Application of oridonin in preparation of poultry vaccine adjuvant

Abstract

The present application relates to the technical field of veterinary biological products, in particular to application of oridonin in preparation of poultry vaccine adjuvant. The poultry vaccine adjuvant is composed of oridonin, solvent and buffer. Experiments prove that after mixing the poultry vaccine adjuvant with poultry inactivated vaccine (such as Newcastle disease vaccine) for immunization, the humoral immune level of poultry can be significantly improved (HI titer of 21d after the second immunization is increased by more than 1.5 log2), and the secretion level of specific IgY (systemic immunity) and IgA (mucosal immunity) in serum can be extremely significantly up-regulated, and the high level of antibody is maintained for more than 4 weeks longer than that of the conventional vaccine group. Compared with commercial adjuvant ISA 201, the present application has better immune enhancement activity, high safety, no adverse reaction at injection site, and wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, specifically to the application of oridonin in the preparation of avian vaccine adjuvants, and more particularly to an avian immunization vaccine containing oridonin as a vaccine adjuvant, its preparation method, and its application in enhancing the avian vaccine immune response. Background Technology

[0002] Vaccination is the most effective means of preventing and controlling infectious diseases in poultry (such as Newcastle disease and avian influenza). To improve the immunogenicity of inactivated vaccines, adjuvants are usually added. Currently, the most widely used adjuvants for poultry in clinical practice are white oil adjuvants and aluminum adjuvants.

[0003] However, existing adjuvants have significant limitations. While white oil adjuvants can produce high antibody levels, they are water-in-oil emulsions with high viscosity, resulting in slow absorption after injection. This can easily lead to redness, necrosis, or the formation of persistent, non-absorbable granulomas (indurations) at the injection site, harming animal welfare and severely impacting post-slaughter meat quality. Aluminum adjuvants, although relatively safe, tend to induce humoral immunity, making it difficult to induce a strong cellular immune response. Therefore, they offer insufficient protection against certain viral diseases requiring synergistic cellular immune protection. Traditional adjuvants also induce antibody production relatively slowly, making it difficult for animals to rapidly establish an immune barrier during periods of high disease incidence.

[0004] Oridonin, its structural formula is as follows: Oridonin is an enantio-kaurane-type tetracyclic diterpenoid natural organic compound extracted from plants of the genus *Oryza sativa* in the Lamiaceae family. Its chemical formula is C1. 20 H 28 O6. Existing studies have shown that oridonin A possesses pharmacological activities such as antitumor, antibacterial, and anti-inflammatory effects. However, there are currently no publicly reported applications of oridonin A in the field of avian vaccine adjuvants, particularly in enhancing humoral and cellular immune responses and improving the safety of inactivated vaccines such as Newcastle disease vaccines. Summary of the Invention

[0005] The purpose of this invention is to address the problems of low safety, slow absorption, and weak cellular immune activation of existing poultry adjuvants by providing an application of oridonin in the preparation of poultry vaccine adjuvants. Specifically, it provides a novel poultry vaccine adjuvant based on oridonin that is highly effective, safe, and can simultaneously induce strong humoral and cellular immunity.

[0006] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides the application of oridonin in the preparation of avian vaccine adjuvants.

[0007] As a preferred embodiment, the concentration of oridonin A in the poultry vaccine adjuvant is 5-15 mg / mL; more preferably 10 mg / mL.

[0008] As a preferred embodiment, the poultry includes domestic fowl and birds of prey. Specifically, the domestic fowl includes chickens, ducks, geese, turkeys, pigeons, quails, etc.; the birds of prey include geese, eagles, peacocks, egrets, seagulls, etc.

[0009] Secondly, the present invention provides a poultry vaccine adjuvant, the components of which include oridonin, solvent and buffer solution.

[0010] As a preferred embodiment, the concentration of oridonin A in the poultry vaccine adjuvant is 10 mg / mL.

[0011] As a preferred embodiment, the solvent is DMSO; the volume percentage of the solvent in the poultry vaccine adjuvant is <1%.

[0012] As a preferred embodiment, the buffer solution is PBS.

[0013] As a preferred embodiment, the concentration of the PBS is 0.005-0.05 M and the pH is 7.0-8.0.

[0014] Thirdly, the present invention provides a method for preparing the aforementioned poultry vaccine adjuvant, comprising the following steps: dissolving oridonin in a solvent, adding the resulting oridonin solution to a buffer solution, and stirring until homogeneous to obtain the poultry vaccine adjuvant.

[0015] As a preferred embodiment, the specific steps for dissolving oridonin in the solvent are as follows: adding oridonin to the solvent and using intermittent sonication until completely dissolved; the intermittent sonication specifically refers to sonicating for 5-10 minutes with intervals of 3-5 seconds and 2-3 seconds.

[0016] As a preferred embodiment, the oridonin A solution is added dropwise to the buffer solution at a rate of 5-20 μL / min.

[0017] Fourthly, the present invention provides a poultry vaccine, comprising an inactivated poultry vaccine and the aforementioned poultry vaccine adjuvant.

[0018] As a preferred embodiment, the volume ratio of the inactivated poultry vaccine to the poultry vaccine adjuvant is 1:1.

[0019] As a preferred embodiment, the viral antigen of the inactivated poultry vaccine includes at least one of avian Newcastle disease virus, avian influenza virus, and duck serositis virus.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention is the first to discover the application of the small molecule compound oridonin in the preparation of avian vaccine adjuvants. Experimental verification showed that the avian vaccine adjuvant prepared using the small molecule compound oridonin, when mixed with avian inactivated vaccines (such as Newcastle disease vaccines) for immunization, can significantly improve the humoral immunity level of poultry (the HI titer increases by more than 1.5 log2 at 21 days after the second immunization), and can significantly upregulate the secretion levels of specific IgY (systemic immunity) and IgA (mucosal immunity) in serum, and the antibody maintains a high level for more than 4 weeks longer than that in the conventional vaccine group.

[0021] 2) The avian immune adjuvant prepared by the present invention using oridonin has better immune-enhancing activity than commercial adjuvants (such as ISA 201), can induce strong humoral immunity and cellular immunity at the same time, and has high safety with no adverse reactions at the injection site, and has broad clinical application prospects. Attached Figure Description

[0022] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a graph showing the changes in the weight gain rate of chicks in each group after immunization in Example 1; Figure 2 The results of NDV-specific antibody titers in each group of chicks after immunization in Example 2; Figure 3 This is a line graph showing the changes in serum IgY concentration in each group of chicks after immunization in Example 2; Figure 4 This is a line graph showing the changes in serum IgA concentration after immunization of chicks in each group in Example 2. Detailed Implementation

[0023] The present invention will be described in detail below with reference to specific examples. These examples will help those skilled in the art to further understand the invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0024] Example 1: Biosafety evaluation of oridonin A adjuvant vaccine in target animals (1) Preparation of experimental materials Viral antigen: Newcastle disease virus (NDV) LaSota strain, harvested from SPF chicken embryos after inoculation and culture, inactivated by β-propiolactone to obtain NDV inactivated antigen solution, with a hemagglutination (HA) titer ≥2. 6The specific preparation process is as follows: Take 1000 doses of NDV-LaSota lyophilized vaccine, add 1 mL of sterile PBS buffer, and let stand at 4℃ for 1 hour to fully dissolve; add β-propiolactone to a final concentration of 0.10%, and incubate at 37℃ in a shaker for 18 hours to inactivate; after inactivation, hydrolyze at 4℃ for 1-4 hours, and then aliquot the obtained NDV inactivated antigen solution and store at -80℃.

[0025] Immunoadjuvant: Oridonin (purchased from MCE website), purity ≥99% (HPLC detection).

[0026] Excipients: Pharmaceutical grade anhydrous dimethyl sulfoxide (DMSO), 0.01M (pH 7.2) sterile phosphate buffer (PBS).

[0027] Control immune adjuvant: Montanide ISA 201 oil adjuvant (W / O / W type).

[0028] Preparation process of oridonin A adjuvant vaccine: 1.1 Preparation of oridonin mother liquor: Weigh 100 mg of oridonin powder, add 100 μL of anhydrous DMSO, and use an ultrasonic homogenizer (200W, 40kHz) to intermittently sonicate for 5 min (3s sonication, 2s interval) until completely dissolved to form a homogeneous and transparent mother liquor.

[0029] 1.2 Adjuvant working solution dilution: Under constant temperature and magnetic stirring, the stock solution was added dropwise to 9.9 mL of sterile PBS at a rate of 10 μL / min, and stirring was continued for 20 min. The resulting adjuvant working solution concentration was 10 mg / mL, with DMSO volume percentage <1%.

[0030] 1.3 Mixing of Antigen and Adjuvant: Following a dosage standard of 10 mg / kg (equivalent to 3 mg of oridonin per 0.3 mL of vaccine per Babcock chicken based on average chicken weight), the above adjuvant working solution was mixed with the NDV inactivated antigen solution at a 1:1 volume ratio. The mixture was stirred at 500 rpm for 30 min at 4°C to ensure complete adsorption of the induced components, thus obtaining the oridonin adjuvant vaccine.

[0031] Preparation of positive adjuvant vaccine: In accordance with the ISA 201 standard procedure, NDV inactivated antigen solution and Montanide ISA201 oil adjuvant were mixed at a ratio of 50:50 (w / w) and emulsified by high-speed shearing (10000 rpm) for 10 min.

[0032] Preparation of adjuvant-free vaccines: NDV inactivated antigen solution is mixed with sterile PBS at a volume ratio of 1:1 to eliminate solvent and operational interference.

[0033] (2) Experimental design and immunization procedure Healthy Babcock chicks aged 3-5 days (purchased from Jiangsu Boehringer Ingelheim Viton Biotechnology Co., Ltd., Newcastle disease virus negative) were selected and acclimatized to 7 days of age for their first immunization. The experiment was divided into four groups: non-immunized group (PBS), adjuvant-free group, positive adjuvant group, and novel adjuvant group (oridonin A adjuvant vaccine).

[0034] Vaccination schedule: Intramuscular injection, 0.3 mL / bird. The non-immunized group received sterile PBS, the unadjuvanted group received the unadjuvanted vaccine, the positive adjuvanted group received the positive adjuvanted vaccine, and the novel adjuvanted group received the oridonin A adjuvant vaccine; all vaccines were administered for the first time at 7 days of age, and a second dose at 14 days of age.

[0035] (3) Monitoring of clinical response and local reaction Systemic response: During the observation period of 7-42 days, all groups of chicks were in good spirits, with normal feed intake, water consumption, and excretion. There were no records of allergies, respiratory stress, or deaths, and the survival rate was 100%.

[0036] Local reactions: Autopsy results showed that the muscle at the injection site in the novel adjuvant group was normal in color, without necrosis, hemorrhage, or lesions. In the positive adjuvant group, the muscle at the injection site showed slight congestion and more obvious adjuvant cysts. Compared to the positive adjuvant group, no granulomas or adjuvant cysts were observed in the novel adjuvant group after the second immunization, indicating that oridonin has excellent biocompatibility and metabolic efficiency in tissues.

[0037] (4) Growth performance analysis The body weight of chicks in each group was measured at key immunization points (14 days, 21 days, 28 days, and 35 days of age), and the relative weight gain rate (RWGR) was calculated. The results showed ( Figure 1 The relative weight gain (RWGR) of each group showed a physiological decrease with increasing age, consistent with developmental patterns. At 14-21 days, 21-28 days, and 28-35 days of age, the relative weight gain rates of the novel adjuvant group were 60.53%, 44.48%, and 32.31%, respectively. Statistical analysis showed no significant differences between the novel adjuvant group and the control groups (P>0.05), confirming that the oridonine adjuvant had no inhibitory effect on the growth performance of the target animals.

[0038] Example 2: The enhancing effect of oridonin adjuvant on specific humoral immune responses to Newcastle disease in avian birds. (1) Experimental methods Babcock chickens were immunized with the vaccine using the same method described in Example 1. Wing vein serum was collected from chicks in each group before the first immunization (7 days old), 7 days after the second immunization, 7 days after the second immunization, 14 days after the second immunization, 21 days after the second immunization, and 28 days after the second immunization. The fluctuations in NDV-specific antibody titers were determined using a standard microhemagglutination inhibition (HI) test.

[0039] (2) Experimental results and analysis (e.g.) Figure 2 (As shown) Early response: 7 days after the first immunization (14 days of age), the HI titer in the new adjuvant group was significantly higher than that in the non-adjuvant group (P<0.05), effectively shortening the immunization gap period.

[0040] The second immunization resulted in a significant surge: Following the second immunization, the novel adjuvant group exhibited a marked "second surge." The antibody titer reached 4 log2 at 21 days of age (7 days after the second immunization) and peaked at 4.33 log2 at 28 days of age (14 days after the second immunization), which was significantly better than the unadjuvanted group (P<0.01) and numerically exceeded that of the positive adjuvant group.

[0041] Antibody persistence: At 35 days of age (21 days after the second immunization), the antibody titer in the novel adjuvant group was 3.83 log2, which was 1.5 log2 units higher than that in the positive adjuvant group. The antibody decline trend in the novel adjuvant group was significantly slower than that in the positive adjuvant group.

[0042] The results showed that oridonin A can significantly enhance the humoral immune effect of NDV inactivated vaccine, and has the advantages of rapid onset, high titer and long protection period.

[0043] Example 3: The regulatory effect of oridonin adjuvant on serum specific antibody secretion levels (1) Experimental methods Vaccination was performed according to the immunization procedure in Example 1, and venous serum was collected from chicks in each group. The concentrations of antigen-specific immunoglobulin Y (IgY, representing systemic immunity) and immunoglobulin A (IgA, representing mucosal immunity) in the serum were detected using an ELISA kit.

[0044] (2) Experimental results and analysis (results are as follows) Figure 3 and Figure 4 (As shown) Rapid induction of secretion: 7 days after the first immunization (14 days old), the levels of specific IgY and IgA induced by the novel adjuvant group increased rapidly and were significantly higher than those in the no-adjuvant group and the positive adjuvant group (P<0.05).

[0045] Secondary immune response activation: Antibody levels in all groups reached their peak 14 days (28 days of age) after the second immunization, consistent with the pattern of secondary immune response. The novel adjuvant group showed the most outstanding performance, with IgY concentration reaching 1595.9 μg / mL and IgA concentration reaching 382.3 μg / mL, both significantly higher than other groups (P<0.01).

[0046] The results of this embodiment show that oridonin not only significantly enhances systemic immunity (IgY), but also effectively induces mucosal immunity (IgA) secretion.

[0047] Conclusion: Oridonin significantly enhances the overall humoral immune response activated by the vaccine, and has significant technological advantages in enhancing the mucosal immune response.

[0048] It should be noted that, based on the foregoing results, the vaccine adjuvant prepared from oridonin can also be used in other avian virus vaccines (such as avian influenza virus and duck serositis virus), and is not limited to Newcastle disease virus. This invention will not list them all.

[0049] The above description of the embodiments is provided to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. The application of oridonin in the preparation of poultry vaccine adjuvants.

2. The application according to claim 1, characterized in that, In poultry vaccine adjuvants, the concentration of oridonin A is 5-15 mg / mL.

3. A poultry vaccine adjuvant, characterized in that, The components of the poultry vaccine adjuvant include oridonin, solvent, and buffer solution.

4. The poultry vaccine adjuvant according to claim 3, characterized in that, The concentration of oridonin A in the poultry vaccine adjuvant is 10 mg / mL.

5. The poultry vaccine adjuvant according to claim 3, characterized in that, The solvent is DMSO; the volume percentage of the solvent in the poultry vaccine adjuvant is <1%.

6. The poultry vaccine adjuvant according to claim 3, characterized in that, The buffer solution is PBS.

7. A method for preparing an avian vaccine adjuvant according to any one of claims 3-6, characterized in that, The process includes the following steps: dissolving oridonin A in a solvent, adding the resulting oridonin A solution to a buffer solution, and stirring until homogeneous to obtain the poultry vaccine adjuvant.

8. A poultry vaccine, characterized in that, Including inactivated poultry vaccines and poultry vaccine adjuvants as described in any one of claims 3-6.

9. The poultry vaccine according to claim 8, characterized in that, The volume ratio of the inactivated poultry vaccine to the poultry vaccine adjuvant is 1:

1.

10. The poultry vaccine according to claim 8, characterized in that, The viral antigens of the inactivated poultry vaccine include at least one of avian Newcastle disease virus, avian influenza virus, and duck serositis virus.

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