KR101031376B1 - Killed vaccine for preventing salmonella enteritidis - Google Patents

Abstract

PURPOSE: A vaccine containing Salmonella enteritidis isolated from chicken and human body and a vaccine containing the strain and antigen is provided to prevent food poisoning by Salmonella. CONSTITUTION: A Salmonella enteritidis B277 isolated from a chicken is deposited by the number KCTC 1152BP. The Salmonella enteritidis B369 strain isolated from human body is deposited by the number KCTC 1153BP. An inactive vaccine for preventing Salmonella enteritidis contains the Salmonella enteritidis B277 strain as an antigen. The inactive vaccine additionally contains pharmaceutically acceptable adjuvant.

Description

Kill vaccine for preventing Salmonella Enteritidis

The present invention relates to an inactivated vaccine that prevents Salmonella enteritidis infection. More specifically, the inactivated vaccine for preventing Salmonella enteritidis infection, which has excellent protective effect, is selected by inactivating strains B277 and B369 of strongly pathogenic Salmonella enteritidis isolated from chickens and humans, and containing it as an antigen. It is about.

An inactivated vaccine is a vaccine made by killing pathogenic microorganisms by purifying the pathogen and heating it, or by using UV irradiation or formalin or an alkylation reagent, etc., which prevents the virus from replicating when inactivated. It is important to maintain the structure of the antigen.

Inactivated vaccines mainly induce humoral immunity, but have high safety and thus have little side effects of vaccination and have potential application as a multivalent vaccine.

Salmonella is morphologically and physiologically similar to Escherichia coli but has become an independent genus by K. Kauffmann and others for medical convenience. Salmonella has been isolated from enteritis and gastroenteritis patients and animals with various diseases since Salmon and Smith first reported Salmonella choleraesuis in pigs that died of don cholera in 1885. It has also been isolated from chickens, healthy animals such as cows, pigs, goats, dogs, and cats, and from the environment. More than 2,000 serotypes of Salmonella spp. Have been reported to date and can be broadly classified into host-specific species and animal-specific species. Salmonella is a Gram-negative bacillus that does not form spores. Salmonella are all found as parasites in various animals. Salmonella infections occur in several forms, but enteritis is the most common form. Salmonella is a major cause of food poisoning. Salmonella food poisoning is very common in Korea, and its severity continues to increase due to an increase in group meals. In 2000, a study on the causative agents of food poisoning patients found that more than 2,500 food poisonings were caused by Salmonella, which was much higher than other food poisoning agents such as Staphylococcus aureus (about 1,000 cases) and Vibrio (about 200 cases). It is occupying a proportion.

Salmonella infection can cause symptoms such as coarse skin, lack of appetite, conjunctivitis, depression, thinning stools, increased spleen, and death. Salmonella can also be easily delivered to humans because they are present in the intestines of poultry and livestock such as chickens, which humans consume as food. In the United States, the economic damage caused by food poisoning is estimated to be $ 2-40 billion, which can be economically important. However, the effects of vaccines that can prevent Salmonella infection are very limited, and treatments such as antibiotics are not cured and relapse is common, so there are many difficulties in controlling Salmonella.

Therefore, the present inventors screened the highly pathogenic Salmonella enteritidis strains in humans to reduce the incidence of food poisoning caused by Salmonella enteritidis in chickens as well as the highly pathogenic strains for efficient prevention of Salmonella enteritidis. In addition, it was confirmed that the inactivated vaccine prepared by using the strains as an antigen is safe for use and excellent in Salmonella enteritidis defense effect, and completed the present invention.

An object of the present invention to provide a Salmonella enteritidis strain isolated from chickens.

It is also an object of the present invention to provide a Salmonella enteritidis strain isolated from humans.

It is also an object of the present invention to provide a vaccine for preventing Salmonella enteritidis infection, which comprises as an antigen the strain isolated from the chicken or human.

In order to achieve the above object, the present invention provides a Salmonella enteritidis strain isolated from chickens, B277 strain of Accession No. KCTC11552BP.

In another aspect, the present invention provides a Salmonella enteritidis strain isolated from human, B369 strain of Accession No. KCTC11553BP.

Korean representative isolates can be selected through genotyping, phage-type and antibiotic resistance assays in Salmonella enteritidis isolates isolated from humans, eggs and poultry. According to an embodiment of the present invention, a total of 11 strains (B350, 352, 365, 369, 390) isolated from humans and 11 strains (B270, 277, 295, 296, 298, 322) isolated from chickens are Salmonella entertainers. Representative strains of Dys were selected.

In the present invention, the pathogenic test in the mouse can be selected for the pathogenic Salmonella enteritidis strain. In the embodiment of the present invention, the pathogenic strain was selected as a final strain for vaccine production, B277 chicken isolated strain and human derived isolate B369 showing high mortality in the mouse.

The Salmonella enteritidis strain isolated from highly pathogenic chickens was named B277 and was dated September 7, 2009, in accordance with the provisions of the Budapest Treaty on the International Approval of Microbial Deposits under the Patent Procedure. , KCTC11552BP was deposited with Korea Research Institute of Bioscience and Biotechnology.

In addition, the Salmonella enteritidis strain isolated from highly pathogenic individuals was named B369, which was issued on September 7, 2009, in accordance with the provisions of the Budapest Treaty on the International Approval of Microbial Deposits under the Patent Procedure. Cultures, Korea Research Institute of Bioscience and Biotechnology) was deposited with accession number KCTC11553BP.

In another aspect, the present invention provides a vaccine for preventing Salmonella enteritidis infection, which contains the B277 strain isolated from chicken or the B369 strain isolated from human.

The vaccine may be an inactivated vaccine.

The inactivated vaccine can be prepared by purifying pathogens and heating them, or by killing pathogenic microorganisms using drugs such as UV irradiation or formalin or alkylation reagents.

The inactivated vaccine is preferably inactivated at 37 ° C. for 3 days by treatment with formalin.

The vaccine of the present invention may be administered alone but is preferably administered with a pharmaceutically acceptable adjuvant.

Adjuvant is a substance that promotes an immune response to an antigen in a specific way during the initial activation of immune cells, and means an agent, a molecule, etc. that enhances immunity by increasing the activity of cells of the immune system, although not an immunogen to the host (Warren et. al., 1986, Annu. Rev. Immunol ,. 4: 369). Adjuvant selection in the production of inactivated vaccines is an important cause of vaccine efficacy. Adjuvants that can be administered with the vaccine of the present invention to enhance the immune response include any of a variety of adjuvants, and typical adjuvant includes Freund adjuvant, aluminum compound, Muramil di Peptides (muramyl dipeptide), lipopolysaccharides (LPS), monophosphoryl lipid A or coil A and the like are known.

Preferably, the adjuvant that can be used with the Salmonella enteritidis vaccine of the present invention is ISA70.

The vaccine prepared in the present invention shows excellent protective effect in mice and chickens. According to the embodiment of the present invention, when the vaccine was administered to mice and challenged with the strain, the survival rate was about 90% to 100%. In the spleen, bacteria were separated by an average of 1cfu / g, but after 13 days, they were not separated, and cecum was not isolated at all.

The vaccine of the present invention can be used in all species that can be infected with Salmonella enteritidis. For example, it can be used in chicken, pig, beef cattle.

The vaccines of the present invention can be formulated into appropriate formulations with acceptable pharmaceutical carriers and administered by a variety of routes. The route of administration may be administered orally, intraperitoneally, intramuscularly, subcutaneously, intradermally, topically, intranasally, pulmonary, rectally. Preferably, it is formulated as an injection and administered by a method such as intramuscular injection, subcutaneous injection or intradermal injection. Injectables include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, to prevent microbial development Pharmaceutical carriers such as preservatives (eg, mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like).

The vaccine of the present invention is administered in an immunologically effective amount. The term "immunologically effective amount" means an amount sufficient to produce a prophylactic effect and an amount not to cause side effects or serious or excessive immune responses, the exact dosage of which depends on the particular immunogen to be administered, Multiple administrations are possible. In addition, the vaccine of the present invention can be administered in combination with a known vaccine. Preferably, the vaccine of the present invention may be administered twice at four week intervals in an amount of 0.5-1.0 ml per allowance in allowance of 9 × 10 ⁸ to 1.8 × 10 ⁹ cfu / ml.

Salmonella enteritidis infection prevention vaccine prepared in the present invention is excellent in the protective effect by using a strong pathogenic strain, can be used safely as an inactivating vaccine can prevent food poisoning caused by Salmonella early.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention, and modifications, substitutions, and insertions commonly known by those skilled in the art It will be apparent to those skilled in the art that the present invention may be included in the scope of the present invention.

Example 1 Selection of Salmonella Enteritidis Strains

1-1. Selection of strain

Among Salmonella enteritidis isolated from humans and Salmonella enteritidis isolated from chickens, one of the typical pulsed-field gel electrophoresis (PFGE) strains has various phage type (PT) types. Selected. A total of 11 strains isolated from humans (B350, 352, 365, 369, 390) and chicken strains (B270, 277, 295, 296, 298, 322) were selected as representative strains of Salmonella enteritidis. Used in the invention (Table 1).

TABLE 1

Origin of the strain Strain No. PFGE type Phage
type) Antibiotic Resistant Pattern chicken B270 A6 PT35 AM-TIC B277 A6 PT35 AM-TE-S-TIC B295 A1 PT7 AM-TE-S-TIC B296 A6 PT35 B298 A1 PT17 TE-S B322 A1 PT1 Person B350 A1 PT21 AM-TIC B352 A6 RDNC B365 A1 PT3 B369 A1 PT21 AM-TE-S-TIC B390 A6 RDNC

RDNC = Reacted but not confirmed.

1-2. Salmonella Entertainers  Selection of Chick Inoculation and Strains for Pathogenicity Evaluation

Salmonella enteritidis test strain (Table 1) incubated at 37 ° C. for 18 hours was inoculated in nutrient broth. After incubating at 37 ° C. for 5 hours, the number of bacteria in the culture was adjusted to about 6 × 10 ⁷ cfu / 0.2 ml. Each strain was inoculated with 0.2 ml orally in 40 chicks of 1-3 days of age and observed for 10 days. On day 10, chicks surviving in each group were autopsied and spleens, livers, and cecums were harvested uncontaminated. After weighing each organ, 1 ml of sterile peptone water buffer (BPW) was added and then emulsified. After diluting the emulsion 10 times, 0.1 ml was inoculated into XLT-4, a Salmonella separation medium, and then incubated at 37 ° C for 18 hours. Colonies specific to Salmonella grown on cultured XLT4 plates were measured to calculate colonies per gram of organs.

Among the total of 11 Salmonella enteritidis strains, 40 B365 strains were isolated from humans, and the mortality was 100%, and 36 B369 strains were killed, resulting in high mortality of 90%. B277, 295 and 270 strains isolated from chickens were killed 31, 23 and 22, respectively, resulting in high mortality of 77.5%, 57.5% and 55.0%, respectively. In the cecum, B390 was isolated 60% and B277, 369 and 322 were 20%, 40% and 40%, respectively (see Table 2).

TABLE 2

Origin of the strain Strain No. % Mortality Bacterial Separation Rate in Caecum (5%) remark chicken B270 22/40 (55.0) 0.0 B277 31/40 (77.5) 20.0 Selection B295 23/40 (57.5) 0.0 Selection B296 13/40 (32.5) 0.0 B298 10/40 (25.0) 0.0 B322 16/40 (40.0) 40.0 Person B350 30/43 (69.8) 0.0 B352 22/40 (55.0) 0.0 B365 40/40 (100.0) 0.0 Selection B369 36/40 (90.0) 40.0 Selection B390 30/40 (75.5) 60.0

As a result of the first test, it was determined that B277, 295, 365, and 369 among the strains isolated from humans and chickens were highly pathogenic, and these strains were selected and inoculated into chicks, respectively, and retested.

For the second experiment, the selected strains were incubated according to the method mentioned above and then diluted to 3 × 10 ⁷ cfu / 0.2 mL. Thirty-five day-old chicks of each strain were orally inoculated with 0.2 ml of allowance.

B277 and 295 strains isolated from chickens died in 8 cases, respectively, resulting in 26.7% mortality, and B365 and 369 strains isolated from humans died in 7 and 17 cases, respectively, resulting in 23.3% and 56.7% mortality, respectively. ). Among the 4 strains examined, 369 strains isolated from humans showed the highest chick mortality.

In addition, the number of dead chicks was measured up to 2 weeks after inoculation, and organs were collected in the same manner as the first while the live chicks were removed, and the number of colonies of Salmonella enteridis present in the organs was measured per gram. In the case of cecum, the contents and cecum were converted into an emulsion to make an emulsion, which was then placed in a 10 ml SC culture and shaken at 37 ° C. for 24 hours. MacConkey agar medium and XLT4 agar medium (XLT4 agar) were inoculated and incubated at 37 ° C. for 24 hours to determine the growth of Salmonella.

As a result, strain B369 isolated from humans was highly isolated with 4.4 × 10 ⁴ cfu bacteria in soy sauce. B365 was isolated at 3.4 × 10 ⁴ cfu. Strains B277 and B295 isolated from chickens were isolated from 6.1 x 10 ³ cfu and 1.6 x 10 ³ cfu, respectively (Table 3).

Strains B277 and B369 were isolated from the caecum at 60%, and strain B365 was 20%. The strain with the highest mortality rate and the highest intra-organ separation rate was considered as the pathogenic strain, and the isolate B369 in humans and isolate B277 in chickens were used as candidate vaccine candidates for inactivation.

[Table 3]

Strain derived Strain No. Mortality (%) Fungal Separation Rate in Chickens Remark Soy sauce spleen Caecum (%) chicken B277 8/30 (26.7) 5.3 × 10 ² 6.1 × 10 ³ 60.0 Selection B295 8/30 (26.7) 2.0 × 10 ² 1.6 × 10 ³ 0.0 Person B365 7/30 (23.3) 2.9 × 10 ² 3.4 × 10 ⁴ 20.0 B369 17/30 (56.7) 9.8 × 10 ² 4.4 × 10 ⁴ 60.0 Selection

Experimental Example 1 Proliferative Test of Strains for Inactivating Vaccine Preparation

Since the proliferation of the inactivated vaccine strain in the basal medium has a significant effect on the production cost and the manufacturing process, it is necessary to determine the proliferation in the basal medium, LB medium, to investigate the proliferation of the strain. Therefore, B277 and B369 vaccine candidate strains selected as inactivated vaccine strains were inoculated in liquid broth and cultured at 37 ° C., and absorbance (A660) was measured at 660 nm for 5 hours every hour. It was repeated 3-4 times for each strain. As shown in FIG. 1, the proliferation of the bacteria showed rapid growth from 2 hours to 5 hours after inoculation in all three replicates.

Example 2 Preparation of Inactivated Vaccine

Selected inactivated vaccine strains B277 and B369 strains were inoculated in a liquid medium (LB broth), shaken at 37 ℃ culture was adjusted to 1OD ₆₆₀ and 2OD ₆₆₀ . Some of the cultures were harvested for colony counting and the remaining cultures were inactivated at 37 ° C. for 3 days by treatment with 0.3% (v / v) formalin. After inactivation, the bacterial solution was inoculated into nutrient agar, nutrient broth and thioglycolate agar, respectively, and incubated at 22 ° C. and 37 ° C. for 7 days, and then inactivated. . The inactivated bacteria were mixed with adjuvant. The adjuvant used ISA70. 70% (v / v) of the adjuvant ISA70 was added, and 20% of each of the previously adjusted bacterial solutions was added, and the remaining portions were mixed with phosphate buffer (PBS) to prepare a vaccine.

Experimental Example 2 Investigation of the Protective Effect of Salmonella Enteritidis Inactivating Vaccine Using Mice

Mice were used to evaluate the protective effect against Salmonella enteritidis vaccine. Two inactivated vaccines were prepared for each isolate by varying the concentrations of B369 selected from human isolates and B277 strains selected from chicken isolates at 1OD and 2OD. In the case of B369, it is 9 × 10 ⁸ cfu / ml for 1OD, 18 × 10 ⁸ cfu / ml for 2OD, whereas for B277 it is 14 × 10 ⁸ cfu / ml for 1OD and 28 × 10 ⁸ cfu for 2OD In the number of bacteria, the vaccine made of B277 was somewhat higher than that of B369. 100 mice were divided into 10 groups of 10 and as in Table 4, vaccines 1, 2, 3 and 4 were inoculated intraperitoneally at 0.2 ml per horse per group. The control group was inoculated with 0.2 ml of PBS. Two weeks after vaccination, wild type bacteria (wild type_ B369 and B277) were challenged intraperitoneally with 0.2 ml per animal (10LD ₅₀ ) (LD ₅₀ : 1.5 × 10 ⁶ cfu / ml) for 10 days.

Table 4 shows the results of measuring the effects of inactivated test vaccines in mice. In the unvaccinated control group, both challenged with B369 and B277 died. Vaccine 1 prepared with Salmonella enteritidis B369 strain had a survival rate of 88% when wild type B369 challenge was inoculated 2 weeks after inoculation and 90% after wild type B277 inoculation. Vaccine 2 had survival rates of 90% and 100% after challenge with B369 and B277, respectively. Vaccine 3 prepared with Salmonella enteritidis B277 strain showed 100% survival after wild type B369 challenge and 90% survival after wild type B277 challenge. Vaccine 4 showed survival rates of 90% and 70% after B369 and B277 challenge, respectively. There was no difference in cross-protection between different strains of vaccination strains.

[Table 4]

Strain No. vaccine content
(cfu /) Survival rate (%) B369 B277 B369 Vaccine 1 (2OD) 18 x ^{10 8} 8/9 (88%) 9/10 (90%) Vaccine 2 (1OD) 9x10 ⁸ 9/10 (90%) 10/10 (100%) B277 Vaccine 3 (2OD) 28 x ^{10 8} 10/10 (100%) 9/10 (90%) Vaccine 4 (1OD) 14 x ^{10 8} 9/10 (90%) 7/10 (70%) 0/10 (0%) 0/10 (0%)

Experimental Example 3 Result of Bacterial Separation Test Using Chicken

Thirty 6-8 week old chickens were divided into three groups of 10, inactivated vaccines 1 and 2 were inoculated into the thoracic muscles by 0.5 ml each, and the control group was inoculated with PBS. After 3 weeks of inoculation all groups were challenged with wild type S. Enteritidis (B277) and 3, 3 and 4 animals in each group at 6, 13 and 20 days post inoculation. After the chickens were selected to collect blood and the bacteria were separated from the spleen and cecum, the results are shown in FIG. 2. Vaccine 1 had an average of 1 cfu / g of bacteria isolated from the spleen 6 days after challenge, but not after 13 days. Vaccine 2 had an average of 4 cfu / g of bacteria isolated at 6 days after challenge, no isolate at 13 days, and an average of 3 cfu / g at 20 days. In the group vaccinated, no bacteria were isolated from the cecum.

Experimental Example 4 Plate Aggregation Reaction of Inactivated Vaccine

B277 strain was inoculated in tryptose phosphate broth (TPB), shaken for 6-8 hours at 37 ° C, and formalin was added so that the culture concentration was 0.3%. After inactivation for 2-3 days, the mixture was centrifuged at 7,000 rpm for 10 minutes and diluted to an appropriate concentration of PBS, which was used as an antigen for MAT. The antigen stock solution was diluted twice with PBS in 96 well plates, and the pellet was sized at about 1.5-2 mm at 4 ° C. to determine the final dilution factor of the MAT antigen. 50 μl PBS was added to all wells, and 50 μl of test serum was diluted twice. 50 μl of the antigen was added to all the wells, and the serum aggregation value of the aggregation reaction after 24 hours at 37 ° C. was measured.

The result of measuring antibody titers on a commercially available plate agglutination test (MAT) in serum is shown in FIG. 3. Antibody titers of vaccines 1 and 2 increased rapidly after 3 weeks of vaccination. Vaccines 1 and 2 increased without difference in antibody titer values up to 3 weeks of vaccination and showed a small difference 6 days after challenge. The antibody titer value of the control group was lower than that of the vaccinated group.

Experimental Example 5 ELISA Test Results of Inactivated Vaccine

ELISA method was used Salmonella D group ELISA kit (BioChek, Holland). Test serum was diluted 500-fold in the sample dilution solution, and 100 µl was dispensed into each well, and left for 30 minutes at room temperature. Positive antibody was used as the secondary antibody (sheep anti-chicken), and 100 μl was dispensed into each well, followed by reaction at room temperature for 30 minutes. After three successive washes with a washing buffer, the substrate was dissolved in (PNPP: p-nitrophenyl phosphate) substrate buffer (substrate buffer) and 100 μl was dispensed into each well. After 15 minutes, 100 μl of stop solution (sodium hydroxide) was dispensed into each well, and antibody titers were measured using an ELISA reader (OD; 405 nm).

The titers of antibodies of vaccines 1 and 2 increased rapidly after 3 weeks of vaccination. Antibody titers increased up to 6 days after challenge and little difference after 6 days of challenge. The antibody titer value of the control group was much lower than the vaccination group (see FIG. 4).

Experimental Example 6 Inactivated Vaccine Safety Test Results

To investigate the safety of vaccines prepared with adjuvant ISA70, test vaccines were inoculated into the chest muscles of 0.5 ml each of 6-8 week old chicks. The chick survived 100%, and after confirming the inoculation site two weeks after the inoculation, there was an inflammatory scar but no other symptoms (see FIG. 5).

1 shows the proliferation of Salmonella enteritidis B277 and B369 vaccine candidate strains. (A) shows the proliferative property of B277, (B) shows the proliferative property of B369.

Figure 2 shows the results of bacterium separation in the spleen and cecum when inoculated with chickens made with B369 strains and challenged with Salmonella enteritidis B277. vac 1 is a vaccine made of BOD3 strain 1OD, vac 2 means that a B369 strain made of 2OD.

Figure 3 shows the results of measuring the antibody titer against MAT.

Figure 4 shows the results of measuring antibody titers using ELISA antibody kit.

Figure 5 shows the safety test results of Salmonella enteritidis inactivated vaccine used in addition to ISA 70. Two weeks after vaccination, only inflammatory scars remained at the site of inoculation.

Claims (6)

Salmonella enteritidis strain isolated from chicken, B277 strain of Accession No. KCTC1152BP. Salmonella enteritidis strain isolated from human, B369 strain of Accession No. KCTC1153BP. An inactivating vaccine for preventing Salmonella enteritidis infection, comprising the strain of claim 1 or 2 as an antigen. 4. The inactivated vaccine of claim 3 further comprising a pharmaceutically acceptable adjuvant. The inactivated vaccine of claim 4 wherein the adjuvant is ISA70. 4. The inactivated vaccine of claim 3, wherein the vaccine is prepared by inactivating the strain with formalin.

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