Immunogenic composition of african swine fever virus CD2v protein and use thereof

Abstract

An immunogenic fragment of African swine fever virus CD2v protein, a recombinant protein, an immunogenic composition, and a use thereof. The provided immunogenic fragment of African swine fever virus CD2v protein or a variant thereof with immunogenicity can greatly improve the expression quantity while retaining the strong immunocompetence.

Description

Immunogenic composition of African swine fever virus CD2v protein and its application Technical Field

[0001] The present invention relates to immunogenic compositions, and in particular to African swine fever virus CD2v protein or its immunogenic fragment, the corresponding encoding nucleotide sequence, the immunogenic composition and its use. 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 provides an immunogenic composition containing two different domains of the CD2v protein of African swine fever virus (ASFV), and its application.

[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 fragment from positions 18 to 100 of the amino acid sequence as shown in SEQ ID NO: 1, and / or at least the amino acid fragment from positions 117 to 200 of the amino acid sequence as shown in SEQ ID NO: 1.

[0009] In some embodiments, the immunogenic fragment may include at least the amino acid fragment of positions 18 to 100 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the immunogenic fragment of the CD2V protein may include at least the amino acid fragment of positions 18 to 100, 19 to 100, 18 to 101, 19 to 101, 18 to 102, 19 to 102, 18 to 103, 19 to 103, 18 to 104, 19 to 104, 18 to 105, 19 to 105, 18 to 106, 19 to 106, 18 to 107, 19 to 107, 18 to 108, 19 to 108, 18 to 109, or 19 to 109 of the amino acid sequence shown in SEQ ID NO: 1. In a preferred embodiment, the immunogenic fragment may include an amino acid fragment of positions 18 to 100, 18 to 101, 18 to 102, 18 to 103, 18 to 104, 18 to 105, 18 to 106, 18 to 107, 18 to 108 or 18 to 109 of the amino acid sequence shown in SEQ ID NO: 1.

[0010] In some embodiments, the immunogenic fragment includes at least amino acids 18 to 106 of the amino acid sequence shown in SEQ ID NO:1.

[0011] In some embodiments, the immunogenic fragment includes at most amino acids 16 to 110 of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the immunogenic fragment includes at most amino acids 18 to 110 of the amino acid sequence set forth in SEQ ID NO: 1.

[0012] In some embodiments, the immunogenic fragment can have an amino acid sequence as shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and / or SEQ ID NO: 8, or an amino acid sequence having at least 85% sequence identity thereto.

[0013] In some embodiments, the immunogenic fragment may include at least amino acids 117 to 200 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the immunogenic fragment may include at least amino acids 114 to 204 of the amino acid sequence shown in SEQ ID NO: 1.

[0014] In some embodiments, the immunogenic fragment may include at least the amino acid fragments 108-204, 109-204, 110-204, 111-204, 112-204, 113-204, 114-204, 115-204, 116-204, or 117-204 of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the immunogenic fragment may have the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, or an amino acid sequence having at least 85% sequence identity thereto.

[0015] In some embodiments, an immunogenic variant of the immunogenic fragment has an amino acid sequence that is 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% identical to the immunogenic fragment.

[0016] 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 scaffold polypeptide for forming nanoparticles.

[0017] In some embodiments, the second domain serves as a backbone polypeptide, capable of self-assembly or pairing with another backbone polypeptide to assemble into nanoparticles, while displaying the immunogenic fragment of the first domain or a variant thereof on the surface of the nanoparticles.

[0018] 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, LS (dioxotetrahydropteridine synthase), E2P (dihydrosulfonyl acetyltransferase) and I3.

[0019] In a specific embodiment, the second domain can be selected from I52-32A, I52-32B, I53-50A, I53-50B, I32-28A, I32-28B, E2P and I3.

[0020] In some embodiments, the first domain and the second domain form a fusion protein. In some embodiments, the first domain and the second domain are directly connected. In some embodiments, the first domain and the second domain are connected by a linker.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] According to another aspect of the present disclosure, a host cell is provided, which comprises the above-mentioned nucleic acid molecule of the present disclosure, or is capable of expressing the above-mentioned immunogenic fragment or immunogenic variant thereof or the above-mentioned recombinant protein of the present disclosure.

[0025] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0026] In some embodiments, the prokaryotic cell can be selected from Escherichia coli or Bacillus subtilis, such as Escherichia coli BL21, T7E, C41, Arctic, etc.

[0027] 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, Vero cells, or NSO cells.

[0028] 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.

[0029] In some embodiments, the nanoparticles include hexamers self-assembled from scaffold proteins, and the scaffold proteins can be selected from, for example, LS, E2P, and I3.

[0030] In some embodiments, the nanoparticles include a sixty-mer formed by pairing and assembling the backbone protein in the recombinant protein with another backbone protein, for example, I53-34A is paired with I53-34B, I53-40A is paired with I53-40B, I53-47A is paired with I53-47B, I53-50A is paired with I53-50B, I53-51A is paired with I53-51B, I52-03A is paired with I52-03B, I52-32A is paired with I52-32B, I52-33A is paired with I52-33B, I32-06A is paired with I32-06B, I32-19A is paired with I32-19B, and I32-28A is paired with I32-28B.

[0031] In an alternative embodiment, the nanoparticle may include one or more immunogenic fragments or immunogenic variants thereof of the present disclosure. In an exemplary embodiment, the nanoparticle may include two immunogenic fragments or immunogenic variants thereof of the present disclosure, i.e., a fragment comprising at least amino acids 18 to 100 of the amino acid sequence as shown in SEQ ID NO: 1 and a fragment comprising at least amino acids 117 to 200 of the amino acid sequence as shown in SEQ ID NO: 1.

[0032] According to another aspect of the present disclosure, an immunogenic composition is provided, comprising: the above-mentioned immunogenic fragment or immunogenic variant thereof or the above-mentioned recombinant protein, the above-mentioned nucleic acid molecule, the above-mentioned host cell or the above-mentioned nanoparticle of the present disclosure; and a pharmaceutically acceptable carrier.

[0033] In some embodiments, the immunogenic composition may include: one or more of the above-mentioned immunogenic fragments or immunogenic variants thereof or the above-mentioned recombinant proteins disclosed herein, or the above-mentioned nanoparticles, or 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 disclosed herein.

[0034] In some embodiments, the immunogenic composition may further include additional African swine fever virus antigens.

[0035] In some embodiments, the immunogenic composition may further include a pharmaceutically acceptable carrier.

[0036] 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.

[0037] In a preferred embodiment, the saponin is Quil A, QS-21, or GPI-0100.

[0038] 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).

[0039] 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.

[0040] 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.

[0041] In some embodiments, the immunogenic composition can be administered orally, intradermally, intramuscularly, or intranasally.

[0042] 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, nucleic acid molecules or expression vectors encoding one or more of the above-mentioned recombinant proteins, 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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

[0049] Figure 1 exemplifies an electron micrograph of the assembly effect of the 18-100 truncated fusion protein of the extracellular domain 1 of the African swine fever virus CD2v protein.

[0050] Figure 2 exemplifies an electron micrograph of the assembly effect of the 114-204 truncated fusion of the I53-50A protein and the I53-50B protein of the extracellular domain 2 of the African swine fever virus CD2v protein.

[0051] Figure 3 shows the binding strength of different recombinant proteins of the extracellular domain 1 of the African swine fever virus CD2v protein to African swine fever positive serum.

[0052] Figure 4 shows the binding strength of different recombinant proteins of the extracellular domain 2 of the African swine fever virus CD2v protein to African swine fever positive serum.

[0053] FIG5 shows the serum antibody levels of mice after immunization with different recombinant proteins of the extracellular domain 1 of the African swine fever virus CD2v protein.

[0054] FIG6 shows the serum antibody levels of mice after immunization with different recombinant proteins of the extracellular domain 2 of the African swine fever virus CD2v protein. DETAILED DESCRIPTION

[0055] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0056] 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.

[0057] The envelope protein CD2v, encoded by the EP402R gene and also known as the pEP402R protein, consists of 360 amino acids. It is named CD2v because the amino acid sequence of the immunoglobulin-like domain in the encoded extracellular region closely resembles that of CD2 in host cells. The CD2v protein is a glycoprotein composed of a signal peptide, two extracellular immunoglobulin-like domains, a transmembrane region, and an intracellular domain. The intracellular domain contains an acidic domain and a proline-rich repeat sequence. CD2v is a glycoprotein that helps ASFV-infected cells bind to red blood cells in pig blood, promoting the spread of the virus within the host. CD2v plays an important role in promoting viral replication and transmission, as well as immune evasion, and is a hot target for ASFV vaccine research.

[0058] In some embodiments, the full-length CD2V protein of African swine fever virus has the amino acid sequence shown in SEQ ID NO:1.

[0059] To address current issues such as the difficulty in purifying ASFV membrane proteins, low expression levels, and incorrect conformation during prokaryotic expression, this paper analyzed the amino acid sequence of the CD2v protein. First, by analyzing the hydrophobic distribution of the CD2v protein sequence, the extracellular region of the full-length CD2v protein was separated from the transmembrane region and the intracellular region. The resulting fragment sequence of the CD2v protein extracellular region spanned positions 1 to 204. Structural analysis of the CD2v protein then revealed that its extracellular domain primarily consists of two relatively independent domains (Domain 1 and Domain 2).

[0060] While ensuring the integrity of its structural domains, the inventors expressed the two domains of the CD2v extracellular domain separately and screened various truncated CD2v protein fragments to obtain multiple constructs of the CD2v protein capable of eukaryotic expression. The results showed that separating the CD2v extracellular domain into two independent domains and expressing them separately significantly improved the expression of both domains. Furthermore, when fragments of the two CD2v protein domains were simultaneously constructed onto hexameric nanoparticles, the immune response was significantly enhanced, inducing high levels of anti-CD2v antibodies in mice.

[0061] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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 invention. 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.

[0066] 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.

[0067] 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.

[0068] 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. The term "linker" as used herein refers to a (peptide) linker of natural and / or synthetic origin, consisting of linear amino acids. The domains in the bispecific fusion polypeptide of the present invention can be connected by a linker, wherein each linker is fused to at least two polypeptides or domains and / or connected in other ways (e.g., via a peptide bond). In some embodiments, the amino acid sequences of all linkers present in the bispecific fusion polypeptide of the present invention are identical. In other embodiments, the amino acid sequences of at least two linkers present in the bispecific fusion polypeptide of the present invention are different. The linker should have a length suitable for connecting two or more monomeric domains in this manner, and the linker can ensure that the different domains it connects are correctly folded and appropriately presented, thereby exerting their biologically active functions. In different embodiments, the linker has a flexible conformation. Suitable flexible linkers include, for example, glycine, glutamine and / or serine residues.

[0069] "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.

[0070] The variants of the CD2V protein immunogenic fragments disclosed herein can be those that have 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 CD2V protein immunogenic fragments in terms of amino acid sequence by substitution, addition or deletion of one or more amino acids, while retaining comparable immunogenicity.

[0071] The immunogenic variants of the CD2V protein immunogenic fragments disclosed herein can be obtained by replacing one or more conservative amino acids in the CD2V 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 an 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.

[0072] The second domain used herein can self-assemble in vitro or be paired with another scaffold protein to form nanoparticles. The immunogenic fragment of the CD2V protein or its variant used herein forms a fusion protein with the second domain. Through self-assembly or pairing assembly of the second domain, the immunogenic fragment of the CD2V protein or its variant is displayed on the surface of the nanoparticle.

[0073] 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, I3, etc. These polypeptides can self-assemble into nanostructures in pairs in vitro, such as sixty-mer nanostructures.

[0074] In one embodiment, the second domain is a dioxetidine synthase (LS). The monomeric LS subunit can be the full length of the LS protein, a single polypeptide, or any portion thereof, which is capable of directing the self-assembly of the monomeric LS subunits into nanoparticles. Monomeric LS subunits from any known LS protein can be used to produce 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 that display CD2V protein immunofragments on their surface. A representative LS protein has the amino acid sequence shown in SEQ ID NO: 17.

[0075] 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 is capable of directing the self-assembly of the monomeric E2P subunits into 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 CD2V protein immunological fragments on their surface. A representative E2P protein has the amino acid sequence shown in SEQ ID NO: 18.

[0076] 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 nanoparticles. Monomeric I3 subunits from any known I3 protein 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 that display CD2V protein immunofragments on their surface. A representative I3 protein has the amino acid sequence shown in SEQ ID NO: 19.

[0077] In one embodiment, the second domain can be selected from some polypeptides that can be paired and assembled into nanoparticles by in vitro synthesis, 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 -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, and I53-50B4PostT1. These polypeptides can assemble in pairs into nanostructures, such as hexameric nanostructures, in vitro.

[0078] 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."

[0079] 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.

[0080] 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.

[0081] 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.

[0082] The following examples and accompanying drawings are provided to facilitate understanding of the present invention. However, it should be understood that these examples and accompanying drawings are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of protection of the present invention 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 invention.

[0083] In order to solve the current technical problems such as difficult membrane protein purification, low expression level and incorrect prokaryotic expression conformation, and the full-length CD2v is not expressed or is difficult to express. The present invention analyzes the amino acid sequence of the African swine fever virus structural protein CD2V. First, by analyzing the hydrophobic distribution of the protein sequence, the extracellular region of the CD2V full-length protein is separated from the transmembrane region and the intracellular region to obtain the extracellular region fragment sequence of the CD2V protein. Then, by performing a structural analysis on the CD2V protein, its disulfide bond position, structural domain and other information are determined. Under the premise of ensuring the integrity of its structural domain, different truncated CD2V protein fragments were screened, and a variety of constructs of CD2V proteins that can be expressed by eukaryotic secretion were obtained. These CD2V protein fragments can induce the production of high levels of anti-CD2V antibodies after being inoculated into mice. The present invention decomposes the CD2v extracellular segment (18-204) into two independent structural domains for expression, and optimizes the optimal truncated construction respectively:

[0084] Domain 1: When the N-terminus is fixed at position 18 and the C-terminus is located at 100-106, the expression level is significantly increased.

[0085] Domain 2: The C-terminus is fixed at position 204, the N-terminus is located at positions 109-114, and the expression level is significantly improved.

[0086] At the same time, the inventors found that constructing the two domains of CD2v onto hexameric nanoparticles can significantly enhance the immune effect and significantly increase the level of antibodies produced.

[0087] Example 1: Construction of plasmid

[0088] The genes encoding different truncated forms of CD2v (domain 1: 10-100, 10-106, 10-109, 18-100, 18-106, 18-107, 18-109; domain 2: 108-204, 111-204, 114-204, 117-204) were connected to the genes encoding different multimeric scaffold proteins (I53-50A, I53-50B, I52-32A, I32-28B, LS, E2P or I3) through the gene encoding the "linker" (SEQ ID NO: 21). The recombinant eukaryotic gene fragments were obtained by overlapping PCR, and a His tag sequence was introduced at the 5' or 3' end of the gene to facilitate subsequent purification. The recombinant gene fragment was double-digested with Sal I and EcoR I restriction endonucleases, and the digested fragment was ligated with the pCMV-flag linearized vector using DNA ligase to obtain a eukaryotic recombinant plasmid. The sequence information involved is shown in Table 1 below.

[0089] Table 1

[0090] Example 2 Eukaryotic expression of recombinant protein

[0091] The eukaryotic expression of the recombinant protein CD2v includes the following steps:

[0092] (1) Large-scale preparation of eukaryotic recombinant plasmids: Take 1 μg of each eukaryotic recombinant plasmid prepared in Example 1 and mix it with 100 μL Top10 competent cells (purchased from Qingke Bio) 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.

[0093] (2) Eukaryotic recombinant plasmid transfection and cell culture: 1 mg of eukaryotic recombinant plasmid was mixed with transfection reagent PEI (purchased from Polyscience), and then allowed to stand at room temperature for 5-10 minutes. The density was 3-3.5×10 6 To 1 L of 293F cells with a concentration of 100 cells / mL, add the prepared plasmid / PEI mixture dropwise to the cells while shaking, and culture at 130 rpm in an incubator at 37°C with 5% CO2 for 4 days.

[0094] (3) Collection and concentration of culture supernatant: The cells cultured for 4 days as described in (2) were removed, the culture supernatant was harvested by centrifugation, and cell debris was removed by filtration using a 0.45 μm filter. The filtered supernatant was concentrated by ultrafiltration using a membrane coating method (15 kD), and after concentration, diluted with buffer (1× PBS buffer, pH 8.0) and set aside.

[0095] (4) Protein purification: The Ni column was equilibrated with equilibration buffer (1×PBS buffer, pH 8.0), and the supernatant was loaded onto the Ni column. The column was loaded with wash buffer (1×PBS buffer, 20 mM imidazole, pH 8.0) for 5 to 10 column volumes. After the column was loaded with equilibration buffer until baseline equilibrium was reached, the target protein was eluted with elution buffer (1×PBS buffer, 500 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 by NanoDrop. The expression status of each construct is shown in Tables 2 and 3.

[0096] Table 2. Expression of the extracellular domain 1 of the African swine fever CD2v protein (unit: mg / L).

[0097] Table 3. Expression of extracellular domain 2 of African swine fever CD2v protein (unit: mg / L).

[0098] Example 3: Assemble the purified antigens into sixty-mers

[0099] (1) Assembly of two-component nanoparticles: Separately purified recombinant proteins and their corresponding scaffold proteins were mixed; negatively stained grids were prepared, and the assembly of the nanoparticles was examined using a 120 kV electron microscope. The assembly results of recombinant proteins with 18-100 truncation of domain 1 and 114-204 truncation of domain 2 are shown in Tables 4 and 5 below.

[0100] (2) 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 assembly results of recombinant proteins with 18-100 truncation of domain 1 and 114-204 truncation of domain 2 are shown in Tables 4 and 5 below.

[0101] The assembly electron microscopy effect of the exemplary 18-100 truncated fusion I3 protein of domain 1 is shown in Figure 1.

[0102] The electron microscopic effect of the exemplary 114-204 truncated domain 2 after fusion of I53-50A and I53-50B is shown in Figure 2.

[0103] Table 4. Statistics of the effect of assembling sixty-mers of the extracellular domain 1 of the African swine fever CD2v protein. 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.

[0104] Table 5. Statistics of the effect of assembling sixty-mers of the extracellular domain 2 of the African swine fever CD2v protein. 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.

[0105] Example 4 African swine fever positive serum binding experiment

[0106] Take each purified recombinant protein obtained in Example 2 or the assembled particles obtained in Example 3, dilute them with 1×PBS (pH 8.0) to a 1 μg / mL coating working solution, add them to a 96-well ELISA plate, add 100 μL to each well, and place them at 4°C overnight (12-16 hours). Remove the ELISA plate and discard the liquid in the wells, wash three times with PBST (1×PBS plus 0.5‰ Tween 20), and pat dry on absorbent paper. Add 200 μL of blocking buffer (PBST plus 0.2% BSA) to each well and place them at room temperature for 1 hour. Discard the blocking solution, wash three times with PBST, and pat dry on absorbent paper. A 100 μL dilution of African swine fever-positive serum (purchased from the China Veterinary Drug Administration) at 1:1000 in blocking buffer was added to an antigen-coated ELISA plate. The plate was incubated at room temperature for 60 minutes, and the plate was washed three times with PBST. A goat anti-swine IgG-HRP conjugate diluted 1:5000 in blocking buffer was then added to each well. The plate was incubated at room temperature for 60 minutes, and the plate was washed five times with PBST. After patting dry, 100 μL of TMB substrate was added to each well. The plate was incubated at room temperature in the dark for 2-10 minutes, and 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).

[0107] The results of exemplary recombinant proteins or their assembled particles with truncated domain 1 and truncated domain 2 are shown in Figures 3 and 4, respectively. The results show that the purified recombinant proteins or their assembled particles of CD2v obtained in different Examples 2 can be bound by 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.

[0108] Example 5 Antigen immunogenicity determination experiment

[0109] (1) The partially purified CD2v recombinant protein antigen in Example 2 and the assembled CD2v hexameric antigen in Example 3 were used to immunize 6-week-old BALB / c mice using conventional methods. The first primary immunization was performed by subcutaneous injection of 10 μg of antigen in Freund's complete adjuvant; the second immunization was performed 4 weeks later, with 10 μg per mouse in Freund's incomplete adjuvant.

[0110] (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.

[0111] (3) Take the purified CD2V antigen and dilute it with 1×PBS (pH 8.0) 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).

[0112] The serum antibody levels after immunization of mice with exemplary recombinant proteins truncated at 18-100 and 18-106 of domain 1 and recombinant proteins truncated at 114-204 of domain 2 or their assembled particles are shown in Figures 5 and 6, respectively. The results show that compared with monomeric antigens with heterogeneous aggregation states, the CD2V antigen assembled into sixty-mer nanoparticles has higher immunogenicity.

[0113] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. An immunogenic fragment or an immunogenic variant thereof, characterized in that: The immunogenic fragment includes at least the amino acid fragment from positions 18 to 100 of the amino acid sequence shown in SEQ ID NO:1 and / or at least the amino acid fragment from positions 117 to 200 of the amino acid sequence shown in SEQ ID NO:

1.

2. The immunogenic fragment or immunogenic variant thereof according to claim 1, characterized in that: The immunogenic fragment at least includes amino acids 18 to 106 of the amino acid sequence shown in SEQ ID NO: 1; and / or The immunogenic fragment at least includes amino acids 114 to 204 of the amino acid sequence shown in SEQ ID NO:

1.

3. The immunogenic fragment or immunogenic variant thereof according to claim 1, characterized in that: The immunogenic fragment has an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having at least 85% sequence identity thereto; and / or, has an amino acid sequence as shown in SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, or an amino acid sequence having at least 85% sequence identity thereto.

4. A recombinant protein, characterized in that The recombinant protein comprises: A first domain comprising the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3; and The second domain includes a backbone polypeptide for forming nanoparticles.

5. The recombinant protein according to claim 4, 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, LS, E2P and I3, Preferably, the first domain is directly connected to the second domain or connected via a linker.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3, or encodes the recombinant protein according to claim 4 or 5.

7. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 6, or is capable of expressing the immunogenic fragment or immunogenic variant thereof of any one of claims 1 to 3, or the recombinant protein of claim 4 or 5; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

8. A nanoparticle, characterized in that: The nanoparticle comprises the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4 or 5, Preferably, the immunogenic fragment or variant thereof is displayed on the surface of the nanoparticle.

9. An immunogenic composition, characterized in that The immunogenic composition comprises: the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3, the recombinant protein according to claim 4 or 5, the nucleic acid molecule according to claim 6, the host cell according to claim 7 or the nanoparticle according to claim 8; and a pharmaceutically acceptable carrier, Preferably, the immunogenic composition further comprises an additional African swine fever virus antigen.

10. Use of the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3, the recombinant protein according to claim 4 or 5, the nucleic acid molecule according to claim 6, the host cell according to claim 7, the nanoparticle according to claim 8 or the immunogenic composition according to claim 9 in the preparation of a medicament for preventing and / or treating African swine fever virus infection in a subject.

11. The use according to claim 10, 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 includes 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.

12. The use according to claim 10 or 11, 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.

13. A kit for detecting African swine fever virus infection, the kit comprising the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4 or 5.

14. Use of the immunogenic fragment or immunogenic variant thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4 or 5 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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