KR102047910B1 - Fish Oral Vaccine Liposomes and Preparation Method Thereof - Google Patents

Abstract

The present invention relates to liposomes for an oral vaccine for fish and a method of preparing the same, and more specifically, to liposomes consisting of lecithin and cholesterol, and coated with chitosan, and to a composition for an orally administered vaccine for fish, comprising microbial cells of fish streptococcus. The liposomes, consisting of lecithin and cholesterol and coated with chitosan and the composition of the present invention can be easily administered, and show excellent stability when orally administered, and thus can efficiently prevent streptococcus infections in cultured fish.

Description

Fish Oral Vaccine Liposomes and Preparation Method Thereof}

The present invention relates to a liposome for oral vaccines for fish and a method for preparing the same, and more particularly, oral administration of a liposome and a fish streptococci cell which are composed of lecithin and cholesterol and coated with chitosan. It relates to a vaccine composition for.

Due to the development of aquaculture technology and the increase in production, the scale of disease inevitably increases, causing the domestic aquaculture industry to suffer severe obstacles (Cho et al., Fish Pathol . 20: 61-70, 2007). Antibiotics, which are used indiscriminately for the treatment of these diseases, have increased side effects due to misuse, such as causing multidrug-resistant bacteria, and the therapeutic effect is also very low. Therefore, the development of useful vaccines would be the right direction to reduce antibiotic use and increase production. However, the currently developed fisheries vaccine is a very difficult to effectively treat a large amount of fish in the field, such as the injection vaccine causes stress during the vaccine treatment process. In addition, there was a case where the vaccine was mixed with feed for the development of oral vaccine, but the effect was insufficient. Therefore, there is a need for the development of an effective vaccine that can increase the ease of inoculation.

In most fish, infections such as the respiratory (gill), digestive (stomach and intestine), and body surface wounds (skin) are mainly mucosal tissues, and inducing a mucosal immune response is the most effective defense strategy. Due to the nature of the aquatic environment, fish mucosa is always exposed to various environments including pathogens. Thus, the mucosal immune response using oral vaccines can be used to promote a more effective prevention system for pathogens. In addition, due to the nature of the aquaculture industry where vaccination costs are closely related to the economics of the industry, it can be very useful in the field. Mucosal vaccines also have the advantage of being painless and refusal, safe and easy to administer because they do not use needles. Indeed, previous studies have reported that mucosal immunization provides a more effective defense against pathogenic microorganisms infected via mucosal pathways than parenteral immunization (Shim, B.-S., et al. , PloS one, 6 (11): e27953, 2011; Neirynck, S., et al., Nature medicine, 5 (10): 1157-1163, 1999; Rao, SS, et al., PloS one , 5 ( 3): e9812, 2010).

In conclusion, the biggest advantage of mucosal immunization using the mucosal immune system is that injection vaccines, which are commonly used, can only induce a systemic immune response. In contrast, mucosal (nasal or buccal) vaccines produce systemic immunity that produces antigen-specific serum IgG, as well as mucosal immune responses that produce antigen-specific secretory IgA on the mucosal surface. Response can be induced simultaneously (Neutra, M et al., Nature Reviews Immunology, 6 (2): 148-158, 2006; Holmgren, J et al., Nature medicine , 11: S45-S53, 2005).

However, despite the recent development and research by many researchers, mucosal vaccines have been difficult to commercialize due to the efficiency, safety and lack of mucosal immune enhancers. In the case of oral administration of antigen, destruction of the antigen by digestive enzymes is inevitable, and it is necessary to establish a method for safely delivering the antigen to the intestine to induce an effective immune response. In other words, it is urgent to develop a technique for delivering mucosal immunity to the intestinal mucosa without being decomposed and denatured in the stomach.

 Accordingly, the present inventors have made diligent efforts to develop an optimal antigen-delivery system for a new fish oral vaccine that can overcome this problem. As a result, a liposome that encapsulates an immunogen into microparticles is a fish oral vaccine. It was confirmed that the drug carriers exhibit excellent efficacy, and completed the present invention.

An object of the present invention is to efficiently prevent the streptococcal infection of fish through the induction of mucosal immunity by oral administration of the vaccine, liposomes and fish chains composed of lecithin and cholesterol, coated with chitosan The present invention provides a vaccine composition for oral administration of fish comprising a bacterium cell.

Another object of the present invention is streptococci of fish comprising the step of orally administering a feed composition for the prevention or treatment of streptococcal infection of fish comprising the vaccine composition for oral administration of the fish and the vaccine composition for oral administration to the fish It provides a method for preventing or treating an infection.

In order to achieve the above object, the present invention comprises the steps of (a) dissolving lecithin and cholesterol (cholesterol) in an organic solvent in a ratio of 1: 0.1 to 0.3 by weight; (b) hydrating the mixture after concentration under reduced pressure to prepare liposomes through lipid film formation; (c) collecting fish streptococci cells in liposomes by adding and sonicating the inactivated fish streptococci cells to the formed liposomes; (d) coating the liposomes from which the fish streptococci cells are collected with chitosan; And (e) washing the coated liposomes to obtain liposomes for fish oral vaccines. The method also provides a method for preparing liposomes for fish oral vaccines containing fish streptococci.

The present invention also provides an oral vaccine composition for fish comprising liposomes prepared by the above method and in which inactivated fish streptococci cells are collected, wherein the liposomes are formed by lecithin and cholesterol. It provides a vaccine composition for oral administration of fish, characterized in that the coating with chitosan (chitosan).

The present invention also relates to a feed composition for the prevention or treatment of streptococcal infection of fish comprising the vaccine composition for oral administration.

The present invention also relates to a method for treating streptococcal infection of fish comprising the step of orally administering the vaccine composition for oral administration to fish.

Vaccine composition for oral administration of fish consisting of lecithin and cholesterol (cholesterol), coated with chitosan and fish streptococci cells according to the present invention is easy to inoculate, excellent stability during oral administration In this way, streptococcal infection of cultured fish can be prevented efficiently.

1 is a schematic of S. parauberis FKC antigen production.
Figure 2 shows a liposome (left) and FKC staining (right) in which FKC (formalin killed cells) were collected.
Figure 3 analyzes the liposome stability according to the thermal change.
Figure 4 shows the collection rate of liposomes, (a) HPLC results of ibuprofen, (b) HPLC results of gallic acid, (c) HPLC results of the upper liposomes, (d) HPLC results of the lower liposomes.
5 is a liposome prepared in a ratio of 50:10 lecithin and cholesterol selected through in vitro verification, (a) FKC-free control, (b) Probe sonication, (c) reverse phase evaporation method, ( d) manufactured by bubbling method and (e) injection method.
Figure 6 shows the analysis of antigen-specific antibody titers after oral administration of FKC-containing liposomes.
FIG. 7 analyzes antigen-specific antibody titers after 5 weeks of oral administration of FKC-containing liposomes.
8 shows a process for preparing a polysaccharide coated liposome.
Figure 9 shows (a) 0.5 wt% and (b) 1 wt% chitosan coating of liposomes prepared by the Probe sonication method.
Figure 10 shows (a) 0.5 wt% and (b) 1 wt% chitosan coatings of liposomes prepared by the megadose method.
Figure 11 is a comparative analysis of the stability of uncoated liposomes (left) and chitosan coated liposomes (right) for 7 days, (a) liposome stock solution, (b) 10-fold dilution of liposomes, (c) 100-fold dilution of liposomes It is shown.
12 shows a liposome elution graph.
FIG. 13 shows liposome lipid membrane fluidity, and shows zeta potential of (a) Probe sonication method, (b) Probe sonication method-coating, (c) megadose method, and (d) megadose method-coating.
14 is a measurement of liposome size, and shows a) the probe particle method, (b) the probe method method-coating, (c) megadose method, (d) megadose method-the average particle diameter of the coating.
FIG. 15 shows antigen specific antibody titers after 2 weeks of coated and uncoated liposome oral vaccine administration.
FIG. 16 shows antigen specific antibody titers at 1 week of boosting after 3 weeks of coated and uncoated liposome oral vaccine administration.
Figure 17 shows the survival rate according to the period after administration of the coated and uncoated liposome oral vaccine.
Figure 18 shows the relative survival after administration of the coated and uncoated liposome oral vaccine.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

Injectable vaccines are immunized by intramuscular or intraperitoneal vaccines, which can be used with adjuvant to provide a strong immune response, and are very difficult to treat on large scale fish in the field, such as causing stress during vaccine treatment. Immersion vaccine is a method of immersing fish in a high concentration of vaccine solution and immunizing with mucous membranes such as body surface or gills, and has a disadvantage in that a large amount of vaccine is required and the efficacy or duration is very low while handling is less stressed.

Most of the vaccines currently in use are injectable vaccines, which activate the systemic immune system to induce the efficacy of the vaccine. However, there are questions about whether the injected vaccine can effectively induce mucosal immunity of tibia fish. Compared with oral vaccines, parenteral vaccines are generally known to have lower immune immunity than expected, despite their higher protective immunity.

Oral vaccines that induce mucosal immune responses can induce an immune response directly from the mucous membrane of the site of infection, but have strong adhesiveness in the gastrointestinal tract and disrupt the production of antibodies due to the destruction of antigens by strong acids and degrading enzymes. Research is needed to overcome this.

Due to the nature of the aquatic environment, the mucous membrane of fish is always exposed to various environments including pathogens. Thus, the mucosal immune response using oral vaccines can be used to promote a more effective prevention system for pathogens. In addition, due to the characteristics of the aquaculture industry, where vaccination costs are closely related to the economics of the industry, it can be very useful in the field. can do.

Accordingly, in the present invention, to develop a vaccine that can prevent streptococcal infection of fish with excellent mucosal immunity, coating chitosan on liposomes formed of lecithin and cholesterol to collect fish streptococci cells. A vaccine composition for oral administration of fish was prepared to determine the effect of preventing fish streptococcal infection.

Accordingly, the present invention provides a method for preparing a composition comprising: (a) dissolving lecithin and cholesterol in a organic solvent in a ratio of 1: 0.1 to 0.3 by weight; (b) hydrating the mixture after concentration under reduced pressure to prepare liposomes through lipid film formation; (c) collecting fish streptococci cells in liposomes by adding and sonicating the inactivated fish streptococci cells to the formed liposomes; (d) coating the liposomes from which the fish streptococci cells are collected with chitosan; And (e) washing the coated liposomes to obtain liposomes for fish oral vaccines. The method relates to a method for preparing liposomes for fish oral vaccines containing fish streptococci.

In the present invention, the fish is preferably a marine fish or freshwater fish, more preferably flounder, flounder, sea bass, trout, black sea bream, red sea bream, sea bream, other sea bream, minfish, yellowtail, puffer fish, puffer rock, other rockfish It is preferable to be selected from the group consisting of fish, mackerel, trout, trout, mullet, salmon, conger, horse mackerel, eel, reference group, tuna, sturgeon, carp, eel, catfish, mandarin fish and tropical fish. However, the present invention is not limited thereto.

In the present invention, the fish Streptococcus cells are preferably fish pathogenic Gram positive cocci, more preferably Streptococcus parauberis ( Streptococcus parauberus ) , Streptococcus iniae (Streptococcus innia ) , S difficile (Streptococcus difficile ) , L piscium Lactococcus , Vagococcus salmoninarum , Lactococcus garvieae (= Enterococcus seriolicida) (Lactococcus garbier = Enterococcus ceriosaida) , Streptococcus agalactiae ( Streptococcus agalactiae ), Streptococcus dysgalactiae (= Streptococcus difficilis ) (Streptococcus disgalactis dipicactis) , Streptococcus milleri (Streptococcus millery ) , Lactococcus piscium (Lactococcus fish) and Carnobacterium piscicola (Carnobacterium fish cola) is preferably selected from the group consisting of, and most preferably Streptococcus parauberis 19FBSPa0001 (KCTC 13795BP), but is not limited thereto.

Streptococcus parauberis 19FBSPa0001 (KCTC 13795BP) of the present invention was deposited with the Korea Bio Resource Center on January 22, 2019.

In the present invention, the cells are preferably formalin killed cells (FKC), but are not limited thereto.

In the present invention, when the weight ratio of lecithin and cholesterol is less than 1: 0.1 or greater than 1: 0.3, the stability, collection rate and specific antibody formation ability of liposomes are reduced. Therefore, the weight ratio of lecithin and cholesterol is preferably 1: 0.1 to 0.3, more preferably 1: 0.2, but is not limited thereto.

That is, the greater the amount of cholesterol, the higher the stability (hardness) of the liposome, but the lower the fluidity.

In the present invention, the organic solvent is chloroform, methanol, methanol, ethanol, propanol, isopropanol, butanol, acetone, acetone, ether, benzene. (benzene), ethyl acetate, (methylene chloride), hexane (hexane) and cyclohexane (cyclohexane) is selected from the group consisting of, more preferably chloroform (chloroform), but It is not limited.

In the present invention, the reduced pressure concentration may be characterized in that carried out at 400-700 mmHg and 45 ~ 55 ℃ conditions.

In the present invention, the chitosan concentration is preferably 0.5 to 1 w%, but is not limited thereto.

The longer the coating time, and the higher the concentration of chitosan solution, the thicker the resulting coating can be, thus slowing the release of immunogenicity. Therefore, a person skilled in the art can adjust the concentration and coating time of the chitosan solution in consideration of the release rate of the desired drug.

In the present invention, when the chitosan concentration is 1 w% or more, the tendency of entanglement between liposomes increases and productivity decreases, and when it is 0.5 w% or less, stability decreases.

In the present invention, the chitosan solution preferably contains 0.5 wt% lactic acid, but is not limited thereto.

In the present invention, the sonication may be characterized in that it is processed 6 times, 5 seconds each, 30 seconds for a total of 30 seconds using a probe. The probe sonication method can produce a larger size of liposomes than other methods.

In the present invention, the coated liposomes preferably further comprise the step of washing with PBS, but is not limited thereto.

In the present invention, the term "liposome" has a single-layer or multilayered lipid-double membrane structure most similar to a cell membrane having a matrix form of a phospholipid bilayer. Phosphatidylcholine (PC), ethanolamine (PE), serine, sphingomyelins, cardiolipins, plasmalogens, phosphatidic acid, cerebroside. Phospholipids, the basic unit of liposomes, consist of two hydrocarbon chains, anionic or polar, and nonpolar. Hydrocarbon chains vary in length, and in the case of natural phospholipids, hydrocarbons are 16 or more in length and have a degree of unsaturation of about one pair.

Liposomes, which span a wide range of 200-1000 nm in diameter and usually consist of five or more concentric circles, are multilammellar vesicles (MLVs), and the smallest sized liposomes that the phospholipid head can arrange while overcoming curvature are small unilamellar It is a vesicle (SUV) and usually the diameter of SUV is 20-50nm. Liposomes with a diameter of 100-1000 nm and monolayers are called large unilamellar vesicles (LUVs), liposomes with a diameter of about 100 nm and monolayers are intermediate-sized unilamellar vesicles (IUVs) and participate in metabolism as natural or synthetic phospholipids. Interchangeable with biomembrane molecules, biodegradable, minimally toxic to the body, and can capture active substances without chemical bonding.

In addition, it is possible to protect chemically sensitive molecules, has excellent selective permeability and retardation, and is suitable for the production of release rate control system, and by changing the composition, size, charge permeability, surface ligand, etc. of lipids constituting liposomes. The distribution and properties of liposomes in an organism can be adjusted according to circumstances. It has the advantage that it can be selectively used to deliver both hydrophilic and lipophilic drugs, and the drug is released slowly during the slow decomposition, so the potential for application as a drug carrier is very high. In addition, liposomes have many advantages as drug carriers. However, when administered orally, it can be easily degraded or dissolved by low stomach pH and digestive enzymes. Therefore, to protect the membrane of liposomes by coating the surface using a polysaccharide polymer to improve its stability and to effectively deliver the drug to the desired site.

In particular, the size of liposomes is related to the rate of absorption in the body.

In the present invention, the liposome is preferably 800 to 1300nm in size, more preferably 900 to 1100nm, but is not limited thereto.

If the size of the liposome is less than 900nm it is difficult to capture a sufficient amount of fish streptococcus cells (FKC), when the 1100nm or more decreases the absorption rate in the body.

Since the liposome of the present invention is about 1000 nm in size, the antigen can be sufficiently collected. In addition, the size of the liposomes by the chitosan coating is larger, the size of the liposomes produced by the probe method is larger, concentrated at 1㎛, so the absorption is better. The specific size range of liposomes is particularly related to the rate of absorption in the body. In liposome preparation, the rate of capture along with the rate of absorption are the most important.

In another aspect, the present invention is an oral vaccine composition for fish comprising liposomes prepared by the above method, the inactivated fish Streptococcus cells are collected, the liposomes are formed by lecithin and cholesterol (cholesterol) The present invention relates to a vaccine composition for oral administration of fish, which is coated with chitosan.

In the present invention, the term "vaccine" refers to a biological preparation containing an antigenic substance that immunizes a living body. An immunogen that induces immunity to a living body by injecting or injecting a living body to prevent an infectious disease is prevented. Say. In vivo immunization is largely divided into autoimmunity, in which immunity is automatically obtained after infection by pathogens, and passive immunity obtained by an externally injected vaccine. While autoimmunity is characterized by a long period of generation of antibodies related to immunity and continuous immunity, passive immunization with vaccines acts immediately to treat infectious diseases, but has a disadvantage of poor sustainability.

The antigenic substance of the present invention is any one selected from the group consisting of peptides, polypeptides, lactic acid bacteria, proteins expressing the polypeptide, lactic acid bacteria, oligonucleotides, polynucleotides, recombinant bacteria and recombinant viruses expressing the protein. can do. For example, the antigenic substance may be inactivated in the form of whole or partial cell preparations, or in the form of antigenic molecules obtained by conventional protein purification, genetic engineering techniques or chemical synthesis, Streptococcus parauberis , Streptococcus iniae , S difficile , L piscium , Vagococcus salmoninarum (Bago Caucus nareom live monitor), Lactococcus garvieae (= Enterococcus seriolicida ) ( = Enterococcus Lactococcus teach in non-caucus Seri raise cider), Streptococcus agalactiae (Streptococcus agalactia), Streptococcus dysgalactiae (= Streptococcus difficilis ) ( Streptococcus dygalactia = Streptococcus difficilli ) , Streptococcus milleri Streptococcus miliri , Lactococcus piscium (Lactococcus fish) and Carnobacterium piscicola It is preferably selected from the group consisting of (Carnobacterium fish cola), but is not limited thereto. Additional antigens may include pathogenic viruses in the form of inactivated whole or partial cell preparations, or antigenic molecules obtained by conventional protein purification, genetic engineering techniques or chemical synthesis.

In the present invention, the antigenic substance is preferably formalin killed cell (FKC), but is not limited thereto.

The content of the antigenic substance is 1 to 50% by weight, preferably 10 to 30% by weight, based on the total weight of the vaccine composition.

Compositions for vaccines according to the invention include stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylenes, bacterial cell walls, derivatives of bacterial cell walls , Bacterial vaccine, animal poxvirus protein, subviral particle adjuvant, cholera toxin, N, N--dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monoforce And at least one second adjuvant selected from the group consisting of foryl lipid A, dimethyldioctadecyl-ammonium bromide and mixtures thereof.

The vaccine composition of the present invention may also comprise a veterinary acceptable carrier. "Veterinically acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvant, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Carriers, excipients, and diluents that may be included in the composition for vaccines include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the vaccine composition of the present invention can be used in the form of oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions, respectively, according to conventional methods. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used can be prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate and sucrose in the lecithin-like emulsifier. (sucrose), lactose (lactose), gelatin, etc. can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc may also be used. As a liquid preparation for oral administration, suspending agents, liquid solutions, emulsions, syrups, etc. may be used, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. Can be. Formulations for non-oral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations. As the non-aqueous preparation and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used.

The vaccine composition of the present invention may include inactivated cells of the fish pathogen or may further include a buffer solution, and the inactivated cells may be prepared by a formalin treatment method, a heat treatment method or a freeze treatment method, but are not limited thereto. .

In addition, the vaccine composition of the present invention may further comprise an adjuvant or an immune enhancer, preferably the immune enhancer is selected from the group consisting of polysorbate-based nonionic surfactants, aluminum hydroxide gels, squalene and chitosan. It may include one or more selected. Examples of polysorbate-based nonionic surfactants may be Tween 80, Tween60, or Tween 20, and more preferably Tween 80.

The vaccine composition according to the present invention may be prepared by suspending a fish pathogen in a buffer to use as a suspension, or by mixing a buffer suspension containing a fish pathogen and an immunoadjuvant. Alternatively, fish pathogens can be prepared by adding a suspension to a buffer solution in a mixed solution of an adjuvant and a buffer. The vaccine composition according to the invention may further comprise additives such as excipients and / or stabilizers.

In another aspect, the present invention relates to a feed composition for preventing or treating streptococcal infection of fish comprising the vaccine composition for oral administration.

The term "feed" in the present invention means any natural or artificial diet, single meal or single meal ingredients for eating, ingesting and digesting animals.

The feed composition may be prepared by adding a certain amount of the vaccine composition according to the present invention to a medium used in a conventional fish culture.

In another aspect, the present invention relates to a method for treating streptococcal infection of fish, comprising orally administering the vaccine composition for oral administration to fish.

The content of the inactivated cells contained in the vaccine composition according to the present invention may be set by appropriately adjusting the fish or pathogen to be applied. The vaccine composition of the present invention may be prepared in consideration of the content of the inactivated cells injected per fish, and the content of the cells may be administered in batches or divided times.

[ EXAMPLE ]

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Antigen Preparation

Streptococcus parauberis (19FBSPa0001) was isolated from diseased flounder in Wando, 2005, and was deposited on 22 January 2019 at the Korea Institute of Biotechnology and Biotechnology Center (Accession No .: KCTC13795BP).

S. parauberis 19FBSPa0001 is Gram positive cocci with a diameter of 0.5 ~ 2㎛. S. parauberis The bacteria were inoculated in BHIB (Brain heart infusion agar) medium to which 1.5% (v / v) NaCl was added, followed by incubation at 27 ° C for 24 hours. Next, colonies of cultured plate media were inoculated into BHIB to which aseptically added 1.5% NaCl, and then incubated at 150 rpm and 27 ° C. for 48 hours in a stirred incubator (JS Research Inc.). 37% (v / v) formalin (Merck, Germany) was added to the cultured bacteria solution so that 0.5% (v / v) of each culture solution was inactivated at 150 rpm and 20 ° C for 24 hours in a stirred incubator. The inactivated bacteria were centrifuged at 12,000 RCF, washed three times with sterile physiological saline, and the cells were collected and finally weighed by the weight of the wet cells and suspended according to the concentration. The vaccine solution was dispensed into sterile glass bottles and stored at 4 ° C. for use in experiments.

EXAMPLE  2: Fish oral vaccine according to lipid composition Liposome  Produce

In this embodiment, lecithin (Solec FS-B, medifuture, Korea) or phosphatidylcholine (P3556-1G, sigma, Korea) is used as a phospholipid, and cholesterol (cholesterol; C8667-25G, sigma, Korea) is used. Liposomes were prepared. In addition, liposomes were prepared by varying the lipid composition ratio to prepare liposomes in various sizes. Liposomes were prepared by varying the lipid composition of LC (lecithin): CHOL (cholesterol) or PC (phosphatidylcholine): CHOL (cholesterol) in a weight ratio of 50: 1-20.

Known methods for preparing liposomes include Bangham method (MR Mozafari et al., Technology and Health care . 10: 342-344, 2002), Probe type sonication method (Kim et al., The Pharmaceutical Soc . Korea. 38: 131- 139, 1994), Reverse phase evaporation method (S. Channarong et al, Pharm . Sci . Tech. 12: 192-200, 2011), Bubbling method (Madhav and A. Saini. IJRPC, 1 (3): 2231- 2781, 2011), Injection method (M. Pons et al., Int . J. Pharm . 95: 51-56, 1993; C. Charcosset et al., Eng . Res. De s. 508-515, 2015) and Homo mixer emulsion method (Jee et al., J. Kor. Pharm. Sci . 25: 249-264, 1995) and the like.

The antigen used was FKC (formalin-killed cells) antigen prepared in Example 1.

As a result, a large amount of liposomes were produced when prepared with phosphatidylcholine, but lecithin was more suitable in consideration of commercial use. In addition, liposomes prepared at an LC: CHOL = 50: 10 ratio were more stable than liposomes prepared at other ratios, and formed around 1000 nm suitable for sufficiently collecting the cells, thereby forming high-quality liposomes.

EXAMPLE  3: In vitro  Through verification For oral vaccines Optimal Liposomes  Selection

3-1: Liposome  Stability investigation

Since the stability of liposomes is affected by the composition, preparation method, form and size of the lipids used, liposomes with excellent stability were selected for the vaccine.

0.5 g lecithin and 0.1 g cholesterol are placed in a 50 ml round flask and dissolved in chloroform. The solvent was evaporated using a rotary evaporator at a bath temperature of 60 ° C. until a thin lipid film was formed on the flask wall. 3 ml of S. parauberis and 3 μl of 50 mM calcein (calcein; C0875-5G, sigma, Korea) were added to the resulting lipid membrane, and the temperature was fixed at 50 ° C. in a bath type sonication and hydrated for 1 hour. After the sonicator, 10 ml of the buffer was added and washed by centrifugation three times for 10 minutes at 3,000 rpm. After discarding the supernatant, dilute the precipitate 50 times, and again put 9 ml of Triton X-100 (X100-100ML, sigma, Korea) 2 ml (blank; buffer 2 ml) in a test tube, and store it for a certain period of time under various conditions. Separated. The supernatant was filtered through a filter of pore size 0.45 μm (Dismic-25cs, advantec, Tokyo) and the intensity of the fluorescence intensity of the filtrate was measured.

The stability of liposomes was examined by measuring the fluorescence of calcein flowing out over time while leaving a 30 μm-sized liposome enclosed with calcein at 20 ± 0.5 ° C. The amount of calcein encapsulated in liposomes was determined by measuring the absorbance at 495 nm, the maximum absorption wavelength of calcein liberated upon liposome destruction, and determining the concentration, or measuring the fluorescence of calcein liberated at 515 nm.

The liposomes were provided with heat conversion energy (80 ° C., 5 min) to induce liposome degradation, and the stability of the liposomes was measured by measuring the amount of fluorescent material measured.

As a result, when calcein, which is a fluorescent substance, was eluted at 470-509 nm, it was found that the liposome was stable at room temperature, and the stability of the liposome was decreased as the temperature was increased (FIG. 3).

After stability measurement, five excellent liposomes (LC: CHOL = 50: 10 ratio i) FKC-free control, ii) Probe sonication method, iii) reverse phase evaporation method, iv) bubbling method, v) injection method; 5) was selected and analyzed for in vivo antibody formation ability.

3-2: Liposome Capture rate  Measure

Liposomes containing 10% gallic acid (gallic acid; junsei, Japan) were prepared and dissolved in PBS containing ibuprofen (ibuprofen; I4883-1G, sigma, Korea) and used for HPLC experiments. The upper layer and the lower layer supernatant liposomes were evaporated with Triton X-100, and the residual amount was measured by HPLC after the filter at the same concentration. HPLC was quantitatively analyzed using Waters (Alliance e2695 Separations Module), UV detector (Waters 2489 UV / Vis Detector) and Empower3 software. The column was a C18 column (SunFireTM, 4.6 * 300mm, 5μm). Phosphate buffer (pH 6.8): Acetonitrile = 65: 35 was used as HPLC analysis conditions. The flow rate was 1 ml / min, the sample injection amount was 10 μl, and the wavelength was UV 222 nm. Analysis of gallic acid in the test solution showed that the retention time of gallic acid was 1.98 ± 0.02 minutes and 6.01 ± 0.09 minutes for ibuprofen.

The capture rate was calculated by dividing the upper and lower layers of liposomes into the content of gallic acid for ibuprofen as a standard (FIG. 4).

Capture Rate (%) =

As a result, it was found that the liposome upper layer was 84%, the lower layer was 16%, and the liposome capture rate was 16%.

EXAMPLE  4: In vivo  Through verification For oral vaccines Optimal Liposomes  Selection

Non-specific factors and environmental changes were minimized during flounder stocking and incubation , and the results of the disease test before the experiment were negative, and liposome oral administration and specific-antibody formation ability analysis were performed in the flounder without S. parauberis infection history. Was performed.

4-1: Liposomes Oral vaccine  administration

The selected liposomes were mixed with the feed powder and adjusted to a concentration of 3 mg / fish to prepare a liquid. Oral administration was performed by directly injecting liquid oral vaccine solution into the gastric lumen using a syringe with a zona pellucida, and then administered on 1, 3 and 5 days, respectively. PBS and feed mixtures were injected into the control, and S. parauberis FKC was intraperitoneally injected at a concentration of 1 mg / fish for specific-antibody comparison.

Peripheral antigen injection and intraperitoneal antigen injection 2-3 weeks after peripheral blood was extracted from the arrhythmia and intestinal tissue was extracted through dissection. After leaving the blood for 1 hour at 4 ℃ centrifuged for 15 minutes at 4 ℃ at 6,000 rpm and serum was separated. After the intestine was removed and excised, the foreign material on the outer surface was gently removed from the PBS, and the mucous membrane was separated by gently scraping the inside of the intestine using a spatula. Then, 1.5 ml of PBS was added, and then passed through a 0.45 μm syringe filter. Antibodies were used for analysis.

4-2: Specific Antibody Using ELISA Formation ability  analysis

Two to three weeks after oral administration of liposomes, S. parauberis- specific antibody titers in halibut serum and intestinal mucosa were compared and analyzed by ELISA.

Dispense S. parauberis FKC to a concentration of 10 μg / well on a 96-well plate, and attach antigen to the bottom of the plate at 4 ℃ over night. Blocking was performed at room temperature for 1 hour using 3% skim milk, and 10-fold dilutions of halibut serum and intestinal mucus with primary antibody were allowed to react for 1 hour. Thereafter, after washing three times with PBST, the secondary antibody was diluted 1: 1000 with Anti-Japanese flounder IgM Mab and reacted at 37 ° C. for 1 hour. After washing, the aliquots of AP-conjugated anti-mouse IgG (whole molecule) were diluted 1: 1000 and then dispensed and reacted at 37 ° C for 1 hour, washed, and then added with a substrate (PNPP substrate solution) for 20 minutes to absorb 405 nm. It was confirmed at.

As a result, three weeks after oral administration of liposome-coated FKC, the highest antigen-specific antibody titer was identified in No. 2 (liposomes having a lecithin and cholesterol ratio of 50:10 and manufactured by the Probe Sony method) among the liposome candidate groups ( 6), after 5 weeks of oral administration, all antigens of serum and intestinal mucosa had low antigen-specific antibody titers similar to those of the control (FIG. 7), which is similar to previous studies showing that antibody titers fell over time. similar.

Therefore, No. 2 (LC: CHOL = 50: 10 ratio; Probe Sony) was finally selected as a liposome candidate group for oral vaccines. The liposome prepared at this ratio and the LC: CHOL = 50: 10 ratio is suitable as a stable liposome for vaccines having a size of 1 μm. The prepared liposomes were tested as a control.

EXAMPLE  5: polysaccharide coating Liposomes  Produce

5- 1: chitosan  coating Liposomes

It was prepared by coating a polysaccharide in various ratios to liposomes for selected fish oral vaccine through the verification of Example 4.

The polysaccharide coating is hydrated by adding 25 mL of polysaccharide solution with different concentrations of PBS buffer to hydrate the lipid membrane on the flask wall during liposome preparation. In addition, after the liposome particles are formed, the liposome precipitate is mixed in an aqueous polysaccharide solution in the final washing process, and then centrifuged at 12,000 rpm and 4 ° C. for 15 minutes to obtain a liposome precipitate coated with the outside of the liposome membrane. Polysaccharide was mainly used chitosan, alginic acid, polysaccharide solution was 0.5, 1.0, 2.0, 3.0, 4.0, 6.0 wt% concentration and the solvent was PBS buffer solution. The chitosan solution is made at a concentration of 0.5 or 1.0 wt% and likewise using PBS buffer solution as solvent. Chitosan is a water-soluble polymer, but it does not melt well at room temperature, but also does not melt at pH 7.4 of PBS buffer, but only in acidic conditions, so that lactic acid is added to make acidic conditions by adding 0.5wt% of lactic acid to dissolve chitosan. . The procedure for preparing a liposome coated with polysaccharide is shown in FIG. 8.

5-2: Probe Type Sony  Chitosan coating prepared by the method Liposomes

Liposome preparation coated with chitosan was prepared by dissolving 0.5 g lecithin and 0.1 g cholesterol in a 50 ml round flask and dissolving well with chloroform. The solvent was evaporated using a rotary evaporator (N-1000, Rikakikai, Tokyo) at a bath temperature of 60 ° C. until a thin lipid film was formed on the flask wall. 3 ml of S. parauberis FKC was added to the resulting lipid membrane, and the temperature was fixed at 50 ° C. in a bath type sonicator (branson 5210, Korea) and hydrated for 1 hour. After hydration, ultrasound was applied 6 times (30 seconds total) for 5 seconds using a probe sonicator (sonic dismembrator model 500, fisher scientific, Japan), and 0.5 or 1.0 wt% of polymeric chitosan (448869-50G, sigma aldrich, Korea) The solution was added and coated in a stiller for 1 hour. Centrifugation and washing three times for 10 minutes at 3000 rpm to obtain a liposome precipitate coated with the outside in the liposome membrane. The supernatant was discarded and placed in 2 ml of the buffer and checked under a microscope (Fig. 9). Polymeric chitosan aqueous solution is not only soluble at room temperature, but also soluble in buffer and not only in acidic conditions, so that lactic acid (junsei, Japan) is added to make acidic conditions by adding 0.5 wt% of dissolved acid and chitosan.

5-3: Megadose  Manufactured by Liposomes

9g lecithin was added to the beaker, and 50ml of PBS was dissolved in a stiller for 1 hour and 30 minutes. After 1 minute in a homo mixer, 10 ml of S. parauberis FKC was added and mixed again for 1 minute. After centrifugation three times for 10 minutes at 3,000 rpm, washed with a polymer chitosan solution, coated for 1 hour in a stiller and washed again to obtain a coated liposome precipitate. The supernatant was discarded and 2 ml of buffer was added and checked under a microscope (Fig. 10).

EXAMPLE  6: polysaccharide coating Liposome  Stability verification

6-1: chitosan coating Liposome  stability

Liposomes are severely unstable from leaking hydrophilic drugs from liposomes, and the physical stability of the membrane bilayer is stabilized by the addition of cholesterol. To compare the membrane stability of liposomes with and without coating, the uncoated liposomes prepared by the probe type sonication method and chitosan coated liposomes were compared with the storage time.

Stability test results from day 1 to day 7 to produce liposomes, both uncoated liposomes and coated liposomes were stable for 7 days (Fig. 11).

6-2: Gastric juice, antigen dissolution test according to pH change

Antigen dissolution test was performed in simulated gastric fluid TS (pH 1.2) using a dissolution test apparatus (KDIT-200, Kukje Engineering Co., Korea) according to the KPIT method 2 (paddle method). Place the eluate in a container, rotate the paddle at a speed of 100 rpm, add liposomes, filter the eluate after 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 minutes Analyzed by HPLC. The mobile phase was a mixture of acetonitrile, methanol, 0.01 M phosphate buffer (pH 7.4) (30; 20; 50), and the flow rate was 1.0 mL / min. Lichrospher RP-18 was used for the column, and the injection volume was 10 μL and the detection wavelength was 275 nm.

HPLC results were calculated as (A-B / A) * 100 (A: liposomes before elution in gastric juice, B: liposomes by elution time in gastric juice). The results showed that Probe- uncoated was eluted at 5 minutes, Probe-coated at 15 minutes, megadose-uncoated at 20 minutes, and megadose-coated at 25 minutes. At 25 minutes, all liposomes showed the same elution pattern without significant difference in elution at each time period.

Therefore, chitosan-coated liposomes were found to have lower elution rate in gastric juice than uncoated liposomes (FIG. 12). In addition, liposomes prepared by the megadose method proceeded more slowly in acid than liposomes prepared by the probe method.

6-3: Geological membrane  Liquidity measurement

The electrodynamic potential difference resulting from the difference in positive charge density based on repulsive force or attraction between particles is called zeta potential, and the degree of dispersion or aggregation of particles in solution can be evaluated. In general, the greater the absolute value of the potential difference, the better the dispersion.

The zeta potential between the zeta potential -200 and 200 mV at a semiconductor wavelength of 664.5 nm was measured (zeta-potential & particle size analyzer ELSZ-2000ZS, photal otsuka electronics, Japan).

As a result, the zeta potential of Probe type-coated liposome was -11.97mV, Probe type-coated -14.04mV, megadose-coated -16.31mV, and megadose-coated -16.36mV. That is, the absolute value of the zeta potential of the coated liposomes was large and was well dispersed (FIG. 13).

6-4: Liposomes  Size measurement

When nanoscale particles become small particulates, they are affected by the molecular motion of the solvent, causing Brownian motion. Brownian motion is a phenomenon in which microparticles move irregularly in a liquid or gas. The speed of motion is affected by the size of particles. Small particles move faster and larger particles move slowly. The dynamic light scattering method occurs when light is emitted to the particles and the phase difference scattering phenomenon according to the particle motion speed is used. The particle size and distribution were analyzed using the principle of dynamic light scattering.

Liposomal size was measured by the Korea Institute of Polymer Science (KSM) using zeta-potential & particle size analyer ELSZ-2000ZS. PBS was used as a solvent and the average particle diameter was analyzed at a particle size of 0.1 nm at 10 μm using the principle of dynamic light scattering at 25 ° C.

As a result, Probe type-coated liposomes had an average particle size of 980 nm, Probe type-coated liposomes had an average of 1164 nm, megadose-coated 289 nm, and megadose-coated 318 nm (FIG. 14). That is, it can be seen that the average particle diameter of the coated liposome is larger.

The liposomes of the megadose method had a high dispersion of various sizes, and the probe type liposomes had a higher concentration of 1 μm, resulting in better absorption. Therefore, the higher the stability of the probe-type liposome with good absorption rate is expected to be better in vivo .

EXAMPLE  7: polysaccharide coating Liposome Immunogenic capacity  analysis

7-1: Polysaccharide Coating Liposomes Oral vaccine  administration

Polysaccharide-coated and uncoated liposomes were mixed with feed powder and prepared in a liquid state by adjusting the dose to a concentration of 3 mg / fish. The composition of the liposome oral vaccine is shown in Table 1 below.

S. parauberis FKC Coating One dose of vaccine Blank - - - Probe soni ○ - FKC-3 mg / fish Probe soni-coating ○ ○ FKC-3 mg / fish Megados ○ - FKC-3 mg / fish Megados-coating ○ ○ FKC-3 mg / fish Intraperitoneal ○ - FKC-1 mg / fish Oral ○ - FKC-3 mg / fish Control - - -

For oral administration, a liquid oral vaccine solution was directly injected into the gastric lumen using a syringe with a zona pellucida. 1, 3 and 5 days respectively. PBS and feed mixtures were injected into the control, and S. parauberis FKC was intraperitoneally injected at a concentration of 1 mg / fish for specific-antibody comparison.

Experiments were carried out by receiving 30 flounder in each of the 1 liposomes + FKC, ② coated liposomes + FKC, ③ liposomes (without FKC), ④ FKC intraperitoneal administration, ⑤ FKC oral administration, ⑥ control six tanks.

7-2: Specific Antibody Using ELISA Formation ability  analysis

S. parauberis FKC was prepared at 10 ⁸ cfu / ml, and 100 µl was dispensed into each well of a 96-well plate and the antigen was attached to the plate bottom at 4 ° C over night. Blocking is performed at room temperature for 1 hour using 5% skim milk, and 10-fold dilutions of halibut serum and intestinal mucus with primary antibody are allowed to react for 1 hour. After washing three times with PBST, the secondary antibody was diluted with 1: 1000 Anti-Japanese flounder IgM Mab and then reacted at 37 ° C. for 1 hour. After washing, the aliquots of AP-conjugated anti-mouse IgG (whole molecule) were diluted 1: 1000 and then dispensed, and then reacted at 37 ° C. for 1 hour. After washing, the substrate was added with PNPP substrate solution, and the absorbance was 405 nm after 20 minutes. It was confirmed at.

Two weeks after the coated and uncoated liposome oral vaccine administration, the antigen-specific antibody titer was analyzed by ELISA. Appeared (FIG. 15). In addition, the antigen-specific antibody titer was analyzed by boosting the vaccine 3 weeks after the oral vaccine administration, and the highest antibody titer was found in the serum and intestinal mucosa of the Probe Sony coating-coated group. Significantly high antibody titers were seen (FIG. 16).

EXAMPLE  7: Coating for vaccine Liposomes  Survival rate analysis for oral administration

Two weeks after oral administration of coated and uncoated liposomes, S. parauberis specific antibody titers in halibut serum and intestinal mucosa were compared and analyzed by ELISA method. In addition, three weeks after oral administration of coated and uncoated liposomes and antigen intraperitoneal administration, S. parauberis (KSP28) was injected subcutaneously at a concentration of 10 ⁶ CFU / fish, and then analyzed for survival and relative survival for 20 days.

S. parauberis artificial infection test after coating and uncoated liposome oral vaccine to flounder showed the highest survival rate (83%) in the Probe Sony-coated test group, intraperitoneal (IP, 70%) and general oral administration (Oral, 53%) showed a higher survival rate (FIG. 17). Meagados and megados-coatings had survival rates of 43% and 46%, respectively. All flounder did not show mortality after 12 days of infection, and total mortality did not occur in the control (control-13%, blank-10).

In addition, the relative survival analysis showed that the Probe Sony-coated test group had the highest survival rate (70%), intraperitoneal administration (57%), Probe Sony-uncoated (44%), and oral administration (40%). ), The relative survival rate was high (FIG. 18).

In other words, the antigen-specific antibody formation ability after oral administration of liposomes and viability after artificial infection showed that Probe Sony-coated liposomes were most suitable as oral vaccine drug carriers for fish. Increased antigen-specific antibody titers in both serum and intestinal mucosa could also be identified, indicating that oral vaccines can activate mucosal immunity or induce systemic immune responses via the oral route.

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology KCTC13795BP 20190122

Claims (19)

Method for preparing a liposome for oral vaccines containing fish streptococcus cells comprising the following steps:
(a) dissolving lecithin and cholesterol in an organic solvent in a ratio of 1: 0.1 to 0.3 by weight;
(b) hydrating the mixture after concentration under reduced pressure to prepare liposomes through lipid film formation;
(c) adding the inactivated fish streptococci cells to the formed liposomes and performing sonification using a probe to collect the fish streptococci cells in the liposomes;
(d) coating the liposomes from which the fish streptococci cells are collected with chitosan; And
(e) washing the coated liposomes to obtain liposomes for fish oral vaccines of 900-1100 nm size.
According to claim 1, wherein the fish is flounder, flounder, perch, trout, black sea bream, red sea bream, rock bream, other sea bream, sea fish, yellowtail, puffer fish, sea bass rockfish, other rockfish, mackerel, songme, trout, mullet Or salmon, conger eel, horse mackerel, eel, reference group, rodent, sturgeon, carp, eel, catfish, mandarin and tropical fish.
According to claim 1, wherein the fish Streptococcus cells Streptococcus parauberis ( Streptococcus parauberus ) , Streptococcus iniae (Streptococcus innia ) , S difficile (Streptococcus difficile ) , L piscium Lactococcus , Vagococcus salmoninarum , Lactococcus garvieae (Lactococcus Garbier ), Streptococcus agalactiae Streptococcus dygalactiae , Streptococcus dysgalactiae , Streptococcus milleri (Streptococcus milleri) , Lactococcus piscium (Lactococcus fish) and Carnobacterium piscicola ( Carnobacterium fish) A method for preparing a liposome for oral fish, characterized in that selected from the group consisting of fish.
The method according to claim 1, wherein the cells are formalin killed cells (FKC).
The method of claim 1, wherein the organic solvent is chloroform, methanol, methanol, ethanol, propanol, isopropanol, butanol, acetone, acetone, ether, Method for producing a liposome for oral fish fish, characterized in that selected from the group consisting of benzene, ethyl acetate, ethyl acetate, methylene chloride, hexane and cyclohexane.
The method of claim 1, wherein the reduced pressure concentration is performed at 400-700 mmHg and 45 ° C. to 55 ° C. 2.
The method of claim 1, wherein the chitosan is a fish liposome for oral vaccines, characterized in that 0.5 ~ 1 w%.
The method of claim 1, wherein the sonication using the probe is performed 6 times at 5 seconds for a total of 30 seconds.
delete An oral vaccine composition for fish prepared by the method of claim 1 and comprising liposomes in which inactivated fish streptococci cells are collected, wherein the liposomes are formed by lecithin and cholesterol, and chitosan ) Is a vaccine composition for oral administration of fish, characterized in that 900 to 1100nm size is coated with.
The fish of claim 10, wherein the fish is flounder, flounder, perch, trout, black sea bream, red sea bream, rock bream, other sea bream, sea fish, yellowtail, puffer fish, sea bass, rockfish, mackerel, mackerel, trout, trout, mullet A vaccine composition for oral administration of fish, characterized in that the fish selected from the group consisting of salmon, conger eel, horse mackerel, eel, reference group, rodent, sturgeon, carp, eel, catfish, mandarin and tropical fish.
The Streptococcus parauberis of claim 10, wherein the fish Streptococcus cell is ( Streptococcus parauberus ) , Streptococcus iniae (Streptococcus innia ) , S difficile (Streptococcus difficile ) , L piscium Lactococcus , Vagococcus salmoninarum , Lactococcus garvieae (Lactococcus Garbier ), Streptococcus agalactiae Streptococcus dygalactiae , Streptococcus dysgalactiae , Streptococcus milleri (Streptococcus milleri) , Lactococcus piscium (Lactococcus fish) and Carnobacterium piscicola ( Carnobacterium fish cola) is a vaccine composition for oral administration of fish, characterized in that selected from the group consisting of.
delete A feed composition for preventing or improving streptococcal infection of fish, comprising the vaccine composition for oral administration of claim 10.
15. The method of claim 14, wherein the fish is flounder, flounder, perch, squid, black sea bream, red sea bream, rock bream, other sea bream, sea fish, yellowtail, puffer fish, sea bass, rockfish, mackerel, mackerel, trout, trout, mullet Feed composition, characterized in that selected from the group consisting of salmon, conger eel, horse mackerel, eel, reference group, rodent, sturgeon, carp, eel, catfish, mandarin and tropical fish.
The method of claim 14, wherein the fish streptococcal infectionStreptococcus parauberis (Streptococcus parauberus), Streptococcus iniae (Streptococcus innia), S difficile (Streptococcus difficile), L piscium (Lactococcus fish), Vagococcus salmoninarum (Bagococcus Salmonanarum),Lactococcus garvieae (Lactococcus Garbier),Streptococcus agalactiae (Streptococcus agalactia),Streptococcus dysgalactiae Streptococcus Disgalactia, Streptococcus milleri(Streptococcus milleri), Lactococcus piscium (Lactococcus fish) andCarnobacterium piscicola A feed composition, characterized in that the infection by the cells selected from the group consisting of (Carnobacterium fish cola).
A method for preventing or treating streptococcal infection of a fish, comprising the step of orally administering the vaccine composition for oral administration of claim 10 to a fish.
According to claim 17, The fish is flounder, flounder, perch, tuna, black sea bream, red sea bream, rock bream, other sea bream, sea fish, yellowtail, puffer fish, sea bass, rockfish, mackerel, mackerel, trout, trout, mullet Prevents or treats streptococcal infections of fish, characterized in that they are selected from the group consisting of fish, salmon, conger eel, horse mackerel, eel, reference group, rodent, sturgeon, carp, eel, catfish, mandarin and tropical fish How to.
18. The method of claim 17, wherein the fish streptococcal infection is Streptococcus parauberis ( Streptococcus parauberus ) , Streptococcus iniae (Streptococcus innia ) , S difficile (Streptococcus difficile ) , L piscium (Lactococcus pium ) , Vagococcus salmoninarum (Bagoccus Salmonanarum), Lactococcus garvieae (Lactococcus Garbier ), Streptococcus agalactiae (Streptococcus agalactia), Streptococcus dysgalactiae Streptococcus disgalactia, Streptococcus milleri , Lactococcus piscium (Lactococcus) and Carnobacterium piscicola A method for preventing or treating a streptococcal infection of fish, characterized in that the infection is caused by a bacterial cell selected from the group consisting of (Carnobacterium fish cola).

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