AU746311B2 - Compositions derived from (mycobacterium vaccae) and methods for their use - Google Patents

Description

WO 99/32634 PCT/NZ98/00189 COMPOSITIONS DERIVED FROM MYCOBACTERIUM VACCAE AND METHODS FOR THEIR USE Technical Field 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. In particular, 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 S cancer.

Background of the Invention Tuberculosis is a chronic, infectious disease, that is caused by infection with i Mycobacterium tuberculosis 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, Ssignificant morbidity and death may result.

Although 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 WO 99/32634 PCTINZ98/00189 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. Currently, 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 ofMycobacterium bovis bovis). However, 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.

A less well-known mycobacterium that has been used for immunotherapy for tuberculosis and also leprosy, by subcutaneous or intradermal injection, is Mycobacterium vaccae vaccae), which is non-pathogenic in humans. However, there is less information on the efficacy of M. vaccae compared with BCG, and it has not been used widely to vaccinate the general public. M bovis BCG and M vaccae are believed to contain antigenic compounds that are recognised by the immune system of individuals exposed to infection with M. tuberculosis.

Several patents and other publications disclose treatment of various conditions by administering mycobacteria, including M vaccae, or certain mycobacterial fractions. 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.

WO 99/32634 PCT/NZ98/00189 International Patent Publication WO 91/01751 discloses the use of antigenic and/or immunoregulatory material from M. vaccae as an immunoprophylactic to delay and/or prevent the onset of AIDS. International Patent Publication WO 94/06466 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for therapy of HIV infection, with or without AIDS and with or without associated tuberculosis.

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. Among the disorders mentioned in this publication are 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 heatkilled preparations retain vaccine and immunotherapeutic properties. For example, 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 3 WO 99/32634 PCT/NZ98/00189 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.

There are two general classes of compounds which have been isolated from mycobacterial species that exhibit adjuvant properties. The first are water soluble wax D fractions White, I. Bemstock, R.G.S. Johns and E. Lederer, Immunologyv 1:54, 1958; US Patent 4,036,953). The second are muramyl dipeptide-based substances (N-acetyl glucosamine and N-glycolymuramic acid in approximately equimolar amounts) as described in U.S. Patents 3,956,481 and 4,036,953. These compounds differ from the delipidated and deglycolipidated M. vaccae (DD-M vaccae) of the present invention in the following aspects of their composition: 1. They are water-soluble agents, whereas DD-M vaccae is insoluble in aqueous solutions.

2. They consist of a range of small oligomers of the mycobacterial cell wall unit, either extracted from bacteria by various solvents, or digested from the cell wall by an enzyme. In contrast, DD-M vaccae contains highly polymerised cell wall.

3. All protein has been removed from their preparations by digestion with proteolytic enzymes. The only constituents of their preparations are the components of the cell wall peptidoglycan structure, namely alanine, glutamic acid, diaminopimelic acid, N-acetyl glucosamine, and N-glycolylmuramic acid. In contrast, DD-M vaccae contains 50% w/w protein, comprising a number of distinct protein species.

The delivery of vaccines by nasal aerosols to reach lung tissue, or by oral delivery to the gastrointestinal tract has been generally limited to attenuated strains of virus. For example, vaccination against poliovirus has employed oral delivery of attenuated strains of this virus since the development of the Sabin vaccine. Aviron Incorporated and the National Institute of Allergy and Infectious Diseases in the United States have recently reported the WO 99/32634 PCT/NZ98/00189 successful use of an influenza vaccine administered in a nasal spray. In this case, a live attenuated influenza strain provided 93% protection against influenza in young children.

Vaccines consisting of killed viruses or bacteria, or of recombinant proteins have not been delivered by nasal aerosol or oral delivery. There are several reasons for this. There are few reports of successful immunisation resulting in T cell immunity or antibody synthesis employing these agents administered nasally. Further, oral delivery of proteins and killed organisms often results in the development of tolerance, which is exactly the reverse outcome sought in successful immunisation.

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. In patients with pulmonary sarcoidosis, 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-y 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 i WO 99/32634 PCT/NZ98/00189 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 Th2type 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. 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. In 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 WO 99/32634 PCT/NZ98/00189 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.

Another disorder with a similar immune abnormality to asthma is allergic rhinitis.

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. Nonseasonal 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. Like asthma, local eosinophilia in the affected tissues is a major feature of allergic rhinitis. As a result of this inflammation, patients develop sneezing, nasal discharge and congestion. In more severe cases, 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%. 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 WO 99/32634 PCT/NZ98/00189 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 year survival rate for NSCLC is only 5-10%.

The factors leading to the development of lung cancer are complex and multiple.

Environmental and genetic factors interact and cause sequential and incremental abnormalities which lead to uncontrolled cell proliferation, invasion of adjacent tissues and spread to distant sites.

Both cell-mediated and humoral immunity have been shown to be impaired in patients with lung cancer. Radiotherapy and chemotherapy further impair the immune function of patients. Attempts have been made to immunise patients with inactivated tumour cells or tumour antigens to enhance host anti-tumor response. Bacillus Calmette-Guerin (BCG) has been administered into the chest cavity following lung cancer surgery to augment non-specific immunity. Attempts have been made to enhance anti-tumor immunity by giving patients lymphocytes treated ex vivo with interleukin-2. These lymphokine-activated lymphocytes acquire the ability to kill tumor cells. The current immunotherapies for lung cancer are still at a developmental stage and their efficacies yet to be established for the standard management of lung cancer.

In one aspect, this invention deals with treatment of disorders of skin which appear to be associated with factors that influence the balance of thymus-derived immune cells known as Thl and Th2. These T cells are identified by their cytokine secretion phenotype. 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 WO 99/32634 PCT/NZ98/00189 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.

There are two major hypotheses concerning the pathogenesis of psoriasis. 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.

A second hypothesis is that T cells interacting with antigen-presenting cells in skin release pro-inflammatory and keratinocyte-stimulating cytokines (Hancock, G.E. et al., J. Exp.

Med. 168:1395-1402, 1988). Only T cells of genetically predetermined individuals possess the capacity to be activated under such circumstances. 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, Br. J. Dermatol. 110:555-564, 1984).

As the majority of psoriasis patients have limited forms of the disease, topical treatments which include dithranol, tar preparations, corticosteroids and the recently introduced vitamin D3 analogues (calcipotriol, calcitriol) can be used. A minority of psoriasis patients have a more serious condition, for which a number of systemic therapeutic modalities are available. Specific systemic therapies include UVB, PUVA, methotrexate, vitamin A derivatives (acitretin) and immuno-suppressants such as Cyclosporin A. The effectiveness of Cyclosporin and FK-506 for treating psoriasis provides support for the T cell 1 1 WO 99/32634 PCT/NZ98/00189 hypothesis as the prime cause of the disease (Bos, J.D. et al., Lancet II: 1500-1502, 1989; Ackerman, C. et al., J. Invest. Dermatol. 96:536 [abstract], 1991).

Atopic dermatitis is a chronic pruritic inflammatory skin disease 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. In contact dermatitis, 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).

Only a small proportion of the T cells in a lesion of allergic contact dermatitis are specific for the relevant antigen. Activated T cells probably migrate to the sites of inflammation regardless of antigen-specificity. Delayed-type hypersensitivity can only be transferred by T cells (CD4' cells) sharing the MHC class II antigens. The 'response' to contact allergens can be transferred by T cells sharing either MHC class I (CD8' cells) or class II (CD4' cells) molecules (Sunday, M.E. et al., J. Immunol. 125:1601-1605, 1980).

Keratinocytes can produce interleukin-1 which can facilitate the antigen presentation to T cells. The expression of the surface antigen intercellular adhesion molecule-1 (ICAM-1) is induced both on keratinocytes and endothelium by the cytokines tumor necrosis factor (TNF) and interferon-gamma (IFN-y).

I WO 99/32634 PCT/NZ98/00189 If the causes can be identified, removal alone will cure allergic contact dermatitis.

During active inflammation, topical corticosteroids are useful. 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. 14:314-318, 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.

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. This evidence is based on consistent histopathological findings of a lymphocytic T cell infiltrate in and around the hair follicles with increased numbers of Langerhans cells, the observation that alopecia areata will respond to treatment with immunomodulating agents, and that there is a statistically significant association between alopecia areata and a wide variety of autoimmune diseases (Mitchell, A.J. et al., J. Am. Acad. Dermatol. 11:763-775, 1984).

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. 85:569-576, 1985; Gupta, A.K. et al., J. Am.

Acad. Dermatol. 22:242-250, 1990).

f WO 99/32634 PCT/NZ98/00189 The large variety of therapeutic modalities in alopecia areata can be divided into four categories: 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 h 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. 296:13-17, 1988). Basal cell carcinomas are the most common of all cancers.

WO 99/32634 PCT/NZ98/00189 Lesions are usually surgically excised. Alternate treatments include retinoids, 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).

Squamous cell carcinoma (SCC) is the second most common cutaneous malignancy, and its frequency is increasing. There are an increasing number of advanced and metastatic cases related to a number of underlying factors. Currently, metastatic SCC contributes to over 2000 deaths per year in the United States; the 5 year survival rate is 35%, with 90% of the metastases occurring by 3 years. Metastasis almost always occurs at the first lymphatic drainage station. The need for medical therapy for advanced cases is clear. A successful medical therapy for primary SCC of the skin would obviate the need for surgical excision with its potential for scarring and other side effects. This development may be especially desirable for facial lesions.

Because of their antiproliferative and immunomodulating effects in vitro, interferons (IFNs) have also been used in the treatment of melanoma (Kirkwood, J.M. et al., J. Invest.

Dermatol. 95:180S-4S, 1990). Response rates achieved with systemic IFN-a, in either high or low dose, in metastatic melanoma were in the range 5-30%. Recently, encouraging results response) were obtained with a combination of IFN-a and DTIC. Preliminary observations indicate a beneficial effect of IFN-a in an adjuvant setting in patients with high risk melanoma. Despite the low efficacy of IFN monotherapy in metastatic disease, several randomised prospective studies are now being performed with IFNs as an adjuvant or in combination with chemotherapy (McLeod, G.R. et al., J. Invest. Dermatol. 95:185S-7S, 1990; Ho, V.C. et al., J. Invest. Dermatol. 22:159-76, 1990).

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

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-I WO 99/32634 PCT/NZ98/00189 significant economic cost (Kraus, S.J. et al., Review oflnfectious Diseases 2(6):S620-S632, 1990; Frazer, Current Opinion in Immunology 8(4):484-491, 1996).

Summary of the Invention Briefly stated, the present invention provides compositions present in or derived from M. vaccae and methods for their use in the prevention, treatment and diagnosis of diseases, including mycobacterial infection, immune disorders of the respiratory system, and skin disorders. The 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.

In a first aspect, isolated polypeptides derived from Mycobacterium vaccae are provided comprising an immunogenic portion of an antigen, or a variant of such an antigen.

In specific embodiments, the antigen includes an amino acid sequence selected from the group consisting of: 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; 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, 197, 199, 201, 203, 205 and 207; (c) sequences having at least about 75% 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, 197, 199, 201, 203, 205 and 207; and sequences having at least about 95% 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, 1 i WO 99/32634 PCT/NZ98/00189 197, 199, 201, 203, 205 and 207, measured using alignments produced by the computer algorithm BLASTP, as described below.

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. In another aspect, the present invention provides fusion proteins comprising at least one polypeptide of the present invention.

Within other aspects, the present invention provides pharmaceutical compositions 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. In certain embodiments, the non-specific immune response enhancer is selected from the group consisting of: delipidated and deglycolipidated M vaccae cells; inactivated M vaccae cells; delipidated 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.

In yet another aspect, methods are provided 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. In one embodiment, the immune response is a Thl response. In further aspects of this invention, methods are provided for the treatment of a disorder in a patient, comprising administering to the patient a pharmaceutical composition or vaccine of the present invention. In certain embodiments, the disorder is selected from the group consisting of immune disorders, infectious diseases, skin diseases and diseases of the respiratory system. Examples of such diseases include mycobacterial infections, asthma and psoriasis.

In other aspects, the invention provides methods for the treatment of immune disorders, infectious diseases, skin diseases or diseases of the respiratory system, comprising administering a composition comprising inactivated M vaccae cells, delipidated and deglycolipidated M vaccae cells or M. vaccae culture filtrate.

_1 WO 99/32634 PCT/NZ98/00189 Methods for enhancing an immune response to an antigen are also provided. In one embodiment, such methods comprising administering a polypeptide that comprises an immunogenic portion ofa M vaccae antigen which includes a sequence of SEQ ID NO: 89 or 201, or a variant thereof. In a further embodiment, 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.

In further aspects of this invention, methods and diagnostic kits are provided for detecting mycobacterial infection in a patient. In a first embodiment, 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. In a second embodiment, the method comprises contacting a biological sample with at least one of the above polypeptides; and detecting in the sample the presence of 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.

In yet another aspect, 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.

These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

WO 99/32634 PCT/NZ98/00189 Brief Description of the Drawings Figs. 1A and 1B 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 pg of heat-killed M vaccae or 200-100 pg 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 pg of heat-killed M vaccae or 200 pig of DD-M vaccae, respectively, as late as one week prior to challenge with ovalbumin. Fig. 2E shows the percentage of eosinophils in mice immunized either intranasally or subcutaneously 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 jIg 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 ofM. 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).

WO 99/32634 PCT/NZ98/00189 Fig. 7A(i) (iv) illustrate the non-specific immune amplifying effects of 10 Pg, 100 utg and Img autoclaved M vaccae and 75 pg 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. 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 aPT cells, y6T 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-I TNF-a, IL-12 and IFN-y, 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-13, TNF-a and IL-12, respectively, by human PBMC.

Figs. 11A-D illustrate the stimulation of IL-l p, TNF-c, IL-12 and IFN-y 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.

WO 99/32634 PCT/NZ98/00189 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.

Detailed Description of the Invention As noted above, 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 incorporated 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. tuberculosis and M bovis BCG.

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 .iI. I_ i_ -i i WO 99/32634 PCT/NZ98/00189 antigen to elicit a specific immune response and as an adjuvant to enhance immune responses to unrelated proteins.

Certain 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. These different classes of immune attack (T cell or B cell) are controlled by different subpopulations of CD4 T cells, commonly referred to as 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.

The two types of 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-y 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-y 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. In particular, the majority of T-cell clones from atopic human lymphocytes resemble the murine Th2 cell that produces IL-4, whereas very few clones produce IFN-y.

Therefore, the selective expression of the Th2 subset with subsequent production of IL-4 and decreased levels of IFN-y-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 WO 99/32634 PCT/NZ98/00189 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. As shown below, 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.

In one aspect, the present invention provides methods for the immunotherapy of respiratory and/or lung disorders, including tuberculosis, sarcoidosis, asthma, allergic rhinitis and lung cancers, in a patient to enhance Thl-type immune responses. In one embodiment, the compositions are delivered directly to the mucosal surfaces of airways leading to and/or within the lungs. However, 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. As illustrated below, administration of such 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.

As used herein the term "respiratory system" refers to the lungs, nasal passageways, trachea and bronchial passageways.

As used herein the term "airways leading to or located in the lung" includes the nasal passageways, mouth, tonsil tissue, trachea and bronchial passageways.

As used herein, 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 I- i WO 99/32634 PCT/NZ98/00189 words, the inventive methods may be employed to induce protective immunity for the prevention or treatment of disease.

In another aspect, 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. As detailed below, multiple administrations of such compositions, preferably by intradermal injection, have been shown to be highly effective in the treatment of psoriasis.

As used herein the term "inactivated M vaccae" refers to M vaccae that have either been killed by means of heat, as detailed below in Example 7, or subjected to radiation, such as 6 Cobalt at a dose of 2.5 megarads. As used herein, 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).

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.

In yet a further aspect, 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. In specific embodiments, 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.

WO 99/32634 PCTINZ98/00189 As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full length proteins antigens), wherein the amino acid residues are linked by covalent peptide bonds. Thus, 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. As detailed below, 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," as used herein, refers to the ability to elicit an immune response in a patient, such as a human, or in a biological sample. In particular, immunogenic antigens are capable of stimulating cell proliferation, interleukin-12 production or interferon-y 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 cell proliferation, interferon-y production or interleukin-12 production) that is substantially similar to that generated by the full-length antigen. In other words, an immunogenic portion of an antigen may generate at least about 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 WO 99/32634 PCT/NZ98/00189 about 65% and most preferably about 100%, of the interferon-y 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-y 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.

The term "polynucleotide(s)," as used herein, 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.

The compositions and methods of this invention also encompass variants of the above polypeptides and polynucleotides. As used herein, the term "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.

WO 99/32634 PCT/NZ98/00189 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 BLASTN algorithm version 2.0.4 [Feb-24-1998], 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 BLAST family of algorithms, including BLASTN and BLASTP, is described at NCBI's website at URL http://www.ncbi.nlm.nih.gov/BLAST/newblast.html and in the publication of Altschul, Stephen et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402. 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 85:2444-2448 (1988) and W.R. Pearson, "Rapid and Sensitive Sequence Comparison with FASTP and FASTA," Methods in Enzymology 183:63-98 (1990).

The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity: Unix running command: blastall -p blastn -d embldb -e 10 -G 1 -E 1 -r 2 -v 50 -b 50 -i queryseq o results; and parameter default values: -p Program Name [String] -d Database [String] -e Expectation value [Real] -G Cost to open a gap (zero invokes default behavior) [Integer] WO 99/32634 PCT/NZ98/00189 -E Cost to extend a gap (zero invokes default behavior) [Integer] -r Reward for a nucleotide match (blastn only) [Integer] -v Number of one-line descriptions [Integer] -b Number of alignments to show [Integer] -i Query File [File In] -o BLAST report Output File [File Out] Optional For BLASTP the following running parameters are preferred: blastall -p blastp -d swissprotdb -e 10 -G 1 -E 1 -v 50 -b 50 -i queryseq -o results -p Program Name [String] -d Database [String] -e Expectation value [Real] -G Cost to open a gap (zero invokes default behavior) [Integer] -E Cost to extend a gap (zero invokes default behavior) [Integer] -v Number of one-line descriptions [Integer] -b Number of alignments to show [Integer] -I Query File [File In] -o BLAST report Output File [File Out] Optional 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 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, indicates true similarity. For example, an E value of 0.1 assigned to a hit is interpreted 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 WO 99/32634 PCT/NZ98/00189 similar score simply by chance. By this criterion, the aligned and matched portions of the sequences then have a probability of 90% of being the same. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in the EMBL database is 1% or less using the BLASTN or FASTA algorithm.

According to one embodiment, "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.

According to a preferred embodiment, 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% 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. As used herein, "stringent conditions" refers to prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65 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.

Portions and other variants of 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. For example, 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, JAm. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied WO 99/32634 PCT/NZ98/00189 BioSystems, Inc. (Foster City, CA), and may be operated according to the manufacturer's instructions. 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 poly- His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

In general, M vaccae antigens, and DNA sequences encoding such antigens, may be prepared using any of a variety of procedures. For example, 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. Preferably, the host cells employed are E. coli, mycobacteria, insect, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner 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

I

;I i- WO 99/32634 PCTINZ98/00189 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 rabbit or monkey) raised specifically against M. vaccae antigens.

Regardless of the method of preparation, the antigens described herein have the ability to induce an immunogenic response. More specifically, the antigens have the ability to induce cell proliferation and/or cytokine production (for example, interferon-y and/or interleukin-12 production) in T cells, NK cells, B cells or macrophages derived from an M. tuberculosisimmune individual. 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 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.

For example, a preparation of peripheral blood mononuclear cells (PBMCs) may be employed without further separation of component cells. PBMCs may be prepared, for example, using density centrifugation through Ficoll TM (Winthrop Laboratories, NY). T cells for use in the assays described herein may be purified directly from PBMCs. Alternatively, 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 proteinspecific T cells, resulting in a line composed solely of such cells. These cells may then be I WO 99/32634 PCT/NZ98/00189 cloned and tested with individual proteins, using methods well known in the art, to more accurately define individual T cell specificity.

In general, regardless of the method of preparation, the polypeptides disclosed herein are prepared in an isolated, substantially pure, form. Preferably, the polypeptides are at least about 80% pure, more preferably at least about 90% pure and most preferably at least about 99% pure. In certain preferred embodiments, described in detail below, the substantially pure polypeptides are incorporated 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 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 incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: their ability to adopt a flexible extended conformation; their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide _I r WO 99/32634 PCT/NZ98/00189 linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 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.

As detailed below, the inventors have demonstrated that heat-killed 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. Furthermore, the inventors have demonstrated similarities between the immunological properties of the inventive M vaccae protein GV-23 and those of two known Thl-inducing adjuvants. GV-23 may thus be employed in the treatment of diseases that involve enhancing a Thl immune response. Examples of such diseases include allergic diseases (for example, asthma and eczema) autoimmune diseases (for example, systemic lupus erythematosus) and infectious diseases (for example, tuberculosis and leprosy). In addition, GV-23 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.

For use in the inventive therapeutic methods, 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 WO 99/32634 PCT/NZ98/00189 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, incorporated into a fusion protein or present within a separate polypeptide.

Alternatively, 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. In such vaccines, 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. In a preferred embodiment, the DNA may be introduced using a viral expression system vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic, or defective, replication competent virus. Techniques for incorporating 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. For example, 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.

1 I L i~ WO 99/32634 PCT/NZ98/00189 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. In general, the pharmaceutical compositions and vaccines may be administered by injection intradermal, intramuscular, intravenous or subcutaneous), intranasally by aspiration) or orally. Between 1 and 3 doses may be administered for a 1-36 week period. Preferably, 3 doses are administered, at intervals of 3-4 months, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. 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. In general, the amount of polypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 p.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.

In one embodiment, 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. For example, 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. In other embodiments, 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. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a lipid, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, Smagnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable j_ WO 99/32634 PCT/NZ98/00189 biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.

Any of a variety of 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.

In another aspect, this invention provides methods for using one or more of the inventive polypeptides to diagnose tuberculosis using a skin test. As used herein, 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. Preferably, 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 the immunogenic portion of the polypeptide employed, or a variant thereof). The response may be measured visually, using a ruler. In general, a response that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter, is a positive response, indicative of tuberculosis infection.

For use in a skin test, the polypeptides of the present invention are preferably formulated, as pharmaceutical compositions containing a polypeptide and a physiologically acceptable carrier, as described above. Such compositions typically contain one or more of the above polypeptides in an amount ranging from about 1 utg to about 100 ig, preferably from about 10 jtg to about 50 jig in a volume of 0.1 ml. Preferably, the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween 8 0TM.

WO 99/32634 PCT/NZ98/00189 In a preferred embodiment, 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. In general, a 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.

In another aspect, methods are provided for detecting mycobacterial infection in a biological sample, using one or more of the inventive polypeptides, either alone or in combination. In embodiments in which multiple polypeptides are employed, polypeptides other than those specifically described herein, such as the 38 kDa antigen described above, may be included. As used herein, a "biological sample" is any antibody-containing sample obtained from a patient. Preferably, the sample is whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid or urine. More preferably, the sample is a blood, serum or plasma sample obtained from a patient or a blood supply. The polypeptide(s) are used in an assay, as described below, to determine the presence or absence of antibodies to the polypeptide(s) in the sample, relative to a predetermined cut-off value. The presence of such antibodies indicates the presence of mycobacterial infection.

In embodiments in which more than one polypeptide is employed, the polypeptides used are preferably complementary 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.

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WO 99/32634 PCT/NZ98/00189 A variety of assay formats employing one or more polypeptides to detect antibodies in a sample are well known in the art. See, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In a preferred embodiment, 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 in a semi-competitive assay). Alternatively, 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. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, 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. In the context of the present invention, the term "bound" refers to both noncovalent association, such as adsorption, 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 adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption 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. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of polypeptide ranging from about 10 ng to about 1 utg, and preferably about 100 ng, is sufficient to bind an adequate amount of antigen.

i WO 99/32634 PCT/NZ98/00189 Covalent attachment of polypeptide to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the polypeptide. For example, the 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, Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).

In certain embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA).

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.

More specifically, once the polypeptide is immobilized on the support as described above, 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. In general, an appropriate contact time, or incubation time, is that period of time that is sufficient to detect the presence of antibody within a M tuberculosis-infected sample. Preferably, the contact time is sufficient to achieve a level of binding that is at least 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 minutes is generally sufficient.

L- i- WO 99/32634 PCT/NZ98/00189 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. Preferably, 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. Preferred 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 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.

To determine the presence or absence of anti-mycobacterial antibodies in the sample, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value is the average mean signal obtained when the immobilized antigen is incubated with samples from an uninfected patient. In an alternate

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WO 99/32634 PCTNZ98/00189 preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Sciencefor 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. In the flow-through test, antibodies within the sample bind to the immobilized polypeptide as the sample passes through the membrane. A detection reagent protein A-colloidal gold) 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. In 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 antimycobacterial antibodies in the sample. Typically, the 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. In general, 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. Preferably, the amount of polypeptide immobilized on the membrane ranges from about 25 ng to about 1 pg, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount one drop) of patient serum or blood.

Numerous other assay protocols exist that are suitable for use with the polypeptides of the present invention. The above descriptions are intended to be exemplary only.

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, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogen comprising the antigenic ~L .i.

i WO 99/32634 PCT/NZ98/00189 polypeptide is initially injected into any of a wide variety of mammals mice, rats, rabbits, sheep and goats). The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating 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.

6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity 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 supematants of the resulting hybridoma colonies. In addition, 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. For example, primers comprising at least 10 contiguous oligonucleotides of an inventive polynucleotide may be used in polymerase chain reaction (PCR) based tests. Similarly, probes comprising at least 18 contiguous oligonucleotides of an inventive polynucleotide may ~1 iI L;~ WO 99/32634 PCT/NZ98/00189 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.

The word "about," when used in this application with reference to a percentage by weight composition, contemplates a variance of up to 10 percentage units from the stated percentage. When used in reference to percentage identity or percentage probability, the word "about" contemplates a variance of up to one percentage unit from the stated percentage.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1 EFFECT OF IMMUNIZATION OF MICE WITH M VACCAE ON TUBERCULOSIS 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, g/l; tryptone, 5 g/l; glucose, 1 g/l) at 37 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 10'1 M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120 The culture filtrate was passaged through a 0.45 pm filter into sterile bottles.

As shown in Fig.lA, when mice were immunized with 1 mg, 100 pg or 10 ig 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. In this example, spleen, I WO 99/32634 PCT/NZ98/00189 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. Immunization of mice with heat-killed M tuberculosis H37Rv had no significant protective effects on mice subsequently infected with live M. tuberculosis H37Rv.

Fig.lB shows that when mice were immunized with 100 gig of M vaccae culture filtrate, and infected three weeks later with 5x105 CFU of M tuberculosis H37Rv, significant protection was also seen. When spleen, liver and lung tissue was harvested from mice three weeks after infection, and live bacilli numbers (CFU) determined, a 1-2 log reduction in numbers, as compared to non-immunized control mice, was observed.

EXAMPLE 2 EFFECT OF INTRADERMAL AND INTRA-LUNG ROUTES OF IMMUNISATION WITH M VACCAE ON TUBERCULOSIS IN CYNOMOLGOUS MONKEYS 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. Prior to immunisation, all monkeys were weighed (Wt kg), body temperature was measured (temp), and a blood sample taken for determination of erythrocyte sedimentation rate (ESR mm/hr) and lymphocyte proliferation (LPA) to an in vitro challenge WO 99/32634 PCT/NZ98/00189 with purified protein (PPD) prepared from Mycobacterium bovis. Both ESR and LPA have been used as indicators of inflammatory T cell responses. At day 33 post-immunisation these measurements were repeated. At day 34, all monkeys received a second immunisation using the same amount of M vaccae and route of immunisation as the initial immunisation. On day 62, body weight, temperature, ESR and LPA to PPD were measured, then all monkeys were infected with 103 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 1 COMPARISON OF INTRADERMAL AND INTRALUNG ROUTES OF IMMUNISATION Group Identification Amount of Route of Number Number of Immunogen Immunisation Monkey 1 S3101-E 0 (Controls) 3144-B 0 2 4080-B 500 jlg intradermal (Immunised 3586-B 500 tg intradermal with heat-killed M vaccae) 3 3534-C 500 tg intralung (Immunised 3160-A 500 ig intralung with heat-killed M. vaccae) 4 (Immunised 3564-B 100 g intradermal with culture filtrate) 3815-B 100 Rg intradermal (Immunised 4425-A 100 g intralung with culture filtrate) 2779-D 100 gg intralung _r WO 99/32634 PCT/NZ98/00189 The results of these studies are provided below in Tables 2A-E and are summarized below: 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 xrays 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 2B The two monkeys immunised twice with 500 jtg M vaccae intradermally showed no sign of lung disease 84 days post-infection with M tuberculosis. The LPA responses to PPD indicated there was immune reactivity to M tuberculosis, and both animals continued to gain weight, a consistent indication of a lack of disease.

Table 2C The two monkeys immunised twice with 500 jig 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.

Immunisation twice with 500 jig of whole M vaccae has consistently shown protective effects against subsequent infection with live M tuberculosis. The data presented in Tables 2D and 2E show the effects of immunisation with 100 pg of M vaccae culture filtrate.

Monkeys immunised intradermally showed signs of developing disease 84 days postinfection, while in those immunised intralung, one animal showed disease after 56 days and one animal showed disease 84 days post-infection. This was a significant delay in disease onset indicating that the immunisation process had resulted in some protective immunity.

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-i WO 99/32634 WO 9932634PCT/NZ98/OOI 89 TABLE 2A CONTROL MONKEYS ESR LPA LPA.

ID# Days Wt.Kgs Temp. 'Mm/hr PPD10 PPD1 X-Ray Remarks S3101E 0 2.17 1 37.0 1 0 10.47 1. 1 Negative 34 1.88 37.3 ND 0.85 1.4 FND 62 2.02 36.0 ND 1.3 1.5 ND -*Time of Infection 28 2.09 38.0 J 2 1.31 3.7 Positive 56 1.92 37.2 20 5.6 9.1 Positive 84 1.81 37.5 8 5.6 Positive 121 DIED LPA LPA.

JD# Dys.t.g Temp, ESR PPD -PPD. X-Ray Remarks _Mm/hr -l:1pg- 3144-B 1 01 2.05 1 36.7 1 0 0.87 1.8 Negative 34 1 1.861 37.6 1 ND 2.2 1.4 ND 621 1.87 1 36.5[ ND 1.6 1.6 ND ->Time of Infection 28 2.10 38.0 0 12J 8.7 Positive 56 1.961 37.6 0 29. 21.1 Positive 84 1.82 J 37.3 45.3 23.4 Positive 131 DIED ND Not Done WO 99/32634 WO 9932634PCT/NZ98/O1 89 TABLE 2B MONKEYS IMMUNISED WITH WHOLE HEAT-KILLED M. VACCAE (500 tig)

INTRADERMAL

LPA LPA: IDN Days WtKgs Temp. ESR PPD PPD X-Ray Remarks 10Aig Ij4g 4080-B 1 01 2.05 1 37.1 1 1 1.1 1 0.77 1Negative 341 1.971 38.0 1 ND 1 1.71 1.4 1ND 621 2.091 36.7 1_ ND 1 1.5 1.5 ND -Time of Infection 28 2.15 37.6 J 0 2.6 2.1 Negative 56 2.17 37.61 0 8.2 7.6 Negative 84 2.25 37.3 j 0 3.8 2.8 jNegative 178 DIED 'LPA: LPA; D#Days WL g Temp..', ESR PPD:: PPD X-Ray:Remarks 1O119____ 3586-B 0. 2.29. 37.0. 0 1.1 1.4 Negative 341 2.221 38.0 1 ND 1 1.91 1.6 ND 621 2.391 36.01 ND 1 1.3 1 1.6 ND -*Time of Infection 28 2.31 38.2 0 J 3.2 1 2.6 Negative 56 2.32 37.2 0 J 7.8 4.2 Negative 84 2.81 37.4 0 J3.4 1.8 Negative 197. DIED ND Not Done WO 99/32634 PTN9/08 PCT/NZ98/00189 TABLE 2C MONKEYS IMMUNISED WITH WHOLE HEAT-KILLED M. VACCAE (500 gg)

INTRALUNG

1DM Day WtKg Tep. SR LPA LPA ID# Das t.gs em. SR PPD PPD X-Ray Remarks mm/hr lOgg.In 3534-C 0 12.15 1 36.8 1 0 1 1.71 1.3 Negative 1341 2.001 37.8 1ND 1 4.41 1.4 ND 162 2.13 1 36.41 ND I .3.21 1.9 ND -~Time of Infection____ 28 2.38 J 37.7 J 0o 1.9 2.6 Negative 56 2.421 37.81 01 5.3 4.7 Negative 84 2.46 J 37.1 1 i 3.1 3.2 Negative 210, No sign of lung disease Negative L PA LPA- I# Days. Wt.Kgs Tep ESR PPD PPD X-Ray :Remark ~mm/hr lOpg 3160-A 0. 2.17 37.3 0 1.2. 0.79 Negative 34 1.98 37.1 ND 3.9 7.8 ND 621 2.17 36.9 ND 1.71 2.4 ND -Time of Infection 2.38 37.7 0 2.6 Negative 2.42 37.8 0 5. 4.7 Negative 2.46 37.1 1 3.1 3.2 Negative 210 Stable lung disease Positive ND Not Done WO 99/32634 WO 9932634PCT/NZ98/OO1 89 TABLE 2D MONKEYS IMMUNISED WITH CULTURE FILTRATE (100 g.g)

INTRADERMAL

-LPA LPA IM# Days Wt.Kgs Tem p. '.ESR PPD: PPD X-Ray Remarks mm/hr 1ljig ljig 3564-B 0 12.40 37.2 0 1.41 1.4 Negative 34 2.42 38.1 ND 3.3 2.7 ND 62 2.31 37.1 ND 3.1 3.4 ND ->Time of Infection 28 2.41 38.6 13 J 24 13.6 Negative 56 I2.38 38.61 01 12.7 12.0 Negative 84 2.41 38.6 2J 21.1 11.8 Positive 140._ Died .LPA

LPA

ID Dy W.~g em. ESR PD PPD X-Ray Remarks m /r O±1g 3815-B 0. 2.31 36.3 0 1.0, 1.4 Negative 34 2.36 38.2 ND 1.9 2.0 ND 621 2.361 36.41 ND- 3.71 2.8 ND -Time of Infection 28 2.45 37.8 J 0 2.1 3.3 JNegative 56 j 2.28 37.3 4 8.0 5.6 jNegative 84 J 2.32 37. 0 1.9 2.21 Positive Positive ND =Not Done WO 99/32634 WO 9932634PCT/NZ98/00189 TABLE 2E MONKEYS IMMUNISED WITH CULTURE FILTRATE (100 .Lg)

INTRALUNG

LPA LPA 1D# Days WtKgs Temp ESR PPD PPD X-Ray Remarks mm/hr I0ig I 4425-A 1 01 2.05 1 36.0 1 0 0.35 1.2 Negative 34 2.0 376IDn. 2.4 ND 62 2.11 3 7.6 r ND 2.21 1.6 ND -+Time of Infection 28 2.21 38.0 0 8.4 4.1 Negative 561 2.11 37.61 0 23.91 17.7 Negative 841 2.18 37.9 0 8.41 7.21 Positive 210. Stable lung disease Positive LPJA. LPA DasWt.Kgs Temp.,- R P PPD X-Ray Remarks.

2779-D 0. 2,56 38.6 2 1.9. 1.4 Negative 28 2.55 37.9 ND 0.78 1.1 ND 561 2.691 3 8.4 -ND 1.31 1.5 ND Time of Infection 1_ 961 24 N1N Died ND Not Done WO 99/32634 PCT/NZ98/00189 EXAMPLE 3 EFFECT OF IMMUNISATION WITH M VACCAE ON ASTHMA IN MICE This example demonstrates that both heat-killed M vaccae and DD-M vaccae, when administered to mice via the intranasal route, are able to inhibit the development of an allergic immune response in the lungs. This was demonstrated in a mouse model of the asthma-like allergen specific lung disease. The severity of this allergic disease is reflected in the large numbers of eosinophils that accumulate in the lungs.

C57BL/6J mice were given 2 pg ovalbumin in 100 ul alum adjuvant by the intraperitoneal route at time 0 and 14 days, and subsequently given 100 pg ovalbumin in 50 pl phosphate buffered saline (PBS) by the intranasal route on day 28. 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.

As shown in Figs. 2A and B, groups of seven mice administered either 10 or 1000 jpg of heat-killed M. vaccae (Fig. 2A), or 10 100 or 200 gg 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 105 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 gg of heat-killed M. vaccae (Fig. 2C) or 200 ug 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.

As shown in Fig. 2E, immunisation with either 1 mg of heat-killed M vaccae or 200 p.g of DD-M vaccae, given either intranasally or subcutaneously reduced lung -I l_ WO 99/32634 PCT/NZ98/00189 eosinophilia following challenge with ovalbumin when compared to control animals given PBS. In the same experiment, 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.

DD-M.vaccae depleted of mycolic acids and arabinogalactan Mycolic acids were depleted from DD-M.vaccae by treatment with potassium hydroxide KOH) in ethanol for 48 hours at 37°C. Mycolic acid depleted DD-M.vaccae cells were then washed with ethanol and ether and dried. Arabinogalactans were depleted from the KOH treated DD-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. As shown in Table 3, both mycolate depleted DD-M.vaccae as well as mycolic acid and arabinogalactan depleted DD-M.vaccae, given intranasally to ovalbumin sensitized mice reduced the accumulation of eosinophils in the bronchoalveolar lavage fluid following challenge with ovalbumin.

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

In addition, serum samples were collected from mice in the experiment shown in Fig.

2E and antibodies to ovalbumin was measured by standard enzyme-linked immunoassay (EIA). As shown in Table 3A below, sera from mice infected with BCG had higher levels of ovalbumin specific IgGI than sera from PBS controls. In contrast, mice II; i WO 99/32634 PCT/NZ98/00189 immunized with M vaccae or DD-M vaccae had similar or lower levels of ovalbuminspecific IgGl. As IgG1 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.

TABLE 3 DECREASED LUNG EOSINOPHILIA IN MICE TREATED WITH MYCOLIC ACID DEPLETED DD-M VACCAE OR MYCOLIC ACID AND ARABINOGALACTAN DEPLETED DD-M. VACCAE.

Treatment Group Eosinophils in BAL Mean S.E.M.

PBS 58.8 8.4 Mycolic acid depleted DD-M vaccae 21.8 17.4 Mycolic acid and arabinogalactan 16.8 0.3 depleted DD-M. vaccae Note: At least 7 mice per group.

TABLE 3A LOW ANTIGEN-SPECIFIC IgG SERUM LEVELS IN MICE IMMUNIZED WITH HEAT-KILLED M VACCAE OR DD-M VACCAE Treatment Group Serum IgG Mean SEM M.vaccae i.n. 185.00 8.3 M. vaccae s.c. 113.64 DD-M vaccae i.n. 96.00 8.1 DD-M vaccae s.c. 110.00 4.1 BCG, Pasteur 337.00 27.2 BCG, Connaught 248.00 46.1 PBS 177.14 11.4 I~ i~ WO 99/32634 PCT/NZ98/00189 Note: Ovalbumin-specific IgG1 was detected using anti-mouse IgGI (Serotec). Group means are expressed as the reciprocal of the EU50 end point titre.

EXAMPLE 4 EFFECT OF IMMUNIZING MICE WITH M VACCAE, DD-M VACCAE OR RECOMBINANT M VACCAE PROTEINS ON TUBERCULOSIS This example illustrates the effect of immunization with heat-killed M.vaccae, DD- 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) DD-M.vaccae (50 ug); d) A pool of recombinant proteins containing 15 ug of each of GV4P, GV7, GV9, GV27B, GV33 protein (prepared as described below); and e) Heat-killed M.vaccae plus the pool of recombinant proteins Three weeks after the last intraperitoneal immunization, the mice were infected with X 105 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.

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). As shown in Fig. 3A, mice immunized with heat-killed M.vaccae or DD-Mvaccae showed a mean reduction of >1 or logs CFU, respectively.

53 WO 99/32634 PCT/NZ98/00189 As shown in Fig. 3B, 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. However, when GV4P, GV7, GV9, GV27B 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-Mvaccae and recombinant proteins may be developed as vaccines against tuberculosis.

EXAMPLE EFFECT OF INTRADERMAL INJECTION OF HEAT-KILLED MYCOBACTERIUM VACCAE ON PSORIASIS IN HUMAN PATIENTS This example illustrates the effect of two intradermal injections of heat-killed Mycobacterium vaccae on psoriasis in human patients.

M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, tryptone, 5g/l; glucose, 1 g/l) at 37 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 1010 M vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120 OC and stored frozen at -20 oC. 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 oC and 0.2 ml aliquoted under sterile conditions into vials for use in patients.

Twenty-four volunteer psoriatic patients, male and female, 15-61 years old with no other systemic diseases were admitted to treatment. Pregnant patients were not included. The patients had PASI scores of 12-35. The PASI score is a measure of the location, size and degree of skin scaling in psoriatic lesions on the body. A PASI score of above 12 reflects 54 I II~~ ;i WO 99/32634 PCT/NZ98/00189 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 24 patients were then injected intradermally with 0.1 ml M. vaccae (equivalent to 500 This was followed three weeks later with a second intradermal injection with the same dose of M. vaccae (500 tg). Psoriasis was evaluated from four weeks before the first injection of heat-killed M. vaccae to twelve weeks after the first injection as follows: A. The PASI scores were determined at 0, 3, 6 and 12 weeks; B. Patient questionnaires were completed at 0, 3, 6 and 12 weeks; and C. Psoriatic lesions and each patient were photographed at 0, 3, 6, 9 and 12 weeks.

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

i~ WO 99/32634 WO 9932634PCT/NZ98/OO1 89 TABLE 4 Patient Data in the Study of the Effect of M. vaccae in Psoriasis Code Duration of No. Patient Age/Sex Disorder Admission PASI Score PS-01 D.C. 49/F 30 years 28.8 PS-002 E.S. 41/F 4 months 19.2 PS-003 M.G. 24/F 8 months 18.5 PS-004 D.B. 54/M 2 years 12.2 PS-005 C.E. 58/F 3 months 30.5 PS-006 M.G. 18/F 3 years 15.0 PS-007 L.M. 27/M 3 years 19.0 PS-008 C.C 21/F 1 month 12.2 PS-009 E.G 42/F 5 months 12.6 PS-010 J.G 28/M 7 years 19.4 IlI J.U 39/M 1 year 15.5 PS-012 C.S 471M 3 years 30.9 PS-013 H.B 44/M 10 years 30.4 PS-014 N.J 41/M 17 years 26.7 15 J.T 61/F 15 years 19.5 PS-016 L.P 44/M 5 years 30.2 PS-017 E.N 45/M 5 years 19.5 PS-018 E.L 28/F 19 years 16.0 PS-019 B.A 38/M 17 years 12.3 PS-020 P.P 58/F 1 year 13.6 PS-021 L.1 27/F 8 months 22.0 PS-022 A.C 20/F 7 months 26.5 PS-023 C.A 61/F 10 years 12.6 PS-024 FT 39/M 15 years 29.5 WO 99/32634 PCT/NZ98/00189 All patients demonstrated a non-ulcerated, localised erythematous soft indurated reaction at the injection site. No side effects were noted, or complained of by the patients.

The data shown in Table 5, below, are the measured skin reactions at the injection site, 48 hours, 72 hours and 7 days after the first and second injections of heat-killed M. vaccae. The data shown in Table 6, below, are the PASI scores of the patients at the time of the first injection ofM. vaccae (Day 0) and 3, 6, 9, 12 and 24 weeks later.

It can clearly be seen that, by week 9 after the first injection of M vaccae, 16 of 24 patients showed a significant improvement in PASI scores. Seven of fourteen patients who have completed 24 weeks of follow-up remained stable with no clinical sign of redevelopment of severe disease. These results demonstrate the effectiveness of multiple intradermal injections of inactivated M. vaccae in the treatment of psoriasis. PASI scores below 10 reflect widespread healing of lesions. Histopathology of skin biopsies indicated that normal skin structure is being restored. Only one of the first seven patients who have completed 28 weeks follow-up has had a relapse.

WO 99/32634 WO 9932634PCTNZ98/OOI 89 TABLE Skin Reaction Measurements in Millimeter Code No.

Time of Measurement First Injection Second Injection 48 hours 72 hours 7 days 48 hours 72 hours 7 days PS-001 12x10 12x10 10x8 15x14 15x14- lOxlO PS-002 18x14 20A18 18x14 16x12 18x12 15x10 PS-003 lOxlO 14x10 I10x8 15x12 15x10 lOXlO PS-004 14x12 22x18 20x15 20x20 20x18 14x10 PS-005 lOxlO 13x10 DNR DNR DNR DNR PS-006 10x8 lOxlO 6x4 12x10 15x15 10Ox6 PS-007 15x15 18x16 12x10 15x13 15x12 12x10 PS-008 18x18 13x12 12x10 18x17 15x10 15x10 PS-009 13x13 18x1 5 12x8 15x13 12x12 1207 10 13x11I 15x15 8x 12x12 12x12 PS-01i1 17x13 14x12 12x11I 12x10 12x10 12x10 PS-012 17x12 15x12 9x9 lOxlO 10Ox6 8x6 PS-013 18xl 1 15A1I 15x10 15x10 15x13 14x6 PS-014 15x12 15x11I 15x10 13x12 14x10 PS-015 15x12 16x12 15x10 7x6 14x12 6A4 PS-016 6x5 6x6 6x5 Wx 9x8 9x6 17 20x15 15x14 14x10 15x15 17x16 DNR PS-018 14x10 10x8 10x8 12x12 lOXlO lOXlO P5-019 lOXlO 14x12 10x8 DNR 15x14 15x14 PS-020 15x12 15x15 12x15 15x15 14x12 13x12 PS-021 15x12 15x12 7x4 llxlO 11X10 1 1x8 PS-022 12x10 10x8 I10x8 15x12 13x10 10x8 PS-023 13x12 14x12 lOxlO 17x17 15x15 DNR WO 99/32634 WO 9932634PCTNZ98OOI 89 DNR TABLE 6 Clinical Status of Patients after Injection of M. vaccae (PASI Scores) Code No. Day 0 Week 3 Week 6 Week 9 Week 12 Week 24 PS-001 28.8 14.5 10.7 2.2 0.7 0 PS-002 19.2 14.6 13.6 10.9 6.2 0.6 PS-003 18.5 17.2 10.5 2.7 1.6 0 PS-004 12.2 13.4 12.7 7.0 1.8 0.2 PS-005* 30.5 DNR 18.7 DNR DNR 0 PS-006 15.0 16.8 16.4 2.7 2.1 PS-007 19.0 15.7 11.6 5.6 2.2 0 PS-008 12.2 .11.6 11.2 11.2 5.6 0 PS-009 12.6 13.4 13.9 14.4 15.3 13.0 PS-010 18.2 16.0 19.4 17.2 16.9 19.3 PS-oil1 17.2 16.9 16.7 16.5 16.5 15.5 PS-0 12 30.9 36.4 29.7 39.8** PS-013 19.5 19.2 18.9 17.8 14.7 17.8 PS-014 26.7 14.7 7.4 5.8 9.9 24.4*** PS-015 30.4 29.5 28.6 28.5 28.2 24.3 PS-016 30.2 16.8 5.7 3.2 0.8 3.3 PS-017 12.3 12.6 12.6 12.6 8.2 8.7 PS-018 16.0 13.6 13.4 13.4 13.2 12.8 PS-019 19.5 11.6 7.0 DNR DNR DNR PS-020 13.6 13.5 12.4 12.7 12.4 4.4 WO 99/32634 PCT/NZ98/00189 Patient PS-005 received only one dose of autoclaved Mvaccae.

Patient PS-012 removed from trial, drug (penicillin) induced dermatitis Patient PS-014 was revaccinated DNR Did not report Patients treated with M.vaccae may achieve remission (PASI score The remission or improvement of PASI score may be long lasting. By example, 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 was re-vaccinated with injections of Mvaccae and subsequently improved with PASI falling from 17.8 (Week 60) to 0.8 (week 84). Thus patients may benefit from repeated treatment.

EXAMPLE 6 EFFECT OF INTRADERMAL INJECTION OF DD-M. VACCAE ON PSORIASIS IN HUMAN PATIENTS This example illustrates the effect of two intradermal injections of DD-M vaccae on psoriasis.

Seven volunteer psoriatic patients, male and female, 18-45 years old with no other systemic diseases were admitted to treatment. Pregnant patients were not included. The patients had PASI scores of 12-24. As discussed above, the PASI score is a measure of the location, size and degree of skin scaling in psoriatic lesions on the body. A PASI score of WO 99/32634 PCT/NZ98/00189 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 tg). This was followed three weeks later with a second intradermal injection with the same dose of DD-M vaccae (100 p.g).

Psoriasis was evaluated from four weeks before the first injection of M vaccae to six weeks after the first injection as follows: A. the PASI scores were determined at 0, 3 and 6 weeks; B. patient questionnaires were completed at 0, 3 and 6 weeks; and C. psoriatic lesions and each patient were photographed at 0 and 3 weeks.

The data shown in Table 7 describe the age, sex and clinical background of each patient.

TABLE 7 Patient Data in the Study of the Effect of DD-M. vaccae in Psoriasis Code Duration of No. Patient Age/Sex Disorder Admission PASI Score PS-025 A.S 25/F 2 years 12.2 PS-026 M.B 45/F 3 months 14.4 PS-027 A.G 34/M 14 years 24.8 PS-028 E.M 31/M 4 years 18.2 PS-029 A.L 44/M 5 months 18.6 PS-030 V.B 42/M 5years 21.3 PS-031 R.A 18/M 3 months 13.0 All patients demonstrated a non-ulcerated, localised erythematous soft indurated reaction at the injection site. No side effects were noted, or complained of by the patients.

The data shown in Table 8 are the measured skin reactions at the injection site, 48 hours, 72 hours and 7 days after the first injection of DD-M. vaccae, and 48 hours and 72 hours after the second injection.

61 WO 99/32634 WO 9932634PCTNZ98/OOI 89 TABLE 8 Skin Reaction Measurements in Millimeters Code No. Time of Measurement First Injection Second Injection 48 hours 72 hours 7 days 48 hours 72 hours PS-025 8x8 8x8 3x2 lOxlO lOXlO PS-026 12x12 12x12 8x8 DNR 14x14 PS-027 9x8 lOxlO I10x8 9x5 9x8 PS-028 lOxlO lOxlO I10x8 lOXlO lOxlO PS-029 8x6 8x6 5x5 8x8 8x8 PS-030 14x12 14x14 lOXlO 12x10 12x10 PS-031 lOXlO 12x12 I10x6 14xl2 12x1 DNR Did not report 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 3, 6, 12 and 24 weeks later.

TABLE 9 Clinical Status of Patients after Injection of DD-M vaccae (PASI Scores) Code No. Day 0 Week 3 Week 6 Week 12 Week 24 PS-025 12-2 4-1 1.8 1.4 1.7 PS-026 14-4 11-8 6.0 6.9 1.4 PS-027 24-8 23-3 18.3 9.1 10.6 PS-028 18-2 24-1 28.6 Dropped PS-029 18.6 9.9 7.4 3.6 0.8 PS-030 21.3 15.7 13.9 16.5 13.6 PS-031 13.0 5.1 2.1 1.6 0.3 WO 99/32634 PCT/NZ98/00189 It can clearly be seen that by week 3 after the first injection of DD-M vaccae, five patients showed a significant improvement in PASI scores. By week 24, six of seven patients showed a significant improvement in PASI score.

By way of example, Patient PS-031 went into remission (PASI score 0) at week 32 and remained in remission when seen at week 48. The PASI score of patient PS-025 was reduced to less than 1 for more than 12 weeks. Upon an exacerbation of psoriasis (PASI 15.8) at week 48, the patient was re-treated with DD-M.vaccae and improveded promptly with PASI scores falling to 6.8 and 0.6 at weeks 52 and 56 respectively.

Thus treatment of psoriasis with DD-M.vaccae may lead to disease remission or provide prolonged benefit. Patients may also benefit with repeated treatment.

EXAMPLE 7 PREPARATION OF COMPOSITIONS FROM M VACCAE This example illustrates the processing of different constituents of M vaccae.

Preparation of Delipidated and Deglycolipidated M.vaccae and Compositional Analysis Heat-killed M. vaccae was prepared as described as above in Example 1. To prepare delipidated Mvaccae, the autoclaved Mvaccae was pelleted by centrifugation, the pellet washed with water, collected again by centrifugation and then freeze-dried. An aliquot of this freeze-dried Mvaccae was set aside and referred to as lyophilised Mvaccae. When used in experiments it was resuspended in PBS to the desired concentration. Freeze-dried M vaccae was treated with chloroform/methanol 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 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 WO 99/32634 PCT/NZ98/00189 M.vaccae used. For bioassay, the delipidated and deglycolipidated M vaccae (DD-M vaccae), was resuspended in phosphate-buffered saline by sonication, and sterilised by autoclaving.

The 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 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.

TABLE 9 Compositional analyses of heat-killed M. vaccae and DD-M. vaccae MONOSACCHARIDE COMPOSITION sugar alditol M. vaccae DD-M. vaccae Inositol 3.2% 1.7% Ribitol 1.7% 0.4% Arabinitol 22.7% 27.0% Mannitol 8.3% 3.3% Galactitol 11.5% 12.6% Glucitol 52.7% 55.2% FATTY ACID COMPOSITION Fatty acid M. vaccae DD-M. vaccae C14:0 3.9% 10.0% C16:0 21.1% 7.3% C16:1 14.0% 3.3% C18:0 4.0% C18:1* 1.2% 2.7% C18:1w9 20.6% 3.1% C18:1w7 12.5% 5.9% C22:0 12.1% 43.0% C24:1* 6.5% 22.9% WO 99/32634 PCT/NZ98/00189 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.

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

Comparison of composition of DD-M. vaccae with delipidated and deglycolipidated forms ofM. tuberculosis and M. smegmatis 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 1- C ill WO 99/32634 PCT/NZ98/00189 DD-M vaccae contained 34% and 55% protein, whereas DD-M tuberculosis and DD- M. smegmatis contained 79% and 72% protein, respectively.

TABLE Amino Acid Composition of Delipidated and Deglycolipidated Mycobacteria Amino Acid Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Total Protein DD-M.vaccae Batch 1 9.5 6.0 5.3 11.1 6.1 9.9 14.6 0.5 6.3 1.9 3.6 7.8 1.4 4.2 1.9 4.1 5.8 55.1 DD-M.vaccae Batch 2 9.5 5.9 5.3 11.2 5.9 9.7 14.7 0.5 6.4 1.9 3.5 7.9 1.7 4.0 1.8 4.0 5.9 33.8

DD-

M.smegmatis 9.3 5.0 4.2 11.1 7.5 9.4 14.6 0.3 7.2 1.9 4.1 8.2 1.8 3.2 2.0 4.1 6.2 72.1

DD-

M.uberculosis 9.1 5.3 3.3 12.5 5.2 9.8 14.2 7.8 1.9 4.7 8.3 1.8 1.9 4.2 6.4 78.5 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 66 WO 99/32634 WO 9932634PCT/NZ98/OO1 89 glucose while both DD-M tuberculosis and M smegmatis contain glycerol, as shown in Table TABLE 11I Alditol Acetate wt% MOlM DD-M.vaccae Batch 1 Inositol 0.0 0.0 Arabinose 54.7 59.1 Mannose 1.7 Glucose 31.1 28.1 Galactose 12.5 .11.3 100.0 100.0 DD-M.vaccae Batch 2 Inositol 0.0 0.0 Arabinose 51.0 55.5 Mannose 2.0 1.8 Glucose 34.7 31.6 Galactose 12.2 11.1 100.0 100.0 DD-M.smeg Inositol 0.0 0.0 Glycerol 15.2 15.5 Arabinose 69.3 70.7 Xylose 3.9 Mannose 2.2 1.9 Glucose 0.0 0.0 Galactose 9.4 100.0 100.0 DD-Mtb Inositol 0.0 0.0 Glycerol 9.5 9.7 Arabinose 69.3 71.4 Mannose 3.5 Glucose 1.5 1.3 Galactose 12.4 .10.7 96.2 96.0 M. vaccae glycolipids 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 WO 99/32634 PCT/NZ98/00189 starting wet weight of M vaccae used. For bioassay, the glycolipids were dissolved in phosphate-buffered saline.

EXAMPLE 8 IMMUNE MODULATING PROPERTIES OF DELIPIDATED AND DEGLYCOLIPIDATED M VACCAE AND RECOMBINANT PROTEINS FROM M VACCAE This example illustrates the immune modulating properties of different constituents of M. vaccae.

Production of Interleukin-12 from macrophages Whole heat-killed M vaccae and DD-M vaccae were shown to have different cytokine stimulation properties. The stimulation of a Thl immune response is enhanced by the production of interleukin-12 (IL-12) from macrophages. The ability of different M.

vaccae preparations to stimulate IL-12 production was demonstrated as follows.

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 vaccae, lyophilized M vaccae, DD-M vaccae and M. vaccae glycolipids, prepared as described above, were added. After a further three days at 37 the culture supernatants were assayed for the presence of IL-12 produced by macrophages. As shown in Fig. 4, the M. vaccae preparations stimulated the production of IL-12 from macrophages.

By contrast, these same M vaccae preparations were examined for the ability to stimulate interferon-gamma production from Natural Killer (NK) 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 vaccae, DD-M vaccae, or M vaccae glycolipids. The data 68

L

WO 99/32634 PCT/NZ98/00189 shown in Fig. 5 demonstrates that, while heat-killed M vaccae and M vaccae glycolipids stimulate production of interferon-gamma, DD-M vaccae stimulated relatively less interferon-gamma. The combined data from Figs. 4 and 5 indicate that, compared with whole heat-killed M vaccae, DD-M vaccae is a better stimulator of IL-12 than interferon gamma.

These findings demonstrate that removal of the lipid glycolipid constituents from M vaccae results in the removal of molecular components that stimulate interferon-gamma from NK cells, thereby effectively eliminating an important cell source of a cytokine that has numerous harmful side-effects. DD-M. vaccae thus retains Thl immune enhancing capacity by stimulating IL-12 production, but has lost the non-specific effects that may come through the stimulation of interferon-gamma production from NK cells.

The adjuvant effect of DD-M vaccae and a number of M vaccae recombinant antigens of the present invention, prepared as described below, was determined by measuring stimulation of IL-12 secretion from murine peritoneal macrophages. Figs. 6A, 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 interferongamma for three hours. The culture medium was replaced and various concentrations of M vaccae recombinant proteins GVs-3 GV-4P (GV-4P), GVc-7 GV-23, GV- 27, heat killed M vaccae, DD-M vaccae (referred to as delipidated M vaccae in Figs. 6A, B and M vaccae glycolipids or lipopolysaccharide were added. After three days at 37 °C, the culture supernatants were assayed for the presence of IL-12 produced by macrophages. As shown in Figs. 6A, B and C, the recombinant proteins and M vaccae preparations stimulated the production of IL-12 from macrophages.

In a subsequent experiment, IFNy-primed peritoneal macrophages from BALB/c mice were stimulated with 40 ug/ml ofM. vaccae recombinant proteins in culture for 3 days and the presence of IL-12 produced by macrophages was assayed. As shown in Fig. 7, in these experiments IFNy-primed macrophages produced IL-12 when cultured with a control protein, ovalbumin (ova). However, the recombinant proteins GV 24B, 38BP, 38AP, 27, 5, 27B, 3, 23 1 WO 99/32634 PCT/NZ98/00189 and 22B stimulated more than twice the amount of IL-12 detected in control macrophage cultures.

Detection of Nonspecific Immune Amplifier from Whole M. vaccae and the Culture Filtrate ofM. Vaccae M. vaccae culture supernatant killed M vaccae, delipidated M vaccae and delipidated and deglycolipidated M vaccae (DD-M vaccae), prepared as described above, were tested for adjuvant activity in the generation of a cytotoxic T cell immune response to ovalbumin, a structurally unrelated protein, in the mouse. This anti-ovalbumin-specific cytotoxic response was detected as follows. C57BL/6 mice (2 per group) were immunized by the intraperitoneal injection of 100 pg of ovalbumin with the following test adjuvants: autoclaved M 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 heatkilled M tuberculosis or heat-killed M. bovis BCG, M. phlei or M smegmatis or M vaccae culture filtrate. After 10 days, spleen cells were stimulated in vitro for a further 6 days with 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 [pCi per 2x10 6 prior to the killing assay. Killing or cytolytic activity is expressed as specific lysis using the formula: cpm in test cultures cpm in control cultures x100% total cpm cpm in control cultures It is generally known that ovalbumin-specific cytotoxic cells are generated only in mice immunized with ovalbumin with an adjuvant but not in mice immunized with ovalbumin alone.

WO 99/32634 PCT/NZ98/00189 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.

As shown in Fig. 7A, cytotoxic cells were generated in mice immunized with 10 jg, (ii) 100 ug or (iii) 1 mg of autoclaved M vaccae or (iv) 75 gg of M vaccae culture filtrate. Fig.

7B shows that cytotoxic cells were generated in mice immunized with 1 mg whole autoclaved M vaccae or (ii) 1 mg delipidated and deglycolipidated M vaccae. As shown in Fig. 7C(i), 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. By way of comparison, 100 pg 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 (Fig. 7D(i)) 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.

These findings demonstrate that heat-killed M. vaccae and DD-M vaccae have adjuvant properties not seen in other mycobacteria. Furthermore, delipidation and deglycolipidation of M vaccae removes an NK cell-stimulating activity but does not result in a loss of T-cell stimulating activity.

In a separate experiment, 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 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 71 WO 99/32634 PCT/NZ98/00189 demonstrated in fractions corresponding to pi of 4.2-4.32 (fraction nos. 4.49-4.57 (fraction nos. 13-17) and 4.81-5.98 (fraction nos. 23-27).

Identifcation of proteins in DD-M. vaccae by antibodies BALB/c 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. vaccae-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 vaccae-derived GV 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.

TABLE 12 Reactivity of DD-M. vaccae antiserum with M.vaccae-derived GV antigens GV Antigen 3 5P 5 7 9 22B 24 27 27A27B 33 Reactivity* 103 10 103 102 104 103 104 10 6 10s 106 104 1 104 *Expressed as highest dilution of serum from DD-M.vaccae immunised mice showing greater activity than serum from non-immunised mice.

Proteins in DD-M.vaccae identified by T cell responses BALB/c mice were injected in each footpad with 100 ug DD-M.vaccae 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. vaccae-derived GV proteins. After 3 days, cell proliferation and IFNy production were assessed.

72 WO 99/32634 PCT/NZ98/00189 T cell proliferative responses of lymph node cells from DD-M.vaccae immunized mice to GV proteins.

Lymph node cells from DD-M vaccae-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 Mvaccae derived proteins GV 3, 7, 9, 23, 27, 27B and 33 are likely to be present in DD-M.vaccae.

TABLE 13 Proliferative responses of lymph node cells from DD-M vaccae-immunised mice and control mice to GV proteins in vitro Stimulation index* observed in the GV protein presence of GV proteins at 50 pg/ml DD-M.vaccae immunised Control mice mice GV3 4.63 1.52 GV7 3.32 1.27 GV9 6.48 2.64 GV23 4.00 1.76 GV27 5.13 1.40 GV27B 7.52 1.48 GV33 3.31 1.45 *Stimulation index cpm from tritiated Thymidine uptake in presence of GV protein/cpm in absence of GV protein IFNy production by lymph node cells from DD-M. vaccae immunized mice following in vitro challenge with GV proteins WO 99/32634 PCT/NZ98/00189 Lymph node cells from non-immunized mice did not produce IFNy upon stimulation with GV proteins. As shown in Table 14 below, lymph node cells from DD-M.vaccae immunized mice secrete IFNy 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 IFNy 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, and 46 are thus likely to be present in DD-M. vaccae.

TABLE 14 Production of IFNy by popliteal lymph node cells from DD-M vaccae-immunised mice following in vitro challenge with GV protein IFNy (ng/ml) GV protein Dose of GV protein used in vitro (Jg/ml) or control 50 10 2 GV-3 8.22 3.73 ND ND GV-4P ND ND ND 8.90 4.54 0.57 0.40 ND ND ND ND GV-7 ND ND ND GV-9 ND ND ND GV-13 1.64 ±0.40 ND ND GV-14 ND ND ND GV-14B ND ND ND GV-22B 20.15 1.96 4.34 0.02 ND GV-23 41.38 6.69 6.97 2.78 ND GV-24B ND ND ND GV-27 46.86 17.14 33.06 17.61 10.14 ±3.01 GV-27A 7.25 4.36 ND ND GV-27B 100.36 37.84 33.03 7.54 14.33 1.01 GV-29 5.93 0.47 ND ND GV-33 9.82 4 4.64 ND ND GV-38AP 1.44 1.20 ND ND GV-38BP 5.62 0.70 ND ND GV-42 ND ND ND GV-44 ND ND ND WO 99/32634 PCT/NZ98/00189 DD-M.vaccae 109.59 15.48 90.23 6.48 65.16 3.68 M. vaccae 68.89 4.38 67.91 7.92 48.92 3.86 ND Not Detectable Proteins in DD-M.vaccae as non-specific immune amplifiers In subsequent experiments, the five proteins GV27, 27A, 27B, 23 and 45 were used as non-specific immune amplifiers with ovalbumin antigen to immunize mice as described above in Example 6. As shown in Figure 12, 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.

EXAMPLE 9 AUTOCLAVED M VACCAE GENERATES CYTOTOXIC CD8 T CELLS AGAINST M TUBERCULOSIS INFECTED MACROPHAGES 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 lig of killed M vaccae which was prepared as described in Example 1. Two weeks after immunization, 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 51Chromium at 2 jCi per 10" macrophages. The macrophages were then cultured with CD8 T cells overnight (16 hours) at killer to target ;i LI.

WO 99/32634 PCT/NZ98/00189 ratios of 30:1. Specific killing was detected by the release of 51 Chromium and expressed as specific lysis, calculated as in Example The production of IFN-y and its release into medium after 3 days of co-culture of CD8 T cells with macrophages was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with a rat monoclonal antibody directed to mouse IFN-y (Pharmigen, San Diego, CA, USA) in PBS for 4 hours at 4 oC. Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. The plates were then washed four times in PBS containing 0.2% Tween 20, and samples diluted 1:2 in culture medium in the ELISA plates were incubated overnight at room temperature. The plates were again washed, and a biotinylated monoclonal rat anti-mouse IFN-y antibody (Pharmigen), diluted to 1 pg/ml in PBS, was added to each well. The plates were then incubated for 1 hour at room temperature, washed, and horseradish peroxidase-coupled avidin D (Sigma A-3151) was added at a 1:4,000 dilution in PBS. After a further 1 hour incubation at room temperature, the plates were washed and OPD substrate added. The reaction was stopped after 10 min with HC1. The optical density was determined at 490 nm. Fractions that resulted in both replicates giving an OD two-fold greater than the mean OD from cells cultured in medium alone were considered positive.

As shown in Table 15, CD8 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-y when cocultured with infected macrophages. The amount of IFN-y produced in coculture was greater with CD8 T cells derived from M. vaccae immunized mice.

WO 99/32634 PCT/NZ98/00189 TABLE EFFECT WITH M TUBERCULOSIS INFECTED AND UNINFECTED MACROPHAGES Specific Lysis IFN-y (ng/ml) of Macrophages CD8 T cells uninfected infected uninfected infected Control 0 0 0.7 24.6 M. vaccae Immunized 0 95 2.2 43.8 EXAMPLE PURIFICATION AND CHARACTERIZATION OF POLYPEPTIDES FROM M VACCAE CULTURE FILTRATE This example illustrates the preparation of M. vaccae soluble proteins from culture filtrate. 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 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 pm 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 0.45p.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 77 WO 99/32634 PCT/NZ98/00189 sample was less than 1.0 mS. This procedure reduced the 20 1 volume to approximately 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 10mM Tris HCI 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.

Fractions from isoelectric focusing were combined, and the polypeptides were purified on a Vydac C4 column (Separations Group, Hesperia, CA, USA) 300 Angstrom pore size, micron particle size (10 x 250 mm). The polypeptides were eluted from the column with a linear gradient of acetonitrile (0-80% v/v) in 0.05% trifluoroacetic acid (TFA). The flow-rate was 2.0 ml/min and the HPLC eluent was monitored at 220 nm. Fractions containing polypeptides were collected to maximize the purity of the individual samples.

Relatively abundant polypeptide fractions were rechromatographed on a Vydac C4 column (Separations Group) 300 Angstrom pore size, 5 micron particle size (4.6 x 250 mm).

The polypeptides were eluted from the column with a linear gradient from 20-60% of acetonitrile in 0.05% TFA at a flow-rate of 1.0 ml/min. The column eluent was 78 WO 99/32634 PCT/NZ98/00189 monitored at 220 nm. Fractions containing the eluted polypeptides were collected to maximise the purity of the individual samples. Approximately 20 polypeptide samples were obtained and they were analysed for purity on a polyacrylamide gel according to the procedure of Laemmli (Laemmli, U. Nature 277:680-685, 1970).

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 mm) or a Vydac Diphenyl column (Separations Group) 300 Angstrom pore size, 5 micron particle size (4.6 x 250 mm). From a Mono Q column, polypeptides were eluted with a linear gradient from 0-0.5 M NaCl in 10 mM Tris HCI pH 8.0. From a Vydac Diphenyl column, 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 polyacrylamide gel as described above.

For sequencing, the polypeptides were individually dried onto Biobrene T (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.

Internal sequences were also determined on some antigens by digesting the antigen with the endoprotease Lys-C, or by chemically cleaving the antigen with cyanogen bromide.

Peptides resulting from either of these procedures were separated by reversed-phase HPLC on a Vydac C18 column using a mobile phase of 0.05% trifluoroacetic acid with a gradient of acetonitrile containing 0.05% TFA The eluent was monitored at 214 nm.

Major internal peptides were identified by their UV absorbance, and their N-terminal sequences were determined as described above.

Using the procedures described above, six soluble M vaccae antigens, designated GVc-1, GVc-2, GVc-7, GVc-13, GVc-20 and GVc-22, were isolated. Determined N-terminal 79 ;i- WO 99/32634 PCT/NZ98/00189 and internal sequences for GVc-1 are shown in SEQ ID NOS: 1, 2 and 3, respectively; the Nterminal 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).

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. 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. 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 nonreducing sample buffer and applied to the gel. The separated proteins were transferred to a i--l WO 99/32634 PCT/NZ98/00189 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-17 and GVc-19 are provided in SEQ ID NOS: 16-20, respectively.

All of the above amino acid sequences were compared to known amino acid sequences in the SwissProt data base (version R32) using the GeneAssist system. No significant homologies to the amino acid sequences GVc-2 to GVc-22 were obtained. The amino acid sequence for GVc-1 was found to bear some similarity to sequences previously identified from M bovis and M tuberculosis. In particular, GVc-1 was found to have some homology with M tuberculosis MPT83, a cell surface protein, as well as MPT70. These proteins form part of a protein family (Harboe et al., Scand. J. Immunol. 42:46-51, 1995).

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

Further studies with GVc-22 suggested that only a part of the gene encoding GVc-22 was cloned. When sub-cloned into the expression vector pET16, no protein expression was obtained. Subsequent screening of the M vaccae BamHI genomic DNA library with the incomplete gene fragment led to the isolation of the complete gene encoding GVc-22. To distinguish between the full-length clone and the partial GVc-22, the antigen expressed by the full-length gene was called GV-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.

81 I WO 99/32634 PCT/NZ98/00189 Amplifications primers AD86 and AD112 (SEQ ID NO: 60 and 61, respectively) were designed from the amino acid sequence of GVc-1 (SEQ ID NO: 1) and the M tuberculosis 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 SEQ ID NO: 62. The insert of this clone was used to screen a M. vaccae genomic DNA library constructed in lambda ZAP- Express (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 SEQ ID NOS: 146 and 147 respectively.

From the amino acid sequences provided in SEQ ID NOS: 1 and 2, degenerate oligonucleotides EV59 and EV61 (SEQ ID NOS: 148 and 149 respectively) were designed.

Using PCR, 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.

For expression and purification, 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-y 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% autologous serum, penicillin (60 gg/ml), streptomycin (100 tg/ml), and glutamine (2 mM).

WO 99/32634 PCT/NZ98/00189 After 3 days, 50 il of medium was removed from each well for the determination of IFN-y levels, as described below. The plates were cultured for a further 4 days and then pulsed with 1 pCi/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-y was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with a mouse monoclonal antibody directed to human IFN-y (Endogen, Wobural, MA) 1 pjg/ml phosphate-buffered saline (PBS) for 4 hours at 4 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. The plates were again washed, and a biotinylated polyclonal rabbit anti-human IFN-y serum (Endogen), diluted to 1 p.g/ml in PBS, was added to each well. The plates were then incubated for 1 hour at room temperature, washed, and horseradish peroxidase-coupled avidin A (Vector Laboratories, Burlingame, CA) was added at a 1:4,000 dilution in PBS. After a further 1 hour incubation at room temperature, the plates were washed and orthophenylenediamine (OPD) substrate added. The reaction was stopped after 10 min with 10% HC1. The optical density (OD) was determined at 490 nm. Fractions that resulted in both replicates giving an OD two-fold greater than the mean OD from cells cultured in medium alone were considered positive.

Examples of polypeptides containing sequences that stimulate peripheral blood mononuclear cells (PBMC) T cells to proliferate and produce IFN-y 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.

83 1- WO 99/32634 PCT/NZ98/00189 TABLE 16 Antigen Proliferation IFN-y GVc-1 GVc-2 GVc-7 GVc-13 GVc-14 EXAMPLE 11 PURIFICATION AND CHARACTERISATION OF POLYPEPTIDES FROM M VACCAE CULTURE FILTRATE BY 2-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS 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 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 gtm 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 tim membrane filter.

84 WO 99/32634 PCT/NZ98/00189 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 10mM TrisHCl buffer pH 8.0. Polypeptides were eluted with a linear gradient of NaCl 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 lg of polypeptide were made 8M in urea and applied to polyacrylamide isoelectric focusing rod gels (diameter 2mm, length 150 mm, pH After the isoelectric focusing step, the first dimension gels were equilibrated with reducing buffer and applied to second dimension gels (16% polyacrylamide). Polypeptides from the second dimension separation were transferred to PVDF membranes by electroblotting in 10mM 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 Nterminal sequences for these polypeptides are shown in SEQ ID NOS: 21-29, 63 and 64, respectively. Using the purification procedure described above, more protein was purified to extend the amino acid sequence previously obtained for GVs-9. The extended amino acid sequence for GVs-9 is provided in SEQ ID NO: 65. Further studies resulted in the isolation -l WO 99/32634 PCT/NZ98/00189 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.

All of these amino acid sequences were compared to known amino acid sequences in the SwissProt data base (version R35 plus update). No significant homologies were obtained, with the exceptions of GVs-3, GVs-4, GVs-5 and GVs-9. GVs-9 was found to bear some homology to two previously identified M. tuberculosis proteins, namely M. tuberculosis cutinase precursor and an M. tuberculosis hypothetical 22.6 kDa protein. GVs-3, GVs-4 and 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 proteins from M leprae (SEQ ID NO: 36) and M tuberculosis (SEQ ID NO: 37).

EXAMPLE 12 DNA CLONING STRATEGY FOR THE M VACCAE ANTIGEN 85 SERIES 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 DNA. An appropriately-sized 485 bp band was identified, purified, and cloned into T-tailed pBluescript II SK (Stratagene, La Jolla, CA). Twenty-four individual colonies were screened at random for the presence of the antigen 85 PCR product, then sequenced using the Perkin Elmer/Applied Biosystems Model 377 automated sequencer and the M13-based primers, T3 and T7. Homology searches of the GenBank databases showed that twenty-three clones contained insert with significant homology to published antigen 85 genes from M T-1 WO 99/32634 PCT/NZ98/00189 tuberculosis and M bovis. Approximately half were most homologous to antigen 85C gene sequences, with the remainder being more similar to antigen 85B sequences. In addition, these two putative M vaccae antigen 85 genomic sequences were 80% homologous to one another. Because of this high similarity, the antigen 85C PCR fragment was chosen to screen M. vaccae genomic libraries at low stringency for all three antigen 85 genes.

An M. vaccae genomic library was created in lambda Zap-Express (Stratagene, La Jolla, CA) by cloning BamHI partially-digested M vaccae genomic DNA into similarlydigested X vector, with 3.4 x 105 independent plaque-forming units resulting. For screening purposes, twenty-seven thousand plaques from this non-amplified library were plated at low density onto eight 100 cm 2 plates. For each plate, duplicate plaque lifts were taken onto Hybond-N+ nylon membrane (Amersham International, United Kingdom), and hybridised under reduced-stringency conditions (55 oC) to the radiolabelled antigen 85C PCR product.

Autoradiography demonstrated that seventy-nine plaques consistently hybridised to the antigen 85C probe under these conditions. Thirteen positively-hybridising plaques were selected at random for further analysis and removed from the library plates, with each positive clone being used to generate secondary screening plates containing about two hundred plaques. Duplicate lifts of each plate were taken using Hybond-N+ nylon membrane, and hybridised under the conditions used in primary screening. Multiple positively-hybridising plaques were identified on each of the thirteen plates screened. Two well-isolated positive phage from each secondary plate were picked for further analysis. Using in vitro excision, twenty-six plaques were converted into phagemid, and restriction-mapped. It was possible to group clones into four classes on the basis of this mapping. 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 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 87 ~ii- WO 99/32634 PCT/NZ98/00189 of mycobacterial species. To determine the remaining nucleotide sequence for each gene, appropriate subclones were constructed and sequenced. Overlapping sequences were aligned using the DNA Strider software. The determined DNA sequences for M vaccae antigens 85B and 85C are shown in SEQ ID NOS: 40-42, respectively, with the predicted amino acid sequences being shown in SEQ ID NOS: 43-45, respectively.

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, and the sequences of the primers for GVs-5 are provided in SEQ ID NO: 68 and 69. A XhoI restriction site was added to the primers for GVs-3, and EcoRI and BamHl 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 pProEX 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.

Expression of a fragment of the M vaccae antigen GVs-4 (antigen 85B homolog) was performed as follows. The primers AD58 and AD59, described above, were used to amplify a 485 bp fragment from M vaccae genomic DNA. This fragment was gel-purified using standard techniques and cloned into EcoRV-digested pBluescript containing added dTTP residues. The base sequences of inserts from five clones were determined and found to be identical to each other. These inserts had highest homology to Ag85B from M tuberculosis.

The insert from one of the clones was subcloned into the EcoRI/XhoI sites of pProEX 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 SEQ ID NO: 70 and 106, respectively.

88 -I WO 99/32634 PCT/NZ98/00189 Similar to the cloning of GV-4P, 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.

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

The ability of purified recombinant GVs-3, GV-4P and GVs-5 to stimulate proliferation of T cells and interferon-y production in human PBL from PPD-positive, healthy donors, was assayed as described above. The results of this assay are shown in Table 17, 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, indicates polypeptides having activity greater than four times above background, and ND indicates not determined.

Table 17 Donor Donor:> i: Donor: Donor :.Donor Donor G97005-1i: G97006 :i G97007 G97008 G97009.. G97010 Prolif: -IFN Prolif IFN :Prolif IFN Prolif IFN Prolif IFN Prolif IFN S -Y -7 -v Y. -Y GVs- ND ND GV- ND ND 4P GVs- EXAMPLE 13 DNA CLONING STRATEGY FOR M. VACCAE ANTIGENS 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: This probe was used to screen a M vaccae genomic DNA library as described in Example 89 WO 99/32634 PCTINZ98/00189 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.

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 XhoI restriction site was added to the primers for cloning convenience. Following amplification from genomic M. vaccae DNA, fragments were cloned into the Xhol-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).

The ability of purified recombinant GVc-7 to stimulate proliferation of T-cells and stimulation of interferon-y production in human PBL, from PPD-positive, healthy donors, was assayed as described above. The results are shown in Table 18, 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.

TABLE 18 Donor Proliferation Interfeoni-yr G97005 G97008 G97009 G97010 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 l WO 99/32634 PCT/NZ98/00189 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. GVcontains 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: 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 EcoRvdigested pBluescript II SK' (Stratagene), and then transferred to pET16 expression vector.

Recombinant protein was purified following the manufacturer's protocol.

The ability of purified recombinant GV-33 to stimulate proliferation of T-cells and stimulation of interferon-y production in human PBL was assayed as described above. The results are shown in Table 19, 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.

91 WO 99/32634 PCT/NZ98/00189 TABLE 19 Stimulatory Activity of Polypeptides Donor Proliferation Interferon-y G97005 G97006 G97007 G97008 G97009 G97010 EXAMPLE 14 ISOLATION OF PROTEINS FROM DD-M VACCAE 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. A delipidated form of M. vaccae was prepared by sonicating autoclaved M. vaccae for four bursts of 30 seconds on ice using the Virsonic sonicator (Virtis, Disa, USA). The material was then centrifuged (9000 rpm, 20 minutes, JAl0 rotor, brake The resulting pellet was suspended in 100 ml of chloroform/methanol 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 mg (dry weight) per ml as delipidated M. vaccae.

Glycolipids were removed from the delipidated M. vaccae preparation by refluxing in v/v ethanol for 2 hours. The insoluble material was collected by centrifugation (10,000 rpm, JA20 rotor, 15 mins, brake The extraction with 50% v/v ethanol under reflux was repeated twice more. 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 pooled SDS extracts were cooled to 4 °C, and precipitated SDS was removed by centrifugation (10,000 rpm, JA20 rotor, 15 mins, brake WO 99/32634 PCT/NZ98/00189 Proteins were precipitated from the supernatant by adding an equal volume of acetone and incubating at -20 oC for 2 hours. The precipitated proteins were collected by centrifugation, washed in 50% v/v acetone, dried in vacuo, and redissolved in PBS.

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 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.

In subsequent studies, more of the SDS-extracted proteins described above were prepared by preparative SDS-PAGE on a BioRad Prep Cell (Hercules, CA). Fractions corresponding to molecular weight ranges were precipitated by trichloroacetic acid to remove SDS before assaying for adjuvant activity in the anti-ovalbumin-specific cytotoxic response assay in C57BL/6 mice as described above. The adjuvant activity was highest in the 60-70 kDa fraction. The most abundant protein in this size range was purified by SDS-PAGE blotted on to a polyvinylidene difluoride (PVDF) membrane and then sequenced. 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.

An expression library of M vaccae genomic DNA in BamH1-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 93 WO 99/32634 PCT/NZ98/00189 the manufacturer's protocol. The base sequence of the 5' end of the insert of this clone, hereinafter referred to as GV-27, 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: In subsequent studies, an extended (full-length except for the predicted 51 terminal nucleotides) DNA sequence for GV-27 was obtained (SEQ ID NO: 113). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 114. Further studies led to the isolation of a full-length DNA sequence for GV-27 (SEQ ID NO: 159). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 160. GV-27 was found to be 93.7% identical to the M tuberculosis GroEL at the amino acid level.

Two peptide fragments, comprising the N-terminal sequence (hereinafter referred to as GV-27A) and the carboxy terminal sequence of GV-27 (hereinafter referred to as GV-27B) were prepared using techniques well known in the art. 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. Subsequent studies led to the isolation of an extended DNA sequence for GV-27B. This sequence is provided in SEQ ID NO: 161, with the corresponding amino acid sequence being provided in SEQ ID NO: 162.

The sequence of GV-27A 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 Following the same protocol as for the isolation of GV-27, pBK-CMV phagemid 3-1 was isolated. The antigen encoded by this DNA was named GV-29. The determined nucleotide 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 94

_I=I~

WO 99/32634 PCT/NZ98/00189 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.

EXAMPLE DNA CLONING STRATEGY FOR THE M VACCAE ANTIGENS GV-23, GV-24. GV-25. GV-26. GV-38A AND GV-38B M. vaccae (ATCC Number 15483) was grown in sterile Medium 90 at 37 °C for 4 days and harvested 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 80 T and oleic acid/ albumin/dextrose/catalase additive (Difco Laboratories, Detroit, Michigan) at 37 oC 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 23S ribosomal RNA (rRNA) by hybridisation of the total RNA fraction to oligonucleotides AD 10 and AD 11 (SEQ ID NO: 81 and 82) complementary to M tuberculosis rRNA. These oligonucleotides were designed from mycobacterial 16S rRNA sequences published by Bottger (FEMS Microbiol. Lett. 65:171-176, 1989) and from sequences deposited in the databanks. Depletion was done by hybridisation of total RNA to oligonucleotides AD10 and AD11 immobilised on nylon membranes (Hybond N, Amersham International, United Kingdom). Hybridisation was repeated until rRNA bands were not visible on ethidium bromide-stained agarose gels. An oligonucleotide, AD12 (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.

WO 99/32634 PCT/NZ98/00189 The M. tuberculosis and M. vaccae cDNA was used as template for single-sidedspecific PCR (3S-PCR). For this protocol, a degenerate oligonucleotide AD1 (SEQ ID was designed based on conserved leader sequences and membrane protein sequences.

After 30 cycles of amplification using primer AD1 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 AD1 and AD7. After gel purification, bands were cloned into pGEM-T (Promega) and the base sequence determined.

Searches with the determined nucleotide arid predicted amino acid sequences of band 12B21 (SEQ ID NOS: 86 and 87, respectively) showed homology to the pota gene of E.coli encoding the ATP-binding protein of the spermidine/putrescine ABC transporter complex published by Furuchi et al. (Jnl. Biol. Chem. 266: 20928-20933, 1991). The spermidine/putrescine transporter complex of E.coli consists of four genes and is a member of the ABC transporter family. The ABC (ATP-binding Cassette) transporters typically consist of four genes: an ATP-binding gene, a periplasmic, or substrate binding, gene and two transmembrane genes. The 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, 270:397-403, 1995).

An M. vaccae genomic DNA library constructed in BamHl-digested lambda ZAP Express (Stratagene) was probed with the radiolabelled 238 bp band 12B21 following standard protocols. A plaque was purified to purity by repetitive screening and a phagemid containing a 4.5 kb insert was identified by Southern blotting and hybridisation. 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 gene consisted of 1449 bp including an untranslated 5' region of 320 bp containing putative -10 and promoter elements. 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 96 I WO 99/32634 PCT/NZ98/00189 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-P-galactoside (IPTG). The recombinant protein was named GV-23 and purified from inclusion bodies according to the manufacturer's protocol. In subsequent studies, GV-23 (SEQ ID NO: 88) was re-cloned into the alternative vector pET16 (Novagen). The amino acid sequence of SEQ ID NO: 89 contains an ATP binding site at residues 34 to 41. At residues 116 to 163 of SEQ ID NO: 89, there is a conserved region that is found in the ATP-transporter family of proteins.

These findings suggest that GV-23 is an ATP binding protein.

A 322 bp Sall-BamH1 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. To identify the gene, the radiolabelled insert of this subclone was used to probe a M.

vaccae genomic DNA library constructed in the Sail-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.

For expression cloning, primers EV-26 and EV-27 (SEQ ID NOS: 95-96) 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. In subsequent studies, GV-24 (SEQ ID NO: 93) was re-cloned into the alternative vector pET16 (Novagen).

To improve the solubility of the purified recombinant antigen, the 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 97 WO 99/32634 PCTINZ98/00189 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.

The ability of purified recombinant protein GV-23 and GV-24 to stimulate proliferation of T cells and interferon-y production in human PBL was determined as described above. The results of these assays are provided in Table 20, 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, indicates polypeptides having activity greater than four times above background, and (ND) indicates not determined.

TABLE Donor Donor Donor Donor Donor Donor G97005 G97006 :G97007 G97008 G97009 G97010.:: Prolif IFN-y Prolif IFN-y Prolif IFN-y Prolif IFN-y Prolif IFN-y Prolif IFN-y GV-23 GV-24 ND ND 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.

Further subcloning and base sequence analysis of the adjacent 509 bp failed to reveal significant homology to PotC, the second transmembrane protein of E. coli, and suggests that a second transmembrane protein is absent in the M. vaccae homologue of the ABC transporter.

An open reading frame with homology to M. tuberculosis acetyl-CoA acetyl transferase, however, was identified starting 530 bp downstream of the transmembrane protein and the translated protein was named GV-26. The determined partial nucleotide sequence and predicted amino acid sequence for GV-26 are shown in SEQ ID NO: 99 and 100, respectively.

I ~I i I" WO 99/32634 PCT/NZ98/00189 Using a protocol similar to that described above for the isolation of GV-23, 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. Subsequent studies led to the isolation of an extended DNA sequence for GV-38A, provided in SEQ ID NO: 171. 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).

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).

Both the 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. To express the antigens without the hydrophobic 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 99 II:. WO 99/32634 PCT/NZ98/00189 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.

EXAMPLE 16 PURIFICATION AND CHARACTERISATION OF POLYPEPTIDES FROM M. VACCAE CULTURE FILTRATE BY PREPARATIVE ISOELECTRIC FOCUSING AND PREPARATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS 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 pm 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 trichloroacetic acid. The precipitated proteins were re-dissolved in 100 mM Tris.HCl pH 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 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 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.

100 WO 99/32634 PCTINZ98/00189 Pools of fractions containing proteins of interest were identified by analytical polyacrylamide gel electrophoresis and fractionated by preparative polyacrylamide gel electrophoresis. Samples were fractionated on 12.5% SDS-PAGE gels, and electroblotted onto nitrocellulose membranes. Proteins were located on the membranes by staining with Ponceau Red, destained with water and eluted from the membranes with acetonitrile/0.lM ammonium bicarbonate pH 8.9 and then concentrated by lyophilisation.

Eluted proteins were assayed for their ability to induce proliferation and interferon-y secretion from the peripheral blood lymphocytes 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 Nterminal sequences for these polypeptides are shown in SEQ ID NOS: 101-105, respectively.

Subsequent studies led to the isolation of a 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.

Following standard DNA amplification and cloning procedures as described in Example 13, the genes encoding 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. Within the full-length sequence of GV-42, 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.

101 i WO 99/32634 PCT/NZ98/00189 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 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.

With further sequencing, 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-3phosphate dehydrogenase.

EXAMPLE 17 ISOLATION OF THE DD-M VACCAE ANTIGENS GV-45 AND GV-46 Proteins were extracted from DD-M vaccae (500 mg; prepared as described above) by suspension in 10 ml 2% SDS/PBS and heating to 50 OC for 2 h. The insoluble residue was removed by centrifugation, and proteins precipitated from the supernatant by adding an equal volume of acetone and incubating at -20 oC for 1 hr. The precipitated proteins were collected by centrifugation, dissolved in reducing sample buffer, and fractionated by preparative SDSpolyacrylamide gel electrophoresis. The separated proteins were electroblotted onto PVDF membrane in 10 mM CAPS/0.01% SDS pH 11.0, and N-terminal sequences were determined in a gas-phase sequenator.

From these experiments, a protein represented by a band of approximate molecular weight of 30 kDa, designated GV-45, was isolated. The determined N-terminal sequence for is provided in SEQ ID NO: 187. From the same experiments, a protein of 102 WO 99/32634 PCT/NZ98/00189 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 S and M smegmatis.

From the amino acid sequence of GV-45, 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 BamHI-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. With additional sequencing, 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.

EXAMPLE 18 IMMUNOGENICITY AND IMMUNOMODULATING PROPERTIES

OF

RECOMBINANT PROTEINS DERIVED FROM M. VACCAE A. INDUCTION OF T CELL PROLIFERATION AND IFN-y PRODUCTION 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

(IFA).

Control mice received phosphate buffered saline in IFA. 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 103 ~I WO 99/32634 PCTINZ98/00189 thymidine. The amount of interferon gamma (IFNy) produced and secreted by these cells into the culture supernatants was assayed by standard enzyme-linked immunoassay.

As shown in Table 21 summarising proliferative responses, all GV proteins were found to induce a T cell proliferative response. The lymph node T cells from an immunized mouse proliferated in response to the specific GV protein used in the immunization. Lymph node cells from non-immunised mice did not proliferate in response to GV proteins. The data in Table 22 showing IFNy production, indicate that most of the GV proteins stimulated IFNy production by lymph node cells from mice immunised with the corresponding GV protein.

When lymph node cells from non-immunized mice were cultured with individual GV proteins, IFNy production was not detectable.

The GV proteins are thus immunogenic in being able to stimulate T cell proliferation and/or IFNy production when administered by subcutaneous injection. The antigen-specific stimulatory effects on T cell proliferation and IFNy production are two advantageous properties of candidate vaccines for tuberculosis.

104 T WO 99/32634 WO 9932634PCT/NZ98/00189 TABLE 21 immunogenic Properties of GV proteins: Proliferation Proliferation (cpm) GV protein Dose of GV protein used in vitro ig/m1) 2 0.08 GV-1/70 31,550± 803 19,058 ±2,449 5,596 ±686 GV-1/83 -~18,549±2,716 23,932 ±1,964 11,787±1,128 GV-3 34,751 ±1,382 6,379+ 319 4,590± 1,042 -GV-4P 26,460±1,877 10,370 ±667 6,685 ±673 42,418±2,444 23,902 ±2,312 13,973±772 35,691±159 14,457 1,185 8,340±725 GV-7 38,686 ±974 22,074 3,698 115,906 1,687 GV-9 30,599 ±2580 15,260±2,764 4,531_± 1,240 GV-13 15,296±2,006 7,163±833 3,701±243 GV-14 27,754 ±1,872 13,001 ±3,273 9,897±2,833 GV-14B 10,761 ±h 485 5,075 ±1,470 2,341 ±289 GV-22B 3,199±771 -F 3,255±386 1 1841±318 GV-23 35,598.± 1330 15,423 2,858 7,393 ±2,188 GV-24B 43,678 ±2,190 30,307 ±_1,533 15,375 ±2,594 GV-27 18,165 ±3,300 16,329± 1,794 6,107± 1,773 GV-27A 23,723 ±850 -~6,860±746 -4,295± 780 GV-27B 31,602 ±1,939 29,468 3,867 30,306 ±1,912 GV-29 20,034 ±3,328 8,107±488 2,982±897 GV-33 41,529 ±1,919 27,529 ±1,238 8,764 ±256 29,163±2,693 9,968± 314 1,626±406 GV-38AP 28,971 ±4,499 17,396 ±878 8,060 810 GV-38BP_ 19,746±245 11,732±3,207 6,264± 875 25,185 ±2,877 19,292 ±2,294 10,883 ±893 GV-41B 24,646±2,714 12,627 ±3,622 5,772 ±1,041 GV-42 25,486±3,029 20,591±2,021 13,789±775 GV-44 2,684 ±1,995 3,577± 1,725 1,499± 959 9,554 482 3,683 127 1,497±19 105 WO 99/32634 WO 9932634PCT/NZ98/OO1 89 TABLE 22 Immunogenic properties of GV proteins: IFNy production IFNy (nglml) GV protein Dose of GY protein used in vitro (jig/ml) _50 I10 2 GV-1/70 24.39 ±6.66 6.19+L 1.42 1.90 ±0.53 GV-1/83 11.34 5.46 5 36 1.34 2.73:L 1.55 GV-3 3.46 0.30 i 1.57:1: 0.04 not detectable GV-4P 6.48 ±0.37 3.00±0.52 1.38 ±0.50 4.08 1.41 6.10 ±2.72 2.35 0.40 34.98 ±15.26 9.95 ±3.42 5.68 0.79 GV-7 33.52± 3.08 25.47:±4.14 9.60 1.74 GV-9 92.27±45.50 88.54 ±16.48 30.46±1.77 GV-13 11.60±2.89 2.04±:L0. 58 1.46±0.62 GV-14 8.28 ±1.56 3.19 ±L 0.56 0.94 ±0.24 GV-1I4B not detectable not detectable not dete ctable GV-22B not detectable Inot detectable not detectable GV-23 59.67 14.88 3 0.70 ±4.4 8 9.17±= 1.51 GV-24B 6.76 ±0.58 3.20±0.50 _J 1.97±=L0.03 GV-27 72.22 14. 30.86 ±10.55 21.38 ±3.12 GV-27A 2.32 1.51±0.73 _not detectable GV-27B 87.98 15.78 44.43 ±8.70 21.49 5.60 GV-29 7.56 2.5 1.22 ±0.56 not detectable GV-33 7.71 0.26 1 8.44 ±2.35 1.52 0.24 GV-38AP 23.49 ±5.89 8.87 ±1.62 4.17 1.72 GV-38BP 5.30± 0.95 I 3.10± 1.19 1.91 1.01 15.65±7.89 10jl .58±1.31 3.57± 1.53 GV-41B 16.73 ±1.61 5.08 ±1.08 2.13 ±1.10 GV-42 95.97 ±23.86 I52.88 ±5.79 30.06 ±8.94 GV-44 not detectable jnot detectable not detectable WO 99/32634 PCT/NZ98/00189 B. ACTIVATION OF LYMPHOCYTE SUBPOPULATIONS The ability of recombinant M vaccae proteins of the present invention, heat-killed M.

vaccae and DD-M vaccae to activate lymphocyte subpopulations was determined by examining upregulation of expression of CD69 (a surface protein expressed on activated cells).

PBMC from normal donors (5 x 106 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, apT cells or y6T cells, in combination with monoclonal antibodies against CD69, followed by flow cytometry analysis Table 23 shows the percentage of ac3T cells, yST cells and NK cells expressing CD69 following stimulation with heat-killed M vaccae, DD-M vaccae or recombinant M vaccae proteins. These results demonstrate that heat-killed M vaccae, DD-M vaccae and GV-23 stimulate the expression of CD69 in the lymphocyte subpopulations tested compared with control (non-stimulated cells), with particularly high levels of CD69 expression being seen in NK cells. GV-45 was found to upregulate CD69 expression in capT cells.

107 ~i _I WO 99/32634 PCT/NZ98/00189 TABLE 23 Stimulation of CD69 Expression a4pT cells y6T cells NK cells Control 3.8 6.2 4.8 Heat-killed M. 8.3 10.2 40.3 vaccae DD-M. vaccae 10.1 17.5 49.9 GV-22B 5.6 3.9 8.6 GV-23 5.8 10.0 46.8 GV-27 5.5 4.4 13.3 GV-27A 5.5 4.4 13.3 GV-27B 4.4 2.8 7.1 11.7 4.9 6.3 The ability of the recombinant protein GV-23 (20 ug/ml) to induce CD69 expression in lymphocyte subpopulations was compared with that of the known Thl-inducing adjuvants MPL/TDM/CWS (Monophosphoryl Lipid A/ Trehalose 6'6' dimycolate; Sigma, St. Louis, MO; at a final dilution of 1:20) and CpG ODN (Promega, Madison, WI; 20 ug/ml), and the known Th2-inducing adjuvants aluminium hydroxide (Superfos Biosector, Kvistgard, Denmark; at a final dilution of 1:400) and cholera toxin (20 ug/ml), using the procedure described above. MPL/TDM/CWS and aluminium hydroxide were employed at the maximum concentration that does not cause cell cytotoxicity. Figs. 8A-C show the stimulation of CD69 expression on apT cells, y8T cells and NK cells, respectively. GV-23, MPL/TDM/CWS and CpG ODN induced CD69 expression on NK cells, whereas aluminium hydroxide and cholera toxin did not.

WO 99/32634 PCT/NZ98/00189 C. STIMULATION OF CYTOKINE PRODUCTION The ability of recombinant M vaccae proteins of the present invention to stimulate cytokine production in PBMC was examined as follows. PBMC from normal donors (5 x 106 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. Culture supernatants were harvested and tested for the production of IL- 13, TNF-ca, IL-12 and IFN-y using standard ELISA kits (Genzyme, Cambridge, MA), following the manufacturer's instructions. Figs. 9A-D show the stimulation of IL-1 TNF-a, IL-12 and IFN-y 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 GVwere found to stimulate the production of IL-1p, TNF-a and IL-12. Figs. 10A-C show the stimulation of IL-lp, TNF-a and IL-12 production, respectively, in human PBMC (determined as described above) by varying concentrations of GV-23 and Figs. 11A-D show the stimulation of IL-11, TNF-a, IL-12 and IFN-y 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-13 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.

D. ACTIVATION OF ANTIGEN PRESENTING CELLS The ability of heat-killed M. vaccae, DD-M vaccae and recombinant M vaccae proteins to enhance the expression of the co-stimulatory molecules CD40, CD80 and CD86 on B cells, monocytes and dendritic cells was examined as follows.

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), 109 j- WO 99/32634 PCT/NZ98/00189 GV-27 (SEQ ID NO: 160), GV27A (SEQ ID NO: 117), GV-27B (SEQ ID NO: 162) or GV- (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, and 26 show the fold increase in mean fluorescence intensity from control (non-stimulated cells) for dendritic cells, monocytes, and B cells, respectively.

TABLE 24 Stimulation of CD40, CD80 and CD86.Expression on Dendritic Cells CD80 CD86 Control 0 0 0 Heat-killed M 6.1 3.8 1.6 vaccae DD-M. vaccae 6.6 4.2 1.6 GV-22B 4.6 1.9 1.6 GV-23 6.0 4.5 1.8 GV-27 5.2 1.9 1.6 GV-27A 2.3 0.9 GV-27B 2.6 1.1 1.1 5.8 3.0 3.1 i WO 99/32634 WO 9932634PCT/NZ98/OO1 89 TABLE Stimulation of CD40, CD80 and CD86 Expression on Monocytes TABLE 26 Stimulation of CD40, CD80 and CD86 Expression on B Cells WO 99/32634 PCT/NZ98/00189 As shown above, increased levels of CD40, CD80 and CD86 expression were seen in dendritic cells, monocytes and B cells with all the compositions tested. Expression levels were most increased in dendritic cells, with the highest levels of expression being obtained with heat-killed M vaccae, DD-M vaccae, GV-23 and GV-45. Figs. 12A-C show the stimulation of expression of CD40, CD80 and CD86, respectively, in dendritic cells by varying concentrations of GV-23 and 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.

E. DENDRITIC CELL MATURATION AND FUNCTION The effect of the recombinant M vaccae protein GV-23 on the maturation and function of dendritic cells was examined as follows.

Purified dendritic cells (5 x 104 105 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.

TABLE 27 Stimulation of CD83 Expression in Dendritic Cells Treatments %CD83-positive dendritic cells dendritic cells Control 15 8 9 6.6 GV-23 35 13.2 24.7 14.2 LPS 36.3 14.8 27.7 13 WO 99/32634 PCT/NZ98/00189 Data mean SD (n=3) The ability of GV-23 to enhance dendritic cell function as antigen presenting cells was determined by mixed lymphocyte reaction (MLR) assay. 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.

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, changes and modifications can be carried out without departing from the scope of the invention which is intended to be limited only by the scope of the appended claims.

113 Editorial Note File No 18936/99 The following sequence listing is numbered 1 to 99 followed by page number 114.

I WO 99/32634 PCT/NZ98/00189 SEQUENCE LISTING <110> Tan, Paul L.J.

Watson, James D.

Visser, Elizabeth S.

Skinner, Margot A.

Prestidge, Ross L.

<120> Compositions Derived Methods for Their Use from Mycobacterium Vaccae and <130> 11000.1002C2PCT <150> 09/205,426 <151> 1998-12-04 <150> 09/156,181 <151> 1998-09-17 <150> 09/095,855 <151> 1998-06-11 <150> 08/996,624 <151> 1997-12-23 <150> 08/997,362 <151> 1997-12-23 <150> 08/997,080 <151> 1997-12-23 <160> 208 <170> FastSEQ for Windows Version <210> <211> <212> <213> <220> <221> <222> <400> Pro Val 1

PRT

Mycobacterium vaccae

UNSURE

(7) 1 Gly Pro Gly Xaa Ala Ala Tyr Val Gin Gin Val Pro Asp 5 10 Ala 1 Gly Pro Gly Ser Val Gin Gly Met Ala WO 99/32634 WO 9932634PCTNZ98/OO1 89 <210> 2 <211> <212> PRT <213> Mycobacterium vaccae <220> <221> UNSURE <222> Met 1 <400> 2 Xaa Asp Gin Leu Lys Val Asn Asp Asp 5 <210> <211> <212> <213> <220> <221> <222> 3 11

PRT

Mycobacterium vaccae

UNSURE

(2) Met 1 <400> 3 Xaa Pro Val Pro Val Ala Thr Ala Ala Tyr 5 <210> 4 <211> 21 <212> PRT <213> Mycobacterium vaccae Thr 1 Lys <400> 4 Pro Ala Pro Ala Pro Pro Pro Tyr Val Asp His Val Glu Gin Ala 5 10 Phe Gly Asp Leu <210> <211> 29 <212> PRT <213> Mycobacterium vaccae <22 0> <221> UNSURE <222> (25) <400> Met Gin Ala Phe Asn Ala Asp Ala Tyr Ala Phe Ala Lys Arg Glu Lys 1 5 10 Val Ser Leu Ala Pro Gly Val Pro Xaa Val Phe Glu Thr <210> 6 WO 99/32634 WO 9932634PCT/NZ98/OO1 89 <211> <212> <213> <220> <221> <222> 21

PRT

Mycobacterium vaccae

UNSURE

Met 1 Gly <400> 6 Ala Asp Pro Asn Xaa Ala Ile Leu Gin Val Ser Lys Thr Thr Arg 5 10 Gly Gin Ala Ala <210> 7 <211> 11 <212> PRT <213> Mycobacterium vaccae <400> 7 Pro Ile Leu Gin Val Ser Gin Thr Gly Arg <210> 8 <211> 14 <212> PRT <213> Mycobacterium vaccae <220> <221> UNSURE <222> (2) <221> UNSURE <222> (6) <400> 8 Xaa Asp Pro Ile Xaa Leu Gin Leu Gin Val Ser Ser Thr <210> 9 <211> 16 <212> PRT <213> Mycobacterium vaccae Lys 1 <400> 9 Ala Thr Tyr Val Gin Gly Gly Leu Gly Arg Ile Glu Ala Arg Val 5 10 <210> <211> 9 <212> PRT <213> Mycobacterium vaccae WO 99/32634 PCT/NZ98/00189 <220> <221> <222>

UNSURE

(2) Lys 1 <400> Xaa Gly Leu Ala Asp Leu Ala Pro <210> 11 <211> 14 <212> PRT <213> Mycobacterium vaccae <220> <221> <222> <223>

UNSURE

(12) (12) Residue can be either Glu or Ile <221> UNSURE <222> (2) Lys 1 <400> 11 Xaa Tyr Ala Leu Ala 5 Leu Met Ser Ala Val Xaa Ala Ala <210> 12 <211> 11 <212> PRT <213> Mycobacterium vaccae <220> <221> UNSURE <222> (10) <400> 12 Lys Asn Pro Gin Val Ser Asp Glu Leu Xaa Thr 1 5 <210> <211> <212> <213> <220> <221> <222> 13 21

PRT

Mycobacterium vaccae

UNSURE

(9) <400> Ala Pro Ala 1 Ala Ala Met 13 Pro Ala Ala Pro Ala Xaa Gly Asp Pro Ala Ala Val Val 5 10 Ser Thr WO 99/32634 WO 9932634PCT/NZ98/OOI 89 <210> 14 <211> <212> PRT <213> Mycobacterium vaccae <220> <221> <222>

UNSURE

<400> 14 Glu Ala Glu Val Xaa Tyr Leu Gly Gin Pro Gly Glu Leu Val Asn 1 5 10 <210> <211> <212> <213> <220> <221> <222> <223> <221> <222> <223>

PRT

Mycobacterium vaccae

UNSURE

(2) Residue can

UNSURE

Residue can be either Gly or Ala be either Pro or Ala <221> UNSURE <222> (7) Ala 1 <400> Xaa Val Val Pro Pro 5 Xaa Gly Pro Pro Ala Pro Gly Ala Xaa 10 <210> <211> <212> <213> 16

PRT

Mycobacterium vaccae <400> 16 Ala Pro Ala Pro Asp Leu Gin Gly Pro Leu. Val Ser Thr Leu Ser 1 5 10 <210> 17 <211> <212> PRT <213> Mycobacterium vaccae <400> 17 Ala Thr Pro Asp Trp Ser Gly Arg Tyr Thr Val Val Thr Phe Ala Ser 1 5 10 Asp Lys Leu Gly Thr Ser Val Ala Ala WO 99/32634 WO 9932634PCT/NZ98/OO1 89 <210> <211> <212> <213> <220> <221> <222> <223> <221> <222> <223> 18

PRT

mycobacterium vaccae

UNSURE

(15) Residue can

UNSURE

(23) (23) Residue can be either Ala or Arg be either Val or Leu <221> UNSURE <222> (16) (16) <400> 18 Ala Pro Pro Tyr Asp Asp 1 5 Ala Ser Pro Pro Thr Leu Arg Gly Tyr Val Asp Ser Thr Ala Xaa Xaa 10 Xaa Val Val <210> 19 <211> 8 <212> PRT <213> Mycobacterium vaccae <400> 19 Pro Glu Gly Val Ala Pro Pro Glu 1 <210> <211> <212> <213> <220> <221> <222>

PRT

Mycobacteriumn vaccae

UNSURE

(21) (22) Glu 1 Ala <400> Pro Ala Gly Ile Pro Ala Gly Phe Pro Asp Val Ser Ala Tyr Ala 5 10 Val Asp Pro Xaa Xaa Tyr Val Val <210> <211> <212> <213> 21

PRT

Mycobacterium vaccae WO 99/32634 PCT/NZ98/00189 <220> <221> UNSURE <222> (7) <400> 21 Ala Pro Val Gly Pro Gly Xaa Ala Ala Tyr Val Gin Gin Val Pro 1 5 10 <210> 22 <211> <212> PRT <213> Mycobacterium vaccae <400> 22 Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Met Val Pro Ser 1 5 10 <210> 23 <211> 19 <212> PRT <213> Mycobacterium vaccae <400> 23 Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Met Val Pro Ser Pro 1 5 10 Ser Met Gly <210> 24 <211> <212> PRT <213> Mycobacterium vaccae <400> 24 Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Asp Val Phe Ser 1 5 10 <210> <211> 14 <212> PRT <213> Mycobacterium vaccae <220> <221> UNSURE <222> <400> Xaa Xaa Thr Gly Leu His Arg Leu Arg Met Met Val Pro Asn 1 5 <210> 26 <211> <212> PRT WO 99/32634 PCT/NZ98/00189 <213> Mycobacterium vaccae <220> <221> <222> <223> <221> <222> <223>

UNSURE

(16) (16) Residue can

UNSURE

(17) (17) Residue can be either Ser or Val be either Gin or Val Gly Ala Ala Ala Gin Ala Glu Pro Ala Xaa 10 <400> 26 Val Pro Ala Asp Pro Val 1 Xaa Arg Ile Asp <210> 27 <211> 14 <212> PRT <213> Mycobacterium vaccae <220> <221> <222> <223> <221> <222> <223> <221> <222> <223>

UNSURE

(4) Residue can be

UNSURE

(8) Residue can be

UNSURE

(9) Residue can be either Tyr or Pro either Val or Gly either Ile or Tyr <221> UNSURE <222> Asp 1 <400> 27 Pro Xaa Xaa Asp Ile 5 Glu Xaa Xaa Phe Ala Arg Gly Thr <210> <211> <212> <213> 28

PRT

Mycobacterium vaccae <400> 28 Ala Pro Ser Leu Ser Val Ser Asp Tyr Ala Arg Asp Ala Gly Phe 1 5 10 <210> 29 <211> 16 WO 99/32634 WO 9932634PCT/NZ98/001 89 <212> <213> <220> <221> <222> <223> <221> <222> <221> <222> <221> <222> <221>

PRT

Mycobacterium vaccae

UNSURE

Residue can be either Leu or Pro

UNSURE

(1)

UNSURE

UNSURE

(7)

UNSURE

<222> (10) <400> Xaa Leu Xaa 1 29 Ala Xaa Ala Xaa Leu Gly Xaa Thr Val Asp Ala Asp Gin 5 10 Met Arg Gly Pro Lys Leu Thr Pro Cys Ser 145 Gly <210> <211> 33( <212> PR' <213> My <400> Lys Phe Va.

Leu Val Va Val Val Gi Val Giu Ty Val Gin Ph Asp Gly Le Thr Ala Ph Val Gly Gi 115 Gly Lys Al 130 Glu Leu Pr Ser Ala Al 1 r e

U

e 0 y a *0 Asp Giu Ser Leu Gin Arg Glu Gin Gly Giu Val Arg Ala Al a Gin Asn 70 Al a Trp Ser Cys Tyr 150 Gly Phe Met Pro Val 55 Gly Gin Tyr Ser Gin 135 Leu Leu Arg Gly Ala 40 Pro Gly Asp Tyr Phe 120 Thr Gin Ser 0

T

cobacterium leprae Gly Val 25 Glu Ser Ala Asp Gin 105 Tyr Tyr Ser Met Ala 10a Ala Pro Asn Phe 90 Ser Ser Lys Asn Al a Val Leu Phe Ser Ser 75 Ser Gly Asp Trp Lys Gly Ala Leu Ser Met Pro Gly Ile Trp Giu 140 Gin Leu Gly Ser Arg Gly Ala Trp Ser Tyr 125 Thr Ile Ser Met Ala Pro Arg Leu Asp Val 110 Ser Phe Lys Ala Leu is Leu Gly Asp Tyr Ile Val Pro Leu Pro Leu 175 Arg Ile Leu Ile Leu Asn Met Ala Thr Thr 160 Thr 165 170 Leu Ala Ile Tyr His Pro Asp Gin Phe Ile Tyr Val Gly Ser Met Ser WO 99/32634 WO 9932634PCT/NZ98/OO1 89 180 Gly Leu Leu Asp Pro 195 Ala Met Gly Asp Ala 210 185 Ser Asn Ala Met 200 Gly Gly Tyr Lys 215 190 Gly Pro Ser Leu Ile Gly Leu 205 Ala Ala Asp Met Trp, Gly Pro 220 Ser Thr Asp Pro Ala Trp Lys Arg Asn Asp Pro Thr Val Asn Val Gly 225 Thr Lys Gly Gly Ser 305 Gin Leu Pro Leu Gly 290 Trp Gin Ala Glu 260 Arg His Tyr Leu Asn 245 Leu Thr Asn Trp Gly 325 230 Asn Giy Ser Ala Gly 310 Ala Thr Gly Asn Val 295 Giu Thr Arg Asn Ile 280 Phe Gin Pro Ile Asn 265 Lys Asn Leu Gly 235 Met Pro Gin Pro Asp 315 Tyr Al a Asp Asp 300 Met Gly Leu 270 Tyr Gly Pro Asn 255 Leu Asn Thr Asp 240 Gly Glu Al a His Leu 320 Met Val Ala Leu Gin Arg Glu Gin Gly Lys 145 Val His Ser <210> 31 <211> 32 <212> PR <213> My <400> 31 Ile Asp Va Gly Ala Al Ala Thr Al.

Gln Val Pr Asn Gly G1 Ala Gin As Trp Tyr T) Ser Ser Ph: 115 Cys Thr T1 130 Trp Leu SE Gly Leu SE Pro Asp G3 1E Ser Gin G 7

T

cobacterium leprae 1 a a 'p 10 Le Ln 30 Ly Ser Ala Ser Ser Asn Asp Gin Tyr Tyr Ala Met 165 Phe Ile Gly Thr Al a Glu Gly 70 Tyr Ser Ser Lys Asn 150 Ala Ile Glu Lys Leu Phe Ala 55 Ser Asn Gly Asp Trp, 135 Arg Gly Tyr Pro Ile Pro Ser 40 Met Pro Gly Leu Trp 120 Giu Ser Ser Ala Gin Arg Ser 25 Arg Giy Ala Trp Ser 105 Tyr Thr Val Ser Gly 185 Leu Ala 10 Leu Pro Arg Val Asp 90 Val Ser Phe Lys Ala 170 Ser Ile Trp Ile Gly Thr Tyr 75 Ile Val Pro Leu Ser 155 Leu Leu Gly Gly S er Leu Ile Leu Asn Met Ala Thr 140 Thr Ile Ser Leu Arg Leu Pro Lys Leu Thr Pro Cys 125 Ser Gly Leu Ala Ala Trp Ala Val Val Asp Ser Val 110 Gly Giu Ser Ala Leu 190 Met Leu Gly Giu Gin Gly Al a Gly Lys Leu Ala Ala 175 Met Gly Leu Gly Tyr Phe Leu Phe Gly Ala Pro Val 160 Tyr Asp Asp 195 200 205 Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Pro Asn Asp Pro WO 99/32634 WO 9932634PCTNZ98/OOI 89 Ala 225 Asn Leu Gly Asn Trp 305 Met 210 Trp Asn Gly Ser Ala 290 Gly Ala Gin Thr Gly Asn 275 Val Al a Val Arg His Thr 260 Leu Phe Gin Pro Asn Leu 245 Asn Lys Asn Leu Arg 325 Asp 230 Trp Val Phe Leu Asn 310 Ser 215 Pro Val Pro Gin Asn 295 Ala Gly Ile Tyr Ala Asp 280 Al a Met Leu Cys Glu 265 Ala Asp Lys Gin Gly 250 Phe Tyr Gly Pro Ala 235 Asn Leu Asn Thr Asp 315 220 Gly Lys Gly Thr Giu Asn Gly Ala 285 His Ser 300 Leu Gin Leu Pro Phe 270 Gly Trp Asn Val1 Ser 255 Val Gly Giu Thr Al a 240 Giu His His Tyr Leu 320 <210> 32 <211> 338 <212> PRT <213> Mycobacteriumn tuberculosis <400> 32 Met Arg Gly Leu Ile Leu Asn Met Al a Thr 145 Thr Thr Ser Leu Pro Gin Leu Ala Pro so Lys Leu Thr Pro Cys 130 Ser Gly Leu Gly Ala 210 Lys Leu Val Val Val Val1 Asp Pro Val Gly Giu Ser Ala Leu 195 Met Glu Val Val Gly Giu Gin Gly Ala 100 Gly Lys Leu Al a Ile 180 Leu Gly Asp Asp Gly Gly Tyr Phe Leu Phe Gly Ala Pro Val 165 Tyr Asp Asp Pro krg kla Thr Leu Gin 70 Arg Giu Gin Gly Gly 150 Val His Pro Al a Ala Val Val Ala Gin 55 Ser Ala Trp Ser Cys 135 Trp Gly Pro *Ser *Gly 215 Trp Arg Gly Al~ 10 Gly Ala Al~ 25 Thr Ala Gi' 40 Val Pro Se: Gly Gly Al Gin Asp As: 90 Tyr Asp Gi: 105 Ser Phe Ty 120 Gin Thr Ty Leu Gin Al Leu Ser Me 17 Gin Gin Ph 185 Gin Ala Me 200 *Gly Tyr L *Gin Arg As iVal Thr Gly Met Ser Arg a nl r r a t 0 e5 Leu Al a Pro Asn 75 Phe Ser Ser Lys Asn 155 Ala Val Gly Ala Asp Val Phe Ser Ser Ser Gly Asp Trp, 140 Arg Ala Tyr Pro Ser 220 Pro Ser Ser Met Pro Gly Leu Trp 125 Giu His Ser Al a Thr 205 Asp Leu Sly Arg Gly Ala Trp Ser 110 Tyr Thr Val Ser Gly 190 Leu Met Leu Leu Pro Arg Leu Asp Val Gin Phe Lys Ala 175 Ala Ile Trp Val Gly Asp Tyr Ile Val Pro Leu Pro 160 Leu Met Gly Gly Val 225 230 235 240 Gly Lys Leu Ile Ala Asn Asn Thr Arg Val Trp Val Tyr Cys Gly Asn WO 99/32634 WO 9932634PCT/NZ98/OO1 89 Gly Glu Al a His 305 Leu Gly Lys Gly Gly 290 Ser Gin Al a Pro Phe 275 Gly Trp Arg 245 Asp Leu Gly Gly Asn 265 Arg Thr Ser Asn Ile 280 His Asn Gly Val Phe 295 Tyr Trp Giy Ala Gin 310 Leu Giy Ala Thr Pro 325 250 Asn Lys Asp Leu Asn 330 Leu Pro Ala Lys 270 Phe Gin Asp Ala 285 Phe Pro Asp Ser 300 Asn Ala Met Lys 315 Thr Gly Pro Ala 255 Phe Leu Tyr Asn Gly Thr Pro Asp 320 Pro Gin 335 Met Ile Gly Giu Gin Gly Ala Gly Lys Leu 145 Ala Ala Leu Gly Asp 225 Val <210> 33 <211> 32.

<212> PR <213> My <400> 33 Thr Asp Va.

Gly Thr Al Gly Ala Al Tyr Leu G1 Phe Gin Se Leu Arg Al Phe Glu Tr Gly Gin Se 115 Ala Gly Cy 130 Pro Gin Tr Ala Ile G1 Tyr His Pr 18 Asp Pro Se 195 Asp Ala G1 210 Pro Ala Tr Ala Asn As 1 a a n r a p 0 r '5 p y *0 0 Ser Ala Thr Val Gly Gln Tyr Ser Gin Leu Leu 165 Gin Gin Gly Glu Thr Arg Al a Al a Pro Gly 70 Asp Tyr Phe Thr Ser 150 Ser Gln Gly Tyr Arg 230 Arg

T.

cobacterium tuberculosis Lys Val Gly Ser 55 Asn Asp Gin Tyr Tyr 135 Ala Met Phe Met Lys 215 Asn Leu Ile Val1 Al a 40 Pro Asn Tyr Ser Ser 120 Lys Asn Al a Ile Gly 200 Ala Asp Trp Arg Leu 25 Phe Ser Ser Asn Gly 105 Asp Trp Arg Gly Tyr 185 Pro Ala Pro Val Al a Pro Ser Met Pro Gly 90 Leu Trp Giu Ala Ser 170 Al a Ser Asp Thr Tyr Trp Gly Arg Gly Al a 75 Trp Ser Tyr Thr Val 155 Ser Gly Leu Met Gln 235 Cys Gly Leu Pro Arg Val Asp Ile Ser Phe 140 Lys Ala Ser I le Trp 220 Gin Gly Arg Vai Gly Asp Tyr Ile Val Pro 125 Leu Pro Met Leu Gly 205 Gly Ile Asn Arg Gly Leu Ile Leu Asn Met 110 Al a Thr Thr Ile Ser 190 Leu Pro Pro Gly Leu Met Leu Ala Pro Val Lys Val Leu Asp Thr Pro Pro Val Cys Gly Ser Glu Gly Ser 160 Leu Ala 175 Ala Leu Ala Met Ser Ser Lys Leu 240 Thr Pro 245 250 255 Asn Glu Leu Gly Gly Ala Asn Ile Pro Ala Giu Phe Leu Glu Asn Phe WO 99/32634 Val Gly Glu 305 Ser Arg Ser 275 His Asn 290 Tyr Trp, Leu Gly 260 Ser Ala Gly Ala PCTNZ9/OO1 89 13 265 270 Asn Leu Lys Phe Gin Asp Ala Tyr Asn Ala Ala Gly 280 285 Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp 295 300 Ala Gin Leu Asn Ala Met Lys Gly Asp Leu Gin Ser 310 315 320 Gly 325 Met Arg Gly Leu Ile Leu Asn Met Al a Thr 145 Thr Thr Ser Leu Pro 225 Gly Giy Glu <210> 34 <211> 331 <212> PR <213> My <400> 34 Gin Leu Va Leu Val Va, Ala Val G1 Pro Val G1 Lys Val Gi1 Leu Asp G1 Thr Pro Al Pro Val G1 115 Cys Gly Ly 130 Ser Glu Le Gly Ser Al Leu Ala Il 18 Gly Leu Le 195 Ala Met Gi 210 Lys Giu As Lys Leu Ii Lys Pro Se 26 Gly Phe Va 1 n y a 0 y 5 a e 0

U

y 'p Asp Gly Gly Tyr Phe Leu Phe Gly Al a Pro Val 165 Tyr Asp Asp Pro Ala 245 Asp Arg 8

T

cobacterium bovis Arg Ala Thr Leu Gin 70 Arg Giu Gin Gly Gly 150 Val His Pro Ala Ala 230 Asn Leu Thr Val Val Al a Gin 55 Ser Ala Trp Ser Cys 135 Trp Gly Pro Ser Gly 215 Trp, Asn Gly Ser Arg Gly Thr 40 Val Giy Gin Tyr Ser 120 Gin Leu Leu Gin Gin 200 Giy Gin Thr Gly Asn 280 Gly Ala 25 Aia Pro Gly Asp Asp 105 Phe Thr Gin Ser Gin 185 Ala Tyr Arg Arg Asn 265 Ile Ala Val Ala Leu Gly Ala Ser Pro Ala Asn 75 Asp Phe 90 Gin Ser Tyr Ser Tyr Lys Ala Asn 155 Met Ala 170 Phe Val Met Gly Lys Ala Asn Asp 235 Val Trp 250 Asn Leu Lys Phe Thr Val Phe Ser Ser Ser Gly Asp Trp 140 Arg Ala Tyr Pro Ser 220 Pro Val Pro Gin Gly Met Ser Gly Ser Arg Met Gly Pro Ala Gly Trp Leu Ser 110 Trp Tyr 125 Glu Thr His Val Ser Ser Ala Gly 190 Thr Leu 205 Asp Met Leu Leu Tyr Cys Ala Lys 270 Asp Ala Ser Arg Leu Val Pro Gly Arg Asp Leu Tyr Asp Ile Val Val Gin Pro Phe Leu Lys Pro 160 Ala Leu 175 Ala Met Ile Gly Trp Gly Asn Val 240 Gly Asn 255 Phe Leu Tyr Asn 275 285 Ala Gly Gly Gly His Asn Gly Val Phe Asp Phe Pro Asp Ser Gly Thr WO 99/32634 PCTINZ98/00189 His 305 Leu Gly 290 Ser Trp Gin Arg Al a 14 295 300 Giu Tyr Trp Gly Ala Gin Leu Asn Ala Met Lys Pro Asp 310 315 320 Ala Leu Gly Ala Thr Pro Asn Thr Gly Pro Ala Pro Gin 325 330 335 Met Ile Gly Giu Gin Gly Ala Gly Lys Leu 145 Ala Ala Leu Ala Ala 225 Asn Leu Ser Asn <210> <211> 32: <212> PR~ <213> Myc <400> Thr Asp Va Gly Thr Al Gly Ala Al Tyr Leu Gl Phe Gin Se: Leu Arg Al Phe Glu Tr] Gly Gin Se: 115 Ala Gly Cy 130 Pro Gin Tr Ala Ilie Gi1 Tyr His Pr 18 Asp Pro Se 195 Gly Gly Ty 210 Trp Glu Ar Asn Thr Ar Gly Gly Al.

26 Ser Asn Le 275 Ala Val Ph2 a a 0 r p

Y

0 0 r r g a 0 Ser Ala.

Thr Val Gly Gin Tyr Ser Gin Leu Leu 165 Gin Gin Lys Asn Leu 245 Asn Lys Asn Arg Ala Ala Pro Gly 70 Asp Tyr Phe Thr Ser 150 Ser Gin Gly Ala Asp 230 Trp Ile Phe Phe Lys Val Gly Ser 55 Asn Asp Gin Tyr Tyr 135 Ala Met Phe Met Al a 215 Pro Val Pro Gin Pro 295 :obacterium bovis Ile Arg Ala Trp Gly Arg Arg Leu Met Val Ala 40 Pro Asn Tyr Ser Ser 120 Lys Asn Al a Ile Gly 200 Asp Thr Tyr Ala Asp 280 Pro Leu 25 Phe Ser Ser Asn Gly 105 Asp Trp Arg Gly Tyr 185 Leu Met Gin Cys Giu 265 Ala Asn Pro Ser Met Pro Gly 90 Leu Trp Giu Ala Ser 170 Al a Ile Trp Gin Gly 250 Phe Tyr Gly Gly Arg Gly Ala 75 Trp Ser Tyr Thr Val 155 Ser Gly Gly Gly Ile 235 Asn Leu Lys Thr Leu Pro Arg Val Asp Ile Ser Leu 140 Lys Ala Ser Leu Pro 220 Pro Gly Glu Pro His Val Gly Asp Tyr Ile Val Pro 125 Leu Pro Met Leu Ala 205 Ser Lys Thr Asn Ala 285 Ser Giy Leu Ile Leu Asn Met 110 Ala Thr Thr Ile Ser 190 Met Ser Leu Pro Phe 270 Gly Trp Leu Pro Lys Leu Thr Pro Cys Ser Gly Leu 175 Al a Gly Asp Vai Asn 255 Val Gly Glu Ala Val Val Asp Pro Val Gly Giu Ser 160 Al a Leu Asp Pro Ala 240 Glu Arg *His *Tyr 290 Trp Gly Ala Gin Leu Asn Ala Met Lys Gly Asp Leu Gin Ser Ser Leu WO 99/32634 WO 9932634PCT/NZ98/OO1 89 305 Gly Ala Gly 315 Met Phe Gly Lys Gly Tyr Ile Ile Pro Leu 145 Pro Leu Leu Gly Gly 225 Ile Asn Leu Ala Thr 305 <210> 36 <211> 33: <212> PR' <213> My <400> 36 Lys Phe Le Pro Ala Ar Leu Val Gi Pro Gly Le' His Asp Ii Leu Leu As Asn Thr Pr Met Pro Va 115 Ser Gin Gi 130 Thr Gin G1 Thr Gly As Ile Leu Al 18 Ser Gly Ph 195 Leu Ala Me 210 Pro Ser T1 Pro Arg Le Gly Ala Pi 26 Giu Ser LE 275 Aia Ser GI 290 His Ser T~ u y

U

e p 0 0 1 y u n a 0 eo Gin Leu Val Pro Lys Gly Al a Gly Asn Met Al a 165 Ser Leu Asn Asp Val 245 Asn Thr Gly Pro Ile 325 GinI Thr Val Val Ile 70 Leu Phe Gly Gly Pro 150 Ala Tyr Asn Asp Pro 230 Ala Giu Leu Arg Tyr 310 Miet Ile Giy Glu 55 Glm Arg Glu Gin Gln 135 Ser Val Tyr Pro Ser 215 Ala Asn Leu Ser Asn 295 Trp Arg Ala Asp 40 Tyr Phe Al a Giu Ser 120 His Trp Gly Pro Ser 200 Gly Trp Asn Gly Thr 280 Gli Asr Lys Val 25 Thr Leu Gin Gin Tyr 105 Ser Tyr Leu Leu Gin 185 Giu Gly Lys *Thr *Giy 265 *Asn Val Gin Leu Ile Ala Gin Gly Giu 90 Tyr Phe Thr Gin Ser 170 Gin Gly Tyr Arg Arg 250 Asp Giu Phe Gin Asn 330 Phe GilS Ile Val Gly 75 Asp His Tyr Tyr Al a 155 Met Phe Trp Asn Asn 235 Ile Asn Ile Asn Leu 315 Gly Thr Ala Pro Gly Tyr Ser Ser Lys 140 Asn Ser Pro Trp Al a 220 Asp Trp Ile Phe Phe 300 Val Leu Ala Vai Ser Gin Asn Giy Asn 125 Trp Lys Gly Tyr Pro 205 Asn Pro Val Pro Gin 285 Pro Ala Ala Leu Ala Pro His Gly Leu 110 Trp Glu Asn Ser Ala 190 Thr Ser Met Tyr Ala 270 Asn Pro Met Al a Leu Phe Ser Al a Trp Ser Tyr Thr Val Ser 175 Ala Met Met Vai Cys 255 Lys Thr Asn Lys Lys Al a Ser Met Val Asp Val Gin Phe Leu 160 Ala Ser Ile Trp Gin 240 Gly Phe Tyr Gly Pro 320 3

T?

cobacterium ieprae Asp Ile Gin Gin Leu Asn Gly Set Asn Asn Ala WO 99/32634 WO 9932634PCT/NZ98/OOI 89 Met Leu Gly Arg Gly Tyr Ile Ile Pro Leu 145 Pro Leu Leu Gly Gly 225 Ile Asn Leu Ala Thr 305 Asp Ala Thy.

PrC Lei.

Prc Arc Let Asi Met Se~ 13C Th: Il Se: Le 21 Pr Pr Gi Gi Al 29 Hi Il Pr :210> :211> :212> :213> :400> Phe Phe Arg Arg 1Val Gly Gly Leu Asp Ile iLeu Asp i Thr Pro 100 :Pro Val 115 r Gin Ser 0 Arg Glu Gly Asn e Leu Ala 180 r Gly Phe 195 u Ala Met 0 Ser Ser Arg Leu y Thr Pro 260 u Giy Leu 275 a Asp Gly 0 s Ser Trp e Gin His o Ala Ala 340 <210> 38 <211> Glu Val Thr Pro Lys Gly Al a Gly Asn Met Ala 165 Ala Leu Asn Asp Val 245 Ser Thr Gly Pro Val 325 37 340

PRT

Mycobacterium tuberculosis Gin Ala Phe Val Val 70 Leu Phe Gly Gly Pro iso Ala Tyr Asn Asp Pro 230 Ala Asp Leu Arg Tyr 310 Val Ile Gly Glu 55 Gin Arg Glu Gin Gin 135 Ala Val Tyr Pro Ser 215 Al a Asn Leu Arg Asn 295 Trp Arg Ala Gly 40 Tyr Phe Ala Giu Ser 120 Asn Trp Gly Pro Ser 200 Gly Trp Asn Gly Thr 280 Gly Asn Arg Ala 25 Pro Leu Gin Gin Tyr 105 Ser Tyr Leu Leu Gin 185 Glu Gly Lys Thr Gly 265 Asn Val Glu Leu 10 Met Al a Gin Giy Asp 90 Tyr Phe Thr Gin Ser 170 Gin Giy Tyr Arg Arg 250 Asp Gin Phe Gin Arg Ser Ala Ala Thr Thr Gly Thr Val Gly 75 Asp Gin Tyr Tyr Aia 155 Met Phe Trp Asn Asn 235 Ile Asn Thr Asn Leu 315 Ala Aia Pro Gly Tyr Ser Thr Lys 14 0 Asn Ser Pro Trp Ala 220 Asp Trp Ile Phe Phe 300 Val Val Gly Ser Pro Asn Gly Asp 125 Trp Lys Gly Tyr Pro 205 Asn Pro Val Pro Arg 285 Pro Aila Leu Al a Ala His Gly Leu 110 Trp Giu Gly Gly Ala 190 Thr Ser Met Tyr Al a 270 Asp Pro Met Val Phe Ser Ala Trp Ser Tyr Thr Vai Ser 175 Ala Leu Met Val Cys 255 Lys Thr Asn Lys Tyr Ser Met Val Asp Vai Gin Phe Ser i6 0 Ala Ser Ile Trp Gin 240 Gly Phe Tyr Gly Ala 320 Leu Asn Gly Ala Thr Pro Pro Ala Ala Pro Ala 330 335 WO 99/32634 WO 9932634PCT/NZ98/00189 <212> DNA <213> Artificial Sequence <220> <223> Probe made in a lab <400> 38 agcggctggg acatcaacac <210> 39 <211> <212> DNA <213> Artificial Sequence <220> <223> Probe made in a lab <400> 39 cagacgcggg tgttgttggc <210> <211> 1211 <212> DNA <213> Mycobacterium vaccae <400> ggtaccggaa gcacgccgtt gctggagggt atggtgcctt aac tcg ccgg gacatcaaca gtcggtggcc accgtgacdt aaccgcgcgg gcgctgaacc ttcctgaacc ggcggcttca cagcgcaacg gtctactgcg ctcgaaggtc ggccacaacg cgcgagctgc acgaagcccc taaccgaaat cacgaggtgg tccagcgtga gctggaggat tcggcgtcgt cggcgaccgc cgccgtcgat ctctctacct ctcaggcttt agtccagctt acaagtggga tcaagccgac tggcgacctg cctccgaggg aggccgacga atccgatgct gtaacggcca tgaccatccg gtgtgttcaa aggcgatgaa cggccgattg caacgcgatg gcgagcaatc tgacggtatg ggctgtcgcg cggagcattc ggggcgcgac gctcgacggc cgagtggttc ctacaccgac gaccttcctg cggcagcggc gc acc cggag ctggtggccg catgtggggc gaacatcccg gcccaccgag caccaacgag cttcccggcc gcctgacctg cggccgaggg gtggctcatc cttcctgccc agacttcttg acagcgatga tcccggccag atcaagatcc ctgcgtgcgc ctcgacagcg tggtacgccc acccaggagc cctgtcggtc cagttcatct ttcctgatca aagaccgagg accctggtcg ctcggcggcg accttccgcg aacggcacgc caggcgcac c tttcgtcgtc aggaacgccg gacggagagg acaggattcg tgcctgcttt gtctgccggt agttccagag aggaggactt gcatctccgt ccgcccgtaa tcccgggctg tgtcgatggc acgcgggctc acatctcgat ggatcccaac ccaacaacac gcgacctgcc acaactacat acaactgggc ttctctgacg cggggctact agggggtcat tcaacatcca tgggccttgg ggtgggcctg ggagtacctg cggtggcgag caacggctgg ggtgatgccg caagggcccg gctgcaggcc gggttcggcc gatgtccggc gggtgacgcc agcggttgga ccgtatctgg cgccacgttc cgccgcgggt gtactggggt gttgcacgaa gtggccgaca tgcgctacga cgtcgagtac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1211 <210> 41 <211> 485 <212> DNA <213> Mycobacterium. vaccae WO 99/32634 WO 9932634PCT/NZ98/OOI 89 <400> 41 agcggctggg acatcaaCaC atcatgcccg tcggcgggca aaggccggct gccagaccta ctcgccgcca acaagggggt ggttcggcgg cgctgacgct ctgtcgggct acctgaaccc ggtgacgcgg gcggctacaa gcctggaagc gcaacgaccc ctctc cgccgccttc gtccagcttc caagtgggag cgacccgaac ggcgatctac gtccgagggg ggc caacgac gatggtcaac gagtggtacg tacagcgact acgttcctga cgcaacgcgg cacccgcagc tggtggccga atgtggggtc at cggcaagc tcgactcggg ggtacagccc cccaggagct ccgtcggtct agttccagta tgctgatcaa caccgaagga tggtggccaa tctcgcggtg ggcctgcggt gccggcctac gtccatggcc cgccgggt cg catctcgatg cccgagcagc caacaccccc <210> 42 <211> 1052 <212> DNA <213> Mycobacterium vaccae <400> 42 gttgatgaga gcaaaggcgg ctgatcggct gtcgagtacc ggtggcggta ggctgggaca atgccggtcg gggcagaact gaggccaacc agcgcggcgc tcaggcttcc gacgcaggcg cgcaacgacc tactgcggca gccgcgcagt atcgcagccg gggtactggg caggccaccg aaggtgggt t cgatgcaccg tcgccggggg tcgacgtgtt ctcatgcggt tcaacacccc gcggacagtc acacctacaa gcggagtgtc tgacctacgc tgaacccgtc gcttcaacgc cgatggtcaa ccggcacccc tcctcgaagg gcggcaccaa ggcagcagct cc tag ccac c gtttgccgtt ggtgggcgtt ttcggcaacg ctcgccgtcg ctacctgctc tgcgttcgag cagct tctac gtgggagacg gcgcaccggc gatccatcac cgagggctgg cgagagcatg catcaaccag gtcggagctg attcacgttg cggtgtcttc gcagcagatg caccccacac atgaagttca gccgatatgg gccggggcat atgggccgcg gacggtctgc tggttctacg agcgactggt ttcctgaccc aacgcgttcg ccgcagcagt tggccgatgc tggggc ccgt ctggtggcca gacaccggga cggaccaaca aacttcccgg aagcccgaca cc cagagaagtg ccgccgttgc t Ct CCcggc c acatccgggt gtgcccagga agt ccggct t accagccgtc aggagctgcc t cgg CC tgt c tcatctacgc tgatcgggct cctcggaccc acaacacccg ccccgggcca tcgccttccg cctcgggcac tccagcgggt gcggggctcc gc tgcccgga cggt cttcct ccagttccag cgactacaac gtcgacgatc tcggggcaac gacgtggctg gatggcgggc ctcgtcgctg ggcgatgaac ggcgtggaag gatctggatc gaacctgatg tgacaactac ccacagctgg tctgggagct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1052 <210> <211> <212> <213> 43 326

PRT

Mycobacterium vaccae <400> 43 Met Arg Leu Leu Asp Arg Ile Arg Gly 1 Val Pro 10 Pro Trp, Ala Arg Arg Phe Gly Val Ala Val Ala Thr Ala Met Met Phe Ala Leu Val Gly Leu Ala Leu Pro Val Gly Gly Ser Ala Thr Ala Gly Al a 40 Pro Ser Arg Pro Gly Giu Tyr Gin Phe Leu Met Val Pro Ser 55 Gin Ser Gly Gly Giu 70 Arg Ala Gin Glu Asp Ser Met Gly Gly Asn Ser Pro Phe Asn Gly Arg Asp Ile Leu Tyr Leu Asp Ile Asn Lys Ile Leu Asp Thr Gin Leu WO 99/32634 WO 9932634PCT/NZ98/OO1 89 Ala Phe Gly Gly Lys Giy 130 Leu Pro 145 Gly Pro Thr Trp Leu Asn Gly Asp 210 Giy Ile 225 Pro Thr Gly Gin Giu Giy Ala Ala 290 His Asn 305 Leu Gin Glu Gin 115 Pro Giy Val His Pro 195 Ala Pro Leu Pro Leu 275 Giy Trp Ala Trp Phe Leu 100 Ser Ser Phe Thr Vai Thr Trp Leu Gin 150 Gly Leu Ser 165 Pro Giu Gin 180 Ser Glu Giy Gly Gly Phe Thr Ala Val 230 Val Ala Asn 245 Thr Giu Leu 260 .Thr Ile Arg Gly His Asn Ala Tyr Trp 310 His Leu Leu 325 Asp Tyr Tyr 135 Ala Met Phe Trp Lys 215 Gly Asn Gly Thr Gly 295 Gly Ser Thr 120 Lys Asn Al a Ile Trp 200 Ala Gin Thr Gly Asn 280 Val Gly 105 Asp Trp Arg Gly Tyr 185 Pro Asp Arg Arg Gly 265 Glu Phe Ile Trp Glu Aila Ser 170 Ala, Phe Asp Asn Ile 250 Asp Thr Asn Ser Val Val Met Pro Val 110 Tyr Thr Val 155 Ala Gly Lea Met Asp 235 Trp Leu Phe Phe Aia Phe 140 Lys Al a Ser Ile Trp 220 Pro Val Pro Arg Pro 300 Ala Pro 125 Leu Pro Leu Met Asn 205 Giy Met Tyr Al a Asp 285 Al a Met Ala Thr Thr Asn Ser 190 Ile Lys Leu Cys Thr 270 Asn Asn Lys Arg Asn Gin Glu Gly Ser 160 Leu Ala 175 Gly Phe Ser Met Thr Glu Asn Ile 240 Giy Asn 255 Phe Leu Tyr Ile Gly Thr Pro Asp 320 Arg Glu Leu Gin <210> 44 <211> 161 <212> PRT <213> Mycobacterium vaccae <400> 44 Ser Gly Trp Asp Ile Asn Thr Ala Ala Phe Glu Trp Tyr Val Asp Ser 1 5 10 Gly Asp Trp Lys Gly Tyr Pro Leu Trp Glu Giy Ser Ala Met Ala Tyr Thr Val Ala Gly Leu Val Ser Phe Asp Ala Ser 100 Ile Ile Pro Leu Pro Leu Leu Asn Met Ala Thr Asn 70 Thr S er Ile Pro Val Cys Gly 40 Gin Glu 55 Arg Asn Leu Ala Gly Tyr Ser Met Gly Gly 25 Lys Ala Leu Pro Ala Ala Ile Tyr 90 Leu Asn 105 Gly Asp Gin Giy Ala Val 75 His Pro Ala Ser Cys Tyr Gly Pro Ser Gly Ser Phe Gin Thr Leu Aia Leu Ser Gin Gin Glu Gly 110 Giy Tyr Tyr Tyr Al a Met Phe Trp Lys Ser Lys Asn Ala Gin Trp Ala WO 99/32634 WO 9932634PCTINZ98/OOI 89 115 Asn Asp Met 130 Asn Asp Pro 145 Leu 120 125 Trp Gly Pro Pro Lys Asp Pro Ser Ser Ala Trp Lys Arg 135 140 Met Val Asn Ile Gly Lys Leu Val Ala Asn Asn Thr Pro 150 155 160 Met Arg Gly Leu Ile Asp Pro Val Gly Glu 145 Asn Al a Phe Met Ser 225 Leu Pro Gin Asn <210> <211> 33~ <212> PR <213> My( <400> Lys Phe Th Val Gly Va Phe Ala Gl' Pro Val Gli Arg Val Gl Gly Leu Ar Ala Phe Gi Gly Gly G1 115 Asn Gly Gi 130 Leu Pro Th Ala Phe Va.

Ile His Hi 18 Leu Asn Pr 195 Asn Asp Al 210 Asp Pro Al Val Ala As Ser Glu Le 26 Phe Leu G1 275 Tyr Ile A) ri 1 0 n n r 1 0 *0 a -u Glu Ala Gly Tyr Phe Ala Trp Ser Asn Trp Gly 165 Pro Ser Gly Trp Asn 245 Asp *Gly *Ala Lys Asp Ser Leu Gin 70 Gin Phe Ser Tyr Leu 150 Leu Gin Glu Gly Lys 230 Thr Thr Phe Gly Trp Met Ala Asp 55 Gly Asp Tyr Phe Thr 135 Glu Ser Gin Gly Phe 215 Arg Arg Gly Thr Gly krg Al1a rhr 40 Val1 Gly Asp Giu Tyr 120 Tyr Ala Met Phe Trp 200 Asn Asn Ile Thr Leu 280 Thr Gly Ala 25 Ala Phe Gly Tyr Ser 105 Ser Lys Asn Ala Ile 185 Trp, Al a Asp Trp Pro 265 Arg Asn S er 10 Val Gly Ser Thr Asn 90 Gly Asp Trp Arg Gly 170 Tyr Pro Glu Pro Ile 250 Gly Thr Gly cobacterium vaccae Ala Ala Ala Pro His 75 Gly Leu Trp Giu Gly 155 Ser Ala Met Ser Met 235 Tyr Gin Asn Val Lys Leu Phe Ser Ala Trp Ser Tyr Thr 140 Val Al a Ser Leu Met 220 Val Cys Asn Ile Phe Al a Pro Ser Met Val Asp Thr Gin 125 Phe Ser Ala Ser Ile 205 Trp Asn Gly Leu Ala 285 Asn Ala Gly Arg Gly Tyr Ile Ile 110 Pro Leu Arg Leu Leu 190 Gly Gly Ile Thr Met 270 Phe Phe M4et Leu Pro Arg Leu Asn Met Ser Thr Thr Thr 175 Ser Leu Pro Asn Gly 255 Ala Arg Pro His Ile Gly Asp Leu Thr Pro Arg Gin Gly 160 Tyr Gly Al a Ser Gin 240 Thr Ala Asp Ala 290 295 300 Ser Gly Thr His Ser Trp Gly Tyr Trp Gly Gin Gin Leu Gin Gin Met WO 99/32634 WO 9932634PCT/NZ98/001 89 305 Lys Pro Asp <210> <211> <212> <213> 310 315 Ile Gin Arg Val. Leu Giy Ala Gin Ala Thr Ala 325 330 320 46 795

DNA

Mycobacterium vaccae <400> 46 ctgccgcggg tttgccatct ccggtaccgt ccggcgatgt ctcggcgcag cagcattggt caggaagggc accaggtccg ttctatctga cgacgcagcc gccaagcggg agaaggtcag gccgacccga actgggcgat ccgaacgcgc actgcgacat ccctacaacg tgcggtgcca gcgccggcgg cagcggctcg cgacgtaaag atcgctggcc ccagcacggc gtctaactcc cgggcgggcc gatcaccacg cgatggcacc ggcga cttgggtcct gaccaacatg ggccgccacg ttacacgctc gccgagcatg cctcgccccg ccttcaggtc cgccgtcgat gctcggtcag cggtgcagca c cgcgcggc c agacccttgg ctggtgccct gggt cgggag cgaacagcga gggatggtc~j acctcggccg caggcgttca ggtgtgccgt agcagcacca ggccaggagg tggtgagtca ccccgaggcg cctcggcgag tctgcgtggg cccggtccgc gccatgttct caacgaagct qcgcqgcgac gcgcttacga acgccgacgc gggtcttcga cccgcggtgg tgctcagcca cctcgccgag ctgggtcgcg gatctgctcc tgccaccgcg ctccgcacgc gggtaacgat aggagcggcg ggcgaacgc c gttcgacctg gtatgcgttc aaccacgatg gcaggccgcc gcacgacgac agtccggcca cgggtcagcg gggtagacca cccgggacac acgaaatcgt 120 180 240 300 360 420 480 540 600 660 720 780 795 <210> 47 <211> 142 <212> PRT <213> mycobacterium vaccae <400> 47 Arg Thr Ala Thr Thr Lys Leu Gly Met 1 Leu Al a 10 Ala Ala Leu Gly Ala Ala Ala Val Ala Ala Thr Gly Met Val Ser 25 Leu Ala Thr Ala Asn Ala Gin Ala Tyr Glu Giu Gly His Phe Asp Leu Asn Ala Asp Gin Val Arg Tyr Thr 40 Thr Thr Ser Ala Gly Phe Tyr Leu Ala Tyr Ala 70 Pro Trp Val Thr 55 Phe Gin Pro Pro Ser Lys Met Gin Ala Phe Val Ser Leu Ala Pro Gly Val Phe Ala Lys Arg Giu Thr Thr 90 Thr Thr Arg Giu 75 Met Ala Asp Pro Ala Ile Leu Asn Ala His 115 His Asp Asp 130 Gin 100 Cys Val Asn Trp Ala Pro Ser Ser Gly Gly Gin Ala 105 Asp Asp Ile Ala Gly Gin Giu 110 Val Leu Ser Gin 125 Gin Trp Pro Tyr Asn Val 135 Cys Gin Leu Gly 140 <210> 48 <211> 300 <212> DNA PCTINZ98/00189 Wo 99/32634 <213> mycobacteriuM vaccae <400> 48 gccagtgcgc caacggtttt cgaaggctgc gcgacgagtt gacaaggacg tggacgggat ctgttcgtct cgggcgacaa gagctgctgg ccgcccagtt catcgatgcc cgacttccgg ccgccaggac ccttcgaaag gtaagtgttc gcacacaacc tatctcgtCg ccgggCgtgC ggtgCggCgC cgccgaaatt ccggtgggCC gcgtcgtctc cggacgggcg tcaacacgat gcattccacg ctgcgcttgc ggtgatgggg cggtctcgca ccagatcgcc ccgataatcg 120 180 240 300 <210> 49 <211> 563 <212> DNA <213> mycobacterium vaccae <400> 49 ggatcctcgg ccggctcaag agtccgcgcc ttcagtcggg cctgcgaggc gctgtaccac gtcgagcttg ccaaagtgca actgggtgaa accgtgctcg atgtgctgct caagcttctg ctgtggaagg ccctgaccgg gcgggccggc ctggtcgtcg cggactggcc cacgcccacc cggatcgccg acacccagaa gttgatcacc ctggccgacc gccagcgggt gcctgcccgg gcccatgtcc cggcggtgcg cgcgctggcc cgcgtccgaa tcggtcgagg tgc gaggtggatg ttcgcctggg ggtttctCgC cacccggtca gcgagcgaac ggatacgcgc gaggtgcgcc ttgtccggca tggcttgacc tgacgctgga acgagttctg acaccacggc tgccgttCgt gtctgggaaa tggatcaggc ggttccgcag tcgacaccgc gagggtgatg cggctacgag cgactggtat cgtgttggcc caccgaggtg tgtggagtca tgccgcacaa cgatcagggt gggtctggac agggcttcac 120 180 240 300 360 420 480 540 563 <210> <211> 434 <212> DNA <213> mycobacterium vaccae <400> 50 gggCgg9CC gccgacgcgc acgaacgtgg tacctcggtg gccgacgacc atggaggtgc gagtgcgcgg tcggtggcgt cgaggatgag ccatcgacgt tgaacgcacc acacgatcgc tggtgccgac tgctggccca tgctgggCCg ctac caagttcgaa cgccgtCgtC ggt cgcggtC cgagatcgcc cgacaccgtc ggcggtgCgC tcaggtcgcc gtcgtcaccg gaggtcgggc atcaccccga ggggagaagg gccgtgctgg tcggatgcgg atcggcggca ggatggcgtt tcggtggtCg tcggggtgga ccggaaatca cgcggcaggt ctgtagcgcg gctgctCCgg cgcggctttc ctgggacgcg ccacaccgac tcacccgcca tcccgaggcc cgaggattcg ttgcaggggc 120 180 240 300 360 420 434 <210> <211> <212> <213> 51 438

DNA

Mycobacteriumn vaccae <400> 5i ggatcccact cccgcgccgg ggtcgacggc taccgcgtgc ggcggtcgga ccgcagcgca ggacgcggtg gtgatcgcca gttggcgcac acccagcggg caagtggggc gtccccgagg cggcggccag tggccgaccc tgcacgacgt acgaccacta ccccgttcgt cgcggatcgt ctggtacggc ggtgtggagc cccggtgcCg cgaccacctc ggtgccgttg cgagttggac cattccagcg aacagatgtt ctggaggcgc gacatcgaca ggcatcggcg tggcacgaag tgctgatcga cgccctcacg ttcccgccgt ccatcgtcgc cacacctgcg cccaccgcat 120 180 240 300 360 WO 99/32634 PCT/NZ98/00189 23 cgacgacctg acgctggtct gcacccccgc ccggcacttc tccggccggt tgttctcccg cgactcgacg ctgtgggc 420 438 <210> 52 Ala Pro Val Gin Gly Glu <211> <212> <213> <400> Ser Ala Cys Ala Gly Val Asp Pro Asp Asn Leu Leu 87

PRT

Mycobacteriumn vaccae 52 Pro Thr Vai Phe Ile Asp Ala Ala His Asn Pro Gly Gly 10 Cys Arg Arg Leu Arg Asp Giu Phe Asp Phe Arg Tyr Leu 25 Val Ser Val Met Gly Asp Lys Asp Val Asp Gly Ile Arg 40 Gly Val Pro Asp Gly Arg Gly Leu Ala Leu Phe Val Ser 55 Leu Arg Lys Gly Ala Ala Leu Asn Thr Ile Gin Ile Ala 70 75 Ala Ala Gin Leu Gly Asp Trp Gly Val Leu Asn Ala Ile Gin 145 Al a 53 <211> 17 <212> PR <213> My <400> 53 Ser Ser Ai Gly Tyr Gi Asp Glu Ph Giu Gly Ph Leu Leu Ly Trp Lys Al Val Glu Se Leu Asp Gi 115 Thr Giu Va 130 Arg Val Pr His Val Pr

T

cobacterium vaccae a

U

e e s a r 0 n .1 *0 *0 Gly Phe Cys Ser Leu Leu Leu Al a Arg Aia Ala 165 Ser Ser Asp His Leu 70 Thr Val Ala Arg Arg 150 Val Arg Val Arg Ala Trp, Tyr 40 Thr Thr 55 His Pro Gly Arg Val Ala Ala Gln 120 Phe Arg 135 Leu Ser Arg Ala Arg Al~ 10 Cys Gli 25 Val Gli Ala Va.

Val Mel Ala Gi' 90 Asp Trj 105 Arg Il Ser As Gly Il Leu Al 17 i Glu Val Asp Val Thr Leu t p e a 0 Al a Leu Leu Pro 75 Ala Pro Ala Gin Asp 155 Trp Leu Ala Al a Phe Ser Thr Asp Gly 140 Thr Leu Tyr His Lys Val Thr Val Val Thr Glu Arg Pro Thr 110 Thr Gln 125 Leu Ala Ala Gly Asp Arg Phe Gin Leu Glu Leu Gly Lys Asp Leu Gly 175 Ala Leu Asp Val Gly Tyr Leu Arg Asp 160 <210> 54 <211> 144 WO 99/32634 WO 9932634PCTNZ98/OO1 89 Gly Phe Gly Ala Thr Ala Val Al a Val <212> <213> <400> Pro Gly Ala Ala Leu Gly Val Ile Ile Ala Asp Asp Pro Glu Ala Val 115 Ala Ile 130 <210> <211> <212> <213> 54 Pro Phe Gly Thr Glu Leu Ala 100 Ala Gly Arg Ala Arg Pro Ile Val Asn Arg Gly Asn Asp Trp Ile Al a 70 Pro Glu Glu Ser Ser Ala Asp Gly 55 Gly Thr Val Asp Cys 135 Lys Pro Ala 40 Val Glu Asp Leu Ser 120 Ser Phe Ile 25 Thr Asp Lys Thr Leu 105 Glu Gly Giu 10 Asp Asn His Ala Val 90 Ala Cys Cys Val Val Val Thr Gly 75 Ala Gin Al a Arg Val Thr Ala Val Val Asn Asp Tyr Asn His Val Leu Ala Val Val Leu 125 Gly Ser 140 Gly Val Al a Leu His Ala Arg 110 Gly Val Met Glu Pro Gly Pro Arg Ser Arg Ala

PRT

Mycobacterium vaccae Ala Val Val Asp Pro Gln Asp Gin Ser 145

PRT

Mycobacterium vaccae <400> Asp Pro 1 Val Leu Ser Asn Asp Val Ile Ser Leu Ala Ala His Asp Trp Pro Ala 130 Trp, 145 Thr Ile Arg Pro Asn His Leu His 115 Arg Pro Glu Cys Vai Asp Thr Arg 100 Glu His Ala 5 Val Ser Pro His Gln Lys Ala Phe Pro Ala Ala Asp Gly Tyr Pro Ser Arg 40 Leu Glu Ala 55 Tyr Asp His 70 Arg Ala Pro Trp Gly Val His Arg Ilie 120 Ser Gly Arg 135 Ala Arg 25 Ala Leu Leu Phe Pro 105 Asp Leu Ser 10 Val Val Pro Asp Val 90 Glu Asp Phe Trp Leu Gly Al a Ile 75 Val Al a Leu Ser Tyr Ala Pro Val Asp Pro Arg Thr Arg 140 Gly Asp Gin Asp Thr Leu Ile Leu 125 Asp His Pro Arg Ala Ile Gly Val 110 Val Ser Ser Val Met Val Val Ile Glu Cys Thr Ser Trp His Val Ala Gly Leu Thr Leu <210> 56 <211> <212> PRT WO 99/32634 WO 9932634PCTNZ98/OO1 89 <213> Mycobacteriumf vaccae <220> <221> <222> <223> <221> <222> <223>

UNSURE

Residue can

UNSURE

(2) Residue can be either Gly, Ile, Leu or Val be either Ile, Leu, Gly, or Ala <221> UNSURE <222> <221> UNSURE <222> (9) <400> 56 Xaa Xaa Ala Pro Xaa Gly 1 5 Asp Ala Xaa Arg <210> <211> <212> <213> <220> <221> <222> <223> 57 8

PRT

Mycobacterium vaccae

UNSURE

Residue can be either Ile or Leu Pro 1 <400> 57 Glu Ala Glu Ala Asn Xaa Arg <210> <211> <212> <213> <220> <221> <222> <223> <221> <222> <223> 58 11

PRT

Mycobacteriumn vaccae

UNSURE

(4) Residue can

UNSURE

Residue can be either Gin or Gly be either Gly or Gin Tyr Tyr Asp Asn Arg <400> 58 Ala Asn Xaa Xaa Glu Thr 1 WO 99/32634 WO 9932634PCTINZ98/OOI 89 <210> 59 <211> 34 <212> PRT <213> mycobacterium vaccae <400> 59 Asn Ser Pro Arg Ala Glu Ala Glu 1 5 Ala Asn Pro Ala Glu Tyr Tyr Asp Ala Asn Leu Arg Gly Tyr Phe Thr 10 Leu Arg Gly Ile Leu Ala Pro Ile Gly Asp <210> <211> <212> <213> <220> <223>

DNA

Artificial Sequence Made in a lab <400> ccggtgggcc cgggctgcgc <210> <211> <212> <213> 61

DNA

Artificial Sequence <220> <223> made in a lab <400> 61 tggccggcca ccacgtggta <210> <211> <212> <213> 62 313

DNA

Mycobacterium vaccae <400> 62 gccggtgggc ccgggctgcg cggaatacgc gcagggaatg tcgcaggacc cggtcgcggt gctgtacggc tgcactgtcg ggccagctca acagcggtca gtacacggtg ttcgcaccga ccacgatcga cgagctcaag accaattcgt tggtggccgg cca <210> 63 <211> 18 <212> PRT <213> Mycobacterium vaccae ggcagccaat ggcggcctcg atccgcaagt c caacgcggc cactgctgac cccactgggc aacaatccgg aaacctggtg atttagcaag cagcatcctg cggcctcggt agttgacaac gacaccctca ctgccggcat acctaccacg 120 180 240 300 313 WO 99/32634 WO 9932634PCT/NZ98/00189 <220> <221> <222>

UNSURE

.(17) <400>' 63 Giu Pro Ala Gly Pro Leu Pro Xaa Tyr Asn Glu Arg Leu His Thr Leu 1 5 10 Xaa Gin <210> <211> <212> <213:' <22 0> <221> <222> 64

PRT

Mycobacterium vaccae

UNSURE

(21) (21) <400> 64 Gly Leu Asp Asn Giu Leu Ser Leu Val Asp Gly Gin Gly Arg Thr Leu 1 5 10 Thr Val Gin Gin Xaa Asp Thr Phe Leu <210> <211> <212> <213> <220> <221> <222> 26

PRT

Mycobacteriumn vaccae

UNSURE

(3) <221> UNSURE <222> (21) (22) <221> UNSURE <222> (24) (24) <400> Pro Xaa Pro Asp Ile Glu Val Giu Phe Ala Arg Gly Thr Gly Ala 10 Pro Gly Leu Xaa Xaa Val Xaa Asp Ala <210> 66 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab WO 99/32634 WO 9932634PCT/NZ98/OO1 89 ':400> 66 accgccctcg agttctcccg gccaggtctg cc <210> 67 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 67 aagcacgagc tcagtctctt ccacgcggac gt <210> 68 <211> <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 68 catggatcca ttctcccggc ccggtcttcc <210> 69 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 69 tttgaattct aggcggtggc ctgagc <210> <211> 161 <212> PRT <213> Mycobacterium vaccae <400> Ser Gly Trp, Asp Ile Asn Thr Ala Ala Phe Glu Trp Tyr Val Asp Ser 1 5 10 Gly Leu Ala Val Ile Met Pro Val Gly Gly Gin Ser Ser Phe Tyr Ser 25 Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gin Thr Tyr Lys 40 Trp Glu Thr Phe Leu Thr Gin Glu Leu Pro Ala Tyr Leu Ala Ala Asn 55 Lys Gly Val Asp Pro Asn Arg Asn Ala Ala Val Gly Leu Ser Met Ala 70 75 WO 99/32634 PCT/NZ98/00189 Gly Tyr Pro Asn Asn 145 Leu Ser Ala Ala Gly Met Leu 115 Asp Met 130 Asp Pro Ala Leu Thr Leu Ala Ile Tyr 90 Ser Leu Ser Gly Tyr Leu Asn 100 105 Ile Asn Ile Ser Met Gly Asp 120 Trp Gly Arg Thr Glu Asp Pro 135 Met Val Asn Ile Gly Lys Leu 150 His Pro Gin Gin Phe Gin Pro Ser Glu Gly Trp Trp 110 Ala Gly Gly Tyr Lys Ala 125 Ser Ser Ala Trp Lys Arg 140 Val Ala Asn Asn Thr Pro 155 160 <210> 71 <211: 33 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 71 gagagactcg agaacgccca ggaagggcac cag <210> 72 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 72 gagagactcg agtgactcac cactgaccga gc <210> 73 <211> <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <221> unsure <222> <221> unsure <222> <221> unsure <222> <221> unsure i- ~i~ WO 99/32634 WO 9932634PCT/NZ98/OO1 89 <222> (15) <400> 73 ggngcngcnc argcngarcc <210> 74 <211> 825 <212> DNA <213> Mycobacterium vaccae <400> 74 ttggatccca gaggtcgacg cgggcggtcg gtggacgcgg gcgttggcgc cgcaagtggg atagacgacc cgcgactcga ggtggcgaca gatctgaccc cccgaggagg cccatccact ctgctgaccg gtggacccgg ctcccgcgcc gctaccgcgt gaccgcagcg tggtgatcag aca cc cagcg gcgtccccga tgacgctggt cgctgtgggc ccggatacac tgctgccgat cggtgcgcgc gggcgacatt ctgccgacgc agtcgacgtt ggcggcggcc gctggccgac catgcacgac ccacgaccac ggccccgttc ggcgcggat c ctgcaccccc gtcgtgggtg gaagagcttc cggggcctac cc at ctgga c ccgcctcgcc cgagcgggta cgacccgtgg agctggtacg c cggtgtgga gtcccggtgZ: tacgaccacc gtggtgccgt gtcgagttgg gcccggcact gtcaccggct gccgagatcg catcccgcgt ctgaccgagg ccgcatccgt cgcctgaccg tggcggttct gccattccag gcaacagatg cgctggaggc tcgacatcga tgggcatcgg actggcacga tctccggacg cgtcgcacaa gcgacgagta tcgccgacat tggacaacag ggtccgagcc tgccgattcc gaacc cgtgctgatc ttcgccctca gcttcccgcc caccatcgtc cgcacacctg agcccaccgc gttgttctcc ggcgttcttc cggtccgttc ccacatgaac cctgatggtg cgccgaacgc cggt cagcgg 120 180 240 300 360 420 480 540 600 660 720 780 825 <210> <211> <212> <213> 273

PRT

Mycobacterium vaccae Leu 1 Ser Trp <400> Asp Pro Thr Val Leu Ile Ser Asn Arg Pro Ala Pro Ala Ala Ala Ser Trp, Tyr Gly His Ser Glu Val Asp Gly Tyr 25 Arg Val Leu Ala His Asp Val Val Ile Ser Pro Cys Ser Pro Val Pro Leu Ser 40 Glu Ala Val Gly Pro Val Asp Pro Val Gln Arg Met Asp Ala Val Ala Leu Pro 55 Tyr Ala Ile His Asp His Gln Asp His Leu Asp 75 Val Asp Thr Ile Val Leu Ala His Thr Arg Arg Ala Pro Phe 90 Pro Val Pro Leu Gly Ile Gly Ala His Leu Asp Trp 115 Thr Pro Ala 130 Leu Trp Ala Leu 100 His Lys Trp, Gly Val 105 Ile Glu Ala Arg Glu Ala His Arg 120 Gly Asp Asp Leu Thr 125 Arg Ile Val Glu 110 Leu Val Cys Asp Ser Thr Arg His Phe Ser Trp Val 150 Thr Gly Tyr Ser 135 Val Arg Leu Phe Ser 140 His Thr Gly Ser 145 Gly Ser 155 Al a Lys Ala Phe Phe 160 Glu Gly Asp Thr Lys Ser Phe Glu Ile Gly Asp WO 99/32634 WO 9932634PCTNZ98/OO1 89 165 Asp Tyr Gly Pro Ala Phe Ala 195 Leu Asp Leu Phe 180 Asp Leu Thr Leu Leu Pro Ile Gly Ala 185 Pro 175 Tyr His Pro 190 Arg Ala His Ile His Trp Ile His Met Asn 200 Asfl Glu Giu Ala Val 205 Pro Thr Glu Val Ser Leu Met 210 Ala Thr Val 220 Giu Phe Arg Leu His Pro Trp Ser 235 Arg Pro Ala Glu Arg 240 Leu Thr Ala Al a 245 Val Ala Glu Arg Val 250 Thr Leu Thr Val Pro Ile 255 Trp Arg Pro Gly Gin Arg 260 Asp Pro Glu Phe Asp Pro Trp, 270 <210> 76 <211> <212> PRT <213> Mycobacterium vaccae <400> 76 Lys Thr Ile Ala Tyr Asp Giu Giu Ala Ala

I

<210> 77 <211> 337 <212> DNA <213> Mycobacterium vaccae <400> 77 gatccctaca ctggagaagg gaggccctgt aaggctccgg ggtggtcagg ctgggccagg tcctgctggt tcatccaggc ccacgctggt gcttcggtga tcgtcagcga cccgcaaggt cagctccaag cggcaagccg ggtcaacaag ccgccgcaag aagagtcggg cgtcgtcacc gtgtcgaccg ctgctgatca atccgcggca gcgatgctgc ctgtccctgg aaggaca tcaaggatct t cgc cgagga c ctt caagt c aggacatggc agaccgccga gctcccgctg cgtcgagggc cgtcgccgtc catcctcacc cgtctcgctg 120 180 240 300 337 <210> <211> <212> <213> 78 112

PRT

Mycobacterium vaccae <400> 78 Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys Val Ser 1 5 10 Leu Leu Pro Leu Leu Glu Lys Val Ile Gin Ala Gly 25 Ile Ile Ala Glu Asp Val Giu Gly Glu Ala Leu Ser 40 Asn Lys Ile Arg Gly Thr Phe Lys Ser Val Ala Val 55 Phe Gly Asp Arg Arg Lys Ala Met Leu Gin Asp Met Thr Val Lys Asp Lys Pro Leu Leu Thr Leu Val Val Lys Ala Pro Gly Ala Ile Leu Thr WO 99/32634 PCT/NZ98/OO1 89 32 70 75 Gly Gly Gin Val Val Ser Glu Arg Val Gly Leu Ser Leu Giu Thr Ala 90 Asp Val Ser Leu Leu Gly Gin Ala Arg Lys Val Val Val Thr Lys Asp 100 105 110 <210> 79 <211> 360 <212> DNA <213> Mycobacterium vaccae <400> 79 ccgtacgaga gcgggcgacg cgcaacgtcg gaggctgtca tctgccaccg atggacaagg agatcggcgc gcaccaccac cagccggcgc cccagtcgct cggcgatctc tcggcaacga tgagctggtc cgccaccgtg caacccgctc gctgaagtcg cgccggcgac gggtgtcatc aaagaggtcq ctcgctcagg ggcctcaagc gccaaggagg acccagatcg accgtcgagg ccaagaagac ctctggttcg gtggcatcga tcgagaccaa gcgagctcat agtcgaacac cgacgacgtc cgaaggcctg gaaggctgtc ggagcagatt cgccgaggcc cttcggcctg <210> <211> <212> <213> 120

PRT

Mycobacterium vaccae <400> Pro Tyr Glu Lys Ile Gly Ala Giu Leu Val Lys Glu Val Ala Lys Lys Leu Ala Thr Asp Gin Ala Pro Leu Gin Ser Asp Val Ala Gly Leu Val Arg Giu Giy Leu Lys Arg Leu Leu Lys Ser Asp Gly Gly Leu 40 Gly Ile 55 Ala Lys Ser Ala Thr 25 Arg Thr Thr Ala Thr Val Asn Val Ala Ala Glu Gly Ala Asn Ala Val Thr Giu Lys Ala Giu Val Glu 75 Giv Asp Thr Val Thr Lys Giu Gin Ser 70 Ile Ile Ala Thr Ala Ala Met Gin Ile Gly Ile Ala Giu Giu Giu Ser 115 Al a 100 Asn Asp Lys Val Gly 105 90 Asn Glu Gly Vai Glu Leu Thr Val Ile 110 Thr Phe Giy Leu 120 <210> 81 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 81 actgacgctg aggagcgaaa gcgtggggag cgaacaggat tag WO 99/32634 PCT/NZ98/00189 33 <210> 82 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 82 cgacaaggaa cttcgctacc ttaggaccgt catagttacg ggc 43 <210> 83 <211> <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 83 aaaaaaaaaa aaaaaaaaaa <210> 84 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 84 ggaaggaagc ggccgctttt tttttttttt t 31 <210> 8S <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> gagagagagc ccgggcatgc tsctsctsct s 31 <210> 86 <211> 238 <212> DNA <213> Mycobacterium vaccae <400> 86 ctcgatgaac cgctcggagc gctcgacctg aagctgcgcc acgtcatgca gttcgagctc aagcgcatcc agcgggaggt cgggatcacg ttcatctacg tgacccacga ccaggaagag 120 gcgctcacga tgagtgaccg catcgcggtg atgaacgccg gcaacgtcga acagatcggc 180 WO 99/32634 WO 9932634PCT/NZ98/00189 agcccgaccg agatctacga ccgtcccgcg acggtgttcg tcgccagctt catcgaat 238 <210> 87 <211> 79 <212> PRT <213> mycobacterium vaccae Leu Gin Tyr Ala Ile <400> 87 Asp Giu Pro Leu Giy Ala Leu Asp Leu Lys Leu 10 Phe Glu Leu Lys Arg Ile Gin Arg Glu Val Gly 25 Vai Thr His Asp Gin Giu Giu Ala Leu Thr Met 40 Val Met Asn Ala Gly Asn Val Giu Gin Ile Gly 55 Tyr Asp Arg Pro Ala Thr Vai Phe Vai Ala Ser 70 Arg His Val Met Ile Thr Phe Ile Ser Asp Arg Ile Ser Pro Thr Giu Phe Ile Giu <210> 88 <211> 1518 <212> DNA <213> Mycobacterium vaccae <400> 88 cactcgccat acatccagct catcagtaca aatccattgt gaaggtttac cacatcaccg gcgccggtga gacgcagact gggaagacga cgcctcgaag ttccagcact cgcagcaaga gtccggctga cgggtggcgt ctcggagcgc cgggaggtcg agtgaccgca atctacgacc gcgggccggt acgctgaagg gtgcgtccgg gcctgcgtgc ctggccgctc ctgctgcgcc cccggcgacg acgcttcccg gggtgttaca gagaaaatat gcgcgcttt c cgaaatgtaa cccacagcca c tgcagaac c tcgagatcga tctccatcgc ccacgttgcg gcgccgacgt acgcgctgtt aactcggcaa ccgaatttgc tggcccgggc tcgacctgaa ggatcacgtt tcgcggtgat gtcccgcgac gcaccggccg cacgcccggg aacgcatccg gtgccaccgt cggacgact c ccggcgacga acatccccac attgccga ataccccacc tcacagcgac ctgcgcggat attcgttgcg cgacggctgt tgcagaacag ccatgtcacg gcccggggag catgatcgcg gtcgaggac cccgcacatg aggcgaggtc cgagcgcagg actggtgaac gctgcgccac catctacgtg gaacgc cggc ggtgttcgtc ctccaaccgc cgagaccacg ggtcaccccg caccgacctg gaccgtgatc cgtgtacgtc caccgaggac agttcctcga gaagcccggc tctattgtcg gaatcacttg ccccgaggag acggcggatt aagcgcttcg ttcttctcca ggattcgaga ccacccaaca acggtctggg cgcaagcgcg cccgcccagc taccccagcg gtcatgcagt acccacgacc aacgtcgaac gccagcttca gattacgtcg atcgagcccg ggctcccagg accttccaag gcccacgtcg agctgggcac ctcgaagaga agtaaacgaa cgatgcctga agtccggggt cataggtccg gacctgccct ccgcggcacc gcgactacct tgctcggccc ccccgactga agcgcaacgt acaacgtcgc tcgacgagct tgtccggcgg cgctgctgct tcgagctcaa aggaagaggc agatcggcag tcggacaggc agatcgacgt gcgggcacgc acgcgccgac gtccggtggt gccccgagca cggaagcctc tgctcgacga cagaaccgtg tggggtccgg gtgacgaagg tcagatccgc gaccggcac a gcccaagggc ggccgtcgcg gtccgggtgt aggggcgat c c aac acggtg gtacggcccg gctggagatc gcagcagcag cgatgaaccg gcgcatccag gctcacgatg cccgaccgag caacctctgg tctcggCtcg caccctgatg cggtgacgtc gcggctctcg ggatctgccg cctggtgctt ct CCtgagtc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1518 <210> 89 WO 99/32634 WO 9932634PCT/NZ98/00189 Val Val Leu Gly Val His Gly Asp Pro Al a 145 Ala Ile Giu Asn Thr 225 Arg Gly Gly Gly Val 305 Ala Leu Glu <211> 37I <212> PR' <213> Myl <400> 89 Ile Glu Ill Ala Asp Al Gly Pro Se: Phe Glu Th: Ser Arg Th: Tyr Ala Lei Pro Arg Se: Glu Leu Le 115 Ala Gin Le' 130 Leu Val As: Leu Asp Le Gin Arg Gl 18 Glu Ala Le 195 Val Glu Gl 210 Val Phe Va Cys Thr Gl Ser Thr Le 26 His Ala Th 275 Ser Gln As 290 Thr Asp Le Pro Asp As Pro Leu Le 34 Ala Ser Le 355 e a r 0 Li Li n Li

U

0 u n 1 y

U

0 r p u p

U

0

U

Asp Asp Gly Pro Pro Phe Lys Giu Ser Tyr Lys 165 Val Thr Ile Al a Arg 245 Lys Leu Ala Thr Ser 325 Arg Val His Phe Cys Thr Pro 70 Pro Lys Ile Gly Pro 150 Leu Gly Met Gly Ser 230 Ser Al a Met Pro Phe 310 Thr Pro Leu Val Ser Gly Giu 55 Asn His Leu Val Gly 135 Ser Arg Ile Ser Ser 215 Phe Asn Arg Val Thr 295 Gln Val Gly Pro Thr Ile Lys 40 Gly Lys Met Gly Arg 120 Gin Ala His Thr Asp 200 Pro Ile Arg Pro Arg 280 Gly Gly Ile Asp Gly 360 Lys Ar 10 Ala Prc 25 Thr Th: Aia Ill Arg As Thr Va 90 Lys Gl' 105 Leu Th: Gin Gl Leu Le Val Me' 17 Phe Ii.

185 Arg Il Thr Gi Gly Gi Asp Ty 25 Gly Gi 265 Pro Gi Asp Va Pro Va Ala Hi 33 Asp Va 345 Asp As r cobacterium vaccae Phe Gly Asp Tyr Leu Ala 0 1~ r t 0 e e

U

n r 0

U

1 p Gly Thr Arg Val 75 Trp Giu Giu Arg Leu 155 Gin Tyr Al a Ile Ala 235 Val Thr Arg Ala Val 315 Val Tyr Ile Giu Phe Leu Arg Leu Glu Asn Thr Asp Asn Val Arg Phe Ala 125 Val Ala 140 Asp Glu Phe Glu Val Thr Val Met 205 Tyr Asp 220 Asn Leu Giu Ile Thr Ile Ile Arg 285 Cys Val 300 Arg Leu Gly Pro Val Ser Pro Thr 365 Phe Met Gly Val Val Lys i110 Giu Leu Pro Leu His 190 Asn Arg Trp Asp Giu 270 Vai Arg Ser Giu Trp 350 Thr Ser Ile Ala Phe Ala Arg Arg Al a Leu Lys 175 Asp Ala Pro Ala Val 255 Pro Thr Ala Leu Gin 335 Ala Glu Met Ala Asp Gin Tyr Val Arg Arg Gly 160 Arg Gin Gly Ala Gly 240 Leu Gly Pro Thr Ala 320 Asp Pro Asp Leu Glu Giu Met Leu Asp Asp Ser 370 375 WO 99/32634 WO 9932634PCT/NZ98/OO1 89 <210> <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <40-0> gagagactcg aggtgatcga gatcgaccat gtc <210> 91 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 91 agagactcga gcaatcggga agcgtgactc a <210> <211> <212> <213> 92 323

DNA

mycobacterium vaccae <400> 92 gtcgactaca aagaagactt ttgtcgcgca agcaggacat cgcgtcaagg gcctgggatg aatctgcgtc aggacctgtt tacatgaccg gcatggtcgg accatcgacg acctctggga caacgacaac aggcgccgac gctcaatgag ggactcgagc tctcgcctac tcc gagcagtggt ctggtgatcc atcagcgaag at cgacgagg aa caaggc ag tcgccaaggt ccaccgagtt ccggcgtgc gccgcaagtt ccaccggacg caaggagccg catggccgcg caatcgcaag caccgcgccg cgatatccgc <210> 93 <211> 1341 <212> DNA <213> mycobacterium vaccae <400> 93 ccccaccccc ccacctgctg cgc cgcggc c cagtgggacc ctccaactgg catcacggtc ggagccgttg ggccgcgcgc tcgcaagaat cgcgccgtac ttccctggag gcccgaatga gccgcgggcc tcgagcacca ccgctctata gactacaaag tcgcgcaagc gtcaagggcc ctgcgtcagg atgac cggc a ccgacgaaag ccgcacgccg tgaccctcgg cgt cacagga tggccgacgg aagacttcaa aggacatagg tgggatggct acctgttgga tggtcggtct gcacccgcac caccttgcgt ttcgtcgttc cagcggcccc tttcatcgca cgacaacgag cgccgacctg caatgagatc ctcgagcatc cgcctacaac atgtcccgtg cgccgcttca c tggcggcgt gccagcggcg gcgttccaga cagtggttcg gtgatcccca agcgaagccg gacgagggc aaggcagcca ggccgcgtca a cat cgat cc tcggcggtgg gcgggtccga ccctgcgcgt ccgcctcggg ccaaggtcaa ccgagttcat gcgtgcccaa gcaagttcac ccggacgcga gtctgttctc 120 180 240 300 360 420 480 540 600 660 tatccgcacc atcgacgacc tctgggatcc cgcgttcaag WO 99/32634 WO 9932634PCT/NZ98/O0l 89 cgacgtccag gaccaccgag agatccgtcg cgcgcaggcg catcgttccc gcagaaccag caagctggtc caaggtcgat gaacctgaag cgccgccgtc tgcggacgcg cgctccgtat gacggcctcg tccattcagc cttcaccggc tactccggtg gaatccggcg aaggccgccg gcgttcaccc cctgcatcgg tcgtgggcgg accggcggct aggagcataa ctgatggtcc gcatgatcat aggcggt cga aacgactacg acgtcgtgca gcgactggtt aggcgtggat agttcgtgc cggagaaccc cactgaccga gacgcggtgg atggccggtg gctctcgcag tctggtccgc ccgacgacct gctgcaggcg cgtcgacacg cgactacatc cgcactctcg gctgatcaac cgagcagacg tagtgccgat tcgccaccag ggcaactcgc gaacagaacg ggccgcagaa gacaaccccg atggtgatcc tacgaccgag gacatgaccg ccgtcggccg caggagttca gcgaggggca cagccgtcag cggagaatcc acagggggtc acatcgccat atctgcagtt cgtacaccac ccaactacgc acgaactcgc aggtgcaggc acactgcgta taaatggccc cggacaaggt 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1341 <210> <211> <212> <213> 94 393

PRT

Mycobacteriumf vaccae <400> 94 Met Ser Arg Asp Ile Asp 1 5 Arg Thr Leu Arg Arg Arg Giv Leu Thr Leu Gly Ser Pro His Leu Phe Ile Gly 25 Ser Phe Leu Leu 10 Gly Ala Arg Met Thr Ala Arg Gly Ala Ala Ala Ala Ala Ser Asp Ser Ser Gly Ala Ala Ala Cys Gly Thr Ser Leu Arg Val 40 Gin Gly Ala Ser Thr Thr Ser Asn Trp 70 Thr Ala Ser Ser Pro Asp Ser Gly Leu Tyr Met Ala Ala Asp 75 Asp Tyr Gly Phe Ile Al a Phe Gin Gly Ile Thr Asn Asp Asn Lys Gin Asp 115 Ala Arg Val Giu 100 Ile Gin Val 90 Val Trp Phe Ala Lys 105 Val Lys Glu Gly Ala Asp Leu 120 Trp, Lys Glu Asp Phe Ile Pro Thr Pro Glu 125 Ser Leu Ser Arg 110 Phe Met Ala Glu Ala Gly Lys Gly Leu Leu Asn Giu 130 Val Pro Ile 140 Leu Asn Arg Lys Arg Gin Asp Asp Ser Ser 145 Asp Ile 160 Giu Gly Arg Lys 165 Lys Thr Ala Pro Tyr 170 Arg Thr Gly Met Val Gly 175 Leu Ala Tyr Asp Leu Trp 195 Val Gin Asp Asn 180 Asp Ala Ala Thr Gly 185 Gly Asp Ile Arg Pro Ala Phe Lys 200 Ile Arg Val Ser Leu 205 Gly 190 Phe Ser Asp Asn Ser Pro Gly Leu Gly 210 Glu Asn Met 215 Ser Met Leu Ser Gin 220 Val Pro Thr Thr 225 Giu Gin Asn Asp Arg 245 Gly Giu 230 Gly Arg Ile Gin Gin Ser Asp Pro Arg Asn Ile 265 His Arg Gin Asp Leu Val Arg 240 Arg Leu 255 Tyr Ser Arg Arg Arg Pro 260 Ile Ala Gin Al a 270 WO 99/32634 WO 9932634PCTJNZ98/OO1 89 Gly Asp Val 275 Val Pro Giu Val Gin Leu Gin Asp Asn Pro Asp Leu 285 Met Gin Phe Ile Val Ile Pro Ser Gly Gly 290 Tvr Thr Asp 295 Lys Phe Vai Asp Thr 300 Trp Thr Gin Asn Gin 310 Tyr Ala Ala Giu Aia 31i5 Al a Ile Asp Tyr Ile 320 Asp Arg Aia Aia Lys Leu Val 330 Leu Phe Thr Gin Phe Vai 335 Pro Ala Leu Ser Ala Giu 355 Ser 340 Asn Met Thr Asp Giu 345 Pro Ala Lys Vai Pro Leu Ile Asn 360 Ser Ala Giu Asp Pro Aia 350 Gin Aia Asn Giu Phe Asn Leu Lys 370 Thr Aia 385 Ser Trp Ala Ala Tyr Aia Ala Vai 390 Leu Thr Asp 375 Thr Gly Gly Giu Gin Thr 380 <210> <211> <212> <213> 22

DNA

Mycobacteriumn vaccae <400> atgtcccgtg acatcgatcc cc <210> 96 <2ii> 2i <2i2> DNA <213> Mycobacterium vaccae <400> 96 atcggcacta ccaccgcgtc a <210> 97 <21i> 86i <212> DNA <213> Mycobacteriumn vaccae <400> 97 gccggcgctc cctcgttgtc tcggcaacta acgcgttcgt cgttcaaggc tcacgttcct tcaccgcgct gggcggtcat tcagcctgga cgccgcgcag gcatgctggt cccagaccac cggcggcggC acac acgggc gcatatctcg ggagaccggc cgtcgacgcg cgccacggtg cggccggttc gatccgcacc gggcgccatc cggcggtctg gaagatcgac cttcggcaag gttcatcccg catgatcggc cgcgctgagt gctgggttcg cgatcttctt ggctcggtgt ttcacgatgt ctgtgcctgt aagaacctga attgcgtgga gggctgctgc acctacaact ccgcgtctgc gtgatcctgc gccgtcggcg aacgtgatcc ctggggctga gaggatctgg ccgtggtgcc tcatgccgac accacgagca tgctggcgtt tcctggggct agacgatcct ctgacgaggg ggatcatctt tggaggcct c cgatggcgat acttcatcaa agaagcagtt tgttgctgat tatgaccacc gttcttctcg gctgacgttc gatcttccgc cccgctggcc ggtgatcctg ggccgacgaa ccggctgctg catgatcctg c caggacct c gcccggggtg cgccgactat cctggtcgtC cctgatcggc caggcaggcg ctggcacgca gcctgggact tcgttcggCt tacgtcatcg ccgttcttcg ggctgggtgg tccaccagct ccgctgtacg tactcgtcgg ctggccggga ctcggcagta aaggactatc gtgctcctct c cgcactggc 120 180 240 300 360 420 480 540 600 660 720 780 840 WO 99/32634 WO 9932634PCT/NZ98/OO1 89 caccgccgcc cagcaggatc c Val Gly Tyr Gly Leu Leu Ilie Gly Trp Leu 145 Al a Ile Phe Thr Val1 225 Leu Asp <210> 98 <211> 25: <212> PR' <213> My <400> 98 Val Pro Ph Ser Val Ph Val Asp Al.

Tyr Ala Ph Ala Tyr Va Gly Leu Va Ala Trp Th Ala Ile Gi 115 Ala Val 11 130 Pro Leu Ty Ser Gin As Leu Pro Me 18 Ilie Pro Al 195 Gin Thr T1h 210 Lys Asp Ty Ile Leu Il Leu Val 9

T

cobacterium vaccae e a e r 0 y e p t 0 a Phe Met Phe Val Ile Ile Ile Leu Gly Val Leu 165 Ala Val Met Pro Gly 245 Ser Pro Thr Al a Al a 70 Leu Leu Leu Gly Ser 150 Tyr Met Gly Ile Ala 230 Val Leu Ala Thr Leu Met Tyr 40 Thr Val 55 Phe Lys Pro Phe Ala Asp Pro Asp 120 Leu Thr 135 Leu Glu Ser Ser Pro Gly Asp Phe 200 Gly Asn 215 Aia Ala Leu Leu Arg Thr Thr Phe 25 His Glu Leu Cys Aia Gly Phe Val 90 Glu Gly 105 Glu Gly Tyr Asn Lys Ile Ala Pro 170 Val Leu 185 Ile Asn Val Ile Ala Leu Tyr Thr 250 Ser Ala G1i Leu Arg 75 Thr Trp Arg Trp Asp 155 Arg Al a Al a Gin Ser 235 Arg Leu Trp Ile Leu Phe Phe Val Leu Ile 140 Pro Ser Gly Asp Lys 220 Leu Al a Ser Glu Asp Phe Phe Arg Leu Ala Lys Asn Leu Ile Val Thr 110 Leu Ser 125 Ile Phe Arg Leu Phe Gly Ser Met 190 Tyr Leu 205 Gin Phe Gly Leu Leu Gly Thr Gly Ser Phe Leu Arg Ala Thr Met Leu Lys 175 Leu Gly Leu Met Ser 255 Gly Asn Phe Pro Ile Thr Leu Ser Ile Giu 160 Val Vai Ser Val Leu 240 Glu <210> <211> <212> <213> 99 277

DNA

mycobacterium vaccae <400> 99 gtaatctttg ctggagcccg tacgccggta ggcaaactca tgggttcgct caaggacttc aagggcagcg atctcggtgc cgtggcgatc aagggcgccc tggagaaagc cttccccggc gtcgacgacc ctgctcgtct cgtcgagtac gtgatcatgg gccaagtgct ctccgccggc gccggccaga tgcccgcccg ccaggccgcc gtcgccgccg gcatcccgtg ggacgtcgcc 120 180 240 WO 99/32634 WO 9932634PCT/NZ98/OO1 89 tcgctgacga tcaacaagat gtgcctgtcg ggcatcg <210> 100 <211> 92 <212> PRT <213> Mycobacteriumn vaccae <400> 100 Val Ile Phe Ala Gly Ala Arg Thr Pro Val Gly 1 5 10 Leu Lys Asp Phe Lys Gly Ser Asp Leu Gly Ala 25 Ala Leu Giu Lys Ala Phe Pro Gly Val Asp Asp 40 Giu Tyr Vai Ile Met Gly Gin Val Leu Ser Ala 55 Pro Ala Arg Gin Ala Ala Val Ala Ala Gly Ile 70 75 Ser Leu Thr Ile Asn Lys Met Cys Leu Ser Gly Lys Leu Met Gly Ser Val Ala Ile Lys Gly Pro Ala Arg Leu Val Gly Ala Gly Gin Met Pro Trp Asp Val Ala Ile <210> <211> <212> <213> <220> <221> <222> <223> <221> <222> <223> <221> <222> <221> <222> <400> Xaa Xaa Ala 1 101 12

PRT

Mycobacteriuim vaccae

UNSURE

Residue can be either Glu or Pro

UNSURE

(2) Residue can be either Pro or Glu

UNSURE

(7)

UNSURE

(12) (12) 101 Asp Arg Gly Xaa Ser Lys Tyr Arg Xaa 5 102 24

PRT

mycobacterium vaccae

*UNSURE

<210> <211> <212> <213> <220> <221i> <222> WO 99/32634 PCT/NZ98/00189 41 <400> 102 Xaa Ile Asp GJlu Ser Leu Phe Asp Ala Glu Glu Lys Met Glu Lys Ala 1 5 10 Val Ser Val Ala Arg Asp Ser Ala <210> <211> <212> <213> <220> <221> <222> 103 23

PRT

Mycobacterium vaccae

UNSURE

<221> UNSURE <222> (15) <221> UNSURE <222> (17) (17) <400> 103 Xaa Xaa Ile Ala Pro Ala Thr Ser Gly Thr Leu Ser Glu Phe Xaa Ala 1 5 10 Xaa Lys Gly Val Thr Met Glu <210> <211> <212> <213> 104

PRT

Mycobacterium vaccae <400> 104 Pro Asn Val Pro Asp Ala Phe Ala Val Leu Ala Asp Arg Val Gly 1 5 10 <210> 105 <211> 9 <212> PRT <213> Mycobacterium vaccae <220> <221> <222>

UNSURE

<400> 105 Xaa Ile Arg Val Gly Val Asn Gly Phe 1 <210> 106 <211> 485 <212> DNA WO 99/32634 WO 9932634PCT/NZ98/OOI 89 <213> Mycobacterium vaccae <400> 106 agcggctggg acatcaacac atcatgcccg tcggcgggca aaggccggct gccagaccta ctcgccgcca acaagggggt ggttcggcgg cgctgacgct ctgtcgggct acctgaaccc ggtgacgcgg gcggctacaa gcctggaagc gcaacgaccc ctctc cgccgccttc gtccagcttc caagtgggag cgacccgaac ggcgatctac gtccgagggg ggccaacgac gatggtcaac gagtggtacg tacagcgact acgttcctga cgcaacgcgg cacccgcagc tggtggccga atgtggggtc atcggcaagc tcgactcggg ggtacagccc cccaggagct ccgtcggtct agttccagta tgctgatcaa gcaccgagga tggtcgccaa tc tcgcggtg ggcctgcggt gccggcctac gtccatggcc cgccgggtcg catctcgatg cccgagcagc caacaccccc 120 180 240 300 360 420 480 485 <210> 107 <211> 501 <212> DNA <213> Mycobacterium vaccae <220> <221> unsure <222> (441) (441) <221> unsure <222> (450) (450) <400> atgccggtgc gtgggcgctg gtgacgttcg ttcagcggtc gacggcccgg aggcagtggg cgcgagtacg ttcggcacct gtcgcggcct 107 gacgtgcgcg agggcaccgc cctccgacaa agt acacct t cgccgtcgaa tgttcaacta ccgccgcgcg ggcgcaccga atccggcgta cagtgcgctt actggcggcg actcggcacg cagcacgtcc cccgacgatt caactggcag ttcgctggtg natcctggan gcgtccgtga acgccggact agtgtggccg tgtgtgggca ccgcagcccg tgggagtgct ttctacgccc ggcct ctgca ccttcgtcgc ggccgcgtgc ggagcgggcg, cccgccagcc c ctgcgtggc cgcgctacac tccgcggcgc cgaccgccga agggcaccgt ctacacggtg agaacccgac caccgcgtcc ctgggacggc cgacgtcccg cgggtcgatg gatcatgccg 120 180 240 300 360 420 480 501 <210> <211> <212> <213> 108 180

DNA

Mycobacterium vaccae <400> 108 atgaaccagc cgcggcccga IV gcggagtact acgacctgcg aacatcaccg tgctgccggt ggc cgaggcg gggcatcctc agagctgcag aacctgcggg gctacttcac cgccaacccg gccccgatcg gtgacgcgca gcgcaactgc acggcctacg acacgttcat ggccggctga <210> 109 <211> 166 <212> PRT <213> Mycobacterium vaccae <400> 109 Met Pro Val Arg Arg Ala Arg Ser Ala Leu Ala Ser Val Thr Phe Val WO 99/32634 WO 9932634PCT/NZ98/OO1 89 1~ Ala Asp Gly Tyr Asp Thr Cys Leu Arg 145 Val Al a Trp Thr Thr Gly Trp Phe Val 130 Thr Al a Ala Ser Ser Phe Pro Asp Arg 115 Phe Asp Al a Cys Gly Val Ser Al a Gly 100 Gly Tyr Ile Tyr 5 Val Arg Al a Thr Pro Arg Ala Al a Leu Pro 165 Gly Tyr Ala Ser 70 Ser Gln Asp Pro Asp 150 Ala Ala Thr Arg 55 Cys Asn Trp Val Thr 135 Gly Glu Val 40 Gln Val Pro Val Pro 120 Ala Leu Gly 25 Val Pro Gly Thr Phe 105 Arg Asp Cys 10 Thr Thr Glu Thr I le 90 Asn Glu Gly Lys Ala Phe Pro Cys Pro Tyr Tyr Ser Gly 155 Leu Ala Asp Val Gln Asn Al a Met 140 Thr Al a Ser Phe Ala Pro Trp Ala 125 Phe Val Al a Asp Ser Thr Ala Gln 110 Al a Gly Ile Thr Lys Gly Ala Arg Trp Arg Thr Met Pro

I

Asn Ala Gly Gln <210> <211> <212> <213> <400> Arg Asp Gin Pro Asn Pro Asp Ala Thr Ala 110 74

PRT

Mycobacterium vaccae 110 Thr His Pro Gly Ala Asn Gln Ala Val. Thr Ala Ala Met 5 10 Arg Pro Glu Ala Glu Ala Asn Leu Arg Gly Tyr Phe Thr 25 Ala Glu Tyr Tyr Asp Leu Arg Gly Ile Leu Ala Pro Ile 40 Gin Arg Asn Cys Asn Ile Thr Val Leu Pro Val Glu Leu 55 Tyr Asp Thr Phe Met Ala Gly <210> 111 <211> 503 <212> DNA <213> Mycobacterium vaccae <220> <221> unsure <222> (358) (358) <400> 111 atgcaggtgc ggcgtgttct gggcagtgtc ggtgcagcag tcgcggtttc ggccgcgtta tggcagacgg gggtttcgat accgaccgcc tcagcggatc cgtgtccgga catcgaggtg atcttcgcgc gcgggaccgg tgcggaaccc ggcctcgggt gggtcggtga tgcgttcgtc aacgcgctgc ggcccaaggt cggtgagcag tcggtgggca. cctacgcggt gaactacccg WO 99/32634 WO 9932634PCTNZ98/OO1 89 gcaggattcg gacttcgaca gcagtggatg gccgacaact cgccggcgtc atcgacctga cccgatgccg ccccgcgtcg gcgcgacatc cgtggtggcg aatCggCgCC catgggcgcg gccgacgcat cggggcgggt gcccggacac caagcttgtc ctgggcggca tgtcgcangg tcaccgtcga tccgcgaccg ctgggccggt tcacccccac ccgaccacgt ggccgccgtt gtggtcttcg gaaatccgtt gtc 300 360 420 480 503 <210> <211> <212> <213> 112 167

PRT

Mycobacterium vaccae <220> <221> UNSURE <222> (119). (119) <400> 112 Gin Val Arg Met 1 Ser Arg 5 Val Leu Gly Ser Val Gly Ala Ala Val Ala Val Ala Ala Leu Trp Gin Thr Gly Val Ile Ile Pro Thr Asp Pro Cys Glu Pro Gly Pro Asp Ile Glu Leu Gly Trp Val Glv Glu Gin Ser Val1 40 Gly Phe Ala Arg Gly Asn Ala Ser Ala Thr Gly Ala Ala Leu Arg Asp Ala Phe Pro Lys Val Val Gly Thr Val Asn Tyr Ala 70 Asp Pro Gly Phe Asp Phe Gln Lys Ser Ala Pro 90 Asn Gly Ala Ala Asp Ala Ser Gly Arg Val Leu Gly 115 Val Asp Pro Val 100 Gly Trp Met Ala Cys Pro Asp Met Ser Xaa Gly 120 Arg Gly Val Ile Asp 125 Pro Thr Lys Leu 110 Leu Ile Thr Met Pro Pro 130 Arar Val Al a Arg Pro Leu Asp His Val Gly 135 Ala Phe Thr Pro Thr 140 Phe Ala Val Val Val 155 Gly Asn Pro Leu 160 150 Gly Gly Asp Ile Arg Gly 165 <210> <211> <212> <213> 113 1569

DNA

Mycobacterium vaccae <400> 113 atggccaaga gccctcgcag aagaagtggg ctggaggacc gacgacgtcg gaaggcctgc aaggctgtcg gagcagattt caattgcgta acgccgtaaa gcgcccccac cgtacgagaa cgggcgacgg gcaacgtcgc aggctgtcac ctgccaccgc tgacgaagag ggtgacgttg gatcaccaac gatcggcgct caccaccacc agccggcgcc ccagtcgctg ggcgatttcc gcccgccgtg ggcccgaagg gatggtgtgt gagctggtca gccaccgtgc aacccgctcg ctgaagtcgg gccggcgaca gcctcgagcg gtcgcaacgt ccatcgccaa aagaggtcqc tcgctcaggc gcctcaagcg ccaaggaggt cc cagat cgg gggcctcaac cgtgctggag ggagatcgag caagaagacc tctggttcgc tggcatcgag cgagaccaag cgagctcatc 120 180 240 300 360 420 480 WO 99/32634 WO 9932634PCTNZ98/001 89 gccgaggcca ttcggcctgc tacttcgtga gtcagctcca gccggcaagc gtggtcaaca gaccgccgca gaaagagtcg gtcgtcgtca gccggccggg gagaagctgc gctgccaccg gcgaaggctg gctcctgcgc cgcgtggcgc gtcgttgccg gagtacgagg ctgcagaacg aagc cggag tggacaaggt agctcgagct ccgacgccga aggtgtcgac cgctgctgat agatccgcgg aggcgatgct ggctgtccct ccaaggacga tggctcagat aggagcgcct aggtggagc t ccgtcgaaga tggacgacct tgtcggctcc agaaggtgt c acctgctcaa cggcgtccat cggcaacgag caccgagggt gcgccaggaa cgtcaaggat catcgccgag caccttcaag gcaggacatg ggagaccgcc gaccaccatc ccgcgccgag ggccaagctg caaggagcgc gggcatcgtc cggcctgacg gctcaagcag caacctgccc ggccggcgtc cgcgqctctg ggtgtcatca atgcgcttcg gccgtcctgg ctgctcccgc gacgtcgagg tccqtcgccg gccatcctca gacgtctcgc gt cgagggc t atcgagaaca gccqgcggtg aagcaccgca gccggtggcg ggcgacgagg atcgccttca gcgggtcacg gccgacccgg ttcctcacca ccgtcgagga acaagggcta aggatcccta tgctggagaa gcgaggccct tcaaggctcc ccggtggtca tgctgggcca cgggcgattc gcgactccga ttgcggtgat tcgaggacgc gcgtggctct ccaccggtgc acggcggcct gcctcaacgc tgaaggt cac ccgaggccgt gtcgaacacc catctcgggt catcctgctg ggtcatccag gtccacgctg gggcttcggt ggtcgtcagc ggcc cgcaag cgatgccatc ctacgaccgc caaggccgga cgtccgcaac gctgcagtcg caacatcgtc ggagcccggc cgcgaccggt ccgctcggcg cgtcgccgac 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1569 <210> <211> <212> <213> 114 523

PRT

Mycobacterium vaccae Met 1 Arg Lys <400> 114 Ala Lys Thr Ile 5 Gly Leu Asn Ala Gly Arg Asn Val Ala Tyr Asp Giu Glu 10 Val Ala Arg Arg Gly Leu Glu Leu Ala Asp Val Leu Glu Ser Ile Ala Ala 25 Lys Lys Vai Thr Lys Trp Gly Leu Gly Pro Pro Thr Ile Glu Asp Pro Thr Asn Asp Gly Val Lys Glu Ile Giu s0 Tyr Glu Lys Ile Gly Ala 70 Asp 55 Glu Leu Val Lys Val Asp Giu 75 Ala Ala Lys Lys Thr Asp Val Ala Gly Thr Thr Thr Val Thr Val Leu Ala Gin Ala Leu Vai Leu Gly Leu 115 Ser Leu Leu 130 Ala Thr Ala Arg 100 Lys Gly Leu Arg Asn 105 Lys Aia Ala Gly Arg Gly Ile Ala Val Giu Al a 125 Glu Ala Asn Pro 110 Vai Thr Gin Gin Ile Ser Lys Ser Ala Ala Ile Ser 150 Met Asp Lys Lys 135 Ala Val Glu Gly Asp Thr Thr Lys 140 Gin Ile 155 Gly Val Gly Giu Leu 145 Ala Ile 160 Glu Ala Val Gly Asn Giu Ile Thr Giu Ser Asn Phe Asp Lys 195 Gin Giu Ala 165 Thr Phe 180 Gly Tyr Val Leu Val Glu 175 Gly Leu Gin Leu Leu Thr Glu Gly Met Arg 190 Ala Glu Arg Ile Ser Gly 200 Giu Asp Pro Tyr Phe Val Thr Asp Tyr Ile Leu Leu Val Ser Ser Lys WO 99/32634 PCT/NZ98/00189 210 Ser 215 Leu 220 Glu Val 225 Ala Thr Vai Lys Asp 230 Leu Leu Pro Leu Lys Val Ile Gin 240 Gly Lys Pro Leu 245 Val Ile Ile Ala Glu 250 Arg Val Giu Gly Glu Ala 255 Ser Val Leu Ser Thr Ala Val Lys 275 Asp Met Ala Val Asn Lys Ile 265 Asp Gly Thr Phe Lys Pro Gly Phe Gly 280 Gly Arg Arg Lys Ala 285 Glu 270 Met Leu Gin Arg Val Gly Ile Leu Thr Gin Val Val 290 Leu Ser Ser 300 Gly Leu Giu Thr 305 Val Ala 310 Asp Vai Ser Leu Leu 315 Val Gin Ala Arg Val Val Thr Lys 325 Ala Glu Thr Thr Glu Gly Ser Gly Asp 335 Ser Asp Ala Asn Ser Asp 355 Lys Leu Ala 370 Val Giu Leu 385 Ala Lys Ala Leu Leu Gin Glu Ala Thr 435 Lys Gin Ile Gly Arg Val Ala 345 Glu Ile Arg Ala Glu Ile Glu 350 Arg Leu Ala Asp Tyr Asp Arg 360 Val Lys Leu Gin Glu 365 Ala Gly Gly Val Ala 375 Lys Ile Lys Ala Gly 380 Asp Ala Thr Glu Lys Glu Ala Val 405 Ser Ala Arg 390 Glu His Arg Ile Glu 395 Ala Ala Val Arg Asn 400 Glu Gly Ile Val 410 Asp Gly Gly Gly Val Ala 415 Pro Ala Leu Leu Gly Leu 420 Gly Ala Asn Ile Val 440 Gly Val Ala Leu Thr Gly Asp 430 Ala Pro Leu Val Ala Glu Ala Phe Asn 450 Lvs Val Gly 455 Ala Leu Glu Pro Gly 460 Asn Ser Asn Leu 465 Glu Pro 470 Leu Gly His Gly Ala Ala Thr Gly 480 Tyr Giu Asp Leu 485 Leu Lys Ala Gly Val 490 Asp Pro Val Lys Val 495 Phe Leu Thr Arg Ser Thr Thr Glu 515 Gin Asn Ala Ala Ser Ile 505 Asp Lys Pro Glu 520 Ala Ala Leu 510 Val Val Ala <210> 115 <211> 647 <212> DNA <213> Mycobacterium vaccae <400> 115 atggccaaga gccctcgcag aagaagtggg ctggaggacc gacgacgtcg gaaggcctgc aaggctgtcg caattgcgta acgccgtaaa gcgcccccac cgtacgagaa cgggcgacgg gcaacgtcgc aggctgtcac tgacgaagag ggtgacgttg gatcaccaac gatcggcgct caccaccacc agccggcgcc ccagtcgctg gcccgccgtg ggcccgaagg gatggtgtgt gagctggtca gccaccgtgc aacccgctcg ctgaagtcgg gcctcgagcg gtcgcaacgt ccatcgccaa aagaggtcgc tcgctcaggc gcctcaagcg ccaaggaggt gggcctcaac cgtgctggag ggagatcgag caagaagacc tctggttcgc tggcatcgag cgagaccaag 120 180 240 300 360 420 i 1 I~lli; 1;L-~ WO 99/32634 WO 9932634PCTINZ98/00189 gagcagattt ctgccaccgc ggcgatttcc gccgaggcca tggacaaggt cggcaacgag ttcggcctgc agctcgagct caccgagggt tacttcgtga ccgacgccga gcgccaggaa gccggcgaca cccagatcgg cgagctcatc ggtgtcatca ccgtcgagga gtcgaacacc atgcgcttcg acaagggcta catctcgggt gccgtcctgg aggatcc <210> <211> <212> <213> 116 927

DNA

Mycobacterium vaccae <400> 116 gatccctaca ctggagaagg gaggccctgt aaggctccgg ggtggtcagg ctgggccagg ggcgattccg gactccgact gcggtgatca gaggacgccg gtggct ctgc accggtgcca ggcggcctgg ctcaacgccg aaggtcaccc gaggccgtcg t cctgctggt tcatccaggc ccacgctqgt gcttcggtga tcgtcagcga cccgcaaggt atgccatcgc acgaccgcga aggccggagc tccgcaacgc tgcagtcggc acatcgtccg agcccggcgt cgaccggtga gctcggcgct tcgccgacaa cagctccaag cggcaagccg ggtcaacaag ccgccgcaag aagagtcggg cgtcgtcacc cggccgggtg gaagctgcag tgccaccgag gaaggctgcc tcctgcgctg cgtggcgctg cgttgccgag gtacgaggac gcagaacgcg gccggag gtgtcgaccg ctgctgatca atccgcggca gcgatgctgc ctgtccc tgg aaggacgaga gctcagatcc gagcgcctgg gtggagctca gtcgaagagg gacgacctcg tcggCtccgc aaggtgtcca ctgctcaagg gcgtccatcg tcaaggatct tcgccgagga ccttcaagtc aggacatggc agaccgccga ccaccatcgt gcgccgagat ccaagctggc aggagcgcaa gcatcgtcgc gcctgacggg tcaagcagat acctgcccgc ccggcgtcgc cggCt ctgtt gctcccgctg cgtcgagggc cgtcgccgtc catcctcacc cgtCtcgctg cgagggctcg cgagaacagc cggcggtgtt gcaccgcatc cggtggcggc cgacgaggcc cgccttcaac gggtcacggc cgacccggtg cctcaccacc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 927 <210> <211> <212> <213> 117 215

PRT

Mycobacterium vaccae <400> 117 Met Ala Lys Thr 1 Arg Gly Leu Asn Lys Gly Arg Asn Thr Asn Asp Gly Tyr Giu Lys Ile Asp Asp Val Ala Ile Ala Tyr Asp Glu Giu 10 Val Ala Arg Arg Gly Leu Glu Ala Leu Ala Val Val Leu Val Ser Ile 55 Gly Ala Giu 70 Gly Asp Gly Glu Gly Leu Arg Gly Ile Asp Ala 25 Glu Lys Ala Lys Lys Val Thr Lys Trp Gly Ala Leu Gly Pro Pro Thr Ile Glu Asp Pro Giu Ile Giu Leu Val Lys Giu Val 75 Thr Thr. Thr Ala Thr 90 Arg Asn Val Ala Ala Ala Lys Lys Thr Ala Leu Val Leu Gly Leu 115 Arg 100 Lys Val Leu Ala Gin Gly Ala Asn Pro 110 Ala Val Thr Gin Glu 120 Glu Ala Val Glu Ser Leu Leu Lys Ser Ala Lys Val Glu Thr Lys 140 Gin Ile Ser Ala Thr Ala Ala Ile Ser Ala Gly Asp Thr Gin Ile Gly Glu Leu Ile WO 99/32634 PCT/NZ98/00189 145 Ala Glu Phe Gin Asp 1 Leu Ile Asn Phe Gly Asp Glu Arg Asp 145 Ala Lys Glu Ala Ile 225 Gly Ala Lys Glu Ala Met Ser Asn Thr 180 Asp Lys Gly 195 Glu Ala Val 210 <210> 118 <211> 309 <212> PRT <213> Myco <400> 118 Pro Tyr Ile Leu Pro Leu Ile Ala Glu Lys Ile Arg Gly Asp Arg Gly Gin Val Val Ser Leu 100 Thr Thr Ile 115 Val Ala Gin 130 Arg Glu Lys Val Ile Lys His Arg Ile 180 Gly Ile Val 195 Leu Asp Asp 210 Val Arg Val Gly Leu Glu Gly His Gly 260 Ala Gly Val Asp 165 Phe Tyr Leu 150 Lys Gly Ile Glu Val Leu Ser Asp 215 155 160 Gly Asn Glu Gly Val Ile Thr Val Glu 170 175 Gin Leu Glu Leu Thr Glu Gly Met Arg 185 190 Gly Tyr Phe Val Thr Asp Ala Glu Arg 200 205 bacterium vaccae Leu 5 Leu Asp Gly Arg Val Leu Val Ile Leu Ala 165 Glu Ala Leu Ala Pro 245 Leu Ala Leu Glu Val Thr Lys 70 Ser Gly Glu Arg Gin 150 Gly Asp Gly Gly Leu 230 Gly Asn Asp Val Lys Glu Phe 55 Ala Glu Gin Gly Ala 135 Glu Ala Ala Gly Leu 215 Ser Val SAla SPro Ser Val Gly 40 Lys Met Arg Ala Ser 120 Glu Arg Ala Val Gly 200 Thr Ala Val Ala Val Ser Lys 10 Ile Gin 25 Glu Ala Ser Val Leu Gin Val Gly 90 Arg Lys 105 Gly Asp Ile Glu Leu Ala Thr Glu 170 Arg Asn 185 Val Ala Gly Asp Pro Leu Ala Glu 250 Thr Gly 265 Lys Val Val Ser Ala Gly Leu Ser Ala Val Asp Met 75 Leu Ser Val Val Ser Asp Asn Ser 140 Lys Leu 155 Val Glu Ala Lys Leu Leu Glu Ala 220 Lys Gin 235 Lys Val Glu Tyr Thr Arg Thr Lys Thr Lys Ala Leu Val Ala 125 Asp Ala Leu Ala Gin 205 Thr Ile Ser Glu Ser Val Pro Leu Ala Ile Glu Thr 110 Ile Ser Gly Lys Ala 190 Ser Gly Ala Asn Asp 270 Ala Lys Leu Val Pro Leu Thr Lys Ala Asp Gly Glu 175 Val Ala Ala Phe Leu 255 Leu Leu Asp Leu Val Gly Thr Ala Asp Gly Tyr Val 160 Arg Glu Pro Asn Asn 240 Pro Leu Gin 275 280 285 Asn Ala Ala Ser Ile Ala Ala Leu Phe Leu Thr Thr Glu Ala Val Val WO 99/32634 WO 9932634PCTNZ98/OO1 89 290 Ala Asp Lys Pro Glu 305 295 300 <210> 119 <211> 162 <212> DNA <213> Mycobacterium vaccae <400> 119 ctcgtacagg cgacggagat ctccgacgac gccacgtcgg tacggttggt cgccaccctg ttcggcgtcg tgttgttgac gttggtgctg tccgggctca acgccaccct catccagggc gcaccagaag acagctggcg caggcggatt ccgtcgatct tc 120 162 <210> 120 <211> 1366 <212> DNA <213> Mycobacterium vaccae <220> <221> unsure <222> (955) (955) <221> unsure <222> (973) (973) <400> 120 gatgagcagc cccggtgctg gctggcccgc cctgctggta cctgttcggc gggcgcacca cttcgcgctg ggggggcctg ttcggtcggt cgactggatc agtcaactgg cgaactcgcc cgtcgtcacc ggtcgcggcg tgcggccgaa gagcacgtac cgtcgccgac cacactgcgc ttacggcaac cgtagacggc gtgctcgag gaccgcgcac cctggtgcac qtgctgaact ctggtcgtgc ccggtgcaac caggcgatgg gtcgtgttgt gaagacagct atcgcggtcg ttcaccgcac cagatcatct accgtcccca cgtgcaacac ggcgcgtcgt accttcaacg tcgctgcccg tacgagaagt ctgcgatggg ganttcgaca t tggcagacg ggggaacgcc agggtgagtc cgtggcgac t gcgctggagg cgaaagccga cgacctggtt tgaccgaggt tcctgcgtac agatctccga tgacgttggt ggcgcaggcg gtatcaccgt tgggcgtcac cgggtctgct ccgcggcggg atatcgacac tcaccaatta ccgcggacac aactgcgcac cgatcccgtt tctggtacgc cgccggaacg acgaacagca tccagcagcc tgtccgtgat tcctggggca aagtcaccgt tcctgctgca ggcctgggcc gcacaacgcg ctacatcctg cgacgccacg gctgtccggg gattccgtcg gatcatggcc ttccatcgtt gctgctgttc ccggccgtcc cggcggcaac cagccggccc ccccgatgat cgacggacag gcacacaccc cgcgcgccgg gatcgcctcg ggagatcgcc gggt caggta cgatcaggac gaccacgctg gctggagatg cgtgatcggg gtcgcggtcg t tgcgt cggc ccgctgggcg tcggtacggt ctcaacgcca atcttcctcg tatgtctggg cttggcctgg gagcaaccgt gcccacggcc ctgctggtaa gtgggagagc gtctgcgaga atcgccacgc gcggtggacg caggaacttc gccatgcggg gacgtggtgc ccgaccggga ggcgacgtga acgcgggaac gcccgtgacg gccgtg cggtcgggtt gcggcagcgc cgttgctgct tggtcgccac ccctcatcca acgtcgcgcg gcgcgaacgt ctctgcagaa tccggctcgg gcgtggtgga tgcccaacgc accggctgac tgctgtcgtc tctatctcgg actcggtcag gcctnaacgg ctgtggcgtc gtctggtccg tgaggttcat tcccggcgcg cggtactggc agat cgagcg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1366 <210> 121 <211> 455 Met

I

Al a Al a Arg Ala Leu Thr Ser Thr Thr 145 Ser Phe Ser Asp Al a 225 Val Met Gin Pro Arg 305 WO 99/32634 <212> PRT <213> Myco <220> <221> UNSU <222> (318 <221> UNSU <222> (324 <400> 121 Ser Ser Val Val Gly Phe Leu. Arg Arg Thr Tyr Ilie Met Glu Ile Phe Gly Val Leu Ile Gin 100 Ile Phe Leu 115 Vai Ile Met 130 Ala Leu Gly Val Gly Gin A-rg Leu Giy 180 Ala His Gly 195 Thr Gly Gly 210 Ser Phe Thr Vai Thr Thr Leu Ser Ser 260 Ile Ala Thr 275 Leu. His Thr 290 Trp Val Trp, bacterium vaccae

RE

.(318)

RE

(324) PCTINZ98/OOI 89 Leu.

5 Pro Arg Leu Ser Val Gly Asp Al a Val Ile 165 Asp Arg Asn Asn Phe 245 Val Leu Pro Tyr Asn Val Gly Pro Asp 70 Leu Ala Val Tyr Thr 150 Ile Trp Val Leu Tyr 230 Asn Ala Tyr Ala Ala 310 Ser Leu Ser Leu 55 Asp Leu Pro Ala Val 135 Ser Ser Ile Val Leu 215 Ser Ala Ala Leu Val 295 Ala Thr Leu Ala 40 Gly.

Ala Thr Glu Arg 120 Trp Ile Gly Thr Glu 200 Val Arg Ala Ser *Gly 280 *Asp Arg Trp Val 25 Leu Al a Thr Leu Asp 105 Phe Gly Val Leu Val 185 Val Met Pro Asp Leu 265 Ala Asp Arg Leu.

10 Val Ala Leu Ser Val 90 Ser Ala Ala Leu Leu.

170 Pro Asn Pro Val Thr 250 Pro Ala Ser Gin Al a Leu Arg Leu Val 75 Leu Trp Leu Asn Gly 155 Leu.

Thr Trp Asn Gly 235 Pro Glu Glu Val Glu 315 Trp Thr Pro Leu Arg Ser Arg Ile Val 140 Leu Leu Ala Arg Ala 220 Glu Asp Leu Tyr Arg 300 Leu Al a Glu Val Leu Leu Gly Arg Ala 125 Gly Ala Phe Ala Ala 205 Glu His Asp Arg Glu 285 Ser Arg Val Val Gin Leu Val Leu Arg 110 Val Gly Leu Glu Gly 190 Thr Leu Arg Val Thr 270 Lys Thr Xaa Ala His Leu Val Ala Asn Ile Gly Leu Gln Gin 175 Arg His Al a Leu Cys 255 Asp Ser Tyr Asn Val Asn Leu Gin Thr Ala Pro Ile Phe Asn 160 Pro Pro Ile Gly Thr 240 Glu.

Gly Ile Leu Gly 320 Val Ala Asp Xaa Phe Asp Thr Pro Glu Arg Ile Ala Ser Ala Met Arg 325 330 335 WO 99/32634 WO 9932634PCT/NZ98/OOI 89 Ala Val Ala Ser Thr Leu Arg Len 340 Asp Asp Gin Gin Ala Asp Val 355 Gin Pro Gly 370 Val Ser Val Arg Len Val Gin Val Pro Thr 375 Ser Val Ile Asp 390 Arg Giy Asp Phe Arg 360 Gly Gly Asn Gly Glu 365 Val Met Arg Phe Ile 380 Val Gin Gin Ile 350 Arg Leu Gin Asp Gly Arg Leu 385 Val Gin Asp Giy Leu Gly .Gin Asp 395 Thr Ile Pro Ala Arg 400 Giu Leu Gin Thr Len Thr Arg 405 Thr 410 Giu Pro Val Len Met Ala Arg 435 Leu His Val 450 Al a 420 Asp Ala His Ala Leu 425 Lpn Glu Val Thr 415 Val Len Gin 430 Pro Ile Leu Gin Ile Glu Val His Arg Ile Gly Ala Val 455 <210> 122 <211> 898 <212> DNA <213> Mycobacterinm vaccae <400> 122 atgacaattc cgctaccacc accagcattc ctgcgcgcat gagaatcagt acggaggcga accgggcagg gacgacagcg tatctgcagg gacgcgcgcg gagatcgtgc gtgtactccg aaccgggaac ggtgtcaccg ccggt cgggt tgccctggaa tcctgtcgcg tctcggctgc cggtgttcga tcgcggacct tcggcgcgtt cggcgtcatt gaaaccgcgt cgctctatac acggcagcgc accgcttcaa cctacaaggg tgtcggaagc acttcgggtg tgaaggaccg tgcgcgaacg gatgagcatc ggtggtcggt ccgcctcacc gaagaact cg cagcgacggt gcgccgttac cgacgtccgc cccgccgttt ctggtcggcc cttcgaggat gccggatctc c tacgagaag gtacctgcct agtcgacggt tctgaacacc cagtccaagt ttcatcggct gacatccgcg atggtgattt ttccgtcagc tacgaccgga gcgctcatcc cagaactggg gccaatgcca ctgatgctgc gggacaaaca gcggtcgcgt gccgaggaac gtgatggcgg cgacgcgaaa tgctgctgat atcagtccgg agtcgcagtc actcgcgcgg tcggcgatgc cgttcgccaa cgaaatccaa agaaggcgat gattcaacga tcgacctcga tcgt caacgg cgaactcgat cgaccgcctg tccagttccc aagacgcggg gctgcttctg acggtcctcg gcgcgggttg cagcactgcc gacgatcaat caccaccctc cccccagcgc cgcgttcgac gttcttccgc gggcaacgtg cccctatcgc cgactatgtc gttcctgtc cggaattc 120 180 240 300 360 420 480 540 600 660 720 780 840 898 <210> 123 <211> 1259 <212> DNA <213> Mycobacterium vaccae <400> 123 cgcaattgat gacggcgcgg ccatcctggt cggaccggac gggagaagtt cctggccgac cggttgaccg ccgcggcacc 6ccaacgcgg caacaccggg aggcgtactc accggtggac ccgacgaggc gttcgccccg tcatcatctt cggcgtgtcg ggacagtggc aatctgatgc gtcgtcgagg acgctggtgc acgacgatcg ctgccgggac gtggcgcagt ctggcggcca gtgacaccgg gctcggactc ggggaacccc agccggtgac aggacgacta tgcactgggt tcaccaggac tgctgctggc gatgggagac ccggctgttc gccggaggtc cacccgctcc tctcggccac gatcgtggcc cctggtgctg gcggttgttc accggtgaga cgcgagaacc gccgacgaat gtcgaggagg gaggcgttac aagatcgaca tcgacggtga gtccgtccga 120 180 240 300 360 420 480 WO 99/32634 WO 9932634PCTNZ98/OO1 89 tccggcggtt cggtgttgtc atctgtcgat tgtccctgat aggac cacaa gcatgttgac ccgccgccga gctgcgggtt aaatggaccg cgggcatcga acatgtgggg gcatctacgt ccggggaggt gcaggccggc tcgtgacgaa caaggacgag gcccgaaccg gaacgtcacg ctccgaggaa gagtctcggg aggcgtgccg catcatcgac caccgggtcg ttcggcggtc cacctcgcgg cgtcggcgag gcccagcaga ttcggcgatc ctgctcggcg gtgatgcagc gtgatcttcg ctgatggtgg gtcgaccacg cggctggaca cggcacgccg gcggccagcg gatgtcgcct gtgcacgagg cgcggcgtcg tcagcggcgg tgacaacagc aggagcgcgc gctacctcga ccgacatgat tggtcaacga tgcggacgct acgtccggcg ccgagtccgg ggctggtggg accaggtgca tcatgcagga agacggtctg tgactaccgc tttcaacgac cgagaaccaa cggggaggag gggcctcgac cctgacccgc gcacgacggg cacggtcaat gcacgacctg gcggtccacg gcgcggctcc aactctcgac gcggttgcag ctcgctctgc atgagtcgca cggctgatgc acgatcgccc gagttgtcgc cagttcgacg tacctggcca ttcgcgatcg cggctccgcg ttggcgtacg ccccagcccg ttcgtcgccg ggccacccg 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1259 <210> <211> <212> <213> 124 299

PRT

Mycobacterium vaccae <400> 124 Met Thr Ile Leu Pro Trp Asn Ala Arg Thr Ser Giu His Pro Thr Arg 1 Lys 10 Ser Arg Arg Gly Arg Leu Met Tyr His Leu Leu 25 Leu Leu Leu Thr 40 Arg Met Ser Ile Gin Ser Ala Ala Val Arg Ala Ser Lys Leu Leu Val Gly Phe Val Phe Asp Ile Gly Tyr Gin 55 Arg Leu Thr Asp 70 Phe Ala Asp Leu Ser Gly Ile Arg Lys Asn Ser Ile Leu Arg Ser Ser Glu Ser Gin Ser Leu Ser Arg Gly Glu 75 Met Leu Asn Gin Val Ile Tyr Thr Ser Arg Gly Ser Thr Gin Leu Gly 115 Arg Tyr Tyr Ala 100 Asp Giu Ala Ile Gly 105 Thr Phe Ser Asp Ala Thr Ile Asn 120 Ala Gly Gin Ala Giy Phe Arg 110 Ser Leu Arg Asp Ser Gly Asp Arg Thr Asn Thr Thr 130 Asn Arg Leu 140 Ser Vai Asp Val 145 Tyr Arg 150 Tyr Leu Ile Pro Lys 155 Gin Asn Pro Gin Arg 160 Leu Gin Ala Leu 165 Asp Thr Pro Pro Phe 170 Ser Asn Trp Giu Lys Ala 175 Ile Ala Phe Ala Arg Phe 195 Giu Asp Leu Asp 180 Asn Ala Arg Asp Gly 185 Glu Ala Trp Ser Giu Phe Phe Arg 200 Ile Val His Arg 205 Val Ala Aia Asn 190 Phe Asn Phe Tyr Ser Ala Met Leu Leu 210 Asp Leu 215 Giu Gly Asn Asn Ile Val 235 Val 220 Asn Tyr Lys Gly 225 Asn Arg Giu Pro Asp Leu Ser 245 Leu Gly Thr 230 Glu Ala Tyr Gly Pro Tyr Giu Lys Ala Val Ala Ser Asn Ser 250 255 Ilie Asp Tyr Val Gly Val Thr Asp Phe Gly Trp Tyr Leu Pro Ala Giu WO 99/32634 PCT/NZ98/00189 260 Glu Pro Thr Ala 275 Asp Gly Val Met I 290 <210> 125 <211> 419 <212> PRT <213> Mycol <400> 125 Gin Leu Met Thr 1 Thr Gly Glu Thr Ser Arg Leu Phe Glu Gly Gly Thr Gly Thr Thr Leu Gin Arg Gly Asn Glu Ala Leu Gin 100 Val Ile Val Ala 115 Gin Phe Thr Arg 130 Val Ser Leu Ala 145 Arg Arg Leu Gin Leu Ala Leu Pro 180 Ala Phe Asn Asp 195 Gly Glu Glu Arg 210 Glu Pro Val Met 225 Asp His Lys Asn Glu Leu Ser Arg 260 Asp Leu Thr Arg 275 His Val Arg Thr 290 Val Pro Arg Leu 305 265 270 rrp Phe Leu Ser Pro Val Gly Leu Lys Asp Arg Val 280 285 Ala Val Gin Phe Pro Gly Ile 295 bacterium vaccae Ala Ile Arg Pro Val Thr Ala Lys Thr Ala Ala 165 Val Met Ala Gin Val 245 Met Gin Leu Asp Arg Leu Glu Pro Gin 70 Gly Tyr Ile Leu Met 150 Gly Leu Ser Glu Arg 230 Thr Leu Phe His Asn 310 Gly Val Asn Glu 55 Pro Thr Ser Asp Val 135 Leu Ala Ser Arg Asn 215 Tyr Val Thr Asp Asp 295 Val Gin Trp Gly Pro 25 Arg Glu 40 Val Ala Val Thr Thr Ile Pro Va] 101 Thr Asl 120 Leu Se Leu Al Gin Gl1 Arg As

I

18! Asn Lei 200 Gin Ar Leu As Ile Ph Ser Gl 26 Ala Al 280 Gly Ty Arg Ar SArg Asp Thr Gly Met Gly Asp 5 p a p

P

e u 5 a r 9g Asp Lys Asp Thr Glu 90 Asp Glu Thr Arg Ile 170 Glu Ser Leu Gly Ala 250 Glu Ala Leu Thr Asn Phe Glu Arg 75 Asp Leu Ala Val Leu 155 Ser Phe Ile Met Glu 235 Asp Leu Glu Ala Val 315 Leu Leu Ser Ser Asp Pro Phe Ile 140 Phe Gly Gly Lys Leu 220 Glu SMet SMet SSer Ser 300 SAsn Met Ala Val Val Tyr Gly Ala 125 Ile Val Gly Asp Asp 205 Ser Thr Met Val Leu 285 Cys Phe Arg Asp Asp Glu Leu Leu 110 Pro Ile Arg Asp Leu 190 Glu Leu Ile Gly Val 270 SGly SGly SAla Ser Val Arg Glu Gly His Val Phe Pro Tyr 175 Thr Leu Met Ala Leu 255 Val Val Leu Ile Asp Val Arg Ala His Trp Ala Gly Ile 160 Arg Thr Leu Pro Gin 240 Asp Asn Asp Gly SGlu 320 Met Asp Arg Ile Ile Asp Arg His Ala Ala Glu Ser Gly His Asp Leu i I WO 99/32634 PCT/NZ98/00189 Arg Gly Ala Ser 385 Gly Gly Leu Arg Tyr 370 Arg Glu His Arg Ser 355 Gin Val Val Pro Ala 340 Thr Val His Val 325 Gly Ile Asp Thr Gly 345 Leu Ala Tyr Asp Met 360 Gin Arg Gly Ser Pro 375 Glu Val Met Gin Glu 390 Gly Glu Arg Gly Val 405 54 330 Ser Ala Ala Ser Gly 350 Trp Gly Ser Ala Val 365 Gin Pro Gly Ile Tyr 380 Thr Leu Asp Phe Val 395 Glu Thr Val Trp Arg 410 335 Leu Asp Val Ala Leu 415 Val Val Thr Ala 400 Gin <210> 126 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 126 ccggatccga tgagcagcgt gctgaac <210> 127 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 127 gcggatccca cggccccgat cacgtg <210> 128 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 128 ccggatccaa tgacatttct gccctggaat gcg <210> 129 <211> 32 <212> DNA <213> Artificial Sequence <220>

I~

WO 99/32634 WO 9932634PCTNZ98/OO1 89 <223> Made in a lab <400> 129 ccggatccat tcggtggccc tgcaaccgcc ag <210> 130 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 130 ccggatccgg agcaaccgtt ccggctc <210> 131 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 131 ccggatcccg gctatcagtc cggacgg <210> 132 <211> 844 <212> DNA <213> Mycobacterium vaccae <400> 132 gagcaaccgt tccggctcgg gcccacggcc gcgtggtgga ctgctggtaa tgcccaacgc gtgggagagc accggctgac gtctgcgaga tgctgtcgtc atcgccacgc tctatctcgg gcggtggacg actcggtcag caggaacttc gcctaacggc catgcgggct gtggcgtcca cgtggtgcgt ctggtccgtt gaccgggatg aggttcatcg cgacgtgatc ccggcgcggg gcgggaaccg gtactggcga ccgtgacgag atcgagcgcc cgtg cgactggatc agtcaactgg cgaactcgcc cgtcgtcacc ggtcgcggcg tgcggccgaa gagcacgtac gtcgccgacg cactgcgctt acggcaacgg tagacggcag tgctcgagcg ccgcgcacgc tggtgcaccg accgtcccca cgtgcaacac ggcgcgtcgt accttcaacg tcgctgcccg tacgagaagt ctgcgatggg attcgacacg ggcagacgac ggaacgcctc ggtgagtctg tggcgacttc gctggaggaa aaagccgatc ccgcggcgg atatcgacac tcaccaatta ccgcggacac aactgcgcac cgatcccgtt tctggtacgc ccggaacgga gaacagcagg cag cagccgg tccgtgatcg ctggggcaga gtcaccgtgc ctgctgcacg ccggccgtcc cggcggcaac cagccggccc ccccgatgat cgacggacag gcacacaccc cgcgcgccgg tcgcctcggc agatcgccga gtcaggtacc atcaggacgg ccacgctgac tggagatggc tgat cggggc 120 180 240 300 360 420 480 540 600 660 720 780 840 844 <210> 133 <211> 742 <212> DNA <213> Mycobacterium vaccae WO 99/32634 WO 9932634PCT/NZ98/OOI 89 <400> 133 ggctat cagt cgcgagtcgc atttactcgc cagctcggcg cggacgttcg atcccgaaat tgggagaagg gccagattca ctgctcgacc aacatcgtca gcgtcgaact gaaccgaccg gcggtccagt ccggacggtc agtcgcgcgg gcggcagcac atgcgacgat ccaacaccac ccaaccccca cgatcgcgtt acgagttctt tcgagggcaa acggccccta cgatcgacta cctggttcct tccccggaat ctcgctgcgc gttggagaat tgccacggag caataccggg cctcgacgac gcgctatctg cgacgacgcg ccgcgagatc cgtggtgtac tcgcaaccgg tgtcggtgt c gtccccggtc tc gcatcggtgt cagttcgcgg gcgatcggcg caggcggcgt agcggaaacc caggcgctct cgcgacggca gtgcaccgct tccgcctaca gaactgtcgg accgacttcg gggttgaagg tcgaccgcct acctgaagaa cgttcagcga cattgcgccg gcgtcgacgt ataCcccgcc gcgcctggtc tcaacttcga aggggccgga aagcctacga ggtggtacct accgagtcga caccgacatc ctcgatggtg cggtttccgt ttactacgac ccgcgcgctc gtttcagaac ggccgccaat ggatctgatg tctcgggaca gaaggcggt c gcctgccgag cggtgtgatg 120 180 240 300 360 420 480 540 600 660 720 742 <210> <211> <212> <213> <220> <221> <222> 134 282

PRT

Mycobacterium vaccae

UNSURE

(145) (145) <221> UNSURE <222> (151) (151) <400> 134.

Giu Gin Pro Phe Arg Leu Gly Asp Trp Ile 10 Thr Val Pro Thr Ala Ala 1 Gly Thr Arg Pro Ser Ala His Gly Arg Val 25 His Ile Asp Thr Gly Gly Asn Leu Val Glu Val Asn Leu Val Met Leu Ala Gly 40 Asn Pro Val Trp Arg Ala Asn Ala Giu Gly Giu His Ala Ser Phe Arg Leu Thr 55 Thr Tyr Ser Arg Pro Asp Thr Val Val Val Thr 70 Ser Phe Asn Ala Ala 75 Ser Thr Pro Asp Asp Cys Giu Met Leu Ile Ser Val Ala Ala 90 Leu Leu Pro Glu Leu Arg Thr Asp Gly Lys Ser Ile 115 Thr Tyr Leu Gin 100 Pro Ala Thr Leu Tyr 105 Ala Gly Ala Ala Leu His Thr Pro 120 Tyr Val Asp Asp Ser 125 Gin Glu Tyr Glu 110 Val Arg Ser Glu Leu Arg Arg Trp Val 130 Xaa Asn Trp 135 Xaa Ala Ala Arg Arg 140 Glu Gly Val Ala 145 Ala Asp 150 Ala Phe Asp Thr Pro 155 Leu Arg Ile Met Arg Ala Val 165 Asp Ser Thr Leu Arg 170 Val Ala Asp Asp Ala Ser 160 Giu Gin 175 Gly Glu Gin Giu Ile Al a 180 Val Val Arg Leu 185 Arg Tyr Gly Asn 190 WO 99/32634 WO 9932634PCTNZ98/OO1 89 Arg Asp Pro 225 Thr Val Pro Gly Leu Ala Thr Ala Arg Val Leu Asp Giu 145 Leu Asp Ser Gly Trp 225E Ala Leu Gin Gin 195 Gly Arg Val 210 Ala Arg Val Arg Giu Pro Leu Giu Met 260 Ile Leu Leu 275 <210> 135 <211> 247 <212> PRT <213> Myco <400> 135 Tyr Gin Ser Thr Asp Ile Asp Leu Lys Glu Ala Ile Thr Ile Asn Thr Phe Ala Arg Ala Leu 100 Tyr Thr Pro 115 Ala Arg Asp 130 Phe Phe Arg Leu Asp Leu Leu Gly Thr 180 *Giu Ala Tyr 195 *Val Thr Asp 210 Phe Leu Ser Val Gin Phe Pro Ser Leu Val 245 Ala His Val 200 Val Gly Thr Glu Gly 280 Pro Ile Asp Al a Ile 265 Al a Gly Met Arg Phe Ile Val 205 Gin Asp Gly Asp Val Ile 220 Leu Gly Gin Thr Thr Leu 235 240 Ala Leu Giu Giu Val Thr 255 Arg Leu Val His Arg Lys 270 bacterium vaccae Gly Arg Asn Gly Thr Asn Ile Pro Gly Giu Glu 165 Asn Giu Phe Pro Pro 245 Arg Giu Ser Ala Giy 70 Thr Pro Phe Ser Ile 150 Gly Ile Lys Gly Val 230 Gly S er Ser Met Phe 55 Gin Thr Lys Gin Ala 135 Val Asn Val Ala Trp 215 Gly Ile Ser Gin Val 40 Ser Ala Leu Ser Asn 120 Trp His Val Asn Val 200 Tyr Leu Leu Ser 25 Ile Asp Al a Asp Asn 105 Trp Ser Arg Val Gly 185 Ala *Leu *Lys Arg 10 Arg Tyr Gly Ser Asp 90 Pro Glu Ala Phe Tyr 170 Pro Ser Pro Asp Ala Gly Ser Phe Leu 75 Ser Gin Lys Ala Asn 155 Ser Tyr Asn Ala Arg 235 Ser Val Leu Glu Arg Giy Arg Gin Arg Arg Gly Asn Arg Tyr Ala Ile 125 Asn Ala 140 Phe Giu Ala Tyr Arg Asn Ser Ile 205 Glu Giu 220 Val Asp Phe Asp Asn Gin Ser Thr Leu Gly Tyr Tyr Arg Val Leu Gin 110 Ala Phe Arg Phe Asp Leu Lys Gly 175 Arg Giu 190 Asp Tyr Pro Thr Gly Val Arg Phe Ala Asp Asp Asp Ala Asp Asn Met 160 Pro Leu Vai Al a Met 240 <210> 136 WO 99/32634 WO 9932634PCT/NZ98/O1 89 <211> <212> DNA <213> Mycobacterium vaccae <220> <221> unsure <222> (18) (18) <400> 136 atgagcgaaa tcgcccgncc ctggcgggtt ctggcatgtg gcatc <210> 137 <211> 340 <212> DNA <213> mycobacterium vaccae <220> <221> unsure <222> (273) (273) <221> unsure <222> (286) (286) <400> 137 gccaccggcg gcgccgccgc cccgcgatgc ccgcccgccc gagtttttcg ccgccaaggg ctcaacatcg tgctgccgaa gacgcgttcg cggtgctggc cacgtggtgg tcgacaaaca ggtgcccgcc ggtgtccacg cgtcacgatg gccgcggggc cgaccgggtc cgtaggcgag ggggtgagcg atcgcgccgg gagccgcagt tgggagcaca agnggtaaag ttcgacggca ccccggcggt cgacctcggg ccagccgcga tcccggaccc gtcagnagtc cgcgccggc c cacgctcagc cttccgcgcc gaacgtgccg gacaaacgcc 120 180 240 300 340 <210> 138 <211> 235 <212> DNA <213> Mycobacterium vaccae <220> <221> unsure <222> (16) (16) <400> 138 ggtgaccacc agcgtngaac aggtcgttgc cgaagccgcg gaggccaccg acgcgattgt caacggcttc aaggtcagcg ttccgggtcc gggtccggcc gcaccgccac ctgcacccgg tgcccccggt gtcccgcccg cccccggcgc cccggcgctg ccgctggccg tcgcaccacc cccggctccc gctgttcccg ccgtggcgcc cgcgccacag ctgctgggac tgcag <210> 139 <211> <212> PRT <213> Mycobacterium vaccae <400> 139 Met Ser Glu Ile Ala Arg Pro Trp Arg Val Leu Ala Cys Gly Ile WO 99/32634 WO 9932634PCT/NZ98/00189 Al a Val Pro Thr Leu Asp Ser Gly <210> <211> <212> <213> <220> <221> <222> <400> Thr Gly Ala Pro Ala Thr Met Glu s0 Pro Lys Ala Phe Thr Asn

UNSURE

140 Gly Ala Ala Ala Pro Ala Ser Gly Thr Pro Gin Ser Pro Arg Gly 70 Ala Val Leu Ala His Val 100 Ala Met Leu Ser 55 Trp Ala Val Val Pro Ser 40 Arg Glu Asp Val Pro Al a 25 Glu Asp His Arg Asp 105 Ala 10 Arg Phe Phe Ile Val 90 Lys Gly Pro Phe Arg Pro 75 Gly His Val Val Ala Ala Asp Gly Val Ser Ser Ala Leu Pro Lys Gly Ala Thr Lys Asn Asn Gly Glu 110 140 113

PRT

Mycobacterium vaccae Pro Ile Gly Ile Val Gln Phe Ala Ala Val Val Pro Xaa Asp Val 1l Al a Ala Pro <210> <211> <212> <213> <400> Thr Thr Ile Val Pro Pro 35 Pro Ala Ala Val 141 73

PRT

Mycobacterium vaccae 141 Ser Val Glu Gln Val Val Ala Ala Ala Asp Ala Thr Glu 5 10 Asn Gly Phe Lys Val Ser Val Pro Gly Pro Gly Pro Ala 20 25 Pro Ala Pro Gly Ala Pro Gly Val Pro Pro Ala Pro Gly 40 Leu Pro Leu Ala Val Ala Pro Pro Pro Ala Pro Ala Val 55 Ala Pro Ala Pro Gin Leu <210> 142 <211> 273 <212> DNA <213> Mycobacterium vaccae <400> 142 gcgacctacg tgcagggggg tctcggccgc atcgaggccc gggtggccga cagcggatac WO 99/32634 PCTINZ98/00189 agcaacgccg cggccaaggg ctacttcccg ctgagcttca ccgtcgccgg catcgaccag aacggtccga tcgtgaccgc caacgtcacc gcggcggccc cgacgggcgc cgtggccacc cagccgctga cgttcatcgc cgggccgagc ccgaccggat ggcagctgtc caagcagtcc qcactggccc tgatgtccgc ggtcatcgcc gca 120 180 240 273 <210> 143 <211> 91 <212> PRT <213> Mycobacteriumf vaccae <400> 143 Ala Thr Tyr 1 Asp Ser Gly Phe Thr Vai Val Thr Ala Phe Ile Ala Val Tyr Ala Gln Gly Gly Leu Gly Arg 10 Lys Ile Giu Ala Arg Val Ala Ser Asn Ala Ala Ala 25 Asn Giy Tyr Phe Gly Ile Asp Gin 40 Gly Gly Pro Ile Val Gin Pro Leu Ser Thr Ala Asn Pro Leu Thr Aia Ala Pro Thr Gly Pro Ser Pro 70 Ala Val Ala Thr Thr Gly Trp, Leu Ser Lys Gin Ser Ala Leu Ala Leu Met Ser Ala Val Ile Ala Ala <210> 144 <211> 554 <212> DNA <213> Mycobacterium vaccae <400> 144 gatgtcacgc gtttgagcac caagat tcga gcgccc tggc cggcacagat tcagctcggt ccgtcaccgc tcaccgccaa cgcagcgcaa tcatggccgg ccggagaatg ttcagatctc aggacccaaa cggcgtcggg ggcgggcgcc gaccggtcag ggcgatgaac cccggcggag ctgcaacatc ctga taacgttcga ggttaccttg caacatgaaa gcggcatgtc cagccggccg gcgcgtcagt cagccgcggc tactacgacc accgtgctgc ccggagaacg gatttcaggc ttcactggaa tgttcggcgg agtgcaacgc acctagacac ccgaggccga tgcggggcat cggtagagct ccgtcggcac gggggaagca tgaccgtgcg cgtggccgcg cagctcactc cc acc cgggc ggcgaacctg cctcgcccc~g gcagacggcc aacgagttac gtaaccgatc cgcaagccgc gcaaccgtgg accggcaccg gc caa cc agg cggggctact atcqgtgacg tacgacacgt 120 180 240 300 360 420 480 540 554 <210> 145 <211> 136 <212> PRT <213> mycobacterium vaccae <400> 145 Met Lys Phe Thr Gly Met Thr Val Arg 1 5 Gly Val Giy Ala Ala Cys Leu Phe Gly 25 Ala Ala Gin Met Ala Gly Ala Gin Pro 40 Ala Ser Arg Arg Ala Leu Ala 10 Gly Val Ala Ala Ala Thr Val Ala Glu Cys Asn Ala Ser Ser WO 99/32634 WO 9932634PCT/NZ98/OOI 89 Leu Thr Gly Asp Thr His Thr Val Ser Ser Val Thr Gly Gin Arg Gin Tyr Leu Pro Gly Ala Gin Ala Vai Ala Met Asn Pro Gin Arg Pro Giu Ala Tyr Giu Ala Asn Leu Arg 90 Ile Tyr Phe Thr Ala Asn Pro Ala Giu Ala Gin Arg 115 Ala Tyr Asp 130 Tyr 100 Asn Asp Leu Arg Leu Ala Pro Ile Gly Asp 110 Leu Gin Thr Cys Asn Ile Thr 120 Gly Leu Pro Val Giu 125 Thr Phe Met <210> 146 <211> 808 <212> DNA <213> <220> <221> <222> Mycobacterium vaccae unsure (15) <400> ccaagtgtga gaatcacccg aatgatgaca cggtgcggcc ggcctcctcc ggccgaccc t cgaaggtccc caacccgatg tctcgtcgac cgccaagatc catcctgacc tgtgacggtg cgacgcgtcg caccgtgctg 146 cgcgngtgtg tgtgccaatt actcgccgga gcatgttcga gcgatggagt gcggccaacc gggtcggtgg ctgcagacgc accctcgacg gatccggcca taccacgtcg gagggggcgc gtggtgtgcg atgccgccgg acggtagacg cagtgcgggc agtcagccgc gtgaggacgg ccgcgaccga tgatcggct c ccgggatggc tgtcccaggc gcggtgagtt cgctggagac tgcccggcca cggtcacggt gtggggtgca cagcgtag ttccgaccaa aacggtgtcc agtggcggga tgggagcacg cgagatgacc cggctgcgcg agccgatccq gctgtccggc caccgtgttc cctcaagacg ggccgCgcCC tccaacgacg gtccacgaag atcgctgcgg gcctcgtcgg acgtcgtcgg gcctacgccg gtgacggtgg cagctcaatc gcgccgaccg gactccgaca gatcaggtgg ccgcagctgg ggattcagga tggccatcct ccagcagcac cggc cc ctt C agcaggtccc cggcgtcgaa cgcaggtcaa acgacgcgt t tgctgaccaa tcggcgagca tcaaggtcaa atctgatcga 120 180 240 300 360 420 480 540 600 660 720 780 808 gtccgggatg gccgaccagc gaccgccaac gcgacggtgt <210> 147 <211> 228 <212> PRT <213> mycobacterium vaccae <400> 147 Met Met Thr Thr Arg Arg Lys Ser Ala 1 5 Val Ala Ile Leu Gly Ala Ala Thr Ala Ser Ser Ala Ser Ser Thr Asp Giu Met Thr Thr Ser 55 Ala Asn Leu Ile Gly Ser Gly Ala Cys 25 Thr Ala 40 Ser Ala Ala 10 Ser Ser Ala Val Ala Gly Ile Ala Ala Ser Giu Asp Gly Gly Ser Ser Ala Met Giu Ser Ala Pro Ser Ala Asp Pro Ala Ala Tyr Ala Cys Ala Glu Gln Val Pro WO 99/32634 PCT/NZ98/001 89 62 70 75 Glu Gly Pro Gly Ser Val Ala Gly Met Ala Ala Asp Pro Val Thr Val 90 Ala Ala Ser Asn Asn Pro Met Leu Gin Thr Leu Ser Gin Ala Leu Ser 100 105 110 Gly Gin Leu Asn Pro Gin Val Asn Leu Val Asp Thr Leu Asp Gly Gly a 115 120 125 Glu Phe Thr Val Phe Ala Pro Thr Asp Asp Ala Phe Ala Lys Ile Asp 130 135 140 Pro Ala Thr Leu Glu Thr Leu Lys Thr Asp Ser Asp Met Leu Thr Asn 145 150 155 160 Ile Leu Thr Tyr His Val Val Pro Gly Gin Ala Ala Pro Asp Gin Val 165 170 175 Val Gly Glu His Val Thr Val Glu Gly Ala Pro Val Thr Val Ser Gly 180 185 190 Met Ala Asp Gin Leu Lys Val Asn Asp Ala Ser Val Val Cys Gly Gly 195 200 205 Val Gin Thr Ala Asn Ala Thr Val Tyr Leu Ile Asp Thr Val Leu Met 210 215 220 Pro Pro Ala Ala 225 <210> 148 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <221> unsure <222> <221> unsure <222> <400> 148 gcsccsgtsg gnccggntgy gc 22 <210> 149 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <221> unsure <222> <221> unsure <222> WO 99/32634 WO 9932634PCTINZ98/OO1 89 <221> unsure <222> (16) (16) <221> unsure <222> (20) <400> 149 rtasgcsgcn gtngcnacng g <210> 150 <211> 102 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 150 gcccccgtcg gccccggctg tgcggcctac gtgcaacagg tgceggacgg gccgggatcg gtgcagggca tggcgagctc gcccgtagcg accgccgcgt at <210> <211> <212> <213> 151 683

DNA

Mycobacterium vaccae <400-, gcccgccaac cagcatgaaa gggtaccgca acaggtgccg ggcggccgac gaacgtcaat tgacgccttc gctgaccaag cggcgagcat caaggtcaac tctgatcgac tcccccgcac 1151 taaaaccgcc actcttgccg ggcagcgccg gacgggccgg aacccgctgc ctcgtcgaca gccaagatcg atcctcacct gtgacggtgg gacgcgt cgg accgtgctga ccggcctccc gatcatccac gagcgggttt cagccgcgcc gatcggtgca tcaccacgct cgttcaacgg atccggccac accacgtcgt agggggcgcc tggtgtgcgg tgccgccggc ccg tgcaggaagg cgcgatgacc ggtcggaccg gggcatggcg ctcgcaggcg cggccagttc gctggagacc gc ccggc cag ggtcacggtg tggggtgcag agcgtagccg aatctcacga gccgCCgtCg gggtgtgCgg agctcgccgg atctcgggtc accgtgttcg ctcaagaccg gccgcgcccg tccgggatgg accgccaacg ggcggcacca tcatgaacat gtctgtcgct cctacgtgca tggccaccgc agctcaaccc cgccgaccaa attccgacct atcaggtggt ccgaccagct cgacggtgta cagaagaggg 120 180 240 300 360 420 480 540 600 660 683 <210> 152 <211> 231 <212> PRT <213> Mycobacterium vaccae <400> 152 Asp Thr Val Leu Met Pro Pro Ala Asn Asn 1. 5 10 Gly Arg Asn Leu Thr Ile Met Asn Ile Ser 25 Ala Gly Phe Ala Met Thr Ala Ala Val Gly 40 Gly Ser Ala Ala Ala Ala Pro Val Gly Pro Arg Arg Ser Ser Thr Ala Met Lys Thr Leu Ala Gly Leu Ser Leu Gly Thr Ala Gly Cys Ala Ala Tyr Val WO 99/32634 WO 9932634PCT/NZ98/OO1 89 Gin Gly Gin Pro Vai Pro Asp Gly 70 Ala Pro Gly Ser Val Gin Met Ala Ser Ser Val Ala Thr Ala Ala Asp Asn Pro Leu Leu Thr Thr Leu Ser Gly 90 Asn Gin Ala Ile Phe Asn Gly 115 Ala Lys Ile Ser 100 Gly Gin Leu Asn Pro 105 Phe Val Asn Leu Gin Phe Thr Val 120 Leu Ala Pro Thr Asn 125 Thr Vai Asp Thr 110 Asp Ala Phe Asp Ser Asp Asp Pro Ala Glu Thr Leu 130 Leu Leu Lys 140 Pro Thr Lys Ile 145 Pro Leu 150 Giy Tyr His Val Val 155 Val Gly Gin Ala Al a 160 Asp Gin Val Val 165 Met Glu His Val Thr 170 Lys Glu Gly Ala Pro Val 175 Ser Val Thr Val Ser Val Cys Gly 195 Thr Val Leu Gly 180 Gly Ala Asp Gin Val Asn Asp Val Gin Thr Al a 200 Al a Ala Thr Val Tyr 205 Thr Ile Asp Met Pro Pro 210 Pro Pro 225 Ala 215 Pro Pro Gly Gly Giu Giu Gly His Pro Ala Ser 2310 <210> <211> <212> <213> <220> <221> <222> 153 1125

DNA

Mycobacterium vaccae unsure (358) (358) <400> 153 atgcaggtgc tggcagacgg atcttcgcgc aacgcgctgc gcaggattcg gcagtggatg cgccggcgtc cccgatgccg gcgcgacatc gatcqatctg cttcgcctac gggccagagc ggtaacgctg gggcggccag gggactcggt gatgcacatc agagcgcacc acgcgccgtt gggcgcggac ggcgtgttct gggtttcgat gcgggaccgg ggcccaaggt gacttcgaca gccgacaact atcgacctga ccccgcgtcg cgtggtggcg tgtgcgctcg gccgacaacg gtcgagctgc gaggacgccg cgggtgaccg gcggcataag cgaacctgga cggtcccgga cgaccagccc cgcccggccg gggcagtgtc accgaccgcc tgcggaaccc cggtgagcag aatcggcgcc gcc cggacac tcaccgtcga ccgaccacgt gtccgctgcc acgatccgtt gcatggtgga ccgaggcgcc gaccgctgcg ccaccgcgcc cgaataggag c ccggg ccgt ctcgtcccgg cgggaggccc cggctggccg ggtgcagcag tcagcggatc ggcctcgggt tcggtgggca catgggcgcg caagcttgtc tccgcgaccg ggccgccgtt gcagatgagc ctgctcgccc ggaagccgcg ctacctgcac cgaaggcgac cgcggagat c tcctgctggc cggcggcacc tgaccgtcgc tggtgccgca cgtgggcccc tcgcggtttc cgtgtccgga gggtcggtga cctacgcggt gccgacgcat ctgggcggca ctgggccggt gtggtcttcg ggcacctacg ggcttcaacc aacttcgccc ctgttcgtcc gcagtgcgtt ctcgtctggg cggcgcagca ggccccgacg ggtcgacgaa gggttggacg ggacg ggccgcgtta catcgaggtg tgcgttcgtc gaactacccg cggggcgggt tgtcgcangg tcacccccac gaaatccgtt ggccgaagtc tgccggccca gcctggaacc cgcggggcga tcaccgcatc agatgcatgc ctgctcgccg agcacaaccg cctctggccg ctgtcggtgt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1125 WO 99/32634 WO 9932634PCT/NZ98/00189 Met 1 Ser Asp Glu Pro Ala Ser Val Val Arg 145 Arg Met Leu Leu Pro 225 Pro Pro Ser Leu Gly 305 Val <210> 154 <211> 748 <212> PRT <213> Myco <220> <221> UNSU <222> (119 <400> 154 Gin Val Arg Ala Ala Leu Pro Cys Pro Pro Gly Leu Lys Val Gly Gly Phe Asp Giy Arg Vai 100 Leu Gly Gly 115 Asp Pro Arg 130 Val Ala Asp Asp Ile Arg Giu Thr Ser 180 Tyr Pro Arg 195 Giu Cys Tyr 210 Arg Pro His His Giu Ala His Giu Ala 260 Asn Gly Leu 275 Ala Ala Leu 290 Leu Glu Gly Ala Leu Gly Arg 5 Trp Asp Gly Giu Phe Gin Met Pro His Gly 165 Glu Leu Ser Glu Ser 245 Leu Tyr Ala Leu Leu 325 Val Gin Ile Trp Gin 70 Asp Trp Ser Leu Val 150 Gly Arg Tyr Ala Cys 230 Asn Ala Met Ala Pro 310 Leu Leu Thr Giu Val 55 Ser Lys Met Xaa Gly 135 Ala Gly Gly Ser Leu 215 Tyr Leu Thr Giu Ser 295 Arg Glu Giy Giy Val 40 Gly Val Ser Ala Gly 120 Arg Al a Pro Leu Ser 200 Ala Ser Glu Tyr Thr 280 Asn Gly Pro Ser Val 25 Ile Asp Gly Ala Asp 105 Ala Phe Val Arg Tyr 185 Glu Leu Ser Pro Arg 265 Val Pro Leu Arg Val 10 Ser Phe Ala Thr Pro 90 Asn Gly Thr Val Leu 170 Thr Arg Giu Glu Arg 250 Ala Ala His Tyr Gly 330 Gly Ile Ala Phe Tyr 75 Met Cys Val Pro Val 155 Giu His Ile Ala Arg 235 Ala Leu Leu Glu Gly 315 Leu Ala.

Pro Arg Val Al a Gly Pro Ile Thr 140 Phe Pro Arg Leu Ser 220 Pro Leu Ala Gly Ala 300 Leu Ala A~la Thr Gly Asn Val Ala Asp Asp 125 Pro Gly Arg Thr Giu 205 Pro Arg Ala Ala Leu 285 Leu Asn Leu Val Ala Thr Ala Asn Ala Thr 110 Leu Met Asn Gly Tyr 190 Ala Ala Gly His Ser 270 Gly Ala Ser Ala Al a Ser Gly Leu Tyr Asp Lys Ile Pro Pro Leu 175 Arg Ser Ser Leu Ile 255 Pro Leu Ala Giu Pro 335 bacterium vaccae

RE

(119) Val Ala Ala Arg Pro Ala Leu Thr Pro Leu 160 Asn Gly Pro Pro Tyr 240 Ser Ala Ala Arg Arg 320 Arg WO 99/32634 WO 9932634PCT/NZ98/00189 Thr Pro Leu Glu.

385 Ala Arg Thr Ala Met 465 Tyr Ser Gly Met Arg 545 Tyr Leu His Gly Leu 625 Tyr Pro Leu Ala Leu 705 Ala Tyr Arg Glu 370 Al a Leu Gly His Gly 450 Glu Al a Glu Ser Glu 530 Pro Gly His Arg Pro 610 Tyr Ala Arg Tyr Leu 690 Ala Leu Arg Ala 355 Gly Arg Ala.

Leu Arg 435 Leu Thr Leu Arg Glu 515 Thr Thr Leu Ile Ala 9*5 Arg Ala Arg Ala Al a 675 Ala Ala Ala Leu 340 Arg L~eu Gly Arg Tyr 420 Ala Ile His Al a Pro 500 Arg His His Tyr Ser 580 Arg Gly Arg Gly Leu 660 Leu Val Ser Gly Glu His Ile Ser Leu Glu Pro His Glu Val Ala Leu Gly Ala Gly Gly 405 Gly Leu Leu Ile Ala 485 Arg Thr Ile Arg Thr 565 Ala Gly Leu Gly Ala 645 Al a Ala Ala *Pro *Leu 725 Gly Ser Leu 390 Pro Leu Ala Glu Ser 470 Leu Ala His Ser Ala 550 His S er Ser Tyr Ala 630 Arg Pro Ala Leu Ala 710 Tyr Leu Pro 375 Gly His Tyr Thr Leu 455 Ala Ala.

Leu Arg Ile 535 Arg Arg Asn Glu Ala 615 Arg Gly Arg Leu Gly 695 Arg Ala Tyr 360 Ala Leu G1u Gly Hi s 440 Glu Leu Ala Al a Al a 520 Leu Gly Gly Al a Arg 600 Ser Gly Ala Gly Ala 680 Leu Gly Arg 345 Gly Leu Tyr Thr Leu 425 Arg Val Ala Leu Gly 505 Leu Glu Ala Leu Arg 585 Ala Pro Thr Leu Leu 665 Gly Tyr Pro Gly Leu Al a Ala His 410 Asn Ala Ala Gly Ala 490 Leu Ala Ala Leu Tyr 570 Gly Arg Ala His Ala 650 Tyr Leu Val *Arg *Val 730 Vlal Gly Ser 395 Arg Ala Lepi Leu Leu 475 Al a Tyr Ala Arg Ala 555 Pro Gly Gly Arg Arg 635 Val Gly Tyr Ala Ala 715 Ala Ala Leu 380 Pro Ala Arg Ala Thr 460 Tyr Ser Ala Arg Gly 540 Val Arg Leu Thr Gly 620 Ser Ala Leu Leu Leu 700 Leu Lei: Leu 365 Tyr Al a Leu Gly Pro 445 Arg Leu Asn Arg Gly 525 Thr Ala Ala Ala His 605 Al a Glu Leu Tyr Glu 685 Gly Ala Gly 350 Thr Pro Leu Ala Val 430 Arg Pro Glu Ala Gly 510 Ala His Leu Ser Arg 590 Arg Arg Arg Al a Pro 670 Ala Leu Al a Leu His Arg Ala Ser 415 Ala Ala Gly Gly Arg 495 Ala Arg Arg Gly Pro 575 Gly Ala Gly Gly Arg 655 Arg Ser Tyr Leu Tyr Arg Leu Val 400 Glu Leu Leu Leu Leu 480 Gly Arg Gly Thr Leu 560 Gly Thr Arg Gly Leu 640 Gly Gly Pro Ala Ala 720 Pro Arg Gly Leu Tyr Ala Arg Gly Pro Arg Gly Leu Tyr 740 745 WO 99/32634 WO 9932634PCT/NZ98/OOI 89 <210> <211> <212> <213> 155 666

DNA

mycobacterium vaccae <400> atgaaggcaa ccgctttgtt ggcatccacg ggcgtcttcc acctacgtcg caggtgggct atcacgtacg atcgtgaccc ggcatccagg gtgtccaacg gcccgcctga aactga 155 atcattcggg cgcccgcact gccagggccc cgttggaccg tggccggtga ttccgtggt c acggttacgg cgccgctgtt aggtcgcgac cgcacggcac tctcgtcgac atgctacaaa ggcaccatct ggaacgactg caaccggttg aggtgccgac gctgggcgtg cctcaacttc cccgggtgtc cttctccgtg ggtcaccggt cggcgacagc tccgccggcc catgcaggt c accattcagc acccgggagt gagttcgagg ggcatcaact gccgacccgC tcgatcacgg gacgtggccg gctgccggtg gtcaccacct cgatatggtc tggacaatga agtgggacac ggttccactc gcacgctgga tcagctacac tgctgggctt cggacctggg gccccggtgg gtgtgctgct acggcgcacc gcatccatcg gctgagcctg cttcctcaac gggcaaggcg gctgggctac caccccgaac cggtgattcc caa cggc ccc ttccgtggtg gcgtccgttc ctggaacatg 120 180 240 300 360 420 480 540 600 660 666 <210> <211> <212> <213> 156 221

PRT

Mycobacteriumn vaccae <400> 156 Met Lys Ala Asn His Ser Gly Cys Tyr 1 Ser Gly His Pro Ser Leu Asp Asn 5 Pro Glu Lys 10 Ala Leu Cys Ser Leu Ser Leu 40 Pro 25 Gly Ser Ala Gly Pro Ile Trp Leu Ala Pro Ser His Ala Gly Pro Glu Val Phe Pro Val His Gly His Leu Thr Gin Gly Leu Asp Arg Ile Gin Gin Trp 55 Asn Arg Leu Thr 70 Val Ala Gly Giu Asp Thr Phe Leu Arg Giu Trp Thr Asn His Phe 75 Glu Ser Gly Lys Ala Tyr Val Gly Ala Asp 90 Trp Phe Giu Gly Thr Leu Giu Leu Gly Asn Phe Ser 115 Asn Phe Ala Tyr His Val 100 Tyr Thr Thr Asp Pro Leu Gly Phe Pro Asn 120 Leu Gly Pro 105 Ile Ser Leu Gly Thr Tyr Asp Gly 125 Ile Val Gly Ile 110 Tyr Gly Leu Val Thr Pro Phe Gly Asp 130 Pro Leu 135 Ser Ser 140 Leu Phe Pro Gly 145 Gly Val 150 Ala Ile Thr Ala Asp 155 Gly Asn Gly Ile Gin Giu Val Thr Phe Ser Gly Ser Val Val Ser Asn Ala His 185 Ala Val Asp 170 Gly Thr Arg Leu Val Thr Gly Ala Ala 190 Ile Ser Ser Thr Gly 205 Val Ala Gly Gly Gly Val 195 Asp Ser Val 210 Leu Leu Arg Pro Thr Thr Tyr Gly 215 Phe 200 Ala Pro Trp Asn Met Asn 220 WO 99/32634 WO 9932634PCTNZ98/OO1 89 <210> <211> <212> <213> 157 480

DNA

Mycobacterium vaccae <400> 157 aacggctggg acatcaacac atcatgccgg tcggcggaca aacgggcaga actacaccta ctggaggcca accgcggagt ggcagcgcgg cgctgaccta ctgtcaggct tcctgaaccc aacgacgcag gcggcttcaa aagcgcaacg acccgatggt ccctgcgttc gtccagcttc caagtgggag gtcgcgcacc cgcgatccat gtccgagggc cgc cgagagc caacatcaac gagtggttct tacagcgact acgttcctga ggcaacgcgt cacccgcagc tggtggccga atgtggggcc cagctggtgg acgagtccgg ggtaccagcc cccaggagct tcgtcggcct.

agttcatcta tgctgatcgg cgtcctcgga ccaacaacac cttgtcgacg gtctcggggc gccgacgtgg gtcgatggcg cgcctcgtcg gctggcgatg cccggcgtgg ccggatctgg 120 180 240 300 360 420 480 <210> <211> <212> <213> 158 161

PRT

mycobacterium vaccae Asn 1 Gly Asp <400> 158 Gly Trp Asp Leu Ser Thr Trp Tyr Gin Ile Asn Thr Pro Ala Phe Giu Trp Phe Tyr Glu Ser 10 Gly Ile Met Pro Val Gly 25 Asn Gin Ser Ser Trp Glu Arg Gly Thr Phe Pro Ser Arg Leu Thr Gin 55 Arg Thr Giy Gly 40 Giu Gly Gin Asn Tyr Leu Phe Tyr Ser Thr Tyr Lys Glu Ala Asn Leu Pro Thr Trp Gly Val Ser Asn Ala Phe Leu Ser Met Gly 70 Thr Ala Ser Ala Ala Leu Leu Tyr Ala Ile His 90 Asn Pro Gin Gin Phe Ile Tyr Al a Ser Pro Met Leu 115 Giu Ser Met Ser 100 Ile Ser Gly Phe Leu 105 Asn Pro Ser Glu Gly Leu Ala Asp Ala Gly Gly 125 Lys Gly Trp Trp 110 Phe Asn Ala ALrg Asn Asp Trp Gly Pro Ser Asp Pro Ala 130 Pro Met 145 Ile Trp 140 Asn Val Asn Ile Asn 150 Leu Val Ala Asn 155 Thr Arg Ile Trp 160 <210> <211> <212> <213> 159 1626

DNA

Mycobacterium vaccae <400> 159 atggccaaga caattgcgta tgacgaagag gcccgccgtg gcctcgagcg gggcctcaac gccctcgcag acgccgtaaa ggtgacgttg ggcccgaagg gtcgcaacgt cgtgctggag 120 WO 99/32634 WO 9932634PCT/NZ98/OOI 89 aagaagtggg ctggaggacc gacgacgtcg gaaggcctgc aaggctgtcg gagcagattt gccgaggcca ttcggcctgc tacttcgtga gtcagctcca gccggcaagc gtggtcaaca gaccgccgca gaaagagtcg gtcgtcqtca gCC9gccggg gagaagctgc gctgccaccg gcgaaggctg gctcctgcgc cgcgtggcgc gtcgttgccg gagtacgagg ctgcagaacg aagccggaga ttctaa gcgcccccac cgtacgagaa cgggcgacgg gcaacgtcgc aggctgtcac ctgccaccgc tggacaaggt agctcgagct ccgacgccga aggtgtcgac cgctgctgat agatccgcgg aggcgatgct ggctgtcct ccaaggacga tggctcagat aggagcgcct aggtggagct ccgtcgaaga tggacgacct tgtcggCtcc agaaggtgtc acctgctcaa cggcgtccat aggcgtccgc gatcaccaac gatcggcgct caccaccacc agccggcgcc ccagtcgctg ggcgatttcc cggcaacgag caccgagggt gcgccaggaa cgtcaaggat catcgccgag caccttcaag gcaggacatg ggagaccgcc gaccaccatc ccgcgccgag ggccaagctg caaggagcgc gggcatcgtc cggcctgacg gctcaagcag caacctgccc ggccggcgtc cgcggCtctg acccgcgggc gatggtgtgt gagctggtca gccaccgtgc aacccgctcg ctgaagtcgg gc cggcgaca ggtgtcatca atgcgcttcg gccgtcctgg ctgctcccgc gacgtcgagg tccgtcgccg gccatcctca gacgtctcgc gtcgagggct atcgagaaca gccggcggtg aagcaccgca gccggtggcg ggcgacgagg atcgccttca gcgggtcacg gccgacc cgg ttcctcacca gacccgaccg ccatcgccaa aagaggtcgc tcgctcaggc gccicaagcg ccaaggaggt cccagatcgg ccgtcgagga acaagggcta aggatcccta tgctggagaa gcgaggccct tcaaggctcc ccggtggtca tgctgggcca cgggcgattc gcgactccga ttgcggtgat tcgaggacgc gcgtggctct ccaccggtqc acggcggcct gcctcaacgc tgaaggtcac ccgaggccgt gtggcatggg ggagatcgag caagaagac c tctggttcgc tggcat cgag cgagaccaag cgagctcatc gtcgaacacc catctcgggt catcctgctg ggtcatccag gtccacgctg gggcttcggt ggtcgtcagc ggcccgcaag cgatgccatc ctacgaccgc caaggccgga cgtccgcaac gctgcagtcg caacatcgtc ggagcccggc cgcgaccggt ccgctcggcg cgtcgccgac cggtatggac 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1626 <210> 160 <211> 541 <212> PRT <213> Mycobacterium vaccae <400> 160 Met Ala Lys -Thr Ile Ala Tyr Asp Glu 1 Arg Lys Giu 10 Val Ala Arg Arg Gly Leu Glu Gly Leu Asn Ala Leu Ala Asp Ala 25 Lys Val Thr Gly.

Thr Asn Tyr Glu Arg Asn Val Val Leu Glu Lys Lys Trp 40 Asp Gly Val Ser Ile Ala Lys Glu Ile 55 Lys Ile Gly Ala Glu Leu Val Lys Giu 70 75 Val Ala Gly Asp Gly Thr Thr Thr Ala 90 Val Arg Glu Gly Leu Arg Asn Val Ala Gly Glu Val Ala Leu Leu Gly Pro Pro Thr Ile Glu Asp Pro Ala Lys Lys Asp Thr Asp Thr Val Leu Ala Gly Ala Ala Gin Asn Pro Ala Leu Leu Gly Leu 100 Lys 105 Lys 110 Val Thr Gin Arg Gly Ile Glu Ala Val Glu 115 120 Ser Leu Leu Lys Ser Ala Lys Glu Val 130 135 Ala Thr Ala Ala Ile Ser Ala Gly Asp 145 150 Giu Thr Lys Glu Gin Ile Ser 140 Thr Gln Ile Gly Glu Leu Ile 155 160 WO 99/32634 WO 9932634PCTINZ98/OOI 89 Ala Glu Phe Gin Vai 225 Ala Leu Ala Asp Leu 305 Val Ser Asn Lys Val 385 Ala Leu Glu Lys Lys 465 Glu Thr Thr Ala Giu Ala Ser Asn Asp Lys 195 Giu Ala 210 Ser Thr Gly Lys Ser Thr Val Lys 275 Met Ala 290 Ser Leu Val Val Asp Ala Ser Asp 355 Leu Ala 370 Glu Leu Lys Ala Leu Gin Ala Thr 435 Gin Ile 450 Val Ser Tyr Glu Arg Ser Thr Glu 515 Gly Asp 530 <210> <211> <212> <213> Met Thr 180 Gly Val Val Pro Leu 260 Ala Ile Giu Thr Ile 340 Ser Gly Lys Ala Ser 420 Gly Ala Asn Asp Ala 500 Ala Pro Asp 165 Phe Tyr Leu Lys Leu 245 Val Pro Leu Thr Lys 325 Ala Asp Gly Glu Val 405 Ala Ala Phe Leu Leu 485 Leu Val Lys Gly Ile Glu Asp 230 Leu Val Gly Thr Ala 310 Asp Gly Tyr Val Arg 390 Glu Pro Asn Asn Pro 470 Leu Gin Val Val Leu Ser Asp 215 Leu Ile Asn Phe Gly 295 Asp Giu Arg Asp Ala 375 Lys Glu Ala Ile Gly 455 Ala Lys Asn Ala Gly Gin Gly 200 Pro Leu Ile Lys Gly 280 Gly Val Thr Val Arg 360 Val His Gly Leu Val 440 Gly Gly Al a Al a Asp 520 Asn Leu 185 Tyr Tyr Pro Ala Ile 265 Asp Gin Ser Thr Ala 345 Glu Ile Arg Ile Asp 425 Arg Leu His Gly Ala 505 Lys Giu 170 Glu Phe Ile Leu Glu 250 Arg Arg Val Leu Ile 330 Gin Lys Lys Ile Val 410 Asp Val Giu Gly Val 490 Ser Pro Gly Leu Val Leu Leu 235 Asp G li Arg Val Leu 315 Val Ile Leu Ala Glu 395 Ala Leu Ala Pro Leu 475 Ala Ile Glu Val Thr Thr Leu 220 Glu Val Thr Lys Ser 300 Gly Glu Arg Gin Gly 380 Asp Gly Gly Leu Gly 460 Asn Asp Ala Lys Ile Glu Asp 205 Val Lys Glu Phe Al a 285 Giu Gin Giy Ala Glu 365 Ala Ala Gly Leu Ser 445 Val Ala Pro Ala Ala 525 Thr Gly 190 Ala Ser Val Gly Lys 270 Met Arg Ala Ser Giu 350 Arg Ala Val Gly Thr 430 Ala Val Ala Val Leu 510 Ser Val 175 Met Glu Ser Ile Giu 255 Ser Leu Val Arg Gly 335 Ile Leu Thr Arg Val 415 Gly Pro Ala Thr Lys 495 Phe Ala Glu Arg Arg Lys Gin 240 Al a Val Gin Gly Lys 320 Asp Glu Ala Giu Asn 400 Ala Asp Leu Giu Gly 480 Val Leu Pro Thr Gly Gly Met Gly Gly Met 535 Asp Phe 540 161 985

DNA

Mycobacterium vaccae WO 99/32634 WO 9932634PCT/NZ98/OOI 89 '400, gga tccc tac gctggagaag cgaggccctg caaggctccg cggtggtcag gctgggccag gggcgattcc cgactccgac tgcggtgatc cgaggacgcc cgtggctctg caccggtgcc cggcggcctg cctcaacgcc gaaggtcacc cgaggccgtc tggcatgggc .161 atcctgctgg gtcatccagg tccacgctgg ggcttcggtg gtcgtcagcg gcccgcaagg gatgccatcg tacgaccqcg aaggccggag gtccgcaacg ctgcagtcgg aacatcgtcc gagcc cggcg gcgac cggtg cgctcggcgc gtcgccgaca ggtatggact tcagctccaa ccggcaagcc tggt caac aa accgccgcaa aaagagtcgg tcgtcgtcac ccggccgggt agaagctgca ctgccaccga cgaaggctgc ctcctgcgct gcgtggcgct tcgttgccga agtacgagga tgcagaacgc agccggagaa tctaa ggtgtcgacc gctgctgatc gatccgcggc ggcgatgctg gctgtccctg caaggacgag ggctcagatc ggagcgcctg ggtggagctc cgt cgaagag ggacgac ctg gtcggctccg gaaggtgtcc cctgctcaag ggcgtccatc ggcgtccgca gtcaaggatc atcgccgagg accttcaagt caggacatgg gagaccgccg accaccatcg cgcgccgaga gccaagctgg aaggagcgca ggcatcgtcg ggc ctgacgg ctcaagcaga aacctgcccg gccggcgtcg gcggctctgt c ccgcgggcg tgctcccgct acgtcgaggg ccgtcgccgt ccatcctcac acgtctcgct tcgagggctc tcgagaacag ccggcggtgt agcaccgcat ccggtggcgg gcgacgaggc tcgccttcaa cgggtcacgg ccgacccggt tcctcaccac acccgaccgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 985 <210> <211> <212> <213> 162 327

PRT

Mycobacterium vaccae <400> 162 Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys Val Ser Thr Val 5 10 Lys Asp 1 Leu Ile Leu Pro Leu Ile Ala Glu Leu Giu Lys Val Asp Val Glu Asn Lys Ile Giy Lys Ile Gin Ala 25 Giu Ala Leu Ser Val Ala Gly Lys Ser Thr Pro Leu Leu Leu Val Val Arg Gly Thr Phe Gly Phe 55 Ala Val Met Ala Pro Gly Asp Arg Arg Gly Lys 70 Ser Met Leu Gin Asp 75 Ala Ile Leu Thr Gly Gin Val Val Leu Giu Arg Val Gly 90 Lys Leu Ser Leu Giu Thr Ala Asp Val Ser Giu Thr Thr 115 Arg Val Ala Leu 100 Ile Gly Gin Ala Arg 105 Gly Val Val Val Val Giu Giy Ser 120 Glu Asp Ser Asp Al a 125 Asp Thr Lys A sp 110 Ile Ala Giy Ser Asp Tyr Gin Ile Arg 130 Asp Arg Ala 135 Glu Ile Giu Asn Ser 140 Leu Giu Lys Leu 145 Ala Gin 150 Gly Arg Leu Ala Lys 155 Ala Gly Gly Val 160 Val Ile Lys Ala 165 Glu Ala Ala Thr Lys His Arg Ile Asp Ala Val Arg 185 Val Giu Val 170 Asn Ala Ala Leu Glu Leu Lys Giu Arg 175 Lys Ala Ala Val Glu 190 Leu Gin Ser Ala Pro Giu Gly Ile 195 Ala Leu Asp Ala Gly Gly Gly 200 Thr 205 Thr Asp Leu Gly Leu Asp LeuGly Leu Gly Asp Glu Ala GyAlAs Gly Ala Asn WO 99/32634 WO 9932634PCT/NZ98/00189 210 Val1 215 Ser 220 Gin Ile 225 Gly Arg Val Ala Ala Pro Leu Lys 235 Lys Ile Ala Phe Asn 240 Gly Leu Giu Pro 245 Leu Val Val Ala Val. Ser Asn Leu Pro 255 Ala Gly His Lys Ala Gly 275 Asn Ala Ala Gly 260 Val Asn Ala Ala Thr 265 Lys Glu Tyr Glu Ala Asp Pro Val 280 Leu Val Thr Arg Ser 285 Giu Asp Leu Leu 270 Ala Leu Gin Ala Val Val Ser Ile Ala 290 Ala Asp Ala 295 Phe Leu Thr Thr 300 Gly Lys Pro Glu 305 Gly Lys Ala 310 Asp Phe Ser Ala Pro Ala 315 Asp Pro Thr Gly 320 Met Giy Giy Met 325 <210> i63 <211> 403 <212> DNA <213> Mycobacteriumn vaccae <400> 163 ggatccgcgg gaactccgtc ctatcagttc cgaacccgga ggccgaggag caccgacggc catcgccgcg caccggctgg ggcatcqgcg gtcggccgca agtccctggc ctgctggaat gtgttctccc ttccgttcc tgacgaccaa gcgcgtacct ccacccaggt tgctgcggtt tgcgagccga tcgccgagca ggcaggcggc gtacaacccg gtgcatctac gtggagt cgt tttcgaccga catggccgca cgaacggttc cgcgttCtcc gcccgcacct gggatggagg taccgccaca atttcgtggt ggccggggct ctggccgaca gcc ggacggccga gccccggcgg cggcgccgt t atccggtgtc cggtcgacat acgc cgacga 120 180 240 300 360 403 <210> i64 <21i> 336 <212> DNA <213> Mycobacterium vaccae <400> i64 cggaccgcgt gggcggccgc accgacgccg ataccgggga tcgcgtcgag cgtgtggaag cgttgctggc gctggaggcg tggtcaccca gatcctggtc tggtcggcac cggagtgcgc cggcgagttc cctggtgctc gtcgacgtcg atgaagatgg tccgctgggc gcatgagcgc gaccgcgccg tacgacggtg ccgtcggtga agaccgtgct atctcgtcga cgtcga agaaagccgc cgagcgggtc ccgggtggtg gcgcgccccg tcccgqcacc gtcgaaggcc gacgctccgt gccggacagc gccgacgggg ccactggtcg 120 180 240 300 336 <210> 165 <2i1> 134 <212> PRT <213> mycobacteriumn vaccae <400> 165 Asp Pro Arg His Arg Leu Val Thr Thr Lys Tyr Asn Pro Ala Arg Thr 1. 5 10 Trp, Thr Ala Glu Asn Ser Val. Gly Ile Gly Gly Ala Tyr Leu Cys Ile 25 WO 99/32634 WO 9932634PCTINZ98/OOI 89 Tyr Gin Pro Ala Ser Phe Ala Arg 1 Ala Gly Asp Leu Val Thr Met Trp Leu Glu Asp Ala 115 Ala Glu Ser Leu Leu Ile 100 Asp Phe Gly Arg Arg Leu Thr Asn Ser Gly Arg Phe Leu Gly Asp Gly 40 His Asp Arg Val Asp 120

'I

<210> <211> <212> <213> <400> Thr Al a Ser Lys Asp Glu Val Ala Giu Ala Val Thr Pro Leu 166 108

PRT

Mycobacteriumn vaccae 166 Trp Ala Ala Ala Gly 5 Ala Thr Asp Ala Asp Arg Vai Asp Ala Pro 40 Val Gly Asp Arg Vai 55 Met Lys Met Glu Thr 70 Gin Ile Leu Val Ser Val Val Vai Gly Thr 100 yr 'hr ~rg l1a ~he .05 Ele .1u rhr 25 Phe Val VTal Ala Gly 105 Gin Phe Ala Pro Ile Ser 75 Asp Met 90 Ser Leu Ala Ala Phe Asp 10 Gly Asp Ala Ser Ala Gly Leu Arg 75 Gly His 90 Val Arg Val Gly Phe Giu Trp Tyr Ala Ala Ala Giu Phe Arg 125 Arg Ala Leu Val Ser Val Gin Pro Ala Pro Leu Val Al a Arg Pro Pro Gly His 110 Ser Glu Leu Trp Leu Ala Asp Thr Gly Val Arg Giu Arg Lys Tyr Lys Leu Asp Pro Thr Ser Ser Gly Arg Gin Ala Asp Val Ala Gly Gly <210> 167 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 167 atagaattcg tccgacagtg ggacctcgag c <210> 168 <211> 27 <212> DNA <213> Artificial Sequence <220> WO 99/32634 WO 9932634PCT/NZ98/OOI 89 <223> Made in a lab <400> 168 atagaattcc caccgcgtca gccgccg <210> 169 <211> 1111 <212> DNA <213> Mycobacterium vaccae <400> 169 gtccgacagt gcgcgtctcc ctcgggcatc ggtcaaggag gttcatggcc gcccaatcgc gttcaccgcg acgcgatatc gttctccgac gaatccgacc ggggtcagat cgccat cgcg gcagtt cat c caccacgcag ctacgccaag actcgccaag gcaggcgaac tgcgtacgcc tggccctgcg gggacctcga aactggccgc acggtcgact ccgttgtcgc gcgcgcgtca aagaatctgc ccgtacatga cgcaccatcg gtccaggacg accgagtcca ccgtcgcttc caggcgtact gttcccgaat aaccagaagg ctggtcgcgt gtcgatcctg ctgaagtcgt gccgtcaccg gacgcgagga gcaccacgtc tctatatggc acaaagaaga gcaagcagga agggcctggg gtcaggacct ccggcatggt acgacctctg gcctcggcat ttcagcaggc accggcaacg ccggtgacgt ccggcggcga ccgccgaggc tcacccagtt catcggcgga gggcggcact gcggctgacg gcataaatgg acaggacagc cgacggtttc cttcaacgac cat aggcgcc atggctcaat gttggactcg cggtctcgcc ggatcccgcg gatcatgctc ggtcgatctg actacgccga cgtgcagctg ctggttcgtc gtggatcgac cgtgcccgca gaacccgc tg gaccgacgag cggtggtagt

C

ggccccgcca atcgcagcgt aacgagcagt gacctggtga gagatcagcg agcatcgacg tacaacaagg ttcaagggcc tcgcagggca gtccgcgaac cga cc tggc c caggcggaca gacacgatgg tacatctacg ctctcggaca atcaacccgt cagacgcagg gccgatgcga gcggcgccct tccagaccgc ggttcgccaa tccccaccga aagc cggcgt agggccgcaa cagccaccgg gcgtcagtct actcgccgga agaacgacag gcagaaacat accccgatct tgatcccgta accgagccaa tgaccgacga cggc cgaggt agttcaacac ggggcataaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1111 <210> 170 <211> 348 <212> PRT <213> Mycobacterium vaccae <400> 170 Ser Asp Ser Gly Thr Ser Ser Thr Thr 1 5 Ser Gly Ala Leu Arg Val Ser Asn Trp 25 Phe Ile Ala Ala Phe Gln Thr Ala Ser 40 Glu Asp Phe Asn Asp Asn Glu Gin Trp 55 Leu Ser Arg Lys Gin Asp le Gly Ala 70 Phe Met Ala Ala Arg Val Lys Gly Leu Glu Ala Gly Val Pro Asn Arg Lys Asn 100 105 Ser Gin 10 Asp Ser Gly Pro Ala Pro Leu Tyr Met Ala Asp Gly Gly Ile Thr Val Asp Tyr Lys Phe Ala Lys Val Lys Glu Pro Asp Leu Val Ile Pro Thr Glu 75 Gly Trp Leu Asn Glu Ile Ser 90 Leu Arg Gln Asp Leu Leu Asp 110 Ser Ser Ilie Asp Glu Gly Arg Lys Phe Thr Ala Pro Tyr Met Thr Gly 115 120 125 WO 99/32634 WO 9932634PCT/NZ98/00189 Met Val 130 Thr Ile Gly Leu Ala Tyr Asp Asp Leu Trp 150 Asp Val Gin Asp Asn 135 Asp Lys Ala Ala Thr Giy 140 Gly Arg Asp Ile Arg Pro Ala Phe 145 Phe Lys 155 Ile Arg Val Ser Leu 160 Ser Giy Leu Gly 165 Asn Met 170 Ser Met Leu Ser Gin Gly 175 Asn Ser Pro Leu Vai Arg 195 Asn Asp Tyr Pro Thr Thr Giu 185 Gly Ile Gin Gin Gin Asn Asp Arg 200 Al a Gin Ilie Arg Ala Val Asp 190 Phe Thr Gly Ile Ala Gin Ala Asp Asp 210 Tyr Leu 215 Val Ala Gly Asn Ile 220 Asp Al a 225 Gin Ser Gly Asp Gin Leu Gin Al a 235 Trp Asn Pro Asp Leu 240 Phe Ile Val Pro 245 Thr Ser Gly Gly Asp 250 Lys Phe Val Asp Thr Met 255 Val Ile Pro Asp Tyr Ile 275 Gin Phe Val Thr Gin Asn Gin 265 Tyr Ala Ala Giu Asp Axg Ala Asn 280 Asp Ala Lys Leu Ala Trp Ile 270 Ala Phe Thr Ala Lys Val Pro Ala Leu 290 Pro Ser 295 Asn Met Thr Asp Giu 300 Pro Asp 305 Gin Ala Ser Ala Giu 310 Ser Pro Leu Ile Asn 315 Ser Ala Giu Vai 320 Gin Ala Asn Leu Lys 325 Ala Trp, Ala Ala Leu Thr Asp 330 Thr Gly Gly Giu Gin Thr 335 Giu Phe Asn Thr 340 Tyr Ala Ala Val 345 <210> 171 <211> 1420 <212> DNA <213> Mycobacterium vaccae <220> <221> unsure <222> (955) (955) <221> unsure <222> (973) (973) <400> 171 gatgagcagc cccggtgctq gctggcccgc cctgctggta cctgttcggc gggcgcacca cttcgcgctg ggggggcctg ttcqgtcggt cgactggatc agtcaactgg gtgctgaact ctggtcgtgc ccggtgcaac caggcgatgg gtcgtgttgt gaagacagct atcgcggtcg ttcaccgcac cagatcatct accgtcccca cgtgcaacac cgacctggtt tgaccgaggt tcctgcgtac agatctccga tgacgttggt ggcgcaggcg gtatcaccgt tgggcgtcac cgggtctgct ccgcggcggg atatcgacac gg cC tgggc c gcacaacgcg ctacatcctg cgacgccacg gctgtccggg gattccgtcg gatcatgqcc ttccatcgtt gctgctgttc ccqgccgtcc cggcggcaac gtcgcggtcg ttgcgtcggc ccgctgggcg tcggtacggt ctcaacgcca atcttcctcg tatgtctggg cttggcctgg gagcaaccgt gc ccacggc c ctgctggtaa cgqtcgggtt gcggcagcgc cgttgctgct tggtcgccac ccctcatcca acgtcgcgcg gcgcgaacgt ctctgcagaa tccggctcgg gcgtggtgga tgcccaacgc 120 180 240 300 360 420 480 540 600 660 WO 99/32634 WO 9932634PCTNZ98OOI 89 cgaactcgcc cgtcgtcacc ggtcgcggcg tgcggccgaa gagcacgtac cgtcgccgac cacactgcgc ttacggcaac cgtagacggc ggtgctcgag gaccgcgcac cctggtgcac cgcgcacgaa ggcgcgtcgt accttcaacg tcgctgcccg tacgagaagt ctgcgatggg ganttcgaca ttggcagacg ggggaacgcc agggtgagtc cgtggcgact gcgctggagg cgaaagccga cttcggttga tcaccaatta ccgcggacac aactgcgcac cgatcccgtt tctggtacgc cgccggaacg acgaacagca tccagcagcc tgtccgtgat tcctggggca aagtcaccgt tcctgctgca tggcggactc cagccggccc ccccgatgat cgacggacag gcacacaccc cgcgcgc cgg gatcgcctcg ggagat cgcc gggtcaggta cgatcaggac gaccacgctg gctggagatg cgtgatcggg gcaggactgh gtgggagagc gtctgcgaga atcgccacgc gcggtggacg caggaacttc gccatgcggg gacgtggtgc ccgaccggga ggcgacgtga acgcgggaac gcccgtgacg gccgtgatcg accggctgac tgctgtcgtc tctatctcgg actcggtcag gcctnaacgg ctgtggcgtc gtctggtccg tgaggttcat tcccggcgcg cggtactggc agat cgagcg ccgaccggcg 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1420 <210> <211> <212> <213> <220> <221> <222> 172 471

PRT

Mycobacterium vaccae

UNSURE

(318) (318) <221> UNSURE <222> (324) (324) Met 1 Ala Ala <400> 172 Ser Ser Val Val Gly Phe Leu Arg Arg Leu Asn 5 Pro Val Ser Thr Trp Leu 10 Val Ala Trp Ala Val Ala Val Leu Leu Val 25 Leu Leu Thr Glu Arg Gly Ser Arg Thr Tyr Ala 40 Gly Ala Arg Pro Val Leu Val His Asn Gin Leu Leu Leu Val Gin Ile Leu Pro Ala Met Leu 55 Asp Ala Leu Leu Leu Arg Giu Ile Ser Leu Asp 70 Leu Ala Thr Ser Val 75 Leu Leu Val Ala Thr Phe Gly Val Val Gly Leu Thr Leu Val 90 Ser Ser Gly Leu Asn Ala Thr Leu Ilie Ser Ilie Phe 115 Thr Val Ile Gin 100 Leu Ala Pro Glu Asp 105 Phe Trp, Arg Arg Arg Ile Pro 110 Asp Val Ala Arg 120 Trp Ala Leu Ile Ala Val Gly Ile 125 Gly Gly Leu Phe Met Ala Tyr 130 Thr Ala Val 135 Ser Gly Ala Asn Val 140 Leu Leu Gly Val 145 Ser Thr 150 Ile Ile Val Leu Gly 155 Leu Ala Leu Gin Asn 160 Val Gly Gin Ile Ser Gly Leu 165 Asp Phe Arg Leu Ser Ala His 195 Gly 180 Gly Trp Ile Thr Val Leu 170 Pro Asn Thr Ala Ala Trp, Arg Ala 205 Gly Arg Pro 190 Thr His Ile Leu Phe Glu Gin Pro 175 Arg Val Val Glu WO 99/32634 WO 9932634PCT/NZ98/O1 89 Asp Thr 210 Ala Ser Gly Gly Asn Leu Leu 215 Ser Val Met Pro Asn Ala 220 Giu Phe Thr Asn 225 Val Tyr 230 Arg Pro Val Gly 235 Glu Leu Ala Gly His Arg Leu Thr 240 Val Thr Thr Phe 245 Val Asn Ala Ala Asp Met Leu Ser Gin Ile Ala 275 Pro Leu His Ser 260 Thr Ala Ala Ser Leu 265 Al a Thr Pro Asp 250 Pro Glu Leu Ala Glu Tyr Asp Val Cys Giu 255 Leu Tyr Leu Gly 280 Asp Arg Giu 285 Ser Thr Asp Gly 270 Lys Ser Ile Thr Tyr Leu Thr Pro Ala Asp Ser Val 290 Arg Trp, Arg 300 Leu Val Trp Tyr 305 Val Ala 310 Asp Arg Arg Gin Giu 315 Ile Arg Xaa Asn Gly 320 Ala Asp Xaa Phe 325 Thr Thr Pro Glu Arg 330 Asp Ala Ser Ala Met Arg 335 Ala Val Ala Ala Asp Val 355 Gin Pro Gly Ser 340 Val Leu Arg Leu Asp Giu Gin Arg Leu Val Arg 360 Gly Gly Asn Gly Glu 365 Val Gin Giu Ile 350 Arg Leu Gin Asp Gly Arg Gin Val Pro Met Arg Phe 370 Vai Ser Ile 380 Val Leu Ser Val Gin Asp Gly 385 Val Asp 395 Thr Ile Pro Ala Arg 400 Leu Giu Arg Gly 405 Thr Phe Leu Gly Gin 410 Giu Thr Leu Thr Arg Giu 415 Pro Vai Leu Met Ala Arg 435 Leu His Val Ala 420 Asp Ala His Ala Leu 425 Leu Glu Val Thr Glu Ile Glu Arg 440 Ile Val His Arg Lys 445 Al a Val Leu Giu 430 Pro Ile Leu His Giu Leu Ile Gly Ala 450 Arg Leu 465 Val 455 Asp Ala Asp Arg Arg 460 Met Asp Ser <210> <211> <212> <213> 173 2172

DNA

Mycobacterium vaccae <400> 173 tagatgacaa gggcgctacc ctgaccagca tcgctgcgcg ttggagaatc gccacggagg aataccgggc ct cgacgaca cgctatctgc gacgacgcgc cgcgagatcg gtggtgtact ttctgccctq acctcctgtc ttctctcggc catcggtgtt agttcgcgga cgatcggcgc aggcggcgtc gcggaaaccg aggcgctcta gcgacggcag tgcaccgctt ccgcctacaa gaatgcgcga gcggatgagc tgcggtggtc cgaccgcctc cctgaagaac gttcagcgac attgcgccgt cgtcgacgtc taccccgccg cgcctggtcg caa ct tcgag ggggccggat acgtctgaac atccagtcca ggtttcatcg accgacatcc tcgatggtga ggttt ccgtc tactacgacc cgcgcgctca tttcagaact gccgccaatg gatctgatgc ctcgggacaa acccgacgcg agttgctgct qctatcagtc gcgagtcgca tttactcgcg agctcggcga ggacgttcgc tcccgaaatc gggagaaggc ccagattcaa tgctcgacct acatcgtcaa aaaaagacgc gatgctgctt cggacggt cc gtcgcgcggg cggcagcact tgcgacgat c caacaccacc caacccccag gatcgcgttc cgagttcttc cgagggcaac cggcccctat 120 180 240 300 360 420 480 540 600 660 720 WO 99/32634 WO 9932634PCT/NZ98/OO1 89 cgcaac cggg gtcggtgtca tccccggtcg cgga t caacg ggtgagacca gagaac cggg gacgaatcgg gaggaggccc gcgttacagg atcgacaccg acggtgatca cgtccgatcc gctctgccgg agtcgcaatc ctgatgctgt atcgcccagg ttgtcgcgca ttcgacgccg ctggccagct gcgatcgaaa ctccgcgcgg gcgtacgaca cagcccggca gtcgccgccg caccggcgat aactgtcgga agcctacgag aaggcggtcg cgtcgaactc gatcgactat ccgacttcgg ggttgaagga aattgatgac tcctggtcgg agaagttcct ttgaccgccg aacgcggcaa cgtactcacc acgaggcgtt tcatcttcgg ggcggttgca tgttgtCtcg tgtcgatcaa ccctgatgcc accacaagaa tgttgacctc ccgccgagag gcgggttagg tggaccgcat gcatcgacac tgtggggttc tctacgtcac gggaggtcgt ga gtggtacctg ccgagtcgac ggcgcgggga accggacaat ggccgacgtc cggcaccacg caccgggacg ggtggacctg cgccccggtg cgtgtcgctg ggccggcgcc tgacgaattc ggacgagctg cgaac cggtg cgtcacggtg cgaggaactg tctcggggt c cgtgccgcgg catcgaccgg cgggtcggCg ggcggtcgat ctcgcgggtg cggcgagcgc cctgccgagg ggtgtgatgg cagtggcgtg ctgatgcgct gtcgaggggg ctggtgcagc acgatcgagg ccgggactgc gcgcagttca gcggccatgc cagcagatca ggcgatctga ctcggcgagg atgcagcgct atcttcgccg atggtggtgg gaccacgtgc ctggacaacg cacgccgccg gccagcgggc gtcgctaacc cacgaggtca ggcgtcgaga aaccgaccgc cggtccagtt acaccgggat cggactcccg gaaccccgcc cggtga ccac acgactatct actgggtgat ccaggaccct tgctggcgcg gcggcggtga caacagcttt agcgcgccga acctcgacgg acatgatggg tcaacgacct ggacgctgca tccggcgcac agtccgggca tggtggggcg aggtgcagcg tgcaggaaac cggtctggcg ctggttcctg cccgatcgcg gggagacacc gctgttccgc ggaggtcgcc ccgctccgtc cggccacgag cgtggccaag ggtgctgtcg gttgttcgtc ctaccgcctc caacgacatg gaaccaacgg ggaggagacg cctcgacgag gacccgccag cgacgggtac ggtcaatttc cgacctgcgg gtccacgttg cggctccccc tctcgacttc gttgcagggc 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2172 <210> 174 <211> 722 <212> PRT <213> Mycobacterium vaccae Met 1 Lys <400> 174 Thr Ile Leu Pro 5 Arg Arg Gly Arg Trp Asn Ala Arg Thr Ser Glu His Pro Thr Arg 10 Ser Arg Met Ser Tyr His Leu Leu 25 Thr Lys Leu Leu Leu Met Val Gly Phe Ile Gly Val Phe Asp Arg Leu Leu Leu Tyr Gin 55 Thr Asp 70 Asp Leu Leu 40 Ser Ser Ile Leu Ser Leu Ile Gin Ser Ala Ala Val Arg Ala Ser Gly Arg Ser Ser Gin Glu Phe Ala Asn Gin Thr Gly Ser Thr Gin Leu Gly 115 Arg Tyr Tyr Ala 100 Asp Asp Giu Ala Ile Arg Giu Ser Lys Asn Ser Met 90 Ile Gly Ala Phe 105 Asn Thr Gly Gin 120 Ala Asn Thr Thr Val Ile Tyr Ser Arg Gly Ser Arg Ser Asp Gly Phe Arg 110 Ala Ala Ser Leu Arg Ala Thr Ile 130 Asn Arg Val Asp 145 Arg Thr Val Arg 150 125 Leu Asp 140 Leu Ile Pro Asp Ser Gly Lys Ser Asn Pro Gin Arg 155 160 Gin Asn Trp Giu Lys Ala Tyr Leu Gin Ala Leu Tyr Thr Pro Pro Phe WO 99/32634 WO 9932634PCTINZ98/OOI 89 Ile Ala~ Giu Tyr 225 Asn Ile GJlu Asp Met 305 Giu Leu Giy Thr Gly 385 Leu Val Thr Leu Leu 465 Leu Asn Glu Val Lys 545 Ser Ala Arg Asp 210 Lys Arg A.sp Pro Gly 290 Thr Thr Phe Thr Leu 370 Asn Gin Al a Arg Al a 450 Gin Pro Asp Arg Met 530 Asn Arg Phe Phe 195 Leu Gly Giu Tyr Thr 275 Val Ala Ile Arg Pro 355 Val Thr Al a Lys Thr 435 Ala Ala Val Met Ala 515 Gin Val Met Asp 180 Asn Met Pro Leu Val 260 Al a Met Arg Leu Glu 340 Pro Gin Gly Tyr Ile 420 Leu Met Gly Leu Ser 500 Glu Arg Thr Leu 165 Asp Giu Leu Asp Ser 245 Giy' Trp Ala Gly Val 325 Asn Glu Pro Thr Ser 405 Asp Val Leu Ala Ser 485 Arg Asn Tyr Val Thr 565 Ala Phe Leu Leu 230 G1u VJal Phe Vai Gin 310 Gly Arg Val Val Thr 390 Pro Thr Leu Leu Gin 470 Arg Asn Gin Leu Ile 550 Ser Arg Phe Asp 215 Gly Ala Thr Leu Gin 295 Trp Pro Giu Ala Thr 375 Ile Val Asp Ser Ala 455 Gin Asp Leu Arg Asp 535 Phe Glu 170 Asp Gly Ser 185 Arg Giu Ile 200 Leu Glu Gly Thr Asn Ile Tyr Giu Lys 250 As p Phe Gly 265 Ser Pro Val 280 Phe Pro Ile Arg Asp Thr Asp Asn Leu 330 Lys Phe Leu 345 Asp Glu Ser 360 Thr Arg Ser Giu Asp Asp Asp Leu Pro 410 Giu Ala Phe 425 Thr Val Ile 440 Arg Leu Phe Ile Ser Giy Giu Phe Gly 490 Ser Ile Lys 505 Leu Met Leu 520 Gly Glu Glu Ala Asp Met Giu Leu Met 570 Ala Val Asn Vai 235 Ala Trp Giy Ala Gly 315 Met Ala Val Val Tyr 395 Gly Ala Ile Val Gly 475 Asp Asp Ser Thr Met 555 Val Trp His Val 220 Asn Vai Tyr Leu Arg 300 Met Arg Asp Asp Giu 380 Leu Leu Pro Ile Arg 460 Asp Leu Glu Leu Slie 540 *Gly *Val Ser 205 JIal Gly Pkla Leu Lys 285 Ile G1y Ser Val1 Arg 365 Giu Gly His Val Phe 445 Pro Tyr Thr Leu Met 525 Al a Leu Val Ala 190 Phe Tyr Pro Ser Pro 270 Asp Asn Asp Asp Val 350 Arg Ala His Trp Al a 430 Giy Ile Arg Thr Leu 510 Pro Gin Asp Asn 175 Ala Asn Ser Tyr Asn 255 Ala Arg Glu Thr Ser 335 Giu Gly Gin Glu Val 415 Gin Val Arg Leu Ala 495 Gly Glu Asp Giu Asp Asn Phe Ala Arg 240 Ser Giu Val Leu Gly 320 Arg Gly Thr Arg Al a 400 Ile Phe Ser Arg Ala 480 Phe Glu Pro His Leu 560 Leu 575 Thr Arg Gin Phe Asp Ala Ala Ala Giu Ser Leu Gly Val Asp His Val WO 99/32634 WO 9932634PCT/NZ98/00189 Arg Thr Leu 595 Arg Leu Asp His Asp Gly Tyr Leu 600 Thr Ala Ser Cys Gly Leu 605 Ile Gly Val Pro Giu Met Asp Asn Val Arg 610 Arg Ile Arg 615 Ala Val Asn Phe Al a 620 His Ile Asp Arg 625 Arg His 630 Thr Ala Glu Ser Asp Leu Arg Leu 640 Ala Gly Ile Asp 645 Tyr Gly Ser Ala Al a 650 Ser Gly Leu Val Ser Thr Leu Gin Val Gin 675 Val His Giu Al a 660 Arg Asp Met Trp Gly 665 Pro Ala Val Asp Val 670 Thr Gly Arg 655 Ala Asn Ser Arg Gly Ser Pro Gin 680 Thr Giy Ile Tyr Val 685 Ala Val Met Gin 690 Val Val 705 Arg Arg Glu 695 Val Leu Asp Phe Val1 700 Arg Ala Gly Glu Gly Glu Arg Gly 710 Giu Thr Val Leu Gin Gly His 720 <210> <211> <212> <213> 175 898

DNA

Mycobacterium vaccae <400> 175 gagcaaccgt gcccacggcc ctgctggtaa gtgggagagc gt ctgcgaga atcgccacgc gcggtggacg caggaacttc catgcgggct cgtggtgcgt gaccgggatg cgacgtgat c gcgggaaccg ccgtgacgag cgtgatcgcc tccggctcgg gcgtggtgga tgcccaacgc accggctgac tgctgtcgtc tctatctcgg actcggtcag gc ctaa cgg c gtggcgtcca ctggtccgtt aggttcatcg ccggcgcggg gtactgqcga atcgagcgcc gaccggcgcg cgactggatc agtcaactgg cgaactcgcc cgtcgtcacc ggt cgcggcg tgcggccgaa gagcacgtac gtcgccgacg cactgcgctt acggcaacgg tagacggcag tgctcgagcg ccgcgcacgc tggtgcac cg cgcacgaact accgtcccca cgtgcaacac ggcgcgtcgt accttcaacg tcgctgcccg tacgagaagt ctgcgatggg at tcgacacg ggcagacgac ggaacgcct C ggtgagtctg tggcgacttc gctggaggaa aaagccgatc t cggttgatg c cgcggcggg atatcgacac tcaccaatta c cgcggacac aactgcgcac cgatcccgtt tctggtacgc ccggaacgga gaacagcagg cagcagccgg tccgtgatcg c tggggcaga gtcaccgtgc ctgctgcacg gcggactcgc ccggccgtcc cggcggcaac cagccggccc ccccgatgat cgacggacag gcacacaccc cgcgcgccgg tcgc ctcggc agat cgccga gtcaggtacc atcaggacgg ccacgctgac tggagatggc tgatcggggc aggactga 120 i8 0 240 300 360 420 480 540 600 660 720 780 840 898 <210> 176 <211> 2013 <212> DNA <213> Mycobacterium vaccae <400> 176 ggCtatcagt ccggacggtc cgcgagtcgc agtcgcgcgg atttactcgc gcggcagcac cagctcggcg atgcgacgat cggacgttcg ccaacaccac atcccgaaat ccaaccccca ctcgctgcgc gttggagaat tgccacggag caataccggg cCtcgacgac gcgctatctg gcatcggtgt cagttcgcgg gcgatcggcg caggcggcgt agcggaaacc caggcgctct tcgaccgcct acctgaagaa cgttcagcga cattgcgccg gcgtcgacgt ataccccgcc caccgacatc ctcgatggtg cggtttccgt ttactacgac ccgcgcgctc gttt cagaac 120 180 240 300 360 WO 99/32634 WO 9932634PCTNZ98/OO1 89 tgggagaagg gccagattca ctgctcgacc aacatcgtca gcgtcgaact gaaccgaccg gcggtccagt gacaccggga tcggactccc ggaaccccgc ccggtgacca gacgactatc cactgggtga accaggaccc ctgctggcgc agcggcggtg acaacagctt gagcgcgccg tacctcgacg gacatgatgg gtcaacgacc cggacgctgc gtccggcgca gagt ccgggc ctggtggggc caggtgcagc atgcaggaaa acggtctggc cgatcgcgtt acgagttctt t cgagggcaa a cggc ccct a cgatcgacta cctggttcct tcccgatcgc tgggagacac ggctgttccg cggaggtcgc cccgctccgt tcggccacga tcgtggccaa tggtgctgt c ggttgttcgt actaccgcct tcaacgacat agaac caacg gggaggagac gcctcgacga tgacccgcca acgacgggta cggtcaattt acgac ctgcg ggtccacgtt gcggctcccc ctctcgactt ggttgcaggg cgacgacgcg ccgcgagatc cgtggtgtac tcgcaaccgg tgtcggtgtc gtccccggtc gcggatcaac cggtgagacc cgagaaccgg cgacgaatcg cgaggaggcc ggcgttacag gatcgacacc gacggtgatc ccgtccgatc cgctctgccg gagtcgcaat gctgatgctg gatcgcccag gttgtcgcgc gttcgacgcc c ctggc cagc cgcgatcgaa gctccgcgcg ggcgtacgac ccagcccggc cgtcgccgc ccaccggcga cgcgacggca gtgcaccgct tccgcctaca gaactgtcgg accgacttcg gggttgaagg gaattgatga atcctggtcg gagaagttcc gttgaccgcC caacgcggca gcgtactcac gacgaggcgratcatcttcg cggcggttgc gtgttgtctc ctgtcgatca tccctgatgc gaccacaaga atgttgacct gccgccgaga tgcgggttag atggaccgca ggcatcgaca atgtggggtt atctacgtca ggggaggtcg tga gcgcctggtc tcaacttCga aggggccgga aagcctacga ggtggtacct accgagtcga cggcgcgggg gaccggacaa tggccgacgt gcggcaccac acaccgggac cggtggacct tcgccccggt gcgtgtcgct aggccggcgc gtgacgaatt aggaCgagct ccgaaccggt acgtcacggt ccgaggaact gtctcggggt gcgtgccgcg tcatcgaccg ccgggtCggc cggcggtcga cctcgcgggt tcggcgagcg ggccgccaat ggatctgatg tctcgggaca gaaggcggtc gcctgccgag cggtgtgatg acagtggcgt tctgatgcgc cgtcgagggg gctggtgcag gacgatcgag gccgggactg ggcgcagttc ggcggccatg ccagcagatc cggcgatctg gctcggcgag gatgcagcgc gatcttcgcc gatggtggtg cgaccacgtg gctggacaac gcacgccgcc ggccagcggg tgtcgctaac gcacgaggtc cggcgtcgag 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2013 <210> 177 <211> 297 <212> PRT <213> <220> <221> <222> Mycobacterium vaccae

UNSURE

(145) (145) <221> UNSURE <222> (151) (151) Glu 1 Gly Thr <400> 177 Gin Pro Phe Arg 5 Arg Pro Ser Ala His Ile Asp Thr Leu Gly Asp Trp Thr Val Pro Thr Ala Ala His Gly Arg Glu Val Asn Gly Gly Leu Ala Gly Asn 40 Asn Leu Val Met Pro Trp Arg Ala Asn Ala Glu Gly Glu His Val Ala Ser Phe Tyr Ser Arg Arg Leu Pro Asp Thr Val Val Val Thr 70 Ser Phe Asn Ala Al a 75 Ser Thr Pro Asp Asp Arg Cys Glu Met Leu SeVaAlAl LuPrGlLu Ser Val Ala Ala Leu Pro Glu Leu WO 99/32634 PCT/NZ98/00189 82 90 Thr Asp Gly Gin Ile Ala Thr Leu Tyr Leu Gly Ala Ala Glu Tyr Glu 100 105 110 Lys Ser Ile Pro Leu His Thr Pro Ala Val Asp Asp Ser Val Arg Ser 115 120 125 Thr Tyr Leu Arg Trp Val Trp Tyr Ala Ala Arg Arg Gin Glu Leu Arg 130 135 140 Xaa Asn Gly Val Ala Asp Xaa Phe Asp Thr Pro Glu Arg Ile Ala Ser 145 150 155 160 Ala Met Arg Ala Val Ala Ser Thr Leu Arg Leu Ala Asp Asp Glu Gin 165 170 175 Gin Glu Ile Ala Asp Val Val Arg Leu Val Arg Tyr Gly Asn Gly Glu 180 185 190 Arg Leu Gin Gin Pro Gly Gin Val Pro Thr Gly Met Arg Phe Ile Val 195 200 205 Asp Gly Arg Val Ser Leu Ser Val Ile Asp Gin Asp Gly Asp Val Ile 210 215 220 Pro Ala Arg Val Leu Glu Arg Gly Asp Phe Leu Gly Gin Thr Thr Leu 225 230 235 240 Thr Arg Glu Pro Val Leu Ala Thr Ala His Ala Leu Glu Glu Val Thr 245 250 255 Val Leu Glu Met Ala Arg Asp Glu Ile Glu Arg Leu Val His Arg Lys 260 265 270 Pro Ile Leu Leu His Val Ile Gly Ala Val Ala Asp Arg Arg Ala His 275 280 285 Glu Leu Arg Leu Met Asp Ser Gin Asp 290 295 <210> 178 <211> 670 <212> PRT <213> Mycobacterium vaccae <400> 178 Gly Tyr Gin Ser Gly Arg Ser Ser Leu Arg Ala Ser Val Phe Asp Arg 1 5 10 Leu Thr Asp Ile Arg Glu Ser Gin Ser Arg Gly Leu Glu Asn Gin Phe 25 Ala Asp Leu Lys Asn Ser Met Val Ile Tyr Ser Arg Gly Ser Thr Ala 40 Thr Glu Ala Ile Gly Ala Phe Ser Asp Gly Phe Arg Gin Leu Gly Asp 55 Ala Thr Ile Asn Thr Gly Gin Ala Ala Ser Leu Arg Arg Tyr Tyr Asp 70 75 Arg Thr Phe Ala Asn Thr Thr Leu Asp Asp Ser Gly Asn Arg Val Asp 90 Val Arg Ala Leu Ile Pro Lys Ser Asn Pro Gin Arg Tyr Leu Gin Ala 100 105 110 Leu Tyr Thr Pro Pro Phe Gin Asn Trp Glu Lys Ala Ile Ala Phe Asp 115 120 125 Asp Ala Arg Asp Gly Ser Ala Trp Ser Ala Ala Asn Ala Arg Phe Asn 130 135 140 Glu Phe Phe Arg Glu Ile Val His Arg Phe Asn Phe Glu Asp Leu Met i~ I WO 99/32634PC/Z8O19 PCT/NZ98/00189 145 Leu Asp Ser Gly Trp 225 Al a Gly Val Asn Giu 305 Pro Thr Ser Asp Val 385 Leu Ala Ser Arg Asn 465 Tyr Val Thr Asp Asp 545 Val Leu Leu Glu Val 210 Phe Val Gin Gly Arg 290 Val Val Thr Pro Thr 370 Leu Leu Gin Arg Asn 450 Gin Leu Ile Ser Al a 530 Gly Arg Asp Gly Al a 195 Thr Leu Gln Trp Pro 275 Glu Al a Thr Ile Val 355 Asp Ser Ala Gin Asp 435 Leu Arg Asp Phe Glu 515 Ala Tyr Arg Leu Thr 180 Tyr Asp Ser Phe Arg 260 Asp.

Lys Asp Thr Giu 340 Asp Giu Thr Arg Ile 420 Glu Ser Leu Gly Ala 500 Glu Ala Leu Thr Giu 165 Asn Giu Phe Pro Pro 245 Asp Asn Phe Glu Arg 325 Asp Leu Ala Val Leu 405 Ser Phe Ile Met Giu 485 Asp Leu Giu Al a Val 565 150 Gly Ile Lys Gly Val 230 Ile Thr Leu Leu Ser 310 Ser Asp Pro Phe Ile 390 Phe Gly Gly Lys Leu 470 Glu Met Met Ser Ser 550C Asn Asn Val Ala Trp 215 Gly Ala Gly Met Ala.

295 Val Val Tyr Gly Ala 375 Ile Val Gly Asp Asp 455 Ser Thr Met Val *Leu 535 *Cys Phe VTal ksn Val 200 Tyr Leu Arg M1et Arg 280 A.sp Asp Glu Leu Leu 360 Pro Ile Arg Asp Leu 440 Glu Leu Ile Gly Val 520 Gly Gly Ala Val Gly 185 Ala Leu Lys Ile Gly 265 Ser Val Arg.

Glu Gly 345 His Val Phe Pro Tyr 425 Thr Leu Met Ala Leu 505 Val Val Leu le ryr 170 Pro Ser Pro Asp Asn 250 Asp Asp Val Arg Ala 330 His Trp Ala Gly Ile 410 Arg Thr Leu Pro Gin 490 Asp Asn Asp Gly Glu 570 155 Ser Tyr Asn Ala Arg 235 Glu Thr Ser Glu Gly 315 Gin Glu Val Gin Val 395 Arg Leu Al a Gly Glu 475 Asp Glu Asp His *Val 555 *Met Ala Arg Ser Glu 220 Val Leu Gly Arg Gly 300 Thr Arg Ala Ile Phe 380 Ser Arg Ala Phe Giu 460 Pro His Leu Leu Val 540 Pro Asp Tyr Asn Ile 205 Glu Asp Met Giu Leu 285 Gly Thr Gly Leu Val 365 Thr Leu Leu Leu Asn 445 Giu Val Lys Ser Thr 525 Arg Arg IArg Lys Arg 190 Asp Pro Gly Thr Thr 270 Phe Thr Leu Asn Gin 350 Ala Arg Ala Gin Pro 430 Asp Arg Met Asn Arg 510 Arg Thr Leu Ile Gly 175 Glu Tyr Thr Val Al a 255 Ile Arg Pro Val Thr 335 Ala Lys Thr Ala Ala 415 Val Met Ala Gin Val 495 Met Gin Leu Asp Ile 575 160 Pro Leu Val Ala Met 240 Arg Leu Glu Pro Gin 320 Gly Tyr Ile Leu Met 400 Gly Leu Ser Glu Arg 480 Thr Leu Phe His Asn 560 Asp WO 99/32634 WO 9932634PCT/NZ98/00189 Arg His Ala Asp Thr Gly 595 Tvr Asm Met Al a 580 Ser Giu Ser Gly His Asp Lev. Arg Leu Arg Ala Ala Ser Gly 600 Val Leu Val Giy Arg Ser 605 Gin Ala Gly Ilie 590 Thr Leu Ala Vai Gin Arg Trp Gly Ser 610 Gly Ser Ala 615 Ile Asp Val Ala Asn 620 Arg Pro Gin Pro Tyr Vai Thr Val His Giu 625 Met Val 640 Giu Gin Glu Thr Leu 645 Thr Phe Val Ala Al a 650 Glu Val Val Gly 655 Arg Gly Val Val Trp Arg Leu 665 Gin Gly His Arg Arg 670 <210> <211> <212> <213> 179 520

DNA

Mycobacterium vaccae <400> 179 gtgatcgacg cccgacgacc aacatcgact gaggcgcgca gcgatccgca tcgatcccgc ggcgaggacg atcgcaccgg aacggtattg aaaccctctt tggcgtcgat actacqgcgc tggtggtgat actccgacct agctcaccga ccaaggtgtc tacggctgcc gccacgcctt ccatgccgag tcgtaccggc ct Ccaccc cg caagccctac cggcgtcaat ggagcgccgc ggtgcgcaac gacgccaccg ctgtggcggg gagaaga tgg cgcgcgaacc atcacgcagc gaggcgagcc ccgaccaacg cgcgacctgg atccgtcgca ccgtcgtaga ccgacaccac agaaggccgt ccggcatgtt tgtccagcat agctgcgcct acggcaacat tcaagcaggc acgatatgaa agcgacagag ctcggtggca caaccggatc caacgtgccc catcgaggat catccgggtg caaggccaag cacctttcgc gatcgcaggt 120 180 240 300 360 420 480 520 <210> 180 <211> 1071 <212> DNA <213> Mycobacterium vaccae <400> 180 cgtggggaag ggcatcggtg tcggcccagc gtcacggttg accggcggcg gcgctgcccg ttcttcgccg aacatcqtgc gcgttcgcgg gtggtggtct gtcgacagc ggaatgccgt tcccggcgcc accaccagcg ggcttcaagg C Ccggtgt cc gctcccgctg tcgtcgtccc gattgcactc cctgcgccgc cgggcctCc cgcccaacgc ccgccgcggt cccgcccggt ccaagggcgt tgccgaagcc tgctggccga acaaactcgt agaagctgc c cctcgctgat acgtcattgc tcgaacaggc tcagcgttcc cgcccgcccc ttCccgccgt ccgggcgaag tatgagcgaa gggtatcgc gcagcccccg cgcgccacaa gcCcgCCggg gtccacgatc cacgatggag gcggggctgg ccgggtcggc cggcgagttc ggcgtgqcgt cgagggcacc cac cgcgggg cgtggccgaa gggtccgggt cggcgccccg ggcgCCcgcg cctggcgccc atcqcccgtc ggggtgctga ctgcccgccc ctcatcccgc gtgagcgccc gccccggcca ccgcagtcca gagcacatcc ggcaacqgcc gaccccaagg tccaccgacg taccgcgaga cccgaccact gc cgcggagg ccggccgcac gcgctgccgc ccacagctgc gggggacgac cctggcgggt gcatcgcggt ctgccacagt gccccggtgt cqgcggtcgc cctcgggcac gccgcgactt cggacccgaa tgtactcgtc aagcgatcag cgtcgctggc acaacatgaa acctggtgtc ccaccgacgc cgccacctgc tggccqtcgc tgggactgca ggcccctttc tctggcaggt cac cacggcg gacgcaaacc gacgcctgcc gccggccccc gctcagcgag ccgcgccctc cgtgccggac gaacgcccag ccacggcttc cgacttcggc gctgaacacg gctgtcggtg gattgtcaac acccggtgc c accacccccg gggatagacg t 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1071 WO 99/32634 WO 9932634PCT/NZ98/OOI 89 Val Val Asn Thr Val Ala Ile Leu Arg Arg 1~45 <210> 181 <211> 152 <212> PRT <213> MycC <400> 181 Ile Asp Giu Ser Val Ala Pro Gly Met Pro Ile Thr Val Ile Lys Ile Arg Asn Ile Arg Val 100 Val Lys Gin 115 Asn Ile Arg 130 Leu Pro Thr bacterium vaccae Thr Pro Phe Gin Pro Ser Ser Ala Leu Asp Asn Leu Tyr Asp Ile Lys Phe His Asp Leu Arg Ile 40 Ser Ser 55 Giu Ala Leu Gly Pro Gin Aia Lys 120 Asp Met 135 Pro Ser Al a Ala Asn Ile Ser Val Leu 105 Gly Asn Giu 10 Ser Ile Asn Gin Asn 90 Thr Glu Thr Lys Arg Tyr Pro Arg Thr Giu Al a Arg 140 Met Thr Tyr Giu Leu Asn Arg Lys 125 Ile Lys Arg Al a Arg Glu Gly Arg Ser Pro Arg Asn Pro Pro 150 Met 1 Ala Ala Thr Ile Pro Ala Glu Asp <210> <211> <212> <213> <400> Ser Giu Cys Ala Ser Ala Val Thr Pro Arg Ala Gly Arg Pro Phe Phe 115 Phe Arg 182 331

PRT

Mycobacteriumn vaccae 182 Ile Ala Arg Pro Trp Arg 5 Ala Gly Ile Ala Giy Val 25 Gin Pro Gly Leu Pro Gin 40 Gin Thr Val Thr Val Ala 55 Pro Gly Val Thr Pro Ala 70 Val Ser Ala Pro Ala Val Val Ser Thr Ile Ala Pro 100 105 Ala Ala Lys Gly Val Thr 120 Ala Leu Asn Ile Val Leu 135 Val Leu 10 Leu Ser Pro Pro Pro Asn Thr Gly 75 Ala Pro 90 Ala Thr Met Glu Pro Lys Ala Ile Leu Ala Gly Ala Ser Pro Pro 140 Gly Ala Pro Ala Al a Pro Gly Gin 125 Arg Gly Val Ala Pro Ala Ala Thr 110 Ser Gly Ile Thr Pro Gin Ala Leu Leu Ser Trp Gly Thr Ala Leu Val Pro Ser Arg Glu 130 His Ile Pro Asp Pro Asn Val Pro Asp Ala Phe Ala Val Leu Ala Asp WO 99132634 WO 9932634PCT/NZ98/OO1 89 145 Arg Tyr Phe Leu Arg 225 Thr Val Asn Pro Leu 305 Ala Gly Leu Asp 195 Asp Asn Gly Gin Phe 275 Pro Leu Ala Gly Asn Gly Leu Tyr Ser Val 180 Ser Phe Asn Pro Ala 260 Lys Gly Ala Pro 165 Gly Gin Gly Met Asp 245 Val Val Ala Val Gin 325 Glu Lys Gly Lys 230 His Ala Ser Pro Ala 310 Leu Phe Leu Met 215 Leu Tyr Glu Val Gly 295 Pro Leu Asp Pro 200 Pro Asn Leu Ala Pro 280 Val Pro Gly Pro 185 Al a Ser Thr Val Ala 265 Gly Pro Pro Leu Ser 170 Lys Trp Ser Ser Ser 250 Giu Pro Pro Ala Gin 330 Asn Glu Arg Leu Arg 235 Leu Ala Gly Ala Pro 315 Gly Al a Ala Ser Ile 220 Arg Ser Thr Pro Pro 300 Ala Gin Ile Thr 205 Glu His Val1 Asp Al a 285 Gly Val Val Ser 190 Asp Gly Val Thr Al a 270 Ala Al a Pro Val 175 His Ala Thr Ile Thr 255 Ile Pro Pro Ala 160 Val Gly Ser Tyr Ala 240 Ser Val Pro Al a Val 320 <210> <211> <212> <213> 183 207

DNA

Mycobacterium vaccae <400> 183 acctacgagt tcgagaacaa ggtcacgggc ggccgcatcc cgcgcgagta catcccgtcg gtggatgccg gcgcgcagga cgccatgcag tacggcgtgc tggccggcta cccgctggtt aacgtcaagc tgacgctgct cgacggtgcc taccacgaag tcgactcgtc ggaaatggca ttcaaggttg ccggctccca ggtcata <210> 184 <211> 69 <212> PRT <213> Mycobacterium vaccae <400> 184 Thr Tyr Glu Phe Glu Asn Lys Val Thr Gly Gly Arg Ile Pro Arg Giu 1 5 10 Tyr Ile Pro Ser Val Asp Ala Gly Ala Gin Asp Ala Met Gin Tyr Gly 25 Val Leu Ala Gly Tyr Pro Leu Val Asn Val Lys Leu Thr Leu Leu Asp 40 Gly Ala Tyr His Glu Vai Asp Ser Ser Glu Met Ala Phe Lys Val Ala 55 Gly Ser Gin Val Ile 120 180 207 <210> 185 WO 99/32634 WO 9932634PCTNZ98/OO1 89 '211> 898 <212> DNA <213> Mycobacterium vaccae <220> <221> unsure <222> (637) (637) <221> unsure <222> (662) (662) <400> 185 cgacctccac ccgggcgtga ttcaccgaaa aaatgaggac ttcggccgta tcggacgcaa aacaaggaca tcgagatcgt ctgctgaagt tcgactcgat gacaccatcg tcgtcggcag gcgctgccct ggggcgacct aagcgcgaca aggcccaggg ccggccaccg atgaggacat agccagaaca tcatctccaa gtcatcaacg acgagttcgg cnggtccaga acctgcagga ctgaacatcg tgccgacctc ctgaagggca agctcgacgg gacctgaccg ccgagctggg ggccaaccac agaggagaca cttcttccgc cgcggtcaac cctgggccgg caccaagatc gggcgtcgac ccacctcgac caccatcgtg cgcgtcgtgc catcgtcaag cggcccgcac caccggtgcc ctacgcgctg caagtcggcc taggctggtg cccgtgacga gcgctggacg gacctcaccg ctgccctacg aaggcgctcg gtcgtcgtcg gcgggcgcca ct cggcgtca accacgaact ggcctgntga aaggatctgc gccaaggcca cgggtgccga accgtggacg accagtagtc tccgtgttgg cgcagaaggc acaacgccac acgtgagcct aggtcaagga agtccaccgg agaaggtcat acgacgacaa gcctcggccc ccaccatcca gccgggcccg t cggactggt tccccaccgg agatcaacgc gacggcacac tgtgaacggc cgaaggcaag gctggcgcac cgaaggcgag aggcccggcg catcttcacc catctccgcg gtacgacggc gctggcgaag cgcctacacc cgccgccgcg gctgcccgag ctcggtcacc cgcgatga 120 180 240 300 360 420 480 540 600 660 720 780 840 898 <210> <211> <212> <213> <226> <221> <222> 186 268

PRT

Mycobacterium vaccae

UNSURE

(182) (182) <221> UNSURE <222> (190) (190) Val 1 Phe <400> 186 Thr Ile Arg Phe Arg Ala Val 5 Leu Giy Val Asn Gly Phe Gly Arg Ile Giy Arg Asn 10 Ala Lys Asp Asp Ala Gin Giu Gly Lys Asn Ile Giu Ile His Leu Leu Ser Leu Giu Ala Leu Glu Vai Ala Val Asn Lys Phe Asp Ser 55 Giy Glu Asp Thr 70 Val Lys Giu Gly Asp 40 Ile Ilie Pro Thr Asp Asn Ala Pro Thr Leu Ala Tyr Asp Val Leu Gly Arg Leu Val Val Gly Ser 75 Ala Ala Leu Pro Thr Lys Ile Trp Gly Asp Lys Leu 90 WO 99/32634 WO 9932634PCTNZ98/OOI 89 Gly Val Lys Ala Ala Pro 130 Asp Lys 145 Thr Asn Ile Val Asn Leu Ala Leu 210 Leu Val 225 Vai Pro Lys Ser Asp Vai 100 Gin Giy 115 Ala Thr Tyr Asp Cys Leu Lys Giy 180 Gin Asp 195 Asn Ile Leu Pro Ile Pro Ala Thr 260 210> 187 Val Val Giu Ser His Asp Gly Gly 165 Leu Gly Val Giu Thr 245 Val Leu Glu Ser 150 Pro Xaa Pro Pro Leu 230 Gly Asp Asp 135 Gin Leu Thr His Thr 215 Lys S er Ala 120 Ile Asn Ala Thr Lys 200 Ser Gly Val Thr Gly 105 Gly Ala Thr Ile Ile Ile Lys Val 170 Ile His 185 Asp Leu Thr Gly Lys Leu Thr Asp 250 Ile Lys Val Ser 155 Ilie Al a Arg Al a Asp 235 Leu Phe Lys Leu 140 Asn Asn Tyr Arg Ala 220 Gly Thr Thr Val 125 Gly Al a Asp Thr Al a 205 Lys Tyr Al a Lys 110 Ile Val Ser Giu Xaa 190 Arg Al a Ala Glu Arg Ile Asn Cys Phe 175 Val Al a Ile Leu Leu 255 Asp Ser Asp Thr 160 Gly Gin Ala Gly Arg 240 Gly Asp Giu Ile Asn Ala Ala Met <211> 41 <212> PRT <213> Myc <220> <221> UNSi <222> (39 <400> 187 obacterium vaccae

URE

Met Asn Lys Ala Glu Leu Ile Asp Val Leu Thr Giu Lys Leu Gly Ser 1 C;10 Asp Arg Arg Gin Ala Thr Ala A~ Val Ala Ala Val Pro Lys Xaa 4 <210> 188 <211> 26 <212> DNA <213> Artificial Sequel <220> <223> Made in a lab <221> unsure <222> (12) (12) <400> 188 atgaayaarg cngarctsat ygaygt lia Val Giu Asn Val Val Asp Thr Ile 25 ral Val ice WO 99/32634 WO 9932634PCT/NZ98/OOI 89 <210> 189 <211> <212> DNA <213> Artificial Sequence <220> <223> Made in a lab <400> 189 atsgtrtgva cvacgttytc <210> <211> <212> <213> 190 84

DNA

Artificial Sequence <220> <223> Made in a lab <221> unsure <222> (2) <400> 190 gnactcattg acgtactcac tgagaagctg ggctcggatt gtcggcaagc gactgcggca atggagaacg tggtccacac cata <210> <211> <212> <213> 191 337

DNA

Mycobacterium vaccae <220> <221> unsure <222> (2) <400> 191 gnactcattg acgtactcac gtggagaatg ttgtcgacac acgggcttcg gtgttttcga accggcgaga ccgtgaaggt ttcaaggctg ttgtctctgg ggtgtgaccg cgacgagcac tgagaagctg catcgtgcgc gcagcgtcgt caagcccacc cgcacagaag cgcccgcaag ggctcggatt gccgtgcaca cgcgcagcac tcagtcccgg cttccggccg gcagcca gt cggcaagc agggtgagag gcgtggcacg cattccgtcc agggtccggc gactgcggcg cgtcaccatc caatccgcgc cggcgctcag ggt caagcgc 120 180 240 300 337 <210> 192 <211> 111 <212 PRT <213> Myc <220> <221> TiNS' <222> (1) obacterium vaccae

URE

WO 99/32634 WO 9932634PCT/NZ98/0O1 89 <400> 192 Xaa Leu Ile Asp 1 Thr Ala Ala Val Lys Giv Giu Ser Vai Leu Thr Giu Lys Leu 10 Thr Giy Ser Asp Arg Gin Ala Giu Asn Val Val Val. Thr Ile Thr Asp 25 Gly Ile Val Arg Ala Vai His Giu Gin Arg Phe Gly Val Phe Arg Arg Ala Lys Val Lys Arg Ala Arg Val Pro Thr Ser Val Ser Gly Asn Pro Arg Thr Pro Gly Giu Thr Val Gly Ala Gin Phe Pro Ala Ala Vai Ala Gin Lys Phe Arg Leu Pro 90 Thr Ala Val Ala Giu Giy Arg Lys Ala 110 Pro Ala Val Lys Arg Gly 100 Thr Ala Thr <210> <211> <212> <213> 193 1164

DNA

Mycobacterium vaccae <400> 193 ggtggcgcgc atcgagaagc gcgctgcgct ctgcagggca cgctcgccga atggatcgcg aagccgcccg gcggcggcca ctggccgacc atgcacgacg cacgaccact gccccgttcg gcgcggatcg tgcacccccg tcgtgggtgg aagagcttcg ggggcctacc catc tggacc cgcctcgccc gagcgggtac gacccgtggt acgqcttcgg cgcctgccgc actaccgcga acctgcagcg gaccgatccc gctggtacgg cggtgtggag tcccggtgcc acgaccacct tggtgccgtt tcgagttgga cccggcactt tcaccggctc ccgagatcgg atcccgcgtt tgaccgaggt cgcat ccgtg gcctgaccgt ggcggttctg gcccgccccg gacggcctca cctcggcgcg cggcaagttc gatgctgttg gctggccgag ccattccagc caacagatqt gctggaggcg cgacatcgac gggcatcggc ctggcacgaa ctccggacgg gtcgcacaag cgacgagtac cgccgacatc ggacaacagc gtccgagccc gccgattccc aac c gttcacgggc ctgctggccg actccgggcg qtcaacctgg cgcgatctgg ccgccgaagg gtgctgatcg tcgccctcac cttcccgccg accatcgtcg gcacacctgc gcccaccgca ttgttctccc gcgttcttcg ggtccgttcg cacatgaacc ctgatggtgc gccgaacgcc ggtcagcggg gcctgatcat gcgggttcgt aggt cgcgcc agcccccgtc ccaacgccgc gggatcccac aggtcgacgg gggcggtcgg tggacgcggt cgttggcgca gcaagtgggg tagacgacct gcgactcgac gtggcgacac atctgaccct ccgaggaggc ccatccactg tgctgaccgc tggacccgga ggtgcgggcg gctgcgcgcc ggtggcgcgc gggcatcacg atcccagggc tcccgcgccg ctaccgcgtg accgcagcgc ggtgatcagc cacccagcgg cgtccccgag gacgctggtc gctgtgggcg cggatacacg gctgccgatc ggtgcgcgc c ggcgacattc tgccgacgcc gtcgacgttc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1164 <210> 194 <211> 370 <212> PRT <213> Mycobacterium vaccae <400> 194 Met Val Arg Ala Ala Leu Arg Tyr Gly Phe Gly Thr Ala Ser Leu Leu 1 5 10 Ala Gly Gly Phe Val. Leu Arg Ala Leu Gin Gly Thr Pro Ala Ala Leu 25 WO 99/32634 WO 9932634PCT/NZ98/00189 Gly Ala Tyr Arg Met Asp Ala Ser Lys Gly Ser Ser Val Trp 130 Met His 145 Val Val Val Ala Ile Gly Giu Leu 210 Cys Thr 225 Thr Leu Phe Gly Giu Tyr Pro Ala 290 His Leu 305 Trp Ala Arg Leu Ile Pro Arg Phe 370 Thr Asp Arg Gin Asp Val 115 Ser Asp Ile Leu Ala 195 Asp Pro Trp Gly Gly 275 Phe Asp Thr Leu Gly 355 Pro Gly Asp Gly Pro 100 Leu Asn Val Ser Al a 180 His Trp Ala Ala Asp 260 Pro Ala Leu Phe Thr 340 Gly Lys Leu Lys Thr Ile Arg Pro His 165 His Leu His Arg Ser 245 Thr Phe Asp Thr Arg 325 Ala Giu Val Phe Val 55 Gin Arg 70 Pro Pro Pro Ala Giu Val Cys Ser 135 Vai Pro 150 Asp His Thr Gin Arg Lys Glu Ala 215 His Phe 230 Trp Val Gly Tyr Asp Leu Ile His 295 Giu Val 310 Leu Ala Ala Asp Ala Pro Val 40 Asn Leu Giu Met Leu Leu Giy Pro Ile 90 Pro Ala Ala 105 Asp Gly Tyr 120 Pro Ser Arg Leu Giu Ala Tyr Asp His 170 Arg Ala Pro 185 Trp Gly Val 200 His Arg Ile Ser Gly Arg Val Thr Gly 250 Thr Lys Ser 265 Thr Leu Leu 280 Met Asn Pro Asp Asn Ser Pro His Pro 330 Ala Glu Arg 345 Ala Arg Pro Pro Arg Asp 75 Pro Leu Ala Ser Arg Val Alit Val 140 Leu Pro 155 Leu Asp Phe Val Pro Giu Asp Asp 220 Leu Phe 235 Ser. Ser Phe Ala Pro Ile Glu Glu 300 Leu Met 315 Trp Ser Val Arg Arg Ser Pro Ser Gly Ile Leu Ala Trp Leu 125 Gly Ala Ile Vai Al a 205 Leu Ser His Glu Gly 285 Ala Val Giu Leu Asp 365 Ala Glu Tyr 110 Ala Pro Val Asp Pro 190 Arg Thr Arg Lys Ile 270 Ala Val Pro Pro Thr 350 Asn Pro Gly Asp Gin Asp Thr 175 Leu Ile Leu Asp Ala 255 Gly Tyr Arg Ile Ala 335 Val Asn Thr Ala Pro His Pro Arg Ala 160 Ile Gly Val Val Ser 240 Phe Asp His Ala His 320 Glu Pro Gin Arg Val Asp Pro Glu Ser Thr Phe 360 Pro Trp Trp <210> <211> <212> <213> 195 650

DNA

Mycobacterium vaccae <400> 195 gacacaccag caccactgtt aacctcgcta gatcagtcgg ccgaacggaa ggacagccgt gaccctgaaa accctagtca ccagcatgac cgctggggca gcagcagccg caacactcgg WO 99/32634 WO 9932634PCT/NZ98/00189 cgctgccgcc cgcggtgctg ctccaccttg ccagggcggt ggccaagggc cgtgaccgcc gttcatcgcc gatgtccgcg cggcgctcgg gtgggtgtga aacgcaccgc agcgcgctgt ctcggccgca tacttcccgc aacgtcaccg gggccgagcc gtgggtgatc gctgggtggg cctcgattgc tgctttccgc cgggcccggg tcgaggcccg tgagcttcac cggcggcccc cgaccggatg tcccgcacga tgcctgggcg cgtcggtgcg ccctgccccc ctccttcgcc ggtggccgac cgtcgccggc gacgggcgcc gcagctgtcc ttctggtccg gctgcgcgca ggtgtcgccg gatctgcagg ggcgccaagg agcggataca atcgaccaga gtggc cac cc aagcagt ccg cagcgc cgt c agatgaacat gcgcgtcgcc gaccgctggt ccacctacgt gcaacgccgc acggtccgat agccgctgac cactggccct acaigtgtgg 180 240 300 360 420 480 540 600 650 <210> <211> <212> <213> 196 159

PRT

Mycobacterium vaccae met 1 Gly Ala <400> 196 Thr Ala Gly Val Thr Ser Val Leu Asn Ala Ala Ala Ala Ala 5 Ile Ala Val Gly Ala Thr 10 Gly Leu Gly Ala Ala Ala Val Val Ala Gly 25 Ser Ala Pro Leu Gly Pro Leu Leu 40 Ser Ala Pro Ala Pro Pro Ala Ser Pro Asp Leu Gin Gly Ser Phe Val Ser Thr Ala Gly Leu 55 Tyr Ala Leu Ser Gly Leu Ala Lys Ala Ala Thr 70 Ser Val Gin Gly Gly Arg Ile Giu Arg Val Ala Asp Phe Gly Tyr Ser Asn 90 Ile Ala Ala Lys Gly Tyr Phe Pro Leu Val Thr Ala 115 Ser 100 Asn Thr Val Ala Gly 105 Ala Asp Gin Asn Val Thr Ala Pro Thr Gly Al a 125 Gly Gly Pro Ile 110 Val Ala Thr Trp Gin Leu Gln Pro 130 Ser Lys 145 Leu Thr Phe Ile Gin Ser Ala Leu 150 Pro Ser Pro Leu Met Ser Ala 155 Ile Ala Ala <210> <211> <212> <213> 197 285

PRT

Mycobacterium vaccae <400> 197 Gln Val Arg Met 1 Ser Arg 5 Val Leu Gly Ser Val 10 Ser Gly Ala Ala Val Ala Val Ala Ala Leu Trp Gin Thr Gly Vai 25 Asp Ile Glu Val Ile Ile Pro Thr Ala Ser Ala Asp Pro Cys Giu Pro Gly Pro Lys Val Pro Phe Ala Arg Gly Thr Gly Ala 40 Leu Gly Trp Val Gly Asp Ala Phe Val Asn Ala Leu Arg 55 Gly Glu Gin Ser Val Gly Thr Tyr Ala Val Asn Tyr Pro 70 75 PCTNZ98/OO1 89 WO 99/32634 Ala Gly Phe Asp Phe Asp Lys Ser Ala Pro Met Gly Ala Ala Asp Ala Ser Gly Arg Val Leu Gly 115 Val Asp Pro Val 100 Gly Gin Trp Met Ala Asp Asn 105 Cys Pro Asp Thr Lys Leu 110 Leu Ile Thr Met Pro Pro Met Ser Gin Gly 120 Arg Ala Gly Val Ile Asp 125 Pro Arg Pro Leu 130 Val Gly 135 Ala Phe Thr Pro Arg 145 Arg Ala Asp His Val 150 Gly Ala Val Val Val 155 Gin Gly Asn Pro Leu 160 Asp Ile Arg Gly 165 Ile Gly Pro Leu Pro 170 Leu Met Ser Gly Thr Tyr 175 Gly Pro Lys Pro Gly Phe 195 Val Giu Glu Ser 180 Asfl Asp Leu Cys Ala 185 Phe Asp Asp Pro Leu Pro Ala His 200 Al a Ala Tyr Ala Asp 205 Gly 190 Asn Gly Met Gin Ser Val Ala Ala Asn 210 Glu Leu 225 Val Thr Phe 215 Tyr Arg Leu Glu Pro 220 Val Pro Glu Ala Leu Glu Asp 245 Ala Ser Gly Leu His Leu Phe 235 Glu Pro Arg Gly Gly Pro Leu Arg 250 Thr Gly Asp Ala Val Arg 255 Ala Glu Glu 240 Phe Thr Gly Gin Arg Ile Leu Val 275 260 Trp Val 265 Gly Ala Thr Ala Pro 270 Glu Met His Al a 280 Leu Gly Ala Al a 285 <210> <211> <212> <213> 198 743

DNA

Mycobacterium vaccae <400> 198 ggatccgcgg gaactccgtc ctatcagttc cgaacccgga ggccgaggag caccgacggc catcgccgcg ggccgCCggc cggggacctg gtggaaggtc ggaggcgatg cctggt ctcc agtgcgcgca caccggctgg ggcatcggcg gtcggccgca agtccctggc ctgctggaat gtgttctccc ttccgttccc gagttcgacc gtgctctacg gacgtcgccg aagatggaga gctgggcatc tgagcgccgt tgacgaccaa gcgcgtacct ccacccaggt tgctgcggtt tgcgagccga tcgccgagca ggcaggcggc gcgccgagaa acggtgacga tcggtgaccg ccgtgctgcg tcgtcgatcc cga gtacaacccg gcccgcacct gtggagt cgt tttcgaccga catggccgca cgaacggttc cgcgttctcc agccgcgtcg gcgggtcgac ggtggtggcc cgccccggcc cggcacccca taccgccaca atttcgtggt ggccggggct ctggccgaca gccgagcgga aaggccaccg gctccgttcg ggacagccgt gacggggtgg ctggtcgtgg ggacggccga gccccggcgg cggcgccgtt atccggtgtc cggtcgacat acgccgacga ccgcgtgggc acgccgatac cgtcgagcgt tgctggcgct tcacccagat tcggcaccgg 120 180 240 300 360 420 480 540 600 660 720 743 <210> 199 <211> 243 <212> PRT <213> Mycobacterium vaccae WO 99/32634 WO 9932634PCTNZ98/O1 89 <400> 199 Asp Pro Arg His Arg Leu Val Thr Thr Lys Tyr Asn Pro Ala Arg Thr Gly Trp Thr Ala Tyr Gly Met Gin Vai Trp Giu Glu Asn Ser Val Gly Gly Ala Tyr Gly Pro Gly Gly 40 His Gin Phe Val Gly Glu Leu Cys Ile Arg Thr Thr Pro Gly Ser Ser Arg Tyr Pro Trp Arg 55 Phe Thr Ala Pro Phe Leu Leu Arg Ala Phe 70 Giu Asp Arg Ile Ser 75 Met Trp Tyr Pro Vai Ser Giu Giu Leu Leu Arg Ala Asp Ser Ala Ala Gly Arg Gly Giu Arg Ser Val Asp Phe Leu Ala Ile 100 Asp Asp Gly Val Phe 105 Ile Leu Ala Giu His Asn Ala Asp 115 Ala Asp 120 Arg Ala Ala Phe Arg 125 Al a 110 Ser Arg Gin Ala Gly Giu Ala Ala 130 Phe Asp Phe Ser Ala Giu 135 Ala Thr Ala Trp Ala Arg Ala Giu Aia Ser Lys 145 Gly Ala 155 Arg Asp Ala Asp Thr 160 Asp Leu Val Leu 165 Trp Asp Gly Asp Val Asp Ala Pro Phe 175 Ala Ser Ser Ala Gly Gin 195 Leu Arg Ala 210 Gly His Leu 225 Val Arg Ala Val 180 Pro Lys Val Asp Val 185 Glu Val Gly Asp Leu Leu Ala Leu 200 Val1 Ala Met Lys Met 205 Leu Arg Vai Val 190 Glu Thr Val Val Ser Ala Pro Ala Asp Gly 215 Gly Val Thr Gin Val Asp Pro 230 Thr Pro Leu Val 235 Val Gly Thr Gly 240 <210> 200 <211> 858 <212> DNA <213> Mycobacterium vaccae <400> 200 gaaatcccgc cggattgaaa gcgatgggtt tgcatgaaca caagcgactg gagagcgtca gcacgcaatc cgtcccggcg ccggcggtca ccggccaaga gctgcgacca aagaaggcca gcgaccaagg ccggccaaga gtctgaaacc aatgttcgct taccgtgtcc aagcagagct cggcggtgga ccatcacggg cgcgcaccgg ctcagttcaa agcgcggtgt aggctgccgc aggctgcacc ctgccgccaa ctgcaccggc aggctccggc ctcttttcgc gaatgagcct actagtcggt catcgacgta gaacgttgtc cttcggtgtt cgagaccgtg ggctgttgtc gaccgcgacg gaagaaggcc ggccaagaag gaaggctgca caagaaggct cgccaagaag ggcgcccctc gaaattgcgc ccaaagagga.

ctcactgaga.

gacaccatcg ttcgagcagc aaggtcaagc tctggcgcac agcaccgccc gcgccggcca, gccactgccg ccggccaaga ccggccaaga gcgcccgcca aggacggtaa gtggctcttg ccactggttt agctgggctc tgcgcgccgt gtcgtcgcgc ccacctcagt agaagc ttcc gcaaggcagc agaaggctcc ccaagaaggc aggctccggc aggccgcgac agaaggctcc gggggccaag gaaatcagca tcggaggttt ggatcgtcgg gcacaagggt agcacgcgtg cccggcattc ggccgagggt caagaaggct ggcgaagaag cgcgccggcc caagaaggct caaggctgca ggccaagcgc 120 180 240 300 360 420 480 540 600 660 720 780 840

V.

WO 99/32634 PCT/NZ98/00189 ggcggacgca agtaagtc <210> <211> <212> <213> 201 223

PRT

Mycobacterium vaccae <400> 201 Met Asn Lys Ala Glu Leu Ile Asp Val 1 Asp Arg Arg Val Arg Ala Val Phe Glu Leu 10 Glu Thr Glu Lys Leu Gly Ser Gin Val Ala Thr Ala Ala Val 25 Ser Asn Val Val His Lys Gly Glu 40 Ala Val Thr Ile Thr Asp Thr Ile Gly Phe Gly Asn Pro Arg Gin Arg Arg Thr Gly Arg 55 Val Ala Arg Vai Ala Val Arg Glu Thr Val Lys Pro Thr Pro Ser 75 Gly Pro Ala Phe Arg Gly Ala Gin Ala Val Val Ser Val Ala Gin Lys Leu Pro Ala Glu Gly Arg Lys Ala 115 Ala Ala Pro Pro 100 Ala Val Lys Arg Gly 105 Ala Thr Ala Thr Lys Lys Ala Pro 120 Pro Lys Lys Ala Ala 125 Ala Ser Thr Ala 110 Ala Lys Lys Thr Lys Ala Ala Lys Lys 130 Ala Pro Ala 135 Thr Ala Lys Lys Ala 140 Ala Ala Lys Lys 145 Lys Ala 150 Lys Ala Ala Lys Lys 155 Ala Ala Pro Ala Ala Thr Ala Ala 165 Thr Lys Ala Ala Pro 170 Ala Lys Lys Ala Pro Ala 175 Lys Lys Ala Lys Ala Ala 195 Lys Ala Pro 210 Ala 180 Thr Lys Ala Ala Pro 185 Ala Lys Lys Ala Pro Ala Lys 190 Lys Ala Ala Lys Lys Ala Pro Ala Ala 205 Gly Arg Lys Lys Ala Lys Lys Ala 215 Ala Lys Arg Gly 220 <210> 202 <211> 570 <212> DNA <213> Mycobacterium vaccae <400> 202 agacagacag tcggtggcac aaccggatca aacgtgcccg atcgaggatg atccgggtgt aaggccaagg gaactctccc aaggatctcg tgatcgacga ccgacgacct acatcgacta aggcgcgcat cgatccgcaa cgatcccgca gcgaggacgc ggatcaagaa acaagagcac aaccctcttc ggcgtcgatt ctacggcgcc ggtggtgatc ctccgacctc gctcaccgag caaggtgtcg ggacggcgac ccaccagtac ggtctgacca catgccgagg cgtaccggcc tccaccccga aagccctacg ggcgtcaatc gagcgccgcc gtgcgcaaca gccggcgaag acgaatcaga agaagatgga gcgcgaaccc tcacgcagct aggcgagcca cgaccaacga gcgacctggt tccgtcgcaa accaagtgac tcgacgaact gaaggccgtc cggcatgttc gtccagcatc gctgcgcctc cggcaacatc caagcaggcc ggcgatggag ccgcgccgag ggtcaagcac 120 180 240 300 360 420 480 540 570 aaggaaggcg agttgctgga WO 99/32634 WO 9932634PCT/NZ98/001 89 <210> <211> <212> <213> <220> <221> <222> 203 187

PRT

Mycobacterium vaccae

UNSURE

(186) (186) <400> 203 Val Ile Asp Giu 1 Val Ser Val Ala Thr Leu Phe His Ala Glu Giu Lys Met Giu Lys Ala Met Pro Asp Asp Phe Asn Arg Leu Ala 25 Ile Asn Ile Arg Thr Asn Pro Gly Ile Asp Tyr 40 Ser Tyr Giu Gly Arg Ala Gly Ala Ser Ala Arg Met Thr Pro Val Val Ala Ile Ile Thr Gin Leu Ser Giu Ile Asn Val Ile Lys Pro Ala Ser Gin Leu 75 Pro Leu Ile Giu Asp Arg Asn Leu Gly Val Asn Thr Thr Asn Asp Gly Asn Ile Ile Arg Leu Val Lys 115 Arg Asn Ile 130 Asp Gly Asp Val 100 Gin Ile Pro Gin Leu 105 Gly Giu Giu Arg Ala Lys Ala Lys 120 Met Giu Asp Ala Lys 125 Arg Arg Arg Asp 110 Vai Ser Val Ile Lys Lys Arg Arg Lys Ala Gly Giu Ala 135 Asp Glu Glu Leu Giu Val Thr 145 Asp His Arg 155 Giu Lys Asp Lys Ser Thr His 165 Lys Giu Gly Giu 180 Tyr Thr Asn Gin Ile 170 Xaa Pro Asp Glu Leu Val 175 Leu Leu Glu Val 185 <210> <211> <212> <213> 204 1364

DNA

Mycobacterium vaccae <400> 204 cgacctccac ccgggcgtga ttcaccgaaa aaatgaggac ttcggccgta tcggacgcaa aacaaggaca tcgagatcgt ctgctgaagt tcgactcgat gacaccatcg tcgtcggcag gcgctgccct ggggcgacct aagcgcgaca aggcccaggg ccggccaccg atgaggacat agccagaaca tcatctccaa gtcatcaacg acgagttcgg ggccaaccac agaggagaca cttcttccgc cgcggtcaac cctgggccgg caccaagatc gggcgtcgac ccacctcgac caccatcgtg cgcgtcgtgc catcgtcaag taggctggtc cccgtgacga gcgctggacg gacctcaccg ctgccctacg aaggcgctcg gtcgtcgtcg gcgggcgcca ctcggcgtca accacgaact ggcctgatga ac cagtagtc tccgtgttgg cgcagaaggc acaacgccac acqtgagcct aggtcaagga agtccaccgg agaaggtcat acgacgacaa gcctcggccc ccaccatcca gacggcacac tgtgaacggc cgaaggcaag gctggcgcac cgaaggcgag aggcccggcg catcttcacc catctccgcg gtacgacggc gctgqcgaag cgcctacacc 120 180 240 300 360 420 480 540 600 660 WO 99/32634 WO 9932634PCT/NZ98/00189 caggtccaga ctgaacatcg ctgaagggca gacctgaccg gctgcggccg agcgacatcg gacaaccagg gtcgacctgg agaggcgcca ggcgtactcg gggcgc at ca gtcgtcaccg acctgcagga tgccgacctc agctcgacgg ccgagctggg agggcccgct tcaccgatcc ccaaggtcgt tcgccctggt tggcgatcaa tgcgctccga tcgcctcggt cgcatctggg cggcccgcac caccggtgcc ctacgcgctg caagt cgg cc caagggcatc gcacagctcg gtcctggtac cggcaagtcg gtcactcgac cctgaacgtc gccgacgttg caggcccaag aaggatctgc gccaaggcca cgggtgccga a ccgtgg acg ctcaagtact atcttcgact gacaacgagt ctgtaggggc gaccttctgt cccctcgacg aaggcgttga ggtgagccgg gccgggcccg tcggactggt t ccc ca ccgg agatcaacgc acgacgcccc cgggt ctgac ggggctactc gagcgaagcg ccgaaggggt gcgacacgat gtgacgccgg atcc cgccgccgcg gctgcccgag ctcggtcacc cgcgatgaag gatcgtgtcc caaggtcatc caaccgcctc acgggagaac gacggggcgg caccgacccg cgccaaggtg 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1364 <210> <211> <212> <213> 205 340

PRT

mycobacterium vaccae <400> 205 Val Thr Ile Arg Val Gly Val Asn Gly Phe Giy Arg Ile Gly Arg Asn 1 Phe Phe Arg Ala Leu Asp Ala Gin 10 Lys Ala 25 Leu Thr Giu Giy Lys Asn Lys Asp Asp Asn Ala Thr Leu Ala Val Ala Ile Giu Ile His Leu Leu Val Asn Asp 40 Ile Pro Lys Phe Asp Ser Leu Ser 55 Thr Leu Gly Arg Leu Ser Tyr Asp Val Giu Gly Giu Ala Asp 70 Glu Ile Val Val Gly 75 Leu Thr Lys Ile Lys Leu Glu Val Lys Val Gly Pro Ala Ala 90 Gly Pro Trp Gly Asp Leu Gly Val Asp Lys Ala Gin 115 Ala Pro Ala Val 100 Gly Val Glu Ser Thr 105 Gly Ile Phe Thr His Leu Asp Ala 120 Ile Ala Lys Lys Val 125 Gly Lys Arg Asp 110 Ile Ile Ser Val Asn Asp Thr Asp Giu Asp 135 Thr Ile Val Leu 140 Asp 145 Thr Tyr Asp Gly Asn Cys Leu Gly 165 Leu Ser Gin Asn 150 Pro Leu Ala Met Thr Thr Ile Ile Lys Val 170 Ile His Ser 155 Ile Asn Ala Ser Asn Asp Giu Cys Thr 160 Phe Gly 175 Ile Val Lys Asn Leu Gin 195 Ala Leu Asn Gly 180 Asp Ala Tyr Thr 185 Asp Gin Val Gin 190 Arg Ala Ala Gly Pro His Lys 200 Ser Leu Arg Arg Al a 205 Lys Ile Val Pro 210 Leu Val Thr 215 Thr Gly Ala Al a 220 Gly Ala Ile Gly Leu Pro Glu 225 Val Pro Ile Pro Thr 245 Val Leu Lys Gly Lys 230 Gly Ser Vai Thr Asp Giu Ile Asn 265 Leu Asp 250 Ala Asp 235 Leu Thr Ala Giu Tyr Ala Leu Leu Gly 255 Ala Ala Lys Ser Ala Thr 260 Ala Met Lys Ala 270 WO 99/32634 WO 9932634PCT/NZ98/OO1 89 Giu Gly Pro 275 Ser Ser Asp Leu Lys Gly Ile Lys Tyr Tyr His Ser Ser Asp Ala 285 Ile Phe Pro Ile Val Asp Ser Gly Ile Val Thr 290 Thr 300 Val Leu 305 Asn Lys Val Ile Asp 310 Ser Gin Ala Lys Vai 315 Asp Ser Trp Tyr Giu Trp Giy Asn Arg Leu Vai 330 Leu Val Ala Giy Lys Ser <210> <211> <212> <213> 206 522

DNA

Mycobacterium vaccae <400> 206 acctacgagt gtggatgccg aacgt caagc ttcaaggttg ctggagccag ggcgacctga gtcgtgaagg aagacccagg aacgtgtcga tcgagaacaa gcgcgcagga tgacgctgct ccggctccca tgatggcggt actcccgccg cgcaggttcc gccgggccaa aggagatcat ggtcacgggc cgccatgcag cgacggtgcc ggtcatgaag cgaggtcacg tggtcagatc gctgtcggag ctactccatg cgcgaaggcg ggccgcatcc tacggcgtgc taccacgaag aaggctgccg acgcccgagg caggccatgg atgttcggct gtgttcgact acgggccagt cgcgcgagta tgqccggcta tcgactcgtc cccaggcgca attacatggg aggagcggag acgtcggaga cgtacgccga aa catcccgtcg cccgctggtt ggaaatggca gccggtgatc tgaagtgagc cggtgctcgt ccttcggtcg agttccggcg 120 180 240 300 360 420 480 522 <210> <211> <212> <213> 207 173

PRT

Mycobacterium vaccae <400> 207 Tyr Giu Phe Giu Asn Lys Val Thr Thr 1 Tyr Gly 10 Gin Gly Arg Ile Pro Arg Giu Ile Pro Ser Val Asp Ala Leu Ala Gly Tyr Pro Leu Val Giy Ala 25 Val Asn 40 Ser Ser Val Lys Leu Thr Asp Ala Met Gin Tyr Gly Leu Leu Asp Lys Val Ala Gly Ala Tyr s0 Giv Ser Gin Phe His Glu Vai Val Met Lys 70 Val Met Ala Asp 55 Lys Ala Ala Giu Met Ala Ala Gin Ala Gin Pro Val Leu 75 Thr Ile Giu Pro Val Giu Val Gly Glu Val Met Giu Giu 115 Ile 100 Arg Gly Thr 90 Arg Pro Giu Asp Asp Leu Asn Ser 105 Val Arg Gly Gin Ser Gly Ala Arg 120 Val Lys Ala Gin 125 Lys Ile Gin Ala 110 Val Pro Leu Thr Gin Gly Ser Glu Met Phe 130 Arg Ala Asn Tyr 145 Gly Tyr Ser Met 150 Val Gly 135 Val Phe Asp Leu Arg Asp Ser Tyr 155 Ser 140 Ala Glu Val Pro Ala 160 WO 99/32634 PCT/NZ98/OO1 89 Asn Val Ser Lys Glu Ile Ile Ala Lys Ala Thr Gly Gin 165 170 <210> <211> <212> <213> 208 12

PRT

mycobacterium vaccae <400> 208 Ala Leu Pro Gin Leu Thr Asp Giu Gin Arg Ala Ala 1 5 T7

Claims (3)

1. A polypeptide comprising an immunogenic portion of an isolated M. vaccae antigen, wherein the antigen includes a sequence selected from the group consisting of: sequences recited in SEQ ID NOS: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205 and

207. 2. A polypeptide comprising an immunogenic portion of an isolated M. vaccae antigen, wherein the antigen includes a sequence selected from the group consisting of: sequences having at least about 50% identical residues to a sequence recited in SEQ ID NOS: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205 S. and 207 as measured by computer algorithm BLASTP; sequences having at least about 75% identical residues to a sequence recited in SEQ ID NOS: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205 and 207 as measured by computer algorithm BLASTP; and sequences having at least about 95% identical residues to a sequence recited in SEQ ID NOS: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, 194, 196, 197, 199, 201, 203, 205 and 207 as measured by computer algorithm BLASTP. 3. A polypeptide comprising an immunogenic portion of an isolated M. vaccae antigen, wherein the antigen comprises an amino acid sequence encoded by a polynucleotide selected from the group consisting of: sequences recited in SEQ ID NOS: 142, 144, 146, 151, 153, 155, 157, 159, 161, 163, 164, 169, 171, 173, 175, 176, 179, 180, 183, 185, 191, 193, 195, 198 and 200; 0AL 114 complements of the sequences recited in SEQ ID NOS: 142, 144, 146, 151, 153, 155, 157, 159, 161, 163, 164, 169, 171, 173, 175, 176, 179, 180, 183, 185, 191, 193, 195, 198 and 200; and sequences having at least about a 99% probability of being the same as a sequence of or as measured by computer algorithm BLASTN. 4. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide according to any one of claims 1-3. An expression vector comprising a polynucleotide according to claim 4. 6. A host cell transformed with an expression vector according to claim 7. The host cell of claim 6, wherein the host cell is selected from the group consisting ofE. coli, mycobacteria, insect, yeast and mammalian cells. 8. A fusion protein comprising at least one polypeptide according to any one of claims 1-3. 9. A pharmaceutical composition comprising a polypeptide according to any one of claims 1-3 and a physiologically acceptable carrier. 10. A pharmaceutical composition comprising a polynucleotide according to claim 4 and a physiologically acceptable carrier. 11. A pharmaceutical composition comprising a fusion protein according to claim 8 and a physiologically acceptable carrier. 12. A vaccine comprising a polypeptide according to any one of claims 1-3 and a non-specific immune response amplifier. 115 7777 116 13. A vaccine comprising a polynucleotide according to claim 4 and a non- specific immune response amplifier. 14. A vaccine comprising a fusion protein according to claim 8 and a non- specific immune response amplifier. A vaccine according to any one of claims 12-14 wherein the non-specific immune response amplifier is an adjuvant. 16. A vaccine according to any one of claims 12-14 wherein the non-specific immune response amplifier is selected from the group consisting of: delipidated and deglycolipidated M. vaccae cells; inactivated M. vaccae cells; M. vaccae culture filtrate; and constituents present in or derived from any of the compositions of *i 17. A method for enhancing an immune response in a patient, comprising administering to a patient a pharmaceutical composition according to any one of claims 9-11. 18. A method for enhancing an immune response in a patient, comprising administering to a patient a vaccine according to any one of claims 12-14. 19. The method of any one of claims 17 and 18, wherein the immune response is a Thl response. 08/01/02 0vro 117 A method for the treatment of a disorder in a patient, comprising administering to the patient a pharmaceutical composition according to any one of claims 9-11. 21. A method for the treatment of a disorder in a patient, comprising administering to the patient a vaccine according to any one of claims 12-14. 22. The method of any one of claims 20 and 21, wherein the disorder is selected from the group consisting of immune disorders, infectious diseases, skin diseases and diseases of the respiratory system. The method of claim 22, wherein the disorder is selected from the group consisting of mycobacterial infections, asthma, psoriasis, dermatitis, allergic rhinitis, sarcoidosis, alopecia areata, lung cancer, and carcinomas of the skin. 24. A method for the treatment of a disorder in a patient comprising administering a composition comprising a component selected from the group delipidated and deglycolipidated 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 delipidated and deglycolipidated M. vaccae cells treated with an acid; 28/12/01 028/12/01 L T o i. L, 117a the disorder being selected from the group consisting of immune disorders, infectious diseases, skin diseases and diseases of the respiratory system. The method of claim 24, wherein the disorder is selected from the group consisting of mycobacterial infections, asthma psoriasis, dermatitis, allergic rhinitis, sarcoidosis, alopecia areata, lung cancer, and carcinomas of the skin. 26. A method for enhancing a non-specific immune response to an antigen comprising administering a polypeptide, the polypeptide comprising an immunogenic portion of a M. vaccae antigen, wherein the M. vaccae antigen includes a sequence selected from the group consisting of: sequences recited in SEQ ID NO: 89 and 201; and 08/01/02 iLi i-ii.i.ilfiiYlli? i~ sequences having at least about 80% identical residues to a sequence recited in SEQ ID NO: 89 and 201 as determined by computer algorithm BLASTP. 27. A method for detecting mycobacterial infection in a patient, comprising: contacting dermal cells of a patient with one or more polypeptides according to any one of claims 1-3; and detecting an immune response on the patient's skin. 28. The method of claim 27 wherein the immune response is induration. A diagnostic kit comprising: a polypeptide according to any one of claims 1-3; and apparatus sufficient to contact the polypeptide with the dermal cells of a patient. A method for detecting mycobacterial infection in a biological sample, a a. a. a comprising: contacting the biological sample with a polypeptide according to any one of claims 1-3; and detecting in the sample the presence of antibodies that bind to the polypeptide. *g 31. The method of claim 30 wherein the polypeptide(s) are bound to a solid support. 32. The method of claim 30 wherein the biological sample is selected from the group consisting of whole blood, serum, plasma, saliva, cerebrospinal fluid and urine. 33. A method for detecting mycobacterial infection in a biological sample, comprising: (a) contacting the biological sample with a binding agent which is capable of binding to a polypeptide according to any one of claims 1-3; and 118 nTs--.i- detecting in the sample a protein or polypeptide that binds to the binding agent. 34. The method of claim 33 wherein the binding agent is a monoclonal antibody. The method of claim 33 wherein the binding agent is a polyclonal antibody. 36. A diagnostic kit comprising: at least one polypeptide according to any one of claims 1-3; and a detection reagent. 37. The kit of claim 36 wherein the polypeptide is immobilized on a solid support. 38. The kit of claim 36 wherein the detection reagent comprises a reporter group conjugated to a binding agent. 39. The kit of claim 38 wherein the binding agent is selected from the group consisting of anti-immunoglobulins, Protein G, Protein A and lectins. The kit of claim 38 wherein the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles. 41. A monoclonal antibody that binds to a polypeptide according to any one of claims 1-3. 42. A polyclonal antibody that binds to a polypeptide according to any one of claims 1-3. 43. A method for enhancing a non-specific immune response to an antigen comprising administering a composition comprising a component selected from the group consisting of: 119 120 delipidated and deglycolipidated M. vaccae cells depleted of mycolic acids; delipidated and deglycolipidated M. vaccae cells depleted of mycolic acids and arabinogalactan; and delipidated and deglycolipidated M. vaccae cells treated with an acid. 44. A composition comprising a component selected from the group consisting of: delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids and arabinogalactan; and delipidated and deglycolipidated mycobacterial cells treated with an acid. A composition of claim 44, wherein the mycobacterial cells comprise M. vaccae cells. 46. A composition comprising treated M. vacccae cells having an amino acid content of at least 40% w/w. 47. A composition comprising treated M. vaccae cells having a lipid content of less than 3% w/w. 08/01/02 121 48. A composition of any of claims 44-47 in a formulation suitable for delivery to the airways leading to or located in the lungs. 49. A method for modulating the balance of a population of Thl and Th2 cells in a sample population, by administering a composition comprising a component selected from the group consisting of: delipidated and deglycolipidated mycobacterial cells; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids and arabinogalactan; and delipidated and deglycolipidated mycobacterial cells treated with an acid. A method for activating T cells in a sample population, by administering a composition comprising a component selected from the group consisting of: S(a) delipidated and deglycolipidated mycobacterial cells; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids and arabinogalactan; and delipidated and deglycolipidated mycobacterial cells treated with an acid. 21 O 28/12/01 O'4 o 122 51. A method for activating NK cells in a sample population, by administering a composition comprising a component selected from the group consisting of: delipidated and deglycolipidated mycobacterial cells; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids and arabinogalactan; and delipidated and deglycolipidated mycobacterial cells treated with an acid.

550. S S 52. A method for stimulating production of cytokines in a sample population, by administering a composition comprising a component selected from the group consisting of: delipidated and deglycolipidated mycobacterial cells; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids and arabinogalactan; and delipidated and deglycolipidated mycobacterial cells treated with an acid. 53. A method for enhancing expression of co-stimulatory molecules on at least one of dendritic cells and monocytes in a sample population, by administering a composition comprising a component selected from the group consisting of: 28/12/01 122a delipidated and deglycolipidated mycobacterial cells; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids and arabinogalactan; and delipidated and deglycolipidated mycobacterial cells treated with an acid. 54. A method for enhancing dendritic cell maturation and function in a sample population, by administering a composition comprising a component selected from the group consisting of: delipidated and deglycolipidated mycobacterial cells; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids; *i delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids and arabinogalactan; and delipidated and deglycolipidated mycobacterial cells treated with an acid. 55. A method for reducing the accumulation of eosinophils in a sample cell or tissue population, by administering a composition comprising a component selected from the group consisting of: delipidated and deglycolipidated mycobacterial cells; O 08/01/02 122b delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids; delipidated and deglycolipidated mycobacterial cells depleted of mycolic acids and arabinogalactan; and delipidated and deglycolipidated mycobacterial cells treated with an acid. 56. A method according to any of claims 49-55, wherein the mycobacterial cells are M. vaccae cells. *b 4 .4 *0 o49° 4 00 0 4 *oo 08/01/02 57. The use of a composition of any of claims 44-47 in the treatment of a disorder selected from the group consisting of immune disorders, infectious diseases, skin diseases and diseases of the respiratory system. 58. The use of a composition of any of claims 44-47 in the manufacture of a medicament for treatment of a disorder selected from the group consisting of immune disorders, infectious diseases, skin diseases and diseases of the respiratory system. 59. The use of a composition of any of claims 44-47 in the manufacture of a medicament for treatment of a disorder selected from the group consisting of: mycobacterial infections, asthma, psoriasis, dermatitis, allergic rhinitis, sarcoidosis, alopecia areata, lung cancer, and carcinomas of the skin. Dated this 14 December 2000 Genesis Research Development Corporation Limited Patent Attorneys for the Applicant PETER MAXWELL ASSOCIATES *1 1 123 -i