Immunogenic composition of African swine fever virus E248R protein and application thereof

Abstract

The present disclosure relates to immunogenic fragments, recombinant proteins, immunogenic compositions and uses thereof of African swine fever virus E248R protein. The immunogenic fragment of the African swine fever virus E248R protein or the variant with immunogenicity of the immunogenic fragment can greatly improve the expression quantity, and meanwhile, the strong immunocompetence is reserved.

Description

Technical Field

[0001] This invention relates to immunogenic compositions, and more particularly to African swine fever virus E248R protein or its immunogenic fragment, the corresponding encoding nucleotide sequence, immunogenic compositions, and their uses. Background Technology

[0002] African swine fever is a viral disease caused by infection with 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 deadly, with a morbidity and mortality rate that can reach 100%.

[0003] Due to ASFV's large genome and complex immune escape mechanisms, developing an effective vaccine is extremely difficult, and to date, there is no safe and effective vaccine for epidemic prevention and control. Previous studies on African swine fever vaccines have shown that while inactivated vaccines can induce a high level of humoral immune response, they do not provide immune protection. Therefore, current African swine fever vaccine design methods mainly focus on attenuated vaccines and subunit vaccines.

[0004] Live attenuated vaccines are progressing more rapidly in various countries, but their problems are also becoming increasingly apparent. The protection provided by live attenuated vaccines typically only targets homologous strains of the same genotype and cannot protect against attacks from heterologous viruses. Live attenuated vaccines also often have associated adverse side effects, such as skin lesions and joint swelling. Furthermore, live attenuated vaccines may cause chronic or persistent infections and may potentially revert to their original virulence.

[0005] Compared to live attenuated vaccines, subunit vaccines offer a targeted approach with fewer side effects and greater safety. Furthermore, previous research has shown that various African swine fever virus antigens can induce neutralizing antibodies, providing partial immune protection, which makes the development of safe and effective African swine fever vaccines possible. With in-depth research into the structure and immunology of African swine fever virus, designing effective subunit vaccines that can generate protective antibodies and specific cellular immune responses has become a hot topic in the field. Summary of the Invention

[0006] In order to solve one of the aforementioned technical problems in the prior art, this disclosure targets the African swine fever virus (ASFV) E248R protein. By screening various E248R protein fragments obtained by deleting fragments of different lengths from the carboxyl (C-) terminus, it was found that the extracellular region fragment containing the E248R protein can greatly increase the expression level while retaining strong immune activity.

[0007] According to one aspect of this disclosure, an immunogenic fragment or an immunogenic variant thereof is provided, said immunogenic fragment being a fragment comprising at least amino acids 1 to 150 of the amino acid sequence shown in SEQ ID NO:1.

[0008] In some embodiments, the immunogenic fragment may be a fragment comprising at least amino acids 1 to 150 of the amino acid sequence shown 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 shown 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 shown 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 shown in SEQ ID NO:1.

[0009] 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 the first to 175th amino acids of the amino acid sequence shown in SEQ ID NO:1.

[0010] 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.

[0011] In some embodiments, the immunogenic fragment may 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 with it.

[0012] According to another aspect of this disclosure, a recombinant protein is provided, the recombinant protein comprising: a first domain comprising the immunogenic fragment described above or an immunogenic variant thereof; and a second domain comprising a backbone polypeptide for forming nanoparticles.

[0013] In some embodiments, the second domain serves as a scaffold polypeptide capable of self-assembling or pairing with another scaffold polypeptide to assemble into nanoparticles, while simultaneously displaying an immunogenic fragment of the first domain or a variant thereof on the surface of the nanoparticles.

[0014] In some embodiments, the second structural domain may 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, I32-0 6B, 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, I53-50B, I53-50AB1 NegT2, I53-50B4 PostT1, LS (tetrahydropteridine synthase), E2P (dihydrothioacetyltransferase), and I3.

[0015] In a specific implementation, the second structural domain may be selected from I52-32A, I52-32B, I53-50A, I53-50B, I32-28A, I32-28B, E2P, and I3.

[0016] 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 via a linker.

[0017] According to another aspect of this disclosure, a nucleic acid molecule is provided that encodes the immunogenic fragment of the present disclosure described above, or an immunogenic variant thereof, or the recombinant protein described above.

[0018] According to another aspect of this disclosure, an expression vector is provided that includes the nucleic acid molecules described above.

[0019] In some embodiments, the expression vector may be selected from viral or bacterial vectors, such as, but not limited to, African swine fever virus vectors, lentiviral vectors, avian poxvirus vectors, canine measles virus vectors, herpesvirus vectors, varicella virus vectors, adenovirus vectors, adeno-associated virus vectors, etc.

[0020] According to another aspect of this disclosure, a host cell is provided that includes the nucleic acid molecules described above, or that can express the immunogenic fragments described above or their immunogenic variants or the recombinant proteins described above.

[0021] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0022] In some embodiments, the prokaryotic cells may be selected from Escherichia coli or Bacillus subtilis, such as Escherichia coli BL21, T7E, C41, Arctic, etc.

[0023] In some embodiments, the eukaryotic cells may 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.

[0024] According to another aspect of this disclosure, a nanoparticle is provided, the nanoparticle comprising the immunogenic fragments of this disclosure described above or immunogenic variants thereof, or the recombinant protein described above. The immunogenic fragments or variants thereof are displayed on the surface of the nanoparticle.

[0025] In some embodiments, the nanoparticles comprise hexacomers formed by the self-assembly of skeletal proteins, which may be selected from, for example, LS, E2P, and I3.

[0026] In some embodiments, the nanoparticles comprise hexadecimators formed by pairing a skeletal protein in the recombinant protein with another skeletal protein, such as I53-34A paired with I53-34B, I53-40A paired with I53-40B, I53-47A paired with I53-47B, I53-50A paired with I53-50B, I53-51A paired with I53-51B, I52-03A paired with I52-03B, I52-32A paired with I52-32B, I52-33A paired with I52-33B, I32-06A paired with I32-06B, I32-19A paired with I32-19B, and I32-28A paired with I32-28B.

[0027] According to another aspect of this disclosure, an immunogenic composition is provided comprising: the immunogenic fragment of the present disclosure or an immunogenic variant thereof or the recombinant protein thereof, the nucleic acid molecule thereof, the host cell thereof or the nanoparticle thereof; and a pharmaceutically acceptable carrier.

[0028] In some embodiments, the immunogenic composition may include: one or more of the immunogenic fragments disclosed herein or immunogenic variants thereof or the recombinant protein thereof, a nucleic acid molecule or expression vector encoding one or more of the immunogenic fragments disclosed herein or immunogenic variants thereof or the recombinant protein thereof.

[0029] In some embodiments, the immunogenic composition may further include additional African swine fever virus antigen.

[0030] In some embodiments, the pharmaceutically acceptable carrier includes an adjuvant comprising: a polymer of acrylic acid or methacrylic acid, maleic anhydride, and alkenyl derivatives; an immunostimulatory sequence (ISS), such as an oligodeoxyribonucleotide sequence (CpG ODN) having one or more unmethylated CpG units; a water-in-oil (W / O), oil-in-water (O / W), or water-in-oil-in-oil (W / O / W) adjuvant, such as Freund's adjuvant, SPT emulsion, MF59, ISA206, ISA72, adjuvant-65, SAF, etc.; a cationic lipid containing a quaternary ammonium salt such as DDA; cytokines; aluminum hydroxide or aluminum phosphate; saponins (e.g., Quil A, QS-21, GPI-0100); or any combination or mixture thereof.

[0031] In a preferred embodiment, the saponin is Quil A, QS-21, or GPI-0100.

[0032] In a preferred embodiment, the adjuvant comprises an emulsion; the emulsion is an SPT emulsion, an MF59 emulsion, or an emulsion formed from a combination of oil and emulsifier, and the emulsion may be based on light liquid paraffin oil, isoprene-like oils resulting from olefin oligomerization (such as squalane or squalene oil, oils resulting from the oligomerization of olefins, particularly isobutylene or decene), linear alkyl esters of acids or alcohols (more particularly vegetable oils, ethyl oleate, propylene glycol di-(octanoate / caprylate), glyceryl tri-(octanoate / caprylate), or propylene glycol dioleate), branched fatty acids, or Esters of alcohols (especially isostearates); emulsifiers are nonionic surfactants (especially esters of polyoxyethylene fatty acids (e.g., oleic acid), esters of sorbitan, esters of dimannitol (e.g., anhydrous mannitol oleate), esters of aliphatic diols, esters of glycerol, esters of polyglycerol, esters of propylene glycol, and esters of oleic acid, esters of isostearic acid, esters of castor oil, or esters of hydroxystearic acid, which may be ethoxylated, ethers of fatty alcohols and polyols (e.g., oleyl alcohol), and polyoxypropylene-polyoxyethylene block copolymers (especially L121).

[0033] In a preferred embodiment, the polymer of acrylic acid or methacrylic acid is a cross-linked acrylic acid or methacrylic acid polymer, especially a carbomer compound cross-linked with polyolefin ethers or polyols of sugars, preferably Carbomer 974P, 934P and 971P.

[0034] In a preferred embodiment, the copolymer of maleic anhydride and alkenyl derivative is a copolymer of maleic anhydride and ethylene, EMA. In a preferred embodiment, the adjuvant is Gel 01 adjuvant.

[0035] In some embodiments, the immunogenic composition may be administered orally, intradermally, intramuscularly, or intranasally.

[0036] According to another aspect of this disclosure, the use of one or more of the above-described immunogenic fragments of this disclosure or immunogenic variants thereof or the above-described recombinant proteins, nucleic acid molecules or expression vectors encoding one or more of the above-described recombinant proteins of this disclosure in the preparation of a medicament for the prevention and / or treatment of African swine fever virus infection in a subject is provided.

[0037] According to another aspect of this disclosure, a method for preventing and / or treating African swine fever virus infection in a subject is provided.

[0038] In some embodiments, the subject refers to a mammal. In some embodiments, the subject is a Suidae animal, 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.

[0039] In some embodiments, the African swine fever virus infection may be a pathogenic African swine fever virus infection. In some embodiments, the symptoms or diseases of pathogenic African swine fever virus infection may be selected from the group consisting of: African swine fever, acute African swine fever, chronic African swine fever, death, sudden death, fever, high fever, anorexia, lethargy, weakness, loss of appetite, lying down, erythema, cyanotic skin nevus, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, dyspnea, nasal discharge and conjunctival secretions, bleeding, nosebleeds, abortion, leukopenia, and thrombocytopenia.

[0040] According to another aspect of this disclosure, a kit for detecting African swine fever virus infection is provided, the kit comprising the immunogenic fragments of the present disclosure or immunogenic variants thereof or the recombinant protein thereof.

[0041] According to another aspect of this disclosure, the use of the above-described immunogenic fragments of this disclosure or immunogenic variants thereof or the above-described recombinant proteins in the preparation of a kit for detecting African swine fever virus infection in samples from subjects is provided.

[0042] In some embodiments, the sample is selected from bodily fluids or tissue samples from the subject. In some embodiments, the sample may be selected from blood, saliva, or serum samples. Attached Figure Description

[0043] Figure 1 An electron micrograph of the assembled I3 protein, a truncated fusion of African swine fever virus E248R protein 1-161, is shown.

[0044] Figure 2 An electron micrograph of the assembled truncated LS protein of African swine fever virus E248R protein 1-161 is shown.

[0045] Figure 3 The strength of different constructs of the African swine fever virus protein E248R in recognizing African swine fever-positive serum is shown.

[0046] Figure 4 The relative antibody titers in mouse serum at different time points after immunizing mice with different constructs of African swine fever virus protein E248R are shown. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0048] The genome of African swine fever virus (ASFV) exceeds 170 kb, containing more than 150 open reading frames (ORFs). The viral particle diameter exceeds 200 nm. The virus forms a viral factory around the cell nucleus for replication and assembly. ASFV particles are icosahedral in shape and have a multi-layered envelope structure: internal core (also called nucleoid or viral nucleus), core shell, inner envelope, capsid, and external envelope (primarily composed of lipids and a small amount of protein). ASFV-encoded proteins play crucial roles in viral assembly, DNA replication and repair, and gene expression. Furthermore, the ASFV genome encodes many proteins related to immune escape, including proteins that inhibit type I interferon and induce apoptosis, such as DP96R, MGF-505-7R, and pE199L.

[0049] The E248R protein is a structural protein expressed late in the African swine fever virus (ASFV) and is located on the inner envelope of the viral particle, mediating viral membrane fusion. The E248R protein contains intramolecular disulfide bonds, and its amino acid sequence includes a putative myristylation site. During viral infection, the E248R protein is myristylated and establishes a connection with membrane components of the infected cell. As an important membrane protein in the later stages of ASFV replication, it is located on the inner envelope of the viral particle within the viral factory of the infected cell and plays a crucial role in viral replication.

[0050] In some embodiments, the full-length E248R protein of African swine fever virus has the amino acid sequence shown in SEQ ID NO:1.

[0051] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0052] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0053] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0054] The ASFV immunogenic compositions described herein are preferably subunit vaccines. The “subunit vaccine” described herein comprises one or more polypeptides or proteins derived from ASFV, or immunogenic fragments of said polypeptides or proteins, or one or more nucleic acid molecules encoding said immunogenic fragments of said polypeptides or proteins, and said nucleic acid molecules are expressible in pigs. These polypeptides or proteins, said immunogenic fragments of said polypeptides or proteins, or one or more nucleic acid molecules encoding said immunogenic fragments of said polypeptides or proteins can be prepared using techniques known in the art.

[0055] 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. This immunological response may be a cellular and / or antibody-mediated immune response to the immunogenic composition of the present 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 mentioned herein is an animal.

[0056] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include 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.

[0057] In some embodiments, the linker of this disclosure may 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 may be selected from (G... n S) mThe term n and m are each independently selected from integers from 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. As used herein, the term "linker" refers to a (peptide) linker of natural and / or synthetic origin, composed of linear amino acids. The domains in the bispecific fusion polypeptide of the present invention can be linked by linkers, wherein each linker is fused to and / or otherwise linked (e.g., via peptide bonds) with at least two polypeptides or domains. In some embodiments, all linkers present in the bispecific fusion polypeptide of the present invention have the same amino acid sequence. In other embodiments, at least two linkers present in the bispecific fusion polypeptide of the present invention have different amino acid sequences. The linker should have a length suitable for linking two or more monomeric domains in this manner, ensuring that the different domains to which it is linked fold correctly and are properly presented to perform their biologically active functions. In various embodiments, the linker has a flexible conformation. Suitable flexible linkers include, for example, those having glycine, glutamine, and / or serine residues.

[0058] The "percentage (%) sequence identity" relative to a reference amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the reference amino acid sequence after sequence alignment and (if necessary) introducing gaps to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, alignment can be performed in various ways within the art, such as using BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0059] Variants of the E248R protein immunogenic fragment disclosed herein may be obtained by substitution, addition, or deletion of one or more amino acids, thereby achieving 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 E248R protein immunogenic fragment while retaining equivalent immunogenicity.

[0060] Immunogenic variants of the E248R protein immunogenic fragment of this disclosure can be obtained by substituting one or more conserved amino acids into the E248R protein immunogenic fragment. In some embodiments, the substitution of a conserved amino acid can mean replacing an amino acid residue with a biologically similar residue. Particularly preferred substitutions are generally conserved in nature, i.e., those that occur within amino acid families. For example, amino acids are generally classified into four families: (1) acidic – aspartic acid and glutamic acid; (2) basic – lysine, arginine, histidine; (3) nonpolar – 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 conserved changes include replacing one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another hydrophobic residue; or replacing one polar residue, such as replacing lysine with arginine, aspartic acid with glutamic acid, or asparagine with glutamine; or similar conserved substitutions of amino acids with structurally related amino acids, which do not significantly affect biological activity. Therefore, proteins having a substantially identical amino acid sequence to the reference molecule but with a few amino acid substitutions that substantially do not affect the protein's immunogenicity fall within the definition of the reference polypeptide.

[0061] The second domain used in this paper can self-assemble or pair with another skeletal protein to assemble into nanoparticles in vitro. An immunogenic fragment of the E248R protein or a variant thereof used in this paper forms a fusion protein with the second domain. Through self-assembly or pairing assembly of the second domain, the immunogenic fragment of the E248R protein or a variant thereof is displayed on the surface of the nanoparticles.

[0062] In one embodiment, the second domain is dioxetine synthase (LS). The monomeric LS subunit can be the full-length LS protein, a single polypeptide, or any portion thereof, capable of directing the self-assembly of the monomeric LS subunit into nanoparticles. Monomeric LS subunits from any known LS protein can be used to generate the recombinant protein of this disclosure, provided that the monomeric LS subunit can direct the recombinant protein to self-assemble into nanoparticles displaying the E248R protein immunofragment on their surface. Representative LS proteins have the amino acid sequence shown in SEQ ID NO:14.

[0063] In one embodiment, the second domain is a pyruvate dehydrogenase complex (PDC) dihydrolipoamide acetyltransferase (E2P). The monomeric E2P subunit can be the full-length E2P protein, a single polypeptide, or any portion thereof, capable of directing the self-assembly of the monomeric E2P subunit into nanoparticles. Monomeric E2P subunits from any known E2P protein can be used to generate the recombinant protein of this disclosure, provided that the monomeric E2P subunit can direct the recombinant protein to self-assemble into nanoparticles displaying an E248R protein immunofragment on its surface. Representative E2P proteins have the amino acid sequence shown in SEQ ID NO:15.

[0064] In one embodiment, the second domain may be selected from some peptides that are synthesized in vitro and capable of pairing and assembling into nanoparticles, such as I53-34A, I53-34B, I53-40A, I53-40B, I53-47A, I53-47B, I53-50A, I53-50B, I53-51A, I53-51B, I52-03A, I52-03B, I52-32A, I52-32B, I52-33A, and I52-33B. The peptides are 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, I53-50B, I53-50AB1 NegT2, and I53-50B4 PostT1. These peptides can assemble in pairs into nanostructures in vitro, such as hexacomer nanostructures.

[0065] As used herein, the term "immunogenic composition" generally refers to a composition having a substance containing at least one antigen or its immunogenic portion, which elicits an immune response in the host against the composition, either a cellular immune response or an antibody-mediated immune response. 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. The immunogenic composition may also be referred to as a "vaccine" when the host exhibits a protective immune response that enhances resistance to novel infections and / or reduces the clinical severity of the disease.

[0066] As used herein, the term "pharmaceuticalally acceptable carrier" refers to a component of a pharmaceutical preparation that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0067] As used herein, the term "prevention and / or treatment" 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 "prevention and / or treatment" 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 commonly associated with or caused by ASFV infection, compared to a group of animals that have received an effective amount of the immunogenic composition provided herein, in a group of animals that have not received such an immunogenic composition (i.e., reducing the incidence of ASFV infection), or reducing the severity of clinical symptoms commonly associated with or caused by ASFV infection.

[0068] The term "effective dose" as used in this article refers to the effective dose and time period required to achieve the desired therapeutic or preventative effect.

[0069] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0070] To address the technical challenges of purifying membrane proteins, low expression levels, and incorrect prokaryotic expression conformations, this invention analyzed the amino acid sequence of the African swine fever virus structural protein E248R. First, by analyzing the hydrophobic distribution of the protein sequence, the extracellular, transmembrane, and intracellular regions of the full-length E248R protein were separated, obtaining the extracellular region fragment sequence of the E248R protein. Then, structural analysis of the E248R protein was performed to determine its disulfide bond positions, domains, and other information. While ensuring the integrity of its domains, different truncated E248R protein fragments were screened, resulting in multiple constructs of E248R protein that can be secreted and expressed in eukaryotes. Inoculation of mice with these E248R protein fragments induced high levels of anti-E248R antibodies.

[0071] Example 1: Construction of plasmids

[0072] 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 linked with genes encoding different multimeric backbone proteins (I53-50A, I53-50B, I52-32A, I32-28B, LS, E2P, I3) using nucleic acid sequences encoding "linkers". Overlap PCR was used to obtain recombinant eukaryotic gene fragments, and His tag sequences were introduced at the 5' or 3' 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. The digested fragment was then ligated with the pCMV-flag linearized vector using DNA ligase to obtain a eukaryotic recombinant plasmid.

[0073] Table 1

[0074] Builder number Cut-off range Serial Number E248R - Full Length 1-248 SEQ ID NO:1 E248R-115 1-115 SEQ ID NO:2 E248R-157 1-157 SEQ ID NO:3 E248R-159 1-159 SEQ ID NO:4 E248R-161 1-161 SEQ ID NO:5 E248R-163 1-163 SEQ ID NO:6 E248R-166 1-166 SEQ ID NO:7 E248R-169 1-169 SEQ ID NO:8 E248R-179 1-179 SEQ ID NO:9 E248R-189 1-189 SEQ ID NO:10 E248R-199 1-199 SEQ ID NO:11 I53-50A Skeletal proteins SEQ ID NO:12 I53-50B Skeletal proteins SEQ ID NO:13 I52-32A Skeletal proteins SEQ ID NO:14 I32-28B Skeletal proteins SEQ ID NO:15 LS Skeletal proteins SEQ ID NO:16 E2P Skeletal proteins SEQ ID NO:17 I3 Skeletal proteins SEQ ID NO:18 His Tag protein SEQ ID NO:19 connector / SEQ ID NO:20

[0075] Example 2: Eukaryotic expression system for recombinant proteins

[0076] (1) Large-scale preparation of eukaryotic recombinant plasmids: Mix 1 μg of each eukaryotic recombinant plasmid prepared in Example 1 with 100 μl of Top10 competent cells (purchased from Qingke Biotechnology) and place on ice for 15 minutes. After heat shock at 42℃ for 90 seconds, place on ice for 5 minutes, add antibiotic-free liquid LB medium, and incubate at 37℃ and 220 rpm for 40 minutes. After incubation, centrifuge at 2000×g for 5 minutes, discard most of the supernatant, resuspend the competent cells in the remaining medium, and spread evenly on solid LB (Amp+) culture dishes. Incubate at 37℃ for 12–16 hours. After incubation, single colonies with good growth status on the culture dishes can be picked for expansion culture, and plasmid extraction can be performed according to the instructions of the Tiangen plasmid large-scale extraction kit.

[0077] (2) Eukaryotic recombinant plasmid transfection and cell culture: Mix 1 mg of eukaryotic recombinant plasmid with PEI transfection reagent (purchased from Polyscience), and let stand at room temperature for 5-10 minutes. Take a sample with a density of 3-3.5 × 10⁻⁶. 6 1L of 293F cells were prepared and the plasmid / PEI mixture was added dropwise to the cells while shaking. The cells were cultured 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, and the culture was centrifuged to collect the supernatant. Cell debris was removed by filtration using a 0.45 μm filter membrane. The filtered supernatant was concentrated by ultrafiltration using a membrane encapsulation method (15 kD). After concentration, it was diluted with buffer (1×PBS buffer, pH 8.0) for later use.

[0079] (4) Protein purification: The Ni affinity chromatography column was equilibrated with equilibration buffer (1×PBS buffer, pH 8.0). The supernatant was loaded onto the Ni affinity chromatography column. The column was loaded with 5-10 column volumes of washing buffer (1×PBS buffer, 20mM imidazole, pH 8.0). After the column was equilibrated with equilibration buffer to baseline, the target protein was eluted with elution buffer (1×PBS buffer, 500mM imidazole, pH 8.0). The eluted protein solution was filtered through a 0.22μm filter membrane for sterilization. The protein concentration was determined by NanoDrop. The expression of each construct is shown in Table 2.

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

[0081] 1-199 1-189 1-179 1-169 1-166 1-163 1-161 1-159 1-157 1-115 His ＜0.1 ＜0.1 ＜0.1 ＜0.1 ＜0.5 ＞3 ＞3 ＞2 ＜0.5 ＜0.1 I53-50A ＜0.1 ＜0.1 ＜0.1 ＜0.5 ＜0.5 ＞4 ＞4 ＞3 ＜0.5 ＜0.1 I53-50B ＜0.1 ＜0.1 ＜0.1 ＜0.5 ＜0.5 ＞6 ＞3 ＞3 ＜0.5 ＜0.1 I52-32A ＜0.1 ＜0.1 ＜0.1 ＜0.5 ＜0.5 ＜0.5 ＜0.5 ＜0.5 ＜0.1 ＜0.1 I32-28B ＜0.1 ＜0.1 ＜0.1 ＜0.5 ＜0.5 ＞8 ＞5 ＞2 ＜0.5 ＜0.1 LS ＜0.1 ＜0.1 ＜0.1 ＜0.5 ＜0.5 ＞6 ＞4 ＞4 ＜1 ＜0.1 E2P ＜0.1 ＜0.1 ＜0.1 ＜0.5 ＜0.5 ＜0.5 ＜0.5 ＜0.5 ＜0.1 ＜0.1 I3 ＜0.1 ＜0.1 ＜0.1 ＜0.5 ＜0.5 ＞3 ＞3 ＞2 ＜0.5 ＜0.1

[0082] Example 3: Assembly of the purified recombinant protein into hexadecimators

[0083] (1) Assembly of two-component nanoparticles: The recombinant protein purified in Example 2 and the corresponding paired backbone protein were mixed; a negative staining support network was prepared, and the assembly effect of the nanoparticles was detected by 120kV electron microscopy. The assembly results of the recombinant protein truncated to 1-161 are summarized in Table 3.

[0084] (2) Assembly of single-component nanoparticles: Single-component nanoparticles spontaneously assemble without a separate assembly step. A negatively stained sample support network was prepared, and the assembly effect of the nanoparticles was detected using a 120kV electron microscope. The assembly results of recombinant proteins truncated to 1-161 are summarized in Table 3.

[0085] The assembly electron microscopy results of exemplary truncated fusion I3 proteins 1-161 are shown in [reference needed]. Figure 1 This shows that it can self-assemble into nanoparticles, and the polymerization state is uniform.

[0086] The assembly electron microscopy results of exemplary truncated fused LS proteins 1-161 are shown in [reference needed]. Figure 2 This indicates that it cannot self-assemble into a hexameric polymer.

[0087] Table 3. Statistical analysis of the assembly effect of African swine fever E248R protein into hexameric polymers.

[0088] Cut off skeleton Aggregation state Hexacomer particle assembly method Assembly effect 1-161 I53-50A Trimer Assembled with 50B + 1-161 I53-50B pentamer Assembled with 50A + 1-161 I52-32A Dimer Assembled with 32B + 1-161 I32-28B pentamer Assembled with 28A + 1-161 LS hexacomer Self-assembly - 1-161 E2P hexacomer Self-assembly + 1-161 I3 hexacomer Self-assembly +

[0089] Note: In the assembly effect, + indicates that it can be assembled to form hexacomer particles, and - indicates that it cannot be assembled to form hexacomer particles.

[0090] Example 4: African swine fever positive serum binding test

[0091] Take the purified E248R recombinant proteins obtained in Example 2, dilute them with 1×PBS (pH 8.0) to prepare a 1 μg / mL coating working solution, and add 100 μl to each well of a 96-well microplate. Incubate overnight (12-16 hours) at 4°C. Remove the microplate and discard the liquid in the wells. Wash three times with PBST (1×PBS with 0.5‰ Tween 20) and pat dry on absorbent paper. Add 200 μl of blocking buffer (PBST with 0.2% BSA) to each well and incubate at room temperature for 1 hour. Discard the blocking buffer, wash three times with PBST, and pat dry on absorbent paper. African swine fever positive serum (purchased from the China Institute of Veterinary Drug Control) was diluted 1:1000 with blocking buffer. 100 μl of this solution was added to an antigen-coated ELISA plate and incubated at room temperature for 60 minutes. The plate was washed three times with PBST. Then, goat anti-swine IgG-HRP marker diluted 1:5000 with blocking buffer was added to each well. The plate was incubated at room temperature for 60 minutes, washed five times with PBST, and dried. 100 μl of substrate TMB was added to each well, and the plate was incubated at room temperature in the dark for 2-10 minutes. 50 μl of stop solution (2M sulfuric acid) was added to each well. The absorbance was measured at 450 nm using 630 nm as the reference wavelength. A positive result was defined as an absorbance value greater than 2.0 times that of the negative control (cell culture medium).

[0092] The results are as follows: Figure 3 As shown, the results indicate that the recombinant E248R proteins obtained from different Examples 2 were all able to bind to African swine fever positive serum, indicating that the recombinant E248R proteins expressed in this disclosure have the correct spatial conformation.

[0093] Example 5: Antigen Immunogenicity Assay

[0094] (1) Take the E248R recombinant protein antigen purified in Example 2 and the E248R hexamer antigen assembled in Example 3, and immunize 6-week-old BALB / c mice using conventional methods. For the first basal immunization, subcutaneous injection of 10 μg of antigen was used with Freund's complete adjuvant. Four weeks later, a second immunization was performed with 10 μg / mouse using Freund's incomplete adjuvant.

[0095] (2) Blood samples were collected before the first primary 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) to measure antibody levels using ELISA.

[0096] (3) Take the purified E248R antigen and dilute it with 1×PBS (pH 8.0) to prepare a 1 μg / mL coating working solution. Add 100 μl to each well of a 96-well microplate and incubate at 4°C overnight (12–16 hours). Remove the microplate and discard the liquid in the wells. Wash three times with PBST (1×PBS with 0.5‰ Tween 20) and pat dry on absorbent paper. Add 200 μl of blocking buffer (PBST with 0.2% BSA) to each well and incubate at room temperature for 1 hour. Discard the blocking buffer, wash three times with PBST, and pat dry on absorbent paper. Serum from immunized mice at each time point was collected and diluted 1:1000 with blocking buffer. 100 μl of each solution was added to an ELISA plate coated with the antigen and incubated at room temperature for 60 minutes. The plate was washed three times with PBST. Then, goat anti-mouse IgG-HRP-labeled antibody diluted 1:10000 with blocking buffer was added to each well. The plate was incubated at room temperature for 60 minutes, washed five times with PBST, and dried. 100 μl of substrate TMB was added to each well, and the plate was incubated at room temperature in the dark for 2-10 minutes. 50 μl of stop solution (2M sulfuric acid) was added to each well. The absorbance was measured at 450 nm using 630 nm as the reference wavelength. A positive result was defined as an absorbance value greater than 2.0 times that of the negative control (cell culture medium).

[0097] Exemplary antigen immunogenicity assay results are as follows: Figure 4 As shown in the figure. The results show that the recombinant E248R protein and its assembled particles of this disclosure have high immunogenicity, and the E248R antigen assembled into hexamethylenetetramer nanoparticles has higher immunogenicity than the monomeric antigen with a heterogeneous polymerization state.

[0098] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An immunogenic fragment or a variant thereof having immunogenicity, characterized in that, the immunogenic fragment comprises at least the amino acid sequence of positions 1 to 150 of SEQ ID NO:

1.

2. The immunogenic fragment or immunogenic variant thereof according to claim 1, characterized in that, the immunogenic fragment has a length of no more than 175 amino acid residues; Preferably, the immunogenic fragment comprises at most the amino acid sequence of positions 1 to 175 of 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 set forth in at least one of SEQ ID NOs: 3-8, 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 of any one of claims 1 to 3, or an immunogenic variant thereof; and, a second domain comprising a scaffold polypeptide for forming a nanoparticle.

5. The recombinant protein of claim 1, wherein, the second domain is selected from at least one of 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, I53-50B, I53-50AB1 NegT2, I53-50B4 PostT1, LS, E2P, and I3; Preferably, the first domain is directly linked to the second domain or linked via a linker.

6. A nucleic acid molecule, characterized in that, the nucleic acid molecule encodes the immunogenic fragment of any one of claims 1 to 3, or an immunogenic variant thereof, or the recombinant protein of 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 of any one of claims 1 to 3, or an immunogenic variant thereof, 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 of any one of claims 1 to 3, or an immunogenic variant thereof, or the recombinant protein of claim 4 or 5, Preferably, the immunogenic fragment or variant thereof is displayed on the surface of the nanoparticle.

9. An immunogenic composition comprising, the immunogenic composition comprises: the immunogenic fragment of any one of claims 1 to 3, or an immunogenic variant thereof, the recombinant protein of claim 4 or 5, the nucleic acid molecule of claim 6, the expression vector of claim 7, or the nanoparticle of claim 8; and, a pharmaceutically acceptable carrier, Preferably, the immunogenic composition further comprises additional African swine fever virus antigens.

10. Use of the immunogenic fragment of any one of claims 1 to 3, or of a variant thereof having immunogenicity, of the recombinant protein of any one of claims 4 to 5, of the nucleic acid molecule of claim 6, or of the expression vector of claim 7, for the manufacture of a medicament for the prevention and / or treatment of an African swine fever virus infection in a subject.

11. Use according to claim 10, characterized in that, The subject includes a mammal, Preferably, the subject includes a Suidae animal or a pig, More preferably, the subject includes a Sus scrofa, a Sus scrofa domesticus, a Potamochoerus, a Hylochoerus, a Giant forest hog, a bushpig, and a feral pig.

12. 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 the African swine fever virus infection is selected from the group consisting of African swine fever, acute African swine fever, chronic African swine fever, moribundity, death, sudden death, fever, high fever, anorexia, lethargy, weakness, anorexia, recumbency, erythema, cyanotic cutaneous mottling, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, dyspnea, nasal and conjunctival discharge, hemorrhage, nosebleeds, abortion, leukopenia, thrombocytopenia.

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

14. Use of the immunogenic fragment of any one of claims 1 to 3, or a variant thereof having immunogenicity, or the recombinant protein of claim 4 or 5, for the manufacture of a kit for detecting an African swine fever virus infection, Preferably, the sample is selected from a bodily fluid or a tissue sample from a subject, More preferably, the sample is selected from a blood, saliva, or serum sample.

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