EP1044273A2 - COMPOSITIONS DERIVEES DE $i(MYCOBACTERIUM VACCAE) ET LEURS METHODES D'UTILISATION - Google Patents

COMPOSITIONS DERIVEES DE $i(MYCOBACTERIUM VACCAE) ET LEURS METHODES D'UTILISATION

Info

Publication number
EP1044273A2
EP1044273A2 EP98963665A EP98963665A EP1044273A2 EP 1044273 A2 EP1044273 A2 EP 1044273A2 EP 98963665 A EP98963665 A EP 98963665A EP 98963665 A EP98963665 A EP 98963665A EP 1044273 A2 EP1044273 A2 EP 1044273A2
Authority
EP
European Patent Office
Prior art keywords
vaccae
cells
polypeptide
seq
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98963665A
Other languages
German (de)
English (en)
Inventor
Paul Tan
James Watson
Elizabeth S. Visser
Margot A. Skinner
Ross L. Prestidge
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genesis Research and Development Corp Ltd
Original Assignee
Genesis Research and Development Corp Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/997,080 external-priority patent/US5968524A/en
Priority claimed from US08/997,362 external-priority patent/US5985287A/en
Priority claimed from US09/095,855 external-priority patent/US6160093A/en
Priority claimed from US09/205,426 external-priority patent/US6406704B1/en
Application filed by Genesis Research and Development Corp Ltd filed Critical Genesis Research and Development Corp Ltd
Publication of EP1044273A2 publication Critical patent/EP1044273A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to compositions which are present in or may be derived from Mycobacterium vaccae and their use in the treatment, prevention and detection of disorders including infectious diseases, immune disorders and cancer.
  • the invention is related to compounds and methods for the treatment of diseases of the respiratory system, such as mycobacterial infections, asthma, sarcoidosis and lung cancers, and disorders of the skin, such as psoriasis, atopic dermatis, allergic contact dermatitis, alopecia areata, and the skin cancers basal cell carcinoma, squamous cell carcinoma and melanoma.
  • the invention is further related to compounds that function as non-specific immune response amplifiers, and the use of such non-specific immune response amplifiers as adjuvants in vaccination or immunotherapy against infectious disease, and in certain treatments for immune disorders and cancer.
  • Tuberculosis is a chronic, infectious disease, that is caused by infection with Mycobacterium tuberculosis (M. tuberculosis). It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about 8 million new cases and 3 million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as a chronic inflammation of the lungs, resulting in fever and respiratory symptoms. If left untreated, significant morbidity and death may result.
  • tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behaviour is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistant mycobacteria.
  • Inhibiting the spread of tuberculosis requires effective vaccination and accurate, early diagnosis of the disease.
  • vaccination by subcutaneous or intradermal injection with live bacteria is the most efficient method for inducing protective immunity.
  • the most common mycobacterium employed for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of Mycobacterium bovis (M. bovis).
  • BCG Bacillus Calmette-Guerin
  • M. bovis Mycobacterium bovis
  • the safety and efficacy of BCG is a source of controversy and some countries, such as the United States, do not vaccinate the general public.
  • Diagnosis of M. tuberculosis infection is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative).
  • Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, thereby indicating exposure to mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.
  • M. vaccae Mycobacterium vaccae
  • U.S. Patent 4,716,038 discloses diagnosis of, vaccination against and treatment of autoimmune diseases of various types, including arthritic diseases, by administering mycobacteria, including M. vaccae.
  • U.S. Patent 4,724,144 discloses an immunotherapeutic agent comprising antigenic material derived from M. vaccae for treatment of mycobacterial diseases, especially tuberculosis and leprosy, and as an adjuvant to chemotherapy.
  • International Patent Publication WO 91/01751 discloses the use of antigenic and or immunoregulatory material from M.
  • U.S. Patent 5,599,545 discloses the use of mycobacteria, especially whole, inactivated M. vaccae, as an adjuvant for administration with antigens which are not endogenous to M. vaccae. This publication theorises that the beneficial effect as an adjuvant may be due to heat shock protein 65 (hsp 65).
  • International Patent Publication WO 92/08484 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of uveitis.
  • International Patent Publication WO 93/16727 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of mental diseases associated with an autoimmune reaction initiated by an infection.
  • International Patent Publication WO 95/26742 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for delaying or preventing the growth or spread of tumors.
  • International Patent Publication WO 91/02542 discloses the use of autoclaved M. vaccae in the treatment of chronic inflammatory disorders in which a patient demonstrates an abnormally high release of IL-6 and/or TNF or in which the patient's IgG shows an abnormally high proportion of agalactosyl IgG.
  • psoriasis rheumatoid arthritis
  • mycobacterial disease Crohn's disease
  • primary biliary cirrhosis sarcoidosis
  • ulcerative colitis systemic lupus erythematosus
  • multiple sclerosis Guillain-Barre syndrome
  • primary diabetes mellitus and some aspects of graft rejection.
  • M. vaccae is apparently unique among known mycobacterial species in that heat- killed preparations retain vaccine and immunotherapeutic properties.
  • M. tuberculosis BCG vaccines used for vaccination against tuberculosis, employ live strains. Heat-killed M. bovis BCG and M. tuberculosis have no protective properties when employed in vaccines.
  • a number of compounds have been isolated from a range of mycobacterial species which have adjuvant properties. The effect of such adjuvants is essentially to stimulate a particular immune response mechanism against an antigen from another species.
  • DD-M vaccae contains highly polymerised cell wall.
  • DD-M vaccae contains 50% w/w protein, comprising a number of distinct protein species.
  • Sarcoidosis is a disease of unknown cause characterised by granulomatous inflammation affecting many organs of the body and especially the lungs, lymph nodes and liver.
  • Sarcoid granulomata are composed of mononuclear phagocytes, with epithelioid and giant cells in their centre, and T lymphocytes.
  • CD4 T lymphocytes are closely associated with the epithelioid cells while both CD4 and CD8 T lymphocytes accumulate at the periphery.
  • the characteristic immunological abnormalities in sarcoidosis include peripheral blood and bronchoalveolar lavage hyper-globulinaemia and depression of 'delayed type' hypersensitivity reactions in the skin to tuberculin and other similar antigens, such as Candida and mumps.
  • Peripheral blood lymphocyte numbers are reduced and CD4: CD8 ratios in peripheral blood are depressed to approximately 1-1.5:1. These are not manifestations of a generalised immune defect, but rather the consequence of heightened immunological activity which is 'compartmentalised' to sites of disease activity.
  • the total number of cells recovered by bronchoalveolar lavage is increased five- to ten-fold and the proportion of lymphocytes increased from the normal of less than 10-14% to between 15% and 50%. More than 90% of the lymphocytes recovered are T lymphocytes and the CD4-.CD8 ratio has been reported to be increased from the value of 1.8:1 in normal controls to 10.5:1.
  • the T lymphocytes are predominantly of the Thl class, producing IFN- ⁇ and IL-2 cytokines, rather than of the Th2 class. Following treatment, the increase in Thl lymphocytes in sarcoid lungs is corrected.
  • sarcoidosis involves the lungs in nearly all cases. Even when lesions are predominantly seen in other organs, subclinical lung involvement is usually present. While some cases of sarcoidosis resolve spontaneously, approximately 50% of patients have at least a mild degree of permanent organ dysfunction. In severe cases, lung fibrosis develops and progresses to pulmonary failure requiring lung transplantation.
  • the mainstay of treatment for sarcoidosis is corticosteroids. Patients initially responding to corticosteroids often relapse and require treatment with other immunosuppressive drugs such as methotrexate or cyclosporine.
  • Asthma is a common disease, with a high prevalence in the developed world. Asthma is characterised by increased responsiveness of the tracheobronchial tree to a variety of stimuli, the primary physiological disturbance being reversible airflow limitation, which may be spontaneous or drug-related, and the pathological hallmark being inflammation of the airways. Clinically, asthma can be subdivided into extrinsic and intrinsic variants.
  • Extrinsic asthma has an identifiable precipitant, and can be thought of as being atopic, occupational and drug-induced.
  • Atopic asthma is associated with the enhancement of a Th2- type of immune response with the production of specific immunoglobulin E (IgE), positive skin tests to common aeroallergens and/or atopic symptoms. It can be divided further into seasonal and perennial forms according to the seasonal timing of symptoms.
  • IgE immunoglobulin E
  • the airflow obstruction in extrinsic asthma is due to nonspecific bronchial hyperesponsiveness caused by inflammation of the airways. This inflammation is mediated by chemicals released by a variety of inflammatory cells including mast cells, eosinophils and lymphocytes. The actions of these mediators result in vascular permeability, mucus secretion and bronchial smooth muscle constriction.
  • atopic asthma the immune response producing airway inflammation is brought about by the Th2 class of T cells which secrete IL-4, IL-5 and IL-10. It has been shown that lymphocytes from the lungs of atopic asthmatics produce IL-4 and IL-5 when activated. Both IL-4 and IL-5 are cytokines of the Th2 class and are required for the production of IgE and involvement of eosinophils in asthma.
  • Occupational asthma may be related to the development of IgE to a protein hapten, such as acid anhydrides in plastic workers and plicatic acid in some western red cedar-induced asthma, or to non-IgE related mechanisms, such as that seen in toluene diisocyanate-induced asthma.
  • Drug-induced asthma can be seen after the administration of aspirin or other non-steroidal anti-inflammatory drugs, most often in a certain subset of patients who may display other features such as nasal polyposis and sinusitis.
  • Intrinsic or cryptogenic asthma is reported to develop after upper respiratory tract infections, but can arise de novo in middle-aged or older people, in whom it is more difficult to treat than extrinsic asthma.
  • Asthma is ideally prevented by the avoidance of triggering allergens but this is not always possible nor are triggering allergens always easily identified.
  • the medical therapy of asthma is based on the use of corticosteroids and bronchodilator drugs to reduce inflammation and reverse airway obstruction. In chronic asthma, treatment with corticosteroids leads to unacceptable adverse side effects.
  • Allergic rhinitis is a common disorder and is estimated to affect at least 10% of the population. Allergic rhinitis may be seasonal (hay fever) caused by allergy to pollen. Non- seasonal or perennial rhinitis is caused by allergy to antigens such as those from house dust mite or animal dander.
  • the abnormal immune response in allergic rhinitis is characterised by the excess production of IgE antibodies specific against the allergen.
  • the inflammatory response occurs in the nasal mucosa rather than further down the airways as in asthma.
  • local eosinophilia in the affected tissues is a major feature of allergic rhinitis.
  • patients develop sneezing, nasal discharge and congestion.
  • the inflammation extends to the eyes (conjunctivitis), palate and the external ear. While it is not life threatening, allergic rhinitis may be very disabling, prevent normal activities, and interfere with a person's ability to work.
  • Current treatment involves the use of antihistamines, nasal decongestants and, as for asthma, sodium cromoglycate and corticosteroids.
  • Lung cancer is the leading cause of death from cancer.
  • the incidence of lung cancer continues to rise and the World Health Organisation estimates that by 2000AD there will be 2 million new cases annually.
  • Lung cancers may be broadly classified into two categories: small cell lung cancer (SCLC) which represents 20-25% of all lung cancers, and non-small cell lung cancer (NSCLC) which accounts for the remaining 75%.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • the majority of SCLC is caused by tobacco smoke.
  • SCLC tends to spread early and 90% of patients present at diagnosis with involvement of the mediastinal lymph nodes in the chest.
  • SCLC is treated by chemotherapy, or a combination of chemotherapy and radiotherapy. Complete response rates vary from 10% to 50%. For the rare patient without lymph node involvement, surgery followed by chemotherapy may result in cure rates exceeding 60%.
  • the prognosis for NSCLC is more dismal, as most patients have advanced disease by the time of diagnosis. Surgical removal of the tumor is possible in a very small number of patients and the five
  • this invention deals with treatment of disorders of skin which appear to be associated with factors that influence the balance of thymus-derived (T) immune cells known as Thl and Th2. These T cells are identified by their cytokine secretion phenotype.
  • T thymus-derived
  • a common feature of treatment is the use of compounds prepared from M vaccae which have immunomodulating properties that alter the balance of activities of these T cells as well as other immune cells.
  • Psoriasis is a common, chronic inflammatory skin disease which can be associated with various forms of arthritis in a minority of patients.
  • the defect in psoriasis appears to be overly rapid growth of keratinocytes and shedding of scales from the skin surface.
  • Drug therapy is directed at slowing down this process.
  • the disease may become manifest at any age. Spontaneous remission is relatively rare, and life-long treatment is usually necessary.
  • Psoriasis produces chronic, scaling red patches on the skin surface.
  • Psoriasis is a very visible disease, it frequently affects the face, scalp, trunk and limbs. The disease is emotionally and physically debilitating for the patient, detracting significantly from the quality of life. Between one and three million individuals in the United States have psoriasis with nearly a quarter million new cases occurring each year. Conservative estimates place the costs of psoriasis care in the United States currently at $248 million a year.
  • the first is that genetic factors determine abnormal proliferation of epidermal keratinocytes. The cells no longer respond normally to external stimuli such as those involved in maintaining epidermal homeostasis. Abnormal expression of cell membrane cytokine receptors or abnormal transmembrane signal transduction might underlie cell hyperproliferation. Inflammation associated with psoriasis is secondary to the release of pro-inflammatory molecules from hyperproliferative keratinocytes.
  • T cells interacting with antigen-presenting cells in skin release pro-inflammatory and keratinocyte-stimulating cytokines Hancock, G.E. et al, J. Exp. Med. 765:1395-1402, 1988.
  • the keratinocytes themselves may be the antigen-presenting cell.
  • the cellular infiltrate in psoriatic lesions show an influx of CD4+ T cells and, more prominently, CD8+ T cells (Bos, J.D. et al., Arch. Dermatol. Res. 281:23-3, 1989; Baker, B.S., Br. J. Dermatol. 110:555-564, 1984).
  • Atopic dermatitis is a chronic pruritic inflammatory skin disease which usually occurs in families with an hereditary predisposition for various allergic disorders such as allergic rhinitis and asthma.
  • Atopic dermatitis occurs in approximately 10% of the general population.
  • the main symptoms are dry skin, dermatitis (eczema) localised mainly in the face, neck and on the flexor sides and folds of the extremities accompanied by severe itching. It typically starts within the first two years of life. In about 90% of the patients this skin disease disappears during childhood but the symptoms can continue into adult life. It is one of the commonest forms of dermatitis world-wide. It is generally accepted that in atopy and in atopic dermatitis, a T cell abnormality is primary and that the dysfunction of T cells which normally regulate the production of IgE is responsible for the excessive production of this immunoglobulin.
  • Allergic contact dermatitis is a common non-infectious inflammatory disorder of the skin.
  • immunological reactions cannot develop until the body has become sensitised to a particular antigen.
  • Subsequent exposure of the skin to the antigen and the recognition of these antigens by T cells result in the release of various cytokines, proliferation and recruitment of T cells, and finally in dermatitis (eczema).
  • T cells CD4 + cells
  • MHC class II antigens CD4 + cells
  • Keratinocytes can produce interleukin- 1 which can facilitate the antigen presentation to T cells.
  • IFN- ⁇ interferon-gamma
  • TNF tumor necrosis factor
  • IFN- ⁇ interferon-gamma
  • An inhibitory effect of cyclosporin has been observed in delayed-type hypersensitivity on the pro-inflammatory function(s) of primed T cells in vitro (Shidani, B. et al, Eur. J. Immunol. 74:314-318, 1984). The inhibitory effect of cyclosporin on the early phase of T cell activation in mice has also been reported (Milon, G. et al., Ann. Immunol. (Inst. Pasteur) 135d: 237-245, 1984).
  • Alopecia areata is a common hair disease, which accounts for about 2% of the consultations at dermatological outpatient clinics in the United States.
  • the hallmark of this disease is the formation of well-circumscribed round or oval patches of non-scarring alopecia which may be located in any hairy area of the body.
  • the disease may develop at any age. The onset is usually sudden and the clinical course is varied.
  • alopecia areata At present, it is not possible to attribute all or indeed any case of alopecia areata to a single cause (Rook, A. and Dawber, R, Diseases of the Hair and Scalp; Blackwell Scientific Publications 1982: 272-30). There are many factors that appear to be involved. These include genetic factors, atopy, association with disorders of supposed autoimmune etiology, Down's syndrome and emotional stress. The prevalence of atopy in patients with alopecia areata is increased. There is evidence that alopecia areata is an autoimmune disease.
  • Immunophenotyping studies on scalp biopsy specimens shows expression of HLA-DR on epithelial cells in the presumptive cortex and hair follicles of active lesions of alopecia areata, as well as a T cell infiltration with a high proportion of helper/inducer T cells in and around the hair follicles, increased numbers of Langerhans cells and the expression of ICAM- 1 (Messenger, A.G. et al., J. Invest. Dermatol. 85:569-516, 1985; Gupta, A.K. et al., J. Am. Acad. Dermatol. 22:242-250, 1990).
  • alopecia The large variety of therapeutic modalities in alopecia areata can be divided into four categories: (i) non-specific topical irritants; (ii) 'immune modulators' such as systemic corticosteroids and PUVA; (iii) 'immune enhancers' such as contact dermatitis inducers, cyclosporin and inosiplex; and (iv) drugs of unknown action such as minoxidil (Dawber, R.P.R. et al., Textbook of Dermatology, Blackwell Scientific Publications, 5 th Ed, 1982:2533- 2638).
  • Non-specific topical irritants such as dithranol may work through as yet unidentified mechanisms rather than local irritation in eliciting regrowth of hair.
  • Topical corticosteroids may be effective but prolonged therapy is often necessary.
  • Intralesional steroids have proved to be more effective but their use is limited to circumscribed patches of less active disease or to maintain regrowth of the eyebrows in alopecia totalis.
  • Photochemotherapy has proved to be effective, possibly by changing functional subpopulations of T cells.
  • Topical immunotherapy by means of induction and maintenance of allergic contact dermatitis on the scalp may result in hair regrowth in as many as 70% of the patients with alopecia areata. Diphencyprone is a potent sensitiser free from mutagenic activity.
  • Oral cyclosporin can be effective in the short term (Gupta, A.K. et al., J. Am. Acad. Dermatol. 22:242-250, 1990).
  • Inosiplex an immunostimulant, has been used with apparent effectiveness in an open trial.
  • Topical 5% minoxidil solution has been reported to be able to induce some hair growth in patients with alopecia areata. The mechanism of action is unclear.
  • Carcinomas of the skin are a major public health problem because of their frequency and the disability and disfigurement that they cause. Carcinoma of the skin is principally seen in individuals in their prime of life, especially in fair skinned individuals exposed to large amounts of sunlight. The annual cost of treatment and time loss from work exceeds $250 million dollars a year in the United States alone. The three major types - basal cell cancer, squamous cell cancer, and melanoma - are clearly related to sunlight exposure.
  • Basal cell carcinomas are epithelial tumours of the skin. They appear predominantly on exposed areas of the skin. In a recent Australian study, the incidence of basal cell carcinomas was 652 new cases per year per 100,000 of the population. This compares with 160 cases of squamous cell carcinoma or 19 of malignant melanoma (Giles, G. et al., Br. Med. J. 296:13- 1, 1988). Basal cell carcinomas are the most common of all cancers. Lesions are usually surgically excised. Alternate treatments include retinoids, 5-fluorouracil, cryotherapy and radiotherapy.
  • Alpha or gamma interferon have also been shown to be effective in the treatment of basal cell carcinomas, providing a valuable alternative to patients unsuitable for surgery or seeking to avoid surgical scars (Cornell et al., J Am. Acad. Dermatol. 23:694-700, 1990; Edwards, L. et al., J. Am. Acad. Dermatol. 22:496-500, 1990).
  • SCC Squamous cell carcinoma
  • interferons have also been used in the treatment of melanoma (Kirkwood, J.M. et al., J. Invest. Dermatol. °5:180S-4S, 1990).
  • Response rates achieved with systemic IFN- were in the range 5-30%.
  • Recently, encouraging results (30%) response were obtained with a combination of IFN- ⁇ and DTIC.
  • Preliminary observations indicate a beneficial effect of IFN- ⁇ in an adjuvant setting in patients with high risk melanoma.
  • interferon Of all the available therapies for treating cutaneous viral lesions, only interferon possesses a specific antiviral mode of action, by reproducing the body's immune response to infection. Interferon treatment cannot eradicate the viruses however, although it may help with some manifestations of the infection. Interferon treatment is also associated with systemic adverse effects, requires multiple injections into each single wart and has a significant economic cost (Kraus, SJ. et al., Review of Infectious Diseases 2(6):S620-S632, 1990; Frazer, I.H., Current Opinion in Immunology 5(4):484-491, 1996).
  • the present invention provides compositions present in or derived from M vaccae and methods for their use in the prevention, treatment and diagnosis of diseases, including mycobacterial infection, immune disorders of the respiratory system, and skin disorders.
  • the inventive methods comprise administering a composition having antigenic and/or adjuvant properties.
  • Diseases of the respiratory system which may be treated using the inventive compositions include mycobacterial infections (such as infection with M tuberculosis and/or M avium), asthma, sarcoidosis and lung cancers.
  • disorders of the skin which may be treated using the inventive compositions include psoriasis, atopic dermatis, allergic contact dermatitis, alopecia areata, and the skin cancers basal cell carcinoma, squamous cell carcinoma and melanoma.
  • Adjuvants for use in vaccines or immunotherapy of infectious diseases and cancers are also provided.
  • isolated polypeptides derived from Mycobacterium vaccae comprising an immunogenic portion of an antigen, or a variant of such an antigen.
  • the antigen includes an amino acid sequence selected from the group consisting of: (a) sequences recited in SEQ ID NO: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205 and 207; (b) sequences having at least about 50% identical residues to a sequence recited in SEQ ID NO: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 196,
  • DNA sequences encoding the inventive polypeptides, expression vectors comprising these DNA sequences, and host cells transformed or transfected with such expression vectors are also provided.
  • the present invention provides fusion proteins comprising at least one polypeptide of the present invention.
  • the present invention provides pharmaceutical compositions that comprise at least one of the inventive polypeptides, or a DNA molecule encoding such a polypeptide, and a physiologically acceptable carrier.
  • the invention also provides vaccines comprising at least one of the above polypeptides, or at least one DNA sequence encoding such polypeptides, and a non-specific immune response amplifier.
  • the non-specific immune response enhancer is selected from the group consisting of: delipidated and deglycolipidated M. vaccae cells; inactivated M.
  • vaccae cells delipidated and deglycolipidated M.vaccae cells depleted of mycolic acids; delipidated and deglycolipidated M.vaccae cells depleted of mycolic acids and arabinogalactan; and M vaccae culture filtrate.
  • methods for enhancing an immune response in a patient, comprising administering to a patient an effective amount of one or more of the above pharmaceutical compositions and/or vaccines.
  • the immune response is a Thl response.
  • methods are provided for the treatment of a disorder in a patient, comprising administering to the patient a pharmaceutical composition or vaccine of the present invention.
  • the disorder is selected from the group consisting of immune disorders, infectious diseases, skin diseases and diseases of the respiratory system. Examples of such diseases include mycobacterial infections, asthma and psoriasis.
  • the invention provides methods for the treatment of immune disorders, infectious diseases, skin diseases or diseases of the respiratory system, comprising administering a composition comprising inactivated M vaccae cells, delipidated and deglycolipidated M vaccae cells or M vaccae culture filtrate.
  • Methods for enhancing an immune response to an antigen are also provided.
  • such methods comprising administering a polypeptide that comprises an immunogenic portion of a M vaccae antigen which includes a sequence of SEQ ID NO: 89 or 201, or a variant thereof.
  • such methods comprise administering a composition comprising a component selected from the group consisting of: delipidated and deglycolipidated M.vaccae cells depleted of mycolic acids, and delipidated and deglycolipidated M.vaccae cells depleted of mycolic acids and arabinogalactan.
  • the method comprises contacting dermal cells of a patient with one or more of the above polypeptides and detecting an immune response on the patient's skin.
  • the method comprises contacting a biological sample with at least one of the above polypeptides; and detecting in the sample the presence of antibodies that bind to the polypeptide or polypeptides, thereby detecting M tuberculosis infection in the biological sample.
  • suitable biological samples include whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid and urine.
  • Diagnostic kits comprising one or more of the above polypeptides in combination with an apparatus sufficient to contact the polypeptide with the dermal cells of a patient are provided.
  • the present invention also provides diagnostic kits comprising one or more df the inventive polypeptides in combination with a detection reagent.
  • the present invention provides antibodies, both polyclonal and monoclonal, that bind to the polypeptides described above, as well as methods for their use in the detection of mycobacterial infection.
  • Figs. 1A and IB illustrate the protective effects of immunizing mice with autoclaved M vaccae or unfractionated M vaccae culture filtrates, respectively, prior to infection with live M tuberculosis H37Rv.
  • Figs. 2A and B show the percentage of eosinophils in mice immunized intranasally with either 10 or 1000 ⁇ g of heat-killed M vaccae or 200-100 ⁇ g of DD-M vaccae, respectively, 4 weeks prior to challenge with ovalbumin, as compared to control mice.
  • Figs. 2C and D show the percentage of eosinophils in mice immunized intranasally with either 100 ⁇ g of heat-killed M vaccae or 200 ⁇ g of DD-M vaccae, respectively, as late as one week prior to challenge with ovalbumin.
  • Fig. 2A and B show the percentage of eosinophils in mice immunized intranasally with either 10 or 1000 ⁇ g of heat-killed M vaccae or 200-100 ⁇ g of DD-M vaccae, respectively, 4 weeks prior to challenge with ovalbumin, as compared to control mice.
  • Figs. 2C and D
  • 2E shows the percentage of eosinophils in mice immunized either intranasally (i.n.) or subcutaneously (s.c.) with either BCG of the Pasteur strain (BCG-P), BCG of the Connought strain (BCG-C), 1 mg of heat-killed M. vaccae, or 200 ⁇ g of DD-M vaccae prior to challenge with ovalbumin.
  • BCG-P BCG of the Pasteur strain
  • BCG-C BCG of the Connought strain
  • 1 mg of heat-killed M. vaccae or 200 ⁇ g of DD-M vaccae prior to challenge with ovalbumin.
  • Fig. 3A illustrates the effect of immunizing mice with heat-killed M vaccae or delipidated and deglycolipidated M vaccae (DD-M vaccae) prior to infection with tuberculosis.
  • Fig. 3B illustrates the effect of immunizing mice with heat-killed M vaccae, recombinant M vaccae proteins, or a combination of heat-killed M vaccae and M vaccae recombinant proteins prior to infection with tuberculosis.
  • Fig. 4 illustrates the induction of IL-12 by autoclaved M vaccae, lyophilized M vaccae, delipidated and deglycolipidated M vaccae and M vaccae glycolipids.
  • Fig. 5 compares the in vitro stimulation of interferon-gamma production in spleen cells from Severe Combined ImmunoDeficient (SCID) mice by different concentrations of heat-killed (autoclaved) M vaccae, delipidated and deglycolipidated M vaccae, and M vaccae glycolipids.
  • Figs. 6A, B and C illustrate the stimulation of interferon-gamma production by different concentrations of M vaccae recombinant proteins, heat-killed M vaccae, delipidated and deglycolipidated M vaccae (referred to in the figure as "delipidated M vaccae' ' '), M. vaccae glycolipids and lipopolysaccharide, in peritoneal macrophages from C57BL/6 mice (Fig. 6A), BALB/C mice (Fig. 6B) or C3H/HeJ mice (Fig. 6C).
  • Fig. 6A C57BL/6 mice
  • Fig. 6B BALB/C mice
  • Fig. 6C C3H/HeJ mice
  • FIG. 7A(i) - (iv) illustrate the non-specific immune amplifying effects of 10 ⁇ g, 100 ⁇ g and lmg autoclaved M vaccae and 75 ⁇ g unfractionated culture filtrates of M vaccae, respectively.
  • Fig. 7B(i) and (ii) illustrate the non-specific immune amplifying effects of autoclaved M vaccae, and delipidated and deglycolipidated M vaccae, respectively.
  • Fig. 7C(i) illustrates the non-specific immune amplifying effects of whole autoclaved M vaccae.
  • Fig. 7C(ii) illustrates the non-specific immune amplifying effects of soluble M vaccae proteins extracted with SDS from delipidated and deglycolipidated M vaccae.
  • Fig. 7C(iii) illustrates that the non-specific amplifying effects of the preparation of Fig. 7C(ii) are destroyed by treatment with the proteolytic enzyme Pronase.
  • Fig. 7D illustrates the nonspecific immune amplifying effects of heat-killed M vaccae (Fig. 7D(i)), whereas a nonspecific immune amplifying effect was not seen with heat-killed preparations of M tuberculosis (Fig. 7D(ii)), M bovis BCG (Fig. 7D(iii)), M phlei (Fig. 7D(iv)) and M. smegmatis (Fig. 7D(v)).
  • Figs. 8A and B illustrate the stimulation of CD69 expression on ⁇ T cells, ⁇ T cells and NK cells, respectively, by the M vaccae protein GV23, the Thl -inducing adjuvants MPL/TDM/CWS and CpG ODN, and the Th2 -inducing adjuvants aluminium hydroxide and cholera toxin.
  • Figs. 9A-D illustrate the effect of heat-killed M vaccae, DD-M vaccae and M vaccae recombinant proteins on the production of IL-l ⁇ , TNF- ⁇ , IL-12 and IFN- ⁇ , respectively, by human PBMC.
  • Figs. 10A-C illustrate the effects of varying concentrations of the recombinant M vaccae proteins GV-23 and GV-45 on the production of IL-l ⁇ , TNF- ⁇ and IL-12, respectively, by human PBMC.
  • Figs. 11A-D illustrate the stimulation of IL-l ⁇ , TNF- ⁇ , IL-12 and IFN- ⁇ production, respectively, in human PBMC by the M vaccae protein GV23, the Thl -inducing adjuvants MPL/TDM/CWS and CpG ODN, and the Th2-inducing adjuvants aluminium hydroxide and cholera toxin.
  • Figs. 12A-C illustrate the effects of varying concentrations of the recombinant M vaccae proteins GV-23 and GV-45 on the expression of CD40, CD80 and CD86, respectively, by dendritic cells.
  • Fig. 13 illustrates the enhancement of dendritic cell mixed leukocyte reaction by the recombinant M vaccae protein GV-23.
  • the present invention is generally directed to compositions and methods for preventing, treating and diagnosing infectious diseases and immune disorders.
  • Disorders which may be effectively treated using the inventive compositions include diseases of the respiratory system, such as mycobacterial infections, asthma, sarcoidosis and lung cancers, and disorders of the skin, such as psoriasis, atopic dermatis, allergic contact dermatitis, alopecia areata, and the skin cancers basal cell carcinoma, squamous cell carcinoma and melanoma.
  • Effective vaccines that provide protection against infectious microorganisms contain at least two functionally different components.
  • the first is an antigen, which may be polypeptide or carbohydrate in nature, and which is processed by macrophages and other antigen-presenting cells and displayed for CD4 + T cells or for CD8 + T cells. This antigen forms the "specific" target of an immune response.
  • the second component of a vaccine is a non-specific immune response amplifier, termed an adjuvant, with which the antigen is mixed or is inco ⁇ orated into.
  • An adjuvant amplifies either cell-mediated or antibody immune responses to a structurally unrelated compound or polypeptide.
  • Several known adjuvants are prepared from microbes such as Bordetella pertussis, M.
  • Adjuvants may also contain components designed to protect polypeptide antigens from degradation, such as aluminum hydroxide or mineral oil. While the antigenic component of a vaccine contains polypeptides that direct the immune attack against a specific pathogen, such as M tuberculosis, the adjuvant is often capable of broad use in many different vaccine formulations. Certain known proteins, such as bacterial enterotoxins, can function both as an antigen to elicit a specific immune response and as an adjuvant to enhance immune responses to unrelated proteins.
  • pathogens such as M tuberculosis, as well as certain cancers, are effectively contained by an immune attack directed by CD4 + and CD8 + T cells, known as cell-mediated immunity.
  • Other pathogens such as poliovirus, also require antibodies, produced by B cells, for containment.
  • T cell or B cell are controlled by different subpopulations of CD4 + T cells, commonly referred to as Thl and Th2 cells.
  • Thl and Th2 cells are controlled by different subpopulations of CD4 + T cells, commonly referred to as Thl and Th2 cells.
  • Thl and Th2 cells A desirable property of an adjuvant is the ability to selectively amplify the function of either Thl or Th2 populations of CD4 + T cells.
  • Many skin disorders including psoriasis, atopic dermatitis, alopecia, and skin cancers appear to be influenced by differences in the activity of these Th cell subsets.
  • Th cell subsets have been well characterized in a murine model and are defined by the cytokines they release upon activation.
  • the Thl subset secretes IL-2, IFN- ⁇ and tumor necrosis factor, and mediates macrophage activation and delayed-type hypersensitivity response.
  • the Th2 subset releases IL-4, IL-5, IL-6 and IL-10, which stimulate B cell activation.
  • the Thl and Th2 subsets are mutually inhibiting, so that IL-4 inhibits Thl -type responses, and IFN- ⁇ inhibits Th2-type responses. Similar Thl and Th2 subsets have been found in humans, with release of the identical cytokines observed in the murine model.
  • Th2 subset a subset of T-cell clones from atopic human lymphocytes resemble the murine Th2 cell that produces IL-4, whereas very few clones produce IFN- ⁇ . Therefore, the selective expression of the Th2 subset with subsequent production of IL-4 and decreased levels of IFN- ⁇ -producing cells could lead to preferential enhancement of IgE production.
  • Amplification of Thl -type immune responses is central to a reversal of disease state in many disorders, including disorders of the respiratory system such as tuberculosis, sarcoidosis, asthma, allergic rhinitis and lung cancers.
  • Inactivated M vaccae and many compounds derived from M vaccae have both antigen and adjuvant properties which function to enhance Thl -type immune responses.
  • the methods of the present invention employ one or more of these antigen and adjuvant compounds from M vaccae and/or its culture filtrates to redirect immune activities of T cells in patients. Mixtures of such compounds are particularly effective in the methods disclosed herein. While it is well known that all mycobacteria contain many cross-reacting antigens, it is not known whether they contain adjuvant compounds in common.
  • inactivated M vaccae and a modified (delipidated and deglycolipidated) form of inactivated M vaccae have been found to have adjuvant properties of the Thl -type which are not shared by a number of other mycobacterial species. Furthermore, it has been found that M vaccae produces compounds in its own culture filtrate which amplify the immune response to M vaccae antigens also found in culture filtrate, as well as to antigens from other sources.
  • the present invention provides methods for the immunotherapy of respiratory and/or lung disorders, including tuberculosis, sarcoidosis, asthma, allergic rhinitis and lung cancers, in a patient to enhance Thl -type immune responses.
  • the compositions are delivered directly to the mucosal surfaces of airways leading to and/or within the lungs.
  • the compositions may also be administered via intradermal or subcutaneous routes.
  • Compositions which may be usefully employed in such methods comprise at least one of the following components: inactivated M vaccae cells; M vaccae culture filtrate; delipidated and deglycolipidated M vaccae cells (DD-M vaccae); and compounds present in or derived from M vaccae and/or its culture filtrate.
  • compositions results in specific T cell immune responses and enhanced protection against M tuberculosis infection, and is also effective in the treatment of asthma. While the precise mode of action of these compositions in the treatment of diseases such as asthma is unknown, they are believed to suppress an asthma-inducing Th2 immune response.
  • the term "respiratory system” refers to the lungs, nasal passageways, trachea and bronchial passageways.
  • airways leading to or located in the lung includes the nasal passageways, mouth, tonsil tissue, trachea and bronchial passageways.
  • a "patient” refers to any warm-blooded animal, preferably a human. Such a patient may be afflicted with disease or may be free of detectable disease. In other words, the inventive methods may be employed to induce protective immunity for the prevention or treatment of disease.
  • the present invention provides methods for the immunotherapy of skin disorders, including psoriasis, atopic dermatitis, alopecia, and skin cancers in patients, in which immunotherapeutic agents are employed to alter or redirect an existing state of immune activity by altering the function of T cells to a Thl -type of immune response.
  • Compositions which may be usefully employed in the inventive methods comprise at least one of the following components: inactivated M vaccae cells; M vaccae culture filtrate; modified M vaccae cells; and constituents and compounds present in or derived from M vaccae and/or its culture filtrate.
  • multiple administrations of such compositions preferably by intradermal injection, have been shown to be highly effective in the treatment of psoriasis.
  • the term “inactivated M vaccae” refers to M vaccae that have either been killed by means of heat, as detailed below in Example 7, or subjected to radiation, such as 60 Cobalt at a dose of 2.5 megarads.
  • the term “modified M vaccae” includes delipidated M vaccae cells, deglycolipidated M vaccae cells and M vaccae cells that have been both delipidated and deglycolipidated (DD-M vaccae).
  • Example 7 The preparation of DD-M vaccae and its chemical composition are described below in Example 7. As detailed below, the inventors have shown that removal of the glycolipid constituents from M vaccae results in the removal of molecular components that stimulate interferon-gamma production in natural killer (NK) cells, thereby significantly reducing the non-specific production of a cytokine that has numerous harmful side-effects.
  • NK natural killer
  • the present invention provides isolated polypeptides that comprise at least one immunogenic portion of a M vaccae antigen, or a variant thereof, or at least one adjuvant porition of an M. vaccae protein.
  • such polypeptides comprise an immunogenic portion of an antigen, or a variant thereof, wherein the antigen includes a sequence selected from the group consisting of SEQ ID NO: 1-4, 9-16, 18-21, 23, 25, 26, 28, 29, 44, 45, 47, 52-55, 63, 64, 70, 75, 89, 94, 98, 100-105, 109, 110, 112, 121, 124, 125, 134, 135, 140, 141, 143, 145, 147, 152, 154, 156, 158, 160, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 201, 203, 205 and 207.
  • polypeptide encompasses amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.
  • a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences.
  • the additional sequences may be derived from the native M vaccae antigen or may be heterologous, and such sequences may (but need not) be immunogenic.
  • polypeptides of the present invention may be isolated from M vaccae cells or culture filtrate, or may be prepared by synthetic or recombinant means.
  • Immunogenic refers to the ability to elicit an immune response in a patient, such as a human, or in a biological sample.
  • immunogenic antigens are capable of stimulating cell proliferation, interleukin- 12 production or interferon- ⁇ production in biological samples comprising one or more cells selected from the group of T cells, NK cells, B cells and macrophages, where the cells are derived from an M tuberculosis-immune individual. Exposure to an immunogenic antigen generally results in the generation of immune memory such that upon re-exposure to that antigen, an enhanced and more rapid response occurs.
  • Immunogenic portions of the antigens described herein may be prepared and identified using well known techniques, such as those summarised in Paul, Fundamental Immunology, 3d ed., Raven Press, 1993, pp. 243-247. Such techniques include screening polypeptide portions of the native antigen or protein for immunogenic properties. The representative proliferation and cytokine production assays described herein may be employed in these screens.
  • An immunogenic portion of an antigen is a portion that, within such representative assays, generates an immune response (e.g., cell proliferation, interferon- ⁇ production or interleukin- 12 production) that is substantially similar to that generated by the full-length antigen.
  • an immunogenic portion of an antigen may generate at least about 20%, preferably about 65%, and most preferably about 100% of the proliferation induced by the full-length antigen in the model proliferation assay described herein.
  • An immunogenic portion may also, or alternatively, stimulate the production of at least about 20%, preferably about 65% and most preferably about 100%, of the interferon- ⁇ and/or interleukin- 12 induced by the full length antigen in the model assay described herein.
  • a M vaccae adjuvant is a compound found in M vaccae cells or M vaccae culture filtrates which non-specifically stimulates immune responses.
  • Adjuvants enhance the immune response to immunogenic antigens and the process of memory formation. In the case of M vaccae proteins, these memory responses favour Thl -type immunity.
  • Adjuvants are also capable of stimulating interleukin- 12 production or interferon- ⁇ production in biological samples comprising one or more cells selected from the group of T cells, NK cells, B cells and macrophages, where the cells are derived from healthy individuals. Adjuvants may or may not stimulate cell proliferation.
  • Such M vaccae adjuvants include, for example, polypeptides comprising a sequence recited in SEQ ID NO: 89, 117, 160, 162 or 201.
  • polynucleotide(s), means a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding RNA molecules, including HnRNA and mRNA molecules, both sense and anti-sense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides.
  • An HnRNA molecule contains introns and corresponds to a DNA molecule in a generally one-to-one manner.
  • An mRNA molecule corresponds to an HnRNA and DNA molecule from which the introns have been excised.
  • a polynucleotide may consist of an entire gene, or any portion thereof.
  • Operable anti-sense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of "polynucleotide” therefore includes all such operable anti-sense fragments.
  • compositions and methods of this invention also encompass variants of the above polypeptides and polynucleotides.
  • variant covers any sequence which has at least about 40%, more preferably at least about 60%, more preferably yet at least about 75% and most preferably at least about 90% identical residues (either nucleotides or amino acids) to a sequence of the present invention.
  • the percentage of identical residues is determined by aligning the two sequences to be compared, determining the number of identical residues in the aligned portion, dividing that number by the total length of the inventive, or queried, sequence and multiplying the result by 100.
  • Polynucleotide or polypeptide sequences may be aligned, and percentage of identical nucleotides in a specified region may be determined against another polynucleotide, using computer algorithms that are publicly available.
  • Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms.
  • the similarity of polypeptide sequences may be examined using the BLASTP algorithm. Both the BLASTN and BLASTP software are available on the NCBI anonymous FTP server (ftp://ncbi.nlm.nih.gov) under /blast executables/.
  • the computer algorithm FASTA is available on the Internet at the ftp site ftp://ftp.virginia.edu/pub/fasta/. Version 2.0u4, February 1996, set to the default parameters described in the documentation and distributed with the algorithm, is preferred for use in the determination of variants according to the present invention.
  • the use of the FASTA algorithm is described in W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Analysis,” Proc. Natl. Acad. Sci. USA ⁇ 5:2444-2448 (1988) and W.R. Pearson, “Rapid and Sensitive Sequence Comparison with FASTP and FASTA," Methods in Enzymology 183:63-98 (1990).
  • the "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, FASTA, or a similar algorithm align and identify similar portions of sequences.
  • the hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
  • the BLASTN and FASTA algorithms also produce "Expect" values for alignments.
  • the Expect value (E) indicates the number of hits one can "expect” to see over a certain number of contiguous sequences by chance when searching a database of a certain size.
  • the Expect value is used as a significance threshold for determining whether the hit to a database, such as the preferred EMBL database, mdicates true similarity. For example, an E value of 0.1 assigned to a hit is inte ⁇ reted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance.
  • the aligned and matched portions of the sequences then have a probability of 90% of being the same.
  • the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm.
  • variant polynucleotides with reference to each of the polynucleotides of the present invention, preferably comprise sequences having the same number or fewer nucleic acids than each of the polynucleotides of the present invention and producing an E value of 0.01 or less when compared to the polynucleotide of the present invention. That is, a variant polynucleotide is any sequence that has at least a 99% probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at the default parameters.
  • a variant polynucleotide is a sequence having the same number or fewer nucleic acids than a polynucleotide of the present invention that has at least a 99%o probability of being the same as the polynucleotide of the present invention, measured as having an E value of 0.01 or less using the BLASTN or FASTA algorithms set at the default parameters.
  • Variant polynucleotide sequences will generally hybridize to the recited polynucleotide sequence under stringent conditions.
  • stringent conditions refers to prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65 °C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65 °C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65 °C.
  • M vaccae polypeptides may be generated by synthetic or recombinant means.
  • Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids may be generated using techniques well known to those of ordinary skill in the art.
  • such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems, Inc.
  • Variants of a native antigen or adjuvant may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site specific mutagenesis. Sections of the DNA sequence may also be removed using standard techniques to permit preparation of truncated polypeptides.
  • a polypeptide of the present invention may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein.
  • the polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly- His), or to enhance binding of the polypeptide to a solid support.
  • a polypeptide may be conjugated to an immunoglobulin Fc region.
  • M vaccae antigens and DNA sequences encoding such antigens, may be prepared using any of a variety of procedures.
  • soluble antigens may be isolated from M vaccae culture filtrate as described below.
  • Antigens may also be produced recombinantly by inserting a DNA sequence that encodes the antigen 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 employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells.
  • the host cells employed are E. coli, mycobacteria, insect, yeast or a mammalian cell line such as COS or CHO.
  • the DNA sequences expressed in this manner may encode naturally occurring antigens, portions of naturally occurring antigens, or other variants thereof.
  • DNA sequences encoding M vaccae antigens may be obtained by screening an appropriate M vaccae cDNA or genomic DNA library for DNA sequences that hybridize to degenerate oligonucleotides derived from partial amino acid sequences of isolated soluble antigens. Suitable degenerate oligonucleotides may be designed and synthesized, and the screen may be performed as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989. As described below, polymerase chain reaction (PCR) may be employed to isolate a nucleic acid probe from genomic DNA, or a cDNA or genomic DNA library. The library screen may then be performed using the isolated probe. DNA molecules encoding M vaccae antigens may also be isolated by screening an appropriate M vaccae expression library with anti-sera (e.g., rabbit or monkey) raised specifically against M vaccae antigens.
  • anti-sera e.g., rabbit or monkey
  • the antigens described herein have the ability to induce an immunogenic response. More specifically, the antigens have the ability to induce cell proliferation and/or cytokine production (for example, interferon- ⁇ and/or interleukin- 12 production) in T cells, NK cells, B cells or macrophages derived from an M tuberculosis- immune individual.
  • cytokine production for example, interferon- ⁇ and/or interleukin- 12 production
  • An M tuberculosis-immune individual is one who is considered to be resistant to the development of tuberculosis by virtue of having mounted an effective T cell response to M tuberculosis.
  • Such individuals may be identified based on a strongly positive (i.e., greater than about 10 mm diameter induration) intradermal skin test response to tuberculosis proteins (PPD), and an absence of any symptoms of tuberculosis infection.
  • Assays for cell proliferation or cytokine production in T cells, NK cells, B cells or macrophages may be performed, for example, using the procedures described below.
  • the selection of cell type for use in evaluating an immunogenic response to an antigen will depend on the desired response. For example, interleukin- 12 production is most readily evaluated using preparations containing T cells, NK cells, B cells and macrophages derived from M tuberculosis-immune individuals may be prepared using methods well known in the art.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs may be prepared, for example, using density centrifugation through FicollTM (Winthrop Laboratories, NY).
  • T cells for use in the assays described herein may be purified directly from PBMCs.
  • an enriched T cell line reactive against mycobacterial proteins, or T cell clones reactive to individual mycobacterial proteins may be employed.
  • Such T cell clones may be generated by, for example, culturing PBMCs from M tuberculosis-immune individuals with mycobacterial proteins for a period of 2-4 weeks. This allows expansion of only the mycobacterial protein- specific T cells, resulting in a line composed solely of such cells. These cells may then be cloned and tested with individual proteins, using methods well known in the art, to more accurately define individual T cell specificity.
  • the polypeptides disclosed herein are prepared in an isolated, substantially pure, form.
  • the polypeptides are at least about 80%) pure, more preferably at least about 90% pure and most preferably at least about 99% pure.
  • the substantially pure polypeptides are inco ⁇ orated into pharmaceutical compositions or vaccines for use in one or more of the methods disclosed herein.
  • the present invention also provides fusion proteins comprising a first and a second inventive polypeptide or, alternatively, a polypeptide of the present invention and a known M tuberculosis antigen, such as the 38 kDa antigen described in Andersen and Hansen, Infect. Immun. 57:2481-2488, 1989, together with variants of such fusion proteins.
  • the fusion proteins of the present invention may also include a linker peptide between the first and second polypeptides.
  • a DNA sequence encoding a fusion protein of the present invention is constructed using known recombinant DNA techniques to assemble separate DNA sequences encoding the first and second polypeptides into an appropriate expression vector.
  • the 3' end of a DNA sequence encoding the first polypeptide is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide so that the reading frames of the sequences are in phase to permit mRNA translation of the two DNA sequences into a single fusion protein that retains the biological activity of both the first and the second polypeptides.
  • a peptide linker sequence may be employed to separate the first and the second polypeptides by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures.
  • Such a peptide linker sequence is inco ⁇ orated into the fusion protein using standard techniques well known in the art.
  • Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
  • Preferred peptide linker sequences contain Gly, Asn and Ser residues.
  • linker sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Mu ⁇ hy et al., Proc. Natl. Acad. Sci. USA 53:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180.
  • the linker sequence may be from 1 to about 50 amino acids in length.
  • Peptide linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • the ligated DNA sequences encoding the fusion proteins are cloned into suitable expression systems using techniques known to those of ordinary skill in the art.
  • M vaccae, DD-M. vaccae and recombinant M vaccae proteins of the present invention may be employed to activate T cells and NK cells; to stimulate the production of cytokines (in particular Thl class of cytokines) in human PBMC; to enhance the expression of co-stimulatory molecules on dendritic cells and monocytes (thereby enhancing activation); and to enhance dendritic cell maturation and function.
  • cytokines in particular Thl class of cytokines
  • GV-23 may thus be employed in the treatment of diseases that involve enhancing a Thl immune response.
  • GV-23 may be employed as a dendritic cell or NK cell enhancer in the treatment of immune deficiency disorders, such as HIV, and to enhance immune responses and cytotoxic responses to, for example, malignant cells in cancer and following immunosuppressive anti-cancer therapies, such as chemotherapy.
  • the inactivated M vaccae, M. vaccae culture filtrate, modified M vaccae cells, M vaccae polypeptide, fusion protein (or polynucleotides encoding such polypeptides or fusion proteins) is generally present within a pharmaceutical composition or a vaccine.
  • Pharmaceutical compositions may comprise one or more components selected from the group consisting of inactivated M vaccae cells, M vaccae culture filtrate, modified M vaccae cells, and compounds present in or derived from M vaccae and/or its culture filtrate, together with a physiologically acceptable carrier.
  • Vaccines may comprise one or more components selected from the group consisting of inactivated M vaccae cells, M vaccae culture filtrate, modified M vaccae cells, and compounds present in or derived from M vaccae and/or its culture filtrate, together with a non-specific immune response amplifier.
  • Such pharmaceutical compositions and vaccines may also contain other mycobacterial antigens, either, as discussed above, inco ⁇ orated into a fusion protein or present within a separate polypeptide.
  • a vaccine of the present invention may contain DNA encoding one or more polypeptides as described above, such that the polypeptide is generated in situ.
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminator signal).
  • Bacterial delivery systems involve the administration of a bacterium (such as Bacillus- Calmette-Guerin) that expresses an immunogenic portion of the polypeptide on its cell surface.
  • the DNA may be introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic, or defective, replication competent virus.
  • a viral expression system e.g., vaccinia or other poxvirus, retrovirus, or adenovirus
  • Techniques for inco ⁇ orating DNA into such expression systems are well known in the art.
  • the DNA may also be "naked,” as described, for example, in Ulmer et al., Science 259: 1745- 1749, 1993 and reviewed by Cohen, Science 259: 1691 -1692, 1993.
  • the uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
  • a DNA vaccine as described above may be administered simultaneously with or sequentially to either a polypeptide of the present invention or a known mycobacterial antigen, such as the 38 kDa antigen described above.
  • administration of DNA encoding a polypeptide of the present invention may be followed by administration of an antigen in order to enhance the protective immune effect of the vaccine.
  • Routes and frequency of administration, as well as dosage will vary from individual to individual and may parallel those currently being used in immunization using BCG.
  • the pharmaceutical compositions and vaccines may be administered by injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally.
  • a suitable dose is an amount of polypeptide or DNA that, when administered as described above, is capable of raising an immune response in a patient sufficient to protect the patient from mycobacterial infection for at least 1-2 years.
  • the amount of polypeptide present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 ⁇ g.
  • Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 ml to about 5 ml.
  • the pharmaceutical composition or vaccine is in a form suitable for delivery to the mucosal surfaces of the airways leading to or within the lungs.
  • the pharmaceutical composition or vaccine may be suspended in a liquid formulation for delivery to a patient in an aerosol form or by means of a nebulizer device similar to those currently employed in the treatment of asthma.
  • the pharmaceutical composition or vaccine is in a form suitable for administration by injection (intracutaneous, intramuscular, intravenous or subcutaneous) or orally. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will depend on the suitability for the chosen route of administration.
  • the carrier preferably comprises water, saline, alcohol, a lipid, a wax or a buffer.
  • a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate.
  • Biodegradable microspheres e.g., polylactic galactide
  • Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
  • adjuvants may be employed in the vaccines of this invention to non-specifically enhance the immune response.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis, M. tuberculosis, or, as discussed below, M vaccae.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, MI), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ).
  • Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and Quil A.
  • this invention provides methods for using one or more of the inventive polypeptides to diagnose tuberculosis using a skin test.
  • a skin test is any assay performed directly on a patient in which a delayed-type hypersensitivity (DTH) reaction (such as swelling, reddening or dermatitis) is measured following intradermal injection of one or more polypeptides as described above.
  • DTH delayed-type hypersensitivity
  • the reaction is measured at least 48 hours after injection, more preferably 48-72 hours.
  • the DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to the test antigen (i.e., the immunogenic portion of the polypeptide employed, or a variant thereof).
  • the response may be measured visually, using a ruler.
  • a response that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter, is a positive response, indicative of tuberculosis infection.
  • the polypeptides of the present invention are preferably formulated, as pharmaceutical compositions containing a polypeptide and a physiologically acceptable carrier, as described above.
  • Such compositions typically contain one or more of the above polypeptides in an amount ranging from about 1 ⁇ g to about 100 ⁇ g, preferably from about 10 ⁇ g to about 50 ⁇ g in a volume of 0.1 ml.
  • the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween 80TM.
  • a polypeptide employed in a skin test is of sufficient size such that it remains at the site of injection for the duration of the reaction period.
  • polypeptide that is at least 9 amino acids in length is sufficient.
  • the polypeptide is also preferably broken down by macrophages or dendritic cells within hours of injection to allow presentation to T-cells.
  • Such polypeptides may contain repeats of one or more of the above sequences or other immunogenic or nonimmunogenic sequences.
  • a biological sample is any antibody-containing sample obtained from a patient.
  • 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(s) in the sample, relative to a predetermined cut-off value. The presence of such antibodies indicates the presence of mycobacterial infection.
  • the polypeptides used are preferably complementary (i.e., one component polypeptide will tend to detect infection in samples where the infection would not be detected by another component polypeptide).
  • Complementary polypeptides may generally be identified by using each polypeptide individually to evaluate serum samples obtained from a series of patients known to be infected with a Mycobacterium. After determining which samples test positive (as described below) with each polypeptide, combinations of two or more polypeptides may be formulated that are capable of detecting infection in most, or all, of the samples tested. For example, approximately 25-30% of sera from tuberculosis-infected individuals are negative for antibodies to any single protein, such as the 38 kDa antigen mentioned above.
  • Complementary polypeptides may, therefore, be used in combination with the 38 kDa antigen to improve sensitivity of a diagnostic test.
  • a variety of assay formats employing one or more polypeptides to detect antibodies in a sample are well known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • the assay involves the use of polypeptide immobilized on a solid support to bind to and remove the antibody from the sample. The bound antibody may then be detected using a detection reagent that contains a reporter group.
  • Suitable detection reagents include antibodies that bind to the antibody /polypeptide complex and free polypeptide labelled with a reporter group (e.g., in a semi-competitive assay).
  • a competitive assay may be utilized, in which an antibody that binds to the polypeptide is labelled with a reporter group and allowed to bind to the immobilized antigen after incubation of the antigen with the sample. The extent to which components of the sample inhibit the binding of the labelled antibody to the polypeptide is indicative of the reactivity of the sample with the immobilized polypeptide.
  • the solid support may be any solid material to which the antigen may be attached. Suitable materials are well known in the art.
  • the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
  • the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride.
  • the support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.
  • the polypeptides may be bound to the solid support using a variety of techniques well known in the art.
  • the term "bound” refers to both noncovalent association, such as adso ⁇ tion, and covalent attachment, which may be a direct linkage between the antigen and functional groups on the support or a linkage by way of a cross-linking agent. Binding by adso ⁇ tion to a well in a microtiter plate or to a membrane is preferred. In such cases, adso ⁇ tion may be achieved by contacting the polypeptide, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and 1 day.
  • contacting a well of a plastic microtiter plate such as polystyrene or polyvinylchloride
  • an amount of polypeptide ranging from about 10 ng to about 1 ⁇ g, and preferably about 100 ng
  • Covalent attachment of polypeptide to a solid support may generally be achieved by first reacting the support with a bifiinctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the polypeptide.
  • polypeptide may be bound to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the polypeptide (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
  • the assay is an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • This assay may be performed by first contacting a polypeptide antigen that has been immobilized on a solid support, with the sample, such that antibodies to the polypeptide within the sample are allowed to bind to the immobilized polypeptide. 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 a method appropriate for the specific detection reagent.
  • the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20TM (Sigma Chemical Co., St. Louis, MO) may be employed.
  • the immobilized polypeptide is then incubated with the sample, and antibody is allowed to bind to the antigen.
  • the sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation.
  • PBS phosphate-buffered saline
  • an appropriate contact time, or incubation time is that period of time that is sufficient to detect the presence of antibody within a M tuberculosis-infected sample.
  • the contact time is sufficient to achieve a level of binding that is at least 95%o of that achieved at equilibrium between bound and unbound antibody.
  • the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient. Unbound sample may be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20TM. Detection reagent may then be added to the solid support.
  • An appropriate detection reagent is any compound that binds to the immobilized antibody-polypeptide complex and that can be detected by any of a variety of means known in the art.
  • the detection reagent contains a binding agent (such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen) conjugated to a reporter group.
  • a binding agent such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen
  • reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
  • the conjugation of binding agent to reporter group may be achieved using standard methods known in the art. Common binding agents may also be purchased conjugated to a variety of reporter groups from many commercial sources (e.g., Zymed Laboratories, San Francisco, CA, and Pierce, Rockford, IL).
  • the detection reagent is incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound antibody.
  • An appropriate amount of time may generally be determined from the manufacturer's instructions or by assaying the level of binding that occurs over a period of time.
  • Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group.
  • the method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
  • the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value.
  • the cut-off value is the average mean signal obtained when the immobilized antigen is incubated with samples from an uninfected patient.
  • the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, pp. 106-107. In general, signals higher than the predetermined cut-off value are considered to be positive for mycobacterial infection.
  • the assay may also be performed in a rapid flow-through or strip test format, wherein the antigen is immobilized on a membrane, such as nitrocellulose.
  • a membrane such as nitrocellulose.
  • a detection reagent e.g., protein A-colloidal gold
  • a detection reagent then binds to the antibody-polypeptide complex as the solution containing the detection reagent flows through the membrane.
  • the detection of bound detection reagent may then be performed as described above.
  • the strip test format one end of the membrane to which polypeptide is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing detection reagent and to the area of immobilized polypeptide.
  • Concentration of detection reagent at the polypeptide indicates the presence of anti- mycobacterial antibodies in the sample.
  • concentration of detection reagent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result.
  • the amount of polypeptide immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of antibodies that would be sufficient to generate a positive signal in an ELISA, as discussed above.
  • the amount of polypeptide immobilized on the membrane ranges from about 25 ng to about 1 ⁇ g, and more preferably from about 50 ng to about 500 ng.
  • Such tests can typically be performed with a very small amount (e.g. , one drop) of patient serum or blood.
  • the present invention also provides antibodies to the inventive polypeptides.
  • Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • an immunogen comprising the antigenic polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep and goats).
  • the immunogen is injected into the animal host, preferably according to a predetermined schedule inco ⁇ orating one or more booster immunizations, and the animals are bled periodically.
  • Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
  • Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. (5:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells may then be immortalized by fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal, using one of a variety of techniques well known in the art.
  • Monoclonal antibodies may be isolated from the supernatants of the resulting hybridoma colonies.
  • various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood.
  • Antibodies may be used in diagnostic tests to detect the presence of mycobacterial antigens using assays similar to those detailed above and other techniques well known to those of skill in the art, thereby providing a method for detecting mycobacterial infection, such as M tuberculosis infection, in a patient.
  • Diagnostic reagents of the present invention may also comprise polynucleotides encoding one or more of the above polypeptides, or one or more portions thereof.
  • primers comprising at least 10 contiguous oligonucleotides of an inventive polynucleotide may be used in polymerase chain reaction (PCR) based tests.
  • probes comprising at least 18 contiguous oligonucleotides of an inventive polynucleotide may be used for hybridizing to specific sequences. Techniques for both PCR based tests and hybridization tests are well known in the art.
  • Primers or probes may thus be used to detect M tuberculosis and other mycobacterial infections in biological samples, preferably sputum, blood, serum, saliva, cerebrospinal fluid or urine.
  • DNA probes or primers comprising oligonucleotide sequences described above may be used alone, in combination with each other, or with previously identified sequences, such as the 38 kDa antigen discussed above.
  • This example illustrates the effect of immunization with heat-killed M vaccae or M vaccae culture filtrate in mice prior to challenge with live M tuberculosis.
  • M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5 g/1; tryptone, 5 g/1; glucose, 1 g/1) at 37 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 °C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10 mg/ml, equivalent to 10 10 M vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120 °C. The culture filtrate was passaged through a 0.45 ⁇ m filter into sterile bottles.
  • sterile Medium 90 yeast extract, 2.5 g/1; tryptone, 5 g/1; glucose, 1 g/1
  • the cells were harvested by centrifugation, and
  • Fig.l A when mice were immunized with 1 mg, 100 ⁇ g or 10 ⁇ g of M vaccae and infected three weeks later with 5x10 5 colony forming units (CFU) of live M tuberculosis H37Rv, significant protection from infection was seen.
  • CFU colony forming units
  • spleen, liver and lung tissue was harvested from mice three weeks after infection, and live bacilli determined (expressed as CFU).
  • the reduction in bacilli numbers when compared to tissue from non-immunized control mice, exceeded 2 logs in liver and lung tissue, and 1 log in spleen tissue.
  • Fig. IB shows that when mice were immunized with 100 ⁇ g of M vaccae culture filtrate, and infected three weeks later with 5x10 5 CFU of M tuberculosis H37Rv, significant protection was also seen.
  • CFU live bacilli numbers
  • This example illustrates the effect of immunisation with heat-killed M vaccae or M vaccae culture filtrate through intradermal and intralung routes in cynomolgous monkeys prior to challenge with live M tuberculosis.
  • Heat-killed M vaccae and M vaccae culture filtrate were prepared as described above in Example 1.
  • Five groups of cynomolgous monkeys were used, with each group containing 2 monkeys. Two groups of monkeys were immunised with whole heat-killed M vaccae either intradermally or intralung; two groups of monkeys were immunised with M vaccae culture filtrate either intradermally or intralung; and a control group received no immunisations. All immunogens were dissolved in phosphate buffered saline. The composition employed for immunisation, amount of immunogen, and route of administration for each group of monkeys are provided in Table 1.
  • ESR mm/hr erythrocyte sedimentation rate
  • LPA lymphocyte proliferation
  • body weight, temperature, ESR and LPA to PPD were measured, then all monkeys were infected with 10 3 colony forming units of the Erdman strain of Mycobacterium tuberculosis by inserting the organisms directly in the right lungs of immunised animals. Twenty eight days following infection, body weight, temperature, ESR and LPA to PPD were measured in all monkeys, and the lungs were x-rayed to determine whether infection with live M tuberculosis had resulted in the onset of pneumonia.
  • Table 2A Twenty-eight days after infection with M tuberculosis Erdman, chest x-rays of control (non-immunised) monkeys revealed haziness over the right suprahilar regions of both animals, indicating the onset of pneumonia. This progressed and by day 56 post-infection x- rays indicated disease in both lungs. As expected, as disease progressed both control animals lost weight and showed significant LPA responses to PPD, indicating strong T cell reactivity to M tuberculosis. The ESR measurements were variable but consistent with strong immune reactivity.
  • Table 2C The two monkeys immunised twice with 500 ⁇ g M vaccae intralung showed almost identical results to those animals of Table 2B. There was no sign of lung disease 84 days post infection with M tuberculosis, with consistent weight gains. Both animals showed LPA response to PPD in the immunisation phase (day 0-62) and post-infection, indicating strong T cell reactivity had developed as a result of using the lung as the route of immunisation and subsequent infection.
  • mice were given 2 ⁇ g ovalbumin in 100 ⁇ l alum adjuvant by the intraperitoneal route at time 0 and 14 days, and subsequently given 100 ⁇ g ovalbumin in 50 ⁇ l phosphate buffered saline (PBS) by the intranasal route on day 28.
  • PBS phosphate buffered saline
  • the mice accumulated eosinophils in their lungs as detected by washing the airways of the anaesthetised mice with saline, collecting the washings (broncheolar lavage or BAL), and counting the numbers of eosinophils.
  • mice groups of seven mice administered either 10 or 1000 ⁇ g of heat-killed M vaccae (Fig. 2A), or 10 , 100 or 200 ⁇ g of DD-M vaccae, prepared as described below (Fig. 2B) intranasally 4 weeks before intranasal challenge with ovalbumin, had reduced percentages of eosinophils in the BAL cells collected 5 days after challenge with ovalbumin compared to control mice.
  • Control mice were given intranasal PBS.
  • Live M bovis BCG at a dose of 2 x 10 5 colony forming units also reduced lung eosinophilia.
  • the data in Figs. 2A and B show the mean and SEM per group of mice.
  • Figs. 2C and D show that mice given either 1000 ⁇ g of heat-killed M vaccae (Fig. 2C) or 200 ⁇ g of DD-M vaccae (Fig. 2D) intranasally as late as one week before challenge with ovalbumin had reduced percentages of eosinophils compared to control mice. In contrast, treatment with live BCG one week before challenge with ovalbumin did not inhibit the development of lung eosinophilia when compared with control mice.
  • immunization with BCG of the Pasteur (BCG-P) and Connought (BCG-C) strains prior to challenge with ovalbumin also reduced the percentage of eosinophils in the BAL of mice.
  • Eosinophils are blood cells that are prominent in the airways in allergic asthma.
  • the secreted products of eosinophils contribute to the swelling and inflammation of the mucosal linings of the airways in allergic asthma.
  • the data shown in Figs. 2A-E indicate that treatment with heat-killed M vaccae or DD-M vaccae reduces the accumulation of lung eosinophils, and may be useful in reducing inflammation associated with eosinophilia in the airways, nasal mucosal and upper respiratory tract.
  • Mycolic acids were depleted from DD-Mv ⁇ cc ⁇ e by treatment with potassium hydroxide (0.5% KOH) in ethanol for 48 hours at 37°C.
  • Mycolic acid depleted OO-M.vaccae cells were then washed with ethanol and ether and dried.
  • Arabinogalactans were depleted from the KOH treated OO-M.vaccae by further treatment with 1% periodic acid in 3% acetic acid for 1 hr at room temperature followed by treatment with sodium borohydride 0.1M for 1 hour at room temperature. After arabinogalactan depletion, samples were washed with water and lyophilized.
  • mice infected with BCG had higher levels of ovalbumin specific IgGl than sera from PBS controls.
  • mice immunized with M vaccae or DD-M vaccae had similar or lower levels of ovalbumin- specific IgGl.
  • IgGl antibodies are characteristic of a Th2 immune response, these results are consistent with the suppressive effects of heat-killed M vaccae and DD-M. vaccae on the asthma-inducing Th2 immune responses.
  • mice per group At least 7 mice per group.
  • Ovalbumin-specific IgGl was detected using anti-mouse IgGl (Serotec). Group means are expressed as the reciprocal of the EU50 end point titre.
  • This example illustrates the effect of immunization with heat-killed M.vaccae, OO- M.vaccae or recombinant M vaccae proteins without additional adjuvants, or a combination of heat-killed M.vaccae with a pool of recombinant proteins derived from M.vaccae.
  • mice were injected intraperitoneally with one of the following preparations on two occasions three weeks apart: a) Phosphate buffered saline (PBS, control); b) Heat-killed M vaccae (500 ug); c) OO-M.vaccae (50 ug); d) A pool of recombinant proteins containing 15 ug of each of GV4P, GN7, GN9, GN27B, GN33 protein (prepared as described below); and e) Heat-killed M vaccae plus the pool of recombinant proteins
  • mice were infected with 5 X 10 5 live H37Rv M. tuberculosis organisms. After a further three weeks, the mice were sacrificed, and their spleens homogenized and assayed for colony forming units (CFU) of M.tuberculosis as an indicator of severity of infection.
  • CFU colony forming units
  • Figs. 3A and 3B show data in which each point represents individual mice.
  • the numbers of CFU recovered from control mice immunised with PBS alone were taken as the baseline. All data from experimental mice were expressed as number of logarithms of CFUs below the baseline for control mice (or log protection).
  • mice immunized with heat-killed M.vaccae or OO-M.vaccae showed a mean reduction of >1 or 0.5 logs CFU, respectively.
  • the spleens of mice immunized with the pool of recombinant proteins containing GV4P, GV7, GV9, GV27B and GV33 had CFUs slightly less than control mice.
  • GV4P, GV7, GN9, GN27B and GV33 were given in combination with heat-killed M.vaccae, the reduction in CFUs exceeded a mean of > 1.5 logs.
  • the data demonstrates the effectiveness of immunization with M.vaccae, DD- M.vaccae or recombinant proteins derived from M.vaccae against subsequent infection with tuberculosis, and further indicates that M.vaccae, DD-M.vaccae and recombinant proteins may be developed as vaccines against tuberculosis.
  • This example illustrates the effect of two intradermal injections of heat-killed Mycobacterium vaccae on psoriasis in human patients.
  • M vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5g/l; tryptone, 5 g/1; glucose, 1 g/1) at 37 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 °C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10 mg/ml, equivalent to 10 10 M vaccae organisms per ml.
  • sterile Medium 90 yeast extract, 2.5g/l; tryptone, 5 g/1; glucose, 1 g/1
  • the cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 °C for one day. The medium was then centr
  • the cell suspension was then autoclaved for 15 min at 120 °C and stored frozen at -20 °C. Prior to use the M vaccae suspension was thawed, diluted to a concentration of 5 mg/ml in phosphate buffered saline, autoclaved for 15 min at 120 °C and 0.2 ml aliquoted under sterile conditions into vials for use in patients.
  • PASI scores are a measure of the location, size and degree of skin scaling in psoriatic lesions on the body.
  • a PASI score of above 12 reflects widespread disease lesions on the body. The study commenced with a washout period of four weeks where the patients did not have systemic anti-psoriasis treatment or effective topical therapy.
  • the PASI scores were determined at -4, 0, 3, 6 and 12 weeks;
  • PASI score 0
  • the remission or improvement of PASI score may be long lasting.
  • Patient PS-003 achieved remission by week 20 and was still in remission at week 80.
  • Overall 13 of 24 patients showed a greater than 50% improvement in PASI scores.
  • Patient PS-001 achieved remission at week 16, relapsed at week 48 (PASI 2.7), was re- vaccinated with injections of M.vaccae and subsequently improved with PASI falling from 17.8 (Week 60) to 0.8 (week 84). Thus patients may benefit from repeated treatment.
  • This example illustrates the effect of two intradermal injections of DD-M. vaccae on psoriasis.
  • PASI scores are a measure of the location, size and degree of skin scaling in psoriatic lesions on the body.
  • a PASI score of above 12 reflects widespread disease lesions on the body.
  • the study commenced with a washout period of four weeks where the four patients did not have systemic antipsoriasis treatment or effective topical therapy.
  • the seven patients were then injected intradermally with 0.1 ml DD-M vaccae (equivalent to 100 ⁇ g). This was followed three weeks later with a second intradermal injection with the same dose of DD-M vaccae (100 ⁇ g).
  • Psoriasis was evaluated from four weeks before the first injection of M vaccae to six weeks after the first injection as follows:
  • Table 9 The data shown in Table 9 are the PASI scores of the seven patients at the time of the first injection of DD-M. vaccae (Day 0), 3, 6, 12 and 24 weeks later.
  • the PASI score of patient PS-025 was reduced to less than 1 for more than 12 weeks.
  • PASI 15.8
  • PASI 15.8
  • treatment of psoriasis with DD-Mv ⁇ cc ⁇ e may lead to disease remission or provide prolonged benefit. Patients may also benefit with repeated treatment.
  • Heat-killed M vaccae was prepared as described as above in Example 1. To prepare delipidated M.vaccae, the autoclaved M.vaccae was pelleted by centrifugation, the pellet washed with water, collected again by centrifugation and then freeze-dried. An aliquot of this freeze-dried M.vaccae was set aside and referred to as lyophilised M.vaccae. When used in experiments it was resuspended in PBS to the desired concentration. Freeze-dried M vaccae was treated with chloroform methanol (2:1) for 60 mins at room temperature to extract lipids, and the extraction was repeated once.
  • the delipidated residue from chloroform/methanol extraction was further treated with 50% ethanol to remove glycolipids by refluxing for two hours.
  • the 50% ethanol extraction was repeated two times.
  • the pooled 50% ethanol extracts were used as a source of M vaccae glycolipids (see below).
  • the residue from the 50% ethanol extraction was freeze-dried and weighed.
  • the amount of delipidated and deglycolipidated M.vaccae prepared was equivalent to 11.1% of the starting wet weight of M.vaccae used.
  • the delipidated and deglycolipidated M vaccae (DD-M. vaccae) was resuspended in phosphate-buffered saline by sonication, and sterilised by autoclaving.
  • compositional analyses of heat-killed M vaccae and DD-M. vaccae are presented in Table 9. Major changes are seen in the fatty acid composition and amino acid composition of DD-M vaccae as compared to the insoluble fraction of heat-killed M vaccae.
  • the data presented in Table 9 show that the insoluble fraction of heat-killed M.vaccae contains 10% w/w of lipid, and the total amino acid content is 2750 nmoles/mg, or approximately 33% w/w.
  • DD-M. vaccae contains 1.3% w/w of lipid and 4250 nmoles/mg amino acids, which is approximately 51% w/w.
  • the insoluble fraction of heat-killed M vaccae contains 10% w/w of lipid, and DD-M. vaccae contains 1.3% w/w of lipid.
  • the total amino acid content of the insoluble fraction of heat-killed M vaccae is 2750 nmoles/mg, or approximately 33% w/w.
  • the total amino acid content of DD-M vaccae is 4250 nmoles/mg, or approximately 51% w/w.
  • Delipidated and deglycolipidated M tuberculosis and M smegmatis were prepared using the procedure described above for delipidated and deglycolipidated M vaccae. As indicated in Table 10, the profiles of the percentage composition of amino acids in DD-M. vaccae, DD-M. tuberculosis and DD-M smegmatis showed no significant differences. However, the total amount of protein varied - the two batches of DD-M vaccae contained 34% and 55% protein, whereas DD-M tuberculosis and DD- M smegmatis contained 79% and 72% protein, respectively.
  • DD-M vaccae Analysis of the monosaccharide composition shows significant differences between DD-M vaccae, and DD-M tuberculosis and DD-M. smegmatis.
  • the monosaccharide composition of two batches of DD-M. vaccae was the same and differed from that of DD-M tuberculosis and M smegmatis. Specifically, DD-M. vaccae was found to contain free glucose while both DD-M tuberculosis and M smegmatis contain glycerol, as shown in Table 11.
  • the pooled 50% ethanol extracts described above were dried by rotary evaporation, redissolved in water, and freeze-dried.
  • the amount of glycolipid recovered was 1.2% of the starting wet weight of M vaccae used.
  • the glycolipids were dissolved in phosphate-buffered saline.
  • a group of C57BL/6J mice were injected intraperitoneally with DIFCO thioglycolate and after three days, peritoneal macrophages were collected and placed in cell culture with interferon-gamma for three hours.
  • the culture medium was replaced and various concentrations of whole heat-killed (autoclaved) M v ⁇ cc ⁇ e, lyophilized M. v ⁇ cc ⁇ e, DD-M v ⁇ cc ⁇ e and M v ⁇ cc ⁇ e glycolipids, prepared as described above, were added.
  • the culture supernatants were assayed for the presence of IL-12 produced by macrophages.
  • the M v ⁇ cc ⁇ e preparations stimulated the production of IL-12 from macrophages.
  • NK cells Natural Killer cells
  • Spleen cells were prepared from Severe Combined Immunodeficient (SCID) mice. These populations contain 75-80% NK cells. The spleen cells were incubated at 37 °C in culture with different concentrations of heat-killed M v ⁇ cc ⁇ e, DD-M. v ⁇ cc ⁇ e, or M v ⁇ cc ⁇ e glycolipids. The data shown in Fig. 5 demonstrates that, while heat-killed M vaccae and M.
  • v ⁇ cc ⁇ e glycolipids stimulate production of interferon-gamma, DD-M v ⁇ cc ⁇ e stimulated relatively less interferon-gamma.
  • the combined data from Figs. 4 and 5 indicate that, compared with whole heat-killed M v ⁇ cc ⁇ e, DD-M.
  • v ⁇ cc ⁇ e is a better stimulator of IL-12 than interferon gamma.
  • Figs. 6 A, B, and C show data from separate experiments in which groups of C57BL/6 mice (Fig. 6A), BALB/c mice (Fig. 6B) or C3H/HeJ mice (Fig. 6C) were given DIFCO thioglycolate intraperitoneally. After three days, peritoneal macrophages were collected and placed in culture with interferon- gamma for three hours.
  • the culture supernatants were assayed for the presence of IL-12 produced by macrophages.
  • the recombinant proteins and M v ⁇ cc ⁇ e preparations stimulated the production of IL-12 from macrophages.
  • IF ⁇ -primed peritoneal macrophages from BALB/c mice were stimulated with 40 ug/ml of M v ⁇ cc ⁇ e recombinant proteins in culture for 3 days and the presence of IL-12 produced by macrophages was assayed.
  • IF ⁇ -primed macrophages produced IL-12 when cultured with a control protein, ovalbumin (ova).
  • the recombinant proteins GV 24B, 38BP, 38AP, 27, 5, 27B, 3, 23 and 22B stimulated more than twice the amount of IL-12 detected in control macrophage cultures.
  • M. vaccae culture supernatant S/N
  • killed M vaccae delipidated M vaccae and delipidated and deglycolipidated M vaccae (DD-M vaccae)
  • DD-M vaccae deglycolipidated M vaccae
  • vaccae delipidated M vaccae; delipidated M vaccae with glycolipids also extracted (DD-M vaccae) and proteins extracted with SDS; the SDS protein extract treated with Pronase (an enzyme which degrades protein); whole M vaccae culture filtrate; and heat- killed M tuberculosis or heat-killed M bovis BCG, M phlei or M smegmatis or M vaccae culture filtrate.
  • E.G7 cells which are EL4 cells (a C57BL/6-derived T cell lymphoma) transfected with the ovalbumin gene and thus express ovalbumin.
  • the spleen cells were then assayed for their ability to kill non-specifically EL4 target cells or to kill specifically the E.G7 ovalbumin expressing cells. Killing activity was detected by the release of 51 Chromium with which the EL4 and E.G7 cells have been labelled (100 ⁇ Ci per 2x10 6 ), prior to the killing assay. Killing or cytolytic activity is expressed as % specific lysis using the formula:
  • cytotoxic cells are generated only in mice immunized with ovalbumin with an adjuvant but not in mice immunized with ovalbumin alone.
  • the diagrams that make up Fig. 7 show the effect of various M vaccae derived adjuvant preparations on the generation of cytotoxic T cells to ovalbumin in C57BL/6 mice.
  • cytotoxic cells were generated in mice immunized with (i) 10 ⁇ g, (ii) 100 ⁇ g or (iii) 1 mg of autoclaved M vaccae or (iv) 75 ⁇ g of M vaccae culture filtrate.
  • Fig. 7 A cytotoxic cells were generated in mice immunized with (i) 10 ⁇ g, (ii) 100 ⁇ g or (iii) 1 mg of autoclaved M vaccae or (iv) 75 ⁇ g of M vaccae culture filtrate.
  • FIG. 7B shows that cytotoxic cells were generated in mice immunized with (i) 1 mg whole autoclaved M vaccae or (ii) 1 mg delipidated and deglycolipidated (DD-) M vaccae.
  • cytotoxic cells were generated in mice immunized with 1 mg whole autoclaved M vaccae;
  • Fig. 7C(ii) shows the active material in M vaccae soluble proteins extracted with SDS from DD-M vaccae.
  • Fig. 7C(iii) shows that active material in the adjuvant preparation of Fig. 7C(ii) was destroyed by treatment with the proteolytic enzyme Pronase.
  • 100 ⁇ g of the SDS-extracted proteins had significantly stronger immune-enhancing ability (Fig. 7C(ii)) than did 1 mg whole autoclaved M vaccae (Fig. 7C(i)).
  • mice immunized with 1 mg heat-killed M vaccae generated cytotoxic cells to ovalbumin, but mice immunized separately with 1 mg heat-killed M tuberculosis (Fig. 7D(ii)), 1 mg M bovis BCG (Fig. 7D(iii)), 1 mg M phlei (Fig. 7D(iv)), or 1 mg M smegmatis (Fig. 7D(v)) failed to generate cytotoxic cells.
  • mice immunised with ovalbumin plus 200 ug of DD- M.vaccae depleted of mycolic acids and arabinogalactan were also able to generate cytotoxic cells (28% to 46% maximum specific lysis compared with ⁇ 8% specific lysis for control mice immunised with ovalbumin alone).
  • the M vaccae culture filtrate described above was fractionated by iso-electric focusing and the fractions assayed for adjuvant activity in the anti-ovalbumin-specific cytotoxic response assay in C57BL/6 mice as described above. Peak adjuvant activities were demonstrated in fractions corresponding to pl of 4.2-4.32 (fraction nos. 7-9), 4.49-4.57 (fraction nos. 13-17) and 4.81-5.98 (fraction nos. 23-27).
  • mice were immunised intra-peritoneally with 50 ug of DD-M. vaccae once a week for 5 weeks. At the 6 th week mice were sacrificed and their serum collected. The sera were tested for antibodies to recombinant M v ⁇ cc ⁇ e-derived proteins, prepared as described below, in standard enzyme-linked immunoassays.
  • the antisera did not react with several M vaccae recombinant proteins nor with ovalbumin, which served as an irrelevant negative control protein in the enzyme-linked assays (data not shown).
  • Antisera from mice immunised with DD-M vaccae reacted with 12 M. v ⁇ cc ⁇ e-derived GN antigens.
  • the results are shown in Table 12 below.
  • the antisera thus identified GV3, 5P, 5, 7, 9, 22B, 24, 27, 27A, 27B, 33 and 45 as being present in DD-M. vaccae.
  • mice were injected in each footpad with 100 ug DD-M v ⁇ cc ⁇ e in combination with incomplete Freund's adjuvant and 10 days later were sacrificed to obtain popliteal lymph node cells.
  • the cells from immunized and non-immunized control mice were stimulated in vitro with recombinant M v ⁇ cc ⁇ e-derived GV proteins. After 3 days, cell proliferation and IF ⁇ production were assessed. T cell proliferative responses of lymph node cells from OO-M.vaccae immunized mice to GV proteins.
  • Lymph node cells from DD-M. v ⁇ cc ⁇ e-immunized mice did not proliferate in response to an irrelevant protein, ovalbumin, (data not shown). As shown in Table 13, lymph node cells from immunized mice showed proliferative responses to GV 3, 7, 9, 23, 27, 27B, and 33. The corresponding cells from non-immunized mice did not proliferate in response to these GV proteins suggesting that mice immunized with DD-M vaccae have been immunized with these proteins. Thus, the M.vaccae derived proteins GV 3, 7, 9, 23, 27, 27B and 33 are likely to be present in DD-M v ⁇ cc ⁇ e.
  • Stimulation index cpm from tritiated Thymidine uptake in presence of GV protein/cpm in absence of GV protein
  • lymph node cells from DD-M vaccae immunized mice did not produce IFN ⁇ upon stimulation with GV proteins.
  • lymph node cells from DD-M v ⁇ cc ⁇ e immunized mice secrete IFN ⁇ in a dose dependent manner when stimulated with GV 3, 5, 23, 27A, 27B, 33, 45 or 46, suggesting that the mice have been immunized with these proteins.
  • No IFN ⁇ production was detectable when cells from immunized mice were stimulated with the irrelevant protein, ovalbumin (data not shown).
  • the proteins GV 3, 5, 23, 27A, 27B, 33, 45 and 46 are thus likely to be present in DD-M vaccae.
  • the five proteins GV27, 27A, 27B, 23 and 45 were used as non-specific immune amplifiers with ovalbumin antigen to immunize mice as described above in Example 6.
  • 50 ug of any one of the recombinant proteins GV27, 27A, 27B, 23 and 45 when injected with 50-100 ug of ovalbumin, demonstrated adjuvant properties in being able to generate cytotoxic cells to ovalbumin.
  • This example illustrates the ability of killed M vaccae to stimulate cytotoxic CD8 T cells which preferentially kill macrophages that have been infected with M tuberculosis.
  • mice were immunized by the intraperitoneal injection of 500 ⁇ g of killed M vaccae which was prepared as described in Example 1.
  • the spleen cells of immunized mice were passed through a CD8 T cell enrichment column (R&D Systems, St. Paul, MN, USA).
  • the spleen cells recovered from the column have been shown to be enriched up to 90% CD8 T cells.
  • These T cells, as well as CD8 T cells from spleens of non-immunized mice were tested for their ability to kill uninfected macrophages or macrophages which have been infected with M tuberculosis.
  • Macrophages were obtained from the peritoneal cavity of mice five days after they have been given 1 ml of 3% thioglycolate intraperitoneally.
  • the macrophages were infected overnight with M tuberculosis at the ratio of 2 mycobacteria per macrophage. All macrophage preparations were labelled with 51 Chromium at 2 ⁇ Ci per 10 4 macrophages.
  • the macrophages were then cultured with CD8 T cells overnight (16 hours) at killer to target ratios of 30:1. Specific killing was detected by the release of 51 Chromium and expressed as % specific lysis, calculated as in Example 5.
  • ELISA enzyme-linked immunosorbent assay
  • CD 8 T cells from spleens of mice immunized with M vaccae were cytotoxic for macrophages infected with M tuberculosis and did not lyse uninfected macrophages.
  • the CD8 T cells from non-immunized mice did not lyse macrophages.
  • CD8 T cells from naive or non-immunized mice do produce IFN- ⁇ when cocultured with infected macrophages. The amount of IFN- ⁇ produced in coculture was greater with CD8 T cells derived from M vaccae immunized mice.
  • M vaccae (ATCC Number 15483) was cultured in sterile Medium 90 at 37 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37 °C for one day. The medium was then centrifuged (leaving the bulk of the cells) and filtered through a 0.45 ⁇ m filter into sterile bottles.
  • the culture filtrate was concentrated by lyophilization, and redissolved in MilliQ water. A small amount of insoluble material was removed by filtration through a 0.45 ⁇ m membrane.
  • the culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell which contained a 3kDa 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. This procedure reduced the 20 1 volume to approximately 50 ml. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA).
  • the desalted culture filtrate was fractionated by ion exchange chromatography on a column of Q-Sepharose (Pharmacia Biotech, Uppsala, Sweden) (16 X 100 mm) equilibrated with lOmM Tris HC1 buffer pH 8.0. Polypeptides were eluted with a linear gradient of NaCI from 0 to 1.0 M in the above buffer system. The column eluent was monitored at a wavelength of 280 nm.
  • the pool of polypeptides eluting from the ion exchange column was concentrated in a 400 ml Amicon stirred cell which contained a 3 kDa MWCO membrane. The pressure was maintained at 50 psi using nitrogen gas. The polypeptides were repeatedly concentrated by membrane filtration and diluted with 1% glycine until the conductivity of the sample was less than 0.1 mS.
  • the purified polypeptides were then fractionated by preparative isoelectric focusing in a Rotofor device (Bio-Rad, Hercules, CA, USA).
  • the pH gradient was established with a mixture of Ampholytes (Pharmacia Biotech) comprising 1.6% pH 3.5-5.0 Ampholytes and 0.4% pH 5.0 - 7.0 Ampholytes.
  • Acetic acid 0.5 M was used as the anolyte, and 0.5 M ethanolamine as the catholyte.
  • Isoelectric focusing was carried out at 12W constant power for 6 hours, following the manufacturer's instructions. Twenty fractions were obtained.
  • the polypeptide fractions which were shown to contain significant contamination were further purified using a Mono Q column (Pharmacia Biotech) 10 micron particle size (5 x 50 mm) or a Vydac Diphenyl column (Separations Group) 300 Angstrom pore size, 5 micron particle size (4.6 x 250 mm).
  • Mono Q column polypeptides were eluted with a linear gradient from 0-0.5 M NaCI in 10 mM Tris HC1 pH 8.0.
  • polypeptides were eluted with a linear gradient of acetonitrile (20-60% v/v) in 0.1% TFA.
  • the flow-rate was 1.0 ml/min and the column eluent was monitored at 220 nm for both columns.
  • the polypeptide peak fractions were collected and analysed for purity on a 15% polyacrylamide gel as described above.
  • polypeptides were individually dried onto BiobreneTM (Perkin Elmer/ Applied BioSystems Division, Foster City, CA)-treated glass fiber filters.
  • the filters with polypeptide were loaded onto a Perkin Elmer/Applied BioSystems Procise 492 protein sequencer and the polypeptides were sequenced from the amino terminal end using traditional Edman chemistry.
  • the amino acid sequence was determined for each polypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards.
  • GVc-1 six soluble M vaccae antigens, designated GVc-1, GVc-2, GVc-7, GVc-13, GVc-20 and GVc-22, were isolated.
  • N-terminal and internal sequences for GVc-1 are shown in SEQ ID NOS: 1, 2 and 3, respectively; the N- terminal sequence for GVc-2 is shown in SEQ ID NO: 4; internal sequences for GVc-7 are shown in SEQ ID NOS: 5-8; internal sequences for GVc-13 are shown in SEQ ID NOS: 9-11; internal sequence for GVc-20 is shown in SEQ ID NO: 12; and N-terminal and internal sequences for GVc-22 are shown in SEQ ID NO: 56-59, respectively.
  • Each of the internal peptide sequences provided herein begins with an amino acid residue which is assumed to exist in this position in the polypeptide, based on the known cleavage specificity of cyanogen bromide (Met) or Lys-C (Lys).
  • GVc-16, GVc-18 and GVc-21 Three additional polypeptides, designated GVc-16, GVc-18 and GVc-21, were isolated employing a preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) purification step in addition to the preparative isoelectric focusing procedure described above. Specifically, fractions comprising mixtures of polypeptides from the preparative isoelectric focusing purification step previously described were purified by preparative SDS-PAGE on a 15% polyacrylamide gel. The samples were dissolved in reducing sample buffer and applied to the gel.
  • SDS- PAGE preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • the separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane by electroblotting in 10 mM 3- (cyclohexylamino)-l-propanesulfonic acid (CAPS) buffer pH 11 containing 10% (v/v) methanol.
  • PVDF polyvinylidene difluoride
  • CAPS cyclohexylamino-l-propanesulfonic acid
  • the transferred protein bands were identified by staining the PVDF membrane with Coomassie blue. Regions of the PVDF membrane containing the most abundant polypeptide species were cut out and directly introduced into the sample cartridge of the Perkin Elmer/ Applied BioSystems Procise 492 protein sequencer. Protein sequences were determined as described above.
  • the N-terminal sequences for GVc-16, GVc-18 and GVc-21 are provided in SEQ ID NOS: 13, 14 and 15, respectively.
  • Additional antigens designated GVc-12, GVc-14, GVc-15, GVc-17 and GVc-19, were isolated employing a preparative SDS-PAGE purification step in addition to the chromatographic procedures described above. Specifically, fractions comprising a mixture of antigens from the Vydac C4 HPLC purification step previously described were fractionated by preparative SDS-PAGE on a polyacrylamide gel. The 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. The transferred protein bands were identified by staining the PVDF membrane with Coomassie blue.
  • Regions of the PVDF membrane containing the most abundant polypeptide species were cut out and directly introduced into the sample cartridge of the Perkin Elmer/Applied BioSystems Procise 492 protein sequencer. Protein sequences were determined as described above. The determined N-terminal sequences for GVc-12, GVc-14, GVc-15, GVc-17 and GVc-19 are provided in SEQ ID NOS: 16-20, respectively.
  • GVc-22B The determined nucleotide sequence of the gene encoding GV-22B and the predicted amino acid sequence are provided in SEQ ID NOS: 144 and 145 respectively.
  • Amplifications primers AD86 and AD112 were designed from the amino acid sequence of GVc-1 (SEQ ID NO: 1) and the M tuberculosis MPT70 gene sequence. Using these primers, a 310 bp fragment was amplified from M vaccae genomic DNA and cloned into EcoRV-digested vector pBluescript II SK + (Stratagene). The sequence of the cloned insert is provided in S ⁇ Q ID NO: 62. The insert of this clone was used to screen a M vaccae genomic DNA library constructed in lambda ZAP- ⁇ xpress (Stratagene, La Jolla, CA).
  • the clone isolated contained an open reading frame with homology to the M. tuberculosis antigen MPT83 and was re-named GV-1/83. This gene also had homology to the M bovis antigen MPB83.
  • the determined nucleotide sequence and predicted amino acid sequences are provided in S ⁇ Q ID NOS: 146 and 147 respectively.
  • degenerate oligonucleotides ⁇ V59 and EV61 (SEQ ID NOS: 148 and 149 respectively) were designed.
  • a 100 bp fragment was amplified, cloned into plasmid pBluescript II SK + and sequenced (SEQ ID NO: 150) following standard procedures (Sambrook et al. Ibid).
  • the cloned insert was used to screen a M vaccae genomic DNA library constructed in lambda ZAP-Express.
  • the clone isolated had homology to M tuberculosis antigen MPT70 and M bovis antigen MPB70, and was named GV-1/70.
  • the determined nucleotide sequence and predicted amino acid sequence for GV-1/70 are provided in SEQ ID NOS: 151 and 152 respectively.
  • the genes encoding GV1/83, GV1/70, GVc-13, GVc- 14 and GV-22B were sub-cloned into the expression vector pET16 (Novagen, Madison, WI). Expression and purification were performed according to the manufacturer's protocol.
  • the purified polypeptides were screened for the ability to induce T-cell proliferation and IFN- ⁇ in peripheral blood cells from immune human donors. These donors were known to be PPD (purified protein derivative from M tuberculosis) skin test positive and their T cells were shown to proliferate in response to PPD.
  • Donor PBMCs and crude soluble proteins from M vaccae culture filtrate were cultured in medium comprising RPMI 1640 supplemented with 10% (v/v) autologous serum, penicillin (60 ⁇ g/ml), streptomycin (100 ⁇ g/ml), and glutamine (2 mM). After 3 days, 50 ⁇ l of medium was removed from each well for the determination of IFN- ⁇ levels, as described below.
  • the plates were cultured for a further 4 days and then pulsed with l ⁇ Ci/well of tritiated thymidine for a further 18 hours, harvested and tritium uptake determined using a scintillation counter. Fractions that stimulated proliferation in both replicates two-fold greater than the proliferation observed in cells cultured in medium alone were considered positive.
  • IFN- ⁇ was measured using an enzyme-linked immunosorbent assay (ELISA).
  • ELISA plates were coated with a mouse monoclonal antibody directed to human IFN- ⁇ (Endogen, Wobural, MA) 1 ⁇ g/ml phosphate-buffered saline (PBS) for 4 hours at 4 °C.
  • Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. The plates were then washed four times in PBS/0.2% Tween 20, and samples diluted 1 :2 in culture medium in the ELISA plates were incubated overnight at room temperature.
  • polypeptides containing sequences that stimulate peripheral blood mononuclear cells (PBMC) T cells to proliferate and produce IFN- ⁇ are shown in Table 16, wherein (-) indicates a lack of activity, (+/-) indicates polypeptides having a result less than twice higher than background activity of control media, (+) indicates polypeptides having activity two to four times above background, and (++) indicates polypeptides having activity greater than four times above background.
  • M vaccae soluble proteins were isolated from culture filtrate using 2-dimensional polyacrylamide gel electrophoresis as described below. Unless otherwise noted, all percentages in the following example are weight per volume.
  • M vaccae (ATCC Number 15483) was cultured in sterile Medium 90 at 37 °C.
  • M tuberculosis strain H37Rv (ATCC number 27294) was cultured in sterile Middlebrook 7H9 medium with Tween 80 and oleic acid/albumin/dextrose/catalase additive (Difco Laboratories, Detroit, Michigan). The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37 °C for one day. The medium was then centrifuged (leaving the bulk of the cells) and filtered through a 0.45 ⁇ m filter into sterile bottles. The culture filtrate was concentrated by lyophilisation, and redissolved in MilliQ water.
  • a small amount of insoluble material was removed by filtration through a 0.45 ⁇ m membrane filter.
  • the culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell which contained 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. This procedure reduced the 20 1 volume to approximately 50 ml. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA).
  • the desalted culture filtrate was fractionated by ion exchange chromatography on a column of Q-Sepharose (Pharmacia Biotech) (16 x 100 mm) equilibrated with lOmM TrisHCl buffer pH 8.0. Polypeptides were eluted with a linear gradient of NaCI from 0 to 1.0 M in the above buffer system. The column eluent was monitored at a wavelength of 280 nm.
  • the pool of polypeptides eluting from the ion exchange column were fractionated by preparative 2D gel electrophoresis.
  • Samples containing 200-500 ⁇ g of polypeptide were made 8M in urea and applied to polyacrylamide isoelectric focusing rod gels (diameter 2mm, length 150 mm, pH 5-7).
  • the first dimension gels were equilibrated with reducing buffer and applied to second dimension gels (16% poly aery lamide).
  • Polypeptides from the second dimension separation were transferred to PVDF membranes by electroblotting in lOmM CAPS buffer pH 11 containing 10% (v/v) methanol.
  • the PVDF membranes were stained for protein with Coomassie blue.
  • Regions of PVDF containing polypeptides of interest were cut out and directly introduced into the sample cartridge of the Perkin Elmer/ Applied BioSystems Procise 492 protein sequencer.
  • the polypeptides were sequenced from the amino terminal end using traditional Edman chemistry.
  • the amino acid sequence was determined for each polypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards. Using these procedures, eleven polypeptides, designated GVs-1, GVs-3, GVs-4, GVs-5, GVs-6, GVs-8, GVs-9, GVs-10, GVs-11, GV-34 and GV-35 were isolated.
  • the determined N- terminal sequences for these polypeptides are shown in SEQ ID NOS: 21-29, 63 and 64, respectively.
  • SEQ ID NOS: 21-29, 63 and 64 The determined N- terminal sequences for these polypeptides are shown in SEQ ID NOS: 21-29, 63 and 64, respectively.
  • the extended amino acid sequence for GVs-9 is provided in SEQ ID NO: 65.
  • Further studies resulted in the isolation of DNA sequences for GVs-9 (SEQ ID NO: 111) and GV-35 (SEQ ID NO: 155).
  • the corresponding predicted amino acid sequences are provided in SEQ ID NO: 112 and 156, respectively.
  • An extended DNA sequence for GVs-9 is provided in SEQ ID NO: 153, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 154.
  • the predicted amino acid sequence for GVs-9 has been amended in SEQ ID NO: 197.
  • GVs-3, GVs-4 and GVs-5 were found to bear some similarity to the antigen 85A and 85B proteins from M leprae (SEQ ID NOS: 30 and 31, respectively), M tuberculosis (SEQ ID NOS: 32 and 33, respectively) and M bovis (SEQ ID NOS: 34 and 35, respectively), and the antigen 85C proteins from M. leprae (SEQ ID NO: 36) and M tuberculosis (SEQ ID NO: 37).
  • Probes for antigens 85A, 85B, and 85C were prepared by polymerase chain reaction
  • PCR using degenerate oligonucleotides (SEQ ID NOS: 38 and 39) designed to regions of antigen 85 genomic sequence that are conserved between family members in a given mycobacterial species, and between mycobacterial species. These oligonucleotides were used under reduced stringency conditions to amplify target sequences from M vaccae genomic
  • An M vaccae genomic library was created in lambda Zap-Express (Stratagene, La Jolla, CA) by cloning BamHl partially-digested M vaccae genomic DNA into similarly- digested ⁇ vector, with 3.4 x 10 5 independent plaque-forming units resulting.
  • Twenty-seven thousand plaques from this non-amplified library were plated at low density onto eight 100 cm 2 plates.
  • duplicate plaque lifts were taken onto Hybond-N + nylon membrane (Amersham International, United Kingdom), and hybridised under reduced-stringency conditions (55 °C) to the radiolabelled antigen 85C PCR product. Autoradiography demonstrated that seventy-nine plaques consistently hybridised to the antigen 85C probe under these conditions.
  • Sequence data from the 5' and 3' ends of inserts from several representatives of each group was obtained using the Perkin Elmer/Applied Biosystems Model 377 automated sequencer and the T3 and T7 primers. Sequence homologies were determined using BLASTN analysis of the EMBL database. Two of these sets of clones were found to be homologous to M bovis and M tuberculosis antigen 85A genes, each containing either the 5' or 3' ends of the M vaccae gene (this gene was cleaved during library construction as it contains an internal BamHI site). The remaining clones were found to contain sequences homologous to antigens 85B and 85C from a number of mycobacterial species.
  • the M vaccae 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 NO: 40 and 42, respectively) using sequence data downstream from the putative leader sequence and the 3' end of the clone.
  • the sequences of the primers for GVs-3 are provided in SEQ ID NO: 66 and 67
  • the sequences of the primers for GVs-5 are provided in SEQ ID NO: 68 and 69.
  • a Xhol restriction site was added to the primers for GVs-3, and EcoRI and BamRl restriction sites were added to the primers for GVs-5 for cloning convenience. Following amplification from genomic M.
  • vaccae DNA fragments were cloned into the appropriate site of pPro ⁇ X HT prokaryotic expression vector (Gibco BRL, Life Technologies, Gaithersburg, MD) and submitted for sequencing to confirm the correct reading frame and orientation. Expression and purification of the recombinant protein was performed according to the manufacturer's protocol.
  • the insert from one of the clones was subcloned into the EcoRI/ATzoI sites of pPro ⁇ X HT prokaryotic expression vector (Gibco BRL), expressed and purified according to the manufacturer's protocol.
  • This clone was renamed GV-4P because only a part of the gene was expressed.
  • the amino acid and DNA sequences for the partial clone GV-4P are provided in S ⁇ Q ID NO: 70 and 106, respectively.
  • the amplification primers AD58 and AD59 were used to amplify a 485 bp fragment from a clone containing GVs-5 (SEQ ID NO:42). This fragment was cloned into the expression vector pET16 and was called GV-5P.
  • the determined nucleotide sequence and predicted amino acid sequence of GV-5P are provided in SEQ ID NOS: 157 and 158, respectively.
  • GVs-3, GV- 4P and GVs-5 were re-cloned into the alternative vector pET16 (Novagen, Madison, WI).
  • An 84 bp probe for the M vaccae antigen GVc-7 was amplified using degenerate oligonucleotides designed to the determined amino acid sequence of GVc-7 (SEQ ID NOS: 5-
  • This probe was used to screen a M vaccae genomic DNA library as described in Example 12.
  • the determined nucleotide sequence for GVc-7 is shown in SEQ ID NO: 46 and predicted amino acid sequence in SEQ ID NO: 47. Comparison of these sequences with those in the databank revealed homology to a hypothetical 15.8 kDa membrane protein of M tuberculosis.
  • SEQ ID NO: 46 The sequence of SEQ ID NO: 46 was used to design amplification primers (provided in SEQ ID NO: 71 and 72) for expression cloning of the GVc-7 gene using sequence data downstream from the putative leader sequence. A Xhol restriction site was added to the primers for cloning convenience. Following amplification from genomic M vaccae DNA, fragments were cloned into the .ATzoI-site of pProEX HT prokaryotic expression vector (Gibco BRL) and submitted for sequencing to confirm the correct reading frame and orientation. Expression and purification of the fusion protein was performed according to the manufacturer's protocol. In subsequent studies, GVc-7 was re-cloned into the vector pET16 (Novagen).
  • a redundant oligonucleotide probe (SEQ ID NO 73; referred to as MPG15) was designed to the GVs-8 peptide sequence shown in SEQ ID NO: 26 and used to screen a M vaccae genomic DNA library using standard protocols. Two genomic clones containing genes encoding four different antigens was isolated. The determined DNA sequences for GVs-8A (re-named GV-30), GVs-8B (re-named GV-31), GVs-8C (re-named GV-32) and GVs-8D (re-named GV-33) are shown in SEQ ID NOS: 48-51, respectively, with the corresponding amino acid sequences being shown in SEQ ID NOS: 52-55, respectively.
  • GV- 30 contains regions showing some similarity to known prokaryotic valyl-tRNA synthetases; GV-31 shows some similarity to M smegmatis aspartate semialdehyde dehydrogenase; and GV-32 shows some similarity to the H influenza folylpolyglutamate synthase gene. GV-33 contains an open reading frame which shows some similarity to sequences previously identified in M tuberculosis and M leprae, but whose function has not been identified.
  • the determined partial DNA sequence for GV-33 is provided in SEQ ID NO: 74 with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 75. Sequence data from the 3' end of the clone showed homology to a previously identified 40.6 kDa outer membrane protein of M tuberculosis. Subsequent studies led to the isolation of a full-length DNA sequence for GV-33 (SEQ ID NO: 193). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 194.
  • the gene encoding GV-33 was amplified from M vaccae genomic DNA with primers based on the determined nucleotide sequence. This DNA fragment was cloned into EcoRv- digested pBluescript II SK + (Stratagene), and then transferred to p ⁇ T16 expression vector. Recombinant protein was purified following the manufacturer's protocol.
  • M. vaccae bacteria were cultured, pelleted and autoclaved as described in Example 1.
  • Culture filtrates of live M vaccae refer to the supernatant from 24 hour cultures of M vaccae in 7H9 medium with glucose.
  • the resulting pellet was suspended in 100 ml of chloroform/methanol (2:1), incubated at room temperature for 1 hour, re-centrifuged, and the chloroform/methanol extraction repeated.
  • the pellet was obtained by centrifugation, dried in vacuo, weighed and resuspended in PBS at 50 mg (dry weight) per ml as delipidated M vaccae.
  • Glycolipids were removed from the delipidated M vaccae preparation by refluxing in 50% v/v ethanol for 2 hours.
  • the insoluble material was collected by centrifugation and washed in PBS. Proteins were extracted by resuspending the pellet in 2% SDS in PBS at 56 °C for 2 hours. The insoluble material was collected by centrifugation and the extraction with 2% SDS/PBS at 56 °C was repeated twice more.
  • the SDS-extracted proteins derived from DD-M vaccae were analysed by polyacrylamide gel electrophoresis. Three major bands were observed after staining with silver. In subsequent experiments, larger amounts of SDS-extracted proteins from DD- M.vaccae, were analysed by polyacrylamide gel electrophoresis. The proteins, on staining with Coomassie blue, showed several bands.
  • a protein represented by a band of approximate molecular weight of 30 kDa was designated GV-45.
  • the determined N-terminal sequence for GV-45 is provided in SEQ ID NO: 187.
  • a protein of approximate molecular weight of 14 kDa was designated GV-46.
  • the determined N-terminal amino acid sequence of GV-46 is provided in SEQ ID NO: 208.
  • the sequence of the first ten amino acid residues is provided in SEQ ID NO: 76. Comparison of this sequence with those in the gene bank as described above, revealed homology to the heat shock protein 65 (GroEL) gene from M tuberculosis, indicating that this protein is an M vaccae member of the GroEL family.
  • RhoEL heat shock protein 65
  • An expression library of M vaccae genomic DNA in 5 ⁇ mHl -lambda ZAP-Express (Stratagene) was screened using sera from cynomolgous monkeys immunised with M vaccae secreted proteins prepared as described above. Positive plaques were identified using a colorimetric system. These plaques were re-screened until plaques were pure following standard procedures.
  • pBK-CMV phagemid 2-1 containing an insert was excised from the lambda ZAP Express (Stratagene) vector in the presence of ExAssist helper phage following the manufacturer's protocol.
  • the base sequence of the 5' end of the insert of this clone was determined using Sanger sequencing with fluorescent primers on Perkin Elmer/Applied Biosystems Division automatic sequencer.
  • the determined nucleotide sequence of the partial M vaccae GroEL-homologue clone GV-27 is provided in SEQ ID NO: 77 and the predicted amino acid sequence in SEQ ID NO: 78.
  • This clone was found to have homology to M tuberculosis GroEL.
  • a partial sequence of the 65 kDa heat shock protein of M vaccae has been published by Kapur et al. (Arch. Pathol. Lab. Med. 119 :131-138, 1995).
  • the nucleotide sequence of the Kapur et al. fragment is shown in SEQ ID NO: 79 and the predicted amino acid sequence in SEQ ID NO: 80.
  • GV-27A Two peptide fragments, comprising the N-terminal sequence
  • GV-27B the carboxy terminal sequence of GV-27
  • SEQ ID NO: 115 and 116 The nucleotide sequences for GV-27A and GV-27B are provided in SEQ ID NO: 115 and 116, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO: 117 and 118.
  • SEQ ID NO: 161 is provided in SEQ ID NO: 161, with the corresponding amino acid sequence being provided in SEQ ID NO: 162.
  • the sequence of GV-27 A is 95.8% identical to the M tuberculosis GroEL sequence and contains the shorter M vaccae sequence of Kapur et al. discussed above.
  • the sequence for GV-27B shows about 92.2% identity to the corresponding region of M tuberculosis HSP65.
  • pBK-CMV phagemid 3-1 was isolated.
  • the antigen encoded by this DNA was named GV-29.
  • the determined nucleotide sequences of the 5' and 3' ends of the gene are provided in SEQ ID NOS: 163 and 164, respectively, with the predicted corresponding amino acid sequences being provided in SEQ ID NOS: 165 and 166 respectively.
  • GV-29 showed homology to yeast urea amidolyase.
  • the determined DNA sequence for the full-length gene encoding GV-29 is provided in SEQ ID NO: 198, with the corresponding predicted amino acid sequence in SEQ ID NO: 199.
  • the DNA encoding GV-29 was sub-cloned into the vector pET16 (Novagen, Madison, WI) for expression and purification according to standard protocols.
  • M vaccae (ATCC Number 15483) was grown in sterile Medium 90 at 37 °C for 4 days and harvested by centrifugation. Cells were resuspended in 1 ml Trizol (Gibco BRL, Life Technologies, Gaithersburg, Maryland) and RNA extracted according to the standard manufacturer's protocol. M tuberculosis strain H37Rv (ATCC Number 27294) was grown in sterile Middlebrook 7H9 medium with Tween 80TM and oleic acid/ albumin/dextrose/catalase additive (Difco Laboratories, Detroit, Michigan) at 37 °C and harvested under appropriate laboratory safety conditions. Cells were resuspended in 1 ml Trizol (Gibco BRL) and RNA extracted according to the manufacturer's standard protocol.
  • Total M tuberculosis and M vaccae RNA was depleted of 16S and 23 S ribosomal RNA (rRNA) by hybridisation of the total RNA fraction to oligonucleotides AD 10 and AD11 (SEQ ID NO: 81 and 82) complementary to M tuberculosis rRNA.
  • oligonucleotides were designed from mycobacterial 16S rRNA sequences published by Bottger (FEMS Microbiol. Lett. 65:11 - 16, 1989) and from sequences deposited in the databanks. Depletion was done by hybridisation of total RNA to oligonucleotides AD 10 and AD11 immobilised on nylon membranes (Hybond N, Amersham International, United Kingdom).
  • oligonucleotide, AD 12 (SEQ ID NO: 83), consisting of 20 dATP -residues, was ligated to the 3' ends of the enriched mRNA fraction using RNA ligase.
  • First strand cDNA synthesis was performed following standard protocols, using oligonucleotide AD7 (SEQ ID NO: 84) containing a poly(dT) sequence.
  • the M tuberculosis and M vaccae cDNA was used as template for single-sided- specific PCR (3S-PCR).
  • a degenerate oligonucleotide ADl (SEQ ID NO:85) was designed based on conserved leader sequences and membrane protein sequences. After 30 cycles of amplification using primer ADl as 5'-primer and AD7 as 3'-primer, products were separated on a urea polyacrylamide gel. DNA bands unique to M vaccae were excised and re-amplified using primers ADl and AD7. After gel purification, bands were cloned into pGEM-T (Promega) and the base sequence determined.
  • transmembrane genes encode proteins each characteristically having six membrane-spanning regions. Homologues (by similarity) of this ABC transporter have been identified in the genomes of Haemophilus influenza (Fleischmann et al. Science 269 :496-512, 1995) and Mycoplasma genitalium (Fraser, et al. Science, 270:391-403, 1995).
  • the nucleotide sequence of the full-length M vaccae homologue of pota (ATP-binding protein) was identified by subcloning of the 4.5 kb fragment and base sequencing.
  • the nucleotide and predicted amino acid sequences of the M vaccae pota homologue are provided in SEQ ID NO: 88 and 89, respectively.
  • the nucleotide sequence of the M vaccae pota gene was used to design primers EV24 and EV25 (SEQ ID NO: 90 and 91) for expression cloning.
  • the amplified DNA fragment was cloned into pProEX HT prokaryotic expression system (Gibco BRL) and expression in an appropriate E.coli host was induced by addition of 0.6 mM isopropylthio- ⁇ -galactoside (IPTG).
  • IPTG isopropylthio- ⁇ -galactoside
  • the recombinant protein was named GV-23 and purified from inclusion bodies according to the manufacturer's protocol. In subsequent studies, GV-23 (SEQ ID NO: 88) was re-cloned into the alternative vector pET16 (Novagen).
  • SEQ ID NO: 89 contains an ATP binding site at residues 34 to 41. At residues 116 to 163 of SEQ ID NO: 89, there is a conserved region that is found in the ATP -transporter family of proteins. These findings suggest that GV-23 is an ATP binding protein.
  • a 322 bp Sall-BamRl subclone at the 3'-end of the 4.5 kb insert described above showed homology to the potd gene, (periplasmic protein), of the spermidine/putrescine ABC transporter complex of E. coli.
  • the nucleotide sequence of this subclone is shown in SEQ ID NO:92.
  • the radiolabelled insert of this subclone was used to probe a M vaccae genomic DNA library constructed in the S ⁇ /1-site of lambda Zap Express (Stratagene) following standard protocols.
  • a clone was identified of which 1342 bp showed homology with the potd gene of E. coli.
  • the potd homologue of M vaccae was identified by subcloning and base sequencing. The determined nucleotide and predicted amino acid sequences are shown in SEQ ID NO: 93 and 94.
  • primers EV-26 and EV-27 were designed from the determined M vaccae potd homologue.
  • the amplified fragment was cloned into pProEX HT Prokaryotic expression system (Gibco BRL). Expression in an appropriate E. coli host was induced by addition of 0.6 mM IPTG and the recombinant protein named GV-24.
  • the recombinant antigen was purified from inclusion bodies according to the protocol of the supplier.
  • GV-24 SEQ ID NO: 93
  • GV-24 was re-cloned into the alternative vector pET16 (Novagen).
  • the gene encoding GV- 24, but excluding the signal peptide was re-cloned into the expression vector, employing, amplification primers EV101 and EV102 (SEQ ID NOS: 167 and 168).
  • the construct was designated GV-24B.
  • the nucleotide sequence of GV-24B is provided in SEQ ID NO: 169 and the predicted amino acid sequence in SEQ ID NO: 170. This fragment was cloned into pET16 for expression and purification of GV-24B according to the manufacturer's protocols.
  • M vaccae potd gene-homologue Base sequence adjacent to the M vaccae potd gene-homologue was found to show homology to the potb gene of the spermidine/putrescine ABC transporter complex of E.coli, which is one of two transmembrane proteins in the ABC transporter complex.
  • the M vaccae potb homologue (referred to as GV-25) was identified through further subcloning and base sequencing. The determined nucleotide and predicted amino acid sequences for GV-25 are shown in SEQ ID NOS: 97 and 98, respectively.
  • the 3S- PCR band 12B28 (SEQ ID NO: 119) was used to screen the M vaccae genomic library constructed in the BamHI-site of lambda ZAP Express (Stratagene).
  • the clone isolated from the library contained a novel open reading frame and the antigen encoded by this gene was named GV-38A.
  • the determined nucleotide sequence and predicted amino acid sequence of GV-38A are shown in SEQ ID NO: 120 and 121, respectively.
  • the corresponding amino acid sequence is provided in SEQ ID NO: 172. Comparison of these sequences with those in the gene bank, revealed some homology to an unknown M tuberculosis protein previously identified in cosmid MTCY428.12. (SPTREMBL:P71915).
  • GV-38B 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.
  • the determined 5' and 3' nucleotide sequences for GV-38B are provided in SEQ ID NO: 122 and 123, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 124 and 125, respectively. Further studies led to the isolation of the full-length DNA sequence for GV- 38B, provided in SEQ ID NO: 173. The corresponding amino acid sequence is provided in SEQ ID NO: 174.
  • This protein was found to show homology to an unknown M tuberculosis protein identified in cosmid MTCY428.11 (SPTREMBL: P71914).
  • GV-38A and GV-38B antigens were amplified for expression cloning into pET16 (Novagen).
  • GV-38A was amplified with primers KR11 and KR12 (SEQ ID NO: 126 and 127) and GV-38B with primers KR13 and KR14 (SEQ ID NO: 128 and 129).
  • Protein expression in the host cells BL21(DE3) was induced with 1 mM IPTG, however no protein expression was obtained from these constructs. Hydrophobic regions were identified in the N-termini of antigens GV-38A and GV-38B which may inhibit expression of these constructs.
  • the hydrophobic region present in GV-38A was identified as a possible transmembrane motif with six membrane spanning regions.
  • primers KR20 for GV-38A, (SEQ ID NO: 130) and KR21 for GV-38B (SEQ ID NO: 131) were designed.
  • the truncated GV-38A gene was amplified with primers KR20 and KR12, and the truncated GV-38B gene with KR21 and KR14.
  • the determined nucleotide sequences of truncated GV38A and GV-38B are shown in SEQ ID NO: 132 and 133, respectively, with the corresponding predicted amino acid sequences being shown in SEQ ID NO: 134 and 135, respectively.
  • Extended DNA sequences for truncated GV-38A and GV- 38B are provided in SEQ ID NO: 175 and 176, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO: 177 and 178, respectively.
  • M vaccae soluble proteins were isolated from culture filtrate using preparative isoelectric focusing and preparative polyacrylamide gel electrophoresis as described below. Unless otherwise noted, all percentages in the following example are weight per volume.
  • M vaccae (ATCC Number 15483) was cultured in 250 1 sterile Medium 90 which had been fractionated by ultrafiltration to remove all proteins of greater than 10 kDa molecular weight. The medium was centrifuged to remove the bacteria, and sterilised by filtration through a 0.45 ⁇ m filter. The sterile filtrate was concentrated by ultrafiltration over a 10 kDa molecular weight cut-off membrane.
  • Proteins were isolated from the concentrated culture filtrate by precipitation with 10% trichloroacetic acid. The precipitated proteins were re-dissolved in 100 mM Tris.HCl pH 8.0. and re-precipitated by the addition of an equal volume of acetone. The acetone precipitate was dissolved in water, and proteins were re-precipitated by the addition of an equal volume of chloroform: methanol 2:1 (v/v). The chloroform:methanol precipitate was dissolved in water, and the solution was freeze-dried.
  • the freeze-dried protein was dissolved in iso-electric focusing buffer, containing 8 M deionised urea, 2% Triton X-100, 10 mM dithiothreitol and 2% ampholytes (pH 2.5 - 5.0).
  • the sample was fractionated by preparative iso-electric focusing on a horizontal bed of Ultrodex gel at 8 watts constant power for 16 hours. Proteins were eluted from the gel bed fractions with water and concentrated by precipitation with 10% trichloroacetic acid. Pools of fractions containing proteins of interest were identified by analytical polyacrylamide gel electrophoresis and fractionated by preparative polyacrylamide gel electrophoresis.
  • Eluted proteins were assayed for their ability to induce proliferation and interferon- ⁇ secretion from the peripheral blood lymphocytejs of immune donors as detailed above. Proteins inducing a strong response in these assays were selected for further study.
  • Selected proteins were further purified by reversed-phase chromatography on a Vydac Protein C4 column, using a trifluoroacetic acid-acetonitrile system. Purified proteins were prepared for protein sequence determination by SDS-polyacrylamide gel electrophoresis, and electroblotted onto PVDF membranes. Protein sequences were determined as in Example 3. The proteins were named GV-40, GV-41, GV-42, GV-43 and GV-44. The determined N- terminal sequences for these polypeptides are shown in SEQ ID NOS: 101-105, respectively. Subsequent studies led to the isolation of a 5', middle fragment and 3' DNA sequence for GV- 42 (SEQ ID NO: 136, 137 and 138, respectively). The corresponding predicted amino acid sequences are provided in SEQ ID NO: 139, 140 and 141, respectively.
  • GV-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 in SEQ ID NOS: 181 and 182. Further experiments lead to the cloning of the full-length gene encoding GV-41, which was named GV-41B.
  • the determined nucleotide sequence and the predicted amino acid sequence of GV-41B are provided in SEQ ID NOS: 202 and 203, respectively.
  • GV-41 had homology to the ribosome recycling factor of M tuberculosis and M leprae, and GV-42 had homology to a M avium fibronectin attachment protein FAP-A.
  • the amino acid sequence determined for GV-43 (SEQ ID NO: 104) was identified, indicating that the amino acid sequences for GV-42 and GV-43 were obtained from the same protein.
  • Murine polyclonal antisera were prepared against GV-40 and GV-44 following standard procedures. These antisera were used to screen a M vaccae genomic DNA library consisting of randomly sheared DNA fragments. 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 in SEQ ID NO :184.
  • the complete gene encoding GV-40 was not cloned, and the antigen encoded by this partial gene was named GV-40P.
  • An extended DNA sequence for GV-40P is provided in SEQ ID NO: 206 with the corresponding predicted amino acid sequence being provided in SEQ ID NO 207.
  • the determined nucleotide sequence of the gene encoding GV-44 is provided in SEQ ID NO: 185, and the predicted amino acid sequence in SEQ ID NO: 186.
  • the determined DNA sequence for the full-length gene encoding GV-44 was obtained and is provided in SEQ ID NO 204, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 205.
  • Homology of GV-40 to M leprae Elongation factor G was found and GV-44 had homology to M leprae glyceraldehyde-3- phosphate dehydrogenase.
  • GV-45 a protein represented by a band of approximate molecular weight of 30 kDa, designated GV-45, was isolated.
  • the determined N-terminal sequence for GV-45 is provided in SEQ ID NO: 187.
  • a protein of approximate molecular weight of 14 kDa, designated GV-46 was obtained.
  • the determined N-terminal amino acid sequence of GV-46 is provided in SEQ ID NO: 208.
  • GV-46 is homologous to the highly conserved mycobacterial host integration factor of M tuberculosis and M smegmatis.
  • degenerate oligonucleotides KR32 and KR33 (SEQ ID NOS: 188 and 189, respectively) were designed.
  • a 100 bp fragment was amplified, cloned into plasmid pBluescript II SK + (Stratagene, La Jolla, CA) and sequenced (SEQ ID NO:190) following standard procedures (Sambrook, Ibid).
  • the cloned insert was used to screen a M vaccae genomic DNA library constructed in the 5 ⁇ rnHI-site of lambda ZAP -Express (Stratagene).
  • the isolated clone showed homology to a 35 kDa M tuberculosis and a 22 kDa M leprae protein containing bacterial histone-like motifs at the N-terminus and a unique C-terminus consisting of a five amino acid basic repeat.
  • the determined nucleotide sequence for GV-45 is provided in SEQ ID NO: 191, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 192.
  • the determined DNA sequence for the full-length gene encoding GV-45 was obtained and is provided in SEQ ID NO: 200, with the corresponding predicted amino acid sequence in SEQ ID NO: 201.
  • GV recombinant proteins The immunogenicity of Mycobacterium vaccae recombinant proteins (GV recombinant proteins) was tested by injecting female BALB/cByJ mice in each hind foot-pad with 10 ug of recombinant GV proteins emulsified in incomplete Freund's adjuvant (IF A). Control mice received phosphate buffered saline in IF A. The draining popliteal lymph nodes were excised 10 days later and the cells obtained therefrom were stimulated with the immunizing GV protein and assayed for proliferation by measuring the uptake of tritiated thymidine. The amount of interferon gamma (IFN ⁇ ) produced and secreted by these cells into the culture supernatants was assayed by standard enzyme-linked immunoassay.
  • IFN ⁇ interferon gamma
  • the GV proteins are thus immunogenic in being able to stimulate T cell proliferation and/or IFN ⁇ production when administered by subcutaneous injection.
  • the antigen-specific stimulatory effects on T cell proliferation and IFN ⁇ production are two advantageous properties of candidate vaccines for tuberculosis.
  • PBMC from normal donors (5 x 10 6 cells/ml) were stimulated with 20 ug/ml of either heat-killed M vaccae cells, DD-M vaccae or recombinant GV-22B (SEQ ID NO: 145), GV- 23 (SEQ ID NO: 89), GV-27 (SEQ ID NO: 160), GV27A (SEQ ID NO: 117), GV-27B (SEQ ID NO: 162) or GV-45 (SEQ ID NO: 201) for 24 hours.
  • CD69 expression was determined by staining cultured cells with monoclonal antibody against CD56, ⁇ T cells or ⁇ T cells, in combination with monoclonal antibodies against CD69, followed by flow cytometry analysis
  • Table 23 shows the percentage of ⁇ T cells, ⁇ T cells and NK cells expressing CD69 following stimulation with heat-killed M vaccae, DD-M. vaccae or recombinant M vaccae proteins.
  • PBMC from normal donors (5 x 10° cells/ml) were stimulated with 20 ug/ml of either heat-killed M vaccae cells, DD-M vaccae, or recombinant GV-22B (SEQ ID NO: 145), GV-23 (SEQ ID NO: 89), GV-27 (SEQ ID NO: 160), GV27A (SEQ ID NO: 117), GV-27B (SEQ ID NO: 162) or GV-45 (SEQ ID NO: 201) for 24 hours.
  • Figs. 9A-D show the stimulation of IL-l ⁇ , TNF- ⁇ , IL-12 and IFN- ⁇ production, respectively. Heat-killed M vaccae and DD-M vaccae were found to stimulate the production of all four cytokines examined, while recombinant GV-23 and GV- 45 were found to stimulate the production of IL-l ⁇ , TNF- ⁇ and IL-12.
  • Figs. 10A-C show the stimulation of IL-l ⁇ , TNF- ⁇ and IL-12 production, respectively, in human PBMC (determined as described above) by varying concentrations of GV-23 and GV-45.
  • Figs. 11A-D show the stimulation of IL-l ⁇ , TNF- ⁇ , IL-12 and IFN- ⁇ production, respectively, in PBMC by GV-23 as compared to that by the adjuvants MPL/TDM/CWS (at a final dilution of 1 :20), CpG ODN (20 ug/ml), aluminium hydroxide (at a final dilution of 1:400) and cholera toxin (20 ug/ml).
  • GV-23, MPL/TDM CWS and CpG ODN induced significant levels of the four cytokines examined, with higher levels of IL-l ⁇ production being seen with GV-23 than with any of the known adjuvants. Aluminium hydroxide and cholera toxin induced only negligible amounts of the four cytokines.
  • Peripheral blood mononuclear cells depleted of T cells and comprising mainly B cells, monocytes and dendritic cells were stimulated with 20 ug/ml of either heat-killed M vaccae cells, DD-M vaccae, or recombinant GV-22B (SEQ ID NO: 145), GV-23 (SEQ ID NO: 89), GV-27 (SEQ ID NO: 160), GV27A (SEQ ID NO: 117), GV-27B (SEQ ID NO: 162) or GV- 45 (SEQ ID NO: 201) for 48 hours.
  • Stimulated cells were harvested and analyzed for upregulation of CD40, CD80 and CD86 using 3 color flow cytometric analysis. Tables 24, 25 and 26 show the fold increase in mean fluorescence intensity from control (non-stimulated cells) for dendritic cells, monocytes, and B cells, respectively.
  • Figs. 12A-C show the stimulation of expression of CD40, CD80 and CD86, respectively, in dendritic cells by varying concentrations of GV-23 and GV-45.
  • GV-23 The ability of GV-23 to stimulate CD40, CD80 and CD86 expression in dendritic cells was compared to that of the Thl -inducing adjuvants MPL/TDM/CWS (at a final dilution of 1:20) and CpG ODN (20 ug/ml), and the known Th2-inducing adjuvants aluminium hydroxide (at a final dilution of 1 :400) and cholera toxin (20 ug/ml).
  • GV23, MPL/TDM/CWS and CpG ODN caused significant up-regulation of CD40, CD80 and CD86, whereas cholera toxin and aluminium hydroxide induced modest or negligible dendritic cell activation, respectively.
  • Purified dendritic cells (5 x 10 4 - 10 5 cells/ml) were stimulated with GV-23 (20 ug/ml) or LPS (10 ug/ml) as a positive control. Cells were cultured for 20 hour and then analyzed for CD83 (a maturation marker) and CD80 expression by flow cytometry. Non-stimulated cells were used as a negative control. The results are shown below in Table 27.
  • GV-23 The ability of GV-23 to enhance dendritic cell function as antigen presenting cells was determined by mixed lymphocyte reaction (MLR) assay.
  • MLR mixed lymphocyte reaction
  • Purified dendritic cells were culture in medium alone or with GV-23 (20 ug/ml) for 18-20 hours and then stimulated with allogeneic T cells (2 x 10 5 cells/well). After 3 days of incubation, ( 3 H)-thymidine was added. Cells were harvested 1 day later and the uptake of radioactivity was measured.
  • Fig. 13 shows the increase in uptake of ( 3 H)-thymidine with increase in the ratio of dendritic cells to T cells. Significantly higher levels of radioactivity uptake were seen in GV-23 stimulated dendritic cells compared to non-stimulated cells, showing that GV-23 enhances dendritic cell mixed leukocyte reaction.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Pulmonology (AREA)
  • Immunology (AREA)
  • Communicable Diseases (AREA)
  • Dermatology (AREA)
  • Oncology (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des compositions présentes dans Mycobacterium vaccae ou pouvant être dérivées de cette dernière, ainsi que leurs méthodes d'utilisation dans le traitement, la prévention et la détection d'affections, y compris de maladies infectieuses, d'affections immunitaires et du cancer. L'invention concerne également des méthodes d'activation de la réponse immunitaire à un antigène, y compris l'administration d'un filtrat de culture de M. vaccae, de cellules de M. vaccae délipidées, de cellules de M. vaccae dépourvues d'acides mycoliques délipidées et déglycolipidées et de cellules de M. vaccae dépourvues d'acides mycoliques et d'arabinogalactane, délipidées et déglycolipidées.
EP98963665A 1997-12-23 1998-12-23 COMPOSITIONS DERIVEES DE $i(MYCOBACTERIUM VACCAE) ET LEURS METHODES D'UTILISATION Withdrawn EP1044273A2 (fr)

Applications Claiming Priority (13)

Application Number Priority Date Filing Date Title
US156181 1993-11-22
US99662497A 1997-12-23 1997-12-23
US08/997,080 US5968524A (en) 1997-12-23 1997-12-23 Methods and compounds for the treatment of immunologically-mediated psoriasis
US997362 1997-12-23
US08/997,362 US5985287A (en) 1996-08-29 1997-12-23 Compounds and methods for treatment and diagnosis of mycobacterial infections
US997080 1997-12-23
US996624 1997-12-23
US95855 1998-06-11
US09/095,855 US6160093A (en) 1996-08-29 1998-06-11 Compounds and methods for treatment and diagnosis of mycobacterial infections
US15618198A 1998-09-17 1998-09-17
US205426 1998-12-04
US09/205,426 US6406704B1 (en) 1996-08-29 1998-12-04 Compounds and methods for treatment and diagnosis of mycobacterial infections
PCT/NZ1998/000189 WO1999032634A2 (fr) 1997-12-23 1998-12-23 Compositions derivees de mycobacterium vaccae et leurs methodes d'utilisation

Publications (1)

Publication Number Publication Date
EP1044273A2 true EP1044273A2 (fr) 2000-10-18

Family

ID=27557521

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98963665A Withdrawn EP1044273A2 (fr) 1997-12-23 1998-12-23 COMPOSITIONS DERIVEES DE $i(MYCOBACTERIUM VACCAE) ET LEURS METHODES D'UTILISATION

Country Status (15)

Country Link
EP (1) EP1044273A2 (fr)
JP (1) JP2002514385A (fr)
CN (1) CN1294632A (fr)
AU (1) AU746311B2 (fr)
BR (1) BR9814432A (fr)
CA (1) CA2315539A1 (fr)
HU (1) HUP0100352A2 (fr)
ID (1) ID26327A (fr)
IL (1) IL136821A0 (fr)
MX (1) MXPA00006168A (fr)
NO (1) NO20003261L (fr)
NZ (1) NZ505834A (fr)
PL (1) PL341697A1 (fr)
TR (1) TR200001948T2 (fr)
WO (1) WO1999032634A2 (fr)

Families Citing this family (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6569436B1 (en) 1998-10-05 2003-05-27 The Malaghan Institute Of Medical Research Method of using a vaccine
JP4415200B2 (ja) * 1999-01-29 2010-02-17 大塚製薬株式会社 遅発育性抗酸菌ポリペプチド
GB9903539D0 (en) * 1999-02-16 1999-04-07 Stanford Rook Ltd Therapy using M.Vaccae
US6350457B1 (en) * 1999-06-02 2002-02-26 Genesis Research & Development Corporation Limited Methods and compounds for the treatment of immunologically-mediated diseases using mycobacterium vaccae
MXPA01013097A (es) * 1999-07-12 2002-06-04 Genesis Res & Dev Corp Ltd Compuestos para el tratamiento del sistema infeccioso e inmune y metodos para su uso..
US7217554B2 (en) 1999-08-31 2007-05-15 Novozymes A/S Proteases and variants thereof
EP2336331A1 (fr) * 1999-08-31 2011-06-22 Novozymes A/S Nouvelles protéases et variantes associées
CA2381139A1 (fr) * 1999-08-31 2001-03-08 Michael Niederweis Procede pour produire une proteine formant un canal
US6361776B1 (en) * 1999-12-06 2002-03-26 Genesis Research & Development Corp. Ltd. Compounds isolated from M. vaccae and their use in modulation of immune responses
US20030108527A1 (en) * 1999-12-28 2003-06-12 Tsukasa Seya Maturation-promoting agent for immature dendrtic cells
AUPQ761200A0 (en) * 2000-05-19 2000-06-15 Hunter Immunology Limited Compositions and methods for treatment of mucosal infections
US20030104012A1 (en) * 2001-05-11 2003-06-05 Corixa Corporation Vaccines for the treatment of autoimmune disease
US20030147861A1 (en) * 2001-07-26 2003-08-07 Genesis Research And Development Corporation Limited Compounds and methods for the modulation of immune responses
WO2003049751A1 (fr) * 2001-12-10 2003-06-19 Bakulesh Mafatlal Khamar Procede de fabrication d'une composition pharmaceutique utile pour combattre le cancer
GB0303507D0 (en) * 2003-02-14 2003-03-19 Novartis Ag Organic compounds
WO2015104380A1 (fr) * 2014-01-09 2015-07-16 Transgene Sa Fusion d'antigènes mycobactériens hétérooligomères

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8919321D0 (en) * 1989-08-25 1989-10-11 Univ London Treatment of chronic inflammatory conditions
GB9203814D0 (en) * 1992-02-21 1992-04-08 Univ London Treatment of long term auto-immune conditions
US6284255B1 (en) * 1996-08-29 2001-09-04 Genesis Research & Development Corporation Limited Compounds and methods for treatment and diagnosis of mycobacterial infections

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9932634A2 *

Also Published As

Publication number Publication date
HUP0100352A2 (hu) 2001-06-28
JP2002514385A (ja) 2002-05-21
TR200001948T2 (tr) 2001-02-21
WO1999032634A2 (fr) 1999-07-01
BR9814432A (pt) 2000-10-10
CN1294632A (zh) 2001-05-09
AU1893699A (en) 1999-07-12
AU746311B2 (en) 2002-04-18
ID26327A (id) 2000-12-14
MXPA00006168A (es) 2005-02-24
CA2315539A1 (fr) 1999-07-01
NO20003261L (no) 2000-08-22
NO20003261D0 (no) 2000-06-22
WO1999032634A3 (fr) 1999-12-02
IL136821A0 (en) 2001-06-14
PL341697A1 (en) 2001-04-23
NZ505834A (en) 2002-12-20

Similar Documents

Publication Publication Date Title
US6410720B1 (en) Compounds and methods for treatment and diagnosis of mycobacterial infections
EP1523331B1 (fr) Vaccin therapeutique contre la tuberculose
US10519202B2 (en) Tuberculosis TB vaccine to prevent reactivation
AU2006261445B2 (en) Tuberculosis vaccines comprising antigens expressed during the latent infection phase
WO1999032634A2 (fr) Compositions derivees de mycobacterium vaccae et leurs methodes d'utilisation
JP2001501832A (ja) 結核の免疫治療および診断用の化合物および方法
US5985287A (en) Compounds and methods for treatment and diagnosis of mycobacterial infections
WO2005061534A2 (fr) Vaccins antituberculeux ameliores
US6406704B1 (en) Compounds and methods for treatment and diagnosis of mycobacterial infections
US20030007976A1 (en) Methods and compounds for the treatment of immunologically-mediated skin disorders
US6328978B1 (en) Methods for the treatment of immunologically-mediated skin disorders
US7041295B2 (en) Compounds for treatment of infectious and immune system disorders and methods for their use
AU741016B2 (en) Compounds and methods for treatment and diagnosis of mycobacterial infections
CZ20002321A3 (cs) Kompozice získané z Mycobacterium vaccae a způsoby jejich použití
US7192590B2 (en) Compounds for treatment of infectious and immune system disorders and methods for their use
KR20010033132A (ko) 미코박테리움 바카이에서 유도한 조성물 및 이의 이용방법
US20020197265A1 (en) Methods and compounds for the treatment of immunologically - mediated diseases of the respiratory system using mycobacterium vaccae
AU2013206297A1 (en) Tuberculosis vaccines comprising antigens expressed during the latent infection phase
Coler Identification and characterization of novel antigen genes of Mycobacterium tuberculosis

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20000714

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20040406

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040817