African swine fever virus polyepitope fusion protein and ferritin nanoparticle vaccine and application thereof

Abstract

The application belongs to the technical field of animal vaccine preparation, and discloses an African swine fever virus (ASFV) multi-epitope fusion protein, a ferritin nanoparticle vaccine of the ASFV multi-epitope fusion protein and application. The application obtains 16 dominant B cell epitopes by using an ASFV phage display library in combination with reverse screening of clinical positive serum, and constructs a nanoparticle vaccine by linking the 16 epitopes with ferritin self-assembly. The vaccine has excellent biosafety, can induce high-level and sustained specific antibodies and significant IFN-gamma cellular immune response in pigs, can effectively delay the clinical onset and death process of pigs in ASFV virulent challenge experiments, and can significantly reduce the virus load in whole-body organ tissues, thereby providing 25% survival protection rate. The application provides an effective new strategy for the development of a subunit vaccine for responding to various ASFV mutant strains.

Description

Technical Field

[0001] This invention belongs to the field of animal vaccine preparation technology, specifically relating to an African swine fever virus multi-epitope fusion protein and its ferritin nanoparticle vaccine and its application. Background Technology

[0002] African swine fever (ASF) is an acute, febrile, and highly contagious disease caused by the African swine fever virus (ASFV). Due to the large size of the ASFV genome and the complexity of its immune evasion mechanisms, traditional vaccine development faces significant challenges, making subunit vaccines a current research focus due to their promising safety profile. However, existing ASF subunit vaccine development still has significant limitations:

[0003] 1. Biased and unstable protective efficacy in immune mechanism research: Current mainstream academic view holds that protective immunity against ASFV mainly relies on specific T-cell responses. Therefore, existing multi-epitope vaccine development tends to focus on the screening and design of T-cell epitopes. Some literature also reports that T-cell epitope vaccines can produce a certain degree of immune protection. For a long time, the role of B-cell epitopes (humoral immunity) in ASF immune protection has been underestimated and lacks systematic validation.

[0004] 2. Epitope screening methods lack validation against innate immunity: Furthermore, existing epitope screening primarily relies on bioinformatics software for "positive prediction," i.e., prediction followed by validation. This approach struggles to simulate the actual dominant immune response during viral infection, resulting in low immune recognition rates for screened epitope combinations in pigs and difficulty in inducing sufficiently strong immune protection.

[0005] 3. Narrow antigen coverage and insufficient broad-spectrum response: Current technologies typically target only a few structural proteins such as p72, p30, and CD2v, neglecting other key neutralizing sites that may exist within the vast ASFV proteome. Furthermore, existing vaccines lack sufficient coverage against genotypes I and II, as well as the recombinant strains that have frequently emerged in recent years, making it difficult to address complex epidemic situations.

[0006] To address the aforementioned problems, this invention proposes a novel vaccine strategy based on reverse screening of the whole proteome: This invention constructs a phage display library covering the entire ASFV proteome, and uses 59 clinical positive sera for "reverse screening" to comprehensively and systematically identify dominant B cell epitopes. Summary of the Invention

[0007] The purpose of this invention is to provide a multi-epitope fusion protein of African swine fever virus, the fusion protein being shown in SEQ ID NO.5.

[0008] Another object of the present invention is to provide the application of the above-mentioned fusion protein in the preparation of nanoparticle proteins.

[0009] The final objective of this invention is to provide the application of the above-mentioned fusion protein in the preparation of African swine fever vaccines.

[0010] To achieve the above objectives, the present invention adopts the following technical measures:

[0011] The applicant has provided a novel vaccine strategy based on reverse screening of the whole proteome: by constructing a phage display library covering the entire ASFV proteome, 59 clinical positive sera were subjected to "reverse screening" to directly identify dominant B cell epitopes from the perspective of the host's innate immune response. Finally, the 16 epitopes screened out were tandemly linked, and a SpyTag sequence was added to their C-terminus to obtain an African swine fever virus multi-epitope fusion protein, which is shown in SEQ ID NO.5.

[0012] The scope of protection of this invention also includes:

[0013] The recombinant protein obtained by fusing the above fusion protein with a protein purification tag.

[0014] The recombinant protein described above is preferably the one shown in SEQ ID. 1.

[0015] The genes encoding the aforementioned fusion proteins or recombination proteins.

[0016] Expression cassettes, recombinant vectors, recombinant microorganisms, or in vitro recombinant cells containing the above-mentioned coding genes.

[0017] The application of the above-mentioned fusion proteins, recombinant proteins, genes encoding fusion proteins or recombinant proteins, or expression cassettes containing the above-mentioned encoding genes, recombinant vectors, recombinant microorganisms, or ex vivo recombinant cells in the preparation of nanoparticle proteins.

[0018] In the above-described applications, preferably, the nanoparticle protein is obtained by displaying a fusion protein or recombinant protein on ferritin nanoparticles.

[0019] Ferritin nanoparticles of a multi-epitope fusion protein of African swine fever virus, wherein the ferritin nanoparticles are obtained by mixing the protein shown in SEQ ID NO.1 and the protein shown in SEQ ID NO.3.

[0020] The use of the above-mentioned fusion protein, recombinant protein, gene encoding fusion protein or recombinant protein, expression cassette having the above-mentioned encoding gene, recombinant vector, recombinant microorganism or in vitro recombinant cell, or the above-mentioned nanoparticle protein in the preparation of drugs for preventing African swine fever virus infection.

[0021] The application of the above-mentioned fusion protein, recombinant protein, gene encoding fusion protein or recombinant protein, expression cassette having the above-mentioned encoding gene, recombinant vector, recombinant microorganism or in vitro recombinant cell, or the above-mentioned nanoparticle protein in the preparation of African swine fever vaccine.

[0022] An African swine fever vaccine, wherein the African swine fever vaccine contains the aforementioned ferritin nanoparticles.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] This invention utilizes an ASFV phage display library combined with reverse screening of clinically positive serum. After repeatable knockout and conservation comparison with 10 representative strains (including type I, type II, and recombinant strains), 16 dominant B-cell epitopes derived from ASFV structural proteins were preferentially obtained. These 16 tandem epitopes were then self-assembled with ferritin using the SpyTag / SpyCatcher system to construct the nanoparticle vaccine Fer-ASFV-16E.

[0025] Experiments have demonstrated that the 16 highly conserved B-cell epitope peptides selected, after being displayed using ferritin nanoparticles, not only induced extremely high levels of specific antibodies but also induced a significant and durable IFN-γ cell immune response (lasting for more than 11 weeks). Challenge experiments further confirmed that the vaccine Fer-ASFV-16E successfully delayed the onset of fever caused by virulent strains by 3-6 days, delayed the onset of viremia by 6 days, and provided a 25% survival protection rate. This result proves that, under highly efficient display conditions, dominant B-cell epitopes selected based on innate immunity can exert a protective effect similar to traditional pure T-cell epitope vaccines, providing new theoretical support and technical pathways for the development of broad-spectrum, highly effective subunit vaccines against African swine fever. Attached Figure Description

[0026] Figure 1 shows the ultrastructure of packaged T7 phage particles observed under a transmission electron microscope (TEM).

[0027] Figure 2 is a schematic diagram of the three-dimensional structure simulation of the ASFV-16E recombinant protein predicted using AlphaFold3.

[0028] Figure 3 shows the SDS-PAGE identification results of the ASFV-16E recombinant protein after purification by nickel column affinity chromatography;

[0029] Where M is the protein marker, lanes 1, 2, and 3 are in the order of whole bacteria, supernatant, and precipitate, respectively; lanes 4-10 are samples eluted with different concentrations of imidazole.

[0030] Figure 4 shows the SDS-PAGE identification results of the carrier protein SC-Ferritin after purification by nickel column affinity chromatography;

[0031] Where M is the protein marker, lanes 1, 2, and 3 are in the order of whole bacteria, supernatant, and precipitate, respectively; lanes 4-11 are samples eluted with different concentrations of imidazole.

[0032] Figure 5 Figure showing the design, assembly, and characterization results of the Fer-ASFV-16E nanoparticle vaccine;

[0033] Wherein: A is a schematic diagram of the structural design of SC-Ferritin carrier protein and ST-Epitope (ASFV-16E) recombinant protein; B is a schematic diagram of the self-assembly principle of nanoparticle vaccine; C is an SDS-PAGE analysis and identification image before and after assembly, with lane 1 being SC-Ferritin, lane 2 being ASFV-16E monomer, and lane 3 being the assembled Fer-ASFV-16E complex protein; D is a morphological observation image of the carrier protein SC-Ferritin under TEM; E is a morphological observation image of the assembled nanoparticle vaccine Fer-ASFV-16E under TEM.

[0034] Figure 6 shows the safety assessment results of Fer-ASFV-16E in pigs; where: A is a schematic diagram of the immunization experiment and sampling time flow in pigs; B is a graph of rectal body temperature monitoring in pigs after immunization; C is a graph of growth and weight gain changes in pigs during immunization; D is a gross anatomical image of the heart, liver, spleen, lungs, kidneys, and tonsils of pigs at the experimental endpoint.

[0035] Figure 7 shows the level of anti-ASFV specific antibodies (OD) in porcine serum at different time points after immunization, detected by ELISA. 450 The trend chart of changes.

[0036] Figure 8 shows the results of detecting the number of IFN-γ secretory cells in pigs using the ELISpot method at week 7 post-immunization.

[0037] Wherein: A is the statistics and dot plot of IFN-γ secretory cells (SFCs) in porcine peripheral blood mononuclear cells (PBMCs); B is the statistics and dot plot of IFN-γ secretory cells (SFCs) in porcine spleen cells.

[0038] Figure 9 shows the flow cytometry analysis results of T cell subset frequencies in pig spleen at week 7 post-immunization (3 weeks post-second immunization).

[0039] Where A is CD3 + CD8 + Flow cytometry quadrant plots and percentage statistics of T cells; B represents CD3. + CD4 + Flow cytometry quadrant plots and percentage statistics of T cells.

[0040] Figure 10 shows the flow cytometry analysis results of T cell subset frequencies in pig spleen at week 11 post-immunization (7 weeks post-second immunization).

[0041] Where A is the flow cytometry quadrant diagram and percentage statistics of CD3+CD8+ T cells; B is the flow cytometry quadrant diagram and percentage statistics of CD3+CD4+ T cells.

[0042] Figure 11 The clinical protective efficacy of the Fer-ASFV-16E nanovaccine after challenge with a virulent strain is shown in the figure.

[0043] Wherein: A is a schematic diagram of the immunization and challenge program for pigs; B is a statistical curve of the survival rate of pigs in each group within 29 days after challenge; C is a curve of rectal temperature monitoring of pigs in each group after challenge.

[0044] Figure 12 shows the viral load and distribution in pigs after challenge with a potent virus.

[0045] Wherein: A is a curve showing the viral nucleic acid load in blood samples at different time points after challenge; B is a curve showing the viral nucleic acid load in anal swab samples at different time points after challenge; C is a curve showing the viral nucleic acid load in oropharyngeal swab samples at different time points after challenge; D is a statistical chart showing the viral nucleic acid load in various organs and tissues (heart, liver, spleen, lung, kidney, tonsils, etc.) at the experimental endpoint or when the pigs die. Detailed Implementation

[0046] To enable those skilled in the art to better understand the technical content of this invention, the invention will be further described below with reference to specific embodiments and appendices. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial sources.

[0047] Example 1:

[0048] Construction of a phage display library covering the entire African swine fever virus (ASFV) proteome and screening of ASFV dominant epitopes:

[0049] 1) We requested the download of ASFV proteome sequences with a host of pig and a sequence identity ≥ 0.9 from the Uniprot database (search date: August 30, 2023), obtaining a representative sequence set containing 1200 proteins. Based on this, we constructed an ASFV whole-proteome phage display library. The phage library was amplified and its titer was determined; the results showed a phage library titer of 5.5 × 10⁻⁶. 9 pfu / ml. Ninety-six phage plaques were randomly selected and subjected to PCR amplification, which showed that all phages inserted the designed ASFV antigen fragment.

[0050] Simultaneously, negative staining was used to further observe the morphology of the packaged bacteriophages using transmission electron microscopy (TEM). 5 μL of sample suspension was dropped onto a copper grid, allowed to stand for several minutes, then excess liquid was removed with filter paper, followed by the addition of uranyl acetate solution for 1 minute. After drying, the sample was observed under an electron microscope (Hitachi Scientific Instruments Co., Ltd., 120Kvvht 7800). Results are as follows... Figure 1 As shown, the phage exhibits the classic T7 phage morphology, indicating that the phage was successfully packaged.

[0051] 2) In this embodiment, the constructed phage display library was used for immunoprecipitation with African swine fever positive serum, and dominant antigenic epitopes were screened through bioinformatics analysis. Based on the significance criterion, 196 dominant candidate peptides were screened, and 75 non-redundant epitopes were obtained through sequence alignment and overlap analysis. The above epitopes were compared with 10 representative ASFV strains (see Table 1), and the 40 epitopes screened were highly conserved among the strains.

[0052] Table 1 Information on 10 representative strains

[0053] .

[0054] Finally, protein function annotation was performed on 40 conserved epitopes, and 16 dominant epitopes located on ASFV structural proteins were selected (Table 2). These 16 epitopes were used as the core immunogenic components for the subsequent construction of ferritin nanoparticle vaccines.

[0055] Example 2:

[0056] Preparation of Fer-ASFV-16E nanoparticle vaccine:

[0057] This embodiment utilizes the dominant antigenic epitopes obtained through screening to develop an African swine fever nanoparticle vaccine using ferritin self-assembly technology. The specific steps are as follows:

[0058] (I) Design of the core antigen component of the African swine fever virus multi-epitope fusion protein

[0059] The 16 epitopes screened in Example 1 were used as antigen components, and their immunoinformatics predictions were performed. The results are shown in Table 2. Six epitopes also contained T cell recognition sites.

[0060] Table 2. B-cell antigenic epitopes of 16 ASFVs screened using Virscan technology.

[0061] .

[0062] Sixteen epitopes were tandemly linked, and an 8His affinity tag was added to the N-terminus to facilitate purification. A SpyTag sequence was added to the C-terminus. Linkers were inserted between the epitopes and the SpyTag sequence. The final recombinant protein antigen ASFV-16E with the HIS tag is shown in SEQ ID NO.1, and the polynucleotide encoding it is shown in SEQ ID NO.2. The recombinant protein antigen without the HIS tag is shown in SEQ ID NO.5. Those skilled in the art can add other protein purification tags to the N-terminus of the protein shown in SEQ ID NO.5 to obtain different recombinant proteins for the preparation of Fer-ASFV-16E nanoparticle vaccines.

[0063] The physicochemical properties and structure of the fusion protein were predicted, and the results are shown in Table 3. The molecular weight of the ASFV-16E recombinant protein is approximately 64.48 kDa, with a water solubility probability of 0.917 and an immunogenicity probability of 0.925. Secondary structure analysis showed that it is predominantly composed of random coils (54.02%).

[0064] Table 3 Physicochemical properties, secondary structure, and predicted functional characteristics of ASFV-16E recombinant protein

[0065] .

[0066] Simultaneously, AlphaFold3 was used to model the three-dimensional structure of the ASFV-16E recombinant protein, and the results are as follows: Figure 2 As shown, the recombinant protein exhibits an extended tandem conformation, with each antigenic epitope being independent and fully exposed in spatial folding, which is beneficial for recognition and presentation by the immune system.

[0067] (II) Expression of African swine fever virus multi-epitope fusion protein

[0068] The polynucleotide sequence of the African swine fever virus multi-epitope fusion protein shown in SEQ ID NO.2 was synthesized and cloned into the pET28a expression vector (Qingke Biotechnology). After verification by sequencing, it was transformed into Escherichia coli BL21(DE3) competent cells.

[0069] The transformed strain was cultured on LB medium and induced at 37°C for 4 h with IPTG at a final concentration of 0.5 mM. Results showed that ASFV-16E protein was highly expressed intracellularly mainly in a soluble form. After extensive expression, the cells were lysed, and the supernatant was loaded onto a nickel column and eluted with different concentrations of imidazole. SDS-PAGE electrophoresis confirmed the expression. Figure 3 As shown, the elution product bands are relatively simple and of high purity. After ultrafiltration concentration, the final yield of ASFV-16E recombinant protein can reach 30 mg per L of fermentation broth. In this invention, ASFV-16E recombinant protein is also referred to as ST-16E fusion protein.

[0070] (III) Construction and Optimization of Expression Vector Protein (sc-fe)

[0071] The sumo lysin was linked with SpyCatcher and ferritin, with a linker inserted between SpyCatcher and ferritin. The recombinant ferritin sequence is shown in SEQ ID NO.3, and the polynucleotide encoding it is shown in SEQ ID NO.4.

[0072] The polynucleotide sequence of recombinant ferritin shown in SEQ ID NO.4 was synthesized and cloned into the pET28a expression vector (Qingke Biotechnology). After verification by sequencing, it was transformed into E. coli BL21(DE3) competent cells.

[0073] The transformed strain was cultured on LB medium and induced at 37°C for 4 h with IPTG at a final concentration of 0.5 mM. Results showed that the Sc-Fe recombinant protein was highly expressed intracellularly mainly in a soluble form. After extensive expression, the cells were lysed, and the supernatant was loaded onto a nickel column and eluted with different concentrations of imidazole. SDS-PAGE electrophoresis confirmed the expression. Figure 4 As shown, the elution product bands are relatively simple and of high purity. After ultrafiltration concentration, 30 mg of sc-fe recombinant protein can be obtained from 1 L of fermentation broth. In this invention, sc-fe recombinant protein is also referred to as SC-Ferritin carrier protein.

[0074] (IV) Assembly and Characterization of Fer-ASFV-16E Nanoparticle Vaccine Nanoparticle Vaccine Assembly

[0075] Utilizing the spontaneous covalent coupling property of the SpyTag-SpyCatcher system ( Figure 5 In step A and step B), the purified ST-16E fusion protein and SC-Ferritin carrier protein were mixed in a buffer system at a molar ratio of 1:24, so that the antigenic epitope was stably displayed on the ferritin surface through heteropeptide bonds.

[0076] SDS-PAGE analysis was performed on protein samples before and after assembly to verify the coupling effect. Figure 5 As shown in Figure C, lane 1 contains the purified carrier protein SC-Ferritin, with a distinct band at approximately 35 kDa; lane 2 contains the ASFV-16E fusion protein monomer, with a specific band at approximately 65 kDa, consistent with the expected molecular weight of 64.48 kDa; lane 3 contains the product Fer-ASFV-16E after proportional mixing and coupling. The results show a new high-molecular-weight band at approximately 100 kDa. This increase in molecular weight (approximately 35 kDa + 65 kDa) is completely consistent with the expected value after covalent binding of the two components, demonstrating that the Fer-ASFV-16E protein has been successfully covalently coupled to the ferritin carrier surface using the SpyTag / SpyCatcher system.

[0077] The morphology of the SC-Ferritin carrier and the Fer-ASFV-16E nanovaccine was characterized using transmission electron microscopy (TEM), as shown in Figures D and E in Figure 5. Observation revealed that both the SC-Ferritin carrier and Fer-ASFV-16E exhibited a highly uniform spherical cage-like structure, with excellent monodispersity and uniform spatial distribution without significant aggregation. Comparative analysis showed that the particle size of the Fer-ASFV-16E nanoparticles loaded with multi-epitope antigens was significantly shifted compared to the empty carrier, with a marked increase in outer diameter. This morphological change, at the ultrastructural level, confirms that the dominant ASFV epitopes have been successfully loaded and displayed on the surface of the ferritin nanoscaffold, demonstrating that this invention successfully constructed the physically stable African swine fever multi-epitope nanoparticle vaccine Fer-ASFV-16E.

[0078] Example 3:

[0079] Safety and immunogenicity evaluation of the African swine fever multi-epitope nanoparticle vaccine Fer-ASFV-16E in pigs

[0080] The inventors conducted further animal immunization experiments on the Fer-ASFV-16E nanoparticle vaccine prepared in Example 2. The relevant experimental process and results are briefly described below.

[0081] (I) Experimental Design and Safety Assessment: Animal Grouping and Immunization Procedures

[0082] Twelve healthy piglets aged 28±2 days were purchased from Wuhan Zhuletianyuan Ecological Agriculture Co., Ltd. All piglets were clinically healthy before the experiment, with body temperatures ranging from 38.0-39.5℃, normal feed intake, and formed feces. qPCR tests for classical swine fever virus, porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 were all negative. No antibiotics or vaccines were used in the seven days prior to and during the experiment.

[0083] The piglets were randomly divided into three groups: the Fer-ASFV-16E nanovaccine group, the ASFV-16E monomeric protein group, and the PBS control group, with four piglets in each group. Each piglet was injected with 1 ml of vaccine. The ISA 206 mineral oil adjuvant (Seppic) in the vaccine was mixed with the antigen at a volume ratio of 1:1. The single immunization dose of the protein antigen was 200 μg. All piglets were housed in a clean-grade animal room at a temperature of 24±2℃ and fed three times a day.

[0084] Immunization procedures such as Figure 6 As shown in Figure A, the first immunization (1st dose) was administered at 0 weeks, and a booster immunization was administered at 4 weeks, with continued observation until 11 weeks.

[0085] Following immunization (Day Post Vaccination, DPV), the pigs' body temperature was recorded daily, and their weight was recorded every three days. The experimental results are as follows: Figure 6 As shown in Figure B, after immunization, the rectal temperature of the three experimental groups of pigs remained within the normal range of 38.5-39.5°C, and no abnormal fever was observed.

[0086] The growth and weight gain curve of pigs is as follows Figure 6 The results showed no significant difference between the experimental group and the control group, and both groups were in good growth condition.

[0087] At the end of the experiment, gross necropsy was performed on the heart, liver, spleen, lungs, kidneys, and tonsils (tonsil) of the pigs. The results are as follows: Figure 6 The results showed that all the solid organs of the pigs in the Fer-ASFV-16E group were morphologically normal, and no pathological damage was found.

[0088] The above results demonstrate that the Fer-ASFV-16E nanoparticle vaccine has excellent biocompatibility.

[0089] (II) Assessment of vaccine-induced humoral immune response and detection of specific antibody levels

[0090] Serum samples were collected from pigs at 0, 2, 4, and 6 weeks of gestation. The level of anti-ASFV specific antibodies in the serum was dynamically monitored using ELISA. 450(Value). Result as follows Figure 7 As shown, antibody levels in each group rose slowly after the initial immunization; after the booster immunization, antibody levels in the Fer-ASFV-16E group rose rapidly and were higher than those in the ASFV-16E group.

[0091] Experiments showed that the level of specific antibodies induced by the nanovaccine group was significantly higher than that of the monomer group, demonstrating that the ferritin nanocarrier significantly enhanced the humoral immune induction ability of the antigen.

[0092] (III) Assessment of vaccine-induced cellular immune response

[0093] Three weeks after the second immunization (week 7), the number of IFN-γ secretory cells (SFCs) in porcine peripheral blood mononuclear cells (PBMCs) and splenocytes was detected using the ELISpot method.

[0094] The results are as follows Figure 8 As shown, the Fer-ASFV-16E group induced a significant increase in the level of IFN-γ secreting cells (p < 0.05), demonstrating that nanovaccines can induce a more potent cellular immune response.

[0095] (iv) Flow cytometry analysis of T cell subset frequencies at different time points

[0096] Pig spleens were collected 3 weeks (w7) and 7 weeks (w11) after the second immunization, and changes in CD3+CD4+ and CD3+CD8+ T cell subsets were analyzed by flow cytometry.

[0097] CD8+ subgroup analysis results are as follows Figure 9 China A Figure 10 As shown in Figure A, the results indicated that at week 7, Fer-ASFV-16E significantly promoted the proliferation of CD3+CD8+ effector T cells (p < 0.05). At week 11, the frequency of CD8+ positive cells remained higher than that of the control group.

[0098] CD4+ subgroup analysis results are as follows Figure 9 B, Figure 10 As shown in Figure B, the proportion of CD3+CD4+ helper T cells in the nano-vaccine group showed a significant upward trend at 3 and 7 weeks after the second immunization (p < 0.05), indicating that the vaccine can induce a durable and potent helper T cell response.

[0099] Example 4:

[0100] Evaluation of protective efficacy against viral challenge in pigs

[0101] This embodiment uses a highly pathogenic ASFV strain to challenge immunized pigs, aiming to evaluate the clinical protective effect of the Fer-ASFV-16E nanoparticle vaccine of the present invention against lethal challenge and its inhibitory effect on viral replication.

[0102] (I) Experimental Design and Virus Challenge Procedure: Virus Strain and Dosage

[0103] Fourteen healthy piglets, aged 28±2 days, were purchased from Wuhan Zhuletianyuan Ecological Agriculture Co., Ltd. Prior to the experiment, all piglets were clinically healthy, with body temperatures ranging from 38.0-39.5℃, normal feed intake, and formed feces. qPCR tests for classical swine fever virus, porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 were all negative. No antibiotics or vaccines were used in the seven days prior to and during the experiment.

[0104] Piglets were randomly divided into three groups: the Fer-ASFV-16E nanovaccine group (n=4), the ASFV-16E monomeric protein group (n=5), and the PBS control group (n=5). Immunization was administered via intramuscular injection, with each piglet receiving 1 ml of vaccine. The vaccine contained ISA 206 mineral oil adjuvant (Seppic) mixed with the antigen at a 1:1 volume ratio. The single immunization dose of the protein antigen was 200 μg. All piglets were housed in a P3 high-level animal facility at a temperature of 24±2℃ and fed three times daily.

[0105] The highly pathogenic ASFV genotype II SNJ strain (GenBank: OL622042.1) was selected, and the challenge dose was 1 TCID50. The immunization and challenge procedures are as follows: Figure 11 As shown in Figure A, the first and second immunizations were administered intramuscularly at weeks 0 and 4, respectively; and an intramuscular injection was given at week 7 for viral challenge.

[0106] Experimental pigs were divided into three groups: the Fer-ASFV-16E nanovaccine group (n=4), the ASFV-16E monomeric protein group, and the PBS negative control group (n=5). Observation indicators: The pigs were observed continuously for 29 days after challenge. Rectal body temperature and survival status were monitored and recorded daily, and nucleic acid load was measured in anal and oropharyngeal swabs every 3 days. At the end of the experiment or when the pigs were near death, tissue samples were collected for viral load testing.

[0107] (II) Analysis of the Delay in Clinical Onset and Mortality: Body temperature monitoring results are shown in Figure 11C. Most pigs in the PBS and monoclonal vaccine groups began to show significant fever symptoms (body temperature > 40°C) on days 5-8 post-challenge (dpi 5-8). However, the onset of fever in the Fer-ASFV-16E nanovaccine group was significantly delayed, with the onset time concentrated at dpi 12-14, approximately 3-6 days later than the control group. Survival results are shown in Figure 11B. Pigs in the PBS and monoclonal vaccine groups began to die 10 days post-challenge, with a survival rate of 0%. The mortality time in the Fer-ASFV-16E nanovaccine group was delayed by 4-6 days compared to the control group, and at the end of the observation period (dpi 29), 25% of the pigs in this group were still alive, demonstrating a clear clinical protective effect.

[0108] (III) Evaluation of viral load inhibition by qPCR: Blood viral load and oropharyngeal and anal swabs from pigs were tested every 3 days. Results are as follows: Figure 12 As shown in the AC results, viremia in the Fer-ASFV-16E nanovaccine group was delayed by 6 days, and viral load in oropharyngeal and anal swabs was delayed by 3 days. Heart, liver, spleen, lung, kidney, and tonsil tissues were collected from pigs at death or at the end of the experiment, and ASFV genome copy number was detected using qPCR technology. Results analysis: The results are as follows... Figure 12 As shown in Figure D, compared with the PBS group and the monomeric protein group, the viral nucleic acid load in all tested organs and tissues of pigs in the Fer-ASFV-16E nanovaccine group showed a decreasing trend. This demonstrates that the nanovaccine assembly system has the effect of inhibiting viral replication and dissemination in vivo.

[0109] In summary, the Fer-ASFV-16E nanoparticle vaccine maintains an immune advantage in pigs for up to 11 weeks, significantly enhancing overall immunity against the ASFV core epitope. Furthermore, when facing highly pathogenic ASFV strains, this vaccine demonstrates clear clinical protective efficacy, significantly delaying fever, viremia, and viral shedding in pigs, effectively prolonging the disease progression and significantly inhibiting viral replication and dissemination in various organs and tissues. This proves that a conserved multi-epitope combination nanoassembly system based on innate immune screening is a reliable pathway for developing novel and highly effective vaccines against African swine fever.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A synthetically produced multi-epitope fusion protein of African swine fever virus, the fusion protein being shown in SEQ ID NO.

5.

2. The recombinant protein obtained by fusing the fusion protein of claim 1 with a protein purification tag.

3. The recombinant protein according to claim 2, wherein the amino acid sequence is shown in SEQ ID NO.

1.

4. The gene encoding the fusion protein of claim 1 or the recombinant protein of claim 2.

5. An expression cassette, recombinant vector, recombinant microorganism, or ex vivo recombinant cell having the gene encoding as described in claim 4.

6. The use of the fusion protein of claim 1, the recombinant protein of claim 2, the encoding gene of claim 4 or an expression cassette having the encoding gene of claim 4, a recombinant vector, a recombinant microorganism or an ex vivo recombinant cell in the preparation of nanoparticle proteins.

7. Ferritin nanoparticles of African swine fever virus multi-epitope fusion protein, wherein the ferritin nanoparticles are obtained by mixing and assembling the protein shown in SEQ ID NO.1 and the protein shown in SEQ ID NO.

3.

8. The use of the ferritin nanoparticles according to claim 7 in the preparation of a drug for preventing African swine fever virus infection.

9. The use of the ferritin nanoparticles according to claim 7 in the preparation of African swine fever vaccine.

10. An African swine fever vaccine, wherein the African swine fever vaccine contains the ferritin nanoparticles as described in claim 7.

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