Vaccine preparation having long-term storage stability

Abstract

The present disclosure relates to a technique for stably storing an inactivated vaccine against type II streptococcosis in fish. The present disclosure relates to, for example, a lyophilizate of an inactivated vaccine against type II streptococcosis in fish, the lyophilizate comprising: inactivated cells of a causative bacterium of type II streptococcosis; and a cryoprotectant. The present disclosure relates to, for example, a method for storing an inactivated vaccine against type II streptococcosis in fish, the method comprising refrigerating the inactivated vaccine at 10°C or lower, the inactivated vaccine comprising inactivated cells of a causative bacterium of type II streptococcosis, and being in a lyophilized form.

Description

Vaccine formulation with long-term storage stability

[0001] The present disclosure relates to a vaccine formulation having long-term storage stability, for example, a technique for stably storing an inactivated vaccine against streptococcosis.

[0002] Vaccines for alpha-hemolytic streptococcosis have been developed and are commercially available. For example, Marine Genner (registered trademark) Strain 1 is an inactivated vaccine for alpha-hemolytic streptococcosis in yellowtail, which is contained in phosphate-buffered saline (Non-Patent Document 1). Inactivated vaccines for fisheries are generally known to have high storage stability (Non-Patent Document 2).

[0003] Non-Patent Document 3 discloses freeze-drying Aeromonas hydrophila and Streptococcus agalactiae together with skim milk. Non-Patent Document 3 does not disclose anything about the long-term storage stability of the freeze-dried product. Patent Document 1 discloses an inactivated vaccine of Lactococcus garvieae, but does not disclose anything about the long-term storage stability of the inactivated vaccine or the long-term storage stability of the freeze-dried product.

[0004] JP6355512B

[0005] Marine Genner® Strain 1 Package Insert Y. Du et al., 2022, Frontiers in Immunology, 13:1040336 D. Sugiani et al., E3S Web of Conferences 442, 02010 (2023)

[0006] The present disclosure discloses vaccine formulations that have long-term storage stability.

[0007] According to the present disclosure, for example, the following inventions are provided: (1) A freeze-dried product of an inactivated vaccine against type II streptococcosis in fish, the freeze-dried product comprising inactivated cells of the causative bacterium of type II streptococcosis and a cryoprotectant. (2) The freeze-dried product according to (1) above, which has been refrigerated and stored at 10°C or below. (3) The freeze-dried product according to (1) or (2) above, in which the causative bacterium has a capsule. (4) The freeze-dried product according to any of (1) to (3) above, in which the causative bacterium is a bacterium of the genus Lactococcus. (5) The freeze-dried product according to any of (1) to (4) above, in which the inactivated vaccine comprises formalin-inactivated cells. (6) The freeze-dried product according to any of (1) to (5) above, which has been stored for 24 months or more. (7) The freeze-dried product according to any one of (1) to (6) above, which has an efficacy of 70% or more, preferably 80% or more, when reconstituted and used as a vaccine after refrigerated storage at 10°C or below for 31 months. (8) A kit comprising a combination of the freeze-dried product according to any one of (1) to (7) above and water for reconstitution. (9) A freeze-dried product of an inactivated vaccine against type II streptococcosis in fish, the composition comprising the inactivated bacterial cells for use in preparing a freeze-dried product containing the inactivated bacterial cells of the causative bacterium of type II streptococcosis. (10) A method for preserving an inactivated vaccine against type II streptococcosis in fish, the inactivated vaccine comprising inactivated bacterial cells of the causative bacterium of type II streptococcosis, the vaccine being in a freeze-dried form, the method comprising refrigerating the inactivated vaccine at 10°C or below. (11) A method for inducing immunity against streptococcosis in fish, comprising: reconstituting the vaccine by mixing a freeze-dried vaccine containing inactivated cells of the causative bacterium of streptococcosis with water for reconstitution; and administering an effective amount of the reconstituted vaccine to the fish.

[0008] The above are examples of the inventions included in this disclosure, but are not all inclusive. The inventions included in this disclosure are as described below.

[0009] Figure 1 shows the relationship between the efficacy and storage period of a vaccine containing inactivated bacterial cells in phosphate buffered saline after refrigeration at 10°C or below. Figure 2 shows electron microscope images of a vaccine containing inactivated bacterial cells in phosphate buffered saline after long-term storage at 10°C or below. For comparison, electron microscope images of the same species of bacteria with and without a capsule are shown. Figure 3 shows the relationship between the efficacy and storage period of a freeze-dried inactivated vaccine after refrigeration at 10°C or below.

[0010] As used herein, "fish" refers to a group of animals belonging to the subphylum Vertebrata that are not tetrapods. Fish include hagfish, lampreys, Chondrichthyes, and Osteichthyes. Osteichthyes are broadly divided into the subclass Sarcopterygii and Actinopterygii. Actinopterygii includes, for example, Brachiopoda, Porophii, and Neopterygii. Examples of the Neopterygii include the Holostei and Teleostei. The Teleostei include the order Ophiodontiformes (e.g., Pseudoceratopogoniformes, Scrophulariformes, and Anguilliformes); the order Ophiodontiformes (e.g., Ophiodontiformes and Pyododontiformes); the order Clupeiniformes (e.g., Clupeiniformes, Scrophulariformes, Scrophulariformes, Cypriniformes, and Siluriformes); and the order Euthynosteum (e.g., Prospinata, Osmeriformes, Ornithodontiformes, Coleoptera, Myctophiformes, Myctophiformes, Paraspinata, and Echinopterygiiformes). Fish are broadly classified into freshwater fish, saltwater fish, and fish that can live in both freshwater and saltwater, depending on the aquatic environment in which they grow. Fish that can live in both freshwater and saltwater include marine amphidromous fish, catadromous fish, anadromous fish, and amphidromous fish.

[0011] As used herein, "piscine streptococci" refers to bacteria that cause infectious diseases in fish. Among farmed fish, the most commonly produced species are, in descending order of production value, Seriola spp., red sea bream, tiger pufferfish, flounder, coho salmon, striped jack, and horse mackerel, which form a large farmed fish market. Streptococcus infection, which causes streptococcosis in fish such as Seriola spp., for example, streptococcosis caused by streptococci such as alpha-hemolytic streptococci (e.g., Lactococcus garvieae and Lactococcus hormocensis), is one of the most important diseases. Lactococcus garvieae and Lactococcus hormocensis have been isolated from fish of the order Perciformes, such as yellowtail, amberjack, yellowtail amberjack, striped jack, horse mackerel, chub mackerel, spotted jack, and bluefin tuna, fish of the order Flatfish, such as flounder, and fish of the order Tetraodontiformes, such as filefish and black filefish, that have developed piscine streptococcosis. Furthermore, type II Lactococcus garvieae and Lactococcus hormocensis have been isolated mainly from fish of the order Yellowtail, amberjack, and striped jack that have developed piscine streptococcosis.

[0012] As used herein, "Lactococcus garvieae" refers to a type of alpha-hemolytic streptococcus. When alpha-hemolytic streptococci infect fish, they can typically cause symptoms such as cloudy, bleeding, and protruding eyes; redness and abscesses inside the gill opercula; as well as pericarditis, frantic swimming, and distortion of the body. Type I Lactococcus garvieae is Lactococcus garvieae that agglutinates with anti-type Ia rabbit serum. Type II Lactococcus garvieae is Lactococcus garvieae that agglutinates with anti-type II rabbit serum and is characterized as being non-KG+ and non-KG-. Recently, some or all of type II Lactococcus garvieae have been reclassified as L. formosensis. Type III Lactococcus garvieae is Lactococcus garvieae that does not agglutinate with any of the above sera.

[0013] As used herein, the term "capsule" refers to a layer that some bacteria possess and that exists on the outer side of the cell wall. The capsule is composed of components secreted by the cell and is thought to act as a defensive wall that protects the bacterial body from the host's immune cells. When capsulated strains are subjected to Giemsa staining, the area around the bacterial body is not stained. Vaccines against encapsulated bacteria must have a capsule.

[0014] As used herein, "immunity" refers to a biological defense mechanism against non-self (mainly invaders such as pathogens). Immunity can be divided into innate immunity and adaptive immunity. Innate immunity is considered to be non-specific immunity against non-self, and is carried out by immune cells such as macrophages, granulocytes, and NK cells that utilize mechanisms such as pattern recognition. Adaptive immunity is considered to be specific immunity against non-self, and is carried out by lymphocytes such as T cells and B cells. In fish, adaptive immunity is thought to be induced because antisera are produced against pathogens. Among adaptive immune cells, there are cells that remember information about past pathogens and are responsible for immunological memory.

[0015] As used herein, a "vaccine" refers to a composition administered to an individual to induce immunity against a pathogen and its analogs. Vaccines include live vaccines and inactivated vaccines. Live vaccines generally use attenuated strains of pathogens, such as viruses or bacteria, that have reduced toxicity or pathogenicity. Inactivated vaccines can be produced by inactivating a pathogen or its strain through physical and / or chemical treatment. Physical treatments include, for example, heat treatment, ultrasonic treatment, or irradiation with ultraviolet light, X-rays, or gamma rays. Chemical treatments include, for example, treatment with organic solvents such as formalin and chloroform, acid treatment with an acid (e.g., a weak acid) such as acetic acid, alcohol treatment, chlorine treatment, or mercury treatment. Other vaccines include recombinant protein vaccines. Vaccines may also contain adjuvants.

[0016] As used herein, "isolation" means separation from at least one or more components that coexist in a natural environment. A component can be isolated from a bacterial cell by separating it from the bacterial cell and then removing the bacterial cell or other components of the bacterial cell, or by concentrating or increasing the relative concentration of the component.

[0017] As used herein, "freeze-drying" refers to a technique for drying a substance by freezing it and then directly evaporating the ice contained in the frozen material. Freeze-drying is typically performed through a freezing step, a depressurizing step, and a drying step. In the freezing step, the substance is frozen at a very low temperature (usually -40°C or lower). In the depressurizing step, a vacuum is created in the container containing the frozen material. This reduces the vapor pressure of the ice, promoting its sublimation. In the drying step, the temperature in the container is gradually increased under vacuum. This causes the ice to convert directly to water vapor before becoming liquid, promoting drying. A cryoprotectant may be used in freeze-drying.

[0018] As used herein, "efficacy" in the context of vaccines refers to the ability to improve the survival rate of fish exposed to a challenge strain. For example, if fish are challenged with a challenge strain at an intensity that would result in a 0% survival rate, and the survival rate is increased to n% (n is a number between 0 and 100) by prior administration of the vaccine to the fish, the efficacy of the vaccine is n%.

[0019] <Vaccine of the Present Disclosure> According to the present disclosure, a lyophilized vaccine (also referred to as a "lyophilized inactivated vaccine" or a "lyophilized vaccine formulation") is provided. According to the present disclosure, the vaccine is a vaccine against a bacterial infection (including a fish bacterial infection).

[0020] In one aspect, the vaccine is a vaccine against a bacterial infection (including fish bacterial infections) and comprises inactivated cells of a causative bacterium of the bacterial infection (including fish bacterial infections), the inactivated cells having a capsule. In one aspect, the inactivated vaccine of the present disclosure is a vaccine against streptococcosis and comprises inactivated cells of Streptococcus as the inactivated cells. In one aspect, the vaccine of the present disclosure is a vaccine against fish streptococcosis and comprises inactivated cells of fish Streptococcus as the inactivated cells. In one aspect, the vaccine of the present disclosure is a vaccine against fish type II streptococcosis and comprises inactivated cells of fish type II Streptococcus as the inactivated cells. In one aspect, the vaccine of the present disclosure is a vaccine against lactococcus (e.g., fish lactococcus) and comprises inactivated cells of the genus Lactococcus as the inactivated cells. In one aspect, the vaccine of the present disclosure is a vaccine against piscine alpha-hemolytic streptococcosis, and comprises inactivated cells of Lactococcus garvieae (or a strain classified as Lactococcus hormocensis) as the inactivated bacterial cells. In one aspect, the streptococcus is not of the genus Streptococcus or Streptococcus agalactiae. In one aspect, the streptococcus is not of the genus Aeromonas or Aeromonas hydrophila.

[0021] In a preferred embodiment, the vaccine of the present disclosure is an inactivated vaccine. Inactivated vaccines can be produced by techniques well known to those skilled in the art. For example, inactivated vaccines can be produced by subjecting a culture solution containing bacterial cells (which may be separated or isolated bacterial cells, or subsequently cultured bacterial cells) to physical or chemical treatment. Physical treatments include, for example, ultraviolet irradiation, X-ray irradiation, heat treatment, and ultrasonic treatment. Chemical treatments include, for example, treatment with an inactivating agent. Examples of inactivating agents include treatment with organic solvents such as formalin or chloroform, acid treatment with acids or weak acids such as acetic acid, and treatment with alcohol or chlorine. In a preferred embodiment, inactivated bacterial cells are obtained by treatment with organic solvents such as formalin or chloroform. Inactivated bacterial cells have substantially lost their pathogenicity.

[0022] For example, chemical treatment can be performed by adding formalin to the culture solution. The amount of inactivated bacteria contained in the inactivated vaccine is, for example, 10 7 ~10 12 CFU / mL, or 10 8 ~10 11 The concentration of the inactivated bacterial cells may be in the range of CFU / mL. The inactivating agent used in the chemical treatment may be removed by centrifugation or the like, and the inactivating agent may be concentrated as needed, but the vaccine may also contain the inactivating agent.

[0023] In a preferred embodiment, the inactivated bacterial cells of the inactivated vaccine comprise formalin-inactivated bacterial cells.

[0024] Inactivated vaccines may further contain adjuvants, preservatives, stabilizers, cryopreservatives, etc. that are acceptable for vaccine formulations. Inactivated vaccines include, for example, saline (e.g., phosphate-buffered saline). Inactivated vaccines may also contain preservatives such as low concentrations of formalin.

[0025] In one aspect, the inactivated vaccine and water for reconstitution do not contain milk components. In one aspect, the inactivated vaccine and water for reconstitution do not contain milk (e.g., skim milk or nonfat milk) and its derivatives (containing milk components), or do not contain them at a concentration of 2%, 3%, 4%, or 5% by dry weight or more. The dry weight is the weight measured after removing as much of the moisture as possible, for example, to a moisture content of 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, or 1% by weight or less.

[0026] According to the present disclosure, inactivated vaccines lack long-term storage stability when stored refrigerated at temperatures below 10°C. Specifically, according to the examples described below, vaccine efficacy declined approximately 24 months after refrigerated storage, dropping to approximately 10% after 31 months and dropping to approximately 0-15% after 37 months. In the technical field of aqueous vaccines, inactivated vaccines have been known for their high storage stability (Y. Du et al., 2022, Frontiers in Immunology, 13: 1040336). Therefore, it was unexpected that inactivated vaccines containing encapsulated streptococci (particularly Lactococcus spp.) exhibited a significant decline in efficacy after long-term storage. Furthermore, no method existed to address the issue of the lack of long-term storage stability (e.g., more than 2 years, more than 30 months, or more than 3 years) of inactivated vaccines containing encapsulated streptococci (particularly Lactococcus spp.).

[0027] Vaccines are generally sensitive to freezing, raising concerns that freezing may impair or destroy vaccine efficacy (National Vaccine Storage Guidelines - Strive for 5, 3rd Edition, Department of Health, Australian Government, 2019). For example, freeze-drying of the Bacillus Calmette-Guérin (BCG) vaccine is known to significantly impair the viability of BCG after freezing (YL. Wong et al., PNAS, 104(8):2591-2595, 2007). Freeze-drying is not thought to resolve the issue of lack of long-term storage stability (e.g., more than 2 years, more than 30 months, or more than 3 years).

[0028] According to the examples described below, it has been found that when the bacteria that cause bacterial infections (including bacterial infections in fish) have a capsule, the capsule of the inactivated bacterial cells is destroyed, resulting in a problem of reduced vaccine efficacy after long-term storage, and that when inactivated bacterial cells with a capsule are stored frozen, their vaccine efficacy is maintained even after long-term storage.

[0029] According to the present disclosure, the inactivated bacterial cells have a capsule, and the inactivated vaccine is provided in a freeze-dried state. Freeze-drying can be performed by conventional methods. Freeze-drying typically includes a freezing step, a depressurizing step, and a drying step. The freezing step involves freezing the inactivated vaccine in a low-temperature environment to obtain a frozen inactivated vaccine. The depressurizing step involves reducing the pressure (or creating a vacuum) inside the container containing the frozen inactivated vaccine, thereby promoting sublimation of water contained in the frozen product. The drying step involves sublimating the water contained in the frozen product and gradually increasing the temperature inside the container to promote water removal. A cryoprotectant may be used in freeze-drying. The cryoprotectant is mixed with the inactivated vaccine before freeze-drying to protect the inactivated vaccine from damage caused by freezing. Commonly used cryoprotectants can also be used in the present disclosure. The cryoprotectant can be, for example, a sugar (e.g., a monosaccharide such as glucose and fructose; a disaccharide such as sucrose, lactose, and maltose; an oligosaccharide such as raffinose and stachyose; a polysaccharide such as dextran), a sugar alcohol (e.g., sorbitol, xylitol, erythritol, maltitol, mannitol, isomalt), or a polyol (e.g., glycerol, propylene glycol, pentaerythritol, trimethylolpropane, neopentyl glycol). In some embodiments, the cryoprotectant comprises a sugar or sugar alcohol. In some embodiments, the sugar comprises a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide. In some embodiments, the sugar comprises a disaccharide, and in some embodiments, the disaccharide comprises lactose. In some embodiments, the cryoprotectant comprises a sugar alcohol. In some embodiments, the sugar alcohol comprises mannitol.

[0030] In some embodiments, the cryoprotectant comprises lactose.

[0031] In some embodiments, the cryoprotectant comprises mannitol.

[0032] In one aspect, the lyophilized inactivated vaccine of the present disclosure comprises inactivated bacterial cells and a cryoprotectant.

[0033] In one aspect, the inactivated bacterial cells in the lyophilized inactivated vaccine of the present disclosure have a capsule.

[0034] In one aspect, a lyophilized inactivated vaccine of the present disclosure, when reconstituted and used as a vaccine after 31 months of refrigeration at 10°C or below (e.g., 2°C to 8°C, etc.), has an efficacy of 70% or more, preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, particularly preferably 90% or more, and especially preferably 93% or more.

[0035] In certain aspects, the inactivated vaccines of the present disclosure may have an efficacy of 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less after 31 months of refrigerated storage at 10° C. or less.

[0036] In certain aspects, an inactivated vaccine of the present disclosure has an efficacy of 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less after 31 months of refrigerated storage at 10°C or less, and a lyophilized inactivated vaccine of the present disclosure, when reconstituted and used as a vaccine after 31 months of refrigerated storage at 10°C or less, has an efficacy of 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, preferably 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, particularly preferably 90% or more, and especially preferably 93% or more.

[0037] In one aspect, an inactivated vaccine of the present disclosure has an efficacy of 20% or less after 31 months of refrigerated storage at 10°C or below, and a lyophilized version of the inactivated vaccine of the present disclosure has an efficacy of 80% or more, preferably 90% or more, when reconstituted and used as a vaccine after 31 months of refrigerated storage at 10°C or below.

[0038] In some embodiments, the storage period of the lyophilized product may be 5 years or less, 4 years or less, 3 years or less, 2 years or less, or 1 year or less. In some embodiments, the storage period may be 1 year or less, 1 year or more but 2 years, 1.5 years or more but 3 years, 2 years or more but 3 years, or 3 years or more but 4 years. In some embodiments, the lyophilized product has been stored for 1 year or less, 1 year or more but 2 years, 1.5 years or more but 3 years, 2 years or more but 3 years, or 3 years or more but 4 years.

[0039] In some embodiments, the lyophilisate has been stored for a period of 18 months or more, 19 months or more, 20 months or more, 21 months or more, 22 months or more, 23 months or more, 24 months or more, 25 months or more, 26 months or more, 27 months or more, 28 months or more, 29 months or more, or 30 months or more. In some embodiments, the lyophilisate has been stored for a period of 40 months or less, 39 months or less, 38 months or less, 37 months or less, 36 months or less, 35 months or less, 34 months or less, 33 months or less, or 32 months or less.

[0040] Efficacy can be measured by inoculating a subject (e.g., a target fish) with the vaccine, then challenging the subject (e.g., the target fish) with the challenge strain, and determining the subsequent survival rate. For example, if the inactivated bacterial cell is Lactococcus garvieae (or a strain classified as Lactococcus hormocensis), yellowtail fry can be used as the fish, and 14 days after administration of the vaccine, the fish can be challenged with the challenge strain, and the efficacy can be determined from the mortality and survival rate over the following 14 days. In the above case, the challenge can be carried out with type II streptococcus BSLG15203-3-4-4 strain. For developed vaccines, the challenge strain already exists, and methods for evaluating efficacy have also been established.

[0041] The lyophilized product of the present disclosure can be stored or refrigerated at or below 10° C. Thus, the lyophilized product of the present disclosure has a temperature of 10° C. or below (e.g., 0° C. to 10° C. or 2° C. to 8° C.), and can be, for example, in a refrigerated or frozen state of 10° C. or below, and is preferably in a refrigerated state of 10° C. or below.

[0042] The lyophilized product of the present disclosure can be reconstituted with water for reconstitution before administration. The water for reconstitution can be, for example, sterile, aseptic water. The water for reconstitution can have a temperature of seawater. The water for reconstitution can further contain additives acceptable in vaccines.

[0043] According to the present disclosure, a ready-to-use vaccine preparation kit may be provided, comprising the lyophilized product of the present disclosure and water for reconstitution.

[0044] The present disclosure provides a composition for use in preparing a freeze-dried product of an inactivated vaccine against streptococcosis, the composition comprising the inactivated bacterial cells of the causative bacterium of streptococcosis. For example, the present disclosure provides a composition for use in preparing a freeze-dried product of an inactivated vaccine against type II streptococcosis in fish, the composition comprising the inactivated bacterial cells of the causative bacterium of type II streptococcosis.

[0045] According to the present disclosure, there is provided a method for storing an inactivated vaccine against streptococcosis, the inactivated vaccine comprising inactivated cells of the causative bacterium of streptococcosis, the vaccine being in a freeze-dried form, and the method comprising refrigerating the inactivated vaccine at or below 10° C. According to the present disclosure, there is provided, for example, a method for storing an inactivated vaccine against type II streptococcosis in fish, the inactivated vaccine comprising inactivated cells of the causative bacterium of type II streptococcosis, the vaccine being in a freeze-dried form, and the method comprising refrigerating the inactivated vaccine at or below 10° C.

[0046] The present disclosure provides a method for inducing immunity against streptococcosis in fish, the method comprising: reconstituting a vaccine by mixing a lyophilized vaccine containing inactivated cells of the causative bacterium of streptococcosis with water for reconstitution; and administering an effective amount of the reconstituted vaccine to the fish. Administration can be by, for example, injection. Administration can be by, for example, parenteral administration (e.g., intraperitoneal administration, intramuscular administration, intravenous administration, etc.).

[0047] The present disclosure provides use of a lyophilized product containing inactivated cells of the causative bacterium of streptococcosis in the manufacture of an inactivated vaccine against streptococcosis.The present disclosure also provides use of inactivated cells of the causative bacterium of streptococcosis in the manufacture of a lyophilized product of an inactivated vaccine against streptococcosis.

[0048] 1. Storage stability of inactivated vaccines We investigated the storage stability of inactivated vaccines against bacterial infections, focusing on a vaccine against type II streptococcal infections in fish.

[0049] Method: A encapsulated strain of Lactococcus garvieae (121941-B24 strain; currently classified as Lactococcus formocensis) was cultured and inactivated with 0.3% formalin to produce formalin-inactivated cells. After inactivation, the supernatant was replaced with 0.2% formalin-containing phosphate-buffered saline (PBS) and 1.2 × 10 9 The concentration was adjusted to CFU / mL to obtain inactivated 121941-B24 strain cells. To confirm reproducibility, three independently prepared lots of inactivated microbial cells were prepared and stored refrigerated at 2 to 10°C. The efficacy of each lot was confirmed immediately after the start of storage (0 month) and after 3, 6, 12, 18, 24, and 37 months from the start of storage.

[0050] The efficacy was evaluated by an artificial infection test using yellowtail juveniles (0 to 24 months old). 8 cfu / tail) and 0.1 mL of PBS (control group) were intraperitoneally administered to the yellowtail fry, and then the fish were reared for 14 days. 14 days after administration of each inactivated bacterial cell, the yellowtail fry were challenged by intraperitoneal injection of type II streptococcus BSLG15203-3-4-4 strain (challenge strain), and the number of dead fish was counted daily for 14 days after challenge. The challenge strain was injected in an amount that resulted in the death of 80% to 100% of the control group. Specifically, 1.5 x 10 6 ~3 x 10 7 The dose ranged from 0.01 to 0.01 cfu / tail and varied depending on the fish age and weight, but was considered to be a reasonable dose. Mortality and survival rates were calculated from the number of dead fish in each test group, and the difference from the PBS-administered control group was evaluated using Fisher's exact probability calculation method (p<0.05). The effectiveness of the vaccine is determined from the survival rate of yellowtail fry. If the survival rate is higher than that of the negative control group (PBS-administered group), it is suggested that the vaccine is effective. The efficacy rate can also be calculated using the following formula: (Efficacy rate) = [1 - (mortality rate in vaccine-administered group) / (mortality rate in negative control group)] x 100

[0051] Results In the 0-month test, all lots demonstrated extremely high efficacy, with a survival rate of 100% (i.e., an efficacy rate of 100%) (see Figure 1). As the storage period increased, efficacy declined in all lots, and particularly at 37 months, no significant difference was observed in the survival rates of any of the three lots compared to the PBS control group (see Table 1, Figure 1). Thus, although vaccination resulted in a high survival rate of 100% initially at 0 months, there was a tendency for the survival rate and vaccine efficacy to decline over time, with a particularly significant decline in survival rate and vaccine efficacy after 24 months. The survival rates are quantified and shown in Table 1.

[0052]

[0053] These results suggest that inactivated type II streptococcal cells lack long-term storage stability.

[0054] 2. Observation of inactivated bacterial cells after long-term storage A encapsulated, immunogenic strain (121941-B24-1), a capsule-less, non-immunogenic strain (121941-B24-56), and inactivated encapsulated, immunogenic strain 121941-B24 were stored for 1 year and 8 months under the same storage conditions as above.

[0055] Agar-cultured or inactivated bacteria were fixed in 5% glutaraldehyde containing 0.15% ruthenium red for 2 hours at room temperature. The fixed bacteria were collected by centrifugation and embedded in 4% agarose to prepare embedded blocks. The embedded blocks were cut into small pieces and washed five times with cacodylate buffer containing 0.05% ruthenium red. The blocks were post-fixed with 2% osmium tetroxide using standard methods, then washed, dehydrated, embedded in resin, thin-sectioned, and subjected to standard double staining with uranium and lead.

[0056] As shown in Figure 2, immunogenic strains have a layer thought to be a capsule in the outermost layer, whereas non-immunogenic strains have a smooth outermost layer, suggesting that they do not have a capsule. The outermost layer of the inactivated bacterial cells stored for a long time was smooth, suggesting that the capsule structure had disappeared due to long-term storage, resulting in a loss of immunogenicity.

[0057] These results clearly demonstrate that type II streptococcal inactivated vaccines do not have excellent long-term storage stability, and suggest that long-term storage causes capsule loss from the inactivated vaccine cells.

[0058] 3. Study of preservation method We attempted to freeze-dry the inactivated bacterial cells.

[0059] The 121941-B24 strain was cultured and then inactivated with 0.3% formalin to produce formalin-inactivated bacterial cells. After inactivation, the supernatant was replaced with PBS containing 0.2% formalin and 50 mg / mL lactose or PBS containing 0.2% formalin and 50 mg / mL mannitol, and then freeze-dried to obtain dried inactivated 121941-B24 bacterial cells. Meanwhile, the supernatant of the formalin-inactivated bacterial cells was replaced with PBS containing 0.2% formalin, and 1.2 × 10 9 The dried 121941-B24 inactivated cells were adjusted to CFU / mL and used as the 121941-B24 inactivated cells. The prepared vaccine was stored in a refrigerator at 10°C or below. The dried 121941-B24 inactivated cells were used to prepare the vaccine with an antigen content of 1.2 x 10 before use. 9 The cells were suspended in PBS to a concentration of CFU / mL and used. Lactose and mannitol are cryoprotectants.

[0060] Efficacy was evaluated through an artificial infection test using yellowtail fry. 0.1 mL of each vaccine or PBS (control group) was administered intraperitoneally, and the fish were then raised for 14 days. 14 days after administration of each vaccine, the yellowtail fry were challenged intraperitoneally with type II streptococcus BSLG15203-3-4-4 strain, and dead fish were collected and counted daily for 14 days after challenge. Mortality and survival rates were calculated from the number of dead fish in each test group, and differences from the mortality and survival rates of the PBS-administered control group were evaluated using Fisher's exact probability calculation (p<0.05).

[0061] Each inactivated bacterial cell was evaluated immediately after the start of storage (0 months) and at 13, 17, and 31 months after the start of storage. The results are shown in Figure 3 and Table 2. As shown in Figure 3 and Table 2, immediately after the start of the test, all inactivated bacterial cells were confirmed to have significantly higher efficacy than the PBS control group. When lactose was used as an excipient, the effect was high, and a decrease in efficacy was observed at 17 months without lyophilization. However, dried inactivated bacterial cells using lactose were significantly more effective than inactivated bacterial cells. At 31 months, inactivated bacterial cells without lyophilization almost lost their efficacy (efficacy rate: 10.0%). On the other hand, lyophilized inactivated bacterial cells using lactose maintained 95.0% efficacy, and lyophilized inactivated bacterial cells using mannitol maintained 75.0% efficacy, both of which were significantly higher than inactivated bacterial cells. This value was almost the same as the value at month 0, indicating that the freeze-drying method is effective in improving the shelf life of type II streptococcosis vaccines. This result was surprising, considering that the effectiveness of inactivated bacteria declines significantly after storage for more than 24 months. The survival rates are quantified and shown in Table 2.

[0062]

[0063] Inactivated vaccines of encapsulated bacteria show reduced efficacy after long-term storage due to poor long-term capsule stability. The capsule is considered to be an essential structure for vaccine efficacy, and the examples demonstrate that maintaining the capsule is an important issue for inactivated vaccines. This example also demonstrates that the capsule can be stabilized for long periods by cryopreservation. The present invention can be applied to inactivated vaccines of encapsulated bacteria and may be beneficial for the long-term storage of such vaccines.

Claims

1. A freeze-dried inactivated vaccine against type II streptococcosis in fish, comprising inactivated cells of the causative bacterium of type II streptococcosis and a cryoprotectant.

2. The freeze-dried product according to claim 1, which is stored refrigerated at 10°C or below.

3. The freeze-dried product according to claim 1 or 2, wherein the causative bacterium has a capsule.

4. The freeze-dried product according to any one of claims 1 to 3, wherein the causative bacterium is a bacterium of the genus Lactococcus.

5. The freeze-dried product according to any one of claims 1 to 4, wherein the inactivated vaccine contains formalin-inactivated bacterial cells.

6. The freeze-dried product according to any one of claims 1 to 5, which has been stored for 24 months or more.

7. A freeze-dried product according to any one of claims 1 to 6, which has an efficacy of 70% or more, preferably 80% or more, when reconstituted and used as a vaccine after 31 months of refrigeration at 10°C or below.

8. A kit comprising a combination of the lyophilisate according to any one of claims 1 to 7 and water for reconstitution.

9. A freeze-dried product of an inactivated vaccine against type II streptococcosis in fish, comprising inactivated bacterial cells of the causative bacterium of type II streptococcosis, for use in producing the freeze-dried product.

10. A method for storing an inactivated vaccine against type II streptococcosis in fish, the inactivated vaccine containing inactivated cells of the causative bacterium of type II streptococcosis, the vaccine being in a freeze-dried form, and the method comprising refrigerating the inactivated vaccine at 10°C or below.

11. A method for inducing immunity against streptococcosis in fish, comprising reconstituting the vaccine by mixing a lyophilized vaccine containing inactivated cells of the causative bacterium of streptococcosis with water for reconstitution, and administering an effective amount of the reconstituted vaccine to the fish.

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