KR101569151B1 - A Vaccine Composition For cultured fish - Google Patents

Abstract

The present invention relates to a vaccine composition for aquaculture fish, and more particularly to a vaccine composition for aquaculture fish containing nanoliposome and an antigenic substance containing glyceryl cocoate / citrate / lactate, an oil component, a higher alcohol and an aqueous component ≪ / RTI >

The vaccine composition for aquaculture of the present invention induces a stronger immune response, has a sustained release time of the efficacy, and is greatly improved in safety when applied to fish, thereby increasing the shelf life of the composition, So that the effect as a vaccine formulation is excellent.

Nano-liposomes, vaccines, fish culture, bacterial, antigens

Description

{Vaccine composition for cultured fish}

The present invention relates to a vaccine composition for aquaculture.

A fish is a thermophilic animal that has some adaptability to the environment, but if it exceeds that limit, it will cause physiological disorder. In fish, the disease is a state in which the internal and external environment, such as land animals, can no longer maintain a healthy state. Disease is a phenomenon that occurs as a result of a correlation between a host's factor, an onset factor, and the environment. Of the disease-causing factors, only the onset factor does not necessarily cause the disease, I never do that. In other words, the disease does not occur in the condition that the factor of the onset, the factor of the host and the environment are well balanced, and it occurs when the balance is broken. In most cases, the occurrence of the disease is greatly influenced by the environmental condition.

Pathways of invading pathogens into the body of the fish can be seen in skin, gills, nasal passages, and digestive tracts. These organs are protected from mucus, enzymes, antimicrobial and bactericidal substances that enter by invasion and action of antibodies, leukocytes, macrophages, etc. These protective materials are damaged by mechanical damage or parasitic injuries during the selection process, insufficient management of breeding, etc., where the pathogens penetrate into the body and then reach the organs or tissues in the bloodstream to form lesions .

There are protozoa (parasites), fungi (molds), bacteria, viruses and the like. Parasites are very small, ranging from several centimeters to several hundreds of micrometers, bacteria to a few micrometers, and viruses to a few thousandths of a micrometer. Therefore, the ways in which they damage fish and how to counteract them are of course different.

Korean marine aquaculture has continuously improved productivity with the development of aquaculture technology. However, the aquaculture industry is currently suffering from deterioration of coastal aquaculture environment, aging of fisheries due to long-term intensive farming, Productivity is deteriorating. Especially, various infectious diseases and their complications that frequently occur in fish species due to diversification of aquaculture species are accompanied by the emergence of drug resistant bacteria due to frequent use of chemotherapeutic agents, and the production cost due to the degradation of cultured fish, , And measures against such infections are currently the most important challenge in the aquaculture industry. In addition, since the proportion of damage caused by disease occurrence tends to increase, there is a fundamental method of treating diseases by preventing disease occurrence in advance and taking countermeasures.

On the other hand, bacterial diseases of aquaculture are caused by conditional pathogens that cause fish to die from pathogenic bacteria and secondary infections caused by other causes. The breeding population of a farm is rich in organic matter, so it is an environment where many germs can reproduce, causing diseases when fish are not healthy or their defense is weakened. Bacterial diseases are classified into diseases according to the type and symptoms of infecting bacteria, and they are highly infectious and highly infectious, and once the infection is done, there is a large cumulative mortality, which leads to a great economic loss in a farm.

Currently, there are various bacterial diseases in domestic fish farms such as Vibrio disease, Edwardian disease, Streptococcus, and Slide bacterial disease. However, Edwardian disease and Streptococcus are the most common diseases in flounder farming among bacterial diseases.

Edwardsiella tarda, a gram-negative bacillus, has been reported to be present in aquaculture environments as well as other bacteria and causes disease in many tibia fishes (Wyatt et al., 1979; Yasunaga et al., 1982) (1982), flounder (Yasunaga et al., 1982), tilapia (Chanook et al., 1976), chinook salmon (Amandi et al. (Miyashita, 1984; Oh and Chun, 1988). When looking at the water tank in which the disease occurs, these individuals are blackened in color, dying around the central drainage pipe, floating around the drainage pipe and dying. External symptoms include ascites, fins and abdominal flares and redness, abdominal distension, and hernias. Pathogens that survive in migratory fish, reared water, and poor quality of fish are infected orally through the fish or through damaged areas of the skin or gill. Although this disease tends to occur throughout the year, it is more prominent in the summer when the water temperature is maintained at 20 to 30 ° C, and especially when it exceeds 25 ° C, the incidence rate and the damage rate also increase together. (Aoki et al., 1985), it has been suggested that Edward's disease in flounder farms may cause serious damage, but the treatment effect is poor due to the occurrence of resistant bacteria (Mizono, 1993), but the effect is generally not high.

Streptococcosis is a disease that causes a lot of damage not only in Korea but also in neighboring countries such as Asia, USA, South Africa, Australia, Israel and Europe. Streptococcus iniae (syn. Shilot) , S. difficile, Lactococcus garvieae (syn. Enterococcus seriolicida), L. piscium and Vagococcus salmoninarum. Flounder infected with Streptococcus pneumoniae (S. iniae) protrudes with clouding of body color, clouding of eyes, redness of the eye margin and fins. In addition, when it is dissected, it is known that redness, enlargement or reduction of liver, inflammation and hemorrhage of intestinal tract, intracerebral hemorrhage and agglutination inside the lid of gill are formed.

Flounder, which is a major aquaculture fish in Korea, is fast growing in the domestic aquaculture tanks and high in feed efficiency, and is cultivated in the east, west, south sea and Jeju island coast, and is the most dominant species of aquaculture fish production . The flounder has been suffering from various diseases from the beginning of the mulch, and the cumulative mortality of the flounder is 20 ~ Among them, the damage caused by bacterial diseases is the largest, and among the bacterial diseases, the damage caused by Edward's disease and Streptococcus is more than 80%. Among them, Edward's disease is caused by a long period of time, especially in the flounder farm, especially in the high temperature. Therefore, various measures such as antibiotics for bacterial diseases have been prepared. However, (Aoki et al., 1985), but the effect is not so high (Mizo et al., 1993). Mekuchi et al., 1995).

According to a recent study by Jin et al. (2005), bacterial diseases accounted for 45.6% of bacterial diseases in the Jeju Island during 9 years from 1996 to 2004 (10,187 cases), and among the bacterial diseases, 44.3% , Staphylococcus aureus (24.9%), Slug bacteremia (19.7%) and Edwards disease (11.1%), and the monthly incidence of streptococcosis was in the order of 8, 9, 10, and July. As a result of investigation of the occurrence of streptococci by fish size, 13 cases were found in 20 ㎝ or less feces, 61 cases were found in 20 to 30 cm size flora, 133 cases were found in 30 to 40 cm flounder and 83 cases were found in more than 40 cm flounder And 30 cm or more.

As a result of multiplex PCR, 187 strains isolated from the test samples were found to have 44.9% of S.iniae and 55.1% of S.parauberis. Recently, Kim et al. (2005) reported the difference in pathogenicity according to the morphological characteristics of S. iniae isolated from flounder. S. iniae isolated from domestic cultured flounder was isolated from non-viscous colony hemolytic Streptococcus spp., which is a mucinous viscous form with a higher number of cells per chain than the type, and has a capsule reported by Yoshida et al. (1996). Also, it is reported that the strains with capsules are more resistant to host killing than the strains without capsules, and this capsule plays an important role in the pathogenicity of the bacteria.

 On the other hand, the vaccine is prepared to induce an immune response upon antibody formation to a specific disease by administering microorganisms (bacteria, viruses), weakened organisms and the like, which have been sterilized, to the body and have been developed for the purpose of preventing a specific infectious disease The disease can be prevented by the immunity made in the body where the antigen (pathogen) is administered. However, since the immune responses of individuals are different from each other, 100% efficacy is not necessarily guaranteed. However, the increase in the incidence of intractable malignant infectious diseases, The need for appropriate vaccines is increasing due to increased adoption and concerns about the spread of disease due to the movement of infected seeds.

Adjuvants are needed to increase the efficacy and persistence of the vaccine and to induce an appropriate immune response. Although the vaccine immunity enhancer can maximize the specific immune response to the antigen and the efficacy of the vaccine, there is no universal immunity enhancer, and an appropriate immunity enhancer is selected according to parameters such as target animal, disease, etc. The choice of immunostimulant without immunity enhancement and side effects is very important in vaccine development.

As a preventive vaccine against streptococci, Ghittino et al. (1995) investigated the efficacy of formalin-inactivated whole-cell vaccines of S. iniae and L. garvieae in rainbow trout, and Eldar et al. (1995) Reported the immunity of whole cell vaccines and bacterial protein extracts against S. difficile in tilapia, and Klesius et al. (2000) reported that the singleinfected cells of formalin-killed cells and concentrated extracellular products (ECPs) of S. iniae There is a report on the immune response of tilapia to combined vaccine. Evans et al. (2004) compared the efficacy of formalin-killed cells and ECPs in S. agalactiae by intraperitoneal injection and immersion immunization of tilapia, and recently, the possibility of live attenuated vaccines in addition to whole-cell vaccines and ECPs Research is also being attempted.

Accordingly, the present inventors have made intensive efforts to maintain the safety and efficacy of the medicines such as the generation of resistant bacteria by the abuse of drugs by minimizing the damage of the domestic aquaculture environment by reducing the occurrence of bacterial diseases in fishes. As a result, The present inventors have found that a vaccine composition for fish including a potentiator can prevent and treat the occurrence of bacterial diseases in aquaculture fish.

Accordingly, the present invention provides a vaccine composition for aquaculture fish comprising a nanoliposome and an antigenic material containing glyceryl cocoate / citrate / lactate, an oil component, a higher alcohol and an aqueous component.

Other objects and advantages of the present invention will become more apparent from the following examples and claims.

The vaccine composition for aquaculture of the present invention induces a stronger immune response, has a sustained release time of the efficacy, and is greatly improved in safety when applied to fish, thereby increasing the shelf life of the composition, So that the effect as a vaccine formulation is excellent.

In the present invention, "glyceryl cocoate / citrate / lactate" is a nonionic surfactant represented by the following chemical structure, similar in chemical structure to lecithin, a natural surfactant contained in large amounts in egg yolk and soybean. Unlike phosphatidyl choline coupled lecithin, the chemistry of the following structure is characterized by the ester linkage of citric acid lactate glycerol coconut oil, where R is composed of an alkyl group of 12-16 carbons. The above-mentioned glyceryl cocoate / citrate / lactate can be chemically synthesized, and can be extracted in a natural state, or commercialized ones can be purchased and used

≪ Structure of glyceryl cocoate / citrate / lactate >

In the above formula, R is a C12-C16 alkyl group.

The content of glyceryl cocoate / citrate / lactate of the present invention is 0.01 to 20% by weight, preferably 0.1 to 5% by weight based on the total weight of the nanoliposome.

Polysorbate and sorbitan oleate may be used as a secondary emulsifier of glyceryl cocoate / citrate / lactate in the nanoliposome of the present invention. The polysorbate may be, for example, Twin-80, Twin-60 , Tween-40 and Tween-20, the sorbitan oleate is Span 80, and the content of the auxiliary emulsifier is 0.1 to 2% by weight, preferably 0.2 to 1% by weight based on the total weight of the nanoliposome.

As used herein, the term "oil component" refers to an oil that can not be metabolized by fish, refers to an inorganic oil, and the "inorganic oil" referred to above refers to a mixture of liquid hydrocarbons obtained from petroleum through a distillation technique . The term encompasses "inorganic diesel, " oil that is similarly obtained by distillation of petroleum, but whose specific gravity is slightly lower than liquid paraffin. Such oils include alkanes, alkenes, alkanes, and their corresponding acids and alcohols, their ethers and esters, and mixtures thereof. Preferably, the individual compounds of the oil are light hydrocarbon compounds, i.e., those having 6 to 30 carbon atoms. The oils can be prepared synthetically or purified from petroleum products. The oil is a non-metabolizable oil, and as an inorganic oil preferred for use in the nanoliposome and vaccine composition of the present invention, for example, mineral oil, squalane, hydrogenated polydecene, hydrogenated polydecene, castrol, paraffin oil, and non-polar oils such as cycloparaffin.

The term "fish" used in the present invention refers to flounder, rockfish, rockfish, rhododendron, red sea bream, lily fish, mullet, perch fish, whale fish, mackerel fish, horse mackerel, , Red sea bream, lion fish, mullet, sea bass and whale fish, and more preferably, it is not limited to flounder or rockfish.

 The oil component in the nanoliposome of the present invention is 0.1 to 50% by weight, preferably 1 to 20% by weight, and more preferably 1 to 10% by weight based on the total weight of the nanoliposome.

The aqueous component of the present invention can be water, buffered saline or any other suitable aqueous solution and is present in an amount of 1 to 90% by weight based on the total volume of the nanoliposome.

The term "nanoliposome" in the present invention means a nano-liposome having a typical liposome form and having an average droplet size of 50 to 500 nm, preferably 80 to 300 nm, most preferably 100 to 200 nm . The term "liposome" as referred to above refers to mutually spontaneous binding and aligned colloidal particles of amphiphilic molecules with water-soluble hair and insoluble tail, with a hydrophilic space inside and a closed double lipid membrane outside (A kind of pouch) having a bilayer structure similar to a cell membrane. Nano-liposomes can contain water-soluble molecules (including DNA) or drugs in the central hydrophilic space, attach lipid soluble drugs to external lipid bilayers, or bind positively charged and negatively charged substances. The greatest characteristic of nanoliposomes is that they are easy to change due to their diversity and elasticity, and they can be provided with completely new properties, functions and applications by different structures and components. Nano-liposomes have excellent biocompatibility, degradability and stability, and can be widely used as a delivery material for numerous substances and drugs. In general, liposomes containing nanoliposomes are useful for drug and gene transfer in four main ways: 1) wrapping and protecting drugs or DNA to prevent them from being destroyed by enzymes in the blood; 2) the circular state has a function of dragging a substance which is difficult to enter into cells, such as DNA, into cells; 3) serve as a kind of drug delivery system (DDS) that regulates drug release to be delayed; 4) Pointing the therapeutic substance to the target cell, or going to a specific site, can prevent it. Thus, the nanoliposome of the present invention has the advantage of minimizing adverse effects while maximizing the effect of the target drug.

Examples of the substance that can be contained in the nanoliposome of the present invention include peptides, proteins, fats and DNAs such as antibacterial agents, immunomodulators, antigens (including antigenic substances) and antibodies, cytokines, Such as preserving agents, osmotic agents, bioadhesive molecules, and immunostimulatory molecules. Also, the nanoliposome of the present invention is similar to a vital cell membrane, and thus can be widely used as a model of biological membrane function studies and drug permeation.

The nano-liposome of the present invention has a W / O / W form and can penetrate smoothly into blood vessels because it is a water phase. It has a longer duration than W / O type and is safe as a biodegradable material, It is smaller in size than O / W nanoliposome, and the outer membrane is strong, thereby securing the stability of the drug and maximizing the effect of the drug.

The "average droplet size" of the nanoliposomes of the present invention refers to the volume average diameter (VMD) particle size within the volume distribution of particle size. The VMD is calculated by multiplying the particle diameter of each particle by the volume of all the particles of the above-mentioned size. This is then divided by the total volume of all particles.

The term "vaccine" in the present invention refers to a biological agent containing an antigenic substance that immunizes a living body, and includes an immunogen that immunizes a living body by injection or injection into an organism It says. In vivo immunity is largely divided into autoimmunity, which is obtained automatically in vivo after infection with pathogenic bacteria, and passive immunization, which is obtained by externally injected vaccine. While autoimmunity has long duration of immune-related antibodies and is characterized by persistent immunity, passive immunization by vaccine acts immediately for the treatment of infectious diseases but has a disadvantage that its persistence is poor.

The antigenic substance of the present invention is any one selected from the group consisting of a peptide, a polypeptide, a lactic acid bacterium expressing the polypeptide, a protein, a lactic acid bacterium expressing the protein, an oligonucleotide, a polynucleotide, a recombinant bacterium and a recombinant virus can do. As a specific example, the antigenic substance may be in the form of an inactivated whole or partial cytostatic form, or in the form of antigenic molecules obtained by conventional protein purification, genetic engineering techniques or chemical synthesis, such as Edwardsiella tarda, (Streptococcus iniae (syn S. shilot), S. difficile, Lactococcus garvieae (syn. Enterococcus seriolicida), L. piscium, Bagococus salmonarum Vagococcus salmoninarum, Mycoplasma MYO, Mycoplasma hypo-pneumoniae, Haemophilus somnus, Haemophilus parasuis, Bordetella bronchiseptica, Actino bacillus flavorum pneumoniae, Pasteurella mule Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, , Streptococcus uberis, Streptococcus suis, Staphylococcus aureus, Erysipelotrix rusopathiae, Campylobacter sp., Fusobacterium necropolum, Escherichia coli, Salonella entericarparcaris , A pathogenic bacterium such as Leptospira sp., A pathogenic fungus such as Candida, a protozoan such as cryptosporidium parbium, neosporacanum, Toxoplasma gondii, oremeria spp., Ostertagia, couperias, There are antigens produced from intestinal parasites such as E. coli, preferably Edwards salata, Streptococcus, S. diffire, Lactococcus japonica, L. piscium, and Bago corus salmonarum. Activated whole or partial cellular preparation, or by conventional protein purification, genetic engineering techniques or chemical synthesis It may contain the pathogenic virus of the antigen type molecule.

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.

The composition for a vaccine according to the present invention can be used as a stabilizer, emulsifier, aluminum hydroxide, aluminum phosphate, pH adjuster, surfactant, liposome, iscom adjuvant, synthetic glycopeptide, extender, carboxypolymethylene, bacterial cell wall, , A bacterial vaccine, an animal poxvirus protein, a subviral particle adjuvant, a cholera toxin, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, And at least one second auxiliary agent selected from the group consisting of formaldehyde A, dimethyl dioctadecyl-ammonium bromide, and mixtures thereof.

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

In addition, the vaccine composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like in the form of oral preparations and sterilized injection solutions according to a conventional method. In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. As the liquid preparation for oral administration, suspensions, solutions, emulsions, syrups and the like may be used. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, . Formulations for non-oral administration include sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, and freeze-drying agents. As the non-aqueous preparation and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

As a surfactant stabilizer for strengthening nanoliposomes in the preparation of the nanoliposome contained in the vaccine composition of the present invention, higher alcohol and higher fatty acid may be further used as an immersion agent in consideration of sea habit.

Examples of the higher alcohol or higher fatty acid include myristyl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, etc. The content of the higher alcohol or higher fatty acid is 0.1 to 5% by weight based on the total weight of the nanoliposome , Preferably 0.2 to 1 wt%, based on the total weight of the composition.

 Also provided is a method for increasing the antibody production rate to an antigen by injecting the vaccine composition into a fish.

The following experimental examples and examples illustrate the present invention, but the present invention is not limited thereto.

Example 1. Preparation of nanoliposome

Nano liposomes were prepared with the compositions shown in Table 1 below.

Preparation of nanoliposome (content:% by weight) ingredient Comparative Example 1 Comparative Example 2 Comparative Example 3 Experimental Example 1 Experimental Example 2 Experimental Example 3 Experimental Example 4 Experimental Example 5 Experimental Example 6 Experimental Example 7 Experimental Example 8 Experimental Example 9 Experimental Example 10 Experimental Example 11 Experimental Example 12 A Purified water to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to
100 lecithin One 3 4 - - - - - - - - - - - - Glyceryl cocoate / citrate / lactate - - - 4 4 4 4 4 4 4 4 4 4 4 4 B Mineral oil 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Twin 60 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Myristyl alcohol 0.1 0.5 1.0 Cetyl alcohol 0.1 0.5 1.0 Stearyl alcohol 0.1 0.5 1.0 Behenyl alcohol 0.1 0.5 1.0 C Edward test antigen (5 mg / ml) 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

The A phase and the B phase were completely dissolved by heating to 80 ° C and the B phase was mixed into the A phase, and the mixture was emulsified at 3500 rpm using a homomixer (TK-homomixer, Mark-2-F). The emulsified mixture was slowly cooled to 40 ° C., and when the temperature reached 40 ° C., an antigen of C phase was mixed. Then, the mixed solution of A, B and C phases was stirred at 3500 rpm using the same homomixer as above, (Niro, Soavi, Italy) at a pressure of 500 bar.

The glyceryl citrate / lactate / linolate / oleate on phase A was purchased from Sasol, Germany and the Edward test antigen on C, the antigenic component, was used as a test antigen (5 mg / ml) was used.

Example 2. Energy filtration transmission electron microscope (EF-TEM) photograph of nanoliposome particles

The nanoliposome interface was photographed using an energy-filtration transmission electron microscope (EF-TEM, Carl Zeiss) in order to discriminate whether the nanoliposome was formed in the multilayer of Experimental Example 5 prepared in the present invention. Respectively.

As can be seen from the results of FIG. 3, it was confirmed that the interface of the nanoliposome particles made of glyceryl cocoate / citrate / lactate of the present invention was composed of a double layer. It is considered that the shape is not circular because the strength of the particles is flexible.

Example 3: Stability test of nanoliposome

Stability tests of the compositions of Comparative Examples 1 to 3 and Experimental Examples 1 to 12 prepared in Example 1 were carried out. The stability evaluation was carried out for 8 weeks under the conditions of temperature cycling in which the precipitation state, hue, and extent of composition of the composition were circulated at 4 占 폚, 25 占 폚, 42 占 폚, and 24 hour period from 4 占 폚 to 50 占 폚. Respectively.

Stability test of nano liposome stability Settling state Fling color (Temperature) 25 ℃ 42 ° C Temperature cycling 4 ℃ 42 ° C 42 ° C Comparative Example 1 stability stability Sedimentation Sedimentation Sore Discoloration (yellow) Comparative Example 2 stability stability Sedimentation Sedimentation Sore Discoloration (yellow) Comparative Example 3 stability stability Sedimentation Sedimentation Sore Discoloration (yellow) Experimental Example 1 stability stability stability stability stability Stable (white) Experimental Example 2 stability stability stability stability stability Stable (white) Experimental Example 3 stability stability stability stability stability Stable (white) Experimental Example 4 stability stability stability stability stability Stable (white) Experimental Example 5 stability stability stability stability stability Stable (white) Experimental Example 6 stability stability stability stability stability Stable (white) Experimental Example 7 stability stability stability stability stability Stable (white) Experimental Example 8 stability stability stability stability stability Stable (white) Experimental Example 9 stability stability stability stability stability Stable (white) Experimental Example 10 stability stability stability stability stability Stable (white) Experimental Example 11 stability stability stability stability stability Stable (white) Experimental Example 12 stability stability stability stability stability Stable (white)

As can be seen from the results of Table 2, in the case of nanoliposomes (Comparative Examples 1 to 3) prepared with lecithin, precipitation and coalescence were observed at 4 ° C, 42 ° C and 24- But in the case of the nanoliposomes containing the glyceryl cocoate / citrate / lactate and the higher alcohol of the present invention, they were stable without precipitation, discoloration or alteration.

Example 4. Safety test of nanoliposome

Experimental Examples 2, 5, 8, and 11 prepared in Example 1 were used to test 80 days after the vaccination with the test vaccine by intraperitoneal or immersion method for 10 days to evaluate clinical activities such as mobility, anorexia, abdominal distension, And the symptoms were observed.

Results of the safety test for flounder by inoculation route by test vaccine Test group inoculation Number of clinical manifestations after vaccination (cumulative number of deaths) Route One 2 3 4 5 6 7 8 9 10 Experimental Example 2 Abdominal cavity 0 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) Immersion 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Experimental Example 5 Abdominal cavity 0 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) Immersion 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Experimental Example 8 Abdominal cavity 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Immersion 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Experimental Example 11 Abdominal cavity 0 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) Immersion 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

As can be seen from the results of the above Table 3, in the cases of the infusion of the experimental group 2, 5, 8, and 11, the number of the clinical symptoms was shown on the 2nd day from the intraperitoneal administration, In addition, in the case of the nanoliposome prepared by adding the stearyl alcohol to the higher alcohol of Experimental Example 8, there were no clinical symptoms or mortality in all cases of immersion and abdominal (Table 1).

Example 5: Safety and efficacy test for target animal in experiment

The safety and efficacy tests were performed on the target animals in the experiment. The test was carried out in the same manner as in Experimental Example 8 except that the same amount of the streptococcal test vaccine was used in place of the C-phase Edward test vaccine in Experimental Example 8 and Experimental Example 8 of Example 1, and the combination vaccine and Edward vaccine as the commercial vaccine, The following experiment was conducted by designating as Example 5. The safety test was carried out by inoculating the peritoneal cavity with streptococci and by overdosing with twice the amount of the Edward antigens as the immersion method.

Test vaccine: Experimental example 13, streptococcal vaccine (commercial vaccine, comparative example 4), Edward vaccine (using vaccine, comparative example 5) in which Streptococcus test antigen was added instead of Edward test antigen in Experimental examples 8 and 8,

Test animals: 270 infertile flounder, 15-20 cm in mean body size, negative for S.iniae and E.tarda

- Attack strain: S. iniae JSL0208 strain

1) Safety test - Overdose / 2 times

: 120 flounders were divided into four groups of 30 individuals per group. Four groups were divided into four groups: Experimental Example 8 (Edward test vaccine, Group I), Experimental Example 13 (Streptococcal test vaccine, Group II), Comparative Example 4 (Commercial vaccine, Group III) and Comparative Example 5 (Edward commercial vaccine, Group IV) were intraperitoneally or subcutaneously inoculated with twice the recommended dose, respectively. After 21 days, clinical symptoms such as abdominal distension, hernia, motility, And whether or not they were dead.

 2) Effectiveness test

 : The efficacy test was conducted by observing changes in the cohesive antibody and mortality, and the vaccine antigen was administered only as streptococci. Experiments were carried out in Experiment 13 (Streptococcus test vaccine, Group A) at 60 meals, and Comparative Example 4 (Streptococcus commercial vaccine, Group B) at 60 meals. (Non-vaccinated group, C group) that did not receive any vaccine. At the 8th week after the vaccination, blood samples were collected from the flounder that was not inoculated with the vaccine at a rate of at least 60% for 2 weeks. . Iniae (JSL0208) were inoculated peritoneally to all test groups. When the cumulative mortality rate of the control group was at least 60%, the relative survival rate of the remaining vaccine groups was calculated according to the following equation (1), and it was judged that the relative survival rate was more than 60%.

Cumulative mortality rate (%) = (1 - cumulative mortality rate of vaccine group / cumulative mortality rate of control group) X 100

<Results of Safety Test>

: All the test groups survived healthy without any clinical symptoms such as abdominal distension, hernia, decreased motility, decreased appetite during the observation period (see Tables 4 and 5).

Results of the safety test on flounder of test vaccine (1) Test group Daily Clinical Symptoms and Current Status (Number of clinical manifestations (cumulative number of deaths)) One 2 3 4 5 6 7 8 9 10 Group I (n = 30)
(Experimental Example 8) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Group II (n = 30)
(Experimental Example 13) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Group III (n = 30)
(Comparative Example 4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Group IV (n = 30)
(Comparative Example 5) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

Results of the safety test on flounder of test vaccine (2) Test group Daily Clinical Symptoms and Current Status (Number of clinical manifestations (cumulative number of deaths)) 11 12 13 14 15 16 17 18 19 20 21 Group I (n = 30)
(Experimental Example 8) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Group II (n = 30)
(Experimental Example 13) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Group III (n = 30)
(Comparative Example 4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Group IV (n = 30)
(Comparative Example 5) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

 2) Effectiveness test

Antibody titers were detected at 2 weeks after vaccination, and antibody titers peaked at 6 weeks (see FIG. 1). In the case of group A (vaccination with streptococcal vaccine) to which Experimental Example 13 was applied, the highest average was 1: 2.8 times (p <0.01) (P <0.05), respectively.

The cumulative mortality rate of the control group (untreated group) reached 60% at the 11th day after the inoculation, and the group A The cumulative mortality rate of the group B (streptococcal vaccination group) was 23.3% (FIG. 2). Based on this, the relative survival rates of group A and B were 66.7% and 61.2%, respectively.

Safety and efficacy were evaluated by inoculating the flounder, a commercially available flounder - streptococcal vaccine, with the adjuvant - containing test vaccine and adjuvant - containing newly developed nanoliposome form, respectively. As a result of the safety test, the test vaccine and the commercial vaccine were safe for the flounder even when overdosed. In immunogenicity, both vaccines induced significant levels of antibody in flounder, and vaccine containing adjuvant induced a somewhat higher level of antibody. The results showed that the relative survival rate of the test group vaccinated with adjuvant was higher than that of the test group vaccinated with the commercial vaccine at 8 weeks after the vaccination with the nano liposome Form of adjuvant has been shown to have the effect of enhancing the immunogenicity and protective effect of the vaccine.

Example 6. Safety and efficacy test in application of outdoor flounder farm

1) Vaccination

: Experimental Example 13, in which 3,600 flounder was added and streptococcal test antigen was added, was inoculated once with 0.1 ml of peritoneal flounder. For comparison, 3,000 mice were assigned to a non-vaccine control group (non-vaccinated group).

2) Observations

: Two months after the vaccination, the status of vaccination and the clinical manifestations, weight gain, and cohesive antibody titers at 2, 4, and 8 weeks were examined weekly in the vaccinated and non - vaccinated groups. The clinical manifestations and the weight gain test were randomly selected in groups of 10 mice, and the concentration of S. iniae was measured.

3) Results

  <Clinical Symptoms and Current Status>

: As a result of observing the status of Pesa in each group, mortalities were confirmed every week in Experiment 13 (vaccinated group) and control group from the 1st week after vaccination, and mortalities showed symptoms such as blackening and abdominal distension Reference). Bacteria were isolated from dead organisms and identified as S. iniae. Our trends were similar in both vaccination and control groups during the first 2 weeks after vaccination, but the number of deaths in the vaccinated group tended to decrease sharply over 3 weeks. The weekly cumulative mortality rate in the control group was also significantly irregular (Fig. 4).

 <Body weight>

: Body weight gain for vaccination was randomly assigned to each vaccine group and control group randomly before vaccination, 4 and 8 weeks after vaccination. The mean weight of unvaccinated group before vaccination was 79.4g and that of control group was 81.2g. In addition, the mean weight of the unvaccinated group at the 4th week after the vaccination was 145.3g, 143.8g in the control group, 198.3g in the vaccinated group and 181.3g in the control group (see FIG. 5) .

<Change of antibody>

: The change in antibody in the vaccinated group began to increase from the second week after vaccination, with an average of two-fold or less negative pre-vaccination level. In addition, the vaccine was induced to the highest level at 4 weeks after vaccination and maintained for a certain period of time. The reason why the coagulant antibody was induced at a higher level in the laboratory immunogenicity test is considered to be the boosting effect due to the natural infection in the field. In the control group, the antibody was induced at a high level by the individual for 8 weeks, but the average antibody value was lower than 2 times as compared with the vaccinated group.

<Conclusion>

When a streptococcal vaccine containing a nanoliposomal adjuvant was applied to flounder fish, the safety and efficacy of the flounder were evaluated through an 8-week trial period. We observed and compared the status of our patients and the presence of clinical symptoms in the vaccine group (vaccination group with streptococcal strain vaccination) and the control group without vaccination for 8 weeks after the vaccination. As a result, The mortality rate of the vaccinated group was about 1.5%, but the vaccinated group gradually decreased from the 3rd week, and it was confirmed that the vaccine was also safe for the flounder raised in the outdoor farm. In addition, the weight gain rate was similar to that of the vaccine before vaccination, but at 4 and 8 weeks after vaccination, the vaccine group was 1.6 g and 17 g higher than the control group, . In the vaccinated group, the antibody titer was 1: 4 times higher than that of the control group at 4 weeks after vaccination. In contrast, the control group showed induction ability of 1: 2 or less as much antibody to immunogenicity.

FIG. 1 is a graph showing the change of the aggregated antibody per test group after vaccination (A: streptococcal test vaccine (Experimental Example 13), inoculation group, B: streptococcal commercial vaccine (Comparative Example 4) ).

FIG. 2 shows the cumulative mortality of the test group after the inoculation (A: streptococcal test vaccine (Experimental Example 13) inoculation group, B: streptococcus commercial vaccine (Comparative Example 4) inoculation group, and D: nonconjugated group).

3 is an electron micrograph of the nanoliposome of the present invention.

Fig. 4 shows the weekly cumulative mortality change by test group after vaccination (vaccine group: streptococcal test vaccine (experiment 13) inoculation group, control group: non-vaccine group).

FIG. 5 shows changes in the groups of the test groups before and after vaccination (vaccine group: streptococcal test vaccine (Experimental Example 13) inoculation group, control group: non-vaccine group).

FIG. 6 shows changes in antibody per cycle before and after the test vaccination (vaccination group: streptococcal test vaccine (Experimental Example 13) inoculation group, control group: non-vaccination group).

Claims (7)

A vaccine composition for aquaculture fish comprising a nanoliposome and an antigenic material, wherein the composition comprises glyceryl cocoate / citrate / lactate, an oil component, a higher alcohol and an aqueous component, <Structure 1>

In the above formula 1, R is a C12-16 alkyl group 2. The method of claim 1 wherein the antigenic material is selected from the group consisting of an inactivated whole or partial cytoprotective form, or an antigenic molecule form of Eddie Salvatore, Streptococcus iniae ), S. diffire, Lactococcus garvieae, L. pisum, and Bago corus salmonarum. 3. The vaccine composition according to claim 2, wherein the antigenic substance is Streptococcus iniae in the form of an inactivated whole or partial cell preparation. The vaccine composition for aquacultural fish according to claim 1, wherein the higher alcohol is any one selected from the group consisting of myristyl alcohol, cetyl alcohol, stearyl alcohol and behenyl alcohol. The fish according to claim 1, wherein the fish refers to a flounder, a rockfish, a lobster, a lobster, a red sea bream, a lemur, a mullet, a perch, a mackerel, a horse mackerel, , Red sea bream, lush fish, mullet, perch, and codfish. 6. The vaccine composition according to claim 5, wherein the fish is a flounder. A method for preventing or treating bacterial diseases of aquacultured fish by inoculating the vaccine composition according to any one of claims 1 to 6 by intraperitoneal administration or immersion in a fish culture.

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