CZ20002321A3 - Composition obtained from Mycobacterium vaccae and application methods thereof - Google Patents

Abstract

The present invention relates to compositions which are present in or derived from Mycobacterium vaccae, and methods of using them in the treatment, prevention and diagnosis of diseases, including infectious diseases, immune disorders and cancer, and methods of enhancing the antigenic response, including administering M. vaccae culture filtrate, lipid-free or lipid-depleted glycolipid-free M. vaccae cells.

Description

Compositions obtained from Mycobacterium vaccae and methods of using them

Technical field

The invention relates to compositions which can be obtained from or present in Mycobacterium vaccae and their use in the treatment, prevention and detection of disorders including infectious disorders, immune disorders and cancer. In particular, the invention relates to compounds and methods of treating respiratory diseases such as mycobacterial infections, asthma, benign lymphogranuloma and lung and skin disorders such as psoriasis, atopic dermatosis, allergic contact dermatitis, alopecia areata, carcinoma of the base and squamous skin cells and melanoma. The invention is further directed to compounds that serve as enhancers of the organism's non-specific immune response, and to their use as agents that enhance the effect of antigen in vaccination or immunotherapy against infectious diseases and in certain therapies in the treatment of immune disorders and cancer.

BACKGROUND OF THE INVENTION

Tuberculosis is a chronic, infectious disease that is caused by infection with Mycobacterium tuberculosis (M. tuberculosis). This disease is a serious problem in developing countries, as well as an increasing problem in developing regions around the world. There are 8 million new cases and 3 million deaths per year. Although the infection may be symptom free for a considerable period of time, the disease is usually manifested as chronic pneumonia resulting in fever and respiratory problems. If the disease is not treated, it may result in death.

Although the course of tuberculosis can be significantly influenced by the use of widespread antibiotic therapy, this treatment is not sufficient to prevent the spread of the disease. Infected individuals may be symptom free but are contagious for some time. Moreover, it is difficult to monitor the patient's behavior even if the treatment regimen is strictly followed. Some patients do not complete treatment, which may lead to ineffective treatment and to the development of drug resistance of mycobacterium.

Stopping the spread of tuberculosis requires effective vaccination and accurate and timely diagnosis of the disease. At present, the most effective method for creating protective immunity is by subcutaneous or intradermal injection with live bacteria. The most common myliobacter used for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent km / sn of Mycobacterium bovis (M. bovis).

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However, the safety and efficacy of BCG is a source of controversy and some states, such as the United States, do not subject vaccination to the general public. M. tuberculosis infection is usually diagnosed by a skin test involving intradermal action of tuberculin PPD (protein-purified derivative). The antigen-specific T cell response causes measurable hardening of the injection site 48 to 72 hours after injection, demonstrating the action of mycobacterial antigens. However, the sensitivity and accuracy of this test is problematic, and individuals vaccinated with BCG cannot be distinguished from infected subjects.

A less known mycobacterium that has been used for immunotherapy against tuberculosis as well as leprosy by subcutaneous or intradermal injection is Mycobacterium vaccae (M. vaccae), which is not pathogenic to humans. However, there is less information on the efficacy of M. vaccae compared to BCG and M. vaccae has not been widely used to vaccinate the general public. M. bovis BCG and M. vaccae are known to contain antigenic compounds that are recognized by the immune system of an individual who has been injected with M. tuberculosis.

Some patents and other publications describe the treatment of various conditions by the administration of mycobacteria, including M. vaccae or certain mycobacterial fractions. U.S. Pat. U.S. Patent 4,716,038 discloses the diagnosis, vaccination against and treatment of various types of autoimmune diseases including arthritic diseases, including mycobacterium including M. vaccae.

U.S. Pat. U.S. Patent 4,724,144 discloses an immunotherapeutic agent comprising an antigenic material derived from M. vaccae suitable for the treatment of mycobacterial diseases, in particular tuberculosis and leprosy, and as an agent for increasing the effect of antigens in chemotherapy. International Patent Application WO 91/01751 discloses the use of antigenic and / or immunoregulatory material derived from M. vaccae as immunoprophylaxis to slow and / or stop AIDS. International patent application WO 94/06466 discloses the use of antigenic and / or immunoregulatory material obtained from M. vaccae for therapy for HIV infection with or without AIDS and with or without tuberculosis.

U.S. Pat. U.S. Patent 5,599,545 discloses the use of mycobacterium, in particular complete, inactivated M. vaccae, as an antigen enhancer for administration with antigens that are not endogenous to M. vaccae. It is contemplated in this publication that the good effect of this antigen-enhancing agent may be due to the heat shock protein 65 (hsp 65). International Publication No. WO 92/08484 discloses the use of antigenic and / or immunoregulatory material obtained from M. vaccae for the treatment of uveitis. International Application No. WO 93/16727 discloses the use of antigenic and / or immunoregulatory material obtained from M. vaccae for the treatment of mental illnesses associated with an autoimmune response induced by infection. International Application No. WO 95/26742 discloses use

The antigenic and / or immunoregulatory material obtained from M. vaccae is used to slow or stop the growth or spread of tumors. International Patent Application No. WO 91/02542 discloses the use of autoclaved M. vaccae in the treatment of chronic inflammatory disorders in which a patient exhibits an abnormally high release of IL-6 and / or INF, or wherein the patient's IgG exhibits abnormally high agalactosyl IgG values. Among the disorders mentioned in this publication are psoriasis, rheumatoid arthritis, mycobacterial disease, Crohns disease, primary bile cirrhosis, benign lymphogranuloma, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, Guillain-Barr syndrome, primary aspects of transplant rejection, and some transplant diabetes mellitus.

M. vaccae is apparently unique among known mycobacterial species in that even after the heat preparation of the vaccine, where the bacteria are killed, the vaccine does not lose its immunotherapeutic properties. For example, M. tuberculosis BCG vaccines used for vaccination against tuberculosis utilize living strains. Heat-killed M. bovis BCG and M. tuberculosis have no protective effects when used in a vaccine. Many compounds having antigen enhancing properties have been isolated from a number of mycobacterial species. In particular, the effect of such antigen enhancers is to stimulate the partial response of the immune mechanism against antigen from another species.

There are two main classes of compounds that have been isolated from mycobacterial species that exhibit antigen enhancing properties. The first are water-soluble wax D fractions (R.G. White, I. Bemstock, R.G.S. Johns and E. Lederer, Immunology, 1:54, 1958; US Patent 4,036,953). In the second group, muramyl dipeptide-based substances (Nacetyl glucosamine and N-glycolymuramic acid in approximately equimolar amounts) are described in U.S. Pat. Nos. 3,956,481 and 4,036,953. These compounds differ from lipid and glycolipid-free M. vaccae (DDM. Vaccae) in the following aspects of their composition:

1. These are water-soluble agents while DD-A /. vaccae is insoluble in aqueous solution.

2. They are composed of a series of small cell wall oligomers of mycobacterial cells, which are either extracted from the bacterium by various solvents or recovered from the cell wall by enzymes. In contrast to DD-A / vaccae, which contains a highly polymerized cell wall.

3. All proteins were removed from their preparation by digestion with proteolytic enzymes. Only components of their preparation are parts of the peptidoglycan structure of the cell wall, namely alanine, glutamic acid, diaminopimelic acid, N-acetylglucosamine and N-glycolylmuramic acid. In contrast, DD-A /.

vaccae contains 50% by weight of proteins, which include many different types of proteins.

Administration of vaccines in the form of nasal aerosols to treat lung tissue or via the oral route into the gastrointestinal tract has usually been limited to attenuated virus strains. For example, for vaccination against poliovirus, attenuated strains of the virus administered orally until the Sabin vaccine was developed were used. Aviron Incorporated and the National Institute of Allergy and Infectious Diseases in the United States recently reported successful use of a flu vaccine administered in a nasal spray. In this case, the live attenuated influenza virus strain provided 93% protection of children against influenza. Vaccines consisting of killed viruses or bacteria or recombinant proteins were not administered as a nasal aerosol or orally. There are several reasons for this. Few successful immunizations causing T-cell immunity or synthesis of antibodies using these agents administered nasally are known. In addition, oral administration of proteins and dead organisms often causes tolerance, which is the exact opposite of what is expected from successful immunization

Benign lymphogranuloma is a disease of unknown origin characterized by granulomatous inflammation that affects many body organs, particularly the lungs, lymph nodes and liver. Sarcoid granulomas are composed of mononuclear phagocytes by septal and giant cells in the middle and T-lymphocytes. CD4 T-lymphocytes are closely associated with epithelial cells while both CD4 and CD8 T-lymphocytes accumulate at the periphery.

Characteristic immunological disorders in benign lymphogranuloma include peripheral blood and bronchoalveolar lavage hyper-globulinanemia and reduction of delayed skin hypersensitivity to tuberculin and other similar antigens such as Candida and mumps.

The levels of lymphocytes in the peripheral blood are reduced and the CD4: CD8 ratios in the peripheral blood are reduced to approximately 1: 1 to 1.5: 1. These are not manifestations of an overall immune disorder but rather a consequence of increased immunological activity . In patients with pulmonary benign lymphogranuloma, the total number of cells recovered by bronchoalveolar lavage is five to ten times higher and the lymphocyte percentage increases from normal (less than 10 to 14%) to between 15% and 50%. More than 90% of the recovered lymphocytes are T lymphocytes, and the CD4: CD8 ratio is reported to have increased from 1.8: 1 in normal control to 10.5: 1. T lymphocytes are predominantly Thi classes, forming IFN-γ and IL -2 cytokines, rather than from the Th2 class. Subsequent treatment adjusts the increase in the amount of Thi lymphocytes in the sarcoid lungs.

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Benign lymphogranuloma affects the lungs in almost all cases. Even in cases where disorders are mainly observed in other organs, non-clinical pulmonary complaints usually occur. While some cases of benign lymphogranuloma resolve spontaneously, approximately 50% of patients have at least a moderate degree of persistent organ dysfunction. In some cases, pulmonary fibrosis develops and develops into pulmonary failure requiring lung transplantation. The basis of treatment of benign lymphogranuloma is corticosteroids. Patients initially respond to corticosteroids with frequent recurrence and require treatment with other immunosuppressive drugs such as methotrexate or cyclosporin.

Asthma is a common disease prevalent in developed countries. Typical symptoms of asthma are: increased sensitivity of tracheobronchial pathways to various stimuli, primary physiological airflow disorder in both directions, which may be spontaneous or drug-induced, and a typical pathological feature - airway inflammation. Clinically, asthma can be divided into external and internal variants.

External asthma has an identifiable origin and may be atopic, profession-related or drug-induced. Atopic asthma is characterized by an increase in the Th2-type immune response with formation of specific immunoglobulins E (IgE), positive skin tests for common aeroallergens and / or atopic symptoms. It can be further subdivided into seasonal and permanent forms according to the seasonal timing of symptoms. Impaired airflow in external asthma is due to non-specific bronchial hypersensitivity, which is caused by airway inflammation. This infection is transmitted by chemicals that are released by various inflammatory cells including mast cells, eosinophils and lymphocytes. The action of these mediators causes vascular permeability, mucus secretion, and bronchial smooth muscle contraction. In atopic asthma, the airway inflammatory immune response is caused by a class of Th2 T cells that secrete IL-4, IL-5 and IL-10. Lymphocytes from lungs afflicted with atopic asthma have been shown to form IL-4 and IL-5 when activated. Both IL-4 and IL-5 are Th2-class cytokines and are required for IgE production and eosinophil involvement in asthma. Occupational asthma may be related to the development of IgE against protein hapten, for example, acid anhydrides in people working with plastics and induced by asthma by some western red juniper asthma, or non-IgE mechanisms, such as observed in toluene diisocyanate-induced asthma. Drug-induced asthma may be observed after aspirin or other non-steroidal anti-inflammatory drugs, more commonly in a group of patients who may experience other symptoms such as nasal polyps and sinusitis. It is reported that intrinsic or cryptogenic asthma occurs after upper respiratory tract infections, but may develop de novo in middle-aged or elderly people who are more difficult to treat this type of asthma than external asthma.

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The best prevention against asthma is to avoid triggering allergens, but this is not always possible because allergens are not always easily identifiable. Therapeutic therapy for asthma is based on the use of corticosteroids and bronchial augmentation drugs that alleviate inflammation and subsequent respiratory problems. In chronic asthma, corticosteroid treatment leads to unacceptable adverse side effects.

Another disease with a similar immune disorder to asthma is allergic rhinitis. Allergic rhinitis is a common disease and is estimated to affect at least 10% of the population. Allergic rhinitis may be seasonal (hay fever) caused by pollen allergens. Non-seasonal or persistent rhinitis is caused by allergy to antigens such as dust mites in the home or in domestic animals.

The abnormal immune response in allergic rhinitis is characterized by an increase in the production of allergen-specific IgE antibodies. Inflammatory response occurs in nasal mucus rather than in the lower airways as in asthma. As with asthma, local eosinophilia in the affected tissues is a major feature of allergic rhinitis. This infection causes sneezing, runny nose and nasal congestion in patients. In more severe cases, the infection spreads to the eyes (conjunctivitis), to the palate and to the outer ear. Although not life-threatening, allergic rhinitis can be very unpleasant, limiting the patient's daily life and reducing the ability to work. Current treatments include the use of antihistamines, nasal suppressants and, as in asthma, sodium cromoglycate and corticosteroids.

Most cancer deaths are caused by lung cancer. The incidence of lung cancer continues to increase and the World Health Organization estimates that there will be 2 million new cases per year by 2000. Lung cancer can generally be divided into two categories: small cell lung cancer (SCLC), which accounts for 20-25% of all lung cancer cases, and non-small cell lung cancer (NSCLC), which includes the remaining 75%. Most cases of SCLC are caused by tobacco smoke. SCLC tends to spread at the outset and 90% of patients with this diagnosis have mediastinal lymph nodes in the chest. SCLC is treated with chemotherapy or a combination of chemotherapy and radiotherapy. The overall response rate varies between 10% and 50%. On rare occasions, if the patient is free of lymph nodes, surgery with supplementary chemotherapy may have an unusually high resultant recovery rate of 60%. The prognosis for NSCLC is worse as most patients have an advanced stage of the disease at the time they are first diagnosed. Surgical removal of the tumor is only possible in a very small number of patients and the 5-year average survival for NSCLC is only 5-10%.

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The causes of lung cancer are complex and diverse. The interaction of environmental and genetic factors causes gradual and increasing disorders that lead to uncontrolled cell proliferation, invasion of adjacent tissue, and spread to more remote areas.

Lung cancer patients have been shown to have decreased cellular and humoral immunity. Radiotherapy and chemotherapy further reduce the immunity of patients. Attempts have been made to increase the immunity of patients with inactivated tumor cells or tumor antigens, which would increase the host's response to the tumor. Bacillus Calmette-Guerin (BCG) was administered to the thoracic cavity after surgery to increase non-specific immunity. Attempts have been made to increase the immune response against the tumor by administering ex vivo interleukin-2 treated lymphocytes. These lymphokine-activated lymphocytes acquire the ability to destroy tumor cells. Immunotherapies for the treatment of lung cancer are currently under development and their efficacy has yet to be confirmed before they are routinely used to treat lung cancer.

The invention also relates to the treatment of skin diseases that are likely to be related to factors that affect the balance of thymus (T) immune cells known as Th1 and Th2. These T cells are determined by their cytokine secretion phenotype. A common method of treatment is to use a compound prepared from M. vaccae which has immunomodulatory properties that alter the balance of the efficacy of these T cells as well as other immune cells.

Psoriasis is a widespread chronic inflammatory skin disease that may be associated with various forms of arthritis in a small number of patients. This disorder is manifested by too rapid growth of keratinocytes and peeling of scales from the skin surface. Drug treatment is aimed at slowing down this process. The disease can occur at any age. Spontaneous remission is relatively rare and therefore lifelong treatment is necessary. Psoriasis causes chronic peeling red spots on the skin surface. Psoriasis is a very observable disease, often affecting the face, scalp, torso and limbs. The disease mentally and physically weakens the patient, significantly reduces the quality of life. In the United States, one to three million people suffer from psoriasis, and a quarter of a million new cases occur each year. Moderate estimates suggest that US $ 248 is currently spent on psoriasis treatment in the United States.

There are two main hypotheses concerning the pathogenesis of psoriasis. The first assumes that genetic factors determine abnormal proliferation of epidermal keratinocytes. The cells no longer respond normally to external stimuli, such as those required to maintain epidermal homeostasis. Abnormal increases in cell membrane cytokine response or abnormal transmembrane signal transduction (signal transduction between membranes) may be the cause of increased cell proliferation. Psoriasis-related inflammation is caused by the release of pro-inflammatory molecules from over proliferating keratinocytes.

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The second hypothesis states that T cells secrete pro-inflammatory and keratinocytes stimulating cytokines in response to antigen-recognizing cells in the skin. (Hancock, G. E. et al., J. Exp. Med. 168: 1395-1402, 1988). Only T cells of genetically determined individuals have the capacity to be activated under such conditions. Keratinocytes themselves may be antigen-recognition cells. Cellular infiltration in psoriasis-affected sites shows the influx of CD4 + T cells, more often CD8 + T cells (Bos, JD et al., Arch. Dermatol. Res. 281: 23-3, 1989; Baker, BS, Br. Dermatol. 110 : 555-556, 1984).

Because most (90%) of psoriasis patients have a limited form of the disease, topical treatment including ditranol, tar preparations, corticosteroids and the recently reported vitamin D3 analogues (calcipotriol, calcitriol) may be used. A smaller number (10%) of patients have a more severe form of the disease and many systemic therapeutic methods are suitable for treatment. Typical systemic therapies include UVB, PUVA, methotrexate, vitamin A derivatives (acitretin), and immunosuppressants such as cyclosporin A. et al., Lancet II: 1500-1502, 1989; Ackerman, C. et al., J. Invest. Dermatol. 96: 536 [abstracts], 1991).

Atopic dermatitis is a chronic skin condition that causes itching, which usually occurs in families with a hereditary disposition to various forms of allergic diseases such as allergic rhinitis and asthma. Atopic dermatitis affects approximately 10% of the total population. The main symptoms are dry skin, dermatitis (eczema) located mainly on the face, neck and bent areas of the skin and in the folds where it causes particularly unpleasant itching. The disease typically begins in the first two years of life. In 90% of patients, the skin disease disappears during childhood, but symptoms may continue until adulthood. This is one of the most common forms of dermatitis in the world. There is a generally accepted theory that in atopy and atopic dermatitis there is a primary T cell disorder and that T cell dysfunction that normally regulates IgE production is responsible for the excessive production of this immunoglobulin.

Allergic contact dermatitis is a widespread non-infectious inflammatory disorder of the skin. Contact dermatitis does not develop an immunological reaction until the body becomes hypersensitive to a particular antigen. Repeated exposure of the skin to the antigen and recognition of this antigen by T cells results in the release of various cytokines, proliferation and replenishment of T cells, and finally dermatitis (eczema).

Only a small fraction of the T cells at the affected sites of allergic contact dermatitis are antigen-specific. Activated T cells are likely to migrate to sites of infection regardless of antigen specificity. Delayed hypersensitivity can only be transmitted by T cells (CD4 ⁺ cells) that share MHC class II antigens. Response to contact allergens ·· ············ · 000 · · 0 0 0 · · 0 • 0 00 Φ 0 · «0 ·

0 · 0 · 0 · 000000 0000 0 0 0 0 0 0 • 0 00 000 000 · 0 0 * can be transmitted by T cells that share either MHC class I molecules (CD8 ⁺ cells) or class II molecules (CD4 ⁺ cells) (Sunday, ME et al., J. Immunol. 125: 1601-1605, 1980). Keratinocytes can produce interleukin-1, which can facilitate antigen recognition by T cells. The expression of surface antigenic intracellular adhesion molecule 1 (ICAM-1) is induced on both keratinocytes and endothelium by tumor necrosis factor (TNF) cytokines and interferon gamma (IFN-γ).

If the causes can be identified, they will cure the allergic contact dermatitis itself by removing them. The use of corticosteroids is appropriate in the inflammatory phase. In the delayed type of hypersensitivity, an inhibitory effect of cyclosporine on pre-inflammatory function (s) of T cells prepared in vitro was observed (Shidani, B. et al., Eur. J. Immunol. 14: 314 318, 1984). It has also been reported that cyclosporin has an inhibitory effect on the early phase of T cell activation in mice. (Milon, G. et al., Ann. Immunol. (Inst. Pasteur) 135d: 237-245, 1984).

Alopecia areata is a common hair condition. 2% of patients in outpatient clinics in the USA are patients with this disorder. A characteristic feature of this disease is the emergence of clearly circumscribed circular or oval bald spots that may occur on any hairy part of the body. The disease can occur at any age. The onset of the disease is usually sudden and the clinical course varies.

At present it is not yet possible to attribute a single cause to all or any case of alopecia areata (Rook, A. and Dawber, R, Illnesses of the Hair and Scalp; Blackwell Scientific Publications 1982: 272-30). There are many factors that must be taken into account. These are genetic factors, atopy, association with diseases suspected of having an autoimmune origin, Down syndrome and psychological stress. There is an increased incidence of atopy in patients with alopecia areata. There is evidence that alopecia areata is an autoimmune disease. This evidence is based on consistent histopathological findings of lymphocytic T cell infiltrate in and around hair follicles with an increased number of Langerhans cells, observation that alopecia areata responds to immunomodulatory agents treatment, and the fact that there is a statistically significant association between alopecia areata and a broad spectrum of autoimmune diseases (Mitchell, AJ et al., J. Am. Acad. Dermatol. 77: 763-775, 1984).

Immuno-pheno type studies of scalp biopsy specimens show HLA-DR expression on epithelial cells in the predicted coat of hair follicles at sites of active alopecia areata, as well as T cell infiltration with high helper / inducer in and around hair follicles, increasing amounts of Langerhans cells and ICAM-1 elevation (Messenger, AG et al., J. Invest. Dermatol. 85: 569-576, 1985; Gupta, AK et al., J. Am. Acad. Dermatol. 22: 242-250, 1990 ).

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A large number of different therapies for alopecia areata can be divided into four categories: (i) non-specific local stimulants; (ii) immunomodulators such as systemic corticosteroids and PUVA; (iii) immune enhancing agents such as dermatitis-causing contact agents, cyclosporine and inosiplex; and (iv) drugs of unknown effect, for example minoxidil (Dawber, R.P.R. et al., Textbook of Dermatology, Blackwell Scientific Publications, 5th Edition, 1982: 2533-2638). Contemporary nonspecific stimulators, such as dithranol with a hitherto unknown mechanism, may be used to stimulate hair regrowth rather than local irritation. Although local corticosteroids may be effective, long-term therapy is often required. Steroids directly applicable to the affected areas have been shown to be more effective, but their use is limited to the limited patches of the affected areas of the active disease or to support the regrowth of alopecia totalis. Photochemotherapy has been shown to be effective, perhaps because it has an effect on altering functional T cell subpopulations. Local immunotherapy by inducing support for allergic contact dermatitis on the scalp can cause hair regrowth in more than 70% of patients with alopecia areata. Difencyprone is an effective sensitizing agent that has no mutagenic effects. Cyclosporin administered orally may act for a short time (Gupta, A.K. et al., J. Am. Acad. Dermatol. 22: 242-250, 1990). Inosiplex, an immunostimulant, has shown overt efficacy in an open-label experiment. It was reported that topically applied 5% minoxidil solution was able to induce some hair growth in patients with alopecia areata. The mechanism of action is unclear.

Skin cancers are a major general health problem because of their frequency and disability and the disfigurement they cause. Skin cancer occurs mainly in people of active age, especially those with fair skin who have been exposed to sunlight for a long time. In the US alone, annual spending on treatment and incapacity for work exceeds $ 250 million. Three main types - basocellular carcinoma, squamous cell carcinoma and melanoma are clearly associated with sun exposure to the skin.

Basocellular carcinomas are epithelial skin tumors. They occur primarily in areas of skin exposed to radiation. According to a recent Australian study, the incidence of basocellular carcinoma was 652 new cases per year per 100,000 population. This can be compared to 160 cases of squamous cell cancer or 19 cases of malignant melanoma (Giles, G. et al., Br. Med. J. 296: 13-17, 1988). Basal cell carcinomas are the most common of all cancers. The affected areas are usually surgically removed. Alternative treatments include retinoids, 5-fluorouracil, cryotherapy, and radiotherapy. Alpha or gamma interferons have also been shown to be effective in the treatment of basal cell carcinomas and thus provide a suitable alternative to patients who are unable to undergo surgery or are panically afraid of such surgery. (Comell and «« 9 «4 · · · 444 ·· · 4 * 4 · · 4 44 4 · · 4 4 * ···· · 4 · 9 4 4 4 4

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44 444 444 44 44 col., J. Am. Acad. Dermatol. 23: 694-700 (1990); Edwards, L. et al., J. Am. Acad. Dermatol. 22: 496-500 (1990).

Squamous cell carcinoma (SCC) is the second most common skin cancer and its frequency is increasing. The number of advanced cases of metastasis is increasing due to an increase in significant factors. There are currently over 2000 SCC deaths per year in the United States; the average five-year survival is 35% with 90% development of metastases occurring within three years. Metastases almost always occur at the nearest lymph node. The need for advanced therapy is clear. Successful drug therapy of primary SCC skin would avoid the need for surgical removal that carries scars and other side effects. This method may be particularly suitable for affected areas of the face.

Interferons (IFNs) have also been used in the treatment of melanoma (Kirkwood, J.M. et al., J. Invest. Dermatol. 95: 180S - 4S, 1990) for their antiproliferative and immunomodulating effects in vitro. Response rates with systemic IFN-α, both at high and low dose, ranged from 5 to 30% in metastatic melanoma. Encouraging results (30% response) with a combination of IFN-α and DTIC have now been achieved. Preliminary observations indicate the beneficial effect of IFN-α in the antigen enhancing agent administered to patients at high risk of melanoma. Despite the low effectiveness of IFN monotherapy for metastatic disease, several studies are currently underway to address IFN as an antigen enhancer or in combination with chemotherapy (McLeod, GR et al., J. Invest. Dermatol. 95: 185S - 7S, 1990; Ho, VC et al., J. Invest. Dermatol. 22: 159-76, 1990).

Of all suitable therapies for the treatment of skin viral diseases, only interferons have a specific antiviral mode of action, creating an immune response of the body to infection. However, interferon treatment cannot counteract viruses, it can only help against some symptoms of infection. Treatment with interferons is also associated with systemic adverse effects, requires numerous injections at each individual site affected, and is relatively expensive. (Kraus, S.J. et al., Review of Infectious Diseasses 2 (6): S620-S632, 1990; Frazer, I.H., Current Opinion in Immunology 5 (4): 484-491, 1996).

SUMMARY OF THE INVENTION

The invention relates to compositions that are comprised or obtained from M. vaccae and methods of using them in the prevention, treatment and diagnosis of diseases including mycobacterial infections, respiratory immune system disorders, and skin diseases. The methods of the invention include administration and compositions having antigenic and / or enhancer properties

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4 4 4 * • 4 4 4 4 «· the effect of the antigen. Respiratory diseases that can be treated using the compositions of the invention include mycobacterial infections (e.g., M. tuberkulosis and / or Mavium infection), asthma, benign lymphogranuloma, and lung cancer. Skin diseases that can be treated using the compositions of the invention include psoriasis, atopic dermatitis, allergic contact dermatitis, alopecia areata and cutaneous basocellular carcinoma, squamous cell carcinoma, and melanoma. The invention also encompasses antigen enhancers for use in vaccination or immunotherapy of infectious diseases and cancer.

In particular, the invention relates to isolated polypeptides derived from Mycobacterium vaccae which comprise an immunogenic portion of an antigen or a variant of such an antigen. In particular embodiments, the antigen comprises an amino acid sequence selected from the group consisting of: (a) the sequences set forth in SEQ ID NOs: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205, and 207; (b) sequences having at least about 50% of the same residues as those set forth in SEQ ID NO: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205, and 207; (c) sequences having at least about 75% of the same residues as those set forth in SEQ ID NO: 143,145,147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205, and 207; and (d) sequences having at least about 95% of the same residues as those set forth in SEQ ID NO: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174 , 177, 178, 181, 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205, and 207, measured using the BLASTP computer algorithm as described below.

Further, the invention relates to DNA sequences encoding the polypeptides of the invention, expression vectors comprising these DNA sequences, and host cells transformed or transferred by such expression vectors. Furthermore, the invention relates to a protein mixture comprising at least one polypeptide of the invention.

Furthermore, the invention relates to pharmaceutical compositions comprising at least one of the polypeptides of the invention or a DNA molecule encoding such a polypeptide and a physiologically acceptable carrier. The invention also relates to vaccines comprising at least one of the above polypeptides and a non-specific immune response enhancer. In particular embodiments, the immune response enhancer is selected from the group consisting of: lipid and glycolipid-depleted M. vaccae cells; M. vaccae inactivated cells; lipid-free and mycolic acid-depleted glycolipid-depleted M. vaccae cells; lipid and glycolipid depleted mycolic acid and arabinogalactan depleted M. vaccae cells; and M vaccae culture filtrate.

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In addition, the invention also relates to methods of enhancing the immune response of a patient, comprising administering to the patient an effective amount of one or more of the aforementioned pharmaceutical compositions and / or vaccines. In one embodiment, the immune response is a Th1 response. The invention further includes methods of treatment comprising administering to a patient a pharmaceutical composition or vaccine of the invention. In certain embodiments, the disease is selected from the group consisting of immune diseases, infectious diseases, skin diseases, and respiratory diseases. Examples of such diseases include mycobacterial infections, asthma and psoriasis.

The invention further includes methods of treating immune, infectious, skin, or respiratory diseases, comprising administering a composition comprising inactivated M. vaccae cells, lipid and glycolipid-free M. vaccae cells, or M. vaccae culture filtrate.

The invention also encompasses methods of enhancing the immune response to an antigen. In one embodiment, the method comprises administering a polypeptide comprising an immunogenic portion of a M. vaccae antigen comprising the sequences of SEQ ID NO: 89 or 201, or a variant thereof. In another embodiment of the invention, the method comprises administering a composition comprising components selected from the group consisting of M. vaccae cells depleted of lipids and mycolic acid depleted glycolipids and arabinogalactan.

Further, the invention relates to methods and diagnostic kits for determining a mycobacterial infection in a patient. In a first embodiment, the method comprises contacting a patient's skin cells with one or more of the above-mentioned polypeptides and determining an immune response to the patient's skin. In a second embodiment, the method comprises contacting the biological sample with at least one of the above polypeptides and determining the presence of antibodies that bind the polypeptide or polypeptides in the sample to determine M. tuberculosis infection in the biological sample. Suitable biological samples include blood, sputum, blood serum, blood plasma, saliva, cerebrospinal fluid, and urine.

The invention also relates to diagnostic kits comprising one or more of the above polypeptides in combination with apparatuses that are suitable for effecting contact of the polypeptide with the patient's dermal cells. The invention further relates to diagnostic kits comprising one or more inactive polypeptides in combination with a detection reagent.

Further, the invention relates to antibodies, both polyclonal and monoclonal, that bind to the above polypeptides, as well as methods of using them to determine mycobacterial infection.

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These and other aspects of the invention will be apparent in conjunction with the following detailed description and the accompanying drawings. All references cited herein are incorporated herein by reference in their entirety as if each were incorporated individually.

Overview of pictures

Giant. 1A and 1B illustrate the protective effects of immunization of mice with autoclaved M. vaccae or non-fractionated M. vaccae culture filtrates, both applied prior to infection with a live culture of M. tuberkulosis H37Rv.

Giant. Figures 2A and 2B show the percentage of eosinophils in mice immunized intranasally with either 10 or 1000 µg heat-killed M. vaccae or 200 to 100 µg DD-M vaccae, both 4 weeks prior to ovalbumin challenge compared to the control group of mice. Giant. 2C and 2D show the percentage of eosinophils in mice intranasally immunized with either 100 µg heat-killed M. vaccae or 200 µg DD-A1. vaccae, both only one week before ovalbumin challenge. Giant. 2E show the percentage of eosinophils in mice immunized either intranasally (in) or subcutaneously (sc) with either BCG Pasteur strain (BCG-P), BCG Connought strain (BCG-C), 1 mg heat-killed M. vaccae, or 200 pg DD-AI vaccae prior to ovalbumin stimulation.

Giant. 3A illustrate the effect of immunizing mice with heat-killed M. vaccae or lipid and glycolipid-free M. vaccae (DD-A1 vaccae) prior to tuberculosis infection. Giant. 3B illustrates the effect of immunizing mice with heat-killed M. vaccae, recombinant M. vaccae proteins, or a combination of heat-killed M. vaccae and recombinant M. vaccae proteins prior to tuberculosis infection.

Giant. 4 illustrates the induction of IL-12 by autoclaved M. vaccae, lyophilized M. vaccae, M. vaccae devoid of lipids and glycolipids, and M. vaccae glycolipids.

Giant. 5 compares in vitro stimulation of interferon-gamma production in spleen cells of mice suffering from Severe Combined Immunodeficiency (SCID) with different concentrations of heat-killed (autoclaved) M. vaccae, lipid and glycolipid-free M. vaccae and M. vaccae glycolipids.

Giant. Figures 6A, 6B and 6C illustrate the stimulation of interferon-gamma production by different concentrations of recombinant M. vaccae proteins, heat-killed M. vaccae, lipid-free and M.-vaccae-depleted M. vaccae (shown in the figure as "delipidated M. vaccae"), M. vaccae glycolipids and lipopolysaccharide, in peritoneal macrophages of C57BL / 6 mice (Fig. 6A), BALB / C mice (Fig. 6B) or C3H / HeJ mice (Fig. 6C).

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Giant. Figures 7A (i) to (iv) illustrate non-specific immunity enhancing effects of 10 µg, 100 µg and 1 mg autoclaved M. vaccae and 75 µg fractionated undistilled M. vaccae culture filtrates, respectively. Giant. 7B (i) and (ii) illustrate the effects of enhancing the nonspecific immunity of autoclaved M. vaccae and M. vaccae devoid of lipids and glycolipids, respectively. Giant. 7C (i) illustrate non-specific immunity enhancing effects of complete autoclaved M. vaccae. Giant. 7C (ii) illustrate the effects of enhancing the non-specific immunity of soluble M. vaccae proteins extracted with SDS from M. vaccae devoid of lipids and glycolypids. Giant. 7C (iii) shows that the non-specific immunity enhancing effects of the formulation of FIG. 7C (ii) are abolished by the administration of the proteolytic enzyme pronase. Giant. Figure 7D illustrates the effects of non-specific immunity for heat-killed M. vaccae (Fig. 7D (i)), while for heat-killed preparations of M. tuberkulosis (Fig. 7D (ii)), M. bovis BCG (Fig. 7D (iii)), M. phlei (Fig. 7D (iv)) and M. smegmatis (Fig. 7D (v)), no non-specific immunity enhancing effect was observed.

Giant. Figures 8A and B illustrate the stimulation of CD69 expression in αβΤ cells, γδΤ cells and NK cells, respectively, with the M. vaccae GV23 protein, Thl-inducers MPL / TDM / CWS and CpG ODN, and Th2-inducers the effect of the aluminum hydroxide and choleratoxin antigens.

Giant. Figures 9A-9D illustrate the effect of recombinant proteins of heat-killed M. vaccae, DD-M. vaccae and M. vaccae for the production of IL-β, TNF-α, IL-12 and IFN-γ, respectively, by human PBMCs.

Giant. 10A-10C illustrate the effects of various concentrations of recombinant M. vaccae proteins GV-23 and GV-45 on the production of IL-β, TNF-α, and IL-12, respectively, by human PBMCs.

Giant. Figures 11A-11D illustrate the stimulation of IL-β, TNF-α, IL-12 and IFN-γ production, respectively, in human PBMCs by the M. vaccae GV23 protein, Thl-inducers MPL / TDM / CWS and CpG ODN , and Th2-inducing agents that enhance the effect of the aluminum hydroxide and choleratoxin antigens.

Giant. 12A to 12C illustrate the effects of various concentrations of recombinant M. vaccae proteins GV-23 and GV-45 on the formation of CD40, CD80 and CD86, respectively, by dendritic cells.

Giant. 13 illustrates the enhancement of the reaction of dendritic cells with leukocytes by the recombinant M. vaccae protein GV-23.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the invention is generally directed to compositions and methods for preventing, treating and diagnosing infectious diseases and immune disorders. Disorders that can be effectively treated using the compositions of the invention include respiratory diseases such as, for example, diseases of the respiratory tract, e.g. 9 9 9 9 9

Mycobacterial infections, asthma, benign lymphogranuloma and lung cancer, and skin disorders such as psoriasis, atopic dermatitis, allergic contact dermatitis, alopecia areata, and skin cancers: basocellular carcinoma, squamous cell carcinoma, and melanoma.

Effective vaccines that provide protection against infectious microorganisms contain at least two distinctly different components. The first is an antigen, which may inherently be a polypeptide or carbohydrate, and which is processed by macrophages and other antigen-recognizing cells and prepared for CD4 ⁺ T cells or for CD8 ⁺ T cells. This antigen constitutes a specific target of the immune response. The second component of the vaccine is a non-specific immune response enhancer known as an antigen enhancer with which the antigen is mixed or incorporated. An antigen enhancer enhances the immune response mediated by either cells or antibodies to non-structurally related compounds or polypeptides. Several known antigen enhancers are prepared from microbes such as Bordetella pertussis, M. tuberculosis and M. bovis BCG. Antigenic enhancers may also contain components designed to protect polypeptide antigens from degradation; for example aluminum hydroxide or mineral oil. While the antigenic component of a vaccine contains polypeptides that are directed to stimulate an immune attack on specific pathogens, for example M. tuberkulosis, the antigen enhancer is often capable of broad action in the formulations of many different vaccines. Certain known proteins, for example bacterial toxins acting on the small intestine, may act both as an antigen eliciting a specific immune response and as an antigen enhancing agent enhancing a non-specific immune response to proteins.

Certain pathogens, such as M. tuberkulosis, as well as certain cancers, can be effectively affected by immune attack driven by CD4 ⁺ and CD8 ⁺ T cells, known as cell-mediated immunity. B cell antibodies are also required for other pathogens, such as polio virus. These different classes of immune attack (T cell or B cell) are controlled by different subpopulations of CD4 ⁺ cells, typically Th1 and Th2 cells. A desirable property of an antigen enhancer is the ability to selectively enhance the function of either Th1 or Th2 populations of CD4 ⁺ T cells. Many skin diseases including psoriasis, atopic dermatitis, alopecia areata, and skin cancers are likely to be affected by differences in the activity of these Th cell subsets.

These two types of Th cell subgroups have been well established in the mouse model and are defined by the cytokines that they release upon activation. The Thi subgroup secretes IL-2, IFN-γ, and tumor necrosis factor and transmits macrophage activation and delayed type of hypersensitivity. The subgroup secretes IL-4, IL-5, IL-6 and IL-10, which stimulate B cell activation. The Th1 and Th2 subgroups are:

inhibit each other, such that IL-4 inhibits Th1 responses and IFN-γ inhibits Th2 responses. Th1 and Th2-like subgroups releasing cytokines identical to those seen in the mouse model were found in humans. In particular, most T cell clones from atopic human lymphocytes resemble mouse Th2 cells that make IL-4, while very few clones make IFNγ. Selective expression of the Th2 subgroup with subsequent IL-4 production and decreased levels of IFN-γ-producing cells may lead to peripheral increases in IgE production. Enhancement of the Th-1-type immune response is critical to reversing the course of many diseases including diseases of the respiratory system such as tuberculosis, benign lymphogranuloma, asthma, allergic rhinitis and lung cancer.

Deactivated M. vaccae and many M. vaccae derived compounds have both antigen and antigen enhancing properties, thus enhancing the Th1 immune response. The methods of the invention utilize one or more of these antitgens and antigen enhancing agents derived from M. vaccae and / or culture filtrates thereof to control T cell immune activity in patients. Mixtures of such compounds are partially effective in the methods included herein. While it is well known that all mycobacteria contain many cross-reacting antigens, it is not known whether they also contain compounds of an antigen enhancer. As mentioned above, it has been shown that inactivated M. vaccae and modified (lipid and glycolipid-depleted) forms of inactivated M. vaccae have the properties of a Th1-type antigen enhancer that do not have many mycobacterial species. In addition, M. vaccae has been shown to form compounds that enhance the immune response not only to M. vaccae antigens from its own culture filtrate, but also to antigens from other sources.

The invention also relates to methods of immunotherapy of respiratory and / or pulmonary disorders, including tuberculosis, benign lymphogranuloma, asthma, allergic rhinitis and lung cancer, enhancing Th-1 type immune responses in patients. In one embodiment of the invention, the compositions are applied directly to the surface of the mucosa of the airways that lead to the lungs and / or the lungs. However, the compositions may be administered intradermally or subcutaneously. Compositions suitable for such use include at least one of the following: inactivated M. vaccae cells; M. vaccae culture filtrate; lipid and glycolipid-free M. vaccae cells (DD-vac vaccae); and compounds contained in or obtained from M. vaccae and / or its culture filtrate. As illustrated below, the administration of such compositions provokes a specific T cell immune response and enhances protection against M. tuberculosis infection, and is also effective in treating asthma. Although the exact mode of action of these compositions in the treatment of diseases such as asthma is not known, they are believed to suppress a Th2-type immune response that causes asthma.

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The term respiratory system as used herein refers to the lungs, nasal cavity, trachea, and bronchi.

As used herein, the term airway to or in the lungs includes the nasal cavity, oral cavity, tonsillar tissue, trachea, and bronchi.

As used herein, the term patient means any homoothermal animal, especially a human. Such a patient may be affected by the disease or may be free from detectable symptoms of the disease. In other words, the proposed methods can be used to elicit protective immunity for the prevention or treatment of disease.

The invention further relates to methods of immunotherapy applied to skin diseases, including psoriasis, atopic dermatitis, alopecia and skin cancer, in patients in whom the immunotherapeutic agents are to alter or control the current state of immune activity by altering T cell function to a Th-1 type of immune response. Compositions that can be used in the proposed methods include at least one of the following components: inactivated M. vaccae cells; M. vaccae culture filtrate; modified M. vaccae cells; and the components and compounds contained in or obtained from M. vaccae and / or its culture filtrate. As discussed below, repeated administration of such compositions, preferably via intradermal injection, has proven very effective in treating psoriasis.

As used herein, the term inactivated M. vaccae means M. vaccae that has been either killed by heat as described in Example 7 below or exposed to radiation, such as ⁶⁰ cobalt at a dose of 2.5 megarads. As used herein, the term modified M. vaccae includes M. vaccae cells depleted of lipids, M. vaccae cells depleted of glycolipids, and M. vaccae cells which have been depleted of both lipids and glycolipids (DD-αΤ vaccae).

The preparation of DD-A1 vaccae and its chemical compositions is described further in Example 7. As detailed below, the inventors have shown that the removal of glycolipidic components from M. vaccae causes the removal of molecular components that stimulate the production of interferon gamma in natural killer (NK) cells thereby significantly reducing non-specific cytokine production, which has numerous undesirable side effects.

Further, the invention relates to isolated polypeptides comprising at least one immunogenic portion of a M. vaccae antigen, or a variant thereof, or at least one portion of a M. vaccae antigen enhancer. In certain exemplary embodiments, such polypeptides comprise an immunogenic portion of an antigen, or a variant thereof, wherein the antigen comprises sequences selected from the group consisting of: SEQ ID Nos. 1-4, 9-16, 18-21, 23, 25, 26, 28, 29, 44.45.47, 52-55, 63, 64, 70, 75, 89, 94, 98, 100-105, 109, 110, 112, 121, 124, 125, 134, 135, 140, 141, 143 145, 147, 152, 154, 156, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9

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158, 160, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 201, 203, 205, and 207.

As used herein, the term polypeptide includes amino acid chains of any length, including full-length proteins (i.e., antigens), wherein amino acid residues are linked by covalent peptide bonds. Thus, polypeptides comprising an immunogenic portion of one of the aforementioned antigens may comprise the entire immunogenic portion or may contain additional sequences. Additional sequences may be obtained from living M. vaccae antigen or may be heterologous and such sequences may (but need not) be immunogenetic. As detailed below, the polypeptides of the invention may be isolated from M. vaccae cells or culture filtrate, or may be prepared synthetically or by recombination.

As used herein, the term "immunogenic" means the ability to provoke an immune response in a patient, such as a human, or in a biological sample. In particular, immunogenic antigens are capable of stimulating cell proliferation, interleukin 12 production, or interferon-γ production in biological samples comprising one or more cells selected from the group consisting of: T cells, NK cells, B cells and macrophages, wherein the cells are obtained from an individual immune to M. tuberkulosis. Exposure to immunogenic antigens generally results in the generation of immune memory, so that upon re-exposure to the antigen, a stronger and faster response occurs.

The immunogenic portions of the antigens described herein can be prepared and identified using well known procedures, such as those summarized in Paul, Fundamental Immunology, 3rd Edition, Raven Press, 1993, pages 243-247. These procedures include testing for polypeptide portions of the natural antigen. neo protein for immunogenic properties. The representative cytokine proliferation and production assays described herein may be used in this assay. The immunogenic portion of an antigen is that portion that induces an immune response (such as cell proliferation, interferon-γ production or interleukin-12 production) in such representative assays. , which is substantially similar to the immune response that the whole antigen would cause. In other words, the immunogenic portion of the antigen may induce at least about 20%, preferably about 65%, and most preferably about 100%, of the whole antigen-induced proliferation in the model proliferation assay described herein, the immunogenic portion may also or alternatively stimulate at least about 20% preferably about 65%, and most preferably about 100%, of the whole antigen induced interferon-γ and / or interleukin-12 in the model test described in the invention.

M. vaccae An antigen enhancing agent is a compound found in M. vaccae cells or in a M. vaccae culture filtrate that non-specifically stimulates an immune response.

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Antigenic enhancers enhance the immune response to immunogenic antigens and the process of cellular memory formation. In the case of M. vaccae proteins, this commemorative response lies mainly in Th1-type immunity. Antigen enhancers are also capable of stimulating interleukin-12 production or interferon-γ production in biological samples comprising one or more cells selected from the group consisting of: T cells, NK cells, B cells and macrophages, where the cells are derived from healthy cells. individuals. Antigen enhancers may or may not stimulate cell proliferation. Such M. vaccae antigen enhancers include, for example, polypeptides comprising the sequences set forth in SEQ ID Nos. 89, 117, 160, 162 or 201.

As used herein, the term "polynucleotide (s)" means a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding RNA molecules, including HnRNA and mRNA molecules, both 5 '-> 3' and antisense 3 '-> 5' and further includes cDNA, genomic DNA, and recombinant DNA, as well as wholly or partially synthesized polynucleotides. The HnRNA molecule contains introns and corresponds to the DNA molecule generally one to one. The mRNA molecule corresponds to the HnRNA and DNA molecule from which the introns were excised. The polynucleotide may consist of all or part of the gene. The operable antisense polynucleotides may comprise a fragment corresponding to the polynucleotide, and the definition of a "polynucleotide" in the invention includes all such operable antisense fragments. The compositions and methods of the invention also include variants of the above polypeptides and polynucleotides. As used herein, the term "variant" means any sequence having at least about 40%, preferably at least about 60%, more preferably at least about 75%, and most preferably at least about 90% of residues (either nucleotides or amino acids) identical to the sequence of the invention. The percentage of identical residues is determined by flanking the two sequences to be compared, determining the amount of identical residues in the bounded portion, dividing this number by the total length of the proposed or tested sequence, and multiplying the result by one hundred.

The sequences of the polynucleotides or polypeptides can be aligned and the percentage of identical nucleotides relative to another nucleotide can be determined in a given region using computer algorithms that are publicly available. Two exemplary algorithms for comparing and determining polynucleotide sequence similarity are the BLASTN and FASTA programs. Similarity of polypeptide sequences can be ascertained by BLASTP. The BLASTN and BLASTP programs are available on the anonymous NCBI FTP server (ftp: //ncbi.nlm. Nih. Gov) in the / blast / executables / directory. The BLASTN program version 2.0.4 [Feb-24-1998] with default settings described in the documentation distributed as part of the program is preferred for detecting differences according to the invention. Use of BLAST family algorithms including BLASTN and »00 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

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BLASTP is described on the NCBI server with the URL http: //www.ncbi.nlm.nih. gov / BLAST / newblast.html and Altschul, Stephen F., et al. (1997), Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Programs, Nucleic Acids Res. 25: 3389-3402. The FASTA program is available on the Internet via the ftp: //ftp.virginia FTP server. edu / pub / fasta /. Version 2.0.4, February 1996, with the default parameter settings described in the documentation distributed as part of the program, is preferred to detect the difference according to the invention.

The use of FASTA is described in W.R. Pearson and D.J. Lipman, “Improved Tools for Biological Sequence Analysis,” Why. Nati. Acad. Sci. USA 55: 2444-2448 (1988) and W.R. Pearson, "Rapid and Sensitive Sequence Comparison with FASTP and FASTA," Methods in Enzymology 183: 63-9% (1990).

The following trigger parameters are preferred for detecting groupings of similarity using BLASTN that contribute to E percent identity values: Unix command line: blastall -p blastn -d embldb -e 10 -G 1 -E 1 -r 2 -v 50 - b 50 -i queryseq -o results;

Default parameter values:

-p Program Name [String]

-d Database [String]

-e Hope value (E) [real number (Real)]

-G Price for opening space (zero value causes default behavior) [integer (Integer)]

-E Price for gap extension (zero value causes default behavior) [integer (Integer)]

-r Nucleotide Finder Reward Value (blastn program only) [integer (Integer)]

-v Number of single line descriptions (V) [integer (Integer)]

-b Number of displayed groupings (B) [integer (Integer)]

-i Processing input file [Filé In]

-o BLAST output file [Filé Out] optional parameter

The following startup parameters are preferred for BLASTP:

blastall -p blastp -d swissprotdb -e 10 -G 1 -E 1 -v 50 -b 50 -i queryseq -o results -p Program Name [String]

-d Database [String]

-e Hope value (E) [real number (Real)]

-G Price for opening space (zero value causes default behavior) [integer (Integer)]

-E Price for gap extension (zero value causes default behavior) [integer (Integer)]

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-b Number of displayed groupings (b) [integer (Integer)]

-I Processing input file [Filé In]

-o BLAST output file [Filé Out] optional parameter

BLASTN, BLASTP, FASTA, or a similar algorithm aligns one or more database sequences from a given sequence to determine similar parts of the sequences. The sequences found are sorted by the degree of similarity and length of sequence overlap. Finding a sequence in a database generally represents an overlap of only a portion of the sequence of interest.

BLASTN and FASTA also determine the value of “hope” for comparison. The value of hope (E) indicates the number of sequences that can be expected to be found in a random search for a certain number of continuous sequences when searching in a database of a certain size. The hope value is used to determine the boundary indicating whether finding a sequence in a database, such as a preferred EMBL database, indicates true similarity. For example, a value of E = 0.1 assigned to a database finding says that in a database of the size of the EMBL database, 0.1 findings can be expected across the matched portion of a sequence with a similar result only by chance. According to this criterion, the aligned and found part of the sequence then has a probability of 90% that it is identical. For sequences having an E value of 0.01 or less over the matched and found portion, the chance of randomly finding in the EMBL database is 1% or less using LASTN or FASTA.

According to one exemplary embodiment of the invention, "variant" polynucleotides, with reference to each of the polynucleotides of the invention, preferably comprise sequences having the same or lesser number of nucleic acids than each of the polynucleotides of the invention and having E values of 0.01 or less compared to polynucleotides according to the invention. Thus, a variant polynucleotide is any sequence that has at least 99% probability of being identical to a polynucleotide of the invention measured by an E value of 0.01 or less using BLASTN or FASTA programs with default parameters.

The altered polynucleotide sequence will generally hybridize to said polynucleotide sequence under stringent conditions. As used herein, the term strict conditions refers to an initial wash in 6X SSC, 0.2% SDS; hybridization at 65 ° C, 6X SSC, 0.2% SDS overnight; followed by two washes lasting 30 minutes each in 1X SSC, 0.1% SDS at 65 ° C and two washes lasting 30 minutes each in 0.2X SSC, 0.1% SDS at 65 ° C.

Parts and other altered M. vaccae polypeptides can be made synthetically or by recombination. Synthetic polypeptides having less than about 100 amino acids, and generally less than about 50 amino acids, can be made using procedures well known to those skilled in the art. For example, such polypeptides can be synthesized using

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Any of the commercially available solid phase processes, for example the Merrifield method of solid phase synthesis, consisting in that amino acids are gradually added to the growing amino acid chain. See Merrifield, Jm. Chem. Soc. 85: 2149-2146, 1963. Equipment for automated polypeptide synthesis is commonly available from suppliers such as Perkin Elmer / Applied BioSystems, Inc. (Foster City, CA) and can be customized to production requirements. Variants of the natural antigen or antigenic enhancer can be prepared using standard mutagenetic techniques, for example, directed site-specific mutagenesis of the oligonucleotide. The DNA sequence regions can also be removed using standard procedures, allowing the production of truncated polypeptides.

The polypeptide of the invention may be attached to a signaling (or leader) sequence at the N-terminus of a protein which, during or after translation, directs protein transfer. The polypeptide may also be attached to a linker or other sequence to facilitate synthesis, purification or identification of polypeptides (e.g., poly-His) or to enhance binding of the polypeptides to a solid support. For example, the polypeptide may be attached to an immunoglobulin Fc region.

Thus, M. vaccae antigens and DNA sequences encoding such antigens can be prepared using any method. For example, soluble antigens may be isolated from M. vaccae culture filtrate as discussed below. Antigens can also be produced by recombination by inserting a DNA sequence that encodes an antigen into an expression vector and expressing the antigen in a suitable host. Any of the vector expression techniques known to those skilled in the art can be used. Expression can take place in any suitable host cell that has been transformed or transferred into an expression vector containing a DNA molecule encoding a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells are E. coli, mycobacteria, insect, yeast or mammalian cell lines, such as COS or CHO. DNA sequences expressed in this manner can encode naturally occurring antigens, portions of naturally occurring antigens, or other variants thereof.

DNA sequences encoding M. vaccae antigens can be obtained by screening a suitable M. vaccae cDNA or genomic DNA library to find a DNA sequence that hybridizes to degenerative oligonucleotides derived from the partial amino acid sequences of isolated soluble antigens. Suitable degenerate oligonucleotides may be designed and synthesized and screened as described for example in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989. A chain reaction (PCR) can be used to isolate a nucleic acid sample from genomic DNA or cDNA or from a genomic DNA library. Library searches can then be performed using an isolated sample. DNA molecules encoding M vaccae antigens may also be isolated by screening a suitable M. vaccae expression library with an antiserum (e.g., rabbit or monkey) growing specifically against M. vaccae antigens.

Regardless of the method of preparation, the antigens described in the invention have the ability to induce an immune response. More specifically, antigens have the ability to induce cell proliferation and / or cytokine production (e.g., interferon-γ and / or interleukin-12 production) in T cells, NK cells, B cells, or macrophages derived from individuals immune to M. tuberkulosis. An M. tuberculosis immune individual is an individual who is considered resistant to the development of tuberculosis because its T cells are capable of responding effectively to M. tuberculosis. Such individuals can be identified based on a strong positive response to (eg, hardness greater than about 10 mm in diameter) intradermal skin test for tuberculosis proteins (PPD) and the absence of any symptoms of tuberculous infection. Assays for cell proliferation or cytokine production in T cells, NK cells, B cells, or macrophages can be performed using, for example, the procedures described below. The choice of cell type to be used to evaluate the immune response to the antigen will depend on the desired response. For example, the formation of interleukin-12 is much more readily determined using a procedure that utilizes T cells, NK cells, B cells, and macrophages obtained from individuals resistant to M. tuberculosis, which can be prepared according to procedures well known to those skilled in the art. For example, peripheral blood mononuclear cells (PBMCs) can be used without further separation of cell components. PBMCs can be prepared, for example, using Ficoll ™ density centrifugation (Winthrop Laboratories, NY). The T cells used in the assays described herein can be purified directly from PBMCs. Alternatively, enriched T cell lines responsive to macobacterial proteins or T cell clones responsive to individual mycobacterial proteins may be used. Such T cell clones can be obtained, for example, by culturing PBMCs from individuals immune to M. tuberkulosis with mycobacterial proteins for 2-4 weeks. This will only allow the growth of mycobacterial protein-specific T cells, resulting in a line composed of only such cells. These cells can then be cloned and assayed with individual proteins using procedures well known to those skilled in the art to more accurately determine T cells specifically.

In general, regardless of the method of preparation, the polypeptides of the invention are prepared in isolated, substantially pure form. The polypeptides are preferably prepared in at least about 80% purity, more preferably in at least about 90% purity, and most preferably in at least about 99% purity. In one exemplary embodiment, considerably pure polypeptides are included

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The pharmaceutical compositions or vaccines for use in one or more of the methods of the invention.

The invention also relates to compound proteins comprising the first and second polypeptides of the invention or, optionally, a polypeptide of the invention and a known M tuberculosis antigen, such as the 38 kDa antigen described in Aersen and Hansen, Infect. Immun. 57: 2481-2488, 1989, along with variants of such fusion proteins. The fusion proteins of the invention may also include linking peptides between the first and second polypeptides.

DNA sequences encoding the fusion proteins of the invention are produced using well known DNA recombination techniques by assembling separate DNA sequences encoding the first and second polypeptides into a suitable expression vector. The 3 'end of the DNA sequence encoding the first polypeptide is linked with or without a peptide linker to the 5' end of the DNA sequence encoding the second polypeptide, so that the reading fragments of the sequence are in a phase that allows translation of the mRNA of two DNA sequences into one fusion protein and a second polypeptide.

Peptide linkers can be used to separate the first and second polypeptides at a distance that allows both polypeptides to assume their secondary and tertiary structure. Such a polypeptide linker sequence can be inserted into the linked proteins using procedures well known to those skilled in the art. Suitable peptide linkers may be selected based on the following factors: (1) their ability to adopt a flexible expanded conformation; (2) their inability to adopt a secondary structure that could react with functional regions of the first or second polypeptide; and (3) lack of hydrophobic or charged moieties that could react with functional regions of the polypeptides. Preferably, the peptide linker comprises Gly, Asn, and Ser residues. Other near-neutral amino acids such as Thr and Ala can also be used in linker sequences. Amino acid sequences that can be used as linkers include those described in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Nati. Acad. Sci. USA 53: 8258-8262 (1986); U.S. Pat. Patent No. No. 4,935,233 and U.S. Pat. Patent No. 4,751,180. The linker sequences can be from 1 to about 50 amino acids in length. Peptide joining sequences are not required if the first and second polypeptides have non-essential amino acid regions at the N-terminus that can serve to separate functional domains and prevent steric interference. Bound DNA sequences encoding linked proteins can be cloned into suitable expression systems using techniques well known to those skilled in the art.

As discussed in more detail below, the inventors show that heat-killed M. vaccae, DDM. vaccae and recombinant M. vaccae proteins of the invention can be used to activate T

• · 9 9 « 9 9 9 • · • • • 9 • · 9 • • • • • 9 • · · 9 • • · • • • • 9 9 9 9 • • • • • • 9 9 9 999 • · · • · ··

cells and NK cells; to stimulate the production of cytokines (particularly Th1-class cytokines) in human PBMCs; to increase expression of co-stimulatory molecules on dendritic cells and monocytes (and thereby to increase activation); this enhances maturation and cell function. Furthermore, the inventors show similarities between the immunological properties of the M. vaccae GV-23 protein of the invention and the two known Th1-produced agents that increase the effect of the antigen. Thus, GV-23 can be used in the treatment of diseases that exhibit an enhanced Th1 immune response. Examples of such diseases include allergic diseases (for example, asthma and eczema), autoimmune diseases (for example systemic lupus erythematosus) and infectious diseases (for example tuberculosis and leprosy). In addition, GV-23 can be used to support dendritic cells or NK cells in the treatment of diseases causing immune disorders such as HIV, and to support immune responses and cytotoxic responses to, for example, cancer malignancies and subsequent immunosuppressive anti-cancer therapies such as chemotherapy.

For use in the therapeutic methods of the invention, inactivated M. vaccae, M. vaccae culture filtrate, modified M. vaccae cells, M. vaccae polypeptide, fusion proteins (or polynucleotides encoding such polypeptides or fusion proteins) are generally presented in a pharmaceutical composition or vaccine. The pharmaceutical compositions may comprise one or more ingredients selected from the group consisting of: inactivated M. vaccae, M. vaccae culture filtrate, modified M. vaccae cells and compounds contained in or obtained from M. vaccae and / or their culture filtrate together with a physiologically acceptable carrier. The vaccines may comprise one or more components selected from the group consisting of: inactivated M. vaccae, M. vaccae culture filtrate, modified M. vaccae cells and compounds contained in or obtained from M. vaccae and / or their culture filtrate together with a non-specific immune enhancer answers. Such pharmaceutical compositions and bags may also contain other mycobacterial antigens either as mentioned above, incorporated into the fusion proteins or present in a separate polypeptide.

Alternatively, the vaccine of the invention may comprise DNA sequences encoding one or more polypeptides as described above, such that the polypeptide is formed in situ. In such vaccines, the DNA sequences may be present in any of the delivery systems known to those skilled in the art, including nucleic acid expression systems, bacterial and viral expression systems. A suitable nucleic acid expression system comprises the DNA sequence necessary for expression in a patient (such as a suitable promoter and terminator). Bacterial transfer systems include administration of bacteria (such as Bacillus-CalmetteGuerin) that express an immunogenic portion of the polypeptide on its cell surface. In an exemplary embodiment, the DNA sequences may advantageously be produced using a viral expression ############################# until C- A system (such as a vaccine or other poxvirus, retrovirus, or adenovirus), which may include the use of a non-pathogenic or incomplete replication capable virus. Methods for introducing DNA into such an expression system are well known to those skilled in the art. DNA may also be stripped, as described, for example, by Ulmer et al., Science 259: 1745-1749, 1993 and expanded in Cohen, Science 259: 1691-1692, 1993. The uptake of the exposed DNA may be improved by coverage on biodegradable carriers that are well transmitted to cells.

The DNA vaccine described above may be administered concurrently with or subsequent to administration of either the polypeptides of the invention or known mycobacterial antigens such as the 38 kDa antigen mentioned above. For example, administration of DNA encoding a polypeptide of the invention may be followed by administration of antigens to enhance the protective immune effect of the vaccine.

The methods and frequencies administered as well as the dosage will be individual for each individual and may correspond to the frequency and dosage methods currently used in immunization using BCG. In general, pharmaceutical compositions and vaccines may be administered by injection (e.g., intradermally, intramuscularly, intravenously or subcutaneously), intranasally (e.g., by inhalation), or orally. 1 to 3 doses may be administered at intervals of 1 to 36 weeks. Preferably, the 3 doses may be administered 3 to 4 months apart and the booster vaccines may be administered periodically. An alternative procedure may be chosen for a particular patient. A suitable dose is the amount of polypeptide or DNA that, administered as described above, is capable of enhancing the immune response in a patient such that it is sufficient to protect the patient from mycobacterial infection for at least two years. Generally, the amount of polypeptide in the dose (or formed in situ from the DNA dose) varies from about 1 pg to 100 mg per kg of host organism, typically from about 10 pg to 1 mg, preferably from about 100 pg to 1 pg. A suitable dose size will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

In one embodiment, the pharmaceutical composition or vaccine is in a form suitable for application to the surface of the mucosa of the airways leading to the lungs or directly in the lungs. For example, the pharmaceutical composition or vaccine may be dissolved in a liquid formulation for administration to a patient in the form of an aerosol or with a nebulizer similar to those used in the treatment of asthma. In other embodiments, the pharmaceutical composition or vaccine is in a form suitable for injection (intracutaneous, intramuscular, intravenous or subcutaneous) or orally. Any suitable carrier known to those skilled in the art may be used in the pharmaceutical composition of the invention, but its type will depend on its suitability for the mode of administration selected. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, brine, alcohol, some lipid, some wax or some buffer. For oral administration, any of the above may be used

9 · «9 9 9 · · ·

9 ··· 9 9 · 9 9 9 · 9 9 9 · 9

9999 9 9 99 ·· ··· 99 999 999 »9« 9 or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose and magnesium bicarbonate. Biodegradable microspheres (e.g., polylactic galactides) can also be used as carriers for the pharmaceutical compositions of the invention. Suitable biodegradable microspheres are described, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

Any type of antigen enhancer can be used in the vaccines of the invention to enhance the non-specific immune response. Most antigen enhancers contain agents designed to protect the antigen against rapid catabolism, such as aluminum hydroxide or mineral oil, and some non-specific immune response stimulator such as lipid A, Bordetella pertussis, M. tuberkulosis, or, as shown below, M vaccae. Suitable antigen enhancers are commercially available, for example, Freuns Incomplete Adjuvant and Freuns Complete Adjuvant (Difco Laboratories, Detroit, MI), and Merck Adjuvant 65 (Merck and Company, Inc, Rahway, NJ). Other suitable antigen enhancers include saltpeter, biodegradable microspheres, monophosphoryl lipid A and Quil A.

Further, the invention provides methods for using one or more polypeptides of the invention to diagnose tuberculosis by a skin test. By skin test is meant any test performed directly on a patient who is measuring a delayed-type hypersensitivity (DTH) response (such as swelling, flushing or dermatitis) to intradermal injection of one or more polypeptides as described above. The reaction is measured preferably at least 48 hours after injection, more preferably 48 to 72 hours after injection.

A DTH response is a cell-mediated immune response that is greater in patients who have previously been exposed to a test antigen (for example, a portion of an immunogen of a polypeptide used or some variant thereof). The response can be measured visually using a ruler. Generally, a response that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter is a positive response indicative of tuberculosis infection.

For use in the skin test, the polypeptides of the invention are preferably formulated as pharmaceutical compositions comprising the polypeptide and a physiologically acceptable carrier as mentioned above. Such compositions typically comprise one or more of the above polypeptides in an amount ranging from about 1 pg to about 100 pg, preferably from about 10 pg to about 50 pg in a volume of 0.1 ml. The carrier used in such pharmaceutical compositions is preferably a saline solution with a suitable preservative such as phenol and / or Tween 80 ™.

In a preferred embodiment, the polypeptide used in the skin test is sized to remain at the injection site for the duration of the reaction. A polypeptide of at least 9 amino acids in length is generally sufficient. The polypeptide is also preferably degraded by macrophages or dendritic cells. 9 9 9 9

9 9 9 9 9 9 9 9 9 9

9 9 9 9 9

99 999 999 9 9 99 during hours of injection to allow its effect on T cells. Such polypeptides may comprise a repeat of one or more of the above sequences, or other immunogens or other non-immunogenic sequences.

The invention further relates to methods for recognizing a mycobacterial infection in a biological sample using one or more polypeptides of the invention, either alone or in combination. In an exemplary embodiment in which different polypeptides are used, polypeptides other than those specifically described in the invention, such as the above-mentioned 38 kDa antigen, may be included. As used herein, the term biological sample is any sample comprising an antibody obtained from a patient. The sample is preferably complete blood, sputum, serum, plasma, saliva, cerebrospinal fluid, or urine. More preferably, the sample is a blood, serum or plasma sample obtained from a patient or blood supply. The polypeptide (s) is used in the assays as described below to determine the presence or absence of its (s) antibodies in the sample relative to a predetermined cut-off value. The presence of such antibodies indicates a mycobacterial infection.

In an exemplary embodiment that uses more than one polypeptide, complementary polypeptides are preferably used (i.e., one component of the polypeptides will determine the presence of infection in samples in which it would not be determined by the other component of the polypeptides). Complementary polypeptides can generally be determined using each polypeptide separately in the evaluation of serum samples obtained from groups of patients known to be infected with Mycobacterium. After determining which test samples are positive (as discussed below) with each polypeptide, combinations of two or more polypeptides that are capable of detecting infection in most or all of the test samples can be formulated. For example, approximately 25% to 30% of serum samples from tuberculosis infected individuals are negative for antibodies to any single protein, such as the aforementioned 38 kDa antigen. Complementary polypeptides can therefore be used in combination with the 38 kDa antigen to increase the sensitivity of the diagnostic assay.

Those of skill in the art are familiar with many different assays that use one or more polypeptides to determine antibodies in a sample. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In a preferred embodiment, the assay involves the use of polypeptides mobilized on a solid support to bind and remove antibody from the sample. The bound antibody can then be determined using a detection reagent that contains an identifying moiety. Suitable detection reagents include antibodies that bind to the antibody / polypeptide complex and release a polypeptide labeled with an identifying group (for example, in a comparative assay). Alternatively, comparative assays can be performed in which the antibody that binds to the polypeptides is labeled with an identifying group and allows the antibody to bind to the polypeptide. Binding to the immobilizing agent after incubation of the antigen with the sample. Depending on the extent to which sample components inhibit the binding of labeled antibodies to the polypeptide, the potency of the sample with the bound polypeptide is determined.

The solid support may be any solid material to which the antigen can attach. Suitable materials are well known to those skilled in the art. For example, the solid support may be a test well on a microtiter plate or nitrocellulose or other suitable membrane. The substrate may alternatively be frames or discs, for example, glass, fiberglass, latex or some plastic material such as polystyrene or polyvinyl chloride. The support may also be a magnetic particle or a fiber optic sensor such as described in U.S. Pat. Patent No. 5,359,681. Polypeptides can be bound to a solid support using a variety of procedures well known to those skilled in the art. As used herein, the term bonded means both a non-covalent linkage such as adsorption and a covalent linkage, which may be directly between the antigen and functional groups on the support or using a cross-linked reagent. The binding is preferably adsorption to a well in a microtiter plate or membrane. In these cases, adsorption can be achieved by contacting the polypeptide in a suitable buffer with a solid support for a suitable period of time. The contact time depends on the temperature and normally ranges between about 1 hour and 1 day. Generally, contacting a well in a plastic microtiter plate (such as polystyrene or polyvinyl chloride) with an amount of polypeptide in the range of about 10ng to about 1 µg, suitably about 100 ng, is sufficient to bind an appropriate amount of antigen.

Covalent association of a polypeptide to a solid support can generally be achieved by reacting the support with a bifunctional reagent that will react with both the support and any functional group of the polypeptide, such as a hydroxyl group or an amino group. Polypeptides may be coupled to the support, for example, by having a suitable benzoquinone-containing polymer on the surface or by condensing the aldehyde group of the support with an amine and some effective hydrogen of the polypeptide (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at AI 2 -A13).

In certain exemplary embodiments, the enzyme attachment assay is an immunosorbent (ELISA). This assay can be performed by linking a polypeptide antigen that has been immobilized on a solid support with a sample such that the antibodies of the polypeptide in the sample can bind to the immobilized polypeptide. The unbound sample is then removed from the immobilized polypeptide and some detection reagent is added which binds to the immobilized antibody-polypeptide complex. The amount of detection reagent that remains bound to the solid support is then determined using a procedure appropriate for the reagent used.

· 9 9 »» 9 4 »· 9 9 I» · · «« · · ·

More specifically, once the polypeptide is immobilized on the support, as noted above, the remaining protein binding regions of the support are usually blocked. Any suitable blocking agent known to those skilled in the art, such as bovine serum albumin, or Tween 20 ™ (Sigma Chemical Co., St. Louis, MO) can be used. The immobilized polypeptide is then incubated with the sample to allow the antibody to bind to the antigen. The sample may be diluted with a suitable diluent such as phosphate-buffered saline (PBS) prior to incubation. Generally, a suitable contact or incubation time is a time sufficient to determine the presence of antibody in a M. tuberculosis-infected sample. The time sufficient to reach the binding level is preferably at least 95% of the time to reach equilibrium between bound and unbound antibodies. The time required to reach equilibrium can simply be determined by testing the level of binding that is performed during the experiment time. An incubation time of 30 minutes is generally sufficient at room temperature.

Unbound sample can be removed by washing the solid support with a suitable buffer, for example PBS containing 0.1% Tween 20 ™. The detection reagent can then be added to the solid support. A suitable detection reagent is any compound that binds to an immobilized antibody-polypeptide complex and can then be determined by any means known in the art. Preferably, the detection reagent comprises a binding agent (e.g., protein A, protein G, immunoglobulin, lectin or free antigen) conjugated to an identifying moiety. The identification moiety preferably comprises enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups, and biotin. Conjugation of the binding agent to the identifying moiety can be achieved using standard procedures known in the art. Conventional binding agents may also be purchased already conjugated to various identifying groups from a variety of commercial sources (e.g., Zymed Laboratories, San Francisco, CA, and Pierce, Rockford, IL).

The detection reagent is incubated with the immobilized antibody-polypeptide complex for a time sufficient to determine antibody binding. A suitable time can be given in the manufacturer's instructions or can be determined by testing the level of binding that is performed during the experiment. The unbound detection reagent is then removed and the bound detection reagent is determined using an identifying moiety. The procedure used to determine the identification group depends on the nature of the identification group. For radioactive groups, scintillation counting or autoradiographic methods are appropriate. Spectroscopic methods can be used to determine dyes, luminescent groups, fluorescent groups. Biotin can be determined using avidin, which is linked to another identifying group (usually a radioactive or fluorescent group or an enzyme).

το · · · · ···· · · · ·

JZ · · · · * * * * * * * * * * * * * * * * * 9 9 9 9 9 9

Enzyme identification groups can be determined by adding a substrate (usually for a period of time), followed by spectroscopic or other analysis of the reaction products.

The presence or absence of antimycobacterial antibodies in the sample is usually determined by comparing the signal determined from the identification group that remains bound to the solid support with a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value is determined as the mean of the signal obtained when the immobilized antigen is incubated with samples obtained from an uninfected patient. In another preferred embodiment, the cut-off value is determined using the Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, pp. 106-107. Signals greater than a predetermined cut-off value are generally considered to be evidence of mycobacterial infection.

The assay may also be performed using a rapid flow or strip test where the antigen is immobilized on a membrane, such as nitrocellulose. In the flow assay, antibodies in the sample bind to the immobilized polypeptide while the sample passes through the membrane. The detection reagent (e.g., protein A - colloidal gold) then binds to the antibody-polypeptide complex while the solution containing the detection reagent flows through the membrane. The determination of the bound detection reagent can then be carried out as described above.

In the strip test, one end of the membrane to which the polypeptide is bound is immersed in a solution containing the sample. The sample soaks the membrane across the region containing the detection reagent into the immobilized polypeptide region. The concentration of the detection agent in the polypeptides indicates the presence of antimycobacterial antibodies in the sample. The concentration of the detection reagent in the strip usually forms a pattern, for example a line, that can be viewed visually. The absence of such a pattern indicates a negative result. Generally, an amount of membrane-immobilized polypeptides is selected to produce a visually recognizable pattern when the biological sample contains an antibody level that would be sufficient to generate a positive signal in the ELISA as described above. The amount of membrane-immobilized polypeptides is preferably from about 25 ng to about 1 µg, and more preferably from about 50 ng to about 500 ng. These assays can be performed with very small amounts (for example, one drop) of the patient's serum or blood.

There are a number of assays that are suitable for use with the polypeptides of the invention. The above descriptions are meant to be exemplary only.

The invention also relates to antibodies to the polypeptides of the invention. The antibodies can be prepared by any of the methods well known to those skilled in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one method, an immuno-antigen containing antigenic polypeptide is first injected into any subject

• • 9 ·· • • • • • · • 9 • • • * 9 9 9 • • · • • 9 « • 9 9 « • • 9 9 • « • · · «· · • 9 • 9

from a wide class of mammals (e.g., mice, rats, rabbits, sheep, and goats). The immunogen is injected into the animal host, preferably according to a predetermined schedule comprising one or more booster immunizations, and animals are bled repeatedly. Polyclonal antibodies specific for the polypeptide can then be purified from such antisera, for example, by affinity chromatography using polypeptides associated with a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptides of interest can be prepared, for example, using the procedure of Kohler and Milstein, Eur. J, Immunol. 6: 511-519, 1976 and other improvements. Briefly, these methods involve preparing immortal cell lines capable of generating antibodies having the desired properties (e.g., the ability to react with polypeptides of interest). These cell lines can be made, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells can then be immortalized by combining with myeloma cells, preferably those that are syngeneic with the immunized animal, using various techniques known in the art.

Monoclonal antibodies can be isolated from supernatants of the resulting hybrid colonies. In addition, various yield-increasing techniques may be used, such as injecting a hybrid cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. The monoclonal antibodies can then be obtained from the ascital fluid or blood.

The antibodies can be used in diagnostic assays to detect the presence of mycobacterial antigens using assays similar to those described in detail above and other procedures well known to those skilled in the art, thereby providing a method for detecting mycobacterial infection, such as M. tuberkulosis infection in a patient. .

The diagnostic agents of the invention may also include polynucleotides encoding one or more of the above polypeptides, or one or more portions thereof. For example, primers containing at least 10 contiguous oligonucleotides of the polynucleotides of the invention may be used in basic polymerase chain reaction (PCR) assays. Similarly, test sections comprising at least 18 contiguous oligonucleotides of the polynucleotides of the invention can be used to hybridize a particular sequence. Both PCR and hybridization procedures are well known to those skilled in the art. Thus, primers or regions can be used to detect M. tuberculosis and other mycobacterial infections in biological samples, preferably mucus, blood, serum, saliva, cerebrospinal fluid, or urine. The DNA regions or primers containing the oligonucleotide sequences listed above can be used alone, in combination with other or previously mentioned sequences such as the 38 kDa antigen mentioned above.

·· 00 • · 0 0 • 0 0 0 • 0 0 0 0

0 0 0

The word about used herein in reference to the weight percent of the composition means a deviation of up to 10% of the units from said value. In the context of a percent match or percent probability, the word means about a deviation of up to 1 percent unit from that value.

DETAILED DESCRIPTION OF THE INVENTION

The following examples are intended for illustration only and are not intended to limit the invention.

Example 1

Effect of immunization of M.vaccae mice on tuberculosis

This example illustrates the effect of immunization with heat-killed M. vaccae or M. vaccae culture filtrate on mice previously infected with live M. tuberculosis.

M. vaccae (ATCC No. 15483) was cultured in sterile medium 90 (fermentation extract, 2.5 g / l; trypton, 5 g / l; glucose, 1 g / l) at 37 ° C. Cells were harvested by centrifugation and transferred to sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 ° C for one day. The medium was then centrifuged to recover the bacteria and the culture filtrate was discarded. The bacteria were resuspended in phosphate buffered saline at a concentration of 10 mg / ml, which is equivalent to 10 ^1θ M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 minutes at 120 ° C. The culture filtrate was filtered through a 0.45 µm filter into sterile bottles.

As shown in Figure 1A, mice that were immunized with 1 mg, 100 pg or 10 pg M. vaccae and infected three times later with 5x10 ⁵ colony forming units (CFU) of living M. tuberculosis H37Rv were observed to be significant. protection against infection. In this example, tissue from the pancreas, liver, and lungs of mice that were three weeks post infection and detected live bacilli (expressed in CFUs) was used. The decrease in bacillus count compared to tissue from unimmunized control mice exceeded 2 logs in liver and lung and 1 log in pancreas tissue. Immunization of mice with heat-killed M. tuberculosis H37Rv has no significant protective effect on mice subsequently infected with live M. tuberculosis H37Rv.

As shown in Figure 1B, significant protection from infection was observed in mice that were immunized with 100 µg M. vaccae culture filtrate and three weeks later infected with 5x10 ⁵ CFU of M. tuberculosis H37Rv. After pancreatic, liver and lung tissue was removed from mice that were three weeks post infection, the number of live bacilli (CFU) was detected and a 1-2 log reduction was observed compared to a group of unimmunized control mice.

· 9

9 9 9

9

9 9 9

Example 2

Effect of intradermal and intra-pulmonary immunization of M. vaccae on tuberculosis in cynomolgus? monkeys

This example illustrates the effect of immunization with heat-killed M. vaccae or M. vaccae culture filtrate by the intradermal and intra-pulmonary routes in cynomolgus? monkeys that were later infected with live M. tuberculosis.

Heat-killed M. vaccae and M. vaccae and culture filtrate were prepared as described in Example 1 above.

Five cynomolgic groups were used? monkeys, there were two monkeys in each group. Two groups of monkeys were immunized with complete heat-killed M. vaccae either intradermally or nitroplically; two groups of monkeys were immunized with M. vaccae culture filtrate either intradermally or nitroplically and the control group was not immunized. All immunogens were dissolved in phosphate buffered saline. The compositions used for immunization, the amount of immunogens, and the route of administration are listed in Table 1 for each group of monkeys. Prior to immunization, all monkeys were weighed (weight in kg), body temperature (temperature) measured and blood samples were taken to determine rate response. erythrocyte sedimentation (RSE in mm / hour) and lymphocyte proliferation (PL) to in vitro blended purified protein (PPD) prepared from Mycobacterium bovis. Both RSE and PL were used as indicators of T cell response to infection. 33 days after immunization, these measurements were repeated. On day 34, all monkeys received a second immunization with the same amount of M. vaccae and the same route as the first immunization. On day 62, body weight, temperature, RSE and PL were measured for PPD and then all monkeys were infected with 10 ³ CFU of Erdman strain Mycobacterium tuberculosis by placing the organisms directly in the right lung of immunized animals. 28 days post infection, body weight was measured, RSE and PL temperature for PPD in all monkeys and lungs were X-rayed to see if the infection with live M. tuberculosis developed in incipient pneumonia.

Table 1

Comparison of intradermal and intra-pulmonary immunization pathways

Group number Identification Monkey number

The amount of immunogen immunization route

S3101-E • 4

44 • 4 4 • 4

4 4

44 • 4 44

4 4

4 4

4 4

4 4 4

44

(Checks) 3144-B 0 - 2 4080-B 500 pg intradermal (Immunized with heat - killed M. vaccae) 3586-B 500 pg intradermally 3 3534-C 500 pg nitroplicní (Immunized with heat - killed M. vaccae) 3160-A 500 pg nitroplicní 4 (Immunized 3564-B 100 pg intradermally culture filtrate) 3815-B 100 pg intradermally 5 (Immunized 4425-A 100 pg nitroplicní culture filtrate) 2779-D 100 pg nitroplicní

The results of these studies are presented in Tables 2A-E and summarized below:

Table 2A - 28 days after infection with the Erdman strain of M. tuberculosis, vague shadows appeared on the right suprahilar region of both animals after chest X-ray of control (non-immunized) monkeys, indicating early pneumonia. This continued and within 56 days after infection, X-ray showed disease in both lungs. As the disease progressed, both control animals lost weight and showed a significant change in PL for PPD, indicating strong T cell activity against M. tuberculosis. RSE measurements were variable but corresponding to a strong immune response.

Table 2B - Two monkeys twice intradermally immunized with 500 µg M. vaccae showed no signs of lung disease even 84 days after M. tuberculosis infection. PL, relative to PPD, indicated an immune response to M. tuberculosis and both animals gained weight and did not show disease symptoms accordingly.

Table 2C - Two monkeys twice nitrophic immunized with 500 µg M. vaccae showed almost the same results as the animals in Table 2B. They had no symptoms of the disease even 84 days after infection with M. tuberculosis and continuously gained weight. Both animals showed LP proliferation to PPD in the immunization phase (days 0 to 62) and in the post-infection phase, indicating that a strong T cell response developed as a result of lung utilization as a route of immunization and subsequent infection.

Double immunization with 500 µg of complete M. vaccae as expected showed a protective effect against subsequent infection with live M. tuberculosis. The data in Tables 2D and 2E show the effects of immunization with 100 µg M. vaccae culture filtrate. Monkeys immunized intradermally • 4 showed signs of disease development 84 days after infection, whereas those that were immunized intramuscularly showed one sign of disease at 56 days and one at 84 days after infection. This was a significant delay in the onset of the disease showing that the immunization process caused some protective immunity.

Table 2

Monkeys control group

IDENTIFICATION Days Weight kg temperature RSE mm / hour PL PPD10 PL PPD1 X-ray examination notes S3101E 0 2.17 37.0 0 0.47 1.1 Negative 34 1.88 37.3 N 0.85 1.4 N 62 2.02 36.0 N 1.3 1.5 N -> Time of infection 28 2.09 38.0 2 1.3 3.7 Positive 56 1.92 37.2 20 May 5.6 9.1 Positive 84 1.81 37.5 8 4.7 5.6 Positive 121 she died

IDENTIFICATION days wt. kg temperature RSE mm / hour PL PPD 10pg PL PPD Ipg X-ray examination notes 3144-B 0 2.05 36.7 0 0.87 1,8 Negative 34 1.86 37.6 N 2.2 1.4 N 62 1.87 36.5 N 1.6 1.6 N -> Time of infection 28 2.10 38.0 0 12 8.7 Positive 56 1.96 37.6 0 29.6 21.1 Positive 84 1.82 37.3 4 45.3 23.4 Positive 131 she died

N = Not performed

Table 2B • 4 4

4 44

4 4

4 4

4 4 4

4 4

4 4

4 4

4 4 4

4 4 4

Monkeys IMMUNIZED

FULL TEMPERATURE KILLED BY M. VACCAE (500 pg) INTRADERMAL ADMINISTRATION

IDENTIFICATION Days wt. kg temperature RSE mm / hour PL PPD 10pg PL PPD Ipg X-ray examination notes 4080-B 0 2.05 37.1 1 1.1 0.77 Negative 34 1.97 38.0 N 1.7 1.4 N 62 2.09 36.7 N 1.5 1.5 N - »Time of infection 28 2.15 37.6 0 2.6 2.1 Negative 56 2.17 37.6 0 8.2 7.6 Negative 84 2.25 37.3 0 3.8 2.8 Negative 178 she died

IDENTIFICATION days wt. kg temperature RSE mm / hour PL PPD ιθμδ PL PPD Ιμβ X-ray examination notes 3586-B 0 2.29 37.0 0 1.1 1.4 Negative 34 2.22 38.0 N 1.9 1.6 N 62 2.39 36.0 N 1.3 1.6 N -> Time of infection 28 2.31 38.2 0 3.2 2.6 Negative 56 2.32 37.2 0 7.8 . 4.2 Negative 84 2.81 37.4 0 3.4 1,8 Negative 197 she died

N = Not performed • ·

Table 2C ·· ·· ·

49 9

9 94 4

4 4 4 4 4

4 4 4 4

49 444

4 4

4 4

4 4

4 4 4

44

Monkeys IMMUNIZED

FULLY KILLED BY M. VACCAE (500 pg) NON-EUROPEAN ADMINISTRATION

IDENTIFICATION days wt. kg temperature RSE mm / ho d PL PPD 10pg PL PPD / -gp ipg X-ray examination notes 3534-C 0 2.15 36.8 0 1.7 1.3 Negative 34 2.00 37.8 N 4.4 1.4 N 62 2.13 36.4 N 3.2 1.9 N -> Time of infection 28 2.38 37.7 0 1.9 2.6 Negative 56 2.42 37.8 0 5.3 4.7 Negative 84 2.46 37.1 1 3.1 3.2 Negative 210 No sign of lung disease Negative

IDENTIFICATION days wt. kg temperature RSE mm / ho d PL PPD 10pg PL PPD Ipg X-ray examination notes 3160-A 0 2.17 37.3 0 1,2 0.79 Negative 34 1.98 37.1 N 3.9 7.8 N 62 2.17 36.9 N 1.7 2.4 N - »Time of infection 28 2.38 37.7 0 1.9 2.6 Negative 56 2.42 37.8 0 5.3 4.7 Negative 84 2.46 37.1 1 3.1 3.2 Negative 210 Stabilized lung disease Positive

N = Not carried out · · · · · 9 · 9 9

9 99 9

9

9

99 999

9 9 • 9 9

9 9

9 9

9

Table 2D

MONKEY IMMUNIZED BY CULTIVATION FILTER (100 pg) administered INTRADERMAL

IDENTIFICATION days wt. kg temp and RSE mm / hour PL PPD 10pg PL PPD / -gp ipg X-ray examination notes 3564-B 0 2.40 37.2 0 1.4 1.4 Negative 34 2.42 38.1 N 3.3 2.7 N 62 2.31 37.1 N 3.1 3.4 N -> Time of infection 28 2.41 38.6 13 24 13.6 Negative 56 2.38 38.6 0 12.7 12.0 Negative 84 2.41 38.6 2 21.1 11.8 Positive 140 She died

IDENTIFICATION days wt. kg temperature RSE mm / hour PL PPD 10pg PL PPD / -gp ipg X-ray examination notes 3815-B 0 2.31 36.3 0 1.0 1.4 Negative 34 2.36 38.2 N 1.9 2,0 N 62 2.36 36.4 N 3.7 2.8 N - »Time of infection 28 2.45 37.8 0 2.1 3.3 Negative 56 2.28 37.3 4 8.0 5.6 Negative 84 2.32 37.4 0 1.9 2.2 Positive 210 Positive

N = Not performed

99 • ·

Table 2E

9 9 9 9 9

9 9 9

9

MONKEY IMMUNIZED BY CULTIVATIVE FILTER (100 pg) NON-EUROPEAN

IDENTIFICATION days wt. kg temperature RSE mm / hour PL PPD 10pg PL PPD / -gp ipg X-ray examination notes 4425-A 0 2.05 36.0 0 0.35 1,2 Negative 34 2,0 37.6 N 3.0 2.4 N 62 2.11 37.6 N 2.2 1.6 N -> Time of infection 28 2.21 38.0 0 8.4 4.1 Negative 56 2.11 37.6 0 23.9 17.7 Negative 84 2.18 37.9 0 8.4 7.2 Positive 210 Stabilized lung disease Positive

IDENTIFICATION days wt. kg temperature RSE mm / hour PL PPD 10pg PL PPD / -gp ipg X-ray examination notes 2779-D 0 2.56 38.6 2 1.9 1.4 Negative 28 2.55 37.9 N 0.78 1.1 N 56 2.69 38.4 N 1.3 1.5 N in -> Time of infection 56 2.25 39.0 24 N not specified Positive 96 She died

N = Not performed

Example 3

Effect of M. vaccae immunization on asthma in mice

This example demonstrates that both heat-killed M. vaccae and DD-β vaccae administered to mice intranasally are capable of inhibiting the development of an allergic immune response in the lungs. This has been shown in a mouse model of asthma-like lung disease. The severity of this allergic disease is reflected in the large number of eosinophils deposited in the liver.

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C57BL / 6J mice were administered intraperitoneally with 2 µg of ovalbumin in 100 µl of a glacial adjuvant at 0-14 days, followed by intranasal administration of 100 µg of ovalbumin in 50 µl of phosphate buffered saline (PBS) on day 28.

Eosinophils accumulated in the lungs of the mice, as determined by washing the airways of anesthetized mice with saline, pooling the lavage (bronchoalveolar lavage or BAL) and counting the eosinophils.

As seen in Figures 2A and 2B, groups of seven mice that were intranasally administered either 10 or 1000 µg of heat-killed M. vaccae (Fig. 2A) or 10, 100 or 200 µg DD-A1 vaccae 4 weeks prior to intranasal ovalbumin administration (Fig. 2A). , prepared as below (Fig. 2B), had a reduced percentage of eosinophils in BAL cells taken 5 days after ovalbumin administration compared to a control group of mice. A control group of mice was intranasally administered with PBS. Also, live M. bovis BCG at a dose of 2 x 10 ⁵ CFU reduces pulmonary eosinophilia. Figures 2A and 2B show mean SEM per group of mice.

Giant. 2C and 2D show that mice that were either intranasally administered either 1000 µg of heat-killed M. vaccae (Fig. 2C) or 200 µg of DD-AL vaccae (Fig. 2D) only one week prior to ovalbumin had a reduced percentage of eosinophils compared to the group control mice. In contrast, administration of live BCG a week before ovalbumin administration did not stop the development of pulmonary eosinophilia compared to the control group of mice.

As shown in Figure 2E, immunization with either 1 mg heat-killed M. vaccae or 200 µg DD-M vaccae by intranasal (in) or subcutaneous (sc) administration reduces pulmonary eosinophilia resulting from ovalbumin administration compared to a control group of animals in which was administered with PBS. In the same experiment, immunization with BCG Pasteur (BCG-P) and Connought (BCG-C) strains prior to ovalbumin administration also reduced the percentage of eosinophils in BAL mice.

Eosinophils are blood cells that are observed in the airways of allergic asthma. Substances that secrete eosinophils contribute to swelling and infection of the mucosal lining of the airways in allergic asthma. The data in Figures 2A-2E show that treatment with heat-killed M. vaccae or DD-AL vaccae reduces pulmonary eosinophil accumulation and may be useful for reducing eosinophilia-related infection in the airways, nasal mucosa and upper airways.

ΌΌ-M.vaccae free of mycolic acids and arabinogalactan

Mycolic acids were removed from DD-ΑΤ. vaccae by treatment with potassium hydroxide (0.5% KOH) in ethanol for 48 hours at 37 ° C. Mycolic acids removed from DD-M cell. The vaccae were then obtained by extraction from ether. Arabinogalactans have been removed from DD43 ··

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M. vaccae with KOH and subsequent reaction with 1% periodic acid in 3% acetic acid for 1 hour at room temperature and subsequent reaction with sodium borohydride 0.1M for 1 hour at room temperature. As seen in Table 3, mycolate depleted DD-A / vaccae as well as mycolic acid depleted DD-A / vaccae and arabinogalactan, administered intranasally to ovalbumin-sensitive mice, reduce eosinophil accumulation in the bronchoalveolar fluid that occurs upon ovalbumin stimulation.

Administration of heat-killed A (vaccae, DD-A). Thus, mycolic acid-depleted vaccae or DD-M.vaccae and arabinogalactan may reduce the severity of asthma and diseases that involve similar immune abnormalities such as allergic rhinitis.

In addition, serum samples were pooled from mice in the experiment described in Fig. 2E and antibodies to ovalbumin were measured by conventional enzyme-mediated immunoassay (EIA) immunoassays. As shown in Table 3A below, sera from BCG infected mice had higher levels for ovalbumin specific IgG1 than sera from control mice treated with PBS.

In contrast, mice immunized with M. vaccae or DD-A / vaccae had similar or lower levels of IgG1 specific covalbumin. Since IgG1 antibodies are characteristic of the Th2 immune response, these results correspond to the suppressive effect of heat-coated M. vaccae and DD-A /. vaccae to the Th2 immune response caused by asthma.

Table 3

Description of pulmonary eosinophilia in mice administered DD-A /. mycolic acid-depleted vaccae or DD-A / mycolic acid-depleted vaccae and arabinogalactan.

group % Eosinophils in BAL diameter PULL. PBS 58.8 8.4 DD-A / mycalated depleted vaccae acid 21.8 17.4 DD-A / mycalated depleted vaccae acid and arabinogalactan. 16.8 0.3

Note: At least 7 mice per group.

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Table 3Α

Low levels of antigen specific IgG1 in sera of mice immunized with heat-killed M. vaccae or DD- M. vaccae

Group Serum IgG1 diameter PULL M.vaccae i.n. 185.00 8.3 M. vaccae s.c. 113.64 8.0 DD-ΑΤ. vaccae i.n. 96.00 8.1 DD-AT. vaccae s.c. 110.00 4.1 BCG, Pasteur 337,00 27.2 BCG, Connaught 248.00 46.1 PBS 177.14 11.4

Note: Ovalbumin-specific IgG1 was detected with anti-mouse IgG1 (Serotec).

Group averages are expressed as reciprocal EU50 endpoint titer.

Example 4

Effect of immunization with M. vaccae, DD-AT vaccae or recombinant M. vaccae proteins on mouse tuberculosis.

This example illustrates the effect of immunization with heat-killed M.vaccae DDM.vaccae or recombinant M. vaccae proteins without the addition of an antigen enhancer or any combination of heat-killed M.vaccae with a group of recombinant proteins obtained from M. vaccae.

Mice were injected intraperitoneally with one of the following formulations at two doses three weeks apart.

a) Phosphate buffered saline (PBS, control);

b) Heat-killed M. vaccae (500 pg);

c) DD-Afvaccae (50 µg);

d) A group of recombinant proteins comprising: 15 µg of each of the following proteins: GV4P, GV7, GV9, GV27B, GV33 (prepared as described below); and

e) Heat-killed M. vaccae plus a group of recombinant proteins.

• ·

Three weeks after the last intraperitoneal immunization, mice were injected with 5 X 10 ⁵ living organisms H37Rv M.tuberculosis. After an additional three weeks, the mice were sacrificed and their spleen approved and tested for M. tuberculosis colony forming units (CFUs), which are indicative of the severity of the infection.

3A and 3B, each point represents one mouse. The baseline counts were the numbers of CFU obtained from control mice immunized with PBS only. All values obtained from experimental mice were expressed as the number of CFU logarithms below the baseline level of control mice (or log protection). As shown in Figure 3A, mice immunized with heat-killed M.vaccae or OD-M.vaccae show an average decrease of> 1 or 0.5 log CFU, respectively.

As shown in Figure 3B, the spleens of mice immunized with a group of recombinant proteins comprising: GV4P, GV7, GV9, GV27B and GV33 had CFU slightly lower than control mice. However, when GV4P, GV7, GV9, GV27B and GV33 were administered in combination with heat-killed M. vaccae, the reduction in CFU exceeded an average value of > 1.5 logs.

Based on these data, the efficacy of immunization of M.vaccae, OD-M.vaccae or recombinant proteins derived from M.vaccae against subsequent tuberculosis infection is evident, and it is further seen that M.vaccae, DD-M.vaccae and recombinant proteins may be treated as tuberculosis vaccines.

Example 5

Effect of intradermal injection of heat-treated Mycobacterium vaccae on psoriasis in human patients.

This example illustrates the effect of two intradermal injections of heat-killed Mycobacterium vaccae on psoriasis in human patients.

M. vaccae (ATCC No. 15483) was cultured in sterile medium 90 (fermentation yield 2.5 g / l; trypton 5 g / l; glucose 1 g / l) at 37 ° C. Cells were harvested by centrifugation and transferred to sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 ° C for one day. The medium was then centrifuged to obtain bacterial droplets and the culture filtrate was discarded. The bacterial droplets were then resuspended in phosphate buffered saline at a concentration of 10 mg / ml, equivalent to ΙΟ ¹⁰ M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120 ° C and stored frozen at -20 ° C. Prior to use, the M. vaccae suspension was reconstituted and dissolved. Dilute to a concentration of 5 mg / ml with phosphate buffered saline, autoclaved for 15 min at 120 ° C. C and, under sterile conditions, dispensed 0.2 ml into ampoules for use in patients.

Twenty-four volunteers with psoriasis, both men and women aged 15 to 61, who had no other systemic disease, received treatment. Pregnant women were not included in the trial. Patients had a PASI score of 12 to 35. The PASI score is a measurement of the location, size, and degree of peeling of skin in areas affected by psoriasis. A PASI score of more than 12 means a disease that is widespread throughout the body. The study was initiated over a four-week period with no systemic anti-psoriasis treatment or effective local therapy. Thereafter, 24 patients were injected intradermally with 0.1 ml M. vaccae (equivalent to 500 µg). A further three weeks followed by a second intradermal injection with the same dose of M. vaccae (500 µg). Psoriasis was evaluated four weeks before the first injection of heat-killed M. vaccae up to twelve weeks after the first injection as follows:

A. PASI scores were determined at weeks 4, 0, 3, 6 and 12;

B. The questionnaire was completed with patients at weeks 0, 3, 6 and 12; and

C. The affected areas of each patient were photographed at weeks 0, 3, 6, 9 and 12.

The data in Table 4 describe the age, sex and clinical profile of each patient.

Table 4

Patient data in the study of the effect of M. vaccae on psoriasis

Code patient age / gender duration of illness entry pass score PS-001 D.C. 49 / ž 30 years 28.8 PS-002 E.S. 41 / m 4 months 19.2 PS-003 M.G. 24 / ž 8 months 18.5 PS-004 D.B. 54 / m 2 years 12.2 PS-005 C.E. 58 / ž 3 months 30.5 PS-006 M.G. 18 / ž 3 years 15.0 PS-007 L.M. 27 / m 3 years 19.0 PS-008 C.C 21 / ž 1 months 12.2 PS-009 E.G 42 / ž 5 months 12.6 PS-010 J.G 28 / m 7 years 19.4 PS-011 J.U 39 / m 1 years 15.5 PS-012 C.S 47 / m 3 years 30.9 PS-013 H.B 44 / m 10 years 30.4

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PS-014 N.J 41 / m 17 years 26.7 ’ PS-015 J.T 61 / ž 15 years 19.5 PS-016 L.P 44 / m 5 years 30.2 PS-017 E.N 45 / m 5 years 19.5 PS-018 E.L 28 / ž 19 years 16.0 PS-019 B.A 38 / m 17 years 12.3 PS-020 P.P 58 / ž 1 years 13.6 PS-021 IF 27 / ž 8 months 22.0 PS-022 A.C 20 / ž 7 months 26.5 PS-023 C.A 61 / ž 10 years 12.6 PS-024 F.T 39 / m 15 years 29.5

A non-suppurative, localized reddened, slightly hardened injection site reaction was observed in all patients. No side effects or complaints were reported by patients. The data presented in Table 5 below are measurements of skin reactions at the injection site 48 hours, 72 hours and 7 days after the first and second injections of heat-killed M. vaccae. day 0) and 3, 6, 9, 12 and 24 weeks later.

It is clearly seen that by 9 weeks after the first injection of M. vaccae, 16 of the 24 patients showed a significant shift in PAHI score. Seven of the 14 patients who completed the 24-week follow-up were still free of clinical signs of severe disease recurrence. These results demonstrate the efficacy of multiple intradermal injection of inactivated M. vaccae in the treatment of psoriasis. A PASI score of less than 10 indicates a large area of healing of the affected areas. Histopathology of skin biopsies shows that normal skin structure is restoring. Only one of the first seven patients who completed the 28-week follow-up had a relapse.

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Table 5

Measurement of skin reaction in millimeters

code time measurement first injection second injection 48 hours 72 hours 7 days 48 hours 72 hours 7 days PS-001 12x10 12x10 10x8 15x14 15x14 10x10 PS-002 18x14 20x18 18x14 16x12 18x12 15x10 PS-003 10x10 14x10 10x8 15x12 15x10 10x10 PS-004 14x12 22x18 20x15 20x20 20x18 14x10 PS-005 10x10 13x10 neuvedeno neuvedeno neuvedeno neuvedeno PS-006 10x8 10x10 6x4 12x10 15x15 10x6 PS-007 15x15 18x16 12x10 15x13 15x12 12x10 PS-008 18x18 13x12 12x10 18x17 15x10 15x10 PS-009 13x13 18x15 12x8 15x13 12x12 12x7 PS-010 13x11 15x15 8x8 12x12 12x12 5x5 PS-011 17x13 14x12 12x11 12x10 12x10 12x10 PS-012 17x12 15x12 9x9 10x10 10x6 8x6 PS-013 18x11 15x11 15x10 15x10 15x13 14x6 PS-014 15x12 15x11 15x10 13x12 14x10 8x5 PS-015 15x12 16x12 15x10 7x6 14x12 6x4 PS-016 6x5 6x6 6x5 8x8 9x8 9x6 PS-017 20x15 15x14 14x10 15x15 17x16 neuvedeno PS-018 14x10 10x8 10x8 12x12 10x10 10x10 PS-019 10x10 14x12 10x8 neuvedeno 15x14 15x14 PS-020 15x12 15x15 12x15 15x15 14x12 13x12 PS-021 15x12 15x12 7x4 11x10 . 11x10 11x8 PS-022 12x10 10x8 10x8 15x12 13x10 10x8 PS-023 13x12 14x12 10x10 17x17 15x15 neuvedeno PS-024 10x10 10x10 10x8 10x8 8x7 8x7

Table 6

Clinical condition of the patient after M. vaccae injection (PASI score)

code den 0 week 3 week 6 week 9 week 12 week 24 PS-001 28.8 14.5 10.7 2.2 0.7 0 PS-002 19.2 14.6 13.6 10.9 6.2 0.6 PS-003 18.5 17.2 10.5 2.7 1.6 0 PS-004 12.2 13.4 12.7 7.0 1,8 0.2 PS-005 * 30.5 neuvedeno 18.7 neuvedeno neuvedeno 0 PS-006 15.0 16.8 16.4 2.7 2.1 3.0 PS-007 19.0 15.7 11.6 5.6 2.2 0 PS-008 12.2 11.6 11.2 11.2 5.6 0 PS-009 12.6 13.4 13.9 14.4 15.3 13.0 PS-010 18.2 16.0 19.4 17.2 16.9 19.3 PS-011 17.2 16.9 16.7 16.5 16.5 15.5 PS-012 30.9 36.4 29.7 39,8 ** PS-013 19.5 19.2 18.9 17.8 14.7 17.8 PS-014 26.7 14.7 7.4 5.8 9.9 24.4 *** PS-015 30.4 29.5 28.6 28.5 28.2 24.3 PS-016 30.2 16.8 5.7 3.2 0.8 3.3 PS-017 12.3 12.6 12.6 12.6 8.2 8.7 PS-018 16.0 13.6 13.4 13.4 13.2 12.8 PS-019 19.5 11.6 7.0 neuvedeno neuvedeno neuvedeno PS-020 13.6 13.5 12.4 12.7 12.4 4.4 PS-021 22.0 20.2 11.8 11.4 15.5 15.7 PS-022 26.5 25.8 20.7 11.1 8.3 5.6 PS-023 12.6 9.2 6.6 5.0 4.8 12.6 PS-024 29.5 27.5 20.9 19.0 29.8 21.2

Patient PS-005 received only one dose of autoclaved M. vaccae.

• ** Patient PS-012 excluded from trial, drug (penicillin) dermatitis caused *** *** Patient PS-014 was revaccinated

Patients receiving M.vaccae could achieve remission (PASI score = 0). Remission or improvement of the PASI score could be long-term. For example, patient PS-003 achieved remission by week 20 and was still in remission by week 80. A total of 13 of 24 patients showed greater than 50% improvement in PAHI score.

Patient PS-001 achieved remission at week 16, relapse occurred at week 48 (PAHI 2.7), the patient was revaccinated with M.vaccae injection, after which an improvement in PAHI occurred from 17.8 (week 60) to 0.8 ( week 84). Thus, improvement can be achieved by repeated treatment.

Example 6

Effect of intradermal injection of DD-A1. vaccae on psoriasis in human patients

This example illustrates the effect of intradermal injection of DD-A. Vaccae on psoriasis.

Seven volunteers suffering from psoriasis, men and women aged 18 to 45, who had no other systemic disease, were treated. Pregnant women were not included in the trial. Patients had a PASI score of 12 to 24. The PASI score is, as mentioned above, a measurement of the location, size and degree of peeling of skin at sites on the body affected by psoriasis. A PASI score of more than 12 means a disease that is widespread throughout the body. The study was initiated over a four-week period with no systemic anti-psoriasis treatment or effective local therapy. Then, 7 patients were injected intradermally with 0.1 ml DDM vaccae (equivalent to 100 µg). A further three weeks followed by a second intradermal injection with the same dose of DD-M. vaccae (100 µg). Psoriasis was evaluated four weeks prior to the first injection of M. vaccae up to six weeks after the first injection as follows:

A. PASI scores were determined at weeks -4, 0, 3 and 6;

B. patients were completed a questionnaire at weeks 0, 3 and 6; and

C. The affected areas of each patient were photographed at weeks 0 and 3.

The data in Table 7 describe the patient's age, sex and clinical profile.

Table 7

Patient data in a study of the effect of DD-M vaccae on psoriasis.

Code patient age / gender Duration of illness entry pass score PS-025 A.S 25 / ž 2 years 12.2 PS-026 M.B 45 / ž 3 months 14.4 PS-027 A.G 34 / m 14 years 24.8

PS-028 E.M 31 / m 4 years 18.2 PS-029 A.L 44 / m 5 months 18.6 PS-030 Great 42 / m 5 years 21.3 PS-031 R.A 18 / m 3 months 13.0

A non-suppurative, localized reddened, slightly hardened injection site reaction was observed in all patients. No side effects or complaints were reported by patients. The data presented in Table 8 below are measurements of skin reactions at the injection site 48 hours, 72 hours and 7 days after the first injection of DD-Ar. vaccae, and 48 hours and 72 hours after the second injection.

Table 8 skin reaction measurements in millimeters

Code time measurement first injection second injection 48 hours 72 hours 7 days 48 hours 72 hours PS-025 8x8 8x8 3x2 10x10 10x10 PS-026 12x12 12x12 8x8 neuvedeno 14x14 PS-027 9x8 10x10 10x8 9x5 9x8 PS-028 10x10 10x10 10x8 10x10 10x10 PS-029 8x6 8x6 5x5 8x8 8x8 PS-030 14x12 14x14 10x10 12x10 12x10 PS-031 10x10 12x12 10x6 14x12 12x10

The data in Table 9 are the PAI scores of seven patients at the time of the first injection of DD-A1. vaccae (day 0), and 3, 6.12 and 24 weeks later.

Table 9

Clinical status of patients after DD-A1 injection. vaccae (PASI score)

Code den 0 week 3 week 6 week 12 week 24 PS-025 12-2 4-1 1,8 1.4 1.7 PS-026 14-4 11-8 6.0 6.9 1.4

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PS-027 24-8 23-3 18.3 9.1 10.6 PS-028 18-2 24-1 28.6 decreasing PS-029 18.6 9.9 7.4 3.6 0.8 PS-030 21.3 15.7 13.9 16.5 13.6 PS-031 13.0 5.1 2.1 1.6 0.3

It is clearly seen that by 3 weeks after the first injection of DD-AL vaccae, five patients showed a significant improvement in the PAI score. By week 24, six of the seven patients showed significant improvement in PAHI scores.

Example 7

Preparation of M. vaccae compositions

This example illustrates the production of various M. vaccae components.

Preparation of (DD-) lipid and glycolipid-free M.vaccae and composition analysis

Heat-killed M. vaccae was prepared according to the procedure outlined in Example 1. To prepare lipid-free M. vaccae, the M.vaccae autoclave was collected in droplets by centrifugation, the droplets were washed with water, pooled again by centrifugation and then lyophilized. All lyophilized M. vaccae was stored and labeled as lyophilized M. vaccae. Before use in the experiment, it was resuspended in PBS to the desired concentration. The lyophilized M. vaccae was treated with chloroform / methanol (2; 1) for 60 minutes at room temperature to extract the lipids and repeat the extraction once. The delipidized residue after extraction from chloroform / methanol was further mixed with 50% ethanol for reflux for two hours to remove the glycolipids. The extraction from 50% ethanol was repeated twice. A group of 50% ethanol extracts was used as a source of M. vaccae glycolipids (see below). The residue from 50% ethanol extraction was lyophilized and weighed. Quantity prepared

M. vaccae free of lipids and glycolipids corresponded to 11.1% of the initial weight of M.vaccae used before drying For bioassays, M. vaccae (DD-M. Vaccae) free of lipids and glycolipids was resuspended in phosphate buffered saline by sonication and sterilized in autoclave.

The analyzes of the heat-killed M. vaccae and DD-AL vaccae compositions are shown in Table 9. Greater changes are seen in the fatty acids of the DD-AL vaccae compositions and the fatty acid compositions of DD-AL vaccae. The amino acids of the DD-M compositions. vaccae compared to the insoluble fractions of heat-killed M. vaccae.

The data in Table 9 show that the insoluble fraction of heat-killed M. vaccae contains 10% lipid by weight and the total amino acid content is 2750 nmol / mg or about 33% by weight. DD-M vaccae contains 1.3 wt% lipid and 4250 nmol / mg amino acids, which is approximately 51 wt%.

Table 9

Analysis of heat-killed M. vaccae and DD-M vaccae compositions

MONOSACCHARIDE COMPOSITIONS

sugar alditol M vaccae DD-M. vaccae Inositol 3.2% 1,7% Ribitol * 1,7% 0.4% Arabinitol 22.7% 27.0% Mannitol 8.3% 3.3% Galactitol 11.5% 12.6% Glucitol 52.7% 55.2%

COMPOSITION WITH FATTY ACIDS

fatty acid M vaccae DD-M vaccae 04: 0 3.9% 10.0% 06: 0 21.1% 7.3% 06: 1 14.0% 3.3% 08: 0 4.0% 1.5% 08: 1 * 1.2% 2.7% C18: lw9 20.6% 3.1% C18: lw7 12.5% 5.9% C22: 0 12.1% 43.0% C24: l * 6.5% 22.9%

The insoluble fraction of heat-killed M. vaccae contains 10% lipid and DD-M. vaccae contains 1.3% lipid by weight.

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Amino acid compositions

nmol / mg M. vaccae DD-AL vaccae ASP 231 361 THR 170 266 SER 131 199 GLU 319 505 FOR 216 262 GLY 263 404 ALA 416 621 CYS * 24 26 WALL 172 272 MET * 72 94 ILE 104 171 LEU 209 340 TYR 39 75 PHE 76 132 GlcNH2 5 6 HIS 44 77 LYS 108 167 ARG 147 272

Total amino acid content in the insoluble fraction of heat-killed M. vaccaeje 2750 nmol / mg or about 33%. Total amino acid content of DD-A7. vaccae is 4250 nmol / mg, or about 51 wt. %.

Comparison of DD-A1 compositions. vaccae with lipid and glycolipid-free forms of M. tuberculosis and M. smegmatis.

M. tuberculosis and M. smegmatis deprived of lipids and glycolipids were prepared using the previously described procedures for depleting M. vaccae lipids and glycolipids. As shown in Table 10, the percentage of amino acids contained in DD-M compositions. vaccae, DD-A /. tuberculosis and DD-AL smegmatis are not very different. However, the total amount of protein varies - two doses of DD-M. vaccae contained 34% and 55% protein, while DD-Af. tuberculosis and DD-AL. smegmatis contained 79% and 72% protein, respectively.

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Table 10

Amino acid composition of Mycobacteria free of lipids and glycolipids

Amino DD-M.Vaccae DD-AI. vaccae DD- DD-AI. tuberculosis acid dose 1 dose 2 M.smegmatis Asp 9.5 9.5 9.3 9.1 Thr 6.0 5.9 5.0 5.3 Ser 5.3 5.3 4.2 3.3 Glu 11.1 11.2 11.1 12.5 For 6.1 5.9 7.5 5.2 Gly 9.9 9.7 9.4 9.8 Ala 14.6 14.7 14.6 14.2 Cys 0.5 0.5 0.3 0.5 Wall 6.3 6.4 7.2 7.8 Met 1.9 1.9 1.9 1.9 Ile 3.6 3.5 4.1 4.7 Leu 7.8 7.9 8.2 8.3 Tyr 1.4 1.7 1,8 1,8 Phe 4.2 4.0 3.2 3.0 His 1.9 1,8 2,0 1.9 Lys 4.1 4.0 4.1 4.2 Arg 5.8 5.9 6.2 6.4 total% 55.1 33.8 72.1 78.5

protein

Analyzes of monosaccharide compositions show significant differences between DD-AZ. vaccae, and ΌΌ-Μ. tuberculosis and DD-AZ smegmatis. Monosaccharide compositions of two doses of DD-AZ. vaccae were the same and different from DD-AZ. tuberculosis and AZ smegmatis. Specifically, DD-AZ. vaccae contained free glucose while DD-AZ. tuberculosis and AZ smegmatis contained glycerol, as shown in Table 11

Table 11

Alditol Acetate Weight% mol% DD-M. vaccae group 1 inositol 0.0 0.0 arabinose 54.7 59.1 mannose 1.7 1.5 glucose 31.1 28.1 galactose DD-M. vaccae group 2 12.5 100.0 11.3 100.0 inositol 0.0 0.0 arabinose 51.0 55.5 mannose 2,0 1,8 glucose 34.7 31.6 galactose 12.2 100.0 11.1 100.0 DD-M.smeg inositol 0.0 0.0 glycerol 15.2 15.5 arabinose 69.3 70.7 xylose 3.9 4.0 mannose 2.2 1.9 glucose 0.0 0.0 galactose 9 £ 100.0 10 100.0 DD-Mtb inositol 0.0 0.0 glycerol 9.5 9.7 arabinose 69.3 71.4 mannose 3.5 3.0 glucose 1.5 1.3 galactose 12.4 96.2 10.7 96.0

M. vaccae glyco lipids

The above combined 50% ethanol extracts were dried by rotary evaporation, redissolved in water and lyophilized. The amount of glycolipids obtained constituted 1.2% of the original weight of M. vaccae used before drying. For bioassays, glycolipids were dissolved in phosphate buffered saline.

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Example 8

Immune properties of M. vaccae deprived of lipids and glycolipids and properties of recombinant M. vaccae proteins.

This example illustrates immune properties of various M. vaccae components.

Production of interleukin-12 from macro phages

Complete heat-killed M. vaccae and DD-A /. vaccae have shown different cytokine stimulating properties. Stimulation of the Th1 immune response was enhanced by the production of interleukin-12 (IL-12) from macro phages. The ability of various M. vaccae preparations to stimulate IL-12 production is apparent from the following.

A group of C57BL / 6J mice were injected intraperitoneally with DIFCO thioglycolate and after three days peritoneal macrophages were pooled and placed in cell culture medium with interferon gamma for three hours. The culture medium was removed and various concentrations of complete heat-killed (autoclaved) M. vaccae, lyophilized M. vaccae, DD-A) were added. vaccae and M. vaccae glycolipids prepared according to the foregoing procedures. After another three days at 37 ° C, the culture suspension was tested for the presence of IL-12 produced by macrophages.

In contrast, the same preparations of M. vaccae were tested for their ability to stimulate the production of interferon gamma from natural killer-NK cells. Spleen cells were prepared from Severe Combined Immunodeficient (SCID) mice. These populations contained 75-80% NK cells. Spleen cells were incubated at 37 ° C in culture with various concentrations of heat-killed M. vaccae, DD-A / vaccae, or M. vaccae glycolipids. The data in Fig. 5 shows that while heat-killed M. vaccae and M. vaccae glycopeptides stimulate the production of interferon gamma, DD-M. vaccae stimulates the production of interferon gamma relatively little. The data collected in Figures 4 and 5 show that DD-A is compared to complete heat-killed M. vaccae. vaccae better stimulator of IL-12 than interferon gamma.

These results show that removal of lipid and glycolipid components from A, vaccae removes molecular components that stimulate interferon gamma from NK cells, effectively removing an important cellular source of a cytokine that has many undesirable side effects. DD-A /. Thus vaccae maintains an increased capacity of Th1 immunity by stimulating IL-12 production but loses non-specific effects that may be caused by stimulating the production of interferon gamma from NK cells.

The effect of the DD-A / vaccae antigen enhancer and the many recombinant A / vaccae antigens of the invention produced according to the methods below depend on the measurement of stimulation of IL-12 secretion from mouse peritoneal macrophages. Figure 6A and 6B and 6C contain data from separate experiments in which the group of C57BL / 6 mice (Fig. 6A), BALB / c mice (Fig. 6B) • 9 • · 99

9 9 9 9 • 9 99 • · · ·

9 or 9 9 9 9 9 or C3H / HeJ mice (Fig. 6C) administered DIFCO thioglycolate intraperitoneally. After three days, peritoneal macrophages were pooled and placed in interferon gamma culture medium for three hours. Culture medium was removed and various concentrations of M. vaccae recombinant proteins GVs-3 (GV-3), GV-4P (GV-4P), GVc-7 (GV-7), GV-23, GV27, heat-killed M were added. vaccae, DD-M. vaccae (shown in the figures as delipidized M. vaccae in Figs. 6A, 6B and 6C), M. vaccae glycolipids or lipopolysaccharides.

After three days at 37 ° C, the culture supernatants were tested for the presence of macrophage IL-12. As shown in Figure 6A, 6B and 6C, recombinant proteins and preparations from M. vaccae stimulate the production of IL-12 from macrophages.

In the following experiment, IFNγ prepared peritoneal macrophages from BALB / c mice were stimulated with 40 pg / ml recombinant M. vaccae proteins in culture medium for 3 days and then tested for the presence of IL-12 produced by macrophages. As shown in Figure 7, in these experiments IFNγ prepared macrophages produced IL-12 during culturing with a control protein, ovalbumin (s). However, recombinant GV proteins 24B, 38BP, 38AP, 27, 5, 27B, 3, 23 and 22B stimulated more than twice the amount of IL-12 that was observed in macrophage control cultures.

Detection of nonspecific immune enhancer from complete M. vaccae and M.vaccae culture filtrate

Culture supernatant (S / N) of M. vaccae, M.vaccae killed, lipid-free M. vaccae, and

Lipid and glycolipid-free M. vaccae prepared as mentioned previously were tested for the efficacy of an antigen enhancer in eliciting a cytotoxic T cell immune response to ovalbumin and a structurally independent protein in mice. This specific cytotoxic response against albumin was determined as follows. C57BL / 6 mice (2 per group) were immunized by intraperitoneal injection of 100 µg ovalbumin and subsequently tested for antigen enhancers: autoclaved M. vaccae ·, lipid cleared M. vaccae, lipid cleared M. vaccae with glycolipids, extracted (DD - M (vaccae) and proteins extracted with SDS; pronase (protein degraded enzyme) SDS protein extract; culture filtrate of complete M. vaccae and heat-killed M. tuberculosis or heat-killed M. bovis BCG, M. phlei or M. smegmatis, or culture filtrate M. vaccae. After 10 days, spleen cells were stimulated in vitro for an additional 6 days with E.G7 cells which are EL4 cells (T cell lymphoma derived from C57BL / 6) inoculated with the ovalbumin gene and thus producing ovalbumin. The spleen cells were then tested for the ability to kill non-specifically EL4 target cells or kill specifically E.G7 ovalbumin-producing cells. The killing efficiency was determined by 51 chromium release by which EL4 and E.G7 cells were labeled (100 pCi at 2x10 ⁶ ) before 9 9 9 9 9 9

9 9 9 9 9 9 9 9

9 9 9 9 9 9 9 9 9 9

9 9 9 9 9

99 999 999 99 99 killing test. Lethal or cytolytic efficacy is expressed as a percentage of specific cell breakdown using the formula:

cpm in test cultures - cpm in control cultures x 100% cpm total - cpm in control cultures

It is generally known that ovalbumin-specific cytotoxic cells form only in mice immunized with ovalbumin with an antigen booster, but not in mice immunized with ovalbumin alone.

The scheme of Figure 7 shows the effect of various formulations of M. vaccae-derived antigens on the formation of cytotoxic T cells against ovalbumin in C57BL / 6 mice. As seen in Figure 7A, cytotoxic cells were generated in mice immunized with (i) 10 µg, (ii) 100 µg or (iii) 1 mg autoclaved M. vaccae or (iv) 75 µg M. vaccae culture filtrate. Figure 7B shows that cytotoxic cells were generated in mice immunized with (i) 1 mg uptake autoclaved M. vaccae or (ii) 1 mg lipids and glycolipids depleted of (DD-) M. vaccae. As shown in Figure 7C (i), cytotoxic cells were generated in mice immunized with 1 mg of total autoclaved M. vaccae. Figure 7C (ii) shows the active material in a soluble protein extract with SDS of DD-M. vaccae. Figure 7C (iii) shows that the active material in the antigen booster formulation of Figure 7C (ii) has been destroyed by treatment with the proteolytic enzyme pronase. The comparison shows that 100 µg of extracted SDS proteins had significantly greater immune enhancement capacity (Fig. 7C (ii)) than 1 mg of complete autoclaved M. vaccae (Fig. 7C (i)).

Mice immunized with 1 mg heat-killed M. vaccae (Fig. 7D (i)) formed cytotoxic cells to ovalbumin, but mice immunized separately with 1 mg heat-killed M. tuberculosis (Fig. 7D (ii)), 1 mg M. bovis BCG ( Figure 7D (iii)), 1 mg M. phlei (Figure 7D (iv)) or 1 mg M. smegmatis (Figure 7D (v)) did not form cytotoxic cells.

These results show that heat-killed M. vaccae and DD-M. vaccae have antigen enhancing properties that have not been observed in other mycobacteria. In addition, M. vaccae deprived of lipids and glycolipids prevents the stimulatory activity of NK cells but does not reduce the stimulatory activity of T cells.

In a separate experiment, mice immunized with ovalbumin and 200 µg of mycolic acid-depleted ΌΌ-M.vaccae and arabinogalactan were also able to produce cytotoxic cells (maximum specific cell breakdown was 28% to 46% compared to <8% specific breakdown in control mice immunized alone) ovalbumin).

• 4 ·· 4 4 »4 45

4444 44 44

4444 444444

44443 4 4 4 * 4 4 4

4444 4 · 4444

44 444 444

Culture filtrate Μ. The vaccae previously described was fractionated by isoelectric focusing and the fractions were tested for the efficacy of the antigen enhancing agent in an assay for specific cytotoxic anti-ovalbumin response in C57BL / 6 mice as previously reported. The greatest potency of antigen enhancers was seen in fractions corresponding to pIs of 4.2 - 4.32 (fractions 7 - 9), 4.49 - 4.57 (fractions 13 - 17) and 4.81 - 5.98 (Fractions 23-27).

Identification of proteins in DD-A /. vaccae using antibodies

BALB / c mice were immunized intraperitoneally with 50 µg DD-A /. vaccae once a week for 5 weeks. At week 6, the mice were sacrificed and their sera collected. Sera were tested for antibodies against recombinant M. vaccae-derived proteins prepared as described below in standard enzyme-linked immunity assays.

Antisera did not react with several recombinant M. vaccae proteins or ovalbumin, which served as an independent negative control protein in enzyme-linked assays (results not shown). Antisera from mice immunized with DD-A /. vaccae reacted with GV antigens derived from 12 M. vaccae. The results are shown in Table 12 below. The antisera described by GV3, 5P, 5, 7, 9, 22B, 24, 27, 27A, 27B, 33 and 45 are therefore present in DD-A /. vaccae.

Table 12

Ability of antiserum DD-A /. vaccae react with GV antigens derived from M.vaccae

GV Antigen 3 5P 5 7 9 22B 24 27 Mar: 27A 27B 33 45 ability reaeovat * 10 ³ 10 ³ 10 ³ 10 ² 10 ⁴ 10 ³ 10 ⁴ 10 ⁶ 10 ⁵ 10 ⁶ . 10 ⁴ 10 ⁴

Expressed as the highest serum dilution from DD-A /. vaccae immunized mice showing greater responsiveness than serum from unimmunized mice

Proteins in DD-A /. vaccae based on T cell responses

BALB / c mice were injected into each foot with 100 µg DD-A / vaccae in combination with incomplete Freund's antigen-enhancing substance, and 10 days later the mice were sacrificed to obtain lymph node populations. Cells from immunized and non-immunized control mice were stimulated in vitro with recombinant M. vaccae-derived GV proteins. After three days, cell proliferation and IFNγ production were tested.

· 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4

Response to GV proteins consisting in proliferation of lymph node T cells obtained from mice immunized with DD-A /. vaccae

Lymph Node Cells DD-A /. vaccae immunized mice did not respond to proliferation to an irrelevant protein, ovalbumin (data not shown). As shown in Table 13, lymph node cells of immunized mice responded to proliferation to GV 3, 7, 9, 23, 27, 27B, and 33. Corresponding cells from unimmunized mice did not respond to proliferation to said GV proteins, suggesting that mice immunized with DD -AND/. vaccae were immunized with these proteins.

Thus, GV 3, 7, 9, 23, 27, 27B and 33 proteins derived from M. vaccae are likely to be present in DD-A /. vaccae.

Table 13

Proliferation of lymph node cells DD-A /. vaccae immunized and control mice in response to GV proteins in vitro

GV protein Stimulation index * observed in the presence of 50 µg / ml GV proteins DD-A /. vaccae immunized mice control mice GV3 4.63 1.52 GV7 3.32 1,27 GV9 6.48 2.64 GV23 4.00 1.76 GV27 5.13 1.40 GV27B 7.52 1.48 GV33 3.31 1.45

* Stimulation index = cpm from increases in tritiolated thymidine in the presence of GV protein / cpm in the absence of GV protein

Development of IFNγ by lymph node cells DD-A /. vaccae immunized mice after in vitro stimulation with GV proteins

Lymph node cells from unimmunized mice do not produce IFNγ upon stimulation with GV proteins. As shown in Table 14 below, lymph node cells from mice immunized with DDM. vaccae secrete IFNγ in a dose-dependent manner when stimulated with GV 3, 5, 23, 27A, 27B, 33, 45, or 46, indicating that mice were immunized with these proteins. When cells of immunized mice were stimulated with an irrelevant protein, ovalbumin (data not 9 9 9 9 9 9 9 9 9

999 999 99 99), no IFNγ production was detectable. Thus, GV proteins 3, 5, 23, 27A, 27B, 33, 45, and 46 are probably included in DD-A /. vaccae.

Table 14

IFNγ production in DD-A lymph node cells. vaccae immunized mice after in vitro stimulation with GV protein

GV protein or control IFNγ (ng / ml) GV protein dose used in vitro (pg / ml) 50 10 2 GV-3 8.22 ± 3.73 immeasurable immeasurable GV-4P immeasurable immeasurable immeasurable GV-5 8.90 ± 4.54 0.57 ± 0.40 immeasurable GV-5P immeasurable immeasurable immeasurable GV-7 immeasurable immeasurable immeasurable GV-9 immeasurable immeasurable immeasurable GV-13 1.64 ± 0.40 immeasurable immeasurable GV-14 immeasurable immeasurable immeasurable GV-14B immeasurable immeasurable immeasurable GV-22B 20.15 ± 1.96 4.34 ± 0.02 immeasurable GV-23 41.38 ± 6.69 6.97 immeasurable GV-24B immeasurable immeasurable immeasurable GV-27 46.86 ± 17.14 33.06 ± 17.61 10.14 ± 3.01 GV-27A 7.25 ± 4.36 immeasurable immeasurable GV-27B 100.36 ± 37.84 33.03 ± 7.54 14.33 ± 1.01 GV-29 5.93 ± 0.47 immeasurable immeasurable GV-33 9.82 ± 4.64 immeasurable immeasurable GV-38AP 1.44 ± 1.20 immeasurable immeasurable GV-38BP 5.62 ± 0.70 immeasurable immeasurable GV-42 immeasurable immeasurable immeasurable GV-44 immeasurable immeasurable immeasurable DD-M.Vaccae 109.59 ± 15.48 90.23 ± 6.48 65.16 ± 3.68 M. vaccae 68.89 ± 4.38 67.91 ± 7.92 48.92 ± 3.86

Proteins in DD-A /. vaccae as non-specific immune enhancers

In the following experiments, five GV27, 27A, 27B, 23 and 45 proteins were used as non-specific ovalbumin antigen immune enhancers to immunize mice as previously described in Example 6. As shown in Figure 12, 50 µg of any of the recombinant 44-44.

4 · * «4 4« • 4 · 4

4 4 4

44

4

4 «4

4 4 4 • 4V>

· 4 4 4

The 44 proteins GV27, 27A, 27B, 23 and 45, when injected with 50-100 µg ovalbumin, demonstrated antigen-enhancing properties of their ability to form cytotoxic cells against ovalbumin.

Example 9

Autoclaved M. vaccae forms cytotoxic CD8 T cells against M. tuberculosis infected macrophages

This example demonstrates the ability of killed M. vaccae to stimulate cytotoxic CD8 T cells that preferentially kill macrophages that have been infected with M. tuberculosis.

Mice were immunized by intraperitoneal injection of 500 µg of killed M. vaccae prepared as previously described in Example 1. Two weeks after immunization, the spleen cells of immunized mice were passed through a column supplemented with CD8 T cells (R&D Systems, St. Paul, MN, USA).

The splenic cells recovered from the column showed an enrichment of 90% CD8 T cells. These T cells as well as CD8 T cells from the spleen of unimmunized mice were tested for their ability to kill uninfected macrophages, or macrophages that were infected with M. tuberculosis.

Macrophages were obtained from the peritoneal cavity of mice five days after intraperitoneal administration of 1 ml of 3% thioglycolate. Macrophages were infected overnight with M. tuberculosis at a rate of two mycobacteria per macrophage. All macrophage preparations were labeled with 51 chromium 2 pCi per 10 ⁴ macrophages. Then, the macrophages were cultured with CD8 T cells overnight (16 hours) until the killing ratio reached 30: 1. Specific killing was monitored by chromium release and expressed as a percentage of specific disintegration determined as in Example 5.

After 3 days of co-culture of CD8 T cells with macrophages, the production of IFN-γ and its release into the medium was measured by ELISA. ELISA plates were coated with monoclonal antibody from rats targeting mouse IFN-γ (Pharmigen, San Diego, CA, USA) in PBS for 4 hours at 4 ° C. Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. Plates were then washed four times in PBS containing 0.2% Tween 20 and samples were diluted 1: 2 in culture medium, incubated overnight at room temperature in ELISA plates. The plates were washed again and biotinylated monoclonal rat anti-mouse IFN-γ antibody (Pharmigen) diluted to 1 µg / ml in PBS was added to each well. Plates were then incubated for one hour at room temperature, washed and washed

Horseradish peroxidase savidine D (Sigma A-3151) at a 1: 4000 dilution in PBS was added. After an additional hour of incubation at room temperature, the plates were washed and the OPD substrate was added. After 10 minutes, the reaction was quenched with 10% (v / v) HCl. The optical density at 490 nm was determined. It was positively found that the fractions obtained from both replicas gave twice the OD of the average OD of the cells cultured in the medium alone.

As shown in Table 15, CD8 T cells from the spleen of mice vaccinated with M. vaccae were cytotoxic to macrophages infected with M. tuberculosis and did not cause disintegration of uninfected macrophages. CD8 T cells from unimmunized mice did not cause macrophage disintegration. CD8 T cells from natural or unimmunized mice generated IFN-γ when cultured together with infected macrophages. The amount of IFN-γ produced in co-culture was greater with CD 8 T cells obtained from M. vaccae immunized mice.

Table 15

Effect with infected and uninfected M. tuberculosis macrophages

CD8 T cells% Specific breakdown of IFN-γ (ng / ml) macrophages uninfected infected uninfected infected control 0 immunized with M. vaccae 0

0,7 24,6

2,2 43,8

Example 10

Purification and characterization of polypeptides from M. vaccae culture filtrate

This example illustrates the preparation of soluble M. vaccae proteins from the culture filtrate. Unless otherwise indicated, all percentages in the following are weight per volume.

M. vaccae (ATCC No. 15483) was cultured in sterile medium 90 at 37 ° C. Cells were harvested by centrifugation and transferred to sterile Middlebrook 7H9 glucose medium at 37 ° C for one day. The medium was subjected to centrifugation (to remove cell volume) and filtered through a 0.45 µm filter into a sterile bottle.

The culture filtrate was concentrated by lyophilization and redissolved in MilliQ water. A small amount of insoluble material was removed by filtration through a 0.45 µm membrane. Cultivation

I • 9 • 9

9 «9

9 9 9 · «9 9 9 9» 999

999 99 9 · The filtrate was stripped of salt by membrane filtration in a 400 ml Amicon mixer containing a 3 kDa cut-off membrane (MWCO). The pressure was maintained at 50 psi using nitrogen. The culture filtrate was repeatedly concentrated by membrane filtration and diluted with water until the conductivity of the sample was less than 1.0 mS. This procedure reduced the volume of 20 L to approximately 50 mL. Protein concentration was determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA).

The salt-free culture filtrate was separated into fractions by ion exchange chromatography on a Q-Sepharose column (Pharmacia Biotech, Uppsala, Sweden) (16 x 100 mm) equilibrated with 10 mM Tris HCl buffer pH 8.0. The polypeptides were purified by elution with a linear NaCl gradient from 0 to 1.0 M in the above-mentioned buffer system. The column eluent was monitored at 280 nm.

A group of polypeptides eluted from the ion exchange column was encapsulated in a 400 ml Amicon mixer containing a 3 kDa MWCO membrane. The pressure was maintained at 50 psi using nitrogen. Polypeptides were repeatedly concentrated by membrane filtration and diluted with 1% glycine until the conductivity of the sample was less than 0.1 mS.

Purified polypeptides were then fractionated by preparative isoelectric focusing in a Rotofor (Bio-Rad, Hercules, CA, USA). The pH gradient was determined with a mixture of ampholytes (Pharmacia Biotech) containing 1.6% pH of 3.5 to 5.0 ampholytes and 0.4% pH of 5.0 to 7.0 ampholytes. Acetic acid (0.5 M) was used as the anolyte and 0.5 M ethanolamine as the catholyte. Isoelectric focusing was performed at a constant power of 12W for 6 hours according to the manufacturer's instructions. 20 fractions were obtained.

The isoelectric focusing fractions were pooled and the polypeptides purified on a Vydac C4 column (Separations Group, Hesperia, CA, USA) having a pore size of 300 Angstrom, a particle size of 5 microns (10 x 250 mm). Polypeptides were purified by elution on a linear gradient of acetonitrile (0% to 80% v / v) in 0.05% (v / v) trifluoroacetic acid (TFA). The flow rate was 2.0 mL / min and the HPLC eluent was monitored at 220 nm. Fractions containing polypeptides were pooled to achieve maximum purity of individual samples.

Relatively abundant amounts of polypeptide fractions were re-chromatographed on a Vydac C4 column (Separations Group, Hesperia, CA, USA) having a pore size of 300 Angstrom, a particle size of 5 microns (4.6 x 250 mm). Polypeptides were purified by elution from a linear gradient column from 20% to 60% (v / v) acetonitrile in 0.05% (v / v) TFA at a flow rate of 1.0 mL / min. The eluent column was monitored at 220 nm. Fractions containing the elution of purified polypeptides were pooled to maximize the purity of the individual samples.

• 4 « «4 · 4 * · 4 4 • 4 • & 4 «4 4 • 4 • 4 4 · • 4 • 4 • • 4 4 4 4 ··· e 4

Approximately 20 samples with polypeptides were obtained and analyzed for purity on a polyacrylamide gel according to the Laemmli procedure (Laemmli, U. K., Nature 277: 680-685, 1970).

Polypeptide fractions that were significantly contaminated were further purified using a Mono Q column (Pharmacia Biotech), a 10 micron (5 x 50 mm) particle size, or a Vydac Diphenyl column (Separations Group), a 300 Angster pore size, particle size 5 microns (4.6 x 250 mm). From the Mono Q column, the polypeptides were purified by elution with a linear gradient from 0 to 0.5 M NaCl in 10 mM Tris HCl pH 8.0. From the Vydac Diphenyl column, the polypeptides were purified by eluting with a linear gradient of acetonitrile (20% to 60% v / v) in 0.1% TFA. The flow rate was 1.0 ml / min and the column eluent was monitored at 220 nm for both columns. The best fractions of the polypeptides were pooled and analyzed for purity on a 15% polyacrylamide gel as previously reported.

To determine the sequence, the polypeptides were individually dried on Biobrene ™ (Perkin Elmer / Applied BioSystems Division, Foster City, CA) - using a glass wool filter. Polypeptide filters were inserted into a Perkin Elmer / Applied BioSystems Procise 492 protein sequencing apparatus and the polypeptides were sequenced from the amino terminus using traditional Edman chemistry. The amino acid sequence was determined for each polypeptide by comparing the time delay of the PTH amino acid derivative relative to the corresponding standard PTH derivative.

Internal sequences were also determined on some antigens by digesting antigens with Lys-C endoprotease, or by chemically cleaving the antigen with cyanogen bromide. The peptides obtained by these procedures were separated by reverse phase HPLC on a Vydac C1 column using 0.05% (v / v) trifluoroacetic acid mobile phase with an acetonitrile gradient containing 0.05% (v / v) TFA (1% / min). The eluent was monitored at 214 nm. The major internal peptides were determined by their UV absorbance and their N-terminal sequences were determined as previously described.

Six soluble M. vaccae antigens labeled GVc-1, GVc-2, GVc-7, GVc-13, GVc-20 and GVc-22 were isolated using the above procedures. The determined N-terminal and internal sequences for GVc-1 are set forth in SEQ ID NOs: 1, 2 and 3, respectively; the N-terminal sequence for GVc-2 is shown in SEQ ID NO: 4; the internal sequences for GVc-7 are set forth in SEQ ID NOs: 5-8; the internal sequences for GVc-13 are set forth in SEQ ID NOs: 9-11; the internal sequence for GVc-20 is set forth in SEQ ID NO: 12; and the N-terminal sequences for GVc-22 are set forth in SEQ ID NOs: 56 to 59, respectively. Each of the peptide sequences described herein begins with an amino acid residue that is likely to be at this position in the polypeptide, according to known specific cyanogen bromide (Met) or Lys-C (Lys) cleavage.

• * φ φ

In addition to the aforementioned preparatory isoelectric focusing procedure, three additional polypeptides designated GVc-16, GVc-18 and GVc-21 were isolated using a preparative electrophoresis (SDS-PAGE) gel cleaning step containing sodium polyacrylamide dodecyl sulfate. Specifically, the fractions containing the polypeptide mixtures of the isoelectric purification focusing step described previously were purified by SDS-PAGE on a 15% polyacrylamine gel. The samples were dissolved in the sample precipitating buffer and loaded onto the gel. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane by electroblotting in 10 mM 3- (cyclohexylamino) 1-propanesulfonic acid (CAPS) buffer at pH 11 containing 10% (v / v) methanol. The transferred protein groups were determined by Coomassie blue PVDF membrane staining. The PVDF membrane regions covered with the most abundant polypeptide species were excised and directly inserted into a sample cassette of a Perkin Elmer / Applied BioSystems Procise 492 protein sequencing apparatus. Protein sequences were determined as previously described. The N terminus sequences for GVc-16, GVc-18 and GVc-21 are shown in SEQ ID NOs: 13,14 and 15, respectively.

Other antigens designated GVc-12, GVc-14, GVc-15, GVc-17 and GVc-19 were isolated using a preparative SDS-PAGE purification step in addition to the chromatographic procedures described above. More specifically, the fractions containing the antigen mixture of the previously described Vydac C4 HPLC purification step were separated into fractions by a polyacrylamide gel SDS-PAGE method. The samples were dissolved in a non-precipitating buffer and applied to the gel. The separated proteins were transferred to a PVDF membrane by electroblotting in 10 mM CAPS buffer, pH 11 containing 10% (v / v) methanol. The transferred protein groups were determined by staining with Coomassie blue PVDF membrane. PVDF membrane regions covered with the most abundant polypeptide species were excised and directly inserted into a sample cassette of a Perkin Elmer / Applied BioSystems Procise 492 protein sequencing device. Protein sequences were determined as previously described. The N terminus sequences for GVc-12, GVc-14, GVc-15, GVc-17 and GVc-19 are shown in SEQ ID NOS: 16 to 20, respectively.

All of the above amino acid sequences were compared to known amino acid sequences listed in the SwissProt database (version R32) using the GeneAssist system. No significant homologous amino acid sequences with sequences GVc-2 to GVc-22 were obtained. The amino acid sequence for GVc-1 was found to be somewhat similar to the previously determined sequence from M. bovis and M. tuberculosis. Particularly for GVc-1, some homology to M. tuberculosis MPT83 and cell surface protein as well as MPT70 has been found. These proteins form part of a family of proteins (Harboe et al., Sca. J. Immunol. 42: 4651, 1995).

• 4 · 4

I ti ti ti I ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti

Subsequent studies have led to the isolation of DNA sequences for GVc-13, GVc-14 and GVc-22 (SEQ ID Nos. 142, 107 and 108, respectively). The corresponding putative amino acid sequences for GVc-13, GVc-14 and GVc-22 are set forth in SEQ ID NOs: 143, 109 and 110, respectively. The DNA sequence determined for the full length of the gene encoding GVc-13 is set forth in SEQ ID NO: 195 with the corresponding putative amino acid sequence set forth in SEQ ID NO: 196.

Further studies with GVc-22 indicate that only part of the gene that encodes GVc-22 has been cloned. No protein expression occurred when inserted into the pET10 expression vector. Subsequent screening of the M. vaccae BamH1 genomic DNA library with an incomplete gene fragment resulted in the isolation of the full length gene encoding GVc-22. To distinguish between the full-length clone and part of GVc-22, the antigen expressed by the full-length gene was termed GV22B. The determined nucleotide sequence of the gene encoding GV-22B and the predicted amino acid sequence are set forth in SEQ ID NOS: 144 and 145, respectively.

The amplification of primers AD86 and ADI 12 (SEQ ID Nos. 60 and 61, respectively) was designed according to the amino acid sequence GVc-1 (SEQ ID No. 1) and the M. tuberculosis MPT70 gene sequence. Using these primers, a 310 bp fragment from M. vaccae genomic DNA was amplified and cloned into the pBluescript II SK ⁺ (Stratagene) vector from EcoBN. The sequence of the inserted clone is shown in SEQ ID NO: 62. The insert portion of this clone was used to screen the M, vaccae genomic DNA library generated in lambda ZAP-Express (Stratagene, La Jolla, CA). The isolated clone contained an open reading frame homologous to the M. tuberculosis MPT83 antigen and was renamed GV-1/83. This gene was also homologous to the M. bovis MPB83 antigen. The determined nucleotide sequence and predicted amino acid sequence are set forth in SEQ ID NOS: 146 and 147, respectively.

The degenerate oligonucleotides EV59 and EV61 (SEQ ID NOs: 148 and 149, respectively) were designed from the amino acid sequence shown in SEQ ID NOs: 1 and 2.

Using PCR, a 100 bp fragment was amplified, cloned into plasmid pBluescript II SK ⁺ and sequenced (SEQ ID NO: 150) according to standard procedures (Sambrook et al. Ibid). The cloned insert was used to map the M. vaccae genomic DNA library generated in lambda ZAP-Express. The isolated clone showed homology to the M. tuberculosis MPT70 antigen and the M. bovis MPB70 antigen and was named GV-1/70. The determined nucleotide sequence and predicted amino acid sequence for GV-1/70 are shown in SEQ ID NOs: 151 and 152, respectively. For expression and purification, genes encoding GV1 / 83, GV1 / 70, GVc-13, GVc-14 and GV-22B were cloned into the pET10 expression vector (Novagen, Madison, WI). Expression and purification were performed according to the manufacturer's instructions.

• • 4 • 4 « • 4 4 4 « 4 • • • 4 4 9 • 4 4 • • • 4 · 4 4 9 4 · • 4 4 • • 4 · 9 • 4 4 • 4 4 9 4 4 4 4 4 4 4 4 4 4 4 ·

Purified polypeptides were examined for the ability to cause T cell proliferation and IFN-γ production in peripheral blood cells of immune human donors. These donors are known to have a positive skin test of PPD (purified M. tuberculosis protein derivative) and their T cells showed proliferation in response to PPD. Donated PBMCs and crude soluble proteins from M. vaccae culture filtrate were cultured in medium containing RPMI 1640 supplemented with 10% (v / v) autologous serum, penicillin (60 pg / ml), streptomycin (100 pg / ml), and glutamine (2 mM). .

After 3 days, 50 μΐ of media was removed from each well to determine IFN-γ levels as described below. The plates were cultured for an additional 4 days and then supplemented with IpCi / well of tritiated thymidine for an additional 18 hours, from which a tritiated supplement determined using a scintillation counter was obtained. It was positively found that the proliferation-stimulating fractions obtained from both replicas caused twice as much proliferation as that observed in cells cultured in the medium alone.

IFN-γ was measured using an ELISA assay. ELISA plates were coated with a monoclonal mouse antibody directed against human IFN-γ (Endogen, Wobural, MA) with 1 µg / ml phosphate buffered saline (PBS) for 4 hours at 4 ° C.

Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. Plates were then washed four times with PBS / 0.2% Tween 20 and samples were diluted 1: 2 in culture medium in ELISA plates and incubated overnight at room temperature. The plates were washed again and biotinized polyclonal rabbit serum against human IFN-γ (Endogen) diluted to 1 µg / ml in PBS was added to each well. The plates were then incubated for 1 hour at room temperature, washed, and horseradish peroxidase with avidin A (Vector Laboratories, Burlingame, CA) was added at a 1: 4000 dilution in PBS. After an additional hour of incubation at room temperature, the plates were washed and orthophenylenediamine (OPD) substrate was added. The reaction was stopped after 10 minutes with 10% (v / v) HCl. The optical density (OD) was determined to be 490 nm. It was positively found that the fractions obtained from both replicas have twice the OD of the average OD of the cells cultured in the medium alone.

Examples of polypeptides that contain peripheral blood monoclonal (PBMC) T cell proliferation stimulating sequences and IFN-γ production are shown in Table 16, where (-) indicates inefficiency, (+/-) means polypeptides with a result less than twice as high as The control medium efficiency (+) means polypeptides two to four times more potent than the control medium, and (++) means polypeptides having an activity greater than four times the potency.

Table 16

antigen proliferation GVc-1 ++ GVc-2 + GVc-7 +/- GVc-13 + GVc-14 ++ GVc-15 + GVc-20 +

IFN-γ + / ++ ++ +

+ +

Example 11

Purification and characterization of polypeptides from M. vaccae culture filtrate by electrophoresis on a 2 dimensional polyacrylamide gel.

Soluble M. vaccae proteins were isolated from the culture filtrate using two-dimensional polyacrylamide gel electrophoresis as described below. Unless otherwise indicated, all percentages in the following are by weight per volume.

M. vaccae (ATCC No. 15483) was cultured in sterile Med 90 at 37 ° C. M. tuberkulosis strain H37Rv (ATCC No. 27294) was cultured in sterile Middlebrook 7H9 medium with Tween 80 and oleic acid / albumin / dextrose / catalase additives (Difco Laboratories, Detroit, Michigan). Cells were harvested by centrifugation and transferred to sterile Middlebrook 7H9 glucose medium at 37 ° C for one day. The medium was then subjected to centrifugation (to separate the cell contents) and filtered through a 0.45 µm filter into sterile bottles. The culture filtrate was concentrated by lyophilization, diluted again with MilliQ water. A small amount of insoluble material was removed by filtration through a 0.45 µm membrane filter. The culture filtrate was de-salted by membrane filtration in a 400 ml Amicon mixing unit containing a 3 kDa membrane (MWCO). The pressure was maintained at 60 psi using nitrogen. The culture filtrate was repeatedly concentrated by membrane filtration and diluted with water until the conductivity of the sample was less than 1.0 mS. This procedure reduced the volume of 20 L to approximately 50 mL. Protein concentration was determined by Bradford protein assay (Bio-Rad, Hercules,

CA, USA).

The salt-free culture filtrate was separated into fractions by ion exchange chromatography on a Q-Sepharose column (Pharmacia Biotech) (16 x 100 mm) equilibrated with 10 mM Tris HCl.

4 · · ·· ·· · * · *

V 9 9 9 9 9 9 9 9 9

9 9 9 9 9

9 99 999 999 «· ·· pH 8.0 buffer. The polypeptides were purified by elution with a linear NaCl gradient from 0 to 1.0 M in the above-mentioned buffer system. The column eluent was monitored at 280 nm.

A group of polypeptides purified by ion exchange column elution was separated into fractions by preparative 2D gel electrophoresis. Samples containing 200-500 µg of polypeptide were treated with 8M urea and placed in polyacrylamide gel columns (2 mm diameter, 150 mm length, pH 5-7) for isoelectric focusing. After the isoelectric focusing step, the first dimension gels were balanced by reducing the buffer and placed in the second dimension gels (16% polyacrylamide). Polypeptides from the second dimension separation were transferred to PVDF membranes by electroblotting in 10 mM CAPS buffer pH 11 containing 10% (v / v) methanol. PVDF membranes were stained for Coomassie blue proteins. PVDF regions containing the polypeptides were excised and directly inserted into a sample cassette of a Perkin Elmer / Applied BioSystems Procise 492 protein sequencing apparatus. The polypeptides were sequenced from the amino terminus using traditional Edman chemistry. The amino acid sequence of each polypeptide was determined by comparing the delay of the PTH amino acid derivative with the corresponding standard for the PTH derivative. Twelve polypeptides designated as: GVs-1, GVs-3, GVs-4, GVs-5, GVs-6, GVs-8, GVs-9, GVs-10, GVs-11, GV34 and GV- were isolated using these procedures. 35. The N-terminal sequences for these polypeptides are set forth in SEQ ID NOS: 21-29, 63 and 64, respectively. To extend the amino acid sequence previously obtained for GVs-9, multiple proteins were purified using the above procedures. The extended amino acid sequence for GVs-9 is set forth in SEQ ID NO: 65. Further studies resulted in the isolation of the DNA sequence for GVs-9 (SEQ ID NO: 11) and GV-35 (SEQ ID NO: 155). The corresponding putative amino acid sequences are set forth in SEQ ID NOs: 12 and 156, respectively. The extended DNA sequence for GVs-9 is set forth in SEQ ID NO: 153 and the corresponding putative amino acid sequence is set forth in SEQ ID NO: 154. The putative amino acid sequence for GVs-9 has been altered in SEQ ID NO: 197. All of these amino acid sequences were compared to known amino acid sequences in the SwissProt database (version R35 plus update). No significant homologous regions except GVs-3, GVs-4, GVs-5 and GVs-9 were found. GVs-9 showed some homology to the two previously mentioned M. tuberkulosis proteins, namely the M. tuberkulosis cutinase precursor and the hypothetical M. tuberkulosis 22.6 kDa protein. Again, GVs-3, GVs-4 and GVs-5 showed some similarity to the M. leprae 85A and 85B proteins (SEQ ID NOs: 30 and 31, respectively), M. tuberkulosis (SEQ ID NOs: 32 and 33). , respectively) and M. bovis (SEQ ID NOs: 34 and 35, respectively) and with the 85C protein antigen of M. leprae (SEQ ID NO. 36) and M. tuberkulosis (SEQ ID NO. 37).

4

4 4 4 4

4 4 · · · ·

4 44 4

4 4 ·· ·

4 4 4 4 4 4444

Example 12

DNA cloning procedure for M. vaccae antigen series 85.

Experimental samples for antigens 85A, 85B, and 85C were prepared by polymerase chain reaction (PCR) using degenerate oligonucleotides (SEQ ID NOs: 38 and 39) designed for regions of the genomic sequences of antigen 85 that are retained among family members in given mycobacterial species. and among mycobacterial species. These oligonucleotides were used under less stringent conditions to amplify the target sequence of M. vaccae genomic DNA. A suitable size, 485 bp, was determined, purified and cloned into the T-terminal pBluescript II SK (Stratagene, La Jolla, CA). Twenty-four individual colonies were randomly tested for the presence of antigen 85 as a result of PCR, then sequenced using a Perkin Elmer / Applied Biosystems model 377 automated sequencing device and M13-base primers, T3 and T7. Searching for homologous sequences in a gene bank has shown that twenty-three clones contain an inserted sequence that exhibits significant homology with published genes of M. tuberkulosis and M. bovis antigen 85. Approximately half were more homologous to the 85C antigen gene sequence and the remainder were more similar to the 85B antigen sequence. Furthermore, the two putative genomic sequences of the 85 M. vaccae antigen were 80% homologous to each other. Because of this high similarity, this PCR fragment of antigen 85 was selected for the low resolution screening of the M. vaccae genomic library for all three genes of antigen 85.

The M. vaccae genomic library was processed by lambda Zap-Express (Stratagene, La Jolla, CA) by cloning / LzmHI of partially aligned genomic DNA into a single-aligned λ vector, with 3.4 x 10 6, producing independent plaque forming units. For the intended search, twenty-seven thousand plaques from this non-amplification library were coated at low resolution to eight 100 cm @ 2 plates. For each plate, a double plaque lift was taken on a Hybond-N ⁺ nylon membrane (Amersham International, United Kingdom) and hybridized under conditions of reduced sensitivity (55 ° C) to produce the 85C radiolabeled PCR product. Auroradiography showed that seventy-nine plaques consistently hybridized to the 85C antigen under these conditions. Thirteen positively hybridizing plaques were randomly selected for further analysis and removed from the library plates, each positive clone being used to generate secondary search plates containing about 200 plaques. Duplicate lifts of each plate were taken using a Hybond-N ⁺ nylon membrane and hybridized under the conditions used in the previous screening. Multiply positive hybridizing plaques were detected on each of the thirteen scanned plates. Two well-isolated positive phages were taken from each secondary plate for further analysis. Using in vitro excision was • 4

44 • 5 · «

4 4

• 4 4

4 * ·

4 4 4 4

Twenty-six plaques were converted to phage plasmid and restriction mapped. Based on this mapping, clones could be divided into four classes. Using the Perkin Elmer / Applied Biosystems model 377 automatic sequencing device, sequence data of the 5 'and 3' ends of the inserted regions of several representatives of each group and primers T3 and T7 were obtained. Sequence homology was determined using BLASTN analysis according to the EMBL database. Two of these clone sets were homologous to the M. bovis and M. tuberculosis 85A antigen genes, each containing either the 5 'or 3' end of the M. vaccae gene (This gene was cleaved during library formation because it contains the internal region of jBíztwHI). The remaining clones were found to contain sequences homologous to the 85B and 85C antigens from the mycobacterial species group. Appropriate subclones were created and sequenced to determine the remaining nucleotide sequences for each gene. The overlapping sequences were aligned using the DNA Strider computer program. The DNA sequences for the M. vaccae antigens 85A, 85B and 85C detected are set forth in SEQ ID NOs: 40 to 42, respectively, the predicted amino acid sequence is set forth in SEQ ID NOs: 43 to 45, respectively.

The M. vaccae GVs-3 and GVs-5 antigens were expressed and purified as described below. The amplification primers were designed from the inserted sequences of GVs-3 and GVs-5 (SEQ ID Numbers 40 and 42, respectively) using sequence data in the direction of the putative leader sequence at the 3 'end of the clone. The primer sequences for GVs-3 are set forth in SEQ ID Nos. 66 and 67 and the primer sequences for GVs-5 are set forth in SEQ ID Nos. 68 and 69. A XhoI restriction site was added to the primers for GVs-3, and ZcoRI, and Site restriction sites were added to primers for GVs-5 for preferred cloning.

Following amplifications from M. vaccae genomic DNA, the fragments were cloned into a suitable pProEX HT site for a prokaryotic expression vector (Gibco BRL, Life Technologies, Gaithersburg, MD) and subjected to sequences to confirm correct reading frame and orientation. Expression and purification of the recombinant protein was performed according to the manufacturer's instructions.

Expression of the M. vaccae fragment of GVs-4 antigen (85B homolog) was performed as described below. The above-mentioned primers AD58 and AD59 were used to amplify a 485 base pair fragment from M. vaccae genomic DNA. This fragment was gel purified according to standard procedures and cloned into pBluescript from pCoRV containing the added dTTP residues. These sequences of the inserted region of the five clones were determined and found to be identical. These insert regions had the highest homology to that of M. tuberkulosis Ag85B. The inverted region from one of the clones was cloned into the EcoR1 / XhoI sites of the pProEX HT prokaryotic expression vector (Gibco BRL), expressed and purified according to the manufacturer's instructions. This clone was renamed GV-4P because only part of the gene was expressed.

• ·

9 · ♦ ·· · · 9 9 9 · 9 9 9 ·

9 9 9

9 9 9 ·

The amino acid and DNA sequences for the incomplete GV-4P clone are shown in SEQ ID NOS: 70 and 106, respectively.

Similar cloning of GV-4P, amplification of primers AD58 and AD59, a 485 bp fragment from a clone containing GVs-5 (SEQ ID NO: 42) was used for amplification. This fragment was cloned into the pET16 expression vector and was termed GV-5P. The determined nucleotide sequence and predicted amino acid sequence of GV-5P are provided in SEQ ID NOS: 157 and 158, respectively.

Using procedures similar to those previously described, GVs-3, GV-4P, and GVs-5 were re-cloned into the alternative vector pET16 (Novagen, Madison, WI) in subsequent studies.

The ability of purified recombinant GVs-3, GV-4P, and GVs-5 to stimulate T cell proliferation and interferon-γ production in human PBLs from PPD-positive, healthy donors was tested as described below. The results of this assay are shown in Table 17, where (-) means insufficient potency, (+/-) means polypeptides with a result less than twice as high as the control medium (+) means polypeptides two to four times more potent than the control medium, and (++) refers to polypeptides having potency greater than four times the environmental potency.

Table 17

donor G97005 donor G97006 donor G97007 donor G97008 donor G97009 donor G97010 Prol if IFN Prolif IFN-γ Prol if IFN -7 Prolif IFN-γ Prolif IFN-γ Prolif IFN-γ GVs-3 ++ + N N ++ ++ ++ ++ ++ +/- + ++ GV-4P + +/- N N + ++ ++ +/- +/- +/- ++ GVs-5 ++ ++ ++ ++ ++ ++ + ++ ++ + + ++

N - not determined

Example 13

DNA cloning procedure for M. vaccae antigens

The 84 base pair test sample for M. vaccae GVc-7 antigen was amplified using degenerate oligonucleotides that encode the amino acid sequence of GVc-7 (SEQ ID NO: 58). This test sample was used to view a M. vaccae genomic DNA library as described in Example 12. The nucleotide sequence found for GVc-7 is shown in SEQ ID NO: 46 and the predicted amino acid sequence in SEQ ID NO: 47. homology with the putative membrane protein of 15.8 kDaM tuberculosis was found by sequences in the database.

4 4

4 4

44 • * 4 4 • 4 4 4

4 «4 • 4 4 4 • · 44

SEQ ID NO: 46 was used to design amplification primers (shown in SEQ ID NOs: 71 and 72) for expression cloning of the GVc-7 gene using sequence data downstream of the putative leader sequence. For preferred cloning, an XhoI restriction site was added to the primers. Following amplification from M. vaccae genomic DNA, the fragments were cloned into the XhoI site of the prokaryotic expression vector pProEX HT (Gibco BRL) and subjected to sequencing to confirm the correct reading frame and orientation. Expression and purification of the fusion protein was performed according to the manufacturer's instructions. In subsequent studies, GVc-7 was again cloned into the pET10 vector (Novagen).

The ability of purified recombinant GVc-7 to stimulate T cell proliferation and to stimulate the production of interferon-γ in human PBLs from PPD positive healthy donors was tested as previously reported. The results are shown in Table 18, where (-) means insufficient potency, (+/-) means polypeptides with a result less than twice as high as the control medium environment (+) means polypeptides two to four times more potent than the control medium, and (+ +) means polypeptides having an activity greater than four times the environmental performance.

Table 18

Donor Proliferation Interferon-γ G97005 ++ +/- G97008 ++ + G97009 + +/- G97010 +/- ++

A redundant oligonucleotide test sample (SEQ ID No. 73; referred to as MPG15) was designed for the GVs-8 peptide sequence set forth in SEQ ID No. 26 and used to screen a M. vaccae genomic DNA library using standard procedures. Two genomic clones containing genes encoding four different antigens were isolated. The DNA sequences found for GVs-8A (renamed GV-30), GVs-8B (renamed GV-31), GVs-8C (renamed GV-32) and GVs-8D (renamed GV-33) are shown in SEQ ID NOS: 4851, respectively, with the corresponding amino acid sequences as set forth in SEQ ID NOS: 52-55, respectively. GV-30 contains regions showing some similarity to known prokaryotic valyl-tRNA synthetases; GV-31 shows some similarity to aspartate semialdehyde dehydrogenase M. smegmatis; GV-32 shows some similarity to the H. influenza synthase gene for folyl polyglutamate synthase. The GV-33 contains an open reading frame that exhibits 9 9 · 9

9 9 9 9 9 9 9 9 some similarity to the sequence previously found in M. tuberculosis and M. leprae, but complete function was not found.

The determined partial DNA sequence for GV-33 is set forth in SEQ ID NO: 74 and the corresponding putative amino acid sequence is set forth in SEQ ID NO: 75. Sequence data from the 3 'end of the clone showed homology to the previously determined M. tuberculosis 40.6 kDa outer membrane protein. Subsequent studies led to the isolation of the complete DNA sequence for GV-33 (SEQ ID No. 193). The corresponding putative amino acid sequence is set forth in SEQ ID NO: 194.

The gene encoding GV-33 was amplified from M. vaccae genomic DNA with primers based on the determined nucleotide sequence. This DNA fragment was cloned into pBluescript II SK ⁺ (Stratagene) derived from EcoRv and then transferred to the expression vector pET16. The recombinant protein was purified according to the manufacturer's instructions.

The ability of purified recombinant GV-33 to stimulate T cell proliferation and stimulate the production of interferon γ in human PBL was tested as previously described.

The results are shown in Table 19, where (-) means insufficient potency, (+/-) means polypeptides with a result less than twice as high as the control medium environment (+) means polypeptides two to four times more potent than the control medium, and (+ +) means polypeptides having an activity greater than four times the environmental performance.

Table 19

Stimulating activity of polypeptides

Donor Proliferation Interferon-γ G97005 ++ + G97006 ++ 4-4- G97007 - +/- G97008 +/- - G97009 +/- - G97010 +/- 4-4 “

Example 14

Isolation of proteins from DD-A /. vaccae

M. vaccae bacteria were cultured, pelletized and autoclaved as described in Example 1. The culture filtrates of live M. vaccae corresponded to the supernatant produced after 24 hours cultivation of M. vaccae in 7H9 glucose medium. The delipidated form of M. vaccae was • 9 99 9 · 99 ♦ ·

9 9 9 99 99 999

99 · · 9999

99999 9 999999 9999 9 9999 999999 999999 prepared by sonication of autoclaved M. vaccae in four 30 second doses on ice using Virsonic sonicator (Virtis, Disa, USA). The material was then centrifuged (9000 rpm, 20 minutes, JA10 rotor, brake = 5). The resulting pellet was suspended in 100 ml of chloroform / methanol (2: 1), incubated at room temperature for 1 hour, centrifuged again, and the extraction from chloroform / methanol was repeated. The pellet obtained by centrifugation was dried under vacuum, weighed and resuspended in PBS 50 mg (dry weight) per ml as lipid-free M. vaccae.

Glyco lipids were removed from lipid-free M. vaccae by refluxing in 50% (v / v) ethanol for 2 hours. Insoluble material was eliminated by centrifugation (10,000 rpm, JA20 rotor, 15 minutes, brake = 5). The reflux extraction with 50% (v / v) ethanol was repeated two more times. Insoluble material was removed by centrifugation and washed in PBS. Proteins were extracted by resuspending the pellet in 2% SDS in PBS at 56 ° C for 2 hours. Insoluble material was eliminated by centrifugation and extraction with 2% SDS / PBS at 56 ° C was repeated two more times. The resulting SDS extracts were cooled to 4 ° C, the SDS precipitate was removed by centrifugation (10,000 rpm, JA20 rotor, 15 minutes, brake = 5). Proteins were precipitated from the supernatant by adding an equal volume of acetone and incubating at -20 ° C for 2 hours. The precipitated proteins were pooled by centrifugation, washed in 50% v / v acetone, dried under vacuum, and dissolved in PBS.

SDS extracted proteins obtained from DD-A /. vaccae were analyzed by polyacrylamide gel electrophoresis. Three silver bands were observed after silver staining. In subsequent experiments, larger amounts of SDS extracted proteins from DD-A / were analyzed. vaccae by polyacrylamide gel electrophoresis. Coomassie blue stains showed several bands. The protein represented by the 30 kDa average molecular weight band was designated GV-45. The determined N-terminal sequence for GV-45 is set forth in SEQ ID NO: 187. The 14 kDa average molecular weight protein was designated GV-46. The determined N-terminal amino acid sequence for GV-46 is set forth in SEQ ID NO: 208.

In the following studies, multiple SDS extracted proteins described previously were prepared, preparation: SDS-PAGE on BioRad Prep Cell (Hercules, CA). The fractions corresponding to the molecular weight ranges were precipitated with trichloroacetic acid to remove SDS prior to testing for the efficacy of the antigen enhancer in the C57BL / 6 specific cytotoxic response assay as previously reported. The potency of the antigen enhancer was highest in the 60-70 kDa fractions. The most abundant protein in this size range was purified by SDS-PAGE on a polyvinylidene difluoride (PVDF) membrane and then sequenced. The sequence of the first ten amino acid residues is shown in SEQ ID NO: 76. By comparing these sequences to those in the gene bank as described previously, homology to the heat shock protein 65 (GroEL) gene from M. tuberculosis was indicated indicating that this protein is a M. vaccae member of the GroEL protein family.

M. vaccae genomic DNA expression library in ΒαηίΆΪ-lambda ZAP-Express (Stratagene) was screened using cynomolgus serum? monkeys immunized with proteins secreted by M. vaccae prepared as previously described. Positive plaques were determined using a colorimetric system. These plaques were repeatedly scanned according to standard procedures until they were clean. The pBK-CMV phage plasmid 2-1 containing the inserted region was excised from the lambda ZAP Express (Stratagene) vector in the presence of ExAssist helper phage according to the manufacturer's instructions. The base sequence of the 5 'end of the inserted region of this clone, hereinafter referred to as GV-27, was determined using Sanger sequences with fluorescent primers on automatic sequencing of the Perkin Elmer / Applied Biosystems Division. The determined nucleotide sequence of the partial M. vaccae GroEL homologous clone GV-27 is shown in SEQ ID NO 77 and the predicted amino acid sequence in SEQ ID NO 78. This clone was found to be a homolog of M. tuberculosis GroEL. A partial sequence of the M. vaccae heat shock protein of 65 kDa has been published by Kapur et al. (Arch. Pathol. Lab. Med. 119: 131-138, 1995). The nucleotide sequence of the fragment of Kapur et al. is shown in SEQ ID NO: 79 and the predicted amino acid sequence is shown in SEQ ID NO: 80.

In subsequent studies, the extended (full-length except assumed 51 terminal nucleotides) DNA sequences of GV-27 (SEQ ID NO: 113) were obtained. The corresponding putative amino acid sequence is shown in SEQ ID NO 114. The following studies resulted in the isolation of the full length GV-27 DNA sequence (SEQ ID NO 159). The corresponding putative amino acid sequence is set forth in SEQ ID NO: 160. GV-27 was shown to be 93.7% identical to M. tuberculosis GroEL at the amino acid level.

Two peptide fragments containing N-terminal sequences (hereinafter referred to as GV-27A) and GV-27 carboxyl-terminal sequences (hereinafter referred to as GV-27B) were prepared using procedures well known to those skilled in the art. The nucleotide sequences for GV27A and GV-27B are set forth in SEQ ID Nos 115 and 116, respectively, and the corresponding amino acid sequences are set forth in SEQ ID Nos 117 and 118. Subsequent studies led to the isolation of the extended DNA sequence for GV-27B . This sequence is set forth in SEQ ID NO: 161, and the corresponding amino acid sequence is set forth in SEQ ID NO: 162. The GV-27A sequence is 95.8% identical to the M. tuberculosis GroEL sequence and contains the shorter M. vaccae according to Kapur et al., as previously discussed. The sequence for GV-27B shows about 92.2% identity

9 99

9 9 9

99 ♦ 9

9 9 9 • 9 9

9 9

9 9

9 9 9

99 with the corresponding region of M. tuberculosis HSP65. pBK-CMV plasmid phage 3-1 was isolated according to the same instructions as GV-27.

pBK-CMV plasmid phage 3-1 was isolated according to the same procedure as GV-27. The antigen encoded by this DNA was named GV-29. The determined nucleotide sequences of the 5 'end and 3' end of the gene are set forth in SEQ ID NOs: 163 and 164, respectively, and the corresponding putative amino acid sequences are set forth in SEQ ID NOs: 165 and 166, respectively. GV-29 shows homology to yeast urea amidolyase.

The determined DNA sequence for the full length gene encoding GV-29 is set forth in SEQ ID NO 198, and the corresponding predicted amino acid sequence is set forth in SEQ ID NO 199. The DNA encoding GV-29 was cloned into the pET16 vector (Novagen, Madison, WI) for expression and purification according to standard procedures.

Example 15

DNA cloning procedure for M. vaccae antigens GV-23, GV-24, GV-25, GV-26, GV-38A, and GV38B

M. vaccae (ATCC No. 15483) was grown in sterile medium 90 at 37 ° C for 4 days and harvested by centrifugation. Cells were resuspended in 1 ml Trizol (Gibco BRL, Life Technologies, Gaithersburg, Maryla) and RNA was extracted according to the manufacturer's standard instructions. M. tuberculosis strain H37Rv (ATCC No. 27294) was grown in sterile Middlebrook 7H9 medium with Tween 80 ™ and oleic acid / albumin / dextrose / additive catalase (Difco Laboratories, Detroit, Michigan) at 37 ° C and obtained under appropriate laboratory conditions. security conditions. Cells were resuspended in 1 ml Trizol (Gibco BRL) and RNA was extracted according to the manufacturer's standard instructions.

M. tuberculosis and M. vaccae RNAs were deprived of the ribosomal RNA (rRNA) of 16S and 23S units by hybridizing a fraction of the total RNA to AD10 and AD11 oligonucleotides (SEQ ID Nos. 81 and 82), which are complementary to M. tuberculosis krRNA. These oligonucleotides were designed from the mycobacterial 16S unit rRNA sequence described in Bottger (FEMS Microbiol. Lett. 65: 171-176, 1989) and from a sequence stored in databases. Depletion was accomplished by hybridizing total RNA to oligonucleotides AD10 and AD11 immobilized on nylon membranes (Hybond N, Amersham International, United Kingdom). Hybridization was repeated until the rRNA bands were visible on an ethidium bromide stained agarose gel. The AD12 oligonucleotide (SEQ ID NO: 83) consisting of 20 residues of dATP was fused to the 3 'ends of the enriched mRNA fraction using RNA ligase. First strand cDNA synthesis was performed according to standard procedures using oligonucleotides AD7 (SEQ ID NO: 84) containing a poly (dT) sequence.

M. tuberculosis and M. vaccae cDNAs were used as templates for unilateral specific PCR (3S-PCR). For this procedure, a degenerate ADI oligonucleotide (SEQ ID NO: 85) was designed based on the stored leader sequence and membrane protein sequence. After 30 cycles of amplification using ADI primer as 5 'primer and AD7 as 3' primer, the products were separated on urea / polyacrylamide gel. M. vaccae-specific DNA bands were excised and re-amplified using primers AD1 and AD7. After gel purification, the bands were cloned into pGEM-T (Promega) and the base sequence was determined.

Searching with the determined nucleotides and predicted amino acid sequences of the 12B21 band (SEQ ID NOS: 86 and 87, respectively) showed homology to the E. coli genupot that encodes the ATP binding protein of the ABC spermidine / putrescine transport complex described by Furuchi et al. (Jnl. Biol. Chem. 266: 20928-20933, 1991). The E.coli spermidine / putrescine transport complex consists of the Four Genes and is a member of the ABC transporter family. ABC transporters (ATP-binding cassette) commonly consist of four genes: the ATP-binding gene, the periplasmic or substrate-binding gene, and two transmembrane genes. Transmembrane genes encode proteins characterized in that each has six membrane joining regions.

Homologues (according to similarity) of this ABC transporter were found in the genomes of Haemophilus influenza (Fleischmann et al. Science 269: 496-512, 1995) and Mycoplasma genitalium (Fraser, et al Science, 270: 397-403, 1995).

The M. vaccae genomic DNA library generated in the BamHI-digested lambda ZAP Express (Stratagene) was probed with a radiolabeled band of 238 base pairs of 12B21 according to standard procedures. The plaque was purified by repeated screening and a phage plasmid containing a 4.5 kb insert was identified using Southern blotting and hybridization. The nucleotide sequence of the complete M. vaccae pota homolog (ATP binding protein) was determined by subcloning the 4.5 kb fragment and base sequencing. The gene consisted of 1449 base pairs including a 5 'region where 320 base pairs were not translated, containing probably -10 and -35 promoter elements.

The nucleotide and putative amino acid sequences of the M. vaccae poto homolog are set forth in SEQ ID Nos 88 and 89, respectively.

The nucleotide sequence of the M. vaccae pota gene was used to design primers EV24 and EV25 (SEQ ID Nos. 90 and 91) for expression cloning. The amplified DNA fragment was cloned into the prokaryotic pProEX HT expression system (Gibco BRL) and expression was induced in appropriate E. coli hosts by the addition of 0.6 mM isopropylthio-β-galactoside (IPTG).

4 4

4 4

4 4

4 4 4

4 4 4 4

4 4 4 4

4 44 4

4 4 4 4 4 • 4 4 4 4

The recombinant protein was named GV-23 and purified from inclusion bodies according to the manufacturer's instructions. In subsequent studies, GV-23 (SEQ ID No. 88) was re-cloned into the alternative vector pET16 (Novagen). The amino acid sequence of SEQ ID NO: 89 has an ATP binding site at residues 34-41. At residues 166-163 of SEQ ID NO: 89, a region found in the family of ATP transporter proteins is retained. These findings suggest that GV-23 is an ATP binding protein.

The 322 bp SalI-BamDA subclone at the 3 'end of the inserted 4.5 kb sequence described previously exhibits homology to the potd (periplasmic protein) spermidine / putrescine ABC gene of the E. coli transport complex. The nucleotide sequence of this subclone is set forth in SEQ ID NO: 92. To determine this gene, a radiolabeled insertion region of this subclone was used to search a M. vaccae genomic DNA library generated in the lambda Zap Express site (Stratagene) using standard procedures. A clone whose 1342 base pairs showed homology to the potd E. coli gene was determined. The pot vac M homcae homolog was determined by subcloning and base sequencing. The determined nucleotide and predicted amino acid sequence are set forth in SEQ ID NOs: 93 and 94.

For expression cloning, primers EV-26 and EV-27 (SEQ ID NOS: 95-96) were designed from the designated potd homolog of M. vaccae. The amplified fragment was cloned into the pProEX HT prokaryotic expression system (Gibco BRL). Expression in a suitable E. coli host was induced by the addition of 0.6 mM IPTG and the recombinant protein was termed GV-24. Recombinant antigen was purified from inclusion bodies according to the supplier's instructions. In subsequent studies, GV-24 (SEQ ID NO: 93) was again cloned into the alternative vector pET16 (Novagen).

To increase the solubility of the purified recombinant antigen, the gene encoding GV-24 outside the signal peptide was re-cloned into an expression vector using the amplification primers EV101 and EV 102 (SEQ ID NOS: 167 and 168). The result was named GV-24B. The nucleotide sequence of GV-24B is set forth in SEQ ID NO: 169 and the predicted amino acid sequence is set forth in SEQ ID NO: 170. This fragment was cloned into pET16 for expression and purification of GV24B according to the manufacturer's instructions.

The ability of the purified recombinant GV-23 and GV-24 proteins to stimulate T cell proliferation and interferon-γ production in human PBL was determined as previously described. The results of these assays are shown in Table 20, where (-) means insufficient potency, (+/-) means polypeptides with results less than twice as high as the control medium (+) means polypeptides two to four times more potent than the control medium, and (++) means polypeptides having potency greater than four times the environmental and (unspecified) means that the result has not been determined.

• 0 00 00 ·· ·· 0 0 0 0 • 0 0 0 0 • 0 0 0 0 0 0 • 0 0 0 0 0

000 000 00 00 0

0 0 0

0 0 0

00

Table 20

Donor G97005 Donor G97006 Donor G97007 Donor G97008 Donor G97009 Donor G97010 Prolif IFN-γ Prolif IFN-γ Prolif IFN-γ Prolif IFN-γ Prolif IFN-γ Prolif IFN-γ GV-23 ++ ++ ++ H — h + + 4 — l · ++ + - + ++ GV-24 + ++ 4 ~ N N + +/- + +/- +/- ++

Ν - not specified

A base sequence flanking the M. vaccae potd gene homolog was found to determine homology to the potb gene of the spermidine / putrescin E.coli transporter complex, which is one of the two transmembrane proteins in the ABC transporter complex. The M. vaccae potb homolog (referred to as GV-25) was determined by further subcloning and base sequencing. The determined nucleotide and predicted amino acid sequence for GV-25 are set forth in SEQ ID NOS: 97 and 98, respectively.

Further subcloning and base sequencing analysis of the adjacent 509 base pairs revealed no significant homology to sPotC, with the second transmembrane protein E.coli, and suggests that the second transmembrane protein does not occur in the M. vaccae ABC transporter homolog. However, an open reading frame with homology to M. tuberculosis acetylCoA acetyl transferase starting with 530 base pairs downstream of the transmembrane protein was determined and the translational resulting protein was named GV-26. The determined partial nucleotide sequence and predicted amino acid sequence for GV-26 are set forth in SEQ ID NOs: 99 and 100, respectively.

Using a similar procedure to isolate the GV-23, 3S-PCR band 12B28 (SEQ ID No. 119) described previously, the M. vaccae genomic library assembled at the BamHI site of the lambda ZAP Express (Stratagene) was screened. The clone isolated from the library contained a new open reading frame and the antigen encoded by this gene was named GV-38A. The determined nucleotide sequence and predicted amino acid sequence of GV-38A are set forth in SEQ ID NOs: 120 and 121, respectively. Subsequent studies led to the isolation of the extended DNA sequence for GV-38A set forth in SEQ ID No. 171. The corresponding amino acid sequence is set forth in SEQ ID NO. 172. Comparison of these sequences with those stored in the gene bank showed some homology to the unknown protein M. tuberculosis previously identified in cosmid MTCY428,12. (SPTREMBL.-P71915).

Upstream of the GV-38A gene, a second new open reading frame was determined and the antigen encoded by this gene was named GV-38B. The determined nucleotide sequences of the 5 'and 3' ends for GV-38B are set forth in SEQ ID Nos. 122 and 123, respectively, and the corresponding putative amines from the liner sequences are set forth in SEQ ID Nos. 124 and 125, respectively. Other studies

· ΦΦ Φ »· 9 ΦΦ

ΦΦΦ Φ Φ · · · ·

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Úplné ΦΦ ΦΦΦ ΦΦΦ ΦΦ ΦΦ resulted in the isolation of the complete DNA sequence for GV-38B, which is set forth in SEQ ID NO: 173. The corresponding amino acid sequence is set forth in SEQ ID NO: 174. This protein was found to exhibit homology to the unknown protein of M. tuberculosis identified in cosmid MTCY428.11 (SPTREMBL: P71914).

Both GV-38A and GV-38B antigens were amplified for expression cloning in pET16 (Novagen). GV-38A was amplified with primers KR11 and KR12 (SEQ ID Nos. 126 and 127) and GV-38B with primers KR13 and KR14 (SEQ ID Nos. 128 and 129). Protein expression in BL21 (DE3) host cells was induced with 1 mM IPTG, but no protein expression occurred. At the N-terminus of the GV-38A and GV-38B antigens, hydrophobic regions have been identified that may prevent expression of these proteins. The hydrophobic regions present in GV-38A were identified as a possible transmembrane motif with six membrane joining regions. Primers KR20 for GV-38A (SEQ ID No. 130) and KR21 for GV-38B (SEQ ID No. 131) have been designed to express antigens without hydrophobic regions. The GV-38A truncated gene was amplified with KR20 and KR12 primers and the GV-38B truncated gene with KR21 and KRM primers. The determined nucleotide sequences of the truncated GV38A and GV-38B are set forth in SEQ ID Nos. 132 and 133, respectively, and the corresponding predicted amino acid sequence is set forth in SEQ ID Nos. 134 and 135, respectively. The extended DNA sequences for the truncated GV-38A and GV-38B are set forth in SEQ ID NO. 175 and 176, respectively, and the corresponding predicted amino acid sequences are set forth in SEQ ID NO. 177 and 178, respectively.

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Example 16

Purification and characterization of polypeptides from M. vaccae culture filtrate by preparative isoelectric focusing and preparative polyacrylamide gel electrophoresis

Soluble M. vaccae proteins from the culture filtrate using pre-isoelectric focusing and pre-polyacrylamide gel electrophoresis as described below. Unless otherwise indicated, all percentages in the following example are weight units per volume.

M. vaccae (ATCC No. 15483) was cultured in 250 L of sterile medium 90, which was fractionated by ultrafiltration to remove all proteins having a molecular weight greater than 10 kDa. The medium was centrifuged to remove bacteria and sterilized by filtration through a 0.45 µm filter. The sterile filtrate was concentrated by ultrafiltration through a membrane with a molecular weight cut-off of 10 kDa.

Proteins were isolated from the concentrated culture filtrate by precipitation with 10% trichloroacetic acid. The precipitated proteins were redissolved in 100 mM Tris.HCl pH 8.0 and reprecipitated with the addition of an equal volume of acetone. The precipitate formed with acetone was dissolved in water, and the proteins were reprecipitated by adding the same amount of chloroform / methanol 2: 1 (v / v). The precipitate formed from the chloroform / methanol mixture was dissolved in water and the solution was lyophilized.

The lyophilized protein was dissolved in isoelectric focusing buffer containing 8 M deionized urea, 2% Triton X-100, 10 mM dithiotreitol and 2% ampholytes (pH 2.5 5.0). The sample was separated into fractions by preparative isoelectric focusing in a horizontal tank containing Ultrodex gel at a constant power of 8 Watts for 16 hours. The proteins were eluted from the gel gel fractions with water and concentrated by precipitation with 10% trichloroacetic acid.

Fractions of proteins containing proteins of interest were determined by analytical polyacrylamide gel electrophoresis and separated into fractions by preparative polyacrylamide gel electrophoresis. Samples were separated into 12.5% SDS-PAGE gels and electroblotted on a nitrocellulose membrane. The position of the proteins on the membranes was determined by Ponceau Red staining, then the proteins were stained with water and eluted from the membranes with 40% acetonitrile / Ο, am ammonium bicarbonate pH 8.9 and then concentrated by lyophilization.

Eluted proteins were tested for their ability to induce proliferation and secretion of interferon γ in peripheral blood lymphocytes of immune donors as previously reported. Proteins producing a strong response in these assays were selected for further investigation.

• 4 »* 4 · © 4 4 4 O

Selected proteins were further purified by reverse phase chromatography on a Vydac Protein C4 column using a trifluoroacetic acid-acetonitrile system. Purified proteins were prepared for protein sequence determination by SDS-polyacrylamide gel electrophoresis and electroblotting on PVDF membranes. Protein sequences were determined as described in Example 3. The proteins were named GV-40, GV-41, GV-42, GV-43 and GV-44. The determined N-terminal sequences for these polypeptides are set forth in SEQ ID NOS: 101-105, respectively. Subsequent studies led to the isolation of the DNA sequences of the 5 'end, middle fragment and 3' end for GV-42 (SEQ ID NOs: 136, 137 and 138, respectively). The corresponding putative amino acid sequences are set forth in SEQ ID Nos. 139,140 and 141, respectively.

Following standard DNA amplification and DNA cloning procedures as described in Example 13, the genes encoding GV-41 and GV-42 were cloned. The determined nucleotide sequences are set forth in SEQ ID NOs: 179 and 180 and the corresponding putative amino acid sequences are set forth in SEQ ID NOs: 181 and 182. The following experiments led to the cloning of the full length gene encoding GV-41 and named GV-41B . The determined nucleotide sequence and predicted amino acid sequence of GV-41 B are set forth in SEQ ID NOS: 202 and 203, respectively. GV-41 exhibits homology to the ribosomal recycling factor of M. tuberculosis and M. leprae, and GV-42 exhibits homology to the M. avium FAP-A protein. From the full-length sequence of GV-42, the amino acid sequence determining GV-43 (SEQ ID NO: 104) was determined, indicating that the amino acid sequence for GV-42 and GV-43 was obtained from the same protein.

Mouse polyclonal antisera against GV-40 and GV-44 were prepared according to standard procedures. These antisera were used to view a library of M. vaccae genomic DNA consisting of randomly cut DNA fragments. Clones encoding GV-40 and GV-44 were determined and sequenced. The determined nucleotide sequence of the partial gene that encodes GV-40 is set forth in SEQ ID NO: 183 and the predicted amino acid sequence is set forth in SEQ ID NO: 184. The complete gene encoding GV-40 was not cloned and the antigen encoded by this partial gene was named GV-40P. The extended DNA sequence for GV-40P is set forth in SEQ ID NO: 206 and the corresponding putative amino acid sequence is set forth in SEQ ID NO: 207. The nucleotide sequence of the gene encoding GV-44 is set forth in SEQ ID NO: 185 and the putative amino acid sequence is as shown in SEQ ID No. 186. Further sequencing, the determined DNA sequence for the full length gene encoding GV-44 was obtained and is set forth in SEQ ID No. 204 and the corresponding putative amino acid sequence is set forth in SEQ ID No. 205. GV- homology was found. 40 to M. leprae elongation factor G and homology to M. leprae glyceraldehyde-3-phosphate dehydrogenase.

·· ··

Example 17

Isolation of antigens GV-45 and GV-46 DD-A /. vaccae

Proteins were extracted from DD-A /. vaccae (500 mg; prepared as above) by suspending in 10 ml 2% SDS / PBS and heating to 50 ° C for 2 h. Insoluble residue was removed by centrifugation and proteins precipitated from the supernatant by addition of an equal volume of acetone and incubation at temperature - 20 ° C for 1 hour. The precipitated proteins were pooled by centrifugation, dissolved in sample reduction buffer and separated into fractions by SDS polyacrylamide gel electrophoresis. The separated proteins were electroblotted on a PVDF membrane in a mixture of 10 mM CAPS / 0.01% SDS pH 11.0 and the N-terminal sequence was determined in sequencing of the gas phase device.

From these experiments, a protein represented by a band of approximately 30 kDa, designed as GV-45, was isolated. The determined N-terminal sequence for GV-45 is shown in SEQ ID NO. 187. A protein of approximately 14 kDa, designed as GV-46, was obtained from the same experiment. The determined N-terminal amino acid sequence for GV-46 is set forth in SEQ ID NO: 208. GV-46 is homologous to the highly conserved mycobacterial host integration factor of M. tuberculosis and M. smegmatis.

According to the amino acid sequence of GV-45, degenerate oligonucleotides KR32 and KR33 were designed (SEQ ID NOS: 188 and 189, respectively). Following standard procedures (Sambrook, Ibid), the 100 base pair fragment was amplified, cloned into plasmid pBluescript II SK ⁺ (Stratagene, La Jolla, CA) and sequenced (SEQ ID NO. 190). The cloned insert was used to screen a M. vaccae genomic DNA library generated at the Tta / nHI-site of the lambda ZAP-Express (Stratagene). The isolated clone showed homology to M. tuberkculosis 35 kDa and M. leprae 22 kDa proteins containing bacterial histone-like motifs at the N terminus and a unique C terminus consisting of five amino acid base repeats. The determined nucleotide sequence for GV-45 is set forth in SEQ ID NO 191 and the corresponding putative amino acid sequence is set forth in SEQ ID NO: 192. An additional DNA sequence determined for the full length gene encoding GV-45 was obtained by SEQ ID NO: 200 and the corresponding putative amino acid sequence is shown in SEQ ID NO: 201.

Example 18

Immunogenic and immunomodulating properties of recombinant proteins obtained from M. vaccae

A. Induction of T cell proliferation and IFN production γ • <

• 4 • 4 • 4 • 4 • 4

Immunogenicity of recombinant Mycobacterium vaccae proteins (GV recombinant proteins) was tested by inoculating BALB / cByJ female mice in both hind feet with 10 µg of recombinant GV proteins emulsified in incomplete Freund's antigen enhancer (IFA). Control mice received phosphate buffered saline in IFA. Cells obtained 10 days later from popliteal lymph nodes were stimulated with immunizing GV protein and assayed for proliferation by measuring the increase in tritiated thymidine. The amount of interferon gamma (IFNγ) produced and secreted by these cells into the culture supernatant was tested using a standard ELIS A assay. As shown in Table 21, which summarizes the proliferation responses, all GV proteins were found to induce a T cell response to proliferation. The lymph node T cells of the immunized mice responded to immunization with a specific GV protein proliferation. Lymph node cells of unimmunized mice did not respond to GV proteins proliferation. The data in Table 22 show IFNγ production, which shows that most GV proteins stimulated IFNγ production by lymph node cells of mice immunized with the corresponding GV protein. When lymph node cells of unimmunized mice were cultured with individual GV proteins, IFNγ production was not detected.

Thus, GV proteins are immunogens for the ability to stimulate T cell proliferation and / or IFNγ production when administered by subcutaneous injection. Cantigen-specific stimulatory effects on T cell proliferation and IFNγ production are two advantageous properties of tuberculosis vaccines.

· · · · · · · · · · · ·

Table 21

Immunogenic properties of GV proteins: proliferation

GV protein Proliferation (cpm) Batch GV 50 protein used in vitro (pg / ml) 2 0.08 GV-1/70 31.550 ± 803 19.058 ± 2.449 5.596 ± 686 GV-1/83 18.549 ± 2.716 23.932 ± 1.964 11.787 ± 1.128 GV-3 34.751 ± 1.382 6.379 ± 319 4.590 ± 1.042 GV-4P 26.460 ± 1.877 10.370 ± 667 6.685 ± 673 GV-5 42.418 ± 2.444 23.902 ± 2.312 13.973 ± 772 GV-5P 35.691 ± 159 14.457 ± 1.185 8.340 ± 725 GV-7 38.686 ± 974 22.074 ± 3.698 15.906 ± 1.687 GV-9 30.599 ± 2580 15.260 ± 2.764 4.531 ± 1.240 GV-13 15.296 ± 2.006 7.163 ± 833 3.701 ± 243 GV-14 27.754 ± 1.872 13.001 ± 3.273 9.897 ± 2.833 GV-14B 10.761 ± 485 5.075 ± 1.470 2,341 ± 289 GV-22B 3.199 ± 771 3.255 ± 386 1.841 ± 318 GV-23 35.598 ± 1.330 15.423 ± 2.858 7.393 ± 2.188 GV-24B 43.678 ± 2.190 30.307 ± 1.533 15.375 ± 2.594 GV-27 18.165 ± 3.300 16.329 ± 1.794 6.107 ± 1.773 GV-27A 23.723 ± 850 6.860 ± 746 4.295 ± 780 GV-27B 31.602 ± 1.939 29.468 ± 3.867 30.306 ± 1.912 GV-29 20.034 ± 3.328 8.107 ± 488 2.982 ± 897 GV-33 41.529 ± 1.919 27.529 ± 1.238 8.764 ± 256 GV-35 29.163 ± 2.693 9.968 ± 314 1.626 ± 406 GV-38AP 28.971 ± 4.499 17.396 ± 878 8,060 ± 810 GV-38BP 19.746 ± 245 11.732 ± 3.207 6.264 ± 875 GV-40P 25.185 ± 2.877 19.292 ± 2.294 10.883 ± 893 GV-41B 24.646 ± 2.714 12.627 ± 3.622 5.772 ± 1.041 GV-42 25.486 ± 3.029 20.591 ± 2.021 13.789 ± 775 GV-44 2.684 ± 1.995 3.577 ± 1.725 1.499 ± 959 GV-45 9.554 ± 482 3.683 ± 1.127 1.497 ± 199

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Table 22

Immunogenic properties of GV proteins: IFNγ production

GV protein IFNγ (ng / ml) Batch GV] 50 protein used in vitro (pg / ml) 10 2 GV-1/70 24.39 ± 6.66 6.19 ± 1.42 1.90 ± 0.53 GV-1/83 11.34 ± 5.46 5.36 ± 1.34 2.73 ± 1.55 GV-3 3.46 ± 0.30 1.57 ± 0.04 not specified GV-4P 6.48 ± 0.37 3.00 ± 0.52 1.38 ± 0.50 GV-5 4.08 ± 1.41 6.10 ± 2.72 2.35 ± 0.40 GV-5P 34.98 ± 15.26 9.95 ± 3.42 5.68 ± 0.79 GV-7 33.52 ± 3.08 25.47 ± 4.14 9.60 ± 1.74 GV-9 92.27 ± 45.50 88.54 ± 16.48 30.46 ± 1.77 GV-13 11.60 ± 2.89 2.04 ± 0.58 1.46 ± 0.62 GV-14 8.28 ± 1.56 3.19 ± 0.56 0.94 ± 0.24 GV-14B not specified not specified not specified GV-22B not specified not specified not specified GV-23 59.67 ± 14.88 30.70 ± 4.48 9.17 ± 1.51 GV-24B 6.76 ± 0.58 3.20 ± 0.50 1.97 ± 0.03 GV-27 72.22 ± 11.14 30.86 ± 10.55 21.38 ± 3.12 GV-27A 4.25 ± 2.32 1.51 ± 0.73 not specified GV-27B 87.98 ± 15.78 44.43 ± 8.70 21.49 ± 5.60 GV-29 7.56 ± 2.58 1.22 ± 0.56 not specified GV-33 7.71 ± 0.26 8.44 ± 2.35 1.52 ± 0.24 GV-38AP 23.49 ± 5.89 8.87 ± 1.62 4.17 ± 1.72 GV-38BP 5.30 ± 0.95 3.10 ± 1.19 1.91 ± 1.01 GV-40P 15.65 ± 7.89 10.58 ± 1.31 3.57 ± 1.53 GV-41B 16.73 ± 1.61 5.08 ± 1.08 2.13 ± 1.10 GV-42 95.97 ± 23.86 52.88 ± 5.79 30.06 ± 8.94 GV-44 not specified not specified not specified

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B. Stimulation of lymphocyte subpopulation

The ability of recombinant M. vaccae proteins of the invention, heat-killed M. vaccae, and DD-M vaccae to stimulate lymphocyte subpopulations was determined by investigating an increase in CD69 expression (a surface protein expressed on stimulated cells).

PBMCs from normal donors (5 x 10 ⁶ cells / ml) were stimulated with 20 µg / ml of either heat-killed M. vaccae cells or DD-M vaccae or recombinant GV-22B (SEQ ID NO. 145), GV-23 (SEQ ID NO. GV-27 (SEQ ID NO. 160), GV27A (SEQ ID NO. 117), GV-27B (SEQ ID NO. 162), or GV-45 (SEQ ID NO. 201) for 24 hours . CD69 expression was determined by staining cultured cells with a monoclonal anti-CD56 antibody, αβΤ cell, or γδΤ cell in combination with a monoclonal anti-CD69 antibody and subsequent flow cytometric analysis.

Table 23 shows the percentage of αβΤ cells, γδΤ cells and NK cells expressing CD69 after stimulation with heat-killed M vaccae, DD-M. vaccae or recombinant M vaccae proteins. These results show that heat-killed M vaccae, DD-M vaccae and GV-23 stimulate CD69 expression in lymphocyte subpopulations as compared to control (unstimulated cells), particularly high levels of CD69 expression were observed in NK cells. GV-45 was found to increase CD69 expression by αβΤ cells.

Table 23

αβΤ cells γδΤ cells NK cells control 3.8 6.2 4.8 heat-killed M.vaccae 8.3 10.2 40.3 DD-M vaccae 10.1 17.5 49.9 GV-22B 5.6 3.9 8.6 GV-23 5.8 10.0 46.8 GV-27 5.5 4.4 13.3 GV-27A 5.5 4.4 13.3 GV-27B 4.4 2.8 7.1 GV-45 11.7 4.9 6.3

The ability of the recombinant GV-23 protein (20 µg / ml) to induce CD69 expression in the lymphocyte subpopulation was compared with the known ability of the MPL / TDM / CWS antigen enhancer (monophosphoryl lipid A / trehalose 6'6 'dimycolate; Sigma, St. Louis , MO; final dilution 1:20) and CpG ODN (Promega, Madison, WI; 20 µg / ml) induce Th-1 and se • 9 • 9 9 9 * 9 · 9

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9 9 · 9 99

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999 999

9 9

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99 known ability of aluminum hydroxide enhancer (Superfos Biosector, Kvistgard, Denmark; 1: 400 final dilution) and choleratoxin (20 µg / ml) to induce Th-2 using the above procedures. MPL / TDM / CWS and hydroxide Aluminum was used at the maximum concentration that did not cause cellular toxicity. Giant. 8A to 8C show stimulation of CD69 expression by αβΤ cells, γδΤ cells, and NK cells, respectively. GV-23, MPL / TDM / CWS, and CpG ODN induced CD69 expression by NK cells, whereas aluminum hydroxide and choleratoxin did not.

C. Stimulation of cytokine production

The ability of the recombinant M. vaccae proteins of the invention to stimulate cytokine production in PBMCs was tested as follows. PBMCs from normal donors (5 x 10 ⁶ cells / ml) were stimulated with 20 µg / ml either heat-killed M. vaccae cells, DD-A). vaccae or recombinant GV-22B (SEQ ID No. 145), GV-23 (SEQ ID No. 89), GV-27 (SEQ ID No. 160), GV-27A (SEQ ID No. 117), GV-27B (SEQ ID No. 162) or GV-45 (SEQ ID No. 201) for 24 hours. Culture supernatants were obtained and tested for IL-β, TNF-α, IL-12 and IFN-γ production using a standard ELISA kit (Genzyme, Cambridge, MA) according to the manufacturer's instructions. Giant. Figures 9A-9D show stimulation of IL-1β, TNF-α, IL-12 and IFN-γ production, respectively. It was found that heat-killed M. vaccae and DD-A /. vaccae stimulates the production of all four cytokines tested, while recombinant GV-23 and GV-45 stimulate the production of IL-β, TNF-α, and IL-12. Giant. Figures 10A-10C show the stimulation of IL-β, TNF-α, and IL-12 production, respectively, in human PBMCs (as determined previously) by different concentrations of GV-23 and GV-45.

Giant. 11A-1DD show stimulation of IL-β, TNF-α, IL-12 and IFN-γ production, respectively, in PBMC by GV-23 compared to stimulation with the following antigen enhancing agents: MPL / TDM / CWS (in final dilution 1:20), CpG ODN (20 µg / mL), aluminum hydroxide (final dilution 1: 400), and choleratoxin (20 µg / mL). GV-23, MPL / TDM / CWS, and CpG ODN induced significant levels of all four cytokines tested, the levels of IL-ββ formation observed for GV-23 were higher than using any other known antigen enhancer. Aluminum hydroxide and choleratoxin induced only negligible amounts of these four cytokines.

D. Stimulation of antigen-recognizing cells

The ability of heat-killed M. vaccae, DD-A / vaccae, and recombinant M. vaccae proteins to increase expression of CD40, CD80 and CD86 co-stimulatory molecules on B cells, monocytes and dendritic cells was determined as described below.

T cell-depleted peripheral blood monoclonal cells containing predominantly B cells, monocytes and dendritic cells were stimulated with 20 µg / ml of either heat-killed M. vaccae cells, DD-M. vaccae or recombinant proteins GV-22B (SEQ ID No. 145), GV-23 (SEQ ID No. 89), GV-27 (SEQ ID No. 160), GV-27A (SEQ ID No. 117), GV- 27B (SEQ ID NO. 162) or GV-45 (SEQ ID NO. 201) for 48 hours. Stimulated cells were obtained and analyzed for enhancement of CD40, CD80 and CD86 using 3-color current cytometric analysis. Tables 24, 25 and 26 show a strongly increased fluorescence intensity for dendritic cells, monocytes, and B cells, respectively, compared to the low fluorescence intensity of the control (unstimulated cells).

Table 24

Stimulation of CD40, CD80, and CD86 expression in dendritic cells.

CD40 CD80 CD86 control 0 0 0 Heat-killed M. vaccae 6.1 3.8 1.6 DD-M vaccae 6.6 4.2 1.6 GV-22B 4.6 1.9 1.6 GV-23 6.0 4,5 1,8 GV-27 5.2 1.9 1.6 GV-27A 2.3 0.9 1.0 GV-27B 2.6 1.1 1.1 GV-45 5.8 3.0 3.1

Table 25

Stimulation of CD40, CD80, and CD86 expression in monocytes

CD40 CD80 CD86 control 0 0 0 Heat killedM vaccae 2.3 1,8 0.7 DD-M. vaccae 1.9 1.5 0.7 GV-22B 0.7 0.9 1.1 GV-23 2.3 1.5 0.7 GV-27 1.5 1.4 1,2

9 9

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4 4 4

9 9 4

4 4 4

4 4 4

GV-27A 1.4 1.4 1.4 GV-27B 1.6 1,2 1,2 GV-45 1.6 1,2 1.0

Table 26

Stimulation of CD40, CD80, and CD86 expression in B cells

CD40 CD80 CD86 control 0 0 0 Heat-killed M. vaccae 1.6 1.0 1.7 DD-A /. vaccae 1.5 0.9 1.7 GV-22B 1.1 0.9 1,2 GV-23 1,2 1.1 1.4 GV-27 1.1 0.9 1.1 GV-27A 1.0 1.1 0.9 GV-27B 1.0 0.9 0.9 GV-45 1,2 1.1 1.3

As shown previously, increased expression levels of CD40, CD80 and CD86 were observed in dendritic cells, monocytes and B cells of all compositions tested. Expression levels were most elevated in dendritic cells, with the highest expression levels being obtained for heat-killed M. vaccae, DD-A /. vaccae, GV-23 and GV-45. Giant. 12A to 12C show stimulation of CD40, CD80, and CD86 expression, respectively, in dendritic cells at various concentrations of GV-23 and GV-45.

The ability of GV-23 (20 µg / ml) to stimulate CD40, CD80, and CD86 expression in dendritic cells was compared to the ability of MPL / TDM / CWS antigen enhancers (1: 20 final dilution) and CpG ODN (20 µg / ml) induce Th-1 and with the known ability of aluminum hydroxide (1: 400 final dilution) and choleratoxin (20 µg / ml) to induce Th-2. GV-23, MPL / TDM / CWS, and CpG ODN caused a significant increase in CD40, CD80 and CD86, while choleratoxin and aluminum hydroxide induced slight or negligible stimulation of dendritic cells, respectively.

D. Maturation and function of dendritic cells

The effect of recombinant M. vaccae GV-23 protein on maturation and dendritic cell function was investigated as follows.

Purified dendritic cells (5 x 10 ⁴ to 10 ⁵ cells / ml) were stimulated with GV-23 (20 µg / ml) or LPS (10 µg / ml) as a positive control. Cells were cultured for 20 hours and then analyzed for expression of CD83 (maturation marker) and CD80 by flow cytometry. Unstimulated cells were used as a negative control. The results are shown in Table 27 below.

Table 27

Stimulation of CD83 expression in dendritic cells

administered substance % CD83-positive dendritic cells % CD80-positive dendritic cells control 15 ± 8 9 ± 6.6 GV-23 35 ± 13.2 24.7 ± 14.2 LPS 36.3 ± 14.8 27.7 ± 13

data = mean ± SD (n = 3)

The ability of GV-23 to increase the activity of dendritic cells as antigen-recognizing cells was determined by assay (MLR). Purified dendritic cells were cultured in medium alone or with GV-23 (20 µg / ml) for 18-20 hours and then stimulated with allochthonous T cells (2 x 10 ⁵ cells / well). After 3 days of incubation, ( ³ H) thymidine was added. One day later, cells were harvested and increased radioactivity was measured. Giant. 13 shows an increase in ( ³ H) -thymidine increase with an increase in dendritic cell diameter to T cells. Significantly higher levels of increase in radioactivity were observed in GV-23 cells stimulated compared to unstimulated cells, suggesting that GV-23 enhances mixed dendritic cell and leukocyte responses.

Although certain details and some specific examples have been used to illustrate the invention for clarity, changes and modifications may be made therein without departing from the scope of the invention, which is to be limited only by the appended claims.

Claims (60)

PATENT CLAIMS A polypeptide comprising an immunogenic portion of an isolated M. vaccae antigen comprises sequences selected from the group consisting of: sequences SEQ ID NOs: 1 to 4, 9 to 16, 18 to 21, 23, 25, 26, 28, 29, 44, 45, 47, 52-55, 63, 64, 70, 75, 89, 94, 98, 100 to 105, 109, 110, 112, 121, 124, 134, 135, 140, 141, 143, 145, 147, 152, 154, 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205 and 207 . A polypeptide comprising an immunogenic portion of an isolated M. vaccae antigen, wherein the antigen comprises sequences selected from the group consisting of: (a) sequences having at least about 50% residues identical to SEQ ID NOs; 1 to 4, 9 to 16, 18 to 21, 23, 25, 26, 28, 29, 44, 45, 47, 52-55, 63, 64, 70, 75, 89, 94, 98, 100 to 105, 109, 110, 112, 121, 124, 134, 135, 140, 141, 143, 145, 147, 152, 154, 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181 , 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205 and 207, as measured by the BLASTP computer algorithm; (b) sequences having at least about 75% residues identical to SEQ ID NOs: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205 and 207 as measured by the BLASTP computer algorithm; and (c) sequences having at least about 95% residues identical to SEQ ID NOs: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181 , 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205 and 207 as measured by the BLASTP computer algorithm. A polypeptide comprising an immunogenic portion of an isolated M. vaccae antigen, wherein the antigen comprises an amino acid sequence encoded by a polynucleotide selected from the group consisting of: (a) SEQ ID NOs: 40 to 42, 46, 48 to 51, 74, 88, 93, 97, 99, 106 to 108, 111, 120, 122, 123, 132, 133, 136 to 138, 142, 144, 146, 151, 153, 155, 157, 159, 161, 163, 164, 169, 171, 173, 175, 176, 179, 180, 183, 185, 191, 193, 195, 198, and 200; (B) complements of SEQ ID NOs: 40 to 42, 46, 48 to 51, 74, 88, 93, 97, 99, 106 to 108, 111, 120, 122, 123, 132, 133, 136 to 138 142, 144, 146, 151, 153, 155, 157, 159, 161, 163, 164, 169, 171, 173, 175, 176, 179, 180, 183, 185, 191, 193, 195, 198 and 200 ; and (c) sequences that are at least about 99% likely to be the same as (a) or (b) as measured by a BLASTN computer algorithm. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide of any one of claims 1 to 3. An expression vector comprising the polynucleotide of claim 4. A host cell transformed with an expression vector according to claim 5. The host cell of claim 6, wherein the host cell is selected from the group consisting of E. coli, mycobacterial, insect, yeast and mammalian cells. A fusion protein comprising at least one polypeptide of any one of claims 1 to 3. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 3 and a physiologically acceptable carrier. A pharmaceutical composition comprising the polynucleotide of claim 4 and a physiologically acceptable carrier. A pharmaceutical composition comprising the fusion protein of claim 8 and a physiologically acceptable carrier. A vaccine comprising the polypeptide of any one of claims 1 to 3 and a non-specific immune response enhancer. A vaccine comprising the polynucleotide of claim 4 and a non-specific immune response enhancer. · Φ φ φ • • • • • • • • • φ A vaccine comprising the fusion protein of claim 8 and a non-specific immune response enhancer. The vaccine of any one of claims 12 to 14, wherein the non-specific immune response enhancer is an adjuvant. The vaccine of any one of claims 12 to 14, wherein the non-specific immune response enhancer is selected from the group consisting of: (a) lipid and glycolipid-free M. vaccae cells; (b) inactivated M. vaccae 'cells, and (c) culture filtrate Up to vaccae ·, (a) components present or derived from any mixture of (a) to (d). A method of enhancing an immune response in a patient, comprising administering to the patient a pharmaceutical composition according to any one of claims 9 to 11. A method of enhancing an immune response in a patient, comprising administering to the patient a vaccine according to any one of claims 12 to 14. The method of any one of claims 17 and 18, wherein the immune response is a Thi response. A method of treating a disease in a patient, comprising administering to the patient a pharmaceutical composition according to any one of claims 9 to 11. A method of treating a disease in a patient, comprising administering to the patient a vaccine according to any one of claims 12 to 14. The method of any one of claims 20 and 21, wherein the disease is selected from the group consisting of an immune disorder, an infectious disease, a skin disease, and a respiratory disease. • 99 99 9 9 9 99 99 9 9 9 9 9 9 9 · · · · ······ · · · · · · 999 99 99 99 The method of claim 22, wherein the disease is selected from the group consisting of mycobacterial infection, asthma, psoriasis, dermatitis, allergic rhinitis, sarcoidosis, alopecia areata, lung cancer and skin cancers. A method of treating a disease in a patient, comprising administering a composition comprising a component selected from the group consisting of (a) a lipid and glycolipid-free M. vaccae cell; (b) lipid and glycolipid-depleted mycolic acid-depleted M. vaccae cells; (c) lipid and glycolipid-depleted mycolic acid-depleted M. vaccae cells and arabinogalactan; (d) acid treated M. vaccae lipid and glycolipid cells; and (e) components present or derived from any mixture of (a) to (d). The method of claim 24, wherein the disease is selected from the group consisting of mycobacterial infection, asthma, psoriasis, dermatitis, allergic rhinitis, sarcoidosis, alopecia areata, lung cancer and skin cancers. A method of enhancing a non-specific immune response to an antigen comprising administering the polypeptide, the polypeptide comprising an immunogenic portion of a M. vaccae antigen, wherein the M. vaccae antigen comprises a sequence selected from the group consisting of (a) SEQ ID NOS: 89,114,117,118 and 201; (b) a sequence having at least about 97% residues identical to the sequence set forth in SEQ ID NOs 114, 117, 118 as measured by a BLASTP computer algorithm. (c) a sequence having at least about 80% residues identical to SEQ ID NO. 89 and 201 as measured by the BLASTP computer algorithm. A method of detecting a mycobacterial infection in a patient, comprising: (a) contacting the patient's dermal cells with one or more polypeptides of any one of claims 1 to 3; and (b) detecting an immune response to the patient's skin. The method of claim 27, wherein the immune response is induration. 4 44 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 44 44 29. A diagnostic kit comprising: (a) a polypeptide according to any one of claims 1 to 3; and (b) a device allowing sufficient contact of the polypeptides with the dermal cells of the patient. A method for detecting a mycobacterial infection in a biological sample, comprising: (a) contacting the biological sample with the polypeptide of any one of claims 1 to 3; and (b) detecting the presence of antibodies that bind to the polypeptides in the sample. The method of claim 30, wherein the polypeptide (s) are bound to a solid support. The method of claim 30, wherein the biological sample is selected from the group consisting of whole blood, serum, plasma, saliva, cerebrospinal fluid, and urine. A method of detecting a mycobacterial infection in a biological sample, comprising: (a) contacting the biological sample with a binding agent capable of binding the polypeptide of any one of claims 1 to 3; and (b) detecting a protein or polypeptide that binds to the binding agent in the sample. The method of claim 33, wherein the binding agent is a monoclonal antibody. The method of claim 33, wherein the binding agent is a polyclonal antibody. 36. A diagnostic kit comprising: (a) at least one polypeptide of any one of claims 1 to 3; and (b) a detection reagent. The kit of claim 36, wherein the polypeptide is immobilized on a solid support. The kit of claim 36, wherein the detection reagent comprises an identification moiety attached to the binding agent. 100 titi ·· • ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti ti titi The kit of claim 38, wherein the binding agent is selected from the group consisting of anti-immunoglobulins, protein G, protein A, and lactins. The kit of claim 38, wherein the identifying moiety is selected from radioisotopes, fluorescent moieties, luminescent moieties, enzymes, biotin, and staining particles. A monoclonal antibody that binds to the polypeptides of any one of claims 1 to 3. A polyclonal antibody that binds to the polypeptides of any one of claims 1 to 3. A method of enhancing a non-specific immune response to an antigen, comprising administering a composition comprising a component selected from the group consisting of: (a) lipid and glycolipid-free M. vaccae cells depleted in mycolic acids; (b) lipid and glyco lipid-depleted M. vacacid cells depleted in mycolic acids and arabinogalactan; (c) lipid and glyco lipid-free M.vaccae cells; (d) a culture filtrate of M. vaccae cells. 44. A polypeptide comprising an immunogenic portion of an M.vaccae antigen, wherein the antigen comprises a sequence selected from the group consisting of: (a) the sequences shown in SEQ ID NOS: 114,117 and 118; and (b) a sequence having at least about 97% of residues identical to SEQ ID NOS: 114, 117 and 118. 45. A composition comprising a component selected from the group consisting of: (a) lipid and glyco lipid-free M.vaccae cells; (b) lipid and glycol lipid-depleted mycolic acid-depleted M.vaccae cells; (c) lipid-free and glycol-lipid-depleted M. vaccae cells depleted in mycolic acids and arabinogalactan; (d) lipid and glycolipid-free M. vaccae cells treated with acid; and (e) components present or derived from any mixture of (a) to (e). 101 • 9 10 » 9 9 9 · 9 9 99 99 9 9 9 9 9 9 9 9 9 9 9 9 9 9 The composition of claim 45, wherein the mycobacterial cells comprise M. vaccae cells. A composition comprising lipid and glycolipid-free M. vaccae cells having an amino acid content of at least 40% by weight. A composition comprising lipid and glycolipid-free M.vaccae cells having a lipid content of less than 3% by weight. A composition according to any one of claims 45 to 48 in a form suitable for delivery to the airways or localized in the lungs. 50. A method of adjusting the equilibrium of a population of Th1 and Th2 cells in a population sample, by administering a composition comprising a component selected from the group consisting of: (a) lipid-free mycobacterial cells; (b) glycolipid-free mycobacterial cells; (c) lipid and glycolipid-free mycobacterial cells; (d) lipid and glycolipid-free mycobacterial cells depleted of mycolic acids; (e) mycobacterial cells depleted of lipids and glycolipids, depleted of mycolic acids and arabinogalactan; (f) acid-treated mycobacterial cells depleted of lipids and glycolipids; and (g) components present or derived from any mixture of (a) to (f). 51. A method of activating T cells in a population sample by administering a composition comprising a component selected from the group consisting of: (a) lipid-free mycobacterial cells; (b) glycolipid-free mycobacterial cells; (c) lipid and glycolipid-free mycobacterial cells; (d) lipid and glycolipid-free mycobacterial cells depleted of mycolic acids; (e) mycobacterial cells depleted of lipids and glycolipids, depleted of mycolic acids and arabinogalactan; (i) acid-treated mycobacterial cells depleted of lipids and glycolipids; and (g) components present or derived from any mixture of (a) to (f). 102 • 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 00 00 000 · 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 52. A method of activating NK cells in a population sample by administering a composition comprising a component selected from the group consisting of: (a) lipid-free mycobacterial cells; b) glycolipid-free mycobacterial cells; c) lipid and glycolipid-free mycobacterial cells; d) lipid and glycolipid-depleted mycobacterial cells depleted of mycolic acids; e) mycobacterial cells depleted of lipids and glycolipids, depleted of mycolic acids and arabinogalactan; f) acid-treated mycobacterial cells depleted of lipids and glycolipids; and g) components present or derived from any mixture of (a) to (f). 53. A method of stimulating cytokine production in a population sample by administering a composition comprising a component selected from the group consisting of: (a) lipid-free mycobacterial cells; b) glycolipid-free mycobacterial cells; c) lipid and glycolipid-free mycobacterial cells; d) lipid and glycolipid-depleted mycobacterial cells depleted of mycolic acids; e) mycobacterial cells depleted of lipids and glycolipids, depleted of mycolic acids and arabinogalactan; (i) acid-treated mycobacterial cells depleted of lipids and glycolipids; and g) components present or derived from any mixture of (a) to (f). 54. A method of increasing expression of co-stimulatory molecules on at least one of dendritic cells and monocytes in a population sample, by administering a composition comprising a component selected from the group consisting of: (a) lipid-free mycobacterial cells; b) glycolipid-free mycobacterial cells; c) lipid and glycolipid-free mycobacterial cells; d) lipid and glycolipid-depleted mycobacterial cells depleted of mycolic acids; e) mycobacterial cells depleted of lipids and glycolipids, depleted of mycolic acids and arabinogalactan; 4 4 103 4 · 4 4 4 44 44 f) acid-treated mycobacterial cells depleted of lipids and glycolipids; and g) components present or derived from any mixture of (a) to (f). 55. A method for accelerating the development and function of a dendritic cell in a population sample, by administering a composition comprising a component selected from the group consisting of: (a) lipid-free mycobacterial cells; b) glycolipid-free mycobacterial cells; c) lipid and glycolipid-free mycobacterial cells; d) lipid and glycolipid-depleted mycobacterial cells depleted of mycolic acids; e) mycobacterial cells depleted of lipids and glycolipids, depleted of mycolic acids and arabinogalactan; f) acid-treated mycobacterial cells depleted of lipids and glycolipids; and g) components present or derived from any mixture of (a) to (f). 56. A method of reducing the accumulation of eosinophils in a sample of a cell or tissue population by administering a composition comprising a component selected from the group consisting of: (a) lipid-free mycobacterial cells; b) glycolipid-free mycobacterial cells; c) lipid and glycolipid-free mycobacterial cells; d) lipid and glycolipid-depleted mycobacterial cells depleted of mycolic acids; e) mycobacterial cells depleted of lipids and glycolipids, depleted of mycolic acids and arabinogalactan; f) acid-treated mycobacterial cells depleted of lipids and glycolipids; and g) components present or derived from any mixture of (a) to (f). The method of any one of claims 50 to 56, wherein the mycobacterial cells are M. vaccae cells. Use of a composition according to any one of claims 45 to 48, wherein the disease is selected from the group consisting of immune disorders, inflammatory diseases, skin diseases and diseases of the respiratory system. 104 • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • a · a ·· a · a * Use of a composition according to any one of claims 45 to 48, for the manufacture of a medicament for the treatment of a disease selected from the group consisting of immune disorders, inflammatory diseases, skin diseases and respiratory diseases. Use of a composition according to any one of claims 45 to 48, in the manufacture of a medicament for the treatment of a disease selected from the group consisting of mycobacterial infection, asthma, psoriasis, allergic rhinitis, sarcoidosis, alopecia areata, lung cancer and skin cancers.