KR101705098B1 - Vaccine composition for preventing or treating porcine pleuropneumonia comprising attenuated Salmonella mutant - Google Patents

Abstract

The present invention relates to a salmonella mutant strain in which the lon , cpxR and asd genes have been deleted, which further comprises a major pathogenic factor of the causative agent of pleural pneumonia in pigs , a method for producing the mutant strain of Salmonella, a method for preventing the pleural pneumonitis of pigs including the Salmonella mutant strain Or therapeutic vaccine compositions and vaccination methods for the prevention or treatment of swine pneumonia.

Description

[0001] The present invention relates to a vaccine composition for prevention or treatment of swine pneumonia, including an attenuated Salmonella mutant,

The present invention, more specifically, the main pathogenic factor of pleuropneumonia organisms more, lon, cpxR and asd genes containing porcine relates to prophylaxis or therapeutic vaccine composition for swine pleuropneumonia comprising a live attenuated Salmonella mutant is A vaccine composition for prevention or treatment of swine pneumonia of swine including the Salmonella mutant strains, and a vaccination method for prevention or treatment of swine pneumonia.

Actinobacillus pleuropneumoniae is the cause of swine pneumonia. Actinobacillus pleuropneumoniae causes acute bacterial infection, which causes poor appetite, difficulty breathing, vomiting, cyanosis, high fever, nose and mouth bleeding, It is a disease in which animals die. The disease is one of the most important bacterial respiratory diseases of swine, and it occurs most often in the swine industry. The outbreak of this disease is a disease that needs to be paid attention to in the swine industry because it leads to decrease in productivity, productivity and cost of treatment or vaccine.

Pig pleural pneumonia caused by Actinobacillus furopneumoniae is an infectious, fibroplastic, haemorrhagic, and necrotic disease that causes acute morbidity and mortality in pigs, and hemorrhagic, necrotizing pneumonia and fibrosing pleurisy It is a major feature of the disease. However, chronic infections are economically disastrous to the pig farming industry due to reduced growth rate, low feed efficiency and delayed shipment. Actinobacillus furopneumoniae can be used for a variety of pathogenic factors such as endotoxins, lipopolysaccharide (LPS), transferrin-binding proteins, outer membrane proteins (OMPs, outer membrane proteins, and proteases. Of these, Apx I, Apx II and Apx III belonging to the RTX (repeats in toxins) toxin group are the most important pathogenic factors.

Inactivated inactivated vaccine vaccine used to prevent livestock disease does not induce the immune response to the specific antigen. In the case of the purified intestinal vaccine, induction and purification process is troublesome. In the case of recombinant subunit purified vaccine, And a rigorous manufacturing process that requires increased immunity. In addition, it should be kept stable to prevent degeneration and to reduce the side effects of vaccines. In addition to the inconvenience that each individual must be inoculated with a syringe, when an antigen is administered using a syringe, The mucosal immunity can not be strongly activated and there is a problem that it is difficult to maintain immunogenicity due to denaturation in a pH condition or the like in a digestive tract even when a purified vaccine is administered to the oral cavity.

In order to solve such a problem, development of animal disease prevention vaccine using live virus vaccine can be introduced. Microorganisms used for live vaccine development are animal pathogens, which attach to lymphatic tissue or mucosa-associated lymphoid tissue, Should be able to. Compared with the use of a syringe, a live bacterial vaccine can effectively induce an immune response in a mucosal site such as oral or nasal cavity, thereby effectively preventing infection of a pathogen. In order to develop Salmonella as a multivalent in the use of a live bacterial vaccine, a method of weakening the pathogenicity of Salmonella itself, a method of preparing a secretion system capable of expressing an external antigen and secreting it to the outside of the cell, And approaches to increase penetration have been studied. For the expression of foreign antigens and selection of strains, a system based on the asd gene was developed. The aspartate β-semialdehyde dehydrogenase ( asd ) gene necessary for the synthesis of the peptidoglycan of the attenuated Salmonella strain was deleted and the DAP (diaminopimelic acid) And then transformed with an asd plasmid having an external antigen gene to express an external antigen, so that the strain can be selected without antibiotics. However, in order to sufficiently induce the immune response to the expressed antigen, the extracellular secretory ability should be further improved so that it can be secreted in a large amount.

Korean Patent No. 1164045 discloses a 'live vaccine for weakening toxicity against pig pleural pneumonia', Korean Patent Publication No. 2012-0054037 discloses 'a vaccine derived from pig pleural pneumonia and a method for obtaining such a vaccine' However, there is no description of a vaccine composition for the prevention or treatment of swine pneumonia that includes the attenuated salmonella mutant of the present invention.

The present invention has been made in view of the above-mentioned needs, and the present inventors have found that Salmonella which is attenuated by gene deletion is a transgenic Salmonella which expresses a major pathogenic factor of Actinobacillus furopneumoniae, As a result of inoculating the mice with the live mutant strains prepared as the live bacterial vaccine, the live bacterial vaccine excellently induces the immune response of the animal, and the infection of Actinobacillus furopneumoniae was effectively prevented The present invention has been completed.

In order to solve the above problems, the present invention provides a mutant strains of Salmonella strains in which lon, cpxR and asd genes have been deleted. The present invention provides a mutant strains of Salmonella strains isolated from the strains of Ap, Wherein the Salmonella mutant strains further comprise one or more of the factors selected from the group consisting of:

The present invention also provides a Salmonella mutant mixture comprising any one or more of the mutants.

In addition, the present invention provides a vaccine composition for preventing or treating pleural pneumonia of swine comprising the Salmonella mutant mixture as an active ingredient.

The present invention also relates to a method for the treatment of pigs expressing any one of genes selected from the group consisting of Apx Ⅰ A, Apx Ⅱ A, Apx Ⅲ A, OMPA, OmlA and ApfA, which are major pathogenic factors of porcine pleural fluid pneumococci using attenuated salmonella A method for producing Salmonella mutant strains for the prevention or treatment of pleuropneumonia.

In addition, the present invention provides a vaccination method for prevention or treatment of swine pneumonia of swine characterized by inoculating said vaccine composition into sows or piglets.

In addition, the present invention provides a feed additive for immunizing a porcine comprising the above Salmonella mutant mixture.

The Salmonella mutant strain of the present invention secretes the major antigen to the pigs' pleural pneumonia to the outside of the cell and effectively activates the cellular and humoral immune responses of the inoculated animal, thus being useful as a vaccine for preventing swine pneumonia. In addition, vaccine production and mass inoculation are possible, and the inoculation cost is reduced, and production and storage are economical.

FIG. 1 shows the result of Western blotting that Salmonella mutants of the present invention express antigens (Apx I A, Apx II A, Apx II A and OMPA) of pig pleural fluid pneumonia.
FIG. 2 shows the results of ELISA analysis of the serum IgG antibody titers of each Salmonella mutant expressing the protein antigen of the present invention to each antigen induced after oral and nasal inoculation. Intranasal immunization: nasal inoculation, oral immunization: oral vaccination.
FIG. 3 shows the results of ELISA analysis of the serum IgG antibody titer of each antigen after inoculation into the nasal cavity after mixing the Salmonella mutant expressing the protein antigen of the present invention.
FIG. 4 is a graph showing the mucosal IgA antibody titer for each antigen in the sample after fecal sample and vaginal washing were collected after the salmonella mutant expressing the protein antigen of the present invention was mixed and inoculated into the nasal cavity The results were analyzed by ELISA.
FIG. 5 shows SP (splenocyte proliferation assay) results of salmonella mutant antigen by inoculating salmonella mutant expressing the Apx IIA or OMPA antigen of the present invention into a nasal cavity, extracting spleen and extracting spleen cells.
FIG. 6 shows the results of splenocyte analysis for each antigen of Salmonella mutant strain of the present invention.
FIG. 7 shows the results of confirming gene expression levels of IFN-y and IL-4 using real-time PCR from spleen cells stimulated with Salmonella mutant antigens of the present invention.
FIG. 8 shows the results of comparative analysis of the serum IgG antibody titers according to the number of vaccinations after inoculation with a mouse in order to determine the optimal number of vaccination times for the vaccine composition in which the Salmonella mutant expressing the protein antigen of the present invention was mixed . A, 1 dose of vaccine; B, twice vaccination; C, 3 doses of vaccine; D, control group.
FIG. 9 shows the results of analysis of the IgA antibody titer of each antigen in the feces after inoculating mice with a vaccine composition prepared by mixing salmonella mutants expressing the protein antigen of the present invention. A, 1 dose of vaccine; B, twice vaccination; C, 3 doses of vaccine; D, control group.
FIG. 10 is a graph showing changes in feed intake for 21 days after inoculating mice with different numbers of vaccine compositions prepared by mixing salmonella mutants expressing the protein antigen of the present invention. Group A, 1 dose of vaccine; Group B, 2 doses of vaccine; Group C, 3 doses of vaccine.
11 is a graph showing the mouse weights of each group according to the week after inoculating the mice with the vaccine composition prepared by mixing Salmonella mutants expressing the protein antigen of the present invention at different times. Group A, 1 dose of vaccine; Group B, 2 doses of vaccine; Group C, 3 doses of vaccine.
12 is a graph showing the mucosal IgA antibody titer for each antigen in a lung lavage solution after inoculation of a vaccine composition prepared by mixing a salmonella mutant expressing the protein antigen of the present invention in a mouse. Group A, 1 dose of vaccine; Group B, 2 doses of vaccine; Control, control group.

In order to accomplish the object of the present invention, the present invention provides a mutant strains of Salmonella strains in which lon , cpxR and asd genes have been deleted, Apx Ⅰ A, Apx Ⅱ A, Apx Ⅲ A, OMPA, OmlA, Lt; RTI ID = 0.0 > Salmonella < / RTI > mutant strain further comprising one or more factors selected from the group consisting of ApfA.

Salmonella mutants lacking the lon, cpxR, and asd genes in the Salmonella mutant strains according to one embodiment of the present invention were produced so as to be able to select an antigen recombinant strain without an antibiotic as a DAP requiring strain in which the asd gene was deleted , cpxR is deleted, thereby enhancing lymphocyte infiltration ability , increasing immunogenicity, lon gene deletion, and attenuating pathogenicity.

In the Salmonella mutant strains of the present invention, the causative organism of swine pleuropneumonia may be Actinobacillus pleuropneumoniae , but is not limited thereto.

In a Salmonella mutant strain according to another embodiment of the present invention, the major pathogenic factor of the causative agent of swine pleuropneumonia is Apx toxin I-IV (repetitive in RTX toxin), outer membrane protein (OMP), LPS, Rye, and the like, preferably, but not limited to, one or more factors selected from the group consisting of Apx I A, Apx II A, Apx II A, OMPA, OmlA and ApfA. In addition, the major pathogenic factor was recombined into a plasmid vector in which lepB (signal peptidase), secA (ATPase) and secB (chaperone) genes were expressed and the extracellular secretory efficiency of the recombinant antigen was improved. The recombinant plasmid vector was transformed into the above-described attenuated salmonella.

Further, in the 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 Salmonella gallinarium or Salmonella enteritidis , and the like, preferably, but not limited to, Salmonella typhimurium .

The present invention also provides a Salmonella mutant mixture comprising any one or more of the mutants.

The salmonella mutant mixture of the present invention is characterized in that the salmonella in which the lon, cpxR and asd genes have been deleted is selected from the group consisting of Apx Ⅰ A, Apx Ⅱ A, Apx Ⅲ A, OMPA, OmlA and ApfA It is a mixture containing at least one mutant strain of Salmonella that expresses and ex vivo exerts a pathogenic factor.

In addition, the present invention provides a vaccine composition for preventing or treating pleural pneumonia of swine comprising the Salmonella mutant mixture as an active ingredient.

The vaccine composition of the present invention comprises a mixture containing Salmonella mutant strains expressing the major pathogenic factors Apx Ⅰ A, Apx Ⅱ A, Apx Ⅲ A, OMPA, OmlA or ApfA of pig pleural fluid pneumonia in salmonella bacteria attenuated by gene deletion As an effective ingredient, the vaccine composition can be treated in an animal to prevent or treat swine pleurisy pneumonia.

In the vaccine composition according to an embodiment of the present invention, the Salmonella mutant strain mixture may be prepared in the form of a mutant strain or a dead organism, preferably in the form of a mutant strain, but is not limited thereto.

The term "vaccine " in the present invention refers to a biological agent containing an antigen that immunizes a living body. The term " vaccine " refers to an immunogenic or antigenic substance that causes immunization to a living body by injection or oral administration to a human or animal for the prevention of infection. In vivo immunity is largely divided into autoimmunity, which is obtained automatically in vivo after infection with pathogenic bacteria, and passive immunization, which is obtained by externally injected vaccine. While autoimmunity has a long period of immune-related antibody production and is characterized by persistent immunity, passive immunization by vaccine acts immediately for the treatment of infectious diseases but has a disadvantage that its persistence is poor.

The vaccine composition may contain one or more of a stabilizer, an emulsifier, aluminum hydroxide, an aluminum phosphate, a pH adjuster, a surfactant, a liposome, an iscom adjuvant, a synthetic glycopeptide, an extender, a carboxy polymethylene, a subviral particle adjuvant, , N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, dimethyl dioctadecyl- ammonium bromide, The second auxiliary agent may be further contained.

In addition, the vaccine composition may comprise a veterinarily acceptable carrier. The term "veterinarily acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, Examples of the carrier, excipient and diluent which can be contained in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, each of the above-mentioned compositions for the vaccine may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, and nasal formulations such as drips or sprays, And the like. In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. As the liquid preparation for oral administration, suspensions, solutions, emulsions, syrups and the like may be used. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, and freeze-drying agents. Examples of the suspending agent include propylene glycol, polyethyleneglycol, vegetable oil such as olive oil, injectable ester such as ethylolate, and the like, but the present invention is not limited thereto. Penetrants suitable for formulation for intranasal administration are generally known to those skilled in the art. Such suitable formulations are preferably formulated to be sterile, emollient and buffered for stability and compliance. Formulations for intranasal administration are also prepared to stimulate mucus secretion in several ways to maintain normal ciliary action and are described in Remington's Pharmaceutical Science, 18th Ed., Mack Publishing Co., Easton, PA (1990) As noted, suitable formulations are preferably isotonic, slightly buffered formulations that maintain a pH of 5.5 to 6.5, and most preferably comprise an antimicrobial preservative and a suitable drug stabilizing agent.

In addition,

(a) amplifying any gene selected from the group consisting of Apx IA, Apx IIA, Apx IIIA, OMPA, OmlA and ApfA, which are major pathogenic factors of the causative organism of swine flu pneumonia;

(b) cloning the amplified gene of step (a) into a plasmid having lepB, secA and secB genes;

(c) deleting the lon, cpxR and asd genes from the chromosomal DNA of Salmonella strains to produce attenuated Salmonella strains;

(d) transforming the cloned plasmid of step (b) into the Enterobacteriaceae Salmonella strain of step (c); And

(e) selecting the transformed Salmonella mutant strains of step (d). The present invention also provides a method for producing Salmonella mutant strains for the prevention or treatment of swine pneumonia in pigs.

In the method according to an embodiment of the present invention, the porcine pleurisy causing pneumonia may be Actinobacillus pleuropneumoniae , but is not limited thereto.

Further, in the method according to one embodiment, the Salmonella is Salmonella typhimurium (Salmonella typhimurium), Salmonella Thai Phi (Salmonella typi), Salmonella para Thai Phi (Salmonella paratyphi , Salmonella sendai , Salmonella gallinarium or Salmonella enteritidis , and the like, preferably Salmonella typhimurium , but is not limited thereto.

In addition, the present invention provides a vaccination method for prevention or treatment of swine pneumonia of swine characterized by inoculating said vaccine composition into sows or piglets.

As described above, the vaccine composition of the present invention is a Salmonella mutant strain expressing the major pathogenic factors Apx ⅠA, Apx ⅡA, Apx ⅢA, OMPA, OmlA or ApfA of pig pleural fluid pneumonia in salmonella bacteria attenuated by gene deletion Or more as an active ingredient and may be prepared in the form of living cells of the Salmonella mutant strain, but the present invention is not limited thereto.

In the vaccination method according to an embodiment of the present invention, the inoculation of the vaccine composition is administered by oral, nasal, percutaneous, intramuscular, intraperitoneal, intravenous or subcutaneous routes with primary, secondary and tertiary administration And can be inoculated in primary, secondary and tertiary administrations, preferably orally or intranasally, and most preferably, intranasal administration, but not limited thereto.

In addition, the present invention provides a feed additive for immunizing a porcine comprising the above Salmonella mutant mixture.

The feed additive of the present invention includes a Salmonella mutant strain which expresses and secretes major pathogenic factors of the causative organism of swine pneumoniae pneumoniae as an active ingredient and is useful for preventing the infectious agent causative bacteria from being harmful to the organism, Since it induces the humoral immune response effectively, it can contribute to the immune enhancement of pigs when used as a feed additive.

The feed additive of the present invention may be prepared by using the Salmonella mutant strain mixture as it is or adding a known carrier such as cereal grains and its by-products, stabilizer and the like which are allowed to be added to livestock and, if necessary, adding citric acid, fumaric acid, Phosphate such as lactic acid and malic acid, organic acid such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polymerized phosphate), polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan Natural antioxidants such as tannic acid and phytic acid, antibiotics, antimicrobial agents and other additives may be added, and the form thereof may be a proper state such as powder, granule, pellet, suspension, etc. In the case of feeding the feed additive Livestock, etc. can be supplied alone or mixed with feed.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Preparation of protein antigen

Commercially available expression vectors (pQE31 and pET28a) and host cells ( E. coli (Apx ⅠA, Apx ⅡA, Apx ⅢA and OMPA) were inserted into the expression vector using DH5α and E. coli BL21, respectively, and transformed into the corresponding host cell to obtain a protein antigen expression strain. The purified PCR amplification products and the pQE31 and pET28a vectors were digested with restriction enzymes and then electrophoresed on agarose gels. Each slice was cut with AccuPrep ^? gel purification kit were purified using a (Bioneer, Korea), and then T4 DNA ligase (Takara, Japan) then ligated with the two products E. coli DH5? Or E. coli BL21. These transformants were spread evenly on LB agar plates supplemented with ampicillin (when pQE31 was used as an expression vector) or kanamycin (when pET28a was used as an expression vector), and then cultured overnight at 37 DEG C to show antibiotic resistance The transformed E. coli DH5? Or E. coli BL21 were selected. The vector in which each protein antigen gene is inserted is E. coli DH5 ^? Or E. coli BL21 from AccuPrep ^? The plasmid containing each gene was extracted using a plasmid extraction kit (Bioneer, Korea), digested with restriction enzymes and confirmed by electrophoresis on agarose gel. The thus identified colonies were inoculated into 5 ml of LB liquid medium supplemented with antibiotics for each expression vector and cultured overnight at 37 ° C. 1 ml of the cultured mycobacteria was inoculated into 200 ml of LB liquid medium and cultured overnight at 30 ° C with shaking at a rate of 150 rpm. The final concentration of IPTG was added to this culture to a concentration of 1 mM, and the cells were cultured for 4 hours under the same conditions.

The culture was centrifuged at 8,000 rpm at 4 DEG C for 15 minutes. The supernatant was discarded and the precipitate was resuspended in 10 ml of sterile PBS added to give 1 mM of PMSF and frozen at -70 ° C. The frozen suspension was thawed again in a 37 ° C water bath and then frozen at -70 ° C for 2 or 3 times. After thawing, the supernatant was sonicated, and the cells were pulverized. Then, the supernatant was transferred to a sterilization tube by centrifugation at 15,000 rpm for 20 minutes at 4 ° C. When the expressed protein is soluble, ₄ ml of buffer B (100 mM NaH ₂ PO ₄ , 10 mM Tris-Cl, 8 M Urea, pH 8.0) is mixed with the supernatant and resuspended with 6 ml of buffer B if insoluble Followed by stirring at room temperature for 1 hour. The reaction solution was centrifuged at 15,000 rpm at 4 ° C for 20 minutes. The supernatant was transferred to a tube containing resin and stirred for 30 minutes so that the protein bound to the resin. The reaction solution was charged into a prepared column and slowly flowed. The column was recharged with the liquid that was poured out and then flowed slowly again. From the reaction liquid, and then flow to the column with buffer _{C (100mM NaH 2 PO 4,} 10mM Tris-Cl, 8M Urea, pH 6.3) 6㎖ washed three times to remove any other unnecessary proteins are not combined with the resin. An elution buffer _{(100mM NaH 2 PO 4, 10mM} Tris-Cl, 8M Urea, pH 4.5) to a protein by 20 minutes of reaction after charging the column with 2㎖ separating the expressed protein from the resin, and then by the addition of elution buffer 1㎖ Respectively. The recovered proteins were electrophoresed on SDS-PAGE, and the expressed proteins were identified. The proteins were stored at -70 ° C and analyzed by enzyme-linked immunosorbent assay (ELISA), sphenocyte proliferation assay (SPA), flow cytometry (FACS) And used as an antigen for analysis.

2. Preparation of attenuated Salmonella vaccine strain

Antigen of interest (Apx ⅠA, Apx ⅡA, Apx ⅢA and OMPA) for each gene, and then cloned into the vector in pBP244 improved secretion of proteins expressed, pYA232 E. coli 6212 strains that have (a lac I ^q insert) ≪ / RTI > Vectors were extracted from this E. coli strain and transformed into attenuated Salmonella strains (JOL912) by electroporation, respectively, to prepare vaccine candidate strains. The characteristics of the strains and plasmids used in the experiments are shown in Table 1 below.

The strain and plasmid information used in the present invention Strain / plasmid Explanation source Strain  Escherichia coli   DH5α F 'Φ80 lac ZΔM15 f (lac ZYA- arg F) U169 deoR rec A1 end A1 hsdR 17 (rk -, mk +) phoA supE44 thi-1 gyr A96 relA1 Laboratory holdings   BL21 (DE3) pLysS F ^- , omp T, hsd S _B (r _B ^- , m _B ^- ), dcm, gal , lambda (DE3), pLysS, Cm ^r Laboratory holdings   JOL1076 BL21 with pET28a-Apx IA gene   JOL1000 BL21 with pET28a-Apx IIA gene   JOL1007 DH5? With pQE31-Apx IIA gene   JOL997 BL21 with pET28a-OMPA gene   HJL192 BL21 with pET28a-Omla gene   HJL135 BL21 with pET28a-ApfA gene   χ 6212 F ^- λ ^- φ80 Δ ( lac ZYA- arg F) end A1 rec A1 had R17 deo R thi -1 gln V44 gyr A96 rel A1 Δasd A4 Samonella typhimurium   JOL401 S. typhimurium wild type Laboratory holdings   JOL912 S. typhimurium JOL401 derivative ? Lon ? CpxR? Asd Laboratory holdings   JOL1300 JOL912 with pBP244-Apx IA   JOL1301 JOL912 with pBP244-Apx IIA   JOL1302 JOL912 with pBP244-Apx IIIA   JOL1303 JOL912 with pBP244-OMPA   JOL1304 JOL912 with pBP244-Omla   HJL72 JOL912 with pBP244-ApfA   JOL1075 JOL912 with pBP244 Actinobacillus pleuropneumoniae   JOL981 Actinobacillus pleuropneumoniae serotype 2 Laboratory holdings   JOL982 Actinobacillus pleuropneumoniae serotype 5 Laboratory holdings Plasmid  pQE31 IPTG-inducible expression vector; Am ^r Qiagen  pET28a IPTG-inducible expression vector; Km ^r Novagen  pBP244 pYA3493 derivative containing lepB , secA and secB genes Laboratory holdings  pMMP65 Asd + vector, pBR ori, β-lactamase signal sequence-based periplasmic secretion plasmid , 6 × His Laboratory holdings

3. Confirmation of expression of target antigen by Western blotting

Each prepared vaccine candidate strain is cultured overnight in LB liquid medium and centrifuged to prepare supernatant and pellet samples. The supernatant was suspended in an SDS-PAGE sample buffer with 10% TCA (trichloroacetic acid) overnight, boiled at 94 ° C for 5 minutes, and then subjected to SDS-PAGE, transferred to PVDF membrane and blocked with blocking buffer (1% bovine serum albumin in PBST) overnight, then diluted with 1: 500 rabbit polyclonal antibody to each antigen and allowed to react for 1 hour. The secondary antibody (goat anti-rabbit IgG (H + L) HRP) was diluted to 1: 40,000 and allowed to react for 1 hour, followed by color development to confirm expression of the target antigen protein.

Four strains, JOL1300, JOL1301, JOL1302 and JOL1303, whose expression of the target antigen was confirmed by Western blotting, were used as vaccine candidate strains.

[Mouse experiment]

4. Preparation of experimental animals

Five-week-old BALB / c mice were purchased (Koatech, Korea) and used for the experiment after one week of adaptation period.

5. Live vaccine preparation and vaccination

5-1. Preparation of live vaccine for oral vaccination

The attenuated Salmonella, which has confirmed the expression of each antigen protein, is inoculated into LB liquid medium and incubated overnight at 37 ° C. The mixture is then added at 1:20 magnification to a new LB liquid medium to obtain an optical density OD ₆₀₀ of 0.8 Lt; / RTI > The cultured strains were centrifuged at 4,000 rpm for 20 minutes. The precipitated strains were resuspended in sterile PBS and centrifuged again under the same conditions. The precipitated strains were resuspended so that the strains expressing each antigen in PBS (PBS-sucrose) added to 20% sucrose were 2.0x10 ⁹ CFU (colony-forming units) / ml. The experimental animals were inoculated into the oral cavity on the day when the live cells were prepared.

5-2. Preparation of live vaccine for intranasal inoculation

In order to prepare the live vaccine as in the case of oral vaccination, the attenuated Salmonella strain, in which the expression of each antigen protein was confirmed, was inoculated into LB liquid medium and cultured at 37 ° C for 16 hours at a speed of 200 rpm. LB liquid medium and incubated for 4 hours until the OD ₆₀₀ value reached 0.8 under the same conditions. The culture was centrifuged at 4,000 rpm for 20 minutes at 4 ° C, washed three times with sterile PBS, and suspended again in sterile PBS to a total bacterial count of 1.0 × 10 ⁵ CFU / ml. .

5-3. inoculation

Each mouse in the experimental group was fasted overnight and then adjusted to 2 × 10 ⁹ CFU (in PBS-sucrose) per 20 μl per mouse to be inoculated or adjusted to 10 × 10 ⁵ CFU (in PBS) And the control group was inoculated into the oral or nasal cavity with sterile PBS, respectively, and fasted for an additional 30 minutes.

6. Preparation and inoculation of outdoor strain for challenge infection

6-1. Culture of the strain

One colony of Actinobacillus pleuropneumoniae ( hereinafter AP) type 2 and 5 was inoculated into LB liquid medium and cultured overnight at 37 ° C with shaking at 200 rpm.

Each colony of AP type 2 and 5 was inoculated on chocolate agar and incubated at 37 ° C for 16 hours. The cells were suspended in sterile PBS to a concentration of 5 × 10 ⁷ CFU / 20 μl, (Outdoors).

6-2. inoculation

Each mouse was fasted overnight and then inoculated intranasally with 5 × 10 ⁷ CFU to 20 μL per mouse.

7. Inoculation route determination experiment

After immunization with Salmonella typhimurium mutant strain in which each antigen gene was inserted orally or intranasally, immune response and related cytokine induction was confirmed. And the appropriate inoculation route was determined.

7-1. Oral and intranasal inoculation of live vaccine

Each mouse was fasted overnight, adjusted to 2 × 10 ⁹ CFU per mouse, and inoculated into the nasal cavity by inoculation or 10 μl to 1 × 10 ⁵ CFU. The control group was inoculated with sterile PBS-sucrose and PBS Oral inoculation or intranasal inoculation followed by an additional 30-minute fasting (Table 2 and Table 3). (Table 4). In addition, the mice were inoculated one more time on the 21st day after inoculation with the same method.

Oral Inoculation Experiment Plan group Head Strain Antigen name Inoculation number Challenge infection One 15


Samonella typhimurium Apx IA First - 2 × ^{10 9} CFU / 20 μl
Second-order -2 × ^{10 9} CFU / 20 μl
AP type 2 and AP type 5 were mixed in equal amounts to induce a challenge infection 2 15 Apx IIA First - 2 × ^{10 9} CFU / 20 μl
Second-order -2 × ^{10 9} CFU / 20 μl 3 15 Apx IIA First - 2 × ^{10 9} CFU / 20 μl
Second-order -2 × ^{10 9} CFU / 20 μl 4 15 OMPA First - 2 × ^{10 9} CFU / 20 μl
Second-order -2 × ^{10 9} CFU / 20 μl 5 15 Control Sterile PBS-sucrose

Nasal Inoculation Experiment Plan group Head Strain Antigen name Inoculation number Challenge infection One 10


Samonella typhimurium Apx IA 1 > -10 < ⁵ > CFU /
Second order -1x10 ⁵ CFU / 10 μl
AP type 2 and AP type 5 were mixed in equal amounts to induce a challenge infection 2 10 Apx IIA 1 > -10 < ⁵ > CFU /
Second order -1x10 ⁵ CFU / 10 μl 3 10 Apx IIA 1 > -10 < ⁵ > CFU /
Second order -1x10 ⁵ CFU / 10 μl 4 10 OMPA 1 > -10 < ⁵ > CFU /
Second order -1x10 ⁵ CFU / 10 μl 5 10 Control Sterile PBS

Outline of experiment 0 weeks 3 weeks 6 weeks 9 weeks 10 weeks First inoculation Second inoculation Challenge infection Autopsy Blood before inoculation Blood before inoculation Three weeks after 2nd vaccination See Table 3 and Table 4 Mouse subjects surviving challenge infection Sampling Sampling Sampling Sampling Separation of bacteria (lung)

7-2. Sampling

Before the first inoculation, before the second inoculation, and before the challenge infection, the blood was collected separately and the serum was separated and stored at -80 ° C.

7-3. Antibody titration through ELISA

Serum samples taken before inoculation, 3 weeks after the first inoculation and 3 and 6 weeks after the second inoculation were used to determine the activity of antibodies formed against each antigen after oral and nasal inoculation in mice. IgG Were measured by ELISA

7-4. Challenge infection

Each mouse was fasted overnight and then inoculated into the nasal cavity by adjusting the outdoors to be 5 × 10 ⁷ CFU per 20 μl / mouse.

8. Immunization of each antigen after vaccination with vaccine strain expressing protein antigen and defense against challenge infection

After the inoculation of the attenuated Salmonella typhimurium vaccine strain into which each of the corresponding antigen genes is inserted, the vaccine is inoculated into the nasal cavity, and the possibility of vaccination is determined by whether or not the immune response to each individual antigen is induced, Respectively.

8-1. Experimental animals and inoculation

1) Experimental animals: Five week old BALB / c mice were purchased and used for the experiment after 1 week of adaptation period.

2) Inoculation: Each mouse in the experiment group was inoculated intranasally by adjusting the total number of bacteria to 1 × 10 ⁵ CFU per 10 μl / mouse after overnight fasting, and the control group was inoculated intranasally with sterilized PBS (Table 5 and Table 6) .

 Mixed vaccine strain nasal inoculation test plan group Head Antigen name Inoculation number Route Challenge infection A 30 Apx IA, Apx IIA, Apx IIA, OMPA 1 x 10 ⁵ CFU / 10 μl sterile PBS Intranasal inoculation AP Type 2 AP type 5 B 30 Sterile PBS Intranasal inoculation AP Type 2 AP type 5

[※ 5μl inoculated on one side of the nostril.

※ PBS was prepared so that the number of each antigen-expressing vaccine strain was 1 × 10 ⁵ CFU in 10 μl of PBS, and 2.5 μl of each strain was inoculated on the day of manufacture.

Summary of Nasal Inoculation Experiment of Mixed Vaccine Strain 0 weeks 2 weeks 4 weeks 4 weeks 6 weeks 8 weeks 9 weeks First inoculation Primary autopsy Challenge infection Second autopsy Blood before inoculation IgG, sIgA IgG, sIgA LPA, FACs, cytokine measurement IgG, sIgA IgG, sIgA Mouse subjects surviving challenge infection Sampling Sick
Extraction Sick
Extraction Sampling for CD3, CD4, CD8, B cell measurements
(5 per group) Sick
Extraction Sick
Extraction Separation of bacteria (lung) Splenocyte separation

8-2. Sampling

1) Fecal sampling: At 2 weeks, 4 weeks, 6 weeks, and 8 weeks after the first inoculation, the feces were collected and weighed, and 100 mg / ml of PBS containing 0.1% sodium azide And then stored at -80 ° C.

2) Sampling of vaginal lavage: 100 μl of sterile PBS (pH 7.2) was added to the vagina at the same time as the above-mentioned fecal sampling, and the vial was collected using a micropipette and stored at -80 ° C.

3) Blood collection: Blood samples were collected at the same time as the above fecal and vaginal washings, and the serum was separated and stored at -80 ° C.

Splenocyte isolation (for SLP): Spleens were aseptically collected at 4 weeks after inoculation of 5 mice in each group and collected in RPMI 1640. The red blood cells were dissolved in 0.8% ammonium chloride (w / v), centrifuged at 380 xg at 4 ° C for 10 minutes, and the precipitate was washed 3 times with sterile PBS. After the final centrifugation, the cells were resuspended in complete medium (RPMI 1640 supplemented with 100 IU / ml penicillin, 100 μg / ml streptomycine and 10% FCS). The number of cells was calculated, and the cells were divided into 5 × 10 ⁶ cells / 100 μl. Each antigen was diluted to a concentration of 10 to 15 μg / ml in 100 μl, and the mixture was reacted at 37 ° C. and 5% CO _{2 for} 48 hours. Plus Kit (LONZA, Switzerland) was used to measure splenocyte cell proliferation.

5) Splenocyte separation (for FACS and ELISA) : 5 mice per group were aseptically collected at 4 weeks after the first inoculation and collected in RPMI 1640. The red blood cells were dissolved in 0.8% ammonium chloride (w / v), centrifuged at 380 xg at 4 ° C for 10 minutes, and the precipitate was washed 3 times with sterile PBS. After the final centrifugation, the cells were resuspended in complete medium. The number of cells was counted and reacted with each antigen for 12 hours, and CD3, CD4, CD8 and B cells were measured. The cells were counted and reacted with each antigen for 48 hours. The cells were centrifuged at 380 xg for 4 minutes at 4 ° C. The resulting pellet was used to isolate mRNA from the cells, and cDNA was prepared and analyzed by real-time PCR.

8-3. Antibody titer measurement

1) IgG: To measure the activity of the antibody formed against each antigen after inoculating the vaccine strain into the nasal cavity of the mouse, the serum collected at 2 weeks, 4 weeks, 6 weeks, and 8 weeks after the inoculation and after the first inoculation Were assayed for IgG titers by ELISA.

2) sIgA: To confirm the mucosal immune response during the mucosal and systemic immune response after vaccination with the vaccine strain in mice, the mice were injected 2 weeks, 4 weeks, 6 weeks after the inoculation and after the first inoculation At week 8, sIgA titers were measured by ELISA for fecal and vaginal washes.

8-4. Spenocyte proliferation assay

Splenocytes that responded to each antigen for 48 hours at 37 ° C and 5% CO ₂ were measured using a ViaLight Plus Kit (LONZA, Switzerland) (Matsuda et al., 2010, Veterinary Research , 41:51; Qu et al , ≪ / RTI > 2008, Vaccine , 26: 4541-4548).

8-5. Flow cytometry

To observe immune cells closely related to immunization induction after vaccination, 5 mice were sacrificed at 4 weeks after the first inoculation and aseptically spleen was excised and spleen cells were separated and used in the experiment.

8-6. Cytokine IL-4, IL-6 and IFN-gamma measurement

At 4 weeks after vaccination, the level of gene expression of each cytokine was measured by real-time PCR on mRNA isolated from cell pellet obtained by aspirating each corresponding antigen into sterile isolated spleen cells and centrifuging.

8-7. Challenge infection

Each mouse was fasted overnight and then inoculated into the nasal cavity by adjusting the amount of 5x10 ⁷ CFU to 20 μl of the outdoors per bird.

9. Optimization experiment with booster

After the combination of each antigen-expressing vaccine strain, each vaccine is administered to the nasal cavity once, twice, and three times, and the number of inoculations is determined through the defense against the induced immune response and the challenge infection.

9-1. vaccination

6-week-old mice were mixed with a strain expressing each antigen and then inoculated into the nasal cavity once, twice, and three times. In detail, each mouse in the experiment group was inoculated into the nasal cavity by adjusting the total number of bacteria to 1 × 10 ⁵ CFU per 10 μl / mouse after overnight fasting, and the control group was inoculated with sterilized PBS (Table 7 and Table 8).

Optimized experiment planning with booster group Head Antigen name Inoculation number Challenge infection Primary Secondary Third A 15 Apx IA, Apx IIA,
Apx IIA, OMPA ¹ × 10 ⁵ CFU / 10 μl AP type 5 B 15 Apx IA, Apx IIA,
Apx IIA, OMPA ¹ × 10 ⁵ CFU / 10 μl ¹ × 10 ⁵ CFU / 10 μl AP type 5 C 15 Apx IA, Apx IIA,
Apx IIA, OMPA ¹ × 10 ⁵ CFU / 10 μl ¹ × 10 ⁵ CFU / 10 μl ¹ × 10 ⁵ CFU / 10 μl AP type 5 D 15 Sterile PBS - - - AP type 5

[※ 5μl inoculated on one side of the nostril.

※ Prepare 10 μl of PBS so that the number of each antigen-expressing vaccine strain becomes 1 × 10 ⁵ CFU. 2.5 μl of each strain is mixed and inoculated on the day of production.]

Overview of optimization experiment with booster 0 weeks 2 weeks 3 weeks 4 weeks 6 weeks 7 weeks 8, 10, 12, 14 weeks 10 weeks 16-17 weeks First inoculation Second inoculation Primary autopsy 3 doses Second autopsy Third autopsy Challenge infection Blood before inoculation After 2 weeks of inoculation SLA, real-time PCR, FACS, 4 weeks after inoculation After 6 weeks of inoculation SLA, real time PCR, FACS Blood drawing SLA, real time PCR, FACS Collecting pre-challenge blood and collecting blood Sampling Sampling Spleen, sperm sampling Sampling Splenectomy Sampling Splenectomy Lung extraction Splenocyte separation Splenocyte separation Splenocyte separation Mortality and clinical findings

9-2. Sampling

1) Fecal sampling: Feces were collected and weighed at 2 weeks, 4 weeks, 6 weeks, 8 weeks, and 10 weeks after inoculation and after the first inoculation, and 100 mg / ml of PBS containing 0.1% sodium azide And then stored at -80 ° C.

2) Blood collection: Blood samples were collected at the same time as above, and the serum was separated and stored at -80 ° C.

3) Splenocyte Separation: Five mice in each group were aseptically collected at 4 weeks after the last inoculation and collected in RPMI 1640. The red blood cells were dissolved in 0.8% ammonium chloride (w / v), centrifuged at 380 xg at 4 ° C for 10 minutes, and the precipitate was washed 3 times with sterile PBS. After the final centrifugation, the cells were resuspended in complete medium. After counting, the cells were reacted with each antigen for 72 hours, centrifuged at 380 × g, 4 ° C. for 10 minutes, and the supernatant was stored at -80 ° C. and cytokines secreted from the splenocytes were measured by ELISA. After resuspension of the pellet, mRNA was isolated using a kit, cDNA was prepared, and the gene expression levels of IL-4 and IFN-γ were measured by real-time PCR.

9-3. Antibody titers and cytokine measurements

Serum, feces, and vaginal secretions were measured by ELISA for IgG and IgA antibodies, and after 4 weeks of vaccination, aseptically separated splenocytes were stimulated with respective antigens and centrifuged to separate them from the pellet The level of gene expression of IL-4 and IFN-γ was measured by real-time PCR on mRNA.

9-4. Challenge infection

Each mouse was fasted overnight and then inoculated into the nasal cavity by adjusting the amount of 5x10 ⁷ CFU to 20 μl of the outdoors per bird.

10. Safety and IgA measurement of the mucosa in the lungs

(Diarrhea, depression, weight loss) and vaccine strains in the substantive organs (liver, spleen, lung, intestines) after vaccination with the respective antigen-expressing vaccine strains And to confirm the stability of the vaccine.

10-1. vaccination

6-week-old mice were inoculated with ¹ × 10 ⁵ CFU / 10 μl (in PBS) of each strain expressing each antigen once and twice in the nasal cavity (Table 9).

Experiment plan group Head Inoculation number Autopsy Primary Secondary A 12 1 x 10 < ⁵ > CFU / 10 [mu] l ^a 1 day, 1 week, 2 weeks, 3 weeks after vaccination B 12 ¹ × 10 ⁵ CFU / 10 μl ¹ × 10 ⁵ CFU / 10 μl 1 day, 1 week, 2 weeks, 3 weeks after vaccination C 12 Sterile PBS Sterile PBS 1 day, 1 week, 2 weeks, 3 weeks after vaccination

[※ 5μl inoculated on one side of the nostril.

※ ^a Prepare the vaccine strain so that the number of each antigen-expressing vaccine strain is 1 × 10 ⁵ CFU in 10 μl of PBS, and 2.5 μl of each strain is inoculated on the day of manufacture.

10-2. Clinical symptom observation

1) After vaccination, clinical symptoms such as diarrhea, fever and depression, depression and loss of appetite were observed twice daily (morning and evening).

a. A certain amount of feed per day was fed in the same amount as the control group, and the feed consumption was determined.

b. The presence of diarrhea was confirmed by collecting the feces.

c. Body temperature was measured at fixed times daily for one week after vaccination.

2) Weighing: All groups were weighed and recorded at 1, 2, and 3 weeks after vaccination.

10-3. Sampling

At the first, second, third, first, second, and third weeks after the first vaccination, 5 mice randomly selected for each group were weighed, weighed, and sterilized with BPW (Buffered Peptone Water) 100 μg / ml, and 100 μl of the supernatant was directly streaked on a BGA agar plate and an LB agar plate to isolate the vaccine strain, and then the vaccine strain was confirmed by PCR. The remaining suspension was incubated overnight, then 100 를 was added to 10 ml of RV liquid medium and cultured for 24 hours. 100 μl of the culture was inoculated onto a BGA agar plate to isolate the vaccine strain, and then the vaccine strain was finally confirmed by PCR.

10-4. Separation of vaccine strains and preparation of lung lavage fluid in parenchyma after sacrifice

1) Period: Anesthesia was administered to the nasal cavity at 1 day, 1 week, 2 weeks, and 3 weeks after inoculation, and autopsied.

2) Clinical findings of the organs: The gross appearance of each organ was observed at autopsy, weighed and recorded.

3) Target organ: lung, liver and spleen.

4) Preparation of lung lavage solution: Whole lung was incised, and it was disintegrated in 2 ml of sterilized PBS. The resulting lysate was stored at 4 째 C overnight, and centrifuged at 4 째 C and 12,000 × g for 10 minutes. The supernatant was recovered and the protein concentration was measured by using a protein assay kit (BIO-RAD, USA) of 1 ml, adjusted to 1 mg / ml, stored at -20 ° C, For isolating the vaccine strains.

5) Isolation of the vaccine strains: The above tissues were placed in a 50 ml test tube containing BPW and stored in ice, and the actual weight of each organs was recorded by weighing the tube containing each organ. Each organ was finely pulverized using a sterilized disposable Petri dish and a syringe and transferred to a 50 ml test tube. 100 μl of this solution was directly stained on a BGA agar plate and LB agar plate to isolate the vaccine strain, And it was confirmed whether or not the strain was released. The remaining suspension was added to 100 ml of RV liquid medium after overnight culture and cultured for 24 hours. 100 μl of the culture was sprinkled on a BGA agar medium to isolate the vaccine strain, and then the vaccine strain was finally confirmed by PCR.

10-5. Measurement of immune response

The sIgA for each antigen was measured in the prepared bronchial lung lavage using an ELISA kit.

[Objective Animal In the pig  Efficacy experiment]

11. Assessment of vaccine efficacy by defending against challenging strains in piglets after sowing in sows

Sows that were not vaccinated against sows and pleural pneumonia that were not infected with Salmonella and pleural pneumonia between 8 and 9 weeks prior to delivery were used in the experiment.

After the inoculated Salmonella typhimurium vaccine strain in which each antigen gene is inserted, the vaccine is orally inoculated. Then, whether or not the vaccine is determined through the induction of the immune response to each individual antigen and the defense against the challenge infection Respectively.

11-1. Preparation of live vaccine for oral vaccination

To prepare the vaccine for oral vaccination, the attenuated Salmonella strain, in which the expression of each antigen was confirmed, was inoculated into LB broth and cultured at 37 ° C for 16 hours at 200 rpm. The culture was added to the LB broth at a ratio of 1/20 (volume) And incubated for 2 to 4 hours until the OD ₆₀₀ value became 0.8 to 1.0 under the same conditions. The culture was centrifuged at 4,000 rpm at 4,000 rpm for 10 minutes to 20 minutes, washed three times with sterile PBS, and suspended in sterile PBS (PBS-sucrose) containing 20% sucrose to make 2 × 10 ¹⁰ CFU / The vaccine was prepared.

11-2. inoculation

Experimental pigs (sows) were orally inoculated with ¹⁰ ml CFU (in PBS-sucrose) at ¹⁰ ml per mouse, and the control group was inoculated orally with sterile PBS. (Table 10, Table 11).

Oral Inoculation Experiment Plan group Head Strain Antigen name Inoculation number Challenge Infection (piglet) Sow first
(8 weeks pregnant) Second sow
(11 weeks of pregnancy) One 3 Samonella typhimurium Apx IA, Apx IIA, Apx IIIA, OMIA,
ApfA 2 x ¹⁰ < 10 ^> CFU / 10 ml 2 x ¹⁰ < 10 ^> CFU / 10 ml AP type 2 and AP type 5 were mixed in equal amounts to induce a challenge infection 2 3 Control Sterile PBS-sucrose Sterile PBS-sucrose

Outline of experiment Sow Pigeon 8 weeks before delivery 5 weeks before delivery 4 weeks 5 weeks First inoculation Second inoculation Challenge infection Autopsy Pigs surviving after challenge infection Separation of bacteria (lung)

11-3. Preparing and inoculating an outdoor strain for challenge infection

11-3-1. Culture of the strain

Each of the colonies of AP type 2 and 5 was inoculated on chocolate agar and incubated at 37 ° C. for 16 hours. The cells were suspended in sterile PBS to a concentration of 1 × ^{10 8} CFU, Isolate).

11-3-2. Challenge infection

When the piglets were 4 weeks old in each experimental group, the toxic outdoor strain prepared above was inoculated with IN at a dose of 1,000 μl twice.

12. Assessment of vaccine efficacy by defense against challenging strains in piglets after vaccination against sows and piglets

A mutant strain of Salmonella typhimurium , in which each antigen gene was inserted, was inoculated orally into sows and piglets, respectively.

12-1. Oral vaccination of live vaccine

The mice were inoculated orally inoculated with ¹⁰ x ^{10 10} CFU per pig (sows) in the experimental group, or inoculated into the nasal cavity by adjusting to 700 μl to 1 × 10 ⁷ CFU. The control group was orally inoculated with sterilized PBS-sucrose and PBS, respectively . Inoculation was carried out once more at 21 days after inoculation in the same manner as the same bacteria. Piglets were orally inoculated at 5 weeks of age, adjusted to a concentration of 2 × 10 ⁹ CFU per 10 ml.

12-2. Challenge infection

The offspring was inoculated into the nasal cavity by adjusting the outdoors to be 2 × 10 ⁸ CFU per pig in the experimental pigs.

※ Inoculate one of the nostrils with 1,000 μl twice.

※ In 10 ml of PBS, the number of App 2 and 5 strains is 1 × 10 ⁷ CFU, and 50 ml of each strain is mixed and inoculated on the day of manufacture.

13. Isolation of vaccine strains in parenchyma after sacrifice

1) Period: One week after challenge infection,

2) Clinical findings of the organs: At the time of autopsy, gross findings of the pleura and lungs were observed, and pictures were taken and recorded.

3) Target organ: pleural and lung.

4) Isolation of vaccine strains: Extruded into extracorporeal lungs with lesions and checked with chocolate agar for each colony by PCR.

Example  1. Inoculation route determination

1-1. Serum IgG antibody titers by ELISA

It was confirmed that the serum IgG titer increased from 3 weeks after the first inoculation of the protein antigen expression vaccine against each protein antigen regardless of oral or nasal inoculation route (FIG. 2).

1-2. After the challenge,

The results were as shown in Table 12 below. The results are shown in Table 12 below. [Table 12] < tb > < tb > < TABLE >

The number of deaths by each vaccination route after challenge infection Inoculation route Apx IA Apx IIA Apx IIA OMPA Control Oral administration 2 ^a / 6 ^b 1/6 3/6 3/6 3/6 Intranasal administration 1/9 1/9 0/9 1/9 3/8

^a Our head lice / ^b Inoculation head lice

1-3. conclusion

Serum test results showed that the antibody titer was higher than that of oral inoculation rather than oral inoculation. As a result, it was observed that the protective effect of nasal inoculation was better than oral inoculation .

Example 2: Analysis of whether the protein antigen expression vaccine strains were mixed and nasal inoculated and then defended

2-1. Antibody titers to each antigen by ELISA

1) Serum IgG

As a result of ELISA assay for antibody induction in serum after each of the antigens were inoculated into the nasal cavity by mixing each of the protein antigen expression vaccine strains, it was found that the vaccine strain , It was confirmed that serum IgG for each antigen was remarkably increased from 2 weeks after the first inoculation for each protein antigen compared to the control (FIG. 3).

2) mucosal IgA

After immunization with each protein antigen-expressing vaccine strain, the IgA and vaginal washing of the fecal samples for each antigen were tested for IgA antibody induction by ELISA. As a result, From 2 weeks after the first vaccination of the vaccine strain, mucosal IgA of each antigen was significantly increased compared to the control. The highest antibody titer was shown at 4 weeks after vaccination against all the antigens. (Fig. 4).

2-2. Splenocyte proliferation assay

To measure the cellular immune response, spleen cells isolated from the mouse spleen were stimulated with purified Apx IIA and OMPA antigens at 4 weeks after vaccination with the vaccine strain. As a result, as shown in FIG. 5, all of the vaccine strain inoculated group increased about 1.5 times as compared with the control group, which was statistically significant ( P <0.05).

2-3. Flow cytometry

In order to measure cellular and humoral immune responses, mouse spleens were harvested at 4th week after inoculation of the vaccine strains to isolate spleen cells and to examine the composition of T lymphocytes and B lymphocytes using flow cytometry to detect CD4 +, CD8 + and CD45R + B cells) were investigated. As a result, it was observed that CD4 +, CD8 +, and B cells for each antigen were increased compared to the control (FIG. 6).

2-4. Real-time PCR

To determine the secretion level of interleukin-4 (hereinafter referred to as IL-4) and interferon-gamma (hereinafter referred to as INF-y) which are cytokines involved in cellular and humoral immunity, Splenocytes from the inoculated and control mice were separated, splenocytes were isolated, mRNA was obtained by stimulating splenocytes with each protein antigen, cDNA was synthesized, and real-time PCR was performed using primers specific to the respective cytokines. As a result, in the vaccinated group, the level of INF-γ gene expression involved in the Th1-type immune response was increased by at least 180-fold compared with that in the control group, while the expression level of IL-4, a cytokine involved in the Th2- (Fig. 7). &Lt; tb &gt;&lt; TABLE &gt; The above results show that when the vaccine of the present invention is inoculated into the nasal cavity, the Th1 type of immune response is mainly induced but the Th2 type of immune response is also induced.

2-5. After the challenge,

In the vaccinated group, 3 out of 10 animals died and 30% of the mortality was observed, whereas in the control group, 6 out of 10 animals died and 60% of the mortality was observed. Through the vaccination, It was confirmed that the defense was done.

Example 3 Optimization of Vaccine Effect by Booster

3-1. Immune response induced after vaccination

1) Serum IgG

As shown in Table 7, each of the antigen-expressing vaccine strains was mixed and then inoculated into the nasal cavity once, twice or three times. Then, in order to examine the induced immune response, the antibody titer of each antigen was measured by ELISA Respectively. As a result, there was a significant increase from 2 weeks after inoculation regardless of the number of inoculation compared to the control group. However, higher antibody titer was observed in the case of once inoculated with the booster (Fig. 8).

2) mucosal IgA

As shown in FIG. 9, a significant increase in the antibody titer was observed at 8 weeks and 10 weeks in Group B of the feces IgA antibody titer of the twice inoculated group, whereas in the group of 3 times inoculation, , And a significant increase was observed. This result indicates that the 2-dose vaccination is more effective in inducing mucosal IgA (Fig. 9).

3-2. Cytokine analysis

As a result of comparing the expression of IFN-γ and IL-4 according to the number of times of inoculation using real-time PCR with the control group, as shown in Tables 13 and 14, IFN-γ closely related to the induction of Th1 type immunity The highest incidence rate was observed at the first inoculation. It was confirmed that the incidence gradually decreased as the number of inoculation times increased. In contrast, the expression of IL-4, which is closely related to the induction of Th2-type immune response, was increased as the number of inoculations increased. However, in the case of both Apx ⅠA and Apx ⅢA, the level of IL-4 was low at the third inoculation, because the response to the antigen stimulus rapidly started at the third inoculation and the IL-4 was rapidly produced and disappeared .

IFN-y: Increase magnification compared to control Antigen name One dose 2 doses 3 times inoculation Apx IA About 320 times About 38 times About 18 times Apx IIA About 210 times About 49 times About 5 times Apx IIA About 320 times About 65 times About 14 times OMPA About 180 times About 51 times About 20 times

IL-4: Increase magnification compared to control Antigen name One dose 2 doses 3 times inoculation Apx IA About 11 times About 13 times About 18 times Apx IIA About 24 times About 76 times About 100 times Apx IIA About 15 times About 53 times About 18 times OMPA About 31 times About 51 times About 150 times

3-3. After the challenge,

As shown in Table 15, it was confirmed that the vaccine effect through the booster was more effective in the case of twice inoculation (group B) than in the case of one-time inoculation (group A) and three times of inoculation (group C).

After the challenge, group Total number of inoculations Our Head Mortality (%) Control 15 9 60.0 A 14 5 35.7 B 6 One 16.7 C 6 2 33.3

Example 4. Safety of vaccine and IgA analysis of mucosa in lung

4-1. Clinical symptoms after vaccination

Diarrhea and fever were observed once a day for three weeks after vaccination and as a result, diarrhea and fever were not observed in all vaccinated mice. As shown in FIG. 10, the amount of the feed after the vaccination was determined to be 3 days after the vaccination (group A) Feed intake decreased, but then gradually increased, indicating normal feed intake level from day 9 after vaccination. In the case of two vaccinations (group B), the recovery rate was faster than that of the first vaccination, and normal feed intake was obtained from 3 days after the vaccination, and normal feed intake levels were confirmed after 4 days. In addition, the weight of the experimental animals was analyzed. As a result, only one dose of vaccine showed some weight loss after 1 week of inoculation and then recovered afterwards. In vaccination 2 times and 3 times of vaccination, weight change was observed (Fig. 11).

4-2. Whether the vaccine strain is released into the feces

The results of confirming whether the vaccine strain was released from the feces after inoculation of the live bacterial vaccine are shown in Table 16 below. From 1 week after the vaccination, no discharge of the vaccine strain was observed in the feces regardless of the number of vaccinations.

Whether or not shedding occurs in the feces after vaccination group 1 day 2 days 3 days 1 week 2 weeks 3 weeks Direct Enrichment Direct Enrichment Direct Enrichment Direct Enrichment Direct Enrichment Direct Enrichment One dose 4 ^a / 5 ^b 1/1 0/5 0/5 1/5 1/4 0/5 4/5 0/5 0/5 0/5 0/5 2 doses 1/3 0/2 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Control group 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

[Number of vaccine strains identified in ^a feces / ^b total number of inoculations]

4-3. Confirmation of detection of vaccine strains in real organs after vaccination

The isolation of vaccine strains from major organs after nasal inoculation of live vaccine was confirmed. As a result, as shown in Table 17, the vaccine strain was isolated from the main organs until 3 weeks after the vaccination in the 1-time inoculation group, but the vaccine strain was not isolated in the 2-time inoculation group after 3 weeks of inoculation.

Results of isolation of vaccine strains from major organs after vaccination group 1 day 1 week 2 weeks 3 weeks Direct Enrichment Direct Enrichment Direct Enrichment Direct Enrichment One dose lungs 0/3 2/3 0/3 1/3 0/3 1/3 0/3 0/3 liver 0/3 1/3 0/3 1/3 0/3 3/3 0/3 1/3 spleen 0/3 2/3 0/3 2/3 1/3 2/2 1/3 2/2 bowels 0/3 2/3 0/3 1/3 0/3 1/3 0/3 0/3 Feces 4/5 0/1 0/5 4/5 0/5 0/5 0/5 0/5 2 doses lungs 0/3 2/3 0/3 1/3 0/3 0/3 0/3 0/3 liver 1/3 0/2 0/3 0/3 0/3 0/3 0/3 0/3 spleen 0/3 3/3 0/3 1/3 0/3 1/3 0/3 0/3 bowels 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 Feces 1/3 0/2 0/5 0/5 0/5 0/5 0/3 0/5 Control group lungs 0/3 0/3 0/3 0/3 0/3 0/3 0/5 0/3 liver 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 spleen 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 bowels 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 Feces 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

4-4. Of the mucous membrane in the lung lavage fluid IgA  Measure

The IgA antibody titer in the lung lavage fluid was found to be slightly higher than that in the first immunization (Group A) (Fig. 12). This result implies that inoculation twice more than one inoculation leads to better induction of IgA in lungs, which is very important for infectious defense.

Taken together, the above results indicate that when a vaccine strain expressing Apx ⅠA, Apx ⅡA, Apx ⅢA and OMPA of the present invention is mixed with a total of two inoculations into the nasal cavity, both the cellular and humoral immune responses can be induced well , And 2 times inoculation was observed to be more effective than 1 and 3 times inoculation.

Example  5. Objective Animal In the pig  Evaluation of efficacy of vaccine

5-1. Evaluation of vaccine efficacy in piglets after vaccination in sows

As shown in Table 18, in the control group, symptoms of pleural pneumonia were observed in all five animals, while symptoms of pleural pneumonia were observed in three of five pigs vaccinated with sows and challenged in piglets .

Preventive vaccination of sows and challenge infections in piglets and clinical signs by autopsy group Challenge Infection Head Clinical symptom number Normal two Remarks Control group 5 5 0 Vaccinated group 5 3 2

5-2. Evaluation of vaccine efficacy in piglets after vaccination against sows and piglets

As shown in Table 19, in the control group, symptoms of pleural pneumonia were observed in all five poultry. In contrast, among five piglets inoculated with vaccine in sows and piglets and challenged in piglets, one piglet Symptoms were observed.

Preventive vaccination of sows and piglets, challenge infected piglets and clinical signs by autopsy group Challenge Infection Head Clinical symptom number Normal two Remarks Control group 5 5 0 Vaccinated group 5 One 4

5-3. conclusion

In summary, the clinical symptoms of pleural pneumonia were observed in all piglets used as a control group, whereas the clinical symptoms of pleural pneumonia were observed only in 3 cases when the vaccine was vaccinated against sows, I could confirm. In addition, in the case of vaccination of sows and piglets and challenge infection in piglets, mild pleural pneumonia was observed in only 1 case, which could protect pigs from pleural pneumonia more effectively than vaccination of sows I could confirm.

Claims (11)

delete delete delete A vaccine composition for prevention or treatment of swine pneumonia in pigs, which comprises as an active ingredient a mixture of Salmonella typhimurium mutants containing all of Salmonella typhimurium mutants,
Each Salmonella typhimurium mutant contains one of the factors selected from the group consisting of Apx ⅠA, Apx ⅡA, Apx ⅢA and OMPA, which are the major pathogenic factors of the causative bacteria of the swine pneumonia of the pigs in which the lon, cpxR and asd genes are deleted, Wherein the vaccine composition is a Salmonella typhimurium mutant strain. 5. The vaccine composition according to claim 4, wherein the Salmonella typhimurium mutant mixture is a live cell. delete delete delete A vaccination method for prevention or treatment of swine pneumonia in pigs or piglets, characterized in that the vaccine composition of claim 4 or 5 is inoculated into sows or piglets. 10. The vaccine according to claim 9, wherein said inoculation is administered orally or intranasally. delete

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