Immunogenic composition of african swine fever virus p15 protein and use thereof

Abstract

Provided are an immunogenic fragment, a recombinant protein and an immunogenic composition of an African swine fever virus p15 protein and the use thereof. The immunogenic fragment of the African swine fever virus p15 protein or an immunogenic variant thereof has a higher expression level, and the strong immune activity is maintained.

Description

Immunogenic composition of African swine fever virus p15 protein and its application Technical Field

[0001] The present disclosure relates to immunogenic compositions, and in particular to African swine fever virus p15 protein or an immunogenic fragment thereof, the corresponding encoding nucleotide sequence, the immunogenic composition and uses thereof. Background Art

[0002] African swine fever is a viral disease caused by infection with the African swine fever virus (ASFV). The acute form clinically manifests as high fever, depression, anorexia, cyanosis of the skin, and bleeding in various organs. The disease is highly contagious and fatal, with morbidity and mortality rates reaching 100%.

[0003] Due to the large genome structure and complex immune escape mechanisms of ASFV, developing an effective vaccine is extremely difficult. To date, no safe and effective vaccine exists for epidemic prevention and control. Previous research on African swine fever vaccines has shown that inactivated vaccines can induce a high level of humoral immune response but do not provide immune protection. Therefore, current approaches to African swine fever vaccine design primarily focus on attenuated and subunit vaccines.

[0004] Research on attenuated vaccines is progressing more rapidly across various countries, but their challenges are becoming increasingly apparent. Attenuated vaccines typically only protect against homologous strains of the same genotype and offer no protection against heterologous virus challenges. Attenuated vaccines are also often associated with adverse side effects, such as skin lesions and joint swelling. Furthermore, attenuated vaccines can lead to chronic or persistent infection and potentially revert to virulence.

[0005] Compared to attenuated vaccines, subunit vaccines offer a targeted approach with fewer side effects and greater safety. Previous research has shown that various ASFV antigens can induce neutralizing antibodies and provide partial immune protection, paving the way for the development of a safe and effective ASF vaccine. With in-depth research into the structure and immunology of ASFV, the design of effective subunit vaccines capable of generating protective antibodies and specific cellular immune responses has become a hot topic in the field.

[0006] Summary of the Invention

[0007] In order to solve one of the above-mentioned technical problems existing in the prior art, the present disclosure discovered that the African swine fever virus (ASFV) p15 protein has a high expression level while retaining a fragment with strong immune activity.

[0008] According to one aspect of the present disclosure, an immunogenic fragment or an immunogenic variant thereof is provided, wherein the immunogenic fragment comprises at least the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity thereto.

[0009] According to another aspect of the present disclosure, a recombinant protein is provided, comprising: a first domain comprising the above-mentioned immunogenic fragment or an immunogenic variant thereof; and a second domain comprising a backbone polypeptide for forming nanoparticles.

[0010] In some embodiments, the second domain serves as a scaffold polypeptide and is capable of being assembled into nanoparticles, while displaying the immunogenic fragment of the first domain or a variant thereof on the surface of the nanoparticles.

[0011] In some embodiments, the second domain is capable of forming hexameric nanoparticles.

[0012] In some embodiments, the second domain can be selected from, for example, I53-34A, I53-34B, I53-40A, I53-40B, I53-47A, I53-47B, I53-50A, I53-50B, I53-51A, I53-51B, I52-03A, I52-03B, I52-32A, I52-32B, I52-33A, I52-33B, I32-06A, I32-06B, I32-19A, I32-19B, I32-28A, I32-28B, I53-40A.1, I53-40B.1, I53-47A.1, I53-47A.1NegT2, I53-47B.1, I53-47B.1NegT2, I53-50A.1, I53-50A.1NegT2, I53-50A.1PostT1, I53-50B4 PostT1, Encapsulin, LS (dioxotetrahydropteridine synthase), E2P (dihydrosulfonyl acetyltransferase), and I3.

[0013] In a specific embodiment, the second domain can be selected from I52-32A, I52-32B, I53-50A, I53-50B, I32-28A, I32-28B, Encapsulin, LS, E2P and I3.

[0014] In some embodiments, each of the second domains (e.g., LS, E2P, and I3) can independently self-assemble into sixty-mer nanoparticles. In some embodiments, the second domains can be paired and assembled into sixty-mer nanoparticles, such as I52-32A paired with I52-32B, and I53-50A paired with I53-50B.

[0015] In some embodiments, the first domain is directly connected to the second domain or is connected through a linker.

[0016] According to another aspect of the present disclosure, a nucleic acid molecule is provided, which encodes the above-mentioned immunogenic fragment or immunogenic variant thereof or the above-mentioned recombinant protein of the present disclosure.

[0017] According to another aspect of the present disclosure, an expression vector is provided, which includes the above-mentioned nucleic acid molecule of the present disclosure.

[0018] In some embodiments, the expression vector can be selected from a viral or bacterial vector, such as, but not limited to, an African swine fever virus vector, a lentiviral vector, an avian pox virus vector, a canine measles virus vector, a herpes virus vector, a varicella virus vector, an adenovirus vector, an adeno-associated virus vector, and the like.

[0019] According to another aspect of the present disclosure, a host cell is provided, which comprises the aforementioned nucleic acid molecule, or is capable of expressing the aforementioned immunogenic fragment or immunogenic variant thereof or the aforementioned recombinant protein of the present disclosure.

[0020] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0021] In some embodiments, the prokaryotic cell can be selected from Escherichia coli or Bacillus subtilis, such as Escherichia coli BL21 (DE3), T7E, C41, Arctic, etc.

[0022] In some embodiments, the eukaryotic cells can be selected from yeast cells, insect cells, plant cells, animal cells, etc., such as yeast cells, CHO cells, 293 cells, or NSO cells.

[0023] According to another aspect of the present disclosure, a nanoparticle is provided, comprising the immunogenic fragment or immunogenic variant thereof or the recombinant protein disclosed herein, wherein the immunogenic fragment or variant thereof is displayed on the surface of the nanoparticle.

[0024] In some embodiments, the second domain serves as a scaffold polypeptide and is capable of being assembled into nanoparticles, while displaying the immunogenic fragment of the first domain or a variant thereof on the surface of the nanoparticles.

[0025] In some embodiments, the second domain is capable of forming hexameric nanoparticles.

[0026] In some embodiments, the second domain can be selected from, for example, I53-34A, I53-34B, I53-40A, I53-40B, I53-47A, I53-47B, I53-50A, I53-50B, I53-51A, I53-51B, I52-03A, I52-03B, I52-32A, I52-32B, I52-33A, I52-33B, I32-06A, I32-06B, I32-19A, I32-19B, I53-40A.1, I53-40B.1, I53-47A.1, I53-47A.1NegT 2. I53-47B.1, I53-47B.1NegT2, I53-50A.1, I53-50A.1NegT2, I53-50A.1PostT1, I53-50B4 PostT1, LS, E2P and I3.

[0027] In a specific embodiment, the second domain can be selected from I52-32A, I52-32B, I53-50A, I53-50B, I32-28A, I32-28B, Encapsulin, LS, E2P and I3.

[0028] In some embodiments, each of the second domains (e.g., LS, E2P, and I3) can independently self-assemble into sixty-mer nanoparticles. In some embodiments, the second domains can be paired and assembled into sixty-mer nanoparticles, such as I52-32A paired with I52-32B, and I53-50A paired with I53-50B.

[0029] According to another aspect of the present disclosure, an immunogenic composition is provided, comprising: the above-mentioned immunogenic fragment or immunogenic variant thereof, the above-mentioned recombinant protein, the above-mentioned nucleic acid molecule, the above-mentioned host cell or the above-mentioned nanoparticle; and a pharmaceutically acceptable carrier.

[0030] In some embodiments, the immunogenic composition may include: one or more of the above-mentioned immunogenic fragments or immunogenic variants thereof, the above-mentioned recombinant proteins, the above-mentioned nanoparticles, or nucleic acid molecules or expression vectors encoding one or more of the above-mentioned immunogenic fragments or immunogenic variants or recombinant proteins disclosed herein.

[0031] In some embodiments, the immunogenic composition may further include additional African swine fever virus antigens.

[0032] In some embodiments, the pharmaceutically acceptable carrier includes an adjuvant, which includes: a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative polymer; an immunostimulatory sequence (ISS), such as an oligodeoxyribonucleotide sequence (CpG ODN) having one or more unmethylated CpG units; a water-in-oil (W / O) adjuvant, an oil-in-water (O / W) adjuvant or an oil-in-water-in-oil (W / O / W) adjuvant, such as Freund's adjuvant, SPT emulsion, MF59, ISA 206, ISA72, adjuvant-65, SAF, etc.; a cationic lipid containing a quaternary ammonium salt such as DDA; a cytokine; aluminum hydroxide or aluminum phosphate; a saponin (e.g., Quil A, QS-21, GPI-0100); or any combination or mixture thereof.

[0033] In a preferred embodiment, the saponin is Quil A, QS-21, or GPI-0100.

[0034] In a preferred embodiment, the adjuvant comprises an emulsion; the emulsion is an SPT emulsion, an MF59 emulsion, or an emulsion formed by combining an oil with an emulsifier, the emulsion being based on light liquid paraffin oil, isoprenoid oils produced by olefin oligomerization (such as squalane or squalene oil, olefins, especially oils produced by oligomerization of isobutylene or decene), linear alkyl esters of acids or alcohols (more especially vegetable oils, ethyl oleate, propylene glycol di-(octanoate / caprylate), glycerol tri-(octanoate / caprylate) or propylene glycol dioleate), branched fatty acids or esters of alcohols (especially isostearates); emulsifiers are nonionic surfactants (especially esters of polyoxyethylated fatty acids (for example oleic acid), esters of sorbitan, esters of mannide (such as anhydrous mannitol oleate), esters of aliphatic diols, esters of glycerol, esters of polyglycerols, esters of propylene glycol and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which esters may be ethoxylated, ethers of fatty alcohols and polyols (for example oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers (especially L121).

[0035] In a preferred embodiment, the polymer of acrylic acid or methacrylic acid is a cross-linked acrylic acid or methacrylic acid polymer, in particular a compound cross-linked with a polyalkenyl ether of a sugar or a polyol, carbomer, preferably Carbopol 974P, 934P and 971P.

[0036] In a preferred embodiment, the copolymer of maleic anhydride and an alkenyl derivative is a copolymer of maleic anhydride and ethylene, EMA. In a preferred embodiment, the adjuvant is Gel 01 adjuvant.

[0037] In some embodiments, the immunogenic composition can be administered orally, intradermally, intramuscularly, or intranasally.

[0038] According to another aspect of the present disclosure, provided is the use of one or more of the above-mentioned immunogenic fragments or immunogenic variants thereof or the above-mentioned recombinant proteins of the present disclosure, nucleic acid molecules or expression vectors encoding one or more of the above-mentioned immunogenic fragments or immunogenic variants thereof or the above-mentioned recombinant proteins of the present disclosure, the above-mentioned host cells, the above-mentioned nanoparticles or the above-mentioned immunogenic compositions in the preparation of a medicament for preventing and / or treating African swine fever virus infection in a subject.

[0039] According to yet another aspect of the present disclosure, a method for preventing and / or treating African swine fever virus infection in a subject is provided.

[0040] In some embodiments, the subject is a mammal. In some embodiments, the subject is an animal of the Suidae family, such as a pig. In some embodiments, the individual or subject can be a wild boar (Sus scrofa), a domestic pig (Sus scrofa domesticus), a warthog (Potamochoerus), a forest pig (Hylochoerus), a giant forest pig (Hylochoerus), an African wild boar (Potamochoerus), or a feral pig.

[0041] In some embodiments, the African swine fever virus infection can be a pathogenic African swine fever virus infection. In some embodiments, the symptoms or diseases of pathogenic African swine fever virus infection can be selected from the group consisting of: African swine fever, acute African swine fever, chronic African swine fever, death from illness, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, recumbency, erythema, cyanotic skin malignant disease, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, difficulty breathing, nasal and conjunctival discharge, bleeding, nosebleed, abortion, leukopenia, and thrombocytopenia.

[0042] According to another aspect of the present disclosure, a kit for detecting African swine fever virus infection is provided, wherein the kit comprises the above-mentioned immunogenic fragment or immunogenic variant thereof or the above-mentioned recombinant protein of the present disclosure.

[0043] According to another aspect of the present disclosure, provided is the use of the above-mentioned immunogenic fragment or immunogenic variant thereof or the above-mentioned recombinant protein in the preparation of a kit for detecting African swine fever virus infection.

[0044] In some embodiments, the sample is selected from a body fluid or tissue sample from a subject. In some embodiments, the sample can be selected from a blood, saliva or serum sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG1 shows the SDS-PAGE results of prokaryotic expression of p15-His recombinant protein.

[0046] FIG2 shows the molecular sieve purification results of prokaryotically expressed p15-His recombinant protein.

[0047] FIG3 shows the SDS-PAGE results of eukaryotic expression of p15-I3 recombinant protein.

[0048] FIG4 shows the electron microscopic results of eukaryotic expression of p15-I3 recombinant protein.

[0049] FIG5 shows the results of the binding intensity of the prokaryotic expressed p15 recombinant protein to African swine fever positive serum.

[0050] FIG6 shows the results of the binding intensity of the eukaryotically expressed p15 recombinant protein to African swine fever positive serum.

[0051] FIG. 7 shows the results of the p15 prokaryotic antigen immunogenicity assay.

[0052] FIG8 shows the results of the immunogenicity assay of p15 eukaryotic antigen. DETAILED DESCRIPTION

[0053] To make the objectives, technical solutions, and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below with reference to the following embodiments. The specific embodiments described herein are intended only to illustrate the present disclosure and are not intended to limit the present disclosure in any way. In addition, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0054] The African swine fever virus (ASFV) genome is over 170 kb and contains over 150 open reading frames (ORFs). Viral particles exceed 200 nm in diameter. The virus forms a viral factory around the cell nucleus, where viral replication and assembly occur. ASFV particles are icosahedral and possess a multilayered envelope structure, consisting of an internal core (also called a nucleoid or viral nucleoid), a core shell, an inner envelope, a capsid, and an outer envelope (primarily composed of lipids and a small amount of protein). ASFV-encoded proteins play important roles in viral assembly, DNA replication and repair, and gene expression. Furthermore, the ASFV genome encodes numerous proteins involved in immune evasion, including proteins that inhibit type I interferon and induce apoptosis, such as DP96R, MGF-505-7R, and pE199L.

[0055] The ASFV precursor polyprotein pp62 is a structural component of the core capsid, encoded by the gene CP530R, and processed into mature viral structural proteins p15 and p35 by the pS273R protease. Among them, p15 is the main component antigen of the viral core capsid (Core shell), which can bind to phospholipids and DNA and participate in the assembly process of the virus. Constructing and expressing p15 protein is of great significance for studying the biological function of p15 protein and developing candidate vaccines for African swine fever. The present invention aims at the characteristics of heterogeneous polymerization state of p15 protein expressed in vitro, and successfully expresses a variety of African swine fever core capsid key antigens p15 with uniform polymerization state by fusing polymer backbone proteins. It has high immunogenicity and can be used for the development of vaccines, drugs and diagnostic reagents for African swine fever.

[0056] In some embodiments, the p15 protein of the African swine fever virus has the amino acid sequence shown in SEQ ID NO:1.

[0057] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0058] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0059] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0060] The ASFV immunogenic compositions described herein are preferably subunit vaccines. The "subunit vaccines" described herein comprise one or more polypeptides or proteins derived from ASFV, or immunogenic fragments thereof, or one or more nucleic acid molecules encoding immunogenic fragments thereof, and wherein the nucleic acid molecules are capable of being expressed in pigs. These polypeptides or proteins, immunogenic fragments thereof, or one or more nucleic acid molecules encoding immunogenic fragments thereof can be prepared using techniques known in the art.

[0061] As used herein, the term "immunogenic composition" refers to a composition comprising at least one antigen that induces an immunological response in a host or individual to which the immunogenic composition is administered. The immunological response may be a cellular and / or antibody-mediated immune response to the immunogenic composition of the present disclosure. Preferably, the immunogenic composition induces an immune response and, more preferably, confers protective immunity against one or more clinical signs of ASFV infection. Preferably, any host or individual referred to herein is an animal.

[0062] As used herein, numerical ranges are to be understood as including all numbers within the range. For example, a range of 1 to 20 is to be understood as including any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0063] As used herein, the term "host cell" refers to a eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a single-cell entity in vivo or in vitro, wherein the eukaryotic or prokaryotic cell can be, or has been, used as a nucleic acid recipient, including the descendants of the original cell genetically modified by the nucleic acid. It should be understood that the descendants of a single cell are not necessarily completely identical to the original parent in morphology or in terms of genes or complete set of DNA due to natural, accidental, or deliberate mutations. For example, a prokaryotic host cell of the present invention refers to a genetically modified prokaryotic host cell (e.g., bacteria) produced by introducing a heterologous nucleic acid, such as an exogenous nucleic acid that is foreign to the prokaryotic host cell (not naturally occurring) or a recombinant nucleic acid that is not normally present in the prokaryotic host cell, into a suitable prokaryotic host cell; a eukaryotic host cell of the present invention refers to a genetically modified eukaryotic host cell produced by introducing a heterologous nucleic acid, such as an exogenous nucleic acid that is foreign to the eukaryotic host cell or a recombinant nucleic acid that is not normally present in the eukaryotic host cell, into a suitable eukaryotic host cell.

[0064] The term "connector" as used herein refers to a (peptide) linker of natural and / or synthetic origin, consisting of linear amino acids. In some embodiments, the amino acid sequences of all the connectors present in the recombinant protein of the present invention are identical. In other embodiments, the amino acid sequences of at least two connectors present in the recombinant protein of the present invention are different. The connector should have a length suitable for connecting two or more monomer domains in this way, and the connector can ensure that the different domains it is connected to are correctly folded and appropriately presented, thereby exerting the function of its biological activity. In different embodiments, the connector has a flexible conformation. Suitable flexible connectors include, for example, having glycine, glutamine and / or serine residues.

[0065] In some embodiments, the linker of the present disclosure can be a flexible peptide linker. In some embodiments, the peptide linker is rich in glycine, serine, alanine, proline and / or glutamine residues. In some embodiments, the peptide linker can be selected from (G n S) m , wherein n and m are each independently selected from integers of 0 to 5. For example, n is selected from 0, 1, 2, 3, 4 or 5, and m is selected from 1, 2, 3, 4 or 5.

[0066] "Percent (%) sequence identity" relative to a reference amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference amino acid sequence, after aligning the sequences and (if necessary) introducing gaps to obtain maximum percent sequence identity, but without considering any conservative substitutions as part of the sequence identity. To determine percent amino acid sequence identity, alignment can be performed in various ways within the scope of the art, for example, using BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm that achieves maximum alignment over the full length of the compared sequences.

[0067] The variants of the immunogenic fragments of the p15 protein disclosed herein can be obtained by substitution, addition or deletion of one or more amino acids, thereby having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the immunogenic fragments of the p15 protein in terms of amino acid sequence, while retaining immunogenicity comparable thereto.

[0068] The immunogenic variants of the p15 protein immunogenic fragments disclosed herein can be obtained by replacing one or more conservative amino acids in the p15 protein immunogenic fragments. In some embodiments, the replacement of conservative amino acids can mean that an amino acid residue is replaced with a residue with similar biological properties. Particularly preferred substitutions are generally conservative in nature, that is, those that occur within the amino acid family. For example, amino acids are generally divided into four families: (1) acidic - aspartic acid and glutamic acid; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) uncharged polar - glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative changes include substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue, or substitution of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, or similar conservative substitutions of amino acids with structurally related amino acids that do not significantly affect biological activity. Thus, proteins having an amino acid sequence substantially identical to a reference molecule but with minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are within the definition of a reference polypeptide.

[0069] The second domain used herein can self-assemble in vitro or be paired with another scaffold protein to form nanoparticles, for example, self-assemble alone or in pairs to form sixty-mer nanoparticles. The immunogenic fragment of the p15 protein used herein or its variant forms a fusion protein with the second domain, and the immunogenic fragment of the p15 protein or its variant is displayed on the surface of the nanoparticle through self-assembly or paired assembly of the second domain.

[0070] In some embodiments, the second domain can be selected from some polypeptides synthesized in vitro that can self-assemble into nanoparticles, such as LS, E2P and I3, etc. These polypeptides can self-assemble into nanostructures in pairs in vitro, such as sixty-mer nanostructures.

[0071] In one embodiment, the second domain is LS. The monomeric LS subunit can be the full-length LS protein, a single polypeptide, or any portion thereof, which is capable of directing the self-assembly of the monomeric LS subunits into hexameric nanoparticles. Monomeric LS subunits from any known LS protein can be used to generate the recombinant protein of the present disclosure, as long as the monomeric LS subunit is capable of directing the self-assembly of the recombinant protein into nanoparticles displaying immunological fragments of the p15 protein on their surface. A representative LS protein has the amino acid sequence shown in SEQ ID NO:4.

[0072] In one embodiment, the second domain is E2P. The monomeric E2P subunit can be the full length of the E2P protein, a single polypeptide, or any portion thereof, which can direct the self-assembly of the monomeric E2P subunits into hexameric nanoparticles. Monomeric E2P subunits from any known E2P protein can be used to produce the recombinant protein of the present disclosure, as long as the monomeric E2P subunit is capable of directing the self-assembly of the recombinant protein into nanoparticles displaying p15 protein immunological fragments on their surface. A representative E2P protein has the amino acid sequence shown in SEQ ID NO: 5.

[0073] In one embodiment, the second domain is encapsulin. The monomeric encapsulin subunit can be the full-length encapsulin protein, a single polypeptide, or any portion thereof. Any known monomeric encapsulin subunit can be used to produce the recombinant protein of the present disclosure. A representative encapsulin protein has the amino acid sequence shown in SEQ ID NO: 3.

[0074] In one embodiment, the second domain is I3. The monomeric I3 subunit can be the full-length I3 protein, a single polypeptide, or any portion thereof, which is capable of directing the self-assembly of the monomeric I3 subunits into hexameric nanoparticles. Any known monomeric I3 subunit can be used to produce the recombinant protein of the present disclosure, as long as the monomeric I3 subunit is capable of directing the self-assembly of the recombinant protein into nanoparticles displaying the p15 protein immunological fragment on its surface. A representative I3 protein has the amino acid sequence shown in SEQ ID NO:6.

[0075] In one embodiment, the second domain can be selected from some polypeptides synthesized in vitro that can self-assemble into nanoparticles, such as I53-34A, I53-34B, I53-40A, I53-40B, I53-47A, I53-47B, I53-50A, I53-50B, I53-51A, I53-51B, I52-03A, I52-03B, I52-32A, I52-32B, I52-33A, I52 These peptides can assemble in pairs into hexameric nanostructures in vitro.

[0076] As used herein, the term "immunogenic composition" generally refers to a composition comprising at least one antigen or an immunogenic portion thereof that elicits an immune response in a host, either a cellular immune response or an antibody-mediated immune response, against the composition. Preferably, the immunogenic composition induces an immune response, and more preferably, confers protective immunity against one or more of the clinical symptoms of ASFV infection. In cases where the host exhibits a protective immune response resulting in increased resistance to the novel infection and / or reduced clinical severity of the disease, the immunogenic composition may also be referred to as a "vaccine."

[0077] The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0078] As used herein, the term "prevent and / or treat" refers to reducing the incidence of a specific ASFV infection, or reducing the severity of clinical symptoms caused by or associated with a specific ASFV infection. Additionally, the term "prevent and / or treat" may also refer to reducing the number of animals infected with a specific ASFV (i.e., reducing the incidence of ASFV infection), or reducing the severity of clinical symptoms typically associated with or caused by an ASFV infection, in a group of animals that have received an effective amount of an immunogenic composition as provided herein, compared to a group of animals that have not received the immunogenic composition.

[0079] As used herein, the term "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic effect.

[0080] The following examples and figures are provided to facilitate understanding of the present disclosure. However, it should be understood that these examples and figures are intended to illustrate the present disclosure only and are not intended to limit the present disclosure in any way. The actual scope of protection of the present disclosure is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present disclosure.

[0081] Example 1: Construction of plasmid

[0082] (1) Construction of prokaryotic recombinant plasmid

[0083] The gene encoding the truncated fragment of amino acids 1 to 152 of the p15 protein (SEQ ID NO: 1) was ligated with genes encoding different multimeric scaffold proteins (I53-50A, I53-50B, I52-32A, I52-32B, I32-28A, I32-28B, Encapsulin, LS, E2P, and I3) via a nucleic acid sequence encoding a "linker." Overlapping PCR was then used to generate the recombinant prokaryotic gene fragments. A his-tag sequence was introduced at the 5' or 3' end of the gene to facilitate subsequent purification. The recombinant gene fragments were double-digested with Nde I and Xho I restriction endonucleases, and the digested fragments were ligated with a pET-42b linearized vector using DNA ligase to generate prokaryotic recombinant plasmids.

[0084] (2) Construction of eukaryotic recombinant plasmids

[0085] The gene encoding the p15 protein (SEQ ID NO: 1) was ligated with genes encoding different multimeric scaffold proteins (I53-50A, I53-50B, I52-32A, I52-32B, I32-28A, I32-28B, Encapsulin, LS, E2P, and I3) via a nucleic acid sequence encoding a "linker." Overlapping PCR was then used to generate recombinant eukaryotic gene fragments. A his-tag coding sequence was introduced at the 5' or 3' end of the gene to facilitate subsequent purification. The recombinant gene fragments were double-digested with BamHI and XhoI restriction endonucleases. The digested fragments were then ligated with a pcDNA 3.1 linearized vector using DNA ligase to generate eukaryotic recombinant plasmids.

[0086] The sequence information involved in this example is shown in Table 1 below.

[0087] Table 1

[0088] Example 2 Prokaryotic expression of recombinant protein

[0089] (1) Transformation of BL-21 competent cells with prokaryotic recombinant plasmids: 1 μg of each prokaryotic recombinant plasmid prepared in Example 1 was mixed with 100 μl of BL-21 competent cells (purchased from Qingke Biotechnology) and placed on ice for 15 minutes. After heat shock at 42°C for 90 seconds, the cells were placed on ice for 5 minutes. Non-resistant liquid LB medium was added and incubated at 37°C and 220 rpm on a shaking platform for 40 minutes. After the incubation period, the cells were centrifuged at 2000 × g for 5 minutes, most of the supernatant was discarded, and the competent cells were resuspended in the remaining medium and evenly spread on a solid LB (Kana+) culture dish. The cells were incubated in a 37°C incubator for 12 to 16 hours.

[0090] (2) Expression and purification: A single colony was picked from the LB solid culture dish described in step (1), inoculated into 10 ml of LB liquid medium (Kana+), and cultured overnight at 37°C, 220 rpm constant temperature shaking incubator. The next day, the bacterial liquid was inoculated into 1 L of liquid LB medium (Kana+) at a ratio of 1%, and cultured at 37°C, 220 rpm constant temperature shaking incubator until the OD600 value was between 0.6 and 0.8. The culture temperature was then lowered to 16°C, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM to induce expression for 16 to 18 hours. The induced cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in Tris buffer (pH 8.0) and ultrasonically disrupted. After the cells were disrupted, the supernatant was obtained by high-speed centrifugation, and the harvested supernatant was filtered through 0.22 μm. The filtered supernatant was purified using a Ni affinity chromatography column. The chromatography column was equilibrated with equilibration buffer (Tris buffer, pH 8.0), and the supernatant was loaded onto the column. Contaminants were eluted with wash buffer (Tris buffer, 20 mM imidazole, pH 8.0) for 5 to 10 column volumes. The target protein was eluted with elution buffer (Tris buffer, 300 mM imidazole, pH 8.0). The eluted protein solution was sterilized by filtration through a 0.22 μm filter membrane. The protein concentration was determined using NanoDrop, and the yield per liter of purified bacterial solution was determined. The results are shown in Table 2. The SDS-PAGE results of the prokaryotic expression of p15-His are shown in Figure 1, and the molecular sieve purification results of the prokaryotic expression of p15-His recombinant protein are shown in Figure 2, showing that its aggregation state is heterogeneous.

[0091] (3) Assembly of two-component nanoparticles: The purified recombinant proteins and the paired scaffold proteins were mixed; negative staining grids were prepared for the samples, and the assembly effect of the nanoparticles was detected using a 120 kV electron microscope. The results are summarized in Table 2.

[0092] (4) Assembly of single-component nanoparticles: Single-component nanoparticles spontaneously assemble without the need for a separate assembly step. Negatively stained grids were prepared and the assembly of the nanoparticles was examined using a 120 kV electron microscope. The results are summarized in Table 2.

[0093] Table 2. Yield, aggregation state and assembly effect of each prokaryotic construct of p15 Note: In the assembly effect, + represents that the particles can be assembled into sixty-mer particles, and - represents that the particles cannot be assembled into sixty-mer particles.

[0094] Example 3 Eukaryotic expression system of recombinant protein

[0095] (1) Preparation of large-scale eukaryotic recombinant plasmids: Take 1 μg of each eukaryotic recombinant plasmid prepared in Example 1 and mix it with 100 μl of DH5α competent cells (purchased from Qingke Biotechnology) and place it on ice for 15 minutes. After heat shock at 42°C for 90 seconds, place it on ice for 5 minutes, add liquid LB medium without resistance, and culture it on a shaker at 37°C and 220 rpm for 40 minutes. After the culture is completed, centrifuge at 2000×g for 5 minutes, discard most of the supernatant, resuspend the competent cells in the remaining medium and evenly spread them on a solid LB (Amp+) culture dish, and culture it in a constant temperature box at 37°C for 12 to 16 hours. After the culture is completed, a single colony with good growth status on the culture dish can be picked for expansion and plasmid extraction can be performed according to the instructions of the Tiangen Plasmid Extraction Kit.

[0096] (2) Eukaryotic recombinant plasmid transfection and cell culture: Take 1 mg of eukaryotic recombinant plasmid and mix it with transfection reagent PEI in a specific ratio and let it stand at room temperature for 5-10 minutes. The density is about 2×10 6 The prepared plasmid / PEI mixture was added dropwise to the cells with shaking, and cultured at 130 rpm for 4 days.

[0097] (3) Protein purification: The cells cultured for 4 days as described in (2) were removed and the culture was centrifuged. The chromatography column was equilibrated with equilibration buffer (Tris buffer, pH 8.0), and the sample was loaded onto a Ni affinity chromatography column. The column was loaded with wash buffer (Tris buffer, 20 mM imidazole, pH 8.0) for 5 to 10 column volumes to elute the impurities. The target protein was eluted with elution buffer (Tris buffer, 300 mM imidazole, pH 8.0). The eluted protein solution was sterilized by filtration through a 0.22 μm filter membrane, and the protein concentration was determined using NanoDrop. The yield per liter of purified cells was also determined. The results are shown in Table 3. The SDS-PAGE results of an exemplary eukaryotic expression of p15-I3 recombinant protein are shown in Figure 3.

[0098] (4) Assembly of two-component nanoparticles: The purified recombinant proteins and the corresponding paired scaffold proteins were mixed; a negative staining grid was prepared for the samples, and the assembly effect of the nanoparticles was detected using a 120 kV electron microscope. The results are summarized in Table 3.

[0099] (5) Assembly of single-component nanoparticles: Single-component nanoparticles spontaneously assemble without the need for a separate assembly step. Negatively stained grids were prepared and the assembly of the nanoparticles was examined using a 120 kV electron microscope. The results are summarized in Table 3.

[0100] The electron microscopy results of an exemplary eukaryotic expression of p15-I3 recombinant protein are shown in FIG4 , which show that the protein can self-assemble into nanoparticles with a single aggregation state.

[0101] Table 3. Yield, aggregation state and assembly effect of each eukaryotic construct of p15 Note: In the assembly effect, + represents that the particles can be assembled into sixty-mer particles, and - represents that the particles cannot be assembled into sixty-mer particles.

[0102] Example 4 African swine fever positive serum binding test

[0103] Take the p15 recombinant protein in Examples 2 and 3 as an antigen, dilute it with 1×PBS to a 1μg / mL coating working solution, add it to a 96-well ELISA plate, add 100μl to each well, and place it at 4°C overnight (12-16 hours). Remove the ELISA plate and discard the liquid in the wells, wash it three times with PBST (1×PBS plus 0.5‰ Tween 20), and pat it dry on absorbent paper. Add 200μl of blocking buffer (PBST plus 0.2% BSA) to each well and place it at room temperature for 1 hour. Discard the blocking solution, wash it three times with PBST, and pat it dry on absorbent paper. Take African swine fever-positive serum (purchased from the China Veterinary Drug Administration) and dilute it 1:1000 in blocking buffer. Add 100 μl to an antigen-coated ELISA plate. Incubate at room temperature for 60 minutes. Wash the plate three times with PBST. Then, add a goat anti-swine IgG-HRP-labeled antibody diluted 1:10,000 in blocking buffer to each well. Incubate at room temperature for 60 minutes. Wash the plate five times with PBST. After patting dry, add 100 μl of the substrate TMB to each well. Incubate at room temperature in the dark for 2-10 minutes. Add 50 μl of stop solution (2 M sulfuric acid) to each well. Measure the absorbance at 450 nm with a reference wavelength of 630 nm. A positive result is determined if the absorbance is 2.0 times greater than that of the negative control (cell culture medium).

[0104] The results of binding of exemplary prokaryotically expressed p15 recombinant proteins and eukaryotically expressed p15 recombinant proteins or their assembled particles to African swine fever-positive serum are shown in Figures 5 and 6, respectively. The results show that all p15 recombinant proteins or their assembled particles have high binding strength to African swine fever-positive serum, indicating that the above-mentioned recombinant proteins or their assembled particles expressed in the present disclosure have the correct spatial conformation.

[0105] Example 5 Antigen immunogenicity determination experiment

[0106] (1) The p15 recombinant proteins in Examples 2 and 3 were used as p15 prokaryotic antigen and p15 eukaryotic antigen, and 6-week-old BALB / c mice were immunized using conventional methods. The first primary immunization was performed by subcutaneous injection of 10 μg of antigen in Freund's complete adjuvant. Four weeks later, a secondary immunization was performed with 10 μg / mouse in Freund's incomplete adjuvant.

[0107] (2) Blood samples were collected before the first basic immunization (week 0), two weeks after the first immunization (week 2), four weeks after the first immunization (week 4), and two weeks after the second immunization (week 6), and subsequent ELISA was performed to determine the antibody levels.

[0108] (3) Take the purified p15 recombinant protein or its assembled particles, dilute it with 1×PBS to a 1μg / mL coating working solution, add 100μl to each well of a 96-well ELISA plate, and place it at 4°C overnight (12-16 hours). Remove the ELISA plate and discard the liquid in the wells, wash it three times with PBST (1×PBS plus 0.5‰ Tween 20), and pat it dry on absorbent paper. Add 200μl of blocking buffer (PBST plus 0.2% BSA) to each well and place it at room temperature for 1 hour. Discard the blocking solution, wash it three times with PBST, and pat it dry on absorbent paper. Sera from immunized mice at each time point were diluted 1:1000 in blocking buffer. 100 μl was added to an antigen-coated ELISA plate and incubated at room temperature for 60 minutes. The plate was washed three times with PBST. Goat anti-mouse IgG-HRP-labeled antibody diluted 1:10,000 in blocking buffer was then added to each well. The reaction was continued at room temperature for 60 minutes. The plate was washed five times with PBST. After patting dry, 100 μl of TMB substrate was added to each well. The reaction was carried out at room temperature in the dark for 2-10 minutes. 50 μl of stop solution (2 M sulfuric acid) was added to each well. The absorbance at 450 nm was measured with a reference wavelength of 630 nm. A positive result was determined if the absorbance was 2.0 times greater than that of the negative control (cell culture medium).

[0109] The results of the immunogenicity tests of exemplary p15 prokaryotic antigens and p15 eukaryotic antigens are shown in Figures 7 and 8, respectively. The results showed that the p15 recombinant protein assembled into sixty-mer nanoparticles had higher immunogenicity than the p15 recombinant protein with heterogeneous aggregation.

[0110] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. An immunogenic fragment or an immunogenic variant thereof, characterized in that: The immunogenic fragment at least includes the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85% sequence identity thereto.

2. A recombinant protein, characterized in that The recombinant protein comprises: A first domain comprising the immunogenic fragment of claim 1 or an immunogenic variant thereof; and, The second domain includes a backbone polypeptide for forming nanoparticles.

3. The recombinant protein according to claim 2, characterized in that The second domain is selected from I53-34A, I53-34B, I53-40A, I53-40B, I53-47A, I53-47B, I53-50A, I53-50B, I53-51A, I53-51B, I52-03A, I52-03B, I52-32A, I52-32B, I52-33A, I52-33B, I32-06A, I32-06B, I32-19A, I32-19B, I32-28A, I32-28B, I53-40A.1, I53-40B.1, I53-47A.1, I53-47A.1 NegT2, I53-47B.1, I53-47B.1 At least one of NegT2, I53-50A.1, I53-50A.1 NegT2, I53-50A.1 PostT1, I53-50B4 PostT1, Encapsulin, LS, E2P and I3, Preferably, the first domain is directly connected to the second domain or connected via a linker.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the immunogenic fragment or immunogenic variant thereof according to claim 1, or encodes the recombinant protein according to claim 2 or 3.

5. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 4, or is capable of expressing the immunogenic fragment or immunogenic variant thereof of claim 1, or the recombinant protein of claim 2 or 3; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

6. A nanoparticle, characterized in that: The nanoparticle comprises the immunogenic fragment or immunogenic variant thereof according to claim 1, or the recombinant protein according to claim 2 or 3, Preferably, the immunogenic fragment or variant thereof is displayed on the surface of the nanoparticle.

7. An immunogenic composition, characterized in that The immunogenic composition comprises: the immunogenic fragment or immunogenic variant thereof according to claim 1, the recombinant protein according to claim 2 or 3, the nucleic acid molecule according to claim 4, the host cell according to claim 5 or the nanoparticle according to claim 6; and a pharmaceutically acceptable carrier, Preferably, the immunogenic composition further comprises an additional African swine fever virus antigen.

8. Use of the immunogenic fragment or immunogenic variant thereof according to claim 1, the recombinant protein according to claim 2 or 3, the nucleic acid molecule according to claim 4 or the host cell according to claim 5 in the preparation of a medicament for preventing and / or treating African swine fever virus infection in a subject.

9. The use according to claim 8, characterized in that: The subject includes a mammal, Preferably, the subject comprises an animal of the family Suidae or a pig, More preferably, the subject comprises wild boar (Sus scrofa), domestic pig (Sus scrofa domesticus), warthog (Potamochoerus), forest pig (Hylochoerus), giant forest pig (Hylochoerus), African wild boar (Potamochoerus) and feral pig.

10. The use according to claim 8 or 9, characterized in that: The African swine fever virus infection is a pathogenic African swine fever virus infection, Preferably, the disease or symptom of African swine fever virus infection is selected from the group consisting of: African swine fever, acute African swine fever, chronic African swine fever, death from illness, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, prostrate, erythema, cyanotic skin maculopathy, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, dyspnea, nasal and conjunctival secretions, bleeding, nosebleeds, abortion, leukopenia, and thrombocytopenia.

11. A kit for detecting African swine fever virus infection, the kit comprising the immunogenic fragment or immunogenic variant thereof according to claim 1, or the recombinant protein according to claim 2 or 3.

12. Use of the immunogenic fragment or immunogenic variant thereof according to claim 1, or the recombinant protein according to claim 2 or 3 in the preparation of a kit for detecting African swine fever virus infection, Preferably, the African swine fever virus is from a body fluid or tissue sample of a subject. More preferably, the body fluid sample is selected from a blood, saliva or serum sample.

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