WO2016017620A1

WO2016017620A1 - Salmonella vaccine

Info

Publication number: WO2016017620A1
Application number: PCT/JP2015/071332
Authority: WO
Inventors: 江口　正浩; 小川　洋介; 和真白岩; 善弘下地; 英司大石
Original assignee: 国立研究開発法人農業・食品産業技術総合研究機構; 株式会社微生物化学研究所
Priority date: 2014-07-28
Filing date: 2015-07-28
Publication date: 2016-02-04
Also published as: JP2019199478A; JP6712760B2; JPWO2016017620A1

Abstract

It was discovered that when a combination consisting of a protein secreted by a bacterium belonging to the genus Salmonella with dead cells of a bacterium belonging to the genus Salmonella was administered to a living organism, a highly excellent defense effect against the infection with a bacterium belonging to the genus Salmonella was achieved compared with the case of administering either the protein or the dead cells singly. Thus, provided is a vaccine that has a high safety and an excellent defense capability against salmonellosis.

Description

Salmonella vaccine

The present invention relates to a Salmonella vaccine comprising a combination of secreted proteins of Salmonella and dead bacteria.

Since the occurrence of salmonellosis in the livestock industry is extremely economic loss, infection prevention measures for livestock and poultry are important. However, until now, the occurrence of salmonellosis in domestic livestock and poultry has not been completely suppressed.

The use of vaccines has been attempted to prevent Salmonella infection. For example, in Salmonella oral infection, it has been reported that when live bacteria such as attenuated strains are used as an immunogen, infection protection is exhibited. Vaccine development using this attenuated strain has an idea based on immunological and bacteriological findings and an excellent strategy to achieve the goal, but with the risk of side reactions and reversal of pathogenicity There is a problem.

For this reason, attenuated strain vaccines are not currently commercially available, and killed vaccines consisting exclusively of formalin and heat-killed Salmonella spp. Are used (for example, “Bovine Salmonella bivalent vaccine (Scientific Feed Co., Ltd.). Research institute), "chicken Salmonella inactivation 3 blend, KS (Kyoritsu Pharmaceutical Co., Ltd.)).

In addition, a vaccine using a culture supernatant of Salmonella spp. Cultured under specific conditions has been reported as a vaccine not using an attenuated strain. For example, in Patent Document 1, the culture supernatant of Salmonella cultivated in a medium containing low magnesium and low phosphate under SPI-2 induction conditions is used to spread the whole body of Salmonella infected with birds. It has been disclosed to have a protective effect.

However, the current situation is that a component vaccine that is excellent in safety and has an excellent protective effect against infection with Salmonella spp. Has not yet been developed.

Special table 2010-501599

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

In order to solve the above problems, the present inventor has intensively studied the use of components other than attenuated strains as the components of Salmonella vaccine. As a result, when the protein secreted from Salmonella and the killed Salmonella each were administered alone, there was no significant protective effect against Salmonella infection compared to the control treated with no treatment or adjuvant. I was not able to admit. However, it was surprisingly found that when a protein secreted from Salmonella and a killed Salmonella were administered in combination, they exhibited an extremely excellent protective effect against Salmonella infection. This protective effect was observed in all Salmonella infections examined. From these facts, the present inventors have found that the combination of a protein secreted from Salmonella and a killed bacteria of Salmonella is a Salmonella vaccine that has both high safety and excellent effects. As a result, the present invention has been completed.

More specifically, the present invention provides the following aspects.

(1) A vaccine for protecting a living body from salmonellosis, comprising a combination of a protein secreted from Salmonella and a dead fungus of Salmonella.

(2) The protein has 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, pH 5 The vaccine according to (1), which is secreted from Salmonella spp. Cultured in a medium of 0.0.

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

The vaccine of the present invention comprising a combination of a protein secreted from Salmonella and killed bacteria of Salmonella as an active ingredient provides 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 was administered alone or when the killed Salmonella was administered alone, no significant effect was 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.

In addition, when the above protein and killed bacteria were administered alone, abnormal symptoms were observed before the individual died, but in the administration of the vaccine of the present invention relating to those combinations, such abnormal symptoms were 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 species having different serotypes. Therefore, it can be widely applied in salmonellosis.

It is a graph which shows the protective effect from the infection of the Salmonella genus bacteria in the mouse | mouth immunized with the protein secreted from Salmonella genus and / or the dead bacteria of Salmonella genus. As controls, the results of no treatment and adjuvant administration are shown. It is a graph which shows the protective effect from the infection of 3 types of Salmonella in the mouse | mouth which immunized the mixture of the protein secreted from Salmonella and the dead bacteria of 3 types of Salmonella. As a control, no treatment results were shown.

The present invention provides a vaccine for protecting a living body from salmonellosis, comprising as an active ingredient a combination of a protein secreted from Salmonella and killed bacteria of Salmonella. The present invention also provides a method for protecting a living body from salmonellosis, wherein the vaccine is administered to a living body infected with Salmonella.

“Salmonellosis” in the present invention means an infection caused by Salmonella spp. Examples of “Salmonella spp.” That cause salmonellosis include, but are not limited to, Salmonella enterica subsp. In the production of the vaccine of the present invention, a multivalent vaccine can be obtained using two or more Salmonella species.

In the culture for secreting proteins to Salmonella, it is preferable to use an acidic minimal medium. Examples of acidic minimal medium include 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 suitably used.

In the present invention, “protein is 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. `` Is secreted from Salmonella cultivated in a medium of pH 5.0 with a composition of '' means that the protein has the `` property '' that it is secreted from Salmonella cultivated in the medium However, it does not limit the “acquisition method” of the protein.

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 Salmonellosis may be excluded from the vaccine components. Whether a specific secreted protein contributes to the control effect against salmonellosis is determined by administering a combination of the specific protein and a dead fungus of Salmonella to the mouse, infecting the mouse with Salmonella, It is possible to evaluate by testing the survival rate (see this example).

In the vaccine of the present invention, the “dead bacteria” of Salmonella genus used in combination with the secreted protein is, for example, heat treatment, chemical disinfectant treatment, irradiation of radiation or ultraviolet rays, mutation promoting substance It can be prepared by treatment or the like. In the preparation of dead bacteria, heat treatment or treatment with formalin which is a chemical disinfectant is preferably used. The dead bacteria may be a mixture of different Salmonella species (for example, a mixture of Salmonella species of different species or different serotypes). This makes it possible to expand the range of Salmonella that can be protected against infection.

In the vaccine of the present invention, “including a combination” of a protein secreted from Salmonella and a dead fungus of Salmonella means that the vaccine of the present invention contains a protein secreted from Salmonella and Salmonella. Even in the form of a single agent containing both killed bacteria as active ingredients, a combination of a preparation containing a protein secreted from Salmonella as an active ingredient and a preparation containing Salmonella killed as an active ingredient It means that it may be a 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, suspensions, surfactants, stabilizers, flavoring agents, fragrances, excipients, vehicles, preservatives. , Binders, diluents, tonicity agents, soothing agents, extenders, disintegrants, buffers, coating agents, lubricants, colorants, sweeteners, thickeners, flavoring agents, solubilizing agents, or solubilizing agents Although other additives etc. are mentioned, it is not restrict | limited to these. The active ingredient can be in various forms such as injections, aerosols, capsules, tablets, powders, granules, etc. depending on the administration method and therapeutic purpose.

Further, in the vaccine of the present invention, an adjuvant can be further added 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, and vitamin E, but are not particularly limited as long as the effect is exhibited.

The “living body” to which the vaccine of the present invention is administered means living animals and human bodies. The animal is not particularly limited as long as it can be infected by Salmonella, and it may be a domestic animal, a pet animal, a laboratory animal, or any other animal. It may be. Examples of animals include, but are not limited to, cows, pigs, horses, sheep, goats, chickens, ducks, geese, ducks, quails, 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 immunization to give immunity to the living body. When the vaccine preparation of the present invention is a concomitant drug, the preparations may be administered at the same time, or may be administered with a time difference within a range that does not diminish the effect of the combination.

The dose of the vaccine of the present invention may be an amount that can induce an immune response in a living body, such as the age and weight of animals and humans, the type of animal, the type of pathogenic bacteria (for example, the difference in the degree of pathogenicity, etc.) ), And the method and route of administration. The dose of the secreted protein as the active ingredient is usually 0.05 μg to 1500 μg, preferably 5 μg to 500 μg. A single dose of dead bacteria as an active ingredient is usually 10 ³ to 10 ¹⁰ and preferably 10 ⁶ to 10 ⁹ as the number of dead bacteria. Administration may be performed multiple times, and the administration interval in this case 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 spp. Salmonella Typhimurium (S. Typhimurium) (O4 group), Salmonella Choleraesuis (S. Choleraesuis) (O7 group), and Salmonella Dublin (S. Dublin) belonging to Salmonella enterica subsp. (O9 group) was used.

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

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

(3) Preparation of dead bacteria S. Typhimurium, S. Choleraesuis and S. Dublin were statically cultured overnight at 37 ° C. in LB medium, and the cells were centrifuged at 3,000 g for 20 minutes to collect the cells. Then, adjust to 1x10 ⁴ CFU / 500μl PBS, sterilize by boiling at 100 ° C for 5 minutes, and repeat ultrasonic crushing and resting state at 30 second intervals for 10 minutes using Biorutor (BM equipment). As a result, the cells were destroyed.

(4) Preparation of Secreted Protein S. Typhimurium was statically cultured overnight at 37 ° C. in LB medium, followed by shaking culture (160 rpm) at 37 ° C. overnight in a 50-fold amount of LB medium. Thereafter, the cells were collected by centrifugation at 3,000 g for 20 minutes, and statically cultured at 37 ° C. overnight in a pH 5.0 N-minimal medium that is half the amount of the medium used for shaking culture. The supernatant of the pH 5.0 N-minimal medium cultured in static ground was centrifuged at 3,000 g for 30 minutes, and the cells were further removed using a 0.22 μm filter. To the resulting supernatant, a final concentration of 25% trichloroacetic acid (Nacalai Tesque) was added and reacted overnight at 4 ° C., followed by centrifugation at 20,000 g for 45 minutes to recover the protein. Acetone was added to the obtained protein and washed by centrifugation at 20,000 g for 15 minutes, followed by dialysis with PBS for 2 days to remove trichloroacetic acid.

(5) Immunization method A 5-week-old female BALB / c mouse (Japan SLC) is mixed with a secreted protein solution (10 μg of secreted protein and an equivalent amount of Freund's complete adjuvant (Nippon Becton Dickinson Co., Ltd.). 100 μl of the obtained solution) and a dead bacteria solution (100 μl solution obtained by adjusting 1 × 10 ⁴ CFU dead bacteria to 50 μl and mixing with an equal amount of complete adjuvant) were subcutaneously immunized to mice. As controls, secreted protein alone, killed bacteria alone, complete adjuvant alone, or no treatment was also tested.

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

(6) Test of survival rate (a) Fasting for 6 hours before infection was performed, and then 50 μl of 10% sodium bicarbonate was orally administered. Fifteen minutes later, 1 × 10 ⁶ S. Typhimurium was orally infected and then fasted for 30 minutes. Observations were made for 40 days after infection (FIG. 1).

As a result, in the untreated group and the adjuvant immunized group, deterioration of hair gloss was observed from the third day after infection, and energy was lost from the fifth day. In addition, in the secreted protein immunity group and the killed bacteria immunity group, deterioration of hair gloss was observed from the 5th day after infection, and energy was lost from the 7th day. Mice in the untreated, adjuvanted, secreted protein, killed immunized group died from day 6 after infection and all died by day 10 after infection. On the other hand, half of the group administered the combination of killed bacteria and secreted protein survived until the 40th day after infection.

From the above results, it was found that a combination of killed Salmonella spp. And secreted protein is effective for oral infection protection.

(B) Next, a vaccine comprising a combination of a secreted protein and a dead bacterium is present in serotype Salmonella (S. Choleraesuis: O7 group, S. Dublin: O9 group) other than S. Typhimurium (04 group). It was also examined whether or not the infection protective effect was exhibited (FIG. 2). Until now, as a vaccine against Salmonellosis, a mixture of various serotypes of Salmonella inactivated (dead bacteria using formalin treatment) has been used. Therefore, a combination of a dead strain of three strains (S. Typhimurium, S. Choleraesuis, S. Dublin) used for infection and the Salmonella secreted protein prepared in (4) above as a vaccine, the above (6) (a The experiment was conducted in the same manner as in the experiment. In this experiment, 100LD ₅₀ cells (1 × 10 ⁶ S. Typhimurium, 1 × 10 ⁷ S. Choleraesuis, 1 × 10 ⁶ S. Dublin) were used for oral infection. As a control, verification without treatment was also performed.

As a result, when S. Typhimurium was orally infected, the group immunized with a combination of 3 types of dead bacteria and a secreted protein showed a significant protective effect (FIG. 2), consistent with the results of FIG. left). When S. Choleraesuis was orally infected, the untreated group all died in about 20 days, whereas the group immunized with a combination of 3 types of killed bacteria and secreted protein had a significant protective effect. Shown (middle of FIG. 2). In addition, even when S. Dublin was orally infected, the untreated group died in about 10 days, whereas the group immunized with 3 types of killed bacteria and secreted protein had a significant protective effect. (Right of FIG. 2). From the above results, it has been found that the vaccine of the present invention exhibits an excellent protective effect against Salmonella spp. With different serotypes.

An attenuated strain vaccine is known as a Salmonella vaccine with a high infection-protecting effect, but it is not commercially available due to safety issues. From the viewpoint of safety, vaccines using dead bacteria or specific proteins are desirable. The vaccine of the present invention using a combination of a secreted protein and dead bacteria is highly safe and can exert an excellent protective effect against infection with Salmonella 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

A vaccine for protecting a living body from salmonellosis, comprising a combination of a protein secreted from Salmonella and a killed Salmonella.
The protein is composed 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, pH 5.0 The vaccine according to claim 1, which is secreted from Salmonella cultivated in a medium.
A method for protecting a living body from salmonellosis, comprising administering the vaccine according to claim 1 or 2 to a living body infected with Salmonella.