Use of octyl gallate in the preparation of vaccine adjuvants

Abstract

The application discloses application of octyl gallate in preparation of a vaccine adjuvant and belongs to the technical field of vaccine adjuvants.The experiment proves that after immunization by mixing the vaccine adjuvant prepared by taking octyl gallate as an active ingredient with antigens, the body fluid immunity, cellular immunity and mucosal immunity levels of the organism can be improved, the production of specific IgM, IgG, IgY and secretory IgA in serum can be promoted, and the T cell proliferation index and CD4 + , CD8 + T cell related immune indexes can be improved.The vaccine adjuvant prepared by taking octyl gallate has the potential of promoting immune regulation, activating natural immunity and enhancing vaccine immune response, and the vaccine adjuvant prepared by taking octyl gallate is used for preparation of vaccines taking virus antigens, recombinant protein antigens, subunit antigens or inactivated antigens as antigens.

Description

Technical Field

[0001] This invention belongs to the field of vaccine adjuvant technology, specifically relating to the application of octyl gallate in the preparation of vaccine adjuvants. Background Technology

[0002] Vaccination is a crucial means of preventing and controlling infectious diseases in humans and animals. For inactivated vaccines, attenuated vaccines, subunit vaccines, and recombinant protein vaccines against viruses and bacteria, the immunogenicity of a single antigen is often insufficient, usually requiring the addition of adjuvants to enhance immunization efficacy. Existing vaccine adjuvants mainly include oil adjuvants, mineral salt adjuvants, polysaccharide adjuvants, saponin adjuvants, and pattern recognition receptor agonists. However, these adjuvants still have certain limitations. For example, oil adjuvants can produce high antibody levels, but their high viscosity and slow absorption can easily cause irritation at the injection site, leading to redness, swelling, induration, and pain at the injection site, as well as systemic reactions such as fever, fatigue, and muscle aches. Aluminum salt and emulsion adjuvants primarily activate humoral immunity (antibody production), but their ability to induce cellular immunity is poor, and their activation of innate immune signals and induction of early immune regulation are insufficient, especially in activating CD8. + The ability to kill T cells is extremely weak; potent saponin adjuvants (such as QS-21) are highly toxic when used alone and must be combined with systems such as liposomes (such as AS01) to reduce toxicity; some new compound adjuvants (such as AS01, which contains liposomes + MPL + QS-21) have complex production processes and high costs, which limits the popularization of adjuvants in resource-scarce areas.

[0003] In summary, the core challenges currently facing vaccine adjuvants are as follows: traditional adjuvants (such as aluminum salts), while exhibiting good safety profiles, struggle to induce potent cellular and mucosal immunity, failing to meet the demands of intracellular pathogen vaccines; while novel, potent adjuvants are limited by unclear mechanisms, complex manufacturing processes, high costs, and significant local reactions. Therefore, adjuvant development urgently needs breakthroughs in precisely regulating the direction of the immune response, reducing toxicity, simplifying manufacturing processes, and expanding mucosal delivery pathways. The goal is to find novel adjuvants that are precise, efficient, low-toxic, simple to manufacture, and widely applicable, especially those capable of activating cellular and mucosal immune systems, in order to develop a next-generation novel adjuvant system that is highly efficient, low-toxic, widely applicable, and controllable.

[0004] Octyl gallate (OG) is a small molecule compound of the gallic acid ester class, possessing a well-defined chemical structure and good formulation operability. Previous studies have demonstrated that gallic acid esters exhibit antioxidant, antibacterial, antiviral, and immune-related pharmacological activities. However, there are currently no publicly reported applications of octyl gallate in the field of vaccine adjuvants, particularly in promoting immunomodulation, activating innate immunity, and enhancing antigen-specific humoral and cellular immune responses. Summary of the Invention

[0005] The purpose of this invention is to provide an application of octyl gallate in the preparation of vaccine adjuvants. Vaccine adjuvants prepared using octyl gallate have the potential to promote immune regulation, activate innate immunity, and enhance vaccine immune responses. These adjuvants can be used in the preparation of vaccines using viral antigens, recombinant protein antigens, subunit antigens, or inactivated antigens as antigens.

[0006] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is to provide the application of octyl gallate in the preparation of vaccine adjuvants.

[0007] The second technical solution of the present invention provides a vaccine adjuvant, the components of which include octyl gallate, solvent and buffer solution.

[0008] Preferably, the concentration of octyl gallate in the vaccine adjuvant is 5-20 mg / mL.

[0009] Preferably, the solvent is dimethyl sulfoxide (DMSO), and the solvent accounts for less than 1% of the volume of the vaccine adjuvant.

[0010] Preferably, the buffer solution is a phosphate buffer solution.

[0011] More preferably, the phosphate concentration in the phosphate buffer solution is 0.005~0.05M, and the pH value is 7.0~8.0.

[0012] The third technical solution of the present invention provides a method for preparing the above-mentioned vaccine adjuvant, comprising the following steps: Octyl gallate is dissolved in a solvent to obtain an octyl gallate solution. The octyl gallate solution is then mixed evenly with a buffer solution to obtain the vaccine adjuvant.

[0013] The fourth technical solution of the present invention provides an application of the above-mentioned vaccine adjuvant in the preparation of a vaccine, wherein the antigens of the vaccine include: viral antigens, recombinant protein antigens, subunit antigens, inactivated antigens, or model protein antigens.

[0014] The beneficial technical effects of the present invention are as follows: This invention marks the first discovery of the application of octyl gallate in the preparation of vaccine adjuvants. Experimental verification revealed that vaccine adjuvants prepared with octyl gallate, when mixed with antigens for immunization, can increase the production of specific IgG, IgM, IgY, and secretory IgA, and also enhance T cell proliferation index and CD4 count. + CD8 + T-cell-related immune markers.

[0015] The vaccine adjuvant provided by this invention can promote immune regulation and activation of innate immunity, and help improve the synergistic response of vaccine-induced humoral and cellular immunity, especially suitable for viral antigens, recombinant protein antigens, subunit antigens or inactivated antigens with insufficient immunogenicity.

[0016] The vaccine adjuvant preparation process provided by this invention is simple, uses low amounts of solvent, has a clear working solution concentration range, is easy to mix with different antigen systems, and has good prospects for vaccine development and industrial application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This shows the changes in body weight of mice in each group after immunization in Example 1.

[0019] Figure 2 The results of T cell proliferation index detection in mice 32 days after immunization in Example 2 are shown.

[0020] Figure 3 The results show the detection results of specific IgG levels at each immunization time point in each group of mice in Example 3; where A is day 0, B is day 7, C is day 14, D is day 21, and E is day 32.

[0021] Figure 4 The results show the detection of specific IgM levels at each immunization time point in each group of mice in Example 3; where A is day 0, B is day 7, C is day 14, D is day 21, and E is day 32.

[0022] Figure 5 The results show the detection results of IgG (A) and IgM (B) titers in the serum of mice in each group at each immunization time point in Example 3.

[0023] Figure 6 The mice in each group of Example 4 were immunized with CD4 for 32 days. + T cell flow cytometry detection and statistical results; where A~F represent CD4+ in each group. + T cell flow cytometry results: A is the PBS group, B is the OVA group, C is the OVA+Alum group, D is the OVA+OG-20mg / mL group, E is the OVA+OG-10mg / mL group, F is the OVA+OG-5mg / mL group, and G is the CD4 count detected by flow cytometry in each group. + percentage of T cells.

[0024] Figure 7 The mice in each group of Example 4 were immunized with CD8 for 32 days. + T cell flow cytometry detection and statistical results; where A~F represent CD8+ in each group. + T cell flow cytometry results: A is the PBS group, B is the OVA group, C is the OVA+Alum group, D is the OVA+OG-20mg / mL group, E is the OVA+OG-10mg / mL group, F is the OVA+OG-5mg / mL group, and G is the CD8+ result obtained by flow cytometry for each group. + percentage of T cells.

[0025] Figure 8 The results show the HI antibody titer detection results for each group of SPF chicks at each immunization time in Example 5.

[0026] Figure 9 The results show the antibody levels of IgY (A) and IgA (B) in each group of SPF chicks at each immunization time in Example 5.

[0027] Figure 10 The proliferation activity of T cells from SPF chicks 14 days after secondary immunization in each group in Example 5.

[0028] Figure 11 The CD4 count of SPF chicks in each group in Example 5 was 14 days after their second immunization. + / CD3 ratio. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0030] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0031] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] In this invention, room temperature refers to a temperature of 20±10℃.

[0035] Example 1 Preparation and safety evaluation of octyl gallate adjuvant vaccine (1) Preparation of experimental materials Antigen: The model protein antigen ovalbumin (OVA) was used as the antigen to evaluate the enhancement effect of adjuvant on antigen-specific immune response.

[0036] Immunoadjuvant: Octyl gallate, purity ≥99% (HPLC detection).

[0037] Control immune adjuvant: Alhydrogel 2% aluminum adjuvant.

[0038] Excipients: pharmaceutical grade anhydrous dimethyl sulfoxide, 0.01 M (pH 7.2) sterile phosphate buffer.

[0039] Preparation process of octyl gallate adjuvant vaccine: 1.1 Preparation of mother liquor: Weigh 100mg of octyl gallate powder and add it to 100μL of anhydrous DMSO. Use an ultrasonic disruptor (200W, 40kHz) to intermittently sonicate for 5min (3s sonication, 2s interval) until completely dissolved to form a homogeneous and transparent mother liquor.

[0040] 1.2 Dilution of adjuvant working solution: Under constant temperature and magnetic stirring, the mother solution was added dropwise to 9.9 mL of sterile PBS at a rate of 10 μL / min and stirred continuously for 20 min to prepare a 10 mg / mL octyl gallate adjuvant working solution with DMSO volume percentage <1%.

[0041] 1.3 Antigen and Adjuvant Mixing: Based on an average mouse weight of 30g and an immunization volume of 200μL per mouse, the dosage of octyl gallate was set at 5, 10, and 20 mg / kg, corresponding to 0.15, 0.30, and 0.60 mg of octyl gallate per mouse, respectively. OVA was diluted to 1 mg / mL with sterile PBS. For an immunization dose of 50 μg OVA per mouse, 50 μL of OVA antigen solution was taken, and 15, 30, and 60 μL of 10 mg / mL octyl gallate adjuvant working solution were added, respectively. The volume was then brought to 200 μL with sterile PBS. The mixture was stirred continuously at 500 rpm for 30 min at 4℃ to ensure sufficient contact between the antigen and adjuvant, thus preparing high-dose (20 mg / mL), medium-dose (10 mg / mL), and low-dose (5 mg / mL) octyl gallate adjuvants.

[0042] Preparation of the positive aluminum adjuvant group: Following the standard operating procedure for aluminum adjuvant, OVA was diluted to 1 mg / mL with sterile PBS. At an immunization dose of 50 μg OVA per mouse, 50 μL of OVA antigen solution was mixed with 50 μL of Alhydrogel 2% aluminum adjuvant at a 1:1 (v / v) ratio. The mixture was stirred continuously at 500 rpm for 2 h at 4°C to ensure sufficient adsorption of the antigen onto the aluminum adjuvant surface. Then, 100 μL of sterile PBS was added to bring the total volume to 200 μL to obtain the standard aluminum adjuvant control group. Each mouse received an immunization volume of 200 μL, containing 50 μg of OVA.

[0043] Preparation of adjuvant-free group: Mix 50 μL of OVA antigen solution with 150 μL of sterile PBS to make up to 200 μL to prepare adjuvant-free vaccine (PBS group) to eliminate solvent and operation interference.

[0044] (2) Experimental design and immunization procedure Mice were randomly assigned to four groups: a PBS group, an adjuvant-free group (OVA group), a conventional aluminum adjuvant control group (OVA+Alum group), a high-dose octyl gallate group (OVA+OG-20mg / mL), a medium-dose octyl gallate group (OVA+OG-10mg / mL), and a low-dose octyl gallate group (OVA+OG-5mg / mL). Each mouse received a 200μL immunization volume via subcutaneous injection at two points on either side of the back, 100μL at each point. The first immunization was administered on day 0, and a second booster immunization was given on day 14. During the immunization period, the mice's mental status, food and water intake, and weight changes were continuously monitored. Whole blood samples were collected at 7, 14, 21, and 32 days after the first immunization, and serum was separated for subsequent detection of immune indicators such as antibody titers and cytokine levels. On day 32, fresh spleen tissue was collected under sterile conditions, and lymphocytes were separated within 2 hours for subsequent flow cytometry analysis to assess the proportion and proliferative capacity of T lymphocyte subsets.

[0045] (3) Experimental results and analysis The changes in body weight of mice in each group after immunization in Example 1 are shown in the figure. Figure 1 .

[0046] Figure 1 The results showed that the overall weight of the animals in each group showed a stable growth trend. No weight loss or growth inhibition was observed in any of the octyl gallate dosage groups, and no symptoms such as redness, swelling, or ulceration were observed at the injection site. This indicates that the vaccine adjuvant prepared in this invention has good safety in all three experimental dosage ranges.

[0047] Example 2 The promoting effect of octyl gallate adjuvant on T cell proliferation response (1) Experimental methods Immunization was performed according to the immunization procedure in Example 1. Spleen cells were isolated on day 32 post-immunization. To evaluate the antigen-specific T cell proliferation response, the spleen T cell proliferation index (SI) was measured using the CCK-8 assay after in vitro restimulation with OVA antigen, in order to evaluate the promoting effect of octyl gallate adjuvant on OVA-induced cellular immune response and memory T cell proliferation.

[0048] (2) Experimental results and analysis The results of T cell proliferation index detection in each group of mice 32 days after immunization in Example 2 are shown below. Figure 2 .

[0049] Figure 2The results showed that, compared with the PBS group, the unadjuvanted group, and the aluminum adjuvanted group, the T cell proliferation index of the octyl gallate adjuvant group was significantly increased. Among them, the high-dose octyl gallate group had the most prominent promoting effect and the highest stimulation index, which was significantly higher than that of the unadjuvanted group and the aluminum adjuvanted group, and the difference was statistically significant. P <0.0001). The medium- and low-dose groups of octyl gallate also showed strong T-cell proliferation-promoting effects, which were generally higher than those of the aluminum adjuvant group. These results indicate that octyl gallate can effectively enhance the T-cell response after antigen stimulation, with significantly better effects than the currently widely used aluminum adjuvant, and has a role in promoting cellular immunity.

[0050] Example 3 The enhancing effect of octyl gallate adjuvant on specific humoral immune responses (1) Experimental methods Immunization was performed according to the immunization procedure in Example 1. Peripheral blood was collected from mice before immunization and on days 7, 14, 21, and 32 after immunization. After the blood was allowed to stand at room temperature to coagulate, the serum was separated by centrifugation. The levels of OVA-specific IgG and IgM antibodies in the serum were detected by enzyme-linked immunosorbent assay (ELISA), and the serum antibody titer was detected by serial dilution.

[0051] (2) Experimental results and analysis The results of the detection of specific IgG levels at each immunization time point in each group of mice in Example 3 are shown in the figure below. Figure 3 Among them, A is 0 days, B is 7 days, C is 14 days, D is 21 days, and E is 32 days.

[0052] The results of the detection of specific IgM levels in each group of mice at each immunization time point in Example 3 are shown in the figure below. Figure 4 Among them, A is 0 days, B is 7 days, C is 14 days, D is 21 days, and E is 32 days.

[0053] Figure 3 The results of IgG antibody detection showed no significant differences among the groups before immunization. From day 7 post-immunization, IgG levels in the octyl gallate adjuvant group began to increase significantly, with the most significant increase observed in the high-dose octyl gallate group. By days 14, 21, and 32 post-immunization, the IgG levels in the high-dose octyl gallate group remained at their highest levels, significantly higher than those in the PBS group, the unadjuvanted group, and the aluminum adjuvant group. P <0.0001). The IgG levels in the medium-dose and low-dose octyl gallate groups were also significantly higher than those in the unadjuvanted group, showing a good dose-related enhancement trend.

[0054] Figure 4The results of IgM antibody detection showed that IgM levels increased in all immunization groups on day 7 post-immunization, with the most significant increase observed in the high-dose octyl gallate group, which was significantly higher than that in the PBS group, the unadjuvanted group, and the aluminum adjuvant group. P <0.0001). Subsequently, IgM levels gradually decreased, but the octyl gallate adjuvant group maintained relatively high levels on days 14 and 21, suggesting that it can effectively enhance the early humoral immune response and maintain the antibody response intensity for a certain period of time.

[0055] The results of detecting IgG (A) and IgM (B) titers in the serum of mice at each immunization time point in Example 3 are shown in the figure. Figure 5 .

[0056] Figure 5 The results of serum antibody titer testing showed that IgG titers gradually increased with the extension of immunization time, with the highest titer in the high-dose octyl gallate group, followed by the medium-dose group, both significantly higher than the unadjuvanted group and the aluminum adjuvanted group. By day 32 post-immunization, the highest IgG antibody titer in the high-dose octyl gallate group reached 1:102400, significantly higher than the unadjuvanted group and the aluminum adjuvanted group. P <0.001). This indicates a strong antibody response and good antibody persistence. IgM titer peaked on day 7 post-immunization, with the octyl gallate group showing higher early antibody titers than the unadjuvanted and aluminum adjuvanted groups. These results suggest that octyl gallate adjuvant can significantly increase OVA-specific IgG and IgM antibody levels, enhance antibody titers, and provide good persistence of the immune response.

[0057] In summary, octyl gallate adjuvant significantly enhances the specific humoral immune response induced by OVA, promoting both early IgM production and rapid increase and maintenance of high IgG levels. Compared with aluminum adjuvant, octyl gallate adjuvant shows superior effects in increasing antibody levels, enhancing antibody titers, and maintaining antibody persistence, suggesting a better humoral immune-enhancing effect.

[0058] Example 4 Octyl gallate adjuvant for CD4 + and CD8 + Regulation of T cell response (1) Experimental methods Mice were immunized according to the immunization procedure in Example 1. On day 32 post-immunization, mice were sacrificed, and spleen tissue was collected under aseptic conditions. The spleen tissue was ground and filtered in pre-chilled PBS to prepare a spleen single-cell suspension. After erythrocyte lysis and washing with PBS, the cells were resuspended and counted, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6Cells / mL. Approximately 100 μL of spleen lymphocyte suspension was added to each group into a flow cytometry tube, followed by the addition of APC-labeled anti-mouse CD3 monoclonal antibody (0.1 mg / mL), PE-labeled anti-mouse CD4 monoclonal antibody (0.2 mg / mL), and FITC Plus-labeled anti-mouse CD8 monoclonal antibody (0.5 mg / mL). CD3 count was then measured by flow cytometry. + CD4+ cells in T lymphocytes + and CD8 + The proportions of T cell subsets were analyzed using FlowJo software. CD4 counts were compared among the PBS group, the unadjuvanted group, the aluminum adjuvant group, and different doses of octyl gallate. + and CD8 + The study evaluated the changes in T cell proportions and the regulatory effect of octyl gallate adjuvant on T cell subset distribution.

[0059] (2) Experimental results and analysis In Example 4, mice in each group were immunized for 32 days with CD4. + T cell flow cytometry detection and statistical results are shown in [link to data]. Figure 6 Where A~F represent the CD4 groups respectively. + T cell flow cytometry results: A is the PBS group, B is the OVA group, C is the OVA+Alum group, D is the OVA+OG-20mg / mL group, E is the OVA+OG-10mg / mL group, F is the OVA+OG-5mg / mL group, and G is the CD4 count detected by flow cytometry in each group. + percentage of T cells.

[0060] In Example 4, mice in each group were immunized for 32 days with CD8. + T cell flow cytometry detection and statistical results are shown in [link to data]. Figure 7 Where A~F represent the CD8 groups respectively. + T cell flow cytometry results: A is the PBS group, B is the OVA group, C is the OVA+Alum group, D is the OVA+OG-20mg / mL group, E is the OVA+OG-10mg / mL group, F is the OVA+OG-5mg / mL group, and G is the CD8+ result obtained by flow cytometry for each group. + percentage of T cells.

[0061] Figure 6 CD4 + T-cell assay results showed that, compared with the PBS group and the unadjuvanted group, the aluminum adjuvant group significantly increased CD4+ in splenic lymphocytes. + T cell ratio ( P <0.0001), while the octyl gallate adjuvant group had CD4 + The proportion of T cells was further increased compared to the aluminum adjuvant group. Among them, the high-dose octyl gallate group had CD4... +The proportion of T cells was the highest, significantly higher than that of the unadjuvanted group and the aluminum adjuvanted group. P <0.0001); the medium-dose group of octyl gallate also showed a significant increasing trend ( P <0.001).

[0062] Figure 7 CD8 + T-cell assay results showed that the CD8+ group in the octyl gallate adjuvant group... + The proportion of T cells was higher in the PBS group, the unadjuvanted group, and the aluminum adjuvanted group, with the high-dose octyl gallate group showing the highest CD8 count. + T cell counts were significantly higher in the aluminum adjuvant group than in the aluminum adjuvant group. P <0.0001), suggesting that it can promote cytotoxic T cell-related immune responses. Compared with aluminum adjuvant, octyl gallate adjuvant has a greater effect on CD4. + T cells and CD8 + T cells all showed a stronger promoting effect.

[0063] Further analysis suggests that CD4 + T cells are important regulatory cells in the adaptive immune response, providing key auxiliary signals for B cell activation, antibody class switching, and the production of high-affinity antibodies; CD8 + T cells are closely related to cytotoxic immune responses. Octyl gallate adjuvant promotes CD4... + and CD8 + The increased proportion of T cells indicates that it not only enhances humoral immune responses but also effectively activates cellular immune responses. This result provides cellular immunological evidence for the application value of octyl gallate as a highly effective vaccine adjuvant.

[0064] Example 5 The role of octyl gallate adjuvant in Newcastle disease vaccine immunization (1) Experimental group design SPF chicks were confirmed to have no maternal antibodies against Newcastle disease (ND) before enrollment, and the hemagglutination titer of the NDV vaccine before inactivation was 10. 8The ratio of vaccine to adjuvant (prepared according to the method in Example 1) was 1:1 (V:V). Ten birds were randomly assigned to each group, with the following specific groupings and treatments: PBS blank group (non-immunized group), NDV vaccine inactivated antigen group (NDV + no adjuvant), NDV inactivated antigen + ISA 201 (NDV + ISA 201), and NDV inactivated antigen + octyl gallate (NDV + OG). The first immunization was administered at 7 days of age, and a second booster immunization was administered at 21 days of age. Immunization was carried out via intramuscular injection into the pectoral cavity, with the dosage strictly controlled at 0.3 mL per bird. Whole blood samples were collected via wing vein at four key time points: 7 days and 14 days after the first immunization, and 7 days and 14 days after the second immunization, and serum was separated. This serum was used for subsequent detection of immune indicators such as antibody titers and cytokine levels. Fourteen days after the second immunization (35 days old), fresh spleen tissue was collected under aseptic conditions, and lymphocytes were separated within 2 hours for subsequent flow cytometry analysis to assess the proportion and proliferative capacity of T lymphocyte subsets.

[0065] (2) Detection method The hemagglutination (HA) and hemagglutination inhibition (HI) assays were used to determine the NDV-specific antibody IgG in chicken serum. Enzyme-linked immunosorbent assay (ELISA) was used to quantitatively detect the concentrations of NDV-specific antibody Y (represented by IgY) and antibody A (represented by IgA) in serum.

[0066] To assess the level of vaccine-induced cellular immunity, spleen lymphocytes were isolated from chickens at the final sampling point (35 days of age). The proliferative activity of lymphocytes was detected by the CCK-8 assay to assess the formation of memory T cells and the intensity of antigen-specific cellular immune responses in vivo.

[0067] Flow cytometry was used to detect specific CD4 groups in chicken spleen samples. + and CD8 + Lymphocyte subsets. Take 100 μL of spleen cell suspension (approximately 1 × 10⁻⁶ cells / mL). 6 (Each cell type) was treated with appropriate amounts of fluorescently labeled mouse anti-chicken CD3-FITC, CD4-PE, and CD8a-APC monoclonal antibodies, and CD3 was analyzed. + CD4+ cells in T lymphocytes + and CD8 + Subpopulation proportions. In proliferation analysis, at CD3 + The fluorescence intensity of CFSE was detected within the T cell population. Each decrease in fluorescence intensity by half represented one cell division. The proliferation index and number of divisions were calculated using FlowJo V10 software.

[0068] (3) Experimental results and analysis The results of HI antibody titer detection at each immunization time for SPF chicks in Example 5 are shown below. Figure 8 .

[0069] The results of IgY(A) and IgA(B) antibody level detection at each immunization time in each group of SPF chicks in Example 5 are shown in the figure below. Figure 9 .

[0070] Figure 8 and Figure 9 The results showed that at 28 and 35 days of age, the HI antibody titer in the octyl gallate adjuvant group was significantly higher than that in other experimental groups (unadjuvant group and ISA201 group), with statistically significant differences. P <0.0001). The antibody level in this group increased rapidly with age, reaching a peak at 28 days of age and remaining at a high level thereafter. Figure 8 Simultaneously, octyl gallate adjuvant induced higher levels of specific IgY and IgA (…). Figure 9 The results suggest that octyl gallate adjuvant can significantly enhance humoral and mucosal immune responses induced by NDV vaccine and has good antibody persistence.

[0071] In Example 5, the proliferative activity of T cells in SPF chicks 14 days after secondary immunization was observed. Figure 10 .

[0072] Figure 10 The results of cell viability assays showed that the T cell proliferation activity in the NDV + octyl gallate adjuvant group was significantly higher than that in the NDV without adjuvant group and the NDV + ISA 201 group, indicating that octyl gallate adjuvant had a prominent promoting effect on T cell proliferation. The T cell proliferation activity in the NDV + ISA 201 group and the NDV without adjuvant group was significantly lower than that in the octyl gallate adjuvant group, suggesting that the ISA 201 adjuvant had a weaker effect on enhancing cellular immunity than octyl gallate in this experimental system. Further analysis of previous time-point data revealed that the octyl gallate adjuvant group maintained the highest lymphocyte proliferation activity at multiple observation time points after primary and secondary immunization. This indicates that the octyl gallate adjuvant group not only effectively activated cellular immunity but also induced durable immune memory, demonstrating its potent and stable immune activation ability.

[0073] In Example 5, each group of SPF chicks received their second vaccination and were 14 days old with CD4+. + The / CD3 ratio is shown in [link / details]. Figure 11 .

[0074] Figure 11 Quantitative analysis by mid-flow cytometry showed that, compared with the control group that received only unadjuvanted NDV vaccine, the octyl gallate adjuvant group significantly increased CD4 count in the spleen. + The proportion of T lymphocytes ( P <0.05). This indicates that the addition of adjuvant effectively promoted antigen-specific CD4.+ T cell activation and clonal expansion. CD4 + T cells are key regulators of the adaptive immune response, providing crucial auxiliary signals for B cells to produce high-affinity antibodies. Octyl gallate adjuvant therapy works by preferentially promoting CD4+. + The activation and proliferation of T cells, in turn, more efficiently assist B cells, ultimately driving a more robust humoral immune response. This discovery provides solid cellular immunological support for the application value of octyl gallate as a "highly effective immune adjuvant."

[0075] It should be noted that, based on the aforementioned results, the vaccine adjuvant prepared by octyl gallate can also be used in vaccines containing other viral antigens, recombinant protein antigens, subunit antigens, or inactivated antigens, which will not be listed in this invention.

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of octyl gallate in the preparation of vaccine adjuvants.

2. A vaccine adjuvant, characterized in that, The components include octyl gallate, solvent, and buffer solution.

3. The vaccine adjuvant according to claim 2, characterized in that, The concentration of octyl gallate in the vaccine adjuvant is 5-20 mg / mL.

4. The vaccine adjuvant according to claim 2, characterized in that, The solvent is dimethyl sulfoxide, and the solvent accounts for less than 1% of the volume of the vaccine adjuvant.

5. The vaccine adjuvant according to claim 2, characterized in that, The buffer solution is a phosphate buffer.

6. The vaccine adjuvant according to claim 5, characterized in that, The phosphate buffer solution has a phosphate concentration of 0.005~0.05M and a pH value of 7.0~8.

0.

7. A method for preparing the vaccine adjuvant according to any one of claims 2 to 6, characterized in that, Includes the following steps: Octyl gallate is dissolved in a solvent to obtain an octyl gallate solution. The octyl gallate solution is then mixed evenly with a buffer solution to obtain the vaccine adjuvant.

8. The use of the vaccine adjuvant according to any one of claims 2 to 6 in the preparation of a vaccine, characterized in that, The antigens in the vaccine include: viral antigens, recombinant protein antigens, subunit antigens, inactivated antigens, or model protein antigens.

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