MXPA99005553A - Mycobacterium vaccae - Google Patents

Abstract

Antigenic and/or immunogenic material derived from Mycobacterium vaccae is used to down-regulate Th2 activity of the immune system without up-regulation of Th1 activity. Disorders such as Chronic Fatigue Syndrome, Gulf War Syndrome and Total Allergy Syndrome are treated. The material preferably comprises dead M. vaccae cells in a composition which does not include a non-M. vaccae antigen, immunogen or allergen.

Description

OYCOBAC ERIÜM VACCAE FOR THE REGULATION DESCENDING FROM THE TH2 ACTIVITY OF THE SYSTEM. IMMUNE DESCRIPTION OF THE INVENTION The present invention relates to the treatment of disorders that are characterized by a change in the activity of the immune system from Thl activity to Th2 activity. It is based on the surprising discovery that preparations of Mycobacterium vaccae are capable of effecting a non-specific systemic deviation of Thl, in particular by down-regulation of Th2 activity, without the concomitant up-regulation of Thl activity. The disorders to which the present invention can be applied include Chronic Fatigue Syndrome, Gulf War Syndrome and Total Allergy Syndrome. Patients with Chronic Fatigue Syndrome (CFS) (Turnberg et al.) May have a higher incidence of allergic phenomena, twice as many allergic skin reactions and a lower NK cell activity and a low production of IFN-α. and IL-2 (Interferon-α and Interleukin 2, respectively) (Straus, 1996). Some have high levels of antibodies against Epstein-Barr virus (EB virus), which is also a characteristic of individuals with a decrease in REF .: 30459 activity Thl. They also have changes in mood and depression. All these characteristics are compatible with a change from Thl to Th2 in the cytokine profile. This deviation may be a consequence of the modern lifestyle, which does not expose the population to sufficient Thl-inducing stimuli and rather tends to expose it to immunizations and Th2-inducing allergens. The inventors of the present believe that the Gulf War Syndrome represents a special case of CFS, where the Th.2 inducers can be identified, because the Gulf War personnel were administered multiple inducing vaccines. of Th2. Vaccines or infections can exert a systemic effect and increase or reduce non-specifically the Thl / Th2 cytokine balance of the response against other unrelated antigens (Shaheen et al., 1996, Shirakawa et al. , 1996) and affect (positively or negatively) survival due to unrelated diseases (Aaby, 1996, Aaby et al., 1995). The inventors' belief that these vaccines used in the Gulf •. induced a systemic switching of Th.2, is supported by four characteristics of the vaccination protocol: (i) Pertussis was used as an adjuvant in the British troops in the Gulf and its adjuvant function is potent in Th2 (Mu and Sewell, 1993, Ramiya et al., 1996, S it et al., 1996). (ii) The Gulf War troops were given Th2-inducing immunogens against plague, anthrax, typhoid, tetanus and cholera. Furthermore, such a large load of antigens tends to direct the immune response towards Th.2 (Aaby, 1995, Bretscher et al., 1992, Hernández-Pando and Rook, 1994). (iii) The vaccines were administered after the troops arrived in the war zone or together before they traveled there, at the time when the stress levels were high. Immunization in the presence of high levels of glucocorticoids (i.e. cortisol) directs the response to th.2 (Bernton et al., 1995, Brinkmann and Kristofic, 1995, Ramírez et al., 1996). ,., (iv) Troops were also exposed to carbamate and organophosphorus insecticides and these inhibit the IL-2-driven phenomena essential for normal Thl function (Cásale et al., 1993). The importance of this component is uncertain. However, it is rumored that insecticides were often purchased from local sources in the Gulf, so purity was unknown and more toxic contaminants may have been present. Thus, the multiple vaccines administered under these circumstances could cause a long-term systemic cytokine imbalance. This same effect will occur sporadically in the general population, due to vaccines or other environmental stimuli that induce Th.2 and infection and may account for the widespread incidence of chronic fatigue syndrome. These points are explained in more detail later. The inventors of the present have made the surprising observation that preparations of Mycobacterium vaccae (dead) are potentially able to non-specifically redirect a Thl-Th2 systemic deviation. The effect is to divert the activity of the Th.2 immune system in a manner that encompasses the immune response against antigens that are not present within the injected M. vaccae preparation. This has been demonstrated experimentally in both animal models and humans. Representative experiments will be described in more detail later. In particular, they surprisingly demonstrated a down-regulation of Th2 activity, which may not be coupled to up-regulation of Th1 activity. Briefly, in experimental animals a non-specific systemic deviation can be observed away from Th2 activity when administering M. vaccae, in the form of a reduction in the titer of an IL-4 (Th2) -dependent antibody response against ovalbumin (an allergen). not related to M. vaccae itself), in prein unized mice to establish a Th.2 response. Nail single injection of_. vaccae is capable of causing this effect and additional injections may intensify it. The effect is not specific because it does not require the presence of any component of the ovalbumin in the injected preparation. In humans (patients with cancer) the effect has been demonstrated by the_appearance_ in peripheral blood of lymphocytes that spontaneously secrete IL-2 (a characteristic cytokine of Thl) and a decrease in the T cells that secrete IL-4 (a cytokine characteristic of Th2), after a stimulation with phorbol myristate acetate and calcium ionophore. The percentage of lymphocytes that demonstrate this activated Thl phenotype increases progressively after each successive injection of M. vaccae, reaching a plateau in many individuals after 3 to 5 injections of 10 microorganisms (days 0, 15, 30 and then monthly). In addition, reports of human beings suffering from Chronic Fatigue Syndrome who have been administered preparations of M. vaccae with beneficial effects are included below. M. vaccae has been previously described by the present inventors to be used in the treatment of tuberculosis (GB-A-2156673), cancer (ZA 95/2644), HIV (WO94 / 06466) and chronic inflammation (GB-B- 2252044). International Publication WO92 / 08488 (also of the present inventors) describes its use as an adjuvant for its administration with an antigen of interest, observing a conversion of the T cell component of the antigen response of the Th2 standard to the Thl standard. In International Publication WO92 / 08488 there is no suggestion that M. vaccae can cause a non-specific Th2 / Thl change in the activity of the immune system.; that is, no change other than in the response specifically mounted against the antigen administered with M. vaccae. Furthermore, in International Publication WO92 / 08488 and in no other publication there is a suggestion that Th2 activity can be downregulated without concomitant up-regulation of Thl activity. Reviews of M. vaccae as Thl adjuvant include Abou-Zeid et al. (1997), Skinner et al. (1997a) and Skinner et al. (1997b). See also Mosmann and Sad (1996) whose reviews describe that the upregulation of the Thl cytokine has an inhibitory effect on the proliferation of Th2 cells. The present invention relates in general to the down regulation of Th.2 activity of the immune system of an individual, without the up-regulation of Thl activity, particularly in the treatment of a disorder characterized by a change in the activity of the system immune activity Th1 to Th2 activity, in particular where down-regulation of Th2 activity is beneficial without the concomitant up-regulation of Th1 activity. In one aspect, the present invention provides a method for the treatment of an individual, wherein the method includes administering to the individual antigenic and / or incoregulatory material of Mycobacterium vaccae. Another aspect of the present invention provides the use of antigenic and / or immunoregulatory material of Mycobacterium vaccae in the manufacture of a medicament for the treatment of an individual. Another aspect of the present invention provides a substance or composition for use in the treatment of an individual, wherein the substance or composition includes antigenic and / or immunoregulatory material of Mycobacterium vaccae. Preferably the preparation of M. vaccae is provided without any foreign antigen, immunogen or allergen. The disorders to be treated may be characterized by a general non-specific deviation of the patient's immune system from Thl activity to Th2 activity. These can be evaluated or diagnosed with reference to the decreased production of IL-2 or the increase in the production of IL-4 or IL-5 in the individual, or by the detection of IL-13, as representative of the cytokines of Th2, and interferon? (IFN-y) as representative of Thl cytokines. Other Thl / Tb.2 cytokines can also be taken into account. A non-specific change of Th1 activity to Th2 activity of the immune system, could not be attributed or caused by an exposure of the individual to a particular antigen, or to an infection by a particular pathogen. The individuals to be treated may not suffer from tuberculosis or other infections caused by mycobacteria. Particular disorders that can be treated in accordance with the present invention include Chronic Fatigue Syndrome, Gulf War Syndrome and Total Allergy Syndrome (Straus, 1996). Other disorders in which down-regulation of Th.sub.2 activity without upregulation of Thl activity is beneficial, can be treated in accordance with the present invention. Chronic Fatigue Syndrome can be defined operationally, for example, using the so-called 1994 CDC criteria (Fukuda et al.) Or the so-called Oxford criteria (Sharpe et al.). Current knowledge about Chronic Fatigue Syndrome is summarized and described in Turnberg et al. (published in October 1996). The material of Mycobacterium vaccae can be or include dead cells of M. vaccae. Such cells can be killed for example using radiation, e.g. with 60Cobalt at a dose of 2.5 megarads, chemically or by any other means, although it is preferred to use the autoclave, e.g. at 69 kPa for 10 minutes at 115-125 ° C. In an autoclave, a more effective preparation can be obtained than by irradiation. Before killing the M. vaccae cells, they are grown on. an adequate solid medium. A modified Sauton liquid medium (Boyden et al.) Solidified with agar, preferably 1.3% agar, may be preferred. After aerobic incubation, usually at 32 ° C for 10 days, the microorganisms are harvested, then weighed and suspended in diluent ready for administration. If required, they can be stored before use at 4 ° C. Instead of growing the cells in a solid medium, a liquid medium such as modified Sauton's medium can be used (Boyden et al.), for example in a fer enter. The diluent can be unregulated, pyrogen-free saline solution. Preferably, the pH of the diluent is regulated with borates, preferably containing a surfactant such as Tween 80®. A suitable borate regulatory solution is: Na2B4? 7 * 10H2? - 3.63 g, H3BO3 - 5.25 g, NaCl - 6.19 g, Tween 80® at 0.0005%, distilled water up to 1 liter. These diluents are pharmaceutically acceptable. The results in human beings previously mentioned have been obtained by the administration of M. vaccae in a preparation according to the Good Manufacturing Practices (GMP), SRL172, which is available for human use under several IND for research in the Federal Administration of Medicines. of the United States under and several CTX in the Medicines Control Agency of Great Britain. LPG of acute toxicology has been performed at Huntingdon Research. Phase 1 and Phase 2 safety data were obtained in the United States and are filed with the FDA. The SRL172 is in Phase 3 studies for the immunotherapy of tuberculosis. SRL172 may be preferred for use in the present invention. SRL172 is a formulation of M. vaccae derived from the strain called R877R, which was deposited under the Budapest Convention at the National Type Culture Convention (NCTC) of Great Britain, Central Public Health Laboratory, Colindale Avenue, London NW9 5HT, United Kingdom, on February 13, 1984 under number NCTC 11659. Strain R877R was originally isolated from mud samples from Lango district of Central Uganda, (Stanford and Paul). Other strains of M. vaccae can be used instead of the SRL172. A microorganism can be identified as belonging to M. vaccae by biochemical and antigenic criteria (Bonicke et al.). For the present invention, it is preferred that the M. vaccae material be administered free or substantially free of antigenic or immunoregulatory material that does not belong to M. vaccae. In other words, the medicament or composition to be administered may include, or may consist essentially of, antigenic and / or immunoregulatory material of M. vaccae, such as dead cells, an extract or derivative thereof and a pharmaceutically acceptable diluent. The administration of preference is in "a therapeutically effective amount", this being sufficient to show a benefit to the patient. Such a benefit may be when- less the improvement of at least one symptom. The actual amount administered and the rate and duration of the administration will depend on the nature and severity of what is being treated. The prescription of treatment, for example, decisions on dosage, etc., falls within the responsibility of general practitioners and other medical professionals. A single dose (when given dead cells) will usually contain 10 7 to 1010 killed M. vaccae microorganisms. Patients can be given a single dose of 10 8 to 10 9 killed M. vaccae cells, although the dose can be repeated if necessary, for example at intervals of 2 weeks to 6 months. A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the disorder being treated. The pharmaceutical compositions according to the present invention and for use in accordance with the present invention, may include, in addition to the active ingredient, an excipient, vehicle, pH regulator, stabilizer or other pharmaceutically acceptable material, which are well known to technicians. in the matter. Such materials should not be toxic and should not interfere with the effectiveness of the active ingredient. The precise nature of the vehicle or any other material will depend on the route of administration, which is preferably by injection, for example cutaneous, subcutaneous or intradermal injection. For injection, the active ingredient will be in the form of an aqueous solution acceptable parenterally, which should be free of pyrogens and have a suitable pH, isotonicity and stability. Suitable diluents, which are pharmaceutically acceptable and may be preferred, have already been described above. Oral administration can be used, in which case the pharmaceutical composition will be in the form of tablets, capsules, powders or liquid. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil or synthetic oil. Physiological saline, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may also be included. M. vaccae can be administered by aerosol to the respiratory tract, using a suitable formulation, e.g. including particles of a size that travel to the appropriate parts of the airways. This may be a dehydrated powder rather than an aqueous suspension. Instead of dead cells, material derived from M. vaccae can be used, in particular an extract or a synthetic molecule having the required activity. As already briefly described in previous paragraphs, multiple vaccines, stress and other factors can cause a long-term systemic cytokine imbalance in veterans of the Gulf War. The same effect will occur sporadically in the general population due to vaccinations or other environmental stimuli of Th.2 and infections, and may explain the wide incidence of Chronic Fatigue Syndrome. The potent immunogens can have non-specific long-term systemic effects on the nature of the immune response against unrelated antigens. For example, a measles infection reduces the incidence of atopy and other allergic reactions to the Homemade Dust Mite (Shaheen et al., 1996). Similarly, Japanese children who are positive for the tuberculin skin test are less likely to be atopic compared to children who are tuberculin negative, and their ratio of circulating Thl / Th2 cytokines is higher. In addition, after repeated injections of BCG (Bacillus de Cal ette-Guerin), those in whom tuberculin conversion occurs are more likely to lose their atopic symptoms (Shirakawa et al., 1996). The measles vaccine is another example. This vaccine, when used at the standard dose, reduced mortality by a factor considerably greater than what could be explained by the incidence of measles in the unvaccinated population. Vaccines against diphtheria, tetanus and pertussis (Th2 inducers) do not show this non-specific protective effect (Aaby et al., 1995). However, when a high titre of measles vaccine was used, mortality increased, although the protection against measles itself remained (Aaby, 1995).; Aaby et al. , nineteen ninety five). There is evidence that this increase in mortality was accompanied by .ppr a switch to Th.2 and dose-related increases in the induction of a Th2 component were well established for other immunogens (Bcher et al., 1992; Hernández-Pando and Rook , 1994). The vaccines used in the Gulf were Th2 inducers (plague, anthrax, typhoid, tetanus, cholera) and cumulatively constituted a large antigenic load, favoring Th2. In addition, this trend will have been increased by the use of Pertussis as an adjuvant, since it is a potent inducer of Th2 (Mu and Sewell, 1993, Ramiya et al., 1996, Smit et al., 1996). This property of pertussis has recently led to the discussion of the possibility that its use in children contributes to the contemporary increase in the prevalence of atopy (Nilson et al., 1996, Odent et al., 1994). The innate tendency of these vaccines to direct a Th2 systemic response will have been intensified by endocrine factors secondary to the stress to which Gulf War personnel were exposed at the time of vaccination. Several hormones - steroids modulate T cell responses. Dihydroepiandrostérona (DHEA) or unknown metabolites of DHEA, tend to promote a Thl pattern. Thus, DHEA can restore immune functions in elderly mice and correct the release of deregulated cytokines in older animals (Daynes et al., 1993, Garg and Bondada, 1993). Similar properties of this compound have been tested in elderly humans (Morales et al., 1994). It intensifies the production of Thl cytokines such as IL-2 and IFN-y (Daynes and Araneo, 1989, Daynes et al., 1990, Daynes et al., 1995, Daynes et al., 1991). DHEA * also enhances the secon of IL-2 from T cells of human peripheral blood (Suzuki et al., 1991). These effects of DHEA are the inverse effect of glucocorticoids such as cortisol, which intensify Th2 activity and have a synergistic effect with Th2 cytokines (Fischer and Konig, 1991, Guida et al., 1994, Padgett et al. 1995, Wu et al., 1991). If the proliferation of "naive" T lymphocytes is directed in the presence of a non-specific stimulus (Brinkmann and Kristofic, 1995) or by an antigen (such as after a vaccine), T lymphocytes with a Th2 cytokine profile will develop. This has been clearly demonstrated with spleen cells from "clean" laboratory rodents, which have few memory cells under normal circumstances (Ramírez et al., 1996). The general "baseline" is that cortisol favors the development of a Th2 cytokine profile from naive cells (Brinkmann and Kristofic, 1995). This point should not be confused with the fact that the secry activity of established Th2 cell cytokines is easily inhibited by cortisol. Thus, the use of cortisol analogs for conventional treatments for Th2-mediated diseases, such as eczema, asthma and hay fever, could work through anti-inflammatory effects and by reducing the production of cytokines by Th2 cells (Corrigan et al. , 1995) and at the same time the use of cortisol will motivate the perpetuation of the underlying problem by directing newly recruited T cells towards Th2 activity. Psychological and physical stress activates the hypothalamo-pituitary-adrenal axis and also causes a variety of changes including an increase in cortisol production. Excessive exercise and lack of food and sleep resulted in a drop in the ratio of DHEA to cortisol (DHEA / cortisol ratio). This correlated directly with a decrease in the delayed hypersensitivity (HR) response (a Thl marker) and there was a simultaneous elevation of serum IgE levels. IgE was totally dependent on the production of Th2 cytokines (Bernton et al., 1995). This is to be expected in light of the known effects of DHEA and cortisol mentioned above. Another "example of the switching effect of Thl -» Th2 of stress, is the increase of antibodies against EB virus in students who reacted in a stressed way to their exams. This virus is usually controlled by a Thl response and cytotoxic T cells and loss of control results in virus replication and an increase in antibodies (Zwilling, 1992). Similarly, peripheral blood leukocytes of medical students during their examination periods demonstrated less mRNA for IFN-y, which is a Thl cytokine (Glaser et al., 1993).

Similar points can be demonstrated in a more controlled way in animals. Stress due to overpopulation or restrictions may increase the growth of mycobacteria in tuberculous mice (Brown et al., 1993, Tobach and Bloch, 1956). This is a model very sensitive to the presence of even a small component of Th2 (Rook and Hernández-Pando, 1996). Other examples have also been described (Moynihan, 1994). There is considerable evidence that depression can be associated with excessive effects mediated by cortisol in the brain (reviewed in (Raven et al., 1996)) and stress can cause depression. Thus, depression (as seen in CFS and GWS) tends to be associated with Th2-mediated disorders, such as asthma, eczema and some endocrine changes are common to Th2 disorders and depression (Holsboer et al. ., 1984, Rupprecht et al., 1995). In tuberculosis there is a systemic change of Th.2 (Rook and Hernández-Pando, 1996) and a unusual pattern of adrenal steroid metabolites is exerted in the urine (Rook et al., 1996). Thus, there is an increase in cortisol metabolites in relation to cortisone metabolites. The treatment of the disease restores the domain of Thl and corrects the pattern of steroid metabolites, such that the metabolites of cortisone are increased in relation to cortisol metabolites (Rook et al., 1996). The treatment of depression with. the drug metyrapone causes the same change in steroid metabolites (e.g., an increase in metabolites or cortisone in relation to cortisol metabolites) (Raven et al., 1995). The preparation of M. vaccae SRL172 is effective in the immunotherapy of tuberculosis. As described above, vaccines can induce non-specific systemic changes of the Thl / Th2 balance, as well as the specific antigen immunization that is normally sought. The treatment of CFS may require the induction of a systemic non-specific Thl deviation in order to restore the Thl domain on Th2 that is characteristic in normal healthy individuals. This is the opposite of the deviation that could have been induced by vaccines received by personnel in the Gulf. Experimental evidence provided in the present, now shows surprisingly that preparations of Mycobacterium vaccae can reduce the unfavorable * '- deviation from Thl to Th.2 that can be observed in veterans of the Gulf War. The present invention will now be illustrated without limitations with reference to experimental Examples and to the following Figures: Figure 1 shows the change in total serum IgE (ng / ml ± SD) in mice, several days after the first immunization with ovalbumin, calculated with respect to the value on day 46. The black circles are for M receptors. vaccae the white squares are for the controls with saline solution. For convenience, the days of immunization with ovalbumin and administration of M. vaccae or saline are indicated. Figure 2 shows the measurements of IL-5 (pg / ml ± SD) released in vivo - from spleen cells taken from mice challenge with ovalbumin. The minimum concentration detected by the assay used was 15.6 pg / ml. Figure 3 shows the effect of the treatment with subcutaneous injections of saline, («0, in comparison with preparations of 10 cells of M. vaccae (o) on total serum IgE (ng / ml), in BALB / c mice previously immunized with OVA (Ovalbumin). (Fig. 3A) 10 unmodified M. vaccae cells (Mv), M. vaccae cells conjugated with OVA (Mv-OVA) or (Fig. 3C) control of M. vaccae cells washed with NaCl and treated with periodate (NaCl-Mv). The data are expressed as the relative change of the readings obtained with the samples taken on day 46, ± SD. The immunization and treatment programs are indicated in Figs. (OVA: immunization with OVA, 50 μg in AIF). Figure 4 shows the effect of treatment with subcutaneous injections of saline (•), in comparison with preparations of 10 cells of M. vaccae (o) on specific IgE against OVA, in BALB / c mice previously immunized with OVA (Fig. 4A) unmodified M. vaccae cells (Mv) (OD 405 nm), (Fig. 4B) M. vaccae cells conjugated with OVA (Mv-OVA) (OD 405 nm), or (Fig. 4C) M. vaccae control cells washed with NaCl and treated with periodate (NaCl-Mv) (ng / ml). The data are expressed as the relative change of the readings obtained with the samples taken on day 46, ± SD. The immunization and treatment programs are indicated in the Figures. Figure 5 shows the production of IL-2 by splenocytes cultured with OVA. On day 109 spleens were obtained from mice that had been immunized with OVA on days 0 and 24, and treated at days 53 and 81 7 with saline, 10 unmodified M. vaccae (Mv) cells, M. vaccae conjugated with OVA (Mv-OVA) or the corresponding control of M. vaccae cells washed with NaCl and treated with periodate (NaCl-Mv). The culture supernatants were collected for stimulation with IL-2 after "24 hours." The data are presented as the mean levels of cytokine ± SD (pg / ml) Figure 6 shows the production of IL-5 by cultured splenocytes OVA On day 109 spleens were obtained from mice that had been immunized with OVA on days 0 and 24, and treated on days 53 and 81 with • • saline, 10 unmodified M. vaccae cells (Mv), M. vaccae cells conjugated with OVA (Mv-OVA) or the corresponding control of M. vaccae cells washed with NaCl and treated with periodate (NaCl-Mv). The supernatants of the cultures were collected for estimation of IL-5 after 48 hours. The data are presented as the average levels of IL-5 ± SD (pg / ml). Figure 7 shows the effect of treatment with a single subcutaneous injection of different doses of M. vaccae compared to saline on serum IgE levels in BALB / c mice previously immunized with OVA. Serum IgE is expressed as the change (ng / ml) relative to the readings obtained with the samples taken on day 32, before treatment with M. vaccae or saline solution (mean ± SD). Serum samples were collected after treatment on days 52 and 70 (i.e., 10 and 28 days after treatment with saline or M. vaccae on day 42). Immunization and treatment programs are indicated in the experimental methods. The comparison between the different groups was carried out by the Mann-Whitney U test. Figure 8 shows the production of IL-2 (Fig. 8A) and IL-4 (Fig. 8B) by splenocytes with OVA.BALB / ca mice who were administered OVA twice before the different doses of M. vaccae: 10 7, 108 or 109 (pg / ml). The splenocytes were harvested and cultured with OVA on day 82. Immunization and treatment programs are indicated in the experimental methods. The supernatants of the cultures were collected for the IL-4 estimation after 48 hours and for the estimation of IL-2 after 24 hours. The data are presented as the average cytokine levels ± SD. The comparison between the different groups was carried out by the Student's T test (saline: immunization twice with OVA before 7 treatment with saline, 10 Mv: immunization two times with OVA before treatment with 10 M. vaccae). Figure 9 shows the effect of the treatment with 7 two subcutaneous injections (days 53 and 81) of 10 cells of M. vaccae (°) compared to saline (•) on total serum IgE (Fig. 9A) and OVA specific IgE (Fig. 9B). The immunization and treatment programs are indicated in the Figure. The serum IgE is expressed as the ± SD change in relation to the readings obtained with the sample taken on day 46 (ng / ml). The comparison between the different groups was carried out using the Mann-Whitney U test (OVA: immunization with ovalbumin, Mv: immunization with M. vaccae). Figure 10 shows the production of IL-5 (Fig. 10A), IL-2 (Fig. 10B) and IFN-? (Fig. 10C) by splenocytes cultured with OVA (pg / ml). The splenocytes were harvested and cultured with OVA on day 109. The immunization and treatment programs are indicated in the Figure. The supernatants of the cultures were collected for the estimation of IL-5 after 48 hours and for the estimation of IFN-y and IL-2 after 24 hours. The data are presented as the average cytokine levels ± SD. The comparison between the different groups was performed by the Student test (saline: immunization twice with OVA before treatment with saline solution two 7 times; 10 Mv: immunization twice with OVA before 7 treatment twice with 10 M. vaccae). All documents mentioned herein are incorporated by reference. EXAMPLE 1 Administration of M. vaccae to experimental animals and demonstration of the non-specific decrease resulting from Th2 activity of the immune system.

BALB / c mice from 6 to 8 weeks of age were immunized with 50 μg ovalbumin emulsified in oil (incomplete Freund's adjuvant) on days 0 and 24. This is known to evokes a strong Th2 pattern of response, accompanied by production of IgE and inducing the release of two Th2 cytokines, IL-4 and IL-5. Afterwards, the animals received saline solution 7 or 10 cells of M. vaccae activated in autoclave, on days 53 and 81 (by subcutaneous injection). Serum samples were taken on days 46 (i.e., before treatment with saline or M. vaccae) and on days 67, 91 and 109. Spleen cells were then harvested to challenge with ovalbumin in an in vi tro culture system. The supernatants of the cultures were harvested at 48 hours for the cytokine assay by ELISA. Capture assays were used with Pharmingen antibody pairs. IL-5 # 18051D capture; # 18062D (biotinylated) detection. The binding of the detection antibody was revealed with streptavidin-horseradish peroxidase conjugate (Dako P0397) and the CHO was ABTS (Sigma A-1888). IgE # 02111D capture; # 02122D (biotinylated) detection. The binding of the detection antibody was revealed with avidin-alkaline phosphatase conjugate (Sigma E-2636) and the chromogen was pNPP (Sigma N-2770). The results of the IgE levels are expressed as the change of ng / ml compared to the serum concentration on day 46 (the last value before treatment). So, the value of day 46 is subtracted from the value in each subsequent indentation. Results Injections of M. vaccae reduced the elevation of IgE levels caused by immunization with ovalbumin. The reduction caused by the treatment with M. vaccae was significant in all the points tested with respect to time. The results are shown in Figure 1. Similarly, the spleen cells of the immunized animals did not release IL-5 in vitro in response to ovalbumin if the donor animals had been treated with M. vaccae, whereas the cells of Spleen from immunized animals treated with saline, released large amounts of IL-5 in response to ovalbumin. The results are shown in Figure 2. No IL-5 was detected in the control wells grown without ovalbumin (data not shown). Conclusion M. vaccae will reduce a Th2 response pattern, even when administered: (i) after immunization with a potent allergen (in this case ovalbumin), and (ii) without epitopes of the Th2-inducing molecule. Therefore, there is a non-specific systemic down regulation of the Th2 response pattern, which does not depend on a direct adjuvant effect on the allergen itself. EXAMPLE 2 Effect of different preparations of M. vaccae on serum IgE. In another experiment to determine the effect of the M. vaccae-OVA conjugate on the serum IgE titer, BALB / c mice were immunized with OVA in AIF twice, to induce an IgE response; then they were treated twice with saline, with unmodified M. vaccae cells, with M. vaccae-OVA conjugate, or with the corresponding "imitation" that does not contain OVA-conjugated preparation, M. vaccae washed with NaCl, treated with periodate (NaCl-Mv). On day 46, mice immunized with OVA had 112.9 ± 10 (SD) ng / ml IgE, compared to 55.4 ± 1.7 (SD) ng / ml in non-immunized animals (p <0.1). For each mouse, the value of day 46 was used to normalize the data to an initial value of O (i.e., day 46) and subtracted from each subsequent value. Therefore, the plotted values are the ng / ml changes in relation to day 46. The total serum IgE in the control mice (treated with saline) increased steadily during the duration of the experiment (Figs 3A-C ). There was a suppression of total IgE in mice treated with unmodified M. vaccae (Fig. 3A). In contrast, the preparations washed with NaCl and treated with periodate were not effective for suppression of IgE, either conjugated with OVA (Fig. 3B) or unconjugated (Fig. 3C), although a dose of the NaCl-Mv control demonstrated a transient suppression of total serum IgE (p <0.05) (Fig. 3C). These results indicated that the ability to down-regulate IgE was abolished by the preparation washed with NaCl or by the conjugation procedure. In contrast, neither the unmodified M. vaccae cells nor the NaCl-Mv control had a down-regulation effect on the OVA-specific IgE (Figs 4A and 4C). In addition, the OVA-specific IgE response was actually enhanced by treatment with the Mv-OVA conjugate (Fig. 4B). EXAMPLE 3 Effect of different preparations of M. vaccae on the production of IL-2 in spleen cells.

Spleen cells from mice immunized with OVA were harvested on day 109 and cultured with OVA in vitro. Spleen cells from OVA-sensitized mice that were treated with saline did not produce IL-2 in response to OVA in vitro. Spleen cells from immunized mice treated with the M. vaccae preparation that did not contain OVA components, produced detectable levels of IL-2 in response to OVA, but the increase was not significant. In contrast, spleen cells from mice treated with the M. vaccae preparation containing the OVA conjugate released high levels of IL-2 in response to OVA (p <0.05 compared to saline receptors) ( Fig. 5). These results revealed that the conjugation of OVA in M. vaccae increased the production of IL-2 in response to OVA. Thus, there is no significant increase in the Thl response to ovalbumin, unless ovalbumin is included (conjugated with) in M. vaccae. In addition, such a dome was indicated, when ovalbumin is included, although the Thl response then becomes highly significant, the suppression of Th.2 does not improve and could be diminished (see other Examples herein).

EXAMPLE 4 Effect of different preparations of M. vaccae on the production of IL-5 by spleen cells. Spleen cells from mice sensitized with OVA produced high levels of IL-5 in response to OVA (Fig. 6). However, cells from mice immunized with OVA that had been treated with M. vaccae, either unmodified, washed with NaCl, or conjugated with OVA, all released significantly less IL-5 in response to OVA. Thus, in contrast to the results observed when the IgE was measured, neither the conjugation procedure itself nor the presence of OVA conjugated with M. vaccae. had no effect on down-regulation of IL-5 release. If anything, the ability to stop IL-5 was reduced by the conjugation of the allergen with M. vaccae, since the relevant control for Mv-OVA was the preparation NaCl-Mv (conjugate imitation). EXAMPLE 5 Effect of different doses of M. vaccae on the Serum IgE in animals. To determine the effect of different doses of M. vaccae on total serum IgE, mice were immunized BALB / c with OVA in AIF twice (days 0 and 21) to induce IgE responses. These were mixed on day 32 and then treated with saline and then with 107, 108 or Q 10 cells of M. vaccae on day 42. On day 32, mice immunized with OVA had 117.09 ± 35.81 (SD) ng / ml IgE, compared with 69.27 ± 6.09 (SD) ng / ml in animals not immunized (p <0.001). For each mouse, the value of day 32 was used to normalize the data to an initial value (i.e. day 32) of 0 and subtracted from each subsequent value. Therefore, the graphed values are the ng / ml changes in relation to day 32. The IgE response in control mice (treated with saline) had increased further to days 52 and 70, in which samples were taken additional (ie 10 and 28 days after the treatment of day 42) (Fig. 7). In contrast, the increase in IgE level was suppressed in mice treated with M. vaccae at 10, 8 and 109 (Fig. 7). All p values are less than 0.01 among mice treated with saline and treated with different doses of M. vaccae. EXAMPLE 6 Effect of treatment with M. vaccae on the production of cytokines by spleen cells. BALB / c mice were subjected to the same protocol used for Example 5. Then, on day 82, the spleen cells were harvested and cultured with OVA, M. vaccae and concanavalin A (ConA) in vitro. Spleen cells from the group treated with saline produced IL-4, but not IL-2, in response to OVA (Figs 8A-B). Splenic cells from mice immunized with OVA that had been treated with 10 inactivated autoclaved M. vaccae cells demonstrated IL-2 synthesis and a decrease in IL-4 production in response to OVA. The synthesis of IL-2 in response to OVA was also observed using spleen cells from mice treated with 10 or 10 cells of M. vaccae. Note that the increase in IL-2 production in mice treated with M. vaccae was not significant (the ED being greater than the answers), involving only some of the mice in the group. Most of the treated mice did not produce any amount of IL-2. EXAMPLE 7 Effect of two doses of M. vaccae on serum IgE. 7 Since it had previously been shown that 10 M. vaccae cells were the optimal dose to elicit Thl responses against their own antigens and that it had been the most effective dose in the pilot experiments (Fig. 7), this dose was selected for the later studies.

BALB / c mice were immunized with OVA twice, ie on day 0 and day 24, to induce IgE responses and were bled on day 46 and then treated with saline p with 10 cells of M. vaccae twice On days 46 and 81. On day 46, mice immunized with OVA had 112.9 ± 10 (SD) ng / ml IgE, compared to 55.4 ± 1.7 (SD) ng / ml in non-immunized animals (p <0.01) . For each mouse, the value of day 46 was used to normalize the data to an initial value (i.e. day 46) of 0 and subtracted from each subsequent value. Therefore, the plotted values are the ng / ml changes in relation to day 46. The total serum IgE response in the control mice (treated with saline) increased steadily during the duration of the experiment. In contrast, the increase in IgE was suppressed in mice treated with 10 inactivated M. vaccae cells in an autoclave (Fig. 9A). Meanwhile, the suppression of IgE antiOVA by the treatment of M. vaccae was not significant in this trial (Fig. 9B). However, additional experiments showed that BALB / c mice sensitized to OV that received M. vaccae 4 times had significantly lower OVA-specific IgE titers. IgG1 and IgG2a antibodies against OVA were not affected by treatment with M. vaccae. E EMPLO 8 Effect of treatment with M. vaccae on the production of IFN-? IL-2 and IL-5 by the spleen cells. BALB / c mice were subjected to the same protocol used for Example 7. Later in the day 109, the spleen cells were harvested and cultured with OVA in vitro. Spleen cells from the group treated with saline produced high levels of IL-5, but not IL-2, in response to OVA. However, cells from mice immunized with OVA that had been treated twice with 10 inactivated M. vaccae cells in autoclave, did not release IL-5 in response to OVA (Fig. 10A). The production of IL-4 in response to OVA by spleen cells from mice had received two doses of 10 cells of M. vaccae, appeared to be reduced, but the levels of this cytokine were too close to the detection limits of the immunoassay as to be reliable. As demonstrated previously (Fig. 8A), the production of IL-2 induced by OVA was again detectable in the supernatant of spleen cells of some mice treated with M. vaccae (Fig. 10B), although in a statistically non-significant way . A decrease in Th2 was observed in animals that did not show increases in IL-2. There were no differences in IFN- and OVA-induced synthesis by spleen cells from different groups (Fig. 10C). DISCUSSION BALB / c mice that received two immunizations with OVA in AIF, developed a typical Th2 response. There was an increase in serum IgE and the spleen cells from these mice released IL-5 but not IL-2 in response to OVA in vitro. The most surprising result is that this continuous allergen-specific response in BALB / c mice was downregulated by a treatment with a low dose of killed M. vaccae cells without the need for OVA or OVA epitopes to be present in the preparation of M. vaccae. Similarly, our experiments revealed that high levels of serum IgE induced by OVA were suppressed by a wide range of doses of M. vaccae. This was unexpected because previous dose-response studies in mice identified a 7 titer of 10 as the optimal one to induce a Thl response against the mycobacterial antigens contained within M. vaccae, without any detectable Th2 component, while a concentration of 10 induces a mixed Th1 + Th2 response. For all these reasons, it is likely that. The effects of M. vaccae in this model may not work through the suppressive effects of Thl cytokines. An additional level of regulation of IgE levels may be attributable to the induction of factors that bind to IgE. There is no significant increase in the Thl response against ovalbumin, unless ovalbumin (conjugated with) is included in the preparation of M. vaccae and, when included, although the Thl response becomes highly significant, the suppression of Th2 does not improve, and could be diminished. EXAMPLE 9 Measurement of the Th2 and Thl cytokine profiles. There are some data on the cytokine profiles in CFS and Gulf War Syndrome, but these are conflicting and use methods that are not reliable, although the general clinical picture suggests an increase in the Th2 / Thl ratio as described above ( Straus, 1996). The optimal technology to demonstrate this is flow cytometry to accurately enumerate numbers and percentages of T cells from peripheral blood that spontaneously secrete IL-2 (Thl) or IL-4 or IL-5 (both Th2), or that can be induced to secrete these cytokines after in vitro stimulation with calcium ionophore and phorbol myristate acetate (AMF). (The secretion is in fact blocked by an inhibitor (monesin), in such a way that the cytokine accumulates inside the cell, where it is detected (after fixation and permeabilization) with a monoclonal antibody labeled with a fluorochrome that is appropriate) . Using this technology, Th.2 cells are extremely rare in the peripheral blood of normal donors, but may be common in the circulation of patients with chronic infection or cancer (unpublished observations). A second technique is the direct measurement of cytokine levels in plasma or serum. Interleukin 13 (IL-13) can be detected as a representative of Th2 cytokines, and interferon and (IFN-?) As a representative of Thl cytokines. The cytokine profiles of Gulf War veterans are examined using one of these techniques. The determination of a Thl-Th2 deviation is indicative of the applicability of the administration of M. vaccae to improve the symptoms.

EXAMPLE 10 Preparation of M. vaccae for administration. M. vaccae was grown on a solid medium including modified Sauton's medium (Boyden et al.) Solidified with 1.3% agar. The medium was inoculated with the microorganisms and incubated aerobically for 10 days at 32 ° C, to make possible the growth of the microorganism in the plate. The microorganisms were then harvested and weighed and suspended in diluent, to obtain 100 mg of -microorganisms / ml of diluent. Subsequently, the suspension was further diluted with pH regulating saline to obtain a suspension containing 10 mg by wet weight (about 10 cells) of the microorganism / ml of diluent and distributed in 5 ml multiple dose vials. The vials containing live microorganisms were subjected to autoclave sterilization (115-125 ° C) for 10 minutes at 69 kPa, to kill the microorganisms. The therapeutic agent thus produced was stored at 4 ° C before use. EXAMPLE 11 Use of M. vaccae in the treatment of a disorder characterized by a change of Thl / Th2 cytokines. 0.1 ml of the suspension prepared according to Example 3 is vigorously agitated, containing 1 mg by wet weight (approximately 10 cells) according to claim M. vaccae, and immediately afterwards it is administered by intradermal injection on the left deltoid muscle. The Thl and Th2 cytokine levels in the patient can be measured periodically to confirm the change from Th2 to Th1, particularly down-regulation of Th2 activity, while an improvement in the patient's symptoms is observed. E-JEMPLO 12 Treatment of patients with Chronic Fatigue Syndrome using M. vaccae. (1) A 70-year-old patient who was being treated with SRL-172 for squamous cell lung cancer and with a diagnosis of Chronic Fatigue Syndrome, reported with excitement, the improvement of symptoms of the latter after two injections of SRL-172. She felt much better about herself and "her miseries were gone". As an organ teacher, she had taken several additional students - indicating her improvement. This patient had been treated with SRL-172 for more than 1 year and reports continued wellness. (2) One patient had a history of CFS for 7-8 years with symptoms that included "general fatigue, migraines, depression, food allergies, and lymphadenopathy." After two injections of SRL-172 she reported improvement.Their throat and gland irritations Her swelling and food allergies had improved a lot and she needed a lower dose of thyroxine, she spoke much brighter and said she was less depressed. (3) A patient's symptoms took the form of headaches, depression and multiple food allergies. Many other forms of treatment without effect were tried in the treatment with SRL-172. In an attempt to control its symptoms, it came to take large quantities of Nalcron regularly (80 capsules of Nalcron, 100 mg, before and after a meal each Since she has taken SRL-172 at 2-month intervals, her symptoms of CFS, as well as her food allergy, have improved considerably. She believes that she is doing very well as long as she continues with her regular injections. References Aaby, P. (1995) Soc. Sci. Med. 41: 673-86. Aaby et al. , (1995) Brit. Med. J. 311: 481-5. Abou-Zeic et al. , (1997) Infect. I mun. 65: 1856-1862. Bernton et al. , (1995) Ann. NY Acad. Sci. 774: 217-231. Bonicke et al. , (1964) Zentr albl. Bakteriol. Parasitenkd Infection skr. Hyg. Abt. 1, Orig., 192: 133.

Boyden et al., (1955) J. Im unol. 75: 15. Bretscher et al., (1992) Science 257: 539-542. Brinkmann et al., (1995) J. Immunol. 155: 3322-8 Brown et al., (1993) Infect. Im a. 61: 4793-4800. Cásale et al., (1993) Immunopharmacol Immunotoxicol 15: 199-215. Corrigan et al., (1995) Am. J. Respir. Cell. Mol. Biol. 12: 567-78. Daynes et al., (1989) Eur. J. Im unol. 19: 2319-2325. Daynes et al., (1990) J. Ex. Med. 171: 979-996. Daynes et al., (1993) J. Im unol. 150: 5219-5230. Daynes et al., (1995) J. Invest. Dermatol. 105: 14E-19S.

Daynes et al., (1991) Res. Immunol. 142: 40-45. Fischer et al., (1991) Immunology 74: 228-233. Fukuda et al., (1994) Ann. Int. Med 121: 953-9. Garg et al., (1993) Infect. Impiun 61: 2238-2241. Glaser et al., (1993) Behav. Neurosci. 107: 525-9. Guida et al., (1994) Blood 84: 2733-40. Hernández-Pando et al :, (1994) I munology 82: 591-595. • Holsboer et al., (1984) New Eng. J. Med. 311: 1127-. Morales et al., (1994) J. Clin. Endocrinol Metab. 78: 1360-1367. Moynihan, J. 81994) Advances: Mind Body Health 10: 23-28.

Mu et al., (1993) Infect. I mun. 61: 2834-2840.

Nilsson et al., (1996) JAMA 275: 760. Odent et al., (1994) JAMA 272: 592-593. Padgett et al., Ann. NY Acad. Sci. 774: 323-325. Ramírez et al., (1996) J. I munol. 156: 2406-2412. Ramiya et al., (1996) J. Autoimmun. 9: 349-356. Raven et al., (1995) Clin. Endocrinol 43: 637-644. Raven et al., (1996) Psychoneuroendocrinology 21: 277-286. Rook et al., (1996) Annu. Rev. Microbiol. 50: 259-284. Rook et al., (1996) Q. J. Med. 89: 333-341. Rupprecht et al., (1995) Psychoneuroendocrinology 20: 543-551. Shaheen et al., (1995) Lancet 347: 1792-6. Skinner et al., (1997a) Vaccines beyond 2000, July 6-9, Queensland, Australia, Summary. Skinner et al., (1997b) Tnjfect. I mun. , 65: 4525-4530. Stanford and Paul, (1973) Ann. Soc. Beige Med. Trop. 53: 141-389. Sharp et al., (1991) J. R. Soc. Med. 84: 118-21. Shirakawa et al., (1996) Science, in press. S it et al., (1996) Transplant Proc. 28: 665-666. Straus, S. (1996) NIH Publication No. 90-3059: 1-5. Suzuki et al., (1991) Clin. Immunol. Immunopathol. 61: 202-211. Tobach et al., (1956) Am J. Physiol. 187: 399-402.

Turberg et al. , (1996) Report of a joint working group of the Royal Colleges of Physicians, Psychiatrists and General Practitioners. London: Royal College of Physicians 58: vol .. 1). Wu et al. , (1991) J. Clin. Invest. 87: 870-877. Zwilling, B. S. (1992) ASM News 58: 23-25. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Claims (9)

1. RESINS The invention having been described as an antecedent, the content of the following claims is claimed as property: 1. The use of antigenic and / or immunoregulatory material derived from Mycobacterium vaccae in the manufacture of a medicament, characterized in that the medicament does not include any antigen, immunogen or allergen that does not belong to M. vaccae, for the treatment of Chronic Fatigue Syndrome by the down regulation of the Th2 activity of the immune system of an individual, without the up-regulation of the Thl activity of the individual's immune system.
2. 2. The use according to claim 1, characterized in that the antigenic and / or immunoregulatory material derived from M. vaccae comprises dead cells of M. vaccae.
3. 3. The use according to claim 2, characterized in that the cells have been killed by subjecting them to autoclaving.
4. 4. A composition characterized in that it comprises antigenic and / or immunoregulatory material derived from Mycobacterium vaccae and that has no antigen, immunogen or allergen that does not belong to M. vaccae, to be used in the treatment of Chronic Fatigue Syndrome, by means of the regulation descending Th2 activity of the immune system of an individual, without up-regulation of the Thl activity of the individual's immune system.
5. 5. A composition according to claim 4, characterized in that the antigenic and / or immunoregulatory material derived from M. vaccae, comprises dead cells of M. vaccae.
6. 6. A composition according to claim 5, characterized in that the cells have been killed by subjecting them to an autoclave sterilization.
7. 7. A method of treating Chronic Fatigue Syndrome by down-regulating the Th.2 activity of the immune system of an individual, without the up-regulation of the Thl activity of the immune system of an individual, characterized in that said method comprises administering to the individual is a composition comprising antigenic and / or immunoregulatory material derived from Mycobacterium vaccae and having no antigen, immunogen or allergen that does not belong to M. vaccae.
8. 8. A method according to claim 7, characterized in that the antigenic and / or immunoregulatory material derived from M. vaccae comprises dead cells of M. vaccae.
9. 9. A method according to claim 8, characterized in that the cells have been killed by subjecting them to autoclaving.