Use of amino acid sequences from Mycobacterium tuberculosis or corresponding nucleic acids for diagnosis and prevention of tubercular infection, diagnostic kit and vaccine therefrom
09829490 · 2017-11-28
Assignee
Inventors
- Francesca Mariani (Rome, IT)
- Massimo Amicosante (Rome, IT)
- Vittorio Colizzi (Rome, IT)
- Cesare Saltini (Grottaferrata, IT)
Cpc classification
International classification
A61K38/16
HUMAN NECESSITIES
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 tuberculosis infection in a test subject, comprising: incubating in vitro a blood sample or a lavage sample that comprises lymphocyte from the test subject with at least one isolated protein or peptide that comprises at least one Mycobacterium tuberculosis T-cell epitope, said isolated protein or peptide being an isolated protein that is selected from SEQ ID NO: 13 (Rv 0724A), SEQ ID NOS:21-22 (Rv 1251c), SEQ ID NO:24 (Rv 1478), SEQ ID NOS:70, 71 and 87 (Rv3479) wherein said step of incubating is for a time and under conditions sufficient to stimulate the lymphocytes to produce an effector molecule; and 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 tuberculosis.
2. The method of claim 1, wherein the subject is a human or a non human animal, and either or both of: (1) the isolated protein or peptide consists of an amino acid sequence selected from TAWITAWPGLMV (SEQ ID NO:24), AVIVRSELLTQYL (SEQ ID NO:22), GSVRQLPSVLKPPLITLRTLTLSG (SEQ ID NO:71), RPVRRVLLFWPSSGPAP (SEQ ID NO:70), GEIIFISGRLNGaa (SEQ ID NO: 13), ELMARAAVLGSAH (SEQ ID NO:21), and SALLRRLSTCPPES (SEQ ID NO:87), and (2) in the step of incubating there is present one or more Mycobacterium protein, wherein the Mycobacterium protein is selected from ESAT6, CFP 10, TB7.7 and PPD.
Description
(1) 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:
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EXAMPLE 1
Identification of M. tuberculosis Expressed Proteins in Human Infected Macrophages from Both Vitro and Ex Vivo Analyzed Biological Samples
(13) Materials and Methods
(14) ELISPOT Immunodiagnostic Test
(15) 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.
(16) ELISPOT procedure is carried out according to the following step:
(17) 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.
(18) 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.
(19) Cell Preparation and Incubation
(20) 1. isolation of mononucleated cells (PBMC) from venous blood (7 ml with EDTA) by density gradient centrifugation (Ficoll-Hypaque, Pharmacia; Uppsala; Sweden), 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, 2 mM L-Glutamine, 10 U/mL penicillin/streptomycin)) in order to have 2×10.sup.5 cells in 100 μL.
2. add 100 μL/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.
(21) Incubation with Biotinilated Antibody
(22) 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.
(23) Detection
(24) 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.
(25) 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.
(26) ELISA tests for the identification of human and animal IFN-gamma in whole blood samples stimulated with selected peptides and proteins (CMI test protocol).
(27) Results
(28) 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.
(29) 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.
(30) TABLE-US-00001 TABLE 1 Amino In acid vivo sequence modu- ID Rv lation (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 NO: 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
(31) 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.
(32) 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
(33) Table 2 reports MTB selected genes, peptides selected for T CD4+ cell assay and corresponding identification number thereof, respectively.
(34) TABLE-US-00002 TABLE 2 In vivo modu- No Peptide Gene lation SEQ ID TAWITAVVPGLMV Consensus Induced in AM NO: 24 VIR and MDM vs (Rv1478) Sauton's SEQ ID AVIVRSELLTQYL Rv1251c Induced in MDM NO: 22 vs Sauton's SEQ ID GSVRQLPSVLKPP Rv3479 Induced in MDM NO: 71 LITLRTLTLSG vs Sauton's SEQ ID RPVRRVLLF Rv3479 Induced in MDM NO: 70 VVPSSGPAP vs Sauton's SEQ ID GEIIFISGRLNGaa Rv0724A Expressed in NO: 13 AM and MDM SEQ ID ELMARAAVLGSAH Rv1251c Induced in MDM NO: 21 vs Sauton's
(35) 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 3/58 2/63 0/21 0/15 subjects (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 12/55 14/61 6/21 0/15 #1 (Seq ID No: 24) Peptide #2 11/55 8/61 1/21 0/15 (Seq ID No: 22) Peptide #3 27/55 20/61 7/21 0/15 (Seq ID No: 71) Peptide #4 10/55 13/61 6/21 0/15 (Seq ID No: 70) Peptide #5 18/55 11/61 5/21 0/15 (Seq ID No: 13) Peptide #6 14/55 6/61 3/21 0/15 (Seq ID No: 21)
(36) 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
(37) 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 (in 22/35 10/31 4/10 1/10 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
(38) 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.
(39) 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:
(40) Genes always expressed during intracellular replication in MDM and AM: Rv0724A.
(41) Genes induced in AM and/or MDM vs Sauton's medium culture: Rv1251c, Rv1478 and Rv3479.
(42) 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.
(43) 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, Altschul et al., 1997 Nucl. Ac. Res. 25:3389; URL: world wide web.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.
(44) 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.
(45) 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.
(46) 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.
(47) 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 (
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