Nanometer propolis composite adjuvant for poultry and application thereof

Abstract

The application discloses an avian nano propolis composite adjuvant and application thereof, and belongs to the field of veterinary drugs. The avian nano propolis composite adjuvant is obtained by compounding propolis ethanol extract, an immune enhancer and a surfactant. The avian nano propolis composite adjuvant provided by the application has the characteristics of no toxic side effect, extremely low residual amount and good immune effect, and can be used as an adjuvant of poultry inactivated vaccine and a diluent of live vaccine. The avian nano propolis composite adjuvant can be used to prepare vaccines with envelope viruses such as newcastle disease virus and avian influenza virus, and the prepared vaccines have good stability and excellent characteristics of simultaneously inducing cellular immunity and humoral immunity.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drugs, specifically relating to a nano-propolis compound adjuvant for poultry and its application. Background Technology

[0002] Vaccination is one of the effective measures for preventing animal diseases. Its purpose is to induce an immune response against specific pathogens through immunization with vaccines, including live attenuated vaccines, inactivated vaccines, and subunit vaccines, thereby preventing specific diseases. Live attenuated vaccines can stimulate comprehensive systemic and local immunity, providing long-lasting immunity; however, these vaccines carry the risk of shedding the virus and reversion to virulence. Inactivated vaccines are prepared by inactivating pathogens through physicochemical methods. These vaccines induce a weaker immune response, require larger doses, and have higher production costs; therefore, they require adjuvants to enhance their immune effect. Especially in recent years, with the continuous development and introduction of weakly immunogenic vaccines such as genetically engineered subunit vaccines, recombinant vaccines, and synthetic peptide vaccines, the high purity of their antigens necessitates even greater adjuvant support. However, existing adjuvants are no longer sufficient to meet the demands of the rapid development of new vaccines, making the development of novel adjuvants with high immunogenicity and low side effects particularly urgent.

[0003] Propolis adjuvants refer to propolis solutions obtained after ethanol extraction. These solutions contain flavonoids, phenolic acids, and their esters, among other immunomodulatory substances, making them a natural and highly effective immune enhancer. They can increase phagocytic cell activity, promote an increase in white blood cell count, and stimulate the production of specific antibodies, thereby improving the body's immune function. Currently, most developed propolis adjuvants are ethanol extracts of propolis. Vaccines prepared with this adjuvant and enveloped viruses exhibit low antibody titers after use, possibly because a component in the propolis adjuvant disrupts the outer membrane antigens of the enveloped viruses. Furthermore, vaccines prepared by mixing propolis ethanol extract as an adjuvant with inactivated bacterial antigens tend to precipitate during storage. This precipitate accumulates at the bottom of the vaccine vial, is difficult to disperse, and requires vigorous shaking before use, causing inconvenience. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a poultry nano-propolis compound adjuvant and its applications. The poultry nano-propolis compound adjuvant provided by this invention is characterized by being non-toxic, having extremely low residue levels, and exhibiting good immunizing effects. It can be used as an adjuvant for inactivated poultry vaccines and as a diluent for live vaccines. Furthermore, the poultry nano-propolis compound adjuvant can be combined with enveloped viruses such as Newcastle disease virus and avian influenza virus to prepare vaccines. The resulting vaccines exhibit good stability and possess the excellent characteristic of simultaneously inducing cellular and humoral immunity.

[0005] The first aspect of this invention provides a poultry nano-propolis compound adjuvant, which is composed of the following components by volume percentage: 10%~30% propolis ethanol extract, 30%~45% immune enhancer, and 30%~50% surfactant; wherein the dry matter content of propolis in the propolis ethanol extract is 50mg / mL~70mg / mL.

[0006] Furthermore, the immune enhancer is composed of immune enhancer A and immune enhancer B; the volume ratio of immune enhancer A to immune enhancer B is 5~8:1.

[0007] Furthermore, the immune enhancer A is white oil or vitamin D, the immune enhancer B is a mixture of Mycobacterium tumefaciens extract and dimethyl dioctadecyl ammonium bromide, or the immune enhancer B is a saponin.

[0008] Furthermore, the volume ratio of the Mycobacterium tumefaciens extract to dimethyl dioctadecyl ammonium bromide is 100:1~30.

[0009] Furthermore, the Mycobacterium tumefaciens extract is obtained by lysing inactivated Mycobacterium tumefaciens cells at 800-1000 bar and then centrifuging at 11000-13000 rpm for 25-35 minutes.

[0010] Furthermore, the pyrolysis process needs to be repeated twice.

[0011] Furthermore, the saponin is saponin A or saponin QS-21.

[0012] Furthermore, the surfactant is a polyglycerol fatty acid ester, Span-80, and polyoxyethylene hydrogenated castor oil.

[0013] Furthermore, the volume ratio of the polyglycerol fatty acid ester, Span-80, and polyoxyethylene hydrogenated castor oil is 7~9:3~5:2.

[0014] A second aspect of the present invention provides the application of the above-described poultry nano-propolis composite adjuvant in the preparation of poultry inactivated vaccine, subunit vaccine or live vaccine diluent.

[0015] A third aspect of the present invention provides a poultry nano-propolis compound adjuvant vaccine, the vaccine comprising the poultry nano-propolis compound adjuvant described above.

[0016] Furthermore, the vaccine also includes inactivated antigens or subunit antigens of pathogens that infect poultry.

[0017] Furthermore, the antigen is Newcastle disease virus, avian influenza virus, or Haemophilus paragallinarum.

[0018] Furthermore, the volume ratio of the antigen to the poultry nano-propolis compound adjuvant is 2-4:1.

[0019] Preferably, the volume ratio of the antigen to the poultry nano-propolis compound adjuvant is 3:1.

[0020] In summary, compared with the prior art, the present invention has the following beneficial effects: This invention provides a poultry nano-propolis compound adjuvant composed of propolis ethanol extract, an immune enhancer, and a surfactant. The propolis ethanol extract is rich in bioactive components such as flavonoids and terpenes, which can effectively regulate the poultry immune system, stimulate immune cell proliferation, and enhance specific and non-specific immune functions. The addition of the immune enhancer further strengthens the immune response and works synergistically with the propolis extract to significantly improve poultry's resistance to pathogens and enhance vaccine efficacy. The surfactant added to the poultry nano-propolis compound adjuvant can reduce the surface tension of the adjuvant, forming a stable nano-emulsion structure, improving the stability of vaccine preservation, and the nano-scale formulation significantly increases the specific surface area, making the binding of the adjuvant to immune cells more efficient and improving bioavailability.

[0021] By employing a specific nonionic surfactant compound system of hydrogenated castor oil with polyoxyethylene, Span-80, and polyglycerol fatty acid esters, a stable nanoscale emulsion structure is formed, effectively preventing precipitation and aggregation after the propolis extract is mixed with inactivated bacterial antigens, ensuring good stability of the vaccine during storage and preventing precipitation.

[0022] The poultry nano-propolis compound adjuvant, by adding vitamin D, white oil, dimethyl dioctadecyl ammonium bromide, saponins or Mycobacterium tumefaciens extract, can synergistically activate the multi-pathway response of the poultry immune system with propolis ethanol extract, significantly improve antibody titers against enveloped viruses (such as avian influenza virus and Newcastle disease virus), avoid propolis components from damaging the virus outer membrane, and enhance the immune effect of vaccines.

[0023] The poultry nano-propolis compound adjuvant provided by this invention has the characteristics of being non-toxic and having no side effects, extremely low residue, good immune effect, and good safety. It can be used as an adjuvant for poultry inactivated vaccines and a diluent for live vaccines. Attached Figure Description

[0024] Figure 1 The images show the appearance of propolis ethanol extract. From left to right, the two samples are distilled water and propolis ethanol extract, respectively.

[0025] Figure 2 This is the absorption spectrum of the ethanol extract of propolis.

[0026] Figure 3The results show the particle size and particle size distribution of the nano-propolis composite adjuvant prepared in Example 1.

[0027] Figure 4 The results show the particle size and particle size distribution of the nano-propolis composite adjuvant prepared in Example 2.

[0028] Figure 5 The results show the particle size and particle size distribution of the nano-propolis composite adjuvant prepared in Example 3.

[0029] Figure 6 The results show the stability test results of the poultry nano-propolis compound adjuvant vaccine. Figures A, B, C, D, E, and F show the stability test results of the poultry nano-propolis compound adjuvant vaccines prepared in Examples 4, 5, 6, Comparative Examples 1, 2, and 3, respectively. Detailed Implementation

[0030] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0031] In some embodiments of the present invention, propolis was purchased from Shandong Lvdu Biotechnology Co., Ltd.; Span 80, polyglycerol fatty acid esters, polyoxyethylene hydrogenated castor oil, and Tween 80 were all purchased from Croda Ltd. (UK); polyoxyethylene hydrogenated castor oil was purchased from BASF Ltd. (Germany); vitamin D was purchased from Shanxi Jinyang Pharmaceutical Excipients Co., Ltd.; white oil was purchased from Mobil AG; saponin A, saponin QS-21, and dimethyl dioctadecyl ammonium bromide (DDA) were purchased from Sigma-Aldrich Ltd.; and other chemical reagents were all analytical grade and purchased from Sinopharm Group.

[0032] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0033] Most currently developed propolis adjuvants are directly derived from the ethanol extract of propolis. Vaccines prepared with this adjuvant and enveloped viruses have low antibody titers after use, possibly because some component in the propolis adjuvant destroys the outer membrane antigens of the enveloped viruses. In addition, vaccines prepared by mixing propolis ethanol extract as an adjuvant with inactivated bacterial antigens will produce precipitation during storage. The precipitation accumulates at the bottom of the vaccine bottle and is not easy to disperse, requiring vigorous shaking before use, which is inconvenient.

[0034] This invention provides a poultry nano-propolis compound adjuvant and its application. The invention obtains a poultry nano-propolis compound adjuvant by compounding propolis ethanol extract, an immune enhancer, and a surfactant. The properties of the poultry nano-propolis compound adjuvant are characterized, and its safety is tested. Furthermore, this invention utilizes the poultry nano-propolis compound adjuvant to prepare a poultry nano-propolis compound adjuvant vaccine, and the vaccine's properties, safety, and antibody titer after immunization are tested. It is found that the poultry nano-propolis compound adjuvant of this invention can be used to prepare vaccines against enveloped viruses such as Newcastle disease virus and avian influenza virus. The prepared vaccines not only have good stability but also significantly improve antibody titers against enveloped viruses.

[0035] Example 1: A method for preparing a poultry nano-propolis composite adjuvant, comprising the following preparation steps: S1. Preparation of propolis ethanol extract The preparation of propolis ethanol extract was carried out in accordance with GB / T 24283-2018 Propolis. The specific preparation steps are as follows: Crude propolis was frozen at -15℃ for 36 hours, then ground and sieved using a cryogenic grinder. 95% v / v ethanol was added to the sieved crude propolis. The weight ratio of the sieved crude propolis to 95% v / v ethanol was 1:4. The extract was extracted at 37℃ for 60 hours. After cooling, the extract was filtered and centrifuged to remove the sediment. Then, ultrafiltration and concentration were performed to obtain pure propolis ethanol extract.

[0036] This invention involves the determination of the properties, pH value, relative density, oxidation time, dry matter content, and total flavonoid content of propolis ethanol extract.

[0037] Tests for propolis ethanol extract: (1) Characteristic test like Figure 1 As shown, when the prepared propolis ethanol extract was placed in a colorless and transparent container, it was observed under natural light to be a brownish-brown or dark brown glossy transparent liquid with the characteristic aroma of propolis.

[0038] like Figure 2 As shown, the propolis ethanol extract solution was scanned in the wavelength range of 300~600nm, and the results showed that the propolis ethanol extract solution had a maximum absorption peak at 490nm.

[0039] Take an appropriate amount of this product and test the propolis ethanol extract sample using the light scattering method according to the detection method in the General Chapter 0982 of the Pharmacopoeia of the People's Republic of China (2020 Edition) (Part IV). The particle size should be less than 100 nm.

[0040] Testing revealed that the particle size of all three batches of samples (propolis ethanol extract Lot.2021001, propolis ethanol extract Lot.2021002, and propolis ethanol extract Lot.20210033) was below 100 nanometers, meeting the standard.

[0041] (2) pH value measurement According to Appendix 3101 of the "Veterinary Pharmacopoeia of the People's Republic of China (2020 Edition) (Part III)," the pH value of the propolis ethanol extract sample should be within the range of 6.0 to 7.0.

[0042] The results are shown in Table 1. The pH values ​​of the three batches of samples were in the range of 6.2 to 6.6, which met the standard.

[0043] Table 1. pH values ​​of propolis ethanol extract (3) Relative density measurement The relative density of propolis ethanol extract samples should be determined according to Appendix 0601 of the "Veterinary Pharmacopoeia of the People's Republic of China (2020 Edition)" (Part I) using the method of determination of relative density, and should be within the range of 0.75 to 0.95.

[0044] The results are shown in Table 2. The relative density values ​​of the three batches of samples are in the range of 0.80 to 0.86, which meets the standard.

[0045] Table 2. Relative density of propolis ethanol extract (4) Oxidation time The content of reducing substances in propolis is indicated by the time it takes for the purple-red potassium permanganate solution to fade. The ethanol extract of propolis is tested according to the People's Republic of China National Standard (GB / T 24283-2018) for the determination of propolis oxidation time. The oxidation time should be ≤22 seconds.

[0046] The results are shown in Table 3. The oxidation time of the three batches of samples was within the range of 18 to 20 minutes, which meets the standard.

[0047] Table 3 Oxidation time of propolis ethanol extract (5) Dry matter content of propolis ethanol extract Take a propolis ethanol extract solution, weigh the dry matter after ethanol evaporation, obtain the weight of the ethanol extract, and calculate its percentage of the sample weight.

[0048] The results are shown in Table 4. The dry matter content of propolis in the three batches of samples was in the range of 55~62 mg / mL, which met the standard.

[0049] Table 4. Dry matter content of propolis ethanol extract (6) Determination of total flavonoid content According to the People's Republic of China National Standard (GB / T 24283-2018) for propolis, the total flavonoid content in propolis was determined by spectrophotometric colorimetry. The total flavonoid content in each 100 ml of propolis should be 20-40 mg.

[0050] The results are shown in Table 5. The total flavonoid content in the propolis of the three batches of samples was in the range of 29~35 mg / 100 mL, which met the standard.

[0051] Table 5 Total flavonoid content of propolis ethanol extract S2, Extraction of Mycobacterium tumefaciens extract: Mycobacterium tumefaciens ( Mycolicibacterium phlei (Purchased from Shenzhen Aiyi Biotechnology Co., Ltd.) The culture medium was inoculated into 2% glycerol-added nutrient broth (purchased from Qingdao Haibo Biotechnology Co., Ltd.), and cultured at 37℃ with aeration for 72 hours. The bacterial solution was then inactivated by heating at 100℃ for 30 minutes. The inactivated bacterial solution was centrifuged at 8000 rpm for 30 minutes to obtain bacterial cells. The bacterial cells were resuspended in physiological saline and centrifuged twice to remove impurities. The weight of the bacterial cells was calculated, and the solution was resuspended in physiological saline to a concentration of 10%. The bacterial cells were then homogenized using a high-pressure homogenizer (model SCIENTZ-150A, Ningbo Xinzhi Biotechnology Co., Ltd.) at 900 bar, and the process was repeated twice. The homogenized bacterial cells were then centrifuged at 12000 rpm for 30 minutes, and the supernatant was collected as the Mycobacterium tumefaciens extract.

[0052] S3. Preparation of poultry nano-propolis composite adjuvant Mix 30 mL of white oil, 20 mL of Span-80, 10 mL of polyglycerol fatty acid ester, and 5 mL of polyoxyethylene hydrogenated castor oil until homogeneous. Then add 20 mL of the propolis ethanol extract prepared in step S1 and mix until homogeneous. Add 0.1 g of saponin A and finally add water for injection to make up to 100 mL. Stir at 800 rpm for 20 min to fully mix and emulsify. Filter with a 0.22 μm filter to remove bacteria and obtain poultry nano-propolis compound adjuvant.

[0053] Example 2: A method for preparing a poultry nano-propolis composite adjuvant, comprising the following preparation steps: Mix 30 mL of vitamin D, 20 mL of Span-80, 10 mL of polyglycerol fatty acid ester, and 5 mL of polyoxyethylene hydrogenated castor oil until homogeneous. Then add 20 mL of propolis ethanol extract prepared in step S1 of Example 1 and mix until homogeneous. Add 0.1 g of saponin QS-21 and finally add water for injection to make up to 100 mL. Stir at 800 rpm for 30 min to fully mix and emulsify. Filter with a 0.22 μm filter to remove bacteria and obtain poultry nano-propolis compound adjuvant.

[0054] Example 3: A method for preparing a poultry nano-propolis composite adjuvant, comprising the following preparation steps: Mix 30 mL of vitamin D, 20 mL of Span-80, 10 mL of polyglycerol fatty acid ester, and 5 mL of polyoxyethylene hydrogenated castor oil until homogeneous. Then add 20 mL of propolis ethanol extract prepared in step S1 of Example 1 and mix until homogeneous. Next, add 0.1 g of DDA and 5 mL of Mycobacterium tumefaciens extract prepared in step S2 of Example 1. Finally, add water for injection to make up to 100 mL. Stir at 800 rpm for 30 min to fully mix and emulsify. Filter with a 0.22 μm filter to remove bacteria and obtain poultry nano-propolis compound adjuvant.

[0055] The present invention tested the particle size, safety and other indicators of the poultry nano-propolis composite adjuvants prepared in Examples 1 to 3, and the test results are as follows.

[0056] (1) Determination of particle size and particle size distribution of adjuvants According to the detection method provided in the Pharmacopoeia of the People's Republic of China (2020 Edition) (Part IV) General Chapter 0982, the particle size and particle size distribution of the poultry nano-propolis compound adjuvants prepared in Examples 1 to 3 of this invention are determined.

[0057] Test results as follows Figure 3 , Figure 4 and Figure 5 As shown, the particle sizes of the poultry nano-propolis composite adjuvants prepared in Examples 1 to 3 are 145~221nm, 145~255nm and 126~294nm, respectively, all within the range of 100~300nm, indicating that the poultry nano-propolis composite adjuvants prepared in this invention are nanoscale products.

[0058] (2) Sterility test According to the sterility test method for semi-finished products in Appendix 3306 of the Veterinary Pharmacopoeia of the People's Republic of China (2020 Edition) (Part III), the poultry nano-propolis compound adjuvants prepared in Examples 1 to 3 were subjected to sterility testing.

[0059] The results are shown in Table 6; no bacterial growth was observed.

[0060] Table 6. Sterility test of nano-propolis compound adjuvant Note: TG represents thioglycolate fluid medium, GA represents casein agar medium, TSB represents tryptic soy broth medium, and - represents aseptic growth.

[0061] (3) Safety inspection Safety tests were conducted on the poultry nano-propolis composite adjuvants prepared in Examples 1 to 3.

[0062] Mouse method experiment: Ten female mice, weighing 18-22 g, were randomly divided into a blank control group and an experimental group. Each mouse in the blank control group was injected intramuscularly with 0.5 mL of sterile saline. Each mouse in the experimental group was injected intramuscularly with 0.5 mL of the poultry nano-propolis composite adjuvant solution prepared in Examples 1-3. The mice were observed for 7 days. Animal weight was measured before and at the end of the experiment.

[0063] Guinea pig method experiment: Four guinea pigs, weighing 250-350 g, were randomly divided into a blank control group and an experimental group. Each guinea pig in the blank control group was injected intramuscularly with 1.0 mL of sterile physiological saline. Each guinea pig in the experimental group was injected with 1.0 mL of the poultry nano-propolis composite adjuvant solution prepared in Examples 1-3, and observed for 7 days.

[0064] The experimental results are shown in Tables 7-10. During the observation period after injection of the avian nano-propolis compound adjuvant solution, all mice and guinea pigs survived without any abnormal reactions; no swelling, necrosis, or abscesses were observed at the injection site. At the end of the observation period, the body weight of each mouse and guinea pig significantly increased, and there was no significant difference compared to the blank control group, indicating that the adjuvant is safe in mice and guinea pigs.

[0065] Table 7 Safety test results in mice Table 8 Safety tests on guinea pigs Table 9 Safety test results in mice Table 10 Safety tests on guinea pigs Example 4: A method for preparing a poultry nano-propolis compound adjuvant vaccine, comprising the following preparation steps: S1. Preparation of Newcastle disease virus solution 1. Preparation of Newcastle disease virus antigen solution The strain used in production is the Newcastle disease virus LaSota strain, which is identified, stored, and supplied by the China Institute of Veterinary Drug Control.

[0066] (1) Inoculation: Take the production strain and dilute it with sterile physiological saline at 10 ppm. 3 0.1 mL of the solution was injected into the allantoic cavity of 10-day-old susceptible chicken embryos. After injection, the needle hole was sealed and the embryos were incubated at 37°C.

[0067] (2) Incubation and observation: After the chicken embryos are inoculated, they are candled once every 24 hours. Chicken embryos that die within 48 hours are discarded. After that, they are candled once every 24 hours. Dead embryos are removed at any time. At 96 hours, regardless of whether they are dead or not, all embryos are removed, with the air cell facing upward, and placed at 4°C to cool for 12 hours.

[0068] (3) Harvesting: Remove the cooled chicken embryos and harvest the embryonic fluid (harvest live embryos first, then dead embryos). Place the harvested embryonic fluid in a sterile container and sample to determine the HA titer. Discard any embryos with an HA titer lower than 1:256. Perform sterility testing according to the current Chinese Veterinary Pharmacopoeia; no sterile growth should be observed. Store the harvested embryonic fluid below -15℃ before inactivation.

[0069] (4) Concentration: After centrifuging the harvested chicken embryo virus solution at 2-8℃ to remove residues and other particulate impurities, concentrate it using an ultrafiltration concentrator until the HA titer is not less than 1:2048. At the same time, perform sterility testing according to the current Chinese Veterinary Pharmacopoeia, and no sterile growth should be observed. A sample is retained for virus titer testing. The concentrated chicken embryo virus solution is then inactivated.

[0070] (5) Inactivation: The concentrated chicken embryo virus solution of Newcastle disease was introduced into an inactivation tank, and 10% formaldehyde solution was added. The mixer was turned on to stir and mix thoroughly. The final concentration of formaldehyde was 0.1%. After adding the formaldehyde solution, the solution was transferred to another inactivation tank to prevent the virus adhering near the tank opening from failing to come into contact with the inactivating agent. Inactivation was carried out at 37°C for 16 hours (starting the timer when the temperature inside the tank reached 37°C, and stirring was turned on continuously). After that, the solution was removed and stored at 4°C.

[0071] 2. Newcastle disease virus antigen solution testing (1) HA titer: Take Newcastle disease virus antigen solution and determine it according to the appendix of the current Chinese Veterinary Pharmacopoeia. The HA titer should not be less than 1:1024.

[0072] (2) Virus content: The concentrated chicken embryo virus solution taken before inactivation was serially diluted 10 times, and 10 were taken. -7 10 -8 10 -9Three dilutions were applied, with 0.1 mL inoculated into each allantoic cavity of five 10-day-old SPF chicken embryos. The embryos were incubated at 37°C. Embryos that died within 48 hours were discarded. Embryos that died between 48 and 120 hours were removed immediately, and the embryonic fluid was harvested. Observation continued until 120 hours. HA titers were measured in both dead and live embryos. An HA titer of at least 1:128 was considered an infection. The EID was calculated using the Reed-Muench method. 50 Virus content ≥10⁸.³ EID per 0.1 mL 50 Only when the time is right can it be used for seedling production.

[0073] (3) Sterility test: Take the inactivated concentrated chicken embryo virus solution and perform a sterility test according to the appendix of the current Chinese Veterinary Pharmacopoeia. It should show no sterile growth.

[0074] (4) Inactivation test: Take 5 10-day-old SPF chicken embryos, inoculate the allantoic cavity with 0.2 mL of inactivated virus solution per embryo, and incubate at 37°C. Candle the embryos twice a day and observe for 120 hours. No more than 1 chicken embryo should die non-specifically. Measure the HA titer of all chicken embryo solutions. The titer should be negative. Perform one blind passage and observe for 120 hours. Measure the HA titer again. If there is still no hemagglutination, the inactivation is considered complete.

[0075] S2, Avian Influenza Virus (H9 subtype) Antigen Solution 1. Preparation of avian influenza virus (H9 subtype) liquid The avian influenza virus (H9 subtype) JY strain was isolated, identified, stored and supplied by Shandong Lvdou Biotechnology Co., Ltd.

[0076] (1) Inoculation: Take the production strain and dilute it with sterile physiological saline at 10 ppm. 3 0.1 mL of the solution was injected into the allantoic cavity of 10-day-old susceptible chicken embryos. After injection, the needle hole was sealed and the embryos were incubated at 37°C.

[0077] (2) Incubation and observation: After the chicken embryos are inoculated, they are candled once every 24 hours. Chicken embryos that die within 48 hours are discarded. After that, they are candled once every 24 hours. Dead embryos are removed at any time. At 96 hours, regardless of whether they are dead or not, all embryos are removed, with the air cell facing upward, and placed at 4°C to cool for 12 hours.

[0078] (3) Harvesting: Remove the cooled chicken embryos and harvest the embryonic fluid (harvest live embryos first, then dead embryos). Place the harvested embryonic fluid in a sterile container and sample to determine the HA titer. Discard any embryos with an HA titer lower than 1:256. Perform sterility testing according to the current Chinese Veterinary Pharmacopoeia; no sterile growth should be observed. Store the harvested embryonic fluid below -15℃ before inactivation.

[0079] (4) Concentration: After centrifuging the harvested chicken embryo virus solution at 2-8℃ to remove residues and other particulate impurities, concentrate it using an ultrafiltration concentrator until the HA titer is not less than 1:2048. At the same time, perform sterility testing according to the current Chinese Veterinary Pharmacopoeia, and no sterile growth should be observed. A sample is retained for virus titer testing. The concentrated chicken embryo virus solution is then inactivated.

[0080] (5) Inactivation: The concentrated chicken embryo virus solution of avian influenza virus (H9 subtype) is introduced into the inactivation tank, and 10% formaldehyde solution is added in measured amounts. The mixer is turned on to stir and mix thoroughly. The final concentration of formaldehyde is 0.1%. After adding the formaldehyde solution, the solution is transferred to another inactivation tank to prevent the virus adhering near the tank opening from failing to come into contact with the inactivating agent. Inactivate at 37°C for 16 hours (start timing from when the temperature inside the tank reaches 37°C, and stir continuously with the mixer on), then remove and store at 4°C.

[0081] 2. Avian influenza virus (H9 subtype) antigen test (1) HA titer: Take concentrated chicken embryo virus solution and determine it according to the appendix of the current Chinese Veterinary Pharmacopoeia. The HA titer should not be less than 1:1024 before it can be used for vaccine production.

[0082] (2) Virus content: The concentrated chicken embryo virus solution before inactivation was serially diluted 10 times, and 10 were taken. -6 10 -7 and 10 -8 Three dilutions were used, with 0.1 mL of 0.1 mL inoculated into each 10-day-old SPF chicken embryo intravenously. The embryos were incubated at 37°C. Embryos that died within 24 hours were discarded. Candling was performed twice daily. Embryos that died within 24-120 hours were removed, and the embryonic fluid was harvested. Observation continued until 120 hours. The HA titer was measured for both live and dead embryos. An HA titer of at least 1:16 was considered an infection. The EID was calculated using the Reed-Muench method. 50 The viral load per 0.1 mL is ≥107.7 EID. 50 Only when the time is right can it be used for seedling production.

[0083] (3) Sterility test: Take the inactivated concentrated chicken embryo virus solution and perform a sterility test according to the appendix of the current Chinese Veterinary Pharmacopoeia. It should show no sterile growth.

[0084] (4) Inactivation test: Take 5 10-day-old SPF chicken embryos, inoculate the allantoic cavity with 0.2 mL of inactivated virus solution per embryo, and incubate at 37°C. Candle the embryos twice a day and observe for 120 hours. No more than 1 chicken embryo should die nonspecifically. Measure the HA titer of all chicken embryo solutions. The titer should be negative. Perform one blind passage and observe for 120 hours. Measure the HA titer again. If there is no hemagglutination, the inactivation is considered complete.

[0085] S3. Preparation of poultry nano-propolis compound adjuvant vaccine The Newcastle disease virus antigen solution prepared in step S1 and the avian influenza (H9 subtype) antigen solution prepared in step S2 were mixed at a volume ratio of 1:1 to form the antigen phase.

[0086] The antigen phase and the poultry nano-propolis compound adjuvant prepared in Example 1 were stirred and mixed evenly at a volume ratio of 3:1 to obtain the Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, namely the poultry nano-propolis compound adjuvant vaccine, each bottle containing 250mL.

[0087] Example 5: A method for preparing a poultry nano-propolis compound adjuvant vaccine, comprising the following preparation steps: The antigen phase prepared in step S3 of Example 4 and the poultry nano-propolis compound adjuvant prepared in Example 2 were mixed evenly at a volume ratio of 3:1 to obtain the Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, namely the poultry nano-propolis compound adjuvant vaccine, each bottle containing 250mL.

[0088] Example 6: A method for preparing a poultry nano-propolis compound adjuvant vaccine, comprising the following preparation steps: The antigen phase prepared in step S3 of Example 4 and the poultry nano-propolis compound adjuvant prepared in Example 3 were stirred and mixed evenly at a volume ratio of 3:1 to obtain the Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, namely the poultry nano-propolis compound adjuvant vaccine, each bottle containing 250mL.

[0089] Comparative Example 1: A method for preparing a poultry nano-propolis compound adjuvant vaccine, comprising the following preparation steps: The antigen phase and physiological saline prepared in step S3 of Example 4 were mixed with the propolis ethanol extract prepared in step S1 of Example 1 at a volume ratio of 75:20:5 to obtain a Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, namely, a poultry nano-propolis compound adjuvant vaccine, each bottle containing 250mL.

[0090] Comparative Example 2: A method for preparing a poultry nano-propolis compound adjuvant vaccine, comprising the following preparation steps: S1. Preparation of poultry nano-propolis compound adjuvant: Mix 30 mL of white oil, 20 mL of Span-80 and 15 mL of Tween-80 evenly, then add 20 mL of propolis ethanol extract prepared in step S1 of Example 1 and mix evenly. Then add 0.1 g of saponin A, and finally add water for injection to make up to 100 mL. Stir at 800 rpm for 30 min to fully mix and emulsify. Filter with a 0.22 μm filter to remove bacteria and obtain poultry nano-propolis compound adjuvant.

[0091] S2. Preparation of poultry nano-propolis compound adjuvant vaccine: The antigen phase prepared in step S3 of Example 4 and the poultry nano-propolis compound adjuvant prepared in step S1 of this comparative example are mixed evenly at a volume ratio of 3:1 to obtain a Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, namely poultry nano-propolis compound adjuvant vaccine, each bottle containing 250mL.

[0092] Comparative Example 3: A method for preparing a poultry nano-propolis compound adjuvant vaccine, comprising the following preparation steps: S1. Preparation of poultry nano-propolis composite adjuvant: Mix 30 mL of vitamin D, 20 mL of Span-80 and 15 mL of Tween-80 evenly, then add 20 mL of the propolis ethanol extract prepared in step S1 of Example 1 and mix evenly. Then add 0.1 g of saponin QS-21 and finally add water for injection to make up to 100 mL. Stir at 800 rpm for 30 min to fully mix and emulsify. Filter with a 0.22 μm filter to remove bacteria and obtain poultry nano-propolis compound adjuvant.

[0093] S2. Preparation of poultry nano-propolis compound adjuvant vaccine: The antigen phase prepared in step S3 of Example 4 and the poultry nano-propolis compound adjuvant prepared in step S1 of this comparative example are mixed evenly at a volume ratio of 3:1 to obtain a Newcastle disease-avian influenza (H9 subtype) bivalent inactivated vaccine, namely poultry nano-propolis compound adjuvant vaccine, each bottle containing 250mL.

[0094] The present invention tested the properties and other indicators of the poultry nano-propolis compound adjuvant vaccines prepared in Examples 4-6 and Comparative Examples 1-3, and the results are as follows: The results of the property test and the fill quantity test are as follows: Figure 6 As shown, the poultry nano-propolis compound adjuvant vaccines of Examples 4 to 6 exhibit a pale yellow, uniform, and semi-transparent suspension appearance; while the poultry nano-propolis compound adjuvant vaccine of Comparative Example 1 is pale yellow in appearance and shows precipitation after being placed at 4°C for 2 weeks; the poultry nano-propolis compound adjuvant vaccines of Comparative Examples 2 and 3 show stratification after being placed at 4°C, which does not meet the vaccine appearance test standards. Therefore, the vaccines of Comparative Examples 2 and 3 were not further subjected to subsequent tests such as viscosity, formaldehyde residue, fill volume, safety, and efficacy.

[0095] (1) The viscosity of the poultry nano-propolis composite adjuvant vaccines prepared in Examples 4 to 6 and Comparative Example 1 is less than 20 cP, the formaldehyde residue is less than 0.05%, and the filling volume is between 251.3 mL and 252.0 mL.

[0096] (2) Sterility test: The poultry nano-propolis compound adjuvant vaccines prepared in Examples 4 to 6 and Comparative Example 1 were all sterile.

[0097] (3) Safety test: 1 mL of the poultry nano-propolis compound adjuvant vaccine prepared in Examples 4-6 and Comparative Example 1 was injected subcutaneously into the neck of 14-day-old SPF chickens to conduct a safety test. A blank control group without treatment was also set up. Chickens were observed for 14 days after injection. During the observation period, all experimental chickens showed normal spirit, feed intake, and water consumption. After 14 days, the chickens were culled. No abnormalities were observed in any organs, and there was no unabsorbed vaccine, swelling, or ulceration at the injection site. The results indicate that the poultry nano-propolis compound adjuvant vaccines prepared in Examples 4-6 and Comparative Example 1 all exhibited good safety.

[0098] (4) Efficacy test: The efficacy of the poultry nano-propolis compound adjuvant vaccine was tested using 21-day-old SPF chickens.

[0099] After immunizing 10 SPF chickens with the poultry nano-propolis compound adjuvant vaccines prepared in Examples 4-6, the HI antibody titers against Newcastle disease virus (NDV) ranged from 1:16 to 1:64, with average values ​​of 1:29.9, 1:27.9, and 1:26, respectively, meeting the protection standard for NDV vaccines. However, after immunizing SPF chickens with the vaccine prepared in Comparative Example 1, the HI antibody titers against NDV ranged from 1:2 to 1:8, with an average value of 1:4, failing to meet the protection standard for NDV vaccines. Statistical t-test analysis showed no significant difference between Examples 4-6, and no significant difference between Examples 4 and 5. p The value is 0.773, between Example 4 and Example 6. p The value is 0.535, between Example 5 and Example 6. p The value is 0.764. Furthermore, Examples 4-6 and Comparative Example 1... p The values ​​were all less than 0.001, indicating that the immunizing effect of the poultry nano-propolis compound adjuvant vaccine prepared in Examples 4-6 was significantly different from that of the poultry nano-propolis compound adjuvant vaccine prepared with propolis ethanol extract in Comparative Example 1. In addition, the HI antibody titers of the blank control group were all ≤1:2 (Table 11).

[0100] Table 11. HI antibody titer of Newcastle disease virus in chickens after vaccination. Note: In the table, the same letter indicates no significant difference between groups, different letters indicate significant differences between groups, and *** indicates... p <0.001.

[0101] After immunizing SPF chickens with the poultry nano-propolis compound adjuvant vaccines prepared in Examples 4-6, the HI antibody titers against avian influenza (H9) virus ranged from 1:64 to 1:256, with average values ​​of 1:104, 1:97, and 1:147, respectively, meeting the protection standard for avian influenza (H9) vaccines. However, after immunizing SPF chickens with the vaccine prepared in Comparative Example 1, the HI antibody titers against avian influenza (H9) virus ranged from 1:1 to 1:4, with an average value of 1:3, failing to meet the protection standard for avian influenza (H9) vaccines. Statistical analysis using t-tests showed no significant differences between Examples 4-6, and no significant differences between Examples 4 and 5. p The value is 0.79, between Example 4 and Example 6. p The value is 0.105, between Example 5 and Example 6. p The value was 0.06. Furthermore, Examples 4-6 and Comparative Example 1... p The values ​​were all less than 0.001, indicating that the immunizing effect of the poultry nano-propolis compound adjuvant vaccine prepared in Examples 4-6 was significantly different from that of the poultry nano-propolis compound adjuvant vaccine prepared with propolis ethanol extract in Comparative Example 1. In addition, the HI antibody titers of the blank control group were all ≤1:2 (Table 12).

[0102] Table 12. HI antibody titers after avian influenza (H9) vaccination Note: In the table, the same letter indicates no significant difference between groups, different letters indicate significant differences between groups, and *** indicates... p <0.001.

[0103] Example 7: A method for preparing a poultry nano-propolis compound adjuvant vaccine, comprising the following preparation steps: S1. Preparation of inoculum for chicken infectious coryza (type A) vaccine The strain used for production and testing is Haemophilus paragallinarum type A C-Hpg-8, which is identified, stored and supplied by the China Institute of Veterinary Drug Control.

[0104] Seed preparation for production: The bacterial strain was streaked onto chicken broth agar plates and incubated at 37°C for 16-18 hours in an environment containing 5% CO2. Typical colonies with strong fluorescence were then selected and inoculated into the yolk sacs of 5-day-old chicken embryos. The embryos were incubated at 37°C for an additional 30 hours. The yolk fluid from chicken embryos that died within 30 hours was collected as primary seed and stored at -20°C.

[0105] Take the yolk fluid from infected chicken embryos, streak it onto chicken broth agar plates, and incubate at 37°C for 16 hours under conditions containing 5% CO2. Select typical colonies with strong fluorescence and inoculate them into chicken broth medium, then incubate at 37°C for 16 hours as secondary seed culture.

[0106] The secondary seed culture was added to a semi-synthetic culture medium (1000mL of semi-synthetic culture medium contains 5g of polypeptone, 30g of casein, 15g of monosodium glutamate, 5g of yeast extract, 3g of glucose, 15g of sodium chloride, and the remainder of distilled water; the pH was adjusted to 7.2-7.4; before use, 5% inactivated healthy chicken serum and 0.8% of 0.5% coenzyme I were added). The culture was incubated at 37℃ for 18 hours, with the culture flask shaken twice during the incubation period. After purity testing confirmed the absence of contamination, 0.05% formaldehyde solution was added and the culture was stored at 2℃.

[0107] Collect bacterial cells by centrifugation or concentrate them using a hollow fiber ultrafiltration device, and then prepare a suspension with phosphate buffer at pH 7.2 so that each 1 mL contains at least 5 billion bacterial cells (compare with the national standard for biological products, "Chinese Bacterial Turbidity Standard", assuming the standard tube contains 2.8 billion Haemophilus paragallinarum / mL). After passing the purity test, inactivate the bacteria.

[0108] Add 0.15% formaldehyde solution and 0.01% thimerosal to the concentrated bacterial culture solution by volume, sterilize at 2°C for 7 days, and use the sterile growth result as the vaccine antigen.

[0109] Semi-finished product inspection: Sterility test: Take the inactivated concentrated vaccine culture solution and perform a sterility test according to the appendix of the current Chinese Veterinary Pharmacopoeia. No sterile growth was found.

[0110] Test results of bacterial culture for seedling preparation: After 18 hours of incubation, two batches (batch 001 and batch 002) of bacterial culture were randomly selected for testing. The harvest volumes of the bacterial culture for batch 001 and batch 002 were 2,700 mL and 11,000 mL, respectively, and their viable cell counts were 12 × 10⁻⁶. 8 CFU / mL and 18×10 8 CFU / mL, with a total bacterial count of 56×10⁻⁶. 8 CFU / mL and 58×10 8 CFU / mL. After centrifugation, the bacteria were collected and resuspended in PBS, resulting in concentrated bacterial cultures of batch 001 and batch 002 of 180 mL and 730 mL, respectively. The total bacterial count (turbidity count) was 840 × 10⁻⁶ CFU / mL. 8 CFU / mL and 870×10 8 The CFU / mL level meets the requirements for seedling preparation.

[0111] Inactivation test results of concentrated bacterial solution for seedling preparation: After inactivation with formaldehyde solution, the concentrated bacterial solution for seedling preparation was inoculated into semi-synthetic liquid culture medium and solid culture medium, and no sterile growth was observed after incubation.

[0112] The sterility test results of the semi-finished product of the bacterial solution for seedling production were as follows: According to the sterility test of the semi-finished product in the appendix of the current Chinese Veterinary Pharmacopoeia, the concentrated bacterial solution for seedling production, which was inactivated by formaldehyde solution, was inoculated into Fluid Thioglycollate Medium (TG), Peptone from Casein Agar Medium (GA), and Trypticase Soy Broth (TSB) for sterility test of the semi-finished product. No sterile growth was observed in all cases.

[0113] S2. The nano-propolis composite adjuvant prepared in Example 3 and the chicken infectious coryza inactivated antigen prepared in step S1 of this Example are added to the mixing tank in a ratio of 1:3. The stirring motor of the mixing tank is turned on and stirred for 40 minutes until the mixture is uniform.

[0114] Comparative Example 4: A method for preparing a poultry nano-propolis compound adjuvant vaccine, comprising the following preparation steps: The propolis ethanol extract prepared in step S1 of Example 1, the chicken infectious coryza inactivated antigen prepared in step S1 of Example 7, and physiological saline were added to the mixing tank at a volume ratio of 5:75:20. The stirring motor of the mixing tank was turned on and stirred for 40 minutes until the mixture was uniform.

[0115] The present invention further tested the relevant traits of the poultry nano-propolis compound adjuvant vaccines prepared in Example 7 and Comparative Example 4, and the results are as follows: (1) Inspection of vaccine characteristics and dosage The poultry nano-propolis compound adjuvant vaccines prepared in Example 8 and Comparative Example 4 were both uniform, pale yellow, and semi-transparent water-soluble suspensions with viscosities of 30.2 cP and 36.3 cP, respectively; formaldehyde content was below 0.04%, and the dosage met the standard requirements.

[0116] (2) Sterility test results The aseptic test of the avian nano-propolis compound adjuvant vaccine was performed according to the method in the appendix of the current Chinese Veterinary Pharmacopoeia, and the results showed no bacterial growth.

[0117] (3) Safety test results Eight 70-day-old SPF chickens were subcutaneously injected with 1.0 mL of avian nano-propolis compound adjuvant vaccine. After 14 consecutive days of observation, no local or systemic adverse reactions caused by the avian nano-propolis compound adjuvant vaccine were observed, and the results were deemed qualified.

[0118] (4) Results of efficacy test Twenty 70-day-old SPF chickens were randomly divided into three groups: experimental group 1 and experimental group 2, each with 8 chickens; and a blank control group with 4 chickens. Each chicken in experimental group 1 was subcutaneously injected with 0.5 mL of the poultry nano-propolis compound adjuvant vaccine from Example 8; each chicken in experimental group 2 was subcutaneously injected with 0.5 mL of the poultry nano-propolis compound adjuvant vaccine from Comparative Example 4; and the untreated control group served as the blank control group. One month later, each chicken in experimental groups 1 and 2, along with the blank control group under identical conditions, was injected into the subframe sinus with 0.2 mL of C-Hpg-8 bacterial solution (5.0 × 10⁻⁶ viable bacteria). 5 (CFU), observed for 14 days. In the blank control group, all 4 chickens developed the disease, showing swelling of the infraorbital sinus and surrounding area on one or both sides, along with nasal discharge or tearing; in experimental group 2, 1 out of 8 chickens developed the disease, while the remaining 7 chickens showed no clinical symptoms after immunization; while in experimental group 1, all 8 chickens showed no clinical symptoms and were completely protected; indicating that the immunization effect of the poultry nano-propolis compound adjuvant vaccine prepared in Example 8 is stronger than that of the poultry nano-propolis compound adjuvant vaccine prepared in Comparative Example 4.

[0119] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, preferred embodiments are described in this invention. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0120] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A nano-propolis compound adjuvant for poultry, characterized in that, The poultry nano-propolis compound adjuvant is composed of the following components by volume percentage: 10%~30% propolis ethanol extract, 30%~45% immune enhancer, and 30%~50% surfactant. The dry matter content of propolis in the propolis ethanol extract is 50 mg / mL to 70 mg / mL; The immune enhancer is composed of immune enhancer A and immune enhancer B mixed in a volume ratio of 5-8:1; immune enhancer A is white oil or vitamin D, and immune enhancer B is a mixture of Mycobacterium tumefaciens extract and dimethyl dioctadecyl ammonium bromide; the Mycobacterium tumefaciens extract is obtained by lysing inactivated Mycobacterium tumefaciens cells at 800-1000 bar and centrifuging at 11000-13000 rpm for 25-35 minutes to obtain the supernatant; the volume ratio of the Mycobacterium tumefaciens extract to dimethyl dioctadecyl ammonium bromide is 100:1-30; The surfactant is obtained by mixing polyglycerol fatty acid ester, Span-80 and polyoxyethylene hydrogenated castor oil in a volume ratio of 7~9:3~5:

2.

2. The application of the poultry nano-propolis composite adjuvant as described in claim 1 in the preparation of poultry inactivated vaccines or subunit vaccines.

3. A poultry nano-propolis compound adjuvant vaccine, characterized in that, The vaccine comprises the poultry nano-propolis compound adjuvant as described in claim 1 and an inactivated antigen or subunit antigen of a pathogenic microorganism infecting poultry; the antigen is Newcastle disease virus, avian influenza virus, or Haemophilus paragallinarum.

4. The vaccine according to claim 3, characterized in that, The volume ratio of the antigen to the avian nano-propolis adjuvant is 2-4:1.

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