WO2022270953A1 - Souche de salmonella typhimurium à gène yjek supprimé et composition de vaccin contre les salmonelles la comprenant - Google Patents

Souche de salmonella typhimurium à gène yjek supprimé et composition de vaccin contre les salmonelles la comprenant Download PDF

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WO2022270953A1
WO2022270953A1 PCT/KR2022/008979 KR2022008979W WO2022270953A1 WO 2022270953 A1 WO2022270953 A1 WO 2022270953A1 KR 2022008979 W KR2022008979 W KR 2022008979W WO 2022270953 A1 WO2022270953 A1 WO 2022270953A1
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strain
culture
yjek
salmonella
gene
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PCT/KR2022/008979
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Korean (ko)
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한태욱
윤현진
박소연
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주식회사 이노백
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • 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
    • 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 YjeK gene-deleted Salmonella typhimurium strain and a use thereof, and more particularly to a novel Salmonella typhimurium strain; And vaccine compositions, immunogenic compositions and feed compositions comprising the same; it relates to.
  • Salmonella is a gram-negative facultative anaerobic bacterium belonging to Enterobacteriaceae, and is a bacillus that does not form spores. Salmonella is a pathogenic microorganism that infects humans as well as various livestock including pigs, cows, chickens, etc. to cause diseases. It infects humans to cause food poisoning and causes various types of Salmonellosis in animals.
  • Salmonella enterica is classified by serological classification as Salmonella Typhi, which causes typhoid fever, Salmonella Typhimurium, which is the causative agent of typhoid fever, Salmonella Enteriditis, which is an enteritis bacterium, and poultry It includes serospecies such as Salmonella Gallinarum, the causative bacterium of typhus, and Salmonella Pullorum, the causative bacterium of Chubaekri. Salmonella includes zoonotic agents that, depending on the serotype, can cause disease not only in humans but also in livestock.
  • Salmonella typhimurium and Salmonella enteritidis are zoonotic infectious agents that cause food poisoning in humans and cause salmonellosis in livestock such as cattle, chickens and pigs, resulting in acute or chronic enteritis and sepsis, further pneumonia, It causes arthritis, miscarriage, etc.
  • Salmonella galinarum and Salmonella florum are poultry-specific infections that can cause poultry typhus and chubaekri, which can cause mass mortality in poultry. Salmonella florum has no clinical symptoms even when infected in the sex system, but is transmitted from egg to hatched chicks, causing chubaekri, and the highest mortality rate occurs at 1-2 weeks of age.
  • antibiotics are mainly used to prevent and treat Salmonella infection, but Salmonella often penetrates into cells when animals are infected and proliferates, making it difficult for antibiotics, drugs, and other probiotics to penetrate and act.
  • the problem of antibiotic susceptibility has been occurring, and the cause of this has been identified as the misuse of antibiotics, and antibiotics have not been used sufficiently.
  • antibiotics used as growth promoters in compound feeds for industrial animals began to be banned, and antibiotic use began to be regulated in the European Union from 2006 and in Korea from the second half of 2011.
  • the use of antibiotics is gradually decreasing.
  • the use of antibiotics to control bacteria is reduced, the occurrence of bacterial diseases is increasing, so there is an increasing demand for methods of controlling bacterial diseases without using antibiotics.
  • the present inventors completed the present invention by developing a Salmonella typhimurium strain in which the YjeK gene was deleted and confirming its remarkable Salmonella infection preventive effect.
  • an object of the present invention is to provide a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide sequence of SEQ ID NO: 1 is deleted.
  • Another object of the present invention is a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a group consisting of a concentrate of the culture and a dry product of the culture To provide a composition comprising at least one selected from.
  • Another object of the present invention is a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dried product of the culture
  • a method for preventing or treating salmonellosis comprising; administering at least one selected from the group to a subject in need thereof.
  • the present invention provides a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide sequence of SEQ ID NO: 1 is deleted.
  • the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of Provided is a vaccine composition for preventing or treating Salmonellosis, including more than one species.
  • the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of An immunogenic composition against Salmonella comprising more than one species is provided.
  • the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of It provides a feed composition for preventing or improving salmonellosis comprising more than one species.
  • the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of It provides a feed additive composition for preventing or improving salmonellosis comprising more than one species.
  • the present invention is a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dry product of the culture. 1 selected from the group consisting of It provides a method for preventing or treating salmonellosis, including the step of administering at least one species to a subject in need thereof.
  • the YjeK gene-deleted Salmonella typhimurium strain according to the present invention can effectively prevent Salmonella infection and significantly increase the survival rate of individuals. This means that diseases related to Salmonella infection can be effectively prevented when the YjeK gene-deleted Salmonella typhimurium strain of the present invention is used as a vaccine. It can be used in various fields of control.
  • Figure 1A is a diagram showing the results of analyzing the growth of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • Figure 1B is a diagram showing the results of analyzing the motility of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • Figure 1C is a diagram showing the results of lipopolysaccharide and outer membrane protein profile analysis of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • Figure 1D is a diagram showing the antibiotic susceptibility of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • Figure 2A is a diagram showing the results of analyzing the cell invasion and macrophage viability of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • Figure 2B is a diagram showing the results of analyzing the expression of the gene associated with SPI-1 and SPI-2 T3SS of the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • Figure 3A is a diagram showing the results of measuring the spleen weight of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • Figure 3B is a diagram showing the results of measuring the number of bacteria in the spleen of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • Figure 3C is a diagram showing the results of measuring the body weight of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • Figure 4A is a diagram showing the results of measuring serum antibody IgG levels of mice infected with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • Figure 4B is a diagram showing the results of measuring the level of serum antibody IgM in mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • 4C is a diagram showing the results of measuring the IgG2a/IgG1 ratio of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • 5A is a diagram showing the results of measuring the level of the cytokine IFN- ⁇ in splenocytes of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • 5B is a diagram showing the results of measuring the level of the cytokine IL-6 in splenocytes of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • 5C is a diagram showing the results of measuring the level of the cytokine TNF- ⁇ in splenocytes of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • 6A is a diagram showing an immunization test schedule of mice immunized with a ⁇ yjeK mutant strain and a wild-type strain according to the present invention.
  • Figure 6B is a diagram showing the results of measuring the body weight of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • Figure 6C is a diagram showing the results of analyzing the liver weight and the number of bacteria in the liver of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • 6D is a diagram showing the results of analyzing the spleen weight and the number of bacteria in the spleen of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • 6E is a diagram showing the results of survival analysis of mice immunized with the ⁇ yjeK mutant strain and the wild-type strain according to the present invention.
  • the present invention provides a Salmonella typhimurium strain containing a YjeK gene in which the nucleotide sequence of SEQ ID NO: 1 is deleted.
  • the YjeK gene-deleted Salmonella typhimurium strain of the present invention is represented by the nucleotide sequence of SEQ ID NO: 1 in the yjeK gene of Salmonella typhimurium ST1120 (Kim et al., 2017) isolated in Korea through the ⁇ red recombination method. nucleotides are deleted.
  • a variant of the YjeK gene represented by the nucleotide sequence of SEQ ID NO: 1 is included within the scope of the present invention.
  • the gene has a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: It means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by 1.
  • the "percentage of sequence homology" for polynucleotides is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. may include additions or deletions (i.e., gaps) compared to (not including).
  • the strain preferably blocks biochemical activities related to arginine dihydrolase and lysine decarboxylase, but the scope of the present invention is not limited thereto. .
  • the strain is AMC (amoxicillin / clavulanic acid), ampicillin (ampicillin, AM), cephalothin (cf), gentamicin (GM), kanamycin (kanamycin, K), neo It is preferable to exhibit antibiotic sensitivity to at least one antibiotic selected from the group consisting of neomycin (N) and ampicillin/sulbactam (SAM).
  • the strain preferably has a lower expression of flagellin FliC than the wild-type strain and an increased expression of porin OmpD than the wild-type strain.
  • the strain does not induce splenomegaly in the host.
  • spleen enlargement means that the volume or weight of the spleen is increased beyond the normal range. Since the spleen is the largest of the lymphoid tissue in the body, when there is a systemic disease of the hematopoietic organ, it reacts with other hematopoietic organs in the body and expands. Splenomegaly is mainly seen in (1) infections (malaria, subacute endocarditis, salmonellosis, etc.), (2) blood diseases (leukemia, anemia, etc.), and (3) metabolic abnormalities.
  • the culture means that the YjeK gene-deleted Salmonella typhimurium strain is cultured in a culture medium or culture medium, and the culture is a culture containing the strain.
  • the formulation of the culture or culture concentrate is not limited, and for example, the formulation may be liquid or solid.
  • the medium contains nutrients required by the microorganism to be cultured, that is, the microorganism to be cultured, and may be a mixture in which a substance for a special purpose is additionally added.
  • the medium may also be referred to as an incubator or a culture medium, and is a concept that includes all of a natural medium, a synthetic medium, or a selective medium. It may be obtained by separating from the cultured medium, and the medium may be used without limitation as long as the YjeK gene-deleted Salmonella typhimurium strain can grow.
  • the medium is, for example, normal agar medium (or nutrient agar medium; Nutrient agar), TSA (tryptic soy agar) medium, standard agar medium (Standard Methods Agar; Plate Count Agar), lactose medium (lactose broth), BGlB medium ( Brilliant Green lactose Bile Broth), double strength BGlB medium, Endo agar medium (Endo agar), EMB agar medium (Eosin methylene blue agar), normal medium (or nutrient medium; Nutrient Broth), desoxycholate lactose agar medium (Desoxycholate medium) lactose Agar), LB medium (Luria-bertani Broth) or EC medium (EC Broth).
  • normal agar medium or nutrient agar medium; Nutrient agar
  • TSA tryptic soy agar
  • standard agar medium Standard agar medium (Standard Methods Agar;
  • the composition may be formulated and used as a wettable powder, granule or capsule, but is not limited to the above type.
  • the hydrating agent of the present invention may be prepared by drying and pulverizing the solid medium inoculated with the strain, and then adding and mixing a surfactant, an extender or a nutrient.
  • a surfactant polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene alkyl
  • the extenders and nutrients include One or two or more selected from the group consisting of soy flour, rice, wheat, ocher, diatomaceous earth, dextrin, glucose,
  • the granules of the present invention may use the same surfactants, extenders and nutrients as described above after drying and pulverizing the solid medium inoculated with the strain.
  • the granule of the present invention is one or two or more selected from the group consisting of a surface active agent of the strain, an inert carrier, a preservative, a wetting agent, a supply promoter, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protective agent, a buffer and a flow agent It can be prepared by adding more.
  • prevention of the present invention means any action that suppresses or delays the onset of a disease by administering a composition.
  • treatment of the present invention refers to all activities that improve the symptoms of the disease or inhibit or alleviate the disease and change beneficially by the administration of the composition.
  • the composition is intraperitoneal administration, intravenous administration, intramuscular administration, intranasal administration, intra-articular administration, intra-synovial administration, intrathecal administration, intrahepatic administration (intrahepatic) administration, intralesional administration, or intracranial administration is preferable, and more preferably, it may be intraperitoneal administration.
  • the vaccine is a veterinary vaccine containing an antigenic material, which is specific for Salmonella and is administered for the purpose of inducing active or passive immunity.
  • an antigenic material which is specific for Salmonella and is administered for the purpose of inducing active or passive immunity.
  • the vaccine composition according to the present invention can be administered in an immunologically effective amount.
  • the "immunologically effective amount” means an amount sufficient to exhibit a preventive effect against a disease associated with a YjeK gene-deleted strain of Salmonella typhimurium and an amount sufficient to not cause side effects or serious or excessive immune reactions, and is administered accurately.
  • the concentration varies depending on the specific immunogen to be administered, and can be easily determined by a person skilled in the art according to factors well known in the medical field, such as the age, weight, health, sex of the subject to be vaccinated, sensitivity to drugs of the individual, administration route, and administration method. It can be administered once or several times.
  • the vaccine composition according to the present invention may include, in addition to the YjeK gene-deleted Salmonella typhimurium strain as an active ingredient, one or more immune enhancers or excipients or carriers suitable for constituting the vaccine composition.
  • An adjuvant that may be included in the vaccine composition of the present invention refers to a substance that enhances the immune response of an injected animal, and many different adjuvants are known to those skilled in the art.
  • the immune enhancers include Freund's complete and incomplete immune enhancers, vitamin E, nonionic blocking polymers, muramyl dipeptide, Quil A, mineral oil and non-mineral oil and Carbopol, water-in-oil emulsion immune enhancers, etc., but are limited thereto it is not going to be
  • Carriers that may be included in the vaccine composition of the present invention are known to those skilled in the art, and include, but are not limited to, proteins, sugars, and the like.
  • the above carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Carriers for intravenous injection include electrolyte replenishers, liquid and nutritional supplements, and the like, such as those based on Ringer's dextrose.
  • the vaccine composition of the present invention may further contain preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like.
  • the preservatives include formalin, thimerosal, neomycin, polymyxin B and amphotericin B, and the like.
  • the vaccine composition of the present invention may include one or more suitable emulsifiers, such as Span or Tween.
  • the vaccine composition of the present invention may include a protecting agent, and a protecting agent known in the art may be used without limitation, which may include lactose (LPGG) or trehalose (TPGG), It is not limited thereto.
  • a feed composition for preventing or improving salmonellosis comprising at least one member selected from the group consisting of; Or a feed additive composition for preventing or improving salmonellosis; it may be.
  • feed means any natural or artificial composition, one meal, etc., or a component of the one meal meal eaten by animals, and includes the YjeK gene-deleted Salmonella typhimurium strain according to the present invention as an active ingredient.
  • the feed may be prepared with various types of feed known in the art, and preferably may include concentrated feed, roughage and / or special feed, but is not limited thereto.
  • the feed additive is aimed at various effects such as alleviation of disease symptoms in animals, nutrient supplementation and weight loss prevention, improvement of digestibility of fiber in feed, oil quality improvement, reproduction disorder prevention and conception rate improvement, and summer high temperature stress prevention.
  • the feed composition and feed additive composition of the present invention correspond to supplementary feed under the Feed Control Act, and include mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, and trace minerals such as zinc, copper, cobalt, and selenium.
  • Mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, and trace minerals such as zinc, copper, cobalt, and selenium.
  • Mineral preparations such as kerotene, vitamins A, D, E, nicotinic acid, and vitamin B complex
  • protective amino acids such as methionine and lysine
  • protective fatty acids such as calcium salts of fatty acids
  • probiotics lactic acid bacteria
  • yeast cultures lactic acid bacteria
  • molds Live bacteria such as fermentation products, yeast agents, and the like may be further included.
  • the enriched feed includes seed fruits including grains such as wheat, oats and corn, bran including rice bran, bran, barley bran, etc. as a by-product obtained by refining grains, soybeans, fluids, sesame seeds, linseed, coco Fish meal, which is a by-product obtained from oil extraction of palms, residual starch, which is the main component of starch residue, which is the remainder after removing starch from sweet potatoes, potatoes, etc., fish meal, fish residue, and fresh liquid obtained from fish Fish soluble, meat meal, blood meal, feather meal, skim milk powder, animal feed such as dry whey, yeast, chlorella, seaweed but not limited thereto.
  • seed fruits including grains such as wheat, oats and corn, bran including rice bran, bran, barley bran, etc.
  • bran including rice bran, bran, barley bran, etc.
  • by-product obtained by refining grains soybeans, fluids, sesame seeds, linseed, coco Fish meal
  • Forage among the feed includes grass feed such as wild grass, grass, green cutting, turnip for feed, beet for feed, root vegetables such as Lutherbearer, a type of turnip, raw grass, green crops, grains, etc. are filled in a silo and fermented with lactic acid It includes, but is not limited to, silage, which is a stored feed, grass, hay made by cutting down grass, straw of breeding crops, and leaves of leguminous plants.
  • Special feeds include mineral feeds such as oyster shells and rock salt, urea feeds such as urea or its derivative, diureide isobutane, and supplements for ingredients that are likely to be insufficient when only natural feed ingredients are mixed, or formulated feeds to improve the storability of feeds.
  • feed additives and dietary supplements which are substances added in small amounts, but are not limited thereto.
  • a Salmonella typhimurium strain containing the YjeK gene in which the nucleotide represented by the nucleotide sequence of SEQ ID NO: 1 is deleted, a culture of the strain, a concentrate of the culture, and a dried form of the culture It provides a method for preventing or treating salmonellosis comprising; administering at least one selected from the group consisting of to a subject in need thereof.
  • the YjeK gene-deleted Salmonella typhimurium strain is intravenous administration, intramuscular administration, intranasal administration, intra-articular administration, intra-synovial administration, water Intrathecal administration, intrahepatic administration, intralesional administration or intracranial administration is preferred, and intramuscular or intranasal administration is more preferred, but is not limited thereto.
  • the method for preventing or treating salmonellosis of the present invention it is preferable to increase the immune response in vivo by administering a Salmonella typhimurium strain in which the YjeK gene is deleted, and more specifically, the antibody regulates the expression of factors related to the immune response. It is desirable to do
  • Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.
  • ST1120 wild-type Salmonella enterica serovar Typhimurium 1120 isolated in Korea was used.
  • S. Typhimurium 14028 strain ST2173 transformed with the plasmid pBBR1-MCS4 was used.
  • the ST2173 strain contains an ampicillin resistance gene.
  • Bacterial strains were cultured in Luria-Bertani (LB) broth (Duchefa, Haarlem, The Netherlands) at 37°C with 150 ⁇ g/ml ampicillin (Duchefa) when necessary unless otherwise specified. All antibiotics were purchased from Sigma-Aldrich (St. Louis, MO, USA). The biochemical properties of the bacteria were confirmed using the API 20E kit (bioMerieux, Inc., Durham, NC, USA).
  • a ⁇ yjeK mutant strain in which the yjeK gene was deleted was generated through site-directed mutagenesis using the ⁇ red recombination method using plasmids pTP233, pKD3 and pCP20. Specifically, ST1120 containing pTP233 was transformed with a chloramphenicol resistance gene cassette flanked by sequences homologous to the yjeK gene to prepare ST2160 strain. Homologous recombination between the chromosome yjeK and the CM resistance cassette in strain ST2160 was confirmed by diagnostic polymerase chain reaction after plating on LB agar containing 40 ⁇ g/mL chloramphenicol.
  • a chloramphenicol-sensitive strain was selected, and the chloramphenicol-resistance gene was removed from the chromosome to obtain strain ST2161.
  • the pCP20 plasmid was removed by culturing the chloramphenicol-sensitive strain at 42° C., and deletion of the yjeK and CM cassettes was further verified using diagnostic PCR.
  • Bacterial antibiotic susceptibility was assessed using the disk diffusion test on Mueller-Hinton agar (Becton Dickinson, Sparks, MD, USA). Bacterial cells were incubated in LB broth at 37°C for 16-18 hours. The optical density (OD 600 ) at 600 nm of the cultured cells was 0.6-0.7, and the number of cultured cells corresponded to 5 ⁇ 10 7 cells. Colony forming units (CFU)/mL were smeared on Mueller-Hinton (MH) agar using sterile swabs prior to placement of antibiotic discs (BBLTM Sensi-DiscTM, Becton Dickinson).
  • MH Mueller-Hinton
  • antibiotics AMC amoxicillin/clavulanic acid
  • 10 ⁇ g of ampicillin (AM) ampicillin
  • CF cephalothin
  • 10 ⁇ g of gentamicin (GM) 10 ⁇ g of gentamicin (GM), kanamycin, K
  • AMC ampicillin/clavulanic acid
  • CF cephalothin
  • GM gentamicin
  • K Antibiotic susceptibility to 30 ⁇ g, nalidixic acid (NA) 30 ⁇ g, neomycin (N) 30 ⁇ g
  • SAM ampicillin/sulbactam
  • SXT sulfamethoxazole/trimethoprim
  • lysing solution 0.1 M sodium dodecyl sulfate, 50 mM Tris base, 0.128 M NaOH
  • P/C/I solution phenol:chloroform:isoa
  • the LPS fraction was precipitated overnight at -20 °C and centrifuged at 10,000 xg. LPS precipitated for 20 minutes was resuspended in sterile water and treated with DNase (Sigma-Aldrich) and RNase (Promega, Madison, WI, USA) at 37°C for 1 hour.
  • DNase Sigma-Aldrich
  • RNase Promega, Madison, WI, USA
  • the LPS solution was mixed with the P/C/I solution and centrifuged at 10,000 xg for 20 minutes.
  • Purified LPS was suspended in sterile water and quantified using the Pierce LAL Chromogenic Endotoxin Quantification Kit (Thermo Scientific Inc., IL, USA). A 5 ⁇ g LPS sample was loaded on a 12% DOC-PAGE (deoxycholate-polyacrylamide gel electrophoresis) gel and the LPS profile was visualized.
  • Bacterial OMPs were isolated and analyzed. Specifically, bacterial cells cultured in LB broth were harvested by centrifugation at 2,500 xg for 20 minutes. Bacterial cell pellets were suspended in 10 mM N-2-hydroxyethylpiperazine N′-2-ethanesulfonic acid and sonicated with a Vibra-cell ultrasonic liquid processor (Sonics & Materials Inc., Newtown, CT, USA). The bacterial membrane fraction of cell lysates was collected by ultracentrifugation using a 45 Ti rotor (Beckman Coulter, Brea, CA, USA) at 100,000 xg.
  • the membrane fraction was resuspended in a buffer containing 1% N-lauroylsarcosine solution (Sarkosyl, Sigma-Aldrich) to solubilize and remove inner membrane proteins.
  • the sarcosyl-treated OMP fraction was purified using ultracentrifugation as described above and finally resuspended in phosphate buffered saline.
  • the concentration of OMP was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific Inc.). 10 ⁇ g of OMP was resolved using 12% SDS-PAGE and visualized by staining with Coomassie Brilliant Blue.
  • Protein bands of interest were analyzed by peptide mass fingerprinting method. Briefly, proteins were extracted from SDS-PAGE gels and then digested with trypsin (Promega). digested peptide; and ⁇ -cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid (Sigma-Aldrich), and the peptide mixture was subjected to matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Microflex LRF 20, Bruker Daltonics , MA, USA).
  • Mass spectra were collected in the m/z range of 600-3,000 and internally calibrated by the trypsin autodigestion peak (m/z 842.510, 2211.1046). Mass spectra were analyzed using the MASCOT server version 2.3 (Matrix Science, London, UK). Proteins with a MASCOT score >73 were considered significantly matched to the target protein (P ⁇ 0.05).
  • RNA protect Bacteria Reagent (Qiagen, Hilden, Germany) or RNAlater stabilizing solution (Ambion, Austin, TX, USA) according to the manufacturer's instructions. processed. Bacterial total RNA was isolated using the RNeasy mini kit (Qiagen) and residual chromosomal DNA was removed using the TURBO DNA-free kit (Ambion). RNA was reverse transcribed into cDNA using RNA to cDNA EcoDry Premix with random hexamers (Clontech Laboratory, CA, USA), and cDNA corresponding to 10 ng of input RNA was used as a template in each qPCR. Primers were designed using Primer Express Software version 3.0 (Applied Biosystems, MA, USA), and the designed primer sequences are shown in Table 2 above.
  • qPCR was performed using a StepOnePlus real-time PCR instrument (Applied Biosystems) with SYBR Green reagent (Power SYBR Green PCR Master Mix, Applied Biosystems) to detect amplified PCR products.
  • SYBR Green reagent Power SYBR Green PCR Master Mix, Applied Biosystems
  • the relative expression level of each gene was normalized to that of gyrB and expressed as the average of three tests using independently extracted RNA samples.
  • HeLa human epithelial cells and RAW264.7 murine macrophages were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS (fetal bovine serum), at densities of 1 ⁇ 10 5 and 2 ⁇ 10 5 cells/well. were inoculated into 24-well culture plates, respectively.
  • Salmonella cells cultured in LB broth overnight were added to animal cells at a multiplicity of infection of 100. After 30 min of bacterial infection, cells were washed 3 times with PBS and supplemented with fresh DMEM containing gentamicin (100 ⁇ g/mL) for 1.5 h. The medium was replaced with fresh DMEM containing 10 ⁇ g/mL gentamicin for the remainder of the infection period to remove extracellular bacteria.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • infected HeLa cells were lysed using 1% Triton X-100 2 hours post infection, cell lysates were diluted and plated on LB agar.
  • RAW264.7 cells infected in the survival assay were lysed 10 hours after infection, and intracellular bacteria were counted in the same way as described for the invasion assay.
  • LD 50 half-maximal lethal dose
  • In vivo toxicity testing was performed using groups of 4 BALB/c mice infected intraperitoneally with bacterial cells at 10 4 CFU/dose. Mice were sacrificed on day 14 post infection (dpi) and body and spleen weights were measured. Spleens were subsequently homogenized using a TissueLyser II (Qiagen, USA) at 30 Hz for 1 minute, and lysates were serially diluted. To enumerate bacterial cells, serial dilutions were plated on Salmonella-Shigella (SS) agar (Difco, Becton Dickinson, MD, USA).
  • SS Salmonella-Shigella
  • mice Female BALB/c mice were divided into groups of 6 mice each and immunized with wild-type and ⁇ yjeK mutant strains by intraperitoneal injection with 10 3 CFU/dose and 10 4 CFU/dose, respectively.
  • Negative control (N control) was inoculated with PBS instead of the strain. Blood was collected at 0, 7, 14, 28 and 35 days after mouse immunization and serum was stored at -20 °C until use. After 28 days, mice were orally administered with 10 8 CFU/dose (protection assay) or 10 10 CFU/dose (survival assay) of the ST2173 strain.
  • a positive control was challenged with the same dose of ST2173 after administration of PBS.
  • mice In the mouse protection assay, antigen-challenged mice were sacrificed 35 days after immunization. Organs including liver and spleen were collected from sacrificed mice, and plated on SS agar. In mouse survival assays, challenged mice were monitored for an additional 24 days and mortality was recorded.
  • IgG and IgM antibody responses in serum were measured using ELISA as previously described. Optimal concentrations of serum and enzyme conjugate were determined by a checkerboard test using triplicate sera from immunized and non-immunized mice. S. by selecting the best binding ratio between serum and enzyme conjugates. Typhimurium-specific IgG, IgM and IgG subclass (IgG1 and IgG2a) antibody responses were measured. wild type S. OMP prepared from Typhimurium was added to a microplate (Thermo Scientific Inc.) at 0.5 ⁇ g/well and incubated overnight at 4°C. Serum from immunized mice was diluted 1:200 and added to the coated wells.
  • microplate was washed with PBS (TBST) containing Tween 20, and horseradish peroxidase-labeled goat anti-mouse IgG (1:30,000 dilution; Bethyl Laboratories, TX, USA), anti-mouse IgM (1 :35,000 dilution, Bethyl Laboratories), anti-mouse IgG1 (1:25,000 dilution, Bethyl Laboratories) or anti-mouse IgG2a (1:30,000 dilution, Bethyl Laboratories) secondary antibody was treated. Unbound secondary antibody was washed 3 times with 0.05% PBST.
  • spleen cells were isolated from the spleen and counted. 2X10 5 cells/well were plated in complete medium consisting of RPMI-1640 (GenDEPOT, TX, USA), 10 mM HEPES (Duchefa) and 10% FBS. The plated cells were killed by LPS (5 ⁇ g/mL, Sigma-Aldrich) or heat. Typhimurium cells (HKC; 10 8 CFU/0.1 mL) were treated and cultured for 48 hours at 37°C and 5% CO 2 conditions. The supernatant of the cultured cells was collected and cytokines were measured. The supernatant was stored at -20 °C until cytokine measurement.
  • IFN- ⁇ , IL-6 and TNF- ⁇ were determined using a cytokine ELISA kit (MAXTM Standard, BioLegend, Inc., CA, USA) according to the manufacturer's instructions. Cytokine concentrations were calculated using Four Parameter Logistic Curve software (MyAssays Ltd., https://www.myassays.com/).
  • the yjek gene of the wild-type strain ST1120 is represented by the nucleotide sequence of SEQ ID NO: 2, and the nucleotide sequence of SEQ ID NO: 1 was deleted from the wild-type yjek gene using the ⁇ red recombination method. That is, the ⁇ yjeK mutant strain includes the yjek gene in which the nucleotide of SEQ ID NO: 1 is deleted. Since the efp gene is located near the deletion site, the expression of efp was compared between the wild-type and deletion strains. Transcription of efp was hardly affected by the deletion in the 1yjeK mutant strain, ruling out the possibility of a polar effect on EF-P activity in the ⁇ yjeK mutant strain.
  • FIGS. 1A and B The results of analyzing the growth and motility of the ⁇ yjeK mutant strain are shown in FIGS. 1A and B, respectively.
  • the ⁇ yjeK mutant strain blocked the biochemical activities related to arginine dihydrolase and lysine decarboxylase, thereby reducing arginine and lysine decapsulants in the mutant strain. It was confirmed that it represents an altered metabolic pathway of reboxylation.
  • the OmpA induces strong immunogenicity in Salmonella infection, and less porin OmpD is produced, reducing bacterial permeability to oxidizing molecules and promoting bacterial survival in the host.
  • Bacterial OMPs that form porins and efflux pumps affect bacterial resistance to antibiotics by reducing permeability (porins) or increasing drug export (efflux pumps). Therefore, as an increase in porin OmpD was observed in the ⁇ yjeK mutant strain, resistance to antibiotics was further confirmed. The results of confirming resistance to antibiotics are shown in FIG. 1D.
  • the ⁇ yjeK mutant strain was susceptible to other antibiotics except NA, SXT and TE.
  • the inhibitory regions of GM, N, K, and AMC were up to 77% (21.0 ⁇ 0.0 mm), 45% (18.9 ⁇ 0.5 mm), 36% (21.8 ⁇ 1.1 mm), and 34%, respectively. % (27.5 ⁇ 0.7 mm).
  • the ⁇ yjeK mutant strain entered host epithelial cells 3.73 times faster than wild-type bacteria at 2 hours after infection, but failed to fully proliferate in macrophages 10 hours after infection.
  • mice infected with the ⁇ yjeK mutant strain After sacrifice of mice infected with the ⁇ yjeK mutant strain, spleen and body weights were measured. The number of bacteria in the isolated spleen was also counted. The results of confirming the weight of the mouse spleen and the number of bacteria in the spleen are shown in FIGS. 3A and B, respectively. In addition, the result of measuring the body weight of the sacrificed mouse is shown in Figure 3C.
  • mice infected with the ⁇ yjeK mutant strain were also clearly different from those of the wild-type infected group.
  • the weight of the spleen was significantly reduced ( ⁇ yjeK, 0.34 ⁇ 0.05 g vs. wild-type, 0.72 ⁇ 0.12 g), whereas splenomegaly caused by the Salmonella strain was observed in the infection with the wild-type strain. .
  • the number of bacteria in the spleen of the ⁇ yjeK mutant strain treatment group was 4.6X10 4 CFU/g, which was significantly lower than that of the wild-type strain treatment group (2.6X10 5 CFU/g).
  • the immunological effect of the ⁇ yjeK mutant was measured by the level of serum antibodies (IgG and IgM) produced after infection in BALB/c mice.
  • the results of analyzing the expression levels of IgG and IgM are shown in FIGS. 4A and B, respectively.
  • the levels of IgG and IgM antibodies were similar for groups infected with wild-type and ⁇ yjeK mutant strains up to 14 dpi. At 28 dpi, immunization with the ⁇ yjeK mutant strain resulted in a 1.7- and 1.5-fold increase in the levels of IgG and IgM antibodies compared to 14 dpi, respectively.
  • the IgG2a/IgG1 ratio is commonly used as a surrogate marker for the immune balance between T helper (Th)1 and Th2 responses to microbial infections.
  • the IgG2a/IgG1 ratio of the immunized mice was measured, and the results are shown in FIG. 4C.
  • the IgG2a/IgG1 ratio of the mice immunized with the ⁇ yjeK mutant strain ranged from 2.32 (28 dpi) to 3.66 (14 dpi), and the IgG2a/IgG1 ratio of the group infected with the wild-type strain was 5.16 (28 dpi). ) to 5.65 (14 dpi).
  • the spleen of the mouse was collected at 28 dpi, and the splenocytes isolated from the spleen were treated with S. Typhimurium LPS and HKC to estimate the cytokine production level for the Salmonella immunogen.
  • S. Typhimurium LPS and HKC The results of analyzing the cytokines IFN- ⁇ , IL-6 and TNF- ⁇ in the immunized mouse splenocytes are shown in FIGS. 5A to C, respectively.
  • the cytokines IFN- ⁇ , IL-6 and TNF- ⁇ were all increased in response to LPS or HKC, regardless of the bacterial strain used for immunization.
  • immunization with the ⁇ yjeK mutant strain promoted IFN- ⁇ production to a greater extent for LPS (2.24-fold) and HKC (1.5-fold) than was observed for wild-type Salmonella (Fig. 5A).
  • production of the pro-inflammatory cytokines IL-6 and TNF- ⁇ was induced to a lesser extent after immunization with the ⁇ yjeK mutant strain for both LPS and HKC treatment (FIGS. 5B and C).
  • mice immunized with the ⁇ yjeK mutant strain induce IgG and IgM antibodies without signs of systemic infection in the host organ, and at the same time modulate cytokine production in response to challenge with immunogenic antigens of Salmonella.
  • the preventive effect of immunization with the ⁇ yjeK mutant strain was evaluated in BALB/c mice.
  • the ⁇ yjeK mutant strain which is a candidate vaccine, was immunized by intraperitoneal administration to mice.
  • Immunized mice 28 dpi
  • ST2173 wild-type S. Typhimurium 2173
  • ST2173 wild-type S. Typhimurium 2173
  • ST2173 Clinical symptoms of mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain were observed. As a result, ST2173-infected mice without prior vaccination showed typical clinical signs of Salmonella infection, including coarse fur, hard, closed eyes, anorexia, hunched posture, tremors, weight loss and death.
  • mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain was measured, and the results are shown in FIG. 6B.
  • mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain was reduced by about 77% by ST2173 infection.
  • mice immunized with the ⁇ yjeK mutant strain showed weight gain for 7 days after infection with ST2173, similar to healthy mice not injected with the Salmonella strain.
  • mice infected with the wild-type S. Typhimurium 2173 (ST2173) strain were sacrificed, the weight of the liver and spleen was measured, and the number of bacteria in the liver and spleen was analyzed.
  • the results of analyzing the weight and number of bacteria in the liver are shown in FIG. 6C, and the results of analyzing the weight and number of bacteria in the spleen are shown in FIG. 6D.
  • mice immunized with the candidate vaccine showed no signs of liver and spleen enlargement, whereas challenged mice without prior vaccination had enlarged livers and spleens.
  • mice immunized with the candidate vaccine (3.0 X 10 3 CFU/g liver and 4.8 X 10 3 CFU/g spleen) was higher than that of mice immunized with wild-type Salmonella (2.3 X 10 4 CFU/g liver and 7.5 ⁇ 10 4 CFU/g spleen) and unimmunized mice (positive challenge control: 3.9 X 10 5 CFU/g liver and 2.5 X 10 6 CFU/g spleen).
  • the prophylactic effect of the candidate vaccine was evaluated by monitoring the survival of immunized mice after oral infection with ST2173 at a bacterial dose of 100 LD 50 .
  • the evaluation results are shown in Fig. 6E.

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Abstract

La présente invention concerne une souche de Salmonella typhimurium à gène YjeK supprimé et ses utilisations. Plus particulièrement, la présente invention concerne les éléments suivants : une nouvelle souche de Salmonella typhimurium; et une composition vaccinale, une composition immunogène, et une composition alimentaire la comprenant. Il a été confirmé que la souche Salmonella typhimurium à gène YjeK supprimé selon la présente invention peut prévenir efficacement l'infection par Salmonella, augmentant ainsi considérablement le taux de survie d'un individu. Cela signifie que lorsque la souche de Salmonella typhimurium à gène YjeK supprimé de la présente invention est utilisée comme vaccin, les maladies liées à l'infection par Salmonella peuvent être efficacement prévenues. Ainsi, la présente invention peut être utilisée de diverses manières dans le domaine de la lutte contre les maladies chez le bétail.
PCT/KR2022/008979 2021-06-23 2022-06-23 Souche de salmonella typhimurium à gène yjek supprimé et composition de vaccin contre les salmonelles la comprenant WO2022270953A1 (fr)

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