MXPA99002879A - Methods for the production of bacteria containing eukaryotic genes - Google Patents

Abstract

Bacteria containing eukaryotic and/or viral genes, and often having highly pleiomorphic morphology, are obtained by culturing virally-infected eukaryotic cells under aseptic, low oxygen conditions. The bacteria so produced express products encoded by the eukaryotic genes. Analyses indicate that several isolates obtained from culturing retrovirally-infected human brain capillary endothelial cells express human-specific genes previously mapped to widely separated human chromosomes.

Description

METHODS FOR THE PRODUCTION OF BACTERIA CONTAINING EUCARIAN GENES BACKGROUND OF THE INVENTION CROSS REFERENCE TO RELATED REQUESTS This application is a continuation in part of the application with serial number 08/261, 977, filed on June 17, 1994.

FIELD OF THE INVENTION The present invention relates to methods for the production of bacteria containing eukaryotic genes. More specifically, the invention relates to methods for culturing eukaryotic cells transformed retrovirally under conditions in which the bacterium containing the eukaryotic genes subsequently is isolatable from the culture.

DESCRIPTION OF THE BACKGROUND TECHNIQUE During the last century, highly pomorphic bacteria have been isolated from human patients with a variety of diseases including cancer, acquired immunodeficiency syndrome (AIDS) and Hodgkin's disease. In the 19th century, researchers believed that cancer was caused by an infection. But around 1920, after numerous microorganisms were isolated and tested for their potential as vaccines, the researchers discovered that cancer metastasis could be caused by the spread of cancer cells within the host.

Therefore, attention on cancer research moved away from the isolation of microorganisms. However, the microorganisms continued to be isolated from blood and tumors of humans and animals with cancer (see Young, Br. Med. J. (1925) 1:60; Nuzum, Surq. Gynecol. Obstet. (1925) 1J.:343; Glover, Canada Lancet Pract. (1920) 75:92; Glover et al., Canada Lancet Pract. (1926) 66:49; Scott, Northwestern Med. (1925) 24: 162; Stearns et al., J. Bacterial (1929) 18: 227). These bacteria frequently showed characteristics of bacteria with cell wall deficiency and could be observed in the blood of cancer patients by darkfield microscopy. Apparently cancer could be induced by the injection of these bacteria in experimental animals, and it was shown that some forms of cancer were prevented by pre-vaccination with dead bacteria isolated from experimental animals affected with the specific cancer. For example, Diller vaccinated a group of mice with dead bacteria that had originally been isolated in mice with Sarcoma 180; another group of mice was maintained as a control without vaccination. All these mice were then inoculated with Sarcoma 180. Sixty percent of the mice vaccinated with the bacterium rejected the implants after 10 days and lived indefinitely, but all the control mice died due to their tumors (Ann., NY Acad. Sci. (1970) 174: 65). Similarly, Seibert vaccinated young female mice produced inbred, from a strain of mice that had a high incidence of breast cancer with heat killed bacteria, isolated from a mouse with breast cancer from that same strain. These mice demonstrated a statistically significant delay in the development of that apparently inherited breast cancer as compared to unvaccinated female controls (J. Reticuloendothelial Soc. (1977) 21: 279). The bacteria showed a remarkable tendency towards pleomorphism in culture, sometimes appearing as cocci, sometimes as curved or right sticks, sometimes as mobile bacilli, and sometimes mimicking fungi by the production of pseudohyphae or larger spore sacs. Some stages of the bacteria could be made to pass through filters designed to retain all the ordinary bacteria. By cultivating these filtrates *, the original bacteria could grow again. Later researchers confirmed and extended these findings (see Wuerthle-Caspe et al., Ann. NY Acad. Sci. (1970) 174: 636: Alexander-Jackson, Growth (1966) 30: 199; Diller et al., Ann. NY Acad. Sci. (1970) 174: 655; Seibert et al., N.Y. Acad. Sci .. Series II (1972) 34: 504; Inoue and Singer, Nature (1965) 205: 408). When they were sent to reference laboratories for identification, the organisms were classified as common bacteria such as Staphylococcus or Corynebacterium species. But the long time often required for their primary isolation, their sensitivity to the composition of the media, the fried egg appearance of many of their primary isolated colonies, and their marked pleomorphism in culture suggested that their in vivo forms were those of deficient bacteria of cell wall (Mattman, "Cell Wall Deficient Microorganisms", CRC Press: Philadelphia, 1974). More recently, similar highly pleomorphic bacteria from the blood and urine of AIDS patients have been isolated. AIDS is a complex disease in which patients infected with the human immunodeficiency virus (HIV) experience a decrease in CD4-positive lymphocytes and suffer from a series of opportunistic infections and unusual tumors. The progressive loss of CD4-positive type T cells and their subsequent clinical deterioration are directly related to increased levels of DNA from HIV. Some researchers have attributed the loss of control over HIV expression to a number of cofactors, including a variety of heterologous viruses and mycoplasma (Chowdhury et al., Biochem. Biophvs, Res. Commun. (1990) 170: 1365). It has been shown, in particular that the microorganism Mycoplasma fermentans, is present in a high percentage of individuals infected with HIV, but the role of the microbe in AIDS is still not well defined. Researchers are trying to find the relationship between mycoplasma and AIDS (Macon et al, Human Patholoqy (1993) 24: 554; Lo et al., Lancet (1991) 338: 1415; Wang et al., Lancet (1992) 340: 1312). In addition, virus-like infectious agents (VLIAs) have been isolated from AIDS patients and have been shown to cause a systemic infection. These VLIAs are heterogeneous in both size and shape (Lo et al., Am. J. Trop. Med. Hyq. (1989) 41: 364) and have been shown to have a well-defined outer limiting membrane but lack a cell wall. (Lo et al., Am. J. Trop. Med. Hyq. (1989) 40: 399). Hodgkin's disease is another tumor disease with evidence of infectious cause and perhaps that it is contagious. Deficient cell wall bacteria have been isolated from patients. Bunting first isolated some bacteria from glands from untreated cases of Hodgkin's disease. The organism was extremely pleomorphic (Bunting, Bull, Johns Hopkins Hosp. (1914) 25: 173). Subsequently, Mazet isolated 26 strains from Hodgkin patients which were also quite pleomorphic (Mazet, Montpelíer Med. 1941 (1941) 316. Deficient cell wall bacteria ("CWD") are bacteria that are highly pleomorphic, exhibit cell walls. poorly developed or absent, and include not only mycoplasma or PPLO's, but also L-shaped bacteria which have the ability to revert to cell wall-producing bacteria in culture Some CWD bacteria produce a protein that looks like the hormone chorionic gonadotropin, a substance that seems to protect trophoblastic cancer cells from immune recognition There is some evidence that a plasmid may be responsible for this property and even that these bacteria may in some way be intimately associated with retroviruses (Macomber, Medical Hvpothesis (1990) 32: 1-9.) Studies of deficient cell wall bacteria have They have been impeded by difficulties encountered in their isolation and way of cultivating them. Specific strains are often difficult to re-isolate. In addition, many questions regarding the evolutionary origins of bacteria, cell wall deficient or not, and their role in human and animal diseases remain unanswered.

BRIEF DESCRIPTION OF THE INVENTION An objective of the present invention is to provide novel bacteria that contain and express eukaryotic genes. Another object of the invention is to provide a method for producing such bacteria by culturing eukaryotic cells infected with virus under conditions in which the bacteria subsequently are isolated from the cell culture. A further objective of the present invention is to provide a process for producing biological products, in particular human biological products, by cultivating said bacteria under conditions in which such products are expressed by bacteria and are recoverable from the bacterial culture medium.

A further objective of the present invention is to provide vaccines derived from such bacteria. An object of the present invention is to provide systems for diagnosing and detecting diseases mediated by bacteria, retroviruses and retrovirals comprising antibodies to such bacteria. A further object of the present invention is to provide vectors and / or expression systems, derived from such bacteria, that express animal or eukaryotic genes. A further object of the present invention is to provide industrial enzymes and other useful biochemical products from such bacteria. A further object of the present invention is to provide therapeutically useful agents including antibiotics, derivatives of such bacteria. These and other objects of the present invention, which will be apparent during the following detailed description, have been achieved or are readily available as a result of the inventor's findings that it is possible to grow eukaryotic cells infected with viruses under low oxygen conditions. in such a way that bacteria are produced which contain and preferably express animal and / or viral genes.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES In a first embodiment, the present invention provides a bacterium that contains eukaryotic and / or viral genes. The bacteria of the invention are typically highly pleomorphic, can contain both viral and eukaryotic genes and preferably express at least one eukaryotic gene in such a way that the gene product is recoverable by culturing the cells. By "eukaryotic gene" is meant functional genetic information that is present in the eukaryotic cell that is being cultured and preferably, that encodes a protein that has commercial value and which is expressed by the bacterium. The "eukaryotic gene" present in the bacterium need not be identical to the gene present in the eukaryotic cell. For example, although genes in many eukaryotic organisms have sequences (such as introns) that do not code for the polypeptide sequence of the product of that gene, and which may not play any important role in the normal expression of the gene product, the "gene" eukaryotic "present in a bacterium of the present invention need not contain such" non-coding "sequences. There may be other differences between the gene present in the eukaryotic cell and the gene present in the bacterium, which do not affect the ability of the bacterium to express a desired gene product. In some physical or morphological aspects, the bacterium obtained in accordance with the present invention may resemble the bacteria reported as isolated from cancer patients and patients with AIDS, that is, the bacteria deficient in cell wall or also called pleomorphic. Specific examples of bacteria obtained in accordance with the present invention include: These bacteria were deposited in accordance with the Budapest treaty of June 13, 1994, at the American Type Crop Depot, 12301 Parkiawn Drive, Rockville Md 200852 under the deposit numbers shown above. Additionally, the bacteria obtained according to the present invention have been isolated and typified as Bacillus licheniformis, which is a GRAS type microorganism (generally recognized as safe). The present invention further provides a method for the production of bacteria that contains at least one eukaryotic gene (preferably animal and, more preferably, human). The method of this invention, sometimes called de novo speciation, can be divided into the following steps: i) Cultivating virally infected eukaryotic cells under low oxygen conditions to produce a bacterium containing a eukaryotic and / or viral gene; and ii) Select and replicate at least one of such bacteria. Preferably, low oxygen conditions comprise alternating anaerobic culture conditions for at least a brief period of exposure to an aerobic or microaerophilic condition. The step of selecting and replicating the bacteria preferably is carried out under standard bacteriological (aerobic) cell culture conditions. Each of the steps is preferably carried out under aseptic conditions, thereby eliminating or reducing the possibility of contamination.

I. - Cultivating virally infected cells under low oxygen conditions to produce bacteria. Appropriate virally infected eukaryotic cells such as retrovirally infected animal cells can be obtained from a variety of sources including the American Type Culture Deposit (ATCC). Alternatively, appropriate retrovirally infected cells can be prepared by, for example, infecting an animal cell with a retrovirus using conventional techniques such as those described by Robinson et al., Blood (1991) 77: 294. The term "animal" as used herein means a yeast cell or a cell isolated from one of the following phyla: Poniera, Coelenterata, Platyhelminthes, Nematoda, Rotifera, Bastrotricha, Mollusca, Annelida, Onychophora, Arthropoda, Echinodermata, Hemichordata, Chordata These cells are preferably isolated from phylum Chordata, preferably from mammals, more preferably from humans. Suitable mammalian cells include endothelial cells, including brain capillary endothelial cells, monocytic macrophages, hepatoma cells and fibroblasts. Endothelial cells are preferred. Capillar brain endothelial cells are particularly preferred, and cerebral capillary endothelial cells are most preferred. Any infectious retrovirus can be used. Preferably a retrovirus such as Moloney murine leukemia virus, L-cell virus, SIV. HIV, or the Abelson murine leukemia virus are used (Dickson et al., In "RNA Tumor Viruses: Molecular Biology of Tumor Viruses :, Vol. 1, Weiss et al., Eds, Cold Spring Harbor Laboratory Press; N: Y :, 1984) Alternatively, a eukaryotic or animal cell which contains a proviral element can be used Additionally, DNA viruses or vectors derived therefrom can be used (eg SV-40 vector). with the present invention are appropriately produced in accordance with a preferred embodiment of the invention by incubation of retrovirally infected animal cells under low oxygen conditions, such as anaerobic culture conditions with at least one intermittent exposure to microaerophilic or aerobic conditions (hereinafter further referred to as alternating anaerobic / aerobic conditions or "anaerobically / aerobically grown") in eukaryotic media such as DMEM, RPMI, F12, F-10, M199, BME (Middle Basal Eagle), Lib-15 from Leibovitz, Fischer's medium, McCoy's or Weymouth's media, or in the cell culture medium described in the following examples. The anaerobic / aerobic alternating incubation is typically conducted for at least 24 hours, preferably 24-72 hours, at 20-50 ° C, preferably 30-40 ° C, more preferably around 37 ° C. Appropriate bacteriological media (in which cells are cultured using standard bacteriological cell culture conditions following the anaerobic / aerobic culture steps, as described here) include Staphylococcus-Medium 110 agar, a decoction of sunflower seeds, moss Iceland, Irish moss (see Gloves, Can Lancet Pract. (1930) 75:92); ascitic fluid: nutrient agar 3: 1 (see Nuzum, Surv Gvnecol., Obstet. (1925) H: 343), Brain Heart Infusion, Bromotimol Blue Lactose Agar, Dubos Medium, Blood Dextrose Agar, Yeast Extract Broth- Peptone, Staphylococcus Broth, Medium PPLO with or without Violet Glass, Mannitol Salt, Thioglycolate Medium, Modified Brewer Medium, Peptone Glucose Peptone Yeast Extract, Phenol Mannitol Red Agar, Phenylethanol Blood Agar, Lamb Blood Agar, Salt Mannitol Broth , Luria-Bertani Broth, and Tripticasa Soya Broth. The appropriate anaerobic conditions include an atmosphere of 0-2% v / v oxygen, preferably 0-1% v / v oxygen, more preferably less than 0.1% v / v oxygen, more preferably 0% v / v oxygen. The anaerobic atmosphere is typically an inert gas such as N2 or Ar. Appropriate aerobic conditions typically include an atmosphere that contains more oxygen than the atmosphere used for anaerobic culture, for example, air with 5% carbon dioxide, an atmosphere of air, or an atmosphere containing up to 21% v / v oxygen .

II.- Selection and replication of bacteria After cultivation in alternating anaerobic / aerobic conditions, the medium (or cells thereof) is cultivated under conditions that help the growth and replication of the bacteria, such as the standard bacteriological cell culture conditions. For example, the medium containing the cells is subjected to the step of anaerobically / aerobically cultivating, or the cells obtained therefrom, are incubated aerobically at a temperature between 4 and 50 ° C, preferably at 20-40 ° C, for at least 24 hours, preferably for several days or more preferably for several weeks. In this context, "aerobically incubated" means growing in an atmosphere containing 2% v / v O2, preferably more than 5% v / v O2. Good results can be obtained by growing in air. In one embodiment, the medium containing the eukaryotic cells and the eukaryotic residues from the anaerobic / aerobic culture step is resuspended, diluted and aerobically recultivated. Any suitable conventional medium for culturing bacteria can be used including Mannitol Salt agar, Staphylococcus medium, Heart Brain Infusion, Bromotimol Lactose Blue agar, Dubos medium, Blood Dextrose agar, Peptone Broth-Yeast extract, Staphylococcal broth, medium PPLO with o.sin Violet Crystal (growth is tolerated by any means of enrichment). However, media with high salt content tolerate the growth of L-forms and mainly staphylococcal species), Mannitol Salt agar, Thioglycollate medium, Modified Brewer medium, Peptone Glucose agar Yeast extract, Mannitol phenol red agar, Phenylethanol Blood agar, Ram Blood agar, Salt Mannitol broth, Luria-Bertani broth, and Tripticasa Soy broth. The medium can be the same or different from the medium used during the anaerobic / aerobic eukaryotic cell culture step. Alternatively, the anaerobically / aerobically grown medium containing the eukaryotic cells is first filtered prior to the aerobic (or "bacteriological") passage. Conventional techniques can be used to filter the medium such as those described by Mattman ("Cell Wall-Deficient Forms: Stealth Pathogens," 2nd Ed., CRC Press: Boca Raton, Florida, 1993. See chapter 24 in particular). The medium is filtered appropriately through a 0.1-0.8 μm filter. Suitable filters include membranes, diatomaceous earth, porcelain, asbestos and concrete glass. Membrane filters are preferably used. The filtrate is then transferred to a bacteriological culture medium described above. Depending on the porosity of the filter, several forms of bacteria can be isolated. For example to isolate cell wall deficient bacteria, the anaerobically / aerobically grown medium is first filtered through a 0.20-0.45 μm filter, preferably a 0.22 μm filter. Bacteria with cell walls can be obtained using a filter with a larger pore size. The present invention further provides a method for producing biological products by culturing bacteria produced by the present process. The bacteria of the present invention can be used to produce recoverable amounts of biological products that are "encoded" by eukaryotic genes, such as cytokines and receptors (such as inteleukin 1-10 and interferons, and their receptors), growth factors and receptors (such as epidermal growth factor (EGF), acid fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), growth factor obtained from platelets AA, AB and BB (PDGF AA, AB and BB ), insulin-like growth factor (IGF), transforming growth factor (TGF), and its receptors, human serum albumin alpha-fetoprotein, immunoglobulins, hematopoietic growth factors (such as GM-CSF), G-CSF, etc.), coagulation factors, complement factors, steroidal hormones and their receptors (such as glucocorticoid hormones, mineralocortical hormones, sex steroidal hormones, etc., and their receptors), matrix proteins (such as fibronectin, collagen, vitronectin, etc.), other bioactive peptides (such as adrenocorticotropic hormone and fragments, angiotensin and related peptides, natriuretic atrial peptides, bradykinin and related peptides, chemotactic peptides, dynorphin and related peptides, endorphins and β-lipotropin fragments, enkephalin and related peptides, inhibitors of gastrointestinal peptide enzymes, growth hormone releasing peptides, luteinizing hormone releasing hormone and related peptides, melanocyte stimulating hormone and related peptides, neurotensin and related peptides, opioid peptides, oxytocin, vasopressin and related peptides, hormone parathyroid and fragments, peptides related to protein kinase (including PKC), somatostatin and related peptides, and substance P and related peptides (such as isoleucine, threonine, tryptophan, etc.). The bacteria of the present invention can be screened to identify and select them for the production of specific biological producers, using conventional techniques. For example, commercially available antibody probes can be used to screen these microorganisms: Appropriate antibodies are conventionally available from sources such as Sigma Chemical Co. (St. Louis, MO) and ICN Biomedical (Irvine, CA). Other techniques for identifying and / or selecting bacteria of the present invention based on the nature of the expressed eukaryotic gene product will be apparent. For example, bacteria that overproduce an amino acid can be isolated by culturing the cells, followed by the passage of the cultivar at low oxygen conditions, in a medium containing inhibitory concentrations of that amino acid. The bacteria of the present invention can also be used to understand the interrelationships between complex human gene clusters. Several of the bacteria produced by the present invention express gene products which are known to be located on several different chromosomes. Bacteria containing gene clusters provide a unique opportunity to study the function and effect of various stimuli on gene clusters in an organism with a smaller genome than the human genome. In a fourth aspect, the bacteria of the present invention can also be used to generate vaccines against retroviruses or other viruses. Since bacteria can contain both animal and viral genes, microorganisms can be used as a "modified" form of the virus to raise an immune response against the virus in a host animal. Conventional techniques can be used to generate live vaccines using bacteria. Alternatively, the bacteria can be destroyed and used to formulate dead vaccines using conventional techniques. Still further, in another embodiment, polypeptides, or fragments thereof, can be isolated from bacteria and formulated into synthetic vaccines using conventional techniques. Conventional techniques can be used to prepare vaccines such as those written in New Generation Vaccines (Woodrow and Levine, Eds., Marcel Dekker, Inc .: New York, 1990). In this aspect of the invention, a retrovirally infected animal cell which is anaerobically / aerobically cultured is preferably an animal cell of phylum Chordata, more preferably either bird, fish or mammal, useful vaccines are most preferably generated against bovine, porcine diseases. , feline, human, canine, equine, bird and fish. For example, vaccines against Staphylococcus infections in cattle can be formulated and vaccines against feline infectious viruses (such as feline infectious leukemia) can be generated. Still more in another modality, the bacteria of the present invention can be used to provide systems for the diagnosis and detection of bacteria-mediated, retrovirally and retrovirally mediated diseases including antibodies to bacterial and / or retroviral antigens. Suitable antibodies, including both monoclonal and polyclonal antibodies, can be prepared in accordance with conventional techniques using the bacteria, fragments thereof or products thereof as antigens. Appropriate techniques are described in Antibodies: A Laboratory Notebook (Harlow, E. And Lane, D., Cold Sprint Harbor Laboratory, Cold Spring Harbor, NY, 1988). The bacteria of the present invention can also be used to provide expression systems or expression vectors for the production of various animal or eukaryotic proteins for both therapeutic and diagnostic purposes. Background and appropriate techniques are described in Plasmids: A Practical Approach (2nd Edition, Hardy, K. G., IRL Press, Oxford, 1993) and Genetic Engineering of Microorganisms (Puhler, A., VCH Verlagsgesellschaft, Winheim, 1993). In addition to the biological products described above, the bacteria of the present invention can be used to provide industrially useful biological molecules, such as enzymes. "Industrial" or "bulk" enzymes include amylases, cellulases, lignocellulose degrading enzymes, pectinases, proteases, and ligases. Bacterial sources and applications of these enzymes are described in Protein Biotechnology (Walsh, G. And Headon, D., Wiley, Chichester, 1994). The bacteria of the present invention can also be used to provide antibiotics. Bacillus bacteria produce antibiotics such as bacitracin and polymyxin. Actinomycetes of the genus Streptomyces produce antibiotics such as streptomycin, chloramphenicol, tetracycline and erythromycin. The microbiology of these bacteria is described in Biology of Microorganisms (Brock, T. D., Smith, D.W., and Madigan, M.T., Pretice-Hall, 1984). Having generally described this invention, a later understanding may be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended as limiting unless otherwise specified.

EXAMPLES EXAMPLE 1 Flasks with gelatinized culture containing 5-6 x 107 endothelial cells RT-HCMV (grown to confluent aerobically) are anaerobically incubated in 50 ml of a medium consisting of 1: 1 proportions of Medium 199 and Ham's F12 medium containing 80 mM DMSO (tissue culture grade), 11 mM fructose, 35 mM succinate, and 800 mg / L glutamate. In the first anaerobic phase, anaerobic conditions are prepared by purging the medium with nitrogen for 10-15 minutes prior to cell incubation. The cultures fed with anaerobic medium are then placed in an anaerobic bottle (obtained from Becton Dicknson and modified in such a way that it has both a gas inlet and a gas outlet). The gas line to the anaerobic bottle connected to a sterile filter of 0.20 μm to properly filter the possible microbial contaminants found in the nitrogen gas container or in the gas line. The anaerobic bottle, containing the culture flasks sterilely ventilated with eukaryotic cells, is periodically sealed and flushed with sterile nitrogen gas for 2-3 hours until the effluent gas contains 0% oxygen as measured by an oximeter (available from Teledyne) to ensure an anaerobic atmosphere. The gas lines of the anaerobic bottle are then held tightly and the bottle is placed for approximately 18-24 hours in a cell incubator set at a temperature of 30 ° C. After 18-24 hours, the anaerobic vial is removed from the cell incubator. The bottle is open to the atmosphere and the sterile ventilated culture flasks are quickly sealed to prevent exposure to aerobic conditions. Each flask is examined under a tissue culture microscope at room temperature in a well-lit laboratory (fluorescent lighting). After 1-2 hours samples of 2-3 ml of the medium from each flask are aspirated under sterile conditions for subsequent microscopic examination by briefly exposing the contents of the flask to aerobic (microaerophilic) conditions. The sterile ventilated culture flasks are placed in the anaerobic flask, which is purged with sterile nitrogen gas as previously described. During this reinstitution of anaerobic conditions, the contents of the culture flask are exposed to aerobic conditions or, more likely, microaerophilic conditions. This procedure is repeated after another period of 18-24 hours. After approximately 72 hours of the anaerobic / aerobic alternating incubation as described above, the RT-HCMV endothelial cell suspension is filtered through sterile Millipore filters of 0.22 μm (available from Millipore, Bedford, MA) to exclude adequately eukaryotic bacteria and any other bacteria with a cell wall. The filtrates are seeded on Mannitol Sal agar (MSA) or in Staphylococcal Medium 110 and incubated in air at 37 ° C. After several days, different colonies appear on the plates. For the isolation of Micrococcus luteus, a sterile filter of 0.8 μm is used to exclude eukaryotes prior to aerobic bacteriological culture.

COMPARATIVE EXAMPLE 1 / A Controls containing only medium are treated in the same manner as in Example 1. Microbial growth is not observed after bacteriologically aerobically growing the medium of these control flasks.

COMPARATIVE EXAMPLE 1 / B To properly determine whether or not microbial contamination "manifests" in the endothelial cell line RT-HCMV, the endothelial cells RT-HCMV are cultured directly in a variety of bacteriological media. These media include broth Tripticase Soy Caldo Staphylococci, and the standard medium used to culture endothelial cells. RT-HCMV. The cultures are incubated in an agitator, aerobically and at 37 ° C. After 24 hours, aliquots of the three liquid cultures are sown on Staphylococcus Agar 1 10, the plates being incubated in air and at 37 ° C. After 10 days no growth is observed in these plates. After 72 hours and without observing any crude bacterial growth in liquid cultures, the medium staphylococcal culture 1 10 is filtered through a Millipore 0.22 μm filter, and the filtrates are seeded on medium staphylococcal agar plates 110. After incubation for 8 days in air at 37 ° C no bacterial growth is observed in any of these plates. In addition, the endothelial cells RT-HCMV are cultured aerobically in the medium used for the anaerobic incubation of endothelial cells RT-HCMV, and in Broth PPLO with Supplement S Bacto Mycoplasma for 14 days. These cultures are maintained under appropriate conditions for the aerobic isolation of mycoplasmas (10% CO2; 35 ° C), which are the common contaminants of cultured eukaryotic cells (Animal Cell Culture: A Practical Approach, 2nd Edition, Freshney, R. I., Ed., IRL Press Oxford, 1992). We did not isolate microplasmas in this way a highly sensitive molecular double-step PCR method using degenerate, nested primers is used to detect genetic sequences that encode the gene 16 S rARN evolutionarily conserved from some 25 different species of mycoplasma including those commonly found in cell cultures (Hopert et al., J. Immunol., Meth. (1993) 164: 91). The 16 S gene sequence of mycoplasma predicted from positive control samples, and the DNA of Mycoplasma fermentans (ATCC 19989) and Mycoplasma pirum (ATCC 25960) are amplified. No sequences of the 16 S rRNA gene from mycoplasma were amplified from the DNA of lines / strains of aerobically cultured cells, including porcine brain and non-transformed human microvascular endothelial cells, and porcine brain microvascular endothelial cells (RVTE) and human cells transformed with virus. L cells (RT-HCMV). Subsequent phylogenetic analyzes of the gene 16 S mycoplasma rRNA through Genbank indicated that the initiator / PCR method should detect the sequences of the corresponding 16 S rRNA gene from mycoplasma originating from the DNA of any anaerobic mycoplasma (s) which could be asleep in RT-HCMV endothelial cells cultured aerobically. These experiments indicated that bacterial isolates are non-contaminating in cultures of RT-HCMV endothelial cells that are actively propagated. The experiments regulate the possibility that the contamination of endothelial cells RT-HCMV is presented coincidentally, thus demonstrating the importance of the present process to obtain the bacteria.

COMPARATIVE EXAMPLE 1 / C The endothelial line RT-HCMV is subjected to rigorous sterility and tests for mycoplasma to adequately exclude the possibility that the cell line has mycoplasmic, fungal or bacterial contaminants. The methods for testing the fungal and bacterial sterility meets or exceeds the requirements USP XXII and / or 21 C: F: R: § 58. Mycoplasma assay tests for the presence of mycoplasma arable and non-culturable (cell test Vero). The sterility and mycoplasma tests used are similar to those described in Animal Cell Culture: A Practical Approach, 2a. Edition (Freshney, R.I., Ed., IRL Press, Oxford, 1992). It was found that the endothelial cell line RT-HCMV is negative in the presence of fungal or bacterial mycoplasma contaminants.

COMPARATIVE EXAMPLE 1 / D The capillary endothelial cells of the human brain are transformed with L-cell virus, a murine retrovirus closely related to Moloney murine leukemia virus, using techniques similar to those used for the establishment of retrovirally transformed porcine brain microvascular endothelial cell lines (Robinson et al. ., Blood (1991) 77: 294) to produce human capillary microvascular endothelial cells (RT-HCMV) transformed retovirally. These cells are available from the American Type Culture Deposit, Rockville, Maryland, USA, under accession number ATCC CRL 1655. RT-HCMV cells are grown to confluent in flasks of gelatinized tissue cultures with an air-containing atmosphere with 5% CO2 and at a temperature of 37 ° C. Subsequently, culture flasks containing 5-6 X107 endothelial cells RT-HCMV are subjected to various concentrations of sodium chloride, dimethylsulfoxide or hydrogen sulfide added to the standard culture media and incubated under aerobic conditions (without fluctations of the oxygen concentrations) at temperatures between 30-37 ° C. The cultures are examined daily for up to a week looking for any sign of microbial growth. In addition, samples of eukaryotic cells subjected to these conditions are examined by electron microscopy. Bacteria are not isolated from these experiments.

EXAMPLE COMPARAT ATIVO D The endothelial cells RT-HCMV are cultured in Medium 199 supplemented with 100 μg heparin / ml, 2 mM L-glutamine and 10% heat inactivated bovine serum 10 *% (FBS). Prior to sowing all cell culture flasks are pretreated for 15 minutes (followed by a 1x PBS wash) with 1% gelatin (2% sterile gelatin solution from Sigma with an equal volume of sterile water-grade tissue culture) ). Twelve T-75 flasks with standard caps (without filter) in passage 6 are used. Each flask contains approximately 2.23 x 107 viable cells (excluding trypan blue) / flask. The medium is aspirated from 6 of the flasks, and each of these flasks is fed back with half purged with sterile nitrogen (N2) and Ham / F12 (proportions 1: 1 supplemented with 80mM DMSO, 1 1mM of 25mM succinic acid fructose , 800mg of L-glutamic acid, and 100ug of heparin / ml All the medium is filtered using a Corning sterile filter unit of 0.22 μm Prior to feeding, the medium is purged with sterile nitrogen gas for 10-15 minutes. A cellless flask containing growth medium purged with sterile nitrogen is used as a medium control flask.The seven flasks are transferred to the anaerobic chamber, and the chamber is sealed and purged with sterile filtered nitrogen 4 times for approximately one period. Two hours for a total purge time of approximately one hour The air in the chamber is analyzed in terms of its oxygen concentration with a Fryrite analyzer until the reading of the O2 level is 0% for at least 2 consecutive 02 Fryrite tests. The pipe to the chamber is clamped and the chamber is placed for approximately 72 hours in an incubator at 36 ° C + - 2 ° C. Six control flasks are aspirated and fed back with the same growth medium (25ml / flask), which is not purged with N2. A flask (without cells) containing only medium is included as a medium control flask. These 7 flasks are incubated aerobically for approximately 72 hours at 36 ° C + - 2 ° C in the same incubator where the experimental chamber is housed. Approximately 24 and 48 hours after the initiation of the experiment, the chamber is opened at room temperature in a well-lit room and the caps of the flask are quickly sealed. The flasks are observed microscopically and the observations are recorded. These observations are made in a course of 10 minutes the flasks are then placed without stowing in a laminar flow chamber under fluorescent light for approximately 30 minutes. After 30 minutes the lids are loosened, and the flasks remain in the chamber for another 2-3 minutes. The flasks are then transferred to the experimental anaerobic chamber which is sealed and purged with sterile N2 until the affluent gas measures 0% oxygen. The pipe to the chamber is clamped and the chamber is placed back in the incubator. The control flasks are similarly handled and returned to the incubator. After 72 hours of incubation, the chamber and control flasks are removed from the chamber. The contents of each experimental flask are scraped and rigorously mixed and aspirated several times with a 10 ml pipette for approximately 2 minutes followed by whirling action for approximately 2 minutes. The contents of the experimental flasks are mixed, and the samples are collected for sterility and mycoplasma tests. The remnant of these mixed samples is then sterilized by filtration through a 0.22 um filter, and the filtrate samples are collected for sterility and mycoplasma tests. The control cell flasks are treated in the same manner, and the samples are collected for mycoplasma and sterility tests. The rest of the control material is filtered through a 0.22 um filter, and the filtrate is collected for sterility tests. A duplicate experiment is developed using procedures similar to those described above. One part of the bacteriological culture phase of the procedure involves the seeding of both experimental and control samples on Mannitol Sal (MSA) agar, Staph 100 agar (S100), or Blood agar (BDL), followed by incubation at 36 ° C + - 1 ° C for 14-21 days. Another part of the bacteriological culture phase involves extensive sterility tests (aerobic and anaerobic) with both positive and negative controls. Positive controls include Bacillus subtilis, Bacteroides vulgatus, Staphylococcus aureus, and Candida alblcans. These sterility tests meet or exceed USP XXIII and / or 21 C.F.R. § 610. The tests for mycoplasma (aerobic and anaerobic) are designed to detect mycoplasmas, both cultivable in agar and non-culturable in large sample volumes using 2 test systems: agar isolation and Hoescht staining of Vero cells (monkey kidney). inoculated with control and experimental samples together with controls for positive and negative mycoplasma. The tests for mycoplasma and sterility used are similar to those described in Animal Cell Culture: A Practical Approach, 2a. Edition (Freshnei, R. I., Ed., IRL Press, Oxford, 1992). After one to several weeks of incubation, five gram-positive bacilli from bacteriological cultures are isolated from samples of experimental eukaryotic cells subjected to anaerobic eukaryotic cell culture conditions with periodic introductions of an aerobic atmosphere during the eukaryotic cell culture phase. Four different colony morphologies are observed. All isolates are typified as Bacillus licheniformis. All the eukaryotic control cells (anaerobic culture only) and controls of the medium (aerobic and anaerobic / aerobic culture) are negative in terms of bacterial growth during bacteriological culture. These experiments indicate that bacteria are raised from de novo eukaryotic cells.

COMPARATIVE EXAMPLE 2 / A Two experiments are developed in which RT-HCMV cells are subjected to approximately 72 hours of anaerobic conditions in the experimental anaerobic chamber without the periodic introduction (or reintroduction) of an aerobic atmosphere during the eukaryotic cell culture phase. The controls of eukaryotic cells (only aerobic) and the controls of the medium (aerobic and anaerobic only) are developed in the same way. Bacteria or mycoplasmas are not isolated from the bacteriological cultures of the experimental and control samples.

EXAMPLE 3 To properly determine the subsequent filterability of these bacteria, the isolated microorganism, Micrococcus luteus, obtained from Example 1 after filion through a 0.8 μm filter is returned to the same initial anaerobic / aerobic alternating culture conditions used for its isolation from RT endothelial cells -HCMV. After 72 hours, the suspension of bacterial cells is filtered through a Millipore filter of 0.22 μm, seeded in MSA, and incubated in air of 37 ° C. In a period of several days, colonies of bacteria are observed growing on MSA. One of these colonies is classified as a Staphylococcus hemoliticus.

EXAMPLE 4 To demonse that bacterial isolation is not peculiar to the endothelial cell system RT -HCMV, cultured retrovirally sformed porcine brain microvascular endothelial cells are being actively propagated (Robinson et al., Blood (1991) 77: 294), L929 cells ( ATCC CCL 1) and murine lymphoma cells (ATCC TIB52) in the manner described above in Example 1. Gram positive bacteria are isolated from cultures that originally contained retrovirally sformed porcine brain microvascular endothelial cells. In L929 cells sformed with another murine retrovirus, gram positive bacteria were observed in Stem Calcium after 24 hours of inoculation of an anaerobic / aerobic experiment. In the murine lymphoma cells sformed with Abelson MuLV, gram positive bacilli were obtained.

COMPARATIVE EXAMPLE 4 / A To determine adequately whether the presence or absence of an infectious virus is integral to this procedure, similar experiments were developed with two other sformed cell lines, a human colon cell line sformed with SV40 (ATCC CRL 1807) and a cell line from human colon adenocarcinoma (ATCC HTB 38, HT-29 cells). Example 1 repeated using these cell lines in place of the endothelial cells RT-HCMV. Two gram-positive cocci and one gram-positive bacillus were isolated from the SV40 cells during the bacteriological culture phase. Bacteria were not isolated in the human colon adenocarcinoma cell line.

EXAMPLE 5 CHARACTERIZATION OF BACTERIA A. Morphology As nine bacterial isolates are filtered through a Millipore filter of 0.20 or a Millipore filter of 0.22 um prior to seeding on solid medium, by definition, these were exhibited an L-shape or wall deficiency cellular during some phase of their life cycles (Mattman, "Cell Wall - Deficient Forms: Stealth Pathogens", 2nd Ed., CRC Press: Boca Raton, Florida, 1993). Based on fermentation patterns of carbohydrates and MSA, these microorganisms are isolated and categorized into three groups, designated as I, II and III. Five of these bacterial isolates were subjected to extensive bacteriological analysis in the ATCC. Microorganisms isolated individually were classified as Micrococus luteus (isolateol, Group III), Staphylococcus aureus (isolate 2P, Group II), Staphylococcus epidermidis (isolate 5, Group I), Staphylococcus hemoliticus (isolate 1c, Group II). A fluid mobility was documented in some of the sns. In addition, several sns grew well on PPLO agar exhibiting a classic "fried egg" colony morphology. Five other bacteria obtained with the present process were classified as different isolates of Bacillus licheniformis, a GRAS microorganism. No filtration is done prior to the bacteriological culture phase in the production of these bacteria. The morphology studies of the isolates with light microscopy revealed an ultra structure that was extremely pleomorphic. In the case of Staphylococcal isolates, the bacteria exhibited a scarcely uniform coconut type morphology when cultured in Staphylococcus broth. In many samples examined, extra cellular material is present abundantly. Morphologies that do not appear to be prokaryotic or eukaryotic in nature were frequently observed in culture made in Staphylococcus broth. When they were cultured in the standard medium used for the culture of endothelial cells RT-HCMV, ie with a low salt concentration, several bizarre morphologies were observed.

B. Presence of Retroviral DNA The presence of retroviral DNA in the bacterial isolates, obtained as described above in example 1, was demonstrated by PCR amplification of a 500 bp portion of the retroviral gao gene. The gag gene of the L-cell virus is detected by DNA PCR analysis of L929 cells with primers designed to amplify a 500 bp fragment of Moloney muLV gag gene followed by restriction enzyme analysis. The endothelial cell DNA RT-HCMV and the DNA of the 2P isolate ("Staphylococccus aureus") are analyzed via PCR primers designed to amplify a 500 bp fragment of the Moloney muLV gag gene followed by restriction enzyme analysis. Using the published sequence of the Moloney MuLV (Shinnick et al., Nature (1981) 293: 543), the following oligonucleotide PCR primers were synthesized. Towards the 5 'end, bp 1561 to bp 1585; towards the 3 'end, bp 2057 to bp 2035. PCR conditions for each reaction include the use of a polymerase regulator 1 x Taq (BRL), 2.0 mM MgCL2, 200 uM dNTPs (Perkin Elmer), 1 ug of each primer , 0.5 units of TAQ polymerase (BRL), and 2 ug of genomic DNA. Cycling parameters are as follows: 40 cycles of 95 ° C for 40 seconds followed by 55 ° C for 1 minute, 72 ° for 1 minute and a final extension at 72 ° C for 10 minutes. The PCR products are subjected to electrophoresis on a 3% agarose gel (Perkin Elmer) and 500 bp products are isolated using GeneClean (Bio 101). One ug of each purified product is restricted with either Mapl (BRL) or Bgl ll (BMB). The selection of the restriction enzymes, Mspl and Bgl III, for analysis of the PCR products of the 500 bp gag gene is based on the Moloney MuLV restriction map. The sizes of the restriction fragments correspond to those predicted by a MuLV restriction map of Moloney.

Using western blot analysis, it is shown that RT-HCMC endothelial cells express the central protein of gag p30. The 2P isolated microorganism expresses significant amounts of the related core p30 protein and protein. The isolated 2W microorganism expresses small amounts of the core protein p30 with apparently faster electrophoretic mobility, a previously documented physical-chemical feature of the p30 core protein (Dickerson et al., 1984). Other CWD microorganisms isolated from two cultures also express the core protein p30. For the immunoassay analysis equal amounts of protein from endothelial cells RT-HCMV, the 2P and 2W isolates, the two microbes historically related to cancer, and the Woods strain of Staphylococcus aureus 46 were loaded on an SDS-PAGE gel. After gel electrophoresis and transfer to a polyvinylidene fluoride (BIO-RAD) membrane, the spot is probed with a 1: 500 dilution of antiserum produced against the core p30 protein of MLV gag from Moloney. The detection is developed with an alkaline phosphatase method.

C. Presence of animal DNA, animal genes, proteins or animal gene products. Subsequently, all isolates were examined for the presence of human gene products using western blott analysis or indirect fluorescent immunochemistry. The antibodies used to analyze the microbes included those directed against human serum albumin (HSA), protein C kinase (PKC), basic fibroblast growth factor (bFGF) and its receptor, and platelet growth factor dimers. AB (PDGF-AB), the PDGF receptor, α-fetoprotein, transforming growth factor ßi and HLS-DS (α chain). For western blott analysis equal amounts of protein from endothelial cells RT-HCMV, CWD 2P and 2W bacteria and several other isolated microorganisms, the two microbes historically related to cancer, and staphylococcal a-negative control staphylococcal protein were loaded in each lane The microorganisms described by Livingston-Wheeler and others, (in "The Microbiology of Cancer: Compendium," Livingston Wheeler Medical Clinic Publication: San Diego, 1977), which was isolated from a patient with metastatic cancer, produces an HCG-like protein and is identified by the ATCC as a Staphylococcus hemolyticus (ATCC 43253). The microorganism described by Seibert et al. (Ann. NY Acad. Sci. (1979) 174: 690), which was directly isolated from breast adenocarcinoma tissue, also produces an HCG-like protein, and is identified by the ATCC as a Staphylococcus warneri (ATCC 25614). Staphylococcus aureus Woods strain 46 (ATCC 10832) (Miele et al., Am. J. Vet. Res. (1981) 42: 2065) is used as an A-negative staphylococcal protein control. After gel electrophoresis and transfer to polyvinylidene difluoride (PVDF) membranes, the spots are probed with specific antibodies. The detection is carried out with an alkaline phosphatase method. Western blott analyzes for both protein kinase C and PDGF using pellet-type protein samples extracted from several of the isolated microorganisms, two control microorganisms historically related to cancer and a staphylococcal A (SPA) -negative protein from Staphylococcus aureus Wood strain 46, as a control for the pseudo immune Fc SPA reaction (Miele et al., Am. J. Vet. Res. (1981) 42: 2065) are performed. Equal amounts of total protein are placed in each lane for SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The membranes are then probed by the respective polyclonal antibodies recognizing pancreatic PKC, p.m. 77-85 kDa, and dimers of PDGF-AB, p.m. 28-34 kDa (UBI, Lake Placid, NY). The presence of these proteins was observed in several or more of the isolates. Isolated 2W expressed pancreatic PKC (p.m. approximately 80 kDa) in significant amounts. Little was detected, if any of PKC was detected in group III by western blot analysis which indicated a variation of its species in either genomic content or gene expression. The 2P isolate appeared to produce significant amounts of PDGF-AB dimers (p.m. approximately 28-34 kDa) even when compared to the positive control. As in the previous observations concerning the expression of the p30 core protein, Staphylococcus warneri isolated by Seibert et al. Appeared to produce recombinant forms of PKC and PDGF. In addition, some HSA was detected in some of the CWD bacteria (staphylococcal) by immunoblotting. The pellet protein sample of isolate 1 C contained the 63 kD form of HSA and related polyproteins and degraded proteins. The supernatant protein sample contained the 63 kD form and a significant amount of the 66 kD form. To confirm the presence of the HSA gene (cDNA form) in isolate 1 C, PCR analysis and restriction enzyme analysis of a 1.95kb cDNA fragment for HSA from the genomic DNA of isolate 1 C (Watkins and others, Proc. Nat'l Acad. Sci USA (1991) 88, 5959). The cDNA form of the HSA gene was found in the genome of isolate 1 C (Staphylococcus hemolyticus). The presence of human serum proteins was documented in an isolate classified as Bacillus licheniformis. Using immuno-fluorescent cytochemistry and a polyclonal human serum antiprotein antiserum, this isolate exhibited a further immunofluorescence, while a control of Bacillus licheniformis (source: ATCC) exhibited zero-immunofluorescence to one more. Therefore, this indirect fluorescent immunocytochemistry technique documents the presence of human serum proteins expressed in a Bacillus licheniformis isolate obtained with the present method. Finally, the inter-repeat elements Alu and LINES obtained from human eukaryotic cells were found in genomic DNA samples from the 2P and 2W isolates using the PCR technique (polymerase chain reaction). DNA extensions in gel electrophoresis of the amplification of inter-repeat elements are observed when samples of human placenta, endothelial cells and genomic DNA of the 2P and 2W isolates are used but are absent when genomic DNA samples are used as negative controls. Therefore, the presence of multiple gene products for several human genes previously mapped to widely separated chromosomes (Nierman et al., ATCC / NIH Repository Catalog of Human and Mouse DNA Probes and Librarles, American Type Culture Collection: Rockville, MD, 1992) and the presence of inter-repeating "human" elements indicate that the genomes of the bacteria obtained with the present invention are obtained from the human genome. Having already fully described the invention, it will be apparent to one skilled in the art that many changes and modifications can be made the same without departing from the essence or field of the invention as set forth herein.

Claims (25)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for producing a bacterium containing a viral and / or eukaryotic gene, which comprises culturing virally infected eukaryotic cells under low oxygen conditions to produce a bacterium containing a eukaryotic and / or viral gene.

2. The method according to claim 1, further characterized in that said low oxygen conditions comprise anaerobic culture conditions with at least one exposure of the cells to aerobic or microaerophilic culture conditions.

3. The method according to claim 2 which further comprises subjecting the cells to an aerobic culture step.

4. The method according to claim 2, further characterized in that said anaerobic culture conditions comprise an atmosphere containing less than or equal to 1% v / v oxygen, based on the total volume of atmosphere.

5. The method according to claim 4, further characterized in that said atmosphere conditions contain less than 0.1% v / v oxygen, based on the total volume of the atmosphere.

6. - The method according to claim 1, further characterized in that said virally infected eukaryotic cells are retrovirally infected mammalian cells.

7. The method according to claim 1, further characterized in that said mammalian cells are human cells.

8. The method according to claim 1, further characterized in that said eukaryotic cell is a mammalian, bird or fish cell.

9. The method according to claim 8, further characterized in that said eukaryotic cell is an endothelial cell.

10. The method according to claim 1, further characterized in that said eukaryotic cell is a mammalian cerebral capillary endothelial cell.

11. The method according to claim 1, further characterized in that said virally infected cell is infected with a virus selected from the group consisting of L-cell virus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV). ), Abelson murine leukemia virus and Moloney murine leukemia virus.

12. The method according to claim 1, further characterized in that said virus is the L cell virus.

13. The method according to claim 1, further characterized in that said culture step is performed at a temperature between 20 and 50 ° C.

14. - The method according to claim 1, characterized in that said cultivation step is carried out at a temperature of about 37 ° C.

15. A method for producing a bacterium containing a eukaryotic and / or viral gene, which comprises a) culturing virally infected eukaryotic cells under anaerobic conditions with at least one exposure to aerobic or microaerophilic conditions, b) culturing cells from the passage from a) under aerobic conditions, and c) isolating at least one bacterium containing a eukaryotic or viral gene.

16. The method according to claim 15, further characterized in that step b) of aerobic culture is performed in an atmosphere containing at least 0.1% v / v oxygen, based on the total volume of atmosphere.

17. The method according to claim 16, further characterized in that said atmosphere contains more than 1% v / v of oxygen, based on the total volume of the atmosphere.

18. The method according to claim 15, further characterized in that said virally infected eukaryotic cell is a retrovirally infected mammalian endothelial cell.

19. The method according to claim 15, further characterized in that said virally infected eukaryotic cell is an endothelial cell of the human brain capillary infected with the L-cell virus.

20. - The method according to claim 1, further characterized in that it comprises filtering the cultured cells in step a) prior to said step b) of aerobic culture.

21. The method according to claim 20, which comprises filtering the cells through a filter of 0.1 to 0.8 μm.

22. The method according to claim 21, further characterized in that said filter is 0.1 to 0.45 μm.

23. The method according to claim 22, further characterized in that said filter is 0.22 μm.

24. A bacterium containing a eukaryotic gene prepared by a method according to claim 1. 25.- A bacterium containing a eukaryotic gene prepared by a method according to claim 15.