WO1995006726A2 - Dna molecule encoding for cellular uptake of mycobacterium tuberculosis - Google Patents
Dna molecule encoding for cellular uptake of mycobacterium tuberculosis Download PDFInfo
- Publication number
- WO1995006726A2 WO1995006726A2 PCT/US1994/009863 US9409863W WO9506726A2 WO 1995006726 A2 WO1995006726 A2 WO 1995006726A2 US 9409863 W US9409863 W US 9409863W WO 9506726 A2 WO9506726 A2 WO 9506726A2
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- WIPO (PCT)
- Prior art keywords
- polypeptide
- protein
- mycobacterium tuberculosis
- isolated
- assay
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
Definitions
- the present invention relates to a DNA molecule encoding for uptake of Mycobacterium tuberculosis and its use in drugs, vaccines, and diagnostic tests.
- Tuberculosis is the leading cause of death in the world with an estimated 9 million new cases of tuberculosis and 2.9 million deaths occurring from the disease each year. In the United States, the steadily declining incidents of tuberculosis has been reversed since 1985. This problem is compounded by the increasing incidence of drug-resistant strains of MycoJbac erium tuberculosis .
- tuberculosis There are two basic clinical patterns that follow infection with Mycobacterium tuberculosis . In the majority of cases, inhaled tubercle bacilli ingested by phagocytic alveolar macrophages are either directly killed or grow intracellularly to a limited extent in local lesions called tubercles. Infrequently in children and immunocompromised individuals, there is early hematogenous dissemination with the formation of small miliary (millet-like) lesions or life-threatening meningitis.
- the second pattern is the progression or breakdown of infection to active disease.
- Individuals infected with Mycobacterium tuberculosis have a 10% lifetime risk of developing the disease.
- the bacilli spread from the site of initial infection in the lung through the lymphatics or blood to other parts of the body, the apex of the lung and the regional lymph node being favored sites.
- Extrapulmonary tuberculosis of the pleura, lymphatics, bone, genito-urinary system, meninges, peritoneum, or skin occurs in about 15% of tuberculosis patients.
- Treatment for multi-drug resistant tuberculosis requires addition of ethambutol and/or streptomycin in the initial regimen, or second line drugs, such as kanamycin, amikacin, capreomycin, ethionamide, cyclcoserine, PAS, and clofazimin.
- second line drugs such as kanamycin, amikacin, capreomycin, ethionamide, cyclcoserine, PAS, and clofazimin.
- New drugs such as ciprofloxacin and ofloxacin can also be used.
- chemoprophylaxis with isoniazid has been about 90% effective in preventing the disease. Tuberculosis and these treatments are discussed in more detail in B. Bloom et . al.
- tuberculosis Although the currently used treatments for tuberculosis have a relatively high level of success, the need remains to improve the success rate for treating this disease. Moreover, in view of the ever-increasing level of MycoJ acterium tuberculosis strains which are resistant to conventional treatment regimens, new types of treatment must be developed. In high tuberculosis endemic areas, both in the United States and abroad, such resistant strains are becoming increasingly present.
- the present invention relates to an isolated DNA molecule conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages as well as an isolated protein or polypeptide encoded by that isolated DNA molecule.
- the molecule can be inserted as a heterologous DNA in an expression vector forming a recombinant DNA expression system for producing the protein or peptide.
- the heterologous DNA usually inserted in an expression vector to form a recombinant DNA expression system can be incorporated in a cell to achieve this objective.
- the isolated protein or polypeptide of the present invention can be combined with a pharmaceutically-acceptable carrier to form a vaccine or used alone for administration to mammals, particularly humans, for preventing infection by Mycobacterium tuberculosis .
- the protein or polypeptide of the present invention can be used to raise an antibody or a binding portion thereof.
- the antibody or binding portion thereof may be used alone or combined with a pharmaceutically-acceptable carrier to treat mammals, particularly humans, already exposed to Mycobacterium tuberculosis to induce a passive immunity to prevent disease occurrence.
- the protein or polypeptide of the present invention or the antibodies or binding portions thereof raised against them can also be utilized in a method for detection of MycoJbacterium tuberculosis in a sample of tissue or body fluids.
- the protein or polypeptide are utilized, they are provided as an antigen. Any reaction with the antigen or the antibody is detected using an assay system which indicates the presence of Mycobacterium tuberculosis in the sample.
- Mycobacterium tuberculosis can be detected in such a sample by providing a nucleotide sequence of the gene conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages or a fragment thereof as a probe in a nucleic acid hybridization assay or a gene amplication detection procedure (e.g., using a polymerase chain reaction procedure) . Any reaction with the probe is detected so that the presence of Mycobacterium tuberculosis in the sample is indicated.
- the protein or polypeptide of the present invention can also be used for purposes unrelated to the treatment or detection of Mycobacterium tuberculosis . More particularly, the ability of that protein or polypeptide to confer on Mycobacterium tuberculosis an ability to enter mammalian cells can be utilized to permit such cells to uptake other materials. This can be achieved with a product that includes a material for uptake by mammalian cells and the protein or polypeptide of the present invention associated with that material.
- Isolation of the DNA molecule of the present invention constitutes a significant advance in the treatment and detection of such bacteria. It also provides the basis for a vaccine to prevent infection by Mycobacterium tuberculosis and a pharmaceutical agent for passive immunization for those exposed to Mycobacterium tuberculosis .
- the protein utilized in the vaccine or to produce the pharmaceutical agent can be produced at high levels using recombinant DNA technology.
- the protein or polypeptide of the present invention as well as antibodies and binding portions thereof against them permit rapid determination of whether a particular individual is infected with Mycobacterium tuberculosis . Moreover, such detection can be carried out without requiring an examination of the individual being tested for an antibody response.
- the present invention's ability to confer entry of such organisms into mammalian cells has significant utility in therapeutic treatments requiring the introduction of materials into cells, particularly to macrophages.
- By associating the protein or polypeptide of the present invention with pharmaceutical agents such agents can be rapidly introduced into cells for treatment thereof. The enhanced cellular uptake of such products can reduce drug dosages, thus reducing toxicity and cost.
- binding the protein or polypeptide of the present invention to DNA fragments can be utilized in conjunction with gene therapy regimens.
- the ability of the encoded product of the DNA molecule of the present invention to augment uptake into macrophages provides an opportunity to deliver genes specifically to macrophages.
- Such a system can be used to induce not only humoral immunity but cell-mediated immunity.
- Figures 1A, IB, and IC are thin-section electron micrographs of HeLa cells infected with Mycobacterium tuberculosis strain, including H37Ra (ATCC25177) ( Figure 1A) , and the invasive recombinant strain E. coli
- XLl-Blue (pZX7) ( Figure IB and IC) .
- An electron-transparent zone surrounds the Mycobacterium tuberculosis organism (arrow in Figure 1A) .
- the cells were incubated with Mycobacterium tuberculosis strain for 72 hours in Figure 1A and with XLl-Blue (pZX7) for 7.5 hours in Figures IB and IC. Multiple organisms are visible in Figure IC, suggesting bacterial proliferation inside phagosomes. The bars represent 0.5 ⁇ m.
- Figure 2 shows the construction of unidirectional deletional subclones (pZX7.3, pZX7.4, pZX7.5, and pZX7.6) and Bam Hl-Pst I (pZX7.1) , Pst I-HinD III (pZX7.2) , and Bam HI-Eco Rl (pZX7.7) subclones from the original vector pZX7.
- the black bars represent the Afycobacterium tuberculosis DNA sequences, and the white bars represent pBluescript sequences.
- the subclone vectors were transferred into E. coli XLl-Blue and then incubated with these transformed strains for 6 hours with a HeLa cell monolayer.
- Figures 3A, 3B, and 3C are thin-section electron micrographs of human macrophages exposed to the invasive recombinant E. coli clone XLl-Blue (pZX7) for 3 hours ( Figure 3A) and 24 hours (Figure 3B) compared with cells exposed to nonpathogenic E. coli XLl-Blue (pBluescript) for 24 hours ( Figure 3C) .
- the bacteria become compartmentalized, surrounded by layers of membrane inside the macrophage ( Figure 3B) . No bacteria were visible after 24 hours by electron microscopy in macrophages exposed to XLl-Blue (pBluescript) .
- the bars represent 1 ⁇ m.
- Figure 4 shows the SDS-polyacrylamide gel electrophoresis of an acetone-precipitated soluble fraction of bacterial cell sonicate.
- the polypeptides were analyzed in a 9% gel (left) : molecular size standards (lane 1) , E. coli XLl-Blue with a vector (pZN7) containing an unrelated Mycobacterium tuberculosis DNA fragment between the Bam HI- Eco Rl pBluescript cloning sites (lane 2) , and XL1- Blue(pZX7) (lane 3) .
- the present invention relates to an isolated DNA molecule conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages.
- This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 as follows: GGATCGAATT GCTGGCCTTT GGCGGGCGAT TCGTGGAGAT CGCCCGTAGA AAGGTTCGCG 60
- CAGTCGCGCC ACGACATTCA GCAATTGGCG GCTCTGGGCG ACGTCTACGC CGACGCGGCG 540
- GAACGCGCCC GCAAACGCGG CATCACCCTG AGCAACCAGC AATACGACGG CATGTCACGG 1080
- the DNA molecule encodes for a polypeptide having a molecular weight of about 50 to 55 kilodaltons, preferably 52 kilodaltons.
- the amino acid sequence, deduced from the nucleotide sequence corresponding to SEQ. ID. No. 1, 5 represents a highly hydrophilic protein with a hydrophobic region at its carboxy terminus. It could be a secreted protein, a cytoplasmic protein, or a surface protein with its carboxy terminus attached to the outer membrane of the organism. It is believed that this protein or polypeptide 10 has the deduced amino acid sequence corresponding to SEQ. ID. No. 2 as follows:
- Pro Asp Pro Arg Tyr lie His Leu lie Pro Ala Asn Val Asn Ala Asp 50 55 60
- Ala Ala Ala lie Asp Arg Asp Thr Arg Ser Gin Ala Gin Arg Asn His
- Cys Ser lie Ala Met Pro Ala Ala Val Ala Ala Ala Leu Thr Ser Arg 465 470 475 480 Thr Asn Ala Ser Cys Ser Ser Thr Pro Ala Thr Pro Tyr Cys Ala His
- Xaa signifies a stop codon.
- Production of this isolated protein or peptide is preferably carried out using recombinant DNA technology.
- the protein or peptide is believed to have one or more antigenic determinants conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages.
- the protein or polypeptide of the present invention is preferably produced in purified form by conventional techniques.
- the protein of the present invention is secreted into the growth medium of recombinant E. coli .
- the E. coli host cell carrying a recomninant plasmid is propagated, homogenized, and the homogenate is centrifuged to remove bacterial debris. The supernantant is then subjected to sequential ammonium sulfate precipitation.
- the fraction containing the protein of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC.
- the DNA molecule conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present) .
- the heterologous DNA molecule is inserted into the expression system or vector in proper orientation and correct reading frame.
- the vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
- Recombinant genes may also be introduced into viruses, such as vaccina virus.
- Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
- Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtll, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems" Catalog
- Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation.
- the DNA sequences are cloned into the vector using standard cloning procedures in the art, as described ' by Maniatis et al. , Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982) , which is hereby incorporated by reference.
- host-vector systems may be utilized to express the protein-encoding sequence (s) .
- the vector system must be compatible with the host cell used.
- Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.) ; insect cell systems infected with virus (e.g., baculovirus) .
- the expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
- eucaryotic promotors differ from those of procaryotic promotors. Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promotors are not recognized and do not function in eucaryotic cells.
- SD Shine-Dalgarno
- This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein.
- the SD sequences are complementary to the 3'-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome.
- Promotors vary in their "strength" (i.e. their ability to promote transcription) .
- strong promotors for the purposes of expressing a cloned gene, it is desirable to use strong promotors in order to obtain a high level of transcription and, hence, expression of the gene.
- any one of a number of suitable promotors may be used. For instance, when cloning in E.
- promotors such as the T7 phage promoter, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the P R and P L promotors of coliphage lambda and others, including but not limited, to lacUV5, oipF, Jbla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 ( ac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
- a hybrid trp-lacUV5 ( ac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
- Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced.
- the addition of specific inducers is necessary for efficient transcription of the inserted DNA.
- the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside) .
- IPTG isopropylthio-beta-D-galactoside
- Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.
- the DNA expression vector which contains a promotor, may also contain any combination of various "strong" transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (SD) sequence about 7-9 bases 5' to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed.
- SD Shine-Dalgarno
- Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
- Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, and the like.
- the human immune system responds to infection by pathogenic bacteria by producing antibodies that bind to specific proteins or carbohydrates on the bacterial surface. The antibodies stimulate binding to macrophages which have receptors that bind to the F c region of the antibodies.
- Some organisms are ingested (i.e. undergo uptake) by macrophages but are not killed. Amongst these is Mycobacterium tuberculosis . As a result, such organisms are able to survive indefinitely within macrophages and, when they escape from the macrophage, cause active tuberculosis.
- the molecular basis for Mycobacterium tuberculosis uptake is suggested.
- an effective amount of the protein or polypeptide of the present invention can be administered alone or in combination with a pharmaceutically-acceptable carrier to humans, as a vaccine, for preventing infection by Mycobacterium tuberculosis .
- a pharmaceutically-acceptable carrier to humans, as a vaccine, for preventing infection by Mycobacterium tuberculosis .
- Such antibodies or binding portions thereof are administered alone or in combination with a pharmaceutically-acceptable carrier to effect short term treatment of individuals who may have been recently exposed to Mycobacterium tuberculosis .
- Antibodies suitable for use in inducing passive immunity can be monoclonal or polyclonal.
- Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest (i.e. the protein or peptide of the present invention) either in vivo or in vi tro .
- the antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line.
- the resulting fused cells, or hybridomas are cultured and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vi tro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature 256:495 (1975), which is hereby incorporated by reference.
- Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. The virus is carried in appropriate solutions or adjuvants. Following the last antigen boost, the animals are sacrificed and spleen cells removed.
- Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (PEG) or other fusing agents (See Milstein and Kohler, Eur. J. Immunol . 6:511 (1976) , which is hereby incorporated by reference) .
- PEG polyethylene glycol
- This immortal cell line which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.
- Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum.
- the antigens can be injected at a total volume of 100 ⁇ l per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis.
- the rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost.
- Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Ultimately, the rabbits are euthenized with pentobarbitol 150 mg/Kg IV. This and other procedures for raising polyclonal antibodies are disclosed in E. Harlow, et . al. , editors, Antibodies: A Laboratory Manual (1988) , which is hereby incorporated by reference.
- the vaccines and passive immunization agents of this invention can be administered orally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. They may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.
- the solid unit dosage forms can be of the conventional type.
- the solid form can be a capsule, such as an ordinary gelatin type containing the protein or peptide of the present invention or the antibody or binding portion thereof of the present invention and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch.
- these compounds are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents such as, cornstarch, potato starch, or alginic acid, and a lubricant like stearic acid or magnesium stearate.
- the protein or polypeptide of the present invention or the antibody or binding portion thereof of this invention may also be administered in injectable dosages by solution or. suspension of these materials in a physiologically acceptable diluent with a pharmaceutical carrier.
- a pharmaceutical carrier include sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
- Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
- water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.
- the protein or polypeptide of the present invention or the antibody or binding portion thereof of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
- suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
- the materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
- the protein or polypeptide of the present invention can be used as antigens in diagnostic assays for the detection of Mycobacterium tuberculosis body fluids.
- the detection of that bacillus can be achieved with a diagnostic assay employing antibodies or binding portions thereof raised by such antigens.
- Such techniques permit detection of Mycobacterium tuberculosis in a sample of the following tissue or body fluids: blood, spinal fluid, sputum, pleural fluids, urine, bronchial alveolor lavage, lymph nodes, bone marrow, or other biopsied materials.
- the assay system has a sandwich or competitive format.
- suitable assays include an enzyme-linked immunosorbent assay, a radioimmunoassay, a gel diffusion pricipitan reaction assay, an immunodiffusion assay, an agglutination assay, a florescent immunoassay, a protein A immunoassay, or an immunoelectrophoresis assay.
- the nucleotide sequence of the isolated DNA molecule conferring on Mycobacterium tuberculosis an ability to enter mammalian cells and to survive within macrophages may be used as a probe in nucleic acid hybridization assays for the detection of Mycobacteriiur* tuberculosis in various patient body fluids.
- the nucleotide sequence of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, J. Mol . Biol . , 98:508 (1975)) ; Northern blots (Thomas et al. , Proc. Nat'l Acad. Sci.
- the isolated DNA molecule of the present invention can be used in a gene amplication detection procedure (e.g., a polymerase chain reaction) . See H.A. Erlich et. al. , "Recent Advances in the Polymerase Chain Reaction", Science 252:1643-51 (1991), which is hereby incorporated by reference.
- a gene amplication detection procedure e.g., a polymerase chain reaction
- the molecular basis for the uptake phenomenon achieved by Mycobacterium tuberculosis can be utilized to effect uptake of other materials into mammalian cells. This is achieved by utilizing the protein or polypeptide of the present invention in association with such materials for uptake by mammalian cells.
- This phenomenon can be used to introduce a wide variety of materials into such cells, including antibiotics, DNA fragments, anti-neoplastic agents, and mixtures thereof.
- antibiotics including antibiotics, DNA fragments, anti-neoplastic agents, and mixtures thereof.
- the ' opportunity for direct cell entry of antibiotics constitutes a substantial advance, because they will be able to kill intracellular Mycobacterium tuberculosis .
- microspheres are impregnating microspheres with antibiotics and then coating the spheres with the protein or polypeptide of the present invention in order to achieve such uptake.
- therapeutics can be chemically linked to the protein or polypeptide of the present invention.
- This technology can be used to treat a wide array of diseases caused by intracellular pathogens.
- a repertoire of antibiotics having themselves poor cellular penetration but high activity against extracellular Mycobacterium tuberculosis when tested in vitro, can be utilized in conjunction with the protein or polypeptide of the present invention.
- intracellular delivery of anti-neoplastic agents can be greatly enhanced by conjugating such agents to the protein or polypeptide of the present invention. This will enable reductions in dosages for such agents and in their resulting toxicity.
- Another aspect of the present invention is to utilize the protein or polypeptide of the present invention in gene therapy or in a genetic vaccine where pieces of therapeutically or prophylactically useful DNA are conjugated at their thymine residues to the protein or polypeptide of the present invention via linker arms.
- genetic material can be introduced into cells to correct genetic defects or to produce a desired characteristic or products that serve as immunogens.
- Mycobacterium tuberculosis H37Ra strain ATCC 25177 genome was digested with restriction enzymes Sau3 Al and Eco Rl, and the DNA fragments were ligated into the Bam HI-Eco Rl restriction sites of a phagemid vector pBluescript II (Stratagene, La Jolla, CA) .
- the recombinant vectors were introduced into E. coli ELl-Blue (Stratagene) by electroporation.
- E. coli transformant XLl-Blue (pZX7) which harbored a plasmid (pZX7) containing a 1535-base insert in the Bam HI-Eco Rl restriction enzyme sites of the pBluescript vector, was found by the screening procedure to associate consistently with HeLa cells. It was confirmed by transmission electron microscopy that this clone entered HeLa cells (Fig.l) .
- Figure 1A shows HeLa cells infected with Mycobacterium tuberculosis strain H37Ra (ATCC 25177) , while the invasive recombinant strain E. coli XLl-Blue (pZX7) is shown in Figures IB and IC.
- the cells were incubated with Mycobacterium tuberculosis strain for 72 hours in Figure 1A and with XLl-Blue (pZX7) for 7.5 hours in Figure IB and Figure IC.
- Internalization of this clone by HeLa cells was time-dependent (Fig. IB) , with intracellular organisms visible as early as 3.5 hours after infection. Some phagosomes contained multiple organisms (Fig. IC) , which suggested that the bacteria proliferated intracellularly.
- Some of the internalized bacilli were surrounded by a distinct ETZ, similar in appearance to the clear zone surrounding Mycobacterium tuberculosis inside HeLa cells (Fig. 1A, arrow) .
- this zone represents the ETZ often seen around other pathogenic intracellular my- cobacterial organisms (See P. Draper and R.J.W. Rees, Nature 228, 860 (1970); N. Rastogi, Res. Microbiol. 141, 217 (1990) ; T. Yamamoto, M. Nishimura, N. Harada, T. Imaeda, Int. J. Lepr. 26, 111 (1958) , which are hereby incorporated by reference) or is an artifact of the preparation is not clear.
- Nonpathogenic E. coli XLl-Blue strains containing the vector pBluescript or another pBluescript-derived recombinant vector (pZN7) showed no association with HeLa cells after 7.5 hours.
- the plasmid pZX7 was double-digested with HinD III and Kpn I restriction enzymes downstream from the Eco Rl site of the Ban HI-Eco Rl DNA insert to generate a 5' protruding end adjacent to the insert and a four-base 3' protruding end adjacent to the insert and a four-base 3' protrusion at the opposite strand to protect it from Exo III digestion.
- the digested plasmid was mixed with 300 U of Exo III at 37°C, and every 30 s 2.5 ⁇ l aliquots of the Exo III digestion were transferred to tubes containing SI nuclease to remove the remaining single- stranded tails.
- the SI nuclease was inactivated by neutralization and heating at 70°C for 10 min. Klenow DNA polymerase was added to create blunt ends which were ligated to circularize the deletion-containing vectors. The ligation mixture was then used to transform the competent E. coli XLl-Blue strain by electroporation. These transformed strains were incubated for 6 hours with a HeLa cell monolayer.
- Macrophage monolayers infected with the E. coli recombinant clones of Example 1 were established on glass cover slips at the bottom of polystyrene wells. They were initially infected with ⁇ 10 over-night-growth bacteria per macrophage cell for 1 or 2 hours followed by washing with phosphate-buffered saline (pH 7.4) and incubation for an additional 1, 6, or 22 hours. Cultures were performed at 37°C in RPMI-1640 medium (Gibco) with 2% AB heat-inactivated human serum containing gentamicin (10 ⁇ g/ml) . The gentamicin was included to kill the extracellular bacteria.
- the macrophage monolayer was washed again and then lysed with sterile, distilled water. The lysate was plated on tryptic soy agar medium to obtain colony counts. For microscopy, the macrophage monolayer was fixed with 100% methanol, stained with 10% Giemsa stain, and examined by light microscopy or processed for electron microscopy.
- the monolayer that was infected for 1 hour only was examined by light microscopy immediately after it was washed, fixed, and stained.
- the macrophage lysate culture and light microscopy results are shown in Table 1, infra .
- the percentage of infected macrophages was calculated from counts or infected macrophages per 100 to 200 macrophage cells on a cover slip monolayer.
- Each E. coli strain was tested four to six times for each time point, and the means of the percentages of the cells infected by the E. coli recombinant clone and the control strains XLl- Blue (pBluescript) and XLl-Blue (pZX7.3) were compared by students T test.
- Figure 3 shows thin-section electron micrographs of human macrophages exposed to the invasive recombinant E. coli clone XLl-Blue (pZX7) for 3 hours ( Figure 3A) and 24 hours ( Figure 3B) .
- the thin-section micrograph is of human macrophages exposed to nonpathogenic E. coli XLl-Blue (pBluescript) for 24 hours. After 24 hours, bacilli were more numerous inside the cells, compartmentalized, surrounded by multiple layers of a membrane presumably of host origin (Fig. 3B) . No bacteria could be seen inside macrophages infected with E. coli (pBluescript) after 24 hours ( Figure 3C) .
- Table 1 shows the results obtained from this light microscopy and culture study of human macrophage monolayer 5 cells infected with the HeLa cell-invasive E. coli XLl-Blue (pZX7) , subclone XLl-Blue (pZX7.3), and noninvasive XLl-Blue (p. Bluescript) .
- the colony-forming units (CFU) were determined per milliliter of cell culture lysate.
- the percentage of cells infected 10 by the recombinant clone (82 ⁇ 8%) was more than five times that of cells infected by XLl-Blue (pBluescript) (15 + 6%, P ⁇ 0.001) .
- E. coli XLl-Blue pZX7.3
- E. coli XLl-Blue pZX7.3
- the DNA sequences associated with HeLa cell invasion are responsible for increased uptake by the macrophage, and the sequences that confer survival within the macrophage are located downstream of those necessary for mammalian cell entry.
- the Bam Hi-Eco Ri DNA fragment was sequenced by the chain termination method, described in F. Sanger, et. al . , "DNA Sequencing with Chain-Terminating Inhibitors," Proc. Nat. Acad. Sci., 74:5463-67, which is hereby incorporated by reference, and found to have 1535 base pairs [European Molecular Biology Laboratory (EMBL) accession number X70901] .
- the sequence showed no homology with any of the DNA sequences in the database of GenBank (R72.0) or EMBL (R31.0) . No obvious procaryotic promoter consensus sequence could be discerned.
- amino acid sequence homologies can be identified.
- a region near the NH 2 -terminus of the deduced sequence of one potential open reading frame was found to share (i) 27% identity with an 80-residue NH 2 -terminus region of internalin, a protein encoded by Listeria monocytogenes that is associated with mammalian cell entry (A.B. Hartman, M. Venkatesan, E.V. Oaks, J.M. Buysse, J. Bacteriol.
- Protein fractions analyzed by SDS-polyacrylamide gel electrophoresis were prepared as follows: A 5-ml aliquot of bacterial overnight growth (adjusted to absorbance at 550 nm at optical density 600) in tryptic soy broth containing ampicillin (100 ⁇ g/ml) was harvested by centrifugation. We then sonicated the bacterial pellet in 1.5 ml of 10 mM tris-HCI buffer (pH 8.0) containing 5 mM MgCI 2 . The sonicate was centrifuged for 25 min at 12,000 rpm in a microcentrifuge (Eppendorf model 5415C) at 4°C.
- Acetone was added to 600 ⁇ l of the supernatant in a fresh microcentrifuge tube (60% v/v) , and the mixture was centrifuged for 25 min. at 14,000 rpm at 4°C. The pellet was resuspended in 20 ⁇ l of distilled water and 20 ⁇ l of Laemmli's boiling buffer, heated over boiling water for 5 min. and analyzed by SDS-PAGE.
- the bacterial debris containing the outer membrane fraction after the first centrifugation was resuspended in 100 ⁇ l of water and 100 ⁇ l of 15 mM tris-HCI buffer (pH 8.0) containing 7.5 mM MgCI 2 and 3% (v/v) Triton X-100 and centrifuged for 25 min. at 14,000 rpm. The pellet was resuspended in 25 ⁇ l of water and 25 ⁇ l of boiling buffer and boiled it and analyzed a 20- ⁇ l aliquot of the sample by SDS-PAGE.
- the SDS-PAGE i.e., SDS-polyacrylamide gel electrophoresis
- the polypeptides were analyzed in a 9% gel (left) : molecular size standards (lane 1) , E. coli XLl-BBlue with a vector (pZN) containing an unrelated Mycobacterium tuberculosis DNA fragment between the Bam HI- Eco Rl pBluescript cloning sites (lane 2) , and XL1- Blue(pZX7) (land 3) .
- a soluble fraction of the bacterial cell sonicate of XLl-Blue contained a 52-kD polypeptide that was not detected in the soluble fraction of XLl-Blue with a pBluescript-derived vector (pZN7) harboring an unrelated Mycobacterium tuberculosis DNA fragment.
- pZX7 a soluble fraction of the bacterial cell sonicate of XLl-Blue
- pZN7 a pBluescript-derived vector harboring an unrelated Mycobacterium tuberculosis DNA fragment.
- a two- base frameshift introduced by blunt-end ligation after the 5' protruding end had been filled with Klenow DNA polymerase at the Xba I site 12 bases upstream from the Bam HI cloning site in pZX7 (confirmed by sequencing) , led to loss of association with HeLa cells of the E.
- the cloned 1535-bp fragment has more than one open reading frame or if a -single gene product mediates both cell invasion and survival inside the macrophage.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP7508259A JPH09502095A (en) | 1993-09-02 | 1994-09-01 | DNA molecule encoding the cellular uptake of Mycobacterium tuberculosis |
CA002170148A CA2170148A1 (en) | 1993-09-02 | 1994-09-01 | Dna molecule encoding for cellular uptake of mycobacterium tuberculosis |
BR9407527A BR9407527A (en) | 1993-09-02 | 1994-09-01 | Isolated dna molecule isolated protein or polypeptide recombinant dna expression system host cell vaccine to prevent infection and disease in mammals isolated antibody or binding portion of it and product for uptake of materials into mammalian cells and mammalian vaccination processes against passive immunization infection of infected mammals for detection of mycobacterium tuberculosis in a sample and cell uptake |
EP94926657A EP0724635A1 (en) | 1993-09-02 | 1994-09-01 | Dna molecule encoding for cellular uptake of mycobacterium tuberculosis |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11844293A | 1993-09-02 | 1993-09-02 | |
US08/118,442 | 1993-09-02 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1995006726A2 true WO1995006726A2 (en) | 1995-03-09 |
WO1995006726A3 WO1995006726A3 (en) | 1995-04-27 |
Family
ID=22378619
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1994/009863 WO1995006726A2 (en) | 1993-09-02 | 1994-09-01 | Dna molecule encoding for cellular uptake of mycobacterium tuberculosis |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0724635A1 (en) |
JP (1) | JPH09502095A (en) |
CN (1) | CN1133063A (en) |
BR (1) | BR9407527A (en) |
CA (1) | CA2170148A1 (en) |
WO (1) | WO1995006726A2 (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996026275A1 (en) * | 1995-02-22 | 1996-08-29 | Cornell Research Foundation, Inc. | Dna molecule encoding for cellular uptake of mycobacterium tuberculosis and uses thereof |
WO1997041252A2 (en) * | 1996-04-29 | 1997-11-06 | GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) | Dna, rna and a protein useful for detection of a mycobacterial infection |
WO1998001559A1 (en) * | 1996-07-10 | 1998-01-15 | Connaught Laboratories Limited | Genes encoding mycobacterial proteins associated with cell binding and cell entry and uses thereof |
WO1998005784A1 (en) * | 1996-08-07 | 1998-02-12 | Cornell Research Foundation, Inc. | Dna molecule fragments encoding for cellular uptake of mycobacterium tuberculosis and uses thereof |
WO1999010475A2 (en) * | 1997-08-21 | 1999-03-04 | Connaught Laboratories Limited | Attenuated strains of mycobacteria |
US6008201A (en) * | 1993-09-02 | 1999-12-28 | Cornell Research Foundation, Inc. | DNA molecule encoding for cellular uptake of mycobacterium tuberculosis and uses thereof |
US6072048A (en) * | 1997-03-10 | 2000-06-06 | Cornell Research Foundation, Inc. | DNA molecule encoding for cellular uptake of Mycobacterium tuberculosis and uses thereof |
US6177086B1 (en) | 1997-05-06 | 2001-01-23 | Cornell Research Foundation, Inc. | DNA molecule conferring on Mycobacterium tuberculosis resistance against antimicrobial reactive oxygen and nitrogen intermediates |
EP1950221A3 (en) * | 1997-08-14 | 2009-07-15 | Institut Pasteur | Genomic sequences of exported mycobacteria polypeptides, vectors containing them and applications to the diagnosis and prevention of tuberculosis |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112899329B (en) * | 2021-02-02 | 2022-05-10 | 成都可恩生物科技有限公司 | Method for producing tuberculin pure protein derivative |
-
1994
- 1994-09-01 JP JP7508259A patent/JPH09502095A/en active Pending
- 1994-09-01 WO PCT/US1994/009863 patent/WO1995006726A2/en not_active Application Discontinuation
- 1994-09-01 CA CA002170148A patent/CA2170148A1/en not_active Abandoned
- 1994-09-01 BR BR9407527A patent/BR9407527A/en not_active Application Discontinuation
- 1994-09-01 CN CN94193768.2A patent/CN1133063A/en active Pending
- 1994-09-01 EP EP94926657A patent/EP0724635A1/en not_active Withdrawn
Non-Patent Citations (2)
Title |
---|
ABSTR GEN MEET AM SOC MICROBIOL, vol.92, 1992 page 41 ARRUDA S;BOMFIM G;JOHNSON W D JR;RILEY L W; 'CLONING OF A MYCOBACTERIUM-TUBERCULOSIS GENE NECESSARY FOR INVASION OF CULTURED EPITHELIAL CELLS' * |
SCIENCE, vol.261, 10 September 1993, LANCASTER, PA pages 1454 - 1457 ARRUDA S;BOMFIN G;KNIGHTS R;HUIMA-BYRON T;RILEY L W ; 'Cloning of an M. tuberculosis DNA fragment associated with entry and survival inside cells' * |
Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6008201A (en) * | 1993-09-02 | 1999-12-28 | Cornell Research Foundation, Inc. | DNA molecule encoding for cellular uptake of mycobacterium tuberculosis and uses thereof |
US6214543B1 (en) | 1993-09-02 | 2001-04-10 | Cornell Research Foundation, Inc. | DNA molecule encoding for cellular uptake of Mycobacterium tuberculosis and uses thereof |
WO1996026275A1 (en) * | 1995-02-22 | 1996-08-29 | Cornell Research Foundation, Inc. | Dna molecule encoding for cellular uptake of mycobacterium tuberculosis and uses thereof |
WO1997041252A2 (en) * | 1996-04-29 | 1997-11-06 | GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) | Dna, rna and a protein useful for detection of a mycobacterial infection |
WO1997041252A3 (en) * | 1996-04-29 | 1997-12-11 | Biotechnolog Forschung Gmbh | Dna, rna and a protein useful for detection of a mycobacterial infection |
WO1998001559A1 (en) * | 1996-07-10 | 1998-01-15 | Connaught Laboratories Limited | Genes encoding mycobacterial proteins associated with cell binding and cell entry and uses thereof |
AU731979B2 (en) * | 1996-07-10 | 2001-04-12 | Connaught Laboratories Limited | Genes encoding mycobacterial proteins associated with cell binding and cell entry and uses thereof |
WO1998005784A1 (en) * | 1996-08-07 | 1998-02-12 | Cornell Research Foundation, Inc. | Dna molecule fragments encoding for cellular uptake of mycobacterium tuberculosis and uses thereof |
US6224881B1 (en) | 1996-08-07 | 2001-05-01 | Cornell Research Foundation, Inc. | DNA molecule fragments encoding for cellular uptake of Mycobacterium tuberculosis and uses thereof |
US6072048A (en) * | 1997-03-10 | 2000-06-06 | Cornell Research Foundation, Inc. | DNA molecule encoding for cellular uptake of Mycobacterium tuberculosis and uses thereof |
US6399764B1 (en) | 1997-03-10 | 2002-06-04 | Cornell Research Foundation, Inc | DNA molecule encoding for cellular uptake of mycobacterium tuberculosis and uses thereof |
US6177086B1 (en) | 1997-05-06 | 2001-01-23 | Cornell Research Foundation, Inc. | DNA molecule conferring on Mycobacterium tuberculosis resistance against antimicrobial reactive oxygen and nitrogen intermediates |
EP1950221A3 (en) * | 1997-08-14 | 2009-07-15 | Institut Pasteur | Genomic sequences of exported mycobacteria polypeptides, vectors containing them and applications to the diagnosis and prevention of tuberculosis |
US6136324A (en) * | 1997-08-21 | 2000-10-24 | Connaught Laboratories Limited | Attenuated strains of mycobacteria |
WO1999010475A3 (en) * | 1997-08-21 | 1999-05-20 | Connaught Lab | Attenuated strains of mycobacteria |
WO1999010475A2 (en) * | 1997-08-21 | 1999-03-04 | Connaught Laboratories Limited | Attenuated strains of mycobacteria |
US6503514B1 (en) | 1997-08-21 | 2003-01-07 | Aventis Pasteur Limited | Attenuated strains of mycobacteria |
US6548070B1 (en) | 1997-08-21 | 2003-04-15 | Aventis Pasteur Limited | Attenuated strains of mycobacteria |
Also Published As
Publication number | Publication date |
---|---|
BR9407527A (en) | 1997-11-11 |
EP0724635A1 (en) | 1996-08-07 |
JPH09502095A (en) | 1997-03-04 |
CA2170148A1 (en) | 1995-03-09 |
CN1133063A (en) | 1996-10-09 |
WO1995006726A3 (en) | 1995-04-27 |
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