Vaccine stabilizer and application

Abstract

The invention provides a vaccine stabilizer, the stabilizer comprises carbomer and a polysaccharide immunopotentiator, and the invention also provides a preparation method of an inactivated vaccine containing the stabilizer. The vaccine prepared by the method can effectively reduce immune side reactions, reduce immune stimulation and improve the immune effect of the vaccine.

Description

Technical Field

[0001] The present invention belongs to the field of vaccine preparation, and specifically relates to a vaccine stabilizer and application thereof. Background Art

[0002] Commonly used adjuvant formulations for inactivated vaccines include oil-in-water, water-in-oil, water-in-oil-in-water, and polymer adjuvants. Oil-in-water adjuvant vaccines have low viscosity, are easy to inject, are less irritating to animals, and are more in line with animal welfare, but vaccine immunity lasts for a short time and requires multiple immunizations. The antigens of oil-in-water emulsion adjuvants are stored for a long time at the injection site and can continuously stimulate the body, promote the aggregation and proliferation of macrophages, lymphocytes, etc., and enhance the immune protection effect, but the viscosity is relatively high, which is not conducive to injection and is highly irritating to animals after injection; the water-in-oil-in-water type is between the water-in-oil type and the oil-in-water type. Because the outer layer is the water phase, the stimulating effect on the body is significantly weakened. At the same time, the emulsion has low viscosity, is easy to inject, and is less irritating to animals; the inner layer is still the oil-in-water type, which is equivalent to the oil-in-water adjuvant in terms of body stimulation and antibody production. The water-in-oil-in-water emulsion comprises an inner water phase, an intermediate oil phase for encapsulating the inner water phase, and an outer water phase for encapsulating the intermediate oil phase. The encapsulation technology is complex, the preparation process is cumbersome, and the stability of the emulsion is easily affected. Summary of the invention

[0003] In view of this, the present invention aims to overcome the defects in the prior art and provide a vaccine stabilizer that can improve the stability of water-in-oil-in-water adjuvants and enhance the immune effect of the vaccine.

[0004] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0005] A vaccine stabilizer comprises: carbomer and a polysaccharide immunopotentiator.

[0006] Preferably, the carbomer is carbomer 941 or carbomer TR1.

[0007] Preferably, the polysaccharide immunoenhancer is astragalus polysaccharide or β-glucan or wolfberry polysaccharide.

[0008] The application of the vaccine stabilizer in vaccine preparation.

[0009] The preparation method of the vaccine comprises the following steps:

[0010] 1) Preparation of aqueous phase: Take 100 mL of inactivated antigen, add a carbomer solution with a final concentration of 0.075% (V / V), add a polysaccharide immunopotentiator solution with a final concentration of 3% (V / V), mix thoroughly, stir at 300-800 r / min for 2-5 minutes, and preheat in a water bath at 28-32°C;

[0011] 2) Preparation of oil phase: Take the biphasic adjuvant, mix thoroughly and preheat in a water bath at 28-32°C;

[0012] 3) Mix the preheated water phase and oil phase at a ratio of 1:0.8 to 1:1.2 (w / w) for 10 to 20 minutes;

[0013] 4) Then place in a cold water bath at 4-8°C for 2-5 hours, avoid moving and stirring, and divide into quantitative portions.

[0014] Step 1) The preparation method of the carbomer solution is as follows: weigh 0.30 g of carbomer, dissolve it in 100 mL of purified water, stir it thoroughly, swell it overnight, adjust the pH value to 7.0-7.2 with 1.0 mol / L sodium hydroxide, dilute it 2 times with 0.1 mol / L PBS, and sterilize it by high pressure at 121° C. for 20-30 minutes.

[0015] The preparation method of the polysaccharide immunopotentiator solution described in step 1) is as follows: weigh 1.0 g of astragalus polysaccharide, dissolve it in 100 mL of purified water, stir it thoroughly to dissolve the polysaccharide, and sterilize it under high pressure at 116° C. for 20 to 30 minutes.

[0016] The antigen of the vaccine is any one of porcine circovirus, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The stabilizer of the present invention contains carbomer in its formula. The carbomer molecule contains an acid group. After being neutralized by an alkaline substance, the molecular chain diffuses, expands and swells, and the adsorbed antigen molecules are permanently wrapped in the skeleton system, thereby effectively increasing the stability of the antigen.

[0019] The polysaccharide enhancer in the formula of the stabilizer of the present invention plays a significant role in promoting cellular immunity, humoral immunity and non-specific immunity, and effectively increases the immune effect of the vaccine.

[0020] In the disease prevention and control strategy where prevention is more important than cure, the use of biphasic vaccines can not only reduce the immune side effects of oil adjuvant vaccines and reduce immune stimulation, but also improve the immune effect of vaccines, effectively improving the economic effects of animal husbandry.

[0021] Carbomer is an acrylic cross-linked resin, and its main functions are thickening, suspending and emulsifying. Carbomer can be divided into three types according to its viscosity: low viscosity, medium viscosity and high viscosity. Carbomer 941 has long flowability, good transparency and low viscosity of the colloid formed; it has good ion resistance in the ion system; and it has high emulsion stabilization effect.

[0022] Polysaccharide immunoenhancers have obvious immunoenhancing effects. Among them, Astragalus polysaccharide is one of the main active ingredients of Astragalus. It is a polysaccharide obtained through high-tech extraction and separation of Astragalus. It can promote the proliferation and differentiation of lymph node B cells and the formation of plasma cell antibodies, and significantly enhance humoral immunity and nonspecific immune function. DETAILED DESCRIPTION

[0023] The following specific descriptions are exemplary and are intended to provide further explanation of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention.

[0024] The experimental methods used in the following experimental examples are conventional methods unless otherwise specified, and the reagents and drugs are from Sigma-Aldrich.

[0025] Example 1 Preparation method of each component in the stabilizer

[0026] Component 1: Weigh 0.30 g of Carbomer 941, dissolve in 100 mL of purified water, stir thoroughly, and swell overnight. Adjust the pH value to 7.0-7.2 with 1.0 mol / L sodium hydroxide, then add an equal volume of 0.1 mol / L PBS, mix thoroughly, and obtain a 0.15% Carbomer stock solution. Sterilize the Carbomer stock solution at 121°C for 20 minutes for later use.

[0027] Component 2: Weigh 1.0 g of Astragalus polysaccharide, dissolve it in 100 mL of purified water, stir thoroughly to dissolve the polysaccharide, and sterilize under high pressure at 116° C. for 20 to 30 minutes to obtain a 1% polysaccharide mother solution.

[0028] Example 2 Preparation of a biphasic adjuvant vaccine for pseudorabies virus

[0029] 1. Proliferation of Pseudorabies Virus Antigen

[0030] Take ST suspension cells, count the cells, and observe the cell status. When the cell density is ≥8.0×10 6 When the number of cells / mL is 2.0-4.0×10 6 cells / mL, inoculate PRV virus at 5% to 10% of the inoculum, culture for 30 to 40 hours, harvest when the cell viability is less than 80%, and freeze-thaw repeatedly once at -40°C for later use.

[0031] 2. Purification and inactivation of pseudorabies virus antigen

[0032] The obtained PRV antigen was centrifuged at 6000-8000r / min for 10 minutes, the supernatant was filtered with a hollow fiber filter column with a pore size of 0.45μm, and then concentrated 10-15 times by an ultrafiltration concentration device of 100-300KD, purified by a molecular sieve gel process, and the purified antigen was inactivated at 35-37°C for 20-24 hours with BEI at a final concentration of 0.1%. The virus content before inactivation was ≥10 8.7 TCID 50 / mL.

[0033] 3. Vaccine preparation

[0034] 3.1 Improved vaccine preparation method with stabilizer added

[0035] 1) Preparation of antigen aqueous phase: 100 mL of inactivated porcine pseudorabies antigen was added to stabilizer component 1 of Example 1 to a final concentration of 0.075% (V / V), and stabilizer component 2 of Example 1 was added to a final concentration of 3%; the components were mixed according to the proportion, stirred at 300-800 r / min for 2-5 minutes to obtain an antigen aqueous phase, and preheated in a water bath at 28-32° C.;

[0036] 2) Preparation of the oil phase: Take a biphasic adjuvant (such as Seppic 206, 201), mix thoroughly, and place in a 28-32°C water bath to preheat to the water bath temperature, which is the oil phase;

[0037] 3) Vaccine preparation: The preheated water phase and oil phase were mixed in a 1:1 (w / w) ratio, premixed using a magnetic stirring device for 15 minutes, placed in a cold water bath at 4-8°C for 3 hours to avoid movement and stirring, and packaged in a quantitative manner.

[0038] 3.2. Preparation of conventional vaccines

[0039] 1) Preparation of antigen aqueous phase: Take 100 mL of inactivated antigen, add sterile PBS, stir at 300-800 r / min for 2-5 minutes to obtain antigen aqueous phase, and preheat in a water bath at 28-32°C;

[0040] 2) Preparation of oil phase: Take the biphasic adjuvant, mix thoroughly and place in a water bath at 28-32°C to preheat to the water bath temperature, which is the oil phase;

[0041] 3) Vaccine preparation: The preheated water phase and oil phase were mixed in a 1:1 (w / w) ratio, premixed using a magnetic stirring device for 15 minutes, placed in a cold water bath at 4-8°C for 3 hours to avoid movement and stirring, and packaged in a quantitative manner.

[0042] Example 3 Vaccine stability comparison test

[0043] The improved and conventional vaccines prepared in Example 2 were placed at 25°C and 4°C for observation, respectively. The stability of the vaccine was observed by regular sampling (test results are shown in Tables 1 and 2). The test results show that the stabilizer can significantly increase the storage time of the biphasic adjuvant vaccine at 25°C and 4°C.

[0044] Table 1 Stability observation days at 25℃

[0045]

[0046] Table 2 Stability observation days at 4℃

[0047]

[0048]

[0049] “-” indicates a milky white uniform emulsion.

[0050] “-+” means that there is a trace amount of oil phase precipitated on the upper layer of the vaccine liquid / or a trace amount of water phase stratified at the bottom.

[0051] “+” indicates that the vaccine emulsion is clearly stratified and there is an aqueous phase or precipitate at the bottom.

[0052] Example 4 Immune efficacy test of pseudorabies vaccine in pigs

[0053] 1. Vaccine immunization: 15 piglets aged 21 to 28 days were selected, including pigs negative for pathogens such as classical swine fever virus, African swine fever, porcine reproductive and respiratory syndrome virus, and mycoplasma suis, and negative for pseudorabies gB antibody; they were randomly divided into 3 groups, 5 pigs in each group, and the first group was vaccinated with 1 dose of improved vaccine in the neck muscle; the second group was vaccinated with 1 dose of conventional vaccine in the neck muscle, and the third group was used as a blank control (i.e., non-immunized group). Blood was collected from each group of piglets before immunization and on the 21st and 28th days after immunization, and serum was separated to detect the level of neutralizing antibodies.

[0054] 2. Virus challenge: All piglets were challenged with PRV 2.0 mL (containing 10 6.5 TCID 50 , 1.0 mL in each nostril) and observed continuously for 14 days after the challenge. All groups were tested for temperature changes, clinical symptoms, and viral load of nasal swabs (3, 5, and 7 days after the challenge) from 1 to 14 days.

[0055] 3. Neutralizing antibody test: Conduct according to the notes of the current "Chinese Pharmacopoeia of Veterinary Medicine".

[0056] 4. Observation after attack:

[0057] A. Temperature symptoms: The body temperature of non-immunized piglets increases (≥40.5℃) and should last for at least 3 days;

[0058] B: The non-immune group showed obvious respiratory and digestive tract symptoms, and in severe cases, neurological symptoms or even death;

[0059] 5. Nasal swab virus detection: refer to Liaoning Provincial Landmark DB21 / T 3273-2020 "TaqMan Real-time Fluorescence Quantitative PCR Identification Method for Pseudorabies Virus Wild Strain and gE Gene Deficient Vaccine Strain".

[0060] The results of the challenge protection test show that the neutralizing antibody of the improved vaccine group is significantly better than that of the conventional vaccine group (see Table 3 for test results); the excretion rate of the improved vaccine group after challenge is lower than that of the conventional vaccine group; the overall immune effect of the improved vaccine group (5 / 5 protection) is better than that of the conventional vaccine group (5 / 5 protection), while the morbidity rate of the non-immune group after challenge is 5 / 5, and 4 / 5 deaths (see Table 4 for test results). Therefore, it can be seen that the addition of stabilizers can significantly improve the neutralizing antibody level of the biphasic adjuvant vaccine after immunization, can effectively protect piglets, and significantly improve the vaccine immune efficacy test.

[0061] Table 3 Neutralizing antibody assay results

[0062]

[0063]

[0064] Table 4 Comparison results after virus attack

[0065]

[0066] Note: “-” indicates no clinical symptoms; “+” indicates clinical symptoms; Ct>38 is negative, Ct value ≤36 is positive, and 36<Ct≤38 is suspicious.

[0067] In the examples of this experiment, pseudorabies virus was used as an example to verify the stability and immune protection efficacy of the vaccine after the addition of stabilizer. At the same time, porcine circovirus, pseudorabies virus, porcine parvovirus, porcine blue ear virus, and classical swine fever virus were also verified one by one according to this method. Their stability and immune protection were stronger than those of conventional vaccines without stabilizers, which are not described in detail here.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A vaccine stabilizer, characterized in that The stabilizer comprises: carbomer and polysaccharide immunopotentiator.

2. The vaccine stabilizer according to claim 1, characterized in that The carbomer is carbomer 941 or carbomer TR1.

3. The vaccine stabilizer according to claim 1, characterized in that The polysaccharide immunoenhancer is astragalus polysaccharide or β-glucan or wolfberry polysaccharide.

4. Use of the vaccine stabilizer as described in claims 1-3 in vaccine preparation.

5. Use of the stabilizer according to claim 4 in vaccine preparation, characterized in that: The preparation method of the vaccine comprises the following steps: 1) Preparation of aqueous phase: Take 100 mL of inactivated antigen, add a carbomer solution with a final concentration of 0.075% (V / V), add a polysaccharide immunopotentiator solution with a final concentration of 3% (V / V), mix thoroughly, stir at 300-800 r / min for 2-5 minutes, and preheat in a water bath at 28-32°C; 2) Preparation of oil phase: Take the biphasic adjuvant, mix thoroughly and preheat in a water bath at 28-32°C; 3) Mix the preheated water phase and oil phase at a ratio of 1:0.8 to 1:1.2 (w / w) for 10 to 20 minutes; 4) Then place in a cold water bath at 4-8°C for 2-5 hours, avoid moving and stirring, and divide into quantitative portions.

6. The use of the vaccine stabilizer according to claim 4 in vaccine preparation, characterized in that: Step 1) The preparation method of the carbomer solution is as follows: weigh 0.30 g of carbomer, dissolve it in 100 mL of purified water, stir it thoroughly, swell it overnight, adjust the pH value to 7.0-7.2 with 1.0 mol / L sodium hydroxide, dilute it 2 times with 0.1 mol / L PBS, and sterilize it by high pressure at 121° C. for 20-30 minutes.

7. The use of the vaccine stabilizer according to claim 4 in vaccine preparation, characterized in that: The preparation method of the polysaccharide immunopotentiator solution described in step 1) is as follows: weigh 1.0 g of polysaccharide, dissolve it in 100 mL of purified water, stir it thoroughly to dissolve the polysaccharide, and sterilize it by high pressure at 116° C. for 20 to 30 minutes.

8. The use of the vaccine stabilizer according to claim 4 in vaccine preparation, characterized in that The antigen of the vaccine is any one of porcine circovirus, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus.

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