EA041840B1 - DIAGNOSTIC REAGENTS FOR IMPROVED IN VIVO OR IN VITRO CELL-MEDIATED IMMUNOLOGICAL DIAGNOSIS OF TUBERCULOSIS - Google Patents

Description

The present invention discloses compositions for use as a pharmaceutical or diagnostic reagent for improved in vivo or in vitro cell-mediated immunological diagnosis of tuberculosis in a human or animal. The present invention relates to antigenic combinations that increase sensitivity (resulting in fewer false negatives) over existing antigenic combinations without compromising specificity (false positive rates). In particular, the present invention relates to antigenic compositions, not including the 6 kDa secretory early antigen (ESAT-6) antigen currently used in registered products to detect Mycobacterium tuberculosis infection. Alternatively, novel diagnostic or immunogenic compositions may be used in combination with ESAT-6 to further enhance the sensitivity of cell-mediated diagnosis of tuberculosis.

Background of the invention

Tuberculosis (TB) is the leading cause of morbidity and mortality in the world. It is estimated that one person falls ill with TV every four seconds, and every 20-30 seconds someone will die from the disease. In addition, approximately 1/3 of the world's population is latently infected with Mycobacterium tuberculosis (M.tuberculosis), the causative agent of TB.

Immunocompetent individuals infected with M.tuberculosis typically have a 10% lifetime risk of developing active TB disease, with the risk greatly increased, for example, if the individual is co-infected with HIV (Sonnenberg, 2005) or has diabetes (Young, 2009). If left untreated, each person with active pulmonary TB will infect 10-15 people each year (World Health Organization Tuberculosis Fact Sheet No 104, 2002). Thus, it is important to be able to identify M. tuberculosis infected individuals at an early stage of infection in order to prevent the development of M. tuberculosis infection into an active contagious form of pulmonary TB. This can be achieved by prophylactic treatment at the earliest time point after diagnosis. Thus, rapid and accurate diagnosis of M. tuberculosis infection is an important element of world health efforts to control the disease.

Diagnostic assays currently used to determine M. tuberculosis infection include culture, microscopy and PCR of the relevant patient, chest x-ray, standard tuberculin skin test (TST) and interferon gamma release assays (IGRA). The first three methods are used to diagnose active contagious TB and are based on the identification of M. tuberculosis bacteria and thus depend on the presence of bacteria in the sample. This requires a certain bacterial load and access to infectious material, and these methods are therefore not applicable for early diagnosis of infection, i.e. before the onset of clinical manifestations of the disease. The chest x-ray is insensitive and is only useful for pulmonary TB and for detecting TB at a later stage.

The standard tuberculin skin test, which shows a delayed-type hypersensitivity (DTH) reaction, is a simple and inexpensive test based on immunological recognition of mycobacterial antigens in exposed individuals. However, it is far from ideal in detecting M.tuberculosis infection. It uses the intradermal administration of a purified protein derivative (PPD), which is a crude and poorly detectable mixture of mycobacterial antigens, some of which are shared with Bacillus Calmette-Guerin (BCG) proteins, to vaccinate the M. bovis substrain and non-tuberculous environmental mycobacteria. This broad cross-reactivity of PPD is the reason for the poor specificity of TST, leading to a situation where vaccination with BCG and exposure to non-tuberculous mycobacteria gives a test result similar to that observed in M. tuberculosis infected individuals.

M. tuberculosis infection mediates a pronounced cell-mediated immune (CMI) response and detection of immune cells and any product derived from such cells that form as part of a specific response to M. tuberculosis infection would be an appropriate way to detect infection (Andersen, 2000 ). In order to obtain such a specific response, the reagent must 1) be widely recognized by M. tuberculosis infected individuals, and 2) be specific for M. tuberculosis, thereby distinguishing between TB infection, BCG vaccination, and exposure to non-tuberculous mycobacteria in the environment.

The M.tuberculosis genome suggests 4018 open reading frames (http://tuberculist.epfl.ch/, issue R27 - March 2013). However, only a minority of these are T-cell antigens with strong and broad recognition by peripheral blood mononuclear cells (PBMCs) from human patients with TB. Algorithms have been developed to predict T cell epitopes, but experimental validation is needed to identify immunodominant CFP10 and ESAT-6 antigens. The extracellular or culture filtrate (CF) proteins of M. tuberculosis constitute a protein fraction enriched in T-cell antigens (Andersen 1994) and separation of CF proteins using a two-dimensional proteomics approach led to the identification of 59 human T-cell antigens, of which 35 have been described previously (Deenadayalan, 2010). Although the list may not be exhaustive, it highlights that only a small fraction of the approximately 900 CF proteins (Albrethsen,

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2013) are T cell antigens. Logically, it would be expected that the most immunodominant antigens encoded by the M.tuberculosis genome would represent the most expressed subset, and it would be possible with current technologies to rank genes according to transcription levels under certain growth conditions. However, the contribution that transcription level makes to immunogenicity is low and cannot be used systematically to determine exactly which genes encode relevant antigens (Sidders, 2008).

A candidate for a highly specific reagent could be sought among antigens from the RD regions (deletion regions) of the M. tuberculosis genome. These regions represent genomic deletions from the BCG vaccine M. bovis strain against the virulent M. tuberculosis strain (Behr, 1999). Thus, theoretically, proteins from these regions (RD proteins) would be excellent candidates as TB diagnostic reagents, i.e. they would not be recognized by healthy, uninfected individuals, regardless of their BCG vaccination status or exposure to non-pathogenic mycobacterial strains. However, of all the predicted genomic ORFs (open reading frames) removed from BCG, it is essentially unknown which are actually expressed as proteins, and furthermore, immunoreactivity remains unknown until tested with sensitized lymphocytes from M. tuberculosis individuals. This can be done, for example, by testing whole blood, by restimulating the PBMC, or by injecting into the skin substances like those injected in the PPD/Mantoux test. For example, when evaluating Rv3872, small interferon gamma (IFN-γ) responses were shown in human test subjects with TB (n=7) 1-4 months after diagnosis (WO 99/24577), indicating that this protein RD is often not recognized, and Rv3872 has thus not been further investigated as a CMI-based diagnostic test.

Potential specific M. tuberculosis proteins are not limited to RD proteins, as for example the EspC protein (Rv3615c) is specifically recognized in human TB patients and M. bovis infected cattle, but not in BCG vaccinated/infected, even though the gene present in BCG (WO2009060184; Sidders, 2008; Millington, 2011). The lack of response in BCG vaccinated individuals is most likely due to the fact that Rv3615c secretion is aborted in BCG, since it depends on the ESX-1 secretion system, which is partly located at the RD1 locus and which is absent in BCG. This antigen is a CMI diagnostic reagent that is considered to be as strong as ESAT-6 and CFP10 in inducing a pronounced IFN-γ response (ibid.).

Once a potential specific T cell antigen has been identified, it must be verified that the antigen is specifically recognized in M. tuberculosis infected individuals, but not in BCG vaccinated individuals. With respect to Rv3873 from RD1, the protein appeared to be a member of a family of proteins with a conserved motif at amino acids 118-135 also present in other M. tuberculosis proteins. Since a widely recognized T cell epitope was present in this motif, BCG vaccinated individuals also responded to a peptide comprising this sequence (Liu, 2004). However, cross-reactivity was also observed for Rv3878 and Rv3879c, although no homology was found when compared with other known mycobacterial protein sequences (Liu, 2004), emphasizing that the specificity of a potential diagnostic candidate needs to be tested experimentally. Similarly, Rv2653c from RD13 was recognized in both BCG-vaccinated donors and TB patients, despite the fact that BLAST database searches did not reveal any apparent mycobacterial protein that could explain the observed cross-reactivity (Aagaard, 2004).

Having identified specific M.tuberculosis proteins with the potential to diagnose M.tuberculosis infection using CMI-based assay, it remains to be explored whether a pool of such proteins/peptides would provide the preferred sensitivity for the diagnostic test. It is not possible to predict the contribution of a particular antigen to susceptibility after combination with already defined diagnostic antigens; this needs to be experimentally evaluated for each antigen, and preferably in TB patients from different regions with different genetic backgrounds.

The diagnostic potential of CFP10 (Rv3874), ESAT-6 (Rv 3875), two small molecular weight proteins from the RD1 region and TB7.7 (Rv2654) is very well described (Brock, 2004; Moon, 2013, WO2004099771) and is currently used in various diagnostic reagents registered for use in humans. Peptides comprising CFP10 and ESAT-6 are used in the T-SPOT®.TB assay, which is a blood cell assay that detects the immune response of T cells found in PBMCs that have been restimulated by ESAT-6 and CFP10. This response is detected by a highly sensitive enzyme immunostain assay, designated ELISPOT, and commercialized as the T-SPOT.TB assay. This test is highly sensitive and independent of BCG vaccination status. Another registered test for the detection of M. tuberculosis infection is QuantiFERON®-TB Gold, which is an in-vitro diagnostic technology that allows the detection of immune responses in whole blood samples when re-stimulated with peptides including ESAT-6, CFP10 and one peptide from TV7.7. Both of these tests measure interferon-gamma production.

- 2 041840 (IFN-γ) in response to exposure to pools of selective specific antigenic peptides and are currently considered the most modern. The T-SPOT.TB assay and QuantiFERON®-TB are generally recognized as IFN-γ release assays (IGRAs).

Other cytokines and chemokines other than IFN-γ have also been of relevance in monitoring the immunological response to mycobacterial antigens. IFN-γ induced protein (IP10) is expressed at 100-fold levels compared to IFN-γ, and diagnostic assays based on IP-10 secretion have comparable diagnostic capabilities to IFN-γ release assays (Ruhwald, 2009).

In addition to these in vitro assays, which are already registered for use in humans and used worldwide, ESAT-6 and CFP10 have also proven to be effective as intradermal test reagents. Clinical studies have shown that the intradermal test, used in the same way as PPD, but using ESAT-6 and CFP10 produced and delivered as recombinant proteins, can be used to diagnose M. tuberculosis infection and is not affected by BCG vaccination status (Aggerbeck , 2013).

Despite the widespread use of BCG and several diagnostic modalities, including IGRA, TB continues to cause nearly two million deaths per year, and thus there is a further need for the development of immunodiagnostic tests with increased sensitivity. Immunocompromised patients are at increased risk of developing TB and, unfortunately, both TST and IGRA in their present form exhibit suboptimal properties in these groups. Patient groups with the greatest need for improved testing include: HIV-infected patients, patients with immune-mediated inflammatory diseases, patients receiving immunosuppressive treatment (eg, prednisolone or TNF-α inhibitors), and patients with chronic renal failure. For example, among those infected with HTV, low CD4 cell counts (eg, <250 cells/µl) are well known to be significantly associated with higher levels of test indeterminacy, impaired sensitivity of active TB tests, and reduced likelihood of positive test responses in exposed individuals. as described in (Redelman-Sidi, 2013).

Children are another very relevant group for targeted testing. Diagnosis of latent TB infection (LTBI) and TB in children is difficult, microbiological confirmation of infection is often not obtained, and treatment is directed only at clinical manifestations. In both actively and presumably latently infected young children, the immune system is immature, and this is a likely cause of less cytokine release and impaired IGRA conduction. The QuantiFERON©-TB Gold test was recently shown to have a sensitivity of 53% in 81 children with microbiologically confirmed TB, indicating the need for improved immunodiagnostic tests for M. tuberculosis infection in children (Schopfer, 2013).

The main problem with performing the IGRA test in the high-risk patient populations mentioned above (eg, immunocompromised, HTV-infected children) is that the underlying immunosuppressive condition that leads to an increased risk of TB disease is itself characterized by low CMI responses. and low release of IFN-γ in response to antigens. Since the IGRA result is determined by comparing the amount of IFN-γ release with the cutoff value, impaired IFN-γ release increases the risk that the test result will become a false negative. Thus, one skilled in the art would appreciate that the incorporation of more specific antigens will recruit more specific T cells and result in an enhanced CMI response and IFN-γ release and subsequently lower risk of a response below the cutoff. Thus, the addition of additional specific antigens addresses the major limitation in IGRA tests of improved diagnostic sensitivity.

Another benefit of diagnosing M. tuberculosis infection based on higher response values is improved analytical accuracy and more reliable test results. In the QuantiFERON®-TB Gold assay, the positive test cutoff is 0.35 IU/mL or 17.5 pg/mL, i.e. very low concentration, which is difficult to determine with high accuracy - even with sensitive methods such as ELISA. For example, the largest IGRA accuracy study to date found significant variability in response in TB as measured by the QuantiFERON-TB Gold In-Tube in a repeat study of the same patient sample. Within-individual variability included differences of up to 0.24 IU/mL in each direction, while the initial response ranged from 0.25 to 0.80 IU/mL. This led to the conclusion that positive QuantiFERON TB Gold In-Tube test results of less than 0.59 IU/mL should be interpreted with caution (Metcalfe AJRCCM 2012).

Modeling studies indicate that without new vaccines, TB cannot be eradicated, and new and more effective vaccines are an international priority. The general idea is to supplement the currently used BCG vaccine with a booster subunit vaccine or create a new live TB vaccine to replace BCG. There is an increasing number of experimental vaccines in clinical development and the promising general consensus is that ESAT-6 appears to be a necessary vaccine antigen. Thus, many of the new vaccines currently in preclinical or clinical trials contain ESAT-6. Recently, the Aeras Foundation announced the first human trial of a vaccine containing ESAT-6 designed to protect people already latently infected with TB from developing active TB disease (Aagaard, 2011). Some live vaccine candidates have also been developed directly by recombinant engineering to express ESAT-6, such as rBCG:GE (Yang, 2011), rM.S-e6c10 (Zhang, 2010), Salraonella/Ag85B-ESAT-6 (Hall, 2009), rBCG-A(N)-EA(C) (Xu, 2009) or fusion proteins including ESAT-6, such as HI (van Dissel, 2010; van Dissel, 2011). Unfortunately, the use of ESAT-6 based diagnostics in the IGRA test and vaccination with an ESAT-6 containing vaccine is a replica of the cross-reaction problem associated with the concurrent use of TST and BCG.

Therefore, there is a great need for a specific diagnostic reagent that can be used in parallel with vaccines containing both BCG and ESAT-6. When used in vivo or in vitro assays, the reagent should be able to detect M. tuberculosis infections in humans and animals and distinguish not only between TB infection and vaccination with BCG or new ESAT-6 containing vaccines, but also exposure to non-pathogenic mycobacteria of the environment. The diagnostic reagent should be at least as sensitive as the currently used combination of ESAT-6, CFP10 and in some TB7.7 diagnostic assays.

EP 2417456 describes such a system where the use of Rv3615c in conjunction with CFP-10 provides a diagnostic sensitivity that is very similar to the ESAT-6/CFP-10 combination.

Because the unique characteristics of ESAT-6 are highly immunogenic and specific for M. tuberculosis infection, it is unlikely that substituting ESAT-6 with a single antigen will increase sensitivity over ESAT when studying different population groups. This has been demonstrated, for example, by Brock et al., who showed that antigen recognition alone was 1443% in TB patients compared to ESAT-6 which responded in 75% in the same group of patients. Given that most antigens are less immunogenic compared to ESAT-6, it is more likely that an antigen pool is needed for higher magnitude responses and improved diagnostic sensitivity.

According to the examples, it is difficult to predict the sensitivity and specificity of antigenic combinations; on the contrary, this requires detailed development of specific antigenic combinations. In addition, with an increase in the number of peptides in the diagnostic pool, an additional risk of reduced specificity with an increase in the number of false positives is introduced, emphasizing that diagnostic or immunogenic compositions for the specific diagnosis of TB need careful selection and research.

Thus, there is an urgent need for improved in vivo or in vitro cell-mediated immunological diagnosis of M. tuberculosis infection in humans or animals. There is a need for antigenic combinations that increase sensitivity (leading to fewer false negatives) over existing antigenic combinations without compromising specificity (false positive rates). The desired improved antigen combinations include both antigenic compositions that do not include the ESAT-6 antigen to predict the situation when a vaccine containing ESAT-6 is administered, and antigenic combinations containing ESAT-6 to improve existing diagnostic reagents.

The data demonstrate that combinations of CFP10/ESAT6 and CFP10/Rv3615 can be further improved by adding peptides derived from three novel antigens with diagnostic potential. This new result is unexpected for two reasons:

a) Most (>99%) antigens for the TB genome are non-specific and common across different mycobacterial species, so identifying highly recognizable antigens that are specific for Mycobacterium tuberculosis has been very difficult

b) The sensitivity of the diagnostic combination CFP10/RV3615c and CFP10/ESAT6 is already very high, so additional sensitivity enhancement becomes increasingly difficult due to non-specific responses.

The present invention is thus very encouraging as it describes peptides with the ability to not only sensitize the CFP10/RV3615c combination, but also the currently used diagnostic cocktail that includes ESAT6 without compromising specificity.

Brief description of the invention

The present invention relates to improved detection of infections caused by species of the TB complex (M. tuberculosis, M. bovis, M. africanum) and the distinction between infection with TB and vaccination. The improved diagnostic composition should not interfere with the effect of antigens from 1) a new ESAT-6 containing TB vaccine, 2) BCG, or 3) exposure to non-pathogenic mycobacteria in the environment. infusion

- 4 041840 The present invention discloses improved diagnostic or immunogenic compositions that can be used either in vivo or in vitro to detect a cellular response to M. tuberculosis infection and thereby be used to diagnose TB. Using a cocktail or pool of antigens, or a cocktail or pool of peptides comprising these antigens, a highly sensitive test was developed despite the absence of ESAT-6 in diagnostic or immunogenic compositions. In addition, ESAT-6-containing diagnostic immunogenic compositions currently in use have been further improved.

Detailed description of the invention

This diagnostic method is based on cell-mediated immunological (CMI) recognition of antigens expressed by M. tuberculosis bacteria (or other mycobacteria from the tuberculosis complex) during infection. Thus, the test does not require the presence of bacteria in the form of traditional culture, microscopic or PCR methods. This means that the test can be used early in the infection and that the test is applicable regardless of the anatomical location of the infection. The method is ideal for contact tracing as a replacement for the currently used TST.

By selecting M. tuberculosis specific antigens with theoretical diagnostic potential and examining recognition in a range of human TB patients, it has been possible to identify three diagnostic pools that 1) lack ESAT-6 and thus can also be used in ESAT-6 vaccinated individuals. to distinguish between M. tuberculosis infection and vaccination, 2) had the same high specificity as diagnostic pools containing ESAT-6, and 3) had superior sensitivity to M. tuberculosis infection than that obtained with the combination of ESAT-6, CFP10 and TV7.7.

The present invention discloses a diagnostic or immunogenic composition comprising a mixture of substantially pure polypeptides consisting of amino acid sequences selected from:

a) Rv3874 (SEQ ID NO1), Rv3615 (SEQ ID NO 2) and additional compositions selected from Rv3865 (SEQ ID NO 3), Rv2348 (SEQ ID NO 4), Rv3614 (SEQ ID NO 5), Rv2654 (SEQ ID NO 6) and Rv3877 (SEQ ID NO 7); or b) mixtures of fragments of said polypeptides; or c) where in the selected mixture the sequences of polypeptides or fragments of these polypeptides are at least 80% identical to any of the polypeptides from set a) or b), and it is immunogenic.

In circumstances where vaccines containing ESAT-6 will not be approved for use in humans, in areas where vaccines containing ESAT-6 are not expected to be used, and in other circumstances, for example, to further increase the sensitivity of a diagnostic test, any of the above diagnostic or immunogenic compositions can be supplemented with ESAT-6 (SEQ ID NO 51) or one or more fragments thereof.

A preferred diagnostic composition contains a mixture of fragments containing immunogenic epitopes of Rv3874, Rv3615 and optionally ESAT-6, where fragments containing immunogenic epitopes from SEQ ID NO 1 are selected from SEQ ID NO 9-14 and fragments containing immunogenic epitopes from SEQ ID NO 2 are selected from SEQ ID NOs 15-18 or SEQ ID NOs 59-63 and fragments containing immunogenic epitopes from SEQ ID NO 51 are selected from SEQ ID NOs 52-58, where fragments containing immunogenic epitopes from SEQ ID NO 3, selected from SEQ ID NO 19-21 and fragments containing immunogenic epitopes from SEQ ID NO 4 are selected from SEQ ID NO 22-25 and fragments containing immunogenic epitopes from SEQ ID NO 5 are selected from SEQ ID NO 26-45 and where fragments containing immunogenic epitopes from SEQ ID NO 6 are SEQ ID NO 8 and fragments containing immunogenic epitopes from SEQ ID NO 7 are selected from SEQ ID NO 46-50.

The polypeptides in the diagnostic or immunogenic composition may be present as separate structures or when some or all of the polypeptides are fused together, optionally via linkers or spacers.

A preferred diagnostic or immunogenic composition comprises a pool or mixture of SEQ ID NOs 9-14, SEQ ID NOs 15-18, SEQ ID NOs 19-21, and SEQ ID NOs 22-25 mentioned in the examples as peptide pool A.

Detailed Description of Preferred Polypeptides and Fragments of Said Polypeptides

CFP10 (SEQ ID NO 1) is the core protein of ESX-1. The following 6 peptides were selected, including all amino acid sequences of CFP10 (SEQ ID NO: 9-14)

Rv3615c (SEQ ID NO: 2) is a protein secreted by the ESX-1 system. Were selected 4 peptides, including amino acids 55-103 (SEQ ID NO: 15-18). Alternative peptides comprising the C-terminal portion of Rv3615 are the five peptides with the amino acid sequence of SEQ ID NOs: 59-63.

Rv3865 (SEQ ID NO: 3) is a protein associated with the secretion of ESX-1: 3 peptides were selected, including amino acids 9-44 (SEQ ID NO: 19-21). Rv2348c (SEQ ID NO: 4) is located in a region of RD7 that was shown to be absent from BCG: 4 peptides were selected including

- 5041840 amino acids 56-109 from the full length protein sequence (SEQ ID NOs: 22-25).

Rv3614c (SEQ ID NO: 5) is a secreted protein: 20 peptides were selected to include the entire sequence (SEQ ID NO 26-45). Rv2654c (SEQ ID NO: 6)) is a protein of unknown function encoded by the RD11 region: 4 peptide (SEQ ID NO: 8) was selected.

Rv3877 (SEQ ID NO: 7) is located in the RD1 region and is not present in BCG: 5 peptides were selected comprising amino acids 220-284 in the full length protein (511 aa) (SEQ ID NO: 46-50)

ESAT-6 (Rv3875; SEQ ID NO: 51) is the core protein of ESX-1. Were selected 7 peptides, including the entire sequence (SEQ ID NO: 52-58).

The present invention further discloses the use of diagnostic or immunogenic compositions for the preparation of a pharmaceutical composition for diagnosing TB caused by virulent mycobacteria, for example, M. tuberculosis, Mycobacterium bovis or Mycobacterium africanum, or a CMI diagnostic agent or kit containing the diagnostic or immunogenic composition mentioned above, for in vitro or in vivo TB diagnostics.

The present invention also discloses in vitro and in vivo methods for diagnosing TB caused by virulent mycobacteria, for example, M. tuberculosis, Mycobacterium africanum or Mycobacterium bovis, in an animal, including a human, using the diagnostic or immunogenic compositions mentioned above.

An in vivo method for diagnosing TB involves intradermal injection of an animal, including a human, with a pharmaceutical composition as defined above, wherein a positive skin reaction at the injection site indicates that the animal has TB and a negative skin reaction at the injection site indicates that that the animal does not have a TV.

An in vitro method for diagnosing TB involves contacting a sample, eg, whole blood, with a diagnostic composition of the present invention to detect a positive response, eg, cell proliferation or release of cytokines such as IFN-γ.

The present diagnostic and immunogenic compositions can replace compositions currently used in conventional IGRA tests (CFP10/Rv3874 and ESAT-6/Rv3875 in TB.SPOT®.TB and TB7.7/Rv2654c, CFP10/Rv3874 and ESAT-6 /Rv3875 in QuantiFERON®-TB Gold.

The method further includes the following improvement over CFP10/Rv3874 and ESAT6/Rv3875 in TB.SPOT®.TB and TB7.7/Rv2654c, CFP10/Rv3874 and ESAT-6/Rv3875 in QuantiFERON®-TB Gold:

If an individual has been vaccinated with an ESAT-6 containing vaccine, such as a subunit protein vaccine containing ESAT-6 or an engineered recombinant live vaccine or expressing ESAT-6 by itself, then the composition avoids the use of ESAT-6 and is thus, it remains specific for M. tuberculosis infection. This does not apply to CFP10 and ESAT-6 in TB.SPOT®.TB and TB7.7, CFP10 and ESAT-6 in QuantiFERON®-TB Gold or any other ESAT-6 based test.

Compositions containing ESAT-6 are used in CMI tests against M. tuberculosis. The present test avoids the use of ESAT-6 and has the advantage of broad recognition resulting from the use of several M. tuberculosis-specific antigens. In a test with a combination of CFP10, Rv3615c, Rv3865 and Rv2348 (peptide pool A), a sensitivity of 87% and specificity of 98% is obtained compared with a sensitivity of 74% and a specificity of 96% when using quantiferon antigens. Thus, despite the absence of ESAT-6, which is known to be a highly sensitive antigen and recognized by a large proportion of individuals with M. tuberculosis infection, the compositions tested in this document are characterized by a sensitivity level that is 10% higher compared to well-known compositions. based on ESAT-6 and currently used in IGRA assays.

This document also presents data showing that by adding ESAT-6 to the peptide pool consisting of CFP10, Rv3615c, Rv3865 and Rv2348, diagnostic capabilities can be further improved. By adding ESAT-6 to a specific peptide pool, one can increase the magnitude of responses that might be relevant for diagnosis in individuals with various immunosuppressive complications, eg HIV, or for use in, eg, children. Also, when using a combination of peptides from all five antigens (CFP10, ESAT-6, Rv3865, Rv2348, and Rv3615c), it would be possible to increase the incidence of patients with a confirmed diagnosis of TB by 3% compared with using only a pool of CFP10, Rv3615c, Rv2348, and Rv3865.

The present invention also discloses an in vivo TB diagnostic assay. It could be carried out in the form of a skin test on an animal, including a human, using the compositions mentioned above. When this skin test is carried out by intradermal injection of the animal or application to the skin of animals, for example, using a patch or dressing, compositions according to the present invention. A positive skin reaction at the site of injection or application indicates that the animal or human has TB, and a negative skin reaction at the site of injection or application indicates that the animal does not have TB.

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It is not necessary that the peptide pools of the present invention contain full length proteins, since a sequence of only 6-9 amino acids (T cell epitope) is sufficient to elicit an immune response, however, full length proteins will also be applicable. Since it is possible for a person skilled in the art to determine the exact and minimum amino acid sequence for a T-cell epitope included in a protein, the present invention also relates to fragments (immunogenic regions) of polypeptides containing these T-cell epitopes (or their analogues) without specific additional amino acids in the form of proteins. full length and fusion proteins containing these T-cell epitopes (optionally connected by a linker or spacer), and to cocktails or pools containing such polypeptides or fusion proteins.

Additional embodiments of the present invention are described in the examples and in the claims.

Definitions Polypeptides

The word polypeptide in the present invention should have its conventional meaning. It means an amino acid chain of any length, including primary translation product, oligopeptides, short peptides and fragments thereof, where amino acid residues are linked by covalent peptide bonds. The polypeptide can be chemically modified by glycosylation, lipid fusion, for example by chemical lipidation with palmitoyloxysuccinimide as described by Mowat et al. (Mowat, 1991), or with dodecanoyl chloride as described (Lustig, 1976), inclusion of prosthetic groups , or the content of additional amino acids, such as, for example, labeled with histidine or a signal peptide.

Thus, each polypeptide can be characterized by specific amino acids, and encoded by specific nucleic acid sequences. It will be understood that such sequences include analogs and variants produced by recombinant or synthetic methods, in which such polypeptide sequences are modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide, and still be immunogenic in any of the biological assays described in this document. Substitutions are preferably conservative. They are defined according to the following table. Amino acids in the same block in the second column, and preferably in the same row in the third column, can be substituted for each other. Amino acids in the third column are indicated by a one-letter code.

Aliphatic non-polar gap ILV polar-uncharged CSTM NQ Polar-charged DE KR aromatic HFWY

A preferred polypeptide for the purposes of the present invention is an immunogenic antigen fragment from M. tuberculosis. Such an antigen can be obtained, for example, from a M. tuberculosis cell and/or a culture filtrate from M. tuberculosis. Thus, a polypeptide containing an immunogenic region of one of the antigens mentioned above may consist entirely of the immunogenic region or may contain additional sequences. Additional sequences may be derived from the native M. tuberculosis antigen or they may be heterogeneous, and such sequences may, but need not, be immunogenic.

In the present context, the expression substantially pure polypeptide fragment means a preparation of a polypeptide that contains at least 10% by weight of the substance of other polypeptides to which it is naturally associated (lower percentage of substance of other polypeptides is preferred, for example, at least 4%, at most 3%, at most 2%, and at most 1). Preferably, the substantially pure polypeptide is at least 96% pure, i. that the polypeptide makes up at least 96% by weight of the total polypeptides present in the formulation, and a higher percentage is preferred, such as at least 97%, at least 98%, at least 99%, at least at least 99.25%, at least 99.5%, and at least 99.75%. It is particularly preferred that the polypeptide fragment is in substantially pure form, i.e. so that the polypeptide fragment contains practically no other antigen with which it is naturally associated, i.e. did not contain any other antigen from bacteria belonging to the tuberculosis complex or virulent mycobacteria. This can be done by obtaining a polypeptide fragment by recombinant methods in a non-mycobacterial host cell, as will be detailed.

- 7 041840 described below, or by synthesizing the polypeptide fragment using conventional methods of solid or liquid phase peptide synthesis, for example, using the method described (Merrifield 1963) or variations thereof.

Tuberculosis (TB) is understood to mean an infection caused by a virulent mycobacterium from the tuberculosis complex, capable of causing TB infection and disease in an animal or human. Examples of virulent mycobacteria are M. tuberculosis, M. africanum and M. bovis. Examples of relevant animals are cattle, possums, badgers and kangaroos.

A TB patient is understood to mean an individual with a cultured or microscopically proven infection with virulent mycobacteria and/or an individual clinically diagnosed with TB and who is responding to anti-TB chemotherapy. Cultural, microscopic and clinical diagnosis of TB is well known to any person skilled in the art.

Delayed type hypersensitivity reaction (DTH) is understood to mean a T-cell mediated inflammatory response following injection of a polypeptide into or application to the skin, said inflammatory response occurring 72-96 hours after injection or application of the polypeptide.

By the term cytokine is meant an immunomodulatory agent such as interleukins and interferons, which can be used as an indicator of an immunological response. It includes, for example, interferon-gamma IFN-γ, interferon-gamma inducible protein 10, also known as CXCL10 or IP-10, and interleukin 2 (IL-2).

Throughout this specification, unless the context otherwise requires, the expression contain, or variations thereof, such as contains or containing, is to be understood as including a particular element or whole, or a group of elements or wholes, but without excluding any other element or whole , or groups of elements or integers.

Sequence identity

The expression sequence identity indicates a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. The two sequences to be compared must be aligned to the closest match possible by gap insertion or alternatively by truncation at the ends of the protein sequences. Sequence identity can be calculated as (N _η/ ·N^)100 as , where N _d j _f is the number of non-identical residues in the two sequences after alignment, and where N _ref is the number of residues in one of the sequences. Therefore, the DNA sequence of AGTCAGTC will be 75% identical to the AATCAATC sequence (N _dif =2 and N _re f=8). The gap is calculated as the non-identity of the specific residue(s), i.e. the DNA sequence of AGTGTC will be 75% identical to the DNA sequence of AGTCAGTC (N _dif =2 and N _re f=8). Sequence identity can alternatively be calculated using a BLAST program, such as the BLASTP program (Pearson, 1988) (www.ncbi.nim.nih.gov/cgi-bin/BLAST). According to one aspect of the present invention, alignment is performed using the ClustalW sequence alignment method with standard parameters described by Thompson, et al (Thompson, 1994), available at http://www2.ebi.ac.uk/clustalw/.

The preferred minimum percent sequence identity is at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.

Immunogenic epitope

An immunogenic epitope of a polypeptide is a portion of a polypeptide that elicits an immune response in an animal or human and/or in a biological sample, as determined by any of the biological assays described herein. The immunogenic epitope of a polypeptide may be a T-cell epitope or a B-cell epitope. An immunogenic epitope may be associated with one or more relatively small portions of the polypeptide, they may be scattered throughout the polypeptide sequence, or located at specific regions of the polypeptide. For several polypeptides, it has even been shown that epitopes are scattered throughout the polypeptide, spanning the entire sequence (Ravn, 1999).

In order to identify the relevant T-cell epitopes that are recognized in the course of the immune response, a brute-force method can be used: value in immune recognition, for example, by conducting for these deletion mutants, for example, IFN-γ analysis, described in this document. In another method, overlapping peptides, preferably synthetic, having a length of, for example, 20 amino acid residues derived from a polypeptide, are used to detect MHC class II epitopes. These peptides can be assayed using biological assays (eg, the IFN-γ assay described herein) and some of them will respond positively (and thus be immunogenic) as evidence for the presence of a T-cell epitope in the peptide. To detect MHC class I epitopes, it is possible to predict the peptides that will bind (Stryhn, 1996) and then produce these peptides synthetically, and examine them using appropriate biological assays, such as the IFN-γ assay described herein. The peptides are preferably 8 to 11 amino acid residues in length, derived from the polypeptide.

Although the minimum length of a T cell epitope has been shown to be at least 6 amino acids, it is normal for such epitopes to consist of longer stretches of amino acids. Therefore, it is preferred that the polypeptide fragment of the invention is at least 7 amino acid residues in length, e.g., at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, and at least 30 amino acid residues. Therefore, according to important embodiments of the method of this invention, it is preferred that the polypeptide fragment is at least 50 amino acid residues in length, for example, at least 40, 35, 30, 25 and 20 amino acid residues. Peptides having a length of 10 to 30 amino acid residues are expected to be most effective as class II MHC epitopes, and therefore particularly preferred lengths of the polypeptide fragment used in the method of this invention are 18, for example, 15, 14, 13, 12 and even 11 amino acid residues. Peptides of 7 to 12 amino acid residues in length are expected to be most effective as MHC class I epitopes, and therefore particularly preferred polypeptide fragment lengths used in the method of this invention are 11, e.g. 10, 9, 8, and even 7 amino acid residues. .

Immunogenic regions (fragments containing immunogenic epitopes) of polypeptides containing an immunogenic epitope may be recognized by a large part (high frequency) or a small part (low frequency) of a genetically heterogeneous human population. In addition, some immunogenic regions induce strong immunological responses (dominant), while others induce weaker, but still significant, responses (subdominant). High frequency <low frequency can refer to an immunogenic site that binds to widespread MHC molecules (such as HLA) or even a few MHC molecules (Sinigaglia, 1988; Kilgus, 1991). Fragments containing immunogenic epitopes from said polypeptides may be present as overlapping peptides of at least 10 amino acids in length, thereby including multiple epitopes.

Options

A common feature of the polypeptides of the compositions of the present invention is their ability to induce an immunological response, as shown in the examples. It is understood that a variant of a polypeptide of the present invention obtained by substitution, insertion, addition or deletion is also immunogenic, as determined by any of the assays described herein.

immune individual

An immune individual is defined as a human or animal that has cleared or controls infection with virulent mycobacteria or has received a BCG M. bovis vaccination.

Immunogenic

An immunogenic polypeptide is defined as a polypeptide that induces an immune response in a biological sample or individual currently or previously infected with a virulent mycobacterium. Immunogenic polypeptide is a synonym for antigen or antigenic polypeptide, and the two expressions immunogen and antigen are used without distinction in this disclosure; the narrow definition for an antigen is that it is able to bind specifically to a T or B cell receptor, and the narrow definition for an immunogen is that it is capable of inducing an immune response, however, as far as diagnosis is concerned, the content of the two expressions is the same and thus they are used in this document without distinction.

CMI diagnostics

The immune response can be monitored in one of the following ways: in vitro CMI response is determined by the release of a relevant cytokine, such as IFN-γ, from lymphocytes isolated from an animal or human currently or previously infected with virulent mycobacteria, or by detecting the proliferation of these T- cells. The induction is performed by adding the immunogenic composition to a blood cell suspension containing preferably 1x105 cells to 1x106 cells per well. Cells are isolated from blood, spleen, liver, or lung and the immunogenic composition is added to a concentration of, for example, 1-200 μg per ml of suspension, and stimulation is performed for two to five days. To control cell proliferation, cells are labeled with radioactive thymidine and after 16-22 hours of incubation

Proliferation is measured by liquid scintillator counting or other methods to detect a proliferative response. The release of IFN-γ can be determined by ELISA, well known to the person skilled in the art. Other cytokines and chemokines other than IFN-γ might be relevant in monitoring the immunological response to the polypeptide, such as IL-2, IL-12, TNF-α, IL4, TGF-β, IP-10, MIP-1e, MCP-1, IL-1RA and MIG. Another and more sensitive method for determining the presence of a cytokine (eg IFN-γ) is the ELISPOT method, in which cells isolated from, for example, blood are diluted to a concentration of preferably 1-4 x 10 ⁶ cells/ml and incubated for 18-22 hours in the presence of a diagnostic or immunogenic composition to a concentration of preferably 1-200 μg per ml. Thereafter, cell suspensions are diluted to 1-2 x 10 ⁶ /ml and transferred to anti-IFN-γ antibody-coated PVDF microtiter plates and incubated preferably for 4-16 hours. IFN-γ producing cells are detected using a labeled secondary antibody to IFN-γ and a relevant substrate that forms spots that can be counted using a dissecting microscope. The FluoroSpot assay is a modification of the ELISPOT assay and is based on the use of multiple fluorescent anti-cytokines, making it possible to detect two cytokines in the same assay, potentially contributing to improved disease risk prediction, as described below for co-detection of IL-2 and IFN-γ. It is also possible to determine the presence of a cytokine or chemokine response using immunochromatographic technology. This type of assay, well known from rapid pregnancy tests, provides rapid detection of the level of released cytokine or chemokine, and enables the diagnosis of infections and diseases, moreover, in resource-limited settings. Other immunoassays, including colorimetric assays such as turbidimetry, are well known to those skilled in the art and can be used for high throughput detection of cytokine or chemokine levels. It is also possible to detect the presence of an MRNA encoding a relevant cytokine using a polymerase chain reaction (PCR) technique. Detection of a cytokine or chemokine by mRNA level is usually faster than by protein level, since mRNA transcription precedes protein synthesis. For example, mRNA levels of the cytokine IFN-γ and chemokine IP-10 are optimal at shorter incubation periods compared to protein levels. Cytokine and chemokine signals detected by mRNA level can be determined as early as 2 hours after stimulation, and maximum levels are reached after 6-10 hours. Typically, one or more cytokines will be measured using, for example, PCR, immunochromatographic assay, ELISPOT or ELISA. One skilled in the art will appreciate that a significant increase or decrease in the amount of any of these cytokines induced by a specific peptide can be used in assessing the immunological activity of a polypeptide. It will also be appreciated by one skilled in the art that certain patterns of cytokine release are associated with certain clinical conditions. In particular, the predominance of IFN-γ over IL-2 was assumed to indicate early active TB disease, while the predominance of IL-2 over IFN-γ indicates infection control and a low risk of developing TB disease despite the presence of infection. in the mammal under study (Biselli, 2010; Sester, 2011).

The in vitro CMI response can be enhanced by the addition of cytokines such as IL-7 and/or IL-15, and increased release can also be achieved by blocking inhibitory substances such as IL-10, IL4, IL-5 and/or IL-13. Such CMI responses can be detected more reliably if the in vitro culture conditions are optimal for the cells undergoing stimulation. These conditions can be accelerated by the addition of nutrients, for example in the form of simple or complex sugars.

A simpler and more sensitive way is to use whole blood samples without prior isolation of mononuclear cells. Using this method, a sample of heparinized whole blood (with or without pre-lysis of erythrocytes) in an amount of 50-1000 ml is incubated for 18 hours to 6 days with a diagnostic or immunogenic composition according to the present invention to a concentration of preferably 1200 μg / ml of suspension . The supernatant is harvested and the release of IFN-γ (or any other relevant released cytokine, eg IP-10, IL-2 or others) can be determined using an ELISA method which is well known to one of skill in the art.

Another, also simple and at the same time sensitive, in vitro method for determining the CMI response is to stain the sample after incubation with a diagnostic or immunogenic composition on filter paper, such as Whatman 903 or Whatman FTA. After drying, the stained sample is stabilized and the levels of cytokines and chemokines in the sample can be determined at a later stage. CMI responses are readily detected using the aforementioned protein or mRNA measurement techniques. This method is particularly applicable in resource-limited environments or for high-throughput sample preparation and analysis.

Another in vitro method involves drawing blood into vacuum tubes previously coated with immunogenic polypeptides or fusion proteins, optionally also supplemented with a blood stabilizer such as heparin and/or nutrients. Pre-coated incubation tubes provide easy blood collection and eliminate the risk of exposure to blood-borne infection during sample preparation for in vitro incubation. These vacuum tubes are ideal for high throughput processing and automated analysis.

Thus, the present invention also provides an in vitro method for diagnosing current or previous sensitization in an animal or human infected with a virulent mycobacterium, the method comprising obtaining a blood sample from the animal or human, and contacting the animal sample with polypeptides or a composition according to the present invention. of the invention, wherein a significant intercellular phase release of at least one cytokine by mononuclear cells in a blood sample indicates that the animal is sensitized. In this case, a positive response is a response that is greater than the release from a blood sample obtained from a patient without a diagnosis of TB, plus two standard deviations.

In vitro CMI response can also be determined using T cell lines derived from an immune individual or M. tuberculosis infected person, where the T cell lines are activated with live mycobacteria, extracts from a bacterial cell or culture filtrate for 10-20 days with the addition of IL -2. Induction is performed by adding preferably 1-200 μg of the polypeptide per ml of suspension to T cell lines containing, for example, 1x105 to 3x105 cells per well, and incubation is performed for two to six days. The induction of IFN-γ or the release of another relevant cytokine is determined using ELISA. Stimulation of T cells can also be monitored by detecting cell proliferation with radiolabeled thymidine as described above. For both assays, a positive response is a response above background plus two standard deviations.

In vivo CMI response (e.g., skin test, intradermal patch test, post-patch skin test), which can be defined as a positive DTH response after intradermal injection or topical patch application, preferably 1-200 µg of each polypeptide in the diagnostic or immunogenic composition of the present invention an individual who is clinically or subclinically infected with a virulent bacterium, wherein a positive response is at least 5 mm in diameter 72-96 hours after injection or application.

The sensitivity of any particular diagnostic test determines the proportion of individuals with a positive response that are correctly identified or diagnosed by the test, for example, sensitivity is 100% if all individuals with a particular condition are characterized by a positive test. The specificity of a particular screening test reflects the proportion of individuals without the condition who are correctly identified or diagnosed by the test, eg, specificity is 100% if all individuals without the condition test negative.

Susceptibility is defined as the proportion of individuals with a particular condition (eg, active TB infection) who are correctly identified by the methods described herein (eg, test positive for IFN-γ).

Specificity is defined herein as the proportion of individuals without the condition (eg, not exposed to active TB infection) that are correctly identified by the methods described herein (eg, test negative for IFN-γ).

Sensitivity versus False Positive Rate Plot

The accuracy of a diagnostic test is best described by its sensitivity versus false positive rate (ROC) plot (Zweig, 1993). The ROC plot is a plot of all sensitivity/specificity pairs resulting from continuously varying decision threshold over the entire range of observed data.

The clinical utility of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish between two different states of the subjects being tested. Such conditions are, for example, health and disease, latent or recent infection or no infection, or benign or malignant disease.

In each case, the ROC plot shows the overlap between the two distributions when plotted with sensitivity and 1 specificity for the full range of decision thresholds. The y-axis represents sensitivity, or the true positive proportion [defined as (number of true positives) (number of true positives + number of false negatives]. It has also been denoted as a positive test in the presence of a disease or condition. It is calculated solely from The x-axis represents the proportion of false positives, or 1 is the specificity [defined as (number of false positives)/(number of true negatives + number of false positives)]. .

Since true and false positive proportions are calculated exclusively separately, when

- 11 041840 using test results from two different subgroups, the ROC plot does not depend on the frequency of the disease in the sample. Each point on the ROC graph represents a sensitivity/specificity pair corresponding to a certain decision threshold. A test at perfect discrimination (no overlap between the two distributions of results) is characterized by an ROC plot that runs through the upper left corner, where the true positive rate is 1.0, or 100% (absolute sensitivity), and the false positive rate is 0 (absolute specificity). The theoretical plot for the test without delimitation (same distribution of results for the two groups) is the 45th diagonal line from the lower left corner to the upper right corner. Most charts fall between these extremes. (If the ROC plot is located completely under the 45th diagonal, then this is easily corrected by reversing the positive test criterion from more than to less than or vice versa.) Qualitatively, the closer the plot is to the upper left corner, the greater the overall accuracy of the test.

One convenient goal for quantifying the diagnostic accuracy of a laboratory test is to express its capabilities as a single number. The most common overall measure is the area under the ROC chart. For convenience, this area is always > 0.5 (if not, the decision rule can be reversed to make it so). Values are between 1.0 (absolute separation of test values of the two groups) and 0.5 (no apparent difference in distributions between the two groups of test values). The area depends not only on a certain part of the graph, such as the point closest to the diagonal, or sensitivity at 90% specificity, but also on the entire graph. It is a quantitative, descriptive expression of how close the ROC plot is to the absolute plot (area = 1.0).

The clinical utility of novel antigen pools can be assessed in comparison and in combination with other diagnostic tools for a particular infection. In the case of M. tuberculosis infection, the clinical utility of the CMI result can be assessed by comparison with conventional diagnostic tests such as IGRA or TST by analysis of a sensitivity versus false positive rate curve.

How to get

Typically, M. tuberculosis antigens and DNA sequences encoding such antigens can be obtained using any of a variety of procedures.

They can be purified as native proteins from M. tuberculosis cells or culture filtrate using procedures such as those described above. Immunogenic antigens can also be generated recombinantly using a DNA sequence encoding an antigen that has been inserted into an expression vector and expressed in a suitable host. Examples of host cells are E. coli. Polypeptides, or an immunogenic portion thereof, can also be made synthetically having less than about 100 amino acids, and typically less than 50 amino acids, and can be made using techniques well known to those skilled in the art, such as commercially available solid phase techniques, where the amino acids sequentially added to the growing amino acid chain.

A host strain such as E. coli can be used in the construction and preparation of plasmid DNA encoding a fusion polypeptide. Plasmid DNA can then be obtained from daily cultures of the host strain carrying the plasmid of interest and purified using, for example, a column from the Qiagen Giga Plasmid Isolation Kit (Qiagen, Santa Clarita, CA, USA), including the step removal of endotoxin.

Fusion proteins

Although existing as separate structures, two or more of the immunogenic polypeptides can also be produced as fusion proteins, with these methods the best performance of the polypeptides of the present invention can be achieved. For example, all fusion partners that facilitate export of a polypeptide when produced recombinantly, fusion partners that facilitate purification of a polypeptide, and fusion partners that enhance the immunogenicity of a polypeptide fragment of the present invention are interesting possibilities. Thus, the present invention also provides a fusion polypeptide comprising at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) polypeptide or immunogenic fragments as defined above and optionally at least one additional fusion partner, and compositions containing fusion proteins. The fusion partner may, for the purpose of enhancing immunogenicity, be another polypeptide derived from M. tuberculosis, for example, a polypeptide fragment derived from a bacterium belonging to the tuberculosis complex, such as ESAT-6, TB10.4, CFP10, RD1-ORF2, Rv1036, MPB64, MPT64, Ag85A, Ag85B (MPT59), MPB59, Ag85C, 19 kDa lipoprotein, MPT32 and alpha-crystallin, or at least one T-cell epitope of any of the antigens mentioned above (WO0179274; WO01041519; (Nagai, 1991 ; Rosenkrands, 1998; Skjot, 2000) The present invention also relates to fusion polypeptides containing mutual fusions of two or more (for example, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the polypeptides (or their immunogenic parts) of the present invention.

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Figure captions

Fig. 1. Heat map showing immune recognition in 34 Egyptian volunteer donors based on a cutoff value of 100 pg/ml IFN-γ. Two patients were characterized by latent TB (subjects 1 and 2) and 32 patients were diagnosed with TB disease (subjects 3-34). A white symbol indicates no response, a gray symbol indicates a response, and a black symbol indicates a response to a specific antigen in the absence of a response to either ESAT-6 or CFP10.

Fig. 2. Heat map showing immune recognition in 31 Greenlandic volunteer donors based on a cutoff value of 50 pg/ml IFN-γ. 14 were diagnosed with TB disease (subjects 1-14) and 17 were characterized by latent TB (subjects 15-31). A white symbol indicates no response, a gray symbol indicates a response, and a black symbol indicates a response to a specific antigen in the absence of a response to either ESAT-6 or CFP10.

Fig. 3. Heat map showing immune recognition in 30 endemic control donors from Egypt, based on a cutoff value of 100 pg/ml IFN-γ. All donors were vaccinated with BCG and had no history of TB disease or known contact with a TB patient. The donors were identified as endemic controls because they lived in Egypt, which is considered an intermediate endemic country. A white symbol indicates no response and a gray symbol indicates a response. All antigens tested were highly specific, in contrast to PPD, which was included as an example of non-specific antigen challenge. Both donors 31 and 77 recognized a wide range of M. tuberculosis antigens, indicating latent infection, despite the selection criteria mentioned.

Fig. 4. IP-10 responses to quantiferon peptide pool (ESAT-6, CFP10, Rv2654c (peptide 4)) and peptide pool A in 73 Egyptian TB patients. The dotted line indicates median responses of 6 ng/mL for quantiferon antigens and 5.5 ng/mL for peptide pool A.

Fig. 5. IP-10 responses to quantiferon peptide pool (ESAT-6, CFP10, Rv2654c (peptide 4)) and peptide pool A in 100 M.tuberculosis-naive Danish individuals. The dotted line indicates median responses at 0 ng/mL for both antigen pools. It shows high specificity (few false positives) for the entire pool of peptides, indicating that each peptide is highly specific.

Fig. 6. IFN-γ responses to quantiferon peptide pool (ESAT-6, CFP10, Rv2654c (peptide 4)) and peptide pool A in 100 M. tuberculosis-naive Danish individuals. The dotted line indicates a median response of 0 pg/ml for peptide pool A and 4.9 pg/ml for quantiferon antigens. It shows high specificity (few false positives) for the entire pool of peptides, indicating that each peptide is highly specific.

Fig. 7. Sensitivity versus false positive rate (ROC) curve analysis comparing the diagnostic potential of peptide pool A for quantiferon antigens in 100 M.tuberculosis-naive Danish individuals and 73 TB patients. It shows high specificity (few false positives) for the entire pool of peptides, indicating that each peptide is highly specific.

Fig. 8. IP-10 responses (ng/mL) to quantiferon peptide pool (ESAT-6, CFP10 and Rv2654c) and peptide pool A in 68 microbiologically confirmed TB patients and 36 endemic controls from Tanzania.

Fig. 9. IP-10 responses (ng/ml) to peptide pool A and peptide pool A enriched with ESAT-6 in 73 confirmed TB patients from Cairo, Egypt. The line indicates the median.

Examples

Example 1 Initial Selection of Antigens

The T cell antigens chosen for TB immunodiagnosis had to be specific for M. tuberculosis infection in order to avoid interference from BCG vaccination and the most common atypical mycobacteria. At the same time, it was important to avoid ESAT-6 given that ESAT-6 is present in many of the new TB vaccines. As described above, in the Background of the Invention, through a process of careful and direct selection based on theory, practical research and a literature search of hundreds of potential antigens, no more than 9 M. tuberculosis antigens were selected for further investigation. They are CFP10 (Rv3874). The 10 kD culture filtrate antigen, together with ESAT-6, is the basis of the currently used cellular diagnostic blood tests for M. tuberculosis infection using IFN-γ release assays (IGRAs). CFP10 is an immunodominant M. tuberculosis antigen, and the diagnostic specificity of CFP10 and ESAT-6 is due to their genomic location in gene difference region 1 (RD1), a region that is absent in all BCG strains (Behr, 1999) and is included in the pathogenesis of M. tuberculosis . The genes encoding components of the ESX-1 secretion pathway are also located in RD1. A review of interferon-y assay studies described a CFP10 sensitivity of 6171% in patients with TB (Pai, 2004). In contrast to ESAT-6, CFP10 is not part of any of the currently used vaccine candidates in the evaluation process.

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Rv3877

Like CFP10, the Rv3877 gene is located in the RD1 region on the M. tuberculosis chromosome and has no close homologs elsewhere in the M. tuberculosis genome. The protein is not present in M. bovis BCG or M. avium medium mycobacteria and thus can be used to specifically diagnose M. tuberculosis without interference from possible prior BCG vaccination or potent exposure to medium mycobacteria such as M. avium. Rv3877 is a transmembrane protein and a key component of ESX-1 secretion as it forms a pore through which ESX-1 substrates are secreted (Abdallah, 2007). A pool of synthetic peptides including the Rv3877 protein induced positive responses in 33% of PBMCs isolated from human patients with TB (Mustafa, 2008).

Rv3614c and Rv3615c

The espA-espC-espD gene cluster (Rv3616c-Rv3615c-Rv3614c) is required for ESX-1-dependent protein secretion and virulence of M. tuberculosis (Fortune, 2005; MacGurn, 2005), and these three genes have recently been described as being co-transcribed (Chen , 2012). Rv3616c and Rv3615c are co-secreted with ESAT-6 and CFP10 (Fortune, 2005; MacGurn, 2005), while Rv3614c secretion does not directly require ESX-1 functions (Chen, 2012). In cattle, M. bovis Rv3615c partner, Mb3645c, stimulated IFN-γ responses in 37% of M. bovis-infected animals, but not in naive and BCG-vaccinated animals (Sidders, 2008). Mb3645c and Rv3615c were characterized by 100% amino acid identity. In cattle, the C-terminal part of the Mb3645c protein (amino acids 57–103) was the most immunogenic (Sidders, 2008). In humans, Rv3615c has also been identified as a potential candidate for T-cell-based specific immunodiagnosis of M. tuberculosis with case recognition of TB and low responses in BCG vaccinated (Millington, 2011). In patients with active TB, most of the commonly recognized peptides were located in the C-terminal part of the molecule (amino acids 66-90). Although the gene encoding Rv3615c is present in BCG, the Rv3615c protein is specifically recognized in M. tuberculosis infected individuals, but with limited recognition in BCG vaccinated individuals.

EspF (Rv3865)

EspF secretion-associated protein ESX-1 or M. bovis Mb3895 (identical to Rv3865 from M. tuberculosis) was identified by Ewer et al. (Ewer, 2006) as a promising diagnostic marker in experimentally or naturally infected cattle. M. bovis. Fifty percent of experimentally infected cattle responded to the Mb3895 peptide pool, while BCG-vaccinated calves did not respond to this peptide pool.

Rv2348c

Rv2348c is a hypothetical protein with an unknown function. The Rv2348c gene is located in the RD7 region. This region has been shown to be absent from BCG (Behr, 1999) and the protein can thus be used to diagnose TB without interference from prior BCG vaccination. The gene is highly transcribed in vitro (Arnvig, 2011), and the protein has been identified in proteomic studies (de Souza, 2011). A fragment of amino acids 23-50 in the ORF Rv2348c (open reading frame) is characterized by high homology with the M. avium Mav_2040 gene.

Rv3873 A portion of the amino acid sequence of Rv3873, including amino acids 1270, was covered with overlapping peptides. Among several RD peptide pools evaluated, this pool of Rv3873 peptides, named Rv3873A, was identified as one of the most promising pools, recognized by 46% of PBMCs from TB patients (Brock, 2004). Potential cross-reactive segments were not present in this part of the molecule.

Rv3878

As described above for Rv3873, a peptide pool named Rv3878B comprising amino acids 122-189 of this RD1 protein was identified and evaluated. It was recognized by 32% of PBMCs from human TB patients and was proposed as a peptide cocktail or pool that could be combined with ESAT-6 and CFP10 to maximize sensitivity (Brock, 2004).

Rv2654c

The Rv2654c gene was encoded by the RD11 region and encoded a possible PhiRv2 prophage protein with an unknown function. When screening for overlapping peptides comprising the entire Rv2654c protein product (designated TB7.7), Brock et al. found no cross-recognition in BCG vaccinated individuals and furthermore showed a sensitivity of 47% (Brock, 2004). The selected peptide (SEQ ID NO 8) was included in the QuantiFERON® TB Gold test.

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Table 1. Sequence listing for selected peptides

Protein Peptide SEQID NO. Rv2654 P4 8 CFP10 (Rv3874) P1-P6 9-14 Rv3615c P1-P4 15-18 Rv3865 P1-P3 19-21 Rv2348 P1-P4 22-25 Rv3614 P1-P20 26-45 Rv3877 P1-P5 46-50

Example 2 Selection of Antigens

Seven of the antigens listed above were tested for recognition in TB patients or latently infected individuals in two independent studies in Egypt and Greenland. In both studies, ESAT-6 (Rv3875) was also included as a comparison antigen and reference antigen. In addition, PPD was included in Egypt as an example of non-specific antigen stimulation.

Freshly drawn diluted whole blood was restimulated with selected peptides from antigens as described, and the response to ESAT-6 and CFP10 peptide pools was included as a reference.

The Egyptian study included 34 volunteer donors (8 women and 26 men) as positive controls. Thirty-two of them were diagnosed with TB disease with confirmed positive sputum culture (subjects 3-34). Two patients were characterized by a latent form of TB (subjects 1 and 2). In addition, 30 negative control endemic donors (5 women and 25 men) were included. All were presumed to have been vaccinated with BCG, had no history of TB disease, and had no known contact with a TB patient. The Greenland study included 31 subjects (15 women and 16 men). Fourteen were diagnosed with TB disease (subjects 1-14); in 11 cases with confirmed positive sputum culture and in 4 cases, the diagnosis of TB was made on the basis of clinical findings. The remaining 17 subjects were characterized by a latent form of TB (subjects 15-31).

In both studies, freshly drawn diluted whole blood was stimulated in plates with selected antigens (10 μg/ml of each peptide). Synthetic peptides (derived from Genecust) from ESAT-6, CFP10, Rv3873, Rv3878, Rv3615c, Rv3865, Rv3877 and Rv2348 antigens were screened in both studies and also included positive (PHA) and negative (medium only) controls and (Egypt only ) PPD (not shown). Diluted whole blood was incubated at 37 degrees Celsius for 5 days, and then the supernatants were collected and examined for (IFN-γ) using intralaboratory ELISA. A positive response in these studies was defined as an IFN-γ concentration of 100 or 50 pg/ml for the Egyptian and Greenland studies, respectively.

In FIG. 1, 2 and 3 are graphical representations (heat maps) of the data where the individual values contained are represented by color, with white indicating no response, gray indicating response to a specific antigen and black representing antigen response where the same donor does not respond to ESAT-6 and/or CFP10. As shown, several of these antigens studied were recognized in TB patients or latently infected donors, with the most common responses resulting from Rv3615c stimulation (recognized in 49% of donors). Importantly, only ESAT-6 recognized three patients, while CFP10 was not recognized by them when stimulated (patient #9 and 17 in Fig. 1 and patient #3 in Fig. 2) and of these, Rv3615 could be recognized by all three patients. In addition, Rv3615c restimulation was characterized by recognition in 11 and 9 donors not recognized by ESAT-6 and CFP10, respectively. In contrast, two of the antigens were recognized in a very limited number of donors; Rv3873 was recognized in only two of 65 donors, and Rv3878 was recognized in seven of 65 donors. Thus, despite previous data on these antigens in TB patients from Denmark and the Netherlands with intermediate susceptibility (Rv3873 at 32% for Rv3878 and 46% for Rv3873 (Brock, 2004), the data obtained in this document indicated that that not all antigens expected to be sensitive were expressed in all situations Rv3865, Rv3877 and Rv2348 were intermediately sensitive and were recognized in 16, 12 and 15 of 65 donors Significantly, all Rv3615c, Rv3865 and Rv2348 antigens elicited responses in a number of donors that did not recognize ESAT-6 and/or CFP10, further demonstrating the diagnostic potential of these antigens.The specificity of the selected antigens was confirmed in a panel of 30 negative control endemic donors from Egypt (Figure 3).As shown, all antigens examined were highly specific in contrast to PPD, which was included as an example of non-specific antigen stimulation Both donors 31 and 77 recognized a wide range of A wide range of M. tuberculosis antigens, including ESAT-6 and CFP-10, is strongly indicative of latent infection despite the selection criteria mentioned.

Example 3 CFP10 and 3615c are comparable to CFP10 and ESAT-6

The diagnostic capabilities of the combination of CFP10 and Rv3615c were then compared with those of the combination of CFP10 and ESAT-6. Whole blood samples were included from 35 individuals from Greenland, of whom were characterized by a latent form of M. tuberculosis infection, defined as a positive quantiferon test and/or confirmed exposure to M. tuberculosis and a change in tuberculin skin test, and 17 patients were characterized by microbiologically confirmed TB.

Separate aliquots of 200 µl of undiluted whole blood were stimulated with overlapping peptides representing CFP10 (SEQ ID 1) or Rv3615 (SEQ ID 15-18) or ESAT-6 (SEQ ID 51) at a final concentration of 5 µg/ml in a humidified 37°C incubator within 7 days. In parallel, a negative control sample (nil) was prepared. After incubation, the plasma supernatant was isolated and the level of IFN-γ was determined using ELISA.

The diagnostic ability of the three antigens was assessed by adding the measured level of antigen-specific production of IFN-γ (subtracted from the level of stimulated whole blood level in the unstimulated well) in response to stimulation with individual antigen(s), followed by comparison of this sum with the cutoff value. The cut-off value was defined as a pooled antigen-specific response of at least 50 pg/ml and 4-fold higher than the null value in an individual patient. Antigen-specific levels above the cut-off value classified the individual patient as antigen-positive, and antigen-specific levels below the cut-off value as antigen-negative.

In table. 2 shows the sensitivity of individual antigens CFP10, Rv3615c, ESAT-6 and combinations. As shown, the diagnostic capabilities of the combination of CFP10 and Rv3615c were comparable to those of CFP10 and ESAT-6, with both combinations characterized by 60% sensitivity. The combination of CFP10, Rv3615c and ESAT-6 further improved sensitivity from 60% to 69%.

Table 2. Comparison of sensitivity of CFP10, Rv3615c and ESAT-6 and their combinations.

Antigen % sensitive- news CFP10 49 Rv3615c 34 ESAT-6 31 CFP10+Rv3615c 60 CFP10 + ESAT-6 60 CFP10+Rv3615c+ ESAT-6 69

Example 4 Enrichment of the combination of CFP10 and Rv3615c with three antigens: Rv2348, Rv3865 and Rv3877

Using the same whole blood samples as described above (35 individuals from Greenland; 18 with latent M. tuberculosis infection and 17 patients with microbiologically confirmed TB) and the same assay conditions, the effect of combining CFP10 and Rv3615c with three different antigens; Rv2348, RV3865 and RV3877.

The diagnostic ability of the three antigens was assessed by adding the measured level of antigen-specific IFN-γ production (subtracted from the stimulated whole blood level in the unstimulated well) in response to the individual(s) antigens), followed by comparison of the sum with a breakpoint. The cut-off value was defined as a pooled antigen-specific response of at least 50 pg/ml and 4-fold higher than the null value in an individual patient. Antigen-specific levels above the cut-off value classified the individual patient as antigen-positive, and antigen-specific levels below the cut-off value as antigen-negative.

In table. 3 shows the sensitivity of individual Rv3865, CFP10, Rv3615c antigens and combinations. The sensitivity of Rv3865 was relatively low at only 20%, but the addition of Rv3865 to CFP10 and Rv3615c increased the overall diagnostic sensitivity by 6% compared to CFP10 and Rv3865 alone.

Table 3. Comparison of CFP10, Rv3615c, Rv3865 and their combinations for the diagnosis of infection

M.tuberculosis

Antigen % sensitive- news Rv3865 20 CFP10+Rv3615c 60 CFP10+Rv3615c+ Rv3865 66

Similarly, the diagnostic ability of Rv3877 was only 11%, however, this antigen also increased the overall sensitivity of CFP10 and Rv3615c from 60% to 69% (Table 4).

Table 4. Comparison of CFP10, Rv3615c, Rv3877 and their combinations for the diagnosis of M. tuberculosis infection

Antigen % sensitive- news Rv3877 eleven CFP10+Rv3615c 60 CFP10+Rv3615c+ Rv3877 69

Finally, the ability of Rv2348 to increase the diagnostic ability of CFP10 and Rv3615c was also evaluated (Table 5). As shown, the sensitivity of Rv2348 was 29%, and the addition of Rv2348 to CFP10 and Rv3615c increased diagnostic sensitivity by 23% compared to using CFP10 and Rv3615c alone.

Table 5. Comparison of CFP10, Rv3615c, Rv2348 and their combinations for the diagnosis of M. tuberculosis infection.

Antigen % sensitive- news Rv2348 29 CFP10+Rv3615c 60 CFP10+Rv3615c+ Rv2348 83

For further evaluation of sensitivity and specificity, the following antigens (CFP10, Rv3615c, Rv3865 and Rv2348) were selected by combining these 4 antigens into one peptide pool (peptide pool A).

Example 5 Sensitivity and Specificity Study of Peptide Pool A

It is well known in the art that an immunodiagnostic cocktail containing ESAT-6, CFP10 and TB7.7p4 is the preferred method for diagnosing M. tuberculosis infection. The antigen cocktail is considered both sensitive and specific and forms the basis of the quantiferon test. As was clear from previous examples, the combination of antigens improves the diagnostic sensitivity and reliability of the test because the underlying magnitude of IFN-γ responses is higher and more strongly detected compared to the presence of antigens alone.

A very convenient approach to use is the use of vacuum tubes pre-coated with antigens in a cocktail. For example, in the quantiferon test, lyophilized peptides representing an antigenic cocktail are coated with heparin in a vacuum tube. Blood is collected in such a tube, allowing the peptides to interact with antigen-specific CD4 and CD8 T cells. After 16-24 hours of incubation, the tube is centrifuged and the resulting IFN-γ level can be measured in the plasma supernatant and compared with negative and positive control samples. Test subjects may be further classified as either infected or non-infected if the level is above the cutoff for a positive test result.

It is well known that other immune effector molecules associated with IFN-γ signaling are useful in the diagnosis of M. tuberculosis infection (Chego ERJ 2014). Chemokine IP-10 is produced at very high levels and has comparable diagnostic capabilities to IFN-γ.

To demonstrate the utility of combining several antigens into one antigen cocktail, the present inventors combined the following antigens into peptide pool A. Peptide pool A consisted of the following peptides:

CFP10: 6 peptides comprising the entire amino acid sequence of CFP10 (SEQ ID NO: 9-14)

Rv3615c: 4 peptides comprising amino acids 55-103 (SEQ ID NOs: 15-18)

Rv3865: 3 peptides comprising amino acids 9-44 (SEQ ID NOs: 19-21)

Rv2348c: 4 peptides comprising amino acids 56-109 from the full length protein sequence (SEQ ID NOs: 22-25).

A second and independent study in Egypt was conducted to investigate the susceptibility of 4 antigens when pooled in peptide pool A. The study included 73 TB patients with confirmed positive sputum culture and each subject provided a blood sample taken directly into pre-prepared antigen-coated tubes under vacuum. The tubes were coated with either ESAT-6+CFP10+Rv2654c peptides (i.e. the same peptides as in the quantiferon test and used as a reference, designated quantiferon peptide pool) or peptide pool A (CFP10 + Rv3516c + Rv3865 + Rv2348 , as mentioned above). After 16-24 hours

- 17 041840 incubation supernatants were collected and examined for the release of the cytokine IP-10 using an intralaboratory ELISA. As shown in FIG. 4, most TB patients recognized both the A peptide pool and the quantiferon peptide pool. Median responses were 5.5 ng/ml IP10 for peptide pool A and 6.0 for quantiferon peptide pool.

In parallel, an independent study was carried out in Denmark in order to investigate the specificity of the A peptide pool. The study included 100 subjects living in an area with a very low incidence of TB (Denmark) and no known exposure to M. tuberculosis. In 17 cases, subjects were confirmed vaccinated with BCG, and in 19 cases, BCG vaccination status was unknown/unconfirmed. The remaining participants were not vaccinated with BCG. Similar to the susceptibility study, fresh whole blood was collected directly into vacuum tubes precoated with either peptide pool A (CFP10 + Rv3615c + Rv3865 + Rv2348) or quantiferon reference peptide pool (ESAT-6 + CFP10 + Rv2654c). After 16-24 hours of incubation, supernatants were collected and analyzed for cytokine content IP-10 and IFN-γ using an in-house ELISA assay. Although both median IP-10 responses to peptide pool A and quantiferon peptide pool were 0 ng/mL (Figure 5), several unexposed donors responded positively to restimulation with quantiferon antigens at IP-10 levels of approximately 5 ng/mL. The same trend with unexposed donors showing false positive responses was observed in the analysis of IFN-γ secretion (Fig. 6; peptide pool A - median 0 pg/ml, interquartile range (IQR) -0.5-5, 2 pg/ml and quantiferon peptide pool - median 4.9 pg/ml, IQR -0.6-32.45 pg/ml.

Combining data from sensitivity and specificity studies allowed a sensitivity vs. false positive rate (ROC) curve analysis comparing the diagnostic potential of the A peptide pool with the quantiferon antigen pool (FIG. 7). The area under the curve (AUC) was 0.979 for the peptide pool A and 0.947 for the quantiferon antigen pool. By analyzing the ROC curve, the optimal cut-off values for both peptide pool A and quantiferon peptide pool were determined as 1.4 ng/mL for peptide pool A (sensitivity 87.7% with specificity 98.1%), and 2. 3 ng/ml for the quantiferon peptide pool (sensitivity 75.3% with specificity 96.2%).

Using these boundary values directly compared the number of positive and negative responses during restimulation peptide pool quantiferon and peptide pool A (table 6 and 7). Of 73 patients with TB, 54 (74%) who recognized quantiferon peptide pools were within the indicated sensitivity range of 64-89% quantiferon antigens (Dewan, 2007). In comparison, peptide pool A recognized the majority of TB patients in this study (64 out of 73 patients), corresponding to an estimated sensitivity of 88%. Based on the McNiemar test, peptide pool A was significantly more sensitive in this study compared to quantiferon peptide pool (p<0.012).

Table 6. Direct comparative study of quantiferon peptide pool and peptide pool A in 73 patients with TB.

Peptide pool A Negative Positive Total Antigenic pool of quantiferon Negative 6 13 19 Positive 3 51 54 Total 9 64 73

Table 7. Direct comparative study of the quantiferon peptide pool and the peptide pool

And in 100 presumably infected control patients

Peptide pool A Negative Positive Total Antigenic pool of quantiferon Negative 95 1 96 Positive 2 2 4 Total 97 3 100

In conclusion, pool A was significantly more sensitive (more true positives, Table 6) than quantiferon antigens and was at least as specific (comparable false positives, Table 6). .7). These results clearly show that it is possible to 1) develop peptide pools for TB diagnosis lacking ESAT-6 with higher sensitivity compared to currently used antigens for quantiferon, 2) develop an antigen pool lacking ESAT-6 with specificity , comparable with the currently used quantiferon.

Example 6. Validation of the peptide pool A.

The skilled artisan is well aware that the validation of breakpoints for immunodiagnostic tests requires validation in independent cohorts. For this purpose, 68 cases of patients with microbiologically confirmed TB and 36 endemic controls were included; individuals, some of whom had pre-existing but controlled M. tuberculosis infection from Tanzania.

From each donor, 1 ml of blood was taken into 5 vacuum tubes containing lyophilized heparin (18 IU) and peptides (5 μg/peptide) as follows: quantiferon peptide pool (ESAT-6, CFP10 and TB7.7p4 (tube 1), comparable to the quantiferon test), peptide pool A (CFP10, Rv3615, Rv3865 and Rv2348B (tube 2)) and negative control tube (tube 3).

In FIG. 8 shows the responses for the negative control tube (tube 3) minus IP-10 (ng/mL) in disease cases and controls from tube 1 and tube 2. Clearly, the quantiferon peptide pool and the A peptide pool are comparable in terms of high response magnitude in cases of TB. As expected, responses from endemic controls were more heterogeneous, indicating that some of the individuals examined were infected.

Using the predetermined breakpoint identified in Example 5 (1.4 ng/mL), diagnostic accuracy was compared for both TB patients (Table 8) and endemic controls (Table 9). In the TB patient group, the diagnostic sensitivity of the standard quantiferon peptide pool was 66% (45 of 68 included patients identified as positive) and was higher for peptide pool A with a sensitivity of 72% (49 of 68 patients identified as positive). As expected, the agreement between the two tests was very high, with an agreement of 91% (44 were positive in both tests, 18 were negative in both tests, with an overall agreement of 62 of 68).

Table 8 Correspondence between quantiferon peptide pool and peptide pool A after classifying antigen challenge responses from 68 confirmed TB patients using a predetermined breakpoint for a positive test

TV patients Peptide pool of quantiferon Positive Negative Sum Peptide pool A Positive 44 5 49 Negative 1 18 19 Sum 45 23 68

In the endemic control population, there was no gold standard for infection, so the frequency was presented as positive responders. With the help of peptide pool A, 39% (14/36) were detected as positive, and with the help of the standard peptide pool of quantiferon 31% (11/36), which once again indicated a higher sensitivity. Conformity was also very high (92% concordance in 33 cases out of 36 included).

Table 9 Correspondence between quantiferon peptide pool and peptide pool A after classifying antigen challenge responses from 36 endemic controls using a predetermined breakpoint for a positive test

TV patients Peptide pool of quantiferon Positive Negative Sum Peptide pool A Positive eleven 0 AND Negative 3 22 25 Sum 14 22 36

Example 7 Peptide pool A can be further improved when combined with ESAT-6

Additionally, the possibility of adding ESAT-6 to the peptide pool A was evaluated in order to further improve diagnostic capabilities. In this regard, the A + ESAT-6 peptide pool and the A peptide pool were examined in a cohort of 73 cases of confirmed TB in Cairo, Egypt, and using the same assay conditions as described in Example 5. Based on FIG. 9, it is clear that the magnitude of responses is increased when peptide pool A is combined with ESAT-6, with peptide pool A having a median response of 5.50 ng/mL IP-10 compared to peptide pool A with ESAT-6 where the median was 6 .86 ng/mL IP-10. Using a cut-off value of 0.75 ng/mL, the frequency of responders in the two groups was compared. In peptide pool A, the responder rate was 93%, with 68 of 73 patients examined being positive, while the rate for peptide pool A with ESAT-6 was 96% (70 of 73 patients - 96%). Thus, combining peptide pool A with ESAT-6 reduced the false-negative rate from 7 to 4%.

- 19 041840

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Claims (21)

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Brock, et al. (2004). Mapping immune reactivity toward Rv2653 and Rv2654: two novel low-molecular-mass antigens found specifically in the Mycobacterium tuberculosis complex. The Journal of infectious diseases 189(5): 812-819. Aagaard, C., T. Hoang, et al. (2011). A multistage tuberculosis vaccine that confers efficient protection before and after exposure. Nature medicine 17(2): 189-194. CLAIM 1. Diagnostic composition for the diagnosis of tuberculosis, containing a mixture of practically pure polypeptides consisting of amino acid sequences selected from: a) Rv3874 (SEQ ID NO: 1), Rv3615 (SEQ ID NO: 2) and one or more of Rv3865 (SEQ ID NO: 3), Rv2348 (SEQ ID NO: 4), Rv3614 (SEQ ID NO: 5) , Rv2654 (SEQ ID NO: 6) and Rv3877 (SEQ ID NO: 7) and b) at least one fragment from Rv3874 (SEQ ID NO: 1), at least one fragment from Rv3615 (SEQ ID NO: 2), and at least one fragment from one or more of Rv3865 (SEQ ID NO: 3) , Rv2348 (SEQ ID NO: 4), Rv3614 (SEQ ID NO: 5), Rv2654 (SEQ ID NO: 6), and Rv3877 (SEQ ID NO: 7), wherein said fragments are at least 7 amino acid residues in length and contain immunogenic epitopes from said amino acid sequences; and c1) an amino acid sequence having at least 80% sequence identity - 22 041840 the identity of Rv3874 (SEQ ID NO: 1), an amino acid sequence having at least 80% sequence identity to Rv3615 (SEQ ID NO: 2), and one or more amino acid sequences having at least 80% sequence identity to one or several of Rv3865 (SEQ ID NO: 3), Rv2348 (SEQ ID NO: 4), Rv3614 (SEQ ID NO: 5), Rv2654 (SEQ ID NO: 6), and Rv3877 (SEQ ID NO: 7), wherein said amino acids the sequences are immunogenic; or c2) an amino acid sequence having at least 80% sequence identity to at least one fragment from Rv3874 (SEQ ID NO: 1), an amino acid sequence having at least 80% sequence identity to at least one fragment from Rv3615 (SEQ ID NO: 2), and amino acid sequences having at least 80% sequence identity to at least one fragment from one or more of Rv3865 (SEQ ID NO: 3), Rv2348 (SEQ ID NO: 4), Rv3614 (SEQ ID NO: : 5), Rv2654 (SEQ ID NO: 6) and Rv3877 (SEQ ID NO: 7), wherein said fragments are at least 7 amino acid residues in length and contain immunogenic epitopes from the indicated amino acid sequences. 2. Diagnostic composition according to claim 1, containing Rv3874 (SEQ ID NO: 1), Rv3615 (SEQ ID NO: 2) and Rv3865 (SEQ ID NO: 3) or fragments containing their immunogenic epitopes. 3. Diagnostic composition according to claim 1, containing Rv3874 (SEQ ID NO: 1), Rv3615 (SEQ ID NO: 2) and Rv2348 (SEQ ID NO: 4) or fragments containing their immunogenic epitopes. 4. Diagnostic composition according to claim 1, containing Rv3874 (SEQ ID NO: 1), Rv3615 (SEQ ID NO: 2) and Rv3877 (SEQ ID NO: 7) or fragments containing their immunogenic epitopes. 5. Diagnostic composition according to claim 1, containing Rv3874 (SEQ ID NO: 1), Rv3615 (SEQ ID NO: 2), Rv3865 (SEQ ID NO: 3) and Rv2348 (SEQ ID NO: 4) or fragments containing them immunogenic epitopes. 6. Diagnostic composition according to any one of claims 1-4, wherein fragments containing immunogenic epitopes from SEQ ID NO: 1 are selected from SEQ ID NO: 9-14. 7. Diagnostic composition according to any one of claims 1-4, wherein fragments containing immunogenic epitopes from SEQ ID NO: 2 are selected from SEQ ID NO: 15-18 or SEQ ID NO: 59-63. 8. Diagnostic composition according to any one of claims 1-7, wherein fragments containing immunogenic epitopes from SEQ ID NO: 3 are selected from SEQ ID NO: 19-21. 9. Diagnostic composition according to any one of claims 1-7, wherein fragments containing immunogenic epitopes from SEQ ID NO4 are selected from SEQ ID NOs: 22-25. 10. Diagnostic composition according to any one of claims 1-7, wherein fragments containing immunogenic epitopes from SEQ ID NO: 7 are selected from SEQ ID NO: 46-50. 11. Diagnostic composition according to any one of claims 1 to 9, further comprising Rv3875 (SEQ ID NO: 51) or one or more fragments thereof. 12. The diagnostic composition according to claim 11, wherein said fragments and fragments containing immunogenic epitopes from SEQ ID NO: 51 are selected from SEQ ID NO: 52-58. 13. The diagnostic composition of claim 1, wherein the fragments containing immunogenic epitopes from said polypeptides are present as overlapping peptides of at least 10 amino acids in length. 14. Diagnostic composition according to claim 5, where the mixture contains SEQ ID NO: 9-25. 15. A diagnostic composition according to any one of the preceding claims, wherein some or all of the polypeptides are fused together, optionally via linkers or spacers. 16. The use of diagnostic compositions according to any one of the preceding claims for the preparation of a pharmaceutical composition for the diagnosis of TB caused by virulent mycobacteria, for example Mycobacterium tuberculosis, Mycobacterium bovis or Mycobacterium africanum. 17. A CMI diagnostic kit comprising a diagnostic composition according to any one of claims 1-15. 18. The CMI diagnostic kit according to claim 17 for in vitro or in vivo diagnosis of tuberculosis. 19. An in vitro or in vivo method for diagnosing tuberculosis caused by virulent mycobacteria, for example Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, in an animal, including a human, using a diagnostic composition according to any one of claims 1-15. 20. A method for diagnosing tuberculosis according to claim 19, comprising intradermal injection of an animal with a diagnostic composition as defined above, where a positive skin reaction at the injection site indicates that the animal has tuberculosis, and a negative skin reaction at the injection site indicates that the animal does not have tuberculosis. 21. A method for diagnosing tuberculosis according to claim 19, comprising contacting a sample, for example a blood sample, with a diagnostic composition according to the present invention to detect a positive reaction, for example, cell proliferation or release of cytokines, such as IFN-γ or IP-10. -