Patent Application
20100255015
The present invention relates to the therapeutic use of actinomycete glycerol acyl derivatives antigens, their process of extraction and their use in the treatment or the prophylaxis of tuberculosis.
Each year, tuberculosis is the estimated cause of 2 million deaths. The causative agent of this disease is a bacterium, Mycobacterium tuberculosis, by which one of every three people is infected worldwide. It is transmitted through the air by sneezes or coughs of infected persons. The bacterium may be harboured in an inactive state by infected persons who will never develop the disease. However, under certain conditions, such as age or depression of the immune system, the bacterium may become active and cause the onset of active tuberculosis.
It seems that the mycobacterial envelope accounts for an important part of the virulence of bacteria of the Mycobacterium genus. Indeed, up to 40% of mycobacteria dry weight is constituted by lipids. These lipids have been shown to be involved in the modulation of the host immune responses. Some lipids are antigens which stimulate unconventional αβT cells in the context of CD1 proteins. The majority of mycobacterial lipid antigens are presented by the CD1b isoform. This is the case for a large panel of structurally very different molecules, such as mycolic acids, glucose monomycolate, sulfoglycolipids and phosphatidyl-myo-inositol mannosides (PIM).
The existence of CD1-restricted T cells specific for several of the most abundant mycobacterial lipids strongly suggests that CD1 proteins are important in the host response to M. tuberculosis.
At present, there are two ways of fighting tuberculosis: antibiotic therapy and vaccination.
The vaccine used for the prophylaxis of tuberculosis consists of a live attenuated bacterium of the Mycobacterium bovis species. It is named BCG, Bacillus of Calmette and Guerin, after the two scientists who first devised it, at the beginning of the 20th century. The different strains of BCG currently used for vaccination are notably described in Behr M. A. et al. Science (1999) 284:1520-1523.
In addition to its flaw as being a live vaccine, which precludes its use in immunodepressed patients, its protection against tuberculosis is controversial. Thus, BCG protective efficacy in adults range from 0% to 80% according to varying studies, besides, it fails to protect against pulmonary tuberculosis, the most prevalent disease form in adults.
Moreover, antibiotic resistant strains of M. tuberculosis have been found in over 35 countries.
Hence, it is a subject of the present invention to provide a more effective way of treating and/or preventing tuberculosis.
It is thus highly desirable to provide further new immunogenic compounds.
The invention notably provides new antigens, which have been shown to stimulate CD1-restricted T lymphocytes in vitro. Said antigens are glycerol-monomycolate derivatives and have been isolated from actinomycetes, such as mycobacteriae.
The characterization of the mycolic acid derivatives has been largely reported (Laval et al., Anal Chem 2001, 73(18):4537-4544; Villeneuve M. et al., Biochim Biophys Acta 2005, 1715(2):71-80; Villeneuve M. et al., Biochim Biophys Acta 2007, 1768(7):1717-1726; Watanabe M. et al., Microbiology 2001, 147(Pt 7):1825-1837).
These compounds are known although their therapeutic use, and this is one object of the present invention, has neither been disclosed nor suggested. Additionally, the corresponding acylated forms and reduced forms thereof as well as the specific (R) enantiomer of such compounds are novel, according to a further object of the present invention.
More particularly, the invention relates to the compounds of the following general formula (I):
wherein:
R is a residue of a α-alkylated, β-hydroxylated carboxylic acid comprising 20 to 90 carbon atoms;
R′ represents a hydrogen atom or acyl group (—C(═O)-Alkyl) where Alkyl comprises 1 to 10 carbon atoms; and
* denotes an asymmetric carbon atom;
and its enantiomers, diastereoisomers, mixtures thereof, and their therapeutic uses.
The asymmetric carbon of the glycerol skeleton (*) preferably exhibits a (R) configuration.
Preferably, R is of formula:
where (I) is attached to R through ----; and
T represents a methyl group or a branched or linear C10-C60 hydrocarbon chain, or a derivative thereof which additionally comprises one or more double bond(s), —(C═O)— group, cyclopropyl and/or epoxy, and/or is additionally substituted with one or more groups chosen from OH, OMethyl;
** denotes an asymmetric carbon;
n1, n2 identical or different, independently represent an integer.
Preferably, n1 is an integer comprised between 5 and 40; more preferably 5 to 30;
Preferably, n2 is an integer comprised between 5 and 30; more preferably 10 to 20;
Preferably, both **C asymmetric carbons exhibit the (R) configuration.
Preferably, T is chosen from a methyl group or a branched or linear C10-C60, preferably C15-050 hydrocarbon chain which optionally comprises one to five double bond(s), and/or represents a group of formula:
where
X is chosen from:
Y is chosen from:
and n3, n4 identical or different, independently represent an integer comprised between 5 and 30; preferably n3 represents an integer comprised between 12 to 16 and n4 is chosen from 11, 13, 15, 17, 18 and 19,
provided that when Y is —C(═O)—OH, then T is —(CH2)n3—X—C(═O)—OH.
Mycolic acid derivatives are characterized by Laval et al., Anal Chem 2001, 73(18):4537-4544; Villeneuve M. et al., Biochim Biophys Acta 2005, 1715(2):71-80; Villeneuve M. et al., Biochim Biophys Acta 2007, 1768(7):1717-1726; Watanabe M. et al., Microbiology 2001, 147(Pt 7):1825-1837.
According to a preferred embodiment, the present invention concerns per se the compounds of formula (I) as defined above where R′ represents a —C(═O)-Alkyl group.
Such compounds may be obtained from the corresponding compounds of formula (I) as described above by application or adaptation of any known acylation methods generally used, in particular by reacting acetic anhydride.
According to another preferred embodiment, the present invention concerns per se the compounds of formula (I) as defined above where T represents:
where Y is
The corresponding compounds are herein referred to as “reduced compounds”.
Such compounds may be obtained form the corresponding compounds of formula (I) as described above by application or adaptation of any known reduction methods generally used. Preferably, this reaction may be carried out by means of NABH4 treatment.
According to another preferred embodiment, the present invention concerns per se the compounds of formula (I) as defined above where the C* exhibits the (R) configuration.
GroMM herein refers to glycerol monomycolate.
According to a further embodiment, the invention relates to a pharmaceutical composition comprising a compound of formula I such as defined above.
The invention further relates to a pharmaceutical composition characterized in that it comprises a mixture of compounds selected from the compounds of formula I.
The invention also relates to a pharmaceutical composition comprising at least one compound as defined above, or a composition as defined above, in association with a pharmaceutically acceptable vehicle.
Said pharmaceutical compositions are for example vaccines.
Further, the invention also relates to a combination of one or more compound(s) of formula I with one or more mycobacterial lipids, such as compound(s) chosen from the group consisting in mycobacterial phospholipids, mycobacterial lipoglycans, mycobacterial glycolipids and phospholipids, as well as the pharmaceutical composition comprising the same.
Such lipids are known in the art and were disclosed in Nigou et al. (2003) Biochimie, 85, 153-166.
It has been unexpectedly discovered that the above combinations exhibit a synergistic activity, as apparent from the results below.
A pharmaceutically acceptable carrier comprises in particular liposomes.
Said pharmaceutical compositions are useful for treating and/or preventing affections by actinomycetes, in particular mycobacteriae, such as tuberculosis or cutaneous affections.
The composition may also comprise vaccine adjuvants. Vaccine adjuvants for use in human individuals or animals are well known to the man skilled in the art, a list of such salts can be found for instance in “A compendium of vaccine adjuvants and excipients” 2nd edition, Vogel et al. Particular vaccine adjuvants notably comprise aluminum salts or M59, for example.
The invention particularly relates to a pharmaceutical composition as defined above, characterized in that it is presented in a form intended for administration by oral or injectable route.
The invention more particularly relates to a pharmaceutical composition as defined above, characterized in that it comprises one or more other products useful for the treatment or the prophylaxis of tuberculosis, such as BCG or mycobacterial proteins or actinomycete lipids.
The expression “actinomycete glycerol acyl derivatives” refers to lipids found in bacteria of the Cornybacterium, Mycobacterium, and Nocardia (CMN) genus, and preferably in Mycobacterium bovis BCG, Mycobacterium tuberculosis, Mycobacterium smegmatis, Mycobacterium gastri, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium xenopi, Mycobacterium kansasii, Cornybacterium glutamicum and Nocardia asteroids. According to a preferred embodiment said lipids are antigens of M. bovis BCG.
Other products useful for the treatment or the prophylaxis of tuberculosis notably comprise immunomodulators, such as cytokines, DNA fragments encoding M. tuberculosis antigens, live M. tuberculosis deletion mutants, such as mutants in which virulence genes have been deleted, or live recombinant BCG, such as BCG expressing antigens of M. tuberculosis.
As used herein, tuberculosis refers to the disease caused in humans by the bacterium Mycobacterium tuberculosis, and also to the corresponding disease in animals.
According to another embodiment, the invention relates to products comprising:
as a combined preparation for simultaneous, separate or sequential use in the treatment or the prophylaxis of tuberculosis.
The invention also relates to the use of at least one compound as defined above, or of an above mentioned composition, for the preparation of a medicament, notably a vaccine, intended for the treatment or the prophylaxis of tuberculosis.
The invention also relates to a compound as defined above as a medicament for the treatment or the prophylaxis of tuberculosis.
Optionally, the vaccine may comprise a vaccine adjuvant such as described above.
The invention relates to the use of at least one compound as defined above, or of a composition according as defined above, as an immune reaction activator, and more particularly an inflammatory reaction activator.
By “immune reaction activator” is meant a compound which has the ability to activate components or processes of the immune reaction, in vitro or in vivo, in particular cells of the immune system, such as T lymphocytes, B lymphocytes, antigen presenting cells (APCs), such as dendritic cells or macrophages, monocytes or granulocytes.
By “inflammatory reaction activator” is meant a compound which has the ability to activate components or processes of the inflammatory reaction, in vitro or in vivo, such as diapedesis, capillary permeabilization, macrophage activation or fever onset for instance.
The invention also relates to the use of at least one compound as defined above, or of a composition as defined above, to induce the activation of T lymphocytes, notably CD1-restricted T lymphocytes. The activation can proceed in vitro or in vivo.
The activation of T lymphocytes can be assessed by several methods, such as measuring cell proliferation or cytokine production such as IFN-γ (interferon-γ), IL-2 (interleukine-2), IL-4 (interleukine-4), or TNF-α (tumor necrosis factor α), or GM-CSF for instance.
CD1-restricted T lymphocytes are T lymphocytes which are activated by antigens presented by CD1 molecules.
The invention also relates to the use of at least one compound as defined above, or of a composition as defined above, to induce the production of cytokines such as IFN-γ, TNF-α, IL-2, IL-4, or GM-CSF.
This production induction can be done in vitro or in vivo. The production of cytokines such as IFN-γ, TNF-α, IL-2, IL-4 or GM-CSF can be measured for instance by immunoassays, such as ELISA (enzyme linked immunosorbent assay) or EIA (enzyme immunoassay).
According to another embodiment, the invention relates to a process for generating a T cell clone specific to the compounds of formula (I), characterized in that it comprises the following stages:
1) incubating antigen presenting cells (APCs), notably dendritic cells, with an actinomycete lipidic preparation, with the exception of Mycobacterium tuberculosis lipidic preparation, or a compound of formula (I),
2) contacting peripheral blood mononuclear cells with the envelope antigen loaded APCs to obtain proliferating T cells,
3) cloning proliferating T cells by limiting dilution and selecting the clone releasing a molecule selected from the group comprising cytokines such as IFN-γ, TNF-α, GM-CSF, IL-2 or IL-4 when contacted by a compound of formula (I).
Preferably, in step 1), the actinomycete lipidic preparation is a Mycobacterium bovis BCG lipidic preparation.
The invention further relates to T cell clones such as obtained by the above-mentioned process. Such a T cell clone is described in the examples as Z5B71.
According to another embodiment, the invention relates to a process for screening products, such as actinomycete glycerol acyl derivatives extracted from actinomycetes, such as Mycobacterium, Corynebacterium and Nocardia, characterized in that it comprises the following stages:
The invention also relates to a process for the extraction of compounds as defined above, or of a composition as defined above, from actinomycetes, such as Mycobacterium, Corynebacterium and Nocardia characterized in that it comprises the following stages:
According to a preferred aspect, said process further comprises the following additional steps:
The present invention also concerns the compositions obtainable by the above process as well as the pharmaceutical compositions comprising the same, and the use of said composition for the preparation of a medicament for the prophylaxis or the treatment of tuberculosis, the use of said composition as an immune reaction activator, the use of said composition to induce the activation of T lymphocytes, the use of said composition to induce the production of cytokines such as IFN-γ, TNF-α, IL-2, IL-4, or GM-CSF as defined above.
Corynebacterium glutamicum (▪), Nocardia (), M. bovis BCG (▴), M. tuberculosis H37Rv (♦), M. smegmatis (□), M. fortuitum (∘), M. tuberculosis H37Ra (Δ)
TLC analysis of eight representative fractions (fractions 1, 3, 5, 10, 18, 19, 28 and 34) obtained by silica chromatography purification of “methanol insoluble” fraction is presented in (A). The encircled compound corresponds to the active compound. The same fractions were tested for stimulation of the Z5B71 T cell clone by measuring IFN-γ release (B). Bars indicated SD.
The structures presented in (B) and (C) correspond to (S)-1-O-mycoloyl-glycerol (1-O-mycoloyl-sn-glycerol) and (R)-1-O-mycoloyl-glycerol (3-O-mycoloyl-sn-glycerol), respectively. R corresponds to mycolic acid.
The purification was followed by antigenic potency of the lipidic fractions (B and C). Released IFN-γ by the Z5B71 T cell clone was measured by ELISA. Bars indicate SD. The lipidic fraction refers to the chloroform phase.
Reagents. L-1,3-O-isopropylidene-sn-glycerol and D-1,2-O-isopropylidene-sn-glycerol, glyceryl tripalmitate, 1,2 dipalmitoyl-sn-glycerol and monopalmitoyl glycerol were purchased from Sigma. Cardiolipids were purified from M. bovis BCG lipidic extract, as described in Gilleron et al. (2001) J Biol Chem 276, 34896-904.
Bacterial Strain and Culture Conditions. Nocardia asteroides and Corynebacterium glutamicum were grown in suspension during 2 to 3 days at 32° C. on Luria Broth and Brain Heart Infusion medium, respectively. Mycobacteria were grown during 4 to 8 weeks at 37° C. on Sauton's medium as surface biofilm. Cells were harvested, separated from the culture media, and left in chloroform/methanol (2:1, v/v) at room temperature in order to kill bacteria.
Cell Culture. Human promyelocytic THP-1 were transfected with cDNA of human CD1A, or CD1B, or CD1C or double-transfected with CD1B and CD1E using the BCMGS-Neo and -Hygro vectors by electroporation. Cells were grown in RPMI-1640 supplemented with 10% fetal calf serum (FCS), 10 mM HEPES, 2 mM UltraGlutamine II, MEM nonessential amino acids, 1 mM Na-pyruvate (all from Cambrex, Brussels, Belgium) and 100 μg/ml kanamycin (Invitrogen, Basel, Switzerland). DCs were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors by culturing in the presence of recombinant IL-4 and GM-CSF, as described previously Porcelli et al. (1992), Nature 360, 593-7. T cell clones were generated as described De Libero (1997), ed. Butcher, N. F. a. G. (IRL, Oxford), pp. 123-40, and grown in RPMI-1640 supplemented with 5% AB human serum (Swiss Red Cross), 10 mM HEPES, 2 mM UltraGlutamine II, MEM nonessential amino acids, 1 mM Na-pyruvate, 100 μg/ml kanamycin and 100 units/ml human recombinant IL-2.
T Cell Activation Assays. DCs (3×104/well) or CD1-transfected THP-1 cells (3×104/well) in RPMI-1640 medium containing 10% FCS were incubated for 2 hr at 37° C. with different concentrations of sonicated antigens before the addition of T cells (105/well in triplicate). Supernatants were harvested after 36 hr of incubation, and cytokine release was measured by using enzyme-linked immunosorbent assay (ELISA) kits (IFN-γ from Instrumentation Laboratory, Schlieren, Switzerland; TNF-α from BD Pharmingen, Basel, Switzerland). Data are expressed as mean pg/ml±standard deviation (SD) of triplicates.
Analysis of CD1 Restriction. CD1 restriction was investigated by inhibiting T cell activation using the following monoclonal antibodies (mAb) at 10 μg/ml 30 min before the addition of T cells: OKT6 (anti-CD1a, American Type Culture Collection ATCC CRL-8019), WM-25 (anti-CD1b; Immunokontakt, Lugano, Switzerland), L161 (anti-CD1c; Instrumentation Laboratory), W6-32 (anti-MHC class I, ATCC) and L243 (anti-MHC class II, ATCC). Anti-TCRVγ9 (B3) mAb was used as irrelevant mAb.
Pulsing and infection of DCs with M. tuberculosis. M. tuberculosis was killed at 80° C. for 20 min and incubated in RPMI-1640 medium containing 10% FCS for 2 hours at 37° C. with DCs (3×104/well) before addition of T cells (105/well in triplicate). DCs were infected as previously described De Libero et al. (2005) Immunity 22, 763-72 and IFN-γ release was monitored by ELISA.
Lipidic Fractions Used for Generation of T Cell Clone. The Z5B71 T cell clone was generated with a M. bovis BCG PIM2-enriched fraction, obtained previously Gilleron et al. (2001) J Biol Chem 276, 34896-904. Briefly, M. bovis BCG cells were suspended in chloroform/methanol (1:1, v/v) and filtered four times. The chloroform/methanol extract was concentrated and constituted the lipidic fraction, which was further partitioned between water and chloroform. The chloroform phase was evaporated and suspended in a minimum volume of chloroform. The addition of acetone overnight at 4° C. led to formation of a precipitate, which was centrifuged (3,000 g at 4° C. for 15 min) to generate an “acetone-soluble” phase and an “acetone-insoluble” phase. The “acetone-soluble” phase was applied to a QMA-Spherosil M (BioSepra SA, Villeneuve-la-Garonne, France) column eluted successively with chloroform, chloroform/methanol (1:1, v/v), methanol to elute neutral compounds and then with chloroform/methanol (1/:2, v/v) containing 0.1 M ammonium acetate to elute negatively charged compounds (as PIMA Lipidic fractions from several actinomycetes. Different lipidic fractions were prepared (as described above) from several actinomycetes species and tested against the Z5B71 T cell clone: the lipidic fraction from M. smegmatis, M. tuberculosis H37Ra, M. fortuitum, M. chelonae, M. xenopi, N. asteroides and C. glutamicum, the “acetone-soluble” phase from M. smegmatis, M. tuberculosis H37Rv, M. gastri, M. kansasii, M. marinum and M. bovis BCG and the “acetone-insoluble”phase from M. tuberculosis H37Rv, M. gastri, M. kansasii, M. marinum and M. bovis BCG.
Purification of the GroMM from M. bovis BCG. The “acetone-insoluble” fraction from M. bovis BCG, obtained as described above, was subjected to methanol precipitation giving “methanol soluble” and “methanol insoluble” fractions. The “methanol-insoluble” fraction (150 mg) was fractionated on a silica acid column (1.3×23 cm) eluted with 7×175 ml of chloroform (fractions 1-7), 8×200 ml of chloroform-methanol (95:5, v/v) (fractions 8-15), 9×225 ml of chloroform-methanol (9:1, v/v) (fractions 16-24), 7×175 ml of chloroform-methanol (85:15, v/v) (fractions 25-31) and 7×175 ml of chloroform-methanol (7:3, v/v) (fractions 32-38). Fractions 5 to 8 were found to contain GroMM and were pooled.
Purifications were checked by TLC on aluminium-backed silica gel plates (Alugram Sit G; Macherey-Nagel) using as migration solvent chloroform/methanol (95:5, v/v). Anthrone (9,10-dihydro-9-oxo-anthracene) (Sigma) at 0.2% dissolved in H2SO4 at 85% was used to detect glycolipids.
GroMM Acetylation. Peracetylation was realized on 200 μg of GroMM which were dissolved in acetic anhydride/anhydrous pyridine (1:1, v/v) and heated at 80° C. for 30 min. The reaction mixture was dried under a stream of nitrogen and dissolved in chloroform for MALDI-Tof-MS analysis. For selective acetylation of GroMM hydroxyl functions, 16 μl of acetic anhydride were added to 10 mg of GroMM dissolved in 150 μl of pyridine and kept 3 hours at 0° C. Mono- and di-acetylated GroMM were extracted by CHCl3 and purified on silica column eluted successively by CHCl3 and CHCl3/CH3OH 95/5 (v/v). Number of acetyl substituents and their location on GroMM were determined by MALDI-Tof-MS and NMR analysis.
GroMM Saponification. 800 μl of methoxyethanol/KOH 20% (7:1, v/v) were added to 500 μg of GroMM. The mixture was then heated in an oven at 100° C. for 3 h. After cooling and acidification by aqueous sulphuric acid pH 3, mycolic acids were extracted three times by diethyl ether (v/v). Etheral phases were pooled, washed five times with water and dried under a stream of nitrogen. Mycolic acids were redissolved in 100 μl chloroform for MALDI-Tof-MS analysis.
GroMM Reduction. 1 mg of GroMM purified from BCG was reacted with 1,3 mg of NaBH4 in 140 μl THF, under stirring for 15 minutes at room temperature. Reaction was stopped by adding acetic acid. The reaction mixture was evaporated, stirred with dichloromethane and washed twice with water.
GroMM Synthesis. Reactions were performed under argon in anhydrous solvents.
1-O-(4-Methylbenzenesulfonyl)-2,3-O,O-isopropylidene-sn-glycerol (Sowden et al. (1942) J Am Chem Soc 42, 1291-3): p-toluenesulfonyl chloride (0.3 g, 1.57 mmol) was added at 0° C. to 2,3-O,O-isopropylidene-sn-glycerol (230 mg, 1.74 mmol) dissolved in anhydrous pyridine (0.8 ml). After 24 h at room temperature, diethyl ether was added to the reaction mixture and the organic phase was washed sequentially with cold, 1 M hydrochloric acid, water, saturated sodium bicarbonate solution and water. The ether extract was dried on magnesium sulphate and concentrated. Chromatography on silica gel (elution with petroleum ether/ethyl acetate 4/1, v/v) gave the product (around 100 mg, 0.35 mmol). In the same manner, 3-O-(4 methylbenzenesulfonyl)-1,2-O,O-isopropylidene-sn-glycerol was obtained from 1,2-O,O-isopropylidene-sn-glycerol.
Potassium mycolates: Mycolic acids (gift from M. A. Lanéelle, IPBS, Toulouse) were released from M. tuberculosis H37Rv cells according to the method described in Laval et al. (2001) Anal Chem 73, 4537-44. Potassium mycolates were obtained by two successive washings of mycolic acids dissolved in chloroform with 10 mM hydrochloric acid and 5 M potassium hydroxide. After extraction with chloroform, potassium mycolates were dried on magnesium sulphate and concentrated.
1-O-Mycoloyl-2,3-O,O-isopropylidene-sn-glycerol (Defaye et al. (1956) Bull Soc Chim Biol (Paris) 38, 1301-4): Potassium mycolates (8 mg, around 6 μmol) were added to 1-O-(4 methylbenzenesulfonyl)-2,3-O,O-isopropylidene-sn-glycerol (2 mg, 7 μmol) in anhydrous dimethylformamide (0.150 ml) and stirred for 3 days at 135° C. The reaction mixture was acidified by 1M hydrochloric acid and extracted by diethyl ether. Ether extract was then washed with water, dried on magnesium sulphate and concentrated. Chromatography on silica gel (elution with petroleum ether/diethyl ether 5/1, v/v) gave the product (around 4 mg, 2.9 μmol). In the same way, 3-O-mycoloyl-1,2-O,O-isopropylidene-sn-glycerol was obtained from 3-O-(4 methylbenzenesulfonyl)-1,2-O,O-isopropylidene-sn-glycerol.
1-O-Mycoloyl-sn-glycerol (GroMM) (Sowden et al. (1942) J Am Chem Soc 42, 1291-3): 100 μl of concentrated hydrochloric acid (d=1.017) were added to 1-O-Mycoloyl-2,3-O,O-isopropylidene-sn-glycerol (4 mg, 2.9 μmol) dissolved in diethyl ether (0.1 ml). After 10 min of stirring at room temperature, water was added to the reaction mixture and the product (3 mg, 2.3 μmol) was extracted with diethyl ether. The ether extract was dried on magnesium sulphate and concentrated. Supplemental purification was not necessary. 3-O-mycoloyl-sn-glycerol was also obtained using 3-O-mycoloyl-1,2-O,O-isopropylidene-sn-glycerol.
MALDI-Tof-MS. Analysis by MALDI-Tof-MS were performed on a 4700 Proteomics Analyser (with Tof-Tof Optics, Applied Biosystems) using the reflectron mode. Ionization was effected by irradiation with pulsed UV light (355 nm) from an Nd:YAG laser. GroMM samples were analyzed by the instrument operating at 20 kV in the positive ion mode using an extraction delay time set at 20 ns. Typically, spectra from 100 to 250 laser shots were summed to obtain the final spectrum. The HABA (2-[4-hydroxy-phenylazo]-benzoic acid) matrix (Sigma-Aldrich) was used at a concentration of ˜10 mg/ml in ethanol/water (1:1, v/v). Then, 0.5 μl sample solution and 0.5 μl of the matrix solution were deposited on the target, mixed with a micropipette and dried under a gentle stream of warm air. The measurements were externally calibrated at two points with mycobacterial PIM.
Nuclear Magnetic Resonance (NMR) Analysis. NMR spectra were recorded with an Avance DMX500 spectrometer (Bruker) equipped with an Origin 200 SGI using Xwinnmx 2.6. GroMM was dissolved in CDCl3-CD3OD, (9:1, v/v) and analyzed in 200×5 mm 535-PP NMR tubes at 298 K. Proton chemical shifts are expressed in parts per million downfield from the signal of the chloroform (δH/TMS 7.27). All the details concerning correlation spectroscopy and homonuclear Hartmann-Hahn spectroscopy sequences used and experimental procedures were as previously described Gilleron et al. (2003) J Biol Chem 278, 29880-9.
Recognition of GroMM by Lymphocytes from Tuberculosis Patients and Healthy Donors. PBMCs were purified from blood of healthy donors and tuberculosis patients after informed consent was given. All patients included in this study suffered from culture proven pulmonary tuberculosis and were undergoing treatment with anti-tuberculosis drugs for 2 to 8 weeks at the time of blood donation. PBMCs (105/well) were cultured with autologous DCs (2×104/well) in the presence or absence of 10 μg/ml GroMM. After 6 days supernatants were collected and tested for the presence of IFN-γ by sandwich ELISA. PBMCs were kept at 37° C. in supplemented RPMI 1640 medium (see above) during the 2 days of autologous monocytes differentiation to DCs. In parallel with GroMM PBMCs were stimulated with 10 μg/ml PPD (Chiron) and recombinant ESAT6 (Lionex). Healthy donors that responded by releasing at least 250 pg/ml IFN-γ in response to PPD were scored as BCG immunized. Donors that responded to both antigens were considered as latently M. tuberculosis infected. Those that did not respond to PPD were scored PPD-negative. For individual experiments, a cocktail of CD1b- and CD1c-blocking antibodies (BCD1b3.1 and F10—10 μg/ml each) was added 30 min prior to GroMM to block CD1 presentation.
In order to identify the M. tuberculosis CD1-restricted T cell lipid antigen repertoire, a strategy was developed based on the generation of T cell lines stimulated by three different types of crude mycobacterial lipid fractions, according to their polarity and charge (Gilleron et al. (2004) J Exp Med 199, 649-59). From these lines, derived from PBMC of one highly PPD-reactive healthy donor, T cell clones were established by limiting dilution and 10 individual clones which were found CD1-restricted, were further investigated. One of these clones, called Z5B71, which is CD4+ was investigated. It was obtained against a fraction enriched in phosphatidyl-myo-inositol dimannosides (PIM2), obtained from M. bovis BCG cell wall extraction. This clone reacts to lipidic extracts from different mycobacterial species (M. bovis BCG, M. smegmatis, M. gastri, M. marinum, M. fortuitum, M. xenopi, M. kansasii and M. tuberculosis H37Ra) and from Corynebacterium glutamicum and Nocardia asteroides (
Purification of the lipid antigen was performed using M. bovis BCG lipidic extract because it showed the strongest stimulatory activity and also because the diversity of glycolipids in the cell envelope of this species is lower than in that of M. tuberculosis. Lipidic extract was partitioned by acetone precipitation, a solvent known to precipitate phospholipids. Both “acetone-soluble” and “acetone-insoluble” fractions (
“Methanol-insoluble” fraction was then fractionated on a silica gel column eluted by chloroform containing increasing amounts of methanol (
The positive mode MALDI mass spectrum showed a complex pattern of peaks assigned to cationized sodiated molecular ions [M+Na]+ (
The mass difference (Δ 74 amu) between the native product and the resulting mycolic acid after alkaline treatment suggested that the mycolic acid esterifies a glycerol residue. The number of alcohol functions on the molecule was proved by peracetylation of the molecule, and analysis of the reaction products by MALDI MS (not shown). The displacement of 126 mass unit proved the presence of three alcohol functions. Then, the assignment of the novel antigen to glycerol monomycolate (GroMM) was further supported by 1D 1H NMR analysis (
Hemi-Synthetic GroMM (sGroMM) is Immunogenic
In order to confirm the structure of the antigen, GroMM analogues were chemically synthesized (
The chemical synthesis was started using (R)- and (S)-isopropylidene-glycerol, activated by p-toluene-sulfonyl (Sowden et al. (1942) J Am Chem Soc 42, 1291-3) (
The s(R)-GroMM stimulated the Z5B71 T cells more efficiently than the s(S)-GroMM, while the purified, natural compound has an intermediate activity (
GroMM is Presented by CD1B and does not Require the Presence of CD1E
The CD1 restriction element of GroMM was investigated by stimulating the T-cell clone using a panel of CD1-transfected APCs. Only CD1b-expressing cells were able to stimulate T cells (
An important issue is whether mycobacterial lipid antigens maintain their antigenicity during infection, i.e. when they are released from the cell wall of the bacteria resident in the phagosome. This was investigated by using DCs which were pulsed either with heat-killed M. tuberculosis, or which were infected with living bacteria. DCs pulsed with heat-killed bacteria were used to stimulate three T cell clones recognizing three different M. tuberculosis lipidic antigens, namely GroMM (Z5B71), Ac2SGL (Z4B27) and PIM6 (Z5B11). The Ac2SGL and PIM6-reactive cells (de la Salle et al. (2005) Science 310, 1321-4; Gilleron et al. (2004) J Exp Med 199, 649-59) were previously described. High doses of heat-killed bacteria (100 μg/ml) induced a strong T cell response (
T Cells from PPD-Positive Healthy Donors Recognize GroMM
To directly test whether T cells in the peripheral blood of human donors recognize the novel lipid compound, blood samples were collected from tuberculosis patients (n=11), PPD-negative (n=10) and BCG immunized (n=22) and latently M. tuberculosis infected (n=19) healthy donors and measured IFN-γ release after GroMM stimulation. Significant responses were solely observed in BCG immunized or latently infected, healthy donors (with a mean average of 73 pg/ml and 117 pg/ml, respectively;
To test the activity of the reduced compounds, the stimulation of the Z5B71 T cell clone by measuring IFN-γ release of GroMM and reduced GroMM was measured. The results shown in
Synergistic Activity of Combinations of Mycobacterial GroMM with Lipids
To test whether lipids would potentialize the acitivity of mycobacterial GroMM, the stimulation of the Z5B71 T cell clone by measuring IFN-release of both agents alone or in combination was measured for various mycobacterial lipids (A) or phospholipids (B). The results shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 07291249.6 | Oct 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP08/63736 | 10/13/2008 | WO | 00 | 6/24/2010 |
