USE OF AMINO ACID SEQUENCES FROM MYCOBACTERIUM TUBERCULOSIS OR CORRESPONDING NUCLEIC ACIDS FOR DIAGNOSIS AND PREVENTION OF TUBERCULAR INFECTION, DIAGNOSTIC KIT AND VACCINE THEREFROM

Abstract

The present invention refers to the use of gene sequences or portions thereof characterized in that the same belong to the classes of in vitro and ex vivo induced, repressed or conserved genes in Mycobacterium tuberculosis currently infected human macrophages and to corresponding peptides or consensus peptides or proteins for the preparation of specific bio-markers for the diagnosis and prevention of active or latent disease.

Claims

1. A method for detecting a Mycobacterium species infection in a test subject, comprising: (a) incubating in vitro a blood sample or a lavage sample that comprises lymphocytes from the test subject with at least one isolated protein or peptide that comprises at least one Mycobacterium T-cell epitope, said isolated protein or peptide being selected from: (i) SEQ ID NO:1 (Rv0023), SEQ ID NOS:2-3 (Rv0182c), SEQ ID NOS: 4-5 (Rv0290), SEQ ID NOS:6-10 (Rv0601c), SEQ ID NOS:11-12 (Rv0647c), SEQ ID NO:13 (Rv0724A), SEQ ID NOS:14-20 (Rv0890c), SEQ ID NOS:21-22 (Rv1251c), SEQ ID NO:23 (Rv1398c), SEQ ID NO:24 (Rv1478), SEQ ID NOS:25-26 (Rv1497), SEQ ID NO:27 (Rv1575), SEQ ID NO:28 (Rv1578c), SEQ ID NOS:29-31 (Rv1899c), SEQ ID NO:32 (Rv2137c), SEQ ID NOS:33-35 (Rv2333c), SEQ ID NOS:36-37 (Rv2548), SEQ ID NOS:38-40 (Rv2557), SEQ ID NOS:41-42 (Rv2816c), SEQ ID NOS:43-47 (Rv2990), SEQ ID NOS:48-51 (Rv3094c), SEQ ID NO:52 (Rv3107c), SEQ ID NO:53 (Rv3188), SEQ ID NOS:54-61 (Rv3239c), SEQ ID NOS:62-63 (Rv3296), SEQ ID NOS:64-66 (Rv3425), SEQ ID NOS:67-69 (Rv3446c), SEQ ID NOS:70, 71, and 87 (Rv3479), SEQ ID NOS:72-73 (Rv3482c), and SEQ ID NO:74 (Rv3780); (ii) a homolog of any one of the proteins or peptides of (i) having an amino acid sequence with at least 80% similarity to said protein or peptide of (i) after optimal alignment and comprising at least one Mycobacterium T-cell epitope; and (iii) a peptide fragment of the protein or peptide as defined in (i) or (ii), said peptide fragment having a T-cell epitope, or a chemical analog thereof, wherein said step of incubating is for a time and under conditions sufficient to stimulate the lymphocytes to produce an effector molecule; and (b) detecting a test level of the effector molecule, wherein detection of a test level that is higher than a control level that is detected following incubation of control lymphocytes from a healthy control subject, indicates that the test subject is infected with, or has been previously exposed to, the Mycobacterium species.

2. The method of claim 1 wherein the Mycobacterium species is selected from M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium paratuberculosis, M. avium silvaticum, M. avium “hominissuis”, M. colombiense, M. asiaticum, M. gordonae, M. gastri, M. kansasii, M. hiberniae, M. nonchromogenicum, M. terrae, M. triviale, M. ulcerans, M. pseudoshottsii, M. shottsii, M. triplex, M. genavense, M. florentinum, M. lentiflavum, M. palustre, M. kubicae, M. parascrofulaceum, M. heidelbergense, M. interjectum, M. simiae, M. branderi, M. cookii, M. celatum, M. bohemicum, M. haemophilum, M. malmoense, M. szulgai, M. leprae, M. lepraemurium, M. lepromatosis, M. africanum, M. botniense, M. chimaera, M. conspicuum, M. doricum, M. farcinogenes, M. heckeshornense, intracellulare, M. lacus, M. marinum, M. monacense, M. montefiorense, M. murale, M. nebraskense, M. saskatchewanense, M. scrofulaceum, M. shimoidei, M. tusciae, M. xenopi, M. intermedium, M. abscessus, M. chelonae, M. bolletii, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M. boenickei, M. peregrinum, M. porcinum, M. senegalense, M. septicum, M. neworleansense, M. houstonense, M. mucogenicum, M. mageritense, M. brisbanense, M. cosmeticum, M. parafortuitum, M. austroafricanum, M. diernhoferi, M. hodleri, M. neoaurum, M. frederiksbergense, M. aurum, M. vaccae, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madagascariense, M. phlei, M. smegmatis, M. goodii, M. wolinskyi, M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M. arosiense, M. aubagnense, M. caprae, M. chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense, M. parmense, M. phocaicum, M. poriferae, M. rhodesiae, M. seoulense and M. tokaiense.

3. The method of claim 1 wherein one or more of: (a) the Mycobacterium species is Mycobacterium tuberculosis, (b) either the subject is a human or the subject is a non-human animal, and (c) one of: (1) the at least one isolated protein or peptide that comprises at least one Mycobacterium T-cell epitope is an Rv0890c peptide that is selected from SEQ ID NOS: 14-19 and 20, or an Rv2333c peptide that is selected from SEQ ID NOS: 33, 34 and 35, or an Rv2548 peptide that is selected from SEQ ID NOS: 36 and 37, (2) in the step of incubating there is present one or more Mycobacterium protein, or a peptide fragment thereof or a chemical analog derived therefrom, wherein the one or more Mycobacterium protein is selected from ESAT6, CFP10, TB7.7 and PPD, and wherein the detected test level of the effector molecule is greater than a detectable effector molecule level that can be produced by said lymphocytes incubated only with said one or more Mycobacterium protein selected from ESAT6, CFP10, TB7.7 and PPD, or (3) both (1) and (2).

4. A method for detecting a Mycobacterium species infection in a test subject, comprising: (a) incubating in vitro a biological sample that comprises lymphocytes from the test subject with at least one protein or peptide that is selected from: (i) an isolated protein or peptide that comprises at least one T-cell epitope and that comprises an amino acid sequence from a Mycobacterium species and is selected from SEQ ID NO:1 (Rv0023), SEQ ID NOS:2-3 (Rv0182c), SEQ ID NOS: 4-5 (Rv0290), SEQ ID NOS:6-10 (Rv0601c), SEQ ID NOS:11-12 (Rv0647c), SEQ ID NO:13 (Rv0724A), SEQ ID NOS:14-20 (Rv0890c), SEQ ID NOS:21-22 (Rv1251c), SEQ ID NO:23 (Rv1398c), SEQ ID NO:24 (Rv1478), SEQ ID NOS:25-26 (Rv1497), SEQ ID NO:27 (Rv1575), SEQ ID NO:28 (Rv1578c), SEQ ID NOS:29-31 (Rv1899c), SEQ ID NO:32 (Rv2137c), SEQ ID NOS:33-35 (Rv2333c), SEQ ID NOS:36-37 (Rv2548), SEQ ID NOS:38-40 (Rv2557), SEQ ID NOS:41-42 (Rv2816c), SEQ ID NOS:43-47 (Rv2990), SEQ ID NOS:48-51 (Rv3094c), SEQ ID NO:52 (Rv3107c), SEQ ID NO:53 (Rv3188), SEQ ID NOS:54-61 (Rv3239c), SEQ ID NOS:62-63 (Rv3296), SEQ ID NOS:64-66 (Rv3425), SEQ ID NOS:67-69 (Rv3446c), SEQ ID NOS:70, 71, and 87 (Rv3479), SEQ ID NOS:72-73 (Rv3482c), and SEQ ID NO:74 (Rv3780); (ii) a homolog of the protein or peptide as defined in (i) having an amino acid sequence with at least 80% similarity to said protein or peptide after optimal alignment and comprising at least one Mycobacterium T-cell epitope, and (iii) a peptide fragment of the protein or peptide as defined in (i) or (ii) having a T-cell epitope, or a chemical analog thereof, or (b) incubating in vitro a biological sample that comprises lymphocytes from the test subject with at least one protein or peptide of (a)(i), (a)(ii) or (a)(iii) and further with a Mycobacterium protein, or a fragment thereof or a chemical analog thereof, said Mycobacterium protein being selected from: (a) ESAT, (b) CFP10, (c) TB7.7, and (d) PPD, wherein said step (a) or (b) of incubating is for a time and under conditions sufficient to stimulate the lymphocytes to produce an effector molecule; and (c) detecting a test level of the effector molecule, wherein following step (b) the detected test level of the effector molecule is greater than a detectable effector molecule level that can be produced by said lymphocytes incubated only with said one or more Mycobacterium protein selected from ESAT6, CFP10, TB7.7 and PPD, and wherein following either step (a) or step (b), detection of a test level that is higher than a control level that is detected following incubation of control lymphocytes from a healthy control subject, indicates that the test subject is infected with, or has been previously exposed to, the Mycobacterium species.

5. The method of claim 4 wherein the Mycobacterium species is selected from M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium paratuberculosis, M. avium silvaticum, M. avium “hominissuis”, M. colombiense, M. asiaticum, M. gordonae, M. gastri, M. kansasii, M. hiberniae, M. nonchromogenicum, M. terrae, M. triviale, M. ulcerans, M. pseudoshottsii, M. shottsii, M. triplex, M. genavense, M. florentinum, M. lentiflavum, M. palustre, M. kubicae, M. parascrofulaceum, M. heidelbergense, M. interjectum, M. simiae, M. branderi, M. cookii, M. celatum, M. bohemicum, M. haemophilum, M. malmoense, M. szulgai, M. leprae, M. lepraemurium, M. lepromatosis, M. africanum, M. botniense, M. chimaera, M. conspicuum, M. doricum, M. farcinogenes, M. heckeshornense, M. intracellulare, M. lacus, M. marinum, M. monacense, M. montefiorense, M. murale, M. nebraskense, M. saskatchewanense, M. scrofulaceum, M. shimoidei, M. tusciae, M. xenopi, M. intermedium, M. abscessus, M. chelonae, M. bolletii, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M. boenickei, M. peregrinum, M. porcinum, M. senegalense, M. septicum, M. neworleansense, M. houstonense, M. mucogenicum, M. mageritense, M. brisbanense, M. cosmeticum, M. parafortuitum, M. austroafricanum, M. diernhoferi, M. hodleri, M. neoaurum, M. frederiksbergense, M. aurum, M. vaccae, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madagascariense, M. phlei, M. smegmatis, M. goodii, M. wolinskyi, M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M. arosiense, M. aubagnense, M. caprae, M. chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense, M. parmense, M. phocaicum, M. poriferae, M. rhodesiae, seoulense and M. tokaiense.

6. The method of claim 4 wherein the at least one isolated protein or peptide that comprises at least one Mycobacterium T-cell epitope is an Rv0890c peptide that is selected from SEQ ID NOS: 14-19 and 20, or an Rv2333c peptide that is selected from SEQ ID NOS: 33, 34 and 35, or an Rv2548 peptide that is selected from SEQ ID NOS: 36 and 37.

7. An isolated protein selected from: (a) an isolated protein that comprises an amino acid sequence from a Mycobacterium species and that is selected from SEQ ID NO:1 (Rv0023), SEQ ID NOS:2-3 (Rv0182c), SEQ ID NOS: 4-5 (Rv0290), SEQ ID NOS:6-10 (Rv0601c), SEQ ID NOS:11-12 (Rv0647c), SEQ ID NO:13 (Rv0724A), SEQ ID NOS:14-20 (Rv0890c), SEQ ID NOS:21-22 (Rv1251c), SEQ ID NO:23 (Rv1398c), SEQ ID NO:24 (Rv1478), SEQ ID NOS:25-26 (Rv1497), SEQ ID NO:27 (Rv1575), SEQ ID NO:28 (Rv1578c), SEQ ID NOS:29-31 (Rv1899c), SEQ ID NO:32 (Rv2137c), SEQ ID NOS:33-35 (Rv2333c), SEQ ID NOS:36-37 (Rv2548), SEQ ID NOS:38-40 (Rv2557), SEQ ID NOS:41-42 (Rv2816c), SEQ ID NOS:43-47 (Rv2990), SEQ ID NOS:48-51 (Rv3094c), SEQ ID NO:52 (Rv3107c), SEQ ID NO:53 (Rv3188), SEQ ID NOS:54-61 (Rv3239c), SEQ ID NOS:62-63 (Rv3296), SEQ ID NOS:64-66 (Rv3425), SEQ ID NOS:67-69 (Rv3446c), SEQ ID NOS:70, 71, and 87 (Rv3479), SEQ ID NOS:72-73 (Rv3482c), and SEQ ID NO:74 (Rv3780); said isolated protein comprising a mycobacterial epitope that is recognized by T-cells, and (b) the isolated protein of (a) which consists of an amino acid sequence selected from SEQ ID NOS: 14, 15, 16, 17, 18, 19, 20, 33, 34, 35, 36 and 37.

8. A composition comprising at least one of: (a) an isolated nucleic acid molecule encoding the protein of claim 7, (b) a vector comprising the nucleic acid molecule of (a), and (c) an isolated cell comprising the vector of (b).

9. The method of claim 4 wherein the biological sample comprises a blood sample or a lavage sample.

10. The method of claim 9 wherein at least one of: (a) heparin is present in the step of incubating, (b) heparin and an added carbohydrate are present in the step of incubating, and (c) at least one Mycobacterium protein, or a peptide fragment thereof or a chemical analog thereof, is present in the step of incubating, said Mycobacterium protein being selected from ESAT6, CFP10, TB7.7 and PPD.

11. The method of claim 9 wherein the Mycobacterium species is selected from M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium paratuberculosis, M. avium silvaticum, M. avium “hominissuis”, M. colombiense, M. asiaticum, M. gordonae, M. gastri, M. kansasii, M. hiberniae, M. nonchromogenicum, M. terrae, M. triviale, M. ulcerans, M. pseudoshottsii, M. shottsii, M. triplex, M. genavense, M. florentinum, M. lentiflavum, M. palustre, M. kubicae, M. parascrofulaceum, M. heidelbergense, M. interjectum, M. simiae, M. branderi, M. cookii, M. celatum, M. bohemicum, M. haemophilum, M. malmoense, M. szulgai, M. leprae, M. lepraemurium, M. lepromatosis, M. africanum, M. botniense, M. chimaera, M. conspicuum, M. doricum, M. farcinogenes, M. heckeshornense, M. intracellulare, M. lacus, M. marinum, M. monacense, M. montefiorense, M. murale, M. nebraskense, M. saskatchewanense, M. scrofulaceum, M. shimoidei, M. tusciae, M. xenopi, M. intermedium, M. abscessus, M. chelonae, M. bolletii, M. fortuitum, M. fortuitum subsp. acetamidolyticum, M. boenickei, M. peregrinum, M. porcinum, M. senegalense, M. septicum, M. neworleansense, M. houstonense, M. mucogenicum, M. mageritense, M. brisbanense, M. cosmeticum, M. parafortuitum, M. austroafricanum, M. diernhoferi, M. hodleri, M. neoaurum, M. frederiksbergense, M. aurum, M. vaccae, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madagascariense, M. phlei, M. smegmatis, M. goodii, M. wolinskyi, M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M. arosiense, M. aubagnense, M. caprae, M. chlorophenolicum, M. fluoroanthenivorans, M. kumamotonense, M. novocastrense, M. parmense, M. phocaicum, M. poriferae, M. rhodesiae, M. seoulense and M. tokaiense.

12. The method of claim 9 wherein in the step of incubating, the at least one protein or peptide comprises an amino acid that is selected from SEQ ID NOS: 14, 15, 16, 17, 18, 19, 20, 33, 34, 35, 36 and 37.

13. The method of claim 9 wherein the effector molecule is selected from interferon-γ, TNF-α, and an interleukin.

14. The method of claim 9 wherein the effector molecule is interferon-γ.

Description

[0098] The present invention now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to enclosed drawings wherein:

[0099] FIG. 1. Analysis of IFN-gamma production as PPD response comparing four subject populations: a. first test pulmonary TB patients; b. healthy contacts, TB exposed, PPD positive; c. healthy controls, professionally TB exposed, Quantiferon positive; d. negative controls, Quantiferon negative, BCG vaccinated.

[0100] FIG. 2. Analysis of IFN-gamma production as TAWITAVVPGLMV (SEQ ID NOT 24) peptide response from 4 tested subject groups.

[0101] FIG. 3. Analysis of IFN-gamma production as AVIVRSELLTQYL (SEQ ID NO 22) peptide response from 4 tested subject groups.

[0102] FIG. 4. Analysis of IFN-gamma production as GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO 71) peptide response from 4 tested subject groups.

[0103] FIG. 5. Analysis of IFN-gamma production as RPVRRVLLFVVPSSGPAP (SEQ ID NO 70) peptide response from 4 tested subject groups.

[0104] FIG. 6. Analysis of IFN-gamma production as GEIIFISGRLNGaa (SEQ ID NO 13) peptide response of tested samples.

[0105] FIG. 7. Analysis of IFN-gamma production as ELMARAAVLGSAH (SEQ ID NO 21)peptide response of tested samples.

[0106] FIG. 8. Analysis of IFN-gamma production as ESAT6 (QQWNFAGIEAAASAIQGNVTSIHSL-SEQ ID NO:84) response of tested samples.

[0107] FIG. 9. Frequency of positive tests using only SEQ ID NO: 24,21,71,70,13,22 six peptides.

[0108] FIG. 10. Sensitivity increase of Quantiferon TB-plus commercial used test after SEQ ID NO: 24,21,70,71,13,22 peptide addition thereto.

[0109] FIG. 11. Combination use of 1-6 peptides, which allow in panel a 76,3%, 62% and 66,7%, compared to 55%, 33% and 45.5% sensitivity obtained using RD1 (ESAT6) multiepitope peptide, for TB+ subjects, PPD+esposed contacts and HCWs, respectively, to be obtained.

EXAMPLE 1

[0110] Identification of M. tuberculosis expressed proteins in human infected macrophages from both vitro and ex vivo analyzed biological samples

Materials and Methods

[0111] ELISPOT Immunodiagnostic Test

[0112] The global procedure for test carrying out demands for: 96 well plates (MAIPS45, Millipore, Sunnyvale, Calif., USA); primary antibody (IFN-gamma coating monoclonal, M-700A, Pierce-Endogen Inc, Rockford, USA); biotynilated antibody (M-701B, Pierce-Endogen Inc); streptavidin-HRP (Pierce-Endogen); substrate (AEC Staining kit, Sigma); stimulus (peptides, PHA and other antigens) at ready to use concentration.

[0113] ELISPOT procedure is carried out according to the following step:

[0114] Coating: treatment of a 96 well plate using 5 μg/mL sterile phosphate buffer (PBS) solution of primary antibody delivered at 100 μL/well; coat the plate and incubate at 4° C. for 20 hours; wash the plate 4 times using 200 μL/well of sterile PBS, at final washing eliminate excess liquid by tapping plate on adsorbent paper.

[0115] Blocking: addition of 200 μL/well of “blocking solution” [sterile PBS containing 10% fetal calf serum (FCS)], in order to prevent not specific protein binding; plate incubation for 2 hours at room temp.; “blocking solution” suction.

[0116] Cell Preparation and Incubation

1. isolation of mononucleated cells (PBMC) from venous blood (7 ml with EDTA) by density gradient centrifugation (Ficoll-Hypaque, Pharmacia; Uppsala; Swedewn), using a quick method based on the use of filtering tubes for leucocytes separation (LeucoSep™, ARNIKA, Milan). After two washings with PBS (phosphate buffered saline) 1×, the pellet is resuspended in complete medium (RPMI 1640 containing HEPES 25 mM, 10% v/v FCS, 2mM L-Glutamine, 10 U/mL penicillin/streptomycin)) in order to have 2×10.sup.5 cells in 100 μL.
2. add 100 pL/well of cellular suspension together with 100 μL of different stimula;
3. incubate the plate for 40 hours at 37° C. in incubator at 5% CO.sub.2;
4. remove the cells;
5. wash the plate 4 times with 200 μL/well of PBS and 4 times with 200 μL/well of “Wash buffer” [PBS/0.05% Tween 20 (Sigma)];
6. at the final washing eliminate the excess liquid by tapping the plate on absorbent paper.

[0117] Incubation With Biotinilated Antibody

[0118] 100 μl/well of biotinilated antibody diluted in PBS/4% bovine serum albumin (fraction V, Sigma) at concentration of 1 μg/ml have been delivered. Then the plate has been incubated for 100 minutes at 37° C. in incubator at 5% CO.sub.2; the plate has been washed 4 times with “Wash buffer”. At final washing excess liquid has been eliminated by tapping plate on adsorbent paper.

[0119] Detection

[0120] For the detection 100 μl/well of “Streptavidin-HRP” 1:1000 diluted in “Wash buffer”. The plate has been incubated for 30 minutes at room temp. in the dark, then washed 4 times with “Wash buffer”. At final washing excess liquid has been eliminated by tapping plate on adsorbent paper. 100 μL/well of substrate have been delivered. In parallel as a control for enzyme-substrate reaction occurrence 100 μl of as prepared substrate with 100 μl of “diluted Streptavidin-HRP” has been incubated for few minutes. If the reaction is successful the substrate turns out from light brown to pink colour.

[0121] Finally the plate has been incubated for 10-20 minutes at room temp. in the dark. The substrate has been discarded, the plate washed with water eliminating excess and air dried for 20 hours.

[0122] ELISA tests for the identification of human and animal IFN-gamma in whole blood samples stimulated with selected peptides and proteins (CMI test protocol).

Results

[0123] The authors have identified the group of M. tuberculosis expressed proteins in human infected macrophages from both in vitro and ex vivo analyzed biological samples. M.tuberculosis gene expressions in three different growth environments have been compared: synthetic medium (Sauton's), monocyte-derived-human macrophage (MDM) infected in culture with M. tuberculosis, alveolar macrophage (AM) from bronchoalveolar lavage (BAL) samples of pulmonary TB affected patients before the antibiotic therapy.

[0124] From thus obtained 9 groups of genes, first 100 proteins, according to a combination criteria (immunogenicity, tubercular Complex specificity, etc), have been selected. From these 100 proteins, again a 30 protein group has been selected, for which a positive response in immunological tests on TB patient whole blood had been obtained.

TABLE-US-00001 TABLE 1 Amino acid sequence In vivo ID Rv modulation (UNIPROT) Already tested peptides Rv0023 B, E P67704 EMWDIRNRGVIPAGALPRVR (SEQ ID NO: 1) Rv0182c E O07426 AKFRSVRVVVITGSVTAAPVRVSETLRRLI (SEQ ID NO: 2), ESVRLAFVAALQH (SEQ ID NO: 3) Rv0290 D O86362 GLLITIRSPRSGIA (SEQ ID NO: 4), AQLLWQLPLLSIG (SEQ ID NO: 5) Rv0601c C, E O07777 ADLVRELVTILPIVLVIAAVAAYLLSR (SEQ ID NO: 6) AAYLLSRAALRPVDRIRAAA (SEQ ID NO: 7) TTLNTMLTRLQRALAHEQQF (SEQ ID NO: 8) DLFVSIDPDHLRRILTAVLDN (SEQ ID NO: 9) SGLGLAIVAALTTTHGG (SEQ ID NO: 10) Rv0647c E P96936 GRLPRKGPWQQKVIKELPQ (SEQ ID NO: 11), GKIVVLMGAVGTMKPETQAA (SEQ ID NO: 12) Rv0724A B Q79FX1 GEIIFISGRLNGaa (SEQ ID NO: 13) Rv0890c C, E Q10550 ARVRSMSPLEIAD (SEQ ID NO: 14), EQILFRRLAPFVGGF (SEQ ID NO: 15), AALVRALTACGCSS (SEQ ID NO: 16), DKWTLCQILYWRGVGTCISGD (SEQ ID NO: 17), TKVLGLYTQAQVLAYCG (SEQ ID NO: 18), DQVTMHQVLMAQLALAGG(SEQ ID NO: 19), EGVRLLGAAAALRQQTRQVRFK (SEQ ID NO: 20) Rv1251c C, E O50466 ELMARAAVLGSAH (SEQ ID NO: 21), AVIVRSELLTQYL (SEQ ID NO: 22) Rv1398c D P64835 GTLRHLDPPVRRSGGREQHL (SEQ ID NO: 23) Rv1478 E O53169 TAWITAVVPGLMV (SEQ ID NO: 24) Rv1497 C, E P71778 APMVFSATKGMTA (SEQ ID NO: 25), TCAMRRLAHRFSGG(SEQ ID NO: 26) Rv1575 C, E O06615 SVVRRKQTLLSAQ (SEQ ID NO: 27) Rv1578c E O06612 GVVHRNPAVTVAE (SEQ ID NO: 28) Rv1899c D O07733 PGVVATHAVRTLGTTGSRAIGL (SEQ ID NO: 29), PQWRRARVRLCGRWWRRSNTTRGAS (SEQ ID NO: 30), ARLMVGAVRRHRPGSLQR (SEQ ID NO: 31) Rv2137c D O06238 aaMRNMKSTSHE (SEQ ID NO: 32) Rv2333c E P71879 QTIVMLWTAAVGCA (SEQ ID NO: 33), LCMLMLGLLMLIFSEHRSS(SEQ ID NO: 34), SALVLVGLGLCGSGVALCLT (SEQ ID NO: 35) Rv2548 A, E P95005 SELVRFELLAGVRESE (SEQ ID NO: 36), VDYLIAATAIVVDA (SEQ ID NO: 37) Rv2557 A, E P65003 QGIEYYKSSVLPQIE (SEQ ID NO: 38), EGWIVYARSTTIQAQS (SEQ ID NO: 39), TRRMYSNYGF (SEQ ID NO: 40) Rv2816c A P71637 FGYRVQESAFEAMLTKGQLAKLV (SEQ ID NO: 41), DNIRIYKIRGVAAVTFYGRGRLVSAE (SEQ ID NO: 42) Rv2990 E O53239 RSYILRAGISSLFRYIEGVHGER (SEQ ID NO: 43), SAMRPQDRLLVGNWVDDSLL (SEQ ID NO: 44), LYLVGLEPYVQFE (SEQ ID NO: 45), AGFRILEARRFPI (SEQ ID NO: 46), IRYRARYVNGQLNMCLARI (SEQ ID NO: 47) Rv3094c E O05773 ALLVAYLPARSREEMF (SEQ ID NO: 48), NRLRLAATHAVRT(SEQ ID NO: 49), APLQRRFRDAFTATAHFQVNE(SEQ ID NO: 50), SRELPGRVLLDQPADVSM (SEQ ID NO: 51) Rv3107c A, E O05784 EPVVTVDVTAMSAVLEID (SEQ ID NO: 52) Rv3188 C, E O53334 AVIQVSDRAVRGWRTGDIRPERY (SEQ ID NO: 53) Rv3239c C, E O05884 PDLRGALLLAVTLGLVT (SEQ ID NO: 54), PDWGWLSVATVGSFLA(SEQ ID NO: 55) GAVLGVAVMVILIGKPEHGTA (SEQ ID NO: 56), AAICFIAVAVAAAVL(SEQ ID NO: 57), TKLVRLTKAQFDEIA(SEQ ID NO: 58), ADLVLAGPAASREH(SEQ ID NO: 59), YAYEYFIRHNPLSDYA(SEQ ID NO: 60), FPVRGLVRGRRTLTLLEA (SEQ ID NO: 61) Rv3296 A, E P96901 EVLRILRRRSLAALRA (SEQ ID NO: 62), RVILHSPYGLRVHGPLAL (SEQ ID NO: 63) Rv3425 E Q50703 AAWVINGLANAYNDT (SEQ ID NO: 64), DQYRARNVAVMNAYVSWTRSALSDLPR (SEQ ID NO: 65), SDLLADAVERYLQWLSKSSSQLKHA (SEQ ID NO: 66) Rv3446c C, E O06263 GPVVVHPSWWSAA (SEQ ID NO: 67), ITAVVLIDVPSTVAGA (SEQ ID NO: 68), AAVVRHGATTLQRP (SEQ ID NO: 69) Rv3479 C, E O06342 RPVRRVLLFVVPSSGPAP (SEQ ID NO: 70), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO: 71), SALLRRLSTCPPES (SEQ ID N: 87) Rv3482c E O06345 GAVLRLVVRFAEPLPPSP (SEQ ID NO: 72), AGYLLTYTIANNGKEFAEL (SEQ ID NO: 73) Rv3780 D P65091 aaVRKRMVIGLSTGSDDD (SEQ ID NO: 74) Cons FS ALLLRDVLQWKSAEVADAIG (SEQ ID NO: 75) NSLLQRARSQLQTVRPSAADRLSAA (SEQ ID NO: 76) Cons MSWVMVSPELVVAAAADLAG (SEQ ID NO: 77) PE_PGRS AAFYAQFVQALTSGGAY (SEQ ID NO: 78) Cons REG ALLVRMPTSLPAVA (SEQ ID NO: 79) Cons CW SRLRTHVRPDAPLVPLALRVDGLRSRW (SEQ ID NO: 80) AAVLTMLGVAGYGW (SEQ ID NO: 81) GLFMIFLDALIVNVALPDIQR (SEQ ID NO: 82) SWVVASYSLGMAVFIMSAGTLADLL (SEQ ID NO: 83) Legend of modulation: A: up-regulated in AM vs MDM; B: always expressed in AM and MDM; C: up-regulated in MDM vs AM; D: up-regulated in Sauton vs MDM and/or AM; E: up-regulated in MDM and/or AM vs Sauton’s

[0125] After an ulterior selection initially 43 peptides from 4 groups of subjects: Pulmonary TB before antibiotic therapy (n=13), recently exposed healthy contacts (TB patient relations) PPD+ (n=8); long TB patient exposed healthy contacts (professional exposure of hospital workers) PPD+ (n=5); BCG vaccinated negative controls, PPD− (n=4), have been designed, synthesized and tested.

[0126] Then 6 most sensible and specific peptides have been selected (see table 2) and the study has been repeated using an extended subject sample (see tables 3-4 and FIGS. 1-7).

[0127] Table 2 reports MTB selected genes, peptides selected for T CD4+ cell assay and corresponding identification number thereof, respectively.

TABLE-US-00002 TABLE 2 In vivo No Peptide Gene modulation SEQ ID TAWITAV Consensus Induced in AM and  NO: 24 VPGLMV VIR(Rv1478) MDM vs Sauton's SEQ ID AVIVRSE Rv1251c Induced in MDM  NO: 22 LLTQYL vs Sauton's SEQ ID GSVRQLPSVLKP Rv3479 Induced in MDM  NO: 71 PLITLRTLTLSG vs Sauton's SEQ ID RPVRRVLL Rv3479 Induced in MDM  NO: 70 FVVPSSGPAP vs Sauton's SEQ ID GEIIFISGRL Rv0724A Expressed in AM  NO: 13 NGaa and MDM SEQ ID ELMARAAVLG Rv1251c Induced in MDM  NO: 21 SAH vs Sauton's

TABLE-US-00003 TABLE 3 Microbiologically Recently checked TB MTB BCG- patients (before exposed Sanitary vaccinated therapy start) subjects workers controls Examined 58 63 21 15 subject number Anergic subjects  3/58  2/63  0/21  0/15 (mitogene low response or no response to all stimula) Quantiferon 41/55 25/61 11/21  3/15 TB-Gold in-Tube PPD 55/55 61/61 21/21 15/15 ESAT-6 control 28/55 17/61  7/21  0/15 peptide Peptide #1 12/55 14/61  6/21  0/15 (Seq ID N°: 24) Peptide #2 11/55  8/61  1/21  0/15 (Seq ID N°: 22) Peptide #3 27/55 20/61  7/21  0/15 (Seq ID N°: 71) Peptide #4 10/55 13/61  6/21  0/15 (Seq ID N°: 70) Peptide #5 18/55 11/61  5/21  0/15 (Seq ID N°: 13) Peptide #6 14/55  6/61  3/21  0/15 (Seq ID N°: 21)

TABLE-US-00004 TABLE 4 Microbiologically Recently checked TB MTB BCG- patients (before exposed Sanitary vaccinated therapy start) subjects workers controls Examined 58 63 21 15 subject number Anergic subjects  3/58  2/63  0/21 0/15 (mitogene low response or no response to all stimula) Quantiferon 41/55 25/61 11/21 3/15 TB-Gold in-Tube Panel 1-6 40/55 30/61 11/21 0/15 peptides QFT Gold + 49/55 34/61 13/21 3/15 panel 1-6 peptides

TABLE-US-00005 TABLE 5 Microbiologically Recently checked TB MTB BCG- patients (before exposed Sanitary vaccinated therapy start) subjects workers controls Examined 38 32 10 10 subject number Anergic  3  1  0  0 1-6 peptides 22/35 10/31 4/10 1/10 (in pool) QFT Gold 25/35 11/31 4/10 2/10 QFT Gold + 1-6 29/35 13/31 5/10 2/10 peptides in same well

[0128] Results obtained with said six peptides and peptide belonging to ESAT6 protein, i.e. a highly immunogenic protein occurring in both above mentioned commercial kits, have been compared.

[0129] MTB genes observed as induced, both in course of human macrophage infection and/or in alveolar macrophage samples from active pulmonary TB patients, are indicated in the following list:

[0130] Genes always expressed during intracellular replication in MDM and AM: Rv0724A.

[0131] Genes induced in AM and/or MDM vs Sauton's medium culture: Rv1251c, Rv1478 and Rv3479.

[0132] Two groups of MTB genes share probable role in the survival inside human host cell (both primary macrophages from healthy in vitro infected donors and TB patient alveolar macrophages) resulting in design thereof as MTB intracellular survival bio-markers, whereas MTB virulence definition is just based on the pathogen ability to invade, survive and replicate within the host cell.

[0133] Moreover, the authors of the present invention have designed peptides of some gene groups belonging to same metabolic category, in order to find “consensus” protein sequences for said categories. The research is based on the assumption that functional domains of similar function exploiting proteins occurring in various bacterial species, are conserved. In order to find these conserved motifs the sequence multiple alignment (PSSM), using PSI-BLAST (Position Specific Iterated Basic Local Alignment Search Tool, http://www.ncbi.nlm.nih.gov/BLAST), is generated. After the sequences with higher similarity grades with the inserted sequence has been detected, it is possible to select the proteins suitable to contribute to the generation of the profile used for the successive data bank search; in this way the number of the sequences contributing to the generation of the profile is different for the different sequence positions.

[0134] A multi-alignment allows structurally and functionally important, because extremely conserved, residues to be detected and said residues as a whole will constitute the “consensus” sequence or sequences for each MTB protein functional group.

[0135] Therefore proteins (induced or repressed in human macrophage) of metabolic functional groups (for example regulatory proteins, lipid metabolism involved proteins, etc.) detected as “modulated” by M. tuberculosis during infection course, have been analyzed for search of conserved sequences. Using PSI-BLAST sequence various multiple alignments, from which we have reached the best “consensus” sequences for the peptide synthesis, have been obtained.

[0136] The peptides derived from selected proteins have been synthesized and used for the detection and quantification of MTB specific T CD4+ lymphocytes using detection system for IFN-γ producing cells both with ELISPOT technique and with TB diagnosis high sensitivity ELISA assay, Quantiferon TB-Plus and Quantiferon CMI. This technique allows the frequency of T cells producing a determined cytokine (for example, IFN-γ) as a response to a specific antigenic stimulus suggesting that the immune system of treated subjects has been able to evoke an immune response towards said peptides when infectious agent (MTB) encoding for the same occurs, to be quantified. The second technique allows total IFN-gamma production resulting from specific T lymphocytes as a response to selected antigens, to be quantified.

[0137] Although this test does not represent the evidence of ability thereof to induce protection from MTB infection, the occurred detection of the presence of lymphocytes recognizing these peptides specifically and differently in MTB infected subjects or with active tuberculosis, is an index of their immunogenicity, indispensable minimal characteristic in order a vaccine and a diagnostic test to be proved effective. Further these peptides, alone or in addition to other mycobacterial antigens, allow a sensitive and specific test for TB diagnosis to be provided and the sensitivity of commercial test, i.e. current reference gold standard for the tubercular diagnosis, to be enhanced (FIG. 10). When panel evaluated and directly tested within same well, 6 selected peptides allow Quantiferon TB gold in-Tube response from 75% to 89% (+14%) and from 71% to 83% (+13%), respectively, for subjects with active TB, to be enhanced, without decreasing the assay specificity.

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