Cat triple vaccine immunologic adjuvant containing two cat interleukinins and metabolism regulating molecules, composition and application

Abstract

The invention relates to the technical field of biological medicine, in particular to a cat triple vaccine immunologic adjuvant containing two cat interleukinins and a metabolism regulating molecule, a composition and application. The composition comprises cat interleukin 15, cat interleukin 23 and metabolic regulation molecules, the metabolic regulation molecules comprise beta-nicotinamide mononucleotide, beta-nicotinamide adenine dinucleotide disodium salt, N-acetylcysteine and L-carnitine, and the composition 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 capacity of vaccines, prolong the protection time of the vaccines and reduce adverse reactions; the vaccine can reduce or control reproduction infection of viruses in a more durable and higher level mode, the specific humoral immunity level and the memory response capacity of animals are remarkably improved, and invasion and diffusion of FPV, FCV and FHV-1 are better resisted.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and specifically, to a feline triple vaccine immune adjuvant, composition and application containing two feline interleukins and a metabolic regulatory molecule. Background Art

[0002] With the development of the Chinese pet market, the number of pet cats has increased unprecedentedly, and the status of pet cats in families has been greatly improved. In the face of the continuously growing scale of pet cats, the prevention and epidemic prevention of cat infectious diseases are particularly important. Feline panleukopenia, feline calicivirus disease and feline viral rhinotracheitis are three common cat infectious diseases, which are caused by feline parvovirus (FPV), feline calicivirus (FCV) and feline herpesvirus (FHV-1) infections respectively, and can cause diseases in the cat respiratory tract and intestines, etc. In severe cases, they can even lead to death. The feline triple vaccine is fully called "feline panleukopenia, rhinotracheitis, calicivirus disease triple vaccine", that is, a feline vaccine specifically for these three types of infectious diseases. At present, the penetration rate of the feline triple vaccine in China is about 10%, which still has a large gap compared with developed countries. Moreover, China's feline triple vaccine has long relied on imports, and imported vaccines have the problems of low similarity between the virus strains and the domestic prevalent virus strains and weak protective power.

[0003] The use effect of domestic feline triple vaccines is also restricted by various factors, and there are problems such as low protective power, short protection time, and adverse reactions. At the same time, there is currently no highly efficient vaccine adjuvant that can improve the protective power and reduce the vaccination cost. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a feline triple vaccine immune adjuvant, composition and application containing two feline interleukins and a metabolic regulatory molecule.

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

[0006] The present invention provides a composition, including a feline interleukin mixture and a metabolic regulatory molecule, the feline interleukin mixture includes feline interleukin 15 and feline interleukin 23, and the metabolic regulatory molecule includes β-nicotinamide mononucleotide, β-nicotinamide adenine dinucleotide disodium salt, N-acetylcysteine and L-carnitine.

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

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

[0009] Furthermore, the mass ratio of the feline interleukin mixture to β-nicotinamide mononucleotide, disodium β-nicotinamide adenine dinucleotide, N-acetylcysteine, and L-carnitine is 0.1 - 0.5:0.1 - 0.5:0.1 - 0.5:50 - 1200.

[0010] The present invention also provides an application of the composition as described above, and the composition can be used to prepare a feline triple vaccine or an immunizing agent for a feline triple vaccine.

[0011] Furthermore, the feline triple vaccine is a combined inactivated vaccine.

[0012] Furthermore, the immunizing agent includes a vaccine adjuvant and a vaccine potentiator.

[0013] The present invention also provides a feline triple vaccine immunoadjuvant containing two feline interleukins and a metabolic regulatory molecule, including the composition as described above.

[0014] The present invention also provides a combined feline triple inactivated vaccine, including the feline triple vaccine immunoadjuvant and feline triple vaccine antigen as described above, and the volume ratio of the feline triple vaccine immunoadjuvant to the feline triple vaccine antigen is 1:0.5 - 1.5.

[0015] Furthermore, it also includes a solvent; in each milliliter of the combined feline triple inactivated vaccine, the feline triple vaccine antigen includes 10 7.0 TCID 50 / dose for strain WH - 2017, 10 7.5 TCID 50 / dose for strain LZ - 2016, and 10 5.5 TCID 50 / dose for strain CS - 2016; the solvent includes one or more of sterilized physiological saline and sterilized buffer solution.

[0016] The beneficial effects of the present invention are as follows:

[0017] (1) The composition of the present invention can be used to prepare a feline triple vaccine or an auxiliary agent for a feline triple vaccine, which can effectively improve the protective power of the vaccine, extend the vaccine protection time, and reduce adverse reactions;

[0018] (2) The composition of the present invention provides an application of interleukin in a feline triple vaccine adjuvant, provides new ideas for the development and research of feline triple vaccine adjuvants, and also expands new fields for the application of feline interleukin;

[0019] (3) The inactivated feline triple vaccine containing an immune adjuvant of the present invention has high safety, does not affect the growth and development of animals, and can significantly promote animals to produce more blood-specific TcEM and TcCM cells, improve the specific cellular immunity and memory ability of animals, and reduce or control the reproductive infection of feline panleukopenia virus (FPV), feline calicivirus (FCV) and feline herpesvirus (FHV-1) more persistently and at a high level;

[0020] (4) The inactivated feline triple vaccine containing an immune adjuvant of the present invention can increase the number of lymphoid follicle B cells and activated B cells in 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a comparison chart of the changes in the body weights of mice in each experimental group for the immune adjuvant of the feline triple vaccine of the present invention;

[0022] Figure 2 It is a comparison chart of the changes in the neutralization titer of feline calicivirus neutralizing antibody in the sera of mice in each experimental group in Example 1 for the immune adjuvant of the feline triple vaccine of the present invention;

[0023] Figure 3 It is a comparison chart of the changes in the level of feline herpesvirus specific antibody (FHV-Ab) in the plasma of mice in each experimental group in Example 2 for the immune adjuvant of the feline triple vaccine of the present invention;

[0024] Figure 4 It is a comparison chart of the changes in the level of feline parvovirus specific antibody (FPV-Ab) in the plasma of mice in each experimental group in Example 2 for the immune adjuvant of the feline triple vaccine of the present invention;

[0025] Figure 5 It is a comparison chart of the changes in the red blood cell volume distribution width (RSD-CV) in the peripheral blood of mice in each experimental group in Example 3 for the immune adjuvant of the feline triple vaccine of the present invention;

[0026] Figure 6 It is a comparison chart of the changes in red blood cells (RBC) in the peripheral blood of mice in each experimental group in Example 3 for the immune adjuvant of the feline triple vaccine of the present invention;

[0027] Figure 7 It is a comparison chart of the changes in the percentage of lymphocytes (LYM%) in the peripheral blood of mice in each experimental group in Example 3 for the immune adjuvant of the feline triple vaccine of the present invention;

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

[0029] Figure 9 For the feline triple vaccine immune adjuvant of the present invention, in Example 4, the comparison chart of the proportions 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 feline triple vaccine immune adjuvant of the present invention, in Example 4, the comparison chart of the proportions of T lymphocyte subsets in the spleens of experimental mice on the 56th day after the first immunization;

[0031] Figure 11 For the feline triple vaccine immune adjuvant of the present invention, 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 In it, a is treatment group B2. Figure 11 In it, b is vaccine control group C1. Figure 11 In it, c is blank control group C3.

[0032] Figure 12 For the feline triple vaccine immune adjuvant of the present invention, in Example 4, the scatter plot of central memory T cells in the spleens of mice in each experimental group on the 56th day after the first immunization. Figure 12 In it, a is treatment group B2. Figure 12 In it, b is vaccine control group C1. Figure 12 In it, c is blank control group C3.

[0033] Figure 13 For the feline triple vaccine immune adjuvant of the present invention, in Example 4, the scatter plot of tissue-resident memory T cells in the spleens of mice in each experimental group on the 56th day after the first immunization. Figure 13 In it, a is treatment group B2. Figure 13 In it, b is vaccine control group C1. Figure 13 In it, c is blank control group C3.

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

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

[0036] Figure 16For the feline triple vaccine immunoadjuvant of the present invention, in Example 4, the comparison chart of the proportions of B lymphocyte subsets in mice of each experimental group on the 56th day after the first immunization. Detailed implementation mode

[0037] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0038] The composition of the present invention includes a feline interleukin mixture and a metabolic regulatory molecule. The feline interleukin mixture includes feline interleukin-15 (interleukin-15, IL-15) and feline interleukin-23 (interleukin-23, IL-23). The metabolic regulatory molecule includes β-nicotinamide mononucleotide (NMN), β-nicotinamide adenine dinucleotide disodium salt (NADH), N-acetylcysteine (N-Acetylcysteine, N-Ace), and L-carnitine (L-carnitne).

[0039] The composition of the present invention can be used to prepare a feline triple vaccine or an immunopreparation of a feline triple vaccine. Among them, the feline triple vaccine specifically refers to a vaccine that can prevent feline panleukopenia virus (FPV), feline calicivirus (FCV), and feline herpesvirus (FHV-1).

[0040] The composition of the present invention can effectively improve the protective power of the vaccine, extend the vaccine protection time, and reduce adverse reactions. This composition can synergistically enhance the immune response level of the feline triple inactivated vaccine, improve and maintain a high level of immune protection and protection period of the feline triple inactivated vaccine, and is safe and non-toxic. It can stimulate animals to obtain specific humoral and cellular immune protection against feline panleukopenia virus (FPV), feline calicivirus (FCV), and feline herpesvirus (FHV-1) earlier, faster, and more persistently.

[0041] In addition, the above composition of the present invention provides the application of interleukin in the adjuvant of the feline triple vaccine, providing new ideas for the development and research of the adjuvant of the feline triple vaccine, and also expanding new fields for the application of feline interleukin.

[0042] Preferably, the mass ratio of feline interleukin-15 to feline interleukin-23 is 1:1.

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

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

[0045] Preferably, during use, calculated based on the average initial immunization body weight of mice at 18 g per mouse, the dosages of the above components are as follows: 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] Furthermore, the feline triple vaccine is a combined inactivated vaccine.

[0047] Furthermore, the immunopotentiator includes a vaccine adjuvant and a vaccine synergist.

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

[0049] The feline triple vaccine immunopotentiator containing two interleukins and metabolic regulatory molecules of the present invention includes the composition as described above, and also includes pharmaceutically and immunologically acceptable excipients.

[0050] Specifically, the pharmaceutically and immunologically acceptable excipients include solvents, and the solvents can be one or more of sterilized normal saline and sterilized buffer solutions.

[0051] The combined feline triple inactivated vaccine of the present invention includes the feline triple vaccine immunopotentiator and the feline triple vaccine antigen as described above, 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 combined feline triple inactivated vaccine of the present invention, 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 β-nicotinamide mononucleotide is 220 - 280 μg / ml, the concentration of disodium β-nicotinamide adenine dinucleotide 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 combined feline triple-inactivated vaccine of the present invention, 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, it further includes a solvent; in each milliliter of the combined feline triple-inactivated vaccine, the feline triple vaccine antigen includes 10 7.0 TCID 50 / dose of WH-2017 strain, 10 7.5 TCID 50 / dose of LZ-2016 strain, and 10 5.5 TCID 50 / dose of CS-2016 strain. The solvent includes one or more of sterilized water, sterilized normal saline, and sterilized buffer solution.

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

[0056] Preferably, the feline triple-inactivated vaccine containing an immunoadjuvant can be administered by intramuscular or subcutaneous injection.

[0057] Verified by experiments, the feline triple-inactivated vaccine containing an immunoadjuvant of the present invention has high safety, does not affect the growth and development of animals, and can significantly promote the production of more blood-specific TcEM (cytotoxic Teffector memory cell) and TcCM (Tc central memory) cells in animals, improving the specific cellular immunity and its memory ability of animals, and reducing or controlling the reproductive infection of feline panleukopenia virus (FPV), feline calicivirus (FCV), and feline herpesvirus (FHV-1) more persistently and at a high level. At the same time, inoculating the combined vaccine can increase the number of follicular B cells (FOB) and activated B cells in the immune system, significantly promote the production of more specific FPV, FCV, and FHV-1 antibodies in animals, significantly improve the specific humoral immunity level and its memory response ability of animals, and better resist the invasion and spread of FPV, FCV, and FHV-1.

[0058] The present invention will be specifically described below through specific examples.

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

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

[0061] In the following embodiments, the grouping and immunization procedures of the mice were as follows:

[0062] (1) Thirty 6-week-old female Kunming mice were randomly divided into 3 groups, with 10 mice in each group.

[0063] (2) Each group of mice was immunized subcutaneously in the back according to the specific treatment method in Table 1, and boosted immunization was carried out once in the third week.

[0064] Table 1 Immunization grouping of mouse experiments

[0065]

[0066] The specific sources of the reagents in Table 1 were: Feline panleukopenia virus, feline calicivirus, feline rhinotracheitis virus vaccine antigen (10 7.0 TCID 50 / dose WH-2017 strain + 10 7.5 TCID 50 / dose LZ-2016 strain + 10 5.5 TCID 50 / The strain CS-2016 in Toufen 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 produced and provided by RnD Systems, USA. β-Nicotinamide mononucleotide (NMN, 1094-61-7, produced by Xi'an Tianfeng Biotechnology Co., Ltd.), β-Nicotinamide adenine dinucleotide disodium salt (NADH, 606-68-8, produced by Bangtai Biotechnology Engineering (Shenzhen) Co., Ltd.), N-Acetylcysteine (616-91-1, provided by Shanghai Jizhi Biochemical Technology Co., Ltd.), L-carnitine (541-15-1, provided by MedChemExpress LLC (Shanghai)).

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

[0068] Data collection was carried out on the mice in each of the above groups. The specific collection method was as follows:

[0069] (1) Body weight index: The mice in each group were weighed once a week for eight consecutive weeks, and the dynamic changes in the body weights of the mice in each group were recorded. The results are shown in Figure 1 . According to Figure 1 It can be seen that there were no significant differences in the body weights of the mice in each group at 8 time points (P>0.05), and there were no significant differences in growth and weight gain, proving that the vaccine adjuvant used in the present invention has good biosafety.

[0070] (2) Blood immune index: The mice in each group were collected with venous anticoagulant blood (200 μL / mouse) by tail cutting once a week.

[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 vaccine neutralizing antibody level by fixed virus diluted serum method

[0073] Neutralizing antibody level detection was carried out on the days before the first immunization, and on the 7th, 14th, 28th, 42nd, and 56th days after the first immunization. The feline calicivirus diluted to contain 100 TCID 50 per unit dose was mixed with an equal volume of the test serum serially diluted 2-fold, and reacted at 37 °C for 60 minutes. Each dilution was inoculated into 3-6 cell wells. After inoculation, the number of cell wells with or without CPE in each group was recorded, and the 50% protective dose (PD 50 ) of each group was calculated by the Reed-Muench method, and then the serum neutralization titer was calculated.

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

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

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

[0077] The determination results of the levels of feline herpesvirus-specific antibody (FHV-Ab) and feline parvovirus-specific antibody (FPV-Ab) are shown in Figure 3 and Figure 4 .

[0078] According to Figure 3 It can be seen that the levels of feline herpesvirus-specific antibody (FHV-Ab) in both group B2 and group C1 showed a gradually increasing trend. The specific antibody levels in the experimental group (B2) were higher than those in the control group (C1) on days 7, 28, 42, and 56 after the first immunization, and the specific antibody levels in the experimental group (B2) increased significantly at the measurement time points after the second immunization on day 21.

[0079] According to Figure 4 It can be seen that the levels of feline parvovirus-specific antibody (FPV-Ab) in the experimental group (B2) showed a gradually increasing trend. The specific antibody levels in B2 were higher than those in the negative control group (C1) at the five measurement time points, and the specific antibody levels in the experimental group (B2) increased significantly at the measurement time points after the second immunization on day 14. It shows that the adjuvant has obvious and continuous immune enhancement effects.

[0080] Example 3 Routine immunological analysis of mouse whole blood

[0081] Blood samples of each group were subjected to routine blood analysis before the first immunization and on days 7, 14, 28, 42, and 56 after the first immunization. 50 μL of EDTA anticoagulated blood samples were taken from each group, and the routine blood analysis of mouse whole blood was carried out according to the operating procedures of the Tekang veterinary automatic five-class blood analyzer (TEK-VET5). The analysis results are shown in 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 cells, lymphocyte percentage, and mean corpuscular hemoglobin concentration of the mice in each experimental group (P > 0.05), indicating that the vaccine adjuvant has reliable biological safety.

[0083] Verification of the immune effect in Example 4

[0084] In this example, the changes of immune cells in the venous blood of mice in each experimental group at different times were analyzed by blood flow cytometry to verify the effect of the adjuvant of the present invention on the immune response and long-term immune protection after the primary infection of mice.

[0085] In this example, the changes of immune cells were detected by flow cytometry on the blood samples of each group of mice at 28, 42, and 56 days after the first immunization. The specific detection steps are as follows:

[0086] (1) Add 1 μl of each of the flow antibodies against mouse CD45 (CD45 Monoclonal Antibody (30-F11), Super Bright 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 ) to a 1.5 ml EP tube 1 containing 100 μl of mouse peripheral anticoagulated blood (EDTA·2K anticoagulant), and incubate 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, then centrifuge at 1500 rpm and 4°C for 5 min, and discard the supernatant.

[0088] (3) Add 200 μl of PBS (containing 0.5% BSA) to the cell pellet in step 2, pipette to mix well, centrifuge at 1500 rpm and 4 °C for 5 min, and discard the supernatant.

[0089] (4) Repeat step 3 once.

[0090] (5) Resuspend the cells with 500 μl of PBS (containing 0.5% BSA), then add paraformaldehyde to a final volume of 2% for fixation, place in the dark at 4 °C, and load onto the machine.

[0091] Perform the same treatment for the following staining protocol:

[0092] (1) Add 1 μl of each of the following flow antibodies to tube 2: anti-mouse CD45 (CD45 Monoclonal Antibody (30-F11), SuperBright 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 (CD69 Monoclonal Antibody (H1.2F3), PE, eBioscience TM ), and CD103 (CD103 (Integrin alpha E) Monoclonal Antibody (2E7), APC, eBioscience TM ).

[0093] (2) Add 1 μl of each of the following flow antibodies to tube 3: anti-mouse CD19 (CD19 BUV395), IgM (lgM APC), IgD (lgD FITC), and CD38 (PE / Dazzle TM 594 anti-mouse CD38).

[0094] In this example, the preparation process of the single-cell suspension of the spleen is as follows:

[0095] (1) After the mouse is sacrificed, place the spleen on a 70-μm cell strainer, then place it in a 6-cm dish, add 3 ml of PBS (containing 0.5% BSA), and directly grind it with the handle of a 2-ml syringe on the 70-μm cell strainer. After grinding, rinse the cell strainer with 2 ml of PBS (containing 0.5% BSA).

[0096] (2) Add the spleen cell suspension to a 15-ml centrifuge tube and centrifuge at 1500 rpm at 4°C for 5 min. Discard the supernatant.

[0097] (3) Lyse red blood cells: Add 3 ml of 1× RBC lysis buffer, vortex and pipette to mix well, lyse at room temperature for 5 min, centrifuge at 1500 rpm at 4°C for 5 min, and discard the supernatant.

[0098] (4) Secondary sieving: Add 3 ml of PBS (containing 0.5% BSA), vortex and pipette to mix well. Then place a 70-μm cell sieve on top of a 50-ml centrifuge tube and filter the spleen cells again. Collect the filtered cells in a 50-ml centrifuge tube, centrifuge at 1500 rpm at 4°C for 5 min, discard the supernatant, wash once with PBS containing BSA and then once with PBS without BSA.

[0099] (5) Resuspend spleen cells: Add 1 ml of PBS (containing 0.5% BSA), vortex and pipette to mix well. Use a pipette to pick out connective tissue, and then add 100 μl of spleen cells to each test. The preparation of the single-cell suspension of spleen cells is completed and can be used for subsequent staining procedures.

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

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

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

[0103] (1) Gating logic for immune memory T lymphocytes:

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

[0105] Table 2 Flow cytometry gating logic

[0106]

[0107] Figure 9 Percentage of CD8+ effector memory T lymphocytes in the peripheral blood of mice on day 56 in groups B2, C1, and C3 Figure 10 Percentage of T lymphocyte subsets in the spleen of mice on day 56 in groups B, C1, and C3. Figure 11 , Figure 12 , Figure 13 Scatter plots of CD8+ effector memory T cells in the peripheral blood of mice on day 56, central memory T cells in the spleen, and tissue-resident memory T cells in the spleen, respectively.

[0108] According to the above results, it can be seen that in the blood T cell analysis 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 spleen T cell analysis 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 cytotoxic effects to clear virus-infected cells. Central memory T cells can exist in the body for a long time and can respond rapidly when encountering the same antigen again, providing long-term immune effects, while effector memory T cells provide rapid immune protection after primary infection.

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

[0111] (2) Gating logic for immune memory B lymphocytes:

[0112] Referring to the blood flow cytometry sample preparation and instrument operation method of this example, add 1 μl of each of the flow antibodies anti-mouse CD19 (CD19 BUV395, BD), IgM (lgM APC, ThermoFisher, 17-5790-82), and IgD (lgD FITC, ThermoFisher, 11-5993-85) to the tube; B cells are defined as the CD19+ population.

[0113] When analyzing IgM and IgD in B cells, four different subsets can be distinguished: 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 subsets in the peripheral blood of mice on the 28th, 42nd, and 56th days in groups B, C1, and C3 are shown in Figure 14 , Figure 15 , Figure 16 , reflecting the changes in B lymphocyte subsets in the blood samples of mice in each group at the three measurement time points.

[0115] According to the above results, it can be seen that at the three measurement time points, 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 content of follicular B cells (IgM-, IgD+) and activated B cells (IgM-, IgD-) is significantly higher than that in the control groups (C1, C3).

[0116] Transitional cells represent the final stage before differentiating into a more mature population of pre-immune B cells, indicating that the addition of adjuvant promotes the activation and differentiation of B cells, provides a basis for the immune system to produce more antibodies with high affinity for antigens, and promotes 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 description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A composition, characterized in that The invention comprises a cat interleukin mixture and a metabolic regulating molecule, wherein the cat interleukin mixture comprises cat interleukin 15 and cat interleukin 23, and the metabolic regulating molecule comprises beta-nicotinamide mononucleotide, beta-nicotinamide adenine dinucleotide disodium salt, N-acetylcysteine ​​and L-carnitine.

2. A composition according to claim 1, characterized in that The mass ratio of the feline interleukin 15 to the feline interleukin 23 is 1:

1.

3. A composition according to claim 2, characterized in that 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.

4. A composition according to claim 3, characterized in that The mass ratio of the cat interleukin mixture to beta-nicotinamide mononucleotide, beta-nicotinamide adenine dinucleotide disodium salt, N-acetylcysteine ​​and L-carnitine is 0.1-0.5:0.1-0.5:0.1-0.5:50-1200.

5. Use of the composition according to any one of claims 1 to 4, characterized in that: The composition can be used to prepare a cat triple vaccine or an immune preparation of a cat triple vaccine.

6. The use of a composition according to claim 5, characterized in that: The cat triple vaccine is a compound inactivated vaccine.

7. The use of a composition according to claim 5, characterized in that: The immune preparation includes vaccine adjuvants and vaccine enhancers.

8. A cat triple vaccine immune adjuvant containing two cat interleukins and a metabolic regulatory molecule, characterized in that: The invention comprises the composition according to any one of claims 1 to 4.

9. A composite cat triple inactivated vaccine, characterized in that: It comprises the cat triple vaccine immune adjuvant and the cat triple vaccine antigen as claimed in claim 8, and the volume ratio of the cat triple vaccine immune adjuvant to the cat triple vaccine antigen is 1:0.5-1.

5.

10. A composite cat triple inactivated vaccine according to claim 9, characterized in that: In each milliliter of the composite cat triple inactivated vaccine, the cat triple vaccine antigen includes WH-2017 strain 10 7.0 TCID 50 / Toufen, LZ-2016 strain 10 7.5 TCID 50 / Toufen and CS-2016 strain 10 5.5 TCID 50 / head portion; also includes a solvent, and the solvent includes one or more of sterile saline and sterile buffer.

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