (virulent), M. flavescens, M. gastri, M. kansasii, M. leprae, M. marinum, M. smegmatis and M. szulgai.

22. Use of Claim 21 wherein the Mycobacterium species is M. tubuerculosis.

23. Use of Claim 22 wherein the Mycobacterium antigen is selected from the list consisting of early secretary antigenic target-6 (ESAT-6), clture filtrate protein- 10 (CFP- 10), tuberculosis protein 7.7 (TB7.7), a peptide comprising an amino acid sequecne having at least about 80% similarity to ESAT-6, CFP-10 or TB7.7 after optimal alignment and carrying a T-cell epitope, Purified protein derivative (PPD) and a killed M. tuberculosis preparation (MTBk) or other killed mycobacterial preparation.

24. Use of Claim 23 wherein a subject is stratified as having an active M. tuberculosis infection when the antigen stimulates an elevation in production of an effector molecule selected from the list consisting of IFN-γ, TNFa, IL-6, IL-lra, MCP-1 , IL-2, IL-10, IL-13, IP-lO and MIP-Ιβ.

25. Use of Claim 24 wherein a subject is stratified as having an active M. tuberculosis infection when the antigen stimulates an elevation in production of an effector molecule elected from the list consisting of TNFa, IL-lra, IL-6, IL-10 and MCP-1.

26. Use of Claim 31 wherein a subject is stratified as having an active M. tuberculosis infection when the antigen stimulates an elevation in production of an effector molecule elected from the list consisting of TNFa.

27. A method for identifying a subject with an active infection by M. tuberculosis, said method comprising screening for the capacity for immune cells in a fluid sample from the subject in vitro to produce an elevated level of TNFa in response to ESAT-6 or CFP-10 or a peptide having at least 80% amino acid sequence similarity to ESAT-6 or CFP-10 and which peptide carries a T-cell epitope wherein an elevated amount of TNFa is indicative of an active infection.

28. The method of Claim 27 wherein the fluid sample is whole blood or a cellular fraction thereof.

29. A kit comprising one or more Mycobacterial antigens and reagents to detect one or more effector molecules when used in the method of any one of Claims 1 to 19, 27 or 28.

30. A therapeutic protocol to treat a subject infected with M. tuberculosis, said protocol comprising determining whether the subject has an active or latent infection by the method of any one of Claims 1 to 19 and selecting a treatment regime on the basis of whether the infection is active or latent.