MXPA99008868A - Mycobacterium recombinant vaccines - Google Patents
Mycobacterium recombinant vaccinesInfo
- Publication number
- MXPA99008868A MXPA99008868A MXPA/A/1999/008868A MX9908868A MXPA99008868A MX PA99008868 A MXPA99008868 A MX PA99008868A MX 9908868 A MX9908868 A MX 9908868A MX PA99008868 A MXPA99008868 A MX PA99008868A
- Authority
- MX
- Mexico
- Prior art keywords
- mycobacterium
- vector
- cells
- coding region
- selection marker
- Prior art date
Links
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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
RECOMBINANT VACCINES OF MYCOBACTERIUM
TECHNICAL CAIVIPO OF THE INVENTION
The present invention relates to DNA constructs for cloning and methods for cloning Mycobacterium genes.
BACKGROUND OF THE INVENTION
The immune system of mammals comprises both humoral and cellular components that are interrelated but play different roles. Although both branches of the immune system include helper T cells, the appearance of the immune response depends on which subclass of T cells is involved. The auxiliary T lymphocytes are produced by two maturation pathways (TH-1 and TH-2), are grouped according to a cluster differentiation (CD4 and CD8), and secrete different cytokines. Both components of the immune system constantly scrutinize and investigate what unfolds in association with the molecules of the major histocompatibility complex (MHC) on the cell surface. The humoral immune response includes helper T lymphocytes produced by the TH-2 T cell maturation pathway. Cells in 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 the proliferation of macrophages, contributing to a down regulation of the TH-1 response. FNT causes tissue inflammation and necrosis when it is released at high levels, which are indications of complete immune system failure in many diseases. CD4 + T lymphocytes are activated by contacting antigens deployed in association with MHC class II molecules (MHC II), on the surface of macrophages and antigen presenting cells. Antibodies are produced by B cells when they interact with these activated CD4 + T lymphocytes. MHC II molecules reside in vesicles that engulf and destroy extracellular materials. In this way, their location within the cell gives them their specific function to monitor the content of these vesicles. They bind specifically to antigens that have been enzymatically processed in the lysosomes of immune cells after phagocytosis. The humoral immune response is required to protect the extracellular environment against antigens and extracellular parasites through antibodies that may be effective in neutralizing infectious agents. However, the humoral immune response can not eliminate whole cells that become diseased, cause tissue destruction and necrosis, and is not effective in combating intracellular diseases. Consequently, the body relies on the cellular immune response for the protection of pathologies that start in the intracellular environment. The cellular immune response is carried out through cytotoxic immune cells that are capable of killing diseased cells. The cellular immune response includes auxiliary T lymphocytes produced by the T-cell maturation pathway, TH-1. Cells in this pathway secrete cytokines such as IL-2, IL-12, IL-15, interferon gamma (IFN), lymphotoxin and granulocyte macrophage colony stimulation factor (GMCSF). These cytokines activate macrophages. Cytotoxic T lymphocytes are CD8 + T cells that are activated by contact with antigens associated with p-class MHC molecules (MHC I). The MHC I molecules reside around the * producers of proteins such as the endoplastic reticulum. In this way,
their location within the cell gives them their specific function of monitoring the output and transport of materials produced within the cell. They bind specifically to antigens that have been synthesized in the intracellular environment as in the case of cancer or intracellular diseases. The cellular immune response protects against chronic intracellular diseases
such as intracellular infection, parasitism and cancer, activating macrophages and facilitating the detection and lysis of diseased cells. The result is the formation of a granuloma that is a paradigm of protective immunity in intracellular diseases. Although the immune system has evolved to be efficient in
To select the target antigens against which an immune response is provided, it is not always successful 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 diseases despite advances in detection, diagnosis and treatment. Many of these diseases are able to surround the immune system and progress without attack. For others with a long latency period, the diagnosis is usually made very late. Some have multiresistance profiles against the treatment of drugs 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. Treatment with chemotherapy is expensive and can be carried out only after a significant expansion of the pathological process, or if there is a transmission of infection and damage to the host. Although these diseases can develop an immune response, they usually compromise their effectiveness by suppressing or mimicking MHC molecules. In this type of disease, an immune response of TH-1 favors protection, while the down-regulation of its pathway, the conversion to TH-2 during the chronic course of the disease, or the upregulation of the TH-2 pathway. harmful to the host. Consequently, a shift to the TH-1 response or down-regulation of the TH-1 pathway should be beneficial on its own, and when associated with appropriate chemotherapy, it would be equivalent to an effective response to resistance, chronicity, and disease. Therefore, treatment methods for intracellular diseases that favor an immune response of TH-1 rather than a TH-2 response are required. Cancers are caused by genetic alterations that interrupt the metabolic activities of the cell. These genetic changes can result from hereditary and / or environmental factors including infections by pathogenic viruses. As in other intracellular diseases, cellular immunity plays a major role in host defense against cancer. Traditionally, immunotherapies for cancer were designed to boost the cellular immune response using specific and non-specific stimuli, including: 1) passive cancer immunotherapies where the antibodies have been administered to patients, showing success only in rare cases; 2) active cancer immunotherapies wherein the materials expressing cancer antigens have been administered to patients (e.g., the injection of whole parts or fractions of cancer cells that have been irradiated or chemically or genetically modified), showing very little impact on models of experimental tumors and 3) The combination of adoptive lymphocytes and IL-2, which caused the regression of tumors in mice and metastatic melanoma in humans. Tumor infiltrating lymphocytes (TIL) capable of mediating tumor regression are lymphoid cells that can grow from suspensions of individual cells of the tumor incubated with IL-2. In this way, the antigens recognized by TIL are more likely to be involved in the immune response against cancer in vivo, and the cDNA and amino acid sequences of several of these antigens have been identified. Although these discoveries have opened new opportunities for the development of specific immunotherapies for cancer, treatment methods based on mixing cancer antigens or the cloning and expression of genes encoding these antigens are required in a delivery system that favors a response of TH-1 more than a TH-2 response to these antigens. Intracellular infections are caused by bacteria, viruses, parasites and fungi. These infectious agents are present either free in the environment or are carried by untreated hosts. Humans, animals and plants can serve as hosts, and if left untreated, can act as receptacles facilitating further dispersion of such agents to others. Intracellular pathogens such as M. tuberculosis, M. leprae, and tumor viruses cause diseases worldwide in millions of people each year. It is estimated that M. tuberculosis infects at least 30 million people per year and will cause an average of three million deaths per year during this decade, making tuberculosis (TB) the number one cause of death from the point of view of a single infectious agent (World Health Organization, 1996). TB occurs most commonly in developing countries, but the prevalence of TB has recently increased in the United States, as well as in developing countries, due to an increase in the number of immunologically compromised individuals with HIV infection. The risk of TB infection has also increased in individuals with diabetes, hemophilia, lymphomas, leukemias and other malignant neoplasms, since these individuals have compromised immune systems. Leprosy and viruses that cause neoplasia are also important intracellular pathogens worldwide. Leprosy currently causes diseases in more than 12 million people, and it is believed that at least 15% of cancers in humans are caused by the neoplastic transformation of cells by viruses. Intracellular infections with highly virulent strains resolve quickly resulting in the death or cure of the patient. However, organisms of lower virulence may persist in the host and develop chronic diseases. Mycobacterium infections develop through a spectrum that varies from a state of high resistance associated with cellular immunity to an opposite state and extreme low resistance associated with humoral immunity. For example, leprosy is caused by Mycobacterium leprae that remains uncultivable. The disease manifests an immuno-histological spectrum with six groups. At one end of the spectrum is polar tuberculous leprosy (TT), a paucibacillary form of the disease characterized by a strong immune response of TH-1 and a bacteriolytic effect that leads to granuloma formation and restricts the growth of M. leprae, respectively. At the opposite end of the spectrum is polar lepromatous leprosy (LL), a multibasilar form of the disease that 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, levels of IL-2 and cells with IL-2 receptors decrease, T cells become defective in their functions, and M. leprae proliferates without restriction within macrophages and Schwann cells. With this immune failure the elimination of the bacterium is markedly delayed, and the patient continues to carry bacilli from the tissues even after a prolonged therapy with drugs. 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 that reflect the different balances achieved by the body between the immune responses of TH-1 and TH-2. Likewise, tuberculosis married to Mycobacterium tuberculosis also manifests itself in an immunoclinical spectrum with multiple groups (four). The polar reactive group (RR) is associated with an immune response of TH-1 while the opposite pole (UU) is non-reactive and is associated with an immune response of TH-2. Therefore, there are clear indications that the immune response of TH-1 is the main defense mechanism against leprosy and tuberculosis. In this way, treatment and immunoprophylaxis against these diseases should focus on improving the TH-1 pathway. Allergic diseases are characterized by prolonged production of IgE molecules against common environmental antigens. This production depends on IL-4 and is inhibited by gamma interferon. In this way, allergic reactions involve an immune response of TH-2 that requires a low level of stimulation by allergens. Therefore, the preferable treatment for allergies would be to include the following: switch to an immune response of TH-1, which requires a high level of stimulation; activate CD8 + T cells and the production of interferon gamma; reduce IgE production and recruit eosinophils and mast cells; and increase 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 that 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. In this way, vaccinated individuals would have circulating cytotoxic T lymphocytes (CTL) that could interact and lyse stressed cells, whereas the expression of putative antigenic domains of autoimmunity in a susceptible host could 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 var fortuitum plasmid, pAL 5000," Plasmid 27: 130-140).
The methods available for the 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 mycobacterium is impermeable to antibiotics. Likewise, antibiotics have no effect on viral pathogenesis. Chemotherapy as a means of prophylaxis for high-risk individuals may be effective against M. tuberculosis or M. leprae, but it has its disadvantages. Chemotherapeutic agents have undesirable side effects in the patient, are expensive, and can lead to the potential existence of Mycobacterium strains resistant to multiple drugs. In addition to these disadvantages, chemotherapy as a means of treating active TB, leprosy and neoplasms induced by viruses has a minimal effect since it is used only after a significant progression of the disease. 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. Initial vaccines administered as protection against acute infections were developed using antigens to initiate an immune response regardless of their nature or mechanism. The purpose was to protect against watery infections in which an immune response of TH-2 could be efficient. These vaccines were made from a variety of crude antigens including deleted or attenuated whole cells, toxins and other structural components derived from the pathogen. Bacterial products such as peptidoglycan, lipoproteins, lipopolysaccharides and mycolic acid were used as therapeutic and prophylactic agents in various diseases. The administration of nonspecific stimulants derived from Corynebacterium paryum, Streptococcus, Serratia marcescens and Mycobacterium to cancer patients showed some efficacy and concomitantly improved the immune response against the disease. The adjuvants were developed to stimulate the immune response to antigenic material. One of these adjuvants was Freund's complete adjuvant, which consisted of Mycobacterium tuberculosis eliminated suspended in oil and emulsified with an aqueous antigen solution. This preparation was found to be very toxic for human use (Riott, et al., Immunology, 5th ed., Mosby, Philadelphia, pp. 332, 370 (1998).) After these first steps, efforts have been made to isolate and develop individual antigens. even individual epitopes in vaccines Molecular techniques have been used for the last two decades to clone the genes and map the domains of the corresponding proteins, however, the individual antigens or cytokines did not reproduce the same physiological effects as a whole bacterial adjuvant. For example, the development of antigens for M. tuhberculosis, M. leprae and other intracellular parasites was unsuccessful due to the dogma that specific protective antigens or ethyrtopes could not precisely define a protective antigen for these diseases. Ignored the fact that the immune response to a pathogen is a 1
coherent response to a complex mosaic of epitopes displayed by the pathogen with certain epitopes conferring protection and other epitopes mediating virulence and immunopathology. These vaccines have not been successful in establishing the favored TH-1 response over the TH-2 response. Initial vaccines were also not potent against intracellular diseases. The vaccines were not efficient, had short lives or triggered inadequate immune responses similar to hypersensitivity reactions in allergic diseases that result in necrosis, which worsens the onset of the disease process in many chronic infections such as tuberculosis and leprosy. For example, BCG (Bacille-Calmette Guérin) is a vaccine that has been used for the prophylaxis of TB and leprosy, but has a questionable efficacy. BCG is a live attenuated vaccine derived from M. bovis, a strain of Mycobacterium that is closely related to M. tuberculosis. BCG has only been marginally effective against leprosy and is not currently recommended for prophylaxis. The results of controlled studies to determine the efficacy of BCG vaccines for TB prophylaxis have been conflicting. Estimates of the effectiveness of BCG 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 did not provide a protective effect against the onset of pulmonary TB. Other studies have shown that BCG produces an inconsistent and fluctuating immunity. Since an effective vaccine has not been developed to protect against leprosy or virus-induced cancers, and because BCG is not reliable for prophylaxis of TB, a more effective vaccine is required. An example of such novel vaccines would combine selective antigens with potent adjuvants and stimulate the cellular immune response to provide a lasting protective immunogen. The patent of E.U. A. No. 3,956,481, Jolles et al., Discloses a water-soluble extract of mycobacteria suitable as an adjuvant, in which delipidated bacterial residues undergo either a mild extraction procedure or treatment with pyridine followed by treatment with ethanol or water. It was found that these extracts were toxic in humans, discouraging their use as a vaccine. In the patent of E.U.A. No. 4,036,953, Adam et al. Describes an adjuvant to improve the effects of a vaccine, in which the adjuvant is prepared by interrupting 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. It was also noted that adjuvants of this type were toxic in humans. The patent of E.U.A. No. 4,724,144, Rook et al., Discloses an immunotherapeutic agent comprising antigenic material from deleted 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 that survived the long-term exposure of chemotherapeutic agents. Although the vaccine shows an improved immune response, it is limited only to antigens endogenous to Mycobacterium vaccae. In the patent of E.U.A. No. 5,599,545, Stanford, et al. Discloses an immunotherapeutic agent comprising Mycobacterium vaccae cells removed in combination with an antigen exogenous to mycobacteria that promotes a TH-1 response. The exogenous antigen can be combined with the Mycobacterium vaccae removed by combination, chemical conjugation or absorption, or alternatively produced by the expression of an exogenous gene in Mycobacterium vaccae vector by means of a plasmid, cosmid, viral or other expression vector , or inserted into the genome. Although these compositions promote the immune response of TH-1, they were limited only to cells of
Mycobacterium vaccae eliminated. In addition, the patent does not provide any guidance on how to make Mycobacterium expression vectors, or how to incorporate the plasmid, cosmid or viral expression vectors, or how to integrate the expression vector into the genome. In the patent of E.U.A. No. 5,583,038, Stover discloses an expression vector for expressing a protein or polypeptide in a bacterium comprising a first DNA sequence encoding at least one secretion signal of a lipoprotein and a second DNA sequence encoding a protein desired, fragment of protein, polypeptide or peptide heterologous to the bacterium expressing the desired protein, etc. Stover demonstrated the use of an origin of replication recognized in Mycobacterium and the desire to eliminate sequences not necessary for plasmid replication, for example, to reduce a pALmid fragment pAL5000 containing said origin of replication to 1910 base pairs. Stover also describes the use of an attP-integrase gene fragment of mycobacteriophage L5 to transform M. smegmatis and BCG. WO 92/01783 describes a DNA that includes a first DNA sequence that contains a phage integration agent and a second DNA sequence that codes for a protein or polypeptide heterologous to Mycobacterium in which the DNA will be integrated to integrate DNA into a Mycobacterium chromosome and then administer the mycobacteria as a vaccine and / or therapeutic agent. WO 92/01783 also describes the use of a recognized origin of replication in Mycobacterium and the desire to eliminate sequences that are not necessary for plasmid replication, for example, to reduce a fragment of plasmid pAL5000 containing said origin of repiication to 1910 pairs of bases, and the use of an attP-integrase gene fragment from mycobacteriophage L5 to transform Mycobacterium and BCG. David et al., (David et al., 1992. Plasmid 28: 267-271) describes a promiscuous plasmid vector for E. coli and mycobacteria constructed from an E. cou plasmid containing the ColE1 origin, a Ptl fragment of 2.6 Kb of the bacteriophage D29 and kanamycin resistance gene, which successfully transformed Mycobacterium smegmatis. By erroneously reporting that the transformation was achieved due to an origin of replication of the D29 fragment, David and others did not teach the use of a minimum functional component of D29 comprising a binding site and an integrase gene. With respect to Mycobacterium diseases, the advances made in the area of genetic tools and vaccine strategies included: Isolation, characterization and sequencing of plasmid pAL5000 of Mycobacterium; the identification of the kanamycin resistance gene as a selection marker for Mycobacterium; the development of the first promiscuous vectors of Escherichia coli (E. coli'Mycobacterium), the construction of genomic libraries of M. Tuberculosis and M. Leprae, and the expression of Mycobacterium DNA in E. 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. others, 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 ", summary U6 of the 88th annual meeting of the American Society of 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; Labid i A. January, 1986, "Contribution to aplan of action for research in molecular biology and immunology of mycobacteria", doctoral thesis, University of Paris and Pasteur Institute, Paris, France). These advances have opened the way for the application of recombinant DNA technology to Mycobacterium (Lazraq and others, 1990. Conjugative transfer of a shuttle plasmid from Escherichia coli to Mycobacteirum smegmatis, FEMS Microbiol, Lett 69, 135-138; } Konicek and others. 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 of Microbiology Washington, D.C.). The Mycobacterium expression vectors resulting from these advances are not suitable for the development of vaccines 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 by microgram of vector DNA), 2) the vectors lack multiple cloning sites, 3) the protocols for the transformation of mycobacteria with these expression plasmids result in an inefficient transformation, 4) the spectrum of mycobacteria transformed by the vectors are restricted because the transformation depends on the host, and 5) the current expression plasmids do not transform mycobacteria in a stable manner. Therefore, suitable Mycobacterium expression vectors that can provide efficient transformation and stable expression of multiple protective immunogens in mycobacteria are required. We have now found suitable antigen delivery systems using non-pathogenic Mycobacterium strains, cloning vectors and Mycobacterium expression vectors that contain protective immunogens that specifically stimulate a cell-mediated immune response by inducing TH-1, O cells. cytotoxic T lymphocytes, and provide a consistent and prolonged immunity to intracellular pathogens.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates a sequence of the origin of replication in E. coli (695 bp). The underlined base indicates the point of replication. Figure 2 illustrates a sequence for the kanamycin gene (932 bp). The underlined sequences are in the order 5 'to 3': the region (-35) for the gene, the region (-10) for the gene, the region of the ribosomal binding site for the gene, the start codon (ATG ) and the stop codon (TAA). Figure 3A illustrates a sequence of the origin of replication of pAL 5000 (1463 bp) obtained by analysis with restriction enzymes. 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. Fotuitum plasmid, pAL 5000", Plasmid 27: 130-140). The underlined sequences indicate in order 5 'to 3': the position of the primers towards the 5 'end (Fc, Fi, F2 and F3), and towards the 3' end (R4, R3, R2, R1 and Rc) used in the PCR analysis, respectively. Figure 3B illustrates a sequence of the origin of replication of pAL 500 (1392 bp) obtained after PCR analysis. The superscript numbers 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 order 5 'to 3': the position of the primers towards the 5 'end (F1, F2 and F3) and towards the 3' end (R, R3, R2 and R1) used in the analysis of PCR, respectively. Figure 4A illustrates a sequence of the binding site (attP) and the integrase gene (inf) of mycobacteriophage D29, obtained by restriction enzyme 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 order 5 'to 3': the position of the initiators towards the 5 'end (Fc, F1, F2, F3 and F4) and towards the 3' end (R, R3, R2 , R1 and Rc) used in the PCR analysis, respectively. The underlined sequences not delimited by numbered nucleotides indicate in order 5 'to 3': the binding site (attP), the region (-35) for the gene (int), the region (-10) for the integrase gene (int), the ribosomal binding site for the integrase gene (inf) and the start codon (ATG) for the integrase gene (in.) The stop codon for the integrase gene (int) is the
TGA1531_ Figure 4B illustrates a sequence of the binding site (attP) and integrase gene (int) of 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 order 5 'to 3': the position of the primers towards the 5 'end (Fc, Fi, F2, F3 and F) and towards the 3' end (R4, R, R2 , Ri and Rc) used in the PCR analysis, respectively. The underlined sequences not delimited by numbered nucleotides indicate in order 5 'to 3': the binding site (attP), the region (-35) for the gene (int), the region (-10) for the integrase gene (int), the region of the ribosomal binding site for the integrase gene (int), and the start codon (ATG) for the integrase gene (int). The stop codon for the integrase gene (int) is TGA1537 Figure 4C illustrates a sequence of the binding site (attP) and the integrase (int) gene of mycobacteriophage D2g, 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 order 5 'to 3': the position of the inkers towards the 5 'end (Fc, Fi, F2, F3 and F4) and towards the 3' end (R, R3, R2 , Ri and Rc) used in the PCR analysis, respectively. The underlined sequences not delimited by numbered nucleotides indicate in order 5 'to 3': the binding site (attP), the region (-35) for the gene (inf), the region (-10) for the integrase gene (int), the region of the ribosomal binding site for the integrase gene (inf) and the start codon (ATG) for the integrase gene (inf). The stop codon for the integrase gene (nt) is TGA1537. Figure 5 illustrates a sequence for the kanamycin gene promoter (102 bp) and the first ATG codon. The underlined sequences are in order 5 'to 3': the region (-35) for the gene, the region (-10) for the gene, the region of the ribosomal binding site for the gene and the start codon (ATG ). Figure 6 illustrates a sequence of the fragment of pAL 5000 containing the open reading frame ORF 2 (2096 bp). The superscript numbers indicate the position of the nucleotides in the published sequence of pAL 5000 (Labidi et al., 1992 Plasmid 27: 130-140). The underlined sequence (GGATCC) is the only Bam Hl site that is encompassed by the ORF 2 promoter. Figure 7 is a genetic map of a representative genetic transfer system, where "C-ter / anch / seq" = sequence End Anchor C; "MCS / express" = multiple cloning site for expression; "N-ter / lead / seq" = N-end guide sequence; "Myco / Prom" = Mycobacterium promoter; "Rep / Inted / yco" = origin of replication of Mycobacterium or phage binding site and integrase gene (either one or the other but not both are present in a certain vector); "MCS / gene / clone" = multiple cloning site for general cloning; "univ / select / mark" = universal selection marker; and "ori / E.co //" = origin of replication of E. coli.
DETAILED DESCRIPTION OF THE INVENTION
The therapeutic or prophylactic vaccines of the present invention combine a protective immunogen with one or more strains of Mycobacterium that act as a delivery system and an adjuvant, preferably in addition to cytokines and suitable chemotherapy. The rational is that the Mycobacterium cells will be ingested by macrophages and will remain inside the macrophage, blocking the mechanism of elimination of the macrophage and synthesizing at the same time the protective immunogen. The immunogen will be processed and presented on the surface of the macrophage cell to T cells, resulting in the activation of TH-1 cells and a cell-mediated immune response that is protective against intracellular disease. One aspect of the present invention uses an antigen delivery system in the form of a non-pathogenic Mycobacterium strain to provide products that combine Mycobacterium adjuvants, non-toxic immunoregulators, nontoxic immunostimulatory protective immunogens specific for a variety of diseases and non-toxic amounts. of cytokines that strengthen the TH-1 pathway. Preferably, the present invention uses a protective immunogen delivery system in the form of a non-pathogenic 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 granulomas. Said immunogens may be protein antigens or other immunogenic products produced by culturing and killing the diseased cell or infectious microorganism, separating and purifying the immunogens from natural or recombinant sources, or by cloning and expression in a Mycobacterium delivery system of the genes that code for 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, ovaries, esophagus, oral and pharynx, kidney, non-Hodgkin's , brain, cervix, larynx, myeloma, corpus uteri, melanoma, thyroid, Hodgkin's and testicles; 2) bacterial infections including but not limited to mycobacteriosis (eg, tuberculosis and leprosy), Neisseria infections (eg, gonorrhea and meningitis), brucellosis, plague, spirochetosis (eg, trypanosomiasis, Lyme disease and tularemia), rickettsiosis (eg typhus, Rickettsia rash and anaplasmosis), chlamydiosis (eg, 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, infectious anemia in equines, avian influenza, heartworm and flea allergy in canines; and 8) other diseases including but not limited to leukemia, multiple sclerosis, bovine spongiform encephalitis (BSE), and myoencephalitis (ME). These antigens can be used individually or in combination in a 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. The protective endogenous lipid immunogens that are preferred for the treatment of tuberculosis, leprosy and other mycobacterials include but are not limited to complex lipid heteropolymers such as phenolic glycolipids PGL I and PGL, Tb1, the sulpholipid SL I, the diacyl trehalose DAT and the lipo-oligosaccharide LOS. These immunogens lipids 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 an expression vector is used, the expression system must provide the necessary genes coding for the enzymes necessary to modify the lipid to a stage where it is immunogenic. The mycobacterial adjuvant of the present invention is one that reinforces the immune response of TH-1, and preferably down-regulates the TH-2 response. Mycobacterium strains are characterized by their lack of pathogenicity to mammals and their ability to be ingested mammalian macrophages. The Mycobacterium strains of the present invention can be live or dead after their administration. When the vaccines of the present invention are administered to immunocompromised patients, only killed Mycobacterium strains are used. Preferably, the Mycobacterium strains can be obtained from American Type Culture Collection (Rockville, MD). One or more types of Mycobacterium species can be used in the preparation of a vaccine. Examples include but are not limited to Mycobacterium vaccae, Mycobacterium gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium chitae, Mycobacterium duvalii, Mycobacterium flavescens, Mycobacterium nonchromogenicum,
Mycobacterium neoaurum and Mycobacterium bovis BCG non-pathogenic. M. bovis BCG and M. gastri are the only known Mycobacterium species that have precursors to produce lipids of M. tuberculosis and M. leprae; therefore, M. gastri should be used if the exogenous lipid precursors are to be expressed in a TB or leprosy vaccine. 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 use either a Mycobacterium strain or several 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 are associated 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). In addition, the vaccine of the present invention can also be administered in combination with appropriate chemotherapy for the treatment of patients with active diseases. If a live Mycobacterium strain is used as an adjuvant, appropriate chemotherapy should be selected that does not interfere with the adjuvant function of live Mycobacterium. Examples of suitable concomitant chemotherapy is Taxol-R for the treatment of cancer or protein inhibitors for the treatment of AIDS. The protective immunogens, cytokines and concomitant chemotherapy can be produced separately in synthetic or recombinant form, purified by any conventional technique. They can be used in parallel with, mixed with, or conjugated to living or dead Mycobacterium cells of interest.
Genetic transfer system
The genetic transfer system of the present invention comprises cloning vectors in which the genes of interest are cloned and the transformation technique is used to introduce and express the recombinant molecules in the delivery system. The above cloning vectors that have been used in Mycobacterium species include the extra fortheromic plasmid of M. fortuitum pAL 5000 (Labidi et al., 1992. "Cloning and DNA sequencing of the Mycobacterium fortuitum var fortuitum plasmid, pAL 5000", Plasmid 27 : 130-140) that replicates extrachromosomally and 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 broad-spectrum virulent phage capable of efficiently infecting and reproducing itself in cultivated Mycobacterium species and binding to non-cultivated M. leprae. New cloning vectors have now been developed which are generally made from any replication origin (s) or integrative system (s), selection marker (s) and multiple cloning site (s) (MCS). The cloning vectors are comprised of the minimum functional sizes of several components, including the following components: the E. coli replicon, the kanamycin selection marker, the origin of replication of pAL 5000 and the binding site of D29 (attP) ) and integrase gene (inf). Using conventional deletion techniques, the coding region for component has been reduced to the point where the additional loss of base pairs resulted in the loss of function, hence the minimum functional size designation. The sequences for each minimal functional component are given as follows: origin of replication in E. coli (695 bp) as SEQ ID NO: 1 and Fig. 1; kanamycin gene (932 bp) as SEQ ID NO: 2 and Fig. 2; origin of replication in pAL 5000 (1463 bp) obtained by analysis with restriction enzymes as SEQ ID NO: 3 and Fig. 3A; origin of replication in pAL 5000 (1382 bp) obtained after PCR analysis as SEQ ID NO: 4 and Fig. 3B; D2g binding site of mycobacteriophage and integrase gene (1631 bp) obtained by restriction enzyme analysis as SEQ ID NO: 5 and Fig. 4A; mycobacteriophage D29 binding site and integrase gene (1413 bp) obtained after PCR analysis as SEQ ID NO: 6 and Fig. 4B; and mycobacteriophage D29 binding site and integrase gene (1374 bp) obtained after PCR analysis as SEQ ID NO: 7 and Fig. 4C. It is well understood in the art of deletion techniques that although the sequences identified above provide the coding regions for each minimal functional component, an additional loss of few base pairs from the minimum functional component could still result in a functional component. of the present invention. Numerous origins of replication of E. coli are commercially available and can it? used in the present invention. For example, the ColE1 replication origin of E. coli is found in most plasmid vectors designed for commercially available E. coli. Although the point of replication is usually indicated for these vectors, the smallest fragment that can support efficient replication in E. coli or has been specified to date. Using the commercially available plasmid vector pNEB 193 (Guan C, New England Biolabs Inc. USA, 1993) as starting material, it has now been determined through deletions with restriction endonuclease, cloning and transformation analysis that the most fragment of DNA small that can support efficient ColE1 replication in E. coli is limited to a sequence of 695 bp given in SEQ ID NO: 1 and Fig. 1. This origin of replication of E. coli at minimum functional size has been successfully used in the construction of cloning vectors of E. coli and promiscuous vectors of E coli-Mycobacterium of the present invention. Although a variety of selection markers is available for the selection of transformed cells and can be used in the present invention, the 1489 bp Streptococcus faecalis gene coding for kanamycin resistance 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. Instl. Pasteur / Microbiol. 136B, 209-215). Although 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 been established. It has now been found that the minimum functional sequence for this gene measures approximately 932 bp as shown in SEQ ID NO: 2 and Fig. 2. The minimal functional size kanamycin gene described herein has been successfully used in the construction of cloning vectors of E. coli and promiscuous vectors of E. coli-Mycobacterium of the present invention. Vectors containing a plasmid origin of replication are not normally integrated into the chromosome of the host cell. In this way, they are extrachromosomal vectors. The replication and maintenance in Mycobacterium strains of the extrachromosomal vectors developed in this study are supported by the origin of replication of plasmid pAL 5000 of Mycobacterium fortuitum (Labidi et al., 1984. "Plasmid profiles of Mycobacterium fortuitum complex isolates", Curr. Microbiol 11, 235-240). Plasmid pAL 5000 is the most conscientiously studied Mycobacterium plasmid, and has been used worldwide to develop vectors for gene transfer in Mycobacterium (Falkinham, III, JO and JT Crawford, 1994. Plasmids, pp. 185-198. Barry Bloom (ed.), Tuberculosis: Pathogenesis, protection and control, American Society for Microbiology, Washington, DC). functional analysis of plasmid pAL 5000 has indicated the location of two open reading frames encoding 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 losing its function (SEQ ID NO: 3 and Fig. 3A) . The 1247 bp fragment extending from nucleotide 4439 to nucleotide 863 has been found, and the 1315 bp fragment extending from nucleotide 4587 to nucleotide 1079 does not support replication in Mycobacterium (SEQ ID NO: 3 and Fig. 3A). In this way, the role of sequences extending from nucleotide 4439 to nucleotide 4587, and from nucleotide 863 to nucleotide 1079 has now been investigated. In the absence of suitable restriction sites in these two areas of the pAL 5000 sequence , sets of primers have been designed towards the 5 'and 3' end that span the two areas. Then PCR is used to amplify the different fragments that are subsequently cloned into an E. coli replicon containing the kanamycin gene. Using the PCR analysis technique, the origin of replication of minimal functional pAL 5000 has been reduced to a 1382 bp fragment extending from nucleotide 4468 to nucleotide 1027 as shown 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 a part of the 51 bp sequence extending from nucleotide 4468 to nucleotide 4518 and the 55 bp sequence extending from nucleotide 973 to nucleotide 1027 could also not be necessary for replication. This origin of replication of pAL 5000 of minimal functional size described herein has been successfully used in the cloning vectors of Mycobacterium and in the construction of the promiscuous vectors of E. coli-Mycobacterium of the present invention. Vectors can also include a phage binding site (attP) and its accompanying integrase gene. A preferred embodiment of the present invention comprises the binding site (attP) and the integrase (inf) gene of mycobacteriophage D29 (Forman et al., 1954. Am J Public Health 44: 1326-1333). Phage D29 is a broad-spectrum virulent phage capable of infecting cultivated Mycobacterium species and of reproducing itself efficiently. To develop integrative vectors, a map of this binding site (attP) and integrase gene (int) has been determined by constructing a set of hybrid plasmids containing overlapping fragments of the D29 genome. The recombinant plasmids were then electroporated into Mycobacterium strains placed on plates of LB medium containing 50 ug / ml kanamycin. A plasmid containing a 2589 bp fragment generated Mycobacterium transformants. The 2589 bp fragment was isolated and analyzed further. After establishing its restriction map, another set of hybrid plasmids containing overlapping segments of the 2589 bp fragment was constructed. These recombinant plasmids were electroporated in the Mycobacterium strains and then placed on plates with selective media. The smallest fragment still capable of generating kanamycin-resistant Mycobacterium transformants was isolated and sequenced using a double-stranded plasmid template and sequenase version 2.0 (USB, Cleveland, Ohio, USA). Sequence analysis indicated that the size of the fragment was 1631 bp, which comprised the phage binding site (attP), the gene promoter and the fat gene (int) from 5 'to 3'. (SEQ ID NO: 5 and Fig. 4A). Subsequent deletion studies were carried out with reference to the 1631 bp fragment. A 1413 bp fragment originating from base pair 1 19 to 1531, illustrated in Figure 4B, produced a high transformation efficiency. Additional deletion studies resulted in a fragment of 1374 bp that originated from base pair 158 through 1531, illustrated in Figure 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 integration efficiency and stability. It is believed that a part of the 39 bp sequence extending from nucleotide 119 to nucleotide 157 might not be necessary for integration. These (AttP), (inf) of D29 and the above sequence as described above are the smallest phage DNA fragment used so far in the construction of integrative Mycobacterium expression vectors and integrative promiscuous vectors of E. coli -Mycobacterium. The MCS is a synthetic fragment of DNA that contains the recognition sites for certain restriction enzymes that do not cut into the vector sequence. The choice of enzymes that will be included in the MCS is based on its frequent use in cloning and its availability. Representative enzymes include SamH I, EcoR V and Pst I. From these minimal functional components, e have developed cloning vectors that maximize the capacity for several cloning sites. Preferably, the cloning vectors comprise each component in its minimum functional size. For example, extrachromosomal cloning vectors have been constructed by assembling the minimal functional fragments for the origin of replication of E. coli, the origin of replication of pAL 5000, the kanamycin gene and the MCS. Exemplary integrative cloning vectors have the same structure, except that the origin of pAL 5000 is replaced by 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 approximately 3 Kb and a transformation efficiency of approximately 108. Each vector has a theoretically unlimited cloning capacity and is capable of transforming species of Mycobacterium Each cloning vector is presented in Table I. Figure 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.
In addition, 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 minimum functional components are used in each cloning vector is ultimately governed by the application of the vaccine and the maximum transformation size. For example, an integrative cloning vector may contain the minimum functional component for the binding site and integrase gene while the selection marker is larger than its minimum functional size. Such an arrangement may originate because the cloning vector contains only one site to clone a protective immunogen, thereby allowing other components of the vector to vary in size as long as the vector is small enough to allow efficient transformation in Mycobacterium cells. Preferably, the present invention uses a promiscuous vector of E. coli-Mycobacterium constructed by applying various recombinant DNA techniques. The constructed vector can efficiently be transformed into an E. coli or Mycobacterium host, allowing the selected mycobacterial genes to be cloned and expressed exponentially. Preferably, the promiscuous vector of E. coli-Mycobacterium uses a selection marker that can be expressed in both genders. A promiscuous vector comprises a kanamycin selection marker, an origin of replication for E. coli and an origin of replication for plasmid pAL 5000 of Mycobacterium. Another promiscuous vector comprises a kanamycin selection marker, an origin of replication for E. coli and a binding site and integrase gene of bacteriophage D29. Each component of the promiscuous constructed vector has been reduced to its smallest functional size, thus increasing its efficiency of cloning and transformation. 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
Strains of Mycobacterium have been successfully transformed by electroporation, (Labidi et al., 1992. "Cloning and DNA sequencing of the Mycobacterium fortuitum 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 described vector designs, culture medium and transformation technique have significantly improved the transformation efficiency for species of Mycobacterium and have taken it for the first time to a level comparable to that obtained with E. coli. It has been found that integrative vectors containing the binding site (attP) and the fattening gene (int) of phage D29 are integrated into the chromosomes of their hosts in a region complementary to the region (attP). This region is the bacterial binding site (attB) and is located between the genes encoding the proline transfer RNA (tRNAPr0) and the glycine transfer RNA (tRNAGly)
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Indicates the marker used to select transformants, Ap, Tc and Km were used at 100 ug / ml, 15 ug / ml and 50 ug / ml, 00 respectively. Indicates the remaining functional replication origin and / or integration in the vector. It indicates the transformation efficiencies obtained by electroporation for E co // 'and Mycobacterium, respectively. The efficiencies for D29 and pAL 5000 were arbitrarily set at 100% when these vectors were used with their host strains respectively. It indicates ORFs that are not those involved in replication, expressed as pAL 5000 in minicells of E. coli. Abbreviations: PM = Molecular Weight, pb = base pairs, Ap = Ampicillin, Tc = Tetracycline, Km = canamlcina, ORF = Open Reading Frame.
Expression vectors
The expression vectors of the present invention are made by inserting functional promoters of plasmid or chromosomal origin into the cloning vectors that serve as base structures. Expression vectors are designed to carry and express selected genes in the delivery system. They contain in their structures the genetic information necessary for their self-replication in the cytoplasm, or their integration in the chromosome of the host. They provide the promoter and regulatory sequences necessary for 1) gene expression, and if necessary, 2) the secretion of the gene product out of the cytoplasm to the structure of the cell membrane or to the extracellular environment. Although the kanamycin gene is a selection marker that is preferred 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 strains of E. coli and Mycobacterium, 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. For the first time, the use of a kanamycin promoter to construct promiscuous vectors of E. coli-Mycobacterium expression is reported. Another expression vector that is preferred in the present invention used the open reading frame (ORF) 2 promoter of pAL 5000. An open reading frame (ORF 2) that encodes a 60-65 KDa protein in E-cell mini-cells. coli was identified in plasmid pAL 5000. To map the promoter region of this ORF, the 2096 bp fragment containing this open reading frame was isolated (SEQ ID NO: 9 and Fig. 6). Through deletions with restriction endonuclease, cloning and transformation analysis, a set of hybrid plasmids containing overlapping segments of the 2096 bp fragment was constructed. These recombinant plasmids were electroporated in DS410 of E. coli. Minicells were prepared from transformants and proteins encoded with plasmids were analyzed as indicated in Example 4. The ORF promoter was found in the sequence spanning the unique Bam Hl site in the fragment indicated in Figure 6.
The products of the invention are administered by injection applied intradermally or by other routes (eg, oral, nasal, subcutaneous, intraperitoneal and intramuscular) in a volume of approximately 100 microliters containing 107 to 1011 of live or killed Mycobacterium cells. recombinant, or the same amount of non-recombinant Mycobacterium cells mixed with, or conjugated to, predetermined amounts of the exogenous antigens, cytokines, and / or drugs. If the products are 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 used separately, they can be administered in any order, in the same or in different places, and using the same or different routes. The invention takes into consideration that the products are designed for use in humans or animals, and therefore must be effective and safe with or without some additional pharmaceutical formulation that could add other ingredients.
In summary, the cloning and expression vectors that are preferred in the present invention comprise a promiscuous vector of E. coli-Mycobacterium that contains the following: an origin of replication for both E. coli (replicon for E. coli) and Mycobacterium (origin of replication for pAL 5000), a marker of resistance to kanamycin, multiple cloning sites, promoters and regulatory sequences for the secretion of gene products outside of bacteria and for their insertion into the cell membrane, and the site of union (att P) and integrase gene (int) of phage D29. Another type of cloning and expression vectors that are preferred contain all of these elements listed above, except for the binding site of phage D29 and the integrase gene. Multiple cloning sites allow the cloning of a variety of DNA fragments. The replicon for E. coli, the origin of replication for pAL 5000, the resistance marker to kanamycin and the attP site of D29 and the int genes have been mapped and reduced to their minimum functional sizes to maximize the cloning capacity of the vector and increase transformation efficiency. A new transformation protocol was developed so that the efficiency with which these vectors transform strains of Mycobacterium (108 transformants of Mycobacterium I μg of DNA) approaches the efficiency of transformation for E. coli. The vaccine system of the present invention has a number of advantages over current vaccines. The main advantage of such a system over current vaccines is the ability to specifically express immunogens that develop a consistent and protective immune response, that is, a prolonged activation of TH-1 cells with the concomitant activation of macrophages. Additional advantages include: 1) protective immunogens for more than one intracellular disease can be incorporated into a vaccine, 2) said genetically engineered vaccine is flexible since new technology can be easily incorporated to improve the vaccine, and 3) large amounts of immunogen can be synthesized. using a genetically engineered expression vector to induce protective immunity, 4) Mycobacterium itself acts as an adjuvant injected together with the immunogen to induce immunity, 5) the vaccine is naturally sent to macrophages because Mycobacterium infects these cells, 6) and the result will be a prolonged immunity since a strain of Mycobacterium remains alive inside the macrophages for a long time. The methodologies for carrying out various aspects of the present invention are presented below.
DNA, RNA and oligonucleotide primers
DNA and RNA were extracted and purified in Cytoclonal Pharmaceutics, Inc. Dallas, Tecas. Oligonucleotide primers were purchased from National Biosciences Inc., PIymouth, MN., Or from Integrated DNA Technologies Inc., Coralville, IA.
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 Fermentas Inc., Lithuania and TaKaRa Biomedicals Inc., Kyoto, Japan. The DNA ligase was purchased from Boehringer Mannheim Biochemica Inc., Indianapolis, IN .; Gibco-BRL Inc., Gaithersburg, MD. and New England Biolabs. The RNasa was purchased from
Prime > 3 First Inc., Boulder, CO. The deoxyribonucleotides and DNA polymerase I (Klenow fragment) were purchased from New England Biolabs. The 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 programs
The Oligo computer programs (National Biosciences Inc., PIymouth, 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 and bacteriophage strains were used from the Vaccine Program collection at Cytoclonal Pharmaceutics Inc., Dallas, TX. The 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 of E. coli strains. Accordingly, a general culture medium, hereinafter referred to as a Labidi 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 medium per liter contains: approximately 0.25% protease 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 (.) amino acid complex (approximately 0.126% aianine, 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, approximately 0.023% histidine, approximately 0.021% tyrosine, approximately 0.02% methionine, approximately 0.0 14% tryptophan and approximately 0.01% cysteine), approximately 0.306% Na2HPO4 > about 0.055% KH2PO4, about 0.05% NH4CI, about 0.335% NaCl, about 0.0001% ZnSO4, about 0.0001% CUSO4, about 0.0001% FeC, about 0.012% MgSO4) about 0.05% Tween 80 and about 0.8% glycerol (except for M. bovis), pH 7.0. A solid form of this medium can be obtained by adding 2.0% agar. Whenever necessary, this medium can be complemented with preferred selection markers and / or with special factors required for the growth of certain species, such as mycobactin for M. paratuberculosis and hemin factor X for M. haemophilium.
For the transformation, cultures were made in Labidi medium. The cultures were incubated at the appropriate temperature for each strain. The cultures in liquid media were shaken at 150 rpm on a Gyromax 730 gyratory shaker (Amerex Instruments Inc., Hercules CA). In the cultivation of Mycobacterium cells for the vaccine, cultures were made on protein-free media: [per liter: 6.0% glycerol, 0.75% glucose, 0.4% aspargin, 0.25% Na2HPO4, 0.2% citric acid , 0.1% KH2PO4, 0.05% ferric ammonium citrate, 0.05% MgSO4, 0.02% Teween 80, 0.0005% CaCl2, 0.0001% ZnSO4 and 0.0001% CuSO4 at a final pH of 7]. Whenever necessary, this means can be complemented with the selection markers and / or the necessary growth factors. For the routine culture of E. coli strains, the bacteria were grown in Luria Broth medium (LB) [per liter of medium: 1% tryptone, 1% NaCl and 0.5% yeast extract in distilled or deionized water ] The solid form of the LB medium was obtained by adding 2.0% agar to the above formula. When necessary, the medium was supplemented with selection markers. The cultures were incubated at 37 ° C except if the culture required otherwise. The cultures in liquid media were shaken at 280 rpm on a Gyromax 703 rotary shaker (Amerex Instruments Inc., Hercules, CA). The spheroplasts were prepared from Mycobacterium cultures as previously described (Labidi, et al. 1984, Curr. Microbiol. 11, 235-240). Briefly, the spheroplast solution [per ml of Mycobacterium culture (14 mg of glycine, 60 μg of D-cycloserine, 1 mg of lithium chloride, 200 μg of lisizime and 2 mg of EDTA)] was added to the cultures of Mycobacterium in exponential growth phase, and incubation was continued for three generations to induce the formation of spheroplasts. The spheroplasts were subsequently collected by centrifugation for 20 min. at 300 rpm, at 4 ° C, washed and resuspended in the storage solution of spheroplasts [per liter, (6.05 gm of tris, 18.5 gm of EDTA, 250 gm of sucrose and the pH was adjusted to 7)].
Cultivation of Mvcohacteriu for adjuvants
The adjuvants were made from harvested Mycobacterium cells after preferably cultivating the corresponding Mycobacterium strains in a protein-free liquid medium. The medium was inoculated and incubated at the appropriate temperature. The culture was stirred at 150 rpm for adequate aeration. The ODßoo of the crop was monitored daily to determine when the crop reached the stationary phase. In the stationary phase, the number of cells per milliliter was determined through serial dilutions and plating each dilution in triplicate. The culture was centrifuged in a sterile manner for 30 minutes, at 5000 rpm, at 4 ° C. The pelleted cells were washed twice with sterile distilled water cooled with ice and pelleted as indicated above. The pellet was resuspended in pyrogen-free saline solution (for injection only), to form a cell suspension varying from 108-1012 cells per ml. The Mycobacterium cell suspension was provided in suitable multi-dose containers and used either live or dead. The methods that are preferred to kill Mycobacterium cells include the use of chemicals, radiation or intense heat (autoclave for 30 minutes at 104-124 kPa at 120-122 ° C).
DNA and RNA preparations
Plasmid DNA from E. coli strains was prepared, as described in the previous text (Labidi, et al., 1984. "Plasmid profiles of Mycobacterium fortuitum complex isolates7 Curr. Microbiol., 11, 235-240) 300 μl of spheroplasts microcentrifuged in another preferred method of the invention The pellet was resuspended in 360 μl of freshly prepared SI solution [250 mM tris (pH7), 50 mM EDTA (pH8), 50 mM glucose and 2.5 μl / ml losozyme]. 240 μl of S II [10% SDS (pH7)] was added and the pellet was incubated at 65 ° C for 15 minutes Subsequently, 300 μl of S III [7.5 ammonium acetate (pH 7.5) or NaCl 5 was added. M or 3M potassium acetate (pH 5.2) or 3M sodium acetate (pH 5.2)] and the pellet was incubated on ice for 15 minutes and microcentrifuged for 15 minutes at 0 ° C to 14 Krpm. 2.5 μl of proteinase K (20 mg / ml) and incubated at 37 ° C for 15 minutes The aqueous phase was extracted three times by adding 250 μl of phenol regulated in pH and 250 μl of chloroform / isoamyl alcohol (24: 1, v / v) each time.
The pellet was vortexed, microcentrifuged for 15 minutes at 14 Krpm at room temperature and the aqueous phase was recovered. To the last aqueous phase, 1 ml of isopropanol was added, briefly vortexed and microcentrifuged for 10 minutes at 14 Krpm at room temperature. The pellet was dried at 37 ° C for 5 minutes and the DNA was dissolved in 50 μl of sterile distilled water. Total DNA from Mycobacterium strains was prepared as described above (Labidi, A., 1986). Another method that is preferred is to add sterile glass spheres to the pellet obtained from 20 ml of spheroplasts. The pellet vigorously vortexed to have a homogeneous suspension. The suspension was treated with 20 ml of SI, 8 ml of Sil and 14 ml of SIII. The aqueous phase was extracted several times, each time with 10.5 ml of a pH-regulated phenol / chloroform / isoamyl alcohol solution. The total DNA was precipitated with a volume of 0.6 of isopropanol, then dissolved in a gradient of cesium chloride and ethidium bromide. The gradient was centrifuged and treated according to techniques that are well established in the art. The plasmid DNA was separated after the chromosomal DNA. Total RNA from E. coli strains containing the appropriate plasmids and the application was prepared in a preferred two-step protocol. A crude preparation of total RNA was made using the protocol provided with the "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 "Qiagen Total RNA Isolation" kit (Qiagen Inc, Chatsworth, CA). The combination of the two systems efficiently separated the total RNA from other contaminating nucleic acids.
Preparation of electro-competent cells
Strains of Mycobacterium can be transformed only by electroporation (Labidi, A., 1986). Therefore, bacterial cells must become electro-competent before being subjected to this procedure. The E. coli strains were made electro-competent following the protocol provided with the BRL Cell Porator apparatus (BRL Life Technologies, Gaithersburg, MD). For the Mycobacterium strains, a single culture colony of Mycobacterium was inoculated in 25 ml of Labidi medium in a 250 ml screw cap flask. The culture was stirred at 150 rpm at the appropriate temperature until OD600 reached 0.7. The culture was checked to verify contamination by staining. If there was no contamination, a second culture was started by inoculating 50 μl of the first culture in 200 ml of Labidi medium in a 2000 ml screw-cap flask. The culture was stirred at 150 rpm at the appropriate temperature until OD600 reached 0.7. The culture was chilled 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 in 31 ml of 3.5% sterile cold glycol and centrifuged (5 Krpm) for 10 minutes at 4 ° C. The second pellet was suspended in 12 ml of 7% sterile cold glycerol and centrifuged (3 Krpm) for 10 minutes at 4 ° C. The third pellet was suspended in 6 ml of sterile 10% cold glycerol and centrifuged (3 Krpm) for 10 minutes at 4 ° C. The fourth pellet was suspended in a minimum volume of approximately 2.0 ml of sterile cold giicerol at 10.0%, aliquots of fractions of 25.0 μl were formed and then used immediately or stored at minus 80 ° C.
Transformation
The electroporation technique was applied to strains of E. coli and
Mycobacterium Electro-competent E. coli or Mycobacterium cells (25 μl) were mixed with vector DNA (10 ng in 1 μl), incubated on ice / water for 1 minute and transferred to an electroporation vessel (0.15 cm space). ). The electroporation was carried out with a BRL Cell Porator cat. 1600 equipped with a Voltage Booster Unit cat. 1612 (BRL Life Techonologies, Gaithersburg, MD). The Voltage Booster Unit was set at a resistance of 4 kilohms and the Power Supply Unit was set at a capacitance of 330 microfarad, a fast charge speed index and a low ohm mode to eliminate the additional resistance. Once the containers were in the security camera, the "load / arm" button was set to "charge", the "top button" was held down until the voltage of the capacitors deployed in the Power Supply Unit it will reach 410 volts for strains of E. coli and 330 volts for strains of Mycobacterium. The "charge / arm" button was set to "arm" and the voltage of the capacitors dropped to 400 volts for the E. coli strains and 316 volts for the Mycobacterium strains. The "activate button" was pressed to provide approximately 2.5 kilovolts for strains of E. coli and Mycobacterium, respectively. These voltage values were displayed in the Voltage Booster Unit. Each voltage value corresponds to 2.5 kilovolts that divided by 0.15 cm give 16.66 kilovolts / cm through the container space for the E. coli strains and 1.9 kilovolts divided by 0.15 cm they give 12.66 kilovolts / cm through the container space for the Mycobacterium strains. The electroporated cells of each sample were immediately collected with 1 ml of Labidi medium, transferred to a 15 ml round bottom falcon tube (Becton Dickenson Inc., Lincoln Park, NJ) and incubated for a generation time under temperature. and suitable agitation conditions. The cultures were diluted 1: 102 to 1: 105 in sterile distilled water. Diluted cultures were plated (100 μl) in triplicate on LB containing kanamycin and Labidi media, respectively. The plates were incubated at suitable temperatures until the colonies were visible and easy to count. Counted numbers were averaged and used to calculate transformation efficiencies. A positive control and a negative control were included for each species and each experiment.
DNA sequencing
DNA was sequenced using a double-stranded plasmid template and the protocol provided with the "Sequenase Version 2.0" device (USB, Cleveland, Ohio, USA). The sequence was analyzed by computer using the Mac Vector program (Oxford Molecular Group Inc., Campbell, CA).
In vitro analysis of the stability of the vector
Individual Mycobacterium transformant colonies were grown to saturation on Labidi medium containing kanamycin (50 μg / ml). The number of generations required to reach saturation is significantly different between mycobacteria of slow and rapid growth. The saturated cultures were diluted to 1: 102 and 1: 106 in Labidi medium free of antibiotics. The 1: 106 dilution was plated immediately (0.1 ml per plate) on Labidi medium containing antibiotic to determine the number of kanamycin-resistant colonies per my culture at the start of the experiment. For calculation reasons, the number of kanamycin-resistant colonies per my culture was considered as 100%. 0.1 ml fractions of the 1: 102 dilution were used to inoculate fresh antibiotic-free Labidi medium and allowed to grow to saturation. This procedure was repeated for six months. The number of kanamycin resistant colonies was determined each time. The proportion of antibiotic-resistant colonies in the culture after a period of six months was found to be 96%.
DNA and RNA transactions
The DNA and RNA were treated with the appropriate enzymes respectively, as recommended by the manufacturers.
Integration analysis
The integration of the vectors containing the binding site (attP) and integrase gene (int) of the mycobacteriophage D29 in the chromosomes of the host strains of Mycobacterium was analyzed by the preparation of plasmid DNA and by hybridization using the cloned fragment of the genome. of D29 as a probe.
Mini-cell analysis
The minicell cell analysis was carried out using DS410 from E. coli, which is a mutant strain of E. coli (MinA and MinB). This mutant splits asymmetrically and produces normal cells and small non-nucleated cells called mini-cells. Minicells are easily separated from normal cells by their differential sedimentation on a sucrose gradient. If the mini-cell-producing strain contains a multiple-copy plasmid, each of its mini-cells will not have a chromosome but will carry at least one copy of the plasmid. Since mini-cells 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. The Nutrient Broth broth is the medium used in this technique. The preparation of the minicells originated with the preparation of electrocompetent cells of DS410 from E. coli with the appropriate recombinant plasmids. Each clone containing plasmid is grown overnight in 400 ml of NB having the appropriate selection markers. A clone of the untransformed DS410 was cultured on 400 ml of NB alone to serve as a control. Three gradients of 35 ml of sucrose (10-30% w / v) were prepared per clone using M9-mm-S [per liter of medium: 200 g of sucrose, 100 ml of 10X I-M9-mm sterile, 10 ml of CaCl2 at 10 mM sterile and 10 ml of MgSO4 at 100 mM sterile]. The gradients were then placed at minus 70 ° C for at least one hour or until the gradients were completely frozen. The gradients were then placed at 4 ° C overnight to allow the gradient to thaw and settle. The bacterial cultures were centrifuged for 5 minutes at 2 Krpm at 4 ° C. The supernatants were then centrifuged for 15 minutes at 8 Krpm at 4 ° C.
Each pellet was resuspended subsequently in 6 ml of M9-mm [per 10X liters of medium: 400 mM of NaH2PO4, 200 mM KH2PO4, 80 mM NaCl and 200 mM NH4CI)]. Each 3 ml of cell suspension is placed on a sucrose gradient. The gradients are then centrifuged for 18 minutes at 5 Krpm at 4 ° C. The upper third of the band of transparent white minicellules is recovered from each gradient. An equal volume of M9-mm is added to each tube and centrifuged for 10 minutes at 4 ° C. Each pellet is resuspended subsequently in 3 ml of M9-mm and the suspension is placed on the last gradient and centrifuged for 18 minutes at 5 Krpm at 4 ° C. The upper third of the band of the white transparent minicellules is recovered and the optical density is read at 600 nm. The number of cells in the mini-cell preparation was calculated using the equation of 2 ODßoo, which is equal to 10 10 minicells per ml. Preferably, the level of whole cell contamination in the preparation of minicells is determined. The mini-cell suspension is centrifuged for 10 minutes at 10 Kfm at 4 ° C and resuspended in M9-mm-G [per 100 ml of medium: 300 ml of sterile glycerol (100%), 1 ml of 10 mM sterile CaCl2. , 1 ml of sterile 100 mM MgSO and 10 ml of sterile 10X l-M9-mm]. The labeling of the proteins encoded by plasmids with d35 of methionine is achieved by placing 100 μl of minicells in the microcentrifuge 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 test medium per 100 ml of medium]. The pellet is incubated at 37 ° C for 90 minutes and 25 μCi of S35-methionine are added. The pellet is incubated at 37 ° C for 60 minutes. Add 10 μl of unlabeled MS (0.8 gm of L (-) methionine in 100 ml of distilled water] and incubate at 37 ° C for 5 minutes, micro-centrifugation for 3 minutes at room temperature, the pellet is resuspended in 50 μl of BB [per 100 ml of solution, (0.71 gm of Na2HPO4, 0.27 gm of KH2PO4, 0.41 gm of NaCl and 400 μl of MgSO4) at 100 mM sterile] and 50 μl of SDS-SB [per 10 ml of solution, ( 12.5 ml of tris at 1 M (pH 6.8), 20 ml of sterile glycerol (100%), 10 ml of 20% SDS (pH 7.2), 5 ml 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 12% SDS-polyacrylamide gel.
Initiator extension analysis
The primer extension analysis was carried out in accordance with the protocol provided with the "First Extension System" (Promega Inc. Madison, Wl).
Analysis of ribonuclease protection test
The ribuonase protection test (RPA) was carried out in accordance with the protocol provided with the "Ambion HypSpeed RPA Kit" (Ambion Inc. Austin, TX).
Amplification of DNA by polymerase chain reaction
The DNA fragments of the mycobacteriophage D29 genome and the Mycobacterium chromosomal and plasmid DNA were amplified by polymerase chain reaction using a Progene Programmable Dri-Block Cycler (Techne Inc. Princeton, NJ). The reaction mixture was subjected 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 described for the first time in this report. Examples 1-3 demonstrate the present invention in terms of the use of specific antigens in the treatment of various diseases. These examples are illustrative and are not intended to be limiting with respect to the selected Mycobacterium antigen and strain, nor with the application of the promiscuous vector of E. coli-Mycobacterium.
EXAMPLE 1
Exemplary vaccine against AIDS
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 for IL-2, gamma INF and GMCSF (Cytoclonal Pharmaceutics, Inc. Dallas, Texas) are electroporated into a recipient strain of M. aurum. The transformants are checked to verify their plasmid content. A clone containing the expected hybrid plasmid is cultured in the protein-free liquid medium. The inoculated medium is incubated at a temperature of 35 to 37 ° C. The culture is stirred at 150 rpm for adequate aeration. The ODβ of the culture is measured daily, and a growth curve is established that shows optical densities against time. As in the stationary phase, the number of cells per milliliter is determined through serial dilutions (1: 10 to 1: 1010), and plated in triplicate plates of each dilution in Labidi medium. The culture is centrifuged sterile for 30 minutes at 5000 rpm at 4 ° C. The pelleted cells are washed twice with sterile distilled water cooled with ice and pelleted as indicated above. The pellet is resuspended in pyrogen-free saline for injection only, to have a suspension of 108 to 1012 cells per ml. The Mycobacterium cell suspension is placed in suitable multi-dose vials. The product is administered by injection applied intradermally in a volume of approximately 100 ul containing 107 to 1011 recombinant Mycobacterium cells. If a deleted form of the vaccine is preferred, the cells can be removed either chemically, by radiation or by autoclaving for 30 minutes at 104-124 kPa at 120-122 ° C. If a deleted form of the vaccine is used, those antigens or cytokines that could be inactivated during the procedure are added to the product separately, or the recombinant cells are removed 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 PSA prostate cancer antigen (National Institutes of Health, Bethesda, MD), is cloned according to with the procedure given in example 1. The product is prepared and administered according to the procedure of example 1.
EXAMPLE 3
Exemplary vaccine against allergies
If the product is being used for vaccination against allergies such as reactions to the main birch pollen allergen, the gene coding for the allergen such as the birch pollen allergen Bet V1a (University of Vienna, Austria) is cloned in accordance with the procedure given in example 1. The product is prepared and administered according to the procedure given in example 1.
LIST OF SEQUENCES
(1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Cytoclonal Pharmaceutics, Inc. (B) STREET: 9000 Harry Lines BIvd., Suite 330 (C) CITY: Dallas (D) STATE: Texas (E) COUNTRY : USA (F) POSTAL CODE: 75235 (G) TELEPHONE: (214) 353-2923 (H) TELEFAX: (214) 350-9514 (I) TELEX:
(ii) TITLE OF THE INVENTION: Mycobacterium recombinant vaccines
(iii) NUMBER OF SEQUENCES: 9
(iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: Sidley & Austin (B) STREET: 717 N. Harwood, Suite 3400 (C) CITY: Dallas (D) STATE: Texas (E) COUNTRY: United States (F) ZIP CODE: 75201
(v) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM COMPATIBLE PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30
(vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION:
(vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 60/042849 (B) DATE OF SUBMISSION: MARCH 28, 1997
(viii) INFORMATION OF THE APPORTER / AGENT: (A) NAME: Hansen, Eugenia S. (B) REGISTRATION NUMBER: 31, 966 (C) REFERENCE NUMBER / CASE: 10365/05602 (ix) INFORMATION BY TELECOMMUNICATIONS: (A) ) TELEPHONE: 214-981-3300 (B) TELEFAX: 214-981-3400
(2) INFORMATION FOR SEQ. ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 695 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 1:
GTTTTTTCCAT AGOCTCCGCC CCCCTGACOA GCATCACAAA AATCGACGCT CAAGTCAGAG 60
GTGGCQAAAC CCGACAGGAC TATAAAGATA CC? GGCGTTT CCCCCTGGAA GCTCCCTCGT 120 ßCGCTCTCCT GTTCCGACCC TGCCGCTTAC COGATACCTG TCCGCCTTTC TCCCTTCSGG 180
AAGCGTGGCG CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG 240
CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG 300
TAACTATCGT CTTGAGTCCA ACCCGGTAAß ACACGACTTA TCGCCAC GG CAGCAGCCAC 360
TGQTAACAGG ATTAGCAGAO CGMOTATGT AGGCGGTGCT ACASAGTTCT TQAAOTGOTG 420
GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT 480
TACCTTCGOA AAAAGAQTTs GTAOCTCTTG ATCCGSCAAA CAAACCftCCG CTGGTAGCßß 540
TßßTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC 600
TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGQATTTT 660
GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGA 69S (2) INFORMATION FOR SEQ.ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 932 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double ( D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 2: GTTGTGTCTC AAAATCTCTG ATGTTACATT GCACAAGATA AAAATATATC ATCATGAACA 60
ATAAAACTGT CTGCTTACAT AAACAGTAAT ACAAGGGGTG TTATGAGCCA TATTCftACGG 120
GAAACGTCTT GCTCGAGGCC GCGATTAAAT TCCAACATGG ATGCTGATTT ATATGGGTAT 180
AAATGGGCTC GCGATAATGT CGGGCAATCA GGTGCGACAA TCTATCGATT GTATGGGAAG 240
CCCGATGCGC CAGAGTTGTT TCTGAAACAT GGCAAAGGTA GCGTTGCCAA TGATGTTACA 300
GATGAGATGG TCAOACTAAA CTGGCTGACG GAATTTATGC CTCTTCCGAC CATCAAGCAT 360
TTTATCCGTA CTCCTGATGA TGCATGGTTA CTCACCACTG CGATCCCCGG GAAAACAGCA 420
TTCCAGSTAT TAGAAGAATA TCCTGATTCA QGTGAAAATA TTSTTGATGC GCTGGCAGTG 480
TTCCTGCGCC GGTTGCATTC GATTCCTGTT TGTAATTGTC CTTTTAACAG CGATCGCGTA 540
TTTCGTCTCG C? CAGGCGCA ATCACGAATG AATAACGGTT TGGTTGATGC GAGTOATTTT 600
GATGACGAGC GTAATGGCTG GCCTGTTGAA CAAGTCTGGA AAGAAATGCA TAAGCTTTTG 660
CCATTCTCAC CGGATTCAßT CGTCACTCAT GGTGATTTCT CACTTGATAA CCTTATTTTT 720
GACGAGGGGA AATTAATAGG TTGTATTGAT GTTGGACGAG TCGGAATCGC AGACCGATAC 780
CAßGATCTTß CCATCCTATß GAACTGCCTC GGTGAGTTTT CTCCTTCATT ACAGAAACGG 840
CTTTTTCAAA AATATGGTAT TGATAATCCT GATATGAATA AATTGCAGTT TCATTTGATG 900
CTCGATOAGT TTTTCTAATC AGAATTGGTT AA 932 (2) INFORMATION FOR SEQ.ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1463 base pairs (B) (B) TYPE: nucleic acid (C) TYPE OF CHAIN : double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 3: TGTTCCTCCT GGTTGsTACA GGTOSTTGGß GGTGCTCGGC TGTCßCGGTT GTTCCACCAC 60
C & ß8CTCQA COGGAOAOCQ GßaO? ßTßTO CSßTTßTßOß OTCKJCCCCTC? SCC3 AATAT 120
CTG¡ACTTGGA GCTCQTQTCß GACCATACAC CßßTGATTAA TCßTGGTCTA CTACCAASCQ 180
TS? GCCXCGT COCCßACOFTA TTTOAGCAGC TCTßßCTOCC GTA TQOCCC CTOQQVAßCß 240
ACGATCTOCT CGAGGGOAT TAC GCCAAA GCCGCQCGTC GQCCCTAGGC CGCCGGTACA 300
TCORßßCßAA CCCRACROOG CT8CA? ACC TßCTQßTCßT GßAOSTSßAC CATCC? OACa 350 caacßcrcca «20
ATCßCQCC? A CßßCCACßCA CACßCftßTßT GOGOACTCAA CßCCCCTOTT CCXCOCACCO 410
AATACGCGCa GCOTAAQCCG CTCGCATACA TGSCßßCßTß CGCCGAftGßC CTTCGOCG G 540
CCOTCG? CGG COAG GCAGT T? CTC? GGCC TC? TGACCAA AAACCCCCG CACATCGCCT S00
GGOAAACGG? ATGGCTCCAC TCAGA.TCTCT ACACACT AG CCA ATCGAG GCCGAGCTCG 660
OCGCGAACAT OCCftC8CGQ CSCTGOCsTC AGCAOACCAC OTACA? MCa GCTCCOACßC 720
CßCTAOGGCß GAATTQCGCA CTßTTCSATT CCGTCAGG GTGGG CXA CßTCCCGCCC 780
TC? TGCQßAT CTACCTUCUJ ACCCOOftACG TGOACGGACT CaaCCßCOCO? TCTATS CO 840
AßTGCCACOC GCGAAACGCC OAATTCCCßT GCAACGACGT OTOTCCCGSA CCQC ACCßO 900
ACAGCGAOOt C GCG ATC GCCAAC? QCA TTTßSCßTTO GATCACAACC? AOTCßCQCA 9 (0
TTreßecßOA coaßftxcera TGCßcaccaa 1020 cocoaMoao cocftßcxaca cßcacßccGß COTCAGCCAT GGAGGCñTTG CTATQASCQA CGC3CTACAGC GACGOCTACA GCSAC8GCTA 1140 aucccßcAs ccß? Crßrcc ßra? Cacßcc aMßocacrc 1200
ACTATCCGAA CGCCACOT G TCCGGC COT GGCOCAJ30AA CGCAGCGAOT GGCTCGCCGA 1260
GCAQOCTßCA CGCG GCGAA GCATCCGCOC CTATC? CQAC QACOAGSGCC ACTCTTGOCC 1320
GCAAACGGCC AAACATTTCG GGCTGCATCT GQACACCGT AAC3CGACTCQ GCTATCGQGC 13 BO
(Waß? AAO? ß CßtßCOOCAß AACAOGAAGC OOCTCAAAA OCCCACMCO AAOCCß? Cft? 1440 CC? CCQCTQ TTCTA? CGC? ATT 1463 (2) INFORMATION FOR SEQ.ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1382 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 4: GßGTQCTCQQ C OTCßCßßT TOTTCCACCA CCAGßGCTCQ ACGGGAGAGC GSGßßAGTOT 60 TCTOACTTSG AOCTCOTGTC GGAC ATA A 120
CCQQTQATTA ATCOTSaTCT ACTACCAAQC GT AGCCACß TCßCCGACOA ATTT6AGCAG 180
CTCTQGCTGC COTACTOSCC GCTQOCAAGC GACCATCTGC TCOAGOGOAT CTACCGCCAA 240
AOCCGCGCGT CßßCCCTAGß CCG CGGTAC ATCGAGGCGA ACC AACAGC GCTGGCAAAC 300
CTßCTGOTCG TGG? CßTSßA CCATCCAOAC GCAGCOCTCC Q? ßCSCTC? ß COCCCGßSQQ 3S0
TCC ATCCGC TGC AACGC GATCGTGGGC AATCGCGCCA ACGGCCACGC ACACGCAOTO 420
TOßG ACTCA ACaCCCCIGT TCCACQ A C GAATACGCGC GGCsTAAGCC ßCTCQCATAC 480
ATSGCGGCGT GCGCCßAAßß CCTTCOOCQC GCCGTCßACO GCGACCßCAG TTACTCAGaC S40
CTCATQACCA A ??? CCCCßß CCACATCGCC TOßßAAACOß AATßßCTCCA CTCAGATCTC 600
T? CACAC CA GCCACATCGA GßCCCAGCTC QGCGCGAACA TßCCACCGCC GCGCTGQCGT 660
CAOCAOACCA COTACAAAOC QßCTCCOACa CCOCWOOßC OMATTOOGC ACTOTTCß? T 720
TCCßTCAGßT TQTQßaCCTA TCOTCCCGCC CTCATGCGOA TCTACCTQCC OACCCGSAAC 780 ßTOSACGQ? C TCGGCCGCC3C OAT TATGCC OAGTGCCACa CGCGAAACGC COAATTCCCG 140
TGCAACGACQ TQTOTCCCQ8 ACCGCTACCG GACAGCGAGG TCCGCGCCAT CGCCA? CAGC 900
ATTTßGCSIT GOATCACAAC OAOTCG OC ATXTBGOCTß ACOßßATCGT GOTCTACGAß 9 (0
GCCACACTCA OTQCGCGCCA GTC0C2CCA.TC TCßCßOAAGG GCGCAGCAGC GCOCACGGCG 1020 aocßoc? CAß TTQca8ßca CGCAAAOTCC GCOTCAGCCA TCOAGOCATT GcraiGaacQ bear
A8aCTACAG CßACßßCTAC AGCOACGQCT ACAACCGGCA GCCGACTG C CG AAAAAGC 1140 cßTOAcacac ßrccßßcTca 1200
TOGCGCAGGA ACOCAOCGAS TGS TCOCCO AGCAGGC GC ACSCGCGCOA AGCATCCGCG 1260
CCTATCACGA COACGAOGOC CACTCrTGGC CßCAAACßSC CAAACATTTC GGGCTGCATC 1320
TOGA? CCGT TAAßCGACTC GGCTATCQQG CGAGGAAAGA GCOTQCGßCA OAACAGOAAQ 1380
CO 13i2 (2) INFORMATION FOR SEQ.ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1631 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY : linear
(ii) TYPE OF MOLECULE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 5: GTGAGAßAAT CTTCACTGCA CCAGCTCCGA TCTGGTGTAC CGCCCCTCGT CTGTTGCAGC 60
AOGCGGGGGG CTTTCTTCGT CTGTCGGAGs TCGAAGGTAG CAGATGTGTC GCTGTATCCG 120
GGCAGCATAA ATGCAGßTCA TTAGTGTCGC TCTAAGGTCG CGGCCCCCTC TCGGGGATCC 180
GGTCCTCGGG CTAAAAACCA CCTCTGACCT GTGGAGCGGG CGACGGGRAT CßAACCCGCG 240
TAGCTAGTTT GGAAGTAAGG GGGTCGGCGT GTCACATTCT CCCAGCTCAG ACCCTGTTTT 300
TftQCTCTGAC CCTGTGCGAC CTTGAAGTGG ACAAAAATGC CTGTTCACGG ACACGCAAAG 360
ACGTCTGAAG GTCGCAATAA GGTCGCATTC CGGtAGCCTG TTTCGCATGG CAGCAAGACG 420
GAGAGGATGG GGATCGCTGC GGACCCAGCG CAGCGGTCGA GTGCAAGCGT CGTACGTCAG 480
CCCGATCGAC GGGCAGCGGT ACTTCGGGCC GAGßAACTAC GACAACCßOA TGGACGCCGA 540
AßCGTGGCTC GCGTCTGAGA AGCGGCTGAT CGACAACGAG GAGTGGACCC CGCCGGCCGA 600
GCGCGAGAAG AAGGCTGCGG CGAGTGCCAT CACGGTCGAG GAGTACACCA AGAAGTGGAT 660
CGCCGAGCGA GACCTCQCTß GCGGCACCAA GGATCTCTAC AGCACGCACG CTCGCAAQCG 720
GATCTACCCG GTGTTGGGCG ACACCCCGGT CGCCGAGATG ACCCCCGCCC TTGTCCGGGC 780
GTGGTGGGCC GGGATGGGTA AGCAGTACCC OACGGCACGG CGOCACGCCT ACAACGTACT 840
CCGGGCGGTC ATGAATACCG CTGTAGAGGA CAAGCTGGG TCGGAG? ACC CGTGCCGGAT 900
CGAGCAGAAG GCACCCGCTG AGCGCGACGT GGAAGCCCTC ACACCGGASG AOCTßG? CGT 960
ASTGGCCGGG GASGTGTTCß AGCACTACCG CGTGGCCGTC TACATCCTGG CGTGGACCAG 1020
CCTGCGGTTC GGTGAGCTGA TCGAGATCCG CCGCAAGOAC ATCGTGßATG ACGGCGAGAC 1080
GATGAAGCTC CGCGTGCGCC GGGGCGCGGC CCGCGTCGGC GASAAGATCß TCGTCGGCAA 1140
CACCAAGACC GTCAGOTCCA AGCGGCCGGT GACCGTGCCG CCTCACGTCG CGGCOATGAT 1200 CCGCGAGCAC ATGGCTGACC GGACGAAGAT GAACAAGGGG CCGGAAGCTC TCCTGGTsAC 1260
CACCACGCGG ßßGCAGCGGC TGTCGAAGTC TGCGTTCACT CGCTCOCTGA 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 TTACCCCCTß AAAOGCAAAA AGCCCCCCTC CCAAGGCCAT seo
ACAGCCTCAA GAGGGGGGTT TCTTGTCACT CAQTCCACAC G3TCCATTGG ATCTTGGGCß 1620
TGTAGACGAT C 1631
(2) INFORMATION FOR SEQ.ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1413 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 6:
CGGGCAGCAT AAATGCAGGT CATTAGTGTC GCTCTAAGGT CGCGGCCCCC TCTCGGGGAT 60
CCGGTCCTCs 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 GTACTTCG6G CCGAGGAACT ACGACAACCG GATGGACGCC 420
GAAGCGTGGC TCGCGTCTGA. G? AGCGGCTG ATCGACAACG AGGAGTGGAC CCCGCCGGCC 480
GAGCGCGAGA AGAAGGCTGC GGCGAGTGCC ATCACGGTCG AGGAGTACAC CAAGAAGTGG S40
ATCGCCGAGC GAGACCTCGC TGGCGGCACC AAGGATCTCT ACAGCACGCA CGCTCGCAAG 600 rfK »T < "T &rT! PCW3TGTTC« 3ß CGACACCCCG GTCGCCGAGA TGACCCCCGC CCTTGTCCGG 660
GCGTGGTGGG CCGGGATGGG TAAGCAGTAC CCGACGGCAC GGCGGCACGC CTACAACGTA 720
CTCCGGGCGG TCATGAATAC CGCTGTAGAG GACAAGCTGG TGTCGGAGAA CCCGTGCCGG 780
ATCGAGCSGA ASGCACCCGC TGAGCGCGAC GT6GAAGCCC TCACACCGGA GßAGCTGGAC 840
GTAGTGGCCG GGGAGGTGTT CGAGCACTAC CGCGTGGCCG TCTACATCCT sGCGTGGACC 900
AGCCTGCGGT TCGGTGAGCT GATCGAGATC CGCCGCAA ß ACATCGTGGA TGACGGCGAG 960
ACGATGAAGC TCCGCGTGCG CCGGGGCGCs GCCCGCGTCG GCGAGAAGAT CGTCGTCGGC 1020
AACACCAAGA CCGTCAGGTC CAAGCGGCCG GTGACCGTGC CGCCTCACGT CGCGGCGATG 1080
ATCCGCGAGC ACATGGCTGA CCGGACGAAG ATGAACAAGG GGCCGGAAGC TCTCCTGGTG 1140
ACCACCACGC GGßßßCAGCG GCTGTCGAAG TCTßCGTTCA CTCGCTCGCT GAAGAAGGGC 1200
TACGCCAAGA TCGGTCGACC GGACCTCCGC ATCCACGACC TCCGGGCCGT GGGAGCCACG 1260
CTGGCGGCTC AGGCCGGTGC GACGACCAAG GAGCTGATGG TGCGCCTCGG GCACACGACT 1320
CCGCGCATGG CGATGAAGTA CCAGATGGCC TCAGCAGCCC STGACGAGGA GATAGCGAGG 1380
CGAATGTCGG AQCTGGCAGG OATTACCCCC TGA 1413
(2) INFORMATION FOR SEQ. ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1374 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: 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 TTCCGGTAßC 240
CTGTTTCGCA TGGCAGCAAG ACGGAGAGGA TGGGGATCGC TGCGGACCCA GCGCAGCGGT 300
CGAßTGCAAG CGTCGTACGT CAGCCCGATC GACßßßCAGC G TACTTCGG GCCGAGGAAC 360
TACGACAACC GGATGGACGC CGAAGCGTGG CTCGCGTCTG AGAAGCGGCT GATCGACAAC 420
GAGGAßTGßA CCCCGCCGGC CGAGCGCGAG AAGAAGGCTß 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 AAGGCACCCß CTGAGCGCGA CGTßßAAßCC 780
CTCACACCGG AGGAGCTGGA CGTAGTGGCC GGGGAGGTGT TCGAGCACTA CCGCGTGGCC 840
GTCTACATCC TGGCGTGGAC CAGCCTGCGG TTCGGTGAGC TGATCGAGAT CCGCCGCAAG 900
GACATCGTGG ATGACGGCGA GACGATGAAG CTCCGCGTGC GCCGGQGCGC GGCCCGCGTC 960
GGCGAGAAGA TCGTCGTCGO CAACACCAAG ACCGTCAGGT CCAAßCßßCC GGTGACCSTG 1020
CCGCCTCACG TCGCGGCGAT GATCCGCGAG CACATGGCTG ACCGGACGAA ßATGAACAAs 1080
GßßCCGGAAG CTCTCCTGGT GACCACCACG CGGGGGCAGC GGCTGTCGAA GTCTGCGTTC 1140
ACTCGCTCGC TGAAGAAGGG CTACGCCAAG ATCGGTCGAC CGGACCTCCG CATCCACGAC 1200
CTCCGGGCCG TGGGAGCCAC GCTGGCGGCT CAGGCC6GTG CGACGACCAA GGAGCTGATG 1260
GTGCGCCTCG GGCACACGAC TCCGCGCATG GCGATGAAGT ACCAGATGGC CTCAGCAGCC 1320
CGTGAC6AGG AGATAGCGAG GCG? ATGTC? GAGCTGGCAG GGATTACCCC CTGA 1374
(2) INFORMATION FOR SEQ. ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 105 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 8:
GTTGTGTCTC AAAATCTCTG ATGTTACATT GCACAAGATA AAAATATATC ATCATGAACA 60 ATAAAACTGT CTGCTTACAT AAACAGTAAT ACAAGGGGTG TTATG IOS
(2) INFORMATION FOR SEQ. ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2096 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear
(ii) TYPE OF MOLECULE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ. ID NO: 9:
GGTCACCTGC GATCACACCG AGCGTGCAGG TAGCGAAGTC CTCATCACCA CCAGGACGGG 60
CCTGGGCGAT ACCAGCGCCG GGGGCGATCC CGCCAGGAAA TGCCGTCCAA TCßßTGTCCß 120
CGACTGCGGC GGAGCGGACA CTCCGACCAA CACAACAACC AACGTCGTCA TAGCGACGAC 180
GAACCACGAT CGGATGATCC GAATCACTGC GCTGTCCATA CAGGCGGCCA CCCCTCQAAC 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 CGATCCAßCG CCTGCCTCAC CACTTTTCGT S40
CGTCGCGGGG CTCACCCGGG TACTGSACCG GATCGCCACT ATCGAAACGG CTACGCGCGG 600
CGGCAGCGGC GGCGCTGGCG GCGGCACGTT CATCACCACC GGACCGGGAA CCAGCGTCGA 660
TTCATCGATG GCCGGCTGAA TCGGCCGGCG TTCGTCGGsC AGCAGGTCCG CGAGCTCGTC 720
GQCATCGATß TACTGCCGGC CGGCGGATCG TCGTCACGCA GAATGTGGGA CACCAGCGCC 780
TTGTCGCGGG CCTCTTCGCC GGTGAGGATC CGCTCGGAGG CGCGGTCGCG GCGCGGCTGT 840
GGCATGTCGG GGCGTGCCGC TCCCCCGGCß CCGCCCATCß GCCCGCCCAT TGGCATTCCG 900
CCCATGCCGC CCATCATTCC TGTGGAGCCA GCTGGCCCGG TCTTCAATGG AGGCAGGCCC 960
GCTGACGGCG ACGTGGAGGC GSTGCßCCCC GAAATCTGGG CCGGATCAAC TCGSCCACCG 1020
GTCACGGTCG GATTGGCGGC CGGTGTTGTC GGTGCGACAA CACCGCCGAC AACGCCGCGC 1080
CCCGCCATCG CCGAACCACG GGGTGGTGGG TGCGTCCGAC CTGCCAGAAT C6TCCC6QCG 1140
TCGCGGCTGC TGCTGAACAC CGCCGAGCCC GCCGCCAGTC GGGAAAGCGC TGGGCATCAT 1200
GGTCGGGCCG GßßßCCATCG GAGCGGGTGC ACCTGTCGGG GCTGGTGGCG GCGTCAGCGC 1260
CGTCGCCTGC ACCATCGGCC GTGGGCCGCC GACACCTCCG GGTCGCACC GCCGCCGCCG 1320
ACGATCGTGT CGTCAGCGCC GCCGCCSACG ATQGTGTCGT CCCAACCGTC GCGCGGCTGG 13B0
AGßTCGCßßG GCGACCGGAA AATGCCrTTA TCGTGGCCGG ACACCTTGGA ATCGGTGTCC 1440
GGCTCGTCGG GCAGGCCTTC CGTCGCTGAC GTGCACGCGC GCTCCAATCG CTCCAGCGCC 1500
GCCTGGACCT CGGGATCGGC AGCCGTCCCG CCCCGAATGA CCGGGCGGCC GCGGCCGGCC 1560
TCTCCCACCG CACGCAGGGC CGTCGGCGAT TTTCAGCAGG TCGCCßCCCA TTTCCOACAT 1620
CTTTTCCTCG GCGGCCGATC GCCGCACCGG ACCCAATGTC GTCCGGAAAC GGCTCGGCCG 1680
CßATCGACTC CAGCAACGCG GCCATGTCGA TGCGCTCCTG AAACTCGGCC TCGTTGGTCA 1740
OCGAATCGCC GTCATAACßß ATGGCGCCCß GGCCGCCGCG CGATATCGAG CCGAGAACGT 1800
TATCGAAGTT GGTCATGTGT AATCCCCTCG TTTGAACTTT GGATTAAGCG TAGATACACC 1860
CTTGGACAAG CCAGTTGGAT TCßGAGACAA GCAAATTCAG CCTTAAAAAG GGCGAGGCCC 1920 TGCGQTGGTG GAACACCGCA GGGCCTCTAA CCGCTCGACG CGCTGCACCA ACCAG
Claims (131)
- NOVELTY OF THE INVENTION CLAIMS 1. - A pharmaceutical composition for administration to a human or animal, which provides a continuous source of a protein of interest in said human or animal after its administration thereto, and which stimulates the cellular immunity of said human or animal after its administration ai itself, made in accordance with the steps of: (a) growing an inoculum of at least one strain of living Mycobacterium, which is not pathogenic to said human or animal and is capable of maintaining a symbiotic relationship with macrophages in said commensal human or animal, in a liquid culture medium capable of providing sufficient nutrients for the growth of said Mycobacterium strain at a suitable temperature to obtain a liquid cell culture; (b) cooling said culture to about 4 ° C; (c) centrifuging said culture cooled to approximately 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 it in sterile cold glycerol and centrifuging at about 4 ° C to obtain electrocompetent Mycobacterium cells; (f) mixing said electrocompetent Mycobacterium cells with integrative vector DNA, said integrative vector DNA comprising a first coding region for a protein of interest cloned under the control of a recognized promoter sequence in Mycobacterium, a second coding region for a binding site and an integrase gene for mycobacteriophage D29, and a third coding region for a suitable selection marker for said Mycobacterium strain to form a transformation mixture; (g) carrying out electroporation in said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said transformed Mycobacterium cells having said integrative vector DNA incorporated into the genome of said Mycobacterium strain at an efficiency index of transformation greater than or equal to 107 transformants per microgram of vector DNA and being capable of expressing said protein of interest after its administration in said human or animal; and (h) isolating said transformed Mycobacterium cells from non-transformed Mycobacterium cells by cultivating said electroporated culture in the presence of a substance in which said selection marker allows the transformed Mycobacterium cells to be distinguished from untransformed Mycobacterium cells.
- 2. The composition according to claim 1, made by a method further comprising the step of transferring the culture of transformed Mycobacterium cells from step (h) of said culture medium to a protein-free liquid culture medium and cultivating said cultures. Mycobacterium cells transformed into said protein-free liquid culture medium under conditions suitable for the production of a vaccine.
- 3. The composition according to claim 1, further characterized in that said second coding region for said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 5.
- 4. The composition according to claim 1, further characterized in that said second coding region for said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 6.
- 5. The composition according to claim 1, further characterized in that said second coding region for said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 7.
- 6. The composition according to claim 1, further characterized in that said third coding region for said selection marker suitable for said Mycobacterium strain comprises a kanamycin selection marker consisting essentially of the sequence provided in SEQ ID NO: 2.
- 7. The composition according to claim 1, further characterized in that said second coding region for a binding site and an integrase gene are derived from a variant of mycobacteriophage D29.
- 8. The composition according to claim 1, further characterized in that said strain of Mycobacterium 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.
- 9. The composition according to claim 1, further comprising a cytokine associated with cellular immunity.
- 10. The composition according to claim 1, further comprising a chemotherapeutic agent.
- 11. A pharmaceutical composition for administration to a human or animal, which provides a continuous source of a protein of interest in said human or animal after its administration thereto, and which stimulates the cellular immunity of said human or animal after its administration to the same, made in accordance with the steps of: (a) growing an inoculum of at least one strain of living Mycobacterium, which is not pathogenic to said human or animal and is capable of maintaining a symbiotic relationship with macrophages in said human or animal, in a liquid culture medium capable of providing sufficient nutrients for the growth of said Mycobacterium strain at a suitable temperature to obtain a liquid cell culture; (b) cooling said culture to about 4 ° C; (c) centrifuging said culture cooled to approximately 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 it in sterile cold glycerol and centrifuging at about 4 ° C to obtain electrocompetent Mycobacterium cells; (f) mixing said electrocompetent Mycobacterium cells with integrative vector DNA, said integrative vector DNA comprising a first coding region for a protein of interest cloned under the control of a recognized promoter sequence in Mycobacterium, a second coding region for the minimal functional component of a binding site and an integrase gene for mycobacteriophage D29, and a third coding region for a suitable selection marker for said Mycobacterium strain to form a transformation mixture; (g) carrying out electroporation in said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said transformed Mycobacterium cells having said integrative vector DNA incorporated into the genome of said Mycobacterium strain at an efficiency index of transformation greater than or equal to 107 transformants per microgram of vector DNA and being capable of expressing said protein of interest after its administration in said human or animal; and (h) isolating said transformed Mycobacterium cells from non-transformed Mycobacterium cells by cultivating said electroporated culture in the presence of a substance in which said selection marker allows the transformed Mycobacterium cells to be distinguished from untransformed Mycobacterium cells.
- 12. The composition according to claim 11, made by a method further comprising the step of transferring the culture of transformed Mycobacterium cells from step (h) of said culture medium to a protein-free liquid culture medium and cultivating said cultures. Mycobacterium cells transformed into said protein-free liquid culture medium under conditions suitable for the production of a vaccine.
- 13. The composition according to claim 11, further characterized in that said second coding region for said minimum functional component of said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 5.
- 14. The composition according to claim 11, further characterized in that said second coding region for said minimum functional component of said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 6.
- 15. The composition according to claim 11, further characterized in that said second coding region for said minimum functional component of said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 7.
- 16. The composition according to claim 11, further characterized in that said second coding region for a binding site and an integrase gene come from a variant of mycobacteriophage D29.
- 17. The composition according to claim 11, further characterized in that said strain of Mycobacterium 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 according to claim 11, further comprising a cytokine associated with cellular immunity.
- 19. The composition according to claim 11, further comprising a chemotherapeutic agent.
- 20. A pharmaceutical composition for administration to a human or animal, which provides a continuous source of a protein of interest in said human or animal after its administration thereto, and which stimulates the cellular immunity of said human or animal after its administration to the same, made in accordance with the steps of: (a) growing an inoculum of at least one strain of living Mycobacterium, which is not pathogenic to said human or animal and is capable of maintaining a symbiotic relationship with macrophages in said human or animal, in a liquid culture medium capable of providing sufficient nutrients for the growth of said Mycobacterium strain at a suitable temperature to obtain a liquid cell culture; (b) cooling said culture to about 4 ° C; (c) centrifuging said culture cooled to approximately 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 it in sterile cold glycerol and centrifuging at about 4 ° C to obtain electrocompetent Mycobacterium cells; (f) mixing said electrocompetent Mycobacterium cells with integrative vector DNA, said integrative vector DNA comprising a first coding region for a protein of interest cloned under the control of a promoter recognized in Mycobacterium, a second coding region for a site of binding and an integrase gene for mycobacteriophage D29, and the minimum functional component of a third coding region for a suitable selection marker for said Mycobacterium strain to form a transformation mixture; (g) carrying out electroporation in said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said transformed Mycobacterium cells having said integrative vector DNA incorporated into the genome of said Mycobacterium strain at an efficiency index of transformation greater than or equal to 107 transformants per microgram of vector DNA and being able to express said protein of interest after its administration in said human or animal; and (h) isolating said transformed Mycobacterium cells from non-transformed Mycobacterium cells by cultivating said electroporated culture in the presence of a substance in which said selection marker allows the transformed Mycobacterium cells to be distinguished from untransformed Mycobacterium cells. 21. - The composition according to claim 20, made by a method further comprising the step of transferring the culture of transformed Mycobacterium cells from step (h) of said culture medium to a protein-free liquid culture medium and cultivating said cultures. Mycobacterium cells transformed into said protein-free liquid culture medium under conditions suitable for the production of a vaccine. 22. The composition according to claim 20, further characterized in that said third coding region for said minimum functional component of said selection marker suitable for said Mycobacterium strain comprises a kanamycin selection marker consisting essentially of the sequence provided in SEQ. ID NO: 2 23. The composition according to claim 20, further characterized in that said second coding region for a binding site and an integrase gene are derived from a variant of mycobacteriophage D29. 24. The composition according to claim 20, further characterized in that said strain of Mycobacterium 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 according to claim 20, further comprising a cytokine associated with cellular immunity. 26. The composition according to claim 20, further comprising a chemotherapeutic agent. 27. A pharmaceutical composition for administration to a human or animal, which provides a continuous source of a protein of interest in said human or animal after its administration thereto, and which stimulates the cellular immunity of said human or animal after its administration to the same, made in accordance with the steps of: (a) growing an inoculum of at least one strain of living Mycobacterium, which is not pathogenic to said human or animal and is capable of maintaining a symbiotic relationship with macrophages in said human or animal, in a liquid culture medium capable of providing sufficient nutrients for the growth of said Mycobacterium strain at a suitable temperature to obtain a liquid cell culture; (b) cooling said culture to about 4 ° C; (c) centrifuging said culture cooled to approximately 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 it in sterile cold glycerol and centrifuging at about 4 ° C to obtain electrocompetent Mycobacterium cells; (f) mixing said electrocompetent Mycobacterium cells with an extrachromosomal DNA vector, said extrachromosomal DNA vector comprising a first coding region for a protein of interest cloned under the control of a recognized promoter sequence in Mycobacterium, a second region supporting the replication in Mycobacterium, and a third coding region for a suitable selection marker for said Mycobacterium strain to form a transformation mixture; (g) carrying out electroporation in said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said Mycobacterium cells comprising said transformed extrachromosomal DNA vector at an efficiency index of transformation greater than or equal to 107 transformants per microgram of vector DNA and being able to express said protein of interest after its administration in said human or animal; and (h) isolating said transformed Mycobacterium cells from non-transformed Mycobacterium cells by cultivating said electroporated culture in the presence of a substance in which said selection marker allows the transformed Mycobacterium cells to be distinguished from untransformed Mycobacterium cells. 28. The composition according to claim 27, made by a method further comprising the step of transferring the culture of transformed Mycobacterium cells from step (h) of said culture medium to a protein-free liquid culture medium and cultivating said cultures. Mycobacterium cells transformed into said protein-free liquid culture medium under conditions suitable for the production of a vaccine. 29. The composition according to claim 27, further characterized in that said second region that supports replication in Mycobacterium consists essentially of the sequence of an origin of replication of a Mycobacterium plasmid provided in SEQ ID NO: 3. 30. The composition according to claim 27, further characterized in that said second region that supports replication in Mycobacterium consists essentially of the sequence of an origin of replication of a Mycobacterium plasmid provided in SEQ ID NO: 4. 31. The composition according to claim 27, further characterized in that said coding region for a suitable selection marker for said Mycobacterium strain comprising a kanamycin selection marker consists essentially of the sequence provided in SEQ ID NO: 2. 32. The composition according to claim 27, further characterized in that said Mycobacterium plasmid is pAL 5000. 33. The composition according to claim 27, further characterized in that 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 according to claim 27, further comprising a cytokine associated with cellular immunity. 35.- The composition according to claim 27, further comprising a chemotherapeutic agent. 36.- A pharmaceutical composition for administration to a human or animal, which provides a continuous source of a protein of interest in said human or animal after its administration thereto, and which stimulates the cellular immunity of said human or animal after its administration to the same, made in accordance with the steps of: (a) growing an inoculum of at least one strain of living Mycobacterium, which is not pathogenic to said human or animal and is capable of maintaining a symbiotic relationship with macrophages in said human or animal, in a liquid culture medium capable of providing sufficient nutrients for the growth of said Mycobacterium strain at a suitable temperature to obtain a liquid cell culture; (b) cooling said culture to about 4 ° C; (c) centrifuging said culture cooled to approximately 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 it in sterile cold glycerol and centrifuging at about 4 ° C to obtain electrocompetent Mycobacterium cells; (f) mixing said electrocompetent Mycobacterium cells with an extrachromosomal DNA vector, said extrachromosomal DNA vector comprising a first coding region for a protein of interest cloned under the control of a recognized promoter sequence in Mycobacterium, a second region for the component functional minimum that supports replication in Mycobacterium, and a third coding region for a suitable selection marker for said Mycobacterium strain to form a transformation mixture; (g) carrying out electroporation in said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said Mycobacterium cells comprising said transformed extrachromosomal DNA vector at an efficiency index of transformation greater than or equal to 107 transformants per microgram of vector DNA and being able to express said protein of interest after its administration in said human or animal; and (h) isolating said transformed Mycobacterium cells from non-transformed Mycobacterium cells by cultivating said electroporated culture in the presence of a substance in which said selection marker allows the transformed Mycobacterium cells to be distinguished from untransformed Mycobacterium cells. 37. - The composition according to claim 36, made by a method further comprising the step of transferring the culture of transformed Mycobacterium cells from step (h) of said culture medium to a protein-free liquid culture medium and cultivating said cultures. Mycobacterium cells transformed into said protein-free liquid culture medium under conditions suitable for the production of a vaccine. 38.- The composition according to claim 36, further characterized in that said second region for the minimal functional component that supports replication in Mycobacterium consists essentially of the sequence of an origin of replication of a plasmid of Mycobacterium provided in SEQ ID NO: 3. 39.- The composition according to claim 36, further characterized in that said second region for the minimal functional component that supports replication in Mycobacterium consists essentially of the sequence of an origin of replication of a plasmid of Mycobacterium provided in SEQ ID NO: 4. 40.- The composition according to claim 36, further characterized in that said Mycobacterium plasmid is pAL 5000. 41. The composition according to claim 36, further characterized in that 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 according to claim 36, further comprising a cytokine associated with cellular immunity. 43. The composition according to claim 36, further comprising a chemotherapeutic agent. 44.- A pharmaceutical composition for administration to a human or animal, which provides a continuous source of a protein of interest in said human or animal after its administration thereto, and which stimulates the cellular immunity of said human or animal after its administration to the same, made in accordance with the steps of: (a) growing an inoculum of at least one strain of living Mycobacterium, which is not pathogenic to said human or animal and is capable of maintaining a symbiotic relationship with macrophages in said human or animal, in a liquid culture medium capable of providing sufficient nutrients for the growth of said Mycobacterium strain at a suitable temperature to obtain a liquid cell culture; (b) cooling said culture to about 4 ° C; (c) centrifuging said culture cooled to approximately 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 it in sterile cold glycerol and centrifuging at about 4 ° C to obtain electrocompetent Mycobacterium cells; (f) mixing said electrocompetent Mycobacterium cells with an extrachromosomal DNA vector, said extrachromosomal DNA vector comprising a first coding region for a protein of interest cloned under the control of a recognized promoter sequence in Mycobacterium, a second region supporting the replication in Mycobacterium, and a third coding region for the minimum functional component of a suitable selection marker for said Mycobacterium strain to form a transformation mixture; (g) carrying out electroporation in said transformation mixture to form an electroporated culture comprising transformed Mycobacterium cells, said Mycobacterium cells comprising said transformed extrachromosomal DNA vector at an efficiency index of transformation greater than or equal to 107 transformants per microgram of vector DNA and being able to express said protein of interest after its administration in said human or animal; and (h) isolating said transformed Mycobacterium cells from non-transformed Mycobacterium cells by cultivating said electroporated culture in the presence of a substance in which said selection marker allows the transformed Mycobacterium cells to be distinguished from untransformed Mycobacterium cells. 45. The composition according to claim 44, made by a method further comprising the step of transferring the culture of transformed Mycobacterium cells from step (h) of said culture medium to a protein-free liquid culture medium and cultivating said cultures. Mycobacterium cells transformed into said protein-free liquid culture medium under conditions suitable for the production of a vaccine. 46. The composition according to claim 44, further characterized in that said coding region for the minimum functional component of said selection marker suitable for said Mycobacterium strain comprising a kanamycin selection marker consists essentially of the sequence provided in SEQ ID NO: 2 47. The composition according to claim 44, further characterized in that said Mycobacterium plasmid is pAL 5000. 48. The composition according to claim 44, further characterized in that 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 according to claim 44, further comprising a cytokine associated with cellular immunity. 50.- The composition according to claim 44, further comprising a chemotherapeutic agent. 51.- A pharmaceutical composition for administration to a human or animal, said composition being able to stimulate the cellular immunity of said human or animal after its administration to said human or animal, comprising the steps of: (a) cultivating a inoculum of at least one strain of living Mycobacterium, which is not pathogenic to said human or animal and is capable of maintaining a commensal symbiotic relationship with macrophages in said human or animal, in a liquid culture medium capable of providing sufficient nutrients for the growth of said Mycobacterium strain at a suitable 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 it in sterile cold saline and centrifuging at about 4 ° C to obtain washed live Mycobacterium cells; and (f) mixing said living Mycobacterium cells with an antigen of interest. 52. The composition according to claim 51, further characterized in that said strain of Mycobacterium 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 according to claim 51, further comprising a cytokine associated with cellular immunity. 54. The composition according to claim 51, further comprising a chemotherapeutic agent. 55. - A pharmaceutical composition for administration to a human or animal, said composition being able to stimulate the cellular immunity of said human or animal after its administration to said human or animal, comprising the steps of: (a) cultivating an inoculum of two or more strains of Mycobacterium alive, which are not pathogenic for said human or animal and are capable of maintaining a commensal symbiotic relationship with macrophages in said human or animal, in a liquid culture medium capable of providing sufficient nutrients for the growth of said Mycobacterium strains at a suitable temperature to obtain a liquid cell culture; (b) centrifuging said culture to obtain a pellet of live Mycobacterium cells and a supernatant; (c) separating said pellet from said supernatant; (d) washing said pellet and centrifuging it to obtain washed Mycobacterium cells; (e) killing said washed Mycobacterium cells to obtain a Mycobacterium adjuvant and (f) mixing said Mycobacterium adjuvant with an antigen of interest. 56. - The composition according to claim 55, further characterized in that said strains of Mycobacterium 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 according to claim 55, further comprising a cytokine associated with cellular immunity. 58. The composition according to claim 55, further comprising a chemotherapeutic agent. 59. A promiscuous vector comprising a first region that supports replication in Escherichia coli, a second coding region for a binding site and a mycobacteriophage integrase gene D29, and a third coding region for a suitable selection marker both for Escherichia coli and for Mycobacterium, said first region that supports replication in Escherichia coli comprising the minimum functional component of said origin of replication for Escherichia coli, where said promiscuous vector is capable of transforming bacteria at a rate of transformation efficiency of more than or equal to 107 transformants per microgram of vector DNA. 60.- The promiscuous vector according to claim 59, further characterized in that said minimum functional component of said origin of replication for Escherichia coli consists essentially of the sequence provided in SEQ ID NO: 1. 61.- The promiscuous vector according to claim 59, further characterized in that said second coding region is for a binding site and an integrase gene of a variant of mycobacteriophage D29. 62. - A promiscuous vector comprising a first region that supports replication in Escherichia coli, a second coding region for a binding site and a mycobacteriophage D29 gene, and a third coding region for a suitable selection marker both for Escherichia coli as for Mycobacterium, said second coding region for a binding site and integrase gene of said mycobacteriophage D29 comprises the minimum functional component of said binding site and integrase gene of said mycobacteriophage D29, wherein said promiscuous vector is capable of transforming bacteria at an efficiency index of transformation of more than or equal to 107 transformants per microgram of vector DNA. 63. - The promiscuous vector according to claim 62, further characterized in that said minimum functional component of said binding site and the fat gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 5. 64. - The promiscuous vector according to claim 62, further characterized in that said minimum functional component of said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 6. 65. - The promiscuous vector according to claim 62, further characterized in that said minimum functional component of said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 7. 66. - The promiscuous vector according to claim 62, further characterized in that said second coding region is for a binding site and an integrase gene of a variant of mycobacteriophage D29. 67. - A promiscuous vector comprising a first region that supports replication in Escherichia coli, a second coding region for a binding site and a mycobacteriophage integrase gene D29, and a third coding region for a selection marker suitable for both Escherichia coli as for Mycobacterium, said third coding region for said selection marker suitable for both Escherichia coli and Mycobacterium comprising the minimum functional component of said selection marker suitable for both Escherichia coli and Mycobacterium, wherein said promiscuous vector is capable of transforming bacteria at an efficiency index of transformation of more than or equal to 107 transformants per microgram of vector DNA. 68. - The promiscuous vector according to claim 67, further characterized in that said minimum functional component of said selection marker suitable for both Escherichia coli and Mycobacterium comprises a kanamycin selection marker consisting essentially of the sequence provided in SEQ ID NO: 2. 69. - The promiscuous vector according to claim 67, further characterized in that said second coding region is for a binding site and an integrase gene of a variant of mycobacteriophage D29. 70. - A promiscuous vector comprising a first region that supports replication in Escherichia coli, a second coding region that supports replication in Mycobacterium and a third coding region for a selection marker suitable for both Escherichia coli and Mycobacterium, said first region that supports replication in Escherichia coli comprising the minimum functional component of said origin of replication for Escherichia coli, wherein said promiscuous vector is capable of transforming bacteria at an efficiency index of transformation of more than or equal to 107 transformants per microgram of Vector DNA. 71. The promiscuous vector according to claim 70, further characterized in that said minimum functional component of said origin of replication for Escherichia coli consists essentially of the sequence provided in SEQ ID NO: 1. 72.- A promiscuous vector comprising a first region that supports replication in Escherichia coli, a second region that supports replication in Mycobacterium and a third coding region for a selection marker suitable for both Escherichia coli and Mycobacterium, said second The region that supports replication in Mycobacterium essentially comprises the minimum functional component of said origin of replication of said Mycobacterium plasmid, wherein said promiscuous vector is capable of transforming bacteria at an efficiency index of transformation of more than or equal to 107 transformants per microgram of vector DNA. 73. - The promiscuous vector according to claim 72, further characterized in that said minimum functional component of said origin of replication of said Mycobacterium plasmid consists essentially of the sequence provided in SEQ ID NO: 3. 74. - The promiscuous vector according to claim 72, further characterized in that said minimum functional component of said origin of replication of said Mycobacterium plasmid consists essentially of the sequence provided in SEQ ID NO: 4. 75. - A promiscuous vector comprising a first region that supports replication in Escherichia coli, a second region that supports replication in Mycobacterium and a third coding region for a selection marker suitable for both Escherichia coli and Mycobacterium, said third region of coding for a selection marker suitable for both Escherichia coli and Mycobacterium comprises the minimum functional component of said selection marker suitable for both Escherichia coli and Mycobacterium, wherein said promiscuous vector is capable of transforming bacteria at an index of transformation efficiency of more than or equal to 107 transformants per microgram of vector DNA. 76. - The promiscuous vector according to claim 75, further characterized in that said minimum functional component of said selection marker suitable for both Escherichia coli and Mycobacterium comprises a kanamycin selection marker consisting essentially of the sequence provided in SEQ ID NO: 2. 77.- A vector comprising a first coding region for a binding site and mycobacteriophage integrase gene D29, and a second coding region for a selection marker suitable for Mycobacterium, said first coding region for a binding site and integrase gene of said mycobacteriophage D29 comprising the minimal functional component of said binding site and integrase gene of said mycobacteriophage D29, wherein said promiscuous vector is capable of transforming bacteria at an efficiency index of transformation of more than or equal to 107 transformants per microgram of vector DNA. 78. The vector according to claim 77, further characterized in that said minimum functional component of said binding site and fat gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 5. 79. The vector according to claim 77, further characterized in that said minimum functional component of said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 6. 80.- The promiscuous vector according to claim 77, further characterized in that said minimum functional component of said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 7. 81. - The promiscuous vector according to claim 77, further characterized in that said second coding region is for a binding site and an integrase gene of a variant of mycobacteriophage D29. 82.- A vector comprising a first coding region for a binding site and mycobacteriophage integrase gene D29, and a second coding region for a selection marker suitable for Mycobacterium, said second coding region for a selection marker Suitable for Mycobacterium comprises the minimum functional component of said suitable selection marker for said Mycobacterium strain, wherein said promiscuous vector is capable of transforming bacteria at an efficiency index of transformation of more than or equal to 107 transformants per microgram of DNA of vector. 83. The vector according to claim 82, further characterized in that said minimum functional component of said selection marker suitable for said Mycobacterium strain comprises a kanamycin selection marker consisting essentially of the sequence provided in SEQ ID NO: 2 . 84. The promiscuous vector according to claim 82, further characterized in that said second coding region is for a binding site and an integrase gene of a variant of mycobacteriophage D29. 85.- A vector for carrying and expressing genes selected from a strain of Mycobacterium, comprising a first coding region for a binding site and mycobacteriophage integrase gene D29, and a second coding region for a suitable selection marker for Mycobacterium, said first coding region for a binding site and mycobacteriophage integrase gene D29 comprises the minimum functional component of said binding site and integrase gene of said mycobacteriophage D29, wherein said vector is capable of transforming bacteria at an index of transformation efficiency of more than or equal to 107 transformants per microgram of vector DNA. 86. - The vector according to claim 85, further characterized in that said minimum functional component of said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 5. 87. - The vector according to claim 85, further characterized in that said minimum functional component of said binding site and integrase gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 6. 88. - The vector according to claim 85, further characterized in that said minimum functional component of said binding site and fat gene of said mycobacteriophage D29 consists essentially of the sequence provided in SEQ ID NO: 7. 89. - The promiscuous vector according to claim 85, further characterized in that said second coding region is for a binding site and an integrase gene of a variant of mycobacteriophage D29. 90. - A vector for carrying and expressing genes selected from a strain of Mycobacterium, comprising a first coding region for a binding site and a mycobacteriophage D29 gene, and a second coding region for a suitable selection marker for Mycobacterium , said second coding region for said selection marker suitable for Mycobacterium comprises the minimum functional component of said selection marker suitable for said strain of Mycobacterium, wherein said vector is capable of transforming bacteria at an index of transformation efficiency of more than or equal to 107 transformants per microgram of vector DNA. 91.- The vector according to claim 90, further characterized in that said minimum functional component of said selection marker suitable for said Mycobacterium strain comprises a kanamycin selection marker consisting essentially of the sequence provided in SEQ ID NO: 2 . 92.- The promiscuous vector according to claim 90, further characterized in that said second coding region is for a binding site and an integrase gene of a variant of mycobacteriophage D29. 93.- A vector comprising a first region that supports replication in Mycobacterium and a second coding region for a selection marker suitable for Mycobacterium, said first region that supports replication in Mycobacterium comprises the minimum functional component of said origin of replication of said Mycobacterium plasmid, wherein said vector is capable of transforming bacteria at an efficiency index of transformation of more than or equal to 107 transformants per microgram of vector DNA. 94. - The vector according to claim 93, further characterized in that said minimum functional component of said origin of replication of said Mycobacterium plasmid consists essentially of the sequence provided in SEQ ID NO: 3. 95. - The promiscuous vector according to claim 93, further characterized in that said minimum functional component of said origin of replication of said Mycobacterium plasmid consists essentially of the sequence provided in SEQ ID NO: 4. 96. - A vector comprising a first region that supports replication in Mycobacterium and a second coding region for a selection marker suitable for Mycobacterium, said second coding region for a selection marker suitable for Mycobacterium comprises the minimum functional component of said marker of suitable selection for Mycobacterium, wherein said vector is capable of transforming bacteria at an index of transformation efficiency of more than or equal to 107 transformants per microgram of vector DNA. 97. - The vector according to claim 96, further characterized in that said minimum functional component of said selection marker suitable for said Mycobacterium comprises a kanamycin selection marker consisting essentially of the sequence provided in SEQ ID NO: 2. 98.- A vector comprising a first region that supports replication in 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 comprises the component functional minimum of said origin of replication for said Escherichia coli. The vector according to claim 98, further characterized in that said minimum functional component of said origin of replication of said Escherichia coli consists essentially of the sequence provided in SEQ ID NO: 1. 100.- A vector comprising a first region that supports replication in Escherichia coli and a second coding region for a suitable selection marker for said Escherichia coli, said second coding region for a suitable selection marker for said Escherichia coli comprises the minimum functional component of said selection marker suitable for said Escherichia coli, wherein said vector is capable of transforming bacteria at an efficiency index of transformation of more than or equal to 107 transformants per microgram of vector DNA. 101. The vector according to claim 100, further characterized in that said minimum functional component of said selection marker suitable for said Escherichia coli comprises a kanamycin selection marker for said Escherichia cou consisting essentially 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 that supports replication in Escherichia coli, and a second coding region for a suitable selection marker for said Escherichia coli, said first region That supports replication in Escherichia coli comprises the minimum functional component of said origin of replication for said Escherichia coli, wherein said vector is capable of transforming bacteria at an efficiency index of transformation of more than or equal to 107 transformants per microgram of DNA of vector. 103. The vector according to claim 102, further characterized in that said minimum functional component of said origin of replication of said Escherichia coli consists essentially of the sequence provided in SEQ ID NO: 1. 104.- A vector for carrying and expressing selected genes in Escherichia coli, comprising a first region that supports replication in Escherichia coli, and a second coding region for a suitable selection marker for said strain of Escherichia coli, said second coding region for a suitable selection marker for said strain of Escherichia coli comprises the minimum functional component of said selection marker suitable for said Escherichia coli, wherein said vector is capable of transforming bacteria at an efficiency index of transformation of more than or equal to 107 transformants per microgram of vector DNA. The vector according to claim 104, further characterized in that said minimum functional component of said selection marker suitable for said Escherichia coli comprises a kanamycin selection marker for said Escherichia coli consisting essentially of the sequence provided in SEQ ID NO: 2 106.- The use of a pharmaceutical composition according to claim 1, in the preparation of a drug to stimulate cell immunity in an animal. The use according to claim 106, wherein the pharmaceutical composition is in combination with a cytokine associated with cellular immunity. 108. The use according to claim 106, wherein the pharmaceutical composition is in combination with a chemotherapeutic agent. 109.- The use of a pharmaceutical composition according to claim 11, in the preparation of a drug to stimulate cellular immunity in an animal. 110. The use according to claim 109, wherein the pharmaceutical composition is in combination with a cytokine associated with cellular immunity. 111. The use according to claim 109, wherein the pharmaceutical composition is in combination with a chemotherapeutic agent. 112. The use of a pharmaceutical composition according to claim 20, in the preparation of a drug for stimulating cellular immunity in an animal. 113. The use according to claim 112, wherein the pharmaceutical composition is in combination with a cytokine associated with cellular immunity. 114. The use according to claim 112, wherein the pharmaceutical composition is in combination with a chemotherapeutic agent. 115. The use of a pharmaceutical composition according to claim 27, in the preparation of a drug for stimulating cellular immunity in an animal. 116. The use according to claim 115, wherein the pharmaceutical composition is in combination with a cytokine associated with cellular immunity. 117. - The use according to claim 115, wherein the pharmaceutical composition is in combination with a chemotherapeutic agent. 118. The use of a pharmaceutical composition according to claim 36, in the preparation of a drug for stimulating cellular immunity in an animal. 119. - The use according to claim 118, wherein the pharmaceutical composition is in combination with a cytokine associated with cellular immunity. 120. - The use according to claim 118, wherein the pharmaceutical composition is in combination with a chemotherapeutic agent. 121. - The use of a pharmaceutical composition according to claim 44, in the preparation of a drug for stimulating cellular immunity in an animal. 122. The use according to claim 121, wherein the pharmaceutical composition is in combination with a cytokine associated with cellular immunity. 123. - The use according to claim 121, wherein the pharmaceutical composition is in combination with a chemotherapeutic agent. 124. The use of a pharmaceutical composition according to claim 51, in the preparation of a drug for stimulating cellular immunity in an animal. 125. The use according to claim 124, wherein the pharmaceutical composition is in combination with a cytokine associated with cellular immunity. 126. The use according to claim 124, wherein the pharmaceutical composition is in combination with a chemotherapeutic agent. 127.- The use of a pharmaceutical composition according to claim 55, in the preparation of a drug to stimulate cellular immunity in an animal. 128. The use according to claim 127, wherein the pharmaceutical composition is in combination with a cytokine associated with cellular immunity. 129. The use according to claim 127, wherein the pharmaceutical composition is in combination with a chemotherapeutic agent. 130.- A culture medium comprising approximately 0.25% proteose peptone; about 0.2% nutrient broth, about 0.075% pyruvic acid, about 0.05% sodium glumamate, about 0.5% albumin fraction V, about 0.7% dextrose, about 0.0004% catalase, about 0.005% oleic acid, L (.) amino acid complex (approximately 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, approximately 0.023% histidine, approximately 0.021% tyrosine, approximately 0.02% methionine, approximately 0.014% tryptophan and approximately 0.01% cysteine), approximately 0.306% Na2HPO4, approximately 0.055% KH2PO4, approximately 0.05% NH4CI, approximately 0.335% NaCl, approximately 0.0001% of ZnSO4, approximately 0.0001% of CUSO4, approximately 0.0001% of FeCl3, approximately 0.012% of MgSO and approximately 0.05% of Tween 80 where the pH of said medium is approximately 7. 131.- The culture medium of compliance with the claim 130, which further comprises about 0.8% glycerol.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US042849 | 1997-03-28 | ||
US60/042849 | 1997-03-28 |
Publications (1)
Publication Number | Publication Date |
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MXPA99008868A true MXPA99008868A (en) | 2000-09-04 |
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