Cat triple vaccine immunologic adjuvant containing three cat interleukinins and manganese ions, composition and application

Abstract

The invention relates to the technical field of biology, in particular to a cat triple vaccine immunologic adjuvant containing three cat interleukinins and manganese ions, a composition and application. The composition comprises feline interleukin 2, feline interleukin 4, feline interleukin 6 and manganese ions, and can be used for preparing a feline triple vaccine or an immune preparation of the feline triple vaccine, and the feline triple vaccine immune adjuvant containing the three feline interleukin comprises the compound. The immunologic adjuvant disclosed by the invention has the characteristics of safety and high efficiency, not only can effectively solve the limitation of the existing vaccine, but also can further enhance the comprehensive protective efficacy of the cat triple vaccine on the three viruses, is long in immunization duration, effectively reduces adverse reactions, remarkably improves the safety and protection rate of inoculation, and has a good application prospect. The method is of great significance to promotion of feline disease prevention and pet health management.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to a feline triple vaccine immune adjuvant, composition and application containing three kinds of feline interleukins and manganese ions. Background Art

[0002] Feline triple vaccine is an important vaccine for preventing high-risk infectious diseases such as feline panleukopenia virus (FPV), feline calicivirus (FCV) and feline herpesvirus (FHV-1). However, existing feline triple vaccines have certain defects in terms of immune efficacy, protection duration and low immunoreactivity to some individuals. Especially when facing individuals with low immune response, the protection effect of existing vaccines may be insufficient. In addition, although traditional immune adjuvants (such as aluminum salts and emulsifiers) can enhance the immunogenicity of antigens, they have problems such as inducing a single immune response and some adjuvants being prone to cause local inflammation or systemic side effects, which limits their application potential.

[0003] Immune adjuvants play an important role in vaccines in enhancing antigen immunogenicity, prolonging the protection period and optimizing the balance of humoral and cellular immunity. With the development of immunology and biotechnology, novel adjuvants are gradually becoming the key means to improve vaccine efficacy. By researching and applying novel immune adjuvants, more efficient antigen delivery can be achieved, a wide range of immune pathways can be activated, and the protection ability of vaccines against complex pathogen mixed infections can be improved. Therefore, researching and developing novel safe and efficient immune adjuvants and applying them to feline triple vaccines can not only effectively solve the limitations of existing vaccines, but also further enhance the comprehensive protection efficacy of vaccines against three viruses, prolong the immune duration, reduce adverse reactions, and improve the safety and protection rate of vaccine inoculation. This is of great significance for promoting the prevention of feline diseases and pet health management. 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 three kinds of feline interleukins and manganese ions.

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

[0006] The present invention provides a composition, including feline interleukin-2, feline interleukin-4, feline interleukin-6 and manganese ions.

[0007] On the basis of the above technical solution, the present invention can also be improved as follows.

[0008] Further, the mass ratio of the feline interleukin-2, the feline interleukin-4, the feline interleukin-6 and the manganese ions is 0.05-1:0.05-1:0.05-1:50-200.

[0009] Furthermore, in the composition, the mass ratio of feline interleukin-2, feline interleukin-4, feline interleukin-6, and manganese ions is 0.1-0.5:0.1-0.5:0.1-0.5:100-200.

[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 immunopotentiator for a feline triple vaccine.

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

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

[0013] The present invention also provides a feline triple vaccine immunoadjuvant containing three feline interleukins, including the composition as described above.

[0014] Furthermore, it also includes pharmaceutically and immunologically acceptable excipients.

[0015] The present invention also provides a combined feline triple inactivated vaccine, including the feline triple vaccine immunoadjuvant containing three feline interleukins as described above and an antigen, and the volume ratio of the feline triple vaccine immunoadjuvant to the antigen is 1:0.5-1.5.

[0016] Furthermore, in each milliliter of the combined feline triple inactivated vaccine, the feline triple vaccine antigen includes 10 7.0 TCID 50 / head portion of WH-2017 strain, 10 7.5 TCID 50 / head portion of LZ-2016 strain, and 10 5.5 TCID 50 / head portion of CS-2016 strain; it also includes a solvent, and the solvent includes one or more of sterilized water, sterilized physiological saline, and sterilized buffer solution.

[0017] The beneficial effects of the present invention are as follows:

[0018] (1) The composition of the present invention can be used to prepare a feline triple vaccine or an auxiliary immunopotentiator for a feline triple vaccine, which can achieve more efficient antigen delivery, activate a wide range of immune pathways, and improve the protection ability of the feline triple vaccine against complex pathogen mixed infections;

[0019] (2) The composition of the present invention provides an application of interleukin in the adjuvant of the feline triple vaccine, provides a new idea for the development and research of the adjuvant of the feline triple vaccine, and also expands a new field for the application of feline interleukin;

[0020] (3) The feline triple vaccine immunoadjuvant containing three feline interleukins of the present invention has the characteristics of safety and high efficiency. It can not only effectively solve the limitations of existing vaccines, but also further enhance the comprehensive protective efficacy of the feline triple vaccine against the above three viruses. It has a long immune duration, effectively reduces adverse reactions, significantly improves the safety of vaccination and the immune protection rate, and is of great significance for promoting the prevention of infectious viral diseases in feline animals and pet health management;

[0021] (4) The combined feline triple inactivated vaccine of the present invention can stimulate a stronger immune response after vaccination, significantly stimulate and improve the animal to produce a high level of specific antibody titer earlier, and protect the animal from infection by feline panleukopenia virus, calicivirus and herpesvirus; and the high-level antibody titer is maintained for a longer period than the control group.

[0022] (5) The combined feline triple inactivated vaccine of the present invention can significantly promote the animal to produce more blood-specific CD4 effector memory T cells (Th effector memory, ThEM), while having less or no significant difference in the impact on other T cell subsets, improving the animal's specific cellular immunity and its memory ability, and resisting the infection of feline panleukopenia virus, calicivirus and herpesvirus persistently and at a high level;

[0023] (6) The combined feline triple inactivated vaccine of the present invention can significantly promote the animal to produce higher levels of specific FPV, FCV and FHV-1 antibodies, follicular B cells (Follicular B cell, B FO ) and the number of activated B cells (Class-switched or Activated B cells, B AM ) show that the immunoadjuvant significantly enhances the activation of B cells and the humoral immune response, significantly improves the animal's specific humoral immunity level and its memory response ability, and better defends against the infection and invasion of feline panleukopenia virus, calicivirus and herpesvirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a comparison chart of the changes in the body weights of mice in each experimental group in Example 1 of the feline triple vaccine immunoadjuvant of the present invention;

[0025] Figure 2 It is a comparison chart of the changes in the mean corpuscular volume in the peripheral blood of mice in each experimental group in Example 2 of the feline triple vaccine immunoadjuvant of the present invention;

[0026] Figure 3 It is a comparison chart of the changes in the lymphocyte percentage of mice in each experimental group in Example 2 of the feline triple vaccine immunoadjuvant of the present invention;

[0027] Figure 4For the feline triple vaccine immune adjuvant of the present invention, in Example 2, comparison chart of changes in mean corpuscular hemoglobin of mice in each experimental group;

[0028] Figure 5 For the feline triple vaccine immune adjuvant of the present invention, in Example 2, comparison chart of changes in mean corpuscular hemoglobin concentration of mice in each experimental group;

[0029] Figure 6 For the feline triple vaccine immune adjuvant of the present invention, in Example 2, comparison chart of changes in mean platelet volume of mice in each experimental group;

[0030] Figure 7 For the feline triple vaccine immune adjuvant of the present invention, in Example 3, comparison chart of percentages of various B lymphocyte subsets in peripheral blood of mice in each experimental group on the 28th day;

[0031] Figure 8 For the feline triple vaccine immune adjuvant of the present invention, in Example 3, comparison chart of percentages of various B lymphocyte subsets in peripheral blood of mice in each experimental group on the 42nd day;

[0032] Figure 9 For the feline triple vaccine immune adjuvant of the present invention, in Example 3, comparison chart of percentages of various B lymphocyte subsets in peripheral blood of mice in each experimental group on the 56th day;

[0033] Figure 10 For the feline triple vaccine immune adjuvant of the present invention, in Example 3, comparison chart of percentages of different T lymphocyte subsets in peripheral blood of mice in each experimental group on the 56th day;

[0034] Figure 11 For the feline triple vaccine immune adjuvant of the present invention, in Example 4, comparison chart of changes in feline calicivirus neutralizing antibody titers in groups A, C1, and C3 on days 0, 7, 14, 28, 42, and 56;

[0035] Figure 12 For the feline triple vaccine immune adjuvant of the present invention, in Example 4, comparison chart of changes in detection results of feline calicivirus antibody (FCV-Ab) levels in each experimental group;

[0036] Figure 13 For the feline triple vaccine immune adjuvant of the present invention, in Example 4, comparison chart of changes in detection results of feline herpesvirus antibody (FHV-Ab) levels in each experimental group;

[0037] Figure 14 For the feline triple vaccine immune adjuvant of the present invention, in Example 4, comparison chart of changes in detection results of feline panleukopenia virus antibody (FPV-Ab) levels in each experimental group. Detailed implementation manners

[0038] The principles and features of the present invention will be described below in conjunction with the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0039] The composition of the present invention includes feline interleukin-2 (IL-2), feline interleukin-4 (IL-4), feline interleukin-6 (IL-6), and manganese ions. Interleukin is abbreviated as interleukin (IL).

[0040] The composition of the present invention can be used to prepare a feline triple vaccine or an adjuvant for 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).

[0041] The above composition of the present invention can achieve more efficient antigen delivery, activate a wide range of immune pathways, and improve the protective ability of the feline triple vaccine in a complex pathogen mixture; at the same time, the adjuvant also has the characteristics of safety and high efficiency. It can not only effectively solve the limitations of existing vaccines, but also further enhance the comprehensive protective efficacy of the feline triple vaccine against the above three viruses. The immune duration is long, the adverse reactions are effectively reduced, and the safety and immune protection rate of vaccination are significantly improved, which is of great significance for promoting the prevention of feline diseases and pet health management.

[0042] In addition, the above composition of the present invention provides the application of feline 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 a new field for the application of feline interleukin.

[0043] Preferably, in the composition of the present invention, the mass ratio of feline interleukin-2, feline interleukin-4, feline interleukin-6, and manganese ions is 0.05-1:0.05-1:0.05-1:50-200.

[0044] More preferably, in the composition, the mass ratio of feline interleukin-2, feline interleukin-4, feline interleukin-6, and manganese ions is 0.1-0.5:0.1-0.5:0.1-0.5:100-200.

[0045] Even more preferably, in the composition, the mass ratio of feline interleukin-2, feline interleukin-4, feline interleukin-6, and manganese ions is 1:1:1:400.

[0046] Preferably, the feline triple vaccine is a combined inactivated vaccine.

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

[0048] The feline triple vaccine immunoadjuvant containing three feline interleukins of the present invention comprises the above-mentioned composition and also comprises pharmaceutically acceptable excipients.

[0049] More preferably, the dosages of each component are calculated based on the average initial immunization body weight of mice being 18 g per mouse: feline IL-2: 0.028 μg / g (w / w), feline IL-4: 0.028 μg / g (w / w), feline IL-6: 0.028 μg / g (w / w); Mn 2+ : 11.11 μg / g (w / w).

[0050] Specifically, pharmaceutically acceptable excipients include solvents, and the solvents can be one or more of sterilized water, sterilized physiological saline, and sterilized buffer solution.

[0051] The composite feline triple inactivated vaccine of the present invention comprises the feline triple vaccine immunoadjuvant containing three feline interleukins as described above.

[0052] Preferably, it further comprises feline triple vaccine antigen and a solvent. The feline triple vaccine antigen comprises 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 physiological saline, and sterilized buffer solution.

[0053] Preferably, the total amount of the antigen is 0.1 - 1 μg.

[0054] Preferably, in the vaccine of the present invention, the concentration contents of each component in the adjuvant are as follows: feline IL-2: 2.5 μg / ml; feline IL-4: 2.5 μg / ml; feline IL-6: 2.5 μg / ml; Mn 2+ : 1 mg / ml.

[0055] Preferably, the inoculation method of the feline triple inactivated vaccine containing the immunoadjuvant includes intramuscular or subcutaneous injection.

[0056] Experimental verification shows that, compared with the separate inoculation of the triple inactivated vaccine, the compound feline triple inactivated vaccine of the present invention significantly increases the numbers of white blood cells, neutrophils and lymphocytes in the peripheral blood of animals (p < 0.05). This indicates that the compound feline triple inactivated vaccine of the present invention stimulates a stronger immune response after inoculation, manifested as a significant increase in the number of immune cells in the peripheral blood. Inoculation with the compound adjuvant vaccine can significantly stimulate and increase the production of high-level specific antibody titers in animals earlier, protecting the animals from feline panleukopenia virus, calicivirus and herpesvirus infections; and the high-level antibody titers are maintained for a longer period than in the control group. Compared with the separate inoculation of the triple inactivated vaccine, inoculation with the compound vaccine can significantly promote the production of more blood-specific CD4 effector memory T cells (Th effector memory, ThEM) in animals, while having less or no significant difference in the effects on other T cell subsets, improving the specific cellular immunity and its memory ability of animals, and resisting the reproductive infections of feline panleukopenia virus, calicivirus and herpesvirus persistently and at a high level. At the same time, inoculation with the compound vaccine can significantly promote the production of higher levels of specific FPV, FCV and FHV-1 antibodies, follicular B cells (Follicular B cell, B FO ) and the number of activated B cells (Class-switched or Activated B cells, B AM ) show that the immune adjuvant significantly enhances the activation of B cells and the humoral immune response, significantly improving the specific humoral immunity level and its memory response ability of animals, and better defending against the infections and invasions of feline panleukopenia virus, calicivirus and herpesvirus.

[0057] The present invention verifies the immune enhancement effects of feline IL-2, IL-4, IL-6 and manganese ions as adjuvants for the feline triple inactivated vaccine, and obtains an effective cytokine combination and its dosage, achieving a synergistic enhancement of the immune response level of the feline triple inactivated vaccine, improving and maintaining a high level of immune protection and protection period of the feline triple inactivated vaccine. No inflammatory damage and lesions appear locally in the body after inoculation with these new compound adjuvants for the feline triple inactivated vaccine, proving that they are safe and non-toxic, and can be used as safe and reliable new and highly effective immune adjuvants for the feline triple inactivated vaccine, which will be able to 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.

[0058] The following specifically describes the present invention through specific examples.

[0059] In the following embodiments, unless otherwise specified, all experimental operations are carried out by 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 can be obtained through commercial channels unless otherwise specified. Unless otherwise specified, the quantitative experiments in the embodiments are carried out with three replicates, and the final results are 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, then 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 immunization procedures and grouping of the mice were as follows:

[0062] Thirty 6-week-old female Kunming mice were selected and randomly divided into three groups, with 10 mice in each group. According to the protocol in Table 1, the mice were immunized subcutaneously on the back, and a booster immunization was carried out on each group in the third week. During the immunization period, the body weight of the mice was measured weekly, and at the same time, 200 μl of venous anticoagulant blood samples were collected from each mouse by the tail cutting method. On the 56th day after the first immunization, the mice were euthanized, and the number of lymphocyte subsets in the peripheral blood and spleen tissues of the mice was detected.

[0063] Table 1 Immunization grouping of mouse experiments

[0064]

[0065] The feline triple vaccine antigens (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) used in each group of experiments in Table 1 were provided by Sichuan Huapai Biotechnology (Group) Co., Ltd. Feline interleukin 2 (IL-2, catalog number: RP0099F-005), IL-4 (catalog number: RP0296F-005) and IL-6 (catalog number: RP2116F-005) were produced and provided by Kingfisher Biotech, USA.

[0066] Mn 2+From manganese chloride MnCl2, produced by Tianjin Fuchen Chemical Reagent Factory, analytical pure reagent.

[0067] Example 1 Detection and Statistics of Mouse Body Weight Index

[0068] During the immunization period of this example, the body weights of mice in each group were measured weekly, and the results are as Figure 1 shown. According to Figure 1 it can be seen that at each time point, there was no significant difference in the body weights of the three groups of mice (P>0.05).

[0069] Example 2 Statistical Analysis of Blood Routine Indexes

[0070] In this example, 50 μL of EDTA-anticoagulated whole blood samples were collected from mice in each group. Subsequently, in accordance with the operating procedures specified by the Tek Veterinary Automatic Five-Category Blood Analyzer (TEK-VET5), a comprehensive blood routine analysis of the whole blood of mice was carried out, and the analysis results are as Figures 2-6 shown.

[0071] Figures 2 to 6 Successively presented the changes in mean corpuscular volume, lymphocyte percentage, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and mean platelet volume in the peripheral blood of mice during the experimental process.

[0072] According to the above experimental results, it can be seen that at 6 time points in the peripheral blood of groups A, C1, and C3, there were no significant differences in mean corpuscular volume, lymphocyte percentage, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and mean platelet volume (P>0.05). This indicates that the immunoadjuvant has good biosafety.

[0073] Example 3 Verification of Immunization Effect

[0074] In this example, the changes in immunocompetent cells in the peripheral blood of mice in each experimental group at different times were analyzed by flow cytometry to verify the effects of the adjuvant of the present invention on T cells and B cells.

[0075] In this example, the specific process of flow cytometry detection is as follows:

[0076] 1) In a 1.5 ml EP tube 1 containing 100 μl of mouse peripheral anticoagulated blood (added with EDTA·2K anticoagulant), 1 μl of each of the following flow antibodies was added in sequence: anti-mouse CD45 (CD45 Monoclonal Antibody(30-F11),SuperBright TM 600,eBioscience TM )、CD3(BD Pharmingen TMFITC Hamster Anti-Mouse CD3e), CD4 (CD4 Monoclonal Antibody(GK1.5),eFluor TM 450,eBioscience TM ), CD8(CD8a MonoclonalAntibody(53-6.7),PerCP-Cyanine5.5,eBioscience TM ), CD44(CD44Monoclonal Antibody(IM7),APC,eBioscience TM ) and CD62L(CD62L(L-Selectin)Monoclonal Antibody(MEL-14),PE,eBioscience TM ). After mixing, incubate at 4°C in the dark for 30 minutes.

[0077] 2) Add 1 ml of 1× RBC lysis buffer (prepared with deionized water) to the above reaction system, lyse at room temperature for 5 minutes, centrifuge at 1500 rpm and 4°C for 5 minutes, and discard the supernatant.

[0078] 3) Washing: Add 200 μl of PBS (containing 0.5% BSA) to the cell pellet obtained in step 2, mix by pipetting, centrifuge at 1500 rpm and 4° C. for 5 minutes, discard the supernatant, and repeat washing twice.

[0079] 4) After adding 500 μl PBS (containing 0.5% BSA) to resuspend the cells, add an appropriate amount of paraformaldehyde to make the final volume reach 2%, place at 4° C. in the dark, and then perform flow cytometry detection.

[0080] The same treatment as above was applied to the following staining schemes:

[0081] 1) Tube 2: Add 1 μl of each of the following flow cytometry antibodies: anti-mouse CD45 (CD45 Monoclonal Antibody (30-F11), Super Bright 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(Integrinalpha E)Monoclonal Antibody(2E7),APC,eBioscience TM );

[0082] 2) Tube 3: Add 1 μl of each of the following flow antibodies: anti-mouse CD19 (CD19 BUV395), IgM (IgM APC), IgD (IgD FITC), and CD38 (PE / Dazzle TM 594 anti-mouse CD38).

[0083] In this example, the specific steps for preparing the single-cell suspension of the spleen are as follows:

[0084] 1) Splenic grinding: After sacrificing the mouse, place the spleen on a 70-μm cell strainer and then put it into a 6-cm culture dish. Add 3 ml of PBS (containing 0.5% BSA). Grind the spleen tissue directly on the strainer with the plunger of a 2-ml syringe, and then rinse the cell strainer with 2 ml of PBS (containing 0.5% BSA) to collect the cell suspension.

[0085] 2) Centrifugal precipitation: Transfer the collected spleen cell suspension into a 15-ml centrifuge tube, centrifuge at 1500 rpm and 4 °C for 5 minutes, discard the supernatant, and retain the cell pellet.

[0086] 3) Lysis of red blood cells: Add 3 ml of 1× RBC lysis buffer to the cell pellet, vortex and mix well, and then lyse at room temperature for 5 minutes. Subsequently, centrifuge at 1500 rpm and 4 °C for 5 minutes, and discard the supernatant.

[0087] 4) Secondary filtration: Add 3 ml of PBS (containing 0.5% BSA), vortex and pipette to mix well, and then filter the suspension through a 70-μm cell strainer into a 50-ml centrifuge tube. After filtration, centrifuge at 1500 rpm and 4 °C for 5 minutes, discard the supernatant. Wash once with PBS (containing 0.5% BSA), and then wash once with PBS without BSA.

[0088] 5) Resuspension of cells: Add 1 ml of PBS (containing 0.5% BSA) to the pellet, vortex and mix well. If connective tissue is found, use a pipette tip to pick it out. Subsequently, add 100 μl of the spleen cell suspension to each experimental system to complete the preparation of the single-cell suspension of the spleen and prepare for subsequent staining.

[0089] 6) Blocking: Resuspend the splenocytes 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 minutes, and directly incubate with surface-labeling antibodies without washing.

[0090] 7) Staining and fixation: Stain according to the aforementioned color-matching scheme, incubate in the dark on ice for 30 minutes. Subsequently, wash twice, add 200 μl of 4% paraformaldehyde, fix in the dark at room temperature for 30 minutes, wash again, and resuspend in 200 μl of PBS for ready-to-use detection on the machine.

[0091] Gating logic of this example:

[0092] (1) Gating logic of immunomemory B lymphocytes

[0093] According to the blood flow cytometry sample preparation and machine loading method of this example, add 1 μl of anti-mouse CD19 (CD19 BUV395, BD), IgM (lgM APC, ThermoFisher, 17-5790-82), and IgD (lgD FITC, ThermoFisher, 11-5993-85) flow antibodies into the tube respectively; among them, B cells are defined as the CD19+ population.

[0094] When analyzing IgM and IgD in B cells, four different subsets can be distinguished: follicular (FO) B cells (naive) (IgM−, IgD+), marginal zone (MZ) B cells (IgM+, IgD+), transitional B cells (IgM+, IgD−), and class-switched or activated B cells (IgM−, IgD−), as shown in Table 2 specifically.

[0095] Table 2 Flow cytometry gating logic

[0096]

[0097] Figure 7 、 Figure 8 、 Figure 9 are the percentages of various B lymphocyte subsets in the peripheral blood of mice in groups A, C1, and C3 on days 28, 42, and 56 respectively, and significant differences are shown at multiple time points (P < 0.05).

[0098] According to Figures 7-9 it can be seen that the B FO cells ( The proportion of IgM+ and IgD+ remained high throughout the experiment, significantly higher than that in Group C1 and Group C3 (P < 0.05). Meanwhile, the proportion of Class-switched or Activated B cells (IgM-IgD-) in Group A gradually increased, especially on Day 42 and Day 56, indicating that the immune adjuvant might enhance B cell activation and humoral immune responses. AM (2) Gating logic for immune memory T lymphocytes

[0099] (2) Gating logic for immune memory T lymphocytes

[0100] T cells were defined as the CD3+ / CD45+ population. According to the different expressions of CD4 and CD8, T cells could be further divided into four major subsets. These subsets were CD4+ T H cells, CD8+ T C cells, CD4+ / CD8+ double-positive (DP) cells, and CD4- / CD8- double-negative (DN) cells. The latter were mainly composed of γδ+ cells under normal circumstances. When analyzing the expressions of CD44 and CD62L in T H and T C (helper T cells and cytotoxic T cells), they could be divided into three different subsets: tissue-resident memory cells, central memory cells, and effector memory cells.

[0101] Figure 10 It showed the percentage changes of different T lymphocyte subsets (central memory T cells and effector memory T cells of CD4+ and CD8+) in the peripheral blood of mice on Day 56 in Group A, Group C1, and Group C3. The results showed that there was no significant difference in CD4+ central memory T cells among the three groups (P > 0.05). Among CD8+ central memory T cells, the proportion in Group C1 was significantly higher than that in Group A (P < 0.05), while there was no statistical difference between Group C3 and Group A and Group C1. For CD8+ effector memory T cells, no significant difference was observed among the groups (P > 0.05). These results indicated that the adjuvant treatment group had a more significant effect on CD4+ effector memory T cells, while had a smaller or no significant effect on other T cell subsets.

[0102] Example 4 Neutralizing / antigen-specific antibody detection

[0103] (1) Feline triple vaccine neutralizing antibody assay - fixed virus diluted serum method: Dilute the virus to contain 100 TCID per unit dose 50The concentration was mixed well with an equal amount of the tested serum serially diluted 2-fold and placed in an environment at 37 °C for reaction for 60 minutes. For each dilution, 3 to 6 wells of cells were inoculated. After the inoculation operation was completed, the number of cell wells showing cytopathic effect (CPE) in each group was carefully recorded, and the 50% protective dose (PD 50 ) was calculated according to the Reed-Muench method, and then the neutralization titer of the serum was calculated.

[0104] Figure 11 Were the feline panleukopenia virus neutralizing antibody titers in groups A, C1, and C3 at days 0, 7, 14, 28, 42, and 56.

[0105] According to Figure 11 It can be seen that at different time points after inoculation, there were significant differences in the feline calicivirus (FCV) neutralizing antibody titers among the three groups. The antibody titer in group A increased rapidly on Day 7, slightly lower than that in group C1, but on Day 28, Day 42, and Day 56, the antibody titer was significantly higher than the other two groups (P < 0.05), showing a strong and persistent immune response, indicating that the synergistic effect of immune adjuvant A and Mn effectively enhanced the intensity and persistence of the immune response. The antibody titer in group C1 reached the peak on Day 7, but the antibody level decreased rapidly thereafter, indicating that Mn had a certain promoting effect on the early immune response, but without the support of an immune adjuvant, it was difficult to maintain a long-term immune effect. In contrast, the antibody titer in group C3 remained at a very low level throughout the observation period with almost no significant change. This indicates that the use of immune adjuvants is a key strategy to enhance the immune response intensity of feline panleukopenia virus vaccine and maintain the antibody effect.

[0106] (2) Detection of feline panleukopenia virus antigen-specific antibodies

[0107] Referring to the operation instructions of the feline panleukopenia virus antibody (FCV-Ab / FHV-Ab / FPV-Ab) detection kit (fluorescence immunochromatography method) (product number: PRG108, Shanghai Jiling Biotechnology Co., Ltd., China), the levels of feline calicivirus antibody (FCV-Ab), feline herpesvirus antibody (FHV-Ab), and feline parvovirus antibody (FPV-Ab) in mouse plasma were detected in vitro. Figure 12 、 Figure 13 、 Figure 14 Were the detection results of the levels of feline calicivirus antibody (FCV-Ab), feline herpesvirus antibody (FHV-Ab), and feline parvovirus antibody (FPV-Ab), respectively.

[0108] According to Figure 12 、 Figure 13 and Figure 14It can be seen that in the antibody detection corresponding to all three antigens (FCV, FHV, FPV), the antibody levels in Group A are higher than those in Groups C1 and C3 (P < 0.05), showing the highest antibody levels, especially in the later stage (Day 56). Group C1 showed a certain immune response in the early stage (Day 7 and Day 28), with antibody levels higher than those in Group C3, but significantly lower than those in Group A in the later stage (Day 56), indicating that the effect of Mn on enhancing the immune response is relatively limited, which shows that the immune adjuvant has a significant effect on enhancing the intensity and persistence of the immune response.

[0109] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A composition, characterized in that Including feline interleukin 2, feline interleukin 4, feline interleukin 6 and manganese ions.

2. A composition according to claim 1, characterized in that In the composition, the mass ratio of the feline interleukin 2, the feline interleukin 4, the feline interleukin 6 and the manganese ion is 0.05-1:0.05-1:0.05-1:50-200.

3. A composition according to claim 2, characterized in that In the composition, the mass ratio of the feline interleukin 2, the feline interleukin 4, the feline interleukin 6 and the manganese ion is 0.1-0.5:0.1-0.5:0.1-0.5:100-200.

4. Use of the composition according to any one of claims 1 to 3, characterized in that: The composition can be used to prepare a cat triple vaccine or an immune preparation of a cat triple vaccine.

5. The use of a composition according to claim 4, characterized in that: The cat triple vaccine is a compound inactivated vaccine.

6. The use of a composition according to claim 4, characterized in that: The immune preparation includes vaccine adjuvants and vaccine enhancers.

7. A cat triple vaccine immune adjuvant containing three cat interleukins and manganese ions, characterized in that: The invention comprises the composition according to any one of claims 1 to 3.

8. A cat triple vaccine immunoadjuvant containing three cat interleukins and manganese ions according to claim 7, characterized in that, Pharmaceutically or immunologically acceptable excipients are also included.

9. A composite cat triple inactivated vaccine, characterized in that: It comprises the cat triple vaccine immune adjuvant and antigen as described in claim 7 or 8, and the volume ratio of the cat triple vaccine immune adjuvant to the 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 compound cat triple inactivated vaccine, the antigen includes 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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