JP2002514385A - COMPOSITIONS DERIVED FROM MYCOBACTERIUM BACKEY AND USE THEREOF - Google Patents

Abstract

  (57) [Summary] The present invention provides compositions that are present in or derived from Mycobacterium bakey, as well as their use in treating, preventing and detecting diseases, including infectious diseases, immune diseases and cancer. M. Bucket culture filtrate, delipidated M. Bucket cells, delipidated and deglycolipidated M without mycolic acid. Delipidated and deglycolipidated M. with the exception of bucke cell, mycolic acid and arabinogalactan. Also provided is a method of enhancing an immune response to an antigen, comprising the administration of a bucket cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention relates generally to either present in Mycobacterium Bakke (Mycobacteriu m vaccae), or compositions can be derived from it, as well as infections, diseases including immune diseases and cancer, treatment, It relates to its use in prevention and detection. In particular, the present invention relates to respiratory diseases such as mycobacterial infection, asthma, sarcoidosis and lung cancer, and basal cell carcinoma which is psoriasis, atopic dermatitis, allergic contact dermatitis, alopecia areata and skin cancer , Squamous cell carcinomas and melanomas. The present invention further provides compounds that function as non-specific immune response amplifiers, and the use of these non-specific immune response amplifiers as adjuvants, for vaccination or immunotherapy against infectious diseases, and for certain immune diseases and cancers. For use in the treatment of

BACKGROUND OF THE INVENTION Tuberculosis is a chronic infectious disease, which is a mycobacterium tuberculosis ( Mycobacterium tubeculosis, (M. Tuberclo
-))) Is caused by infection. It is a major disease in developing countries and
This problem is also increasing in developed countries around the world, with about 8 million new cases each year and 3
One million people have died. There may be no signs of infection for a very long time,
The disease manifests itself as chronic lung inflammation resulting in fever and respiratory symptoms.
Most often manifest. If left untreated, the morbidity was significantly worse
Or death.

[0003] Tuberculosis can generally be controlled using a wide range of antibiotic therapies, but such treatments do not adequately prevent the spread of the disease. Infected individuals are sometimes asymptomatic or contagious. Furthermore, it is essential to follow the treatment regime, but it is difficult to monitor the behavior of the patient. Some patients do not follow the course of treatment, resulting in ineffective treatment or the development of drug-resistant mycobacteria.

[0004] Stopping the spread of tuberculosis requires effective vaccination and accurate early diagnosis of the disease. Currently, live bacterial vaccination by subcutaneous or intradermal injection is the most effective method of inducing protective immunity. The most common Mycobacterium used for this purpose is Mycobacterium. Bobisu (. M bovis, (M Bobisu).) Is a non-toxic strain of Bacillus Calmette - a Guerin (BCG). However, the safety and efficacy of BCG is controversial, and some countries, such as the United States, have not been vaccinated by the general public. M. Diagnosis of tuberculosis infection is generally made using a skin test, which involves intradermal exposure to tuberculin PPD (a purified protein derivative). By 48-72 hours post-injection, the antigen-specific T cell response results in measurable sclerosis at the injection site, suggesting exposure to mycobacterial antigen. However, this test has problems with sensitivity and specificity and cannot distinguish between individuals vaccinated with BCG and those infected.

[0005] It has been used by subcutaneous or intradermal injection in immunotherapy of tuberculosis and also leprosy.
Another less well-known mycobacterium is M. vaccae , M. vaccae , which is not pathogenic to humans. But,
M. compared to BCG. There is little information on the effectiveness of buckets and it has not been widely used for vaccination of the general public. M. Bovis BCG and M.
Bucket is M. It is believed to contain antigenic compounds that are recognized by the immune system of individuals exposed to infection by tuberculosis.

[0006] Several patents and other references are disclosed in M. Disclosed are methods of treating various conditions by administration of mycobacteria, including buckets, or certain mycobacterial fractions. U.S. Pat. No. 4,716,038 is disclosed in US Pat. Disclosed are methods of diagnosing, vaccinating and treating various types of autoimmune diseases, including arthritic diseases, by administration of mycobacteria, including buckets. U.S. Patent No. 4,724,144 discloses M. M. for treating mycobacterial diseases, particularly tuberculosis and leprosy, and as an adjuvant for chemotherapy. Disclosed is an immunotherapeutic agent comprising an antigenic substance derived from a bucket. International Patent Publication No. WO 91/01751 discloses that M.I. Disclosed is the use of antigenic and / or immunomodulatory substances derived from buckets. International Patent Publication No. WO 94/06466 describes that AIDS has or has not developed AIDS, and with or without associated tuberculosis,
M. in the treatment of HIV infection. Disclosed is the use of antigenic and / or immunomodulatory substances derived from buckets.

[0007] US Pat. No. 5,599,545 discloses M.S. As adjuvants for administration with antigens not endogenous to the bucket, mycobacteria, especially M. inactivated completely. Disclose the use of a bucket. This publication theorizes that the beneficial effect as an adjuvant may be due to heat shock protein 65 (hsp65). International Patent Publication No. WO 92/08484 discloses M.E. for the treatment of uveitis. Disclosed is the use of antigenic and / or immunomodulatory substances from a bucket. International Patent Publication No. WO 93/16727 discloses M.E. Disclosed is the use of antigenic and / or immunomodulatory substances from a bucket. International Patent Publication No. WO 95/26742 discloses M.E., which slows or prevents tumor growth or spread. Antigenicity and / or
Alternatively, the use of an immunomodulator is disclosed. International Patent Publication WO 91/0254
2 autoclaved M. in the treatment of chronic inflammatory diseases where the patient exhibits abnormally high release of IL-6 and / or TNF, or the patient's IgG exhibits an abnormally high rate of galactosyl-free IgG. Disclose the use of a bucket. The diseases described in this publication include psoriasis, rheumatoid arthritis, mycobacterial disease, Crohn's disease, primary biliary cirrhosis, sarcoidosis, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, Guillain-Barre syndrome , Primary diabetes, and certain aspects of graft rejection.

[0008] M. Buckets are clearly unique among known mycobacterial species in that heat killed formulations retain vaccine and immunotherapeutic properties. For example, M. used for vaccination against tuberculosis. Tuberculosis BCG vaccine utilizes live strains. Em killed by heat. Bovis BCG and M. Tuberculosis has no protective properties when used in vaccines. Many compounds have been isolated from a range of mycobacterial species with adjuvant properties. The effect of such adjuvants is to essentially stimulate a specific immune response mechanism against antigens from another species.

[0009] There are generally two classes of compounds isolated from mycobacterial species that exhibit adjuvant properties. The first is the water-soluble wax D fraction (RG White, I. Bernstock).
k), RGS Johns and E. Lederer, Immunology, 1: 54,19.
58; U.S. Patent No. 4,036,953). Second, U.S. Pat.
No. 81 and 4,036,953, a muramyl dipeptide-based substance (substantially equimolar amounts of N-acetylglucosamine and N-glycolylmuramic acid). These compounds are used in the lipidation and deglycolipidation of the present invention. It differs from DD (DD-M. BACKET) in that its composition is as follows: Although they are water-soluble agents, DD-M. Buckets are insoluble in aqueous solutions. 2. They consist of a series of small oligomers of mycobacterial cell wall units, either extracted from bacteria by various solvents or digested from cell walls by enzymes.
In contrast, DD-M. Buckets contain highly polymerized cell walls. 3. All proteins have been removed from the preparation by digestion with proteolytic enzymes. The only components of the formulation are components of the cell wall peptidoglycan structure,
That is, alanine, glutamic acid, diaminopimelic acid, N-acetylglucosamine, and N-glycolylmuramic acid. In contrast, DD-M. Buckets contain 50% w / w protein, including many different protein species.

[0010] Delivery of vaccines to lung tissue by nasal aerosol or to the gastrointestinal tract by oral delivery has generally been limited to attenuated strains of the virus. For example, vaccination against poliovirus has used oral delivery of attenuated strains of the virus since the development of the Sabin vaccine. Avilon Incorporated (Avi
ron Incorporated) and the National Institute of Allergy and Infectious Diseases in the United States recently reported the successful use of a flu vaccine administered in a nasal spray. In this case, the live attenuated influenza strain protected 93% of the influenza in children. Consisting of dead viruses or bacteria,
Or vaccines consisting of recombinant proteins have not yet been delivered by nasal aerosol or oral delivery. There are several reasons for this. There are few reports of successful intranasal administration of these agents to produce T cell immunity or antibody synthesis. In addition, oral administration of proteins and dead microorganisms often results in resistance, which is the exact opposite result of successful immunization.

[0011] Sarcoidosis is an unexplained disease characterized by granulomatous inflammation that affects many organs of the body, especially the lungs, lymph nodes, and liver. Sarcoid granulomas contain mononuclear phagocytes, epithelioid and giant cells in the center, and T lymphocytes. Although CD4 T lymphocytes are closely associated with epithelioid cells, both CD4 and CD8 T lymphocytes accumulate in the periphery. The characteristic immunological abnormalities of sarcoidosis, hyperglobulinemia of peripheral blood and bronchoalveolar lavage fluid, and tuberculin and other similar antigens, for example Candida (Candida) and "delayed" of the skin to such mumps virus hypersensitivity Including a diminished response. The peripheral blood lymphocyte count is reduced and the peripheral blood CD4: CD8 ratio is reduced to about 1-1.5: 1. This is not a sign of general immune deficiency, but rather the result of the increased immunological activity "segmenting" into sites of disease activity. In patients with pulmonary sarcoidosis, the total number of cells recovered by bronchoalveolar lavage increased 5-10 fold and the proportion of lymphocytes increased from less than 10-14% of normal to 15% to 50%. More than 90% of the recovered lymphocytes are T lymphocytes, and the CD4: CD8 ratio is reported to have increased from a normal control of 1.8: 1 to 10.5: 1. These T lymphocytes are mainly of the Th1 class, which produces IFN-γ and IL-2 cytokines, rather than the Th2 class. After treatment, the increase in Th1 lymphocytes in the sarcoid lung is corrected.

[0012] Sarcoidosis is involved in the lung in almost all cases. Even when the lesions are found primarily in other organs, they usually accompany the lungs asymptomatically. Although sarcoidosis may disappear spontaneously, about 50% of patients have at least mild permanent organ dysfunction. In severe cases, pulmonary fibrosis develops and progresses to lung failure requiring lung transplantation. The main treatment for sarcoidosis is corticosteroids. Patients who respond early to corticosteroids often relapse and require treatment with other immunosuppressive drugs such as methotrexate or cyclosporine.

[0013] Asthma is a common disease, common in the developed world. Asthma is characterized by increased responsiveness of the tracheobronchial tree to a variety of stimuli, an early physiological impairment of which is reversible airflow restriction, which occurs spontaneously or is drug-related. A prominent pathological feature, though likely to occur, is inflammation of the airways. Clinically, asthma can be further divided into extrinsic and endogenous.

Extrinsic asthma has identifiable sediments and can be considered atopic, occupational, and drug-induced. Atopic asthma has specific immunoglobulin E (Ig
E), associated with enhanced Th2-type immune response with positive skin tests for common air allergens and / or atopic symptoms. The symptom can be further classified as seasonal or year-round. Airflow obstruction in extrinsic asthma
Due to non-specific bronchial hypersensitivity caused by inflammation of the airways. This inflammation is mediated by chemicals released by various inflammatory cells, including mast cells, eosinophils and lymphocytes. The action of these mediators results in vascular penetration, mucus secretion and bronchial smooth muscle contraction. In atopic asthma, the immune response that causes inflammation of the airways is due to the Th2 of T cells secreting IL-4, IL-5 and IL-10.
Brought by the class. Lymphocytes in the lungs of atopic asthma activate I
It has been shown to produce L-4 and IL-5. IL-4 and IL-5
Are both Th2 class cytokines and are required for IgE production and eosinophil involvement in asthma. Occupational asthma is caused by acid anhydrides in plastic workers and by plicatic acid in western red cedar-induced asthma.
cid) or related to non-IgE related mechanisms as seen in toluene diisocyanate induced asthma. Drug-induced asthma is seen after administration of aspirin or other non-steroidal anti-inflammatory drugs and is most common in certain subsets of patients who may exhibit other features such as nasal polyposis and sinusitis. Endogenous or latent asthma
It has been reported to occur after upper respiratory tract infections, but may occur newly in middle-aged or elderly people, and is more difficult to treat than exogenous asthma.

[0015] Ideally, asthma is prevented by avoiding the triggering allergen, but this is not always possible, and identifying the triggering allergen is not always easy. Treatment of asthma is based on the use of corticosteroids and bronchodilators to reduce inflammation and relieve airway obstruction. In chronic asthma, treatment with corticosteroids produces unacceptable side effects.

[0016] Another disease that has immunological abnormalities similar to asthma is allergic rhinitis. Allergic rhinitis is a common disease and it is estimated that at least 10% of the population is affected. Allergic rhinitis can be occasional (hay fever) caused by an allergy to pollen. Occasional or perennial rhinitis results from allergy to antigens such as house dust mites or animal dander.

The abnormal immune response of allergic rhinitis is characterized by overproduction of allergen-specific IgE antibodies. The inflammatory response occurs in the nasal mucosa rather than from the lower airways as in asthma. Similar to asthma, local eosinophils in the affected tissue are the main hallmark of allergic rhinitis. As a result of this inflammation, the patient sneezes, discharges secretions from the nose and becomes hyperemic. In more severe cases, the inflammation spreads to the eyes (conjunctivitis), palate and outer ear. Although not life-threatening, allergic rhinitis can render a person very incapacitated, interfere with normal activity, or impair their ability to work. Current treatments include:
Antihistamines, nasal decongestants, and asthma include the use of sodium cromoglycate and corticosteroids.

[0018] Lung cancer is the leading cause of cancer death. Lung cancer incidence continues to rise and the World Health Organization estimates that by the year 2000, 2 million new cases will be affected each year.
Lung cancer can be divided into two broad categories: small cell lung cancer (SCLC), which accounts for 20-25% of all lung cancers, and non-small cell lung cancer (NSCLC), which accounts for the remaining 75%.
). The majority of SCLC is caused by tobacco smoke. SCLC tends to spread quickly, and 90% of diagnosed patients have complications in the mediastinal lymph nodes of the chest. S
CLC is treated with chemotherapy or a combination of chemotherapy and radiation therapy. The complete response rate varies from 10% to 50%. In rare patients without lymph node involvement, surgery after chemotherapy can result in a cure rate greater than 60%. The prognosis of NSCLC is worse, with most patients progressing to disease by the time of diagnosis. Surgical removal of the tumor is only possible in a very small number of patients, and the 5-year survival rate of NSCLC is only 5-10%.

The factors that cause the development of lung cancer are complex and multiple. Environmental and genetic factors interact, causing continuous and incremental abnormalities, resulting in unlimited cell proliferation, infiltration of adjacent tissues and spread to distant sites (metastasis).

[0020] Both cellular and humoral immunity have been shown to be impaired in lung cancer patients. Radiation therapy and chemotherapy further impair the patient's immune function. Attempts have been made to immunize patients with inactivated tumor cells or tumor antigens to enhance the host's anti-tumor response. Bacillus Calmette-Guerin (BC
G) has been administered to the thoracic cavity after lung cancer surgery to enhance non-specific immunity. Attempts have been made to enhance anti-tumor immunity by administering to patients lymphocytes treated ex vivo with interleukin-2. Lymphocytes activated with these lymphokines have acquired the ability to kill tumor cells. Current immunotherapy of lung cancer is still in development and its efficacy as a standard lung cancer treatment technique has not yet been established.

In one aspect, the present invention relates to the treatment of skin diseases that appear to be related to factors affecting the balance of thymus-derived (T) immune cells known as Th1 and Th2. These T cells are identified by their cytokine secretion phenotype.
A common therapeutic feature is that M. et al. Have immunomodulatory properties that alter the balance of activity of these T cells as well as other immune cells. The use of a compound prepared from a cake.

[0022] Psoriasis is a common chronic inflammatory dermatosis and may be associated with various forms of arthritis in a small number of patients. Defects in psoriasis indicate excessively rapid growth of keratinocytes and detachment of scales from the skin surface. Pharmacotherapy is aimed at slowing down this process. The disease can occur at any age. Spontaneous decline is relatively rare and usually requires long-term treatment. In psoriasis, chronic scale-like erythema develops on the skin surface. Psoriasis is a very visible disease, often affecting the face, scalp, trunk, and extremities. The disease weakens the patient, both mentally and physically, and greatly reduces the quality of life. One to three million people in the United States suffer from psoriasis, and nearly 250,000 new cases each year. The conservative estimate of psoriasis cure in the United States is currently $ 248 million annually.

There are two main hypotheses regarding the etiology of psoriasis. First, genetic factors determine the abnormal growth of epidermal keratinocytes. Cells no longer respond normally to external stimuli, such as those that maintain epidermal homeostasis. Abnormal expression of cell membrane cytokine receptors or abnormal transmembrane signal transduction may underlie cell hyperproliferation. Inflammation associated with psoriasis occurs after release of proinflammatory molecules from hyperproliferating keratinocytes.

The second hypothesis is that T cells that interact with antigen presenting cells in the skin release proinflammatory and keratinocyte stimulating cytokines (Hancock, GE). ), Et al., J. Exp.
1402, 1988). Only genetically predetermined T cells of an individual have the ability to be activated in such an environment. Keratinocytes themselves may be antigen presenting cells. Cellular infiltrates of psoriatic lesions show an influx of CD4 + T cells, more notably CD8 + T cells (Boss, JD, et al., Arch. D.
ermatol. Res. 281: 23-3, 1989; Baker, BS, Br. J. Dermatol. 110: 555-5
64, 1984).

Due to the limited form of disease in most (90%) patients with psoriasis, dithranol, tar formulations, corticosteroids and recently introduced vitamin D3 homologs (
Topical treatments including calcipotriol (calcitriol) can be used. A small number (10%) of psoriasis patients are more severe and utilize many systemic treatment modalities. Specific systemic treatments include immunosuppressants such as UVB, PUVA, methotrexate, vitamin A derivatives (acytretin), and cyclosporin A. The efficacy of cyclosporine and FK-506 in treating psoriasis supports the T cell hypothesis as the primary cause of the disease (Boss, J. D. et al., Lancet.
II: 1500-1502, 1989; Ackerman, Ackerman
, C) et al. Invest. Dermatol. 96: 536 [abstract], 1991).

Atopic dermatitis is a chronic pruritic inflammatory dermatosis that usually occurs in families with a genetic predisposition to various allergic diseases such as allergic rhinitis and asthma. Atopic dermatitis occurs in about 10% of the total population. The main symptom is dermatitis (eczema) mainly localized on the flexed and extended sides of dry skin, face, neck and limbs, with severe itching. Typically starts by the age of two. In about 90% of patients, the skin disease resolves in childhood, but symptoms may persist into adults. It is the most common form of dermatitis worldwide. In atopy and atopic dermatitis, it is generally accepted that T cell abnormalities occur first, and dysfunction of T cells that normally regulates IgE production is responsible for this overproduction of immunoglobulins.

[0027] Allergic contact dermatitis is a common non-infectious inflammatory disease of the skin. In contact dermatitis, no immunological reaction occurs until the body is sensitized to a particular antigen.
When the skin is exposed to antigens, and these antigens are recognized by T cells, various cytokines are released, allowing T cells to proliferate and recruit, ultimately leading to dermatitis (eczema)
Occurs.

In allergic contact dermatitis lesions, only a small percentage of T cells are specific for the relevant antigen. Activated T cells probably migrate to sites of inflammation regardless of antigen specificity. Delayed type hypersensitivity can only be transmitted by T cells (CD4 ⁺ cells) that have MHC type II antigens. The "response" to the contact allergen is MHCI type (C
D8 ⁺ cells) or T cells with either type II (CD4 ⁺ cells) molecules (Sunday, ME, et al., J.I.
mmunol. 125: 1601-1605, 1980). Keratinocytes can produce interleukin-1, which can facilitate antigen presentation to T cells. Surface antigen-cell adhesion molecule-1 (ICAM-1) expression is induced on both keratinocytes and endothelium by cytokines, tumor necrosis factor (TNF) and interferon-gamma (IFN-γ).

If the cause can be identified, allergic contact dermatitis can be cured only by removing the cause.
During periods of inflammation, topical corticosteroids are useful. The inhibitory effect of cyclosporin was observed in vitro with delayed-type hypersensitivity of sensitized T cells to proinflammatory function (s) (Shidani, Shidan).
i, B. ) Et al., Eur. J. Immunol. 14: 314-318, 1984
). The inhibitory effect of cyclosporine on the early phase of T cell activation in mice has also been reported (Milon, G. et al., Ann. Immunol. (Pastur Institute) 135d: 237-245, 1984).

Alopecia areata is a common hair disorder, accounting for approximately 2% of consultations in dermatological outpatient clinics in the United States. A prominent feature of the disease is the formation of demarcated, round or oval scarless alopecia, which can occur in any area of the body's hair. The disease can occur at any age. Its onset is usually sudden, and its clinical history varies.

At present, it is not possible to attribute all or virtually all cases of alopecia areata to a single cause (Look, A. and Dauber, R. (Da)
wber, R), Hair and Scalp Diseases; Blackwell Science Press (Blackw
ell Scientific Publications) 1982: 272.
-30). Many factors are thought to be involved. It includes genetic factors, atopy, associations with diseases of putative autoimmune etiology, Down syndrome and emotional stress. Patients with alopecia areata have a high prevalence of atopy. There is evidence that alopecia areata is an autoimmune disease. This evidence is associated with an increased number of Langerhans cells,
Consistent histopathological findings of lymphocytic T-cell infiltrates in and around hair follicles, observation that alopecia areata responds to treatment with immunomodulators, and that of alopecia areata and a wide variety of autoimmune diseases (Mitchell, AJ) et al., J. Mol.
Am. Acad. Dermatol. 11: 763-775, 1984).

[0032] Immunophenotypic studies of scalp biopsy specimens show that expression of HLA-DR on epithelial cells in the cortex and hair follicles where active alopecia areata lesions are likely to develop, and in and around the hair follicle T cell infiltration is shown with a high percentage of helper / inducer T cells, increased Langerhans cell number and expression of ICAM-1 (Messenger, AG, et al., J. Invest.
. Dermatol. 85: 569-576, 1985; Gupta, AK (
Gupta, A .; K. ) Et al. Am. Acad. Dermatol. 22: 2
42-250, 1990).

The various types of therapeutic agents for alopecia areata can be divided into four categories: (i) non-specific local stimulants; (ii) "immunomodulators" such as systemic corticosteroids and PUVA; (iii) B.) Contact dermatitis-inducing agents, "immunopotentiators" such as cyclosporine and inosiplex; and (iv) drugs of unknown mechanism of action, such as minoxidil (Dawbe, R.P.
r, R. P. R. ) Et al., A dermatology textbook, Blackwell Science Publishing,
Fifth Edition, 1982: 2533-2638). Non-specific local irritants such as dithranol may act by hair regrowth, rather than local irritation, by an as-yet unidentified mechanism. Topical corticosteroids can be effective, but often require long-term treatment. Intralesional steroids have been found to be more effective, but their use has been limited to maintaining less severe disease border plaques or eyebrow regeneration in alopecia areata. Photochemotherapy proved effective, probably due to an altered functional subset of T cells. Local immunotherapy by inducing and maintaining allergic contact dermatitis to the scalp can regenerate hair in 70% of alopecia areata patients. Diphencypron (d
iphencyprone) is a potent sensitizer without mutagenic activity. Oral cyclosporine may be effective in a short period of time (Gupta, Gupta
, A. K. ) Et al. Am. Acad. Dermatol. 22: 242-25
0, 1990). Inosiplex, an immunostimulant, has been shown to be effective in open trials. It has been reported that a topical 5% minoxidil solution was able to induce some hair growth in alopecia areata patients. Its mechanism of action is unknown.

[0034] Skin cancer is a major public health problem because of its frequency and the inconvenience and ugliness it causes. Skin cancer is mainly found in mature individuals, especially those with fair skin that have been exposed to large amounts of sunlight. Annual treatment costs and time lost from work exceed $ 250 million annually in the United States alone. Three main types of basal cell carcinoma, squamous cell carcinoma and melanoma are clearly associated with sun exposure.

[0035] Basal cell carcinoma is an epithelial tumor of the skin. They appear preferentially in exposed skin areas. In a recent Australian study, the incidence of basal cell carcinoma was
There were 652 new cases per 100,000 population. This corresponds to 160 cases of squamous cell carcinoma or 19 cases of malignant melanoma (Gills, G
Giles, G) et al., Br. Med. J. 296: 13-17, 1988). Basal cell carcinoma is the most common of all cancers. Lesions are usually removed by surgery. Other treatments include retinoids, 5-fluorouracil, cryotherapy and radiation therapy. Alpha or gamma interferons have also been shown to be effective in treating basal cell carcinoma and provide a valuable alternative to patients who are inappropriate for surgery or who do not want to leave surgery (Cornell et al.) J. Am.
Acad. Dermatol. 23: 694-700, 1990; Edwards,
Edwards, L. et al. Am. Acad. Dermatol. 22
: 496-500, 1990).

[0036] Squamous cell carcinoma (SCC) is the second most common skin malignancy and is increasing in frequency. The number of advanced and metastatic cases associated with many underlying factors is increasing. Currently, metastatic SCC causes more than 2000 deaths annually in the United States, with a 5-year survival rate of 35% and 90% metastasis within 3 years. Metastasis almost always occurs at the site of the first lymphatic drainage. Clearly, medical treatment is needed for advanced cases. A way to successfully treat early SCC of the skin would eliminate the need for surgical resection with possible scars and other side effects. This development may be particularly desirable for facial lesions.

Interferons (IFNs) have also been used in the treatment of melanoma due to their anti-proliferative and immunomodulatory effects in vitro (Kirkwood,
JK (Kirkwood, JM) et al. Invest. Derm
atol. 95: 180S-4S, 1990). In metastatic melanoma, response rates achieved with high or low doses of systemic IFN-α ranged from 5-30%.
Recently, promising results (30% response) have been obtained with a combination of IFN-α and DTIC. Preliminary observations indicated that adjuvanted IFN-
A beneficial effect of α is suggested. Despite the low efficacy of IFN monotherapy in metastatic disease, several prospective randomized studies using IFN as adjuvants or in combination with chemotherapy are currently being conducted (Macleod, McLeod, GR, et al., J. Invest. Dermato.
l. 95: 185S-7S, 1990; Ho, VC, et al. Invest. Dermatol. 22: 159-76, 1990).

Of all available skin virus lesion treatments, only interferon is
It has a special mechanism of antiviral action that regenerates the body's immune response to infection. Interferon treatment does not cure the virus, but may help with the symptoms of some infections. Interferon therapy is also associated with systemic side effects,
It requires several injections into each wart, which is quite economical (Kraus, SJ, et al., Review of Infe.
ctious Disease 2 (6): S620-S632, 1990; Fraser, IH, Current Opi.
nion in Immunology 8 (4): 484-491, 1996.
).

SUMMARY OF THE INVENTION Briefly stated, the present invention relates to M. Compositions present or derived from a bucket are provided and their use in the prevention, treatment and diagnosis of diseases including mycobacterial infections, respiratory immune disorders and skin disorders. This law
Administering a composition having antigenic and / or adjuvant properties.
Respiratory diseases that can be treated using the compositions of the present invention include mycobacterial infections (
For example, M. Tuberculosis and / or M. Avium infection by (M. Aviu m)), including asthma, sarcoidosis and lung cancers. Skin diseases that can be treated using the compositions of the present invention include psoriasis, atopic dermatitis, allergic contact dermatitis, alopecia areata, and skin cancers, basal cell carcinoma, squamous cell carcinoma and melanoma including. Also provided are vaccines or adjuvants for use in immunotherapy of infectious diseases and cancer.

[0040] In a first aspect, there is provided an isolated polypeptide from Mycobacterium bakke, comprising an immunogenic portion of an antigen or a variant of said antigen. In a specific embodiment, the antigen is a computer algorithm BL as described below.
When measured using an alignment prepared by ASTP, (a) SEQ ID NOs: 143, 145, 147, 152, 154, 156, 158, 1
60, 162, 165, 166, 170, 172, 174, 177, 178, 1
81, 182, 184, 186, 187, 192, 194, 196, 197, 1
Sequences listed in 99, 201, 203, 205 and 207; (b) SEQ ID NOs: 143, 145, 147, 152, 154, 156, 158, 1
60, 162, 165, 166, 170, 172, 174, 177, 178, 1
81, 182, 184, 186, 187, 192, 194, 196, 197, 1
Sequences having at least about 50% identical residues to the sequences listed in 99, 201, 203, 205 and 207; (c) SEQ ID NOs: 143, 145, 147, 152, 154, 156, 158, 1
60, 162, 165, 166, 170, 172, 174, 177, 178, 1
81, 182, 184, 186, 187, 192, 194, 196, 197, 1
Sequences having at least about 75% identical residues to the sequences listed in 99, 201, 203, 205 and 207; and (d) SEQ ID NOs: 143, 145, 147, 152, 154, 156, 158, 1
60, 162, 165, 166, 170, 172, 174, 177, 178, 1
81, 182, 184, 186, 187, 192, 194, 196, 197, 1
99, 201, 203, 205 and 207 comprising an amino acid sequence selected from the group consisting of sequences having at least about 95% identical residues to the sequences recited in

[0041] Also provided are DNA sequences encoding the polypeptides of the present invention, expression vectors containing these DNA sequences, and host cells transformed or transfected with the expression vectors. In another aspect, the present invention provides a fusion protein comprising at least one polypeptide of the present invention.

In another aspect, the present invention provides a pharmaceutical composition comprising the polypeptide of the present invention, or a DNA molecule encoding the polypeptide, and at least one physiologically acceptable carrier. . The invention also provides a vaccine comprising at least one of the above polypeptides, or a DNA sequence encoding the polypeptide, and at least one non-specific immune response enhancer (enhancer).
In certain embodiments, the non-specific immune response enhancer is a delipidated and deglycolipidated MM. Bucket cells; inactivated M. Bucket cells; delipidated and deglycolipidated M without mycolic acid. Bucket cells; delipidated and deglycolipidated M. except for mycolic acid and arabinogalactan. Bucket cells; and M. It is selected from the group consisting of bakey culture filtrates.

In yet another aspect, it is provided to enhance a patient's immune response comprising administering to the patient an effective amount of one or more of the above pharmaceutical compositions and / or vaccines. In one embodiment, the immune response is a Th1 response. In a further aspect of the invention there is provided a method of treating a disease in a patient, comprising administering to the patient a pharmaceutical composition or vaccine of the invention. In certain embodiments, the disease is selected from the group consisting of an immune disease, an infectious disease, a skin disease, and a respiratory disease. Examples of such diseases include mycobacterial infections, asthma and psoriasis.

[0044] In another aspect, the present invention provides an inactivated M.p. Bucket cells, delipidated and deglycolipidated M. Bucket cells or M. Provided is a method for treating an immune disease, an infectious disease, a dermatological disease or a disease of the respiratory system, comprising administering a composition comprising a baket culture filtrate.

[0045] Also provided are methods of enhancing an immune response to an antigen. In one embodiment, the method comprises the steps of: Administering a polypeptide comprising an immunogenic portion of a bucke antigen or a variant thereof. In a further embodiment, the method comprises the steps of delipidating and deglycolipidating MM. Bucket cells; delipidated and deglycolipidated M. except for mycolic acid and arabinogalactan. Administering a composition comprising a component selected from the group consisting of bucke cells.

In a further aspect of the present invention, there are provided methods and diagnostic kits for detecting a mycobacterial infection in a patient. In a first embodiment, the method comprises contacting a patient's skin cells with one or more of the above polypeptides and detecting an immune response in the patient's skin. In a second embodiment, the method comprises contacting a biological sample with at least one of the above polypeptides and detecting in the sample the presence of an antibody that binds the polypeptide or the group of polypeptides, and M of biological sample. Detecting a tuberculosis infection. Suitable biological samples include whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid and urine.

A diagnostic kit comprising one or more of the above polypeptides in combination with a device capable of contacting the polypeptide with the skin cells of a patient is provided. The invention also provides a diagnostic kit comprising one or more polypeptides of the invention in combination with a detection reagent.

[0048] In yet another aspect, the invention provides antibodies (both polyclonal and monoclonal) that bind to the above polypeptides and their use in detecting mycobacterial infections.

[0049] These and other aspects of the invention will become apparent with reference to the following detailed description and accompanying drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if individually incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention generally relates to compositions and methods for preventing, treating and diagnosing infectious and immune diseases. Diseases that can be effectively treated using the compositions of the present invention include respiratory diseases such as mycobacterial infections, asthma, sarcoidosis and lung cancer, and skin disorders such as psoriasis, atopic dermatitis, allergic Includes contact dermatitis, alopecia areata, and skin cancers, basal cell carcinoma, squamous cell carcinoma and melanoma.

[0051] Effective vaccines that provide protection against infectious microorganisms include at least two functionally distinct components. The first is an antigen, which can be a naturally occurring polypeptide or carbohydrate, which is processed by macrophages and other antigen presenting cells to show CD4 ⁺ or CD8 ⁺ T cells. This antigen becomes the "specific" target of the immune response. The second component of the vaccine is a non-specific immune response enhancer, called an adjuvant, which mixes with or incorporates the antigen. Adjuvants amplify a cellular or antibody immune response to structurally unrelated compounds or polypeptides. Some known adjuvants are B. pertussis, M. et al. Tuberculosis and M. Bovis BC
It is prepared from microorganisms such as G. Adjuvants can also include components designed to protect the polypeptide antigen from degradation, such as aluminum hydroxide or mineral oil. The antigenic component of the vaccine is M. Although containing polypeptides that direct an immune attack against certain pathogens, such as tuberculosis, adjuvants are often widely available in many different vaccine formulations. Certain known proteins, such as bacterial enterotoxins, can function as antigens to elicit a specific immune response and as adjuvants to enhance the immune response to unrelated proteins.

M. Certain pathogens, such as tuberculosis, and some cancers are effectively contained by an immune attack directed by CD4 ⁺ and CD8 ⁺ T cells known as cellular immunity. Other pathogens, such as poliovirus, also contain B
It also requires antibodies produced by the cells. These different classes of immune attacks (T
Cells or B cells) are regulated by different subsets of CD4 ⁺ T cells, commonly referred to as Th1 and Th2 cells. A desirable property of the adjuvant is CD4 ⁺ T
The ability to selectively amplify the function of Th1 or Th2 assembly in a cell. psoriasis,
Many skin disorders, including atopic dermatitis, alopecia and skin cancer, are associated with these Th
It appears to be affected by differences in cell subset activity.

The two types of Th cell subsets are well characterized in murine models and are defined by cytokines that release on activation. The Th1 subset is IL-2,
Secretes IFN-γ and tumor necrosis factor and mediates macrophage activation and delayed-type hypersensitivity responses. The Th2 subset releases IL-4, IL-5, IL-6 and IL-10, which stimulates B cell activation. Th1 and Th2 subsets inhibit each other, IL-4 inhibits Th1-type responses, IFN-γ
Inhibits Th2-type responses. Similar Th1 and Th2 subsets are found in humans and release the same cytokines as observed in murine models. In particular,
Most T cell clones of atopic human lymphocytes are similar to murine Th2 cells that produce IL-4, while few clones produce IFN-γ. Therefore, the Th2 subset is selectively expressed, followed by the production of IL-4 and the IF
By reducing the levels of N-γ producing cells, IgE production can be selectively enhanced. Amplification of Th1-type immune responses is central to the reversal of the disease state of many diseases including respiratory diseases, such as tuberculosis, sarcoidosis, asthma, allergic rhinitis and lung cancer.

Inactivated M. Bakke and M. Many compounds from buckets have both antigenic as well as adjuvant properties, which function to enhance a Th1-type immune response. The method of the present invention may be used to re-induce the immune activity of T cells in a patient. Utilize one or more of these antigens and adjuvant compounds from a bucket and / or its culture filtrate. Mixtures of such compounds are particularly effective for the methods disclosed herein. It is known that all mycobacteria contain many cross-reactive antigens, but it is not known whether they commonly contain adjuvant compounds. As shown below, inactivated M.A. Bucket and decoration (
Delipidated and deglycolipidated) forms of inactivated M. Bucket was found to exhibit the Th1 type of adjuvant properties, which is not found in many other mycobacterial species. Furthermore, M. Bucket is not only suitable for antigens of other origin, but also for M.L. It has been found that it also produces compounds that amplify the immune response against the buckeet antigen.

In one aspect, the invention provides immunotherapy of respiratory and / or pulmonary disease, including tuberculosis, sarcoidosis, asthma, allergic rhinitis and lung cancer in a patient to enhance a Th1-type immune response. I do. In one embodiment, the composition is delivered directly to the mucosal surface of the airways to and / or within the lung. However, the compositions may also be administered by the intradermal or subcutaneous route. A composition that may be usefully employed in such a method comprises at least one of the following components:
Bucket cells; M. Bucket culture filtrate; delipidated and deglycolipidated M. M. Bucket cells (DD-M. Bakke); A compound present in or derived from a bucket and / or its culture filtrate. As explained below,
Administration of such compositions results in specific T cell immune responses and M. It provides enhanced protection against tuberculosis infection and is also effective in treating asthma. The exact mechanism of action of these compositions in treating diseases such as asthma is unknown, but is believed to be to suppress the asthma-induced Th2 immune response.

The term “respiratory system” as used herein refers to the lungs, nasal tract, trachea and bronchial tract.

The term “airways to or located in the lungs” as used herein includes the nasal tract, mouth, tonsil tissue, trachea and bronchial tract.

The term “patient” as used herein refers to a homeothermic animal, preferably a human. Such a patient may be suffering from the disease or may be free of a detectable disease.
In other words, the method of the present invention can be used for inducing protective immunity for preventing or treating a disease.

In another aspect, the invention relates to the use of an immunotherapeutic agent to alter or redirect existing immune activity by altering the function of T cells into a Th1-type immune response. Including psoriasis, atopic dermatitis, alopecia, and skin cancer;
Provide immunotherapy for skin diseases. Compositions that can be usefully employed in the methods of the present invention include:
Contains at least one of the following components: inactivated M. Bucket cells; M. Bucket culture filtrate; modified M. Bucket cells; and M. Components and compounds present in or derived from the cake and / or its culture filtrate. As detailed below, multiple administrations of the composition, preferably by intradermal injection, have been shown to be very effective in treating psoriasis.

As used herein, the term “inactivated M. bakke” is used to kill by heat or with a 2.5 megarad dose of ⁶⁰ cobalt, as detailed in Example 7 below. Irradiated, M. Means Bucket. As used herein, the term "modified M. bakey" refers to delipidated M. Bucket cells, deglycolipidated M. Bucket cells, and delipidated and deglycolipidated M. Bucket cells (DD-M.
Bakey).

DD-M. The preparation of the bucket and its chemical composition is described below in Example 7. As described in detail below, the present inventor has described M. Removing the glycolipid component from the bucket removes the molecular component that stimulates natural killer (NK) cell interferon-gamma production, thus significantly reducing non-specific production of cytokines that have a number of deleterious side effects. That was shown.

In yet another aspect, the present invention relates to at least one M.p. An immunogenic portion of a Bucket antigen or a variant thereof, or at least one M.p. Provided is an isolated polypeptide comprising an adjuvant portion of a bucke protein. In specific embodiments, the polypeptide comprises an immunogenic portion of an antigen or a variant thereof, wherein the antigen comprises SEQ ID NOs: 1-4, 9-16, 18-21, 23, 25, 2,
6, 28, 29, 44, 45, 47, 52-55, 63, 64, 70, 75, 8
9, 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.

The term “polypeptide” as used herein encompasses amino acid chains of any length, including full-length proteins (ie, antigens), wherein the amino acid residues are linked by covalent peptide bonds. are doing. Thus, a polypeptide comprising one immunogenic portion of the antigen may be composed entirely of the immunogenic portion or may include additional sequences. Additional sequences can be found in M.E. The sequence may be derived from a Bucket wild-type antigen or heterologous, and the sequence may (but need not) be immunogenic. As described in detail below, the polypeptides of the present invention can be obtained from M.I.
It may be isolated from bucke cells or culture filtrate, or prepared by synthetic or recombinant means.

“Immunogenic” as used herein refers to the ability to elicit an immune response in a patient, such as a human, or in a biological sample. In particular, immunogenic antigens
Cell proliferation in a biological sample comprising one or more cell groups selected from the group consisting of T cells, NK cells, B cells and macrophages, where the cells are from an M. tuberculosis immunized individual. , Can stimulate interleukin-12 production or interferon-γ production. Exposure to immunogenic antigens generally forms immune memory, and re-exposure to antigens results in an enhanced, faster response.

The immunogenic portions of the antigens described herein can be found in Paul, Fundamental Immunology, Third Edition, Raven Press, 1993, pp. 147-146. It can be prepared and identified using known techniques, as summarized in 243-247. This technique involves screening a polypeptide portion of a natural antigen or protein for immunological properties. Representative proliferation and cytokine production assays described herein can be utilized for these screens. The immunogenic portion of the antigen, in such representative assays, elicits an immune response (eg, cell proliferation, interferon-γ production or interleukin-12 production) substantially similar to that produced by the full-length antigen. It is the part that produces. In other words, the immunogenic portion of the antigen will be at least about 20%, preferably about 65%, and most preferably about 10%, of the proliferation induced by the full-length antigen in the model proliferation assays described herein.
0% can be produced. The immunogenic portion may also, or alternatively, be capable of interferon-γ induced by the full-length antigen in the model assays described herein.
And / or may stimulate at least about 20%, preferably about 65%, most preferably about 100% production of interleukin-12.

M. Backey's adjuvant non-specifically stimulates the immune response. Bucket cells or M. It is a compound found in the culture filtrate of a bakey culture. Adjuvants enhance the immune response to immunogenic antigens and the process of memory formation. M. In the case of bucke proteins, these memory responses assist in Th1-type immunity.
The adjuvant can also be used in a biological sample comprising one or more cells selected from the group consisting of T cells, NK cells, B cells and macrophages, wherein the cells are from a healthy individual, in an interleukin. -12 production or interferon-γ production can be stimulated. Adjuvants may also or may not stimulate cell proliferation. This M. Bacquet adjuvants include, for example, SEQ ID NOs: 89, 1
17, 160, 162 or 201.

The term “polynucleotide” as used herein refers to a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, and includes HnRNA and mRNA molecules, both sense and antisense strands. , DNA and the corresponding RNA molecules, cDNA, genomic DNA and recombinant DNA,
As well as fully or partially synthetic polynucleotides. HnRNA molecules contain introns and generally correspond to DNA molecules on a one-to-one basis. mRNA molecules correspond to HnRNA and DNA molecules with truncated introns. A polynucleotide may consist of the entire gene, or any portion thereof. Operable antisense polynucleotides include fragments of the corresponding polynucleotides, and thus include "
The definition of "polynucleotide" includes all such operable antisense fragments.

The compositions and methods of the present invention also include variants of the above polypeptides and polynucleotides. As used herein, the term "variant" refers to at least about 40%, more preferably at least about 60%, even more preferably at least about 75%, and most preferably at least about 90% of the sequence of the invention. The same residue (
Nucleotides or amino acids). Percent identical residues is to align the two sequences to be compared, determine the number of identical residues in the aligned portion, divide that number by the total length of the invention or search sequence, and multiply the result by 100. Determined by

The polynucleotide or polypeptide sequences are aligned, and the percent identical nucleotides in a particular region can be determined relative to another polynucleotide using publicly available computer algorithms. Two exemplary algorithms for aligning polynucleotide sequences and identifying their similarity are BLASTN and FLASTN.
This is the ASTA algorithm. Polypeptide sequence similarity can be determined using the BLASTP algorithm. Both BLASTN and BLASTP software are NCBI anonymous FTP at / blast / executable /
Available on the server (ftp://ncbi.nlm.nih.gov).
BLASTN algorithm version 2.0.4 [February 24, 1998]
Setting the default parameters described in the document and distributing by algorithm is preferred for use in determining variants according to the invention. BLASTN and B
Use of the BLAST family of algorithms, including LASTTP, is available at the NCBI website at URL http: // www. ncbi. nlm. nih. go v / BLAST / newblast. html and Altschul, Stephen F. et al. (1997).
"Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Program" (a new generation of p
protein database search programs), Nuc
leic Acids Res. 25: 3389-3402.
Computer algorithm FASTA is, on the Internet ftp site f tp: // ftp. virginia. Available at edu / pub / fasta / . Version 2.0u4, February 1996, set to the default parameters described in the document and distributed algorithmically is preferred for use in determining variants according to the invention. The use of the FASTA algorithm has been described by W. R. Pearson and D. J. Lipman, "An Improved Means of Biological Sequence Analysis" (Impr.
overed Tools for Biological Sequence A
analysis, Proc. Natl. Acad. Sci. USA 85: 2
444-2448 (1988) and W. R. Pearson (WR
Pearson), "Rapid and Sensitive Sequence Comparison with FASTP and FASTA" (Rapid and Sensitive Sequence Comp).
arison with FASTP and FASTA), Methods
in Enzymology 183: 63-98 (1990).

The following operating parameters are preferred for alignment and similarity determination using BLASTN that gives an E value and% identity: Unix Operating Command: bla
stall -p blastn -d embldb-e 10 -G 1-
E 1-r 2-v 50-b 50-i queryseq -o result (result); and parameter default value: -p program name [string] -d database [string] -e expected value (E) [real number -Cost of opening G gap (zero is default behavior) [integer]-Cost of extending E gap (zero is default behavior) [integer] -r Nucleotide match acquisition (blastn only) [integer] -v Number of lines described (V) [integer] -b Number of alignments shown (B) [integer] -i Search file [read file] -o BLAST report output file [read 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-o results -p program name [string] -d database [string] -e expected value (E) [real number] -G cost of opening a gap (zero means default action and ) [Integer]-Cost of extending E gap (zero is default behavior) [integer] -v Number of single lines described (v) [integer] -b Number of alignments shown (b) [integer]- I Search file [File read] -o BLAST report output file [File read] Optional.

A “hit” to one or more database sequences by a search sequence issued by BLASTN, BLASTP, FASTA, or a similar algorithm aligns and identifies similar parts of the sequence. Hits are ordered by similarity and length of sequence overlap. Hits to database sequences generally indicate overlap with only a portion of the length of the search sequence.

The BLASTN and FASTA algorithms also calculate “expected” values for the alignment. The expected value (E) indicates the number of hits that can be "expected" to accidentally encounter a certain number of contiguous sequences when searching a database of a certain size. The expected value is used as a significance threshold to determine if a hit against a database, eg, a preferred EMBL database, indicates true similarity.
For example, an E value assigned to a hit of 0.1 would mean that a database of the size of the EMBL database would inadvertently find a match of 0.1 on alignments of sequences with similar scores. Interpret with the meaning. By this criterion, the aligned and matching portions of the sequence have a probability of being about 90% identical. For sequences with an E value of 0.01 or less on the aligned and matched portions, the probability of accidentally finding a match in the EMBL database is 1% or less using the BLASTN or FASTA algorithms.

According to one embodiment, the “variant” polynucleotide, for each polynucleotide of the invention, preferably has the same number or fewer nucleic acids as each polynucleotide of the invention, compared to the polynucleotide of the invention. Include sequences with an E value of 0.01 or less. That is, a denatured polynucleotide has a probability of being at least 99% identical to the polynucleotide of the present invention, and has an E value of 0.01 or less when measured using the BLASTN or FASTA algorithm set as default parameters. , Any array. In a preferred embodiment, the variant polynucleotide is a sequence having the same number or fewer nucleic acids as the polynucleotide of the present invention, has a probability of being at least 99% identical to the polynucleotide of the present invention, and has BLASTN or the default parameter set. Sequences with an E value of 0.01 or less as measured using the FASTA algorithm.

The variant polynucleotide sequences will generally hybridize under stringent conditions to the recited polynucleotide sequences. As used herein, “stringent conditions” refers to pre-washing with 6 × SSC, 0.2% SDS solution;
XSSC, hybridized overnight in 0.2% SDS solution;
Wash twice with 1.times.SSC, 0.1% SDS each at 5.degree. C .;
. Washing with 2 × SSC, 0.1% SDS is meant.

M. Some and other variants of the Bucket polypeptide may be prepared by synthetic or recombinant means. Synthetic polypeptides having less than about 100 amino acids, generally less than about 50 amino acids, can be prepared using techniques known to those of ordinary skill in the art. For example, the polypeptide may be synthesized using any of the commercially available solid phase techniques, such as the Merrifield solid phase synthesis method, which adds amino acids to a continuously extending chain of amino acids. Merrifield
d). Am. Chem. Soc. 85: 2149-2146, 1963. An automated polypeptide synthesizer is available from Perkin Elmer / Applied BioSystems, Inc.
Inc. ) (Foster City, CA
)) And can be obtained commercially and operated according to the manufacturer's instructions. Variants of the natural antigen or adjuvant can be prepared using standard mutagenesis techniques, such as oligonucleotide site-directed mutagenesis. Sections of the DNA sequence may also be removed using standard techniques that allow for the preparation of truncated polypeptides.

The polypeptides of the present invention may be conjugated (covalently linked) to a signal (or leader) sequence at the N-terminus of the protein that directs translocation of the protein simultaneously or post-translationally. The polypeptide can also be conjugated to a linker or other sequence to facilitate the synthesis, purification or identification of the polypeptide (eg, poly-His) or to enhance binding of the polypeptide to a solid support. I can make it. For example, a polypeptide can be conjugated to an immunoglobulin Fc region.

In general, M. Bucket antigen, and the DNA sequence encoding the antigen, may be prepared using any of a variety of methods. For example, soluble antigens can be obtained as described below. It can be isolated from the bakey culture filtrate. The antigen may also be a D that encodes the antigen.
It can be produced recombinantly by inserting the NA sequence into an expression vector and expressing the antigen in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be used. Expression can be achieved in a suitable host cell, which has been transformed or transfected with an expression vector containing a DNA molecule encoding the recombinant polypeptide. Suitable host cells include prokaryotic cells, yeast and higher eukaryotic cells. Preferably, the host cell used is E. coli , mycobacteria, insect, yeast or a mammalian cell line such as COS or CHO.
The DNA sequence so expressed may encode a naturally occurring antigen, a portion of a naturally occurring antigen, or a variant thereof.

M. The DNA sequence encoding the bucke antigen is amplified by a suitable M.C. hybridizing with a modified oligonucleotide derived from the partial amino acid sequence of the isolated soluble antigen. It can be obtained by screening a bucke cDNA or genomic DNA library for DNA sequences. Appropriate degenerate oligonucleotides can be designed and synthesized, and screening can be performed as described, for example, in Sambrook et al., Molecular Cloning: Molecular Cloning.
: A Laboratory Manual, Cold Spring Harbor Laboratory
ries), Cold Spring Harbor, New York, 1989. As described below, a nucleic acid probe can be isolated from genomic DNA, or a cDNA or genomic DNA library, using the polymerase chain reaction (PCR). Library screening can then be performed using the isolated probes. M. A DNA molecule encoding a bucke antigen is described in M. et al. Using an antiserum (e.g., rabbit or monkey) specifically raised against a Bucket antigen, the appropriate M.p. It can be isolated by screening a bucket expression library.

[0079] Regardless of the method of preparation, the antigens described herein have the ability to induce an immunogenic response. More specifically, the antigen is M. T from tuberculosis-immunized individuals
It has the ability to induce cell proliferation and / or cytokine production (eg, interferon-γ and / or interleukin-12 production) in cells, NK cells, B cells or macrophages. M. Tuberculosis immunized individuals are M. An individual who is considered to be resistant to the development of tuberculosis because it shows an effective T cell response to tuberculosis. Such an individual has a tuberculosis protein (P
It can be identified on the basis of a strongly positive intradermal skin test response to PD) (ie, a sclerosis diameter of about 10 mm or more) and no signs of tuberculosis infection. T cells, N
Assays for cell proliferation or cytokine production in K cells, B cells or macrophages can be performed, for example, using the methods described below. The choice of cell type to use in assessing the immunogenic response to the antigen depends on the desired response. For example, interleukin-12 production can be prepared using the methods described in M. et al. It is most easily evaluated using preparations containing T cells, NK cells, B cells and macrophages from tuberculosis immunized individuals. For example, peripheral blood mononuclear cells (PB
The preparation of MCs) may be used without further separating the constituent cells. PBM
Cs is, for example, Ficoll (registered trademark) (Winthrop.
Laboratories (Winthrop Laboratories, New York). T cells used in the assays described herein can be purified directly from PBMCs. Alternatively, an enhanced T cell line that is reactive to mycobacterial proteins, or a T cell clone that is reactive to individual mycobacterial proteins, may be used. This T cell clone is
For example, for 2-4 weeks, M.P. It can be produced by culturing PBMCs derived from tuberculosis-immunized individuals. As a result, only T cells specific to the mycobacterial protein proliferate, and as a result, a system consisting only of the cells is formed. These cells are then transformed using methods known in the art.
It can be cloned and tested with individual proteins to more precisely define individual T cell specificity.

In general, regardless of preparation method, the polypeptides disclosed herein are prepared in a fairly pure isolated form. Preferably, the polypeptide is at least about 80% pure, more preferably at least about 90% pure, most preferably at least about 99% pure.
% Pure. In certain preferred embodiments detailed below, a substantially pure polypeptide is incorporated into a pharmaceutical composition or vaccine for use in one or more of the methods disclosed herein.

The present invention also provides first and second polypeptides of the present invention, or alternatively, a polypeptide of the present invention and a known polypeptide as described in M.E. Tuberculosis antigens such as Andersen and Hansen, Infect. Imm
un. 57: 2481-2488, 1989. A fusion protein comprising a 38 kDa antigen is provided along with a variant of the fusion protein. A fusion protein of the invention can also include a linker peptide between the first and second polypeptides.

[0082] The DNA sequence encoding the fusion protein of the present invention can be prepared using known recombinant DNA techniques to associate the separate DNA sequences encoding the first and second polypeptides with an appropriate expression vector. To construct. D encoding the first polypeptide
By linking the 3 'end of the NA sequence to the 5' end of the DNA sequence encoding the second polypeptide, with or without a peptide linker, the reading frame of the sequence is in phase and the two DNA sequences Translation of the mRNA
A single fusion protein is obtained that retains the biological activity of both the first and second polypeptides.

[0083] A peptide linker sequence may be used to separate the first and second polypeptides a sufficient distance to ensure that each polypeptide folds into its secondary and tertiary structure. The peptide linker sequence is incorporated into the fusion protein using standard techniques known in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) be able to assume a flexible and extended conformation;
(2) no secondary structure capable of interacting with functional epitopes on the first and second polypeptide; and (3) no hydrophobic or charged residues capable of reacting with the functional epitope on the polypeptide. It is. A preferred peptide linker sequence is G
Includes ly, Asn and Ser residues. Other nearly neutral amino acids, such as Thr
And Ala may also be used for the linker sequence. Amino acid sequences that can be usefully used as linkers are described in Maratea et al., Gene 40: 39-.
46, 1985; Murphy et al., Proc. Natl. Aca
d. Sci. USA 83: 8258-8262, 1986; U.S. Pat.
35,233 and U.S. Pat. No. 4,751,180. The length of the linker sequence can be 1 to about 50 amino acids. A peptide linker sequence is not required if the first and second polypeptides have a non-essential N-terminal amino acid region that can be used to separate functional domains and prevent steric hindrance.
The ligated DNA sequence encoding the fusion protein is cloned into an appropriate expression system using techniques known to one of ordinary skill in the art.

As described in detail below, the inventor has determined that the heat-killed M. of the present invention. Bucket,
DD-M. Bucket and recombinant M. Activate T cells and NK cells using the Bucket protein; stimulate the production of human PBMC cytokines (particularly Th1 class cytokines); enhance expression of costimulatory molecules on dendritic cells and mononuclear cells (And thus enhanced activation); demonstrated that dendritic cell maturation and function could be enhanced. Further, the present inventor has confirmed that M.I. Bucket protein GV-23
Demonstrated the similarity between the immunological properties of the two known Th1-derived adjuvants. Thus, GV-23 can be used for treating diseases involving enhanced Th1 immune response. Examples of such 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 is used as a dendritic cell or NK cell enhancer in the treatment of immunodeficiencies such as AIDS, and for example to enhance the immune and cytotoxic responses to cancerous malignant cells, and in chemotherapy and the like. For immunosuppressive anticancer therapy.

For use in the methods of treatment of the present invention, an inactivated M.A. Bucket, M. Bucket culture filtrate, modified M. Bucket cells, M. The bakey polypeptide, fusion protein (or polynucleotide encoding the polypeptide or fusion protein) is generally present in a pharmaceutical composition or vaccine. Pharmaceutical compositions may contain inactivated M.p. Bucket cells, M. Bucket culture filtrate, modified M. Buckey cells, and M. The one or more components selected from the group consisting of compounds present in or derived from the bucket and / or culture filtrate thereof may be included with a physiologically acceptable carrier. The vaccine is inactivated M.V. Bucket cells,
M. Bucket culture filtrate, modified M. Buckey cells, and M. One or more components selected from the group consisting of compounds present in or derived from the bucket and / or its culture filtrate may be included with the non-specific immune response amplifying agent.
The pharmaceutical compositions and vaccines may also include other mycobacterial antigens incorporated into the fusion protein or present on separate polypeptides, as described above.

[0086] Alternatively, a vaccine of the invention may comprise DNA encoding one or more of the polypeptides described above, wherein the polypeptide is produced in situ. In the vaccine, the DNA can be in a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Suitable nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (eg, a suitable promoter and terminator signal). Bacterial delivery systems involve the administration of bacteria (eg, Bacillus Calmette-Guerin) that express the immunogenic portion of the polypeptide on the cell surface. In a preferred embodiment, the DNA comprises a viral expression system (
(Eg, vaccinia or other poxvirus, retrovirus, or adenovirus), which may include the use of non-pathogenic, ie, defective, replication competent viruses. Techniques for incorporating DNA into the expression system are known in the art. DNA is described, for example, in Ulmer et al., Science.
259: 1745-1749, 1993; Cohen
, Science 259: 1691-1692, 1993. Uptake of naked DNA can be increased by coating the DNA with biodegradable beads, which are efficiently transported to cells.

The DNA vaccine may be administered simultaneously or sequentially with a polypeptide of the invention or a known mycobacterial antigen, such as the 38 kDa antigen described above. For example, after administration of the DNA encoding the polypeptide of the present invention, an antigen may be administered to enhance the protective immunity effect of the vaccine.

The route and frequency of administration and dosage will vary from individual to individual and may be similar to those currently used for immunization with BCG. Generally, the pharmaceutical compositions and vaccines may be administered by injection (eg, intradermal, intramuscular, intravenous or subcutaneous), intranasally (eg, by inhalation) or orally. 1-3 doses may be administered for 1-36 weeks. Preferably, three doses are administered at 3-4 month intervals, followed by periodic booster vaccinations. Protocols tailored to individual patients may be appropriate. A suitable dose is a polypeptide or DNA amount that, when administered as described above, is capable of raising the patient's immune response enough to protect the patient from mycobacterial infection for at least 1-2 years. Generally, it is present in a single dose (or D
The amount of polypeptide (produced in situ by NA) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, preferably from about 100 pg to about 1 μg. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 ml to about 5 ml.

In one embodiment, the pharmaceutical composition or vaccine is in a form suitable for delivery to the mucosal surface of the airways to or within the lungs. For example, a pharmaceutical composition or vaccine may be suspended in a liquid formulation for delivery to a patient in aerosol form or using a spray device similar to those currently used in asthma treatment. In other embodiments, the pharmaceutical compositions or vaccines are in a form suitable for injection (intradermal, intramuscular, intravenous or subcutaneous) or oral administration. Although any suitable carrier known to those of ordinary skill in the art may be used in the pharmaceutical compositions of the invention, the type of carrier will vary depending on the suitability for the chosen route of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably includes water, saline, alcohol, lipids, waxes or buffers. For oral administration, any of the above or solid carriers may be employed, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose, and magnesium carbonate. Biodegradable microspheres (eg, polylactic galactide may also be used as carriers in the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are described, for example, in US Pat. Nos. 4,897,268 and 5). , 0
No. 75,109.

Any of a variety of adjuvants can be used in the vaccines of the invention to non-specifically enhance an immune response. Most adjuvants include substances designed to protect antigens from rapid catabolism, such as aluminum hydroxide or mineral oil, and lipid A, B. pertussis, M. et al. Tuberculosis, or M. discussed below. Non-specific immune response stimulators, such as buckets, are included. Suitable adjuvants include, for example, Freund's incomplete adjuvant and Freund's complete adjuvant (Difco Laboratories, Detroit, Michigan) and Merck Adjuvant 65 (Merck & Company, Inc.).
Merc and Company), Lowway, New Jersey). Other suitable adjuvants include aluminum, biodegradable microspheres, monophosphoryl lipid A and Quil A.

In another aspect, the present invention provides a method for diagnosing tuberculosis using a skin test.
Methods for using one or more polypeptides of the invention are provided. As used herein, a "skin test" is any assay performed directly on a patient, wherein a delayed type hypersensitivity (DTH) response (eg, swelling, redness or dermatitis) is determined by one of the methods described above. It is measured after intradermal injection of one or more polypeptides. Preferably, the response is measured at least 48 hours after injection, more preferably 48-72 hours.

The DTH response is a cellular immune response that is greater in patients who have been previously exposed to the test antigen (ie, the immunogenic portion of the polypeptide used, or a variant thereof). The response may be measured visually using a ruler. Generally, a diameter of about 0.
A response of 5 cm or more, preferably about 1.0 cm or more in diameter, is a positive response indicating a tuberculosis infection.

For use in skin tests, the polypeptides of the invention may preferably be formulated as pharmaceutical compositions comprising the polypeptide and a physiologically acceptable carrier, as described above. The composition typically comprises from about 1 μg to about 1 μg in a volume of 0.1 ml.
It contains an amount of one or more of the above polypeptides in an amount ranging from 00 μg, preferably from about 10 μg to about 50 μg. Preferably, the carrier used in the pharmaceutical composition is a saline solution containing a suitable preservative, such as phenol and / or Tween 80®.

[0094] In a preferred embodiment, the polypeptide used in the skin test is of a size sufficient to remain at the injection site during the reaction. Generally, a polypeptide of at least 9 amino acids in length is sufficient. The polypeptide is also preferably degraded by macrophages or dendritic cells within hours after injection and presented to T cells.
The polypeptide may comprise a repeat of one or more of the above sequences or other immunogenic or non-immunogenic sequences.

In another aspect, there is provided a method for detecting a mycobacterial infection in a biological sample, using one or more of the polypeptides of the invention, alone or in combination. In embodiments using multiple polypeptides, the 38 kD
Polypeptides other than those specifically noted herein, such as the a antigen, may also be included. As used herein, a "biological sample" is any antibody-containing sample obtained from a patient. Preferably, the sample is whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid or urine. More preferably, the sample is a blood, serum or plasma sample obtained from a patient or a blood supply. The polypeptide (s) are used in an assay as described below to determine the presence or absence of antibodies to the polypeptide in a sample with respect to a predetermined cutoff value. The presence of the antibody indicates the presence of a mycobacterial infection.

In embodiments that use one or more polypeptides, the polypeptides used are preferably complementary (ie, one component of the polypeptide detects an infection in a sample, while the other Is not detected by the polypeptide of the component (1). Complementary polypeptides can generally be identified by using each polypeptide individually to evaluate serum samples from a series of patients known to be infected with mycobacteria. After each polypeptide has been used to determine which sample tested positive (described below), a combination of two or more polypeptides capable of detecting infection can be formulated in most or all of the samples tested. For example, about 25-30% of sera from tuberculosis infected individuals are negative for antibodies to any single protein, such as the 38 kDa antigen described above. Therefore, a complementary polypeptide can be used in combination with a 38 kDa antigen to increase the sensitivity of a diagnostic test.

Various assay formats are known in the art that use one or more polypeptides to detect antibodies in a sample. See, for example, Harlow and Lane, Antibodies: Experimental Manual, Cold Spring Harbor Laboratory, 1988. In a preferred embodiment,
The assay involves the use of a polypeptide immobilized on a solid support that binds to the antibody and removes the antibody from the sample. Thereafter, the bound antibody can be detected using a detection reagent that includes a reporter group. Suitable detection reagents include antibodies that bind to the antibody / polypeptide complex and free polypeptide labeled with a reporter group (eg,
Semi-competitive assay). Alternatively, a competitive assay may be used in which antibodies that bind to the polypeptide are labeled with a reporter group and the antigen is incubated with the sample and then bound to the immobilized antigen. The extent to which the components of the sample block the binding of the labeled antibody to the polypeptide is an indicator of the reactivity of the sample with the immobilized polypeptide.

[0098] The solid support can be any solid material to which the antigen can be attached. Suitable materials are
It is known in the art. For example, the solid support can be a test well in a microtiter plate, or nitrocellulose, or other suitable membrane. Alternatively, the support may be a bead or a disc, for example, glass, glass fiber, latex or a plastic material, for example, polystyrene or polyvinyl chloride.
The support may also be a magnetic particle or fiber optic sensor, for example, US Pat.
, 681.

[0099] Polypeptides can be attached to a solid support using various techniques known in the art. In the context of the present invention, the term "bound" refers to non-covalent associations such as adsorption,
And both covalent attachment, which can be a direct link between the antigen and the functional group on the support or a link using a cross-linking agent. Binding by adsorption to the wells or membranes of a microtiter plate is preferred. In such cases, adsorption can be achieved by contacting the polypeptide with the solid support in a suitable buffer for a suitable time. Contact time varies with temperature, but is typically about 1 hour to 1 day. Generally, contacting the wells (eg, polystyrene or polyvinyl chloride) of a plastic microtiter plate with an amount of polypeptide in the range of about 10 ng to about 1 μg, preferably about 100 ng, is sufficient for binding the appropriate amount of antigen. .

Covalent attachment of a polypeptide to a solid support generally involves first attaching the support to a solid support.
It can be achieved by reacting the polypeptide with a bifunctional reagent that reacts with both the support and the functional group (eg, a hydroxyl or amino group). For example, a polypeptide can be attached to a support with a suitable polymer coating using benzoquinone, or by condensation of an aldehyde group on the support with an amine and active hydrogen on the polypeptide. (See, for example, Pierce Immunology Catalog and Handbook, 1991, A12-A13).

[0101] In one embodiment, the assay is an enzyme-linked immunosorbent assay (ELISA). The assay can be performed by first contacting a polypeptide antigen, which has been immobilized on a solid support, with a sample, and binding an antibody against a polypeptide in the sample 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 detection reagent that remains bound to the solid support is then determined using methods appropriate for the specific detection reagent.

More specifically, once the polypeptide is immobilized on a support as described above,
The remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to one of ordinary skill in the art, such as bovine serum albumin or Tween 20® (Sigma Chemical Co., St. Louis, Mo.) may be used. The immobilized polypeptide is then incubated with the sample, allowing the antibody to bind to the antigen. Prior to incubating the samples, phosphate buffered saline (PB
It may be diluted with a suitable diluent such as S). In general, a suitable contact or incubation time is determined by M.E. The time is sufficient to detect the presence of the antibody in the tuberculosis infected sample. Preferably, the contact time is sufficient to achieve a binding level of at least 95% of the binding level achieved at equilibrium between the bound and unbound antibodies. The time required to achieve equilibrium can be readily determined by assaying the level of binding that occurs over a period of time. An incubation time of about 30 minutes at room temperature is generally sufficient.

[0103] Unbound sample may be removed by washing the solid support with a suitable buffer, such as PBS containing 0.1% Tween20®. The detection reagent can then be added to the solid support. Suitable detection reagents are any compounds that bind to the immobilized antibody-polypeptide complex and can be detected by any of a variety of methods known in the art. Preferably, the detection reagent comprises a binding agent (eg, protein A, protein G, immunoglobulin, lectin or free antigen) conjugated to a reporter group. Preferred reporter groups include enzymes (eg, horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. Conjugation of the binding agent to the reporter group can be achieved using standard methods in the art. Common binders conjugated to various reporter groups are also available from many vendors (eg, Zyme Laboratories (Zyme)
d Laboratories), San Francisco, California, and Pierce, Rockford, Ill.).

The detection reagent is incubated with the immobilized antibody-polypeptide complex for a time sufficient to detect the bound antibody. A suitable time can generally be determined by the manufacturer's instructions or by assaying the level of binding that occurs over a period of time. The unbound reagent is then removed and the bound detection reagent is detected using a reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally suitable. Spectroscopy can be used to detect dyes, luminescent and fluorescent groups. Biotin can be detected using avidin linked to different reporter groups (usually radioactive or fluorescent groups or enzymes). Enzyme reporter groups can be detected by adding a substrate (generally at a specific time), followed by spectroscopic or other analysis of the reaction product.

To determine the presence or absence of anti-mycobacterial antibodies in a sample, the signal detected from the reporter group that remains attached to the solid support is generally compared to the signal corresponding to the predetermined cut-off value. Compare. In one preferred embodiment, the cut-off value is the average signal obtained when the immobilized antigen is incubated with a sample from a non-infected patient. In another preferred embodiment, the cutoff value is determined by the method of Sackett et al., Clinical Epidemi.
ology: Basic Science of Clinical Medicine (A Basic Science f)
or Clinical Medicine), Little Brown and Co., 1985, p. 106
Determined using the receiver operator curve according to the method of -107.
Generally, a signal that is higher than a predetermined cutoff value is considered positive for mycobacterial infection.

The assay can also be performed in a rapid flow-through test or strip test format, where the antigen is immobilized on a membrane such as nitrocellulose. In a flow-through test, the antibodies in the sample bind to the immobilized polypeptide as the sample passes through the membrane. Thereafter, the detection reagent (eg, protein A-colloidal gold) binds to the antibody-polypeptide complex as the solution containing the detection reagent flows through the membrane. Thereafter, detection of the bound detection reagent can be performed as described above. In the strip test format, one end of the membrane to which the polypeptide is attached is immersed in a sample-containing solution. The sample travels along the membrane, through the area containing the detection reagent, to the location of the immobilized polypeptide. The concentration of the detection reagent in the polypeptide indicates the presence of the mycobacterial antibody in the sample. Typically, the concentration of the detection reagent at the site shows a pattern, such as a line, that can be confirmed with the eye. Without such a pattern, the result is negative. Generally, the amount of polypeptide immobilized on the membrane will be identifiable by eye if the biological sample contains sufficient levels of antibody to produce a positive signal in Eliza, as discussed above. The pattern is selected to be produced. Preferably, the amount of polypeptide immobilized on the membrane ranges from about 25 ng to about 1 μg, more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount (eg, a drop) of patient serum or blood.

There are a number of other assay protocols that are suitable for using the polypeptides of the present invention. The above description is exemplary only.

[0108] The present invention also provides antibodies to the polypeptides of the invention. Antibodies can be prepared by any of a variety of techniques known to those of ordinary skill in the art. For example,
Harlow and Lane, Antibodies: Laboratory Manual, Cold
See Spring Harbor Laboratory, 1988. In one such technique, an immunogen comprising an antigenic polypeptide is first injected into any of a wide variety of mammals (eg, mice, rats, rabbits, sheep, and goats). The immunogen is injected into the animal host and blood is periodically bled from the animal, preferably according to a predetermined schedule that has incorporated one or more booster immunizations. Thereafter, a polyclonal antibody specific for the polypeptide can be purified from the serum, for example, by affinity chromatography using the polypeptide linked to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest are described, for example, in Kohler and Milstein, Eur. J.
Immunol. 6: 511-519, 1976 and modifications thereof. Briefly, these methods involve the preparation of immortalized cell lines that can produce antibodies with the desired specificity (ie, reactivity with the polypeptide of interest). The cell line can be produced, for example, from spleen cells obtained from an animal immunized as described above. Thereafter, the spleen cells are derived from one of various techniques known in the art.
One can be used to immortalize by fusion of a myeloma cell fusion partner, preferably an immunized animal and a syngeneic one.

[0110] Monoclonal antibodies can be isolated from the supernatant of the resulting hybridoma colonies. In addition, various techniques may be used to increase the yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. afterwards,
Monoclonal antibodies may be collected from ascites fluid or blood.

The antibodies may be used in diagnostic tests to detect the presence of mycobacterial antigen using assays similar to those described in detail above or other techniques known to those of skill in the art,
M in the patient. Methods are provided for detecting mycobacterial infections, such as tuberculosis infection.

The diagnostic reagents of the present invention may also include a polynucleotide encoding one or more of the above polypeptides, or one or more portions thereof. For example, primers comprising at least 10 conjugated oligonucleotides of a polynucleotide of the invention can be used in a polymerase chain reaction (PCR) based test.
Similarly, probes comprising at least 18 conjugated oligonucleotides of a polynucleotide of the present invention can be used for hybridizing to specific sequences. Techniques for both PCR-based and hybridisation tests are known in the art. Thus, the primers or probes may be used in biological samples, preferably in sputum, blood, serum, saliva, cerebrospinal fluid or urine. It can be used to detect tuberculosis or other mycobacterial infections. DNA probes or primers comprising the above oligonucleotide sequences can be used alone or in combination with each other, or can be used with previously identified sequences such as the 38 kDa antigen discussed above.

The term “about,” as used herein with respect to weight percent of the composition, refers to the percentage of the stated percent.
From 10% to 10%. When used in terms of% identity or% probability, the term "about" contemplates variations from the stated% to the 1% unit.

The following examples are provided for purposes of illustration, not limitation. Example 1 M for tuberculosis. Effect of Immunization of Mice Using Bucket Heat killed M. in mice before testing in tuberculosis. Bucket or M. The effect of immunization using the culture filtrate of a bucket cake will be described.

M. Bucket (ATCC No. 15483) was added to sterile medium 90 (yeast extract,
2.5 g / l; tryptone, 5 g / l; glucose, 1 g / l) at 37 ° C. The cells are harvested by centrifugation and sterile Middlebrook containing glucose (M
idlebrook 7H9 medium (Difco Laboratories (Difco
Laboratories), Detroit, Michigan, USA) at 37 ° C. for 1 day. The medium was then centrifuged to pellet the bacteria and the culture filtrate was removed. Bacterial pellet was placed in phosphate buffered saline at 10 ¹⁰ em. Resuspended at a concentration of 10 mg / ml, equivalent to a Bucket microorganism. The cell suspension was then autoclaved at 120 ° C. for 15 minutes. The culture filtrate was passed through a 0.45 μm filter into a sterile bottle.

As shown in FIG. 1A, mice were injected with 1 mg, 100 μg or 10 μg of M.M. Immunization was performed using a bucket, and 3 weeks later, 5 × 10 ⁵ colony forming units (CFU)
) Raw em. Infection with tuberculosis H37Rv resulted in significant protection from infection. In this example, spleen, liver and lung tissue were
After a week, live bacilli were determined (expressed as CFU). Compared to the tissues of non-immunized control mice, the number of bacilli was reduced by 2 logs in liver and lung tissues and 1 log or more in spleen tissues. Em killed by heat. Immunization of mice with tuberculosis H37Rv was subsequently performed on live M. When infected with tuberculosis H37Rv, the mice did not have any significant protective effects on the mice.

FIG. 1B shows that mice were em. Immunization was carried out using 100 μg of the culture filtrate of the basket, 3
After a week, 5 × 10 ⁵ CFU M. Infection with tuberculosis H37Rv indicates that significant protection was obtained. Spleen, liver and lung tissues were
When collected from mice after a week and the viable bacterial count (CFU) was determined, a 1-2 log reduction was observed compared to non-immunized control mice.

Example 2 M. tuberculosis in Sinomolgus monkeys. Effect of Immunization of Intradermal and Intrapulmonary Routes Using Bucket Vaccine Heat-killed M. by intradermal and pulmonary routes in cynomolgus monkeys before attack with tuberculosis. Bucket or M. The effect of immunization using the culture filtrate of a bucket cake will be described.

M. killed by heat. Bakke and M. Bucket culture filtrate was prepared as described above in Example 1. Five groups of cynomolgus monkeys are used, each group containing two monkeys. Two groups of monkeys were killed by heat either intradermally or intrapulmonarily by heat. Immunized with a bucket, two groups of monkeys were injected intradermally or pulmonarily. Immunization was carried out using the culture filtrate of the basket, and the control group was not immunized. All immunogens were dissolved in phosphate buffered saline. For each group of monkeys, the composition used for immunization, the amount of immunogen, and the route of administration are provided in Table 1. Before immunization, all monkeys were weighed (weight kg), body temperature was measured (temperature), and erythrocyte sedimentation rate (ESR mm
/ Hr) and Mycobacterium. Purified protein (PP
Blood samples were taken to determine lymphocyte proliferation (LPA) upon in vitro challenge by D). ESR and LPA have been used as indicators of inflammatory T cell responses. These measurements were repeated 33 days after immunization. On day 34,
All monkeys received the same amount of em. A second immunization was performed using the bucket and immunization routes. To day 62, body weight, temperature, measuring the LPA on ESR and PPD, then all monkeys, by inserting the microorganisms directly to the right lung of immunized animals, Mycobacterium of 10 ³ colony forming units Infected with the Ertman strain of tuberculosis. Twenty-eight days after injection, LPA for body weight, temperature, ESR and PPD was measured for all monkeys, the lungs were X-rayed and live M.p. It was determined whether infection with tuberculosis caused pneumonia.

[0120]

[Table 1]

The results of these studies are set forth below in Tables 2A-E and are summarized below. Table 2A-M. Twenty-eight days after infection with tuberculosis Erdman, chest x-rays of control (non-immunized) monkeys showed a shadow in the upper right hilar region of both animals, suggesting the development of pneumonia. The condition progressed, and by 56 days after infection, x-ray showed disease in both lungs. As expected, as the disease progressed, both control animals lost weight and showed significant LPA for PPD, which was the It suggests strong T cell reactivity to tuberculosis. ESR measurements varied, but strong immunoreactivity was consistent. Table 2B-500 [mu] g of em. Two monkeys immunized twice intradermally with a bucket were em. 84 days after infection with tuberculosis, there were no signs of lung disease. The LPA response to PPD allows M.P. Although immunoreactive for tuberculosis, both animals continued to gain weight, indicating that they were consistently disease free. Table 2C-500 μg em. Two monkeys immunized twice with lungs using a bucket showed almost the same results as the animals in Table 2B. M. At 84 days after infection with tuberculosis, there were no signs of lung disease and there was consistent weight gain. Both animals exhibited an LPA response to PPD during the immunization phase (0-62 days) and after infection, which resulted in strong T cell responsiveness as a result of using the lung as a route of immunization and subsequent infection. Suggest that.

500 μg of total em. Two immunizations with a bucket were used to give live M.
The protective effect against subsequent infection by tuberculosis was consistently demonstrated. The data presented in Tables 2D and 2E represent 100 μg em. The effect of immunization using the culture filtrate of the basket was shown. Intradermally immunized monkeys showed signs of disease at 84 days post-infection, while intrapulmonary immunized monkeys showed one animal showing disease after 56 days and one animal Showed disease 84 days after infection. The onset of the disease was significantly delayed, suggesting that the immunization process provided protective immunity.

[0123]

[Table 2]

[0124]

[Table 3]

[0125]

[Table 4]

[0126]

[Table 5]

[0127]

[Table 6]

Example 3 M. against asthma in mice. Effect of Immunization with Bucket In this example, M. was killed by heat. Bucket and DD-M. Both buckets demonstrate that when administered to mice by the intranasal route, they can prevent the development of a pulmonary allergic immune response. This has been demonstrated in a mouse model of asthma-like allergen-specific lung disease. The severity of this allergic disease is reflected in the massive eosinophil accumulation in the lungs.

[0129] C57BL / 6J mice were challenged with 100 μl by intraperitoneal administration on days 0 and 14.
1 μl of ovalbumin in aluminum adjuvant, followed by 28 μl
On day 100 μg in 50 μl phosphate buffered saline (PBS) by intranasal route
Ovalbumin was administered. Mice accumulate eosinophils in the lungs, which were detected by washing the airways of anesthetized mice with saline, collecting the lavage fluid (bronchoalveolar lavage or BAL) and counting the number of eosinophils. .

As shown in FIGS. 2A and B, M. killed with 10 or 1000 μg of heat four weeks prior to intranasal challenge with ovalbumin. Bucket (FIG. 2A), or 10, 100 or 200 μg of DD-M. Groups of 7 mice that received either of the buckets (FIG. 2B) intranasally had a lower percentage of eosinophils in BAL cells collected 5 days after ovalbumin challenge compared to control mice. Control mice received PBS intranasally. Raw M. at a dose of 2 × 10 ⁵ colony forming units. Bovis BCG also reduced pulmonary eosinophils. The data in FIGS. 2A and B show the average and standard error per group of mice.

FIGS. 2C and D show 10 weeks later, one week prior to the ovalbumin challenge.
M killed by 00 μg heat. Bucket (FIG. 2C) or 200 μg DD-
M. Mice given either of the buckets (FIG. 2D) intranasally show lower eosinophil percentage compared to control mice. In contrast, treatment with live BCG one week before challenge with ovalbumin did not prevent the development of pulmonary eosinophils when compared to control mice.

As shown in FIG. 2E, M. killed with 1 mg of heat. Bucket or 20
0 μg DD-M. Intranasal (in) or subcutaneous (s.
c. ) Upon immunization, pulmonary eosinophils were lower after challenge with ovalbumin compared to control animals receiving PBS. In the same experiment, before attacking with ovalbumin,
Pasteur (BCG-P) and Connaught (BCG-C)
) Immunization with strain BCG also reduced the eosinophil percentage of BAL in mice.

[0133] Eosinophils are prominent blood cells in the airways of allergic asthma. Eosinophil secretion products are responsible for swelling and inflammation of mucosal lines in the airways of allergic asthma. FIG. 2A
Data shown in -E are heat killed M.E. Bucket or DD-M. Treatment with buckets has been shown to reduce pulmonary eosinophil accumulation and may be useful in ameliorating inflammation associated with eosinophils in the respiratory tract, nasal mucosa and upper respiratory tract. DD-M. Excluding mycolic acid and arabinogalactan. Bacquet mycolic acid was treated with a solution of potassium hydroxide (0.5% KOH) in ethanol at 37 ° C. for 48 hours to give DD-M. Removed from the bucket. Then, DD-M. Bucket cells were washed with ethanol and ether and dried. Arabinogalactan is 1% periodic acid in 3% acetic acid.
KOH treated DD-M. By further treatment at room temperature for 1 hour and then at room temperature for 1 hour with 0.1 M sodium borohydride. Removed from the bucket. After removing the arabinogalactan, the samples were washed with water and lyophilized. As shown in Table 3, DD-M. Minus mycolic acid was administered intranasally to mice sensitized with ovalbumin. DD-M. Excluding bucke and mycolic acid and arabinogalactan. Both buckets reduced eosinophil accumulation in bronchoalveolar lavage fluid after challenge with ovalbumin.

Therefore, M. killed by heat. Bucket, DD-M. DD-M. Without basket or mycolic acid and arabinogalactan. Administration of the bucket may reduce the severity of diseases involving similar immune disorders, such as asthma and allergic rhinitis. In addition, serum samples were collected from the mice of the experiment shown in FIG. 2E, and antibodies to ovalbumin were measured by a standard enzyme-linked enzyme-linked assay (EIA). As shown in Table 3A below, serum from mice infected with BCG was:
Ovalbumin-specific IgG1 levels were higher than sera from PBS controls. In contrast, M. Bucket or DD-M. Mice immunized with the bucket had the same or lower levels of ovalbumin-specific IgG1. I
Since the gG1 antibody is characteristic of the Th2 immune response, these results indicate that the asthma-induced T
heat killed M. h2 immune response. Bucket and DD-M. This is consistent with the effect of suppressing basketball.

[0135]

[Table 7]

[0136]

[Table 8]

Example 4 M. in Mice against Tuberculosis Bucket, DD-M. Bucket or recombinant M. Effect of Immunization with Bucket Protein In this example, M. was killed by heat without adding an adjuvant. Bucket, DD-M. Bucket or recombinant M. Using the Bucket protein or M.P. M. Pool killed by heat with heat from a pool of recombinant proteins from bucket. The effect of immunization by the combination of a bucket is shown.

Mice were injected twice ip three times apart with one of the following formulations: a) phosphate buffered saline (PBS, control); b) heat killed M. Bucket (500 μg); c) DD-M. D) 15 μg each of GV4P, GV7, GV9, GV27B and GV33 proteins
g) a pool of recombinant proteins (prepared as described below); and e) heat-killed M. Bucket and pool of recombinant proteins.

Three weeks after the last intraperitoneal immunization, mice were challenged with 5 × 10 ⁵ live H37RvM. Infected with tuberculosis. After an additional three weeks, the mice were sacrificed and their spleens homogenized to give M. p. Tuberculosis was assayed for colony forming units (CFU).

FIGS. 3A and 3B show data where each point represents an individual mouse. The number of CFUs recovered from control mice immunized with PBS alone was taken as the base dose. All data from experimental mice are expressed as the log of CFU that is below the baseline of control mice (ie, log protection). As shown in FIG. 3A, heat killed M.E. Bucket or DD-M. Mice immunized with the bucket showed on average a log reduction in CFU of> 1 or 0.5, respectively.

As shown in FIG. 3B, GV4P, GV7, GV9, GV27B and GV33
Spleens from mice immunized with a pool of recombinant proteins containing CFU had slightly lower CFU than control mice. However, GV4P, GV7, GV9, GV2
7B and GV33 were heat killed. When used together with a backpack, CFU
The log reduction of was> 1.5 on average.

[0142] This data is based on M.E. Bucket, DD-M. Bucket or M. The effect of immunization using a recombinant protein derived from a bucket was demonstrated. Bucket, DD-M. It suggests that buckets and recombinant proteins could be developed as vaccines against tuberculosis.

Example 5 Effect of Intradermal Injection of Heat Killed Mycobacterium Bacquet on Psoriasis in Human Patients This example demonstrates the heat killed Mycobacterium
Figure 3 shows the effect of two intradermal injections of a bucket.

M. Bucket (ATCC No. 15483) was added to sterile medium 90 (yeast extract,
2.5 g / l; tryptone, 5 g / l; glucose, 1 g / l) at 37 ° C. Cells are harvested by centrifugation and sterile Middlebrook 7H containing glucose.
Nine media (Difco Laboratories, Detroit, Michigan, USA) at 37 ° C for 1 day. The medium was then centrifuged to pellet the bacteria and the culture filtrate was removed. Bacterial pellet was placed in phosphate buffered saline at 10 ¹⁰ em. Resuspended at a concentration of 10 mg / ml, equivalent to a Bucket microorganism. The cell suspension was then autoclaved at 120 ° C for 15 minutes and stored frozen at -20 ° C. Before use, M. Thaw the bucket suspension and add 5 mg / ml in phosphate buffered saline.
And autoclaved at 120 ° C. for 15 minutes and dispensed 0.2 ml into sterile vials under sterile conditions for use by patients.

Twenty-four volunteer volunteer psoriasis men and women aged 15-61 years without any other systemic illness were licensed for treatment. Pregnant patients were not included. The patient's PASI score was 12-35. The PASI score measures the location, size, and degree of skin scaling of a psoriatic lesion in a living body. The above 12 PASI scores are:
It reflects the wide range of disease lesions on the living body. The study began with a 4-week washout period, during which patients did not receive systemic anti-psoriatic treatment or effective local therapy.

The 24 patients were then given 0.1 ml em. Buckets (equivalent to 500 μg) were injected intradermally. Three weeks later, the same amount of M. A second intradermal injection was performed using a bucket (500 μg). M. killed psoriasis with heat. From four weeks before the first injection of the bucket to twelve weeks after the first injection, the evaluation was as follows: PASI scores were determined at -4, 0, 3, 6, and 12 weeks; B. Patient interviews are performed at weeks 0, 3, 6, and 12; Pictures of psoriatic lesions and each patient were taken at 0, 3, 6, 9, and 12 weeks. The data shown in Table 4 describes the age, sex, and medical history of each patient.

[0147]

[Table 9]

All patients had a localized, non-ulcerated erythematous soft-sclerosis reaction at the injection site. No side effects were noted and no patient complained. The data shown in Table 5 below shows the heat killed M.E. Skin reactions measured at the injection site 48 hours, 72 hours, and 7 days after the first and second injections of the bucket. The data shown in Table 6 below are from M.E. At the time of the first injection of the bucket (day 0) and part 3
Is the PASI score of the patient after 6, 9, 12, and 24 weeks.

M. By 9 weeks after the first injection of the bucket, 16 of 24 patients had
It is clearly observed that a significant improvement in the PASI score is shown. Seven of 14 patients who performed a 24-week follow-up were stable with no clinical signs of a relapse of severe disease. These results indicate that inactivated M. in the treatment of psoriasis. 8 demonstrates the efficacy of multiple intradermal injections of a bucket. A PASI score of 10 or less reflects extensive lesion healing. Histopathology of skin biopsy suggested that normal skin structure had been restored. Of the first seven patients who performed a 28-week follow-up, only one relapsed.

[0150]

[Table 10]

[0151]

[Table 11]

M. Bucket-treated patients may have remissions (PASI score = 0)
. Remission or improvement of PASI can be long lasting. As an example, the patient PS-0
03 had a remission by the 20th week and still remained in the 80th week. In a total of 13 of the 24 patients, the PASI score improved by more than 50%.

Patient PS-001 showed remission at week 16 and at week 48 (PASI 2.
7). The vaccination was vaccinated again with the injection of the bucket, after which the PASI dropped from 17.8 (60 weeks) to 0.8 (84 weeks) and improved. Thus, repeated treatment can be beneficial to the patient.

Example 6 DD-M. For psoriasis in human patients. Effect of Intradermal Injection of Bucket This example demonstrates DD-M. Figure 3 shows the effect of two intradermal injections of a bucket.

Seven volunteer psoriasis patients, 18-45 years old, men and women without any other systemic disease were licensed for treatment. Pregnant patients were not included. The patient's PASI score was 12-24. As discussed above, the PASI score measures the location, size, and skin scale of psoriatic lesions in a living organism. 12 above
A person's PASI score reflects the wide range of disease pathology in vivo. The study began with a 4-week washout period, during which four patients did not receive systemic anti-psoriatic treatment or effective local therapy. Then, 7 patients were given 0.1 ml of D
D-M. Buckets (equivalent to 100 μg) were injected intradermally. Three weeks later, the same amount of DD-M. A second intradermal injection was performed using a bucket (100 μg).

Psoriasis was administered to M. From 4 weeks before the first injection of the bucket, 12 weeks after the first injection
Up to a week later, the following was evaluated: A. PASI scores were determined at -4, 0, 3, and 6 weeks; B. Patient interviews are performed at weeks 0, 3, and 6; Pictures of psoriatic lesions and each patient were taken at 0 and 3 weeks. The data shown in Table 7 describes the age, gender, and medical history of each patient.

[0157]

[Table 12]

All patients had a localized, non-ulcerated erythematous soft-sclerotic response at the injection site. No side effects were noted and no patient complained. The data shown in Table 8 is for DD-M. Skin reactions measured at the injection site 48 hours, 72 hours, and 7 days after the first injection of the bucket and 48 hours and 72 hours after the second injection.

[0159]

[Table 13]

The data shown in Table 9 below was obtained from DD-M. PASI scores of 7 patients 3, 6, 12 and 24 weeks after the first injection of the bucket (day 0).

[0161]

[Table 14]

DD-M. By three weeks after the first injection of the bucket, five patients were given PA
It can clearly be seen that a significant improvement in the SI score was shown. By week 24, 6 out of 7 patients had a significant improvement in PASI score.

As an example, patient PS-031 remitted at 32 weeks (PASI score = 0),
It also remained in remission when observed at 48 weeks. PAS of patient PS-025
The I-score dropped below 1 for more than 12 weeks. When psoriasis recurred at 48 weeks (PASI = 15.8), patients received DD-M. When the bucket is treated again, PA
The SI score rapidly dropped to 6.8 and 0.6 at 52 and 56 weeks, respectively, and improved.

Accordingly, DD-M. Treatment of psoriasis with a bucket can ameliorate the disease or confer long-term benefits. Repeat treatments may also be beneficial to the patient.

Example 7 Preparation of a Bucket-Derived Composition 4 illustrates the processing of the different components of the bucket.

Delipidation and Deglycolipidation (DD-) M. Preparation of Bucket and Composition Analysis Heat-killed M. The bucket was prepared as described in Example 1 above. Delipidated M. M. autoclaved to prepare a bucket. The bucket is pelletized by centrifugation, the pellet is washed with water, collected again by centrifugation,
It was then freeze-dried. This freeze-dried M. An aliquot of the bucket was placed on one side and this was lyophilized. Called a bucket. When used in experiments, resuspended in PBS to desired concentration. Lyophilized M. The bucket was treated with chloroform / methanol (2: 1) for 60 minutes at room temperature to extract lipids, and the extraction was repeated one more time. The delipidated residue extracted with chloroform / methanol was further treated with 50% ethanol, and refluxed for 2 hours to remove glycolipids. This 50% ethanol extraction was repeated twice. The pooled 50% ethanol extract was combined with M.E. It was used as a source of bakee glycolipids (see below). The residue extracted with 50% ethanol was lyophilized and weighed. Prepared delipidated and deglycolipidated M. The amount of the bucket was determined by the em. This was 11.1% of the initial wet weight of the bucket. For bioassays, delipidated and deglycolipidated M. The buckets (DD-M. Bucket) were resuspended in phosphate buffered saline under sonication and sterilized in an autoclave.

M. killed by heat. Bucket and DD-M. The composition analysis of the bucket is presented in Table 9. Em killed by heat. Compared to the insoluble fraction of the cake, DD
-M. There were significant changes in the fatty acid and amino acid compositions of the buckeet. According to the data presented in Table 9, M. killed by heat. The insoluble fraction of the bucket was 1
It contains 0% w / w lipid and the total amino acid content is shown to be 2750 nmol / mg, or about 33% w / w. DD-M. Bucket is 1.3% w
/ W lipids and 4250 nmol / mg, or about 51% w / w amino acids.

[0168]

[Table 15]

M. killed by heat. The insoluble fraction of the cake contains 10% w / w lipid,
DD-M. The bucket contains 1.3% w / w lipid.

[0170]

[Table 16]

M. killed by heat. The total amino acid content of the insoluble fraction of the cake was 2750 n
mol / mg, ie about 33% w / w. DD-M. The total amino acid content of the bucket is 4250 nmol / mg, or about 51% w / w.

M. in delipidated and deglycolipidated forms. Tuberculosis and M. Smegma ( M. smegmatis ) DD-M. Comparison of Bucket Composition Delipidated and Deglycolipidated M. Tuberculosis and M. Smegma,
Delipidation and deglycolipidation M. Prepared using the method described above for the bucket.
As shown in Table 10, DD-M. Bucket, DD-M. Tuberculosis
And DD-M. Significant difference in smegma amino acid composition% profile is shown
Did not. However, the total amount of protein is different, DD-M. Two of the bucket
The batch contained 34% and 55% protein, while DD-M. Tsu
Velcrosis and DD-M. Smegma has 79% and 72%
It contained protein.

[0173]

[Table 17]

Analysis of the monosaccharide composition indicated that DD-M. Bucket, and DD-M. Tuberculosis and DD-M. Significant differences are shown between smegma. DD-M.
The monosaccharide composition of the two batches of Bucket was identical, but DD-M. Tuberculosis and M. It was different from the monosaccharide composition of smegma. In particular, as shown in Table 11, DD-M. The bucket contains free glucose, but DD-M. Tuberculosis and M. Both smegmas were found to contain glycerol.

[0175]

[Table 18]

M. Backey Glycolipids The pooled 50% ethanol extracts were dried on a rotary evaporator, redissolved in water and lyophilized. The amount of glycolipids recovered was determined by the em. 1.2% of the initial wet weight of the bucket. Glycolipids were dissolved in phosphate buffered saline for bioassays.

Example 8 Delipidation and Deglycolipidation M. Bakke and M. Immunomodulatory Properties of Recombinant Proteins from Bucket This example demonstrates the different components M.M. Figure 4 shows the immunomodulatory properties of a bucket. Production of interleukin-12 from macrophages All M. killed by heat. Bucket and DD-M. Buckets were shown to have different cytokine stimulating properties. Stimulation of the Th1 immune response is enhanced by the production of interleukin-12 (IL-2) from macrophages. A different M. The IL-12 stimulating ability of the bucket preparations is shown below.

A group of C57BL / 6J mice was injected intraperitoneally with DIFCO thioglycolate, and three days later, peritoneal macrophages were collected and placed in cell culture with interferon-γ for 3 hours. The culture medium was replaced and all the M. killed (autoclaved) heat-killed (autoclaved) prepared as described above. Bucket, freeze-dried em. Bucket, DD-M. Bakke and M. Backey glycolipid was added. After an additional 3 days at 37 ° C, culture supernatants were assayed for the presence of IL-12 produced by macrophages. As shown in FIG. Bucket preparations stimulated the production of IL-12 from macrophages.

On the other hand, these M. Use the natural killer (NK)
The ability to stimulate interferon gamma production from cells was examined. Spleen cells were prepared from severe combined immunodeficiency (SCID) mice. These populations contain 75-80% of NK cells. Spleen cells were killed at 37 ° C. with various concentrations of heat. Bucket, DD-M. Bucket or M. The cells were incubated in a culture solution together with the backey glycolipid. According to the data shown in FIG. Bakke and M. Backey glycolipids stimulate the production of interferon gamma, but DD-
M. Bucket is demonstrated to stimulate interferon gamma relatively weakly. The combined data of FIGS. 4 and 5 show that all heat-killed M. DD-M. Bucket is shown to stimulate IL-12 more than interferon gamma.

These observations are based on M. The removal of lipid glycolipid components from the
The molecular components that stimulate interferon γ from K cells will be removed,
Thus, it indicates that an important cellular source of cytokines with numerous adverse side effects is effectively eliminated. Therefore, DD-M. Bucket retains the ability to enhance Th1 immunity by stimulating IL-12 production, but loses the non-specific effects that can be produced by stimulating interferon gamma production from NK cells.

The DD-M. Of the invention, prepared as follows: Bucket and many em. The adjuvant effect of the Bucket recombinant antigen was that IL from murine peritoneal macrophages
Determined by measuring stimulation of -12 secretion. 6A, B and C show C5
7BL / 6 mouse (FIG. 6A), BALB / c mouse (FIG. 6B) or C3H / H
10 shows separate experimental data of intraperitoneally administering DIFCO thioglycolate to a group of eJ mice (FIG. 6C). Three days later, peritoneal macrophages were collected and placed in culture with interferon gamma for 3 hours. Replace the culture medium with various concentrations of M. Bucket recombinant proteins GVs-3 (GV-3), GV-4P (GV-4P), G
Vc-7 (GV-7), GV-23, GV-27, M. killed by heat. Bucket, DD-M. Bakey (referred to as delipidated M. bakey in FIGS. 6A, B and C); Backey glycolipid or lipopolysaccharide was added. After 3 days at 37 ° C., culture supernatants were assayed for the presence of IL-12 produced by macrophages. As shown in FIGS. 6A, B and C, the recombinant protein and M.A. Bucket preparations stimulated the production of IL-12 from macrophages.

In a subsequent experiment, peritoneal macrophages sensitized with IFNγ from BALB / c mice were cultured at 40 μg / ml em. Stimulation with the Bucket recombinant protein and assayed for the presence of IL-12 produced by macrophages.
As shown in FIG. 7, in these experiments, macrophages sensitized with IFNγ produced IL-12 when cultured with the control protein, ovalbumin. However, the recombinant proteins GV24B, 38BP, 38AP, 27, 5, 2
7B, 3, 23 and 22B show IL- detected in control macrophage cultures.
More than twice the amount of 12 was stimulated.

All M. Bakke and M. Detection of non-specific immunoamplifying substance from culture filtrate of M. Bucket Bucket culture supernatant (S / N), dead M. Bucket, delipidated M. Bucket and delipidated and deglycolipidated M. Bucket (DD-M. Buckey) was tested in mice for adjuvant activity in developing a cytotoxic T cell immune response to ovalbumin, a structurally unrelated protein. This anti-ovalbumin-specific cytotoxic response was detected as follows. C57BL / 6 mice (2 per group) were tested in the following test adjuvant: Autoclaved M.M. Bucket; delipidated M. Bucket; delipidated M from which glycolipids were also extracted. Protein extracted with Bucket (DD-M. Bucket) and SDS; S treated with pronase (proteolytic enzyme)
DS protein extract; Bucket culture filtrate; and heat killed M. Tuberculosis or heat-killed M. Bovis BCG, M. M. phlei or M. Smegma or M. 100 μg ovalbumin was immunized by intraperitoneal injection, along with the bucke culture filtrate. Ten days later, the spleen cells were EL4 cells (C57BL / 6-derived T-cell lymphocytes) transfected with the ovalbumin gene, and thus E. coli expressing ovalbumin. G7 cells were used to stimulate in vitro for an additional 6 days. The spleen cells are then transformed into non-specific EL4 target cells or specific E. coli cells. The cells were assayed for their ability to kill cells expressing G7 ovalbumin. Killing activity was determined prior to killing activity by EL4 and E. coli. G7 cells were detected by the release of labeled chromium 51 (100 μCi per 2 × 10 ⁶ ). Killing or cytolytic activity is determined by the formula

[0184]

(Equation 1)

And expressed as% specific dissolution.

In general, ovalbumin-specific cytotoxic cells are known to be produced only in mice immunized with ovalbumin with an adjuvant, but not in mice immunized with ovalbumin alone. .

[0187] The schematic in Figure 7 shows the various M. pneumoniae cells for the production of cytotoxic T cells against ovalbumin in C57BL / 6 mice. Fig. 4 shows the effect of the adjuvant preparation derived from a bucket. As shown in FIG. 7A, cytotoxic cells consisted of (i) 10 μg, (ii)
) 100 μg or (iii) 1 mg of autoclaved M. Bucket or (iv) 75 μg em. Produced in mice immunized with the Bucket culture filtrate. Figure 7B shows that cytotoxic cells were (i) 1 mg of autoclaved whole M. Bucket or (ii) 1 mg of delipidated and deglycolipidated (DD-) M. This shows that the protein was produced in mice immunized with a bucket.
As shown in FIG. 7C (i), the cytotoxic cells were 1 mg of autoclaved whole M.V. Produced in mice immunized with a bucket; FIG. 7C (ii) shows DD-M. M. extracted from Sakae by SDS. Figure 2 shows the active ingredients of the Bucket soluble protein. FIG. 7C (iii) shows that the active substance of the adjuvant preparation of FIG. 7C (ii) was degraded by treatment with protease pronase. By comparison, 100 μg of SDS extracted protein (FIG. 7C (ii))
1 mg of all emc autoclaved. It showed a significantly stronger immunopotentiating ability than the bucket (FIG. 7C (i)).

1 mg heat killed M. Mice immunized with a bucket (FIG. 7D (i)) produced cytotoxic cells to ovalbumin, but 1 mg of heat-killed M.L.
Tuberculosis (FIG. 7D (ii)), 1 mg em. Bovis BCG (Fig. 7D
(Iii)) 1 mg em. Frey (FIG. 7D (iv)), or 1 mg of M.D. Mice immunized separately with smegma (FIG. 7D (v)) did not produce cytotoxic cells.

These findings indicate that heat killed M. Bucket and DD-M. Buckets demonstrate that they have adjuvant properties not found in other mycobacteria. In addition, delipidated and deglycolipidated M. Buckets remove NK cell stimulating activity but do not lack T cell stimulating activity.

In a separate experiment, DD-M.O.D. Was excluded from ovalbumin and mycolic acid and arabinogalactan. Mice immunized with 200 μg of buckets were also able to produce cytotoxic cells (28% -46% maximum lysis compared to <8% specific lysis of control mice immunized with ovalbumin alone). Dissolution).

The above M. Bucket culture filtrates were fractionated by isoelectric focusing and fractions were assayed for adjuvant activity in the anti-ovalbumin-specific cytotoxic response assay in C57BL / 6 mice described above. Peak adjuvant activity is p
I is 4.2-4.32 (fraction number 7-9), 4.49-4.57 (fraction number 13)
-17) and 4.81-5.98 (fractions 23-27). DD-M. Identification of Proteins in Bucket BALB / c mice were challenged with 50 μg of DD-M. Immunization was performed intraperitoneally using a bucket. Mice were killed at 6 weeks and their sera were collected. Serum was prepared from recombinant M.A. Antibodies to the Bucket-derived proteins were tested in a standard enzyme-linked immunoassay.

The antisera was tested in several enzyme-linked assays by several M. Neither did the Bucket recombinant protein react to ovalbumin, which was used as an unrelated negative control protein (data not shown). DD-M. Antisera from mice immunized with the backpack contained 12 em. Reacted with GV antigen from Bucket. The results are shown in Table 12 below. Therefore, the antiserum was GV3,5P, 5,7
, 9, 22B, 24, 27, 27A, 27B, 33 and 45 as DD-M. Identified as present in the bucket.

[0193]

[Table 19]

DD-M. Identified by T cell response. Bucket protein BALB / c mice in each footpad, combined with incomplete Freund's adjuvant 1
00 μg DD. M. Buckets were injected and killed 10 days later to obtain popliteal lymph node cells. Cells from immunized and non-immunized control mice were transformed with recombinant M.D. Stimulated in vitro with GV protein from Bucket. Three days later, cell proliferation and IFN
Gamma production was assessed.

DD-M. For GV protein. T cell proliferative response of lymph node cells from Bucket immunized mice DD-M. Lymph node cells from Bucket immunized mice did not proliferate in response to an unrelated protein, ovalbumin (data not shown). As shown in Table 13, lymph node cells from immunized mice were GV3,7,
Proliferated in response to 9, 23, 27, 27B and 33. Corresponding cells from non-immunized mice did not proliferate in response to these GV proteins, indicating that DD-M. Mice immunized with the bucket suggest that they have been immunized with these proteins. Therefore, M. Bucket-derived protein GV
3, 7, 9, 23, 27, 27B and 33 are DD-M. It appears to be in the bucket.

[0196]

[Table 20]

After challenge with GV protein in vitro, DD-M. IFNγ production by lymph node cells from Bucket-immunized mice

Lymph node cells from non-immunized mice were stimulated with IFNγ upon stimulation with GV protein.
Did not produce As shown in Table 14 below, DD-M. Lymph node cells from Bucket immunized mice were GV3, 5, 23, 27A, 27B, 33, 4
Upon stimulation with 5 or 46, IFNγ was secreted in a dose-dependent manner, suggesting that mice were immunized with these proteins. When cells from immunized mice were stimulated with an unrelated protein, ovalbumin, I
No FNγ production was detected (data not shown). Thus, the proteins GV3, 5, 23, 27A, 27B, 33, 45 and 46 were produced by DD-M. It appears to be in the bucket.

[0199]

[Table 21]

DD-M. As a non-specific immunoamplifier Proteins in Bucket In subsequent experiments, five proteins GV27, 27A, 27B, 23 and 4
5 was used with the ovalbumin antigen as a non-specific immunoamplifier and mice were immunized as described above in Example 6. As shown in FIG. 12, the recombinant protein GV
50 μg of any one of 27, 27A, 27B, 23 and 45 was added to 50-1
When injected with 00 μg ovalbumin, it showed adjuvant properties capable of producing cytotoxic cells to ovalbumin.

Example 9 M. Autoclaved. Bucket is M. Producing cytotoxic CD8 cells against tuberculosis-infected macrophages. Bucket is M. 9 shows that it can stimulate cytotoxic CD8 T cells that preferentially kill macrophages infected with tuberculosis.

[0202] Mice were killed M. prepared as described in Example 1. Immunization was performed by intraperitoneal injection of 500 μg of a bucket. Two weeks after immunization, the spleen cells of the immunized mice were replaced with CD
Passed through an 8T cell enrichment column (R & D Systems, St. Paul, Minnesota, USA). Spleen cells recovered from the column showed that CD8 T cells were enriched to 90%. These T cells, as well as CD8 T cells from the spleen of a non-immunized mouse, were transfected into uninfected macrophages or M. mori. The ability to kill macrophages infected with tuberculosis was tested.

Macrophages were obtained from the peritoneal cavity of mice 5 days after intraperitoneal administration of 1 ml of 3% thioglycolate. Macrophages were expressed at a ratio of 2 mycobacteria / 1 macrophage by M. Infected overnight using tuberculosis. All macrophage preparation per 10 ⁴ macrophages were labeled with chromium 51 2 .mu.Ci.
Macrophages were then cultured with CD8 T cells overnight (16 hours) at a killer to target ratio of 30: 1. Specific kill was detected by the release of chromium 51, calculated as in Example 5 and expressed as% specific dissolution.

The production of IFN-γ and its release into the medium three days after co-culturing CD8 T cells with macrophages was measured using a solid-phase enzyme-linked immunoassay (ELISA). ELISA plates were incubated with mouse IFN-γ in PBS at 4 ° C. for 4 hours.
Rat monoclonal antibody (Pharmigen) directed against
(San Diego, California, USA). Wells are allowed to stand for 1 hour at room temperature
Blocked with PBS containing 0.2% Tween20. The plate is then reduced to 0.2%
Wash four times in PBS containing Tween 20 and add 1 to the culture medium of the ELISA plate.
: 2 diluted samples were incubated overnight at room temperature. Plates were washed again and a biotinylated monoclonal rat anti-mouse IFN-γ antibody (Pharmigen), diluted to 1 μg / ml in PBS, was added to each well. The plates were then incubated at room temperature for 1 hour, washed and horseradish peroxidase-linked avidin D (Sigma A-3151) was added at a 1: 4,000 dilution in PBS.
After an additional hour of incubation at room temperature, the plates were washed and OPD substrate was added. The reaction was stopped after 10 minutes with 10% (v / v) HCl. Optical density is 4
Determined at 90 nm. As a result, the average O of the cells cultured only in the medium in the repeated experiments was
Fractions giving OD twice higher than D were considered positive.

As shown in Table 15, M. C from spleen of mouse immunized with a bucket
D8T cells were obtained from M. Cytotoxic to macrophages infected with tuberculosis, uninfected macrophages did not lyse. CD8 T cells from non-immunized mice did not lyse macrophages. CD8 T cells from native, ie, non-immunized mice, produce IFN-γ when cultured with infected macrophages, but the amount of IFN-γ produced in the co-culture was determined by M. et al. It was more abundant in CD8 T cells from bucket-immunized mice.

[0206]

[Table 22]

Example 10 Purification and Characterization of a Polypeptide Derived from a Bucket Culture Filtrate 3 shows the preparation of a bagley soluble protein. Unless otherwise specified, all percentages in the following examples are by weight per volume.

M. Buckets (ATCC No. 15483) were cultured in sterile medium 90 at 37 ° C. Cells are harvested by centrifugation and sterile Middlebrook 7H containing glucose.
Transferred to medium 9 at 37 ° C. for 1 day. The medium was then centrifuged (removing the cells) and placed in a sterile bottle through a 0.45 μm filter.

The culture filtrate was concentrated by freeze-drying and dissolved in reverse osmosis water (MilliQ). Small amounts of insoluble material were removed by filtration through a 0.45 μm membrane. The culture filtrate is
Desalting was performed by membrane filtration in a 400 ml Amicon stirred cell containing a 3 kDa molecular weight cut off (MWCO) membrane. The pressure was maintained at 50 psi 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. In this way, the volume of 20 l was reduced to about 50 ml. Protein concentration was determined by the Bradford protein assay (
Bio-Rad, Hercules, California, USA.

[0210] The desalted culture filtrate was Q-equilibrated with 10 mM Tris-HCl buffer (pH 8.0).
-Fractionation by ion-exchange chromatography on a Sepharose column (Pharmacia Biotech, Uppsala, Sweden) (16 x 100 mm). The polypeptide eluted with a linear gradient of 0-1.0 M NaCl in the above buffer system. The column eluent was monitored at a wavelength of 280 nm.

The polypeptide pool eluted from the ion exchange column was concentrated in a 400 ml Amicon stirred cell containing a 3 kDa MWCO membrane. The pressure is 50 using nitrogen gas.
Maintained in Psi. The polypeptide was repeatedly concentrated by membrane filtration and diluted with 1% glycine until the conductivity of the sample was below 0.1 mS.

Next, the purified polypeptide was fractionated by preparative isoelectric focusing on a Rotofor apparatus (Bio-Rad, Hercules, CA, USA). The pH gradient was 1.6% pH 3.5-5.0 ampholyte and 0.4%
The pH was established using a mixture of amphoteric electrolytes (Pharmacia Biotech) containing 5.0-7.0 ampholytes. Acetic acid (0.5M) as anolyte, 0.5M
Of ethanolamine was used as the catholyte. Isoelectric focusing was performed at a constant power of 12 W for 6 hours according to the manufacturer's instructions. 20 fractions were obtained.

The isoelectric focusing fractions were combined and the polypeptide was converted to Vydac C4
Columns (Separations Group)
Hesperia, California, USA) Pore size 300Å
And a particle size of 5 μ (10 × 250 mm). The polypeptide is expressed as 0.05
% (V / v) linear gradient of acetonitrile in trifluoroacetic acid (TFA) (0-8
(0% v / v). The flow rate is 2.0 ml / min, HPLC
The eluent was monitored at 220 nm. Fractions containing the polypeptide were collected to maximize the purity of individual samples.

A relatively large amount of the polypeptide fraction was applied to a Vidak C4 column (Separations
Group) Chromatography again with a pore size of 300 ° and a particle size of 5μ (4.6 × 250 mm). The polypeptide was treated at a flow rate of 1.0 ml / min with a flow rate of 0.05 ml / min.
The column was eluted with a linear gradient of 20-60% (v / v) acetonitrile in% (v / v) TFA. The column eluent was monitored at 220 nm. Fractions containing the eluted polypeptide were collected to maximize individual sample purity. Approximately 20 polypeptide samples were obtained and analyzed for purity on polyacrylamide gels according to the method of Laemmli (Laemml
i, U. K), Nature 277 : 680-685 1970).

[0215] The polypeptide fractions that were shown to contain significant contaminants were further subjected to Mono Q column (Pharmacia Biotech) particle size 10μ (5 x 50 mm) or Vidak diphenyl column (Separations Group) pores. Purification was performed using a size of 300 mm and a particle size of 5μ (4.6 × 250 mm). From the Mono Q column, the polypeptide was eluted with a linear gradient of 0-0.5 M NaCl in 10 mM Tris HCl pH 8.0. From the Vidak diphenyl column, the polypeptide was
Eluted with a linear gradient of acetonitrile in 20% TFA (20-60% v / v). The flow rate was 1.0 ml / min and the column eluent was monitored at 220 nm for both columns. The polypeptide peak fractions were collected and analyzed for purity on a 15% polyacrylamide gel as described above.

For sequencing, polypeptides were individually dried on Biobrene® (PerkinElmer / Applied Biosystems Division, Foster City, Calif.) Treated glass fiber filters. Filters with polypeptides were loaded onto a Perkin Elmer / Applied Biosystems Procise 492 protein sequencer and polypeptides were sequenced from the amino terminus using conventional Edman chemistry.
The amino acid sequence was determined for each polypeptide by comparing the retention time of the PTH amino acid derivative against the appropriate PTH derivative standard.

Internal sequences on the antigen were also determined by digestion of the antigen with the endoprotease Lys-C or by chemically cleaving the antigen with cyanogen bromide. Peptides obtained by either of these methods were purified on a Vidac using a 0.05% (v / v) trifluoroacetic acid mobile phase with a gradient of acetonitrile containing 0.05% (v / v) TFA. Separation by reverse phase HPLC on a C18 column (1% / min). The eluent was monitored at 214 nm. The major internal peptides are identified by their UV absorption,
Its N-terminal sequence was determined as described above.

Using the above method, GVc-1, GVc-2, GVc-7, GVc-13
, GVc-20 and GVc-22. The Bucket antigen was isolated. The determined N-terminal and internal sequences of GVc-1 are SEQ ID NO: 1,
2 and 3; the N-terminal sequence of GVc-2 is shown in SEQ ID NO: 4; the internal sequence of GVc-7 is shown in SEQ ID NOs: 5-8; the internal sequence of GVc-13 is SEQ ID NOs: 9-11
The internal sequence of GVc-20 is shown in SEQ ID NO: 12; and the N-terminal and internal sequence of GVc-22 are shown in SEQ ID NOs: 56-59, respectively. Each of the internal peptides presented herein starts at an amino acid residue that is predicted to have (Met) or Lys-C (Lys) at this position in the polypeptide based on the known cleavage specificity of cyanogen bromide. .

[0219] Three additional polypeptides, termed GVc-16, GVc-18 and GVc-21, were added to the preparative isoelectric focusing method described above, followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (SDS-PAGE) isolated using a purification step. Specifically, the fraction containing the mixture of polypeptides from the preparative isoelectric focusing electrophoresis purification step described above was subjected to preparative SDS-PAGE on a 15% polyacrylamide gel.
Purified by E. Samples were dissolved in reducing sample buffer and applied to the gel.
The separated proteins were electroblotted in 10 mM 3- (cyclohexylamino) -1-propanesulfonic acid (CAPS) buffer pH 11 containing 10% (v / v) methanol to produce polyvinylidene difluoride (PV).
DF) Transferred to membrane. The transferred protein band was PVD by Coomassie Blue.
Identified by staining of F membrane. The region of the PVDF membrane containing the most abundant polypeptide species was excised and introduced directly into the sample cartridge of the PerkinElmer / Applied Biosystems Procise 492 protein sequencer. The protein sequence was determined as described above. N of GVc-16, GVc-18 and GVc-21
The terminal sequences are provided in SEQ ID NOs: 13, 14 and 15, respectively.

GVc-12, GVc-14, GVc-15, GVc-17 and GVc-1
An additional antigen, designated No. 9, was added to the chromatographic method described above, plus preparative SDS-
Isolated using a PAGE purification step. Specifically, the above-mentioned Vidak C4HP
Fractions containing the mixture of antigens from the LC purification step were fractionated on a polyacrylamide gel by preparative SDS-PAGE. Samples were dissolved in non-reducing sample buffer and applied to the gel. The separated proteins were transferred to a PVDF membrane by electroblotting in 10 mM CAPS buffer pH 11 containing 10% (v / v) methanol. Transfer the transferred protein band to Coomassie Blue P
Identified by staining of VDF membrane. A region of the PVDF membrane containing the most abundant polypeptide species was excised and analyzed by PerkinElmer / Applied Biosystems Procise 49.
Two protein sequencers were introduced directly into the sample cartridge. The protein sequence was determined as described above. GVc-12, GVc-14, GVc-15, G
The determined N-terminal sequences of Vc-17 and GVc-19 are SEQ ID NO: 16-
20.

All of the above amino acid sequences were converted to the Gene Assist System (GeneAssists).
t System) was used to compare to the known amino acid sequence of the SwissProt database (version R32). Amino acid sequence GV
No significant homology from c-2 to GVc-22 was obtained. The amino acid sequence of GVc-1 is described in M. Bovis and M. Tuberculosis was found to have some similarity to previously identified sequences. In particular, GVc-1 is a cell surface protein, M.C. Tuberculosis MPT83, and was found to have some homology to MPT70. These proteins form part of a protein family (Harboe et al., Scand. Immunol.
42: 46-51, 1995).

Subsequent studies have shown that GVc-13, GVc-14 and GVc-22 DN
The A sequence was isolated (SEQ ID NOs: 142, 107 and 108, respectively). GVc
The corresponding predicted amino acid sequences for -13, GVc-14 and GVc-22 are provided in SEQ ID NOs: 143, 109 and 110, respectively. The determined DNA sequence of the full length gene encoding GVc-13 is provided in SEQ ID NO: 195, and the corresponding predicted amino acid sequence is provided in SEQ ID NO: 196.

Further studies of GVc-22 suggested that only a portion of the gene encoding GVc-22 was cloned. When subcloned into the expression vector pET16, no protein was expressed. Then, M. using the incomplete gene fragment. Screening of the Backey BamHI genomic DNA library resulted in the isolation of the complete gene encoding GVc-22. To distinguish full-length clones from partial GVc-22, the antigen expressed by the full-length gene was designated GV-22B. The determined nucleotide sequence and predicted amino acid sequence of the gene encoding GV-22B are SEQ ID NOs: 144 and 14
5 respectively.

Amplification primers AD86 and AD112 (SEQ ID NOs: 60 and 61, respectively)
) Is the amino acid sequence of GVc-1 (SEQ ID NO: 1) and M. A tuberculosis MPT70 gene sequence was designed. Using these primers, 310b
p fragment was prepared using the M.p. Amplified from bucke genomic DNA and digested with EcoRV digested vector pBluescript II SK ⁺ (Stratagene
gene)). The cloned insert sequence was SEQ ID NO: 62.
To provide. The insert of this clone was used to construct M. ZAP-Express (Stratagene, La Jolla, CA). The Bucket genomic DNA library was screened. The clone isolated was M.E. It contained an open reading frame homologous to the tuberculosis antigen MPT83 and was renamed GV-1 / 83. This gene also
M. Bovis antigen MPB83 also showed homology. The determined nucleotide sequence and predicted amino acid sequence are provided in SEQ ID NOs: 146 and 147, respectively.

From the amino acid sequences provided in SEQ ID NOs: 1 and 2, degenerate oligonucleotides EV59 and EV61 (SEQ ID NOs: 148 and 149, respectively) were designed.
The 100 bp fragment was amplified using PCR and the plasmid pBlues
Crypt II SK ⁺ was cloned and sequenced according to standard methods (Sambrook et al., supra) (SEQ ID NO: 150). Using the cloned insert, the M.A. The Bucket genomic DNA library was screened. The clone isolated was M.E. Tuberculosis antigen MPT70 and M. It has homology to Bovis antigen MPB70 and has GV-
It was named 1/70. The determined nucleotide sequence and predicted amino acid sequence of GV-1 / 70 are provided in SEQ ID NOS: 151 and 152, respectively.

For expression and purification, GV1 / 83, GV1 / 70, GVc-13, GV
The genes encoding c-14 and GV-22B were transformed into the expression vector pET16
(Novagen, Madison, Wis.). Expression and purification were performed according to the manufacturer's protocol.

Purified polypeptides were screened for their ability to induce T cell proliferation and IFN-γ in peripheral blood cells from immunized human donors. These donors are
(Purified protein derived from M. tuberculosis) It was known that the skin test was positive, and the T cells showed proliferation in response to PPD. Donor PBMC and M.
10% (v / v) autologous serum,
The cells were cultured in a medium containing RPMI1640 supplemented with penicillin (60 μg / ml), streptomycin (100 μg / ml) and glutamine (2 mM).

After three days, 50 μl of medium was removed from each well for determination of IFN-γ levels as described below. Plates were cultured for an additional 4 days, after which 1 μCi / well of tritiated thymidine was applied for an additional 18 hours, collected, and tritium incorporation was measured using a scintillation counter. Fractions that stimulated growth more than twice in both homogenous cultures were considered positive compared to growth observed in cells cultured in medium alone.

[0230] IFN-γ was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with 1 μg / ml phosphate buffered saline (PBS) for 4 hours at 4 ° C. using a mouse monoclonal antibody directed against human IFN-γ (Endogen, Wobral, Mass.). . Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature.
The plate was then washed four times in PBS / 0.2% Tween 20 and the ELI
Samples diluted 1: 2 in SA plate culture medium were incubated overnight at room temperature. Plates were washed again and biotinylated polyclonal rabbit anti-human IFN-γ serum (endogen), diluted to 1 μg / ml in PBS, was added to each well. The plate is then incubated at room temperature for 1 hour, washed, and washed with horseradish peroxidase-linked avidin A (Vector Laboratories (Vecto)
r Laboratories), Burlingame,
(California) at a 1: 4,000 dilution in PBS. After an additional hour of incubation, the plates were washed and orthophenylenediamine (OPD) substrate was added. The reaction was stopped after 10 minutes with 10% (v / v) HCl. Optical density (
OD) was determined at 490 nm. As a result, the fraction giving an OD twice higher than the average OD of the cells cultured in the medium alone in all the repeated experiments was considered positive.

Examples of polypeptides containing sequences that stimulate peripheral blood mononuclear cells (PBMC) T cells and proliferate and produce IFN-γ are shown in Table 16, where (-) indicates no activity. (+/-) indicates a polypeptide having a result of 2 times or less the background activity of the control medium, and (+) indicates a polypeptide having 2 to 4 times the activity of the background. (++) indicates a polypeptide having an activity at least 4 times higher than the background.

[0231]

[Table 23]

Example 11 M. by two-dimensional polyacrylamide gel electrophoresis. Purification and Characterization of Polypeptides from Bucket Culture Filtrate Bucket soluble protein was isolated from the culture filtrate using two-dimensional polyacrylamide gel electrophoresis as described above. Unless otherwise specified, all percentages in the following examples are by weight per volume.

M. Buckets (ATCC No. 15483) were cultured in sterile medium 90 at 37 ° C. M. Tuberculosis strain H37Rv (ATCC No. 27294)
Cultures were performed in sterile Middlebrook 7H9 medium containing ween 80 and an oleic acid / albumin / dextrose / catalase additive (Difco Laboratories, Detroit, Michigan). Cells were collected by centrifugation and transferred to sterile Middlebrook 7H9 medium containing glucose at 37 ° C. for 1 day. The medium was then centrifuged (removing the cells) and placed in a sterile bottle through a 0.45 μm filter. The culture filtrate was concentrated by freeze-drying and dissolved in reverse osmosis water (MilliQ). A small amount of insoluble material was removed by filtration through a 0.45 μm membrane filter.

The culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell containing a 3 kDa MWCO membrane. The pressure was maintained at 60 psi 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. In this way, the volume of 20 l was reduced to about 50 ml. Protein concentration was determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA).

[0235] The desalted culture filtrate was Q-equilibrated with 10 mM Tris-HCl buffer (pH 8.0).
-Sepharose column (Pharmacia Biotech) (16 x 100 mm)
And fractionated by ion exchange chromatography. The polypeptide eluted with a linear gradient of 0-1.0 M NaCl in the above buffer system. Column eluent is 280 nm
Was monitored at the wavelength.

The pool of polypeptides eluted from the ion exchange column was fractionated by preparative 2D gel electrophoresis. A sample containing 200-500 μg of the polypeptide was made 8 M in urea and subjected to polyacrylamide isoelectric focusing gel (diameter 2 mm, length 150 mm).
mm, pH 5-7). After the isoelectric focusing step, the one-dimensional gel was equilibrated with a reducing buffer and applied to a two-dimensional gel (16% polyacrylamide). The polypeptides from the two-dimensional separation were combined with 10 mM CAPS containing 10% (v / v) methanol.
Transferred to PVDF membrane by electroblotting in buffer pH11. PVDF membrane stained proteins with Coomassie blue. The PVDF region containing the polypeptide of interest was excised and directly introduced into a sample cartridge of a Perkin Elmer / Applied Biosystems Procise 492 protein sequencer. Polypeptides were sequenced from the amino acid termini using conventional Edman chemistry. The amino acid sequence is the P
Each polypeptide was determined by comparing the retention times of the TH amino acid derivatives. Using these methods, GVs-1, GVs-3, GVs-4, GVs
s-5, GVs-6, GVs-8, GVs-9, GVs-10, GVs-11,
Eleven polypeptides, designated GV-34 and GV-35, were isolated. The determined N-terminal sequences of these polypeptides are shown in SEQ ID NOs: 21-29, 63 and 64, respectively. Using the purification method described above, more proteins were purified to extend the amino acid sequence previously obtained with GVs-9. The extended amino acid sequence of GVs-9 is provided in SEQ ID NO: 65. Further studies have isolated the DNA sequences of GVs-9 (SEQ ID NO: 111) and GV-35 (SEQ ID NO: 155). The corresponding predicted amino acid sequences are provided in SEQ ID NOs: 112 and 156, respectively. The extended DNA sequence for GVs-9 is provided in SEQ ID NO: 153, and the corresponding predicted amino acid sequence is provided in SEQ ID NO: 154. The predicted amino acid sequence of GVs-9 has been modified by SEQ ID NO: 197.

All of these amino acid sequences were compared to known amino acid sequences in the Swiss Plot Database (updated version R35). GVs-3, GV
No significant homology was obtained except for s-4, GVs-5 and GVs-9.
GVs-9 is one of two previously identified M. Tuberculosis protein, ie, M. Tuberculosis cutinase precursor and M. Tuberculosis was found to have some homology to the putative 22.6 kDa protein. GVs-3, GVs-4 and GVs-5 are available from M.S. Repre (M
. leprae) (SEQ ID NOs: 30 and 31, respectively); Tuberculosis (SEQ ID NOs: 32 and 33, respectively) and M. Antigens 85A and 85B proteins from Bovis (SEQ ID NOs: 34 and 35, respectively), and M.
Repre (SEQ ID NO: 36) and M.E. It was found to have some similarity to the antigen 85C protein from tuberculosis (SEQ ID NO: 37).

Example 12 M.P. DNA Cloning Strategy for Bucket Antigen 85 Series Probes for antigens 85A, 85B, and 85C are used to identify antigen 85 conserved between members of a given family of mycobacterial species and between mycobacterial species.
Prepared by replication chain reaction (PCR) using degenerate oligonucleotides designed for regions of the genomic sequence (SEQ ID NOs: 38 and 39). Using these oligonucleotides under low stringency conditions, M.P. The target sequence from the bucky genomic DNA was amplified. An appropriately sized 485 bp band was identified, purified, and cloned into pBluescript II SK with T tail (Stratagene, La Jolla, CA). Twenty-four individual colonies were randomly screened for the presence of the antigen-85 PCR product and then sequenced using a PerkinElmer / Applied Biosystems model 377 automatic sequencer and M3 based primers T3 and T7. . By homology search of the gene bank database, 23 clones were published in M. Tuberculosis and M. Bovis-derived antigen 85
It was shown to contain an insert with significant homology to the gene. About half are almost homologous to the antigen 85C gene sequence, and the rest are more similar to the antigen 85B sequence. In addition, these two putative em. Bucket antigen 85 genomic sequences were 80% homologous to each other. Due to this high similarity, the antigen 85C P
The CR fragment was low in stringency for all three antigen 85 genes. Selected to screen the Bucket Genomic Library.

M. The Backey genomic library was obtained from M. partially digested with BamHI. By cloning the bucky genomic DNA into a similarly digested lambda vector, it was created in lambda Zap-Express (Stratagene, La Jolla, Calif.), Giving 3.4 × 10 ⁵ independent plaque forming units. Was. For screening, 27,000 plaques from this unamplified library were spread at low density on eight 100 cm ² plates. For each plate,
The duplicated plaque is picked up and Hybond-N ⁺ nylon membrane (
(Amersham International, UK) and hybridized to the radiolabeled antigen 85C PCR product under low stringency conditions (55 ° C). Autoradiography demonstrated that 79 plaques hybridized tightly to the antigen 85C probe under these conditions. 13 for further analysis
Positive hybridized plaques were randomly selected, removed from the library plate, and each positive clone was used to generate a secondary screening plate containing approximately 200 plaques. Picking up of duplicates of each plate was performed using a Hybond-N ⁺ nylon membrane and hybridized under the conditions used for the primary screening. A number of positive hybridized plaques were identified on each of the 13 plates screened. Two well-isolated positive phages on each secondary plate were picked for further analysis. Using in vitro excision, 26 plaques were converted to phagemids and restriction maps were made. Clones could be classified into four classes based on this map. Sequence data at the 5 'and 3' ends of several representative inserts in each group were obtained using a Perkin Elmer / Applied Biosystems model 377 automated sequencer and T3 and T7 primers. Sequence homology was determined using BLASTN analysis of the EMBL database. Two of the sets of these clones were M.E. Bovis and M. H. tuberculosis antigen 85A gene. It was found to contain the 5 'or 3' end of the bucke gene, which was truncated during library construction as it contains an internal BamHI site. The remaining clones
It was found to contain sequences homologous to antigens 85B and 85C of many mycobacterial species. Appropriate subclones were constructed and sequenced to determine the remaining nucleotide sequence of each gene. Duplicate sequences are based on DNA walking (stride).
r) Aligned using software. M. The determined DNA sequences of the Bucket antigens 85A, 85B and 85C are shown in SEQ ID NOs: 40-42, respectively, and the predicted amino acid sequences are shown in SEQ ID NOs: 43-45, respectively.

M. Bucket antigens GVs-3 and GVs-5 were expressed and purified as follows. Amplification primers were designed from the insert sequences of GVs-3 and GVs-5 (SEQ ID NOS: 40 and 42, respectively) using sequence data downstream and 3 'of the putative leader sequence of the clone. The primer sequences for GVs-3 are provided in SEQ ID NOs: 66 and 67, and the primer sequences for GVs-5 are provided in SEQ ID NOs: 66 and 67. To facilitate cloning, an XhoI restriction site was added to GV
In addition to the s-3 primer, EcoRI and BamHI restriction sites were added to GVs-5.
Of primers. Genome M. After amplification from bucke DNA, the fragment was cloned into the appropriate site of the pProEX HT prokaryotic expression vector (Gibco BRL, Life Technologies, Gaithersburg, MD) and sequenced for the correct reading frame and orientation. confirmed. Expression and purification of the recombinant protein was performed according to the manufacturer's protocol.

M. Expression of a fragment of the Bucket antigen GVs-4 (antigen 85B homolog) was performed as follows. Using the primers AD58 and AD59 described above,
M. A 485 bp fragment was amplified from bucke genomic DNA. This fragment was gel purified using standard techniques and Ec containing added dTTP residues.
It was cloned into oRV digested pBluescript. The nucleotide sequences of the inserts of the five clones were determined and found to be identical to each other. These inserts are provided by M.E. It showed high homology to Ag85B from tuberculosis. The insert of one of these clones was transferred to the pProEX HT prokaryotic expression vector (Gibco B
RL) at the EcoRI / XhoI site, expressed and purified according to the manufacturer's protocol. This clone was renamed GV-4P because it expressed only part of the gene. The amino acid and DNA sequences of the partial clone GV-4P are provided in SEQ ID NOs: 70 and 106, respectively.

As with the cloning of GV-4P, amplification primers AD58 and AD58
A 485 bp fragment was amplified from a clone containing GVs-5 (SEQ ID NO: 42) using 59. This fragment was cloned into the expression vector pET16 and named GV-5P. The determined nucleotide sequence and predicted amino acid sequence of GV-5P are provided in SEQ ID NOs: 157 and 158, respectively.

In a subsequent study, GVs-3, GV-4P and GVs-5 were recloned into another vector pET16 (Novagen, Madison, Wis.) Using methods similar to those described above.

The ability of purified recombinant GVs-3, GV-4P and GVs-5 to stimulate the proliferation of T cells and interferon-γ production in human PBL (peripheral blood lymphocytes) of PPD positive healthy donors Was assayed as described above. The results of this assay are shown in Table 17, where (-) indicates no activity and (+/-) indicates a polypeptide with a result less than twice the background activity of the control medium. In the table, (+) indicates a polypeptide having 2-4 times the activity of the background, (++) indicates a polypeptide having an activity 4 times or more higher than the background, and ND was not determined. Indicates that

[0245]

[Table 24]

Example 13 M.P. DNA Cloning Strategy for Bucket Antigen M. An 84 bp probe of the Bucket antigen GVc-7 was amplified using a degenerate oligonucleotide designed against the determined amino acid sequence of GVc-7 (SEQ ID NOS: 5-8). Using this probe, M.I. The Bucket genomic DNA library was screened. The determined nucleotide sequence of GVc-7 is shown in SEQ ID NO: 46, and the predicted amino acid sequence is shown in SEQ ID NO: 47. When these sequences are compared with those in the data bank, M. Estimation of tuberculosis 1
Homology to the 5.8 kDa membrane protein was found.

Using the sequence of SEQ ID NO: 46, an amplification primer (SEQ ID NO: 71) for expression cloning of the GVc-7 gene using sequence data downstream of the putative leader sequence
And 72). An XhoI restriction site was added to facilitate cloning. Genome M. After amplification from bucke DNA, the fragment was cloned into the XhoI site of the pProEX HT prokaryotic expression vector (Gibco BRL) and sequenced to confirm the correct reading frame and orientation. Expression and purification of the fusion protein was performed according to the manufacturer's protocol. In a subsequent study, GVc-7 was converted to the vector pET16 (Novagen).
Recloned.

Stimulation of T cell proliferation and interferon-γ production in human PBLs of PPD positive healthy donors was assayed for the ability of purified recombinant GVc-7 to stimulate as described above. The results are shown in Table 18, where (-) indicates no activity and (+)
(/-) Indicates a polypeptide having a result of not more than twice the background activity of the control medium, (+) indicates a polypeptide having 2-4 times the activity of the background, and (++) Indicates a polypeptide having an activity at least 4 times higher than the background.

[0249]

[Table 25]

The overlapping oligonucleotide probe (SEQ ID NO: 73; referred to as MPG15)
Designed against the GVs-8 peptide sequence set forth in SEQ ID NO: 26 and em. It was used to screen a bucke genomic DNA library. Two genomic clones containing genes encoding four different antigens were isolated. GVs-8A (renamed GV30), GVs-8B (GV-31
GVs-8C (renamed GV-32) and GVs-8D (GV-33).
And the corresponding amino acid sequences are shown in SEQ ID NOs: 52-55, respectively. GV-30 contains a region that shows some similarity to known prokaryotic valyl-tRNA synthase; Shows some similarity to Smegma aspartate semialdehyde dehydrogenase; Influenza / Horyl polyglutamate (
Folylpolyglutamate) shows some similarity to the synthase gene. GV-33 is available from M.M. Tuberculosis and M. It contains an open reading frame previously identified in the reprep, but whose function is somewhat similar to unidentified sequences.

The determined partial DNA sequence of GV-33 is provided in SEQ ID NO: 74, and the corresponding predicted amino acid sequence is provided in SEQ ID NO: 75. Sequence data for the 3 'end of the clone was obtained from the previously identified M.V. It showed homology to a 40.6 kDa outer membrane protein of tuberculosis. Subsequent studies indicated that the full-length DN of GV-33 (SEQ ID NO: 193)
The A sequence was isolated. The corresponding predicted amino acid sequence is provided in SEQ ID NO: 194.

The gene encoding GV-33 was prepared using the primers based on the determined nucleotide sequence as described in M.E. Amplified from bucky genomic DNA. This DNA fragment was cloned into EcoRV digested pBluescript II SK ⁺ (Stratagene) and then transferred to a pET16 expression vector. Recombinant protein was purified according to the manufacturer's protocol.

Stimulation of T cell proliferation and interferon-γ production on human PBL was assayed for the ability of purified recombinant GV-33 to stimulate as described above. The results are shown in Table 19, where (-) indicates no activity, (+/-) indicates a polypeptide with a result less than twice the background activity of the control medium, and ( (+) Indicates a polypeptide having an activity 2 to 4 times the background, and (++) indicates a polypeptide having an activity 4 times or more higher than the background.

[0254]

[Table 26]

Example 14 M.P. Isolation of Bucket-Derived Protein M. Bucket bacteria were cultured, pelleted, and autoclaved as described in Example 1. Raw M. The culture filtrate of the Bucket was em. It means the supernatant obtained by culturing the bucket for 24 hours. M. The delipidated form of the bucket is a Virsonic sonicator (Vertis (
Virtis), Disa, USA) for 30 seconds on ice.
M. autoclaved once. It was prepared by sonicating the bucket. The material was then centrifuged (9000 rpm, 20 minutes, JA10 rotor, brake = 5). The obtained pellet was suspended in 100 ml of chloroform / methanol (2: 1), incubated at room temperature for 1 hour, centrifuged again, and chloroform / methanol extraction was repeated. The pellet was obtained by centrifugation, vacuum dried, weighed and delipidated. Resuspended in PBS at 50 mg per ml (dry weight) as a bucket.

Glycolipids were re-lipidated by refluxing in 50% v / v ethanol for 2 hours. Removed from the bucket preparation. Insoluble material was collected by centrifugation (1
0,000 rpm, JA20 rotor, 15 minutes, brake = 5). Under reflux 50
The extraction with% v / v ethanol was repeated two more times. Insoluble material was collected by centrifugation and washed in PBS. Pellets protein at 56 ° C for 2 hours
Extracted by resuspension in 2% SDS in S. The insoluble material was collected by centrifugation and extraction with 56% 2% SDS / PBS was repeated twice more. The pooled SDS extract was cooled to 4 ° C and the precipitated SDS was removed by centrifugation (10
2,000 rpm, JA20 rotor, 15 minutes, brake = 5). The protein was precipitated from the supernatant by adding an equal volume of acetone and incubating at -20 ° C for 2 hours. The precipitated protein was collected by centrifugation, washed with 50% v / v acetone, dried in vacuo and redissolved in PBS.

DD. M. Proteins extracted with SDS from the bucket were analyzed by polyacrylamide gel electrophoresis. Three major bands were observed after silver staining. In subsequent experiments, DD. M. Larger amounts of SDS-extracted proteins from the bucket were analyzed by polyacrylamide gel electrophoresis. The protein showed several bands when stained with Coomassie Blue. The protein presented by a band with a molecular weight of about 30 kDa was called GV-45. The determined N-terminal sequence of GV-45 is provided in SEQ ID NO: 187. The protein having a molecular weight of about 14 kDa was designated as GV-46. The determined N-terminal amino acid sequence of GV-46 is provided in SEQ ID NO: 208.

In subsequent studies, other SDS-extracted proteins were prepared by preparative SDS-PAGE on a BioRad Prep Cell (Hercules, CA). Fractions corresponding to the molecular weight range were precipitated with trichloroacetic acid, and assayed for C57BL / 6 mouse anti-ovalbumin-specific cytotoxic response assay for adjuvant activity as described above.
DS was removed. Adjuvant activity was highest in the 60-70 kDa fraction.
The most abundant proteins in this size range were purified by SDS-PAGE, blotted onto polyvinylidene difluoride (PVDF) membrane, and then sequenced. The sequence of the first 10 amino acid residues is provided in SEQ ID NO: 76. When this sequence is compared with the sequence of the above gene bank, M. Homology to the heat shock protein 65 (GroEL) gene from tuberculosis was found, indicating that this protein is a member of the GroEL family M.E. Suggests that you are a buckeet member.

M. in BamHI-λZAP-Express (Stratagene). The expression library of bucke genomic DNA was prepared as described above. Screening was performed using serum from cynomolgus monkeys immunized with the Bucket secreted protein. Positive plaques were identified using a colorimetric system. These plaques were rescreened until the plaques were pure by standard methods. The pBK-CMV phagemid 2-1 containing the insert was excised from the λZAP Express (Stratagene) vector in the presence of ExAssist helper phage according to the manufacturer's instructions. This clone (hereinafter G
V-27) was determined using the Sanger sequencing method with a fluorescent primer on a Perkin Elmer / Applied Biosystems Division automated sequencer. Partial M. The determined nucleotide sequence of clone GV-27, homologous to Buckey GroEL, is provided in SEQ ID NO: 77, and the predicted amino acid sequence is provided in SEQ ID NO: 78. This clone was obtained from M.E. Tuber Closis G
It was found to have homology to roEL. M. Bucket 65kDa
The partial sequence of the heat shock protein is described in Kapur et al. (Arch.
atol. Lab. Med. 119: 131-138, 1995). The nucleotide sequence of the fragment of Kappa et al. Is shown in SEQ ID NO: 79, and the predicted amino acid sequence is shown in SEQ ID NO: 80.

In a subsequent study, the extension of GV-27 (full length except for the predicted 51 terminal nucleotide)
The DNA sequence was obtained (SEQ ID NO: 113). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 114. Further studies have isolated the full length DNA sequence of GV-27 (SEQ ID NO: 159). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 160. GV-27 is expressed at the amino acid level by M.E. Tuberculosis was found to be 93.7% identical to GroEL.

Two peptide fragments containing the N-terminal sequence of GV-27 (hereinafter GV-27A) and the carboxy-terminal sequence (hereinafter GV-27B) were prepared using techniques known in the art. . The nucleotide sequences of GV-27A and GV-27B are provided in SEQ ID NOs: 115 and 116, respectively, and the corresponding amino acid sequences are provided in SEQ ID NOs: 117 and 118. Subsequent research has shown that GV-
An extended DNA sequence of 27B was isolated. This sequence is provided in SEQ ID NO: 161, and the corresponding amino acid sequence is provided in SEQ ID NO: 162. The sequence of GV-27A is described in M. The M. tuberculosis GroEL sequence is 95.8% identical and has a shorter M.E. Includes bucke array. The sequence of GV-27B is described in M. It shows about 92.2% identity to the corresponding region of the tuberculosis HSP65. Following the same protocol as for the isolation of GV-27, pBK-CMV phagemid 3-1 was isolated. The antigen encoded by this DNA was named GV-29. The determined nucleotide sequence at the 5 ′ and 3 ′ ends of the gene is SEQ ID NO: 16.
3 and 164, respectively, and the corresponding predicted amino acid sequence is SEQ ID NO: 165
And 166 respectively. GV-29 showed homology to yeast urea amidolyase. The determined DNA sequence of the full length gene encoding GV-29 is provided in SEQ ID NO: 198, and the corresponding predicted amino acid sequence is provided in SEQ ID NO: 199. GV
DNA encoding -29 was subcloned into the vector pET16 (Novagen, Madison, Wis.) For expression and purification according to standard protocols.

Example 15 M.E. GV-23, GV-24, GV-25, GV-26, which are
DNA cloning strategy for GV-38A and GV-38B Buckets (ATCC No. 15483) were grown in sterile medium 90 at 37 ° C. for 4 days and collected by centrifugation. Transfer cells to 1 ml Trizol
(Gibco BRL, Life Technologies, Gaithersburg, MD) and RNA extracted according to standard manufacturer's protocols. M. Tuberculosis strain H37Rv (ATCC No. 27294) was transformed into Tween8.
0.RTM. And sterile Middlebrook 7H9 medium containing oleic acid / albumin / dextrose / catalase additive (Difco Laboratories, Detroit, Mich.) At 37.degree. C. and harvest under appropriate experimentally safe conditions did. Cells were resuspended in 1 ml Trizol (Gibco BRL) and RNA was extracted according to the manufacturer's standard protocol.

All M. Tuberculosis and M. From the bucke RNA, the total RNA fraction was purified by M.E. Oligonucleotide AD10 complementary to tuberculosis rRNA
And by hybridization to AD11 (SEQ ID NOs: 81 and 82), 16S and 23S ribosomal RNA (rRNA) was removed. These oligonucleotides are available from Botger (FEMS Microb)
iol. Lett. 65: 171-176, 1989), and from the sequences deposited in the databank, and from the mycobacterial 16S rRNA sequence. Removal was performed by hybridizing total RNA to oligonucleotides AD10 and AD11 immobilized on a nylon membrane (Hybond N, Amersham International, UK). Hybridization was repeated until no rRNA band was visible on ethidium bromide stained agarose gel.
AD12 of an oligonucleotide consisting of 20 dATP residues (SEQ ID NO: 83)
Was ligated to the 3 'end of the enriched mRNA fraction using RNA ligase. First strand cDNA synthesis was performed according to standard protocols using oligonucleotide AD7 (SEQ ID NO: 84) containing a poly (dT) sequence.

M. Tuberculosis and M. Bucket cDNA is one-sided specific PCR
(3S-PCR) was used as a template. For this protocol, a degenerate oligonucleotide AD1 (SEQ ID NO: 85) was designed based on the conserved leader sequence and the membrane protein sequence. After 30 cycles of amplification using primer AD1 as the 5'-primer and AD7 as the 3'-primer, the products were separated on a urea / polyacrylamide gel. M. DNA bands unique to the bucket were excised and re-amplified using primers AD1 and AD7. After gel purification, the band was cloned into pGEM-T (Promega) and the nucleotide sequence was determined.

A search using the determined nucleotides and predicted amino acid sequence of band 12B21 (SEQ ID NOs: 86 and 87, respectively) revealed Furuchi et al. (Jn.
l. Biol. Chem. 266: 20928-20933, 1991), which encodes the ATP binding protein of the spermidine / putrescine ABC transporter complex. E. coli showed homology to the pota gene. E
. The E. coli spermidine / putrescine transporter complex is composed of four genes and is a member of the ABC transporter family. The ABC (ATP binding cassette) transporter typically consists of four genes: an ATP binding gene, a periplasmic or substrate binding gene, and two transmembrane genes. The transmembrane gene encodes a protein that each characteristically has six transmembrane regions. A homologue of this ABC transporter (by similarity) is Haemophilus influenza (Fleischmann).
Fleischmann) et al., Science 269: 496-512,19.
95) and Mycoplasma genitalium
talium) (Fraser et al., Science, 270: 3).
97-403, 1995).

M. constructed with BamHI digested λZAP Express (Stratagene).
The Bucket genomic DNA library was searched using radiolabeled 238 bp band 12B21 according to standard protocols. Plaques were purified pure by repeated screening and phagemids containing the 4.5 kb insert were identified by Southern blot and hybridization. pota (ATP binding protein). The nucleotide sequence of the Bucket homolog was identified by subcloning a 4.5 kb fragment and by base sequencing. The gene has an estimated -10
And 1449 bp containing a 320 bp untranslated 5 'region containing the -35 promoter element. M. The nucleotide and predicted amino acid sequences of the Bucket pota homolog are provided in SEQ ID NOs: 88 and 89, respectively.

M. Primers EV24 and EV25 (SEQ ID NOs: 90 and 91) for expression cloning were designed using the nucleotide sequence of the Bucket pota gene. The amplified DNA fragment was cloned into the pProEX HT prokaryotic expression system (Gibco BRL) and Expression in the E. coli host was induced by the addition of 0.6 mM isopropylthio-β-galactoside (IPTG). The recombinant protein was named GV-23 and was purified from inclusion bodies according to the manufacturer's protocol. In a subsequent study, GV-23 (SEQ ID NO: 88) was transferred to another vector, pET1.
6 (Novagen). The amino acid sequence of SEQ ID NO: 89 is 34-
The ATP binding site is contained at residue 41. At residues 116-163 of SEQ ID NO: 89 there is a conserved region found in the ATP transporter family of proteins. These findings are
This suggests that GV-23 is an ATP binding protein.

The 322 bp SalI-BamHI subclone at the 3 ′ end of the 4.5 kb insert described above was obtained from E. coli. E. coli showed homology to the potd gene (periplasmic protein) of the spermidine / putrescine ABC transporter complex. The nucleotide sequence of this subclone is shown in SEQ ID NO: 92. To identify the gene, the radiolabeled insert of this subclone was used and λZap
M. constructed at the SalI site of Express (Stratagene). The Bucket genomic DNA library was searched. 1342 bp is E. coli. A clone showing homology to the E. coli potd gene was identified. M. Bucket's potd homologue is
Identified by subcloning and base sequencing. The determined nucleotides and predicted amino acid sequences are shown in SEQ ID NOs: 93 and 94.

Primers EV-26 and EV-27 for expression cloning (SEQ ID NO: 95)
-96) is determined. Designed from the Bucket potd homolog. The amplified fragment was cloned into the pProEX HT prokaryotic expression system (Gibco BRL).
Appropriate E. Expression in the E. coli host was induced by the addition of 0.6 mM IPTG, and the recombinant protein was named GV-24. Recombinant antigen was purified from inclusion bodies according to the manufacturer's protocol. In a subsequent study, GV-24 (SEQ ID NO: 93)
) Was recloned into another vector pET16 (Novagen).

To increase the solubility of the purified recombinant protein, the amplification primers EV101 and EV102 (SEQ ID NOs: 167 and 168) were used to convert a gene encoding GV-24 but without the signal peptide into an expression vector. Again. The construct was called GV-24B. The nucleotide sequence of GV-24B is provided in SEQ ID NO: 169, and the predicted amino acid sequence is provided in SEQ ID NO: 170. This fragment was used for expression and purification of GV-24B according to the manufacturer's instructions.
It was cloned into ET16.

The ability of purified recombinant proteins GV-23 and GV-24 to stimulate proliferation of T cells and interferon-γ production on human PBL was assayed as described above. The results of these assays are shown in Table 20, where (-) indicates no activity and (+/-) indicates a polypeptide with a result less than twice the background activity of the control medium. (+) Indicates a polypeptide having an activity 2 to 4 times the background, (++) indicates a polypeptide having an activity 4 times or more higher than the background, and (ND) indicates a determination. Indicates that it did not.

[0272]

[Table 27]

M. The nucleotide sequence adjacent to the Bucket potd gene homolog is one of the two transmembrane proteins of the ABC transporter complex, E. coli. It was found to show homology to the potb gene of the E. coli spermidine / putrescine ABC transporter complex. M. Bucket potb homolog (designated GV-25)
Was identified by further subcloning and base sequencing. GV-
The 25 determined nucleotide and predicted amino acid sequences are shown in SEQ ID NOs: 97 and 98, respectively.

The flanking 509 bp was further subcloned and sequenced.
. No significant homology to PotC, a second transmembrane protein of E. coli, was observed, indicating that the second transmembrane protein was the ABC transporter, M.C. It suggests that it is not present in the Bucket homolog. However, M. An open reading frame with homology to the tuberculosis acetyl-CoA acetyltransferase is found in the 530 transmembrane protein.
The translated protein, identified as beginning bp downstream, was designated GV-26. The definitive partial nucleotide sequence and predicted amino acid sequence of GV-26 are shown in SEQ ID NOs: 99 and 100, respectively.

Using a protocol similar to that described above for the isolation of GV-23, 3S-PCR
M. constructed using the band 12B28 (SEQ ID NO: 119) at the BamHI site of λZAP Express (Stratagene). The Bucket genomic library was screened. Clones isolated from the library contain a novel open reading frame, the antigen encoded by this gene being GV-38.
A. The determined nucleotide sequence and predicted amino acid sequence of GV-38A are shown in SEQ ID NOs: 120 and 121, respectively. Subsequent research has shown that GV-3
The 8A extended DNA sequence was isolated and provided in SEQ ID NO: 171. The corresponding amino acid sequence is provided in SEQ ID NO: 172. Comparing these sequences with the sequences in the gene bank, previously the cosmid MTCY428.12 (SPTREMBL: P719)
Unknown M identified in 15). Homology to the tuberculosis protein was determined.

Upstream of the GV-38A gene, a second novel open reading frame was identified, and the antigen encoded by this gene was named GV-38B. GV-
The determined 5 'and 3' nucleotide sequences of 38B are shown in SEQ ID NOs: 122 and 123.
And the corresponding predicted amino acid sequences are set forth in SEQ ID NOs: 124 and 125, respectively.
To each. Further studies have isolated the full length DNA sequence of GV-38B and provide it in SEQ ID NO: 173. The corresponding amino acid sequence is provided in SEQ ID NO: 174. This protein is cosmid MTCY428.11 (SPTREMB
L: P71914). Homology to the tuberculosis protein was determined.

[0277] Both GV-38A and GV-38B antigens were amplified for expression cloning into pET16 (Novagen). GV-38A was amplified using primers KR11 and KR12 (SEQ ID NOS: 126 and 127), and GV-38B was amplified using primers KR13 and KR14 (SEQ ID NOs: 128 and 129).
Protein expression in host cell BL21 (DE3) was induced with 1 mM IPTG, but no protein was expressed from these constructs. A hydrophobic region has been identified at the N-terminus of the antigens GV-38A and GV-38B, which may prevent the expression of these constructs. The hydrophobic region present in GV-38A has been identified as a potential transmembrane motif with six transmembrane regions. To express an antigen that does not contain a hydrophobic region, GV-3
Primer KR20 (SEQ ID NO: 130) for 8A and KR2 for GV-38B
1 (SEQ ID NO: 131) was designed. The truncated GV-38A gene was amplified using primers KR20 and KR12, and the truncated GV-38B gene was amplified using KR21 and KR14. The determined nucleotide sequences of truncated truncated GV38A and GV-38B are shown in SEQ ID NOs: 132 and 133, respectively, and the corresponding predicted amino acid sequences are shown in SEQ ID NOs: 134 and 135, respectively. Cutting shortened GV-3
The extended DNA sequences for 8A and GV-38B are provided in SEQ ID NOs: 175 and 176, respectively, and the corresponding amino acid sequences are provided in SEQ ID NOs: 177 and 178, respectively.

Example 16 M.P. by preparative isoelectric focusing and preparative polyacrylamide gel electrophoresis. Purification and Characterization of Polypeptides from Bucket Culture Filtrate Bucket soluble proteins were isolated from the culture filtrate using preparative isoelectric focusing and preparative polyacrylamide gel electrophoresis as described below. Unless otherwise specified, all percentages in the following examples are by weight per volume.

M. The bucket (ATCC No. 15483) was fractionated by ultrafiltration and cultured in 250 l of sterile medium 90 from which all proteins having a molecular weight of 10 kDa or more had been removed. The medium was centrifuged to remove bacteria and sterilized by filtration through a 0.45 μm filter. The sterile filtrate was concentrated by ultrafiltration on a 10 kDa molecular weight cutoff membrane.

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

The lyophilized protein was purified using 8M deionized urea, 2% Triton.
) X-100, 10 mM dithiothreitol and 2% ampholyte (pH 2.5
-5.0) in a buffer solution containing isoelectric focusing. Samples were taken for 16 hours, 8
Fractionation was performed by preparative isoelectric focusing on a horizontal bed of Ultrodex gel at a constant power of watts. The protein was eluted from the gel bed fraction with water and precipitated by 10% trichloroacetic acid and concentrated.

The pool of the fraction containing the protein of interest was identified by qualitative polyacrylamide gel electrophoresis and fractionated by preparative poracrylamide gel electrophoresis. Samples were fractionated on a 12.5% SDS-PAGE gel and electroblotted onto a nitrocellulose membrane. The protein was located on the membrane by staining with Ponceau Red, decolorized with water, eluted from the membrane with 40% acetonitrile / 0.1 M ammonium bicarbonate pH 8.9, and then concentrated by lyophilization .

The eluted proteins were assayed for their ability to induce proliferation and interferon-γ secretion from peripheral blood lymphocytes of immunized donors as described in detail above.
Proteins that elicit a strong response in these assays were selected for further study.

The selected proteins were prepared using the trifluoroacetic acid-acetonitrile system
Further purification was achieved by reverse phase chromatography on a Vidak protein C4 column. Purified proteins were prepared for protein sequencing by SDS polyacrylamide gel electrophoresis and electroblotted onto PVDF membrane. The protein sequence was determined as in Example 3. Proteins are GV-40, GV-412, GV
-42, GV-43 and GV-44. The determined N-terminal sequences of these polypeptides are shown in SEQ ID NOs: 101-105, respectively. Through subsequent research,
The 5 ', intermediate fragment and 3' DNA sequences of GV-42 (SEQ ID NOs: 136, 137 and 138, respectively) were isolated. The corresponding predicted amino acid sequences are provided in SEQ ID NOs: 139, 140 and 141, respectively.

According to the standard DNA amplification and cloning methods described in Example 13, GV-
The genes encoding 41 and GV-42 were cloned. The determined nucleotide sequences are provided in SEQ ID NOs: 179 and 180, respectively, and the predicted amino acid sequences are provided in SEQ ID NOs: 181 and 182, respectively. Further experiments cloned the full-length gene encoding GV-41 and named it GV-41B. GV-41
The determined nucleotide sequence and predicted amino acid sequence of B are SEQ ID NOs: 202 and 2
03 respectively. GV-41 is a M.V. Tuberculosis and M.
Shows homology to replisome ribosome recycling factor, and GV-42 is It showed homology to avium fibronectin adhesion protein FAP-A. G
Within the full length sequence of V-42, the amino acid sequence determined by GV-43 (SEQ ID NO: 104) was identified, suggesting that the amino acid sequences of GV-42 and GV-43 were obtained from the same protein. .

The murine polyclonal antiserum was prepared according to standard methods using GV-40 and GV-40.
Prepared for V-44. Using these antisera, randomly sheared DN
A consisting of the A fragment. The Bucket genomic DNA library was screened. Clones encoding GV-40 and GV-44 were identified and sequenced. The determined nucleotide sequence of the partial gene encoding GV-40 is provided in SEQ ID NO: 183, and the predicted amino acid sequence is provided in SEQ ID NO: 184. GV-
The complete gene encoding 40 was not cloned, and the antigen encoded by this partial gene was designated GV-40P. The extended DNA sequence for GV-40P is provided in SEQ ID NO: 206, and the corresponding predicted amino acid sequence is provided in SEQ ID NO: 207. The determined nucleotide sequence of the gene encoding GV-44 is SEQ ID NO: 1.
85, the predicted amino acid sequence is provided in SEQ ID NO: 186. Further sequencing yields the determined DNA sequence of the full length gene encoding GV-44, provided in SEQ ID NO: 204, and the corresponding predicted amino acid sequence in SEQ ID NO: 205.
GV40 M. Homology to repli elongation factor G was determined, and GV-44 was identified as M. It had homology to repreglyceraldehyde triphosphate dehydrogenase.

Example 17 DD-M. Isolation of Bucket antigens GV-45 and GV-46 Proteins were suspended in 10 ml of 2% SDS / PBS and heated at 50 ° C. for 2 hours to obtain DD-M. Extracted from bucket (500 mg; prepared as described above). The insoluble residue was removed by centrifugation and the protein was sedimented from the supernatant by adding an equal volume of acetone and incubating at -20 ° C for 1 hour. Precipitated proteins were collected by centrifugation, dissolved in reducing sample buffer, and fractionated by preparative SDS polyacrylamide gel electrophoresis. The separated proteins were electroblotted onto PVDF membrane in 10 mM CAPS / 0.01% SDS pH 11.0, and the N-terminal sequence was determined with a gas phase sequencer.

From these experiments, a protein designated as GV-45 and isolated by a band with a molecular weight of approximately 30 kDa was isolated. The determined N-terminal sequence of GV-45 is SEQ ID NO: 187
To provide. The same experiment yielded a protein called GV-46 with a molecular weight of about 14 kDa. The determined N-terminal amino acid sequence of GV-46 is provided in SEQ ID NO: 208. GV-46 is a M.V. Tuberculosis and M. Homology to Smegma's highly conserved mycobacterial host integration factor.

From the amino acid sequence of GV-45, the modified oligonucleotides KR32 and KR32
Thirty-three (SEQ ID NOs: 188 and 189, respectively) were designed. The 100 bp fragment was amplified, cloned into plasmid pBluescript II SK + (Stratagene, La Jolla, CA) and sequenced (SEQ ID NO: 190) according to standard methods (Sambrook, supra). Em. Constructed at the BamHI site of λZAP-Express (Stratagene) using the cloned insert. The Bucket genomic DNA library was screened. The isolated clone is a 35 kDa M.p. containing a unique C-terminus consisting of a bacterial histone-like motif at the N-terminus and a basic repeat of 5 amino acids. Tuberculosis and 22 kDa M. Shows homology to the repreprotein. The determined nucleotide sequence of GV-45 is provided in SEQ ID NO: 191, and the corresponding predicted amino acid sequence is provided in SEQ ID NO: 192. Further sequencing yielded the determined DNA sequence of the full length gene encoding GV-45, which is provided in SEQ ID NO: 200,
The corresponding predicted amino acid sequence is provided in SEQ ID NO: 201.

Example 18 Immunogenicity and immunomodulatory properties of recombinant proteins from bucke A. Induction of T Cell Proliferation and IFN-γ Production The immunogenicity of the Mycobacterium bucke recombinant protein (GV recombinant protein) was determined in female BALB / cByJ mice in each hind footpad in incomplete Freund's adjuvant (IFA). Tested by injecting 10 μg of recombinant GV protein emulsified in 1. Control mice received phosphate buffered saline in IFA. Drained popliteal lymph node cells were excised 10 days later, and cells obtained therefrom were stimulated with immunizing GV protein and assayed for proliferation by measuring tritiated thymidine incorporation. The amount of interferon gamma (IFNγ) produced by these cells and secreted into the culture supernatant was assayed by a standard enzyme-linked immunoassay.

As shown in Table 21, which summarizes the proliferative response, all GV proteins were found to induce a T cell proliferative response. Lymph node T cells from immunized mice proliferated in response to the specific GV protein used for immunization. Lymph node cells from non-immunized mice did not proliferate in response to GV protein. The data in Table 22 showing IFNγ production suggest that most GV proteins stimulated IFNγ production by lymph node cells from mice immunized with the corresponding GV protein. IFNγ production was not detectable when lymph node cells from non-immunized mice were cultured with individual GV proteins.

Thus, GV proteins are immunogenic in that when administered by subcutaneous injection, they can stimulate T cell proliferation and / or IFNγ production. T cell proliferation and IF
The antigen-specific stimulatory effect on Nγ production is a beneficial property for two tuberculosis vaccine candidates.

[0293]

[Table 28]

[0294]

[Table 29]

B. Activation of Lymphocyte Subassembly The recombinant M. of the present invention. Bucket protein, heat killed M. Bucket and DD-M. The ability of Buckey to activate lymphocyte subpopulations was demonstrated by CD69 (
It was determined by examining the up-regulation of expression of surface protein (expressed on activated cells).

PBMCs from normal donors (5 × 10 ⁶ cells / ml) were treated with 20 μg / ml heat killed M. Bucket cells, DD-M. Bucket or recombinant GV-22
B (SEQ ID NO: 145), GV-23 (SEQ ID NO: 89), GV-27 (SEQ ID NO: 1)
60), GV27A (SEQ ID NO: 117), GV-27B (SEQ ID NO: 162) or GV-45 (SEQ ID NO: 201) for 24 hours. CD69 expression is CD5
6. Cultured cells were stained using a monoclonal antibody against αβ T cells or γδ T cells in combination with a monoclonal antibody against CD69, and then determined by flow cytometry analysis.

Table 23 shows heat-killed M.E. Bucket, DD-M. Bucket or recombinant M. Αβ T cells expressing CD69, γδ T after stimulation with
% Of cells and NK cells. These results indicate that M. killed by heat. Bucket, DD-M. Bucket and GV-23 demonstrated that they stimulated CD69 expression in the tested lymphocyte subsets compared to controls (unstimulated cells), with particularly high CD69 expression levels being found in NK cells. GV-45 was found to up-regulate CD69 expression on αβ T cells.

[0298]

[Table 30]

Using the method described above, recombinant protein GV-23 (20 μg / ml)
The ability of lymphocyte subpopulations to induce CD69 expression was assessed using the known Th1-inducing adjuvant MPL / TDM / CWS (monophosphoryl lipid A / trehalose 6′6 ′).
Dimycolate; Sigma, St. Louis, MO; final dilution 1:20) and C
pG ODN (Promega, Madison, Wis .; 20 μg / ml), and the known Th2-derived adjuvant aluminum oxide (Superfos Biosector), Kvistguard (Kvis)
tgard), Denmark; final dilution 1: 400) and cholera toxin (20 μg)
/ Ml). MPL / TDM / CWS and aluminum oxide were used at the highest concentration that did not cause cytotoxicity. 8A-C show αβ
FIG. 9 shows stimulation of CD69 expression on T cells, γδ T cells and NK cells. GV-
23, MPL / TDM / CWS and CpG ODN induced CD69 expression in NK cells, but not aluminum oxide and cholera toxin. C. Stimulation of cytokine production The recombinant M. of the present invention. The ability of the buckeet protein to stimulate cytokine production in PBMC was examined as follows. PBMCs from normal donors (5 × 10 ⁶ cells / ml) were killed with 20 μg / ml heat. Bucket, DD-M. Bucket 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), G
The cells were stimulated with V-27B (SEQ ID NO: 162) or GV-45 (SEQ ID NO: 201) for 24 hours. The culture supernatant is collected and standard ELISA kit (Genzyme (GenGen)
zyme) (Cambridge, Mass.) according to the manufacturer's instructions for the stimulation of production of IL-1β, TNF-α, IL-12 and IFN-γ. 9A-D show IL-1β, TNF-α, and IL-12, respectively.
And stimulation of IFN-γ production. Em killed by heat. Bucket and DD
-M. Bucket was found to stimulate the production of all four cytokines examined, while recombinant GV-23 and GV-45 stimulated the production of IL-1β, TNF-α and IL-12. There was found. FIGS. 10A-C show stimulation of IL-1β, TNF-α and IL-12 production in human PBMC (as determined above) by various concentrations of GV-23 and GV-45, respectively.

FIGS. 11A-D show IL-1β and T, respectively, in PBMC by GV-23.
The stimulation of NF-α, IL-12 and INF-γ production was stimulated with the adjuvant MPL /
Shown are those compared with TDM / CWS (final dilution 1:20), CpG ODN (20 μg / ml), aluminum oxide (final dilution 1: 400) and cholera toxin (20 μg / ml). GV-23, MPL / TDM / CWS and C
pG ODN induced significant levels of the four cytokines examined, indicating that IL-1β production levels were higher with GV-23 than with any of the known adjuvants. Aluminum oxide and cholera toxin induced very little of the four cytokines. D. Activation of antigen presenting cells. M. killed by heat. Bucket, DD-M. Bucket and recombinant M. Bucket protein co-stimulatory molecule CD40 on B cells, mononuclear cells and dendritic cells
, CD80 and CD86 were tested for their ability to enhance expression as follows.

Peripheral blood mononuclear cells containing T cells removed and predominantly containing only B cells, mononuclear cells and dendritic cells were killed by heat at 20 μg / ml. Bucket cells, DD-M. Bucket 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
The cells were stimulated with -27B (SEQ ID NO: 162) or GV-45 (SEQ ID NO: 201) for 48 hours. Collect the stimulated cells and use three-color flow cytometry analysis
CD40, CD80 and CD86 were analyzed for upregulation. Tables 24, 25 and 26 show the fold increase in mean fluorescence intensity from control (unstimulated cells) in dendritic cells, mononuclear cells, and B cells, respectively.

[0302]

[Table 31]

[0303]

[Table 32]

[0304]

[Table 33]

As indicated above, increased expression levels of CD40, CD80 and CD86 were seen in dendritic cells, mononuclear cells and B cells of all compositions tested. The expression levels are highest in dendritic cells, with the highest expression levels in heat-killed M. aureus. Bucket, DD-M. Obtained with Bucket, GV-23 and GV-45.
FIGS. 12A-C show stimulation of dendritic cell expression of CD40, CD80 and CD86, respectively, by varying concentrations of GV-23 and GV-45.

The ability of GV-23 to stimulate CD40, CD80 and CD86 expression on dendritic cells was determined by the Th1 inducing adjuvant MPL / TDM / CWS (final dilution 1:20).
And CpG ODN (20 μg / ml), and the known Th2-derived adjuvant aluminum oxide (final dilution 1: 400) and cholera toxin (20 μg / m2).
l). GV23, MPL / TDM / CWS and CpG OD
N caused significant up-regulation of CD40, CD80 and CD86, while cholera toxin and aluminum oxide induced little or negligible dendritic cell activation, respectively. E. FIG. Dendritic cell maturation and function The effect of the Bucket protein GV-23 on maturation and function of dendritic cells was examined as follows.

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

[0308]

[Table 34]

The ability of GV-23 to enhance the function of dendritic cells as antigen presenting cells was determined by a mixed lymphocyte reaction (MLR) assay. Purified dendritic cells, alone or with GV-23 (20 μg / ml), were cultured in media for 18-20 hours and then stimulated with allogeneic T cells (2 × 10 ⁵ cells / well). Three days after incubation, (< ³ > H) -thymidine was added. Cells were harvested one day later and radioactivity uptake was measured. FIG. 13 shows that as the ratio of dendritic cells to T cells increases, ( ³ H) -thymidine incorporation increases. Significantly higher levels of radioactivity uptake were seen in GV-23 stimulated dendritic cells compared to unstimulated cells, indicating that GV-2
3 shows that dendritic cell mixed lymphocyte reaction is enhanced.

Although the present invention has been described in some detail by way of illustration and example in order to provide a clear understanding of the invention, changes and modifications may be made without departing from the scope of the invention and the scope of the invention Should be limited only by the scope of the appended claims.

[Sequence list]

[Brief description of the drawings]

1A and 1B show raw M. Each of the autoclaved M. tuberculosis H37Rv-infected M. Bucket or unfractionated M. [Fig. 4] Fig. 4 shows the protective effect against mice immunized using a culture filtrate of a basket.

1A and 1B show raw M. Each of the autoclaved M. tuberculosis H37Rv-infected M. Bucket or unfractionated M. [Fig. 4] Fig. 4 shows the protective effect against mice immunized using a culture filtrate of a basket.

FIGS. 2A and 2B show that M. oryzae were killed with 10 or 1000 μg of heat, respectively, 4 weeks prior to ovalbumin testing, compared to control mice. Bucket or 200-100 μg DD-M. Figure 5 shows% eosinophils in mice immunized intranasally with a bucket. FIGS. 2C and 2D show that 100 μg each of heat-killed M. a.m., one week prior to the ovalbumin test. Bucket or 200 μg DD-M. Figure 5 shows% eosinophils in mice immunized intranasally with a bucket. FIG. 2E shows the BCG of the Pasteur strain before testing for ovalbumin.
(BCG-P), Connaught strain BCG (BCG-C), M. killed with 1 mg heat. Bucket, or 200 μg DD-M. Intranasal (i.
. n. ) Or subcutaneous (sc) immunized mice.

FIGS. 2A and 2B show that M. aliquots were killed with 10 or 1000 μg of heat, respectively, 4 weeks prior to testing for ovalbumin compared to control mice. Bucket or 200-100 μg DD-M. Figure 5 shows% eosinophils in mice immunized intranasally with a bucket. FIGS. 2C and 2D show that 100 μg each of heat-killed M. a.m., one week prior to the ovalbumin test. Bucket or 200 μg DD-M. Figure 5 shows% eosinophils in mice immunized intranasally with a bucket. FIG. 2E shows the BCG of the Pasteur strain before testing for ovalbumin.
(BCG-P), Connaught strain BCG (BCG-C), M. killed with 1 mg heat. Bucket, or 200 μg DD-M. Intranasal (i.
. n. ) Or subcutaneous (sc) immunized mice.

FIGS. 2A and 2B show that M. aliquots were killed with 10 or 1000 μg of heat, respectively, 4 weeks prior to ovalbumin testing, as compared to control mice. Bucket or 200-100 μg DD-M. Figure 5 shows% eosinophils in mice immunized intranasally with a bucket. FIGS. 2C and 2D show that 100 μg each of heat-killed M. a.m., one week prior to the ovalbumin test. Bucket or 200 μg DD-M. Figure 5 shows% eosinophils in mice immunized intranasally with a bucket. FIG. 2E shows the BCG of the Pasteur strain before testing for ovalbumin.
(BCG-P), Connaught strain BCG (BCG-C), M. killed with 1 mg heat. Bucket, or 200 μg DD-M. Intranasal (i.
. n. ) Or subcutaneous (sc) immunized mice.

FIGS. 2A and 2B show that E. m., Killed with 10 or 1000 μg of heat, respectively, 4 weeks prior to testing for ovalbumin compared to control mice. Bucket or 200-100 μg DD-M. Figure 5 shows% eosinophils in mice immunized intranasally with a bucket. FIGS. 2C and 2D show that 100 μg each of heat-killed M. a.m., one week prior to the ovalbumin test. Bucket or 200 μg DD-M. Figure 5 shows% eosinophils in mice immunized intranasally with a bucket. FIG. 2E shows the BCG of the Pasteur strain before testing for ovalbumin.
(BCG-P), Connaught strain BCG (BCG-C), M. killed with 1 mg heat. Bucket, or 200 μg DD-M. Intranasal (i.
. n. ) Or subcutaneous (sc) immunized mice.

FIGS. 2A and 2B show 10 and 1000 μg of heat-killed Em. 4 weeks prior to ovalbumin testing, respectively, compared to control mice. Bucket or 200-100 μg DD-M. Figure 5 shows% eosinophils in mice immunized intranasally with a bucket. FIGS. 2C and 2D show that 100 μg each of heat-killed M. a.m., one week prior to the ovalbumin test. Bucket or 200 μg DD-M. Figure 5 shows% eosinophils in mice immunized intranasally with a bucket. FIG. 2E shows the BCG of the Pasteur strain before testing for ovalbumin.
(BCG-P), Connaught strain BCG (BCG-C), M. killed with 1 mg heat. Bucket, or 200 μg DD-M. Intranasal (i.
. n. ) Or subcutaneous (sc) immunized mice.

FIG. 3A shows that heat-killed M. pneumoniae prior to tuberculosis infection. Bucket or delipidated and deglycolipidated M. The effect of immunizing a mouse with a bucket (DD-M. Bucket) is shown. FIG. 3B shows that heat-killed M. et al. Prior to tuberculosis infection. Bucket, recombinant M. M. killed by bakey protein or heat. Bakke and M. Figure 4 shows the effect of immunizing mice with a combination of the Bucket recombinant proteins.

FIG. 3A shows that heat-killed M. et al. Prior to tuberculosis infection. Bucket or delipidated and deglycolipidated M. The effect of immunizing a mouse with a bucket (DD-M. Bucket) is shown. FIG. 3B shows that heat-killed M. et al. Prior to tuberculosis infection. Bucket, recombinant M. M. killed by bakey protein or heat. Bakke and M. Figure 4 shows the effect of immunizing mice with a combination of the Bucket recombinant proteins.

FIG. 4 shows autoclaved M.P. Bucket, freeze-dried em. Bucket,
Delipidation and deglycolipidation M. Bakke and M. I by Bucket glycolipid
4 shows induction of L-12.

FIG. 5 shows heat-killed (autoclaved) M. at various concentrations. Bucket, delipidated and deglycolipidated M. Bucket, and M. FIG. 4 compares in vitro stimulation of interferon-γ production in spleen cells from severe combined immunodeficiency (SCID) mice by glycolipids in Buckey.

FIGS. 6A, B and C show various concentrations of em. In peritoneal macrophages from C57BL / 6 mice (FIG. 6A), BALB / C mice (FIG. 6B) or C3H / HeJ mice (FIG. 6C). Bucket recombinant protein, heat killed M. Bucket, delipidated and deglycolipidated M. Bakey (referred to as "lipidated M. bakey" in the figure), M. FIG. 9 shows stimulation of interferon-γ production by baccay glycolipids and lipopolysaccharide.

FIGS. 6A, B and C show various concentrations of em. In peritoneal macrophages from C57BL / 6 mice (FIG. 6A), BALB / C mice (FIG. 6B) or C3H / HeJ mice (FIG. 6C). Bucket recombinant protein, heat killed M. Bucket, delipidated and deglycolipidated M. Bakey (referred to as "lipidated M. bakey" in the figure), M. FIG. 9 shows stimulation of interferon-γ production by baccay glycolipids and lipopolysaccharide.

FIGS. 6A, B and C show various concentrations of em. In peritoneal macrophages from C57BL / 6 mice (FIG. 6A), BALB / C mice (FIG. 6B) or C3H / HeJ mice (FIG. 6C). Bucket recombinant protein, heat killed M. Bucket, delipidated and deglycolipidated M. Bakey (referred to as "lipidated M. bakey" in the figure), M. FIG. 9 shows stimulation of interferon-γ production by baccay glycolipids and lipopolysaccharide.

FIGS. 7A (i)-(iv) show 10 μg, 100 μg and 1 mg of autoclaved M.E. Bucket and 75 μg em. Fig. 3 shows the non-specific immunoamplification effect of the non-fractionated culture filtrate of a basket. FIG. 7B (i) and (ii)
M. respectively autoclaved. Bucket, and delipidated and deglycolipidated M. 3 shows the non-specific immunoamplification effect of a bucket. FIG. 7C (i) shows all autoclaved M.M. 3 shows the non-specific immunoamplification effect of a bucket. FIG. 7C (i
i) is delipidated and deglycolipidated M. Soluble M. extracted from the cake by SDS. 3 shows the non-specific immunoamplification effect of the Bucket protein. FIG. 7C (iii) shows that the non-specific immunoamplification effect of the preparation of FIG. 7 (ii) is degraded by treatment with protease pronase. FIG. 7D shows M. killed by heat. FIG. 7D shows the non-specific immunoamplification effect of the bucket (FIG. 7D (i)). Tuberculosis (FIG. 7D (ii)), M.C. Bovis BCG (Fig. 7D
(Iii)), M. Frey (FIG. 7D (iv)) and M.E. Smegmatis (
It was not found in the heat-killed preparation of FIG. 7D (v)).

7A (i)-(iv) show 10 μg, 100 μg and 1 mg of autoclaved M. Bucket and 75 μg em. Fig. 3 shows the non-specific immunoamplification effect of the non-fractionated culture filtrate of a basket. FIGS. 7B (i) and (ii) show autoclaved M.E. Bucket, and delipidated and deglycolipidated M. 3 shows the non-specific immunoamplification effect of a bucket. FIG. 7C (i) shows all autoclaved M.M. 3 shows the non-specific immunoamplification effect of a bucket. FIG. 7C (ii
) Are delipidated and deglycolipidated M. Soluble M. extracted from the cake by SDS. 3 shows the non-specific immunoamplification effect of the Bucket protein. FIG. 7C (iii)
The non-specific immunoamplification effect of the preparation of FIG. 7 (ii) indicates that it is degraded by treatment with protease pronase. FIG. 7D shows M. killed by heat. FIG. 7D shows the non-specific immunoamplification effect of the bucket (FIG. 7D (i)).
M. Tuberculosis (FIG. 7D (ii)), M.C. Bovis BCG (Fig. 7D (
iii)), M. Frey (FIG. 7D (iv)) and M.E. It was not found in the heat killed preparation of Smegmatis (FIG. 7D (v)).

7A (i)-(iv) show 10 μg, 100 μg and 1 mg of autoclaved em. Bucket and 75 μg em. Fig. 3 shows the non-specific immunoamplification effect of the non-fractionated culture filtrate of a basket. FIGS. 7B (i) and (ii) show autoclaved M.E. Bucket, and delipidated and deglycolipidated M. 3 shows the non-specific immunoamplification effect of a bucket. FIG. 7C (i) shows all autoclaved M.M. 3 shows the non-specific immunoamplification effect of a bucket. FIG. 7C (ii
) Are delipidated and deglycolipidated M. Soluble M. extracted from the cake by SDS. 3 shows the non-specific immunoamplification effect of the Bucket protein. FIG. 7C (iii)
The non-specific immunoamplification effect of the preparation of FIG. 7 (ii) indicates that it is degraded by treatment with protease pronase. FIG. 7D shows M. killed by heat. FIG. 7D shows the non-specific immunoamplification effect of the bucket (FIG. 7D (i)).
M. Tuberculosis (FIG. 7D (ii)), M.C. Bovis BCG (Fig. 7D (
iii)), M. Frey (FIG. 7D (iv)) and M.E. It was not found in the heat killed preparation of Smegmatis (FIG. 7D (v)).

7A (i)-(iv) show 10 μg, 100 μg and 1 mg of autoclaved M.E. Bucket and 75 μg em. Fig. 3 shows the non-specific immunoamplification effect of the non-fractionated culture filtrate of a basket. FIGS. 7B (i) and (ii) show autoclaved M.E. Bucket, and delipidated and deglycolipidated M. 3 shows the non-specific immunoamplification effect of a bucket. FIG. 7C (i) shows all autoclaved M.M. 3 shows the non-specific immunoamplification effect of a bucket. FIG. 7C (ii
) Are delipidated and deglycolipidated M. Soluble M. extracted from the cake by SDS. 3 shows the non-specific immunoamplification effect of the Bucket protein. FIG. 7C (iii)
The non-specific immunoamplification effect of the preparation of FIG. 7 (ii) indicates that it is degraded by treatment with protease pronase. FIG. 7D shows M. killed by heat. FIG. 7D shows the non-specific immunoamplification effect of the bucket (FIG. 7D (i)).
M. Tuberculosis (FIG. 7D (ii)), M.C. Bovis BCG (Fig. 7D (
iii)), M. Frey (FIG. 7D (iv)) and M.E. It was not found in the heat killed preparation of Smegmatis (FIG. 7D (v)).

FIG. 8A and FIG. Αβ T cells, respectively, by the Bucket protein GV23, Th1 derived adjuvants MPL / TDM / CWS and CpG ODN, and Th2 derived adjuvants aluminum hydroxide and cholera toxin, respectively.
Figure 4 shows stimulation of CD69 expression on γδ T cells and NK cells.

FIGS. 9A-D show IL-1β, TNF-α, I by human PBMC, respectively.
Heat killed M. for production of L-12 and IFN-γ. Bucket, D
D-M. Bakke and M. 3 shows the effect of the buckeet recombinant protein.

FIGS. 10A-C show various concentrations of M. pneumoniae for production of IL-1β, TNF-α and IL-12, respectively, by human PBMC. Bucket protein GV-
23 and 23 show the effects of GV-45.

FIGS. 11A to 11D are views of M.E. IL-1β, TNF-α, IL-, respectively, on human PBMCs with the bucke protein GV-23, Th1 derived adjuvants MPL / TDM / CWS and CpG ODN, and Th2 derived adjuvants aluminum hydroxide and cholera toxin, respectively. 12 shows stimulation of 12 and IFN-γ production.

FIGS. 12A-C show CD40, CD80 and CD by dendritic cells, respectively.
86 at various concentrations for the expression of recombinant M.86. Bucket protein GV-23
And the effects of GV-45.

FIG. 13 shows recombinant M. FIG. 9 shows enhancement of dendritic cell mixed lymphocyte reaction by the Bucket protein GV-23.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 11/06 A61P 17/00 4C084 17/00 17/06 4C085 17/06 31/00 4C086 31/00 31 / 06 4H045 31/06 37/04 37/04 C07K 14/35 C07K 14/35 16/12 16/12 19/00 19/00 C12N 1/19 C12N 1/19 1/21 1/21 C12P 21/08 5/10 C12Q 1/02 C12P 21/08 G01N 33/569 A C12Q 1/02 33/68 G01N 33/569 C12R 1:32) 33/68 C12N 15/00 ZNAA // (C12N 15/09 ZNA 5 / (00B C12R 1:32) (31) Priority claim number 08 / 996,624 (32) Priority date December 23, 1997 (December 23, 1997) (33) Priority claim country United States (US) ( 31) Priority claim number 09 / 095,855 (32) Priority date Heisei 1 June 11, 1998 (June 11, 1998) (33) Priority claiming country United States (US) (31) Priority claim number 09 / 156,181 (32) Priority date September 17, 1998 (1998) (9.17) (33) Priority country United States (US) (31) Priority number 09 / 205,426 (32) Priority date December 4, 1998 (1998.12.4) (33) Priority country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD) , SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA , CH, C , CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Bizer, Elizabeth S. New Zealand Auckland Blockhouse Bay Lynbrook Avenue 3 (72) Inventor Skinner, Margot A New Zealand Auckland West Tomia West End Road 113 (72) Inventor Prestige, Los El New Zealand Auckland Freemans Bay Head Baan Street 20 F-term (reference) 2G045 AA25 AA28 AA40 BB20 CB01 CB09 CB21 DA36 DA77 DA78 FB03 FB07 FB08 FB12 FB13 FB15 4B024 AA01 AA12 AA13 BA31 BA43 CA01 CA07 DA02 DA05 DA06 DA12 GA11 HA15 4B063 QA19 QQ02 QQ79 QQ96 QR48 QR75 QS24 QX01 4B064 AG27 AG31 CA19 CC24 DA01 DA15 4B065 AA26X AA36X AA36Y AA72X AA90X AC14 BA02 BD50 CA24 CA25 CA44 CA45 CA46 CA60 4C084 AA02 AA03 AA13 BA01 BA08 BA20 BA21 BA22 BA23 BA41 BA42 CA04 DA40 NA14 ZA591AZA1BZA1ZA1BZA1ZA1BZA1ZA1BZA1ZA1BA1 FF24 4C086 AA01 AA02 EA16 MA01 MA02 MA03 MA04 MA05 NA14 ZA59 ZA89 ZB02 ZB05 ZB07 ZB09 ZB35 4H045 AA10 AA11 BA10 BA41 CA11 CA40 DA76 DA86 EA31 EA52 FA74

Claims (43)

[Claims] 1. Isolated M. A polypeptide comprising an immunogenic portion of a bucke antigen, wherein the antigen comprises SEQ ID NOs: 143, 145, 147, 152, 154, 15
6, 158, 160, 162, 165, 166, 170, 172, 174, 17
7, 178, 181, 182, 184, 186, 187, 192, 194, 19
6, 197, 199, 201, 203, 205, and 207. A polypeptide comprising a sequence selected from the group consisting of: 2. Isolated M. A polypeptide comprising an immunogenic portion of a bucke antigen, comprising: (a) SEQ ID NO: 143, as measured by the computer algorithm BLASTP;
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,
A sequence having at least about 50% identical residues to the sequences listed in 205 and 207; (b) SEQ ID NO: 143, as measured by the computer algorithm BLASTP
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,
A sequence having at least about 75% identical residues to the sequences recited in 205 and 207; and (c) SEQ ID NO: 143, as measured by the computer algorithm BLASTP.
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,
A polypeptide comprising a sequence selected from the group consisting of: a sequence having at least about 95% identical residues to the sequences recited in 205 and 207. 3. An isolated M. A polypeptide comprising an immunogenic portion of a Bucket antigen, comprising: (a) SEQ ID NOs: 142, 144, 146, 151, 153, 155, 157, 1
59, 161, 163, 164, 169, 171, 173, 175, 176, 1
79, 180, 183, 185, 191, 193, 195, 198 and 200
(B) SEQ ID NOs: 142, 144, 146, 151, 153, 155, 157, 1
59, 161, 163, 164, 169, 171, 173, 175, 176, 1
79, 180, 183, 185, 191, 193, 195, 198 and 200
(C) as determined by the computer algorithm BLASTN, (a) or (b)
A) a sequence that has at least about 99% probability of being identical to the sequence of (a). The polypeptide comprising an amino acid sequence encoded by a polynucleotide selected from the group consisting of: 4. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide according to any one of claims 1 to 3. 5. An expression vector comprising the polynucleotide according to claim 4. 6. A host cell transformed with the expression vector according to claim 5. 7. The host cell of claim 6, wherein the host cell is selected from the group consisting of E. coli, mycobacteria, insects, yeast, and mammalian cells. A fusion protein comprising at least one polypeptide according to any one of claims 1 to 3. 9. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 3 and a physiologically acceptable carrier. 10. A pharmaceutical composition comprising the polynucleotide according to claim 4 and a physiologically acceptable carrier. 11. A pharmaceutical composition comprising the fusion protein according to claim 8 and a physiologically acceptable carrier. A vaccine comprising the polypeptide according to any one of claims 1 to 3 and a non-specific immune response amplifying substance. 13. A vaccine comprising the polynucleotide according to claim 4 and a non-specific immune response amplifying substance. 14. A vaccine comprising the fusion protein according to claim 8 and a non-specific immune response amplifying substance. 15. The vaccine according to any one of claims 12 to 14, wherein the non-specific immune response amplifying substance is an adjuvant. 16. A non-specific immune response amplifying substance comprising: (a) delipidated and deglycolipidated M. (B) inactivated M. B.c. cells; and (c) M. The vaccine according to any one of claims 12 to 14, wherein the vaccine is selected from the group consisting of: 17. A method of enhancing a patient's immune response, comprising administering a pharmaceutical composition according to any one of claims 9 to 11 to a patient. 18. A method for enhancing an immune response in a patient, comprising administering to the patient the vaccine according to any one of claims 12 to 14. 19. The method according to any one of claims 17 and 18, wherein the immune response is a Th1 response. 20. A method for treating a disease in a patient, comprising administering the pharmaceutical composition according to any one of claims 9 to 11 to the patient. 21. A method for treating a disease in a patient, comprising administering the vaccine according to any one of claims 12 to 14 to the patient. 22. The method according to any one of claims 20 and 21, wherein the disease is selected from the group consisting of an immune disease, an infectious disease, a skin disease and a respiratory disease. 23. The method of claim 23, wherein the disease is selected from the group consisting of mycobacterial infection, asthma and psoriasis. (A) Inactivated M. (B) delipidated and deglycolipidated M. (C) Delipidated and deglycolipidated M. with mycolic acid removed. (D) Delipidated and deglycolipidated M. with mycolic acid and arabinogalactan removed. B. Cell; and (e) M. A disease of the patient, wherein the disease is selected from the group consisting of an immune disease, an infectious disease, a skin disease, and a disease of the respiratory system, which comprises administering a composition comprising a component selected from the group consisting of a bucke culture filtrate. Cure. 25. The method of claim 24, wherein said disease is selected from the group consisting of mycobacterial infection, asthma and psoriasis. 26. A method of enhancing a non-specific immune response to an antigen, comprising administering a polypeptide, wherein the polypeptide comprises M. The immunogenic portion of a Bucket antigen. The Bucket antigen has (a) the sequences listed in SEQ ID NOs: 89 and 201; and (b) has at least about 80% identical residues to the sequences listed in SEQ ID NOs: 89 and 201, as measured by the computer algorithm BLASTP. A method comprising a sequence selected from the group consisting of: 27. (a) skin cells of a patient according to any one of claims 1 to 3;
Contacting with one or more of the polypeptides of paragraphs above; and (b) detecting an immune response in the patient's skin. 28. The method of claim 27, wherein the immune response is sclerosis. 29. A diagnostic kit comprising: (a) the polypeptide according to any one of claims 1 to 3; and (b) a device for bringing the polypeptide into sufficient contact with skin cells of a patient. 30. (a) contacting a biological sample with a polypeptide according to any one of claims 1 to 3; and (b) determining in the sample the presence of antibodies that bind to said polypeptide. A method of detecting a mycobacterial infection in a biological sample, comprising detecting. 31. The polypeptide (s) attached to a solid support.
0 method. 32. The method of claim 30, wherein the biological sample is selected from the group consisting of whole blood, serum, plasma, saliva, cerebrospinal fluid, and urine. 33. (a) contacting a biological sample with a binding agent capable of binding to the polypeptide of any one of claims 1 to 3; and (b) a protein binding to the binding agent. Alternatively, a method for detecting a mycobacterial infection in a biological sample, comprising detecting the polypeptide in the sample. 34. The method of claim 33, wherein said binding agent is a monoclonal antibody. 35. The method of claim 33, wherein said binding agent is a polyclonal antibody. 36. A diagnostic kit comprising: (a) at least one polypeptide according to any one of claims 1 to 3; and (b) a detection reagent. 37. The polypeptide according to claim 3, wherein the polypeptide is immobilized on a solid support.
Kit of 6. 38. The kit of claim 36, wherein the detection reagent comprises a reporter group conjugated to a binding agent. 39. The kit of claim 38, wherein said binding agent is selected from the group consisting of anti-immunoglobulins, protein G, protein A and lectins. 40. A reporter group comprising a radioisotope, a fluorescent group, a luminescent group, an enzyme,
39. The kit of claim 38, wherein the kit is selected from the group consisting of biotin, and dye particles. 41. A monoclonal antibody that binds to the polypeptide according to any one of claims 1 to 3. 42. A polyclonal antibody that binds to the polypeptide according to any one of claims 1 to 3. 43. (a) Delipidated and deglycolipidated em., Excluding mycolic acid. (B) delipidated and deglycolipidated M, excluding mycolic acid and arabinogalactan. A method of enhancing a non-specific immune response to an antigen, comprising administering a composition comprising a component selected from the group consisting of bucky cells.