Patent Publication Number: US-10309962-B2

Title: Diagnostic reagents

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This patent application is a continuation of U.S. patent application Ser. No. 14/493,950, filed Sep. 23, 2014, which is a divisional of U.S. patent application Ser. No. 13/810,945, filed Mar. 22, 2013, which is a National Stage of International Patent Application No. PCT/GB2011/051343, filed Jul. 18, 2011, which claims benefit of GB 1012072.3, filed Jul. 19, 2010, the disclosure of each is hereby incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to reagents for use in the detection of tuberculosis infections, particularly tuberculosis in mammals such as human beings and cattle, more particularly infection by Mycobacteria such as  M. tuberculosis  and  M. bovis . The reagents are useful to differentiate between animals with a tuberculosis infection and those which have been vaccinated against infection, as a positive result is only obtained from infected animals (or animals exposed to an infectious agent). 
     BACKGROUND 
       M. tuberculosis  and  M. bovis  are important pathogens of man and animals.  M. tuberculosis  is thought to infect up to a third of the world&#39;s human population, remaining undetected during a latent phase of infection and reactivating to cause 10 million cases of tuberculosis and other diseases per year, resulting in 2 million deaths (Corbett et al. (2003) Arch. Intern. Med. vol. 163 pp 1009-1021).  M. bovis , which has more than 99.9% sequence identity with  M. tuberculosis , is the predominant causative agent of bovine tuberculosis (BTB) and also causes disease in human. Cases of bovine tuberculosis in cattle caused by  M. tuberculosis  have also been reported, particularly in developing countries with high incidence rates of human TB (see, for example, Berg et al.(2009) PLoS ONE vol. 4 e5068). BTB represents a significant economic burden to the agricultural industries of various countries including the United Kingdom (Krebs (1997) “Bovine Tuberculosis in Cattle &amp; Badgers” HMSO, London, United Kingdom). 
     The primary diagnostic test used in the control and surveillance of bovine TB is the tuberculin skin-test, a test that has remained in the forefront of TB diagnosis in both man and cattle for over 100 years. The development of the test arose following the preparation of the first ‘tuberculin’ by Robert Koch in 1890. Whilst Koch&#39;s tuberculin failed to live up to its initial claims of having curative properties, its diagnostic potential was quickly realised. The most common formats of the test used in cattle are the caudal fold test (CFT), the single intradermal cervical tuberculin test (SIT) and the single intradermal comparative cervical tuberculin test (SICCT) (Monaghan et al. (1994) Vet. Microbiol. vol. 40 pp 111-24). Both test formats use a purified protein derivative (PPD) tuberculin prepared from a culture of  M. bovis  (PPD-B) as the primary diagnostic antigen. Additionally, the SICCT test includes the use of a  M. avium  derived PPD (PPD-A) to provide a measure of environmental sensitisation. It is the more specific of the two tests (Plum (1931) Cornell Vet. vol. 21 pp 68-76; Stenius (1938) Veterinary Record vol. 50 pp 633-7) and is therefore the adopted test format in the UK. 
     In addition to skin tests, blood-based diagnostic assays that measure antigen-induced lymphokine production such as the interferon gamma (IFN-γ) are also under consideration. The cytokine IFN-γ appears to be critical in the development of immunity to  M. tuberculosis . For example, both mice with a disrupted IFN-γ gene and humans with mutated IFN-γ receptor are highly susceptible to mycobacterial infections. However, specificity constraints are associated with the use of PPD in such assays. These arise due to the crude mixture of  M. bovis  proteins that PPD contains, many of which are cross-reactive with the BCG vaccine strain and environmental mycobacterial species such as  M. avium  and  M. intracellulare.    
     The term “tuberculosis infection assay” used in the present specification may refer to any of these diagnostic tests referred to above. 
     Bovine TB is a significant and ongoing problem in the UK. Cattle vaccination has been identified as one of the most promising long term UK control strategies (Krebs (1997) “Bovine Tuberculosis in Cattle &amp; Badgers” HMSO) and the development of an efficacious vaccine continues to be a research priority. Currently, promising vaccines against bovine TB are based on heterologous prime-boost combinations that include the live attenuated  M. bovis  vaccine strain Bacille Calmette-Guerin (BCG) as one of their components (Hogarth et al. (2006) J. Pharm. Pharmacol. vol. 58 pp 749-57). However, as in humans, vaccination of cattle with BCG compromises the specificity of the tuberculin skin-test since PPD contains cross reactive antigens shared by both pathogenic and vaccine strains (Berggren (1981) Br. Vet. J. vol. 137 pp 88-94; Buddle et al. (1999) Clin. Diagn. Lab. Immunol. vol. 6 pp 1-5; Waddington &amp; Ellwood (1972) Br. Vet. J. vol. 128 pp 541-52). Therefore, the development of diagnostic tests that can differentiate vaccinated from infected animals, so-called DIVA tests, are an essential pre-requisite to allow the inclusion of BCG-based vaccination as part of bovine TB control strategies. 
     Previous studies have demonstrated that diagnostic reagents which distinguish between vaccinated and infected cattle can be developed using specific, defined antigens that are present in virulent  M. bovis  but absent from the BCG. Genetic analysis of BCG has revealed that several large genomic regions have been deleted during attenuation and subsequent prolonged propagation in culture. These regions have been characterised and antigens from one of these regions, RD1, have been studied extensively in several species including humans and cattle. For example, it has been demonstrated that protein or peptide cocktails composed of two RD1 region antigens, ESAT-6 and CFP-10, can be used to distinguish between  M. bovis  infected and BCG-vaccinated cattle. In humans, synthetic ESAT-6 and CFP-10 peptides are used in an interferon-γ release assay (QUANTIFERON®-TB Gold Test) for TB diagnosis. 
     The practical application of such DIVA reagents have so far been largely realised through their use in blood-based interferon-γ (IFN-γ) release assays (IGRAs). For example, WO2009/060184 disclosed several polypeptides including epitopes from Rv3615c which were found to be useful to detect  M. bovis  or  M. tuberculosis  infection, using such an assay. This polypeptide has also been confirmed as useful to detect  M. tuberculosis  infection in humans (Millington et al. (2011) Proc. Natl. Acad. Sci. USA vol. 108 pp 5730-5735). Rv3615c is referred to herein using the  M. tuberculosis  genome annotation (genolist.pasteur.fr/TubercuList/, accessed 14 Jul. 2011). In the  M. bovis  genome, it is annotated as Mb3645c (genolist.pasteur.fr/BoviList/, accessed 14 Jul. 2011). 
     Given the high level of familiarity and wide-spread application of the tuberculin skin-test by veterinarians and clinicians, a DIVA skin-test format would provide a valuable additional test platform. This might especially be the case where the logistics of access to laboratory-based resources is problematic. It is also notable that in recent years there has also been renewed interest in a skin-test based DIVA test for human TB with several reports demonstrating the skin-test potential of ESAT-6 (Aggerbeck &amp; Madsen (2006) Tuberculosis (Edinb.) vol. 86 pp 363-73; Arend et al. (2000) J. Infect. Dis. vol. 181 pp 1850-4; Wu et al. (2008) Clin. Exp. Immunol. vol. 152 pp 81-7). 
     The present invention accordingly addresses the problem of providing discriminatory diagnostic reagents for the detection of mycobacterial infections, particularly in a DIVA skin-test format. 
     SUMMARY OF INVENTION 
     According to a first aspect of the invention, there is provided a diagnostic reagent comprising a polypeptide comprising amino acid sequence SEQ ID NO:7 or a functional variant thereof. The polypeptide may be a polypeptide which is not full length Rv2346c or Rv1793. It may, for example, comprise between 15-65 amino acids, for example, between 15-60 amino acids, 15-50 amino acids, 15-40 amino acids and may, for example, comprise at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. This polypeptide elicits a positive result when used in an assay to determine whether an animal has a tuberculosis infection or has been exposed to a tuberculosis agent. Advantageously, the diagnostic reagent can allow a user to differentiate between an animal having a tuberculosis infection and one which has been vaccinated against such an infection, as is disclosed herein for the first time and as will be explained in further detail below. Therefore, the reagent is characterised in that the 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. A negative result is also obtained when the animal is unvaccinated and uninfected (or unexposed to a tuberculosis agent). The animal may be a mammal, for example a cow, a badger or a human being. 
     The diagnostic reagent may alternatively or further comprise a polypeptide comprising amino acid sequence SEQ ID NO:8 or a functional variant thereof. The polypeptide may be a polypeptide which is not full length Rv3020c. It may, for example, comprise between 15-65 amino acids, for example, between 15-60 amino acids, 15-50 amino acids, 15-40 amino acids and may, for example, comprise at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. This polypeptide also elicits a positive result when used in an assay to determine whether an animal has a tuberculosis infection or has been exposed to a tuberculosis agent. Again, the diagnostic reagent can allow a user to differentiate between an animal having a tuberculosis infection and one which has been vaccinated against such an infection. The reagent is, therefore, characterised in that the 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. A negative result is also obtained when the animal is unvaccinated and uninfected (or unexposed to a tuberculosis agent). 
     None of the polypeptides Rv2346c, Rv1793 or Rv3020c has previously been identified as useful to differentiate between tuberculosis-infected and vaccinated animals. 
     Optionally or additionally, the diagnostic reagent may alternatively or further comprise at least one polypeptide each comprising at least one of the amino acid sequences SEQ ID NO:1, 2, 3, 4, 5, 6, 9 or 10 or functional variants thereof. 
     In one embodiment, the diagnostic reagent may comprise all of amino acid sequences SEQ ID NOs:1-9. This may be in the form of individual polypeptides each having an amino acid sequence selected from SEQ ID NOs:1-9, or may be one or more fusion proteins each comprising two or more of SEQ ID NOs:1-9. These sequences were included in peptide pool Sec#1 described herein and shown in Table 3 below. 
     The term “tuberculosis infection”, as used throughout this specification, indicates an infection in which the causative agent is a  Mycobacterium , for example,  M. tuberculosis, M. bovis , and/or  M. africanum . In some cases, the infection can be the result of exposure to a combination of these bacterial species. Likewise, the term “tuberculosis agent” indicates an organism capable of causing tuberculosic symptoms, typically a  Mycobacterium , for example  M. tuberculosis, M. bovis , and/or  M. africanum.    
     A vaccine which might be administered to an animal to vaccinate against a tuberculosis infection includes the BCG vaccine. 
     The diagnostic reagent may alternatively or further comprise a polypeptide comprising at least one of the amino acid sequences SEQ ID NOs:50-69 or a functional variant thereof. These sequences were included in peptide pools #11 and #14 described herein and shown in Table 2 below. For example, the diagnostic reagent may comprise SEQ ID NOs:7 and 50-59 and/or SEQ ID NOs:8 and 60-69. 
     In some embodiments, the diagnostic reagent may further comprise at least one polypeptide each comprising at least one of the amino acid sequences SEQ ID NOs:11-49 or functional variants thereof. By way of non-limiting example, the diagnostic reagent may comprise the polypeptides having amino acid sequences SEQ ID NOs:11, 18, 22, 30 and 45, or may comprise the polypeptides having amino acid sequences SEQ ID NOs:13, 15, 24, 46; in other words, the diagnostic reagent may comprise any combination of polypeptides which each have an amino acid sequence freely selected from those indicated by SEQ ID NOs: 11-49. 
     Sequences SEQ ID NOs:11-21 are peptides which are fragments of ESAT-6. Sequences SEQ ID NOs:22-31 are peptides which are fragments of CFP-10. Sequences SEQ ID NOs:32-43 are peptides which are fragments of Rv3615c. These sequences are discussed in co-pending application no. PCT/GB2011/050843. SEQ ID NOs:44, 45 and 46 are full-length ESAT-6, CFP-10, and Rv3615c, respectively. SEQ ID NO:47 is the protein MPB83, SEQ ID NO:48 is a fragment of MPB83 and SEQ ID NO:49 is the protein MPB70. 
     In one embodiment, the diagnostic reagent may comprise SEQ ID NOs:7, 11-42 and 50-59. Alternatively or additionally, the diagnostic reagent may comprise SEQ ID NOs:8, 11-42 and 60-69. 
     The diagnostic reagent may comprise individual peptide sequences, or may comprise one or more fusion proteins each comprising at least two amino acid sequences selected from SEQ ID NOs:1-69, preferably comprising at least one of SEQ ID NOs:7 or 8. 
     The diagnostic reagent may be for use in a method of detecting a tuberculosis infection in a mammal, or of detecting exposure of an animal to a tuberculosis agent. Advantageously, the method is capable of confirming such infection or exposure, as differentiated from vaccination. The method may be a skin test such as a caudal fold test (CFT), single intradermal test (SIT) or single intradermal comparative cervical test (SICCT). A positive result is obtained when the animal is infected with (or has been exposed to) a tuberculosis agent and a negative result is obtained if the animal is not so infected or exposed, even if the animal has been vaccinated against infection by a tuberculosis agent. 
     In some embodiments, for example for use in a skin test, the diagnostic reagent may be in the form of a sterile injectable preparation which may be an aqueous or an oleaginous suspension, or a suspension in a non-toxic parenterally-acceptable diluent or solvent. The aqueous suspension may be prepared in, for example, mannitol, water, Ringer&#39;s solution or isotonic sodium chloride solution. Alternatively, it may be prepared in phosphate buffered saline solution. The oleaginous suspension may be prepared in a synthetic monoglyceride, a synthetic diglyceride, a fatty acid or a natural pharmaceutically-acceptable oil. The fatty acid may be an oleic acid or an oleic acid glyceride derivative. The natural pharmaceutically-acceptable oil may be an olive oil, a castor oil, or a polyoxyethylated olive oil or castor oil. The oleaginous suspension may contain a long-chain alcohol diluent or dispersant, for example, Ph. Hely. 
     Therefore, according to a second aspect of the invention, there is provided 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 the first aspect of the invention; and   (ii) determining whether the cells of said population recognise the diagnostic reagent.   

     Such a method is a “tuberculosis infection assay”, as referred to above and described herein. The population of cells may include T-cells. Recognition of the diagnostic reagent by said cells may be by way of, for example, binding of a T cell receptor to the diagnostic reagent, for example, binding of the T cell receptor to at least one polypeptide included in the diagnostic reagent. The method may comprise a cell-mediated immunity (CMI) assay, which may detect interferon gamma (IFN-γ) as described herein. 
     According to a related aspect of the invention, there is provided 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 according to the first aspect of the invention. This is also considered to be a “tuberculosis infection assay” as referred to above. “Using” and “use” of polypeptides and diagnostic reagents in the skin test included in the method typically involves intradermal injection of the polypeptide(s) and/or diagnostic reagent into the animal. The skin test may be a CFT, SIT or SICCT test, as described in the Office International des Epizooties (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (ISBN-10:92-9044-718-4; or oie.int/eng/normes/mmanual/a_summry.htm, accessed 14 Jul. 2011). The manual provides information, definitions and guidelines on positive test criteria. 
     The methods according to this aspect of the invention may be for detecting  M. bovis  or  M. tuberculosis  infection in an animal, for example (but not limited to) a mammal such as a cow, badger or human being. Advantageously, because the diagnostic reagents are able to differentiate between an infected animal and a vaccinated animal, the user can be sure that a positive result from the method is a true positive indicating infection, rather than a false positive resulting from earlier vaccination of the animal. Therefore, the methods in this aspect of the invention provide 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 according to the first aspect of the invention, wherein use of the method on an animal which has been vaccinated against infection by a tuberculosis agent results in a negative skin test being obtained and use of the method on an animal infected with a tuberculosis agent results in a positive skin test being obtained. 
     According to a third aspect of the invention, there is provided a diagnostic kit comprising a diagnostic reagent according to the first aspect of the invention. The diagnostic kit may be for use in one or more methods according to the second aspect of the invention. Particularly where the kit is for use in a skin test method, the diagnostic reagent may be in liquid form, as outlined above, or may be in solid (for example, lyophilised) form. It may be included in the kit in the form of at least one aliquot of 0.05-0.15 ml containing 1-15 μg of each polypeptide contained in the diagnostic reagent. For example, the kit may comprise aliquots of about 0.05 ml, about 0.06 ml, about 0.07 ml, about 0.08 ml, about 0.09 ml, about 0.1 ml, about 0.11 ml, about 0.12 ml, about 0.13 ml, about 0.14 ml or about 0.15 ml, containing 1-15 μg, 3-12 μg, 5-10 μg of each protein, for example, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg or about 10 μg of each polypeptide. Each aliquot may be contained in a disposable injection device. The kit may further comprise at least one sample of PPD. The diagnostic reagent is able to detect a tuberculosis infection in an animal, or exposure of an animal to a tuberculosis agent, for example it may be able to detect infection by or exposure to  M. bovis  or  M. tuberculosis . Particularly when the diagnostic reagent comprises one or more of SEQ ID NOs:1-9 and/or 50-69, it enables a user to differentiate between a tuberculosis infected animal and an animal which has been vaccinated against a tuberculosis infection, by methods such as described herein. As mentioned above, this enables the user to rely on positive test results when using the diagnostic reagent as an indication of tuberculosis infection rather than of vaccination. 
     According to a fourth aspect of the invention, there is provided a polypeptide (which may be isolated) comprising or consisting of the amino acid sequence of any of SEQ ID NOs:1-10 or 50-69 or a functional variant thereof. The full length antigen polypeptides listed in Table 3 may be excluded (e.g., Rv1038c, Rv1197, Rv1792, etc.). Such a polypeptide may form a component of a diagnostic reagent according to the first aspect of the invention. The term “functional variant” indicates a polypeptide in which the amino acid sequence differs from the base sequence from which it is derived in that one or more amino acids within the sequence are substituted for other amino acids. The variant is a functional variant because the functional characteristics of the polypeptide from which the variant is derived are maintained. For example, a similar immune response is elicited by exposure of an animal, or a sample from an animal, to the variant polypeptide as compared to the non-variant. In particular, any amino acid substitutions, additions or deletions must not alter or significantly alter the tertiary structure of one or more epitopes contained within the polypeptide from which the variant is derived. The skilled person is readily able to determine appropriate functional variants without the application of inventive skill. 
     Amino acid substitutions may be regarded as “conservative” where an amino acid is replaced with a different amino acid with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type. 
     By “conservative substitution” is meant the substitution of an amino acid by another amino acid of the same class, in which the classes are defined as follows: 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Class 
                 Amino acid examples 
               
               
                   
                   
               
             
            
               
                   
                 Nonpolar: 
                 Ala, Val, Leu, Ile, Pro, Met, Phe, Trp 
               
               
                   
                 Uncharged polar: 
                 Gly, Ser, Thr, Cys, Tyr, Asn, Gln 
               
               
                   
                 Acidic: 
                 Asp, Glu 
               
               
                   
                 Basic: 
                 Lys, Arg, His. 
               
               
                   
                   
               
            
           
         
       
     
     As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that polypeptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptide&#39;s conformation. 
     As mentioned above, non-conservative substitutions are possible provided that these do not disrupt the tertiary structure of an epitope within the polypeptide, for example, which do not interrupt the immunogenicity (for example, the antigenicity) of the peptide. In particular, the addition or deletion of a small number (for example, up to about 10, or up to about 5, for example about 1, 2, 3, 4 or 5) of amino acids to the N- or C-terminus of a polypeptide may not adversely affect the immunogenicity of the peptide and such variants to an amino acid sequence are particularly envisaged as being included within the term “functional variant” of a polypeptide. 
     Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide. Suitably, variants may be at least about 50% identical, about 60% identical, for example at least about 75% identical, such as at least about 85%, 90%, 95%, 96%, 97%, 98% or about 99% identical to the base sequence, determined as a percentage of the full length of the longest of the polypeptides being compared. 
     Sequence identity between amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties. The percentage sequence identity may be determined using the BLASTP software, publicly available at blast.ncbi.nlm.nih.gov/Blast.cgi (accessible on 14 Jul. 2011), using default parameter settings. Comparison should be determined for the full length sequence of the polypeptide, to avoid high sequence identity over a short fragment of the polypeptide. 
     The invention also encompasses fusion proteins comprising more than one of SEQ ID NOs:1-10 and/or 50-69. The full length antigen proteins listed in Table 3 may be excluded (e.g., Rv1038c, Rv1197, Rv1792, etc.). 
     According to a fifth aspect of the invention, there is provided a nucleic acid encoding a polypeptide comprising at least one of the amino acid sequences SEQ ID NOs:1-10 or 50-69 or a functional variant thereof. The DNA sequence encoding the full-length antigen proteins listed in Table 3 may be excluded (e.g., the Rv1038c gene, the Ry1197 gene, etc.). The nucleic acid may form part of a vector and there is also provided a cell transformed with such a vector. 
     Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to” and do not exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. 
     Features of each aspect of the invention may be as described in connection with any of the other aspects. 
     Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith. 
     Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose. 
    
    
     
       BRIEF DESCRIPTION OF FIGURES 
       Particular non-limiting examples of the present invention will now be described with reference to the following Figures, in which: 
         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; 
         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); 
         FIG. 3A  shows the responder frequency of 22 TB-reactor and 21 BCG-vaccinated animals to the Sec#1 peptide pool (p&lt;0.05, Fisher&#39;s Exact Test) and 
         FIG. 3B  shows IFN-γ responses (ΔOD 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; 
         FIG. 4  shows skin-test responses to peptide cocktails in cattle naturally exposed to  M. bovis  infection; and 
         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&lt;0.05), in cattle naturally exposed to  M. bovis  infection. 
     
    
    
     EXAMPLES 
     Materials and Methods 
     Cattle 
     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: 
     (i) Tuberculosis Reactors (TB-reactors) 
     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 1×10 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. 
     (ii) BCG-vaccinated 
     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 1×10 6  CFU into the side of the neck. 
     Production and Preparation of Peptides and Antigens 
     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 μg/ml, or formulated into additional peptide pools at a concentration of 10μg/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 μg/ml/peptide. This peptide cocktail was used as a ‘gold standard’ with which to compare the immunogenicities of the other antigens. 
     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 μg/ml. Staphylococcal enterotoxin B (SEB; Sigma-Aldritch, UK) was included as a positive control at a final concentration of 1 μg/ml, while whole blood was incubated with RPMI 1640 alone as a negative control. 
     IFN-γ Enzyme-linked Immunosorbent Assay (ELISA) 
     Whole blood aliquots (250 μl were added in duplicate to antigen in 96-well plates and incubated at 37° C. in the presence of 5% CO 2  for 24 hours, following which plasma supernatants were harvested and stored at −80° C. until required. Quantification of IFN-γ 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 450 ) with antigen minus the OD 450  without antigen (ΔD 450 ) was ≥0.1 in both of the duplicate wells. 
     Skin Test Evaluation of Rv2346c (Peptide Pool #11) or Rv3020c (Peptide Pool #14) 
     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. 
     Antigens were administered at 8 sites per animal (4 on each side of the neck). Antigens were administered in a volume of 100 μl. 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. 
     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. 
     Results 
     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-γ 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-γ 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-γ response in any of the BCG-vaccinated animals studied. 
     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 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) 
               
               
                   
               
            
           
         
       
     
       FIG. 1  details the responder frequencies for the top 8 most frequently recognised peptide pools, i.e. those that induced an IFN-γ 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 2 
               
             
            
               
                   
               
               
                 Sequences of peptide pools #11 and #14. 
               
            
           
           
               
               
               
               
            
               
                   
                 SEQ 
                   
                 SEQ 
               
               
                   
                 ID 
                   
                 ID 
               
               
                 Sequence 
                 NO 
                 Sequence 
                 NO 
               
               
                   
               
            
           
           
               
               
            
               
                 Peptide Pool #11 (Rv2346c pool) 
                 Peptide Pool #14 (Rv3020c pool) 
               
            
           
           
               
               
               
               
            
               
                 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 
               
               
                   
               
            
           
         
       
     
     Although 6 out of the top 8 most frequently recognized peptide pools induced IFN-γ 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-γ 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-γ responses in BCG-vaccinated animals with responder frequencies ranging from 4% to 17% ( FIG. 2 ). 
     In order to evaluate whether a combination of individual peptides from different secretome antigens is sufficient to differentially induce an IFN-γ 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 &amp; 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 3 
               
             
            
               
                   
               
               
                 Identification of peptides in pools Sec#1 and Sec#2 
               
            
           
           
               
               
               
               
               
            
               
                 Pool 
                 Peptide 
                 SEQ ID 
                 Sequence 
                 Located in antigens: 
               
               
                   
               
               
                 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 
                 As above but omitting Pep#64 
               
               
                   
               
            
           
         
       
     
     As shown in  FIG. 3A , the responder frequency to the Sec#1 peptide pool was significantly greater in TB-reactor animals (p&lt;0.05, Fisher&#39;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-γ 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-γ 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-γ □ above the cut off ( FIG. 3B ). Overall, 7 out of 13 (54%) TB-reactor animals produced IFN-γ 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 ). 
     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 
     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-γ 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. 
     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. 
     It is unlikely that IFN-γ responses to a single protein antigen will be sufficient for the detection of  M. bovis  infection of cattle. Indeed, the interferon-γ 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. 
     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-γ 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. 
     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. 
     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.