COMPOSITIONS USEFUL IN THE DIAGNOSTIC OF LATENTLY INFECTED MYCOBACTERIUM TUBERCULOSIS

Abstract

The present invention concerns a composition comprising at least three peptides derived from Mycobacterium tuberculosis antigen Rv2626c, its use in the diagnostic of latently infected Mycobacterium tuberculosis (LTBI) subjects, corresponding methods of use and kits.

Claims

1. A composition comprising at least three peptides, in which the amino acid sequences of said at least three peptides consist of distinctive fragments of from 5 to 30 contiguous amino acids of the amino acid sequence of Mycobacterium tuberculosis antigen Rv2626c of SEQ ID No1, the peptides of the amino acid sequence of SEQ ID No40, SEQ ID No41 and SEQ ID No42 being excluded.

2. A composition according to claim 1, comprising between 3 and 10 peptides.

3. A composition according to claim 1, in which the amino acid sequences of said at least three peptides comprise distinctive fragments of from 10 to 30 contiguous amino acids, preferably from 12 to 20 contiguous amino acids of the amino acid sequence of Mycobacterium tuberculosis antigen Rv2626c of SEQ ID No1.

4. A composition according to claim 1 in which said at least three peptides are chosen from: peptide 6 of SEQ ID No2 or a fragment of at least 3 amino acids thereof; peptide 12 of SEQ ID No3 or a fragment of at least 3 amino acids thereof; peptide 18 of SEQ ID No4 or a fragment of at least 3 amino acids thereof; peptide 24 of SEQ ID No5 or a fragment of at least 3 amino acids thereof; peptide 30 of SEQ ID No6 or a fragment of at least 3 amino acids thereof; peptide 36 of SEQ ID No7 or a fragment of at least 3 amino acids thereof; peptide 7 of SEQ ID No8 or a fragment of at least 3 amino acids thereof; peptide 8 of SEQ ID No9 or a fragment of at least 3 amino acids thereof; peptide 9 of SEQ ID No10 or a fragment of at least 3 amino acids thereof; peptide 10 of SEQ ID No11 or a fragment of at least 3 amino acids thereof; peptide 11 of SEQ ID No12 or a fragment of at least 3 amino acids thereof; peptide 19 of SEQ ID No13 or a fragment of at least 3 amino acids thereof; peptide 20 of SEQ ID No14 or a fragment of at least 3 amino acids thereof; peptide 21 of SEQ ID No15 or a fragment of at least 3 amino acids thereof; peptide 22 of SEQ ID No16 or a fragment of at least 3 amino acids thereof; peptide 23 of SEQ ID No17 or a fragment of at least 3 amino acids thereof; peptide 31 of SEQ ID No18 or a fragment of at least 3 amino acids thereof; peptide 32 of SEQ ID No19 or a fragment of at least 3 amino acids thereof; peptide 33 of SEQ ID No20 or a fragment of at least 3 amino acids thereof; peptide 34 of SEQ ID No21 or a fragment of at least 3 amino acids thereof; peptide 35 of SEQ ID No22 or a fragment of at least 3 amino acids thereof; peptide 1 of SEQ ID No25 or a fragment of at least 3 amino acids thereof; peptide 2 of SEQ ID No26 or a fragment of at least 3 amino acids thereof; peptide 3 of SEQ ID No27 or a fragment of at least 3 amino acids thereof; peptide 4 of SEQ ID No28 or a fragment of at least 3 amino acids thereof; peptide 5 of SEQ ID No29 or a fragment of at least 3 amino acids thereof; peptide 13 of SEQ ID No30 or a fragment of at least 3 amino acids thereof; peptide 14 of SEQ ID No31 or a fragment of at least 3 amino acids thereof; peptide 15 of SEQ ID No32 or a fragment of at least 3 amino acids thereof; peptide 16 of SEQ ID No33 or a fragment of at least 3 amino acids thereof; peptide 17 of SEQ ID No34 or a fragment of at least 3 amino acids thereof; peptide 25 of SEQ ID No35 or a fragment of at least 3 amino acids thereof; peptide 26 of SEQ ID No36 or a fragment of at least 3 amino acids thereof; peptide 27 of SEQ ID No37 or a fragment of at least 3 amino acids thereof; peptide 28 of SEQ ID No38 or a fragment of at least 3 amino acids thereof; and peptide 29 of SEQ ID No39 or a fragment of at least 3 amino acids thereof.

5. A composition according to, claim 1, comprising six peptides, in which the amino acid sequences of said six peptides consist of distinctive fragments of from 12 to 20 contiguous amino acids.

6. A composition according to claim 1 comprising: peptides 6, 12, 18, 24, 30 and 36 or fragments of at least 3 amino acids thereof; peptides 7, 8, 9, 10, 11 and 12 or fragments of at least 3 amino acids thereof; peptides 19, 20, 21, 22, 23 and 24 or fragments of at least 3 amino acids thereof; peptides 31, 32, 33, 34, 35 and 36 or fragments of at least 3 amino acids thereof; peptides 1, 7, 13, 19, 25 and 31 or fragments of at least 3 amino acids thereof; peptides 2, 8, 14, 20, 26 and 32 or fragments of at least 3 amino acids thereof; peptides 3, 9, 15, 21, 27 and 33 or fragments of at least 3 amino acids thereof; peptides 4, 10, 16, 22, 28 and 34 or fragments of at least 3 amino acids thereof; peptides 5, 11, 17, 23, 29 and 35 or fragments of at least 3 amino acids thereof; or peptides 13, 14, 15, 16, 17 and 18 or fragments of at least 3 amino acids thereof.

7. A composition as claimed in claim 1 further comprising CFP-10 and/or ESAT-6 antigens.

8-11. (canceled)

12. A method of diagnosing LTBI subjects, said method comprising the use of a composition as claimed in claim 1, by: measuring the level of expression of IFN-gamma in isolated peripheral blood mononuclear cells (PBMC) or in a blood sample from a subject; and deducing therefrom if the subject has a LTBI.

13. A method to discriminate between healthy subjects, subjects with active Tuberculosis and LTBI subjects comprising the use of a composition as claimed in claim 7, by: (i) measuring the level of expression of IFN-gamma in isolated peripheral blood (ii) mononuclear cells (PBMC) or in a blood sample from a subject; and (iii) deducing therefrom if the subject has a LTBI.

14. A method according to claim 13 comprising: (i) culturing isolated peripheral blood mononuclear cells (PBMC) or a blood sample from a subject with the composition; (ii) measuring the level of expression of IFN-gamma of said isolated peripheral blood mononuclear cells (PBMC) or of said blood sample; and (iii) deducing therefrom if the subject has a LTBI.

15. A kit for diagnosing LTBI subjects and/or for discriminating healthy subjects, subjects with active Tuberculosis and LTBI subjects comprising: (i) a composition as claimed in claim 1; and (ii) instructions for use to diagnose LTBI and/or for discriminating healthy subjects, subjects with active Tuberculosis and LTBI subjects.

16. A composition according to claim 1, comprising three peptides, in which the amino acid sequences of said three peptides consist of distinctive fragments of from 12 to 20 contiguous amino acids.

17. A composition according to claim 1, comprising: peptides 1, 2 and 5 or fragments of at least 3 amino acids thereof; or peptides 12, 23 and 24 or fragments of at least 3 amino acids thereof.

18. A method according to claim 12 comprising: (i) culturing isolated peripheral blood mononuclear cells (PBMC) or a blood sample from a subject with the composition; (ii) measuring the level of expression of IFN-gamma of said isolated peripheral blood mononuclear cells (PBMC) or of said blood sample; and (iii) deducing therefrom if the subject has a LTBI.

Description

FIGURES

[0258] FIG. 1 shows the IFN-gamma level of expression measured by ELISA on: A) Peripheral blood mononuclear cells (PBMC) from healthy donors (HD), patients with tuberculosis (TB) and latently M. tuberculosis infected individuals (LTBI) cultured for 5 days with compositions according to the invention and B) Whole blood from healthy donors (HD), patients with tuberculosis (TB) and latently M. tuberculosis infected individuals (LTBI) cultured for 24H with compositions according to the invention. Bars represent the mean±SEM. Mann-Whitney test for unpaired samples * p<0.05, ** p<0.01, *** p<0.001.

[0259] FIG. 2: Peripheral blood mononuclear cells from healthy donors (HD), patients with tuberculosis (TB) and latently M. tuberculosis infected individuals (LTBI) were cultured with compositions according to the invention. After five days, IFN-γ production was evaluated in cell free supernatants by ELISA. Bars represent the mean±SEM. Mann-Whitney test for unpaired samples * p<0.05, ** p<0.01, *** p<0.001.

EXAMPLES

Example 1

Protocols

Subjects

[0260] Healthy adults lacking a history of tuberculosis that had received Bacillus Calmette-Guérin (BCG) vaccination at birth participated in the study. Among this group, the diagnosis of latent tuberculosis (LTBI subjects) was established using QuantiFERON TB In-tube Gold® test (Cellestis Inc.), following the manufacturer's instructions. This test was used to discriminate LTBI subjects among a healthy population. Indeed, as this test, as already mentioned, does not allow to discriminate active TB subjects from LTBI subjects, it was made sure to analyze a healthy population with no possibility of active TB to be able to conclude that QuantiFERON positive individuals were LTBI subjects.

[0261] HIV-uninfected patients with active tuberculosis (TB subjects) were evaluated at the Dr. F. Muñiz Hospital, Buenos Aires, Argentina. Diagnosis of disease was established based on clinical and radiological data together with the identification of acid-fast bacilli (AFB) in sputum. Patients included in this study had received less than 1 week of anti-tuberculosis therapy.

[0262] The control group (HD subjects) included individuals that matched in terms of sex, age and ethnicity with TB patients and LTBI individuals.

[0263] All participants provided a written informed consent for the collection of peripheral blood samples and subsequent analysis.

[0264] The protocols conducted in this work were approved by the Ethical Committee of the Hospital Muñiz and by the International Review Board Fundación Huésped.

[0265] Due to the intensive immigration that Argentina has received from European countries during its history, as well as from other Latin American countries during the last decades, the Argentinean population comprises a very diverse genetic background.

[0266] Moreover, since BCG vaccination in mandatory in this country, it is also possible to test if BCG vaccination causes false positives upon stimulation with the compositions according to the invention.

Peptides

[0267] Synthetic peptides of 13 to 17 amino acids, spanning the sequence of Mycobacterium tuberculosis antigen Rv2626c of SEQ ID No1 were synthesized by Biomatik Corp. using Fmoc chemistry.

[0268] Lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO), aliquoted and stored at −70° C.

[0269] Peptide purity was of more than 80%, as assayed by HPLC, and their composition was verified by mass spectrometry.

[0270] For in vitro stimulation, 4 pools of 6 peptides were prepared (Table 1), with each peptide at a final concentration of 2 mg/ml, following the methodology previously described by Addo et al (J Virol. 2003 February; 77(3):2081-92).

TABLE-US-00001 TABLE 1 Mycobacterium Tuberculosis antigen Rv2626c peptides pools Pool Reference Peptide reference 6  6 12 18 24 30 36 (SEQ ID N.sup.o2) (SEQ ID N.sup.o3) (SEQ ID N.sup.o4) (SEQ ID N.sup.o5) (SEQ ID N.sup.o6) (SEQ ID N.sup.o7) 8  7  8  9 10 11 12 (SEQ ID N.sup.o8) (SEQ ID N.sup.o9) (SEQ ID N.sup.o10) (SEQ ID N.sup.o11) (SEQ ID N.sup.o12) (SEQ ID N.sup.o3) 10 19 20 21 22 23 24 (SEQ ID N.sup.o13) (SEQ ID N.sup.o14) (SEQ ID N.sup.o15) (SEQID N.sup.o16) (SEQ ID N.sup.o17) (SEQ ID N.sup.o5) 12 31 32 33 34 35 36 (SEQ ID N.sup.o18) (SEQ ID N.sup.o19) (SEQ ID N.sup.o20) (SEQ ID N.sup.o21) (SEQ ID N.sup.o22) (SEQ ID N.sup.o7)

Cell Preparation and Reagents

[0271] Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll-Hypaque (GE Healthcare) and cultured (1×10.sup.6 cells/ml) with the different peptide pools (5 μg/ml) with RPMI 1640 (Gibco) supplemented with L-glutamine, penicillin/streptomycin and 10% of human serum (Sigma-Aldrich). After five days, cell free supernatants were collected to determine IFN-γ expression by ELISA (BioLegend).

Results

[0272] A pool matrix in which each pool was composed of 6 different peptides was first designed and IFN-γ production against those pools was then tested.

[0273] The results shown in FIG. 1 indicate that pools 6, 8, 10 and 12 were able to significantly discriminate LTBI individuals from patients with tuberculosis and healthy controls by inducing significantly higher levels of IFN-γ in LTBI individuals compared to active TB patients and healthy donors.

[0274] These results show that the compositions according to the invention can be used in a diagnostic test to discriminate LTBI subjects among TB, LTBI and HD subjects.

[0275] As it was previously mentioned, latent tuberculosis infection represents the main reservoir for M. tuberculosis, making its effective detection key in the struggle against active disease.

[0276] Nowadays, despite their shortcomings, the most commonly used assays for diagnosing latent tuberculosis infection are the tuberculin skin test (TST) and two commercial assays: QuantiFERON TB Gold In Tube, from the firm Cellestis GmbH, and T-Spot TB, from the firm Oxford Immunotec. Both commercial kits are interferon gamma release assays (IGRAs), which use specific M. tuberculosis peptides (mainly CFP-10 and ESAT-6) to induce secretion of this cytokine in individuals infected with the pathogen.

[0277] Thus, while these assays differentiate infected individuals from healthy ones, unlike the compositions according to the invention, they do not discriminate between latent and active infection.

[0278] The TST, most commonly used in less developed countries, presents high variability and is dependent on both the observer and the way of administration. It is not standardizable nor objective and, in addition, can present false positives, especially with BCG vaccination. Both T-Spot TB and QuantiFERON TB Gold In Tube assays are much more specific than the TST, however, they are unable to differentiate latently infected individuals from those with active disease.

[0279] Table 2 illustrates the expected results using the different available diagnostic tests and the compositions of the invention.

TABLE-US-00002 TABLE 2 QuantiFERON TB Gold In Composition of Subjects Tube T-Spot TB TST the invention LTBI + + +/− + TB + + + − HD − −− +/− −

Example 2

Protocols

Subjects.

[0280] BCG-vaccinated healthy adults lacking a history of TB (household contacts and healthcare workers) were recruited. Among this group of individuals, diagnosis of LTBI was established using QuantiFERON-TB Gold In-Tube (QFT-GIT; Qiagen, USA; according to manufacturer's directions) and Tuberculin Skin Test (TST) tests. LTBI diagnosis was assigned to any subject with a positive QFT-GIT/TST and no clinical or radiological evidence of active TB. In the event of discordant QFT-GIT/TST results, individuals were assigned to the corresponding group on the basis of the QFT-GIT result. The group of healthy donors (HD) was comprised by adult individuals without TB disease (tested by chest X-rays and analysis of acid-fast bacilli in sputum) and with negative QFT-G IT/TST.

[0281] HIV-uninfected patients with active TB were evaluated at Dr. F. Muñiz or Dr. E. Tornú Hospitals (Buenos Aires, Argentina). Diagnosis of TB disease was established based on clinical and radiological data together with culture-confirmation and the identification of acid-fast bacilli in sputum. Patients included in this study had received less than one week of anti-TB therapy.

[0282] Information regarding demographic data and prior TB exposure was obtained at the time of recruitment. All participants provided written informed consent for sample collection and subsequent analysis. The protocols conducted were approved by the Ethical Committee of the Dr. F. Muñiz and the Dr. E. Tornú Hospitals and by the International Review Board Fundación Huésped.

[0283] Due to the intensive immigration that Argentina has received from European countries during its history, as well as from other Latin American countries during the last decades, the Argentinean population comprises a very diverse genetic background.

[0284] Moreover, since BCG vaccination in mandatory in this country, it is also possible to test if BCG vaccination causes false positives upon stimulation with the compositions according to the invention.

Study Inclusion and Exclusion Criteria for Individuals Participating in the Study.

[0285] Inclusion criteria: a) adult (over 18 years old) men and women with active pulmonary TB and b) healthy volunteers with high level of exposure to M. tuberculosis (household contacts of TB patients and healthcare workers of National Referral Hospitals for TB). All recruited subjects were BCG-vaccinated. QFT-GIT and TST assays were used to determine the presence of LTBI among individuals without clinical or microbiological diagnosis of active TB. All TB patients included in the study had a positive culture for M. tuberculosis.

[0286] Exclusion criteria: a) HIV positive or positive serology to other viral or bacterial infections; b) patients with diabetes, cancer, autoimmune diseases or other conditions that may affect the immune system of the individual; c) pregnant women and d) children. Among the population of active TB patients were excluded: a) patients with multidrug-resistant tuberculosis (MDR-TB) infection, b) patients with more than seven consecutive days of anti-TB treatment. Individuals with indeterminate QFT-GIT results were also excluded from the study.

Peptides

[0287] Overlapping synthetic peptides (13-17 amino acids, overlapping by 11 amino acids (aa)) spanning the sequence of Rv2626c of SEQ ID No1 were synthesized by Biomatik Corp. using Fmoc chemistry. Peptide purity was superior to 80%, as assayed by HPLC, and their composition was verified by mass spectrometry. Lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO), aliquoted and stored at −70° C. Table 3 displays the peptide pools used. For in vitro stimulation, peptides were arranged in pools of 6 peptides each (shown in Table 3), with each peptide at a final concentration of 2 mg/ml.

TABLE-US-00003 TABLE 3 Mycobacterium Tuberculosis antigen Rv2626c peptides pools Pool Reference Peptide reference 1 1  7 13 19 25 31 (SEQ ID N.sup.o25) (SEQ ID N.sup.o8) (SEQ ID N.sup.o30) (SEQ ID N.sup.o13) (SEQ ID N.sup.o35) (SEQ ID N.sup.o18) 2 2  8 14 20 26 32 (SEQ ID N.sup.o26) (SEQ ID N.sup.o9) (SEQ ID N.sup.o31) (SEQ ID N.sup.o14) (SEQ ID N.sup.o36) (SEQ ID N.sup.o19) 3 3  9 15 21 27 33 (SEQ ID N.sup.o27) (SEQ ID N.sup.o10) (SEQ ID N.sup.o32) (SEQID N.sup.o15) (SEQ ID N.sup.o37) (SEQ ID N.sup.o20) 4 4 10 16 22 28 34 (SEQ ID N.sup.o28) (SEQ ID N.sup.o11) (SEQ ID N.sup.o33) (SEQ ID N.sup.o16) (SEQ ID N.sup.o38) (SEQ ID N.sup.o21) 5 5 11 17 23 29 35 (SEQ ID N.sup.o29) (SEQ ID N.sup.o12) (SEQ ID N.sup.o34) (SEQ ID N.sup.o17) (SEQ ID N.sup.o39) (SEQ ID N.sup.o22) 6 6 12 18 24 30 36 (SEQ ID N.sup.o2) (SEQ ID N.sup.o3) (SEQ ID N.sup.o4) (SEQ ID N.sup.o5) (SEQ ID N.sup.o6) (SEQ ID N.sup.o7) 8 7 8 9 10 11 12 (SEQ ID N.sup.o8) (SEQ ID N.sup.o9) (SEQ ID N.sup.o10) (SEQ ID N.sup.o11) (SEQ ID N.sup.o12) (SEQ ID N.sup.o13) 9 13  14 15 16 17 18 (SEQ ID N.sup.o30) (SEQ ID N.sup.o31) (SEQ ID N.sup.o32) (SEQ ID N.sup.o33) (SEQ ID N.sup.o34) (SEQ ID N.sup.o4) 10 19  20 21 22 23 24 (SEQ ID N.sup.o13) (SEQ ID N.sup.o14) (SEQ ID N.sup.o15) (SEQID N.sup.o16) (SEQ ID N.sup.o17) (SEQ ID N.sup.o5) 12 31  32 33 34 35 36 (SEQ ID N.sup.o18) (SEQ ID N.sup.o19) (SEQ ID N.sup.o20) (SEQ ID N.sup.o21) (SEQ ID N.sup.o22) (SEQ ID N.sup.o7)

Cell Preparation and Reagents.

[0288] Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll-Hypaque (GE Healthcare) and cultured (1×10.sup.6 cells/ml) with the different peptide pools (5 μg/ml) with RPMI 1640 (Gibco) supplemented with L-glutamine, penicillin/streptomycin and 10% human serum (Sigma-Aldrich). After five days, cell free supernatants were collected to determine IFN-γ expression by ELISA (BioLegend).

Results

[0289] A pool matrix in which each pool was composed of 6 different peptides was first designed and IFN-γ production against those pools was then tested.

[0290] As can be observed in FIG. 2, pools 1, 2, 3, 4, 5, 6, 8, 9, 10 and 12 induced high IFN-γ levels, which significantly discriminate LTBI individuals from tuberculosis patients and healthy controls.

[0291] Taken together these data show that the compositions according to the invention can be used in a diagnostic test to discriminate LTBI subjects among TB, LTBI and HD subjects and are very good candidates to be used in a diagnostic test to discriminate among LTBI subjects from TB patients and HD.