WO2014185439A1 - VACCIN CONTRE L'INFECTION PAR LE VIRUS DE L'HERPÈS-2 DES CYPRINIDÉS (CyHV-2), PROCÉDÉ DE PRODUCTION CORRESPONDANT, ET PROCÉDÉ DE PRODUCTION DU VIRUS CyHV-2 - Google Patents

VACCIN CONTRE L'INFECTION PAR LE VIRUS DE L'HERPÈS-2 DES CYPRINIDÉS (CyHV-2), PROCÉDÉ DE PRODUCTION CORRESPONDANT, ET PROCÉDÉ DE PRODUCTION DU VIRUS CyHV-2 Download PDF

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WO2014185439A1
WO2014185439A1 PCT/JP2014/062778 JP2014062778W WO2014185439A1 WO 2014185439 A1 WO2014185439 A1 WO 2014185439A1 JP 2014062778 W JP2014062778 W JP 2014062778W WO 2014185439 A1 WO2014185439 A1 WO 2014185439A1
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cyhv
virus
vaccine
cells
fish
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Japanese (ja)
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尚史 伊東
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独立行政法人水産総合研究センター
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • C12N7/06—Inactivation or attenuation by chemical treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a vaccine for CyHV-2 infection, a method for producing the same, and a method for producing CyHV-2 virus.
  • CyHV-2 infectious disease has caused great damage in goldfish farms in Japan since it first occurred in Aichi Prefecture in 1992 (Aichi Prefectural Government website http://www.pref.aichi.jp/0000009518.html ). Furthermore, the damage caused by the infectious disease has occurred not only in Japan but around the world.
  • Non-patent Documents 1 and 2 goldfish in the United States (Non-patent Documents 1 and 2), Taiwan (Non-patent Document 3), Australia (Non-patent Document) 4), New Zealand (Non-Patent Document 5), and the United Kingdom (Non-Patent Document 6) have been reported, and in Giberiobna Hungary (Non-Patent Document 7), Czech (Non-Patent Document 8), and China (Non-Patent Document) The occurrence report from 9) has been made.
  • CyHV-2 is sometimes called GFHNV (Goldfisholdhematopoietic necrosis virus) and is a kind of fish herpesvirus known to cause necrosis in the hematopoietic tissue of infected fish. It is closely related to CyHV-1 and CyHV-3, which also infects cyprinid fish (Non-patent Document 14).
  • GFHNV Goldfisholdhematopoietic necrosis virus
  • Non-patent Document 10 There is a report of the growth of CyHV-2 using fathead minnow-derived FHM cells (Non-patent Document 10), but in this method, virus passage can be performed only up to four times. In addition, there is a report (Non-patent Documents 6 and 9) on the growth of CyHV-2 using KF-1 cells derived from carp fin, but this method also succeeded in the continuous growth of CyHV-2. Not done.
  • Non-patent Document 11 There has been a report (Non-patent Document 11) that CyHV-2 can be continuously propagated using goldfish fin-derived GFF cells, but the proliferation method is not sufficiently disclosed in the report, Conversely, there is a report that it is difficult to grow CyHV-2 using GFF cells (Non-Patent Document 12), and there is room for debate as to whether this proliferation method is an excellent method.
  • CyHV-2 infection has caused great damage on a global scale, but due to the difficulty in continuously growing CyHV-2, there have been no reports of vaccine development against this infection.
  • CyHV-3 also referred to as KHV
  • CyHV-3 which is genetically close to CyHV-2, uses carp as a host, and caused great damage all over the world (Non-patent Document 13).
  • Attempts have been made to develop vaccines against CyHV-3 infections, but to date, safe and effective vaccines against CyHV-3 infections are not widespread (Non-Patent Documents 14 and 15). In general, administration of an inactivated vaccine by intraperitoneal injection is not effective for CyHV-3 infection (Non-patent Document 15).
  • CyHV-3 inactivated vaccines were thought to be ineffective, and development of attenuated live vaccines has been attempted (Non-Patent Documents 14 and 15). CyHV-3 inactivated vaccines are generally considered to be ineffective, but the inactivated CyHV-3 is encapsulated in liposomes and orally administered to carp in a certain proportion. (Non-Patent Document 16).
  • An object of the present invention is to provide a vaccine against CyHV-2 infection and a method for producing the same, and to prevent damage caused by CyHV-2 infection that has caused great damage all over the world. It is also an object of the present invention to provide a method for continuous growth of CyHV-2 virus that is useful for vaccine production.
  • the present inventor has conducted intensive research to solve the above problems, and has developed a method capable of continuously proliferating CyHV-2 in vitro. In this proliferation method, it has been confirmed that at least 12 subcultures are possible using cultured cells. Since it is known that an inactivated vaccine is not effective with CyHV-3, which is a related virus of CyHV-2, the present inventor did not expect an effect on a CyHV-2 inactivated vaccine at first. . However, contrary to expectation, when the present inventor made an inactivated CyHV-2 vaccine using CyHV-2 grown by the above method and ingested it in goldfish, the survival rate against CyHV-2 infection was remarkable. An improvement was found (survival improved from 0% to 57%). The present inventor completed the present invention based on the above results.
  • the present invention relates to the following.
  • CyHV-2 inactivated cyprinid herpesvirus-2
  • a method for preventing CyHV-2 infection comprising administering an effective amount of inactivated CyHV-2 to a target fish.
  • a method for rearing the target fish comprising administering an effective amount of inactivated CyHV-2 to the target fish, and rearing the control fish.
  • a method for producing CyHV-2 virus comprising the following steps (a) to (c): (A) adding CyHV-2 virus to a cell culture medium containing cultured cells derived from cyprinid fish; (B) every 7 to 14 days after the addition of CyHV-2, the cultured cells are dispersed, and 50% or more of the cells and medium used so far remain, and 1 to 2 times the amount of the remaining medium is newly added. And (c) a step of recovering CyHV-2 virus when 50% or more of the cells lose their adherence to the bottom surface of the culture vessel after passage. .
  • a method for producing a vaccine against CyHV-2 infection comprising the following steps (a) to (d): (A) adding CyHV-2 virus to a cell culture medium containing cultured cells derived from cyprinid fish; (B) every 7 to 14 days after the addition of CyHV-2, the cultured cells are dispersed, and 50% or more of the cells and medium used so far remain, and 1 to 2 times the amount of the remaining medium is newly added. Adding a suitable medium and subculture; (C) After passage, when 50% or more of the cells lose adhesion to the bottom of the culture vessel, the step of recovering CyHV-2 virus; and (d) recovered in (c) Inactivating the CyHV-2 virus.
  • the present invention provides an effective vaccine against CyHV-2 infection. Furthermore, it is possible to efficiently produce a vaccine for CyHV-2 infection by using the method for producing CyHV-2 of the present invention capable of continuous growth. The effectiveness of the vaccine of the present invention is confirmed by intraperitoneal administration without using liposomes. Therefore, the vaccine of the present invention does not require a step of encapsulating in liposomes, and provides a method for preparing a vaccine effective against CyHV-2 infection that can be prepared and administered very easily.
  • the present invention provides a vaccine against CyHV-2 infection, which contains inactivated CyHV-2.
  • CyHV-2 is a kind of herpesvirus belonging to the family of herpesviridae, and CyHV-1 and CyHV-3 are known as closely related viruses.
  • CyHV-2 that can be used in the vaccine of the present invention may include variants or subtypes of CyHV-2 introduced with natural or artificial mutations, in addition to wild-type CyHV-2.
  • CyHV-2 may be a virus isolated from a fish infected with CyHV-2, or a virus grown in vitro using cultured cells, preferably a virus grown using cultured cells. .
  • CyHV-2 virus When recovering the CyHV-2 virus from the fish body, it can be isolated from an organ excised or removed from a fish belonging to the cyprinid family that has developed or died due to CyHV-2 infection, such as goldfish and crucifers.
  • organs for example, spleen, kidney, sputum, liver, sputum, intestinal tract, heart and the like can be used.
  • virus isolation materials are, for example, cut into pieces with an ophthalmic scissors, added with a common salt solution or medium, for example, phosphate buffer (PBS) or MEM medium, and ground with a homogenizer.
  • PBS phosphate buffer
  • MEM ground with a homogenizer.
  • a tissue piece that has been cut into pieces with an ophthalmic scissor is fixed in a cell culture flask, and a cell line that has expanded and proliferated / divided from the fixed tissue piece can also be used for vaccine production as a virus isolation material. .
  • the cultured cells that can be used to propagate and pass CyHV-2 in vitro are not particularly limited as long as CyHV-2 can be infected and CyHV-2 virus can replicate in the infected cells.
  • Cultured cells derived from fish belonging to the cyprinidae are preferred. Examples include various primary cells derived from cyprinid fish or their subcultured cells, established cells, and the like. Examples of cell lines include known cell lines such as GFF cells, KF-1 cells, FHM cells, BF-2 cells, CHSE-214 cells, JSKG cells, KRE-3 cells, RTG-2 cells, and YTF cells. be able to.
  • SRTF cells newly established from goldfish fins at the Tamsui Government Office, National Fisheries Research Center.
  • Preferred cell lines are GFF cells, KF-1 cells, FHM cells, and SRTF cells, and particularly preferred are GFF cells and SRTF cells.
  • Examples of a medium known to those skilled in the art that can be used for culturing the cells include, for example, 199 medium, MEM (Eagle's Minimum Essential Medium) medium, BME (Eagle's Basal Medium) medium, Hanks solution, Dulbecco's phosphate.
  • a buffer solution (PBS) or the like can be used.
  • additives conventional substances such as commercially available human serum, fetal bovine serum FCS (Fetal Calf Serum), antibiotics, sodium hydrogen carbonate solution, sodium chloride, non-essential amino acids [manufactured by Dainippon Sumitomo Pharma Co., Ltd.]
  • An appropriate amount can be used.
  • these final concentrations added and mixed per liter of medium are about 3 to 25% (V / V) for serum, about 1 to 6 g for sodium bicarbonate, and about 0.1 to 0.25 for sodium chloride.
  • Molar and non-essential amino acids are preferably standard amounts described in the product catalog.
  • the cultured cells that can be used in virus propagation / passaging are not limited to one type of cell, and can be changed based on the possibility of virus propagation during virus propagation / passaging. That is, one or more types of cells can be used in combination.
  • the culture broth was centrifuged at low speed, the infected cell fraction was collected and sonicated, the disrupted cell suspension was frozen and thawed with dry ice and organic solvent, and the suspension of the infected cells.
  • a supernatant obtained by low-speed centrifugation of such a suspension, a resuspended solution of the sediment cells, and the like can be used as a vaccine stock solution.
  • this vaccine stock solution can be sterilized by a membrane filtration method, and antibiotics can also be added.
  • the virus recovered by the above method can be concentrated and purified by ultracentrifugation using a sucrose density gradient.
  • the collected virus solution is centrifuged at about 100,000 G for 90 minutes, and the precipitate containing the virus is resuspended in an appropriate salt solution such as Tris-sodium chloride-EDTA buffer (TNE) solution.
  • TNE Tris-sodium chloride-EDTA buffer
  • the virus-containing solution is layered on a density gradient solution having a sucrose concentration of 10-60% (W / W), and subjected to ultracentrifugation to precipitate the virus antigen. Then, the precipitate is recovered and purified virus antigen as a resuspended solution.
  • the sucrose concentration that can be employed in the present invention is in the range of about 10 to 60% (W / W), and the centrifugation conditions are about 50,000 to 150,000 G and about 60 to 1000 minutes.
  • CyHV-2 grown using cultured cells there can be mentioned CyHV-2 produced by the production method of the present invention described later.
  • virions that are complete virus particles, incomplete virus particles, virion components and their post-translational modifications, infection protective antigens, epitopes for neutralization reactions, and the like can be used as vaccine antigens.
  • the virion component and its post-translational modification may be, for example, a protein, peptide, nucleic acid, sugar chain, glycopeptide, glycoprotein, lipid, glycolipid, or a modification thereof derived from CyHV-2 Any component of the viral particle may be used as long as it can elicit an immune response in the subject and achieve a desired effect.
  • the constituent component may be a naturally-derived component derived from CyHV-2 virus virion, or may be artificially synthesized.
  • the vaccine of the present invention may contain one or a combination of two or more of the components.
  • CyHV-2 contained in the vaccine of the present invention is an inactivated virus.
  • CyHV-3 a related virus of CyHV-2
  • inactivated virus vaccines were not effective, so it was expected that inactivated vaccines would not be effective for CyHV-2 as well.
  • the present inventors have revealed that inactivated CyHV-2 exhibits a high vaccine effect.
  • the inactivated virus include an inactivated whole particle virus that has been inactivated while maintaining the structure of the virus particle, and an inactivated split virus that has been inactivated by destroying the virus particle.
  • the inactivated whole particle virus can be obtained by chemical treatment such as ⁇ -propiolactone, phenol, aldehyde (formalin, glutaraldehyde, etc.), ultraviolet irradiation, radiation irradiation, heating treatment, and the like.
  • Inactivated split virus can be obtained by chemical treatment with ether or the like.
  • the inactivated virus used in the present invention is preferably all particles of CyHV-2 inactivated by aldehyde from the viewpoint of maintaining antigenicity and high vaccine effect.
  • the inactivation method can be appropriately set depending on the type of inactivation treatment and the inactivating agent to be used, and is not particularly limited as long as the vaccine effect against CyHV-2 infection is achieved.
  • formalin is used for inactivation.
  • the amount added is about 0.1 to 0.3% (V / V) at the final concentration
  • the temperature for inactivation is 4 to 20 ° C
  • inactivation treatment The time for performing the step is 24 to 72 hours.
  • Such relaxation can be achieved, for example, by reducing the amount of the inactivating agent, adding neutral amino acids or basic amino acids, reducing the inactivation temperature, shortening the inactivation time, and the like.
  • the free formaldehyde remaining in the inactivation step can be neutralized by adding an equal amount of sodium bisulfite, if necessary, or removed by dialysis.
  • an effective amount of inactivated CyHV-2 is diluted with a salt solution or a medium, such as a medium MEM. That is, by such dilution, the amount of inactivated CyHV-2 in the vaccine is adjusted to an amount necessary for antibody production. Further, at that time, a stabilizer for enhancing the heat resistance of the vaccine and an adjuvant as an adjuvant for enhancing the immunogenicity can be added and mixed.
  • a stabilizer for enhancing the heat resistance of the vaccine and an adjuvant as an adjuvant for enhancing the immunogenicity can be added and mixed.
  • saccharides and amino acids can be used as stabilizers, and mineral oil, vegetable oil, alum, aluminum phosphate, bentonite, silica, muramyl dipeptide derivatives, thymosin, interleukin, and the like can be used as adjuvants.
  • a vaccine is not only liquid, but can be used as a dry preparation by lyophilization after dispensing.
  • an assay is based on an existing preparation similar to the vaccine according to the present invention, for example, “Japanese encephalitis inactivated vaccine” defined in “Animal Biologics Standards” based on the Pharmaceutical Affairs Law (Act No. 145 of 1960), “ It can be performed in accordance with regulations such as “rabies tissue culture inactivated vaccine”.
  • inactivated CyHV-2 may be encapsulated in liposomes for the preparation of the vaccine of the present invention. Since there is a report (Non-patent Document 16) that inactivated CyHV-3 has been found to be effective as a vaccine by encapsulating in liposomes, the vaccine effect can be recognized in the same manner for inactivated CyHV-2. However, in one embodiment, the inactivated CyHV-2 used in the vaccine of the present invention can exert a desired effect even when it is not encapsulated in liposomes.
  • the vaccine of the present invention can be used for fish of any age that may be infected with CyHV-2. From the viewpoint of fish farming conservation, it is preferable to use it for juvenile or fry, but it can also be used for adult fish.
  • the method of use for example, intraperitoneal, subcutaneous, or intramuscular inoculation, immersion method, oral administration, mixed food administration and the like are possible.
  • the method of administering the vaccine of the present invention comprises intraperitoneal, Subcutaneous or intramuscular inoculation, immersion method, and the like.
  • the amount to be used needs to be set appropriately depending on the type, size, age, administration method, etc. of the fish to be administered. For example, in the case of inoculation, it is desirable to use about 0.05 to 1.0 mL of the above antigen amount.
  • the vaccine can be diluted about 10 to 100 times with breeding water or hypotonic breeding water.
  • the vaccine can be stored in a cold, non-freezing environment, for example, in a cool dark place of about 2-8 ° C.
  • CyHV-2 infection in fish is found in all organs such as sputum, sputum, heart, intestine, spleen, brain and kidney.
  • the subject to which the vaccine of the present invention is administered is not particularly limited as long as it is a fish that is infected with CyHV-2 and may exhibit the above pathological condition, but a preferred subject to be administered is a cyprinid fish, and a more preferred subject to be administered is goldfish, They are fungi, and the most preferable administration subject is goldfish.
  • the duration of the effect of the vaccine of the present invention may vary depending on the dose, method and frequency of administration of the vaccine, and the type, size and age of the fish to be administered.
  • the vaccine of the present invention The effects of can be sustained for more than 8 weeks with a single dose.
  • the present invention provides a method for preventing CyHV-2 infection and a method for raising the target fish, comprising administering the vaccine to the target fish.
  • the vaccine of the present invention is administered to target fish not infected with CyHV-2 by, for example, intraperitoneal, subcutaneous, or intramuscular inoculation, immersion method, oral administration, mixed food administration, and the like. By doing so, it is possible to impart resistance to CyHV-2 infection and prevent the onset of pathological conditions due to the infection.
  • the present invention also provides a method for breeding fish while administering the vaccine of the present invention in a similar manner, preventing the onset of CyHV-2 infection, or curing fish suffering from the infection. I will provide a.
  • Administration in the prevention method and breeding method can be performed one or more times, and multiple administration is preferred from the viewpoint of imparting sufficient resistance. Multiple administrations can be performed by one administration method, or a combination of administration methods. Examples of the administration method include intraperitoneal, subcutaneous, or intramuscular inoculation, a dipping method, oral administration, and mixed food administration. Administration by intraperitoneal, subcutaneous, or intramuscular inoculation, immersion method or the like, which has not been confirmed to be effective with an inactivated CyHV-3 vaccine, is particularly preferred.
  • the administration interval needs to be set appropriately depending on the size, type, age, breeding environment, etc. of the target fish. For example, the vaccine is administered intraperitoneally to goldfish, and the vaccine is administered again 6 to 12 days after administration. The method of administration is mentioned.
  • the age of the target fish may be any of adult fish, juvenile fish, and juvenile fish, but juvenile fish or juvenile fish are preferred from the viewpoint of fish farming conservation.
  • the present invention provides a method for producing CyHV-2 virus.
  • the manufacturing method includes the following steps: (A) adding CyHV-2 virus to a cell culture medium containing cultured cells derived from cyprinid fish; (B) every 7 to 14 days after the addition of CyHV-2, the cultured cells are dispersed, and 50% or more of the cells and medium used so far remain, and 1 to 2 times the amount of the remaining medium is newly added. And (c) a step of recovering CyHV-2 virus when 50% or more of the cells lose their adherence to the bottom surface of the culture vessel after passage. .
  • the cultured cells derived from cyprinid fish that can be used in the method for producing CyHV-2 virus of the present invention are particularly limited as long as CyHV-2 can be infected and CyHV-2 virus can replicate in infected cells.
  • GFF cells, KF-1 cells, FHM cells, SRTF cells are preferred, with GFF cells and SRTF cells being most preferred. In culturing these cells, media and additives known to those skilled in the art can be used. CyHV-2 virus is added to the culture solution of cultured cells derived from cyprinid fish that have been cultured by the above method.
  • the addition amount of the virus is not limited as long as the virus in an amount necessary for vaccine production is obtained, usually, the virus solution to 10 2 to 10 4 in terms to the logarithm log of the TCID 50 (TCID 50 / mL) , medium Add 1/10 to 1/20 of the amount.
  • the cells at the time of virus addition may be in a state of being adhered to the bottom surface of the culture vessel, or may be dispersed and suspended in the culture solution before fixing. Preferably, the cells at the time of virus addition are in a dispersed and floating state.
  • the production method includes a step of dispersing and subculture cells every 7 to 14 days after the addition of CyHV-2 virus.
  • a step of dispersing and subculture cells every 7 to 14 days after the addition of CyHV-2 virus At the time of the passage, 50% or more of the medium used before the passage is left, and a new medium of 1 to 2 times the remaining medium is added. Since the residual medium contains a virus produced before passage, the step of allowing most of the medium to remain is important in order to increase the virus titer.
  • the step of allowing the medium before passage to remain at 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% may be performed before passage. Since it results in leaving much of the virus produced, it can be employed in the method for producing a virus of the present invention.
  • CyHV-2 virus can be recovered when it has lost adhesion to the bottom of the container.
  • the cell that has lost its adhesion may be a dead cell or a living cell.
  • the virus can be recovered from the medium alone, the cultured cells alone, or the culture medium and the cultured cells.
  • the virus is a supernatant obtained by low-speed centrifugation of the culture solution, a disrupted cell suspension obtained by collecting and sonicating the infected cell fraction, a suspension of virus and its infected cells frozen and thawed with dry ice and an organic solvent, such suspension.
  • the solution can be collected from the supernatant obtained by low-speed centrifugation, the resuspended solution of the sediment cells, and the like.
  • the present invention further provides a method for producing a vaccine against CyHV-2 infection using the CyHV-2 virus produced by the above method.
  • the vaccine production method further includes a step of inactivating the CyHV-2 virus produced by the above method.
  • the inactivation can be performed by any method as long as a desired vaccine effect is obtained, but in a preferred embodiment, the inactivation is performed by ⁇ -propiolactone, phenol, aldehyde (formalin, glutaraldehyde, etc.) Can be carried out by chemical treatment such as ultraviolet irradiation, radiation irradiation, and heating treatment. It is also possible to produce a vaccine as an inactivated split virus by chemical treatment with ether or the like.
  • the inactivation method can be appropriately set according to the type of inactivation treatment and the inactivating agent used, and is not particularly limited as long as the vaccine effect against CyHV-2 infection is achieved.
  • formalin is used for inactivation. Is used, the addition amount is about 0.1 to 0.3% (V / V) at the final concentration, the temperature at the time of inactivation is 4 to 20 ° C., and the time for the inactivation treatment is 24 to 72. Time is given.
  • the antigenicity of the vaccine is impaired by inactivation, it is necessary to devise ingenuity to relax the inactivation conditions.
  • Such relaxation can be achieved, for example, by reducing the amount of the inactivating agent, adding neutral amino acids or basic amino acids, reducing the inactivation temperature, shortening the inactivation time, and the like.
  • the free formaldehyde remaining in the inactivation step can be neutralized by adding an equal amount of sodium bisulfite, if necessary, or removed by dialysis.
  • Example 1 Propagation of CyHV-2 virus using cultured cells
  • GFF cells Li and Hukuda Non-Patent Document 14
  • the GFF cells were obtained at 25 ° C. in MEM medium supplemented with 10% fetal calf serum (Equitech Bio, Inc.) and antibiotics (100 U / mL penicillin and 100 mg / mL streptomycin (Wako Pure Chemical Industries, Ltd.)). Cultured.
  • the CyHV-2 virus which is the source of the propagation, was obtained from Mr.
  • the virus titer obtained was 10 3.0 TCID 50 / mL.
  • 7 to 14 days after passage if the proportion of cells not attached to the culture flask was less than 50%, further passage was performed.
  • Example 2 Preparation of vaccine for CyHV-2 infection CyHV-2 virus solution obtained by the above method (virus titer 10 3.0 TCID 50 / mL, formalin 0.1% (V / V) and treated at 4 ° C. for 48 hours.
  • Example 4 Improvement of survival rate after CyHV-2 infection by administration of vaccine for CyHV-2 infection using Ryukin (Fig. 3)
  • 200 CyHV-2-free Ryukins produced at Tamaki Government Building, Inc. were used. Divide the test fish into 8 tanks of 25 fish, respectively, vaccine zone (i) or (ii); vaccine + booster zone (i) or (ii); untreated zone (i) or (ii); non-infected zone It was set as (i) or (ii).
  • CyHV-2 virus (virus titer 10 3. 3) prepared according to the method described in Example 1 .
  • Example 2 prepared by the method of Example 2 was injected intraperitoneally with an inactivated CyHV-2 infection vaccine and immunized.
  • the vaccine + booster sections (i) and (ii) were boosted again in the same manner 4 weeks after the first immunization.
  • goldfish are immersed for 1 hour in a 1000-fold diluted CyHV-2 virus (10 3.0 TCID 50 / mL), which is the same lot virus solution used for vaccine production.
  • virus attack tests were performed on vaccine sections (i) and (ii), vaccine + booster sections (i) and (ii), and untreated sections (i) and (ii). Thereafter, death was observed for 34 days.
  • Non-infected areas (i) and (ii) were reared without vaccination and virus attack and observed for 34 days of death.
  • the average survival rate was 34% in the untreated group, whereas the average survival rate was 72% and 76% in the vaccine group and the vaccine + booster group, respectively.
  • the vaccine of the present invention can prevent damage caused by CyHV-2 infection that causes great damage.
  • the present invention also provides a method for continuous propagation of CyHV-2 virus, thereby enabling mass production of the vaccine.

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  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Biochemistry (AREA)
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Abstract

L'invention concerne l'obtention, par inactivation d'un virus CyHV-2, d'un vaccin hautement efficace contre l'infection par le CyHV-2. L'invention concerne également un procédé de propagation continue du virus CyHV-2, une telle propagation étant jusqu'à présent difficile à réaliser. L'utilisation de ce procédé de propagation permet une production en masse d'un vaccin contre l'infection par le virus CyHV-2.
PCT/JP2014/062778 2013-05-15 2014-05-14 VACCIN CONTRE L'INFECTION PAR LE VIRUS DE L'HERPÈS-2 DES CYPRINIDÉS (CyHV-2), PROCÉDÉ DE PRODUCTION CORRESPONDANT, ET PROCÉDÉ DE PRODUCTION DU VIRUS CyHV-2 WO2014185439A1 (fr)

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JP2014543029A JP6406703B2 (ja) 2013-05-15 2014-05-14 コイ科ヘルペスウイルス−２（Ｃｙｐｒｉｎｉｄｈｅｒｐｅｓｖｉｒｕｓ−２：ＣｙＨＶ−２）感染症用ワクチンおよびその製造方法、ならびにＣｙＨＶ−２ウイルス製造方法

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CN106366187A (zh) * 2016-09-14 2017-02-01 上海海洋大学 一株ii型鲤疱疹病毒orf72蛋白的单克隆抗体及其应用
CN108070678A (zh) * 2018-02-01 2018-05-25 上海海洋大学 恒温实时检测ii型鲤疱疹病毒的rpa试剂盒及其专用引物和探针
CN108752470A (zh) * 2018-06-13 2018-11-06 苏州大学 一种抗鲤疱疹病毒ii型orf72的卵黄抗体及其制备方法
CN110468069A (zh) * 2019-08-19 2019-11-19 中国水产科学研究院长江水产研究所 一种干酪乳杆菌yfi-5及其在抗鲤疱疹病毒ii型中的应用
CN110564750A (zh) * 2019-08-30 2019-12-13 中国水产科学研究院长江水产研究所 一种鲫造血器官坏死症酵母口服疫苗及应用
CN110893235A (zh) * 2019-11-13 2020-03-20 浙江省淡水水产研究所 一种鲫造血器官坏死病灭活疫苗及其制备方法
CN111411087A (zh) * 2020-03-16 2020-07-14 中国水产科学研究院长江水产研究所 一种鲤疱疹病毒ii型弱毒株及其应用
CN112279896A (zh) * 2018-11-05 2021-01-29 共鳞实业(深圳)有限公司 一种用于鱼类预防或治疗CyHV-2感染的试剂及应用

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Cited By (16)

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CN106366187A (zh) * 2016-09-14 2017-02-01 上海海洋大学 一株ii型鲤疱疹病毒orf72蛋白的单克隆抗体及其应用
CN108070678A (zh) * 2018-02-01 2018-05-25 上海海洋大学 恒温实时检测ii型鲤疱疹病毒的rpa试剂盒及其专用引物和探针
CN108752470A (zh) * 2018-06-13 2018-11-06 苏州大学 一种抗鲤疱疹病毒ii型orf72的卵黄抗体及其制备方法
CN108752470B (zh) * 2018-06-13 2021-06-22 苏州大学 一种抗鲤疱疹病毒ii型orf72的卵黄抗体及其制备方法
CN112279898A (zh) * 2018-11-05 2021-01-29 共鳞实业(深圳)有限公司 预防或治疗鱼类感染CyHV-2的试剂及其应用
CN112279896B (zh) * 2018-11-05 2022-06-21 共鳞实业(深圳)有限公司 一种用于鱼类预防或治疗CyHV-2感染的试剂及应用
CN112279898B (zh) * 2018-11-05 2022-06-21 共鳞实业(深圳)有限公司 预防或治疗鱼类感染CyHV-2的试剂及其应用
CN112279896A (zh) * 2018-11-05 2021-01-29 共鳞实业(深圳)有限公司 一种用于鱼类预防或治疗CyHV-2感染的试剂及应用
CN110468069A (zh) * 2019-08-19 2019-11-19 中国水产科学研究院长江水产研究所 一种干酪乳杆菌yfi-5及其在抗鲤疱疹病毒ii型中的应用
CN110468069B (zh) * 2019-08-19 2020-11-06 中国水产科学研究院长江水产研究所 一种干酪乳杆菌yfi-5及其在抗鲤疱疹病毒ii型中的应用
CN110564750A (zh) * 2019-08-30 2019-12-13 中国水产科学研究院长江水产研究所 一种鲫造血器官坏死症酵母口服疫苗及应用
CN110564750B (zh) * 2019-08-30 2020-12-25 中国水产科学研究院长江水产研究所 一种鲫造血器官坏死症酵母口服疫苗及应用
CN110893235A (zh) * 2019-11-13 2020-03-20 浙江省淡水水产研究所 一种鲫造血器官坏死病灭活疫苗及其制备方法
CN110893235B (zh) * 2019-11-13 2023-10-24 浙江省淡水水产研究所 一种鲫造血器官坏死病灭活疫苗及其制备方法
CN111411087B (zh) * 2020-03-16 2021-03-23 中国水产科学研究院长江水产研究所 一种鲤疱疹病毒ii型弱毒株及其应用
CN111411087A (zh) * 2020-03-16 2020-07-14 中国水产科学研究院长江水产研究所 一种鲤疱疹病毒ii型弱毒株及其应用

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