A fish rhabdovirus attenuated vaccine freeze-dried protective agent, vaccine and preparation method thereof

Abstract

The present invention relates to the field of viruses, and specifically to a fish rhabdovirus attenuated vaccine freeze-dried protective agent, vaccine and preparation method thereof. The freeze-dried protective agent includes sucrose, seaweed liquid, PEG, gelatin and casein hydrolyzed protein, and has good heat resistance through compounding of multiple components, has good protective effect when stored at 4°C for a long time, can be used to prepare effective freeze-dried vaccines, and ensures the stability of the product under short-term deviation storage conditions.

Description

Technical Field

[0001] The present invention relates to the field of vaccine protective agents, and in particular to a fish rhabdovirus attenuated vaccine freeze-dried protective agent, a vaccine and a preparation method thereof. Background Art

[0002] Largemouth bass rhabdovirus or fish rhabdovirus (Micropterus salmoides Rhabdovirus, MSRV) can cause explosive deaths of famous fish such as mandarin fish and sea bass. It is extremely easy to be infected from the seedling stage to the breeding process, and the mortality rate can be as high as over 90%. In particular, the mortality rate of seedling infection is almost 100%. There is no effective prevention and control method. However, there is a lack of commercial attenuated vaccines for largemouth bass rhabdovirus. The applicant has isolated a largemouth bass rhabdovirus attenuated strain from nature, which has good immunogenicity and can be used as a candidate strain for attenuated vaccines.

[0003] Rhabdovirus is a single-stranded negative-strand RNA virus with a double lipid envelope. It is not resistant to high temperatures and can be quickly inactivated at 56°C. Most domestic products are live vaccines with conventional protective agents. The main ingredients of the protective agent are skim milk, sucrose, gelatin, etc. The protective agent has a simple formula. If it is stored at 2°C~8°C, the shelf life is only about 4 to 6 months. Most vaccines need to be stored below -15°C. If the cold chain system fails during the transportation and use of the vaccine, the temperature of the vaccine storage environment will rise, which often leads to a decrease in vaccine efficacy or failure, resulting in immunization failure.

[0004] As a widely used pharmaceutical engineering technology, low-temperature vacuum freeze-drying technology includes a series of complex processes such as freezing, sublimation, and drying. All parameters must be well controlled in the entire process to produce qualified vaccines that meet quality standards. However, in the freeze-drying process with many steps, various extreme low temperatures, freezing, and drying conditions will destroy the proteins in the vaccine products to a certain extent; therefore, in order to ensure the good quality of freeze-dried vaccine products, it is necessary to add certain freeze-drying protective agents in the freeze-drying process; according to the results of freeze-dried appearance, virus titer, and thermal stability tests, the optimal formula of freeze-drying protective agents is screened; the selection of appropriate freeze-drying protective agents largely determines the quality of the finished freeze-dried vaccine.

[0005] Common freeze-drying protective agents mainly include sugars, polymers, amino acids, etc. Trehalose, lactose and maltose have stable glassy properties and are more suitable as freeze-drying protective agents. When an organism containing trehalose is in a dry and dehydrated state, or in a hypertonic environment, trehalose automatically forms a glassy protective layer to improve the osmotic pressure tolerance or desiccation resistance of cells in the body, thereby protecting proteins, lipids, sugars, nucleic acids, etc. in the body from damage by the drying state, allowing cells to continue to remain active. Polyethylene glycol has good water solubility, good moisture retention, and is relatively stable. It is often used for freeze-drying protection of clinical drugs. Glycine can maintain the shape of the sample during the freeze-drying process, reduce sample shrinkage, shorten the freeze-drying time, and thus reduce the damage of freeze-drying to the virus structure; the addition of gelatin can increase viscosity, facilitate the formation of a good appearance state, and increase the stability of the antigen; the present invention screens out the best freeze-drying formula through research through the preparation of various freeze-drying protectants, and prepares an effective freeze-dried vaccine, which can be stored for a long time at 4°C and is stable under short-term deviations from the storage conditions, providing supporting data for determining the validity period and storage conditions of the fish rhabdovirus attenuated freeze-dried vaccine. Summary of the invention

[0006] In view of the above problems, the present invention provides a freeze-dried protective agent for attenuated fish rhabdovirus vaccine.

[0007] The technical solution of the present invention is as follows:

[0008] In the first aspect, the present invention provides a fish rhabdovirus attenuated vaccine lyophilization protective agent, the lyophilization protective agent comprises sucrose, seaweed liquid, PEG, gelatin and casein hydrolyzed protein. The lyophilization protective agent is compounded with a variety of auxiliary materials, and the protective agent obtained has good heat resistance, and the titer decreases less than 1 under short-term non-cold chain conditions, and is relatively stable.

[0009] Preferably, the final concentrations of the components of the lyophilized protective agent in the vaccine semi-finished product are: trehalose 5-15%, casein hydrolyzate 5-25%, sucrose 5-25%, PEG 1-3%, and gelatin 1-3%; the vaccine semi-finished product refers to the vaccine stock solution before lyophilization, and the matrix liquid for preparing the protective agent is water for injection, etc.

[0010] Preferably, the final concentration of each component of the lyoprotectant in the vaccine semi-finished product is: the final concentration of each component of the lyoprotectant in the vaccine semi-finished product is: trehalose 5-10%, casein hydrolyzate 10-25%, sucrose 5-15%, PEG 1.5%, and gelatin 1%.

[0011] In a second aspect, the present invention provides a method for preparing the above-mentioned lyoprotectant, characterized in that it comprises the following steps:

[0012] Dissolve sucrose and gelatin in the matrix solution and sterilize by high pressure to obtain solution A;

[0013] Dissolve trehalose, PEG and casein hydrolyzate in water, filter and sterilize to obtain solution B.

[0014] In a third aspect, the present invention provides a fish rhabdovirus attenuated vaccine, which is prepared from fish rhabdovirus virus liquid and the above-mentioned lyophilization protective agent.

[0015] Preferably, the virus strain in the virus liquid is perch rhabdovirus MsRV-0509, which has been deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: V202412.

[0016] In a fourth aspect, the present invention further provides a method for preparing the attenuated fish rhabdovirus vaccine, comprising the following steps:

[0017] Dissolve sucrose and gelatin in water and sterilize by high pressure to obtain solution A;

[0018] Dissolve trehalose, PEG and casein hydrolyzate in water, filter and sterilize to obtain solution B;

[0019] The A solution, the B solution and the fish rhabdovirus solution are mixed evenly to obtain a vaccine stock solution;

[0020] The vaccine stock solution is freeze-dried to obtain the fish rhabdovirus attenuated vaccine.

[0021] Preferably, the freeze drying conditions are: -50°C for 2h, -40°C for 24h, -25°C for 20h. Freeze drying has less moisture, the product has a loose appearance, and is easy to disperse in the solution.

[0022] Preferably, the vaccine stock solution is obtained by mixing evenly in a volume ratio of A solution: B solution: virus solution = 1:1:1.

[0023] Preferably, the virus titer in the virus stock solution is 5.01 gTCID50 / mL, which has a good protection rate, and the survival rate of fish after infection is above 80%.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention screens out the best freeze-dried protective agent formula through research and preparation of various freeze-dried protective agents, and prepares an effective freeze-dried vaccine. The product can be stored for a long time at 4°C, and the product is stable under short-term deviation from the storage conditions. The protection rate is good, and the survival rate of fish after infection can reach more than 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Comparison of viral titer test results of single-ingredient lyophilized protective agent stored at -20℃ for 12 months and at the initial stage;

[0027] Figure 2 Comparison of viral titer test results of single-ingredient lyophilized protective agent stored at 4℃ for 12 months and at the initial stage;

[0028] Figure 3 Comparison of virus titer test results of the compound lyophilized protective agent stored at -20℃ for 12 months and at the initial stage;

[0029] Figure 4 Comparison of virus titer test results of the compound freeze-dried protective agent stored at 4℃ for 12 months and at the initial stage;

[0030] Figure 5 These are the results of fish experiments. DETAILED DESCRIPTION

[0031] The lyophilized protective agent of the present invention can be used with various fish rhabdoviruses for preparing vaccines, and is not limited to the sea bass rhabdovirus MsRV-0509 in the following examples.

[0032] The present invention is described in detail below in conjunction with examples. It should be understood that the following examples are only used as explanations and illustrations of the present invention and do not limit the scope of the present invention in any way.

[0033] 1 Research Materials and Methods

[0034] 1.1 Cells and viruses

[0035] The seabass rhabdovirus MSRV-0509 has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: V202412. That is, the largemouth bass rhabdovirus was isolated and preserved by the laboratory of the fish disease department of the Pearl River Fisheries Research Institute. The relevant deposit certificate has been provided in the prior application 202410440997.6.

[0036] CPB cells: infectious spleen and kidney necrosis virus, which can be isolated in the laboratory or purchased. This example is isolated and stored in the laboratory of the fish disease room of the Pearl River Fisheries Research Institute.

[0037] The sea bass was purchased from Guangdong Dequan Fishery Development Co., Ltd.

[0038] 1.2 Virus proliferation

[0039] The MSRV virus was inoculated into CPB cells grown to a monolayer. After incubation for 1 hour, L15 culture medium with a final concentration of 2% fetal bovine serum was added and cultured in a 28°C incubator. When more than 90% of the cells showed cytopathic effects, the virus solution was harvested, the TCID50 of the virus was determined, and the virus was aliquoted and stored at low temperature for later use.

[0040] 1.3 Preparation of single lyophilized protective agent-vaccine stock solution

[0041] Use water for injection to prepare 1% skim milk powder solution, 2% gelatin solution and 8% sucrose solution by mass volume percentage respectively. After high-pressure sterilization, mix them evenly in a volume ratio of 1:1 of single protective agent: virus solution and then dispense them into vials.

[0042] 1.4 Preparation of compound freeze-dried protective agent-vaccine stock solution

[0043] Using water for injection as the matrix liquid, the concentrations of each component (mass volume percentage) in the vaccine stock solution were prepared in different proportions: sucrose 15-25%, trehalose 5-15%, PEG 1-3%, gelatin 1-3%, and a total of 12 compound lyoprotectants, T1 to T12.

[0044] The specific configuration method is:

[0045] Dissolve sucrose, gelatin, etc. in water for injection, sterilize under high pressure to obtain liquid A for use; dissolve trehalose, PEG, and casein hydrolyzate in water for injection, filter and sterilize to obtain liquid B for use; mix liquid A: liquid B: virus liquid in a volume ratio of 1:1:1 to obtain a vaccine stock solution, and fill the prepared vaccine stock solution into vials.

[0046] Table 1 Formula of compound lyophilized preparation

[0047]

[0048] 1.5 Determination of freeze-drying curve

[0049] In order to determine the optimal freeze-drying curve, the same batch of samples were freeze-dried with freeze-drying curves of -50℃ - 2h, -40℃ - 16h, -20℃ - 10h and -50℃ - 2h, -40℃ - 24h, -25℃ - 20h, and then the freeze-dried moisture content and other indicators were determined.

[0050] 1.6 Moisture determination of freeze-dried samples

[0051] According to the sixth edition of the Chinese Pharmacopoeia of Veterinary Medicine, the residual moisture of the freeze-dried sample was determined. The clean weighing bottle was dried for 2 hours. After cooling and weighing, the vaccine bottle with good vacuum was quickly opened, the product was poured into the weighing bottle, covered, and weighed on a balance. After weighing, the sample was placed in a vacuum drying oven and dried for 3 hours, and then the weighing bottle was taken out and weighed.

[0052] Residual moisture = (sample weight before drying - sample weight after drying) / sample weight before drying × 100%

[0053] 1.7 Heat resistance test of freeze-dried samples

[0054] The freeze-dried samples were stored at 26°C and 37°C, and samples were taken at 1, 3, 5, and 7 days to determine their TCID50.

[0055] 1.8 Storage period test of freeze-dried samples

[0056] The above freeze-dried samples were freeze-dried according to the optimal freeze-drying curve, and stored at 4°C and -20°C, respectively. The samples were taken to determine the TCID 50 .

[0057] 1.9 Fish immune protection experiment

[0058] The following fish experiments were conducted: (1) The above-mentioned freeze-dried vaccine T10 was dissolved in PBS and then 10 -5 TCID 50 After the fish are immersed in water for 1 hour, they are placed in the fish tank for feeding (T10); (2) The non-freeze-dried vaccine is adjusted to 10 -5 TCID 50 ; After the fish were immersed in immunization for 1 hour, they were placed in a fish tank and fed (MSRV-0509); (3) Using PBS as the control, after the fish were immersed in immunization for 1 hour, they were placed in a fish tank and fed (PBS); 21 days later, the virus was challenged and the immune protection was measured.

[0059] 2. Results

[0060] 2.1 Determination of freeze-drying curve

[0061] Freeze-drying was carried out using different procedures. After freeze-drying, the appearance was observed and the residual moisture was measured. Finally, -50℃2h, -40℃24h and -25℃20h were determined to be the best freeze-drying curves.

[0062] The following 2.2-2.5 experimental results are all test results under the optimal freeze-drying curve.

[0063] 2.2 Appearance inspection

[0064] The appearance of each single and compound sample after freeze-drying is shown in Tables 2 and 3. The morphology of each freeze-dried product is different. After being dissolved in water, each formula is clear.

[0065] Table 2 Single lyophilization protective agent

[0066]

[0067] Table 3 Compound lyophilization protective agent

[0068]

[0069] 2.3 Heat resistance test of freeze-dried samples

[0070] The results of the heat-resistant storage experiments at 37°C and 26°C are shown in Tables 4 and 5, respectively. The results confirm that when stored at 26°C, the titers of most samples of compound lyophilized protective agents can be kept relatively stable within 7 days, with a drop in titer of less than 1, but single-formula lyophilized protective agents are relatively unstable, with a relatively large drop in titer. The effect of high temperature at 37°C is not ideal, and the vaccine is relatively stable under short-term storage conditions without cold chain.

[0071] Table 4 Heat resistance test at 37℃

[0072]

[0073] Table 5 Heat resistance test at 26℃

[0074]

[0075] 2.4 Moisture determination of freeze-dried samples

[0076] The residual moisture content of freeze-dried samples was measured according to the sixth edition of the Chinese Pharmacopoeia of Veterinary Medicine. Regardless of whether it was a single or compound prescription, the residual moisture content of each sample was less than 4%, which met the requirements for biological products.

[0077] Table 6 Single freeze-drying protective agent

[0078]

[0079] Table 7 Compound lyophilization protective agent

[0080]

[0081] 2.5 Storage period test of freeze-dried samples

[0082] The above freeze-dried samples were freeze-dried according to the optimal freeze-drying curve, stored at 4°C and -20°C, and samples were taken regularly to determine the TCID 50 , the results are shown in Tables 8 and 9, Figure 1-4 The results also give the comparison of virus titers of single and compound lyophilized protective agents at 12 months and at the beginning. The results show that the long-term storage effect at -20℃ is good, but the virus titer of single lyophilized protective agents stored at 4℃ for a long time decreases rapidly and is not suitable for use; compound lyophilized protective agent formulas T1, T9, T10, and T11 have good protective effects when stored at 4℃ for a long time.

[0083] Table 8 Virus titer during storage period of single lyophilized protective agent (TCID 50 )

[0084]

[0085] Table 9 Virus titer during the storage period of compound lyophilized protective agent (TCID 50 )

[0086]

[0087] 1.9 Fish immune protection experiment

[0088] The results of the fish experiment are as follows Figure 5 As shown, the results confirmed that the vaccine T 10 The liquid vaccine and the virus titer lgTCID50 / mL had a good protection rate after immunization. The survival rate of fish after infection was over 80%, while the survival rate of the control group was only 30%. The results showed that the freeze-dried vaccine prepared by the freeze-dried protective agent of the present invention had a protective effect.

[0089] In summary, this study first screened out the best freeze-drying curve through the preparation of various single and compound freeze-dried protective agents: -50℃2h, -40℃24h, -25℃20h, and then compared the performance tests of different formulas. It can be seen that the heat resistance of the compound freeze-dried agent is significantly better than that of the single formula, and then combined with the freeze-dried storage period test, the best compound freeze-dried agent formula, such as T1, T9, T10, T11, etc., was screened out. Long-term storage at 4℃ has a good protective effect. After each portion is stored for 12 months, the virus titer does not drop by more than 0.5, which can be used to prepare effective freeze-dried vaccines and is stable under short-term deviation storage conditions. Finally, through the fish immunization experiment, it is also verified that the freeze-dried vaccine containing the freeze-dried protective agent of the embodiment of the present invention has a good protective effect. After immunization with a virus titer of lgTCID50 / mL, the survival rate of the fish after the virus attack can reach more than 80%.

[0090] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0091] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. The use of a lyophilized protective agent in the preparation of an attenuated fish rhabdovirus vaccine, characterized in that: The lyophilization protective agent includes trehalose, casein hydrolyzate, sucrose, PEG and gelatin; The final concentrations of the components of the lyophilized protective agent in the vaccine semi-finished product are as follows, in terms of mass volume percentage: trehalose 5-15%, casein hydrolyzate 5-25%, sucrose 5-25%, PEG 1-3%, and gelatin 1-3%; the vaccine semi-finished product refers to the vaccine stock solution before lyophilization.

2. The use according to claim 1, characterized in that: The final concentrations of the components of the lyophilized protective agent in the vaccine semi-finished product are: trehalose 5-10%, casein hydrolyzate 10-25%, sucrose 5-15%, PEG 1.5%, and gelatin 1%.

3. The use according to any one of claims 1-2, characterized in that: The preparation method of the lyophilized protective agent comprises the following steps: Dissolve sucrose and gelatin in water and sterilize by high pressure to obtain solution A; Dissolve trehalose, PEG and casein hydrolyzate in water, filter and sterilize to obtain solution B.

4. A fish rhabdovirus attenuated vaccine, characterized in that: The invention is prepared from fish rhabdovirus liquid and the freeze-dried protective agent described in any one of claims 1 to 3.

5. The attenuated fish rhabdovirus vaccine according to claim 4, characterized in that: The virus strain in the virus liquid is perch rhabdovirus MsRV-0509, which has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: V202412.

6. The method for preparing the attenuated fish rhabdovirus vaccine according to any one of claims 4 to 5, characterized in that: The steps include: Dissolve sucrose and gelatin in water and sterilize by high pressure to obtain solution A; Dissolve trehalose, PEG and casein hydrolyzate in water, filter and sterilize to obtain solution B; The A solution, the B solution and the fish rhabdovirus solution are mixed evenly to obtain a vaccine stock solution; The vaccine stock solution is freeze-dried to obtain the fish rhabdovirus attenuated vaccine.

7. The method for preparing the attenuated fish rhabdovirus vaccine according to claim 6, characterized in that: The freeze-drying conditions are: -50°C for 2h, -40°C for 24h, and -25°C for 20h.

8. The method for preparing the attenuated fish rhabdovirus vaccine according to claim 7, characterized in that: The vaccine stock solution was obtained by mixing evenly in a volume ratio of liquid A: liquid B: virus solution = 1:1:

1.

9. The method for preparing the attenuated fish rhabdovirus vaccine according to claim 8, wherein the virus titer in the virus liquid is 5.0 lgTCID50 / mL.

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