Immunogenic composition of african swine fever virus e248r protein and use thereof

Abstract

Provided are an immunogenic fragment, a recombinant protein, and an immunogenic composition of the African swine fever virus E248R protein, as well as the use thereof. The immunogenic fragment of the African swine fever virus E248R protein or an immunogenic variant thereof can greatly improve expression levels while retaining strong immunological activity.

Description

Immunogenic composition of African swine fever virus E248R protein and its application Technical Field

[0001] The present invention relates to an immunogenic composition, and in particular to the African swine fever virus E248R 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 targets the African swine fever virus (ASFV) E248R protein, and screens a variety of E248R protein fragments obtained by fragments of different lengths lacking the carboxyl (C-) terminus. It is found that fragments containing the extracellular region of the E248R protein can greatly increase the expression level while retaining 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 is a fragment comprising at least amino acids 1 to 150 of the amino acid sequence shown in SEQ ID NO: 1.

[0009] In some embodiments, the immunogenic fragment may be a fragment comprising at least amino acids 1 to 150 of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the immunogenic fragment may be a fragment comprising at least amino acids 1 to 159 of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the immunogenic fragment may be a fragment comprising at least amino acids 1 to 161 of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the immunogenic fragment may be a fragment comprising at least amino acids 1 to 163 of the amino acid sequence set forth in SEQ ID NO: 1.

[0010] In some embodiments, the length of the immunogenic fragment does not exceed 175 amino acid residues. In some embodiments, the immunogenic fragment includes at most amino acids 1 to 175 of the amino acid sequence shown in SEQ ID NO:1.

[0011] In some embodiments, the immunogenic fragment may include a fragment of amino acids 1 to 155, 1 to 156, 1 to 157, 1 to 158, 1 to 159, 1 to 160, 1 to 161, 1 to 162, 1 to 163, 1 to 164, 1 to 165, 1 to 166, 1 to 167, 1 to 168, 1 to 169, 1 to 170, 1 to 171, 1 to 172, 1 to 173, 1 to 174 or 1 to 175 of the amino acid sequence shown in SEQ ID NO:1.

[0012] In some embodiments, the immunogenic fragment can have an amino acid sequence as shown in SEQ ID NO:3, SEQ ID NO:4, 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%, 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0023] In some embodiments, the prokaryotic cell can be selected from Escherichia coli or Bacillus subtilis, such as Escherichia coli BL21, T7E, C41, Arctic, etc.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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, and 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.

[0030] In some embodiments, the immunogenic composition may further include additional African swine fever virus antigens.

[0031] 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.

[0032] In a preferred embodiment, the saponin is Quil A, QS-21, or GPI-0100.

[0033] 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).

[0034] 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.

[0035] 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.

[0036] In some embodiments, the immunogenic composition can be administered orally, intradermally, intramuscularly, or intranasally.

[0037] According to another aspect of the present disclosure, provided are one or more of the above-mentioned immunogenic fragments or immunogenic variants thereof or the above-mentioned recombinant proteins of the present disclosure, and the use of nucleic acid molecules or expression vectors encoding one or more of the above-mentioned recombinant proteins of the present disclosure in the preparation of drugs for preventing and / or treating African swine fever virus infection in subjects.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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 of the present disclosure in preparing a kit for detecting African swine fever virus infection in a sample from a subject.

[0043] 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

[0044] Figure 1 shows an electron micrograph of the assembly of the truncated fusion I3 protein of African swine fever virus E248R protein 1-161.

[0045] Figure 2 shows an electron micrograph of the assembly of a truncated fusion LS protein of African swine fever virus E248R protein 1-161.

[0046] Figure 3 shows the strength of different constructions of African swine fever virus protein E248R in recognizing African swine fever positive serum.

[0047] Figure 4 shows the relative antibody titers in mouse sera at different time points after mice were immunized with different constructions of African swine fever virus protein E248R. DETAILED DESCRIPTION

[0048] 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.

[0049] 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.

[0050] The E248R protein is a structural protein expressed in the late stages of African swine fever virus (ASFV). It is located on the inner envelope of the virion and mediates viral membrane fusion. The E248R protein contains an intramolecular disulfide bond and a putative myristoylation site in its amino acid sequence. During viral infection, the E248R protein becomes myristoylated and establishes associations with membrane components in infected cells. As a key membrane protein in the late stages of ASFV replication, it is localized to the inner envelope of virions within the virus factory in infected cells, playing a crucial role in viral replication.

[0051] In some embodiments, the full-length E248R protein of African swine fever virus has the amino acid sequence shown in SEQ ID NO:1.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] "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.

[0060] The variants of the immunogenic fragments of the E248R protein disclosed herein may 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 immunogenic fragments of the E248R protein in terms of amino acid sequence by substitution, addition or deletion of one or more amino acids, while retaining comparable immunogenicity.

[0061] Immunogenic variants of the immunogenic fragments of the E248R protein disclosed herein can be obtained by replacing one or more conservative amino acids in the immunogenic fragments of the E248R protein. 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.

[0062] The second domain used herein is capable of self-assembly in vitro or paired with another scaffold protein to form nanoparticles. The immunogenic fragment of the E248R protein or its variant used herein forms a fusion protein with the second domain. Through self-assembly or paired assembly of the second domain, the immunogenic fragment of the E248R protein or its variant is displayed on the surface of the nanoparticle.

[0063] 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 the E248R protein immunofragment on its surface. A representative LS protein has the amino acid sequence shown in SEQ ID NO: 14.

[0064] In one embodiment, the second domain is a pyruvate dehydrogenase complex (PDC) dihydrolipoamide acetyltransferase (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 monomeric E2P subunits to self-assemble into nanoparticles. Monomeric E2P subunits from any known E2P protein can be used to produce the recombinant protein of the present invention, as long as the monomeric E2P subunits can direct the recombinant protein to self-assemble into nanoparticles displaying E248R protein immunological fragments on their surface. The representative E2P protein has the amino acid sequence shown in SEQ ID NO: 15.

[0065] 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.

[0066] 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."

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] To address current technical issues such as difficulty in membrane protein purification, low expression levels, and incorrect conformation during prokaryotic expression, the present invention analyzed the amino acid sequence of the African swine fever virus structural protein E248R. By first analyzing the hydrophobic distribution of the protein sequence, the extracellular region of the full-length E248R protein was separated from the transmembrane region and intracellular region, obtaining the sequence of the E248R protein extracellular region fragment. Structural analysis of the E248R protein was then performed to determine information such as the location of disulfide bonds and domain structure. While ensuring the integrity of its domain structure, different truncated E248R protein fragments were screened, resulting in multiple constructs of the E248R protein that can be secreted and expressed in eukaryotes. These E248R protein fragments induced high levels of anti-E248R antibodies after inoculation into mice.

[0072] Example 1: Construction of plasmid

[0073] Different truncated forms of E248R (E248R-199, E248R-189, E248R-179, E248R-169, E248R-166, E248R-163, E248R-161, E248R-159, E248R-157, E248R-115) were connected to genes encoding different multimeric scaffold proteins (I53-50A, I53-50B, I52-32A, I32-28B, LS, E2P, I3) through a nucleic acid sequence encoding a "linker". The recombinant eukaryotic gene fragment was 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 sequence information involved is shown in Table 1 below. 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.

[0074] Table 1

[0075] Example 2: Eukaryotic expression system of recombinant protein

[0076] (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 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.

[0077] (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 1L of 293F cells was prepared by adding the prepared plasmid / PEI mixture dropwise to the cells while shaking, and incubating at 130 rpm for 4 days.

[0078] (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.

[0079] (4) Protein purification: The Ni affinity chromatography column was equilibrated with equilibration buffer (1×PBS buffer, pH 8.0), and the supernatant was loaded onto the Ni affinity chromatography 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 Table 2.

[0080] Table 2. Expression of African swine fever E248R protein in various constructs (unit: mg / L)

[0081] Example 3: Hexameric assembly experiment of purified recombinant protein

[0082] (1) Assembly of two-component nanoparticles: The purified recombinant proteins from Example 2 were mixed with their corresponding scaffold proteins. Negatively stained grids were prepared and the nanoparticle assembly was examined using a 120 kV electron microscope. The assembly results for the 1-161 truncated recombinant protein are shown in Table 3.

[0083] (2) Assembly of Single-Component Nanoparticles: Single-component nanoparticles spontaneously assemble, eliminating 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 of assembling the recombinant protein with the 1-161 truncation are shown in Table 3.

[0084] The assembly electron microscopy effect of the exemplary 1-161 truncated fusion I3 protein is shown in Figure 1, which shows that it can self-assemble into nanoparticles with a single aggregation state.

[0085] The assembly electron microscopy effect of the exemplary 1-161 truncated fusion LS protein is shown in Figure 2, which shows that it cannot self-assemble to form sixty-mers.

[0086] Table 3. Statistics of the effect of African swine fever E248R protein assembly into sixty-mers.

[0087] 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.

[0088] Example 4: African swine fever positive serum binding experiment

[0089] Take each purified E248R recombinant protein obtained in Example 2 and dilute it with 1×PBS (pH 8.0) to a 1 μg / mL coating working solution. Add 100 μl to each well of a 96-well ELISA plate and incubate at 4°C overnight (12-16 hours). Remove the 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 incubate at room temperature for 1 hour. Discard the blocking solution, wash three times with PBST, and pat dry on absorbent paper. A 100 μl sample of African swine fever-positive serum (purchased from the China Veterinary Drug Administration) was diluted 1:1000 in blocking buffer and added to an antigen-coated ELISA plate. The plate was incubated at room temperature for 60 minutes, then washed three times with PBST. A goat anti-swine IgG-HRP marker diluted 1:5000 in blocking buffer was added to each well. The plate was incubated at room temperature for 60 minutes, washed five times with PBST, and patted dry. 100 μl of the substrate, TMB, 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 at 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).

[0090] The results are shown in Figure 3 below, which show that the purified recombinant proteins of E248R obtained in different Examples 2 can all be bound by African swine fever positive serum, indicating that the purified recombinant proteins of E248R expressed in the present disclosure have the correct spatial conformation.

[0091] Example 5: Antigen immunogenicity assay

[0092] (1) The purified E248R recombinant protein antigens in Example 2 and the assembled E248R hexameric antigens 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. Four weeks later, a second immunization was performed with 10 μg per mouse in Freund's incomplete adjuvant.

[0093] (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.

[0094] (3) Take the purified E248R 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 incubate 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 200μl of blocking buffer (PBST plus 0.2% BSA) to each well and incubate at room temperature for 1 hour. Discard the blocking solution, wash three times with PBST, and pat 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).

[0095] The results of exemplary antigen immunogenicity determinations are shown in Figure 4. The results show that the E248R recombinant protein and its assembled particles disclosed herein have high immunogenicity, and that the E248R antigen assembled into hexameric nanoparticles has higher immunogenicity than monomeric antigens with heterogeneous aggregation states.

[0096] 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 at least includes the amino acid fragment from positions 1 to 150 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 length of the immunogenic fragment does not exceed 175 amino acid residues; Preferably, the immunogenic fragment includes at most amino acids 1 to 175 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 at least one of SEQ ID NOs: 3 to 8, or an amino acid sequence having at least 85% sequence identity therewith.

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 1, 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 expression vector 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 any one of claims 4 to 5, the nucleic acid molecule according to claim 6 or the expression vector according to claim 7 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 sample is selected from a body fluid or tissue sample from a subject, More preferably, the sample is selected from a blood, saliva or serum sample.

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