Poultry epidemic disease inactivated vaccine adjuvant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of veterinary biological products. The invention provides a poultry epidemic disease inactivated vaccine adjuvant as well as a preparation method and application thereof. The adjuvant is prepared from the following raw materials in dosage ratio: 70 to 80mL of white oil, 0.5 to 1.5 mL of Tween-80, 1 to 2g of chitosan, 0.5 to 1.5 g of astragalus polysaccharide and 0.5 to 1.5 g of vitamin E. According to the invention, multiple raw materials are utilized, and a triple synergistic mechanism of long-acting slow release, efficient activation and safety protection is formed through emulsion stabilization and targeted delivery at a physical level, multi-channel activation (including natural immunity, adaptive immunity, humoral immunity and mucosal immunity) at an immune level and synergistic oxidation resistance and inflammatory reaction regulation at a safety level; the immune response efficiency, the antibody duration and the safety of the vaccine can be remarkably improved, and the vaccine is particularly suitable for the special immune physiological characteristics of poultry.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, in particular to an adjuvant for an inactivated poultry disease vaccine and a preparation method and application thereof. Background Art

[0002] Poultry farming is a vital component of the livestock industry, but frequent outbreaks of poultry diseases (such as highly pathogenic avian influenza, Newcastle disease, infectious bursal disease, and infectious bronchitis) pose a serious threat to the industry. These diseases spread rapidly and have high mortality rates. For example, the H5N1 subtype of avian influenza can cause up to 100% mortality in chickens. These diseases are also prone to large-scale, cross-regional epidemics, severely impacting the development of the poultry industry.

[0003] Currently, poultry disease prevention and control relies primarily on vaccination, with inactivated vaccines widely used due to their safety and stability. However, inactivated vaccines suffer from weak immunogenicity. Therefore, the development of an effective, safe, and cost-effective adjuvant compound to enhance the protective efficacy of inactivated vaccines against poultry diseases has become an urgent need in the industry. Summary of the Invention

[0004] The purpose of the present invention is to provide an inactivated poultry disease vaccine adjuvant and its preparation method and application, which can significantly enhance the humoral immunity and cellular immune response of the inactivated vaccine, reduce side effects, and prolong the immune protection period, while having the characteristics of simple preparation process and low cost.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an adjuvant for an inactivated vaccine against poultry diseases. The usage ratio of the raw materials in the adjuvant is: 70-80 mL of white oil: 0.5-1.5 mL of Tween-80: 1-2 g of chitosan: 0.5-1.5 g of astragalus polysaccharide: 0.5-1.5 g of vitamin E.

[0007] Preferably, the usage ratio of each raw material in the adjuvant is: white oil 75 mL: Tween-80 1 mL: chitosan 1.5 g: astragalus polysaccharide 1 g: vitamin E 1 g.

[0008] The present invention also provides an inactivated vaccine for poultry diseases, which comprises an inactivated antigen liquid and the adjuvant.

[0009] Preferably, the solvent of the inactivated antigen solution is water, and the titer of the virus in the inactivated antigen solution is 10 7.5 ~10 9 TCID 50 / mL.

[0010] Preferably, the virus in the inactivated antigen solution is any one of avian influenza virus, duck serositis virus and Newcastle disease virus.

[0011] The present invention also provides a method for preparing the vaccine, comprising the following steps:

[0012] (1) mixing chitosan, astragalus polysaccharide, vitamin E and inactivated antigen solution to obtain an aqueous solution;

[0013] (2) mixing white oil and Tween-80 to obtain an oil phase solution;

[0014] (3) The aqueous phase solution and the oil phase solution are mixed, and after mixing, they are emulsified to prepare an inactivated vaccine for poultry diseases.

[0015] Preferably, the volume ratio of the inactivated antigen solution to the oil phase solution is 1:1-3.

[0016] Preferably, the mixing temperature in step (1) is 20-38° C., the mixing speed is 400-600 rpm, and the mixing time is 5-10 min.

[0017] Preferably, the mixing temperature in step (2) is 30-40° C., the mixing speed is 400-800 rpm, and the mixing time is 5-10 min.

[0018] Preferably, the mixing temperature in step (3) is 33-36° C., the mixing speed is 400-800 rpm, and the mixing time is 20-30 min.

[0019] The beneficial effects of the present invention are:

[0020] In the poultry disease inactivated vaccine prepared by the present invention, white oil, Tween-80, chitosan, astragalus polysaccharide, and vitamin E, as adjuvant components, each possess advantages and produce a synergistic effect: white oil, as the main oil phase, forms a stable emulsion, delaying antigen release and building a local immune microenvironment; Tween-80, as an emulsifier, stabilizes the oil-water interface and promotes antigen uptake; chitosan, through its cationic properties, targets mucosal immunity and activates the TLR pathway, enhancing antigen presentation; astragalus polysaccharide, as a natural immunomodulator, activates multiple immune pathways and inhibits immunosuppression, while also providing antioxidant protection; and vitamin E, through antioxidant activity, maintains antigen structure stability, strengthens immune cell function, and alleviates local inflammation. These multiple ingredients, through physical emulsion stabilization and targeted delivery, immune activation of multiple pathways (covering innate and adaptive immunity, humoral immunity, and mucosal immunity), and safety synergistic antioxidant and inflammatory response regulation, form a triple synergistic mechanism of long-lasting sustained release, efficient activation, and safe protection. This significantly improves the vaccine's immune response efficiency, antibody persistence, and safety, making it particularly well-suited to the unique immunophysiological characteristics of poultry. DETAILED DESCRIPTION

[0021] The invention provides an adjuvant for an inactivated vaccine against poultry diseases. The usage ratio of the raw materials in the adjuvant is: 70-80 mL of white oil: 0.5-1.5 mL of Tween-80: 1-2 g of chitosan: 0.5-1.5 g of astragalus polysaccharide: 0.5-1.5 g of vitamin E.

[0022] In the present invention, the usage ratio of each raw material in the adjuvant is preferably: white oil 75 mL: Tween-80 1 mL: chitosan 1.5 g: astragalus polysaccharide 1 g: vitamin E 1 g.

[0023] The present invention also provides an inactivated vaccine for poultry diseases, which comprises an inactivated antigen liquid and the adjuvant.

[0024] In the present invention, the solvent of the inactivated antigen solution is preferably water, and the titer of the virus in the inactivated antigen solution is preferably 10 7.5 ~10 9 TCID 50 / mL, more preferably 10 8 TCID 50 / mL.

[0025] In the present invention, the virus in the inactivated antigen solution is preferably any one of avian influenza virus, duck serositis virus and Newcastle disease virus, and more preferably avian influenza virus.

[0026] The present invention also provides a method for preparing the vaccine, comprising the following steps:

[0027] (1) mixing chitosan, astragalus polysaccharide, vitamin E and inactivated antigen solution to obtain an aqueous solution;

[0028] (2) mixing white oil and Tween-80 to obtain an oil phase solution;

[0029] (3) The aqueous phase solution and the oil phase solution are mixed, and after mixing, they are emulsified to prepare an inactivated vaccine for poultry diseases.

[0030] In the present invention, the volume ratio of the inactivated antigen solution to the oil phase solution is preferably 1:1 to 3, more preferably 1:2.

[0031] In the present invention, the mixing temperature in step (1) is preferably 20-38°C, more preferably 25-32°C, and even more preferably 28°C; the mixing speed is preferably 400-600 rpm, more preferably 450-550 rpm, and even more preferably 500 rpm; and the mixing time is preferably 5-10 min, and even more preferably 8 min.

[0032] In the present invention, the mixing temperature in step (2) is preferably 30-40°C, more preferably 32-38°C, and even more preferably 35°C; the mixing speed is preferably 400-800 rpm, more preferably 500-700 rpm, and even more preferably 600 rpm; and the mixing time is preferably 5-10 min, and even more preferably 8 min.

[0033] In the present invention, the mixing temperature in step (3) is preferably 33-36°C, more preferably 34°C, the mixing speed is preferably 400-800 rpm, more preferably 500-700 rpm, and more preferably 600 rpm, and the mixing time is preferably 20-30 min, more preferably 22-28 min, and more preferably 25 min.

[0034] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] (1) 1.5 g of chitosan, 1 g of astragalus polysaccharide, 1 g of vitamin E, and 76 mL of an inactivated avian influenza H5 vaccine antigen solution that passed the test were mixed at 28° C. and 500 rpm for 8 min to obtain an aqueous solution;

[0037] The virus titer in the inactivated avian influenza H5 vaccine antigen solution is 10 8 TCID 50 / 1mL;

[0038] (2) Mix 75 mL of white oil and 1 mL of Tween-80 at 35°C and 600 rpm for 8 min to obtain an oil phase solution;

[0039] (3) The aqueous phase solution and the oil phase solution were mixed at 34° C. and 600 rpm for 25 min, and after mixing, they were emulsified using an emulsifier to prepare an inactivated vaccine 1.

[0040] Example 2

[0041] (1) 1 g of chitosan, 0.5 g of astragalus polysaccharide, 0.5 g of vitamin E, and 35.3 mL of an inactivated avian influenza H5 vaccine antigen solution that passed the test were mixed at 20° C. and 400 rpm for 5 min to obtain an aqueous solution;

[0042] The virus titer in the inactivated avian influenza H5 vaccine antigen solution is 10 8 TCID 50 / 1mL;

[0043] (2) Mix 70 mL of white oil and 0.5 mL of Tween-80 at 30°C and 400 rpm for 5 min to obtain an oil phase solution;

[0044] (3) The aqueous phase solution and the oil phase solution were mixed at 33° C. and 400 rpm for 20 min. After mixing, they were emulsified using an emulsifier to prepare inactivated vaccine 2.

[0045] Example 3

[0046] (1) 2 g of chitosan, 1.5 g of astragalus polysaccharide, 1.5 g of vitamin E, and 27.2 mL of an inactivated avian influenza H5 vaccine antigen solution that passed the test were mixed at 38° C. and 600 rpm for 10 min to obtain an aqueous solution;

[0047] The virus titer in the inactivated avian influenza H5 vaccine antigen solution is 10 5 TCID 50 / 1mL;

[0048] (2) 80 mL of white oil and 1.5 mL of Tween-80 were mixed at 40°C and 800 rpm for 10 min to obtain an oil phase solution;

[0049] (3) The aqueous phase solution and the oil phase solution were mixed at 36° C. and 800 rpm for 30 min, and after mixing, they were emulsified using an emulsifier to prepare inactivated vaccine 3.

[0050] Comparative Example 1

[0051] The same method as in Example 1 was used, except that the amount of astragalus polysaccharide was increased from 1 g to 4 g to prepare inactivated vaccine 4.

[0052] Comparative Example 2

[0053] The same method as Example 1 was used, except that "1.5 g chitosan" and "1 g astragalus polysaccharide" were removed to prepare inactivated vaccine 5.

[0054] Experimental example

[0055] (1) Quality inspection of inactivated vaccines

[0056] The prepared inactivated vaccines 1 to 5 were subjected to quality inspections including dosage form, stability, viscosity and sterility. For specific methods, please refer to the "Pharmacopoeia of Veterinary People's Republic of China" (2020 edition).

[0057] Results: The inactivated vaccines 1 to 5 prepared by the present invention all have the appearance of uniform emulsions; the dosage forms are all oil-in-water type. Take a clean pipette, draw a small amount of vaccine and drop it on the surface of cold water. Except for the first drop, none of them spread; draw 10 mL of vaccine into a centrifuge tube and centrifuge it at 3000 r / min for 15 minutes. None of them broke the emulsion; the viscosity and sterility tests all met the requirements of the "Pharmacopoeia of Veterinary People's Republic of China" (2020 edition).

[0058] (2) Safety testing of inactivated vaccines

[0059] Experimental subjects: 50 14-day-old SPF chickens. Before the experiment began, the 50 SPF chickens were checked for health to ensure that they were free of disease infection.

[0060] Grouping: 50 SPF chicks were randomly divided into 5 groups: A, B, C, D, E, and F, with 10 chicks in each group to ensure balanced grouping.

[0061] Group A: intramuscular injection of 2.0 mL of inactivated vaccine 1;

[0062] Group B: intramuscular injection of 2.0 mL of inactivated vaccine 2;

[0063] Group C: intramuscular injection of 2.0 mL of inactivated vaccine 3;

[0064] Group D: intramuscular injection of 2.0 mL of inactivated vaccine 4;

[0065] Group E: intramuscular injection of 2.0 mL of inactivated vaccine 5;

[0066] Experimental period: 15 days of observation.

[0067] Results: No chickens in the inactivated vaccine group died within 15 days. Throughout the trial, the chickens remained active and healthy, with no behavioral abnormalities or lethargy. Feed intake remained normal, with no significant difference in daily feed intake compared to pre-vaccination levels.

[0068] (3) Efficacy test of inactivated vaccines

[0069] Experimental subjects: 210 21-day-old SPF chickens. Before the experiment began, the 210 21-day-old SPF chickens were health checked to ensure that they were free of disease infection.

[0070] Grouping: 210 28-day-old SPF chickens were randomly divided into 7 groups: G, H, I, J, K, L, and M, with 10 chickens in each group, and 3 replicates in total to ensure balanced grouping.

[0071] Group G: intramuscular injection of 0.3 mL of inactivated vaccine 1;

[0072] Group H: intramuscular injection of 0.3 mL of inactivated vaccine 2;

[0073] Group I: intramuscular injection of 0.3 mL of inactivated vaccine 3;

[0074] Group J: intramuscular injection of 0.3 mL of inactivated vaccine 4;

[0075] Group K: intramuscular injection of 0.3 mL of inactivated vaccine 5

[0076] Group L: intramuscular injection of 0.3 mL of commercially available inactivated avian influenza H5 vaccine;

[0077] Group M: intramuscular injection of 0.3 mL of normal saline;

[0078] Trial period: 28 days.

[0079] Immunization schedule: Blood samples were collected on the 7th, 14th, 21st and 28th days after immunization.

[0080] Blood collection steps: Use the wing vein blood collection method to ensure standardized operation and reduce stress response to the chicken. The collected blood is centrifuged and serum is extracted.

[0081] Antibody level detection: The hemagglutination test (HA) was used to detect the red blood cell agglutination reaction, and the hemagglutination inhibition test (HI) was used to detect the neutralizing ability of antibodies in the serum. The results were expressed in log2 form, and the average value was calculated.

[0082] Data recording and analysis: The antibody level results of each blood test were recorded in detail. The antibody levels of different groups at each time point were compared and analyzed using statistical analysis software to evaluate the effects of different treatment groups on the immune response of SPF chickens. The HI antibody titers (log2) of SPF chickens after immunization with inactivated vaccines in each treatment group are shown in Table 1 below.

[0083] Table 1 HI antibody titer (log2) after immunization of SPF chickens with inactivated vaccine

[0084] experimental group 7 days after immunization 14 days after immunization 21 days after immunization 28 days after immunization Group G 6.13 8.11 9.21 9.95 Group H 5.95 7.87 9.09 9.84 Group I 6.02 8.04 9.15 9.89 Group J 4.82 6.83 7.79 8.67 Group K 3.70 5.90 6.81 7.59 Group L 3.17 5.55 6.64 7.30 Group M 0.4 0.3 0.5 0.4

[0085] The results show that the vaccine adjuvant prepared in the present application is conducive to the early release of antigens, promotes the early formation of antibodies, and can quickly make up for the antibody level after the attenuation of maternal antibodies in young chickens.

[0086] The vaccine prepared in the examples of the present application has the advantage of increasing immune antibodies 1 to 28 days after immunization compared to the commercially available avian influenza H5 inactivated vaccine.

[0087] As can be seen from the above examples, the present invention provides an inactivated poultry disease vaccine adjuvant, its preparation method, and application. The dosage ratio of each raw material in the adjuvant is: white oil 70-80 mL: Tween-80 0.5-1.5 mL: chitosan 1-2 g: astragalus polysaccharide 0.5-1.5 g: vitamin E 0.5-1.5 g. The present invention utilizes multiple raw materials, through physical stabilization of the emulsion and targeted delivery, immune multi-pathway activation (covering innate immunity and adaptive immunity, humoral immunity and mucosal immunity), and safety synergistic anti-oxidation and regulation of inflammatory responses, to form a triple synergistic mechanism of long-term sustained release, efficient activation, and safety protection. This can significantly improve the immune response efficiency, antibody persistence, and safety of the vaccine, and is particularly adapted to the special immune physiological characteristics of poultry.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An adjuvant for an inactivated poultry disease vaccine, characterized in that: The usage ratio of each raw material in the adjuvant is: white oil 70-80 mL: Tween-80 0.5-1.5 mL: chitosan 1-2 g: astragalus polysaccharide 0.5-1.5 g: vitamin E 0.5-1.5 g.

2. The adjuvant according to claim 1, characterized in that The usage ratio of each raw material in the adjuvant is: white oil 75 mL: Tween-80 1 mL: chitosan 1.5 g: astragalus polysaccharide 1 g: vitamin E 1 g.

3. A poultry disease inactivated vaccine, characterized in that: The vaccine comprises an inactivated antigen solution and the adjuvant according to claim 1 or 2.

4. The vaccine according to claim 3, characterized in that The solvent of the inactivated antigen solution is water, and the titer of the virus in the inactivated antigen solution is 10 7.5 ~10 9 TCID 50 / mL.

5. The vaccine according to claim 4, characterized in that The virus in the inactivated antigen liquid is any one of avian influenza virus, duck serositis virus and Newcastle disease virus.

6. The method for preparing the vaccine according to any one of claims 3 to 5, characterized in that: The following steps are involved: (1) mixing chitosan, astragalus polysaccharide, vitamin E and inactivated antigen solution to obtain an aqueous solution; (2) mixing white oil and Tween-80 to obtain an oil phase solution; (3) The aqueous phase solution and the oil phase solution are mixed, mixed and emulsified to prepare an inactivated vaccine for poultry diseases.

7. The preparation method according to claim 6, characterized in that The volume ratio of the inactivated antigen solution to the oil phase solution is 1:1-3.

8. The preparation method according to claim 7, characterized in that The mixing temperature in step (1) is 20-38° C., the mixing speed is 400-600 rpm, and the mixing time is 5-10 min.

9. The preparation method according to claim 8, characterized in that The mixing temperature in step (2) is 30-40° C., the mixing speed is 400-800 rpm, and the mixing time is 5-10 min.

10. The preparation method according to claim 9, characterized in that The mixing temperature in step (3) is 33-36° C., the mixing speed is 400-800 rpm, and the mixing time is 20-30 min.