African swine fever immunization O-A-Dex-Ap

Abstract

The application belongs to the technical field of animal vaccines, and particularly relates to an O-A-Dex-Ap for African swine fever immunization. Based on the good biocompatibility of Dextran, the application uses the Dextran as a nanoparticle to load a coupled CTL epitope peptide. Preliminary experimental results show that, compared with the epitope peptide alone, the O-A-Dex-Ap after loading and coupling can more effectively promote antigen presentation and activate more persistent CTL immune response, and has good application potential. Based on the results, a good technical foundation can be laid for subsequent preparation of African swine fever and other disease vaccines.

Description

Technical Field

[0001] This application belongs to the field of animal vaccine technology, specifically relating to an OA-Dex-Ap vaccine for African swine fever immunization. Background Technology

[0002] African swine fever virus (ASFV) is the only double-stranded nucleoplasmic large DNA virus in the Asfarviridae family. Infection with this virus can cause systemic hemorrhagic necrosis, splenomegaly, and hemorrhagic diathesis in domestic pigs, with a mortality rate of up to 100%. First reported in 1921, it spread to China and surrounding Asian countries in 2018, causing severe economic losses worldwide. Currently, however, there is no effective commercial vaccine to adequately prevent the spread of African swine fever.

[0003] Studies on ASFV have shown that it may employ multiple strategies to suppress host immunity and evade innate and adaptive immune responses, which may be a major reason for the limitations in vaccine development. Specifically, the two main components of innate immunity, IFN-I activity and the inflammatory response, can be significantly blocked by ASFV proteins. For example, studies have shown that ASFV pM1249L exerts a dual inhibitory effect on IFN-I by inhibiting TBK1 phosphorylation, mediating lysosome-dependent degradation of IRF3; ASFV pH240R and pMGF505-7R inhibit NF-κB activation, restrict NF-κB nuclear translocation, and disrupt the assembly of the NLRP3 inflammasome by targeting these two signaling pathways. Furthermore, regarding adaptive immunity, immunization with recombinantly expressed ASFV proteins (P30 (CP204L), P54 (E183L), P72 (B646L), and P22) showed that generating ASFV-specific neutralizing antibodies only slightly delayed the onset of clinical disease; that is, neutralizing antibodies targeting only ASFV proteins may not provide sufficient protection. Therefore, a CTL response of specific immune cells may be a better strategy for combating ASFV infection.

[0004] However, existing studies have shown that ASFV can significantly impair T-cell immunity. Reports indicate that Armenian pigs infected with highly virulent ASFV exhibit impaired T-cell immune responses, with a reduction in the total number of CD4+ T cells, DP-T cells, and CD8+ T cells. Cytotoxic CD8+ T cells are a key component of cell-mediated immunity, an adaptive immune response initiated by MHC presentation of antigenic peptides to T cells under various pathological conditions. Further research suggests that the ASFV EP153R protein may inhibit MHC-I transport, which undoubtedly limits the development of related vaccines. Furthermore, T-cell epitopes are MHC-restricted and depend on SLA for effective presentation in pigs. Therefore, developing effective vaccines based on CTL immunity still faces numerous technical challenges that need to be addressed. Summary of the Invention

[0005] The purpose of this application is to provide an OA-Dex-Ap for African swine fever immunization, thereby laying a certain technical foundation for the prevention and control of African swine fever.

[0006] The technical solution adopted in this application is described in detail below.

[0007] OA-Dex-Ap for African swine fever immunization is prepared through the following steps. (a) Preparation of T cell CTL epitopes The sequences of the CTL epitopes (AP peptides, antigenic peptides) are shown in SEQ ID Nos. 1-10, as follows: (II) Preparation of OA-Dex nanoparticles In the preparation process, dextran (α1-6 configuration dextran) was first oxidized using the sodium periodate method, followed by reaction with 2-methoxypropene to form a cyclic acetal. Finally, the aldehyde groups on the surface of the oxidized dextran were modified. The specific preparation process is as follows: (1) Dissolve 2.5g of dextran in 10mL of ultrapure water, then add 0.55g of sodium periodate, stir at room temperature (around 18~25℃) and in the dark for about 5 hours to allow for a full reaction; after the reaction is complete, dialyze the product using a dialysis bag with a molecular weight cutoff of 8000, and freeze-dry to obtain oxidized dextran. (2) Take 1g of the oxidized dextran prepared above, dissolve it in 9mL of anhydrous DMSO, and add p-toluenesulfonic acid pyridine (15.6mg of p-toluenesulfonic acid pyridine was dissolved in 1mL of anhydrous DMSO beforehand) and 3.4mL of 2-methoxypropylene under non-reactive atmosphere conditions (e.g., using N2 environment). After reacting for 3 hours (at room temperature, around 18-25°C), 1 mL of triethylamine was added to quench the reaction. After quenching, the reactants were added dropwise to ultrapure water to precipitate the precipitate. After precipitation, centrifuge at 10,000 rpm for 20 min to separate the precipitate. Wash the precipitate at least twice to ensure it is clean, and then freeze-dry it to obtain oxidized acetalized dextran. (3) Take 20 mg of the oxidized acetalized dextran prepared above, dissolve it in 1 mL of dichloromethane, and then perform ultrasonic emulsification on ice (sonicate for 1 min, with 4 s on and 2 s off during the process). Then add 2 mL of the 3% polyvinyl alcohol aqueous solution prepared in advance (prepared by dissolving polyvinyl alcohol in ultrapure water under 70°C water bath conditions), and perform ultrasonic emulsification on ice again (sonicate for 1 min, with 4 s on and 2 s off during the process). Then add 10 mL of the 0.3% polyvinyl alcohol prepared in advance, and stir at room temperature (around 18~25°C) in the dark for 3 h to fully react. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 20 min, the precipitate was collected, and after washing and freeze-drying, the precipitate was obtained as OA-Dex nanoparticles.

[0008] (III) Coupling with antigenic peptides (Preparation of OA-Dex-Ap) Take 2 mg of the antigen peptide prepared in step (I) (preferably, after uniformly mixing the antigen peptides in step (I) in equal mass ratio, the amount of sample after mixing is 2 mg), and dissolve it in 20 μL of DMSO; Simultaneously, 10 mg of OA-Dex prepared in step (II) was dissolved in 200 μL of DMSO; Mix the above antigen peptide solution with OA-Dex solution thoroughly and incubate at room temperature (around 18~25℃) with shaking overnight (around 8~10h) to ensure a complete reaction. After overnight incubation, the antigen peptide was purified using an ultrafiltration tube (30 kDa), and the solution was replaced with ultrapure water. After washing and lyophilization, OA-Dex-Ap, which is adsorbed and coupled with nanoparticles, was obtained.

[0009] The application of OA-Dex-Ap for African swine fever immunization in the preparation of African swine fever vaccine preparations.

[0010] Nanoparticles, due to their unique properties (such as high surface area, tunable size, and the ability to be functionalized to load antigens), have significant application potential in vaccine preparation. In this application, based on the good biocompatibility of Dextran, it is used as a nanoparticle to load and couple related CTL epitope peptides.

[0011] Preliminary experimental results indicate that, compared to epitope peptides alone, the loaded and conjugated OA-Dex-Ap can more effectively promote antigen presentation and activate a more durable CTL immune response, demonstrating good application potential. Based on these results, a solid technical foundation can be laid for the subsequent preparation of vaccines against African swine fever and other diseases. Attached Figure Description

[0012] Figure 1 The results of characterization and detection of OA-Dex nanoparticles and OA-Dex-Ap related to conjugated adsorbed antigen peptides are as follows: A is a schematic diagram of the oxidative acetalization and condensation reaction of OA-Dex; B shows the Fourier transform infrared spectra of Dextran and OA-Dex; C represents the dissolution rate of OA-Dex nanoparticles at different pH values; D is a scanning electron microscope image of the OA-Dex nanoparticles (scale bar: 500 nm). E represents the dynamic light scattering analysis of OA-Dex and OA-Dex-Ap; F represents the toxicity test results of OA-Dex in DC2.4 cells using the CCK-8 colorimetric method; Figure 2 The results are experimental evaluations of the phagocytic effect and organismal distribution of OA-Dex-Ap; among which: A shows the confocal image results of OA-Dex-Ap and DC2.4 cells co-incubated for 24 hours, where DAPI (blue) represents the cell nucleus and FITC (green) represents OA-Dex-Ap labeled with fluorescein isothiocyanate; B represents the secretion of IL-12 and TNF-α in the supernatant of nanoparticles co-cultured with cells using ELISA. C represents the detection of transcriptional levels of IL-12 and TNF-α after the co-culture process of nanoparticles and cells; D represents in vivo imaging of mice injected with Cy7-labeled OA-Dex-Ap and free antigen peptides, and in vitro imaging of major organs 72 hours later. The images show the imaging results of the same mouse in each group at different time points. The organs in vitro are, in order, liver, lung, kidney, heart, inguinal lymph nodes, and spleen (left side). The bar chart shows the comparison of organ fluorescence intensity (right side). Figure 3 The results show the expression levels of CD11c, CD80, and CD86 after OA-Dex-Ap and BMDCs were co-incubated in vitro for 48 hours; (Figure:) A shows the results of the flow scatter plot; B shows the results of the bar graph quantitative analysis; Figure 4 For the assessment of the mouse immune process and in vivo toxicity of OA-Dex-Ap; in the figure: A is a schematic diagram of the immunization process. Balb / c mice were immunized three times at 14-day intervals. The immunization dose was 100μL containing 40μg of antigen. The mice were euthanized on the seventh day after the three immunizations, and serum and organs were collected for subsequent experiments. B is the curve of mouse body weight change (n=5); C is an HE-stained section of the major organs after the third immunization (scale bar: 50 μm). Figure 5 The experimental results related to the significant CTL immune response induced by OA-Dex-Ap are as follows: A represents the results of the MTT assay evaluating cell proliferation after co-incubation of OA-Dex-Ap with the spleen of immunized mice; B represents the secretion of Granzyme B and IL-2 in the serum of immunized mice as detected by ELISA; C represents the number of IFN-γ spots in the spleen of immunized mice after in vitro stimulation by ELISpot. Figure 6 The percentages of helper T cells (CD3+CD4+), cytotoxic T cells (CD3+CD8a+), cytotoxic T lymphocytes (CD3+CD8a+CD107a+), and central memory T cells (CD62L+CCR7+) in spleen cells of immunized mice were determined by flow cytometry; among which: A shows the results of the flow scatter plot; B shows the results of the bar graph quantitative analysis; When statistical data is involved in the figure, all experiments were conducted independently, in triplicate. The bar chart is expressed as mean and standard deviation (mean±SD). The significance level is expressed as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns (not significant). Detailed Implementation

[0013] The present application will be further explained below with reference to the accompanying drawings and embodiments.

[0014] Example 1 Based on the advantages of nanoparticle vaccines in antigen presentation, this application combines the T-cell epitopes obtained through screening to prepare related nanoparticle vaccines. The specific preparation process is briefly described below.

[0015] (a) Screening of immune-dominant T cell epitopes First, using relevant publicly available bioinformatics results, high-frequency SLA class I molecules were selected as the basis to predict potential T-cell epitopes of multiple ASFV proteins. Subsequently, the predicted binding results of the predicted peptides to any MHC molecules with known sequences are combined, the predicted immune epitopes are scored and sorted, and the epitope peptides with strong binding ability are listed. Finally, by combining the validated T-cell epitopes found in relevant databases, specific T-cell epitopes were identified, and finally, the selected epitopes were artificially synthesized.

[0016] (II) Preparation of OA-Dex nanoparticles Clinically, α1-6 dextran is a dextran derived from *L. mesenteroides* subsp. *dextranicum*, commonly used in sodium chloride or glucose injection formulations. Considering its good biocompatibility, this application uses it as a raw material to prepare nanoparticles via double emulsion evaporation. The preparation process involves first oxidizing Dextran using the sodium periodate method, followed by a reaction with 2-methoxypropene to form a cyclic acetal, and finally modifying the aldehyde groups on the surface of the oxidized dextran (reaction flow is as follows). Figure 1 (As shown in Figure A). The specific preparation process is as follows.

[0017] (1) Dissolve 2.5g of dextran in 10mL of ultrapure water, then add 0.55g of sodium periodate, stir at room temperature (around 18~25℃) and in the dark for about 5 hours to allow for a full reaction; after the reaction is complete, dialyze the product using a dialysis bag with a molecular weight cutoff of 8000, and freeze-dry to obtain oxidized dextran. (2) Take 1g of the oxidized dextran prepared above, dissolve it in 9mL of anhydrous DMSO, and add p-toluenesulfonic acid pyridine (15.6mg of p-toluenesulfonic acid pyridine was dissolved in 1mL of anhydrous DMSO beforehand) and 3.4mL of 2-methoxypropylene under non-reactive atmosphere (N2 environment was used in this example); After reacting for 3 hours (at room temperature of approximately 18-25°C), 1 mL of triethylamine was added to quench the reaction. After quenching, the reactants were added dropwise to ultrapure water to precipitate the precipitate. After precipitation, centrifuge at 10,000 rpm for 20 min to separate the precipitate. Wash the precipitate at least twice to ensure it is clean, and then freeze-dry it to obtain oxidized acetalized dextran. (3) Take 20 mg of the oxidized acetalized dextran prepared above, dissolve it in 1 mL of dichloromethane, and then perform ultrasonic emulsification on ice (sonicate for 1 min, with 4 s on and 2 s off during the process). Then add 2 mL of the 3% polyvinyl alcohol aqueous solution prepared in advance (prepared by dissolving polyvinyl alcohol in ultrapure water under 70°C water bath conditions), and perform ultrasonic emulsification on ice again (sonicate for 1 min, with 4 s on and 2 s off during the process). Then add 10 mL of the 0.3% polyvinyl alcohol prepared in advance, and stir at room temperature (around 18~25°C) in the dark for 3 h to fully react. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 20 min, the precipitate was collected, and after washing and freeze-drying, the precipitate was obtained as OA-Dex nanoparticles.

[0018] In the preparation of OA-Dex nanoparticles, dextran is first oxidized by sodium periodate, and then reacted with 2-methoxypropylene to obtain OA-Dex. The hydroxyl groups on the surface of the modified dextran are converted into some acyclic aldehydes and some cyclic aldehydes. Among them, the acyclic aldehydes can undergo a condensation reaction with imino groups to form Schiff bases, which can then covalently bind to the target peptide chains.

[0019] Fourier transform infrared (FTIR) spectroscopy was performed on the prepared OA-Dex. Figure 1 (B) It can be seen that, compared with the unreacted dextran, there is a significant absorption peak at 1730 cm⁻¹ to 1740 cm⁻¹, which is the saturation absorption peak of C=O, indicating that the aldehyde group of the dextran after reaction was successfully constructed.

[0020] Since glucose (C6H12O6) contains a fixed amount of aldehyde molecules on its surface, a standard curve for aldehydes was further constructed using glucose. Calculations showed that each mol of OA-Dex prepared in this application contains an average of 1.5 mol of aldehydes. Further solubility test results showed that ( Figure 1 (c) The OA-Dex prepared in this application remains stable in PBS at pH 7.4, but is completely hydrolyzed within 24 hours at pH 4.6, indicating its acid degradation capability and the ability to release antigens within acidic organelles. This also demonstrates its potential for practical biological applications.

[0021] Scanning electron microscopy (SEM) observation of the prepared OA-Dex particles revealed that, in the dry state, the OA-Dex particles exhibited uniform spherical shapes. Figure 1 D), with a particle size distribution between 100-300 nm. Figure 1 E).

[0022] (III) Coupling with antigenic peptides (Preparation of OA-Dex-Ap) Take 2 mg of the antigen peptide prepared in step (I) (previously, mix the antigen peptides prepared in step (I) in equal mass ratios), and dissolve them in 20 μL of DMSO. Simultaneously, 10 mg of OA-Dex prepared in step (II) was dissolved in 200 μL of DMSO; Mix the above antigen peptide solution with OA-Dex solution thoroughly and incubate at room temperature (around 18~25℃) with shaking overnight (around 8~10h) to ensure a complete reaction. After overnight incubation, the antigen peptide was purified using an ultrafiltration tube (30 kDa), and the solution was replaced with ultrapure water. After washing and lyophilization, OA-Dex-Ap, which is adsorbed and coupled with nanoparticles, was obtained.

[0023] Dynamic light scattering (DLS) analysis of the prepared OA-Dex-Ap showed that, compared with the average particle size of OA-Dex particles of 270 nm, the average particle size of O-A-Dex-Ap reached 352 nm after coupling with the antigen peptide.

[0024] Taking DC2.4 cells as an example, the cytotoxicity of different concentrations of OA-Dex-Ap was analyzed using the CCK-8 colorimetric assay. The results showed that ( Figure 1 F), OA-Dex-Ap is non-toxic and harmless, which lays the foundation for its practical biological applications.

[0025] It should be noted that the specific experimental procedure for CCK-8 colorimetric evaluation is as follows: DC2.4 cells were cultured at low density overnight in 96-well plates. After cell adhesion, different concentrations of OA-Dex-Ap were added and incubated for 24 hours. Cell viability was then assessed according to the CCK8 kit instructions (five replicates were performed for each concentration, and the mean ± SD was calculated).

[0026] Example 2 In order to investigate the uptake efficiency of OA-Dex-Ap by antigen-presenting cells and to actually assess its distribution and flow in vivo, the inventors conducted relevant experimental tests on the OA-Dex-Ap prepared in Example 1. The specific experimental results are briefly described below.

[0027] (a) Evaluation of the uptake efficiency of OA-Dex-Ap by antigen-presenting cells Prior to preparation, OA-Dex-Ap-FITC is obtained by labeling OA-Dex-Ap with FITC (the preparation method can be referred to in the existing technology). DC2.4 cells were cultured at low density overnight in 24-well plates, and after adhesion, OA-Dex-Ap-FITC was added and co-incubated for 24 h. After incubation, the supernatant was discarded, the cells were washed with PBS, fixed with paraformaldehyde, and the cell nuclei were stained with DAPI before being observed using a laser confocal microscope (ZEISS).

[0028] To confirm whether OA-Dex-Ap can be phagocytosed by DC cells and thus internalized as an antigen, we performed the above-mentioned phagocytosis assay using DC2.4 cells. The results showed that ( Figure 2 A) After treatment with FITC-labeled OA-Dex-Ap, DC2.4 cells exhibited abundant green fluorescence distributed around the cell nucleus, with a significantly enhanced green fluorescence compared to the Ap group. This result demonstrates that OA-Dex-Ap can be phagocytosed by DC cells, thereby completing the internalization and presentation of the antigen.

[0029] Based on the above results, after incubating OA-Dex-Ap with DC2.4 cells for 48 h, the cell culture supernatant was collected, and the levels of IL-12 and TNF-α in the supernatant were detected using ELISA. The results showed that ( Figure 2 B): Both Ap and OA-Dex-Ap stimulated DC cells to secrete cytokines, showing a significant increase compared to the PBS group, while the nanoparticle group showed a slightly higher level than the Ap group. Furthermore, the transcriptional levels of IL-12 and TNF-α were detected by qPCR at different incubation time points, finding that they reached their peak at 24h and 12h, respectively. Figure 2 C).

[0030] This result indicates that OA-Dex-Ap not only presents itself as an antigen, but also acts as a strong stimulating signal to stimulate dendritic cells to complete phagocytosis and signal transduction, and drives T cells to differentiate into Th1 cells, thereby activating CTL immunity.

[0031] (II) In vivo imaging observation In advance, using cyanine dye (Cy7) to label the antigen peptide (Ap-Cy7), referring to the description in Example 1 above, cyanine dye (Cy7) labeled nanoparticles were prepared to obtain: OA-Dex-Ap-Cy7; Selected BALB / c female mice (6-8 weeks old) were acclimatized and then subcutaneously injected with labeled nanoparticles (40 μg / mouse) at multiple points on their backs. The flow direction and persistence of the nanoparticles were observed and recorded using an in vivo imaging system at 6h, 12h, 24h, 48h, and 72h. After the last imaging, the mice were euthanized and the main organs were obtained for in vitro imaging.

[0032] Experimental results show that ( Figure 2(D) The OA-Dex-Ap group showed a stronger fluorescence signal than the Ap group. While the fluorescence intensity of both groups began to decrease at 12 hours, the OA-Dex-Ap group maintained fluorescence in vivo for over 72 hours, while the fluorescence of the Ap group almost disappeared at 48 hours. This indicates that OA-Dex-Ap significantly prolonged the in vivo action time of the antigenic peptide. Furthermore, the in vitro imaging results of major mouse organs after 72 hours were consistent with the in vivo experiments. Notably, effective fluorescence was observed in the inguinal lymph nodes and spleen of mice in the OA-Dex-Ap group, further demonstrating that OA-Dex-Ap completed the migration of immune organs in vivo.

[0033] Example 3 Based on Example 2, the inventors conducted a test and evaluation experiment on the stimulation effect of the OA-Dex-Ap provided in this application on actual BMDC. The specific experimental results are briefly described below.

[0034] (a) Isolation and culture of BMDC 6-8 week old female BALB / c mice were used for acclimatization feeding, and stem cells were isolated from their femur and tibia to obtain BMDCs. The specific procedure is as follows: Mice were euthanized by cervical dislocation and immersed in 75% ethanol for 5 min. The femur and tibia were separated under sterile conditions. The bone marrow was washed with PBS and filtered through a 70 μm cell sieve. After lysing red blood cells, the cells were resuspended in RPMI-1640 containing 10% FBS and 1% penicillin / streptomycin and placed in T75 culture flasks. IL-4 (15 ng / mL) and GM-CSF (15 ng / mL) were added to induce differentiation. The cells were cultured at 37°C and 5% CO2. Half of the culture medium was discarded every 3 days and replaced with fresh culture medium and cytokines. Immature BMDCs were harvested on day 7 and used immediately.

[0035] (II) Effects of nanoparticles on the stimulatory maturation of BMDC cells The BMDC obtained in step (I) is converted into 1×10 5 Cells were added to 96-well plates at a density of [number] cells / well. BMDCs were co-incubated with Ap (40 μg / mL) and OA-Dex-Ap (40 μg / mL) for 48 h. Lipopolysaccharide (LPS) (100 ng / mL) served as a positive control. After treatment, cells were digested and separated using 0.25% (v / v) trypsin-EDTA digestion solution. Cells were collected and counted. The collected cells were incubated at room temperature in the dark for 40 min using APC-labeled anti-mouse CD11c, FITC-labeled anti-mouse CD80, and PE-labeled anti-mouse CD86 antibodies. The expression of CD11c, CD80, and CD86 was analyzed by flow cytometry.

[0036] Experimental results show that ( Figure 3 A, Figure 3 B): Compared with the PBS group and the Ap group, the expression of CD80+CD86+ (27.06%) in the OA-Dex-Ap group was significantly upregulated, which was significantly higher than that in the PBS group (8.61%) and the single antigen peptide group (11.40%). This result indicates that the strategy of using OA-Dex loaded with antigen peptides enhances the stimulation ability of BMDC cells, effectively promotes the maturation of BMDCs, and thus enhances the activation of T lymphocytes.

[0037] Example 4 Based on the above experiments, the inventors conducted further animal experiments using mice as an example. The specific experimental details are briefly described below.

[0038] Experimental grouping and procedure: Female BALB / c mice aged 6-8 weeks were randomly divided into three groups (n=5). Each group received a subcutaneous injection of Ap or OA-Dex-Ap (40 μg / 200 μL per mouse) into the back, while the control group received the same dose of PBS. The mice were immunized three times at 14-day intervals (procedure as follows). Figure 4 (as shown in A); During the period, the weight changes of mice were recorded every three days. On the 7th day after the third vaccination, whole blood was collected from the eyeballs to prepare serum for ELISA detection. The major organs (heart, liver, spleen, lung and kidney) were aseptically isolated to prepare HE staining sections, and the spleen was isolated to prepare single cell suspension for FACS analysis.

[0039] Weight statistics results ( Figure 4 B) indicates that compared with PBS, there was no significant difference in body weight in the OA-Dex-Ap group, and the weight showed a steady upward trend. HE staining results of relevant tissue and organ sections showed that ( Figure 4 C): No obvious pathological changes were observed in the major organs and tissues, which indicates that OA-Dex has good biocompatibility and can be used as a vaccine vector.

[0040] (a) MTT and ELISpot During MTT testing: Mice were euthanized by cervical dislocation after three immunizations. Spleens were isolated under sterile conditions, minced, and ground. The splenic pulp and tissue fragments were filtered through a 70 μm cell sieve. Red blood cells were lysed, washed once with PBS, and centrifuged at 1000 rpm for 5 min, discarding the supernatant. Cells were resuspended in RPMI-1640 medium containing 10% FBS and 1% penicillin-dextrin, and quantified to 1 × 10⁶ cells / mL. 6 cells / mL; The cell suspension prepared above was seeded at 100 μL / well into 96-well plates. PBS, Ap, and OA-Dex-Ap were used as stimulants (10 μg / mL), and complete culture medium was used as a negative control. After incubation for 36 h, MTT solution was added, and after another 4 h of incubation, DMSO was added to dissolve the MTT. The absorbance at 490 nm was measured, and the lymphocyte stimulation index of the nanoparticles was calculated (5 parallel experiments were performed for each group).

[0041] The results showed that ( Figure 5 A): The OA-Dex-Ap group induced strong cell proliferation, which was 3.7 times that of the PBS group and much higher than that of the Ap group (2.1 times), while the OA-Dex group showed no change.

[0042] In other words, this result indicates that OA-Dex-Ap can significantly promote lymphocyte proliferation and successfully establish antigen-specific immune memory.

[0043] During testing: Referring to the instruction manual, the proportion of antigen-specific T cells was identified using the IFN-γ ELISpot kit. During the detection, the spleen cell suspension prepared above was seeded onto a pre-coated ELISpot plate, incubated with different stimulants for 24 hours, and then a colorimetric reaction was performed. The spots were imaged using a CTL-ImmunoSpot® S6 analyzer and quantitative analysis was performed.

[0044] ELISA results of relevant serum cytokines showed ( Figure 5 B): OA-Dex-Ap induced higher levels of Gzmb and IL-2, significantly higher than in the PBS and Ap groups. This is an important signal that CTLs have been activated and are exerting a cytotoxic effect (activated CTLs exhibit strong cytotoxic effects, killing target cells via the perforin / granzyme pathway and releasing IFN-γ, which can further promote DC maturation and Th1 immune responses). Further IFN-γ secretion level detection results showed that ( Figure 5 (C) The OA-Dex-Ap group exhibited the most efficient IFN-γ secretion level. This further indicates that the OA-Dex-Ap group can effectively promote the establishment of immune memory.

[0045] (II) FACS Analysis Referring to the foregoing description, a single-cell suspension (1×10⁻⁶) was prepared from mouse spleen. 6 / mL), different antibodies (CD3-APC, CD4-FITC, CD8-FITC, CD107a-PE, CD62L-APC, CCR7-PE) were added, and the cells were incubated at room temperature in the dark for 40 min. After washing with PBS, the changes in mouse spleen lymphocyte populations were analyzed by flow cytometry.

[0046] Experimental results show that ( Figure 6 A, Figure 6 B): Compared with the PBS group and the monomeric antigen Ap group, OA-Dex-Ap induced the highest level of cellular immunity, with helper T cells (CD3+CD4+) at 29.57%, CD3+CD8+ T cells at 9.18%, CTL (CD8+CD107a+) cells at 4.04%, and Tcm (CCR7+CD62L+) cells at 18.57%.

[0047] This result indicates that OA-Dex-Ap can induce strong cellular immunity, in which CD3+CD4+ cells, as helper T cells, participate in the activation of CD8+ T cells, causing them to differentiate into CTL cells. At the same time, depending on the cytokine environment, CD4+ T cells can differentiate into Th1 cells, effectively activating cellular immunity. CTL cells can rapidly kill target cells through the perforin / granzyme pathway, and can also induce the death of target cells through the Fas-FasL and cytokine pathways. The generation of Tcm cells indicates that OA-Dex-Ap can induce the establishment of immune memory.

[0048] The above series of experimental results show that the OA-Dex-Ap provided in this application can effectively promote CTL activation, induce immune signal generation and immune cell establishment, and promote the occurrence of a strong Th1 immune response. This has important technical value for the long-term establishment of CTL immunity in the prevention and treatment of ASFV.

Claims

1. OA-Dex-Ap for African swine fever immunization, characterized in that, It is prepared by the following steps: (a) Preparation of T cell CTL epitopes The CTL epitope antigen peptide sequences are shown in SEQ ID No. 1~10; (II) Preparation of OA-Dex nanoparticles In the preparation process, dextran was first oxidized using the sodium periodate method, followed by reaction with 2-methoxypropene to form a cyclic acetal, and finally the aldehyde groups on the surface of the oxidized dextran were modified. The specific preparation process is as follows: (iii) Coupling with antigenic peptides Take one or more of the antigenic peptides prepared in step (I), mix them in any proportion, and dissolve them in DMSO; Simultaneously, the OA-Dex prepared in step (II) was dissolved in DMSO; Mix the above antigen peptide solution with OA-Dex solution thoroughly and incubate overnight at room temperature with shaking to ensure a complete reaction. After overnight incubation, purification, washing, and freeze-drying, OA-Dex-Ap, which is an adsorption-coupled antigen peptide and nanoparticles, is obtained.

2. The OA-Dex-Ap for African swine fever immunization as described in claim 1, characterized in that, In step (iii), the ten antigenic peptides are used in the same proportion, the total amount of antigenic peptides is 2mg, and the amount of OA-Dex is 10mg.

3. The preparation method of OA-Dex-Ap for African swine fever immunization according to claim 1, characterized in that, Includes the following steps: (a) Preparation of T cell CTL epitopes The CTL epitope antigen peptide sequences are shown in SEQ ID No. 1~10; (II) Preparation of OA-Dex nanoparticles In the preparation process, dextran was first oxidized using the sodium periodate method, followed by reaction with 2-methoxypropene to form a cyclic acetal, and finally the aldehyde groups on the surface of the oxidized dextran were modified. The specific preparation process is as follows: (iii) Coupling with antigenic peptides Take one or more of the antigenic peptides prepared in step (I), mix them in any proportion, and dissolve them in DMSO; Simultaneously, the OA-Dex prepared in step (II) was dissolved in DMSO; Mix the above antigen peptide solution with OA-Dex solution thoroughly and incubate overnight at room temperature with shaking to ensure a complete reaction. After overnight incubation, purification, washing, and freeze-drying, OA-Dex-Ap, which is an adsorption-coupled antigen peptide and nanoparticles, is obtained.

4. The preparation method of OA-Dex-Ap for African swine fever immunization as described in claim 3, characterized in that, In step (ii), the specific preparation operations are as follows: (1) Dissolve 2.5g of dextran in 10mL of ultrapure water, then add 0.55g of sodium periodate, stir at room temperature and in the dark until fully reacted; after the reaction is complete, dialyze the product using a dialysis bag, and freeze-dry to obtain oxidized dextran; (2) Take 1g of the oxidized dextran prepared above, dissolve it in 9mL of anhydrous DMSO, and add p-toluenesulfonic acid pyridine and 3.4mL of 2-methoxypropylene under non-reactive atmosphere conditions; After the reaction is complete, triethylamine is added to quench the reaction; after quenching, the reactants are added dropwise to ultrapure water to precipitate the precipitate. After precipitation, the precipitate is separated, washed clean, and then freeze-dried to obtain oxidized acetalized dextran. (3) Take 20 mg of the oxidized acetalized dextran prepared above, dissolve it in 1 mL of dichloromethane, and then perform ultrasonic emulsification on ice. Then add 2 mL of the 3% polyvinyl alcohol aqueous solution prepared in advance, perform ultrasonic emulsification on ice again, and then add 10 mL of the 0.3% polyvinyl alcohol prepared in advance. Stir at room temperature and in the dark to allow the reaction to proceed fully. After the reaction was completed, the precipitate was centrifuged, collected, washed, and freeze-dried to obtain OA-Dex nanoparticles.

5. The use of the OA-Dex-Ap for African swine fever immunization as described in claim 1 or 2 in the preparation of African swine fever vaccine preparations.

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