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Title:  Mycobacterium vaccae for down-regulation of the Th2 activity of the immune system

United States Patent:  6,878,377

Issued:  April 12, 2005

Inventors:  Rook; Graham A. (London, GB); Stanford; John L. (London, GB); Zumla; Alimuddin L. (London, GB)

Assignee:  Stanford Rook Limited (London, GB)

Appl. No.:  761762

Filed:  January 18, 2001

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 of the Invention

The present invention relates to treatment of disorders which are characterised by a shift in activity of the immune system from Th1 activity to Th2 activity. It is founded on the surprising discovery that preparations of Mycobacterium vaccae are able to effect a non-specific systemic Th1 bias, in particular by down-regulation of Th2 activity without concomitant up-regulation of Th1 activity. Disorders to which the present invention may 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 the number of allergic skin reactions, and low activity of NK cell activity and low production IFN.gamma. and IL-2 (Straus, 1996). Some have high levels of antibody to EB virus, which is also a feature of individuals with a decreased Th1 activity. They also have mood changes and depression. These features are all compatible with a Th1 to Th2 switch in cytokine profile. This bias may be a consequence of the modern life-style, which fails to expose the population to sufficient Th1-inducing stimuli, and rather tends to expose it to Th2-inducing immunisations and allergens.

The present inventors believe that Gulf War Syndrome represents a special case of CFS, where the Th2-inducing stimuli can be identified, because Gulf War personnel were given multiple Th-2-inducing vaccinations. Vaccinations or infections can exert a systemic effect, and non-specifically increase or reduce the Th1/Th2 cytokine balance of the response to other unrelated antigens (Shaheen et al., 1996, Shirakawa et al., 1996), and affect (positively or negatively) survival from unrelated diseases (Aaby, 1996, Aaby et al., 1995). The inventor' belief that these vaccinations used in the Gulf induced a systemic Th2 switch is supported by four features of the vaccination protocol:

(i) Pertussis was used as an adjuvant in British troops in the Gulf and its adjuvanticity is potently Th2 (Mu & Sewell, 1993, Ramiya et al., 1996, Smit et al., 1996).

(ii) Gulf War troops were given Th2-inducing immunogens, against plague, anthrax, typhoid, tetanus and cholera. Furthermore, such a large antigen load tends to drive the response towards Th2 (Aaby, 1995, Bretscher et al., 1992, Hernandez-Pando & Rook, 1994).

(iii) The vaccinations were given after deployment of the troops in the war zone, or just before they travelled there, at a time when stress levels will have been high. Immunisation in the presence of raised levels of glucocorticoids (i.e. cortisol) drives the response towards Th2 (Bernton et al., 1995, Brinkmann & Kristofic, 1995, Ramirez et al., 1996).

(iv) The troops were also exposed to carbamate and organophosphorous insecticides, and these inhibit IL-2-driven phenomena essential for normal Th1 function (Casale et al., 1993). The importance of this component is uncertain. However, it has been rumoured that the insecticides were often obtained from local sources in the Gulf, so purity was not known, and even more toxic contaminants may have been present.

Thus, multiple vaccinations administered under these circumstances may have caused a long-lasting systemic cytokine imbalance. The same effect will occur sporadically in the general population, due to vaccinations or other Th2-inducing environmental stimuli and infections, and can account for the widespread incidence of Chronic Fatigue Syndrome.

These points are explained in greater detail below.

The present inventors have made the surprising observation that preparations of (killed) Mycobacterium vaccae are potently able to redress in a non-specific manner a systemic Th1.fwdarw.Th2 bias. The effect is to bias immune system activity away from Th2 in a way that encompasses the immune response to antigens that are not present within the M. vaccae preparation injected.

This has been demonstrated experimentally both in an animal model and in man. Representative experiments are described in detail below. In particular, they surprisingly show a down-regulation of Th2 activity, which may not be coupled with up-regulation of Th1 activity.

Briefly, in experimental animals a non-specific systemic bias away from Th2 activity on administration of M. vaccae can be seen as a reduction in the titre of an IL-4 (Th2) dependent antibody response to ovalbumin (an allergen unrelated to M. vaccae itself), in mice pre-immunised so as to establish a Th2 response. A single injection of M. vaccae is able to cause this effect, and further injections can enhance it. The effect is non-specific because it does not require the presence of any component of ovalbumin in the injected preparation.

In humans (cancer patients) the effect has been demonstrated by the appearance in the peripheral blood of lymphocytes that spontaneously secrete IL-2 (a characteristic Th1 cytokine) and a decrease in T cells that secrete IL-4 (a characteristic Th2 cytokine) after stimulation with phorbol myrystate acetate and calcium ionophore. The percentage of lymphocytes showing this activated Th1 phenotype increases progressively after each successive injection of M. vaccae, reaching a plateau in many individuals after 3-5 injections of 109 organisms (days 0, 15, 30 and then monthly).

Moreover, reports are included below of human subjects suffering from Chronic Fatigue Syndrome to whom M. vaccae preparations have been administered with beneficial effects.

M. vaccae has previously been disclosed by the present inventors for use in treatment of tuberculosis (GB-A-2156673), cancer (ZA 95/2644), HIV (WO94/06466) and chronic inflammation (GB-B-2252044). WO92/08488 (also from the present inventors) discloses its use as an adjuvant for administration with an antigen of interest, noting a conversion of the T-cell component of the response to the antigen from the Th2 pattern to the Th1 pattern. There is not in WO92/08488 any suggestion that M. vaccae can cause a non-specific Th2/Th1 shift in the activity of the immune system, that is any shift other than in the response specifically mounted to the antigen administered with the M. vaccae. Furthermore there is neither in

WO92/08488 nor elsewhere any suggestion that it can down-regulate Th2 activity without concomitantly up-regulating Th1 activity.

Reviews of M. vaccae as a Th1 adjuvant include Abou-Zeid et al. (1997), Skinner et al. (1997a), and Skinner et al. (1997b). See also Mosmann and Sad (1996) which reviews up-regulation of Th1 cytokine having an inhibitory effect on Th2 cell proliferation.

The present invention generally relates to down-regulation of Th2 activity of the immune system of an individual without up-regulation of Th1 activity, particularly in treatment of a disorder characterised by a shift in immune system activity from Th1 to Th2 activity, in particular where down-regulation of Th2 activity is beneficial without concomitant up-regulation of Th1 activity.

In one aspect the present invention provides a method of treatment of an individual, the method including administration to the individual of antigenic and/or immunoregulatory material of Mycobacterium vaccae.

A further aspect of the present invention provides use of antigenic and/or immunoregulatory material of Mycobacterium vaccae in the manufacture of a medicament for treatment of an individual.

Another aspect of the present invention provides a substance or composition for use in treatment of an individual, the substance or composition including antigenic and/or immunoregulatory material of Mycobacterium vaccae.

Preferably, the M. vaccae preparation is provided without any extraneous ("foreign") antigen, immunogen or allergen being included.

Disorders to be treated may be characterised by a general, non-specific bias of the patient's immune system from Th1 activity to Th2 activity. This may be assessed or diagnosed with reference to decreased IL-2 production or increased IL-4 or IL-5 production in the individual, or by detection of IL-13, as a representative of the Th2 cytokines, and interferon gamma (IFN.gamma.) as a representative Th1 cytokine. Other Th1/Th2 cytokines may be considered. A non-specific shift from Th1 to Th2 activity of the immune system may be not attributed to or caused by exposure of the individual to a particular antigen, or infection by a particular pathogen. Individuals to be treated may be not suffering from tuberculosis or other mycobacterial infection. Particular disorders that may 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 a down-regulation of Th2 activity without up-regulation of Th1 is beneficial may be treated in accordance with the present invention.

Chronic Fatigue Syndrome may be operationally defined, for instance 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 summarised and discussed in Turnberg et al. (published October 1996).

The Mycobacterium vaccae material may be or include dead cells of M. vaccae. Such cells may be killed, for instance using irradiation, e.g. from 60 Cobalt at a dose of 2.5 megarads, chemically, or by any other means, although autoclaving is preferred, e.g. at 69 kPa for 10 minutes at 115oC.-125oC. Autoclaving may yield a more effective preparation than irradiation.

Prior to being killed, M. vaccae cells may be grown on a suitable solid medium. A modified Sauton's liquid medium may be preferred (Boyden et al.), solidified with agar, preferably 1.3% agar. After aerobic incubation, generally at 32oC. for 10 days, the organisms may be harvested, then weighed and suspended in diluent, ready for administration. Storage, if required before use, may be at 4oC.

Instead of growing the cells on a solid medium, a liquid medium, such as the modified Sauton's medium (Boyden et al.), may be employed, for instance in a fermentor.

The diluent may be unbuffered saline, pyrogen-free. Preferably, the diluent is borate-buffered, preferably containing a surfactant such as Tween 80.RTM.. A suitable borate buffer is: Na2 B4 O7. 10H2 O-3.63 g, H3 BO3 -5.25 g, NaCl-6.19 g, Tween 80.RTM. 0.0005%, distilled water to 1 liter. These diluents are pharmaceutically acceptable.

The human results mentioned above have been obtained by administration of M. vaccae as the GMP preparation, SRL172, which is available for human use under several investigator IND's from the Federal Drug Administration, and CTX's from the Medicines Control Agency in the UK. GLP acute toxicology has been performed by Huntingdon Research. Phase 1 and Phase 2 safety data have been obtained in the USA, and lodged with the FDA. SRL172 is in Phase 3 trials for the immunotherapy of tuberculosis. SRL172 may be preferred for use in the present invention.

SRL172 is a M. vaccae formulation derived from the strain denoted R877R which was deposited under the Budapest Convention at the National Collection of Type Cultures (NCTC) Central Public Health Laboratory, Colindale Avenue, London NW9 5HT, United Kingdom, on Feb. 13, 1984 under the number NCTC 11659. R877R was originally isolated from mud samples from the Lango district of Central Uganda (Stanford and Paul).

Other M. vaccae strains may be used instead of SRL172. An organism can be identified as belonging to M. vaccae by biochemical and antigenic criteria (Bonicke et al.).

It is preferred for the present invention that the M. vaccae material is administered free or substantially free from non-M. vaccae antigenic or immunoregulatory material. In other words the medicament or composition to be administered may include, or may consist essentially of, M. vaccae antigenic and/or immunoregulatory material, such as dead cells, an extract or derivative thereof, and a pharmaceutically acceptable diluent.

Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practioners and other medical doctors.

A single dosage (where dead cells are to be administered) will generally contain from 107 to 1010 killed M. vaccae microorganisms. Patients may be administered a single dose of 108 to 109 killed M. vaccae, though the dose may be repeated if need be, for instance at intervals from 2 weeks to 6 months.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which is preferably by injection, e.g. cutaneous, subcutaneous or intra-dermal.

For injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Suitable diluents, which are pharmaceutically acceptable and may be preferred, have been discussed already above.

Oral administration may be used, in which case the pharmaceutical composition may be in tablet, capsule, powder or liquid form. A tablet may 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 oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

M. vaccae may be administered by aerosol to the airways, using a suitable formulation, e.g. including particles of a size which travels to the appropriate parts of the airways. This may be a dried powder rather than aqueous suspension.

Instead of killed cells, material derived from M. vaccae may be used, in particular an extract or a synthetic molecule which has the requisite activity.

As has been discussed briefly already above, multiple vaccinations, stress and other factors may have caused a long-lasting systemic cytokine imbalance in Gulf War veterans. The same effect will occur sporadically in the general population, due to vaccinations or other Th2-inducing environmental stimuli and infections, and can account for the widespread incidence of Chronic Fatigue Syndrome.

Potent immunogens can have systemic long-lasting non-specific effects on the nature of the immune response to unrelated antigens. For instance measles infection reduces the incidence of atopy, and of allergic reactions to House Dust Mite (Shaheen et al., 1996). Similarly, Japanese children that are tuberculin skin-test positive are less likely to be atopic than are tuberculin negative children, and their ratio of circulating Th1/Th2 cytokines is higher. Moreover after repeated injection of BCG, those in whom tuberculin conversion occurs have an increased probability of losing their atopic symptoms in (Shirakawa et al., 1996).

Measles vaccination is another example. This vaccine, when used at the standard dose, reduced mortality by considerably more than can be accounted for by the incidence of measles in the unvaccinated population. Diphtheria, tetanus and pertussis vaccines (Th2-inducing) do not show this non-specific protective effect (Aaby et al., 1995). However when a high titre measles vaccine was used the mortality increased, though protection from measles itself was maintained (Aaby, 1995, Aaby et al., 1995). There is evidence that this increase in mortality was accompanied by a switch towards Th2, and dose-related increases in the induction of a Th2 component are well established for several other immunogens (Bretscher et al., 1992, Hernandez-Pando & Rook, 1994). The vaccines used in the Gulf were Th2-inducing (plague, anthrax, typhoid, tetanus, cholera), and accumulatively constituted a large antigenic load, further favouring Th2. Moreover this tendency will have been increased by the use of pertussis as an adjuvant since it is a potent inducer of Th2 (Mu & Sewell, 1993, Ramiya et al., 1996, Smit et al., 1996). This property of pertussis has led recently to discussion of the possibility that its use in children contributes to the contemporary increased prevalence of atopy (Nilsson et al., 1996, Odent et al., 1994).

The innate tendency for these vaccines to drive a systemic Th2 response will have been greatly enhanced by endocrine factors secondary to the stress to which the Gulf War personnel were exposed at the time of vaccination.

Several steroid hormones modulate T cell responses. Dehydroepiandrosterone (DHEA) or unknown metabolites of DHEA, tend to promote a Th1 pattern. Thus DHEA can restore immune functions in aged mice, and correct dysregulated cytokine release seen in old animals (Daynes et al., 1993, Garg & Bondada, 1993). It has been tested for similar properties in aged humans (Morales et al., 1994). It enhances production of Th1 cytokines such as IL-2 and IFN.gamma. (Daynes & Araneo, 1989, Daynes et al., 1990, Daynes et al., 1995, Daynes et al., 1991). DHEA also enhances IL-2 secretion from human peripheral blood T cells (Suzuki et al., 1991).

These effects of DHEA are the reverse of the effect of glucocorticoids such as cortisol which enhance Th2 activity and synergise with Th2 cytokines (Fischer & Konig, 1991, Guida et al., 1994, Padgett et al., 1995, Wu et al., 1991). If proliferation of "naive" T lymphocytes is driven in the presence of a non-specific stimulus (Brinkmann & Kristofic, 1995), or by an antigen (as follows vaccination) T lymphocytes with a Th2 cytokine profile will develop. This has been rather clearly shown with spleen cells from "clean" laboratory rodents which have few memory cells under normal circumstances (Ramirez et al., 1996).

Overall the "bottom line" is that cortisol favours the development of a Th2 cytokine profile from naive cells (Brinkmann & Kristofic, 1995). This point must not be confused with the fact that the cytokine-secreting activity of established Th2 cells is readily inhibited by cortisol. Thus the use of cortisol analogues for conventional treatments for Th-2-mediated diseases such as eczema, asthma and hay fever may work via anti-inflammatory effects, and by reducing cytokine production by Th2 cells (Corrigan et al., 1995), and yet at the same time the use of cortisol will encourage perpetuation of the underlying problem by driving newly recruited T cells towards Th2.

Psychological and physical stress activate the hypothalamo-pituitary-adrenal axis, and so lead to a variety of changes including increased production of cortisol. Excessive exercise and deprivation of food and sleep resulted in a falling ratio of DHEA to cortisol (DHEA/cortisol ratio). This correlated directly with a fall in delayed hypersensitivity (DTH) responsiveness (a Th1 marker), and there was a simultaneous rise in serum IgE levels. IgE is wholly dependent upon Th2 cytokine production (Bernton et al., 1995). This is to be expected in the light of the known effects of DHEA and cortisol outlined above.

A further example of the Th1.fwdarw.Th2 switching effect of stress is the increase in antibody to EB virus in students reacting in a stressed manner to their exams. This virus is usually controlled by a Th1 response and cytotoxic T cells, and loss of control results in virus replication and increased antibody (Zwilling, 1992). Similarly, peripheral blood leukocytes from medical students during exam periods showed lower mRNA for IFN.gamma., a Th1 cytokine (Glaser et al., 1993).

Similar points can be demonstrated in a more controlled manner in animals. Stress due to crowding or restraint can increase mycobacterial growth in tuberculous in mice (Brown et al., 1993, Tobach & Bloch, 1956). This is a model which is acutely sensitive to the presence of even a small Th2 component (Rook & Hernandez-Pando, 1996). Other examples have been discussed (Moynihan, 1994).

There is considerable evidence that depression can be associated with excessive cortisol-mediated effects in the brain (reviewed in (Raven et al., 1996)), and stress can lead to depression. Thus depression (as seen in CFS and GWS) tends to associate with Th2-mediated disorders, such as asthma, eczema, and some endocrine changes are common to Th2 disorders and to depression (Holsboer et al., 1984, Rupprecht et al., 1995).

In tuberculosis there is a systemic Th2 shift (Rook & Hernandez-Pando, 1996), and an unusual pattern of metabolites of adrenal steroids is excreted in the urine (Rook et al., 1996). Thus there are increased metabolites of cortisol relative to metabolites of cortisone. Treatment of the disease restores the Th1 dominance and corrects the pattern of steroid metabolites, so that metabolites of cortisone increase relative to metabolites of cortisol (Rook et al., 1996). Treatment of depression with the drug metyrapone causes the same change in steroid metabolites (e.g. increase in metabolites or cortisone relative to metabolites of cortisol) (Raven et al., 1995). The M. vaccae preparation SRL172 is effective in the immunotherapy of tuberculosis.

As discussed above, vaccinations can induce systemic non-specific changes in Th1/Th2 balance, as well as the antigen-specific immunisation that is usually sought. The treatment for CFS may require induction of a systemic non-specific Th1 bias in order to restore the dominance of Th1 over Th2 that is characteristic of normal healthy individuals. This is the opposite of the bias that may have been induced by the vaccinations received by personnel in the Gulf.

Experimental evidence provided herein now shows surprisingly that Mycobacterium vaccae preparations can reduce the detrimental Th1 to Th2 bias which may be seen in Gulf War veterans.

Claim 1 of 4 Claims

What is claimed is:

1. A method of treating a disorder characterised by an up-regulation of Th2 activity of the immune system of an individual comprising;

determining the amount of Th2 activity in the immune system of an individual; and,

administering to the individual an amount of a composition comprising antigenic and/or immunoregulatory material of Mycobacterium vaccae and not any non-M. vaccae antigen, immunogen or allergen, sufficient to reduce Th2 activity of said immune system without increasing the Th1 activity thereof.


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