African swine fever mRNA vaccine composition capable of synergistically activating and enhancing humoral immunity and cellular immunity and application thereof

Abstract

The invention discloses an African swine fever mRNA vaccine composition capable of synergistically activating and enhancing humoral immunity and cellular immunity and application of the African swine fever mRNA vaccine composition, and belongs to the technical field of biological medicines. Specifically, a plurality of key antigen proteins of the African swine fever virus are used as target spots and are connected in series through connexons to form single-chain mRNA, so that multiple immunogens can be translated from the same transcript, and multiple antibodies are induced to be generated at the same time; a conservative recombinant T cell immunogen capable of activating cellular immune response is introduced, so that the cellular immune response can be enhanced, the ADE risk can be reduced, and the cross protection capability can also be improved; in addition, a molecular adjuvant IL-12 is added into the mRNA vaccine to enhance the immune response of the African swine fever vaccine, so that stronger immune protection efficacy is induced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an African swine fever mRNA vaccine combination that can synergistically activate and enhance humoral immunity and cellular immunity and its application. Background Technology

[0002] African swine fever virus (ASFV) is a large, linear double-stranded DNA virus with a genome of approximately 170-194 kb, encoding over 150 open reading frames (ORFs). It has a complex hierarchical structure and can cause severe hemorrhagic fever in both domestic and wild pigs, with an acute mortality rate approaching 100%. ASFV can be transmitted through multiple routes, including infected pigs and their secretions, contaminated equipment / feed, and pork products. It is difficult to inactivate in contaminated meat products, easily leading to long-distance spread and sustained regional epidemics. The numerous ASFV genotypes, significant differences in the lineage of circulating strains, and the potential for large-scale genomic variations such as deletions and recombinations pose ongoing challenges to the broad-spectrum and rapid development of vaccines.

[0003] Currently, vaccine development routes for ASFV mainly include live attenuated vaccines, inactivated vaccines, subunit vaccines, viral vector vaccines, DNA vaccines, and mRNA vaccines. Although all of these routes have been researched and attempted, they still face problems such as unstable immune protection, difficulty in balancing safety and efficacy, limited cross-protection against heterologous strains, and insufficient industrial consistency. They are still unable to meet the comprehensive requirements of "safety, effectiveness, scalability, and rapid iteration" for long-term prevention and control of major animal diseases.

[0004] The limitations of live attenuated vaccines lie in the following: they typically rely on deleting specific virulence-related genes to reduce pathogenicity. Due to the complex immune escape mechanism of ASFV, deletion of a single or limited gene does not necessarily guarantee adequate safety; while deletion of multiple genes may lead to decreased immunogenicity, resulting in insufficient or unstable protection. Furthermore, attenuated strains may still pose a risk of genetic instability during continuous passage or in vivo replication, leading to a resurgence of virulence or residual pathogenicity, thus creating pressure on biosafety and regulatory assessments. Live attenuated vaccines are somewhat dependent on the strain background; the attenuation and protective effects between different genotypes / isolated strains may vary significantly, further limiting their universal application in strains circulating in different regions.

[0005] The limitations of inactivated and subunit vaccines are as follows: inactivated vaccines often fail to induce sufficient protective immune responses, especially in cellular immunity, making it difficult to provide stable protection upon challenge. While subunit vaccines offer safety advantages, their immunogen selection and combination are highly dependent on the accurate recognition of protective antigens / epitopes; in the context of ASFV diversity, single or a few structural protein antigens often fail to achieve stable broad-spectrum protection, and cross-protection between different strains is limited.

[0006] The limitations of viral vector vaccines and DNA vaccines lie in their ability to simultaneously induce humoral and cellular immunity, making them a key direction for ASFV vaccines. Our team's previously filed patent ("African Swine Fever Virus Immunogen Composition and Its Application") discloses an African swine fever virus immunogen composition and its application. The vaccine described can be a viral vector vaccine (e.g., recombinant vaccinia virus rTV vaccine) or a nucleic acid vaccine. This approach, based on immunological mechanisms, emphasizes the simultaneous activation of humoral and T-cell immune responses, possessing significant technical and application value. However, while existing viral vector vaccines and DNA vaccines can induce effective immune responses, they still have the following shortcomings: First, ASFV protection exhibits significant strain / region specificity. Faced with dominant circulating strains in different regions, genotypic differences, and recombination / deletion variations, vector vaccines or DNA vaccines have a relatively long iterative cycle of "antigen matching construction, validation, and scale-up," making it difficult to form a rapid response loop against emerging circulating strains. Second, while multi-antigen loading can improve immune coverage, the increased number of exogenous genes may place higher demands on vector construction efficiency, genetic stability, expression balance, and subsequent production consistency. Third, vector vaccines are affected by factors such as vector-related immune background and repeated vaccination strategy design, and their large-scale production and quality consistency control require stricter regulatory requirements. Therefore, given the frequent macro-genomic variations of ASFV, rapid changes in regional circulating strains, and the need for rapid customization and updates, vector vaccines still have room for further improvement.

[0007] ASFV can suppress the host's innate and adaptive immune responses through multiple targets and pathways, meaning that simply pursuing high antibody titers does not necessarily equate to safe and effective protection. Furthermore, recent studies have shown that antibodies targeting certain viral proteins (such as pA137R) can induce antibody-dependent enhancement (ADE), promoting viral entry into immune cells via Fcγ receptors and leading to disease exacerbation. These issues place higher demands on antigen selection, the balance of immune response types, and vaccine safety design. Therefore, how to stably induce cellular immune responses that reduce ADE and are more crucial for combating viremia while ensuring safety, thereby achieving scalable multi-antigen combinations and rapid updates, remains a long-standing technical challenge in this field.

[0008] In summary, existing vaccine technologies generally still struggle to simultaneously meet the following requirements: (i) rapid updating capability against different genotypes, recombinant, or deletion variants; (ii) balanced expression and formulation flexibility under multi-antigen combinations; (iii) stable induction of effective T-cell immune responses while avoiding risks associated with live virus replication; and (iv) process feasibility suitable for large-scale production with good batch-to-batch consistency. Therefore, there is an urgent need in this field for a new vaccine technology that can complete antigen sequence updates and combination optimization in a shorter timeframe, balancing safety and immunogenicity, and adapting to the control challenges posed by the regional prevalence of ASFV and large-scale genomic variations.

[0009] Based on the aforementioned needs, mRNA vaccine technology, with its advantages of not requiring viral replication, flexible sequence design and combination, rapid iterative updates, and suitability for standardized preparation processes with lipid delivery systems, offers a new technical approach to solving the challenges of ASFV vaccines in areas such as rapid updates, multi-antigen combinations, cellular immune induction, and industrial consistency. This invention aims to address the shortcomings of the aforementioned existing technologies by proposing an improved mRNA vaccine technology solution to solve at least some of the above-mentioned technical problems, providing a safe, effective, and commercially viable new strategy for the long-term prevention and control of African swine fever. Summary of the Invention

[0010] The purpose of this invention is to address the technical challenges of insufficient safety of existing live attenuated vaccines, the vector effect of viral vector vaccines making it difficult to induce a balanced immune response, and the tendency of single antigens to induce ADE (antibody-deprivation) effects. This invention aims to provide an African swine fever (ASFV) mRNA vaccine combination and its application. Through an mRNA engineering platform, this invention constructs a novel nucleic acid vaccine that can synergistically activate high-titer neutralizing antibodies and broad-spectrum T-cell immunity, aiming to achieve effective protection against different genotypes of ASFV strains.

[0011] To achieve the above objectives, this application adopts the following technical solution:

[0012] In a first aspect, the present invention provides a nucleic acid composition for inducing an immune response to African swine fever virus (ASFV), said nucleic acid composition comprising a combination of 1) and 2) or a combination of 1) and 2) and 3): 1) At least one mRNA encoding the ASFV immunogen; 2) At least one mRNA encoding a recombinant T-cell antigen; 3) At least one mRNA encoding an immune regulatory molecule; The mRNA is either encapsulated in a delivery system or forms a complex with the delivery system to achieve in vivo delivery.

[0013] The mRNA encoding the ASFV immunogen includes encoding at least one structural protein that primarily induces a humoral immune response. The structural protein comprises at least one or more proteins or fragments selected from the following: p54 / pE183L, P30 / CP204L, p72 / B646L, p49 / B438L, CD2v / pEP402R, p17 / D117L, p72 chaperone / B602L, H240R; or one or more homologous proteins thereof, wherein the homologous proteins share at least 75% identity with the aforementioned proteins and have the same or substantially the same immunogenic amino acid sequence or antigenic fragment thereof. The immunogen of the recombinant T-cell antigen is derived from one or more non-structural proteins of African swine fever virus, which mainly induce cellular immune responses. Preferably, it consists of: F334L, H359L, D250R, D339L, F1055L, G1211R, A528, and MGF-505-7R proteins; or one or more of their homologous proteins, wherein the homologous proteins have at least 75% identity with the above proteins and have the same or substantially the same amino acid sequence or antigenic fragments of the same or substantially the same immunogenicity.

[0014] In some embodiments, the immunogenic proteins p54 / pE183L, p30 / CP204L, p72 / B646L, p49 / B438L, CD2v / pEP402R, p17 / D117L, p72 chaperone / B602L, and Penton / H240R that induce humoral immunity comprise their full-length or a portion thereof amino acid sequences, or amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to and have the same or substantially the same immunogenicity as those sequences, and are further preferably amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively.

[0015] In some embodiments, the immunogen that induces cellular immunity is a recombinant conserved T-cell antigen constructed by analyzing a non-structural protein of African swine fever virus (ASFV) using immunological and bioinformatics methods, and then analyzing the T-cell consensus sequence recognized by the porcine major histocompatibility complex (SLA) molecule, such as the recombinant T-antigen amino acid sequence shown in SEQ ID NO:12.

[0016] Furthermore, to achieve the independent and correct expression of immunogens or structural proteins that induce humoral immunity and cellular immunity respectively, they are combined in different arrangements. Optionally, immunogens that induce humoral immunity and / or immunogens that induce cellular immunity are linked in different linker configurations to construct polycistronic mRNAs, thereby achieving correct expression and translation on the same transcript. Optionally, the linkers have different amino acid lengths, including sequences of 1-28 amino acids. The linker sequences of the further linkers are derived from glycine and serine to provide flexibility, glutamic acid and lysine to improve water solubility, and / or rigid linker peptides in helical form that can effectively separate the fusion proteins, such as EGKSSGSGSESKST, GSAGSAAGSGEF, KESGSVSSEQLAQFRSLD, (GGGS)n, (EAAAK)n sequences, or sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them and have the same or substantially the same amino acid sequence; wherein n = 0, 1, 2, 3, 4, or 5; or derived from 2A self-cleaving peptide sequences, including but not limited to F2A derived from foot-and-mouth disease virus, E2A derived from Equinerhinitis A virus, and Porcine swine virus. The P2A peptide of teschovirus, the T2A peptide derived from the thin-spotted moth virus (Thosea asigna virus), or peptides that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it and have the same or substantially the same amino acid sequence. Furthermore, the immunogens that induce humoral immunity include CD2v / pEP402R, p54 / pE183L, and P30 / CP204L, which are linked by linkers. The linkers are preferably sequences derived from Porcine teschovirus P2A, such as the amino acid sequence ATNFSLLKQAGDVEENPGP shown in SEQ ID NO:13; or preferably sequences derived from Thosea asigna virus T2A, such as the amino acid sequence ERGSLLTCGDVEENPGP shown in SEQ ID NO:14. The two different linker sequences can be either first or last. The final reconstituted fusion protein, as candidate immunogen 1 (abbreviated as CD2v-p54-p30), has the amino acid sequence shown in SEQ ID NO:15. Furthermore, the immunogens inducing humoral immunity include p49 / B438L, p72 / B646L, Penton / H240R, and p72chaperone / B602L, each linked by a linker derived from the P2A sequence of porcine teschovirus. The order of the different immunogens can be before or after the linker, ultimately recombinating into four different fusion proteins, as shown in the amino acid sequences of candidate immunogen 2 (p49-B602L) as shown in SEQ ID NO:16; candidate immunogen 3 (p49-Penton) as shown in SEQ ID NO:17; candidate immunogen 4 (p72-Penton) as shown in SEQ ID NO:18; and candidate immunogen 5 (p72-B602L) as shown in SEQ ID NO:19. In a further preferred embodiment, the p17 / protein encoding D117L located on the inner membrane of ASFV is linked to a conserved T-cell antigen in the order from N-terminus to C-terminus, and linked by a linker derived from the sequence of Porcine teschovirus P2A. The order of different immunogens may be before or after the linker, and finally reassembled into the amino acid sequence of candidate immunogen 6 (abbreviated as p17-T) as shown in SEQ ID NO:20.

[0017] In some embodiments, the mRNA encoding the ASFV immunogen encodes any of the following candidate immunogens: 1) Candidate immunogen 1: CD2v-p54-p30, a fusion protein formed by linking CD2v / pEP402R, p54 / pE183L and P30 / CP204L through a linker, has the amino acid sequence shown in SEQ ID NO:15; 2) Candidate immunogen 2: P49-B602L, a fusion protein formed by linking p49 / B438L and B602L through a linker, has the amino acid sequence shown in SEQ ID NO:16; 3) Candidate immunogen 3: P49-Penton, a fusion protein formed by linking p49 / B438L and Penton / H240R through a linker, has the amino acid sequence shown in SEQ ID NO:17; 4) Candidate immunogen 4: p72-Penton, a fusion protein formed by linking p72 / B646L and Penton / H240R via a linker, has the amino acid sequence shown in SEQ ID NO:18; 5) Candidate immunogen 5: P72-B602L, a fusion protein formed by linking p72 / B646L and B602L via a linker, whose amino acid sequence is shown in SEQ ID NO:19; In some embodiments, the mRNA encoding the recombinant T-cell antigen encodes a fusion protein p17-T formed by linking the recombinant T-cell antigen that induces a cellular immune response with p17 / D117L, the amino acid sequence of which is shown in SEQ ID NO:20.

[0018] In some embodiments, the mRNA encoding the ASFV immunogen comprises any of the following codon-optimized coding sequences: 1) SEQ ID NO:21; 2) SEQ ID NO:22; 3) SEQ ID NO:23; 4) SEQ ID NO:24; 5) SEQ ID NO:25; In some embodiments, the mRNA encoding the recombinant T-cell antigen comprises a codon-optimized coding sequence SEQ ID NO:26.

[0019] In some embodiments, the at least one mRNA encoding the ASFV immunogen is a single-stranded mRNA, and its open reading frame encodes a multi-antigen fusion protein; the arrangement order, linkage mode, linker peptide type and length of each antigen fragment in the fusion protein are not limited.

[0020] In some embodiments, the nucleic acid composition further includes: at least one mRNA encoding an immunomodulatory molecule; the immunomodulatory molecule includes, but is not limited to, one or more of aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotides, manganese ion adjuvant, colloidal manganese adjuvant, Freund's adjuvant, MF59, QS-21, Poly I:C and other TLR ligands, and cytokines; the cytokines include, but are not limited to, one or more of GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21, and IFN-α.

[0021] In some embodiments, the immunomodulatory molecule is the cytokine IL-12.

[0022] The immunomodulatory molecule IL-12, which enhances the immune response, is composed of two different subunits: IL-12A (IL-12 p35) and IL-12B (IL-12 p40). To promote the synergistic effect between IL-12A (IL-12 p35) and IL-12B (IL-12 p40), they are linked by a linker, preferably with the amino acid sequence SGGGGSGGGGSGGGGSPVSVPTASPSASSSSSQCRSS as shown in SEQ ID NO: 1. The two different subunits can be placed before or after the linker. Furthermore, the IL-12 is mouse-derived IL-12 and / or porcine-derived IL-12. The final reconstituted adjuvant molecule has the amino acid sequence shown in SEQ ID NO: 2 for mouse IL-12 and the amino acid sequence shown in SEQ ID NO: 3 for porcine IL-12. In some embodiments, the IL-12 is mouse-derived IL-12 and / or porcine-derived IL-12; the mRNA encoding IL-12 comprises a codon-optimized nucleic acid sequence encoding IL-12, the nucleic acid sequence being: 1) The coding sequence of mouse IL-12: as shown in SEQ ID NO:27; and / or 2) The coding sequence of porcine IL-12: as shown in SEQ ID NO:28; The IL-12 has an immune adjuvant function that promotes Th1 immune responses and / or enhances overall immune responses.

[0023] In some specific embodiments, the mRNA encoding the ASFV immunogen encodes any of the following codon-optimized immunogens: i) Encoding codon-optimized candidate immunogen 1, the codon-optimized nucleic acid sequence is shown in SEQ ID NO:21; ii) The candidate immunogen 2 with optimized codons is shown in SEQ ID NO:22; iii) The candidate immunogen 3 with optimized codons is shown in SEQ ID NO:23; iv) The candidate immunogen 4 with optimized codons is shown in SEQ ID NO:24; v) Encoding codon-optimized candidate immunogen 5, the codon-optimized nucleic acid sequence is shown in SEQ ID NO:25; In some embodiments, the mRNA encoding the recombinant T-cell immunogen encodes a codon-optimized p17-T, the codon-optimized nucleic acid sequence of which is shown in SEQ ID NO:26.

[0024] In some other specific embodiments, the nucleic acid composition further includes at least one mRNA encoding an immunomodulatory molecule, which encodes a codon-optimized adjuvant molecule IL-12, preferably, which encodes codon-optimized mouse IL-12 and / or porcine IL-12, the codon-optimized nucleic acid sequence being shown in SEQ ID NO:27; the codon-optimized porcine IL-12 nucleic acid sequence being shown in SEQ ID NO:28.

[0025] In some embodiments, the immunogenic mRNA and the immunomodulatory molecule mRNA are administered at doses of equal mass or equal molar mass; or the dose is adjusted according to immunogenicity, expression level, and / or immunoprotective effect.

[0026] In some embodiments, the delivery system is a carrier system capable of delivering mRNA into animal cells, including but not limited to liposomes, lipid nanoparticles, polymer nanoparticles, cationic polymer complexes, inorganic nanocarriers, emulsions, or combinations thereof.

[0027] Furthermore, the nucleic acid composition is loaded into an mRNA vector to prepare a nucleic acid vaccine; wherein the mRNA vector includes, but is not limited to, linear, circular, and self-replicating vectors.

[0028] In some embodiments, the administration method of the nucleic acid composition satisfies any one of the following: A) The mRNAs in the combination are individually packaged in a delivery system and administered separately; or B) The mRNAs in the combination are individually encapsulated in the delivery system and then mixed and administered; or C) The mRNAs in the combination are formulated separately and then administered in combination; or D) The mRNAs in the combination are co-encapsulated in the same delivery system before administration.

[0029] In some embodiments, the mRNA is unmodified mRNA or nucleoside-modified mRNA. The arrangement of its 5' cap, 5' / 3' untranslated region, and poly(A) tail is not limited to any specific sequence, type, or length range.

[0030] In embodiments of the present invention, the mRNA vector includes the basic elements required for in vitro transcription from the 5' end to the 3' end, specifically the T7 promoter, the 5' untranslated region (UTR), the coding sequence (CDS), the 3' untranslated region (UTR), and the polyadenylated tail (poly A) sequence; all four basic elements must be included. The 5'-UTR sequence, the 3'-UTR sequence, and the polyadenylated tail (poly A) sequence are in the optimal combination as described in Chinese Patent Application No. 202211129466.2. The coding sequence includes, but is not limited to, an immunogen containing multiple protective epitopes of African swine fever virus that can induce humoral immune responses and a conserved T-cell immunogen composition that induces cellular immune responses.

[0031] Secondly, the present invention provides an immunogenic peptide derived from African swine fever virus (ASFV), said immunogenic peptide being selected from any one or more combinations of the following 1)-6): 1) A fusion protein CD2v-p54-p30 or a variant thereof formed by linking CD2v / pEP402R, p54 / pE183L and p30 / CP204L via a linker, wherein the amino acid sequence of CD2v-p54-p30 is as shown in SEQ ID NO:15, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:15, and retaining the function of the fusion protein as shown in SEQ ID NO:15; 2) A fusion protein P49-B602L formed by linking p49 / B438L and B602L via a linker, wherein the amino acid sequence of P49-B602L is as shown in SEQ ID NO:16, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:16, and retaining the function of the fusion protein as shown in SEQ ID NO:16; 3) A fusion protein P49-Penton formed by linking p49 / B438L and Penton / H240R via a linker, wherein the amino acid sequence of P49-Penton is as shown in SEQ ID NO:17, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:17, and retaining the function of the fusion protein as shown in SEQ ID NO:17; 4) A fusion protein P72-Penton formed by linking p72 / B646L and Penton / H240R via a linker, wherein the amino acid sequence of P72-Penton is as shown in SEQ ID NO:18, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:18, and retaining the function of the fusion protein as shown in SEQ ID NO:18; 5) A fusion protein P72-B602L formed by linking p72 / B646L and B602L via a linker, wherein the amino acid sequence of P72-B602L is as shown in SEQ ID NO:19, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:19, and retaining the function of the fusion protein as shown in SEQ ID NO:19; 6) A fusion protein p17-T formed by linking recombinant conserved T-cell antigen with p17 / D117L via a linker, wherein the amino acid sequence of p17-T is as shown in SEQ ID NO:20, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:20, and retaining the function of the fusion protein as shown in SEQ ID NO:20.

[0032] Thirdly, the present invention provides the use of the nucleic acid composition as described in the first aspect and / or the immunogenic peptide as described in the second aspect in the preparation of a medicament for the prevention and / or treatment of African swine fever virus infection. The nucleic acid composition may be used alone or in combination with antibodies against African swine fever virus and / or antiviral drugs. Combination administration is preferred.

[0033] Preferably, the drug is a vaccine preparation, and its administration methods include, but are not limited to, intramuscular injection, nasal administration, nebulization, microneedle injection, subcutaneous injection, intradermal injection, and intramuscular injection, with intramuscular injection and subcutaneous injection being preferred, and intramuscular injection being more preferred in terms of ease of administration.

[0034] Compared with the prior art, the present invention has the following beneficial effects: This invention targets multiple key antigenic proteins of African swine fever virus (ASFV) to screen for multiple candidate immunogens, which are then linked together to form single-stranded polycistronic mRNAs. This allows for the translation of multiple antigens from a single transcript, simultaneously inducing the production of multiple antibodies. Compared to simply mixing multiple vaccines with each mRNA encoding a single immunogen, this method offers advantages such as lower production costs, simpler production processes, and rapid preparation. Furthermore, the addition of IL-12 as a molecular adjuvant to the mRNA vaccine enhances the immune response, thereby inducing stronger immunoprotective efficacy. In addition, the invention introduces a conserved recombinant T antigen that activates cellular immune responses, which not only enhances cellular immune responses and reduces the risk of adverse drug reactions (ADEs) but also improves cross-protective capabilities. Therefore, the ASFV mRNA vaccine strategy provided by this invention can be rapidly developed for different genotypes and serotypes of ASFV strains, offering an effective response to the ASFV epidemic. Attached Figure Description

[0035] Figure 1 Schematic diagrams illustrating the construction of different multivalent immunogens; Figure 2 HEK293T cells were transfected with p17-T, CD2v-p54-p30, p49-B602L, p49-Penton, p72-Penton, and p72-B602L mRNAs, and their expression was detected by flow cytometry. Figure 3 HEK293T cells were transfected with p17-T, CD2v-p54-p30, p49-B602L, p49-Penton, p72-Penton, and p72-B602L mRNA, and their expression was detected by Western blot. A is a Western blot image of the p17 protein; B is a Western blot image of the T antigen; C represents the Western blot image of the CD2v protein, its extracellular segment, the incompletely disconnected CD2v-p54 and the full-length CD2v-p5-p30 fusion protein; D is a Western blot image of the p54 protein; E represents the Western blot image of p30 protein; F shows the Western blot images of p49 protein, the incompletely disconnected p49-B602L fusion protein, and the p49-Penton fusion protein. G is a Western blot image of B602L and the incompletely disconnected p49-B602L fusion protein; H represents the Western blot bands of Penton protein and the unbroken p49-Penton fusion protein. I is a Western blot image of Penton protein; J represents the Western blot images of p72 and B602L proteins; Figure 4 Immunogenicity of Group 1 (p17-T, CD2v-p54-p30, p49-B602L, p72-Penton) and Group 2 (p17-T, CD2v-p54-p30, p49-Penton, p72-B602L) mRNA vaccine combinations in BALB / c mice was evaluated. The mice used in the experiment were 6-8 week old female BALB / c mice. The immunogen was six mRNA vaccines, which were administered in two doses at three-dose intervals in the form of Group 1 and Group 2. Blood samples were collected one week after the booster dose, and the titers of specific binding antibodies against different ASFV antigens in mouse serum were detected by ELISA. ns: indicates no significant difference. express p <0.01; express p <0.0001.

[0036] Figure 5 Six- to eight-week-old female BALB / c mice were vaccinated with a combination of Group 1 (p17-T, CD2v-p54-p30, p49-B602L, p72-Penton) and Group 2 (p17-T, CD2v-p54-p30, p49-Penton, p72-B602L) mRNA vaccines. One week after a booster dose, the T cell response level in the spleen of the mice was detected by ELISpot. The vertical axis represents the number of IFN-γ secreting cells per million spleen cells, and the horizontal axis represents the immune group; ns or no label: indicates no significant difference. express p <0.05.

[0037] Figure 6 HEK293T cells were transfected with IL-12 mRNA, and expression was verified by Western blot. Figure 7Comparison of antibody titers specifically binding to different ASFV antigens after mice were inoculated with Group 1 (p17-T, CD2v-p54-p30, p49-B602L, p72-Penton) and Group 1 + IL-12; ns or unlabeled: indicates no significant difference. express p <0.05; express p <0.01.

[0038] Figure 8 After mice were inoculated with Group 1 (p17-T, CD2v-p54-p30, p49-B602L, p72-Penton) and Group 1 + IL-12, the T cell response level in the spleen of the mice was detected by ELISpot; the vertical axis represents the number of cells secreting IFN-γ per million spleen cells, and the horizontal axis represents the immunization group; ns or no label: indicates no significant difference. express p <0.05; express p <0.01. Detailed Implementation

[0039] To further illustrate the technical means and effects of this invention, the following detailed description is provided in conjunction with specific embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit its scope.

[0040] Unless otherwise specified, all techniques or conditions used in the examples are conventional methods, performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. All reagents or instruments used are conventional products that can be purchased through legitimate channels.

[0041] Materials, Methods and Animals The mRNA preparation, mRNA vaccine preparation, animal immunization protocols, and detection methods involved in the experiments described in this example are as follows: laboratory animals All experimental mice were specific pathogen-free (SPF) grade. Among them, BALB / c mice (female, 6-8 weeks old) were purchased from Suzhou Huachang Biotechnology Co., Ltd. and were all housed in the SPF experimental area of ​​the laboratory.

[0042] Cells, proteins, antibodies HEK-293T (human embryonic kidney cells 293) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. p17, CD2v, p54, p30, p49, B602L, Penton and p72 proteins and their specific antibodies were purchased from Antibody System; HA tag and flag-tagged antibodies were purchased from Sanying Biotechnology.

[0043] (1) Immunogen design and vaccine preparation Different immunogens p17-T, CD2v-p54-p30, p49-B602L, p49-Penton, p72-Penton, and p72-B602L, along with the adjuvant molecule IL-12, had their corresponding nucleotide sequences optimized using eukaryotic cell codons. The codon-optimized nucleic acid sequences are shown in SEQ ID NO:26 (p17-T), SEQ ID NO:21 (CD2v-p54-p30), SEQ ID NO:22 (p49-B602L), SEQ ID NO:23 (p49-Penton), SEQ ID NO:24 (p72-Penton), SEQ ID NO:25 (p72-B602L), SEQ ID NO:27 (mouse IL-12), or SEQ ID NO:28 (pig IL-12), respectively. These seven nucleic acid sequences were synthesized by Shanghai Qingke Biotechnology Co., Ltd., and then inserted into a constructed high-expression mRNA vector.

[0044] Different immunogens p17-T, CD2v-p54-p30, p49-B602L, p49-Penton, p72-Penton, and p72-B602L, constructed using XbaI restriction endonuclease pairs, were linearized with the adjuvant molecule IL-12 expression vector. Candidate mRNAs were prepared in vitro via co-transcriptional capping. Following the instructions of the nearshore T7 in vitro transcription kit (E131-01A), N1-methylpseudouridine triphosphate (Nanjing Shenji Biotechnology, N1-Me-pUTP, 100mM, NMPUTP001) was used to completely replace uridine triphosphate during in vitro transcription. Additionally, the cap analog CAP GAG (3OMe) (Nanjing Shenji Biotechnology, CAP3111) was added for one-step co-transcriptional capping to generate the native Cap1 structure. The corresponding modified mRNA was synthesized according to the instructions, purified by lithium chloride precipitation, washed three times with 75% ethanol and dried, dissolved in RNAase-free water, and the concentration was determined by nanodrop. The prepared mRNA molecules were sent to Novogene Bioscience Instruments (Shanghai) Co., Ltd. to prepare mRNA liposome nanoparticle vaccines for subsequent mouse experiments.

[0045] (2) Animal immunization The first group of immunological experiments was conducted to compare the differences in humoral and cellular immunity induced by different combinations of immunogens. The following experiments were performed: Using 6-8 week old female BALB / c mice as a model, a "prime-boost" immunization strategy was adopted, with two injections administered 3 weeks apart, and 4-5 mice in each group. The specific grouping is shown in the table below. The experimental group was divided into two groups, group 1 and group 2. Each immunogen was administered by intramuscular injection of 5 µg of vaccine, while the control group received an equal volume of Empty-LNP (100 µL). Blood was collected one week after the booster injection. The specific binding ability of the mice to p17, CD2v, p54, p30, p49, B602L, Penton, and p72 proteins was detected by enzyme-linked immunosorbent assay (ELISA), and the T cell response level in the spleen of mice in different combinations was detected by ELISpot assay. The groups are as follows:

[0046] The second group of immune experiments compared the differences in responses induced by group 1 and group 2, confirming that the design of group 1 was superior. To further verify the enhanced immune response induced by the addition of IL-12, the following experiments were conducted: Using a 6-8 week old female BALB / c mouse model, two injections were administered 3 weeks apart according to a "prime-boost" immunization strategy, with 4-5 mice in each group. Specific groupings are shown in the table below. The experimental group was divided into two groups: Group 1 and Group 1 + IL-12. Each immunogen was administered via intramuscular injection of 5 µg of vaccine. The control group received an equal volume of Empty-LNP (100 µL). Blood was collected one week after the booster injection. The specific binding capacity to p17, CD2v, p54, p30, p49, B602L, Penton, and p72 proteins was detected by enzyme-linked immunosorbent assay (ELISA), and the splenic T cell response levels in different combinations were detected by ELISpot assay.

[0047] (3) Detection method Western blot assay for detecting the expression of the target protein: ① Prepare a separating gel of appropriate concentration according to the size of the target protein and perform SDS-PAGE electrophoresis: load 20 μL of sample into each well, electrophore at 80 V for 0.5 h, then adjust to 100 V and continue for 1.5 h, and stop by judging the position of bromophenol blue.

[0048] ② Transfer (wet transfer): Activate the PVDF membrane in methanol for 30 s, place it in the transfer solution and stack it with the sponge, filter paper and adhesive in the order of "black electrode-sponge-filter paper-adhesive-PVDF-filter paper-sponge-white electrode"; transfer the membrane at a constant current of 200 mA for 1.5-2 h.

[0049] ③ Blocking and antibody incubation: Block the PVDF membrane in 5% skim milk powder for 1-2 h; primary antibody 1:1000, incubate at room temperature for 2 h, wash 5 times with PBST (3 min / time); secondary antibody HRP-IgG 1:5000, incubate at room temperature for 1 h, wash 5 times with PBST (3 min / time).

[0050] ④ Color development and analysis: Incubate with color development solution for 1 min, expose to the analyzer for 2 min, record the bands and analyze the results.

[0051] Flow cytometry detection of target protein expression: ① 24 h after transfection, cells were collected by flow cytometry and centrifuged at 500 g for 5 min; ② Wash once with 0.5 mL of staining buffer, centrifuge at 500 g for 5 min; carefully aspirate the supernatant with a pipette, being careful not to aspirate cells from the bottom of the tube; ③ Prepare the fluorescent antibody mix (specific antibodies against p17, CD2v, p54, p30, p49, B602L, Penton, and p72 proteins diluted 1:100). Mix the antibody mix thoroughly and centrifuge briefly for 10 seconds to remove any remaining antibody from the tube wall. Add 50 μL of the mix / sample to the sample and mix well. Incubate at 4 °C for 30 min. ④ After the reaction, wash once with 0.8 mL of staining buffer, incubate at 500 g for 5 min, and discard the supernatant. Then add mouse anti-rabbit IgG-FITC and goat anti-mouse IgG-FITC antibodies, diluted 1:100, incubate at 4 ℃ for 30 min, wash the cells, and then resuspend them with 250 μL of staining buffer. Detect the cells using a Fortessa instrument.

[0052] Enzyme-linked immunosorbent assay (ELISA) was used to detect the titers of antibodies that specifically bind to each protein: ① Coating and blocking: The eight proteins were diluted to 1 ng / μL with 50 mM carbonate buffer (pH 9.6), 100 μL / well, and coated overnight at 4°C; after washing once with PBST, 200 μL / well of 5% skim milk powder was added and blocked at room temperature for 2 h.

[0053] ② Sample loading and secondary antibody incubation: Using 5% skim milk powder as diluent, serum samples were initially diluted 1:100, then serially diluted 2-fold, loaded into wells, and reacted at 37°C for 1 h; after washing 5 times with PBST, 100 μL / well of HRP-labeled goat anti-mouse IgG (1:5000) was added, and the mixture was reacted at room temperature for 1 h.

[0054] ③ Color development, termination, and reading: After washing 6 times with PBST, add OPD color development solution (protected from light) and react for 5–10 min; stop the reaction with 2 M sulfuric acid and read the value at 490 nm. Result interpretation: Determine the antibody endpoint dilution (titer) by using 2.1 times the OD value of the negative control as the cut-off value; express the titer as geometric mean titer (GMT) and analyze the results using GraphPad Prism 9.

[0055] Mouse IFN- γ ELISPot test: ① Take the IFN of BD. γ The Elispot mouse kit was used for antibody coating according to the instructions: anti-IFN-γ was coated into the appropriate number of wells using sterile PBS according to the experimental requirements. γ The antibody was diluted 1:200 and 100 μL / well was added to an Elispot plate. Incubation was performed overnight at 4 °C. ② Remove the Elispot plate that has been incubated overnight, discard the liquid in the wells, wash once with 200 μL of R10, let stand for 3 min, and discard. Add 200 μL of fresh R10 and incubate at room temperature for 2 h. ③ After blocking, discard the culture medium and add 50 μL of stimulant (stimulating peptide library, final single peptide concentration 5 μg / mL) to each well, along with negative control R10 and positive control [PMA (50 ng / mL) + Ionomycin (1 μg / mL)] or other positive stimulants). Then adjust the cell concentration to 4 × 10⁻⁶ cells / well. 6 Add 50 μL of cells per well in sequence. Gently mix and incubate in a humidified chamber for approximately 20 hours in a CO2 incubator. The first three steps must be performed in a biosafety cabinet; the following steps do not require aseptic conditions.

[0056] ④ After incubation, discard the culture. Wash each well twice with 200 μL of pre-cooled distilled water, and three times with 200 μL of PBST, leaving each wash for 3-5 minutes before discarding. Pat the remaining liquid dry on absorbent paper. ⑤ Dilute the biotin-conjugated detection antibody 1:250 in antibody dilution buffer (PBS + 10% FBS), add 100 μL / well, and incubate at room temperature for 2 h. Discard the liquid, add 200 μL PBST to each well and wash 4 times. After each wash, let stand for 1-2 min and then discard the solution, patting the remaining liquid dry on absorbent paper. ⑥ Dilute Streptavidin-HRP 1:100 in antibody dilution buffer (PBS + 10% FBS), add 100 μL / well, and incubate at room temperature for 1 h. Discard the reaction solution, wash each well 5 times with 200 μL PBST, and discard after 1-2 min. Finally, wash 3 times with 200 μL / well PBS and discard. ⑦ Color Development: AEC colorimetric solution should be prepared fresh before use: Add 1 drop (approximately 20 μL) of the colorimetric substrate to every 1 mL of colorimetric solution, mix thoroughly, and add 100 μL to each well. Incubate at room temperature in the dark for 5-60 min. Monitor the color development; when clearer red dots appear, gently rinse the plate with tap water for 5 min and stop the reaction. After the plate has air-dried, place it on an Elispot plate reader to read the positive dots.

[0057] The stimulating peptide library was synthesized by Suzhou Qiangyao Biotechnology Co., Ltd., and covers the p17, CD2v, p54, p30, p49, B602L, Penton, p72 and T antigen (T) sequences, respectively.

[0058] Each peptide library consists of 9 / 10 single peptides, each single peptide is 15 amino acids, and each peptide has 10 / 11 amino acid overlaps. Specifically: p17 includes 18 peptides, T antigen includes 144 peptides, CD2v includes 48 peptides, p54 includes 27 peptides, p30 includes 30 peptides, p49 includes 72 peptides, Penton includes 72 peptides, p72 includes 59 peptides, and B602L includes 15 peptides.

[0059] The nucleic acid and amino acid sequences involved in this invention are as follows: SEQ ID NO:1, which is the amino acid sequence derived from the linker between the IL-12A subunit (IL-12p35) and the IL-12B subunit (IL-12p40): 5'-SGGGGSGGGGSGGGGSPVSVPTASPSASSSSSQCRSS-3' SEQ ID NO:2, which is the amino acid sequence derived from mouse IL-12: 5’-MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRGGGGSGGGGSGGGGSPVSVPTASPSASSSSSQCRSSMCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA-3’ SEQ ID NO:3, the amino acid sequence derived from porcine IL-12: 5’-MHLQQLVVSWFSLVWLASPIVAIWELEKNVYVVELDWYPNAPGEMVVLTCNTPEEDGITWTSDQSSEVLGTGKTLTIHVKEFGDAGQYTCRKGGAVLSQSLLLLHKKEDGIWSTDILKDQKEPKNKSFLKCEAKNYSGRFTCWWLTAISTDLKFSVKSSRGSTDPRGVTCGTATLSEDLGEYKKYRVECQEGSACPAAEESLPIEVVLEAVHKLKYENYTSSFFIRDIIKPDPPKNLQLNPLKNSRHVEISWEYPDTWSTPHSYFSLMFGVQVQGKNKREKKDKLFTDQISAKVTCHKDANIRVQARDRYYSSSWSEWASVSCNGGGGSGGGGSGGGGSPVSVPTASPSASSSSSQCRSSRSLPATTAGPGMFKCLNHSQNLLKAVSNTLQKAKQTLEFYSCTSEEIDHEDITKDKTSTVEACLPLELATNESCLAARETSLITNGNCLTSGKTSFMTTLCLSSIYEDLKMYHVEFQAMNAKLLMDPKRQIFLDQNMLTAITELMQALNFNSETVPQKPSLEELDFYKTKIKLCILLHAFRIRAVTIDRMMSYLN-3’ SEQ ID NO:4, i.e., the amino acid sequence derived from p54 / pE183L: 5’-MDSEFFQPVYPRHYGECLSPVTTPSFFSTHMYTILIAIVVLVIIIIVLIYLFSSRKKKAAAIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSL-3’ SEQ ID NO:5, i.e., the amino acid sequence derived from P30 / CP204L: 5’-MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKK -3’ SEQ ID NO:6, the amino acid sequence derived from p72 / B646L: 5’-MASGGAFCLIANDGKADKIILAQDLLNSRISNIKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYYQPPLALWIKLRFWFNENVNLAIPSVSIPFGERFITIKLASQKDLVNEFPGLFVRQSRFIAGRPSRRNIRFKPWFIPGVINEISLTNNELYINNLFVTPEIHNLFVKRVRFSLIRVHKTQVTHTNNNHHDEKLMSALKWPIEYMFIGLKPTWNISDQNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREEYQPSGHINVSRAREFYISWDTDYVGSITTADLVVSASAINFLLLQNGSAVLRYST-3’ SEQ ID NO:7, the amino acid sequence derived from p49 / B438L: 5’-MYHDYASKLLADYRSDPPLWESDLPRHNRYSDNILNSRYCGNKNGAAPVYNEYTNSPEKAEKGLQLSDLRNFSFMLNPQHKNIGYGDAQDLEPYSSIPKNKLFNHFKNHRPAFSTHTENLIRRNVVRTEKKTFPQVASLKGTQKNCLTQPSSLPSLKNPKNSSVPSTRFSEHTKFFSYEDLPKLRTKGTIKHEQHLGDQMPGQHYNGYIPHKDVYNILCLAHNLPASVEKGIAGRGIPLGNPHVKPNIEQELIKSTSTYTDVPMLGPLPPKDSQHGREYQEFSANRHMLQVSNILHSVFANHSIKPQILEDIPVLNAQLTSIKPVSPFLNKAYQTHYMENIVTLVPRFKSIANYSSPIPNYSKRNSGQAEYFDTSKQTISRHNNYIPKYTGGIGDSKLDSTFPKDFNASSVPLTSAEKDHSLRGDNSACCISSISPSL-3’ SEQ ID NO:8, the amino acid sequence derived from CD2v / pEP402R: 5’-MIILIFLIFSNIVLSIDYWVSFNKTIILDSNITNDNNDINGVSWNFFNNSFNTLATCGKAGNFCECSNYSTSIYNITNNCSLTIFPHNDVFDTTYQVVWNQIINYTIKLLTPATPPNITYNCTNFLITCKKNNGTNTNIYLNINDTFVKYTNESILEYNWNNSNINNFTATCIINNTISTSNETTLINCTYLTLSSNYFYTFFKLYYIPLSIIIGITISILLISIITFLSLRKRKKHVEEIESPPPESNEEEQCQHDDTTSIHEPSPREPLLPKPYSRYQYNTPIYYMRPSTQPLNPFPLPKPCPPPKPCPPPKPCPPPKPCPSAESYSPPKPLPSIPLLPNIPPLSTQNISLIHVDRII-3’ SEQ ID NO:9, the amino acid sequence derived from p17 / D117L: 5’-MDTETSPLLSHNLSTREGIKQSTQGLLAHTIAKYPGTTAILLGILILLIIILIIVAIVYYNRTIDCKSSIPKPPPSYYVQQPEPHHHFPVFFRKRKNSTSLQSHIPSDEQLAELAHS-3’ SEQ ID NO:10, the amino acid sequence derived from p72 chaperone / B602L: 5’-MAEFNIDELLKNVLEDPSTEISEETLKQLYQRTNPYKQFKNDSRVAFCSFTNLREQYIRRLIMTSFIGYVFKALQEWMPSYSKPTHTTKTLLSELITLVDTLKQETNDVPSESVVNTILSIADSCKTQTQKSKEAKTTIDSFLREHFVFDPNLHAQSAYTCADTNVDTCASMCADTNVDTCASMCADTNVDTCASTCTSTEYTDLADPERIPLHIMQKTLNVPNELQADIDAITQTPQGYRAAAHILQNIELHQSIKHMLENPRAFKPILFNTKITRYLSQHIPPQDTFYKWNYYIEDNYEELRAATESIYPEKPDLEFAFIIYDVVDSSNQQKVDEFYYKYKDQIFSEVSSIQLGNWTLLGSFKANRERYNYFNQNNEIIKRILDRHEEDLKIGKEILRNTIYHKKAKNIQETGPDAPGLSIYNSTFHTDSGIKGLLSFKELKNLEKASGNIKKAREYDFIDDCEEKIKQLLSKENLTPDEESELIKTKKQLDNALEMLNVPDDTIRVDMWVNNNNKLEKEILYTKAEL-3’ SEQ ID NO:11, the amino acid sequence derived from Penton / H240R: 5'-MAANIIATRAVPKMASKKEHQYCLLDSQEKRHGHYPFSFELKPYGQTGANIIGVQGSLTHVIKMTVFPFMIPFPLQKTHIDDFIGGRIYLFFKELDMQAVSDVNGMQYHFEFKVVPVSPN QVELLPVNNKYKFTYAIPVVQYLTPIFYDLSGPLDFPLDTLSVHVDILSNHIQLPIQNHNLTTGDRVFISGYKHLQTIELCKNNKIFIKNIPPLSSEKIKLYILKNRIRIPLYFKSLKTSK-3' SEQ ID NO:12, which is the amino acid sequence derived from the conserved T-cell antigen: SEQ ID NO:13, which is the amino acid sequence derived from Porcine teschovirus P2A: 5'-ATNFSLLKQAGDVEENPGP-3' SEQ ID NO:14, which is the amino acid sequence derived from the T2A of the Thosea asigna virus: 5'-EGRGSLLTCGDVEENPGP-3' SEQ ID NO:15, which is the amino acid sequence derived from candidate immunogen 1 (CD2v-p54-p30): 5’-MIILIFLIFSNIVLSIDYWVSFNKTIILDSNITNDNNDINGVSWNFFNNSFNTLATCGKAGNFCECSNYSTSIYNITNNCSLTIFPHNDVFDTTYQVVWNQIINYTIKLLTPATPPNITYNCTNFLITCKKNNGTNTNIYLNINDTFVKYTNESILEYNWNNSNINNFTATCIINNTISTSNETTLINCTYLTLSSNYFYTFFKLYYIPLSIIIGITISILLISIITFLSLRKRKKHVEEIESPPPESNEEEQCQHDDTTSIHEPSPREPLLPKPYSRYQYNTPIYYMRPSTQPLNPFPLPKPCPPPKPCPPPKPCPPPKPCPSAESYSPPKPLPSIPLLPNIPPLSTQNISLIHVDRIIGSGYPYDVPDYASSGATNFSLLKQAGDVEENPGPEMDSEFFQPVYPRHYGECLSPVTTPSFFSTHMYTILIAIVVLVIIIIVLIYLFSSRKKKAAAIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSLGSGDYKDDDDKDYKDDDDKGGSEGRGSLLTCGDVEENPGPLEMDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKKGSGEQKLISEEDL-3’ SEQ ID NO:16, which is the amino acid sequence derived from candidate immunogen 2 (abbreviated as p49 - B602L): SEQ ID NO:17, the amino acid sequence derived from candidate immunogen 3 (abbreviated as p49-Penton): 5’-MYHDYASKLLADYRSDPPLWESDLPRHNRYSDNILNSRYCGNKNGAAPVYNEYTNSPEKAEKGLQLSDLRNFSFMLNPQHKNIGYGDAQDLEPYSSIPKNKLFNHFKNHRPAFSTHTENLIRRNVVRTEKKTFPQVASLKGTQKNCLTQPSSLPSLKNPKNSSVPSTRFSEHTKFFSYEDLPKLRTKGTIKHEQHLGDQMPGQHYNGYIPHKDVYNILCLAHNLPASVEKGIAGRGIPLGNPHVKPNIEQELIKSTSTYTDVPMLGPLPPKDSQHGREYQEFSANRHMLQVSNILHSVFANHSIKPQILEDIPVLNAQLTSIKPVSPFLNKAYQTHYMENIVTLVPRFKSIANYSSPIPNYSKRNSGQAEYFDTSKQTISRHNNYIPKYTGGIGDSKLDSTFPKDFNASSVPLTSAEKDHSLRGDNSACCISSISPSLGSGEQKLISEEDLATNFSLLKQAGDVEENPGPEMAANIIATRAVPKMASKKEHQYCLLDSQEKRHGHYPFSFELKPYGQTGANIIGVQGSLTHVIKMTVFPFMIPFPLQKTHIDDFIGGRIYLFFKELDMQAVSDVNGMQYHFEFKVVPVSPNQVELLPVNNKYKFTYAIPVVQYLTPIFYDLSGPLDFPLDTLSVHVDILSNHIQLPIQNHNLTTGDRVFISGYKHLQTIELCKNNKIFIKNIPPLSSEKIKLYILKNRIRIPLYFKSLKTSKGSGDYKDHDGDYKDHDIDYKDDDDK-3’ SEQ ID NO:18, the amino acid sequence derived from candidate immunogen 4 (abbreviated as p72-Penton): 5’-MASGGAFCLIANDGKADKIILAQDLLNSRISNIKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYYQPPLALWIKLRFWFNENVNLAIPSVSIPFGERFITIKLASQKDLVNEFPGLFVRQSRFIAGRPSRRNIRFKPWFIPGVINEISLTNNELYINNLFVTPEIHNLFVKRVRFSLIRVHKTQVTHTNNNHHDEKLMSALKWPIEYMFIGLKPTWNISDQNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREEYQPSGHINVSRAREFYISWDTDYVGSITTADLVVSASAINFLLLQNGSAVLRYSTGSGYPYDVPDYASSGATNFSLLKQAGDVEENPGPEMAANIIATRAVPKMASKKEHQYCLLDSQEKRHGHYPFSFELKPYGQTGANIIGVQGSLTHVIKMTVFPFMIPFPLQKTHIDDFIGGRIYLFFKELDMQAVSDVNGMQYHFEFKVVPVSPNQVELLPVNNKYKFTYAIPVVQYLTPIFYDLSGPLDFPLDTLSVHVDILSNHIQLPIQNHNLTTGDRVFISGYKHLQTIELCKNNKIFIKNIPPLSSEKIKLYILKNRIRIPLYFKSLKTSKGSGDYKDHDGDYKDHDIDYKDDDDK-3’ SEQ ID NO:19, which is the amino acid sequence derived from candidate immunogen 5 (p72-B602L): SEQ ID NO:20, which is the amino acid sequence derived from candidate immunogen 6 (p17-T): SEQ ID NO:21 is the codon-optimized nucleic acid sequence derived from candidate immunogen 1 (CD2v-p54-p30): SEQ ID NO:22 is the codon-optimized nucleic acid sequence derived from candidate immunogen 2 (p49-B602L): SEQ ID NO:23 is the codon-optimized nucleic acid sequence derived from candidate immunogen 3 (p49-Penton): SEQ ID NO:24 is the codon-optimized nucleic acid sequence derived from candidate immunogen 4 (p72-Penton): SEQ ID NO:25 is the codon-optimized nucleic acid sequence derived from candidate immunogen 5 (p72-B602L): SEQ ID NO:26, which is the codon-optimized nucleic acid sequence derived from candidate immunogen 6 (p17-T): SEQ ID NO:27 is the codon-optimized nucleic acid sequence derived from mouse IL-12, an adjuvant molecule that enhances the immune response: SEQ ID NO:28 is the codon-optimized nucleic acid sequence derived from porcine IL-12, an adjuvant molecule that enhances the immune response:

[0060] The following detailed description, in conjunction with the accompanying drawings and specific experiments, further illustrates the invention. Unless otherwise specified, all reagents, instruments, equipment, and methods used in this invention are commercially available and conventional in this technical field.

[0061] Example 1: Construction of fusion protein candidate immunogen The fusion immunogens p17-T, CD2v-p54-p30, p49-B602L, p49-Penton, p72-Penton, and p72-B602L were constructed according to the experimental method (1) described above. The schematic diagrams of the construction are shown below. Figure 1 As shown.

[0062] Example 2: Flow cytometry analysis of HEK293T cells after transfection with mRNAs of six fusion protein candidate immunogens. The mRNAs corresponding to the fusion immunogens p17-T, CD2v-p54-p30, p49-B602L, p49-Penton, p72-Penton, and p72-B602L were prepared according to experimental method (2) and transfected into HEK293T cells for verification. One day before transfection, cells were seeded into 12-well plates at a density of 250,000 cells / well. The culture medium was DMEM complete medium diluted with Opti-MEM (invitrogen). 2 μg of mRNA was transfected into each well and mixed with Lipofectamine™ 3000 at a mass-to-volume ratio of 1:2. The mixture was added dropwise to the 12-well plates and incubated at 37 ℃ for 24 h. Samples were then collected and analyzed. Flow cytometry was used to validate the results. The primary antibodies were specific antibodies against CD2v, p54, p30, p49, and p72 proteins. The primary antibodies against B602L, Penton, and T antigens were all specific antibodies with flag tags added after the C-terminus. P17 was a specific antibody with an HA tag added after the C-terminus. The secondary antibody was a fluorescent secondary antibody conjugated with FITC.

[0063] The results are as follows Figure 2As shown, all candidate mRNA molecules were highly expressed after transfection into HEK293T cells; among them, the expression rates of the fusion immunogen p17-T, p17, and T antigens were 99.7% and 99.9%, respectively; after transfection with the trivalent fusion immunogen CD2v-p54-p30, the expression rates of CD2v, p54, and p30 genes were as high as 99.5%, 99.8%, and 87.9%, respectively; after transfection with p49-B602L, the expression rates of p49 and B602L reached 99.6%, respectively. The expression rates of p49 and Penton reached 99.3% and 99%, respectively, after transfection with p49-Penton, respectively; after transfection with p72-Penton, p72 and Penton reached 99.8% and 99.8%, respectively; after transfection with p72-B602L, p72 and B602L reached 99.7% and 83%, respectively. Only in the p72-B602L immunogen was the expression level of B602L below 85%, while the expression levels of other genes remained at around 90% or above. High levels of protein expression indicate that the candidate mRNA molecules have good expression efficacy.

[0064] Example 3: Expression verification of 6 fusion candidate immunogen mRNAs after transfection into HEK293T cells. The mRNAs of the six immunogens prepared according to experimental method (2) were transfected into HEK293T cells for expression verification, using the same method as in Example 2. Samples were collected 24 h after transfection. Western blot verification was performed. The primary antibodies for T antigen, Penton, and B602L were anti-Flag labeled antibodies; the primary antibody for p17 was an anti-HA labeled antibody; and the others were specific antibodies against CD2v, p54, p30, p49, and p72 proteins. The secondary antibody was an HRP-conjugated secondary antibody. Results are as follows: Figure 3 As shown, p17-T mRNA can be highly expressed in HEK293T cells after transfection. The p17 target protein band is located at approximately 20 kDa in monomeric form, and it can also form a trimer at approximately 40 kDa. Figure 3 A); The target protein of the T antigen is located at approximately 130 kDa (A). Figure 3 B), both sizes are correct; CD2v-p54-p30 mRNA transfection into HEK293T cells also showed normal expression. After incubation with CD2v-specific antibody, multiple bands of the target protein were observed. As a transmembrane protein, its full-length band was approximately 40 kDa, with an extracellular segment of approximately 25 kDa. An incompletely broken CD2v-p54 band was observed around 70 kDa, while the full-length CD2v-p5-p30 fusion protein was present around 100 kDa. Figure 3C); p54 target protein mainly exists in dimer form, with its monomer molecular weight around 24-28 kDa under reducing conditions, and the dimer size around 50-60 kDa. Figure 3 D); The theoretical molecular weight of the p30 target protein is 23.6 kDa, but it is a complex membrane phosphorylated protein. After phosphorylation modification, its size is around 30 kDa. Figure 3 E); After transfecting HEK293T cells with p49-B602L mRNA, and after incubation with p49-specific antibody, multiple target protein bands were observed. Under reducing conditions, the p49 protein band was approximately 50 kDa, and an incompletely broken p49-B602L band could be seen at 120 kDa. Figure 3 F); the theoretical molecular weight of B602L is approximately 68 kDa, and its size after glycosylation modification is around 70 kDa; the incompletely broken p49-B602L target band also exists at 120 kDa. Figure 3 G); After transfection of HEK293T cells with p49-Penton mRNA, and incubation with p49-specific antibody, the p49 protein band was approximately 50 kDa under reducing conditions, and an unbroken p49-Penton fusion protein was observed at 80 kDa. Figure 3 F); The target protein band of Penton is around 28-30 kDa, and the unbroken p49-Penton can be seen at 80 kDa. Figure 3 H); After transfection of HEK293T cells with p72-Penton mRNA, and after incubation with p72-specific antibody, the p72 protein band size was approximately 72 kDa under reducing conditions, while the Penton target protein band was approximately 28-30 kDa. Figure 3 I); After transfection of HEK293T cells with p72-B602L mRNA, the p72 protein band size was around 72 kDa under reducing conditions; the B602L protein size after glycosylation modification was around 70 kDa. Figure 3 j); All nine proteins from the above six fusion immunogens were expressed normally and at the expected size, which prepares the groundwork for subsequent animal evaluation.

[0065] Example 4: Immunogenicity evaluation of six fusion candidate immunogen mRNA vaccines in BALB / c mice In this embodiment, the mRNA that was correctly expressed after verification in Examples 2 and 3 was selected and prepared into an mRNA cationic lipid nanoparticle vaccine for animal immune evaluation. According to the experimental methods (1) and (2) above, the six fusion candidate immunogens were divided into two groups: Group 1: p17-T, CD2v-p54-p30, p49-B602L, p72-Penton; Group 2: p17-T, CD2v-p54-p30, p49-Penton, p72-B602L. Two injections were administered at 3-week intervals. Blood was collected one week after the second injection, and the mice were sacrificed. Spleen cells were collected, and the spleen cells were stimulated with p17, CD2v, p54, p30, p49, B602L, Penton, p72, and T antigen (T) peptide libraries, respectively. The T cell immune response level was detected by ELISA. The differences in specific antibody responses induced after vaccination in Group 1 and Group 2 were analyzed by enzyme-linked immunosorbent assay (ELISA).

[0066] The results are as follows Figure 4 As shown, there were significant differences between the two groups in terms of induced binding antibodies, especially in group 1, which showed significantly better results than group 2 in inducing binding antibodies against p72 and B602L. There were no significant differences between the two groups in binding antibodies against p17, CD2v, p54, p30, p49, and Penton, and the levels of binding antibodies against p17, p49, and Penton were similar to those in the control group. Figure 4 In terms of inducing T cell responses, group 1 was slightly better, such as inducing IFN-γ responses against B602L, Penton, and CD2v. γ The number of positive cells in group 1 was higher than that in group 2. Figure 5 In addition, the levels of antibodies binding to p17, p49, and Penton were low because these three proteins have fewer B-cell epitopes, but can induce higher levels of T-cell responses.

[0067] These experimental results indicate that the combination design of Group 1 in this invention is superior to that of Group 2, and that multiple multivalent immunogens can not only induce higher titers of binding antibodies, but also induce stronger T cell responses, thereby achieving a balance between antibody and cell responses.

[0068] Example 5: Immunogenicity evaluation of an IL-12 mRNA vaccine containing four fusion candidate immunogens in BALB / c mice. To further enhance the immune response levels of the nine immunogens, especially to increase the intensity of T cell responses, IL-12 was added in this embodiment to assess the differences in immune responses. This embodiment used four multivalent immunogens from group 1 evaluated in Example 4 for animal experimental immune assessment. Following the experimental methods (1) and (2) described above, the prepared IL-12 mRNA was transfected into HEK293T cells. The supernatant and cells were then collected, and Western blot was used to verify its expression. The results are as follows: Figure 6 As shown, IL-12 was expressed normally in both cell supernatant and cell lysate. The full-length IL-12 and p40 monomer in the cell supernatant were slightly larger than the target protein in the cell lysate, which is presumably due to the influence of post-translational modification.

[0069] Subsequently, mice were injected intramuscularly with 5 µg of each of the four immunogens in Group 1 (p17-T, CD2v-p54-p30, p49-B602L, and p72-Penton) that expressed the correct IL-12. The control group received an equal volume of Empty-LNP (100 µL). One week after a booster injection, blood was collected and the mice were sacrificed. Blood samples were collected to assess antibody response levels, and spleen cells were harvested for ELISApot assay to detect T-cell immune response levels.

[0070] The results are as follows Figure 7 As shown, there was no significant difference between the two groups in the induced binding antibodies against p17, CD2v, p54, p30, p49, B602L, Penton, and p72. Figure 7 However, in inducing T cell responses, the addition of IL-12 significantly increased the cellular response induced by the T antigen, with the number of positive cells (GMT) per million spleen cells increasing from 1187 to 4014. Figure 8 This further suggests that IL-12 plays a significant role in enhancing T cell responses, especially those induced by non-structural proteins, and could be used to prevent African swine fever virus infection.

[0071] As can be seen from the above embodiments, the African swine fever mRNA vaccine combination provided in this invention, which can synergistically activate and enhance antibody and T-cell immunity, can not only solve the problems of poor immunization effect, insufficient safety, and ADE effect that aggravates the disease due to reliance on humoral immunity when used to prepare drugs for the prevention of African swine fever, but also can be rapidly developed according to local conditions for different genotypes and serotypes of the virus, providing an effective response to the African swine fever epidemic and having broad application prospects.

Claims

1. A nucleic acid composition for inducing an immune response against African swine fever virus (ASFV), characterized in that, The nucleic acid composition comprises a combination of 1) and 2) or a combination of 1) and 2) and 3): 1) At least one mRNA encoding the ASFV immunogen 2) At least one mRNA encoding a recombinant T-cell immunogen; 3) At least one mRNA encoding an immune regulatory molecule; The mRNA is either encapsulated in a delivery system or forms a complex with the delivery system to achieve in vivo delivery.

2. The nucleic acid composition according to claim 1, characterized in that, The mRNA encoding the ASFV immunogen encodes any of the following immunogens: 1) The fusion protein (CD2v-p54-p30) formed by linking CD2v / pEP402R, p54 / pE183L and p30 / CP204L via a linker has the amino acid sequence shown in SEQ ID NO:15; 2) The fusion protein P49-B602L formed by linking p49 / B438L and B602L via a linker has the amino acid sequence shown in SEQ ID NO:16; 3) The fusion protein P49-Penton, formed by linking p49 / B438L and Penton / H240R via a linker, has the amino acid sequence shown in SEQ ID NO:17; 4) The fusion protein P72-Penton, formed by linking p72 / B646L and Penton / H240R via a linker, has the amino acid sequence shown in SEQ ID NO:18; 5) The fusion protein P72-B602L formed by linking p72 / B646L and B602L via a linker has the amino acid sequence shown in SEQ ID NO:19; And / or, the mRNA encoding the recombinant T cell immunogen encodes a fusion protein p17-T formed by linking the recombinant conserved T cell antigen with p17 / D117L via a linker, the amino acid sequence of which is shown in SEQ ID NO:

20.

3. The nucleic acid composition according to claim 2, characterized in that, The mRNA encoding the ASFV immunogen encodes any of the following codon-optimized immunogens: i) Encoding codon-optimized candidate immunogen 1, the codon-optimized nucleic acid sequence is shown in SEQ ID NO:21; ii) The candidate immunogen 2 with optimized codons is shown in SEQ ID NO:22; iii) The candidate immunogen 3 with optimized codons is shown in SEQ ID NO:23; iv) The candidate immunogen 4 with optimized codons is shown in SEQ ID NO:24; v) The candidate immunogen 5 with codon optimization is shown in SEQ ID NO:25; And / or, the mRNA encoding the recombinant T cell immunogen encodes a codon-optimized p17-T, the codon-optimized nucleic acid sequence of which is shown in SEQ ID NO:

26.

4. The nucleic acid composition according to any one of claims 1-3, characterized in that, The mRNA encoding the ASFV immunogen is a single-stranded mRNA, and its open reading frame encodes a multi-antigen fusion protein.

5. The nucleic acid composition according to any one of claims 1-4, characterized in that, The immunomodulatory molecule is selected from one or more adjuvants and / or cytokines; Preferably, the adjuvant is selected from one or more of aluminum adjuvants, cholera toxin and its subunits, oligodeoxynucleotides, manganese ion adjuvants, colloidal manganese adjuvants, Freund's adjuvant, MF59, QS-21, Poly I:C and other TLR ligands; Preferably, the cytokine is selected from one or more of GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21 and IFN-α; more preferably, the immunomodulatory molecule is IL-12.

6. The nucleic acid composition according to claim 5, characterized in that, The IL-12 is mouse-derived IL-12 and / or porcine-derived IL-12; Preferably, the mRNA encoding IL-12 comprises a codon-optimized coding sequence, wherein the coding sequence is: 1) The coding sequence of mouse IL-12 is shown in SEQ ID NO:27; and / or 2) The coding sequence of porcine IL-12 is shown in SEQ ID NO:

28.

7. The nucleic acid composition according to any one of claims 1-6, characterized in that, The mRNA encoding the ASFV immunogen and the mRNA encoding the immunomodulatory molecule are administered at doses of equal mass and / or equal molar mass. And / or, the administration method of the nucleic acid composition satisfies any of the following: A) The mRNAs in the combination are individually packaged in a delivery system and administered separately; or B) The mRNAs in the combination are individually encapsulated in the delivery system and then mixed and administered; or C) The mRNAs in the combination are formulated separately and then administered in combination; or D) The mRNAs in the combination are co-encapsulated in the same delivery system before administration.

8. The nucleic acid composition according to any one of claims 1-7, characterized in that, The delivery system is a carrier system capable of delivering mRNA into animal cells, and the carrier system is selected from liposomes, lipid nanoparticles, polymer nanoparticles, cationic polymer complexes, inorganic nanocarriers, emulsions, or combinations thereof; And / or, the mRNA is unmodified mRNA and / or nucleoside-modified mRNA.

9. An immunogenic peptide derived from African swine fever virus (ASFV), characterized in that, The immunogenic peptide is selected from any one or a combination of the following 1), 2), 3), 4), 5), and 6): 1) A fusion protein CD2v-p54-p30 or a variant thereof formed by linking CD2v / pEP402R, p54 / pE183L and p30 / CP204L via a linker, wherein the amino acid sequence of CD2v-p54-p30 is as shown in SEQ ID NO:15, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:15, and retaining the function of the fusion protein as shown in SEQ ID NO:15; 2) A fusion protein P49-B602L formed by linking p49 / B438L and B602L via a linker, wherein the amino acid sequence of P49-B602L is as shown in SEQ ID NO:16, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:16, and retaining the function of the fusion protein as shown in SEQ ID NO:16; 3) A fusion protein P49-Penton formed by linking p49 / B438L and Penton / H240R via a linker, wherein the amino acid sequence of P49-Penton is as shown in SEQ ID NO:17, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:17, and retaining the function of the fusion protein as shown in SEQ ID NO:17; 4) A fusion protein P72-Penton formed by linking p72 / B646L and Penton / H240R via a linker, wherein the amino acid sequence of P72-Penton is as shown in SEQ ID NO:18, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:18, and retaining the function of the fusion protein as shown in SEQ ID NO:18; 5) A fusion protein P72-B602L formed by linking p72 / B646L and B602L via a linker, wherein the amino acid sequence of P72-B602L is as shown in SEQ ID NO:19, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:19, and retaining the function of the fusion protein as shown in SEQ ID NO:19; 6) A fusion protein p17-T formed by linking recombinant conserved T-cell antigen with p17 / D117L via a linker, wherein the amino acid sequence of p17-T is as shown in SEQ ID NO:20, and the variant is a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:20, and retaining the function of the fusion protein as shown in SEQ ID NO:

20.

10. The use of the nucleic acid composition of any one of claims 1-8 or the immunogenic peptide of claim 9 in the preparation of a medicament for the prevention and / or treatment of ASFV infection, characterized in that, The drug may be administered alone or in combination with anti-ASFV antibodies and / or antiviral drugs. Preferably, the drug is administered in combination with anti-ASFV antibodies and / or antiviral drugs.

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