JP6712760B2 - Salmonella vaccine - Google Patents

Description

The present invention relates to a Salmonella vaccine containing a combination of a secreted protein of Salmonella and a dead bacterium.

Since the outbreak of salmonellosis in the livestock industry causes a great economic loss, it is important to take measures to prevent infection of livestock and poultry. However, to date, the occurrence of salmonellosis in domestic livestock and poultry has not been completely suppressed.

The use of vaccines has been attempted to prevent the infection of Salmonella. For example, in oral Salmonella infection, it has been reported that when live bacteria such as attenuated strains are used as an immunogen, protection against infection is exhibited. Vaccine development using this attenuated strain has an idea based on immunological and bacteriological knowledge and excellent strategicity for achieving the purpose, but it involves side reactions and risk of reversion to pathogenicity. There is a problem.

Therefore, the attenuated strain vaccine is not currently on the market, and a killed vaccine composed of Salmonella genus sterilized by formalin or heat is used exclusively (for example, “Bovine Salmonella bivalent vaccine (Science feed Research Institute)", "Chicken Salmonella Inactivation 3 Mixture/KS (Kyoritsu Pharmaceutical Co., Ltd.)".

Further, as a vaccine that does not use an attenuated strain, a vaccine that uses a culture supernatant of Salmonella sp. that has been cultured under specific conditions has also been reported. For example, in Patent Document 1, the culture supernatant of Salmonella sp. cultivated in a low-magnesium/low-phosphate medium under SPI-2 inducing conditions prevents the spread of Salmonella sp. It is disclosed to have a protective effect.

However, a component vaccine having excellent safety and an excellent protective effect against Salmonella infection is not yet developed.

Special table 2010-501599

The present invention has been made in view of such circumstances, and an object thereof is to provide a vaccine having high safety and an excellent protective effect against salmonellosis.

In order to solve the above-mentioned problems, the present inventor diligently studied the use of components other than attenuated strains as components of Salmonella vaccine. As a result, administration of a protein secreted from Salmonella and killed Salmonella alone did not have a significant protective effect against Salmonella infection compared to controls without treatment or with adjuvant. I was not able to admit. However, it was found that, when a protein secreted from Salmonella spp. and a killed strain of Salmonella spp. are combined and administered, surprisingly, an extremely excellent protective effect against Salmonella spp. infection is exerted. This protective effect was observed in all Salmonella infections examined. From these facts, the present inventors have found that a combination of a protein secreted from Salmonella and a killed strain of Salmonella is a Salmonella vaccine having high safety and excellent effects, and is infected by various Salmonella. The present invention has been completed and the present invention has been completed.

The present invention provides the following aspects in more detail.

(1) A vaccine for protecting a living body from Salmonellosis, which comprises a combination of a protein secreted from Salmonella and a dead bacterium of Salmonella.

(2) The protein has a pH of 5 at a composition of 5 mM KCl, 7.5 mM (NH ₄ ) ₂ SO ₄ , 0.5 mM K ₂ PO ₄ , 38 mM glycerol, 100 mM Tris-HCl, 30 μM MgCl ₂ , 0.2% glucose, and 0.1% casamino acid. The vaccine according to (1), which is secreted from Salmonella sp.

(3) A method for protecting a living body from Salmonellosis, which comprises administering the vaccine according to (1) or (2) to a living body infected with Salmonella.

The vaccine of the present invention comprising as an active ingredient a combination of a protein secreted from Salmonella and a dead bacterium of Salmonella produces an extremely high survival rate even when infected with Salmonella when administered to a living body. I was able to. When the protein secreted from Salmonella is administered alone or when the killed Salmonella is administered alone, no significant effect is observed compared to the control (no treatment or adjuvant administration), Considering that all individuals died, the effect of the vaccine of the present invention is a surprising synergistic effect.

Further, when the above protein or killed bacteria were administered alone, abnormal symptoms were observed before the individual died, but with the administration of the vaccine of the present invention related to these combinations, such abnormal symptoms were observed. I was not able to admit. This fact also supports the excellent action of the vaccine of the present invention.

Furthermore, the vaccine of the present invention was able to show a protective effect against infection with various Salmonella spp. Therefore, it can be widely applied in salmonellosis.

It is a graph which shows the protective effect from the infection of Salmonella spp. in the mouse immunized with the protein secreted from Salmonella spp. and/or the killed bacteria of Salmonella spp. As a control, the results of no treatment and adjuvant administration are shown. FIG. 3 is a graph showing the protective effect against infection of three Salmonella spp. in a mouse immunized with a mixture of a protein secreted from Salmonella spp. and a dead strain of three Salmonella spp. As a control, the result of no treatment was shown.

The present invention provides a vaccine for protecting a living body from salmonellosis, which comprises, as an active ingredient, a combination of a protein secreted from Salmonella and a killed strain of Salmonella. The present invention also provides a method for protecting a living body from salmonellosis, which comprises administering the vaccine to a living body infected with Salmonella.

The "salmonellosis" in the present invention means an infectious disease caused by Salmonella. Examples of the "Salmonella genus bacteria" that cause Salmonella disease include, but are not limited to, Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Dublin, Salmonella Enteritidis, Salmonella Gallinarum, and Salmonella Pullorum belonging to Salmonella enterica subsp. enterica. In the production of the vaccine of the present invention, it is also possible to make a multivalent vaccine by using two or more species of Salmonella.

In the culture for allowing Salmonella to secrete the protein, it is preferable to use an acidic minimal medium. The minimum acidic medium, for example, 5 mM KCl, 7.5 mM (NH ₄ ) ₂ SO ₄ , 0.5 mM K ₂ PO ₄ , 38 mM glycerol, 100 mM Tris-HCl, 30 μM MgCl ₂ , 0.2% glucose, 0.1% casamino acid. A medium having a composition of pH 5.0 can be preferably used.

Incidentally, keep the present invention _{"protein, 5mM KCl, 7.5mM (NH 4} ) 2 SO 4, 0.5mM K 2 PO 4, 38mM glycerol, 100mM Tris-HCl, 30μM MgCl 2, 0.2% glucose, 0.1% casamino acids "Is secreted from Salmonella spp. cultured in a medium having a pH of 5.0" means that the protein has the "property" of being secreted from Salmonella spp. However, it does not mean that the “acquisition method” of the protein is limited.

In the present invention, it is not necessary to use all the proteins secreted from Salmonella, and for example, secreted proteins that do not contribute to the control effect against Salmonella may be excluded from the vaccine component. Whether or not a specific secreted protein contributes to the control effect against Salmonellosis is administered to the mouse a combination of the specific protein and killed bacteria of the genus Salmonella, and infected with Salmonella to the mouse, It can be evaluated by testing its survival rate (see this Example).

In the vaccine of the present invention, "killed bacteria" of Salmonella spp. used in combination with the secretory protein include Salmonella spp., for example, heat treatment, chemical disinfectant treatment, irradiation with radiation or ultraviolet rays, and mutation promoting substance. It can be prepared by treatment or the like. In the preparation of killed bacteria, heat treatment or treatment with a chemical disinfectant formalin is preferably used. The killed bacteria may be a mixture of different Salmonella species (eg, a mixture of Salmonella species of different species or different serotypes). This makes it possible to expand the range of Salmonella that can prevent infection.

In the vaccine of the present invention, "including a combination" of a protein secreted from Salmonella and a killed bacterium of Salmonella means that the vaccine of the present invention comprises a protein secreted from Salmonella and a Salmonella bacterium. Even in the form of a single agent containing both killed bacteria as active ingredients, a combination of a formulation containing a protein secreted from Salmonella as an active ingredient and a formulation containing dead bacteria of Salmonella as an active ingredient It means that it may be in the form.

The vaccine of the present invention may contain a pharmacologically acceptable carrier in addition to the above active ingredients. Examples of such carriers include sterilized water and physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, flavors, fragrances, excipients, vehicles, preservatives. , Binders, diluents, isotonic agents, soothing agents, bulking agents, disintegrating agents, buffering agents, coating agents, lubricants, coloring agents, sweetening agents, thickening agents, flavoring agents, solubilizing agents, or Other additives may be mentioned, but not limited thereto. The active ingredient may be in various forms such as injections, aerosols, capsules, tablets, powders and granules, depending on the administration method and therapeutic purpose.

Moreover, in the vaccine of the present invention, an adjuvant can be further added in order to enhance the immune response. Examples of the adjuvant include Freund's complete adjuvant, Freund's incomplete adjuvant, oil adjuvant, aluminum hydroxide, aluminum phosphate, saponin, vitamin E, etc., but are not particularly limited as long as they exhibit the effect.

The "living body" to which the vaccine of the present invention is administered means the body of living animals and humans. The animal is not particularly limited as long as it is an animal that can be infected with Salmonella, and may be domestic animals, pet animals (pets), experimental animals, and animals for other purposes. May be Examples of animals include, but are not limited to, cows, pigs, horses, sheep, goats, chickens, ducks, geese, ducks, quail, pheasants, pigeons, turkeys, guinea fowls, dogs, cats, and the like.

The vaccine of the present invention can be administered to a living body by a known administration route including subcutaneous, intradermal, intravenous, intramuscular, oral, and intranasal to immunize the living body. When the vaccine preparation of the present invention is a concomitant drug, each preparation may be administered simultaneously, or may be administered with a time lag within a range that does not diminish the effect of the combination.

The dose of the vaccine of the present invention may be any amount as long as it can induce an immune response in a living body, such as the age and weight of animals and humans, the type of animals, the type of pathogenic bacteria (for example, the difference in pathogenicity, etc. ), and the method and route of administration. The single dose of the secretory protein as an active ingredient is usually 0.05 μg to 1500 μg, preferably 5 μg to 500 μg. The dose of killed bacteria as an active ingredient is usually 10 ³ to 10 ¹⁰ and preferably 10 ⁶ to 10 ⁹ as the killed bacteria number. The administration may be performed multiple times, in which case the administration interval is usually 1 to 2 weeks.

Hereinafter, the present invention will be described more specifically based on Examples, but the present invention is not limited to the following Examples.

(1) Salmonella spp. Salmonella enterica subsp. enterica belong to Salmonella Typhimurium (S. Typhimurium) (O4 group), Salmonella Choleraesuis (S. Choleraesuis) (O7 group), and Salmonella Dublin (S. Dublin). (O9 group) was used.

These Salmonella spp. are available from the American Type Culture Collection (ATCC).

(2) Medium The following medium was used for culturing Salmonella.

(3) Preparation of dead bacteria
S. Typhimurium, S. Choleraesuis, and S. Dublin were statically cultured overnight in LB medium at 37° C., and the bacterial cells were collected by centrifugation at 3,000 g for 20 minutes. After that, adjust to 1x10 ⁴ CFU/500μl PBS, sterilize by boiling at 100°C for 5 minutes, and use Biorutor (BM equipment), repeat ultrasonic disruption and dormant state in High mode for 10 minutes at 30 second intervals. The cells were destroyed by.

(4) Preparation of secreted protein
S. Typhimurium was cultivated in LB medium at 37° C. overnight in static culture, and was cultured in 50 times the volume of LB medium at 37° C. overnight in shaking (160 rpm). Then, the cells were collected by centrifugation at 3,000 g for 20 minutes, and statically cultured overnight at 37° C. in a medium of pH 5.0 N-minimal medium, which is half the amount of the medium used for shaking culture. The supernatant of the statically cultivated pH 5.0 N-minimal medium was centrifuged at 3,000 g for 30 minutes, and the cells were removed using a 0.22 μm filter. A final concentration of 25% trichloroacetic acid (Nacalai Tesque) was added to the obtained supernatant, the mixture was reacted overnight at 4° C., and the protein was recovered by centrifugation at 20,000 g for 45 minutes. Acetone was added to the obtained protein, washed by centrifugation at 20,000 g for 15 minutes, and dialyzed with PBS for 2 days to remove trichloroacetic acid.

(5) Immunization method
A 5-week-old female BALB/c mouse (Japan SLC) was mixed with a secretory protein solution (10 μg of secretory protein and an equal amount of Freund's complete adjuvant (Becton Dickinson, Japan) to obtain a solution. The mouse was subcutaneously immunized with 100 μl) and a killed bacterium solution (100 μl solution prepared by adjusting 1×10 ⁴ CFU killed bacterium to 50 μl and mixing an equal amount of complete adjuvant). As a control, secretory protein alone, killed bacteria alone, complete adjuvant alone, or no treatment was also verified.

After 2 to 6 weeks after the immunization, the antibody titer to the secreted protein and dead bacteria was confirmed by the ELISA method. Specifically, the secreted protein or killed bacteria was adjusted to 5 μg/ml, and 100 μl/well was immobilized on a plate for 96 ELISA. Then, mouse serum was reacted with the secretory protein on the plate, followed by reaction with HRP-anti-mouse antibody, followed by color development, and the antibody titer was measured based on the color development. When an antibody titer against killed bacteria was not observed after administration of killed bacteria alone, killed bacteria (1×10 ⁴ CFU/200 μl PBS) was intravenously injected into mice for booster immunization.

(6) Assay of survival rate (a) Fasting was performed for 6 hours before infection, and then 50 μl of 10% sodium hydrogen carbonate was orally administered. After 15 minutes, 1×10 ⁶ S. Typhimurium was orally infected, and then fasting was performed for 30 minutes. Observation was carried out for 40 days after infection (Fig. 1).

As a result, in the untreated group and the adjuvant-immunized group, deterioration of the hair gloss was observed from the third day after infection, and loss of energy was observed from the fifth day after infection. Also, in the secretory protein immunized group and the killed bacterial immunized group, deterioration of hair gloss was observed from the 5th day after infection, and loss of energy was observed from the 7th day. Mice in the untreated, adjuvant, secreted protein and killed immunized groups died from the 6th day after the infection, and all died by the 10th day after the infection. On the other hand, in the group to which the combination of killed bacteria and secreted protein was administered, half of them survived until 40 days after infection.

From the above results, it was found that the combination of killed bacteria of Salmonella and secreted protein is effective for the protection against oral infection.

(B) Next, a vaccine consisting of a combination of secretory protein and killed bacteria was applied to serotypes of Salmonella (S. Choleraesuis: O7 group, S. Dublin: O9 group) other than S. Typhimurium (04 group). It was examined whether or not it also showed an infection protective effect (Fig. 2). As a vaccine against salmonellosis, a mixture of inactivated Salmonella of various serotypes (killed bacteria used for formalin treatment) has been used so far. Therefore, a combination of the dead bacteria of three strains (S. Typhimurium, S. Choleraesuis, S. Dublin) used for infection and the Salmonella secreted protein prepared in (4) above is used as a vaccine, and the combination of the above (6) (a) The experiment was conducted by the same method as the experiment of (1). For oral infection in this experiment, 100 LD ₅₀ of each bacterial cell (1×10 ⁶ S. Typhimurium, 1×10 ⁷ S. Choleraesuis, 1×10 ⁶ S. Dublin) was used. In addition, as a control, verification without treatment was also performed.

As a result, when S. Typhimurium was orally infected, the group immunized with the combination of the secretory protein and the mixture of three kinds of dead bacteria showed a significant protective effect against infection, consistent with the result of FIG. 1 (FIG. 2). left). Even when S. Choleraesuis was orally infected, all the untreated groups died in about 20 days, whereas the group immunized with the combination of three types of killed bacteria and secreted proteins had a significant protective effect against infection. It is shown (center of FIG. 2). In addition, when S. Dublin was orally infected, all the untreated groups died in about 10 days, whereas the group immunized with three types of dead bacteria and secretory proteins showed a significant protective effect against infection. It is shown (Fig. 2, right). From the above results, it was revealed that the vaccine of the present invention exhibits an excellent protective effect against Salmonella spp.

As a Salmonella genus vaccine having a high infection protection effect, an attenuated strain vaccine is known, but it is not commercially available due to problems such as safety. From the viewpoint of safety, a vaccine using killed bacteria or a specific protein is desirable. The vaccine of the present invention utilizing a combination of secretory protein and killed bacteria is highly safe and can exert an excellent protective effect against Salmonella infection on animals such as livestock and humans. Therefore, the vaccine of the present invention can be used particularly in the fields of agriculture and medicine.

Claims (2)

(A) Composition of 5 mM KCl, 7.5 mM (NH ₄ ) ₂ SO ₄ , 0.5 mM K ₂ PO ₄ , 38 mM glycerol, 100 mM Tris-HCl, 30 μM MgCl ₂ , 0.2% glucose, 0.1% casamino acid and pH 5.0. Culturing Salmonella in a medium,
(B) recovering the protein secreted from the cultured Salmonella spp., and (c) combining the recovered protein with killed Salmonella spp.
A method for producing a vaccine for protecting a living body from salmonellosis, which comprises: (A) Composition of 5 mM KCl, 7.5 mM (NH ₄ ) ₂ SO ₄ , 0.5 mM K ₂ PO ₄ , 38 mM glycerol, 100 mM Tris-HCl, 30 μM MgCl ₂ , 0.2% glucose, 0.1% casamino acid and pH 5.0. Culturing Salmonella in a medium,
(B) a step of recovering the protein secreted from the cultured Salmonella spp., and (c) a living body infected with Salmonella spp. in combination with the killed Salmonella spp. ) Is administered to
A method of protecting a living body (excluding human) from salmonellosis, including.