WO2018124407A1 - Composition de vaccin contenant un mutant de salmonelle atténuée utilisé comme principe actif pour prévenir ou traiter simultanément une entéropathie proliférative porcine et la salmonelle - Google Patents

Composition de vaccin contenant un mutant de salmonelle atténuée utilisé comme principe actif pour prévenir ou traiter simultanément une entéropathie proliférative porcine et la salmonelle Download PDF

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WO2018124407A1
WO2018124407A1 PCT/KR2017/006150 KR2017006150W WO2018124407A1 WO 2018124407 A1 WO2018124407 A1 WO 2018124407A1 KR 2017006150 W KR2017006150 W KR 2017006150W WO 2018124407 A1 WO2018124407 A1 WO 2018124407A1
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salmonella
attenuated salmonella
strain
antigen
opta
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Korean (ko)
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이존화
원가연
김제형
박수연
문성철
정호경
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주식회사 코미팜
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/195—Antibiotics
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/255—Salmonella (G)
    • 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
    • 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

  • the present invention relates to a vaccine composition for the simultaneous prevention or treatment of swine proliferative ileitis and salmonellosis comprising attenuated Salmonella mutant strain as an active ingredient.
  • Porcine proliferative enteritis is a disease caused by Lawsonia intracellularis , which is widespread all over the world, causing significant damage to pig farmers. The incidence rate is known to be around 30% worldwide, and in 2000, the US National Animal Health Monitoring System (NAHMS) survey reported that more than one-third of all farms and 75% of large farms were infected. In Korea, more than 53% of the positive rates of individual pigs surveyed were reported.
  • NAHMS National Animal Health Monitoring System
  • PPE-based diseases caused by thickening of the ileum wall due to proliferation of enterocytes can be divided into proliferative hemorrhagic enteropathy and chronic incineinal adenomatosis, acute type of pig hemorrhagic intestine.
  • the disease develops in recently stocked pigs or pigs, with red and black tars around the anus before mortality, weakness, anemia, and anorexia.
  • Chronic type has slower feed intake, weight loss, and major clinical symptoms are brown, watery diarrhea that lasts from several days to four weeks.
  • Statistics show that during the PPE epidemic, daily weight gains are reduced by 9-35% and feed efficiency is 6-20%, causing significant economic losses to farmers.
  • Antibiotics susceptible to intracellular proliferation Gram-negative bacteria such as tyrosin, tetratracyclines, and lyomycin, have been used to prevent the treatment of this disease, but have been practiced in Korea since July 2011. Prohibition of antibiotics in feeds has increased the need to develop effective vaccines to control the bacteria present on pig farms.
  • Pigs with diarrhea may not be able to pinpoint the cause of the disease based solely on clinical symptoms or diarrhea.
  • diarrhea caused by fibrous necrotic ulcerative colitis caused by swine salmonellosis which is a representative swine diarrheal disease
  • Salmonella a Gram-negative bacterium that is characterized by intracellular proliferation, such as Lawsonia
  • Salmonella has been shown to have the same clinical symptoms as well as a significant correlation with Salsonella in pigs with Salmonella.
  • Korean Patent No. 0758614 discloses 'Cultivation of Lausonia Intracellulose, Anti-Lausonia Intracellular Vaccine and Diagnostic Reagent'
  • Korean Patent No. 1151004 discloses that the adhesion factor of bovine pathogenic E. coli Transformed attenuated Salmonella mutants and vaccine compositions for preventing and treating Escherichia coli and Salmonella bacterium comprising the same are disclosed, but pig proliferative ileitis comprising the attenuated Salmonella mutants of the present invention as an active ingredient and There is no description of vaccine compositions for simultaneous prophylaxis or treatment of Salmonellosis.
  • the present invention is derived from the above requirements, the inventors of the present invention to enhance the immune response due to the vaccine and to prevent two types of swine diarrhea at the same time four antigens derived from Lawsonia Intracellulose (OptA, OptB, FliC, Hly) was cloned into a recombinant vector for a live vaccine containing a Bla signal sequence to transform the LPS O-antigen-deficient attenuated Salmonella strain and inoculated with the humoral and cellular antigens in the mice inoculated with the transformed Salmonella mixture. By confirming that a mediated immune response is induced, the present invention has been completed.
  • the present invention provides attenuated Salmonella mutants comprising any one antigen selected from the group consisting of OptA, OptB, FliC and Hly antigen derived from Lawsonia intracellularis .
  • the present invention is Lawsonia Intracellulose ( Lawsonia) Preparation of attenuated Salmonella mutant strain for simultaneous prevention or treatment of swine proliferative ileitis and Salmonellosis comprising amplifying any one gene selected from the group consisting of genes encoding OptA, OptB, FliC and Hly antigens derived from intracellularis ) Provide a method.
  • the present invention also provides an attenuated Salmonella mutant mixture comprising two or more of the mutants.
  • the present invention provides a vaccine composition for the simultaneous prevention or treatment of swine proliferative ileitis and Salmonellosis comprising the attenuated Salmonella mutant strain or a mixture thereof as an active ingredient.
  • the present invention provides a feed additive for the simultaneous prevention of pig proliferative ileitis and Salmonellosis comprising the attenuated Salmonella mutant strain or a mixture thereof as an active ingredient.
  • the attenuated Salmonella mutant strain of the present invention has an outer shell structure and shell structure similar to that of the Salmonella spp., And expresses the Lawsonia intracellularis-derived OptA, OptB, FliC or Hly antigen to the outside of the cell, thereby humoral and cellular Since it is possible to induce a mediated immune response, the mutant strain of the present invention is expected to be useful as a vaccine that can simultaneously prevent and treat swine proliferative ileitis and salmonellosis, which can be safely, economically and easily inoculated.
  • the rfaL of the present invention Gene-deleted Salmonella mutants not only enhance the expression of selected Lawsonia antigens outside the cell, but also expand the availability of Salmonella probiotic vaccines, which have been limited in use due to misdiagnosis of serological tests using antibodies with LPS antigen. It is expected to do.
  • FIG. 1 is a simplified illustration of gene conversion when DNA fragments prepared with pKD3 as a template and pKD46 were transformed into a Salmonella strain ( S. Typhimurium ) having a target gene.
  • FIG. 2 is a result of comparing the surface O-antigen expression of JOL912, a strain containing O-antigen synthesis-related rfaL gene, and JOL1800, a strain lacking O-antigen synthesis-related rfaL gene, and is a result of silver staining.
  • Figure 3 is a result of confirming the degree of cell death of wild-type strains JOL401, mutant JOL912 and JOL1800 through the complement-dependent cytotoxicity test using rabbit serum complement.
  • Figure 4 shows the invasion / phagocytosis (4 hours, 24 hours, 48 hours after infection) of the Salmonella strains. Blue is macrophage, fluorescent green is Salmonella strain ( S. Typhimurium ). A) JOL401, B) JOL912, C) JOL1800.
  • FIG. 6 is a result of Western blot analysis of each Lawsonia protein expressed in JOL1800-derived strains (JOL1809, JOL1810, JOL1811 or JOL1812) expressing each Lawsonia antigen. Arrows indicate the expected size of each protein.
  • M Marker, A) Periplasmic sample of control strain, A ') Supernatant sample of control strain, B) Interspace sample of OptA expressing strain JOL1809, C) OptB Intercellular sample of the expression strain JOL1810, C ') Supernatant sample of the OptB expression strain JOL1810, D) Intercellular sample of the FliC expression strain JOL1811, E) Intercellular sample of the Hly expression strain JOL1812, E') Hly expression Supernatant samples of strain JOL1812.
  • Fig. 7 shows the result of confirming the titer of IgG for each antigen in the mouse group immunized by mixing four kinds of JOL1800-derived strains expressing each Lawsonia antigen. Comparison of non-immunized mice (Group A, white bars) and mice immunized with all four antigens (Group J, gray bars). I) IgG that specifically responds to OptA, II) IgG that specifically responds to OptB, III) IgG that specifically responds to FliC, IV) IgG that specifically responds to Hly. * Marked when there is the largest significant difference between control and immunized groups ( P ⁇ 0.02).
  • FIG. 8 shows the results of confirming the titer of secreted IgA for each antigen in the mouse group immunized by mixing four strains of JOL1800 expressing each Lawsonia antigen.
  • FIG. 8A shows the vaginal mucosal secretion IgA (viginal sIgA), and
  • FIG. 8B shows the titer of intestinal secretion IgA (intestinal sIgA).
  • Non-immunized mice Group A, white bars
  • mice immunized with all four antigens Group J, gray bars).
  • Figure 9 shows the results of the T cell immune response of splenocytes isolated from mice using FACS.
  • I Comparison of T cell changes of CD3 + CD4 + T cells and CD3 + CD8 + in non-immunized and immunized mice.
  • II The extent of CD3 + T cells, CD3 + CD4 + T cells and CD3 + CD8 + T cells increase in immunized mice against non-immunized mice.
  • FIG. 10 shows mRNA expression results of cytokine genes measured by re-stimulation of splenocytes isolated from mice immunized by mixing immunized mice with four strains of JOL1800 expressing each of the Lawsonia antigens. The amount of cytokine expressed in non-immunized mouse group A was increased to 1 relative expression level. The spleen cells of mice belonging to group J were divided and stimulated with OptA, OptB, FliC, and Hly, respectively, and the cytokine expression levels were measured.
  • the present invention provides an attenuated Salmonella mutant comprising any one antigen selected from the group consisting of OptA, OptB, FliC and Hly antigens derived from Lawsonia intracellularis .
  • L. intracellularis Gram-negative intracellular parasites are known as causative agents of swine proliferative ileitis.
  • Autotransporter one of the Gram-negative bacteria outer membrane proteins, is an N-terminal signal peptide, a passenger domain, and a ⁇ -domain.
  • the N-terminal signal peptide transports the protein synthesized in the passenger domain to the cytoplasmic membrane and expresses the protein transported by the ⁇ -domain to the cytoplasmic membrane to the outer membrane. It creates a path that can be made.
  • These expressed passenger domain proteins are known to play a role in bacterial adhesion to the host's target organs or cells, invasion, and bacterial virulence, but have been attempted in live vaccines to prevent PPE.
  • the OptA and OptB antigens are 101-200 including a portion of the first site, the passenger domain, which is expected to be the most antigenic of the LI0649 genes (802639-805194) encoding the Lawsonia intracellularis derived autotransporter protein.
  • the first amino acid sequence portion was selected as the first candidate protein (OptA), and the 534-851th amino acid sequence portion including the portion of the passenger domain and the entirety of the ⁇ -domain was determined as the second candidate protein (OptB).
  • the range of OptA antigens according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 1 and functional equivalents thereof.
  • “Functional equivalent” means at least 60%, preferably at least 80%, more preferably at least 90%, even more preferably at least 60% of the amino acid sequence represented by SEQ ID NO: 1 as a result of the addition, substitution, or deletion of the amino acid
  • “Substantially homogeneous physiological activity” means swine proliferative ileitis vaccine activity.
  • the invention also includes fragments, derivatives and analogues of the OptA antigen.
  • the range of OptB antigens according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 2 and functional equivalents thereof. Details of the functional equivalents of the protein are as described above. Also included are fragments, derivatives and analogues of OptB.
  • the FliC antigen was selected from a gene encoding hook associated flagella C (Lasonia PHE / MN gene map LI0570 gene) that synthesizes flagellin, a protein constituting flagella. .
  • Flagella on the cell surface that regulates bacterial movement usually acts as pathogen-associated molecular patterns (PAMPs) to activate the acquired immune system as a component of the innate immunity in the host. It interacts with Toll like receptor 5 (TLR5), one of the stimulating factors, and activates NF- ⁇ B in macrophage and contributes to the activation of the acquired immune system.
  • PAMPs pathogen-associated molecular patterns
  • TLR5 Toll like receptor 5
  • the flagella gene has not yet been used as a candidate gene for PPE live vaccines.
  • Lawsonia intracellularis has one long flagella, and in this study, the protein associated with the flagella hook was selected as the antigen.
  • the site of selection of the antigen is the flagellin N-terminal helical portion and the C-terminal spiral (C-terminal helical), which are conserved regions of 885nt (701958-702842). It may be more effective in inducing immune responses due to PAMPs, including the moiety.
  • FliC includes proteins having the amino acid sequence represented by SEQ ID NO: 3 and functional equivalents thereof. Details of the functional equivalents of the protein are as described above. Also included are fragments, derivatives and analogues of FliC.
  • the Hly antigen of the present invention was selected from Lawsonia intracellularis derived hemolysin (Hly) gene (LI0004 gene of the Lawsonia PHE / MN gene map).
  • Lossonia intracellular lyse breaks down the entry vacuole that forms when it enters the cytoplasm and escapes from the host's phagolysosomal fusion.
  • Hemolysin releases membrane-damaging cytolytic enzymes. It is known to produce and help break down inlet vesicles. On the 5th day of infection, the vesicles escaped from the vacuoles in the small intestine villus and gland cells (crypt cells) and grew freely in the cytoplasm.
  • the Hly antigen selection site synthesizes a hemolysin A protein known as bacterial pore forming toxin at a size of 756 nt (3454-4209).
  • a hemolysin gene has been widely used as a candidate gene for vaccine development of other bacteria, but it has not been used as a candidate gene for PPE live vaccine.
  • Hly includes proteins having the amino acid sequence represented by SEQ ID NO: 4 and functional equivalents thereof. Details of the functional equivalents of the protein are as described above. Also included are fragments, derivatives and analogues of Hly.
  • the attenuated Salmonella mutant strain of the present invention may be deleted asd gene.
  • Salmonella mutant strains lon, cpxR and asd genes are deleted, more preferably lon, cpxR , rfaL And Salmonella mutants lacking the asd gene, but are not limited thereto.
  • Salmonella mutants in which lon, cpxR and asd genes are deleted ⁇ cpxR ⁇ asd Salmonella Typhimurium (JOL912) is a DAP (diaminopimellic acid) requester that lacks the asd gene, and is designed to select antigen-recombinant strains without antibiotics.
  • JOL912 is a DAP (diaminopimellic acid) requester that lacks the asd gene, and is designed to select antigen-recombinant strains without antibiotics.
  • DAP diaminopimellic acid
  • lymphocyte penetration is increased to increase immunogenicity
  • lon Deletion of the gene can attenuate pathogenicity. It is known that the strain has no effect on the production of extracellular polysaccharide (EPS), which can act as an antigen, and thus acts as an antigen by itself, resulting in sufficient humoral mucosal cellular immune response.
  • EPS extracellular polysaccharide
  • LPS Salmonella lipopolysaccharide
  • O-PS O-antigen polysaccharide
  • the O-antigen free rough Salmonella mutants ( ⁇ lon) ⁇ cpxR ⁇ asd ⁇ rfaL , JOL1800 maintains the antigenicity of LPS and exerts an immune response stimulated by external antigens by exposing the host's immune system to obscuring the LPS during the expression of the extracellular membrane of the selected Losonia intracellulase antigen gene.
  • ⁇ lon ⁇ cpxR ⁇ asd ⁇ rfaL , JOL1800
  • JOL1800 survived in the spleen of the host to effectively penetrate macrophages in mice and induce an immune response regardless of the inoculation route.
  • serum complement was more easily killed than attenuated Salmonella strain derived from wild strain (JOL912).
  • the attenuated JOL1800 eliminates O-antigens is more sensitive to serum complement but is thought to complement the weakened defense mechanism by avoiding LPS-specific antibodies in individuals that have already been exposed to Salmonella strains.
  • DIVA Differentiation of Infected and Vaccinated Animals
  • the attenuated Salmonella strain of the present invention is any one of the genes selected from the group consisting of Bla ( ⁇ -lactamse) signal sequence, genes encoding OptA, OptB, FliC and Hly antigens linked thereto; And asd gene; but may be transformed with a recombinant vector comprising, but is not limited thereto.
  • the recombinant vector may be pJHL65 (asd + vector, pBR ori, 6xHis) or pJHL80 (asd + vector, p15A ori, 6xHis) having a secretion system based on the Bla signal sequence.
  • Salmonella mutants of the present invention wherein the Salmonella is Salmonella typhimurium (Salmonella typhimurium), Salmonella tie blood (Salmonella typi), Salmonella para tie blood (Salmonella paratyphi), Salmonella Sendai (Salmonella sendai), Salmonella Galina Solarium ( Salmonella gallinarium ) or Salmonella enteritidis ( Salmonella enteritidis ) and the like, preferably Salmonella typhimurium, but is not limited thereto.
  • step (b) cloning the amplified gene of step (a) into a recombinant vector having an asd gene;
  • step (c) transforming the cloned plasmid of step (b) into an attenuated Salmonella strain
  • step (d) provides a method for producing attenuated Salmonella mutant strain for simultaneously preventing or treating swine proliferative ileitis and Salmonellosis comprising the step of selecting the transformed Salmonella mutant strain of step (c).
  • the present invention also provides an attenuated Salmonella mutant mixture comprising two or more of the mutants.
  • Salmonella mutant mixture of the present invention is lon, cpxR , rfaL And asd Salmonella mutants having a gene deleted are mixtures containing two or more Salmonella mutants expressing any one antigen selected from the group consisting of OptA, OptB, FliC and Hly antigens in the extracellular membrane or extracellular.
  • the mixture of Salmonella mutants may be a mixture comprising all of Salmonella mutants expressing OptA, Salmonella mutants expressing OptB, Salmonella mutants expressing FliC and Salmonella mutants expressing Hly, but are not limited thereto.
  • the present invention provides a vaccine composition for the simultaneous prevention or treatment of swine proliferative ileitis and Salmonellosis comprising the attenuated Salmonella mutant strain or a mixture thereof as an active ingredient.
  • the vaccine composition of the present invention is a Lawsonia intracellular cellulose to Salmonella attenuated by gene deletion (lawsonia) Intracellularis ) containing a mixture containing one or more Salmonella mutants expressing OptA, OptB, FliC and Hly antigens as an active ingredient, by treating the vaccine composition in humans or animals to prevent or prevent pig hyperplasia ileitis and Salmonellosis It can be cured.
  • the Salmonella mutant strain mixture may be prepared in the form of mutant strains or dead bacteria, preferably in the form of mutant strains, but is not limited thereto.
  • composition of the present invention may be administered orally or parenterally (eg, applied intramuscularly, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, preferably orally or subcutaneously.
  • parenterally eg, applied intramuscularly, intravenously, subcutaneously, intraperitoneally or topically
  • the dosage of the composition varies depending on the weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of the disease or the like of the human or animal.
  • the vaccine composition may be inoculated in humans or mammals, and the mammal may be cows, deer, goats, goats, dogs, pigs, and the like, and preferably inoculated in pigs, but is not limited thereto.
  • the term “vaccine” refers to a biological agent containing an antigen that immunizes a living body, and refers to an immunogen or antigenic substance that immunizes the living body by injection or oral administration to a human or animal for the prevention of infection.
  • In vivo immunization is largely divided into automatic immunity obtained automatically by the in vivo immunity after infection of a pathogen and passive immunity obtained by an externally injected vaccine. While autoimmunity is characterized by a long period of production of immune-related antibodies and continuous immunity, passive immunization with vaccines acts immediately to treat infectious diseases, but has a disadvantage of poor sustainability.
  • the vaccine composition includes stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylene, subviral particle adjuvants, cholera toxin , N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium bromide and mixtures thereof
  • the second adjuvant may be further contained.
  • the vaccine composition may comprise a veterinary acceptable carrier.
  • veterinary acceptable carrier includes any and all solvents, dispersion media, coatings, antigen adjuvant, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like.
  • Carriers, excipients, and diluents that may be included in the composition for vaccines include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.
  • the vaccine composition is an oral formulation such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and nasal formulations such as drips or sprays and sterile injectable solutions, respectively, according to a conventional method.
  • Formulated in the form of can be used.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used can be prepared.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate and sucrose in the lecithin-like emulsifier. Or lactose, gelatin, etc. can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc may also be used. As a liquid preparation for oral administration, suspending agents, liquid solutions, emulsions, syrups, etc. may be used. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be used.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations.
  • non-aqueous preparation and suspending agent propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like may be used, but are not limited thereto.
  • Suitable penetrants for formulations for intranasal administration are generally known to those skilled in the art. Such suitable formulations are formulated to be preferably sterile, isotonic and buffered for stability and compliance.
  • Formulations for intranasal administration are also formulated to stimulate mucus secretion in several aspects to maintain normal ciliary action, and suitable formulations are preferably slightly buffered formulations that maintain isotonicity, pH 5.5 to 6.5, and most preferably Antimicrobial preservatives and suitable drug stabilizers.
  • the present invention provides a feed additive for the simultaneous prevention of pig proliferative ileitis and Salmonellosis comprising the attenuated Salmonella mutant strain or a mixture thereof as an active ingredient.
  • the feed additive of the invention rosoni ah intra-cell-less (Lawsonia intracellularis) derived OptA, OptB, including a Salmonella mutant, or a mixture thereof to express and secrete FliC and Hly antigen as an active ingredient, a Salmonella mutant, or a mixture thereof rosoni O And as well as the protective effect on Salmonella, effectively induces cellular and humoral immune response in animals, when used as feed additives can contribute to the simultaneous prevention and immune boosting of pig proliferative ileitis and Salmonellosis in livestock.
  • the feed additive of the present invention may use the Salmonella mutant mixture as it is or additionally add known carriers, stabilizers and the like, such as grains and by-products allowed for livestock, citric acid, fumaric acid, adipic acid, lactic acid, if necessary.
  • Organic acids such as malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechins, alpha-tocopherols, rosemary extracts, vitamin C, green tea extracts, licorice extracts, chitosan, Natural antioxidants such as tannic acid and phytic acid, antibiotics, antibacterial agents, and other additives may be added, and the shape may be in a suitable state such as powder, granules, pellets, suspensions, and the like. It may be supplied alone or mixed with the feed.
  • Salmonella typhimurium (hereinafter, referred to as S. Typhimurium ) strain from which the asd gene was removed was cultured by adding 50 ⁇ g / ml DAP at 37 ° C in LB medium. Temperature sensitive strains were cultured in LB medium, 28 ° C. Strains with lambda-red genes were cultured in LB medium containing 0.2% L-arabinose. All strains were stored at ⁇ 80 ° C. in LB liquid medium containing 20% glycerol.
  • the O-antigen ligase gene, rfaL was removed from the existing attenuated S. Typhimurium to develop a strain without O-antigen in LPS on the bacterial surface. Plasmid pKD46 containing lambda red, which induces recombination, was transformed by electric shock into S. Typhimurium JOL912 ( ⁇ cpxR, ⁇ lon, ⁇ asd ) , which is attenuated live bacteria. After culturing at 32 ° C. or lower (28-30 ° C.), PCR was confirmed whether the plasmid was successfully introduced.
  • the pKD3 plasmid as a template was amplified by PCR using a 40-nucleotide-long primer containing the rfaL gene and a portion of the pKD3 plasmid.
  • the amplified PCR product was extracted and transformed into S. Typhimurium competent cells into which pKD46 was introduced. It was plated on LB (Luria-Bertani) solid medium to which arabinos was added and incubated at 37 ° C. Recombined strains were selected in LB solid medium containing 25 ⁇ g / ml chloramphenicol. Knock-out was confirmed by PCR using primers.
  • competent cells were made from colonies confirmed to be transgenic, and the pCP20 plasmid was transformed. Cultured in LB medium, 37 °C was selected bacteria without chloramphenicol resistance. LPS was extracted and confirmed by SDS-PAGE for comparison with wild S. Typhimurium . The finally identified O-antigen deleted Salmonella strain was named JOL1800.
  • Serum complement sensitivity of S. Typhimurium was measured using rabbit serum. Serum free of anti-salmonella antibodies was taken from rabbits and used for analysis. All strains used were incubated until late log phase and diluted to 1 ⁇ 10 7 cfu / 100 ⁇ l.
  • mice The primary peritoneal macrophage of mice was infected with JOL401, JOL912, and JOL1800, and the phagocytosis and infiltration were observed using a fluorescence microscope. Macrophages were harvested by injecting 5% BSA into the abdominal cavity of 4 week old mice. Macrophages were treated aseptically and covered with 0.2% gelatin-coated coverglass slip in a 5 ⁇ 10 6 cell number. Thereafter, RPMI [RPMI 1640, 10% FBS (heat-inactivated fetal bovine serum), 1 ⁇ Antibiotic-Antimycotic (Gibco, Life technologies, USA)] was poured into 6-well cell culture plates.
  • S. Typhimurium was infected with MOI 10: 1. After bacterial treatment, the cells were incubated in 37%, 5% CO 2 incubator for 30 minutes. The phagocytic or non-penetrating bacteria were washed with PBS and placed in the incubator for 48 hours with the addition of RPMI containing antibiotics. Cover glass slip cells were fixed with 3% paraformaldehyde diluted with PBS and subjected to immunofluorescence staining.
  • the primary antibody is chicken anti-912 polyclonal sera when infected with JOL401, JOL912, or chicken anti-1800 polyclonal sera when infected with JOL1800.
  • S. Typhimurium strain of the present invention was carried out to determine how long the remaining in the host organs.
  • Four-week-old mice were orally inoculated with JOL401 and JOL1800 at 1 ⁇ 10 7 cfu, respectively, and the third group was intramuscularly inoculated with JOL1800.
  • the ⁇ asd strain JOL1800 was inoculated by transforming the asd + plasmid pJHL65 by electric shock. Spleens were harvested aseptically by anesthetizing 8 mice of each group every 3, 7, 14, 21, 28 days after inoculation.
  • the spleen was homogenized with 2 ml BPW (buffered peptone water, Becton, MD, USA) using a homogenizer. 100 ⁇ l of the homogenized solution was immediately inoculated evenly into Brilliant green agar (BGA) plates and incubated overnight at 37 ° C. Simultaneously, the remaining BPW samples were inoculated in Rappaport-Vassiliadis (RV) medium and incubated at 37 ° C. for 48 hours. The colonies obtained were finally S. Typhimurium PCR was confirmed using specific primers.
  • BPW Brilliant green agar
  • Purified antigenic protein, LPS was coated on an ELISA plate by dispensing at a concentration of 200 ng / well.
  • Mouse serum, the primary antibody was diluted 1: 100 with PBS, and HRP-condensed anti-mouse IgG, the secondary antibody, was diluted at a ratio of 1: 8,000.
  • HRP-condensed anti-mouse IgG the secondary antibody
  • 100 ⁇ l per well of the OPD (Sigma-Aldrich, US) reaction solution was dispensed for 5 minutes and then stopped with 3M H 2 SO 4 , and the OD value was measured at 492 nm.
  • the value of serum IgG attached to LPS was expressed as the average OD value.
  • the bacterial strain, plasmid used in the present invention is listed in Table 1.
  • Commercially available vectors for expression of E. coli proteins, pET28a and pET32a plasmids have kanamycin resistance, so the strains were cultured in the presence of kanamycin (50 ⁇ g / ml).
  • the Bl21 (DE3) pLysS strain was used for purifying Lawsonia antigen protein.
  • the strain was cultured in the presence of 0.1M IPTG (Isopropyl ⁇ -D-1-thiogalactopyranoside) when inducing overexpression during protein purification.
  • pJHL65 and pJHL80 asd + plasmids were used as vectors to secrete and deliver antigen proteins cloned in a Bla signal sequence-based periplasmic secretion manner. All strains were incubated at 37 ° C. using LB medium and stored at ⁇ 80 ° C. using LB liquid medium containing 20% glycerol. DAP is at a concentration of asd 50 ⁇ g / ml - was added to culture the strain JOL1800.
  • Candidate antigens of L. intracellularis are OptA and OptB, which are involved in autotransporter synthesis, FliC, which synthesizes flagella related proteins, and Hly, which is considered to be involved in cell infiltration. Genes required for the expression of antigenic proteins were all obtained using gene synthesis (Bioneer, Korea). The genes of L. intracellularis that synthesize each protein were examined through GenBank of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). Each L.
  • intracellularis antigen was analyzed by Peptide Property Calculator software (http://www.biosyn.com/peptidepropertycalculator/), and then synthesized by selecting the nucleotide sequence of the high antigenicity.
  • the genes were cloned into the pBHA vector, with the restriction sites identified at each end.
  • the restriction sites added at both ends of the L. intracellularis antigen gene are OptA for Sal I and Xho I, OptB for Sal I and Pst I, FliC for EcoR I and Sal I, and Hly for EcoR I and Hind III.
  • Recombinant plasmids pET32a-OptA, pET28a-OptB, pET28a-FliC, and pET28a-Hly were prepared by cloning into the plasmids pET28a and pET32a, respectively, using the designated restriction enzymes.
  • OptA was not inserted into pET28a and cloned into pET32a, a similar strain of plasmid.
  • the resulting recombinant plasmid was transformed into E. coli BL21 (DE3) pLysS by a thermal shock method to prepare a strain for protein purification overexpressing the antigenic protein in the presence of IPTG. L.
  • OptA, OptB, FliC, and Hly proteins in each strain were purified using Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Valencia, CA).
  • NTA Ni-nitrilotriacetic acid
  • the antigenic protein of L. intracellularis obtained by culturing and purifying each strain was used to detect specific antibodies against each antigen in mice.
  • the antigen genes were cloned into pJHL65 and pJHL80 plasmids that express heterologous antigens in the intercellular space, and then pJHL80-OptA, pJHL65-OptB, pJHL65-FliC. pJHL65-Hly was obtained.
  • JOL1800 was transformed into JOL1800 by electric shock, respectively.
  • the JOL1800 was washed twice with distilled water containing 10% glycerol, placed in a cuvette and mixed with 0.1 ⁇ g of purified plasmid, subjected to electric shock with Bio-Rad MicroPulser (Bio-Rad, USA), and recovered. LB liquid medium without DAP was recovered. 1 ml was incubated at 37 ° C. for 1 hour.
  • Plasmids were recovered from the selected strains, digested with restriction enzymes corresponding to the respective antigens, and confirmed by electrophoresis and PCR on agarose gels.
  • the antigens of L. intracellularis expressed in the outer membrane and periplasmic space of the prepared vaccine strain were confirmed by Western blot.
  • Antigen proteins expressed in the recombinant strains were obtained in cell-free supernatants through TCA precipitation.
  • the cells were cultured at 37 ° C and LB liquid medium until the optical density (OD 600 ) was measured at 600 nm, and centrifuged at 3,400 ⁇ g for 20 minutes. The supernatant was then separated.
  • Each protein sample was separated by SDS-PAGE for Western blot analysis and transferred to a 0.2 ⁇ m PVDF membrane (Millipore, Billerica, Mass., USA) at 4 ° C. with blocking buffer (3% BSA, PBS, 0.1% Tween-20). The reaction was overnight. The next day, the anti-his-tag antibody (IG Therapy Co., Ltd., Korea) was diluted to 1: 5,000 and reacted for 1 hour, followed by HRP (horseradish peroxidase) -condensed goat anti-mouse IgG diluted to 1: 5,000. Time was processed.
  • HRP horseradish peroxidase
  • strains and plasmids used in this experiment were recombinant attenuated Salmonella strains JOL1809, JOL1810, JOL1811, and JOL1812, which express the L. intracellularis antigens in Table 1.
  • JOL1809, JOL1810, JOL1811, and JOL1812 strains were mixed by 5 ⁇ 10 7 CFU, respectively, and a total of 2 ⁇ 10 8 CFU / 100 ⁇ l strains were determined as the final inoculation amount.
  • Each rat feces collected on the plate was treated with 500 ⁇ l of 50mM EDTA, centrifuged at 3,400 ⁇ g for 15 minutes, and the supernatant was separated and stored at -20 ° C. and used for the experiment.
  • Serum was collected by orbital venous blood collection and centrifuged at 4,000 ⁇ g for 5 minutes to separate serum, which was supernatant, and stored at -20 ° C.
  • ELISA was performed to measure sIgA and IgG specific for L. intracellularis OptA, OptB, FliC, and Hly antigens expressed in recombinant vaccine strains.
  • As the coating antigen OptA, OptB, FliC, and Hly antigen proteins purified from JOL1586, JOL1593, JOL1682, and JOL1742 strains were used. Purified antigenic protein was administered at a concentration of 500 ng / well, and standard protein wells were coated with goat-anti mouse IgG or goat anti-mouse sIgA at a concentration of 200 ng / well, respectively, and then coated overnight at 4 ° C.
  • the coated plate was washed three times with PBS (PBST) containing 0.05% of Tween 20 and then blocked with blocking buffer (3% skim milk in PBS) at 37 ° C. for 1 hour.
  • Serum and PBS were diluted 1: 100, vaginal lavage was diluted 1: 3, intestinal lavage was diluted 1: 4, and 100 ⁇ l was dispensed into the wells, followed by reaction at 37 ° C. for 1 hour.
  • In the case of serum, goat anti-mouse IgG HRP and vaginal lavage were diluted 1: 5,000 in goat anti-mouse IgA HRP, and 100 ⁇ l was dispensed into each well, followed by reaction at 37 ° C. for 1 hour.
  • FACS fluorescence activated cell sorting
  • Anti-mouse CD3e-PE, anti-mouse CD4-perCP-vio700, and anti-mouse CD8a-FITC were reacted at 4 ° C. for 15 minutes in dark conditions. Stained cells were washed three times with 200 ⁇ l FACS buffer and analyzed using the MACSQuant ® analyzer (miltenyi Biotec, Germany).
  • RNA samples were divided into 96 well plates in the same manner as in 3.7.
  • OptA, OptB, FliC, and Hly were stimulated at 200 ng / ⁇ l for 1 hour at 5% CO 2 environmental conditions in 1x10 6 cells for 48 hours.
  • total RNA was extracted using GeneAll ® Hybrid-RTM kit and synthesized into cDNA using ReverTra Ace ® qPCR RT Kit.
  • Primers of mouse interferon-gamma (IFN- ⁇ ), interleukin (IL) -4, and IL-17 used for real-time PCR are listed in Table 3.
  • Real-time PCR was measured by using SYBR ® Green Real-Time PCR Master Mix (QPK-201, TOYOBO, Japan). It was measured using a Step One plus Real Time PCR system (Applied Biosystems). For each cytokine, the amount of RT-PCR product was normalized to ⁇ -actin values using internal standards.
  • Primer used in RT-PCR of the present invention antigen Primer direction Sequence (5 ' ⁇ 3') SEQ ID NO: IFN- ⁇ Forward direction TCA AGT GGC ATA GAT GTG GAA GAA 13 Reverse TGG CTC TGC AGG ATT TTC ATG 14 IL-4 Forward direction ACA GGA GAA GGG ACG CCA T 15 Reverse GAA GCC CTA CAG ACG AGC TCA 16 IL-17 Forward direction ACC GCA ATG AAG ACC CTG AT 17 Reverse TCC CTC CGC ATT GAC ACA 18
  • JOL912 has three genes ( ⁇ lon ⁇ cpxR ⁇ asd Auxotrophic mutant strains asd Deletion of the gene necessitates DAP upon replication. Thus, asd + plasmids are used as vectors expressing antigen genes to satisfy balanced-lethal complementation.
  • Exo acts at both ends of the ds DNA to create a 3-overhang, and bet binds to 3 ss DNA, causing strand exchange by RecA.
  • the cat gene cassette exchanged here made the plasmid pKD3 a template.
  • JOL912 with pKD46 and linear DNA was introduced to replace rfaL with the cat gene cassette.
  • pKD46 exits the cell. Removal of the target gene was confirmed by PCR using primers from the rfaL flanking region.
  • wild rfaL is changed to cat gene cassette ( ⁇ 1.1kb)
  • the size of the amplicon which is 1.9kb in total, is shortened to 1.7kb.
  • Plasmid pCP20 was transformed into a strain in which removal of the rfaL gene was confirmed. pCP20 was introduced to remove antibiotic resistance genes and expressed FLP recombinase that acts directly on the FLP recognition target (FRT) sites present at both ends of the antibiotic resistance gene cassette. After culturing at 30 °C to obtain a strain from which the cat gene cassette was removed, and further cultured at 37 °C or 40 °C to obtain a final strain without the plasmid pCP20. The last strain obtained was not resistant to antibiotics derived from JOL912. lon, cpxR , asd , rfaL The mutant strain JOL1800 was deleted.
  • JOL1800 rfaL It is a rough Salmonella strain without O-antigen due to the gene removal. After PAGE separation and purified LPS extraction in JOL1800 it can be confirmed that there is no O-antigen on the surface of the bacteria (Fig. 2). In the S-type JOL912 used as a control, long-expressed O-antigens can be seen.
  • complement sensitivity analysis was performed by culturing wild S. Typhimurium JOL401, attenuated strain JOL912, and JOL1800, a strain derived from JOL912. R-type JOL1800 showed a more sensitive response to rabbit complement than the other S-type strains. JOL1800 showed a marked bacterial count reduction compared to wild JOL401 and JOL912 (FIG. 3).
  • Macrophages of mice were infected with O-antigen-deleted S. Typhimurium JOL1800 from wild S. Typhimurium JOL401, attenuated S. Typhimurium JOL912, JOL912 to identify differences in the ability of the three strains to feed or penetrate macrophages. .
  • JOL401, JOL912, JOL1800 all confirmed the infiltration of bacteria into macrophages 4 hours after infection.
  • Salmonella have an infection pathway that survives in the macrophages of the host and spreads the bacteria systemically.
  • JOL1800 which additionally removed the O-antigen of the bacterial surface LPS by deleting the rfaL gene, was found to have the ability to invade and survive macrophages (FIG. 4). ).
  • Mutant strains produced by the deletion of several genes are very attenuated and sometimes do not reach the real organs or die easily, sometimes causing infection. When attenuated strains are used as vaccines, proper attenuation control is required so that the vaccine strains do not survive for too long in the host to cause disease or to survive too short to cause an immune response.
  • the mice were infected with JOL401, JOL912, and JOL1800 three days later, seven days later, 14 days later, and 21 days later. The spleens were collected and tested for the presence of bacteria (Table 4). All strains were detected in the spleen 3, 7, 14 days after inoculation. JOL401, JOL912 survived in the spleen until 21 days after inoculation. The recovery time of JOL1800 was 12 days and was investigated by PROBIT analysis. It can be seen that JOL1800 can invade the host's immune system and sufficiently induce an immune response.
  • ELISA was performed on the sera of mice inoculated with LPS extracted from S. Typhimurium to investigate the possibility of DIVA of the developed JOL1800 strain.
  • S-type strains with O-antigens, JOL401 and JOL912 showed LPS-specific antibodies, whereas L-type strains without O-antigen were detected in JOL1800 (Fig. 5).
  • Boosting was performed three weeks after the first inoculation, considering that LPS-specific antibodies could not be induced due to rapid in vivo clearance due to attenuation of JOL1800.
  • the plasmids pJHL80-OptA, pJHL65-OptB, pJHL65-FliC, pJHL65-Hly which were prepared by cloning the synthesized L. intracellularis antigens OptA, OptB, FliC, and Hly into the antigen expression plasmids pJHL65 and pJHL80, do not have O-antigens.
  • the vaccine strain was completed by electroshock to DIOL-capable attenuated S. Typhimurium strain JOL1800.
  • pJHL65 and pJHL80 express the cloned antigen in the intercellular space or surface of the bacteria.
  • JOL1809 OptA expression JOL1800
  • JOL1810 OptB expression JOL1800, respectively.
  • JOL1811 FliC expressing JOL1800
  • JOL1812 Hly expressing JOL1800
  • T lymphocyte subpopulation using FACS in splenocytes isolated from all immunized and non-immunized control mice to assess cellular immune responses following inoculation with recombinant attenuated S. Typhimurium live vaccines JOL1809, JOL1810, JOL1811, JOL1812 was analyzed.
  • Total T cells are expressed as CD3 +
  • CD4 + and CD8 + are helper T cells and cytotoxic T cells, respectively.
  • 9 days after the inoculation also CD3 + CD4 + and CD3 + CD8 + with the overall increase in the CD3 + It rose significantly (FIG. 9).
  • CD3 + T cells showed an increase of 50% or more, CD4 + increased by 45% or more, and CD8 + increased by 55% or more compared to the control group.
  • RT-PCR was performed to measure the mRNA expression level of IFN- ⁇ , IL-4, and IL-17 to evaluate the expression level of cytokines that regulate cellular immune responses.
  • cytokines were significantly increased in OptA (FIG. 10).
  • OptB IL-4 and IFN- ⁇ were significantly increased in all antigens.
  • IL-17 is a cytokine secreted by differentiated Th17 cells, indicating that all antigens stimulate secretion. It is believed that CD4 + T cells have differentiated into Th1 and Th2 cells, and it is estimated that the differentiation of Th17 cells occurs.

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Abstract

La présente invention concerne une composition de vaccin contenant un mutant de salmonelle atténuée utilisé comme principe actif pour prévenir ou traiter simultanément une entéropathie proliférative porcine et la salmonelle. Il a été confirmé que la présente invention induit des réponses immunitaires humorales et à médiation cellulaire à des antigènes chez une souris vaccinée avec un mélange du mutant de salmonelle atténuée, lorsque la salmonelle atténuée a été transformée à l'aide d'un vecteur développé pour augmenter la sécrétion d'antigènes OptA, Optb, FliC ou Hly dérivés de Lawsonia intracellularis. Par conséquent, le mutant selon la présente invention est censé être utilisé comme vaccin pour prévenir ou traiter simultanément une entéropathie proliférative porcine et la salmonelle, qui est sûr, économique, et peut être inoculé en toute sécurité.
PCT/KR2017/006150 2016-01-04 2017-06-13 Composition de vaccin contenant un mutant de salmonelle atténuée utilisé comme principe actif pour prévenir ou traiter simultanément une entéropathie proliférative porcine et la salmonelle WO2018124407A1 (fr)

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KR20080105099A (ko) * 2008-09-22 2008-12-03 브리제 유니버시타이트 브루셀 약독화 살모넬라 생백신
KR20110022428A (ko) * 2009-08-27 2011-03-07 주식회사 중앙백신연구소 돼지의 병원성 대장균의 부착인자가 형질전환된 약독화 살모넬라균 변이주 및 이를 포함하는 돼지의 병원성 대장균증 및 살모넬라균증의 예방 및 치료용 백신 조성물
KR20110023502A (ko) * 2009-08-31 2011-03-08 전북대학교산학협력단 소의 병원성 대장균의 부착인자가 형질전환된 약독화 살모넬라균 변이주 및 이를 포함하는 소의 대장균증 및 살모넬라균증의 예방 및 치료용 백신조성물
KR20140130944A (ko) * 2013-05-02 2014-11-12 전북대학교산학협력단 약독화된 살모넬라 변이주를 유효성분으로 포함하는 위축성 비염 및 파스츄렐라성 폐렴의 동시 예방용 생균 백신 조성물
KR20170081569A (ko) * 2016-01-04 2017-07-12 전북대학교산학협력단 약독화된 살모넬라 변이주를 유효성분으로 포함하는 돼지 증식성 회장염 및 살모넬라증 동시 예방 또는 치료용 백신 조성물

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