WO2023160425A1 - 一种鲑鳟鱼传染性造血器官坏死病和传染性胰脏坏死病单价佐剂疫苗及其二联佐剂疫苗和制备方法 - Google Patents

一种鲑鳟鱼传染性造血器官坏死病和传染性胰脏坏死病单价佐剂疫苗及其二联佐剂疫苗和制备方法 Download PDF

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WO2023160425A1
WO2023160425A1 PCT/CN2023/075791 CN2023075791W WO2023160425A1 WO 2023160425 A1 WO2023160425 A1 WO 2023160425A1 CN 2023075791 W CN2023075791 W CN 2023075791W WO 2023160425 A1 WO2023160425 A1 WO 2023160425A1
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infectious
vaccine
necrosis
virus
adjuvant
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PCT/CN2023/075791
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English (en)
French (fr)
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徐黎明
赵景壮
任广明
邵轶智
卢彤岩
林玉杰
赵文闻
刘琪
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中国水产科学研究院黑龙江水产研究所
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Priority claimed from CN202210176019.6A external-priority patent/CN115804837B/zh
Priority claimed from CN202210175989.4A external-priority patent/CN115837074B/zh
Priority claimed from CN202210176219.1A external-priority patent/CN115804839B/zh
Application filed by 中国水产科学研究院黑龙江水产研究所 filed Critical 中国水产科学研究院黑龙江水产研究所
Publication of WO2023160425A1 publication Critical patent/WO2023160425A1/zh

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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
    • 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
    • A61K39/205—Rhabdoviridae, e.g. rabies 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the invention relates to the field of biotechnology, in particular to a monovalent adjuvant vaccine for infectious hematopoietic organ necrosis and infectious pancreatic necrosis of salmon and trout, a dual adjuvant vaccine and a preparation method thereof.
  • Salmon and trout refer to a group of high-quality cold-water fish mainly represented by salmonids, mainly including Atlantic salmon (Salmosalar), rainbow trout (Oncorhynchus mykiss), king salmon (Oncorhynchus tshawytscha), coho salmon (Oncorhynchus kisutch), etc., are international mainstream breeding and trade species.
  • the main breed of salmon and trout in my country is rainbow trout, which is mainly freshwater aquaculture.
  • the aquaculture areas are widely distributed in areas with relatively developed cold water resources such as northeast, northwest, and southwest China. They have played an active role in driving local economic development and increasing fishermen's income. effect.
  • Salmon and trout farming meets my country's modern fishery development requirements of "improving quality, increasing efficiency, and green development".
  • large-scale recirculating aquaculture facilities have been introduced in my country's inland areas, and modern marine pastures have been developed to breed rainbow trout in coastal areas.
  • the salmon and trout aquaculture industry has rapidly transformed and upgraded.
  • the salmon and trout farming industry in my country is facing major disease problems, and the industrial development is seriously hindered.
  • IHN infectious hematopoietic necrosis
  • IPN infectious pancreatic necrosis
  • IPNV can cause the highest damage to salmon trout juveniles such as rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), American char (Salvelinus fontinalis), and leprosy salmon (Brachymystax lenok). More than 80% mortality, IHN can cause the highest mortality of rainbow trout, Atlantic salmon and other juvenile fish more than 90%, which is the primary disease problem that must be solved in the development of my country's salmon and trout industry.
  • the vaccine was used to immunize rainbow trout directly, and it was found that the prepared inactivated vaccine had a relatively ideal early protective effect, but with the extension of time, the immune protection efficiency of the vaccine began to decline from 45 days after immunization, and it did not have long-term protection. In the long term, it cannot meet the market demand, so it is extremely necessary to optimize the existing inactivated vaccines.
  • IHN nucleic acid vaccine targeting North American genotypes, and there is no effective IHN prevention and control drug in my country. Due to the mutation of virus strains and the existence of biological safety hazards, foreign commercial vaccines cannot be directly introduced and applied. Therefore, the problem of salmon and trout diseases in my country can only be solved through the independent research and development of vaccines.
  • the research group carried out the research and development of IHN inactivated vaccine in the early stage. By using formaldehyde and BPL to inactivate, IHN inactivated vaccine was prepared, and the vaccine was used to directly immunize rainbow trout.
  • the prepared inactivated vaccine has a relatively ideal early immune protection effect (referring to Chinese patents CN113144185A and CN113122510A), but as time goes on, when 60 days after immunization, the immune protection efficiency of the vaccine decreases significantly, and it cannot maintain a long protection In the short term, it cannot meet the market demand, so it is extremely necessary to optimize the existing IHN inactivated vaccine.
  • the object of the present invention is to provide a monovalent adjuvant vaccine for infectious hematopoietic organ necrosis disease and infectious pancreatic necrosis disease of salmon and trout, a dual adjuvant vaccine and a preparation method thereof.
  • the present invention claims to protect a dual vaccine of infectious hematopoietic necrosis disease and infectious pancreatic necrosis disease.
  • infectious hematopoietic necrosis disease vaccine and infectious pancreatic necrosis disease vaccine claimed in the present invention are composed of infectious hematopoietic organ necrosis disease vaccine and infectious pancreatic necrosis disease vaccine according to the volume ratio of 1: (1-9) Mixed proportions.
  • the infectious hematopoietic necrosis disease vaccine is composed of infectious hematopoietic necrosis virus inactivated liquid and Montanide TM GEL 02 PR adjuvant;
  • the infectious pancreatic necrosis disease vaccine is composed of infectious pancreatic necrosis virus inactivated liquid And Montanide TM GEL 02 PR adjuvant composition.
  • the volume percentage of Montanide TM GEL 02 PR adjuvant is 10%-20% (that is, the volume percentage of the infectious hematopoietic necrosis virus inactivated solution The content is 80-90%).
  • the volume percentage of Montanide TM GEL 02 PR adjuvant is 10%-20% (that is, the volume percentage of the infectious pancreatic necrosis virus inactivated solution is 80% -90%).
  • the volume ratio of the infectious hematopoietic necrosis virus inactivated solution and Montanide TM GEL 02 PR adjuvant may be 9:1.
  • the volume ratio of the infectious pancreatic necrosis virus inactivated solution and Montanide TM GEL 02 PR adjuvant can be 9:1.
  • volume ratio of the infectious hematopoietic necrosis vaccine and the infectious pancreatic necrosis vaccine may specifically be 1:1 or 1:4 or 1:9.
  • infectious hematopoietic necrosis virus inactivation solution can be obtained by inactivating infectious hematopoietic organ necrosis virus with formaldehyde.
  • infectious pancreatic necrosis virus inactivation solution can be obtained by inactivating infectious pancreatic necrosis virus with formaldehyde.
  • the final concentration of formaldehyde may be 0.25%-2.5% (eg 0.25%) by volume.
  • virus titer of the infectious hematopoietic necrosis virus inactivation solution before inactivation may be 10 7 TCID 50 /0.1ml.
  • the virus titer of the infectious pancreatic necrosis virus inactivation solution before inactivation may be 10 7 TCID 50 /0.1ml.
  • both the infectious hematopoietic necrosis vaccine and the infectious pancreatic necrosis vaccine can be prepared according to the related methods in the fourth aspect as follows.
  • infectious hematopoietic necrosis disease and infectious pancreatic necrosis disease dual vaccine can be Prepared according to the method described in the fourth aspect below.
  • the present invention claims the infectious hematopoietic necrosis vaccine described in the first aspect above.
  • the present invention claims the infectious pancreatic necrosis vaccine described in the first aspect above.
  • the present invention claims a method for preparing a dual vaccine for infectious hematopoietic organ necrosis disease and infectious pancreatic necrosis disease.
  • the method for preparing infectious hematopoietic necrosis disease and infectious pancreatic necrosis disease dual vaccine claimed in the present invention may include the following steps:
  • the infectious hematopoietic necrosis vaccine prepared in (A) and the infectious pancreatic necrosis vaccine prepared in (B) are in a volume ratio of 1: (1-9) Mixed to obtain infectious hematopoietic necrosis disease and infectious pancreatic necrosis disease dual vaccine.
  • the volume percentage of Montanide TM GEL 02 PR adjuvant is 10%-20% (that is, the volume percentage of the infectious hematopoietic necrosis virus inactivated solution The content is 80-90%).
  • the volume percentage of Montanide TM GEL 02 PR adjuvant is 10%-20% (that is, the volume percentage of the infectious pancreatic necrosis virus inactivated solution is 80% -90%).
  • the volume ratio of the infectious hematopoietic necrosis virus inactivated solution and Montanide TM GEL 02 PR adjuvant may be 9:1.
  • the volume ratio of the infectious pancreatic necrosis virus inactivated solution and Montanide TM GEL 02 PR adjuvant can be 9:1.
  • volume ratio of the infectious hematopoietic necrosis vaccine and the infectious pancreatic necrosis vaccine may specifically be 1:1 or 1:4 or 1:9.
  • step (A) the infectious hematopoietic necrosis virus inactivation solution can be obtained by inactivating infectious hematopoietic organ necrosis virus with formaldehyde.
  • step (B) the infectious pancreatic necrosis virus inactivation solution can be obtained by inactivating infectious pancreatic necrosis virus with formaldehyde.
  • step (A) the virus titer of the infectious hematopoietic necrosis virus inactivation solution before inactivation may be 10 7 TCID 50 /0.1ml.
  • step (B) the virus titer of the infectious pancreatic necrosis virus inactivation solution before inactivation may be 10 7 TCID 50 /0.1ml.
  • step (A) the preparation of the infectious hematopoietic necrosis virus inactivation solution can be carried out as follows: take the infectious hematopoietic necrosis virus solution with a virus titer of 10 7 TCID 50 /0.1ml, add formaldehyde to make it final The concentration is 5mM, mix quickly; place the virus solution that has added formaldehyde on a shaker, inactivate at 100r/min for 24h at 24°C, and stop the inactivation with sodium bisulfite solution with a final concentration of 1mM to obtain infection Sexual hematopoietic organ failure Dead virus inactivation solution.
  • step (B) the preparation of the infectious pancreatic necrosis virus inactivation solution can be carried out as follows: take the infectious pancreatic necrosis virus solution with a virus titer of 10 7 TCID 50 /0.1ml, add formaldehyde to make it final The concentration is 0.25%-2.5% (such as 0.25%) volume percentage, mix well; place on a shaker, 24-26°C (room temperature), 100r/min inactivation for 12h, stop the inactivation, and obtain infectious pancreas Necrotic virus inactivation solution.
  • the present invention claims a method for preparing infectious hematopoietic necrosis disease vaccine.
  • the method for preparing the infectious hematopoietic necrosis disease vaccine claimed in the present invention may include the step (A) described in the fourth aspect above.
  • the present invention claims a method for preparing a vaccine for infectious pancreatic necrosis disease.
  • the method for preparing a vaccine for infectious pancreatic necrosis disease as claimed in the present invention may include the step (B) described in the fourth aspect above.
  • the present invention claims to protect infectious hematopoietic necrosis virus inactivation liquid, infectious pancreatic necrosis virus inactivation liquid and Montanide TM GEL 02 PR adjuvant in the preparation of infectious hematopoietic organ necrosis disease and infectious pancreatic necrosis disease Application in the dual vaccine; the infectious hematopoietic necrosis disease vaccine and the infectious pancreatic necrosis disease dual vaccine are 1:(1- 9) The ratio is mixed.
  • the present invention claims to protect the application of infectious hematopoietic necrosis virus inactivated solution and Montanide TM GEL 02 PR adjuvant in the preparation of infectious hematopoietic necrosis virus vaccine.
  • the present invention claims to protect the application of infectious pancreatic necrosis virus inactivated solution and Montanide TM GEL 02 PR adjuvant in the preparation of infectious pancreatic necrosis disease vaccine.
  • the volume percentage of Montanide TM GEL 02 PR adjuvant is 10%-20% (that is, the infectious hematopoietic organ necrosis virus inactivated
  • the volume percent composition of living liquid is 80-90%).
  • the volume percentage of Montanide TM GEL 02PR adjuvant is 10%-20% (that is, the inactivated infectious pancreatic necrosis virus
  • the volume percentage of liquid is 80-90%).
  • the volume ratio of the infectious hematopoietic organ necrosis virus inactivated solution and Montanide TM GEL 02 PR adjuvant may be 9:1.
  • the volume ratio of the infectious pancreatic necrosis virus inactivated solution and Montanide TM GEL 02 PR adjuvant may be 9:1.
  • the volume ratio of the infectious hematopoietic necrosis vaccine and the infectious pancreatic necrosis vaccine may be 1:1 or 1:4 or 1:9.
  • the inactivated liquid for infectious hematopoietic organ necrosis virus can be obtained by inactivating infectious hematopoietic organ necrosis virus with formaldehyde.
  • the infectious pancreatic necrosis virus inactivation solution can be obtained by inactivating infectious pancreatic necrosis virus with formaldehyde. Further, when using formaldehyde to inactivate the infectious pancreatic necrosis virus, the final concentration of formaldehyde may be 0.25%-2.5% (eg 0.25%) by volume.
  • the virus titer of the infectious hematopoietic necrosis virus inactivation solution before inactivation may be 10 7 TCID 50 /0.1ml.
  • the virus titer of the infectious pancreatic necrosis virus inactivation solution before inactivation is 10 7 TCID 50 /0.1ml.
  • the dual vaccine of the present invention can be used to prevent IHNV and IPNV hosts from infecting infectious hematopoietic necrosis virus and/or infectious pancreatic necrosis virus, and to prevent and/or treat IHNV and IPNV hosts due to infection Diseases caused by infectious hematopoietic necrosis virus and/or infectious pancreatic necrosis virus.
  • the infectious hematopoietic necrosis disease vaccine of the present invention can be used to prevent IHNV hosts from being infected with infectious hematopoietic necrosis virus, and to prevent and/or treat diseases caused by IHNV hosts infected with infectious hematopoietic necrosis virus .
  • the present invention claims to protect the effectiveness of the dual vaccine described in the first aspect above in the prevention and/or treatment of IHNV and IPNV hosts caused by infection with infectious hematopoietic necrosis virus and/or infectious pancreatic necrosis virus application.
  • the present invention claims the use of the infectious hematopoietic necrosis disease vaccine described in the second aspect above in the prevention and/or treatment of diseases caused by infection of infectious hematopoietic organ necrosis virus in IHNV hosts.
  • the present invention claims to protect the application of the infectious pancreatic necrosis disease vaccine described in the third aspect above in the prevention and/or treatment of diseases caused by infectious pancreatic necrosis virus infection of IPNV hosts.
  • the present invention also claims a method for preventing and/or treating IHNV and IPNV hosts caused by infection with infectious hematopoietic necrosis virus and/or infectious pancreatic necrosis virus, comprising the following steps: using the preceding
  • the dual vaccine described in the first aspect can prevent and/or treat IHNV and IPNV host diseases caused by infectious hematopoietic necrosis virus and/or infectious pancreatic necrosis virus.
  • the present invention also claims a method for preventing and/or treating IHNV hosts caused by infectious hematopoietic necrosis virus infection, including the following steps: using the infectious hematopoietic organ necrosis virus described in the second aspect above
  • the disease vaccine realizes the prevention and/or treatment of IHNV hosts caused by infectious hematopoietic necrosis virus infection.
  • the present invention also claims a method for preventing and/or treating IPNV hosts caused by infectious pancreatic necrosis virus infection, including the following steps: using the infectious pancreatic necrosis virus described in the third aspect above
  • the virus vaccine realizes the prevention and/or treatment of diseases caused by IPNV hosts infected with infectious pancreatic necrosis virus.
  • the IHNV and/or IPNV host can be fish, such as salmon and trout (Salmon and trout), specifically rainbow trout.
  • the infectious hematopoietic necrosis virus is specifically an isolate of infectious hematopoietic necrosis virus LN15.
  • the infectious pancreatic necrosis virus is specifically the isolate of infectious pancreatic necrosis virus GS2020-1.
  • Montanide TM GEL 02 PR adjuvant is replaced with the same or similar composition
  • Other adjuvants should also belong to the protection scope of the present invention.
  • Montanide TM GEL 02 PR adjuvant contains polyoxyethylene C12-C18 alkyl ether.
  • Figure 1 is an analysis of the relative immune protection efficacy of the IHN adjuvanted vaccine. Different letters on the error line represent significant differences (P ⁇ 0.05).
  • Figure 2 is the immune protection period of IHN formaldehyde inactivated adjuvant B vaccine to rainbow trout.
  • Figure 3 shows the mental state of rainbow trout under different immunization methods of IHN adjuvant vaccine.
  • Fig. 4 is an anatomical diagram of rainbow trout on the 7th day after immunization with IHN adjuvant vaccine.
  • Fig. 5 is an anatomical diagram of rainbow trout on the 14th day after IHN adjuvant vaccine immunization.
  • Figure 6 is HE staining observation of spleen tissue sections 7 days after IHN adjuvant vaccine immunization (40 ⁇ ).
  • Fig. 7 is HE staining observation of spleen tissue sections 14 days after IHN adjuvant vaccine immunization (40 ⁇ ).
  • Fig. 8 is HE staining observation of liver tissue section 7 days after IHN adjuvant vaccine immunization (40 ⁇ ).
  • Fig. 9 is HE staining observation of liver tissue section 14 days after IHN adjuvant vaccine immunization (40 ⁇ ).
  • Figure 10 is HE staining observation of kidney tissue sections 7 days after IHN adjuvant vaccine immunization (40 ⁇ ).
  • Figure 11 is HE staining observation of kidney tissue sections 14 days after IHN adjuvant vaccine immunization (40 ⁇ ).
  • Figure 12 is the analysis of the immune protection efficacy of the IPN adjuvant vaccine. Different letters on the error line represent significant differences (P ⁇ 0.05).
  • Figure 13 is the tissue section of rainbow trout 7 days after immunization with IPN adjuvant B inactivated vaccine.
  • Figure 14 is the tissue section of rainbow trout 14 days after immunization with IPN adjuvant B inactivated vaccine.
  • Figure 15 is an analysis of the protective efficacy of the dual vaccine against IHNV. Different letters on the error line represent significant differences (P ⁇ 0.05).
  • Figure 16 is the tissue section of rainbow trout 7 days after the dual vaccine immunization.
  • Figure 17 is the tissue section of rainbow trout 14 days after the dual vaccine immunization.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.
  • Example 1 Preparation and Effect Testing of Infectious Hematopoietic Necrosis Disease and Infectious Pancreatic Necrosis Disease Monovalent Vaccine and Dual Vaccine
  • Infectious hematopoietic necrosis virus LN15 isolate infectious hematopoietic necrosis virus, IHNV was recorded in "Xu L, Zhao J, Liu M, et al. Phylogeography and evolution of infectious hematopoietic necrosis virus in China [J]. Molecular phylogenetics and evolution, 2019,131:19-28.” an article; contagion Infectious pancreatic necrosis virus GS2020-1 isolate (infectious pancreatic necrosis virus, IPNV) was isolated and preserved in our laboratory, and the strain was recorded in the following documents: Duan K Y, Zhao J Z, Ren G M, et al.
  • Chinook salmon embryo cells (CHSE-214 cells) are preserved by our laboratory, and the cells are recorded in the following documents: Xu L M, Zhao J Z, Liu M, et al.
  • Bivalent DNA vaccine induces significant immune responses against infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout[J].Scientific reports,2017,7(1):5700. The public can obtain it from the applicant, and it can only be used for repeating the experiment of the present invention, and cannot be used for other purposes.
  • IHN formaldehyde inactivation solution take fresh IHNV virus solution (10 7 TCID 50 /0.1ml), add formaldehyde (final concentration is 5mM), and mix quickly. Place the virus solution with added formaldehyde on a shaker, inactivate at 100 r/min for 24 hours at 24°C, add sodium bisulfite solution (final concentration: 1 mM) to terminate inactivation, and obtain IHN formaldehyde inactivated vaccine.
  • each adjuvant (Table 1) was sterilized in a high-temperature autoclave (116° C.) for 20 minutes, mixed with IHNV formaldehyde inactivated solution and according to the proportion (Table 1), to form a uniform solution to obtain formaldehyde inactivated adjuvant vaccine.
  • the specific operation is as follows: take the above-mentioned IPNV inactivation solution and inoculate it on a 6-well cell culture plate (3 ⁇ 10 5 cells/well) covered with CHSE-214 cells, and use 1ml of IPNV inactivation solution in each well. Then culture the cells in a carbon dioxide incubator at 15°C for 7 days. If there is no cytopathic effect within 7 days, place the cells in a -80°C refrigerator and freeze and thaw twice, then centrifuge at 12000r/min at 4°C for 15min to collect the supernatant . The supernatant was inoculated into CHSE-214 cells according to the above method for blind passage, and the blind passage was continued until the third passage, and the cell pathological changes were observed every day to determine the inactivation conditions of IPNV.
  • the optimal inactivation conditions were used to prepare the IPNV inactivation solution.
  • the inactivation solution was mixed with each adjuvant after sterilization (sterilized at 116° C. for 20 minutes in a high-temperature and high-pressure autoclave) according to Table 1, and mixed to form a homogeneous solution to obtain an IPN adjuvant vaccine.
  • the PBS injection group served as the negative control group.
  • the vaccine group includes: inactivated vaccine with adjuvant added and inactivated vaccine without adjuvant (naked vaccine).
  • There are four kinds of adjuvants (Table 1): biphasic GR208 adjuvant (marked as A1), Montanide TM ISA 763 A VG (marked as A2), Montanide TM GEL 02 PR (marked as B), 4% Al(OH) 3 Gels (marked C).
  • the dose used was 50 ⁇ L per tail.
  • the immunization method used was intraperitoneal injection.
  • Immune rainbow trout came from Liaoning Benxi Aigemolin Industrial Co., Ltd.
  • the PBS injection group served as the negative control group.
  • the vaccine group includes: adjuvanted inactivated vaccine and naked vaccine (inactivated vaccine without adjuvant).
  • adjuvants There are four kinds of adjuvants (Table 1): biphasic GR208 adjuvant (marked as A1), Montanide TM ISA 763 A VG (marked as A2), Montanide TM GEL 02 PR (marked as B), 4% Al(OH) 3 Gels (marked C).
  • the immunization dose used was 50 ⁇ L per tail, and the immunization method was intraperitoneal injection.
  • the rainbow trout were cultured in an indoor flowing water culture tank (2 meters in diameter, 80 cm in water depth, and 13 ⁇ 2° C. in water temperature).
  • 30 days after immunization rainbow trout (body weight: 7 ⁇ 1 g) were challenged with IPNV (1 ⁇ 10 6 TCID 50 /0.1 ml) at a dose of 10 ⁇ L/tail by intraperitoneal injection, 10 fish for each treatment.
  • IPNV 1 ⁇ 10 6 TCID 50 /0.1 ml
  • the visceral tissues liver, spleen and head kidney
  • 5 fish were collected, homogenized with PBS buffer at a ratio of 1:10 (g:ml), and then centrifuged at 12000g/min at 4°C for 15min Harvest the supernatant.
  • the supernatant was diluted 10 times (10 -1 ⁇ 10 -10 ), and then inoculated on CHSE-214 cells cultured on 96-well cell culture plates. After 7 days, the tissue concentration was calculated by the Reed-Muench method.
  • Virus titer The immune protective effect of IPN adjuvant vaccine was analyzed by comparing the IPNV titer in rainbow trout tissue of PBS injection group (control group) and the difference of IPNV titer in rainbow trout tissue of vaccine immunized group.
  • a dose of 100 ⁇ L per tail was selected, and rainbow trout (average body weight 10 ⁇ 1 g) were intraperitoneally injected. After the immunization, the rainbow trout was placed in an indoor flowing water culture cylinder (2 meters in diameter, water Deep 80cm, water temperature 13 ⁇ 2 °C) breeding.
  • Rainbow trout (average body weight 10 ⁇ 1g) were immunized intraperitoneally with a single dose (100 ⁇ L), a second single dose (7 days apart, 100 ⁇ L each time), and a superdose (300 ⁇ L) double adjuvant vaccine. Group of 20 tails. Rainbow trout injected with PBS was used as negative control. After immunization, observe the rainbow trout's mental state and whether there is any abnormality at the inoculation site, whether the food intake is normal, whether there are symptoms such as death. On day 7 and day 14 after immunization, 5 rainbow trout were randomly selected from each group, dissected and observed with the naked eye. At the same time, the liver, spleen and kidney were taken to make HE-stained paraffin sections, and the tissue sections were observed with a microscope.
  • the virus was inactivated with different concentrations of formaldehyde for different periods of time, the inactivated virus liquid was harvested, and the inactivation effect was tested using CHSE-214 cells.
  • the results are shown in Table 2.
  • the final concentration of formaldehyde was 0.025% and 0.075% (volume ratio)
  • pathological changes could still be observed on CHSE-214 cells even after 48 hours of incubation, indicating that this concentration failed to completely kill IPNV live
  • the final concentration of formaldehyde is 0.25% and 2.5% (volume ratio)
  • IPNV can be completely inactivated within 12 hours, and no cytopathic changes can be observed on the cells, indicating that this concentration can completely inactivate IPNV.
  • the present invention selects a formaldehyde solution with a final concentration of 0.25% (volume ratio), places it at room temperature (25 ⁇ 1° C.), and incubates on a shaking table at 100 r/min for 12 hours. Prepare IPN virus inactivation solution.
  • adjuvanted inactivated vaccines which are respectively named A1 inactivated vaccine, A2 inactivated vaccine, B inactivated vaccine, and C inactivated vaccine.
  • the inactivated solution without any adjuvant is named naked seedling.
  • Rainbow trout were immunized by intraperitoneal injection with a dose of 50 ⁇ L per tail. During the immunization process, it was found that the A2 inactivated vaccine was too viscous and the particles were too large to be injected. Therefore, no further research was carried out on the A2 inactivated vaccine.
  • the challenge was carried out 30 days after immunization, and the virus load in the tissues of rainbow trout in each group was measured 3 days after the challenge.
  • the protection period of the best adjuvant B inactivated vaccine was studied, and the formaldehyde inactivated solution (bare vaccine) without adjuvant was used for comparison.
  • the results showed that at different times after immunization, the viral loads of rainbow trout in each immunized group were lower than those of the control group, and the viral loads of the B inactivated vaccine group were significantly lower than those of the naked vaccine group (P ⁇ 0.05); After prolongation, the individual rainbow trout gradually increased, and the viral load also showed a downward trend.
  • the virus could not be detected in the rainbow trout in the B inactivated vaccine group, while the rainbow trout in the naked seedling group The virus can still be detected.
  • the protective efficacy against IHNV was analyzed for the prepared 5 combinations of dual vaccines.
  • the results showed that at 1 and 2 months after immunization, the vaccines in each group had extremely high relative immune protection rates, up to 100%, and there was no significant difference among the groups; at 4 months after immunization, the vaccines in each group were still It has a relatively high protection rate, up to over 85%.
  • the immune protection rate of the H1P9 vaccine group was significantly lower than that of the other two groups (P ⁇ 0.05), but still as high as over 78% (Figure 15).
  • the anti-IPNV protective efficacy analysis was carried out on the prepared 5 kinds of combined dual vaccines.
  • the results showed that at 1 and 2 months after immunization, the vaccines in each group could significantly reduce the IPNV titer (P ⁇ 0.05), and the dual vaccine in the H1P9 group had a stronger antiviral effect, and the virus titer was only 10,000 times lower than that of the control group. 1/1 (Table 4); 4 months after immunization, the presence of any virus could not be detected from the H1P1, H1P4, H1P9 immunized groups, and at this time There was no significant difference between the P1H4 and P1H9 vaccine groups and the negative control, and there was no anti-IPNV effect.
  • the dual vaccines of the five combinations designed by the present invention can all produce good protective effects against IHNV ( FIG. 15 ), while only the dual vaccines combined with H1P1, H1P4, and H1P9 can significantly resist IHNV. Therefore, the present invention determines that H1P1, H1P4 and H1P9 are the best dual vaccine combination, that is, mix IHN adjuvant B vaccine and IPN adjuvant B vaccine according to the ratio of 1:1 or 1:4 or 1:9.
  • the present invention selects 4 kinds of adjuvants, and utilizes the method recommended by the adjuvant product manual to be compatible with IHNV inactivated liquid and IPNV inactivated liquid respectively, prepares the adjuvanted inactivated vaccine, immunizes rainbow trout, and measures the relative immunity of the vaccine at different time points Protective effect The best vaccine adjuvants against individual IHNV and IPNV were screened. Then, the best vaccine adjuvants for individual IHNV and IPNV were mixed according to different ratios, the dual adjuvant vaccine with the best effect was screened, and the safety of the dual vaccine was tested.
  • the final gained IHN adjuvant vaccine prolongs the protection period of the IHN inactivated vaccine
  • the final gained IPN adjuvant vaccine prolongs the protection period of the IPN vaccine
  • the final gained dual adjuvant vaccine can simultaneously help the host to effectively resist the infection of IHNV and IPNV, and
  • the protection period is as long as 4 months, and the safety of the double vaccine is good.

Abstract

提供了一种鲑鳟鱼传染性造血器官坏死病和传染性胰脏坏死病单价佐剂疫苗及其二联佐剂疫苗和制备方法。提供的二联疫苗由传染性造血器官坏死病疫苗和传染性胰脏坏死病疫苗按照体积比为 1：（1-9）的比例混合而成；所述传染性造血器官坏死病疫苗由传染性造血器官坏死病毒灭活液和Montanide TM GEL 02 PR 佐剂组成；所述传染性胰脏坏死病疫苗由传染性胰脏坏死病毒灭活液和 Montanide TM GEL 02 PR 佐剂组成。所述二联佐剂疫苗能同时帮助宿主有效抵抗IHNV和IPNV的感染，且保护期长达4个月，并且该二联疫苗安全性良好。

Description

一种鲑鳟鱼传染性造血器官坏死病和传染性胰脏坏死病单价佐剂疫苗及其二联佐剂疫苗和制备方法 技术领域
本发明涉及生物技术领域，具体涉及一种鲑鳟鱼传染性造血器官坏死病和传染性胰脏坏死病单价佐剂疫苗及其二联佐剂疫苗和制备方法。
背景技术
鲑鳟鱼(Salmon and trout)是指以鲑科鱼类为主要代表的一批优质冷水性鱼类，主要包括大西洋鲑(Salmosalar)、虹鳟(Oncorhynchus mykiss)、王鲑(Oncorhynchus tshawytscha)、银鲑(Oncorhynchus kisutch)等，是国际主流养殖和贸易品种。我国鲑鳟鱼主养品种为虹鳟，主要以淡水养殖为主，养殖区域广泛分布在中国东北、西北、西南等冷水资源较发达的地区，在带动当地经济发展、促进渔民增收方面发挥了积极的作用。鲑鳟鱼养殖符合我国“提质增效、绿色发展”的现代渔业发展要求。近些年，我国内陆引进了大型循环水养殖设施，沿海地区也开发了现代海洋牧场养殖虹鳟，鲑鳟鱼产养殖业迅速转型升级。但是我国鲑鳟鱼养殖业正面临重大病害难题，产业发展严重受阻。
目前威胁我国养殖鲑鳟的疫病主要为2种病毒病，即传染性造血器官坏死病(infectious hematopoietic necrosis，IHN)和传染性胰脏坏死病(infectious pancreatic necrosis，IPN)。
根据环境、宿主、毒株等不同，IPNV最高可造成虹鳟(Oncorhynchus mykiss)、褐鳟(Salmo trutta)、美洲红点鲑(Salvelinus fontinalis)、细鳞鲑(Brachymystax lenok)等鲑鳟鱼类幼鱼80％以上的死亡率，IHN最高可造成虹鳟、大西洋鲑等幼鱼90％以上的死亡率，是我国鲑鳟鱼产业发展必须解决的首要病害难题。
目前，我国尚无任何有效的防控IPN药物。由于毒株存在变异及存在生物安全隐患等问题，国外商业化疫苗无法直接引进应用，因此我国鲑鳟鱼病害难题只能通过疫苗的自主研发来解决。课题组在前期利用本土毒株，开展了IPN灭活疫苗的研制工作，通过利用β-丙内酯(BPL)进行灭活，制备了IPN灭活疫苗(参见中国专利申请CN113583968A)。利用该疫苗直接对虹鳟进行免疫，结果发现所制备的灭活疫苗具有较理想的早期保护效力，但是随着时间的延长，在免疫后45天开始，疫苗免疫保护效率开始下降，不具备长保护期，无法满足市场需求，因此极有必要对现有的灭活疫苗进行优化。
目前，全球范围内，仅加拿大上市了一种针对北美基因型的IHN核酸疫苗，我国尚无有效的IHN防控药物。由于毒株存在变异及存在生物安全隐患等问题，国外商业化疫苗无法直接引进应用，因此我国鲑鳟鱼病害难题只能通过疫苗的自主研发来解决。课题组在前期开展了IHN灭活疫苗的研制工作，通过利用甲醛和BPL进行灭活，制备了IHN灭活疫苗，利用该疫苗直接对虹鳟进行免疫，结果 发现所制备的灭活疫苗具有较理想的早期免疫保护效力(参见中国专利CN113144185A和CN113122510A)，但是随着时间的延长，在免疫后60天时，疫苗免疫保护效率显著下降，不能维持较长的保护期，无法满足市场需求，因此极有必要对现有的IHN灭活疫苗进行优化。
目前，全球范围内并无针对IHN和IPN这两种病的二联疫苗。
发明公开
本发明的目的是提供一种鲑鳟鱼传染性造血器官坏死病和传染性胰脏坏死病单价佐剂疫苗及其二联佐剂疫苗和制备方法。
第一方面，本发明要求保护一种传染性造血器官坏死病和传染性胰脏坏死病二联疫苗。
本发明要求保护的传染性造血器官坏死病和传染性胰脏坏死病二联疫苗，由传染性造血器官坏死病疫苗和传染性胰脏坏死病疫苗按照体积比为1：(1-9)的比例混合而成。
其中，所述传染性造血器官坏死病疫苗由传染性造血器官坏死病毒灭活液和MontanideTM GEL 02 PR佐剂组成；所述传染性胰脏坏死病疫苗由传染性胰脏坏死病毒灭活液和MontanideTM GEL 02 PR佐剂组成。
进一步地，在所述传染性造血器官坏死病疫苗中，MontanideTM GEL 02 PR佐剂的体积百分含量为10％-20％(即所述传染性造血器官坏死病毒灭活液的体积百分含量为80-90％)。在所述传染性胰脏坏死病疫苗中，MontanideTM GEL 02 PR佐剂的体积百分含量为10％-20％(即所述传染性胰脏坏死病毒灭活液的体积百分含量为80-90％)。
更进一步地，在所述传染性造血器官坏死病疫苗中，所述传染性造血器官坏死病毒灭活液和MontanideTM GEL 02 PR佐剂的体积比可为9:1。在所述传染性胰脏坏死病疫苗中，所述传染性胰脏坏死病毒灭活液和MontanideTM GEL 02 PR佐剂的体积比可为9:1。
进一步地，所述传染性造血器官坏死病疫苗和所述传染性胰脏坏死病疫苗的体积比具体可为1:1或1:4或者1:9。
进一步地，所述传染性造血器官坏死病毒灭活液可为传染性造血器官坏死病毒经甲醛灭活后所得。所述传染性胰脏坏死病毒灭活液可为传染性胰脏坏死病毒经甲醛灭活后所得。利用甲醛对所述传染性胰脏坏死病毒进行灭活时，甲醛的终浓度可为0.25％-2.5％(如0.25％)体积百分含量。
进一步地，所述传染性造血器官坏死病毒灭活液在灭活前的病毒滴度可为107TCID50/0.1ml。所述传染性胰脏坏死病毒灭活液在灭活前的病毒滴度可为107TCID50/0.1ml。
进一步地，所述传染性造血器官坏死病疫苗和所述传染性胰脏坏死病疫苗均可按照如下第四方面中相关方法制备得到。
更进一步地，所述传染性造血器官坏死病和传染性胰脏坏死病二联疫苗可 按照如下第四方面所述方法制备得到。
第二方面，本发明要求保护前文第一方面中所述的传染性造血器官坏死病疫苗。
第三方面，本发明要求保护前文第一方面中所述的传染性胰脏坏死病疫苗。
第四方面，本发明要求保护一种制备传染性造血器官坏死病和传染性胰脏坏死病二联疫苗的方法。
本发明要求保护的制备传染性造血器官坏死病和传染性胰脏坏死病二联疫苗的方法，可包括如下步骤：
(A)制备传染性造血器官坏死病毒灭活液；然后将所述传染性造血器官坏死病毒灭活液和MontanideTM GEL 02 PR佐剂混合，得到传染性造血器官坏死病疫苗；
(B)制备传染性胰脏坏死病毒灭活液；然后将所述传染性胰脏坏死病毒灭活液和MontanideTM GEL 02 PR佐剂混合，得到传染性胰脏坏死病疫苗；
(C)将(A)中制得的所述传染性造血器官坏死病疫苗和(B)中制得的所述传染性胰脏坏死病疫苗按照体积比为1：(1-9)的比例混合，得到传染性造血器官坏死病和传染性胰脏坏死病二联疫苗。
进一步地，在所述传染性造血器官坏死病疫苗中，MontanideTM GEL 02 PR佐剂的体积百分含量为10％-20％(即所述传染性造血器官坏死病毒灭活液的体积百分含量为80-90％)。在所述传染性胰脏坏死病疫苗中，MontanideTM GEL 02 PR佐剂的体积百分含量为10％-20％(即所述传染性胰脏坏死病毒灭活液的体积百分含量为80-90％)。
更进一步地，在所述传染性造血器官坏死病疫苗中，所述传染性造血器官坏死病毒灭活液和MontanideTM GEL 02 PR佐剂的体积比可为9:1。在所述传染性胰脏坏死病疫苗中，所述传染性胰脏坏死病毒灭活液和MontanideTM GEL 02 PR佐剂的体积比可为9:1。
进一步地，所述传染性造血器官坏死病疫苗和所述传染性胰脏坏死病疫苗的体积比具体可为1:1或1:4或者1:9。
在步骤(A)中，所述传染性造血器官坏死病毒灭活液可为传染性造血器官坏死病毒经甲醛灭活后所得。在步骤(B)中，所述传染性胰脏坏死病毒灭活液可为传染性胰脏坏死病毒经甲醛灭活后所得。
在步骤(A)中，所述传染性造血器官坏死病毒灭活液在灭活前的病毒滴度可为107TCID50/0.1ml。在步骤(B)中，所述传染性胰脏坏死病毒灭活液在灭活前的病毒滴度可为107TCID50/0.1ml。
在步骤(A)中，所述制备传染性造血器官坏死病毒灭活液可按照如下进行：取病毒滴度为107TCID50/0.1ml的传染性造血器官坏死病毒液，加入甲醛使其终浓度为5mM，迅速混匀；将加完甲醛的病毒液置于摇床，24℃条件下，100r/min灭活24h，用终浓度为1mM的亚硫酸氢钠溶液终止灭活，即得传染性造血器官坏 死病毒灭活液。
在步骤(B)中，所述制备传染性胰脏坏死病毒灭活液可按照如下进行：取病毒滴度为107TCID50/0.1ml的传染性胰脏坏死病毒液，加入甲醛使其终浓度为0.25％-2.5％(如0.25％)体积百分含量，混匀；置于摇床，24-26℃(室温)，100r/min灭活12h，终止灭活，即得传染性胰脏坏死病毒灭活液。
第五方面，本发明要求保护一种制备传染性造血器官坏死病疫苗的方法。
本发明要求保护的制备传染性造血器官坏死病疫苗的方法，可包括前文第四方面中所述的步骤(A)。
第六方面，本发明要求保护一种制备传染性胰脏坏死病疫苗的方法。
本发明要求保护的制备传染性胰脏坏死病疫苗的方法，可包括前文第四方面中所述的步骤(B)。
第七方面，本发明要求保护传染性造血器官坏死病毒灭活液、传染性胰脏坏死病毒灭活液和MontanideTM GEL 02 PR佐剂在制备传染性造血器官坏死病和传染性胰脏坏死病二联疫苗中的应用；所述传染性造血器官坏死病和传染性胰脏坏死病二联疫苗由传染性造血器官坏死病疫苗和传染性胰脏坏死病疫苗按照体积比为1：(1-9)的比例混合而成。
第八方面，本发明要求保护传染性造血器官坏死病毒灭活液和MontanideTM GEL 02 PR佐剂在制备传染性造血器官坏死病疫苗中的应用。
第九方面，本发明要求保护传染性胰脏坏死病毒灭活液和MontanideTM GEL 02 PR佐剂在制备传染性胰脏坏死病疫苗中的应用。
在第七和第八方面中，在所述传染性造血器官坏死病疫苗中，MontanideTM GEL 02 PR佐剂的体积百分含量为10％-20％(即所述传染性造血器官坏死病毒灭活液的体积百分含量为80-90％)。
在第七和第九方面中，在所述传染性胰脏坏死病疫苗中，MontanideTM GEL 02PR佐剂的体积百分含量为10％-20％(即所述传染性胰脏坏死病毒灭活液的体积百分含量为80-90％)。
在第七和第八方面中，在所述传染性造血器官坏死病疫苗中，所述传染性造血器官坏死病毒灭活液和MontanideTM GEL 02 PR佐剂的体积比可为9:1。
在第七和第九方面中，在所述传染性胰脏坏死病疫苗中，所述传染性胰脏坏死病毒灭活液和MontanideTM GEL 02 PR佐剂的体积比可为9:1。
在第七方面中，所述传染性造血器官坏死病疫苗和所述传染性胰脏坏死病疫苗的体积比具体可为1:1或1:4或者1:9。
在第七和第八方面中，所述传染性造血器官坏死病毒灭活液可为传染性造血器官坏死病毒经甲醛灭活后所得。
在第七和第九方面中，所述传染性胰脏坏死病毒灭活液可为传染性胰脏坏死病毒经甲醛灭活后所得。进一步地，利用甲醛对所述传染性胰脏坏死病毒进行灭活时，甲醛的终浓度可为0.25％-2.5％(如0.25％)体积百分含量。
在第七和第八方面中，所述传染性造血器官坏死病毒灭活液在灭活前的病毒滴度可为107TCID50/0.1ml。
在第七和第九方面中，所述传染性胰脏坏死病毒灭活液在灭活前的病毒滴度为107TCID50/0.1ml。
在上述相关方面中，本发明所述二联疫苗可用于预防IHNV和IPNV宿主感染传染性造血器官坏死病毒和/或传染性胰脏坏死病毒，用于预防和/或治疗IHNV和IPNV宿主因感染传染性造血器官坏死病毒和/或传染性胰脏坏死病毒所致疾病。
在上述相关方面中，本发明所述传染性造血器官坏死病疫苗可用于预防IHNV宿主感染传染性造血器官坏死病毒，用于预防和/或治疗IHNV宿主因感染传染性造血器官坏死病毒所致疾病。
第九方面，本发明要求保护前文第一方面中所述二联疫苗在预防和/或治疗IHNV和IPNV宿主因感染传染性造血器官坏死病毒和/或传染性胰脏坏死病毒所致疾病中的应用。
第十方面，本发明要求保护前文第二方面中所述传染性造血器官坏死病疫苗在预防和/或治疗IHNV宿主因感染传染性造血器官坏死病毒所致疾病中的应用。
第十一方面，本发明要求保护前文第三方面中所述传染性胰脏坏死病疫苗在预防和/或治疗IPNV宿主因感染传染性胰脏坏死病毒所致疾病中的应用。
第十二方面，本发明还要求保护一种预防和/或治疗IHNV和IPNV宿主因感染传染性造血器官坏死病毒和/或传染性胰脏坏死病毒所致疾病的方法，包括如下步骤：使用前文第一方面中所述二联疫苗实现对IHNV和IPNV宿主因感染传染性造血器官坏死病毒和/或传染性胰脏坏死病毒所致疾病的预防和/或治疗。
第十三方面，本发明还要求保护一种预防和/或治疗IHNV宿主因感染传染性造血器官坏死病毒所致疾病的方法，包括如下步骤：使用前文第二方面中所述传染性造血器官坏死病疫苗实现对IHNV宿主因感染传染性造血器官坏死病毒所致疾病的预防和/或治疗。
第十四方面，本发明还要求保护一种预防和/或治疗IPNV宿主因感染传染性胰脏坏死病毒所致疾病的方法，包括如下步骤：使用前文第三方面中所述传染性胰脏坏死病疫苗实现对IPNV宿主因感染传染性胰脏坏死病毒所致疾病的预防和/或治疗。
其中，所述IHNV和/或IPNV宿主可为鱼类，如鲑鳟鱼(Salmon and trout)，具体如虹鳟。
在本发明的具体实施方式中，所述传染性造血器官坏死病毒具体为传染性造血器官坏死病毒LN15分离株。所述传染性胰脏坏死病毒具体为传染性胰脏坏死病毒GS2020-1分离株。
在本发明中，将MontanideTM GEL 02 PR佐剂替换为组成成分相同或相似的 其他佐剂也应当属于本发明的保护范围。MontanideTM GEL 02 PR佐剂中含有聚氧乙烯C12-C18烷基醚。
附图说明
图1为IHN佐剂疫苗相对免疫保护效力分析。误差线上不同字母代表差异显著(P<0.05)。
图2为IHN甲醛灭活佐剂B疫苗对虹鳟的免疫保护期。
图3为IHN佐剂疫苗不同免疫方式下虹鳟的精神状态。
图4为IHN佐剂疫苗免疫后第7d虹鳟的解剖图。
图5为IHN佐剂疫苗免疫后第14d虹鳟的解剖图。
图6为IHN佐剂疫苗免疫后7d脾脏组织切片HE染色观察(40×)。
图7为IHN佐剂疫苗免疫后14d脾脏组织切片HE染色观察(40×)。
图8为IHN佐剂疫苗免疫后7d肝脏组织切片HE染色观察(40×)。
图9为IHN佐剂疫苗免疫后14d肝脏组织切片HE染色观察(40×)。
图10为IHN佐剂疫苗免疫后7d肾组织切片HE染色观察(40×)。
图11为IHN佐剂疫苗免疫后14d肾组织切片HE染色观察(40×)。
图12为IPN佐剂疫苗免疫保护效力分析。误差线上不同字母代表差异显著(P<0.05)。
图13为IPN佐剂B灭活疫苗免疫后7天虹鳟组织切片。
图14为IPN佐剂B灭活疫苗免疫后14天虹鳟组织切片。
图15为二联疫苗抗IHNV保护效力分析。误差线上不同字母代表差异显著(P<0.05)。
图16为二联疫苗免疫后7天虹鳟组织切片。
图17为二联疫苗免疫后14天虹鳟组织切片。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
实施例1、传染性造血器官坏死病和传染性胰脏坏死病单价疫苗以及二联疫苗的制备及效果检测
一、材料与方法
1、材料
传染性造血器官坏死病毒LN15分离株(infectious hematopoietic necrosisvirus,IHNV)记载于“Xu L,Zhao J,Liu M,et al.Phylogeography and evolution of infectious hematopoietic necrosis virus in China[J].Molecular phylogenetics and evolution,2019,131:19-28.”一文；传染 性胰脏坏死病毒GS2020-1分离株(infectious pancreatic necrosis virus，IPNV)本实验室分离保存，该毒株记载在如下文献中：Duan K Y,Zhao J Z,Ren G M,et al.Molecular Evolution of Infectious Pancreatic Necrosis Virus in China[J].Viruses,2021,13(3):488.。上述两个病毒毒株按照国家生物安全的有关规定公众可从申请人处获得，仅可用于重复本发明试验使用，不得他用。
大鳞大麻哈鱼胚胎细胞(CHSE-214细胞)由本实验室保存，该细胞记载在如下文献中：Xu L M,Zhao J Z,Liu M,et al.Bivalent DNA vaccine induces significant immune responses against infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout[J].Scientific reports,2017,7(1):5700。公众可从申请人处获得，仅可用于重复本发明试验使用，不得他用。
2、IHN佐剂疫苗的制备
IHN甲醛灭活液的制备：取新鲜的IHNV病毒液(107TCID50/0.1ml)，加入甲醛(终浓度为5mM)，迅速混匀。将加完甲醛的病毒液置于摇床，24℃条件下，100r/min灭活24h，加入亚硫酸氢钠溶液(终浓度为1mM)终止灭活，获得IHN甲醛灭活疫苗。
佐剂灭活疫苗的制备：各佐剂(表1)经高温高压灭菌锅(116℃)灭菌20min，与IHNV甲醛灭活液和按照配比(表1)，进行混匀，形成均质溶液，获得甲醛灭活佐剂疫苗。
3、IPN佐剂疫苗的制备
(1)IPNV灭活条件的确定
向新鲜的IPN病毒液(107TCID50/0.1ml)中加入甲醛使其终浓度分别为0.025％、0.075％、0.25％、2.5％(体积比)，置于室温(25±1℃)条件下，摇床100r/min，分别孵育12、24、36、48h进行灭活处理。加入亚硫酸氢钠溶液(终浓度为1mM)终止灭活，收获IPNV灭活液，在细胞水平上检测灭活效果。具体操作如下：取上述IPNV灭活液接种于长满CHSE-214细胞的6孔细胞培养板上(3×105个细胞/孔)，每孔1ml IPNV灭活液。然后将细胞置于15℃二氧化碳培养箱中培养7d，若7d内并未出现细胞病变，则将细胞置于-80℃冰箱反复冻融2次，然后12000r/min 4℃离心15min收集上清液。将该上清液按照上述方法接种CHSE-214细胞进行盲传，连续盲传至第3代，每天观察细胞病变情况，确定IPNV的灭活条件。
(2)IPN佐剂疫苗的制备
利用最佳灭活条件，制备IPNV灭活液。将该灭活液与灭菌(高温高压灭菌锅116℃灭菌20min)后的各佐剂按照表1进行配比，混匀，形成均质溶液，获得IPN佐剂疫苗。
表1、佐剂信息
4、最佳IHN佐剂疫苗的筛选
(1)IHN疫苗最佳佐剂的确定
虹鳟(平均体重10±1g)来自辽宁本溪艾格莫林实业有限公司。PBS注射组作为阴性对照组。疫苗组包括：添加佐剂的灭活疫苗和不加佐剂的灭活疫苗(裸苗)。佐剂为四种(表1)：双相GR208佐剂(标记为A1)、MontanideTM ISA 763 A VG(标记为A2)、MontanideTM GEL 02 PR(标记为B)、4％Al(OH)3凝胶(标记为C)。采用的剂量为50μL每尾。采用的免疫方式为腹腔注射免疫。免疫后将虹鳟置于室内循环水系统(水温13±1℃)养殖。在免疫后30d，对虹鳟进行腹腔注射攻毒，攻毒剂量为50μL 100TCID50的IHNV病毒原液，每种处理60尾，连续观察21d，统计各组虹鳟累积死亡数，计算各组疫苗相对免疫保护率。计算公式为：相对免疫保护率＝[1-(免疫组死亡率/对照组死亡率)]×100％。根据相对保护率结果，确定IHN疫苗最佳佐剂。
(2)最佳IHN佐剂疫苗的长期免疫保护效力分析
选择最佳IHN佐剂疫苗及其裸苗，采用10μL和50μL的剂量，对平均体重为10±1g的虹鳟进行腹腔注射免疫。以PBS注射的虹鳟为阴性对照。免疫后将虹鳟置于室内流水养殖圆缸(2米直径，水深80cm，水温14±1℃)养殖。于免疫后不同时间对虹鳟进行腹腔注射攻毒试验，每种处理60尾虹鳟，置于室内循环水系统(水温13±1℃)养殖。记录各组虹鳟死亡情况，计算疫苗的相对免疫保护率(同步骤(1))，确定IHN佐剂疫苗的长效保护作用。
(3)IHN佐剂灭活疫苗的安全性检验
选择最佳IHN佐剂疫苗，采用一次单剂量(50μL)、二次单剂量(间隔7天，每次50μL)、一次超剂量(250μL)佐剂疫苗，对虹鳟(平均体重10±1g)进行腹腔注射免疫，每个处理20尾。以PBS注射的虹鳟为阴性对照。免疫后每天观察虹鳟精神状态及接种部位有无异常，摄食是否正常，是否出现死亡等症状。于末次免疫后7d和14d分别从各组中随机挑选5尾虹鳟，进行解剖及肉眼观察，同时取肝、脾、肾脏，制作HE染色石蜡切片，利用显微镜观察 组织切片。
5、最佳IPN佐剂疫苗的筛选
(1)IPN疫苗最佳佐剂的确定
免疫虹鳟(平均体重10±1g)来自辽宁本溪艾格莫林实业有限公司。PBS注射组作为阴性对照组。疫苗组包括：添加佐剂的灭活疫苗和裸苗(不加佐剂的灭活疫苗)。佐剂为四种(表1)：双相GR208佐剂(标记为A1)、MontanideTM ISA 763 A VG(标记为A2)、MontanideTM GEL 02 PR(标记为B)、4％Al(OH)3凝胶(标记为C)。采用的免疫剂量50μL每尾，免疫方式为腹腔注射免疫。免疫后将虹鳟置于室内流水养殖圆缸(2米直径，水深80cm，水温13±2℃)养殖。在免疫后30d，以10μL/尾的剂量将IPNV(1×106TCID50/0.1ml)对虹鳟(体重为7±1g)进行腹腔注射攻毒，每个处理10尾。于攻毒后3d，分别取5尾鱼的内脏组织(肝、脾及头肾)，以1:10(g:ml)的比例用PBS缓冲液进行匀浆，然后12000g/min 4℃离心15min收获上清液。上清液经无菌过滤后进行10倍稀释(10-1～10-10)，然后接种于培养在96孔细胞培养板上的CHSE-214细胞上，7d后按Reed-Muench法计算组织中病毒滴度。比较PBS注射组(对照组)虹鳟组织中IPNV滴度与疫苗免疫组虹鳟组织中IPNV滴度差异来分析IPN佐剂疫苗的免疫保护效力。
(2)最佳IPN佐剂疫苗的长期免疫保护效力分析
选择最佳IPN佐剂疫苗及其裸苗，采用100μL的剂量，对平均体重为10±1g的虹鳟进行腹腔注射免疫。以PBS注射的虹鳟为阴性对照。免疫后将虹鳟置于室内流水养殖圆缸(2米直径，水深80cm，水温14±1℃)养殖。参照步骤4中的方法，于免疫后不同时间对虹鳟(n＝10)进行攻毒试验，然后置于室内循环水系统(水温13±1℃)养殖，3d后测定组织中病毒载量。
(3)最佳IPN佐剂疫苗的安全性检验
选择最佳IPN佐剂疫苗，采用单剂量(100μL)、二次单剂量(间隔7天，每次100μL)、一次超剂量(300μL)，对虹鳟(平均体重10±1g)进行腹腔注射免疫,每个处理20尾。以PBS注射的虹鳟为阴性对照。免疫后每天观察虹鳟精神状态及接种部位有无异常，摄食是否正常，是否出现死亡等症状；于末次免疫后7d和14d分别从各组中随机挑选5尾虹鳟，进行解剖及肉眼观察；同时取肝、脾、肾脏，制作HE染色石蜡切片，利用显微镜观察组织切片。
6、二联疫苗的制备及免疫
选择最佳IHN佐剂疫苗和IPN佐剂疫苗制备二联疫苗。首先按照IHN佐剂疫苗和IPN佐剂疫苗1:1、1:4、1:9(v/v)的比例充分混匀，制备出3种二联疫苗，分别命名为H1P1、H1P4、H1P9；同时按照IPN佐剂疫苗和IHN佐剂疫苗1:4、1:9(v/v)的比例充分混匀，再制备出2种二联疫苗，分别命名为P1H4、P1H9；共制备出5种二联疫苗。选择100μL每尾的剂量，对虹鳟(平均体重10±1g)进行腹腔注射。免疫后将虹鳟置于室内流水养殖圆缸(2米直径，水 深80cm，水温13±2℃)养殖。
7、二联疫苗抗IHNV效力分析
于免疫后1、2、4个月，分别对虹鳟进行腹腔注射攻毒，攻毒剂量为50μL100TCID50的IHNV病毒原液，每个处理60尾，连续观察21d，统计各组虹鳟累积死亡数，计算各组疫苗相对免疫保护率。计算公式为：相对免疫保护率＝[1-(免疫组死亡率/对照组死亡率)]×100％。
8、二联疫苗抗IPNV效力分析
于免疫后1、2、4个月，分别以10μL/尾的剂量将IPNV(1×106TCID50/0.1ml)对虹鳟(n＝10)进行腹腔注射攻毒。于攻毒后3d，分别取5尾鱼的内脏组织(肝、脾及头肾)，以1:10(g:ml)的比例用PBS缓冲液进行匀浆，然后12000g/min 4℃离心15min收获上清液。上清液经无菌过滤后进行10倍稀释(10-1～10-10)，然后接种于培养在96孔细胞培养板上的CHSE-214细胞上，7d后按Reed-Muench法计算组织中病毒滴度。
9、二联疫苗的安全性检验
采用一次单剂量(100μL)、二次单剂量(间隔7天，每次100μL)、一次超剂量(300μL)的二联佐剂疫苗，对虹鳟(平均体重10±1g)进行腹腔注射免疫，每组20尾。以PBS注射的虹鳟为阴性对照。免疫后每天观察虹鳟精神状态及接种部位有无异常，摄食是否正常，是否出现死亡等症状。于免疫后7d和14d分别从各组中随机挑选5尾虹鳟，进行解剖及肉眼观察，同时取肝、脾、肾脏，制作HE染色石蜡切片，利用显微镜观察组织切片。
二、结果与分析
1、最佳IHN灭活疫苗
(1)IHN疫苗最佳佐剂的确定
选择不同的商业化佐剂，按照说明书要求，与病毒灭活液配伍，制备出佐剂灭活疫苗，免疫虹鳟。在免疫时发现，A2佐剂疫苗粘稠度过高，无法进行注射免疫，因此后续研究不包括该佐剂疫苗。在免疫后1个月，进行攻毒试验，计算各疫苗的相对免疫保护率。结果显示，各组疫苗均具有显著的免疫保护效力，其中佐剂B灭活疫苗组无任何虹鳟死亡，相对免疫保护效力高达100％，显著高于其他各组疫苗(P<0.05)，其余各组佐剂疫苗的相对免疫保护率在60％以上。因此，选择IHN佐剂B疫苗进行二联疫苗的制备。参见图1。
(2)最佳IHN佐剂疫苗的免疫保护期
对效果最好的甲醛灭活的佐剂B疫苗进行保护期研究，同时以不加佐剂的甲醛灭活裸苗进行对比研究。结果发现，不加佐剂的裸苗的相对免疫保护率大大低于加了佐剂B的甲醛灭活疫苗。并且随着时间的增加，裸苗的免疫保护效果急剧下降，而佐剂B甲醛灭活疫苗的相对保护率一直维持在较高水平，在免疫后120天相对免疫保护率仍然高达90％以上，而相同剂量的裸苗随着免疫后时间的增加，相对免疫保护率急剧降低，到免疫后120天时，相对免疫保护效率 降到20％以下。该结果说明佐剂B显著延长了疫苗的保护期，可使疫苗保护效力维持在较高水平长达数月。参见图2。
(3)最佳IHN佐剂疫苗的安全性
对效果最好的甲醛灭活的佐剂B疫苗进行了免疫动物安全性研究。采用不同剂量对虹鳟进行免疫，然后对虹鳟的行为状态及组织生理变化进行了观察。通过观察发现，各组免疫虹鳟均摄食正常，精神状态正常，无任何不良反应，无任何死亡情况(图3)。于免疫后第7d和14d分别从各组中随机挑选5尾虹鳟，解剖观察状态。如图所示，一次单剂量、多次单剂量和一次超剂量方式免疫虹鳟，免疫后第7d和14d，虹鳟的各器官均正常，没有明显的损伤(图4和图5)。对各时期的肝、脾、肾组织进行石蜡切片HE染色观察，结果发现与对照组相比，各剂量组的虹鳟组织均无病理变化(图6至图11)。该结果表明，该佐剂灭活疫苗对虹鳟具有理想的安全性。
2、IPNV灭活条件的确定
利用不同浓度的甲醛对病毒进行不同时间的灭活处理，收获灭活病毒液，利用CHSE-214细胞进行灭活效果检验。结果如表2所示，当甲醛终浓度为0.025％、和0.075％(体积比)时，即使经过48h孵育，在CHSE-214细胞上依旧能观察到病变，说明该浓度未能将IPNV彻底灭活；当甲醛终浓度为0.25％和2.5％(体积比)时，12h即可将IPNV完全灭活，在细胞上观察不到任何细胞病变，说明该浓度可将IPNV彻底灭活。考虑到尽可能降低疫苗产品中外源物质含量，本发明选择终浓度为0.25％(体积比)的甲醛溶液，置于室温(25±1℃)条件下，摇床100r/min，孵育12h，来制备IPN病毒灭活液。
表2、甲醛最佳灭活剂量的筛选

注:“+++”为首次接毒后便可观察到细胞病变；“---”代表各代细胞均未发生病变。
3、最佳IPN佐剂疫苗
(1)IPN疫苗最佳佐剂的确定
将IPN病毒灭活液，分别与4种佐剂进行配伍，制备出佐剂灭活疫苗，分别命名为A1灭活疫苗、A2灭活疫苗、B灭活疫苗、C灭活疫苗。不加任何佐剂的灭活液命名为裸苗。采用50μL每尾的剂量对虹鳟进行腹腔注射免疫，在免疫过程中发现，A2灭活疫苗粘稠度过高，颗粒过大，无法进行注射操作，因此未对A2灭活疫苗进行进一步研究。于免疫后30d进行攻毒，攻毒后3d，测定各组虹鳟组织中病毒载量。结果如图12所示，与阴性对照组相比，各疫苗免疫 组虹鳟组织中病毒滴度均显著下降，其中B佐剂灭活疫苗组病毒滴度降低最多，病毒滴度仅为对照组的十万分之一(图12)。因此，本发明选择IPN佐剂B疫苗制备二联疫苗。
(2)最佳IPN佐剂疫苗的免疫保护期
对效果最好的佐剂B灭活疫苗进行保护期研究，同时以不加佐剂的甲醛灭活液(裸苗)进行对比。结果显示，在免疫后不同时间，各免疫组虹鳟体内病毒载量均低于对照组，B灭活疫苗组的病毒载量显著低于裸苗组(P<0.05)；随着攻毒时间的延长，虹鳟个体逐渐增大，病毒载量也出现下降的趋势，在免疫后3、4个月攻毒时，B灭活疫苗组虹鳟体内已经无法检测到病毒的存在，而裸苗组虹鳟体内仍然能检测出病毒。该结果说明佐剂B显著延长了疫苗的保护期，可使疫苗保护效力维持在较高水平长达数月。参见表3。
表3、免疫后不同时期攻毒后虹鳟组织中的病毒载量
(3)最佳IPN佐剂疫苗的安全性
对效果最好的甲醛灭活的佐剂B疫苗进行了免疫动物安全性研究。采用不同剂量对虹鳟进行免疫，然后对虹鳟的行为状态及组织生理变化进行了观察。通过观察发现，各组免疫虹鳟均摄食正常，精神状态正常，无任何不良反应，无任何死亡情况。解剖发现，各组虹鳟组织内脏均无显著的病理性改变，组织切片HE染色观察显示，免疫虹鳟的肝脾肾器官均无显著的病理变化(图13、图14)。
4、二联疫苗抗IHNV免疫保护效力
对制备的5种组合的二联疫苗进行抗IHNV保护效力分析。结果显示，在免疫后1、2个月时，各组疫苗均具有极高的相对免疫保护率，最高达100％，各组间无显著差异；在免疫后4个月时，各组疫苗仍然具有较高的保护率，最高达85％以上，此时H1P9疫苗组免疫保护率显著低于其余两组(P<0.05)，但是仍然高达78％以上(图15)。该结果说明，上述5种组合二联疫苗均能对IHNV产生良好的保护作用。
5、二联疫苗抗IPNV免疫保护效力
对制备的5种组合的二联疫苗进行抗IPNV保护效力分析。结果显示，在免疫后1、2月，各组疫苗均能显著降低IPNV滴度(P<0.05)，且H1P9组二联疫苗具有更强的抗病毒效果，病毒滴度仅为对照组的万分之一(表4)；在免疫后4个月时，无法从H1P1、H1P4、H1P9免疫组中检测到任何病毒的存在，而此时 P1H4、P1H9疫苗组与阴性对照无显著差异，已无抗IPNV效果。在以上任何时间点，阴性对照组中均检测出大量的病毒。该结果说明，H1P1、H1P4、H1P9组合的二联疫苗均能显著抵抗IPNV，其中H1P4、H1P9组合具有更好抗IPNV效果。
表4、免疫后不同时期攻毒后虹鳟组织中的病毒载量
6、二联疫苗免疫保护效力综合判定
本发明设计的5种组合的二联疫苗均能对IHNV产生良好的保护作用(图15)，而仅有H1P1、H1P4、H1P9组合的二联疫苗能显著抵抗IPNV。因此，本发明判定H1P1、H1P4和H1P9为最佳二联疫苗组合，即按照1:1或1:4或1:9的比例混合IHN佐剂B疫苗和IPN佐剂B疫苗。
7、二联疫苗的安全性
对二联疫苗进行了免疫动物安全性研究。采用不同剂量对虹鳟进行免疫，然后对虹鳟的行为状态及组织生理变化进行了观察。通过观察发现，各组免疫虹鳟均摄食正常，精神状态正常，无任何不良反应，无任何死亡情况。于免疫后第7d和14d分别从各组中随机挑选5尾虹鳟，解剖观察。结果显示各器官均正常，没有明显的损伤。对各时期的肝、脾、肾组织进行石蜡切片HE染色观察，结果发现与对照组相比，各剂量组的虹鳟组织均无病理变化(图16至图17)。该结果表明，该二联疫苗对虹鳟具有理想的安全性。
工业应用
本发明选择了4种佐剂，利用佐剂产品说明书推荐的方法分别与IHNV灭活液和IPNV灭活液进行配伍，制备佐剂灭活疫苗，免疫虹鳟，通过测定不同时间点的疫苗相对免疫保护效果筛选出针对单独的IHNV和IPNV最佳的疫苗佐剂。然后将针对单独的IHNV和IPNV最佳的疫苗佐剂按照不同比例混合，筛选效果最佳的二联佐剂疫苗，并对二联疫苗进行安全性检验。最终所得IHN佐剂疫苗延长了IHN灭活疫苗的保护期，最终所得IPN佐剂疫苗延长了IPN疫苗的保护期，最终所得二联佐剂疫苗能同时帮助宿主有效抵抗IHNV和IPNV的感染，且保护期长达4个月，并且该二联疫苗安全性良好。

Claims (45)

  1. 一种传染性造血器官坏死病和传染性胰脏坏死病二联疫苗，由传染性造血器官坏死病疫苗和传染性胰脏坏死病疫苗按照体积比为1：(1-9)的比例混合而成；
    所述传染性造血器官坏死病疫苗由传染性造血器官坏死病毒灭活液和MontanideTMGEL 02 PR佐剂组成；
    所述传染性胰脏坏死病疫苗由传染性胰脏坏死病毒灭活液和MontanideTMGEL 02 PR佐剂组成。
  2. 根据权利要求1所述的二联疫苗，其特征在于：在所述传染性造血器官坏死病疫苗中，MontanideTMGEL 02 PR佐剂的体积百分含量为10％-20％。
  3. 根据权利要求2所述的二联疫苗，其特征在于：在所述传染性造血器官坏死病疫苗中，所述传染性造血器官坏死病毒灭活液和MontanideTMGEL 02 PR佐剂的体积比为9:1。
  4. 根据权利要求1-3中任一所述的二联疫苗，其特征在于：在所述传染性胰脏坏死病疫苗中，MontanideTMGEL 02 PR佐剂的体积百分含量为10％-20％。
  5. 根据权利要求4所述的二联疫苗，其特征在于：在所述传染性胰脏坏死病疫苗中，所述传染性胰脏坏死病毒灭活液和MontanideTMGEL 02 PR佐剂的体积比为9:1。
  6. 根据权利要求1-5中任一所述的二联疫苗，其特征在于：所述传染性造血器官坏死病疫苗和所述传染性胰脏坏死病疫苗的体积比为1:1或1:4或者1:9。
  7. 根据权利要求1-6中任一所述的二联疫苗，其特征在于：所述传染性造血器官坏死病毒灭活液为传染性造血器官坏死病毒经甲醛灭活后所得。
  8. 根据权利要求1-7中任一所述的二联疫苗，其特征在于：所述传染性胰脏坏死病毒灭活液为传染性胰脏坏死病毒经甲醛灭活后所得。
  9. 根据权利要求8所述的二联疫苗，其特征在于：利用甲醛对所述传染性胰脏坏死病毒进行灭活时，甲醛的终浓度为0.25％-2.5％体积百分含量。
  10. 根据权利要求1-9中任一所述的二联疫苗，其特征在于：所述传染性造血器官坏死病毒灭活液在灭活前的病毒滴度为107TCID50/0.1ml。
  11. 根据权利要求1-10中任一所述的二联疫苗，其特征在于：所述传染性胰脏坏死病毒灭活液在灭活前的病毒滴度为107TCID50/0.1ml。
  12. 权利要求1-11任一中所述的传染性造血器官坏死病疫苗。
  13. 权利要求1-11任一中所述的传染性胰脏坏死病疫苗。
  14. 一种制备传染性造血器官坏死病和传染性胰脏坏死病二联疫苗的方法，包括如下步骤：
    (A)制备传染性造血器官坏死病毒灭活液；然后将所述传染性造血器官坏 死病毒灭活液和MontanideTMGEL 02 PR佐剂混合，得到传染性造血器官坏死病疫苗；
    (B)制备传染性胰脏坏死病毒灭活液；然后将所述传染性胰脏坏死病毒灭活液和MontanideTMGEL 02 PR佐剂混合，得到传染性胰脏坏死病疫苗；
    (C)将(A)中制得的所述传染性造血器官坏死病疫苗和(B)中制得的所述传染性胰脏坏死病疫苗按照体积比为1：(1-9)的比例混合，得到传染性造血器官坏死病和传染性胰脏坏死病二联疫苗。
  15. 根据权利要求14所述的方法，其特征在于：在所述传染性造血器官坏死病疫苗中，MontanideTMGEL 02 PR佐剂的体积百分含量为10％-20％。
  16. 根据权利要求15所述的方法，其特征在于：在所述传染性造血器官坏死病疫苗中，所述传染性造血器官坏死病毒灭活液和MontanideTMGEL 02 PR佐剂的体积比为9:1。
  17. 根据权利要求14-16中任一所述的方法，其特征在于：在所述传染性胰脏坏死病疫苗中，MontanideTMGEL 02 PR佐剂的体积百分含量为10％-20％。
  18. 根据权利要求17所述的方法，其特征在于：在所述传染性胰脏坏死病疫苗中，所述传染性胰脏坏死病毒灭活液和MontanideTMGEL 02 PR佐剂的体积比为9:1。
  19. 根据权利要求14-18中任一所述的方法，其特征在于：所述传染性造血器官坏死病疫苗和所述传染性胰脏坏死病疫苗的体积比为1:1或1:4或者1:9。
  20. 根据权利要求14-19中任一所述的方法，其特征在于：步骤(A)中，所述传染性造血器官坏死病毒灭活液为传染性造血器官坏死病毒经甲醛灭活后所得。
  21. 根据权利要求14-20中任一所述的方法，其特征在于：步骤(B)中，所述传染性胰脏坏死病毒灭活液为传染性胰脏坏死病毒经甲醛灭活后所得。
  22. 根据权利要求21所述的方法，其特征在于：利用甲醛对所述传染性胰脏坏死病毒进行灭活时，甲醛的终浓度为0.25％-2.5％体积百分含量。
  23. 根据权利要求14-22中任一所述的方法，其特征在于：步骤(A)中，所述传染性造血器官坏死病毒灭活液在灭活前的病毒滴度为107TCID50/0.1ml。
  24. 根据权利要求14-23中任一所述的方法，其特征在于：步骤(B)中，所述传染性胰脏坏死病毒灭活液在灭活前的病毒滴度为107TCID50/0.1ml。
  25. 根据权利要求14-24中任一所述的方法，其特征在于：步骤(A)中，所述制备传染性造血器官坏死病毒灭活液按照如下进行：取病毒滴度为107TCID50/0.1ml的传染性造血器官坏死病毒液，加入甲醛使其终浓度为5mM，混匀；置于摇床，24℃，100r/min灭活24h，终止灭活，即得传染性造血器官坏死病毒灭活液。
  26. 根据权利要求14-25中任一所述的方法，其特征在于：步骤(B)中， 所述制备传染性胰脏坏死病毒灭活液按照如下进行：取病毒滴度为107TCID50/0.1ml的传染性胰脏坏死病毒液，加入甲醛使其终浓度为0.25％-2.5％体积百分含量，混匀；置于摇床，24-26℃，100r/min灭活12h，终止灭活，即得传染性胰脏坏死病毒灭活液。
  27. 一种制备传染性造血器官坏死病疫苗的方法，包括权利要求14-26任一中所述的步骤(A)。
  28. 一种制备传染性胰脏坏死病疫苗的方法，包括权利要求14-26任一中所述的步骤(B)。
  29. 传染性造血器官坏死病毒灭活液、传染性胰脏坏死病毒灭活液和MontanideTMGEL 02 PR佐剂在制备传染性造血器官坏死病和传染性胰脏坏死病二联疫苗中的应用；
    所述传染性造血器官坏死病和传染性胰脏坏死病二联疫苗由传染性造血器官坏死病疫苗和传染性胰脏坏死病疫苗按照体积比为1：(1-9)的比例混合而成。
  30. 传染性造血器官坏死病毒灭活液和MontanideTMGEL 02 PR佐剂在制备传染性造血器官坏死病疫苗中的应用。
  31. 传染性胰脏坏死病毒灭活液和MontanideTMGEL 02 PR佐剂在制备传染性胰脏坏死病疫苗中的应用。
  32. 根据权利要求29或30所述的应用，其特征在于：在所述传染性造血器官坏死病疫苗中，MontanideTMGEL 02 PR佐剂的体积百分含量为10％-20％。
  33. 根据权利要求32所述的应用，其特征在于：在所述传染性造血器官坏死病疫苗中，所述传染性造血器官坏死病毒灭活液和MontanideTMGEL 02 PR佐剂的体积比为9:1。
  34. 根据权利要求29或31所述的应用，其特征在于：在所述传染性胰脏坏死病疫苗中，MontanideTMGEL 02 PR佐剂的体积百分含量为10％-20％。
  35. 根据权利要求34所述的应用，其特征在于：在所述传染性胰脏坏死病疫苗中，所述传染性胰脏坏死病毒灭活液和MontanideTMGEL 02 PR佐剂的体积比为9:1。
  36. 根据权利要求29和32-35中任一所述的应用，其特征在于：在所述二联疫苗中，所述传染性造血器官坏死病疫苗和所述传染性胰脏坏死病疫苗的体积比为1:1或1:4或者1:9。
  37. 根据权利要求29、30和32-36中任一所述的应用，其特征在于：所述传染性造血器官坏死病毒灭活液为传染性造血器官坏死病毒经甲醛灭活后所得。
  38. 根据权利要求29和31-37中任一所述的应用，其特征在于：所述传染性胰脏坏死病毒灭活液为传染性胰脏坏死病毒经甲醛灭活后所得。
  39. 根据权利要求38所述的应用，其特征在于：利用甲醛对所述传染性胰 脏坏死病毒进行灭活时，甲醛的终浓度为0.25％-2.5％体积百分含量。
  40. 根据权利要求29、30和32-39中任一所述的应用，其特征在于：所述传染性造血器官坏死病毒灭活液在灭活前的病毒滴度为107TCID50/0.1ml。
  41. 根据权利要求29和31-40中任一所述的应用，其特征在于：所述传染性胰脏坏死病毒灭活液在灭活前的病毒滴度为107TCID50/0.1ml。
  42. 根据权利要求1-41中任一所述的二联疫苗或传染性造血器官坏死病疫苗或传染性胰脏坏死病疫苗或方法或应用，其特征在于：所述传染性造血器官坏死病毒为传染性造血器官坏死病毒LN15分离株。
  43. 如下任一应用：
    P1、权利要求1-11中任一所述二联疫苗在预防和/或治疗IHNV和IPNV宿主因感染传染性造血器官坏死病毒和/或传染性胰脏坏死病毒所致疾病中的应用；
    P2、权利要求12所述传染性造血器官坏死病疫苗在预防和/或治疗IHNV宿主因感染传染性造血器官坏死病毒所致疾病中的应用；
    P3、权利要求13所述传染性胰脏坏死病疫苗在预防和/或治疗IPNV宿主因感染传染性胰脏坏死病毒所致疾病中的应用。
  44. 如下任一方法：
    Q1、一种预防和/或治疗IHNV和IPNV宿主因感染传染性造血器官坏死病毒和/或传染性胰脏坏死病毒所致疾病的方法，包括如下步骤：使用权利要求1-11中任一所述二联疫苗实现对IHNV和IPNV宿主因感染传染性造血器官坏死病毒和/或传染性胰脏坏死病毒所致疾病的预防和/或治疗；
    Q2、一种预防和/或治疗IHNV宿主因感染传染性造血器官坏死病毒所致疾病的方法，包括如下步骤：使用权利要求12所述传染性造血器官坏死病疫苗实现对IHNV宿主因感染传染性造血器官坏死病毒所致疾病的预防和/或治疗；
    Q3、一种预防和/或治疗IPNV宿主因感染传染性胰脏坏死病毒所致疾病的方法，包括如下步骤：使用权利要求13所述传染性胰脏坏死病疫苗实现对IPNV宿主因感染传染性胰脏坏死病毒所致疾病的预防和/或治疗。
  45. 根据权利要求1-44中任一所述的二联疫苗或传染性造血器官坏死病疫苗或传染性胰脏坏死病疫苗或方法或应用，其特征在于：所述传染性胰脏坏死病毒为传染性胰脏坏死病毒GS2020-1分离株。
PCT/CN2023/075791 2022-02-24 2023-02-14 一种鲑鳟鱼传染性造血器官坏死病和传染性胰脏坏死病单价佐剂疫苗及其二联佐剂疫苗和制备方法 WO2023160425A1 (zh)

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