Veterinary vaccine composite adjuvant, and preparation method and application thereof

Abstract

The application discloses a kind of veterinary vaccine complex adjuvant and its preparation method and application, and the active ingredient in the vaccine complex adjuvant is manganese complex and Mincle agonist.The veterinary vaccine complex adjuvant of the application combines Mn with TDB, so that the immune enhancement effect of TDB adjuvant.The complex adjuvant can induce strong immune response reaction, significantly improve the immune effect, and use no residual damage.In the application, H7N9 subtype avian influenza inactivated virus is used as antigen, and the H7N9 vaccine prepared by mixing with the complex adjuvant can enhance the humoral immune and cellular immune response reaction of the body compared with DDA-TDB used alone, reduce viral load, improve the protection level, and provide product and technical support for the production of veterinary vaccine.

Description

Technical Field

[0001] This invention relates to a veterinary vaccine adjuvant, its preparation method, and its application, belonging to the field of animal vaccines. Background Technology

[0002] Vaccination remains a crucial measure for animal disease prevention and control. However, many candidate vaccines still suffer from poor immunogenicity, hindering their widespread application. To enhance vaccine immunogenicity and clinical application, there is an urgent need to research and discover safe and effective adjuvants to compensate for vaccine shortcomings and improve and enhance vaccine efficacy. Currently, many types of adjuvants are available for vaccines, but their clinical application is limited by factors such as high toxicity, high cost, and single function. In particular, oil-emulsion adjuvants (white oil) commonly used in veterinary vaccines suffer from poor metabolic capacity, limited enhancement of immune efficacy, and the need to improve the sustainability of antigen release. Furthermore, many veterinary oil-adjuvant vaccines induce short-term immune protection, failing to achieve long-term immunity. This is mainly due to weak cellular immunity induced by the vaccine or the lack of T cell helpers. Therefore, there is an urgent need to invent a novel adjuvant that is low-cost, has few side effects, high safety, and can simultaneously induce long-term immunity and enhance humoral and cellular immune responses to compensate for the shortcomings of oil-emulsion adjuvants, thereby improving the protective efficacy of existing veterinary vaccines.

[0003] Macrophage-inducible C-type lectin (Mincle) is primarily expressed on the surface of antigen-presenting cells, including macrophages, monocytes, dendritic cells, and neutrophils. It belongs to the atypical C-type lectin family and is encoded by Clec4e. Mincle plays multiple roles in the immune response, recognizing a range of different endogenous and exogenous ligands, including damaged cells, fungi, bacteria, viruses, and parasites, activating them to lead to the production of cytokines and chemokines, promoting or inhibiting inflammation. More importantly, Mincle can also induce adaptive immune responses, such as antigen-specific T-cell responses and antibody production. Trehalose 6,6'-disorbate (TDB), an agonist of Mincle, has been shown to produce an immunomodulatory effect in animal vaccines, although this effect is not yet strong enough.

[0004] Manganese (Mn) is an essential trace element that participates in various physiological processes and plays a vital role in life activities. In particular, the function of Mn as an inorganic salt adjuvant in vaccines is increasingly being explored. Studies have confirmed that Mn can activate the cGAS-STING pathway through interaction with cyclic GMP-AMP synthase (cGAS) to promote the production of type I interferon. Furthermore, Mn can promote the maturation and differentiation of antigen-presenting cells, thereby enhancing immunity by strengthening cellular and humoral immunity.

[0005] To date, there have been no reports on the application of Mincle agonists in combination with Mn as adjuvants for veterinary vaccines. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a veterinary vaccine compound adjuvant, its preparation method, and its application.

[0007] Technical solution: The present invention provides a veterinary vaccine adjuvant, wherein the active ingredients in the vaccine adjuvant are a manganese-containing complex and a Mincle agonist.

[0008] Furthermore, the manganese-containing complex contains divalent manganese ions.

[0009] Furthermore, the manganese-containing complex includes one or more of manganese chloride, manganese bromide, manganese iodide, manganese sulfate, manganese nitrate, manganese perchlorate, manganese acetate, manganese borate, manganese oxide, manganese dioxide, manganese tetroxide, manganese phosphate, or manganese carbonate.

[0010] Furthermore, the manganese-containing complex is preferably manganese chloride.

[0011] Furthermore, the Mincle agonist is derived from an extracellular ligand recognized by Mincle, which includes one or more of the following: trehalose 6,6'-dicarboxylate, trehalose 6,6'-dibenzyl ester, glycerol glycolipid, mannitol-coupled with mannitol, glycerol monomycinate, β-gentiobiose diglyceride, β-glucose diglyceride, α-glucosyldiacylglycerol, glucosyl-2-tetradecyl stearate (GlcC14C18), mannitol-2-tetradecyl stearate, glucosyl monomycinate, arabinose monomycinate, cholesterol sulfate, and β-glucosylceramide.

[0012] Furthermore, the preferred Mincle agonist is trehalose 6,6'-dibenzyl ester (TDB).

[0013] Furthermore, the concentration of the Mincle agonist is 0.2~2 mg / mL, the concentration of manganese ions is 1.0~10 mg / mL, and the ratio of manganese ions to Mincle agonist concentration is 2~10:1.

[0014] Furthermore, the compound adjuvant also includes a cationic polymer for delivering the vaccine compound adjuvant.

[0015] Further, the cationic polymer is one or more of the following: trimethyl-2,3-diolenooxypropylammonium chloride, lysophosphatidylcholine, dimethyl dioctadecylammonium, trimethyl dodecylammonium bromide, 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl]cholesterol, polyamines, polyamide-amines, polymethacrylates, polyamino acids, polyesters, or natural polysaccharides.

[0016] Furthermore, the cationic polymer is preferably dimethyl dioctadecylammonium (DDA).

[0017] Furthermore, the concentration of the cationic polymer is 1.0-5.0 mg / mL.

[0018] The present invention also provides a method for preparing the above-mentioned veterinary vaccine adjuvant, comprising the following steps: dissolving the cationic polymer and Mincle agonist in an organic solvent, mixing thoroughly and then removing the organic solvent to obtain a lipid film; adding PBS to hydrate the lipid film to obtain a liposome adjuvant; dissolving a manganese-containing reagent in PBS and mixing it with the liposome adjuvant to obtain the veterinary vaccine adjuvant.

[0019] Specifically, the preparation steps include: 1) Take an appropriate amount of organic solvents methanol and chloroform in a ratio of 1:9 to prepare an organic solvent, and dissolve DDA and TDB separately; add DDA and TDB in a vial at a mass ratio of 5:1, dissolve and mix thoroughly, place in a fume hood and air dry overnight to form a uniform lipid film at the bottom of the vial. 2) Add an appropriate amount of PBS buffer and hydrate in a 60 ℃ water bath for 20 minutes, taking it out and vortexing it for 1 minute every 5 minutes; use an ultrasonic cleaner to perform sonication twice, 1 minute each time, to obtain DDA-TDB liposome adjuvant; 3) Weigh an appropriate amount of MnCl2·4H2O into PBS, dissolve it completely at room temperature, and then mix it thoroughly with DDA-TDB liposome adjuvant to obtain DDA-TDB-Mn composite adjuvant.

[0020] The present invention also provides a veterinary vaccine comprising the above-mentioned veterinary vaccine adjuvant and antigen.

[0021] Furthermore, the antigen concentration is 0.2~2.0 mg / mL.

[0022] Furthermore, the volume ratio of the antigen to the veterinary vaccine adjuvant is 1:1.

[0023] Furthermore, the mass ratio of the cationic polymer, Mincle agonist, manganese-containing complex, and vaccine antigen is 5:1:5:1.

[0024] The present invention also provides an inactivated vaccine for H7N9 subtype avian influenza, wherein the vaccine comprises the above-mentioned veterinary vaccine adjuvant and H7N9 subtype avian influenza immunogen.

[0025] Furthermore, the manganese-containing complex in the veterinary vaccine adjuvant is manganese chloride, the mincle agonist is trehalose 6,6'-dibenzene ester, and the cationic polymer is dimethyl dioctadecyl ammonium.

[0026] Furthermore, the H7N9 subtype avian influenza immunogen is an inactivated A / chicken / Guangdong / GD15 / 2016 H7N9 subtype avian influenza virus.

[0027] Furthermore, the method of using the vaccine composition is one or any combination of intravenous injection, intramuscular injection, intradermal injection, subcutaneous injection, eye drops or nasal drops.

[0028] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: The veterinary vaccine adjuvant of the present invention combines Mn with TDB, thereby enhancing the immune effect of the TDB adjuvant. The adjuvant can induce a strong immune response, significantly improving the immunization effect, and leaves no residual damage after use. The H7N9 vaccine prepared by mixing the inactivated H7N9 subtype avian influenza virus as an antigen with the adjuvant, compared with using DDA-TDB alone, can enhance the humoral and cellular immune responses, reduce viral load, and improve the level of protection, providing complete protection against lethal H7N9 subtype avian influenza virus challenge in SPF chickens after immunization. This provides product and technical support for the production of veterinary vaccines. Attached Figure Description

[0029] Figure 1 The expression of inflammatory factors in chicken spleen cells after treatment with Mn and TDB according to the present invention; Figure 2 Electron micrographs (A(H7N9 WIV), B(DDA-TDB-Mn+H7N9 WIV)), particle size (C), and zeta potential (D) of the DDA-TDB-Mn combined inactivated avian influenza virus vaccine complex of the present invention. Figure 3 This invention enhances the HI antibody after immunization; Figure 4 This invention provides a gating strategy for flow cytometry staining of chicken T cells. Figure 5 This invention enhances the CD4 levels in the spleen (A) and lungs (B) after immunization. +T cells and CD8 + The absolute number of T cells; Figure 6 To enhance the detection of IFN-γ secretion levels by chicken T cells after immunization, this invention was developed. Figure 7 This invention describes the changes in clinical symptoms in SPF chickens after viral challenge. Figure 8 The survival rate of SPF chickens after viral challenge according to this invention; Figure 9 This refers to the viral load in the lungs of SPF chickens after challenge with the virus according to this invention. Figure 10 This invention relates to the pathological changes in lung tissue of SPF chickens after viral challenge. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0031] Example 1: Verification of adjuvant activity of Mn and TDB in chicken spleen cells by in vitro stimulation 1. Mn at different concentrations 2+ Detection of immune-related genes expressed in chicken spleen cells To verify in vitro whether Mn has adjuvant activity against primary chicken cells, mononuclear cell suspensions were prepared from the spleen of SPF chickens and resuspended to an appropriate concentration in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were then seeded at 10 × 10⁶ cells per well in a 12-well cell culture plate. 6 Cells were collected. An appropriate amount of MnCl2·4H2O was weighed, dissolved and diluted with culture medium, and added to cell wells at different concentrations, creating a concentration gradient of Mn (unit: μg / mL): 0, 2.5, 12.5, 25, 50, 100. The plated cells were incubated at 37℃ in a 5% CO2 incubator for 12 hours. Cells were then collected, and total RNA was extracted using the TRIPure Reagent kit (Aidlab Biotechnologies Co., Ltd., RN01). The relative expression levels of TNF-α, IL-1β, and IL-6 were detected using quantitative real-time PCR (ChamQ SYBR qPCR Master Mix, Vazyme, Q311). Each reaction system consisted of 20 μL, containing 10 μL of 2 × ChamQ SYBR qPCR Master Mix, 0.4 μL of forward and reverse primers (see Table 1), 2 μL of cDNA, and 7.4 μL of ddH2O. The reaction procedure was as follows: pre-denaturation at 95℃ for 30 seconds; cyclic reaction (95℃ for 10 seconds, 60℃ for 30 seconds, 40 cycles); and melting curve acquisition (95℃ for 15 seconds, 60℃ for 1 minute).

[0032]

[0033] The results are as follows Figure 1 As shown in Figure A, different concentrations of Mn The levels of cytokines secreted by stimulated cells showed significant differences compared to other concentrations, with Mn... At a concentration of 25 μg / mL, the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 genes were significantly increased (p<0.01). The experimental results indicate that Mn exerts a dose-dependent effect on the stimulation of primary chicken cells, with the optimal concentration of 25 μg / mL demonstrating the best efficacy, suggesting that Mn exhibits adjuvant activity in the in vitro stimulation of chicken cells.

[0034] 2. TDB and Mn 2+ Detection of immune-related gene expression in chicken spleen cells under combined action To verify in vitro whether Mn combined with TDB has adjuvant activity against primary chicken cells, TDB was dissolved in isopropanol, and 100 μL of TDB solutions with concentrations of 2.5 μg / mL, 5.0 μg / mL, and 12.5 μg / mL were uniformly coated onto the bottom of 12-well cell culture plates and air-dried. Peripheral blood and spleen from SPF chickens were used to prepare mononuclear cell suspensions, which were resuspended to appropriate concentrations in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured at 10 × 10⁻⁶ cells / well. 6 Each cell was seeded into one well of the aforementioned 12-well plate, and Mn-containing cells were added. Prepare 25 μg / mL culture medium, with wells for individual and combined treatments, and mix well. After stimulating the prepared cell plates in a 37°C, 5% CO2 incubator for 12 hours, collect cells to extract total RNA, and use quantitative real-time PCR to detect the relative expression levels of TNF-α, IL-1β, and IL-6.

[0035] The results are as follows Figure 1 As shown in Figure B, based on the optimal Mn concentration (25 μg / mL) combined with different TDB concentrations, the results of treating chicken spleen cells indicate that the relative expression levels of TNF-α, IL-1β, and IL-6 were highest when the Mn:TDB ratio was 5. The comparison results of the optimal concentrations for each group are shown below. Figure 1 As shown in Figure C, compared with the Mn group alone (25 μg / mL) and the TDB group (5.0 μg / mL), the expression levels of TNF-α, IL-1β, and IL-6 genes in the TDB+Mn group (Mn:TDB=5) were significantly increased (P<0.05). The experimental results suggest that Mn can synergistically enhance the adjuvant activity of TDB.

[0036] Example 2: Preparation of DDA-TDB-Mn combined inactivated avian influenza virus vaccine complex 1. Preparation of DDA-TDB-Mn composite adjuvant by thin-film hydration method Dissolve DDA and TDB in an appropriate amount of methanol-chloroform solution (volume ratio 1:9). Add the solution to a vial at a DDA to TDB mass ratio of 5:1, mix thoroughly, and air dry overnight in a fume hood to form a uniform lipid film at the bottom of the vial. Add 1-3 mL of PBS buffer and hydrate at 60°C for 20 minutes, vortexing for 1 minute every 5 minutes. Finally, sonicate three times at 60°C for 1 minute each time to obtain a liposome adjuvant with a TDB concentration of 0.5 mg / mL and a DDA concentration of 2.5 mg / mL. Store at 4°C for later use and name it DDA-TDB. Weigh an appropriate amount of MnCl2·4H2O into PBS and dissolve it thoroughly at room temperature. 2+ The concentration was 2.5 mg / mL, and then it was thoroughly mixed with DDA-TDB to obtain the DDA-TDB-Mn complex adjuvant.

[0037] 2. Formaldehyde inactivation method for preparing vaccine immunogens Formaldehyde was diluted with PBS at a ratio of 1:50. The viral allantoic fluid was then added dropwise to the diluted formaldehyde solution at a ratio of 43:7 and mixed thoroughly. The mixture was incubated overnight at 4°C. The next day, the inactivated virus solution was placed in a bacterial shaker at 37°C and incubated with shaking for 12 hours. The resulting inactivated virus solution was inoculated into SPF chicken embryos. On the fourth day, the allantoic fluid from the chicken embryos was collected for a hemagglutination test. No hemagglutination indicated complete inactivation. The concentration of the inactivated virus was determined using a BCA protein concentration assay kit.

[0038] 3. Preparation and in vitro characterization of the vaccine composition 3.1 The DDA-TDB and DDA-TDB-Mn adjuvants were mixed thoroughly with the A / chicken / Guangdong / GD15 / 2016 strain (patent number: CN117886901A) H7N9 inactivated virus at a ratio of 1:1 to achieve a final concentration of 0.2 mg / mL of H7N9 inactivated virus. The resulting vaccine compositions were named DDA-TDB+H7N9 WIV and DDA-TDB-Mn+H7N9 WIV, respectively. The one without adjuvants was named H7N9 WIV.

[0039] 3.2 Take appropriate amounts of H7N9 WIV or DDA-TDB-Mn+H7N9 WIV and add them dropwise to a copper grid. After negative staining with 1% phosphotungstic acid for 1-2 minutes and drying indoors, observe the morphology and photograph it using a TEM (Tecnai 12) transmission electron microscope. Figure 2 As shown in A (H7N9WIV) and B (DDA-TDB-Mn+H7N9 WIV), the DDA-TDB-Mn+H7N9 WIV complex adjuvant encapsulates the vaccine immunogen and still maintains an intact vesicle morphology.

[0040] 3.3 Pipette 0.1 mL of H7N9 WIV or DDA-TDB-Mn+H7N9 WIV into the particle size and potential sample cells, respectively. Dilute each sample 10-fold with 0.9 mL of PBS buffer, mix thoroughly, and then analyze using a Malvern particle size analyzer at room temperature. Figure 2 As shown in C and D, the average particle size of H7N9 WIV is 200 nm and the average Zeta potential is -5 mV; the average particle size of DDA-TDB-Mn+H7N9 WIV is 800 nm and the average Zeta potential is 45 mV.

[0041] Example 3: Immunogenicity Analysis 1. Experimental Grouping Twenty-one-day-old SPF chickens (purchased from Boehringer Ingelheim Viton Biotechnology Co., Ltd.) were randomly divided into four groups of 25 birds each. Group 1 was the PBS control group, receiving 100 µL of PBS via subcutaneous injection and 100 µL via intranasal administration; Group 2 was the H7N9 WIV group, receiving 100 µL of inactivated H7N9 virus via subcutaneous injection and 100 µL via intranasal administration; Group 3 was the DDA-TDB+H7N9 WIV group, receiving 100 µL of DDA-TDB+H7N9 WIV via subcutaneous injection and 100 µL via intranasal administration; and Group 4 was the DDA-TDB-Mn+H7N9 WIV group, receiving 100 µL of DDA-TDB-Mn+H7N9 WIV via subcutaneous injection and 100 µL via intranasal administration. Booster immunizations were administered 14 days after the initial immunization, using the same dose and route of administration.

[0042] 2. Hemagglutination inhibition (HI) test to detect serum antibody levels To evaluate the role of the compound adjuvant in the humoral immune response of chickens, peripheral blood was collected from the wing veins of chickens in each group on days 7, 14, and 21 after the initial immunization. Serum was used for HI antibody detection. 25 µL of PBS was added to each well of the hemagglutination plate. 25 µL of serum was diluted from well 1 to well 11, discarded, and 25 µL of PBS was added to well 12. 25 µL of four units of antigen was added to wells 1-10, and 25 µL of PBS was added to wells 11 and 12. After incubation at room temperature for 30 minutes, 25 µL of 1% chicken erythrocyte suspension was added, and incubation was continued at room temperature for 30 minutes. Results were then interpreted: coagulation indicated an ineffective titer, and no coagulation indicated an effective titer. Results are as follows: Figure 3 As shown, compared with the DDA-TDB+H7N9 WIV group, the serum antibody level of the DDA-TDB-Mn+H7N9 WIV group was significantly increased, indicating that the TDB combined with Mn adjuvant can assist the vaccine immunogen and significantly enhance the humoral immune response level of immunized chickens.

[0043] 3. Changes in T cell subtypes in chicken immune tissues and organs On day 7 after booster immunization, spleen, lungs, and peripheral blood were collected from chickens in each group, and mononuclear cells were prepared. 2 × 10⁻⁶ cells were collected from each cell. 6 Chicken T cells were stained with mouse anti-chicken CD45, CD3, CD4, CD8, TCR γδ, CD25, and flow cytometry antibodies. CD45 in chicken T cells was detected by flow cytometry. + T cells and CD8 + The absolute number of T cells. According to... Figure 4 The gating strategy was used to analyze the flow cytometry data, and the absolute number of T cells in the chicken spleen and lungs was analyzed. Figure 5 The results showed that, compared with the DDA-TDB+H7N9 WI group, the DDA-TDB-Mn+H7N9 WIV group had lower CD4 counts. + T cells and CD8 + The absolute number of T cells increased significantly, and the difference was extremely significant. P <0.05). Experimental results indicate that the addition of TDB combined with Mn adjuvant can promote CD4. + T cells, CD8 + The proliferation and differentiation of T cells promote the body's and cells' immune responses, resulting in a higher proportion of CD4+. + T cells and CD8 + T cells.

[0044] 4. Enzyme-linked immunospot assay (ELISpot) to detect IFN-γ secretion levels in chicken spleen cells. On day 7 after booster immunization, chicken spleens from each group were harvested to prepare mononuclear cells. Chicken IFN-γ antibody (0.5 μg / ml) was coated onto MultiScreen™ - IP 96-well plates (Millipore, MSIPN4W50) and incubated overnight at 4°C. The plates were washed twice with sterile PBS and blocked at 37°C and 5% CO2 for 1 hour. The cells to be tested (2 × 10⁻⁶ cells / well) were then added. 5 / well), then add inactivated toxin, positive peptide (patent number: CN117886901A) and other stimulants to a final concentration of 10 μg / ml. After culturing at 37℃ and 5% CO2 for 24-48 hours, wash 5 times with PBST, add biotin-labeled chicken IFN-γ antibody (0.5 μg / ml) and incubate at room temperature for 1 hour. Add alkaline phosphatase-labeled streptavidin ALP (Mabtech, 3310-10-1000) and incubate at room temperature for 1 hour, then wash 5 times with PBST. Finally, add BCIP / NBT-plus for ALP substrate solution (Mabtech, 3650-10) and incubate in the dark for 10 minutes. Blue-black spots form at the locations where cytokines appear, with each spot representing a single IFN-γ-secreting cell. Finally, scan and analyze using an ELISpot analyzer to calculate the number of spots per well.

[0045] The aforementioned positive peptides are immunogenic single epitope polypeptides P4, P10, P12, and P15 (patent number: CN117886901A). When these peptides stimulated the spleen cells of vaccine-immunized SPF chickens, the IFN-γ secretion level of chicken T cells in the DDA-TDB-Mn+H7N9 WIV group was significantly higher than that in the DDA-TDB+H7N9 WIV group (p<0.001). Figure 6 (B) The experimental results show that, compared with the DDA-TDB+H7N9 WIV group, the DDA-TDB-Mn+H7N9 WIV group induced a stronger HA-specific cellular immune response. A representative graph of the IFN-γ ELISPOT assay results is shown below. Figure 6 As shown in Figure A.

[0046] Example 4: Virus Challenge Protection Experiment 1. Clinical symptoms and survival rate after challenge The experimental chickens in Example 3 were challenged with the virus on day 14 after booster immunization, via nasal administration of 200 µL of 10 5.0 EID 50 Chickens were inoculated with highly pathogenic H7N9 avian influenza virus (A / chicken / Guangdong / GD15 / 2016) at appropriate doses, and clinical symptoms (such as swollen and watery eyes, lethargy, ruffled feathers, bleeding leg scales, and purplish comb) and survival rates were recorded after 14 days. Results are as follows: Figure 7 and 8As shown, the clinical scores of the DDA-TDB+H7N9 WIV+GD15 group and the DDA-TDB-Mn+H7N9 WIV+GD15 group were significantly lower than those of the H7N9 WIV+GD15 group and the PBS+GD15 group. Furthermore, the clinical score of the DDA-TDB-Mn+H7N9 WIV+GD15 group was significantly lower than that of the DDA-TDB+H7N9 WIV+GD15 group. The survival rate of the DDA-TDB-Mn+H7N9 WIV+GD15 group and the PBS group was 100%, significantly higher than that of the DDA-TDB+H7N9 WIV+GD15 group and the H7N9 WIV+GD15 group and the PBS+GD15 group. The survival rate of the H7N9 WIV+GD15 group and the PBS+GD15 group was 0%, while the survival rate of the DDA-TDB+H7N9 WIV+GD15 group was 90% (see...). Figure 8 The above results indicate that the addition of TDB combined with Mn adjuvant can effectively resist lethal attacks from the H7N9 subtype avian influenza virus, and that Mn can synergistically enhance the protective efficacy of the TDB combined vaccine.

[0047] 2. Changes in viral load in chicken lung tissue after challenge On day 4 post-infection, lung tissue was collected from each group of chickens. 0.1 g of tissue was weighed, added to 1-2 mL of PBS, and thoroughly ground. The mixture was subjected to three freeze-thaw cycles, centrifuged at 800 g for 1 minute, and the supernatant was collected. The supernatant was filtered through a 0.22 µm filter membrane, serially diluted 10-fold with PBS, and inoculated into 9-day-old SPF chicken embryos. The embryos were incubated at 37°C for 48 hours. Embryos that died within 24 hours were considered non-specific deaths. After excluding dead embryos, allantoic fluid from live embryos was collected for hemagglutination titer determination. 25 µL of PBS was added to each well of the hemagglutination plate. 25 µL of chicken embryo allantoic fluid was diluted from well 1 to well 11, thoroughly mixed, and the 25 µL mixture was discarded. 25 µL of PBS was added to well 12. 25 µL of 1% chicken erythrocyte suspension was added to each well, and the plate was incubated at 37°C for 30 minutes. Results were then interpreted, and the EID was calculated using the Reed-Muench method. 50 The non-immunized PBS group was set as the control group, such as... Figure 9 As shown, the viral titer was significantly lower in the DDA-TDB-Mn+H7N9 WIV group compared to the DDA-TDB+H7N9 WI group.

[0048] 3. Pathological changes in chicken lung tissue after viral challenge Seven days after viral challenge, lung tissue from chickens in each group was collected, immersed in tissue fixative, embedded in paraffin, stained with hematoxylin and eosin (HE), and sectioned for observation of tissue lesions. For example... Figure 10As shown, the lesion severity in the DDA-TDB+H7N9 WIV+GD15 and DDA-TDB-Mn+H7N9 WIV+GD15 groups was significantly lower than that in the H7N9 WIV+GD15 and PBS+GD15 groups. Furthermore, the lesion severity in the DDA-TDB-Mn+H7N9 WIV+GD15 group was significantly lower than that in the DDA-TDB+H7N9 WIV+GD15 group. Both the PBS+GD15 and H7N9 WIV+GD15 groups showed significant hemorrhage and extensive inflammatory cell infiltration, indicating severe lung damage. However, the lung tissue morphology of the chickens challenged in the DDA-TDB-Mn+H7N9 WIV+GD15 group was clearer, with only a very small amount of inflammatory cell infiltration in the atrial and bronchiolar regions.

Claims

1. An inactivated vaccine against H7N9 subtype avian influenza, characterized in that, The vaccine comprises a vaccine adjuvant and an inactivated H7N9 subtype avian influenza virus. The active ingredients in the vaccine adjuvant are a manganese-containing complex and a Mincle agonist. The manganese-containing complex contains divalent manganese ions. The concentration ratio of manganese ions to Mincle agonist is 5:

1. The adjuvant also includes a cationic polymer for delivering the vaccine adjuvant. The Mincle agonist is trehalose 6,6'-dibenzyl ester, and the cationic polymer is dimethyl dioctadecyl ammonium. The vaccine adjuvant is for chickens.

2. The H7N9 subtype avian influenza inactivated vaccine according to claim 1, characterized in that, The manganese-containing complex in the vaccine adjuvant is manganese chloride.

3. The H7N9 subtype avian influenza inactivated vaccine according to claim 1, characterized in that, The concentration of the Mincle agonist in the vaccine adjuvant is 0.2~2 mg / mL, and the concentration of manganese ions is 1.0~10.0 mg / mL.

4. The H7N9 subtype avian influenza inactivated vaccine according to claim 1, characterized in that, The concentration of the cationic polymer in the vaccine adjuvant is 1.0~5.0 mg / mL.

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