Veterinary vaccine composite adjuvant as well as preparation method and application thereof

Abstract

The invention discloses a veterinary vaccine composite adjuvant as well as a preparation method and application thereof. Active ingredients in the vaccine composite adjuvant are a manganese-containing compound and a Mincle agonist. According to the veterinary vaccine composite adjuvant disclosed by the invention, Mn and TDB are combined, so that the immunological enhancement effect of the TDB adjuvant is achieved. The composite adjuvant can induce a strong immune response reaction, significantly improves the immune effect, and is free of residual damage in use. Compared with the H7N9 vaccine prepared by singly using DDA-TDB, the H7N9 vaccine prepared by mixing the H7N9 subtype avian influenza inactivated virus as the antigen with the composite adjuvant can enhance humoral immunity and cellular immune response of a body, reduce virus load and improve protection level, and provides a product and technical support for production of veterinary vaccines.

Description

Technical Field

[0001] The invention relates to a composite adjuvant for veterinary vaccines and a preparation method and application thereof, belonging to the field of animal vaccines. Background Art

[0002] Vaccination is still an important measure for animal disease prevention and control. However, many candidate vaccines still have problems such as poor immunogenicity, which makes it difficult to further promote their application. In order to increase the immunogenicity of vaccines and enable them to be used in clinical practice, it is urgent to study and discover safe and effective immune adjuvants to make up for the shortcomings of vaccines and improve and enhance the effects of vaccines. Adjuvants directly restrict the improvement of animal vaccine quality. The lag in the invention of veterinary vaccine adjuvants has led to the fact that most domestic veterinary vaccine adjuvants still rely on foreign imports. There are many types of adjuvants that can be used for vaccines. Due to the shortcomings of high toxicity, high cost and single function, it is difficult to achieve clinical application. In particular, oil emulsion adjuvants (white oil) are commonly used in veterinary vaccines, but they have poor metabolic capacity, limited improvement in immune effects and the sustainability of antigen release needs to be improved. In addition, many veterinary oil adjuvant vaccines induce a short immune protection period and it is difficult to achieve long-term immunity. The main reason is that the cellular immunity induced by the vaccine is weak or lacks T cell helper. Therefore, there is an urgent need to invent a new adjuvant with low cost, few side effects, high safety, and the ability to simultaneously induce long-lasting immunity and enhance humoral and cellular immune responses, in order to make up for the shortcomings of oil-emulsion adjuvants and thus improve the protective effect of existing veterinary vaccines.

[0003] Macrophage-inducible C-type lectin (Mincle) is mainly expressed on the surface of antigen-presenting cells, including macrophages, monocytes, dendritic cells, and neutrophils. It is a member of the atypical C-type lectin family and is encoded by Clec4e. Mincle has multiple functions in the immune response process and can recognize a series of different endogenous or exogenous ligands, including damaged cells, fungi, bacteria, viruses, and parasites, activating the production of cytokines and chemokines, promoting inflammation or inhibiting inflammation. More importantly, Mincle can also induce adaptive immune responses, such as antigen-specific T cell responses and antibody production. As an agonist of Mincle, trehalose 6,6'-dibehenate (TDB) was used in the study of TDB for enhancing the immune effect of animal vaccines. It was found that TDB can produce an immune enhancement effect in animals, but this effect is not strong enough.

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

[0005] So far, there has been no report on the application of Mincle agonist combined with Mn in veterinary vaccine adjuvants. Summary of the invention

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

[0007] Technical solution: The present invention provides a veterinary vaccine composite adjuvant, wherein the active ingredients in the vaccine composite 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 compound 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 tetraoxide, manganese phosphate or manganese carbonate.

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

[0011] Furthermore, the Mincle agonist is derived from the extracellular ligand recognized by Mincle, and the extracellular ligand includes one or more of trehalose 6,6'-dimycolate, trehalose 6,6'-dibehenate, glyceroglycolipid, mannosyl fatty acid-coupled mannitol, glycerol monomycolate, β-gentiobiose diglyceride, β-glucose diglyceride, α-glucosyl diacylglycerol, glucosyl-2-tetradecanoate (GlcC14C18), mannosyl-2-tetradecanoate, glucose monomycolate, arabinose monomycolate, cholesterol sulfate, and β-glucosylceramide.

[0012] Furthermore, the Mincle agonist is preferably trehalose 6,6'-dibehenate (TDB).

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

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

[0015] Furthermore, the cationic polymer is one or more of trimethyl-2,3-dioleyloxypropylammonium chloride, lysolecithin, dimethyldioctadecylammonium, trimethyldodecylammonium bromide, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol, polyamines, polyamide-amines, polymethacrylates, polyamino acids, polyesters or natural polysaccharides.

[0016] Furthermore, the cationic polymer is preferably dimethyldioctadecyl ammonium (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 composite adjuvant, comprising the following steps: dissolving the cationic polymer and the Mincle agonist with an organic solvent, removing the organic solvent after sufficient mixing to obtain a lipid film; adding PBS to hydrate the lipid film to obtain a liposome adjuvant; dissolving the manganese-containing reagent with PBS and mixing it with the liposome adjuvant to obtain the veterinary vaccine composite adjuvant.

[0019] Specifically, the preparation steps include:

[0020] 1) Take appropriate amount of organic solvent methanol and chloroform in the ratio of 1:9 to make an organic solvent, dissolve DDA and TDB respectively; add DDA to TDB in the mass ratio of 5:1 into the vial, fully dissolve and mix, place in a fume hood to air dry overnight, and form a uniform lipid film at the bottom of the vial;

[0021] 2) adding an appropriate amount of PBS buffer and hydrating in a 60° C. water bath for 20 minutes, wherein the mixture was taken out every 5 minutes and vortexed for 1 minute; ultrasonic cleaning was performed twice, each time for 1 minute, to obtain a DDA-TDB liposome adjuvant;

[0022] 3) Weigh an appropriate amount of MnCl 2 ·4H 2 O to PBS, fully dissolved at room temperature, and then fully mixed with DDA-TDB liposome adjuvant to obtain DDA-TDB-Mn composite adjuvant.

[0023] The present invention also provides a veterinary vaccine, which comprises the veterinary vaccine composite adjuvant and antigen.

[0024] Furthermore, the antigen concentration is 0.2-2.0 mg / mL.

[0025] Furthermore, the volume ratio of the antigen and the veterinary vaccine composite adjuvant is 1:1.

[0026] Furthermore, the mass ratio of the cationic polymer, Mincle agonist, manganese-containing complex, and vaccine antigen is 5:1:5:1.

[0027] The present invention also provides an H7N9 subtype avian influenza inactivated vaccine, which comprises the above-mentioned veterinary vaccine composite adjuvant and an H7N9 subtype avian influenza immunogen.

[0028] Furthermore, the manganese-containing complex in the veterinary vaccine composite adjuvant is manganese chloride, the Mincle agonist is trehalose 6,6'-dibehenate, and the cationic polymer is dimethyl dioctadecyl ammonium.

[0029] Furthermore, the H7N9 subtype avian influenza immunogen is an inactivated A / chicken / Guangdong / GD15 / 2016H7N9 subtype avian influenza virus.

[0030] Furthermore, the vaccine composition is used by intravenous injection, intramuscular injection, intradermal injection, subcutaneous injection, eye drops or nasal drops, or any combination thereof.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The composite adjuvant for veterinary vaccines of the present invention combines Mn with TDB to enhance the immune enhancement effect of the TDB adjuvant. The composite adjuvant can induce a strong immune response, significantly improve the immune effect, and there is no residual damage after use. In this application, the H7N9 vaccine prepared by mixing the inactivated H7N9 subtype avian influenza virus as an antigen and mixing it with the composite adjuvant can enhance the body's humoral immunity and cellular immune response, reduce the viral load, and improve the protection level compared to the use of DDA-TDB alone. After immunization, SPF chickens are completely protected from lethal attacks by H7N9 subtype avian influenza viruses. Provide product and technical support for the production of veterinary vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The expression of inflammatory factors in chicken spleen cells after the action of Mn and TDB of the present invention;

[0033] Figure 2 The electron micrographs (A (H7N9WIV), 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 are shown;

[0034] Figure 3 The HI antibody after boosting immunization of the present invention;

[0035] Figure 4This is the gating strategy for chicken T cell flow cytometry staining of the present invention;

[0036] Figure 5 CD4 in spleen (A) and lung (B) after booster immunization of the present invention + T cells and CD8 + Absolute number of T cells;

[0037] Figure 6 The ELISPOT method was used to detect the level of IFN-γ secretion by chicken T cells after booster immunization in the present invention;

[0038] Figure 7 The clinical symptom changes of SPF chickens after virus attack of the present invention;

[0039] Figure 8 is the survival rate of SPF chickens after virus challenge of the present invention;

[0040] Fig. 9 is the viral load in the lungs of SPF chickens after virus challenge of the present invention;

[0041] Fig.10 The figure shows the pathological changes in the lung tissues of SPF chickens after being challenged with poison. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0043] Example 1: In vitro stimulation of chicken spleen cells by Mn and TDB to verify their adjuvant activity

[0044] 1. Different concentrations of Mn 2+ Detection of immune-related genes expressed in spleen cells of infected chickens

[0045] In order to verify in vitro whether Mn has adjuvant activity on primary chicken cells, mononuclear cell suspension was prepared from the spleen of SPF chickens, resuspended to an appropriate concentration in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and 10×10 mononuclear cells were plated in each well of a 12-well cell culture plate. 6 cells. Weigh an appropriate amount of MnCl 2 ·4H 2 O, dissolved and diluted with culture medium, added to the cell wells at different concentrations and mixed to form a concentration gradient of Mn in the wells (unit: μg / mL): 0, 2.5, 12.5, 25, 50, 100. The plated cell plates were placed at 37°C and 5% CO 2After 12 hours incubation, cells were collected and total RNA was extracted using the TRIpure Reagent kit (Aidlab Biotechnologies Co., Ltd, RN01). The relative expression of TNF-α, IL-1β, and IL-6 was detected using fluorescent quantitative PCR (ChamQ SYBR qPCR Master Mix, Vazyme, Q311). Each reaction system was 20 μL, which contained 10 μL 2×ChamQ SYBR qPCR Master Mix, 0.4 μL upstream and downstream primers (see Table 1), 2 μL cDNA, and 7.4 μL ddH 2 O. The reaction procedure was as follows: pre-denaturation at 95°C for 30 seconds; cycle reaction (95°C for 10 seconds, 60°C for 30 seconds, 40 cycles); melting curve acquisition (95°C for 15 seconds, 60°C for 1 minute).

[0046] Table 1

[0047]

[0048] The results are as follows Figure 1 As shown in A, there are significant differences in the levels of cytokine secretion by cells stimulated by different concentrations of Mn. Compared with other concentrations, when the Mn concentration is 25 μg / mL, the gene expression of inflammatory factors TNF-α, IL-1β, and IL-6 significantly increased (p<0.01). The experimental results show that Mn has a certain dose-dependent effect on chicken primary cells, among which the Mn concentration is 25 μg / mL, and the effect is the best, indicating that Mn has adjuvant activity in chicken cells stimulated in vitro.

[0049] 2. TDB and Mn 2+ Detection of immune-related genes expressed in spleen cells of chickens treated with combined

[0050] In order to verify in vitro whether Mn combined with TDB has adjuvant activity on 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 evenly coated on the bottom of a 12-well cell culture plate and dried. Mononuclear cell suspensions were prepared from SPF chicken peripheral blood and spleen, and resuspended to an appropriate concentration in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. 6 Place 100 cells per well in the above 12-well plate, add medium containing Mn (25 μg / mL), set up single treatment and combined treatment wells, and mix well. Place the plated cell plate at 37°C and 5% CO 2 After 12 hours of stimulation in the incubator, the cells were collected to extract total RNA, and the relative expression levels of TNF-α, IL-1β, and IL-6 were detected by fluorescence quantitative PCR.

[0051] The results are as follows Figure 1 As shown in B, according to the above optimal Mn concentration (25 μg / mL), the results of combining different TDB concentrations to act on chicken spleen cells show that when the Mn:TDB ratio is 5, the relative expression levels of TNF-α, IL-1β, and IL-6 are the highest. The comparison results of the optimal concentrations of each group are shown in Figure 1 As shown in Figure C, compared with the Mn group (25 μg / mL) and the TDB group (5.0 μg / mL), the gene expression levels of TNF-α, IL-1β, and IL-6 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.

[0052] Example 2: Preparation of DDA-TDB-Mn combined inactivated avian influenza virus vaccine complex

[0053] 1. Preparation of DDA-TDB-Mn composite adjuvant by thin film hydration method

[0054] Take an appropriate amount of organic solvent methanol-chloroform solution (volume ratio 1:9) to dissolve DDA and TDB, add it to the cillin bottle according to the mass ratio of DDA to TDB of 5:1, fully dissolve and mix, place it in a fume hood to air dry overnight, and form a uniform lipid film at the bottom of the bottle. Add 1-3mL PBS buffer and place it at 60℃ for hydration for 20 minutes, vortexing for 1 minute every 5 minutes, and finally perform ultrasound in a 60℃ water bath 3 times, each for 1 minute, to obtain a liposome adjuvant, in which the TDB concentration is 0.5mg / mL and the DDA concentration is 2.5mg / mL, and it is stored at 4℃ for use, named DDA-TDB. Weigh an appropriate amount of MnCl 2 ·4H 2 O to PBS and fully dissolved at room temperature. 2+ The concentration is 2.5 mg / mL, and then it is fully mixed with DDA-TDB to obtain a DDA-TDB-Mn composite adjuvant.

[0055] 2 Formaldehyde inactivation method to prepare vaccine immunogen

[0056] Dilute formaldehyde and PBS at a ratio of 1:50, add the virus allantoic fluid and the diluted formaldehyde solution at a ratio of 43:7, mix well, and place at 4°C overnight. Take it out on the second day and place it in a 37°C bacterial shaker for 12 hours. Inoculate the inactivated virus solution into SPF chicken embryos, collect the chicken embryo allantoic fluid on the 4th day for hemagglutination test, and the absence of hemagglutination indicates complete inactivation. The concentration of the inactivated virus is tested by the BCA protein concentration test kit.

[0057] 3 Preparation of vaccine composition and its in vitro characterization

[0058] 3.1 The DDA-TDB and DDA-TDB-Mn composite adjuvants were respectively mixed with the A / chicken / Guangdong / GD15 / 2016 strain (patent number: CN117886901A) H7N9 inactivated virus in a ratio of 1:1 to make the final concentration of the H7N9 inactivated virus 0.2 mg / mL. The obtained vaccine compositions were named DDA-TDB+H7N9 WIV and DDA-TDB-Mn+H7N9 WIV, and the one without adjuvant was named H7N9WIV.

[0059] 3.2 Take an appropriate amount of H7N9 WIV or DDA-TDB-Mn+H7N9 WIV and drop it onto the copper grid, stain it with 1% phosphotungstic acid for 1 to 2 minutes, dry it indoors, and observe the morphology with a TEM transmission electron microscope (Tecnai 12) and take pictures. Figure 2 As shown in A (H7N9WIV) and B (DDA-TDB-Mn + H7N9 WIV), the DDA-TDB-Mn + H7N9 WIV composite adjuvant still maintains a complete vesicle morphology after encapsulating the vaccine immunogen.

[0060] 3.3 Pipette 0.1mL H7N9 WIV or DDA-TDB-Mn+H7N9 WIV and add it to the particle size and potential sample pools, respectively. Add 0.9mL PBS buffer to dilute 10 times, mix thoroughly, and then test with a Malvern particle size analyzer at room temperature. Figure 2 As shown in C and D, the average particle size of H7N9 WIV is 200nm, and the average Zeta potential is -5mV; the average particle size of DDA-TDB-Mn+H7N9 WIV is 800nm, and the average Zeta potential is 45mV.

[0061] Example 3: Immunogenicity Analysis

[0062] 1 Experimental Grouping

[0063] 21-day-old SPF chickens (purchased from Boehringer Ingelheim Weitong Biotech Co., Ltd.) were randomly divided into 4 groups, with 25 in each group. Group 1 was the PBS control group, which was immunized with 100 μL of PBS by subcutaneous injection and 100 μL of nasal drops; Group 2 was the H7N9 WIV group, which was immunized with 100 μL of H7N9 inactivated virus by subcutaneous injection and 100 μL of nasal drops; Group 3 was the DDA-TDB+H7N9 WIV group, which was immunized with 100 μL of DDA-TDB+H7N9WIV by subcutaneous injection and 100 μL of nasal drops; Group 4 was the DDA-TDB-Mn+H7N9 WIV group, which was immunized with 100 μL of DDA-TDB-Mn+H7N9 WIV by subcutaneous injection and 100 μL of nasal drops. After the first immunization, booster immunization was performed with the same dose and inoculation route 14 days later.

[0064] 2. Hemagglutination inhibition test (HI) to detect serum antibody levels

[0065] In order to evaluate the role of the composite adjuvant in the humoral immune response of chickens, peripheral blood was collected from the wing vein of each group of chickens on the 7th, 14th and 21st days after the initial immunization, and serum was taken for HI antibody detection. 25 μL PBS was added to each well of the blood coagulation plate, 25 μL serum was aspirated and diluted from the 1st well to the 11th well, 25 μL serum was discarded and 25 μL PBS was added to the 12th well. 25 μL of four-unit antigen was added to wells 1 to 10, and 25 μL PBS was added to wells 11 and 12. After 30 minutes at room temperature, 25 μL of 1% chicken red blood cell suspension was added and allowed to react at room temperature for 30 minutes. The results were judged. Coagulation indicates invalid titer, and non-coagulation indicates valid titer. The results are as follows Figure 3 As shown, compared with the DDA-TDB+H7N9 WIV group, the serum antibody level in the DDA-TDB-Mn+H7N9 WIV group was significantly increased, indicating that the TDB combined with Mn composite adjuvant can assist the vaccine immunogen and significantly enhance the humoral immune response level of immunized chickens.

[0066] 3. Changes of T cell subtypes in chicken immune tissues and organs

[0067] On the 7th day after booster immunization, spleen, lung and peripheral blood of chickens in each group were collected and mononuclear cells were prepared. 2×10 6 The cells were stained with mouse anti-chicken CD45, CD3, CD4, CD8, TCRγδ, CD25, and flow cytometry antibodies, and CD4 in chicken T cells was detected by flow cytometry. + T cells and CD8 + The absolute number of T cells. Figure 4 The flow cytometry data were analyzed using the gating strategy in to analyze the absolute number of T cells in the spleen and lung of chickens. Figure 5 The results showed that compared with the DDA-TDB+H7N9 WI group, the DDA-TDB-Mn+H7N9 WIV group had a significantly higher CD4 + T cells and CD8 + The absolute number of T cells increased significantly, and the difference was extremely significant (P<0.05). The experimental results show that the addition of TDB combined with Mn composite adjuvant can promote CD4 + T cells, CD8 + T cells proliferate and differentiate, promote the body and cellular immune response, and produce a higher proportion of CD4 + T cells and CD8 + T cells.

[0068] 4 ELISpot assay to detect IFN-γ secretion level in chicken spleen cells

[0069] On the 7th day after booster immunization, the spleens of chickens in each group were taken to prepare mononuclear cells. Chicken IFN-γ antibody (0.5 μg / ml) was coated onto MultiScreenTM-IP 96-well plates (Millipore, MSIPN4W50) and incubated overnight at 4°C; washed twice with sterile PBS and incubated at 37°C, 5% CO 2 The cells to be tested (2×10 5 / well), and then add inactivated toxin, positive peptide (patent number: CN117886901A) and other stimulants, the final concentration is 10μg / ml, 37℃, 5% CO 2 After culturing for 24-48 hours under the same conditions, the cells were washed with PBST for 5 times, and biotin-labeled chicken IFN-γ antibody (0.5 μg / ml) was added and incubated at room temperature for 1 hour; alkaline phosphatase-labeled streptavidin ALP (Mabtech, 3310-10-1000) was added and incubated at room temperature for 1 hour, and then washed with PBST for 5 times. Finally, BCIP / NBT-plus for ALP substrate solution (Mabtech, 3650-10) was added and incubated in the dark for 10 minutes. Blue-black spots were formed at the location where the cytokines appeared, and each spot represented a single cell secreting IFN-γ. Finally, the number of spots in each well was scanned and analyzed using an ELISpot analyzer.

[0070] The above positive peptides are single epitope peptides P4, P10, P12, and P15 (patent number: CN117886901A) with immunogenicity. After stimulating the spleen cells of SPF chickens immunized with the vaccine, the secretion level of IFN-γ by 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. Representative figures of IFN-γ ELISPOT test results are shown in Figure 6 As shown in A.

[0071] Example 4: Challenge protection experiment

[0072] 1 Clinical symptoms and survival rate after challenge

[0073] The experimental chickens in Example 3 were challenged with 200 μL of 10 5.0 EID 50The chickens were inoculated with highly pathogenic H7N9 avian influenza virus (A / chicken / Guangdong / GD15 / 2016) at 100 mg / kg doses, and clinical symptoms (such as swollen eyes and tears, depression, ruffled hair, bleeding foot scales, and purple combs) were scored and the survival rate was recorded for 14 days. Figure 7 and 8 As 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, and the clinical scores of the DDA-TDB-Mn+H7N9 WIV+GD15 group were significantly lower than those of the DDA-TDB+H7N9 WIV+GD15 group. The survival rates of the DDA-TDB-Mn+H7N9 WIV+GD15 group and the PBS group were 100%, which was significantly higher than those of the DDA-TDB+H7N9 WIV+GD15 group and the H7N9 WIV+GD15 group and the PBS+GD15 group, while the survival rates of the H7N9 WIV+GD15 group and the PBS+GD15 group were 0, and the survival rate of the DDA-TDB+H7N9WIV+GD15 group was 90% (see Figure 8 ). The above results show that the addition of TDB combined with Mn adjuvant can effectively resist the lethal attack of H7N9 subtype avian influenza virus and Mn can synergistically enhance the protective power of TDB combined vaccine.

[0074] 2 Changes in viral load in chicken lung tissue after virus challenge

[0075] On the 4th day after the virus attack, the lung tissue of each group of chickens was collected, 0.1g of tissue was weighed, 1-2mL PBS was added and fully ground, and the mixture was repeatedly frozen and thawed 3 times, centrifuged at 800g for 1 minute, the supernatant was taken, filtered through a 0.22μm filter membrane, diluted 10 times with PBS and inoculated into 9-day-old SPF chicken embryos, and incubated in an incubator at 37°C for 48 hours. Among them, chicken embryos that died within 24 hours were judged as non-specific deaths. After excluding dead chicken embryos, live chicken embryo allantoic fluid was collected for hemagglutination titer determination. 25μL PBS was added to each well of the hemagglutination plate, 25μL chicken embryo allantoic fluid was aspirated and diluted from the 1st well to the 11th well, and after sufficient mixing, 25μL of the mixed solution was discarded, and 25μL PBS was added to the 12th well; 25μL 1% chicken red blood cell suspension was added to each well, and the reaction was performed at 37°C for 30 minutes. The results were determined and the EID was calculated according to the Reed-Muench method. 50 The non-immunized PBS group was set as the control group. Fig. 9 As shown, the virus titer in the DDA-TDB-Mn+H7N9 WIV group was significantly reduced compared with that in the DDA-TDB+H7N9WI group.

[0076] 3 Pathological changes in lung tissue of chickens after infection

[0077] On the 7th day after infection, the lung tissues of chickens in each group were collected and immersed in tissue fixative, embedded in paraffin, stained with HE, and sliced ​​to observe the tissue pathological changes. Fig.10 As shown in the figure, the degree of lesions in the DDA-TDB+H7N9 WIV+GD15 group and the DDA-TDB-Mn+H7N9 WIV+GD15 group were significantly lower than those in the H7N9 WIV+GD15 group and the PBS+GD15 group, and the degree of lesions 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 group and the H7N9 WIV+GD15 group showed obvious bleeding and a large number of inflammatory cell infiltrations, showing severe lung damage. However, the lung tissue morphology of the chickens in the DDA-TDB-Mn+H7N9WIV+GD15 group was clearer, and only a very small amount of inflammatory cell infiltration was present in the lung chambers and bronchioles.

Claims

1. A composite adjuvant for veterinary vaccines, characterized in that: The active ingredients in the vaccine composite adjuvant are manganese-containing complex and Mincle agonist.

2. The veterinary vaccine composite adjuvant according to claim 1, characterized in that: The manganese-containing complex contains divalent manganese ions.

3. The veterinary vaccine composite adjuvant according to claim 1, characterized in that: The concentration of the Mincle agonist is 0.2 to 2 mg / mL, the concentration of manganese ions is 1.0 to 10.0 mg / mL, and the concentration ratio of manganese ions to the Mincle agonist is 2 to 10:

1.

4. The veterinary vaccine composite adjuvant according to claim 1, characterized in that: The composite adjuvant also includes a cationic polymer for delivering the vaccine composite adjuvant.

5. The veterinary vaccine composite adjuvant according to claim 4, characterized in that: The concentration of the cationic polymer is 1.0-5.0 mg / mL.

6. The method for preparing the composite adjuvant for veterinary vaccines according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: dissolving a cationic polymer and a Mincle agonist with an organic solvent, mixing them 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 with PBS and then mixing it with the liposome adjuvant to obtain a veterinary vaccine composite adjuvant.

7. A veterinary vaccine, characterized in that: The vaccine comprises the veterinary vaccine composite adjuvant and antigen according to claim 1.

8. The veterinary vaccine according to claim 7, characterized in that The antigen concentration is 0.2-2 mg / mL.

9. An inactivated vaccine for H7N9 subtype avian influenza, characterized in that: The vaccine comprises the veterinary vaccine composite adjuvant according to any one of claims 1 to 5 and an H7N9 subtype avian influenza immunogen.

10. The H7N9 subtype avian influenza inactivated vaccine according to claim 9, characterized in that: The manganese-containing complex in the veterinary vaccine composite adjuvant is manganese chloride, the Mincle agonist is trehalose 6,6'-dibehenate, and the cationic polymer is dimethyl dioctadecyl ammonium.

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