DIAGNOSTIC REAGENTS

Abstract

There is provided a diagnostic reagent useful to determine whether an animal has a tuberculosis infection or has been exposed to a tuberculosis agent, for example a Mycobacterium. The reagent is useful to distinguish between such an animal and an animal which has been vaccinated against a tuberculosis infection.

Claims

1. A diagnostic reagent comprising a polypeptide comprising amino acid sequence SEQ ID NO:8 or a functional variant thereof, characterised in that the diagnostic reagent elicits a negative diagnostic assay result when a tuberculosis infection assay is carried out on a sample from an animal which has been vaccinated against infection by a tuberculosis agent.

2. The diagnostic reagent of claim 1 further comprising: (i) amino acid sequence SEQ ID NO:7 or functional variants thereof; and/or (ii) at least one of the amino acid sequences SEQ ID NO:1, 2, 3, 4, 5, 6 or 9 or functional variants thereof; and/or (iii) all of SEQ ID NOs:1-7 and 9.

3. The diagnostic reagent of claim 1 further comprising: (i) amino acid sequence SEQ ID NO:10 or a functional variant thereof; and/or (ii) at least one of the amino acid sequences SEQ ID NOs:11-21 or functional variants thereof; and/or (iii) at least one of the amino acid sequences SEQ ID NOs:22-31 or functional variants thereof; and/or (iv) at least one of the amino acid sequences SEQ ID NOs:32-43 or functional variants thereof; and/or (v) at least one of the amino acid sequences SEQ ID NOs:44-49 or functional variants thereof; and/or (vi) at least one of the amino acid sequences SEQ ID NOs:50-59 or functional variants thereof; and/or (vii) at least one of the amino acid sequences SEQ ID NOs:60-69 or functional variants thereof.

4. The diagnostic reagent of claim 1 comprising amino acid sequences SEQ ID NOs:8 and 60-69, optionally comprising amino acid sequences SEQ ID NOs:7, 8, 11-42 and 50-69.

5. A method of detecting a tuberculosis infection in an animal, or exposure of an animal to a tuberculosis agent, comprising the steps of (i) contacting a population of cells from the animal with at least one diagnostic reagent as defined in claim 1; and (ii) determining whether the cells of said population recognise the diagnostic reagent; optionally wherein the population of cells includes T-cells.

6. The method of claim 5, comprising a cell-mediated immunity (CMI) assay, optionally wherein the CMI assay detects interferon gamma (IFN-y).

7. A method of detecting a tuberculosis infection in an animal, or exposure of an animal to a tuberculosis agent, comprising conducting a skin test on the animal using at least one diagnostic reagent as defined in claim 1.

8. A diagnostic kit comprising at least one diagnostic reagent as defined in claim 1.

9. The kit of claim 8 in which the diagnostic reagent is able to detect a tuberculosis infection in an animal, or exposure of an animal to a tuberculosis agent.

10. The kit of claim 9 in which the diagnostic reagent is suitable for use to differentiate between an animal having a tuberculosis infection and an animal vaccinated against tuberculosis infection.

11. A polypeptide comprising the amino acid sequence of SEQ ID NO:8 or a functional variant thereof.

12. A nucleic acid encoding the polypeptide of claim 11.

13. A vector comprising the nucleic acid of claim 12.

14. A cell transformed with the vector of claim 13.

Description

BRIEF DESCRIPTION OF FIGURES

[0048] Particular non-limiting examples of the present invention will now be described with reference to the following Figures, in which:

[0049] FIG. 1 shows the responder frequency of 23 TB-reactor (TB) and 8 BCG-vaccinated (BCG) animals to the most frequently recognized secretome peptide pools;

[0050] FIG. 2 shows the responder frequencies of 22 TB-reactor animals (open bars) and 23 BCG-vaccinated animals (filled bars) to individual secretome peptides (* indicates peptides selected for inclusion into the Sec#1 peptide pool);

[0051] FIG. 3 (A) shows the responder frequency of 22 TB-reactor and 21 BCG-vaccinated animals to the Sec#1 peptide pool (p<0.05, Fisher's Exact Test) and FIG. 3(B) shows IFN- responses (D.sub.450) from 8 TB-reactor and 3 BCG-vaccinated animals to both the Sec#1 and Sec#2 peptide pools, with the dashed horizontal line representing the cut off for a positive response;

[0052] FIG. 4 shows skin-test responses to peptide cocktails in cattle naturally exposed to M. bovis infection; and

[0053] FIG. 5 shows skin-test responses of a reference peptide cocktail containing peptides of ESAT6, CFP10 and Rv3615c either alone or in combination with a peptide cocktail of either Rv2346c (peptide pool #11) or Rv3020c (peptide pool #14) (significance between groups determined by repeated measure ANOVA, * p<0.05), in cattle naturally exposed to M. bovis infection.

EXAMPLES

Materials and Methods

Cattle

[0054] All animals were housed at the Veterinary Laboratories Agency at the time of blood sampling and procedures were conducted within the limits of a United Kingdom Home Office Licence under the Animal (Scientific Procedures) Act 1986, which were approved by the local ethical review committee. The following groups of animals were used in this study:

[0055] (i) Tuberculosis Reactors (TB-Reactors)

[0056] Heparinised blood samples were obtained from naturally infected, SICCT-positive reactors from herds known to have bovine tuberculosis (BTB) as determined by the Animal Health Agency. Heparinised blood samples were also obtained from 4 animals who were experimentally infected ca. 6 months with an M. bovis field strain from Great Britain (AF 2122/97) by intratracheal instillation of 110.sup.3 CFU as previously described (Dean et al. (2005) Infect. Immun. vol. 73 pp 6467-6471). A detailed post mortem examination of 36 TB-reactor animals revealed visible TB-lesions in all but four animals, confirming the presence of active disease.

[0057] (ii) BCG-Vaccinated

[0058] Heparinised blood samples were obtained from animals vaccinated with BCG as previously described (Vordermeier et al. (1999) Clin. Diagn. Lab. Immunol. vol. 6 pp 675-682). Briefly, calves (ca. 6 months of age) from BTB-free herds were vaccinated with BCG Pasteur by subcutaneous injection of 110.sup.6 CFU into the side of the neck.

Production and Preparation of Peptides and Antigens

[0059] 119 proteins secreted, or potentially secreted, from Mycobacterium bovis were selected for antigen screening as previously described (Jones et al. (2010) Infect. Immun. vol. 78 pp 1326-1332). Peptides were synthesized (JPT Peptide Technologies GmbH, Berlin, Germany) in pools of 20-mers overlapping by 12 amino acids for each of the genes of interest. In total, 379 peptide pools containing a total of 4129 peptides were evaluated. These peptide pools were dissolved in RPMI 1640 (Gibco, UK) containing 20% dimethyl sulfoxide (DMSO) to obtain a concentration of 1 mg/ml/peptide, and the peptide pools were used to stimulate whole blood at a final concentration of 5m/ml/peptide. Peptides that comprised the pools for some antigens were synthesized individually (Mimotopes Pty Ltd, Clayton, Australia), dissolved in RPMI 1640 containing 20% DMSO to obtain a concentration of 5mg/m1 and used individually to stimulate whole blood at a final concentration of 10 g/ml, or formulated into additional peptide pools at a concentration of 10g/ml/peptide. Peptides from ESAT-6 and CFP-10 were formulated to obtain a peptide cocktail as previously described (Vordermeier et al. (2001) Clin. Diagn. Lab. Immunol. vol. 8 pp 571-578) and were used at a final concentration of 5 g/ml/peptide. This peptide cocktail was used as a gold standard with which to compare the immunogenicities of the other antigens.

[0060] Bovine tuberculin (PPD-B) was supplied by the Tuberculin Production Unit at the Veterinary Laboratories Agency, Weybridge, Surrey, UK and was used at a final concentration of 10 g/ml. Staphylococcal enterotoxin B (SEB; Sigma-Aldritch, UK) was included as a positive control at a final concentration of 1 g/ml, while whole blood was incubated with RPMI 1640 alone as a negative control.

IFN- Enzyme-Linked Immunosorbent Assay (ELISA)

[0061] Whole blood aliquots (250 l were added in duplicate to antigen in 96-well plates and incubated at 37 C. in the presence of 5% CO.sub.2 for 24 hours, following which plasma supernatants were harvested and stored at 80 C. until required. Quantification of IFN- in the plasma supernatants was determined using the Bovigam ELISA kit (Prionics AG, Switzerland). A result was considered positive if the optical density at 450 nm (OD.sub.450) with antigen minus the OD.sub.450 without antigen (D.sub.450) was 0.1 in both of the duplicate wells.

Skin Test Evaluation of Rv2346c (Peptide Pool #11) or Rv3020c (Peptide Pool #14)

[0062] Skin-test evaluation of defined protein and peptide antigens was performed in cattle naturally exposed to M. bovis infection (n=17). These cattle were recruited as a result of providing positive responses to the single intradermal cervical comparative tuberculin (SICCT) skin-test during routine field surveillance operations.

[0063] Antigens were administered at 8 sites per animal (4 on each side of the neck). Antigens were administered in a volume of 100 l. All defined protein or peptide antigen cocktails were administered at a concentration of 5 ug/ml per cocktail component. The skin thickness was measured at the injection site immediately prior to intradermal administration of antigen. Skin thickness at the injection site was re-measured 72 hours after antigen administration and the increase in skin-thickness over this time is determined. This method is also suitable for determining the reaction in cattle uninfected with M. bovis, whether vaccinated or unvaccinated against such infection.

[0064] Purified recombinant protein antigens were supplied by Lionex GmbH. The composition of the cocktail of ESAT-6, CFP-10 and Rv3615c was SEQ ID NOs:11-43. A combination of 11 peptides providing a complete sequence overlap for each of the antigens Rv3020c and Rv2346c was used, as listed in Table 2 below (SEQ ID NOs:7 and 50-59 provide complete sequence overlap for Rv2346c and SEQ ID NOs:8 and 60-69 provide complete sequence overlap for Rv3020c). The Rv3020c and Rv2346c cocktails (i.e. peptide pools #14 and #11, respectively, as shown in Table 2) were tested individually and in combination with a cocktail containing the overlapping peptides of ESAT-6, CFP-10 and Rv3615c (i.e., SEQ ID NOs: 11-43), which is itself the subject of co-pending patent application PCT/GB2011/050843.

[0065] Results

[0066] To test the hypothesis that M. bovis secreted proteins are likely to contain immunogenic antigens that can be used to increase the specificity of diagnostic tests, 379 pools of overlapping peptides (4129 peptides in total representing 119 antigens) were screened for their ability to stimulate an IFN- response in vitro using whole blood from both TB-reactor (n=23) and BCG-vaccinated animals (n=8). As expected, all TB-reactor and BCG-vaccinated animals responded to PPD-B and to the positive control antigen SEB, whilst 22 TB-reactor animals (96%) and 2 BCG-vaccinated animals (25%) responded to the ESAT-6/CFP-10 peptide cocktail (data not shown). Of the 379 peptide pools, approximately half (n=184) failed to induce IFN- in any of the TB-reactor or BCG-vaccinated animals. For the remaining 195 peptide pools, 163 and 77 were recognized by TB-reactor and BCG-vaccinated animals respectively, with 45 being recognised by both groups of animals (Table 1). Encouragingly, with regards to differential diagnostic reagents, 118 different peptide pools were recognized by TB-reactor animals but failed to induce an IFN- response in any of the BCG-vaccinated animals studied.

[0067] A hierarchy of responses to the different peptide pools was noted, with responder frequencies ranging from 4% to 65% in the TB-reactor animals, and 13% to 38% in the BCG-vaccinated animals (Table 1).

TABLE-US-00002 TABLE 1 Recognition of the secreted antigen peptide pools. Number of peptide Number of peptide pools recognized pools not recognized (%) (%) BCG- BCG- TB-reactors vaccinated TB-reactors vaccinated All peptide pools 163 (43%) 77 (20%) 216 (57%) 302 (80%) (379 in total) TB-reactor pools 163 (100%) 45 (28%) N.A. 118 (72%) (163 in total)

[0068] FIG. 1 details the responder frequencies for the top 8 most frequently recognised peptide pools, i.e. those that induced an IFN- response in more than half of the TB-reactor animals studied. Strikingly, all but one peptide pool (#30-2) represented antigens belonging to the ESAT-6 protein family. Interestingly, peptide pools #11 and #14 were not recognized by any of the BCG-vaccinated animals, suggesting that they contain peptides with potential application as DIVA reagents. The peptides included in these pools are indicated in Table 2.

TABLE-US-00003 TABLE2 Sequencesofpeptidepools#11and#14. SEQ SEQ ID ID Sequence NO Sequence NO PeptidePool#11(Rv2346cpool) PeptidePool#14(Rv3020cpool) MTINYQFGDVDAHGAMIRAQ 50 MSLLDAHIPQLIASHTAFAA 60 DVDAHGAMIRAQAGLLEAEH 51 PQLIASHTAFAAKAGLMRHT 61 IRAQAGLLEAEHQAIVRDVL 52 AFAAKAGLMRHTIGQAEQQA 62 EAEHQAIVRDVLAAGDFWGG 53 MRHTIGQAEQQAMSAQAFHQ 63 RDVLAAGDFWGGAGSVACQE 7 EQQAMSAQAFHQGESAAAFQ 64 FWGGAGSVACQEFITQLGRN 54 AFHQGESAAAFQGAHARFVA 65 ACQEFITQLGRNFQVIYEQA 55 AAFQGAHARFVAAAAKVNTL 66 LGRNFQVIYEQANAHGQKVQ 56 RFVAAAAKVNTLLDIAQANL 67 YEQANAHGQKVQAAGNNMAQ 57 VNTLLDIAQANLGEAAGTYV 68 QKVQAAGNNMAQTDSAVGSS 58 QANLGEAAGTYVAADAAAAS 8 VQAAGNNMAQTDSAVGSSWA 59 EAAGTYVAADAAAASSYTGF 69

[0069] Although 6 out of the top 8 most frequently recognized peptide pools induced IFN- responses in some BCG-vaccinated animals, the inventors next reasoned that a fine detail investigation of the immunogenicity of the components of these pools may reveal additional individual peptides with potential use as DIVA reagents. To this end, overlapping peptides contained within these peptide pools were screened individually for their ability to induce IFN- production in both TB-reactor (n=22) and BCG-vaccinated (n=23) animals. In these experiments, 19 TB-reactor animals (86%) but no BCG-vaccinated animals (0%) responded to the peptide cocktail (data not shown). Fifty-three individual peptides were identified as immunogenic in TB-reactor animals, with responder frequencies ranging from 5% to 50% (FIG. 2). Of these peptides, six (peptides #17, #32, #48, #58, #67 and #69) also induced IFN- responses in BCG-vaccinated animals with responder frequencies ranging from 4% to 17% (FIG. 2).

[0070] In order to evaluate whether a combination of individual peptides from different secretome antigens is sufficient to differentially induce an IFN- response in TB-reactor animals, a peptide pool (Sec#1) consisting of 10 peptides was constructed. Firstly, peptides #42 and #55 (SEQ ID NOs:7 & 8, respectively) were selected as they were the two most frequently recognised peptides (responder frequencies of 50% and 36% respectively) and also because they belonged to peptide pools not recognised by BCG-vaccinated animals (pools #11 and #14 respectively, FIG. 1). A further four peptides (peptides #20, #29, #33 and #64) were next selected as they were recognised in TB-reactor animals that failed to respond to peptides #42 or #55 (data not shown). Lastly, a further four peptides (peptides #16, #19, #25 and #57) were included due to their location in regions of homology between multiple ESAT-6 proteins (see Table 3).

TABLE-US-00004 TABLE3 IdentificationofpeptidesinpoolsSec#1andSec#2 Pool Peptide SEQID Sequence Locatedinantigens: Sec#1 Pep#16 1 MWASAQNISGAGWSGMAEAT Rv1038c,Rv1197, Rv1792,Rv2347c, Rv3620c Pep#19 2 MTQMNQAFRNIVNMLHGVRD Rv1038c,Rv3620c Pep#20 3 RNIVNMLHGVRDGLVRDANN Rv1038c,Rv1197, Rv1792,Rv2347c, Rv3620c Pep#25 4 MAQMNQAFRNIVNMLHGVRD Rv1197,Rv2347c Pep#29 5 EAEHQAIIRDVLTASDFWGG Rv1198 Pep#33 6 LGRNFQVIYEQANAHGQKVQ Rv1198,Rv2346c, Rv3619c,Rv1037c, Rv1793 Pep#42 7 RDVLAAGDFWGGAGSVACQE Rv2346c,Rv1793 Pep#55 8 QANLGEAAGTYVAADAAAAS Rv3020c Pep#57 9 DVDAHGAMIRAQAGSLEAEH Rv3619c,Rv1037c Pep#64 10 SAELPDWLAANRGLAPGGHA Rv3803c Sec#2 AsabovebutomittingPep#64

[0071] As shown in FIG. 3A, the responder frequency to the Sec#1 peptide pool was significantly greater in TB-reactor animals (p<0.05, Fisher's Exact Test), with 14 out of 22 (64%) TB-reactor animals recognising the peptide pool compared with 6 out of 21 (29%) BCG-vaccinated animals. In order to optimise the peptide pool for use as a DIVA reagent, the individual peptide components of the Sec#1 peptide pool were re-screened for their ability to induce an IFN- response in BCG-vaccinated animals. These experiments identified only a single peptide (peptide #64) as being immunogenic in some BCG-vaccinated animals (data not shown). Thus, a second peptide pool (Sec#2) was constructed that lacked this peptide and the ability of both Sec#1 and Sec#2 to induce IFN- was compared in both TB-reactor animals (n=8) and BCG-vaccinated animals (n=3) which had previously demonstrated to recognise the former peptide pool. Omitting peptide #64 from the pool had little effect on the responder frequency for TB-reactor animals, with 7 of the 8 animals still producing IFN- above the cut off (FIG. 3B). Overall, 7 out of 13 (54%) TB-reactor animals produced IFN- in response to Sec#2 (data not shown). In contrast, removal of peptide #64 completely abrogated the response in all BCG-vaccinated animals tested (FIG. 3B).

[0072] The skin-test data is shown in FIG. 4. One animal failed to induce a skin-reaction in the comparative tuberculin skin-test and, similarly, this same animal provided no skin-test response to any of the defined antigen combinations. Addition of the peptide cocktail of Rv3020c (SEQ ID NOs:8 and 60-69) to the reference cocktail containing peptides of ESAT-6, CFP-10 and Rv3615c (SEQ ID NOs: 11-43) demonstrated significantly stronger responses than the reference cocktail alone, see FIG. 5.

Discussion

[0073] The results presented herein have significant importance with regards to the development of DIVA reagents. Screening of 119 proteins secreted, or potentially secreted, by M. bovis revealed three unique peptide pools that were frequently recognized by M. bovis-infected cattle but failed to induce an IFN- response in any BCG-vaccinated animals studied. Two of these peptide pools consisted of overlapping peptides that represented the full amino acid sequence for two individual antigens, Rv2346c and Rv3020c, whilst the third (Sec#2) consisted of a cocktail of 9 peptides derived from multiple antigens.

[0074] The underlying mechanism for the differential recognition of Rv2346c and Rv3020c remains unclear. Firstly, both genes are located in the genomes of M. bovis (strain AF2122/97) and, more importantly, in BCG Pasteur (strain 1173P2) which was used to immunise the cattle. Secondly, genome analysis revealed identical amino acid sequences of the two proteins between the two strains. Thus, the lack of immune responses to Rv2346c and Rv3020c seen in BCG-vaccinated animals is unlikely to be explained by deletions or amino acid sequence alterations within these two proteins in the BCG Pasteur strain used for vaccination.

[0075] It is unlikely that IFN- responses to a single protein antigen will be sufficient for the detection of M. bovis infection of cattle. Indeed, the interferon- release assay (IGRA) test for human M. tuberculosis infection utilizes synthetic peptides from two different M. tuberculosis antigens (ESAT-6 and CFP-10). With this in mind, the inventors developed a cocktail (Sec#2) containing individual peptides isolated from various peptide pools representing the most frequently recognised antigens. Interestingly, these antigens were found to belong to the ESAT-6 protein family, highlighting the immunodominance of these antigens. The Sec#2 cocktail contained several immunodominant peptides with restricted expression amongst the ESAT-6 proteins; for example, peptide #55 is located only within Rv3020c while peptide #42 is located in Rv2346c and Rv1793 (Table 3). However, given the high degree of amino acid similarity between the members of the ESAT-6 protein family, several of these peptide sequences represented multiple antigens. For example, peptides #16 and #20 are located in Rv1038c, Rv1197, Rv1792, Rv2347c and Rv3620c, while peptide #33 is located in Rv1198, Rv2346c, Rv3619c, Rv1037c and Rv1793. Thus, without wishing to be bound by theory, targeting these shared sequences not only reduces the number of different components within the DIVA reagent but may also exploit a potential greater antigenic load for these regions.

[0076] The ESAT-6/CFP-10 peptide cocktail used in the studies presented herein has been developed as a DIVA reagent in cattle, with reported sensitivities of approximately 78% in M. bovis-infected animals (Sidders et al. (2008) Infect. Immun. vo. 76 pp 3932-3939; Vordermeier et al. (2001) Clin. Diagn. Lab. Immunol. vol. 8 pp 571-578). Thus, one area of research of high importance is the identification of reagents that may complement the ESAT-6/CFP-10 peptide cocktail in the diagnosis of bovine TB. Recently, the inventors have demonstrated that 4 out of 7 (57%) M. bovis-infected animals that failed to recognise the ESAT-6/CFP-10 peptide cocktail did mount an IFN- response to the antigen Rv3615c, theoretically increasing diagnostic sensitivity to 91% without compromising specificity in BCG-vaccinated animals (Sidders et al. (2008) Infect. Immun. vo. 76 pp 3932-3939). In the current study, 5 out of 13 (38%) TB-reactor animals recognized Rv3615c (data not shown), results similar to those previously reported (Sidders et al. (2008)). All of these 5 animals recognized the Sec#2 peptide cocktail, which also induced responses in a further two animals (overall responder frequency of 54%), suggesting that the Sec#2 peptide cocktail may be as good, if not better, at complementing ESAT-6/CFP-10 in the diagnosis of bovine TB without compromising specificity in BCG-vaccinated animals.

[0077] Finally, skin test data showed that the peptides improved the sensitivity of skin test detection of M. bovis infected cattle when using a cocktail of ESAT-6, CFP-10 and Rv3615c peptides.

[0078] In summary, the results of this study demonstrate that cocktails of synthetic peptides derived from secreted or potentially secreted antigens have the capacity to distinguish between M. bovis-infected and BCG-vaccinated animals in blood-based screening assays.