EP0973881A2 - Impfstoffe aus rekombinanten mycobakterien - Google Patents

Impfstoffe aus rekombinanten mycobakterien

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Publication number
EP0973881A2
EP0973881A2 EP98913194A EP98913194A EP0973881A2 EP 0973881 A2 EP0973881 A2 EP 0973881A2 EP 98913194 A EP98913194 A EP 98913194A EP 98913194 A EP98913194 A EP 98913194A EP 0973881 A2 EP0973881 A2 EP 0973881A2
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European Patent Office
Prior art keywords
mycobacterium
coding region
animal
cells
selection marker
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EP98913194A
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English (en)
French (fr)
Inventor
Abdel Hakim Labidi
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Exegenics Inc
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Cytoclonal Pharmaceutics Inc
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Publication of EP0973881A2 publication Critical patent/EP0973881A2/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins

Definitions

  • the present invention relates to DNA constructs for cloning and methods of cloning mycobacterium genes.
  • the mammalian immune system comprises both humoral and cellular components which are interrelated but have different roles. Although both arms of the immune system involve helper T cells, the outcome of the immune response depends on which subclass of T cells is involved.
  • Helper T lymphocytes are produced by two maturation pathways (TH-1 and TH-2), are grouped according to cluster differentiation (CD4 and CD8), and secrete different cytokines. Both components of the immune system constantly scan and survey what is displayed in association with the molecules of the major histocompatibility complex (MHC), at the cell surface.
  • MHC major histocompatibility complex
  • the humoral immune response involves helper T lymphocytes produced by the T cell maturation pathway TH-2.
  • Cells of this pathway secrete cytokines such as Interleukin 4 (IL-4), IL-5, IL-6, IL-9, IL-10 and tumor necrosis factor (TNF).
  • IL-4 Interleukin 4
  • IL-6 IL-6
  • IL-9 tumor necrosis factor
  • TNF tumor necrosis factor
  • cytokines inactivate macrophage proliferation, contributing to a down-regulation of the TH-1 response.
  • TNF causes tissue inflammation and necrosis when released at high levels, which are the indications of failure of the overall immune system in many diseases.
  • CD4+ T lymphocytes become activated through contact with antigens displayed in association with MHC class II molecules (MHC II), at the surface of macrophages and antigen presenting cells.
  • MHC II MHC class II
  • Antibodies are produced by B cells when they interact with these activated CD4+ T lymphocytes.
  • the MHC II molecules reside in the vesicles that engulf and destroy extracellular materials. Thus, their location within the cell gives them their specific function in monitoring the content of these vesicles. They specifically bind to antigens that have been enzymatically processed in the lysosomes of the immune cells after phagocytosis.
  • the humoral immune response is required to protect the extracellular environment against extracellular antigens and parasites through antibodies which can be effective in neutralizing infectious agents.
  • the humoral immune response cannot eliminate whole cells that become diseased, it causes tissue destruction and necrosis, and it is not effective in fighting intracellular diseases.
  • the body relies on the cellular immune response for protection from pathologies that start in the intracellular environment.
  • Cellular immune response is carried out through cytotoxic immune cells which are capable of killing diseased cells.
  • the cellular immune response involves helper T lymphocytes produced by the T cell maturation pathway TH-1. Cells of this pathway secrete cytokines such as IL-2, IL-12, IL-15, gamma Interferon (IFN), lymphotoxin, and Granylocyte Macrophage Colony Stimulating Factor (GMCSF). These cytokines activate macrophages.
  • the cytotoxic T lymphocytes are CD8+ T cells that become activated through contact with antigens associated with MHC class I molecules (MHC I).
  • MHC I molecules reside around the protein factories such as the endoplasmic reticulum. Thus, their location within the cell gives them their specific function of monitoring the output and transport of materials produced inside the cell. They specifically bind to antigens that have been synthesized in the intracellular environment like in the case of cancer or intracellular diseases.
  • the cellular immune response protects against chronic intracellular diseases such as intracellular infection, parasitism and cancer, by activating the macrophages and facilitating the detection and lysis of diseased cells. The result is the formation of a granuloma which is the paradigm of protective immunity in intracellular diseases.
  • the immune system has evolved to be efficient in selecting the target antigens against which an immune response is delivered, it does not always succeed in selecting the appropriate combination of the humoral and cellular immune components necessary to contain or eliminate the disease.
  • intracellular diseases resulting from genetic disorders, cancer, infections, allergies and autoimmune reactions are particularly difficult to treat and continue to be life threatening illnesses despite the advances in detection, diagnosis and treatment.
  • Many of these diseases are able to circumvent the immune system and progress without challenge. For others with a long latency period, diagnosis is often made too late.
  • these diseases may elicit an immune response, they usually compromise its effectiveness by suppressing or mimicking the MHC molecules.
  • a TH-1 immune response favors protection, while down- regulation of this pathway, conversion to TH-2 during the chronic course of the disease, or up-regulation of the pathway TH-2 is detrimental to the host. Accordingly, a shift to TH-1 response or up-regulation of the TH-1 pathway should be beneficial on its own, and when associated with appropriate chemotherapy, would mount an effective response to resistance, chronicity, and disease. Therefore, treatment methods for intracellular diseases are needed which favor a TH-1 immune response rather than a TH-2 response.
  • cancers are caused by genetic alterations that disrupt the metabolic activities of the cell. These genetic changes can result from hereditary and/or environmental factors including infections by pathogenic viruses. Like in other intracellular diseases, cellular immunity plays a major role in the host defense against cancer.
  • cancer immunotherapies were designed to boost the cellular immune response by using specific and non-specific stimuli, including: 1) passive cancer immunotherapies where antibodies have been administered to patients, showing success only in rare cases; 2) active cancer immunotherapies where materials expressing cancer antigens have been administered to patients (e.g., the injection of whole or fractions of cancer cells that have been irradiated, modified chemically, or genetically) showing little impact in experimental tumor models; and 3) the combination of adoptive lymphocytes and IL-2, which caused regression of tumors in mice and metastic melanoma in humans.
  • Tumor infiltrating lymphocytes capable of mediating tumor regression are lymphoid cells that can be grown from single cell suspensions of the tumor incubated with IL-2.
  • TIL Tumor infiltrating lymphocytes
  • antigens recognized by TIL are more likely to be involved in vivo in anticancer immune response, and the cDNA and the amino-acid sequences of several of these antigens have been identified. While these findings have opened new opportunities for the development of cancer specific immunotherapies, treatment methods based on mixing cancer antigens or the cloning and expression of the genes encoding these antigens into a delivery system that favors a TH-1 response rather than a TH-2 response to these antigens are needed.
  • Intracellular infections are caused by bacteria, viruses, parasites, and fungi. These infectious agents are either present free in the environment or carried by untreated hosts. Humans, animals and plants can serve as hosts, and if not treated, they can act as reservoirs facilitating the further spreading of such agents to others. Intracellular pathogens such as M tuberculosis, M. leprae, and tumor viruses cause disease worldwide in millions of people each year. It is estimated that M. tuberculosis infects at least thirty million people/year and will cause an average of three million deaths/year during this decade, making tuberculosis (TB) the number one cause of death from a single infectious agent (World Health Organization, 1996).
  • TB occurs most commonly in developing countries, but the prevalence of TB has increased recently in the U.S., as well as in developing countries, due to an increase in the number of immune compromised individuals with HIN infection.
  • the risk of TB infection has also increased in individuals with diabetes, hemophilia, lymphomas, leukemias, and other malignant neoplasms, because these individuals have compromised immune systems.
  • Leprosy and viruses which cause neoplasia are also important intracellular pathogens worldwide. Leprosy presently causes disease in more than twelve million people, and at least 15% of human cancers are thought to be caused by neoplastic transformation of cells by viruses.
  • Intracellular infections with highly virulent strains are quickly resolved resulting in death or cure of the patient.
  • organisms of lower virulence can persist in the host and develop chronic diseases.
  • Mycobacterium infections develop through a spectrum that ranges from a state of high resistance associated with cellular immunity to an opposite extreme of low resistance associated with humoral immunity.
  • leprosy is caused by Mycobacterium leprae which remains uncultivable. The disease manifests an immuno-histological spectrum with six groups.
  • TT tuberculous leprosy
  • LL polar lepromatous leprosy
  • the reactive polar group (RR) is associated with a TH-1 immune response while the opposite pole (UU) is unreactive and is associated with a TH-2 immune response. Therefore, there are clear indications that the TH-1 immune response is the main defense mechanism in leprosy and tuberculosis. Thus, treatment and immuno- prophylaxis against these diseases should be aimed at enhancing the TH-1 pathway.
  • Allergic diseases are characterized by the sustained production of Ig E molecules against common environmental antigens. This production is dependent of IL-4 and is inhibited by gamma interferon.
  • the allergic reactions involve a TH-2 immune response which requires a low level of stimulation by allergens. Therefore, preferable treatment for allergies would include the following: switching to a TH-1 immune response, which requires a high level of stimulation; activating CD8+ T cells and the production of gamma interferon; reducing the production of Ig E and recruitment of eosinophils and mast cells; and increasing the threshold concentration of the allergen to trigger a reaction.
  • Mycobacterium gene products especially heat shock proteins, show homologies with bacterial, viral, parasitic, mycotic, and tumor antigens suggesting that these similarities may reflect regions in Mycobacterium antigens which can serve as potential inducers of cross immunity to different diseases.
  • Heat shock proteins are overexpressed by stressed cells in many pathologies including infections, cancer, and autoimmune diseases.
  • vaccinated individuals would have circulating cytotoxic T lymphocytes (CTL) that can interact and lyse the stressed cells, while the expression of putative autoimmunity antigenic domains in a susceptible host may lead to the suppression of the immune response and the chronicity of the disease.
  • CTL cytotoxic T lymphocytes
  • the available methods for prophylaxis and treatment of intracellular diseases include antibiotics, chemotherapy, and vaccines.
  • Antibiotics have not been effective in treating diseases caused by M. tuberculosis or M. leprae because the lipid-rich cell wall of a mycobacteria is impermeable to antibiotics.
  • antibiotics have no effect on viral pathogenesis.
  • Chemotherapy as a means of prophylaxis for high-risk individuals can be effective against M. tuberculosis o M. leprae, but it has disadvantages.
  • Chemotherapeutic agents have undesirable side-effects in the patient, are costly, and iead to the potential existence of multi-drug resistant Mycobacterium strains.
  • Bacterial products such as peptidoglycan, lipoproteins, lipopolysaccharides, and mycolic acids were used as therapeutic and prophylactic agents in several diseases.
  • the administration of non-specific stimulants derived from Corynebacterium parvum, Streptococci, Serratia marcescens, and Mycobacterium, to cancer patients showed some efficacy and concomitantly enhanced the immune response against the disease.
  • Adjuvants were developed to stimulate the immune response to antigenic material.
  • One such adjuvant was complete Freund's adjuvant, which consisted of killed Mycobacterium tuberculosis suspended in oil and emulsified with aqueous antigen solution. This preparation was found to be too toxic for human use. (Riott, et al., Immunology, 5th ed., Mosby, Philadelphia, pp. 332, 370 (1998).
  • the dogma furthermore, has ignored the fact that the immune response to a pathogen is a coherent response to a mosaic complex of epitopes displayed by the pathogen with some epitopes conferring protection and other epitopes mediating virulence and immunopathology. These vaccines have been unsuccessful in establishing the favored TH-1 response over the TH-2 response. Early vaccines were also not potent against intracellular diseases. The vaccines were inefficient, short-lived, or triggered inappropriate immune responses similar to hypersensitivity reactions in allergic diseases that result in necrosis, which worsens the outcome of the pathological process in many chronic infections such as tuberculosis and leprosy. For example, BCG
  • BCG Bacille-Calmette Guerin
  • M. bovis Mycobacterium strain that is closely related to M. tuberculosis.
  • BCG has been only marginally effective against leprosy and is not currently recommended for leprosy prophylaxis.
  • Results from controlled studies to determine the efficacy of BCG vaccines for TB prophylaxis have been conflicting.
  • Estimates of BCG efficacy from placebo-controlled studies range from no efficacy to 80% efficacy.
  • Jolles et al. discloses a hydrosoluble extract of mycobacteria suitable as an adjuvant, wherein delipidated bacterial residues are subjected either to a mild extraction process or treatment with pyridine followed by treatment with ethanol or water. These extracts were found to be toxic in humans, discouraging their use as a vaccine.
  • the exogenous antigen may be combined with the killed Mycobacterium vaccae by admixture, chemical conjugation or absorption, or alternatively produced by expression of an exogenous gene in Mycobacterium vaccae via plasmid, cosmid, viral or other expression vector, or inserted into the genome. While these compositions promote the TH-1 immune response, they were limited only to killed Mycobacterium vaccae cells. Further, the patent provides no guidance as to how to make Mycobacterium expression vectors, or how to incorporate the expression vectors into either a plasmid, cosmid, or viral expression vector, or how to integrate the expression vector into the genome.
  • Mycobacterium diseases advances made in the area of genetic tools and vaccine strategy included: the isolation, characterization and sequencing of the Mycobacterium plasmid pAL 5000; the identification of the kanamycin resistance gene as a selection marker for Mycobacterium, the development of the first Escherichia coli (E. coli) I Mycobacterium shuttle vectors; the construction of M. tuberculosis and M. leprae genomic libraries; and the expression of Mycobacterium DNA inE. coli. (Labidi, et al. 1984. "Plasmid profiles of Mycobacterium fortuitum complex isolates," Curr.
  • the Mycobacterium expression vectors resulting from such advancements are not suitable for vaccine development because: 1) the expression vectors are large so the vectors have limited cloning capacity and low transformation efficiency (calculated as the number of transformants obtained per microgram of vector DNA), 2) the vectors lack multiple-cloning sites, 3) the protocols for transformation of mycobacteria with these expression plasmids result in inefficient transformation, 4) the spectrum of mycobacteria transformed by the vectors is restricted because transformation is host- dependent, and 5) the current expression plasmids do not stably transform mycobacteria. Therefore, suitable Mycobacterium expression vectors are needed which can provide efficient transformation and stable expression of multiple protective immunogens in mycobacteria.
  • Suitable antigen delivery systems using nonpathogenic Mycobacterium strains, cloning vectors, and Mycobacterium expression vectors have now been found which contain protective immunogens that specifically stimulate a cell- mediated immune response by the induction of TH-1 cells, or cytotoxic T lymphocytes, and provide a consistent, prolonged immunity to intracellular pathogens.
  • Fig. 1 depicts a sequence of the origin of replication in E. coli (695 bp). The underlined base indicates the replication point.
  • Fig. 2 depicts a sequence for the kanamycin gene (932 bp).
  • the underlined sequences are in the 5' to 3' order: the (-35) region for the gene, the (-10) region for the gene, the ribosomal binding site region for the gene, the starting codon (ATG), and the stop codon (TAA).
  • Fig. 3 A depicts a sequence of the pAL 5000 origin of replication (1463 bp) obtained by restriction enzymes analysis. The numbers in superscript indicate the position of the nucleotides in the published sequence of pAL 5000
  • Fig. 3B depicts a sequence of the pAL 5000 origin of replication (1382 bp) obtained after PCR analysis.
  • the numbers in superscript indicate the position of the nucleotides in the published sequence of pAL 5000 (Labidi, et al. 1992. Plasmid 27: 130-140).
  • the underlined sequences indicate in the 5' to 3' order: the position of the forward ( F l5 F 2 and F 3 ), and the reverse (R 4 , R 3 ,
  • Fig. 4 A depicts a sequence of the attachment site (attP) and the integrase gene (mt) of the Mycobacteriophage D 29 , obtained by restriction enzymes analysis (1631 bp).
  • the numbers in superscript indicate the position of the nucleotides in the sequence.
  • the underlined sequences delimited by numbered nucleotides indicate in the 5' to 3' order: the position of the forward (F c , F l5 F 2 , F 3 , and F 4 ) and the reverse (R 4 , R 3 , R 2 , R and R primers used in PCR analysis, respectively.
  • the underlined sequences not delimited by numbered nucleotides indicate in the 5' to 3' order: the attachment site (attP), the (-35) region for the gene ( t), the (-10) region for the integrase gene (int), the ribosomal binding site region for the integrase gene (int), and the starting codon (ATG) for the integrase gene (int).
  • the stop codon for the integrase gene (int) is the TGA 1531 .
  • Fig. 4B depicts a sequence of the attachment site (attP) and the integrase gene (int) of the Mycobacteriophage D 29 , obtained after PCR analysis ( 1413 bp).
  • the numbers in superscript indicate the position of the nucleotides in the sequence.
  • the underlined sequences delimited by numbered nucleotides indicate in the 5' to 3' order: the position of the forward (F 3 , and F 4 ) and the reverse (R 4 , R 3 , and R 2 ) primers used in PCR analysis, respectively.
  • the underlined sequences not delimited by numbered nucleotides indicate in the 5' to 3' order: the attachment site (attP), the (-35) region for the gene (int), the
  • the stop codon for the integrase gene (int) is the TGA 1531 .
  • Fig. 4C depicts a sequence of the attachment site (attP) and the integrase gene (mt) of the Mycobacteriophage D 29 , obtained after PCR analysis
  • the underlined sequences not delimited by numbered nucleotides indicate in the 5' to 3' order: the attachment site (attP), the (-35) region for the gene (int), the (-10) region for the integrase gene (int), the ribosomal binding site region for the integrase gene (mt), and the starting codon (ATG) for the integrase gene (int).
  • the stop codon for the integrase gene (int) is the TGA 1531 .
  • Fig. 5 depicts a sequence for the kanamycin gene promoter(102 bp) and the first ATG codon.
  • the underlined sequences are in the 5' to 3' order: the (-35) region for the gene, the (-10) region for the gene, the ribosomal binding site region for the gene, and the starting codon (ATG).
  • Fig. 6 depicts a sequence of the pAL 5000 fragment containing the open reading frame ORF 2 (2096 bp).
  • the numbers in superscript indicate the position of the nucleotides in the published sequence of pAL 5000 (Labidi, et al. 1992. /7 ⁇ -w ⁇ /27: 130-140).
  • the underlined sequence (GGATCC is the unique Bam HI site which is spanned by the ORF 2 promoter.
  • MycofP ⁇ om Mycobacterium promoter;
  • Rep/Integ/Myc ⁇ Mycobacterium origin of replication or phage attachment site and integrase gene (either one or the other but not both is present in a given vector);
  • MCS/gen clon.” multiple cloning site for general cloning;
  • univ/select/mark.” universal selection marker;
  • o ⁇ /E.coli E. coli origin of replication.
  • the therapeutic or prophylactic vaccines of the present invention combine a protective immunogen with one or more Mycobacterium strains acting as a delivery system and an adjuvant, preferably in addition to cytokines and appropriate chemotherapy.
  • the rationale is that the Mycobacterium cells will be ingested by macrophages and remain within the macrophage, blocking the killing mechanism of the macrophage while synthesizing the protective immunogen.
  • the immunogen will be processed and presented on the macrophage cell surface to T cells, resulting in TH-1 cell activation and a cell- mediated immune response that is protective against the intracellular disease.
  • One aspect of the present invention uses an antigen delivery system in the form of a nonpathogenic Mycobacterium strain to provide products combining nontoxic immuno-regulating Mycobacterium adjuvants, nontoxic immuno-stimulating protective immunogens specific for a variety of diseases, and nontoxic amounts of cytokines that boost the TH-1 pathway.
  • the present invention uses a protective immunogen delivery system in the form of a nonpathogenic Mycobacterium strain, a genetic transfer system in the form of cloning vectors, and expression vectors to carry and express selected genes in the delivery system.
  • Protective immunogen delivery system in the form of a nonpathogenic Mycobacterium strain to provide products combining nontoxic immuno-regulating Mycobacterium adjuvants, nontoxic immuno-stimulating protective immunogens specific for a variety of diseases, and nontoxic amounts of cytokines that boost the TH-1 pathway.
  • the present invention uses a protective immunogen delivery system in the form of a nonpathogenic Mycobacterium strain, a genetic transfer system in the form of
  • the protective immunogens of the present invention form pure non- necrotizing complete granuloma.
  • Such immunogens can be protein antigens or other immunogenic products produced by culturing and killing the diseased cell or infectious microorganism, by separating and purifying the immunogens from natural or recombinant sources, or by the cloning and expression into a
  • the protective immunogens of the present invention include antigens associated with: 1) cancer including but not limited to lung, colorectum, breast, stomach, prostate, pancreas, bladder, liver, ovary, esophagus, oral and pharynx, kidney, non-Hodgkin's, brain, cervix, larynx, myeloma, corpus uteri, melanoma, thyroid, Hodgkin's, and testis; 2) bacterial infections including but not limited to mycobacteriosis (e.g., tuberculosis and leprosy), Neisseria infections (e.g., gonorrhea and meningitis), brucellosis, plague, spirochetosis (e.g., trypanosomiasis, Lyme disease and tularemia
  • mycobacteriosis e.g., tuberculosis and leprosy
  • Neisseria infections e.g., gonor
  • lipid protective immunogens for the treatment of tuberculosis, leprosy, and other mycobacterioses include but are not limited to complex lipid heteropolymers such as the phenolic glycolipids PGL I and PGL Tbl, the sulfolipid SL I, the diacyl-trehalose DAT and the lipo-oligosaccharide LOS. These lipid immunogens are not synthesized, or modified to their final forms by all Mycobacterium species.
  • the host strain must provide the necessary precursors to synthesize the desired final immunogenic products.
  • the expression system When using an expression vector, the expression system must provide the necessary genes that encode the necessary enzymes to modify the lipid to a stage where it is immunogenic.
  • the mycobacterial adjuvant of the present invention is one that boosts the TH-1 immune response, and preferably down-regulates the TH-2 response.
  • the Mycobacterium strains are characterized by their lack of pathogenicity to mammals and their capacity to be ingested mammalian macrophages
  • the Mycobacterium strains of the present invention may be live or dead upon administration When the vaccines of the present invention are administered to immunocompromised patients, only dead Mycobacterium strains are used
  • Mycobacterium strains can be obtained from the American Type Culture Collection (Rockville, MD)
  • One or more types of Mycobacterium species may be utilized in the preparation of a vaccine Examples include but are not limited to nonpathogenic Mycobacterium vaccae, Mycobacterium gastri, Mycobacterium triviale, Mycobacterium aurum, Mycobacterium thermoresistible, Mycobacterium chitae, Mycobacterium duvaln, Mycobacterium flavescens, Mycobacterium nonchromogenicum, Mycobacterium neoaurum, and Mycobacterium bovis BCG M. bovis BCG and M. gastri are the only known Mycobacterium species that have precursors for producing M. tuberculosis and M.
  • M. gastri must be used if the precursors of exogenous lipids are to be expressed in a vaccine for TB or leprosy M. gastri and M. triviale can be found in the gastrointestinal tract, and are, thus, important for use in oral vaccines
  • the Mycobacterium adjuvants of the present invention can utilize either one Mycobacterium strain or multiple strains, however, when killed Mycobacterium vaccae is used, it is preferably administered in combination with other Mycobacterium species
  • the vaccine of the present invention also comprises cytokines that associate with the TH-1 pathway
  • cytokines include but are not limited to gamma interferon (IFN), interleukin(IL)-2, IL-12,
  • IL-15 IL-15 and granulocyte macrophage colony stimulating factor (GMCSF)
  • the vaccine of the present invention may also be administered in combination with appropriate chemotherapy for treatment of patients with active diseases If a live Mycobacterium strain is used as an adjuvant, appropriate chemotherapy must be selected that does not interfere with the adjuvant function of the live Mycobacterium Examples of appropriate concommitant chemotherapy is Taxol-R for the treatment of cancer or protein inhibitors for AIDS treatment.
  • the protective immunogens, cytokines, and concommitant chemotherapy may be produced separately in a synthetic or in a recombinant form, purified by any conventional technique. They may be used in parallel with, mixed with, or conjugated to live or dead Mycobacterium cells of interest. Genetic transfer system
  • the genetic transfer system of the present invention comprises cloning vectors where the genes of interest are cloned and the transformation technique is used to introduce and express the recombinant molecules into the delivery- system.
  • Previous cloning vectors which have been used in Mycobacterium species include the extrachromosomal M. fortuitum plasmid pAL 5000 (Labidi, et al. 1992. "Cloning and DNA sequencing of the Mycobacterium fortuitum var. fortuitum plasmid, pAL 5000," Plasmid 27: 130-
  • Mycobacteriophage D 29 is a large spectrum virulent phage able to infect and efficiently reproduce itself in cultivated Mycobacterium species and attach itself to uncultivated M. leprae.
  • the cloning vectors are comprised of the minimum functional sizes of various components including the following components: the E. coli replicon, the kanamycin selection marker, the pAL 5000 origin of replication, and the D 29 attachment site (attP) and integrase gene (int).
  • the coding region for each component have been reduced to the point that further loss of base pairs resulted in loss of function, hence the designation of minimum functional size.
  • the sequences for each minimum functional component are given as follows: origin of replication in E.
  • coli (695 bp) as S ⁇ Q ID NO: 1 and Fig. 1 ; kanamycin gene (932 bp) as S ⁇ Q ID NO:2 and Fig. 2; origin of replication in pAL 5000 (1463 bp) obtained by restriction enzyme analysis as S ⁇ Q ID NO: 3 and Fig. 3A; origin of replication in pAL 5000 (1382 bp) obtained after PCR analysis as S ⁇ Q ID NO:4 and Fig. 3B; Mycobacteriophage D 29 attachment site and integrase gene (1631 bp) obtained by restriction enzyme analysis as S ⁇ Q ID NO: 5 and Fig.
  • coli origin of replication Col ⁇ l is found in most commercially available plasmid vectors designed for E. coli. Although the replication point is usually indicated for these vectors, the smallest fragment that can support an efficient replication in E. coli has not heretofore been specified.
  • pN ⁇ B 193 Guan C, New England Biolabs Inc., USA, 1993
  • This E. coli origin of replication of minimum functional size has been successfully utilized in the construction o ⁇ E.coli cloning vectors and E. coli-Mycobacterium shuttle vectors of the present invention.
  • Streptococcus faecalis 1489 bp gene coding for resistance to kanamycin has been selected as a representative selection marker for Mycobacterium (Labidi, et al. 1992. "Cloning and DNA sequencing of the Mycobacterium fortuitum var. fortuitum plasmid, pAL 5000," Plasmid 27: 130-140; Labidi, et al. 1985. "Restriction endonuclease mapping and cloning of Mycobacterium fortuitum var ⁇ . fortuitum plasmid pAL 5000," Ann. Insti.
  • Pasteur/Microbiol. 136B, 209-215) While this gene is well established as the selection marker for Mycobacterium (Konicek, et al . 1991. Folia Microbiol. 36(5), 411 -422), the smallest fragment capable of supporting kanamycin selection in Mycobacterium has not heretofore been established. It has now been found that the minimal functional sequence for this gene is about 932 bp as shown in SEQ ID:NO2 and Fig. 2.
  • the kanamycin gene of minimum functional size described herein has been successfully utilized in the construction of E. coli cloning vectors and E. coli- Mycobacterium shuttle vectors of the present invention.
  • Vectors containing a plasmid origin of replication do not usually integrate in the chromosome of the host strain. Thus, they are extra- chromosomal vectors.
  • the replication and maintenance in Mycobacterium strains of the extra-chromosomal vectors developed in this study, are supported by the origin of replication o ⁇ t e Mycobacterium fortuitum plasmid pAL 5000. Labidi, et al. 1984. "Plasmid profiles of Mycobacterium fortuitum complex isolates," Curr. Microbiol. 11, 235-240.
  • the pAL 5000 plasmid is the most thoroughly studied Mycobacterium plasmid and has been used worldwide to develop vectors for genetic transfer in Mycobacterium (Falkinham, III, J.O. and IT. Crawford. 1994. Plasmids, p. 185-198. In Barry Bloom (ed), Tuberculosis: Pathogenesis, protection and control. American Society for Microbiology, Washington, D.C.). Functional analysis of the pAL 5000 plasmid has indicated the location of two open reading frames coding for a 20 KDa and a 65 KDa protein, respectively, and a 2579 bp fragment containing its origin of replication (Labidi, et al. 1992. Plasmid 27: 130-140). In the present invention, the 2579 bp fragment was reduced through deletions with restriction enzymes to a 1463 bp fragment extending from nucleotide 4439 to nucleotide
  • Vectors can also include a phage attachment site (attP) and its accompanying integrase gene.
  • a preferred embodiment of the present invention comprises the attachment site (attP) and the integrase gene (int) of the Mycobacteriophage D 29 (Forman, et al. 1954. Am J Public Health 44: 1326-1333).
  • the phage D 29 is a large spectrum virulent phage able to infect cultivated Mycobacterium species and efficiently reproduce itself.
  • a map of its attachment site (attP) and integrase gene (int) has been determined by constructing a set of hybrid plasmids containing overlapping fragments of D 29 genome.
  • the recombinant plasmids were then electroporated into the Mycobacterium strains and plated on LB medium containing 50 uglrc ⁇ kanamycin.
  • a plasmid containing a 2589 bp fragment generated Mycobacterium transformants.
  • the 2589 bp fragment was isolated and further analyzed.
  • Another set of hybrid plasmids were constructed containing overlapping segments of the 2589 bp fragment.
  • the smallest fragment still able to generate kanamycin resistant Mycobacterium transformants were isolated and sequenced using a double strand plasmid template and sequenase version 2.0 (USB, Cleveland, Ohio, USA).
  • Subsequent deletions studies regarding the 1631 bp were performed.
  • a 1413 bp originating from base pair 119 to 1531 illustrated in Fig. 4B afforded a high transformation efficiency.
  • the MCS is a synthetic fragment of DNA containing the recognition sites for certain restriction enzymes that do not cut in the vector sequence.
  • the choice of enzymes to be included in the MCS is based on their frequent use in cloning and their availability. Representative enzymes include Bam I, EcoR V, and Pst I.
  • cloning vectors have been developed which maximize the capacity for multiple cloning sites.
  • the cloning vectors comprise each component at its minimal functional size.
  • extra-chromosomal cloning vectors have been constructed by assembling the minimum functional fragments for the E. coli origin of replication, the pAL 5000 origin of replication, the kanamycin gene, and the MCS.
  • Exemplary integrative cloning vectors have the same structure except the origin of pAL 5000 is replaced by the attP and the integrase gene of D 29 .
  • the vectors When each component of the cloning vector is reduced to its smallest functional size, the vectors have a size of about 3 Kb and a transformation efficiency about 10 8 .
  • Each vector has a theoretically unlimited cloning capacity and is capable of transforming Mycobacterium species.
  • Each cloning vector is presented in Table I.
  • Fig.7 presents a genetic map of an exemplary cloning and expression vector.
  • the present invention does not require any particular ordering of the functional components within the cloning vector.
  • the cloning vectors of the present invention do not require that each component contained in the vector be reduced to its minimum functional size.
  • the degree to which the minimal functional components are utilized in each cloning vector is dictated ultimately by the application of the vaccine and the maximum transformation size.
  • an integrative cloning vector may contain the minimal functional component for the attachment site and integrase gene while the selection marker is larger than its minimal functional size.
  • Such an arrangement can arise because the cloning vector contains only one site for cloning a protective immunogen, thereby allowing other components of the vector to range in size as long as the vector is of a small enough size to allow for efficient transformation into Mycobacterium cells.
  • the present invention uses an E. coli-Mycobacterium shuttle vector constructed by applying various recombinant DNA techniques.
  • the constructed vector can be efficiently transformed into either an E. coli or
  • the E. coli-Mycobacterium shuttle vector uses a selection marker that can be expressed in both genera.
  • One shuttle vector is comprised of a kanamycin selection marker, an origin of replication for E. coli, and an origin of replication for the Mycobacterium plasmid pAL 5000.
  • Another shuttle vector is comprised of a kanamycin selection marker, an origin of replication for E. coli, and an attachment site and integrase gene of the Bacteriophage D29.
  • Each component of the constructed shuttle vector has been reduced to its smallest functional size thereby enhancing its cloning and transformation efficiency.
  • the genetic transfer system of the present invention preferably comprises cloning vectors for more than one protective immunogen.
  • the genetic transfer system of each Mycobacterium strain comprises cloning vectors for one or more protective immunogens. Transformation
  • Mycobacterium strains have been successfully transformed through electroporation. (Labidi, et al. 1992. "Cloning and DNA sequencing of the Mycobacteriumfortuitum var. fortuitum plasmid, pAL 5000," Plasmid 27: 130-
  • MW Molecular Weight
  • bp base pair
  • Ap Ampicillin
  • Tc Tetracycline
  • Km Kanamycin
  • the expression vectors of the present invention are made by inserting functional promoters from plasmid or chromosomal origin into the cloning vectors which serve as backbones.
  • the expression vectors are tailored to carry and express selected genes in the delivery system. They contain in their structures the genetic information necessary for their auto-replication in the cytoplasm, or their integration into the chromosome of the host. They provide the promoter and the regulatory sequences necessary for 1) gene expression, and if necessary, 2) the secretion of the gene product out of the cytoplasm to the cell membrane structure or to the extracellular environment.
  • kanamycin gene is a preferred selection marker for the present invention, it is also well expressed in a wide range of hosts including Mycobacterium and E. coli species, and therefore, vectors containing the promoter of this gene can express foreign genes in E. coli and Mycobacterium strains, respectively.
  • vectors containing the promoter of this gene can express foreign genes in E. coli and Mycobacterium strains, respectively.
  • SEQ ID NO: 8 and Fig. 5 The use of a kanamycin promoter to construct E. coli- Mycobacterium expression shuttle vectors is reported for the first time.
  • Another preferred expression vector in the present invention used the promoter of pAL 5000 open reading frame (ORF) 2.
  • ORF open reading frame
  • ORF 2 encoding a 60 - 65 KDa protein in E. coli minicells was identified in the plasmid pAL 5000.
  • the 2096 bp fragment containing this open reading frame (SEQ ID NO:9 and Fig. 6) has been isolated.
  • SEQ ID NO:9 and Fig. 6 The promoter of the ORF was found in the sequence spanning the unique Bam HI site in the fragment indicated in Fig. 6.
  • the products of the invention are administered by injection given intradermal or via other routes (e.g., oral, nasal, subcutaneous, intraperitoneal, intramuscular) in a volume of about 100 microliters containing 10 7 to 10 11 live or killed cells of recombinant Mycobacterium, or the same amount of nonrecombinant Mycobacterium cells mixed with, or conjugated to predetermined amounts of the exogenous antigens, the cytokines, and/or the drugs. If the products are being used with patients with active diseases, they should be associated with drug treatments that do not interfere with the live form of the vaccine if it is being used. If the products of the invention are being used separately, they can be administered in any order, at the same or at different sites, and using the same or different routes.
  • the invention takes in consideration that the products are designed to be used in humans or in animals and therefore they must be effective and safe with or without any further pharmaceutical formulation that may add other ingredients.
  • the preferred cloning and expression vectors of the present invention comprise an E. coli-Mycobacterium shuttle vector which contains the following: an origin of replication for both E. coli (E. coli replicon) and Mycobacterium (pAL 5000 origin of replication), a kanamycin resistance marker, multiple cloning sites, promoters and regulatory sequences for secretion of gene products out of the bacteria and for insertion into the cell membrane, and the attachment site (attP) and integrase gene (int) of phage D 29 .
  • Another type of preferred cloning and expression vectors contain all of these elements listed above except the phage D 29 attachment site and integrase gene.
  • the multiple cloning sites allow cloning of a variety of DNA fragments.
  • the E. coli replicon, the pAL 5000 origin of replication, the kanamycin resistance marker, and the D 29 attP site and int genes have been mapped and reduced to their minimum functional sizes to maximize the cloning capacity of the vector and to increase the transformation efficiency.
  • a new transformation protocol was developed so that the efficiency with which these vectors transform Mycobacterium strains (10 8 Mycobacterium transformants/ ⁇ g DNA) approaches the transformation efficiency for E. coli.
  • the vaccine system of the present invention has a number of advantages over current vaccines.
  • the major advantage of such a system over current vaccines is the ability to specifically express immunogens that elicit a consistent, protective immune response, i.e., a prolonged activation of TH-1 cells with concomitant activation of macrophages.
  • Additional advantages include: 1) protective immunogens for more than one intracellular disease can be incorporated into one vaccine, 2) such a genetically engineered vaccine is flexible in that new technology can be easily incorporated to improve the vaccine, and 3) large amounts of immunogen can be synthesized by using a genetically engineered expression vector to induce protective immunity, 4) the Mycobacterium itself acts as an adjuvant injected along with the immunogen to induce immunity, 5) the vaccine is naturally targeted to macrophages because t e Mycobacterium infect these cells, 6) and prolonged immunity will result since a Mycobacterium strain remains live within by the macrophages for a long time.
  • DNA RNA and oligonucleotide primers.
  • DNA and RNA were extracted and purified at Cytoclonal Pharmaceutics, Inc., Dallas, Texas.
  • the oligonucleotide primers were purchased from National Biosciences Inc., Madison, MN., or from Integrated DNA Technologies Inc., Coralville, I A. Enzymes.
  • Restriction endonucleases were purchased from United States Biochemical Inc., Cleveland, OH.; New England Biolabs Inc., Beverly, MA.; Promega Inc., Madison, WL; Stratagene Inc., La Jolla, CA.; MBI Fermantas Inc., Lithuania.; and TaKaRa Biomedicals Inc., Kyoto, Japan. DNA ligase was purchased from Boehringer Mannheim Biochemica Inc., Indianapolis, IN.;
  • Deoxyribonucleotides and DNA polymerase I were purchased from New England Biolabs Alkaline phosphatase was purchased from Boehringer Mannheim Biochemica and New England Biolabs Taq polymerase was purchased from Qiagen Inc , Chatsworth, CA AMV reverse transcriptase was purchased from Promega Inc DNase-free RNase and RNase-free DNase were purchased from Ambion Inc , Austin, TX Computer software
  • Bacterial strains and bacteriophages were used from the collection of the Vaccine Program at Cytoclonal Pharmaceutics Inc , Dallas, TX Antibiotics ampicillin, kanamycin and tetracycline were purchased from
  • Labidi's medium The requirements for Mycobacterium species to grow are usually more complex and more diversified than those for E. coli strains Consequently, a general culture medium, hereinafter designated Labidi's medium, has been developed which can support the growth of all Mycobacterium species and which contributes to the increased transformation rate of the present invention
  • the composition of the Labidi's medium per liter contains about 0 25% proteose peptone No 3, about 0 2% nutrient broth, about 0 075% pyruvic acid, about 0 05%) sodium glutamate, about 0 5% albumin fraction V, about 0 7% dextrose, about 0 0004% catalase, about 0 005% oleic acid, L H amino-acid complex (about 0 126% alanine, about 0 097% leucine, about 0 089% glycine, about 0 086%) valine, about 0 074% arginine, about 0 06% threonine, about 0
  • this medium can be supplemented with preferred selection markers and/or with special factors required for the growth of certain species such as mycobactin for M. paratuberculosis and hemin X factor forM haemophilium.
  • mycobactin for M. paratuberculosis and hemin X factor forM haemophilium.
  • cultures were grown on Labidi's medium. The cultures were incubated at the appropriate temperature for each strain. Cultures in liquid media were shaken at 150 rpm in a rotatory shaker Gyromax 703 (Amerex Instruments Inc., Hercules, CA).
  • LB Luria Broth
  • tryptone 1% NaCl
  • yeast extract in distilled or deioninzed water
  • the solid form of the LB medium was obtained by adding 2.0% agar to the previous formula. When necessary, the medium was supplemented with selection markers.
  • the cultures were incubated at 37°C except if the culture required otherwise. Cultures in liquid media were shaken at 280 rpm in a rotatory shaker Gyromax 703 (Amerex Instruments Inc., Hercules, CA).
  • Spheroplasts were prepared from Mycobacterium cultures as previously described (Labidi, et al. 1984. Curr. Microbiol. 11, 235-240).
  • the spheroplast solution [for every ml of Mycobacterium culture (14 mg of glycine, 60 ⁇ g of D-cycloserine, 1 mg of lithium chloride, 200 ⁇ g of lysizyme, and 2 mg of EDTA)] was added to the Mycobacterium cultures in exponential growth phase, and the incubation was continued for three generations to induce spheroplast formation
  • the spheroplasts were subsequently collected by centrifugation for 20 min, at 3000 rpm, at 4°C, washed and resuspended in the spheroplast storage solution [per liter, (6 05 gm of tris, 18 5 gm of EDTA, 250 gm of sucrose, and pH adjusted to 7)] Culturing Mycobacterium for Adjuvants
  • the adjuvants are made of Mycobacterium cells harvested after preferably growing the corresponding Mycobacterium strains in a liquid protein free medium
  • the medium is inoculated and incubated at the appropriate temperature
  • the culture is shaken at 150 rpm for appropriate aeration
  • the OD 600 of the culture is monitored daily to determine when the culture reaches stationary phase
  • the number of cells per milliliter is determined through serial dilutions and plating each dilution in triplicate
  • the culture is sterilely centrifuged for 30 minutes, at 5000 rpm, at 4°C
  • the pelleted cells are washed twice with ice cold sterile distilled water and pelleted as indicated above
  • the pellet is re-suspended into pyrogen-free saline (for injection only), to form a suspension of cells ranging from 10 8 - 10 12 cells per ml
  • the Mycobacterium cell suspension is dispensed into suitable multi-dose vials and used alive, or dead P
  • the aqueous phase is extracted three times by adding 250 ⁇ l of buffered phenol and 250 ⁇ l of chloroform/iso- amyl-alcohol (24:1, v/v) each time.
  • the pellet is vortexed, microcentrifuged for 15 minutes at 14 Krpm at room temperature and the aqueous phase recovered.
  • To the last aqueous phase is added 1 ml of isopropanol, vortex briefly and microcentrifuge for 10 minutes at 14 Krpm at room temperature.
  • the pellet is dried at 37 ° C for 5 minutes and the DNA is dissolved in 50 ⁇ l of sterile distilled water.
  • Total DNA was prepared from Mycobacterium strains as described before (Labidi, A., 1986). Another preferred method is to add sterile glass beads to the pellet obtained from 20 ml of spheroplasts. The pellet is vortexed vigorously to have a homogeneous suspension. The suspension is treated with 20 ml of SI, 8 ml of SII, and 14 ml of SIII. The aqueous phase is extracted several times, each time with 10.5 ml of a buffered phenol/chloroform/iso- amyl-alcohol solution. The total DNA is precipitated with 0.6 volume of isopropanol, then dissolved in a cesium chloride gradient and ethidium bromide. The gradient is centrifuged and treated according to techniques that are well established in the art. The plasmid DNA then be separated from the chromosomal DNA.
  • Total RNA was prepared from E. coli strains containing the appropriate plasmids and application of a preferred two step protocol.
  • a crude preparation of total RNA was made using the protocol provided with the kit "Ultraspec RNA Isolation System” (Biotex Laboratories Inc., Houston, TX). Since the latter was always contaminated with plasmid DNA, the total RNA was further purified using the protocol provided with the kit "Qiagen Total RNA Isolation" (Qiagen Inc., Chatsworth, CA). The combination of the two systems efficiently separated total RNA from other contaminating nucleic acids.
  • Mycobacterium strains can be transformed only through electroporation (Labidi, A., 1986). Therefore, the bacterial cells must be made electro- competent before being subject to this procedure. E. coli strains were made electro-competent following the protocol provided with the BRL Cell Porator apparatus ( BRL Life Technologies, Gaithersburg, MD).
  • Mycobacterium strains For Mycobacterium strains, a single colony of Mycobacterium culture was inoculated into 25 ml of Labidi's medium in a 250 ml screw capped flask.
  • the culture was shaken at 150 rpm at appropriate temperature until the OD 600 reached 0.7.
  • the culture was checked for contamination by staining. If there was no contamination, a second culture was started by inoculating 50 ⁇ l of the first culture into 200 ml of Labidi's medium in a 2000 ml screw capped flask. The culture was shaken at 150 rpm at appropriate temperature until the OD ⁇ reached 0.7.
  • the culture was cooled on ice/water for 2 hours, and then the bacterial cells were harvested by centrifugation (7.5 Krpm) for 10 minutes at 4°C.
  • the first pellet was suspended into 31 ml of 3.5% sterile cold glycerol and centrifuged (5 Krpm) for 10 minutes at 4°C.
  • the second pellet was suspended into 12 ml of 7% sterile cold glycerol and centrifuged (3 Krpm) for
  • the third pellet was suspended into 6 ml of 10% sterile cold glycerol and centrifuged (3 Krpm) for 10 minutes at 4°C.
  • the fourth pellet was suspended in a minimum volume of about 2.0 ml of 10.0% sterile cold glycerol, aliquoted into 25.0 ⁇ l fractions then used immediately or stored at minus 80°C.
  • E. coli and Mycobacterium strains The technique of electroporation was applied to E. coli and Mycobacterium strains.
  • E. coli ox Mycobacterium electro-competent cells 25 ⁇ l were mixed with vector DNA (10 ng in 1 ⁇ l), incubated on ice/water for 1 minute then transferred to an electroporation cuvette (0.15 cm gap).
  • the electroporation was conducted with a BRL Cell Porator apparatus Cat. series 1600 equipped with a Voltage Booster Unit Cat. series 1612 (BRL Life Technologies, Gaithersburg, MD).
  • the Voltage Booster Unit was set at a resistance of 4 kiloohms and the Power Supply Unit was set at a capacitance of 330 microfarad, a fast charging speed rate and a low Ohm mode to eliminate extra-resistance.
  • charge/arm button was set to “charge”, the “up button” was held down until the capacitors voltage displayed in the Power Supply Unit reached 410 volts for E. coli and 330 volts for Mycobacterium strains.
  • the “charge/arm button” was set to “arm” and the capacitors voltage was allowed to fall down to 400 volts for E. coli and to 316 volts for Mycobacterium strains.
  • the “trigger button” was pushed to deliver about 2.5 kilovolts for E. coli and Mycobacterium strains, respectively.
  • These voltage values were displayed on the Voltage Booster Unit. Each voltage value corresponds to 2.5 kilovolts divided by 0.15 cm equals 16.66 kilovolts/cm across the cuvette gap forE.
  • the DNA was sequenced using a double strand plasmid template and the protocol provided with the kit "Sequenase Version 2.0" (USB, Cleveland, Ohio, USA). The sequence was computer analyzed using Mac Vector program
  • Fractions of 0.1 ml of the dilution 1 : 10 2 were used to inoculate fresh antibiotic-free Labidi's medium and allowed to grow to saturation. This procedure was repeated for six months. Each time the number of Kanamycin- resistant colonies was determined. The proportion of antibiotic-resistant colonies in the culture after the six month period was found to be 96%.
  • DNA and RNA were treated with the appropriate enzymes respectively, as recommended by the manufacturers.
  • minicells analysis was performed using the E. coli DS410, which is a mutant strain of E. coli (MinA and MinB). This mutant divides asymmetrically and produces normal cells and small anucleated cells called minicells. The minicells are easily separated from normal cells by their differential sedimentation on a sucrose gradient. If the minicells producing strain contains a multi-copy plasmid, each of its minicells will not have a chromosome but will carry at least one copy of the plasmid. Since minicells are capable of supporting DNA, RNA and protein synthesis for several hours, they are used as an in vivo gene expression system for prokaryotes. The expression product is labeled with S 35 -methionine and analyzed by protein gel electrophoresis. Nutrient Broth is the medium used in this technique.
  • Preparation of minicells originated with the preparation of electrocompetent cells of E. coli DS410 with the appropriate recombinant plasmids. Each plasmid containing clone is grown overnight in 400 ml NB having the appropriate selection markers. One clone of the non transformed DS410 was grown on 400 ml NB alone to serve as a control.
  • sucrose gradients (10-30% w/v) were prepared per clone using M9-mm-S[per liter of medium: 200 gm of sucrose, 100 ml of sterile 10X I- M9-mm, 10 ml of sterile 10 mM CaCl 2 , and 10 ml of sterile 100 mM Mg SO 4 ].
  • the gradients are then placed at minus 70 ° C for at least one hour or until the gradients are completely frozen.
  • the gradients are then placed at minus 70 ° C for at least one hour or until the gradients are completely frozen.
  • the gradients are then placed at
  • Each 3 ml of cell suspension is layered on top of a sucrose gradient. The gradients are then centrifuged for 18 minutes at 5 Krpm at 4° C. The top one- third of the white transparent minicells band are recovered from each gradient. An equal volume of M9-mm is added to each tube and centrifuged for 10 minutes at 10 Krpm at 4° C. Each peilet is subsequently resuspended in 3 ml of M9-mm and the suspension is layered on top of the last gradient and centrifuged for 18 minutes at 5 Krpm at 4° C. The top one-third of the white transparent minicells band is recovered and the optical density is read at 600 nm.
  • the number of cells in the minicells preparation is calculated using the equation of 2 OD 600 , which equals 10 10 minicells per ml.
  • the level of whole cell contamination is determined in the minicells' preparation.
  • the minicell suspension is centrifuged for 10 minutes at 10 Krpm at 4° C and resuspended in M9-mm-G [per 100 ml of medium: 30 ml of sterile (100%) glycerol, 1 ml of sterile 10 mM CaCl 2 , 1 ml of sterile 100 mM MgSO 4 , and 10 ml of sterile 10X I-M9-mm].
  • the labeling of the plasmid encoded proteins with 5 35 methionine is achieved by placing 100 ⁇ l of minicells in the microcentriuge for 3 minutes at 4° C. The pellet is resuspended in 200 ⁇ l of M9-mm and 3 ⁇ l of MAM [10.5 gm of methionine assay medium per 100 ml of medium]. The pellet is incubated at 37° C for 90 minutes and 25 ⁇ Ci of S 35 -methionine is added. The pellet is incubated at 37° C for 60 minutes. 10 ⁇ l of unlabeled MS (0.8 gm of
  • RPA Ribonuclease protection assay
  • DNA fragments from the Mycobacteriophage D 29 genome and Mycobacterium plasmid and chromosomal DNA were amplified by polymerase chain reaction using a Progene Programmable Dri-Block Cycler (Techne Inc.,
  • E. coli- Mycobacterium expression vectors containing genes encoding HIV env, rev, and gag/pol proteins (National Institutes of Health, Bethtesda MD), and genes encoding IL-2, gamma INF and GMCSF (Cytoclonal Pharmaceutics, Inc., Dallas, Texas) are electroporated into a recipient strain M. aurum. The transformants are checked for their plasmid content. A clone containing the expected hybrid plasmid is grown in the protein-free liquid medium. The inoculated medium is incubated at a temperature of 35 to 37°C. The culture is shaken at 150 rpm for appropriate aeration.
  • the OD 600 of the culture is measured daily, and a growth curve featuring optical densities versus time is established.
  • the number of cells per milliliter is determined through serial dilutions (1: 10 to 1 :10 10 ), and plating in triplicates of each dilution on Labidi's medium.
  • the culture is sterilely centrifuged for 30 minutes, at 5000 rpm, at 4°C.
  • the pelleted cells are washed twice with ice cold sterile distilled water and pelleted as indicated above.
  • the pellet is resuspended into pyrogen-free saline for injection only, to have a suspension of 10 8 to 10 12 cells per ml.
  • the Mycobacterium cell suspension is dispensed into suitable multi-dose vials.
  • the product is administered by injection given intradermal in a volume of about 100 ul containing 10 7 to 10 11 cells of recombinant Mycobacterium.
  • the cells can be killed either chemically, by radiation, or by autoclaving for 30 min, at 15 - 18 psig (104 - 124 kPa) at 120 - 122°C. If a killed form of the vaccine is used, those antigens or cytokines that may be inactivated during the process are added to the product separately, or the recombinant cells are killed by radiation.
  • Example 2 Exemplary Cancer Vaccine
  • the gene encoding the cancer antigen such as the prostate cancer antigen PSA (National Institutes of Health, Bethesda, MD), is cloned according to the procedure given in Example 1.
  • the product is prepared and adminstered according to the procedure given in Example 1.
  • Example 3 Exemplary Allergy Vaccine If the product is being used for vaccination against allergies such as reactions to the major allergen of birch pollen, the gene encoding the allergen such as the birch pollen allergen BetVla (Univeristy of Vienna, Austria) is cloned according to the procedure given in Example 1. The product is prepared and adminstered according to the procedure given in Example 1.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • AAATGGGCTC GCGATAATGT CGGGCAATCA GGTGCGACAA TCTATCGATT GTATGGGAAG 240
  • MOLECULE TYPE DNA (genomic)
  • CAGGGCTCGA CGGGAGAGCG GGGGAGTGTG CAGTTGTGGG GTGGCCCCTC AGCGAAATAT 120
  • AATACGCGCG GCGTAAGCCG CTCGCATACA TGGCGGCGTG CGCCGAAGGC CTTCGGCGCG 540
  • CTCATGACCA AAAACCCCGG CCACATCGCC TGGGAAACGG AATGGCTCCA CTCAGATCTC 600
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CTCCGGGCGG TCATGAATAC CGCTGTAGAG GACAAGCTGG TGTCGGAGAA CCCGTGCCGG 780
  • MOLECULE TYPE DNA (genomic)
  • GTCTACATCC TGGCGTGGAC CAGCCTGCGG TTCGGTGAGC TGATCGAGAT CCGCCGCAAG 900
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • TCTCCCACCG CACGCAGGGC CGTCGGCGAT TTTCAGCAGG TCGCCGCCCA TTTCCGACAT 1620

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