Immunogenic composition of African swine fever virus EP153R protein and application thereof

Abstract

The invention relates to an immunogenic fragment of African swine fever virus EP153R protein, a recombinant protein, an immunogenic composition and application thereof. The immunogenic fragment of the African swine fever virus EP153R 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] The present disclosure relates to immunogenic compositions, and in particular to African swine fever virus EP153R protein or its immunogenic fragments, corresponding encoding nucleotide sequences, immunogenic compositions and uses thereof. Background Art

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

[0003] Due to the large genome structure and complex immune escape mechanism of ASFV, it is very difficult to develop an effective vaccine. So far, there is no safe and effective vaccine for epidemic prevention and control. Previous studies on African swine fever vaccines have shown that "inactivated vaccines" can induce a high level of humoral immune response, but cannot provide immune protection. Therefore, the current design methods of African swine fever vaccines mainly focus on attenuated vaccines and subunit vaccines.

[0004] The research on attenuated vaccines is progressing faster in various countries, but the problems they have are also increasingly exposed. The protection provided by attenuated vaccines is usually only against homologous strains of the same genotype, and cannot resist heterologous virus attacks. Attenuated vaccines also often have associated adverse side effects, such as skin lesions and joint swelling. In addition, attenuated vaccines may also cause chronic or persistent infections and may restore virulence.

[0005] Compared with attenuated vaccines, subunit vaccines provide a targeted approach with fewer side effects and higher safety. In addition, previous research results have shown that a variety of African swine fever virus antigens can induce the production of neutralizing antibodies and provide partial immune protection, which provides the possibility of developing a safe and effective African swine fever vaccine. With the in-depth study of the structure and immunology of African swine fever virus, designing an effective subunit vaccine that can produce protective antibodies and specific cellular immune responses has become one of the hot topics in the field. Summary of the invention

[0006] In order to solve one of the above-mentioned technical problems existing in the prior art, the present invention targets the African swine fever virus (ASFV) EP153R protein and finds that a fragment containing the extracellular region of the EP153R protein can greatly increase the expression level while retaining strong immune activity.

[0007] According to one aspect of the present disclosure, an immunogenic fragment or an immunogenic variant thereof is provided, wherein the immunogenic fragment at least comprises an amino acid fragment from positions 50 to 146 of the amino acid sequence shown in SEQ ID NO:1.

[0008] In some embodiments, the immunogenic fragment is no more than 120 amino acid residues in length.

[0009] In some embodiments, the immunogenic fragment may include a fragment of amino acids 1 to 146, 1 to 147, 1 to 148, 1 to 149, 1 to 150, 1 to 151, 1 to 152, 1 to 153, 1 to 154, 1 to 155, 1 to 156, 1 to 157 or 1 to 158 of the amino acid sequence shown in SEQ ID NO:1.

[0010] In some embodiments, the immunogenic fragment includes at most amino acids 50 to 158 of the amino acid sequence shown in SEQ ID NO:1.

[0011] In some embodiments, the immunogenic fragment includes at most amino acids 1 to 158 of the amino acid sequence as shown in SEQ ID NO:1. In some embodiments, the immunogenic fragment includes at most amino acids 1 to 154 of the amino acid sequence as shown in SEQ ID NO:1. In some embodiments, the immunogenic fragment includes at most amino acids 1 to 152 of the amino acid sequence as shown in SEQ ID NO:1. In some embodiments, the immunogenic fragment includes at most amino acids 1 to 150 of the amino acid sequence as shown in SEQ ID NO:1. In some embodiments, the immunogenic fragment includes at most amino acids 1 to 148 of the amino acid sequence as shown in SEQ ID NO:1. In some embodiments, the immunogenic fragment includes at most amino acids 1 to 146 of the amino acid sequence as 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: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 backbone polypeptide for forming nanoparticles.

[0014] In some embodiments, the second domain acts as a backbone polypeptide, capable of being assembled into nanoparticles, while displaying the immunogenic fragment of the first domain or a variant thereof on the surface of the nanoparticles.

[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, I32-0 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 (dioxotetrahydropteridine synthase), E2P (dihydrosulfide 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, Encapsulin and LS.

[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 connector.

[0018] According to another aspect of the present disclosure, a nucleic acid molecule is provided, which encodes the above-mentioned immunogenic fragment or its immunogenic variant 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, or is capable of expressing the above-mentioned immunogenic fragment or its immunogenic variant 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 cell 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, etc.

[0025] According to another aspect of the present disclosure, a nanoparticle is provided, the nanoparticle comprising the above-mentioned immunogenic fragment or its immunogenic variant or the above-mentioned recombinant protein of the present disclosure, wherein the immunogenic fragment or its variant 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 and Encapsulin.

[0027] In some embodiments, the nanoparticle includes a sixty-mer formed by pairing and assembling a skeleton protein in the recombinant protein with another skeleton 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, which includes: the above-mentioned immunogenic fragment or its immunogenic variant, the above-mentioned recombinant protein, the above-mentioned nucleic acid molecule, the above-mentioned host cell or the above-mentioned nanoparticle; and a pharmaceutically acceptable carrier.

[0029] In some embodiments, the immunogenic composition may include: one or more of the above-mentioned immunogenic fragments or immunogenic variants thereof, the above-mentioned recombinant proteins, the above-mentioned nanoparticles, or nucleic acid molecules or expression vectors encoding one or more of the above-mentioned immunogenic fragments or immunogenic variants or recombinant proteins of the present disclosure.

[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 non-methylated 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; an 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 can be 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-(caprylate / caprylate), glycerol tri-(caprylate / 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, especially a compound carbomer cross-linked with a polyalkenyl ether of a sugar or a polyol, 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 is the use of one or more of the above-mentioned immunogenic fragments or immunogenic variants thereof or the above-mentioned recombinant proteins of the present disclosure, nucleic acid molecules or expression vectors encoding one or more of the above-mentioned immunogenic fragments or immunogenic variants thereof or the above-mentioned recombinant proteins of the present disclosure, the above-mentioned host cells, the above-mentioned nanoparticles or the above-mentioned immunogenic compositions in the preparation of a medicament for preventing and / or treating African swine fever virus infection in a subject.

[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 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), and a wild 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, prostrate, erythema, cyanotic skin mole disease, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, dyspnea, nasal discharge and conjunctival secretions, bleeding, nosebleeds, abortion, leukopenia, thrombocytopenia.

[0041] According to another aspect of the present disclosure, a kit for detecting African swine fever virus infection is provided, the kit comprising the above-mentioned immunogenic fragment or its immunogenic variant 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 its immunogenic variant or the above-mentioned recombinant protein of the present disclosure in the preparation of a kit for detecting African swine fever virus infection.

[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 The results show the strength of binding of some EP153R recombinant proteins to African swine fever positive serum.

[0045] Figure 2 The figure shows the serum antibody levels of mice immunized with some EP153R recombinant proteins. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the present disclosure clearer, the present disclosure is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of the well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0047] The genome of African swine fever virus (ASFV) is more than 170kb, contains more than 150 open reading frames (ORFs), and the diameter of virus particles is more than 200nm. The virus forms a virus factory around the cell nucleus to replicate and assemble the virus. ASFV particles are icosahedral and have a multilayer envelope structure, namely the inner core (also called the nucleoid, viral nucleoid), the core shell, the inner envelope, the capsid, and the outer envelope (mainly composed of lipids and a small amount of protein). The proteins encoded by ASFV play an important role in virus assembly, DNA replication and repair, and gene expression. In addition, 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.

[0048] The EP153R gene is 474bp in length and encodes a lectin membrane protein containing 158 amino acids. It is one of the few glycosylated membrane proteins of ASFV. The protein contains N-glycosylation sites, phosphorylation sites, acylation sites, transmembrane regions, C-type animal agglutination domains, and cell attachment sequences, and is expressed in both the early and late stages of viral infection. The EP153R protective antigen synergizes with the ASFV outer membrane protein CD2v and participates in the adsorption of blood cells by virus-infected cells. The proliferation of the virus in cells is not affected by the loss of EP153R, but the blood cell adsorption phenomenon caused by ASFV-infected cells basically disappears. At the same time, the C-type animal agglutination domain of the EP153R protein has an inhibitory effect on the expression of MHC-I class antigens. The construction and expression of the EP153R protein is of great significance for studying the biological function of the EP153R protein and developing candidate vaccines for African swine fever.

[0049] In some embodiments, the full-length EP153R protein of African swine fever virus has an amino acid sequence as shown in SEQ ID NO:1.

[0050] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0051] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein 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.

[0052] 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.

[0053] The ASFV immunogenic composition described herein is preferably a subunit vaccine. The "subunit vaccine" described herein comprises one or more polypeptides or proteins derived from ASFV, or immunogenic fragments of the polypeptides or proteins, or one or more nucleic acid molecules encoding the immunogenic fragments of the polypeptides or proteins, and the nucleic acid molecules can be expressed in pigs. These polypeptides or proteins, immunogenic fragments of the polypeptides or proteins, or one or more nucleic acid molecules encoding the immunogenic fragments of the polypeptides or proteins can be prepared using techniques known in the art.

[0054] The term "immunogenic composition" as used herein refers to a composition comprising at least one antigen that induces an immunological response in a host or individual to which the immunogenic composition is administered. The immunological response may be a cellular and / or antibody mediated immune response to the immunogenic composition of the present disclosure. Preferably, the immunogenic composition induces an immune response and more preferably confers protective immunity against one or more clinical signs of ASFV infection. Preferably, any host or individual referred to herein is an animal.

[0055] Numerical ranges used herein should be understood to include all numbers within the range. For example, a range of 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.

[0056] The term "connector" as used herein refers to a (peptide) linker of natural and / or synthetic origin, consisting of linear amino acids. In some embodiments, the amino acid sequences of all the connectors present in the recombinant protein of the present invention are identical. In other embodiments, the amino acid sequences of at least two connectors present in the recombinant protein of the present invention are different. The connector should have a length suitable for connecting two or more monomer domains in this way, and the connector can ensure that the different domains connected thereto are correctly folded and appropriately presented, thereby exerting the function of its biological activity. In different embodiments, the connector has a flexible conformation. Suitable flexible connectors include, for example, having glycine, glutamine and / or serine residues.

[0057] 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.

[0058] "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 to the amino acid residues in the reference amino acid sequence, after aligning the sequences and (as needed) introducing gaps to obtain maximum percentage sequence identity, but without considering any conservative substitutions as part of the sequence identity. To determine the amino acid sequence identity percentage, alignment can be performed in various ways within the scope of the art, such as using BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0059] The variants of the immunogenic fragments of the EP153R protein disclosed herein may be substituted, added or deleted with one or more amino acids, thereby having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the immunogenic fragments of the EP153R protein in terms of amino acid sequence, while retaining comparable immunogenicity thereto.

[0060] The immunogenic variants of the EP153R protein immunogenic fragments disclosed herein can be obtained by replacing one or more conservative amino acids in the EP153R protein immunogenic fragments. In some embodiments, the replacement of conservative amino acids can represent the replacement of an amino acid residue with a biologically similar residue. Particularly preferred substitutions are generally conservative in nature, i.e., those substitutions that occur within an amino acid family. For example, amino acids are generally divided into four families: (1) acidic - aspartic acid and glutamic acid; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) uncharged polar - glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative changes include substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, or substitution of one polar residue for another polar residue, such as arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine, etc.; or similar conservative substitutions of amino acids with structurally related amino acids that do not have a significant effect on biological activity. Therefore, proteins having an amino acid sequence substantially identical to a reference molecule but having a small number of amino acid substitutions that do not substantially affect the immunogenicity of the protein are within the definition of a reference polypeptide.

[0061] The second domain used herein can be self-assembled in vitro or paired with another scaffold protein to form nanoparticles. The immunogenic fragment of the EP153R protein used herein or its variant forms a fusion protein with the second domain, and the immunogenic fragment of the EP153R protein or its variant is displayed on the surface of the nanoparticle through the self-assembly or paired assembly of the second domain.

[0062] In some embodiments, the second domain can be selected from some polypeptides synthesized in vitro that can self-assemble into nanoparticles, such as LS, Encapsulin, etc. These polypeptides can self-assemble into nanostructures in pairs in vitro, such as sixty-mer nanostructures.

[0063] In one embodiment, the second domain is Encapsulin. The monomeric Encapsulin subunit can be the full length, single polypeptide or any portion thereof of the Encapsulin protein, which can direct the monomeric Encapsulin subunits to self-assemble into nanoparticles. Any known monomeric Encapsulin subunit can be used to produce the recombinant protein of the present disclosure, as long as the monomeric Encapsulin subunit can direct the recombinant protein to self-assemble into nanoparticles displaying EP153R protein immune fragments on its surface. The representative Encapsulin protein has an amino acid sequence as shown in SEQ ID NO: 14.

[0064] In one embodiment, the second domain is (LS). The monomeric LS subunit can be the full length, single polypeptide or any portion thereof of the LS protein, which can direct the monomeric LS subunits to self-assemble 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 can direct the recombinant protein to self-assemble into nanoparticles displaying EP153R protein immune fragments on its surface. A representative LS protein has an amino acid sequence as 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.1 NegT2, I53-47B.1, I53-47B.1NegT2, I53-50A.1, I53-50A.1NegT2, I53-50A.1PostT1, I53-50B, I53-50AB1 NegT2 and I53-50B4 PostT1.

[0066] As used herein, the term "immunogenic composition" generally refers to a composition having a substance containing at least one antigen or an immunogenic portion thereof, which substance 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 novel infections 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 preparation other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0068] 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. In addition, 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 typically associated with or caused by 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 preventive effect.

[0070] Examples and drawings are provided below to help understand the present disclosure. However, it should be understood that these examples and drawings are only used to illustrate the present disclosure and do not constitute any limitation. The actual scope of protection of the present disclosure 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 disclosure.

[0071] In order to solve the current technical problems such as the difficulty in purifying membrane proteins, low expression levels, and incorrect conformation of prokaryotic expression, the present invention analyzes the amino acid sequence of the African swine fever virus structural protein EP153R. While ensuring the integrity of its structural domain, different truncated EP153R protein fragments are screened to obtain a variety of constructs of EP153R protein that can be expressed by eukaryotic secretion. These EP153R protein fragments can induce the production of high levels of anti-EP153R antibodies after inoculation into mice.

[0072] Example 1: Construction of plasmid

[0073] After connecting the genes of different truncated forms of EP153R (50-158, 50-156, 50-154, 50-152, 50-150, 50-148 and 50-146) with the genes of different polymeric skeleton proteins (I53-50B, I52-32A, I52-32B, I32-28A, I32-28B, LS, Encapsuli) through the nucleic acid sequence encoding the "linker", the recombinant eukaryotic gene fragments were obtained by overlapping PCR, and His tag sequences were introduced at the 5' or 3' of the gene to facilitate subsequent purification. The recombinant gene fragments were double-digested with Sal I and EcoR I restriction endonucleases, and the fragments after digestion were connected with the pCMV-flag linearized vector using DNA ligase to obtain eukaryotic recombinant plasmids.

[0074] The sequence information involved in this example is shown in Table 1 below.

[0075] Table 1

[0076] Protein name describe Sequence number EP153R full length SEQ ID NO:1 EP153R(50-158) Truncation range 50-158 SEQ ID NO:2 EP153R(50-156) Truncation range 50-156 SEQ ID NO:3 EP153R(50-154) Truncation range 50-154 SEQ ID NO:4 EP153R(50-152) Truncation range 50-152 SEQ ID NO:5 EP153R(50-150) Truncation range 50-150 SEQ ID NO:6 EP153R(50-148) Truncation range 50-148 SEQ ID NO:7 EP153R(50-146) Truncation range 50-146 SEQ ID NO:8 I53-50B Skeleton protein SEQ ID NO:9 I52-32A Skeleton protein SEQ ID NO:10 I52-32B Skeleton protein SEQ ID NO:11 I32-28A Skeleton protein SEQ ID NO:12 I32-28B Skeleton protein SEQ ID NO:13 Encapsulin Skeleton protein SEQ ID NO:14 LS Skeleton protein SEQ ID NO:15 his Tag protein SEQ ID NO:16 Linker - SEQ ID NO:17

[0077] Example 2 Eukaryotic expression of recombinant protein

[0078] (1) Preparation of large-scale eukaryotic recombinant plasmids: Take 1 μg of each eukaryotic recombinant plasmid prepared in Example 1 and mix it with 100 μL 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 220rpm 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 incubator 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 culture, and plasmid extraction can be performed according to the instructions of the Tiangen Plasmid Extraction Kit.

[0079] (2) Eukaryotic recombinant plasmid transfection and cell culture: Mix the eukaryotic recombinant plasmid with the transfection reagent PEI (purchased from Polyscience) and let it stand at room temperature for 5-10 minutes. Take 1L of 293 cells with moderate density and add the prepared plasmid / PEI mixture dropwise to the cells while shaking. Culture with shaking for 4 days.

[0080] (3) Collection and concentration of culture supernatant: The cells cultured for 4 days as described in (2) were taken out, the culture was centrifuged to harvest the supernatant, and the cell debris was removed by filtration using a 0.45 μm filter membrane. The filtered supernatant was concentrated by ultrafiltration using a membrane packing method (15 kD), and after concentration, it was diluted with a buffer (1×PBS buffer, pH 8.0) and set aside.

[0081] (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 chromatography column was loaded with wash buffer (1×PBS buffer, 20 mM imidazole, pH 8.0) for 5 to 10 column volumes. After the chromatography 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 yield per liter of cells after purification was also determined. The results are shown in Table 2.

[0082] Table 2. Eukaryotic expression levels of each recombinant plasmid of EP153R, unit: mg / L

[0083] 50-158 50-156 50-154 50-152 50-150 50-148 50-146 His ＜0.1 ＜0.1 ＜0.5 ＜1 ＜1 ＜1 ＜1 I32-28A ＜0.1 ＜0.1 ＜0.5 ＜1 ＜1 ＜1 ＜1 I32-28B ＜0.1 ＜0.1 ＜0.5 ＞6 ＞8 ＞8 ＞8 I52-32A ＜0.1 ＜0.1 ＜0.5 ＞6 ＞6 ＞6 ＞6 I52-32B ＜0.1 ＜0.1 ＜0.5 ＞8 ＞10 ＞10 ＞10 I53-50B ＜0.1 ＜0.1 ＜0.5 ＜0.5 ＜0.5 ＜0.5 ＜0.5 Encapsulin ＜0.1 ＜0.1 ＜0.5 ＞2 ＞3 ＞5 ＞5 LS ＜0.1 ＜0.1 ＜0.5 ＜0.5 ＜0.5 ＜0.5 ＜0.5

[0084] Example 3: The purified antigens were subjected to a sixty-mer assembly experiment

[0085] (1) Assembly of two-component nanoparticles: The purified recombinant proteins and the corresponding paired backbone proteins were mixed; negative staining grids were prepared for the samples, and the assembly effect of the nanoparticles was detected using a 120 kV electron microscope. The assembly results of the 50-152 truncated recombinant protein are shown in Table 3 below.

[0086] (2) Assembly of single-component nanoparticles: Single-component nanoparticles assemble spontaneously without the need for a separate assembly step. Prepare negatively stained grids for the samples and use a 120 kV electron microscope to detect the assembly effect of the nanoparticles. The assembly results of the recombinant protein truncated with 50-152 are shown in Table 3 below.

[0087] Table 3 Statistics of the assembly effect of the truncated recombinant protein of African swine fever EP53R protein 50-152.

[0088]

[0089]

[0090] 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.

[0091] Example 4 African swine fever positive serum binding experiment

[0092] Take the purified recombinant protein obtained in Example 2, dilute it with 1×PBS (pH 8.0) to a 1μg / mL coating working solution, add it to a 96-well ELISA plate, add 100μL to each well, and place it at 4°C overnight (12-16 hours). Take out the ELISA plate and discard the liquid in the wells, wash it three times with PBST (1×PBS plus 0.5‰ Tween 20), and pat it dry on absorbent paper. Add 200μL of blocking buffer (PBST plus 0.2% BSA) to each well and place it at room temperature for 1 hour. Shake off the blocking solution, wash it three times with PBST, and pat it dry on absorbent paper. Take African swine fever positive serum (purchased from China Veterinary Drug Administration) and dilute it with blocking solution at 1:1000, take 100 μL and add it to the ELISA plate coated with antigen, react at room temperature for 60 minutes, wash the plate 3 times with PBST, then add goat anti-swine IgG-HRP marker diluted with blocking solution 1:5000 to each well, react at room temperature for 60 minutes, wash the plate 5 times with PBST, pat dry, add 100 μL substrate TMB to each well, react at room temperature in the dark for 2-10 minutes, and add 50 μL of stop solution (2M sulfuric acid) to each well. Take 630nm as the reference wavelength and measure the absorbance value at 450nm. The absorbance value is 2.0 times greater than the negative control (negative control is cell culture medium) as the positive judgment standard.

[0093] The results of EP153R eukaryotic protein binding to African swine fever positive serum are as follows Figure 1 As shown, the results show that the purified recombinant proteins of EP153R obtained in different Examples 2 can be bound by African swine fever positive serum, indicating that the above-mentioned recombinant proteins expressed in the present disclosure have the correct spatial conformation.

[0094] Example 5 Antigen immunogenicity determination experiment

[0095] (1) Using the purified partial African swine fever virus EP153R recombinant protein or assembled particles in Example 2 and Example 3 as antigens, 6-week-old BALB / c mice were immunized by conventional methods. For the first basic immunization, 10 μg of antigen was injected subcutaneously in Freund's complete adjuvant; and a second immunization was performed 4 weeks later, 10 μg / mouse in Freund's incomplete adjuvant.

[0096] (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 the ELISA method was used to analyze the antibody levels in the serum.

[0097] (3) Take the purified EP153R recombinant protein, 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 place it at 4°C overnight (12 to 16 hours).

[0098] (4) Remove the ELISA plate and discard the liquid in the wells, wash three times with PBST (1×PBS plus 0.5‰ Tween 20), and pat dry on absorbent paper. Add 200 μL of blocking buffer (PBST plus 0.2% BSA) to each well and leave at room temperature for 1 hour.

[0099] (5) Discard the blocking solution, wash three times with PBST, and pat dry on absorbent paper. Take the immune mouse serum at each time point, dilute it 1:1000 with blocking solution, take 100 μL and add it to the ELISA plate coated with antigen, and react at room temperature for 60 minutes.

[0100] (6) Discard the liquid in the ELISA plate, wash the plate three times with PBST, and then add goat anti-swine IgG-HRP marker diluted 1:10000 in blocking solution to each well and react at room temperature for 60 minutes.

[0101] (7) Discard the liquid in the ELISA plate, wash the plate five times with PBST, pat dry, add 100 μL of substrate TMB to each well, react at room temperature in the dark for 2-10 minutes, and add 50 μL of stop solution (2 M sulfuric acid) to each well.

[0102] (8) Using 630 nm as the reference wavelength, measure the absorbance at 450 nm. A positive result is judged as an absorbance value that is 2.0 times greater than the negative control (the negative control is the cell culture medium).

[0103] (9) After the secondary antibody treatment, add 1 mL of 1× permeabilization solution and wash the cells twice with 1 mL of 1× permeabilization solution. Resuspend the cells in 1× PBS (containing 1% FBS) and use flow cytometry to detect the fluorescence intensity of each sample, which is the relative antibody level in mouse serum.

[0104] The results of serum antibody levels after immunization of mice with recombinant proteins truncated with EP153R 50-152 and 50-146 are shown in Figure 2 As shown, the results showed that each recombinant protein of EP153R had high immunogenicity, and the immunogenicity of the particle antigen was significantly higher than that of the non-particle antigen.

[0105] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments, and all technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. An immunogenic fragment or an immunogenic variant thereof, characterized in that: The immunogenic fragment at least includes the amino acid fragment from positions 50 to 146 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 120 amino acid residues; Preferably, the immunogenic fragment includes at most amino acids 50 to 154 of the amino acid sequence shown in SEQ ID NO:

1.

3. The immunogenic fragment or immunogenic variant thereof according to claim 1, characterized in that: The immunogenic fragment has an amino acid sequence as shown in SEQ ID NO: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% sequence identity thereto.

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

5. The recombinant protein according to claim 4, characterized in that The second domain is selected from I53-34A, I53-34B, I53-40A, I53-40B, I53-47A, I53-47B, I53-50A, I53-50B, I53-51A, I53-51B, I52-03A, I52-03B, I52-32A, I52-32B, I52-33A, I52-33B, I32-06A, I32-06B, I32 -19A, I32-19B, I32-28A, I32-28B, I53-40A.1, I53-40B.1, I53-47A.1, I53-47A.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 connected to the second domain or connected via a linker.

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

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

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

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

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

11. The use according to claim 10, characterized in that: The subject includes a mammal, Preferably, the subject comprises an animal of the family Suidae or a pig, More preferably, the subject includes wild boar (Sus scrofa), domestic pig (Sus scrofa domesticus), warthog (Potamochoerus), forest pig (Hylochoerus), giant forest pig (Hylochoerus), African wild boar (Potamochoerus) and feral pig.

12. The use according to claim 10 or 11, characterized in that: The African swine fever virus infection is a pathogenic African swine fever virus infection, Preferably, the disease or symptom of African swine fever virus infection is selected from the group consisting of: African swine fever, acute African swine fever, chronic African swine fever, death from illness, death, sudden death, fever, high fever, anorexia, lethargy, weakness, lack of appetite, prostrate, erythema, cyanotic skin maculopathy, dysentery, constipation, abdominal pain, respiratory symptoms, cough, vomiting, dyspnea, nasal and conjunctival secretions, bleeding, nosebleeds, abortion, leukopenia, and thrombocytopenia.

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

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

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