Compositions useful in the diagnostic of latently infected <i>Mycobacterium tuberculosis </i>

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 method of diagnosing latently infected Mycobacterium tuberculosis (LTBI) in a human subject, said method comprising: (i) culturing isolated peripheral blood mononuclear cells (PBMC) or a blood sample from the subject with a composition, wherein the composition comprises peptide 1 consisting of SEQ ID NO: 25, peptide 2 consisting of SEQ ID NO: 26, and peptide 5 consisting of SEQ ID NO: 29; and/or peptide 12 consisting of SEQ ID NO: 3, peptide 23 consisting of SEQ ID NO: 17, and peptide 24 consisting of SEQ ID NO: 5; and (ii) measuring the level of expression of IFN-gamma of said cultured PBMC or said cultured blood sample; and (iii) diagnosing the LTBI in said human subject when the level of the IFN-gamma in said human subject is higher compared to the level of IFN-gamma in healthy human subjects and human patients with active tuberculosis.

2. A method of discriminating a human subject with LTBI from a healthy human subject and from a human patient with active tuberculosis comprising: (i) culturing isolated peripheral blood mononuclear cells (PBMC) or a blood sample from the subject with a composition, wherein the composition comprises peptide 1 consisting of SEQ ID NO: 25, peptide 2 consisting of SEQ ID NO: 26, and peptide 5 consisting of SEQ ID NO: 29; and/or peptide 12 consisting of SEQ ID NO: 3, peptide 23 consisting of SEQ ID NO: 17, and peptide 24 consisting of SEQ ID NO: 5, wherein the composition further comprises CFP-10 antigen and/or ESAT antigen of Mycobacterium tuberculosis; (ii) measuring the level of expression of IFN-gamma of said cultured PBMC or said cultured blood sample; and wherein a higher level of the IFN-gamma in said human subject compared to the level of IFN-gamma in the healthy human subject and in the human patient with active tuberculosis discriminates the human subject with LTBI from the healthy human subject and the human patient with active tuberculosis.

Description

FIGURES

(1) FIGS. 1A and 1B show 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.

(2) FIGS. 2A and 2B: 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

(3) Protocols

(4) Subjects

(5) Healthy adults lacking a history of tuberculosis that had received Bacillus Calmette-Guerin (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.

(6) 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.

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

(8) All participants provided a written informed consent for the collection of peripheral blood samples and subsequent analysis.

(9) The protocols conducted in this work were approved by the Ethical Committee of the Hospital Muñiz and by the International Review Board Fundación Huésped.

(10) 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.

(11) 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.

(12) Peptides

(13) Synthetic peptides of 13 to 17 amino acids, spanning the sequence of Mycobacterium tuberculosis antigen Rv2626c of SEQ ID No 1 were synthesized by Biomatik Corp. using Fmoc chemistry.

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

(15) Peptide purity was of more than 80%, as assayed by HPLC, and their composition was verified by mass spectrometry.

(16) 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).

(17) TABLE-US-00001 TABLE 1 Mycobacterium Tuberculosis antigen Rv2626c peptides pools Pool Reference Peptide reference 6  6 12 18 24 30 36 (SEQ ID No 2) (SEQ ID No 3) (SEQ ID No 4) (SEQ ID No 5) (SEQ ID No 6) (SEQ ID No 7) 8  7  8  9 10 11 12 (SEQ ID No 8) (SEQ ID No 9) (SEQ ID No 10) (SEQ ID No 11) (SEQ ID No 12) (SEQ ID No 3) 10 19 20 21 22 23 24 (SEQ ID No 13) (SEQ ID No 14) (SEQ ID No 15) (SEQ ID No 16) (SEQ ID No 17) (SEQ ID No 5) 12 31 32 33 34 35 36 (SEQ ID No 18) (SEQ ID No 19) (SEQ ID No 20) (SEQ ID No 21) (SEQ ID No 22) (SEQ ID No 7)
Cell Preparation and Reagents

(18) 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).

(19) Results

(20) 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.

(21) The results shown in FIGS. 1A and 1B indicate that pools 6, 8, 10 and 12 were able to significantly discriminate LTB1 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.

(22) 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.

(23) 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.

(24) 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.

(25) 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.

(26) 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.

(27) Table 2 illustrates the expected results using the different available diagnostic tests and the compositions of the invention.

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

Example 2

(29) Protocols

(30) Subjects.

(31) 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-GIT/TST.

(32) 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.

(33) 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.

(34) 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.

(35) 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.

(36) Study Inclusion and Exclusion Criteria for Individuals Participating in the Study.

(37) 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.

(38) 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.

(39) Peptides

(40) Overlapping synthetic peptides (13-17 amino acids, overlapping by 11 amino acids (aa)) spanning the sequence of Rv2626c of SEQ ID No 1 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.

(41) TABLE-US-00003 TABLE 3 Mycobacterium Tuberculosis antigen Rv2626c peptides pools Pool Reference Peptide reference 1  1  7 13 19 25 31 (SEQ ID No 25) (SEQ ID No 8) (SEQ ID No 30) (SEQ ID No 13) (SEQ ID No 35) (SEQ ID No 18) 2  2  8 14 20 26 32 (SEQ ID No 26) (SEQ ID No 9) (SEQ ID No 31) (SEQ ID No 14) (SEQ ID No 36) (SEQ ID No 19) 3  3  9 15 21 27 33 (SEQ ID No 27) (SEQ ID No 10) (SEQ ID No 32) (SEQ ID No 15) (SEQ ID No 37) (SEQ ID No 20) 4  4 10 16 22 28 34 (SEQ ID No 28) (SEQ ID No 11) (SEQ ID No 33) (SEQ ID No 16) (SEQ ID No 38) (SEQ ID No 21) 5  5 11 17 23 29 35 (SEQ ID No 29) (SEQ ID No 12) (SEQ ID No 34) (SEQ ID No 17) (SEQ ID No 39) (SEQ ID No 22) 6  6 12 18 24 30 36 (SEQ ID No 2) (SEQ ID No 3) (SEQ ID No 4) (SEQ ID No 5) (SEQ ID No 6) (SEQ ID No 7) 8  7  8  9 10 11 12 (SEQ ID No 8) (SEQ ID No 9) (SEQ ID No 10) (SEQ ID No 11) (SEQ ID No 12) (SEQ ID No 13) 9 13 14 15 16 17 18 (SEQ ID No 30) (SEQ ID No 31) (SEQ ID No 32) (SEQ ID No 33) (SEQ ID No 34) (SEQ ID No 4) 10 19 20 21 22 23 24 (SEQ ID No 13) (SEQ ID No 14) (SEQ ID No 15) (SEQ ID No 16) (SEQ ID No 17) (SEQ ID No 5) 12 31 32 33 34 35 36 (SEQ ID No 18) (SEQ ID No 19) (SEQ ID No 20) (SEQ ID No 21) (SEQ ID No 22) (SEQ ID No 7)
Cell Preparation and Reagents.

(42) 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).

(43) Results

(44) 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.

(45) As can be observed in FIGS. 2A and 2B, pools 1, 2, 3, 4, 5, 6, 8, 9, 10 and 12 induced high IFN-γ levels, which significantly discriminate LTB1 individuals from tuberculosis patients and healthy controls.

(46) 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.