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WO1997008955A1 - Systeme d'apport bacterien - Google Patents

Systeme d'apport bacterien Download PDF

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Publication number
WO1997008955A1
WO1997008955A1 PCT/US1996/014190 US9614190W WO9708955A1 WO 1997008955 A1 WO1997008955 A1 WO 1997008955A1 US 9614190 W US9614190 W US 9614190W WO 9708955 A1 WO9708955 A1 WO 9708955A1
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WO
WIPO (PCT)
Prior art keywords
shigella
cell
attenuated
flexneri
dna
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PCT/US1996/014190
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English (en)
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WO1997008955A9 (fr
Inventor
Arthur A. Branstrom
Donata R. Sizemore
Jerald C. Sadoff
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Department Of The Army, Us Government
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Filing date
Publication date
Priority claimed from US08/523,855 external-priority patent/US5824538A/en
Application filed by Department Of The Army, Us Government filed Critical Department Of The Army, Us Government
Priority to EP96932169A priority Critical patent/EP0881884A4/fr
Priority to IL123569A priority patent/IL123569A/en
Priority to CA002231332A priority patent/CA2231332C/fr
Priority to AU71059/96A priority patent/AU731061B2/en
Priority to JP9511361A priority patent/JP2000500734A/ja
Publication of WO1997008955A1 publication Critical patent/WO1997008955A1/fr
Publication of WO1997008955A9 publication Critical patent/WO1997008955A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0283—Shigella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • a bacterial vector capable of delivering functional nucleic acids to cells can be produced by introducing a bacterial plasmid containing promoters and other instructions recognized by eukaryotic cells into bacteria capable of invading cells, or being taken up by cells, or capable of releasing the nucleic acids such that they are taken up by cells.
  • the bacteria used in this delivery system do not have to be alive in order to deliver the nucleic acids of choice.
  • the nucleic acids delivered to the cell in this way can direct the eukaryotic cell to produce antigens or other functional molecules.
  • bacterial delivery systems therefor can be used as vaccines to prevent or treat infectious diseases and cancer, down regulate the immune system in the case of tissue rejection in transplantation, prevent or treat autoimmune diseases and other diseases related to dysregulation of the immune system.
  • the bacterial delivery systems can be used for gene therapy or gene replacement for treatment or amelioration of disease such as hereditary genetic diseases, cancers and virus infections.
  • Direct DNA-mediated immunization is another approach to the introduction of functional nucleic acids and vaccine development.
  • Highly purified bacterial plasmid DNAs expressing desired proteins under the control of viral promoters have been injected primarily into muscle or skin by traditional needle and syringe or by other more exotic methods such as biolistic transfection with DNA-coated gold microparticles (for review see Donnelly, J.J. et al . J. Immunol . Methods (1994)176: 145) .
  • Investigators using this technology have been able to elicit neutralizing antibodies, cytotoxic T lymphocytes and protection to challenge in several animal models of infection ranging from influenza to malaria.
  • bacteria as a delivery system as described in this invention is a unique method of delivering DNA to mammalian cells and has the potential to provide a simple, inexpensive way of extending DNA immunization to the local immune system and beyond through oral and other mucosal routes of immunization.
  • live bacteria have been utilized as vaccines in order to protect against subsequent infection. Attenuated or less virulent Shigella, Salmonella, Listeria , and other bacteria have been given orally to immunize against subsequent infection with more virulent forms of these bacteria. Likewise, attenuated bacterial and mycobacterial organisms such as Bacille Calmette-Guerin (BCG) have been administered parenterally to protect against related organisms such as M. tuberculosis. Genes from bacteria, viruses and parasites have been cloned into a variety of bacteria and mycobacteria for the purpose of directing the bacteria to express the foreign antigen or impart on the bacteria certain desired properties for use as a live vaccine.
  • BCG Bacille Calmette-Guerin
  • Examples include cloning the invasion genes of Shigella into the normally non-invasive E. coli rendering the E. coli invasive and therefore more suitable for use as a vaccine strain, or cloning of P. falciparum malaria genes into Salmonella typhimurium which subsequently express these malaria proteins and, following oral administration of the bacteria, induce specific cytotoxic T cell immunity and protection in mice against malaria challenge (Sadoff et al . Science (1988) 240:336-338; Aggrawal et al . J. Exp. Med. (1990) 172:1083-1090).
  • the bacterial delivery system of the present invention is designed to deliver functional nucleic acids which direct eukaryotic cells to produce antigens and other functional molecules. In this case, toxicity to the carrier is eliminated because plasmid-encoded gene expression is dependent upon the machinery of the eukaryotic cell allowing proper folding of the antigen for presentation or direction of cell functions. In addition, if desired, it can be used to deliver prokaryotically produced antigens and functional molecules.
  • Shigella was chosen as an example of a bacterial delivery system because of its ability to invade cells, escape from the phagosome, and enter into the cytoplasm of eukaryotic cells. These properties are not required of a bacteria chosen for application of the present invention, but simplified the experimental system. Shigella serves as an example of both nucleic acid delivery and bacterial antigen delivery with vaccine utility. Shigellae are enteric pathogens that invade the human colonic epithelium and multiply intracellularly, causing bacillary dysentery. Bacillary dysentery is caused by all members of the genus Shigella (S. boydii , S. dysenteriae, S. flexneri , and S. sonnei) .
  • Shigellosis is prevalent in developing countries, but is also found in industrialized nations, especially in institutional settings. It has been estimated that Shigellosis is the cause of half a million deaths a year, mostly among children, making the development of a safe and effective Shigella vaccine important (Stole, B. J. et al . J. Infect . Dis . (1982) 146: 177). All documents cited herein supra or infra are hereby incorporated by reference. To cause dysentery, Shigella strains must be able to recognize, invade and multiply within epithelial cells of the colon (LaBrec, E. H. et al . J. Bacteriol . (1964) 88: 1503).
  • an attenuated Shigella strain that can deliver functional nucleic acids to cells and deliver heterologous and homologous antigens. Even though a specific bacteria is described herein and is shown to deliver nucleic acids to eukaryotic cells whether the bacteria were alive or inactivated, this invention is applicable to all bacteria and mycobacteria. Plasmids introduced into other cells such as plant cells may also render these cells capable of delivering nucleic acids. Specifically, the attenuated Shigella strain of the present invention is capable of delivering functional nucleic acids and serving as a vaccine candidate itself against Shigella infections. The attenuated Shigella strain of the present invention enters the cell but, once inside the host cell, dies releasing its contents.
  • the attenuated Shigella strain described herein is sufficiently attenuated to not cause disease, while still maintaining the ability to enter mammalian cells.
  • This strain is shown to be protective against Shigella flexneri 2a strain 2457T challenge in the guinea pig keratoconjunctivitis model, an animal model wherein the invasion of the corneal epithelium by Shigella mimics the process seen in the intestinal epithelium of the human or primate host (Mackel et al . Am. J. Hyg. (1961) 73: 219-223; Sereny, B. Acta Microbiol . Acad. Sci . Hung. (1962) 9: 55-60).
  • Shigellae was placed in Shigella flexneri 2a strain 2457T for the specific purpose of delivering DNA to mucosal epithelial cells of the gut.
  • DAP diaminopimelate
  • This mutant strain of Shigella represents a highly attenuated bacterial vector, which is capable of invading mammalian cells and providing protective immunity against strain specific Shigella infection, as well as serving as a delivery vehicle for oral and other mucosal DNA immunization and gene therapy strategies.
  • an attenuated strain of Shigella which retains the ability to enter a cell, but dies once inside the cell.
  • the attenuated strain of Shigella can be used as a vaccine for treatment or reduction of the severity or symptoms of disease caused by Shigella or for protection against Shigella infections.
  • an attenuated and inactivated strain of Shigella which retains the ability to enter a cell, but dies once inside the cell.
  • the attenuated and inactivated strain of Shigella can be used as a vaccine for treatment or reduction of the severity or symptoms of disease caused by Shigella or for protection against Shigella infections.
  • It is still another object of the invention to provide a method for attenuating different strains of Shigella for use as a protective vaccine against infection or for ameliorating disease symptoms caused by Shigella infection.
  • the DNA encoding desired gene(s) or antigen(s) can be introduced into the described attenuated Shigella strain of the present invention or an attenuated/inactivated Shigella strain and the recombinant attenuated Shigella strain allowed to enter mammalian cells.
  • the recombinant attenuated Shigella will die once inside the cell, successfully delivering functional foreign DNA to mammalian cells.
  • Such a delivery vehicle could be used for oral and other mucosal immunization and gene therapy strategies.
  • Still another object of the invention is to provide an attenuated strain of S. flexneri which is mutant in the asd gene for use as a vaccine against infection by S. flexneri , for reducing the symptoms in an individual caused by such an infection, or as a delivery vehicle for heterologous antigens or DNA.
  • a further object of the present invention is to provide a safer strain which can be used in diagnostic assays for detecting of disease caused by Shigella or determining exposure to Shigella in an individual and a kit therefor. It is yet another object of the invention to provide Shigella components for the production of antibodies for use in a diagnostic assay for the detection of Shigella in a sample.
  • Figure 1 shows the construction of a ⁇ asd derivative of Shigella flexneri 2a strain 2457T
  • Figure 2 represents results from the use of strain 15D as a carrier to deliver pCMV ⁇ , a mammalian DNA expression plasmid, to BHK cells.
  • the number of surviving 15D (o) and 15D(pCMV ⁇ ) ( ⁇ ) were determined over a 48 hour time course.
  • Units of ⁇ -galactosidase activity per mg protein were also determined for BHK cells alone (o) , BHK cells infected with 15D (•) and BHK cells infected with 15D(pCMV ⁇ ) (—) .
  • a flask of semi-confluent BHK cells consists of approximately 0.5-1 x IO 7 cells. Determinations of ⁇ -galactosidase activity were made on an estimated 0.5 x 10 7 cells; and
  • Figure 3 shows results of intracellular immunostaining to detect expression of ⁇ -galactosidase in BHK cells infected with 15D and 15D(pCMV ⁇ ) .
  • Immunostained infected BHK cells after the addition of gentamicin containing medium (B) 15D(pCMV ⁇ ) 30 minutes, (C) 15D 4 hours, (D) 15D(pCMV ⁇ ) 4 hours, (E) 15D(pCMV ⁇ ) 24 hours, (F) 15D(pCMV ⁇ ) 48 hours, (G) 15D 24 hours and (H) BHK cells alone; (B-H 10X fluorescence phase lens) .
  • Figure 4 shows lymphoproliferative responses induced by ConA ( Figure 4A) , E. coli LPS ( Figure 4B) , heat-killed 2457T ( Figure 4C) , and purified ⁇ -galactosidase ( Figure 4D) from mice receiving a concentrated bacterial suspension intranasally.
  • Splenocytes (1 xl ⁇ 5 /well) were cultured in the presence of 5 ⁇ g/ml ConA, 2.5 ⁇ g/ml E.
  • Figure 5 is a Western showing antibody responses to ⁇ -galactosidase of intranasally inoculated mice. Groups of mice were inoculated with either 15D, 15D(pCMV ⁇ ) , or 15D(pCMV ⁇ ) containing 50 ⁇ g/ml of DAP. Sera were tested for reactivity to ⁇ -galactosidase. Lane A, coomassie stained SDS-PAGE gel.
  • Immunoblot lanes B-G were exposed to 1:50 dilution of pooled sera from mice inoculated with: B, IO 6 15D; C, 10 7 15D; D, 10 7 15D(pCMV ⁇ ) ; E, 10 6 15D(pCMVB) ; F, 10 7 15D(pCMV ⁇ ) + DAP; and G, IO 6 15D(pCMV ⁇ ) + DAP.
  • the present invention describes an attenuated Shigella strain and a process for the production of an attenuated Shigella strain for use as an immunogen for protection against Shigella infections, and for use as a carrier for the delivery of heterologous antigens, for the delivery of DNA to mucosal surfaces, or for use in a diagnostic assay.
  • This process is generally applicable to all bacteria and mycobacteria.
  • the present invention describes the construction of an isolate of Shigella flexneri containing a deletion in the gene encoding aspartate b-semialdehyde dehydrogenase (ASD) , an essential enzyme required for synthesizing the bacterial cell wall constituent diaminopi elic acid (DAP) .
  • ASD aspartate b-semialdehyde dehydrogenase
  • DAP diaminopi elic acid
  • the Shigella flexneri 2a strain 2457T was mutated by integration of a deleted E. coli asd gene containing a 553 bp deletion from position 439 to 991 of the structural gene (SEQ ID NO: 1) into its chromosome.
  • a kanamycin resistance cassette containing the complete Tn5 kanamycin gene was cloned between the flanking sequences of the mutant asd gene.
  • any Shigella strain can be mutated to provide an asd mutant as an attenuated strain.
  • the strain does not need to be virulent, but preferably should have the ability to enter or be taken up by the target cell.
  • the asd mutation will facilitate the destruction of the bacteria once the bacteria is inside the cell.
  • any gene other than asd can be mutated to have the same effect on the bacteria, namely retain the ability to enter the cell and die once inside the cell or be attenuated to such an extent that clinical symptoms be acceptable. Examples of such genes include, but are not limited to, thyA, genes for LPS production, htrA and htrB, and dut .
  • a mutation in the gene of choice can be any chemical change in the DNA leading to a change in the genetic character such that the function of the gene product is lost or altered resulting in the inability of the bacteria to survive inside the host cell.
  • Chemical changes in DNA include, but are not limited to, single or multiple deletion, single or multiple point mutation, integration of another gene or genes or portions of genes into the structural portion of the gene to be mutated, and the addition or deletion of transposons (Please see review by Kleckner et al . J. Mol . Biol . (1977) 116: 125). strains which include mutations in addition to the asd mutation are contemplated, and are within the scope of the invention.
  • the attenuated Shigella 15D strain was prepared as follows.
  • a gene encoding E. coli asd was amplified using PCR in order to incorporate restriction sites necessary for cloning into a vector.
  • any homologous asd gene could be used to generate an asd deletion in Shigella .
  • Homologous genes include, but are not limited to, asd sequences obtained from Corynebacterium giutamicum, Bacillus subtilis, Mycobacterium smegmatis, Pseudomonas aeruginosa, Leptospira interrogans, Bordetella pertussis, Corynebacterium flavum, Neisseria meningitidis, Vibrio cholera, Mycobacterium bovis, Streptomyces skiyoshiensis, Streptococcus mutans, Vibrio mimicus, and Brucella species.
  • any method of incorporating the necessary restriction sites for cloning into a vector of choice can be used such as the use of linkers or adaptors, blunt end cloning into a polylinker and other DNA cloning techniques known to a person of ordinary skill in the art (For review, please see Current Protocols in Molecular Biology r F. M. Ausubel et al. Eds. Greene Publishing Associates and Wiley-Interscience, New York) .
  • any vector which can be linearized for the insertion of the fragment of interest can be used for cloning and are known to people in the art. Examples of vectors include, but are not limited to, high copy plasmids, phagmids, single copy vectors, expression vectors, and phages.
  • the resulting plasmid with E. coli asd was reverse PCR amplified to delete 553 bp of the E. coli asd structural gene (position 439 to 991) to produce a mutant E. coli asd or ⁇ asd (SEQ. ID. NO:2).
  • Any other method known to people in the art for introducing mutations, deleting genes or portions of genes can be used, such as, for example Bal 31 digestion, multiple restriction digestion or recombination.
  • the kanamycin resistance (Kan r ) cassette from the commercial plasmid pUC4K-KIXX (Pharmacia) was purified and cloned between the flanking ⁇ asd sequences producing ⁇ asd: :Kan r .
  • any gene or genes, whether for antibiotic resistance, or for the purpose of gene therapy or antigen production can be inserted in the asd deletion. Methods for the formation of proper ends for fragment ligation are known to people in the art. Furthermore, it is not necessary to insert a gene in the asd deletion, the deletion itself is sufficient to confer the mutant phenotype and produce an attenuated Shigella .
  • the vector pCVD442 is a mobilizable suicide vector containing sacB as a positive counter selection system for recombination.
  • Any vector with an origin of replication that does not function in Shigella would serve as an acceptable suicide vector.
  • a counter selective gene such as sacB, EF-G, klah, B or C, ⁇ P gene, or the T7 bacteriophage genes 1.2 or 10 is preferable but not necessary, for selection of transformants.
  • E. coli strain SMIO ⁇ pir was used for transformations using the ligations of ⁇ asd: :Kan r into the pCVD442. Any strain which allows for the propagation of the suicide vector, and is a suitable strain for conjugations in Shigella can be used. Vectors and suitable bacteria are within the knowledge of people in the art.
  • the SMIO ⁇ pir (pCVD422: : ⁇ asd: :Kan r ) was conjugated to S. flexneri 2a strain 2457T (pAB322[Tet r , Amp 3 ]) and Amp r /Tet r conjugants selected. Conjugation of Shigella is well known to a person with ordinary skill in the art. Any method for tagging the recipient strain could be used. An auxotrophic marker or antibiotic marker allows for selection over the donor strain. Similarly, the suicide vector could be introduced directly into Shigella by transformation or electroporation. Growing the conjugants on sucrose, a standard protocol for sacB containing plasmids, resulted in a second recombination event producing the isolate 15D, given ATCC accession number ATCC 55710.
  • the isolate of choice was obtained by screening for Kan r and a requirement for DAP.
  • the isolate of choice can be screened for a requirement for DAP if the mutation is in the ASD gene, or for a requirement for the product of the gene which was deleted, or for the presence of a gene inserted into the bacteria.
  • Other screening methods are known to people in the art and dependent on the particular specifics of the strain. For example, positive selection could also be performed by scoring for a marker gene such as xyiE which would be maintained between the recombining fragments.
  • the present invention relates to a method for the delivery of a desired gene or genes into a cell, the method comprising the steps of:
  • any gene or genes can be introduced into the Shigella chromosome or virulence plasmid by methods described above, or alternatively can be carried by Shigella in a replicating or nonreplicating plasmid.
  • the vectors of interest can be introduced via transformation, electroporation, transfection or conjugation.
  • Genes for immunizations would include genes encoding foreign antigens from organisms causing, for example, diarrheal diseases such as rotavirus, sexually transmitted diseases such as human immunodeficiency virus, Neisseria gonorrhoeae, and human papilloma virus, and gastrointestinal diseases such as the ulcer causing Helicobacter pylori .
  • the attenuated Shigella was shown to deliver DNA and antigens to cells whether the bacteria was alive or inactivated.
  • Inactivation of bacteria is known in the art and can be achieved, for example, by heating to 56°C for 30 minutes. Inactivation can only be performed to the extent that delivery of functional nucleic acids is not unduly compromised.
  • DNA encoded antigens to the mucosal immune system by Shigella may permit mucosal immunization simultaneously with multiple antigens that can be directed for class I and/or class II presentation, stimulation of Thl or Th2 help, or secreted while maintaining the proper folding and conformational epitopes for IgA and IgG antibody production. Similar methods can be used for the delivery of DNA for gene therapy and correction of inborn errors of metabolisms.
  • Such genes would include, for example, replacement of defective genes such as the CFTR gene for cystic fibrosis or introduction of new genes such as reverse transcriptase or protease antisense genes for the treatment of HIV or genes to upregulate Thl immune responses such as interleukin-12 (IL-12) or genes to up- or down-regulate certain receptors, metabolites or hormones such as cholesterol and cholesterol receptors, insulin and insulin receptors, or genes encoding products that can kill cancer cells such as Tumor Necrosis Factor (TNF) , or genes to upregulate systems that have decreased for a variety of reasons including aging such as secretion of growth hormone, stimulation of osteocytes to promote bone growth and down regulation of osteoclasts to decrease bone desorption.
  • TNF Tumor Necrosis Factor
  • Similar methods can be used for delivery of nucleic acids to down regulate the immune system in an antigen specific manner or general manner in order to prevent or control autoimmune diseases or other diseases involved in dysregulation of the immune system or for prevention or treatment of specific diseases or conditions including transplantation.
  • autoimmune diseases or other diseases involved in dysregulation of the immune system or for prevention or treatment of specific diseases or conditions including transplantation.
  • Examples include the prevention or treatment of autoimmune encephalitis, multiple sclerosis, lupus erythematosis, diabetes mellitus, Crohn's disease and other inflammatory bowel diseases, and rheumatoid arthritis and other inflammatory joint and skin diseases.
  • Other examples include down regulation of immune responses that inhibit appropriate protective or curative immune responses such as down regulation of immune responses that distract from protective and curative immune responses to cancer and other diseases.
  • Th2 responses For example, down regulation of Th2 responses when Thl responses are appropriate for prevention and treatment of cancer, Leishmania , Mycobacteri um tuberculosis, and HIV. This can be accomplished using this methodology througQN, and HIV. This manipulation of the unique immunosuppressive properties of the gut and other local immune systems in combination with the ability to code for production of the appropriate cytokine milieu for induction of the appropriate immune response and suppression of inappropriate responses.
  • the present invention relates to a method for the introduction of antigens of interest into cells.
  • a method for the introduction of antigens of interest into cells would comprise introduction of the desired DNA or antigen into attenuated or attenuated/inactivated Shigella such that the desired antigens are produced, and administering said Shigella to an individual.
  • Said antigens can be produced during the life cycle of the Shigella prior to entering said cells.
  • These antigens can be expressed from a prokaryotic promoter, and can either be constitutively expressed or induced.
  • genes include those from parasitic organisms from which an immune response is desired.
  • the present invention relates to a method for the introduction of DNA or antigens of interest into cells in vi tro. Such a method would comprise introduction of the desired DNA or antigen into attenuated or attenuated/inactivated Shigella such that the desired antigens are produced, and administering said Shigella to cells.
  • Shigella infects several different cells types, such as BHK (baby hamster kidney cells) , HeLa (Human cervical epitheloid carcinoma) , CaCo-2 (human colonic adenocarcinoma) and therefor is capable of delivering desired DNA or antigens into cells wherein said DNA can be expressed.
  • Cells following DNA delivery can be transplanted for therapeutic purposes, for gene therapy or used as reagents in diagnostic assays.
  • the present invention relates to a method for the production of invasive bacterial strains.
  • the invasion genes that shigellae utilize can be inserted into other bacteria, such as E. coli , for example.
  • E. coli a bacteria found in the natural flora of the intestine, is that the body will not raise an immune response against the bacteria, allowing multiple doses of the desired antigen or DNA to be introduced, and the immune response to be raised against the desired antigen and not against the bacteria delivering the foreign antigen.
  • the virG gene, or other chromosomally encoded factors, and the virulence plasmid containing the virulence genes found in Shigella may be used to engineer an invasive strain from a non-invasive candidate (Please see Sansonetti et al . Infect . Immun. (1983) 39:1392).
  • the present invention relates to a vaccine against Shigella infection.
  • the attenuated S. flexneri strain of the present invention can be used as an immunizing agent against S. flexneri infection. This strain has been shown to elicit a protective immune response in a guinea pig keratoconjunctivitis animal model.
  • Other Shigellae strains can be attenuated similarly to the S. flexneri by introducing a mutation in a Shigellae gene as described above such that the resultant Shigella enters the cell and subsequently dies.
  • Such a mutation can be in the asd gene for example, and the resulting attenuated strains used as a vaccine against infection with the specific serotype of shigellae strain used, for example, S. boydii , S. dysenteriae, S. flexneri , and S. sonnei .
  • the attenuated Shigella vaccine can be prepared in the form of a mixed vaccine which contains one strain or several different strains of attenuated Shigella . Further, the vaccine can include at least one other antigen as long as the added antigen does not interfere with the effectiveness of the attenuated Shigella vaccine and the side effects and adverse reactions, if any, are not increased additively or synergistically.
  • Vaccines are prepared for oral administration, either as liquid solutions or suspensions; solid form suitable for solution in, or suspension in, liquid prior to administration.
  • the preparation may also be emulsified, or the ingredients are often mixed with excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, nose drops or powders and contain about 10 - 10 12 attenuated and/or attenuated/inactivated Shigella .
  • Vaccines can also be in the form of injectables.
  • Suitable excipients would include, for example, saline or buffered saline (pH about 7 to about 8) , or other physiologic, isotonic solutions which may also contain dextrose, glycerol or the like and combinations thereof. However, agents which disrupt or dissolve lipid membranes such as strong detergents, alcohols, and other organic solvents should be avoided.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) , N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1•-2'-di palmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP
  • MTP-PE monophosphoryl lipid A
  • TIBI cell wall skeleton
  • the effectiveness of an adjuvant may be determined by measuring the level of desired immune response directed against the Shigella , carried antigen, or DNA encoded antigen resulting from administration of the attenuated Shigella , in vaccines which are also comprised of the various adjuvants.
  • the vaccine can be administered in the form of a liquid or suspension prepared as discussed above. Additional formulations which are suitable for other modes of administration include suppositories. Additionally, the vaccine can be lyophilized.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the attenuated Shigella enough to generate the desired immune response, i.e., protection or reduction of disease incidence or severity without causing undesirable, adverse side affects, generally in a range of 10 10 12 colony forming units of attenuated Shigella per dose.
  • the vaccine may be administered orally, subcutaneously, intradermally, or intramuscularly in a dose effective for the production of the desired immune response.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.
  • the quantity to be administered which is generally in the range of or 10 to 10 12 colony forming units of attenuated and/or attenuated/inactivated Shigella per dose, depends on whether it is acting as a vaccine to Shigella or a carrier of heterologous antigens or DNA, on the subject to be treated, capacity of the subject's immune system to develop the desired immune response, and the degree of protection desired.
  • Precise amounts of the vaccine to be administered may depend on the judgement of the practitioner and may be peculiar to each subject, antigen, or use of the Shigella as a vaccine or carrier.
  • the vaccine may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • the dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
  • suitable immunization schedules include: (I) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to elicit the desired immune responses expected to confer protective immunity, or reduce disease symptoms or reduce severity of disease.
  • the generation of protective immunity against Shigella with an attenuated Shigella vaccine may reasonably be expected after a primary course of immunization consisting of 1 to 3 inoculations. These could be supplemented by boosters at intervals (e.g., every two years) designed to maintain a satisfactory level of protective immunity.
  • the present invention relates to a method of detecting the presence of Shigella antigens or an immune response against Shigella , in particular, S. flexneri , in a sample.
  • Shigella antigens or an immune response against Shigella in particular, S. flexneri
  • One advantage of using the attenuated Shigella of the present invention is the reduction in cumbersome safety procedures necessary with highly infective natural Shigella ; the attenuated Shigella presents a reduced risk to the operator due to the inability of the bacteria to survive inside the host cell.
  • Detection protocols may be based, for example upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example use solid supports, or may be by immunoprecipitation.
  • a label may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules.
  • Assays which amplify the signals from the probe are also known examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA or ELISPOT assays.
  • a diagnostic assay can be constructed, for example, by coating a surface (i.e. a solid support) for example, a microtitration plate or a membrane (e.g.
  • nitrocellulose membrane , with said attenuated Shigella described above or purified bacterial components from attenuated Shigella , for example, LPS and membrane or cellular components, and contacting it with the serum of a person suspected of having a Shigella infection.
  • the presence of a resulting complex formed between the attenuated Shigella and antibodies specific therefor in the serum can be detected by any of the known methods common in the art, such as fluorescent antibody spectroscopy or colorimetry. This method of detection can be used, for example, for the diagnosis of Shigella infection, detection of immune responses, and determination of previous exposures to specific Shigella components.
  • bacterial components for example, LPS and membrane or cellular components, can safely be purified from attenuated Shigella , and may be used for the production of antibodies, monoclonal or polyclonal, for the detection of Shigella in a sample.
  • the antibodies may be used to identify Shigella in the tissues or body fluids of individuals infected with Shigella , thus permitting rapid and accurate immunological diagnosis of such infections.
  • the antibodies are also useful for the immunological detection of Shigella present as contaminants in water, biologicals, pharmaceuticals, or food. Detection is rapid, sensitive, and highly specific.
  • a diagnostic composition can contain a concentration of the antibody effective to detect Shigella .
  • the antibody can be packaged and sold in freeze-dried or other acceptable form for diagnostic use. It may be mixed with a suitable carrier, attached to an appropriate solid phase (e.g., latex particle, or plastic microtiter plate), conjugated with an enzyme or dye, or radiolabeled, depending on what immunological method is employed. If the antibody is found to neutralize Shigella , or reduce infection, it can be used for immunoprophylaxis or therapy of Shigella infections, or their consequences.
  • a suitable carrier e.g., latex particle, or plastic microtiter plate
  • the present invention relates to a diagnostic kit which contains the attenuated Shigella and ancillary reagents that are well known in the art and that are suitable for use in detecting the presence of Shigella as contaminants in food, water, biologicals and pharmaceuticals, or for the detection of immune responses to Shigella in samples.
  • Samples for detection of immune responses to Shigella would be serum and tissue samples from human, monkeys, or other mammal.
  • the appropriate reagents and materials required for the conduct of the assay can be packaged along with a suitable set of assay instructions. Described below are examples of the present invention which are provided only for illustrative purposes, and not to limit the scope of the present invention. In light of the present disclosure, numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art.
  • EXAMPLE 1 Construction of an attenuated S. flexneri 2a strain
  • a deletion mutation was made in the gene encoding ASD, an essential enzyme required for synthesizing the bacterial cell wall constituent diaminopimelic acid (DAP) (Nakayama et al . BioTechnology (1988) 6: 693).
  • Figure 1 illustrates the construction of 15D, a Das isolate of Shigella flexneri 2a strain 2457T. The gene encoding for E. coli asd (Haziza et al . EMBO J.
  • the kanamycin resistance cassette from the commercial plasmid pUC4K-KIXX (Pharmacia) was purified as a Smal fragment and cloned between the flanking asd sequences. Using forward and reverse primers containing restriction sites SacI and Sail , respectively, PCR amplification resulted in a 2 kb PCR fragment containing the asd gene with an internal deletion and the Kan r cassette. The entire Dasd: :Kan r PCR fragment was cloned into the Sad/ Sail site of the positive selection suicide vector pCVD442 (Donnenberg and Kaper, Jn.fect. Immun. (1991) 59: 4310). Ligations were transformed into SMIO ⁇ pir (Simon et al .
  • Strain 15D was able to maintain the commercially available eukaryotic expression vector pCMV ⁇ without antibiotic selection.
  • pCMV ⁇ expresses E. coli ⁇ -galactosidase under the control of the immediate early promoter and enhancer from the human cytomegalovirus (CMV) in mammalian cells, which permitted us to easily analyze mammalian-mediated gene expression after delivery (MacGregor and Caskey, Nucl . Acids Res. (1989) 17: 2365).
  • Strain 15D was screened to ensure that the large plasmid essential for bacterial invasion of mammalian cells had not been lost during the genetic manipulations. Strain 15D was found to express the virulence associated polypeptides, IpaB and IpaC, as determined by immunoblotting (Mills et al . Infect . Immun . (1988) 56: 2933) showing no loss of the invasion plasmid. It was important to demonstrate that
  • Strains 15D and 15D(pCMV ⁇ ) were each tested for the ability to invade cultured baby hamster kidney (BHK) cells with and without supplementation of DAP during the 90 minutes allowed for invasion (Oaks et al . Infect . Immun. (1985) 48: 124) . After this period of interaction, monolayers were extensively washed and treated with gentamicin (50 ⁇ g/ml) containing medium for at least 30 minutes to eliminate extracellular bacteria.
  • Intracellular bacterial viability and ⁇ -galactosidase activity were followed over a 48 hour time course.
  • viable bacteria recovered from infected BHK cells the following protocol was followed. 1 x 10 5 BHK cells were plated in wells of a 24-well plate. This assay was adapted from those described previously for Shigella plague analysis (Mills et al . Infect . Immun . (1988) 56: 2933; Oaks et al . Infect . Immun. (1985) 48:124).
  • a single congo red-binding positive colony (denoting the expression of plasmid-encoded Shigella virulence determinants) of each strain was used to inoculate overnight LB broth cultures containing 50 ug/ml DAP [15D] or DAP plus 250 ug/ml of ampicillin [ (15D(pCMV ⁇ ) ] . Overnight cultures were diluted 1:50 and grown to approximately mid-log phase in the presence of DAP. Two hundred microiiters of a 10X bacterial solution in HBSS with or without the addition of 50 ug/ml DAP were added to three wells of semi-confluent BHK cells, which had been washed with DMEM (BioWhittaker) , at approximately 50:1.
  • Bacteria were allowed to interact with the BHK cells in this minimal volume for 90 minutes at 37°C, 5% C0 2 .
  • Non-adherent bacteria were removed by extensive washes with HBSS. Extracellular bacteria were then killed by the addition of DMEM with 10% heat inactivated FBS (BioWhittaker) and 50 ⁇ g/ml gentamicin.
  • DMEM heat inactivated FBS
  • 50 ⁇ g/ml gentamicin 50 ⁇ g/ml gentamicin.
  • cells were lysed with a 0.2% Triton-X-100 solution and appropriate dilutions plated on TSA congo red DAP plates for determination of viable bacterial counts.
  • IX IO 5 BHK cells were plated in Nunc chamber slides and infected with 15D and 15D(pCMV ⁇ ) as described above. At the appropriate times, chamber slides were extensively washed, fixed and stained with a Leukostain set (Fisher) . At least 450 cells were visually examined by light microscopy for data analysis. An Instat statistical program (Graphpad, San Diego, CA) was used to calculate means and standard deviations.
  • EXAMPLE 3 Expression of DNA delivered to cells by strain 15D Bacteria were grown as described in Example 1 except that the bacterial suspensions were concentrated 10-fold and 2 mis were added to each flask. In this assay, 50 ⁇ g/ml of DAP was added to bacterial suspensions prior to their addition to flasks of semi-confluent BHK cells. Bacteria were added at a ratio of approximately 100:1. At the indicated time points, BHK cells were removed by trypsinization and washed in PBS. A portion of the cell suspension was lysed with a 0.2%
  • the remainder of the cells were assayed for ⁇ -galactosidase activity, ⁇ -galactosidase activity was measured in the remaining cell extract by a standard biochemical assay that uses the conversion of o-nitrophenyl- ⁇ -D-galactoside (ONPG) to galactose and the chromophore o-nitrophenol to quantitatively detect activity spectrophotometrically (Nolan et al . in Methods in Molecular Biology, E. J. Murray and J. M. Walker, Eds. (Humana Press Inc., Clifton, N. J., 1991) Vol.
  • ONPG o-nitrophenyl- ⁇ -D-galactoside
  • infected monolayers were immunostained to visually detect intracellular ⁇ -galactosidase expression within individual cells.
  • 3 wells of a 4-well chamber slide of BHK cell monolayers infected with either 15D or 15D(pCMV ⁇ ) were immunostained to detect ⁇ -galactosidase expression (Sander et al . J. i munol . Methods (1993) 166:201) .
  • monolayers were fixed in phosphate-buffered 4% paraformaldehyde for 5 min. and subsequently blocked with 3% goat serum (Gibco-BRL) in HBSS for 30 min.
  • BHK cells were then permeabilized for 1 min. with HBSS containing 0.1% saponin (Sigma) solution.
  • Monoclonal anti- ⁇ -galactosidase (Sigma) was diluted 1:2000 in 0.1% saponin/HBSS and applied for 30 min. at 37°C in a humidified chamber.
  • Secondary anti-mouse IgG (Fc specific) FITC conjugated (Sigma) was diluted 1:32 and applied for 30 min. at room temperature. Between each step chamber slides were washed extensively with 0.1% saponin/HBSS solution. A final wash step of HBSS alone was used to close permeabilized cells. Fluorescent images were visualized with either a Nikon microphot with Epi-fluorescence attachment or an Olympus-VAN04-S with fluorescence attachment. Results are shown in Figure 3.
  • each bacterium is estimated to contain about 3.93 (10 "g ) mg of DNA.
  • Intracytoplasmic delivery of approximately 4-20 x IO -9 mg of DNA by Shigella is sufficient for expression of ⁇ -galactosidase.
  • Table 2 Visual examination of infected BHK cells.
  • P815 cells were infected with 15D(pCMV ⁇ ) .
  • Bacteria used to infect P815 cells were grown as described in Example 1. After the addition of the bacteria with DAP to the non-adherent P815 cells cultured in 6-well plates, the plate was spun at 500 X g for 5 minutes. Bacteria and P815 cells were allowed to interact for 90 minutes. The cells were then extensively washed with DMEM and resuspended in DMEM containing 100 ⁇ g/ml gentamicin for a one hour incubation at 37°C, 5% C0 2 .
  • ⁇ -galactosidase activity and protein concentrations were determined at 24 hours as described (Nolan et al . , supra).
  • P815 cells which express H-2 d class I MHC molecules, have been successfully infected with 15D(pCMV ⁇ ) and experiments are currently underway to determine if these cells can present Shigella delivered DNA encoded foreign antigens in the context of class I.
  • 15D provides protection against infection by shigella in vivo
  • Heat-killed heat to 56°C for 30 minutes.
  • Eyes from animals in experiment C were also stained for ⁇ -galactosidase activity. Eyes from animals inoculated with
  • the purpose of this experiment was to determine the immune responsiveness of animals at the time of challenge as well as during the recovery period.
  • the spleens or cervical nodes of two animals were pooled for testing. Two challenged animals from each group were sacrificed 3 and 4 weeks post challenge for testing. Proliferative responses were tested on animals being analyzed for protection. Pre-challenge-animals were vaccinated as described and organs tested at the time other animals were being challenged.
  • Spleens and cervical nodes were processed to a single cell suspension and plated in 96 well plates at a concentration of 1-2 XlO 5 cells per well in 100 ml. Ten ml of each stimulus was added to the appropriate wells. After three days in culture, the amount of proliferation that had taken place was measured using a non-radioactive kit. Responses are presented in Table 5 below.
  • EXAMPLE 8 Mouse Intranasal Challenge Proliferation The purpose of this experiment was to measure in an alternative model (i.e. murine intranasal) the ability of 15D to deliver DNA in vivo. In addition, immune responses to the carrier were also determined.
  • mice Groups of five mice each were inoculated twice intranasally 4 weeks apart. For each strain or treatment, three different doses were also given. Amounts are indicated below.
  • One treatment group consisted of mice given 15D(pCMV ⁇ ) with 50 ⁇ g/ml of DAP added to the culture prior to inoculation.
  • spleens were removed, processed to a single cell suspension and plated in 96 well plates at 2 x 10 s cells per well in 100 ml.
  • Ten ml of the stimuli were added to the appropriate wells. Plates were incubated for three days, and the amount of proliferation that had taken place was measured using a non-radioactive kit.
  • Stimulation index for ConA, E. coli LPS and heat killed 2457T was calculated by dividing the average experimental O.D. value by that of the naive control. Results are shown in Table 6 below. Stimulation Index for b-gal is experimental (pCMV ⁇ ) O.D. value divided by that of 15D.
  • A' polymixin B was added to the b-gal protein to chelate any contaminating LPS.
  • mice that have been inoculated with 15D(pCMV ⁇ ) with or without the addition of DAP are capable of proliferating in response to b-gal protein.
  • Ly phoproliferative and antibody responses directed against the plasmid expressed ⁇ -galactosidase were measured after bacterial delivery of plasmid DNA to the nasal tissue of mice. Two intranasal inoculations were administered on days 0 and 28. Four weeks after the last inoculation, splenocytes from mice receiving 15D(pCMV ⁇ ) showed lymphoproliferative responses directed against ⁇ -galactosidase.
  • mice Eight to 10 week-old female BALB/c mice (Harlan Sprague Dawley, Indianapolis, IN) were sedated by intramuscular injection of a mixture of 0.3 mg xylazine hydrochloride (Rompun; Mobay Corp., Shawnee, KA) and 1.0 mg of ketamine hydrochloride (Ketaset; Aveco Company, Fort Dodge, IA) in 50ml of saline. A concentrated bacterial suspension (15ml) was dropped onto the external nares of each mouse. Mice in groups of 5 to 10 were administered either IO 6 or IO 7 viable bacteria on day 0 and 4 weeks.
  • mice received inocula of 15D(pCMV ⁇ ) supplemented with 50 ⁇ g/ml of DAP. Blood for serum analysis was collected 4 weeks after the last inoculation. At that time, spleens were also removed for in vi tro determination of lymphoproliferative responses induced by ConA, E. coli LPS, heat-killed 2457T, and purified ⁇ -galactosidase (Sigma, St. Louis, MO) . Splenocytes ( lxl0 5 /vtell) were cultured in the presence of 5 ⁇ g/ml ConA, 2.5 ⁇ g/ml E.
  • coli LPS 5 ⁇ g/ml heat-killed 2457T, and 2.5 ⁇ g/ml ⁇ -galactosidase with 10 ⁇ g/ml poly ixin B (Burroughs Wellcome, Research Triangle Park, NC) for 3 days.
  • Levels of proliferation were determined using a Cell Titer 96TM AQ ueous non-radioactive cell proliferation kit (Promega, Madison, WI) .
  • Reported OD490 values were calculated by subtracting the mean value of unstimulated cells from the mean value of stimulated cells.
  • mice inoculated with 15D(pCMV ⁇ ) with or without the addition of DAP are capable of proliferating in response to ⁇ -galactosidase, up to five-fold higher than controls ( Figure 4D) .
  • Sera from groups of mice inoculated with either 15D, 15D(pCMV ⁇ ), or 15D(pCMV ⁇ ) containing 50 ⁇ g/ml of DAP were tested for reactivity to ⁇ -galactosidase.
  • One microgram of purified ⁇ -galactosidase was electrophoresed on 7.5% SDS-polyacrylamide gels. After electrophoresis, gels were electroblotted to nitrocellulose. Casein blocked blots were then sectioned before overnight exposure to pooled sera samples (diluted 1:50 in casein buffer). Bound antibody was detected with a 1:500 dilution of secondary rabbit anti-mouse Ig conjugated with alkaline phosphatase (BMB, Indianapolis, IN) .
  • Alkaline phosphatase activity was detected by substrates BCIP/NBT (Sigma) .
  • Immunoblot analysis revealed antibody responses specific for ⁇ -galactosidase in sera samples from mice infected with 15D(pCMV ⁇ ) .
  • results presented here represent the first evidence that attenuated bacteria can be used to deliver plasmid DNA to mucosal surfaces with subsequent stimulation of immune responses directed against the plasmid-encoded foreign gene product.
  • This approach to vaccine development should simplify production and delivery of DNA-based vaccines, while expanding the technology to allow stimulation of often desired mucosal immune responses.
  • Any bacterial vector DNA delivery system will need to strike a balance between cell invasion with its subsequent reactogenicity and efficiency of delivery.
  • the genes responsible for invasion also cause invasion and apoptosis of macrophages followed by inflammation (Zychlinsky et al . Nature (1992) 358:167).
  • the bacterial DNA delivery system which we describe has several advantages for certain applications. Delivery of DNA encoded antigens to the mucosal immune system should permit mucosal immunization simultaneously with multiple antigens that can be directed for class I and/or II presentation, stimulation of Thl or Th2 help, or secreted maintaining the proper folding and conformational epitopes for IgA and IgG antibody production.
  • Diarrheal diseases such as rotavirus; sexually transmitted diseases such as human immunodeficiency virus, Neisseria gonorrhoeae, and human papilloma virus; and gastrointestinal diseases such a ⁇ the ulcer causing Helicobacter pylori , to name a few, may be especially responsive to this approach.
  • CTGCGTGCTA ACAAAGCAGG ATAAGTCGCA TTACTCATGG 120
  • CTGCGTGCTA ACAAAGCAGG ATAAGTCGCA TTACTCATGG 120

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Abstract

Cette invention se rapporte à un procédé pour introduire des acides nucléiques fonctionnels dans des cellules au moyen d'un système d'apport bactérien. Ce système d'apport peut être utilisé comme vaccin pour prévenir ou traiter des maladies infectieuses. Cette invention peut s'appliquer à n'importe quelle bactérie désirée, y compris des souches atténuées de Shigella.
PCT/US1996/014190 1995-09-06 1996-09-06 Systeme d'apport bacterien WO1997008955A1 (fr)

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EP96932169A EP0881884A4 (fr) 1995-09-06 1996-09-06 Systeme d'apport bacterien
IL123569A IL123569A (en) 1995-09-06 1996-09-06 Attenuated shigella strain for delivering a mamalian expression plasmid into a mammalian cell
CA002231332A CA2231332C (fr) 1995-09-06 1996-09-06 Systeme d'apport bacterien
AU71059/96A AU731061B2 (en) 1995-09-06 1996-09-06 Bacterial delivery system
JP9511361A JP2000500734A (ja) 1995-09-06 1996-09-06 細菌送達系

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WO1999018221A1 (fr) * 1997-10-07 1999-04-15 University Of Maryland Biotechnology Institute Procede d'introduction et d'expression d'arn dans des cellules animales
WO1999034007A1 (fr) * 1997-12-29 1999-07-08 Schering Aktiengesellschaft Introduction d'adn plasmidique codant pour un polypeptide dans le cytosol de macrophages par des bacteries suicidaires attenuees
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US7115269B2 (en) 1997-04-18 2006-10-03 Gesellschaft Fuer Biotechnologische Forschung Mbh (Gbf) Attenuated Salmonella strain used as a vehicle for oral immunization
WO1998048026A1 (fr) * 1997-04-18 1998-10-29 Gesellschaft für Biotechnologische Forschung mbH Souche attenuee de salmonella utilisee en tant que vehicule d'immunisation orale
US7514089B2 (en) 1997-09-10 2009-04-07 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
US7354592B2 (en) 1997-09-10 2008-04-08 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
US6368604B1 (en) 1997-09-26 2002-04-09 University Of Maryland Biotechnology Institute Non-pyrogenic derivatives of lipid A
US6841345B1 (en) 1997-09-26 2005-01-11 University Of Maryland Biotechnology Institute Treatment and prevention of immunodeficiency virus infection by administration of non-pyrogenic derivatives of lipid A
WO1999018221A1 (fr) * 1997-10-07 1999-04-15 University Of Maryland Biotechnology Institute Procede d'introduction et d'expression d'arn dans des cellules animales
US6500419B1 (en) 1997-10-07 2002-12-31 University Of Maryland Biotechnology Institute Method for introducing and expressing RNA in animal cells
WO1999029884A3 (fr) * 1997-12-11 1999-08-12 Von Eichel Streiber Christoph Procede de conditionnement genetique cible pour l'induction d'une transgenese ciblee somatique
US7700091B2 (en) 1997-12-11 2010-04-20 Christoph Von Eichel-Streiber Modified bacteria and methods of use to transform eukaryotic cells
DE19754938B4 (de) * 1997-12-11 2006-04-20 Christoph von Dr. Eichel-Streiber TGC-Verfahren zur Induktion einer zielgerichteten, somatischen Transgenität
WO1999034007A1 (fr) * 1997-12-29 1999-07-08 Schering Aktiengesellschaft Introduction d'adn plasmidique codant pour un polypeptide dans le cytosol de macrophages par des bacteries suicidaires attenuees
JP2002500047A (ja) * 1997-12-30 2002-01-08 セントロ ナシオナル デ インベスチガシオネス シエンテイフイカス 新規なコレラ菌ワクチン候補と構築方法
US6596477B1 (en) 1998-09-28 2003-07-22 University Of Maryland Biotechnology Institute Treatment and prevention of immunodeficiency virus infection by administration of non-pyrogenic derivatives of lipid A
US6825028B1 (en) * 1998-12-11 2004-11-30 Christoph Von Eichel-Streiber Recombinant listeria
EP1261369A4 (fr) * 1999-10-04 2006-02-01 Vion Pharmaceuticals Inc Compositions et methodes d'administration ciblees sur les tumeurs de molecules effectrices
US7452531B2 (en) 1999-10-04 2008-11-18 Vion Pharmaceuticals, Inc. Compositions and methods for tumor-targeted delivery of effector molecules
US10857233B1 (en) 2010-02-09 2020-12-08 David Gordon Bermudes Protease inhibitor combination with therapeutic proteins including antibodies
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria

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AU7105996A (en) 1997-03-27
JP2000500734A (ja) 2000-01-25
AU731061B2 (en) 2001-03-22
IL123569A0 (en) 1998-10-30
EP0881884A1 (fr) 1998-12-09
IL123569A (en) 2006-10-05
CA2231332C (fr) 2007-04-17
CA2231332A1 (fr) 1997-03-13
EP0881884A4 (fr) 2004-07-14

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