MXPA00006168A - Compositions derived from mycobacterium vaccae. - Google Patents

Abstract

The present invention provides compositions which are present in or may be derived from Mycobacterium vaccae, together with methods for their use in the treatment, prevention and detection of disorders including infectious diseases, immune disorders and cancer. Methods for enhancing the immune response to an antigen including administration of M. vaccae culture filtrate, delipidated M. vaccae cells, delipidated and deglycolipidated M. vaccae cells depleted of mycolic acids, and delipidated and deglycolipidated M.vaccae cells depleted of mycolic acids and arabinogalactan are also provided.

Description

"COMPOSITIONS DERIVED FROM" MYCOBACTERIUM VACCAE "AND METHODS FOR USE" TECHNICAL FIELD The present invention relates generally to compositions that are present in, or may be derived from, "Micobacterium vaccae" and its use in the treatment, prevention and arrest of disorders including infectious diseases, immune disorders and cancer. In particular, the invention relates to compounds and methods for the treatment of diseases of the respiratory system, such as mycobacterial infections, asthma, sarcoidosis and lung cancers and skin disorders, such as psoriasis, atopic dermatitis, allergic contact dermatitis, alopecia aerata and the basic cell carcinoma of skin cancers, carcinoma and squamous cell melanoma. The invention further relates to compounds that function as non-specific immune response amplifiers, and the use of these non-specific immune response amplifiers as adjuvants in vaccination or immunotherapy against infectious disease, and in certain treatments for immune disorders and cancer. BACKGROUND OF THE INVENTION Tuberculosis is a chronic infectious disease that is caused by infection with "Mycobacteri m tuberculosis" (M. tuberculosis). It is a major disease in developing countries, as well as an increased problem in developed areas of the world, with approximately 8 million new cases and 3 million deaths each year. Even though the infection may be asymptomatic for a considerable period of time, the disease manifests more commonly as a chronic inflammation of the lungs, resulting in fever and respiratory symptoms. If left untreated, significant morbidity and death can result. Even though tuberculosis can usually be controlled using prolonged antibiotic therapy, this treatment is not enough to prevent the spread of the disease. Infected people may be asymptomatic but contagious for a period of time. In addition, even when compliance in the treatment regimen is critical, the patient's compartment is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of mycobacteria resistant to the drug. Inhibiting the spread of tuberculosis requires effective vaccination and accurate early diagnosis of the disease. Currently, vaccination by subcutaneous or intradermal injection with active bacteria is the most efficient method to induce protective immunity. The common mycobacterium used for this purpose ^ .. is Bacillus Calmette-Guerin (BCG), an avirulent strain of ^ Mycobacterium bovis (M. jbovis). However, security and The effectiveness of BCG is of course controversial and some countries, such as the United States, do not vaccinate the general public. The diagnosis of M. tuberculosis infection is commonly achieved using a skin test, which involves intradermal exposure to tuberculin ^ x 10 PPD (purified derivative with protein). Antigen-specific T cell responses in measurable induration at the site of injection by 48-72 hours after injection, thus indicating exposure to mycobacterial antigens. Sensitivity and specificity, 15 however, have been a problem with this test, and people vaccinated with BCG can not be distinguished from infected people. f ^ Mycobacteria have been used for immunotherapy > less well known for tuberculosis and also leprosy, by subcutaneous or intradermal injection, is Mycobacterium vaccae [M. vaccae), which is nonpathogenic in humans. However, there is less information on the effectiveness of M. vaccae compared to BCG, and it has not been used extensively to vaccinate the general public. It is believed that M. bovis BCG and M. vaccae contain antigenic compounds that are recognized by the immune system of persons exposed to infection with M. tuberculosis. Several patents and other publications disclose the treatment of the different conditions 5 administering mycobacteria, including M. vaccae, or certain mycobacterial fractions. U.S. Patent No. 4,716,038 discloses the diagnosis of, vaccination against and treatment of autoimmune diseases of various types, including arthritic diseases, by administering mycobacteria, including M. vaccae. U.S. Patent No. 4,724,144 discloses an immunotherapeutic agent comprising antigenic material derived from M. vaccae for the treatment of mycobacterial diseases, especially tuberculosis and leprosy, and is an adjuvant for chemotherapy. International Patent Publication WO 91/01751 discloses the use of an antigenic and / or immunoregulatory material of M. vaccae f as an immunoprophylactic to retard and / or prevent the initiation of AIDS. International Patent Publication 20 Number WO 94/06466 discloses the use of an antigenic and / or immunoregulatory material derived from M. vaccae for HIV infection therapy with or without AIDS and with or without associated tuberculosis. U.S. Patent Number 5,599,545 gives 25 the use of mycobacteria, especially Ai. vaccae, whole, inactivated, such as an adjuvant for administration with antigens that are not endogenous to M. vaccae. This publication has the theory that the beneficial effect as an adjuvant can be due to the heat shock protein 65 (hsp 65). International Patent Publication Number WO 92/08484 discloses the use of an antigenic and / or immunoregulatory material derived from M. vaccae for the treatment of uveitis. International Patent Publication Number WO 93/16727 discloses the use of an antigenic and / or immunoregulatory material derived from M. vaccae for the treatment of mental illnesses associated with an autoimmune reaction initiated by an infection. International Patent Publication Number WO 95/26742 discloses the use of an antigenic and / or immunoregulatory material derived from M. vaccae to retard or prevent the growth or spread of tumors. International Patent Publication Number WO 91/02542 discloses the use of autoclaved M. vaccae in the treatment of chronic inflammatory disorders in which a patient demonstrates an abnormally high release of IL-6 and / or TNF or where the patient's IgG shows an abnormally high proportion of agalactosyl IgG. Among the disorders mentioned in this publication are psoriasis, rheumatoid arthritis, mycobacterial diseases, Crohn's disease, primary biliary cirrhosis, sarcoidosis, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, Guillain-Barre syndrome, primary diabetes mellitus, and some aspects of graft rejection. M. vaccae is apparently unique among the known mycobacterial species in those heat-killed preparations that retain the vaccine and immunotherapeutic properties. For example, M. tuberculosis BCG vaccines used for tuberculosis vaccination use active strains. M. bovis BCG and? G. Tuberculosis eliminated by heat does not have protective properties when used in vaccines. A number of compounds have been isolated from a scale of mycobacterial species that have adjuvant properties. The effect of these adjuvants is essentially to stimulate a mechanism of specific immune response against an antigen of other species. V ... There are two classes of general compounds that have been isolated from the mycobacterial species that exhibit adjuvant properties. The first ones are water soluble D-wax fractions (R.G. White, I. Bernstock, R.G.S. Johns and E. Lederer, Immunoloqv. 1:54, 1958; U.S. Patent Number 4,036,953). The second are the substances based on the muramyl dipeptide (N-acetyl glucosamine and N-glycolimuric acid in approximately eguimolar amounts) as described in US Pat. Nos. 3,956,481 and 4,036,953. These compounds differ from the delipidated and deglycolipidated M. vaccae (DD-M vaccae) of the present invention, in the following aspects of its composition: 1. They are water-soluble agents where ?? - ?. vaccae is insoluble in aqueous solutions. 2. It consists of a scale of small oligomers of the wall unit of the mycobacterial cell, either extracted from bacteria by different solvents or digested from the cell wall by an enzyme. In contrast, DD-M. vaccae contains a highly polymerized cell wall. 3. All protein has been removed from its preparations by digestion with proteolytic enzymes. The only constituents of its preparation are the components of the peptidoglycan structure of the cell wall, namely alanine, glutamic acid, diaminopimelic acid, N-acetyl glucosamine and N-glycolylmuramic acid. In contrast, DD-JÍ. vaccae contains 50% w / w protein, which comprises a number of different protein species. The delivery of vaccines by nasal sprays to reach the lung tissue or by oral delivery to the gastrointestinal tract has generally been limited to strains of attenuated virus. For example, poliovirus vaccination has employed the oral supply of attenuated strains of this virus since the development of the Sabin vaccine. Aviron Incorporated and The National Institute of Allergy and Infectious Diseases in the United States have recently reported the successful use of a flu vaccine administered in a nasal spray. In this case, a live attenuated influenza strain provided 93% protection against influenza in young children. Vaccines consisting of virus or deleted bacteria or recombinant proteins have not been delivered by nasal spray or oral supply. There are many reasons for this. There are a few satisfactory immunization reports resulting in T cell immunity or antibody synthesis using these nasally administered agents. In addition, the oral supply of proteins and eliminated organisms frequently results in the development of tolerance, which is exactly the reverse result sought in satisfactory immunization. Sarcoidosis is a disease of unknown cause, characterized by granulomatous inflammation that affects many organs of the body, and especially the lungs, lymph nodes and liver. The granulomata V of sarcoid is composed of mononuclear phagocytes, with 5 giant cells and epitheloids in its center, and T lymphocytes. CD4 T lymphocytes are closely associated with epitheloid cells while CD4 and CD8 T lymphocytes accumulate in the periphery. The characteristic immunological abnormalities in sarcoidosis include peripheral blood and hyper-globulinemia of bronchoalveolar lavage and of pressure of hypersensitivity reactions of the "delayed type" in the skin to tuberculin and other similar antigens, such as Candida and orejones. Peripheral blood lymphocyte numbers are reduced and the CD4: CD8 ratios in the peripheral blood are depressed to approximately 1-1.5: 1. These are not manifestations of a generalized immune defect, but rather the consequence of increased immunological activity that is "compartmentalized" with 20 sites of disease activity. In patients with pulmonary sarcoidosis, the total number of cells recovered by bronchoalveolar lavage is increased by 5 to 10 times and the proportion of lymphocytes increased from normal to less than 10% to 14% to between 15% and 50%. More than 25-90% of the recovered lymphocytes are t-lymphocytes and the ratio of CD4: CD8 has been reported as having increased from a value of 1.8: 1 in normal controls to 10.5: 1. The T lymphocytes are predominantly the Thl class producing lFN-? and IL-2 cytokines, instead of the Th2 class. After treatment, the increase in Thl lymphocytes in the sarcoid lungs is corrected. Sarcoidosis involves the lungs in almost all cases. Even when the lesions are seen predominantly in other organs, there is usually a subclinical lung involved present. Even though some cases of sarcoidosis resolve spontaneously, approximately 50% of patients have at least a mild degree of permanent organ malfunction. In serious cases, fibrosis of the lung develops and progresses to lung failure requiring lung transplantation. The main stay of treatment for sarcoidosis is eroid corycos. Patients who initially respond to corticosteroids often relapse and require treatment with other immunosuppressive drugs such as methotrexate or cyclosporine. Asthma is a common disease, with high prevalence in the developed world. Asthma is characterized by an increased response of the tracheobronchial tree to a variety of stimuli, with the primary physiological alteration being a reversible airflow limitation that may be spontaneous or related to the drug, the pathological hallmark being airway inflammation. Clinically, asthma can be subdivided into extrinsic and intrinsic variants. Extrinsic asthma has an identifiable precipitant and can be believed to be atopic, occupational, and drug induced. Atopic asthma is associated with the improvement of a Th2 immune response with the production of specific immunoglobulin E (IgE), skin tests positive to common aeroallergenic and / or atopic symptoms. It can be further divided into seasonal and perennial forms according to synchronization of the expression of symptoms. The obstruction of the air flow in the extrinsic asthma is due to non-specific bronchial hyperresponsiveness caused by the inflammation of the respiratory tract. This inflammation is mediated by chemical substances released by a variety of inflammatory cells including master cells, eosinophils and lymphocytes. The actions of these mediators result in vascular permeability, mucous secretion and bronchial smooth muscle constriction. In atopic asthma, the immune response that causes inflammation of the airway is effected by the Th2 class of T cells that secrete IL-4, IL-5 and IL-10. It has been shown that lymphocytes in the lungs of atopic asthmatics produce IL-4 and IL-5 when they are activated. Both IL-4 and IL-5 are cytokines of the Th2 class and are required for IgE replication and the complication of eosinophils in asthma. Occupational asthma may be related to the development of IgE to a protein hapten, such as acid anhydrides in plastic workers and has been plicatic in some of the asthma induced by western red cedar or to non-related IgE mechanisms such as seen in asthma induced by toluene diisocyanate. Drug-induced asthma can be seen after the administration of aspirin or other non-steroidal anti-inflammatory drugs, most often in a certain subset of patients that may exhibit other peculiarities such as nasal polyposis and sinusitis. Intrinsic or cryptogenic asthma is reported to develop after upper respiratory tract infections, but it may occur de novo in middle-aged or older people where it is more difficult to treat than extrinsic asthma. Asthma is ideally prevented by avoiding trigger allergens, but this is not always possible nor can trigger allergens always be easily identified. The medical therapy of asthma is based on the use of corticosteroids and bronchodilator drugs to reduce inflammation and obstruction of the respiratory tract.

In chronic asthma, treatment with corticosteroids leads to unacceptable detrimental side effects. Another disorder with an immune abnormality similar to asthma is allergic rhinitis. Allergic rhinitis is a common disorder and is estimated to affect at least 10% of the population. Allergic rhinitis can be seasonal (hay fever) caused by allergy to pollen. Perennial or non-seasonal rhinitis is caused by allergy to antigens such as those of house dust mite or animal dander. The abnormal immune response in allergic rhinitis is characterized by excessive production of specific IgE antibodies against allergens. The inflammatory response occurs in the nasal mucosa instead of further down the airway as in asthma. Similar to asthma, local eosinophilia in affected tissues is a major feature of allergic rhinitis. As a result of this inflammation, patients develop sneezing, discharge and nasal congestion. In some more serious cases, the inflammation extends to the eyes (conjunctivitis), palate or outer ear. Even when it is not a life threatening, allergic rhinitis can be very invalid, prevent normal activities and interfere with a person's ability to work. The current treatment involves the use of antihistamines. nasal deagulants and for asthma, sodium cromoglycate and corticosteroids. Lung cancer is the leading cause of cancer death. The incidence of lung cancer continues to rise and the World Health Organization estimates that by the year 2000 AD there will be two million new cases annually. Lung cancers can be broadly classified into two categories: small cell lung cancer (SCLC) which accounts for 20% to 25% of all lung cancers, and non-small cell lung cancer (NSCLC) which is the responsible for the remaining 75%. The majority of SCLC is caused by tobacco smoke. The SCLC tends to spread early and 90% of the patients present during the diagnosis the involvement of the mediastinal lymph nodes in the chest. SCLC is treated with chemotherapy, or a combination of chemotherapy and radiation therapy. Complete response regimens vary from 10% to 50%. For the rare patient without involvement with lymph nodes, surgery followed by chemotherapy can result in cure regimens that exceed 60%. The prognosis for NSCLC is more fatal, since most patients have advanced disease by the time the diagnosis is made. Surgical removal of the tumor is possible in a very small number of patients and the five-year survival rate for NSCLC is only 5% to 10%. The factors that lead to the development of lung cancer are complicated and multiple. Environmental and genetic factors interact and cause sequence and incremental abnormalities that lead to uncontrolled cell proliferation, invasion of adjacent tissues and spread to different sites. It has been shown that both humoral and cell-mediated immunity does not worsen in patients with lung cancer. Radiotherapy and chemotherapy also deteriorate the immune function of patients. Attempts have been made to immunize patients with non-activated tumor cells or tumor antigens to improve the counter-tumor response of the host. Bacillus Calmette-Guerin (BCG) has been administered in the chest cavity after lung cancer surgery to increase non-specific immunity. Attempts have been made to improve immunity against tumors by providing patients with lymphocytes treated ex vivo with interleukin-2. These lymphocytes activated with lymphokine acquire the ability to kill or eliminate tumor cells. Current immunotherapies for lung cancer are still in a stage of development and their efficacy has yet to be established for the normal administration of lung cancer. In one aspect, this invention deals with the ^ '' treatment of skin disorders that appear to be associated with factors that influence the rest of the immune cells derived from the thymus (T) known as Thl and Th2. These T cells are identified by their cytokine secretion phenotype. A common feature of the treatment is the use of compounds prepared from M. vaccae f "· 10 that have immunomodulation properties that alter the rest of the activities of these T cells as well as other immune cells." Psoriasis is an inflammatory skin disease. Chronic common that can be associated with 15 different forms of arthritis in a minority of patients.The defect in psoriasis appears as being an extra-rapid growth of keratinocytes and the shedding of scales on the surface of the skin. It is aimed at slowing down this process.The disease can become manifest at any age.Spontaneous remission is relatively rare and lifelong treatment is usually necessary.Psoriasis produces red patches of chronic sealing on the surface of the skin. It is a very visible disease, frequently affecting the face, skull, trunk and limbs.The disease is debilitating to the patient emotionally and physically, deducting themselves significantly from the quality of life. Between one and three million people in the United States have psoriasis with almost a quarter of a million new cases occurring each year. Conservative calculations put the costs of psoriasis care in the United States at present to $ 248 million dollars per year. There are two main hypotheses related to the pathogenesis of psoriasis. The first is that genetic factors determine the abnormal proliferation of epidermal keratinocytes. Cells no longer respond normally to external stimuli such as those involved to maintain epidermal homeostasis. Abnormal expression of cytokine receptors on the cell membrane or transduction of the abnormal transmembrane signal may be below the hyperproliferation of the cell. The inflammation associated with psoriasis is secondary to the release of the pro-inflammatory molecules of the hyperproliferative ceratinocytes. A second hypothesis is that T cells that interact with cells that present an antigen in the skin release of the pro-inflammatory cytokines and keratinocyte stimulants (Hancock, G.E. and others, J ".

Exp. Med. 168: 1395-1402, 1988). Only the T cells of genetically predetermined individuals possess the ability to activate under these circumstances. The keratinocytes themselves can be a cell that presents an antigen. The cellular infiltrate in psoriasis lesions shows an influx of CD4 + T cells and more prominently CD8 + T cells (Bos, JD et al., Arch. Dermatol. Res. 281: 23-3, 1989; Baker, BS , Br. J. Dermatol 110: 555-564, 1984). Since most (90%) of psoriasis patients have limited forms of the disease, topical treatments including ditaranol, tar preparations, corticosteroids and recently introduced vitamin D3 analogs (calcipotriol, calcitriol) can be used. A minority (10% of psoriasis patients have a more serious condition, for which a number of systematic therapeutic modalities are available.) Specific systematic therapies include UVB, PUVA, methotrexate, vitamin A derivatives (acitretin) and immunosuppressants such as Cyclosporin A. The efficacy of Cyclosporin and FK-506 to treat psoriasis provide support for the T cell hypothesis as the main cause of the disease (Bos, J.D. and others, Lancet II: 1500-1502, 1989; Ackerman, C et al., J. Invest. Dermatol. 96: 536 [abstract], 1991).

Atopic dermatitis is a chronic pruritic inflammatory skin disease that usually occurs in families with a hereditary predisposition to (various allergic disorders such as allergic rhinitis 5 and asthma.) Atopic dermatitis occurs in approximately 10% of the general population.The main symptoms are dry skin, dermatitis (eczema) localized mainly on the face, neck and on the sides and folds of the extremities accompanied by serious itching.It typically begins within the first two years of life.In approximately 90% of patients this skin disease disappears during childhood but the symptoms may continue into adulthood. the most common forms of dermatitis worldwide, it is generally accepted that atopy and atopic dermatitis, the normality of the T cell is primary and that the malfunction of the T cells that normally regulate the production of IgE fx is responsible for the production excessive of this immunoglobulin 20 Allergic contact dermatitis is a non-infectious inflammatory skin disorder. To contact dermatitis, immune reactions can not develop until the body has been sensitized to a specific antigen. The exhibition Subsequent skinning of the antigen and recognition of these antigens by T cells results in the release of different cytokines, proliferation and recruitment of T cells and finally dermatitis V. (eczema) 5 Only a small proportion of the T cells in an allergic contact dermatitis lesion are specific for the related antigen. Activated T cells probably m large towards the sites of inflammation regardless of the specificity of the antigen. Delayed type hypersensitivity can only be transferred by T cells (CD4 + cells) that share MHC class II antigens. The 'response' for contact allergens can be transformed by the T cells that share either the MHC class I (CD8 + cells) or class II (CD4 + cells) molecules (Sunday, ME et al., J. Immunol. 125: 1601-1605, 1980). The ceratinocytes can produce interleukin-1 which can facilitate the presentation of the antigen to the T cells. The expression of the intracellular adhesion molecule-1 of surface antigen (ICAM-1) is induced both in the ceratinocytes and endothelium by the factor of cytokine tumor necrosis (TNF) and interferon-gamma (IFN-?). If causes can be identified, removal will only cure allergic contact dermatitis. During active inflammation, topical corticosteroids are useful. An inhibitory effect of cyclosporin has been observed in delayed-type hypersensitivity in the pro-inflammatory function (s) of T cells in vitro (Shidani, B. et al., Eur. J., Jmmunol., 14: 314-318, 1984) The inhibitory effect of cyclosporine in the early phase of T cell activation in mice has also been reported (Milton, G. et al., Ann. Immunol. (Inst. Pasteur) 135d: 237-245 , 1984) .Aceased alopecia is a common hair disease, accounting for approximately 2% of consultations in outpatient dermatological clinics in the United States.The hallmark of this disease is the formation of well-round or oval patches. Circumscribed alopecia that does not form a scar that can be placed in any hair area of the body.The disease can develop at any age.Initiation is usually sudden and the clinical course is varied.At present, it is not possible to attribute all or then any A case of alopecia sent to a single cause (Rook, A. and Dawber, R. Diseases of the Hair and Scalp; Blackwell Scientific Publications 1982: 272-30). There are many factors that seem to be involved. These include genetic factors, atopy, association with disorders of suspected autoimmune etiology. Down syndrome and emotional effort. The prevalence of atopy in patients with alopecia aereated has been increased. There is evidence that airborne alopecia is an autoimmune disease. This evidence is based on consistent histopathological findings of a lymphocytic T cell infiltrate in and around the hair follicles with increased numbers of Langerhans cells., the observation that airborne alopecia will respond to treatment with immunomodulating agents, and that there is a statistically significant association between aerated alopecia and a wide variety of autoimmune diseases (Mitchell, AJ et al., J. Am. Acad. Dermatol. 763-775, 1984). Immunophenotype studies in hull biopsy specimens show the expression of HLA-DR in the epithelial cells in the cortex and hair follicles of active lesions of alopecia aereate, as well as the infiltration of the T cell with a high proportion of T cells of help / induction in and around the hair follicles, increased numbers of Langerhans cells and the expression of ICAM-1 (Messenger, AG et al., i7.Invest.Dermatol.85: 569-576; Gupta, A.K. and others, J. Am. Acad. Dermatol. 22: 242-250, 1990). The great variety of therapeutic modalities in alopecia aereada can be divided into four categories: (i) non-specific topical irritants; (ii) 'immune modulators' such as systemic corticosteroids and PUVA; (iii) 'immune enhancers' such as inducers of contact dermatitis, cyclosporin and inosiplex; and (iv) drugs of unknown action such as minoxidil (Dawber, R.P.R., et al., Textbook of Dermatology, Blackwell Scientific Publications, Fifth Edition, 1982: 2533-2638). Non-specific topical irritants such as dithranol can work through yet-unidentified mechanisms instead of local irritation to allow new hair growth. Topical corticosteroids may be effective but prolonged therapy is often necessary. Intralesional steroids have been shown to be more effective but their use is limited to circumscribed patches of a less active disease or to maintain re-growth of the eyebrows in alopecia totalis. Photochemotherapy has been shown to be effective, possibly changing the functional subpopulations of T cells. Topical immunotherapy by induction and maintenance of allergic contact dermatitis in the skull may result in the regrowth of the hair, as well as 70% of patients with alopecia aereated. Diphencyprone is a potent sensitizer free of mutagenic activity. Oral cyclosporine may be effective in the short term (Gupta, A.K. and others, J. Am. Acad.

Dermatol. 22: 242-250, 1990). Inosiplex, an immunostimulant, has been used with apparent efficacy in an open test. A solution of topical 5% minoxidil has been reported to be able to induce some hair growth in patients with alopecia aereated. The mechanism of action is unclear. Carcinomas of the skin are a major public health problem due to their frequency and disability and disfigurement that can cause. Carcinoma of the skin is mainly seen in individuals at the beginning of their lives, especially in clear-skinned individuals exposed to large amounts of sunlight. The annual cost of treatment and the loss of work time exceeds $ 250 million dollars per year in the United States alone. The three main types - basic cell cancer, squamous cell cancer and melanoma - are clearly related to exposure to sunlight. Basic cell carcinomas are epithelial tumors of the skin. They appear predominantly in the exposed areas of the skin. In a recent Australian study, the incidence of basal cell carcinomas was 652 new cases per year per 100,000 population. This compares with 160 cases of squamous cell carcinoma or 19 of malignant melanoma (Giles, G. et al., Br. Med. J. 296: 13-17, 1988). Basic cell carcinomas are the most common of all cancers. The lesions are usually surgically excited. Alternative treatments include retinoids, 5-fluorouracil, cryotherapy, and radiation therapy. Interferon alpha or gamma have also been shown to be effective in the treatment of basal cell carcinomas by providing a valuable alternative to patients not suitable for surgery or trying to avoid surgical scars (Cornell et al., 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 common cutaneous malignancy and its frequency is increasing. There is an increased number of advanced and metastatic cases related to a number of underlying factors. Currently, metastatic SCC contributes to more than 2000 deaths per year in the United States; the 5-year survival rate is 35%, with 90% of the metastases occurring over 3 years. Metastasis almost always occurs in the first season of lymphatic drainage. The need for medical therapy for advanced cases is clear. Satisfactory medical therapy for the primary SCC of the skin would avoid the need for surgical excision with its potential for scarring and other side effects. This development may be especially desirable for facial injuries. Because of their antiproliterative and in vitro immunomodulation effects, interferons (IFNs) have also been used in the treatment of melanoma (Kirkwood, J.M., et al., J. Invest, Dermatol 95: 180S-4S, 1990). The response regimens achieved with systematic IFN-a in either a high or low dose, in metastatic melanoma were within the range of 5% to 30%. Recently, encouraging results (30% response) were obtained with a combination of IFN-a and DTIC. Preliminary observations indicate a beneficial effect of IFN-a in adjuvant adjustment in patients at high risk of melanoma. Despite the low efficacy of IFN monotherapy in metastatic disease, several studies of randomized leaflets with IFNs are now being carried out as an adjuvant 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 the therapies available for the treatment of viral skin lesions, only interferon has a mode of specific antiviral action, reproducing the immune response of the body to the infection.The treatment of interferon can not eradicate the virus, however, even though it can help with certain manifestations of the infection.The treatment of interferon is also associated with detrimental effects ^ systematic, requires multiple injections in each ^ wart and has a significant economic cost (Kraus, S.J. and others, Review of Infections Diseases 2 (6): S620-S632, 1990; Fraze, I.H., Current Opinion in Imnamology ß (4): 484-491, 1996). COMPENDIUM OF THE INVENTION Briefly stated, the present invention provides compositions present in or derived from M. vaccae and methods for their use in the prevention, treatment and diagnosis of diseases, including mycobacterial infection, immune disorders of the respiratory system and skin disorders. The methods of the invention comprise administering a composition having antigenic and / or adjuvant properties. Diseases of the respiratory system that can be treated using the "compositions of the invention include mycobacterial infections (such as infection with M. tuberculosis. and / or M. avium), asthma, sarcoidosis and lung cancers. Skin disorders that can be treated using the compositions of the invention include psoriasis, atopic dermatitis, allergic contact dermatitis, alopecia aereate and basal cell carcinoma. cancers of the skin, carcinoma and squamous cell melanoma. Adjuvants for use in vaccines or immunotherapy of infectious diseases and cancers are also provided. In a first aspect, isolated polypeptides derived from Mycobacterium vaccae are provided comprising an immunogenic portion of an antigen, or a variant of this antigen. In specific embodiments, the antigen includes an amino acid sequence that is selected from the group consisting of: (a) the sequences mentioned 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; (b) sequences having at least about 50% residues identical to a sequence mentioned 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) the sequences that have at least. ^ approximately 75% residues identical to a sequence 20 mentioned 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% residues identical to a sequence mentioned 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, which are measured ^ N using the alignments produced by the BLASTP computer algorithm, as described below. The DNA sequences encoding the polypeptides of the invention, the expression vectors comprising the DNA sequences and the host cells transformed or transferred with expression vectors are also provided. In another aspect, the present invention provides fusion proteins comprising at least one polypeptide of the present invention Within other aspects, the present invention provides pharmaceutical compositions comprising at least one of the polypeptides of the invention, or one DNA molecule encoding this polypeptide, and a physiologically acceptable carrier. The invention also provides vaccines comprising at least one of the aforementioned polypeptides or at least one DNA sequence encoding these polypeptides, and a non-specific immune response amplifier. In certain embodiments, the non-specific immune response enhancer is selected from the group consisting of delipidated and deglycolipidated M. vaccae cells; inactivated M. vaccae cells; delipidated M. vaccae cells and deglycolipidates depleted of mycolic acids; delipidated M. vaccae and deglycolipidase cells depleted of mycolic acids and arabinogalactana; and a culture filtrate of M. vaccae. In still another aspect, methods are provided for improving an immune response in a patient, comprising administering to a patient an effective amount of one or more of the aforementioned pharmaceutical compositions and / or vaccines. In one embodiment, the immune response is a Thl response. In further aspects of this invention, methods are provided for the treatment of a disorder in a patient, which comprises administering to the patient a pharmaceutical composition or a vaccine of the present invention. In certain embodiments, the disorder is selected from the group consisting of immune disorders, infectious diseases, skin diseases and diseases of the respiratory system. Examples of these diseases include mycobacterial infections, asthma and psoriasis. In other aspects, the invention provides methods for the treatment of immune disorders, infectious diseases, skin diseases or diseases of the respiratory system, which comprises administering a composition consisting of the? G cells. inactivated vaccines, delipidated and diglycolipidated M. vaccae cells or a culture filtrate of M. vaccae.

Methods for improving an immune response to an antigen are also provided. In one embodiment, these methods comprise administering a polypeptide comprising an immunogenic portion of an M. vaccae antigen that includes a sequence of SEQ ID NO: 89 or 201, or a variation thereof. In a further embodiment, these methods comprise administering a composition comprising a component that is selected from the group consisting of: delipidated M. vaccae and deglycolipidase cells depleted of mycolic acids, and delipidated M. vaccae and deglycolipidase cells depleted of mycolic acids and arabinogalactan. In additional aspects of this invention, diagnostic methods and kits for detecting mycobacterial infection in a patient are provided. In a first embodiment, the method comprises contacting the dermal cells of a patient with one or more of the aforementioned polypeptides and detecting an immune response in the skin of the patient. In a second embodiment, the method comprises contacting a biological sample with at least one of the aforementioned polypeptides; and detecting in the sample the presence of antibodies that bind to the polypeptide or polypeptides, thereby detecting M. tuberculosis infection in the biological sample. Appropriate biological samples include whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid and urine. "Diagnostic kits comprising one or more of the above-mentioned polypeptides in combination with an apparatus sufficient to contact the polypeptide with the dermal cells of a patient are also provided.The present invention also provides diagnostic kits that comprise one or more of the polypeptides of the invention in combination with a detection reagent In yet another aspect, the present invention provides antibodies, both polyclonal and monoclonal which bind to the polypeptides described above as well as methods for their Use in the Detection of a Mycobacterial Infection These and other aspects of the present invention will become apparent upon reference to the following detailed description and the accompanying drawings.All references herein disclosed are incorporated herein by reference. in its entirety as if each one were incorporated indiv IDENTIFIED BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB illustrate the protective effects of immunizing mice with M. vaccae subjected to autoclave treatment or M. culture filtrates. vaccate unfractionated, respectively, before infection with active M. tuberculosis H37v. Figures 2A and B show the percentage of eosinophils in mice immunized intranasally with either 10 or 1000 g of M. vaccae removed with heat or 200 to 100 μg of ?? - ?. vaccinate, respectively, four weeks before challenge with ovalbumin, as compared to control mice. Figures 2C and D show the percentage of eosinophils in mice immunized intransally with either 100 μg of heat-killed M. vaccae or 200 μg of ?? - ?. Vaccinate, respectively as late as one week before challenge with ovalbumin. Figure 2E shows the percentage of eosinophils in mice immunized either intranasally (in) or subcutaneously (sc) with either BCG of the Pasteur strain (BCG-P), BCG of the Connought strain (BCG-C), 1 mg of M. vaccae removed by heat or 200 μg of DD-M. Vaccinate before the challenge with ovalbumin. Figure 3A illustrates the effect of mouse immunized with heat-killed M. vaccae or delipidated or vaclyllated M. vaccae (DD-M vaccae) before infection with tuberculosis. Figure 3B illustrates the effect of immunizing mice with heat-killed M. vaccae, γ-proteins. Vaccinate recombinants or a combination of? vaccae removed by heat and recombinant M. vaccae proteins before infection with tuberculosis. Figure 4 illustrates the induction of IL-12 by autoclaved M. vaccae, M. vaccae 5 lyophilized, M. vaccae delipidada and deglicolipidada and glycolipids of M. vaccae. Figure 5 compares the in vitro stimulation of interferon-gamma production in the spleen cells of the Serum Combined Immuno Deficient (SCID) mice by different concentration of heat-killed M. vaccae (autoclaved), M vaccae delipidado and deglicolipidado and glycolipids of M. vaccae. Figures 6A, B and C illustrate the stimulation of interferon-gamma production by different concentrations of recombinant M. vaccae proteins, heat-killed M. vaccae, delipidated M. vaccae and deglycolipidated (referred to in Figure f "" as "M. vaccae delipidate"), glycolipids of M. vaccae and lipopolysaccharide in peritoneal macrophages of C57BL / 6 mice (Figure 6A), BALB / C mice (Figure 6B) or C3H / HeJ mice (Figure 6C) . Figure 7A (i) - (iv) illustrates the non-specific immune amplification effects of 10 μg, 100 μg and 1 mg of γG. vaccae subjected to autoclaving and 25 75 μg of unfractionated culture filtrates of M. vaccae, respectively. Figure 7B (i) and (ii) illustrate the non-specific immune amplification effects of M. vaccae subjected to autoclaving treatment and delipidated and deglycolipidated M. vaccae, respectively. Figure 5C (i) illustrates the effects of non-specific immune amplification of M. vaccae subjected to whole autoclave treatment. Figure 7C (ii) illustrates the non-specific immune amplification effects of the soluble M. vaccae proteins extracted with SDS from delipidated and deglycolipidated M. vaccae. Figure 7C (iii) illustrates the non-specific amplification effects of the preparation of Figure 7C (ii) which are destroyed by treatment with the proteolytic enzyme Pronasa. Figure 7D illustrates the effects of non-specific immune amplification of heat-cleared M. vaccae (Figure 7D (i)) f while a non-specific immune amplification effect is not seen with heat-killed preparations of M. tuberculosis ^ ( Figure 7D (ii), M. bovis BCG (Figure 7D (iii)) # M. phlei (Figure 7D (iv)) and M. smeg atis (Figure 7D (v)). FIGS. 8A and B illustrate the stimulation of CD69 expression in aβ cells, α cells? and NK cells, respectively, by the M. vaccae GV23 protein, the Thl MPL / TDM / CWS and CpG ODN induction adjuvants, and the Th2 inducing adjuvants of aluminum hydroxide and cholera toxin.

Figures 9A-D illustrate the effect of heat-killed M. vaccae, the recombinant DD-M proteins. vaccae and M. vaccae in the production of l-? ß, TNF-a, IL-12 and IFN- ?, respectively, by means of human PBMC. Figures 10A-C illustrate the effects of varying the concentrations of the recombinant M. vaccae proteins GV-23 and GV-45 in the production of IL-? Β, TNF-a and IL-12, respectively, by means of human PBMC . Figures 11A-D illustrate the stimulation of IL-? ß ?, f "10 TNF-a, IL-12 and IFN-α production, respectively, in human PBMC by the M. vaccae GV23 protein, the induction adjuvants of Thl MPL / TDM / CWS and CpG ODN, and the Th2-inducing adjuvants of aluminum hydroxide and cholera toxin 15 Figures 12A-C illustrate the effects of varying the concentrations of the recombinant M. vaccae GV-23 proteins and GV-45 in the expression of CD40, CD80 and - ^ CD86, respectively, by dendritic cells Figure 13 illustrates the improvement of dendritic cell 20 mixed with leukocyte reaction by the recombinant M. vaccae protein GV-23. DETAILED DESCRIPTION OF THE INVENTION As mentioned above, the present invention is generally directed with positions and methods for preventing, treating and diagnosing infectious diseases and immune disorders, which can be effectively treated using the compositions of the invention. of the invention include diseases of the respiratory system such as mycobacterial infections, asthma, sarcoidosis and lung cancers, and skin disorders, such as psoriasis, atopic dermatitis, allergic contact dermatitis, alopecia aereate and basal cell carcinoma of cancers of the lungs. Skin, carcinoma and squamous cell melanoma. Effective vaccines that provide protection against infectious microorganisms contain at least two functionally different components. The first is an antigen, which may be a polypeptide or carbohydrate in nature that is processed by macrophages and other antigen presenting cells and is disclosed for CD4 + T cells or CD8 * T cells. This antigen forms the "specific" target of the immune response. The second component of a vaccine is a non-specific immune response amplifier, called an adjuvant with which the antigen is mixed or incorporated. An adjuvant amplifies either the immune responses mediated by a cell or antibody in a structurally unrelated compound or a polypeptide. Various known adjuvants are prepared from microbes such as Bordetella pertussia, M. tuberculosis and M. bovis BCG.

The adjuvants may also contain components designed to protect the degradation polypeptide antigens such as aluminum hydroxide or mineral oil. Although the antigenic component of a vaccine contains polypeptides that direct the immune attack against a specific pathogen, such as M. tuberculosis, the adjuvant is often capable of wide use in many different vaccine formulations. Certain known proteins such as bacterial enterotoxins can function both as an antigen to carry out a specific immune response and as an adjuvant to improve immune responses for unrelated proteins. Certain pathogens such as M. tuberculosis as well as certain cancers are effectively contained by targeted immune attack by CD4 * and CD8 + T cells, known as cell-mediated immunity. Other pathogens such as poliovirus also require antibodies produced by B cells for containment. These different classes of immune attack (T cell or B cell) are controlled by their different populations of CD4 + T cells which are commonly referred to as Th1 and Th2 cells. A desirable property of an adjuvant is the ability to selectively amplify the function of either Th1 or Th2 populations of CD4 * T cells. Many skin disorders, including psoriasis, atopic dermatitis, alopecia and skin cancers appear to be influenced by differences in subgame activity of the Th cell. In two types of Th cell subgames are well characterized in a murine model and are defined by cytokines that release during activation. The subset of Thl secretes IL-2, IFN-? and the tumor necrosis factor, and mediates macrophage activation and the delayed-type hypersensitivity response. The subset Th2 releases IL-4, IL-5, IL-6 and IL-10 that stimulate B cell activation. Thl and Th2 subgames are mutually inhibitory so that IL-4 inhibits Thl type responses, and IFN-? inhibits Th2 type responses. The subgames of Thl and Th2 alike have been found in humans with the release of identical cytokines observed in the murine model. In particular, most clones of the T cell of atopic human lymphocytes resemble the murine Th2 cell that produces IL-4, while very few clones produce IFN-α. Therefore, the selective expression of subset Th2 with the subsequent production of IL-4 and decreased levels of IFN-α producing cells. could lead to preferential improvement of IgE production. The amplification of Thl-type immune responses is central to an inversion of the disease state in many disorders, including respiratory system disorders such as tuberculosis, sarcoidosis, asthma, allergic rhinitis and lung cancers. Ai. vaccae inactivated and many compounds derived from? G. vaccae have both antigen and adjuvant properties that work to improve the immune response of the Thl type. The methods of the present invention employ one or more of these antigen and adjuvant compounds of M. vaccae and / or their culture filtrates to redirect the immune activities of T cells in patients. Mixtures of these compounds are particularly effective in the methods disclosed herein. Although it is well known that mycobacteria contain many transverse reaction antigens, it is not known whether they contain adjuvant compounds in common. As shown below, the inactivated M. vaccae and the modified form (delipidate and deglycolipidate) of inactivated M. vaccae have been found to have Thl-type adjuvant properties that are not shared by a number of other mycobacterial species. It has also been found that M. vaccae produces compounds in its own culture filtrate that amplify the immune response to the M. vaccae antigens that are also found in culture filtrate as well as antigens from other sources. In one aspect, the present invention provides methods for the immunotherapy of respiratory and / or lung disorders including tuberculosis, sarcoidosis, asthma, allergic rhinitis and lung cancers, in a patient to improve Thl-type immune responses. In one embodiment, the compositions are delivered directly to the mucosal surface of the airways leading to and / or into the lungs. However, the compositions can also be administered through intradermal or subcutaneous routes. The compositions that can be usefully employed in these methods comprise at least one of the following components: inactivated M. vaccae cells; culture filtrate of M. vaccae; delipidated and deglycolipidated M. vaccae (? -? vaccae) cells and the compounds present in or derived from M. vaccae and / or their culture filtrate. As will be illustrated below, the administration of these compositions results in specific T cell immune responses and improved protection against M. tuberculosis infection and is also effective in the treatment of asthma. Although the precise mode of action of these compositions in the treatment of diseases such as asthma is unknown, it is believed that they suppress an immune response of Th2 inducing asthma. As used herein, the term "respiratory system" refers to the lungs, the nasal passages, the bronchial passages and the trachea. As used herein, the term "respiratory passages that lead to or are placed in the lung" includes the nasal passages, the mouth, the tonsil tissue, the trachea, and the bronchial passages. As used herein, a "patient" refers to any warm-blooded animal, preferably a human being. This patient may suffer from a disease or may be exempt from a detectable disease. In other words, the methods of the invention can be employed to induce protective immunity for the prevention or treatment of diseases. In another aspect, the present invention provides methods for the immunotherapy of skin disorders, including psoriasis, atopic dermatitis, alopecia and skin cancers in patients, wherein the immunotherapeutic agents are used to alter or redirect an existing state of Immune activity altering the function of T cells towards a Thl type of immune response. The compositions that can be usefully employed in the methods of the invention comprise at least one of the following components: inactivated M. vaccae cells; culture filtrate of M. vaccae; modified M. vaccae cells; and the constituents and compounds present in or derived from M. vaccae and / or their culture filtrate. As detailed below, multiple administrations of these compositions preferably by intradermal injection have been shown to be highly effective in the treatment of psoriasis. As used herein, the term "inactivated M. vaccae" refers to M. vaccae which has either been removed by heat, as will be detailed below in Example 7, or has been subjected to radiation such as S0Cobalto at a dose of 2.5 megaradios. As used herein, the term "modified M. vaccae" includes the cells of M. vaccae delipidates, the cells of M. vaccae deglicolipidadas and the cells of M. vaccae that have both delipidated and deglycolipidated (DD-2V. vaccae). The preparation of DD-M. vaccae and its chemical composition will be described below in Example 7. As will be detailed below, the inventors have shown that the removal of the glycolipid constituents of M. vaccae results in the removal of molecular components that stimulate the production of interferon. -gamma in the natural cells of elimination (NK), thus significantly reducing the non-specific production of a cytokine that has numerous harmful side effects. In still a further aspect, the present invention provides isolated polypeptides comprising at least an immunogenic portion of an M. vaccae antigen or a variation thereof or at least a portion of adjuvant of the M. vaccae protein. In specific embodiments, these polypeptides comprise an immunogenic portion of an antigen, or a variant thereof wherein the antigen includes a sequence that is selected from the group consisting of SEQ ID NO: 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, 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" encompasses amino acid chains of any length, including full-length proteins, i.e., antigens (ie, antigens) wherein the amino acid residues are linked by covalent peptide bonds. In this manner, a polypeptide comprising an immunogenic portion of one of the aforementioned antigens may consist entirely of an immunogenic portion, or may contain additional sequences. Additional sequences can be derived from inactive M. vaccae antigen or can be heterologous, and these sequences can (but need not be) immunogenic. As detailed below, the polypeptides of the present invention can be isolated from M. vaccae cells or culture filtrate or can be prepared by synthetic or recombinant means. The term "immunogenic" as used herein refers to the ability to provide 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, the production of interleukin-12 or the production of interferon-? in biological samples comprising one or more cells that are selected from the group of T cells, NK cells, B cells and macrophages, wherein the cells are derived from a person immune to Af. tuberculosis. Exposure to an immunogenic antigen usually results in the generation of an immune memory in such a manner that during re-exposure to that antigen, an improved and faster response occurs. Immunogenic portions of the antigens described herein can be prepared and identified using well-known techniques, such as those summarized in Paul, Fundamental Immunolog, Third Edition, Raven Press, 1993, pages 243-247. These techniques include screening polypeptide portions of the native antigen or protein for immunogenic properties. The representative proliferation and cytokine production assays described herein can be employed in these screens. An immunogenic portion of an antigen is a portion that, within these representative assays, generates an immune response (cell proliferation, interferon-α production or interleukin-12 production) that is essentially similar to that generated by the length antigen. complete In other words, an immunogenic portion of an antigen can generate at least about 20%, preferably about 65% and most preferably about 100% of the proliferation induced by the full-length antigen in the proliferation assay model, which is described in the present. An immunogenic portion may also stimulate or alternatively effect the production of at least about 20%, preferably about 65% and most preferably about 100% of the interferon-? and / or interleukin-12 induced by the full length antigen in the model assay described herein.

An adjuvant M. vaccae is a compound found in M. vaccae cells or the filtrates of the Af culture. vaccae that non-specifically stimulates immune responses. Adjuvants improve the immune response for immunogenic antigens and the memory formation process. In the case of M. vaccae proteins, these memory responses favor Thl type immunity. The adjuvants are also capable of stimulating the production of interleukin-12 or the production of interferon-? in biological samples that comprise a V .. or more cells that are selected from the group of T cells, NK cells and B cells and macrophages, where the cells are derived from healthy people. The adjuvants may or may not stimulate the proliferation of the cell. These adjuvants of M. vaccae include for example polypeptides comprising a sequence mentioned in SEQ ID NO: 89, 117, 160, 162 or 201. The term "polynucleotide (s)" as used in V. present, means a single or double chain polymer of deoxyribonucleotide or ribonucleotide bases and includes the corresponding DNA and RNA molecules, including hRNA and mRNA, both sense and antisense strands, comprises the cDNA and the genomic DNA and the recombinant DNA, as well as the synthesized polynucleotides complete or partially. An RNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner. An mRNA molecule corresponds to a molecule of A NHn and DNA from which the introns have been cut. A polynucleotide may consist of an entire gene or any portion thereof. The antisense polynucleotides capable of functioning can comprise a fragment of the corresponding polynucleotide and the definition of "polynucleotide", therefore, includes all these operable anti-sense fragments. The compositions and methods for this invention also encompass variants of the aforementioned polypeptides and polynucleotides. As used herein, the term "variant" covers any sequence having at least about 40%, more preferably at least about 60%, and most preferably still about 75%, and preferably at least less about 90% identical residues (either nucleotides or amino acids) to a sequence of the present invention. The percentage of identical residues is determined by aligning the two sequences to be compared, determining the number of identical residues in the aligned portion, dividing the number by the total length of the sequence of the invention, or requested, and multiplying the result by Polynucleotide or polypeptide sequences can be aligned and the percentage of identical nucleotides r in a specified region can be determined against another V. polynucleotide, using computer algorithms that can be obtained publicly. Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms. The similarity of the polypeptide sequences can be examined using the BLASTP algorithm. Both the BLASTN and BLASTP software are available on the anonymous NCBI FTP server (ftp://ncbi.nlm.nih.gov) under / blast / executables /. The version of the algorithm BLASTN 2.0.4 [February-24-1998], which conforms to the failure parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of variants, in accordance with the present invention. The use of the BLAST family of algorithms, including r BALSTN and BLASTP, is described on the NCBI network side at URL http: // www. ncbi. nlm. nih.s ?? / BLAST / newblast. html and in the publication of Stephen F. Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein data searc programs", Nucleic Acide Res. 25: 3389-3402. The FASTA computer algorithm is available online at ftp://ftp.virginia.edu/pub/fasta/. The 25 version 2.0u4, February 1996, which conforms to the failure parameters described in the documentation and which is distributed with the algorithm, is preferred for use in the determination of the variants according to the present invention. The use of the FASTA algorithm is described in the 5 article by W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Analysis", Proc. Nati Acad. Sci. USA 85: 2444-2448 (1998) and the article by W.R. Pearson, "Rapid and Sensitive Sequence Comparison with FASTP and FASTA," Methoda in Enzymology 183: 63-98 (1990). "10 The following operating parameters are preferred for the determination of the alignments and similarities using BLASTN that contribute to the values of E and the identity of the percentage: Unix operating command: blastall -p -d embldb -e 10 -G 1 -E 1 -r 2 -v 50 -b 15 50 -i queryseq - or results; and parameter failure values: -p Program name [String] f N -d Database [String] v > -? -e Expectation value (E) [Real] 20 -G Cost to open a space (zero invokes the failure behavior) [Integer] -E Cost to extend a space (zero invokes the failure behavior) [Integer] -r Prize for a nucleotide match (blastn 25 only) [Integer] -v Number of descriptions of a line (V) [Integer] -b Number of alignments to show (B) [Integer] -i Request file [Input file ] -o BLAST report output file [output file] optional For BLASTP the following operating parameters are preferred: blastall -p blastp -d swissprotdb -e 10 -G 1 -E 1 -v 50 -b 50 -i queryseq -or results -p Program name [String] -d Database [String] -e Expectation value (E) [Real] -G Cost to open a space (zero invokes the failure behavior) [Integer] -E Cost to extend a space (zero invokes a failure behavior) [Integer] -v Number of descriptions of a line (v) [Integer] -b Number of alignments to display (b) [Integer] -I Request File [Archive] -o BLAST Report Output File [Output File] Optional. These "hits" to one or more database sequences by means of a requested sequence produced by BLASTN, BLASTP, FASTA or a similar algorithm, align and identify the similar portions of the sequences. The hits are placed in the order of the degree of similarity and the length of the overlap of the sequence. Successes to a database sequence usually represent an overlap through only a fraction of the length of the sequence of the entire requested sequence. The BLASTN and FASTA algorithms also produce "Expected" values for alignments. The value of Expected (E) indicates the number of hits that can be "expected" to see through a certain number of contiguous sequences by any possibility when looking for a database of a certain size. The expected value is used as a significant threshold to determine whether the hit to a database such as preferred EMBL indicates a true similarity. For example, an E value of 0.1 that is assigned to a hit is interpreted as implying that in a database of the size of the EMBL database, one might expect to see 0.1 match through the aligned portion of the sequence with a similar score simply by chance. By this criterion, the aligned and coincidental portions of the sequences have a 90% probability of being equal. For sequences that have an E value of 0.01 or less through the aligned and coincident portions, the probability of finding a coincidence by chance in the EMBL database is 1% or less, using the BLASTN or FASTA algorithm. According to one embodiment, the "variant" polynucleotides with reference to each of the polynucleotides of the present invention preferably comprise sequences having the same or a lesser number of nucleic acids than each of the polynucleotides of the present invention, and which produce an E-value of 0.01 or less when compared to the polynucleotide of the present invention. That is, a polynucleotide variant in any sequence having at least a 99% probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less, using the BLASTN or FASTA algorithms adjusted to the failure parameters. According to a preferred embodiment, a variant polynucleotide is a sequence having the same number or less number of nucleic acids as a polynucleotide of the present invention having at least a 99% probability of being the same as the polynucleotide of the present invention. invention that is measured having an E value of 0.01 or less using the BLASTN or FASTA algorithms adjusted to the failure parameters. Variant polynucleotide sequences usually hybridize to the mentioned polynucleotide sequence under stringent conditions. As used herein, "stringent conditions" refers to pre-washing in a 6X SSC solution, 0.2% SDS; Hybridizing av "· 65 ° C, 6X SSC, 0.2% SDS overnight, followed by two 5 washes of 30 minutes each in IX SSC, 0.1% SDS at 65 ° C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65eC. The portions and other variants of M. vaccae polypeptides can be generated by . { N 10 synthetic or recombinant. The synthetic polypeptides that v-| > they have less than about 100 amino acids, and generally, less than about 50 amino acids can be generated using techniques well known to those skilled in the art. For example, these Polypeptides can be sintered using any of the commercially available solid-phase techniques such as the Merrifield solid-phase synthesis method, where f the amino acids are added in sequence to a chain of growing amino acids. See in Merrifield, J. Am.

C ejn. Soc. 85: 2149-2146, 1963. The equipment for automatic synthesis of the polypeptides can be obtained commercially from the suppliers of Perkin / Elmer Applied BioSystems, Inc. (Foster City, CA), and can be operated in accordance with the instructions of maker.

Variants of a native antigen or adjuvant can be prepared using normal mutagenesis techniques such as site-specific mutagenesis driven by oligonucleotide. Sections of the DNA sequence can also be removed using standard techniques to allow preparation of the truncated polypeptides. A polypeptide of the present invention can be conjugated to a signal (or leader) sequence at the N-terminal end of the protein that co-translationally or post-transiently directs the transfer of the protein. The polypeptide can also be conjugated in a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g. poly-His) or to improve the binding of the polypeptide to a solid support. For example, a polypeptide can be conjugated to an immunoglobulin Fe region. In general, M. vaccae antigens and the DNA sequences encoding these antigens can be prepared using any of a variety of methods. For example, soluble antigens can be isolated from the culture filtrate of M. vaccae, as will be described below. The antigens can also be produced recombinantly by inserting the DNA sequence encoding the antigen into an expression vector and expressing the antigen in an appropriate host. Any of a variety of expression vectors known to those skilled in the art may be employed. Expression can be achieved by any appropriate host cell that has been transformed or transferred with 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 employed are E. coli, mycobacteria, insect, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner can encode naturally occurring antigens, naturally occurring portions or other variants thereof. The DNA sequences encoding the M. vaccae antigens can be obtained by screening an appropriate M. vaccae cDNA or genomic DNA library for DNA sequences that hybridize to degenerate oligonucleotides derived from the partial amino acid sequences of the isolated soluble antigens. Appropriate degenerate oligonucleotides can be designed and synthesized and the screen can be carried out, as described for example, in the article by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989 As will be described below, polymerase chain reaction (PCR) can be employed to isolate a nucleic acid probe from the genomic DNA, or a cDNA or genomic DNA library. The library screen can then be carried out using the isolated probe. The DNA molecules encoding the M. vaccae antigens can also be isolated by screening an appropriate M. vaccae expression library with an antiserum (e.g., rabbit or monkey) that specifically elevates against the M. vaccae antigens. Regardless of the method of preparation, the antigens described herein have the ability to induce an immunogenic response. More specifically, the antigens have the ability to induce cell proliferation and / or cytokine production (e.g., the production of interferon-α and / or interleukin-12) in T cells, NK cells, B cells or macrophages derived from a person immune to M. tuberculosis. A person immune to M. tuberculosis is one that is considered to be resistant to the development of tuberculosis due to having mounted an effective T cell response to M. tuberculosis. These individuals can be identified based on an intensely positive intradermal skin test response (i.e., greater than approximately 10 mm diameter duration) to tuberculosis proteins (PPD) and an absence of any of the symptoms of the infection of tuberculosis. Assays for cell proliferation or cytokine production in T cells, NK cells, B cells or macrophage can be carried out for example using the methods described below. The selection of the type of cell to be used to evaluate an immunogenic response to an antigen will depend on the desired response. For example, the production of interleukin-12 is more easily evaluated using preparations containing T cells, NK cells, B cells and macrophages derived from persons immune to M. tuberculosis, can be prepared using the methods well known in the art. technique. For example, a preparation of peripheral blood mononuclear cells (PBMCs) can be employed without further separation of the component cells. PBMCs can be prepared, for example, using density centrifugation through Ficoll ™ (Winthrop Laboratories, NY). T cells for use in the assays described herein can be purified directly from PBMCs. Alternatively, an enriched T cell line reactive against mycobacterial proteins or T cell clones reactive to individual mycobacterial proteins can be employed, of course. These T cell clones can be generated for example by culturing the PBMCs of persons immune to M. tuberculosis with mycobacterial proteins for a period of 2 to 4 weeks. This allows the expansion of only the T cells specific for mycobacterial protein, resulting in a line composed solely of these cells. These cells can then be cloned and tested with individual proteins using methods well known in the art, in order to more accurately define the T cell individual specificity. Generally, regardless of the method of preparation, the polypeptides disclosed in the present they are prepared in an isolated, essentially pure form. Preferably, the polypeptides are at least about 80% pure, more preferably at least about 90% pure and most preferably at least about 99% pure. In certain preferred embodiments that are described in detail below, the essentially pure polypeptides are incorporated into the pharmaceutical compositions or vaccines for use in one or more of the methods disclosed herein. The present invention also provides fusion proteins comprising a first and a second polypeptide of the invention or alternatively, a polypeptide of the present invention and a known M. tuberculosis antigen, such as the 38 kDa antigen described in the Andersen article and Hansen, Infect.

Jiranun 57: 2481-2488, 1989, together with the variants of these fusion proteins. The fusion proteins of the present invention also include a peptide binding between the first and second polypeptides. A DNA sequence encoding a fusion protein of the present invention is constructed using known recombinant DNA techniques to assemble the separated DNA sequences encoding the first and second polypeptides into an appropriate expression vector. The 3 'end of a DNA sequence encoding the first polypeptide is ligated, with or without a peptide linker to the 5' end of a DNA sequence encoding the second polypeptide so that the reading frames of the sequences remain in phase to allow the transfer of mRNA from the two DNA sequences into a single fusion protein that retains the biological activity of both the first and second polypeptides. A peptide linker sequence can be used to separate the first and second polypeptides by a sufficient distance to ensure that each polypeptide is bent into its secondary and tertiary structures. This sequence of the peptide linker is incorporated into the fusion protein using standard techniques well known in the art. The appropriate peptide linker sequences can be selected based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) its inability to adopt a secondary structure that could interact with the functional epitopes in the first and second polypeptides; and (3) the lack of hydrophobic or charged residues, which could react with the functional epitopes of the polypeptide. Preferred peptide linker sequences contain residues of Gly, Asn and Ser. Other almost neutral amino acids such as Thr and Ala ("can also be used in the linker sequence." The amino acid sequences that can be usefully employed as linkers include those disclosed in the Maratea et al. article, Gene 40: 39-46, 1985, Murphy et al., Proc. Nati, Acad. Sci. USA 33: 8258-8262, 1986; North American Patent Number 4,935,233 and the North American Patent Number 4,751,180. The linker sequence may be from 1 to about 50 amino acids in length. Peptide linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent steric interference. The ligated DNA sequences encoding the fusion proteins are cloned into appropriate expression systems using known techniques for those people who know the technique.

As detailed below, the inventors have demonstrated that heat-killed M. vaccae, DD-M. vaccae and recombinant M. vaccae proteins of the present invention can be used to activate T cells and NK cells; to stimulate the production of cytokines (in particular the class of Thl cytokines) in human PBMC; to improve the expression of co-stimulatory molecules in dendritic cells and monocytes (thus improving activation); and to improve the maturation and function of the dendritic cell. In addition, the inventors have demonstrated similarities between the immunological properties of the M. vaccae protein of the invention GV-23 and those of two known Thl-inducing adjuvants. GV-23 can thus be used in the treatment of diseases that involve improving a Thl immune response. Examples of these diseases include allergic diseases (eg, asthma and eczema), autoimmune diseases (eg, systemic lupus erythematosus) and infectious diseases (eg, tuberculosis and leprosy). In addition, GV-23 can be used as a dendritic cell or an NK cell enhancer in the treatment of immune deficiency disorders such as HIV, and to improve immune responses and cytotoxic responses to for example, malignant cells in cancer and following immunosuppressive cancer therapies such as chemotherapy. For use in the therapeutic methods of the invention, inactivated M. vaccae, the M. vaccae culture filtrate, modified M. vaccae cells, M. vaccae polypeptide, the fusion protein (or polynucleotides encoding these polypeptides or fusion) is generally present within a pharmaceutical composition or a vaccine. The pharmaceutical compositions may comprise one or more components that are selected from the group consisting of inactivated M. vaccae cells, M. vaccae culture filtrate, modified M. vaccae cells or compounds present in or derived from M. vaccae and / or its culture filtrate, together with a physiologically acceptable carrier. The vaccines may comprise one or more of the components that are selected from the group consisting of inactivated M. vaccae cells, M. vaccae culture filtrate, modified M. vaccae cells and compounds present in or derived from M. vacce and / or its culture filtrate, together with a non-specific immune response amplifier. These pharmaceutical compositions and vaccines may also contain other mycobacterial antigens either as discussed above, which are incorporated into a fusion protein or which are present within a separate polypeptide.

Alternatively, a vaccine of the present invention may contain DNA coding for one or more of the polypeptides as described above / such that the polypeptide is generated in itself. In these vaccines DNA may be present within any of a variety of delivery systems known to those skilled in the art including nucleic acid expression systems, bacterial and viral expression systems. Suitable nucleic acid expression systems contain the DNA sequences necessary for expression in the patient (with an appropriate promoter and a terminator signal). The bacterial delivery system involves the administration of a bacterium (such as Bacillus-Calmette-Guerin) that expresses an immunogenic portion of the polypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., a vaccine or other pox virus, retrovirus, or adenoviruts), which may involve the use of a nonpathogenic virus competent or replication defective. Techniques for incorporating DNA into these expression systems are well known in the art. The DNA can also be "naked" as described for example, in the article by Ulmer et al., Science 259: 1745-1749, 1993 and reviewed by Cohen, Science 259: 1691-1692, 1993. Acceptance of naked DNA can increase by coating the DNA in biodegradable beads, which are transported efficiently to the cells. A DNA vaccine as described above may be administered simultaneously or sequentially with any polypeptide of the present invention or a mycobacterial antigen known as 38 kDa antigen described above. For example, administration of DNA encoding a polypeptide of the present invention can be followed by administration of an antigen in order to improve the protective immune effect of the vaccine. The routes and frequency of administration, as well as the dosage, will vary from person to person and may be parallel to those currently being used in immunization using BCG. In general, the pharmaceutical compositions and vaccines can be administered by injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. They can be administered between 1 and 3 doses during a period of 1 to 36 weeks. Preferably, 3 doses are administered at intervals of 3 to 4 months, and booster vaccines can be periodically provided. Alternative protocols may be appropriate for individual patients. An appropriate dose is an amount of polypeptide or DNA which, when administered as described above, is capable of raising an immune response to a patient sufficiently to protect the patient from mycobacterial infection for at least 1 year. to 2 years. In general, the amount of polypeptide present in a dose (or produced in situ by DNA in a dose) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 pg. Appropriate dose sizes will vary with the size of the patient, but will typically vary from about 0.1 ml to about 5 ml. In one embodiment, the pharmaceutical composition or vaccine is in an appropriate form to be delivered to mucosal surfaces of the airways leading to or into the lungs. For example, the pharmaceutical composition or vaccine can be suspended in a liquid formulation to be delivered to a patient in an aerosol form or by means of a nebulization device similar to those currently used in the treatment of asthma. In other modalities, the pharmaceutical composition or vaccine is in an appropriate form for administration by injection (intracutaneous, intramuscular, intravenous or subcutaneous) or orally. Although any suitable carrier known to those skilled in the art can be employed in the pharmaceutical compositions of the invention, the type of carrier will depend on the suitability for the selected route of administration. For parenteral administration, such as a subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a lipid, a wax or a stabilizer. For oral administration, any of the aforementioned carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) can also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109. Any of a variety of adjuvants can be employed in the vaccines of this invention to non-specifically enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bor etella pertussi, M. tuberculosis, or , as discussed below, M. vaccae. Suitable adjuvants can be obtained commercially, for example, from Freund's 5 Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, MI), and Merck Adjuvant 65 (Merck and Company, Inc., from Rahway, NJ). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and Quil A. ^ In another aspect, this invention provides methods for using one or more of the polypeptides of the invention to diagnose tuberculosis using a skin test. As used herein, a "skin test" in any test carried out directly in a patient in whom the delayed-type hypersensitivity reaction (DTH) (such as swelling, redness or dermatitis) is measured after the intradermal injection of one or more of the polypeptides as described above. Preferably, the The reaction is measured at least 48 hours after the injection, more preferably 48 to 72 hours. The DTH reaction is a cell-mediated immune response that is greatest in patients who have been previously exposed to the test antigen (ie, the Immunogenic portion of the polypeptide employed, or a variant thereof). The response can be measured visually using a regulator. In general, 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 a skin test, the polypeptides of the present invention are preferably formulated as pharmaceutical compositions containing a polypeptide and a physiologically acceptable carrier, as described above. These compositions typically contain one or more of the aforementioned polypeptides in an amount ranging from about 1 μg to about 100 μg, preferably from about 10 to about 50 μg in a volume of 0.1 ml. Preferably, the carrier employed in these pharmaceutical compositions is a saline solution with appropriate preservatives such as phenol and / or Tween 80 ™. In a preferred embodiment, a polypeptide employed in a skin test is of sufficient size such that it remains at the site of injection throughout the duration of the reaction period. Generally, a polypeptide that is at least 9 amino acids in length is sufficient. The polypeptide is also preferably disintegrated by macrophages or dendritic cells within hours of injection to allow presentation to T cells. These polypeptides may contain repeats of one or more of the above-cited sequences or other immunogenic or non-immunogenic sequences. In another aspect, methods are provided for detecting mycobacterial infection in a biological sample using one or more of the polypeptides of the invention, either alone or in combination. In embodiments in which multiple polypeptides are employed, polypeptides other than those specifically described herein can be included, such as the 38 kDa antigen described above. As used herein, a "biological sample" is any sample that contains an antibody obtained from a patient. Preferably, the sample is whole blood, a sputum, serum, plasma, saliva, cerebrospinal fluid or urine. Most preferably, the sample is a sample of blood, serum or plasma that is obtained from a patient or a blood supply. The polypeptide (s) is used in an assay, as will be described below, to determine the presence or absence of antibodies to the polypeptide (s) in the sample, relative to a predetermined cut-off value. The presence of these antibodies indicates the presence of mycobacterial infection.

In the embodiment in which more than one polypeptide is employed, the polypeptides used are preferably complementary (ie, a component polypeptide will tend to detect infection in samples where the infection would not be detected by another component polypeptide). The complementary polypeptides can usually be identified using each polypeptide individually to evaluate serum samples obtained from a series of patients known to be infected with a Mycobacterium. After determining which of the samples have a positive test (as will be described below) with each polypeptide, combinations of two or more polypeptides can be formulated which are capable of detecting the infection in the majority, but in all, the samples tested. For example, approximately 25% to 30% of the serum of persons infected with tuberculosis are negative for antibodies to a single protein, such as the 38 kDa antigen mentioned above. The complementary polypeptides can therefore be used in combination with the 38 kDa antigen to improve the sensitivity of a diagnostic test. A variety of assay formats employing one or more polypeptides to detect antibodies in a sample are well known in the art. See, e.g., Harlow and Lane, Antibiotics: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In a preferred embodiment, the assay involves the use of a polypeptide immobilized on a solid support to bind to and remove the antibody from the sample. The ligated antibody can then be detected using a detection reagent containing a reporter group. Appropriate detection reagents include antibodies that bind to the antibody / polypeptide complex and free polypeptide labeled with a reporter group (e.g., in a semi-competitive assay). Alternatively, a competitive assay can be used, in which an antibody that binds to the polypeptide is labeled with a reporter group that is allowed to bind to the immobilized antigen after incubation of the antigen with the sample. The degree to which the components of the sample inhibit the binding of the labeled antibody in the polypeptide is indicative of the reactivity of the sample with the immobilized polypeptide. The solid support can be any solid material to which the antigen can be fixed. Suitable materials are well known in the art. For example, the solid support can be a test well in a microtiter plate or a microcellulose or other appropriate membrane. Alternatively, the support may be an account or disk, such as a glass, fiberglass, latex or a plastic material such as polystyrene or polyvinyl chloride. The support can also be a magnetic particle or an optical fiber sensor, such as those disclosed / for example, in U.S. Patent Number 5,359, 681. The polypeptides can be ligated to the solid support using a variety of well-known techniques. the bouquet In the context of the present invention, the term "ligate" refers to the non-covalent association, such as adsorption, and covalent attachment, which may be a direct link between the antigen and the functional groups on the support or a bond to through an entanglement agent. Bonding by adsorption in a well in a microtiter plate or membrane is preferred. In these cases, adsorption can be achieved by contacting the polypeptide, in an appropriate stabilizing agent, with the solid support for an appropriate amount of time. The contact time varies with temperature, but is typically between about 1 hour and 1 day. Generally, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinyl chloride) with an amount of the polypeptide ranging from about 10 ng to about 1 μg, and preferably about 100 ng, is enough to bind an adequate amount of antigen. The covalent attachment of the polypeptide to a solid support can usually be achieved by first reacting the support with a bifunctional reagent that will react both with the support and a functional group, such as a hydroxyl or amino group, in the polypeptide. For example, the polypeptide can be ligated to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen in the polypeptide (see, eg, Pierce Immunotechnology Catalog and Handbook, 1991, in A12-A13). In certain embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA). This assay can be carried out by first contacting a polypeptide antigen that has been immobilized on a solid support with the sample, such that antibodies to the polypeptide within the sample are allowed to bind to the immobilized polypeptide. The unbound sample is then removed from the immobilized polypeptide and a detection reagent capable of binding to the immobilized antibody-polypeptide complex is added. The amount of the detection reagent remaining bound to the solid support is then determined using an appropriate method for the specific detection reagent. More specifically, once the polypeptide is immobilized on the support as described above, the remaining protein binding sites in the site are typically blocked. Any suitable blocking agent known to those skilled in the art such as bovine serum albumin or Tween 20 ™ (Sigma Chemical Co., of St. Louis, O) can be employed. The immobilized polypeptide is then incubated with the sample, an antibody is allowed to bind to the antigen. The sample can be diluted with an appropriate diluent, such as phosphate stabilized salt (PBS) before incubation. In general, an appropriate contact time, or incubation time, is that period of% time that is sufficient to detect the presence of antibody within a sample infected with M. tuberculosis. Preferably, the contact time is sufficient to achieve a binding level that is at least 95% of that achieved at equilibrium between the bound and unbound antibody. The time needed to achieve equilibrium can be easily determined by testing the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient. The unbound sample can be removed by washing the solid support with an appropriate stabilizer, such as PBS containing 0.1% Tween 20 ™. The detection reagent can then be added to the solid support. An appropriate detection reagent in any compound that binds to the immobilized antibody-polypeptide complex and that can be detected by any of a variety of means known in the art. Preferably, the detection reagent contains a binding agent (such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen) that is conjugated to a reporter group. Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. The conjugation of the binding agent to the reporter group can be achieved using the normal methods known in the art. Common binding agents can also be purchased conjugates to a variety of reporter groups from many commercial sources (e.g., Zymed Laboratories, San Francisco, CA, and Pierce, Ockford, IL). The detection reagent is incubated with the immobilized antibody-polypeptide complex for a sufficient amount of time to detect the bound antibody. An appropriate amount of time can usually be determined from the manufacturer's instructions or by testing the level of binding that occurs over a period of time. The unbound detection reagent is then removed and the bound detection reagent is detected using the reporter group. The method used to detect the reporting group depends on the nature of the reporting group. For radioactive groups, methods of scintillation or autoradiographic counting are generally appropriate. Spectroscopic methods can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin, coupled with a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups can be detected by addition on a substrate (usually for a specific period), followed by spectroscopic analysis or other analysis of the reaction products. To determine the presence or absence of the anti-mycobacterial antibodies in the sample, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal corresponding to a predetermined cut-off value. In a preferred embodiment, the cut-off value is an average average signal obtained when the immobilized antigen is incubated with samples from an uninfected patient. In an alternative preferred embodiment, the cutoff value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A basic Science for Clinical Medicine, Little Brown and Co., 1985, pages 106- 107 In general, signals higher than the predetermined cutoff value are considered as positive for mycobacterial infection. The assay can also be carried out in a rapid through flow or strip test format, wherein the antigen is immobilized on a membrane, such as nitrocellulose. In the through-flow test, antibodies within the sample bind to the immobilized polypeptide as the sample passes through the membrane. A detection reagent (e.g., protein A-colloidal gold) is then ligated to the antibody-polypeptide complex as the solution containing the detection rectifier flows through the membrane. The detection of the bound detection reagent can then be carried out as described above. In the strip test format, one end of the membrane to which the polypeptide binds is immersed in a solution containing the sample. The sample is migrated along the membrane through a region containing the detection reagent and the area of the immobilized polypeptide. The concentration of the detection reagent in the polypeptide indicates the presence of antimicrobial antibodies in the sample. Typically, the concentration of the detection reagent in the site generates a pattern, such as a line that can be read visually. The absence of this pattern indicates a negative result. Generally, the amount of the polypeptide immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of antibodies that would be sufficient to generate a positive signal in an ELISA, as discussed above. Preferably, the amount of the polypeptide immobilized on the membrane ranges from about 25 ng to about 1 μg and more preferably from about 50 ng to about 500 ng. These tests can typically be carried out with a very small amount (e.g., one drop) of the patient's serum or blood. There are numerous other assay protocols that are suitable for use with the polypeptides of the present invention. The aforementioned descriptions are intended to be exemplary only.

The present invention also provides antibodies to the polypeptides of the invention. The antibodies can be prepared by any of a variety of techniques known to those skilled in the art. See, eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one of these techniques / an immunogen comprising the antigenic polypeptide is initially injected into any of a plurality of mammals (eg, mice, rats). , rabbits, sheep and goats). The immunogen is injected into the host animal, preferably according to a predetermined schedule that incorporates one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide can then be purified from these antisera by, for example, affinity chromatography using the polypeptide coupled with an appropriate solid support. Monoclonal antibodies specific for the antigenic polypeptide of interest can be prepared for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6: 511-519, 1976, and the improvements to it. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity in the polypeptide of interest). These cell lines can be produced, for example, from spleen cells that are obtained from an immunized animal as described above. Spleen cells can then be immortalized by fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal, using one of a variety of techniques well known in the art. The monoclonal antibodies can be isolated from the supernatants of the resulting hybridoma colonies. In addition, various techniques can be employed to improve performance, such as an injection of the hybridoma cell line into the peritoneal cavity of an appropriate vertebrate host, such as a mouse. The monoclonal antibodies can then be harvested from the fluid or blood. The antibodies can be used in diagnostic tests to detect the presence of mycobacterial antigens using assays similar to those detailed above and other techniques well known to those skilled in the art, thus providing a method for detecting mycobacterial infection. , such as M. tuberculosis infection, in a patient.

The diagnostic reagents of the present invention may also comprise polynucleotides that encode one or more of the aforementioned polypeptides or one or more portions thereof. For example, primers comprising at least 10 continuous oligonucleotides of a polynucleotide of the invention can be used in tests based on polymerase chain reaction (PCR). Similarly, probes comprising at least 18 contiguous oligonucleotides of the polynucleotide of the invention can be used to hybridize to specific sequences. The techniques for both PCR-based tests and hybridization tests are well known in the art. The primers or tests can therefore be used to detect M. tuberculosis and other mycobacterial infections in biological samples preferably sputum, blood, serum, saliva, cerebrospinal fluid or urine. DNA test probes or primers comprising the oligonucleotide sequences described above can be used alone, in combination with one another or with previously identified sequences such as the 38 kDa antigen discussed above . The word "approximately", when used in this application with reference to a percentage by weight of the composition, proposes a variation up to units of percentage of 10 of the percentage shown. When used with reference to the percentage identity or percentage probability, the word "approximately" proposes a variety of up to one unit of one percent of the percentage shown. The following examples are offered by way of illustration and not by way of limitation. EXAMPLE 1 EFFECT OF IMMUNIZATION OF MICE WITH AF. VACCAE IN TUBERCULOSIS This example illustrates the effect of immunization with heat-killed M. vaccae or culture filtrate of M. vaccae in mice before challenge with Af. active tuberculosis. M. vaccae (ATCC number 15483) was cultured in a Sterile medium 90 (yeast extract, 2.5 g / 1, tryptone, 5 g / 1, glucose, 1 g / 1) at 37 ° C. The 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 granulate the bacteria, and the culture filtrate was removed. The bacterial granule was resuspended in phosphate-stabilized saline at a concentration of 10 mg / ml, equivalent to IO1 ^ Af organisms. vaccae for me. The cell suspension was then subjected to autoclaving for 15 minutes at 120 ° C. The culture filtrate was passed through a 0.45 μm filter to sterile bottles. As shown in Figure 1A, when mice immunized with 1 mg, 100 μg or 10 μg of M. vaccae were infected three weeks later with 5x10 ^ colony forming units (CFU) or active M. tuberculosis H37Rv, a significant protection from infection. In this example, the spleen, liver and lung tissue were harvested from the mice three weeks after infection, and the active bacilli were determined (expressed as CFU). The reduction in the numbers of bacilli, when compared to the tissue of unimmunized control mice, exceeded 2 plots in liver and lung tissue, and a diagram in the spleen tissue. Immunization of mice with heat-killed M. tuberculosis H37Rv had no significant protective effects in mice subsequently infected with M. tuberculosis active H37Rv. Figure IB shows that when the mice were immunized with 100 μg of the M. vaccae culture filtrate, and were infected three weeks later with 5x10 ^ CFU of M. tuberculosis H37Rv, significant protection was also seen. When the spleen, liver and lung tissue were harvested from mice three weeks after infection, and the numbers of active bacilli (CFU) were determined, a reduction of 1-2 diagrams in numbers was observed compared to the control mice not immunized. EXAMPLE 2 EFFECT OF INTRADERMICA AND INTRA-PULMONARY VIAS OF IMMUNIZATION WITH M. VACCAE IN TUBERCULOSIS IN POCKET MONOS This example illustrates the effect of immunization with M. vaccae removed by heat or culture filtrate. v. M. vaccae through intradermal and intrapulmonary pathways in cynomolgus monkeys before challenge with active M. tuberculosis. M. vaccae removed by heat and the culture filtrate of M. vaccae were prepared as described above in Example 1. Five groups of cynomolgus monkeys were used, with each group consisting of 2 monkeys. Two groups of monkeys were immunized with whole M. vaccae removed by heat either intradermally or intrapulmonary; two groups of monkeys were immunized with M. vaccae culture filtrate either intradermally or intrapulmonary; and a control group did not receive immunizations. All immunogens were dissolved in phosphate-stabilized saline. The composition used for immunization, the amount of immunogen and the route of administration for each group of monkeys are given in Table 1. Prior to immunization, all monkeys were weighed (weight in kg), body temperature was measured (temp), and a blood sample was taken to determine the erythrocyte sedimentation rate (ESR mm / hr) and lymphocyte proliferation (LPA) to a challenge in vi tro with purified protein (PPD) prepared from Mycobacterium bovis. Both the ESR and LPA have been used as indicators of the responses of the inflammatory T cell. On day 33 of post-immunization, these measurements were repeated. On day 34, all monkeys received a second immunization using the same amount of M. vaccae and immunization route as the initial immunization. On day 62, body weight, temperature, ESR and LPA were measured to PPD, and then all monkeys were infected with 10 ^ colony forming units of the Erdman strain of Mycobacterium tuberculosis by inserting the organisms directly into the lungs rights of immunized animals. Twenty-eight days after infection, body weight, temperature, ESR and LPA to PPD were measured in all monkeys, and the lungs were subjected to x-rays to determine if infection with active M. tuberculosis had resulted the initiation of pneumonia.

TABLE 1 COMPARISON OF INTRADERMIC AND INTRAPULMONARY ADMINISTRATION ROUTES V No. of No. of Via Amount of Immunogenic Immunogenicity Group Mono 10 S3101-E 0 (Controls) 3144-B 0 4080-B 500 μg intradermal (Immunized with 3586-B 500 μg intradermal V 15 M. vaccae removed by heat) 3534-C 500 μg intrapulmonary (Immunized with 3160-A 500 μg intrapulmonary 20 M. vaccae removed by heat) (Immunized 3564-B 100 μg intradermal with filtered 3815-B 100 μg intradermal culture) (Immunized 4425-A 100 μg intrapulmonary 30 with filtered 2779-D 100 μg intrapulmonary culture) The results of these studies are given below in Tables 2A-E and are summarized below: Table 2A - Twenty-eight days after the Infection with Erdman tuberculosis, the chest x-rays of the control monkeys (not immunized) revealed nebulosity through the right suprahilar regions of both animals indicating the initiation of pneumonia. This progressed and by day 56 post-infection the x-rays indicated disease 5 in both lungs. As expected, as the disease progressed, both control animals lost weight and showed significant LPA responses to PPD, indicating intense T cell activity to M. tuberculosis. The ESR measures were variable but rs 10 compatible with the intense immune reactivity. Table 2B - The two monkeys immunized twice with 500 μg of M. vaccae intradermally did not show signs of lung disease 84 days post-infection with M. tuberculosis. The responses of LPA to PPD indicated that 15 had immune reactivity to M. tuberculosis, and both animals continued to gain weight, a consistent indication of a lack of disease. Table 2C - The two monkeys immunized twice with 500 μg of intrapulmonary M. vaccae showed almost identical results to those animals of Table 2B. There were no signs of lung disease 84 days post-infection with M. tuberculosis, with consistent weight gains. Both animals showed response of LPA to PPD in the immunization phase (day 0-62) and post-infection, indicating intense T-cell reactivity that had developed as a result of using the lung as the route of immunization and infection subsequent Immunization twice with 500 μ? of M. vaccae whole has consistently shown protective effects against subsequent infection with active M. tuberculosis. The data presented in Tables 2D and 2E show the effects of immunization with 100 μg of culture filtrate of M. vaccae. Monkeys immunized intradermally showed signs of developing the disease 84 days post-infection, whereas in those immunized intrapulmonally, one animal showed the disease after 56 days and one animal showed the disease 84 days post-infection. This was a significant delay in the initiation of the disease indicating that the immunization process had resulted in some protective immunity.

TABLE 2A MONOS DE CONTROL ID # Days Weight Temp. ESR LPA LPA Observe Mm / hr PPD10 PPDl Kgs x ray S3101 E 0 2.17 37.0 0 0.47 1.1 Negative 34 1.88 37.3 ND 0.85 1.4 ND 62 2.02 36.0 ND 1.3 1.5 ND - > Infection Time 28 2.09 38.0 2 1.3 3.7 Positive 56 1.92 37.2 20 5.6 9.1 Positive 84 1.81 37.5 8 4.7 5.6 Positive 121 MURIERON ID # Days Weight Temp. ESR LPA LPA Observe Mm / hr PPD PPD Kgs? Μ? X-rays 3144-B 0 2.05 36.7 0 0.87 1.8 Negative 34 1.86 37.6 ND 2.2 1.4 ND 62 1.87 36.5 ND 1.6 1.6 ND ? Infection Time 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 MURIERON ND = Not Done TABLE 2B IMMUNIZED MONKEYS WITH M VACCAE WHOLE ELIMINATED BY HEAT (500 μg) INTRADERMIC ID # Days Weight Temp. ESR LPA LPA Observe Mm / hr PPD PPD Kgs 10 \ ig? ? X-rays 4080-B 0 2.05 37.1 1 1.1 0.77 Negative 34 1.97 38.0 ND 1.7 1.4 ND 62 2.09 36.7 ND 1.5 1.5 ND ? Infection Time 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 MURIERON ID # Days Weight Temp. ESR LPA LPA Observe mm / hr PPD PPD Kgs? Μ? i g x rays 3586-B 0 2.29 37.0 0 1.1 1.4 Negative 34 2.22 38.0 ND 1.9 1.6 ND 62 2.39 36.0 ND 1.3 1.6 ND ? 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 NURIERON ND = Not Done TABLE 2C IMMUNIZED MONKEYS WITH M VACCAE ENTIRE ELIMINATED BY HEAT (500 μg) INTRAPULMONAR ID # Days Weight Temp. ESR LPA LPA Observa¬ V, in mm / hr PPD PPD Kgs 10μ¾ ^ g x-ray 3534-C 0 2.15 36.8 0 1.7 1.3 Negative 34 2.00 37.8 ND 4.4 1.4 ND 62 2.13 36.4 ND 3.2 1.9 ND fifteen ? Time of Infection 28 2.38 37.7 0 1.9 2.6 Negative l 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 disease Negative lung 25 ID # Days Weight Temp. ESR LPA LPA Observe mm / hr PPD PPD Kgs l (^ g iMg x-ray 3160-A 0 2.17 37.3 0 1.2 0.79 Negative 34 1.98 37.1 ND 3.9 7.8 ND 35 62 2.17 36.9 ND 1.7 2.4 ND - »Time of Infection 28 2.38 37.7 0 1.9 2.6 Negative 40 56 2.42 37.8 0 5.3 4.7 Negative 84 2.46 37.1 1 3.1 3.2 Negative 45 Stable lung disease Positive CHART 2D IMMUNIZED MONKEYS WITH CULTIVATION FILTRATION (100 μg) INTRADERMIC IDtt Days Weight Temp. ESR LPA LPA Observe mm / hr PPD PPD Kgs 10μ5 x rays 3564-B 0 2.40 37.2 0 1.4 1.4 Negative 34 2.42 38.1 ND 3.3 2.7 ND 62 2.31 37.1 ND 3.1 3.4 ND - > 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 Died ID # Days Weight Temp. ESR LPA LPA Observe mm / hr PPD PPD Kgs? Μ? X-rays 3815-B 0 2.31 36.3 0 1.0 1.4 Negative 34 2.36 38.2 ND 1.9 2.0 ND 62 2.36 36.4 ND 3.7 2.8 ND - Infection time 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 ND = Not Done TABLE 2E IMMUNIZED MONKEYS WITH CULTURE FILTRATION (100 μg) INTRAPULMONARY ID # Days Weight Temp. ESR LPA LPA Observe mm / hr PPD PPD Kgs lOMg? Μ? X-rays 4425-A 0 2.05 36.0 0 0.35 1.2 Negative 34 2.0 37.6 ND 3.0 2.4 ND 62 2.11 37.6 ND 2.2 1.6 ND - Infection time 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 Disease. of stable lung Positive ID # Days Weight Temp. ESR LPA LPA Observe mm / hr PPD PPD Kgs 10μ5? Μ? X-rays 3160-A 0 2.56 38.6 2 1.9 1.4 Negative 28 2.55 37.9 ND 0.78 1.1 ND 56 2.69 38.4 ND 1.3 1.5 ND? Infection Time 56 2.25 39.0 24 ND ND Positive 96 They died ND = Not Done EXAMPLE 3 EFFECT OF IMMUNIZATION WITH M. VACCAE ASTHMA IN THE MICE This example demonstrates that both M. vaccae removed by heat and DD-M. vaccae, when administered to mice through the intranasal route, were able to inhibit the development of the allergic immune response in the pulomons. This was demonstrated in a mouse model of asthma-like allergen-specific lung disease. The seriousness of this allergic disease is reflected in the large numbers of eosinophils that accumulate in the lungs. C57BL / 6J mice were provided with 2 μg of ovalbumin in 100 μ? of the alum adjuvant by the intraperitoneal route for the time of 0 and 14 days, and were subsequently administered with 100 μg of ovalbumin in 50 μ? of phosphate-stabilized saline (PBS) by the intranasal route on day 28. The mice accumulated eosinophils in their lungs as detected by washing the airways of mice anesthetized with saline, collecting washings (bronchiolar lavage or BAL), and counting the numbers of eosinophils. As shown in Figures 2A and B, groups of seven mice were administered with either 10 or 1000 μg of M. vaccae removed by heat (Figure 2A), or 10, 100 or 200 μg of? -? - vaccae, prepared as described below (Figure 2B) intranasally 4 weeks before the intranasal challenge with ovalbumin, had reduced the percentages of eosinophils in the BAL cells collected 5 days after challenge with ovalbumin compared to control mice. Control mice were provided with intranasal PBS. M. bovis activA BCG at a dose of 2 x 105 colony forming units also reduced eosinophilia of the lung. The data in Figures 2A and B show the medium and the SEM per group of mice. Figures 2C and D show that the mice that were administered with either 1000 μg of M. vaccae removed by heat (Figure 2C) or 200 μg of ?? - ?. Vaccinate (Figure 2D) intranasally as late as well as one week before challenge with ovalbumin had reduced percentages of eosinophils compared to control mice. In contrast, treatment with BCG active a week prior to the challenge with ovalbumin did not inhibit the development of lung eosinophilia when compared with the control mice. As shown in Figure 2E, immunization with either 1 mg of ?? vaccae removed by heat or 200 μg of ?? - ?. vaccae, which was administered either intranasally (i.n.) or subcutaneously (s.c.), reduced eosinophilia after challenge with ovalbumin when compared to control animals administered PBS. In the same experiment, immunization with BCG with the Pasteur (BCG-P) and Connought (BCG-C) strains before challenge with ovalbumin also reduced the percentage of eosinophils in the BAL of the mice. Eosinophils are blood cells that are prominent in the airways in allergic asthma. The secreted products of eosinophils contribute to the swelling and inflammation of the lining of the mucosa of the respiratory tract in allergic asthma. The data shown in Figures 2A-E indicate that treatment with M. vaccae eliminated by heat or UD-M. vaccae reduces the accumulation of eosinophils in the lung, and may be useful in reducing the inflammation associated with eosinophilia in the respiratory tract, nasal mucosa, and upper respiratory tract. DD-M. vaccae drained of mycolic acids and arabinogalactana The mycolic acids were depleted of DD-M. Vaccinate by treatment with potassium hydroxide (0.5% KOH) in ethanol for 48 hours at 3 ° C. Mycolic acid depleted DD-M. vaccum and the cells were then washed with ethanol and ether and dried. The arabinogalactans were depleted of KOH treated with ?? - ?. Vaccinate by additional treatment with 1% periodic acid in 3% acetic acid for 1 hour at room temperature followed by treatment with 0.1M sodium borohydride for 1 hour at room temperature. After exhaustion of arabinogalactan, the samples were washed with water and lyophilized. As shown in Table 3, both the depleted mycolate of ?? - ?. vaccae as well as mycolic acid and arabinogalactana depleted the ??? vaccae, administered intranasally to mice sensitized with ovalbumin reduced with ovalbumin reduced the accumulation of eosinophils in the bronchoalveolar lavage fluid after challenge with ovalbumin. The administration of M. vaccae, eliminated by heat, DD-M. vaccae or DD-M. Vaccination depleted of mycolic acids and arabinogalactan, can therefore reduce the seriousness of asthma and diseases involving similar immune abnormalities, such as allergic rhinitis. In addition, serum samples were collected from the mice in the experiment shown in Figure 2E and antibodies to ovalbumin were measured by normal enzyme-linked immunoassay (EIA). As shown in Table 3A below, sera from mice infected with BCG had higher levels of ovalbumin-specific IgGl than sera from PBS controls. In contrast, mice immunized with M. vaccae or DD-M. vaccae had similar or more low levels of ovalbumin-specific IgGl. Since IgG1 antibodies are characteristic of a Th2 immune response, these results are compatible with the suppressive effects of Af. vaccae removed by heat and DD-M. Vaccinate in the Th2 immune responses that induce asthma. TABLE 3 LUNG EOSINOPHILIES DECREASED IN MICE TREATED WITH SOLD MYCOLIC ACID OF DD-M. VACCAE OR MYCOLIC ACID AND ARABINOGALACTANA SOLD OUT OF DD-M. VACCAE.

Treatment Group% of Eosinophils in BAL Medio S.E.M. PBS 58.8 8.4 Mycolic acid depleted of DD-M. Vacate 21.8 17.4 Mycolic acid and arabinogalactan depleted of DD-M. vaccae 16.8 0.3 Note: At least 7 mice per group.

TABLE 3A LEVELS OF SPECIFIC IgGl SERUM OF THE LOW ANTIGEN IN IMMUNIZED MICE WITH M. VACCAE ELIMINATED BY HEAT OR DD-M. VACCAE Serum IgGl Medium SEM Treatment Group M. vaccae i.n. 185.00 8.3 M. vaccae s.c. 113.64 8.0 DD-M. vaccae i.n. 96.00 8.1 DD-M. 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 IgGl was detected using anti-mouse IgGl (Serotec). The means of the group are expressed as the reciprocal of the valuation of the final point EU50. EXAMPLE 4 EFFECT OF IMMUNIZING MICE WITH PROTEINS FROM M. VACCAE, DD-M. VACCAE OR M. VACCAE RECOMBINANT IN TUBERCULOSIS This example illustrates the effect of immunization with M. vaccae proteins, heat-killed, DD-M. vacillate or recombinant M. vaccae without additional adjuvants, or a combination of heat-killed M. vaccae with a pool of recombinant proteins derived from M. vaccae. Mice were injected intraperitoneally with one of the following preparations on two separate occasions at three weeks: a) Saline stabilized with phosphate (PBS, control); b) M. vaccae removed by heat (500 μg); c) DD-AT. vaccae (50 μg); d) A pool of recombinant proteins containing 15 ug of each of the proteins GV4P, GV7, GV9, GV27B, GV33 (prepared as will be described below); and e) heat-killed M. vaccae plus puddle of recombinant proteins Three weeks after the last intraperitoneal immunization, the mice were infected with 5 X 105 live M. tuberculosis H37Rv organisms. After three additional weeks, the mice were sacrificed, and their spleens homogenized and tested for colony forming units (CFU) of M. tuberculosis as an indicator of seriousness or infection. Figures 3A and 3B show the data in which each point represents individual mice. The CFU numbers recovered from the control mice immunized with PBS were only taken as the baseline. All data from the experimental mice were expressed as the number of logarithms of the CFUs per '? below the baseline for the control mice (or diagram protection). As shown in Figure 3A, mice immunized with M. vaccae removed by heat or? - ?. vaccae showed an average reduction of > 1 or 0.5 CFU diagrams, respectively. As shown in Figure 3B, the spleens of the mice immunized with the pool of recombinant proteins containing GV4P, GV7, GV9, GV27B and GV33, had slightly lower CFU than the control mice. However, when GV4P, GV7, GV9, GV27B and GV33 were administered in combination with heat-killed M. vaccae, the CFU reduction exceeded a means of > 1.5 diagrams The data demonstrates the efficacy of immunization with M. vaccae, ?? - ?. vaccae or recombinant proteins derived from M. vaccae against subsequent infection with tuberculosis, and also indicates that M. vaccae, ?? - ?. Vaccinate and recombinant proteins can be developed as vaccines against tuberculosis. EXAMPLE 5 EFFECT OF INTRADERMIC INJECTION OF MYCOBACTERIUM VACCAE ELIMINATED WITH HEAT IN PSORIASIS IN HUMAN PATIENTS 25 This example illustrates the effect of two intradermal injections of Mycobac er um vaccae eliminated by heat in psoriasis in human patients. M. vaccae (ATCC number 15483) was cultured in sterile medium 90 (yeast extract, 2.5 g / 1, tryptone, 5 g / 1, glucose, 1 g / 1) at 37 ° c. The cells were harvested by centrifugation, and transferred to sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 3 ° C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate was removed.The bacterial granule was re-suspended in phosphate-stabilized saline at a concentration of 10 mg / ml, equivalent to 10 * 0 M organisms. Vaccinate for me The cell suspension was autoclaved for 15 minutes at 120 ° C and stored frozen at -20 ° C. Before use the M. vaccae suspension was thawed, diluted to a concentration of 5 mg / ml in phosphate-stabilized saline, was treated in an autoclave for 15 minutes at 120 ° C and formed in aliquots of 0.2 20 milliliter under sterile conditions in small bottles for use in patients. Male and female, 15 to 61 years of age without other systematic diseases were admitted to the 25th treatment.Patient patients were not included.

Patients had PASI scores of 12-35. The PASI score is a measure of the location / size and degree of skin flakes in psoriatic lesions in the body. An earlier PASI score of 12 reflects widespread disease lesions in the body. The study began with a four-week washout period in which patients did not have systematic anti-psoriasis treatment or effective topical therapy. The 24 patients were then injected intradermally with 0.1 ml of M. vaccae (equivalent to 500 g). This was followed three weeks later with a second intradermal injection with the same dose of M. vaccae (500 μg). Psoriasis was assessed four weeks before the first injection of heat-killed M. vaccae twelve weeks after the first injection as follows: A. PASI scores were determined at -4, 0, 3, 6 and 12 weeks; B. The patient's questionnaires were completed at 0, 3, 6 and 12 weeks; and C. Psoriatic lesions and each patient were photographed at 0 # 3, 6, 9 and 12 weeks. The data shown in Table 4 describe the age, sex and clinical history of each patient.

TABLE 4 Patient Data in the Effect Study of M. vaccae in Psoriasis No. of Patient Age / Sex Duration of 1 Admission Code Disorder of the PASI Score PS-001 D.C. 49 / f 30 years 28.8 PS-002 E.S. 41 / F 4 months 19.2 PS-003 M.G. 24 / F 8 months 18.5 PS-004 D.B. 54 / M 2 years 12.2 PS-005 CE. 58 / F 3 months 30.5 PS-006 M.G. 18 / F 3 years 15.0 PS-007 L.M. 27 / M 3 years 19.0 PS-008 C.C 21 / F 1 month 12.2 PS-009 E.G 42 / F 5 months 12.6 PS-010 J.G 28 / M 7 years 19.4 PS-011 J.U 39 / M 1 year 15.5 PS-012 C.S 47 / M 3 years 30.9 PS-013 H.B 44 / M 10 years 30.4 PS-014 N. J 41 / M 17 years 26.7 PS-015 J.T 61 / F 15 years 19.5 PS-016 L.P 44 / M 5 years 30.2 PS-017 EN 45 / M 5 years 19.5 PS-018 EL 28 / F 19 years 16.0 PS-019 BA 38 / M 17 years 12.3 PS-020 PP 58 / F 1 year 13.6 PS-021 LI 27 / F 8 months 22.0 PS-022 AC 20 / F 7 months 26.5 PS-023 CA 61 / F 10 years 12.6 PS-024 FT 39 / M 15 years 29.5 All patients demonstrated a mild localized non-ulcerated induration at the injection site. No side effects were observed or claimed by patients. The data shown in Table 5, given below), are the skin reactions measured at the site of injection, 48 hours, 72 hours and 7 days after the first and second injections of M. vaccae removed by heat . The data shown in Table 6, presented below, are PASI and patient scores at the time of the first injection of M. vaccae (Day 0) and 3, 6, 9, 12 and 24 weeks later. It can be clearly seen that, by week 9 after the first injection of M. vaccae, 16 of the 24 patients showed a significant improvement in PASI scores. Seven of the fourteen patients who had completed 24 weeks of follow-up remained stable without clinical signs of new development of the serious disease. These results demonstrate the efficacy of multiple intradermal injections of inactivated M. vaccae in the treatment of 5 psoriasis. PASI scores less than 10 reflect healing of extensive lesions. The histopathology of the skin biopsies indicated that the normal skin structure is being restored. Only one of the first seven patients who have completed 28 weeks 10 of follow-up has had a relapse. v TABLE 5 Skin Reaction Measures in Millimeters First Injection Code Second Injection Time 48 hours 72 hours 7 days 48 hours 72 hours 7 days PS-001 12x10 12x10 lOx 8 15x14 15x14 10x10 r PS-002 18x14 20x18 18x14 16x12 18x12 15x10 PS-003 10x10 14x10 lOx 8 15x12 15x10 10x10 PS-004 14x12 22x18 20x15 20x20 20x18 14x10 PS-005 10x10 13x10 DNR DNR DNR DNR 30 PS-006 lOx 8 10x10 6x 4 12x10 15x15 lx 6 PS-007 15x15 18x16 12x10 15x13 15x12 12x10 PS-008 18x18 13x12 12x10 18x17 15x10 15x10 PS-009 13x13 18x15 12x 8 15x13 12x12 12x 7 PS-010 13x11 15x15 8x 8 12x12 12x12 5x5 PS-011 17x13 14x12 12x11 12x10 12x10 12x10 PS-012 17x12 15x12 9x 9 10x10 lOx 6 8x 6 PS-013 18x11 15x11 15x10 15x10 15x13 14x 6 PS-014 15x12 15x11 15x10 13x12 14x10 8x5 PS- 015 15x12 16x12 15x10 7x 6 14x12 6x 4 PS-016 6x 5 6x 6 6x 5 8x 8 9x 8 9x 6 PS- • 017 20x15 15x14 14x10 15x15 17x16 DN PS-|018 14x10 lOx 8 lOx 8 12x12 10x10 10x10 PS- 019 10x10 14x12 lOx 8 DNR 15x14 15x14 PS- 020 15x12 15x15 12x15 15x15 14x12 13x12 PS-|021 15x12 15x12 7x 4 11x10 11x10 llx 8 PS- 022 12x10 lOx 8 lOx 8 15x12 13x10 lOx 8 PS- 023 13x12 14x12 10x10 17x17 15x15 DNR PS- 024 10x10 10x10 lOx 8 10x8 8x 7 8x 7 DNR = Not reported TABLE 6 Current Clinical Status of Patients after Injection M. vaccae (PASI Scores) No. Day 0 Week Week Week Week Week of 3 6 9 12 24 Code 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 DNR 18.7 DNR DNR 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 DNR DNR DNR 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 M. vaccae subjected to autoclaving.

• ** Patient PS-012 was removed from the test, drug-induced dermatitis (penicillin) • *** Patient PS-014 was re-vaccinated • DNR = Not reported Patients treated with M. vaccae can achieve remission ( PASI score = 0). The remission or improvement of the PASI score can be long-lasting. For example, Patient PS-003 achieved remission through week 20 and is still in remission at week 80. In total 13 of the 24 patients showed a greater than 50% improvement in PASI scores. Patient PS-001 achieved remission at week 16, relapsed at week 48 (PASI 2.7), was re-vaccinated with M. vaccae injections and subsequently improved with PASI falling from 17.8 (Week 60) to 0.8 (week 84).

In this way patients can benefit from repeated treatment. EXAMPLE 6 EFFECT OF INTRADERMIC INJECTION OF DD-M. VñCCAE IN PSORIASIS IN HUMAN PATIENTS This example illustrates the effect of two intradermal injections of ?? - ?. vaccae in psoriasis. Seven voluntary psoriatic patients, male and female, aged 18 to 45 years without other systematic illnesses admitted to the treatment. Pregnant patients were not included. The patients had PASI scores of 12-24. As discussed above, the PASI score is a measure of the location, size, and degree of skin flaking in psoriatic lesions in the body. A PASI score of more than 12 reflects disease lesions widely disseminated in the body. The study began with a four-week washout period after all four patients had not undergone treatment for systemic psoriasis or effective topical therapy. The seven patients were then injected intradermally with 0.1 ml of DD-M. vaccae (equivalent to 100 μg). This was followed three weeks later with a second intradermal injection with the same dose of DD-M. Vacate (100 \ ig).

Psoriasis was assessed four weeks before the first injection of M. vaccae at six weeks after the first injection as follows: A. PASI scores were determined at -4, 0, 3 and 6 weeks; B. the patient's questionnaires were completed at 0, 3 and 6 weeks; and C. psoriatic lesions and each patient were photographed at 0 and 3 weeks. The data shown in Table 7 describe the age, sex and clinical history of each patient. TABLE 7 Patient Data in the Study of the Effect of DD-M. Vaccinate in the Psoariasis No. Patient Age / Sex Duration Admission of the of the Score of Code Disorder PASI 20 PS-025 A.S 25 / F 2 years 12.2 V.. PS-026 MB 45 / F 3 months 14.4 PS-027 AG 34 / M 14 years 24.8 25 PS-028 EM 31 / M 4 years 18.2 PS-029 AL 44 / M 5 months 18.6 30 PS-030 VB 42 / M 5 years 21.3 PS-031 RA 18 / M 3 months 13.0 All patients demonstrated an indurated soft localized erythematous non-ulcerated reaction at the injection site. No side effects were observed, nor were they claimed by the patients. The data shown in Table 8 are the skin reactions measured at the injection site, at 48 hours, 72 hours and 7 days after the first injection of DD-M. Vaccinate, and 48 hours and 72 hours after the second injection. TABLE 8 Measures of Skin Reaction in Millimeters No. First Code Injection Second Injection Time 48 hours 72 hours 7 days 48 hours 72 hours PS-025 8x 8 8x 8 3x 2 10x10 10x10 PS-026 12x12 12x12 8x 8 DNR 14x14 PS-027 9x 8 10x10 lOx 8 9x 5 9x 8 PS-028 10x10 10x10 lOx 8 10x10 10x10 PS-029 8x 6 8x 6 5x 5 8x 8 8x 8 PS-030 14x12 14x14 10x10 12x10 12x10 PS-031 10x10 12x12 lOx 6 14x12 12x10 DNR = Not reported The data shown in Table 9 are the PASI scores of the seven patients at the time of the first injection of DD-M. vaccae (Day 0), 3, 6, 12 and 24 weeks later. TABLE 9 Current Clinical Status of Patients after Injection of? - ?. vaccae (PASI Scores) Day No. 0 Week 3 Week 6 Week 12 Week 24 Code PS-025 12.2 4.1 1.8 1.4 1.7 PS-026 14.4 11.8 6.0 6.9 1.4 PS-027 24.8 23.3 18.3 9.1 10.6 PS-028 18.2 24.1 28.6 PS-029 18.6 was abandoned 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 can be seen that for week 3 after the first injection of DD-M. Vaccinate, five patients showed a significant improvement in PASI scores. By week 24, six of the seven patients showed a significant improvement in the PASI score.

As an example, Patient PS-031 went into remission (PASI score = 0) at week 32 and remained in remission when it was seen at week 48. The PASI score of patient PS-025 was reduced to less than 1 for more than 12 weeks. During an exacerbation of psoriasis (PASI = 15.8) at week 48, the patient was again treated with ?? - ?. Vaccinated and improved immediately with PASI scores remaining up to 6.8 and 0.6 at week 52 and 56, respectively. Therefore of the treatment of psoriasis with DD-M. Vaccination can lead to remission of the disease or provide prolonged benefit. Patients can also benefit from repeated treatment. EXAMPLE 7 PREPARATION OF M. VACCAE COMPOSITIONS This example illustrates the processing of the different constituents of M. vaccae. Preparation of the (DD-) M. vaccae Delipidate and Deglicopidate and the Assay Analysis The heat-killed M. vaccae was prepared as described above in Example 1. To prepare the delipidated M. vaccae, the M. vaccae subjected to treatment in the autoclave was granulated by centrifugation, the granule was washed with water, collected again by centrifugation and then dried by freezing. An aliquot of this freeze-dried M. vaccae was removed and referred to as freeze-dried M. vaccae. When used in the experiments it was resuspended in PBS at the desired concentration. The Ai. Freeze-dried vaccae was treated with chloroform / methanol (2: 1) for 60 minutes at room temperature to extract the lipids, and the extraction was repeated once. The delipidated residue from the chloroform / methanol extraction was further treated with 50% ethanol to remove the glycolipids by refluxing for two hours. The 50% ethanol extraction was repeated twice. The 50% ethanol extracts were used as a source of M. vaccae glycolipids (see below). The residue of the 50% ethanol extraction was freeze dried and weighed. The amount of delipidated and deglycolipidated M-vaccae prepared was equivalent to 11.1% of the initial wet weight of M-vaccae used. For the bioassay, the delipidated and deglycolipidated M. vaccae (DD-M vaccae) was resuspended in phosphate-stabilized saline by sonication and sterilized by autoclaving. The analysis of the composition of M.vaccae and DD-. vaccae removed by heat are presented in Table 9. The main changes are seen in the composition of the fatty acid and the amino acid composition of DD-AI. vaccae compared to the insoluble fraction of M. vaccae removed by heat. The data presented in Table 9 show that the insoluble fraction of M. vaccae removed by heat contains 10% w / w lipid, and the total content of the amino acid is 2750 nmoles / mg, or about 33% w / w . The DD-M. vaccae contains 1.3% w / w of the lipid and 4250 nmoles / mg of amino acids, which is approximately 51% w / w. TABLE 9 Analysis of the composition of M. vaccae and DD-M. vaccae removed by heat COMPOSITION OF MONOSACARIDO 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 OF FATTY ACID Fatty acid M. vaccae OD-M. vaccae C14: 0 3.9% 10.0% C16: 0 21.1% 7.3% C16: l 14.0% 3.3% 10 C18: 0 4.0% 1.5% C18: 1 * 1.2% 2.7% C18: lw9 20.6% 3.1% f V. C18: lw7 12.5% 5.9% C22: 0 12.1% 43.0% 20 C24: l * 6.5% 22.9% The insoluble fraction of M. vaccae removed by heat contains 10% w / w of lipid, and the DD-AÍ. vaccae contains 1.3% w / w lipid. AMINO ACID COMPOSITION Nmoles mg M, vaccae DD-M. Vacate ASP 231 361 THR 170 266 SER 131 199 35 GLU 319 505 PRO 216 262 GLY 263 404 ALA 416 621 CYS * 24 26 VAL 172 272 ET * 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 The total amino acid content of the insoluble fraction M.vaccae removed by heat is 2750 nmoles / mg, or about 33% w / w. The total content of the amino acid of ?? -? vaccae is 4250 nmol / mg, or approximately 51% w / w. Comparison of DD-M composition. vaccae with the delipidated and deglycolipidate forms of M. tuberculosis and M. soiecpaiat s M. tuberculosis and M. smegmatis delipidates and deglycolipidates were prepared using the procedure described above for M. vaccae delipidae and deglycolipidate. As indicated in Table 10, the profiles of the percentage of the amino acid composition in ?? - ?. vaccae, DD-? G. tuberculosis and DD-? G. smegmatis did not show significant differences. However, the total amount of protein varied - the two batches of DD-Af. vaccae contained 34% and 55% protein, whereas DD-? G. tuberculosis and DD-. Smegmatis contained 79% and 72% protein, respectively. TABLE 10 Amino Acid Composition of Delipidated and Deglycolipidated Mycobacterxas Amino DD - M. empty DD-M. goes DD-DD- Acid Lot 1 Lot 2 M. smegmatis M .tuberculosis 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 Pro 6.1 5.9 7.5 5.2 Gly 9.9 9.7 9.4 9.8 Wing 14.6 14.7 14.6 14.2 Cys 0.5 0.5 0.3 0.5 Val 6.3 6.4 7.2 7.8 Met 1.9 1.9 1.9 1.9 lie 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 The analysis of the monosaccharide composition shows significant differences between DD-M.vaccae, and DD-Af. tuberculosis and DD-Af. smegmatis. The composition of the monosaccharide of two batches of DD-Af. vaccae was the same and differed from that of ?? -? tuberculosis and Af. Smegmatis. Specifically, DD-M.vaccae was found to contain free glucose while both DD-Af. tuberculosis as Ai. smegmatis contain glycerol, as shown in Table 11. TABLE 11 Alditol acetate% by weight% molar DD-M.vaccae Lot 1 Inositol 0.0 0.0 Arabinose 54.7 59.1 Mañosa 1.7 1.5 Glucose 31.1 28.1 Galactose 12.5 11.3 100.0 100.0 DD-M. vaccae Lot 2 Inositol 0.0 0.0 Arabinose 51.0 55.5 Mañosa 2.0 1.8 Glucose 34.7 31.6 Galactose 12.2 11.1 100.0 100.0 DD- .smeg Inositol 0.0 0.0 Glycerol 15.2 15.5 Arabinose 69.3 70.7 Xylose 3.9 4.0 Maffein 2.2 1.9 Glucose 0.0 0.0 Galactose 9.4 8.0 100.0 100.0 DD-Mtb Inositol 0.0 0.0 Glycerol 9.5 9.7 Arabinose 69.3 71.4 Mañosa 3.5 3.0 Glucose 1.5 1.3 Galactose 12.4 10.7 96.2 96.0 Glycolipids from M. vaccae The soaked 50% ethanol extracts described above were dried by rotary evaporation, redissolved in water, and freeze-dried. The amount of the glycolipid recovered was 1.2% of the initial wet weight of M. vaccae used. For the bioassay, the glycolipids were dissolved in phosphate-stabilized saline. EXAMPLE 8 IMMUNE MODULATION PROPERTIES OF M. VACCAE DELIPIPATE AND DEGLICOLIPIPATE AND RECOMBINANT PROTEINS M. VACCAE This example illustrates the immune modulation properties of the different constituents of M. vaccae. Production of Interleukin-12 from macrophages The M. vaccae and ?? - ?. vaccae removed by heat is shown as having different cytokine stimulation properties. The stimulation of an immune response of Thl is improved by the production of interleukin-12 (IL-12) of macrophages. The ability of different M.vaccae preparations to stimulate the production of IL-12 was demonstrated as follows. A group of C57BL / 6J mice were injected intraperitoneally with thioglycolate DIFCO and after three days, the peritoneal macrophages were collected and placed in a cell culture with interferon-gamma for three hours. The culture medium was replaced and various concentrations of whole M. accae removed by heat (autoclaved), M. vaccae, lyophilized M. vaccae, DD-M were added. vaccae and M. vaccae glycolipids, prepared as described above. After an additional three days at 37 ° C, the culture supernatants were tested for the presence of IL-12 produced by the macrophages. As shown in Figure 4, preparations of M. vaccae stimulated the production of IL-12 from macrophages. By contrast, these same M.vaccae preparations were examined for the ability to stimulate interferon-gamma production of Natural Killer (NK) cells. Spleen cells were prepared from Serious Combined Immunodeficient (SCID) mice. These populations contain 75% to 80% of NK cells. Spleen cells were incubated at 37 ° C in a culture with 5 different concentrations of M. vaccae removed by heat, ?? - ?. vaccae or DD-M glycolipids. vacillate The data shown in Table 5 show that, even when M. vaccae removed by heat and the glycolipids of M. vaccae stimulate the production of interferon-gamma, the DD-M. vaccae stimulated relatively less interferon-gamma. The combined data of Figures 4 and 5 indicate that, in comparison to whole M. vaccae removed by heat, DD-W. vaccae is a better stimulator of IL-12 than interferon-gamma. 15 These discoveries demonstrate that the removal of the constituents of the lipid glycolipid from M. vaccae results in the removal of the molecular components that stimulate the interferon-gamma of the NK cells, effectively eliminating in this way a important cell source of a cytokine that has numerous peri-legal side effects. The DO-M.vaccae therefore retains the ability to improve immune Thl by stimulating the production of IL-12, but has lost the non-specific effects that can be achieved through the stimulation of the interferon-gamma production of the cells NK The adjuvant effect of DD-M. vaccae and a number of recombinant M. vaccae antigens of the present invention, which is prepared as will be described below, was determined by measuring the stimulation of IL-12 secretion from murine peritoneal macrophages. Figures 6A, B and C show the data from separate experiments in which groups of mice of C57BL / 6 (Figure 6A), BALB / c mice (Figure 6B) or C3H / HeJ mice (Figure 6C) were administered intraperitoneally DIFCO dithoglycolate. After three days, the peritoneal macrophages were collected and placed in the culture with interferon-gamma for three hours. The culture medium was replaced and different concentrations of the recombinant proteins of Ai were added. vacciae GVs-3 (GV-3), GV-4P (GV-4P), GVc-7 (GV-7), GV-23, GV-27, M. vaccae heat-killed, DD-M. vaccae (referred to as M. vaccae delipidada in Figures 6A, B and C), the glycolipids of M. vaccae or the lipopolysaccharide. After three days at 37 ° C, the culture supernatants were tested for the presence of IL-12 produced by the macrophages. As shown in Figures 6A, B and C, recombinant proteins and preparations of M. vaccae stimulated the production of IL-12 from macrophages. In a subsequent experiment, the peritoneal macrophages primed with IFNy of the BALB / c mice were stimulated with 40 ug / ml of recombinant M. vaccae proteins in the culture for 3 days and the presence of the IL-12 produced by the macrophages was tested . As shown in Figure 7, in these experiments the macrophages primed with ???? produced IL-12 when cultured with a control protein, ovalbumin (ova). However, the recombinant proteins GV 24B, 38BP, 38AP, 27, 5, 27B, 3, 23 and 22B stimulated more than twice the amount of IL-12 detected in control macrophyte cultures Detection of Non-Specific Immune Amplifier M. vaccae Entero and the Culture Filtrate of M. vaccae The culture supernatant of M. vaccae (S / N), the M. vaccae eliminated, M. vaccae delipidae and M. vaccae delipidate and deglycolipidated [?? - ?. vaccae), prepared as described above, were tested to determine adjuvant activity in the generation of a cytotoxic T cell immune response to ovalbumin, a protein structurally unrelated in the mouse. This specific cytotoxic response of the anti-ovalbumin was detected in the following manner. C57BL / 6 mice (2 per group) were immunized by intraperitoneal injection of 100 μ? of ovalbumin with the following test adjuvants: M.vaccae; submitted to autoclave treatment; M.vaccae delipidae; M.vaccae delipidated with glycolipids also extracted (DD-M vaccae) and proteins extracted with SDS; the SDS protein extract treated with Pronasa (an enzyme that degrades the protein); the culture filtrate of whole M.vaccae; and M. tuberculosis removed by heat or M. bovis BCG heat-killed M. phlei or M. smeg atis or M.vaccae culture filtrate. After 10 days, the spleen cells were stimulated in vitro for an additional 6 days with E.G7 cells which were EL4 cells (a C57BL / 6-derived T-cell lymphoma) transfected with the ovalabumin gene and therefore expressing ovalbumin. Spleen cells were then assayed for their ability to kill non-specific EL4 target cells or to eliminate cells expressing ovalbumin E.G7. The elimination activity was detected by the release of 51 chromium with which the EL4 and E.G7 cells were labeled (100 μl by 2xl06), before the elimination test. The elimination or cytolytic activity is expressed as% specific lysis using the formula: cpm in test-cpm cultures in control cultures xlOO% Total cpm cpm in control cultures It is generally known that the specific cytotoxic cells of ovalbumin are generated only in mice immunized with ovalbumin with an adjuvant but not in mice immunized with ovalbumin alone. The diagrams in Figure 7 show the effect of several preparations of the adjuvant derived from M. vaccae in the generation of cytotoxic T cells to ovalbumin in C57BL / 6 mice. As shown in Figure 7A, cytotoxic cells were generated in mice immunized with (i) 10 μg / (ii) 100 μg or (iii) 1 mg of autoclaved M. vaccae or (iv) 75 μg of filtrate of cultivation M. vaccae. Figure 7B shows that the cytotoxic cells were generated in mice immunized with (i) 1 mg of M. vaccae subjected to whole autoclave treatment or (ii) 1 mg of M. vaccae delipidada and deglycolipidada (DD-) M. vaccae . As shown in Figure 7C (i), the cytotoxic cells were generated in mice immunized with 1 mg of M. vaccae subjected to whole autoclave treatment; Figure 7C (ii) shows the active material in the soluble proteins of M. vaccae extracted with SDS from DD-M. vacillate Figure 7C (iii) shows that the active material in the preparation of the adjuvant of Figure 7C (ii) was destroyed by treatment with the proteolytic enzyme Pronasa. By way of comparison, 100 of the proteins extracted with SDS had significantly stronger immune-enhancing capacity (Figure 7C (ii) than 1 mg of M. vaccae subjected to whole autoclave treatment (Figure 7C (i). 1 mg of heat-killed M.vaccae (Figure 7D (i)) generated cytotoxic cells to ovalbumin, but mice immunized separately with 1 mg of heat-killed M. tuberculosis (Figure 7D (ii), 1 mg of M. bovis BCG (Figure 7D (iii), 1 mg of M. phlei (Figure 7D (iv)), or 1 mg of M. smegmatis (Figure 7D (v)) failed to generate the cytotoxic cells These discoveries demonstrate that M. vaccae heat-killed and DD-M vaccae have adjuvant properties that are not seen in the other mycobacteria.In addition, the delipidation and deglycolipidation of M.vaccae removes a stimulatory activity from the NK cell but does not result in a loss of stimulatory activity. of the T cell. In an experiment separate Do mice immunized with ovalbumin plus 200 ug of ?? - ?. vaccae depleted of mycolic acids and arabinogalactan, were also able to generate cytotoxic cells (from 28% to 46% maximum specific lysis compared to < 8% lysis V, · specific for control mice immunized with ovalbumin alone). The M. vaccae culture filtrate described above was fractionated by isoelectric focusing and the fractions were tested for adjuvant activity in the anti-ovalbumin-specific cytotoxic response assay in C57BL / 6 mice as described. 10 describes in the foregoing. The maximum adjuvant activities were demonstrated in fractions corresponding to pl of 4.2-4.32 (fractions numbers 7-9), 4.49-4.57 (fractions numbers 13-17) and 4.81-5.98 (fractions numbers 23-27). Identification of proteins in DD-M. vaccae by 15 antibodies BALB / c mice were immunized intraperitoneally with 50 ug of DD-W. Vaccinate once a week for 5 weeks. In the sixth week, the mice were sacrificed and their serum was collected. Sera were probed for antibodies to recombinant M.vaccae-derived proteins, prepared as described below, in normal enzyme-linked immunoassays. The antisera did not react with several recombinant M.vaccae proteins nor with ovalbumin, which served as a negative control protein in the enzyme-linked assays (data not shown). Antisera from mice immunized with DD-M. vaccae reacted with 12 GV antigens derived from M. vaccae. The results are shown in Table 12, which is presented below. The antisera identified in this way GV3, 5P, 5, 7, 9, 22B, 24, 27, 27A, 27B, 33 and 45 as being present in DD-M. vacillate TABLE 12 Reactivity of the DD-M antiserum. Vaccinate with GV antigens derived from M. vaccae Antigen of GV 3 5P 5 7 9 22B 24 27 27A 27B 33 45 Reactivity * 103 103 103 102 104 103 104 106 105 106 104 104 * Expressed as higher dilution of serum from mice immunized with DD-M. vaccae showing greater activity than serum from non-immunized mice. Proteins in DD-M. vaccae identified by T cell responses BALB / c mice were injected into each leg with 100 ug of DD-M. vaccae in combination with the incomplete Freund's adjuvant and 10 days later were sacrificed to obtain the cells of the popliteal lymph node. The cells from the immunized and non-immunized control mice were stimulated in vitro with GV proteins derived from recombinant M. vaccae. After 3 days, cell proliferation and IFNy production was evaluated. Proliferative responses of the T cell of the lymph node cells of mice immunized with DD-M. Vaccinate GV proteins. The lymph node cells of the mice immunized with DD-M. vaccae did not proliferate in response to an unrelated protein, ovalbumin, (data not shown). As shown in Table 13, lymph node cells and immunized mice showed proliferative responses to GV 3, 7, 9, 23, 27, 27B and 33. Corresponding cells from non-immunized mice did not proliferate in response to these proteins of GV suggesting that mice immunized with DD-M. vaccae have been immunized with these proteins. In this way, the proteins derived from M. vaccae GV 3, 7, 9, 23, 27, 27B and 33 are likely to be present in DD-M. vacillate TABLE 13 Proliferative responses of lymph node cells from mice immunized with DD-γ. vaccae and control mice to GV proteins in vitro Stimulation index * observed in Protein presence of GV proteins at 50 μg / ml GV Immunized mice Control mice with DD-M. vaccae 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 of absorption of tritiated Thymidine in the presence of protein / GV cpm in the absence of the GV protein Production of ???? by lymph node cells from mice immunized with DD-. raccae after the in vitro challenge with GV proteins The lymph node cells of the non-immunized mice did not produce IFNy during the stimulation with GV proteins. As shown in Table 14 presented below, lymph node cells from mice immunized with? - ?. vaccae secrete IFNy in a manner that depends on the dose when stimulated with GV 3, 5, 23, 27A, 27B, 33, 45 or 46, suggesting that the mice have been immunized with these proteins. No production of ???? when the cells of the immunized mice were stimulated with the unrelated protein, ovalbumin (data not shown). The proteins GV 3, 5, 23, 27A, 27B, 33, 45 and 46 therefore have the possibility of being present in DD-M. vacillate TABLE 14 Proliferation of IFNy by popliteal lymph node cells from mice immunized with DD-M. Vaccinate after challenge in vivo with GV protein IFNy (ng / ml) GV protein or control Dose of GV protein used in vitro. { μg ml) GV- P ND ND ND GV-5 8.90 + 4.54 0.57 + 0.40 ND GV-5P ND ND ND GV-7 ND ND ND GV-9 ND ND ND GV-13 1.64 + 0.40 ND ND GV-14 ND ND ND GV- 14B ND ND ND GV-22B 20.15 + 1.96 4.34 + 0 .02 ND GV-23 41.38 + 6.69 6.97 + 2 .78 ND GV-24B ND ND ND GV-27 46.86 + 17.14 33.06 + 17 .61 10 .14 + 3 01 GV-27A 7.25 + 4.36 ND ND GV-27B 100.36 + 37.84 33.03 + 7. 54 14 .33 + 1 .o: GV-29 5.93 + 0.47 ND ND GV-33 9.82 + 4.64 ND ND GV-38AP 1.44 + 1.20 ND ND GV-38BP 5.62 + 0.70 ND ND GV- 2 ND ND ND GV- 4 ND ND ND DD-Af. Vacate 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 ND = Not Detectable Proteins in DD-M. Vaccinate as non-specific immune amplifiers In subsequent experiments, the five proteins GV27, 27A, 27B, 23 and 45 were used as non-specific immune amplifiers with the ovalbumin antigen to immunize the mice as described above in Example 6. As shown in Figure 12, 50 ug of any of the recombinant proteins GV27, 27A, 27B, 23 and 45, when injected with 50 to 100 ug of ovalbumin demonstrated adjuvant properties by being able to generate the cytotoxic cells to ovalbumin. EXAMPLE 9 M. VACCAE TREATED IN A AUTOCLAVE GENERATES CD8 CYTOXIC CELLS T AGAINST MACROPHAGES INFECTED WITH M. TUBERCULOSIS This example illustrates the ability of M. vaccae removed to stimulate CD8 cytotoxic T cells that preferentially remove macrophages that have been infected with M. tuberculosis. Mice were immunized by intraperitoneal injection of 500 μg of killed M. vaccae which was prepared as described in Example 1. Two weeks after immunization, the spleen cells of the immunized mice were passed through a column of enrichment of the CD8 T cell (R &D Systems, of St. Paul, MN, USA). Spleen cells recovered from the column have been shown to be enriched up to 90% with CD8 T cells. These T cells, as well as CD8 T cells from the spleens of non-immunized mice were tested for their ability to eliminate uninfected macrophages or macrophages that have been infected with M. tuberculosis. Macrophages were obtained from the peritoneal cavity of the mice five days after 1 ml of 3% thioglycollate had been administered intraperitoneally. The macrophages were infected overnight with M. tuberculosis at a ratio of 2 mycobacteria per macrophage. All macrophage preparations were labeled with 51 chromium at 2 μ? for 10 ^ macrophages. The macrophages were then cultured with CD8 T cells overnight (16 hours) at the eliminator ratios to the target of 30: 1. Specific elimination was detected by the release of 51 chromium and expressed as% specific lysis, which is calculated as in Example 5. The production of IFN-? and their release to the medium after 3 days of co-culture of the CD8 T cells with macrophages was measured using an enzyme-linked immunosorbent assay (ELISA). The ELISA plates were coated with a rat monoclonal antibody directed to the IF -? of the mouse (Pharmigen, San Diego, CA, USA) in PBS for 4 hours at 4 ° C. The wells were blocked with PBS containing 0.2% Tween 20 for 1 hour, at room temperature. The plates were then washed four times in PBS containing 0.2% Tween 20, and the samples were diluted 1: 2 in a culture medium in the ELISA plates that were incubated overnight at room temperature. The plates were again washed, and an IFN-α antibody was added to each well. of anti-mouse biotinylated monoclonal rat (Pharmigen), which is diluted to 1 μg ml in PBS. The plates were then incubated for 1 hour at room temperature, washed, and avidin D coupled with horseradish peroxidase (Sigma A-3151) was added at a dilution of 1: 4000 in PBS. After an additional 1 hour incubation at room temperature, the plates were washed and an OPD substrate was added. The reaction was stopped after 10 minutes with 10% (volume / volume) of HC1. The optical density was determined at 490 nm. The fractions that resulted in both duplicates providing an OD twice higher than the mean OD of the cells grown in the medium alone, were considered positive. As shown in Table 15, the CD8 T cells from the spleens of mice immunized with M. vaccae were cytotoxic for macrophages infected with M. tuberculosis and did not lyse the non-infected macrophages. The CD8 T cells from the non-immunized mice did not lyse the macrophages. CD8 T cells from natural or non-immunized mice produce IF -? when they are co-cultured with infected macrophages. The amount of IFN-? produced the co-culture was greater with the CD8 T cells that are derived from the mice immunized with M. vaccae TABLE 15 EFFECT WITH MACROPHAGES INFECTED AND NOT INFECTED WITH M. TUBERCULOSIS % Specific Lysis IFN-? (ng / ml) of macrophages infected non infected CD8 infected infected CD8 T cells Control 0.7 24.6 Immunized with M. vaccae 95 2.2 43.8 EXAMPLE 10 PURIFICATION AND CHARACTERIZATION OF POLIPEPTIDES OF FILTRATION OF CULTIVATION OF M. VACCAE This example illustrates the preparation of soluble M.vaccae proteins from culture filtrate. Unless it manifests otherwise, all the percentages in the following example are in weight by volume. The Ai. vaccae (ATCC number 15483) was cultured in sterile medium 90 at 37 ° C. Cells were harvested by centrifugation, and transferred to Middlebrook 7H9 medium with glucose at 37 ° C for one day. The medium was then centrifuged (leaving the volume of the cells) and filtered through a 0.45μp filter? in sterile bottles. The culture filtrate was concentrated by lyophilization, and redissolved in MilliQ water. A small amount of insoluble material was removed by filtration through a membrane at 0.45 μp ?. The culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell containing a 3kDa molecular weight cut-off membrane (MWCO). The pressure was maintained at 3.52 kg per cm ^ using nitrogen gas. 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 from 20 1 to about 50 ml. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA). The desalted culture filtrate was fractionated by ion exchange chromatography on a Q-Sepharose column (Pharmacia Biotech, Uppsala, Sweden) (16 x 100 mm) equilibrated with lOmM of a Tris HCl stabilizer of pH 8.0. The polypeptides were eluted with a linear gradient of 0 to 1.0 M NaCl in the aforementioned stabilizer system. The eluent of the column was monitored with a wavelength of 280 nm. The pool of polypeptides eluting from the ion exchange column was concentrated in a 400 ml Amicon stirred cell containing a 3 kDa MWCO membrane. The pressure was maintained at 3.52 kg per cm ^ using nitrogen gas. The polypeptides were repeatedly concentrated by membrane filtration and diluted with 1% glycine until the conductivity of the sample was less than 0.1 mS. The purified polypeptides were then fractionated by isoelectric focusing of preparation on a Rotofor device (Bio-Rad, Hercules, CA, USA). The pH gradient was established with a mixture of Ampholytes (Pharmacia Biotech) comprising 1.6% of Ampholytes of pH 3.5-5.0 and Ampholytes of 0.4% of pH 5.0 - 7.0. Acetic acid (0.5 M) was used as the anolyte, and 0.5 M ethanolamine as the catholyte The isoelectric focusing was carried out at 20 a constant power of 12 W for 6 hours, followed by the manufacturer's instructions. The fractions of the isoelectric focus were combined and the polypeptides were purified on a Vydac C4 column (Separations Group, Hesperia, CA, USA) pore size of 300 Angstrom units, particle size of 5 microns (10 x 250 The polypeptides were eluted from the column with a linear gradient of acetonitrile (from 0 to 80% volume / volume) in trifluoroacetic acid of 0.05% (volume / volume) (TFA) .The flow rate was 2.0 ml. per minute and the HPLC eluent was monitored at 220 nm The fractions containing the polypeptides were collected to maximize the purity of the individual samples The relatively abundant polypeptide fractions were rechromatographed in a Vydac C4 column (Separations Group) with pore size of 300 Angstrom units, particle size of 5 microns (4.6 x 250 mm). The polypeptides were eluted from the column with a linear gradient of 20% to 60% (volume / volume) of acetonitrile at 0.05% (volume / volume) of TFA at a flow rate of 1.0 ml per minute. The eluent of the column was monitored at 220 nm. The fractions containing the eluted polypeptides were collected to maximize the purity of the individual samples. Approximately 20 polypeptide samples were obtained and analyzed for purity on a polyacrylamide gel according to the Laemmli procedure (Laemmli, U. K., Nature 277: 680-685, 1970).

Fractions of the polypeptide that are shown to contain significant contamination were further purified using a Mono Q column (Pharmacia Biotech) from (.10 micron particle size (5 x 50 mm) or a 5 column Vydac Diphenyl (Separations Group) pore size 300 Angstrom units, particle size 5 microns (4.5 x 250 mm). Mono Q, the polypeptides were eluted with a linear gradient of 0-0.5 M NaCl in 10 mM Tris HCl of pH 8.0.Of a column 10 of Vydac Diphenyl, the polypeptides were eluted with a linear gradient of acetonitrile (from 20 % to 60% volume / volume) in 0.1% TFA The flow rate was 1.0 milliliter per minute and the eluent of the column was monitored at 220 nm for both columns Maximum fractions of the polypeptide were collected and analyzed for purity on a 15% polyacrylamide gel or as described above For the sequence, the polypeptides were individually dried in Biobrene ™ (Perkin Elmer / Applied 20 BioSystems Division, Foster City, CA) -which are treated glass fiber filters.The filters with the polypeptide or were loaded into a Perkin Elmer / Applied BioSystems Procise 492 protein sequencing apparatus and the polypeptides were subjected to amino terminal end sequence using traditional Edman chemistry. The amino acid sequence was determined for each polypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards. The internal sequences were also determined in some antigens by digesting the antigen with the endoprotease Lys-C, or chemically separating the antigen with cyanogen bromide. Peptides resulting from any of these procedures were separated by reverse phase HPLC on a Bydac C18 column using a mobile phase of 0.05% (volume / volume) of trifluoroacetic acid with an acetonitrile gradient containing 0.05% (volume / volume ) of TFA (1% / minute). The eluent was monitored at 214 nm. The major internal peptides were identified by their absorbance to ultraviolet light, and their N-terminal sequences were determined as described above. Using the procedures described above, six soluble M.vaccae antigens designated GVc-1, Gvc-2, GVc-7, GVc-13, GVc-20 and GVc-22 were isolated. The N-terminal and internal sequences determined for GVc-1 are shown 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 shown in SEQ ID NOS: 5-8; the internal sequences for GVc-13 are shown in SEQ ID NOS: 9-11; the internal sequence for GVc-20 is shown in SEQ ID NO: 12; and the N-terminal and internal GVc-22 sequences are shown in SEQ ID NO: 56-59, respectively. Each of the internal peptide sequences provided herein begins with an amino acid residue that is presumed to exist at this position in the polypeptide, based on the known cleavage specificity of cyanogen bromide (et) or Lys-C (Lys) Three additional polypeptides designated GVc-16, GVc-18 and GVc-21, were isolated using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which is a purification step in addition to the isoelectric focusing process of preparation described above. Specifically, the fractions comprising polypeptide mixtures of the isoelectric focusing purification step of the preparation described above were purified by SDS-PAGE preparation on a 15% polyacrylamide gel. The samples were dissolved in a reducing sample stabilizer and applied to the gel. The separated proteins were transferred to a polyvinylidene difluoride membrane (PVDF) by electro-drying in 10 mM 3- (cyclohexylamino) -1-propanesulfonic acid (CAPS) pH stabilizer of 11 containing 10% (volume / volume) of methanol. The transferred protein bands were identified by staining the PVDF membrane with Coomassie blue. The PVDF membrane regions containing the most abundant polypeptide species were cut and introduced directly into the sample cartridge of the apparatus that provides Perkin Elmer / Applied BioSyste protein sequences Procise 492. The protein sequences were determined as it is described in the foregoing. The N-terminal sequences for GVc-16, GVc-18 and GVc-21 are provided in SEQ ID NOS: 13, 14 and 15, respectively. Additional antigens designated GVc-12, GVc-14, GVc-15, GVc-17 and GVc-19, were isolated using a purification step of SDS-PAGE preparation in addition to the chromatographic procedures described above. Specifically, the fractions comprising a mixture of antigens from the above-described Vydac C4 HPLC purification step were fractionated by SDS-PAGE preparation on a polyacrylamide gel. The samples were dissolved in a non-reducing sample stabilizer and applied to the gel. The separated proteins were transferred to a PVDF membrane by electro-drying in a CAPS stabilizer of 10 mM, pH 11 containing 10% (volume / volume) of methanol. Transferred protein bands were identified by staining the PVDF membrane with Coomassie blue. PVDF membrane regions containing the most abundant polypeptide species were cut and embedded directly into the sample cartridge of Perkin Elmer protein apparatus / Applied BioSystems Procise 492. Protein sequences were determined as follows. described in the foregoing. The N-terminal sequences determined for GVc-12, GVc-14, GVc-15, GVc-17 and GVc-19 are provided in SEQ ID NOS: 16-20, respectively. All of the aforementioned amino acid sequences were compared to the amino acid sequences known in the SwissProt database (version R32) using the GeneAssist system. No significant homologies were obtained to the amino acid sequences GVc-2 to GVc-22. The amino acid sequence for GVc-1 was found to bear some similarity to the previously identified sequences of M. bovis and M. tuberculosis. In particular, GVc-1 was found to have some homology with M. tuberculosis MPT83, a cell surface protein, as well as MPT70. These proteins are part of a family of proteins (Harboe et al., Scand J. Imunol, 42: 46-51, 1995). Subsequent studies lead to the isolation of the DNA sequences for GVc-13, GVc-14 and GVc-22 (SEQ ID NO: 142, 107 and 108, respectively). The corresponding predicted amino acid sequences for GVc-13 # GVc-14 and GVc-22 are given in SEQ ID NO: 143, 109 and 110, respectively. The DNA sequence determined for the coding of full-length gene GVc-13 is provided in SEQ ID NO: 195, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 196. Additional studies with GVc-22 suggested that only part of the GVc-22 gene coding had been cloned. When sub-cloned into the pET16 expression vector, no protein expression was obtained. Subsequent screening of the M. vaccae BamHI genomic DNA library with the incomplete gene fragment led to the isolation of the complete gene coding GVc-22. To distinguish between full length clone and partial GVc-22, the antigen expressed by the full-length gene was called GV-22B. The nucleotide sequence of the GV-22B gene coding and the predicted amino acid sequence are provided in SEQ ID NOS: 144 and 145, respectively. The amplification primers AD86 and AD112 (SEQ ID NO: 60 and 61, respectively) were designated from the amino acid sequence of GVc-1 (SEQ ID NO: 1) and the gene sequence of M. tuberculosis MPT70. Using these primers, a 310 bp fragment of the M.vaccae genomic DNA was amplified and cloned into the EcoRV-digested vector pBluescript II SK + (Stratagene). The cloned insert sequence provided in SEQ ID NO: 62. The insertion of this clone was used to screen a genomic DNA library of M. vaccae constructed in ZAP-Express lambda (Stratagene, La Jolla, CA). The isolated clone contained an open reading frame with homology to M. tuberculosis antigen MPT83 and was renamed as GV-1/83. This gene also had homology to the M. bovis MPB83 antigen. The determined nucleotide sequence and the predicted amino acid sequences are provided in SEQ ID NOS: 146 and 147 respectively. Of the amino acid sequences that are provided in SEQ ID NOS: 1 and 2, degenerate oligonucleotides EV59 and EV61 (SEQ ID NOS: 148 and 149 respectively) were designated. Using PCR, an lOObp fragment cloned in plasmid pBluescript II SK + and subjected to sequence (SEQ ID NO: 150) was amplified following normal procedures (Sambrook et al., Ibid). The cloned insert used to screen a M.vaccae genomic DNA library constructed in ZAP-Express lambda. The isolated clone had homology to the Af antigen. tuberculosis MPT70 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 given in SEQ ID NOS: 151 and 152, respectively. For expression and purification, the genes encoding GV1 / 83, GV1 / 70, GVc-13, GVc-14 and GV-22B were sucked into the pET16 expression vector (Novagen, Madison, WI). Expression and purification were carried out according to the manufacturer's protocol. The purified polypeptides were screened for the ability to induce proliferation of the T cell and IFN-α. in the peripheral blood cells of immune human donors. These donors were known to be PPD (purified protein derivative of M. tuberculosis) positive skin test and their T cells are shown to be proliferating in response to PPD. The donor PBMCs and the crude soluble proteins from the culture filtrate of M.vaccae were cultured in a medium comprising RPMI 1640 supplemented with 10% (volume / volume) of autologous serum, penicillin (60 μg ml). streptomycin (100 μg ml), and glutamine (2 mM). After 3 days, 50 μ? of the middle of each well for the determination of IFN-α levels, as will be described below. The plates were cultured for an additional 4 days and then pulsed with a? Μ ?? / ???? of tritiated thymidine for an additional 18 hours were harvested and the tritium uptake was determined using a scintillation counter. Fractions that stimulated proliferation in both duplicates twice as large as the observed proliferation(In cells grown in the medium alone, 5 were considered positive.) IFN-γ was measured using an enzyme-linked immunosorbent assay (ELISA) ELISA plates were coated with a mouse monoclonal antibody directed to human IFN-γ. (Endogenous, Wobural, MA) ^ g / ml Saline stabilized with phosphate (PBS) for 4 hours at 4 ° C. The wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. The plates were then washed four times in PBS / 0.2% Tween 20, and the samples were diluted 1: 2 in the culture medium.

ELISA plates that were incubated overnight at room temperature. The plates were again washed, and an IF -? Serum was added to each well. of biotinylated polyclonal rabbit anti-human (Endogenous), diluted to 1 μg ml in PBS. The plates were then incubated for 1 hours at room temperature, washed, and avidin A coupled with horseradish peroxidase (Vector Laboratories, Burlingame, CA) was added at a dilution of 1: 4,000 in PBS. After an additional 1 hour of incubation at room temperature, the plates were washed and the substrate of orthophenylenediamine (OPD) was added. The reaction was stopped after 10 minutes with 10% (volume / volume) of HC1. The optical density (OD) was determined at 490 nm. The fractions that resulted in both duplicates providing an OD twice as high as the OD of the medium of the cells grown in the medium alone, were considered positive. Examples of sequences containing polypeptides that stimulate peripheral blood mononuclear cells T cells (PBMC) to proliferate and produce IF -? are shown in Table 16, where (-) indicates a lack of activity, (+/-) indicates polypeptides that have a result less than twice as high as the background activity of control medium, (+) indicates polypeptides having activity two to four times above the background, and (++) indicates polypeptides having activity greater than four times above the background. TABLE 16 Antigen Proliferation IFN GVc-1 ++ +/- GVc-2 + ++ GVc-7 +/- - GVc-13 + ++ GVc-1 ++ + GVc-15 + + GVc-20 + + EXAMPLE 11 PURIFICATION AND CHARACTERIZATION OF THE POLIPEPTIDES OF M. VACCAE CULTURE FILTRATION USING 2-DIMENSIONAL POLYACRILAMIDE GEL ELECTROPHORESIS 5 The soluble M. vaccae proteins were isolated from culture filtrate using 2-dimensional polyacrylamide gel electrophoresis as will be described below. Unless otherwise mentioned, all percentages in the following example are by weight 10 by volume. M. vaccae (ATCC number 15483) was cultured in sterile medium 90 at 37 ° C. Strain M. tuberculosis H37Rv (ATCC number 27294) was cultured in Middlebrook sterile medium 7H9 with Tween 80 and oleic acid / albumin / dextrose / 15 catalase additive (Difco Laboratories, Detroit, Michigan). The cells were harvested by centrifugation, and transferred to the Middlebrook sterile medium 7H9 with glucose at 37 ° C for one day. The medium was then centrifuged (leaving the volume of the 20 cells) and filtered through a 0.45 μp filter? in sterile bottles. The culture filtrate was concentrated by lyophilization, and redissolved in MilliQ water. A small amount of insoluble material was removed by filtration through a filter membrane of 0.45 Pm.

The culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell containing a 3 kDa MWCO membrane. The pressure is V maintained at 4.22 kg per cm2 using nitrogen gas. 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 from 20 1 to about 50 ml. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USES) . The filtrate from the desalted culture was fractionated by ion exchange chromatography on a Q-Sepharose column (Pharmacia Biotech) (16 x 100 mm) equilibrated with 10 m of the TrisHCl stabilizer of pH 8.0. The polypeptides were eluted with a linear gradient of NaCl from 0 to 1.0 in the aforementioned stabilizer system. The eluent of the column was monitored at a wavelength of 280 nm. The pool of polypeptides that are eluted from the ion exchange column were fractionated by 2D gel electrophoresis of preparation. Samples containing from 200 to 500 g of the polypeptide were made at 8M in urea and applied to the polyacrylamide isoelectric focusing rod gels (diameter of 2 mm, length of 150 miti, pH of 5-7). After the isoelectric focusing step, the gels of the first dimension were equilibrated with a reducing stabilizer and applied to gels of the second dimension. { 16% polyacrylamide). The polypeptides from the second dimension separation were transferred to the PVDF membranes by electro-drying in the CAPS stabilizer of lOmM pH 11 containing 10% (volume / volume) of methanol. The PVDF membranes were stained for protein with Coomassie blue. The polypeptide regions containing PVDF of interest were cut and directly introduced into the sample cartridge of Perkin Elmer / Applied Bio Systems Procise 492 protein sequence apparatus. The polypeptides were sequenced from the amino terminal end using traditional Edman chemistry. The amino acid sequence was determined for each polypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards. Using these procedures, eleven polypeptides designated GVs-1, GVs-3, GVs-4, GVs-5, GVs-6, GVs-8, GVs-9, GVs-10, GVs-11, GV-34 and GV were isolated. -35. The N-terminal sequences determined for these polypeptides are shown in SEQ ID NOS: 21-29, 63 and 64, respectively. Using the purification procedure described above, more protein was purified to amplify the amino acid sequence obtained above for GVs-9. The extended amino acid sequence for GVs-9 was provided in SEQ ID NO: 65. Additional studies resulted in isolation of the DNA sequences for GVs-9 (SEQ ID NO: 111) and GV-35 (SEQ ID NO: 155). The corresponding predicted amino acid sequences were provided in SEQ ID NO: 112 and 156, respectively. An extended or extended DNA sequence for GVs-9 is provided in SEQ ID NO: 153, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 154. The predicted amino acid sequence for GVs-9 had been modified in SEQ ID NO: 197. All of these amino acid sequences were compared with the amino acid sequences known in the SwissProt database (version R35 plus update). Significant homologies were not obtained with the exceptions of GVs-3, GVs-4, GVs-5 and GVs-9. GVs-9 was found to carry some homology to two previously identified M. tuberculosis proteins, namely, the M. tuberculosis cutinase precursor and the hypothetical 22.6 kDa protein of M. tuberculosis. The GVs-3, GVs-4 and GVs-5 were found to carry some similarity in the proteins of antigen 85A and 85B of M. leprae (SEQ ID NOS: 30 and 31, respectively), M. tuberculosis (SEQ ID NOS: 32 and 33, respectively) and M. bovis (SEQ ID NOS: 34 and 35, respectively), and 85C antigen of M. leprae (SEQ ID NO: 36) and M. tuberculosis (SEQ ID NO: 37) . EXAMPLE 12 DNA CLONING STRATEGY FOR M. VACCAE ANTIGEN OF SERIES 85 Test probes for antigens 85A, 85B and 85C were prepared by polymerase chain reaction (PCR) using the degenerate oligonucleotides (SEQ ID NOS: 38 and 39) designated to the regions of the gene antigen gene sequence 85 that were conserved among the members of the family in a determined mycobacterial species and between the mycobacterial species. These oligonucleotides were used under stringent stringent conditions to amplify the genomic DNA target sequences of M. vaccae. A roughly sized band of 485 bp was identified, purified, and cloned into pBluescript II SK tail T (Stratagene, La Jolla, CA). Twenty-four individual colonies were randomly screened for the pre-emergence of the 85 PCR antigen product, and then sequenced using the Perkin Elmer / Applied BioSystems Model 377 automatic sequence apparatus and the MI3, T3 and T7 based primers. The homology searches of the GenBank databases showed that twenty-three clones contained an insertion with significant homology to the published antigen 85 genes of M. tuberculosis and M. bovis. Approximately half were more homologous to the 85C antigen gene sequences with the remainder being more similar to the 85B antigen sequences. In addition / these two genomic sequences of putative M. vaccae 85 antigen were 80% homologous to each other. Due to this high similarity, the 85C PCR antigen fragment was selected to screen genomic libraries of M. vaccae to be less stringent for all 85 antigen genes. A genomic library of M. vaccae was created in Zap-Express lambda (Stratagene, La Jolla, CA) cloning the genomic DNA of M. vaccae Ba RI partially digested in the digested vector similarly, with 3.4 x 10 5 independent plaque forming units. For sieving purposes, twenty-seven thousand plates from this unamplified library were placed in plates at low density towards eight 100 cm¿ plates. For each plate, duplicate plate surveys were taken on the Hybond-N + nylon membrane (Amersham International, UK), and hybridized under stringent reduced conditions (55 ° C) to the 85 C antigen irradiated from the PCR product. The autoradiography showed that seventy new plates were hybridized consistently to the 85 C antigen test probe under these conditions. Thirteen positive hybridization plates were randomly selected for further analysis and plates were removed from the library and each positive clone was used to generate secondary screening plates containing approximately two hundred plates. Duplicate surveys of each plate were taken using the Hybond-N + nylon membrane, and hybridized under the conditions used in the primary screening. The multiple positively hybridized plates were identified in each of the thirteen screened plates. The isolated positive pool of two wells from each secondary plate was collected for further analysis. Using in vitro excision, twenty-six plates were converted to fagemid, and subjected to restriction. It was possible to group the clones into four classes based on this map preparation. Sequence data from the 5 'to 3' ends of several representative insertions of each group was obtained using the Perkin Elmer / Applied BioSystems Model 377 automatic sequence apparatus and primers T3 and T7. Sequence homologies were determined using the BLASTN analysis of the EMBL database. Two of these sets of clones were found to be homologous to the 85A antigen genes of M. bovis and M. tuberculosis each containing either the 5 'or 3' ends of the M. vaccae gene (this gene was separated during construction from the library because it contains an internal BamHI site). The remaining clones were found to contain sequences homologous to antigens 85B and 85C of a number of mycobacterial species. To determine the remaining nucleotide sequence for each gene, the appropriate subclones were constructed and sequenced. The overlap sequences were aligned using the DNA Strider software. The DNA sequences determined for the M. vaccae 85A, 85B and 85C antigens are shown in SEQ ID NOS: 40-42, respectively, with the predicted amino acid sequences having been shown in SEQ ID NOS: 43-54, respectively. The antigens of M. vaccae GVs-3 and GVs-5 were expressed and purified in the following manner. The amplification primers were designated from the insertion sequences of GVs-3 and GVs-5 (SEQ ID NOS: 40 and 42, respectively) using the sequence data downstream of the putative forward sequence and the 3 'end of the clone. The sequences of the primers for GVs-3 were provided in SEQ ID NO: 66 and 67, and the sequences of the primers for GVs-5 were given in SEQ ID NO: 68 and 69. An Xhol restriction site was added the primers for GVs-3 and the restriction sites EcoRI and BamHI were added to the primers for GVs-5 for cloning convenience. After amplification of genomic M. vaccae DNA, the fragments were cloned into the appropriate site of the prokaryotic expression vector pProEX HT (Gibco BRL, Life Technologies, of Gaithersburg, MD) and subjected to the sequence in order to confirm the correct reading frame and orientation. The expression and purification of the recombinant protein was carried out according to the manufacturer's protocol. The expression of a fragment of the M. vaccae antigen of GVs-4 (antigen of homologue 85B) was carried out in the following manner. The primers AD58 and AD59, which are described above, were used to amplify a 485 bp fragment of M. vaccae genomic DNA. This fragment was purified from the gel using standard techniques and cloned into the pBluescript digested with EcoRV containing added dTTP residues. The base sequences of the insertions of five clones were determined and found to be identical to one another. These inserts had the highest homology for Ag85B of M. tuberculosis. The insertion of one of the clones was subcloned into the EcoRI / XhoI sites of the prokaryotic expression vector pProEX HT (Gibco BRL), which is expressed and purified according to the manufacturer's protocol. This clone was renamed GV-4P because only part of the gene was expressed. The amino acid and DNA sequence for the partial clone GV-4P are provided in SEQ ID NO: 70 and 106, respectively. Similar to the cloning of GV-4P, the amplification primers AD58 and AD59 were used to amplify a 485 bp fragment of a clone containing GVs-5 (SEQ ID NO: 42). This fragment was cloned into the expression vector pET16 and was designated GV-5P. The determined nucleotide sequence and the predicted amino acid sequence of GV-5P are given in SEQ ID NOS: 157 and 158, respectively. In subsequent studies, using procedures similar to those described above, GVs-3, GV-4P and GVs-5 were recloned into the alternative vector pET16 (Novagen, Madison, WI). The ability of the purified recombinants GVs-3, GV-4P and GVs-5 to stimulate the proliferation of T cells and the production of interferon-? In human PBL of healthy PPD-positive donors, it was tested, as described above. The results of this test are shown in Table 17, where (-) indicate a lack of activity, (+/-) indicates polypeptides that have a result less than twice as high as the background activity of the control medium (+) indicates polypeptides having activity two to four times above the background, (++) indicates polypeptides having activity greater than four times above the background, and ND indicates not determined. Table 17Donor Donor Donor G97005 G97006 G97007 Prolif IFN Prolif IFN Prolif IFN -y -y -Y GVs-3 ++ + ND ND ++ ++ GV-4P + +/- ND ND + ++ GVs-5 ++ ++ + + ++ ++ ++ Table 17 (continued) Donator Donor G97008 G97009 G97010 Prolif IFN Prolif IFN Prolif IFN -y -Y -Y GVs-3 ++ ++ ++/- + ++ GV-4P ++ ++ +/- + / - +/- ++ GVs-5 + ++ ++ ++ ++ EXAMPLE 13 DNA CLONING STRATEGY FOR M. VACCAE ANTIGENS An 84 bp test probe for the M. vaccae GVc-7 antigen was amplified using degenerate oligonucleotides designated to a determined amino acid sequence of GVc-7 (SEQ ID NOS) : 5-8). This test probe was used to screen a genomic DNA library of M. vaccae as described in Example 12. The nucleotide sequence determined for GVc-7 is shown in SEQ ID NO: 46 and the predicted amino acid sequence in the SEQ ID NO: 47. The comparison of these sequences with those in the homology revealed in databank in a hypothetical membrane protein of 15.8 kDa of M. tuberculosis. The sequence of SEQ ID NO: 46 was used to design amplification primers (which are provided in SEQ ID NO: 71 and 72) for cloning of GVc-7 gene expression using sequence data downstream of the forward sequence putative An XhoI restriction site was added to the primers for cloning convenience. After amplification of the genomic DNA of M. vaccae, the fragments were cloned into the Xhol site of pProEX HT which is the prokaryotic expression vector (Gibco BRL) and subjected to sequence to confirm the correct reading frame and orientation . The expression and purification of the fusion protein was carried out according to the manufacturer's protocol. In subsequent studies, GVc-7 was re-cloned into the vector pET16 (Novagen).

The ability of purified recombinant GVc-7 to stimulate the proliferation of T cells and stimulate the production of interferon-? in the human PBL, from the positive PPD positive donors, it was tested as described above. The results are shown in Table 18, where (-) indicates a lack of activity (+/-) indicates polypeptides that have a result less than twice as high as the background activity of the control medium, (+) indicates polypeptides having activity two to four times above the bottom, and (++) indicates polypeptides having activity greater than four times above the background. TABLE 18 Donor Interferon Proleration- 'G97005 ++ +/- G97008 ++ + G97009 + +/- G97010 +/- ++ A redundant oligonucleotide test probe (EQ ID NO: 73, referred to as MPG15) was designed to sequence the peptide GVs-8 shown in SEQ ID NO: 26 and used to screen a DNA library Af genomics Vaccinate using normal protocols. Two genomic clones containing genes encoding four different antigens were isolated. The r DNA sequences determined for GVs-8A (re-named GV-30), GVs-8B (re-named GV-31), GVs-8C (re-named GV-32) and GVs-8D 5 (re- named GV-33) are shown in SEQ ID NOS: 48-51, respectively, with the corresponding amino acid sequences having been shown in SEQ ID NOS: 52-55, respectively. The regions contain GV-30 showing some similarity to the synthetasis of valyl-tRNA V 10 known prokaryotes; GV-31 shows some similarity to the semialdehyde dehydrogenase of M. smeg atis aspartate; and GV-32 shows some similarity to the folly polyglutamate synthase gene of H. influenza. GV-33 contains an open reading frame that shows certain similarity to the sequences previously identified in M. tuberculosis and M. leprae, but whose function has not been identified. The partial DNA sequence determined for GV-33 is provided in SEQ ID NO: 74 with the sequence of the corresponding predicted amino acid having been provided in SEQ ID NO: "75. Sequence data from the 3 'end of the clone showed homology to the previously identified 40.6 kDa outer membrane protein of M. tuberculosis. led to the isolation of a full-length DNA sequence for GV-33 (SEQ ID NO: 193). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 194. Coding of the GV-33 gene was amplified from M. vaccae genomic DNA with primers based on the determined nucleotide sequence. This DNA fragment was cloned into pBluescrpt II SK + digested with EcoRv (Stratagene), and then transferred to the pET16 expression vector. The recombinant protein was purified following the manufacturer's protocol. The ability of recombinant GV-33 purified to stimulate the proliferation of T cells and stimulate the production of interferon-? in the human PBL it was tested as described above. The results are shown in Table 19, where (-) indicates a lack of activity, (+/-) indicates polypeptides that have a result less than twice as high as the background activity of the control medium, (+) indicates polypeptides having activity two to four times above the background, and (++) indicates polypeptides having activity greater than four times above the background.

TABLE 19 Stimulatory Activity of Polypeptides Donor Proleration Interferon- 'G97005 ++ + G97006 +++ G97007 - +/- G97008 +/- - G97009 +/- - G97010 +/- ++ EXAMPLE 1 ISOLATION OF DD-M PROTEINS. VACCAE Bacteria of M. vaccae were cultured, granulated and autoclaved as described in Example 1. Culture filtrates of active M. vaccae refer to the supernatant of 24-hour cultures of M. vaccae in a 7H9 medium with glucose. A delipidate form of M. vaccae was prepared by sonicating M. vaccae subjected to autoclaving treatment for four 30 second bursts on ice using a Virsonic sonicator (Virtis, Disa, USA). The material was then centrifuged (9000 revolutions per minute, 20 minutes, rotor JA 10, brake = 5). The resulting granule was suspended in 100 ml of chloroform / methanol (2: 1), incubated at room temperature for 1 hour, re-centrifuged, and the chloroform / methanol extraction was repeated. The granule was obtained by centrifugation, dried under vacuum, weighed and resuspended in PBS at 50 mg (dry weight) by me as M. vaccae delipidated. The glycolipids were removed from the delipidated M. vaccae preparation by refluxing in 50% volume / volume of ethanol for 2 hours. The insoluble material was collected by centrifugation (10,000 revolutions per minute, JA20 rotor, 15 minutes, brake = 5). Extraction with 50% volume / volume of refluxing ethanol was repeated twice more. The insoluble material was collected by centrifugation and washed in PBS. The proteins were extracted by resuspending the pellet in 2% SDS in PBS at 56 ° C for 2 hours. The insoluble material was collected by centrifugation and extraction with 2% SDS / PBS at 56 ° C was repeated twice more. The flooded SDS extracts were cooled to 4 ° C, and the precipitated SDS was removed by centrifugation (10,000 revolutions per minute, 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 collected by centrifugation, washed in 50% volume / volume of acetone, dried in vacuo and dissolved in PBS.

The proteins extracted from SDS-derived from ?? - ?. vaccae were analyzed by electrophoresis of polyacrylamide gel. The three main bands were observed after staining with silver. In the subsequent experiments / the largest amounts of proteins extracted with SDS from DD-M. vaccae, were assayed by polyacrylamide gel electrophoresis. The proteins, when stained with Coomassie blue, showed several bands. A protein represented by a band of approximately a molecular weight of 30 kDa was designated GV-45. The N-terminal sequence determined for GV-45 is given in SEQ ID NO: 187. A protein of approximate molecular weight of 14 kDa was designated GV-46. The determined N-terminal amino acid sequence of GV-46 was provided in SEQ ID NO: 208. In subsequent studies, more of the proteins extracted with SDS described above are prepared by SDS-PAGE of Preparation in a BioRad Preparation Cell (Hercules, CA). The fractions corresponding to the molecular weight scales were precipitated by trichloroacetic acid to remove the SDS before being assayed to determine the activity of the adjuvant in the anti-ovalbumin-specific cytotoxic response assay in the C5 * 7BL / 6 mice as described. described in the foregoing. Adjuvant activity was highest in the 60-70 kDa fraction. The most abundant protein in this size scale was purified by SDS-PAGE which was dried to a polyvinylidene difluoride membrane (PVDF) and then subjected to sequence. The sequence of the first ten amino acid residues are provided in SEQ ID NO: 76. Comparison of this sequence with those in the gene bank as described above revealed the homology to the 65 shock protein gene Thermal (GroEL) of M. tuberculosis, indicating that this protein is a member of M. vaccae of the GroEL family. An expression library of M. vaccae genomic DNA in BamHI-lambda ZAP-Express (Stratagene) was screened using sera from cynomolgus monkeys immunized with secreted M. vaccae proteins prepared as described above. Positive plaques were identified using a colorimetric system. These plates were re-screened until the plates were pure following normal procedures. Phagemid 2-1 of pBK-CMV containing an insert was excised from the ZAP Express lambda vector (Stratagene) in the presence of the ExAssist helper phage following the manufacturer's protocol. The base sequence of the 51 end of the insert of this clone, which is referred to below as GV-21, was determined using the Blood sequence with fluorescent primers in the automatic sequence apparatus Perkin-Elmer / Applied Biosytems Division . The de-termined nucleotide sequence of the clone of the partial G. GV-27 M.sub.Ge.a.V. homolog is provided in SEQ ID NO: 77 and the amino acid sequence predicted in SEQ ID NO: 78. This clone was found to have homology to GroEL of M. tuberculosis. A partial sequence of the 65 kDa heat shock protein of M. vaccae has been published by Kapur et al. (Arch. Pathol, Lab. Med. 129: 131-138, 1995). The nucleotide sequence of the Kapur and others fragment is shown in SEQ ID NO: 79 and the amino acid sequence predicted in SEQ ID NO: 80. In the subsequent studies, an extended DNA sequence for GV-27 was obtained. (except the full length for the predicted terminal nucleotides 51) the DNA sequence for GV-27 obtained (SEQ ID NO: 113). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 114. Additional studies led to the isolation of the full-length DNA sequence for GV-27 (SEQ ID NO: 159). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 160. GV-27 was found to be 93.7% identical to GroEl of M. tuberculosis at the amino acid level.

Two peptide fragments comprising the N-terminal sequence (referred to below as GV-27A) and the carboxy-terminal sequence of GV-27 (hereinafter referred to as 5 GV-27B) were prepared using techniques well known in the field. The nucleotide sequences for GV-27A and GV-27B are provided in SEQ ID NO: 115 and 116, respectively, with the corresponding amino acid sequences provided in SEQ ID NO: 117 and 118. Subsequent studies led to the isolation in an extended DNA sequence for GV-27B. This sequence is provided in SEQ ID NO: 161, with the corresponding amino acid sequence being provided in SEQ ID NO: 162. The sequences of GV-27A is 95.8% identical to the sequence of M. tuberculosis GroEL and contains the shorter M. vaccae sequence from Kapur and others, which is discussed above. The sequence for GV-27B shows approximately 92.2% identity to the corresponding region of M. tuberculosis HSP65. Following the same protocol as for the isolation of GV-27, the phagemid pBK-CMV 3-1 was isolated. The antigen encoded by this DNA is named GV-29. The determined nucleotide sequences of the 5 'and 3' ends of the gene are provided in SEQ ID NOS: 163 and 164, respectively, with the sequences of Predicted corresponding amino acids being provided in SEQ ID NO: 165 and 166 respectively. GV-29 showed homology to the yeast urea amidoliaza. The DNA sequence determined for the full-length gene encoding GV-29 is provided in SEQ ID NO: 198, with the corresponding predicted amino acid sequence in SEQ ID NO: 199. The DNA encoding GV-29 it was sub-cloned into the pET16 vector (Novagen, Madison, WI.) for expression and purification according to standard protocols .. EXAMPLE 15 V, DNA CLONING STRATEGY FOR M. VACCAE ANTIGENS GV-23 , GV-24, GV-25, GV-26, GV-38A and GV-38B M. vaccae (ATCC number 15483) was grown in sterile medium 90 at 37 ° C for 4 days and harvested 15 by centrifugation The cells were resuspended in 1 ml of Trizol (Gibco BRL, Life Technologies, Gaitherburg, Maryland) and the RNA was extracted according to the normal manufacturer's protocol.The strain of M. tuberculosis H37Rv (ATCC number 2794) was grown in medium 20 sterile Middlebrook 7H9 with Tween 80TM and an oleic acid / albumin / dextrose / catalase additive (Difco) Laboratories, Detroit, Michigan) at 37 ° C and harvested under appropriate laboratory safety conditions. The cells were resuspended in 1 ml of Trizol (Gibco BRL) and the RNA was extracted according to the manufacturer's normal protocol. ^ The total RNA of M. tuberculosis and M. vaccae was depleted of 16S and 23S of ribosomal RNA (rRNA) by hybridization of the total RNA fraction to oligonucleotides ADIO and AD11 (SEQ ID NO: 81 and 82) complementary to RRNA from M. tuberculosis. These oligonucleotides were designated from the microbacterial 16S rRNA sequences published by Bottger. { FEMS f. 10 Microbiol. Lett. 55: 171-176, 1989) and of the sequences deposited in the data banks. Depletion was carried out by hybridization of the total RNA to the ADIO and AD11 oligonucleotides immobilized on nylon membranes (Hybond N, Amersham International, UK). The Hybridization was repeated until the rRNA bands were not visible on agarose gels stained with ethidium bromide. An oligonucleotide, AD12, (SEQ ID NO: 83), consisting of 20 dATP residues, was ligated to the 3"ends of the enriched mRNA fraction using the ligase from RNA. The first cDNA synthesis chain was carried out following the normal protocols, using the oligonucleotide AD7 (SEQ ID NO: 84) containing a poly (dT) sequence. The cDNA of M. tuberculosis and M. vaccae was used as a template for single-sided specific PCR (3S-PCR). For this protocol, a degenerate oligonucleotide AD1 (SEQ ID NO: 85) was designated based on the r conserved leader sequences and the v protein membrane sequences. After 30 cycles of amplification using primer AD1 as 5 '-primer and AD7 as 3' primer, the products were separated on a urea / polyacrylamide gel. The DNA strands unique to Af. vaccae were cut and re-amplified using primers. AD1 and AD7. After purification of the gel, the '10 bands were cloned into pGEM-T (Promega) and the base sequence was determined. Searches with the determined nucleotide and the predicted amino acid sequences of band 12B21 (SEQ ID NOS: 86 and 87, respectively) showed homology to the E. coli pota gene encoding the ATP binding protein of the spermidine / putrescine ABC transporter complex published by Furuchi et al. (JnJ. ^ Bioi. Chem. 266: 20928-20933, 1991). The E. coli spermidine / putrescine transporter complex consists of of four genes and is a member of the ABC transporter family. The ABC transporters (ATP binding cassette) typically consist of four genes: an ATP binding gene, a periplasmic gene, or a substrate binding, the gene and two transmembrane genes. The genes Transmembrane proteins encode each one having characteristically six regions that span the membrane. Homologs (by similarity) of this ABC transporter have been identified in the genomes of Haemophilus influenza (Fleischmann and others Science 269: 496-512, 1995) and Mycoplasma genitalium (Fraser, et al., Science, 270-397-403, 1995). ). A genomic DNA library of M. vaccae constructed in ZAP Express lambda digested with BamHl- (Stratagene) was tested with the irradiated band of 238 bp 12B21 according to the normal protocols. A plate was purified to obtain purity by repetitive sieving and a phagemid containing a 4.5 kb insert was identified by drying and hybridization. The nucleotide sequence of the full-length murid of M. vaccae of pota (ATP binding protein) was identified by subcloning the 4.5 kb fragment and base sequence. The gene consisted of 1449 bp including a 5 'untranslated 320 bp region containing putative promoter -10 and -35 elements. The predicted nucleotide and amino acid sequences of the M. vaccae pota homologue are provided in SEQ ID NO: 88 and 89, respectively. The nucleotide sequence of the M. vaccae pota gene was used to designate primers EV24 and EV25 (SEQ ID NO: 90 and 91) to express the cloning. The amplified DNA fragment was cloned into the prokaryotic expression system pProEX HT (Gibco BRL) and expression in an appropriate E.coli host was induced by the addition of 0.6 m of isopropylthio-p-galactoside (IPTG). The recombinant protein was named GV-23 and purified from inclusion bodies according to the manufacturer's protocol. 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 contains an ATP binding site at residues 34 to 41. At residues 116 to 163 of SEQ ID NO: 89, there is a conserved region that is in the family of ATP transporter proteins. These findings suggest that GV-23 is an ATP binding protein. A Sacl-BamHI subclone of 322 bp at the 3 'end of the 4.5 kb insert described above showed homology to the potd gene, (periplasmic protein) of the spermidine / putrescine ABC which is an E. transporter complex. coli The nucleotide sequence of this subclone is shown in SEQ ID NO: 92. To identify the gene, the irradiated insert of this subclone was used to test a genomic DNA library of M. vaccae constructed at the Sail site of Zap Express lambda ( Stratagene) following the normal protocols. A clone was identified of which 1342 bp showed homology with potd gene of E. coll. The potd homolog of M. vaccae was identified by subcloning and subjecting the base to sequence. The determined nucleotide and predicted amino acid sequences are shown in SEQ ID NO: 93 and 94. For expression cloning, primers EV-26 and EV-27 (SEQ ID NO: 95-96) were designated from the determined potd homolog of M. vaccae. The amplified fragment was cloned into the prokaryotic expression system pProEX HT (Gibco BRL). Expression in an appropriate E. coli host was induced by addition of 0.6 mM of IPTG and the recombinant protein was designated GV-24. The recombinant antigen was purified from the inclusion bodies according to the supplier's protocol. In subsequent studies, GV-24 (SEQ ID NO: 93) was re-cloned into the alternative vector pET16 (Novagen). To improve the solubility of the purified recombinant antigen, the coding of the GV-24 gene, but excluding the signal peptide, was re-cloned into the expression vector, using the amplification primers EV101 and EV102 (SEQ ID NO: 167 and 168). The construction was designated GV-24B. The nucleotide sequence of GV-24B is provided in SEQ ID NO: 169 and the amino acid sequence predicted in SEQ ID NO: 170. This fragment was cloned into pET16 for expression and purification of GV-24B according to the protocols of maker . The ability of the purified recombinant protein GV-23 and GV-24 to stimulate the proliferation of T cells and the production of interferon-? in the human PBL was determined as described above. The results of these tests are given in Table 20 / where (-) indicates a lack of activity, (+/-) indicates polypeptides that have a result less than twice as high as the background activity of the control medium, (+) indicates polypeptides having activity two to four times above the bottom, (++) indicates polypeptides having an activity greater than four times above the bottom, and (ND) indicates not determined. TABLE 20 Donor Donor Donor G97005 G97006 G97007 Prolif IFN Prolif IFN Prolif IFN -y "Y -Y GV-23 ++ ++ ++ ++ + + GV-24 ++ ++ ND ND Table 20 (continued) Donor Donor Donor G97008 G97009 G97010 Prolif IF Prolif IFN Prolif IFN -Y-Y "And GV-23 ++ ++ + + ++ GV-24 + +/- + +/- +/- ++ The base sequence adjacent to the homolog of the potd GENE of M. vaccae was found to show homology to the potb gene of the ABC transport complex of spermidine / putriscin from E. coli, which is one of the two transmembrane proteins in the ABC transporter complex. The potb homolog of M. vaccae (referred to as GV-25) was identified through additional subcloning and base sequence. The determined nucleotide and the amino acid sequences predicted for GV-25 are shown in SEQ ID NO: 97 and 98, respectively. Additional subcloning and base sequence analysis of 509 bp adjacent failed to reveal significant homology to PotC, the second transmembrane protein of E. coli, and suggest that a second transmembrane protein is absent in the M. vaccae homologue of the transporter ABC. An open reading frame with acetyl homology of M. tuberculosis transferase was CoA acetyl, however it was identified starting at 530 bp downstream of the transmembrane protein and the transfer protein as it was called GV-26. The determined sequence of the partial nucleotide and predicted amino acid sequence for GV-26 is shown in SEQ ID NO: 99 and 100, respectively. Using a protocol similar to that described above for the isolation of GV-23, the 3S-PCR and 12B28 band (SEQ ID NO: 119) was used to screen the M. vaccae genomic library constructed at the BamHI site. of ZAP Express lambda (Stratagene). The clone isolated from the library contained a novel open reading frame and the antigen encoded by this gene was called GV-38A. The determined nucleotide sequence and the predicted amino acid sequence of GV-38A are shown in SEQ ID NO: 120 and 121, respectively. Subsequent studies led to the isolation of the extended DNA sequence for GV-38A, which is provided in SEQ ID NO: 171. The corresponding amino acid sequence is provided in SEQ ID NO: 172. The comparison of these sequences with those in The gene bank revealed some homology to an unknown M. tuberculosis protein identified previously in the cosmid MTCY428.12. (SPTREMBL: p71914).

Upstream of the GV-38A gene, a second novel open reading frame was identified and the antigen encoded by this gene was designated GV-38B. The determined 5 'and 3' nucleotide sequences of GV-38B are provided in SEQ ID NO: 122 and 123, respectively, with the corresponding predicted amino acid sequences having been provided in SEQ ID NO: 124 and 125, respectively. Additional studies led to the isolation of the full length DNA sequence for GV-38B, which is provided in the SEQ ID NO: 173. The corresponding amino acid sequence is provided in SEQ ID NO: 174. This protein is found to show homology to the unknown M. tuberculosis protein identified in the cosmid MTCY428.il (SPTREMBL: p71914). Both the GV-38A and GV-38B antigens were amplified by expression cloning in pET16 (Novagen). The GV-38A was amplified with the primers KR11 and KR12 (SEQ ID NO: 126 and 127) and GV-38B with the primers KR13 and KR14 (SEQ ID NO: 128 and 129). The expression of the protein in BL21 host cells (DE3) was induced with 1 mM IPTG, however, no protein expression was obtained from these constructs. The hydrophobic regions were identified in the N-terms of the GV-38A and GV-38B antigens that can inhibit the expression of these constructs. The hydrophilic region present in GV-38A was identified as a possible transmembrane motif with six regions spanning the membrane. To express the antigen without the hydrophobic regions, the 5 KR20 primers for GV-38A, (SEQ ID NO: 130) and KR21 for GV-38B (SEQ ID NO: 131) were designated. The truncated GV-38A gene was amplified with the KR20 and KR12 primers and the GV-38B gene truncated with KR21 and KR14. The determined nucleotide sequences of truncated GV38A and GV-38B are ^ and 10 show in SEQ ID NO: 132 and 133, respectively, with the corresponding predicted amino acid sequences having been shown in SEQ ID NO: 134 and 135, respectively. Extended DNA sequences for truncated GV-38A and GV-38B are provided in SEQ ID NO: 15 175 and 176, respectively, with the corresponding amino acid sequences provided in SEQ ID NO: 177 and 178, respectively. EXAMPLE 16 PURIFICATION AND CHARACTERIZATION OF POLYPEPTIDES OF M. VACCAE FILTRATION FILTRATION BY PREPARATIVE ISOELECTRIC APPROACH AND PREPARATIVE POLYACRILAMIDE GEL ELECTROPHORESIS The soluble proteins of M. vaccae were isolated from the culture filtrate using preparative isoelectric focusing and gel electrophoresis. of preparative polyacrylamide as will be described below. Unless otherwise mentioned, all percentages in the following f example are weight per volume. M. vaccae (ATCC number 15483) was cultured in 5 250 1 of sterile Medium 90 which was fractionated by ultrafiltration to remove all proteins of molecular weight greater than 10 kDa. The medium was centrifuged to remove the bacteria, and sterilized by filtration through a 0.45 g filter. The sterile filtrate was concentrated by ultrafiltration through a 10 kDa molecular weight cut-off membrane. The proteins were isolated from the concentrated culture filtrate by precipitation with 10% trichloroacetic acid. The precipitated proteins were re-dissolved in 100 mM Tris.HCl of pH 8.0 and re-precipitated by the addition of an equal volume of acetone. The precipitated material of acetone was dissolved in water, and the proteins were re-precipitated by the addition of an equal volume of chloroform: methane1 of 2: 1 (volume / volume). The precipitated chloroform: methanol material was dissolved in water and the solution was freeze-dried. The freeze-dried protein was dissolved in an iso-electric focusing stabilizer, containing 8 M 25 deionized urea, 2% Triton X-100, 10 mM dithiothreitol and 2% ampholytes (pH 2.5 - 5.0). The sample was fractionated by G ~ * preparative isoelectric focusing on a horizontal bed of Ultrodex gel at 8 watts of constant power for 16 hours. The proteins were eluted from the fractions of the gel bed with water and concentrated by precipitation with 10% trichloroacetic acid. The puddles of fractions containing proteins of interest were identified by gel electrophoresis. 10 of analytical polyacrylamide and fractionated by preparative polyacrylamide gel electrophoresis. The samples were fractionated in 12.5% of SDS-PAGE gels, and were electro-dried in nitrocellulose membranes. The proteins were placed on the membranes stained with Ponceau Red 15, faded with water and eluted from the membranes with 40% acetonitrile / 0.1 M ammonium bicarbonate of pH 8.9 and then concentrated by lyophilization. The eluted proteins were tested because of their ability to induce the proliferation and secretion of interferon-? of the peripheral blood lymphocytes of immune donors as detailed above. The proteins that induce an intense response in these trials were selected for further study.

The selected proteins were further purified by reverse phase chromatography on a Vydac column of Protein C4, using a system of trifluoroacetic acid-acetonitrile. The purified proteins were prepared from protein sequence determination by SDS-polyacrylamide gel electrophoresis and were electro-dried on the PVDF membranes. The protein sequences were determined as in Example 3. The proteins were designated GV-40, GV-41, GV-42, GV-43 and GV-44. The N-terminal sequences determined for these polypeptides are shown in SEQ ID NOS: 101-105, respectively. Subsequent studies lead to the isolation of a 5 'intermediate fragment, and the 3 'DNA sequence for GV-42 (SEQ ID NOS: 136, 137 and 138, respectively). The corresponding predicted amino acid sequences are provided in SEQ ID NOS: 139, 140 and 141, respectively. Following normal DNA amplification and cloning procedures as described in Example 13, the genes according to GV-41 and GV-42 were cloned. The determined nucleotide sequences are provided in SEQ ID NOS: 179 and 180, respectively, and the amino acid sequences predicted in SEQ ID NOS: 181 and 182. Additional extracts led to the cloning of the full-length gene encoding GV -41, which was called GV-41B. The determined nucleotide sequence and the predicted amino acid sequence of GV-41B are provided in SEQ ID NOS: 202 and 203, respectively. GV-41 had homology to the ribosome recycling factor of M. tuberculosis and M. leprae, and GV-42 had homology to a M. avium fibronectin binding protein of FAP-A. Within the full length sequence of GV-42, the determined amino acid sequence of GV-43 (SEQ ID NO: 104) was identified, indicating that the amino acid sequence for GV-42 and GV-43 were obtained from it protein. The murine polyclonal antisera were prepared with GV-40 and GV-44 following normal procedures. These antisera were used to screen a genomic DNA library of M. vaccae consisting of randomly cut DNA fragments. The clones are encoded GV-40 and GV-44 were identified and subjected to sequence. The determined nucleotide sequence of the partial gene encoding GV-40 is provided in SEQ ID NO: 183 and the amino acid sequence predicted in SEQ ID NO: 184. The entire gene coding for GV-40 was not cloned, and the antigen encoded by this partial gene was called GV-40P. An extended DNA sequence for GV-40P is provided in SEQ ID NO: 206 with the corresponding predicted amino acid sequence provided in SEQ ID NO: 207. The determined nucleotide sequence of the gene encoding GV- is provided in SEQ. ID NO: 185 and the amino acid sequence predicted in SEQ ID NO: 186. With additional sequence, the DNA sequence determined for the full-length gene encoding GV-44 was obtained and are provided in SEQ ID NO: 204 , with the corresponding predicted amino acid sequence providing in SEQ ID NO: 205. GV-40 homology for the M. leprae Elongation Factor G was found and GV-44 had the homology to the glyceraldehyde-3-phosphate dehydrogenase of M. leprae. EXAMPLE 1 ISOLATION OF THE DD-M ANTIGENS. VACCAE GV-45 and GV-46 Proteins were extracted from DD-M. vaccae (500 mg; which were prepared as described above) by suspension in 10 ml of 2% SDS / PBS and heating at 50 ° C for 2 hours. The insoluble residue was removed by centrifugation, and the proteins were precipitated from the supernatant by adding an equal volume of acetone and incubating at -20 ° C for 1 hour. The precipitated proteins were collected by centrifugation, dissolved in a reducing sample stabilizer, and fractionated by preparative SDS-polyacrylamide gel electrophoresis. The separated proteins were electro-dried on the PVDF membrane in 10 mM CAPS / 0.01% SDS of pH 11.0, and the N-terminal sequences were determined in a gas phase sequence apparatus. From these experiments, a protein represented by a band of an approximate molecular weight of 30 kDa, designated GV-45, was isolated. The N-terminal sequence determined for GV-45 is provided in SEQ ID NO: 187. From the same experiments, a protein of approximate molecular weight of 14 kDa, designated GV-6, was obtained of course. The determined N-terminal amino acid sequence of GV-46 is provided in SEQ ID NO: 208. GV-46 is homologous to the highly conserved mycobacterial host integration factor of M. tuberculosis and M. smegmatis. From the amino acid sequence of GV-45, the degenerate oligonucleotides KR32 and KR33 (SEQ ID NOS: 188 and 189, respectively) were designated.A 100 bp fragment was amplified, cloned into the pBluescript II SK + plasmid (Stratagene, La Jolla , CA) and subjected to sequence (SEQ ID NO: 190) following normal procedures (Sambrook, Ibid.) The cloned insert was used to screen a M. vaccae genomic DNA library constructed at the BamHI site of ZAP-Express Lambda (Stratagene) The isolated clone showed homology to the M. tuberculosis 35 kDa protein and to a 22 kDa protein of M. leprae that contains motifs similar to the bacterial histone in the N-terminus and a unique C-terminus that consists of a basic repeat of five amino acids The nucleotide sequence determined for GV-45 is provided in SEQ ID NO: 191, with the corresponding predicted amino acid sequence having been provided in SEQ ID NO: 192. With sequence In addition, the DNA sequence determined for the full-length gene encoding GV-45 is obtained and is provided in SEQ ID NO: 200, with the corresponding predicted amino acid sequence in SEQ ID NO: 201. EXAMPLE 18 IMMUNOGENICITY AND IMMUNOMODULATION PROPERTIES OF RECOMBINANT PROTEINS DERIVED FROM M. VACCAE A. INDUCTION OF CELL PROLIFERATION AND IFN- PRODUCTION? The immunogenicity of the recombinant proteins Mycobacterium vaccae (GV recombinant proteins) was tested by injecting the female BALB / cByJ mice in each hind paw with 10 g of the recombinant GV proteins emulsified in an incomplete Freund's adjuvant (IFA). Control mice received phosphate-stabilized saline in IFA. Popliteal lymph nodes were drained 10 days later and the cells obtained therefrom were stimulated with the immunization GV protein and assayed for proliferation by measuring the absorption of the tritiated thymidine. The amount of interferon gamma (IFNy) produced and secreted by these cells into the culture supernatants was assayed by normal enzyme linked immunoassay. As shown in Table 21 the proliferative responses are summarized, all GV proteins were found to induce a T cell proliferative response. T cells from the lymph node of an immunized mouse proliferated in response to the specific GV protein used in the immunization. The lymph node cells of the non-immunized mice did not proliferate in response to the GV proteins. The data in Table 22 showing IFNy production indicate that in most of the GV proteins that stimulate IFNy production by aliphatic ganglion cells from non-immunized mice they were cultured with the individual GV proteins, and the production of IFNy was not detectable. The GV proteins are therefore immunogenic, being able to stimulate the proliferation of the T cell and / or the production of IFNy when administered by subcutaneous injection. The specific stimulatory effects of the antigen on T-cell proliferation and the production of IFNy are two advantageous properties of candidate vaccines for tuberculosis. TABLE 21 Immunogenic Properties of GV Proteins: Proliferation Proliferation (cpm) Protein GV Protein dose used in vitro (μ? / P ??) 50 2 0.08 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 detectable 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 undetectable undetectable not detectable GV-22B undetectable undetectable not detectable GV-23 56.67 + 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 detectable GV-27B 87. 98 + 15. 78 44. 43 + 8.70 21. 49 + 5.60 r GV-29 7. 56 + 2. 58 1. 22 + 0.56 not detectable V. 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 undetectable undetectable not detectable TABLE 22 Immunogenic Properties of GV Proteins: Production from ?? ? IFNy (ng / ml) GV protein GV protein dose used in vitro ^ g / ml) 50 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 detectable 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 r GV-9 92 .27 + 45.50 88. 54 + 16.48 30.46 + 1.77 V 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 undetectable undetectable not detectable GV-22B undetectable undetectable not detectable GV-23 59 .67 + 14.88 30 .70 + 4.48 9.17 + 1.51 GV-2 B 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 detectable 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 detectable 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 40 GV-44 non-detectable non-detectable non-detectable B. ACTIVATION OF LYMPHOCYTE SUBPOPULATIONS The ability of the recombinant M. vaccae proteins of the present invention, M. vaccae eliminated by heat and? - ?. vaccae to activate the lymphocyte subpopulations was determined by examining up-regulation of CD69 expression (a surface protein expressed in activated cells). PBMC from normal donors (5 x 106 cells per milliliter) were stimulated with 20 ug / ml of either heat-cleared M. vaccae cells,? - ?. 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. The CD69 expression was determined by staining the cultured cells with the monoclonal antibody against CD56, the aβ cells. or δt cells, in combination with monoclonal antibodies against CD69, followed by flow cytometry analysis. Table 23 shows the percentage of aß? of the cells? d? and NK cells expressing CD69 following the stimulation with heat-killed M. vaccae, DD-? G. vaccae or recombinant M. vaccae proteins. These results show that the M. vaccae eliminated by heat, the ?? - ?. vaccae and GV-23 stimulate the expression of CD69 in subpopulations of lymphocytes that are warranted compared to control (unstimulated cells), with particularly high levels of CD69 expression seen in NK cells. It was found that GV-45 upregulates CD69 expression in β cells. TABLE 23 Stimulation of Expression CD69 cells aßt cells? d? NK cells Control 3.8 6.2 4.8 M. vaccae Eliminated by heat 8.3 10.2 40.3 DD-M. Vacate 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 4.44 13.3 GV-27A 5.5 4.44 13.3 GV-27B 4.4 2.8 7.1 GV- 5 11.7 4.9 6.3 The capacity of the recombinant protein GV-23 (20 ug / ml) to induce CD69 expression in lymphocyte subpopulations was compared with that of Thl MPL / TDM / CWS inducing adjuvants (Monophosphoryl Lipid / Trehalose 6 '6' Dimycolate; Sigma, St. LouiS / MO; at a final dilution of 1:20) and CpG ODN (Promega, Madison, WI; 20 ug / ml), and the known Th2-inducing adjuvants of aluminum hydroxide (Superfos Biosector, Kvistgard, Denmark, at a final dilution of 1: 400) and cholera toxin (20 ug / ml), using the procedure described above. MPL / TDM / CWS and aluminum hydroxide were used at the maximum concentration that does not cause cell cytotoxicity. Figures 8A-C show the stimulation of CD69 expression in cells aß ?, cells? D? and the NK cells, respectively. GV-23, MPL / TDM / CWS and CpG ODN induced CD69 expression in NK cells, whereas aluminum hydroxide and cholera toxin did not. C. STIMULATION OF CYTOKINE PRODUCTION The ability of the recombinant M. vaccae proteins of the present invention to stimulate cytokine production in PBMC was examined in the following manner. PBMC from normal donors (5 x 10 ^ cells per ml) were stimulated with 20 ug / ml of either heat-cleared M. vaccae cells and DD-Af. vaccinating 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. The cultures were harvested and tested for the production of IL-? ß, TNF-a, IL-12 and IFN-? using normal ELISA kits (Genzyme, Cambridge, MA), following the manufacturer's instructions, Figures 9A-D show the stimulation of IL, TNF-α, IL-12 and IFN-α production respectively. M. vaccae and DD-? G. vaccae eliminated by V. Heat was found to stimulate the production of all 5 four cytokines examined, while GV-23 and CV-45 recombinants were found to stimulate the production of IL-? ß, TNF-a and IL-12, Figures 10A-C show the stimulation of IL-? ß, TNF-a and the production of IL-12, respectively, in human PBMC (which was determined as described above) by varying the concentrations of GV-23 and GV-45. Figures 11A-D show the stimulation of IL-β, TNF-α, IL-12 and IFN-α production respectively in PBMC by GV-23 compared to that by means of MPL / TDM / CWS adjuvants (a a final dilution of 1:20), CpG ODN (20 ug / ml), aluminum hydroxide (at a final dilution of 1: 400) and cholera toxin (20 ug / ml). GV-23, "^ MPL / TDM / CWS and CpG ODN induced significant levels of 'v. the four cytokines examined, with higher levels of IL-αβ production seen with GV-23 than with any of the known adjuvants. Aluminum hydroxide and cholera toxin induced only negligible amounts of the four cytokines.

D. ACTIVATION OF CELLS PRESENTING ANTIGEN The capacity of M. vaccae eliminated by heat, ^ -s DD-Af. vaccae and recombinant M. vaccae proteins to improve the expression of the co-stimulatory molecules 5 CD40, CD80 and CD86 in B cells, monocytes and dendritic cells was examined as follows. The peripheral blood mononuclear cells depleted of. T cells and comprising mainly B cells, monocytes and dendritic cells were stimulated with 20 ug / ml of either heat-cleared M. vaccae cells, DD-M. vaccae, or recombinant GV-22B (SEQ ID NO: 145), GV-23 (SEQ ID NO: 89), 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 48 hours. Stimulated cells were harvested and analyzed for upregulation of CD40, CD80 and CD86 using 3-color flow cytometric analysis. Tables 24, 25 and 26 show the increase in the mean fluorescence intensity of the control (unstimulated cells), for the dendritic cells, monocytes and B cells, respectively.

TABLE 2 Stimulation of CD40, CD80 and CD86 Expression in Dendritic Cells CD40 CD80 CD86 Control 0 0 0 M. vaccae eliminated by Heat 6.1 3.8 1.6 V.

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-2 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 in Monocytes CD40 CD80 CD86 Control 0 0 0 M. vaccae eliminated by Heat 2.3 1.8 0.7 ?? -? 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 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 Expression Stimulation of CD40, CD80 and CD86 in B Cells '? CD40 CD80 CD86 Control 0 0 0 M. vaccae eliminated by Heat 1.6 1.0 1.7 Or 10 DD-W. 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-2 A 1.0 1.1 0.9 20 GV-27B 1.0 0.9 0.9 GV-45 1.2 1.1 1.3 As shown above, increased levels of CD40, CD80 and CD86 expression are seen in dendritic cells, monocytes and B cells with all tested compositions. The expression levels were increased more in the dendritic cells with the highest levels of expression having been obtained with heat-killed M. vaccae, DD-. vaccae, GV-23 and GV-45. Figures 12A-C show the stimulation of the expression of CD40, CD80 and CD86, respectively, in the dendritic cells by varying the concentrations of GV-23 and GV-45. The ability of GV-23 to stimulate CD40, CD80 and CD86 expression in dendritic cells was compared with that of adjuvants that induce Thl MPL / TDM / CWS (at a final dilution of 1:20) and CpG ODN (20). ug / ml), and the known Th2 induction adjuvants in aluminum hydroxide (at a final dilution of 1: 400) and cholera toxin (20 ug / ml). GV23, MPL / TDM / CWS and CpG ODN caused significant upregulation of CD40, CD80 and CD86, while cholera toxin and aluminum hydroxide induced modest or insignificant activation of the dendritic cell, respectively. E. MATURATION AND FUNCTION OF THE DENDRITIC CELL The effect of the recombinant M. vaccae GV-23 protein on the maturation and function of the dendritic cells was examined in the following manner.

Purified dendritic cells (5 x 1? 4 -105 cells per ml) were stimulated with GV-23 (20 ug / ml) or LPS (10 uh / ml) as a positive control. The cells were cultured for 20 hours and then analyzed for CD83 (a maturation marker) and CD80 expression by flow cytometry. Unstimulated cells were used as a negative control. The results are shown below in Table 27. TABLE 27 Stimulation of CD83 Expression in Dendritic Cells Treatments% of cells% dendritic cells CD83- dendritic CD80 positive positive 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 improve the function of the dendritic cell as cells presenting the antigen was determined by mixed lymphocyte reaction assay (MLR). The purified dendritic cells were cultured in a medium alone or with GV-23 (20 ug / ml) for 18 to 20 hours and then stimulated with allogeneic T cells (2 x 10 cells / well). After 3 days of incubation, (3H) -thymidine was added. The cells were harvested 1 day later and the absorption of radioactivity was measured. Figure 13 shows the increase in (3H) -thymidine uptake with an increase in the ratio of dendritic cells to T cells. Significantly higher levels of radioactivity were seen in dendritic cells stimulated with GV-23 compared to the unstimulated cells, which show that GV-23 improves the reaction of leukocyte mixed with the dendritic cell. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, changes and modifications may be made without departing from the scope of the invention which is intended to be limited only by the scope of the invention. appended claims.

Claims (43)

1. CLAIMS; A polypeptide comprising an immunogenic portion of an isolated M. vaccae antigen, wherein the antigen includes a sequence that is selected from the group consisting of the sequences mentioned 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
2. 2. A polypeptide comprising an immunogenic portion of an Af antigen. vaccae isolated, wherein the antigen includes a sequence that is selected from the group consisting of: (a) sequences having at least about 50% residues identical to a sequence mentioned 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 as measured by the BLASTP computer algorithm; (b) sequences having at least about 75% residues identical to a sequence mentioned 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 as measured by the BLASTP computer algorithm; and (c) the sequences having at least about 95% residues identical to a sequence mentioned 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 as measured by the BLASTP computer algorithm.
3. 3. A polypeptide comprising an immunogenic portion of an isolated M. vaccae antigen, wherein the antigen comprises an amino acid sequence encoded by a polynucleotide that is selected from the group consisting of: (a) the sequences mentioned in SEQ. ID NOS: 142, 144, 146, 151, 153, 155, 157, 159, 161, 163, 164, 169, 171, 173, 175, 176, 179, 180, 183, 185, 20 191, 193, 195, 198 and 200; (b) complements of the sequences mentioned in SEQ ID NOS: 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) the sequences having at least about 99% probability of being the same as a sequence of (a) or (b) as measured by the BLAS computer algorithm N.
4. 4. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide according to any one of claims 1 to 3.
5. 5. An expression vector comprising a polynucleotide according to claim 4.
6. 6. A host cell transformed with an expression vector in accordance with claim 5.
7. The host cell of claim 6, wherein the host cells are selected from the group consisting of E. coli, mycobacteria, insect, yeast and mammalian cells.
8. 8. A fusion protein comprising at least one polypeptide according to any of claims 1 to 3.
9. 9. A pharmaceutical composition comprising a polypeptide according to any of claims 1 to 3, and a physiologically acceptable carrier.
10. 10. A pharmaceutical composition comprising a polynucleotide according to claim 4, and a physiologically acceptable carrier.
11. 11. A pharmaceutical composition comprising a fusion protein according to claim 8, and a physiologically acceptable carrier.
12. 12. A vaccine comprising a polypeptide according to any of claims 1 to 3, and a non-specific immune response amplifier.
13. A vaccine comprising a polynucleotide according to claim 4, and a non-specific immune response amplifier.
14. 14. A vaccine comprising a fusion protein according to claim 8, and a non-specific immune response amplifier.
15. 15. A vaccine according to any of claims 12 to 14 wherein the non-specific immune response amplifier is an adjuvant.
16. 16. A vaccine according to any of claims 12 to 14, wherein the non-specific immune response amplifier is selected from the group consisting of: (a) delipidated and deglycolipidated M. vaccae cells; (b) inactivated M. vaccae cells, and (c) M. vaccae culture filtrate.
17. 17. A method for improving an immune response in a patient, comprising administering to a patient a pharmaceutical composition according to any of claims 9 to 11. 5
18. 18. A method for improving an immune response in a patient, comprising administering to a patient a vaccine according to any of claims 12 to 14.
19. 19. The method of any of claims 17 and 18, wherein the immune response is a Thl response.
20. 20. A method for the treatment of a disorder in a patient, comprising administering to the patient a pharmaceutical composition in accordance with Any of claims 9 to 11.
21. 21. A method for the treatment of a disorder in a patient, comprising administering to the patient a ^ vaccine according to any of claims 12 to 14. 20
22. 22. The method of any of claims 20 and 21, wherein the disorder is selected from the group consisting of immune disorders, infectious diseases, skin diseases, and Diseases of the respiratory system.
23. 23. The method of claim 23 wherein the disorder is selected from the group consisting of mycobacterial infections, asthma and psoriasis.
24. 24. A method for the treatment of a disorder in a patient comprising administering a composition comprising a component that is selected from the group consisting of: (a) inactivated M. vaccae cells; (b) delipidated and deglycolipidated M. vaccae cells; (c) delipidated and deglycolipidated M. vaccae cells of mycolic acids; (d) cells of M. vaccae delipidates and deglycolipidates depleted of mycolic acids and arabinogalactan; and (e) a culture filtrate of M. vaccae, the disorder is selected from the group consisting of immune disorders, infectious diseases, skin diseases and diseases of the respiratory system.
25. 25. The method of claim 24, wherein the disorder is selected from the group consisting of mycobacterial infections, asthma, and psoriasis.
26. 26. A method for improving a non-specific immune response to an antigen comprising administering a polypeptide, the polypeptide comprising an immunogenic portion of the M. vaccae antigen, while the M. vaccae antigen includes a sequence that is selected from the group consisting of : (a) the sequences mentioned in SEQ ID NO: 89 and 201; and (b) sequences having at least about 80% residues identical to a sequence mentioned in SEQ ID NO: 89 and 201 as determined by the BLASTP computer algorithm.
27. 27. A method for detecting mycobacterial infection in a patient, comprising: (a) contacting the dermal cells of a patient with one or more polypeptides according to any of claims 1 to 3; and (b) detecting an immune response in the patient's skin.
28. 28. The method of claim 27, wherein the immune response is induration.
29. 29. A diagnostic kit comprising: (a) a polypeptide according to any of claims 1 to 3; and (b) an apparatus sufficient to contact the polypeptide with the skin cells of a patient.
30. 30. A method for detecting mycobacterial infection in a biological sample comprising: (a) contacting the biological sample with a polypeptide according to any of claims 1 to 3; and (b) detecting in the sample the presence of the antibodies that bind to the polypeptide.
31. 31. The method of claim 30 wherein the polypeptide (s) is linked to a solid support.
32. 32. The method of claim 30, wherein the biological sample is selected from the group consisting of whole blood, serum, plasma, saliva, fluid 15 cerebrospinal and urine.
33. 33. A method for detecting mycobacterial infection in a biological sample, comprising: (a) contacting the biological sample with a binding agent that is capable of binding to a polypeptide according to any one of claims 1 to 3; and (b) detecting in the sample a protein or polypeptide that binds to the binding agent.
34. 34. The method of claim 33 wherein the binding agent is a monoclonal antibody.
35. 35. The method of claim 33 wherein the binding agent is a polyclonal antibody.
36. 36. A diagnostic kit comprising: v v (a) at least one polypeptide according to any of claims 1 to 3; and (b) a detection agent.
37. 37. The kit of claim 36 wherein the polypeptide is immobilized on a solid support.
38. 38. The kit of claim 36 wherein the detection reagent comprises a reporter group that is conjugated to a binding agent.
39. 39. The kit of claim 38 wherein the binding agent is selected from the group consisting of anti-immunoglobulins, Protein G, Protein A and 15 lectins.
40. 40. The kit of claim 38 wherein the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and particles of a dye.
41. 41. The monoclonal antibody that binds to a polypeptide according to any of claims 1 to 3.
42. 42. A polyclonal antibody that is linked to a polypeptide according to any of claims 1 to 3.
43. 43. A method for improving a non-specific immune response to an antigen comprising administering a composition comprising a component that is selected from the group consisting of: (a) delipidated and vaclyzed M. vaccae cells of mycolic acids; and (b) delipidated and deglycolipidated M. vaccae cells of mycolic and arabinogalactan acids.