EP0973881A2 - Mycobacterium recombinant vaccines - Google Patents

Abstract

Mycobacterium recombinant vaccines for treatment of intracellular diseases have been developed utilizing an antigen delivery system in the form of Mycobacterium strains, a genetic transfer system in the form of cloning nonpathogenic and expression vectors, and related technologies to provide products combining nontoxic immuno-regulating Mycobacterium adjuvants, nontoxic immuno-stimulating exogenous antigens specific for a variety of diseases, and nontoxic amounts of cytokines that boost the TH-1 pathway. Cloning and expression Mycobacterium vectors include both extra-nuclear and integrative vectors.

Description

MYCOBACTERIUM RECOMBINANT VACCINES

TECHNICAL FIELD OF THE INVENTION

The present invention relates to DNA constructs for cloning and methods of cloning mycobacterium genes.

BACKGROUND OF THE INVENTION

The mammalian immune system comprises both humoral and cellular components which are interrelated but have different roles. Although both arms of the immune system involve helper T cells, the outcome of the immune response depends on which subclass of T cells is involved. Helper T lymphocytes are produced by two maturation pathways (TH-1 and TH-2), are grouped according to cluster differentiation (CD4 and CD8), and secrete different cytokines. Both components of the immune system constantly scan and survey what is displayed in association with the molecules of the major histocompatibility complex (MHC), at the cell surface.

The humoral immune response involves helper T lymphocytes produced by the T cell maturation pathway TH-2. Cells of this pathway secrete cytokines such as Interleukin 4 (IL-4), IL-5, IL-6, IL-9, IL-10 and tumor necrosis factor (TNF). These cytokines inactivate macrophage proliferation, contributing to a down-regulation of the TH-1 response. TNF causes tissue inflammation and necrosis when released at high levels, which are the indications of failure of the overall immune system in many diseases. CD4+ T lymphocytes become activated through contact with antigens displayed in association with MHC class II molecules (MHC II), at the surface of macrophages and antigen presenting cells. Antibodies are produced by B cells when they interact with these activated CD4+ T lymphocytes. The MHC II molecules reside in the vesicles that engulf and destroy extracellular materials. Thus, their location within the cell gives them their specific function in monitoring the content of these vesicles. They specifically bind to antigens that have been enzymatically processed in the lysosomes of the immune cells after phagocytosis. The humoral immune response is required to protect the extracellular environment against extracellular antigens and parasites through antibodies which can be effective in neutralizing infectious agents. However, the humoral immune response cannot eliminate whole cells that become diseased, it causes tissue destruction and necrosis, and it is not effective in fighting intracellular diseases. Consequently, the body relies on the cellular immune response for protection from pathologies that start in the intracellular environment. Cellular immune response is carried out through cytotoxic immune cells which are capable of killing diseased cells. The cellular immune response involves helper T lymphocytes produced by the T cell maturation pathway TH-1. Cells of this pathway secrete cytokines such as IL-2, IL-12, IL-15, gamma Interferon (IFN), lymphotoxin, and Granylocyte Macrophage Colony Stimulating Factor (GMCSF). These cytokines activate macrophages. The cytotoxic T lymphocytes are CD8+ T cells that become activated through contact with antigens associated with MHC class I molecules (MHC I). MHC I molecules reside around the protein factories such as the endoplasmic reticulum. Thus, their location within the cell gives them their specific function of monitoring the output and transport of materials produced inside the cell. They specifically bind to antigens that have been synthesized in the intracellular environment like in the case of cancer or intracellular diseases. The cellular immune response protects against chronic intracellular diseases such as intracellular infection, parasitism and cancer, by activating the macrophages and facilitating the detection and lysis of diseased cells. The result is the formation of a granuloma which is the paradigm of protective immunity in intracellular diseases. Although the immune system has evolved to be efficient in selecting the target antigens against which an immune response is delivered, it does not always succeed in selecting the appropriate combination of the humoral and cellular immune components necessary to contain or eliminate the disease. For example, intracellular diseases resulting from genetic disorders, cancer, infections, allergies and autoimmune reactions are particularly difficult to treat and continue to be life threatening illnesses despite the advances in detection, diagnosis and treatment. Many of these diseases are able to circumvent the immune system and progress without challenge. For others with a long latency period, diagnosis is often made too late. Some display multi-resistance profiles against drug treatment or have their disease processes originating in environments accessible only to high doses of existing drugs. Many of these drug treatments have high toxic side effects. Treating with chemotherapy is expensive and may be implemented only after significant expansion of the pathological process, or if there is transmission of infection and damage to the host. Although these diseases may elicit an immune response, they usually compromise its effectiveness by suppressing or mimicking the MHC molecules.

In this type of illness, a TH-1 immune response favors protection, while down- regulation of this pathway, conversion to TH-2 during the chronic course of the disease, or up-regulation of the pathway TH-2 is detrimental to the host. Accordingly, a shift to TH-1 response or up-regulation of the TH-1 pathway should be beneficial on its own, and when associated with appropriate chemotherapy, would mount an effective response to resistance, chronicity, and disease. Therefore, treatment methods for intracellular diseases are needed which favor a TH-1 immune response rather than a TH-2 response.

Cancers are caused by genetic alterations that disrupt the metabolic activities of the cell. These genetic changes can result from hereditary and/or environmental factors including infections by pathogenic viruses. Like in other intracellular diseases, cellular immunity plays a major role in the host defense against cancer. Traditionally, cancer immunotherapies were designed to boost the cellular immune response by using specific and non-specific stimuli, including: 1) passive cancer immunotherapies where antibodies have been administered to patients, showing success only in rare cases; 2) active cancer immunotherapies where materials expressing cancer antigens have been administered to patients (e.g., the injection of whole or fractions of cancer cells that have been irradiated, modified chemically, or genetically) showing little impact in experimental tumor models; and 3) the combination of adoptive lymphocytes and IL-2, which caused regression of tumors in mice and metastic melanoma in humans. Tumor infiltrating lymphocytes (TIL) capable of mediating tumor regression are lymphoid cells that can be grown from single cell suspensions of the tumor incubated with IL-2. Thus, antigens recognized by TIL are more likely to be involved in vivo in anticancer immune response, and the cDNA and the amino-acid sequences of several of these antigens have been identified. While these findings have opened new opportunities for the development of cancer specific immunotherapies, treatment methods based on mixing cancer antigens or the cloning and expression of the genes encoding these antigens into a delivery system that favors a TH-1 response rather than a TH-2 response to these antigens are needed.

Intracellular infections are caused by bacteria, viruses, parasites, and fungi. These infectious agents are either present free in the environment or carried by untreated hosts. Humans, animals and plants can serve as hosts, and if not treated, they can act as reservoirs facilitating the further spreading of such agents to others. Intracellular pathogens such as M tuberculosis, M. leprae, and tumor viruses cause disease worldwide in millions of people each year. It is estimated that M. tuberculosis infects at least thirty million people/year and will cause an average of three million deaths/year during this decade, making tuberculosis (TB) the number one cause of death from a single infectious agent (World Health Organization, 1996). TB occurs most commonly in developing countries, but the prevalence of TB has increased recently in the U.S., as well as in developing countries, due to an increase in the number of immune compromised individuals with HIN infection. The risk of TB infection has also increased in individuals with diabetes, hemophilia, lymphomas, leukemias, and other malignant neoplasms, because these individuals have compromised immune systems. Leprosy and viruses which cause neoplasia are also important intracellular pathogens worldwide. Leprosy presently causes disease in more than twelve million people, and at least 15% of human cancers are thought to be caused by neoplastic transformation of cells by viruses.

Intracellular infections with highly virulent strains are quickly resolved resulting in death or cure of the patient. However, organisms of lower virulence can persist in the host and develop chronic diseases. Mycobacterium infections develop through a spectrum that ranges from a state of high resistance associated with cellular immunity to an opposite extreme of low resistance associated with humoral immunity. For example, leprosy is caused by Mycobacterium leprae which remains uncultivable. The disease manifests an immuno-histological spectrum with six groups. At one end of the spectrum, there is the polar tuberculous leprosy (TT), a paucibacillary form of the disease which is characterized by a strong TH-1 immune response and a bacteriolytic effect that lead to granuloma formation and restrict the growth of M. leprae, respectively. At the opposite end of the spectrum there is the polar lepromatous leprosy (LL), a multibacillary form of the disease which is characterized by a strong but inefficient TH-2 immune response and a down- regulation of the TH-1 pathway. During the chronic course of the disease the levels of IL-2 and cells with IL-2 receptors diminish, the T cells become defective in their functions, and M. leprae proliferates unrestricted within the macrophages and the schwann cells. With this immune failure the clearance of the bacteria is markedly retarded, and the patient continues to harbor bacilli in the tissues even after prolonged drug therapy. The antibodies react with circulating antigens forming immune complexes that lead to tissue damage, necrosis and organ failure. Between these two extremes there are four borderline forms of leprosy reflecting the different balances achieved by the body between TH-1 and TH-2 immune responses. Likewise, tuberculosis caused by Mycobacterium tuberculosis also manifests an immuno-clinical spectrum with multiple (four) groups. The reactive polar group (RR) is associated with a TH-1 immune response while the opposite pole (UU) is unreactive and is associated with a TH-2 immune response. Therefore, there are clear indications that the TH-1 immune response is the main defense mechanism in leprosy and tuberculosis. Thus, treatment and immuno- prophylaxis against these diseases should be aimed at enhancing the TH-1 pathway.

Allergic diseases are characterized by the sustained production of Ig E molecules against common environmental antigens. This production is dependent of IL-4 and is inhibited by gamma interferon. Thus, the allergic reactions involve a TH-2 immune response which requires a low level of stimulation by allergens. Therefore, preferable treatment for allergies would include the following: switching to a TH-1 immune response, which requires a high level of stimulation; activating CD8+ T cells and the production of gamma interferon; reducing the production of Ig E and recruitment of eosinophils and mast cells; and increasing the threshold concentration of the allergen to trigger a reaction.

Mycobacterium gene products, especially heat shock proteins, show homologies with bacterial, viral, parasitic, mycotic, and tumor antigens suggesting that these similarities may reflect regions in Mycobacterium antigens which can serve as potential inducers of cross immunity to different diseases. Heat shock proteins are overexpressed by stressed cells in many pathologies including infections, cancer, and autoimmune diseases. Thus, vaccinated individuals would have circulating cytotoxic T lymphocytes (CTL) that can interact and lyse the stressed cells, while the expression of putative autoimmunity antigenic domains in a susceptible host may lead to the suppression of the immune response and the chronicity of the disease. (Labidi, et al. 1992. "Cloning and DNA sequencing of the Mycobacterium fortuitum vaτ.fortuitum plasmid, pAL 5000," Plasmid 27: 130-140).

The available methods for prophylaxis and treatment of intracellular diseases include antibiotics, chemotherapy, and vaccines. Antibiotics have not been effective in treating diseases caused by M. tuberculosis or M. leprae because the lipid-rich cell wall of a mycobacteria is impermeable to antibiotics. Likewise, antibiotics have no effect on viral pathogenesis. Chemotherapy as a means of prophylaxis for high-risk individuals can be effective against M. tuberculosis o M. leprae, but it has disadvantages. Chemotherapeutic agents have undesirable side-effects in the patient, are costly, and iead to the potential existence of multi-drug resistant Mycobacterium strains. In addition to these disadvantages, chemotherapy as a means of treating active TB, leprosy, and virus-induced neoplasms has minimal effect since it is used only after significant disease progression. Consequently, vaccination is the therapy of choice because it provides the best protection at the lowest cost with the least number of undesirable side-effects. Early vaccines administered as protection against acute infections were developed using antigens to initiate an immune response regardless of its nature or its mechanism. The aim was to protect against acute infections where a TH- 2 immune response may be efficient. These vaccines were made of a variety of crude antigens including killed or attentuated whole cells, toxins, and other structural components derived from the pathogen. Bacterial products such as peptidoglycan, lipoproteins, lipopolysaccharides, and mycolic acids were used as therapeutic and prophylactic agents in several diseases. The administration of non-specific stimulants derived from Corynebacterium parvum, Streptococci, Serratia marcescens, and Mycobacterium, to cancer patients showed some efficacy and concomitantly enhanced the immune response against the disease. Adjuvants were developed to stimulate the immune response to antigenic material. One such adjuvant was complete Freund's adjuvant, which consisted of killed Mycobacterium tuberculosis suspended in oil and emulsified with aqueous antigen solution. This preparation was found to be too toxic for human use. (Riott, et al., Immunology, 5th ed., Mosby, Philadelphia, pp. 332, 370 (1998).

Following these first steps, efforts have been made to isolate and to develop single antigens and even single epitopes into vaccines. Molecular techniques have been used for the last two decades to clone the genes and map the domains of the corresponding proteins. However, individual antigens or cytokines did not reproduce the same physiological effects like a whole bacterial adjuvant. For example, antigen development forM tuberculosis, M. leprae, and other intracellular parasites were fruitless because the dogma of the specific protective antigens or epitopes could not accurately define a protective antigen for these diseases. The dogma, furthermore, has ignored the fact that the immune response to a pathogen is a coherent response to a mosaic complex of epitopes displayed by the pathogen with some epitopes conferring protection and other epitopes mediating virulence and immunopathology. These vaccines have been unsuccessful in establishing the favored TH-1 response over the TH-2 response. Early vaccines were also not potent against intracellular diseases. The vaccines were inefficient, short-lived, or triggered inappropriate immune responses similar to hypersensitivity reactions in allergic diseases that result in necrosis, which worsens the outcome of the pathological process in many chronic infections such as tuberculosis and leprosy. For example, BCG

(Bacille-Calmette Guerin) is a vaccine that has been used for TB and leprosy prophylaxis, but has questionable efficacy. BCG is an attenuated live vaccine derived from M. bovis, a Mycobacterium strain that is closely related to M. tuberculosis. BCG has been only marginally effective against leprosy and is not currently recommended for leprosy prophylaxis. Results from controlled studies to determine the efficacy of BCG vaccines for TB prophylaxis have been conflicting. Estimates of BCG efficacy from placebo-controlled studies range from no efficacy to 80% efficacy. A large scale BCG trial in India (n=360,000 people) showed that BCG failed to provide a protective effect against the onset of pulmonary TB. Other studies have shown that BCG produces an inconsistent, fluctuating immunity. Because no effective vaccine has been developed to protect against leprosy or virus-induced cancers, and because BCG is unreliable for TB prophylaxis, a more effective vaccine is needed. An example of such new vaccines would combine selective antigens with potent adjuvants and stimulate the cellular immune response to deliver a lasting protective immunogen.

In U.S. Patent No. 3,956,481, Jolles et al. discloses a hydrosoluble extract of mycobacteria suitable as an adjuvant, wherein delipidated bacterial residues are subjected either to a mild extraction process or treatment with pyridine followed by treatment with ethanol or water. These extracts were found to be toxic in humans, discouraging their use as a vaccine.

In U.S. Patent No. 4,036,953, Adam et al. discloses an adjuvant for enhancing the effects of a vaccine, wherein the adjuvant is prepared by disrupting mycobacteria or Nocardia cells; separating and removing waxes, free lipids, proteins, and nucleic acids; digesting delipidated material from the cell wall with a murolytic enzyme; and collecting the soluble portion. Adjuvants of this type were also noted to be toxic in humans.

In U.S. Patent No. 4,724,144, Rook, et al. discloses an immuntherapeutic agent comprising antigenic material from killed Mycobacterium vaccae cells useful for the treatment of diseases such as tuberculosis and leprosy. The vaccine has been shown to be effective against persistent microorganisms which survived long exposure to chemotherapeutic agents. Although the vaccine shows improved immune response, it is limited only to antigens endogenous to Mycobacterium vaccae. In U.S. Patent No. 5,599,545, Stanford, et al. discloses an immunotherapeutic agent comprising killed Mycobacterium vaccae cells in combination with an antigen exogenous to mycobacteria which promotes a TH- 1 response. The exogenous antigen may be combined with the killed Mycobacterium vaccae by admixture, chemical conjugation or absorption, or alternatively produced by expression of an exogenous gene in Mycobacterium vaccae via plasmid, cosmid, viral or other expression vector, or inserted into the genome. While these compositions promote the TH-1 immune response, they were limited only to killed Mycobacterium vaccae cells. Further, the patent provides no guidance as to how to make Mycobacterium expression vectors, or how to incorporate the expression vectors into either a plasmid, cosmid, or viral expression vector, or how to integrate the expression vector into the genome.

With respect to Mycobacterium diseases, advances made in the area of genetic tools and vaccine strategy included: the isolation, characterization and sequencing of the Mycobacterium plasmid pAL 5000; the identification of the kanamycin resistance gene as a selection marker for Mycobacterium, the development of the first Escherichia coli (E. coli) I Mycobacterium shuttle vectors; the construction of M. tuberculosis and M. leprae genomic libraries; and the expression of Mycobacterium DNA inE. coli. (Labidi, et al. 1984. "Plasmid profiles of Mycobacterium fortuitum complex isolates," Curr.

Microbiol. 11, 235-240; Labidi, et al. 1985. "Cloning and expression of mycobacterial plasmid DNA in Escherichia coli, " FEMS Microbiol Lett. 30, 221-225; Labidi, et al. 1985. "Restriction endonuclease mapping and cloning of Mycobacterium fortuitum var ^■. fortuitum plasmid pAL 5000," Ann. Insti. Pasteur Microbiol. 136B, 209-215; Labidi, et al. May 8-13, 1988. "Nucleotide sequence analysis of a 5.0 kilobase plasmid from Mycobacterium fortuitum," Abstract U6 of the 88th Annual Meeting of the American Society for Microbiology, Miami, Florida, USA; Labidi, et al. 1992. "Cloning and DNA sequencing of the Mycobacterium fortuitum var. fortuitum plasmid, pAL 5000," Plasmid 27, 130-140; Labidi, A. January, 1986. "Contribution to a plan of action for research in molecular biology and immunology of mycobacteria," Ph.D. Thesis. University of Paris and Pasteur Institute, Paris, France). Such advancements have opened the way for the application of recombinant DNA technology to Mycobacterium. ( Lazraq, et al. 1990. Conjugative transfer of a shuttle plasmid from Escherichia coli to Mycobacterium smegmatis. FEMS Microbiol. Lett. 69, 135-138;

Konicek, et al. 1991. Gene manipulation in mycobacteria. Folia Microbiol. 36(5), 411-422; and Falkinham, III, J O. and J.T. Crawford. 1994. Plasmids, p. 185-198. In Barry Bloom (ed.), Tuberculosis: Pathogenesis, protection and control. American Society for Microbiology, Washington, D.C.). The Mycobacterium expression vectors resulting from such advancements are not suitable for vaccine development because: 1) the expression vectors are large so the vectors have limited cloning capacity and low transformation efficiency (calculated as the number of transformants obtained per microgram of vector DNA), 2) the vectors lack multiple-cloning sites, 3) the protocols for transformation of mycobacteria with these expression plasmids result in inefficient transformation, 4) the spectrum of mycobacteria transformed by the vectors is restricted because transformation is host- dependent, and 5) the current expression plasmids do not stably transform mycobacteria. Therefore, suitable Mycobacterium expression vectors are needed which can provide efficient transformation and stable expression of multiple protective immunogens in mycobacteria. Suitable antigen delivery systems using nonpathogenic Mycobacterium strains, cloning vectors, and Mycobacterium expression vectors have now been found which contain protective immunogens that specifically stimulate a cell- mediated immune response by the induction of TH-1 cells, or cytotoxic T lymphocytes, and provide a consistent, prolonged immunity to intracellular pathogens.

BRIEF DESCRIPTION OF THE DRAWINGS:

Fig. 1 depicts a sequence of the origin of replication in E. coli (695 bp). The underlined base indicates the replication point.

Fig. 2 depicts a sequence for the kanamycin gene (932 bp). The underlined sequences are in the 5' to 3' order: the (-35) region for the gene, the (-10) region for the gene, the ribosomal binding site region for the gene, the starting codon (ATG), and the stop codon (TAA).

Fig. 3 A depicts a sequence of the pAL 5000 origin of replication (1463 bp) obtained by restriction enzymes analysis. The numbers in superscript indicate the position of the nucleotides in the published sequence of pAL 5000

(Labidi, et al. 1992. "Cloning and DNA sequencing of the Mycobacteriumfortuitum var. fortuitum plasmid, pAL 5000," Plasmid 21.130- 140). The underlined sequences indicate in the 5' to 3' order: the position of the forward (F_c, Fj, F₂, and F₃), and the reverse (R₄, R₃, R₂, R and Re) primers used in PCR analysis, respectively.

Fig. 3B depicts a sequence of the pAL 5000 origin of replication (1382 bp) obtained after PCR analysis. The numbers in superscript indicate the position of the nucleotides in the published sequence of pAL 5000 (Labidi, et al. 1992. Plasmid 27: 130-140). The underlined sequences indicate in the 5' to 3' order: the position of the forward ( F_l5 F₂ and F₃), and the reverse (R₄, R₃,

R₂ and R_λ) primers used in PCR analysis, respectively.

Fig. 4 A depicts a sequence of the attachment site (attP) and the integrase gene (mt) of the Mycobacteriophage D₂₉, obtained by restriction enzymes analysis (1631 bp). The numbers in superscript indicate the position of the nucleotides in the sequence. The underlined sequences delimited by numbered nucleotides indicate in the 5' to 3' order: the position of the forward (F_c, F_l5 F₂, F₃, and F₄) and the reverse (R₄, R₃, R₂, R and R primers used in PCR analysis, respectively. The underlined sequences not delimited by numbered nucleotides indicate in the 5' to 3' order: the attachment site (attP), the (-35) region for the gene ( t), the (-10) region for the integrase gene (int), the ribosomal binding site region for the integrase gene (int), and the starting codon (ATG) for the integrase gene (int). The stop codon for the integrase gene (int) is the TGA¹⁵³¹.

Fig. 4B depicts a sequence of the attachment site (attP) and the integrase gene (int) of the Mycobacteriophage D₂₉, obtained after PCR analysis ( 1413 bp). The numbers in superscript indicate the position of the nucleotides in the sequence. The underlined sequences delimited by numbered nucleotides indicate in the 5' to 3' order: the position of the forward (F₃, and F₄) and the reverse (R₄, R₃, and R₂) primers used in PCR analysis, respectively. The underlined sequences not delimited by numbered nucleotides indicate in the 5' to 3' order: the attachment site (attP), the (-35) region for the gene (int), the

(-10) region for the integrase gene (int), the ribosomal binding site region for the integrase gene (int), and the starting codon (ATG) for the integrase gene (int). The stop codon for the integrase gene (int) is the TGA¹⁵³¹.

Fig. 4C depicts a sequence of the attachment site (attP) and the integrase gene (mt) of the Mycobacteriophage D₂₉, obtained after PCR analysis

( 1374 bp). The numbers in superscript indicate the position of the nucleotides in the sequence. The underlined sequences delimited by numbered nucleotides indicate in the 5' to 3' order: the position of the forward (F₄) and the reverse (R₄, R₃, and R₂) primers used in PCR analysis, respectively. The underlined sequences not delimited by numbered nucleotides indicate in the 5' to 3' order: the attachment site (attP), the (-35) region for the gene (int), the (-10) region for the integrase gene (int), the ribosomal binding site region for the integrase gene (mt), and the starting codon (ATG) for the integrase gene (int). The stop codon for the integrase gene (int) is the TGA¹⁵³¹. Fig. 5 depicts a sequence for the kanamycin gene promoter(102 bp) and the first ATG codon. The underlined sequences are in the 5' to 3' order: the (-35) region for the gene, the (-10) region for the gene, the ribosomal binding site region for the gene, and the starting codon (ATG).

Fig. 6 depicts a sequence of the pAL 5000 fragment containing the open reading frame ORF 2 (2096 bp). The numbers in superscript indicate the position of the nucleotides in the published sequence of pAL 5000 (Labidi, et al. 1992. /7α-wικ/27: 130-140). The underlined sequence (GGATCC is the unique Bam HI site which is spanned by the ORF 2 promoter.

Fig. 7 is a gene map of a representative genetic transfer system, wherein "C-ter/anch/seq." = C terminal anchoring sequence; "MCS/express." = multiple cloning site for expression; "N-ter/lead/seq. " = N terminal leading sequence;

"MycofPτom " - Mycobacterium promoter; "Rep/Integ/Mycø" = Mycobacterium origin of replication or phage attachment site and integrase gene (either one or the other but not both is present in a given vector); "MCS/gen clon." = multiple cloning site for general cloning; "univ/select/mark." = universal selection marker; and "oή/E.coli" = E. coli origin of replication.

DETAILED DESCRIPTION OF THE INVENTION

The therapeutic or prophylactic vaccines of the present invention combine a protective immunogen with one or more Mycobacterium strains acting as a delivery system and an adjuvant, preferably in addition to cytokines and appropriate chemotherapy. The rationale is that the Mycobacterium cells will be ingested by macrophages and remain within the macrophage, blocking the killing mechanism of the macrophage while synthesizing the protective immunogen. The immunogen will be processed and presented on the macrophage cell surface to T cells, resulting in TH-1 cell activation and a cell- mediated immune response that is protective against the intracellular disease.

One aspect of the present invention uses an antigen delivery system in the form of a nonpathogenic Mycobacterium strain to provide products combining nontoxic immuno-regulating Mycobacterium adjuvants, nontoxic immuno-stimulating protective immunogens specific for a variety of diseases, and nontoxic amounts of cytokines that boost the TH-1 pathway. Preferably, the present invention uses a protective immunogen delivery system in the form of a nonpathogenic Mycobacterium strain, a genetic transfer system in the form of cloning vectors, and expression vectors to carry and express selected genes in the delivery system. Protective immunogen delivery system

The protective immunogens of the present invention form pure non- necrotizing complete granuloma. Such immunogens can be protein antigens or other immunogenic products produced by culturing and killing the diseased cell or infectious microorganism, by separating and purifying the immunogens from natural or recombinant sources, or by the cloning and expression into a

Mycobacterium delivery system of the genes encoding these protein antigens or the enzymes necessary to modify an endogenous lipid to a stage where it is immunogenic and specific. The protective immunogens of the present invention include antigens associated with: 1) cancer including but not limited to lung, colorectum, breast, stomach, prostate, pancreas, bladder, liver, ovary, esophagus, oral and pharynx, kidney, non-Hodgkin's, brain, cervix, larynx, myeloma, corpus uteri, melanoma, thyroid, Hodgkin's, and testis; 2) bacterial infections including but not limited to mycobacteriosis (e.g., tuberculosis and leprosy), Neisseria infections (e.g., gonorrhea and meningitis), brucellosis, plague, spirochetosis (e.g., trypanosomiasis, Lyme disease and tularemia), rickettsiosis (e.g., typhus, rickettsialpox, and anaplasmosis), chlamydiosis (e.g., trachoma, pneumonia, atherosclerosis, and urethritis), and Whipple's disease; 3) parasitic diseases including but not limited to malaria, leishmania, trypanosomiasis, and toxoplasmosis; 4) viral diseases including but not limited to measles, hepatitis, T-cell leukemia, dengue, AIDS, lymphomas, herpes, and warts; 5) autoimmune diseases including but not limited to rheumatoid arthritis, ankylosing spondylitis, and Reiter's syndrome; 6) allergy diseases including but not limited to asthma, hay fever, atopic eczema, and food allergies; 7) veterinary diseases including but not limited to feline immunodeficiency, equine infectious anemia, avian flue, heartworm, and canine flea allergy; and 8) other diseases including but not limited to leukemia, multiple sclerosis, bovine spongiform (BSE), and myoencephalitis (ME). These antigens can be used singly or in combination in one vaccine. When a combination of antigens is used, they can be administered together at one time or they can be administered separately at different times. Preferred endogenous lipid protective immunogens for the treatment of tuberculosis, leprosy, and other mycobacterioses include but are not limited to complex lipid heteropolymers such as the phenolic glycolipids PGL I and PGL Tbl, the sulfolipid SL I, the diacyl-trehalose DAT and the lipo-oligosaccharide LOS. These lipid immunogens are not synthesized, or modified to their final forms by all Mycobacterium species. Therefore, the host strain must provide the necessary precursors to synthesize the desired final immunogenic products. When using an expression vector, the expression system must provide the necessary genes that encode the necessary enzymes to modify the lipid to a stage where it is immunogenic. The mycobacterial adjuvant of the present invention is one that boosts the TH-1 immune response, and preferably down-regulates the TH-2 response. The Mycobacterium strains are characterized by their lack of pathogenicity to mammals and their capacity to be ingested mammalian macrophages The Mycobacterium strains of the present invention may be live or dead upon administration When the vaccines of the present invention are administered to immunocompromised patients, only dead Mycobacterium strains are used

Preferable Mycobacterium strains can be obtained from the American Type Culture Collection (Rockville, MD) One or more types of Mycobacterium species may be utilized in the preparation of a vaccine Examples include but are not limited to nonpathogenic Mycobacterium vaccae, Mycobacterium gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium chitae, Mycobacterium duvaln, Mycobacterium flavescens, Mycobacterium nonchromogenicum, Mycobacterium neoaurum, and Mycobacterium bovis BCG M. bovis BCG and M. gastri are the only known Mycobacterium species that have precursors for producing M. tuberculosis and M. leprae lipids, therefore, M. gastri must be used if the precursors of exogenous lipids are to be expressed in a vaccine for TB or leprosy M. gastri and M. triviale can be found in the gastrointestinal tract, and are, thus, important for use in oral vaccines The Mycobacterium adjuvants of the present invention can utilize either one Mycobacterium strain or multiple strains, however, when killed Mycobacterium vaccae is used, it is preferably administered in combination with other Mycobacterium species

Preferably, the vaccine of the present invention also comprises cytokines that associate with the TH-1 pathway Examples of such cytokines include but are not limited to gamma interferon (IFN), interleukin(IL)-2, IL-12,

IL-15 and granulocyte macrophage colony stimulating factor (GMCSF)

Additionally, the vaccine of the present invention may also be administered in combination with appropriate chemotherapy for treatment of patients with active diseases If a live Mycobacterium strain is used as an adjuvant, appropriate chemotherapy must be selected that does not interfere with the adjuvant function of the live Mycobacterium Examples of appropriate concommitant chemotherapy is Taxol-R for the treatment of cancer or protein inhibitors for AIDS treatment.

The protective immunogens, cytokines, and concommitant chemotherapy may be produced separately in a synthetic or in a recombinant form, purified by any conventional technique. They may be used in parallel with, mixed with, or conjugated to live or dead Mycobacterium cells of interest. Genetic transfer system

The genetic transfer system of the present invention comprises cloning vectors where the genes of interest are cloned and the transformation technique is used to introduce and express the recombinant molecules into the delivery- system. Previous cloning vectors which have been used in Mycobacterium species include the extrachromosomal M. fortuitum plasmid pAL 5000 (Labidi, et al. 1992. "Cloning and DNA sequencing of the Mycobacterium fortuitum var. fortuitum plasmid, pAL 5000," Plasmid 27: 130-

140) which replicate extrachromosomally and the mycobacteriophage D₂₉. (Forman, et al. 1954. "Bacteriophage active against virulent Mycobacterium tuberculosis: isolation and activity," Am J Public Health 44: 1326-1333) Mycobacteriophage D₂₉ is a large spectrum virulent phage able to infect and efficiently reproduce itself in cultivated Mycobacterium species and attach itself to uncultivated M. leprae.

New cloning vectors have now been developed which are generally made of either origin(s) of replication or integration system(s), selection marker(s), and multiple cloning site(s) (MCS). The cloning vectors are comprised of the minimum functional sizes of various components including the following components: the E. coli replicon, the kanamycin selection marker, the pAL 5000 origin of replication, and the D₂₉ attachment site (attP) and integrase gene (int). Using conventional deletion techniques, the coding region for each component have been reduced to the point that further loss of base pairs resulted in loss of function, hence the designation of minimum functional size. The sequences for each minimum functional component are given as follows: origin of replication in E. coli (695 bp) as SΕQ ID NO: 1 and Fig. 1 ; kanamycin gene (932 bp) as SΕQ ID NO:2 and Fig. 2; origin of replication in pAL 5000 (1463 bp) obtained by restriction enzyme analysis as SΕQ ID NO: 3 and Fig. 3A; origin of replication in pAL 5000 (1382 bp) obtained after PCR analysis as SΕQ ID NO:4 and Fig. 3B; Mycobacteriophage D₂₉ attachment site and integrase gene (1631 bp) obtained by restriction enzyme analysis as SΕQ ID NO: 5 and Fig. 4 A; Mycobacteriophage D₂₉ attachment site and integrase gene (1413 bp) obtained after PCR analysis as SΕQ ID NO:6 and Fig. 4B; and Mycobacteriophage D₂₉ attachment site and integrase gene (1374 bp) obtained after PCR analysis as SΕQ ID NO: 7 and Fig. 4C. It is well understood in the art of deletion techniques that while the above-identified sequences provide the coding regions for each minimum functional component, an additional loss of a few base pairs from the minimum functional component could still result in a functional component of the present invention. Numerous E. coli origins of replication are commercially available and can be utilized in the present invention. For example, the E. coli origin of replication ColΕl is found in most commercially available plasmid vectors designed for E. coli. Although the replication point is usually indicated for these vectors, the smallest fragment that can support an efficient replication in E. coli has not heretofore been specified. Using the commercially available plasmid vector pNΕB 193 ( Guan C, New England Biolabs Inc., USA, 1993) as starting material, it has now been determined through restriction endonuclease deletions, cloning, and transformation analysis that the smallest DNA fragment that can support an efficient ColEl replication in E. coli is limited to a 695 bp sequence given in SEQ ID NO: 1 and Fig. 1. This E. coli origin of replication of minimum functional size has been successfully utilized in the construction oϊE.coli cloning vectors and E. coli-Mycobacterium shuttle vectors of the present invention.

While a variety of selection markers are available for the selection of transformed cells and can be used in the present invention, the Streptococcus faecalis 1489 bp gene coding for resistance to kanamycin has been selected as a representative selection marker for Mycobacterium (Labidi, et al. 1992. "Cloning and DNA sequencing of the Mycobacterium fortuitum var. fortuitum plasmid, pAL 5000," Plasmid 27: 130-140; Labidi, et al. 1985. "Restriction endonuclease mapping and cloning of Mycobacterium fortuitum var ^■. fortuitum plasmid pAL 5000," Ann. Insti. Pasteur/Microbiol. 136B, 209-215). While this gene is well established as the selection marker for Mycobacterium (Konicek, et al . 1991. Folia Microbiol. 36(5), 411 -422), the smallest fragment capable of supporting kanamycin selection in Mycobacterium has not heretofore been established. It has now been found that the minimal functional sequence for this gene is about 932 bp as shown in SEQ ID:NO2 and Fig. 2.

The kanamycin gene of minimum functional size described herein has been successfully utilized in the construction of E. coli cloning vectors and E. coli- Mycobacterium shuttle vectors of the present invention.

Vectors containing a plasmid origin of replication do not usually integrate in the chromosome of the host strain. Thus, they are extra- chromosomal vectors. The replication and maintenance in Mycobacterium strains of the extra-chromosomal vectors developed in this study, are supported by the origin of replication oϊ t e Mycobacterium fortuitum plasmid pAL 5000. Labidi, et al. 1984. "Plasmid profiles of Mycobacterium fortuitum complex isolates," Curr. Microbiol. 11, 235-240. The pAL 5000 plasmid is the most thoroughly studied Mycobacterium plasmid and has been used worldwide to develop vectors for genetic transfer in Mycobacterium (Falkinham, III, J.O. and IT. Crawford. 1994. Plasmids, p. 185-198. In Barry Bloom (ed), Tuberculosis: Pathogenesis, protection and control. American Society for Microbiology, Washington, D.C.). Functional analysis of the pAL 5000 plasmid has indicated the location of two open reading frames coding for a 20 KDa and a 65 KDa protein, respectively, and a 2579 bp fragment containing its origin of replication (Labidi, et al. 1992. Plasmid 27: 130-140). In the present invention, the 2579 bp fragment was reduced through deletions with restriction enzymes to a 1463 bp fragment extending from nucleotide 4439 to nucleotide

1079 without loosing its function (SEQ ID NO:3 and Fig. 3A). It has been found that the 1247 bp fragments extending from nucleotide 4439 to nucleotide 863, and the 1315 bp fragment extending from nucleotide 4587 to nucleotide 1079 do not support replication in Mycobacterium (SEQ ID NO:3 and Fig. 3A). Thus, the role of the sequences extending from nucleotide 4439 to nucleotide 4587, and from nucleotide 863 to nucleotide 1079 have now been investigated. In the absence of usable restrictions sites in these two areas of the pAL 5000 sequence, sets of forward and reverse primers that span the two areas have been designed. PCR is then used to amplify the different fragments which are subsequently cloned into an E. coli replicon containing the kanamycin gene. Using PCR analysis technique, the minimal functional pAL

5000 origin of replication has been reduced to a 1382 bp fragment extending from nucleotide 4468 to nucleotide 1027 as given in SEQ ID NO: 4 and Fig. 3B. Although it has been determined that the 1383 bp fragment extending from nucleotide 4519 to nucleotide 1079, and the 1356 bp fragment extending from nucleotide 4439 to nucleotide 972 did not support replication in

Mycobacterium, it is further believed that some of the 51 bp sequence extending from nucleotide 4468 to nucleotide 4518 and the 55 bp sequence extending from nucleotide 973 to nucleotide 1027 also might not be needed for replication. This pAL 5000 origin of replication of minimum functional size described herein has been successfully utilized in the Mycobacterium cloning vectors and construction of E. coli-Mycobacterium shuttle vectors of the present invention.

Vectors can also include a phage attachment site (attP) and its accompanying integrase gene. A preferred embodiment of the present invention comprises the attachment site (attP) and the integrase gene (int) of the Mycobacteriophage D₂₉ (Forman, et al. 1954. Am J Public Health 44: 1326-1333). The phage D₂₉ is a large spectrum virulent phage able to infect cultivated Mycobacterium species and efficiently reproduce itself. To develop integrative vectors, a map of its attachment site (attP) and integrase gene (int) has been determined by constructing a set of hybrid plasmids containing overlapping fragments of D₂₉ genome. The recombinant plasmids were then electroporated into the Mycobacterium strains and plated on LB medium containing 50 uglrcλ kanamycin. A plasmid containing a 2589 bp fragment generated Mycobacterium transformants. The 2589 bp fragment was isolated and further analyzed. After establishing its restriction map, another set of hybrid plasmids were constructed containing overlapping segments of the 2589 bp fragment. These recombinant plasmids were electroporated into the Mycobacterium strains then plated on selective media. The smallest fragment still able to generate kanamycin resistant Mycobacterium transformants were isolated and sequenced using a double strand plasmid template and sequenase version 2.0 (USB, Cleveland, Ohio, USA). The sequence analysis indicated that the fragment size was 1631 bp, which comprised from 5' to 3' the phage attachment site (attP), the integrase gene promoter and the integrase gene (int) (SEQ ID NO: 5 and Fig. 4A). Subsequent deletions studies regarding the 1631 bp were performed. A 1413 bp originating from base pair 119 to 1531 , illustrated in Fig. 4B afforded a high transformation efficiency. Additional deletion studies resulted in a 1374 bp fragment originating from base pair 158 to 1531, illustrated in Fig. 4C. The 1374 bp fragment generated Mycobacterium transformants, but the transformation efficiency was 100 times lower and the incubation time becomes much longer, probably due to low efficiency of integration and stability. It is believed that some of the 39 bp sequence extending from nucleotide 119 to nucleotide 157 might not be needed for integration. These D₂₉ (AttP), (int) and the preceding sequence as described above are the smallest phage DNA fragment so far used in the construction of Mycobacterium integrative expression vector and E . coli/ Mycobacterium integrative shuttle vectors.

The MCS is a synthetic fragment of DNA containing the recognition sites for certain restriction enzymes that do not cut in the vector sequence. The choice of enzymes to be included in the MCS is based on their frequent use in cloning and their availability. Representative enzymes include Bam I, EcoR V, and Pst I. From these minimal functional components, cloning vectors have been developed which maximize the capacity for multiple cloning sites. Preferably, the cloning vectors comprise each component at its minimal functional size. For example, extra-chromosomal cloning vectors have been constructed by assembling the minimum functional fragments for the E. coli origin of replication, the pAL 5000 origin of replication, the kanamycin gene, and the MCS. Exemplary integrative cloning vectors have the same structure except the origin of pAL 5000 is replaced by the attP and the integrase gene of D₂₉. When each component of the cloning vector is reduced to its smallest functional size, the vectors have a size of about 3 Kb and a transformation efficiency about 10⁸. Each vector has a theoretically unlimited cloning capacity and is capable of transforming Mycobacterium species. Each cloning vector is presented in Table I.

Fig.7 presents a genetic map of an exemplary cloning and expression vector. The present invention does not require any particular ordering of the functional components within the cloning vector.

Further, the cloning vectors of the present invention, do not require that each component contained in the vector be reduced to its minimum functional size. The degree to which the minimal functional components are utilized in each cloning vector is dictated ultimately by the application of the vaccine and the maximum transformation size. For example, an integrative cloning vector may contain the minimal functional component for the attachment site and integrase gene while the selection marker is larger than its minimal functional size. Such an arrangement can arise because the cloning vector contains only one site for cloning a protective immunogen, thereby allowing other components of the vector to range in size as long as the vector is of a small enough size to allow for efficient transformation into Mycobacterium cells.

Preferably, the present invention uses an E. coli-Mycobacterium shuttle vector constructed by applying various recombinant DNA techniques. The constructed vector can be efficiently transformed into either an E. coli or

Mycobacterium host, allowing selected mycobacterial genes to be exponentially cloned and expressed. Preferably, the E. coli-Mycobacterium shuttle vector uses a selection marker that can be expressed in both genera. One shuttle vector is comprised of a kanamycin selection marker, an origin of replication for E. coli, and an origin of replication for the Mycobacterium plasmid pAL 5000. Another shuttle vector is comprised of a kanamycin selection marker, an origin of replication for E. coli, and an attachment site and integrase gene of the Bacteriophage D29. Each component of the constructed shuttle vector has been reduced to its smallest functional size thereby enhancing its cloning and transformation efficiency. By reducing the vector components to their minimum functional size, the cloning vectors have the capacity for a multiple cloning site with a large number of restriction sites. Therefore, the genetic transfer system of the present invention preferably comprises cloning vectors for more than one protective immunogen. When more than one Mycobacterium strain is used in a vaccine, the genetic transfer system of each Mycobacterium strain comprises cloning vectors for one or more protective immunogens. Transformation

Mycobacterium strains have been successfully transformed through electroporation. (Labidi, et al. 1992. "Cloning and DNA sequencing of the Mycobacteriumfortuitum var. fortuitum plasmid, pAL 5000," Plasmid 27: 130-

140) It is understood that other transformation techniques developed for Mycobacterium would be useful in the present invention. The electroporation techniques of the present invention are described in Example 3, and the results are given in Table 1. The vector designs, culture medium, and the transformation technique described have improved significantly the transformation efficiency for Mycobacterium species and brought it for the first time to a level comparable to that obtained with E. coli. The integrative vectors containing the attachment site (attP) and the integrase gene (int) of the phage D₂₉ have been found to integrate into the chromosomes of their hosts at a region complementary of the region (attP). This region is the bacterial attachment site (attB) and is located between the genes encoding the Proline transfer RNA (tRNA¹™) and the Glycine transfer RNA (tRNA^Gly).

Table I: Vectors

to

t oo

to

<- o

-P-

S

σs

t- oo

4--

O

t

-1^

4-.

-P-

4-. σs.

4->

4-- oo

-

t

(-fl

sS>

Indicates the marker used to select transformants. Ap, Tc and Km were used at 100 ug/ml, 15 ug/ml and 50 ug/ml respectively.

2 Indicates the remaining functional origin of replication and/or integration in the vector. 3 Indicates the transformation efficiencies obtained by electroporation for E. coli and Mycobacterium respectively.

The efficiencies for D₂₉ and pAL 5000 were set arbitrarily at 100% when these vectors are used with their host strains respectively.

4 Indicates ORFs other than the one(s) involved in replication, expressed from pAL 5000 in E. coli mini-cells.

5 Abbreviations: MW = Molecular Weight, bp = base pair, Ap = Ampicillin, Tc = Tetracycline, Km = Kanamycin,

ORF = Open Reading Frame.

Expression vectors

The expression vectors of the present invention are made by inserting functional promoters from plasmid or chromosomal origin into the cloning vectors which serve as backbones. The expression vectors are tailored to carry and express selected genes in the delivery system. They contain in their structures the genetic information necessary for their auto-replication in the cytoplasm, or their integration into the chromosome of the host. They provide the promoter and the regulatory sequences necessary for 1) gene expression, and if necessary, 2) the secretion of the gene product out of the cytoplasm to the cell membrane structure or to the extracellular environment.

While the kanamycin gene is a preferred selection marker for the present invention, it is also well expressed in a wide range of hosts including Mycobacterium and E. coli species, and therefore, vectors containing the promoter of this gene can express foreign genes in E. coli and Mycobacterium strains, respectively. Using conventional PCR techniques, the minimum functional component of this promoter was determined and is given in SEQ ID NO: 8 and Fig. 5. The use of a kanamycin promoter to construct E. coli- Mycobacterium expression shuttle vectors is reported for the first time.

Another preferred expression vector in the present invention used the promoter of pAL 5000 open reading frame (ORF) 2. An open reading frame

(ORF 2) encoding a 60 - 65 KDa protein in E. coli minicells was identified in the plasmid pAL 5000. To map the promoter region of this ORF, the 2096 bp fragment containing this open reading frame (SEQ ID NO:9 and Fig. 6) has been isolated. Through restriction endonuclease deletions, cloning, and transformation analysis, a set of hybrid plasmids containing overlapping segments of the 2096 bp fragment were constructed. These recombinant plasmids were electroporated into E. coli DS410. Minicells were prepared from transformants and plasmid encoded proteins were analyzed as indicated in Example 4. The promoter of the ORF was found in the sequence spanning the unique Bam HI site in the fragment indicated in Fig. 6.

The products of the invention are administered by injection given intradermal or via other routes (e.g., oral, nasal, subcutaneous, intraperitoneal, intramuscular) in a volume of about 100 microliters containing 10⁷ to 10¹¹ live or killed cells of recombinant Mycobacterium, or the same amount of nonrecombinant Mycobacterium cells mixed with, or conjugated to predetermined amounts of the exogenous antigens, the cytokines, and/or the drugs. If the products are being used with patients with active diseases, they should be associated with drug treatments that do not interfere with the live form of the vaccine if it is being used. If the products of the invention are being used separately, they can be administered in any order, at the same or at different sites, and using the same or different routes. The invention takes in consideration that the products are designed to be used in humans or in animals and therefore they must be effective and safe with or without any further pharmaceutical formulation that may add other ingredients.

In summary, the preferred cloning and expression vectors of the present invention comprise an E. coli-Mycobacterium shuttle vector which contains the following: an origin of replication for both E. coli (E. coli replicon) and Mycobacterium (pAL 5000 origin of replication), a kanamycin resistance marker, multiple cloning sites, promoters and regulatory sequences for secretion of gene products out of the bacteria and for insertion into the cell membrane, and the attachment site (attP) and integrase gene (int) of phage D₂₉. Another type of preferred cloning and expression vectors contain all of these elements listed above except the phage D₂₉ attachment site and integrase gene. The multiple cloning sites allow cloning of a variety of DNA fragments. The E. coli replicon, the pAL 5000 origin of replication, the kanamycin resistance marker, and the D₂₉ attP site and int genes have been mapped and reduced to their minimum functional sizes to maximize the cloning capacity of the vector and to increase the transformation efficiency. A new transformation protocol was developed so that the efficiency with which these vectors transform Mycobacterium strains (10⁸ Mycobacterium transformants/μg DNA) approaches the transformation efficiency for E. coli.

The vaccine system of the present invention has a number of advantages over current vaccines. The major advantage of such a system over current vaccines is the ability to specifically express immunogens that elicit a consistent, protective immune response, i.e., a prolonged activation of TH-1 cells with concomitant activation of macrophages. Additional advantages include: 1) protective immunogens for more than one intracellular disease can be incorporated into one vaccine, 2) such a genetically engineered vaccine is flexible in that new technology can be easily incorporated to improve the vaccine, and 3) large amounts of immunogen can be synthesized by using a genetically engineered expression vector to induce protective immunity, 4) the Mycobacterium itself acts as an adjuvant injected along with the immunogen to induce immunity, 5) the vaccine is naturally targeted to macrophages because t e Mycobacterium infect these cells, 6) and prolonged immunity will result since a Mycobacterium strain remains live within by the macrophages for a long time.

Methodologies for performing various aspects of the present invention are presented below. DNA. RNA and oligonucleotide primers.

DNA and RNA were extracted and purified at Cytoclonal Pharmaceutics, Inc., Dallas, Texas. The oligonucleotide primers were purchased from National Biosciences Inc., Plymouth, MN., or from Integrated DNA Technologies Inc., Coralville, I A. Enzymes.

Restriction endonucleases were purchased from United States Biochemical Inc., Cleveland, OH.; New England Biolabs Inc., Beverly, MA.; Promega Inc., Madison, WL; Stratagene Inc., La Jolla, CA.; MBI Fermantas Inc., Lithuania.; and TaKaRa Biomedicals Inc., Kyoto, Japan. DNA ligase was purchased from Boehringer Mannheim Biochemica Inc., Indianapolis, IN.;

Gibco-BRL Inc., Gaithersburg, MD., and New England Biolabs. RNase was purchased from 5 Prime >3 Prime Inc , Boulder, CO

Deoxyribonucleotides and DNA polymerase I (Klenow fragment) were purchased from New England Biolabs Alkaline phosphatase was purchased from Boehringer Mannheim Biochemica and New England Biolabs Taq polymerase was purchased from Qiagen Inc , Chatsworth, CA AMV reverse transcriptase was purchased from Promega Inc DNase-free RNase and RNase-free DNase were purchased from Ambion Inc , Austin, TX Computer software

The computer software Oligo (National Biosciences Inc, Plymouth, MN) and Mac Vector (Oxford Molecular Group Inc , Campbell, CA) were used to design primers and to analyze nucleic acid and protein sequences Preparation of Microorganisms

Bacterial strains and bacteriophages were used from the collection of the Vaccine Program at Cytoclonal Pharmaceutics Inc , Dallas, TX Antibiotics ampicillin, kanamycin and tetracycline were purchased from

Sigma Chemical Co , Inc (Saint Louis, MO)

The requirements for Mycobacterium species to grow are usually more complex and more diversified than those for E. coli strains Consequently, a general culture medium, hereinafter designated Labidi's medium, has been developed which can support the growth of all Mycobacterium species and which contributes to the increased transformation rate of the present invention The composition of the Labidi's medium per liter contains about 0 25% proteose peptone No 3, about 0 2% nutrient broth, about 0 075% pyruvic acid, about 0 05%) sodium glutamate, about 0 5% albumin fraction V, about 0 7% dextrose, about 0 0004% catalase, about 0 005% oleic acid, L_H amino-acid complex (about 0 126% alanine, about 0 097% leucine, about 0 089% glycine, about 0 086%) valine, about 0 074% arginine, about 0 06% threonine, about 0 059%) aspartic acid, about 0 057% serine, about 0 056% proline, about 0 05%) glutamic acid, about 0 044% isoleucine, about 0 033% glutamine, about 0 029%) phenylalanine, about 0 025%) asparagine, about 0 024% lysine, about

0 023%) histidine, about 0 021%) tyrosine, about 0 02% methionine, about 0.014% tryptophan, and about 0.01% cysteine), about 0.306% Na₂HPO₄, about 0.055% KH₂PO₄, about 0.05% NH₄C1, about 0.335% NaCl, about 0.0001% ZnSO₄, about 0.0001% CuSO₄, about 0.0001% FeCl₃, about 0.012% MgSO₄, about 0.05% Tween 80, and about 0.8% Glycerol (except forM bovis), pH 7.0. A solid form of this medium can be obtained by adding 2.0% agar. Whenever it is necessary, this medium can be supplemented with preferred selection markers and/or with special factors required for the growth of certain species such as mycobactin for M. paratuberculosis and hemin X factor forM haemophilium. For transformation, cultures were grown on Labidi's medium. The cultures were incubated at the appropriate temperature for each strain. Cultures in liquid media were shaken at 150 rpm in a rotatory shaker Gyromax 703 (Amerex Instruments Inc., Hercules, CA).

In growing Mycobacterium cells for the vaccine, cultures were grown on protein-free media: [per liter: 6.0% glycerol, 0.75%glucose, 0.4% asparagine, 0.25% Na₂HPO₄, 0.2% citric acid, 0.1% KH₂PO₄, 0.05% ferric ammonium citrate, 0.05% MgSO₄, 0.02% Tween 80, 0.0005%) CaCl₂, 0.0001% ZnSO₄, and 0.0001% CuSO₄, at a final pH of 7 ]. Whenever it is necessary, this medium can be supplemented with the required selection markers and/or the growth factors.

For routine culture of E. coli strains, the bacteria were cultivated on Luria Broth (LB) medium [per liter of medium: 1% tryptone, 1% NaCl, and 0.5% yeast extract in distilled or deioninzed water]. The solid form of the LB medium was obtained by adding 2.0% agar to the previous formula. When necessary, the medium was supplemented with selection markers. The cultures were incubated at 37°C except if the culture required otherwise. Cultures in liquid media were shaken at 280 rpm in a rotatory shaker Gyromax 703 (Amerex Instruments Inc., Hercules, CA).

Spheroplasts were prepared from Mycobacterium cultures as previously described (Labidi, et al. 1984. Curr. Microbiol. 11, 235-240).

Briefly, the spheroplast solution [for every ml of Mycobacterium culture (14 mg of glycine, 60 μg of D-cycloserine, 1 mg of lithium chloride, 200 μg of lysizyme, and 2 mg of EDTA)] was added to the Mycobacterium cultures in exponential growth phase, and the incubation was continued for three generations to induce spheroplast formation The spheroplasts were subsequently collected by centrifugation for 20 min, at 3000 rpm, at 4°C, washed and resuspended in the spheroplast storage solution [per liter, (6 05 gm of tris, 18 5 gm of EDTA, 250 gm of sucrose, and pH adjusted to 7)] Culturing Mycobacterium for Adjuvants

The adjuvants are made of Mycobacterium cells harvested after preferably growing the corresponding Mycobacterium strains in a liquid protein free medium The medium is inoculated and incubated at the appropriate temperature The culture is shaken at 150 rpm for appropriate aeration The OD₆₀₀ of the culture is monitored daily to determine when the culture reaches stationary phase At the stationary phase, the number of cells per milliliter is determined through serial dilutions and plating each dilution in triplicate The culture is sterilely centrifuged for 30 minutes, at 5000 rpm, at 4°C The pelleted cells are washed twice with ice cold sterile distilled water and pelleted as indicated above The pellet is re-suspended into pyrogen-free saline (for injection only), to form a suspension of cells ranging from 10⁸ - 10¹² cells per ml The Mycobacterium cell suspension is dispensed into suitable multi-dose vials and used alive, or dead Preferred methods for killing the mycobacterium cells include the use of chemicals, radiation, or intense heat (autoclaving for 30 min, at 15 - 18 psig (104 - 124 kPa) at 120 - 122°C) DNA and RNA Preparations Plasmid DNA was prepared from E. coli strains, as described in prior text (Labidi, et al. 1984. "Plasmid profiles of Mycobacterium fortuitum complex isolates," Curr. Microbiol. 11, 235-240) 300 μl of spheroplasts were microcentrifuged in another preferred method of the invention The pellet was resuspended in 360 μl of freshly prepared SI solution [250 mM tris (pH7), 50mM EDTA (pH8), 50 mM glucose, and 2 5 μg/ml losozyme] 240 μl of S II

[10%) SDS (pH7)] was added and the pellet incubated at 65° C for 15 minutes Subsequently, 300 μl of S III [7.5 ammonium acetate (pH 7.5), or 5 M NaCl, or 3 M potassium acetate (pH 5.2), or 3 M sodium acetate (pH 5.2)] was added and the pellet was incubated on ice for 15 minutes and microcentrifuged for 15 minutes at 0° C at 14 Krpm. 2.5 μl of proteinase K (20 mg/ml) was added and incubated at 37° C for 15 minutes. The aqueous phase is extracted three times by adding 250 μl of buffered phenol and 250 μl of chloroform/iso- amyl-alcohol (24:1, v/v) each time. The pellet is vortexed, microcentrifuged for 15 minutes at 14 Krpm at room temperature and the aqueous phase recovered. To the last aqueous phase is added 1 ml of isopropanol, vortex briefly and microcentrifuge for 10 minutes at 14 Krpm at room temperature.

The pellet is dried at 37 ° C for 5 minutes and the DNA is dissolved in 50 μl of sterile distilled water.

Total DNA was prepared from Mycobacterium strains as described before (Labidi, A., 1986). Another preferred method is to add sterile glass beads to the pellet obtained from 20 ml of spheroplasts. The pellet is vortexed vigorously to have a homogeneous suspension. The suspension is treated with 20 ml of SI, 8 ml of SII, and 14 ml of SIII. The aqueous phase is extracted several times, each time with 10.5 ml of a buffered phenol/chloroform/iso- amyl-alcohol solution. The total DNA is precipitated with 0.6 volume of isopropanol, then dissolved in a cesium chloride gradient and ethidium bromide. The gradient is centrifuged and treated according to techniques that are well established in the art. The plasmid DNA then be separated from the chromosomal DNA.

Total RNA was prepared from E. coli strains containing the appropriate plasmids and application of a preferred two step protocol. A crude preparation of total RNA was made using the protocol provided with the kit "Ultraspec RNA Isolation System" (Biotex Laboratories Inc., Houston, TX). Since the latter was always contaminated with plasmid DNA, the total RNA was further purified using the protocol provided with the kit "Qiagen Total RNA Isolation" (Qiagen Inc., Chatsworth, CA). The combination of the two systems efficiently separated total RNA from other contaminating nucleic acids. Preparation of Electro-competent Cells

Mycobacterium strains can be transformed only through electroporation (Labidi, A., 1986). Therefore, the bacterial cells must be made electro- competent before being subject to this procedure. E. coli strains were made electro-competent following the protocol provided with the BRL Cell Porator apparatus ( BRL Life Technologies, Gaithersburg, MD).

For Mycobacterium strains, a single colony of Mycobacterium culture was inoculated into 25 ml of Labidi's medium in a 250 ml screw capped flask.

The culture was shaken at 150 rpm at appropriate temperature until the OD₆₀₀ reached 0.7. The culture was checked for contamination by staining. If there was no contamination, a second culture was started by inoculating 50 μl of the first culture into 200 ml of Labidi's medium in a 2000 ml screw capped flask. The culture was shaken at 150 rpm at appropriate temperature until the OD^ reached 0.7. The culture was cooled on ice/water for 2 hours, and then the bacterial cells were harvested by centrifugation (7.5 Krpm) for 10 minutes at 4°C. The first pellet was suspended into 31 ml of 3.5% sterile cold glycerol and centrifuged (5 Krpm) for 10 minutes at 4°C. The second pellet was suspended into 12 ml of 7% sterile cold glycerol and centrifuged (3 Krpm) for

10 minutes at 4°C. The third pellet was suspended into 6 ml of 10% sterile cold glycerol and centrifuged (3 Krpm) for 10 minutes at 4°C. The fourth pellet was suspended in a minimum volume of about 2.0 ml of 10.0% sterile cold glycerol, aliquoted into 25.0 μl fractions then used immediately or stored at minus 80°C.

Transformation

The technique of electroporation was applied to E. coli and Mycobacterium strains. E. coli ox Mycobacterium electro-competent cells (25 μl) were mixed with vector DNA (10 ng in 1 μl), incubated on ice/water for 1 minute then transferred to an electroporation cuvette (0.15 cm gap). The electroporation was conducted with a BRL Cell Porator apparatus Cat. series 1600 equipped with a Voltage Booster Unit Cat. series 1612 (BRL Life Technologies, Gaithersburg, MD). The Voltage Booster Unit was set at a resistance of 4 kiloohms and the Power Supply Unit was set at a capacitance of 330 microfarad, a fast charging speed rate and a low Ohm mode to eliminate extra-resistance. Once the cuvettes were in the safety chamber, the

"charge/arm button" was set to "charge", the "up button" was held down until the capacitors voltage displayed in the Power Supply Unit reached 410 volts for E. coli and 330 volts for Mycobacterium strains. The "charge/arm button" was set to "arm" and the capacitors voltage was allowed to fall down to 400 volts for E. coli and to 316 volts for Mycobacterium strains. The "trigger button" was pushed to deliver about 2.5 kilovolts for E. coli and Mycobacterium strains, respectively. These voltage values were displayed on the Voltage Booster Unit. Each voltage value corresponds to 2.5 kilovolts divided by 0.15 cm equals 16.66 kilovolts/cm across the cuvette gap forE. coli strains and 1.9 kilovolts divided by 0.15 cm equals 12.66 kilovolts/cm across the cuvette gap for Mycobacterium strains. The electroporated cells of each sample were immediately collected with 1 ml of Labidi's medium, transferred to a 15 ml falcon tube with a round bottom (Becton Dickenson Inc., Lincoln Park, NJ) and incubated for one generation time under appropriate temperature and shaking conditions. The cultures were diluted 1 : 10² to 1 : 10⁵ into sterile distilled water. The diluted cultures were plated (100 μl) in triplicates on Kanamycin-containing LB and Labidi's media, respectively. The plates were incubated at appropriate temperatures until colonies were visible and easy to count. The numbers counted were averaged and used to calculate transformation efficiencies. A negative and a positive control were included for each species and each experiment. DNA Sequencing

The DNA was sequenced using a double strand plasmid template and the protocol provided with the kit "Sequenase Version 2.0" (USB, Cleveland, Ohio, USA). The sequence was computer analyzed using Mac Vector program

(Oxford Molecular Group Inc., Campbell, CA). In Vitro Analysis of Vector's Stability.

Single Mycobacterium transformant colonies were grown to saturation on Labidi's medium containing kanamycin (50 μg/ml). The number of generations required to reach saturation is significantly different between slow and rapidly growing mycobacteria. The saturated cultures were diluted to

1 : 10² and to 1 : 10⁶ into antibiotic-free Labidi's medium. The dilution 1 : 10⁶ was immediately plated (0.1 ml per plate) on antibiotic containing Labidi's medium to determine the number of Kanamycin-resistant colonies per ml of culture at the start of the experiment. For calculation purposes, the number of Kanamycin-resistant colonies per ml of this culture was considered to be 100%.

Fractions of 0.1 ml of the dilution 1 : 10² were used to inoculate fresh antibiotic-free Labidi's medium and allowed to grow to saturation. This procedure was repeated for six months. Each time the number of Kanamycin- resistant colonies was determined. The proportion of antibiotic-resistant colonies in the culture after the six month period was found to be 96%.

DNA and RNA transactions.

DNA and RNA were treated with the appropriate enzymes respectively, as recommended by the manufacturers.

Integration analysis

The integration of vectors containing the attachment site (attP) and the integrase gene (int) of the Mycobacteriophage D₂₉ into the chromosomes of the Mycobacterium host strains was analyzed by plasmid DNA preparation and by hybridization using the cloned fragment from the D₂₉ genome as a probe. Minicells analysis

Minicells analysis was performed using the E. coli DS410, which is a mutant strain of E. coli (MinA and MinB). This mutant divides asymmetrically and produces normal cells and small anucleated cells called minicells. The minicells are easily separated from normal cells by their differential sedimentation on a sucrose gradient. If the minicells producing strain contains a multi-copy plasmid, each of its minicells will not have a chromosome but will carry at least one copy of the plasmid. Since minicells are capable of supporting DNA, RNA and protein synthesis for several hours, they are used as an in vivo gene expression system for prokaryotes. The expression product is labeled with S³⁵-methionine and analyzed by protein gel electrophoresis. Nutrient Broth is the medium used in this technique.

Preparation of minicells originated with the preparation of electrocompetent cells of E. coli DS410 with the appropriate recombinant plasmids. Each plasmid containing clone is grown overnight in 400 ml NB having the appropriate selection markers. One clone of the non transformed DS410 was grown on 400 ml NB alone to serve as a control.

Three 35 ml sucrose gradients (10-30% w/v) were prepared per clone using M9-mm-S[per liter of medium: 200 gm of sucrose, 100 ml of sterile 10X I- M9-mm, 10 ml of sterile 10 mM CaCl₂, and 10 ml of sterile 100 mM Mg SO₄]. The gradients are then placed at minus 70 ° C for at least one hour or until the gradients are completely frozen. The gradients are then placed at

4° C overnight to allow the gradient to thaw and to be established. The bacterial cultures are centrifuged for 5 minutes at 2 Krpm at 4° C. The supernatants are then centrifuged for 15 minutes at 8 Krpm at 4° C. Each pellet is subsequently resuspended in 6 ml of M9-mm [per 10X liter of medium: 400 mM NaH₂PO₄, 200 mM KH₂PO₄, 80 mM NaCl, and 200 mM NH₄C1)] .

Each 3 ml of cell suspension is layered on top of a sucrose gradient. The gradients are then centrifuged for 18 minutes at 5 Krpm at 4° C. The top one- third of the white transparent minicells band are recovered from each gradient. An equal volume of M9-mm is added to each tube and centrifuged for 10 minutes at 10 Krpm at 4° C. Each peilet is subsequently resuspended in 3 ml of M9-mm and the suspension is layered on top of the last gradient and centrifuged for 18 minutes at 5 Krpm at 4° C. The top one-third of the white transparent minicells band is recovered and the optical density is read at 600 nm. The number of cells in the minicells preparation is calculated using the equation of 2 OD₆₀₀, which equals 10¹⁰ minicells per ml. Preferably, the level of whole cell contamination is determined in the minicells' preparation. The minicell suspension is centrifuged for 10 minutes at 10 Krpm at 4° C and resuspended in M9-mm-G [per 100 ml of medium: 30 ml of sterile (100%) glycerol, 1 ml of sterile 10 mM CaCl₂, 1 ml of sterile 100 mM MgSO₄, and 10 ml of sterile 10X I-M9-mm]. The labeling of the plasmid encoded proteins with 5³⁵ methionine is achieved by placing 100 μl of minicells in the microcentriuge for 3 minutes at 4° C. The pellet is resuspended in 200 μl of M9-mm and 3 μl of MAM [10.5 gm of methionine assay medium per 100 ml of medium]. The pellet is incubated at 37° C for 90 minutes and 25 μCi of S³⁵-methionine is added. The pellet is incubated at 37° C for 60 minutes. 10 μl of unlabeled MS (0.8 gm of

L(-) methionine in 100 ml of distilled water] is added and incubated at 37° C for 5 minutes and microcentrifuged for 3 minutes at room temperature. The pellet is resuspended in 50 μl of BB [per 100 ml of solution, (0.71gm of Na₂HPO₄, 0.27 gm ofKH₂PO₄, 0.41 gm of NaCl, and 400 /A of sterile 100 mM MgSO₄)] and 50 μl of SDS-SB [per 10 ml of solution, (12.5 ml of sterile 1

M tris (pH 6.8), 20 ml of sterile (100%) glycerol, 10 ml of 20% SDS (pH 7.2), 5ml of mercaptoethanol, and 250 μl of 0.4% bromophenol blue)]. The pellet is boiled for 3 minutes, centrifuged, and the top 25 μl of the sample is applied to a 12%) SDS-polyacrylamide slab gel. Primer extension analysis

Primer extension analysis was conducted according to the protocol provided with the kit "Primer Extension System" (Promega Inc., Madison, WI). Ribonuclease protection assay analysis

Ribonuclease protection assay (RPA) was conducted according the protocol provided with the "Ambion HypSpeed RPA Kit" (Ambion Inc.

Austin, TX). DNA amplification by polymerase chain reaction

DNA fragments from the Mycobacteriophage D₂₉ genome and Mycobacterium plasmid and chromosomal DNA were amplified by polymerase chain reaction using a Progene Programmable Dri-Block Cycler (Techne Inc.,

Princeton, NJ). The reaction mixture was subject to denaturation (94 °C for 3 minutes), followed by 10 cycles of amplification (94 °C for 2 minutes, 55 °C for 2 minutes, 72 °C for 2 minutes), followed by 30 cycles of amplification (94 °C for 2 minutes, 63 °C for 2 minutes, 72° C for 2 minutes). The programming described above is disclosed for the first time in this report. Examples 1-3 demonstrate the present invention in terms of use of specific antigens in the treatment of various diseases. These examples are illustrative and are not meant to be limiting with regard to the selected antigen and Mycobacterium strain nor the application of the E.coli-Mycobacterium shuttle. Example 1 : Exemplary AIDS Vaccine

If the product is being used to vaccinate against AIDS, E. coli- Mycobacterium expression vectors containing genes encoding HIV env, rev, and gag/pol proteins (National Institutes of Health, Bethtesda MD), and genes encoding IL-2, gamma INF and GMCSF (Cytoclonal Pharmaceutics, Inc., Dallas, Texas) are electroporated into a recipient strain M. aurum. The transformants are checked for their plasmid content. A clone containing the expected hybrid plasmid is grown in the protein-free liquid medium. The inoculated medium is incubated at a temperature of 35 to 37°C. The culture is shaken at 150 rpm for appropriate aeration. The OD₆₀₀ of the culture is measured daily, and a growth curve featuring optical densities versus time is established. At the stationary phase, the number of cells per milliliter is determined through serial dilutions (1: 10 to 1 :10¹⁰), and plating in triplicates of each dilution on Labidi's medium. The culture is sterilely centrifuged for 30 minutes, at 5000 rpm, at 4°C. The pelleted cells are washed twice with ice cold sterile distilled water and pelleted as indicated above. The pellet is resuspended into pyrogen-free saline for injection only, to have a suspension of 10⁸ to 10¹² cells per ml. The Mycobacterium cell suspension is dispensed into suitable multi-dose vials. The product is administered by injection given intradermal in a volume of about 100 ul containing 10⁷ to 10¹¹ cells of recombinant Mycobacterium. If a killed form of the vaccine is preferred, the cells can be killed either chemically, by radiation, or by autoclaving for 30 min, at 15 - 18 psig (104 - 124 kPa) at 120 - 122°C. If a killed form of the vaccine is used, those antigens or cytokines that may be inactivated during the process are added to the product separately, or the recombinant cells are killed by radiation. Example 2: Exemplary Cancer Vaccine

If the product is being used to vaccinate against cancer such as prostate cancer, the gene encoding the cancer antigen such as the prostate cancer antigen PSA (National Institutes of Health, Bethesda, MD), is cloned according to the procedure given in Example 1. The product is prepared and adminstered according to the procedure given in Example 1.

Example 3 : Exemplary Allergy Vaccine If the product is being used for vaccination against allergies such as reactions to the major allergen of birch pollen, the gene encoding the allergen such as the birch pollen allergen BetVla (Univeristy of Vienna, Austria) is cloned according to the procedure given in Example 1. The product is prepared and adminstered according to the procedure given in Example 1.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Cytoclonal Pharmaceutics, Inc.

(B) STREET: 9000 Harry Hines Blvd, Suite 330

(C) CITY: Dallas

(D) STATE: Texas

(E) COUNTRY: USA

(F) POSTAL CODE (ZIP) : 75235

(G) TELEPHONE: (214) 353-2923 (H) TELEFAX: (214) 350-9514 (I) TELEX:

(ii) TITLE OF INVENTION: Mycobacterium Recombinant Vaccines

(iii) NUMBER OF SEQUENCES: 9

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Sidley ε. Austin

(B) STREET: 717 N. Harwood, Suite 3400

(C) CITY: Dallas

(D) STATE: Texas

(E) COUNTRY: United States

(F) ZIP: 75201

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/042849

(B) FILING DATE: 28-MAR-1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Hansen, Eugenia S.

(B) REGISTRATION NUMBER: 31,966

(C) REFERENCE/DOCKET NUMBER: 10365/05602

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 214-981-3300

(B) TELEFAX: 214-981-3400 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 695 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG 60

GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT 120

GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG 180

AAGCGTGGCG CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG 240

CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG 300

TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC 360

TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG 420

GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT 480

TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG 540

TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC 600

TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT 660

GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGA 695

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 932 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GTTGTGTCTC AAAATCTCTG ATGTTACATT GCACAAGATA AAAATATATC ATCATGAACA 60 ATAAAACTGT CTGCTTACAT AAACAGTAAT ACAAGGGGTG TTATGAGCCA TATTCAACGG 120

GAAACGTCTT GCTCGAGGCC GCGATTAAAT TCCAACATGG ATGCTGATTT ATATGGGTAT 180

AAATGGGCTC GCGATAATGT CGGGCAATCA GGTGCGACAA TCTATCGATT GTATGGGAAG 240

CCCGATGCGC CAGAGTTGTT TCTGAAACAT GGCAAAGGTA GCGTTGCCAA TGATGTTACA 300

GATGAGATGG TCAGACTAAA CTGGCTGACG GAATTTATGC CTCTTCCGAC CATCAAGCAT 360

TTTATCCGTA CTCCTGATGA TGCATGGTTA CTCACCACTG CGATCCCCGG GAAAACAGCA 420

TTCCAGGTAT TAGAAGAATA TCCTGATTCA GGTGAAAATA TTGTTGATGC GCTGGCAGTG 480

TTCCTGCGCC GGTTGCATTC GATTCCTGTT TGTAATTGTC CTTTTAACAG CGATCGCGTA 540

TTTCGTCTCG CTCAGGCGCA ATCACGAATG AATAACGGTT TGGTTGATGC GAGTGATTTT 600

GATGACGAGC GTAATGGCTG GCCTGTTGAA CAAGTCTGGA AAGAAATGCA TAAGCTTTTG 660

CCATTCTCAC CGGATTCAGT CGTCACTCAT GGTGATTTCT CACTTGATAA CCTTATTTTT 720

GACGAGGGGA AATTAATAGG TTGTATTGAT GTTGGACGAG TCGGAATCGC AGACCGATAC 780

CAGGATCTTG CCATCCTATG GAACTGCCTC GGTGAGTTTT CTCCTTCATT ACAGAAACGG 840

CTTTTTCAAA AATATGGTAT TGATAATCCT GATATGAATA AATTGCAGTT TCATTTGATG 900

CTCGATGAGT TTTTCTAATC AGAATTGGTT AA 932

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1463 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

TGTTCCTCCT GGTTGGTACA GGTGGTTGGG GGTGCTCGGC TGTCGCGGTT GTTCCACCAC 60

CAGGGCTCGA CGGGAGAGCG GGGGAGTGTG CAGTTGTGGG GTGGCCCCTC AGCGAAATAT 120

CTGACTTGGA GCTCGTGTCG GACCATACAC CGGTGATTAA TCGTGGTCTA CTACCAAGCG 180

TGAGCCACGT CGCCGACGAA TTTGAGCAGC TCTGGCTGCC GTACTGGCCG CTGGCAAGCG 240 ACGATCTGCT CGAGGGGATC TACCGCCAAA GCCGCGCGTC GGCCCTAGGC CGCCGGTACA 300

TCGAGGCGAA CCCAACAGCG CTGGCAAACC TGCTGGTCGT GGACGTAGAC CATCCAGACG 360

CAGCGCTCCG AGCGCTCAGC GCCCGGGGGT CCCATCCGCT GCCCAACGCG ATCGTGGGCA 420

ATCGCGCCAA CGGCCACGCA CACGCAGTGT GGGCACTCAA CGCCCCTGTT CCACGCACCG 480

AATACGCGCG GCGTAAGCCG CTCGCATACA TGGCGGCGTG CGCCGAAGGC CTTCGGCGCG 540

CCGTCGACGG CGACCGCAGT TACTCAGGCC TCATGACCAA AAACCCCGGC CACATCGCCT 600

GGGAAACGGA ATGGCTCCAC TCAGATCTCT ACACACTCAG CCACATCGAG GCCGAGCTCG 660

GCGCGAACAT GCCACCGCCG CGCTGGCGTC AGCAGACCAC GTACAAAGCG GCTCCGACGC 720

CGCTAGGGCG GAATTGCGCA CTGTTCGATT CCGTCAGGTT GTGGGCCTAT CGTCCCGCCC 780

TCATGCGGAT CTACCTGCCG ACCCGGAACG TGGACGGACT CGGCCGCGCG ATCTATGCCG 840

AGTGCCACGC GCGAAACGCC GAATTCCCGT GCAACGACGT GTGTCCCGGA CCGCTACCGG 900

ACAGCGAGGT CCGCGCCATC GCCAACAGCA TTTGGCGTTG GATCACAACC AAGTCGCGCA 960

TTTGGGCGGA CGGGATCGTG GTCTACGAGG CCACACTCAG TGCGCGCCAG TCGGCCATCT 1020

CGCGGAAGGG CGCAGCAGCG CGCACGGCGG CGAGCACAGT TGCGCGGCGC GCAAAGTCCG 1080

CGTCAGCCAT GGAGGCATTG CTATGAGCGA CGGCTACAGC GACGGCTACA GCGACGGCTA 1140

CAACCGGCAG CCGACTGTCC GCAAAAAGCC GTGACGCGCC GAAGGCGCTC GAATCACCGG 1200

ACTATCCGAA CGCCACGTCG TCCGGCTCGT GGCGCAGGAA CGCAGCGAGT GGCTCGCCGA 1260

GCAGGCTGCA CGCGCGCGAA GCATCCGCGC CTATCACGAC GACGAGGGCC ACTCTTGGCC 1320

GCAAACGGCC AAACATTTCG GGCTGCATCT GGACACCGTT AAGCGACTCG GCTATCGGGC 1380

GAGGAAAGAG CGTGCGGCAG AACAGGAAGC GGCTCAAAAG GCCCACAACG AAGCCGACAA 1440

TCCACCGCTG TTCTAACGCA ATT 1463

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1382 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GGGTGCTCGG CTGTCGCGGT TGTTCCACCA CCAGGGCTCG ACGGGAGAGC GGGGGAGTGT 60

GCAGTTGTGG GGTGGCCCCT CAGCGAAATA TCTGACTTGG AGCTCGTGTC GGACCATACA 120

CCGGTGATTA ATCGTGGTCT ACTACCAAGC GTGAGCCACG TCGCCGACGA ATTTGAGCAG 180

CTCTGGCTGC CGTACTGGCC GCTGGCAAGC GACGATCTGC TCGAGGGGAT CTACCGCCAA 240

AGCCGCGCGT CGGCCCTAGG CCGCCGGTAC ATCGAGGCGA ACCCAACAGC GCTGGCAAAC 300

CTGCTGGTCG TGGACGTAGA CCATCCAGAC GCAGCGCTCC GAGCGCTCAG CGCCCGGGGG 360

TCCCATCCGC TGCCCAACGC GATCGTGGGC AATCGCGCCA ACGGCCACGC ACACGCAGTG 420

TGGGCACTCA ACGCCCCTGT TCCACGCACC GAATACGCGC GGCGTAAGCC GCTCGCATAC 480

ATGGCGGCGT GCGCCGAAGG CCTTCGGCGC GCCGTCGACG GCGACCGCAG TTACTCAGGC 540

CTCATGACCA AAAACCCCGG CCACATCGCC TGGGAAACGG AATGGCTCCA CTCAGATCTC 600

TACACACTCA GCCACATCGA GGCCGAGCTC GGCGCGAACA TGCCACCGCC GCGCTGGCGT 660

CAGCAGACCA CGTACAAAGC GGCTCCGACG CCGCTAGGGC GGAATTGCGC ACTGTTCGAT 720

TCCGTCAGGT TGTGGGCCTA TCGTCCCGCC CTCATGCGGA TCTACCTGCC GACCCGGAAC 780

GTGGACGGAC TCGGCCGCGC GATCTATGCC GAGTGCCACG CGCGAAACGC CGAATTCCCG 840

TGCAACGACG TGTGTCCCGG ACCGCTACCG GACAGCGAGG TCCGCGCCAT CGCCA--CAGC 900

ATTTGGCGTT GGATCACAAC CAAGTCGCGC ATTTGGGCGG ACGGGATCGT GGTCTACGAG 960

GCCACACTCA GTGCGCGCCA GTCGGCCATC TCGCGGAAGG GCGCAGCAGC GCGCACGGCG 1020

GCGAGCACAG TTGCGCGGCG CGCAAAGTCC GCGTCAGCCA TGGAGGCATT GCTATGAGCG 1080

ACGGCTACAG CGACGGCTAC AGCGACGGCT ACAACCGGCA GCCGACTGTC CGCAAAAAGC 1140

CGTGACGCGC CGAAGGCGCT CGAATCACCG GACTATCCGA ACGCCACGTC GTCCGGCTCG 1200

TGGCGCAGGA ACGCAGCGAG TGGCTCGCCG AGCAGGCTGC ACGCGCGCGA AGCATCCGCG 1260

CCTATCACGA CGACGAGGGC CACTCTTGGC CGCAAACGGC CAAACATTTC GGGCTGCATC 1320

TGGACACCGT TAAGCGACTC GGCTATCGGG CGAGGAAAGA GCGTGCGGCA GAACAGGAAG 1380

CG 1382 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1631 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

GTGAGAGAAT CTTCACTGCA CCAGCTCCGA TCTGGTGTAC CGCCCCTCGT CTGTTGCAGC 60

AGGCGGGGGG CTTTCTTCGT CTGTCGGAGG TCGAAGGTAG CAGATGTGTC GCTGTATCCG 120

GGCAGCATAA ATGCAGGTCA TTAGTGTCGC TCTAAGGTCG CGGCCCCCTC TCGGGGATCC 180

GGTCCTCGGG CTAAAAACCA CCTCTGACCT GTGGAGCGGG CGACGGGAAT CGAACCCGCG 240

TAGCTAGTTT GGAAGTAAGG GGGTCGGCGT GTCACATTCT CCCAGCTCAG ACCCTGTTTT 300

TAGCTCTGAC CCTGTGCGAC CTTGAAGTGG ACAAAAATGC CTGTTCACGG ACACGCAAAG 360

ACGTCTGAAG GTCGCAATAA GGTCGCATTC CGGTAGCCTG TTTCGCATGG CAGCAAGACG 420

GAGAGGATGG GGATCGCTGC GGACCCAGCG CAGCGGTCGA GTGCAAGCGT CGTACGTCAG 480

CCCGATCGAC GGGCAGCGGT ACTTCGGGCC GAGGAACTAC GACAACCGGA TGGACGCCGA 540

AGCGTGGCTC GCGTCTGAGA AGCGGCTGAT CGACAACGAG GAGTGGACCC CGCCGGCCGA 600

GCGCGAGAAG AAGGCTGCGG CGAGTGCCAT CACGGTCGAG GAGTACACCA AGAAGTGGAT 660

CGCCGAGCGA GACCTCGCTG GCGGCACCAA GGATCTCTAC AGCACGCACG CTCGCAAGCG 720

GATCTACCCG GTGTTGGGCG ACACCCCGGT CGCCGAGATG ACCCCCGCCC TTGTCCGGGC 780

GTGGTGGGCC GGGATGGGTA AGCAGTACCC GACGGCACGG CGGCACGCCT ACAACGTACT 840

CCGGGCGGTC ATGAATACCG CTGTAGAGGA CAAGCTGGTG TCGGAGAACC CGTGCCGGAT 900

CGAGCAGAAG GCACCCGCTG AGCGCGACGT GGAAGCCCTC ACACCGGAGG AGCTGGACGT 960

AGTGGCCGGG GAGGTGTTCG AGCACTACCG CGTGGCCGTC TACATCCTGG CGTGGACCAG 1020

CCTGCGGTTC GGTGAGCTGA TCGAGATCCG CCGCAAGGAC ATCGTGGATG ACGGCGAGAC 1080

GATGAAGCTC CGCGTGCGCC GGGGCGCGGC CCGCGTCGGC GAGAAGATCG TCGTCGGCAA 1140

CACCAAGACC GTCAGGTCCA AGCGGCCGGT GACCGTGCCG CCTCACGTCG CGGCGATGAT 1200 CCGCGAGCAC ATGGCTGACC GGACGAAGAT GAACAAGGGG CCGGAAGCTC TCCTGGTGAC 1260

CACCACGCGG GGGCAGCGGC TGTCGAAGTC TGCGTTCACT CGCTCGCTGA AGAAGGGCTA 1320

CGCCAAGATC GGTCGACCGG ACCTCCGCAT CCACGACCTC CGGGCCGTGG GAGCCACGCT 1380

GGCGGCTCAG GCCGGTGCGA CGACCAAGGA GCTGATGGTG CGCCTCGGGC ACACGACTCC 1440

GCGCATGGCG ATGAAGTACC AGATGGCCTC AGCAGCCCGT GACGAGGAGA TAGCGAGGCG 1500

AATGTCGGAG CTGGCAGGGA TTACCCCCTG AAACGCAAAA AGCCCCCCTC CCAAGGCCAT 1560

ACAGCCTCAA GAGGGGGGTT TCTTGTCACT CAGTCCACAC GGTCCATTGG ATCTTGGGCG 1620

TGTAGACGAT C 1631

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1413 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

CGGGCAGCAT AAATGCAGGT CATTAGTGTC GCTCTAAGGT CGCGGCCCCC TCTCGGGGAT 60

CCGGTCCTCG GGCTAAAAAC CACCTCTGAC CTGTGGAGCG GGCGACGGGA ATCGAACCCG 120

CGTAGCTAGT TTGGAAGTAA GGGGGTCGGC GTGTCACATT CTCCCAGCTC AGACCCTGTT 180

TTTAGCTCTG ACCCTGTGCG ACCTTGAAGT GGACAAAAAT GCCTGTTCAC GGACACGCAA 240

AGACGTCTGA AGGTCGCAAT AAGGTCGCAT TCCGGTAGCC TGTTTCGCAT GGCAGCAAGA 300

CGGAGAGGAT GGGGATCGCT GCGGACCCAG CGCAGCGGTC GAGTGCAAGC GTCGTACGTC 360

AGCCCGATCG ACGGGCAGCG GTACTTCGGG CCGAGGAACT ACGACAACCG GATGGACGCC 420

GAAGCGTGGC TCGCGTCTGA GAAGCGGCTG ATCGACAACG AGGAGTGGAC CCCGCCGGCC 480

GAGCGCGAGA AGAAGGCTGC GGCGAGTGCC ATCACGGTCG AGGAGTACAC CAAGAAGTGG 540

ATCGCCGAGC GAGACCTCGC TGGCGGCACC AAGGATCTCT ACAGCACGCA CGCTCGCAAG 600

CGGATCTACC CGGTGTTGGG CGACACCCCG GTCGCCGAGA TGACCCCCGC CCTTGTCCGG 660 GCGTGGTGGG CCGGGATGGG TAAGCAGTAC CCGACGGCAC GGCGGCACGC CTACAACGTA 720

CTCCGGGCGG TCATGAATAC CGCTGTAGAG GACAAGCTGG TGTCGGAGAA CCCGTGCCGG 780

ATCGAGCAGA AGGCACCCGC TGAGCGCGAC GTGGAAGCCC TCACACCGGA GGAGCTGGAC 840

GTAGTGGCCG GGGAGGTGTT CGAGCACTAC CGCGTGGCCG TCTACATCCT GGCGTGGACC 900

AGCCTGCGGT TCGGTGAGCT GATCGAGATC CGCCGCAAGG ACATCGTGGA TGACGGCGAG 960

ACGATGAAGC TCCGCGTGCG CCGGGGCGCG GCCCGCGTCG GCGAGAAGAT CGTCGTCGGC 1020

AACACCAAGA CCGTCAGGTC CAAGCGGCCG GTGACCGTGC CGCCTCACGT CGCGGCGATG 1080

ATCCGCGAGC ACATGGCTGA CCGGACGAAG ATGAACAAGG GGCCGGAAGC TCTCCTGGTG 1140

ACCACCACGC GGGGGCAGCG GCTGTCGAAG TCTGCGTTCA CTCGCTCGCT GAAGAAGGGC 1200

TACGCCAAGA TCGGTCGACC GGACCTCCGC ATCCACGACC TCCGGGCCGT GGGAGCCACG 1260

CTGGCGGCTC AGGCCGGTGC GACGACCAAG GAGCTGATGG TGCGCCTCGG GCACACGACT 1320

CCGCGCATGG CGATGAAGTA CCAGATGGCC TCAGCAGCCC GTGACGAGGA GATAGCGAGG 1380

CGAATGTCGG AGCTGGCAGG GATTACCCCC TGA 1413

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1374 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

TCGCGGCCCC CTCTCGGGGA TCCGGTCCTC GGGCTAAAAA CCACCTCTGA CCTGTGGAGC 60

GGGCGACGGG AATCGAACCC GCGTAGCTAG TTTGGAAGTA AGGGGGTCGG CGTGTCACAT 120

TCTCCCAGCT CAGACCCTGT TTTTAGCTCT GACCCTGTGC GACCTTGAAG TGGACAAAAA 180

TGCCTGTTCA CGGACACGCA AAGACGTCTG AAGGTCGCAA TAAGGTCGCA TTCCGGTAGC 240

CTGTTTCGCA TGGCAGCAAG ACGGAGAGGA TGGGGATCGC TGCGGACCCA GCGCAGCGGT 300

CGAGTGCAAG CGTCGTACGT CAGCCCGATC GACGGGCAGC GGTACTTCGG GCCGAGGAAC 360 TACGACAACC GGATGGACGC CGAAGCGTGG CTCGCGTCTG AGAAGCGGCT GATCGACAAC 420

GAGGAGTGGA CCCCGCCGGC CGAGCGCGAG AAGAAGGCTG CGGCGAGTGC CATCACGGTC 480

GAGGAGTACA CCAAGAAGTG GATCGCCGAG CGAGACCTCG CTGGCGGCAC CAAGGATCTC 540

TACAGCACGC ACGCTCGCAA GCGGATCTAC CCGGTGTTGG GCGACACCCC GGTCGCCGAG 600

ATGACCCCCG CCCTTGTCCG GGCGTGGTGG GCCGGGATGG GTAAGCAGTA CCCGACGGCA 660

CGGCGGCACG CCTACAACGT ACTCCGGGCG GTCATGAATA CCGCTGTAGA GGACAAGCTG 720

GTGTCGGAGA ACCCGTGCCG GATCGAGCAG AAGGCACCCG CTGAGCGCGA CGTGGAAGCC 780

CTCACACCGG AGGAGCTGGA CGTAGTGGCC GGGGAGGTGT TCGAGCACTA CCGCGTGGCC 840

GTCTACATCC TGGCGTGGAC CAGCCTGCGG TTCGGTGAGC TGATCGAGAT CCGCCGCAAG 900

GACATCGTGG ATGACGGCGA GACGATGAAG CTCCGCGTGC GCCGGGGCGC GGCCCGCGTC 960

GGCGAGAAGA TCGTCGTCGG CAACACCAAG ACCGTCAGGT CCAAGCGGCC GGTGACCGTG 1020

CCGCCTCACG TCGCGGCGAT GATCCGCGAG CACATGGCTG ACCGGACGAA GATGAACAAG 1080

GGGCCGGAAG CTCTCCTGGT GACCACCACG CGGGGGCAGC GGCTGTCGAA GTCTGCGTTC 1140

ACTCGCTCGC TGAAGAAGGG CTACGCCAAG ATCGGTCGAC CGGACCTCCG CATCCACGAC 1200

CTCCGGGCCG TGGGAGCCAC GCTGGCGGCT CAGGCCGGTG CGACGACCAA GGAGCTGATG 1260

GTGCGCCTCG GGCACACGAC TCCGCGCATG GCGATGAAGT ACCAGATGGC CTCAGCAGCC 13 0

CGTGACGAGG AGATAGCGAG GCGAATGTCG GAGCTGGCAG GGATTACCCC CTGA 1374

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 105 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

GTTGTGTCTC AAAATCTCTG ATGTTACATT GCACAAGATA AAAATATATC ATCATGAACA 60

ATAAAACTGT CTGCTTACAT AAACAGTAAT ACAAGGGGTG TTATG 105 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2096 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

GGTCACCTGC GATCACACCG AGCGTGCAGG TAGCGAAGTC CTCATCACCA CCAGGACGGG 60

CCTGGGCGAT ACCAGCGCCG GGGGCGATCC CGCCAGGAAA TGCCGTCCAA TCGGTGTCCG 120

CGACTGCGGC GGAGCGGACA CTCCGACCAA CACAACAACC AACGTCGTCA TAGCGACGAC 180

GAACCACGAT CGGATGATCC GAATCACTGC GCTGTCCATA CAGGCGGCCA CCCCTCGAAC 240

TCACCAGCTT CAATGCGCGT CTGCAAAGAC TGCCATGGAG CGCTACTCGG GCCGGTCTCA 300

ACGCACTGCT CGAAGAAATC GACAGCGGCC AGTGCACCGA ACTCCTTGTG CTGCTCGGCT 360

TGCAGCTCGG CGCTCCACGT CTTCACCTCG GGCGCGGACA ATTCGACGAC CTTGTTAGCG 420

ATCGACGCAT TGGTCGCCGC AGCAATGCCC GCCACATCCC AGTCCCCTGG ATCGAGGTCG 480

GCGCGGCACA ACAGCTCCGC GATCCGACCC CGATCCAGCG CCTGCCTCAC CACTTTTCGT 540

CGTCGCGGGG CTCACCCGGG TACTGAACCG GATCGCCACT ATCGAAACGG CTACGCGCGG 600

CGGCAGCGGC GGCGCTGGCG GCGGCACGTT CATCACCACC GGACCGGGAA CCAGCGTCGA 660

TTCATCGATG GCCGGCTGAA TCGGCCGGCG TTCGTCGGGC AGCAGGTCCG CGAGCTCGTC 720

GGCATCGATG TACTGCCGGC CGGCGGATCG TCGTCACGCA GAATGTGGGA CACCAGCGCC 780

TTGTCGCGGG CCTCTTCGCC GGTGAGGATC CGCTCGGAGG CGCGGTCGCG GCGCGGCTGT 840

GGCATGTCGG GGCGTGCCGC TCCCCCGGCG CCGCCCATCG GCCCGCCCAT TGGCATTCCG 900

CCCATGCCGC CCATCATTCC TGTGGAGCCA GCTGGCCCGG TCTTCAATGG AGGCAGGCCC 960

GCTGACGGCG ACGTGGAGGC GGTGCGCCCC GAAATCTGGG CCGGATCAAC TCGGCCACCG 1020

GTCACGGTCG GATTGGCGGC CGGTGTTGTC GGTGCGACAA CACCGCCGAC AACGCCGCGC 1080

CCCGCCATCG CCGAACCACG GGGTGGTGGG TGCGTCCGAC CTGCCAGAAT CGTCCCGGCG 1140

TCGCGGCTGC TGCTGAACAC CGCCGAGCCC GCCGCCAGTC GGGAAAGCGC TGGGCATCAT 1200 GGTCGGGCCG GGGGCCATCG GAGCGGGTGC ACCTGTCGGG GCTGGTGGCG GCGTCAGCGC 1260

CGTCGCCTGC ACCATCGGCC GTGGGCCGCC GACACCTCCG TGGTCGCACC GCCGCCGCCG 1320

ACGATCGTGT CGTCAGCGCC GCCGCCGACG ATGGTGTCGT CCCAACCGTC GCGCGGCTGG 1380

AGGTCGCGGG GCGACCGGAA AATGCCTTTA TCGTGGCCGG ACACCTTGGA ATCGGTGTCC 1440

GGCTCGTCGG GCAGGCCTTC CGTCGCTGAC GTGCACGCGC GCTCCAATCG CTCCAGCGCC 1500

GCCTGGACCT CGGGATCGGC AGCCGTCCCG CCCCGAATGA CCGGGCGGCC GCGGCCGGCC 1560

TCTCCCACCG CACGCAGGGC CGTCGGCGAT TTTCAGCAGG TCGCCGCCCA TTTCCGACAT 1620

CTTTTCCTCG GCGGCCGATC GCCGCACCGG ACCCAATGTC GTCCGGAAAC GGCTCGGCCG 1680

CGATCGACTC CAGCAACGCG GCCATGTCGA TGCGCTCCTG AAACTCGGCC TCGTTGGTCA 1740

GCGAATCGCC GTCATAACGG ATGGCGCCCG GGCCGCCGCG CGATATCGAG CCGAGAACGT 1800

TATCGAAGTT GGTCATGTGT AATCCCCTCG TTTGAACTTT GGATTAAGCG TAGATACACC 1860

CTTGGACAAG CCAGTTGGAT TCGGAGACAA GCAAATTCAG CCTTAAAAAG GGCGAGGCCC 1920 TGCGGTGGTG GAACACCGCA GGGCCTCTAA CCGCTCGACG CGCTGCACCA ACCAG

Claims

WE CLAIM:

1. A pharmaceutical composition for administration to an animal providing a continuous source of a protein of interest into said animal upon administration thereto and stimulating the cellular immunity of said animal upon administration thereto made according the steps of:

(a) cultivating an inoculum of at least one live Mycobacterium strain, which is nonpathogenic to said animal and capable of sustaining a commensial symbiotic relationship with macrophages in said animal, in a liquid culture medium capable of providing sufficient nutrients for growth of said Mycobacterium strain at an appropriate temperature to obtain a liquid cell culture;

(b) cooling said culture to about 4┬░C;

(c) centrifuging said cooled culture at about 4┬░C to obtain a pellet of live Mycobacterium cells and a supernatant; (d) separating said pellet from said supernatant;

(e) washing said pellet by suspending in sterile cold glycerol and centrifuging at about 4┬░C to obtain electro-competent Mycobacterium cells;

(f) mixing said electro-competent Mycobacterium cells with vector DNA, said vector DNA comprising a first coding region for a protein of interest, a second coding region for an attachment site and an integrase gene for a Mycobacterium bacteriophage, and a third coding region for a selection marker suitable for said Mycobacterium strain to form a transformation mixture;

(g) performing electroporation on said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said transformed Mycobacterium cells having said vector DNA incorporated into the genome of said Mycobacterium strain and being capable of expressing said protein of interest after administration into said animal; and

(h) isolating said transformed Mycobacterium cells from non- transformed Mycobacterium cells by growing said electroporated culture in the presence of a substance which said selection marker permits transformed Mycobacterium cells but not non-transformed Mycobacteriums cells to survive

2. The composition of claim 1, made by a method further comprising the step of transferring the culture of step (b) from said culture medium to a liquid protein-free culture medium and cultivating said Mycobacterium strain in said liquid protein-free culture medium under appropriate conditions prior to step (c).

3. The composition of claim 1, wherein said second coding region for said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID No: 5.

4. The composition of claim 1, wherein said second coding region for said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID No: 6.

5. The composition of claim 1, wherein said second coding region for said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID No:7.

6. The composition of claim 1, wherein said third coding region for said selection marker suitable for said Mycobacterium strain comprising a kanamycin selection marker essentially consisting of the sequence provided in SEQ ID No:2.

7. The composition of claim 1, wherein said bacteriophage is mycobacteriophage D29.

8. The composition of claim 1, wherein said Mycobacterium strain is selected from the group consisting of Mycobacterium gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium chitae, Mycobacterium duvalu, Mycobacterium flavescens, Mycobacterium nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and Mycobacterium vaccae

9 The composition of claim 1, further comprising a cytokine associated with cellular immunity

10 The composition of claim 1, further comprising a chemotherapeutic agent

11 A pharmaceutical composition for administration to an animal providing a continuous source of a protein of interest into said animal upon administration thereto and stimulating the cellular immunity of said animal upon administration thereto made according the steps of (a) cultivating an inoculum of at least one live Mycobacterium strain, which is nonpathogenic to said animal and capable of sustaining a commensial symbiotic relationship with macrophages in said animal, in a liquid culture medium capable of providing sufficient nutrients for growth of said Mycobacterium strain at an appropriate temperature to obtain a liquid cell culture,

(b) cooling said culture to about 4┬░C,

(c) centrifuging said cooled culture at about 4┬░ C to obtain a pellet of live Mycobacterium cells and a supernatant,

(d) separating said pellet from said supernatant, (e) washing said pellet by suspending in sterile cold glycerol and centrifuging at about 4┬░C to obtain electro-competent Mycobacterium cells, (f) mixing said electro-competent Mycobacterium cells with vector DNA, said vector DNA comprising a first coding region for a protein of interest, a second coding region for the minimal functional component of an attachment site and an integrase gene for a Mycobacterium bacteriophage, and a third coding region for a selection marker suitable for said Mycobacterium strain to form a transformation mixture;

(g) performing electroporation on said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said transformed Mycobacterium cells having said vector DNA incorporated into the genome of said Mycobacterium strain and being capable of expressing said protein of interest after administration into said animal; and

(h) isolating said transformed Mycobacterium cells from nontransformed Mycobacterium cells by growing said electroporated culture in the presence of a substance which said selection marker permits transformed

Mycobacterium cells but not non-transformed Mycobacteriums cells to survive.

12. The composition of claim 11, made by a method further comprising the step of transferring the culture of step (b) from said culture medium to a liquid protein-free culture medium and cultivating said Mycobacterium strain in said liquid protein-free culture medium under appropriate conditions prior to step (c).

13. The composition of claim 11, wherein said second coding region for said minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ED No: 5.

14. The composition of claim 11, wherein said second coding region for said minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID No:6.

15. The composition of claim 11, wherein said second coding region for said minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID No:7.

16. The composition of claim 11, wherein said bacteriophage is mycobacteriophage D29.

17. The composition of claim 11, wherein said Mycobacterium strain is selected from the group consisting of Mycobacterium gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and Mycobacterium vaccae.

18. The composition of claim 11, further comprising a cytokine associated with cellular immunity.

19. The composition of claim 11, further comprising a chemotherapeutic agent.

20. A pharmaceutical composition for administration to an animal providing a continuous source of a protein of interest into said animal upon administration thereto and stimulating the cellular immunity of said animal upon administration thereto made according the steps of:

(a) cultivating an inoculum of at least one live Mycobacterium strain, which is nonpathogenic to said animal and capable of sustaining a commensial symbiotic relationship with macrophages in said animal, in a liquid culture medium capable of providing sufficient nutrients for growth of said Mycobacterium strain at an appropriate temperature to obtain a liquid cell culture;

(b) cooling said culture to about 4┬░C; (c) centrifuging said cooled culture at about 4┬░C to obtain a pellet of live Mycobacterium cells and a supernatant;

(d) separating said pellet from said supernatant;

(e) washing said pellet by suspending in sterile cold glycerol and centrifuging at about 4┬░C to obtain electro-competent Mycobacterium cells;

(f) mixing said electro-competent Mycobacterium cells with vector DNA, said vector DNA comprising a first coding region for a protein of interest, a second coding region for an attachment site and integrase gene for a Mycobacterium bacteriophage, and the minimal functional component of a third coding region for a selection marker suitable for said Mycobacterium strain to form a transformation mixture;

(g) performing electroporation on said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said transformed Mycobacterium cells having said vector DNA incorporated into the genome of sύά Mycobacterium strain and being capable of expressing said protein of interest after administration into said animal; and

(h) isolating said transformed Mycobacterium cells from nontransformed Mycobacterium cells by growing said electroporated culture in the presence of a substance which said selection marker permits transformed Mycobacterium cells but not non-transformed Mycobacteriums cells to survive.

21. The composition of claim 20, made by a method further comprising the step of transferring the culture of step (b) from said culture medium to a liquid protein-free culture medium and cultivating said Mycobacterium strain in said liquid protein-free culture medium under appropriate conditions prior to step (c).

22. The composition of claim 20, wherein said third coding region for said minimal functional component of said selection marker suitable for said Mycobacterium strain comprising a kanamycin selection marker essentially consisting of the sequence provided in SEQ ID No:2.

23. The composition of claim 20, wherein said bacteriophage is mycobacteriophage D29.

24. The composition of claim 20, wherein said Mycobacterium strain is selected from the group consisting of Mycobacterium gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and Mycobacterium vaccae.

25. The composition of claim 20, further comprising a cytokine associated with cellular immunity.

26. The composition of claim 20, further comprising a chemotherapeutic agent.

27. A pharmaceutical composition for administration to an animal providing a continuous source of a protein of interest into said animal upon administration thereto and stimulating the cellular immunity of said animal upon administration thereto made according the steps of:

(a) cultivating an inoculum of at least one live Mycobacterium strain, which is nonpathogenic to said animal and capable of sustaining a commensial symbiotic relationship with macrophages in said animal, in a liquid culture medium capable of providing sufficient nutrients for growth of said Mycobacterium strain at an appropriate temperature to obtain a liquid cell culture;

(b) cooling said culture to about 4┬░C;

(c) centrifuging said cooled culture at about 4┬░C to obtain a pellet of live Mycobacterium cells and a supernatant;

(d) separating said pellet from said supernatant; (e) washing said pellet by suspending in sterile cold glycerol and centrifuging at about 4┬░C to obtain electro-competent Mycobacterium cells;

(f) mixing said electro-competent Mycobacterium cells with an extra- chromosomal DNA vector, said extra-chromosomal DNA vector comprising a first coding region for a protein of interest, a second coding region for an origin of replication for Mycobacterium, and a third coding region for a selection marker suitable for said Mycobacterium strain to form a transformation mixture;

(g) performing electroporation on said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said transformed Mycobacterium cells comprising said extra-chromosomal DNA vector and being capable of expressing said protein of interest after administration into said animal; and

(h) isolating said transformed Mycobacterium cells from nontransformed Mycobacterium cells by growing said electroporated culture in the presence of a substance which said selection marker permits transformed Mycobacterium cells but not non-transformed Mycobacteriums cells to survive.

28. The composition of claim 27, made by a method further comprising the step of transferring the culture of step (b) from said culture medium to a liquid protein-free culture medium and cultivating said Mycobacterium strain in said liquid protein-free culture medium under appropriate conditions prior to step (c).

29. The composition of claim 27, wherein said second coding region for an origin of replication for Mycobacterium essentially consisting of the sequence of an origin of replication of a Mycobacterium plasmid provided in SEQ ED No:3.

30. The composition of claim 27, wherein said second coding region for an origin of replication for Mycobacterium essentially consisting of the sequence of an origin of replication of a. Mycobacterium plasmid provided in SEQ ID No:4.

31. The composition of claim 27, wherein said coding region for a selection marker suitable for said Mycobacterium strain comprising a kanamycin selection marker essentially consisting of the sequence provided in SEQ ID No:2.

32. The composition of claim 27, wherein said Mycobacterium plasmid is pAL 5000.

33. The composition of claim 27, wherein said Mycobacterium strain is selected from the group consisting of Mycobacterium gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and Mycobacterium vaccae.

34. The composition of claim 27, further comprising a cytokine associated with cellular immunity.

35. The composition of claim 27, further comprising a chemotherapeutic agent.

36. A pharmaceutical composition for administration to an animal providing a continuous source of a protein of interest into said animal upon administration thereto and stimulating the cellular immunity of said animal upon administration thereto made according the steps of:

(a) cultivating an inoculum of at least one live Mycobacterium strain, which is nonpathogenic to said animal and capable of sustaining a commensial symbiotic relationship with macrophages in said animal, in a liquid culture medium capable of providing sufficient nutrients for growth of said Mycobacterium strain at an appropriate temperature to obtain a liquid cell culture;

(b) cooling said culture to about 4┬░C; (c) centrifuging said cooled culture at about 4┬░C to obtain a pellet of live Mycobacterium cells and a supernatant;

(d) separating said pellet from said supernatant;

(e) washing said pellet by suspending in sterile cold glycerol and centrifuging at about 4┬░C to obtain electro-competent Mycobacterium cells; (f) mixing said electro-competent Mycobacterium cells with an extra- chromosomal DNA vector, said extra-chromosomal DNA vector comprising a first coding region for a protein of interest, a second coding region for the miminal functional component of an origin of replication for Mycobacterium, and a third coding region for a selection marker suitable for said Mycobacterium strain to form a transformation mixture;

(g) performing electroporation on said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said transformed Mycobacterium cells comprising said extra-chromosomal DNA vector and being capable of expressing said protein of interest after administration into said animal; and

(h) isolating said transformed Mycobacterium cells from nontransformed Mycobacterium cells by growing said electroporated culture in the presence of a substance which said selection marker permits transformed Mycobacterium cells but not non-transformed Mycobacteriums cells to survive.

37. The composition of claim 36, made by a method further comprising the step of transferring the culture of step (b) from said culture medium to a liquid protein-free culture medium and cultivating said Mycobacterium strain in said liquid protein-free culture medium under appropriate conditions prior to step (c).

38. The composition of claim 36, wherein said second coding region for the minimal functional component of said origin of replication for Mycobacterium essentially consisting of the sequence of an origin of replication of a Mycobacterium plasmid provided in SEQ ID No: 3.

39. The composition of claim 36, wherein said second coding region for the minimal functional component of said origin of replication for Mycobacterium essentially consisting of the sequence of an origin of replication of a Mycobacterium plasmid provided in SEQ ID No:4.

40. The composition of claim 36, wherein said Mycobacterium plasmid is pAL 5000.

41. The composition of claim 36, wherein said Mycobacterium strain is selected from the group consisting of Mycobacterium gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and Mycobacterium vaccae.

42. The composition of claim 36, further comprising a cytokine associated with cellular immunity.

43. The composition of claim 36, further comprising a chemotherapeutic agent.

44. A pharmaceutical composition for administration to an animal providing a continuous source of a protein of interest into said animal upon administration thereto and stimulating the cellular immunity of said animal upon administration thereto made according the steps of: (a) cultivating an inoculum of at least one live Mycobacterium strain, which is nonpathogenic to said animal and capable of sustaining a commensial symbiotic relationship with macrophages in said animal, in a liquid culture medium capable of providing sufficient nutrients for growth of said Mycobacterium strain at an appropriate temperature to obtain a liquid cell culture;

(b) cooling said culture to about 4┬░C;

(c) centrifuging said cooled culture at about 4┬░C to obtain a pellet of live Mycobacterium cells and a supernatant;

(d) separating said pellet from said supernatant;

(e) washing said pellet by suspending in sterile cold glycerol and centrifuging at about 4┬░C to obtain electro-competent Mycobacterium cells;

(f) mixing said electro-competent Mycobacterium cells with an extra- chromosomal DNA vector, said extra-chromosomal DNA vector comprising a first coding region for a protein of interest, a second coding region for an origin of replication for Mycobacterium, and a third coding region for the minimal functional component of a selection marker suitable for said Mycobacterium strain to form a transformation mixture;

(g) performing electroporation on said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said transformed Mycobacterium cells comprising said extra-chromosomal DNA vector and being capable of expressing said protein of interest after administration into said animal; and

(h) isolating said transformed Mycobacterium cells from nontransformed Mycobacterium cells by growing said electroporated culture in the presence of a substance which said selection marker permits transformed Mycobacterium cells but not non-transformed Mycobacteriums cells to survive.

45. The composition of claim 44, made by a method further comprising the step of transferring the culture of step (b) from said culture medium to a liquid protein-free culture medium and cultivating said Mycobacterium strain in said liquid protein-free culture medium under appropriate conditions prior to step (c).

46. The composition of claim 44, wherein said coding region for the minimal functional component of said selection marker suitable for said Mycobacterium strain comprising a kanamycin selection marker essentially consisting of the sequence provided in SEQ ID No:2.

47. The composition of claim 44, wherein said Mycobacterium plasmid is pAL 5000.

48. The composition of claim 44, wherein said Mycobacterium strain is selected from the group consisting of Mycobacterium gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and Mycobacterium vaccae.

49. The composition of claim 44, further comprising a cytokine associated with cellular immunity.

50. The composition of claim 44, further comprising a chemotherapeutic agent.

51. A pharmaceutical composition for administration to an animal, said composition capable of stimulating the cellular immunity of said animal upon administration into said animal comprising the steps of:

(a) cultivating an inoculum of at least one five. Mycobacterium strain, which is nonpathogenic to said animal and capable of sustaining a commensial symbiotic relationship with macrophages in said animal, in a liquid culture medium capable of providing sufficient nutrients for growth of said Mycobacterium strain at an appropriate temperature to obtain a liquid cell culture;

(b) cooling said culture to about 4┬░C;

(c) centrifuging said cooled culture to obtain a pellet of live Mycobacterium cells and a supernatant;

(d) separating said pellet from said supernatant;

(e) washing said pellet by suspending in sterile cold saline and centrifuging at about 4┬░C to obtain washed live Mycobacterium cells; and

(f) mixing said 7/ve Mycobacterium cells with a protein of interest.

52. The composition of claim 51, wherein said Mycobacterium strain is selected from the group consisting of Mycobacterium gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium nonchromogenicum, Mycobacterium bovis BCG, Mycobacterium neoaurum, and Mycobacterium vaccae.

53. The composition of claim 51, further comprising a cytokine associated with cellular immunity.

54. The composition of claim 51, further comprising a chemotherapeutic agent.

55. A pharmaceutical composition for administration to an animal, said composition capable of stimulating the cellular immunity of said animal upon administration into said animal comprising the steps of:

(a) cultivating an inoculum of two or more live Mycobacterium strains, which are nonpathogenic to said animal and capable of sustaining a commensial symbiotic relationship with macrophages in said animal, in a liquid culture medium capable of providing sufficient nutrients for growth of said Mycobacterium strains at an appropriate temperature to obtain a liquid cell culture; (b) centrifuging said culture to obtain a pellet of Mycobacterium cells and a supernatant,

(c) separating said pellet from said supernatant,

(d) washing said pellet and centrifuging to obtain washed Mycobacterium cells,

(e) killing said washed Mycobacterium cells to obtain Mycobacterium adjuvant, and

(f) mixing said Mycobacterium adjuvant with a protein of interest

56 The composition of claim 55, wherein said Mycobacterium strains are selected from the group consisting of Mycobacterium gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium nonchromogenicum,

Mycobacterium bovis BCG, Mycobacterium vaccae, and Mycobacterium neoaurum

57 The composition of claim 55, further comprising a cytokine associated with cellular immunity

58 The composition of claim 55, further comprising a chemotherapeutic agent

59 A shuttle vector comprising a first coding region for an origin of replication for Escherichia coli, a second coding region for an attachment site and an integrase gene for a Mycobacterium bacteriophage, and a third coding region for a selection marker suitable for both Escherichia coli and Mycobacterium, said first coding region for said origin of replication for

Escherichia coli comprising the minimal functional component of said origin of replication for Escherichia coli

60. The shuttle vector according to claim 59, wherein said minimal functional component of said origin of replication for Escherichia coli essentially consisting of the sequence provided in SEQ ID NO:l.

61. The shuttle vector of claim 59, wherein said bacteriophage is mycobacteriophage D29.

62. A shuttle vector comprising a first coding region for an origin of replication for Escherichia coli, a second coding region for an attachment site and an integrase gene for a Mycobacterium bacteriophage, and a third coding region for a selection marker suitable for both Escherichia coli and Mycobacterium, said second coding region for said attachment site and integrase gene for said Mycobacterium bacteriophage comprising the minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage.

63. The shuttle vector according to claim 62, wherein minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID NO:5.

64. The shuttle vector according to claim 62, wherein minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID NO:6.

65. The shuttle vector according to claim 62, wherein minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID NO:7.

66. The shuttle vector of claim 62, wherein said bacteriophage is mycobacteriophage D29.

67. A shuttle vector comprising a first coding region for an origin of replication for Escherichia coli, a second coding region for an attachment site and an integrase gene for a Mycobacterium bacteriophage, and a third coding region for a selection marker suitable for both Escherichia coli and Mycobacterium, said third coding region for said selection marker suitable for both Escherichia coli and Mycobacterium comprising the minimal functional component of said selection marker suitable for both Escherichia coli and Mycobacterium .

68. The shuttle vector according to claim 67, wherein said minimal functional component of said selection marker suitable for both Escherichia coli and Mycobacterium comprising a kanamycin selection marker essentially consisting of the sequence provided in SEQ ID NO:2.

69. The shuttle vector of claim 67, wherein said bacteriophage is mycobacteriophage D29.

70. A shuttle vector comprising a first coding region for an origin of replication for Escherichia coli, a second coding region for an origin of replication in Mycobacterium, and a third coding region for a selection marker suitable for both Escherichia coli and Mycobacterium, said first coding region for said origin of replication for Escherichia coli comprising the minimal functional component of said origin of replication for Escherichia coli.

71. The shuttle vector according to claim 70, wherein said minimal functional component of said origin of replication for Escherichia coli essentially consisting of the sequence provided in SEQ ID No: 1.

72. A shuttle vector comprising a first coding region for an origin of replication for Escherichia coli, a second coding region for an origin of replication in Mycobacterium, and a third coding region for a selection marker suitable for both Escherichia coli and Mycobacterium, said second coding region for an origin of replication in Mycobacterium essentially comprising the minimal functional component of said origin of replication of said Mycobacterium plasmid.

73. The shuttle vector according to claim 72, wherein said minimal functional component of said origin of replication of said Mycobacterium plasmid essentially consisting of the sequence provided in SEQ ID No:3.

74. The shuttle vector according to claim 72, wherein said minimal functional component of said origin of replication of sa d Mycobacterium plasmid essentially consisting of the sequence provided in SEQ ID No:4.

75. A shuttle vector comprising a first coding region for an origin of replication for Escherichia coli, a second coding region for an origin of replication in Mycobacterium, and a third coding region for a selection marker suitable for both Escherichia coli and Mycobacterium, said third coding region for a selection marker suitable for both Escherichia coli and Mycobacterium comprising the minimal functional component of said selection marker suitable for both Escherichia coli and Mycobacterium.

76. The shuttle vector according to claim 75, wherein said minimal functional component of said selection marker suitable for both Escherichia coli and Mycobacterium comprising a kanamycin selection marker essentially consisting of the sequence provided in SEQ ID NO:2.

77. A vector comprising a first coding region for an attachment site and integrase gene for a Mycobacterium bacteriophage and a second coding region for a selection marker suitable for Mycobacterium, said first coding region for said attachment site and integrase gene for said Mycobacterium bacteriophage comprising the minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage.

78. The vector according to claim 77, wherein said minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID No: 5.

79. The vector according to claim 77, wherein said minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID No:6.

80. The vector according to claim 77, wherein said minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID No:7.

81. The vector of claim 77, wherein said bacteriophage is mycobacteriophage D29.

82. A vector comprising a first coding region for an attachment site and integrase gene for a Mycobacterium bacteriophage and a second coding region for a selection marker suitable for Mycobacterium, said second coding region for said selection marker suitable for said Mycobacterium strain comprising the minimal functional component of said selection marker suitable for said Mycobacterium strain.

83. The vector according to claim 82, wherein said minimal functional component of said selection marker suitable for said Mycobacterium strain comprising a kanamycin selection marker essentially consisting of the sequence provided in SEQ ID No:2.

84. The vector of claim 82, wherein said bacteriophage is mycobacteriophage D29.

85. A vector for carrying and expressing selected genes of a Mycobacterium strain comprising a first coding region for an attachment site and integrase gene for a Mycobacterium bacteriophage and a second coding region for a selection marker suitable for Mycobacterium, said first coding region for said attachment site and integrase gene for said Mycobacterium bacteriophage comprising the minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage.

86. The vector according to claim 85, wherein said minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID No: 5.

87. The vector according to claim 85, wherein said minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID No: 6.

88. The vector according to claim 85, wherein said minimal functional component of said attachment site and integrase gene for said Mycobacterium bacteriophage essentially consisting of the sequence provided in SEQ ID No: 7.

89. The vector according to claim 85, wherein said bacteriophage is mycobacteriophage D29.

90. A vector for carrying and expressing selected genes of a Mycobacterium strain comprising a first coding region for an attachment site and integrase gene for a Mycobacterium bacteriophage and a second coding region for a selection marker suitable for Mycobacterium, said second coding region for said selection marker suitable for said Mycobacterium strain comprising the minimal functional component of said selection marker suitable for said Mycobacterium strain.

91. The vector according to claim 90, wherein said minimal functional component of said selection marker suitable for said Mycobacterium strain comprising a kanamycin selection marker essentially consisting of the sequence provided in SEQ ID No:2.

92. The vector according to claim 90, wherein said bacteriophage is mycobacteriophage D29.

93. A vector comprising a first coding region for an origin of replication in Mycobacterium and a second coding region for a selection marker suitable for Mycobacterium, said first coding region for said origin of replication in Mycobacterium comprising the minimal functional component of said origin of replication of said Mycobacterium plasmid.

94. The vector according to claim 93, wherein said minimal functional component of origin of replication of said Mycobacterium plasmid essentially consisting of the sequence provided in SEQ ID No:3.

95. The vector according to claim 93, wherein said minimal functional component of origin of replication of said Mycobacterium plasmid essentially consisting of the sequence provided in SEQ ED No:4.

96. A vector comprising a first coding region for an origin of replication in Mycobacterium and a second coding region for a selection marker suitable for Mycobacterium, said second coding region for said selection marker suitable for said Mycobacterium comprising the minimal functional component of said selection marker suitable for said Mycobacterium.

97. The vector according to claim 96, wherein said minimal functional component of said selection marker suitable for said Mycobacterium comprising a kanamycin selection marker essentially consisting of the sequence provided in SEQ ID No:2.

98. A vector comprising a first coding region for an origin of replication for Escherichia coli and a second coding region for a selection marker suitable for said Escherichia coli, said first coding region for an origin of replication for Escherichia coli comprising the minimal functional component of said origin of replication for said Escherichia coli.

99. The vector according to claim 98, wherein said minimal functional component of said origin of replication of said Escherichia coli essentially consisting of the sequence provided in SEQ ED No:l.

100. A vector comprising a first coding region for an origin of replication for Escherichia coli and a second coding region for a selection marker suitable for said Escherichia coli, said second coding region for a selection marker suitable for said Escherichia coli comprising the minimal functional component of said selection marker suitable for said Escherichia coli.

101. The vector according to claim 100, wherein said minimal functional component of said selection marker suitable for said Escherichia coli comprising a kanamycin selection marker for said Escherichia coli essentially consisting of the sequence provided in SEQ ID No:2.

102. A vector for carrying and expressing selected genes in Escherichia coli comprising a first coding region for an origin of replication for Escherichia coli and a second coding region for a selection marker suitable for said Escherichia coli, said first coding region for an origin of replication for Escherichia coli comprising the minimal functional component of said origin of replication for said Escherichia coli.

103. The vector according to claim 102, wherein said minimal functional component of said origin of replication of said Escherichia coli essentially consisting of the sequence provided in SEQ ID No:l.

104. A vector for carrying and expressing selected genes in Escherichia coli comprising a first coding region for an origin of replication for Escherichia coli and a second coding region for a selection marker suitable for said Escherichia coli strain, said second coding region for a selection marker suitable for said Escherichia coli comprising the minimal functional component of said selection marker suitable for said Escherichia coli.

105. The vector according to claim 104, wherein said minimal functional component of said selection marker suitable for said Escherichia coli comprising a kanamycin selection marker for said Escherichia coli essentially consisting of the sequence provided in SEQ ID No:2.

106. A method of administering to an animal an effective amount of the pharmaceutical composition according to claim 1 to stimulate cellular immunity in said animal.

107. The method according to claim 106, further comprising administration of a cytokine associated with cellular immunity.

108. The method according to claim 106, further comprising administration of a chemotherapeutic agent.

109. A method of administering to an animal an effective amount of the pharmaceutical composition according to claim 11 to stimulate cellular immunity in said animal.

110. The method according to claim 109, further comprising administration of a cytokine associated with cellular immunity.

111. The method according to claim 109, further comprising administration of a chemotherapeutic agent.

112. A method of administering to an animal an effective amount of the pharmaceutical composition according to claim 20 to stimulate cellular immunity in said animal.

113. The method according to claim 112, further comprising administration of a cytokine associated with cellular immunity.

114. The method according to claim 112, further comprising administration of a chemotherapeutic agent.

115. A method of administering to an animal an effective amount of the pharmaceutical composition according to claim 27 to stimulate cellular immunity in said animal.

116. The method according to claim 115, further comprising administration of a cytokine associated with cellular immunity.

117. The method according to claim 115, further comprising administration of a chemotherapeutic agent.

118. A method of administering to an animal an effective amount of the pharmaceutical composition according to claim 36 to stimulate cellular immunity in said animal.

119. The method according to claim 118, further comprising administration of a cytokine associated with cellular immunity.

120. The method according to claim 118, further comprising administration of a chemotherapeutic agent.

121. A method of administering to an animal an effective amount of the pharmaceutical composition according to claim 44 to stimulate cellular immunity in said animal.

122. The method according to claim 121, further comprising administration of a cytokine associated with cellular immunity.

123. The method according to claim 121, further comprising administration of a chemotherapeutic agent.

124. A method of administering to an animal an effective amount of the pharmaceutical composition according to claim 51 to stimulate cellular immunity in said animal.

125. The method according to claim 124, further comprising administration of a cytokine associated with cellular immunity.

126. The method according to claim 124, further comprising administration of a chemotherapeutic agent.

127. A method of administering to an animal an effective amount of the pharmaceutical composition according to claim 55 to stimulate cellular immunity in said animal.

128. The method according to claim 127, further comprising administration of a cytokine associated with cellular immunity.

129. The method according to claim 127, further comprising administration of a chemotherapeutic agent.

130. A culture medium comprising about 0.25% proteose peptone; about 0.2%) nutrient broth; about 0.075% pyruvic acid; about 0.05% sodium glutamate; about 0.5% albumin fraction V; about 0.7% dextrose; about 0.0004% catalase; about 0.005% oleic acid; L,_, amino-acid complex ( about 0.126% alanine; about 0.097% leucine; about 0.089% glycine; about 0.086% valine; about 0.074%) arginine; about 0.06% threonine; about 0.059% aspartic acid; about 0.057% serine; about 0.056% proline; about 0.05%) glutamic acid; about 0.044% isoleucine; about 0.033% glutamine; 0.029% phenylalanine; about 0.025%) asparagine; about 0.024% lysine; about 0.023% histidine; about 0.021% tyrosine; about 0.02% methionine; about 0.014% tryptophan; and about 0.01% cysteine); about 0.306% Na₂HPO₄; about 0.055% KH₂PO₄; about 0.05% NH₄C1; about 0.335% NaCl; about 0.0001% ZnSO₄; about 0.0001% CuSO₄; about 0.0001% FeCl₃; about 0.012% MgSO₄; and about╬╕.05% Tween 80; wherein the pH of said medium is about 7.

131. The culture medium according to claim 130, further comprising about 0.8%) glycerol.