CN115804837A - Infectious pancreatic necrosis adjuvant vaccine and preparation method thereof - Google Patents
Infectious pancreatic necrosis adjuvant vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an adjuvant vaccine for infectious pancreatic necrosis and a preparation method thereof. The infectious pancreatic necrosis vaccine provided by the invention comprises infectious pancreatic necrosis virus inactivated liquid and Montanide TM GEL 02PR adjuvant. The invention selects 4 adjuvants, utilizes the method recommended by the adjuvant product specification to be compatible with IPNV inactivated liquid, prepares adjuvant inactivated vaccine, immunizes rainbow trout, and screens out the optimal vaccine adjuvant by measuring the relative immune protection effect of the vaccine at different time points. At the same time, the best performing vaccine was tested for safety. The finally obtained adjuvant vaccine prolongs the protection period of the IPN vaccine and has good safety.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an adjuvant vaccine for infectious pancreatic necrosis and a preparation method thereof.
Background
The etiology of the infectious pancreatic necrosis disease (IPN) is Infectious Pancreatic Necrosis Virus (IPNV), which belongs to the family of birnaviridae, the genus of aquatic birnavirus, and can infect various kinds of freshwater fishes. In China, IPN mainly threatens salmon and trout such as rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trout), javellus trout (Salvelinus fontinalis), brachymystax lenok and the like. According to different environments, hosts, strains and the like, IPNV can cause the death rate of juvenile fish of more than 80 percent at most, and becomes one of the disease problems limiting the development of the salmon and trout industry in China.
At present, no effective medicine for preventing and controlling IPN exists in China. Because the strain has the problems of variation, potential biological safety hazards and the like, foreign commercial vaccines cannot be directly introduced and applied, and therefore, the problem of disease of salmon and trout in China can be solved only through the independent research and development of the vaccine. The subject group developed work for the development of an IPN inactivated vaccine by using a native strain in the early stage, and prepared the IPN inactivated vaccine by inactivating with β -propiolactone (BPL) (see chinese patent application CN113583968 a). The vaccine is directly used for immunizing rainbow trout, and the result shows that the prepared inactivated vaccine has ideal early protection efficacy, but the vaccine immune protection efficiency begins to decline from 45 days after immunization along with the prolonging of time, does not have a long protection period, and cannot meet the market demand, so that the existing inactivated vaccine is extremely necessary to be optimized.
Disclosure of Invention
The invention aims to provide an adjuvant vaccine for infectious pancreatic necrosis and a preparation method thereof.
In a first aspect, the invention claims an infectious pancreatic necrosis vaccine.
The invention claims an infectious pancreatic necrosis vaccine, which is prepared from infectious pancreatic necrosis virus inactivated liquid and Montanide TM GEL 02PR adjuvant.
Further, in the infectious pancreatic necrosis vaccine, montanide TM GEL 0The volume percentage content of the 2PR adjuvant is 10-20% (namely the volume percentage content of the infectious pancreatic necrosis virus inactivation liquid is 80-90%).
Further, the infectious pancreatic necrosis virus inactivation solution and Montanide TM The GEL 02PR adjuvant may have a volume ratio of 9:1.
Further, the infectious pancreatic necrosis virus inactivation solution can be obtained by inactivating infectious pancreatic necrosis viruses by formaldehyde.
Further, where formaldehyde is used to inactivate the infectious pancreatic necrosis virus, the final concentration of formaldehyde may be from 0.25% to 2.5% (e.g., 0.25%) by volume.
Further, the virus titer of the infectious pancreatic necrosis virus inactivation solution before inactivation may be 10 7 TCID 50 /0.1ml。
Further, the infectious pancreatic necrosis vaccine may be prepared according to the method described in the second aspect below.
In a second aspect, the invention claims a method for preparing an infectious pancreatic necrosis vaccine.
The method for preparing the infectious pancreatic necrosis vaccine claimed by the invention can comprise the following steps:
(A) Preparing an infectious pancreatic necrosis virus inactivation solution;
(B) The infectious pancreatic necrosis virus inactivation solution and Montanide are added TM And mixing GEL 02PR adjuvant to prepare the infectious pancreatic necrosis vaccine.
Further, in the infectious pancreatic necrosis vaccine, montanide TM The volume percentage content of the GEL 02PR adjuvant is 10-20% (namely the volume percentage content of the infectious pancreatic necrosis virus inactivation liquid is 80-90%).
Further, in the infectious pancreatic necrosis vaccine, the infectious pancreatic necrosis virus inactivation solution and Montanide TM The volume ratio of GEL 02PR adjuvant may be 9:1.
Further, the infectious pancreatic necrosis virus inactivation solution can be obtained by inactivating infectious pancreatic necrosis viruses by formaldehyde.
Further, when formaldehyde is used to inactivate the infectious pancreatic necrosis virus, the final concentration of formaldehyde may be 0.25% to 2.5% (e.g., 0.25%) by volume.
Further, the virus titer of the infectious pancreatic necrosis virus inactivation solution before inactivation can be 10 7 TCID 50 /0.1ml。
In the step (a), the preparation of the infectious pancreatic necrosis virus inactivation solution may be performed as follows: taking the virus titer as 10 7 TCID 50 0.1ml of infectious pancreatic necrosis virus solution, adding formaldehyde to make the final concentration of the infectious pancreatic necrosis virus solution be 0.25-2.5% (such as 0.25%) by volume percentage, and mixing uniformly; inactivating for 12h at 100r/min in a shaker at 24-26 deg.C (room temperature), and terminating inactivation with sodium bisulfite solution with final concentration of 1mM to obtain inactivating solution of infectious pancreatic necrosis virus.
In the step (B), the infectious pancreatic necrosis virus inactivation solution and Montanide are added TM Montanide can also be added before the GEL 02PR adjuvant is mixed TM And (3) sterilizing the GEL 02PR adjuvant. Such as a autoclave (116 deg.C) for 20min.
In a third aspect, the invention claims an infectious pancreatic necrosis virus inactivation solution and Montanide TM Application of GEL 02PR adjuvant in preparation of infectious pancreatic necrosis vaccine.
Further, in the infectious pancreatic necrosis vaccine, montanide TM The volume percentage content of the GEL 02PR adjuvant is 10-20% (namely the volume percentage content of the infectious pancreatic necrosis virus inactivation liquid is 80-90%).
Further, the infectious pancreatic necrosis virus inactivation solution and Montanide TM The volume ratio of GEL 02PR adjuvant may be 9:1.
Further, the infectious pancreatic necrosis virus inactivation solution can be obtained by inactivating infectious pancreatic necrosis viruses by formaldehyde.
Further, where formaldehyde is used to inactivate the infectious pancreatic necrosis virus, the final concentration of formaldehyde may be from 0.25% to 2.5% (e.g., 0.25%) by volume.
Further, the virus titer of the infectious pancreatic necrosis virus inactivation solution before inactivation can be 10 7 TCID 50 /0.1ml。
In the above aspects, the vaccine of the present invention can be used for preventing the IPNV host from being infected by the infectious pancreatic necrosis virus, and can be used for preventing and/or treating diseases caused by the IPNV host infected by the infectious pancreatic necrosis virus.
The invention also claims the use of said vaccine in the prevention and/or treatment of diseases caused by infection of the IPNV host with infectious pancreatic necrosis virus.
The present invention also claims a method for preventing and/or treating diseases caused by infection of an infectious pancreatic necrosis virus in an IPNV host, comprising the steps of: the vaccine provided by the invention is used for preventing and/or treating diseases caused by infection of IPNV host with infectious pancreatic necrosis virus.
Wherein the IPNV host is fish, such as Salmon and trout, and particularly rainbow trout.
In a particular embodiment of the invention, the infectious pancreatic necrosis virus is in particular the infectious pancreatic necrosis virus GS2020-1 isolate.
In the present invention, montanide is used TM It is also within the scope of the present invention to replace the GEL 02PR adjuvant with other adjuvants of the same or similar composition. Montanide TM The GEL 02PR adjuvant contains polyoxyethylene C12-C18 alkyl ether.
The invention selects 4 adjuvants, utilizes the method recommended by the adjuvant product specification to be compatible with IPNV inactivated liquid, prepares adjuvant inactivated vaccine, immunizes rainbow trout, and screens out the optimal vaccine adjuvant by measuring the relative immune protection effect of the vaccine at different time points. At the same time, the best performing vaccine was tested for safety. The finally obtained adjuvant vaccine prolongs the protection period of the IPN vaccine and has good safety.
Drawings
FIG. 1 shows the viral load in rainbow trout tissues (30 days post-immunization challenge). Different letters on the error bars represent significant differences (P < 0.05).
FIG. 2 shows the rainbow trout tissue sections 7 days after immunization with the adjuvant B inactivated vaccine.
FIG. 3 shows the rainbow trout tissue sections 14 days after immunization with the adjuvant B inactivated vaccine.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of infectious pancreatic necrosis Virus adjuvant vaccine and examination of the Effect thereof
1. Materials and methods
1. Material
The infectious pancreatic necrosis virus GS2020-1 isolate is isolated and preserved by the laboratory, and the strain is described in the following documents: duan K Y, ZHao J Z, ren G M, et al molecular Evolution of extraction fashion viros in China [ J ]. Virus, 2021,13 (3): 488). The public is available from the applicant according to the national biosafety regulations and can only be used for repeated experiments of the invention, but not for other purposes.
Salmon embryo cells (CHES-214 cells) were maintained in the laboratory and were described in the following references: xu L M, zhao J Z, liu M, et al. Bivalent DNA vaccine antigens and antigens induced nutritional ingredients biochemical optoelectronic circuits in raw tissue [ J ] Scientific reports,2017,7 (1): 5700. The applicant is publicly available and can only use the test for repeated invention, and no other use is needed.
2. Determination of IPNV inactivation conditions
To fresh IPNV virus solution (10) 7 TCID 50 0.1 ml) is added with formaldehyde to make the final concentration respectively 0.025%, 0.075%, 0.25%, 2.5% (volume ratio), and is placed in a condition of room temperature (25 +/-1 ℃) and a shaking table for 100r/min, and is respectively incubated for 12h, 24 h, 36 h, and 48h for inactivation treatment. Inactivation was terminated by adding sodium bisulfite solution (final concentration 1 mM), and IPNV inactivation solution was harvested and the effect of inactivation was examined at the cellular level. The specific operation is as follows: the IPNV inactivation solution was inoculated onto 6-well cell culture plates (3X 10) full of CHSE-214 cells 5 Individual cells/well), 1ml of IPNV inactivation solution per well. Then, the cells are placed in a carbon dioxide incubator at 15 ℃ for culture for 7d, if no cytopathic effect occurs in 7d, the cells are placed in a refrigerator at-80 ℃ for repeated freeze thawing for 2 times, and then are centrifuged at 12000r/min at 4 ℃ for 15min to collect supernatant. The supernatant was inoculated with the CHSE-214 cells according to the above method for blind transfer, continuously blind transferred to 3 rd generation, and the conditions of cytopathic effect were observed every day to determine the conditions for inactivating IPNV.
3. Preparation of IPN adjuvant vaccine
And preparing the IPNV inactivation solution by using the optimal inactivation condition. And (3) proportioning the inactivated solution and each sterilized adjuvant (sterilized at 116 ℃ in a high-temperature and high-pressure sterilizing pot for 20 min) according to the table 1, and uniformly mixing to form a homogeneous solution, thereby obtaining the IPN adjuvant vaccine.
TABLE 1 adjuvant information
4. Immunoprotection phase analysis of IPN-adjuvanted vaccines
Immunity rainbow trout (average body weight 7. + -.1 g) was from Nianning Benxi Ai Gemo forest industries, inc. The PBS-injected group served as a negative control group. The vaccine groups included: inactivated vaccine with adjuvant and naked vaccine (inactivated vaccine without adjuvant). The adjuvants were four (table 1): biphasic GR208 adjuvant (labeled A1), montanide TM ISA 763A VG (labeled A2) and Montanide TM GEL 02PR (labeled B), 4% 3 Gel (labeled C). The adopted immunization dose is 100 mu L per tail, and the immunization mode is intraperitoneal injection immunization.After immunization, the rainbow trout is placed in an indoor running water culture round tank (2 m diameter, water depth of 80cm, water temperature of 13 +/-2 ℃) for culture. IPNV (1X 10) was administered at a dose of 10. Mu.l/tail 30d after immunization 6 TCID 50 0.1 ml) to carry out intraperitoneal injection for attacking toxin to rainbow trout (the weight is 7 +/-1 g). Each treatment 20 tails. At 3d after challenge, 5 fish visceral tissues (liver, spleen and head kidney) were individually homogenized with PBS buffer at a ratio of 1. The supernatant was sterile filtered and diluted 10-fold (10) -1 ～10 -10 ) Then, the cells were inoculated onto CHSE-214 cells cultured in a 96-well cell culture plate, and after 7d, the virus titer in the tissue was calculated by the Reed-Muench method. The immunoprotective efficacy of the vaccines was analyzed by comparing the difference in IPNV titer in the tissues of rainbow trout in the PBS-injected group (control group) and the vaccine-immunized group.
5. Long-term immunoprotection efficacy analysis of optimal IPN adjuvant vaccines
The best adjuvant vaccine and naked vaccine are selected, 100 mu L dosage is adopted, and the abdominal injection immunization is carried out on the rainbow trout with the average weight of 10 +/-1 g. Rainbow trout injected with PBS was used as negative control. After immunization, the rainbow trout is placed in an indoor flowing water culture round tank (2 m diameter, water depth of 80cm, water temperature of 14 +/-1 ℃) for culture. Referring to the method in step 4, the rainbow trout (n = 10) was subjected to challenge test at different times after immunization, then cultured in an indoor circulating water system (water temperature 13 ± 1 ℃) and the viral load in the tissues was determined after 3 d.
6. Safety test for optimal IPNV adjuvant vaccine
The best adjuvant vaccine was selected and the rainbow trout (average body weight 10 ± 1 g) was immunized intraperitoneally with a single dose (100 μ L), two single doses (100 μ L each with 7 days intervals), and one overdose (300 μ L), each treatment for 20 tails. Rainbow trout injected with PBS was used as negative control. Observing the mental state of the rainbow trout and the abnormal state of the inoculated part every day after immunization, whether the ingestion is normal, whether the death and other symptoms occur; randomly selecting 5 rainbow trout from each group at 7d and 14d after the last immunization, and carrying out dissection and visual observation; meanwhile, the liver, spleen and kidney were taken, HE stained paraffin sections were made, and the tissue sections were observed with a microscope.
2. Results and analysis
1. Determination of IPNV inactivation conditions
And (3) inactivating the virus by using formaldehyde with different concentrations for different times, harvesting inactivated virus liquid, and performing inactivation effect test by using CHSE-214 cells. The results are shown in table 2, where the final concentration of formaldehyde was 0.025%, and 0.075% (by volume), lesions were still observed on CHSE-214 cells even after 48h incubation, indicating that this concentration failed to completely inactivate IPNV; at final formaldehyde concentrations of 0.25% and 2.5% (by volume), complete inactivation of IPNV was achieved within 12h, and no cytopathic effect was observed on the cells, indicating that this concentration completely inactivated IPNV. In consideration of reducing the content of foreign substances in vaccine products as much as possible, the invention selects formaldehyde with the final concentration of 0.25 percent (volume ratio), places the formaldehyde at room temperature (25 +/-1 ℃) for 12 hours by shaking the bed at 100r/min, and incubates to prepare the IPN virus inactivation solution.
TABLE 2 screening of optimal Formaldehyde inactivation dose
After the first virus inoculation, "+ + + + + +" indicates that cytopathy can be observed; "- - -" indicates that no lesion occurred in each passage of cells.
2. Immune protection rate of vaccine
The IPNV virus solution inactivated by formaldehyde is respectively matched with 4 adjuvants to prepare adjuvant inactivated vaccines which are respectively named as A1 inactivated vaccine, A2 inactivated vaccine, B inactivated vaccine and C inactivated vaccine. The inactivated solution without any adjuvant is named as naked seedlings. The rainbow trout is immunized by intraperitoneal injection with 100 mu L of dose per tail, and in the immunization process, the A2 inactivated vaccine is found to have too high viscosity and too large particles, so that the injection operation cannot be carried out, and further research on the A2 inactivated vaccine is not carried out. And (4) performing challenge at 30d after immunization, and measuring the virus load in tissues of each group of rainbow trout 3 days after challenge. The results are shown in fig. 1, and compared with the control group, the virus titer in the rainbow trout tissues of each vaccine immunization group is significantly reduced, wherein the virus titer of the adjuvant B inactivated vaccine group is reduced most, and the virus titer is only one ten thousandth of that of the control group (fig. 1). Therefore, protective period and safety research is further carried out aiming at the B inactivated vaccine.
3. Immunoprotection phase of best adjuvant vaccines
The protective period of the adjuvant B inactivated vaccine with the best effect is studied, and meanwhile, formaldehyde inactivated solution (naked vaccine) without adjuvant is used for comparison. The results show that the virus load in rainbow trout of each immune group is lower than that of a control group and the virus load of a B inactivated vaccine group is obviously lower than that of a naked vaccine group (P < 0.05) at different times after immunization; with the prolonging of the challenge time, the individual rainbow trout gradually increases, the virus load also tends to decrease, and when the virus is challenged 3 or 4 months after immunization, the virus in the rainbow trout of the B inactivated vaccine group can not be detected, and the virus in the rainbow trout of the nude vaccine group can still be detected. This result indicates that adjuvant B significantly prolongs the protective period of the vaccine, and can maintain the protective efficacy of the vaccine at a high level for several months. See table 3.
TABLE 3 viral load in rainbow trout tissues after different periods of challenge after immunization
4. Safety of optimally adjuvanted vaccines
The adjuvant B vaccine with the best effect of inactivating formaldehyde is subjected to immune animal safety research. The rainbow trout is immunized by adopting different dosages, and then the behavior state and the tissue physiological change of the rainbow trout are observed. Through observation, each group of immune rainbow trout has normal food intake, normal mental state, no adverse reaction and no death condition. The visceral organs of the rainbow trout tissues of all groups are not obviously pathologically changed by dissection, and the liver, spleen and kidney organs of the immune rainbow trout are not obviously pathologically changed by observing tissue section HE staining (figure 2 and figure 3).
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. An infectious pancreatic necrosis vaccine is prepared from inactivated infectious pancreatic necrosis virus solution and Montanide TM GEL 02PR adjuvant.
2. The vaccine of claim 1, wherein: montanide in the infectious pancreatic necrosis vaccine TM The volume percentage content of the GEL 02PR adjuvant is 10-20 percent;
further, the infectious pancreatic necrosis virus inactivation solution and Montanide TM The volume ratio of the GEL 02PR adjuvant is 9:1.
3. The vaccine of claim 1 or 2, characterized in that: the infectious pancreas necrosis virus inactivation solution is obtained by inactivating infectious pancreas necrosis viruses by formaldehyde;
further, when formaldehyde is used for inactivating the infectious pancreatic necrosis virus, the final concentration of the formaldehyde is 0.25-2.5% by volume.
4. The vaccine of any one of claims 1-3, wherein: the virus titer of the infectious pancreatic necrosis virus inactivation solution before inactivation is 10 7 TCID 50 /0.1ml。
5. A method for preparing an infectious pancreatic necrosis disease vaccine, comprising the steps of:
(A) Preparing an infectious pancreatic necrosis virus inactivation solution;
(B) Subjecting the infectious pancreasInactivated necrotic virus solution and Montanide TM And mixing GEL 02PR adjuvant to prepare the infectious pancreatic necrosis vaccine.
6. The method of claim 5, wherein: montanide in the infectious pancreatic necrosis vaccine TM The volume percentage content of the GEL 02PR adjuvant is 10-20 percent;
further, in the infectious pancreatic necrosis vaccine, the infectious pancreatic necrosis virus inactivation solution and Montanide TM The volume ratio of the GEL 02PR adjuvant is 9:1.
7. The method according to claim 5 or 6, characterized in that: the infectious pancreas necrosis virus inactivation solution is obtained by inactivating infectious pancreas necrosis viruses by formaldehyde;
further, when the infectious pancreatic necrosis virus is inactivated by formaldehyde, the final concentration of the formaldehyde is 0.25-2.5% by volume;
and/or
The virus titer of the infectious pancreatic necrosis virus inactivation solution before inactivation is 10 7 TCID 50 /0.1ml。
8. The method according to any one of claims 5-7, wherein: in the step (A), the preparation of the infectious pancreatic necrosis virus inactivation solution is carried out as follows: taking the virus titer as 10 7 TCID 50 0.1ml of infectious pancreatic necrosis virus liquid, adding formaldehyde to make the final concentration of the infectious pancreatic necrosis virus liquid be 0.25-2.5% volume percentage content, and uniformly mixing; inactivating the mixture for 12h at the temperature of 24-26 ℃ at the speed of 100r/min, and stopping inactivating to obtain the infectious pancreatic necrosis virus inactivating solution.
9. Infectious pancreatic necrosis virus inactivation solution and Montanide TM The GEL 02PR adjuvant is applied to the preparation of infectious pancreatic necrosis vaccine;
further, in the infectious pancreatic necrosis vaccine, montanide TM Volume hundred of GEL 02PR adjuvantThe content of each component is 10 to 20 percent;
further, the infectious pancreatic necrosis virus inactivation solution and Montanide TM The volume ratio of the GEL 02PR adjuvant is 9:1;
and/or
Further, the infectious pancreatic necrosis virus inactivation solution is obtained by inactivating infectious pancreatic necrosis viruses by formaldehyde;
further, when the infectious pancreatic necrosis virus is inactivated by formaldehyde, the final concentration of the formaldehyde is 0.25 to 2.5 volume percent;
and/or
Further, the virus titer of the infectious pancreatic necrosis virus inactivation solution before inactivation is 10 7 TCID 50 /0.1ml。
10. The vaccine or method or use according to any one of claims 1 to 9, wherein: the infectious pancreatic necrosis virus is specifically an infectious pancreatic necrosis virus GS2020-1 isolate.
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| CN202210176019.6A CN115804837B (en) | 2022-02-24 | 2022-02-24 | Infectious pancreatic necrosis adjuvant vaccine and preparation method thereof |
| PCT/CN2023/075791 WO2023160425A1 (en) | 2022-02-24 | 2023-02-14 | Monovalent adjuvanted vaccine for infectious hematopoietic necrosis and infectious pancreatic necrosis of salmon and trout, and bivalent adjuvanted vaccine thereof and preparation method therefor |
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