CA2315539A1 - Compositions derived from mycobacterium vaccae and methods for their use - Google Patents

Abstract

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

Description

WO 99/32634 PGT/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 cancer.
Backttround of the Invention Tuberculosis is a chronic, infectious disease, that is caused by infection with Mycobacterium tuberculosis (M. tuberculosis). It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about 8 million new cases and 3 million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as a chronic inflammation of the lungs, resulting in fever and respiratory symptoms. If left untreated, significant morbidity and death may result.
Although tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not su~cient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behaviour is diffcult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective dent 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 e~cient method for inducing protective immunity. The most common mycobacterium employed for this purpose is Bacillus Calinette-Guerin (BCG), an avirulent strain of Mycobacterium bovis (M. 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 intradennal 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 lepmsy, by subcutaneous or intradermal injection, is Mycobacterium vaccae (M. 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 aatigenic compounds that are recognised by the immune system of individuals exposed to infection with M. tuberculosis.
Several patents and other publications disclose dent 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 treatrnent of autoimmune diseases of various types, including arthritic diseases, by administering mycobacteria, including M. vaccae. U.S. Patent 4,724,144 discloses an immunotherapeutic agent comprising antigenic material derived from M. vaccae for treatment of mycobacterial diseases, especially tuberculosis and leprosy, and as an adjuvant to chemotherapy.

International Patent Publication WO 91/01751 discloses the use of antigenic and/or immunoregulatory material from M. 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 gmwth 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, Guillaia-Bane syndrome, primary diabetes mellitus, and some aspects of graft rejection.
M. vaccae is apparently unique among known mycobacterial species in that heat-killed preparations retain vaccine and immunotherapeutic properties. 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 4 PCT/NZ98~00189 species which have adjuvant properties. The elect 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 (R.G. White, I. Bemstock, R.G.S. Johns and E. Lederer, ImmunoloQV.
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, vaccina~on 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 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 IFNJy 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 at9e predominantly seen in other organs, subclinical lung involvement is usually present. While some cases of sarcoidosis resolve spontaneously, approximately 50% of patients have at least a mild degree of permanent organ dysfunction. In severe cases, lung fibrosis develops and progresses to pulmonary failure requiring lung transplantation. The mainstay of treatment for sareoidosis is corticostemids. 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 tracheobmnchial tree to a variety of stimuli, the primary physiological disturbance being reversible airflow limitation, which may be spontaneous or drug-related, and the pathological hallinark being inflammation of the airways. Clinically, asthma can be subdivided into extrinsic and intrinsic variants.
Extrinsic asthma has an identifiable precipitant, and can be thought of as being atopic, occupational and drug-induced. Atopic asthma is associated with the enhancement of a Th2-type of immune response with the production of specific immunoglobulin E
(IgE), positive skin tests to common aeroallergens and/or atopic symptoms. It can be divided further into seasonal and perennial forms according to the seasonal timing of symptoms. 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. Non-seasonal or perennial rhinitis is caused by allergy to antigens such as those from house dust mite or animal dander.
The abnormal immune response in allergic rhinitis is characterised by the excess production of IgE antibodies specific against the allergen. The inflammatory response occurs in the nasal mucosa rather than further down the airways as in asthma. 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 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 eglcacies 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 (T~
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 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 ailects 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 pm-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. Farp.
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 psoriadc lesions show an influx of CD4+
T cells and, more prominently, CD8+ T cells (Bos, J.D. et al., Arch. Dermatol.
Res. 281:23-3, 1989; Baker, B.S., Br. J. Dermatol. ll D:555-564, 1984).
As the majority (90~/0) 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 (10%) of psoriasis patients have a more serious condition, for which a number of systemic therapeutic modalities are available. Specific systemic therapies include UVB, PUVA, methotrexaxe, 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 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 ('INF) and interferon-gamma (IFN-Y).

If the causes can be identified, removal alone will cure allergic contact dermatitis.
During active inflammation, topical corticostemids are useful. An inhibitory effect of cyclosporin has been observed in delayed-type hypersensitivity on the pro-inflammatory functions) 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 denmatological 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).

The large variety of therapeutic modalities in alopecia areata can be divided into four categories: (i) non-specific topical irritants; (ii) 'immune modulators' such as systemic corticosteroids and PUVA; (iii) 'immune enhancers' such as contact dermatitis inducers, cyciosporin and inosiplex; and (iv) drugs of unknown action such as minoxidil (Dawber, R.P.R. et al., Textbook of Dermatology, Blackweil Scientific Publications, 5'~
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 PC"f/NZ98I00189 Lesions are usually surgically excised. Alternate treatments include retinoids, 5-fluorouracil, cryotherapy and radiotherapy. Alpha or gamma interferon have also been shown to be effective in the treatment of basal cell carcinomas, providing a valuable alternative to patients unsuitable for surgery or seeking to avoid surgical scars (Cornell et al., J.
Am. Acad.
Dermatol. 23:694-700, 1990; Edwards, L. et al., J. Am. Acad Dermatol. 22:496-500, 1990).
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. 95:1805-4S, 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 (30% 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.
95:1855-7S, 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 significant economic cost (Kraus, S.J. et al., Review of Infectious Diseases 2(6):S620-S632, 1990; Frazer, LH., Current Opinion in Immunology 8(4):484-491, 1996).
Summary of the Invenpon 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 (a) sequences recited in SEQ ID NO: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192, I94, 196, 197, 199, 201, 203, 205 and 207; (b) sequences having at least about 50% identical residues to a sequence recited in SEQ ID NO: 143, 145, 147, 152, 154 156, 158, 160, 162, 165, 166, i70, 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 (d) 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, WO 99/32634 PCT/NZ98I~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 aad deglycolipidated M. vaccae cells or M. vaccae culture filtrate.

WO 99/32634 PCT/NZ9$/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 of a 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.

Brief Description of the Drawines Figs. 1 A and 1 B illustrate the protective erects 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 p,g of DD-M.
vaccae, respectively, 4 weeks prior to challenge with ovalbumin, as compared to control mice. Figs.
2C and D show the percentage of eosinophils in mice immunized intranasally with either 100 p.g of heat-killed M. vaccae or 200 pg 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 (i.n.) or subcutaneously (s.c.) with either BCG
of the Pasteur strain (BCG-P), BCG of the Connought strain (BCG-C), I mg of heat-killed M.
vaccae, or 200 wg of DD-M. vaccae prior to challenge with ovalbumin.
Fig. 3A illustrates the effect of immunizing mice with heat-killed M. vaccae or delipidated and deglycolipidated M. vaccae (DD-M. vaccae) prior to infection with tuberculosis. Fig. 3B illustrates the effect of immunizing mice with heat-killed M. vaccae, recombinant M. vaccae proteins, or a combination of heat-killed M. vaccae and M. vaccae recombinant proteins prior to infection with tuberculosis.
Fig. 4 illustrates the induction of IL-12 by autoclaved M. vaccae, lyophilized M.
vaccae, delipidated and deglycolipidated M. vaccae and M. vaccae glycolipids.
Fig. 5 compares the in vitro stimulation of interferon-gamma production in spleen cells from Severe Combined ImmunoDeficient (SCID) mice by different concentrations of heat-killed (autoclaved) M. vaccae, delipidated and deglycolipidated M.
vaccae, and M.
vaccae glycolipids.
Figs. 6A, B and C illustrate the stimulation of interferon-gamma production by different concentrations of M. vaccae recombinant proteins, heat-killed M.
vaccae, delipidated and deglycolipidated M. vaccae (referred to in the figure as "delipidated M.
vaccae"), M.
vaccae glycolipids and lipopolysaccharide, in peritoneal macrophages from C57BL/6 mice (Fig. 6A), BALB/C mice (Fig. 6B) or C3H/HeJ mice (Fig. 6C).

Fig. 7A(i) - (iv) illustrate the non-specific immune amplifying effects of 10 pg, 100 p,g and 1 mg autoclaved M. vaccae and 75 p.g unfractionated culture filtrates of M. vaccae, respectively. Fig. 7B(i) and (ii) illustrate the non-specific immune amplifying effects of autoclaved M. vaccae, and delipidated and deglycolipidated M. vaccae, respectively. Fig.
7C(i) illustrates the non-specific immune amplifying effects of whole autoclaved M. vaccae.
Fig. 7C(ii) illustrates the non-specific immune amplifying effects of soluble M. vaccae proteins extracted with SDS from delipidated and deglycolipidated M. vaccae.
Fig. 7C(iii) illustrates that the non-specific amplifying effects of the preparation of Fig. 7C(ii) are destroyed by treatment with the proteolytic enzyme Pronase. Fig. 7D
illustrates the non-specific immune amplifying effects of heat-killed M. vaccae (Fig. 7D(i)), whereas a non-specific 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 a(3T cells, y8T 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-1 Vii, TNF-a, IL-12 and IFN-~y, respectively, by human PBMC.
Figs. l0A-C illustrate the effects of varying concentrations of the recombinant M.
vaccae proteins GV-23 and GV-45 on the production of IL-1 Vii, TNF-a, and IL-12, respectively, by human PBMC.
Figs. 11A-D illustrate the stimulation of IL-1(3, TNF-a, 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.

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 denmatis, 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 Wp 99132634 PCT/NZ98I00189 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 marine 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 marine model. In particular, the majority of T-cell clones from atopic human lymphocytes resemble the marine 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 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 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 ~°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.

As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds. 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 ar 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 (e.g., 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 20%, preferably about 65%, and most preferably about 100% of the proliferation induced by the full-length antigen in the model proliferation assay described herein. An immunogenic portion may also, or alternatively, stimulate the production of at least about 20%, preferably about 65% and most preferably about 100%, of the interferon-y and/or interleukin-I2 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.

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 2Ø4 [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 s~,o_vBLAST/newblast.html and in the publication of Altschul, Stephen F., et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402. The computer algorithm FASTA is available on the Internet at the ftp site ft~://ftn.virginia.edu/pub/fasta/. Version 2.Ou4, 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 -a 10 -G 1 -E 1 -r 2 -v 50 -b SO
-i queryseq -o results; and parameter default values:
-p Program Name [String]
-d Database [String]
-a Expectation value (E) [Real]
-G Cost to open a gap (zero invokes default behavior) [Integer]

-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 (V) [integer]
-b Number of alignments to show (B) [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 --a 10 -G I -E 1 v 50 -b 50 -i queryseq -o results -p Program Name [String]
-d Database [String]
-a Expectation value (E) [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 (v) [Integer]
-b Number of alignments to show (b) [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 (E) indicates the number of hits one can "expect" to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database, such as the preferred EMBL database, 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/OOt89 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 I% 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 poiynucleotide 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 °C, 6X SSC, 0.2%
SDS overnight; followed by two washes of 30 minutes each in 1X 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 Mernfield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain.
See Mernfield, 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 PC"f/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 (e.g., 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 described below, polymerase chain reaction (PCR) may be employed to isolate a nucleic acid probe from genomic DNA, or a cDNA or genomic DNA library. The library screen may then be performed using the isolated probe. DNA molecules encoding M. vaccae antigens may also be isolated by screening an appropriate M. vaccae expression library with anti-sera (e.g., rabbit or monkey) raised specifically against M. vaccae antigens.
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.
tuberculosis-immune 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 (i.e., greater than about 10 mm diameter induration) intradermal skin test response to tuberculosis proteins (PPD), and an absence of any symptoms of tuberculosis infection.
Assays for cell proliferation or cytokine production in T cells, NK cells, B
cells or macrophages may be performed, for example, using the procedures described below. The selection of cell type for use in evaluating an immunogenic response to an antigen will depend on the desired response. For example, interleukin-12 production is most readily evaluated using preparations containing T cells, NK cells, B cells and macrophages derived from M. tuberculosis-immune individuals may be prepared using methods well known in the artt.
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 FicollT"' (Winthrop Laboratories, N~. 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 protein-specific T cells, resulting in a line composed solely of such cells. These cells may then be WO 99/32634 PCTINZ98ro0189 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 5' end of a DNA sequence encoding the second polypeptide so that the reading frames of the sequences are in phase to permit mRNA translation of the two DNA sequences into a single fusion protein that retains the biological activity of both the first and the second polypeptides.
A peptide linker sequence may be employed to separate the first and the second polypeptides by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is 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: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
Preferred peptide linker sequences contain Gly, Asn and Ser residues. 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 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 (e.g., 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.

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 (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., 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 pg. 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, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos.
4,897,268 and 5,075,109.
Any of a variety of adjuvants 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, Nn. 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 (i.e., 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 ~.g to about 100 fig, preferably from about 10 ~g to about 50 p,g 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 80T"".

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 (i.e., one component polypeptide will tend to detect infection in samples where the infection would not be detected by another component polypeptide). Complementary polypeptides may generally be identified by using each polypeptide individually to evaluate serum samples obtained from a series of patients known to be infected with a Mycobacterium. After determining which samples test positive (as described below) with each polypeptide, combinations of two or more polypeptides may be formulated that are capable of detecting infection in most, or all, of the samples tested. For example, approximately 25-30% of sera from tuberculosis-infected individuals are negative for antibodies to any single protein, such as the 38 kDa antigen mentioned above.
Complementary polypeptides may, therefore, be used in combination with the 38 kDa antigen to improve sensitivity of a diagnostic test.

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, e.g., 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 (e.g., 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 ~,g, and preferably about 100 ng, is sufficient to bind an adequate amount of antigen.

WO 99/32634 PCT/NZ98/001$9 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, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A 12-A 13 ).
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 95% of that achieved at equilibrium between bound and unbound antibody. The time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound sample may be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20TM. Detection reagent may then be added to the solid support. An appropriate detection reagent is any compound that binds to the immobilized antibody-poiypeptide 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 (e.g., Zymed Laboratories, San Francisco, CA, and Pierce, Rockford, IL).
The detection reagent is incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound antibody. An appropriate amount of time may generally be determined from the manufacturer's instructions or by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group.
The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
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 predetenmined 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 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 Science for Clinical Medicine, Little Brown and Co., 1985, pp. 106-107. In general, signals higher than the predetermined cut-off value are considered to be positive for mycobacterial infection.
The assay may also be performed in a rapid flow-through or strip test format, wherein the antigen is immobilized on a membrane, such as nitrocellulose. In the flow-through test, antibodies within the sample bind to the immobilized polypeptide as the sample passes through the membrane. A detection reagent (e.g., protein A-colloidal gold) then binds to the antibody-polypepdde 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 anti-mycobacterial 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 su~cient 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 wg, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount (e.g., one drop) of patient serum or blood.
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, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogen comprising the antigenic WO 99/32634 PCT/NZ98ro0189 polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep and goats). The immunogen is injected into the animal host, preferably according to a predetermined schedule 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 (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells may then be immortalized by fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal, using one of a variety of techniques well known in the art.
Monoclonal antibodies may be isolated from the supernatants of the resulting hybridoma colonies. 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 polymerise chain reaction (PCR) based tests.
Similarly, probes comprising at least 18 contiguous oligonucleotides of an inventive polynucleotide may be used for hybridizing to specific sequences. Techniques for both PCR based tests and hybridization tests are well known in the art. Primers or probes may thus be used to detect M. tuberculosis and other mycobacterial infections in biological samples, preferably sputum, blood, serum, saliva, cerebrospinal fluid or urine. DNA probes or primers comprising oligonucleotide sequences described above may be used alone, in combination with each other, or with previously identified sequences, such as the 38 kDa antigen discussed above.
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.

EFFECT OF IMMUNIZATION OF MICE WITH M. YACCAE
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, 2.5 g/1; tryptone, 5 g/I; glucose, 1 g/1) at 37 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 °C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10 mg/ml, equivalent to 10'° M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120 °C. The culture filtrate was passaged through a 0.45 ~,m filter into sterile bottles.
As shown in Fig.lA, when mice were immunized with 1 mg, 100 ~g or 10 ~g of M.
vaccae and infected three weeks later with SxlOs colony forming units (CFU) of live M.
tuberculosis H37Rv, significant protection from infection was seen. In this example, spleen, liver and lung tissue was harvested from mice three weeks after infection, and live bacilli determined (expressed as CFU). The reduction in bacilli numbers, when compared to tissue from non-immunized control mice, exceeded 2 logs in liver and lung tissue, and 1 log in spleen tissue. Immunization of mice with heat-killed M. tuberculosis H37Rv had no significant protective effects on mice subsequently infected with live M.
tuberculosis H37Rv.
Fig. l B shows that when mice were immunized with 100 ~g of M. vaccae culture filtrate, and infected three weeks later with Sx105 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.

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 intraiung 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 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 10' 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.

COMPARISON OF INTRADERMAL AND INTRALUNG
ROUTES OF I1~~SATION
Group Identification Amount of Route of Number Number of Immunogen Immunisation Monkey (Controls) 3144-B 0 -2 4080-B 500 ~g intradermal (Immunised 3586-B 500 pg intradermal with heat-killed M. vaccae) 3 3534-C S00 ~.g intralun g (Immunised 3160-A 500 ~g intralung with heat-killed M, vaccae) (Immunised 3564-B 100 ~tg intradermal with culture filtrate) 3815-B 100 p.g intradermal (Immunised 4425-A 100 pg intralung with culture filtrate) 2779-D 100 ~g intralung 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 x-rays indicated disease in both lungs. As expected, as disease progressed both control animals lost weight and showed significant LPA responses to PPD, indicating strong T
cell reactivity to M. tuberculosis. The ESR measurements were variable but consistent with strong immune reactivity.
Table 2B - The two monkeys immunised twice with 500 pg 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 pg 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 ~,g 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 p.g of M. vaccae culture filtrate.
Monkeys immunised intradermally showed signs of developing disease 84 days post-infection, 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.

CONTROL MONKEYS
ESR I:PA LPA .
ID# ' DsysW~Kgs Tcmp. 11%im/hrPPD10 . X-Ray Remarks PPDl 53101E 0 2.17 37.0 0 0.47 1.1 Negative __ _ 34 1.88 37.3 ND 0.85 1.4 ND

62 2.02 36.0 ND 1.3 1.5 ND

--~ Time ction of Infe 28 2.09 38.0 2 1.3 3.7 Positive 56 1.92 37.2 20 5.6 9.1 Positive 84 1.81 37.5 8 4.7 5.6 Positive ~A LPA
ID# Days W~Kgs :: ~emp,ESR PPD PPD X-Rsy Remarks : Mm/hr lQ~g 1#~8.:

3144-B 0 2.05 36.7 0 0.87 1.8 Negative 34 1.86 37.6 ND 2.2 1.4 ND

62 1.87 36.5 ND 1.6 1.6 ND

-> Time ction of Infe 28 2.10 38.0 0 12 8.7 Positive 56 1.96 37.6 0 29.6 21.1 Positive 84 1.82 37.3 4 45.3 23.4 Positive ND = Not Done MONKEYS IMMiINISED
WITH WHOLE HEAT-KILLED M. VACCAE (500 fig) INTRADERMAL
LPA- LPA.
ID# Days W~Kgs Temp. ESR PPD PPD X-Ray Remarl~s Mm/hr l0ug lp8 4080-B 0 2.05 37.1 1 1.1 0.77 Negative 34 1.97 38.0 ND 1.7 1.4 ND

62 2.09 36.7 ND 1.5 1.5 ND

-~ Time of ction Infe 28 2. 37.6 0 2.6 2.1 Negative i 56 2.17 37.6 0 8.2 7.6 Negative 84 2.25 37.3 0 3.8 2.8 Negative IIf# Days ~ WtK,gs' Temp.ESR PPD PPD X-Rah Remarks _ . mml6r 1Q leg.
...:; !:' 3586-8 0 2.29 37.0 0 1.1 1.4 Negative 34 2.22 38.0 ND 1.9 1.6 ND

62 2.39 36.0 ND 1.3 1.6 ND

-~ Time of ction Infe 28 2.31 38.2 0 3.2 2.6 Negative 56 2.32 37.2 0 7.8 4.2 Negative 84 2.81 37.4 0 3.4 1.8 Negative ND = Not Done MONKEYS IMMUrIISED
WITH WHOLE HEAT-KILLED M. VACCAE (500 fig) INTRALUNG
~A ~A -ID# :Days W~Kgs Temp ESR PPD PPD X-Ray Remarks mm/hr l0~tg leg 3534-C 0 2.15 36.8 0 1.7 1.3- Negative 34 2.00 37.8 ND 4.4 I.4 ND

62 2.13 36.4 ND 3.2 1.9 ND

-~ Time ction of Infe 28 2.38 37.7 0 1.9 2.6 Negative 56 2.42 37.8 0 5.3 4.7 Negative 84 2.46 37.1 1 3.1 3.2 Negative 210 No Negative sign of lung disease LPA LPA .
ID# Days Wt.If~s. ' ESR PPD . X-RayRemarks ., Temp mmlhr 10~,g PPD
: leg 3160-A 0 2.17 37.3 0 1.2 0.79 Negative 34 1.98 37.1 ND 3.9 7.8 ND

62 2.17 36.9 ND 1.7 2.4 ND

~ Time of ction Infe 28 2.38 37.7 0 1.9 2.6 Negative 56 2.42 37.8 0 5.3 4.7 Negative 84 2.46 37.1 1 3.1 3.2 Negative 210 Stable Positive lung disease ND = Not Done WO ~~Z~4 PCT/NZ98/00189 TABLE ZD
MONKEYS nVIMUI~TISED
WITH CULTURE FILTRATE (100 fig) INTRADERMAL
LPA LPA
Dais,Wf:KgsTemp.; LSR PPD PPD X-Ray'Remarks mm/hr l~~g leg 3564-B 0 2.40 37.2 0 1.4 1.4 Negative 34 2.42 38.1 ND 3.3 2.7 ND

62 2.31 37.1 ND 3.1 3.4 ND

Time of Infection 28 2.41 38.6 13 24 13.6 Negative 56 2.38 38.6 0 12:7 12.0 Negative 84 2.41 38.6 2 21.1 11.8 Positive 140 Died LPA IPA
ID#. D~~ys~Wwlt~ "' Temp:ESR PPD PPD X=Ray Remarka ma~ltirl4ug 1~,~
:~.::

3815-B 0 2.31 36.3 0 1.0 1.4 Negative 34 2.36 38.2 ND 1.9 2.0 ND

62 2.36 36.4 ND 3.7 2.8 ND

-~ Time of Infection 28 2.45 37.8 0 2.1 3.3 Negative 56 2.28 37.3 4 8.0 5.6 Negative 84 2.32 37.4 0 1.9 2.2 Positive 210 Positive ND = Not Done MONKEYS IMMUNISED
WITH CULTURE FILTRATE (100 fig) INTRALUNG
IPA LpA
ID# Days Wi,~ Temp. EsR PPD PPD
' -X-Ray: Remarks mmlhr iQUg. 1~$

4425-A 0 2.05 36.0 0 0.35 1.2 Negative 34 2.0 37.6 ND 3.0 2.4 ND

62 2.11 37.6 ND 2.2 1.6 ND

--~ Time ction of Infe 28 2.21 38.0 0 8.4 4.1 Negative 56 2.11 37.6 0 23.9 17.7 Negative 84 2.18 37.9 0 8.4 7.2 Positive 210 Stable Positive lung disease I~BA LPA
~# Days Wt.Kgs.Temp.,.ESR PD pPD X Ray~Remarlcs P Dot ~:~~ ~ , ..
mrnl6r 2779-D 0 2,56 38.6 2 1.9 1.4 Negative 28 2.55 37.9 ND 0.78 1.1 ND

56 2.69 38.4 ND 1.3 1.5 ND
-~ Time of Infection 56 2.25 39.0 24 ND ND Positive ND = Not Done 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 ~g ovalbumin in 100 p.l alum adjuvant by the intraperitoneal route at time 0 and 14 days, and subsequently given 100 ~g ovalbumin in 50 p,l 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 ~tg of heat-killed M. vaccae (Fig. 2A), or 10 , 100 or 200 ~g of DD-M. vaccae, prepared as described below (Fig. 2B) intranasally 4 weeks before intranasal challenge with ovalbumin, had reduced percentages of eosinophils in the BAL cells collected 5 days after challenge with ovalbumin compared to control mice. Control mice were given intranasal PBS.
Live M.
bovis BCG at a dose of 2 x 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 p,g of heat-killed M. vaccae (Fig. 2C) or 200 pg 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 pg of DD-M. vaccae, given either intranasally (i.n.) or subcutaneously (s.c.), reduced lung 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 deuleted of mycolic acids and arabinogalactan Mycolic acids were depleted from DD-M. vaccae by treatment with potassium hydroxide (0.5% 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 0.1 M 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 immunized with M. vaccae or DD-M. vaccae had similar or lower levels of ovalbumin-specific IgGI. As IgGI 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.

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. vaccae21.8 17.4 Mycolic acid and arabinogalactan16.8 0.3 depleted DD-M. vaccae 1V VIG: r» iGam i mice per group.

LOW ANTIGEN-SPECIFIC IgGI SERUM LEVELS
IN MICE IMMUNIZED WITH HEAT-KILLED M. VACCAE OR DD-M. VACCAE
Treatment Group Serum IgGl _ M~ SEM

M.vaccae i.n. 185.00 8.3 M, vaccae s.c. 113.64 8,p DD-M. vaccae i.n. 96.00 8.1 DD-M. vaccae s.c. 110.00 4.1 BCG, Pasteur 337.00 27.2 BCG, Connaught 248.00 46.1 PBS 177.14 11.4 Note: Ovalbumin-specific IgGI was detected using anti-mouse IgGl (Serotec).
Group means are expressed as the reciprocal of the EU50 end point titre.

EFFECT OF IM1~IUNLING MICE WITH M. VACCAE. DD-M. VACCAE OR
RECOMBINANT M YACCAE 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 S
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-M. vaccae showed a mean reduction of > 1 or 0.5 logs CFU, respectively.

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-M. vaccae and recombinant proteins may be developed as vaccines against tuberculosis.

EFFECT OF INTRADERMAL INJECTION OF HEAT-KILLED MYCOBACTERIUM
YACCAE 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, 2.Sg/1; tryptone, Sg/1; glucose, 1 gll) at 37 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, MI, USA) with glucose at 37 °C for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10 mg/ml, equivalent to ? J'° M. vaccae organisms per ml. The cell suspension was then autoc!~~~ed For 15 min at 120 °C and stored frozen at 20 °C. Prior to use the M. vaccae suspension was thawed, diluted to a concentration of 5 mg/ml in phosphate buffered saline, autoclaved for 15 min at 120 °C and 0.2 ml aliquoted under sterile conditions into vials for use in patients.
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 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 pg). This was followed three weeks later with a second intradermal injection with the same dose of M. vaccae (500 fig). 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 -4, 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.

Patient Data in the Study of the Effect of M. vaccae in Psoriasis Code Duration No. Patient Age/Sex of Admission PASI
PS-001 D.C. 49/F Disorder Score PS-002 E.S. 41/F 30 years 28.8 PS-003 M.G. 24/F 4 months 19.2 PS-004 D.B. 54/M 8 months 18.5 PS-005 C.E. 58/F 2 years 12.2 PS-006 M.G. 18/F 3 months 30.5 3 years 15.0 PS-007 L.M. 27/M 3 years I9.0 PS-008 C.C 21/F 1 month 12.2 PS-009 E.G 42/F 5 months 12.6 PS-010 J.G 28/M 7 years 19.4 PS-011 J.U 39/M 1 year 15.5 PS-012 C.S 47/M 3 years 30.9 PS-013 H.B 44/M 10 years 30.4 PS-014 N.J 41/M 17 years 26.7 PS-015 J.T 61/F 15 years 19.5 PS-OI6 L.P 44/M 5 years 30.2 PS-017 E.N 45/M S 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~ I yes, 13.6 ~, PS-021L.I 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 F.T 39/M 15 years 29.5 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 of M. 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.

Skin Reaction Measurements in Millimeter Code No.
_ Time of Measurement First Second Inj~tion Injection 48 hours72 hours 7 days 48 hours 72 hours 7 days PS-001 12x10 12x10 10x8 15x14 15x14 10x10 PS-002 18x14 20x18 18x14 16x12 18x12 15x10 PS-003 10x10 14x10 10x8 15x12 15x10 10x10 PS-004 14x12 22x18 20x15 20x20 20x18 14x10 PS-005 10x10 13x10 DNR DNR DNR DNR

PS-006 10x8 10x10 6x4 12x10 15x15 10x6 PS-007 15x15 18x16 12x10 15x13 15x12 12x10 PS-008 18x18 13x12 12x10 18x17 15x10 15x10 PS-009 13x13 18x15 12x8 15x13 12x12 12x7 PS-010 13x11 15x15 8x8 12x12 12x12 5x5 PS-011 17x13 14x12 12x11 12x10 12x10 12x10 PS-012 17x12 15x12 9x9 10x10 10x6 8x6 PS-013 18x11 15x11 15x10 15x10 15x13 14x6 PS-014 15x12 15x11 15x10 13x12 14x10 8x5 PS-015 15x12 16x12 15x10 7x6 14x12 6x4 PS-016 6x5 6x6 6x5 8x8 9x8 9x6 PS-017 20x15 15x14 14x10 15x15 17x16 DNR

PS-018 14x10 10x8 10x8 12x12 10x10 10x10 PS-019 10x10 14x12 10x8 DNR 15x14 15x14 PS-020 15x12 15x15 12x15 15x15 14x12 13x12 PS-021 15x12 15x12 7x4 11x10 11x10 11x8 PS-022 12x10 10x8 10x8 15x12 13x10 10x8 PS-023 13x12 14x12 10x10 17x17 15x15 DNR

Code No. - -Time of Meaaurement PS-024 _ 10x10 10x8 10x8 8x7 8x7 10x10 DNR
=
Did not report.

Clinical Status of Patients after Injection of M. vaccae (PASI Scores) Code No: Day 0 Week 3 Week Week 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 3.0 ~

PS-007 19.0 15:7 11.6 5.6 -- 2.2 0 PS-008 12.2 11.6 11.2 11.2 5.6 0 -PS-009 12.6 13.4 13.9 14.4 15.3 13.0 PS-010 18.2 16.0 19.4 17.2 16.9 19.3 PS-011 17.2 16.9 16.7 16.5 16.5 15.5 PS-012 30.9 36.4 29.7 39.8**

PS-013 19.5 19.2 18.9 17.8 14.7 17.8 PS-014 26.7 14.7 7.4 5.8 9.9 24.4***

PS-015 30.4 29.5 28.6 28.5 28.2 24.3 PS-016 30.2 16.8 5.7 3.2 0.8 3.3 PS-017 12.3 12.6 12.6 12.6 8.2 8.7 PS-018 16.0 13.6 13.4 13.4 13.2 12.8 PS-019 19.5 11.6 7.0 DNR DNR DNR

PS-020 13.6 13.5 12.4 12.7 12.4 4.4 PS-021 22.0 20.2 11.8 11.4 15.5 15.7 PS-022 2b.5 25.8 20.7 8.3 5.6 PS-023 12.b 9.2 6.6 5.0 4.8 12.b PS-024 29.5 27.5 20.9 19.0 29.8 21.2 ~ * Patient PS-005 received only one dose of autoclaved M. vaccae.
~ ** Patient PS-012 removed from trial, drug (penicillin) induced dermatitis ~ * * * Patient PS-O 14 was revaccinated ~ DNR = Did not report Patients treated with M. vaccae may achieve remission (PASI score = 0). The remission br 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 2.7), was re-vaccinated with injections ofM.vaccae and subsequently improved with PASI
falling from 17.8 (Week b0) to 0.8 (week 84). Thus patients may benefit from repeated treatment.

EFFECT OF )NTRADERMAT ~,~CTTON OF DD MYACC'Ai~' ON PSORIASIS IN HUMAN pA~~rrS
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 above 12 reflects widespread disease lesions on the body. The study commenced with a washout period of four weeks where the four patients did not have systemic antipsoriasis treatment or effective topical therapy. The seven patients were then injected intradermally with 0.1 ml DD-M. vaccae (equivalent to 100 ~,g). This was followed three weeks later with a second intradermal injection with the same dose of DD-M. vaccae (100 pg).
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 -4, 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.

Patient Data in the Study of the Effect of DD-M. vaccae in Psoriasis Code Duration lVo. Patient Age/Sex of Admission PASI
Disorder 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 34nvI 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 Syears 21.3 PS-031 RA 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.

Skin Reaction Measurements in Millimeters Code No. Time of Measurement First Second Injection Injection 48 hours72 hours7 days 48 hours 72 hours PS-025 8x8 8x8 3x2 10x10 10x10 PS-026 12x12 12x12 8x8 DNR 14x14 PS-027 9x8 10x10 10x8 9x5 9x8 PS-028 10x10 10x10 10x8 10x10 10x10 PS-029 8x6 8x6 5x5 8x8 gxg PS-030 14x12 14x14 10x10 12x10 12x10 PS-031 10x10 I2x12 10x6 14x12 12x10 IIATD _. .a ..,..-_~
Tl:.i .".

L1~~\ -' Ll 1 11V1. 1G~JVlI
The data shown in Table 9 are the PASI scores of the seven patients at the time of the first injection of DD-M. vaccae (Day 0), 3, 6, 12 and 24 weeks later.

Clinical Status of Patients after Injection of DD-M. vaccae (PASI Scores) code No. Day 0 Week Week Week Week PS-025 122 4~ 1 1.8 1.4 1.7 PS-026 144 11 ~8 6.0 6.9 1.4 PS-027 248 233 18.3 9.1 10.6 PS-028 182 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 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 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.

PREPARATION OF COMPOSITIONS FROM M YACCAE
This example illustrates the processing of different constituents of M.
vaccae.
Preparation of DeGpidated and Deglycolipidated (DD-) M.vaccae and Compositional Analysis Heat-killed M. vaccae was prepared as described as above in Example 1. To prepare delipidated M. vaccae, the autoclaved M. vaccae was pelleted by centrifugation, the pellet washed with water, collected again by centrifugation and then freeze-dried. An aliquot of this freeze-dried M. vaccae was set aside and referred to as lyophilised M. vaccae.
When used in experiments it was resuspended in PBS to the desired concentration. Freeze-dried M. vaccae was treated with chloroform/methanol {2:1 ) for 60 rains at room temperature to extract lipids, and the extraction was repeated once. The delipidated residue from chloroform/methanol extraction was further treated with 50% ethanol to remove glycolipids by refluxing for two hours. The 50% ethanol extraction was repeated two times. The pooled 50%
ethanol extracts were used as a source of M. vaccae glycolipids (see below). The residue from the 50%
ethanol extraction was freeze-dried and weighed. The amount of delipidated and deglycolipidated M. vaccae prepared was equivalent to 11.1 % of the starting wet weight of M. vaccae used. 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 10%
w/w of lipid, and the total amino acid content is 2750 nmoles/mg, or approximately 33% w/w.
DD-M. vaccae contains 1.3% w/w of lipid and 4250 nmoles/mg amino acids, which is approximately 51 % w/w.

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 C 14:0 3.9% 10.0%

C16:0 21.1% 7.3%

C 16:1 14.0% 3.3%

C18:0 4.0% 1.5%

C 18:1 * 1.2% 2.7%

C 18:1 w9 20.6% 3 .1 _ Cl8:lw7 12.5% 5.9%

C22:0 12.1 % 43.0%

C24:1 * 6.5% 22.9%

PC'T/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 ,A 416 621 CYS* 24 26 V~, 172 272 MET* 72 94 GlcNH2 5 6 ~G 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 of M. 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 DD-M. vaccae contained 34% and 55% protein, whereas DD-M. tubet~culosis and DD-M. smegmatis contained 79% and 72% protein, respectively.

Amino Acid Composition of Delipidated and Deglycolipidated Mycobacteria Amino DD-M.vaccae DD-M.vaccaeDD- DD-Acid Batch 1 Batch M.smegnratisM.tuberculosis Asp 9.5 9.5 9.3 9.1 Thr 6.0 5.9 5.0 5.3 Ser 5.3 5.3 4.2 3.3 Glu 11.1 11.2 11.1 12.5 Pro 6.1 5.9 7.5 5.2 Gly 9.9 9.7 9.4 9.8 Ala 14.6 14.7 14.6 14.2 Cys 0.5 0.5 0.3 0.5 Val 6.3 6.4 7.2 7.8 Met 1.9 1.9 1.9 1.9 Ile 3.6 3.5 4.1 4.7 Leu 7.8 7.9 8.2 8.3 Tyr 1.4 1.7 1.8 1.8 Phe 4.2 4.0 3.2 3.0 His 1.9 1.8 2.0 1.9 Lys 4.1 4.0 4.1 4.2 Arg 5.8 5.9 6.2 6.4 Total % 55.1 33.8 72.1 78.5 Protein Analysis of the monosaccharide composition shows significant differences between DD-M. vaccae, and DD-M. tuberculosis and DD-M. smegmatis. The monosaccharide composition of two batches of DD-M. vaccae was the same and differed from that of DD-M.
tuberculosis and M. smegmatis. Specifically, DD-M. vaccae was found to contain free glucose while both DD-M. tuberculosis and M. smegmatis contain glycerol, as shown in Table 11.

Aldftol Acetate wt% mol%
DD-M.vaccse Batcb 1 Inositol 0.0 0.0 Arabinose 54.7 59.1 Mannose 1.7 1.5 Glucose 31.1 28.1 Galactose 12.5 100.0 100.0 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 Arabinose69.3 70.7 Xylose 3.9 4.0 Mannose 2.2 1.9 Glucose 0.0 0.0 Galactose_9.4 _8.0 100.0 100.0 DD-Mtb Inositol0.0 0.0 Glycerol9.5 9.7 Arabinose69.3 71.4 Mannose 3.5 3.0 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 starting wet weight of M. vaccae used. For bioassay, the glycolipids were dissolved in phosphate-buffered saline.

IMMUNE MODULATING PROPERTIES OF
DELIPIDATED AND DEGLYCOLIPIDATED M. YACCAE 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 °C, 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 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 S 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 stilriulate 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 marine 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 interferon-gamma for three hours. The culture medium was replaced and various concentrations of M.
vaccae recombinant proteins GVs-3 (GV-3), GV-4P (GV-4P), GVc-7 (GV-7), GV-23, GV-27, heat killed M. vaccae, DD-M. vaccae (referred to as delipidated M. vaccae in Figs. 6A, B
and C), 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 of M. 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 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 of M. Vaccae M. vaccae culture supernatant (S/N), 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 heat-killed 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 5 ~ Chromium with which the EL4 and E.G7 cells have been labelled (100 pCi per 2x106), 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.

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 (i) 10 pg, (ii) 100 pg or (iii) 1 mg of autoclaved M. vaccae or (iv) 75 ~tg of M. vaccae culture filtrate. Fig.
7B shows that cytotoxic cells were generated in mice immunized with (i) 1 mg whole autoclaved M. vaccae or (ii) 1 mg delipidated and deglycolipidated (DD-) M.
vaccae. 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 ~g of the SDS-extracted proteins had significantly stronger immune-enhancing ability (Fig. 7C(ii)) than did 1 mg whole autoclaved M. vaccae (Fig. 7C(i)).
Mice immunized with 1 mg heat-killed M. vaccae (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 <8% specific lysis for control mice immunised with ovalbumin alone).
The M. vaccae culture filtrate described above was fractionated by iso-electric focusing and the fractions assayed for adjuvant activity in the anti-ovalbumin-specific cytotoxic response assay in C57BL/6 mice as described above. Peak adjuvant activities were demonstrated in fractions corresponding to pI of 4.2-4.32 (fraction nos. 7-9), 4.49-4.57 (fraction nos. 13-17) and 4.81-5.98 (fraction nos. 23-27).
Identifcation of proteins in DD-M. vaccae by antibodies BALB/c mice were immunised infra-peritoneally with 50 ug of DD-M. vaccae once a week for 5 weeks. At the 6"' 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, SP, 5, 7, 9, 22B, 24, 27, 27A, 27B, 33 and 45 as being present in DD-M.
vaccae.

Reactivity of DD-M. vaccae antiserum with M.vaccae-derived GV antigens GV Antigen3 ~ 5 j 7 ~ 9 i 22B 27A ; 27B 33 _ 5P 24 j 27 i 45 ~ ~ I ~ i i Reactivity*1 1 1 U3 ~ 102 ~ 1 U4 1 U3 U' 1 U' 1 U U6 ~ i ~ ;

US
~

!

U"
j U' ;

"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.

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

Proliferative responses of lymph node cells from DD-M. vaccae-immunised mice and control mice to GV proteins in vitro Stimulation index*
GV protein observed in the presence of GV proteins at 50 pg/ml DD-M.vaccae immunisedControl mice mice GV3 4.63 I .52 GV7 3.32 1.27 GV9 6.48 2.64 GV23 4.00 I .76 GV27 S.I3 1.40 GV27B 7.52 I .48 GV33 3.3 I I .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 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, S, 23, 27A, 27B, 33, 45 and 46 are thus likely to be present in DD-M. vaccae.

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 (pg/ml) or control 50 10 2 GV-3 8.22 ~ 3.73 ND ND
__-_.._.....___.__._._._......_____.__._..___ GV-4P ~ -.- ~ ___..________..._.._._ND
_._.___.....-.._._._.....___.._~~_._____ _.__.._....._...._ GV-5 8.90 t 4.54 0.57 t 0.40 ND

GV-SP ND ND ND
__ _- '____ -_...._._____.-_ _..__..~~___.-___.____~_______-~_._.

GV-9 ND N_D __ _ _ ND _ ~T~~n~a ~i ~

GV-13 1.64 ~ 0.40 ND ND

GV-14 ND _ ND
ND

GV-14B -. _~ ~ ~ ~

GV-22B !_20.15 ~_ ~ 4.34 t 0.02 ND
1.96 ---_.._.__ -~

GV-23 41.38 t 6.69 6.97 t 2.78 ~

GV~24B __ ND _-__ ~ ____..~

__ GV-27. 46_86 t 17.14 _ _33.06 f -_10.14 t 3.01 17.61 __..__ ~ ~

GV-27A 7.25 t 4.36 ND _ __ _ _ ~y ~ ~ ND_ ~

GV-27B 100.36 ~ 37.8433.03 f 7.54 14.33 t 1.01 GV-29 5.93 t 0.47 ND ND

___.__GV-33_._, 9.82 f 4.64.._...._...__. ~ ND
_.____ ____ GV..38AP.___ 1.44 t ND _ __ ND _ _,_ 1.20 ~_ ~

GV-38BP 5.62 f 0.70 ND ND

. _ _._ _..__.~-.._._._.__...._.
__ WO 99/32634 PC"f/NZ98100189 _ DD_-M.vacca_e 109_.59 t 15_.48_ 90._2_3 t _6.4_8 - 65.161.3.68 M. vaccae ~_._..._~_ 68 ~89 t 4 3g ~_~._~-_-_57.91 t 7.92yy~~- 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.

AUTOCLAVED M YACCAE 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 wg 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 51 Chromium at 2 ~Ci per 104 macrophages. The macrophages were then cultured with CD8 T cells overnight (16 hours) at killer to target ratios of 30:1. Specif c killing was detected by the release of 5 ~ Chromium and expressed as specific lysis, calculated as in Example 5.
The production of IFNw 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 °C. Wells were blocked with PBS
containing 0.2% Tween 20 for 1 hour at room temperature. The plates were then washed four times in PBS 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 ~tg/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 10% (v/v) HCI. 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.

cells from naive or non-immunized mice do produce IFN~ when cocultured with infected macrophages. The amount of IFNw produced in coculture was greater with CD8 T
cells derived from M. vaccae immunized mice.

WO 99!32634 PCTMZ98/00189 EFFECT WITH M. TUBERCULOSIS INFECTED
AND UNINFECTED MACROPHAGES
Specific Lysis IFN-ry (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 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 °C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37 °C for one day. The medium was then centrifuged (leaving the bulk of the cells) and filtered through a 0.45 Eun 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 0.45Nxn membrane. The culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell which contained a 3kDa molecular weight cut-off (MWCO) membrane.
The pressure was maintained at 50 psi using nitrogen gas. The culture filtrate was repeatedly concentrated by membrane filtration and diluted with water until the conductivity of the sample was less than 1.0 mS. This procedure reduced the 201 volume to approximately 50 ml. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA).
The desalted culture filtrate was fractionated by ion exchange chromatography on a column of Q-Sepharose (Pharmacia Biotech, Uppsala, Sweden) (16 X 100 mm) equilibrated with l OmM Tris HCl buffer pH 8Ø 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 lcDa 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-S.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, 5 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% {v/v) trifluoroacetic acid ('TFA). The flow-rate was 2.0 ml/min and the HPLC eluent was monitored at 220 nm.
Fractions containing polypeptides were 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% (v/v) of acetonitrile in 0.05% (v/v) TFA at a flow-rate of 1.0 mUmin. The column eluent was 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. K., Nature 277:680-685, 1970).
The polypeptide fractions which were shown to contain significant contamination were further purified using a Mono Q column (Pllarmacia Biotech) 10 micron particle size (5 x 50 mm) or a Vydac biphenyl 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 NaCI in 10 mM Tris HCl pH 8Ø From a Vydac biphenyl 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 1 S%
polyacryiamide gel as described above.
For sequencing, the polypeptides were individually dried onto Biobrene"r' (Perkin ElmerlApplied 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% (v/v) trifluoroacetic acid with a gradient of acetonitrile containing 0.05% (v/v) TFA (1%/min). 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 and internal sequences for GVc-1 are shown in SEQ ID NOS: 1, 2 and 3, respectively; the N-terminal sequence for GVc-2 is shown in SEQ ID NO: 4; internal sequences for GVc-7 are shown in SEQ ID NOS: 5-8; internal sequences for GVc-13 are shown in SEQ ID
NOS: 9-11;
internal sequence for GVc-20 is shown in SEQ ID NO: 12; and N-terminal and internal sequences for GVc-22 are shown in SEQ ID NO: 56-59, respectively. Each of the internal peptide sequences provided herein begins with an amino acid residue which is assumed to exist in this position in the polypeptide, based on the known cleavage specificity of cyanogen bromide (Met) or Lys-C (Lys).
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)-1-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 sequences. 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, fi~actions comprising a mixture of antigens from the Vydac C4 HPLC purification step previously described were fractionated by preparative SDS-PAGE on a polyacrylamide gel. The samples were dissolved in non-reducing sample buffer and applied to the gel. The separated proteins were transferred to a PVDF membrane by electroblotting in 10 mM CAPS buffer, pH 11 containing 10%
(v/v) methanol. The transferred protein bands were identified by staining the PVDF
membrane with Coomassie blue. Regions of the PVDF membrane containing the most abundant polypeptide species were cut out and directly introduced into the sample cartridge of the Perkin Elmer/Applied BioSystems Procise 492 protein sequencer. Protein sequences were determined as described above. The determined N-terminal sequences for GVc-12, GVc-14, GVc-15, GVc-17 and GVc-19 are provided in SEQ ID NOS: 16-20, respectively.
,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 pETl6, 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.

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 MPT70 gene sequence. Using these primers, a 310 by 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 by fragment was amplified, cloned into plasmid pBluescript II
SK+ and sequenced (SEQ ID NO: 150) following standard procedures (Sambrook et al.
Ibicl). 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 respectively.
For expression and purification, the genes encoding GV 1/83, GV 1/70, GVc-13, GVc-14 and GV-22B were sub-cloned into the expression vector pETl6 (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% (v/v) autologous serum, penicillin (60 ~tg/ml), streptomycin (100 ~g/ml), and glutamine (2 mM).

After 3 days, 50 pl 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 pg/ml phosphate-buffered saline (PBS) for 4 hours at 4 °C. Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. The plates were then washed four times in PBS/0.2% Tween 20, and samples diluted 1:2 in culture medium in the ELISA plates were incubated overnight at room temperature. The plates were again washed, and a biotinylated polyclonal rabbit anti-human IFN~y serum (Endogen), 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 A
(Vector Laboratories, Burlingame, CA) was added at a 1:4,000 dilution in PBS. After a furtber 1 hour incubation at room temperature, the plates were washed and orthophenylenediamine (OPD) substrate added. The reaction was stopped after 10 min with 10% (v/v) HCI. 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-r 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.

wo 99r~zs~a Pcrn~z9sroois9 Antigen ProliferationIFN-r GVc-1 ++ +/-GVc-2 + ++

GVc-7 +/- -GVc-13 + ++

GVc-14 ++ +

GVc-1 + +
S

GVc-20 + +

PURIFICATION AND CHARACTERISATION OF POLYPEPTIDES
FROM M VACCAE CULTURE FILTRATE BY

M. vaccae soluble proteins were isolated from culture filtrate using 2-dimensional polyacrylamide gel electrophoresis as described below. Unless otherwise noted, all percentages in the following example are weight per volume.
M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 at 37 °C. M.
tuberculosis strain H37Rv (ATCC number 27294) was cultured in sterile Middlebrook 7H9 medium with Tween 80 and oleic acid/albumin/dextrose/catalase additive (Difco Laboratories, Detroit, Michigan). The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37 °C for one day.
The medium was then centrifuged (leaving the bulk of the cells) and filtered through a 0.45 ~.m filter into sterile bottles. The culture filtrate was concentrated by lyophilisation, and redissolved in MilliQ
water. A small amount of insoluble material was removed by filtration through a 0.45 ~m membrane filter.

The culture filtrate was desalted by membrane filtration in a 400 mI 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 x100 mm) equilibrated with l OmM TrisHCl buffer pH 8Ø Polypeptides were eluted with a linear gradient of NaCI from 0 to 1.0 M in the above buffer system. The column eluent was monitored at a wavelength of 280 nm.
The pool of polypeptides eluting from the ion exchange column were fractionated by preparative 2D gel electrophoresis. Samples containing 200-S00 ~tg of polypeptide were made 8M in urea and applied to polyacrylamide isoelectric focusing rod gels (diameter 2mm, length 150 mm, pH 5-7). 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 lOmM CAPS buffer pH 11 containing 10%
(v/v) methanol. The PVDF membranes were stained for protein with Coomassie blue.
Regions of PVDF containing polypeptides of interest were cut out and directly introduced into the sample cartridge of the Perkin Elmer/Applied BioSystems Procise 492 protein sequencer. The polypeptides were sequenced from the amino terminal end using traditional Edman chemistry.
The amino acid sequence was determined for each polypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards. Using these procedures, eleven polypeptides, designated GVs-1, GVs-3, GVs-4, GVs-5, GVs-6, GVs-8, GVs-9, GVs-10, GVs-11, GV-34 and GV-35 were isolated. The determined N-tensninal 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 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 GVs-5 were found to bear some similarity to the antigen 85A and 85B proteins from M.
leprae (SEQ ID NOS: 30 and 31, respectively), M. tuberculosis (SEQ ID NOS: 32 and 33, respectively) and M. bovis (SEQ ID NOS: 34 and 35, respectively), and the antigen 85C
proteins from M. leprae (SEQ ID NO: 36) and M. tuberculosis (SEQ ID NO: 37).

DNA CLONING STRATEGY FOR THE M. YACCAE

Probes for antigens 85A; 85B, and 85C were prepared by polymerase chain reaction (PCR) using degenerate oligonucleoddes (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 by 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 sequences 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.

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 similarly-digested ~, 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 cm2 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 ~C) 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 sequences and the T3 and T7 primers.
Sequence homologies were determined using BLASTN analysis of the EMBL
database. Two of these sets of clones were found to be homologous to M. bovis and M.
tuberculosis antigen 85A genes, each containing either the 5' or 3' ends of the M. vaccae gene (this gene was cleaved during library construction as it contains an internal BamHI site).
The remaining clones were found to contain sequences homologous to antigens 85B and 85C from a number of mycobacterial species. 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 85A, 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 BamHI 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 homology was performed as follows. The primers AD58 and AD59, described above, were used to amplify a 485 by 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 EcoRIlXhoI 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.

wo 99r~u~a rrrmrz9sroo><s9 Similar to the cloning of GV-4P, the amplification primers AD58 and AD59 were used to amplify a 485 by fragment from a clone containing GVs-5 (SEQ ID N0:42).
This fragment was cloned into the expression vector pETl6 and was called GV-SP. The determined nucleotide sequence and predicted amino acid sequence of GV-SP 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 pETl6 (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 Donoc Donor Donor Donor Donor . :

G9'7.005 .: ..697007 697008 697009 697010 697006 . ' :

ProlifIFN -ProlifIFN;.ProlifIFN.:ProlifIFN_Prolif1FN Prolif1FN
:~ .. . ::

: y. -r , , : Y
-r . . -,~ ;
, ~~

GVs- ++ + ND ND ++ ++ ++ ++ ++ +/- + ++

GV + +/- ND ND + ++ ++ ++ +/- +/- +/- ++
-GVs- ++ ++ ++ ++ ++ ++ + ++ ++ + + ++

DNA CLONING STRATEGY FOR M. VACCAE ANTIGENS
An 84 by 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: S-8). This probe was used to screen a M. vaccae genomic DNA library as described in Example 12. The determined nucleotide sequence for GVc-7 is shown in SEQ ID NO: 46 and predicted amino acid sequence in SEQ ID NO: 47. Comparison of these sequences with those in the databank revealed homology to a hypothetical 15.8 kDa membrane protein of M.
tuberculosis.
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 XhoI-site of pProEX HT prokaryotic expression vector (Gibco BRL) and submitted for sequencing to confirm the correct reading fi~ame 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 pETl6 (Novagen).
The ability of purified recombinant GVc-7 to stimulate proliferation of T-cells and stimulation of interferon-y production in human PBL, finm 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 poiypeptides having activity greater than four times above background.

.. .. ' .b. .f"~. .. .6'.
4....$ vv ~y."
~ ...... .
W,, ..

697005 ~ +/-697008 ++ +

697009 + +/-697010 +/- ++

A redundant oligonucleotide probe (SEQ ID NO 73; referred to as MPG15) was designed to the GVs-8 peptide sequence shown in SEQ ID NO: 26 and used to screen a M.
vaccae genomic DNA library using standard protocols. Two genomic clones containing genes encoding four different antigens was isolated. The determined DNA
sequences for GVs-8A (re-named GV-30), GVs-8B (re-named GV-31), GVs-8C (re-named GV-32) and GVs-8D (re-named GV-33) are shown in SEQ ID NOS: 48-51, respectively, with the corresponding amino acid sequences being shown in SEQ ID NOS: 52-55, respectively. GV-30 contains regions showing some similarity to known prokaryotic valyl-tRNA
synthetases;
GV-31 shows some similarity to M. smegmatis aspartate semialdehyde dehydrogenase; and GV-32 shows some similarity to the H. influenza folylpolyglutamate synthase gene. GV-33 contains an open reading frame which shows some similarity to sequences previously identified in M. tuberculosis and M. leprae, but whose function has not been identified.
The determined partial DNA sequence for GV-33 is provided in SEQ ID NO: 74 with the corresponding predicted amino acid sequence being provided in SEQ ID NO:
75.
Sequence data from the 3' end of the clone showed homology to a previously identified 40.6 kDa outer membrane protein of M. tuberculosis. Subsequent studies led to the isolation of a full-length DNA sequence for GV-33 (SEQ ID NO: 193). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 194.
The gene encoding GV-33 was amplified from M. vaccae genomic DNA with primers based on the determined nucleotide sequence. This DNA fragment was cloned into EcoRv-digested pBluescript II SK+ {Stratagene), and then transferred to pETl6 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.

Stimulatory Activity of Polypeptides Donor ProliferationInterferon-y 697005 ++ +
- _ 697006 .E+ ++
_ 697007 - +/-697008 +/- -G97009 +/- -G97010 +/- - ++

ISOLATION OF PROTEINS FROM DD-M. YACCAE
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 (Virus, Disa, USA). The material was then centrifuged {9000 rpm, 20 minutes, JA10 rotor, brake =
5). The resulting pellet was suspended in 100 ml of chloroform/methanol (2:1), incubated at room temperature for 1 hour, re-centrifuged, and the chloroform/methanol extraction repeated.
The pellet was obtained by centrifugation, dried in vacuo, weighed and resuspended in PBS at 50 mg (dry weight) per ml as delipidated M. vaccae.
Glycolipids were removed from the delipidated M. vaccae preparation by refluxing in 50% v/v ethanol for 2 hours. The insoluble material was collected by centrifugation (10,000 rpm, JA20 rotor, 15 mins, brake = 5). 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 = 5). Proteins were precipitated from the supernatant by adding an equal volume of acetone and incubating at -20 °C for 2 hours. The precipitated proteins were collected by centrifugation, washed in 50% 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 GV-45 is provided in SEQ ID NO: 187. A protein of approximate molecular weight of 14 kDa was designated GV-46. The determined N-terminal amino acid sequence of GV-46 is provided in SEQ ID NO: 208.
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 N0:76.
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 BamHl-lambda ZAP-Express (Stratagene) was screened using sera from cynomolgous monkeys immunised with M. vaccae secreted proteins prepared as described above. Positive plaques were identified using a colorimetric system. These plaques were re-screened until plaques were pure following standard procedures. pBK-CMV phagemid 2-1 containing an insert was excised from the lambda ZAP Express (Stratagene) vector in the presence of ExAssist helper phage following the manufacturer's protocol. The base sequence of the 5' end of the insert of this clone, hereinafter referred to as GV-27, was determined using Sanger sequencing with fluorescent primers on Perkin Elmer/Applied Biosystems Division automatic sequences. 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 pubiished by Kapur et al. (Arch. Pathol.
Lab. Med. 119 :131-138, 1995). The nucleotide sequence of the Kapur et al. fragment is shown in SEQ ID
NO: 79 and the predicted amino acid sequence in SEQ ID NO: 80.
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 HSP65.
Following the same protocol as for the isolation of GV-27, pBK-CMV phagemid 3-1 was isolated. The antigen encoded by this DNA was named GV-29. The determined nucleotide sequences of the 5' and 3' ends of the gene are provided in SEQ ID NOS: 163 and 164, respectively, with the predicted corresponding amino acid sequences being provided in SEQ

ID NOS: 165 and 166 respectively. GV-29 showed homology to yeast urea amidolyase. The determined DNA sequence for the full-length gene encoding GV-29 is provided in SEQ ID
NO: 198, with the corresponding predicted amino acid sequence in SEQ ID NO:
199. The DNA encoding GV-29 was sub-cloned into the vector pETl6 (Novagen, Madison, WI) for expression and purification according to standard protocols.

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'M and oleic acid/
albumin/dextrose/catalase additive (Difco Laboratories, Detroit, Michigan) at 37 °C and harvested under appropriate laboratory safety conditions. Cells were resuspended in 1 ml Trizol (Gibco BRL) and RNA
extracted according to the manufacturer's standard protocol.
Total M. tuberculosis and M. vaccae RNA was depleted of 16S and 23S ribosomal RNA (rRNA) by hybridisation of the total RNA fraction to oligonucleotides AD10 and AD11 (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.

The M. tuberculosis and M. vaccae cDNA was used as template for single-sided-specific PCR (3S-PCR). For this protocol, a degenerate oligonucleotide AD1 (SEQ ID
N0:85) was designed based on conserved leader sequences and membrane protein sequences.
After 30 cycles of amplification using primer AD1 as S'-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 and 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. (Jn1 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 by 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 by including an untranslated 5' region of 320 by containing putative -10 and -35 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 and EV25 (SEQ ID NO: 90 and 91) for expression cloning. The amplified DNA
fragment was cloned into pProEX HT prokaryotic expression system (Gibco BR.L) and expression in an appropriate E.coli host was induced by addition of 0.6 mM isopropylthio-(i-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 pETl6 (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 by Sall-BamHl 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
N0:92. To identify the gene, the radiolabelled insert of this subclone was used to probe a M.
vaccae genomic DNA library constructed in the Sall-site of lambda Zap Express (Stratagene) following standard protocols. A clone was identified of which 1342 by showed homology with the potd gene of E. coli. The potd homologue of M. vaccae was identified by sub-cloning 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 pETl6 (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 EV 101 and EV 102 (SEQ ID NOS: 167 and 168). The construct was designated GV-24B. The nucleotide sequence of GV-24B is provided in SEQ ID NO:

and the predicted amino acid sequence in SEQ ID NO: 170. This fragment was cloned into pETl6 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 PBIJ 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.

Donor Donor Donor Donor Donor Donor - .

697005 G9700~ 697007 697008 697009 G970I0 ProlifIFN-YProlif-IFN-yProlifIFN-YProlifIFN-yProlifIFN-YProlifIFN
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 by 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 by 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.

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 deternvned 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 foil-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 pETl6 (Novagen). GV-38A was amplified with primers KR11 and KR12 (SEQ ID NO:

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 KRI2, and the truncated GV-38B gene with KR21 and KR14. The determined nucleotide sequences of truncated GV38A and GV-38B are shown in SEQ ID NO: 132 and 133, respectively, with the corresponding predicted amino acid sequences being shown in SEQ ID
NO: 134 and 135, respectively. Extended DNA sequences for truncated GV-38A and GV-38B are provided in SEQ ID NO: 175 and 176, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO: 177 and 178, respectively.

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, alI percentages in the following example are weight per volume.
M. vaccae (ATCC Number 15483) was cultured in 2501 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 p.m filter. The sterile filtrate was concentrated by ultrafiltration over a 10 kDa molecular weight cut-off membrane.
Proteins were isolated from the concentrated culture filtrate by precipitation with 10%
trichloroacetic acid. The precipitated proteins were re-dissolved in 100 mM
Tris.HCl pH 8Ø
and re-precipitated by the addition of an equal volume of acetone. The acetone precipitate was dissolved in water, and proteins were re-precipitated by the addition of an equal volume of chloroform:methanol 2:1 (v/v). The chloroform:methanol precipitate was dissolved in water, and the solution was freeze-dried.
The freeze-dried protein was dissolved in iso-electric focusing buffer, containing 8 M
deionised urea, 2% Triton X-100, 10 mM dithiothreitol and 2% ampholytes (pH
2.5 - 5.0).
The sample was fractionated by preparative iso-electric focusing on a horizontal bed of Ultrodex gel at 8 watts constant power for 16 hours. Proteins were eluted from the gel bed fractions with water and concentrated by precipitation with 10%
trichloroacetic acid.

Pools of fractions containing proteins of interest were identified by analytical polyacrylamide gel electrophoresis and fractionated by preparative polyacrylamide gel electrophoresis. 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 40%
acetonitrile/0.1 M 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 N-terminal sequences for these polypeptides are shown in SEQ ID NOS: 101-105, respectively.
Subsequent studies led to the isolation of a 5', middle fragment and 3' DNA
sequence for GV-42 (SEQ ID NO: 136, 137 and 138, respectively}. The corresponding predicted amino acid sequences are provided in SEQ ID NO: 139, 140 and 141, respectively.
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.

Murine polyclonal antisera were prepared against GV-40 and GV-44 following standard procedures. These antisera were used to screen a M. vaccae genomic DNA library consisting of randomly sheared DNA fragments. Clones encoding GV-40 and GV-44 were identified and sequenced. The determined nucleotide sequence of the partial gene encoding GV-40 is provided in SEQ ID NO: 183 and the predicted amino acid sequence in SEQ ID
N0:184. 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-3-phosphate dehydrogenase.

ISOLATION OF THE DD-M. YACCAE 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 °C 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 °C for 1 hr. The precipitated proteins were collected by centrifugation, dissolved in reducing sample buffer, and fractionated by preparative SDS-polyacrylamide gel electrophoresis. The separated proteins were electroblotted onto PVDF
membrane in 10 mM CAPS/0.01% SDS pH 11.0, and 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 lcDa, designated GV-45, was isolated. The determined N-terminal sequence for GV-45 is provided in SEQ ID NO: 187. From the same experiments, a protein of approximate molecular weight of 14 kDa, designated GV-46, was obtained. The determined N-terminal amino acid sequence of GV-46 is provided in SEQ ID NO: 208. GV-46 is homologous to the highly conserved mycobacterial host integration factor of M.
tuberculosis and M. smegmatis.
From the amino acid sequence of GV-45, degenerate oligonucleotides KR32 and KR33 (SEQ ID NOS: 188 and 189, respectively) were designed. A 100 by fragment was amplified, cloned into plasmid pBluescript II SK+ (Stratagene, La Jolla, CA) and sequenced (SEQ ID N0:190) following standard procedures (Sambrook, Ibia~. 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.

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 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.

Immunogenic Properties of GV proteins: Proliferation Proliferation (cpm) GV protein Dose of GV
protein used in vitro (p,g/ml) ~__.____ 50 2 .08 GV-1/70 31,550 t 19,058 t 2,4495,596 f 686 GV- 18,549 ~ 23,932 ~ 1,964~
1/83 2,716 11,787 t 1,128 .. _ _ ___ _ 6,379 ~ 319 ____.
.._ 34,751 t 4,590 f 1,042 GV-3 1,382 G_V-_4P__ 26,4_60 ~ _ 1.0,370._~A667_~._6,685 t _ 1,8_77_ _T_ 673~,_ ~~ ~
~. "~

GV-5 42,418 t 23,902 ~ 2,31213,973 t 772 2,444 : 8 340 ~ 725 GV-SP 35 691 ~ 14 457 f 1 ._._.._._____.._._.._._._159 185 .._...._._.____.
...._._... ~ _..._..__.-......_.._-_.
> >
_-.._ .~...__._._.________.._._._......_~___..__ -_._..._._.._.
_..

GV-7 38,686 f 22,074 t 3,69815,906 t 1,687 GV-9 30,599 t 15,260 f 2,764~ 4,531 t 1,240 _-...~______.___~..- -.__ .__.~____ __.__..~~.____~_...~__~.___..
GV-13 15,296 ~ 7,163 t 833 ' 3,701 t 243__ 2,006 GV-14 27,754 t 13,001 t 3,273~ 9,897 ~ 2,833 ___.____....___1,872 -___.__.. _~ ~___._____.__._-.._.___._._.....__._._.._:.
.~_.___.._..~
___._.__._.

GV-14B 10,761 ~ 5,075 t 1,470 ' 2,341 t 289 GV-22B 3,199 ~ 771 3,255 t 386 1,841 t 318 .._ -_... __.-_._.__.__ ' __~___ . ~_.._.-.-~
__-___ _ GV-23 35,598 ~ 15,423 t 2,858~ 7,393 f 2,188 1,330 . GV-24B! 43,678 1_2,19030,307 t_1.,533~_~ _15,375.,1, 2,594 GV-27 65 t 3,300 16,329 t 1,7946,107 ~ 1,773 18,1 _GV~-27A___._,_ 6,860 t 746 _4,295 t 780 23,723 t _,. _ 850 ~

GV-27B 31,602 t 29,468 f 3,867i 30,306 f 1,912 1,939 _._._ Gu,29__..2_0,034 t _.__ 8,107 _ ' .___ 2,982 3,328 t 488_......._..t 897___......

GV-33 41,529 f 27,529 ~ 1,2388,764 f 256 1,919 GV-35 29,163. t 9,968 f 314 ~ 1,626. 406 2,693"_ ~ _.

- 28,971 t ' 17,396 t ~ 8,060 t 810 GV-38AP 4,499 878 GV-38BP.._.__19,746 t _.11,732..1. _~ 6,264 t, 245 3,207 875_ GV-40P 25,185 ~ ~ 19,292 t 10,883 ~ 893 2,877 2,294 V-41B 24,646 ~ ' 12,627 f .5,772 ~ 1,041 G 2,714 3,622 _.

_ 25,486 t 20,591 f 2,021i 13,789 ~ 775 __ 3,029 _ GV-42 ' GV-44 2,684 t 1,9953,577 ~ 1,725 ' 1,499 f 959 _...___.._..___ _____.....__.__.__...-_-__._.....____....__...__..._..___._...__._..._;._~_._-.__~_.___ GV-45 9,554 f 482 3,683 t 1,127 ~ 1,497 ~ 199 WO 99/32634 PCf/NZ98/00189 Immunogenic properties of GV proteins: IFNy production IFNy (ng/ml) GV protein Dose of GV
protein used in vitro (~tg/ml) r GV-1/70 24.39 t 6.66 ; 6.19 t 1.42_1.90 f 0.53 _,. GV-1/83 11.34_t 5.46 536 t_ 1.34 2.73 t 1.55 _ _~_ __... GV-3 - 3.46 t 0.301.57 t 0.04 _ __ _..._..._._.___...r..,_. ~ _._..
_.___..._.._.~.__.__._._...___..._not detectable ,__GV-4P _ ~___ _-_ _._ .____.._.__.
GV-5 6.48 f 0.37_~,._._ 3..00 x._0_52___.____..._-__...
4.08 f 1.41 ___ .._. 1_.38 ~ 6.10 ~ 2.72 _t 0.50 2.35 ~ 0.40 __ GV~SP 34.98 t 15.26~ 9.95 t_3.42 _ ._ _ _ 5.68 f 0.79 GV-7 33.52 t 3.08 _~ 9.60 f 1.74 GV-9 -_ -.._.__._._~~ 25.47 t 4.14___.._.__.__.__-........~_._.
_...._____...-__-__92.27_t _-____.-~_.___......_30.46 GV-13 45_50 ._.. _..._g8.54,f t 1.77 _ 11.60 t 2.89 16.48 1.46 t 0.62 2.04 f 0.58_ GV-14 8.28 t 1.56 3.19 x_0_.56 0.94 t 0 .24 GV-14B not detectable_ _ GV-22B _ not detectable.-.._ _.. not detectablenot detectableT not detectable_ GV-23 ..__~_._._ ,__.. __. no_t detectable _ 30.70 t 4.48 ___.__._ 59.67 t 14.88 9.17 t ~~1.5~1 __ GV-24B..-_y._6.76 t _ 1.97 t 0.03 0.58__ ' 3.20 f 0_50 GV-27w __ 72_22 t._11~ _.__ GV_27A _1.4..~~ _.._ 3,0_86. _.__._21-38 GV-27B 425 t 2_32 10.55_ ~ 3-12 -_~_~( .~~_..1.51 -.not detectable 87.98 ~ 15.78t_0~73 21.49 t 5.60 ' 44.43 t 8.70 GV-29 7.56 t 2.58 __ not detectable 1.22 t 0.56 __ GV-33-. _.._ 7.71 _ 1_52 t 0;24 t 0.26 8.44 f 2.35_ ' GV-38AP 23.49 t 5_89 _ ,1.._ ~ 8.87 ~ 1.62 4.17 f 1.72 GV-38BP 5.30 t 0.95 3.10 t 1:19 1.91 t 1.01 GV-40P 15.65 ~ 7.89 10.58 t 1.31 _ .__.._.....__._.___...-.. ____ _...__ ' 3.57 ~ 1.53 GV-41 B __ .__......_. _.__~ _-16.73 ~ 1_ . _~. 5.08 2.13 ~ 1.10 61 -~ t 1.08 - GV-42_, __ 95.97 ~ _ ...... 30 23.86 ~; ___,_ 52.88 .0 f 6 f 5. 8.94 GV-44 not detectable_ _ ~ _ _ _ not detectable_ ~ ~~ _ ' not detectable 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, a(3T cells or y8T cells, in combination with monoclonal antibodies against CD69, followed by flow cytometry analysis Table 23 shows the percentage of a~iT cells, y8T cells and NK cells expressing 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 a~iT cells.

WO 99!32634 PCT/NZ98/00189 Stimulation of CD69 Expression a(3T cells y8T cells NK cells Control .8 6.2 4.g 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 S.8 10.0 46.8 GV-27 S.S 4.4 13.3 GV-27A S.S 4.4 13.3 GV-27B 4.4 2.8 7.1 GV-4S 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 a(3T 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.

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: I62) or GV-45 (SEQ ID NO:
201) for 24 hours. Culture supernatants were harvested and tested for the production of IL-1 (3, TNF-a, 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 (3, 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 GV-45 were found to stimulate the production of IL-lei, TNF-a and IL-12. Figs.
l0A-C show the stimulation of IL-1 Vii, TNF-a and IL-12 production, respectively, in human PBMC
(determined as described above) by varying concentrations of GV-23 and GV-45.
Figs. 11A-D show the stimulation of IL-1(3, 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-1 ~i 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 cell$ were stimulated with 20 ug/ml of either heat-killed M. vaccae cells, DD-M. vaccae, or recombinant GV-22B (SEQ ID NO: 145), GV-23 (SEQ ID NO:
89), GV-27 (SEQ ID NO: 160), GV27A (SEQ ID NO: 117), GV-27B (SEQ ID NO: 162) or GV-45 (SEQ ID NO: 201) for 48 hours. Stimulated cells were harvested and analyzed for up-regulation of CD40, CD80 and CD86 using 3 color flow cytometric analysis.
Tables 24, 25 and 26 show the fold increase in mean fluorescence intensity from control (non-stimulated cells) for dendritic cells, monocytes, and B cells, respectively.

Stimulation of CD40, CD80 and CD86Expression on Dendritic Cells CD40 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 1.0 GV-27B 2.6 1.1 1.1 GV-45 5.8 3.0 3.1 Stimulation of CD40, CD80 and CD86 Expression on Monocytes Control 0 0 0 Heat-killed M. 2.3 1.8 0.7 vaccae DD-M. vaccae 1.9 1.5 0.7 GV-22B 0.7 0.9 1.1 GV-23 2.3 1.5 0.7 GV-27 1.5 1.4 1.2 GV-27A 1.4 1.4 1.4 GV-27B 1.6 1.2 1.2 GV-45 1.6 1.2 1.0 Stimulation of CD40, CD80 and CD86 Expression on B Cells Control 0 0 0 Heat-killed M. 1.6 1.0 1.7 vaccae DD-M. vaccae 1.5 0.9 1.7 GV-22B 1.1 0.9 1.2 GV-23 1.2 1.1 1.4 GV-27 1.1 0.9 1.1 GV-27A 1.0 1.1 0.9 GV-27B 1.0 0.9 0.9 GV-45 1.2 1.1 1.3 As shown 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 GV-45.
The ability of GV-23 to stimulate CD40, CD80 and CD86 expression in dendritic cells was compared to that of the Thl-inducing adjuvants MPLfTDM/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.

Stimulation of CD83 Expression in Dendritic Cells Treatments %CD83-positive % CD80-positive dendritic cells dendritic cells Control 15 ~ 8 9 t 6.6 GV-23 35 ~ 13.2 24.7 ~ 14.2 LPS 36.3 t 14.8 27.7 ~ 13 Data = mean t 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 105 cells/well). After 3 days of incubation, (3H)-thymidine was added.
Cells were harvested 1 day later and the uptake of radioactivity was measured.
Fig. 13 shows the increase in uptake of (3H)-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.

SEQUENCE LISTING
<110> Tan, Paul L.J.
Watson, James D.
Visser, Elizabeth S.
Skinner, Margot A.
Prestidge, Ross L.
<120> Compositions Derived from Mycobacterium Vaccae and Methods for Their Use <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 3.0 <210> 1 <211> 25 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (7) . . . (7) <400> 1 Ala Pro Val Gly Pro Gly Xaa Ala Ala Tyr Val Gln Gln Val Pro Asp Gly Pro Gly Ser Val Gln Gly Met Ala <210> 2 <211> 10 <212> PRT
<213> Mycobacterium vaccae <220>
c221> UNSURE
<222> (2)...(2) <400> 2 Met Xaa Asp Gln Leu Lys Val Asn Asp Asp <210> 3 <211> 11 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (2)...(2) <400> 3 Met Xaa Pro Val Pro Val Ala Thr Ala Ala Tyr <210> 4 <211> 21 <212> PRT
c213> Mycobacterium vaccae <400> 4 Thr Pro Ala Pro Ala Pro Pro Pro Tyr Val Asp His Val Glu Gln Ala Lys Phe Gly Asp Leu <210> 5 <211> 29 <212> PRT
<213> Mycobacterium vaccae <220>
c221> UNSURE
<222> (25)...(25) <400> 5 Met Gln Ala Phe Asn Ala Asp Ala Tyr Ala Phe Ala Lys Arg Glu Lys Val Ser Leu Ala Pro Gly Val Pro Xaa Val Phe Glu Thr <210> 6 <211> 21 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (6) . . . (6) <400> 6 Met Ala Asp Pro Asn Xaa Ala Ile Leu Gln Val Ser Lys Thr Thr Arg Gly Gly Gln Ala Ala <210> 7 <211> 11 <212> PRT
<213> Mycobacterium vaccae <400> 7 Met Pro Ile Leu Gln Val Ser Gln Thr Gly Arg <210> 8 <211> 14 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (2)...{2) <221> UNSURE
<222> (6)...{6) <400> 8 Met Xaa Asp Pro Ile Xaa Leu Gln Leu Gln Val Ser Ser Thr <210> 9 <211> 16 <212> PRT
<213> Mycobacterium vaccae <400> 9 Lys Ala Thr Tyr Val Gln Gly Gly Leu Gly Arg Ile Glu Ala Arg Val <210> 10 <211> 9 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (2)...(2) <400> 10 Lys Xaa Gly Leu Ala Asp Leu Ala Pro <210> 11 <211> 14 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> {12)...{12) <223> Residue can be either Glu or Ile <221> UNSURE
<222> (2) . . . (2) <400> 11 Lys Xaa Tyr Ala Leu Ala Leu Met Ser Ala Val Xaa Rla Ala <210> 12 <211> 11 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (10)...{10) <400> 12 Lys Asn Pro Gln Val Ser Asp Glu Leu Xaa Thr <210> 13 <211> 21 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (9) .. . (9) <400> 13 Ala Pro Ala Pro Ala Ala Pro Ala Xaa Gly Asp Pro Ala Ala Val Val Ala Ala Met Ser Thr <210> 14 <211> 15 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (5)...(5) <400> 14 Glu Ala Glu Val Xaa Tyr Leu Gly Gln Pro Gly Glu Leu Val Asn <210> 15 <211> 15 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (2) . . . (2) <223> Residue can be either Gly or Ala <221> UNSURE
<222> (15)...(15) <223> Residue can be either Pro or Ala <221> UNSURE
<222> (7) . . . (7) <400> 15 Ala Xaa Val Val Pro Pro Xaa Gly Pro Pro Ala Pro Gly Ala Xaa <210> 16 <211> 15 <212> PRT
<213> Mycobacterium vaccae <400> 16 Ala Pro Ala Pro Asp Leu Gln Gly Pro Leu Val Ser Thr Leu Ser <210> 17 <211> 25 <212> PRT
<213> Mycobacterium vaccae <400> 17 Ala Thr Pro Asp Trp Ser Gly Arg Tyr Thr Val Val Thr Phe Ala Ser Asp Lys Leu Gly Thr Ser Val Ala Ala <210> is <211> 25 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (15)...(15) <223> Residue can be either Ala or Arg <221> UNSURE
<222> (23)...(23) <223> Residue can be either Val or Leu <221> UNSURE
<222> (16)...(16) <400> 18 Ala Pro Pro Tyr Asp Asp Arg Gly Tyr Val Asp Ser Thr Ala Xaa Xaa Ala Ser Pro Pro Thr Leu Xaa Val Val <210> 19 <211> B
<212> PRT
<213> Mycobacterium vaccae <400> 19 Glu Pro Glu Gly Val Ala Pro Pro <210> 20 <211> 25 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (21)...(22}
<400> 20 Glu Pro Ala Gly Ile Pro Ala Gly Phe Pro Asp Val Ser Ala Tyr Ala Ala Val Asp Pro Xaa Xaa Tyr Val Val <210> 21 <211> 15 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (7) . . . (7) <400> 21 Ala Pro Val Gly Pro Gly Xaa Ala Ala Tyr Val Gln Gln Val Pro <210> 22 <211> 15 <212> PRT
<213> Mycobacterium vaccae <400> 22 Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Met Val Pro Ser <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 Ser Met Gly <210> 24 <211> 15 <212> PRT
<213> Mycobacterium vaccae <400> 24 Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Asp Val Phe Ser <210> 25 <211> 14 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (1) . . . (2) <400> 25 Xaa Xaa Thr Gly Leu His Arg Leu Arg Met Met Val Pro Asn <210> 26 <211> 20 <212> PRT

<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (16)...(16) <223> Residue can be either Ser or Val <221> UNSURE
<222> (17) . . . (17) <223> Residue can be either Gln or Val <400> 26 Val Pro Ala Asp Pro Val Gly Ala Ala Ala Gln Ala Glu Pro Ala Xaa Xaa Arg Ile Asp <210> 27 <211> 14 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (4)...(4) <223> Residue can be either Tyr or Pro <221> UNSURE
<222> (8)...(8) <223> Residue can be either Val or Gly <221> UNSURE
<222> (9)...(9) <223> Residue can be either Ile or Tyr <221> UNSURE
<222> (3) . .. (3) <400> 27 Asp Pro Xaa Xaa Asp Ile Glu Xaa Xaa Phe Ala Arg Gly Thr <210> 28 <211> 15 <212> PRT
<213> Mycobacterium vaccae <400> 28 Ala Pro Ser Leu Ser Val Ser Asp Tyr Ala Arg Asp Ala Gly Phe <210> 29 <211> 16 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (2)...(2) <223> Residue can be either Leu or Pro <221> UNSURE
<222> (1) . . . (1) <221> UNSURE
<222> (5)...(5) <221> UNSURE
<222> (7)...(7) <221> UNSURE
<222> (10)...(10) <400> 29 Xaa Xaa Leu Ala Xaa Ala Xaa Leu Gly Xaa Thr Val Asp Ala Asp Gln <210> 30 <211> 330 <212> PRT
<213> Mycobacterium leprae <400> 30 Met Lys Phe Val Asp Arg Phe Arg Gly Ala Val Ala Gly Met Leu Arg Arg Leu Val Val Glu Ala Met Gly Val Ala Leu Leu Ser Ala Leu Ile Gly Val Val Gly Ser Ala Pro Ala Glu Ala Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Asn Gly Gly Ala Asn Ser Pro Ala Leu Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Phe Ser Gly Trp Asp Ile Asn Thr Thr Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Ile Ser Val Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Glu Tyr Leu Gln Ser Asn Lys Gln Ile Lys Pro Thr Gly Ser Ala Ala Val Gly Leu Ser Met Ala Gly Leu Ser Ala Leu Thr Leu Ala Ile Tyr His Pro Asp Gln Phe Ile Tyr Val Gly Ser Met Ser Gly Leu Leu Asp Pro Ser Asn Ala Met Gly Pro Ser Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Ser Thr Asp Pro Ala Trp Lys Arg Asn Asp Pro Thr Val Asn Val Gly Thr Leu Ile Ala Asn Asn Thr Arg Ile Trp Met Tyr Cys Gly Asn Gly Lys Pro Thr Glu Leu Gly Gly Asn Asn Leu Pro Ala Lys Leu Leu Glu Gly Leu Val Arg Thr Ser Asn Ile Lys Phe Gln Asp Gly Tyr Asn Ala Gly Gly Gly His Asn Ala Val Phe Asn Phe Pro Asp Ser Gly Thr His Ser Trp Glu Tyr Trp Gly Glu Gln Leu Asn Asp Met Lys Pro Asp Leu Gln Gln Tyr Leu Gly Ala Thr Pro Gly Ala <210> 31 <211> 327 <212> PRT
<213> Mycobacterium leprae <400> 31 Met Ile Asp Val Ser Gly Lys Ile Arg Ala Trp Gly Arg Trp Leu Leu Val Gly Ala Ala Ala Thr Leu Pro Ser Leu Ile Ser Leu Ala Gly Gly Ala Ala Thr Ala Ser Ala Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Glu Ala Met Gly Arg Thr Ile Lys Val Gln Phe Gln Asn Gly Gly Asn Gly Ser Pro Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn Thr Ser Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Val Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Thr Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Lys Trp Leu Ser Ala Asn Arg Ser Val Lys Ser Thr Gly Ser Ala Val Val Gly Leu Ser Met Ala Gly Ser Ser Ala Leu Ile Leu Ala Ala Tyr His Pro Asp Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu Met Asp Ser Ser Gln Gly Ile Glu Pro Gln Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Pro Asn Asp Pro Ala Trp Gln Arg Asn Asp Pro Ile Leu Gln Ala Gly Lys Leu Val Ala Asn Asn Thr His Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro Ser Glu Leu Gly Gly Thr Asn Val Pro Ala Glu Phe Leu Glu Asn Phe Val His Gly Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Gly Ala Gly Gly His Asn Ala Val Phe Asn Leu Asn Ala Asp Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Pro Asp Leu Gln Asn Thr Leu Met Ala Val Pro Arg Ser Gly <210> 32 <211> 338 <212> PRT
<213> Mycobacterium tuberculosis <400> 32 Met Gln Leu Val Asp Arg Val Arg Gly Ala Val Thr Gly Met Ser Arg Arg Leu Val Val Gly Ala Val Gly Ala Ala Leu Val Ser Gly Leu Val Gly Ala Val Gly Gly Thr Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Ala Asn Ser Pro Ala Leu Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Phe Ser Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gln Ser Gly Leu Ser Val Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Gln Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gly Trp Leu Gln Ala Asn Arg His Val Lys Pro Thr Gly Ser Ala Val Val Gly Leu Ser Met Ala Ala Ser Ser Ala Leu Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Val Tyr Ala Gly Ala Met Ser Gly Leu Leu Asp Pro Ser Gln Ala Met Gly Pro Thr Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ser Asp Met Trp Gly Pro Lys Glu Asp Pro Ala Trp Gln Arg Asn Asp Pro Leu Leu Asn Val Gly Lys Leu Ile Ala Asn Asn Thr Arg Val Trp Val Tyr Cys Gly Asn Gly Lys Pro Ser Asp Leu Gly Gly Asn Asn Leu Pro Ala Lys Phe Leu Glu Gly Phe Val Arg Thr Ser Asn Ile Lys Phe Gln Asp Ala Tyr Asn Ala Gly Gly Gly His Asn Gly Val Phe Asp Phe Pro Asp Ser Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Pro Asp Leu Gln Arg Ala Leu Gly Ala Thr Pro Asn Thr Gly Pro Ala Pro Gln Gly Ala <210> 33 <211> 325 <212> PRT
<213> Mycobacterium tuberculosis <400> 33 Met Thr Asp Val Ser Arg Lys Ile Arg Ala Trp Gly Arg Arg Leu Met Ile Gly Thr Ala Ala Ala Val Val Leu Pro Gly Leu Val Gly Leu Ala Gly Gly Ala Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp 65 70 75 g0 Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Ile Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gln Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser Ala Ala Ile Gly Leu Ser Met Ala Gly Ser Ser Ala Met Ile Leu Ala Ala Tyr His Pro Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu Leu Asp Pro Ser Gln Gly Met Gly Pro Ser Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Glu Arg Asn Asp Pro Thr Gln Gln Ile Pro Lys Leu Val Ala Asn Asn Thr Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu Gly Gly Ala Asn Ile Pro Ala Glu Phe Leu Glu Asn Phe Val Arg Ser Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn Ala Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu Gln Ser Ser Leu Gly Ala Gly <210> 34 <211> 338 <212> PRT
<213> Mycobacterium bovis <400> 34 Met Gln Leu Val Asp Arg Val Arg Gly Ala Val Thr Gly Met Ser Arg Arg Leu Val Val Gly Ala Val Gly Ala Ala Leu Val Ser Gly Leu Val Gly Ala Val Gly Gly Thr Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Ala Asn Ser Pro Ala Leu Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Phe Ser Gly Trp Asp Ile g5 90 95 Asn Thr Pro Ala Phe Glu Trp Tyr Asp Gln Ser Gly Leu Ser Val Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Gln Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gly Trp Leu Gln Ala Asn Arg His Val Lys Pro Thr Gly Ser Ala Val Val Gly Leu Ser Met Ala Ala Ser Ser Ala Leu Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Val Tyr Ala Gly Ala Met Ser Gly Leu Leu Asp Pro Ser Gln Ala Met Gly Pro Thr Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ser Asp Met Trp Gly Pro Lys Glu Asp Pro Ala Trp Gln Arg Asn Asp Pro Leu Leu Asn Val Gly Lys Leu Ile Ala Asn Asn Thr Arg Val Trp Val Tyr Cys Gly Asn Gly Lys Pro Ser Asp Leu Gly Gly Asn Asn Leu Pro Ala Lys Phe Leu Glu Gly Phe Val Arg Thr Ser Asn Ile Lys Phe Gln Asp Ala Tyr Asn Ala Gly Gly Gly His Asn Gly Val Phe Asp Phe Pro Asp Ser Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Pro Asp Leu Gln Arg Ala Leu Gly Ala Thr Pro Asn Thr Gly Pro Ala Pro Gln Gly Ala <210> 35 <211> 323 <212> PRT
<213> Mycobacterium bovis <400> 35 Met Thr Asp Val Ser Arg Lys Ile Arg Ala Trp Gly Arg Arg Leu Met Ile Gly Thr Ala Ala Ala Val Val Leu Pro Gly Leu Val Gly Leu Ala Gly Gly Ala Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Ile Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Leu Leu Thr Ser Glu Leu Pro Gln Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser Ala Ala Ile Gly Leu Ser Met Ala Gly Ser Ser Ala Met Ile Leu Ala Ala Tyr His Pro Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu Leu Asp Pro Ser Gln G1y Met Gly Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Glu Arg Asn Asp Pro Thr Gln Gln Ile Pro Lys Leu Val Ala Asn Asn Thr Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu Gly Gly Ala Asn Ile Pro Ala Glu Phe Leu Glu Asn Phe Val Arg Ser Ser Asn Leu Lys Phe Gln Asp Ala Tyr Lys Pro Ala Gly Gly His Asn Ala Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu Gln Ser Ser Leu Gly Ala Gly <210> 36 <211> 333 <212> PRT
<213> Mycobacterium leprae <400> 36 Met Lys Phe Leu Gln Gln Met Arg Lys Leu Phe Gly Leu Ala Ala Lys Phe Pro Ala Arg Leu Thr Ile Ala Val Ile Gl~r Thr Ala Leu Leu Ala Gly Leu Val Gly Val Val Gly Asp Thr Ala Ile Ala Val Ala Phe Ser Lys Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly His Asp Ile Lys Ile Gln Phe Gln Gly Gly Gly Gln His Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Glu Asp Tyr Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Glu Tyr Tyr His Ser Gly Leu Ser Val Ile Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asn Trp Tyr Gln Pro Ser Gln Gly Asn Gly Gln His Tyr Thr Tyr Lys Trp Glu Thr Phe Leu Thr Gln Glu Met Pro Ser Trp Leu Gln Ala Asn Lys Asn Val Leu Pro Thr Gly Asn Ala Ala Val Gly Leu Ser Met Ser Gly Ser Ser Ala Leu Ile Leu Ala Ser Tyr Tyr Pro Gln Gln Phe Pro Tyr Ala Ala Ser Leu Ser Gly Phe Leu Asn Pro Ser Glu Gly Trp Trp Pro Thr Met Ile Gly Leu Ala Met Asn Asp Ser Gly Gly Tyr Asn Ala Asn Ser Met Trp Gly Pro Ser Thr Asp Pro Ala Trp Lys Arg Asn Asp Pro Met Val Gln Ile Pro Arg Leu Val Ala Asn Asn Thr Arg Ile Trp Val Tyr Cys Gly Asn Gly Ala Pro Asn Glu Leu Gly Gly Asp Asn Ile Pro Ala Lys Phe Leu Glu Ser Leu Thr Leu Ser Thr Asn Glu Ile Phe Gln Asn Thr Tyr Ala Ala Ser Gly Gly Arg Asn Gly Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp Pro Tyr Trp Asn Gln Gln Leu Val Ala Met Lys Pro Asp Ile Gln Gln Ile Leu Asn Gly Ser Asn Asn Asn Ala <210> 37 <211> 340 <212> PRT
<213> Mycobacterium tuberculosis <400> 37 Met Thr Phe Phe Glu Gln Val Arg Arg Leu Arg Ser Ala Ala Thr Thr Leu Pro Arg Arg Val Ala Ile Ala Ala Met Gly Ala Val Leu Val Tyr Gly Leu Val Gly Thr Phe Gly Gly Pro Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Vai Pro Ser Ala Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Gly Gly Gly Pro His Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Glu Tyr Tyr Gln Ser Gly Leu Ser Val Ile Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Thr Asp Trp Tyr Gln Pro Ser Gln Ser Asn Gly Gln Asn Tyr Thr Tyr Lys Trp Glu Thr Phe Leu Thr Arg Glu Met Pro Ala Trp Leu Gln Ala Asn Lys Gly Val Ser Pro Thr Gly Asn Ala Ala Val Gly Leu Ser Met Ser Gly Gly Ser Ala Leu Ile Leu Ala Ala Tyr Tyr Pro Gln Gln Phe Pro Tyr Ala Ala Ser Leu Ser Gly Phe Leu Asn Pro Ser Glu Gly Trp Trp Pro Thr Leu Ile Gly Leu Ala Met Asn Asp Ser Gly Gly Tyr Asn Ala Asn Ser Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Lys Arg Asn Asp Pro Met Val Gln Ile Pro Arg Leu Val Ala Asn Asn Thr Arg Ile Trp Val Tyr Cys Gly Asn Gly Thr Pro Ser Asp Leu Gly Gly Asp Asn Ile Pro Ala Lys Phe Leu Glu Gly Leu Thr Leu Arg Thr Asn Gln Thr Phe Arg Asp Thr Tyr Ala Ala Asp Gly Gly Arg Asn Gly Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp Pro Tyr Trp Asn Glu Gln Leu Val Ala Met Lys Ala Asp Ile Gln His Val Leu Asn Gly Ala Thr Pro Pro Ala Ala Pro Ala Ala Pro Ala Ala <210> 38 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Probe made in a lab <400> 38 agcggctggg acatcaacac 20 <210> 39 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Probe made in a lab <400> 39 cagacgcggg tgttgttggc 20 <210> 40 <211> 1211 <212> DNA
<213> Mycobacterium vaccae <400> 40 ggtaccggaagctggaggattgacggtatgagacttcttgacaggattcgtgggccttgg60 gcacgccgtttcggcgtcgtggctgtcgcgacagcgatgatgcctgctttggtgggcctg120 gctggagggtcggcgaccgccggagcattctcccggccaggtctgccggtggagtacctg180 atggtgccttcgccgtcgatggggcgcgacatcaagatccagttccagagcggtggcgag240 aactcgccggctctctacctgctcgacggcctgcgtgcgcaggaggacttcaacggctgg300 gacatcaacactcaggctttcgagtggttcctcgacagcggcatctccgtggtgatgccg360 gtcggtggccagtccagcttctacaccgactggtacgcccccgcccgtaacaagggcccg420 accgtgacctacaagtgggagaccttcctgacccaggagctcccgggctggctgcaggcc480 aaccgcgcggtcaagccgaccggcagcggccctgtcggtctgtcgatggcgggttcggcc540 gcgctgaacctggcgacctggcacccggagcagttcatctacgcgggctcgatgtccggc600 ttcctgaacccctccgagggctggtggccgttcctgatcaacatctcgatgggtgacgcc660 ggcggcttcaaggccgacgacatgtggggcaagaccgaggggatcccaacagcggttgga720 cagcgcaacgatccgatgctgaacatcccgaccctggtcgccaacaacacccgtatctgg780 gtctactgcggtaacggccagcccaccgagctcggcggcggcgacctgcccgccacgttc840 ctcgaaggtctgaccatccgcaccaacgagaccttccgcgacaactacatcgccgcgggt900 ggccacaacggtgtgttcaacttcccggccaacggcacgcacaactgggcgtactggggt960 cgcgagctgcaggcgatgaagcctgacctgcaggcgcaccttctctgacggttgcacgaa1020 acgaagcccccggccgattgcggccgagggtttcgtcgtccggggctactgtggccgaca1080 taaccgaaatcaacgcgatggtggctcatcaggaacgccgagggggtcattgcgctacga1140 cacgaggtgggcgagcaatccttcctgcccgacggagaggtcaacatccacgtcgagtac1200 tccagcgtgaa 1211 <210> 41 <211> 485 <212> DNA
<213> Mycobacterium vaccae <400> 41 agcggctgggacatcaacaccgccgccttcgagtggtacgtcgactcgggtctcgcggtg 60 atcatgcccgtcggcgggcagtccagcttctacagcgactggtacagcccggcctgcggt 120 aaggccggctgccagacctacaagtgggagacgttcctgacccaggagctgccggcctac 180 ctcgccgccaacaagggggtcgacccgaaccgcaacgcggccgtcggtctgtccatggcc 240 ggttcggcggcgctgacgctggcgatctaccacccgcagcagttccagtacgccgggtcg 300 ctgtcgggctacctgaacccgtccgaggggtggtggccgatgctgatcaacatctcgatg 360 ggtgacgcgggcggctacaaggccaacgacatgtggggtccaccgaaggacccgagcagc 420 gcctggaagcgcaacgacccgatggtcaacatcggcaagctggtggccaacaacaccccc 480 ctctc 485 <210> 42 <211>- 1052 <212> DNA
<213> Mycobacterium vaccae <400> 42 gttgatgagaaaggtgggttgtttgccgttatgaagttcacagagaagtggcggggctcc 60 gcaaaggcggcgatgcaccgggtgggcgttgccgatatggccgccgttgcgctgcccgga 120 ctgatcggcttcgccgggggttcggcaacggccggggcattctcccggcccggtcttcct 180 gtcgagtacctcgacgtgttctcgccgtcgatgggccgcgacatccgggtccagttccag 240 ggtggcggtactcatgcggtctacctgctcgacggtctgcgtgcccaggacgactacaac 300 ggctgggacatcaacacccctgcgttcgagtggttctacgagtccggcttgtcgacgatc 360 atgccggtcggcggacagtccagcttctacagcgactggtaccagccgtctcggggcaac 420 gggcagaactacacctacaagtgggagacgttcctgacccaggagctgccgacgtggctg 480 gaggccaaccgcggagtgtcgcgcaccggcaacgcgttcgtcggcctgtcgatggcgggc 540 agcgcggcgctgacctacgcgatccatcacccgcagcagttcatctacgcctcgtcgctg 600 tcaggcttcctgaacccgtccgagggctggtggccgatgctgatcgggctggcgatgaac 660 gacgcaggcggcttcaacgccgagagcatgtggggcccgtcctcggacccggcgtggaag 720 cgcaacgacccgatggtcaacatcaaccagctggtggccaacaacacccggatctggatc 780 tactgcggcaccggcaccccgtcggagctggacaccgggaccccgggccagaacctgatg 840 gccgcgcagttcctcgaaggattcacgttgcggaccaacatcgccttccgtgacaactac 900 atcgcagccggcggcaccaacggtgtcttcaacttcccggcctcgggcacccacagctgg 960 gggtactgggggcagcagctgcagcagatgaagcccgacatccagcgggttctgggagct 1020 caggccaccgcctagccacccaccccacaccc 1052 <210> 43 <211> 326 <212> PRT
<213> Mycobacterium vaccae <400> 43 Met Arg Leu Leu Asp Arg Ile Arg Gly Pro Trp Ala Arg Arg Phe Gly Val Val Ala Val Ala Thr Ala Met Met Pro Ala Leu Val Gly Leu Ala Gly Gly Ser Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Met Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Ile Gln Phe Gln Ser Gly Gly Glu Asn Ser Pro Ala Leu Tyr Leu Leu Asp Gly Leu Arg Ala Gln Glu Asp Phe Asn Gly Trp Asp Ile Asn Thr Gln g5 90 95 Ala Phe Glu Trp Phe Leu Asp Ser Gly Ile Ser Val Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Thr Asp Trp Tyr Ala Pro Ala Arg Asn Lys Gly Pro Thr Val Thr Tyr Lys Trp Glu Thr Phe Leu Thr Gln Glu Leu Pro Gly Trp Leu Gln Ala Asn Arg Ala Val Lys Pro Thr Gly Ser Gly Pro Val Gly Leu Ser Met Ala Gly Ser Ala Ala Leu Asn Leu Ala Thr Trp His Pro Glu Gln Phe Ile Tyr Ala Gly Ser Met Ser Gly Phe Leu Asn Pro Ser Glu Gly Trp Trp Pro Phe Leu Ile Asn Ile Ser Met Gly Asp Ala Gly Gly Phe Lys Ala Asp Asp Met Trp Gly Lys Thr Glu Gly Ile Pro Thr Ala Val Gly Gln Arg Asn Asp Pro Met Leu Asn Ile Pro Thr Leu Val Ala Asn Asn Thr Arg Ile Trp Val Tyr Cys Gly Asn Gly Gln Pro Thr Glu Leu Gly Gly Gly Asp Leu Pro Ala Thr Phe Leu Glu Gly Leu.Thr Ile Arg Thr Asn Glu Thr Phe Arg Asp Asn Tyr Ile Ala Ala Gly Gly His Asn Gly Val Phe Asn Phe Pro Ala Asn Gly Thr His Asn Trp Ala Tyr Trp Gly Arg Glu Leu Gln Ala Met Lys Pro Asp Leu Gln Ala His Leu Leu <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 Gly Leu Ala Val Ile Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Gln Glu Leu Pro Ala Tyr Leu Ala Ala Asn Lys Gly Val Asp Pro Asn Arg Asn Ala Ala Val Gly Leu Ser Met Ala Gly Ser Ala Ala Leu Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Gln g5 90 95 Tyr Ala Gly Ser Leu Ser Gly Tyr Leu Asn Pro Ser Glu Gly Trp Trp Pro Met Leu Ile Asn Ile Ser Met Gly Asp Ala Gly Gly Tyr Lys Ala Asn Asp Met Trp Gly Pro Pro Lys Asp Pro Ser Ser Ala Trp Lys Arg Asn Asp Pro Met Val Asn Ile Gly Lys Leu Val Ala Asn Asn Thr Pro Leu <210> 45 <211> 334 <212> PRT
<213> Mycobacterium vaccae <400> 45 Met Lys Phe Thr Glu Lys Trp Arg Gly Ser Ala Lys Ala Ala Met His Arg Val Gly Val Ala Asp Met Ala Ala Val Ala Leu Pro Gly Leu Ile Gly Phe Ala Gly Gly Ser Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Asp Val Phe Ser Pro Ser Met Gly Arg Asp Ile Arg Val Gln Phe Gln Gly Gly Gly Thr His Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Phe Tyr Glu Ser Gly Leu Ser Thr Ile Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Gln Pro Ser Arg Gly Asn Gly Gln Asn Tyr Thr Tyr Lys Trp Glu Thr Phe Leu Thr Gln Glu Leu Pro Thr Trp Leu Glu Ala Asn Arg Gly Val Ser Arg Thr Gly Asn Ala Phe Val Gly Leu Ser Met Ala Gly Ser Ala Ala Leu Thr Tyr Ala Ile His His Pro Gln Gln Phe Ile Tyr Ala Ser Ser Leu Ser Gly Phe Leu Asn Pro Ser Glu Gly Trp Trp Pro Met Leu Ile Gly Leu Ala Met Asn Asp Ala Gly Gly Phe Asn Ala Glu Ser Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Lys Arg Asn Asp Pro Met Val Asn Ile Asn Gln Leu Val Ala Asn Asn Thr Arg Ile Trp Ile Tyr Cys Gly Thr Gly Thr Pro Ser Glu Leu Asp Thr Gly Thr Pro Gly Gln Asn Leu Met Ala Ala Gln Phe Leu Glu Gly Phe Thr Leu Arg Thr Asn Ile Ala Phe Arg Asp Asn Tyr Ile Ala Ala Gly Gly Thr Asn Gly Val Phe Asn Phe Pro Ala Ser Gly Thr His Ser Trp Gly Tyr Trp Gly Gln Gln Leu Gln Gln Met Lys Pro Asp Ile Gln Arg Val Leu Gly Ala Gln Ala Thr Ala <210> 46 <211> 795 <212> DNA
<213> Mycobacterium vaccae <400> 46 ctgccgcgggtttgccatctcttgggtcctgggtcgggaggccatgttctgggtaacgat 60 ccggtaccgtccggcgatgtgaccaacatgcgaacagcgacaacgaagctaggagcggcg 120 ctcggcgcagcagcattggtggccgccacggggatggtc~gcgcggcgacggcgaacgcc 180 caggaagggcaccaggtccgttacacgctcacctcggccggcgcttacgagttcgacctg 240 ttctatctgacgacgcagccgccgagcatgcaggcgttcaacgccgacgcgtatgcgttc 300 gccaagcgggagaaggtcagcctcgccccgggtgtgccgtgggtcttcgaaaccacgatg 360 gccgacccgaactgggcgatccttcaggtcagcagcaccacccgcggtgggcaggccgcc 420 ccgaacgcgcactgcgacatcgccgtcgatggccaggaggtgctcagccagcacgacgac 480 ccctacaacgtgcggtgccagctcggtcagtggtgagtcacctcgccgagagtccggcca 540 gcgccggcggcagcggctcgcggtgcagcaccccgaggcgctgggtcgcgcgggtcagcg 600 cgacgtaaagatcgctggccccgcgcggcccctcggcgaggatctgctccgggtagacca 660 ccagcacggcgtctaactccagacccttggtctgcgtgggtgccaccgcgcccgggacac 720 cgggcgggccgatcaccacgctggtgccctcccggtccgcctccgcacgcacgaaatcgt 780 cgatggcaccggcga 795 <210> 47 <211> 142 <212> PRT
<213> Mycobacterium vaccae <400> 47 Met Arg Thr Ala Thr Thr Lys Leu Gly Ala Ala Leu Gly Ala Ala Ala Leu Val Ala Ala Thr Gly Met Val Ser Ala Ala Thr Ala Asn Ala Gln Glu Gly His Gln Val Arg Tyr Thr Leu Thr Ser Ala Gly Ala Tyr Glu Phe Asp Leu Phe Tyr Leu Thr Thr Gln Pro Pro Ser Met Gln Ala Phe Asn Ala Asp Ala Tyr Ala Phe Ala Lys Arg Glu Lys Val Ser Leu Ala Pro Gly Val Pro Trp Val Phe Glu Thr Thr Met Ala Asp Pro Asn Trp Ala Ile Leu Gln Val Ser Ser Thr Thr Arg Gly Gly Gln Ala Ala Pro Asn Ala His Cys Asp Ile Ala Val Asp Gly Gln Glu Val Leu Ser Gln His Asp Asp Pro Tyr Asn Val Arg Cys Gln Leu Gly Gln Trp <210> 48 <211> 300 <212> DNA

WO 99!32634 PCT/NZ98/00189 <213> Mycobacterium vaccae <400> 48 gccagtgcgccaacggttttcatcgatgccgcacacaaccccggtgggccctgcgcttgc 60 cgaaggctgcgcgacgagttcgacttccggtatctcgtcggcgtcgtctcggtgatgggg 120 gacaaggacgtggacgggatccgccaggacccgggcgtgccggacgggcgcggtctcgca 180 ctgttcgtctcgggcgacaaccttcgaaagggtgcggcgctcaacacgatccagatcgcc 240 gagctgctggccgcccagttgtaagtgttccgccgaaattgcattccacgccgataatcg 300 <210> 49 <211> 563 <212> DNA
<213> Mycobacterium vaccae <400> 49 ggatcctcggccggctcaagagtccgcgccgaggtggatgtgacgctggacggctacgag 60 ttcagtcgggcctgcgaggcgctgtaccacttcgcctgggacgagttctgcgactggtat 120 gtcgagcttgccaaagtgcaactgggtgaaggtttctcgcacaccacggccgtgttggcc 180 accgtgctcgatgtgctgctcaagcttctgcacccggtcatgccgttcgtcaccgaggtg 240 ctgtggaaggccctgaccgggcgggccggcgcgagcgaacgtctgggaaatgtggagtca 300 ctggtcgtcgcggactggcccacgcccaccggatacgcgctggatcaggctgccgcacaa 360 cggatcgccgacacccagaagttgatcaccgaggtgcgccggttccgcagcgatcagggt 420 ctggccgaccgccagcgggtgcctgcccggttgtccggcatcgacaccgcgggtctggac 480 gcccatgtcccggcggtgcgcgcgctggcctggcttgaccgagggtgatgagggcttcac 540 cgcgtccgaatcggtcgaggtgc 563 <210> 50 c211> 434 <212> DNA
<213> Mycobacterium vaccae <400> 50 gggccgggcccgaggatgagcaagttcgaagtcgtcaccgggatggcgttcgcggctttc 60 gccgacgcgcccatcgacgtcgccgtcgtcgaggtcgggctcggtggtcgctgggacgcg 120 acgaacgtggtgaacgcaccggtcgcggtcatcaccccgatcggggtggaccacaccgac 180 tacctcggtgacacgatcgccgagatcgccggggagaaggccggaaatcatcacccgcca 240 gccgacgacctggtgccgaccgacaccgtcgccgtgctggcgcggcaggttcccgaggcc 300 atggaggtgctgctggcccaggcggtgcgctcggatgcggctgtagcgcgcgaggattcg 360 gagtgcgcggtgctgggccgtcaggtcgccatcggcggcagctgctccggttgcaggggc 420 tcggtggcgtctac 434 <210> 51 <211> 438 <212> DNA
<213> Mycobacterium vaccae <400> 51 ggatcccactcccgcgccggcggcggccagctggtacggccattccagcgtgctgatcga 60 ggtcgacggctaccgcgtgctggccgacccggtgtggagcaacagatgttcgccctcacg 120 ggcggtcggaccgcagcgcatgcacgacgtcccggtgccgctggaggcgcttcccgccgt 180 ggacgcggtggtgatcgccaacgaccactacgaccacctcgacatcgacaccatcgtcgc 240 gttggcgcacacccagcgggccccgttcgtggtgccgttgggcatcggcgcacacctgcg 300 caagtggggcgtccccgaggcgcggatcgtcgagttggactggcacgaagcccaccgcat 360 cgacgacctg acgctggtct gcacccccgc ccggcacttc tccggccggt tgttctcccg 420 cgactcgacg ctgtgggc 438 <210> 52 <211> 87 <212> PRT
<213> Mycobacterium vaccae <400> 52 Ala Ser Ala Pro Thr Val Phe Ile Asp Ala Ala His Asn Pro Gly Gly Pro Cys Ala Cys Arg Arg Leu Arg Asp Glu Phe Asp Phe Arg Tyr Leu Val Gly Val Val Ser Val Met Gly Asp Lys Asp Val Asp Gly Ile Arg Gln Asp Pro Gly Val Pro Asp Gly Arg Gly Leu Ala Leu Phe Val Ser Gly Asp Asn Leu Arg Lys Gly Ala Ala Leu Asn Thr Ile Gln Ile Ala Glu Leu Leu Ala Ala Gln Leu <210> 53 <211> 175 <212> PRT
<213> Mycobacterium vaccae <400> 53 Gly Ser Ser Ala Gly Ser Arg Val Arg Ala Glu Val Asp Val Thr Leu Asp Gly Tyr Glu Phe Ser Arg Ala Cys Glu Ala Leu Tyr His Phe Ala Trp Asp Glu Phe Cys Asp Trp Tyr Val Glu Leu Ala Lys Val Gln Leu Gly Glu Gly Phe Ser His Thr Thr Ala Val Leu Ala Thr Val Leu Asp Val Leu Leu Lys Leu Leu His Pro Val Met Pro Phe Val Thr Glu Val Leu Trp Lys Ala Leu Thr Gly Arg Ala Gly Ala Ser Glu Arg Leu Gly Asn Val Glu Ser Leu Val Val Ala Asp Trp Pro Thr Pro Thr Gly Tyr Ala Leu Asp Gln Ala Ala Ala Gln Arg Ile Ala Asp Thr Gln Lys Leu Ile Thr Glu Val Arg Arg Phe Arg Ser Asp Gln Gly Leu Ala Asp Arg Gln Arg Val Pro Ala Arg Leu Ser Gly Ile Asp Thr Ala Gly Leu Asp Ala His Val Pro Ala Val Arg Ala Leu Ala Trp Leu Asp Arg Gly <210> 54 <211> 144 <212> PRT
<213> Mycobacterium vaccae <400> 54 Gly Pro Gly Pro Arg Asn Ser Lys Phe Glu Val Val Thr Gly Met Ala Phe Ala Ala Phe Ala Asp Ala Pro Ile Asp Val Ala Val Val Glu Val Gly Leu Gly Gly Arg Trp Asp Ala Thr Asn Val Val Asn Ala Pro Val Ala Val Ile Thr Pro Ile Gly Val Asp His Thr Asp Tyr Leu Gly Asp Thr Ile Ala Glu Ile Ala Gly Glu Lys Ala Gly Asn His His Pro Pro Ala Asp Asp Leu Val Pro Thr Asp Thr Val Ala Val Leu Ala Arg Gln Val Pro Glu Ala Asn Glu Val Leu Leu Ala Gln Ala Val Arg Ser Asp Ala Ala Val Ala Arg Glu Asp Ser Glu Cys Ala Val Leu Gly Arg Gln Val Ala Ile Gly Gly Ser Cys Ser Gly Cys Arg Gly Ser Val Ala Ser <210> 55 <211> 145 <212> PRT
<213> Mycobacterium vaccae <400> 55 Asp Pro Thr Pro Ala Pro Ala Ala Ala Ser Trp Tyr Gly His Ser Ser Val Leu Ile Glu Val Asp Gly Tyr Arg Val Leu Ala Asp Pro Val Trp Ser Asn Arg Cys Ser Pro Ser Arg Ala Val Gly Pro Gln Arg Met His Asp Val Pro Val Pro Leu Glu Ala Leu Pro Ala Val Asp Ala Val Val Ile Ser Asn Asp His Tyr Asp His Leu Asp Ile Asp Thr Ile Val Ala Leu Ala His Thr Gln Arg Ala Pro Phe Val Val Pro Leu Gly Ile Gly Ala His Leu Arg Lys Trp Gly Val Pro Glu Ala Arg Ile Val Glu Leu Asp Trp His Glu Ala His Arg Ile Asp Asp Leu Thr Leu Val Cys Thr Pro Ala Arg His Phe Ser Gly Arg Leu Phe Ser Arg Asp Ser Thr Leu Trp <210> 56 <211> 10 <212> PRT

<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (1) . . . (1) <223> Residue can be either Gly, Ile, Leu or Val <221> UNSURE
<222> (2)...(2) <223> Residue can be either Ile, Leu, Gly, or Ala <221> UNSURE
<222> (5)...(5) <221> UNSURE
<222> (9)...(9) <400> 56 Xaa Xaa Ala Pro Xaa Gly Asp Ala Xaa Arg <210> 57 <211> 8 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (7)...(7) <223> Residue can be either Ile or Leu <400> 57 Pro Glu Ala Glu Ala Asn Xaa Arg <210> 58 <211> 11 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (4)...(4) <223> Residue can be either Gln or Gly <221> UNSURE
<222> (5)...(5) <223> Residue can be either Gly or Gln <400> 58 Thr Ala Asn Xaa Xaa Glu Tyr Tyr Asp Asn Arg <210> 59 <211> 34 <212> PRT
<213> Mycobacterium vaccae <400> 59 Asn Ser Pro Arg Ala Glu Ala Glu Ala Asn Leu Arg Gly Tyr Phe Thr Ala Asn Pro Ala Glu Tyr Tyr Asp Leu Arg Gly Ile Leu Ala Pro Ile Gly Asp <210> 60 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 60 ccggtgggcc cgggctgcgc 20 <210> 61 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 61 tggccggcca ccacgtggta 20 <210> 62 <211> 313 <212> DNA
<213> Mycobacterium vaccae <400> 62 gccggtgggcccgggctgcgcggaatacgcggcagccaatcccactgggccggcctcggt 60 gcagggaatgtcgcaggacccggtcgcggtggcggcctcgaacaatccggagttgacaac 120 gctgtacggctgcactgtcgggccagctcaatccgcaagtaaacctggtggacaccctca 180 acagcggtcagtacacggtgttcgcaccgaccaacgcggcatttagcaagctgccggcat 240 ccacgatcgacgagctcaagaccaattcgtcactgctgaccagcatcctgacctaccacg 300 tggtggccggcca 313 <210> 63 <211> 18 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (7)...(17) <400> 63 Glu Pro Ala Gly Pro Leu Pro Xaa Tyr Asn Glu Arg Leu His Thr Leu Xaa Gln <210> 64 <211> 25 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (21)...(21) <400> 64 Gly Leu Asp Asn Glu Leu Ser Leu Val Asp Gly Gln Gly Arg Thr Leu Thr Val Gln Gln Xaa Asp Thr Phe Leu <210> 65 <211> 26 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> {3) .. . (3) <221> UNSURE
<222> (21) . . . {22) <221> UNSURE
<222> (24)...(24) <400> 65 Asp Pro Xaa Pro Asp Ile Glu Val Glu Phe Ala Arg Gly Thr Gly Ala Glu Pro Gly Leu Xaa Xaa Val Xaa Asp Ala <210> 66 <211> 32 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 66 accgccctcg agttctcccg gccaggtctg cc 32 <210> 67 <211> 32 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 67 aagcacgagc tcagtctctt ccacgcggac gt 32 <210> 68 <211> 30 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 68 catggatcca ttctcccggc ccggtcttcc 30 <210> 69 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 69 tttgaattct aggcggtggc ctgagc 26 <210> 70 <211> 161 <212> PRT
<213> Mycobacterium vaccae <400> 70 Ser Gly Trp Asp Ile Asn Thr Ala Ala Phe Glu Trp Tyr Val Asp Ser Gly Leu Ala Val Ile Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Gln Glu Leu Pro Ala Tyr Leu Ala Ala Asn Lys Gly Val Asp Pro Asn Arg Asn Ala Ala Val Gly Leu Ser Met Ala Gly Ser Ala Ala Leu Thr Leu Ala Ile Tyr His Pro Gln Gln Phe Gln Tyr Ala Gly Ser Leu Ser Gly Tyr Leu Asn Pro Ser Glu Gly Trp Trp Pro Met Leu Ile Asn Ile Ser Met Gly Asp Ala Gly Gly Tyr Lys Ala Asn Asp Met Trp Gly Arg Thr Glu Asp Pro Ser Ser Ala Trp Lys Arg Asn Asp Pro Met Val Asn Ile Gly Lys Leu Val Ala Asn Asn Thr Pro Leu <210> 71 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 71 gagagactcg agaacgccca ggaagggcac cag 33 <210> 72 <211> 32 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 72 gagagactcg agtgactcac cactgaccga gc 32 <210> 73 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <221> unsure <222> (3) . . . (3) <221> unsure <222> (6)...(6) <221> unsure <222> (9)...(9) <221> unsure <222> (15)...(15) <400> 73 ggngcngcnc argcngarcc <210> 74 <211> 825 <212> DNA
<213> Mycobacterium vaccae <400>

ttggatcccactcccgcgccggcggcggccagctggtacggccattccagcgtgctgatc 60 gaggtcgacggctaccgcgtgctggccgacccggtgtggagcaacagatgttcgccctca 120 cgggcggtcggaccgcagcgcatgcacgacgtcccggtgccgctggaggcgcttcccgcc 180 gtggacgcggtggtgatcagccacgaccactacgaccacctcgacatcgacaccatcgtc 240 gcgttggcgcacacccagcgggccccgttcgtggtgccgttgggcatcggcgcacacctg 300 cgcaagtggggcgtccccgaggcgcggatcgtcgagttggactggcacgaagcccaccgc 360 atagacgacctgacgctggtctgcacccccgcccggcacttctccggacggttgttctcc 420 cgcgactcgacgctgtgggcgtcgtgggtggtcaccggctcgtcgcacaaggcgttcttc 480 ggtggcgacaccggatacacgaagagcttcgccgagatcggcgacgagtacggtccgttc 540 gatctgaccctgctgccgatcggggcctaccatcccgcgttcgccgacatccacatgaac 600 cccgaggaggcggtgcgcgcccatctggacctgaccgaggtggacaacagcctgatggtg 660 cccatccactgggcgacattccgcctcgccccgcatccgtggtccgagcccgccgaacgc 720 ctgctgaccgctgccgacgccgagcgggtacgcctgaccgtgccgattcccggtcagcgg 780 gtggacccggagtcgacgttcgacccgtggtggcggttctgaacc 825 <210> 75 <211> 273 <212> PRT
<213> Mycobacterium vaccae <400> 75 Leu Asp Pro Thr Pro Ala Pro Ala Ala Ala Ser Trp Tyr Gly His Ser Ser Val Leu Ile Glu Val Asp Gly Tyr Arg Val Leu Ala Asp Pro Val Trp Ser Asn Arg Cys Ser Pro Ser Arg Ala Val Gly Pro Gln Arg Met His Asp Val Pro Val Pro Leu Glu Ala Leu Pro Ala Val Asp Ala Val Val Ile Ser His Asp His Tyr Asp His Leu Asp Ile Asp Thr Ile Val Ala Leu Ala His Thr Gln Arg Ala Pro Phe Val Val Pro Leu Gly Ile Gly Ala His Leu Arg Lys Trp Gly Val Pro Glu Ala Arg Ile Val Glu Leu Asp Trp His Glu Ala His Arg Ile Asp Asp Leu Thr Leu Val Cys Thr Pro Ala Arg His Phe Ser Gly Arg Leu Phe 5er Arg Asp Ser Thr Leu Trp Ala Ser Trp Val Val Thr Gly Ser Ser His Lys Ala Phe Phe Gly Gly Asp Thr Gly Tyr Thr Lys Ser Phe Ala Glu Ile Gly Asp Glu Tyr Gly Pro Phe Asp Leu Thr Leu Leu Pro Ile Gly Ala Tyr His Pro Ala Phe Ala Asp Ile His Met Asn Pro Glu Glu Ala Val Arg Ala His Leu Asp Leu Thr Glu Val Asp Asn Ser Leu Met Val Pro Ile His Trp Ala Thr Phe Arg Leu Ala Pro His Pro Trp Ser Glu Pro Ala Glu Arg Leu Leu Thr Ala Ala Asp Ala Glu Arg Val Arg Leu Thr Val Pro Ile Pro Gly Gln Arg Val Asp Pro Glu Ser Thr Phe Asp Pro Trp Trp Arg Phe <210> 76 <211> 10 <212> PRT
<213> Mycobacterium vaccae <400> 76 Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala <210> 77 <211> 337 <212> DNA
<213> Mycobacterium vaccae <400> 77 gatccctacatcctgctggtcagctccaaggtgtcgaccgtcaaggatctgctcccgctg 60 ctggagaaggtcatccaggccggcaagccgctgctgatcatcgccgaggacgtcgagggc 120 gaggccctgtccacgctggtggtcaacaagatccgcggcaccttcaagtccgtcgccgtc 180 aaggctccgggcttcggtgaccgccgcaaggcgatgctgcaggacatggccatcctcacc 240 ggtggtcaggtcgtcagcgaaagagtcgggctgtccctggagaccgccgacgtctcgctg 300 ctgggccaggcccgcaaggtcgtcgtcaccaaggaca 337 <210> 7B
<211> 112 <212> PRT
<213> Mycobacterium vaccae <400> 78 Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gln Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln Asp Met Ala Ile Leu Thr 65 70 75 g0 Gly Gly Gln Val Val Ser Glu Arg Val Gly Leu Ser Leu Glu Thr Ala Asp Val Ser Leu Leu Gly Gln Ala Arg Lys Val Val Val Thr Lys Asp <210> 79 <211> 360 <212> DNA
<213> Mycobacterium vaccae <400> 79 ccgtacgagaagatcggcgctgagctggtcaaagaggtcgccaagaagaccgacgacgtc60 gcgggcgacggcaccaccaccgccaccgtgctcgctcaggctctggttcgcgaaggcctg120 cgcaacgtcgcagccggcgccaacccgctcggcctcaagcgtggcatcgagaaggctgtc180 gaggctgtcacccagtcgctgctgaagtcggccaaggaggtcgagaccaaggagcagatt240 tctgccaccgcggcgatctccgccggcgacacccagatcggcgagctcatcgccgaggcc300 atggacaaggtcggcaacgagggtgtcatcaccgtcgaggagtcgaacaccttcggcctg360 <210> 80 <211> 120 <212> PRT
<213> Mycobacterium vaccae <400> 80 Pro Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Ala Val Thr Gln Ser Leu Leu Lys Ser Ala Lys Glu Val Glu Thr Lys Glu Gln Ile Ser Ala Thr Ala Ala Ile Ser Ala Gly Asp Thr Gln Ile Gly Glu Leu Ile Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu Glu Ser Asn Thr Phe Gly Leu <210> 81 <211> 43 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 81 actgacgctg aggagcgaaa gcgtggggag cgaacaggat tag 43 <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> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 83 aaaaaaaaaa aaaaaaaaaa 20 <210> 84 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 84 ggaaggaagc ggccgctttt tttttttttt t 31 <210> 85 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 85 gagagagagc ccgggcatgc tsctsctsct s 31 <210> 86 <211> 238 <212> DNA
<213> Mycobacterium vaccae <400> 86 ctcgatgaac cgctcggagc gctcgacctg aagctgcgcc acgtcatgca gttcgagctc 60 aagcgcatcc agcgggaggt cgggatcacg ttcatctacg tgacccacga ccaggaagag 120 gcgctcacga tgagtgaccg catcgcggtg atgaacgccg gcaacgtcga acagatcggc 180 agcccgaccg agatctacga ccgtcccgcg acggtgttcg tcgccagctt catcgaat 238 <210> 87 <211> 79 <212> PRT
<213> Mycobacterium vaccae <400> 87 Leu Asp Glu Pro Leu Gly Ala Leu Asp Leu Lys Leu Arg His Val Met Gln Phe Glu Leu Lys Arg Ile Gln Arg Glu Val Gly Ile Thr Phe Ile Tyr Val Thr His Asp Gln Glu Glu Ala Leu Thr Met Ser Asp Arg Ile Ala Val Met Asn Ala Gly Asn Val Glu Gln Ile Gly Ser Pro Thr Glu Ile Tyr Asp Arg Pro Ala Thr Val Phe Val Ala Ser Phe Ile Glu <210> 88 <211> 1518 <212> DNA
<213> Mycobacterium vaccae <400>

cactcgccatgggtgttacaataccccaccagttcctcgaagtaaacgaacagaaccgtg60 acatccagctgagaaaatattcacagcgacgaagcccggccgatgcctgatggggtccgg120 catcagtacagcgcgctttcctgcgcggattctattgtcgagtccggggtgtgacgaagg180 aatccattgtcgaaatgtaaattcgttgcggaatcacttgcataggtccgtcagatccgc240 gaaggtttaccccacagccacgacggctgtccccgaggaggacctgccctgaccggcaca300 cacatcaccgctgcagaacctgcagaacagacggcggattccgcggcaccgcccaagggc360 gcgccggtgatcgagatcgaccatgtcacgaagcgcttcggcgactacctggccgtcgcg420 gacgcagacttctccatcgcgcccggggagttcttctccatgctcggcccgtccgggtgt480 gggaagacgaccacgttgcgcatgatcgcgggattcgagaccccgactgaaggggcgatc540 cgcctcgaaggcgccgacgtgtcgaggaccccacccaacaagcgcaacgtcaacacggtg600 ttccagcactacgcgctgttcccgcacatgacggtctgggacaacgtcgcgtacggcccg660 cgcagcaagaaactcggcaaaggcgaggtccgcaagcgcgtcgacgagctgctggagatc720 gtccggctgaccgaatttgccgagcgcaggcccgcccagctgtccggcgggcagcagcag780 cgggtggcgttggcccgggcactggtgaactaccccagcgcgctgctgctcgatgaaccg840 ctcggagcgctcgacctgaagctgcgccacgtcatgcagttcgagctcaagcgcatccag900 cgggaggtcgggatcacgttcatctacgtgacccacgaccaggaagaggcgctcacgatg960 agtgaccgcatcgcggtgatgaacgccggcaacgtcgaacagatcggcagcccgaccgag1020 atctacgaccgtcccgcgacggtgttcgtcgccagcttcatcggacaggccaacctctgg1080 gcgggccggtgcaccggccgctccaaccgcgattacgtcgagatcgacgttctcggctcg1140 acgctgaaggcacgcccgggcgagaccacgatcgagcccggcgggcacgccaccctgatg1200 gtgcgtccggaacgcatccgggtcaccccgggctcccaggacgcgccgaccggtgacgtc1260 gcctgcgtgcgtgccaccgtcaccgacctgaccttccaaggtccggtggtgcggctctcg1320 ctggccgctccggacgactcgaccgtgatcgcccacgtcggccccgagcaggatctgccg1380 ctgctgcgccccggcgacgacgtgtacgtcagctgggcaccggaagcctccctggtgctt1440 cccggcgacgacatccccaccaccgaggacctcgaagagatgctcgacgactcctgagtc1500 acgcttcccgattgccga 1518 <210> 89 <211> 376 <212> PRT
<213> Mycobacterium vaccae <400> 89 Val Ile Glu Ile Asp His Val Thr Lys Arg Phe Gly Asp Tyr Leu Ala Val Ala Asp Ala Asp Phe Ser Ile Ala Pro Gly Glu Phe Phe Ser Met Leu Gly Pro Ser Gly Cys Gly Lys Thr Thr Thr Leu Arg Met Ile Ala Gly Phe Glu Thr Pro Thr Glu Gly Ala Ile Arg Leu Glu Gly Ala Asp Val Ser Arg Thr Pro Pro Asn Lys Arg Asn Val Asn Thr Val Phe Gln His Tyr Ala Leu Phe Pro His Met Thr Val Trp Asp Asn Val Ala Tyr Gly Pro Arg Ser Lys Lys Leu Gly Lys Gly Glu Val Arg Lys Arg Val Asp Glu Leu Leu Glu Ile Val Arg Leu Thr Glu Phe Ala Glu Arg Arg Pro Ala Gln Leu Ser Gly Gly Gln Gln Gln Arg Val Ala Leu Ala Arg Ala Leu Val Asn Tyr Pro Ser Ala Leu Leu Leu Asp Glu Pro Leu Gly Ala Leu Asp Leu Lys Leu Arg His Val Met Gln Phe Glu Leu Lys Arg Ile Gln Arg Glu Val Gly Ile Thr Phe Ile Tyr Val Thr His Asp Gln Glu Glu Ala Leu Thr Met Ser Asp Arg Ile Ala Val Met Asn Ala Gly Asn Val Glu Gln Ile Gly Ser Pro Thr Glu Ile Tyr Asp Arg Pro Ala Thr Val Phe Val Ala Ser Phe Ile Gly Gln Ala Asn Leu Trp Ala Gly Arg Cys Thr Gly Arg Ser Asn Arg Asp Tyr Val Glu Ile Asp Val Leu Gly Ser Thr Leu Lys Ala Arg Pro Gly Glu Thr Thr Ile Glu Pro Gly Gly His Ala Thr Leu Met Val Arg Pro Glu Arg Ile Arg Val Thr Pro Gly Ser Gln Asp Ala Pro Thr Gly Asp Val Ala Cys Val Arg Ala Thr Val Thr Asp Leu Thr Phe Gln Gly Pro Val Val Arg Leu Ser Leu Ala Ala Pro Asp Asp Ser Thr Val Ile Ala His Val Gly Pro Glu Gln Asp Leu Pro Leu Leu Arg Pro Gly Asp Asp Val Tyr Val Ser Trp Ala Pro Glu Ala Ser Leu Val Leu Pro Gly Asp Asp Ile Pro Thr Thr Glu Asp Leu Glu Glu Met Leu Asp Asp Ser <210> 90 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 90 gagagactcg aggtgatcga gatcgaccat gtc 33 <210> 91 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 91 agagactcga gcaatcggga agcgtgactc a 31 <210> 92 <211> 323 <212> DNA
<213> Mycobacterium vaccae <400> 92 gtcgactacaaagaagacttcaacgacaacgagcagtggttcgccaaggtcaaggagccg 60 ttgtcgcgcaagcaggacataggcgccgacctggtgatccccaccgagttcatggccgcg 120 cgcgtcaagggcctgggatggctcaatgagatcagcgaagccggcgtgcccaatcgcaag 180 aatctgcgtcaggacctgttggactcgagcatcgacgagggccgcaagttcaccgcgccg 240 tacatgaccggcatggtcggtctcgcctacaacaaggcagccaccggacgcgatatccgc 300 accatcgacgacctctgggatcc 323 <210> 93 <211> 1341 <212> DNA
<213> Mycobacterium vaccae <400> 93 ccccacccccttccctggagccgacgaaaggcacccgcacatgtcccgtgacatcgatcc 60 ccacctgctggcccgaatgaccgcacgccgcaccttgcgtcgccgcttcatcggcggtgg 120 cgccgcggccgccgcgggcctgaccctcggttcgtcgttcctggcggcgtgcgggtccga 180 cagtgggacctcgagcaccacgtcacaggacagcggccccgccagcggcgccctgcgcgt 240 ctccaactggccgctctatatggccgacggtttcatcgcagcgttccagaccgcctcggg 300 catcacggtcgactacaaagaagacttcaacgacaacgagcagtggttcgccaaggtcaa 360 ggagccgttgtcgcgcaagcaggacataggcgccgacctggtgatccccaccgagttcat 420 ggccgcgcgcgtcaagggcctgggatggctcaatgagatcagcgaagccggcgtgcccaa 480 tcgcaagaatctgcgtcaggacctgttggactcgagcatcgacgagggccgcaagttcac 540 cgcgccgtacatgaccggcatggtcggtctcgcctacaacaaggcagccaccggacgcga 600 tatccgcaccatcgacgacctctgggatcccgcgttcaagggccgcgtcagtctgttctc 660 cgacgtccaggacggcctcggcatgatcatgctctcgcagggcaactcgccggagaatcc720 gaccaccgagtccattcagcaggcggtcgatctggtccgcgaacagaacgacagggggtc780 agatccgtcgcttcaccggcaacgactacgccgacgacctggccgcagaaacatcgccat840 cgcgcaggcgtactccggtgacgtcgtgcagctgcaggcggacaaccccgatctgcagtt900 catcgttcccgaatccggcggcgactggttcgtcgacacgatggtgatcccgtacaccac960 gcagaaccagaaggccgccgaggcgtggatcgactacatctacgaccgagccaactacgc1020 caagctggtcgcgttcacccagttcgtgcccgcactctcggacatgaccgacgaactcgc1080 caaggtcgatcctgcatcggcggagaacccgctgatcaacccgtcggccgaggtgcaggc1140 gaacctgaagtcgtgggcggcactgaccgacgagcagacgcaggagttcaacactgcgta1200 cgccgccgtcaccggcggctgacgcggtggtagtgccgatgcgaggggcataaatggccc1260 tgcggacgcgaggagcataaatggccggtgtcgccaccagcagccgtcagcggacaaggt1320 cgctccgtatctgatggtcct 1341 <210> 94 <211> 393 <212> PRT
<213> Mycobacterium vaccae <400> 94 Met Ser Arg Asp Ile Asp Pro His Leu Leu Ala Arg Met Thr Ala Arg Arg Thr Leu Arg Arg Arg Phe Ile Gly Gly Gly Ala Ala Ala Ala Ala Gly Leu Thr Leu Gly Ser Ser Phe Leu Ala Ala Cys Gly Ser Asp Ser Gly Thr Ser Ser Thr Thr Ser Gln Asp Ser Gly Pro Ala Ser Gly Ala Leu Arg Val Ser Asn Trp Pro Leu Tyr Met Ala Asp Gly Phe Ile Ala Ala Phe Gln Thr Ala Ser Gly Ile Thr Val Asp Tyr Lys Glu Asp Phe Asn Asp Asn Glu Gln Trp Phe Ala Lys Val Lys Glu Pro Leu Ser Arg Lys Gln Asp Ile Gly Ala Asp Leu Val Ile Pro Thr Glu Phe Met Ala Ala Arg Val Lys Gly Leu Gly Trp Leu Asn Glu Ile Ser Glu Ala Gly Val Pro Asn Arg Lys Asn Leu Arg Gln Asp Leu Leu Asp Ser Ser Ile Asp Glu Gly Arg Lys Phe Thr Ala Pro Tyr Met Thr Gly Met Val Gly Leu Ala Tyr Asn Lys Ala Ala Thr Gly Arg Asp Ile Arg Thr Ile Asp Asp Leu Trp Asp Pro Ala Phe Lys Gly Arg Val Ser Leu Phe Ser Asp Val Gln Asp Gly Leu Gly Met Ile Met Leu Ser Gln Gly Asn Ser Pro Glu Asn Pro Thr Thr Glu Ser Ile Gln Gln Ala Val Asp Leu Val Arg Glu Gln Asn Asp Arg Gly Ser Asp Pro Ser Leu His Arg Gln Arg Leu Arg Arg Arg Pro Gly Arg Arg Asn Ile Ala Ile Ala Gln Ala Tyr Ser Gly Asp Val Val Gln Leu Gln Ala Asp Asn Pro Asp Leu Gln Phe Ile Val Pro Glu Ser Gly Gly Asp Trp Phe Val Asp Thr Met Val Ile Pro Tyr Thr Thr Gln Asn Gln Lya Ala Ala Glu Ala Trp Ile Asp Tyr Ile Tyr Asp Arg Ala Asn Tyr Ala Lys Leu Val Ala Phe Thr Gln Phe Val Pro Ala Leu Ser Asp Met Thr Asp Glu Leu Ala Lys Val Asp Pro Ala Ser Ala Glu Asn Pro Leu Ile Asn $ro Ser Ala Glu Val Gln Ala Asn Leu Lys Ser Trp Ala Ala Leu Thr Asp Glu Gln Thr Gln Glu Phe Asn Thr Ala Tyr Ala Ala Val Thr Gly Gly <210> 95 <211> 22 <212> DNA
<213> Mycobacterium vaccae <400> 95 atgtcccgtg acatcgatcc cc 22 <210> 96 <211> 21 <212> DNA
<213> Mycobacterium vaccae <400> 96 atcggcacta ccaccgcgtc a 21 <210> 97 <211> 861 <212> DNA
<213> Mycobacterium vaccae <400> 97 gccggcgctcgcatatctcgcgatcttcttccgtggtgccgttcttctcgctggcacgca 60 cctcgttgtcggagaccggcggctcggtgttcatgccgacgctgacgttcgcctgggact 120 tcggcaactacgtcgacgcgttcacgatgtaccacgagcagatcttccgctcgttcggct 180 acgcgttcgtcgccacggtgctgtgcctgttgctggcgttcccgctggcctacgtcatcg 240 cgttcaaggccggccggttcaagaacctgatcctggggctggtgatcctgccgttcttcg 300 tcacgttcctgatccgcaccattgcgtggaagacgatcctggccgacgaaggctgggtgg 360 tcaccgcgctgggcgccatcgggctgctgcctgacgagggccggctgctgtccaccagct 420 gggcggtcatcggcggtctgacctacaactggatcatcttcatgatcctgccgctgtacg 480 tcagcctggagaagatcgacccgcgtctgctggaggcctcccaggacctctactcgtcgg 540 cgccgcgcagcttcggcaaggtgatcctgccgatggcgatgcccggggtgctggccggga 600 gcatgctggtgttcatcccggccgtcggcgacttcatcaacgccgactatctcggcagta 660 cccagaccaccatgatcggcaacgtgatccagaagcagttcctggtcgtcaaggactatc 720 cggcggcggccgcgctgagtctggggctgatgttgctgatcctgatcggcgtgctcctct 780 acacacgggcgctgggttcggaggatctggtatgaccacccaggcaggcgccgcactggc 840 caccgccgcc cagcaggatc c <210> 98 <211> 259 <212> PRT
<213> Mycobacterium vaccae <400> 98 Val Val Pro Phe Phe Ser Leu Ala Arg Thr Ser Leu Ser Glu Thr Gly Gly Ser Val Phe Met Pro Thr Leu Thr Phe Ala Trp Asp Phe Gly Asn Tyr Val Asp Ala Phe Thr Met Tyr His Glu Gls~ Ile Phe Arg Ser Phe Gly Tyr Ala Phe Val Ala Thr Val Leu Cys Leu Leu Leu Ala Phe Pro Leu Ala Tyr Val Ile Ala Phe Lys Ala Gly Arg Phe Lys Asn Leu Ile Leu Gly Leu Val Ile Leu Pro Phe Phe Val Thr Phe Leu Ile Arg Thr Ile Ala Trp Thr Ile Leu Ala Asp Glu Gly Trp Val Val Thr Ala Leu Gly Ala Ile Gly Leu Leu Pro Asp Glu Gly Arg Leu Leu Ser Thr Ser Trp Ala Val Ile Gly Gly Leu Thr Tyr Asn Trp Ile Ile Phe Met Ile Leu Pro Leu Tyr Val Ser Leu Glu Lys Ile Asp Pro Arg Leu Leu Glu Ala Ser Gln Asp Leu Tyr Ser Ser Ala Pro Arg Ser Phe Gly Lys Val Ile Leu Pro Met Ala Met Pro Gly Val Leu Ala Gly Ser Met Leu Val Phe Ile Pro Ala Val Gly Asp Phe Ile Asn Ala Asp Tyr Leu Gly Ser Thr Gln Thr Thr Met Ile Gly Asn Val Ile Gln Lys Gln Phe Leu Val Val Lys Asp Tyr Pro Ala Ala Ala Ala Leu Ser Leu Gly Leu Met Leu Leu Ile Leu Ile Gly Val Leu Leu Tyr Thr Arg Ala Leu Gly Ser Glu Asp Leu Val <210> 99 <211> 277 <212> DNA
<213> Mycobacterium vaccae <400> 99 gtaatctttg ctggagcccg tacgccggta ggcaaactca tgggttcgct caaggacttc 60 aagggcagcg atctcggtgc cgtggcgatc aagggcgccc tggagaaagc cttccccggc 120 gtcgacgacc ctgctcgtct cgtcgagtac gtgatcatgg gccaagtgct ctccgccggc 180 gccggccaga tgcccgcccg ccaggccgcc gtcgccgccg gcatcccgtg ggacgtcgcc 240 tcgctgacga tcaacaagat gtgcctgtcg ggcatcg 277 <210> 100 <211> 92 <212> PRT
<213> Mycobacterium vaccae <400> 100 Val Ile Phe Ala Gly Ala Arg Thr Pro Val Gly Lys Leu Met Gly Ser Leu Lys Asp Phe Lys Gly Ser Asp Leu Gly Ala Val Ala Ile Lys Gly Ala Leu Glu Lys Ala Phe Pro Gly Val Asp Asp Pro Ala Arg Leu Val Glu Tyr Val Ile Met Gly Gln Val Leu Ser Ala Gly Ala Gly Gln Met Pro Ala Arg Gln Ala Ala Val Ala Ala Gly Ile Pro Trp Asp Val Ala Ser Leu Thr Ile Asn Lys Met Cys Leu Ser Gly Ile <210> 101 <211> 12 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (1)...(1) <223> Residue can be either Glu or Pro <221> UNSURE
<222> (2)...(2) <223> Residue can be either Pro or Glu <221> UNSURE
<222> (7) . . . (7) <221> UNSURE
<222> (12) . . . (12) <400> 101 Xaa Xaa Ala Asp Arg Gly Xaa Ser Lys Tyr Arg Xaa <210> 102 <211> 24 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (1)...(1) <400> 102 Xaa Ile Asp Glu Ser Leu Phe Asp Ala Glu Glu Lys Met Glu Lys Ala Val Ser Val Ala Arg Asp Ser Ala <210> 103 <211> 23 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (1) . . . (2) <221> UNSURE
<222> (15)...(15) <221> UNSURE
<222> (17) . . . (17) <400> 103 Xaa Xaa Ile Ala Pro Ala Thr Ser Gly Thr Leu Ser Glu Phe Xaa Ala Xaa Lys Gly Val Thr Met Glu <210> 104 <211> 15 <212> PRT
<213> Mycobacterium vaccae <400> 104 Pro Asn Val Pro Asp Ala Phe Ala Val Leu Ala Asp Arg Val Gly <210> 105 <211> 9 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (1)...(1) <400> 105 Xaa Ile Arg Val Gly Val Asn Gly Phe <210> 106 <211> 485 <212> DNA

<213> Mycobacterium vaccae <400>

agcggctgggacatcaacaccgccgccttcgagtggtacgtcgactcgggtctcgcggtg 60 atcatgcccgtcggcgggcagtccagcttctacagcgactggtacagcccggcctgcggt 120 aaggccggctgccagacctacaagtgggagacgttcctgacccaggagctgccggcctac 180 ctcgccgccaacaagggggtcgacccgaaccgcaacgcggccgtcggtctgtccatggcc 240 ggttcggcggcgctgacgctggcgatctaccacccgcagcagttccagtacgccgggtcg 300 ctgtcgggctacctgaacccgtccgaggggtggtggccgatgctgatcaacatctcgatg 360 ggtgacgcgggcggctacaaggccaacgacatgtggggtcgcaccgaggacccgagcagc 420 gcctggaagcgcaacgacccgatggtcaacatcggcaagctggtcgccaacaacaccccc 480 ctctc 485 <210> 107 <211> 501 <212> DNA
<213> Mycobacterium vaccae <220>
<221> unsure <222> (441)...(441) <221> unsure <222> (450)...(450) <400> 107 atgccggtgcgacgtgcgcgcagtgcgcttgcgtccgtgaccttcgtcgcggccgcgtgc60 gtgggcgctgagggcaccgcactggcggcgacgccggactggagcgggcgctacacggtg120 gtgacgttcgcctccgacaaactcggcacgagtgtggccgcccgccagccagaacccgac180 ttcagcggtcagtacaccttcagcacgtcctgtgtgggcacctgcgtggccaccgcgtcc240 gacggcccggcgccgtcgaacccgacgattccgcagcccgcgcgctacacctgggacggc300 aggcagtgggtgttcaactacaactggcagtgggagtgcttccgcggcgccgacgtcccg360 cgcgagtacgccgccgcgcgttcgctggtgttctacgccccgaccgccgacgggtcgatg420 ttcggcacctggcgcaccganatcctgganggcctctgcaagggcaccgtgatcatgccg480 gtcgcggcctatccggcgtag 501 <210> 108 <211> 180 <212> DNA
<213> Mycobacterium vaccae <400> 108 atgaaccagc cgcggcccga ggccgaggcg aacctgcggg gctacttcac cgccaacccg 60 gcggagtact acgacctgcg gggcatcctc gccccgatcg gtgacgcgca gcgcaactgc 120 aacatcaccg tgctgccggt agagctgcag acggcctacg acacgttcat ggccggctga 180 <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 Ala Ala Ala Cys Val Gly Ala Glu Gly Thr Ala Leu Ala Ala Thr Pro Asp Trp Ser Gly Arg Tyr Thr Val Val Thr Phe Ala Ser Asp Lys Leu Gly Thr Ser Val Ala Ala Arg Gln Pro Glu Pro Asp Phe Ser Gly Gln Tyr Thr Phe Ser Thr Ser Cys Val Gly Thr Cys Val Ala Thr Ala Ser Asp Gly Pro Ala Pro Ser Asn Pro Thr Ile Pro Gln Pro Ala Arg Tyr Thr Trp Asp Gly Arg Gln Trp Val Phe Asn Tyr Asn Trp Gln Trp Glu Cys Phe Arg Gly Ala Asp Val Pro Arg Glu Tyr Ala Ala Ala Arg Ser Leu Val Phe Tyr Ala Pro Thr Ala Asp Gly Ser Met Phe Gly Thr Trp Arg Thr Asp Ile Leu Asp Gly Leu Cys Lys Gly Thr Val Ile Met Pro Val Ala Ala Tyr Pro Ala <210> 110 <211> 74 <212> PRT
<213> Mycobacterium vaccae <400> 110 Pro Arg Asp Thr His Pro Gly Ala Asn Gln Ala Val Thr Ala Ala Met Asn Gln Pro Arg Pro Glu Ala Glu Ala Asn Leu Arg Gly Tyr Phe Thr Ala Asn Pro Ala Glu Tyr Tyr Asp Leu Arg Gly Ile Leu Ala Pro Ile Gly Asp Ala Gln Arg Asn Cys Asn Ile Thr Val Leu Pro Val Glu Leu Gln Thr Ala 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 60 tggcagacgg gggtttcgat accgaccgcc tcagcggatc cgtgtccgga catcgaggtg 120 atcttcgcgc gcgggaccgg tgcggaaccc ggcctcgggt gggtcggtga tgcgttcgtc 180 aacgcgctgc ggcccaaggt cggtgagcag tcggtgggca cctacgcggt gaactacccg 240 gcaggattcggacttcgacaaatcggcgcccatgggcgcggccgacgcatcggggcgggt 300 gcagtggatggccgacaactgcccggacaccaagcttgtcctgggcggcatgtcgcangg 360 cgccggcgtcatcgacctgatcaccgtcgatccgcgaccgctgggccggttcacccccac 420 cccgatgccgccccgcgtcgccgaccacgtggccgccgttgtggtcttcggaaatccgtt 480 gcgcgacatccgtggtggcggtc <210> 112 <211> 167 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (119)...(119) <400> 112 Met Gln Val Arg Arg Val Leu Gly Ser Val Gly Ala Ala Val Ala Val Ser Ala Ala Leu Trp Gln Thr Gly Val Ser Ile Pro Thr Ala Ser Ala Asp Pro Cys Pro Asp Ile Glu Val Ile Phe Ala Arg Gly Thr Gly Ala Glu Pro Gly Leu Gly Trp Val Gly Asp Ala Phe Val Asn Ala Leu Arg Pro Lys Val Gly Glu Gln Ser Val Gly Thr Tyr Ala Val Asn Tyr Pro Ala Gly Phe Asp Phe Asp Lys Ser Ala Pro Met Gly Ala Ala Asp Ala Ser Gly Arg Val Gln Trp Met Ala Asp Asn Cys Pro Asp Thr Lys Leu Val Leu Gly Gly Met Ser Xaa Gly Ala Gly Val Ile Asp Leu Ile Thr Val Asp Pro Arg Pro Leu Gly Arg Phe Thr Pro Thr Pro Met Pro Pro Arg Val Ala Asp iiis Val Ala Ala Val Val Val Phe Gly Asn Pro Leu Arg Asp Ile Arg Gly Gly Gly <210> 113 <211> 1569 <212> DNA
<213> Mycobacterium vaccae <400> 113 atggccaagacaattgcgtatgacgaagaggcccgccgtggcctcgagcggggcctcaac 60 gccctcgcagacgccgtaaaggtgacgttgggcccgaagggtcgcaacgtcgtgctggag 120 aagaagtggggcgcccccacgatcaccaacgatggtgtgtccatcgccaaggagatcgag 180 ctggaggacccgtacgagaagatcggcgctgagctggtcaaagaggtcgccaagaagacc 240 gacgacgtcgcgggcgacggcaccaccaccgccaccgtgctcgctcaggctctggttcgc 300 gaaggcctgcgcaacgtcgcagccggcgccaacccgctcggcctcaagcgtggcatcgag 360 aaggctgtcgaggctgtcacccagtcgctgctgaagtcggccaaggaggtcgagaccaag 420 gagcagatttctgccaccgcggcgatttccgccggcgacacccagatcggcgagctcatc 480 gccgaggccatggacaaggtcggcaacgagggtgtcatcaccgtcgaggagtcgaacacc 540 ttcggcctgcagctcgagctcaccgagggtatgcgcttcgacaagggctacatctcgggt 600 tacttcgtgaccgacgccgagcgccaggaagccgtcctggaggatccctacatcctgctg 660 gtcagctccaaggtgtcgaccgtcaaggatctgctcccgctgctggagaaggtcatccag 720 gccggcaagccgctgctgatcatcgccgaggacgtcgagggcgaggccctgtccacgctg 780 gtggtcaacaagatccgcggcaccttcaagtccgtcgccgtcaaggctccgggcttcggt 840 gaccgccgcaaggcgatgctgcaggacatggccatcctcaccggtggtcaggtcgtcagc 900 gaaagagtcgggctgtccctggagaccgccgacgtctcgctgctgggccaggcccgcaag 960 gtcgtcgtcaccaaggacgagaccaccatcgtcgagggctcgggcgattccgatgccatc 1020 gccggccgggtggctcagatccgcgccgagatcgagaacagcgactccgactacgaccgc 1080 gagaagctgcaggagcgcctggccaagctggccggcggtgttgcggtgatcaaggccgga 1140 gctgccaccgaggtggagctcaaggagcgcaagcaccgcatcgaggacgccgtccgcaac 1200 gcgaaggctgccgtcgaagagggcatcgtcgccggtggcggcgtggctctgctgcagtcg 1260 gctcctgcgctggacgacctcggcctgacgggcgacgaggccaccggtgccaacatcgtc 1320 cgcgtggcgctgtcggctccgctcaagcagatcgccttcaacggcggcctggagcccggc 1380 gtcgttgccgagaaggtgtccaacctgcccgcgggtcacggcctcaacgccgcgaccggt 1440 gagtacgaggacctgctcaaggccggcgtcgccgacccggtgaaggtcacccgctcggcg 1500 ctgcagaacgcggcgtccatcgcggctctgttcctcaccaccgaggccgtcgtcgccgac 1560 aagccggag 1569 <210> 114 <211> 523 <212> PRT
<213> Mycobacterium vaccae <400> 114 Met Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Ala Val Thr Gln Ser Leu Leu Lys Ser Ala Lys Glu Val Glu Thr Lys Glu Gln Ile Ser Ala Thr Ala Ala Ile Ser Ala Gly Asp Thr Gln Ile Gly Glu Leu Ile Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu Glu Ser Asn Thr Phe Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg Phe Asp Lys Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Ala Glu Arg Gln Glu Ala Val Leu Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gln Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln Asp Met Ala Ile Leu Thr Gly Gly Gln Val Val Ser Glu Arg Val Gly Leu Ser Leu Glu Thr Ala Asp Val Ser Leu Leu Gly Gln Ala Arg Lys Val Val Val Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ser Gly Asp Ser Asp Ala Ile Ala Gly Arg Val Ala Gln Ile Arg Ala Glu Ile Glu Asn Sex Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg Leu Ala Lys Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala Ala Thr Glu Val Glu Leu Lys Glu Arg Lys His Arg Ile Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val Glu Glu Gly Ile Val Ala Gly Gly Gly Val Ala Leu Leu Gln Ser Ala Pro Ala Leu Asp Asp Leu Gly Leu Thr Gly Asp Glu Ala Thr Gly Ala Asn Iie Val Arg Val Ala Leu Ser Ala Pro Leu Lys Gln Ile Ala Phe Asn Gly Gly Leu Glu Pro Gly Val Val Ala Glu Lys Val Ser Asn Leu Pro Ala Gly His Gly Leu Asn Ala Ala Thr Gly Glu Tyr Glu Asp Leu Leu Lys Ala Gly Val Ala Asp Pro Val Lys Val Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Ala Leu Phe Leu Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu <210> 115 <211> 647 <212> DNA
<213> Mycobacterium vaccae <400>

atggccaagacaattgcgtatgacgaagaggcccgccgtggcctcgagcggggcctcaac60 gccctcgcagacgccgtaaaggtgacgttgggcccgaagggtcgcaacgtcgtgctggag120 aagaagtggggcgcccccacgatcaccaacgatggtgtgtccatcgccaaggagatcgag180 ctggaggacccgtacgagaagatcggcgctgagctggtcaaagaggtcgccaagaagacc240 gacgacgtcgcgggcgacggcaccaccaccgccaccgtgctcgctcaggctctggttcgc300 gaaggcctgcgcaacgtcgcagccggcgccaacccgctcggcctcaagcgtggcatcgag360 aaggctgtcgaggctgtcacccagtcgctgctgaagtcggccaaggaggtcgagaccaag420 WO 99/32634 ~ PCT/NZ98/00189 gagcagattt ctgccaccgc ggcgatttcc gccggcgaca cccagatcgg cgagctcatc 480 gccgaggcca tggacaaggt cggcaacgag ggtgtcatca ccgtcgagga gtcgaacacc 540 ttcggcctgc agctcgagct caccgagggt atgcgcttcg acaagggcta catctcgggt 600 tacttcgtga ccgacgccga gcgccaggaa gccgtcctgg aggatcc 647 <210> 116 <211> 927 <212> DNA
<213> Mycobacterium vaccae <400>

gatccctacatcctgctggtcagctccaaggtgtcgaccgtcaaggatctgctcccgctg 60 ctggagaaggtcatccaggccggcaagccgctgctgatcatcgccgaggacgtcgagggc 120 gaggccctgtccacgctggtggtcaacaagatccgcggcaccttcaagtccgtcgccgtc 180 aaggctccgggcttcggtgaccgccgcaaggcgatgctgcaggacatggccatcctcacc 240 ggtggtcaggtcgtcagcgaaagagtcgggctgtccctggagaccgccgacgtctcgctg 300 ctgggccaggcccgcaaggtcgtcgtcaccaaggacgagaccaccatcgtcgagggctcg 360 ggcgattccgatgccatcgccggccgggtggctcagatccgcgccgagatcgagaacagc 420 gactccgactacgaccgcgagaagctgcaggagcgcctggccaagctggccggcggtgtt 480 gcggtgatcaaggccggagctgccaccgaggtggagctcaaggagcgcaagcaccgcatc 540 gaggacgccgtccgcaacgcgaaggctgccgtcgaagagggcatcgtcgccggtggcggc 600 gtggctctgctgcagtcggctcctgcgctggacgacctcggcctgacgggcgacgaggcc 660 accggtgccaacatcgtccgcgtggcgctgtcggctccgctcaagcagatcgccttcaac 720 ggcggcctggagcccggcgtcgttgccgagaaggtgtccaacctgcccgcgggtcacggc 780 ctcaacgccgcgaccggtgagtacgaggacctgctcaaggccggcgtcgccgacccggtg 840 aaggtcacccgctcggcgctgcagaacgcggcgtccatcgcggctctgttcctcaccacc 900 gaggccgtcgtcgccgacaagccggag 927 <210> 117 <211> 215 <212> PRT
<213> Mycobacterium vaccae <400> 117 Met Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro 100 105 lI0 Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Ala Val Thr Gln Ser Leu Leu Lys Ser Ala Lys Glu Val Glu Thr Lys Glu Gln Ile Ser Ala Thr Ala Ala Ile Ser Ala Gly Asp Thr Gln Ile Gly Glu Leu Ile Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu Glu Ser Asn Thr Phe Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg Phe Asp Lys Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Ala Glu Arg Gln Glu Ala Val Leu Glu Asp <210> 118 <211> 309 <212> PRT
<213> Mycobacterium vaccae <400> 118 Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gln Ala Gly Lys Pro Leu Leu hle Ile Ala Glu Asp Val Glu Gly Glu Ala Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln Asp Met Ala Ile Leu Thr 65 70 75 gp Gly Gly Gln Val Val Ser Glu Arg Val Gly Leu Ser Leu Glu Thr Ala Asp Val Ser Leu Leu Gly Gln Ala Arg Lys Val Val Val Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ser Gly Asp Ser Asp Ala Ile Ala Gly Arg Val Ala Gln Ile Arg Ala Glu Ile Glu Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg Leu Ala Lys Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala Ala Thr Glu Val Glu Leu Lys Glu Arg Lys His Arg Ile Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val Glu Glu Gly Ile Val Ala Gly Gly Gly Val Ala Leu Leu Gln Ser Ala Pro Ala Leu Asp Asp Leu Gly Leu Thr Gly Asp Glu Ala Thr Gly Ala Asn Ile Val Arg Val Ala Leu Ser Ala Pro Leu Lys Gln Ile Ala Phe Asn Gly Gly Leu Glu Pro Gly Val Val Ala Glu Lys Val Ser Asn Leu Pro Ala Gly His Gly Leu Asn Ala Ala Thr Gly Glu Tyr Glu Asp Leu Leu Lys Ala Gly Val Ala Asp Pro Val Lys Val Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Ala Leu Phe Leu Thr Thr Glu Ala Val Val WO 99/32634 PC'f/NZ98/00189 Ala Asp Lys Pro Glu <210> 119 <211> 162 <212> DNA
<213> Mycobacterium vaccae <400> 119 ctcgtacagg cgacggagat ctccgacgac gccacgtcgg tacggttggt cgccaccctg 60 ttcggcgtcg tgttgttgac gttggtgctg tccgggctca acgccaccct catccagggc 120 gcaccagaag acagctggcg caggcggatt ccgtcgatct tc 162 <210> 120 <211> 1366 <212> DNA
<213> Mycobacterium vaccae <220>
<221> unsure <222> (955)...(955) <221> unsure <222> (973)...(973) <400>

gatgagcagcgtgctgaactcgacctggttggcctgggccgtcgcggtcgcggtcgggtt60 cccggtgctgctggtcgtgctgaccgaggtgcacaacgcgttgcgtcggcgcggcagcgc120 gctggcccgcccggtgcaactcctgcgtacctacatcctgccgctgggcgcgttgctgct180 cctgctggtacaggcgatggagatctccgacgacgccacgtcggtacggttggtcgccac240 cctgttcggcgtcgtgttgttgacgttggtgctgtccgggctcaacgccaccctcatcca300 gggcgcaccagaagacagctggcgcaggcggattccgtcgatcttcctcgacgtcgcgcg360 cttcgcgctgatcgcggtcggtatcaccgtgatcatggcctatgtctggggcgcgaacgt420 ggggggcctgttcaccgcactgggcgtcacttccatcgttcttggcctggctctgcagaa480 ttcggtcggtcagatcatctcgggtctgctgctgctgttcgagcaaccgttccggctcgg540 cgactggatcaccgtccccaccgcggcgggccggccgtccgcccacggccgcgtggtgga600 agtcaactggcgtgcaacacatatcgacaccggcggcaacctgctggtaatgcccaacgc660 cgaactcgccggcgcgtcgttcaccaattacagccggcccgtgggagagcaccggctgac720 cgtcgtcaccaccttcaacgccgcggacacccccgatgatgtctgcgagatgctgtcgtc780 ggtcgcggcgtcgctgcccgaactgcgcaccgacggacagatcgccacgctctatctcgg840 tgcggccgaatacgagaagtcgatcccgttgcacacacccgcggtggacgactcggtcag900 gagcacgtacctgcgatgggtctggtacgccgcgcgccggcaggaacttcgcctnaacgg960 cgtcgccgacganttcgacacgccggaacggatcgcctcggccatgcgggctgtggcgtc1020 cacactgcgcttggcagacgacgaacagcaggagatcgccgacgtggtgcgtctggtccg1080 ttacggcaacggggaacgcctccagcagccgggtcaggtaccgaccgggatgaggttcat1140 cgtagacggcagggtgagtctgtccgtgatcgatcaggacggcgacgtgatcccggcgcg1200 ggtgctcgagcgtggcgacttcctggggcagaccacgctgacgcgggaaccggtactggc1260 gaccgcgcacgcgctggaggaagtcaccgtgctggagatggcccgtgacgagatcgagcg1320 cctggtgcaccgaaagccgatcctgctgcacgtgatcggggccgtg 1366 <210> 121 <211> 455 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (318)...(318) <221> UNSURE
<222> (324) . . . (324) <400> 121 Met Ser Ser'Val Leu Asn Ser Thr Trp Leu Ala Trp Ala Val Ala Val Ala Val Gly Phe Pro Val Leu Leu Val Val Leu Thr Glu Val His Asn Ala Leu Arg Arg Arg Gly Ser Ala Leu Ala Arg Pro Val Gln Leu Leu Arg Thr Tyr Ile Leu Pro Leu Gly Ala Leu Leu Leu Leu Leu Val Gln Ala Met Glu Ile Ser Asp Asp Ala Thr Ser Val Arg Leu Val Ala Thr 65 70 75 g0 Leu Phe Gly Val Val Leu Leu Thr Leu Val Leu Ser Gly Leu Asn Ala Thr Leu Ile Gln Gly Ala Pro Glu Asp Ser Trp Arg Arg Arg Ile Pro Ser Ile Phe Leu Asp Val Ala Arg Phe Ala Leu Ile Ala Val Gly Ile Thr Val Ile Met Ala Tyr Val Trp Gly Ala Asn Val Gly Gly Leu Phe Thr Ala Leu Gly Val Thr Ser Ile Val Leu Gly Leu Ala Leu Gln Asn Ser Val Gly Gln Ile Ile Ser Gly Leu Leu Leu Leu Phe Glu Gln Pro Phe Arg Leu Gly Asp Trp Ile Thr Val Pro Thr Ala Ala Gly Arg Pro Ser Ala His Gly Arg Val Val Glu Val Asn Trp Arg Ala Thr His Ile Asp Thr Gly Gly Asn Leu Leu Val Met Pro Asn Ala Glu Leu Ala Gly Ala Ser Phe Thr Asn Tyr Ser Arg Pro Val Gly Glu His Arg Leu Thr Val Val Thr Thr Phe Asn Ala Ala Asp Thr Pro Asp Asp Val Cys Glu Met Leu Ser Ser Val Ala Ala Ser Leu Pro Glu Leu Arg Thr Asp Gly Gln Ile Ala Thr Leu Tyr Leu Gly Ala Ala Glu Tyr Glu Lys Ser Ile Pro Leu His Thr Pro Ala Val Asp Asp Ser Val Arg Ser Thr Tyr Leu Arg Trp Val Trp Tyr Ala Ala Arg Arg Gln Glu Leu Arg Xaa Asn Gly Val Ala Asp Xaa Phe Asp Thr Pro Glu Arg Ile Ala Ser Ala Met Arg Ala Val Ala Ser Thr Leu Arg Leu Ala Asp Asp Glu Gln Gln Glu Ile Ala Asp Val Val Arg Leu Val Arg Tyr Gly Asn Gly Glu Arg Leu Gln Gln Pro Gly Gln Val Pro Thr Gly Met Arg Phe Ile Val Asp Gly Arg Val Ser Leu Ser Val Ile Asp Gln Asp Gly Asp Val Ile Pro Ala Arg Val Leu Glu Arg Gly Asp Phe Leu Gly.Gln Thr Thr Leu Thr Arg Glu Pro Val Leu Ala Thr Ala His Ala Leu Glu Glu Val Thr Val Leu Glu Met Ala Arg Asp Glu Ile Glu Arg Leu Val Hi8 Arg Lys Pro Ile Leu Leu His Val Ile Gly Ala Val <210> 122 <211> 898 <212> DNA
<213> Mycobacterium vaccae <400>

atgacaattctgccctggaatgcgcgaacgtctgaacacccgacgcgaaaaagacgcggg 60 cgctaccacctcctgtcgcggatgagcatccagtccaagttgctgctgatgctgcttctg 120 accagcattctctcggctgcggtggtcggtttcatcggctatcagtccggacggtcctcg 180 ctgcgcgcatcggtgttcgaccgcctcaccgacatccgcgagtcgcagtcgcgcgggttg 240 gagaatcagttcgcggacctgaagaactcgatggtgatttactcgcgcggcagcactgcc 300 acggaggcgatcggcgcgttcagcgacggtttccgtcagctcggcgatgcgacgatcaat 360 accgggcaggcggcgtcattgcgccgttactacgaccggacgttcgccaacaccaccctc 420 gacgacagcggaaaccgcgtcgacgtccgcgcgctcatcccgaaatccaacccccagcgc 480 tatctgcaggcgctctataccccgccgtttcagaactgggagaaggcgatcgcgttcgac 540 gacgcgcgcgacggcagcgcctggtcggccgccaatgccagattcaacgagttcttccgc 600 gagatcgtgcaccgcttcaacttcgaggatctgatgctgctcgacctcgagggcaacgtg 660 gtgtactccgcctacaaggggccggatctcgggacaaacatcgtcaacggcccctatcgc 720 aaccgggaactgtcggaagcctacgagaaggcggtcgcgtcgaactcgatcgactatgtc 780 ggtgtcaccgacttcgggtggtacctgcctgccgaggaaccgaccgcctggttcctgtcc 840 ccggtcgggttgaaggaccgagtcgacggtgtgatggcggtccagttccccggaattc 898 <210> 123 <211> 1259 <212> DNA
<213> Mycobacterium vaccae <400> 123 cgcaattgatgacggcgcggggacagtggcgtgacaccgggatgggagacaccggtgaga 60 ccatcctggtcggaccggacaatctgatgcgctcggactcccggctgttccgcgagaacc 120 gggagaagttcctggccgacgtcgtcgaggggggaaccccgccggaggtcgccgacgaat 180 cggttgaccgccgcggcaccacgctggtgcagccggtgaccacccgctccgtcgaggagg 240 cccaacgcggcaacaccgggacgacgatcgaggacgactatctcggccacgaggcgttac 300 aggcgtactcaccggtggacctgccgggactgcactgggtgatcgtggccaagatcgaca 360 ccgacgaggcgttcgccccggtggcgcagttcaccaggaccctggtgctgtcgacggtga 420 tcatcatctteggcgtgtcgctggcggccatgctgctggcgcggttgttcgtccgtccga 480 WO 99!32634 PCT/NZ98/00189 tccggcggttgcaggccggcgcccagcagatcagcggcggtgactaccgcctcgctctgc 540 cggtgttgtctcgtgacgaattcggcgatctgacaacagctttcaacgacatgagtcgca 600 atctgtcgatcaaggacgagctgctcggcgaggagcgcgccgagaaccaacggctgatgc 660 tgtccctgatgcccgaaccggtgatgcagcgctacctcgacggggaggagacgatcgccc 720 aggaccacaagaacgtcacggtgatcttcgccgacatgatgggcctcgacgagttgtcgc 780 gcatgttgacctccgaggaactgatggtggtggtcaacgacctgacccgccagttcgacg 840 ccgccgccgagagtctcggggtcgaccacgtgcggacgctgcacgacgggtacctggcca 900 gctgcgggttaggcgtgccgcggctggacaacgtccggcgcacggtcaatttcgcgatcg 960 aaatggaccgcatcatcgaccggcacgccgccgagtccgggcacgacctgcggctccgcg 1020 cgggcatcgacaccgggtcggcggccagcgggctggtggggcggtccacgttggcgtacg 1080 acatgtggggttcggcggtcgatgtcgcctaccaggtgcagcgcggctccccccagcccg 1140 gcatctacgtcacctcgcgggtgcacgaggtcatgcaggaaactctcgacttcgtcgccg 1200 ccggggaggtcgtcggcgagcgcggcgtcgagacggtctggcggttgcagggccacccg 1259 <210> 124 <211> 299 <212> PRT
<213> Mycobacterium vaccae <400> 124 Met Thr Ile Leu Pro Trp Asn Ala Arg Thr Ser Glu His Pro Thr Arg Lys Arg Arg Gly Arg Tyr His Leu Leu Ser Arg Met Ser Ile Gln Ser Lys Leu Leu Leu Met Leu Leu Leu Thr Ser Ile Leu Ser Ala Ala Val Val Gly Phe Ile Gly Tyr Gln Ser Gly Arg Ser Ser Leu Arg Ala Ser Val Phe Asp Arg Leu Thr Asp Ile Arg Glu Ser Gln Ser Arg Gly Leu Glu Asn Gln Phe Ala Asp Leu Lys Asn Ser Met Val Ile Tyr Ser Arg Gly Ser Thr Ala Thr Glu Ala Ile Gly Ala Phe Ser Asp Gly Phe Arg Gln Leu Gly Asp Ala Thr Ile Asn Thr Gly Gln Ala Ala Ser Leu Arg Arg Tyr Tyr Asp Arg Thr Phe Ala Asn Thr Thr Leu Asp Asp Ser Gly Asn Arg Val Asp Val Arg Ala Leu Ile Pro Lys Ser Asn Pro Gln Arg Tyr Leu Gln Ala Leu Tyr Thr Pro Pro Phe Gln Asn Trp Glu Lys Ala Ile Ala Phe Asp Asp Ala Arg Asp Gly Ser Ala Trp Ser Ala Ala Asn Ala Arg Phe Asn Glu Phe Phe Arg Glu Ile Val His Arg Phe Asn Phe Glu Asp Leu Met Leu Leu Asp Leu Glu Gly Asn Val Val Tyr Ser Ala Tyr Lys Gly Pro Asp Leu Gly Thr Asn Ile Val Asn Gly Pro Tyr Arg Asn Arg Glu Leu Ser Glu Ala Tyr Glu Lys Ala Val Ala Ser Asn Ser Ile Asp Tyr Val Gly Val Thr Asp Phe Gly Trp Tyr Leu Pro Ala Glu Glu Pro Thr Ala Trp Phe Leu Ser Pro Val Gly Leu Lys Asp Arg Val Asp Gly Val Met Ala Val Gln Phe Pro Gly Ile <210> 125 <211> 419 <212> PRT
<213> Mycobacterium vaccae <400> 125 Gln Leu Met Thr Ala Arg Gly Gln Trp Arg Asp Thr Gly Met Gly Asp Thr Gly Glu Thr Ile Leu Val Gly Pro Asp Asn Leu Met Arg Ser Asp Ser Arg Leu Phe Arg Glu Asn Arg Glu Lys Phe Leu Ala Asp Val Val Glu Gly Gly Thr Pro Pro Glu Val Ala Asp Glu Ser Val Asp Arg Arg Gly Thr Thr Leu Val Gln Pro Val Thr Thr Arg Ser Val Glu Glu Ala Gln Arg Gly Asn Thr Gly Thr Thr Ile Glu Asp Asp Tyr Leu Gly His Glu Ala Leu Gln Ala Tyr Ser Pro Val Asp Leu Pro Gly Leu His Trp Val Ile Val Ala Lys Ile Asp Thr Asp Glu Ala Phe Ala Pro Val Ala Gln Phe Thr Arg Thr Leu Val Leu Ser Thr Val Ile Ile Ile Phe Gly Val Ser Leu Ala Ala Met Leu Leu Ala Arg Leu Phe Val Arg Pro Ile Arg Arg Leu Gln Ala Gly Ala Gln Gln Ile Ser Gly Gly Asp Tyr Arg Leu Ala Leu Pro Val Leu Ser Arg Asp Glu Phe Gly Asp Leu Thr Thr Ala Phe Asn Asp Met Ser Arg Asn Leu Ser Ile Lys Asp Glu Leu Leu Gly Glu Glu Arg Ala Glu Asn Gln Arg Leu Met Leu Ser Leu Met Pro Glu Pro Val Met Gln Arg Tyr Leu Asp Gly Glu Glu Thr Ile Ala Gln Asp His Lys Asn Val Thr Val Ile Phe Ala Asp Met Met Gly Leu Asp Glu Leu Ser Arg Met Leu Thr Ser Glu Glu Leu Met Val Val Val Asn Asp Leu Thr Arg Gln Phe Asp Ala Ala Ala Glu Ser Leu Gly Val Asp His Val Arg Thr Leu His Asp Gly Tyr Leu Ala Ser Cys Gly Leu Gly Val Pro Arg Leu Aap Asn Val Arg Arg Thr Val Asn Phe Ala Ile Glu 305 310 315 ~ 320 Met Asp Arg Ile Ile Asp Arg His Ala Ala Glu Ser Gly His Asp Leu WO 99/32634 PC"T/NZ98/00189 Arg Leu Arg Ala Gly Ile Asp Thr Gly Ser Ala Ala Ser Gly Leu Val Gly Arg Ser Thr Leu Ala Tyr Asp Met Trp Gly Ser Ala Val Asp Val Ala Tyr Gln Val Gln Arg Gly Ser Pro Gln Pro Gly Ile Tyr Val Thr Ser Arg Val His Glu Val Met Gln Glu Thr Leu Asp Phe Val Ala Ala Gly Glu Val Val Gly Glu Arg Gly Val Glu Thr Val Trp Arg Leu Gln Gly His Pro <210> 126 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 126 ccggatccga tgagcagcgt gctgaac 27 <210> 127 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 127 gcggatccca cggccccgat cacgtg 26 <210> 128 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 128 ccggatccaa tgacatttct gccctggaat gcg 33 <210> 129 <211> 32 <212> DNA
<213> Artificial Sequence <220>

<223> Made in a lab <400> 129 ccggatccat tcggtggccc tgcaaccgcc ag ' 32 <210> 130 <2I1> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 130 ccggatccgg agcaaccgtt ccggctc 27 <210> 131 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 131 ccggatcccg gctatcagtc cggacgg 27 <210> 132 <211> 844 <212> DNA
<213> Mycobacterium vaccae <400>

gagcaaccgttccggctcggcgactggatcaccgtccccaccgcggcgggccggccgtcc 60 gcccacggccgcgtggtggaagtcaactggcgtgcaacacatatcgacaccggcggcaac 120 ctgctggtaatgcccaacgccgaactcgccggcgcgtcgttcaccaattacagccggccc 180 gtgggagagcaccggctgaccgtcgtcaccaccttcaacgccgcggacacccccgatgat 240 gtctgcgagatgctgtcgtcggtcgcggcgtcgctgcccgaactgcgcaccgacggacag 300 atcgccacgctctatctcggtgcggccgaatacgagaagtcgatcccgttgcacacaccc 360 gcggtggacgactcggtcaggagcacgtacctgcgatgggtctggtacgccgcgcgccgg 420 caggaacttcgcctaacggcgtcgccgacgattcgacacgccggaacggatcgcctcggc 480 catgcgggctgtggcgtccacactgcgcttggcagacgacgaacagcaggagatcgccga 540 cgtggtgcgtctggtccgttacggcaacggggaacgcctccagcagccgggtcaggtacc 600 gaccgggatgaggttcatcgtagacggcagggtgagtctgtccgtgatcgatcaggacgg 660 cgacgtgatcccggcgcgggtgctcgagcgtggcgacttcctggggcagaccacgctgac 720 gcgggaaccggtactggcgaccgcgcacgcgctggaggaagtcaccgtgctggagatggc 780 ccgtgacgagatcgagcgcctggtgcaccgaaagccgatcctgctgcacgtgatcggggc 840 cgtg 844 <210> 133 <211> 742 <212> DNA
<213> Mycobacterium vaccae <400>

ggctatcagtccggacggtcctcgctgcgcgcatcggtgttcgaccgcctcaccgacatc 60 cgcgagtcgcagtcgcgcgggttggagaatcagttcgcggacctgaagaactcgatggtg 120 atttactcgcgcggcagcactgccacggaggcgatcggcgcgttcagcgacggtttccgt 180 cagctcggcgatgcgacgatcaataccgggcaggcggcgtcattgcgccgttactacgac 240 cggacgttcgccaacaccaccctcgacgacagcggaaaccgcgtcgacgtccgcgcgctc 300 atcccgaaatccaacccccagcgctatctgcaggcgctctataccccgccgtttcagaac 360 tgggagaaggcgatcgcgttcgacgacgcgcgcgacggcagcgcctggtcggccgccaat 420 gccagattcaacgagttcttccgcgagatcgtgcaccgcttcaacttcgaggatctgatg 480 ctgctcgacctcgagggcaacgtggtgtactccgcctacaaggggccggatctcgggaca 540 aacatcgtcaacggcccctatcgcaaccgggaactgtcggaagcctacgagaaggcggtc 600 gcgtcgaactcgatcgactatgtcggtgtcaccgacttcgggtggtacctgcctgccgag 660 gaaccgaccgcctggttcctgtccccggtcgggttgaaggaccgagtcgacggtgtgatg 720 gcggtccagttccccggaattc 742 <210> 134 <211> 282 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (145) .. . (145) <221> UNSURE
<222> (151) . . . (151) <400> 134.
Glu Gln Pro Phe Arg Leu Gly Asp Trp Ile Thr Val Pro Thr Ala Ala Gly Arg Pro Ser Ala His Gly Arg Val Val Glu Val Asn Trp Arg Ala Thr His Ile Asp Thr Gly Gly Asn Leu Leu Val Met Pro Asn Ala Glu Leu Ala Gly Ala Ser Phe Thr Asn Tyr Ser Arg Pro Val Gly Glu His Arg Leu Thr Val Val Thr Thr Phe Asn Ala Ala Asp Thr Pro Asp Asp Val Cys Glu Met Leu Ser Ser Val Ala Ala Ser Leu Pro Glu Leu Arg Thr Asp Gly Gln Ile Ala Thr Leu Tyr Leu Gly Ala Ala Glu Tyr Glu Lys Ser Ile Pro Leu His Thr Pro Ala Val Asp Asp Ser Val Arg Ser Thr Tyr Leu Arg Trp Val Trp Tyr Ala Ala Arg Arg Gln Glu Leu Arg Xaa Asn Gly Val Ala Asp Xaa Phe Asp Thr Pro Glu Arg Ile Ala Ser Ala Met Arg Ala Val Ala Ser Thr Leu Arg Leu Ala Asp Asp Glu Gln Gln Glu Ile Ala Asp Val Val Arg Leu Val Arg Tyr Gly Asn Gly Glu Arg Leu Gln Gln Pro Gly Gln Val Pro Thr Gly Met Arg Phe Ile Val Asp Gly Arg Val Ser Leu Ser Val Ile Asp Gln Asp Gly Asp Val Ile Pro Ala Arg Val Leu Glu Arg Gly Asp Phe Leu Gly Gln Thr Thr Leu Thr Arg Glu Pro Val Leu Ala Thr Ala His Ala Leu Glu Glu Val Thr Val Leu Glu Met Ala Arg Asp Glu Ile Glu Arg Leu Val His Arg Lys Pro Ile Leu Leu His Val Ile Gly Ala Val <210> 135 <211> 247 <212> PRT
<213> Mycobacterium vaccae <400> 135 Gly Tyr Gln Ser Gly Arg Ser Ser Leu Arg Ala Ser Val Phe Asp Arg Leu Thr Asp Ile Arg Glu Ser Gln Ser Arg Gly Leu Glu Asn Gln Phe Ala Asp Leu Lys Asn Ser Met Val Ile Tyr Ser Arg Gly Ser Thr Ala Thr Glu Ala Ile Gly Ala Phe Ser Asp Gly Phe Arg Gln Leu Gly Asp Ala Thr Ile Asn Thr Gly Gln Ala Ala Ser Leu Arg Arg Tyr Tyr Asp Arg Thr Phe Ala Asn Thr Thr Leu Asp Asp Ser Gly Asn Arg Val Asp Val Arg Ala Leu Ile Pro Lys Ser Asn Pro Gln Arg Tyr Leu Gln Ala Leu Tyr Thr Pro Pro Phe Gln Asn Trp Glu Lys Ala Ile Ala Phe Asp Asp Ala Arg Asp Gly Ser Ala Trp Ser Ala Ala Asn Ala Arg Phe Asn Glu Phe Phe Arg Glu Ile Val His Arg Phe Asn Phe Glu Asp Leu Met Leu Leu Asp Leu Glu Gly Asn Val Val Tyr Ser Ala Tyr Lys Gly Pro Asp Leu Gly Thr Asn Ile Val Asn Gly Pro Tyr Arg Asn Arg Glu Leu Ser Glu Ala Tyr Glu Lys Ala Val Ala Ser Asn Ser Ile Asp Tyr Val Gly Val Thr Asp Phe Gly Trp Tyr Leu Pro Ala Glu Glu Pro Thr Ala Trp Phe Leu Ser Pro Val Gly Leu Lys Asp Arg Val Asp Gly Val Met Ala Val Gln Phe Pro Gly Ile <210> 136 <211> 45 <212> DNA
<213> Mycobacterium vaccae <220>
<221> unsure <222> (18)...(18) <400> 136 atgagcgaaa tcgcccgncc ctggcgggtt ctggcatgtg gcatc 45 <210> 137 <211> 340 <212> DNA
<213> Mycobacterium vaccae <220>
<221> unsure <222> (273)...(273) <221> unsure <222> (286)...(286) <400> 137 gccaccggcg gcgccgccgc ggtgcccgcc ggggtgagcg ccccggcggt cgcgccggcc 60 cccgcgatgc ccgcccgccc ggtgtccacg atcgcgccgg cgacctcggg cacgctcagc 120 gagtttttcg ccgccaaggg cgtcacgatg gagccgcagt ccagccgcga cttccgcgcc 180 ctcaacatcg tgctgccgaa gccgcggggc tgggagcaca tcccggaccc gaacgtgccg 240 gacgcgttcg cggtgctggc cgaccgggtc agnggtaaag gtcagnagtc gacaaacgcc 300 cacgtggtgg tcgacaaaca cgtaggcgag ttcgacggca 340 <210> 138 <211> 235 <212> DNA
<213> Mycobacterium vaccae <220>
<221> unsure <222> (16)...(16) <400> 138 ggtgaccacc agcgtngaac aggtcgttgc cgaagecgcg gaggccaccg acgcgattgt 60 caacggcttc aaggtcagcg ttccgggtcc gggtccggcc gcaccgccac ctgcacccgg 120 tgcccccggt gtcccgcccg cccccggcgc cccggcgctg ccgctggccg tcgcaccacc 180 cccggctccc gctgttcccg ccgtggcgcc cgcgccacag ctgctgggac tgcag 235 <210> 139 <211> 15 <212> PRT
<213> Mycobacterium vaccae <400> 139 Met Ser Glu Ile Ala Arg Pro Trp Arg Val Leu Ala Cys Gly Ile <210> 140 <211> 113 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (96)...(96) <400> 140 Ala Thr Gly Gly Ala Ala Ala Val Pro Ala Gly Val Ser Ala Pro Ala Val Ala Pro Ala Pro Ala Met Pro Ala Arg Pro Val Ser Thr Ile Ala Pro Ala Thr Ser Gly Thr Leu Ser Glu Phe Phe Ala Ala Lys Gly Val Thr Met Glu Pro Gln Ser Ser Arg Asp Phe Arg Ala Leu Asn Ile Val Leu Pro Lys Pro Arg Gly Trp Glu His Ile Pro Asp Pro Asn Val Pro Asp Ala Phe Ala Val Leu Ala Asp Arg Val Gly Gly Lys Gly Gln Xaa Ser Thr Asn Ala His Val Val Val Asp Lys His Val Gly Glu Phe Asp Gly <210> 141 <211> 73 <212> PRT
<213> Mycobacterium vaccae <400> 141 Val Thr Thr Ser Val Glu Gln Val Val Ala Ala Ala Asp Ala Thr Glu Ala Ile Val Asn Gly Phe Lys Val Ser Val Pro Gly Pro Gly Pro Ala Ala Pro Pro Pro Ala Pro Gly Ala Pro Gly Val Pro Pro Ala Pro Gly Ala Pro Ala Leu Pro Leu Ala Val Ala Pro Pro Pro Ala Pro Ala Val Pro Ala Val Ala Pro Ala Pro Gln Leu <210> 142 <211> 273 <212> DNA
<213> Mycobacterium vaccae <400> 142 gcgacctacg tgcagggggg tctcggccgc atcgaggccc gggtggccga cagcggatac 60 wo 99r~z63a pcrrnz9siooi89 agcaacgccg cggccaaggg ctacttcccg ctgagcttca ccgtcgccgg catcgaccag 120 aacggtccga tcgtgaccgc caacgtcacc gcggcggccc cgacgggcgc cgtggccacc 180 cagccgctga cgttcatcgc cgggccgagc ccgaccggat ggcagctgtc caagcagtcc 240 gcactggccc tgatgtccgc ggtcatcgcc gca 273 <210> 143 <211> 91 <212> PRT
<213> Mycobacterium vaccae <400> 143 Ala Thr Tyr Val Gln Gly Gly Leu Gly Arg Ile Glu Ala Arg Val Ala Asp Ser Gly Tyr Ser Asn Ala Ala Ala Lys Gly Tyr Phe Pro Leu Ser Phe Thr Val Ala Gly Ile Asp Gln Asn Gly Pro Ile Val Thr Ala Asn Val Thr Ala Ala Ala Pro Thr Gly Ala Val Ala Thr Gln Pro Leu Thr Phe Ile Ala Gly Pro Ser Pro Thr Gly Trp Gln Leu Ser Lys Gln Ser Ala Leu Ala Leu Met Ser Ala Val Ile Ala Ala <210> 144 <211> 554 <212> DNA
<213> Mycobacterium vaccae <400> 144 gatgtcacgcccggagaatgtaacgttcgaccggagaacgccgtcggcacaacgagttac 60 gtttgagcacttcagatctcggttaccttggatttcaggcgggggaagcagtaaccgatc 120 caagattcgaaggacccaaacaacatgaaattcactggaatgaccgtgcgcgcaagccgc 180 gcgccctggccggcgtcggggcggcatgtctgttcggcggcgtggccgcggcaaccgtgg 240 cggcacagatggcgggcgcccagccggccgagtgcaacgccagctcactcaccggcaccg 300 tcagctcggtgaccggtcaggcgcgtcagtacctagacacccacccgggcgccaaccagg 360 ccgtcaccgcggcgatgaaccagccgcggcccgaggccgaggcgaacctgcggggctact 420 tcaccgccaacccggcggagtactacgacctgcggggcatcctcgccccgatcggtgacg 480 cgcagcgcaactgcaacatcaccgtgctgccggtagagctgcagacggcctacgacacgt 540 tcatggccggctga 554 <210> 145 <211> 136 <212> PRT
<213> Mycobacterium vaccae <400> 145 Met Lys Phe Thr Gly Met Thr Val Arg Ala Ser Arg Arg Ala Leu Ala Gly Val Gly Ala Ala Cys Leu Phe Gly Gly Val Ala Ala Ala Thr Val Ala Ala Gln Met Ala Gly Ala Gln Pro Ala Glu Cys Asn Ala Ser Ser Leu Thr Gly Thr Val Ser Ser Val Thr Gly Gln Ala Arg Gln Tyr Leu Asp Thr His Pro Gly Ala Asn Gln Ala Val Thr Ala Ala Met Asn Gln Pro Arg Pro Glu Ala Glu Ala Asn Leu Arg Gly Tyr Phe Thr Ala Asn Pro Ala Glu Tyr Tyr Asp Leu Arg Gly Ile Leu Ala Pro Ile Gly Asp 100 . 105 110 Ala Gln Arg Asn Cys Asn Ile Thr Val Leu Pro Val Glu Leu Gln Thr Ala Tyr Asp Thr Phe Met Ala Gly <210> 146 <211> 808 <212> DNA
<213> Mycobacterium vaccae <220>
<221> unsure <222> (15)...(15) <400> 146 ccaagtgtgacgcgngtgtgacggtagacgttccgaccaatccaacgacgccgcagctgg 60 gaatcacccgtgtgccaattcagtgcgggcaacggtgtccgtccacgaagggattcagga 120 aatgatgacaactcgccggaagtcagccgcagtggcgggaatcgctgcggtggccatcct 180 cggtgcggccgcatgttcgagtgaggacggtgggagcacggcctcgtcggccagcagcac 240 ggcctcctccgcgatggagtccgcgaccgacgagatgaccacgtcgtcggcggccccttc 300 ggccgaccctgcggccaacctgatcggctccggctgcgcggcctacgccgagcaggtccc 360 cgaaggtcccgggtcggtggccgggatggcagccgatccggtgacggtggcggcgtcgaa 420 caacccgatgctgcagacgctgtcccaggcgctgtccggccagctcaatccgcaggtcaa 480 tctcgtcgacaccctcgacggcggtgagttcaccgtgttcgcgccgaccgacgacgcgtt 540 cgccaagatcgatccggccacgctggagaccctcaagacggactccgacatgctgaccaa 600 catcctgacctaccacgtcgtgcccggccaggccgcgcccgatcaggtggtcggcgagca 660 tgtgacggtggagggggcgccggtcacggtgtccgggatggccgaccagctcaaggtcaa 720 cgacgcgtcggtggtgtgcggtggggtgcagaccgccaacgcgacggtgtatctgatcga 780 caccgtgctgatgccgccggcagcgtag 808 <210> 147 <211> 228 <212> PRT
<213> Mycobacterium vaccae <400> 147 Met Met Thr Thr Arg Arg Lys Ser Ala Ala Val Ala Gly Ile Ala Ala Val Ala Ile Leu Gly Ala Ala Ala Cys Ser Ser Glu Asp Gly Gly Ser Thr Ala Ser Ser Ala Ser Ser Thr Ala Ser Ser Ala Met Glu Ser Ala Thr Asp Glu Met Thr Thr Ser Ser Ala Ala Pro Ser Ala Asp Pro Ala Ala Asn Leu Ile Gly Ser Gly Cys Ala Ala Tyr Ala Glu Gln Val Pro 65 70 75 g0 Glu Gly Pro Gly Ser Val Ala Gly Met Ala Ala Asp Pro Val Thr Val Ala Ala Ser Asn Asn Pro Met Leu Gln Thr Leu Ser Gln Ala Leu Ser Gly Gln Leu Aan Pro Gln Val Asn Leu Val Asp Thr Leu Asp Gly Gly Glu Phe Thr Val Phe Ala Pro Thr Asp Asp Ala Phe Ala Lys Ile Asp Pro Ala Thr Leu Glu Thr Leu Lys Thr Asp Ser Asp Met Leu Thr Asn Ile Leu Thr Tyr His Val Val Pro Gly Gln Ala Ala Pro Asp Gln Val Val Gly Glu His Val Thr Val Glu Gly Ala Pro Val Thr Val Ser Gly Met Ala Asp Gln Leu Lys Val Asn Asp Ala Ser Val Val Cys Gly Gly Val Gln Thr Ala Asn Ala Thr Val Tyr Leu Ile Asp Thr Val Leu Met Pro Pro Ala Ala <210> 148 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <221> unsure <222> (12)...(12) <221> unsure <222> (17)...(17) <400> 148 gcsccsgtsg gnccggntgy gc 22 <210> 149 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <221> unsure <222> (10) . . . (10) <221> unsure <222> (13)...(13) <221> unsure <222> (16)...(16) <221> unsure <222> (20)...(20) <400> 149 rtasgcsgcn gtngcnacng g 21 <210> 150 <211> 102 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 150 gcccccgtcg gccccggctg tgcggcctac gtgcaacagg tgccggacgg gccgggatcg 60 gtgcagggca tggcgagctc gcccgtagcg accgccgcgt at 102 <210> 151 <211> 683 <212> DNA
<213> Mycobacterium vaccae <400>

gcccgccaactaaaaccgccgatcatccactgcaggaaggaatctcacgatcatgaacat 60 cagcatgaaaactcttgccggagcgggtttcgcgatgaccgccgccgtcggtctgtcgct 120 gggtaccgcaggcagcgccgcagccgcgccggtcggaccggggtgtgcggcctacgtgca 180 acaggtgccggacgggccgggatcggtgcagggcatggcgagctcgccggtggccaccgc 240 ggcggccgacaacccgctgctcaccacgctctcgcaggcgatctcgggtcagctcaaccc 300 gaacgtcaatctcgtcgacacgttcaacggcggccagttcaccgtgttcgcgccgaccaa 360 tgacgccttcgccaagatcgatccggccacgctggagaccctcaagaccgattccgacct 420 gctgaccaagatcctcacctaccacgtcgtgcccggccaggccgcgcccgatcaggtggt 480 cggcgagcatgtgacggtggagggggcgccggtcacggtgtccgggatggccgaccagct 540 caaggtcaacgacgcgtcggtggtgtgcggtggggtgcagaccgccaacgcgacggtgta 600 tctgatcgacaccgtgctgatgccgccggcagcgtagccgggcggcaccacagaagaggg 660 tcccccgcacccggcctcccccg 683 <210> 152 <211> 231 <212> PRT
<213> Mycobacterium vaccae <400> 152 Asp Thr Val Leu Met Pro Pro Ala Asn Asn Arg Arg Ser Ser Thr Ala Gly Arg Asn Leu Thr Ile Met Asn Ile Ser Met Lys Thr Leu Ala Gly Ala Gly Phe Ala Met Thr Ala Ala Val Gly Leu Ser Leu Gly Thr Ala Gly Ser Ala Ala Ala Ala Pro Val Gly Pro Gly Cys Ala Ala Tyr Val Gln Gln Val Pro Asp Gly Pro Gly Ser Val Gln Gly Met Ala Ser Ser Pro Val Ala Thr Ala Ala Ala Asp Asn Pro Leu Leu Thr Thr Leu Ser Gln Ala Ile Ser Gly Gln Leu Asn Pro Asn Val Asn Leu Val Asp Thr Phe Asn Gly Gly Gln Phe Thr Val Phe Ala Pro Thr Asn Asp Ala Phe Ala Lys Ile Asp Pro Ala Thr Leu Glu Thr Leu Lys Thr Asp Ser Asp Leu Leu Thr Lys Ile Leu Thr Tyr His Val Val Pro Gly Gln Ala Ala Pro Asp Gln Val Val Gly Glu His Val Thr Val Glu Gly Ala Pro Val Thr Val Ser Gly Met Ala Asp Gln Leu Lys Val Asn Asp Ala Ser Val Val Cys Gly Gly Val Gln Thr Ala Asn Ala Thr Val Tyr Leu Ile Asp Thr Val Leu Met Pro Pro Ala Ala Pro Gly Gly Thr Thr Glu Glu Gly Pro Pro His Pro Ala Ser Pro <210> 153 <211> 1125 <212> DNA
<213> Mycobacterium vaccae <220>
<221> unsure <222> (358)...(358) <400> 153 atgcaggtgcggcgtgttctgggcagtgtcggtgcagcagtcgcggtttcggccgcgtta60 tggcagacgggggtttcgataccgaccgcctcagcggatccgtgtccggacatcgaggtg120 atcttcgcgcgcgggaccggtgcggaacccggcctcgggtgggtcggtgatgcgttcgtc180 aacgcgctgcggcccaaggtcggtgagcagtcggtgggcacctacgcggtgaactacccg240 gcaggattcggacttcgacaaatcggcgcccatgggcgcggccgacgcatcggggcgggt300 gcagtggatggccgacaactgcccggacaccaagcttgtcctgggcggcatgtcgcangg360 cgccggcgtcatcgacctgatcaccgtcgatccgcgaccgctgggccggttcacccccac420 cccgatgccgccccgcgtcgccgaccacgtggccgccgttgtggtcttcggaaatccgtt480 gcgcgacatccgtggtggcggtccgctgccgcagatgagcggcacctacgggccgaagtc540 gatcgatctgtgtgcgctcgacgatccgttctgctcgcccggcttcaacctgccggccca600 cttcgcctacgccgacaacggcatggtggaggaagccgcgaacttcgcccgcctggaacc660 gggccagagcgtcgagctgcccgaggcgccctacctgcacctgttcgtcccgcggggcga720 ggtaacgctggaggacgccggaccgctgcgcgaaggcgacgcagtgcgtttcaccgcatc780 gggcggccagcgggtgaccgccaccgcgcccgcggagatcctcgtctgggagatgcatgc840 gggactcggtgcggcataagcgaataggagtcctgctggccggcgcagcactgctcgccg900 gatgcacatccgaacctggacccgggccgtcggcggcaccggccccgacgagcacaaccg960 agagcgcacccggtcccggactcgtcccggtgaccgtcgcggtcgacgaacctctggccg1020 acgcgccgttcgaccagccccgggaggccctggtgccgcagggttggacgctgtcggtgt1080 gggcgcggaccgcccggccgcggctggccgcgtgggccccggacg 1125 <210> 154 <211> 748 <212> PRT
<213> Mycobacterium vaccae <220>
<221> iJNSURE
<222> (119)...(119) <400> 154 Met Gln Val Arg Arg Val Leu Gly Ser Val Gly Ala Ala Val Ala Val Ser Ala Ala Leu Trp Gln Thr Gly Val Ser Ile Pro Thr Ala Ser Ala Asp Pro Cys Pro Asp Ile Glu Val Ile Phe Ala Arg Gly Thr Gly Ala Glu Pro Gly Leu Gly Trp Val Gly Asp Ala Phe Val Asn Ala Leu Arg Pro Lys Val Gly Glu Gln Ser Val Gly Thr Tyr Ala Val Asn Tyr Pro Ala Gly Phe Asp Phe Asp Lys Ser Ala Pro Met GIy Ala Ala Asp Ala Ser Gly Arg Val Gln Trp Met Ala Asp Asn Cys Pro Asp Thr Lys Leu Val Leu Gly Gly Met Ser Xaa Gly Ala Gly Val Ile Asp Leu Ile Thr Val Asp Pro Arg Pro Leu Gly Arg Phe Thr Pro Thr Pro Met Pro Pro Arg Val Ala Asp His Val Ala Ala Val Val Val Phe Gly Asn Pro Leu Arg Asp Ile Arg Gly Gly Gly Pro Arg Leu Glu Pro Arg Gly Leu Asn Met Glu Thr Ser Glu Arg Gly Leu Tyr Thr His Arg Thr Tyr Arg Gly Leu Tyr Pro Arg Leu Tyr Ser Ser Glu Arg Ile Leu Glu Ala Ser Pro Leu Glu Cys Tyr Ser Ala Leu Ala Leu Glu Ala Ser Pro Ala Ser Pro Pro Arg Pro His Glu Cys Tyr Ser Ser Glu Arg Pro Arg Gly Leu Tyr Pro His Glu Ala Ser Asn Leu Glu Pro Arg Ala Leu Ala His Ile Ser Pro His Glu Ala Leu Ala Thr Tyr Arg Ala Leu Ala Ala Ser Pro Ala Ser Asn Gly Leu Tyr Met Glu Thr Val Ala Leu Gly Leu Gly Leu Ala Leu Ala Ala Leu Ala Ala Ser Asn Pro His Glu Ala Leu Ala Ala Arg Gly Leu Glu Gly Leu Pro Arg Gly Leu Tyr Gly Leu Asn Ser Glu Arg Val Ala Leu Gly Leu Leu Glu Pro Arg Gly Leu Ala Leu Ala Pro Arg Thr Tyr Arg Leu Glu His Ile Ser Leu Glu Pro His Glu Val Ala Leu Pro Arg Ala Arg Gly Gly Leu Tyr Gly Leu Val Ala Leu Thr His Arg Leu Glu Gly Leu Ala Ser Pro Ala Leu Ala Gly Leu Tyr Pro Arg Leu Glu Ala Arg Gly Gly Leu Gly Leu Tyr Ala Ser Pro Ala Leu Ala Val Ala Leu Ala Arg Gly Pro His Glu Thr His Arg Ala Leu Ala Ser Glu Arg Gly Leu Tyr Gly Leu Tyr Gly Leu Asn Ala Arg Gly Val Ala Leu Thr His Arg Ala Leu Ala Thr His Arg Ala Leu Ala Pro Arg Ala Leu Ala Gly Leu Ile Leu Glu Leu Glu Val Ala Leu Thr Arg Pro Gly Leu Met Glu Thr His Ile Ser Ala Leu Ala Gly Leu Tyr Leu Glu Gly Leu Tyr Ala Leu Ala Ala Leu Ala Ala Leu Ala Ala Ser Asn Ala Arg Gly Ser Glu Arg Pro Arg Ala Leu Ala Gly Leu Tyr Ala Arg Gly Ala Arg Gly Ser Glu Arg Thr His Arg Ala Leu Ala Ala Arg Gly Ala Arg Gly Met Glu Thr His Ile Ser Ile Leu Glu Ala Arg Gly Thr His Arg Thr Arg Pro Thr His Arg Ala Arg Gly Ala Leu Ala Val Ala Leu Gly Leu Tyr Gly Leu Tyr Thr His Arg Gly Leu Tyr Pro Arg Ala Ser Pro Gly Leu His Ile Ser Ala Ser Asn Ala Arg Gly Gly Leu Ala Arg Gly Thr His Arg Ala Arg Gly Ser Glu Arg Ala Arg Gly Thr His Arg Ala Arg Gly Pro Arg Gly Leu Tyr Ala Ser Pro Ala Arg Gly Ala Arg Gly Gly Leu Tyr Ala Arg Gly Ala Arg Gly Thr His Arg Ser Glu Arg Gly Leu Tyr Ala Arg Gly Ala Arg Gly Ala Leu Ala Val Ala Leu Ala Arg Gly Pro Arg Ala Leu Ala Pro Arg Gly Leu Tyr Gly Leu Tyr Pro Arg Gly Leu Tyr Ala Leu Ala Ala Leu Ala Gly Leu Tyr Leu Glu Ala Ser Pro Ala Leu Ala Val Ala Leu Gly Leu Tyr Val Ala Leu Gly Leu Tyr Ala Leu Ala Ala Ser Pro Ala Arg Gly Pro Arg Ala Leu Ala Ala Leu Ala Ala Leu Ala Gly Leu Tyr Ala Arg Gly Val Ala Leu Gly Leu Tyr Pro Arg Gly Leu Tyr Ala Arg Gly Pro Arg Gly Leu Tyr <210> 155 <211> 666 <212> DNA
<213> Mycobacterium vaccae <400> 155 atgaaggcaaatcattcgggatgctacaaatccgccggcccgatatggtcgcatccatcg 60 ccgctttgttcgcccgcactggcaccatctcatgcaggtctggacaatgagctgagcctg 120 ggcatccacggccagggcccggaacgactgaccattcagcagtgggacaccttcctcaac 180 ggcgtcttcccgttggaccgcaaccggttgacccgggagtggttccactcgggcaaggcg 240 acctacgtcgtggccggtgaaggtgccgacgagttcgagggcacgctggagctgggctac 300 caggtgggctttccgtggtcgctgggcgtgggcatcaacttcagctacaccaccccgaac 360 atcacgtacgacggttacggcctcaacttcgccgacccgctgctgggcttcggtgattcc 420 atcgtgaccccgccgctgttcccgggtgtctcgatcacggcggacctgggcaacggcccc 480 ggcatccaggaggtcgcgaccttctccgtggacgtggccggccccggtggttccgtggtg 540 gtgtccaacgcgcacggcacggtcaccggtgctgccggtggtgtgctgctgcgtccgttc 600 gcccgcctgatctcgtcgaccggcgacagcgtcaccacctacggcgcaccctggaacatg 660 aactga 666 <210> 156 <211> 221 <212> PRT
<213> Mycobacterium vaccae <400> 156 Met Lys Ala Asn His Ser Gly Cys Tyr Lys Ser Ala Gly Pro Ile Trp Ser His Pro Ser Pro Leu Cys Ser Pro Ala Leu Ala Pro Ser His Ala Gly Leu Asp Asn Glu Leu Ser Leu Gly Val His Gly Gln Gly Pro Glu His Leu Thr Ile Gln Gln Trp Asp Thr Phe Leu Asn Gly Val Phe Pro Leu Asp Arg Asn Arg Leu Thr Arg Glu Trp Phe His Ser Gly Lys Ala Thr Tyr Val Val Ala Gly Glu Gly Ala Asp Glu Phe Glu Gly Thr Leu Glu Leu Gly Tyr His Val Gly Phe Pro Trp Ser Leu Gly Val Gly Ile Asn Phe Ser Tyr Thr Thr Pro Asn Ile Thr Tyr Asp Gly Tyr Gly Leu Asn Phe Ala Asp Pro Leu Leu Gly Phe Gly Asp Ser Ile Val Thr Pro Pro Leu Phe Pro Gly Val Ser Ile Thr Ala Asp Leu Gly Asn Gly Pro Gly Ile Gln Glu Val Ala Thr Phe Ser Val Asp Val Ala Gly Pro Gly Gly Ser Val Val Val Ser Asn Ala His Gly Thr Val Thr Gly Ala Ala Gly Gly Val Leu Leu Arg Pro Phe Ala Arg Leu Ile Ser Ser Thr Gly Asp Ser Val Thr Thr Tyr Gly Ala Pro Trp Asn Met Asn <210> 157 <211> 480 <212> DNA
<213> Mycobacterium vaccae <400> 157 aacggctgggacatcaacacccctgcgttcgagtggttctacgagtccggcttgtcgacg 60 atcatgccggtcggcggacagtccagcttctacagcgactggtaccagccgtctcggggc 120 aacgggcagaactacacctacaagtgggagacgttcctgacccaggagctgccgacgtgg 180 ctggaggccaaccgcggagtgtcgcgcaccggcaacgcgttcgtcggcctgtcgatggcg 240 ggcagcgcggcgctgacctacgcgatccatcacccgcagcagttcatctacgcctcgtcg 300 ctgtcaggcttcctgaacccgtccgagggctggtggccgatgctgatcgggctggcgatg 360 aacgacgcaggcggcttcaacgccgagagcatgtggggcccgtcctcggacccggcgtgg 420 aagcgcaacgacccgatggtcaacatcaaccagctggtggccaacaacacccggatctgg 480 <210> 158 <211> 161 <212> PRT
<213> Mycobacterium vaccae <400> 158 Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Phe Tyr Glu Ser Gly Leu Ser Thr Ile Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Gln Pro Ser Arg Gly Asn Gly Gln Asn Tyr Thr Tyr Lys Trp Glu Thr Phe Leu Thr Gln Glu Leu Pro Thr Trp Leu Glu Ala Asn Arg Gly Val Ser Arg Thr Gly Asn Ala Phe Val Gly Leu Ser Met Ala Gly Ser Ala Ala Leu Thr Tyr Ala Ile His His Pro Gln Gln Phe Ile Tyr Ala Ser Ser Leu Ser Gly Phe Leu Asn Pro Ser Glu Gly Trp Trp Pro Met Leu Ile Gly Leu Ala Met Asn Asp Ala Gly Gly Phe Asn Ala Glu Ser Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Lys Arg Asn Asp Pro Met Val Asn Ile Asn Gln Leu Val Ala Asn Asn Thr Arg Ile Trp Ile <210> 159 <211> 1626 <212> DNA
<213> Mycobacterium vaccae <400> 159 atggccaaga caattgcgta tgacgaagag gcccgccgtg gcctcgagcg gggcctcaac 60 gccctcgcag acgccgtaaa ggtgacgttg ggcccgaagg gtcgcaacgt cgtgctggag 120 aagaagtggggcgcccccacgatcaccaacgatggtgtgtccatcgccaaggagatcgag180 ctggaggacccgtacgagaagatcggcgctgagctggtcaaagaggtcgccaagaagacc240 gacgacgtcgcgggcgacggcaccaccaccgccaccgtgctcgctcaggctctggttcgc300 gaaggcctgcgcaacgtcgcagccggcgccaacccgctcggcctcaagcgtggcatcgag360 aaggctgtcgaggctgtcacccagtcgctgctgaagtcggccaaggaggtcgagaccaag420 gagcagatttctgccaccgcggcgatttccgccggcgacacccagatcggcgagctcatc480 gccgaggccatggacaaggtcggcaacgagggtgtcatcaccgtcgaggagtcgaacacc540 ttcggcctgcagctcgagctcaccgagggtatgcgcttcgacaagggctacatctcgggt600 tacttcgtgaccgacgccgagcgccaggaagccgtcctggaggatccctacatcctgctg660 gtcagctccaaggtgtcgaccgtcaaggatctgctcccgctgctggagaaggtcatccag720 gccggcaagccgctgctgatcatcgccgaggacgtcgagggcgaggccctgtccacgctg780 gtggtcaacaagatccgcggcaccttcaagtccgtcgccgtcaaggctccgggcttcggt840 gaccgccgcaaggcgatgctgcaggacatggccatcctcaccggtggtcaggtcgtcagc900 gaaagagtcgggctgtccctggagaccgccgacgtctcgctgctgggccaggcccgcaag960 gtcgtcgtcaccaaggacgagaccaccatcgtcgagggctcgggcgattccgatgccatc1020 gccggccgggtggctcagatccgcgccgagatcgagaacagcgactccgactacgaccgc1080 gagaagctgcaggagcgcctggccaagctggccggcggtgttgcggtgatcaaggccgga1140 gctgccaccgaggtggagctcaaggagcgcaagcaccgcatcgaggacgccgtccgcaac1200 gcgaaggctgccgtcgaagagggcatcgtcgccggtggcggcgtggctctgctgcagtcg1260 gctcctgcgctggacgacctcggcctgacgggcgacgaggccaccggtgccaacatcgtc1320 cgcgtggcgctgtcggctccgctcaagcagatcgccttcaacggcggcctggagcccggc1380 gtcgttgccgagaaggtgtccaacctgcccgcgggtcacggcctcaacgccgcgaccggt1440 gagtacgaggacctgctcaaggccggcgtcgccgacccggtgaaggtcacccgctcggcg1500 ctgcagaacgcggcgtccatcgcggctctgttcctcaccaccgaggccgtcgtcgccgac1560 aagccggagaaggcgtccgcacccgcgggcgacccgaccggtggcatgggcggtatggac1620 ttctaa 1626 <210> 160 <211> 541 <212> PRT
<213> Mycobacterium vaccae <400> 160 Met Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu Glu Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu Gly Pro Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala Pro Thr Ile Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu Leu Glu Asp Pro Tyr Glu Lys Ile Gly Ala Glu Leu Val Lys Glu Val Ala Lys Lys Thr Asp Asp Val Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln Ala Leu Val Arg Glu Gly Leu Arg Asn Val Ala Ala Gly Ala Asn Pro Leu Gly Leu Lys Arg Gly Ile Glu Lys Ala Val Glu Ala Val Thr Gln Ser Leu Leu Lys Ser Ala Lys Glu Val Glu Thr Lys Glu Gln Ile Ser Ala Thr Ala Ala Ile Ser Ala Gly Asp Thr Gln Ile Gly Glu Leu Ile Ala Glu Ala Met Asp Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu Glu Ser Asn Thr Phe Gly Leu Gln Leu Giu Leu Thr Glu Gly Met Arg Phe Asp Lys Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Ala Glu Arg Gln Glu Ala Val Leu Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gln Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gl~r Thr Phe Lys Ser Val Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln Asp Met Ala Ile Leu Thr Gly Gly Gln Val Val Ser Glu Arg Val Gly Leu Ser Leu Glu Thr Ala Asp Val Ser Leu Leu Gly Gln Ala Arg Lys Val Val Val Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ser Gly Asp Ser Asp Ala Ile Ala Gly Arg Val Ala Gln Ile Arg Ala Glu Ile Glu Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg Leu Ala Lys Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala Ala Thr Glu Val Glu Leu Lys Glu Arg Lys His Arg Ile Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val Glu Glu Gly Ile Val Ala Gly Gly Gly Val Ala Leu Leu Gln Ser Ala Pro Ala Leu Asp Asp Leu Gly Leu Thr Gly Asp Glu Ala Thr Gly Ala Asn Ile Val Arg Val Ala Leu Ser Ala Pro Leu Lys Gln Ile Ala Phe Asn Gly Gly Leu Glu Pro Gly Val Val Ala Glu Lys Val Ser Asn Leu Pro Ala Gly His Gly Leu Asn Ala Ala Thr Gly Glu Tyr Glu Asp Leu Leu Lys Ala Gly Val Ala Asp Pro Val Lys Val Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Ala Leu Phe Leu Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu Lys Ala Ser Ala Pro Ala Gly Asp Pro Thr Gly Gly Met Gly Gly Met Asp Phe <210> 161 <211> 985 <212> DNA
<213> Mycobacterium vaccae <400>

ggatccctacatcctgctggtcagctccaaggtgtcgaccgtcaaggatctgctcccgct 60 gctggagaaggtcatccaggccggcaagccgctgctgatcatcgccgaggacgtcgaggg 120 cgaggccctgtccacgctggtggtcaacaagatccgcggcaccttcaagtccgtcgccgt 180 caaggctccgggcttcggtgaccgccgcaaggcgatgctgcaggacatggccatcctcac 240 cggtggtcaggtcgtcagcgaaagagtcgggctgtccctggagaccgccgacgtctcgct 300 gctgggccaggcccgcaaggtcgtcgtcaccaaggacgagaccaccatcgtcgagggctc 360 gggcgattccgatgccatcgccggccgggtggctcagatccgcgccgagatcgagaacag 420 cgactccgactacgaccgcgagaagctgcaggagcgcctggccaagctggccggcggtgt 480 tgcggtgatcaaggccggagctgccaccgaggtggagetcaaggagcgcaagcaccgcat 540 cgaggacgccgtccgcaacgcgaaggctgccgtcgaagagggcatcgtcgccggtggcgg 600 cgtggctctgctgcagtcggctcctgcgctggacgacct~ggcctgacgggcgacgaggc 660 caccggtgccaacatcgtccgcgtggcgctgtcggctccgctcaagcagatcgccttcaa 720 cggcggcctggagcccggcgtcgttgccgagaaggtgtccaacctgcccgcgggtcacgg 780 cctcaacgccgcgaccggtgagtacgaggacctgctcaaggccggcgtcgccgacccggt 840 gaaggtcacccgctcggcgctgcagaacgcggcgtccatcgcggctctgttcctcaccac 900 cgaggccgtcgtcgccgacaagccggagaaggcgtccgcacccgcgggcgacccgaccgg 960 tggcatgggcggtatggacttctaa <210> I62 <211> 327 <212> PRT
<213> Mycobacterium vaccae <400> 162 Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys Val Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gln Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu Ala Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys Ser Val Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met Leu Gln Asp Met Ala Ile Leu Thr Gly Gly Gln Val Val Ser Glu Arg Val Gly Leu Ser Leu Glu Thr Ala Asp Val Ser Leu Leu Gly Gln Ala Arg Lys Val Val Val Thr Lys Asp Glu Thr Thr Ile Val Glu Gly Ser Gly Asp Ser Asp Ala Ile Ala Gly Arg Val Ala Gln Ile Arg Ala Glu Ile Glu Asn Ser Asp Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg Leu Ala Lys Leu Ala Gly Gly Val Ala Val Ile Lys Ala Gly Ala Ala Thr Glu Val Glu Leu Lys Glu Arg Lys His Arg Ile Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val Glu Glu Gly Ile Val Ala Gly Gly Gly Val Ala Leu Leu Gln Ser Ala Pro Ala Leu Asp Asp Leu Gly Leu Thr Gly Asp Glu Ala Thr Gly Ala Asn Ile Val Arg Val Ala Leu Ser Ala Pro Leu Lys Gln Ile Ala Phe Asn Gly Gly Leu Glu Pro Gly Val Val Ala Glu Lys Val Ser Asn Leu Pro Ala Gly His Gly Leu Asn Ala Ala Thr Gly Glu Tyr Glu Asp Leu Leu Lys Ala Gly Val Ala Asp Pro Val Lys Val Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Ala Leu Phe Leu Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu Lys Ala Ser Ala Pro Ala Gly Asp Pro Thr Gly Gly Met Gly Gly Met Asp Phe <210> 163 <211> 403 <212> DNA
<213> Mycobacterium vaccae <400>

ggatccgcggcaccggctggtgacgaccaagtacaacccggcccgcacctggacggccga 60 gaactccgtcggcatcggcggcgcgtacctgtgcatctacgggatggagggccccggcgg 120 ctatcagttcgtcggccgcaccacccaggtgtggagtcgttaccgccacacggcgccgtt 180 cgaacccggaagtccctggctgctgcggtttttcgaccgaatttcgtggtatccggtgtc 240 ggccgaggagctgctggaattgcgagccgacatggccgcaggccggggctcggtcgacat 300 caccgacggcgtgttctccctcgccgagcacgaacggttcctggccgacaacgccgacga 360 catcgccgcgttccgttcccggcaggcggccgcgttctccgcc 403 <210> 164 <211> 336 <212> DNA
<213> Mycobacterium vaccae <400>

cggaccgcgtgggcggccgccggcgagttcgaccgcgccgagaaagccgc gtcgaaggcc60 accgacgccgataccggggacctggtgctctacgacggtgcgagcgggtc gacgctccgt120 tcgcgtcgagcgtgtggaaggtcgacgtcgccgtcggtgaccgggtggtg gccggacagc180 cgttgctggcgctggaggcgatgaagatggagaccgtgctgcgcgccccg gccgacgggg240 tggtcacccagatcctggtctccgctgggcatctcgtcgatcccggcacc ccactggtcg300 tggtcggcaccggagtgcgcgcatgagcgccgtcga 336 <210> 165 <211> 134 <212> PRT
<213> Mycobacterium vaccae <400> 165 Asp Pro Arg His Arg Leu Val Thr Thr Lys Tyr Asn Pro Ala Arg Thr Trp Thr Ala Glu Asn Ser Val Gly Ile Gly Gly Ala Tyr Leu Cys Ile Tyr Gly Met Glu Gly Pro Gly Gly Tyr Gln Phe Val Gly Arg Thr Thr Gln Val Trp Ser Arg Tyr Arg His Thr Ala Pro Phe Glu Pro Gly Ser Pro Trp Leu Leu Arg Phe Phe Asp Arg Ile Ser Trp Tyr Pro Val Ser 65 70 75 g0 Ala Glu Glu Leu Leu Glu Leu Arg Ala Asp Met Ala Ala Gly Arg Gly Ser Val Asp Ile Thr Asp Gly Val Phe Ser Leu Ala Glu His Glu Arg Phe Leu Ala Asp Asn Ala Asp Asp Ile Ala Ala Phe Arg Ser Arg Gln Ala Ala Ala Phe Ser Ala <210> 166 <211> 108 <212> PRT
<213> Mycobacterium vaccae <400> 166 Arg Thr Ala Trp Ala Ala Ala Gly Glu Phe Asp Arg Ala Glu Lys Ala Ala Ser Lys Ala Thr Asp Ala Asp Thr Gly Asp Leu Val Leu Tyr Asp Gly Asp Glu Arg Val Asp Ala Pro Phe Ala Ser Ser Val Trp Lys Val Asp Val Ala Val Gly Aap Arg Val Val Ala Gly Gln Pro Leu Leu Ala Leu Glu Ala Met Lys Met Glu Thr Val Leu Arg Ala Pro Ala Asp Gly Val Val Thr Gln Ile Leu Val Ser Ala Gly His Leu Val Asp Pro Gly Thr Pro Leu Val Val Val Gly Thr Gly Val Arg Ala <210> 167 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 167 atagaattcg tccgacagtg ggacctcgag c 31 <210> 168 <211> 27 <212> DNA
<213> Artificial Sequence <220>

<223> Made in a lab <400> 168 atagaattcc caccgcgtca gccgccg 2~
<210> 169 <211> 1111 <212> DNA
<213> Mycobacterium vaccae <400>

gtccgacagtgggacctcgagcaccacgtcacaggacagcggccccgccagcggcgccct60 gcgcgtctccaactggccgctctatatggccgacggtttcatcgcagcgttccagaccgc120 ctcgggcatcacggtcgactacaaagaagacttcaacgacaacgagcagtggttcgccaa180 ggtcaaggagccgttgtcgcgcaagcaggacataggcgccgacctggtgatccccaccga240 gttcatggccgcgcgcgtcaagggcctgggatggctcaatgagatcagcgaagccggcgt300 gcccaatcgcaagaatctgcgtcaggacctgttggactcgagcatcgacgagggccgcaa360 gttcaccgcgccgtacatgaccggcatggtcggtctcgcctacaacaaggcagccaccgg420 acgcgatatccgcaccatcgacgacctctgggatcccgcgttcaagggccgcgtcagtct480 gttctccgacgtccaggacggcctcggcatgatcatgctctcgcagggcaactcgccgga540 gaatccgaccaccgagtccattcagcaggcggtcgatctggtccgcgaacagaacgacag600 ggggtcagatccgtcgcttcaccggcaacgactacgccgacgacctggccgcagaaacat660 cgccatcgcgcaggcgtactccggtgacgtcgtgcagctgcaggcggacaaccccgatct720 gcagttcatcgttcccgaatccggcggcgactggttcgtcgacacgatggtgatcccgta780 caccacgcagaaccagaaggccgccgaggcgtggatcgactacatctacgaccgagccaa840 ctacgccaagctggtcgcgttcacccagttcgtgcccgcactctcggacatgaccgacga900 actcgccaaggtcgatcctgcatcggcggagaacccgctgatcaacccgtcggccgaggt960 gcaggcgaacctgaagtcgtgggcggcactgaccgacgagcagacgcaggagttcaacac1020 tgcgtacgccgccgtcaccggcggctgacgcggtggtagtgccgatgcgaggggcataaa1080 tggccctgcggacgcgaggagcataaatggc 1111 <210> 170 <211> 348 <212> PRT
<213> Mycobacterium vaccae <400> 170 Ser Asp Ser Gly Thr Ser Ser Thr Thr Ser Gln Asp Ser Gly Pro Ala Ser Gly Ala Leu Arg Val Ser Asn Trp Pro Leu Tyr Met Ala Asp Gly Phe Ile Ala Ala Phe Gln Thr Ala Ser Gly Ile Thr Val Asp Tyr Lys Glu Asp Phe Asn Asp Asn Glu Gln Trp Phe Ala Lys Val Lys Glu Pro Leu Ser Arg Lys Gln Asp Ile Gly Ala Asp Leu Val Ile Pro Thr Glu Phe Met Ala Ala Arg Val Lys Gly Leu Gly Trp Leu Asn Glu Ile Ser Glu Ala Gly Val Pro Asn Arg Lys Asn Leu Arg Gln Asp Leu Leu Asp Ser Ser Ile Asp Glu Gly Arg Lys Phe Thr Ala Pro Tyr Met Thr Gly Met Val Gly Leu Ala Tyr Asn Lys Ala Ala Thr Gly Arg Asp Ile Arg Thr Ile Asp Asp Leu Trp Asp Pro Ala Phe Lys Gly Arg Val Ser Leu Phe Ser Asp Val Gln Asp Gly Leu Gly Met Ile Met Leu Ser Gln Gly Asn Ser Pro Glu Asn Pro Thr Thr Glu Ser Ile Gln Gln Ala Val Asp Leu Val Arg Glu Gln Asn Asp Arg Gly Gln Ile Arg Arg Phe Thr Gly Asn Asp Tyr Ala Asp Asp Leu Ala Ala Gly Asn Ile Ala Ile Ala Gln Ala Tyr Ser Gly Asp Val Val Gln Leu Gln A1~ Asp Asn Pro Asp Leu Gln Phe Ile Val Pro Glu Ser Gly Gly Asp Trp Phe Val Asp Thr Met Val Ile Pro Tyr Thr Thr Gln Asn Gln Lys Ala Ala Glu Ala Trp Ile Asp Tyr Ile Tyr Asp Arg Ala Asn Tyr Ala Lys Leu Val Ala Phe Thr Gln Phe Val Pro Ala Leu Ser Asp Met Thr Asp Glu Leu Ala Lys Val Asp Pro Ala Ser Ala Glu Asn Pro Leu Ile Asn Pro Ser Ala Glu Val Gln Ala Asn Leu Lys Ser Trp Ala Ala Leu Thr Asp Glu Gln Thr Gln Glu Phe Asn Thr Ala Tyr Ala Ala Val Thr Gly Gly <210> 171 <211> 1420 <212> DNA
<213> Mycobacterium vaccae <220>
<221> unsure <222> (955) . . . (955) <221> unsure <222> (973)...(973) <400>

gatgagcagcgtgctgaactcgacctggttggcctgggccgtcgcggtcgcggtcgggtt 60 cccggtgctgctggtcgtgctgaccgaggtgcacaacgcgttgcgtcggcgcggcagcgc 120 gctggcccgcccggtgcaactcctgcgtacctacatcctgccgctgggcgcgttgctgct 180 cctgctggtacaggcgatggagatctccgacgacgccacgtcggtacggttggtcgccac 240 cctgttcggcgtcgtgttgttgacgttggtgctgtccgggctcaacgccaccctcatcca 300 gggcgcaccagaagacagctggcgcaggcggattccgtcgatcttcctcgacgtcgcgcg 360 cttcgcgctgatcgcggtcggtatcaccgtgatcatggcctatgtctggggcgcgaacgt 420 ggggggcctgttcaccgcactgggcgtcacttccatcgttcttggcctggctctgcagaa 480 ttcggtcggtcagatcatctcgggtctgctgctgctgttcgagcaaccgttccggctcgg 540 cgactggatcaccgtccccaccgcggcgggccggccgtccgcccacggccgcgtggtgga 600 agtcaactggcgtgcaacacatatcgacaccggcggcaacctgctggtaatgcccaacgc 660 -cgaactcgccggcgcgtcgttcaccaattacagccggcccgtgggagagcaccggctgac720 cgtcgtcaccaccttcaacgccgcggacacccccgatgatgtctgcgagatgctgtcgtc780 ggtcgcggcgtcgctgcccgaactgcgcaccgacggacagatcgccacgctctatctcgg840 tgcggccgaatacgagaagtcgatcccgttgcacacacccgcggtggacgactcggtcag900 gagcacgtacctgcgatgggtctggtacgccgcgcgccggcaggaacttcgcctnaacgg960 cgtcgccgacganttcgacacgccggaacggatcgcctcggccatgcgggctgtggcgtc1020 cacactgcgcttggcagacgacgaacagcaggagatcgccgacgtggtgcgtctggtccg1080 ttacggcaacggggaacgcctccagcagccgggtcaggtaccgaccgggatgaggttcat1140 cgtagacggcagggtgagtctgtccgtgatcgatcaggacggcgacgtgatcccggcgcg1200 ggtgctcgagcgtggcgacttcctggggcagaccacgctgacgcgggaaccggtactggc1260 gaccgcgcacgcgctggaggaagtcaccgtgctggagatggcccgtgacgagatcgagcg1320 cctggtgcaccgaaagccgatcctgctgcacgtgatcggggccgtgatcgccgaccggcg1380 cgcgcacgaacttcggttgatggcggactcgcaggactg~ 1420 <210> 172 <211> 471 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (318)...(318) <221> UNSURE
<222> (324)...(324) <400> 172 Met Ser Ser Val Leu Asn Ser Thr Trp Leu Ala Trp Ala Val Ala Val Ala Val Gly Phe Pro Val Leu Leu Val Val Leu Thr Glu Val His Asn Ala Leu Arg Arg Arg Gly Ser Ala Leu Ala Arg Pro Val Gln Leu Leu Arg Thr Tyr Ile Leu Pro Leu Gly Ala Leu Leu Leu Leu Leu Val Gln Ala Met Glu Ile Ser Asp Asp Ala Thr Ser Val Arg Leu Val Ala Thr Leu Phe Gly Val Val Leu Leu Thr Leu Val Leu Ser Gly Leu Asn Ala Thr Leu Ile Gln Gly Ala Pro Glu Asp Ser Trp Arg Arg Arg Ile Pro Ser Ile Phe Leu Asp VaI Ala Arg Phe Ala Leu Ile Ala Val Gly Ile Thr Val Ile Met Ala Tyr Val Trp Gly Ala Asn Val Gly Gly Leu Phe Thr Ala Leu Gly Val Thr Ser Ile Val Leu Gly Leu Ala Leu Gln Asn Ser Val Gly Gln Ile Ile Ser Gly Leu Leu Leu Leu Phe Glu Gln Pro Phe Arg Leu Gly Asp Trp Ile Thr Val Pro Thr Ala Ala Gly Arg Pro Ser Ala His Gly Arg Val Val Glu Val Asn Trp Arg Ala Thr His Ile Asp Thr Gly Gly Asn Leu Leu Val Met Pro Asn Ala Glu Leu Ala Gly Ala Ser Phe Thr Asn Tyr Ser Arg Pro Val Gly Glu His Arg Leu Thr Val Val Thr Thr Phe Asn Ala Ala Asp Thr Pro Asp Asp Val Cys Glu Met Leu Ser Ser Val Ala Ala Ser Leu Pro Glu Leu Arg Thr Asp Gly Gln Ile Ala Thr Leu Tyr Leu Gly Ala Ala Glu Tyr Glu Lys Ser Ile Pro Leu His Thr Pro Ala Val Asp Asp Ser Val Arg Ser Thr Tyr Leu Arg Trp Val Trp Tyr Ala Ala Arg Arg Gln Glu Leu Arg Xaa Asn Gly Val Ala Asp Xaa Phe Asp Thr Pro Glu Arg Ile Ala Ser Ala Met Arg Ala Val Ala Ser Thr Leu Arg Leu Ala Asp Asp Glu Gln Gln Glu Ile Ala Asp Val Val Arg Leu Val Arg Tyr Gly Asn Gly Glu Arg Leu Gln Gln Pro Gly Gln Val Pro Thr Gly Met Arg Phe Ile Val Asp Gly Arg Val Ser Leu Ser Val Ile Asp Gln Asp Gly Asp Val Ile Pro Ala Arg Val Leu Glu Arg Gly Asp Phe Leu Gly Gln Thr Thr Leu Thr Arg Glu Pro Val Leu Ala Thr Ala His Ala Leu Glu Glu Val Thr Val Leu Glu Met Ala Arg Asp Glu Ile Glu Arg Leu Val His Arg Lys Pro Ile Leu Leu His Val Ile Gly Ala Val Ile Ala Asp Arg Arg Ala His Glu Leu Arg Leu Met Asp Ser Gln Asp <210> 173 <211> 2172 <212> DNA
<213> Mycobacterium vaccae <400>

tagatgacaattctgccctggaatgcgcgaacgtctgaacacccgacgcgaaaaagacgc60 gggcgctaccacctcctgtcgcggatgagcatccagtccaagttgctgctgatgctgctt120 ctgaccagcattctctcggctgcggtggtcggtttcatcggctatcagtccggacggtcc180 tcgctgcgcgcatcggtgttcgaccgcctcaccgacatccgcgagtcgcagtcgcgcggg240 ttggagaatcagttcgcggacctgaagaactcgatggtgatttactcgcgcggcagcact300 gccacggaggcgatcggcgcgttcagcgacggtttccgtcagctcggcgatgcgacgatc360 aataccgggcaggcggcgtcattgcgccgttactacgaccggacgttcgccaacaccacc420 ctcgacgacagcggaaaccgcgtcgacgtccgcgcgctcatcccgaaatccaacccccag480 cgctatctgcaggcgctctataccccgccgtttcagaactgggagaaggcgatcgcgttc540 gacgacgcgcgcgacggcagcgcctggtcggccgccaatgccagattcaacgagttcttc600 cgcgagatcgtgcaccgcttcaacttcgaggatctgatgctgctcgacctcgagggcaac660 gtggtgtactccgcctacaaggggccggatctcgggacaaacatcgtcaacggcccctat720 cgcaaccgggaactgtcggaagcctacgagaaggcggtcgcgtcgaactcgatcgactat 780 gtcggtgtcaccgacttcgggtggtacctgcctgccgaggaaccgaccgcctggttcctg 840 tccccggtcgggttgaaggaccgagtcgacggtgtgatggcggtccagttcccgatcgcg 900 cggatcaacgaattgatgacggcgcggggacagtggcgtgacaccgggatgggagacacc 960 ggtgagaccatcctggtcggaccggacaatctgatgcgctcggactcccggctgttccgc 1020 gagaaccgggagaagttcctggccgacgtcgtcgaggggggaaccccgccggaggtcgcc 1080 gacgaatcggttgaccgccgcggcaccacgctggtgcagccggtgaccacccgctccgtc 1140 gaggaggcccaacgcggcaacaccgggacgacgatcgaggacgactatctcggccacgag 1200 gcgttacaggcgtactcaccggtggacctgccgggactgcactgggtgatcgtggccaag 1260 atcgacaccgacgaggcgttcgccccggtggcgcagttcaccaggaccctggtgctgtcg 1320 acggtgatcatcatcttcggcgtgtcgctggcggccatgctgctggcgcggttgttcgtc 1380 cgtccgatccggcggttgcaggccggcgcccagcagatcagcggcggtgactaccgcctc 1440 gctctgccggtgttgtctcgtgacgaattcggcgatctgacaacagctttcaacgacatg 1500 agtcgcaatctgtcgatcaaggacgagctgctcggcgaggagcgcgccgagaaccaacgg 1560 ctgatgctgtccctgatgcccgaaccggtgatgcagcgctacctcgacggggaggagacg 1620 atcgcccaggaccacaagaacgtcacggtgatcttcgccgacatgatgggcctcgacgag 1680 ttgtcgcgcatgttgacctccgaggaactgatggtggtggtcaacgacctgacccgccag 1740 ttcgacgccgccgccgagagtctcggggtcgaccacgtgcggacgctgcacgacgggtac 1800 ctggccagctgcgggttaggcgtgccgcggctggacaacgtccggcgcacggtcaatttc 1860 gcgatcgaaatggaccgcatcatcgaccggcacgccgccgagtccgggcacgacctgcgg 1920 ctccgcgcgggcatcgacaccgggtcggcggccagcgggctggtggggcggtccacgttg 1980 gcgtacgacatgtggggttcggcggtcgatgtcgctaaccaggtgcagcgcggctccccc 2040 cagcccggcatctacgtcacctcgcgggtgcacgaggtcatgcaggaaactctcgacttc 2100 gtcgccgccggggaggtcgtcggcgagcgcggcgtcgagacggtctggcggttgcagggc 2160 caccggcgatga 2172 <210> 174 <211> 722 <212> PRT
<213> Mycobacterium vaccae <400> 174 Met Thr Ile Leu Pro Trp Asn Ala Arg Thr Ser Glu His Pro Thr Arg Lys Arg Arg Gly Arg Tyr His Leu Leu Ser Arg Met Ser Ile Gln Ser Lys Leu Leu Leu Met Leu Leu Leu Thr Ser Ile Leu Ser Ala Ala Val Val Gly Phe Ile Gly Tyr Gln Ser Gly Arg Ser Ser Leu Arg Ala Ser Val Phe Asp Arg Leu Thr Asp Ile Arg Glu Ser Gln Ser Arg Gly Leu Glu Asn Gln Phe Ala Asp Leu Lys Asn Ser Met Val Ile Tyr Ser Arg Gly Ser Thr Ala Thr Glu Ala Ile Gly Ala Phe Ser Asp Gly Phe Arg Gln Leu Gly Asp Ala Thr Ile Asn Thr Gly Gln Ala Ala Ser Leu Arg Arg Tyr Tyr Asp Arg Thr Phe Ala Asn Thr Thr Leu Asp Asp Ser Gly Asn Arg Val Asp Val Arg Ala Leu Ile Pro Lys Ser Asn Pro Gln Arg Tyr Leu Gln Ala Leu Tyr Thr Pro Pro Phe Gln Asn Trp Glu Lys Ala lss 170 175 Ile Ala Phe Asp Asp Ala Arg Asp Gly Ser Ala Trp Ser Ala Ala Asn Ala Arg Phe Asn Glu Phe Phe Arg Glu Ile Val His Arg Phe Asn Phe Glu Asp Leu Met Leu Leu Asp Leu Glu Gly Asn Val Val Tyr Ser Ala Tyr Lys Gly Pro Asp Leu Gly Thr Asn Ile Val Asn Gly Pro Tyr Arg Asn Arg Glu Leu Ser Glu Ala Tyr Glu Lys Ala Val Ala Ser Asn Ser Ile Asp Tyr Val Gly Val Thr Asp Phe Gly Trp Tyr Leu Pro Ala Glu Glu Pro Thr Ala Trp Phe Leu Ser Pro Val Gly Leu Lys Asp Arg Val Asp Gly Val Met Ala Val Gln Phe Pro Ile Ala Arg Ile Asn Glu Leu Met Thr Ala Arg Gly Gln Trp Arg Asp Thr Gly Met Gly Asp Thr Gly Glu Thr Ile Leu Val Gly Pro Asp Asn Leu Met Arg Ser Asp ,Ser Arg Leu Phe Arg Glu Asn Arg Glu Lys Phe Leu Ala Asp Val Val Glu Gly Gly Thr Pro Pro Glu Val Ala Asp Glu Ser Val Asp Arg Arg Gly Thr Thr Leu Val Gln Pro Val Thr Thr Arg Ser Val Glu Glu Ala Gln Arg Gly Asn Thr Gly Thr Thr Ile Glu Asp Asp Tyr Leu Gly His Glu Ala Leu Gln Ala Tyr Ser Pro Val Asp Leu Pro Gly Leu His Trp Val Ile Val Ala Lys Ile Asp Thr Asp Glu Ala Phe Ala Pro Val Ala Gln Phe Thr Arg Thr Leu Val Leu Ser Thr Val Ile Ile Ile Phe Gly Val Ser Leu Ala Ala Met Leu Leu Ala Arg Leu Phe Val Arg Pro Ile Arg Arg Leu Gln Ala Gly Ala Gln Gln Ile Ser Gly Gly Asp Tyr Arg Leu Ala Leu Pro Val Leu Ser Arg Asp Glu Phe Gly Asp Leu Thr Thr Ala Phe Asn Asp Met Ser Arg Asn Leu Ser Ile Lys Asp Glu Leu Leu Gly Glu Glu Arg Ala Glu Asn Gln Arg Leu Met Leu Ser Leu Met Pro Glu Pro Val Met Gln Arg Tyr Leu Asp Gly Glu Glu Thr Ile Ala Gln Asp His Lys Asn Val Thr Val Ile Phe Ala Asp Met Met Gly Leu Asp Glu Leu Ser Arg Met Leu Thr Ser Glu Glu Leu Met Val Val Val Asn Asp Leu Thr Arg Gln Phe Asp Ala Ala Ala Glu Ser Leu Gly Val Asp His Val Arg Thr Leu His Asp Gly Tyr Leu Ala Ser Cys Gly Leu Gly Val Pro Arg Leu Asp Asn Val Arg Arg Thr Val Asn Phe Ala Ile Glu Met Asp Arg Ile Ile Asp Arg His Ala Ala Glu Ser Gly His Asp Leu Arg Leu Arg Ala Gly Ile Asp Thr Gly Ser Ala Ala Ser Gly Leu Val Gly Arg Ser Thr Leu Ala Tyr Asp Met Trp Gly Ser Ala Val Asp Val Ala Asn Gln Val Gln Arg Gly Ser Pro Gln Pro Gly Ile Tyr Val Thr Ser Arg Val His Glu Val Met Gln Glu Thr Leu Asp Phi Val Ala Ala Gly Glu 690 695 7p0 Val Val Gly Glu Arg Gly Val Glu Thr Val Trp Arg Leu Gln Gly His Arg Arg <210> 175 <211> 898 <212> DNA
<213> Mycobacterium vaccae <400>

gagcaaccgttccggctcggcgactggatcaccgtccccaccgcggcgggccggccgtcc 60 gcccacggccgcgtggtggaagtcaactggcgtgcaacacatatcgacaccggcggcaac 120 ctgctggtaatgcccaacgccgaactcgccggcgcgtcgttcaccaattacagccggccc 180 gtgggagagcaccggctgaccgtcgtcaccaccttcaacgccgcggacacccccgatgat 240 gtctgcgagatgctgtcgtcggtcgcggcgtcgctgcccgaactgcgcaccgacggacag 300 atcgccacgctctatctcggtgcggccgaatacgagaagtcgatcccgttgcacacaccc 360 gcggtggacgactcggtcaggagcacgtacctgcgatgggtctggtacgccgcgcgccgg 420 caggaacttcgcctaacggcgtcgccgacgattcgacacgccggaacggatcgcctcggc 480 catgcgggctgtggcgtccacactgcgcttggcagacgacgaacagcaggagatcgccga 540 cgtggtgcgtctggtccgttacggcaacggggaacgcctccagcagccgggtcaggtacc 600 gaccgggatgaggttcatcgtagacggcagggtgagtctgtccgtgatcgatcaggacgg 660 cgacgtgatcccggcgcgggtgctcgagcgtggcgacttcctggggcagaccacgctgac 720 gcgggaaccggtactggcgaccgcgcacgcgctggaggaagtcaccgtgctggagatggc 780 ccgtgacgagatcgagcgcctggtgcaccgaaagccgatcctgctgcacgtgatcggggc 840 cgtgatcgccgaccggcgcgcgcacgaacttcggttgatggcggactcgcaggactga 898 <210> 176 <211> 2013 <212> DNA
<213> Mycobacterium vaccae <400> 176 ggctatcagtccggacggtcctcgctgcgcgcatcggtgttcgaccgcctcaccgacatc 60 cgcgagtcgcagtcgcgcgggttggagaatcagttcgcggacctgaagaactcgatggtg 120 atttactcgcgcggcagcactgccacggaggcgatcggcgcgttcagcgacggtttccgt 180 cagctcggcgatgcgacgatcaataccgggcaggcggcgtcattgcgccgttactacgac 240 cggacgttcgccaacaccaccctcgacgacagcggaaaccgcgtcgacgtccgcgcgctc 300 atcccgaaatccaacccccagcgctatctgcaggcgctctataccccgccgtttcagaac 360 tgggagaaggcgatcgcgttcgacgacgcgcgcgacggcagcgcctggtcggccgccaat 420 gccagattcaacgagttcttccgcgagatcgtgcaccgcttcaacttcgaggatctgatg 480 ctgctcgacctcgagggcaacgtggtgtactccgcctacaaggggccggatctcgggaca 540 aacatcgtcaacggcccctatcgcaaccgggaactgtcggaagcctacgagaaggcggtc 600 gcgtcgaactcgatcgactatgtcggtgtcaccgacttcgggtggtacctgcctgccgag 660 gaaccgaccgcctggttcctgtccccggtcgggttgaaggaccgagtcgacggtgtgatg 720 gcggtccagttcccgatcgcgcggatcaacgaattgatgacggcgcggggacagtggcgt 780 gacaccgggatgggagacaccggtgagaccatcctggtcggaccggacaatctgatgcgc 840 tcggactcccggctgttccgcgagaaccgggagaagttcctggccgacgtcgtcgagggg 900 ggaaccccgccggaggtcgccgacgaatcggttgaccgccgcggcaccacgctggtgcag 960 ccggtgaccacccgctccgtcgaggaggcccaacgcggcaacaccgggacgacgatcgag 1020 gacgactatctcggccacgaggcgttacaggcgtactcaccggtggacctgccgggactg 1080 cactgggtgatcgtggccaagatcgacaccgacgaggcg~tcgccccggtggcgcagttc 1140 accaggaccctggtgctgtcgacggtgatcatcatcttcggcgtgtcgctggcggccatg 1200 ctgctggcgcggttgttcgtccgtccgatccggcggttgcaggccggcgcccagcagatc 1260 agcggcggtgactaccgcctcgctctgccggtgttgtctcgtgacgaattcggcgatctg 1320 acaacagctttcaacgacatgagtcgcaatctgtcgatcaaggacgagctgctcggcgag 1380 gagcgcgccgagaaccaacggctgatgctgtccctgatgcccgaaccggtgatgcagcgc 1440 tacctcgacggggaggagacgatcgcccaggaccacaagaacgtcacggtgatcttcgcc 1500 gacatgatgggcctcgacgagttgtcgcgcatgttgacctccgaggaactgatggtggtg 1560 gtcaacgacctgacccgccagttcgacgccgccgccgagagtctcggggtcgaccacgtg 1620 cggacgctgcacgacgggtacctggccagctgcgggttaggcgtgccgcggctggacaac 1680 gtccggcgcacggtcaatttcgcgatcgaaatggaccgcatcatcgaccggcacgccgcc 1740 gagtccgggcacgacctgcggctccgcgcgggcatcgacaccgggtcggcggccagcggg 1800 ctggtggggcggtccacgttggcgtacgacatgtggggttcggcggtcgatgtcgctaac 1860 caggtgcagcgcggctccccccagcccggcatctacgtcacctcgcgggtgcacgaggtc 1920 atgcaggaaactctcgacttcgtcgccgccggggaggtcgtcggcgagcgcggcgtcgag 1980 acggtctggcggttgcagggccaccggcgatga 2013 <210> 177 <211> 297 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (145)...(145) <221> UNSURE
<222> (151)...(151) <400> 177 Glu Gln Pro Phe Arg Leu Gly Asp Trp Ile Thr Val Pro Thr Ala Ala Gly Arg Pro Ser Ala His Gly Arg Val Val Glu Val Asn Trp Arg Ala Thr His Ile Asp Thr Gly Gly Asn Leu Leu Val Met Pro Asn Ala Glu Leu Ala Gly Ala Ser Phe Thr Asn Tyr Ser Arg Pro Val Gly Glu His Arg Leu Thr Val Val Thr Thr Phe Asn Ala Ala Asp Thr Pro Asp Asp Val Cys Glu Met Leu Ser Ser Val Ala Ala Ser Leu Pro Glu Leu Arg Thr Asp Gly Gln Ile Ala Thr Leu Tyr Leu Gly Ala Ala Glu Tyr Glu Lys Ser Ile Pro Leu His Thr Pro Ala Val Asp Asp Ser Val Arg Ser Thr Tyr Leu Arg Trp Val Trp Tyr Ala Ala Arg Arg Gln Glu Leu Arg 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 Gln Gln Glu Ile Ala Asp Val Val Arg Leu Val Arg Tyr Gly Asn Gly Glu Arg Leu Gln Gln Pro Gly Gln Val Pro Thr Gly Met Arg Phe Ile Val Asp Gly Arg Val Ser Leu Ser Val Ile Asp Gln Asp Gly Asp Val Ile Pro Ala Arg Val Leu Glu Arg Gly Asp Phe Leu Gly Gln Thr Thr Leu Thr Arg Glu Pro Val Leu Ala Thr Ala His Ala Leu Glu Glu Val Thr Val Leu Glu Met Ala Arg Asp Glu Ile Glu Arg Leu Val His Arg Lys Pro Ile Leu Leu His Val Ile Gly Ala Val Ala Asp Arg Arg Ala His Glu Leu Arg Leu Met Asp Ser Gln Asp <210> 178 <213> 670 <212> PRT
<213> Mycobacterium vaccae <400> 178 Gly Tyr Gln Ser Gly Arg Ser Ser Leu Arg Ala Ser Val Phe Asp Arg Leu Thr Asp Ile Arg Glu Ser Gln Ser Arg Gly Leu Glu Asn Gln Phe Ala Asp Leu Lys Asn Ser Met Val Ile Tyr Ser Arg Gly Ser Thr Ala Thr Glu Ala Ile Gly Ala Phe Ser Asp Gly Phe Arg Gln Leu Gly Asp Ala Thr Ile Asn Thr Gly Gln Ala Ala Ser Leu Arg Arg Tyr Tyr Asp Arg Thr Phe A3.a Asn Thr Thr Leu Asp Asp Ser Gly Asn Arg Val Asp Val Arg Ala Leu Ile Pro Lys Ser Asn Pro Gln Arg Tyr Leu Gln Ala Leu Tyr Thr Pro Pro Phe Gln Asn Trp Glu Lys Ala Ile Ala Phe Asp Asp Ala Arg Asp Gly Ser Ala Trp Ser Ala Ala Asn Ala Arg Phe Asn Glu Phe Phe Arg Glu Ile Val His Arg Phe Asn Phe Glu Asp Leu Met Leu Leu Asp Leu Glu Gly Asn Val Val Tyr Ser Ala Tyr Lys Gly Pro Asp Leu Gly Thr Asn Ile Val Asn Gly Pro Tyr Arg Asn Arg Glu Leu Ser Glu Ala Tyr Glu Lys Ala Val Ala Ser Asn Ser Ile Asp Tyr Val Gly Val Thr Asp Phe Gly Trp Tyr Leu Pro Ala Glu Glu Pro Thr Ala Trp Phe Leu Ser Pro Val Gly Leu Lys Asp Arg Val Asp Gly Val Met Ala Val Gln Phe Pro Ile Ala Arg Ile Asn Glu Leu Met Thr Ala Arg Gly Gln Trp Arg Asp Thr Gly Met Gly Asp Thr Gly Glu Thr Ile Leu Val Gly Pro Asp Asn Leu Met Arg Ser Asp Ser Arg Leu Phe Arg Glu Asn Arg Glu Lys Phe Leu Ala Asp Val Val Glu Gly Gly Thr Pro Pro Glu Val Ala Asp Glu Ser Val Asp Arg Arg Gly Thr Thr Leu Val Gln Pro Val Thr Thr Arg Ser Val Glu.Glu Ala Gln Arg Gly Asn Thr Gly Thr Thr Ile Glu Asp Asp Tyr Leu Gly His Glu Ala Leu Gln Ala Tyr Ser Pro Val Asp Leu Pro Gly Leu His Trp Val Ile Val Ala Lys Ile Asp Thr Asp Glu Ala Phe Ala Pro Val Ala Gln Phe Thr Arg Thr Leu Val Leu Ser Thr Val Ile Ile Ile Phe Gly Val Ser Leu Ala Ala Met Leu Leu Ala Arg Leu Phe Val Arg Pro Ile Arg Arg Leu Gln Ala Gly Ala Gln Gln Ile Ser Gly Gly Asp Tyr Arg Leu Ala Leu Pro Val Leu Ser Arg Asp Glu Phe Gly Asp Leu Thr Thr Ala Phe Asn Asp Met Ser Arg Asn Leu Ser Ile Lys Asp Glu Leu Leu Gly Glu Glu Arg Ala Glu Asn Gln Arg Leu Met Leu Ser Leu Met Pro Glu Pro Val Met Gln Arg Tyr Leu Asp Gly Glu Glu Thr Ile Ala Gln Asp His Lys Asn Val Thr Val Ile Phe Ala Asp Met Met Gly Leu Asp Glu Leu Ser Arg Met Leu Thr Ser Glu Glu Leu Met Val Val Val Asn Asp Leu Thr Arg Gln Phe Asp Ala Ala Ala Glu Ser Leu Gly Val Asp His Val Arg Thr Leu His Asp Gly Tyr Leu Ala Ser Cys Gly Leu Gly Val Pro Arg Leu Asp Asn Val Arg Arg Thr Val Asn Phe Ala Ile Glu Met Asp Arg Ile Ile Asp WO 99/32634 PCTlNZ98/00189 Arg His Ala Ala Glu Ser Gly His Asp Leu Arg Leu Arg Ala Gly Ile Asp Thr Gly Ser Ala Ala Ser Gly Leu Val Gly Arg Ser Thr Leu Ala Tyr Asp Met Trp Gly Ser Ala Val Asp Val Ala Asn Gln Val Gln Arg Gly Ser Pro Gln Pro Gly Ile Tyr Val Thr Ser Arg Val His Glu Val Met Gln Glu Thr Leu Asp Phe Val Ala Ala Gly Glu Val Val Gly Glu Arg Gly Val Glu Thr Val Trp Arg Leu Gln Gly His Arg Arg <210> 179 <211> 520 <212> DNA
<213> Mycobacterium vaccae <400> 179 gtgatcgacgaaaccctcttccatgccgaggagaagatggagaaggccgtctcggtggca 60 cccgacgacctggcgtcgattcgtaccggccgcgcgaaccccggcatgttcaaccggatc 120 aacatcgactactacggcgcctccaccccgatcacgcagctgtccagcatcaacgtgccc 180 gaggcgcgcatggtggtgatcaagccctacgaggcgagccagctgcgcctcatcgaggat 240 gcgatccgcaactccgacctcggcgtcaatccgaccaacgacggcaacatcatccgggtg 300 tcgatcccgcagctcaccgaggagcgccgccgcgacctggtcaagcaggccaaggccaag 360 ggcgaggacgccaaggtgtcggtgcgcaacatccgtcgcaacgatatgaacacctttcgc 420 atcgcaccggtacggctgccgacgccaccgccgtcgtagaagcgacagaggatcgcaggt 480 aacggtattggccacgccttctgtggcgggccgacaccac 520 <210> 180 <211> 1071 <212> DNA
<213> Mycobacterium vaccae <400>

cgtggggaaggattgcactctatgagcgaaatcgcccgtccctggcgggttctggcaggt 60 ggcatcggtgcctgcgccgcgggtatcgccggggtgctgagcatcgcggtcaccacggcg 120 tcggcccagccgggcctcccgcagcccccgctgcccgcccctgccacagtgacgcaaacc 180 gtcacggttgcgcccaacgccgcgccacaactcatcccgcgccccggtgtgacgcctgcc 240 accggcggcgccgccgcggtgcccgccggggtgagcgccccggcggtcgcgccggccccc 300 gcgctgcccgcccgcccggtgtccacgatcgccccggccacctcgggcacgctcagcgag 360 ttcttcgccgccaagggcgtcacgatggagccgcagtccagccgcgacttccgcgccctc 420 aacatcgtgctgccgaagccgcggggctgggagcacatcccggacccgaacgtgccggac 480 gcgttcgcggtgctggccgaccgggtcggcggcaacggcctgtactcgtcgaacgcccag 540 gtggtggtctacaaactcgtcggcgagttcgaccccaaggaagcgatcagccacggcttc 600 gtcgacagccagaagctgccggcgtggcgttccaccgacgcgtcgctggccgacttcggc 660 ggaatgccgtcctcgctgatcgagggcacctaccgcgagaacaacatgaagctgaacacg 720 tcccggcgccacgtcattgccaccgcggggcccgaccactacctggtgtcgctgtcggtg 780 accaccagcgtcgaacaggccgtggccgaagccgcggaggccaccgacgcgattgtcaac 840 ggcttcaaggtcagcgttccgggtccgggtccggccgcaccgccacctgcacccggtgcc 900 cccggtgtcccgcccgcccccggcgccccggcgctgccgctggccgtcgcaccacccccg 960 gctcccgctgttcccgccgtggcgcccgcgccacagctgctgggactgcagggatagacg 1020 tcgtcgtcccccgggcgaagcctggcgcccgggggacgacggcccctttct 1071 <210> 181 <211> 152 <212> PRT
<213> Mycobacterium vaccae <400> 181 Val Ile Asp Glu Thr Leu Phe His Ala Glu Glu Lys Met Glu Lys Ala Val Ser Val Ala Pro Asp Asp Leu Ala Ser Ile Arg Thr Gly Arg Ala Asn Pro Gly Met Phe Asn Arg Ile Asn Ile Asp Tyr Tyr Gly Ala Ser Thr Pro Ile Thr Gln Leu Ser Ser Ile Asn Val Pro Glu Ala Arg Met Val Val Ile Lys Pro Tyr Glu Ala Ser Gln Leu Arg Leu Ile Glu Asp Ala Ile Arg Asn Ser Asp Leu Gly Val Asn Pro Thr Asn Asp Gly Asn Ile Ile Arg Val Ser Ile Pro Gln Leu Thr Glu Glu Arg Arg Arg Asp Leu Val Lys Gln Ala Lys Ala Lys Gly Glu Asp Ala Lys Val Ser Val Arg Asn Ile Arg Arg Asn Asp Met Asn Thr Phe Arg Ile Ala Pro Val Arg Leu Pro Thr Pro Pro Pro Ser <210> 182 <211> 331 <212> PRT
<213> Mycobacterium vaccae <400> 182 Met Ser Glu Ile Ala Arg Pro Trp Arg Val Leu Ala Gly Gly Ile Gly Ala Cys Ala Ala Gly Ile Ala Gly Val Leu Ser Ile Ala Val Thr Thr Ala Ser Ala Gln Pro Gly Leu Pro Gln Pro Pro Leu Pro Ala Pro Ala Thr Val Thr Gln Thr Val Thr Val Ala Pro Asn Ala Ala Pro Gln Leu Ile Pro Arg Pro Gly Val Thr Pro Ala Thr Gly Gly Ala Ala Ala Val Pro Ala Gly Val Ser Ala Pro Ala Val Ala Pro Ala Pro Ala Leu Pro Ala Arg Pro Val Ser Thr Ile Ala Pro Ala Thr Ser Gly Thr Leu Ser Glu Phe Phe Ala Ala Lys Gly Val Thr Met Glu Pro Gln Ser Ser Arg Asp Phe Arg Ala Leu Asn Ile Val Leu Pro Lys Pro Arg Gly Trp Glu His Ile Pro Asp Pro Asn Val Pro Asp Ala Phe Ala Val Leu Ala Asp Arg Val Gly Gly Asn Gly Leu Tyr Ser Ser Asn Ala Gln Val val Val Tyr Lys Leu Val Gly Glu Phe Asp Pra Lys Glu Ala Ile Ser His Gly Phe Val Asp Ser Gln Lys Leu Pro Ala Trp Arg Ser Thr Asp Ala Ser Leu Ala Asp Phe Gly Gly Met Pro Ser Ser Leu Ile Glu Gly Thr Tyr Arg Glu Asn Asn Met Lys Leu Asn Thr Ser Arg Arg His Val Ile Ala Thr Ala Gly Pro Asp His Tyr Leu Val Ser Leu Ser Val Thr Thr Ser Val Glu Gln Ala Val Ala Glu Ala Ala Glu Ala Thr Asp Ala Ile Val Asn Gly Phe Lys Val Ser Val Pro Gly Pro Gly Pro Ala Ala Pro Pro Pro Ala Pro Gly Ala Pro Gly Val Pro Pro Ala Pro Gly Ala Pro Ala Leu Pro Leu Ala Val Ala Pro Pro Pro Ala Pro Ala Val Pro Ala Val Ala Pro Ala Pro Gln Leu Leu Gly Leu Gln Gly <210> 183 <211> 207 <212> DNA
<213> Mycobacterium vaccae <400> 183 acctacgagt tcgagaacaa ggtcacgggc ggccgcatcc cgcgcgagta catcccgtcg 60 gtggatgccg gcgcgcagga cgccatgcag tacggcgtgc tggccggcta cccgctggtt 120 aacgtcaagc tgacgctgct cgacggtgcc taccacgaag tcgactcgtc ggaaatggca 180 ttcaaggttg ccggctccca ggtcata 207 <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 Glu Tyr Iie Pro Ser Val Asp Ala Gly Ala Gln Asp Ala Met Gln Tyr Gly Val Leu Ala Gly Tyr Pro Leu Val Asn Val Lys Leu Thr Leu Leu Asp Gly Ala Tyr His Glu Val Asp Ser Ser Glu Met Ala Phe Lys Val Ala Gly Ser Gln Val Ile <210> 185 <211> 898 <212> DNA
<213> Mycobacterium vaccae <220>
<221> unsure <222> (637)...(637) <221> unsure <222> (662)...(662) <400> 185 cgacctccacccgggcgtgaggccaaccactaggctggtpaccagtagtcgacggcacac 60 ttcaccgaaaaaatgaggacagaggagacacccgtgacgatccgtgttggtgtgaacggc 120 ttcggccgtatcggacgcaacttcttccgcgcgctggacgcgcagaaggccgaaggcaag 180 aacaaggacatcgagatcgtcgcggtcaacgacctcaccgacaacgccacgctggcgcac 240 ctgctgaagttcgactcgatcctgggccggctgccctacgacgtgagcctcgaaggcgag 300 gacaccatcgtcgtcggcagcaccaagatcaaggcgctcgaggtcaaggaaggcccggcg 360 gcgctgccctggggcgacctgggcgtcgacgtcgtcgtcgagtccaccggcatcttcacc 420 aagcgcgacaaggcccagggccacctcgacgcgggcgccaagaaggtcatcatctccgcg 480 ccggccaccgatgaggacatcaccatcgtgctcggcgtcaacgacgacaagtacgacggc 540 agccagaacatcatctccaacgcgtcgtgcaccacgaactgcctcggcccgctggcgaag 600 gtcatcaacgacgagttcggcatcgtcaagggcctgntgaccaccatccacgcctacacc 660 cnggtccagaacctgcaggacggcccgcacaaggatctgcgccgggcccgcgccgccgcg 720 ctgaacatcgtgccgacctccaccggtgccgccaaggccatcggactggtgctgcccgag 780 ctgaagggcaagctcgacggctacgcgctgcgggtgccgatccccaccggctcggtcacc 840 gacctgaccgccgagctgggcaagtcggccaccgtggacgagatcaacgccgcgatga 898 <210> 186 <211> 268 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (182)...(182) <221> UNSURE
<222> (190)...(190) <400> 186 Val Thr Ile Arg Val Gly Val Asn Gly Phe Gly Arg Ile Gly Arg Asn Phe Phe Arg Ala Leu Asp Ala Gln Lys Ala Glu Gly Lys Asn Lys Asp Ile Glu Ile Val Ala Val Asn Asp Leu Thr Asp Asn Ala Thr Leu Ala His Leu Leu Lys Phe Asp Ser Ile Leu Gly Arg Leu Pro Tyr Asp Val Ser Leu Glu Gly Glu Asp Thr Ile Val Val Gly Ser Thr Lys Ile Lys Ala Leu Glu Val Lys Glu Gly Pro Ala Ala Leu Pro Trp Gly Asp Leu Gly Val Asp Val Val Val Glu Ser Thr Gly Ile Phe Thr Lys Arg Asp Lys Ala Gln Gly His Leu Asp Ala Gly Ala Lys Lys Val Ile Ile Ser Ala Pro Ala Thr Asp Glu Asp Ile Thr Ile Val Leu Gly Val Asn Asp Asp Lys Tyr Asp Gly Ser Gln Asn Ile Ile Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Gly Pro Leu Ala Lys Val Ile Asn Asp Glu Phe Gly Ile Val Lys Gly Leu Xaa Thr Thr Ile His Ala Tyr Thr Xaa Val Gln Asn Leu Gln Asp Gly Pro His Lys Asp Leu Arg Arg Ala Arg Ala Ala Ala Leu Asn Ile Val Pro Thr Ser Thr Gly Ala Ala Lys Ala Ile Gly Leu Val Leu Pro Glu Leu Lys Gly Lys Leu Asp Gly Tyr Ala Leu Arg Val Pro Ile Pro Thr Gly Ser Val Thr Asp Leu Thr Ala Glu Leu Gly Lys Ser Ala Thr Val Asp Glu Ile Asn Ala Ala Met <210> 187 <211> 41 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (39)...(39) <400> 187 Met Asn Lys Ala Glu Leu Ile Asp Val Leu Thr Glu Lys Leu Gly Ser Asp Arg Arg Gln Ala Thr Ala Ala Val Glu Asn Val Val Asp Thr Ile Val Ala Ala Val Pro Lys Xaa Val Val <210> 188 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <221> unsure <222> (12)...(12) <400> 188 atgaayaarg cngarctsat ygaygt 26 <210> 189 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <400> 189 atsgtrtgva cvacgttytc 20 <210> 190 <211> 84 <212> DNA
<213> Artificial Sequence <220>
<223> Made in a lab <221> unsure <222> (2) . . . (2) <400> 190 gnactcattg acgtactcac tgagaagctg ggctcggatt gtcggcaagc gactgcggca 60 atggagaacg tggtccacac cata 84 <210> 191 <211> 337 <212> DNA
<213> Mycobacterium vaccae <220>
<221> unsure <222> (2) . .. (2) <400> 191 gnactcattg acgtactcac tgagaagctg ggctcggatt gtcggcaagc gactgcggcg 60 gtggagaatg ttgtcgacac catcgtgcgc gccgtgcaca agggtgagag cgtcaccatc 120 acgggcttcg gtgttttcga gcagcgtcgt cgcgcagcac gcgtggcacg caatccgcgc 180 accggcgaga ccgtgaaggt caagcccacc tcagtcccgg cattccgtcc cggcgctcag 240 ttcaaggctg ttgtctctgg cgcacagaag cttccggccg agggtccggc ggtcaagcgc 300 ggtgtgaccg cgacgagcac cgcccgcaag gcagcca 337 <210> 192 <211> 111 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (1)...(1) WO 99/3x634 PCT/NZ98/00189 <400> 192 Xaa Leu Ile Asp Val Leu Thr Glu Lys Leu Gly Ser Asp Arg Gln Ala Thr Ala Ala Val Glu Asn Val Val Asp Thr Ile Val Arg Ala Val His Lys Gly Glu Ser Val Thr Ile Thr Gly Phe Gly Val Phe Glu Gln Arg Arg Arg Ala Ala Arg Val Ala Arg Asn Pro Arg Thr Gly Glu Thr Val Lys Val Lys Pro Thr Ser Val Pro Ala Phe Arg Pro Gly Ala Gln Phe Lys Ala Val Val Ser Gly Ala Gln Lys Leu Pro Ala Glu Gly Pro Ala Val Lys Arg Gly Val Thr Ala Thr Ser Thr Ala Arg Lys Ala Ala <210> 193 <211> 1164 <212> DNA
<213> Mycobacterium vaccae <400> 193 ggtggcgcgcatcgagaagcgcccgccccggttcacgggcgcctgatcatggtgcgggcg 60 gcgctgcgctacggcttcgggacggcctcactgctggccggcgggttcgtgctgcgcgcc 120 ctgcagggcacgcctgccgccctcggcgcgactccgggcgaggtcgcgccggtggcgcgc 180 cgctcgccgaactaccgcgacggcaagttcgtcaacctggagcccccgtcgggcatcacg 240 atggatcgcgacctgcagcggatgctgttgcgcgatctggccaacgccgcatcccagggc 300 aagccgcccggaccgatcccgctggccgagccgccgaagggggatcccactcccgcgccg 360 gcggcggccagctggtacggccattccagcgtgctgatcgaggtcgacggctaccgcgtg 420 ctggccgacccggtgtggagcaacagatgttcgccctcacgggcggtcggaccgcagcgc 480 atgcacgacgtcccggtgccgctggaggcgcttcccgccgtggacgcggtggtgatcagc 540 cacgaccactacgaccacctcgacatcgacaccatcgtcgcgttggcgcacacccagcgg 600 gccccgttcgtggtgccgttgggcatcggcgcacacctgcgcaagtggggcgtccccgag 660 gcgcggatcgtcgagttggactggcacgaagcccaccgcatagacgacctgacgctggtc 720 tgcacccccgcccggcacttctccggacggttgttctcccgcgactcgacgctgtgggcg 780 tcgtgggtggtcaccggctcgtcgcacaaggcgttcttcggtggcgacaccggatacacg 840 aagagcttcgccgagatcggcgacgagtacggtccgttcgatctgaccctgctgccgatc 900 ggggcctaccatcccgcgttcgccgacatccacatgaaccccgaggaggcggtgcgcgcc 960 catctggacctgaccgaggtggacaacagcctgatggtgcccatccactgggcgacattc 1020 cgcctcgccccgcatccgtggtccgagcccgccgaacgcctgctgaccgctgccgacgcc 1080 gagcgggtacgcctgaccgtgccgattcccggtcagcgggtggacccggagtcgacgttc 1140 gacccgtggtggcggttctgaacc 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 Ala Gly Gly Phe Val Leu Arg Ala Leu Gln Gly Thr Pro Ala Ala Leu Gly Ala Thr Pro Gly Glu Val Ala Pro Val Ala Arg Arg Ser Pro Asn Tyr Arg Asp Gly Lys Phe Val Asn Leu Glu Pro Pro Ser Gly Ile Thr Met Asp Arg Asp Leu Gln Arg Met Leu Leu Arg Asp Leu Ala Asn Ala 65 70 75 ~ 80 Ala Ser Gln Gly Lys Pro Pro Gly Pro Ile Pro Leu Ala Glu Pro Pro Lys Gly Asp Pro Thr Pro Ala Pro Ala Ala Ala Ser Trp Tyr Gly His Ser Ser Val Leu Ile Glu Val Asp Gly Tyr Arg Val Leu Ala Asp Pro Val Trp Ser Asn Arg Cys Ser Pro Ser Arg Ala Val Gly Pro Gln Arg Met His Asp Val Pro Val Pro Leu Glu Ala Leu Pro Ala Val Asp Ala Val Val Ile Ser His Asp His Tyr Asp His Leu Asp Ile Asp Thr Ile Val Ala Leu Ala His Thr Gln Arg Ala Pro Phe Val Val Pro Leu Gly Ile Gly Ala His Leu Arg Lys Trp Gly Val Pro Glu Ala Arg Ile Val Glu Leu Asp Trp His Glu Ala His Arg Ile Asp Asp Leu Thr Leu Val Cys Thr Pro Ala Arg His Phe Ser Gly Arg Leu Phe Ser Arg Asp Ser Thr Leu Trp Ala-Ser Trp Val Val Thr Gly Ser Ser His Lys Ala Phe Phe Gly Gly Asp Thr Gly Tyr Thr Lys Ser Phe Ala Glu Ile Gly Asp Glu Tyr Gly Pro Phe Asp Leu Thr Leu Leu Pro Ile Gly Ala Tyr His Pro Ala Phe Ala Asp Ile His Met Asn Fro Glu Glu Ala Val Arg Ala His Leu Asp Leu Thr Glu Val Asp Asn Ser Leu Met Val Pro Ile His Trp Ala Thr Phe Arg Leu Ala Pro His Pro Trp Ser Glu Pro Ala Glu Arg Leu Leu Thr Ala Ala Asp Ala Glu Arg Val Arg Leu Thr Val Pro Ile Pro Gly Gln Arg Val Asp Pro Glu Ser Thr Phe Asp Pro Trp Trp Arg Phe <210> 195 <211> 650 <212> DNA
<213> Mycobacterium vaccae <400> 195 gacacaccag caccactgtt aacctcgcta gatcagtcgg ccgaacggaa ggacagccgt 60 gaccctgaaa accctagtca ccagcatgac cgctggggca gcagcagccg caacactcgg 120 cccggtgtcccgcccgcccccggcgccccggcgctgccgctggccgtcgca cgctgccgccgtgggtgtgacctcgattgccgtcggtgcgggtgtcgccggcgcgtcgcc 180 cgcggtgctgaacgcaccgctgctttccgcccctgcccccgatctgcagggaccgctggt 240 ctccaccttgagcgcgctgtcgggcccgggctccttcgccggcgccaaggccacctacgt 300 ccagggcggtctcggccgcatcgaggcccgggtggccgacagcggatacagcaacgccgc 360 ggccaagggctacttcccgctgagcttcaccgtcgccggcatcgaccagaacggtccgat 420 cgtgaccgccaacgtcaccgcggcggccccgacgggcgccgtggccacccagccgctgac 480 gttcatcgccgggccgagcccgaccggatggcagctgtccaagcagtccgcactggccct 540 gatgtccgcggtgggtgatctcccgcacgattctggtccgcagcgccgtcacatgtgtgg 600 cggcgctcgggctgggtgggtgcctgggcggctgcgcgcaagatgaacat 650 <210> 196 <211> 159 <212> PRT
<213> Mycobacterium vaccae <400> 196 Met Thr Ala Gly Ala Ala Ala Ala Ala Thr Leu Gly Ala Ala Ala Val Gly Val Thr Ser Ile Ala Val Gly Ala Gly Val Ala Gly Ala Ser Pro Ala Val Leu Asn Ala Pro Leu Leu Ser Ala Pro Ala Pro Asp Leu Gln Gly Pro Leu Val Ser Thr Leu Ser Ala Leu Ser Gly Pro Gly Ser Phe Ala Gly Ala Lys Ala Thr Tyr Val Gln Gly Gly Leu Gly Arg Ile Glu Ala Arg Val Ala Asp Ser Gly Tyr Ser Asn Ala Ala Ala Lys Gly Tyr Phe Pro Leu Ser Phe Thr Val Ala Gly Ile Asp Gln Asn Gly Pro Ile Val Thr Ala Asn Val Thr Ala Ala Ala Pro Thr Gly Ala Val Ala Thr Gln Pro Leu Thr Phe Ile Ala Gly Pro Ser Pro Thr Gly Trp Gln Leu Ser Lys Gln Ser Ala Leu Ala Leu Met Ser Ala Val Ile Ala Ala <210> 197 <211> 285 <212> PRT
<213> Mycobacterium vaccae <400> 197 Met Gln Val Arg Arg Val Leu Gly Ser Val Gly Ala Ala Val Ala Val Ser Ala Ala Leu Trp Gln Thr Gly Val Ser Ile Pro Thr Ala Ser Ala Asp Pro Cys Pro Asp Ile Glu Val Ile Phe Ala Arg Gly Thr Gly Ala Glu Pro Gly Leu Gly Trp Val Gly Asp Ala Phe Val Asn Ala Leu Arg so ss so Pro Lys Val Gly Glu Gln Ser Val Gly Thr Tyr Ala Val Asn Tyr Pro AlaGlyPhe Phe Asp Lys Ser ProMet Gly Ala Asp Ala Ala Asp Ala SerGlyArg Gln Trp Met Ala Cys Pro Asp Lys Val Asp Asn Thr Leu loo l05 llo ValLeuGly Met Ser Gln Gly GlyVal Ile Asp Ile Gly Ala Leu Thr ValAspPro Pro Leu Gly Arg ThrPro Thr Pro Pro Arg Phe Met Pro ArgValAla,AspHis Val Ala Ala ValVal Phe Gly Pro Val Asn Leu ArgAspIle Gly Gly Gly Pro ProGln Met Ser Thr Arg Leu Gly Tyr GlyProLys Ile Asp Leu Cys LeuAsp Asp Pro Cys Ser Ala Phe Ser ProGlyPhe Leu Pro Ala His AlaTyr Ala Asp Gly Asn Phe Asn Met ValGluGlu Ala Asn Phe Ala LeuGlu Pro Gly Ser Ala Arg Gln Val GluLeuPro Ala Pro Tyr Leu LeuPhe Val Pro Gly Glu His Arg Glu ValThrLeu Asp Ala Gly Pro ArgGlu Gly Asp Val Glu Leu Ala Arg PheThrAla Gly Gly Gln Arg ThrAla Thr Ala Ala Ser Val Pro Glu IleLeu Glu Met His Ala LeuGly Ala Ala Val Gly Trp <210> 198 <211> 743 <212> DNA
<213> Mycobacterium vaccae <400> 198 ggatccgcggcaccggctggtgacgaccaagtacaacccggcccgcacctggacggccga 60 gaactccgtcggcatcggcggcgcgtacctgtgcatctacgggatggagggccccggcgg 120 ctatcagttcgtcggccgcaccacccaggtgtggagtcgttaccgccacacggcgccgtt 180 cgaacccggaagtccctggctgctgcggtttttcgaccgaatttcgtggtatccggtgtc 240 ggccgaggagctgctggaattgcgagccgacatggccgcaggccggggctcggtcgacat 300 caccgacggcgtgttctccctcgccgagcacgaacggttcctggccgacaacgccgacga 360 catcgccgcgttccgttcccggcaggcggccgcgttctccgccgagcggaccgcgtgggc 420 ggccgccggcgagttcgaccgcgccgagaaagccgcgtcgaaggccaccgacgccgatac 480 cggggacctggtgctctacgacggtgacgagcgggtcgacgctccgttcgcgtcgagcgt 540 gtggaaggtcgacgtcgccgtcggtgaccgggtggtggccggacagccgttgctggcgct 600 ggaggcgatgaagatggagaccgtgctgcgcgccccggccgacggggtggtcacccagat 660 cctggtctccgctgggcatctcgtcgatcccggcaccccactggtcgtggtcggcaccgg 720 agtgcgcgcatgagcgccgtcga 743 <210> 199 <211> 243 <212> PRT
<213> Mycobacterium vaccae <400> 199 Asp Pro Arg His Arg Leu Val Thr Thr Lys Tyr Asn Pro Ala Arg Thr Trp Thr Ala Glu Asn Ser Val Gly Ile Gly Gly Ala Tyr Leu Cys Ile Tyr Gly Met Glu Gly Pro Gly Gly Tyr Gln Phe Val Gly Arg Thr Thr Gln Val Trp Ser Arg Tyr Arg His Thr Ala Pro Phe Glu Pro Gly Ser Pro Trp Leu Leu Arg Phe Phe Asp Arg Ile Ser Trp Tyr Pro Val Ser Ala Glu Glu Leu Leu Glu Leu Arg Ala Asp Met Ala Ala Gly Arg Gly Ser Val Asp Ile Thr Asp Gly Val Phe Ser Leu Ala Glu His Glu Arg Phe Leu Ala Asp Asn Ala Asp Asp Ile Ala Ala Phe Arg Ser Arg Gln Ala Ala Ala Phe Ser Ala Glu Arg Thr Ala Trp Ala Ala Ala Gly Glu Phe Asp Arg Ala Glu Lys Ala Ala Ser Lys Ala Thr Asp Ala Asp Thr Gly Asp Leu Val Leu Tyr Asp Gly Asp Glu Arg Val Asp Ala Pro Phe Ala Ser Ser Val Trp Lys Val Asp Val Ala Val Gly Asp Arg Val Val Ala Gly Gln Pro Leu Leu Ala Leu Glu Ala Met Lys Met Glu Thr Val Leu Arg Ala Pro Ala Asp Gly Val Val Thr Gln Ile Leu Val Ser Ala Gly His Leu Val Asp Pro Gly Thr Pro Leu Val Val Val Gly Thr Gly Val Arg Ala <210> 200 <211> 858 <212> DNA
<213> Mycobacterium vaccae <400> 200 gaaatcccgcgtctgaaaccctcttttcgcggcgcccctcaggacggtaagggggccaag 60 cggattgaaaaatgttcgctgaatgagcctgaaattgcgcgtggctcttggaaatcagca 120 gcgatgggtttaccgtgtccactagtcggtccaaagaggaccactggttttcggaggttt 180 tgcatgaacaaagcagagctcatcgacgtactcactgagaagctgggctcggatcgtcgg 240 caagcgactgcggcggtggagaacgttgtcgacaccatcgtgcgcgccgtgcacaagggt 300 gagagcgtcaccatcacgggcttcggtgttttcgagcagcgtcgtcgcgcagcacgcgtg 360 gcacgcaatccgcgcaccggcgagaccgtgaaggtcaagcccacctcagtcccggcattc 420 cgtcccggcgctcagttcaaggctgttgtctctggcgcacagaagcttccggccgagggt 480 ccggcggtcaagcgcggtgtgaccgcgacgagcaccgcccgcaaggcagccaagaaggct 540 ccggccaagaaggctgccgcgaagaaggccgcgccggccaagaaggctccggcgaagaag 600 gctgcgaccaaggctgcaccggccaagaaggccactgccgccaagaaggccgcgccggcc 660 aagaaggccactgccgccaagaaggctgcaccggccaagaaggctccggccaagaaggct 720 gcgaccaaggctgcaccggccaagaaggctccggccaagaaggccgcgaccaaggctgca 780 ccggccaagaaggctccggccgccaagaaggcgcccgccaagaaggctccggccaagcgc 840 ggcggacgca agtaagtc 858 <210> 201 <211> 223 <212> PRT
<213> Mycobacterium vaccae <400> 201 Met Asn Lys Ala Glu Leu Ile Asp Val Leu Thr Glu Lys Leu Gly Ser Asp Arg Arg Gln Ala Thr Ala Ala Val Glu Asn Val Val Asp Thr Ile Val Arg Ala Val His Lys Gly Glu Ser Val Thr Ile Thr Gly Phe Gly Val Phe Glu Gln Arg Arg Arg Ala Ala Arg Val Ala Arg Asn Pro Arg Thr Gly Glu Thr Val Lys Val Lys Pro Thr Ser Val Pro Ala Phe Arg Pro Gly Ala Gln Phe Lys Ala Val Val Ser Gly Ala Gln Lys Leu Pro Ala Glu Gly Pro Ala Val Lys Arg Gly Val Thr Ala Thr Ser Thr Ala Arg Lys Ala Ala Lys Lys Ala Pro Ala Lys Lys Ala Ala Ala Lys Lys Ala Ala Pro Ala Lys Lys Ala Pro Ala Lys Lys Ala Ala Thr Lys Ala Ala Pro Ala Lys Lys Ala Thr Ala Ala Lys Lys Ala Ala Pro Ala Lys Lys Ala Thr Ala Ala Lys Lys Ala Ala Pro Ala Lys Lys Ala Pro Ala Lys Lys Ala Ala Thr Lys Ala Ala Pro Ala Lys Lys Ala Pro Ala Lys Lys Ala Ala Thr Lys Ala Ala Pro Ala Lys Lys Ala Pro Ala Ala Lys Lys Ala Pro Ala Lys Lys Ala Pro Ala Lys Arg Gly Gly Arg Lys <210> 202 <211> 570 <212> DNA
<213> Mycobacterium vaccae <400> 202 agacagacagtgatcgacgaaaccctcttccatgccgaggagaagatggagaaggccgtc 60 tcggtggcacccgacgacctggcgtcgattcgtaccggccgcgcgaaccccggcatgttc 120 aaccggatcaacatcgactactacggcgcctccaccccgatcacgcagctgtccagcatc 180 aacgtgcccgaggcgcgcatggtggtgatcaagccctacgaggcgagccagctgcgcctc 240 atcgaggatgcgatccgcaactccgacctcggcgtcaatccgaccaacgacggcaacatc 300 atccgggtgtcgatcccgcagctcaccgaggagcgccgccgcgacctggtcaagcaggcc 360 aaggccaagggcgaggacgccaaggtgtcggtgcgcaacatccgtcgcaaggcgatggag 420 gaactctcccggatcaagaaggacggcgacgccggcgaagaccaagtgacccgcgccgag 480 aaggatctcgacaagagcacccaccagtacacgaatcagatcgacgaactggtcaagcac 540 aaggaaggcgagttgctggaggtctgacca 570 WO 99I32b34 PCT/NZ98/00189 <210> 203 <211> 187 <212> PRT
<213> Mycobacterium vaccae <220>
<221> UNSURE
<222> (186)...(186) <400> 203 Val Ile Asp Glu Thr Leu Phe His Ala Glu Glu Lys Met Glu Lys Ala Val Ser Val Ala Pro Asp Asp Leu Ala Ser Ile Arg Thr Gly Arg Ala Asn Pro Gly Met Phe Asn Arg Ile Asn Ile Asp Tyr Tyr Gly Ala Ser Thr Pro Ile Thr Gln Leu Ser Ser Ile Asn Val Pro Glu Ala Arg Met Val Val Ile Lys Pro Tyr Glu Ala Ser Gln Leu Arg Leu Ile Glu Asp Ala Ile Arg Asn Ser Asp Leu Gly Val Asn Pro Thr Asn Asp Gly Asn Ile Ile Arg Val Ser Ile Pro Gln Leu Thr Glu Glu Arg Arg Arg Asp Leu Val Lys Gln Ala Lys Ala Lys Gly Glu Asp Ala Lys Val Ser Val Arg Asn Ile Arg Arg Lys Ala Met Glu Glu Leu Ser Arg Ile Lys Lys Asp Gly Asp Ala Gly Glu Asp Glu Val Thr Arg Ala Glu Lys Asp Leu Asp Lys Ser Thr His Gln Tyr Thr Asn Gln Ile Asp Glu Leu Val Lys His Lys Glu Gly Glu Leu Leu Glu Val Xaa Pro <210> 204 <211> 1364 <212> DNA
<213> Mycobacterium vaccae <400> 204 cgacctccacccgggcgtgaggccaaccactaggctggtcaccagtagtcgacggcacac 60 ttcaccgaaaaaatgaggacagaggagacacccgtgacgatccgtgttggtgtgaacggc 120 ttcggccgtatcggacgcaacttcttccgcgcgctggacgcgcagaaggccgaaggcaag 180 aacaaggacatcgagatcgtcgcggtcaacgacctcaccgacaacgccacgctggcgcac 240 ctgctgaagttcgactcgatcctgggccggctgccctacgacgtgagcctcgaaggcgag 300 gacaccatcgtcgtcggcagcaccaagatcaaggcgctcgaggtcaaggaaggcccggcg 360 gcgctgccctggggcgacctgggcgtcgacgtcgtcgtcgagtccaccggcatcttcacc 420 aagcgcgacaaggcccagggccacctcgacgcgggcgccaagaaggtcatcatctccgcg 480 ccggccaccgatgaggacatcaccatcgtgctcggcgtcaacgacgacaagtacgacggc 540 agccagaacatcatctccaacgcgtcgtgcaccacgaactgcctcggcccgctggcgaag 600 gtcatcaacgacgagttcggcatcgtcaagggcctgatgaccaccatccacgcctacacc 660 caggtccagaacctgcaggacggcccgcacaaggatctgcgccgggcccgcgccgccgcg 720 ctgaacatcgtgccgacctccaccggtgccgccaaggccatcggactggtgctgcccgag 780 ctgaagggcaagctcgacggctacgcgctgcgggtgccgatccccaccggctcggtcacc 840 gacctgaccgccgagctgggcaagtcggccaccgtggacgagatcaacgccgcgatgaag 900 gctgcggccgagggcccgctcaagggcatcctcaagtactacgacgccccgatcgtgtcc 960 agcgacatcgtcaccgatccgcacagctcgatcttcgactcgggtctgaccaaggtcatc 1020 gacaaccaggccaaggtcgtgtcctggtacgacaacgagtggggctactccaaccgcctc 1080 gtcgacctggtcgccctggtcggcaagtcgctgtaggggcgagcgaagcgacgggagaac 1140 agaggcgccatggcgatcaagtcactcgacgaccttctgtccgaaggggtgacggggcgg 1200 ggcgtactcgtgcgctccgacctgaacgtccccctcgacggcgacacgatcaccgacccg 1260 gggcgcatcatcgcctcggtgccgacgttgaaggcgttgagtgacgccggcgccaaggtg 1320 gtcgtcaccgcgcatctgggcaggcccaagggtgagccggatcc 1364 <210> 205 <211> 340 <212> PRT
<213> Mycobacterium vaccae <400> 205 Val Thr Ile Arg Val Gly Val Asn Gly Phe Gly Arg Ile Gly Arg Asn Phe Phe Arg Ala Leu Asp Ala Gln Lys Ala Glu Gly Lys Asn Lys Asp Ile Glu Ile Val Ala Val Asn Asp Leu Thr Asp Asn Ala Thr Leu Ala His Leu Leu Lys Phe Asp Ser Ile Leu Gly Arg Leu Pro Tyr Asp Val Ser Leu Glu Gly Glu Asp Thr Ile Val Val Gly Ser Thr Lys Ile Lys Ala Leu Glu Val Lys Glu Gly Pro Ala Ala Leu Pro Trp Gly Asp Leu Gly Val Asp Val Val Val Glu Ser Thr Gly Ile Phe Thr Lys Arg Asp Lys Ala Gln Gly His Leu Asp Ala Gly Ala Lys Lys Val Ile Ile Ser Ala Pro Ala Thr Asp Glu Asp Ile Thr Ile Val Leu Gly Val Asn Asp Asp Lys Tyr Asp Gly Ser Gln Asn Ile Ile Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Gly Pro Leu Ala Lys Val Ile Asn Asp Glu Phe Gly Ile Val Lys Gly Leu Met Thr Thr Ile His Ala Tyr Thr Gln Val Gln Asn Leu Gln Asp Gly Pro His Lys Asp Leu Arg Arg Ala Arg Ala Ala Ala Leu Asn Ile Val Pro Thr Ser Thr Gly Ala Ala Lys Ala Ile Gly Leu Val Leu Pro Glu Leu Lys Gly Lys Leu Asp Gly Tyr Ala Leu Arg Val Pro Ile Pro Thr Gly Ser Val Thr Asp Leu Thr Ala Glu Leu Gly Lys Ser Ala Thr Val Asp Glu Ile Asn Ala Ala Met Lys Ala Ala Ala Glu Gly Pro Leu Lys Gly Ile Leu Lys Tyr Tyr Asp Ala Pro Ile Val Ser Ser Asp Ile Val Thr Asp Pro His Ser Ser Ile Phe Asp Ser Gly Leu Thr Lys Val Ile Asp Asn Gln Ala Lys Val Val Ser Trp Tyr Asp Asn Glu Trp Gly Tyr Ser Asn Arg Leu Val Asp Leu Val Ala Leu Val Gly Lys Ser Leu <210> 206 <211> 522 <212> DNA
<213> Mycobacterium vaccae <400> 206 acctacgagttcgagaacaaggtcacgggcggccgcatcccgcgcgagtacatcccgtcg 60 gtggatgccggcgcgcaggacgccatgcagtacggcgtgctggccggctacccgctggtt 120 aacgtcaagctgacgctgctcgacggtgcctaccacgaagtcgactcgtcggaaatggca 180 ttcaaggttgccggctcccaggtcatgaagaaggctgccgcccaggcgcagccggtgatc 240 ctggagccagtgatggcggtcgaggtcacgacgcccgaggattacatgggtgaagtgagc 300 ggcgacctgaactcccgccgtggtcagatccaggccatggaggagcggagcggtgctcgt 360 gtcgtgaaggcgcaggttccgctgtcggagatgttcggctacgtcggagaccttcggtcg 420 aagacccagggccgggccaactactccatggtgttcgactcgtacgccgaagttccggcg 480 aacgtgtcgaaggagatcatcgcgaaggcgacgggccagtas 522 <210> 207 <211> 173 <212> PRT
<213> Mycobacterium vaccae <400> 207 Thr Tyr Glu Phe Glu Asn Lys Val Thr Gly Gly Arg Ile Pro Arg Glu Tyr Ile Pro Ser Val Asp Ala Gly Ala Gln Asp Ala Met Gln Tyr Gly Val Leu Ala Gly Tyr Pro Leu Val Asn Val Lys Leu Thr Leu Leu Asp Gly Ala Tyr His Glu Val Asp Ser Ser Glu Met Ala Phe Lys Val Ala Gly Ser Gln Val Met Lys Lys Ala Ala Ala Gln Ala Gln Pro Val Ile Leu Glu Pro Val Met Ala Val Glu Val Thr Thr Pro Glu Asp Tyr Met Gly Glu Val Ile Gly Asp Leu Asn Ser Arg Arg Gly Gln Ile Gln Ala Met Glu Glu Arg Ser Gly Ala Arg Val Val Lys Ala Gln Val Pro Leu Ser Glu Met Phe Gly Tyr Val Gly Asp Leu Arg Ser Lys Thr Gln Gly Arg Ala Asn Tyr Ser Met Val Phe Asp Ser Tyr Ala Glu Val Pro Ala Asn Val Ser Lys Glu Ile Ile Ala Lys Ala Thr Gly Gln <210> 208 <211> 12 <212> PRT
<213> Mycobacterium vaccae <400> 208 Ala Leu Pro Gln Leu Thr Asp Glu Gln Arg Ala Ala

Claims (43)

Claims

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:
(a) 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 and 207 as measured by computer algorithm BLASTP;
(b) 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 (c) sequences having at least about 95% identical residues to a sequence recited in SEQ ID NOS: 143, 145, 147, 152, 154 I56, 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:
(a) 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;
(b) 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 (c) sequences having at least about a 99% probability of being the same as a sequence of (a) or (b) 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.

5. An expression vector comprising a polynucleotide according to claim 4.

6. A host cell transformed with an expression vector according to claim 5.

7. The host cell of claim 6, wherein the host cell is selected from the group consisting of E. toll, 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.

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.

15. 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:
(a) delipidated and deglycolipidated M. vaccae cells;
(b) inactivated M. vaccae cells; and (c) M. vaccae culture filtrate.

17. A method for enhancing an immune response it 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.

20. 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 is 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.

23. The method of claim 23 wherein the disorder is selected from the group consisting of mycobacterial infections, asthma, and psoriasis.

24. A method for the treatment of a disorder in a patient comprising administering composition comprising a component selected from the group consisting of:
(a) delipidated and deglycolipiidated M, vaccae cells;
(b) delipidated and deglycolipidated M.vaccae cells depleted of mycolic acids; and (c) delipidated and deglycolipidated M.vaccae cells depleted of mycolic acids and arabinogalactan;
the disorder being selected from the group consisting of immune disorders, infectious diseases, skin diseases and diseases of the respiratory system.

25. The method of claim 24, wherein the disorder is selected from the group consisting of mycobacterial infections, asthma and psoriasis.

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:
(a) sequences recited in SEQ ID NO: 89 and 201; and (b) 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:
(a) contacting dermal cells of a patient with one or more polypeptides according to any one of claims 1-3; and (b) detecting an immune response on the patient's skin.

28. The method of claim 27 wherein the immune response is induration.

29. A diagnostic kit comprising:
(a) a polypeptide according to any one of claims 1-3; and (b) apparatus sufficient to contact the polypeptide with the dermal cells of a patient.

30. A method for detecting mycobacterial infection in a biological sample, comprising:
(a) contacting the biological sample with a polypeptide according to any one of claims 1-3; and (b) detecting in the sample the presence of antibodies that bind to the polypeptide.

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 (b) 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.

35. The method of claim 33 wherein the binding agent is a polyclonal antibody.

36. A diagnostic kit comprising:
(a) at least one polypeptide according to any one of claims 1-3; and (b) 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.

40. The kit of claim 38 wherein the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and 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:
(a) delipidated and deglycolipidated M.vaccae cells depleted of mycolic acids;
and (b) delipidated and deglycolipidated M. vaccae cells depleted of mycolic acids and arabinogalactan.