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WO2025093041A1 - 非洲猪瘟病毒CD2v蛋白的免疫原性组合物及其应用 - Google Patents

非洲猪瘟病毒CD2v蛋白的免疫原性组合物及其应用 Download PDF

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Publication number
WO2025093041A1
WO2025093041A1 PCT/CN2024/129778 CN2024129778W WO2025093041A1 WO 2025093041 A1 WO2025093041 A1 WO 2025093041A1 CN 2024129778 W CN2024129778 W CN 2024129778W WO 2025093041 A1 WO2025093041 A1 WO 2025093041A1
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Prior art keywords
immunogenic
swine fever
african swine
amino acid
seq
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English (en)
French (fr)
Inventor
高光侠
黄林龙
张晓林
张家龙
张云星
聂晓华
范秀丽
张立国
王祥喜
高璞
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Guangdong Lanyu Biotechnology Co Ltd
Institute of Biophysics of CAS
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Guangdong Lanyu Biotechnology Co Ltd
Institute of Biophysics of CAS
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Publication of WO2025093041A1 publication Critical patent/WO2025093041A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to an immunogenic composition, and in particular to African swine fever virus CD2v protein or an immunogenic fragment thereof, a corresponding encoding nucleotide sequence, an immunogenic composition and uses thereof.
  • African swine fever is a viral disease caused by infection with African swine fever virus (ASFV).
  • ASFV African swine fever virus
  • 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%.
  • African swine fever vaccines 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.
  • Attenuated vaccines 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.
  • subunit vaccines provide a targeted approach with fewer side effects and higher safety.
  • 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.
  • 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.
  • the present disclosure provides an immunogenic composition containing two different domains of the CD2v protein of African swine fever virus (ASFV), and applications thereof.
  • ASFV African swine fever virus
  • an immunogenic fragment or an immunogenic variant thereof is provided, wherein the immunogenic fragment includes at least the amino acid fragment of positions 18 to 100 of the amino acid sequence as shown in SEQ ID NO:1, and/or includes at least the amino acid fragment of positions 117 to 200 of the amino acid sequence as shown in SEQ ID NO:1.
  • the immunogenic fragment may include at least the amino acid fragment from positions 18 to 100 of the amino acid sequence shown in SEQ ID NO: 1.
  • the immunogenic fragment of the CD2V protein may be The invention comprises at least the amino acid fragment of positions 18 to 100, 19 to 100, 18 to 101, 19 to 101, 18 to 102, 19 to 102, 18 to 103, 19 to 103, 18 to 104, 19 to 104, 18 to 105, 19 to 105, 18 to 106, 19 to 106, 18 to 107, 19 to 107, 18 to 108, 19 to 108, 18 to 109 or 19 to 109 of the amino acid sequence shown in SEQ ID NO: 1.
  • the immunogenic fragment may include an amino acid fragment at positions 18 to 100, 18 to 101, 18 to 102, 18 to 103, 18 to 104, 18 to 105, 18 to 106, 18 to 107, 18 to 108 or 18 to 109 of the amino acid sequence shown in SEQ ID NO: 1.
  • the immunogenic fragment includes at least amino acids 18 to 106 of the amino acid sequence shown in SEQ ID NO:1.
  • the immunogenic fragment includes at most amino acids 16 to 110 of the amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the immunogenic fragment includes at most amino acids 18 to 110 of the amino acid sequence as shown in SEQ ID NO: 1.
  • the immunogenic fragment can have an amino acid sequence as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and/or SEQ ID NO:8, or an amino acid sequence having at least 85% sequence identity thereto.
  • the immunogenic fragment may include at least the amino acid fragment of positions 117 to 200 of the amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, the immunogenic fragment includes at least the amino acid fragment of positions 114 to 204 of the amino acid sequence as shown in SEQ ID NO: 1.
  • the immunogenic fragment may include at least an amino acid fragment of positions 108 to 204, 109 to 204, 110 to 204, 111 to 204, 112 to 204, 113 to 204, 114 to 204, 115 to 204, 116 to 204, or 117 to 204 of the amino acid sequence shown in SEQ ID NO: 1.
  • the immunogenic fragment may have an amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, or an amino acid sequence having at least 85% sequence identity thereto.
  • an immunogenic variant of the immunogenic fragment has an amino acid sequence that has 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 the immunogenic fragment.
  • a recombinant protein 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.
  • the second domain acts as a backbone polypeptide, capable of self-assembly or pairing with another backbone polypeptide to assemble into nanoparticles, while displaying the immunogenic fragment of the first domain or a variant thereof on the surface of the nanoparticles.
  • the second domain can be selected from, for example, I53-34A, I53-34B, I53-40A, I53-40B, I53-47A, I53-47B, I53-50A, I53-50B, I53-51A, I53-51B, I52-03A, I52-03B, I52-32A, I52-32B, I52-33A, I52-33B, I32-06A, I32-06B, I32-19A, I32-19B, I32-28A, I32-28B, I53-40A.1, I53-40B.1, I53-47A.1, I53-47A.1NegT2, I53-47B.1, I53-47B.1NegT2, I53-50A.1, I53-50A.1NegT2, I53-50A.1PostT1, I53-50B4 PostT1, LS (dihydropteroidine synthase), E2P (dihydrosulfide acet
  • the second domain can be selected from I52-32A, I52-32B, I53-50A, I53-50B, I32-28A, I32-28B, E2P and I3.
  • 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.
  • nucleic acid molecule which encodes the above-mentioned immunogenic fragment or its immunogenic variant or the above-mentioned recombinant protein of the present disclosure.
  • an expression vector which includes the above-mentioned nucleic acid molecule of the present disclosure.
  • 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.
  • 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.
  • a host cell which includes the above-mentioned nucleic acid molecule of the present disclosure, or can express the above-mentioned immunogenic fragment or its immunogenic variant or the above-mentioned recombinant protein of the present disclosure.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cell can be selected from Escherichia coli or Bacillus subtilis, such as Escherichia coli BL21, T7E, C41, Arctic, etc.
  • 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.
  • a 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.
  • the nanoparticles include hexamers self-assembled from scaffold proteins, and the scaffold proteins can be selected from, for example, LS, E2P, and I3.
  • 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.
  • I53-34A is paired with I53-34B
  • I53-40A is paired with I53-40B
  • I53-47A is paired with I53-47B
  • I53-50A is
  • the nanoparticle may include one or more immunogenic fragments of the present disclosure or a fragment thereof.
  • the nanoparticle may include two immunogenic fragments of the present disclosure or immunogenic variants thereof, i.e., a fragment comprising at least amino acids 18 to 100 of the amino acid sequence as shown in SEQ ID NO: 1 and a fragment comprising at least amino acids 117 to 200 of the amino acid sequence as shown in SEQ ID NO: 1.
  • an immunogenic composition which includes: the above-mentioned immunogenic fragment or its immunogenic variant or the above-mentioned recombinant protein, the above-mentioned nucleic acid molecule, the above-mentioned host cell or the above-mentioned nanoparticle of the present disclosure; and a pharmaceutically acceptable carrier.
  • the immunogenic composition may include: one or more of the above-mentioned immunogenic fragments or their immunogenic variants or the above-mentioned recombinant proteins disclosed herein, or the above-mentioned nanoparticles, or a nucleic acid molecule or expression vector encoding one or more of the above-mentioned immunogenic fragments or their immunogenic variants or the above-mentioned recombinant proteins disclosed herein.
  • the immunogenic composition may further include additional African swine fever virus antigens.
  • the immunogenic composition may further include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes an adjuvant, which includes: a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative polymer; an immunostimulatory sequence (ISS), such as an oligodeoxyribonucleotide sequence (CpG ODN) having one or more unmethylated CpG units; an oil-in-water (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
  • ISS
  • the saponin is Quil A, QS-21, or GPI-0100.
  • 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 (especially est
  • 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.
  • the copolymer of maleic anhydride and an alkenyl derivative is a copolymer of maleic anhydride and ethylene EMA.
  • the adjuvant is Gel 01 adjuvant.
  • the immunogenic composition can be administered orally, intradermally, intramuscularly, or intranasally.
  • a method for preventing and/or treating African swine fever virus infection in a subject is provided.
  • 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 feral pig.
  • the African swine fever virus infection can be a pathogenic African swine fever virus infection.
  • 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.
  • kits for detecting African swine fever virus infection comprising the above-mentioned immunogenic fragment or its immunogenic variant or the above-mentioned recombinant protein of the present disclosure.
  • 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.
  • the sample is selected from a body fluid or tissue sample from a subject.
  • the sample can be selected from a blood, saliva or serum sample.
  • Figure 1 exemplarily shows an electron micrograph of the assembly effect of the 18-100 truncated fusion I3 protein of the extracellular domain 1 of the African swine fever virus CD2v protein.
  • Figure 2 exemplarily shows an electron micrograph of the assembly effect of the 114-204 truncated fusion of the I53-50A protein and the I53-50B protein in the extracellular domain 2 of the African swine fever virus CD2v protein.
  • Figure 3 shows the intensity results of binding of different recombinant proteins of the extracellular domain 1 of the African swine fever virus CD2v protein to African swine fever positive serum.
  • Figure 4 shows the intensity results of binding of different recombinant proteins of the extracellular domain 2 of the African swine fever virus CD2v protein to African swine fever positive serum.
  • Figure 5 shows the serum antibodies of mice immunized with different recombinant proteins of the extracellular domain 1 of the African swine fever virus CD2v protein level.
  • FIG. 6 shows the serum antibody levels of mice immunized with different recombinant proteins of the extracellular domain 2 of the African swine fever virus CD2v protein.
  • the genome of African swine fever virus is more than 170kb, containing more than 150 open reading frames (ORFs).
  • the diameter of the virus particle 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 multi-layered envelope structure, namely the internal core (also called the nucleoid, viral nucleoid), core shell, inner envelope, capsid, and external 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.
  • 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.
  • the envelope protein CD2v is encoded by the EP402R gene, also known as the pEP402R protein, and contains 360 amino acids. It is named CD2v because the amino acid sequence of the immunoglobulin-like domain in the extracellular region of the encoded protein is very similar to the CD2 of the host cell.
  • the CD2v protein is a glycoprotein composed of a signal peptide, two extracellular immunoglobulin-like domains, a transmembrane region and an intracellular domain. The intracellular domain contains an acidic domain and a proline-rich repeat sequence.
  • CD2v is a glycoprotein that helps ASFV-infected cells bind to red blood cells in pig blood and promotes the spread of the virus in the host. CD2v plays an important role in promoting viral replication and transmission, viral immune escape, etc., and is a hot target for ASFV vaccine research.
  • the full-length CD2V protein of African swine fever virus has an amino acid sequence as shown in SEQ ID NO:1.
  • the present disclosure analyzes the amino acid sequence of CD2v protein.
  • the extracellular region of the CD2V full-length protein is separated from the transmembrane region and the intracellular region, and the sequence of the extracellular region fragment of the CD2V protein is obtained from position 1 to position 204.
  • the sequence of the extracellular region fragment of the CD2V protein is obtained from position 1 to position 204.
  • structural analysis of the CD2V protein it is determined that its extracellular mainly contains two relatively independent domains (domain 1 and domain 2).
  • the inventors expressed the two domains of the CD2v extracellular domain separately, and screened different truncated CD2v protein fragments to obtain a variety of constructs of CD2v proteins that can be expressed in eukaryotes.
  • the extracellular domain of CD2v protein is divided into two independent domains and expressed separately, the expression of these two domains can be significantly improved.
  • the fragments of the two domains of CD2v protein are simultaneously constructed on sixty-mer nanoparticles, it has a significantly enhanced immune effect and can induce high levels of anti-CD2V antibodies after immunizing mice.
  • 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.
  • 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.
  • immunogenic composition refers to a composition comprising at least one antigen that induces an immunological response in a host or individual to which the immunogenic composition is administered.
  • the immunological response may be a cellular and/or antibody mediated immune response to the immunogenic composition of the invention.
  • the immunogenic composition induces an immune response and more preferably confers protective immunity against one or more clinical signs of ASFV infection.
  • any host or individual referred to herein is an animal.
  • 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.
  • the term "host cell” refers to an in vivo or in vitro eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell strain) cultured as a single-cell entity, wherein the eukaryotic or prokaryotic cell can be, or has been, used as a nucleic acid recipient, including the progeny of an original cell genetically modified by nucleic acid. It should be understood that the progeny of a single cell are not necessarily completely identical to the original parent in morphology or in terms of genes or a complete set of DNA due to natural, accidental or deliberate mutations.
  • a prokaryotic host cell of the present invention refers to a genetically modified prokaryotic host cell (e.g., bacteria) produced by introducing a heterologous nucleic acid, such as an exogenous nucleic acid that is foreign to the prokaryotic host cell (not naturally occurring) or a recombinant nucleic acid that is not normally present in the prokaryotic host cell, into a suitable prokaryotic host cell;
  • a eukaryotic host cell of the present invention refers to a genetically modified eukaryotic host cell produced by introducing a heterologous nucleic acid, such as an exogenous nucleic acid that is foreign to the eukaryotic host cell or a recombinant nucleic acid that is not normally present in the eukaryotic host cell, into a suitable eukaryotic host cell.
  • the linker of the present disclosure can be a flexible peptide linker.
  • the peptide linker is rich in glycine, serine, alanine, proline and/or glutamine residues.
  • the peptide linker can be selected From ( GnS ) m , wherein n and m are each independently selected from integers of 0 to 5. For example, n is selected from 0, 1, 2, 3, 4 or 5, and m is selected from 1, 2, 3, 4 or 5.
  • the term "linker” as used herein refers to a (peptide) linker of natural and/or synthetic origin, consisting of linear amino acids.
  • the domains in the bispecific fusion polypeptide of the present invention can be connected by a linker, wherein each linker is fused to at least two polypeptides or domains and/or connected in other ways (e.g., via a peptide bond).
  • the amino acid sequences of all linkers present in the bispecific fusion polypeptide of the present invention are the same.
  • the amino acid sequences of at least two linkers present in the bispecific fusion polypeptide of the present invention are different.
  • the linker should have a length suitable for connecting two or more monomer domains in this way, and the linker can ensure that the different domains connected to it are correctly folded and appropriately presented, thereby exerting their biologically active functions.
  • the linker has a flexible conformation. Suitable flexible linkers include, for example, glycine, glutamine and/or serine residues.
  • Percent (%) sequence identity 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.
  • 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.
  • Variants of the immunogenic fragments of the CD2V 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 CD2V protein in terms of amino acid sequence, while retaining comparable immunogenicity thereto.
  • the immunogenic variants of the CD2V protein immunogenic fragments disclosed herein can be obtained by replacing one or more conservative amino acids in the CD2V protein immunogenic fragments.
  • 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.
  • 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.
  • 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.
  • 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 CD2V protein used herein or its variant forms a fusion protein with the second domain, and the immunogenic fragment of the CD2V protein or its variant is displayed on the surface of the nanoparticle by self-assembly or paired assembly of the second domain.
  • the second domain can be selected from some polypeptides synthesized in vitro that can self-assemble into nanoparticles, such as LS, E2P, I3, etc. These polypeptides can self-assemble into nanostructures in pairs in vitro, such as sixty-mer nanostructures.
  • the second domain is a dioxotetrahydropteroidine synthase (LS).
  • the monomeric LS subunit can be the full length of the LS protein, a single polypeptide or any portion thereof, 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 CD2V protein immune fragments on its surface.
  • a representative LS protein has an amino acid sequence as shown in SEQ ID NO: 17.
  • the second domain is E2P.
  • the monomeric E2P subunit can be the full length of the E2P protein, a single polypeptide or any portion thereof, which can direct the monomeric E2P subunits to self-assemble into nanoparticles.
  • Monomeric E2P subunits from any known E2P protein can be used to produce the recombinant protein of the present disclosure, as long as the monomeric E2P subunit can direct the recombinant protein to self-assemble into nanoparticles displaying CD2V protein immune fragments on its surface.
  • a representative E2P protein has an amino acid sequence as shown in SEQ ID NO: 18.
  • the second domain is I3.
  • the monomeric I3 subunit can be the full length of the I3 protein, a single polypeptide or any portion thereof, which can direct the monomeric I3 subunits to self-assemble into nanoparticles.
  • Monomeric I3 subunits from any known I3 protein can be used to produce the recombinant protein of the present disclosure, as long as the monomeric I3 subunit can direct the recombinant protein to self-assemble into nanoparticles displaying CD2V protein immune fragments on its surface.
  • a representative I3 protein has an amino acid sequence as shown in SEQ ID NO: 19.
  • the second domain can be selected from some polypeptides that can be paired and assembled into nanoparticles by in vitro synthesis, such as I53-34A, I53-34B, I53-40A, I53-40B, I53-47A, I53-47B, I53-50A, I53-50B, I53-51A, I53-51B, I52-03A, I52-03B, I52-32A, I52-32B, I52-33A, I52-33B , I32-06A, I32-06B, I32-19A, I32-19B, I32-28A, I32-28B, I53-40A.1, I53-40B.1, I53-47A.1, I53-47A.1NegT2, I53-47B.1, I53-47B.1NegT2, I53-50A.1, I53-50A.1NegT2, I53-50A.1PostT1 and I53-50B4 PostT1.
  • These peptides can be
  • 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.
  • 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.”
  • pharmaceutically acceptable carrier 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.
  • 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.
  • 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.
  • 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.
  • the present invention analyzes the amino acid sequence of the African swine fever virus structural protein CD2V, and first separates the extracellular region of the CD2V full-length protein from the transmembrane region and the intracellular region by analyzing the hydrophobic distribution of the protein sequence, and obtains the CD2V protein extracellular region fragment sequence. Then, by performing a structural analysis on the CD2V protein, its disulfide bond position, domain and other information are determined.
  • CD2V protein fragments On the premise of ensuring the integrity of its domain, differently truncated CD2V protein fragments were screened, and a variety of constructs of CD2V proteins that can be expressed by eukaryotic secretion were obtained. These CD2V protein fragments can induce the production of high levels of anti-CD2V antibodies after being inoculated into mice.
  • the present invention decomposes the CD2v extracellular segment (18-204) into two independent domains for expression, and optimizes the optimal truncated constructions respectively:
  • the C-terminus is fixed at position 204, the N-terminus is located at positions 109-114, and the expression level is significantly increased.
  • the inventors found that constructing the two domains of CD2v onto sixty-mer nanoparticles can significantly enhance the immune effect and significantly increase the level of antibodies produced.
  • CD2v The genes encoding different truncated forms of CD2v (domain 1: 10-100, 10-106, 10-109, 18-100, 18-106, 18-107, 18-109; domain 2: 108-204, 111-204, 114-204, 117-204) were connected to the genes encoding different multimeric skeleton proteins (I53-50A, I53-50B, I52-32A, I32-28B, LS, E2P or I3) through the gene encoding the "linker” (SEQ ID NO: 21), and 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.
  • multimeric skeleton proteins I53-50A, I53-50B, I52-32A, I32-28B, LS, E2P or I3
  • linker SEQ ID NO: 21
  • the recombinant gene fragments were double-digested with Sal I and EcoR I restriction endonucleases, and the fragments after digestion were connected to the pCMV-flag linearized vector using DNA ligase to obtain Eukaryotic recombinant plasmid, the sequence information involved is shown in Table 1 below.
  • the eukaryotic expression of the recombinant protein CD2v comprises the following steps:
  • Eukaryotic recombinant plasmid transfection and cell culture 1 mg of eukaryotic recombinant plasmid was mixed with transfection reagent PEI (purchased from Polyscience), and then allowed to stand at room temperature for 5-10 minutes. 1 L of 293F cells with a density of 3-3.5 ⁇ 10 6 cells/mL was taken, and the prepared plasmid/PEI mixture was added dropwise to the cells while shaking, and cultured at 37°C, 130 rpm in an incubator containing 5% CO 2 for 4 days.
  • transfection reagent PEI purchased from Polyscience
  • Example 3 Assembling the purified antigens into sixty-mers
  • Table 4 Statistics of the effect of assembling sixty-mers of the extracellular domain 1 of the African swine fever CD2v protein. Note: In the assembly effect, + represents that the particles can be assembled into sixty-mer particles, and - represents that the particles cannot be assembled into sixty-mer particles.
  • Table 5 Statistics of the effect of assembling sixty-mers of the extracellular domain 2 of the African swine fever CD2v protein. Note: In the assembly effect, + represents that the particles can be assembled into sixty-mer particles, and - represents that the particles cannot be assembled into sixty-mer particles.
  • Example 2 Take each purified recombinant protein obtained in Example 2 or the assembled particles obtained in Example 3, dilute them with 1 ⁇ PBS (pH 8.0) to a 1 ⁇ g/mL coating working solution, add them to a 96-well ELISA plate, add 100 ⁇ L to each well, and place them at 4°C overnight (12-16 hours). Take out 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 ⁇ L of blocking buffer (PBST plus 0.2% BSA) to each well and place at room temperature for 1 hour. Discard the blocking solution, wash three times with PBST, and pat dry on absorbent paper.
  • 1 ⁇ PBS pH 8.0
  • Example 2 The partially purified CD2v recombinant protein antigen in Example 2 and the assembled CD2v hexameric antigen in Example 3 were used to immunize 6-week-old BALB/c mice using conventional methods.
  • 10 ⁇ g of antigen was injected subcutaneously in Freund's complete adjuvant; a second immunization was performed 4 weeks later, 10 ⁇ g per mouse in Freund's incomplete adjuvant.
  • mice after immunization with exemplary recombinant proteins truncated at 18-100 and 18-106 of domain 1 and recombinant proteins truncated at 114-204 of domain 2 or their assembled particles are shown in Figures 5 and 6, respectively.
  • the results show that compared with monomeric antigens with heterogeneous aggregation states, the CD2V antigen assembled into sixty-mer nanoparticles has higher immunogenicity.

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Abstract

非洲猪瘟病毒CD2v 蛋白的免疫原性片段、重组蛋白、免疫原性组合物及其用途。提供的非洲猪瘟病毒CD2v 蛋白的免疫原性片段或其具有免疫原性的变体能够大大提高表达量，同时保留了强免疫活性。

Description

非洲猪瘟病毒CD2v蛋白的免疫原性组合物及其应用 技术领域
本发明涉及免疫原性组合物，具体涉及关于非洲猪瘟病毒CD2v蛋白或其免疫原性片段、对应的编码核苷酸序列、免疫原性组合物及其用途。
背景技术
非洲猪瘟是由非洲猪瘟病毒(African swine fever virus，ASFV)感染引起的一种病毒性疾病，急性型临床上表现为高热、沉郁、厌食、皮肤发绀、各脏器出血，该病呈现出高度的传染性和致命性，发病率和致死率可以达到100％。
由于ASFV具有庞大的基因组结构和复杂的免疫逃逸机制，使得研制有效的疫苗十分困难，至今还没有安全、有效的疫苗用于疫情防控。此前关于非洲猪瘟疫苗的研究表明，“灭活疫苗”可以诱导产生较高水平的体液免疫应答，却不能提供免疫保护。因此，目前非洲猪瘟疫苗设计方法主要集中在减毒疫苗和亚单位疫苗。
减毒疫苗在各个国家的研究进展更快，但其存在的问题也日益暴露。减毒疫苗提供的保护通常仅针对相同基因型的同源毒株，而不能抵抗异源病毒攻击。减毒疫苗通常还具有相关的不良副作用，例如皮肤损伤和关节肿胀。此外，减毒疫苗还可能导致慢性或持续性感染，并有可能恢复毒力。
与减毒疫苗相比，亚单位疫苗提供了一种副作用更少且安全性更高的靶向方法。并且此前研究结果表明，多种非洲猪瘟病毒抗原能够诱导产生中和抗体，提供部分免疫保护，这为开发安全有效的非洲猪瘟疫苗提供了可能。随着对非洲猪瘟病毒结构学、免疫学等方面的深入研究，设计出能够产生保护性抗体和特异性细胞免疫反应的有效亚单位疫苗成为领域内的热点之一。
发明内容
为了解决现有技术中存在的上述技术问题之一，本公开提供了含非洲猪瘟病毒(ASFV)的CD2v蛋白的两个不同结构域的免疫原性组合物，及其应用。
根据本公开的一个方面，提供了一种免疫原性片段或其具有免疫原性的变体，其中所述免疫原性片段至少包括如SEQ ID NO:1所示氨基酸序列的第18至100位的氨基酸片段，和/或至少包括如SEQ ID NO:1所示氨基酸序列的第117至200位的氨基酸片段。
在一些实施方式中，所述免疫原性片段可以至少包括如SEQ ID NO:1所示氨基酸序列的第18至100位的氨基酸片段。在一些实施方式中，所述CD2V蛋白的免疫原性片段可以 至少包括如SEQ ID NO:1所示氨基酸序列的第18～100位、第19～100位、第18～101位、第19～101位、第18～102位、第19～102位、第18～103位、第19～103位、第18～104位、第19～104位、第18～105位、第19～105位第18～106位、第19～106位、第18～107位、第19～107位、第18～108位、第19～108位、第18～109位或第19～109位的氨基酸片段。在优选的实施方式中，所述免疫原性片段可以包括如SEQ ID NO:1所示氨基酸序列的第18～100位、第18～101位、第18～102位、第18～103位、第18～104位、第18～105位、第18～106位、第18～107位、第18～108位或第18～109位的氨基酸片段。
在一些实施方式中，所述免疫原性片段至少包括如SEQ ID NO:1所示氨基酸序列的第18至106位氨基酸。
在一些实施方式中，所述免疫原性片段至多包括如SEQ ID NO:1所示氨基酸序列的第16至110位氨基酸。在一些实施方式中，所述免疫原性片段至多包括如SEQ ID NO:1所示氨基酸序列的第18至110位氨基酸。
在一些实施方式中，所述免疫原性片段可以具有如SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7和/或SEQ ID NO:8所示的氨基酸序列，或与其具有至少85％序列同一性的氨基酸序列。
在一些实施方式中，所述免疫原性片段可以至少包括如SEQ ID NO:1所示氨基酸序列的第117至200位的氨基酸片段。在一些实施方式中，所述免疫原性片段至少包括如SEQ ID NO:1所示氨基酸序列的第114至204位氨基酸。
在一些实施方式中，所述免疫原性片段可以至少包括如SEQ ID NO:1所示氨基酸序列的第108～204位、第109～204位、第110～204位、第111～204位、第112～204位、第113～204位、第114～204位、第115～204位、第116～204位或第117～204位的氨基酸片段。在一些实施方式中，所述免疫原性片段可以具有SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11所示的氨基酸序列，或与其具有至少85％序列同一性的氨基酸序列。
在一些实施方式中，所述免疫原性片段的具有免疫原性的变体与所述免疫原性片段具有至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或至少100％序列同一性的氨基酸序列。
根据本公开的另一方面，提供了重组蛋白，所述重组蛋白包括：第一结构域，其包括上述的免疫原性片段或其具有免疫原性的变体；和，第二结构域，其包括用于形成纳米颗粒的骨架多肽。
在一些实施方式中，所述第二结构域作为骨架多肽，能够自组装或与另一种骨架多肽配对组装成纳米颗粒，同时将第一结构域的免疫原性片段或其变体展示在纳米颗粒的表面。
在一些实施方式中，所述第二结构域可以选自例如I53-34A、I53-34B、I53-40A、I53-40B、I53-47A、I53-47B、I53-50A、I53-50B、I53-51A、I53-51B、I52-03A、I52-03B、I52-32A、I52-32B、 I52-33A、I52-33B、I32-06A、I32-06B、I32-19A、I32-19B、I32-28A、I32-28B、I53-40A.1、I53-40B.1、I53-47A.1、I53-47A.1NegT2、I53-47B.1、I53-47B.1NegT2、I53-50A.1、I53-50A.1NegT2、I53-50A.1PostT1、I53-50B4 PostT1、LS(二氧四氢蝶啶合酶)、E2P(二氢硫酰乙酰转移酶)和I3。
在具体的实施方式中，所述第二结构域可以选自I52-32A、I52-32B、I53-50A、I53-50B、I32-28A、I32-28B、E2P和I3。
在一些实施方式中，所述第一结构域与所述第二结构域形成融合蛋白。在一些实施方式中，所述第一结构域与所述第二结构域直接连接。在一些实施方式中，所述第一结构域与所述第二结构域通过连接子连接。
根据本公开的另一方面，提供了一种核酸分子，其编码本公开的上述免疫原性片段或其具有免疫原性的变体或上述重组蛋白。
根据本公开的又一方面，提供了一种表达载体，其包括本公开的上述核酸分子。
在一些实施方式中，所述表达载体可以选自病毒或细菌载体，例如，但不限于非洲猪瘟病毒载体、慢病毒载体、鸟类痘病毒载体、犬麻疹病毒载体、疱疹病毒载体、水痘病毒载体、腺病毒载体、腺相关病毒载体等。
根据本公开的又一方面，提供了一种宿主细胞，其包括本公开的上述核酸分子，或能表达本公开的上述免疫原性片段或其具有免疫原性的变体或上述重组蛋白。
在一些实施方式中，所述宿主细胞为原核细胞或真核细胞。
在一些实施方式中，所述原核细胞可以选自大肠杆菌或枯草芽孢杆菌等，例如大肠杆菌BL21、T7E、C41、Arctic等。
在一些实施方式中，所述真核细胞可以选自酵母细胞、昆虫细胞、植物细胞、动物细胞等，例如酵母细胞、CHO细胞、293细胞、Vero细胞或NSO细胞等。
根据本公开的又一方面，提供了一种纳米颗粒，所述纳米颗粒包括本公开的上述免疫原性片段或其具有免疫原性的变体或上述重组蛋白。所述免疫原性片段或其变体展示在所述纳米颗粒的表面。
在一些实施方式中，所述纳米颗粒包括骨架蛋白自组装形成的六十聚体，所述骨架蛋白可以选自例如LS、E2P和I3。
在一些实施方式中，所述纳米颗粒包括所述重组蛋白中的骨架蛋白与另一种骨架蛋白配对组装形成的六十聚体，例如I53-34A与I53-34B配对、I53-40A与I53-40B配对、I53-47A与I53-47B配对、I53-50A与I53-50B配对、I53-51A与I53-51B配对、I52-03A与I52-03B配对、I52-32A与I52-32B配对、I52-33A与I52-33B配对、I32-06A与I32-06B配对、I32-19A与I32-19B配对、I32-28A与I32-28B配对。
在可选的实施方式中，所述纳米颗粒可以包括本公开的一种或多种免疫原性片段或其具 有免疫原性的变体。在示例性的实施方式中，所述纳米颗粒可以包括本公开的两种免疫原性片段或其具有免疫原性的变体，即至少包括如SEQ ID NO:1所示氨基酸序列的第18至100位的氨基酸片段和至少包括如SEQ ID NO:1所示氨基酸序列的第117至200位的氨基酸片段。
根据本公开的又一方面，提供了一种免疫原性组合物，其包括：本公开的上述免疫原性片段或其具有免疫原性的变体或上述重组蛋白、上述核酸分子、上述宿主细胞或上述纳米颗粒；和，药学上可接受的载剂。
在一些实施方式中，所述免疫原性组合物可以包括：本公开的一种或多种上述免疫原性片段或其具有免疫原性的变体或上述重组蛋白，或上述纳米颗粒，或编码本公开的一种或多种上述免疫原性片段或其具有免疫原性的变体或上述重组蛋白的核酸分子或者表达载体。
在一些实施方式中，所述免疫原性组合物可以进一步包括额外的非洲猪瘟病毒抗原。
在一些实施方式中，所述免疫原性组合物可以进一步包括药学上可接受的载剂。
在一些实施方式中，所述药学上可接受的载剂包括佐剂，所述佐剂包括：丙烯酸或甲基丙烯酸、马来酸酐和链烯基衍生物聚合物的聚合物；免疫刺激序列(ISS)，诸如具有一个或多个非甲基化CpG单元的寡脱氧核糖核苷酸序列(CpG ODN)；油包水(W/O)佐剂、水包油(O/W)佐剂或油包水包油(W/O/W)佐剂，诸如弗氏佐剂、SPT乳液、MF59、ISA 206、ISA72、佐剂-65、SAF等；含有季铵盐例如DDA的阳离子脂质；细胞因子；氢氧化铝或磷酸铝；皂苷(例如Quil A、QS-21、GPI-0100)；或，其任意组合或混合物。
在优选的实施方式中，所述皂苷为Quil A、QS-21、GPI-0100。
在优选的实施方式中，所述佐剂包括乳剂；所述乳剂为SPT乳剂、MF59乳剂，或乳剂由油与乳化剂组合形成，乳剂可基于轻液体石蜡油、因烯烃寡聚产生的类异戊二烯油(如角鲨烷或角鲨烯油，烯烃，特别是异丁烯或癸烯低聚化产生的油)、酸或醇的含线性烷基的酯(更尤其植物油、油酸乙酯、丙二醇二-(辛酸酯/葵酸酯)、甘油三-(辛酸酯/葵酸酯)或丙二醇二油酸酯)、支链脂肪酸或醇的酯(尤其异硬脂酸酯)；乳化剂为非离子表面活性剂(尤其聚氧乙烯化脂肪酸(例如油酸)的酯、山梨聚糖的酯、二缩甘露醇的酯(如无水甘露醇油酸酯)、脂肪族二元醇的酯、甘油的酯、聚甘油的酯、丙二醇的酯以及油酸的酯、异硬脂酸的酯、蓖麻油酸的酯或羟基硬脂酸的酯，上述酯可经乙氧基化、脂肪醇和多元醇(例如油醇)的醚、聚氧丙烯-聚氧乙烯嵌段共聚物(尤其特别是L121)。
在优选的实施方式中，丙烯酸或甲基丙烯酸的聚合物为交联的丙烯酸或甲基丙烯酸聚合物，尤其是与糖的聚链烯基醚或聚醇交联的化合物卡波姆，优选为卡波普974P、934P和971P。
在优选的实施方式中，顺丁烯二酸酐和链烯基衍生物的共聚物为顺丁烯二酸酐与乙烯的共聚物EMA。在优选的实施方式中，所述佐剂为Gel 01佐剂。
在一些实施方式中，所述免疫原性组合物可以经口、皮内、肌内或鼻内施用。
根据本公开的又一方面，提供了本公开的一种或多种上述免疫原性片段或其具有免疫原性的变体或上述重组蛋白，编码本公开的一种或多种上述重组蛋白的核酸分子或者表达载体，上述宿主细胞，上述纳米颗粒或上述免疫原性组合物在制备用于预防和/或治疗受试者的非洲猪瘟病毒感染的药物中的应用。
根据本公开的又一方面，提供了一种用于预防和/或治疗受试者的非洲猪瘟病毒感染的方法。
在一些实施方式中，所述受试者指哺乳动物。在一些实施方式中，所述受试者为猪科(Suidae)动物，例如猪。在一些实施方式中，所述个体或受试者可以为野猪(Sus scrofa)、家猪(Sus scrofa domesticus)、疣猪(Potamochoerus)、林猪(Hylochoerus)、巨林猪(Hylochoerus)、非洲野猪(Potamochoerus)以及野化猪。
在一些实施方式中，所述非洲猪瘟病毒感染可以是病原性非洲猪瘟病毒感染。在一些实施方式中，病原性非洲猪瘟病毒感染的症状或疾病可以选自由以下组成的组：非洲猪瘟、急性非洲猪瘟、慢性非洲猪瘟、病死、死亡、猝死、发热、高热、厌食、嗜睡、虚弱、食欲缺乏、伏卧、红斑、发绀性皮肤斑痣病、痢疾、便秘、腹痛、呼吸道症状、咳嗽、呕吐、呼吸困难、鼻涕及结合膜分泌物、出血、流鼻血、流产、白血球减少症、血小板减少症。
根据本公开的又一方面，提供了一种检测非洲猪瘟病毒感染的试剂盒，所述试剂盒包括本公开的上述免疫原性片段或其具有免疫原性的变体或上述重组蛋白。
根据本公开的又一方面，提供了本公开的上述免疫原性片段或其具有免疫原性的变体或上述重组蛋白在制备检测非洲猪瘟病毒感染的试剂盒中的应用。
在一些实施方式中，所述样本选自来自受试者的体液或者组织样本。在一些实施方式中，所述样本可以选自血液、唾液或血清样本。
附图说明
图1示例性示出了非洲猪瘟病毒CD2v蛋白胞外结构域1的18-100截短融合I3蛋白后组装效果电镜图。
图2示例性示出了非洲猪瘟病毒CD2v蛋白胞外结构域2的114-204截短融合I53-50A蛋白后与I53-50B组装效果电镜图。
图3示出了非洲猪瘟病毒CD2v蛋白胞外结构域1不同重组蛋白结合非洲猪瘟阳性血清的强度结果。
图4示出了非洲猪瘟病毒CD2v蛋白胞外结构域2不同重组蛋白结合非洲猪瘟阳性血清的强度结果。
图5示出了非洲猪瘟病毒CD2v蛋白胞外结构域1不同重组蛋白免疫小鼠后的血清抗体 水平。
图6示出了非洲猪瘟病毒CD2v蛋白胞外结构域2不同重组蛋白免疫小鼠后的血清抗体水平。
具体实施方式
为使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步的详细说明。此处所描述的具体实施例仅用于解释本发明，并不用于构成对本发明的任何限制。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本公开的概念。这样的结构和技术在许多出版物中也进行了描述。
非洲猪瘟病毒(African swine fever virus，ASFV)的基因组超过170kb，包含了超过150个开放阅读框(ORF)，病毒粒子直径超过200nm，病毒在细胞核周围形成病毒工厂，进行病毒的复制和组装。ASFV颗粒呈二十面体，具有多层囊膜结构，即内核(internal core)(又叫核状体、病毒类核)、核壳(core shell)、内包膜(inner envelope)、衣壳(capsid)、外包膜(external envelope，成分主要是类脂和少量蛋白质)。ASFV编码的蛋白在病毒组装、DNA复制和修复以及基因表达方面发挥着重要作用。此外，ASFV基因组编码许多与免疫逃逸相关的蛋白，包括抑制I型干扰素和诱导细胞凋亡蛋白，如DP96R、MGF-505-7R和pE199L等蛋白。
囊膜蛋白CD2v由EP402R基因编码，也称为pEP402R蛋白，包含360个氨基酸，因编码蛋白胞外区免疫球蛋白样结构域的氨基酸序列与宿主细胞的CD2非常相似，所以命名为CD2v。CD2v蛋白包含一个信号肽、胞外2个免疫球蛋白样结构域、跨膜区及胞内结构域组装而成的糖蛋白，其中胞内结构域包含1个酸性结构域和1个富含脯氨酸的重复序列。CD2v是一种糖蛋白，能帮助ASFV感染细胞与猪血液中的红细胞结合，促进病毒在宿主体内的扩散传播。CD2v在促进病毒复制与传播、病毒免疫逃逸等方面发挥着重要作用，是ASFV疫苗研究的热点靶标。
在一些实施方式中，非洲猪瘟病毒的全长CD2V蛋白具有如SEQ ID NO:1所示的氨基酸序列。
为了解决目前ASFV膜蛋白纯化难、表达量低以及原核表达构象不正确等问题，本公开分析了CD2v蛋白的氨基酸序列。首先，通过分析CD2v蛋白序列的疏水分布，将CD2V全长蛋白的胞外区与跨膜区、胞内区分开，获得CD2V蛋白胞外区片段序列为第1位至204位。然后，通过对CD2V蛋白进行结构学分析，确定其胞外主要包含两个相对独立的结构域(结构域1和结构域2)。
在保证其结构域完整的前提下，发明人将CD2v胞外域的两个结构域分开表达，并筛选了不同截短的CD2v蛋白片段，获得了可真核表达的CD2v蛋白的多种构建体。结果发现将 CD2v蛋白胞外结构域分成两个独立的结构域分别进行表达时，能够明显提高这两个结构域的表达。另外，将CD2v蛋白两个结构域的片段同时构建到六十聚体的纳米颗粒上时，具有显著增强的免疫效果，在免疫小鼠后能诱导产生高水平的抗CD2V抗体。
除非另有定义，否则本发明使用的所有技术术语和科技术语都具有如在本发明所属领域中通常使用的相同含义。出于解释本说明书的目的，将应用以下定义，并且在适当时，以单数形式使用的术语也将包括复数形式，反之亦然。
除非上下文另有明确说明，否则本文所用的表述“一种”和“一个”包括复数指代。例如，提及“一个细胞”包括多个这样的细胞及本领域技术人员可知晓的等同物等等。
本文所用的术语“约”表示其后的数值的±20％的范围。在一些实施方式中，术语“约”表示其后的数值的±10％的范围。在一些实施方式中，术语“约”表示其后的数值的±5％的范围。
本文所述的ASFV免疫原性组合物优选为亚单位疫苗。本文所述的“亚单位疫苗”包含源自ASFV的一个或多个多肽或蛋白质，或所述多肽或蛋白质的免疫原性片段，或编码所述多肽或蛋白质免疫原性片段的一个或多个核酸分子，且所述核酸分子能在猪体内进行表达。这些多肽或蛋白质，所述多肽或蛋白质的免疫原性片段，或编码所述多肽或蛋白质免疫片段的一个或多个核酸分子可采用本领域的公知技术来制备。
本文所用的术语“免疫原性组合物”是指包括至少一种在施用免疫原性组合物的宿主或个体中诱发免疫学反应的抗原的组合物。该免疫学反应可为对本发明的免疫原性组合物的细胞及/或抗体调介的免疫反应。优选地，免疫原性组合物诱导免疫反应且更优选地赋予抵抗ASFV感染的一或多种临床体征的保护免疫性。优选地，本文提及的任一宿主或个体是动物。
本文所使用的数值范围应被理解为已经列举了对该范围内的所有数字。例如，1至20的范围应当理解为包括来自下组的任何数字、数字组合或子范围：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20。
本文所用的术语“宿主细胞”指体内或体外真核细胞、原核细胞或来自以单细胞实体培养的多细胞生物(例如，细胞株)的细胞，其中，真核或原核细胞可以是、或已经用作核酸接受体，包括经核酸遗传修饰的原始细胞的后代。应理解，由于天然、偶然或故意突变，单个细胞的后代在形态或在基因或整套DNA方面并不一定与原始亲代完全相同。例如，本发明原核宿主细胞指，通过将异源核酸，例如对原核宿主细胞外源(不是天然存在)的外源性核酸或原核宿主细胞中不是正常存在的重组核酸引入合适的原核宿主细胞所产生的遗传修饰的原核宿主细胞(例如，细菌)；本发明真核宿主细胞指，通过将异源核酸，例如对真核宿主细胞外源的外源性核酸或真核宿主细胞中不是正常存在的重组核酸引入合适的真核宿主细胞所产生的遗传修饰的真核宿主细胞。
在一些实施方式中，本公开的连接子可以为柔性肽接头。在一些实施方式中，肽接头富含甘氨酸、丝氨酸、丙氨酸、脯氨酸和/或谷氨酰胺残基。在一些实施方式中，肽接头可以选 自(GnS)m，其中n和m各自独立地选自0～5的整数。例如，n选自0、1、2、3、4或5，m选自1、2、3、4或5。本文所用的术语“连接子”指天然和/或合成来源的(肽)接头，由线性氨基酸组成。本发明的双特异性融合多肽中的各结构域可以通过连接子连接，其中每个连接子与至少两个多肽或结构域融合和/或以其他方式连接(例如，经由肽键)。在一些实施方式中，存在于本发明的双特异性融合多肽中的所有连接子的氨基酸序列相同的。在其他实施方式中，存在于本发明的双特异性融合多肽中的至少两个连接子的氨基酸序列是不同的。连接子应该具有适合以这种方式连接两个或更多个单体结构域的长度，连接子能够确保其所连接的不同结构域正确折叠并合适地呈现，从而发挥其生物学活性的功能。在不同的实施方式中，连接子具有柔性构象。合适的柔性连接子包括，例如具有甘氨酸、谷氨酰胺和/或丝氨酸残基。
相对于参照氨基酸序列具有“百分比(％)序列同一性”指在比对序列和(根据需要)引入缺口以获得最大百分比序列同一性后，候选序列中与参照氨基酸序列中的氨基酸残基相同的氨基酸残基的百分比，但不考虑任何保守取代为序列同一性的一部分。为了确定氨基酸序列同一性百分比，可以以本领域范围内的各种方式进行比对，例如使用BLAST、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适当参数，包括在被比较序列全长上实现最大比对所需的任何算法。
本公开的CD2V蛋白免疫原性片段的变体可以是通过一个或多个氨基酸的替换、添加或缺少，从而在氨基酸序列上与CD2V蛋白免疫原性片段具有至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性，同时保留了与其相当的免疫原性。
本公开的CD2V蛋白免疫原性片段的具有免疫原性的变体可以通过对CD2V蛋白免疫原性片段进行一个或多个保守氨基酸的替换来获得。在一些实施方式中，保守氨基酸的替换可以表示一个氨基酸残基替换成生物学上性质相似的残基。特别优选的取代在性质上通常是保守的，即在氨基酸家族内发生的那些取代。例如，氨基酸通常分为四个家族：(1)酸性-天冬氨酸和谷氨酸；(2)碱性-赖氨酸、精氨酸、组氨酸；(3)非极性-丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸；(4)不带电荷的极性-甘氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、丝氨酸、苏氨酸、酪氨酸。苯丙氨酸、色氨酸和酪氨酸有时被分类为芳香族氨基酸。保守变化的实例包括用一个疏水残基诸如异亮氨酸、缬氨酸、亮氨酸或甲硫氨酸取代另一个疏水残基，或用一个极性残基取代另一个极性残基，诸如精氨酸取代赖氨酸、谷氨酸取代天冬氨酸或谷氨酰胺取代天冬酰胺等；或用结构相关的氨基酸对氨基酸的类似保守替代，所述替代对生物活性不会产生重大影响。因此，具有与参考分子基本相同的氨基酸序列但具有基本上不影响蛋白质免疫原性的少量氨基酸取代的蛋白质在参考多肽的定义范围内。
本文所用的第二结构域能够在体外自组装成或与另一种骨架蛋白配对组装成纳米颗粒。本文所使用的CD2V蛋白的免疫原性片段或其变体与所述第二结构域形成融合蛋白，通过所述第二结构域的自组装或配对组装，CD2V蛋白的免疫原性片段或其变体被展示在纳米颗粒的表面。
在一些实施方案中，所述第二结构域可以选自通过涉及，体外合成的一些能够自组装成纳米颗粒的多肽，例如LS、E2P、I3等。这些多肽能够在体外成对的自组装成纳米结构，例如六十聚体的纳米结构。
在一个实施方案中，所述第二结构域是二氧四氢蝶啶合酶(LS)。单体LS亚基可以是LS蛋白的全长、单个多肽或其任意部分，其能够指导单体LS亚基自组装为纳米颗粒。来自任意已知LS蛋白的单体LS亚基均可以用于产生本公开的重组蛋白，只要所述单体LS亚基能够指导所述重组蛋白自组装为在其表面上展示CD2V蛋白免疫片段的纳米颗粒即可。代表性的LS蛋白具有如SEQ ID NO:17所示的氨基酸序列。
在一个实施方案中，所述第二结构域是E2P。单体E2P亚基可以是E2P蛋白的全长、单个多肽或其任意部分，其能够指导单体E2P亚基自组装为纳米颗粒。来自任意已知E2P蛋白的单体E2P亚基均可以用于产生本公开的重组蛋白，只要所述单体E2P亚基能够指导所述重组蛋白自组装为在其表面上展示CD2V蛋白免疫片段的纳米颗粒即可。代表性的E2P蛋白具有如SEQ ID NO:18所示的氨基酸序列。
在一个实施方案中，所述第二结构域是I3。单体I3亚基可以是I3蛋白的全长、单个多肽或其任意部分，其能够指导单体I3亚基自组装为纳米颗粒。来自任意已知I3蛋白的单体I3亚基均可以用于产生本公开的重组蛋白，只要所述单体I3亚基能够指导所述重组蛋白自组装为在其表面上展示CD2V蛋白免疫片段的纳米颗粒即可。代表性的I3蛋白具有如SEQ ID NO:19所示的氨基酸序列。
在一个实施方案中，所述第二结构域可以选自通过涉及，体外合成的一些能够配对组装成纳米颗粒的多肽，例如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和I53-50B4 PostT1。这些多肽能够在体外成对的组装成纳米结构，例如六十聚体的纳米结构。
本文所用的术语“免疫原性组合物”一般指具有含有至少一种抗原或其免疫原部分的物质的组合物，该物质在宿主体内引发针对组合物的细胞免疫反应或抗体介导的免疫反应的免疫反应。优选是，免疫原性组合物诱导免疫反应，且更优选是，赋予针对ASFV感染的临床症状中之一或多种的保护性免疫。在宿主展现保护性免疫反应使得抗新型感染性增强且/或疾病的临床严重程度降低的情况下，免疫原性组合物也可称为“疫苗”。
本文所用的术语“药学上可接受的载体”指药物制剂中除了活性成分之外的对受试者无毒性的成分。药学上可接受的载体包括，但不限于缓冲剂、赋形剂、稳定剂或防腐剂。
本文所使用的术语“预防和/或治疗”指降低特定ASFV感染的发生率，或降低由特定ASFV感染造成或与特定ASFV感染相关联的临床症状的严重程度。另外，术语“预防和/或治疗”也可以指与动物未接受如本文所提供的免疫原性组合物的动物组相比，在动物已接受有效量的该免疫原性组合物的动物组中，减少感染特定ASFV的动物的数目(即，降低ASFV感染的发生率)，或降低通常与ASFV感染相关联或由ASFV感染造成的临床症状的严重程度。
本文所用的术语“有效量”是指为了实现所需的治疗或预防效果，在必要的剂量和时间段内的有效的量。
下面提供实施例和附图以帮助理解本发明。但应理解，这些实施例和附图仅用于说明本发明，但不构成任何限制。本发明的实际保护范围在权利要求书中进行阐述。应理解，在不脱离本发明精神的情况下，可以进行任何修改和改变。
为了解决目前膜蛋白纯化难、表达量低以及原核表达构象不正确等技术问题，且全长的CD2v不表达或者很难表达。本发明分析了非洲猪瘟病毒结构蛋白CD2V的氨基酸序列，先通过分析蛋白序列的疏水分布，将CD2V全长蛋白的胞外区与跨膜区、胞内区分开，获得CD2V蛋白胞外区片段序列。再通过对CD2V蛋白进行结构学分析，确定其二硫键位置、结构域等信息。在保证其结构域完整的前提下，筛选了不同截短的CD2V蛋白片段，获得了可真核分泌表达的CD2V蛋白的多种构建体，这些CD2V蛋白片段接种小鼠后能诱导产生高水平的抗CD2V的抗体。本公开将CD2v胞外段(18-204)分解为两个独立的结构域去表达，并分别优化出了最佳的截短构建：
结构域1：N端固定18位，C端位于100-106时，表达量显著提高。
结构域2：C端固定204位，N端位于109-114位，表达量显著提高。
与此同时，发明人发现将CD2v的两个结构域构建到六十聚体的纳米颗粒上，可以显著的增强免疫效果，产生的抗体水平显著提高。
实施例1：质粒的构建
将编码CD2v不同的截短形式(结构域1：10-100、10-106、10-109、18-100、18-106、18-107、18-109；结构域2：108-204、111-204、114-204、117-204)的基因与编码不同的多聚体骨架蛋白(I53-50A、I53-50B、I52-32A、I32-28B、LS、E2P或I3)的基因通过编码“连接子”(SEQ ID NO:21)的基因连接后，采用重叠PCR的方法得到重组后的真核基因片段，并在基因5’或3’引入His标签序列，方便后续纯化。采用Sal I和EcoR I限制性内切酶对重组基因片段进行双酶切，酶切后片段采用DNA连接酶与pCMV-flag线性化载体连接，获得 真核重组质粒，涉及的序列信息参见下表1。
表1
实施例2重组蛋白的真核表达
重组蛋白CD2v的真核表达包括以下步骤：
(1)真核重组质粒大量制备：取1μg实施例1制备的各真核重组质粒与100μL Top10感受态细胞(购自擎科生物)混合，冰上放置15分钟。42℃热击90秒后置于冰上5分钟，加入无抗性的液体LB培养基，37℃、220rpm摇床培养40分钟。培养结束后，2000×g离心5分钟，弃掉大部分上清，剩余培养基重悬感受态细胞并均匀涂布在固体LB(Amp+)培养皿中，37℃恒温箱培养12～16小时。培养结束后，可挑取培养皿上单一且生长状态良好的菌落扩大培养，并按照天根质粒大提试剂盒说明书进行质粒提取。
(2)真核重组质粒转染及细胞培养：取1mg真核重组质粒与转染试剂PEI(购自polyscience)混合，混合后室温静置5-10分钟。取密度为3～3.5×106个/mL的293F细胞1L，将制备好质粒/PEI混合物逐滴边摇边加到细胞中，37℃，含5％ CO2的培养箱中130rpm转速培养4天。
(3)培养上清收取及浓缩：将(2)中所述培养4天的细胞取出，将培养物进行离心收获上清，采用0.45μm滤膜过滤除去细胞碎片。过滤后的上清液采用膜包法(15kD)进行超滤浓缩，浓缩后采用缓冲液(1×PBS缓冲液，pH8.0)稀释，备用。
(4)蛋白纯化：用平衡缓冲液(1×PBS缓冲液，pH8.0)平衡Ni柱，将上清液加载Ni柱，用洗杂缓冲液(1×PBS缓冲液，20mM咪唑，pH8.0)加载层析柱5～10个柱体积，用平衡缓冲液加载层析柱至基线平衡后，用洗脱缓冲液(1×PBS缓冲液，500mM咪唑，pH8.0)洗脱目的蛋白，洗脱的蛋白液经0.22μm的滤膜过滤除菌后，采用NanoDrop测定蛋白浓度，各构建表达情况见表2和表3。
表2.非洲猪瘟CD2v蛋白胞外结构域1表达情况(单位mg/L)。
表3.非洲猪瘟CD2v蛋白胞外结构域2表达情况(单位mg/L)。
实施例3将纯化后的各抗原进行六十聚体组装
(1)双组分纳米颗粒的组装：对分别纯化的重组蛋白以及相应的配对骨架蛋白进行混合；制备样品负染载网，采用120kV电镜检测纳米颗粒的组装效果。示例性以结构域1的18-100截短和结构域2的114-204截短的重组蛋白的组装结果统计于下表4和表5。
(2)单组分纳米颗粒的组装：单组分纳米颗粒自发组装，无需单独组装步骤。制备样品负染载网，采用120kV电镜检测纳米颗粒的组装效果。示例性以结构域1的18-100截短和结构域2的114-204截短的重组蛋白的组装结果统计于下表4和表5。
示例性的结构域1的18-100截短融合I3蛋白的组装电镜效果见图1。
示例性的结构域2的114-204截短融合I53-50A与I53-50B组装后电镜效果见图2。
表4.非洲猪瘟CD2v蛋白胞外结构域1组装六十聚体效果统计。

注：组装效果中，+代表可以组装形成六十聚体颗粒，-代表无法组装形成六十聚体颗粒。
表5.非洲猪瘟CD2v蛋白胞外结构域2组装六十聚体效果统计。

注：组装效果中，+代表可以组装形成六十聚体颗粒，-代表无法组装形成六十聚体颗粒。
实施例4非洲猪瘟阳性血清结合实验
取实施例2获得的各纯化后的重组蛋白或实施例3获得的组装颗粒，用1×PBS(pH 8.0)稀释成1μg/mL的包被工作液，分别加入到96孔酶标板中，每孔加入100μL，置于4℃过夜(12-16小时)。取出酶标板弃去孔中液体，PBST(1×PBS加0.5‰Tween 20)洗三遍，在吸水纸上拍干。每孔中加入封闭缓冲液200μL(PBST加入0.2％ BSA)，室温放置1小时。甩掉封闭液，PBST洗三遍，在吸水纸上拍干。取非洲猪瘟阳性血清(购自中国兽医药品监察所)用封闭液稀释1:1000稀释，取100μL加至包被有抗原的酶标板中，室温反应60分钟，用PBST洗板3次，然后每孔中加入用封闭液1:5000稀释的山羊抗猪IgG-HRP标记物，室温反应60分钟，用PBST洗板5次，拍干后，向每孔中各加入100μL底物TMB，室温避光反应2-10分钟，向每孔中加入终止液(2M硫酸)50μL。以630nm为参考波长，测量450nm 处的吸光度值。以吸光度值大于阴性对照(阴性对照为细胞培养基)的2.0倍为阳性判断标准。
示例性的结构域1截短和结构域2截短的重组蛋白或其组装颗粒的结果分别如图3和图4所示，结果显示不同的实施例2获得的CD2v各纯化后的重组蛋白或其组装颗粒均能够被非洲猪瘟阳性血清结合，说明本公开表达的上述重组蛋白或其组装颗粒具有正确的空间构象。
实施例5抗原免疫原性测定实验
(1)取上述实施例2中部分纯化的CD2v重组蛋白抗原以及实施例3中组装的CD2v六十聚体抗原，常规方法免疫6周龄BALB/c小鼠，首次基础免疫皮下注射抗原10μg，使用弗氏完全佐剂；隔4周后进行二次免疫，10μg/只，使用弗氏不完全佐剂。
(2)分别在首次基础免疫前(0周)、首次免疫后两周(2周)、首次免疫后四周(4周)和二免后两周(6周)采血，进行后续ELISA测定抗体水平。
(3)取纯化后的CD2V抗原，用1×PBS(pH8.0)稀释成1μg/mL的包被工作液，分别加入到96孔酶标板中，每孔加入100μL，置于4℃过夜(12～16小时)。取出酶标板弃去孔中液体，PBST(1×PBS加0.5‰Tween 20)洗三遍，在吸水纸上拍干。每孔中加入封闭缓冲液200μL(PBST加入0.2％ BSA)，室温放置1小时。甩掉封闭液，PBST洗三遍，在吸水纸上拍干。取各时间点免疫小鼠血清，用封闭液1:1000稀释，取100μL加至包被有抗原的酶标板中，室温反应60分钟，用PBST洗板3次，然后每孔中加入用封闭液1:10000稀释的山羊抗鼠IgG-HRP标记抗体，室温反应60分钟，用PBST洗板5次，拍干后，向每孔中各加入100μL底物TMB，室温避光反应2-10分钟，向每孔中加入终止液(2M硫酸)50μL。以630nm为参考波长，测量450nm处的吸光度值。以吸光度值大于阴性对照(阴性对照为细胞培养基)的2.0倍为阳性判断标准。
示例性的以结构域1的18-100截短和18-106截短的重组蛋白以及结构域2的114-204截短的重组蛋白或其组装颗粒的免疫小鼠后的血清抗体水平分别如图5和图6所示，结果显示相较于聚合状态不均一的单体抗原，组装形成六十聚体纳米颗粒的CD2V抗原具有更高的免疫原性。
本发明的技术方案不限于上述具体实施例的限制，凡是根据本发明的技术方案做出的技术变形，均落入本发明的保护范围之内。

Claims (14)

  1. 一种免疫原性片段或其具有免疫原性的变体，其特征在于，所述免疫原性片段至少包括如SEQ ID NO:1所示氨基酸序列的第18至100位的氨基酸片段和/或至少包括如SEQ ID NO:1所示氨基酸序列的第117至200位的氨基酸片段。
  2. 根据权利要求1所述的免疫原性片段或其具有免疫原性的变体，其特征在于，所述免疫原性片段至少包括如SEQ ID NO:1所示氨基酸序列的第18至106位氨基酸；和/或
    所述免疫原性片段至少包括如SEQ ID NO:1所示氨基酸序列的第114至204位氨基酸。
  3. 根据权利要求1所述的免疫原性片段或其具有免疫原性的变体，其特征在于，所述免疫原性片段具有如SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7或SEQ ID NO:8所示的氨基酸序列，或与其具有至少85％序列同一性的氨基酸序列；和/或，具有如SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11所示的氨基酸序列，或与其具有至少85％序列同一性的氨基酸序列。
  4. 一种重组蛋白，其特征在于，所述重组蛋白包括：
    第一结构域，其包括权利要求1至3中任一项所述的免疫原性片段或其具有免疫原性的变体；和，
    第二结构域，其包括用于形成纳米颗粒的骨架多肽。
  5. 根据权利要求4所述的重组蛋白，其特征在于，所述第二结构域选自I53-34A、I53-34B、I53-40A、I53-40B、I53-47A、I53-47B、I53-50A、I53-50B、I53-51A、I53-51B、I52-03A、I52-03B、I52-32A、I52-32B、I52-33A、I52-33B、I32-06A、I32-06B、I32-19A、I32-19B、I32-28A、I32-28B、I53-40A.1、I53-40B.1、I53-47A.1、I53-47A.1 NegT2、I53-47B.1、I53-47B.1 NegT2、I53-50A.1、I53-50A.1 NegT2、I53-50A.1 PostT1、I53-50B4 PostT1、LS、E2P和I3中的至少一种，
    优选地，所述第一结构域与所述第二结构域直接连接或通过连接子连接。
  6. 一种核酸分子，其特征在于，所述核酸分子编码如权利要求1至3中任一项所述的免疫原性片段或其具有免疫原性的变体，或编码如权利要求4或5所述的重组蛋白。
  7. 一种宿主细胞，其特征在于，所述宿主细胞包括权利要求6所述的核酸分子，或能表达权利要求1至3中任一项所述的免疫原性片段或其具有免疫原性的变体，或权利要求4或5所述的重组蛋白；
    优选地，所述宿主细胞为原核细胞或真核细胞。
  8. 一种纳米颗粒，其特征在于，所述纳米颗粒包括权利要求1至3中任一项所述的免疫原性片段或其具有免疫原性的变体，或权利要求4或5所述的重组蛋白，
    优选地，所述免疫原性片段或其变体展示在所述纳米颗粒的表面。
  9. 一种免疫原性组合物，其特征在于，所述免疫原性组合物包括：权利要求1至3中任一项所述的免疫原性片段或其具有免疫原性的变体、权利要求4或5所述的重组蛋白、权利 要求6所述的核酸分子、权利要求7所述的宿主细胞或权利要求8所述的纳米颗粒；和，药学上可接受的载剂，
    优选地，所述免疫原性组合物进一步包括额外的非洲猪瘟病毒抗原。
  10. 权利要求1至3中任一项所述的免疫原性片段或其具有免疫原性的变体、权利要求4或5所述的重组蛋白、权利要求6所述的核酸分子、权利要求7所述的宿主细胞、权利要求8所述的纳米颗粒或权利要求9所述的免疫原性组合物在制备用于预防和/或治疗受试者的非洲猪瘟病毒感染的药物中的应用。
  11. 根据权利要求10所述的应用，其特征在于，所述受试者包括哺乳动物，
    优选地，所述受试者包括猪科(Suidae)动物或猪，
    更优选地，所述受试者包括野猪(Sus scrofa)、家猪(Sus scrofa domesticus)、疣猪(Potamochoerus)、林猪(Hylochoerus)、巨林猪(Hylochoerus)、非洲野猪(Potamochoerus)和野化猪。
  12. 根据权利要求10或11所述的应用，其特征在于，所述非洲猪瘟病毒感染是病原性非洲猪瘟病毒感染，
    优选地，所述非洲猪瘟病毒感染的疾病或症状选自由以下组成的组：非洲猪瘟、急性非洲猪瘟、慢性非洲猪瘟、病死、死亡、猝死、发热、高热、厌食、嗜睡、虚弱、食欲缺乏、伏卧、红斑、发绀性皮肤斑痣病、痢疾、便秘、腹痛、呼吸道症状、咳嗽、呕吐、呼吸困难、鼻涕及结合膜分泌物、出血、流鼻血、流产、白血球减少症、血小板减少症。
  13. 一种检测非洲猪瘟病毒感染的试剂盒，所述试剂盒包括权利要求1至3中任一项所述的免疫原性片段或其具有免疫原性的变体，或权利要求4或5所述的重组蛋白。
  14. 权利要求1至3中任一项所述的免疫原性片段或其具有免疫原性的变体，或权利要求4或5所述的重组蛋白在制备检测非洲猪瘟病毒感染的试剂盒中的应用，
    优选地，所述非洲猪瘟病毒来自受试者的体液或者组织样本，
    更优选地，所述体液样本选自血液、唾液或血清样本。
PCT/CN2024/129778 2023-11-03 2024-11-04 非洲猪瘟病毒CD2v蛋白的免疫原性组合物及其应用 Pending WO2025093041A1 (zh)

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