BR112016013915B1 - DIAGNOSTIC COMPOSITION; USE OF DIAGNOSTIC COMPOSITIONS; CMI DIAGNOSTIC TOOL OR SET; AND IN VITRO OR IN VIVO DIAGNOSIS METHOD OF TUBERCULOSIS CAUSED BY VIRULENT MYCOBACTERIA - Google Patents

Abstract

DIAGNOSTIC COMPOSITION; USE OF DIAGNOSTIC COMPOSITIONS; CMI DIAGNOSTIC TOOL OR SET; AND IN VITRO OR IN VIVO DIAGNOSIS METHOD OF TUBERCULOSIS CAUSED BY VIRULENT MYCOBACTERIA. The present invention describes in vitro and in vivo diagnostic methods, with improved specificity and sensitivity for the detection of tuberculosis. The diagnostic reagents of the present invention can replace previous mixtures, cocktails, combinations of antigens, comprising ESAT-6, but including ESAT-6 further improves diagnosis.

Description

[001] The present invention describes compositions for use as a pharmaceutical or diagnostic reagent, for the immunological diagnosis of tuberculosis in a human or animal subject, cell-mediated, improved in vivo or in vitro. The invention relates to antigen combinations that increase sensitivity (which gives fewer false negatives), compared to existing antigen combinations, without compromising specificity (the amount of false positives). In particular, the invention relates to antigen compositions that do not include the antigen designated the 6 kDa primitive secretory antigenic target (ESAT-6), which is currently used in products registered to detect Mycobacterium tuberculosis infection. Alternatively, new diagnostic or immunogenic compositions can be used in combination with ESAT-6 to further enhance the sensitivity of cell-mediated diagnosis.

Background of the invention

[002] Tuberculosis (TB) is one of the main causes of morbidity and mortality worldwide. It is estimated that one person develops TB every four seconds and someone will die from the disease every 20-30 seconds. In addition, approximately one third of the world's population is latently infected with Mycobacterium tuberculosis (M. tuberculosis), the causative agent of TB. developing active TB disease during life, and this risk increases drastically, 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). Therefore, it is important to be able to detect individuals infected with M. tuberculosis at an early stage of the infection, in order to prevent the progression of M. tuberculosis infection from activating contagious pulmonary TB. This can be achieved by prophylactic treatment at the earliest possible moment after diagnosis. Consequently, rapid and accurate diagnosis of M. tuberculosis infection is an important element of global health measures to control the disease.

[004] Current diagnostic assays to determine infection with M. tuberculosis include culture, microscopy and PCR of relevant material from patients, chest X-rays, the conventional tuberculin skin test (TST), and interferon-gamma release (IGRAs). The first three methods are used to diagnose active contagious TB and are based on identification of the M. tuberculosis bacteria and accordingly depend on the presence of the bacteria in the sample. This requires a certain bacterial load and access to infectious material, and consequently the methods are not appropriate for early diagnosis of the infection, that is, before the onset of clinical disease. Chest X-rays are insensitive and are only applicable for pulmonary TB and to detect TB at an earlier stage.

[005] The conventional tuberculin skin test, presenting a delayed-type hypersensitivity reaction (DTH), is a simple and inexpensive test, based on the recognition of mycobacterial antigens in exposed individuals. However, it is far from ideal in detecting M. tuberculosis infection. It employs intradermal injection of purified protein derivatives (PPD), which is a crude and ill-defined mixture of mycobacterial antigens, some of which are shared with proteins from the M. bovis bacillus Calmette-Guèrin (BCG) vaccine substrain and environmental mycobacteria non-tuberculous. This broad PPD reactivity causes poor TST specificity, leading to a situation in which BCG vaccination and exposure to nontuberculous mycobacteria gives an assay result similar to that seen in individuals infected with M. tuberculosis.

[006] M. tuberculosis-mediated infection mediates a strong, cell-mediated immune response (CMI) and the detection of immune cells and any products derived from said cells, which are generated as a part of the specific response to the infection by M. tuberculosis would be an appropriate method to detect the infection (Andersen, 2000). In order to generate such a specific response, the reagent must be 1) widely recognized by individuals infected with M. tuberculosis, and 2) be specific for M. tuberculosis, thus discriminating between TB infection, BCG vaccination, and exposure to non-tuberculous environmental mycobacteria.

[007] The M. tuberculosis genome provides 4018 open reading systems (http://tuberculist.epfl.ch/, version R27 - March 2013). However, only a minority of these are T cell antigens, with strong and widespread recognition by peripheral blood mononuclear cells (PBMCs) of human TB patients. Algorithms have been developed to predict T cell epitopes, but experimental verification was essential to identify the immunodominant antigens CFP10 and ESAT-6. 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 by a two-dimensional proteomics-based approach led to the identification of 59 M. tuberculosis antigens. human T cells, of which 35 had been previously described (Deenadayalan, 2010). Although this list may not be exhaustive, it highlights that only a minimal portion of the approximately 900 CF proteins (Albrethsen, 2013) are T cell antigens. Logically, one would expect that the majority of immunodominant antigens encoded from the genome of M. tuberculosis would be the subset with the highest expression and, with current technology, it would be possible to order the 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 accurately determine which genes encode relevant antigens (Sidders, 2008).

[008] A highly specific candidate reagent could be sought among antigens from the RD regions (deletion regions) of the M. tuberculosis genome. These regions represent genomic deletions of the BCG vaccine strain of M. bovis compared to the virulent M. tuberculosis strain (Behr, 1999). Therefore, in theory, proteins from these regions (RD proteins) would be excellent candidates as TB diagnostic reagents, that is, they would not be recognized by healthy, non-infected individuals, regardless of their BCG vaccine status or exposure to non-infected mycobacterial strains. pathogenic. However, of all the predicted genomic ORFs (open reading systems) deleted from BCG, it is not known per se which are, in fact, expressed as proteins and, furthermore, the immunoreactivity remains unknown until tested with sensitized lymphocytes from individuals infected with M. tuberculosis. This can be done, for example, in a complete blood test, by restimulation of PBMCs or by injecting the substances into the skin, analogously to the way the PPD/Mantoux test is administered. For example, evaluation of Rv3872 showed low interferon gamma (IFN-γ) responses in the human TB patients tested (n = 7) 1-4 months after diagnosis (WO 99/24577), demonstrating that this RD protein is not often recognized, and, consequently, Rv 3872 was not studied again as the subject of a diagnostic test based on CMI.

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

[010] When a potentially specific T cell antigen has been identified, it must be verified that the antigen is recognized specifically in individuals infected with M. tuberculosis and not in people vaccinated with BCG. For Rv3873 of RD1 it appeared that the protein was a member of a family of proteins with a conserved motif at amino acids 118-135, also present in other M. tuberculosis proteins. As a widely recognized T cell epitope was present in this motif, individuals also vaccinated with BCG also responded to the peptide that expanded this sequence (Liu, 2004). However, cross-reactivity was also observed with Rv3878 and Rv3879c, although no homology was detected by comparison with sequences of other known mycobacterial proteins (Liu, 2004), showing that the specificity of a potential diagnostic candidate does not have to be verified experimentally. Similarly, Rv2653c of RD13 was recognized in both BCG-vaccinated donors and TB patients, although database searches with the BLAST algorithm did not reveal any obvious mycobacterial proteins that could explain the observed cross-reactivity (Aagaard, 2004).

[011] Having identified specific M. tuberculosis proteins, with potential for the diagnosis of M. tuberculosis infection, by CMI-based assays, it remains to be investigated whether an association of these proteins/peptides will provide the desired sensitivity for a diagnostic test . It is not possible to predict to what extent a given antigen will contribute to sensitivity after combination with already defined diagnostic antigens; This will have to be evaluated experimentally for each antigen, and preferably in TB patients from different parts of the world, with different genetic backgrounds.

[012] The diagnostic potential of CFP10 (Rv3874), ESAT-6 (Rv3875), two low molecular weight proteins from the RD1 region, and TB7.7 (Rv2654) is very well documented (Brock, 2004; Moon, 2013, WO2004099771 ) and are currently used in different diagnostic reagents registered for human use. Peptides covering CFP10 and ESAT-6 are used in the T-SPOT®.TB test, which is a cellular blood test that detects the immune response of T cells found in PBMCs that have been restimulated with ESAT-6 and CFP10. This response is detected by a highly sensitive, enzyme-linked, immunospot methodology called ELISPOT and is marketed as the T-SPOT.TB test. This test is highly sensitive and is independent of BCG vaccination status. Another test registered for the detection of infection with M. tuberculosis is QuantiFERON®-TB Gold, which is an in vitro diagnostic technology, allowing the detection of immune responses in whole blood samples, by restimulation with peptides that cover ESAT-6 , CFP10 and a simple peptide from TB7.7. Both of these tests measure the production of interferon gamma (IFN-γ) in response to exposure to antigen-specific peptide associations, and are currently considered state of the art. The T-SPOT.TB and QuantiFERON®-TB Gold tests are collectively recognized as IFN-γ release assays (IGRAS).

[013] Other cytokines and chemokines, in addition to IFN-y, have also shown relevance when monitoring the immunological response to mycobacterial antigens. The IFN-γ-induced protein (IP-10) is expressed at 100-fold higher levels compared to IFN-γ and diagnostic assays based on IP-10 secretion have shown diagnostic performance comparable to IP-10 release assays. IFN-Y (Ruhwald, 2009).

[014] In addition to these in vitro tests, which are already registered for human use and are used worldwide, ESAT-6 and CFP10 have also demonstrated that they are effective as skin assay reagents. Clinical studies have shown that a skin test, administered in the same way as the PPD but employing ESAT-6 and CFP10, produced and supplied as recombinant proteins, can be used to diagnose M. tuberculosis infection and is a condition unaffected by vaccination. with BCG (Aggerbeck, 2013).

[015] Despite the widespread use of BCG and various diagnostic methods, including IGRA, TB continues to take its toll, with almost two million deaths per year, and there is a continued need to develop diagnostic tests. immunodiagnostics with improved sensitivity. Immunocompromised patients are at high risk of developing TB and unfortunately, both TST and IGRAs, in their present forms, perform less than optimally in these groups. Patient groups with the highest need for improved testing include: HIV-infected patients, patients with immune-mediated inflammatory diseases, patients receiving immunosuppressive medication (e.g., prednisolone or TNF-a inhibitors), and patients with chronic renal failure. . In, for example, HIV-infected individuals, it is well known that a low CD4 cell count (e.g., <250 cells/µL) is strongly associated with high rates of indeterminate test results, compromised test sensitivity for active TB, and a analogous decrease for positive test responses in exposed individuals, reviewed in (Redelman-Sidi, 2013).

[016] Another very relevant group for targeted testing are children. The diagnosis of latent TB infection (LTBI) and TB in children is difficult, microbiological confirmation of the infection is often not obtained and treatment is driven only by clinical presentation. In both active and presumably latently infected children, the immune system is immature and is the likely cause of poor cytokine release and compromised IGRA performance. The QuantiFERON©-TB Gold test was recently shown to have a sensitivity of 53% in 81 children with microbiologically confirmed TB, justifying the need for improved immunodiagnostic tests for M. tuberculosis infection in children (Schopfer, 2013).

[017] The fundamental problem with testing IGRA performance in the high-risk patient groups mentioned above (e.g., children, immunosuppressed people, HIV-infected people) is the fact that the underlying immunosuppressive state that forces the increased risk of TB disease itself is characterized by low CMI responses and low IFN-γ release in response to antigens. As the IGRA result is determined based on comparing the magnitude of IFN-Y release to suppression, impaired IFN-Y release increases the risk of the test result becoming falsely negative. Consequently, it is obvious to recipient experts that the inclusion of more specific antigens will attract more specific T cells and this results in an increased response and CMI and release of IFN-γ and consequently a decreased risk of the response falling below suppression. . Consequently, the addition of additional specific antigens causes a greater limitation of IGRA tests, improving diagnostic sensitivity.

[018] Another benefit of diagnosing M. tuberculosis infection based on higher magnitude responses means increased analytical precision and more reliable test results. In the QuantiFERON®-TB Gold test, the cutoff value for a positive test is 0.35 IU/mL or 17.5 pg/mL, a very low concentration that is difficult to determine with high precision — even with sensitive methods such as ELISA. For example, the most accurate study of an IGRA currently detected considerable variability in the TB response measured by QuantiFERON-TB Gold in the tube when retesting the sample from the same patient. Intersubject variability included differences up to 0.24 IU/mL in either direction when the initial response was between 0.25 and 0.80 IU/mL. This has led to the conclusion that positive QuantiFERON-TB Gold tube test results of less than 0.59 IU/mL should be interpreted with caution (Metcalfe AJRCCM 2012).

[019] Modeling studies suggest that without new vaccines, TB cannot be eliminated, and that new, more effective vaccines are an international priority. The general idea is to complement the current BCG vaccine with a subunit booster vaccine, or create a new live TB vaccine to replace BCG. There are a growing number of experimental vaccines in clinical development and the emerging consensus is that ESAT-6 appears to be an essential vaccine antigen. Thus, many of the new vaccines currently at the preclinical level or in clinical trials contain ESAT-6. Recently, the Aeras Foundation announced the first human experience of a vaccine containing ESAT-6, intended to protect people already latently infected with TB from developing active TB disease (Aagaard, 2011). Several vaccine candidate lines have also been processed by direct recombination to express ESAT-6, e.g., rBCG:GE (Yang, 2011), rM.S-e6c10 (Zhang, 2010), Salmonella/Ag85B-ESAT-6 (Hall, 2009), rBCG-A(N)-E-A(C) (Xu, 2009) or fusion proteins incorporating ESAT-6, for example, to H1 (van Dissel, 2010; van Dissel, 2011). Unfortunately, the use of ESAT-6-based diagnosis in the IGRA test and vaccination with an ESAT-6-containing vaccine are an exact repetition of the cross-reactivity problem associated with the parallel use of TST and BCG.

[020] Consequently, there is a great need for a specific diagnostic reagent that can be used in parallel with vaccines containing either BCG or ESAT-6. Using in vivo or in vitro assays, the reagent should be capable of detecting M. tuberculosis infections in humans and animals, and discriminating not only between TB infection and BCG vaccination or the new vaccines containing ESAT-6, but also the exposure to non-pathogenic environmental mycobacteria. The diagnostic reagent should have at least the same sensitivity as the current combination of ESAT-6, CFP10 and the same TB7.7 diagnostic assays.

[021] Document EP2417456 describes such a system in which the use of Rv3615c, in conjunction with CFP-10, provides a diagnostic sensitivity very similar to that of the ESAT-6/CFP-10 combination.

[022] Because the unique characteristics of ESAT-6 are highly immunogenic and specific for infection with M. tuberculosis, it is not likely that replacing ESAT-6 with a simple antigen will increase sensitivity, compared to ESAT, when studying various population groups. This was demonstrated, for example, by Brock et al., showing recognition of simple antigens between 14-43% in TB patients, compared to ESAT-6, giving rise to a 75% response in the same group of patients. Given that most antigens are less immunogenic compared to ESAT-6, it is more likely that a combination of antigens will be required for large magnitude responses and improved diagnostic sensitivity.

[023] As exemplified, it is not simple to predict the sensitivity and specificity of antigen combinations; more precisely, this requires a detailed design of specific antigen combinations. Furthermore, by increasing the number of peptides in the diagnostic combination, this introduces the risk of further decreasing specificity by increasing the risk of false positives, highlighting that the diagnosis or immunogenic compositions for the specific diagnosis of TB require be carefully chosen and tested.

[024] There is, therefore, an urgent need for immunological diagnosis, in vivo or in vitro, mediated by cells, of infection with M. tuberculosis in a human or animal. In other words, a need for combinations of antigens that increase sensitivity (which gives fewer false negatives), compared to existing combinations of antigens, without compromising specificity (number of false positives). The required improved antigen combinations refer to both antigen compositions that do not include the ESAT-6 antigen, to anticipate the situation when a vaccine comprising ESAT-6 is introduced, and antigen combinations comprising ESAT-6 to improve reagents. diagnostic method of the present state of the art.

[025] Our data demonstrate that the CPF10/ESAT6 combinations and the CPF10/Rv3615 combinations can be further improved by introducing peptides derived from three new antigens with diagnostic potential. This new finding is unexpected for two reasons: a) the majority (>99%) of antigens in the TB genome are non-specific and are shared among several mycobacterial species, and thus the identification of strongly recognized antigens that are specific for TB Mycobacterium tuberculosis, has been very difficult, b) the sensitivity of the diagnostic combinations CPF10/RV3615c and CPF10/ESAT6 are already very high, and therefore, increasing sensitivity further becomes increasingly difficult, due to non-specific responses.

[026] The present invention is therefore very encouraging, as it describes peptides with the ability not only to increase the sensitivity of the CPF10/Rv3615c combination, but also of the current diagnostic "cocktail", which includes ESAT-6 and without compromising specificity.

Summary of the invention

[027] The invention is related to the improved detection of infections caused by species of the TB complex (M. tuberculosis, M. bovis, M. africanum) and discriminating between TB infection and vaccination. The improved diagnostic composition should not interfere with the effect of antigens from neither 1) a new TB vaccine, containing ESAT-6, 2) BCG, nor 3) exposure to non-pathogenic environmental mycobacteria. The invention discloses improved diagnostic or immunogenic compositions, which 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. Through the use of a "cocktail" or an association of antigens, or a "cocktail" or an association of peptides covering these antigens, we made the test highly sensitive, despite the absence of ESAT-6 in the immunogenic diagnostic compositions. Furthermore, we have further improved the diagnostic immunogenic compositions comprising ESAT-6 currently used.

Detailed exposition of the invention

[028] The diagnostic method is based on cell-mediated immunological recognition (CMI) of antigens expressed by M. tuberculosis bacteria (or other mycobacteria from the tuberculosis complex) during infection. Consequently, the test does not require the presence of bacteria like traditional culture, microscopy and PCR methods. This means that the test can be applied in advance, at the stage of infection, and that the test is applicable regardless of the anatomical location of the infection. The method is ideal for contact tracking, replacing the currently used TST.

[029] By selecting M-tuberculosis specific antigens, with theoretical diagnostic potential, and testing recognition in a series of human TB patients, we were able to identify three diagnostic associations, in which 1) ESAT-6 is absent and thus can also be used in individuals vaccinated with ESAT-6 to discriminate between M. tuberculosis infection and vaccination, 2) showed the same high specificity as diagnostic pools containing ESAT-6, and 3) showed exhibited a sensitivity to infection with M. tuberculosis greater than that obtained by a combination of ESAT-6, CPF10, and TB7.7.

[030] The present invention presents a diagnostic or immunogenic composition, comprising a mixture of essentially pure polypeptides, consisting of amino acid sequences chosen from:

[031] a) Rv3874 (SEQ ID NO 1), Rv3615 (SEQ ID NO 2) and additional compositions chosen 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);

[032] or b)

[033] a mixture of fragments of said polypeptides;

[034] or c)

[035] wherein the chosen mixture of polypeptides or fragments of said polypeptides has at least 80% sequence identity with any of the polypeptides of choice in a) or b) and is, at the same time, immunogenic.

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

[037] A preferred diagnostic composition comprises a mixture of fragments comprising the immunogenic epitopes of Rv3874, Rv3615 and optionally ESAT-6, wherein the fragments comprising immunogenic epitopes of SEQ ID NO 1 are chosen from SEQ ID NO 9-14, and the fragments comprising immunogenic epitopes of SEQ ID NO 2 are chosen from SEQ ID NO 15-18 or SEQ ID NO 59-63, and the fragments comprising immunogenic epitopes of SEQ ID NO 51 are chosen from SEQ ID NO 52-58, wherein the fragments comprising immunogenic epitopes of SEQ ID NO 3 are chosen from SEQ ID NO 19-21, and the fragments comprising immunogenic epitopes of SEQ ID NO 4 are chosen from SEQ ID NO 22-25 and the fragments comprising immunogenic epitopes of SEQ ID NO 5 are chosen from SEQ ID NO 26-45 and wherein the fragments comprising immunogenic epitopes of SEQ ID NO 6 is SEQ ID NO 8 and the fragments comprising immunogenic epitopes of SEQ ID NO 7 are chosen from SEQ ID NO 46-50.

[038] The polypeptides in the diagnostic or immunogenic composition may be present as separate entities or in which some or all of the polypeptides are fused together, optionally through linkers or spacers.

[039] A preferred diagnostic or immunogenic composition comprises a combination or mixture of SEQ ID NO 9-14, SEQ ID NO 15-18, SEQ ID NO 19-21 and SEQ ID NO 22-25, mentioned in the examples as a combination of A peptides.

[040] Detailed description of preferred polypeptides and fragments of said polypeptides:

[041] CFP10 (SEQ ID NO 1) is a major ESX-1 protein. The following 6 peptides, covering the entire amino acid sequence of CFP10, were chosen (SEQ IDs NO 9-14)

[042] Rv3615c (SEQ ID NO 2) is a protein secreted by the ESX-1 system. 4 peptides covering amino acids 55-103 (SEQ ID NO 15-18) were chosen. Alternative peptides covering the C-terminal part of Rv3615 are the five peptides with the amino acid sequence SEQ ID NO 59-63.

[043] Rv3865 (SEQ ID NO 3) is a protein associated with ESX-1 secretion: 3 peptides covering amino acids 9-44 were chosen (SEQ IDs NO 19-21)

[044] Rv2348c (SEQ ID NO 4) is located in the RD7 region that has been shown to be absent in BCG: 4 peptides were chosen covering amino acids 56-109 of the full length of the protein sequence (SEQ IDs NO 22-25)

[045] Rv3614c (SEQ ID NO 5) is a secreted protein: 20 peptides, covering the entire sequence, were selected (SEQ IDs NO 26-45)

[046] Rv2654c (SEQ ID NO 6) is a protein with unknown function, encoded by the RD11 region: peptide 4 was chosen (SEQ ID NO 8)

[047] Rv3877 (SEQ ID NO 7) is located in the RD1 region and is not present in BCG: 5 peptides were selected, covering amino acids 220-284 in the total length of the protein (511 aa) (SEQ IDs NO 46-50)

[048] ESAT-6 (Rv3875; SEQ ID NO 51) is a major ESX protein. 7 peptides covering the entire sequence (SEQ ID NO 52-58) were selected.

[049] The invention further features the use of diagnostic or immunogenic compositions for the preparation of a pharmaceutical composition for the diagnosis of TB caused by virulent mycobacteria, for example, by M. tuberculosis, Mycobacterium bovis, or Mycobacterium africanum, and a tool or CMI diagnostic set, comprising a diagnostic or immunogenic composition mentioned above, for the in vitro or in vivo diagnosis of TB.

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

[051] The in vivo TB diagnostic method comprises the intradermal injection, into an animal, including a human being, of a pharmaceutical composition, as defined above, in which a positive skin response at the injection site is indicative of the animal has TB, and a negative skin response at the injection site is indicative of the animal not having TB.

[052] The in vitro method of TB diagnosis comprises contacting a sample, for example, a blood sample, with a diagnostic or immunogenic composition according to the invention, in order to detect a positive reaction, e.g. For example, the proliferation of cells or the release of cytokines, such as IFN-Y.

[053] The present diagnostic or immunogenic compositions can replace compositions that are currently used in established IGRA tests (CFP10/Rv3874 and ESAT-6/Rv3875 in TB.SPOT®.TB test and TB7.7/Rv2654c, CFP10/Rv3874 and ESAT-6/Rv3875 in QuantiFERON®- TB Gold.

[054] Furthermore, the method has the following improvements, compared to CFP10/Rv3874 and ESAT-6/Rv3875 in TB.SPOT®.TB test and TB7.7/Rv2654c, CFP10/Rv3874 and ESAT-6/Rv3875 in QuantiFERON®-TB Gold:

[055] if an individual has been vaccinated with a vaccine containing ESAT-6, such as a protein subunit vaccine comprising ESAT-6, or a live recombinant vaccine engineered to express ESAT-6 or which inherently expresses it, the composition avoids the use of ESAT-6 and, consequently, is still specific for infection with M. tuberculosis. This is not the case for CPF10 and ESAT-6 in the TB.SPOT®.TB test and TB7.7, CFP10 and ESAT-6 in QuantiFERON®-TB Gold or any other ESAT-6 based test.

[056] Compositions containing ESAT-6 are being used in CMI-based M. tuberculosis tests. The presented test can avoid the use of ESAT-6 and takes advantage of the wide recognition obtained from the use of more than one specific M. tuberculosis antigen. In our test, we obtained, with the combination of CFP10, Rv3615c, Rv3865, and Rv2348 (peptide association A), a sensitivity of 87% and a specificity of 98%, compared to a sensitivity of 74% and specificity of 96% , using Quantiferon antigens. Thus, despite the lack of ESAT-6, known to be a highly sensitive antigen and recognized by a high proportion of individuals harboring an M. tuberculosis infection, the compositions tested here obtain >10% high sensitivity rates compared to those well-known compositions based on ESAT-6 and currently used in IGRA assays.

[057] Here we also present data showing that, by adding ESAT-6 to the peptide pool consisting of CFP10, Rv3615c, Rv3865 and Rv2348, we can further improve diagnostic performance. By adding ESAT-6 to the specified peptide pool, we can increase the magnitude of responses that could be relevant for diagnosis in individuals with different immunosuppressive complications, for example, HIV, or for use, for example, in children. Also by using a combination of peptides from all five antigens (CFP10, ESAT-6, Rv3865, Rv2348 and Rv3615c) we could increase the frequency of patients with a confirmed TB diagnosis by 3%, compared to having the association of CFP10 , Rv3615c, Rv2348 and Rv3865 isolates.

[058] The present invention also discloses the performance of in vivo tests for the diagnosis of TB. This could be done in the format of a test on the skin of an animal, including a human being, with the compositions mentioned above. The skin test would be: intradermal injection into an animal or application to the skin of animals, of a composition of the present invention, for example, with a dressing or bandage, with a positive response to the skin test, at the injection site. or application, is an indicator that the animal or human being has TB, and a negative response to the skin test, at the injection or application site, is an indicator that the animal or human being does not have TB.

[059] It is not necessary for the peptide associations of the invention to comprise the proteins in their entire length, since a sequence of just 6-9 amino acids (T cell epitope) is sufficient to infer an immune response, but the proteins of full length will also be useful. As it is possible for those skilled in the art to determine the exact and minimal amino acid sequences for the T cell epitope embedded in a protein, the present invention also relates to fragments (immunogenic portions) of polypeptides comprising said T cell epitopes. (or analogues thereof) without specific additional amino acids, such as full-length proteins and fusion proteins, comprising said T cell epitopes (optionally coupled via a linker or spacer), and to "cocktails" or combinations comprising said polypeptides or fusion proteins.

[060] Other embodiments of the invention will be described in the examples and claims.

Definitions

[061] Polypeptides

[062] The word "polypeptide", in the present invention, should have its usual meaning. It is a chain of amino acids of any length, including a full-length protein, oligopeptides, small peptides, and fragments thereof, in which the amino acid residues are linked by covalent peptide bonds. Polypeptides can be chemically modified, by being glycosylated, by being lipidated, for example, by chemical lipidation with palmitoyloxy-succinimide, as described by Mowat et al (Mowat, 1991), or with didecanoyl chloride, as described by ( Lustig, 1976), because they contain prosthetic groups, or because they contain additional amino acids, such as, for example, a "his-tag" or a signal peptide.

[063] Each polypeptide must therefore be characterized by specific amino acids and be encoded by specific nucleic acid sequences. It will be understood that such sequences include analogues and variants, produced by recombinant or synthetic methods, in which said polypeptide sequences have been modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide, and are further immunogenic. in any of the biological assays described here. Substitutions are preferably "conservative". These 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 in one-letter code.

[064] A preferred polypeptide, within the scope of the present invention, is a fragment of an immunogenic M. tuberculosis antigen. Such an antigen may be, for example, derived from the M. tuberculosis cell and/or M. tuberculosis culture filtrate. Thus, a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences. Additional sequences may be derived from the native M. tuberculosis antigen, or be heterologous, and said sequences may, but not necessarily, be immunogenic.

[065] In the present context, the term "substantially pure polypeptide fragment" means a polypeptide preparation that contains, at most, 10% by weight of another polypeptide material with which it is naturally associated (low percentages of other polypeptide materials are preferred). , for example, a maximum of 4%, a maximum of 3%, a maximum of 2%, and a maximum of 1). It is preferred that the substantially pure polypeptide is at least 96% pure, that is, that the polypeptide constitutes at least 96% by weight of the total polypeptide material present in the preparation, and higher percentages, such as at least 97%, are preferred. at least 98%, at least 99%, at least 99.25%, at least 99.5%, and at least 99.75%. It is especially preferred that the polypeptide fragment is in "essentially pure form", i.e., that the polypeptide fragment is essentially free from any other antigen with which it is naturally associated, i.e., free from any other antigen of bacteria belonging to the tuberculosis complex or a virulent mycobacteria. This can be accomplished by preparing the polypeptide fragment by recombinant methods in a non-mycobacterial host cell, as will be described in detail below, or by synthesizing the polypeptide fragment by well-known solid or liquid phase peptide synthesis methods, e.g. , by the method described by (Merrifield 1963), or variations thereof.

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

[067] A "TB patient" means an individual with an infection with virulent mycobacteria, proven by culture or microscopically, and/or an individual clinically diagnosed with TB and who responds to anti-TB chemotherapy. The culture, microscopy and clinical diagnosis of TB are well known to anyone skilled in the art.

[068] The term "delayed type hypersensitivity reaction" (DTH) means an inflammatory response, mediated by a T cell, elicited after the injection or application of a polypeptide to the skin, with said inflammatory response appearing within 72-96 hours after injection or application of the polypeptide.

[069] The term "cytokine" means any immunomodulatory agent, such as interleukins and interferons, which can be used as an indication of an immunological response. This includes, for example, interferon-gamma "IFN-Y", interferon-gamma inducible protein 10, also known as CXCL10 or "IP-10", and interleukin 2 (IL-2).

[070] Throughout the present invention, unless the context requires otherwise, the word "comprise", or variations thereof, such as "comprises" or "comprising", will be understood as implying the inclusion of an element designated , or an integer, or a group of integers, or groups of elements or integers, but not to the exclusion of any other element or integer or group of elements or integers.

[071] Sequence identity

[072] The term "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 fit as closely as possible to the gap insertion or, alternatively, be truncated at the ends of the protein sequences. sequence identity can be calculated as (Nref-Ndif)100, where N is the total number of identical non-Nref residues in the two sequences when aligned, and where Nref is the number of residues in one of the sequences. Thus, the AGTCAGTC DNA sequence will have 75% sequence identity with the AATCAATC sequence (Ndif = 2 and Nref = 8). A gap is counted as non-identity of the specific residue(s), i.e., the AGTGTC DNA sequence will have 75% sequence identity with the AGTCAGTC DNA sequence (Ndif = 2 and Nref = 8 ). Sequence identity can alternatively be calculated by the BLAST program, for example, the BLASTP program (Pearson, 1988) (www.ncbi.nlm.nih.gov/cgi-bin/BLAST). In one aspect of the invention, the alignment is performed with the ClustalW sequence alignment method with default parameters as described by Thompson, et al (Thompson, 1994), available at http://www2.ebi.ac.uk/ clustalw/.

[073] A preferred minimum percentage of sequence identity is at least 80%, such as 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

[074] An immunogenic epitope of a polypeptide is a part of the polypeptide that triggers an immune response in an animal or a human, and/or in a biological sample 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. The immunogenic epitope may be related to one or a few relatively small parts of the polypeptide, may be dispersed throughout the polypeptide sequence, or may be located in specific parts of the polypeptide. polypeptide. For some polypeptide epitopes, they have been shown to be dispersed throughout the polypeptide, covering the entire sequence (Ravn, 1999).

[075] To identify relevant T cell epitopes that are recognized during an immune response, it is possible to use a "brute force" method: since T cell epitopes are linear, polypeptide deletion mutants, if constructed systematically , will reveal which regions of the polypeptide are essential in immune recognition, for example, by subjecting these deletion mutants, for example, to the described IFN-Yaqui assay. Another method uses overlapping peptides for the detection of MHC class II epitopes, preferably synthetic, having a length of, for example, 20 amino acid residues derived from the polypeptide. These peptides can be tested in biological assays (e.g., the IFN-γ assay as described herein) and some of these will give a positive response (and thus are immunogenic) as evidence of the presence of a T cell epitope in the peptide. For the detection of MHC class I epitopes it is possible to predict peptides that will bind (Stryhn, 1996) and then produce these synthetic peptides and test them in relevant biological assays, for example, the IFN-γ assay, as described here. The peptides preferably have a length of, for example, 8 to 11 amino acid residues derived from the polypeptide.

[076] Although it has been shown that the minimum length of a T cell epitope is at least 6 amino acids, it is normal for such epitopes to be made up of longer amino acid stretches. Thus, it is preferred that the polypeptide fragment of the invention has a length of at least 7 amino acid residues, such as 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, in important embodiments of the method of the invention, it is preferred that the polypeptide fragment has a length of at most 50 amino acid residues, such as at most 40, 35, 30, 25 and 20 amino acid residues. amino acids. It is expected that peptides having a length between 10 and 30 amino acid residues will prove to be more efficient than MHC class II epitopes and, therefore, especially preferred lengths of the polypeptide fragment used in the method of the invention are 18, such as 15 , 14, 13, 12 and even 11 amino acid residues. It is expected that peptides having a length between 7 and 12 amino acid residues will prove to be most efficient as MHC class I epitopes and, consequently, especially preferred lengths of the polypeptide fragment used in the method of the invention are 11, such as 10, 9, 8 and even 7 amino acid residues.

[077] The immunogenic portions (fragments comprising immunogenic epitopes) of the polypeptides, comprising the immunogenic epitope, can be recognized by a large part (high frequency) or a smaller part (low frequency) of the genetically heterogenic human population. Furthermore, some immunogenic moieties induce highly immunological responses (dominant), while others induce smaller but still significant responses (subdominant). High frequency <low frequency can be related to the immunogenic portion binding to widely distributed MHC molecules (HLA type), or even to multiple MHC molecules (Sinigaglia, 1988; Kilgus, 1991). Fragments comprising immunogenic epitopes of said polypeptides may be present as overlapping peptides of at least 10 amino acids in length, thus encompassing several epitopes.

[078] Variants

[079] A common particularity of the polypeptides of the compositions of the invention is their ability to induce an immunological response, as illustrated in the examples. It should be understood that a variant of a polypeptide of the invention, produced by substitution, insertion, addition or deletion, is also immunogenic, as determined by any of the assays described herein.

[080] Immune individual

[081] An immune individual is defined as a person or animal that has eradicated or controlled an infection with virulent mycobacteria, or has received a vaccination with M. bovis BCG.

[082] Immunogenic

[083] An immunogenic polypeptide is defined as a polypeptide that induces an immune response in a biological sample or in an individual habitually or previously infected with a virulent mycobacteria. An immunogenic polypeptide is synonymous with an antigen or an antigenic polypeptide, and the two terms immunogen and antigen are used interchangeably in this presentation; the strict definition of an antigen is that it is capable of specifically binding to a T cell or B cell receptor and the strict definition of an immunogen is that it is capable of provoking an immune response, but when it comes to diagnosis, the effect of the two terms is the same and, therefore, they are used interchangeably here.

[084] CMI Diagnosis

[085] The immune response can be monitored by one of the following methods: an in vitro CMI response is determined by the release of a relevant cytokine, such as IFN-Y, from lymphocytes taken from an animal or human habitually or previously infected with mycobacteria virulent cells, or by detecting the proliferation of these T cells. Induction is carried out by adding an immunogenic composition to a suspension of blood cells, preferably comprising from 1x105 cells to 1x106 cells per well, the cells being isolated either from the blood, or from the spleen, of the liver, or lung, resulting in the addition of an immunogenic composition in a concentration of at least 1-200 µg per mL of suspension, and stimulation being carried out from two to five days. To monitor cell proliferation, cells are vibrated with radioactively labeled thymidine and, after 16-22 hours of incubation, proliferation is detected by liquid scintillation counting or any other methods to detect a proliferative response. The release of IFN-γ can be determined by the ELISA method, which is well known to those skilled in the art. Other cytokines and chemokines, in addition to IFN-γ, may be relevant when monitoring the immunological response to polypeptides, such as IL-2, IL-12, TNF-a, IL-4, TGF-ß, IP-10, MIP -1ß, MCP-1, IL-1RA and MIG. Another, more sensitive, method for determining the presence of a cytokine (for example, IFN-γ), is the ELISPOT method, in which cells isolated, for example, from blood are diluted to a concentration, preferably from 1 to 4 x 106 cells/mL, and incubated for 18-22 hours, in the presence of the diagnostic or immunogenic composition, resulting in a concentration, preferably, of 1-200 µg/mL. The cell suspensions are then diluted to 1 to 2 x 106/ml and transferred to polyvinylidene fluoride membrane microtiter plates coated with anti-IFN-γ and incubated preferably for 4 to 16 hours. Cells that produce IFN-Y are determined by using labeled secondary anti-IFN-γ antibodies, and a relevant stain-generating substrate, which can be enumerated using a dissecting microscope. The FluoroSpot assay is a modification of the ELISPOT assay and is based on the use of multiple fluorescent anticytokines that make it possible to generate two cytokines in the same assay, potentially allowing for improved prediction of disease risk, as will be described below for IL-2 co-determination. and IFN-γ. It is also possible to determine the presence of a cytokine or chemokine response using lateral flow technology. This type of assay — well known for rapid pregnancy tests — enables rapid detection of the level of cytokine or chemokine released and allows diagnosis of infection and disease also in many resource-constrained settings. Other immunoassays, including colorimetric assays, such as turbidimetry assays, which can be used for high-throughput detection of cytokine or chemokine levels are also known to those skilled in the art. It is also possible to determine the presence of mRNA coding for the relevant cytokines, using the polymerase chain reaction (PCR) technique. Detection of cytokines or chemokines at the mRNA level is generally faster than at the protein level, as mRNA transcription follows protein synthesis. For example, the mRNA levels of the cytokine IFN-γ and the chemokine IP-10 are optimal at short incubation periods compared to the protein level. Cytokine and chemokine signals detected at the mRNA level can be realized as early as 2 hours after stimulation, and maximum levels are reached within 6-10 hours. Generally, one or more cytokines will be measured, using, for example, PCR, lateral flow, ELISPOT or ELISA. It will be appreciated by those skilled in the art that a significant increase or decrease in the amount of any of these cytokines, induced by a specific polypeptide, can be used to evaluate the immunological activity of the polypeptide. Recipients of skill will also appreciate that certain patterns of cytokine release are associated with certain clinical conditions. In particular, an IFN-Y domain over IL-2 has been suggested to be indicative of incipient active TB disease, while an IL-2 domain over IFN-γ suggests control of the infection and a low risk of developing TB, despite of the presence of the infection in the mammal subject to the tests (Biselli, 2010; Sester, 2011).

[086] The in vitro CMI response can be increased 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, IL-14 , IL-5 and/or IL-13. Similar CMI responses can be detected more reliably if in vitro culture conditions are optimal for the cells that have been subjected to stimulation. Such conditions can be improved by adding nutrients, for example in the form of simple and complex sugars.

[087] A simpler, although sensitive, method is the use of whole blood samples, without prior isolation of mononuclear cells. With this method, a sample of heparized whole blood (with or without prior lysis of erythrocytes), in an amount of 50-1000 mL and incubation being carried out within 18 hours for up to 6 days, with the diagnosis of the immunogenic composition of the invention, resulting in a concentration of preferably 1-200 µg/mL. The supernatant is collected and the release of IFN-γ (or any other relevant released cytokine, for example, IP-10, IL-2 or others) can be determined by the ELISA method, which is well known to those skilled in the art.

[088] Another in vitro method, also simple, and yet sensitive, to determine a CMI response, is by applying the sample — after incubation with the diagnostic or immunogenic composition — in the form of dots on a paper towel. filter, for example, Whatman 903 or Whatman FTA paper. After drying, the sample, spread as drops, is stabilized and the cytokine and chemokine levels in the sample can be detected at a later stage. CMI responses are readily detected with the techniques mentioned above for protein or mRNA determinations. This method is particularly suitable for resource-poor setups, or for high-throughput sample preparation and analysis.

[089] Another in vitro method comprises "vacutainer" blood collection tubes, previously coated with immunogenic polypeptides or fusion proteins thereof, optionally also adding a blood stabilizer, such as Heparin and/or nutrients. Pre-coated incubation tubes allow simple blood collections and eliminate the risk of exposure to a blood-borne infection while the sample is prepared for in vitro incubation. These "vacutainer" tubes are ideal for high-throughput processing and automation.

[090] Consequently, the invention also relates to an in vitro method for continuous diagnosis or prior sensitization in an animal or human being, with a virulent mycobacteria, the method comprising providing a blood sample from the animal or human being. human, and the animal sample is placed in contact with the polypeptide of the composition of the invention, and a significant release in the extracellular phase of at least one cytokine, by mononuclear cells, in the blood sample, is indicative of the animal being sensitized; a positive response is a response of more than the release of a blood sample derived from a patient without a diagnosis of TB, plus two standard deviations.

[091] An in vitro CMI response can also be determined by using T cell lines derived from an immune individual or a person infected with M. tuberculosis, in which the T cell lines were derived or with live mycobacteria, extracts of bacterial cells or culture filtrates for 10 to 20 days with the addition of IL-2. Induction is carried out by adding, preferably, 1-200 µg of polypeptide per mL of T cell line suspension containing, for example, 1 x 105 cells to 3 x 105 cells per well, and incubation being carried out for 2 to 6 days. . The induction of IFN-Y or the release of another relevant cytokine is detected by ELISA. T cell stimulation can also be monitored by detecting cell proliferation using radioactively labeled thymidine, as described above. For both assays, a positive response is a response of more than the background plus two standard deviations.

[092] An in vivo CMI response (e.g., skin test, transdermal skin test, patch skin test) that can be determined as a positive DTH response after intradermal injection or local patch application, preferably of 1 -200 µg of each polypeptide in the diagnostic or immunogenic composition of the invention, to an individual who is clinically or subclinically infected with a virulent mycobacteria, having a positive response with a diameter of at least 5 mm, 72-96 hours after injection or application .

Diagnostic accuracy and cut-off values

[093] The sensitivity of any given diagnostic test defines the proportion of individuals with a positive response who are correctly identified or diagnosed by the test, for example, the sensitivity is 100% if all individuals with a given condition have a positive test. . The specificity of a given screening test reflects the proportion of individuals without the status who are correctly identified or diagnosed by the test; for example, 100% specificity is if all individuals without the status have a negative test result.

[094] Sensitivity is defined as the proportion of individuals with a given state (for example, active TB infection), who are correctly identified by the described methods of the invention (for example, have a positive result in the IFN-Y test)•

[095] Specificity is defined here as the proportion of individuals without the condition (for example, without exposure to active TB infection), who are correctly identified by the described methods of the invention (for example, have a negative result in the IFN- y).

[096] Receiver-operation characteristics

[097] The accuracy of a diagnostic test is best described by its receiver-operating characteristics (ROC) (Zweig, 1993). The ROC plot is a dot plot of all sensitivity/specificity pairs, resulting from continuous variation of the decision threshold, for the entire range of observed data.

[098] The clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify patients into relevant clinical subgroups. Diagnostic accuracy measures the ability of the test to correctly distinguish two different states of the observed patients. Such states are, for example, health and disease, latent or recent infection versus no infection, or benign disease versus malignant disease.

[099] In each case, the ROC plot represents the overlap between two distributions by representing sensitivity in relation to 1 - specificity for the full range of decision thresholds. On the y-axis is sensitivity, or the true positive fraction [defined as (number of true positive test results)(number of true positive test results + number of false negative test results]. This has also been referred to as positivity in presence of a disease or condition. It is calculated solely from the affected subgroup. On the x-axis is the false positive fraction, or 1 - specificity [defined as (number of false positive results)/(number of true negative results + number of false positives)] It is an index of specificity and is calculated entirely from the unaffected subgroup.

[0100] Because the true and false positive fractions are calculated entirely separately, using test results from two different subgroups, the ROC plot is independent of the prevalence of the disease in the sample. Each point on the ROC plot represents a sensitivity/specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (there is no overlap in the two distributions of results) has a ROC plot that passes through the upper left corner, where the true positive fraction is 1.0 or 100% (perfect sensitivity), and the false positive fraction is 1.0 or 100% (perfect sensitivity), and the false positive fraction is is 0 (perfect specificity). The theoretical graph for a test without discrimination (identical distributions of the results of the two groups) is a diagonal line at 45°, from the lower left corner to the upper right corner. Most points fall between these two extremes. (If the ROC plot falls completely below the diagonal at 45°, this is easily remedied by reversing the "positivity" criterion from "greater than" to "less than", or vice versa). Qualitatively, the closer the point is to the top left corner, the greater the overall accuracy of the test.

[0101] A convenient objective for quantifying the diagnostic accuracy of a laboratory test is to express its performance by a single number. The most common global measurement is the area under the ROC chart. By convention, this area is always > 0.5 (if not, you can always invert the decision rule to make it that way). Values range between 1.0 (perfect separation of the test values of the two groups) and 0.5 (no apparent distribution difference between the two groups of test values). The area depends not only on a particular portion of the graph, such as the point closest to the diagonal, or the sensitivity at 90% specificity, but on the entire graph. This is a quantitative, descriptive expression of how close the ROC graph is to the perfect graph (area = 1.0).

[0102] The clinical utility of new antigen associations can be evaluated in comparison, and in combination, with other diagnostic tools for the given infection. In the case of M. tuberculosis infection, the clinical utility of a CMI result can be evaluated in comparison with established diagnostic tests, such as IGRA or the TST, using a receiver operator curve analysis.

[0103] Preparation methods

[0104] In general, M. tuberculosis antigens, and DNA sequences encoding said antigens, can be prepared using any of a series of procedures.

[0105] They can be purified as native proteins, from cell filtrate or cultures of M. tuberculosis, using procedures such as those described above. Immunogenic antigens can also be produced recombinantly, using a DNA sequence encoding the antigen, which has been inserted into an expression vector and expressed in an appropriate host. Examples of host cells are E. coli. Polypeptides, or the immunogenic portion thereof, may also be produced synthetically, having less than about 100 amino acids, and generally less than 50 amino acids, and may be generated using techniques well known to those skilled in the art, such as techniques in commercially available solid phase, in which amino acids are added sequentially to a growing amino acid chain.

[0106] In the construction and preparation of the plasmid DNA encoding the polypeptide, a host strain, such as E. coli, can be used. Plasmid DNA can then be prepared from overnight cultures of the host strain carrying the plasmid of interest and purified using, for example, the Qiagen Giga-Plasmid Column Kit (Qiagen, Santa Clarita, CA , USA), including an endotoxin removal step.

Fusion proteins

[0107] In addition to being separate entities, two or more of the immunogenic polypeptides can also be produced as fusion proteins, through which methods superior characteristics of the polypeptide of the invention can be achieved. For example, fusion partners that facilitate export of the polypeptide when produced recombinantly, fusion partners that facilitate purification of the polypeptide, and fusion partners that promote immunogenicity of the polypeptide fragment of the invention are all possibilities. interesting. Accordingly, the invention also pertains to a fusion polypeptide, comprising at least two (such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) immunogenic polypeptides or fragments defined above, and optionally by at least one additional fusion partner, and compositions comprising fusion proteins. In order to favor immunogenicity, the fusion partner can be another polypeptide derived from M. tuberculosis, such as 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, 19kDa 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 invention also pertains to a fusion polypeptide, comprising mutual fusions of two or more (such as 3, 4, 5, 6, 7, 8, 9, 10 or more) of the polypeptides (or immunogenic portions thereof) of the invention.

Picture's description

[0108] Figure 1. Heat map, showing immune recognition in 34 volunteer donors from Egypt, based on a cutoff value of 100 pg/mL of IFN-Y. Two cases (patients 1 and 2) had latent TB, and 32 cases were diagnosed with TB disease (patients 3-34). The white color code indicates no response, the gray color code indicates a response, and the black color code indicates a response to the given antigen without response to either ESAT-6 or CFP10.

[0109] Figure 2. Heat map, showing immune response recognition in 31 volunteer donors from Greenland, based on a cutoff value of 50 pg/mL of IFN-y. 14 were diagnosed with TB (patients 1-14) and 17 had latent TB (patients 15-31). The white color code indicates no response, the gray color code indicates a response, and the black color code indicates a response to the given antigen without response to either ESAT-6 or CFP10.

[0110] Figure 3. Heat map, showing immune recognition in 30 endemic control donors from Egypt, based on a cutoff value of 100 pg/mL of 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 defined as "endemic controls" as they were living in Egypt, which is considered an intermediate endemic country. White color code indicates no response and gray color code indicates response. The antigens investigated were all highly specific in contrast to PPD, which was included as an example of a non-specific antigenic stimulation. Donors 31 and 77 both recognized a wide range of M. tuberculosis antigens, indicating latent infection, despite the aforementioned selection criteria.

[0111] Figure 4. IP-10 responses to the association of Quantiferon peptides (ESAT-6, CFP10, Rv2654c (peptide 4)) and the association of A peptides in 73 Egyptian patients with TB. Dotted lines indicate mean responses of 6 ng/mL for Quantiferon antigens and 5.5 ng/mL for A-peptide association.

[0112] Figure 5. IP-10 responses to the association of Quantiferon peptides (ESAT-6, CFP10, Rv2654c (peptide 4)) and the association of A peptides in 100 individuals from Denmark not exposed to M. tuberculosis. Dotted lines indicate mean responses of 0 ng/mL for both antigen combinations. This shows high specificity (few false positives) across the peptide pool, indicating that each peptide had high specificity.

[0113] Figure 6. Responses to IFN-Y for the association of Quantiferon peptides (ESAT-6, CFP10, Rv2654c (peptide 4)) and the association of A peptides in 100 individuals from Denmark not exposed to M. tuberculosis. Dotted lines indicate mean responses of 0 pg/mL for the A peptide association, and 4.9 pg/mL for Quantiferon antigens. This shows high specificity (few false positives) across the peptide pool, indicating that each peptide had high specificity.

[0114] Figure 7. Analysis of the receiver operator characteristic curve (ROC), comparing the diagnostic potential of the association of A peptides with Quantiferon antigens in 100 individuals not exposed to M. tuberculosis and in 73 patients with TB. This shows a high specificity (few false positives) of the entire peptide association, indicating that each peptide has a high specificity.

[0115] Figure 8. IP-10 responses (ng/mL) to the association of Quantiferon peptides (ESAT-6, CFP10 and Rv2654c) and to the association of A peptides in 68 microbiologically confirmed cases of TB patients and 36 controls endemic to Tanzania.

[0116] Figure 9. IP-10 (ng/mL) responses to the A-peptide association, and the A-peptide association enriched with ESAT-6, in 73 patients from Cairo, Egypt, with confirmed TB. The lines indicate the average.

EXAMPLES

[0117] Example 1. Initial selection of antigens

[0118] The T cell antigens chosen for TB immunodiagnosis should be specific for infection with M. tuberculosis, to avoid interference from vaccination with BCG and many prevalent atypical mycobacteria. At the same time, it is important to avoid ESAT-6, as ESAT-6 is present in many of the new TB vaccines. As described above in "Background of the Invention", through an extensive and strict selection and elimination process, based on theoretical considerations, practical tests and literature search of hundreds of potential antigens, we selected no more than 9 M. tuberculosis for further testing. These are:

[0119] CFP10 (Rv3874). The 10 kDa culture filtrate antigen, together with ESAT-6, is the basis of current cell-based blood tests for the diagnosis of M. tuberculosis infection by IFN-Y release assays (IGRAs). CFP10 is an immunodominant M. tuberculosis antigen, and the diagnostic specificity of CFP10 and ESAT-6 is driven by their genomic location in region of difference 1 (RD1), a region that is absent in all BCG strains (Behr , 1999) and is involved in the pathogenesis of M. tuberculosis. Genes encoding components of the ESX-1 secretion pathway are also located in RD1. In a review of interferon-Y assay studies, a sensitivity for CPF10 of 61-71% was reported in patients with TB (Pai, 2004). In contrast to ESAT-6, CFP10 is not part of any of the current vaccine candidates under evaluation.

[0120] Rv3877. As for CFP10, the Rv3877 gene is located in the RD1 region on the M. tuberculosis chromosome and has no close homologues anywhere in the M. tuberculosis genome. The protein is not present in M. bovis BCG or the environmental mycobacteria M. avium, and can therefore be used for the specific diagnosis of M. tuberculosis, without interference from possible previous BCG vaccines, nor from strong exposure to mycobacteria. environmental, such as M. Avium. Rv3877 is a transmembrane protein and a key component of ESX-1 secretion, as it forms the pore through which ESX-1 substrates are secreted (Abdallah, 2007). A combination of synthetic peptides covering the Rv3877 protein induced positive responses in 33% of PBMCs isolated from human TB patients (Mustafa, 2008).

[0121] Rv3614c and Rv3615c. The espA-espC-espD gene cluster (Rv3616c-Rv3615c-Rv3614c) is essential for ESX-1-dependent protein secretion and virulence of M. tuberculosis (Fortune, 2005; MacGurn, 2005), and has recently been shown to the three genes are co-transcribed (Chen, 2012). Rv3616c and Rv3615c are cosegregated with ESAT-6 and CFP10 (Fortune, 2005; MacGurn, 2005), whereas secretion of Rv3614c does not exclusively require the functions of ESX-1 (Chen, 2012). In cattle, the M. bovis counterpart of Rv3615c, Mb3645c, stimulated IFN-Y responses in 37% of M. bovis-infected animals, but not in plain and BCG-vaccinated animals (Sidders, 2008). Mb3645c and Rv3615c show 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 immunodiagnosis, specific for M. tuberculosis, with recognition of TB cases and low response in BCG vaccine recipients (Millington, 2011). In patients with active TB, the most frequently 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 recognized specifically in individuals infected with M. tuberculosis, but with limited recognition in people vaccinated with BCG.

[0122] EspF (Rv3865). The protein associated with the secretion of ESX-1, EspF, or M. bovis Mb3895 (identical to Rv3865 of M. tuberculosis) was identified by Ewer et al (Ewer, 2006), as a promising diagnostic marker in experimentally or naturally infected cattle. with M. bovis. 50% of experimentally infected cattle responded to the combination of the Mb3895 peptide, while calves vaccinated with BCG did not respond to this peptide association.

[0123] Rv2348c. Rv2348c is a hypothetical protein with unknown function. The Rv2348c gene is located in the RD7 region. This region has been shown to be absent in BCG (Behr, 1999) and the protein can therefore be used for the diagnosis of TB without the interference of previous BCG vaccination. The gene is highly transcribed in vitro (Arnvig, 2011) and the protein has been identified in proteome studies (de Souza, 2011). The 23-50 amino acid fragment in the Rv2348c ORF (open reading system) has very high homology to the M. avium Mav_2040 gene.

[0124] Rv3873. From the amino acid sequence of Rv3873, a region covering 1270 amino acids was covered by overlapping peptides. Among several RD peptide associations evaluated, this Rv3873 peptide association, termed Rv3873A, was identified as one of the most promising associations recognized by 46% of PBMCs from TB patients (Brock, 2004). Potential extensions of cross-reactivity were not present in this part of the molecule.

[0125] Rv3878. As described above for Rv3873, a sequence of peptides called Rv3878B, covering amino acids 122-189 of this RD1 protein, was defined and evaluated. It was recognized by 32% of PBMCs from human TB patients, and was suggested as a cocktail or combination of peptides, which could be combined with ESAT-6 and CFP10 to maximize sensitivity (Brock, 2004).

[0126] Rv2654c. The Rv2654c gene is encoded by the RD11 region and encodes a possible PhiRv2 prophage protein, with unknown function. By selecting overlapping peptides that cover the entire protein product of Rv2654c (designated TB7.7), Brock et al found no cross-recognition in BCG-vaccinated individuals and, in addition, showed a sensitivity of 47% (Brock, 2004 ). A selected peptide (SEQ ID #8) is included in the QuantiFERON® TB Gold test.

[0127] Table 1. List of sequences for the selected peptides.

[0128] Example 2. Antigen selection

[0129] Seven of the antigens listed in the text 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 comparator and reference antigen. Furthermore, PPD in Egypt was included as an example of a non-specific antigen stimulation.

[0130] Diluted whole blood, recently collected as a sample, was restimulated with the selected peptides, from the antigens, as outlined, and the response to the ESAT-6 and CFP10 peptide associations were included as references.

[0131] In the study in Egypt, 34 voluntary donors (8 female and 26 male) were included as positive controls. 32 of these were diagnosed with TB disease with documented positive saliva culture (patients 3-34). Two cases had latent TB (patients 1 and 2). In addition, 30 donors were included as endemic negative controls (5 female and 25 male). These were all presumed to be BCG vaccinated, had no history of TB disease, and had no known contact with a TB patient. In the Greenland study, 31 patients were included (15 females and 16 males). 14 were diagnosed with TB disease (patients 1-14); in 11 cases positive saliva culture was documented, and in 4 cases the diagnosis of TB was made on clinical grounds. The remaining 17 patients had latent TB (patients 15-31).

[0132] In both studies, diluted whole blood, freshly collected as a sample, was stimulated in plates with the selected antigens (10 µg/mL of each peptide). Synthetic peptides (obtained from Genecust) from ESAT-6, CFP10, Rv3873, Rv3878, Rv3615c, Rv3865, Rv3877 and Rv2348 antigens were screened in both studies and positive (PHA) and negative (isolated medium) controls were included and ( only in Egypt) PPD (not represented) were also included. Diluted whole blood was incubated at 37°C for 5 days, and subsequently supernatants were collected and tested for (IFN-Y) by a home-made ELISA. A positive response in these studies was defined as an IFN-γ concentration of 100 or 50 pg/mL for the studies in Egypt and Greenland, respectively.

[0133] Figures 1, 2 and 3 are graphical representations (heat maps) of data in which the individual values contained are represented as colors, in which the white color shows lack of response, the gray color indicates a response to the given antigen, and the black color represents a response to an antigen in which the same donor does not respond to ESAT-6 and/or CFP10. As shown, several of the antigens under test were recognized in TB patients or in latently infected donors, where the most predominant responses were derived from stimulation with Rv3615c (recognized in 49% of donors). Important fact, ESAT-6 only recognizes three patients that were not recognized in stimulation with CFP10 (patients no. 9 and 17 in Figure 1, and patient no. 3 in Figure 2) and of those, Rv3615 is capable of recognizing all three patients . Furthermore, restimulation with Rv3615c showed recognition in 11 and 9 donors unrecognized 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 2 of 65 donors and Rv3878 was recognized in 7 of 65 donors. Thus, despite previous data on these antigens in TB patients from Denmark and the Netherlands, with intermediate sensitivity (Rv3873 with 32% for Rv3878 and 46% for Rv3873 (Brock, 2004), the data obtained here highlight that not all antigens which are expected to be sensitive perform well in all settings. Rv3865, Rv3877, and Rv2348 showed intermediate sensitivity and were recognized in 16, 12, and 15 of 65 donors. Importantly, the antigens Rv3615c, Rv3865, and Rv2348 all elicit responses in a number of donors that do 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 donors endemic to Egypt (Figure 3). Such As shown, the antigens investigated were all highly specific, in contrast to PPD, which was included as an example of nonspecific antigen stimulation. Donors 31 and 77 both recognized a wide range of M. tuberculosis antigens, including both ESAT -6 and CFP-10, strongly indicating a latent infection, despite the selection criteria mentioned.

[0134] Example 3. CFP10 and 3615c are comparable to CFP10 and ESAT-6

[0135] The diagnostic performance of the CFP10 and Rv3615c combination was subsequently compared to that of the CFP10 and ESAT-6 combination. We included whole blood samples from 35 individuals from Greenland, of whom 18 had a latent M. tuberculosis infection, defined as a positive Quantiferon test and/or proven exposure to M. tuberculosis conversion tuberculin skin test, and 17 patients who were microbiologically confirmed TB. Individual 200 µL aliquots 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 incubator at 37 °C for 7 days. A negative (zero) control sample was prepared in parallel. After incubation, plasma supernatant was isolated and the level of IFN-Y was determined using ELISA.

[0136] The diagnostic ability of the three antigens was assessed by adding the measured level of antigen-specific production of IFN-Y (stimulated whole blood level subtracted from the level in the unstimulated well) in response to stimulation with individual antigen(s). , followed by comparing this sum with a cutoff value. The cutoff value was defined as a combined antigen-specific response of at least 50 pg/mL and 4-fold greater than the zero value in the individual patient. Specific antigen levels above the cutoff value classified the patient as antigen positive, and specific antigen levels below the cutoff value as antigen negative.

[0137] Table 2 shows the sensitivity of individual antigens CFP10, Rv3615c, ESAT-6 and combinations. As shown, the diagnostic performance of the combination of CFP10 and Rv3615c is comparable to that of CFP10 and ESAT-6, with both combinations showing 60% sensitivity. The combination of CFP10, Rv3615c and ESAT-6 further improves sensitivity from 60% to 69%.

[0138] Table 2. Comparison of the sensitivity of CFP10, Rv3615c and ESAT-6 and combinations thereof.

[0139] Example 4. Enrichment of the combination CFP10 and Rv3615c with three antigens: Rv2348, Rv3865 and Rv3877

[0140] Using the same whole blood samples as previously (35 individuals from Greenland; 18 with latent M. tuberculosis infection and 17 patients with microbiologically confirmed TB) and the same assay conditions, we evaluated the effect of combining CFP10 and Rv3615c with three different antigens: Rv2348, Rv3865 and Rv3877.

[0141] The diagnostic capacity of the three antigens was assessed by adding the measured level of antigen-specific production of IFN-Y (level in stimulated whole blood subtracted from the level in the unstimulated well) in response to the antigen(s) individual values, followed by comparison with the sum at a cutoff value. The cutoff value was defined as a combined antigen-specific response of at least 50 pg/mL and 4-fold higher than the zero value in the individual patient. Specific antigen levels above the cutoff value classified the individual patient as antigen positive, and specific antigen levels below the cutoff value as antigen negative.

[0142] Table 3 shows the sensitivity of individual antigens Rv3865, CFP10, Rv3615c and combinations. The sensitivity of Rv3865 is relatively modest, with only 20% sensitivity, but adding Rv3865 to CFP10 and Rv3615c increases the total diagnostic sensitivity by 6% compared to CFP10 and Rv3865 alone.

[0143] Table 3. Comparison of CFP10, Rv3615c, Rv3865 and combinations thereof, for the diagnosis of M. tuberculosis infection.

[0144] Similarly, the diagnostic capacity of Rv3877 was only 11%, but this antigen also improved the overall sensitivity of CFP10 and Rv3615c from 60% to 69% (Table 4).

[0145] Table 4. Comparison of CFP10, Rv3615c, Rv3877 and combinations thereof for the diagnosis of the invention with M. tuberculosis.

[0146] Finally, we evaluated whether Rv2348 was also capable of increasing the diagnostic capacity of CFP10 and Rv3615c (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 only CFP10 and Rv3615c.

[0147] Table 5. Comparison of CFP10, Rv3615c, Rv2348 and combinations thereof for the diagnosis of infection with M. tuberculosis.

[0148] We chose the following antigens (CFP10, Rv3615c, Rv3865 and Rv2348) for another evaluation of sensitivity and specificity, when combining these 4 antigens in a single peptide association (peptide association A).

[0149] Example 5. Sensitivity and specificity test of the A-peptide association

[0150] It is well known in the field that an immunodiagnostic "cocktail" comprising ESAT-6, CFP10 and TB7.7p4 is the preferred method for diagnosing infection with M. tuberculosis. This cocktail of antigens is considered both sensitive and specific, and forms the basis of the Quantiferon test. As is evident from the previous examples, the combination of antigens improves diagnostic sensitivity and test reliability, as the underlying magnitude of the IFN-Y responses is greater and is detected more strongly, compared to having only the antigens.

[0151] A very "user-friendly" approach to immunodiagnosis is the use of "vacutainer" tubes previously coated with antigens in a cocktail. For example, in the Quantiferon test, lyophilized peptides, representing the antigen cocktail, are coated with heparin in a vacutainer tube. Blood is poured into this 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 level of IFN-Y produced can be measured in the supernatant plasma and compared with positive and negative control samples. Tested patients can be further classified as either infected or not infected if the level is above a cutoff value for positive test results.

[0152] It is well known that other immune determining molecules, associated with IFN-γ signaling, are useful for diagnosing M. tuberculosis infection (Chego ERJ 2014). The chemokine IP-10 is produced at very high levels and has diagnostic performance comparable to that of IFN-γ.

[0153] To demonstrate the utility of combining several antigens into an antigen cocktail, we combined the following antigens in the "A peptide pool". Peptide pool A consisted of the following peptides: CFP10: 6 peptides, covering the entire amino acid sequence of CFP10 (SEQ IDs no. 9-14) Rv3615c: 4 peptides, covering amino acids 55-103 (SEQ IDs no. 15-18) Rv3865: 3 peptides, covering amino acids 9-44 (SEQ IDs no. 19-21) Rv2348c: 4 peptides, covering amino acids 56-109 of the entire length of the protein sequence (SEQ IDs no. 22-25)

[0154] A second, independent study, in Egypt, was carried out in order to test the sensitivity of the 4 antigens, when combined in the association of A peptides. The study included 73 patients with TB, with a documented saliva culture, and each patient gave a blood sample, taken directly into vacuum tubes coated with previously prepared antigens. The tubes were coated with either the ESAT-6 + CFP10 + Rv2654c peptides (i.e., the same peptides as in the Quantiferon test and used as a reference, designated the Quantiferon peptide pool) or with the A peptide pool (CFP10 + Rv3516c + Rv3865 + Rv2348, as indicated above). After 16-24 hours of incubation, supernatants were collected and tested for IP-10 cytokine release with a home-made ELISA assay. As shown in Figure 4, a high proportion of TB patients recognized both the A peptide pool and the Quantiferon peptide pool. The median responses were 5.5 ng/mL IP-10 for the A peptide pool and 6.0 for the Quantiferon peptide pool.

[0155] In parallel, an independent study was carried out in Denmark in order to test the specificity of the A-peptide association. In the study, 100 patients were included who lived in an area with a very low prevalence of TB (Denmark) and without exposure known to M. tuberculosis. In 17 cases, patients were documented as having been vaccinated with BCG, and in 19 cases BCG vaccination status was unknown/undocumented. The remaining participants were not vaccinated with BCG. Analogous to the sensitivity study, fresh whole blood was collected directly into vacuum tubes, previously coated either with the association of A peptides (CFP10 + Rv3615c + Rv3865 + Rv2348), or with the association of Quantiferon reference peptides (ESAT-6 + CFP10 + Rv2654c). After 16-24 hours of incubation, supernatants were collected and tested for IP-10 and IFN-Y cytokine content with a home-made ELISA assay. Although the median IP-10 responses to A-peptide pool and Quantiferon peptide pool were both 0 ng/mL (Figure 5), some unexposed donors exhibited positive responses upon restimulation with Quantiferon antigens with IP-10 levels. approximately 5 ng/mL. The same trend was observed with unexposed donors presenting false positive responses when IFN-Y secretion was analyzed (Figure 6; median A-peptide association of 0 pg/mL, interquartile range (IQR) of -0.5 -5.2 pg/mL and median Quantiferon peptide association 4.9 pg/mL, IQR -0.6-32.45 pg/mL.

[0156] Combining the data for sensitivity and specificity studies allowed us to perform a receiver operator characteristic curve (ROC) analysis, comparing the diagnostic potential of the association of A peptides with the association of Quantiferon antigen peptides (Figure 7) . The area under the curve (AUC) was 0.979 for the A peptide association and 0.947 for the Quantiferon antigen peptide association. By ROC curve analysis, the optimal cutoff values, both for the association of A peptides and for the association of Quantiferon antigen peptides, were identified as 1.4 ng/mL for the association of A peptides (sensitivity 87.7% at 98.1% specificity); and 2.3 ng/mL for the association of Quantiferon antigen peptides (sensitivity 75.3% to 96.2% specificity).

[0157] Using these cutoff values, we compared face to face the number of positive and negative responses by restimulation with the Quantiferon peptide association and the A peptide association (Tables 6 and 7). Of the 73 TB patients, 54 (74%) recognized the Quantiferon peptide pool, which is within the published range of sensitivity of Quantiferon antigens between 64-89% (Dewan, 2007). In comparison, the A-peptide combination recognized a greater proportion of TB patients in this study (64 of 73 patients), corresponding to an estimated sensitivity of 88%. Using a McNemar test, the A-peptide pool demonstrated significantly higher sensitivity in this study compared to the Quantiferon peptide pool (p < 0.012).

[0158] Table 6. Face-to-face comparison of the Quantiferon peptide association and the A-peptide association in 73 TB patients.

[0159] Table 7. Face-to-face comparison of the Quantiferon peptide pool and the A peptide pool in 100 presumably uninfected controls.

[0160] In conclusion, the A-peptide combination exhibited significantly higher sensitivity (more true positives; table 6) compared to Quantiferon antigens and, furthermore, was at least as specific (comparable false positives; table 7). These results clearly demonstrate that it is possible to 1) design pools of ESAT-6-deprived TB diagnostic peptides with a higher sensitivity compared to current Quantiferon antigens, 2) design a pool of non-ESAT-6 containing antigens , with a specificity comparable to current Quantiferon.

[0161] Example 6. Validation of the association of peptides A.

[0162] It is well known to those skilled in the art that the validation of cutoff values for immunodiagnostic tests requires confirmation in independent groups. For this purpose, we included 68 cases of patients with microbiologically confirmed TB and 36 endemic controls, that is, individuals of whom some have previously existing but controlled M. tuberculosis infection from Tanzania.

[0163] From each donor, 1 mL of blood was collected into 5 vacutainer tubes, comprising lyophilized heparin (18 IU) and peptides (5 µg/peptide), as follows: association of Quantiferon peptides (ESAT-6, CFP10 and TB7. 7p4 (tube 1), comparable to the Quantiferon test); association of A peptides (CFP10, Rv3615, Rv3865 and Rv 2348B (tube 2)) and a negative control tube (tube 3).

[0164] In Figure 8 we show the response of the negative control tube (tube 3) subtracted from IP-10 (ng/mL) in the cases, and controls of tube 1 and tube 2. It is evident that the association of Quantiferon peptides and the association of A peptides are comparable in terms of the high magnitude of response in TB cases. As expected, endemic control responses are more heterogeneous confirmation that some tested individuals are infected.

[0165] Using the pre-defined cutoff value, identified in example 5 (1.4 ng/mL), the accuracy of the diagnosis was compared, both for patients with TB (table 8) and for endemic controls (table 9) . In the TB patient group, the diagnostic sensitivity of the association of standard Quantiferon peptides was 66% (45 defined as positive, out of 68 patients included) and higher for the association of A peptides, with 72% sensitivity (49 defined as positive, of 68 patients). As expected, the agreement between these two tests was very high, with 91% agreement (44 being positive in both tests, 18 being negative in both tests, with an overall agreement of 62 out of 68).

[0166] Table 8. Agreement between the Quantiferon peptide pool and the A peptide pool, after classification responses to antigen stimulation of 68 patients with confirmed TB, using predetermined cutoff values for positive testing.

[0167] In the endemic control population there is no gold standard for infection, therefore we present the reason as positive responses. The A peptide pool detected 39% (14/36) as positive, and the standard Quantiferon peptide pool 31% (11/36), again suggesting higher sensitivity. Agreement was also very high (92%, corresponding to agreement in 33 cases out of 36 included).

[0168] Table 9. Agreement between the Quantiferon peptide pool and the A peptide pool, after classification responses to antigen stimulation from 36 endemic controls, using predetermined cutoff values for positive testing.

[0169] Example 7. The association of A peptides can be further improved when combined with ESAT-6.

[0170] We further evaluated the possibility of adding ESAT-6 to the A-peptide association, with the aim of further improving diagnostic performance. Consequently, we tested the A + ESAT-6 peptide association and the A peptide association in the group of 73 confirmed TB cases, in Cairo, Egypt, and using the same assay conditions as those described in example 5. In figure 9 is It is evident that the magnitude of responses is increased when combining the association of A peptides with ESAT-6, with the association of A peptides having a median response of 5.50 ng/mL of IP-10, compared to the association of A peptides with ESAT-6, where the median is 6.86 ng/mL of IP-10. Using a cutoff value of 0.75 ng/mL, we compared responder frequencies in the two groups. In the association of A peptides, the responder frequency was 93%, with 68 positives, of the 73 patients tested, while the frequency for the association of A peptides with ESAT-6 was 96% (70 of 73 patients - 96%) . Thus, combining the association of A peptides with ESAT-6 reduced the false negative rate from 7% to 4%.

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Claims (12)

1. DIAGNOSTIC COMPOSITION, characterized by comprising a mixture of pure polypeptides, consisting of amino acid sequences chosen from: a) Rv3874 (SEQ ID NO 1), Rv3615 (SEQ ID NO 2) and one or more chosen from Rv3865 (SEQ ID NO 3), Rv2348 (SEQ ID NO 4) and Rv3877 (SEQ ID NO 7); or b) at least one fragment of Rv3874 (SEQ ID NO 1) chosen from SEQ ID NO 9 to 14, at least one fragment of Rv3615 (SEQ ID NO 2) chosen from SEQ ID NO 15 to 18 or SEQ ID NO 59 to 63, and at least one fragment of one or more selected from Rv3865 (SEQ ID NO 3) chosen from SEQ ID NO 19 to 21, Rv2348 (SEQ ID NO 4) chosen from SEQ ID NO 22 to 25 and Rv3877 (SEQ ID NO 7) chosen from SEQ ID NO 46 to 50. 2. DIAGNOSTIC COMPOSITION, according to claim 1, characterized by comprising Rv3874 (SEQ ID NO 1), Rv3615 (SEQ ID NO 2) and Rv3865 (SEQ ID NO 3). 3. DIAGNOSTIC COMPOSITION, according to claim 1, characterized by comprising Rv3874 (SEQ ID NO 1), Rv3615 (SEQ ID NO 2) and Rv2348 (SEQ ID NO 4). 4. DIAGNOSTIC COMPOSITION, according to claim 1, characterized by comprising Rv3874 (SEQ ID NO 1), Rv3615 (SEQ ID NO 2) and Rv3877 (SEQ ID NO 7). 5. DIAGNOSTIC COMPOSITION, according to claim 1, characterized by comprising Rv3874 (SEQ ID NO 1), Rv3615 (SEQ ID NO 2), Rv3865 (SEQ ID NO 3) and Rv2348 (SEQ ID NO 4). 6. DIAGNOSTIC COMPOSITION, according to any one of claims 1 to 5, characterized by additionally comprising Rv3875 (SEQ ID NO 51) or fragments of SEQ ID NO 51 chosen from SEQ ID NO 52-58. 7. DIAGNOSTIC COMPOSITION, according to claim 5, characterized in that the mixture comprises SEQ ID NO 9-25. 8. DIAGNOSTIC COMPOSITION, according to any one of the preceding claims, characterized in that some or all of the polypeptides are optionally fused together through linkers and spacers. 9. USE OF DIAGNOSTIC COMPOSITIONS, as defined in claims 1 to 15, characterized in that it is for the preparation of a pharmaceutical composition for the diagnosis of TB caused by a virulent mycobacteria, for example, by Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium africanum . 10. CMI DIAGNOSTIC TOOL OR KIT, characterized in that it comprises a diagnostic composition, as defined in any one of claims 1 to 8. 11. CMI DIAGNOSTIC TOOL OR KIT, according to claim 10, characterized in that it is for diagnosing tuberculosis in vitro or in vivo. 12. METHOD OF IN VITRO DIAGNOSIS OF TUBERCULOSIS CAUSED BY VIRULENT MYCOBACTERIA, for example, by Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium africanum, in an animal, including a human being, characterized by using a diagnostic composition, as defined in claims 1 to 8 comprising contacting a sample, for example, a blood sample, with a diagnostic composition, as defined in claims 1 to 8, in order to detect a positive reaction, for example, the proliferation of cells or the release of cytokines, such as IFN-Y or IP-10.