DIAGNOSTIC REAGENT

Abstract

The invention provides a Mycobacterium Tuberculosis Complex (MTC) (for example, M. bovis and/or M. tuberculosis) diagnostic reagent comprising the reagent components:

a. a Rv3616c antigen polypeptide and/or a Rv3616c antigenic cocktail;

b. a Rv1789 antigen polypeptide and/or a Rv1789 antigenic cocktail;

c. a Rv3810 antigen polypeptide and/or a Rv3810 antigenic cocktail; and

d. a Rv3478 antigen polypeptide and/or a Rv3478 antigenic cocktail.

The diagnostic reagent may further comprise one or more of a ESAT-6 antigen polypeptide and/or a ESAT-6 antigenic cocktail; a CFP-10 antigen polypeptide and/or a CFP-10 antigenic cocktail; a Rv3615c antigen polypeptide and/or a Rv3615c antigenic cocktail; and/or SEQ ID NO:207 (a fusion protein of ESAT-6 and CFP-10 and Rv3615c). There are also provided methods and kits involving use of the diagnostic reagent.

Claims

1. A Mycobacterium Tuberculosis Complex (MTC) diagnostic reagent comprising the reagent components: a. a Rv3616c antigen polypeptide and/or a Rv3616c antigenic cocktail; b. a Rv1789 antigen polypeptide and/or a Rv1789 antigenic cocktail; c. a Rv3810 antigen polypeptide and/or a Rv3810 antigenic cocktail; and d. a Rv3478 antigen polypeptide and/or a Rv3478 antigenic cocktail.

2. The MTC diagnostic reagent according to claim 1, wherein the diagnostic reagent comprises a Mycobacterium bovis (M. bovis) and/or Mycobacterium tuberculosis (M. tuberculosis) diagnostic reagent comprising the reagent components: a. a Rv3616c antigen polypeptide and/or a Rv3616c antigenic cocktail; b. a Rv1789 antigen polypeptide and/or a Rv1789 antigenic cocktail; c. a Rv3810 antigen polypeptide and/or a Rv3810 antigenic cocktail; and d. a Rv3478 antigen polypeptide and/or a Rv3478 antigenic cocktail.

3. The MTC diagnostic reagent according to claim 1 or claim 2 further comprising at least one further reagent component selected from: a. a ESAT-6 antigen polypeptide and/or a ESAT-6 antigenic cocktail; b. a CFP-10 antigen polypeptide and/or a CFP-10 antigenic cocktail; c. a Rv3615c antigen polypeptide and/or a Rv3615c antigenic cocktail; d. SEQ ID NO:207.

4. The MTC diagnostic reagent according to any one of claims 1 to 3 comprising the reagent components: a. a Rv3616c antigen polypeptide and/or a Rv3616c antigenic cocktail; b. a Rv1789 antigen polypeptide and/or a Rv1789 antigenic cocktail; c. a Rv3810 antigen polypeptide and/or a Rv3810 antigenic cocktail; d. a Rv3478 antigen polypeptide and/or a Rv3478 antigenic cocktail; e. a ESAT-6 antigen polypeptide and/or a ESAT-6 antigenic cocktail; f. a CFP-10 antigen polypeptide and/or a CFP-10 antigenic cocktail; g. a Rv3615c antigen polypeptide and/or a Rv3615c antigenic cocktail; and h. a Rv3020c antigen polypeptide and/or a Rv3020c antigenic cocktail.

5. The MTC diagnostic reagent according to any preceding claim comprising a reagent component which is a Rv3616c antigenic cocktail, the cocktail comprising: a. SEQ ID NOs:97-144; b. SEQ ID NOs:97-143 and 145; or c. SEQ ID NOs:187-206; or comprising a reagent component which is a Rv3616c antigen polypeptide having SEQ ID NO:208.

6. The MTC diagnostic reagent according to any preceding claim comprising a reagent component which is a Rv1789 antigen polypeptide having SEQ ID NO:183 or a functional variant thereof, or comprising a reagent component which is a Rv1789 antigenic cocktail comprising SEQ ID NOs:1-48.

7. The MTC diagnostic reagent according to any preceding claim comprising a reagent component which is a Rv3478 antigen polypeptide having SEQ ID NO:185 or a functional variant thereof, or comprising a reagent component which is a Rv3478 antigenic cocktail comprising SEQ ID NOs:49-96.

8. The MTC diagnostic reagent according to any preceding claim comprising a reagent component which is a Rv3810 antigen polypeptide having SEQ ID NO:186 or a functional variant thereof, or comprising a reagent component which is a Rv3810 antigenic cocktail comprising SEQ ID NOs:146-179.

9. The MTC diagnostic reagent according to any preceding claim comprising a reagent component which is an ESAT-6 antigen polypeptide having SEQ ID NO:180 or a functional variant thereof.

10. The MTC diagnostic reagent according to any preceding claim comprising a reagent component which is a CFP-10 antigen polypeptide having SEQ ID NO:181 or a functional variant thereof.

11. The MTC diagnostic reagent according to any preceding claim comprising a reagent component which is a Rv3615c antigen polypeptide having SEQ ID NO:182 or a functional variant thereof.

12. The MTC diagnostic reagent according to any preceding claim comprising a reagent component which is a Rv3020c antigen polypeptide having SEQ ID NO:184 or a functional variant thereof.

13. The MTC diagnostic reagent according to any preceding claim comprising SEQ ID NOs:97-144 and 180-186 and/or a functional variant of any of these in its place.

14. The MTC diagnostic reagent according to any of claims 1-12 comprising SEQ ID NOs:180-206 and/or a functional variant of any of these in its place.

15. The MTC diagnostic reagent according to any preceding claim for use in a method of detecting in an animal infection with or exposure to one or more MTC species comprising contacting the animal with the diagnostic reagent and/or comprising obtaining a sample from the animal and contacting the sample with the diagnostic reagent.

16. The MTC diagnostic reagent according to claim 15, wherein the diagnostic reagent is an M. bovis and/or M. tuberculosis diagnostic reagent for use in a method of detecting in an animal infection with or exposure to M. bovis and/or M. tuberculosis.

17. A method of diagnosing in an animal infection with or exposure to one or more Mycobacterium Tuberculosis Complex (MTC) species, the method comprising contacting the animal or a sample obtained therefrom with: a. a Rv3616c reagent component comprising a Rv3616c antigen polypeptide and/or a Rv3616c antigenic peptide cocktail; b. a Rv1789 reagent component comprising a Rv1789 antigen polypeptide and/or a Rv1789 antigenic cocktail; c. a Rv3810 reagent component comprising a Rv3810 antigen polypeptide and/or a Rv3810 antigenic cocktail; and d. a Rv3478 reagent component comprising a Rv3478 antigen polypeptide and/or a Rv3478 antigenic cocktail.

18. The method according to claim 17, wherein the method is a method of diagnosing in an animal infection with or exposure to M. bovis and/or M. tuberculosis.

19. The method according to claim 17 or claim 18 wherein the Rv3616c, Rv1789, Rv3810 and Rv3478 reagent components are in the form of a diagnostic reagent according to any of claims 1-14.

20. The method according to any one of claims 17 to 19 comprising obtaining a biological sample from the animal and conducting a blood-derived parameter release test on the sample using the Rv3616c, Rv1789, Rv3810 and Rv3478 reagent components.

21. The method according to claim 20 wherein the blood-derived parameter release test is a cytokine release test.

22. The method according to claim 21 wherein the cytokine release test is an interferon gamma release assay (IGRA).

23. The method according to claim 19 comprising conducting a skin test on the animal, the skin test comprising administration of the diagnostic reagent to the animal.

24. A diagnostic kit comprising a. a Rv3616c reagent component comprising a Rv3616c antigen polypeptide and/or a Rv3616c antigenic peptide cocktail; b. a Rv1789 reagent component comprising a Rv1789 antigen polypeptide and/or a Rv1789 antigenic cocktail; c. a Rv3810 reagent component comprising a Rv3810 antigen polypeptide and/or a Rv3810 antigenic cocktail; and d. a Rv3478 reagent component comprising a Rv3478 antigen polypeptide and/or a Rv3478 antigenic cocktail.

25. The diagnostic kit according to claim 24 comprising a diagnostic reagent which comprises the Rv3616c, Rv1789, Rv3810 and Rv3478 reagent components.

26. The diagnostic kit according to claim 24 or 25 for use in the method according to any of claims 17-23.

27. A diagnostic kit according to any of claims 24-26, wherein the diagnostic reagent is able to detect a Mycobacterium Tuberculosis Complex (MTC) species infection in an animal.

28. A diagnostic kit according to any of claims 24-27, wherein the diagnostic reagent is able to detect a M. bovis or M. tuberculosis infection in an animal.

29. A diagnostic kit according to claim 27, wherein the diagnostic reagent is able to differentiate between a MTC species infected animal and an animal vaccinated against a MTC species infection.

30. A diagnostic kit according to claim 28, wherein the diagnostic reagent is able to differentiate between a M. bovis or M. tuberculosis infected animal and an animal vaccinated against M. bovis or M. tuberculosis infection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0110] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0111] FIG. 1 shows the interferon gamma release assay (IGRA) results of stimulation of peripheral blood mononuclear cells (PBMCs) with Rv1789, Rv3478, Rv3616c or Rv3810 induced significantly stronger IFN-γ responses in M. bovis infected cattle compared to uninfected controls. Cryo-preserved PBMC from infected and uninfected animals were stimulated for 3 days with these antigens. IFN-γ production was determined in supernatants by Bovigam ELISA. Statistical analysis using t-test, with p-values shown in the Figure.

[0112] FIG. 2 shows skin test results obtained by testing M. bovis infected animals (left panel) and uninfected control calves (right panel) using various reagents. Reactions were read at 0 and 72 h and data are expressed as increase in skin reactions at the 72 h read point (Δskin thickness in mm). Horizontal bars: median responses. Statistical analysis by Wilcoxon signed ranks test. ****, P<0.0001, NS, not significant.

[0113] FIG. 3 shows a comparison of skin test responses induced by TRT1 and TRT2 in infected and uninfected control animals. Reactions were read at 0 and 72 h and data are expressed as increase in skin reactions at the 72 h read point (Δskin thickness in mm). Horizontal bars: median responses. Statistical analysis by Wilcoxon signed ranks test. ***, P<0.001, NS, not significant.

[0114] FIG. 4 shows Receiver Operator Characteristic (ROC) Analysis of TRT1 (left panel) and TRT2 (right panel) performance. Results are summarised in the tables accompanying the figure panels.

[0115] FIG. 5 shows a comparison of IGRA responses induced by DST and TRT2 reagents in infected and uninfected control animals. Whole blood from 22 infected and 30 uninfected control calves were stimulated for 20 h with DST and TRT2 reagents at different concentrations. IFN-γ production was quantified by ELISA and expressed as nil antigen corrected values (ΔOD). Symbols and solid horizontal lines represent individual animals and group medians respectively. The dotted horizontal line indicates the preliminary cut-off for positivity (ΔOD>0.1).

[0116] FIG. 6 shows skin test results obtained by testing M. bovis experimentally infected (n=20), naturally infected (n=21), uninfected control (n=30) and Gudair-vaccinated (n=29) calves using various reagents. Reactions were read at 0 and 72 h and data are expressed as increase in skin reactions at the 72 h read point (Δskin thickness in mm). Horizontal bars: median responses. Statistical analysis by Friedman test with Dunn's post test. **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0117] FIG. 7 shows a comparison of IGRA responses induced by various reagents in M. bovis experimentally infected (n=20), naturally infected (n=21), uninfected control (n=30) and Gudair-vaccinated (n=29) calves. Whole blood from calves were stimulated for 20 h with PPDA, PPDB, B-A (PPD-B-PPD-A; otherwise referred to as the SICCT-test), DST or TRT2 reagents. IFN-γ production was quantified by ELISA and expressed as nil antigen corrected values (ΔOD). Symbols and solid horizontal lines represent individual animals and group medians respectively. The dotted horizontal line indicates the preliminary cut-off for positivity (ΔOD>0.1). Statistical analysis by Friedman test with Dunn's post test. **, p<0.01; ***, P<0.001; ****, p<0.0001.

DETAILED DESCRIPTION

Materials and Methods

Preparation of Antigens

[0118] (a) for in vitro assay Antigens are referred to herein in accordance with standard M. tuberculosis nomenclature, since sequences found in M. bovis (as well as M. africanum, M. orygis, M. microti, M. canetti, M. caprae, M. pinnipedii, M. suricattae and M. mungi) are identical to those found in M. tuberculosis. Antigen sequences may be obtained via mycobrowser.epfl.ch (accessed 10 Jul. 2019), searching for sequences from M. tuberculosis H37Rv.

[0119] The candidate antigens listed in Table 1, to be screened in the peripheral blood mononuclear (PBMC) assay, were prepared either as as recombinant proteins or as separate pools of overlapping synthetic peptides (20-mers overlapping by 12 amino acids; JPT Peptide Technologies, Germany). Examples of the preparation of peptide pools for various antigens is well understood by the skilled person and is described for certain antigens, for example, in W02009/060184, W02011/135369 and Millington et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108 5730.

[0120] Details of the peptide pools for Rv1789, Rv3478, Rv3616c, and Rv3810 are shown in Table 4 below. The lyophilized peptide pools were reconstituted in RPMI 1640 (Gibco Life Technologies, UK) containing 2.25% DMSO to obtain a concentration of 55 μg of each peptide/ml, with the exception of Rv3616c which was reconstituted in RPMI 1640 containing 25% DMSO to obtain a concentration of 1 mg of each peptide/ml. All peptide pools were used to stimulate cattle PBMC at a final concentration of 5 μg of each peptide/ml. As a control, a recombinant fusion protein consisting of the antigens ESAT-6, CFP-10 and Rv3615c (Rv-EC; Lionex Ltd; SEQ ID NO:207) was used at a final concentration of 5 μg/ml.

[0121] (b) for in vivo skin testing ESAT-6, CFP-10, Rv1789, Rv3020c, Rv3478, Rv3615c and Rv3810 were sourced as recombinant proteins from a commercial manufacturer (Lionex Ltd, Germany, sequences shown in Table 5 below).

[0122] Rv3616c was prepared as either (i) Rv3616c.sub.(JPT): a peptide pool of 48 synthetic peptides (SEQ ID NOs:97-144, each overlapping by 12 amino acids; JPT Peptide Technologies) where the lyophilized peptide pool was reconstituted in PBS to obtain a concentration of 0.8 mg of each peptide/ml; or (ii) Rv3616c.sub.(Gen): a synthetic peptide pool consisting of sixteen 40-mers, three 25-mers and one 20-mer (SEQ ID NOs:187-206; GenScript Biotech, Netherlands) where each individual lyophilized peptide was first reconstituted in PBS to a concentration of 10 mg/ml and then combined together to obtain a peptide pool of 0.5 mg of each peptide/ml. Details of the peptide sequences included in the pools are shown in Table 5 below.

[0123] Skin test reagents TRT1 and TRT2 were then formulated by combining ESAT-6, CFP-10, Rv1789, Rv3020c, Rv3478, Rv3615c and Rv3810 proteins with either Rv3616c.sub.(JPT) (TRT1) or Rv3616c.sub.(Gen) (TRT2), so that each protein or individual peptide was at a concentration of 100 μg/ml. As a control, a skin test reagent (DST) comprised of ESAT-6, CFP-10 and Rv3615c proteins only was also formulated at 100 μg of each protein/ml. Bovine tuberculin (PPD-B) and avian tuberculin (PPD-A) were obtained from a commercial manufacturer (Thermo Fisher). Information on the sequences included in the reagents is shown in Table 6 below.

Animals

[0124] For the in vitro testing of antigens, archived PBMC from the following groups of cattle (Bos taurus taurus) were used: [0125] (i) naturally M. bovis-infected cattle originating from UK herds known to have bTB (natural infection was confirmed by post-mortem and/or culture analysis); [0126] (ii) non-infected control cattle originating from UK herds in the Low Risk Area that were Officially TB Free for over 5 years; and [0127] (iii) Gudair Johne's disease-vaccinated cattle originating from GB herds that were

[0128] Officially TB Free for over 5 years.

[0129] For in vivo testing of skin test reagents, the following groups of cattle were used: [0130] (i) experimentally M. bovis-infected cattle consisting of male calves experimentally infected with approx. 10,000 CFU of a field strain of M. bovis (AF2122/97) via the endobronchial route (infection was confirmed by post-mortem and/or culture analysis); [0131] (ii) non-infected control cattle as described above;

[0132] 1(iii) naturally M. bovis-infected cattle originating from herds from the Republic of Ireland known to have confirmed bTB; and [0133] (iv) Gudair-vaccinated cattle as described above.

[0134] The experimentally M. bovis-infected cattle were skin tested 5 weeks post infection. Tuberculin skin test-positive cattle (based on the comparative cervical tuberculin test) were selected from herds with persistent and confirmed bTB as the naturally M. bovis infected cattle. Animal procedures were approved by the APHA Animal Welfare and Ethical Review Board.

In Vitro Stimulation of PBMC

[0135] Cryo-preserved PBMC were thawed as quickly as possible in a water bath at 37° C. Upon thawing, appropriate volume of complete media (RPMI 1640 containing 2 mM GlutaMax, 25 mM HEPES, 0.1 mM NEAA, 5×10.sup.−5M β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco Life Technologies, UK) and 10% fetal calf serum (Sigma-Aldrich, UK)) was added in a dropwise manner and centrifuged at 350 g for 10 minutes at room temperature. The supernatant was discarded, the cell pellet gently loosened and resuspended in complete media and the cells counted using a hemocytometer. PBMC were plated at 2×10.sup.5 cells/well in 96-well plates and stimulated with and without antigens for 3 days at 37° C. in the presence of 5% CO.sub.2, following which cell supernatants were removed and stored at −80° C. until required.

IFN-γ ELISA

[0136] Quantification of IFN-γ in PBMC culture supernatant was determined using the commercially available BOVIGAM enzyme-linked immunosorbent assay (ELISA) kit (Thermo Fisher Scientific, USA). Results were expressed as the optical density at 450 nm (OD.sub.450) for cultures stimulated with antigen minus the OD450 for cultures without antigen (i.e. ΔOD.sub.450).

Skin Test Procedure

[0137] Injection sites located in the border of the anterior and middle third of the neck on either side of the cow were clipped and skin thickness recorded. PPD-A and PPD-B were administered in a 0.1 ml volume via intradermal injection as per manufacturer's recommendations. DST, TRT1 and TRT2 reagents were administered in a similar manner so that each individual protein or peptide was delivered at a 10 μg dose. To account for potential injection site differences, a Latin Square design was applied with animals randomly assigned to the Latin Square combinations. Skin thickness was measured again by the same operator 72 hours after administration, and the difference in skin thickness (mm) between the pre- and post-skin test readings recorded.

Statistical Analysis

[0138] All statistical analyses were performed using Prism 7 (Graphpad Software, USA).

Results

Example 1

Production of Candidate Antigens

[0139] Eighteen candidate proteins were selected for testing (see Table 1). These proteins were sourced from commercial sources, when available, as recombinant proteins. However, for the majority of proteins, this was not possible. In these cases, overlapping synthetic peptide sets were designed and commercially produced using state-of-the-art high-throughput peptide synthesis chemistry.

Antigen Screening and Complementation

[0140] These antigens were screened in interferon gamma release assays (IGRA) using bio-banked peripheral blood mononuclear cells (PBMC) from previous experiments and projects. Samples were obtained from naturally infected field reactors as well as uninfected controls.

[0141] To down-select to the most promising candidate antigens, the following gating criteria were applied: [0142] Significantly stronger IGRA responses induced by antigens in PBMC from M. bovis infected animals compared to uninfected controls; [0143] Specificity (i.e. no responses in uninfected animals including animals with high avian PPD responses); [0144] Antigens complement responses to the existing DIVA skin test antigens (ESAT-6, CFP-10, and Rv3615c).

[0145] Four antigens fulfilled all three of these criteria: Rv1789, Rv3478, Rv3616c, Rv3810 (italicised in Table 1). This was surprising since several other candidate antigens had been identified previously as being potentially useful in the development of diagnostic reagents (Cockle et al. (2006) Clin. Vaccine Immunol. 13 1119; Jones et al. (2010) Infect. Immun. 78 1326; Jones et al. (2010) Clin. Vaccine Immunol. 17 1344; Jones et al. (2013) Clin. Vaccine Immunol. 20 1675; Mustafa et al. Infect. Immun. 74 4566).

TABLE-US-00002 TABLE 1 Summary of responses to candidate antigens. Significant IFN-γ: Infected > Controls Specific Complementation Rv1789 Yes Yes Yes Rv3478 Yes Yes Yes Rv3616c Yes Yes Yes Rv3810 Yes Yes Yes Rv0288 No N/A No Rv0445c No N/A No Rv1038c No N/A No Rv1195 No N/A No Rv1197 No N/A No Rv1253 No N/A No Rv1387 No N/A No Rv1792 No N/A No Rv1983 No N/A No Rv2608 No N/A No Rv3017c No N/A No Rv3444c No N/A No Rv3872 No N/A No Rv3783 No N/A No

[0146] The IGRA responses for these four antigens are shown in FIG. 1 in comparison with responses induced by the DIVA skin test fusion protein (SEQ ID NO:207, a fusion of Rv3615c, ESAT6, CFP10). Responses to the peptide pools derived from each of the four proteins were significantly higher in PBMC from infected compared to uninfected cattle (p-values<0.0001 to 0.05), with no significant responses induced in T cells from uninfected animals.

[0147] PBMC from seven infected cattle did not respond to ESAT-6 (data not shown). The peptide pool for each of the four antigens described above were recognised by between one and five of these animals demonstrating their potential to complement the DIVA skin test antigens to increase overall signal strength and sensitivity (data not shown). This observation was confirmed when we considered three animals not recognising the DIVA skin test fusion protein, one of which responded to the Rv3616c peptide pool.

[0148] Based on these results, these four antigens were selected for in vivo assessment. This newly formed ‘TRT’ skin test cocktail included the three DST antigens (ESAT-6, CFP-10, Rv3615c), the four antigens listed above (Rv1789, Rv3478, Rv3616c, Rv3810) as well as an additional antigen (Rv3020c) that we had hitherto identified to induce specific immune responses in infected animals, but lacked the high specificity in BCG vaccinated calves which is a requirement for antigens to be used in a DIVA reagent (Jones et al. (2010) Clin. Vaccine Immunol. 17 1344; Jones et al. (2012) Clin. Vaccine Immunol. 19 620).

Example 2

Testing TRT Reagents In Vivo by Skin Testing

[0149] Most of the antigens were produced as recombinant proteins by Lionex GmbH (Braunschweig, Germany). Only the antigen Rv3616c could not be produced as a recombinant protein as it proved to be lytic to hosts utilised for expression. To overcome this technical problem, a set of 48 overlapping synthetic peptides was produced (SEQ ID NOs: 97-144). The TRT1 cocktail (see Table 6) was formed by mixing this Rv3616c overlapping peptide set with the protein antigens (Rv1789, Rv3478, Rv3810, ESAT-6, CFP-10, Rv3615c and Rv3020c).

[0150] We also designed and procured a second Rv3616c cocktail of 20 peptides, composed of 40-, 25- and 20-mer peptides (SEQ ID NOs:187-206) which were combined with the recombinant proteins described above into TRT2 (see Table 6).

[0151] We infected 42 calves via the endobronchial route with around 10,000 CFU M. bovis AF2122/97. Six weeks later, skin tests were performed using PPD-A, PPD-B, DST and TRT1. Injection sites were assigned in individual animals with a Latin Square design with animals randomly assigned to the different sub-groups in the Latin Square applying the double lottery principle. Infection was confirmed at necroscopy by the presence of visible pathology and M. bovis culture. To test for specificity a set of uninfected control calves was skin tested with PPD-B (n=30), PPD-A (n=30), DST (n=30), TRT1 (n=20) and TRT2 (n=30).

[0152] As shown in FIG. 2, statistically highly significantly stronger responses were induced in infected calves following injection of TRT1 compared to DST injection (P<0.0001). In contrast, only 1/20 of the uninfected controls gave rise to a small response (2 mm) with TRT1, whilst none of the DST induced responses were above 1 mm (FIG. 2).

[0153] In a subset of animals (n=22), the TRT2 reagent was tested alongside the other skin test reagents described above. We compared responses induced by TRT2 with those induced with TRT1 in a subset of infected animals as well as in uninfected controls (FIG. 3).

[0154] The results presented in FIG. 3 clearly demonstrate that TRT2 induced a significantly stronger skin response in infected cattle compared to TRT1 (P<0.001), whilst not inducing skin test responses above 1 mm (1/30) in uninfected controls (FIG. 3). Together, the data presented in FIGS. 2 and 3 therefore clearly demonstrate that the addition of additional antigens to the DST protein cocktail can significantly increase the strength of reactivity without compromising specificity. Furthermore, TRT2, which contains fewer but longer peptides representing Rv3616c, is a markedly improved formulation.

[0155] The raw data presented in FIGS. 2 and 3 was used to undertake ROC (Receiver Operating Characteristic) analysis with a view to define the diagnostic performance of TRT1 and TRT2, to define cut-offs for positivity and to estimate their relative sensitivities when we set specificity at 100%. These ROC analyses are shown in FIG. 4 and Table 2. As FIG. 4 shows, both TRT1 and TRT2 are high performance diagnostic antigens when applied in skin tests. This could be demonstrated by the high areas under the curves (TRT1: 0.9869 (95% CI: 0.9589, 1.0); TRT2: 1 (95% CI: 1, 1); P<0.0001 for both analyses).

TABLE-US-00003 TABLE 2 Definition of provisional TRT1 and TRT2 cut-off values at 100% specificity. Sensitivity % (95% CI) Antigen Specificity % Cut-off n/N SICCT, 100% >4 mm 93% (81, 98) standard 39/42 SICCT, 100% >2 mm 98% (88, 100) severe 41/42 SIT 100% 4 mm and 100% (88, 100) greater 42/42 DST 100% 2 mm and 98% (88, 100) greater 41/42 TRT1 100% 3 mm and 95% (84, 99) greater 40/42 TRT2 100% 5 mm and 100% (85, 100) greater 22/22

[0156] We further investigated the outcome of the ROC analyses by assessing relative sensitivity in the test animals after setting test specificity at 100% (Table 2). This allowed us to define cut-points for TRT1 and TRT2 positivity in accordance with routine methods. Compared to the DST cut-off of 2 mm and higher set in previous studies, the cut-off points for TRT1 and TRT2 were 3 mm and higher and 5 mm and higher respectively. These cut-offs maintained high sensitivity values (95% and 100% for TRT1 and TRT2 respectively), comparable to SICCT at standard or severe interpretation, SIT or DST sensitivities (Table 2). Thus, we have achieved the objective of demonstrating significantly stronger skin test responses with TRT1 and TRT2 compared to the DST, thus being able to define higher cut-off values, which lead to a more robust test in terms of reading the skin test results (since the cut-off can be adjusted to increase test sensitivity).

[0157] The TRT2 cocktail was also tested for its diagnostic potential in whole blood IGRA assays. To this end, we performed antigen dose titration experiments for both DST and TRT2 reagents using whole blood from experimentally M. bovis infected (n=22) and uninfected control (n=30) calves (FIG. 5). Using the standard PPD-6 minus PPD-A assay readout, all 22 infected calves tested positive, whereas 2 of the 30 control calves tested positive (data not shown). Using a preliminary cut-off (ΔO.D>0.1), 21 out of 22 infected animals tested positive to the DST reagent at the highest concentration tested (5 μg/ml), which decreased to 19 and 15 test positives at the lower antigen concentrations (1 μg/ml and 0.1 μg/ml respectively). In contrast, all infected animals tested positive to the TRT2 reagent at all antigen concentrations. Indeed, a significantly greater proportion of the infected calves tested positive at the lowest concentration of the TRT2 reagent compared to the DST (P-value=0.0089, Fisher's exact test). None of the control animals gave a positive response to the DST reagent at any of the concentrations tested. At the 1 μg/ml TRT2 concentration used four calves tested positive to the TRT2 reagent. Interestingly, one of these was also an animal that gave a positive PPD-6 minus PPD-A result. However, at the lowest antigen concentration tested (0.1 μg/ml), only one control animal remained positive to the TRT2 reagent, whilst responses were maintained in the infected animals. These results highlight the potential of the TRT2 antigens for use in IGRA as an ancillary test to the skin test, using the provisionally defined dose of 0.1 μg/ml with a cut-off value of ΔOD>0.1; although antigen dosage and appropriate cut-offs may still need to be fully defined and possibly refined using data from future studies.

Example 3

Testing TRT Reagents in Other Settings

[0158] We then investigated the TRT2 cocktail in other in vivo and in vitro animal categories. The other categories were naturally infected cattle, which is more reflective of a field situation, and in animals strongly sensitised to M. a. sso paratuberculosis antigens, which was achieved by vaccinating calves with the Gudair vaccine. These were compared to experimentally infected cattle and non-infected controls, as described above. Skin tests were performed using PPD-A, PPD-B, DST and TRT2. These results are shown in FIG. 6, including B-A, which represents the results of PPD-6 minus PPD-A (SICCT test). As shown in FIG. 6, statistically significantly stronger responses were induced in naturally infected calves following injection of TRT2 compared to DST injection (P<0.01). In contrast, no or low responses were observed in the Gudair-vaccinated calves. Only 3/29 of the vaccinated calves gave rise to a small (2 mm) response to TRT2. Only 1/29 of the vaccinated calves gave rise to a small (3 mm) response to TRT2. No response was observed from the remaining 25/29 vaccinated calves to TRT2 (FIG. 6). The level of reactivity to TRT2 in the Gudair-vaccinated calves was marginally higher than in naive, unvaccinated calves (FIG. 6). These data further demonstrate that the TRT protein cocktail can significantly increase the strength of reactivity as compared to DST, without compromising specificity.

[0159] The higher responses to TRT2 in naturally infected animals compared to the DST, allowed us to re-appraise the cut-off for positivity for the TRT in accordance with routine methods. By applying a TRT2 cut-off of 4 mm and higher, we could achieve the same level of specificity as seen in naïve animals (Table 3), whilst being comparable to the sensitivity achieved with PPD-B alone (SIT, Table 3). Thus, we have shown the significantly stronger skin test response with TRT2 compared to the DST. This has allowed us to define higher cut-off values, which lead to a more accurate interpretation of the skin test.

[0160] The in vitro diagnostic potential of the TRT2 cocktail for naturally infected and vaccinated cattle was also tested using whole blood IGRA assays. Antigen exposure experiments were performed for PPD-A, PPD-B, DST and TRT2 reagents using whole blood from naturally infected cattle and from the Gudair-vaccinated animals. The data of these experiments are shown in FIG. 7 (together with B-A, i.e. PPD-B-PPD-A). As with the skin test, TRT2 induced significantly stronger IGRA responses than the DST in the naturally infected animals (P<0.001). In contrast, the IGRA response to TRT2 in samples from Gudair-vaccinated animals was minimal or absent below the cut-off for positivity (FIG. 7, cut-off used: OD450 with antigen minus OD without antigen>0.1). These results therefore suggest that the TRT will have utility as an auxiliary test to be used alongside skin testing. These results further confirm the potential of the TRT2 antigens for use in IGRA as an ancillary test to the skin test.

TABLE-US-00004 TABLE 3 Comparison of skin test results at defined cut off values. Naturally infected Controls Gudair vaccinated Cut off DST-C TRT-2 DST-C TRT-2 DST-C TRT-2 >=2 mm 81 [60, 92] 100 [85, 100] 0 [0, 11] 0 [0, 11] 10 [4, 26] 14 [5, 31] (17/21) (21/21) (0/30) (0/30) (3/29) (4/29) >=3 mm 71 [50, 86]  95 [77, 100] 0 [0, 11] 0 [0, 11]  0 [0, 12]  3 [0, 17] (15/21) (20/21) (0/30) (0/30) (0/29) (1/29) >=4 mm 48 [28, 68]  76 [55, 89]* 0 [0, 11] 0 [0, 11]  0 [0, 12]  0 [0, 12] (10/21) (16/21) (0/30) (0/30) (0/29) (0/29) >=5 mm 29 [14, 50]   71 [50, 86]** 0 [0, 11] 0 [0, 11]  0 [0, 12]  0 [0, 12] (6/21) (15/21) (0/30) (0/30) (0/29) (0/29) SIT 76 [55, 89] 0 [0, 11] 79 [62, 90] (16/21) (0/30) (23/29) SICCT 14 [5, 35] 0 [0, 11] 0 [0, 12] (Std) (3/21) (0/30) (0/29) SICCT 52 [32, 72] 0 [0, 11] 0 [0, 12] (Sv) (11/21) (0/30) (0/29) *p < 0.05, **p < 0.01 McNemar test (compared to DST-C).

TABLE-US-00005 TABLE 4 Peptide pool details used for in vitro PBMC screening. Mtb SEQ ID Antigen designation Name Amino acid sequence NO. Rv1789 antigenic peptide pool PPE26 Rv1789 Peptide_001 MDFGALPPEVNSVRMYAGPG 1 PPE26 Rv1789 Peptide_002 EVNSVRMYAGPGSAPMVAAA 2 PPE26 Rv1789 Peptide_003 AGPGSAPMVAAASAWNGLAA 3 PPE26 Rv1789 Peptide_004 VAAASAWNGLAAELSSAATG 4 PPE26 Rv1789 Peptide_005 GLAAELSSAATGYETVITQL 5 PPE26 Rv1789 Peptide_006 AATGYETVITQLSSEGWLGP 6 PPE26 Rv1789 Peptide_007 ITQLSSEGWLGPASAAMAEA 7 PPE26 Rv1789 Peptide_008 WLGPASAAMAEAVAPYVAWM 8 PPE26 Rv1789 Peptide_009 MAEAVAPYVAWMSAAAAQAE 9 PPE26 Rv1789 Peptide_010 VAWMSAAAAQAEQAATQARA 10 PPE26 Rv1789 Peptide_011 AQAEQAATQARAAAAAFEAA 11 PPE26 Rv1789 Peptide_012 QARAAAAAFEAAFAATVPPP 12 PPE26 Rv1789 Peptide_013 FEAAFAATVPPPLIAANRAS 13 PPE26 Rv1789 Peptide_014 VPPPLIAANRASLMQLISTN 14 PPE26 Rv1789 Peptide_015 NRASLMQLISTNVFGQNTSA 15 PPE26 Rv1789 Peptide_016 ISTNVFGQNTSAIAAAEAQY 16 PPE26 Rv1789 Peptide_017 NTSAIAAAEAQYGEMWAQDS 17 PPE26 Rv1789 Peptide_018 EAQYGEMWAQDSAAMYAYAG 18 PPE26 Rv1789 Peptide_019 AQDSAAMYAYAGSSASASAV 19 PPE26 Rv1789 Peptide_020 AYAGSSASASAVTPFSTPPQ 20 PPE26 Rv1789 Peptide_021 ASAVTPFSTPPQIANPTAQG 21 PPE26 Rv1789 Peptide_022 TPPQIANPTAQGTQAAAVAT 22 PPE26 Rv1789 Peptide_023 TAQGTQAAAVATAAGTAQST 23 PPE26 Rv1789 Peptide_024 AVATAAGTAQSTLTEMITGL 24 PPE26 Rv1789 Peptide_025 AQSTLTEMITGLPNALQSLT 25 PPE26 Rv1789 Peptide_026 ITGLPNALQSLTSPLLQSSN 26 PPE26 Rv1789 Peptide_027 QSLTSPLLQSSNGPLSWLWQ 27 PPE26 Rv1789 Peptide_028 QSSNGPLSWLWQILFGTPNF 28 PPE26 Rv1789 Peptide_029 WLWQILFGTPNFPTSISALL 29 PPE26 Rv1789 Peptide_030 TPNFPTSISALLTDLQPYAS 30 PPE26 Rv1789 Peptide_031 SALLTDLQPYASFFYNTEGL 31 PPE26 Rv1789 Peptide_032 PYASFFYNTEGLPYFSIGMG 32 PPE26 Rv1789 Peptide_033 TEGLPYFSIGMGNNFIQAAK 33 PPE26 Rv1789 Peptide_034 IGMGNNFIQAAKTLGLIGSA 34 PPE26 Rv1789 Peptide_035 QAAKTLGLIGSAAPAAVAAA 35 PPE26 Rv1789 Peptide_036 IGSAAPAAVAAAGDAAKGLP 36 PPE26 Rv1789 Peptide_037 VAAAGDAAKGLPGLGGMLGG 37 PPE26 Rv1789 Peptide_038 KGLPGLGGMLGGGPVAAGLG 38 PPE26 Rv1789 Peptide_039 MLGGGPVAAGLGNAASVGKL 39 PPE26 Rv1789 Peptide_040 AGLGNAASVGKLSVPPVWSG 40 PPE26 Rv1789 Peptide_041 VGKLSVPPVWSGPLPGSVTP 41 PPE26 Rv1789 Peptide_042 VWSGPLPGSVTPGAAPLPVS 42 PPE26 Rv1789 Peptide_043 SVTPGAAPLPVSTVSAAPEA 43 PPE26 Rv1789 Peptide_044 LPVSTVSAAPEAAPGSLLGG 44 PPE26 Rv1789 Peptide_045 APEAAPGSLLGGLPLAGAGG 45 PPE26 Rv1789 Peptide_046 LLGGLPLAGAGGAGAGPRYG 46 PPE26 Rv1789 Peptide_047 GAGGAGAGPRYGFRPTVMAR 47 PPE26 Rv1789 Peptide_048 GAGPRYGFRPTVMARPPFAG 48 Rv3478 antigenic peptide pool PPE60 Rv3478 Peptide_001 VVDFGALPPEINSARMYAGP 49 PPE60 Rv3478 Peptide_002 PEINSARMYAGPGSASLVAA 50 PPE60 Rv3478 Peptide_003 YAGPGSASLVAAAKMWDSVA 51 PPE60 Rv3478 Peptide_004 LVAAAKMWDSVASDLFSAAS 52 PPE60 Rv3478 Peptide_005 DSVASDLFSAASAFQSVVWG 53 PPE60 Rv3478 Peptide_006 SAASAFQSVVWGLTVGSWIG 54 PPE60 Rv3478 Peptide_007 VVWGLTVGSWIGSSAGLMAA 55 PPE60 Rv3478 Peptide_008 SWIGSSAGLMAAAASPYVAW 56 PPE60 Rv3478 Peptide_009 LMAAAASPYVAWMSVTAGQA 57 PPE60 Rv3478 Peptide_010 YVAWMSVTAGQAQLTAAQVR 58 PPE60 Rv3478 Peptide_011 AGQAQLTAAQVRVAAAAYET 59 PPE60 Rv3478 Peptide_012 AQVRVAAAAYETAYRLTVPP 60 PPE60 Rv3478 Peptide_013 AYETAYRLTVPPPVIAENRT 61 PPE60 Rv3478 Peptide_014 TVPPPVIAENRTELMTLTAT 62 PPE60 Rv3478 Peptide_015 ENRTELMTLTATNLLGQNTP 63 PPE60 Rv3478 Peptide_016 LTATNLLGQNTPAIEANQAA 64 PPE60 Rv3478 Peptide_017 QNTPAIEANQAAYSQMWGQD 65 PPE60 Rv3478 Peptide_018 NQAAYSQMWGQDAEAMYGYA 66 PPE60 Rv3478 Peptide_019 WGQDAEAMYGYAATAATATE 67 PPE60 Rv3478 Peptide_020 YGYAATAATATEALLPFEDA 68 PPE60 Rv3478 Peptide_021 TATEALLPFEDAPLITNPGG 69 PPE60 Rv3478 Peptide_022 FEDAPLITNPGGLLEQAVAV 70 PPE60 Rv3478 Peptide_023 NPGGLLEQAVAVEEAIDTAA 71 PPE60 Rv3478 Peptide_024 AVAVEEAIDTAAANQLMNNV 72 PPE60 Rv3478 Peptide_025 DTAAANQLMNNVPQALQQLA 73 PPE60 Rv3478 Peptide_026 MNNVPQALQQLAQPAQGVVP 74 PPE60 Rv3478 Peptide_027 QQLAQPAQGVVPSSKLGGLW 75 PPE60 Rv3478 Peptide_028 GVVPSSKLGGLWTAVSPHLS 76 PPE60 Rv3478 Peptide_029 GGLWTAVSPHLSPLSNVSSI 77 PPE60 Rv3478 Peptide_030 PHLSPLSNVSSIANNHMSMM 78 PPE60 Rv3478 Peptide_031 VSSIANNHMSMMGTGVSMTN 79 PPE60 Rv3478 Peptide_032 MSMMGTGVSMTNTLHSMLKG 80 PPE60 Rv3478 Peptide_033 SMTNTLHSMLKGLAPAAAQA 81 PPE60 Rv3478 Peptide_034 MLKGLAPAAAQAVETAAENG 82 PPE60 Rv3478 Peptide_035 AAQAVETAAENGVWAMSSLG 83 PPE60 Rv3478 Peptide_036 AENGVWAMSSLGSQLGSSLG 84 PPE60 Rv3478 Peptide_037 SSLGSQLGSSLGSSGLGAGV 85 PPE60 Rv3478 Peptide_038 SSLGSSGLGAGVAANLGRAA 86 PPE60 Rv3478 Peptide_039 GAGVAANLGRAASVGSLSVP 87 PPE60 Rv3478 Peptide_040 GRAASVGSLSVPPAWAAANQ 88 PPE60 Rv3478 Peptide_041 LSVPPAWAAANQAVTPAARA 89 PPE60 Rv3478 Peptide_042 AANQAVTPAARALPLTSLTS 90 PPE60 Rv3478 Peptide_043 AARALPLTSLTSAAQTAPGH 91 PPE60 Rv3478 Peptide_044 SLTSAAQTAPGHMLGGLPLG 92 PPE60 Rv3478 Peptide_045 APGHMLGGLPLGHSVNAGSG 93 PPE60 Rv3478 Peptide_046 LPLGHSVNAGSGINNALRVP 94 PPE60 Rv3478 Peptide_047 AGSGINNALRVPARAYAIPR 95 PPE60 Rv3478 Peptide_048 NNALRVPARAYAIPRTPAAG 96 Rv3616c antigenic peptide pool EspA Rv3616c Peptide_001 MSRAFIIDPTISAIDGLYDL 97 EspA Rv3616c Peptide_002 PTISAIDGLYDLLGIGIPNQ 98 EspA Rv3616c Peptide_003 LYDLLGIGIPNQGGILYSSL 99 EspA Rv3616c Peptide_004 IPNQGGILYSSLEYFEKALE 100 EspA Rv3616c Peptide_005 YSSLEYFEKALEELAAAFPG 101 EspA Rv3616c Peptide_006 KALEELAAAFPGDGWLGSAA 102 EspA Rv3616c Peptide_007 AFPGDGWLGSAADKYAGKNR 103 EspA Rv3616c Peptide_008 GSAADKYAGKNRNHVNFFQE 104 EspA Rv3616c Peptide_009 GKNRNHVNFFQELADLDRQL 105 EspA Rv3616c Peptide_010 FFQELADLDRQLISLIHDQA 106 EspA Rv3616c Peptide_011 DRQLISLIHDQANAVQTTRD 107 EspA Rv3616c Peptide_012 HDQANAVQTTRDILEGAKKG 108 EspA Rv3616c Peptide_013 TTRDILEGAKKGLEFVRPVA 109 EspA Rv3616c Peptide_014 AKKGLEFVRPVAVDLTYIPV 110 EspA Rv3616c Peptide_015 RPVAVDLTYIPVVGHALSAA 111 EspA Rv3616c Peptide_016 YIPVVGHALSAAFQAPFCAG 112 EspA Rv3616c Peptide_017 LSAAFQAPFCAGAMAVVGGA 113 EspA Rv3616c Peptide_018 FCAGAMAVVGGALAYLAVKT 114 EspA Rv3616c Peptide_019 VGGALAYLAVKTLINATQLL 115 EspA Rv3616c Peptide_020 AVKTLINATQLLKLLAKLAE 116 EspA Rv3616c Peptide_021 TQLLKLLAKLAELVAAAIAD 117 EspA Rv3616c Peptide_022 KLAELVAAAIADIISDVADI 118 EspA Rv3616c Peptide_023 AIADIISDVADIIKGILGEV 119 EspA Rv3616c Peptide_024 VADIIKGILGEVWEFITNAL 120 EspA Rv3616c Peptide_025 LGEVWEFITNALNGLKELWD 121 EspA Rv3616c Peptide_026 TNALNGLKELWDKLTGWVTG 122 EspA Rv3616c Peptide_027 ELWDKLTGWVTGLFSRGWSN 123 EspA Rv3616c Peptide_028 WVTGLFSRGWSNLESFFAGV 124 EspA Rv3616c Peptide_029 GWSNLESFFAGVPGLTGATS 125 EspA Rv3616c Peptide_030 FAGVPGLTGATSGLSQVTGL 126 EspA Rv3616c Peptide_031 GATSGLSQVTGLFGAAGLSA 127 EspA Rv3616c Peptide_032 VTGLFGAAGLSASSGLAHAD 128 EspA Rv3616c Peptide_033 GLSASSGLAHADSLASSASL 129 EspA Rv3616c Peptide_034 AHADSLASSASLPALAGIGG 130 EspA Rv3616c Peptide_035 SASLPALAGIGGGSGFGGLP 131 EspA Rv3616c Peptide_036 GIGGGSGFGGLPSLAQVHAA 132 EspA Rv3616c Peptide_037 GGLPSLAQVHAASTRQALRP 133 EspA Rv3616c Peptide_038 VHAASTRQALRPRADGPVGA 134 EspA Rv3616c Peptide_039 ALRPRADGPVGAAAEQVGGQ 135 EspA Rv3616c Peptide_040 PVGAAAEQVGGQSQLVSAQG 136 EspA Rv3616c Peptide_041 VGGQSQLVSAQGSQGMGGPV 137 EspA Rv3616c Peptide_042 SAQGSQGMGGPVGMGGMHPS 138 EspA Rv3616c Peptide_043 GGPVGMGGMHPSSGASKGTT 139 EspA Rv3616c Peptide_044 MHPSSGASKGTTTKKYSEGA 140 EspA Rv3616c Peptide_045 KGTTTKKYSEGAAAGTEDAE 141 EspA Rv3616c Peptide_046 SEGAAAGTEDAERAPVEADA 142 EspA Rv3616c Peptide_047 EDAERAPVEADAGGGQKVLV 143 EspA Rv3616c Peptide_048 RAPVEADAGGGQKVLVRNVV 145 Rv3810 antigenic peptide pool PirG Rv3810 Peptide_001 VPNRRRRKLSTAMSAVAALA 146 PirG Rv3810 Peptide_002 LSTAMSAVAALAVASPCAYF 147 PirG Rv3810 Peptide_003 AALAVASPCAYFLVYESTET 148 PirG Rv3810 Peptide_004 CAYFLVYESTETTERPEHHE 149 PirG Rv3810 Peptide_005 STETTERPEHHEFKQAAVLT 150 PirG Rv3810 Peptide_006 EHHEFKQAAVLTDLPGELMS 151 PirG Rv3810 Peptide_007 AVLTDLPGELMSALSQGLSQ 152 PirG Rv3810 Peptide_008 ELMSALSQGLSQFGINIPPV 153 PirG Rv3810 Peptide_009 GLSQFGINIPPVPSLTGSGD 154 PirG Rv3810 Peptide_010 IPPVPSLTGSGDASTGLTGP 155 PirG Rv3810 Peptide_011 GSGDASTGLTGPGLTSPGLT 156 PirG Rv3810 Peptide_012 LTGPGLTSPGLTSPGLTSPG 157 PirG Rv3810 Peptide_013 PGLTSPGLTSPGLTDPALTS 158 PirG Rv3810 Peptide_014 TSPGLTDPALTSPGLTPTLP 159 PirG Rv3810 Peptide_015 ALTSPGLTPTLPGSLAAPGT 160 PirG Rv3810 Peptide_016 PTLPGSLAAPGTTLAPTPGV 161 PirG Rv3810 Peptide_017 APGTTLAPTPGVGANPALTN 162 PirG Rv3810 Peptide_018 TPGVGANPALTNPALTSPTG 163 PirG Rv3810 Peptide_019 ALTNPALTSPTGATPGLTSP 164 PirG Rv3810 Peptide_020 SPTGATPGLTSPTGLDPALG 165 PirG Rv3810 Peptide_021 LTSPTGLDPALGGANEIPIT 166 PirG Rv3810 Peptide_022 PALGGANEIPITTPVGLDPG 167 PirG Rv3810 Peptide_023 IPITTPVGLDPGADGTYPIL 168 PirG Rv3810 Peptide_024 LDPGADGTYPILGDPTLGTI 169 PirG Rv3810 Peptide_025 YPILGDPTLGTIPSSPATTS 170 PirG Rv3810 Peptide_026 LGTIPSSPATTSTGGGGLVN 171 PirG Rv3810 Peptide_027 ATTSTGGGGLVNDVMQVANE 172 PirG Rv3810 Peptide_028 GLVNDVMQVANELGASQAID 173 PirG Rv3810 Peptide_029 VANELGASQAIDLLKGVLMP 174 PirG Rv3810 Peptide_030 QAIDLLKGVLMPSIMQAVQN 175 PirG Rv3810 Peptide_031 VLMPSIMQAVQNGGAAAPAA 176 PirG Rv3810 Peptide_032 AVQNGGAAAPAASPPVPPIP 177 PirG Rv3810 Peptide_033 APAASPPVPPIPAAAAVPPT 178 PirG Rv3810 Peptide_034 PPIPAAAAVPPTDPITVPVA 179

TABLE-US-00006 TABLE 5 Additional peptide and protein amino acid sequences. SEQ Mtb Peptide/ ID Antigen designation protein Amino acid sequence NO. EspA Rv3616c Peptide EADAGGGQKVLVRNVV 144 ESAT-6 Rv3875 Protein MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATEL 180 NNALQNLARTISEAGQAMASTEGNVTGMFA CFP-10 Rv3874 Protein MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQ 181 KQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF EspC Rv3615c Protein MTENLTVQPERLGVLASHHDNAAVDASSGVEAAAGLGESVAITHGPYCSQFNDTLNVYLTAHNALGS 182 SLHTAGVDLAKSLRIAAKIYSEADEAWRKAIDGLFT PPE26 Rv1789 Protein MDFGALPPEVNSVRMYAGPGSAPMVAAASAWNGLAAELSSAATGYETVITQLSSEGWLGPASAAMA 183 EAVAPYVAWMSAAAAQAEQAATQARAAAAAFEAAFAATVPPPLIAANRASLMQLISTNVFGQNTSAIA AAEAQYGEMWAQDSAAMYAYAGSSASASAVTPFSTPPQIANPTAQGTQAAAVATAAGTAQSTLTEM ITGLPNALQSLTSPLLQSSNGPLSWLWQILFGTPNFPTSISALLTDLQPYASFFYNTEGLPYFSIGMG NNFIQSAKTLGLIGSAAPAAVAAAGDAAKGLPGLGGMLGGGPVAAGLGNAASVGKLSVPPVWSGPLP GSVTPGAAPLPVSTVSAAPEAAPGSLLGGLPLAGAGGAGAGPRYGFRPTVMARPPFAG EsxS Rv3020c Protein MSLLDAHIPQLIASHTAFAAKAGLMRHTIGQAEQQAMSAQAFHQGESAAAFQGAHARFVAAAAKVN 184 TLLDIAQANLGEAAGTYVAADAAAASSYTGF PPE60 Rv3478 Protein VVDFGALPPEINSARMYAGPGSASLVAAAKMWDSVASDLFSAASAFQSVVWGLTVGSWIGSSAGLM 185 AAAASPYVAWMSVTAGQAQLTAAQVRVAAAAYETAYRLTVPPPVIAENRTELMTLTATNLLGQNTPAI EANQAAYSQMWGQDAEAMYGYAATAATATEALLPFEDAPLITNPGGLLEQAVAVEEAIDTAAANQLM NNVPQALQQLAQPAQGVVPSSKLGGLWTAVSPHLSPLSNVSSIANNHMSMMGTGVSMTNTLHSML KGLAPAAAQAVETAAENGVWAMSSLGSQLGSSLGSSGLGAGVAANLGRAASVGSLSVPPAWAAAN QAVTPAARALPLTSLTSAAQTAPGHMLGGLPLGHSVNAGSGINNALRVPARAYAIPRTPAAG PirG Rv3810 Protein VPNRRRRKLSTAMSAVAALAVASPCAYFLVYESTETTERPEHHEFKQAAVLTDLPGELMSALSQGLSQ 186 FGINIPPVPSLTGSGDASTGLTGPGLTSPGLTSPGLTSPGLTDPALTSPGLTPTLPGSLAAPGTTLAP TPGVGANPALTNPALTSPTGATPGLTSPTGLDPALGGANEIPITTPVGLDPGADGTYPILGDPTLGTI PSSPATTSTGGGGLVNDVMQVANELGASQAIDLLKGVLMPSIMQAVQNGGAAAPAASPPVPPIPAAAA VPPTDPITVPVA EspA Rv3616c Peptide MSRAFIIDPTISAIDGLYDLLGIGIPNQGGILYSSLEYFE 187 EspA Rv3616c Peptide LGIGIPNQGGILYSSLEYFEKALEELAAAFPGDGWLGSAA 188 EspA Rv3616c Peptide KALEELAAAFPGDGWLGSAADKYAGKNRNHVNFFQELADL 189 EspA Rv3616c Peptide DKYAGKNRNHVNFFQELADLDRQLISLIHDQANAVQTTRD 190 EspA Rv3616c Peptide DRQLISLIHDQANAVQTTRDILEGAKKGLEFVRPVAVDLT 191 EspA Rv3616c Peptide ILEGAKKGLEFVRPVAVDLTYIPVVGHALSAAFQAPFCAG 192 EspA Rv3616c Peptide YIPVVGHALSAAFQAPFCAGAMAVVGGALAYLVVKTLINA 193 EspA Rv3616c Peptide IISDVADIIKGTLGEVWEFITNALNGLKELWDKLTGWVTG 194 EspA Rv3616c Peptide TNALNGLKELWDKLTGWVTGLFSRGWSNLESFFAGVPGLT 195 EspA Rv3616c Peptide GATSGLSQVTGLFGAAGLSASSGLAHADSLASSASLPALA 196 EspA Rv3616c Peptide SSGLAHADSLASSASLPALAGIGGGSGFGGLPSLAQVHAA 197 EspA Rv3616c Peptide GIGGGSGFGGLPSLAQVHAASTRQALRPRADGPVGAAAEQ 198 EspA Rv3616c Peptide STRQALRPRADGPVGAAAEQVGGQSQLVSAQGSQGMGGPV 199 EspA Rv3616c Peptide VGGQSQLVSAQGSQGMGGPVGMGGMHPSSGASKGTTTKKY 200 EspA Rv3616c Peptide GMGGMHPSSGASKGTTTKKYSEGAAAGTEDAERAPVEADA 201 EspA Rv3616c Peptide KGTTTKKYSEGAAAGTEDAERAPVEADAGGGQKVLVRNVV 202 EspA Rv3616c Peptide AMAVVGGALAYLVVKTLINATQLLK 203 EspA Rv3616c Peptide VKTLINATQLLKLLAKLAELVAAAI 204 EspA Rv3616c Peptide LAKLAELVAAAIADIISDVADIIKG 205 EspA Rv3616c Peptide ESFAGVPGLTGATSGLSQVT 206 Fusion Rv3615c/ Protein MTMITPSLRRDIHMHHHHHHSMDTENLTVQPERLGVLASHHDNAAVDASSGVEAAAGLGESVAITH 207 protein* ESAT-6/ GPYCSQFNDTLNVYLTAHNALGSSLHTAGVDLAKSLRIAAKIYSEADEAWRKAIDGLFTHMTEQQWN CFP-10 FAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLA RTISEAGQAMASTEGNVTGMFAGGMAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGS LQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQ MGFHHHHHH EspA Rv3616c Protein MSRAFIIDPTISAIDGLYDLLGIGIPNQGGILYSSLEYFEKALEELAAAFPGDGWLGSAADKYAGKNR 208 NHVNFFQELADLDRQLISLIHDQANAVQTTRDILEGAKKGLEFVRPVAVDLTYIPVVGHALSAAFQAP FCAGAMAVVGGALAYLVVKTLINATQLLKLLAKLAELVAAAIADIISDVADIIKGTLGEVWEFITNAL NGLKELWDKLTGWVTGLFSRGWSNLESFFAGVPGLTGATSGLSQVTGLFGAAGLSASSGLAHADSLAS SASLPALAGIGGGSGFGGLPSLAQVHAASTRQALRPRADGPVGAAAEQVGGQSQLVSAQGSQGMGGP VGMGGMHPSSGASKGTTTKKYSEGAAAGTEDAERAPVEADAGGGQKVLVRNVV *In the fusion protein, underlined amino acids are from Rv3615c, italicised amino acids are from ESAT-6 and bold amino acids are from CFP-10

TABLE-US-00007 TABLE 6 Content of TRT1, TRT2 and DST reagents Mtb Included in Reagent Antigen designation reagent as: SEQ ID NOs: TRT1 EspA Rv3616c Rv3616c.sub.(JPT) 97-144 antigenic peptide pool ESAT-6 Rv3875 Protein 180 CFP-10 Rv3874 Protein 181 EspC Rv3615c Protein 182 PPE26 Rv1789 Protein 183 EsxS Rv3020c Protein 184 PPE60 Rv3478 Protein 185 PirG Rv3810 Protein 186 TRT2 EspA Rv3616c Rv3616c.sub.(Gen) 187-206 antigenic peptide pool ESAT-6 Rv3875 Protein 180 CFP-10 Rv3874 Protein 181 EspC Rv3615c Protein 182 PPE26 Rv1789 Protein 183 EsxS Rv3020c Protein 184 PPE60 Rv3478 Protein 185 PirG Rv3810 Protein 186 DST ESAT-6 Rv3875 Protein 180 CFP-10 Rv3874 Protein 181 EspC Rv3615c Protein 182