Vaccine against piscine alpha-hemolytic streptococcosis

Abstract

To provide a vaccine against fish α-hemolytic streptococcal disease.SOLUTION: A conditioned medium of Lactococcus bacteria responsible for fish α-hemolytic streptococcal disease is used as an active ingredient of vaccine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a vaccine against piscine alpha-hemolytic streptococcosis. [Background technology]

[0002] Piscine alpha-hemolytic streptococcosis caused by Lactococcus bacteria such as Lactococcus garvieae is known as an infectious disease in the aquaculture of fish such as yellowtail (Patent Documents 1-2 and Non-Patent Documents 1-5).

[0003] For Lactococcus garvieae, conventional serotype diagnostic antisera (which are Lactococcus garvieae KG - There are multiple serotypes that differ in agglutination properties against antisera (antisera against type strains) (Patent Document 1 and Non-Patent Documents 1 to 5), and these serotypes can be distinguished by serotyping PCR (Non-Patent Documents 3 to 4).

[0004] Known vaccines against piscine alpha-hemolytic streptococcosis include those containing as an active ingredient bacterial cells or inactivated bacterial cells of the causative bacteria of piscine alpha-hemolytic streptococcosis, such as Lactococcus garvieae (Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2015-038113 [Patent Document 2] JP 11-332558 [Non-patent literature]

[0006] [Non-Patent Document 1] Fukuda et al., Fish Pathology, 50(4), 200-206, 2015.12 [Non-patent document 2] Yoshida, Fish Pathology, 51(2), 44-48, 2016.6 [Non-patent document 3] Ohbayashi et al., Fish Pathology, 52(1), 46-49, 2017.3 [Non-patent document 4] 2022 Japanese Society of Fish Diseases Conference Abstracts [Non-patent document 5] Ministry of Agriculture, Forestry and Fisheries Consumer Affairs and Safety Bureau Livestock and Fisheries Safety Management Division Director Notice June 9, 2020 Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a vaccine against piscine alpha-hemolytic streptococcosis. [Means for solving the problem]

[0008] The present inventors have discovered that the culture supernatant of Lactococcus bacteria that cause piscine alpha-hemolytic streptococcosis functions as a vaccine against piscine alpha-hemolytic streptococcosis, and have completed the present invention.

[0009] That is, the present invention can be exemplified as follows. [1] 1. A vaccine against piscine alpha-hemolytic streptococcosis, comprising: A vaccine containing a culture supernatant of Lactococcus bacteria that causes piscine alpha-hemolytic streptococcosis. [2] The vaccine as described above, wherein the bacterium is Lactococcus garvieae, Lactococcus formosensis, Lactococcus petauri, or a species closely related to these. [3] The vaccine, wherein the bacterium is Lactococcus garvieae type I, Lactococcus garvieae type II, or serotype unknown Lactococcus garvieae. [4] The vaccine as described above, which is substantially free of bacterial cells. [5] The vaccine, wherein the culture supernatant is obtained by removing bacterial cells from a culture solution of the bacteria, and the bacterial cells have not been inactivated before the removal. [6] The above vaccine further comprises an adjuvant. [7] The vaccine further comprises a detoxifying agent. [8] 1. A method for producing a vaccine against piscine alpha-hemolytic streptococcosis, comprising: a step of culturing Lactococcus bacteria that cause piscine alpha-hemolytic streptococcosis to obtain a culture solution; a step of removing the bacterial cells from the culture solution to obtain a culture supernatant; and concentrating the culture supernatant; A method comprising: [9] The production method as described above, wherein the culture is carried out using a liquid medium.

[10] The production method as described above, which does not include a step of inactivating the bacterial cells before the removal.

[11] The method further comprises the step of adding a detoxifying agent after the removal. [Effects of the Invention]

[0010] The present invention provides a vaccine against piscine alpha-hemolytic streptococcosis, which can be used to prevent the occurrence, transmission, or spread of piscine alpha-hemolytic streptococcosis, such as piscine alpha-hemolytic streptococcosis caused by Lactococcus garvieae type I, Lactococcus garvieae type II, or serotype unknown Lactococcus garvieae. [Brief explanation of the drawings]

[0011] [Figure 1]FIG. 1 shows the results of a test to prevent the onset of piscine alpha-hemolytic streptococcosis using an L. garvieae type II culture supernatant vaccine containing an inactivation step. [Figure 2] FIG. 1 shows the results of a test to prevent the onset of piscine alpha-hemolytic streptococcosis using an L. garvieae type II culture supernatant vaccine that does not include an inactivation step. DETAILED DESCRIPTION OF THE INVENTION

[0012] <1> Vaccine against piscine alpha-hemolytic streptococcosis The vaccines described herein are vaccines against piscine alpha-hemolytic streptococcosis.

[0013] "Fish alpha-hemolytic streptococcosis" refers to an infectious disease in fish caused by alpha-hemolytic streptococci. "Alpha-hemolytic streptococci" refers to streptococci that produce green bands of methemoglobin around colonies on blood agar medium. Examples of alpha-hemolytic streptococci that cause fish alpha-hemolytic streptococcosis include Lactococcus bacteria that cause fish alpha-hemolytic streptococcosis, which will be described later. "Bacteria that cause fish alpha-hemolytic streptococcosis" can be rephrased as "bacteria that cause fish alpha-hemolytic streptococcosis." "Bacteria that cause hemolytic streptococcosis" and "bacteria that cause piscine alpha-hemolytic streptococcosis."

[0014] The vaccines described herein can be applied to piscine alpha-hemolytic streptococcosis. Specifically, the vaccines described herein can be used to prevent piscine alpha-hemolytic streptococcosis. Prevention of piscine alpha-hemolytic streptococcosis includes prevention of the occurrence, transmission, or spread of piscine alpha-hemolytic streptococcosis. The alpha-hemolytic streptococci that cause piscine alpha-hemolytic streptococcosis, to which the vaccines described herein are applied, are also referred to as "alpha-hemolytic streptococci to which the vaccines are applied." The alpha-hemolytic streptococci to which the vaccines are applied may be one type of bacterium, or two or more types of bacteria.

[0015] The fish to which the vaccines described herein are applied are not particularly limited, as long as the effects of the vaccines described herein are desired (for example, prevention of piscine alpha-hemolytic streptococcosis). Examples of fish to which the vaccines described herein are applied include fish that can be infected with alpha-hemolytic streptococcosis. Fish that can be infected with alpha-hemolytic streptococcosis may be saltwater fish, freshwater fish, or diadromous fish. Specific examples of fish that can be infected with alpha-hemolytic streptococcosis include Seriola species (yellowtail, amberjack, yellowtail amberjack, etc.), red sea bream, crimson sea bream, flounder, striped jack, horse mackerel, mackerel, tuna, bluefin tuna, grunt, pufferfish, filefish, eel, and rainbow trout. The fish to which the vaccines described herein are applied may be one type of fish, or two or more types of fish.

[0016] The vaccine described herein contains a culture supernatant of Lactococcus bacteria that cause piscine alpha-hemolytic streptococcosis. This Lactococcus bacterium is also referred to as a "Lactococcus bacterium for vaccine production." This culture supernatant is also referred to as a "culture supernatant of Lactococcus bacteria."

[0017] Lactococcus bacteria that cause piscine alpha-hemolytic streptococcosis (i.e., Lactococcus bacteria for vaccine production) include Lactococcus garvieae, Lactococcus formosensis, Lactococcus petauri, and related species. Lactococcus bacteria for vaccine production particularly include Lactococcus garvieae and related species. Lactococcus bacteria for vaccine production, more particularly, include Lactococcus garvieae. Lactococcus bacteria for vaccine production (particularly Lactococcus garvieae) include Lactococcus garvieae type I, Lactococcus garvieae type II, and serotype unknown Lactococcus garvieae. As the Lactococcus bacteria for vaccine production, one type of Lactococcus bacteria may be used, or two or more types of Lactococcus bacteria may be used. Furthermore, as the culture supernatant, one type of culture supernatant (e.g., culture supernatant of one type of Lactococcus bacteria) may be used, or two or more types of culture supernatant (e.g., culture supernatant of two or more types of Lactococcus bacteria) may be used.

[0018] Examples of alpha-hemolytic streptococci to which the vaccine is applied include the Lactococcus bacteria exemplified above as Lactococcus bacteria for vaccine production. The alpha-hemolytic streptococci to which the vaccine is applied may or may not be the same as the Lactococcus bacteria for vaccine production. The vaccine described herein may be applied, for example, to alpha-hemolytic streptococcus disease caused by at least the same strain as the Lactococcus bacteria for vaccine production. Furthermore, the vaccine described herein may be applied, for example, to alpha-hemolytic streptococcus disease caused by at least a strain of the same species and serotype as the Lactococcus bacteria for vaccine production. Furthermore, the vaccine described herein may be applied, for example, to alpha-hemolytic streptococcus disease caused by at least a strain of the same species as the Lactococcus bacteria for vaccine production.

[0019] The Lactococcus bacteria for vaccine production may be isolated from any of the fish species to which the vaccines described herein are applied, such as those exemplified above. In this case, the fish species to which the vaccines described herein are applied may or may not be the same as the fish from which the Lactococcus bacteria for vaccine production were isolated.

[0020] "A closely related species of bacterial species X" means a bacterium classified in the same genus as bacterial species X but of a different species, or a bacterium classified in the same genus as bacterial species X but of an unidentified species, whose 16S rRNA gene nucleotide sequence has 90% or more, 95% or more, or 97% or more identity with any strain belonging to bacterial species X (e.g., the type strain of bacterial species X).

[0021] "Lactococcus garvieae type I" refers to a bacterium of the genus Lactococcus (particularly Lactococcus garvieae) that exhibits agglutination with a serotype diagnostic antiserum for Lactococcus garvieae and that produces an amplification product of 285±10 bp, 285±5 bp, or 285 bp in a serotype-discriminating PCR for Lactococcus garvieae. "PCR for serotype discrimination for Lactococcus garvieae" refers to the serotype-discriminating PCR for Lactococcus garvieae described by Ohbayashi et al., Fish Pathology, 52(1), 46-49, March 2017 (hereinafter referred to as Ohbayashi et al.). "Antisera for serotype diagnosis ... KG. - The term "antisera" refers to antisera against type strains. Examples of antisera for diagnosing serotypes of Lactococcus garvieae include those described in Oinaka et al., Fish Pathology, 50(2), 37-43, 2015.6.

[0022] "Lactococcus garvieae type II" refers to Lactococcus spp. (especially L. garvieae) that do not agglutinate with L. garvieae serotype diagnostic antisera and that produce amplification products of 1285±10 bp, 1285±5 bp, or 1285 bp in L. garvieae serotype PCR (Ohbayashi et al.). According to Abstract 107 of the 2022 Meeting of the Japanese Society of Fish Pathology, Lactococcus spp. previously considered "Lactococcus garvieae type II" may be classified as Lactococcus formosensis based on phylogenetic analysis, such as whole genome sequencing. However, in this specification, they are referred to as "Lactococcus garvieae type II."

[0023] "Serotype-unknown Lactococcus garvieae" refers to bacteria that are classified as Lactococcus (especially Lactococcus garvieae) because they do not show agglutination with serotype diagnostic antisera for Lactococcus garvieae, or it is difficult to determine agglutination with serotype diagnostic antisera for Lactococcus garvieae, and because amplification products of 628±10bp, 628±5bp, or 628bp are obtained in PCR for serotype discrimination of Lactococcus garvieae (Abstract 107 of the 2022 Meeting of the Japanese Society of Fish Pathology). Serotype-unknown Lactococcus garvieae is a type of bacteria that is classified as Lactococcus (especially Lactococcus garvieae) because it does not show agglutination with serotype diagnostic antisera for Lactococcus garvieae, or it is difficult to determine agglutination with serotype diagnostic antisera for Lactococcus garvieae, and because amplification products of 628±10bp, 628±5bp, or 628bp are obtained in PCR for serotype discrimination of Lactococcus garvieae (Abstract 107 of the 2022 Meeting of the Japanese Society of Fish Pathology). Multilocus sequence analysis (MLSA) using the nucleotide sequences of the rRNA, pheS, recA, rpoA, and rpoB genes showed that it could be located in a cluster distinct from Lactococcus garvieae types I and II.

[0024] The method for obtaining Lactococcus bacteria for vaccine production is not particularly limited. For example, existing strains such as commercially available strains or deposited strains can be used as Lactococcus bacteria for vaccine production. Furthermore, Lactococcus bacteria for vaccine production can be obtained by isolating them from fish, such as yellowtail, suspected of having piscine alpha-hemolytic streptococcosis.

[0025] Lactococcus garvieae type I can be detected by, for example, collecting kidney samples from fish such as yellowtail suspected of having piscine alpha-hemolytic streptococcosis at a fish farm in Japan, cultivating them on agar medium to isolate the bacteria, and then using antisera for diagnosing the serotype of Lactococcus garvieae and / or can be obtained by identifying the strain as Lactococcus garvieae type I using PCR for serotyping Lactococcus garvieae.

[0026] Lactococcus garvieae type II can be obtained by collecting kidney samples from fish, such as yellowtail, suspected of developing piscine alpha-hemolytic streptococcosis despite administration of a conventional inactivated vaccine against Lactococcus garvieae type I at a fish farm in Japan, culturing the samples on agar medium to isolate the bacteria, and identifying the bacteria as Lactococcus garvieae type II using a Lactococcus garvieae serotype diagnostic antiserum and / or Lactococcus garvieae serotype PCR. An example of a conventional inactivated vaccine against Lactococcus garvieae type I is Pisivac® Vibrio+Streptococcus (Kyoritsu Pharmaceutical). Lactococcus garvieae type II can also be obtained by collecting kidneys from fish such as yellowtail, which are suspected of developing piscine alpha-hemolytic streptococcosis despite administering conventional inactivated vaccines against Lactococcus garvieae type I and Lactococcus garvieae type II at fish farms in Japan, such as yellowtail, and culturing them on agar medium to isolate the bacteria, which can then be identified as Lactococcus garvieae type II using a Lactococcus garvieae serotype diagnostic antiserum and / or Lactococcus garvieae serotype PCR. An example of a conventional inactivated vaccine against Lactococcus garvieae type I and Lactococcus garvieae type II is Pisivac (registered trademark) Note 4 (Kyoritsu Pharmaceutical).

[0027] Serotype-unknown Lactococcus garvieae can be obtained by collecting kidney samples from fish such as yellowtail (yellowtail) in Japan, where they are suspected of developing piscine alpha-hemolytic streptococcosis despite administering conventional inactivated vaccines against Lactococcus garvieae type I and Lactococcus garvieae type II at fish farms, and culturing the samples on agar medium to isolate bacteria. Although the bacteria were identified as alpha-hemolytic Lactococcus species, they could not be serotyped using serotype diagnostic antisera and / or PCR for Lactococcus garvieae serotyping (i.e., were determined to be serotype-unknown). An example of a conventional inactivated vaccine against Lactococcus garvieae type I and Lactococcus garvieae type II is Pisivac® Note 4 (Kyoritsu Pharmaceutical).

[0028] Currently available vaccines are all primarily composed of inactivated bacterial cells. Because bacterial cells contain many antigens, they are generally capable of inducing effective immune responses. However, while culture supernatants contain secretions and metabolic products from the bacterial cells, they were expected to contain only a small amount of antigens. Surprisingly, however, the inventors removed Lactococcus cells from a culture of the bacteria, concentrated the culture, and used it as a vaccine antigen. They found that this method induced protective immunity against Lactococcus garvieae type II, a type of Lactococcus that causes piscine alpha-hemolytic streptococcosis, leading to the completion of the present invention.

[0029] "Culture supernatant of Lactococcus bacteria" refers to a liquid fraction obtained by removing the bacterial cells from a culture solution of Lactococcus bacteria. "Culture solution of Lactococcus bacteria" refers to a suspension containing the bacterial cells of Lactococcus bacteria obtained by culturing the bacteria. In the case of liquid culture, the culture solution of Lactococcus bacteria includes a culture obtained by culturing Lactococcus bacteria in a liquid medium. In the case of solid culture, the culture solution of Lactococcus bacteria includes a suspension obtained by culturing Lactococcus bacteria in a solid medium and recovering the bacterial cells on the solid medium. The bacterial cells on the solid medium can be recovered as a suspension by suspending them in a liquid medium, for example. Examples of liquid media include aqueous media such as water and aqueous buffer solutions. In addition to the bacterial cells of Lactococcus bacteria, the culture solution of Lactococcus bacteria may contain various components, such as secretions from the bacterial cells.

[0030] Specifically, the vaccines described herein can be produced, for example, by culturing Lactococcus bacteria that cause piscine alpha-hemolytic streptococcosis (i.e., Lactococcus bacteria for vaccine production) and removing the bacterial cells from the resulting culture. The vaccine production method described herein may be, for example, a method for producing a vaccine against piscine alpha-hemolytic streptococcosis, comprising the steps of culturing Lactococcus bacteria that cause piscine alpha-hemolytic streptococcosis (i.e., Lactococcus bacteria for vaccine production) to obtain a culture (also referred to as the "culturing step") and removing the bacterial cells from the culture to obtain a culture supernatant (also referred to as the "cell removal step"). The culturing step may be, for example, culturing Lactococcus bacteria that cause piscine alpha-hemolytic streptococcosis in a liquid medium to obtain a culture (i.e., a culture). Furthermore, the culture process may be, for example, a process of culturing Lactococcus bacteria that cause piscine alpha-hemolytic streptococcosis in a solid medium, recovering the bacterial cells from the solid medium, and obtaining a suspension containing the bacterial cells (i.e., a culture solution).

[0031] The method for culturing Lactococcus bacteria is not particularly limited. Lactococcus bacteria may be cultured under known conditions used for culturing Lactococcus bacteria, or under appropriately modified conditions. Lactococcus bacteria may be cultured using any medium, such as a solid medium or a liquid medium. Lactococcus bacteria are preferably cultured using a liquid medium, particularly because it allows for large-scale culture. Examples of media, such as solid or liquid media, include known media used for culturing Lactococcus bacteria, or appropriately modified versions thereof. Examples of liquid media include meat extract liquid medium (BHI) and casein-soybean peptone liquid medium (TPB, TSB). A preferred liquid medium is casein-soybean peptone liquid medium, from the viewpoint of reducing production costs. Examples of solid media include the liquid media listed above solidified with a gelling agent such as agar or gellan gum.

[0032] The method for removing bacterial cells from the culture solution is not particularly limited. Examples of methods for removing bacterial cells include filter filtration and centrifugation. The filter used for filter filtration to remove bacterial cells is also called a "sterilization filter." These methods for removing bacterial cells may be used alone or in appropriate combination. Removal of bacterial cells may be carried out, for example, by at least filter filtration. Specifically, removal of bacterial cells may be carried out, for example, by centrifugal filtration of the culture solution through a sterilization filter. Furthermore, removal of bacterial cells may be carried out, for example, by centrifuging the culture solution and then filtering the centrifugal supernatant through a sterilization filter. The sterilization filter preferably has a pore size of 0.45 μm or less. "Bacterial cells have been removed" may mean that the viable cell count in the culture solution (i.e., the culture supernatant) after removal of the bacterial cells is 100 cfu / mL or less, which is the detection limit of the plate culture method, and preferably 10 cfu / mL or less. Furthermore, the total number of bacteria in the culture solution (i.e., culture supernatant) after removal of the bacterial cells may be, for example, 100 cells / mL or less, and preferably 10 cells / mL.

[0033] The bacterial cells may or may not be inactivated before removal from the culture medium. That is, the bacterial cells removed from the culture medium may or may not be inactivated. Furthermore, the vaccine production method described herein may or may not include a step of inactivating the bacterial cells (also referred to as an "inactivation step") before the bacterial cell removal step.

[0034] The inactivation of the bacterial cells can be carried out, for example, by subjecting the culture solution to an inactivation treatment. That is, the culture solution before removal of the bacterial cells may or may not have been subjected to an inactivation treatment. The inactivation step may be, for example, a step of subjecting the culture solution to an inactivation treatment. Inactivation treatments include physical treatments (ultraviolet irradiation, X-ray irradiation, heat treatment, ultrasonic treatment, etc.), chemical treatments (treatment with organic solvents such as formalin or chloroform, treatment with weak acids such as acetic acid, etc.), and the like. (Treatment with other chemicals such as alcohol, chlorine, mercury, etc.) For example, formalin can be added to the culture solution at a final concentration of 0.001 to 2.0 v / v%, more preferably 0.01 to 1.0 v / v%, and the culture solution can be sensitized at 4 to 30°C for 1 to 3 days to inactivate the bacteria with formalin.

[0035] The vaccines described herein may or may not consist of a culture supernatant of Lactococcus bacteria, i.e., the vaccines described herein may contain other components in addition to the culture supernatant of Lactococcus bacteria.

[0036] However, the vaccines described herein do not have to be substantially free of Lactococcus bacteria cells used for vaccine production. "The vaccines described herein are substantially free of Lactococcus bacteria cells used for vaccine production" means that the content of Lactococcus bacteria cells used for vaccine production in the vaccines described herein is 10,000 cells / g or less, 1,000 cells / g or less, 100 cells / g or less, or 10 cells / g or less.

[0037] The content of the Lactococcus culture supernatant in the vaccine described herein is not particularly limited, as long as the vaccine described herein functions as a vaccine against piscine alpha-hemolytic streptococcosis. The content of the Lactococcus culture supernatant in the vaccine described herein can be appropriately set depending on various conditions, such as the amount of antigen derived from the culture supernatant, the vaccine dosage, the vaccine administration method, and the fish to be vaccinated.

[0038] The content of the culture supernatant of Lactococcus bacteria in the vaccine described herein is, for example, 1 x 10 in terms of the viable cell count of the culture solution from which the culture supernatant is derived. 7 cfu / g or more, 1 × 10 8 cfu / g or more, 1 × 10 9 cfu / g or more, 1 × 10 10 cfu / g or more, 1 × 10 11 cfu / g or more, 1 × 10 12 cfu / g or greater, or 1 x 10 13 cfu / g or more, and may be 1 x 10 14 cfu / g or less, 1×10 13 cfu / g or less, 1×10 12 cfu / g or less, 1×10 11 cfu / g or less, 1×10 10 cfu / g or less, 1×10 9 cfu / g or less, or 1 x 10 8 The content of the culture supernatant of Lactococcus bacteria in the vaccine described herein may be, for example, 1 x 10 cfu / g or less, converted into the viable cell count of the culture solution from which the culture supernatant is derived. 7 ~1×10 14 cfu / g, 1 × 10 8 ~1×10 13 cfu / g, or 1 × 10 9 ~1×10 12cfu / g. The phrase "the content of the culture supernatant of Lactococcus bacteria in the vaccine described herein is N cfu / g (N is a positive number) when converted into the viable cell count of the culture solution from which the culture supernatant is derived" means that the total amount of culture supernatant obtained from an amount of culture solution containing N cfu of Lactococcus bacteria (if inactivation treatment is performed, the culture solution before inactivation treatment) is contained in 1 g of the vaccine described herein. For example, 1 x 10 8 100 mL of cultured bacterial solution containing cfu / mL (total viable bacterial count of 1 x 10 10 cfu), and then optionally adding additives such as adjuvants to obtain a final vaccine of 0.5 g, the content of the culture supernatant in the vaccine is 2 x 10 viable bacteria in the culture solution from which the culture supernatant is derived. 10 cfu / g.

[0039] The vaccines described herein may or may not further contain, for example, the components used in the inactivation treatment exemplified above.

[0040] The vaccines described herein may or may not further contain an adjuvant, for example. That is, the method for producing the vaccines described herein may or may not include, for example, a step of adding an adjuvant (also referred to as an "adjuvant addition step"). The method for producing the vaccines described herein may include, for example, an adjuvant addition step after the bacterial cell removal step. The addition of an adjuvant is expected to enhance, for example, the immune response when using the vaccines described herein. Therefore, it is expected that the addition of an adjuvant will make it possible to reduce the amount of culture supernatant of Lactococcus bacteria contained in the vaccines described herein, specifically, to reduce the amount of antigens derived from the culture supernatant contained in the vaccines described herein.

[0041] Adjuvants include precipitating adjuvants, oily adjuvants, and aqueous adjuvants. The term "precipitating adjuvant" refers to a mineral suspending agent that adsorbs peptides. Precipitating adjuvants include aluminum hydroxide (also known as "alum"), calcium phosphate, aluminum phosphate, and alum. The term "oil-based adjuvant" refers to an oil emulsion that emulsifies an aqueous solution containing a peptide. Examples of oil-based adjuvants include liquid paraffin, lanolin, O / W emulsions, W / O emulsions, and W / O / W emulsions. Examples of aqueous adjuvants include saponin, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymers, methacrylic acid copolymers, maleic anhydride copolymers, and alkenyl derivative polymers. More specifically, adjuvants include known aluminum compounds such as aluminum hydroxide and aluminum phosphate, bacterial-derived substances such as AS04 (registered trademark), O / W emulsions such as MF59 (registered trademark) and AS03 (registered trademark), and O / W emulsions and W / O emulsions of the Montanide (trademark) ISA series. Among these, preferred adjuvants include Montanide (trademark) ISA 761VG, 78VG, and 660VG, which are W / O emulsions used as fish adjuvants. One type of adjuvant may be used, or two or more types of adjuvants may be used.

[0042] The vaccines described herein may or may not further contain a detoxifying agent, for example. The vaccines described herein contain a culture supernatant of piscine alpha-hemolytic streptococcus and may therefore contain hemolytic toxins. Hemolytic toxins are generally toxic to cells. Therefore, the vaccines described herein may contain a detoxifying agent, for example, to suppress the action of the hemolytic toxin. Furthermore, the methods for producing the vaccines described herein may or may not include, for example, a step of adding a detoxifying agent (also referred to as a "detoxifying agent addition step"). The methods for producing the vaccines described herein may include, for example, a detoxifying agent addition step after a bacterial cell removal step.

[0043] The detoxifying agent is not particularly limited as long as it inactivates the toxin without impairing the immunogenicity of the vaccine described herein, and examples of the detoxifying agent include preservatives such as formalin.

[0044] The content of a detoxifying agent (e.g., formalin) in the vaccine described herein may be, for example, 0.05 to 0.3 w / w%, preferably 0.1 w / w%. When two or more detoxifying agents are used, the "content of the detoxifying agents" refers to the total content of those detoxifying agents unless otherwise specified. However, when two or more detoxifying agents are used, the content of each of those detoxifying agents may be independently set within the content range of the detoxifying agents exemplified above.

[0045] The culture supernatant may or may not have been subjected to processing such as concentration, as long as it does not impair the immunogenicity of the vaccine described herein. That is, the method for producing the vaccine described herein may or may not include, for example, a step of concentrating the culture supernatant (also referred to as a "concentration step"). The degree of concentration of the culture supernatant is not particularly limited. The degree of concentration of the culture supernatant may be, for example, 2-fold or more, 5-fold or more, 10-fold or more, 100-fold or more, or 10,000-fold or less, 1,000-fold or less, 100-fold or less, 100-fold or less, 10-fold or less, or 5-fold or less, or any compatible combination thereof, in terms of volume ratio. The degree of concentration of the culture supernatant may specifically be, for example, 2-fold to 5-fold, 5-fold to 10-fold, 10-fold to 100-fold, 100-fold to 1,000-fold, or 1,000-fold to 10,000-fold. The degree of concentration of the culture supernatant may be, specifically, for example, 5 to 1000 times by volume. "Concentration" may also include drying. "Concentration of the culture supernatant" may also include concentration of a mixture of the culture supernatant and other components. The vaccine production method described herein may include, for example, a concentration step after the bacterial cell removal step. The vaccine production method described herein may include, for example, a concentration step before the adjuvant addition step. The vaccine production method described herein may include, for example, a concentration step before the detoxifying agent addition step. The vaccine production method described herein may include, for example, a concentration step after a step of adding other components, such as an adjuvant addition step or a detoxifying agent addition step. The concentration step may be performed once or two or more times. For example, the culture supernatant may be concentrated to a certain extent by a concentration step, and then a step of adding other components, such as an adjuvant addition step or a detoxifying agent addition step, may be performed, followed by a further concentration step (e.g., a drying step).

[0046] The method for concentrating the culture supernatant is not particularly limited, and examples of the method for concentrating the culture supernatant include ultrafiltration, vacuum concentration, spray drying, and freeze drying.

[0047] The form of the vaccines described herein is not particularly limited. The vaccines described herein may be in any form, such as a liquid or powder. The vaccines described herein may be produced and provided, for example, in a ready-to-use form, or in a form that requires preparation at the time of use. The vaccines described herein may be produced and provided, for example, in a form that is diluted with a solvent at the time of use. Forms that are diluted with a solvent at the time of use include concentrated forms such as dried products and concentrated liquids. In one aspect, the vaccines described herein (e.g., vaccines in a ready-to-use form) may be, in particular, a liquid. In another aspect, the vaccines described herein (e.g., vaccines in a dry form) may be, in particular, a freeze-dried product.

[0048] In vaccines containing bacterial cells, bacterial components other than antigenic components can cause side effects and allergic reactions. On the other hand, the vaccines described herein are free of bacterial cells, and are therefore expected to suppress side effects and allergic reactions.

[0049] The vaccines described herein may be used alone or in combination with other vaccines. That is, the present invention also discloses combination vaccine formulations combining the vaccines described herein with other vaccines. Multivalent vaccines that combine different antigens may experience reduced immunogenicity due to antigen interference. On the other hand, the vaccines described herein are expected to suppress interference effects because the bacterial cells have been removed. Therefore, the vaccines described herein are expected to function effectively as combination vaccine formulations with other vaccines. The other vaccines may or may not contain inactivated bacterial cells. Examples of other vaccines include vaccines against fish diseases. Specific examples of other vaccines (particularly vaccines against fish diseases) include an inactivated alpha-hemolytic streptococcosis vaccine, an inactivated beta-hemolytic streptococcosis vaccine, an inactivated vibriosis vaccine, an inactivated iridovirus disease vaccine, an inactivated tuberculosis vaccine, and an inactivated Streptococcus dysgalactiae infection vaccine. As the other vaccines, one type of vaccine may be used, or two or more types of vaccines may be used.

[0050] <2> Prevention of piscine alpha-hemolytic streptococcosis The prevention method described herein is a method for preventing piscine alpha-hemolytic streptococcosis, which comprises the step of administering the vaccine described herein to fish.

[0051] The alpha-hemolytic streptococci that cause piscine alpha-hemolytic streptococcosis to which the preventive methods described in this specification are applicable are as described above for the alpha-hemolytic streptococci that cause piscine alpha-hemolytic streptococcosis to which the vaccines described in this specification are applicable.

[0052] The fish to which the preventive methods described herein are applicable are as described above for the fish to which the vaccines described herein are applicable.

[0053] The method of administering the vaccine is not particularly limited as long as it can prevent piscine alpha-hemolytic streptococcosis.

[0054] Vaccine administration methods include injection, immersion, and oral administration. The vaccine dosage can be appropriately determined depending on various conditions, such as the vaccine administration method, the fish to be vaccinated, the content of Lactococcus culture supernatant in the vaccine, and the amount of antigen derived from the culture supernatant. The vaccines described herein can be administered to fish directly or after being appropriately prepared into a form suitable for administration. For example, liquid vaccines described herein may be administered to fish directly or after being appropriately diluted. Furthermore, non-liquid vaccines described herein may be administered to fish directly or after being appropriately prepared into a liquid form. The vaccine may be administered once, twice, or more times.

[0055] In the case of injection, for example, the vaccine described herein may be administered intramuscularly or intraperitoneally to fish. The vaccine dosage may be, for example, 0.05 mL or more, 0.07 mL or more, 0.1 mL or more, 0.3 mL or more, 0.5 mL or more, or 1 mL or more, or 3.0 mL or less, 2.5 mL or less, 2.0 mL or less, 1.5 mL or less, 1.0 mL or less, or 0.5 mL or less, or any compatible combination thereof. Specifically, the vaccine dosage may be, for example, 0.05 to 3.0 mL. The vaccine dosage may be, for example, 1 x 10 viable cells in the culture solution from which the culture supernatant is derived. 7 cfu or more, 1 × 10 8 cfu or more, or 1 × 10 9 cfu or more, 1 x 10 12 less than cfu, 1×10 11 less than cfu, 1×10 10 The dose of the vaccine may be, for example, 1 × 10 cfu or less, calculated as the viable cell count of the culture solution from which the culture supernatant is derived. 7 cfu ~ 1 × 10 12 cfu, 1 × 10 8 cfu ~ 1 × 10 11 cfu, or 1 × 10 9 cfu ~ 1 × 10 10The phrase "the amount of vaccine administered is N cfu (N is a positive number) converted into the viable cell count of the culture solution from which the culture supernatant is derived" means that a vaccine containing the entire amount of culture supernatant obtained from an amount of culture solution containing N cfu of Lactococcus bacteria (if inactivation treatment is performed, the culture solution before inactivation treatment) is administered.

[0056] In the case of the immersion method, for example, fish may be immersed in an immersion solution obtained by diluting the vaccine described herein. The immersion time may be, for example, 0.05 to 48 hours.

[0057] In the case of oral administration, for example, fish may be allowed to freely feed feed mixed with a vaccine described herein. The feed mixed with a vaccine described herein may be continuously fed for, for example, 1 to 20 days.

[0058] Among these, intraperitoneal administration by injection is preferred because it has a high infection prevention effect and provides long-lasting immunity. [Example]

[0059] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0060] Example 1: Production and Use of L. garvieae Type II Culture Supernatant Vaccine Including an Inactivation Step 1. Acquisition of L. garvieae type II strain HY21-1K (Ehime isolate) At a farm in Ehime Prefecture, Japan, Gram-positive bacteria were isolated from the kidneys of yellowtail fish suspected of developing streptococcosis despite administration of a conventional inactivated vaccine against L. garvieae (Kyoritsu Pharmaceutical's Picivac® Note 4). PCR analysis of L. garvieae identified all strains as type II L. garvieae. This isolated and identified strain was designated strain HY21-1K. Type II L. garvieae can be reproducibly obtained using a similar procedure.

[0061] 2.Culture process 1.0 mL of the seed culture (L. garvieae type II strain HY21-1K) was inoculated into 1,000 mL of TPB (Tryptose Phosphate Broth, BD) and cultured at 30°C for 24 hours with agitation (150 rpm). Next, a viable cell count test was performed on the resulting culture solution using the smear plate culture method. That is, a portion of the culture solution was smeared on an agar plate medium, and the viable cell count in the culture solution was determined from the number of colonies that appeared after culture. The result of the viable cell count test was 2.2 x 10 8 cfu / mL.

[0062] 3.Inactivation process Formalin was added to the culture solution at a ratio of 0.3 vol% (3.0 mL / 1,000 mL) and the mixture was stirred (150 rpm) at 30°C for 48 hours for sensitization. A viable bacterial count test was then performed using the smear plate culture method. The results of the viable bacterial count test showed that the number of viable bacteria was below the detection limit, confirming that the bacteria had been inactivated.

[0063] 4. Supernatant collection (centrifugation) process 800 mL of the inactivated cultured bacteria solution was centrifuged (8,000 G for 20 minutes) and the supernatant was collected.

[0064] 5.Supernatant filtration process 800 mL of the collected centrifuged supernatant was filtered (Millex (registered trademark) -HV filter 0.45 μm).

[0065] 6. Filtration supernatant concentration process 770 mL of the filtered supernatant was concentrated to 7.7 mL (100-fold concentration) using a centrifugal ultrafiltration filter (Amicon (registered trademark) Ultra-15 10 kDa Merck). 7.7 mL of the filtered supernatant concentrate was transferred to a 15 mL centrifuge tube and stored at 4°C to serve as the immunization material.

[0066] 7. Prevention test against alpha-hemolytic streptococcosis in fish Thirty-two yellowtail were divided into two groups of 16 fish each (test group and control group). In the test group, the immunizing material was injected intraperitoneally at a volume of 0.1 mL per tail. In the control group, PBS was injected intraperitoneally at a volume of 0.1 mL per tail. Two weeks after the injection, the challenge material (a culture solution of L. garvieae type II strain HY21-1K diluted 100-fold with PBS) was injected intraperitoneally at a volume of 0.1 mL per tail (equivalent to a viable bacterial count immediately after culture) 6 The animals were injected with a dose of 1000 cfu / tail. The survival and death of each group was observed for two weeks after the injection of the challenge material.

[0067] The results are shown in Table 1 and Figure 1. The mortality rate in the test group was significantly lower than that in the control group. Therefore, it was confirmed that the culture supernatant of Lactococcus bacteria, which cause piscine alpha-hemolytic streptococcosis, can function as a vaccine against piscine alpha-hemolytic streptococcosis.

[0068] [Table 1]

[0069] Example 2: Production and Use of an L. garvieae Type II Culture Supernatant Vaccine Without an Inactivation Step 1. Acquisition of L. garvieae type II strain HY21-1K (Ehime isolate) In Example 1, L. garvieae type II strain HY21-1K was obtained.

[0070] 2.Culture process 0.2 mL of the seed culture (L. garvieae type II strain HY21-1K) was inoculated into 200 mL of TPB (Tryptose Phosphate Broth, BD) and allowed to stand at 30°C for approximately 20 hours to prepare the original culture solution. The viable cell count of the original culture solution, as determined by the agar plate dilution method, was 5.5 x 10 8 Next, 4.0 mL of the original culture solution was inoculated into 4,000 mL of TPB and cultured at 30°C for 27 hours with stirring (150 rpm). The viable cell count of the resulting culture solution was 1.4 x 10 9 cfu / mL.

[0071] 3. Supernatant collection (centrifugation) process 4,000 mL of the cultured bacterial solution was centrifuged (8,000 rpm for 20 minutes) and the supernatant was collected.

[0072] 4.Supernatant filtration process 4,000 mL of the collected centrifuged supernatant was filtered (Thermo Scientific (registered trademark) Nalgene (registered trademark) Rapid-Flow (registered trademark) PES membrane filter unit, filter 0.2 μm). A viable cell count test was conducted on the filtered supernatant, and it was confirmed that the viable cell count was below the detection limit.

[0073] 5. Filtration supernatant detoxification process Formalin was added to the filtered supernatant at a ratio of 0.1 vol% (4.0 mL / 4,000 mL), and the mixture was then detoxified by sensitization at 30°C for 18 hours.

[0074] 6. Detoxification filtration supernatant concentration process 4,000 mL of the detoxified filtered supernatant was concentrated (40-fold) by ultrafiltration to 100 mL using a hollow fiber membrane module (Microza pencil-type module series, Asahi Kasei Chemicals Corporation). The detoxified filtered supernatant concentrate was diluted 4-fold with PBS (final detoxified filtered supernatant 10-fold concentrated), stored at 4°C, and used as the immunization material.

[0075] 7. Prevention test against alpha-hemolytic streptococcosis in fish Thirty yellowtail were divided into two groups of 15 fish each (test group and control group). In the test group, the immunizing material was injected intraperitoneally at a volume of 0.1 mL per tail. In the control group, PBS was injected intraperitoneally at a volume of 0.1 mL per tail. Four weeks after the injection, the challenge material (a culture solution of L. garvieae type II strain HY21-1K diluted 100,000 times with PBS) was injected intraperitoneally at a volume of 0.1 mL per tail (equivalent to a viable bacterial count immediately after culture) 2 The animals were injected with a dose of 1000 cfu / tail. The survival and death of each group was observed for two weeks after the injection of the challenge material.

[0076] The results are shown in Table 2 and Figure 2. The mortality rate in the test group was significantly lower than that in the control group. Therefore, it was confirmed that the culture supernatant of Lactococcus bacteria that cause piscine alpha-hemolytic streptococcosis can function as a vaccine against piscine alpha-hemolytic streptococcosis even without an inactivation step.

[0077] [Table 2]

Claims

1. 1. A vaccine against piscine alpha-hemolytic streptococcosis, comprising: Contains culture supernatant of Lactococcus bacteria that cause piscine alpha-hemolytic streptococcosis, A vaccine in which the culture supernatant is obtained by removing bacterial cells from a culture solution of the bacteria, and the bacterial cells have not been inactivated before the removal.

2. 2. The vaccine of claim 1, wherein the bacterium is Lactococcus garvieae, Lactococcus formosensis, Lactococcus petauri, or a closely related species thereof.

3. The vaccine of claim 2, wherein the bacterium is Lactococcus garvieae type I, Lactococcus garvieae type II, or serotype unknown Lactococcus garvieae.

4. The vaccine according to any one of claims 1 to 3, which is substantially free of bacterial cells.

5. The vaccine according to any one of claims 1 to 3, further comprising an adjuvant.

6. The vaccine according to any one of claims 1 to 3, further comprising a detoxifying agent.

7. 1. A method for producing a vaccine against piscine alpha-hemolytic streptococcosis, comprising: a step of culturing Lactococcus bacteria that cause piscine alpha-hemolytic streptococcosis to obtain a culture solution; a step of removing the bacterial cells from the culture solution to obtain a culture supernatant; and concentrating the culture supernatant; Including, The method does not include a step of inactivating the bacterial cells before the removal.

8. The method according to claim 7 , wherein the culturing is carried out using a liquid medium.

9. The method according to claim 7 or 8, further comprising the step of adding a detoxifying agent after the removal.

Images