Methods for the detection of a latent tuberculosis infection

Abstract

The present invention relates to a method for the in vitro detection of a latent tuberculosis infection (LTBI) in a subject, wherein said method comprises determining at least one nucleotide sequence and/or at least one polypeptide of Mycobacterium tuberculosis (Mtb) in a blood cell population of said subject, and wherein the presence of said at least one nucleotide sequence and/or said at least one polypeptide is indicative for said latent tuberculosis infection. In particular, the blood cell population is enriched for hematopoietic stem cells. The invention also relates to a pharmaceutical composition for use in the treatment of the LTBI in the subject, wherein it is determined if the nucleotide sequence and/or the polypeptide of Mtb is/are present in the blood cell population. Further, the invention relates to kits for carrying out the methods of the invention. The invention also relates to the use of the kits.

Claims

1. A method of detecting a latent tuberculosis infection in a subject, wherein said method comprises: testing for the presence of at least one nucleotide sequence of Mycobacterium tuberculosis in a blood sample enriched for CD34.sup.+ cells from the subject, wherein the presence of said at least one nucleotide sequence is indicative of said latent tuberculosis infection.

2. The method of claim 1, wherein said blood sample is enriched for: a) hematopoietic stem cells (HSCs); b) CD34+ cells; c) CD90+ cells; d) lineage negative (Lin.sup.−) cells; e) side population (SP) cells; f) long-term repopulating pluripotent hematopoietic stem cells (LT-pHSCs); and/or g) peripheral blood mononuclear cells (PBMCs) and hematopoietic stem cells (HSCs).

3. The method of claim 2, wherein said blood sample comprises: a) at least about 1×10.sup.3 HSCs; b) at least about 1×10.sup.5 Lin.sup.− cells; c) at least about 1×10.sup.3 SP cells; and/or d) at least about 1×10.sup.3 LT-pHSCs.

4. The method of claim 1, wherein said blood sample is enriched for: a) CD34.sup.+CD90.sup.+ cells; b) CD34.sup.+Lin.sup.−; c) CD90.sup.+CD38.sup.− cells; and/or d) cells being essentially free of one or more cell surface markers selected from the group consisting of CD1c, CD3, CD11c, CD14, CD15, CD16, CD20, CD41, CD56, CD203c, CD235a, BDCA2, and CD45RA.

5. The method of claim 1, wherein said blood sample comprises at least about 1×10.sup.3 CD34.sup.+ cells.

6. The method of claim 5, wherein said blood sample comprises: a) at least about 1×10.sup.3 CD34.sup.+CD90.sup.+ cells; b) at least about 1×10.sup.3 CD34.sup.+CD90.sup.+CD38.sup.− cells; and/or c) at least about 1×10.sup.3 CD34.sup.+CD90.sup.+CD38.sup.low cells.

7. The method of claim 1, wherein said blood sample is a peripheral blood sample.

8. The method of claim 1 wherein said subject a) is immune compromised; b) is immune suppressed; c) suffers from HIV; d) suffers from cancer; and/or e) is or is to be treated with anti-TNFa therapy.

9. The method of claim 1, wherein said subject suffers from HIV, cancer, silicosis, diabetes mellitus, chronic renal failure, chronic renal failure on hemodialysis, gastrectomy jejunoileal bypass, and/or conditions that require prolonged use of corticosteroids or other immunosuppressive agents.

10. The method of claim 1, wherein said subject is an infection drug user, has an excessive alcohol intake, is younger than 5 years, has a low body weight, has a radiographic evidence of prior healed Tuberculosis (TB), is a Tuberculin Skin Test (TST) converter, received or is to receive a hematopoieticstem cell transplantation, received or is to receive a solid organ transplant and/or is an infant or a child under the age of five years with a positive TB test result.

11. The method of claim 1, wherein said subject: a) has no symptoms or physical findings suggestive of tuberculosis; b) has a positive interferon-gamma release assay; c) has a positive tuberculin skin test, d) has a normal chest radiography; and/or e) shows a negative result in a colony formation unit (CFU) assay of a blood sample, a respiratory sample, an urine sample, and/or a stool sample.

12. The method of claim 1, wherein said at least one nucleotide sequence encodes MPB64 and/or IS6110.

13. The method of claim 1, wherein said nucleotide sequence is detected by hybridization and/or Polymerase Chain Reaction (PCR).

14. The method of claim 1, wherein said method further comprises testing for the presence of a Bacillus Calmette-Guérin (BCG) vaccine nucleotide sequence.

15. The method of claim 1, wherein said method further comprises administering to said subject identified as having at least one nucleotide sequence a pharmaceutically effective amount of a Mycobacterium tuberculosis pharmaceutical composition.

16. The method of claim 15, wherein said pharmaceutical composition is an antimicrobial.

17. The method of claim 16, wherein said antimicrobial is an isoniazid, rifampicin or rifapentine.

18. The method of claim 16, wherein said antimicrobial is administered as a co-treatment with isoniazid, rifampin, rifapentine and/or pyridoxine.

19. The method of claim 9, wherein the other immunosuppressive agent is a TNFα antagonist.

20. The method of claim 10, wherein said cancer is head or neck cancer.

Description

(1) The present invention is further described by reference to the following non-limiting figures and examples. The Figures show:

(2) FIG. 1. Human peripheral Lin.sup.−CD34.sup.+ progenitors as well as Lin.sup.−CD34.sup.+CD38.sup.lowCD90.sup.+ and SP.sup.+ pHSCs of IGRA.sup.+ donors harbor Mtb DNA. Lin.sup.+ (n=11), Lin.sup.−CD34.sup.+ (n=15), Lin.sup.−CD34.sup.+CD38.sup.lowCD90.sup.+ (n=2), Lin.sup.−CD34.sup.+CD38+CD90 (n=2) as well as Lin.sup.− SP.sup.+ (n=4; Donors 1, 6, 8 and 9) and Lin.sup.+ SP cells (n=4; Donors 1, 6, 8 and 9) were purified by FACS from blood cells from IGRA.sup.+ (n=8) and IGRA− donors (n=7) (FIG. 7A). In addition, CD1c.sup.+, CD14.sup.+, CD16.sup.+, CD4.sup.+/8.sup.+, CD15.sup.+, CD19.sup.+, and CD56.sup.+ cells were prepared from peripheral blood of IGRA.sup.+ donors (n=3). Genomic DNA was prepared and DNA of 10.sup.3 hematopoietic progenitors as well as 10.sup.5 Lin.sup.+ cells from IGRA.sup.+ and IGRA.sup.− donors were tested by PCR. Panel A and D shows the quantification of Mtb-specific DNA by real-time TaqMan PCR using known Mtb concentrations as reference (see also table 3). Probes used target MPB64 and IS6110 together. TagMan PCRs were performed in 2 independent runs in technical triplicates and normalized to human GAPDH (median+interquartile). Panel B shows a PCR for a 12.7-kb fragment present in Mtb, but not in BCG (Donors 8-13). Panel C shows the quantification of Mtb-specific DNA by serial and limiting dilutions using a single-target PCR for IS6110 on DNA of CD34.sup.+ cells (Donor 13 and 14). In panel D, the results of a real-time SYBR green PCR using primers that target MPB64 alone are shown. Known Mtb concentrations were used as reference. SYBR green PCRs were performed in 2 independent runs in technical triplicates and normalized to human GAPDH (median+interquartile). Note, that MPB64 exists as a single sequence in the Mtb genome, while IS6110 can be present in multiple copies. Panel E shows the quantification of Mtb-specific DNA using probes used target MPB64 and IS6110 together and primers targeting MPB64 alone on DNA purified from Lin.sup.−CD34.sup.+, Lin.sup.−CD34.sup.+CD38.sup.lowCD90.sup.+ and Lin.sup.−CD34.sup.+CD38+CD90 cells (n=2). Panel F shows the monitoring of Mtb infection by CFU Mtb growth on Middlebrook 7H11 agar plates (median+interquartile, n=2-3). Panel G shows the gating strategy for the purification of human peripheral blood Lin.sup.−CD34.sup.+ progenitors as well as Lin.sup.−CD34.sup.+CD38.sup.lowCD90.sup.+ pHSCs by FACS. Panel H shows the gating strategy for the purification of CD45.sup.loCD271.sup.+ mesenchymal stem cells. Panel I shows the quantification of Mtb-specific DNA on DNA of purified MSCs (Donors 10 and 13) by real-time TaqMan PCR using known Mtb concentrations as reference. Probes used target MPB64 and IS6110 together. TagMan PCRs were performed in 2 independent runs in technical triplicates and normalized to human GAPDH (median+interquartile).

(3) TABLE-US-00002 TABLE 3 n Mtb DNA copies/10.sup.3 CD34.sup.+ cells probes/primers Donor 10 Donor 11 Donor 12 Donor 13 Donor 14 Donor 15 IS6110/MPB64 9 ± 5 7 ± 4 10 ± 5   10 ± 3 16 ± 2 10 ± 2 MPB64 4 ± 2 5 ± 3 1 ± 0.5  2 ± 1  7 ± 3  7 ± 4

(4) FIG. 2. Mtb infection in the dermis, detected in different organs and hematopoietic cells of Mtb-infected mice by Mtb DNA PCR and Mtb CFU. C57BL/6 mice were infected with 10.sup.5 CFUs Mtb (strain H37Rv) in the ear dermis. Panel A shows the monitoring of Mtb infection by—real-time TaqMan PCR using probes that targets MPB64 and IS6110 together on genomic DNA of single cell suspensions of 10.sup.5 lung cells (n=8 individual mice), 10.sup.3 FACS-enriched LT-pHSCs, ST-pHSCs and MPPs (FIG. 7B; n=16 individual mice), as well as 10.sup.5 Lin.sup.+ Gr1.sup.+ granulocytes, CD11c.sup.+ dendritic cells, CD19.sup.+ B cells, Mac1.sup.+ macrophages, NK1.1.sup.+ NK cells and CD4.sup.+/8.sup.+ T cells (FIG. 7C; n=4 individual mice) of infected mice day 28 p.i. Real-time TaqMan PCRs were performed in 2 independent runs in technical triplicates and normalized to murine GAPDH (median+interquartile). Panel B shows the quantification of Mtb-specific DNA by serial and limiting dilutions of genomic DNA from purified LT-pHSCs from 3 individual mice by the multiple copy IS6110 PCR (n=3). In panel C results of a real-time SYBR green PCR using primers that target the single copy MPB64 alone are shown (n=4-8 individual mice. Known Mtb concentrations were used as reference. SYBR green PCRs were performed in 2 independent runs in technical triplicates and normalized to murine GAPDH (median+interquartile). Panel D shows the monitoring of Mtb infection by CFU enumeration on Middlebrook 7H11 agar plates in cell homogenates of lung and single cell suspensions of spleen and thymus 28 days p.i. (median+interquartile, n=16). Panel E shows the CFU enumeration on Middlebrook 7H11 agar plates for FACS-enriched Lin.sup.+ Gr1.sup.+ granulocytes, CD11c.sup.+ dendritic cells, Mac1.sup.+ macrophages, NK 1.1.sup.+ NK cells, CD4.sup.+/8.sup.+ T cells, CD19.sup.+ B cells (median+interquartile, n=8). Panel F shows the CFU enumeration on Middlebrook 7H11 agar plates for FACS-enriched Lin− hematopoietic progenitors (median+interquartile, n=16) 28 days p.i. Shown are data of 4 independent experiments. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005 by Mann-Whitney test (unpaired).

(5) FIG. 3. Mtb infection in the dermis, detected in cells of the lung and hematopoietic cells of Mtb-infected mice by histology. Rhodamin-auramin stainings and immunocytochemistry stainings against Mtb (Alexa Fluor 555); LAMP2 (Alexa Flour 647) and EEA1 (Alexa Flour 488) of representative LT-pHSCs (n=6), ST-pHSCs (n=3), MPPs (n=3) as well as cells of the lung (n=5) at day 28 p.i. For each sample 10,000 cells were screened per slide. Rhodamin-auramin stainings were screened on high power (100×) and verified under oil immersion using a fluorescent microscope. Analyses were done using ProGres Capture Pro 2.8.8. (Mtb, red; nuclei, blue). Immunocytochemistry stainings were screened by confocal microscopy using a Zeiss LSM710 with a 100×/0.8 numerical aperture objective lens. Image acquisition was performed using Zen 2010 Version 6.0 and images were analyzed by Zen 2012 Light Edition software (Carl Zeiss Microlmaging; Mtb, red; nuclei, white; LAMP2, blue; EEA1, green). Shown are representative data (cropping of images) for staining of LT-pHSCs, ST-pHSCs, MPPs and cells of the lung. scale bar: 10 μm. *P<0.05 by Mann-Whitney test (unpaired).

(6) FIG. 4. Numbers of Mtb DNA copies (MPB64 PCR) and CFUs in 10.sup.4 cells of different cell populations. (n=4-8; median+interquartile).

(7) FIG. 5. Murine and human pHSCs are infected with Mtb expressing dormancy genes. Panel A shows the expression analyses on RNA isolated from Mtb-infected mouse lung cells and purified LT-pHSCs (median+interquartile, n=8). Panel B shows the expression analyses on RNA isolated from LinCD34.sup.+ pHSCs from IGRA.sup.+ donors (median+interquartile, n=6) and Mtb-infected human monocytic leukemia cell line 96 h p.i. (median+interquartile, n=3). Expression analyses were done by real-time TaqMan PCR for SigA, DosR, c-lat and hspX. SigA was used as reference for Mtb. Real-time TaqMan PCRs were performed in 3 independent runs in technical triplicates. *P<0.05, **P<0.005, ***P<0.005 by Mann-Whitney test.

(8) FIG. 6. Intratracheal transfer of human and murine pHSCs infected with Mtb leads to CFU-forming Mtb and granuloma formation in lungs of transplanted hosts. Panel A shows a scheme of the injection of 1,000 Lin.sup.−CD34.sup.+ and Lin.sup.+ cells from blood of IGRA.sup.+ (Donor 12-14; n=3), Mtb DNA.sup.+ human donors as well as Lin.sup.−CD150.sup.+CD48.sup.− LT-pHSCs (n=3) from bone marrow of infected mice 28 days p.i. into the trachea of Ragr2.sup.−/−Il2rg.sup.−/− mice (3 mice per population). Intratracheal transfer of 100 CFUs Mtb was used as positive (n=3), and uninfected pHSCs and Lin.sup.+ cells of an IGRA.sup.− donor (Donor 3; n=1) as negative, control. Mice were analyzed 3 weeks upon transfer. Panel B shows the monitoring of Mtb infection by TaqMan PCR using probes that targets MPB64 and IS6110 together on genomic DNA of single cell suspensions of 10.sup.5 lung cells from transplanted mice 3 weeks upon transfer. TaqMan PCRs were performed in technical triplicates and normalized to murine GAPDH. Panel C shows CFU Mtb growth on Middlebrook 7H11 agar plates in cell homogenates of lung and single cell suspensions of spleen, thymus and non-separated, 10.sup.5 bone marrow cells 3 weeks upon cell transfer (median+interquartile; n=3 per transferred population). Shown are data from 3 independent experiments. Panel D shows the histopathology of representative lung sections taken from mice 3 weeks upon intratracheal transfer of human and murine Mtb-infected pHSCs, showing evidence of active bacterial growth. Lungs were stained with hematoxilin-and-eosin, screened with 5× objectives and verified using a light microscope. Shown are representative data from 3 independent experiments. Scale bar: 100 μm.

(9) FIG. 7. Sorting strategy in human and mouse. Panel A shows the purification of Lin+, Lin−CD34+, Lin−CD34+CD38−CD90+, Lin−CD34+CD38+CD90− as well as Lin−SP+ and Lin+SP− cells by FACS from blood cells from IGRA+ and IGRA− donors. Panel B and C shows the purification of Lin− hematopoietic progenitors as well as Lin+Gr1+ granulocytes, CD11c+ dendritic cells, Mac1+ macrophages, NK 1.1+NK cells, CD4+/8+ T cells and CD19+/B220+ B cells by FACS from bone marrow of infected mice day 28 p.i. Representative FACS blots are shown.

(10) FIG. 8. Laser setup of the FACS Aria I machine used for sorting human HSC.

(11) FIG. 9. Box plot of the MOTA values of patients shown in table 4 (the red dotted line indicates the cutoff of 3400 for a positive result)

(12) FIG. 10. Amplification plots of the PCR reactions shown in table 2. Only the endogenous controls show a logarithmic amplification.

(13) The present invention is additionally described by way of the following illustrative non-limiting examples that provide a better understanding of the present invention and of its many advantages.

EXAMPLE 1: DETECTION OF LATENT TUBERCULOSIS INFECTION IN PERIPHERAL BLOOD CELLS, SUCH AS HEMATOPOIETIC STEM CELLS

(14) Patient Selection and Human Samples

(15) Latently Mtb infected subjects included in the study were from a Western country, had not been treated previously for tuberculosis (TB) and were not suffering from active TB. Hence, the subjects had no symptoms or physical findings suggestive of active tuberculosis. The subjects also had normal chest radiography. In sputum, urine, stool and blood samples (3 ml of citrate blood) obtained from the patients Mtb was not detectable by Ziehl-Neelsen staining, Bactec culture or conventional PCR. Hence, the only sign of TB infection is a positive reaction to interferon gamma release assay (IGRA).

(16) Collection of blood samples was approved by the Ethics Committee of the Medical University of Vienna (EK 071/2005) and conducted according to the Declaration of Helsinki. LTBI individuals were routinely identified by positive IGRA (Quantiferon-TB Gold® test, Cellestis, Qiagen) and exclusion of active TB. IGRA testing was performed either because of a scheduled treatment with TNF-α inhibitors or because of occupational contact with patients suffering from active pulmonary TB. Informed written consent was obtained from all patients.

(17) Mice

(18) C57BL/6 wild-type mice were purchased from Charles River Laboratories. CD45.1 C57BL/6 and Rag2.sup.−/−Il2rg.sup.−/− mice were bred in our facilities. Infected mice were maintained at biosafety level 3. All animal experiments were approved by the local ethics committee of the German authorities (State Office of Health and Social Affairs Berlin; Landesamtes für Gesundheit und Soziales Berlin, #G0009-14).

(19) Infection with Mtb

(20) Mtb strain H37Rv was cultured in Middlebrook 7H9 broth (BD) supplemented with 0.05% (v/v) Tween 80 and Middlebrook AODC Enrichment (BD) to mid-log phase (OD.sub.600 nm 0.6-0.8). Bacteria were harvested, resuspended in PBS (GIBCO), and frozen at −80° C. until use. For dermal infections, 8- to 10-week-old female C57BL/6 wild-type mice were anesthestized by i.p. administration of ketamine (50 mg/kg) and Rompun (5 mg/kg; Bayer), and 10.sup.5 Mtb in 50 μl PBS were administered into the ear dermis.

(21) For the infection of human monocytic leukemia cells in vitro, THP-1 cells (ATCC®TIB-202™, ATCC cell lines, UK) were used, that were authenticated by STR profiling and tested for mycoplasma contamination. Cell lines from the list of commonly misidentified cell lines (ICLAC) have not been used in the context of this study. THP-1 cells were seeded in T.sub.75 flasks (TPP) in complete RPMI-1640 (cRPMI, RPMI-1640 medium supplemented with 1% L-glutamine, 1% Hepes, 0.1% 2-ME and fetal bovine serum to a final concentration of 10%; GIBCO, Life Technologies). For proper viability of cells, a concentration of 1×10.sup.6 cells/ml was not exceeded. Cells were incubated at 37° C. and 5% CO.sub.2. Differentiation to macrophages was triggered by overnight incubation with PMA (50 ng/ml), followed by two washes in RPMI-1640 and addition of cRPMI-1640 over 48 h post-differentiation. For infection, 10.sup.7 differentiated macrophages were seeded into T.sub.150 flasks in 25 ml cRPMI and 1 ml of medium containing 10.sup.5 Mtb was added. Non-internalized bacteria were washed away 4 h p.i. using PBS and cells were placed back in cRPMI. Cells were harvested for RNA isolation 48 and 96 hours p.i.

(22) Antibodies

(23) For the purification of 2-40×10.sup.3 circulating human hematopoietic precursor cells from 90 ml of peripheral blood, PBMCs were obtained by Ficoll-Paque density gradient centrifugation (Ficoll-Paque Plus; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and incubated with the following lineage antibodies: CD1c (clone AD5-8E7, Miltenyi Biotec), CD3 (UCHT1, Beckman Coulter), CD11c (Bul5, Beckman Coulter), CD14 (RMO52, Beckman Coulter), CD15 (HI98, BioLegend), CD16 (3G8, Beckman Coulter), CD20 (2H7, BioLegend), CD41 (SZ22, Beckman Coulter), CD56 (C218, Beckman Coulter), CD203c (NP4D6, BioLegend), CD235a (KC16, Beckman Coulter), BDCA2 (AC144, Miltenyi Biotec). Secondary staining was done with goat anti-mouse IgG Alexa Fluor 488 (MolecularProbes). Afterwards, immunomagnetic depletion was performed using anti-mouse IgG beads (magnetic cell sorting [MACS]; Miltenyi Biotec, Bergisch Gladbach, Germany). For the positive identification of HSCs, the depleted cellular fraction was subsequently stained with CD34 (8G12, BD Biosciences), CD38 (HIT2, Biolegend) and CD90 (5E10, Biolegend) and CD45 (HI30, BD Biosciences).

(24) MSCs were sorted on a FACS Aria (BD Biosciences) to a purity>98% as CD45.sup.lowCD271.sup.+ cells using the antibodies CD45 (HI30, BD Biosciences) and CD271 (ME20.4-1.H4, BD Biosciences).

(25) For purification of 10.sup.3 cells of mouse bone marrow hematopoietic progenitor cells and 10.sup.5 Lin.sup.+ cells, the following FITC-coupled lineage antibodies were used to separate Lin+(lineage positive cells) from Lin− cells (lineage negative cells): Mac1 (M1/70), Gr1 (RB6-8C5), Ter119 (TER-119), CD19 (1D3), B220 (RA3-6B2), CD5 (53-7.3), CD3ε (145-2C11), CD11c (N418), CD4 (GK1.5), CD8 (53-6.7), and NK1.1 (PK136). For the positive identification of HSCs, the Lin− cell population was stained for antibodies against c-Kit (2B8), Sca1 (D7), CD150 (TC15-12F12.2) and CD48 (HM48-1). Antibodies were obtained from eBioscience, San Diego, Calif. Afterwards, separated cells were screened for purity. Cells were purified to a purity of >98% on an LSRII flow cytometer (Aria II, BD Biosciences). Instrument settings for sorting of human CD34+ HSC on a FACS Aria machine were for example: laser delay: blue: 0.00 red: −34.41 violet: 36.22

(26) area scaling: blue: 1.38 red: 1.24 violet: 1.23

(27) FSC area scaling: 1.07

(28) TABLE-US-00003 Parameters Type Voltage Log FSC A, H, W 230 Off SSC A, H, W 280 Off FITC A 500 On PE A 480 On PE-Texas Red A 500 On PerCP-Cy5-5 A 550 On PE-Cy7 A 600 On APC A 580 On APC-Cy7 A 580 On Violet1 A 440 On Violet2 A 604 On Threshold Parameters Threshold FSC 5 Fluorochromes - % Spectral Overlap Fluorochrome Value(%) PE - FITC 48.00 PE-Texas Red - FITC 11.2 PerCP-Cy5-5 - FITC 3.40 PE-Cy7 - FITC 0.50 APC - FITC 0.30 APC-Cy7 - FITC 0.20 Violet1 - FITC 1.00 Violet2 - FITC 12.00 FITC - PE 0.60 PE-Texas Red - PE 21.00 PerCP-Cy5-5 - PE 7.30 PE-Cy7 - PE 0.90 APC - PE 0.05 APC-Cy7 - PE 0.25 Violet1 - PE 0.00 Violet2 - PE 68.50 FITC - PE-Texas Red 0.00 PE - PE-Texas Red 17.50 PerCP-Cy5-5 - PE-Texas Red 59.00 PE-Cy7 - PE-Texas Red 7.90 APC - PE-Texas Red 2.20 APC-Cy7 - PE-Texas Red 0.10 Violet1 - PE-Texas Red 0.00 Violet2 - PE-Texas Red 50.00 FITC - PerCP-Cy5-5 0.00 PE - PerCP-Cy5-5 2.50 PE-Texas Red - PerCP-Cy5-5 17.80 PE-Cy7 - PerCP-Cy5-5 24.00 APC - PerCP-Cy5-5 18.70 APC-Cy7 - PerCP-Cy5-5 5.60 Violet1 - PerCP-Cy5-5 0.10 Violet2 - PerCP-Cy5-5 1.40 FITC - PE-Cy7 0.18 PE - PE-Cy7 7.00 PE-Texas Red - PE-Cy7 1.40 PerCP-Cy5-5 - PE-Cy7 0.90 APC - PE-Cy7 0.30 APC-Cy7 - PE-Cy7 19.00 Violet1 - PE-Cy7 0.10 Violet2 - PE-Cy7 0.45 FITC - APC 0.00 PE - APC 0.00 PE-Texas Red - APC 0.00 PerCP-Cy5-5 - APC 0.40 PE-Cy7 - APC 0.00 APC-Cy7 - APC 9.20 Violet1 - APC 0.00 Violet2 - APC 0.00 FITC - APC-Cy7 0.20 PE - APC-Cy7 0.40 PE-Texas Red - APC-Cy7 0.40 PerCP-Cy5-5 - APC-Cy7 0.40 PE-Cy7 - APC-Cy7 0.60 APC - APC-Cy7 13.90 Violet1 - APC-Cy7 0.20 Violet2 - APC-Cy7 0.20 FITC - Violet1 0.10 PE - Violet1 0.10 PE-Texas Red - Violet1 0.20 PerCP-Cy5-5 - Violet1 0.00 PE-Cy7 - Violet1 0.00 APC - Violet1 0.20 APC-Cy7 - Violet1 0.15 Violet2 - Violet1 1.90 FITC - Violet2 0.00 PE - Violet2 0.50 PE-Texas Red - Violet2 1.00 PerCP-Cy5-5 - Violet2 1.30 PE-Cy7 - Violet2 1.00 APC - Violet2 0.40 APC-Cy7 - Violet2 0.40 Violet1 - Violet2 3.40

(29) Hoechst Staining

(30) Human PBMC were resuspended in SP buffer (HBSS, 2% FCS, 2 mM HEPES buffer; GIBCO, Life Technologies), prewarmed to 37° C. and incubated with Hoechst 33342 (Molecular Probes, Life Technologies) at 5 μg/ml for 2 h at 37° C. All subsequent steps were carried out on ice. Cells were stained with antibodies against lineage markers as described above. 7-AAD (5 μg/ml, Calbiochem) was added for live-dead cell discrimination. As negative control, PBMCs were preincubated with verapamil (100 μM, Sigma Aldrich). Cells showing a dim staining in the Hoechst blue (450/50 nm band pass filter) and Hoechst red (660/20 nm) channels were sorted to a purity >98% on a FACS ARIA (BD Biosciences).

(31) Mtb DNA Detection

(32) Because DNA extraction using TRIzol has been reported to recover low amounts of pure mycobacterial DNA (Hosek et al., 2006), the DNA extraction procedure first described by van Soolingen et al. (van Soolingen et al., 1991) was used.

(33) The extracted DNA was tested for the presence of nucleotide sequence(s) of Mycobacterium tuberculosis by real-time TaqMan PCR. For this purpose primers were used that either detect both the nucleotide sequences encoding IS6110 and MPB64, or detect IS6110 or MPB64 alone. In detail, 50 ng (pHSCs)—1 μg (Lin.sup.+ and lung cells) of DNA was analyzed by real-time TaqMan® PCR using gene-specific probes targeting sequences of Mtb, namely MPB64 and IS6110 together (Path-M. tuberculosis_MPB64/IS6110, Integrated Science). Each sample was assayed in technical triplicates. TaqMan probes for GAPDH were used as endogenous controls for eukaryotic cells (Human: Hs99999905_m1, Mouse: Mm99999915_g1, Invitrogen). H37Rv DNA was used to construct a standard curve for MPB64 and IS6110. The PCR product was detected as an increase in fluorescence with the ABI PRISM 7700 instrument. DNA was quantified using the SDS software, version 2.2.2.

(34) In addition, 50 ng (pHSCs)—1 μg (Lin.sup.+ and lung cells) of DNA was analyzed by quantitative PCR using the primers with SEQ ID NO: 31 and SEQ ID NO: 32 targeting MPB64 alone (543-bp DNA fragment) (Young J S et al, 2005), using the SYBR green system of detection. Primers targeting human GAPDH (SEQ ID NOs: 33 and 34) and mouse GAPDH (SEQ ID NOs 35 and 36) were used as endogenous controls for eukaryotic cells. Again, H37Rv DNA was used to construct a standard curve for primers used.

(35) PCR products were detected as an increase in fluorescence with the ABI PRISM 7700 instrument. DNA was quantified using the SDS software, version 2.2.2.

(36) In order to reduce amplification backgrounds with primers, as well as to deal with a possible contamination in the PCR master mix, quantitative PCR analyses using a “no template=water control” were also performed for every run. Exponential amplification in the “water control” with Ct values of 51-55 was taken as the limit for Mtb not present in the assay in PCRs targeting IS6110 and MPB64 together. In PCRs targeting MPB64 alone, “water control” Ct values of 48-50 was taken as the detection limit. To ensure that this background did not result from a contamination by genomic DNA, i.e. the amplification of a Mtb specific DNA fragment, the MPB64 qPCR product was analyzed by gel electrophoresis. While the expected PCR product size was detectable in the Mtb.sup.+ samples, such a distinct PCR band were not found in the “water control”. In PCRs targeting IS6110 together with MPB64, samples were evaluated as positive for Mtb with a Ct value of 48 (equivalent to 1 Mtb DNA copy) or lower (equivalent to several Mtb DNA copies). In PCR tests targeting MPB64 alone, samples were considered as Mtb positive with a Ct of 39-40 (equivalent to 1 Mtb DNA copy) or lower (equivalent to several Mtb DNA copies).

(37) For serial and limiting dilution analyses on DNA the primers SEQ ID NO: 5 and SEQ ID NO: 6 were used to amplify a 245-bp DNA fragment encoded by the IS6110 insertion sequence in the Mtb genome (Thierry et al., 1990). At the point where the PCR signal was lost in the serial dilutions, limiting dilution analyses were performed.

(38) Mtb-specific and BCG-specific DNA was also detected using primers previously described (Zumárraga et al., 1999). PCR reactions were performed in a thermal cycler at 95° C. for 15 min, followed by 50 cycles at 95° C. for 30 s, 45 s at different annealing temperatures and 45 s at 72° C. (DNA Engine® PTC2000, Biozym DiagnosticRad). For every reaction uninfected DNA and DNA from H37Rv Mtb were included. PCR products were analyzed by electrophoresis on 2% agarose gels.

(39) Subsequent to the DNA extraction (van Soolingen et al., 1911), the primers SEQ ID NO: 5 and SEQ ID NO: 6 were used to amplify a 245-bp DNA fragment encoded by the IS6110 insertion sequence in the Mtb genome from purified genomic DNA (Thierry et al., 1990). Amplification of GAPDH was chosen as reference gene using the primers SEQ ID NO: 35 and SEQ ID NO: 36 for mouse and SEQ ID NO: 33 and SEQ ID NO: 34 for human. Mtb-specific DNA was also detected by the amplification of an 1135-bp DNA fragment using the primers SEQ ID NO: 7 and SEQ ID NO: 8 as well as a 969-bp DNA fragment using the primers SEQ ID NO: 9 and SEQ ID NO: 10 both detecting an Mtb-specific 12.7-kb genomic sequence. BCG-specific DNA was detected by the amplification of a 2198-bp DNA fragment using the primers SEQ ID NO: 11 and SEQ ID NO: 12.

(40) All described, amplification reactions were performed in a total volume of 25 μl containing 10-50 ng of DNA, 1×Taq polymerase buffer with KCl supplemented with 0.2 mM dNTPMix, 1.5 mM MgCl.sub.2, 1 μM of each primer and 5 U Taq DNA-Polymerase (Thermo Fisher Scientific). PCR reactions were performed in a thermal cycler (DNA Engine® PTC2000, Biozym DiagnosticRad). The mixture was first denatured at 95° C. for 15 min. Then, 50 cycles of PCR were performed with denaturation at 95° C. for 30 s, primer annealing for 45 s at different annealing temperatures and primer extension for 45 s at 72° C. At the end of the last cycle, the mixture was incubated at 72° C. for 10 min. For every reaction a negative control, in which uninfected DNA template was used, and a positive control, containing DNA from H37Rv Mtb were included. PCR products were analyzed by electrophoresis on 2% agarose gels followed by staining with ethidium bromide (10 mg/ml). Limiting dilution analyses were performed at the point where the PCR signal was lost in the serial dilutions.

(41) Colony-Forming Units

(42) Mice were sacrificed at time points described, and organs (spleen, thymus, lung and bone marrow) were aseptically removed and homogenized in 1 ml PBS containing 0.05% Tween 80 (v/v). For pulmonary CFU determination, the lung was removed and incubated in 1 mg/ml collagenase type VIII (Sigma-Aldrich) and 30 μg/ml DNase I (Roche) at 37° C. for 30 min. Thereafter, one half of each of the lung homogenate, the spleen, the thymus and 10.sup.5 bone marrow cells were diluted in PBS containing 0.05% v/v Tween 80 and plated onto Middlebrook 7H11 agar plates supplemented with Middlebrook OADC Enrichment (Dibco). In addition, purified populations of human peripheral blood pHSCs as well as mouse bone marrow hematopoietic progenitor cells were plated. CFUs were enumerated after 4-6 weeks of incubation at 37° C. and 5% CO.sub.2.

(43) RNA/qRT-PCR

(44) Cells were homogenized in TRIzol (Invitrogen) and RNA was isolated via chloroform extraction (Life Technologies), treated with ethanol and dissolved in RNase-free water. RNA fromMtb infected THP-1 cells was isolated as previously described (Dietrich et at, 2000; Rienskma et at, 2015). One hundred ng of total RNA was reverse-transcribed by SuperScript III (Invitrogen) primed with oligodT. The cDNA for the specific target assays was then amplified by pre-amplification reaction using pooled gene-specific primers according to the manufacturer's protocol (Invitrogen). The pre-amplification product was diluted (1:20) and finally analyzed by real-time TaqMan® PCR using the following TaqMan probes: DosR, c-lat, hspX and SigA (Design Batch ID: w1406535517000, order number: 2106064SO, Invitrogen). DosR, c-lat and hspX RNA abundances were normalized to SigA as endogenous controls for Mtb. Each sample was assayed in triplicate. H37Rv DNA was used to construct a standard curve for all inspected genes. The PCR product was detected as an increase in fluorescence with the ABI PRISM 7700 instrument. RNA was quantified using the SDS software, version 2.2.2.

(45) Cytology

(46) Cells were fixed in PBS containing 4% w/v PFA for 24 h at 4° C. Thereafter, cells were immobilized by cytospin on a solid support (Shandon Centrifuge, Modell Cytospin 3). Slides were flooded with auramine-rhodamine for 15 min, fluorescent decolorizer for 2-3 min and potassium permanganate for 3-4 min. A 2-μg/ml working solution of DAPI was used for nuclear visualization.

(47) For immunocytochemistry staining cells were permeabilized with 0.05% Saponin/PBS/1% BSA for 10 min at RT, washed twice with 0.05% Saponin/PBS/1% BSA and labelled with the primary antibody rabbit anti-Mtb (Cat. No. ab905; Abcam, UK), rat anti-LAMP2 (Cat. No. MA5-17861, ThermoFischer Scientific, Germany) and mouse anti-EEA1 (Cat. No. ab70521, Abcam, UK). Rabbit IgG antibody (Cat. No. ab172730; Abcam, UK), rat IgG1 antibody (Cat. No. MA1-90035; ThermoFisher Scientific, Germaniy) and mouse IgG1 antibody (Cat. No. ab91353; Abcarn, UK) were used as isotype controls. Primary antibodies were detected with Alexa Fluor 555 conjugated goat anti-rabbit IgG antibody (Cat. No. A-21428; ThermoFisher Scientific, Germany), Alexa Fluor 647 conjugated goat anti-rat IgG antibody (Cat. No. A-21247; ThermoFischer Scientific, Germany) and a biotin conjugated goat anti-mouse IgG antibody (Cat. No. 31 803, ThermoFisher Scientific, Germany). The biotin conjugated goat anti-mouse IgG antibody was detected by an Alexa Flour 488 conjugated streptavidin antibody (Cat. No. S11223, ThermoFisher Scientifics, Germany). A 2-μg/ml working solution of DAPI was used for nuclear visualization.

(48) Cover slips were mounted in ProLong Gold anti-fade reagent (Cat. No. P36934; Invitrogen, USA) and sealed using adhesives. Slides were screened with either 100× (for images) objectives under oil immersion using a fluorescence microscope (DMRB Fluorescence Microscope, Leica Microsystems) or with confocal microscopy using a Zeiss LSM710 with a 100×/0.8 numerical aperture objective lens under oil immersion. Analyses using the fluorescence microscope were done using ProGres Capture Pro 2.8.8. (Optical Systems, Jenoptick AG). For confocal microscopy image acquisition was performed using Zen 2010 Version 6.0 and images were analyzed by Zen 2012 Light Edition software (Carl Zeiss Microlmaging). For each sample at least 10,000 cells were analyzed.

(49) Histology

(50) For histology, lung caudal lobes were preserved in PBS containing 4% w/v PFA for 24 h at 4° C. and subsequently embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Slides were screened with 5× objectives and verified using a light microscope (Leica DMLB, Leica Microsystems). Analyses were done using ProGres Capture Pro 2.8.8. (Optical Systems, Jenoptick AG).

(51) Intratracheal Administration of Mtb-Infected pHSCs

(52) For challenge, 8- to 10-week-old female Rag2.sup.−/−Il2rg.sup.−/− mice were anesthetized by i.p. administration of ketamine (50 mg/kg) and Rompun (5 mg/kg; Bayer). Thereafter, using a micropipette, 100 CFU of Mtb H37Rv, 1,000 pHSCs of uninfected and Mtb-infected mice at day 28 p.i., or 1,000 Lin.sup.+ cells and pHSCs of human IGRA.sup.+ as well as IGRA donors diluted in 50 μl of sterile PBS were gently placed in the trachea of each mouse. Mice were sacrificed 3 weeks post-transfer and lungs were analyzed for Mtb-specific DNA, CFUs and histologically.

(53) Statistics

(54) For all statistical analyses, PRISM (Version 6, GraphPad, San Diego) software was used. Dispersion is presented as the median+interquartile, unless stated otherwise. Statistical analysis was performed with Mann-Whitney two-tailed test. P values <0.05 were considered significant.

(55) Human Peripheral Blood SP.sup.+ and Lin.sup.−CD34.sup.+CD90.sup.+CD38.sup.lo pHSCs of IGRA.sup.+ Donors Carry Mtb DNA

(56) In order to reveal in which blood cell population Mycobacterium tuberculosis (Mtb or Mtb) is present in a latently infected Mtb subject, blood samples obtained from donors with a latent tuberculosis infection (LTBI) were analyzed for the presence of Mtb. These blood samples were enriched for different blood cell populations, e.g. employing FACS. These blood cell populations were tested for the presence of Mtb. In the following, the detection of Mtb is exemplified by determining at least one nucleotide sequence of Mtb. However, the direct detection of Mtb can also be performed by identifying a polypeptide of Mtb, e.g. IS6110 and/or MPB64.

(57) As an exemplary blood cell population, pluripotent hematopoietic stem cells (pHSCs) of donors with LTBI were analyzed. In order to purify a blood cell population enriched for pHSCs, progenitor cells characterized by Lin.sup.−CD34.sup.+ were enriched by FACS from blood of IGRA.sup.+ donors (subjects with a positive IGRA test) and IGRA donors (subjects with a negative IGRA test) (Table 2). In addition, Lin.sup.−CD34.sup.+CD90.sup.+CD38.sup.− pHSCs (FIG. 7) were purified by FACS from blood and analyzed. Furthermore, Lin.sup.+ cells, e.g. CD1c.sup.+ dendritic cells, CD14+CD16.sup.low and CD141.sup.lowCD16.sup.+ monocytes, CD15.sup.+ granulocytes, CD4.sup.+ or CD8.sup.+ T cells, CD19.sup.+ B cells and CD56.sup.+ NK cells were purified by FACS from blood and analyzed.

(58) Further, pHSCs are highly enriched in the side population (SP) cells (Goodell, 1994; Lin and Goddell, 2006). Therefore, pHSCs were enriched by their drug efflux properties as Hoechst low/negative SP phenotype cells (FIG. 7). In order to determine the at least one nucleotide sequence of Mtb, DNA was isolated from these blood cell populations. DNA of all these potentially Mtb-infected cells was used to PCR-amplify DNA fragments of Mtb-encoded sequences and BCG-encoded sequences to identify possible remnants of a BCG-vaccination (FIG. 1A-E) (Thierry et al., 1990, Zumárraga et al., 1999).

(59) Nucleotides (MPB64, IS6110, 12 kb fragment) of Mtb were selectively detected in ˜1×10.sup.3 Lin−CD34+ as well as SP+ blood cells (Donors 8 and 9) of eight out of eight IGRA.sup.+ donors, while Lin+ cells remained free from Mtb nucleotides (FIGS. 1A and D). DNA of a BCG vaccination was not present in the blood cells obtained from these subjects (Donors 8-13; FIG. 1B). Quantitation of Mtb-specific DNA was done by real-time TaqMan® PCR targeting two Mtb-specific genes, the single copy MPB64 and the multiple copy IS6110 sequence (FIGS. 1A and E), as well as in serial and limiting dilution PCRs targeting IS6110 alone (FIG. 1C). In SP.sup.+ and Lin.sup.−CD34.sup.+ pHSCs from IGRA.sup.+ donors, between seven and twenty copies of Mtb-specific DNA was determined within lysates of 10.sup.3 cells (FIGS. 1A, C and E; table 3). Since these PCR tests detect multiple IS6110 elements in a single Mtb genome (Brosch R G et al., 2000; Fomukong N G et al., 1994; Lok K H et al., 2002; Steensels D et al., 2012), Mtb-specific DNA was also quantitated using primers that target the single copy MPB64 alone. Thereby, we detected between one and seven copies ofMtb DNA within lysates of 10.sup.3 cells (FIG. 1D). In the genome of individual IGRA.sup.+ Mtb.sup.+ donors two to 10-fold higher IS6110 copy numbers were detected than the one MPB64 copy.

(60) In contrast, none of the IGRA donors contained detectable Mtb DNA in any of the Lin.sup.− and Lin.sup.+ blood cells tested by IS6110-MPB64 double-target or MPB64 single-target PCR (FIGS. 1A and D).

(61) From two IGRA.sup.+ donors, CD34.sup.+ progenitors were further FACS-purified as Lin.sup.− CD34.sup.+CD90.sup.+CD38.sup.lo pHSCs (FIG. 7) (Majeti et al., 2007). 10.sup.3 of Lin.sup.−CD34.sup.+CD90.sup.+CD38.sup.lo pHSCs contained between 9 and 14 Mtb DNA copies in the double-target PCR, and between one and five in the MPB64 single-target PCR, while 10.sup.3 of the more differentiated Lin.sup.−CD34.sup.+CD38+CD90 cells contained none (FIG. 1E).

(62) To the contrary, 10.sup.3 of the pool of Lin.sup.+ cells of IGRA.sup.+ donors, as well as FACS-purified dendritic cells, monocytes, granulocytes, T cells, B cells and NK cells, revealed no detectable DNA in all of these PCR assays (FIGS. 1A and D).

(63) Furthermore, Lin-CD45+CD34.sup.+ as well as Lin.sup.−CD34.sup.+CD90.sup.+CD38.sup.−CD45.sup.+ long-term HSCs and for control purposes CD34.sup.+CD90.sup.−CD38.sup.+CD45.sup.+ short-term were sorted on a FACS Aria (BD Biosciences) to a purity>98% (FIG. 1G). Moreover, MSCs purified from peripheral blood were sorted on a FACS Aria (BD Biosciences) to a purity >98% as CD45.sup.loCD271.sup.+ cells using the following antibodies (FIG. 1H): CD45 (HI30, BD Biosciences) and CD271 (ME20.4-1.H4, BD Biosciences). The cell population enriched for CD271.sup.+CD45.sup.low from the peripheral blood of 2 (Donors 10 and 13) out of 8 latently infected subjects was also tested positive for Mtb DNA by qPCR targeting MPB64 and IS6110 together with 1-20 Mtb DNA copies. DNA of Mtb was detected in 10.sup.3 peripheral MSCs (FIG. 1I).

(64) In conclusion, it was surprisingly found that Mtb was detected in a blood cell population of IGRA.sup.+ donors. In particular, the presence of Mtb was demonstrated in a blood cell population enriched for human peripheral Lin.sup.−SP.sup.+ or a blood cell population enriched for CD34.sup.+ cells. Among these blood cells, the LinCD34.sup.+CD90.sup.+CD38.sup.lo pHSCs selectively carried Mtb DNA, while their peripheral Lin.sup.+ cells were consistently free of Mtb.

(65) Replication-competent, active Mtb can form colonies on agar. Thus, the different cell populations from 3 IGRA.sup.+ donors were tested for growth measured by enumerating colony-forming units. Only 1 CFU was formed from lysates of 10.sup.3 Lin.sup.−CD34.sup.+ and Lin.sup.− CD34.sup.+CD90.sup.+CD38.sup.− pHSCs isolated from 2 donors, which, in the two PCR assays, contained between 9 and 14, respectively between one and five, Mtb DNA copies (FIGS. 1A-F and 4). Thus, around one of 4 Mtb DNA copies detected in Lin.sup.−CD34.sup.+ and Lin.sup.−CD34.sup.+CD90.sup.+CD38.sup.− cells were replication-competent, while around 3 of 4 were not in quiescent hematopoietic cells.

(66) TABLE-US-00004 TABLE 2 Patient characteristics # Age Sex IGRA (IU/ml) Comorbidities Medication 1 22 m neg Psoriasis — 2 53 f neg — — 3 38 m neg — — 4 33 m neg allerg RC — 5 45 w neg — — 6 38 w neg — — 7 41 f neg — — 8 29 m 0.87 Psoriasis — 9 43 m 1.8 Psoriasis — 10 27 w 10 Psoriasis — 11 57 m 3.64 — — 12 55 m 4.2 chronic Urticaria Antihistamines 13 37 f 0.65 Eczema — 14 46 f 2.71 — — 15 48 m 9 — — Tablenotes: #: patient number; Sex - m: male; Sex - f: female; neg: negative; allerg RC: allergic rhinoconjunctivitis

(67) A Portion of all CD150.sup.+ LT-pHSCs, but Neither ST-pHSCs Nor Multipotent Progenitors (MPPs) in Bone Marrow of Mtb-Infected Mice Harbour Mtb DNA

(68) Next, mice were infected with Mtb to analyze whether bone marrow-derived LT-pHSCs could become carriers of the bacterium too. A mouse model of intradermal ear infection was employed, in which low numbers of systemic Mtb persist without developing active TB (Reece et al., 2010). A variety of organs, such as the lung, and the spleen, but not the thymus, and hematopoietic cells in them were found to be infected 28 days post-infection (p.i.).

(69) DNA extracted from 10.sup.5 lung cells contained between one and ten copies of Mtb DNA (FIGS. 2A and C).

(70) 10.sup.3 FACS-purified Lin.sup.−CD150.sup.+CD48.sup.− LT-pHSCs (FIG. 7B) (Yilmaz et al., 2006) were found present between 40 and 100 copies of Mtb DNA, as detected by double-target real-time TaqMan® PCR and in serial and limiting dilution analyses targeting IS6110 alone (FIG. 2A-B). In MPB64 single-target PCRs, LT-pHSCs were found to harbor between five and 90 copies of Mtb DNA (FIG. 2C). By contrast, in 10.sup.3 Lin.sup.− Sca1.sup.+ c-Kit.sup.+CD150.sup.+CD48.sup.+ ST-pHSCs and Lin.sup.− Sca1.sup.+ c-Kit.sup.+CD150.sup.−CD48.sup.+ MPPs (FIG. 7B) (Yilmaz et al., 2006), no Mtb DNA could be found in all of these PCR analyses (FIGS. 2A and C).

(71) Furthermore, no Mtb DNA was found in 10.sup.5 FACS-enriched Mac1.sup.+ macrophages, NK1.1.sup.+ NK cells, and CD4.sup.+ as well as CD8.sup.+ T cells (FIG. 2A and FIG. 7C), while between 8 and 60 copies of Mtb DNA were found in double- and single-target PCR analyses of lysates of 10.sup.5 FACS-enriched CD11c.sup.+ dendritic cells, Gr1.sup.+ granulocytes and CD19.sup.+ B cells, i.e. 10 to 100-fold lower numbers of Mtb DNA copies than in LT-pHSCs (FIG. 2A-C).

(72) Consequently, the intradermal infection of mice generated Mtb-infected LT-pHSCs in bone marrow and blood 28 days p.i., and that this infection, like in human LTBI, only affected the LT-pHSCs, but not the ST-pHSC or MPP subpopulations of early hematopoietic progenitors. Nevertheless, the mouse infection model at day 28 p.i. seems also to have limitations. The pathophysiological status of latent infections in LTBI may not entirely be reflected correctly because the lung, and more mature Lin.sup.+ cells in spleen and bone marrow of mice are infected, as seen in our Mtb DNA analyses, but, at least in blood, are not infected in LTBI. However, these infection experiments with mice demonstrate that LT-pHSCs become infected, selectively over ST-pHSCs and MPPs.

(73) Numbers of Replication-Competent, Colony-Forming Mtb in Mtb-Infected Mouse Cells Different Mtb− DNA.sup.+ cell populations were tested for replication-competent, active Mtb measured by enumerating CFUs (FIG. 2D-F). The vast majority, if not all, of the Mtb bacteria detected by PCR in 10.sup.5 lung cells (FIGS. 2A, C, D and 4), as well as in 10.sup.5 FACS-enriched CD11c.sup.+ dendritic cells, Gr1.sup.+ granulocytes and CD19 B-lineage cells in bone marrow (FIGS. 2A, C, D and 4) produced CFUs. Hence, most Mtb bacteria inside these cells were replication-competent. These results also document, that the two assays, for Mtb DNA and for CFUs of active Mtb, detect comparable numbers of bacteria.

(74) By contrast, only 10 CFUs were formed from lysates of 10.sup.3 LT-pHSCs isolated from Mtb-infected mice, which, in MPB64 PCR assays, contained between 5 and 90 Mtb DNA copies (FIGS. 2A, C, E and 4). Thus, up to 80% of Mtb DNA in LT-pHSCs did not form a CFU, therefore, were not replication-competent.

(75) These results may indicate that not every cell of the LT-pHSC pool is infected by Mtb. However, all the infected cells were in a quiescent stage.

(76) Next we directly visualized LT-pHSCs carrying Mtb by rhodamine-auramin staining (Ellis and Zabrowarny, 1993), or with Mtb-specific fluorescent antibodies. In LT-pHSCs, Mtb was readily detectable, whereas ST-pHSCs and MPPs never showed a positive staining (FIG. 3). Approximately one to six of ˜10.sup.3 analyzed LT-pHSCs stained positive for Mtb (FIG. 3). As a control Mtb-infected lung cells were analyzed. In these cells, the number of Mtb positive cells as revealed by rhodamine-auramin staining, qPCR and CFU were showing a good correlation (FIGS. 3 and 4).

(77) Next, the subcellular localization or organelle association of Mtb was attempted to identify in these types of cells, e.g. phagolysosomal structures. Thus, LT-pHSCs and lung cells were stained with fluorescent antibodies against Mtb, the early endosome antigen 1 (EEA1) and the lysosome associated membrane protein 2 (LAMP2). EEA1 is recruited during phagosome maturation, thus, is essential for the phagocytosis of large particles (Araki et al., 1996). LAMP2 is reported to be enriched in late endosomes, late phagosomes and lysosoms. Without LAMP proteins phagolysosomes were shown to lose their microbicidal activity (Binker et al., 2007). Using confocal microscopy, we found that in LT-pHSCs Mtb (in red) was co-localized to the endosomal protein EEA1 (in green) in the cytoplasm of infected pHSCs. However, explicit endosomal or phagolysosomal structures were not detectable and LAMP2 (in blue) could hardly be seen (FIG. 3). In contrast, in infected cells of the lung such structures were identifiable (FIG. 3). Nevertheless, it is known that Mtb do interfere with phagolysosome maturation to avoid degradation (Hasan et al., 1997).

(78) In conclusion, Mtb in LT-pHSCs in mouse bone marrow can be detected by histology. Subcellular localization or organelle association could not be identified, since the pHSC lacked such intracellular structures.

(79) However, the numbers of stained cells are much lower than even the number of replication-competent Mtb. Thus, these stainings are not as sensitive as PCR reactions to allow a quantitative prediction of the actual number of LT-pHSCs harbouring Mtb (Seiler et al., 2003).

(80) Mtb Residing within Human CD34.sup.+ as Well as Mouse CD150.sup.+ pHSCs Express Genes of the Dormancy Regulon

(81) Since 10.sup.3 mouse LT-pHSCs were found to contain between 40 and 100 copies of Mtb genomes, but generated only 10 CFUs (FIGS. 2C, F and 4), it can be concluded that the vast majority, i.e. around 80%, of Mtb bacterial genomes persist in a non-replicating, possibly dormant form. Dormancy of Mtb is induced under hypoxic conditions and is reflected in a change of gene expression (Leistikov et al., 2009; Deb et al., 2009; Jakob and Reichmann 2013). It is controlled by the dormancy regulon and involves transcription of approximately 50 so-called dormancy genes, among themDosR, c-lat and hspX(Sherman et al., 2001). SigA is expressed in non-replicating as well as in replicating, CFU-forming Mtb and thus, can be used as a housekeeping gene (Manganelli et al., 1999). Therefore, it may be hypothesized that the hypoxic niche of LT-pHSCs could induce dormancy of Mtb (Simsek et al., 2010; Parmar et al., 2007). To test for this hypothesis, quantitative RNA expression analyses for Mtb dormancy genes were performed in mouse LT-pHSCs as well as in Mtb-infected lung cells 28 days p.i., in which the vast majority of Mtb organisms form CFUs (FIG. 4).

(82) SigA was detected in both cell types. By contrast, Mtb DNA.sup.+ LT-pHSCs did express DosR, c-lat and hspX RNA, while Mtb from infected lung cells did not express dormancy genes (FIG. 5A). Therefore, it can be concluded that Mtb resides within mouse CD150.sup.+ LT-pHSCs in a non-replicating state, expressing dormancy genes.

(83) In human Mtb-infected DNA.sup.+ SP.sup.+ and Lin.sup.−CD34.sup.+ pHSCs, the SigA expression was detected as well in addition to the the expression of the dormancy regulator genes, DosR, c-lat and hspX, while replicating Mtb isolated from an infected human monocytic leukemia cell line did not express these genes (FIG. 5B).

(84) Consequently, these results document that pHSCs act as an intracellular niche for stressed or dormant non-replicating Mtb in mice and humans.

(85) Intratracheal Transfer of Mtb-Infected Human and Murine pHSCs Leads to Resuscitation of Active TB in Lungs of Transplanted Hosts

(86) The capacity of Mtb-infected pHSCs of human LTBI and of mice 28 days p.i. was analyzed to resuscitate active infection upon intratracheal application into the trachea of Rag2.sup.−/−Il2rg.sup.−/− mice (Woolhiser et al., 2007). Lin.sup.−CD34.sup.+ and Lin.sup.+ cells from blood of latently infected, IGRA.sup.+, Mtb DNA.sup.+ human donors and of IGRA donors (FIGS. 1A and D), Lin.sup.− CD150.sup.+CD48.sup.− LT-pHSCs from bone marrow of infected mice (FIGS. 2A and C), and pure Mtb as control, were administered into the trachea of Rag2.sup.−/−Il2rg.sup.−/− mice and analyzed 1 and 3 weeks later (FIG. 6A). Mtb DNA as well as active, replicating Mtb CFUs were detected in the lungs, spleen, thymus, and bone marrow of recipient mice of pHSCs from IGRA.sup.+ donors and infected mice, but not in recipients of Lin.sup.+ cells from IGRA.sup.+ donors and of cells from IGRA donors (FIG. 6B-C). In histological sections of lungs of the Rag.sup.−/−Il2rg.sup.−/− mice transplanted with either human pHSCs from LTBI or mouse pHSCs from Mtb-infected mice, early granuloma formation became detectable 1 week after pHSC transfer and appeared intensivied 3 weeks after the transfer (FIG. 6D). Transfer of Lin.sup.t cells from IGRA.sup.+ donors and cells from IGRA donors did not induce histological changes in the lung of recipients.

(87) This lung pathology of infection might result from the action of only the bacteria which are replication-competent or from both, the replication competent and stressed, dormant bacteria. In the latter case, stressed dormant Mtb could be resuscitated to active replicating bacteria. In any case, it can be concluded that Mtb-infected human and mouse pHSCs can serve as cellular source that cause TB after intratracheal transfer of these pHSCs.

EXAMPLE 3

(88) Nucleotide sequences of Mtb were not determined in whole blood cells after osmotic red blood cell lysis and peripheral blood mononuclear cells (PBMCs), which were not further enriched, of subjects suffering from latent tuberculosis infection

(89) TABLE-US-00005 TABLE 4 Samples of whole blood cells after osmotic red blood cell lysis to obtain leukocytes that were obtained from patients suffering from latent tuberculosis infection and that were not further enriched failed to show nucleotide sequences of Mtb BD ProbeTec ® COBAS ® Patient Patient Quantiferon (MOTA TaqMan ® Result Nr Nr TB Gold Test values) MTB Test of test a 9 positive 0 negative b 10 positive negative negative c 11 positive negative negative d 12 positive negative negative e 15 positive 0 negative f positive 49 negative g positive 82 negative

(90) The same patients as of the above Example or as described in Tornack J, Reece S T, Bauer W M et al., PLoS One 2017; 12: e0169119 were also tested for the presence of Mtb in their blood to rule out any active infection. Besides culture, this included nucleic acid amplification tests (NAATs). In two independent laboratories, two different tests were used: the BD® ProbeTec ET DTB® (Becton-Dickinson (Little M C, Clin Chem 1999; 45: 777-84))—and the COBAS® TaqMan® MTB (Roche) test (Antonenka U, BMC Infect Dis 2013; 13: 280). Sample preparation in both tests included the osmotic lysis of red blood cells to obtain leukocytes. The BD® ProbeTec® Mtb test is a strand displacement test targeting a 95 bp region of the IS6110 sequence of Mtb, whereas the COBAS® TaqMan® MTB test uses a classical PCR reaction to detect a genus specific sequence within the gene for 16S rRNA. Results in both tests were shown to be comparable when using respiratory secrets of actively infected patients (i.e. patients with active pulmonary TB) as a control (Antonenka U, BMC Infect Dis 2013; 13: 280). In addition to patients number a-e corresponding to patients 9-12 and 15 as described above or in Tornack J, Reece S T, Bauer W M et al., PLoS One 2017; 12: e0169119 that were all tested positive for Mtb in their circulating HSCs, the results of two other patients with LTBI (f and g) are also shown. In the BD® ProbeTec® Mtb test results are depicted in MOTA values, a measurement of the area underneath a relative fluorescent unit curve. The cutoff level for a positive result was a MOTA value of greater than 3400. The box plots of the MOTA levels are shown in FIG. 9.

(91) TABLE-US-00006 TABLE 5 Samples of PBMCs that were not further enriched and that were obtained from patients suffering from latent tuberculosis infection failed to show nucleotide sequences of Mtb Patient Number Target Name C.sub.T C.sub.T Mean C.sub.T SD 101 MPB64/IS6110 Undetermined 101 MPB64/IS6110 Undetermined 101 MPB64/IS6110 Undetermined 101 control 21.20469284 21.20180702 0.024691604 101 control 21.17579842 21.20180702 0.024691604 101 control 21.2249279  21.20180702 0.024691604 102 MPB64/IS6110 Undetermined 102 MPB64/IS6110 Undetermined 102 MPB64/IS6110 Undetermined 102 control 20.5159359  20.49679756 0.032925282 102 control 20.45877838 20.49679756 0.032925282 102 control 20.5156765  20.49679756 0.032925282 103 MPB64/IS6110 57.61778259 57.61778259 103 MPB64/IS6110 Undetermined 57.61778259 103 MPB64/IS6110 Undetermined 57.61778259 103 control 20.51922226 20.5237999 0.143613234 103 control 20.38253021 20.5237999 0.143613234 103 control 20.66964722 20.5237999 0.143613234 c MPB64/IS6110 Undetermined c MPB64/IS6110 Undetermined c MPB64/IS6110 Undetermined c control 20.8352375  20.7854557 0.062206928 c control 20.71572113 20.7854557 0.062206928 c control 20.80541229 20.7854557 0.062206928

(92) Three additional patients with LTBI (Nr. 101-103) and one patient as described above or Tomack J, Reece S T, Bauer W M et al., PLoS One 2017; 12: e0169119 (“c”, same as in table 1) were tested in another PCR reaction using the Mycobacterium tuberculosis complex, targets MPB64 and IS6110 genesig Detection Kit (Integrated Sciences) for the presence of Mtb in their blood. In this experiment PBMC prepared by Ficoll Paque® density gradient centrifugation were used. 200 ng of DNA isolated in the same way as described above were used for the PCR reaction. The target regions amplified in this test include the genes MPB64 and IS6110, and this test was also used for the detection of Mtb in HSC as described above. Depicted are the Ct values of the target regions and of the endogenous control reactions (control). None of the tested patients had a positive result, even though the PCR reaction was extended to 60 cycles. The amplification plots of the PCR reactions are shown in FIG. 10 (A=patient 101, B=patient 102, C=patient 103, D=patient c).

(93) Accordingly, samples of PBMCs or of whole blood cells after osmotic red blood cell lysis, which were not further enriched, did not allow the reliable detection of latent tuberculosis infections. As demonstrated above, the PBMCs that were further enriched for, for example, HSCs, or cells having the cell surface marker(s) CD34 and/or CD90, e.g. by FACS, allowed the reliable detection of nucleotide sequences of Mtb. Such further enriched blood cell populations provide for a reliable detection of subjects suffering from a latent tuberculosis infection.

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