JP2000500734A - Bacterial delivery system - Google Patents

Abstract

(57) [Summary] The present invention relates to a method for introducing a functional nucleic acid into cells using a bacterial delivery system. This delivery system can be used in vaccines for the treatment and prevention of infectious diseases. The present invention can be applied to any desired bacteria, such as the modified strain Shigella .

Description

DETAILED DESCRIPTION OF THE INVENTION                                Bacterial delivery system   The present invention provides functional nucleic acids using a bacterial delivery system. It relates to a method for introduction into cells. Bacteria capable of delivering functional nucleic acids to cells Sex vector can enter a cell or is taken up by a cell Or can release nucleic acids to be taken up by cells Some bacteria contain a promoter and other directives recognized by eukaryotic cells Sex plasmids can be made by introducing. Used for this delivery system The bacteria used need not be alive to deliver the selected nucleic acid. like this Nucleic acids delivered to cells can direct eukaryotic cells to antigens or other functionalities. A molecule can be produced.   Therefore, this unique bacterial delivery system prevents or treats infections and cancer. Down-regulates the immune system in case of tissue rejection in transplantation To prevent or cure autoimmune diseases and other diseases associated with immune system dysregulation. It can be used as a vaccine for treatment. In addition, the bacterial delivery system Treatment or amelioration of diseases such as hereditary diseases, cancer and viral infections. For gene therapy or gene replacement.   Direct DNA-mediated immunization is another approach to the introduction of functional nucleic acids and vaccine development. Roach. High expression of protein of interest under control of viral promoter Purified bacterial plasmid DNA can be obtained by conventional needle and syringe or by D Biolistic transfection with NA coated gold particles Injections, mainly into muscle or skin, by other newer methods, such as For a discussion of J. Donnelly, J.J., et al. Immunol. Me thods (1994) 176: 145). Using this method, researchers have developed a neutralizing anti- Infection from the body, cytotoxic T lymphocytes, and influenza to malaria Protection against antigenic stimuli in some animal models could be triggered. The use of bacteria as the delivery system described in the present invention delivers DNA to mammalian cells And extend DNA immunization to the local immune system, and Provides a simple and inexpensive method that is superior to oral and other mucosal routes of immunization There is a possibility to provide.   Traditionally, live bacteria have been used as vaccines to protect against later infections. Have been. Attenuated or slightly pathogenic Shigella, Salmonella ( Salmonella), Listeria, and other bacteria Administered orally to immunize against subsequent infection with a more virulent form Have been. Similarly, attenuated bacteria, and Calmette-Guerin bacteria (BCG) Unusual mycobacteria have been developed against related organisms such as M. tuberculosis. Has been administered parenterally to provide protection. Of bacteria, viruses and parasites The gene directs the bacterium to express a foreign antigen, or a live vaccine In order to confer some desirable properties to bacteria for use as It has been cloned into bacteria and mycobacteria. Examples are usually non- Cloning the Shigella invasion gene into E. coli Making it invasive, and thus more suitable for use as a vaccine strain, Or Plasmodium falciparum (P. typhimurium) in Salmonella typhimurium.  falciparum malaria) gene, and then Salmonella typhi Expression of the rear protein and oral administration of this bacterium in mice Inducing specific cytotoxic T cell immunity and protection against malaria antigen stimulation (Sadoff et al., Science (1988) 240: 336-338; Agrawal) (Aggrawal) et al. Exp. Med. (1990) 172: 1083-1090). All these bacteria Delivery systems require the bacteria themselves to produce the antigen or functional molecule, and Attenuated enough to be safe for use in humans, but still protected It depends on bacteria that can induce a response. The bacterial delivery system of the invention is eukaryotic Delivering functional nucleic acids that direct biological cells to produce antigens and other functional molecules Designed to be. In this case, the expression of the gene encoded by the plasmid A eukaryotic cell that allows proper folding of the antigen for presentation or direction of function Toxicity to the carrier has been ruled out due to the vesicle mechanism. In addition, it is necessary Used to deliver antigens and functional molecules produced by prokaryotes, if any can do.   The present invention can be applied to any desired bacteria. For eukaryotic cells Due to Shigella's ability to invade, escape the phagosome, and enter the cytoplasm, Shigella was selected as an example of a bacterial delivery system. These properties are useful for the application of the present invention. Although not required for the bacteria selected for, these simplified the experimental system. Red Diarrhea is useful for the delivery of nucleic acids and bacterial antigens, which have utility as vaccines. Serves as both examples. Shigella invades human colonic epithelium and proliferates in cells It is an enteric pathogen that causes bacillary dysentery. Shigellosis is all members of the genus Shigella. Caused by members (Void Shigella (S. boydii), Shiga Shigella (S.  dysenteriae), Shigella flexneri (S. flexneri), and Shigella sonnei ( S. sonnei)). Shigellosis is common in developing nations, but is particularly institutionalized in developed nations. Seen in. Shigellosis causes 500,000 deaths (primarily children) annually Therefore, it is important to develop a safe and effective Shigella vaccine ( J. Stole, B.J., et al. Infect. Dis. (1982) 146: 17 7). All references cited hereinbefore or hereinafter are incorporated herein by reference. Be impregnated.   To cause dysentery, Shigella strains recognize and invade epithelial cells of the colon to It must be able to grow in the vesicle (LaHrec, E.H. ) Et al. Bacteriol. (1964) 88: 1503). Both bacteria and host cells are involved in the invasion process In this process, the host cells actively swallow the bacteria, The bacteria then escape from the phagosome by bacterial-mediated digestion of the phagosome membrane (pi —J. Sansonetti, P.J., et al., Infect. Immun. (1981) 34:75). Once in the cell, the bacteria grow and cause necrosis of the host cell .   Previous studies have shown that parenteral immunization with live or killed Shigella can reduce infection. Proven to not defend (Formal, S.B., et al.) , Proc. Soc. Exp. Bio. Med. (1967) 25: 347; Earl Higgins (Hig) gins, A.R. ) Et al., Am. J. Trop. Med. Hyg. (1955) 4: 281; H. J. Shaugnessy, H.J., et al., JAMA (1946) 132: 362). Recent efforts Focuses on developing attenuated Shigella vaccine to induce mucosal immunity (A. Lindberg, A.A., et al., Vaccine (1988) 6 J.W., et al., Vaccine (1). 992) 10: 766). Some candidates have shown promise, but are safe and effective No vaccine has been found. The Shigella vaccine candidates created so far are: Does not elicit a protective immune response that can protect against subsequent antigenic stimulation, or The strain was not sufficiently attenuated for human use.   Therefore, in view of the above, as a vaccine candidate against Shigella infection, These include the delivery of heterologous and homologous antigens, and DNA-mediated immunization, and gene delivery. Against a properly attenuated strain of Shigella, which serves as a bacterial vector for Needs exist.wrap up   In the present invention, a functional nucleic acid is delivered to a cell, and a heterologous and alloantigen is delivered. Attenuated Shigella strains that can be attenuated are described. Specific bacteria are described herein. Transfer nucleic acids to eukaryotic cells, whether the bacteria are alive or inactivated. The present invention applies to all bacteria and mycobacteria It is possible. Plasmids introduced into other cells, such as plant cells, also Of cells can deliver the nucleic acid.   Specifically, the attenuated Shigella strains of the present invention are capable of delivering functional nucleic acids, In addition, it acts as a vaccine candidate against Shigella infection. Attenuation of the present invention Shigella strains enter cells, but once they enter host cells, they die and die. Release the contents of The attenuated Shigella strains described herein do not cause disease. Fully attenuated, but still maintain the ability to enter mammalian cells I have. In this strain, the invasion of the corneal epithelium by Shigella is restricted to the small intestine of human or primate Guinea pig horns, an animal model mimicking processes found in the epithelium In a conjunctivitis model, Shigella flexneri 2a strain 24 It has been shown to be protective against 57T antigen stimulation (Mackel (Mack el) et al., Am. J. Hyg. (1961) 73: 219-223; Sereny, B., Ac. ta Microbiol. Acad. Sci. Hung. (1962) 9: 55-60).   We use host cells for protein synthesis and processing for antigen presentation. As a method of directing DNA into the cytoplasm of the vesicle, enter the epithelial cells, Utilizing the ability of Shigella to escape from the vesicles (High, N. et al., EMBO J. (19 92) 11: 1991). For the specific purpose of delivering DNA to intestinal mucosal epithelial cells, Mutations in the gene encoding sparginate b-semialdehyde dehydrogenase (ASD) The mutation was introduced into S. flexneri 2a strain 2457T. This makes it an essential peptidoglycan component that constitutes the cell wall of Gram-negative bacteria. Some strains failed to grow in the absence of diaminopimelic acid (DAP). DA P is not present in mammalian tissues and is therefore available for capture by infectious bacteria Never. This mutant strain of Shigella is a highly attenuated bacterial vector. Invading mammalian cells to provide protective immunity against strain-specific Shigella infection. For further oral and other mucosal DNA immunizations and for gene therapy strategies , Can act as a delivery vehicle.   Thus, one object of the present invention is to retain the ability to enter cells, but The aim is to provide an attenuated strain of Shigella that will die once it enters cells. Shigella Attenuated strains are useful for treating the severity or symptoms of disease caused by Shigella. Is used as a vaccine to reduce or protect against Shigella infection be able to.   Another object of the invention is to retain the ability to enter cells, but The purpose of the present invention is to provide an attenuated and inactivated strain of Shigella which is killed when it enters. Shigella weak Poisoned and inactivated strains are used to cure the severity or symptoms of the disease caused by Shigella. As a vaccine for treatment or alleviation or protection against Shigella infection Can be used.   Yet another object of the invention is the use as a protective vaccine against infection, or Reduce different strains of Shigella to improve disease caused by Shigella infection To provide a way to poison.   Yet another object of the invention is to retain the ability to enter cells, but once Contains attenuated Shigella that dies when entering cells, and pharmaceutically acceptable excipients Offers vaccine to reduce symptoms caused by Shigella in individuals Is to provide.   It is a further object of the present invention to provide a delivery vehicle for delivery of DNA to mucosal surfaces. To provide. The DNA encoding the gene or antigen of interest is a mammal The attenuated Shigella strain of the present invention as described above, which is capable of entering cells. Or can be introduced into attenuated / inactivated Shigella strains and recombinant attenuated Shigella strains. Wear. Due to the mutation introduced into the attenuated strain, the recombinant Shigella attenuated once Successful delivery of functional foreign DNA to mammalian cells when they die Can be. Such delivery vehicles are available for oral and other mucosal immunization, and genetic Can be used for child treatment strategies.   Yet another object of the invention is to induce an immune response in a subject against a foreign antigen. Or for the treatment of diseases where foreign antigens are not found or are found in small amounts The goal is to deliver a heterologous foreign antigen expressed by the attenuated Shigella.   Yet another object of the invention is to use the cells for transplantation and gene therapy. Provides a delivery vehicle for delivering DNA and antigen to cells in vitro It is to be.   Yet another object of the present invention is to combat S. flexneri infection. Vulnerable to S. flexneri for use as a vaccine To provide a poisoned and attenuated / inactivated strain.   Yet another object of the present invention is to provide an infection by S. flexneri. Caused by such an infection in an individual for use as a vaccine against To reduce the symptoms caused or with a delivery vehicle for a heterologous antigen or DNA And attenuated S. flexneri, a mutant of the asd gene It is to provide chemical strains.   Yet another object of the present invention is to use a novel species of bacteria as a DNA delivery vehicle. Provide a way to introduce Shigella invasive genes into other bacterial species for use It is.   A further object of the present invention is to detect a disease caused by Shigella, Can be used in diagnostic assays to measure exposure to Shigella in To provide safer strains and kits therefor.   Yet another object of the present invention is to use a diagnostic assay for the detection of Shigella in a sample. It is an object of the present invention to provide a component of Shigella to produce an antibody that produces the antibody.   Yet another object of the invention is to induce protective immunity as a vaccine, Function of the immune system to treat and prevent autoimmune diseases For treatment of all related conditions, for transplantation, gene replacement, and gene therapy In order to introduce functional nucleic acids into cells using bacterial delivery systems, Is to provide a way.BRIEF DESCRIPTION OF THE FIGURES   These and other features, aspects, and advantages of the present invention are described in the following description, claims, and claims. BRIEF DESCRIPTION OF THE DRAWINGS FIG.   FIG. 1 shows the Δasd induction of S. flexneri 2a strain 2457T. 3 illustrates the creation of a conductor.   FIG. 2 shows that pCMVβ, a mammalian DNA expression plasmid, was delivered to BHK cells. This is the result of using strain 15D as a carrier to reach. (A) Living 1 The number of 5D (○) and 15D (pCMVβ) (Σ) was measured over 48 hours . (B) The unit of β-galactosidase activity / mg protein is also BHK alone ( ○), BHK cells infected with 15D (●), and 15D (pCMVβ) The measurement was performed on the stained BHK cells (-). Semi-confluent BHK cells Is about 0.5-1 × 10⁷Consists of cells. β-galactosider The activity is expected to be 0.5 × 10⁷Measured for cells.   FIG. 3 shows β-galase in BHK cells infected with 15D and 15D (pCMVβ). The results of intracellular immunostaining to detect the expression of custosidase are shown. (A) Ge 30 minutes after addition of the medium containing ntamycin, stain with Leukostat Colored, 15D (pCMVβ) infected BHK monolayer (100 × oil immersion lens) . Immunostained infected BHK cells after addition of gentamicin-containing medium: (B ) 15D (pCMVβ) 30 minutes, (C) 15D 4 hours, (D) 15D (pCM Vβ) 4 hours, (E) 15D (pCMVβ) 24 hours, (F) 15D (pC MVβ) 48 hours, (G) 15D 24 hours, and (H) BHK cells alone;   (BH 10x fluorescent phase lens).   FIG. 4 shows that ConA (FIG. 4A), a mouse administered intranasally with a concentrated bacterial suspension. Enterobacter LPS (FIG. 4B), heat killed 2457T (FIG. 4C), and purified β-galacto FIG. 4 shows the lymphoproliferative response induced by the sidase (FIG. 4D). Spleen cells (1 × 10^Five/ Well) with 10 μg / ml of polymyxin B (Burroth Welcome ugs Wellcome), Research Triangle Park , North Carolina), 5 μg / ml ConA, 2.5 μg / ml E. coli. LPS, 5 μg / ml heat-killed 2457T, and 2.5 μg / ml β-galactosida Cultured for 3 days in the presence of. The level of proliferation was determined by Cell Titer ) 96® AQueous non-radioactive cell proliferation kit (Promega, (Madison, Wis.). The reported OD490 value is It was calculated by subtracting the average value of unstimulated cells from the average value of stimulated cells.   FIG. 5 shows the antibody response to β-galactosidase of mice inoculated intranasally. Western. Groups of mice received 15D, 1 containing 50 μg / ml DAP, Either 5D (pCMVβ) or 15D (pCMVβ) was administered. β- Serum was tested for reactivity to galactosidase. Lane A is a bear It is an SDS-PAGE gel stained with C. Immunoblots BG are B, 10⁶ 15D; C, 10⁷15D; D, 10⁷15D (pCMVβ); E, 10⁶15D (PCMVβ); F, 10⁷15D (pCMVβ) + DAP; and G, 10⁶1 To a 1:50 dilution of pooled sera of mice inoculated with 5D (pCMVβ) + DAP Exposed. Immunoblot control lanes; H, 1: 10,000 anti-β-galactos Dase (Promega); I, pooled serum of mice inoculated with saline 1:50 dilution; and J, 1: 500 alkaline phosphatase bound Secondary rabbit anti-mouse.Detailed description   The present invention is directed to use as an immunogen for protection against dysentery infection and To deliver DNA to mucosal surfaces for use as a carrier for delivery of seed antigens Or attenuated Shigella strains for use in diagnostic assays Is described. The method is generally used for all bacteria and mycobacteria. Applies to Rhea (Mycobacteria).   Specifically, the present invention relates to diaminopimelic acid (DA) which is a component of the bacterial cell wall. Dehydrogenation of aspartic acid b-semialdehyde, an essential enzyme required for the synthesis of P) Shigella flexneri (S. f.) Containing a deletion in the gene encoding the enzyme (ASD) lexneri) is described. Without being bound by theory, this mutation The strain retains the ability to enter mammalian cells, but once inside the cell, D Since the AP is not present and is not available from mammals, it cannot be replicated, and It is killed and releases its contents, including complete DNA and antigens already present in the cells.   More specifically, S. flexneri 2a strain 2457T is 553 bp deletion of the structural gene from positions 439 to 991 of the chromosome (SEQ ID NO: Mutation by integration of the deleted Escherichia coli asd gene containing 1) Was. A kanamycin resistance cassette containing the complete Tn5 kanamycin gene Cloned between the sequences on both sides of the mutant asd gene.   In the present invention, any Shigella strain is provided as an attenuated strain as an asd mutant. Mutations can be mutated as provided. The strain does not need to be pathogenic, Preferably have the ability to enter or be taken up by target cells. Bacteria Once in the cell, the asd mutation will promote bacterial destruction. Furthermore, by mutating any gene other than asd, it has the same effect on bacteria. As it has, that is, once inside the cell, it will die or Or, the clinical symptoms can be attenuated to an acceptable degree. like this Examples of genes include thyA, genes for LPS production, htrA and htr B, and dut, but are not limited to these.   One method for making a mutation in the asd gene is described in the following example. is there Alternatively, mutations in the selected gene result in loss or alteration of the function of the gene product. Changes in the genetic properties of bacteria to prevent them from surviving in the host cell. It can be any chemical change in the DNA that occurs. Chemical changes in DNA are simply One or more deletions, one or more point mutations, another one or more Of a gene or part of a gene into a structural part of the gene to be mutated And addition or deletion of transposons, but are not limited to (See the review by Klockner et al., J. Mol. Bio. (1977) 116: 125.) Please see). Strains containing mutations other than the asd mutation are considered. Are also included in the scope of the present invention. Introduce different mutations and these mutations Methods are known to those skilled in the art (Davis,  R.W. )Advanced Bacterial Genetics . A Manual for Genetic Engineerin g  , Cold Spring Harbor Laborator y), New York, 1980).   Specifically, the attenuated Shigella 15D strain was prepared as follows. Cloning Encoding the asd of Escherichia coli in order to incorporate the necessary restriction sites into the vector The gene was amplified using PCR. In the present invention, any homologous asd The gene can be used to create an asd deletion in Shigella. Homologous Genes include Corynebacterium glutamicum ), Bacilluss ubtilis, Mycobacterium smegmatis (My cobacterium smegmatis), Pseudomonas aeruginosa, Leptospira ・ Interrogans (Leptospira interrrogans), pertussis (Bordetella pertu) ssis), Corynebacterium flavum, meningococci (Neisseria meningitidis), Vibrio cholera, Mycobacterium Mycobacterium bovis, Streptomyces schiyoensis ( Streptomyces skiyoshiensis, Streptococcus mutans (Streptoc) occus mutans), Vibrio mimicus and Brucella (Bruc) ella) species, but are not limited thereto. Selected Any method for incorporating restriction sites required for cloning into a vector Use of linkers or adapters, blunt-end cloning into polylinkers, And any other DNA cloning method known to those skilled in the art can be used. To review AboutCurrent Protocols in Molecular Biology, FM Ausberg (F.M. Ausubel) et al., Green Publishing Associates & We Lee Interscience (Greene Publishing Associates and Wiley-Interscie nce), New York, see). Furthermore, for insertion of the desired fragment Any vector that can be linearized can be used for cloning and these will be Is knowledge. Examples of vectors include high copy number plasmids, fur There are dimids, single copy vectors, expression vectors, and phages, etc. It is not limited to these.   E. FIG. E. coli asd produced plasmid was reverse PCR amplified and col Deleting the 553 bp (positions 439-991) of the iasd structural gene and suddenly Mutant E. E. coli asd or Δasd (SEQ ID NO: 2) is prepared. Sudden change Other methods known to those skilled in the art for introducing a mutation and deleting a gene or part of a gene For example, Bal 31 digestion, multiple restriction digestion or recombination can be used.   After the production of Δasd, the commercial plasmid pUC4K-KIXX (Pha rmacia) (Kanmycin resistance)^r) Purify the cassette and place Cloned between the Δasd sequences, Δasd :: Kan^rIs prepared. The present invention According to gene therapy or antigen production, whether due to antibiotic resistance Insertion of any gene (s) into the asd deletion Can be. Appropriate end formation methods for fragment ligation are Is known to those skilled in the art. Furthermore, it is unnecessary to insert the gene into the asd deletion. Because the deletion itself confers a mutant phenotype and produces an attenuated Shigella Is enough.   Δasd :: Kan^rInto the positive selection suicide vector pCVD442 Forward and reverse primers containing the necessary restriction sites (se primers), PCR amplification was performed with the asd gene having an internal deletion and With a unique restriction site^rGenerated a PCR fragment containing the cassette . Again, ligation occurs to insert appropriate restriction sites or Use any method to generate ligated fragment ends be able to. Such methods are well known to those skilled in the art and are described in Maniatis et al. ofMolecular Cloning: A Laboratory Manu al , Cold Spring Harbor Laboratories, 1 982. The vector pCVD442 has a positive counterpart for recombination. Mobility with sacB as a positive counter selection system It is a suicide vector. In Shinella Any vector with a non-functional origin of replication can serve as an acceptable suicide vector stand. Further, for example, sacB, EF-G, klaA, B or C, λP gene Offspring, or opposing selections such as T7 bacteriophage gene 1.2 or 10 The gene is preferred for selection of transformants, but is not required.   Δasd :: Kan^rTransformation of Ligation to pCVD442 For E. E. coli strain SM10λpir was used. Allows suicide vector propagation And a suitable strain for conjugation in Shigella. Any strain can be used. Vectors and suitable bacteria are known to those of skill in the art. Within range. SM10λpir (pCVD422 :: @ asd :: Kan^r) To S. flexneri2a strain 2457T (pAB322 [Tet^r, Amp^s ])^r/ Tet^rA conjugant was selected. Shige Conjugation of lla is well known to those skilled in the art. The recipient strain (recipie Any method can be used to tag the nt strain. Nutrition Requirement markers or antibiotic markers allow selection relative to the donor strain. Similarly, suicide vectors can be transformed or electroporated into Shig. It can also be introduced directly into ella. When the zygote is grown on sucrose (This is a standard protocol for sacB containing plasmids), 2 recombination events occur and ATCC accession number ATCC557 Isolate 15D was obtained, given 10.   Kan^rAnd screening for DAP requirements Isolate was obtained. Isolates to be selected are identified if a mutation is present in the ASD gene. With respect to the requirements for DAP or for the requirements of the deleted gene product , Or can be screened for the presence of the inserted gene in bacteria. You. Other screening methods are known to those skilled in the art, and cs). For example, for xy1E, which is maintained between recombinant fragments Positive selection can also be achieved by scoring such marker genes. Can do it.   In one embodiment, the invention provides a cell with the desired gene (s). The method comprises the following steps:   (I) introducing the gene of interest into an attenuated Shigella strain;   (II) administering the Shigella; including. According to the present invention, any gene (single or plural) can be obtained by the above method. Introduced into the Shigella chromosome or into the virulence plasmid Can be replicated or alternatively replicated or non-replicated by Shigella. It can be carried in rathmid. The vector in question is transformed, electroporated Can be introduced by translation, transfection or conjugation. Exemption Genes for treating epidemics are microorganisms that cause diarrheal disease, such as retroviruses. For example, the human immunodeficiency virus,Neisseria gonorrhoeae And microorganisms that cause sexually transmitted diseases, such as human papillomavirus, and SwellingHelicobacter pyloriCause gastrointestinal diseases, such as Includes genes encoding foreign antigens from microorganisms. Weak poisoning Simulated Shigella, regardless of whether the bacteria are alive or inactivated, It was found to supply DNA and antigen to the cells. Bacterial inactivation is known in the art. For example, by heating to 56 ° C. for 30 minutes. Wear. Inactivation occurs only to the extent that the supply of functional nucleic acid is not unduly impaired. Can be.   Supplying DNA-encoded antigens to the mucosal immune system by Shigella , Class I and / or Class II presentation, Th1 or Th2 help Suitable for the production of IgA and IgG antibodies. And secreted while maintaining conformational epitopes. It is believed to allow for simultaneous mucosal immunization with multiple antigens that can be performed.   A similar approach is to provide DNA for gene therapy and correction of congenital metabolic defects. Can be used. Such genes include, for example, the CFTR gene in cystic fibrosis. Replacement of defective genes, such as offspring, or the treatment of, for example, reverse transcriptase, or HIV. Antisense gene for, for example, interleukin-1 Gene that up-regulates the Th1 immune response, such as Or cholesterol and cholesterol receptor, insulin and insulin Up certain receptors, metabolites and hormones, such as receptors Genes that are down-regulated, such as tumor necrosis factor (TNF) A gene encoding a product capable of killing such cancer cells, or Hormone secretion, stimulation of bone cells to promote bone growth and reduce bone loss Various factors including aging, such as down-regulation of osteoclasts New genes, such as those for up-regulating a depleted system The introduction of   Similar methods prevent autoimmune diseases and other diseases involving immune system dysregulation Or control or prevent or prevent certain diseases or conditions, including transplantation. Or down-regulate the immune system in an antigen-specific or general manner to treat Can be used to supply nucleic acids for Examples are autoimmune encephalitis, multiple sclerosis Disease, lupus erythematosus, diabetes mellitus, Crohn's disease and other inflammatory bowel diseases, and It includes the prevention and treatment of rheumatoid arthritis and other inflammatory joint and skin diseases. Other examples Are immune responses that deviate from protective and therapeutic immune responses to, for example, cancer and other diseases. Inhibits appropriate protective and therapeutic immune responses, such as down-regulation of responses Down-regulation of the immune response. For example, if the Th1 response is cancer, Prevention and cure of Leishmania, Mycobacterium tuberculosis and HIV Down-regulation of Th2 response when appropriate for therapy. This is N And through HIV. Intestinal and other topical immunity The unique immunosuppressive properties of the epidemic system are combined with the ability to code for the production of appropriate cytokines. This combined operation induces an appropriate immune response and suppresses an inappropriate immune response Environment (milieu for).   In another embodiment, the invention relates to a method of introducing the antigen of interest into cells. this Such a method can be used to attenuate Shigella or attenuate / inactivate the desired DNA or antigen. Introducing the desired antigen into Shigella so as to produce it; igella to an individual. The antigen is It can be produced during the life cycle of Shigella before entering. this These antigens are expressed from a prokaryotic promoter and are constitutively expressed or induced. Can be Such genes can be used in parasites where immune responses from them are desirable. Includes genes from organisms.   In another embodiment, the present invention introduces the antigen of interest into cells in vitro. About the method. Such a method involves the attenuated Shigell DNA or antigen. a or introduced into attenuated / inactivated Shigella to produce the desired antigen And administering the Shigella to the cells. Shigell a represents several different cells, such as BHK (baby hamster kidney cells), HeLa (Human uterine epithelial-like cancer), CaCo-2 (human colon adenocarcinoma) DNA or antigen into cells capable of expressing said DNA Can be. Cells after DNA supply are transplanted for therapeutic purposes, for gene therapy Or can be used as reagents for diagnostic analysis.   In yet another embodiment, the invention relates to a method for producing an invasive bacterial strain. Sh The invasion gene used by Igella is, for example, E. coli. Eli Can be inserted into other bacteria such as Such strains, which are now invasive, have It can be used as a carrier for supplying DNA to the intestinal mucosa. Intestinal heaven E. coli, a bacterium found in flora. using a supply vehicle such as E. coli One of the benefits of being able to live is that the body does not mount an immune response ultiple doses) of the desired antigen or DNA, Against the antigens of bacteria and not against bacteria that supply foreign antigens It is. VirG gene or other chromosomally encoded factor  factor) and a virulence gene found in Shigella. Engineering invasive strains from non-invasive candidates using Lurens plasmids Can be engineered (Sansonetti et al., Infect. Im mun. (1983) 39: 1392).   In yet another embodiment, the present invention relates to a vaccine against Shigella infection. Related. The attenuated S. of the present invention. The flexneri strain is S. cerevisiae. flexneri infection It can be used as an immunizing agent. This strain is a guinea pig. It has been shown to elicit a protective immune response in an animal model of keratoconjunctivitis. Other Shigella strains are also As with flexneri, S obtained by introducing a mutation into the higella gene. Shigella can be attenuated so that it dies after entering the cell. This Such mutations can be present, for example, in the asd gene and the resulting attenuated The transformed strain is, for example, S. aureus. boydii, S.M. dysenteriae, S.D. flex neri and S.M. Specific of the Shigella strain used, such as C. sonnei Used as a vaccine against infection with serotypes. Attenuated Shigella The vaccine contains one or several strains of attenuated Shigella It can be formed as a combination vaccine. In addition, the vaccine was added Antigen does not interfere with the effectiveness of attenuated Shigella vaccine, side effects and adverse reactions If there is at least one other, unless it increases additively or synergistically Antigens can be included.   Vaccine as a liquid solution or suspension; dissolved or suspended in liquid prior to administration It is manufactured as a solid form suitable for This formulation can also be emulsified Or components are often, for example, pharmaceutical grades of mannitol, lactose, starch, Magnesium stearate, sodium saccharin, cellulose, magnesium carbonate Mixed with excipients such as These compositions contain solutions, suspensions, tablets, Tablets, capsules, sustained-release preparations, nasal drops or powders,¹²Weak Contains poisoned and / or attenuated / inactivated Shigella.   The vaccine can also be in the form of an injectable drug. Suitable excipients are, for example, Physiological saline or buffered physiological saline (pH about 7 to about 8), or Dext Other physiologies that can also contain loin, glycerol, etc., and combinations thereof. Target, isotonic solutions. However, for example, strong surfactants, alcohols and other Agents that disrupt or dissolve the liquid film, such as organic solvents, should be avoided. You. In addition, if necessary, the vaccine may contain trace amounts of, for example, wetting or emulsifying agents, Adjuvants such as pH-adjusting agents, buffers and / or vaccines Such auxiliary substances can be included. Examples of effective adjuvants are Specifically, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D- Isoglutamine (thr-MDP), N-acetylmuramyl-L-alanyl-D -Isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn -Glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP198) 35 A, called MTP-PE), and 2% squalene / Tween 80 emulsion Three elements extracted from bacteria in the preparation: monophosphoryl lipid A and trehalose TIBI containing sujimycolate, cell wall skeleton and (MPL + TDM + CWS) Is included. The effect of the adjuvant may be Shigella, antigen carried, or species Administration of attenuated Shigella in vaccines also composed of various adjuvants The level of the desired immune response elicited against the DNA-encoded antigen resulting from Can be determined.   The vaccine may be administered as a liquid or suspension prepared as described above. it can. Other formulations suitable for other modes of administration include suppositories. In addition, vaccines Can be lyophilized. For suppositories, conventional binders and carriers are examples For example, polyalkylene glycols or triglycerides can be included; Suppositories such as generate the desired immune response, ie, undesirable adverse side effects Generally sufficient to protect or reduce the incidence or severity of the disease without causing Is 10 to 10 per serving¹²Range of attenuated Shigella colony forming units Within a mixture containing the attenuated Shigella.   Generally, vaccines are administered orally, subcutaneously, at dosages effective for the development of the desired immune response. It can be administered intradermally or intramuscularly. Vaccine suitable for the dosage form Administered in a manner that is prophylactically and / or therapeutically effective. Generally, 10 to 10 per serving¹²Attenuation and / or attenuation / inactivation of colony forming units A dose that is within the range of Shigella is Whether to act as a carrier for cutin or a heterologous antigen or DNA Dependent and subject to be further treated, a pair that produces the desired immune response It depends on the ability of the elephant's immune system and the degree of protection desired. The exact vaccine to administer The amount will depend on the judgment of the practitioner and will be given to each subject, either antigen or Shige. It is unique to the use of lla as a vaccine or as a carrier.   Vaccines may be given in a single dose schedule or, preferably, in the first course of vaccination In 1 to 10 separate doses, followed by maintenance and / or enhancement of the immune response Other doses that are required to do so, such as a second dose in 1-4 months Administer the next dose (s) several months later, if necessary It is administered on a multiple dose schedule. Dosage regimen (dos age regimen) is also determined, at least in part, by the requirements of the individual. Depends on the judgment of the doctor. Examples of suitable immunization schedules include (i) 0, 1, (Ii) 0, 7 days and 1 month schedule; (ii) i) 0 and 1 month schedule; (iv) 0 and 6 month schedule; or protection exemption To give an epidemic or to alleviate the symptoms of the disease or to reduce the severity of the disease Include other schedules that are sufficient. First immunization consisting of 1 to 3 inoculations After course, against Shigella with attenuated Shigella vaccine The occurrence of protective immunity can be reasonably predicted. These should be considered as sufficient levels of protective immunity. For spaced-apart (eg, every two years) boosters designed to maintain bells Therefore, it can be assisted.   In another embodiment, the present invention relates to a method for preparing a Shigella antigen or Sh in a sample. Immune response against iguella (particularly Shigella flexneri) A method for detecting the presence of Use of the attenuated Shigella of the present invention One of the benefits of is the cumbersome security required by highly infectious natural Shigella. Reduction of total treatment; attenuated Shigella survives this bacterium in host cells This indicates that the danger to the operator is reduced due to the inability to operate. The detection protocol is For example, based on competitive analysis, direct reaction analysis or sandwich type analysis Can be. Protocols can use, for example, solid supports or immunoprecipitation It can also be. Most analyzes involve the use of labels; these labels are For example, it can be a fluorescent molecule, a chemiluminescent molecule, a radioactive molecule or a dye molecule. Assays that amplify the signal from the probe are also known, such as biotin or Or avidin and enzyme labeling and enzyme-mediated immunoassays such as E LISA or ELISPOT assay. Using standard methods known in the art For example, microtitration plates or membranes (eg, nitrocellulose The surface (ie, the solid support) such as the membrane) is attenuated by Shigella or attenuated Purified bacterial components from oxidized Shigella, such as LPS And coated with a membrane or cellular element, which has Shigella infection Contact with human suspected serum can constitute a diagnostic assay. Wear. The form between attenuated Shigella and an antibody specific for it in serum The presence of the formed complex is determined by a method such as, for example, fluorescent antibody spectroscopy or colorimetry. It can be detected by any of the methods known in the art. this Detection methods include, for example, diagnosis of Shigella infection, detection of immune response, and specificity. It can be used to determine prior exposure to Shigella elements.   In addition, bacterial components, such as LPS and membrane or cellular components, are attenuated Shigell. a for safe detection of Shigella in samples Can be used for the production of local or polyclonal antibodies. These anti The body identifies Shigella in tissues or body fluids of individuals infected with Shigella. Rapid and accurate immunization of such infections as it can be used to identify Allows for a clinical diagnosis. These antibodies are available in water, biologicals, Elimination of Shigella present as a contaminant in drugs (pharmaceuticals) or food It is also useful for epidemiological detection. Detection is fast, sensitive and highly specific . The diagnostic composition contains a constant concentration of antibody effective to detect Shigella. It can also have. Antibodies can be in lyophilized form or in any other form acceptable for diagnostic use. Can be packaged and sold. This uses any immunological method Depending on the application, a suitable solid phase (for example, a latex) may be mixed with a suitable carrier. Enzymes or dyes attached to microparticles or plastic microtiter plates) Can be conjugated or radioactively labeled. Antibodies in Shigella If found to relieve or reduce infection, this antibody can be used for immune protection. Or for the treatment of Shigella infections or their consequences Can be used.   In yet another embodiment, the present invention relates to an attenuated Shigella, as disclosed in the art. Contain contaminants in foods, water, biologicals and pharmaceuticals. Detection of the presence of Shigella, or immunity to Shigella in a sample Diagnostic kits suitable for use in detecting epidemic responses. Against Shigella A sample for detecting an immune response that occurs may be blood from humans, monkeys or other mammals. These are Qing and tissue samples. Appropriate reagents and materials needed to perform this analysis Can be packaged with a suitable set of analytical instructions. The present invention The examples are further described below, but these examples are provided for illustrative purposes only. And does not limit the scope of the invention. Consider the disclosure in this specification If so, numerous embodiments within the scope of the claims will be apparent to those skilled in the art. U.                                 Example 1 Attenuated S. Construction of flexneri2a strain   Utilize the already known conditionally lethal mutation system when constructing appropriate strains did. Necessary for the synthesis of the bacterial cell wall component diaminopimelic acid (DAP). A deletion mutation was made in the gene encoding ASD, which is an essential enzyme ( Nakayama et al., Biotechnology (1988) 6: 693). Figure 1 shows 15D,Shigella flexneri  D of 2a strain 2457T The construction of the asd isolate is described. E. FIG. E. coli asd-encoding gene (M azabo et al., EMBO J .; (1982) 1: 379) using PCR To incorporate a BglII restriction site. asd is inserted into the previously described vector. (Branstrom et al., 33rd ICAAC (New Orlea) ns, LA, Oct. 20, 1993), Abstract # 11. 36); E. coli x6097 (Nakayama) Et al., Supra). The obtained pAB102 plasmid was subjected to reverse PCR amplification to obtain E. coli. c The 553 bp (positions 439-991) of the oli asd structural gene was deleted [ All primers are supplied in the 5 to 3 orientation, SEQ ID NOs: 3-8]. Mutant E. FIG. E. coli asd or Δasd (SEQ ID NO: 2) is prepared. Commercial plastic Kanamycin-resistant mosquitoes from the Sumid pUC4K-KIXX (Pharmacia) The set was purified as a SmaI fragment and cloned between adjacent asd sequences. It has become. The forward primer and the reverse primer containing the restriction sites SacI and SalI, respectively. Using the primers, PCR amplification was performed using the asd gene with an internal deletion, Kan^r A 2 kb PCR fragment containing the cassette was generated. All Dasd: : Kan^rPCR fragment was selected as Sac of positive selection suicide vector pCVD442. Cloned into I / SalI site (Donnenberg and Kaper, Infect. Immun. (1991) ) 59: 4310). The ligation was transformed into SM10λpir (Si Mon et al., BioTechnology (1983) 1: 784); Were selected according to their tolerance. SM10λpir (pCVD442 :: asd) to S . flexneri2a 2457T (pAB322 [Tet^r/ Amp^s])When Join, Amp^r/ Te^rA zygote was selected. PCR analysis integrates into chromosome The isolateds obtained were isolated on the pCVD442 plasmid asd Was confirmed to be recombined with the downstream part of These isolates form on sucrose. Longer, a second recombination event occurred (Quantt and Hynes, Gene) (1993) 127: 15). Kan^rAnd the DAP requirements To give isolate 15C. Hybridization and PCR analysis This strain was demonstrated to have a deletion in asd. This mutation has a low copy number E. coli cloned into the vector complemented by E. coli asd The phenotype of Naru was restored. 15C to its Tet^rFusaric acid treatment from plasmid (Maloy and Nunn, J. Bacteriol) . (1981) 145: 1110) to give isolate 15D. Example 2Characterization of the isolated 15D   Strain 15D is a commercially available eukaryotic cell expression vector pCMV without antibiotic selection. β could be maintained. pCMVβ is human cytomegal in mammalian cells Under the control of the immediate early promoter and enhancer from Expresses E. coli β-galactosidase, which can be expressed in mammals after release. -Enabled easy analysis of mediated gene expression (MacGregor and Ca Nucl. Acids Res. (1989) 17: 2365). .   Screening of strain 15D, a large plus essential for mammalian cells to enter bacteria It was ensured that the mid was not lost during genetic engineering. Stock 15D is Birren And has been found to express the associated polypeptides, IpaB and IpaC. This is measured by immunoadsorption (Mills et al., Infect. Immun. (1988) 56: 2933), that the invading plasmid is not lost. showed that. Shigel containing mutations in genes required for cell wall synthesis la) still adheres to cells in culture and can also invade cells Proving that it was possible was important. Strains 15D and 15D (pCMVβ) Replenish DAP or replenish DAP during the 90 minute intrusion period, respectively. Ability to enter cultured baby hamster kidney (BHK) cells without supplementation Forces were tested (Oaks et al., Infect. Immun. (1985). 48: 124). After this interaction period, the monolayer is thoroughly washed and then Treated with a medium containing ntamycin (50 μg / ml) for at least 30 minutes, Bacteria were removed. Both these constructs were found to enter BHK cells. However, when DAP was added during the bacterial-cell interaction, the recorded 1 Colony numbers of 5D and 15D (pCMVβ) were significantly increased. Infected BH A fixed and stained chamber slide of a K cell monolayer (chamber slide) es) was examined by light microscopy to demonstrate viable findings. DA during the intrusion phase If P is not present, 15D and 15D (pCMVβ) Just 13% and 10% of BHK cells invaded. On the contrary, DAP contains 33% (15D) and 29% [15D (p CMVβ)] contained bacteria. The purpose of this test is to To determine whether bacteria can be used to release sumid DNA According to the representative data below, during the attachment and invasion phases, the concentrated bacteria Was added to DAP.   Intracellular bacterial viability and β-galactosidase activity were monitored over 48 hours. Traced. To assess viable bacteria recovered from infected BHK cells, The following test method was used. 1 × 10^FiveBHK cells were plated on a plate with 24 recesses. Plates were formed in the recesses. This test method was previously disclosed for Shigella plaque assay The method was modified and adapted (Infect. Im by Mills et al.). mun. (1988) 56: 2933; Infect. Imm un. (1985) 48: 124). Single Congo Red binding positive for each strain Colony (this is the expression of the plasmid-encoded Shigella virulence antigenic determinant) Represents 50 μg / ml DAP [15D] or DAP + 250 In LB broth medium containing μg / ml ampicillin [15D (pCMVβ)] Inoculated overnight. The overnight culture was diluted 1:50 and diluted approximately in the presence of DAP. The cells were grown in the mid-log phase. 50 μg / ml DAP is added 200 My 10X bacterial solution in HBSS with or without added Chloliters were placed in three recesses of sub-confluent BHK cells [these were DMEM (Bio Whitaker)] at about 50: 1. 37 ° C, 5% CO_TwoAt 90 minutes for the bacteria to interact with this minimum volume of BHK cells. Was. Non-adherent bacteria were removed by extensive washing with HBSS. Extracellular Bacteria were then heat-inactivated with 10% FBS (BioWhittaker) and DME containing 50 μg / ml gentamicin Killed by addition of M. At the indicated time points, cells were 0.2% Triton -X-100 solution and dissolve the corresponding dilutions in TSA Congo Red DAP The plate was formed on a plate, and the number of viable bacteria was measured.   For visual inspection of fixed and stained chamber slides, 1 × 10^Five BHK cells were plated on Nunc chamber slides and Were infected with 15D and 15D (pCMVβ) as described. At the corresponding point, Thoroughly wash and fix these chamber slides, then (Fisher). At least 450 cells for data analysis Visual inspection was performed by light microscopy. Instat statistics blog Ram (Graphpad, San Diego, CA) was used to calculate the mean and Standard error was calculated. Example 3Expression of DNA released into cells by strain 15D   The bacterial suspension was concentrated 10-fold, except that 2 ml was added to each flask. The cells were grown as described in Example 1. In this test method, semi-dense BH Before adding the bacterial suspension to the flask of K cells, add 50 μl of DAP to the bacterial suspension. g / ml was added. Bacteria were added at a ratio of about 100: 1. At the time of instruction, BHK Cells are separated by trypsinization and then Washed in PBS. A part of this cell suspension was dissolved in 0.2% Triton-X-100 solution. Dissolve in solution and plate on TSA Congo Red DAP plate The number of bacteria capable of growing was measured. Remaining cells are evaluated for β-galactosidase activity did. β-galactosidase activity was determined by o-nitrophenyl in the remaining cells. Galactose and chromophore o-ni-β-D-galactoside (ONPG) Activity is measured spectrophotometrically as measured by standard biochemical assays using conversion to trophenol. Quantitative analysis by measurement (Methods in Mol by Nolan et al.) ecological Biology, E.C. J. Murray and J.M. M. Walk er editing (Humana Press Inc., Clifton, NJ, 1991) Vol. 7: 217-235). β-galactosidase unit = 380 × OD420 / hour (minute). The total protein concentration of the cell extract was^*Protein Measured with a quality test kit (Pierce).   Initial 1-3 × 10 for each stock⁷Viable bacteria recovered from monolayer of BHK cells However, no β-galactosidase activity was detectable in this cell extract. Attachment Determination of β-galactosidase activity in cell extracts equal to the total number of bacteria added It was negative. After 4 hours, 1 log to 1.5 log of viable cells disappear. Loss and no β-galactosidase activity could be detected. Additional log ~ 1. Loss of 5 log viable cells at both 24 and 48 hour test time points Found in. At both time points, 15D (pCMVβ) from infected BHK cells In cell extracts, an increase in β-galactosidase activity could be easily detected. these The β-galactosidase activity detected at the time of the last detection is Not due. The reason for this is that high levels of viable No β-galactosidase activity was detected despite the presence of cells. And there. In addition, non-invasive isolates of 15D (pCMVβ) (ie, Ipa B and IpaC immunoadsorption negative) were tested for their ability to release plasmid DNA. Was. No β-galactosidase activity was detected at the 24 hour test time .   The finding is that cells must enter mammalian cells to release DNA. And can also escape from the phagocytic vacuole (the phenomenon is usually the first 4 To reinforce the hypothesis that it must be able to I have. At 24 and 48 hours of testing, bacterial killing and plasmid DN Sufficient time had elapsed for the release of A into the cell cytoplasm. Followed by the code Transcription and translation of the reporter gene occurs. Subsequent uptake by eukaryotic cells Extracellular lysis of bacteria, which leads to the release of plasmid DNA with Relationship. This means that non-invasive isolates can induce β-galactosidase activity. Because you can't do that. Example 4Strain 15D as a DNA release vehicle   In order to demonstrate release of pCMVβ DNA into BHK cells, infected monolayers Was immunostained for intracellular β-gas in individual cells. Lactosidase expression was detected visually. 4-concave, as described in Example 1 15D or 15D (pCMVβ) in the three recesses of the head chamber slide ) Were immunostained on BHK cell monolayers and β-galactosidase Expression was detected (Sander et al., J. Immunol. Methods ( 1993) 166: 201). At each time point, the monolayer was washed with a phosphate buffered 4% Fix in laformaldehyde for 5 minutes, then 3% goat serum in HBSS ( Gibco-BRL) for 3 minutes. The BHK cells were then Permeabilization was performed with HBSS containing ponin (Sigma) solution for 1 minute. Monocrona Anti-β-galactosidase (Sigma) in 0.1% sapoin / HBSS Dilution 1: 2000, then kept at 37 ° C. for 30 minutes in a wet chamber . A second anti-mouse IgG (Fc specific) FITC conjugate (Sigma) was : 32 and then kept at room temperature for 30 minutes. Chamber between each step Slides were thoroughly washed with 0.1% sapoin / HBSS solution. Final cleaning process HBSS alone was used to block permeabilized cells. Equipped with fluorescent parts Olympus-VAN04-S or Epi-fluorescent parts Using a Nikon microphoto equipped with It was evaluated visually. The results are shown in FIG.   Monolayers infected with either strain show obvious intracellular immunostaining at 30 minutes of detection. No color was found (FIGS. 3A, B). Monolayer infected with 15D (pCMVβ) Only a few intracellular immunostainings were found at 4 hours of detection (FIGS. 3C, D ). At 24 hours and 48 hours of detection, 15D (pCMVβ) was infected. Several cells stained positive per monolayer area (FIGS. 3E, F). Whole cell cytoplasm Staining of the body was determined by subsequent processing by mammalian cells (ie, transcription and translation). This indicates that the plasmid DNA was released from the bacteria into the cell cytoplasm. Positively stained cells may also be due to the presence of large amounts of β-galactosidase protein. It looked spherical. Fluorescence activated cell sorting (FACS) analysis (by Nolan et al.) 1 to 2% of 5000 cells as measured by At the time of detection, staining was positive for β-galactosidase expression. 15D (pC Leukostat staining of BHK cells infected with MVβ) Visual inspection of the isolated chamber slides revealed that 28% of the cells were 1-5 full fines. Bacteria, and 1.7% were found to contain 5 bacteria (Table 2). ). At 4 hours after gentamicin treatment, 26% of the cells had visually complete cells. Bacteria, and cells containing 4 bacteria were less than 1%. Therefore, 1-5 Bacterial invasion was required for foreign gene expression. pCMVβ is about 500 copies / 7164 bp plasmid with medium to high copy number of bacterial cells Therefore, each bacterium is about 3.93 (10^-9) Containing mg DNA Presumed. About 4-20 × 10 by Shigella^-9mg of DNA in the cytoplasm It is sufficient for expression of β-galactosidase.Percentage of infected BHK cells and bacterial count / infected BHK cells. Cha Member slides and bacteria were prepared as described in Table 1. Data infected Mean percentage of BHK cells and mean of bacteria / infected BHK cells ± standard It is represented by a deviation value (SD). Example 5Gene release by Shigella into different cell types   Shigella bacterial strains invade quite different cells. Gene release to BHK cells To demonstrate that it is not restricted, P815 cells were exposed to 15D (pCMVβ). Dyed. Bacteria used to infect P815 cells were grown as described in Example 1. I let it. 6-Add non-adherent P815 cells cultured in After addition of the bacteria, the plate was spun at 500 × g for 5 minutes. Bacteria and P8 Fifteen cells were allowed to react for 90 minutes. These cells were then thoroughly treated with DMEM. Wash bottom, then in DMEM containing 100 μg / ml gentamicin At 37 ° C, 5% CO_TwoFor 1 hour. These cells Again, wash thoroughly, then DM containing 20 μg / ml gentamicin Resuspend in EM, 37 ° C, 5% CO_TwoOvernight. 24 hours At this point, β-galactosidase activity and protein concentration were measured as described above ( Nolan et al.).   As shown in Table 3, at 24 hours, 10-fold higher compared to basal control Low levels of β-galactosidase were expressed. H-2^dClass I MHC molecule P815 cells that are fully infected with 15D (pCMVβ) Tests show that these cells, in terms of Class I, are not encoded by Shigella-releasing DNA. It is currently being carried out to determine whether a given antigen can be provided. Example 615D is in vivo soProvides protection against Shigella transmission   Tests in the guinea pig keratoconjunctivitis challenge model show that two doses of immunity Shows 100% protection from subsequent Shigella infection for 3 weeks according to the sensitization schedule. Animals On days 0 and 15, 1-4 × 10 per eye⁸Immunized by colony forming units I was sensitized. The challenge was performed 3 weeks after the final immunization. Animals , 3.8 × 10⁸We challenged Virrent 2457T.  Eyes from animals in Experiment C were also stained for β-galactosidase activity. Inoculation with 15D (pCMVβ) and heat-sterilized 15D (pCMVβ) Eyes from the object showed staining. Inoculated with heat-sterilized 15D (pCMVβ) Light staining was detected in the animal eyes. These results indicate that DNA is released. That this highly attenuated strain (attenuated s train) functions in vivo in a guinea pig keratoconjunctivitis model, and And protect against challenge by Shigella even when bacteria are inactivated Prove to provide. Example 7Guinea pig growth test   The purpose of this study was to test the animal's immune response during the challenge and recovery periods. The answer was to evaluate the answer.   Spleens or deep cervical nodes of two animals were collected for testing. 2 charens from each group The died animals were sacrificed for testing at 3 and 4 weeks post-challenge. Proliferative responses were tested in animals evaluated for protection. Preliminary challenge ( pre-challenge) animal, vaccinated as described above, and When was challenged, the organs were tested.   Prepare a single cell suspension from spleen and deep cervical node, plate with 96 recesses And 1-2 × 10 in 100 ml^FivePlated at cell / recess concentration. Each stimulus 10 ml of the agent was added to the corresponding recess. After three days of culture, the amount of growth It was measured using a radioactive kit. These responses are shown in Table 5 below.N. P, when no proliferation is detected * Naive animals showed no detectable response; thus, indeed O. D. Values are shown. ConA: Concanavalin A, 5 μg / ml LPS: E. 250 pg / ml commercial preparation from Coli H. K: Heat sterilized Shigella flexneri ri) 2a strain 2457T 5 μg / ml   All responses were averaged (i.e., 3-4 depressions), and the average basal response was reduced. D . The 490 value was determined. The stimulation index was determined by the mean experiment O.D. D. Values are the average Experiment O. D. Calculated by dividing by value.   These results were obtained at the time of challenge and several weeks after challenge (post challenge), this highly attenuated strain Immune response to mitogen response (specific for mitogen response and for heat-killed antigen) (Including T cells and B cells, as measured by the specific response). β -No growth to the galactosidase protein was detected. Normal immunological of the eye From the properties, this result was expected (Cri by Rocha and Baines). physical Rev. Immun. (1992) 12: 81-100). Example 8Intranasal challenge propagation in mice   The purpose of this experiment was to demonstrate the ability of 15D to release DNA in vivo. (I.e., intranasal mouse). Furthermore, career The immune response to was also measured.   Each group of five mice was inoculated intranasally twice at four week intervals. Each contact For each strain or treatment, three different doses were also administered. These amounts are Shown below. One of the treatment groups consisted of 50 μl of DAP added to the culture before inoculation. Consists of mice fed 15D (pCMVβ) with g / ml. Second contact At 4 weeks post seeding, the spleen was removed, a single cell suspension was prepared, and then 96 In a concave plate, add 2 × 10^FivePlate with cells / recess. phase 10 ml of stimulant was added to the corresponding recess. Incubate these plates for 3 days. The amount of growth that occurred was determined using a non-radioactive kit. These numbers Are averaged, the base value is reduced, and D. The 490 value was determined. Con A, E.C. Col The irritation index for LPS and heat-sterilized 2457T was determined by the mean experiment O.D. D. Values were calculated by dividing by the inexperienced control value. The results are shown in the table below. 6 is shown. The stimulation index for b-gal is O.D. D. Divide by value Experiment (pCMVβ) D. Value. Approximate amount for both inoculations 15D: 3 × 10⁶, 1 × 10⁶And 3 × 10^Five 15D with or without DAP (pCMVβ): 1 × 10⁶ , 5 × 10^FiveAnd 1 × 10^Five * Add polymyxin B to b-gal protein to clean all contaminating LPS Formed.   These results indicate that in this model, 15D was sufficient for pCMVβ DNA Indicates release. At higher inoculum doses, DAP was added. Mice inoculated with 15D (pCMVβ) without or with added b-gal Proliferate in response to proteins. Furthermore, at the specified dose, carry No significant proliferative response to a. Example 9Mouse intranasal response II   Lymphatic proliferative response directed against plasmid-expressed β-galactosidase (Lymphoproliferative responses) and antibodies Response was measured after bacterial release of plasmid DNA into mouse nasal tissue. Twice Was administered on days 0 and 28. 4 weeks after the last inoculation , 15D (pCMVβ) spleen cells from β-galactosidase Ze Showed a lymphoproliferative response directed against BALB / c of 8-10 week old females Mouse (Harlan Sprague Dawley, Indianapol) is, IN) with 0.3 mg of xylazine hydrochloride [Lonpa (Rompun); Mobay Corp. , Shawnee, KA] and 1.0 mg of ketamine hydrochloride [Ketaset; Aveco C ompany, Fort Dodge, IA]. Let it calm down. The concentrated bacterial suspension (15 ml) was dropped onto the nostrils of each mouse . On day 0 and at 4 weeks, 5-10 mice in each group had 10⁶Or 10⁷ Given the surviving bacteria. Several groups of mice were supplemented with 50 μg / ml DAP. A filled 15D (pCMVβ) inoculum was provided. Blood for serum analysis after last inoculation At 4 weeks. At this point, the spleen was also removed from ConA, E. Coli   LPS, heat sterilized 2457T and purified β-galactosidase (Si gma, St .; Louis, MO) induced lymphoproliferative response in vivo (B) Removed for measurement. Spleen cells (1 × 10^Five/ Recess) with polymyxin (Bu) rrows Wellcome, Research Triangle P ark, NC) Purified β-galactosidase containing 10 μg / ml 5 μg / ml, ConA 50 μg / ml, Coli LPS 2.5μg For 3 days in the presence of 5 μg / ml and heat-sterilized 2457T. Was. The degree of proliferation was measured by cell titer 96 (registered trademark) ) AQ_ueousNon-radioactive cell proliferation kit (Promega, Madison, WI) ). The reported OD490 values are the mean of stimulated cells. Calculated by subtracting the mean value of unstimulated cells from the value.   These results indicate that 15D with or without DAP was added. Mice inoculated with (pCMVβ) were up to 5-fold larger than controls, Shows that they can proliferate in response to tosidases (FIG. 4D). . Example 10Antibody response to β-galactosidase in mice intranasally inoculated   15D, 15D (pCMVβ) or 15D containing 50 μg / ml of DAP (p Serum from a group of mice inoculated with CMVβ) was immunized against β-galactosidase. Tested for compliance. 1 microgram of purified β-galactosidase is 7.5 It was subjected to electrophoresis on a% SDS-polyacrylamide gel. After electrophoresis These gels were electroadsorbed on nitrocellulose. These blots are In, then divided into sections, and then overnight collected serum samples ( (Diluted 1:50 in casein buffer). The bound antibody is Phosphatase (BMB, Indianapolis, Ind.) Detection was performed using a 1: 500 dilution of a second rabbit anti-mouse Ig. alkalinity Phosphatase activity was detected by the substrate BCIP / NBT (Sigma) . Immunosorbent analysis was performed on serum samples from mice infected with 15D (pCMVβ). Showed an antibody response specific for β-galactosidase.   Serum samples were also analyzed by ELISA and antibody isotype and And IgG subclasses were measured. Antibodies specific for β-galactosidase Is an antibody of the IgG isotype, which comprises IgG1, IgG2a and IgG2. It is equally represented by b (Table 7). It encompasses both Th1 and Th2 cells. Indicates that it is included.   These results presented here use attenuated bacteria. To release the plasmid DNA on the mucosal surface, -A demonstration of the ability to stimulate an immune response to the encoded foreign gene product This is the first proof. The approach to vaccine development is the development and development of DNA-based vaccines. And supply must be simple, while often stimulating the desired mucosal immune response The technology that makes it possible must be expanded.   We have discovered a new method for releasing functional DNA inside cells. This method should not be restricted to Shigella. This reason is used by Shigella Invading genes can be found in other bacteria, such as E. coli. Because it can be inserted into Coli. (Infect. Immun. (1983) 39 by Sansonetti et al.) : 1392). Similarly, other bacteria, such as Listeria, Can invade cells and escape from the phagocytic vacuole into the cytoplasm ( Portnoy and Jones, Ann. N. Y. Acad. Sci. ( 1994) 730: 15). We show that bacteria can escape phagocytic vacuoles into the cytoplasm. Have no official evidence that export is essential for DNA release, Salmonella typhimurium, which is difficult to reach the cytoplasm la typhimurium). Initial experiments with microorganisms show that It is suggested that this is not an effective DNA release medium.   To achieve a balance between cell entry and release efficiency with its subsequent reactogenicity For this purpose, any bacterial vector DNA release system is required. In the case of Shigella, Genetic response to invasion also indicates macrophage invasion and apoptosis (a poppysis) and then inflammation (Zychlins) Nature (1992) 358: 167). We believe that DAP A Shigella strain that was unable to survive inside the cell in the presence was constructed. This kind of cheap Measuring integrity awaits human testing.   The bacterial DNA release system we disclosed has several advantages for certain applications. Sticky Release of DNA-encoded antigens into the membrane immune system is a class I and / or II Stimulation of Th1 or Th2 help or IgA and IgG antibody production Many can be directed to the hidden maintenance of conformational epitopes and appropriate overlaps Simultaneous immunization with a number of antigens must be possible. A few names Diarrheal diseases, such as retroviruses; diseases transmitted by sexual activity, such as For example, human immunodeficiency virus, Neisseria gonorrhoeae (Neisseria g onorrhoeae), and human papillomavirus; and also gastrointestinal disorders For example, ulcer-forming Helicobacter pylori   pylori) can be particularly responsive to this approach. Intestinal immune tolerance mechanism Suppression of autoimmunity by the Nism mechanism has been proven (Proc by Sun et al. . Natl. Acad. Sci. U. S. A. (1994) 91: 10795). , Should also be acceptable to this approach.   Maybe bacterial release of DNA for vaccine formation and potent gene therapy / replacement The greatest advantage is the ease and acceptance of oral forms and other forms for mucosal delivery In effectiveness. Similarly, this type of DNA vaccine formation (which is Is an attenuated bacterial vector). Vaccines can be produced at the expense of fermentation, cell lysis and packaging. You. Therefore, this type of vaccine is currently being developed and is being developed. At least partially contribute to reducing the price and problems of vaccines Wear.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/04 C12N 9/04 Z 15/09 ZNA G01N 33/569 F G01N 33/569 C12N 15/00 ZNAA (31) Priority claim number 60 / 018,035 (32) Priority date May 21, 1996 (May 21, 1996) (33) Priority claim country United States (US) (81) Designated country EP (AT) , BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA , GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, T ), AL, AM, AT, AU, AZ, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL, IS, JP , KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN (72) Inventor Sizemore, Donata, Earl United States 20879 Steerswick Road, Gaithersburg, MD 10006 Number 301 (72) Inventor Sadov, Gerald, Sea United States 20012 Washington D.C. Double. 1622

Claims (1)

[Claims]   1. Attenuated dysentery that can enter cells and die once inside cells Strain.   2. The claim 1 wherein the strain is Shigella flexneri (S. flexneri). 2. The attenuated Shigella strain according to item 1.   3. The strain is 15D given ATCC accession number ATCC 55710. An attenuated Shigella strain according to claim 2.   4. Aspartate b-semialdehyde dehydrogenase gene present in Shigella A method for producing an attenuated Shigella strain, comprising inactivating bacillus.   5. The attenuation according to claim 4, wherein the inactivation is due to a mutation. Method for producing Shigella strain.   6. Attenuated Shigella can enter cells, but once inside cells The method for producing an attenuated Shigella strain according to claim 4, which is killed.   7. The vaccine is (I) Shigella attenuated, and (Ii) a pharmaceutically acceptable excipient, A vaccine for reducing a disease state caused by Shigella in an individual.   8. 8. The method according to claim 7, wherein the Shigella is S. flexneri. A vaccine for reducing a medical condition in an individual according to claim 1.   9. Attenuated S. flexneri is the ATCC accession number 9. The individual according to claim 8, wherein the individual is 15D given the signal ATCC 55710. To reduce the condition caused by Shigella flexneri (S. flexneri) in the body vaccine.   10. The flexner according to claim 7, wherein Shigella is further inactivated. -A vaccine to reduce the pathology caused by Shigella (S. flexneri) in individuals .   11. An immunologically effective amount of Shigella attenuated in a pharmaceutically acceptable excipient Administering to an individual, reducing the pathology caused by Shigella in the individual. Way.   12. Claim 1 wherein the Shigella is S. flexneri. A method for reducing a disease state caused by Shigella in an individual according to claim 1.   13. 12. The method according to claim 11, wherein the Shigella is further inactivated. A method to reduce the symptoms caused by Shigella.   14． A DNA-containing vehicle containing the attenuated Shigella, into which the DNA is introduced. A delivery vehicle for delivering the drug.   15. Claim 1 wherein the Shigella is S. flexneri. A delivery vehicle for delivering DNA to cells according to claim 4.   16. The cell according to claim 14, wherein the cell is a cell of a small intestinal mucosal epithelial cell. A delivery vehicle for delivering DNA to cells.   17． Claim 1 wherein the Shigella is S. flexneri. A delivery vehicle for delivering DNA to cells according to claim 4.   18. Flexneri (S. flexneri) has ATCC accession number ATCC 18. The cell according to claim 17, wherein the cell is 15D given 55710. A delivery vehicle for delivering NA.   19. 15. The method according to claim 14, wherein the attenuated Shigella is further inactivated. A delivery vehicle for delivering DNA to cells.   20. Consisting of an attenuated Shigella into which the antigen is introduced, for delivering the antigen to cells. Delivery vehicle.   21. Claim 2 wherein the Shigella is S. flexneri. A delivery vehicle for delivering an antigen to a cell according to paragraph 0.   22. Claim 2 wherein the S. flexneri is 15D. A delivery vehicle for delivering an antigen to a cell according to claim 1.   23. 21. The attenuated Shigella according to claim 20, wherein the attenuated Shigella is further inactivated. A delivery vehicle for delivering the antigen to the cells.   24. An immunologically effective amount of Shigella attenuated in a pharmaceutically acceptable excipient For oral immunization of an individual against Shigella, comprising orally administered to the individual the method of.   25. Claim 2 wherein the Shigella is S. flexneri. A method for oral immunization of an individual against Shigella according to claim 4.   26. Claim 2 wherein the S. flexneri is 15D. A method for oral immunization of an individual against Shigella according to claim 5.   27. 25. The red of claim 24, wherein the attenuated Shigella is further inactivated. A method for oral immunization of an individual against Shigella.   28. A method for delivering DNA to a cell, comprising: (I) introducing the DNA into the attenuated Shigella, and (Ii) administering the Shigella to cells.   29. Claim 2 wherein the Shigella is S. flexneri. 9. A method for delivering DNA to a cell according to item 8.   30. Claim 2 wherein the S. flexneri is 15D. 10. A method for delivering DNA to a cell according to item 9.   31. 29. The cell of claim 28, wherein the cell is a mucosal epithelial cell. A method for delivering A.   32. The cell according to claim 28, wherein the mucosal epithelium is a small intestinal mucosal epithelium. A method for delivering NA.   33. 29. The cell of claim 28, wherein the attenuated Shigella is further inactivated. A method for delivering DNA to cells.   34. A method for delivering an antigen to a cell, comprising: (I) introducing the antigen into the attenuated Shigella, and (Ii) administering the Shigella to cells.   35. Claim 3 wherein the Shigella is S. flexneri. A method for delivering an antigen to a cell according to claim 4.   36. Flexneri (S. flexneri) has ATCC accession number ATCC 36. The cell according to claim 35, wherein the antigen is 55D given 55710. A method for delivering.   37. The cell according to claim 34, wherein the cell is a cell of a mucosal epithelium. A method for delivering.   38. The mucosal epithelium is a small intestinal mucosal epithelium. A method for delivering an original.   39. 35. The cell of claim 34, wherein the attenuated Shigella is further inactivated. A method for delivering an antigen to a vesicle.   40. A method for detecting Shigella infection, (I) coating the surface with Shigella attenuated or its components, (Ii) Serum from an individual suspected of having an infection on the coating surface or Contact the tissue sample, and (Iii) the complex formed between Shigella and its specific immune response present in the sample Detecting the presence or absence of infection to detect the presence or absence of infection.   41. Used to detect attenuated Shigella and the presence of an immune response to Shigella in the sample Diagnostic kit for the detection of Shigella infection, comprising an accessory reagent suitable for .   42. Claim 4 wherein the Shigella is S. flexneri. A diagnostic kit for detecting Shigella according to claim 1.   43. Flexneri (S. flexneri) has ATCC accession number ATCC 43. The diagnostic kit of claim 42, which is 15D given 55710. .   44. A method for delivering a functional nucleic acid to cells using bacteria, comprising: (I) introducing the nucleic acid into the attenuated bacterium, and (Ii) administering the bacterium to a cell.