WO2011025344A2 - Mutants de salmonelle atténués dans lesquels l'adhésine de escherichia coli pathogène bovin des transformée et composition vaccinale contenant ces mutants utilisée pour empêcher et traiter la colibacillose et la salmonellose bovines - Google Patents

Mutants de salmonelle atténués dans lesquels l'adhésine de escherichia coli pathogène bovin des transformée et composition vaccinale contenant ces mutants utilisée pour empêcher et traiter la colibacillose et la salmonellose bovines Download PDF

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WO2011025344A2
WO2011025344A2 PCT/KR2010/005895 KR2010005895W WO2011025344A2 WO 2011025344 A2 WO2011025344 A2 WO 2011025344A2 KR 2010005895 W KR2010005895 W KR 2010005895W WO 2011025344 A2 WO2011025344 A2 WO 2011025344A2
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salmonella
vaccine
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coli
escherichia coli
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WO2011025344A3 (fr
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이존화
정구남
김은성
김삼웅
허진
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전북대학교 산학협력단
전라북도
주식회사 중앙백신연구소
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
    • C07K14/245—Escherichia (G)
    • 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)
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    • 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

Definitions

  • the present invention relates to vaccine compositions and methods for vaccination for the prevention and treatment of bovine pathogenic E. coli and Salmonellosis.
  • the delivery of antibodies to the calves via colostrum by vaccination of pregnant cows is the only means of delivery and the most effective immunization of cows.
  • the prevention of the disease by providing colostrum with high antibody content as a vaccine prevention program may be more efficient in terms of time and cost than treatment after the onset.
  • E. coli vaccines are inactivated kill vaccine or K99, F41 linear tablet vaccine, and recombinant subunit tablet vaccine, which are mixed with an adjuvant such as aluminum hydroxide gel and injected into the muscle. It is. Inactivated Bacillus vaccines do not induce an immune response against specific antigens, or induced expression and purification in the case of a linear tablet vaccine, and a complicated manufacturing process that requires enhanced isolation and purification of antigens for recombinant subunit purified vaccines. You have to go through the process.
  • the development of a vaccine for preventing animal diseases using live vaccines can be introduced.
  • the microorganisms used for developing live vaccines can be colonized after invading and infiltrating into the digestive or lymphatic tissues as an animal pathogen. Should be Compared to the case of using a syringe, the live vaccine can sufficiently induce an immune response in the mucosal area such as the oral cavity or the nasal cavity, which is a path of infection, thereby effectively preventing the infection of the pathogen.
  • various methods have been attempted to reduce the pathogenicity of Salmonella itself, and to have a secretion system capable of expressing external antigens and secreting them extracellularly.
  • the present invention recombines a major adhesion factor gene of E. coli to a plasmid vector in which lepB (signal peptidase), secA (ATPase), and secB (chaperone) genes are expressed together with an enhanced extracellular secretion efficiency of recombinant antigens.
  • Vaccines were prepared by transforming attenuated Salmonella with these antigens into recombinant plasmids.
  • the attenuated Salmonella vaccine used for transformation is a DAP requester that lacks the asd gene, and is designed to select an antigen-recombinant strain without antibiotics.
  • the deletion of cpxR increases lymphatic penetration and increases immunogenicity or increases crp or lon. It is characterized by attenuation of pathogenicity by deletion of the gene.
  • LTB heat-labile enterotoxin B subunit
  • CTB cholera-derived cholera toxin B subunit
  • the present invention intends to introduce an oral vaccine that utilizes attenuated Salmonella vaccine as an Escherichia coli-derived antigen carrier using Salmonella, which is not only suitable for live vaccine development conditions but also directly causes small pathogenic diarrhea.
  • Still another object of the present invention is to provide a vaccine composition for preventing and treating pathogenic Escherichia coli and Salmonella bacterium, and a method of vaccination using the same.
  • the present invention is a bovine pathogenic E. coli adhesion factor gene K99 ( fanC ), F41 ( fim41a ), Intimin ( eaeA ), F17 ( f17A ), F17 ( f17G ), CS31A ( clpG ), Afa ( afa8D ) And Afa ( afa8E ) provides any one selected from the group consisting of, and asd gene, Salmonella mutant strains lon , cpxR and asd gene deleted.
  • the present invention is bovine pathogenic Escherichia coli ( E. coli ) LTB (heat-labile enterotoxin B subunit) gene; And, lon, cpxR and asd genes containing the asd gene provides a deletion mutant of Salmonella.
  • E. coli Escherichia coli
  • LTB heat-labile enterotoxin B subunit
  • the present invention is bovine pathogenic E. coli adhesion factor genes K99 ( fanC ), F41 ( fim41a ), Intimin ( eaeA ), F17 ( f17A ), F17 ( f17G ), CS31A ( clpG ), Afa ( afa8D ) and Afa ( afa8E ) as containing either one gene, and asd gene containing, lon, cpxR and asd gene is more than any of the deletion mutant Salmonella one selected from the group consisting of, mixing provides a Salmonella mutant.
  • the present invention is bovine pathogenic Escherichia coli ( E. coli ) LTB (heat-labile enterotoxin B subunit) gene; And including the asd gene, lon, cpxR and asd gene is a deletion Salmonella mutant and cattle E.
  • E. coli Escherichia coli
  • LTB heat-labile enterotoxin B subunit
  • coli adhesion factor gene K99 fanC
  • F41 (fim41a ) Intimin
  • eaeA Intimin
  • F17 (f17A) F17 (f17G)
  • CS31A clpG
  • Afa ( afa8D) and Afa (afa8E) comprises any of the gene, and including the asd gene, lon, cpxR and asd gene is at least one of the deleted Salmonella mutant one selected from the group consisting of
  • the present inventors deposited the mixed Salmonella mutant strain in a depository institution (KCTC) Korea Depositary No. (KCTC11542BP).
  • the present invention provides a vaccine composition for preventing and treating bovine pathogenic E. coli and Salmonella, comprising the Salmonella mutant strain.
  • the bovine pathogenic Escherichia coli is a digestive disease, an edema disease, a respiratory disease or a genitourinary system disease.
  • the digestive disease comprises calf diarrhea or bovine virus diarrheal disease (BVD-MD).
  • BVD-MD bovine virus diarrheal disease
  • the bovine pathogenic E. coli comprises enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), or enteroinvasive E. coli (EIEC).
  • ETEC enterotoxigenic Escherichia coli
  • EPEC enteropathogenic Escherichia coli
  • EIEC enteroinvasive E. coli
  • the Salmonella Salmonella typhimurium Salmonella typhimur (S. typhi), Salmonella paratyphi (S. paratyphi), Salmonella Sendai (S. sendai), Salmonella gallinarium (S. gallinarium) and Salmonella enteritidis.
  • the transformed Salmonella mutant strain is live or dead, and the dead bacterium includes heated or inactivated formalin.
  • the composition may be administered by oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous or nasal route.
  • the present invention provides a feed additive for boosting or boosting the growth of bovine, comprising the vaccine composition.
  • the present invention (a) bovine pathogenic E. coli adhesion factor genes K99 ( fanC ), F41 ( fim41a ), Intimin ( eaeA ), F17 ( f17A ), F17 ( f17G ), CS31A ( clpG ), Afa ( afa8D ) and Afa amplifying any one gene selected from the group consisting of afa8E ; (b) cloning said adherent gene into a plasmid with an asd gene; (c) deleting the lon , cpxR and asd genes from the chromosomal DNA of the Salmonella strain to prepare the attenuated Salmonella strain; (d) transforming the cloned plasmid of step (b) into the attenuated Salmonella strain of step (c); And (e) provides a method for producing a Salmonella mutant strain comprising the step of selecting a transformed Salmonella mutant strain.
  • the present invention comprises the steps of: (a) amplifying a pathogenic E. coli (heat-labile enterotoxin B subunit) LTB gene; (b) cloning said LTB gene into a plasmid with an asd gene; (c) deleting the lon , cpxR and asd genes from the chromosomal DNA of the Salmonella strain to prepare the attenuated Salmonella strain; (d) transforming the cloned plasmid of step (b) into the attenuated Salmonella strain of step (c); And (e) provides a method for producing a Salmonella mutant strain comprising the step of selecting a transformed Salmonella mutant strain.
  • E. coli heat-labile enterotoxin B subunit
  • a vaccine method for the prevention and treatment of bovine pathogenic E. coli and Salmonella characterized in that the Salmonella mutant strain is prepared as a live or dead bacterium vaccine, and then inoculated into cattle or jaws.
  • the vaccine can be inoculated by oral, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous or nasal route.
  • live vaccines can be inoculated orally at the first and second inoculations. More preferably, live vaccines are orally inoculated at the first and second inoculations, but live vaccines other than Salmonella mutants expressing and secreting LTB at the second inoculation may be inoculated.
  • the vaccine composition according to the present invention efficiently delivers E. coli-derived antigens to bovine mucosal lymphoid tissues to induce potent mucosal immunity, as well as vaccine production and mass inoculation, inoculation cost and pain
  • E. coli-derived antigens to bovine mucosal lymphoid tissues to induce potent mucosal immunity, as well as vaccine production and mass inoculation, inoculation cost and pain
  • there is an economic effect that is easy to produce and store. Therefore, it can be used as a multivalent vaccine because it not only prevents infection by inducing an immune response against Salmonella bacteria, but also induces an immune response against an expressing external antigen.
  • 3 and 4 show the results of confirming the extracellular secretion of the recombinant antigen by Western blot.
  • Figure 6 shows the preparation and inoculation method of each vaccine.
  • 7 and 8 show the amount of inoculation determined according to the change of cow feces sIgA and colostrum antibody titer according to LTB addition in the composition of the vaccine.
  • Figure 12 compares the serum antibody titers of cows at delivery by vaccination route.
  • FIG. 13 compares the intestinal sIgA of cows at the time of delivery by vaccination route to fecal antibody titer.
  • 17 to 19 is a comparison of the antibody titer and continuity in the serum, feces of colostrum ingested by each vaccination route.
  • Figure 20 is a diarrhea fecal state caused by pathogenic E. coli challenge infection attempted immediately after birth.
  • Figure 21 shows the change in the antibody of the feces after vaccination of 2 weeks old cow by the vaccination route.
  • Still another object of the present invention is to provide a vaccine composition for preventing and treating pathogenic Escherichia coli and Salmonella bacterium, and a method of vaccination using the same.
  • Adhesion genes for protein expression and recombinant vaccine construction are from the pathogenic E. coli JOL412 (K99 expression), JOL413 (K99 and F41 expression), JOL132 (Intimin expression), JOL426 (F17 and CS31A expression), JOL462 (Afa expression). Secured.
  • pathogenic E. coli isolates JOL412 (K99 expression), JOL695 (F41 expression), JOL681 (Intimin expression), JOL465 (F17 expression), JOL873 (CS31A expression), JOL686 (Afa expression) were used as the challenge strain.
  • the strains were cultured in Luria-Bertani (LB) medium without DAP.
  • the attenuated Salmonella typhimurium that expresses and secretes each pathogenic E. coli attachment factor recombined into the asd plasmid pBP244 was selected from LB agar without DAP (see Table 1, Figure 2).
  • Sequence information of genes deleted in attenuated Salmonella typhimurium can be easily obtained from GeneBank FJ609803.1 for lon, GeneBank AE006468.1 for clon, and GeneBank AF015781.1 for asd. .
  • the present inventors deposited the attenuated Salmonella typhimurium strain ( ⁇ lon ⁇ cpxR ⁇ asd) at the Korea Institute of Bioscience and Biotechnology Center (Accession No. KCTC11540BP, Samonella enterica serovar Typhimurium strain JOL912, deposited August 3, 2009).
  • DNA extraction of Escherichia coli was prepared according to the conventional heating lysis method.
  • Pathogenic Escherichia coli identified by the multiplex PCR reaction of the major subunit gene of the adhesion factor was inoculated in LB agar, incubated overnight at 37 ° C, and then selected by one clone for 16 hours in 1 ml LB medium. Incubated. The supernatant was removed by centrifugation, washed once with sterile distilled water, and heated at 95 ° C. for 20 minutes by adding 50 ⁇ l sterile distilled water. After centrifugation, only the supernatant was transferred to a sterile small tube and extracted and stored as DNA of Escherichia coli.
  • each adhesion factor gene was amplified by PCR using primers (Table 2) designed to have restriction enzyme cleavage sites in the nucleotide sequence for recombination into plasmid vectors. The size of the amplified gene was confirmed by electrophoresis and the sequence was correctly amplified by sequencing. Subsequently, each amplified gene was purified from an agarose gel using an AccuPrep gel purification kit (Bioneer, Korea) after electrophoretic separation.
  • Sequence information of the gene amplified using the primer, K99 ( fanC ) is Genebank M35282.1; F41 ( fim41a ) is GenBank X14354.1; Intimin ( eaeA ) is described in Genebank EF079676.1; F17 ( f17A ) and F17 ( f17G ) are GenBank AF022140.1; CS31A ( clpG ) is found in Genebank M55389.1; Afa ( afa8D ), Afa ( afa8E ) are readily available from GenBank AF072900.3 and LTB from GenBank EU113255.1.
  • PCR amplification products and plasmids, pQE9, pQE10, and pET28a vectors, which were used for overexpression of each of the attached genes, were digested with the corresponding restriction enzymes, separated, purified, and ligated with the T4 DNA ligase (Takara, Japan). It was. E. coli JM109 or E. coli Top 10 was transformed with the recombinant vector as a protein overexpression strain, and the transformed strain was selected by culturing in a selection medium containing the antibiotic marker.
  • Recombinant plasmids in which the attachment factor genes were inserted from each transgenic strain were re-isolated using AccuPrep plasmid extraction kit (Bioneer, Korea), and their size was confirmed by cleavage of the corresponding restriction enzymes in Table 2, and ORF in the vector was determined by sequencing. It was confirmed that (open leading frame) was formed correctly.
  • the recombinant overexpression strains identified above were inoculated in 1ml LB medium containing antibiotics, incubated for 12 hours, and then re-inoculated in 5ml LB broth to confirm whether the protein was expressed from the recombinant adhesion gene. Expression was induced by the addition of (isopropyl- ⁇ D-thiogalactopyranoside). After pulverizing the cells by ultrasound, the samples were prepared by dividing them into supernatant) and resuspension, followed by SDS-PAGE of the samples after induction of overexpression with the control group before induction of expression. It was confirmed whether or not insoluble.
  • each recombinant strain confirmed the adhesion factor expression was isolated from the culture medium cultured with the addition of IPTG and resuspended in 10ml of 1mM phenylmethanesulfonyl fluoride (PMSF) -PBS. After freezing it 2-3 times, the cells were pulverized with ultrasonic waves after repeated thawing in a 37 ° C. water bath, and centrifuged. The supernatant containing soluble expressed protein was 4 ml buffer B (100 mM NaH 2 PO 4).
  • PMSF phenylmethanesulfonyl fluoride
  • Precipitates containing 10 mM Tris.Cl, 8M urea, pH 8.0) and insoluble expressed protein were mixed with 6 ml of buffer B, respectively, and stirred and reacted at room temperature for 1 hour. After centrifugation, the supernatant was stirred for 30 minutes to bind the protein and the resin. After stirring, the supernatant was poured into a separation tube equipped with glass fibers, and then 6 ml buffer C (100 mM NaH 2 PO 4 , 10 mM Tris. Cl, 8 M urea). , pH 6.3), the unnecessary protein was washed and removed. Proteins were separated by 2 ml elution buffer (100 mM NaH 2 PO 4 , 10 mM Tris.
  • Antiserum of each adhesion factor protein was prepared by mixing each attachment factor protein with Freund's adjuvant (Freund's adjuvant), subcutaneously inoculated twice, and then collecting blood to separate serum. One serum was stored at -70 ° C. Anti-adherent antibodies in rabbit serum were identified by Western blot using each subunit protein as an antigen (FIGS. 3 and 4) and used as a coating antigen of ELISA to analyze the immune response induced after bovine vaccination.
  • Freund's adjuvant Freund's adjuvant
  • the plasmid vector of the vaccine strain pBP244 (Kim et al., 2007, Journal of Microbiology and Biotechnology, 17: 1316-1323), expresses SecA, SecB and LepB so that the secretion of foreign antigens is increased and E. coli and Salmonella shuttle plasmids.
  • Each adhesion factor gene and pBP244 fragment digested with the corresponding restriction enzyme were purified from agarose electrophoresis gel using a purification kit, ligated using T4 DNA ligase, and then E. coli ⁇ 7213 with the reaction solution. (JOL767) was transformed and cultured in LB agar without DAP, and then the transforming strains were selected. Recombination of each adhesion factor was confirmed by restriction enzyme digestion by separating plasmid from E. coli ⁇ 7213, and confirmed that the ORF of the recombined gene was correctly formed by sequencing.
  • JOL912 ⁇ asd ⁇ lon ⁇ cpxR
  • JOL 912 was incubated in LB broth containing DAP (50 ⁇ g / ml), washed twice with distilled water containing ice-cold 10% glycerol, mixed with purified plasmid and electroporated in MicroPulser (Bio-Rad, USA). Was implemented. This was incubated for 1 hour with the addition of 1ml LB broth, and then cultured for 16 hours in LB medium without DAP, and transformed strains were selected.
  • the live vaccine strain JOL906 expressing haet-labile toxin subunit B (LTB) of E. coli to be used as an adjuvant was also prepared.
  • the produced soda vaccine strain secreted the main subunit of the Escherichia coli adhesion factor extracellularly without cell destruction, and the final soda was selected as a live vaccine strain by comparing the secretion efficiency.
  • the extracellular secretion of the main subunit protein only the culture supernatant of each vaccine strain 2x10 9 CFU was isolated and filtered, and concentrated by 10% TCA method, followed by Western blot using anti-adherent antibody after SDS-PAGE. Extracellular secretions were compared. As a result, the extracellular secretion efficiency was high in the vaccine strain transformed by recombining the subunit gene into the pBP244 plasmid vector.
  • soda polyvalent vaccine strains were K99-JOL950 ( fanC ), F41-JOL951 ( fim41a ), Intimin-JOL953 ( eaeA ), F17A-JOL825 ( F17A ), F17G-JOL952 ( F17G ), CS31A-JOL954 ( clpG ), AfaD-JOL955 ( afa8D ), AfaE-JOL956 ( afa8E ), LTB-JOL906 ( eltB ) were selected (see Table 3, FIG. 5).
  • the immune antibody against the adherent antigen secreted by Salmonella vaccine in cattle is Escherichia coli. It can contribute to the recognition and defense of antigens.
  • the inventors of the present invention have shown that the bovine pathogenic E. coli adhesion factor genes K99 ( fanC ), F41 ( fim41a ), Intimin ( eaeA ), F17 ( f17A ), F17 ( f17G ), CS31A ( clpG ), Afa ( afa8D ) and Afa ( afa8E ).
  • Mixed Salmonella mutants including mixed Salmonella mutants containing all of the four Salmonella strains and Salmonella mutants containing the heat-labile enterotoxin B subunit (LTB) gene of bovine pathogenic E. coli, were deposited with the Korea Research Institute of Bioscience and Biotechnology. Accession number KCTC11542BP).
  • the same vaccine strain was produced in the form of live oral vaccine, live muscle vaccine, muscle or subcutaneous four vaccine (FIG. 6).
  • the inoculum was made into 10 ml of cows, 5 ml of cows, and 2 ml of the muscle or subcutaneous inoculation.
  • Example 2 After incubating the final selected soda vaccine strain in Example 2 for 16 hours in each LB liquid medium, the live vaccine was inoculated on the day of vaccination, and the dead vaccine was incubated 2 days before vaccination to check the inactivation status. Cultivated vaccine strains were collected according to the number of inoculated bacteria and washed with PBS, and the suspension was resuspended according to the type of vaccine composition.
  • live oral vaccines were resuspended in 0.05% gelatin-PBS, and muscle live vaccines were suspended in 2 ml PBS and used for vaccination.
  • the vaccine was resuspended to a final concentration of 0.5% formalin-PBS and then inactivated at 20 ° C. for 12 hours. Inactivated vaccine cells were washed twice with PBS, incubated for 20 hours to confirm inactivation, and then mixed with aluminum hydroxide gel as an adjuvant and used as a vaccine for four vaccines.
  • the inoculation amount was 5 ⁇ 10 10 CFU, and high antibody titers were induced in colostrum after the vaccination of cows, which was determined as the inoculation amount.
  • Figure 7 shows the increase in fecal sIgA after the first inoculation by LTB strain administration
  • Figure 8 shows the inoculum according to colostrum antibody titers.
  • NC experimental group in Figure 8 did not receive the cow vaccination as a control group
  • 1x10 10 CFU is two oral live vaccine inoculation group
  • 5 ⁇ 10 10 CFU is a group of two oral live vaccine inoculations, which were inoculated with primary 1 (including LTB) 8 weeks before delivery and 5 weeks before delivery.
  • JOL825 inoculation group and JOL953 inoculation group was divided into 1x10 9 CFU and 5x10 9 CFU, respectively, orally inoculated twice. No side effects were observed in both groups of cows by dose. However, from the birth challenge, the diarrhea was induced in the 1x10 9 CFU inoculated group, whereas no diarrhea was induced in the 5x10 9 CFU inoculated group.
  • the cattle vaccination group was divided into A, B, C, D, and NC groups, inoculated with 5 ⁇ 10 10 CFU per dose, and the inoculation was performed with the LTB strain when the first inoculation was performed by live oral vaccine.
  • A was an experimental group inoculated with two live oral vaccines.
  • the first inoculation was performed 8 weeks before delivery and the second inoculation was performed 5 weeks before delivery.
  • test group B was inoculated with the first live oral vaccine 8 weeks before delivery, and then injected with the secondary muscle quadrant five weeks ago, and C was inoculated with the first intramuscular vaccine 8 weeks before delivery by reversing the inoculation path sequence of group B.
  • D Five weeks before the second oral live vaccine was injected, D was the first injection of two muscle four vaccine six weeks before delivery, the second injection three weeks before delivery, and NC was not vaccinated in the cow
  • the experimental group was challenged within 60 hours of birth and challenged at 5 weeks of age in the cows born from the experimental group cows after administration of PBS alone.
  • A1, B1, C1, and NC1 are experimental groups that were challenged within 60 hours after birth among cows born from cows, and A2, B2, and C2 were challenged after 5 weeks of challenge after 2 week vaccination among cows born from cows.
  • the experimental group, NC2 was a test group that was born from cows of the NC test group and then challenged for 5 weeks of age
  • CC was an experimental group that received 5 weeks of challenge after administering a 2 week old vaccine without inoculation of the cow vaccine.
  • the LTB strain was included in the first oral live vaccine inoculation compared with the intestinal sIgA (secreted IgA) antibody titer analyzed by fecal samples compared with that of the experimental group vaccinated without addition of the LTB strain and 3 weeks after the inoculation. This is because the intestinal immune response was higher at 1 week after inoculation with fecal sIgA titers.
  • serum IgG, IgA, and fecal sIgA for 8 weeks before delivery and 1 week after delivery were quantified by week, and serum IgG was expressed for each antigen 8 weeks before delivery.
  • Individual quantitative values varied, but the average level of serum IgG decreased immediately after delivery. Near the scheduled delivery date, the IgG in the blood for all antigens gradually decreased, and the antibodies, which were the lowest immediately after delivery and decreased for about 3-6 weeks after delivery, generally recovered within 1 week.
  • serum IgA increased until delivery, and showed the maximum titer after 3 weeks before delivery, depending on the antigen. The decrease in serum IgG was significantly associated with the point of delivery, while the IgA was characterized by an increase.
  • Intestinal sIgA secretion was increased between 1 and 2 weeks before delivery, and decreased after delivery.
  • the colostrum secreted from cows immediately after delivery and weekly milk IgG and IgA showed significant differences in the total antibody levels and antibody titers against the same antigens, even though the colostrum antibodies were comparable to the antibody levels in the blood at the time of delivery. Individuals with high levels of antibody in their blood did not show high levels of colostrum.
  • colostrum antibody titers were significantly decreased after 3 days of delivery, and there was little difference from normal milk after 1 week.
  • the antibody of the NC experimental group is to analyze the efficacy of the cow vaccination on the basis of the change pattern and antibody titer.
  • the experimental group A the two oral live vaccine inoculation groups, showed evenly high titers by antigen in the colostrum IgG and IgA, serum IgG and IgA, and fecal sIgA at the time of delivery (FIGS. 9, 10, and 11). 12 and 13).
  • NC is the no-vaccine experimental group
  • A is the first oral live vaccine inoculation 8 weeks before delivery, and the second oral live vaccine inoculation 5 weeks before delivery
  • B is the first oral live vaccine inoculation 8 weeks before delivery
  • C is primary intramuscular vaccine at 8 weeks before delivery
  • D is primary intramuscular vaccine at 6 weeks before delivery, and 3 weeks before delivery 2
  • the experimental group of the primary intramuscular vaccination is shown.
  • the experimental group A had high colostrum IgG and IgA antibody titers.
  • the colostrum IgA antibody titer was relatively high even after one week of delivery, which was compared with the decrease in the colostrum IgA antibody titer in the NC experimental group (see FIGS. 10 and 11).
  • Serum IgG showed high titers three weeks after the first inoculation but decreased or similar on average than three weeks after delivery, with higher titers than before inoculation (see FIG. 12).
  • Serum IgA in the NC group which was not vaccinated, slowly increased until delivery, but the A group showed a sharply high titer at delivery.
  • the experimental group A showed a high value in fecal sIgA at the time of delivery as well as one week after the first inoculation (see FIG. 13).
  • the experimental group B which was treated with the second dose of muscle vaccine after the first oral inoculation, showed a lower antibody titer in colostrum IgA than the experimental group A (see FIG. 9).
  • Colostrum IgG antibody titers were as high as A group or higher depending on antigen. Serum IgG of the test group B was increased at the time of delivery, serum IgA increased with antigen, but was different from the test group A, the maximum increase in IgA at the time of delivery after two oral inoculations (see Fig. 12). In addition, the test group B maintained a relatively high titer in blood, unlike the decrease in serum IgA at 1 week after delivery in the test group A. These IgG and IgA titers in the cow's blood could be compared to those with low colostrum IgA antibody titers.
  • the experimental group C which was inoculated with the second oral live vaccine after the first intramuscular vaccination, showed high colostrum IgG, IgA, serum IgG, IgA, and fecal sIgA antibody titers as in the experimental group A (FIGS. 9 to 13).
  • Intramuscular vaccination is generally a method of inoculating pathogen-induced strains in a self-vaccinated form on a farm, and since the recombinant vaccine strain may be composed of a soluble recombinant antigen compared to the open strain, it was inoculated twice with the intramuscular vaccine. As a result, it was found that the experimental group D secreted low colostrum IgG and IgA compared to the experimental group A (see FIG. 9).
  • Table 5 division Emission diarrhea Fever Loss of appetite Deactivation premature birth NC 0 0/9 0/9 0/9 0/9 0/9 White vaccination group A 0 0/5 0/5 0/5 0/5 0/5 B 0 0/5 0/5 0/5 0/5 C 0 0/5 0/5 0/5 0/5 0/5 D 0 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2 0/2
  • the second muscle live vaccine inoculation test group had a preterm birth estimated to be related to inflammation and fever within 2 weeks after vaccination in the range of 5x10 6 to 1x10 10 CFU, and 2 weeks after vaccination. It was decided to exclude the inoculation of live muscle vaccines.
  • live oral vaccination was safe in cows without the side effects of vaccination, and was effective in producing immune antibodies, especially via colostrum.
  • at least one of two doses of oral administration of live vaccines was required for the vaccination of cows.
  • inoculation of live oral vaccines in the second phase was effective in secreting IgA secretion through colostrum.
  • the first inoculation including the LTB strain showed that the immune response was fast and highly induced.
  • each of the cows born from each experimental group was divided into two groups according to the two-week-old vaccination status.
  • the A1, B1, C1 and NC1 JAU experimental groups were challenged within 60 hours of age, and the A2, B2 and C2 JAU experimental groups were challenged 5 weeks after the 2 week vaccination.
  • the NC2 experimental group was challenged at 5 weeks postnatally among the cows born from the cows of the NC test group, and the CC (Calve Control) group was treated with the 5 week old challenge after the vaccine was administered to the 2 week old cow without vaccination. ).
  • the antibody titers of the cows before the cow's colostrum intake group A, B and C were the same as those of the NC experiment group, but serum IgG, IgA, and fecal sIgA were significantly increased after the colostrum intake. Increased.
  • serum of the experimental group A which received the first and second oral live vaccines
  • the C which received the second oral live vaccine after the first intramuscular vaccine, contrasted with the decrease in the level of the jaws of the NC experimental group at 1 to 2 weeks of age.
  • IgG and IgA maintained high titers for up to 2 weeks and then slowly decreased.
  • Fecal sIgA also increased in these experimental groups, showing nearly colostrum antibody titers after colostrum intake.
  • the pathogenic E. coli 1st challenge infection which was performed within 12 hours after confirming the intake of colostrum after birth in FIGS. 14 to 16 had little effect on serum IgG and IgA in the NC1 experimental group, and the 2nd week of 5 weeks of NC1 and NC2 experimental groups. After challenge, IgG and IgA decreased and then recovered.
  • the challenge of 5-week-old challenge showed a temporary increase in sIgA in feces after 1 week of challenge, in contrast to A1 and C1 in FIGS. 17 to 19.
  • the experimental group had high fecal sIgA in 2 to 3 weeks after challenge challenge. That is, the NC1 experimental group was confirmed that the value was higher compared to the increase in the antibody value after 5 weeks of challenge (Fig. 14 to 19).
  • the challenge of JAU is to assess the ability of E. coli to protect against pathogens according to colostrum antibodies.
  • the first infection was performed within 12 hours of birth after confirming the colostrum benefit, and added twice at 24 hour intervals. After oral administration three times in total, the clinical symptoms following infection were observed.
  • a strain causing the diarrhea was determined by oral administration of a pathogenic diarrhea causative bacterium expressing the recombinant Escherichia coli antigen to the cattle vaccine unvaccinated experimental group JAU (NC1), and was prepared by the same method as the live vaccine for oral inoculation (Table 6).
  • the infection test group moved the cows before delivery to the segregator and received them in the form of 1 mo-1-1/1 segregator after delivery and separated to prevent infection through the environment such as oral contact and defecation, salary, water supply after administration of pathogens to the cows. The slope was blocked and the bottom of the barn was kept dry.
  • pathogens were divided into ETEC, EPEC, and EIEC, respectively, in order to infect them, or ETEC, EPEC, and EIEC strains were mixed and administered in one dose for 3 days. All of them were EPEC (JOL681). ), Diarrhea by EIEC (JOL465) was frequent. Diarrhea occurred 3 to 4 days after the last pathogen was administered, and E.
  • coli was isolated from the induced diarrhea, and 20 CFU was randomly selected to confirm the strain by PCR. As a result, 18 to 20 CFU (90% or more) was the same strain. This was regarded as the causative agent of diarrhea in challenge infection. Infected cows were accompanied by inability to stand, lactation and deactivation, requiring antibiotics and fluid treatment. In the NC1 cow group, feces and activity improved after treatment, but severe diarrhea occurred in 3 out of 6 children at 4 to 5 weeks of age.
  • Figure 20 shows water-borne diarrhea, mucous diarrhea, bloody mucus diarrhea mixed with blood induced at 1 week of age due to challenge with pathogenic E. coli challenge immediately after birth.
  • some of the second intramuscular vaccination test group JU (B1) were induced by diarrhea by challenge challenge, whereas both the first and second oral vaccination groups were challenged.
  • the second oral live vaccine group JAU (C1) showed no symptoms of diarrhea from 5 weeks after challenge (see Table 7).
  • the vaccine composition according to the present invention is inoculated twice in pregnant cows, when both are orally administered, inoculated including the primary LTB strain, and paralleled with the muscle vaccine. In the case of administration, it was found that the inoculation should be inoculated with oral live vaccine.
  • the A2, B2, and C2 cows of the cows born from each of the cows who received no challenge immediately after birth were inoculated with 2 weeks old live vaccine, 2x10 9 CFU, and the number of pathogenic E. coli bacteria was administered at 5 weeks of age to determine the efficacy of the vaccine.
  • Experimental groups that received 5 weeks of challenge after the vaccine was administered to 2 weeks old cows without vaccination of cows were divided into CC (Calve Control).
  • the single oral live vaccine inoculation of A2, B2, C2 experimental group 2 weeks old cows born from the cow vaccination test group did not perform vaccination in the serum IgG, IgA titers of the cows. There was no difference between and, but fecal sIgA increased significantly after 1 week of vaccination. Characteristically, the CC cow test group had lower fecal sIgA titers compared to cows born from the cow vaccination group, and showed no significant change due to the cow vaccination.
  • diarrhea did not appear in all A2, B2, C2, and CC groups treated with oral live vaccines.
  • challenge challenge of 5-week-old ZAU in NC experimental group was caused by diarrhea even with a single dose of SAU and once administration.
  • the vaccine composition according to the present invention efficiently delivers E. coli-derived antigens to bovine mucosal lymphoid tissues, induces strong mucosal immunity, and is capable of producing vaccines and mass inoculations, and producing them with reduced side effects such as inoculation costs, pain, and inflammation. Economical and easy to store. Therefore, it can be used as a multivalent vaccine because it not only prevents infection by inducing an immune response against Salmonella bacteria, but also induces an immune response against an expressing external antigen.

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Abstract

La présente invention concerne des mutants de salmonelle qui comprennent un gène d'adhésine de Escherichia coli pathogène bovin et sont transformés en mutants de salmonelle atténués dans lesquels les gènes asd, lon et cpxR ont été délétés; et une composition vaccinale contenant ces mutants, utilisée pour prévenir et traiter la colibacillose et la salmonellose. Les mutants de salmonelle selon la présente invention apportent efficacement un antigène dérivé de Escherichia coli dans des tissus lymphoïdes des muqueuses de bovins et induisent une puissante immunité mucosale, ils permettent également de produire le vaccin associé et d'effectuer l'inoculation en masse. En outre, les mutants de salmonelle selon la présente invention permettent de réduire le coût de l'inoculation et les effets secondaires tels que la douleur, l'inflammation ou similaire. La production et le stockage des mutants de salmonelle selon la présente invention sont aisés et par conséquent, économiques. Ainsi, les mutants de salmonelle selon la présente invention empêchent les infections, en induisant des réponses immunitaires contre la salmonelle et peuvent être utilisés en tant que vaccin polyvalent du fait de l'induction de réponses immunitaires contre l'expression d'antigènes exogènes.
PCT/KR2010/005895 2009-08-31 2010-08-31 Mutants de salmonelle atténués dans lesquels l'adhésine de escherichia coli pathogène bovin des transformée et composition vaccinale contenant ces mutants utilisée pour empêcher et traiter la colibacillose et la salmonellose bovines WO2011025344A2 (fr)

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KR101893827B1 (ko) * 2016-01-04 2018-08-31 전북대학교 산학협력단 약독화된 살모넬라 변이주를 유효성분으로 포함하는 돼지 증식성 회장염 및 살모넬라증 동시 예방 또는 치료용 백신 조성물
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