Preparation and application of bivalent inactivated vaccine composite adjuvant for poultry

Abstract

The invention relates to the technical field of inactivated vaccines for poultry, and discloses a bivalent inactivated vaccine composite adjuvant for poultry, which comprises the following components in percentage by mass: 5-10% of nano-scale chitosan microspheres with the particle size range of 50-100 nanometers; the lipidosome is prepared from phospholipid and cholesterol according to the molar ratio of 3: 1, and the mass fraction is 3%-6%; a cell factor interleukin-2 (IL-2) with a concentration of 10 to 20 [mu] g / mL; the mass fraction of the sodium alginate solution is 1%-3%. According to the preparation and application of the bivalent inactivated vaccine composite adjuvant for poultry, the nanoscale chitosan microspheres, the lipidosome and the like are effectively combined through an innovative formula, the nanoscale chitosan microspheres can promote antigen uptake, the lipidosome realizes slow release of the antigen, meanwhile, the cell factor IL-2 activates immune cells, humoral immunity and cellular immunity are comprehensively enhanced, and the immune effect of the bivalent inactivated vaccine composite adjuvant for poultry is improved. The immunogenicity of the vaccine is greatly improved, the organism can be induced to generate higher-level antibodies and stronger cellular immunity, and a defense line is built for the health of poultry.

Description

Technical Field

[0001] The invention relates to the technical field of poultry inactivated vaccines, in particular to the preparation and application of a composite adjuvant for a bivalent inactivated poultry vaccine. Background Art

[0002] With the booming development of today's large-scale and intensive poultry farming industry, the health of poultry flocks has become a core factor affecting farming efficiency and sustainable development of the industry. Among the many measures to ensure the health of poultry flocks, disease prevention and control undoubtedly occupies a vital position. Once poultry flocks are attacked by diseases, it will not only lead to the stunted growth and development of poultry and increased mortality, but may also cause a series of problems such as reduced meat and egg quality, which will bring direct economic losses to farmers. At the same time, the spread of poultry diseases may also have a chain reaction on the entire poultry farming industry chain, affecting the stable development of related industries such as feed, veterinary drugs, and poultry product processing. Therefore, building a scientific and efficient disease prevention and control system has become the key to the steady development of the poultry farming industry. As a powerful weapon for disease prevention and control, the performance of vaccines is directly related to the effectiveness of prevention and control.

[0003] In the field of poultry vaccines, existing adjuvant problems are prominent. On the one hand, traditional adjuvants have poor immune effects. For example, although aluminum adjuvants are safe, they can only induce limited humoral immunity and are insufficient in stimulating cellular immunity. They are difficult to effectively respond to complex and changeable poultry diseases, making poultry flocks susceptible to infection and seriously affecting breeding efficiency. On the other hand, although oil adjuvants can stimulate strong immune responses, they can easily cause local inflammation and granulomas in poultry, which not only damages the health of poultry and reduces growth performance, but also affects the quality of poultry meat, hindering its widespread application in poultry farming. Therefore, we proposed the preparation and application of a composite adjuvant for a bivalent inactivated poultry vaccine. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies in the prior art, the present invention provides the preparation and application of a composite adjuvant for a bivalent inactivated vaccine for poultry, which has the advantages of good immune effect and high safety, and solves the prominent problems of existing adjuvants in the field of poultry vaccines. On the one hand, traditional adjuvants have poor immune effects. For example, although aluminum adjuvants have good safety, they can only induce limited humoral immunity and are insufficient in stimulating cellular immunity, making it difficult to effectively respond to complex and changeable poultry diseases, making poultry flocks susceptible to infection and seriously affecting breeding benefits. On the other hand, although oil adjuvants can stimulate strong immune responses, they are very likely to cause local inflammation and granulomas in poultry, which not only damages the health of poultry and reduces growth performance, but also affects the quality of poultry meat, hindering their widespread application in poultry breeding.

[0006] (II) Technical solution

[0007] In order to achieve the above-mentioned purpose of good immune effect and high safety, the present invention provides the following technical scheme: a composite adjuvant for a bivalent inactivated vaccine for poultry, comprising the following components: nanoscale chitosan microspheres with a particle size range of 50-100 nanometers and a mass fraction of 5%-10%; liposomes composed of phospholipids and cholesterol in a molar ratio of 3:1, with a mass fraction of 3%-6%; cytokine interleukin-2 (IL-2) with a concentration of 10-20 μg / mL; sodium alginate solution with a mass fraction of 1%-3%.

[0008] Preferably, the deacetylation degree of the nano-scale chitosan microspheres is not less than 90%.

[0009] Preferably, the phospholipid in the liposome is hydrogenated soybean lecithin.

[0010] Preferably, the molecular weight of the sodium alginate is in the range of 100,000-300,000 Daltons.

[0011] A method for preparing a composite adjuvant for a bivalent inactivated vaccine for poultry, comprising the composite adjuvant for a bivalent inactivated vaccine for poultry, and further comprising the following steps:

[0012] S1. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a mass fraction of 2%. Under stirring conditions of 200-300 rpm, slowly add 2% sodium tripolyphosphate solution at a rate of 1-2 drops / second, react at 25-30° C. for 30-60 minutes, and then centrifuge, wash, and freeze-dry to obtain nanoscale chitosan microspheres.

[0013] S2, dissolving phospholipids and cholesterol in chloroform at a molar ratio of 3:1, removing chloroform under rotary evaporation conditions at 100-150 rpm and 40-50° C. to form a lipid film, adding PBS buffer with a pH value of 7.2-7.4, and preparing liposomes by water bath sonication at 60-70° C. and 200-300 watts for 30-45 minutes;

[0014] S3. Mix the prepared nano-scale chitosan microspheres, liposomes, IL-2 and sodium alginate solution in proportion, and stir at 4° C. and 100-150 rpm for 1-2 hours to obtain a composite adjuvant for a bivalent inactivated poultry vaccine.

[0015] A bivalent inactivated poultry vaccine containing a composite adjuvant includes a bivalent inactivated poultry vaccine composite adjuvant. The bivalent inactivated vaccine is prepared by mixing the composite adjuvant with two different poultry antigens in a volume ratio of 1:1:1. The two poultry antigens need to be filtered and sterilized through a 0.22μm microporous filter membrane before mixing.

[0016] Preferably, the prepared bivalent inactivated poultry vaccine needs to be stored at 2-8°C with a shelf life of not less than 12 months, and the content of the active ingredient in the vaccine does not decrease by more than 10% during the shelf life.

[0017] A method for applying a bivalent inactivated poultry vaccine, comprising the bivalent inactivated poultry vaccine containing a composite adjuvant, and further comprising:

[0018] H1. Stir the composite adjuvant and two different poultry antigens at 4°C for 2-4 hours. During the stirring process, perform ultrasonic assisted dispersion for 5-10 minutes at a power of 100-150 watts every 30 minutes to fully combine the adjuvant and the antigen.

[0019] H2. Use intramuscular injection. After disinfecting the injection site of the breast muscle of the poultry with iodine, vaccinate 1-2 week old birds with a vaccination dose of 0.2-0.3 mL per bird.

[0020] (III) Beneficial effects

[0021] Compared with the prior art, the present invention provides a preparation and application of a composite adjuvant for a bivalent inactivated vaccine for poultry, which has the following beneficial effects:

[0022] 1. The preparation and application of the composite adjuvant of the bivalent inactivated vaccine for poultry, through an innovative formula, effectively combines nano-scale chitosan microspheres, liposomes, etc. Nano-scale chitosan microspheres can promote antigen uptake, liposomes can achieve slow release of antigens, and at the same time cytokine IL-2 activates immune cells, comprehensively enhances humoral immunity and cellular immunity, greatly improves the immunogenicity of the vaccine, and can induce the body to produce higher levels of antibodies and stronger cellular immunity, building a strong line of defense for poultry health.

[0023] 2. The preparation and application of the composite adjuvant of the bivalent inactivated poultry vaccine utilizes the biodegradability of nano-chitosan microspheres and the encapsulation effect of liposomes to reduce irritation to the poultry body. Compared with oil adjuvants that are prone to inflammation, the vaccine using this composite adjuvant can significantly reduce the incidence of local inflammation and granuloma in the poultry body, reduce damage to the health of poultry, ensure their normal growth, improve the quality of poultry meat, meet the market demand for high-quality poultry products, and promote the development of the poultry breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention is a flow chart of the preparation method of the composite adjuvant of the bivalent inactivated vaccine for poultry;

[0025] Figure 2 It is a schematic diagram of the application method of the bivalent inactivated vaccine for poultry of the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the embodiments of the present invention and the accompanying drawings to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] See also Figure 1-2 A composite adjuvant for a bivalent inactivated vaccine for poultry, comprising the following components: nanoscale chitosan microspheres with a particle size range of 50-100 nanometers and a mass fraction of 5%-10%; liposomes composed of phospholipids and cholesterol in a molar ratio of 3:1, with a mass fraction of 3%-6%; cytokine interleukin-2 (IL-2) with a concentration of 10-20 μg / mL; sodium alginate solution with a mass fraction of 1%-3%.

[0028] Embodiment 1:

[0029] Nanoscale chitosan microspheres are prepared by ion crosslinking. Chitosan is a natural polysaccharide with a large number of amino and hydroxyl groups in its molecules. In 1% acetic acid solution, the amino groups on the chitosan molecules are protonated, making them positively charged. Sodium tripolyphosphate (TPP) is a multivalent anionic compound. When 2% sodium tripolyphosphate solution is slowly added to the chitosan solution, electrostatic interactions occur between the phosphate anions in TPP and the protonated amino groups on the chitosan molecules, forming an ion crosslinking network, thereby aggregating the chitosan molecules to form microspheres. Under stirring conditions of 200-300 rpm, the chitosan microspheres are prepared by ion crosslinking. , which can ensure that the reaction system is mixed evenly, and the TPP solution is slowly added at 1-2 drops / second to control the growth rate and particle size distribution of the microspheres. The reaction temperature is controlled at 25-30°C because this temperature range can ensure that the reaction proceeds smoothly without causing chitosan degradation or excessively violent reactions that are difficult to control due to excessive temperature. After 30-60 minutes of reaction, the microspheres are separated from the reaction system by centrifugation, washed to remove unreacted substances and impurities, and finally freeze-dried to obtain nanoscale chitosan microspheres. Freeze-drying can maintain the structure and morphology of the microspheres and prevent them from agglomerating during the drying process.

[0030] Liposomes are prepared by a thin film dispersion method. Phospholipids and cholesterol are dissolved in chloroform at a molar ratio of 3:1. Chloroform is a good organic solvent that can fully dissolve phospholipids and cholesterol. Chloroform is removed under rotary evaporation conditions at 100-150 rpm and 40-50°C. At this time, phospholipids and cholesterol will form a uniform lipid film on the container wall. After adding PBS buffer with a pH value of 7.2-7.4, the lipid film is hydrated in the buffer and ultrasonicated in a water bath at 60-70°C and 200-300 watts for 30-45 minutes. The role of ultrasound is to disperse the lipid film to form liposomes. During the ultrasonic process, mechanical energy is converted into heat energy and mechanical energy, which prompts lipid molecules to rearrange to form a closed bilayer structure, namely liposomes. This structure can encapsulate antigens and achieve slow release of antigens.

[0031] Embodiment 2:

[0032] The prepared nano-scale chitosan microspheres, liposomes, IL-2 and sodium alginate solution are mixed in proportion and stirred at 4°C and 100-150 rpm for 1-2 hours. The low temperature environment of 4°C can reduce the denaturation of proteins (such as IL-2) and ensure their biological activity. The purpose of stirring is to fully mix the components. Sodium alginate, as a stabilizer, can increase the stability of the composite adjuvant system and prevent the components from aggregating or precipitating during storage and use.

[0033] The bivalent inactivated vaccine is prepared by mixing a composite adjuvant with two different poultry antigens in a volume ratio of 1:1:1. Before mixing, the two poultry antigens are sterilized by filtering through a 0.22μm microporous filter membrane. This is because the pore size of the filter membrane can effectively retain bacteria and other microorganisms to ensure the sterility of the vaccine and avoid the introduction of contamination during vaccine preparation and use, which affects the quality of the vaccine and the immune effect. When mixing, the composite adjuvant and the two poultry antigens are stirred at 4°C for 2-4 hours. During the stirring process, ultrasonic assisted dispersion is performed every 30 minutes for 5-10 minutes at a power of 100-150 watts. The stirring at 4°C is to prevent the inactivation or denaturation of the antigen and adjuvant due to excessive temperature during the mixing process. Ultrasonic assisted dispersion can further promote the full combination of the adjuvant and the antigen, so that the antigen can be better adsorbed on the surface of nano-scale chitosan microspheres and liposomes, or be encapsulated in liposomes, thereby enhancing the immunogenicity of the vaccine.

[0034] Poultry are vaccinated by intramuscular injection. The breast muscle is chosen as the injection site because the breast muscle is well-developed and rich in blood vessels, which is conducive to the absorption of the vaccine and the stimulation of the immune response. Before vaccination, the breast muscle injection site is disinfected with iodine tincture. Iodine tincture has a broad-spectrum bactericidal effect and can effectively kill bacteria, viruses and other microorganisms on the skin surface of the injection site, reducing the risk of infection during vaccination. For poultry aged 1-2 weeks, the vaccination dose is 0.2-0.3mL per bird. This dose has been determined through a large number of experiments and practices. It can ensure that enough antigens and adjuvants enter the poultry body to stimulate an effective immune response, and will not burden the poultry body or cause adverse reactions due to excessive doses.

[0035] Embodiment three:

[0036] In order to verify the immune effect and safety of the present invention, the following experimental scheme was designed in this example.

[0037] Experimental animals: 300 healthy one-week-old chicks of similar weight were selected and randomly divided into 5 groups, with 60 chicks in each group.

[0038] Experimental vaccine preparation

[0039] Experimental group vaccine: A bivalent inactivated poultry vaccine containing a composite adjuvant was prepared according to the invention, and the two poultry antigens were A antigen and B antigen respectively;

[0040] Control group 1 vaccine: a bivalent inactivated vaccine was prepared using traditional aluminum adjuvant and A and B antigens in the same proportion;

[0041] Control group 2 vaccine: a bivalent inactivated vaccine was prepared using oil adjuvant and A and B antigens in the same proportion;

[0042] Control group 3: no vaccination, serving as blank control;

[0043] Control group 4: Only saline was inoculated as a negative control.

[0044] Immunization program:

[0045] The chicks in the experimental group and each control group were treated according to the following procedures:

[0046] Experimental group: When the chicks were 1 week old, the breast muscle injection site was disinfected with iodine, and each chick was intramuscularly injected with 0.2 mL of a bivalent inactivated poultry vaccine containing a composite adjuvant;

[0047] Control group 1: Also at 1 week of age, each mouse was injected intramuscularly with 0.2 mL of a bivalent inactivated vaccine containing aluminum adjuvant;

[0048] Control group 2: At 1 week of age, each mouse was injected intramuscularly with 0.2 mL of a bivalent inactivated vaccine containing an oil adjuvant;

[0049] Control group 3: no treatment;

[0050] Control group 4: 0.2 mL of normal saline was injected into each muscle.

[0051] Testing indicators and methods:

[0052] Humoral immunity index detection:

[0053] On the 7th, 14th, 21st and 28th days after immunization, 10 chicks were randomly selected from each group, blood samples were collected, serum was separated, and enzyme-linked immunosorbent assay (ELISA) was used to detect the specific antibody levels (IgG, IgM) against A antigen and B antigen in the serum. The absorbance value (OD value) was used to represent the antibody content. The higher the OD value, the higher the antibody level.

[0054] Cellular immune index detection:

[0055] On the 14th and 21st days after immunization, 10 chicks were randomly selected from each group, spleen cells were taken, and the lymphocyte proliferation ability was detected by MTT method. The spleen cells were co-cultured with ConA (concanavalin A, a T lymphocyte mitogen), and the proliferation activity of lymphocytes was reflected by detecting the metabolic capacity of MTT during cell proliferation. The optical density value indicated the degree of proliferation. The higher the optical density value, the stronger the lymphocyte proliferation ability.

[0056] At the same time, flow cytometry was used to detect the proportion of CD4+ and CD8+ T lymphocytes in spleen cells to evaluate the level of cellular immune response.

[0057] Safety index detection:

[0058] After vaccination, the mental state, feeding, drinking and growth and development of the chicks were observed every day, and any adverse reactions, such as local inflammation, granuloma formation, fever, diarrhea, etc., were recorded. On the 28th day after vaccination, each group of chicks was dissected to observe the pathological changes at the injection site and other major organs (such as liver, spleen, kidney, etc.) to evaluate the safety of the vaccine.

[0059] Experimental data recording and analysis:

[0060] Table 1 Humoral immunity experimental data

[0061]

[0062] Table 2 Cellular Immunity Experiment Data

[0063]

[0064] Safety test data:

[0065] Experimental group: After vaccination, the chicks were in good spirits, ate and drank normally, and their growth and development were not affected; after autopsy, there was no obvious inflammatory reaction at the injection site, no granuloma was found, and no pathological changes were found in the main organs;

[0066] Control group 1: The chicks grew and developed normally, but the immune effect was relatively weak;

[0067] Control group 2: 10 chicks had local inflammation and 5 chicks had granulomas, which had a certain impact on growth and development;

[0068] Control group 3 and control group 4: The chicks grew and developed normally, but did not produce an immune response.

[0069] Experimental conclusion:

[0070] In terms of immune effect: the experimental group chicks were significantly better than the control group 1 (aluminum adjuvant group), the control group 3 (blank control group), and the control group 4 (normal saline control group) in terms of humoral immunity and cellular immunity indicators; compared with the control group 2 (oil adjuvant group), the experimental group also had certain advantages in antibody level and lymphocyte proliferation ability, indicating that the composite adjuvant of the present invention can effectively enhance the immunogenicity of the vaccine and induce the body to produce higher levels of antibodies and stronger cellular immune responses;

[0071] Safety: The chicks in the experimental group showed no obvious adverse reactions, while the control group 2 (oil adjuvant group) showed adverse reactions such as local inflammation and granuloma, indicating that the composite adjuvant of the present invention is safer, has less impact on the health of poultry, and is more suitable for use in poultry farming.

[0072] In summary, the preparation and application of the composite adjuvant of the bivalent inactivated poultry vaccine effectively combines nanoscale chitosan microspheres, liposomes, etc. through an innovative formula. Nanoscale chitosan microspheres can promote antigen uptake, and liposomes can achieve slow release of antigens. At the same time, cytokine IL-2 activates immune cells, comprehensively enhances humoral immunity and cellular immunity, greatly improves the immunogenicity of the vaccine, and can induce the body to produce higher levels of antibodies and stronger cellular immunity, building a strong line of defense for poultry health.

[0073] In addition, the preparation and application of the composite adjuvant of the bivalent inactivated poultry vaccine utilizes the biodegradability of nano-scale chitosan microspheres and the encapsulation effect of liposomes to reduce irritation to the poultry body. Compared with oil adjuvants that are prone to inflammation, vaccines using the composite adjuvant can significantly reduce the incidence of local inflammation and granulomas in the poultry body, reduce damage to the health of poultry, ensure their normal growth, improve the quality of poultry meat, meet the market demand for high-quality poultry products, promote the development of the poultry breeding industry, and solve the outstanding problems of existing adjuvants in the field of poultry vaccines. On the one hand, traditional adjuvants have poor immune effects. For example, aluminum adjuvants are safe, but can only induce limited humoral immunity, and are insufficient in stimulating cellular immunity. They are difficult to effectively respond to complex and changeable poultry diseases, making poultry flocks susceptible to infection and seriously affecting breeding benefits. On the other hand, although oil adjuvants can stimulate a strong immune response, they are very likely to cause local inflammation and granulomas in the poultry body, which not only damages the health of poultry and reduces growth performance, but also affects the quality of poultry meat, hindering its widespread application in poultry breeding.

[0074] The relevant modules involved in this system are all hardware system modules or functional modules that combine computer software programs or protocols with hardware in the prior art. The computer software programs or protocols involved in the functional modules are themselves technologies that are well known to those skilled in the art and are not improvements of this system. The improvements of this system are the interaction or connection relationships between the modules, that is, improvements to the overall structure of the system to solve the corresponding technical problems to be solved by this system.

[0075] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composite adjuvant for a bivalent inactivated vaccine for poultry, characterized in that: The invention comprises the following components: nano-scale chitosan microspheres with a particle size range of 50-100 nanometers and a mass fraction of 5%-10%; liposomes composed of phospholipids and cholesterol in a molar ratio of 3:1 and a mass fraction of 3%-6%; cytokine interleukin-2 (IL-2) with a concentration of 10-20 μg / mL; and sodium alginate solution with a mass fraction of 1%-3%.

2. A composite adjuvant for a bivalent inactivated vaccine for poultry according to claim 1, characterized in that: The deacetylation degree of the nano-scale chitosan microspheres is not less than 90%.

3. The composite adjuvant for a bivalent inactivated vaccine for poultry according to claim 1, characterized in that: The phospholipid in the liposome is hydrogenated soybean lecithin.

4. The composite adjuvant for a bivalent inactivated vaccine for poultry according to claim 1, characterized in that: The molecular weight of the sodium alginate ranges from 100,000 to 300,000 Daltons.

5. A method for preparing a composite adjuvant for a bivalent inactivated vaccine for poultry, characterized in that: The method comprises the composite adjuvant of the bivalent inactivated vaccine for poultry according to claims 1 to 4, and further comprises the following steps: S1. Dissolve chitosan in 1% acetic acid solution to prepare a chitosan solution with a mass fraction of 2%. Under stirring conditions of 200-300 rpm, slowly add 2% sodium tripolyphosphate solution at a rate of 1-2 drops / second, react at 25-30° C. for 30-60 minutes, and then centrifuge, wash, and freeze-dry to obtain nanoscale chitosan microspheres. S2, dissolving phospholipids and cholesterol in chloroform at a molar ratio of 3:1, removing chloroform under rotary evaporation conditions at 100-150 rpm and 40-50° C. to form a lipid film, adding PBS buffer with a pH value of 7.2-7.4, and preparing liposomes by water bath sonication at 60-70° C. and 200-300 watts for 30-45 minutes; S3. Mix the prepared nano-scale chitosan microspheres, liposomes, IL-2 and sodium alginate solution in proportion, and stir at 4° C. and 100-150 rpm for 1-2 hours to obtain a composite adjuvant for a bivalent inactivated vaccine for poultry.

6. A bivalent inactivated poultry vaccine containing a composite adjuvant, characterized in that: The invention comprises the composite adjuvant for the bivalent inactivated poultry vaccine according to claims 1 to 4. The bivalent inactivated vaccine is prepared by mixing the composite adjuvant with two different poultry antigens in a volume ratio of 1:1:

1. The two poultry antigens need to be filtered and sterilized by a 0.22 μm microporous filter membrane before mixing.

7. The bivalent inactivated poultry vaccine containing a composite adjuvant according to claim 6, characterized in that: The prepared bivalent inactivated poultry vaccine must be stored at 2-8°C with a shelf life of no less than 12 months, and the content of the vaccine's active ingredients must not decrease by more than 10% during the shelf life.

8. A method for using a bivalent inactivated poultry vaccine, characterized in that: The invention comprises the bivalent inactivated poultry vaccine containing a composite adjuvant as claimed in claim 6, further comprising: H1. Stir the composite adjuvant and two different poultry antigens at 4°C for 2-4 hours. During the stirring process, perform ultrasonic assisted dispersion for 5-10 minutes at a power of 100-150 watts every 30 minutes to fully combine the adjuvant and the antigen. H2. Use intramuscular injection. After disinfecting the injection site of the breast muscle of the poultry with iodine, vaccinate 1-2 week old birds with a vaccination dose of 0.2-0.3 mL per bird.

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