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• • 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
  • A61K39/0275—Salmonella
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • 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
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/42—Salmonella
• • 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

• This invention relates to avirulent microbes, their method of preparation, and their use in vaccines.
• Salmonellosis of swine is one of the most
• S. choleraesuis is host-adapted to swine and is often the etiologic agent of fatal septicemic disease with little involvement of the intestinal tract. This S.
• choleraesuis reservoir in swine is a concern not only because of its disease-causing potential for pigs, but also because of its public health significance for humans.
• the disease caused by S. choleraesuis manifests in many clinical signs.
• the organism is inherently invasive, and does not require the massive luminal
• choleraesuis auxotrophs with requirements for aromatic amino acids due to an aroA mutation have reduced virulence in mice, but were unable, even after three immunizing doses, to induce protective immunity.
• S. choleraesuis vaccine licensed for use in the United States of America is a killed bacterial bactrin, which is not particularly effective in inducing protective immunity.
• the present invention is based, in part, on new avirulent S. choleraesuis derivatives that are not
• choleraesuis and other enteric bacteria with which antibodies to S. choleraesuis cross react. These strains are also useful as carrier microorganisms for the production of expression products encoded on recombinant genes in the bacterial cells.
• one embodiment of the invention is a vaccine for the immunization of an individual comprising an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional adenylate cyclase due to a mutation in a cya gene.
• Another embodiment of the invention is a method for stimulating the immune system to respond to an im- munogenic antigen of S. choleraesuis comprising
• Still another embodiment of the invention is a method for stimulating the immune system to respond to an immunogenic antigen of a pathogen comprising administering to said individual an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially in- capable of producing functional adenylate cyclase and cyclic AMP receptor protein (CRP) and being capable of expressing a recombinant gene encoding the immunogenic antigen, to produce an antigen capable of inducing an immune response in said vertebrate against said pathogen.
• CRP adenylate cyclase and cyclic AMP receptor protein
• Another embodiment of the invention is an isolated avirulent strain of S. choleraesuis which is substantially incapable of producing functional adenylate cyclase.
• Still another embodiment of the invention is a vaccine for the immunization of an individual comprising an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional cyclic AMP receptor protein (CRP) due to a mutation in a crp gene.
• CRP cyclic AMP receptor protein
• Yet another embodiment of the invention is a method for stimulating the immune system to respond to an immunogenic antigen of S. choleraesuis comprising
• Another embodiment of the invention is a method for stimulating the immune system to respond to an immunogenic antigen of a pathogen comprising administering to said individual an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional CRP and being capable of expressing a recombinant gene encoding the immunogenic antigen, to produce an antigen capable of inducing an immune response in said vertebrate against said pathogen.
• Yet another embodiment of the invention is an isolated avirulent strain of S. choleraesuis which is substantially incapable of producing functional CRP due to a mutation in a crp gene.
• Still another embodiment of the invention is a vaccine for the immunization of an individual comprising an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional adenylate cyclase and CRP due to a mutation in the cya and crp genes.
• Yet another embodiment of the invention is a method for stimulating the immune system to respond to an immunogenic antigen of S. choleraesuis comprising
• Another embodiment of the invention is a method for stimulating the immune system to respond to an immunogenic antigen of a pathogen comprising administering to said individual an avirulent derivative of pathogenic S. choleraesuis, said derivative being substantially incapable of producing functional adenylate cyclase and CRP and being capable of expressing a recombinant gene encoding the immunogenic antigen, to produce an antigen capable of inducing an immune response in said vertebrate against said pathogen.
• Still another embodiment of the invention is an isolated avirulent strain of S. choleraesuis which issubstantially incapable of producing functional adenylate cyclase and CRP due to mutations in the cya and crp genes.
• Another embodiment of the invention is a strain selected from the group of strains ATCC 53647, ATCC 53648, ATCC 67923, ATCC 53885, ATCC 67922, and mutants thereof, and derivatives thereof.
• Vaccine means an agent used to stimulate the immune system of a living organism so that protection against future harm is provided .
• Immunization refers to the process of inducing a continuing high level of antibody and/or cellular immune response in which T-lymphocytes can either kill a pathogen and/or activate other cells (e.g., phagocytes) to do so in an organism, which is directed against a pathogen or antigen to which the organism has been previously exposed.
• the phrase is restricted to the anatomical features and mechanisms by which a multicellular organism produces antibodies against an antigenic material which invades the cells of the organism or the extracellular fluid of the organism.
• the antibody so produced may belong to any of the immunological classes, such as immunoglobulins A, D, E, G or M.
• immunoglobulins A, D, E, G or M are particularly interesting.
• vaccines of the nature described herein are likely to produce a broad range of other immune responses in addition to IgA formation, for example, cellular and humoral immunity.
• vertebrate means any member of the subphylum Vertebrata, a primary division of the phylum Chordata that includes the fishes, amphibians, reptiles, birds, and mammals, all of which are characterized by a segmented bony or cartilaginous spinal column. All vertebrates have a functional immune system and respond to antigens by producing antibodies. Thus, all vertebrates are capable of responding to vaccines.
• vaccines are most commonly given to mammals, such as humans or dogs (rabies vaccine), vaccines for commercially raised vertebrates of other classes, such as the fishes and birds if of the nature described herein, are within the scope of the present invention.
• invertebrate any member of the Animal Kingdom, excluding the vertebrates. Such animals constitute the Division Invertebrata and have no backbone or spinal column. This classification includes all animals except fishes, amphibians, reptiles, birds and mammals. Many invertebrates are capable of eliciting a primitive immune response to antigenic stimulation and are susceptible to the same microorganisms which infect vertebrates and which are disclosed herein in accordance with this invention. Examples of such invertebrates are shellfish and mollusks and other related animals.
• An "individual” treated with a vaccine of the invention is defined to include all vertebrates, such as mammals, including domestic animals and humans, and various species of birds, including domestic birds, particularly those of agricultural
• mollusks and certain other invertebrates have a primitive immune system, and are included as “individuals.”
• avirulent does not mean that a microbe of that genus or species cannot ever function as a pathogen, but that the particular microbe being used is avirulent with respect to the particular animal being treated.
• the microbe may belong to a genus or even a species that is normally pathogenic but must belong to a strain that is avirulent.
• “Pathogenic”, as used herein, means capable of causing disease or impairing normal physiological functioning.
• An "avirulent strain” is incapable of inducing the full set of symptoms of the disease that is normally associated with its virulent pathogenic counterpart.
• microbes includes bacteria, protozoa, and unicellular fungi.
• microbes are also contemplated to be within the scope of this invention.
• derivative is meant sexually or asexually derived progeny and mutants of the avirulent strains including single or multiple base substitutions, deletions, insertions or inversions which retain the in-ability to produce functional adenylate cyclase and cAMP receptor protein with or without naturally occurring virulence plasmids.
• strains such as Chi4062 and Chi4064 carry the qyrA mutation conferring nalidixic acid resistance which has been used herein as a convenient marker to follow strains following oral inoculation.
• the gyrA+ mutation can be easily removed by transducing the qyrA+ (conferring sensitivity to nalidixic acid) gene into strains by
• mutant gene refers to genetic material that has been transferred from one organism into a second in such a manner that reproduction of the second organism gives rise to descendants containing the same genetic material.
• gene is used herein in its broadest sense to represent any biological unit of heredity. It is not necessary that the recombinant gene be a complete gene as present in the parent organism, which was capable of producing or regulating the production of a macromolecule, for example, a functioning polypeptide. It is only necessary that the gene be capable of serving as the template in the production of an antigenic product. The product will not necessarily be found in that exact form in the parent organism. For example, a functional gene coding for a polypeptide antigen comprising 100 amino acid residues may be transferred in part into a carrier microbe so that a peptide comprising only 75, or even 10, aminoacid residues is produced by the cellular mechanism of the host cell.
• this gene product is an antigen that will cause formation of antibodies against a similar antigen present in the parent organism, the gene is considered to be within the scope of the term "gene” as defined herein.
• the amino acid sequence of a particular antigen or fragment thereof is known, it is possible to chemically synthesize the DNA fragment or analog thereof by means of automated gene synthesizers or the like and introduce said DNA sequence into the appropriate expression vector.
• a gene as defined and claimed here is any unit of heredity capable of producing an antigen.
• the gene may be of chromosomal, plasmid, or viral origin.
• gene expression means that the information inherent in the structure of the gene is transformed into a physical product in the form of an RNA molecule, polypeptide or other biological molecule by the biochemical mechanisms of the cell in which the gene is located.
• the biological molecule so produced is called the gene product.
• gene product refers to any biological product or products produced as a result of the biochemical reactions that occur under the control of a gene.
• the gene product may be, for example, an RNA molecule, a peptide, or a product produced under the control of an enzyme or other molecule that is the initial product of the gene, i.e., a metabolic product.
• a gene may first control the synthesis of an RNA molecule which is translated by the action of
• RNA molecule the
• glycoproteins and lipoproteins are preferred gene
• allergens means substances that cause allergic reaction, in this case in the animal which will be vaccinated against them. Many different materials may be allergens, such as animal dander and pollen, and the allergic reaction of individual animals will vary for any particular allergen. It is possible to induce tolerance to an allergen in an animal that normally shows an allergic response. The methods of inducing tolerance are well-known and generally comprise administering the allergen to the animal in increasing dosages. Further discussion of tolerance induction is given in the Barrett textbook previously cited.
• This invention is predicated on the discovery that certain mutations can render a microbe avirulent without substantially affecting its immunogenicity. More specifically, this invention is made possible by microbial vaccines in which the microbe carries the deletion (delta) mutations delta-cya and delta-crp. These deletions eliminate the ability to synthesize adenylate cyclase (ATP pyrophosphate lyase (cyclizing) EC 4.6.1.1) and the cyclic AMP receptor protein (CRP), respectively, as described in EPO Pub. No. 315,682.
• EPO Pub. No. 315,862 described how the elimination of cyclic-3',5'-adenosine monophosphate (cAMP), adenylate cyclase and the cyclic AMP receptor protein through delta-cya and delta-crp mutants rendered S.
• cAMP cyclic-3',5'-adenosine monophosphate
• adenylate cyclase adenylate cyclase
• the cyclic AMP receptor protein through delta-cya and delta-crp mutants rendered S.
• the avirulent S. choleraesuis derivative can be used as a carrier bacteria to deliver selected antigens to the gutassociated lymphoid tissue (GALT), for example to the GALT
• a plasmid also called an plasmid
• extrachromosomal element is a hereditary unit that is physically separate from the chromosome of the cell.
• the DNA may be of any size and is often obtained by the action of a restriction endonuclease enzyme which acts to split DNA molecules at specific basepair sites.
• the new recombinant molecules may be transferred into a host cell by various means such as transformation (uptake of naked DNA from the external environment, which can be artificially induced by the presence of various chemical agents, such as calcium ions) or transduction (recombinant DNA packaged and introduced within a phage such as transducing phage or cosmid vectors).
• Recombinant DNA in the carrier cell may continue to exist as a separate piece of DNA or it may insert into the host cell chromosome and be reproduced with the chromosome during cell division. This invention sometimes utilizes transposons as the transferred
• Transposons are highly movable pieces of DNA that insert in DNA and may also be excised. The excision may carry off surrounding genetic material, causing
• the immunogenic component of the vaccine when the immunogenic component of the vaccine is an allergen of the host such a vaccine may be used in an exposure regimen designed to specifically desensitize an allergic host.
• a vaccine for the immunization of a vertebrate or invertebrate animal comprising a live avirulent derivative of S. choleraesuis incapable of producing functional adenylate cyclase and cAMP receptor protein, and capable of expressing a recombinant gene derived from an organism that is a pathogen of or that produces an allergen of said animal.
• a subject of the invention is avirulent strains of S. choleraesuis, which carry mutations in the cya and/or crp genes.
• the S. choleraesuis carrier of the gene product must be properly introduced into the animal.
• introduction of the microbe or gene product directly into the gut or bronchus is preferred, such as by oral administration, gastric intubation or in the form of aerosols, although other methods of administering the vaccine, such as intravenous, intramuscular, subcutaneous injection or intramammary or intrapenial or vaginal administration, are possible.
• the host organism itself can serve as a source of genetic material when immunoregulatory genes or genes for other pharmacologically active substances are being expressed by the vectors.
• Administration of a live vaccine of the type disclosed above to an animal may be by any known or standard technique. These include oral ingestion, gastric intubation, or broncho-nasal spraying. All of these methods allow the live vaccine to easily reach the GALT or BALT cells and induce antibody formation and are the preferred methods of administration. Other methods of administration, such as intravenous injection, that allow the carrier microbe to reach the animal's blood stream may be acceptable. Intravenous, intramuscular or intramammary injection are also acceptable with other embodiments of the invention, as is described later. The dosages required will vary with the
• antigenicity of the gene product and need only be an amount sufficient to induce an immune response typical of existing vaccines. Routine experimentation will easily jestablish the required amount. Multiple dosages used as needed to provide the desired level of protection.
• the pharmaceutical carrier in which the vaccine is suspended or dissolved may be any solvent or solid or encapsulated in a material that is nontoxic to the
• Suitable pharmaceutical carriers include liquid carriers, such as normal saline and other nontoxic salts at or near physiological concentrations, and solid carriers, such as talc or sucrose and which can also be incorporated into feed for farm animals.
• Adjuvants may be added to enhance the antigenicity if desired.
• the vaccine is preferably presented in the form of an aerosol.
• Immunization with a pathogen derived gene product can also be used in conjunction with prior immunization with the avirulent derivative of a pathogenic microorganism acting as a carrier to express the gene product specified by a recombinant gene from a pathogen.
• Such parenteral immunization can serve as a booster to enhance expression of the secretory immune response once the secretory immune system to that pathogen-derived gene product has been primed by immunization with the carrier microbe expressing the pathogen derived gene product to stimulate the lymphoid cells of the GALT or BALT.
• the enhanced response is known as a secondary, booster, or anamnestic response and results in prolonged immune protection of the host.
• Booster immunizations may be repeated numerous times with beneficial results.
• This example illustrates the construction of delta-cya, delta-crp, and delta-cya delta-crp derivatives of S. choleraesuis, the virulence properties of the mutants after peroral (p.o.) inoculation, and the
• S. choleraesuis strains used in this Example are listed in Table 1. Bacterial strains were grown at 37°C in L broth and on L agar (Lennox, Virology 1: 190-206
• Penassay agar Difco antibiotic media #3 + 1.5% BBL agar, Becton Dickinson Microbiology Systems,
• Bacteriol. 145:1110 (1981) were used for fusaric acid selection for deletion mutations in strains with Tn10 insertions. Transformations were performed by the method of Dagert and Ehrlich, Gene 6:23 (1979).
• the plasmid, pSD110 which carries the crp and Amp genes from E. coli, which was described in Schroeder and Dobrogosz, J.
• pYA2028 which has the Inc/Par region of the virulence plasmid cloned into the high copy number plasmid pUC18, as described below.
• mice Male BALB/c mice (Harlan Sprague-Hawley,
• mice were held for one week in a quarantined room prior to being used in
• Virulence of S. choleraesuis strains was determined after p.o. inoculation. Bacteria for inoculation in mice were grown overnight as static cultures at 37oC in L broth. All cultures were diluted 1:20 into prewarmed L broth and aerated at 37oC for approximately 5 h to an optical density at 600 nm of about 0.8 to 1.0. The cells were concentrated 50-fold by centrifugation at 8,000 x g for 10 min at room temperature, followed by suspension in BSG. Dilutions were plated on MacConkey agar with 1% maltose to verify the Cya or Crp phenotype and to enumerate cells.
• mice Prior to p.o. inoculations, mice were deprived of food and water for 4 h before infection. They were then given 30 microliters of 10% (wt/vol) sodium bicarbonate 5- 10 min before being fed a 20 microliter aliquot of S.
• mice/cage Groups of five mice/cage were perorally immunized with various doses of avirulent mutants and then challenged 30 days later with various doses of the wild-type, virulent parent, Chi3246. Morbidity and mortality conditions were observed for at least 60 days.
• the strains which were constructed which were derived from Chi3246 are shown in Table 1. Also shown in Table 1 are the relevant genotypes of the strains, and a description of the method by which the strain was derived, utilizing the methods for transposon insertion via
• bacteriophage can adsorb to and inject DNA into the cells of all S. choleraesuis strains used in this example, but are unable to replicate their DNA in these bacterial cells.
• zhb::Tn10, crp773::Tn10 and cya::Tn10 mutations are in S. typhimurium strains Chi 3711, Chi3773, PP1037 and PP1002, respectively (see Table 1), it was necessary to move the mutations from the smooth-LPS S. typhimurium background into an intermediate Salmonella host in which both
• P22HTint and P1L4 could be propagated.
• the bacteriophagesP22HTint and P1L4 are specific for strains with smooth and rough LPS coats, respectively.
• the hosts, Chi3385 and Chi3477 are restriction-deficient, modification- proficient galE S. typhimurium strains. Growth of Chi3385 and Chi3477 in media with low concentrations of galactose permits synthesis of UDP-galactose, resulting in normal levels of LPS side chains; these conditions are essential for attachment and infection by P22HTint. Growth of
• Chi3385 and Chi3477 in media containing glucose and lacking galactose permits synthesis of a rough or incomplete LPS, and enables the adsorption and replication of P1L4 or P1 clr clm in these rough strains.
• the S. choleraesuis strains Chi3492, Chi3751, Chi3755, Chi3759, Chi3820, and Chi3858 were constructed.
• the Tn10 linked to the delta-cya or delta-crp mutation was eliminated by selection for fusaric acid resistance to yield Chi3860 and Chi3659, respectively.
• pSD110 which encodes the E. coli crp + gene. pSD110 was thus transduced into Chi3752
• Phenotypic analysis of cya and crp mutants Phenotypic analysis of cya and crp mutants.
• the cya mutants (Chi3492, Chi3753, Chi3859), the crp mutants (Chi3751, Chi3752, Chi3820) and the cya crp mutant (Chi3781) were subjected to phenotypic analysis. These strains failed to ferment maltose, mannitol, sorbitol, and melibiose, and slowly fermented galactose. The phenotypes were as expected based on known requirement for cAMP, and for CRP for catabolic activities. The requirements for cAMP, and for CRP for regulation of gene expression are described in the following references. Perlman and
• choleraesuis was accomplished using essentially either the transformation methods described in Dagert and Ehrlich 1979, supra, or the electroporation methods of Feidler and Worth, Anal. Biochem. 170:38 (1988). Transformants were selected for, and maintained by, the inclusion of
• mice choleraesuis strains listed in Table 1, groups of mice were orally inoculated with 100-fold varying doses of each strain. The results are shown in Table 2.
• mice immunized with 10 7 , 10 8 , or 10 9 CFU of the somewhat more virulent strains with mutations in crp exhibited better health and a higher rate of survival after oral challenge with Chi3246 than did mice immunized with the less virulent cya mutant strains.
• animalsimmunized with 10 9 CFU of the cya mutants exhibited better health and a higher rate of survival after oral challenge with Chi3246 than did mice immunized with the less virulent cya mutant strains.
• mice immunized with 10 9 cells of the crp strain survived chal- lenge; in fact, those immunized with 10 7 or 10 8 cells of the crp strain survived challenge with 10 9 cells of the wild-type strain.
• choleraesuis is determined by immunizing the mice with a high, but sublethal dose (8 x 10 8 CFU) of the attenuated strains. Thirty days later, survivors are challenged with Chi3246 at 10 1 , 10 2 , and 10 3 times the LD 50 value of
• Salmonella vaccine strains can serve as carriers to deliver a foreign antigen to the GALT of an animal host by introduction of a gene encoding the antigen into the vaccine strains.
• Nakayama et al., Bio/Tech 6:693 (1988) described a unique system where an Asd + expression-cloning vector was constructed for the purpose of high-level stable expression of foreign antigen genes in delta-cya delta-crp delta-asd S. typhimurium.
• the avirulent properties of the delta-cya delta-crp mutations have been consistently proven with doses administered to mice at approximately 1000 times the LD 50 of the wild-type parent in all Salmonella species previously tested.
• S. typhimurium Chi3656 is grown in L broth containing 5 mM CaCl 2 , and infected with P1L4 to propagate a high titer lysate.
• the PlL4(Chi3656) lysate is then used to transduce S. choleraesuis delta-cya delta-crp Chi3781;
• transductants are screened for by tetracycline resistance. A portion of the tetracycline-resistant transductants are screened for the Asd- phenotype. As a final step, selection on fusaric acid media is performed to identify a tetracycline-sensitive derivative of the S. choleraesuis delta-cya delta-crp delta-asd strain. Additional
• characterization of the final construct is completed by verifying the markers and presence of a complete LPS coat, and by the nonacquisition of additional auxotrophic phenotypes.
• strains provided herein are directly and indirectly suitable for the production of commercial vaccines to prevent diseases caused by S. choleraesuis, and other enteric bacteria with which antibodies to S.
• choleraesuis cross react. These strains are also useful as carrier microorganisms for the production of expression products encoded on recombinant genes in the bacterial cells.

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