A buffer for improving the stability of foot-and-mouth disease vaccine antigen

Abstract

The application provides a buffer for improving the stability of foot-and-mouth disease vaccine antigen, which contains milk serum, sodium chloride, sorbitol, glycine, K2HPO4.3H2O and KH2PO4. The buffer has the effects of improving the thermal stability of 146S antigen and obviously inhibiting the degradation of 146S antigen under the conditions of 4 DEG C and 37 DEG C. The antigen resuspended by the buffer and the prepared vaccine can be stored for a long time under the condition of 4 DEG C. In addition, the buffer is easy to prepare and dissolve, can be used as a medium for resuspending FMD antigen, is a good thermal stability protective agent for FMD antigen and vaccine, can be used in large-scale production, has strong practicability, can obviously reduce the degradation rate of effective antigen in foot-and-mouth disease vaccine under storage conditions and extreme conditions, and obviously improves the product quality.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, and relates to a buffer solution for improving the stability of foot-and-mouth disease vaccine antigens. Background Technology

[0002] Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious animal disease with a rapid onset. Susceptible animals are cloven-hoofed animals, with cattle, sheep, and pigs exhibiting primarily vesicles on the oral epithelium and hoof skin. FMD spreads extremely rapidly; contact between uninfected and infected animals is highly likely to lead to infection. If one animal on a farm is infected, all other farmed animals face significant threats, resulting in substantial economic losses for the farm. Young animals infected often die from myocarditis. FMDV is prevalent worldwide, significantly reducing the export volume of meat products from susceptible animals and hindering international economic exchanges. Therefore, countries worldwide place great importance on FMD prevention and control; it is considered one of the most difficult infectious diseases to eradicate globally. Currently, with the increasing frequency of international trade, the total import and export volume of animal-derived food products is also rising sharply, which makes the prevention and control of FMD even more important. Countries around the world have paid a considerable price to control FMD.

[0003] FMDV consists of a single-stranded positive-sense genomic ribose nucleic acid (RNA) and four structural proteins. FMDV contains small amounts of VP2 and VP4 cleavage precursors, as well as a genome-associated protein (Vpg) consisting of 23–24 amino acid residues, which covalently binds to the 5' end of the viral genomic RNA. FMDV's genome structure is similar to other small RNA viruses, containing approximately 8,500 nucleotides. Its genomic RNA is translated through a long open reading frame (ORF) into a polyprotein. This polyprotein undergoes a series of translational and fission processes to form intermediate products and various complete structural and non-structural proteins.

[0004] Antigen dilution technology, as one of the downstream production technologies for vaccines, is extremely rare in China. Current diluents fail to achieve ideal antigen protection, resulting in rapid loss of effective antigen content within the 12-month shelf life (degradation rate greater than 50%). Improving and maintaining the long-term stability of antigen 146S in inactivated foot-and-mouth disease vaccines has been a key technical challenge for manufacturers. Currently, all vaccine manufacturers are prioritizing antigen diluents in their research and development to enhance product competitiveness. Summary of the Invention

[0005] The purpose of this invention is to provide a buffer solution that improves the stability of foot-and-mouth disease vaccine antigens.

[0006] A buffer solution for improving the stability of foot-and-mouth disease vaccine antigens, wherein the solute of the buffer solution comprises the following components at the following concentrations:

[0007] The emulsion concentration is 55-65 mL / L, sodium chloride concentration is 7-9 g / L, sorbitol concentration is 25-30 g / L, glycine concentration is 0.4-0.5 g / L, K2HPO4·3H2O concentration is 4.32 g / L, and KH2PO4 concentration is 0.144 g / L.

[0008] The solute in the milk emulsion contains the following components at the following concentrations: sodium chloride 6.8 g / L, potassium chloride 0.4 g / L, magnesium sulfate 0.2 g / L, sodium dihydrogen phosphate 0.16 g / L, anhydrous glucose 1 g / L, anhydrous calcium chloride 0.2 g / L, and hydrolyzed milk protein 5 g / L. The above components are dissolved in water, and the pH of the milk emulsion is adjusted to 7.6-7.8 with sodium bicarbonate.

[0009] The pH of the buffer solution is 7.9-8.1 (preferably 8.0). The solvent of the buffer solution is water. The buffer solution is a phosphate buffer. The added component of the buffer solution is an emulsion.

[0010] The preferred solute concentrations of each component in the buffer solution of this invention are:

[0011] The emulsion solution was 60 mL / L, sodium chloride 8 g / L, sorbitol 30 g / L, glycine 0.4 g / L, K2HPO4·3H2O 4.32 g / L, and KH2PO4 0.144 g / L.

[0012] The solute in the milk emulsion contains the following components at the following concentrations: sodium chloride 6.8 g / L, potassium chloride 0.4 g / L, magnesium sulfate 0.2 g / L, sodium dihydrogen phosphate 0.16 g / L, anhydrous glucose 1 g / L, anhydrous calcium chloride 0.2 g / L, and hydrolyzed milk protein 5 g / L. The above components are dissolved in water, and the pH of the milk emulsion is adjusted to 7.6-7.8 with sodium bicarbonate.

[0013] The buffer solution of the present invention can be used as a medium for resuspending FMD antigens and is also an excellent protectant for the thermal stability of FMD antigens and vaccines.

[0014] Improving and maintaining the long-term stability of antigen 146S in inactivated foot-and-mouth disease (FMD) vaccines has always been a key technical challenge for manufacturers. Vaccine quality is crucial to a company's future prospects, and this invention can significantly enhance product competitiveness. With the implementation of this invention, the company's FMD vaccine maintains a leading position in all aspects of quality, possessing strong commercial appeal and guaranteed immunization efficacy. This invention focuses on improving antigen stability and extending shelf life, allowing for the addition of less antigen to the vaccine formulation to reduce product costs, thus possessing significant market value.

[0015] The advantages of this invention over the prior art are:

[0016] The buffer solution of this invention improves the thermostability of 146S antigen and significantly inhibits its degradation at 4°C and 37°C. At 4°C, the antigen resuspended in the buffer solution and the prepared vaccine can be stored for a long time. Furthermore, this buffer solution is easy to prepare and dissolve, serving both as a medium for resuspending FMD antigen and as an excellent thermostability protectant for FMD antigen and vaccine. It is suitable for large-scale production, highly practical, and can significantly reduce the degradation of effective antigen under storage and extreme conditions, thereby improving product quality.

[0017] The 146S decrease in buffer-prepared vaccines under accelerated testing conditions (37℃ water bath for 96 hours) should not exceed 5% (the loss in accelerated testing with current buffer solutions is >40%); the 146S decrease in buffer-prepared vaccines after 12 months of storage should be ≤5% (the 146S decrease in current buffer-prepared vaccines after 12 months of storage should be >60%); compared with existing vaccines, the antibody levels and potency of buffer-prepared vaccines should not be lower than existing levels. Detailed Implementation

[0018] The invention will be further described below through specific implementations.

[0019] Example 1: Preparation of Buffer Solution

[0020] To improve the stability of foot-and-mouth disease (FMD) antigens and vaccines, an orthogonal experimental design L18(3) was used. 5 The addition amounts of emulsion, sodium chloride, sorbitol, and glycine, as well as the pH of the buffer solution (adjusted by the addition amounts of K2HPO4·3H2O and KH2PO4), were set as factors in Table 1. The storage time when the foot-and-mouth disease antigen or vaccine degraded to 0% at 37°C was used as the evaluation index, and 0.02 mol / L PBS buffer (pH 8.0) was used as a control to screen chemical reagents with the best thermostability against the FMD146S antigen.

[0021] The specific contents of each component in the orthogonal experiment are shown in Table 2.

[0022]

[0023]

[0024]

[0025] L18(3) was designed using orthogonal experimental design. 5 The pH value of the solution and the mass concentrations of four components—emulsion, sodium chloride, sorbitol, and glycine—were used as factors of investigation. The storage time of the FMD antigen-prepared vaccine at 37 ℃ until the 146S antigen degradation reached 0% was used as the evaluation index. The results are shown in Table 2. Through orthogonal array range analysis, A2B2C2D3E1 was found to be the optimal combination, which yielded the optimal 1-liter diluent formulation, as shown in Table 4.

[0026]

[0027] Buffer solution preparation method: Add the 6 components to a beaker in any order, add water for injection to dissolve them completely, and bring the volume to 1000 mL.

[0028] Example 2: Accelerated vaccine preparation test using diluent (37°C water bath for 96 hours)

[0029] Vaccine preparation method using buffer solution: Add antigen to the above buffer solution at a final concentration of 30 μg / ml (146S) to form the aqueous phase of the vaccine. Emulsify the aqueous phase and SEPPIC ISA 206 adjuvant at a mass ratio of 1:1 (aqueous phase:oil phase). Preheat the aqueous and oil phases to 32℃±1℃ in a 35℃ constant temperature water bath (shaking several times during 10 minutes to ensure uniform temperature). Then, add the aqueous phase antigen to the oil phase and start timing at 350 RPM, stirring for 10 minutes to complete emulsification. After emulsification, mix the vaccine thoroughly, dispense into containers, and store at 2℃-8℃. The control experiment uses fresh PBS buffer (0.02 mol / L PBS, pH=8.0).

[0030] In the accelerated vaccine test (37°C water bath for 96 hours) using the buffer solution, the 146S antigen decreased by no more than 5%, while the control diluent showed a loss of >40% in the accelerated test (as shown in Table 5). This indicates that the diluent of the present invention has the effect of improving the thermostability of the 146S antigen.

[0031]

[0032] Example 3 Shelf life test of buffer-prepared vaccines

[0033] Vaccine preparation method using buffer solution: Add antigen to the above buffer solution at a final concentration of 30 μg / ml (146S) to form the aqueous phase of the vaccine. Emulsify the aqueous phase and SEPPIC ISA 206 adjuvant at a mass ratio of 1:1 (aqueous phase:oil phase). Preheat the aqueous and oil phases to 32℃±1℃ in a 35℃ constant temperature water bath (shaking several times during 10 minutes to ensure uniform temperature). Then, add the aqueous phase antigen to the oil phase and start timing at 350 RPM with stirring to emulsify. After emulsification, mix the vaccine thoroughly, dispense into containers, and store at 2℃-8℃. The control experiment uses fresh PBS buffer (0.02 mol / L PBS, pH 8.0).

[0034] The 146S antigen degradation rate of the vaccine prepared with the buffer solution was ≤5% after 4°C storage for 12 months, while the 146S antigen degradation rate of the control buffer solution vaccine was >60% after 12 months storage (as shown in Table 6). This indicates that the buffer solution of the present invention has a significant inhibitory effect on the degradation of the 146S antigen.

[0035]

[0036] Example 4 Immunotherapy Test

[0037] Vaccine preparation method using buffer solution: Add antigen 146S at a concentration of 30 μg / ml to the above buffer solution to form the aqueous phase of the vaccine. Emulsify the aqueous phase and SEPPIC ISA 206 adjuvant at a mass ratio of 1:1 (aqueous phase:oil phase). Preheat the aqueous and oil phases to 32℃±1℃ in a 35℃ constant temperature water bath (shaking several times during 10 minutes to ensure uniform temperature). Then, add the antigen from the aqueous phase to the oil phase and start timing at 350 RPM with stirring to emulsify. After emulsification, mix the vaccine thoroughly, dispense into containers, and store at 2℃-8℃. The control experiment uses fresh PBS buffer (0.02 mol / L PBS, pH 8.0).

[0038] Preparation of feeder pigs: 34 healthy pigs weighing approximately 40 kg and around four months of age, and subjected to liquid-phase blocking ELISA.

[0039] The kit diagnoses serum neutralizing antibodies at a ratio not exceeding 1:8 and a negative result for the 3ABC monoclonal antibody test. Supplied by Yongjing Experimental Animal Base.

[0040] Method for detecting antibody levels in primary immunized animals (pigs): Diluted vaccine was administered to the aforementioned growing pigs in three dose groups: 1 dose, 1 / 3 dose, and 1 / 9 dose. Fifteen pigs were immunized in each dose group. 28 days after immunization, blood was aseptically collected from each pig to separate serum, and antibody titers were measured using a liquid-phase blocking ELISA kit. Liquid-phase blocking ELISA antibody titer ≥2. 6 The results were determined according to the standards and requirements of the "2013 National Animal Disease Monitoring and Epidemiological Survey Plan" [Agricultural Medical Document

[2013] No. 9]. See Table 7 for the results.

[0041] Efficacy testing method: 28 days after immunization, 10 pigs from each dose group were randomly selected, along with 2 control pigs under the same conditions, and challenged with 1000 ID50 / 2.0ml of O / Mva98 / BY / strain mouse virus; another 10 pigs, along with 2 control pigs under the same conditions, were challenged with 1000 ID50 / 2.0ml of A / QH strain mouse virus; observation continued for 10 days. The PD50 of the vaccine was calculated according to the Reed-Muench method based on the protection number of immunized pigs. A PD50 / dose ≥ 6 was determined according to the standards required by the "Notice of the General Office of the Ministry of Agriculture on Improving the Quality Standards of Foot-and-Mouth Disease Vaccines" [Agricultural Office Medical

[2013] No. 32]. Results are shown in Table 7.

[0042] The antibody levels and potency of vaccines prepared with antigen diluents are no lower than those of existing vaccines, as compared to existing vaccines.

[0043] level.

[0044]

[0045] The above embodiments 1 to 4 are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any changes, substitutions, or combinations made without departing from the essential principles or composition of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A buffer solution for improving the stability of foot-and-mouth disease vaccine antigens, characterized in that: The solute in this buffer solution contains the following components at the following concentrations: The emulsion concentration is 55-65 mL / L, sodium chloride concentration is 7-9 g / L, sorbitol concentration is 25-30 g / L, glycine concentration is 0.4-0.5 g / L, K2HPO4•3H2O concentration is 4.32 g / L, and KH2PO4 concentration is 0.144 g / L. The solute in the milk emulsion contains the following components at the following concentrations: sodium chloride 6.8 g / L, potassium chloride 0.4 g / L, magnesium sulfate 0.2 g / L, sodium dihydrogen phosphate 0.16 g / L, anhydrous glucose 1 g / L, anhydrous calcium chloride 0.2 g / L, and hydrolyzed milk protein 5 g / L. The above components are dissolved in water, and the pH of the milk emulsion is adjusted to 7.6-7.8 with sodium bicarbonate. The pH of the buffer solution is 7.9 to 8.

1.

2. The buffer solution for improving the stability of foot-and-mouth disease vaccine antigens according to claim 1, characterized in that: The solvent for the buffer solution is water.

3. The buffer solution for improving the stability of foot-and-mouth disease vaccine antigens according to claim 1, characterized in that: The solute in this buffer solution contains the following components at the following concentrations: The emulsion solution contains 60 mL / L of sodium chloride, 8 g / L of sorbitol, 30 g / L of glycine, 0.4 g / L of K2HPO4•3H2O, 4.32 g / L of KH2PO4, and 0.144 g / L of KH2PO4. The solute in the milk emulsion contains the following components at the following concentrations: sodium chloride 6.8 g / L, potassium chloride 0.4 g / L, magnesium sulfate 0.2 g / L, sodium dihydrogen phosphate 0.16 g / L, anhydrous glucose 1 g / L, anhydrous calcium chloride 0.2 g / L, and hydrolyzed milk protein 5 g / L. The above components are dissolved in water, and the pH of the milk emulsion is adjusted to 7.6-7.8 with sodium bicarbonate.

4. The buffer solution for improving the stability of foot-and-mouth disease vaccine antigens according to claim 1, characterized in that: The pH of the buffer solution is 8.

0.

5. The application of the buffer solution for improving the stability of foot-and-mouth disease vaccine antigen according to any one of claims 1-4 in the preparation of resuspension media for foot-and-mouth disease antigen, characterized in that: The antigen is foot-and-mouth disease inactivated vaccine antigen 146S.

6. The application of the buffer solution for improving the stability of foot-and-mouth disease vaccine antigen according to any one of claims 1-4 in the preparation of foot-and-mouth disease antigen and vaccine heat stability protectant, characterized in that: The antigen is foot-and-mouth disease inactivated vaccine antigen 146S.

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