WO2009127971A2

WO2009127971A2 - Control of diseases associated with decrease of t-regulatory cells with a preparation of extended freeze-dried killed bacteria

Info

Publication number: WO2009127971A2
Application number: PCT/IB2009/005498
Authority: WO
Inventors: Gilles Marchal; Micheline Lagranderie; Muhammad Abolhassani; Hervé BERCOVIER; Ayelet Maron
Original assignee: Institut Pasteur; Yissum Research Development Company Of The Hebrew University Of Jerusalem
Priority date: 2008-04-14
Filing date: 2009-04-14
Publication date: 2009-10-22
Also published as: CA2629057A1; WO2009127971A3

Abstract

This invention relates to the use of a preparation of extended freeze- dried (EFD) killed Gram positive bacteria such as Gram positive facultative intracellular bacteria, for example mycobacteria, for the treatment of diseases associated with a decrease of T-regulatory cells (Tregs), particularly CNS inflammatory diseases, and more particularly CNS demyelinating diseases such as multiple sclerosis (MS) and acute-disseminated encephalomyelitis.

Description

CONTROL OF DISEASES ASSOCIATED WITH DECREASE OF T- REGULATORY CELLS WITH A PREPARATION OF EXTENDED FREEZE-

DRIED KILLED BACTERIA

Field of the invention

This invention relates to the use of a preparation of extended freeze-dried (EFD) killed Gram positive bacteria such as Gram positive facultative intracellular bacteria, for example mycobacteria, for the treatment of T- regulatory cells (Tregs)-associated diseases, particularly CNS inflammatory diseases, and more particularly CNS demyelinating diseases such as multiple sclerosis (MS) and acute-disseminated encephalomyelitis. Description of the prior art

Multiple sclerosis (commonly abbreviated as "MS") is a chronic neurological disorder and is often incapacitating. Its clinical manifesta- tions are linked to a demyelination of the nerve fibres of the white matter of the central nervous system (brain, spinal cord and optical nerve) which is secondary to an inflammation of autoimmune origin. Multiple sclerosis is a chronic disease, which is progressively incapacitating and which evolves in episodes. This syndrome, (known as multiple sclerosis or MS) leads to a loss of motor or sensory functions, which are partially recovered after episodes, but unfortunately often progressive. The anti-inflammatory treatments tend to reduce the term or the intensity of the episodes, but are still very insufficient.

The patients are most often young adults (20 to 40 years old) and women, the disease being observed at all ages. About 80,000 people are affected in France (prevalence), with about 2,000 new cases a year (incidence).

The baseline treatments attempt to modify immune responses.

The monoclonal antibody, natalizumab, directed against the alpha chain of leucocyte integrins inhibits the circulation of cells. Another baseline treatment is the anti-CD20 antibody, rituximab, an anti lymphocyte B antibody. Interferon

1-beta is also administered by subcutaneous or intramuscular injection with the objective of "modifying" the immune responses. A new product, FTY720, is undergoing phase 3 clinical trials. By sequestering lymphocytes in the sites of production, it is immunosuppressive and induces important lymphopenia which is apparently well tolerated. Notable secondary effects have been reported: "A first undesired effect is the observation of a "reversible posterior leucoencephalitis" in a patient treated with FTY720. This syndrome is observed in cases of arterial hypertension or in treatments with immunosuppressive agents. It is characterized by an edema of the posterior portion of the brain and is well seen in an MRI. It is associated to confusion, visual disorders and headaches. While reversible if its cause is rapidly removed, it can otherwise evolve towards a definitive toxic edema. On one hand, FTY720 can cause arterial hypertension, especially at high doses. On the other hand, it provokes an immunosuppressive effect. Finally, a case of a macular (part of the retina) edema was observed in a patient treated with FTY720 for a renal transplant." (Comments from a newsletter of the Charcot Foundation).

Experimental autoimmune encephalomyelitis, sometimes Experimental Allergic Encephalomyelitis (EAE), is an animal model of brain inflammation. It is an inflammatory demyelinating disease of the central nervous system (CNS). It is mostly used with rodents and is widely studied as an animal model of the human CNS demyelinating diseases, including the diseases multiple sclerosis and acute-disseminated-encephalomyelitis. EAE can be induced by inoculation with whole CNS tissue, purified myelin basic protein (MBP) or myelin proteolipid protein (PLP), together with adjuvants. It may also be induced by the passive transfer of T cells specifically reactive to these myelin antigens. EAE may have either an acute or a chronic relapsing course. Acute EAE closely resembles the human disease acute disseminated encephalomyelitis, while chronic relapsing EAE resembles multiple sclerosis.

EAE is also the prototype for T-cell-mediated autoimmune disease in general.

The role of T regulatory cells (Treg) in the control of autoagressive immune responses is the subject of intense investigations. A review article, entitled : "Foxp3+ regulatory Tcells in the control of experimental CNS autoimmune disease" summarizes our knowledge on the role of these cells to decrease inflammatory reactions during multiple sclerosis (MS) and during experimental autoimmune encephalomyelitis (EAE). According to these views the Tregs are less abundant in tissues during relapsing periods, while Tregs were accumulated in the CNS during the recovery phases of EAE (O'Connor RA and Anderson S. M. (2008) Journal of Neuroimmunology 193: 1-11)

Furthermore two recent papers report that the role of pertussis toxin (PTx), injected with encephalitogen peptide (MOG_35-55) and 48h later to enhance the severity of EAE, is to reduce the number and function of Tregs (Chen X. et al. (2006) Eur. J. Immunol. 36 : 671-680 ; Cassan et al., The Journal of Immunology, 2006, 177, 1552-1560). Summary of the invention

An object of the present invention is to provide a treatment for T-regulatory cells (Tregs)-associated diseases, and more particularly for CNS inflammatory diseases such as CNS demyelinating diseases like multiple sclerosis (MS) or acute-disseminated encephalomyelitis.

Accordingly, the present invention provides the use of a Gram positive bacteria preparation for the prevention and treatment of a Tregs- associated disease, the preparation being characterized in that the Gram positive bacteria are killed by extended freeze-drying (EFD).

The present invention still provides a method for preventing or treating a Tregs-associated disease, the method comprising the step of administering to a patient an effective amount of an extended freeze-dried

(EFD) killed Gram positive bacteria preparation, thereby stimulating the production of Tregs.

The present invention further provides a preparation of Mycobacterium bovis BCG killed by extended freeze drying for preventing and/or treating a Tregs-associated disease and more particularly for CNS inflammatory diseases such as CNS demyelinating diseases like multiple sclerosis (MS) or acute-disseminateed encephalomyelitis. This preparation is preferably obtained by a process comprising the following steps: a) harvesting a culture of live bacteria cells, b) washing the bacteria cells in water or in an aqueous solution of a salt such as borate, c) freezing the bacteria cells in water or in an aqueous solution of a salt such as borate, d) killing the frozen bacteria cells by drying them in a freeze-dryer, for a time sufficient to remove at least 98.5 % of the water, preferably at least 99% of the water, more preferably at least 99.5% of the water, and e) collecting the extended freeze-dried bacteria cells. The present invention also provides a product comprising a preparation as claimed in anyone of claims 1 to 14 and at least a drug selected from the group consisting of anti-inflammatory and immunoregulatory drugs, as a combined preparation for simultaneous, separate or sequential use in the prevention and/or treatment of a CNS inflammatory disease such as a CNS demyelinating disease like multiple sclerosis (MS) or acute- disseminated encephalomyelitis. Brief description of the drawings

Figure 1 illustrates a marked increase of dendritric cells expressing ICOS-L after EFD treatment. Figure 2 illustrates a selective view of the dendritric cells expressing ICOS-L of Figure 1 after EFD treatment.

Figure 3 is a Western blot showing the marked expression of the Tregs specific marker Foxp-3 in EFD treated mice.

Figure 4 is a panel of 4 Western blots showing that EFD treatment promotes the differentiation or multiplication of naϊve Tcells toward Tregs expressing Foxp3 and increases Tbet expression, whereas it reduces RORγ and GATA3 expression in a mouse model of Crohn disease.

Figure 5 illustrates that EFD treatment induces the maturation of Tregs. Figure 6 illustrates that EFD protects from excessive inflammation in asthma mouse model. Figure 7 illustrates prevention of EAE clinical symptoms by EFD treatment at day 0 of EAE induction (C57BL/9 mice model).

Figure 8 illustrates prevention of EAE clinical symptoms in the C57BL/9 mice model, by EFD treatment at day 0 of EAE induction. Figure 9 illustrates prevention of EAE clinical symptoms in the

C57BL/9 mice model, by EFD treatment at day -14 of EAE induction.

Figure 10 illustrates the effect of EFD treatment at day 0 or day 25 of EAE induction on the clinical symptoms of EAE (SJL/J mice model).

Figure 11 illustrates the effect of EFD treatment at day 0 or day 25 of EAE induction on the survival rate in the SJL/J mice model.

Figure 12 illustrates the effect of EFD treatment at day -30 and day 25 of EAE induction on the clinical symptoms of EAE (SJL/J mice model).

Figure 13 illustrates the effect of EFD treatment at day -30 and day 25 of EAE induction on the survival rate (SJL/J mice model). Detailed description of the invention

The present invention relates to the use of a killed Gram positive bacteria preparation in a pharmaceutical composition for the prevention and/or treatment of a Tregs-associated disease, particularly a CNS inflammatory disease, and more particularly a CNS demyelinating disease such as multiple sclerosis (MS) or acute-disseminated encephalomyelitis. Use of the Gram positive bacteria preparation

One embodiment of the invention relates to the use of a Gram positive bacteria preparation for the prevention and treatment of a Tregs- associated disease, particularly a CNS inflammatory disease, and more particularly a CNS demyelinating disease such as multiple sclerosis (MS) or acute-disseminated encephalomyelitis. The preparation is characterized in that the Gram positive bacteria are killed by Extended Freeze-Drying (EFD). Preferably the preparation contains more than 50 %, or more than 90%, of the bacterial protein components which are in a native structure. The Gram positive bacteria preparation of the invention may also be useful for the preparation of a medicament for the prevention and/or treatment of a Tregs- associated disease, particularly a CNS inflammatory disease such as a CNS demyelinating disease like MS or acute-disseminated encephalomyelitis.

The Gram positive bacteria may be Gram positive facultative intracellular bacteria. Gram positive facultative intracellular bacteria means Gram positive bacteria with a capacity of growing in synthetic medium in vitro as well as of infecting eucaryotic cells from a mammalian or non-mammalian host, in vivo and multiplying in those cells, for example, macrophages.

The bacterial preparation may contain Gram positive facultative intracellular bacteria chosen from the group consisting of Listeria sp., Corynobacterium sp. and Actinomycetes comprising Mycobacteria sp., and even more preferably, e.g. Mycobacterium bovis BCG, Nocardia sp. and Rhodococcus sp.

The Gram positive bacteria preparation according to the invention is preferably a preparation of Mycobacterium bovis BCG killed by extended freeze drying.

The expressions "killed Gram positive bacteria preparation", "Gram positive bacteria preparation", "extended freeze-dried Gram positive bacteria preparation", "extended-freeze-dried killed bacteria", "killed bacterial preparation", "EFD killed Gram positive bacteria preparation", "EFD killed bacteria preparation, "EFD preparation" and "EFD" as used in the context of the present invention refer to a preparation of killed Gram positive bacteria, preferably of killed Mycobacterium bovis BCG bacteria, which contains less than 1.5 % of water, preferably less than 1 % of water, more preferably, less than 0.5 % of water, and in which the bacteria are killed by freeze-drying. The preparation may be prepared according to the method as described in the International PCT Application WO 03/049752, said method comprising the steps of : (i) harvesting a culture of live bacteria cells, (ii) washing the bacteria cells in water or in aqueous solution of a salt, (iii) freezing the bacteria cells in water or in an aqueous solution of salt, (iv) killing the frozen bacteria cells by drying them in a lyophiliser, for a time sufficient to remove at least 98.5% of the water, preferably at least 99% of the water, more preferably at least 99.5% of the water, and (v) collecting the extended freeze-dried killed bacteria cells. An alternative method for the preparation of EFD comprises the steps (i), (iii), (iv) and (v) as defined above; the washing step (ii) is omitted.

The above expressions as used in the context of the present invention refer in other words to killed Gram positive bacteria, preferably killed Mycobacterium bovis BCG bacteria in which the structure of the bacteria molecules and in particular the structure of the bacteria macromolecules (proteins, polysaccharides, lipids) is preserved. Preferably, the structure of the proteins is preserved. An example of preserved protein is Apa which has the same migrating characteristics in a SDS-PAGE gel as the protein extracted from living mycobacteria (Laqueyrerie et al., Infect. Immun., 1995; 63; 4003- 4010).

A fraction of EFD preparation as described above is covered by the expression "EFD" or "EFD preparation" of the invention. As used herein, the fraction of this extended-freeze-dried killed bacteria refers to a fraction selected from the group consisting of : a fraction consisting of an organic solvent extract of said EFD preparation, a fraction consisting of a glycosidase-treated extract of said EFD preparation, a fraction consisting of a DNase and/or a RNase-digested extract of said EFD preparation, a fraction consisting of a protease-treated extract of said EFD preparation, a fraction consisting of said EFD preparation successively treated by an organic solvent, a glycosidase, a DNase and/or a RNase, and finally a protease, and a fraction consisting of said EFD preparation treated by a protease (as subtilisine for example) and a DNase.

The EFD killed bacteria preparation or fractions thereof may be associated with a pharmaceutically acceptable carrier, and/or an immunostimulant, and/or an adjuvant and/or any conventional additives as defined herein below. The Gram positive bacteria preparation and/or the medicament of the invention may be administered by the oral, sublingual, parenteral or intranasal route. Pharmaceutical composition

According to an embodiment of the invention, it is provided a Gram positive bacteria preparation, for the prevention and/or treatment of a Tregs-associated disease. Such a preparation may be obtained by: a) harvesting a culture of live bacteria cells, b) washing the bacteria cells in water or in an aqueous solution of a salt such as borate, c) freezing the bacteria cells in water or in an aqueous solution of a salt such as borate, d) killing the frozen bacteria cells by drying them in a freeze-dryer, for a time sufficient to remove at least 98.5 % of the water, preferably at least 99% of the water, more preferably at least 99.5% of the water, and e) collecting the extended freeze-dried bacteria cells. According to an advantageous embodiment of the invention, the washing step (b) is omitted.

According to another advantageous embodiment of the invention, said preparation is included in a pharmaceutical composition additionally comprising an acceptable carrier and/or an additive and/or an immunostimulant and/or an adjuvant.

The composition of the present invention may be in a form suitable for oral administration. For example, the composition may be in the form of tablets, ordinary capsules, gelatine capsules or syrup for oral administration. These gelatine capsules, ordinary capsules and tablet forms can contain excipients conventionally used in pharmaceutical formulations such as adjuvants or binders like starches, gums and gelatine, adjuvants like calcium phosphate, disintegrating agents like corn starch or algenic acids, a lubricant like magnesium stearate, sweeteners or flavourings. Solutions or suspensions can be prepared in aqueous or non-aqueous media by the addition of pharmacologically compatible solvents. These include glycols, polyglycols, propylene glycols, polyglycol ether, DMSO and ethanol. The composition may additionally contain a pharmaceutically acceptable carrier and/or an additive and/or an immunostimulant and/or an adjuvant such as a liposome containing the bacteria cells or fractions thereof according to the present invention. The additives used for preparing the pharmaceutical composition of the present invention may be chosen among anti-aggregating agents, antioxidants, dyes, flavour enhancers, or smoothing, assembling or isolating agents, and in general among any excipient conventionally used in the pharmaceutical industry.

For parenteral administration, such as subcutaneous injection, the carrier may comprise water, saline buffer, lactose, glutamate, a fat or a wax. For oral administration, any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose and magnesium carbonate may be employed. Biodegradable microspheres (e.g. polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed for example in US patents 4,897,268 and 5,075,109.

Any of the variety of adjuvants may be employed in the compositions of the present invention to enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism or to create controlled inflammatory reactions, such as aluminium hydroxide or mineral oil, and a non-specific stimulator of immune response such as lipid A, Bordetella pertussis toxin. Suitable adjuvants are commercially available as well, for example, Freund's incomplete adjuvant and Freund's complete adjuvant which cannot be used for injection in human. Other suitable adjuvants which can be used in human include aluminium hydroxide, biodegradable microspheres, monophospheryl A and Quil A. Method of treatment

According to an embodiment of the present invention, the EFD killed Gram positive bacteria preparation is used for the prevention and treatment of a Tregs-associated disease, particularly a CNS inflammatory disease, and more particularly a CNS demyelinating disease such as MS or acute-disseminated encephalomyelitis. The method of the invention comprises the step of administering to a patient an effective amount of the EFD killed Gram positive bacteria preparation, to stimulate the production of Tregs, such as CD4+ CD25+ Foxp-3+ T cells. As mentioned above, the disease may be MS or acute- disseminated encephalomyelitis.

The amount of Gram positive bacteria preparation present in the compositions of the present invention may be a therapeutically effective amount. A therapeutically effective amount of Gram positive bacteria prepara- tion is that amount necessary so that the Gram positive bacteria preparation performs its role of stimulating the production of Tregs without causing, overly negative effects in the host to which the composition is administered. The exact amount of Gram positive bacteria preparation to be used and the composition to be administered will vary according to factors such as the type of disease being treated, the mode of administration, as well as the other ingredients in the composition. The composition may be composed of from about 10 μg to about 10 mg of EFD killed Gram positive bacteria preparation.

According to an advantageous embodiment of the present invention, the EFD administration is oral or parenteral. Preferably, said administration is performed every week, every month, every three month or every six month.

For instance, during an oral administration of the composition of the invention, host to be treated could be subjected to a 1 dose schedule of from about 10 μg to about 10 mg of EFD killed Gram positive bacteria prepa- ration per day during 3 consecutive days. The treatment may be repeated once one week later.

For parenteral administration, such as subcutaneous injection, the host to be treated could be subjected to a 1 dose schedule of from about 10 μg to about 10 mg of EFD killed Gram positive bacteria preparation per month or every 6 months.

The treatment may be either preventive or curative. The preventive treatment may be administered to patients as soon as the diagnostic of the disease has been established for a certainty and/or remitting patients. The curative treatment may be given to a patient, just at the beginning of the recurrence of the symptoms of the disease. Example 1 : EFD treatment promotes in vitro the Treg differentiation from a naϊve T lymphocyte population.

Plasmacytoid dendritic cells (pDCs) expressing ICOS-L (Ito et at. 2007), collected from draining lymph nodes of a sub-cutaneous site of EFD injection are able to promote in vitro the Treg differentiation from a naϊve T lymphocyte population. a) Cells were collected from draining lymph nodes 4 days after subcutaneous injection of 100μg of heat-killed BCG (HK-BCG), live BCG, or EFD, or PBS in naϊve mice.

Total number of cells was in the same range (1 to 2 X 10⁷ cells per lymph node). They were analysed for their membrane markers using adequate monoclonal antibodies using FACS analysis.

A slight, (not significant) increase of dendritic cells (CD11c+ Class2+ cells) number was observed after EFD treatment (Fig. 1). A significant increase number of plasmacytoid dendritic cells (CD11c+B220+ cells) was observed after EFD treatment. The number of cells bearing the ICOS-L marker, considered to be a specific marker for plasmacytoid dendritic cells able to promote differentiation and, or proliferation of Treg cells, was 10 fold increased after EFD (Fig. 2). b) When these CD11c+ B220+ cells were sorted and cultivated 72h in vitro in presence of naϊve Tcells the Foxp-3 transcription factor, considered to be specific of Tregs, was highly expressed only in Tcells cultivated in presence of CD11c+B220+ cells sorted from draining lymph nodes of EFD treated mice (Fig. 3). The CD11c+B220+ cells collected from BCG injected mice induced also the Foxp-3, it has to be noted that they were cultivated at the same number than those collected after EFD. In vivo they were at least 5 fold less numerous after BCG than after EFD as indicated previously. Example 2: EFD treatment induces Tregs in a Crohn mouse model.

Rag2-/- mice develop inflammatory bowel disease after intravenous transfer of naϊve CD4+CD45^high Tcells (Fiona POWRIE).

Naϊve cells were collected from 4 spleens of C57BI/6 mice. They were sorted by FACS Aria in order to select only CD4+CD45^high cells.

300 000 of these sorted cells were transferred intravenously 10 Rag2 -/- mice,

5 of them received at the same time 100μg of EFD subcutaneously at the base of the tail.

The spleens were collected 40 days later. The expression of transcription factors (Tbet, Foxp-3, GATA-3 and RORγt) was evaluated in spleen extracts.

EFD treatment promoted the differentiation and/or multiplication of naϊve Tcells toward Tregs expressing Foxp-3 and increased Tbet expression (Th1 cellular immune response) whereas GATA-3 and RORγt were decreased (Th2 and Th 17 cellular immune response) (Fig. 4). Example 3: EFD treatment induces Tregs in normal mice.

Mice were subcutaneaously injected with 100μg of heat-killed BCG, live BCG or EFD. Spleens were harvested 28 days later. Foxp-3 expression was higher in cell extracts from EFD than HK or live BCG injected mice (Fig. 5).

Example 4 : EFD treatment induces Tregs in an asthma mouse model.

In a murine model of asthma, BP2 mice were sensitized with ovalbumin (OVA), injected subcutaneously two times and challenged intra- nasally 42 days later to induce an airway hyperreactivity. Mice treated with EFD 8 days after sensitisation were protected as shown by their decreased sensitivity to methacholine (Penh values) (Fig. 6).

The crucial role of Tregs was demonstrated by lost of the EFD protective effect after injection of a anti-CD25 monoclonal antibody (BD Biosciences, Ref. 553864, J Exp Med (2000), Vol. 192, pp. 303-309). Contrary to this anti-CD25 mAb, an isotype without any affinity for CD25 shows no result on EFD protective effect, demonstrating that EFD acts via Tregs immune response. Example 5: EFD treatment protects mice against paralysis observed in

EAE

(C57BI/6 mice model)

EAE Induction protocol (Cassan et al. and Chen et al.): C57BI/6 mice (female, 8-9 weeks old) were injected at day 0 with 200 μg of MOG 35-55 peptide (MEVGWYRSPFSRWHLYRNGK; Invitrogen Life Technologies, Carlsbad, CA), per mouse, emulsified in CFA (complete Freund's adjuvant; SIGMA # F5506-10ML) supplement with killed Mycobacterium tuberculosis Bacillus (dessicated Mtb H37RA, DIFCO # 231141 ; total 300 μg per mouse) in equal volumes. Injection : subcutaneous , total volume 200 μl

Pertusis toxin (SIGMA, # P159-40μg): 200 ng per mouse (total) in 0.2 ml PBS at 0 and 48 hours, i.v injection Treatment protocol: Mice received EFD 100 μg /100 μl per mouse in PBS or mannitol by subcutaneous injection at day 0 (25 mice) or at day -14 (10 mice) of EAE induction (day 0), 32 mice (day 0) and 7 mice (day -14), received only PBS. Clinical evaluation Paralytic symptoms were recorded daily between days 12 and

28 after EAE induction. Using a clinical scale,

0 no paralysis (healthy)

0.5 weakness or partial tail paralysis

1 complete tail paralysis 2 complete tail paralysis, and uni/bilateral hind limb weakness

3 unilateral hind limb paralysis

4 bilateral hind limb paralysis

5 forelimb paralysis, motionless a mean of paralytic symptoms was established daily. Results

No mice among the 25 EFD treated mice (day 0) developed paralysis during the period of observation while 20 mice among the 32 which received PBS developed paralysis. The difference between the two groups is highly significant using a Chi2 test (Figure 7).

The difference between EFD treated mice, without symptoms and control mice was highly significant (Figures 8 and 9). It can be concluded that EFD treatment prevents symptoms of EAE from appearing when administered at day 0 or day -14 of EAE induction. Example 6: Effect of EFD treatment on paralysis and survival in the SJL/J mice model of EAE EAE Induction protocol

SJL/J mice (female, 8-9 weeks old) were injected at day 0 with 150 μg of PLP 139-151 peptide (HSLGKWLGHPDKF), per mouse, emulsified in CFA (complete Freund's adjuvant; SIGMA # F5506-10ML) supplement with killed Mycobacterium tuberculosis Bacillus (dessicated Mtb H37RA, DIFCO # 231141 ; total 300 μg per mouse) in equal volumes. Injection : subcutaneous , total volume 200 μl Pertusis toxin (SIGMA, # P159-40μg): 100 ng per mouse (total) in 0.2 ml PBS at 0, i.p injection

Treatment protocol:

In a first experiment, mice received EFD 100 μg /100 μl per mouse in PBS by subcutaneous injection in the tail base at day 0 (9 mice) or day 25 (8 mice) of EAE induction. Control mice (14 mice) were injected accordingly with PBS only.

In a second experiment, mice received EFD 100 μg /100 μl per mouse in PBS by subcutaneous injection at day -30 and at day +25 (10 mice) of EAE induction (day 0). Control mice (8 mice) were injected accordingly with PBS only. Clinical evaluation

Paralytic symptoms (scores) and survival were recorded daily between days 0 and 60 after EAE induction, using a clinical scale as described in example 5. A mean of paralytic symptoms and survival was established daily. Results

The results presented in Figures 10 to 13 show that EFD treatment improves the symptoms of EAE and increase the survival rate.

Claims

I . Preparation of Gram positive bacteria killed by extended freeze-drying (EFD) for preventing or treating a T-regulatory cells (Tregs)- associated disease.

2. The preparation according to claim 1 , which contains more than 50 %, and preferentially more than 90%, of the bacterial protein components which are in a native structure.

3. The preparation according to claim 1 or 2, which contains less than 1.5 % of water, preferably less than 1%, more preferably less than 0.5%.

4. The preparation according to anyone of claims 1 to 3, in which the Gram positive bacteria consist of Gram positive facultative intracellular bacteria.

5. The preparation according to claim 4, in which the Gram positive facultative intracellular bacteria consist of mycobacteria.

6. The preparation according to claim 5, in which the mycobacteria consist of Mycobacterium bovis BCG.

7. The preparation according to anyone of claims 1 to 6, wherein the Tregs are CD4+ CD25+ Foxp3+ T cells.

8. The preparation according to anyone of claims 1 to 7, wherein the disease is a central nervous system (CNS) inflammatory disease.

9. The preparation according to claim 8, wherein the disease is a demyelinating disease.

10. The preparation according to claim 9, wherein the disease is multiple sclerosis or acute-disseminated-encephalomyelitis.

I I . The preparation according to anyone of claims 1 to 10, which is composed of from about 10 μg to about 10 mg of Gram positive bacteria killed by extended freeze-drying.

12. The preparation according to anyone of claims 1 to 11 , which is obtainable by a process comprising the following steps: a. harvesting a culture of live bacteria cells, b. washing the bacteria cells in water or in an aqueous solution of a salt such as borate, c. freezing the bacteria cells in water or in an aqueous solution of a salt such as borate, d. killing the frozen bacteria cells by drying them in a freeze- dryer, for a time sufficient to remove at least 98.5 % of the water, preferably at least 99% of the water, more preferably at least 99.5% of the water, and e. collecting the extended freeze-dried bacteria cells.

13. The preparation according to claim 11 , wherein the washing step b. of said method is omitted.

14. The preparation according to anyone of claims 1 to 13, which is included in a pharmaceutical composition comprising an acceptable carrier, an additive, an immunostimulant, and/or an adjuvant.

15. Product comprising a preparation as claimed in anyone of claims 1 to 14, and at least another product selected from the group consisting of anti-inflammatory and immunoregulatory drugs, as a combined preparation for simultaneous, separate or sequential use in the prevention or the treatment of a CNS inflammatory disease as defined in anyone of claims 8 to 10.