Feline triple vaccine immunoadjuvant containing two feline interleukins and a metabolic regulator molecule, compositions and uses

Abstract

The present application relates to the technical field of biological medicine, and in particular to a cat triple vaccine immune adjuvant containing two kinds of cat interleukins and metabolic regulatory molecules, a composition and application. The composition comprises cat interleukin 15, cat interleukin 23 and a metabolic regulatory molecule, the metabolic regulatory molecule comprises beta-nicotinamide mononucleotide, beta-nicotinamide adenine dinucleotide disodium salt, N-acetyl cysteine and L-carnitine, and can be used for preparing a cat triple vaccine or an auxiliary preparation of the cat triple vaccine. The composition can effectively improve the protection of the vaccine, prolong the protection time of the vaccine and reduce adverse reactions; the vaccine can more durably and at a higher level reduce or control the reproductive infection of viruses, significantly improve the specific humoral immune level and memory response ability of animals, and better resist the invasion and spread of FPV, FCV and FHV-1.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a cat triple vaccine immune adjuvant containing two kinds of cat interleukins and metabolic regulatory molecules, a composition and application thereof. BACKGROUND

[0002] With the development of China's pet market, the number of pet cats has grown unprecedentedly, and pet cats have greatly improved their status in the family. In the face of the growing size of pet cats, the prevention and epidemic prevention of cat infectious diseases is particularly important. Feline panleukopenia, feline calicivirus disease and feline viral rhinotracheitis are three common infectious diseases of cats, which are caused by feline panleukopenia virus (FPV), feline calicivirus (FCV) and feline herpesvirus (FHV-1) infection, and can cause diseases such as respiratory tract and intestinal tract of cats, and even death in severe cases. The cat triple vaccine is a cat vaccine specially designed for the three types of infectious diseases. At present, the penetration rate of cat triple vaccine in China is about 10%, which still has a large gap compared with developed countries. Moreover, China's cat triple vaccine has long relied on imports, and the imported vaccine has low similarity to the domestic epidemic strain and weak protection.

[0003] The use effect of domestic cat triple vaccine is also limited by many factors, and there are problems such as low protection, short protection time, and adverse reactions. At the same time, there is no high-efficiency vaccine adjuvant that can improve the protection and reduce the vaccination cost. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a cat triple vaccine immune adjuvant containing two kinds of cat interleukins and metabolic regulatory molecules, a composition and application thereof.

[0005] The technical solution of the present application to solve the above technical problem is as follows:

[0006] The present application provides a composition comprising a cat interleukin mixture and a metabolic regulatory molecule, wherein the cat interleukin mixture comprises cat interleukin 15 and cat interleukin 23, and the metabolic regulatory molecule comprises beta-nicotinamide mononucleotide, beta-nicotinamide adenine dinucleotide disodium salt, N-acetylcysteine and L-carnitine.

[0007] Further, the mass ratio of the cat interleukin 15 and the cat interleukin 23 is 1:1.

[0008] Further, the mass ratio of the cat interleukin mixture to beta-nicotinamide mononucleotide, beta-nicotinamide adenine dinucleotide disodium salt, N-acetylcysteine and L-carnitine is 0.05-1:0.05-1:0.05-1:50-2000.

[0009] Further, the mass ratio of the cat interleukin mixture, beta-nicotinamide mononucleotide, beta-nicotinamide adenine dinucleotide disodium salt, N-acetylcysteine and L-carnitine is 0.1-0.5:0.1-0.5:0.1-0.5:50-1200.

[0010] The application also provides a use of the composition as described above, which is used for preparing a feline triple vaccine or an immune preparation of the feline triple vaccine.

[0011] Further, the feline triple vaccine is a complex inactivated vaccine.

[0012] Further, the immune preparation comprises a vaccine adjuvant and a vaccine synergist.

[0013] The application also provides a feline triple vaccine immune adjuvant containing two cat interleukins and a metabolic regulator, which comprises the composition as described above.

[0014] The application also provides a complex feline triple inactivated vaccine, which comprises the feline triple vaccine immune adjuvant as described above and feline triple vaccine antigens, and the volume ratio of the feline triple vaccine immune adjuvant and the feline triple vaccine antigens is 1:0.5-1.5.

[0015] Further, the complex feline triple inactivated vaccine further comprises a solvent; the feline triple vaccine antigens comprise 10 7.0 TCID 50 / head, the LZ-2016 strain 10 7.5 TCID 50 / head and the CS-2016 strain 10 5.5 TCID 50 / head; and the solvent comprises one or more of sterile physiological saline and sterile buffer.

[0016] The application has the following beneficial effects:

[0017] (1) The composition of the application can be used for preparing a feline triple vaccine or an auxiliary preparation of the feline triple vaccine, which can effectively improve the protection of the vaccine, prolong the protection time of the vaccine and reduce adverse reactions;

[0018] (2) The composition of the application provides the application of interleukins in feline triple vaccine adjuvants, provides a new idea for the development and research of feline triple vaccine adjuvants, and also expands a new field for the application of cat interleukins;

[0019] (3) The feline triple inactivated vaccine containing the immunoadjuvant has high safety, does not affect the growth and development of animals, can obviously promote animals to produce more blood specific TcEM and TcCM cells, improves the specific cellular immunity and memory ability of animals, and more durably and at a high level reduces or controls the reproductive infection of feline panleukopenia virus (FPV), feline calicivirus (FCV) and feline herpesvirus (FHV-1);

[0020] (4) The feline triple inactivated vaccine containing the immunoadjuvant can improve the number of lymphoid follicle B cells and activated B cells of the immune system, obviously promotes animals to produce more specific FPV, FCV and FHV-1 antibodies, significantly improves the specific humoral immunity level and memory response ability of animals, and better resists the invasion and spread of FPV, FCV and FHV-1. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The figure is a comparison chart of the weight change of mice in each experimental group of the feline triple vaccine immunoadjuvant of the present application;

[0022] Figure 2 The figure is a comparison chart of the change of feline calicivirus neutralizing antibody neutralization value in the serum of mice in each experimental group in Example 1 of the feline triple vaccine immunoadjuvant of the present application;

[0023] Figure 3 The figure is a comparison chart of the change of feline calicivirus neutralizing antibody neutralization value in the serum of mice in each experimental group in Example 1 of the feline triple vaccine immunoadjuvant of the present application;

[0024] Figure 4 The figure is a comparison chart of the change of feline calicivirus neutralizing antibody neutralization value in the serum of mice in each experimental group in Example 1 of the feline triple vaccine immunoadjuvant of the present application;

[0025] Figure 5 The figure is a comparison chart of the change of feline calicivirus neutralizing antibody neutralization value in the serum of mice in each experimental group in Example 1 of the feline triple vaccine immunoadjuvant of the present application;

[0026] Figure 6 The figure is a comparison chart of the change of feline calicivirus neutralizing antibody neutralization value in the serum of mice in each experimental group in Example 1 of the feline triple vaccine immunoadjuvant of the present application;

[0027] Figure 7 The figure is a comparison chart of the change of feline calicivirus neutralizing antibody neutralization value in the serum of mice in each experimental group in Example 1 of the feline triple vaccine immunoadjuvant of the present application;

[0028] Figure 8For the cat triple vaccine immunological adjuvant of the present application, in Example 3, the comparison chart of the average mean corpuscular hemoglobin concentration (MCHC) in the peripheral blood of mice in each experimental group;

[0029] Figure 9 For the cat triple vaccine immunological adjuvant of the present application, in Example 4, the comparison chart of the proportion of CD8 effector memory T lymphocytes in the blood of mice in each experimental group on the 56th day after the first immunization;

[0030] Figure 10 For the cat triple vaccine immunological adjuvant of the present application, in Example 4, the comparison chart of the proportion of T lymphocyte subsets in the spleen of each experimental mouse on the 56th day after the first immunization;

[0031] Figure 11 For the cat triple vaccine immunological adjuvant of the present application, in Example 4, the scatter plot of CD8 effector memory T lymphocytes in the peripheral blood of mice in each experimental group on the 56th day after the first immunization, Figure 11 a is the treatment group B2, Figure 11 b is the vaccine control group C1, Figure 11 c is the blank control group C3;

[0032] Figure 12 For the cat triple vaccine immunological adjuvant of the present application, in Example 4, the scatter plot of central memory T cells in the spleen of mice in each experimental group on the 56th day after the first immunization, Figure 12 a is the treatment group B2, Figure 12 b is the vaccine control group C1, Figure 12 c is the blank control group C3;

[0033] Figure 13 For the cat triple vaccine immunological adjuvant of the present application, in Example 4, the scatter plot of tissue-resident memory T cells in the spleen of mice in each experimental group on the 56th day after the first immunization, Figure 13 a is the treatment group B2, Figure 13 b is the vaccine control group C1, Figure 13 c is the blank control group C3;

[0034] Figure 14 For the cat triple vaccine immunological adjuvant of the present application, in Example 4, the comparison chart of the proportion of B lymphocyte subsets in mice in each experimental group on the 28th day after the first immunization;

[0035] Figure 15 For the cat triple vaccine immunological adjuvant of the present application, in Example 4, the comparison chart of the proportion of B lymphocyte subsets in mice in each experimental group on the 42nd day after the first immunization;

[0036] Figure 16The proportion of B lymphocyte subgroups of mice in each experimental group on the 56th day after the first immunization in Example 4 is compared in the following graph. DETAILED DESCRIPTION

[0037] The principles and features of the present application are described below in conjunction with the accompanying drawings, which are only used to explain the present application and are not intended to limit the scope of the present application.

[0038] The composition of the present application comprises a cat interleukin mixture and a metabolic regulator, the cat interleukin mixture comprises cat interleukin 15 (IL-15) and cat interleukin 23 (IL-23), and the metabolic regulator comprises β-nicotinamide mononucleotide (NMN), β-nicotinamide adenine dinucleotide disodium salt (NADH), N-acetylcysteine (N-Ace) and L-carnitne.

[0039] The composition of the present application can be used for preparing a cat triple vaccine or an immune preparation of a cat triple vaccine, wherein the cat triple vaccine specifically refers to a vaccine for preventing feline panleukopenia virus (FPV), feline calicivirus (FCV) and feline herpesvirus (FHV-1).

[0040] The composition of the present application can effectively improve the protection of the vaccine, prolong the protection time of the vaccine, and reduce adverse reactions. The composition can synergistically enhance the immune response level of the cat triple inactivated vaccine, improve and maintain a high level of immune protection of the cat triple inactivated vaccine, and has a specific humoral and cellular immune protection against feline panleukopenia virus (FPV), feline calicivirus (FCV) and feline herpesvirus (FHV-1) earlier, faster and more durable.

[0041] In addition, the above-mentioned composition of the present application provides the application of interleukin in the cat triple vaccine adjuvant, provides a new idea for the development and research of the cat triple vaccine adjuvant, and also expands a new field for the application of cat interleukin.

[0042] Preferably, the mass ratio of cat interleukin 15 and cat interleukin 23 is 1:1.

[0043] Preferably, the mass ratio of the cat interleukin mixture to β-nicotinamide mononucleotide, β-nicotinamide adenine dinucleotide disodium salt, N-acetylcysteine and L-carnitne is 0.05-1:0.05-1:0.05-1:50-2000.

[0044] Preferably, the mass ratio of the feline interleukin mixture to the beta-nicotinamide mononucleotide, the beta-nicotinamide adenine dinucleotide disodium salt, the N-acetylcysteine and the L-carnitine is 0.1-0.5:0.1-0.5:0.1-0.5:50-1200.

[0045] Preferably, in use, the amounts of the above ingredients are, based on an average body weight of 18 g per mouse, feline IL-15: 0.028 μg / g (w / w); feline IL-23: 0.028 μg / g (w / w); NMN: 2.78 μg / g (w / w); NAD: 2.78 μg / g (w / w); N-Ace: 33.33 μg / g (w / w); L-carnitine: 66.67 μg / g (w / w).

[0046] Further, the feline triple vaccine is a complex inactivated vaccine.

[0047] Further, the immunological preparation includes a vaccine adjuvant and a vaccine potentiator.

[0048] The feline triple vaccine immunological adjuvant containing two interleukins and metabolic regulatory molecules of the present application includes the composition as described above.

[0049] The feline triple vaccine immunological adjuvant containing two interleukins and metabolic regulatory molecules of the present application includes the composition as described above, and further includes a pharmaceutically and immunologically acceptable excipient.

[0050] Specifically, the pharmaceutically and immunologically acceptable excipient includes a solvent, which can be one or more of sterile physiological saline, sterile buffer solution.

[0051] The complex feline triple inactivated vaccine of the present application includes the feline triple vaccine immunological adjuvant as described above and the feline triple vaccine antigen, and the volume ratio of the two is 1:0.5-1.5, preferably, the volume ratio of the two is 1:1.

[0052] Preferably, in the complex feline triple inactivated vaccine of the present application, the concentration of feline interleukin 15 is 2-3 μg / ml, the concentration of feline interleukin 23 is 2-3 μg / ml, the concentration of beta-nicotinamide mononucleotide is 220-280 μg / ml, the concentration of beta-nicotinamide adenine dinucleotide disodium salt is 220-280 μg / ml, the concentration of N-acetylcysteine is 2-4 mg / ml, and the concentration of L-carnitine is 5-7 mg / ml.

[0053] Further preferably, in the composite feline triple inactivated vaccine of the present application, the concentration of feline interleukin 15 is 2.5 μg / ml, the concentration of feline interleukin 23 is 2.5 μg / ml, the concentration of β-nicotinamide mononucleotide is 250 μg / ml, the concentration of β-nicotinamide adenine dinucleotide disodium salt is 250 μg / ml, the concentration of N-acetylcysteine is 3 mg / ml, and the concentration of L-carnitine is 6 mg / ml.

[0054] Preferably, the solvent comprises one or more of sterile water, sterile physiological saline, and sterile buffer. 7.0 TCID 50 / fraction, the concentration of LZ-2016 strain is 10 7.5 TCID 50 / fraction, and the concentration of CS-2016 strain is 10 5.5 TCID 50 / fraction.

[0055] Further preferably, 0.1 mL of the antigen corresponds to 0.5 μg of feline IL-15 per cat, 0.5 μg of feline IL-23 per cat, 50 μg of NMN per cat, 50 μg of NAD per cat, 0.6 mg of N-Ace per cat, and 1.2 mg of L-carnitine per cat.

[0056] Preferably, the feline triple inactivated vaccine containing the immunoadjuvant is administered by intramuscular or subcutaneous injection.

[0057] Experiments have verified that the feline triple inactivated vaccine containing the immunoadjuvant of the present application has high safety and does not affect the growth and development of animals, and can significantly promote animals to produce more blood-specific TcEM (cytotoxic Teffector memory cell) and TcCM (Tc central memory) cells, improve the specific cellular immunity and memory ability of animals, and more persistently and at a high level reduce or control the reproductive infection of feline panleukopenia virus (FPV), feline calicivirus (FCV), and feline herpesvirus (FHV-1). At the same time, the administration of the composite vaccine can increase the number of lymphoid follicle B cells (Follicular B cell, FOB) and activated B cells of the immune system, significantly promote animals to produce more specific FPV, FCV, and FHV-1 antibodies, significantly improve the specific humoral immunity level and memory response ability of animals, and better resist the invasion and spread of FPV, FCV, and FHV-1.

[0058] The present application is specifically described below through specific examples.

[0059] In the following examples, unless otherwise specified, all experimental operations are carried out by using conventional methods, referring to the technical means or condition requirements described in the relevant literature or according to the product instruction. The materials and reagents used in the examples, unless otherwise specified, can be obtained by commercial means. Unless otherwise specified, the quantitative experiments in the examples were repeated three times, and the final results were averaged. Data analysis: the experimental data were statistically analyzed using GraphPad Prism 7.0 software, and multiple comparison analysis test was carried out by Tukey's of One way ANOVA; P<0.05 is the significant difference threshold, indicating that there is a significant difference between the data; P>0.05, there is no statistical difference.

[0060] The Kunming mice in the following examples come from the Experimental Animal Center of Sichuan University, with production license number SCXK (Chuan) 2018-026.

[0061] In the following examples, the grouping and immunization procedure of mice are as follows:

[0062] (1) 30 six-week-old female Kunming mice were randomly divided into 3 groups, 10 mice in each group.

[0063] (2) According to the specific treatment method in Table 1, each group of mice was immunized subcutaneously on the back, and was boosted once at the third week.

[0064] Table 1: Immunization grouping of mice

[0065]

[0066] The specific sources of each reagent in Table 1 are as follows: feline triple vaccine antigen (10 7.0 TCID 50 / head WH-2017 strain + 10 7.5 TCID 50 / head LZ-2016 strain + 10 5.5 TCID 50CS-2016 strain) was provided by Sichuan Huapai Biotechnology (Group) Co., Ltd. Feline interleukin 15 (IL-15, catalog number: 7805-FL-010) and IL-23 (catalog number: 2117-FL-025) were provided by RnD Systems, USA. β-nicotinamide mononucleotide (NMN, 1094-61-7, produced by Xi'an Tianfeng Biological Technology Co., Ltd.), β-nicotinamide adenine dinucleotide disodium salt (NADH, 606-68-8, produced by Bondant Biological Engineering (Shenzhen) Co., Ltd.), N-acetylcysteine (616-91-1, provided by Shanghai Jizhisheng Biological Technology Co., Ltd.), and L-carnitne (541-15-1, provided by MedChemExpress LLC (Shanghai)).

[0067] Mn 2+ from manganese chloride MnCl2 (produced by Tianjin Fumian Chemical Reagent Factory, analytical pure reagent).

[0068] Data collection was performed on each group of mice, and the specific collection method was as follows:

[0069] (1) Body weight index: each group of mice was weighed once a week for eight consecutive weeks, and the body weight dynamic changes of each group of mice were recorded. The results are shown in Figure 1 . According to Figure 1 , it can be seen that the body weight of each group of mice at the 8 time points has no significant difference (P>0.05), and the growth weight gain has no significant difference, which proves that the vaccine adjuvant used in the application has good biological safety.

[0070] (2) Blood immune index: the mice in each group were collected once a week by cutting the tail to collect venous anticoagulated blood (200 μL per mouse).

[0071] (3) Lymphocyte subsets: the mice were euthanized on the 56th day after the first immunization, the spleen was taken to prepare a lymphocyte suspension and the number of lymphocyte subsets was detected.

[0072] Example 1 Determination of feline triple neutralizing antibody level by fixed virus dilution serum method

[0073] Neutralizing antibody level detection was performed before the first immunization, on the 7th, 14th, 28th, 42nd and 56th days after the first immunization, 100 TCID 50 of feline calicivirus diluted per unit dose was mixed with an equal amount of 2-fold serially diluted test serum, and the mixture was incubated at 37℃ for 60 minutes. 3-6 cell holes were inoculated for each dilution, and after inoculation, the number of cell holes with CPE in each group was recorded. The half protection dose (PD 50 ) of each group was calculated by Reed-Muench method, and then the neutralizing titer of the serum was calculated.

[0074] The calculation results are shown below. Figure 2 .according to Figure 2 It can be seen that the level of feline calicivirus (FCV) neutralizing antibodies in the adjuvant-treated group (B2) was significantly higher than that in the vaccine control group (C1) at the three measurement time points after the booster immunization in week 3 (P<0.05), indicating that the adjuvant can significantly improve the immune enhancement effect against FCV after booster immunization.

[0075] Example 2: In vitro assay of feline triple antigen-specific antibodies

[0076] Mouse plasma samples were collected and isolated before the first immunization and on days 7, 28, 42, and 56 after the first immunization for in vitro detection of specific antibodies. The levels of feline herpesvirus antibody (FHV-Ab) and feline parvovirus antibody (FPV-Ab) in mouse plasma were determined in vitro according to the instructions of the feline triple antibody (FCV-Ab / FHV-Ab / FPV-Ab) detection kit (fluorescent immunochromatography, catalog number PRG108, Shanghai Jiling Biotechnology Co., Ltd., China).

[0077] The results of feline herpesvirus-specific antibody (FHV-Ab) and feline parvovirus-specific antibody (FPV-Ab) level measurements are shown in [link to data]. Figure 3 and Figure 4 .

[0078] according to Figure 3 It can be seen that the levels of feline herpesvirus-specific antibodies (FHV-Ab) in both groups B2 and C1 showed a gradual increasing trend. On days 7, 28, 42, and 56 after the first immunization, the levels of specific antibodies in the experimental group (B2) were higher than those in the control group (C1). Furthermore, the level of specific antibodies in the experimental group (B2) was significantly higher at the measurement time point after the second immunization on day 21.

[0079] according to Figure 4 It can be seen that the levels of feline parvovirus-specific antibodies (FPV-Ab) in the experimental group (B2) showed a gradual increasing trend. At all five measurement time points, the specific antibody levels in B2 were higher than those in the negative control group (C1), and the specific antibody levels in the experimental group (B2) were significantly higher at the measurement time point after the second immunization on day 14. This indicates that the adjuvant has a significant and sustained immune-enhancing effect.

[0080] Example 3: Routine Immunological Analysis of Whole Blood from Mice

[0081] Blood samples from each group were analyzed for complete blood count (CBC) before the first immunization and on days 7, 14, 28, 42, and 56 after the first immunization. 50 μL of EDTA-anticoagulated blood was collected from each group, and CBC analysis was performed on the mice using the TEK-VET5 fully automated five-part differential blood analyzer. The results are shown below. Figures 5-8 .

[0082] according to Figures 5-8 It can be seen that at each measurement time point, there were no significant differences in the red blood cell volume distribution width, red blood cell and lymphocyte percentages, and mean corpuscular hemoglobin concentration among the experimental groups (P>0.05), indicating that the vaccine adjuvant has reliable biosafety.

[0083] Example 4: Verification of Immunization Effect

[0084] This embodiment uses blood flow cytometry to analyze the changes in immune cells in the venous blood of mice in different experimental groups at different time points to verify the effect of the adjuvant of the present invention on the immune response and long-term immune protection of mice after primary infection.

[0085] In this embodiment, blood samples from each group of mice were analyzed by flow cytometry on days 28, 42, and 56 after the initial immunization to detect changes in immune cells. The specific detection steps were as follows:

[0086] (1) Add anti-mouse CD45 (CD45 Monoclonal Antibody (30-F11), Super Bright) to a 1.5 ml EP tube containing 100 μl of mouse peripheral anticoagulated blood (EDTA·2K anticoagulant). TM 600, eBioscience TM ), CD3 (BDPharmingen TM FITC Hamster Anti-Mouse CD3e), CD4 (CD4 Monoclonal Antibody(GK1.5),eFluor TM 450, eBioscience TM ), CD8(CD8a Monoclonal Antibody(53-6.7),PerCP-Cyanine5.5,eBioscience TM ), CD44(CD44 Monoclonal Antibody(IM7),APC,eBioscience TM ) and CD62L(CD62L(L-Selectin)Monoclonal Antibody(MEL-14),PE,eBioscience TM 1 μl of each flow cytometry antibody was incubated at 4°C in the dark for 30 min.

[0087] (2) Add 1 ml of 1×RBC lysis buffer (prepared with deionized water) to the above reaction system, lyse at room temperature for 5 min, centrifuge at 1500 rpm and 4℃ for 5 min, and remove the supernatant.

[0088] (3) Add 200 μl PBS (containing 0.5% BSA) to the cell pellet from step 2, mix well by pipetting, centrifuge at 1500 rpm and 4℃ for 5 min, and discard the supernatant.

[0089] (4) Repeat step 3.

[0090] (5) After resuspending the cells in 500 μl PBS (containing 0.5% BSA), fix them with 2% paraformaldehyde to a final volume, store in the dark at 4°C, and then run on the instrument.

[0091] The following staining protocols are treated in the same way:

[0092] (1) Add anti-mouse CD45 (CD45 Monoclonal Antibody (30-F11), SuperBright) to tube 2. TM 600, eBioscience TM ), CD3 (BD ​​Pharmingen TM FITC Hamster Anti-Mouse CD3e), CD4 (CD4 Monoclonal Antibody(GK1.5),eFluor TM 450, eBioscience TM ), CD69(CD69Monoclonal Antibody(H1.2F3),PE,eBioscience TM ) and CD103(CD103(Integrin alpha E)Monoclonal Antibody(2E7),APC,eBioscience TM 1 μl of each flow cytometry antibody;

[0093] (2) Anti-mouse CD19 (CD19 BUV395), IgM (lgM APC), IgD (lgD FITC) and CD38 (PE / Dazzle) were added to tube 3. TM 1 μl each of 594 anti-mouse CD38 flow cytometry antibody.

[0094] In this embodiment, the process for preparing spleen single-cell suspension is as follows:

[0095] (1) After the mice were euthanized, the spleen was placed on a 70μm cell sieve and then placed in a 6cm dish. 3ml of PBS (containing 0.5% BSA) was added, and the spleen was directly ground on the 70μm cell sieve by hand with a 2ml syringe. After grinding, the cell sieve was rinsed with 2ml of PBS (containing 0.5% BSA).

[0096] (2) Add spleen cell suspension to 15 ml centrifuge tube, 1500 rpm, 4°C, centrifuge 5 min, remove supernatant.

[0097] (3) Red cell lysis: add 3 ml of 1 x RBC lysis buffer, vortex to mix, lyse at room temperature for 5 min, 1500 rpm, 4°C, centrifuge 5 min, remove supernatant.

[0098] (4) Second straining: add 3 ml PBS (with 0.5% BSA), vortex to mix, then place 70 μm cell strainer on 50 ml centrifuge tube, filter spleen cells again. Collect filtered cells in 50 ml centrifuge tube, 1500 rpm, 4°C, centrifuge 5 min, remove supernatant, wash once with PBS containing BSA and once with PBS without BSA.

[0099] (5) Resuspend spleen cells: add 1 ml PBS (with 0.5% BSA), vortex to mix, remove connective tissue with pipette, then add 100 μl spleen cells to each test; spleen single cell suspension is ready for subsequent staining procedures.

[0100] (6) Resuspend spleen cells with 50 μl PBS (with 0.5% BSA), add 1 μl blocking antibody (BD Pharmingen TM Purified Rat Anti-Mouse CD16 / CD32 (Mouse BD Fc Block TM )), incubate at 4°C for 10 min, without washing, start incubation with surface marker antibodies.

[0101] (7) Stain according to the color scheme for blood flow cytometry, incubate on ice for 30 min, wash twice, add 200 μl 4% paraformaldehyde, fix at room temperature for 30 min in the dark, wash once and resuspend to 200 μl, load onto machine.

[0102] The gate logic of this example is as follows:

[0103] (1) Immune memory T lymphocyte gate logic:

[0104] T cells are defined as the CD3+ / CD45+ population. Depending on the differential expression of CD4 and CD8, T cells can be further divided into four subpopulations: CD4+ TH cells, CD8+ TC cells, CD4+ / CD8+ double positive (DP) cells and CD4- / CD8- double negative (DN) cells. The latter is mainly composed of γδ+ cells under normal circumstances. When analyzing CD44 and CD62L in TH and TC lymphocytes, three different subpopulations can be distinguished: CD62LhiCD44neg / lo Cell, CD62LhiCD44hi central memory (CM) cells and CD62Lneg / loCD44hi effector memory (EM) cells, as shown in Table 2.

[0105] Table 2 Flow cytometry gating logic

[0106]

[0107] Figure 9 The percentage of CD8+ effector memory T lymphocytes in the peripheral blood of mice on day 56, Figure 10 The percentage of T lymphocyte subgroups in the spleen of mice on day 56. Figure 11 、 Figure 12 、 Figure 13 The scatter plots of CD8+ effector memory T cells in the peripheral blood, central memory T cells in the spleen and tissue-resident memory T cells in the spleen of mice on day 56, respectively.

[0108] According to the above results, it can be seen that in the analysis of blood T cells on day 56, the percentage of CD8+ effector memory T cells in the experimental group (B2) was significantly higher than that in the control groups (C1, C3); in the analysis of spleen T cells on day 56, the percentages of CD8+ central memory T cells and tissue-resident memory T cells in the experimental group (B2) were higher than those in the control groups (C1, C3).

[0109] CD8 T cells can directly participate in cytotoxicity to eliminate virus-infected cells. Central memory T cells can exist for a long time in the body and can respond quickly when encountering the same antigen again, providing long-term immune effect, while effector memory T cells provide rapid immune protection after primary infection.

[0110] The above experimental results show that the adjuvant can enhance the immune response of mice after primary infection and provide stronger long-term immune protection effect.

[0111] (2) Immune memory B lymphocyte circle gating logic:

[0112] Referring to the blood flow cytometry sample preparation and machine loading method of the present embodiment, 1 μl of anti-mouse CD19 (CD19 BUV395, BD), IgM (IgM APC, ThermoFisher, 17-5790-82) and IgD (IgD FITC, ThermoFisher, 11-5993-85) flow cytometry antibodies were added to the tube; B cells were defined as the CD19+ population.

[0113] Four different subgroups can be distinguished in the analysis of IgM and IgD in B cells: class switched or activated B cells (IgM-, IgD-), transitional B cells (IgM+, IgD-), marginal zone B cells (IgM+, IgD+) and naive follicular B cells (IgM-, IgD+).

[0114] The percentages of B lymphocyte subgroups in the peripheral blood of mice on the 28th, 42nd and 56th day are shown in Table 1. Figure 14 、 Figure 15 、 Figure 16 The percentages of B lymphocyte subgroups in the peripheral blood of mice on the 28th, 42nd and 56th day are shown in Table 1.

[0115] According to the above results, it can be seen that the content of transitional B cells (IgM+, IgD-) in the experimental group (B2) is less than that in the control groups (C1, C3), while the follicular B cells (IgM-, IgD+) and activated B cells (IgM-, IgD-) are significantly higher than those in the control groups (C1, C3).

[0116] Transitional cells represent the last stage before differentiation into a more mature population of pre-immune B cells, indicating that the addition of adjuvant promotes the activation and differentiation of B cells, and provides a basis for the immune system to produce more antibodies with high affinity to antigens, while promoting the formation of memory B cells, further proving that the addition of adjuvant is more conducive to improving the immune efficiency of vaccines and establishing long-term immune memory.

[0117] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A complex feline triple inactivated vaccine, characterized in that, The cat triple vaccine antigen, a cat interleukin mixture of cat interleukin 15 and cat interleukin 23, and a metabolic regulator of beta-nicotinamide mononucleotide, beta-nicotinamide adenine dinucleotide disodium salt, N-acetyl cysteine and L-carnitine; The cat triple vaccine antigen is 10 7.0 TCID 50 / head, the LZ-2016 strain 10 7.5 TCID 50 / head and the CS-2016 strain 10 5.5 TCID 50 / head; 0.1 mL of the cat triple vaccine antigen corresponds to 0.5 µg per cat for the cat interleukin 15 IL-15, 0.5 µg per cat for the cat interleukin 23, 50 µg per cat for beta-nicotinamide mononucleotide, 50 µg per cat for beta-nicotinamide adenine dinucleotide disodium salt, 0.6 mg per cat for N-acetyl cysteine, and 1.2 mg per cat for L-carnitine; The cat triple vaccine is a cat panleukopenia, rhinotracheitis, calicivirus disease triple vaccine.

2. The inactivated combined vaccine for cats according to claim 1, characterized in that, Each milliliter of the complex cat triple inactivated vaccine also contains one or more of sterile saline, sterile buffer. The cat triple vaccine antigen, a cat interleukin mixture of cat interleukin 15 and cat interleukin 23, and a metabolic regulator of beta-nicotinamide mononucleotide, beta-nicotinamide adenine dinucleotide disodium salt, N-acetyl cysteine and L-carnitine; 0.1 mL of the cat triple vaccine antigen corresponds to 0.5 µg per cat for the cat interleukin 15 IL-15, 0.5 µg per cat for the cat interleukin 23, 50 µg per cat for beta-nicotinamide mononucleotide, 50 µg per cat for beta-nicotinamide adenine dinucleotide disodium salt, 0.6 mg per cat for N-acetyl cysteine, and 1.2 mg per cat for L-carnitine; The cat triple vaccine is a cat panleukopenia, rhinotracheitis, calicivirus disease triple vaccine. Each milliliter of the complex cat triple inactivated vaccine also contains one or more of sterile saline, sterile buffer.

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