WO2024099273A1 - 一种融合蛋白以及包含其的颗粒化的抗原 - Google Patents

一种融合蛋白以及包含其的颗粒化的抗原 Download PDF

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WO2024099273A1
WO2024099273A1 PCT/CN2023/129973 CN2023129973W WO2024099273A1 WO 2024099273 A1 WO2024099273 A1 WO 2024099273A1 CN 2023129973 W CN2023129973 W CN 2023129973W WO 2024099273 A1 WO2024099273 A1 WO 2024099273A1
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seq
sequence
protein
polypeptide
fusion protein
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PCT/CN2023/129973
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English (en)
French (fr)
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李少伟
李婷婷
薛文辉
邓婷婷
方乾骄
顾颖
张军
夏宁邵
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厦门大学
厦门万泰沧海生物技术有限公司
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Publication of WO2024099273A1 publication Critical patent/WO2024099273A1/zh

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  • the present application relates to the field of biomedicine, and more specifically, to a fusion protein and a composition, a kit, a granulated antigen, a vaccine and a pharmaceutical composition comprising the fusion protein.
  • the present application also relates to the use of the fusion protein and a composition, a kit and a granulated antigen comprising the fusion protein in the preparation of a pharmaceutical composition or a vaccine.
  • vaccines are the most economical and effective way to prevent and control infectious diseases. Vaccination can save nearly 6 million lives each year.
  • genetically engineered vaccines mostly use proteins/polysaccharides that are critical to pathogen infection as antigens.
  • the development of such vaccine antigens has the advantages of high safety and mature technology, and can produce immune protection against specific pathogens, they often face problems such as poor humoral immunity, difficulty in achieving long-term protection, and difficulty in activating efficient cellular immunity. Therefore, the development of safe, efficient, and broad-spectrum new immunogens is the key to improving vaccine effectiveness.
  • Granulated vaccines are an important vaccine platform to resist pathogenic threats.
  • HEV hepatitis E
  • HPV human papillomavirus vaccine
  • HBV hepatitis B vaccine
  • Hepatitis E vaccine is a virus-like particles (VLP) vaccine.
  • the hepatitis E antigen fragment expressed by the Escherichia coli expression system - p239 protein (aa368-606) can be assembled into a particle form with a diameter of 25nm after renaturation. It has a structural conformation similar to that of natural virus particles and can well simulate natural virus antigen epitopes.
  • HPV vaccines currently on the market include bivalent vaccines, quadrivalent vaccines and nine-valent vaccines.
  • HPV vaccine is composed of virus-like particles assembled from the main capsid protein L1 of the virus, which can be produced by expression systems such as insect cells, yeast cells and Escherichia coli.
  • Clinical and post-marketing studies have shown that HPV vaccine has good safety and protective effects.
  • the research and development of hepatitis B vaccine has a long history, and there are many hepatitis B vaccines on the market at home and abroad.
  • Hepatitis B vaccine is mainly composed of S protein of different forms of surface antigen HBsAg (SHBs, MHBs or LHBs) spontaneously assembled to form particles, and is currently mainly produced by yeast system and CHO system.
  • SHBs, MHBs or LHBs surface antigen HBsAg
  • hepatitis B vaccine can produce high titers of protective antibodies and has good efficacy and safety.
  • the production of granulated antigens in hepatitis E vaccine, HPV vaccine and hepatitis B vaccine has the advantages of economic efficiency, stable production process, controllable antigen quality, industrial mass production, good safety and strong immunogenicity, which is a good foundation and important reference for the further development of other vaccines.
  • the particle display methods of multivalent antigens mostly use fusion expression with particle carriers or technical methods based on SpyTag/SpyCatcher chemical binding to particle carriers.
  • these methods have great limitations.
  • the limitation of the fusion expression method is that the particle carrier that meets the fusion expression must have an exposed N/C terminus to meet the requirements of foreign protein fusion.
  • the membrane proteins of enveloped viruses are the main immunogenic targets for vaccine development, but these proteins are usually anchored to the membrane structure on the surface of the virus, cannot form particle antigens by themselves, and are usually highly hydrophobic. It is also difficult for intact proteins to directly express and obtain a membrane-containing particle structure.
  • the new coronavirus is an RNA virus with an envelope.
  • the Spike protein is an important molecule on the envelope, which is mainly involved in receptor recognition and cell membrane fusion. Among them, RBD is the protein domain on the Spike protein that directly binds to the host receptor (ACE2) and is also the main target of neutralizing antibodies. Therefore, it is of great value to use RBD protein as a new coronavirus vaccine antigen.
  • gE protein is the most abundant glycoprotein on the surface of varicella-zoster virus (VZV) and is also an important target for vaccine development.
  • VZV varicella-zoster virus
  • Env is the main antigen on the surface of HIV-1 virus and is also a key molecule in the development of AIDS vaccines.
  • the full-length Env molecule is a transmembrane protein, also known as gp160 protein.
  • Env protein By deleting the intracellular segment, the soluble expression of Env protein can be effectively improved.
  • This molecule is called gp140 protein, which forms a natural trimer-like gp140 protein, which has obvious advantages in exposing neutralizing epitopes and neutralizing epitope antibody responses.
  • gp140 protein forms a natural trimer-like gp140 protein, which has obvious advantages in exposing neutralizing epitopes and neutralizing epitope antibody responses.
  • the development of AIDS vaccines based on Env has not been successful.
  • One of the difficulties is that the surface of Env is covered with more than 50% polysaccharide molecules, which significantly reduces the immunogenicity of the antigen.
  • the present invention utilizes the ability of assembled polypeptides (e.g., ORF2 protein of HEV or its fragments or variants thereof, L1 protein of HPV or its fragments or variants thereof, surface antigen of HBV or its fragments or variants thereof) to assemble into VLPs, fuses and expresses nano antibodies that specifically bind to the assembled polypeptides with immunogenic polypeptides, and utilizes the targeted binding properties of nano antibodies to the assembled polypeptides to display a variety of immunogenic polypeptides on the surface of VLPs formed by the assembled polypeptides, thereby obtaining granulated immunogenic polypeptides.
  • the present application provides a system and method for granulating immunogenic polypeptides.
  • the present application also demonstrates that granulated immunogenic polypeptides can induce the body to produce high levels of humoral immunity and cellular immune responses. Therefore, the granulated immunogenic polypeptides of the present invention have the potential to be vaccine candidate molecules, and have the advantages of versatility and convertibility in the design and production of vaccines.
  • the present application provides a fusion protein comprising an immunogenic polypeptide and a nanobody capable of specifically binding to a virus-like particle (VLP);
  • VLP virus-like particle
  • the VLP is a VLP assembled from an assembly polypeptide.
  • the assembly polypeptide is a polypeptide capable of assembling into a VLP.
  • the assembly polypeptide is a coat protein of a natural virus or viroid.
  • the VLP assembled from the assembly polypeptide is similar in structure to a natural virus or viroid, except that the VLP does not contain the genome of a natural virus or viroid.
  • the assembling polypeptide is an artificially prepared and/or screened polypeptide capable of being assembled into a VLP.
  • the VLP assembled into by the assembling polypeptide is similar or dissimilar to the structure of a natural virus or viroid.
  • the assembly polypeptide is selected from a protein of hepatitis E virus (HEV) or a fragment thereof or a variant thereof, a protein of hepatitis B virus (HBV) or a fragment thereof or a variant thereof, a protein of human papillomavirus (HPV) or a fragment thereof or a variant thereof, or any combination thereof; wherein the fragment or variant retains the ability to assemble into VLPs.
  • HEV hepatitis E virus
  • HBV hepatitis B virus
  • HPV human papillomavirus
  • the variant has one or more amino acid replacements, deletions or additions (e.g., 1, 2, 3, 4, 5 amino acid replacements, deletions or additions) compared to the sequence of the polypeptide from which it is derived.
  • the replacement is a conservative replacement.
  • the polypeptide and its variant have the same or similar biological activity.
  • the biological activity is the ability to be assembled into VLPs.
  • the assembly polypeptide is the ORF2 protein of HEV or a fragment or variant thereof.
  • ORF2 protein refers to the protein encoded by the second open reading frame in the genome of the HEV virus.
  • the ORF2 protein or its fragment has the ability to self-assemble into VLPs.
  • the fragment of the ORF2 protein is selected from the group consisting of p239 protein and p495 protein.
  • the assembling polypeptide is selected from p239 protein or a fragment thereof or a variant thereof, p495 protein or a fragment thereof or a variant thereof.
  • sequence of the p239 protein is from amino acid 368 to amino acid 606 corresponding to the amino acid sequence of the ORF2 protein.
  • sequence of the p495 protein is from amino acid 112 to amino acid 606 corresponding to the amino acid sequence of the ORF2 protein.
  • the ORF2 protein has an amino acid sequence as shown in SEQ ID NO: 9.
  • the p239 protein has an amino acid sequence as shown in SEQ ID NO: 40.
  • the p495 protein has an amino acid sequence as shown in SEQ ID NO: 41.
  • the assembling polypeptide is the capsid protein L1 of HPV or a fragment or variant thereof.
  • HPV is composed of a protein coat and core DNA, and the coat is composed of a major capsid protein (L1) and a minor capsid protein (L2).
  • L1 major capsid protein
  • L2 minor capsid protein
  • capsid protein L1 or "HPV L1 protein” or "L1 protein” refers to the L1 protein that constitutes the HPV coat.
  • the assembled polypeptide has an amino acid sequence as shown in SEQ ID NO:74.
  • the assembled polypeptide is a surface protein of HBV (e.g., hepatitis B virus surface antigen) or a fragment thereof or a variant thereof.
  • the assembled polypeptide is selected from LHBs protein or a fragment thereof or a variant thereof of hepatitis B virus surface antigen (HBsAg), MHBs protein or a fragment thereof or a variant thereof, SHBs protein or a fragment thereof or a variant thereof.
  • the assembled polypeptide is selected from SHBs protein or a fragment thereof or a variant thereof of hepatitis B virus surface antigen (HBsAg).
  • Hepatitis B virus surface antigen is composed of large surface protein (LHBs), middle surface protein (MHBs) and small surface protein (SHBs).
  • LHBs large surface protein
  • MHBs middle surface protein
  • SHBs protein refers to the small surface protein constituting HBsAg.
  • the assembled polypeptide has an amino acid sequence as shown in SEQ ID NO:73.
  • the immunogenic polypeptide is a polypeptide obtained from an organism or a non-organism (eg, artificially synthesized) or an immunogenic variant thereof.
  • the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5 substitutions, deletions or additions) compared to the sequence of the polypeptide from which it is derived.
  • the substitution is a conservative substitution.
  • the polypeptide and its variant have the same or similar biological activity.
  • the biological activity is the ability to induce an immune response.
  • the organism is a pathogen (eg, virus, bacteria, fungus, parasite) or a non-pathogen.
  • pathogen eg, virus, bacteria, fungus, parasite
  • non-pathogen e.g, virus, bacteria, fungus, parasite
  • the immunogenic polypeptide is obtained from a non-tumor cell of a mammal (eg, a human).
  • the immunogenic polypeptide is proprotein convertase subtilisin/kexin type 9 (PCSK9).
  • the immunogenic polypeptide is obtained from a tumor cell of a mammal (e.g., a human) (e.g., expressed or overexpressed on the surface of a tumor cell).
  • the immunogenic polypeptide is selected from carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), cancer antigen 125 (CA125).
  • the immunogenic polypeptide is obtained from a virus, a bacterium (e.g., Mycobacterium tuberculosis), a fungus (e.g., Candida albicans), or a parasite (e.g., Plasmodium falciparum).
  • a virus e.g., Mycobacterium tuberculosis
  • a fungus e.g., Candida albicans
  • a parasite e.g., Plasmodium falciparum
  • the virus is selected from varicella-zoster virus (VZV), novel coronavirus (SARS-CoV-2), coronavirus (SARS-COV-1), human immunodeficiency virus type 1 (HIV-1), human papillomavirus, hepatitis B virus, hepatitis A virus, hepatitis C virus, hepatitis E virus, measles virus, mumps virus, influenza virus, and Japanese encephalitis virus.
  • VZV varicella-zoster virus
  • SARS-CoV-2 novel coronavirus
  • SARS-COV-1 coronavirus
  • HAV-1 human immunodeficiency virus type 1
  • human papillomavirus hepatitis B virus, hepatitis A virus, hepatitis C virus, hepatitis E virus, measles virus, mumps virus, influenza virus, and Japanese encephalitis virus.
  • sequence of the assembled polypeptide is derived from a viral pathogen and the sequence of the immunogenic polypeptide is derived from a non-viral pathogen (eg, bacteria, fungi, parasites).
  • sequence of the assembling polypeptide is derived from a viral pathogen and the sequence of the immunogenic polypeptide is derived from another viral pathogen.
  • the immunogenic polypeptide of the present invention can be a polypeptide derived from the surface or core of a virus.
  • the immunogenic polypeptide can be a polypeptide of any structure or function comprising at least 6 amino acid residues.
  • the length of the immunogenic polypeptide is 6 to 10,000 amino acid residues.
  • the length of the immunogenic polypeptide is 25 to 2,000 amino acid residues.
  • the length of the immunogenic polypeptide is 50 to 500 amino acid residues.
  • the immunogenic polypeptide is selected from the RBD protein of SARS-CoV-2 or an immunogenic fragment or variant thereof, the Env protein of HIV-1 or an immunogenic fragment (e.g., gp140, gp160) or variant thereof, and the gE protein of VZV or an immunogenic fragment or variant thereof.
  • the fragment of the Env protein is selected from the group consisting of gp160 protein, gp120 protein, and gp41 protein.
  • the Env protein has an amino acid sequence as shown in SEQ ID NO: 34 or 35.
  • the sequence of the gp160 protein is shown in GenBank with accession numbers AAB05604 and AAD12142.
  • the amino acid sequence of gp41 is shown in GenBank with accession number CAD20975.
  • the RBD protein has an amino acid sequence as shown in any one of SEQ ID NO:1-8.
  • the gE protein has an amino acid sequence as shown in SEQ ID NO:30.
  • Nanobodies that can specifically bind to specific antigens by various methods known in the art, for example, by immunizing alpacas or sharks with specific antigens, screening out positive clones, obtaining the sequence of the heavy chain of the antibody by sequencing, and then constructing a vector containing the sequence of the heavy chain of the antibody, transfecting host cells under specific conditions, and expressing them to obtain Nanobodies.
  • nanobodies of the present application are not limited to the specific forms (e.g., nanobodies) and specific sequences specifically used in the embodiments.
  • the Nanobody is a camelid (eg, alpaca) antibody or a fish (eg, shark) antibody.
  • the Nanobody is a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the fusion protein comprises 2, 3, or more immunogenic polypeptides.
  • each immunogenic polypeptide is independently obtained from the same or a different pathogen (eg, virus).
  • pathogen eg, virus
  • each immunogenic polypeptide is a different polypeptide obtained from the same pathogen (eg, virus).
  • the fusion protein comprises a first immunogenic polypeptide and a second immunogenic polypeptide, and the first immunogenic polypeptide is the RBD protein of SARS-CoV-2, and the second immunogenic polypeptide is the gE protein of VZV.
  • the nanobody specifically binds to the RBD protein and the gE protein.
  • the fusion protein comprises a first immunogenic polypeptide, a second immunogenic polypeptide and a third immunogenic polypeptide
  • the first immunogenic polypeptide is the RBD protein shown in SEQ ID NO: 1
  • the second immunogenic polypeptide is the RBD protein shown in SEQ ID NO: 2
  • the third immunogenic polypeptide is the RBD protein shown in SEQ ID NO: 3.
  • the nanobody specifically binds to the RBD protein shown in SEQ ID NO: 1-3.
  • the fusion protein comprises 1 immunogenic polypeptide.
  • the Nanobody is a Nanobody that specifically binds to a polypeptide of HEV, HBV and/or HPV.
  • the nanobody comprises CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VHH) shown in any one of SEQ ID NO: 10-29, 68, 69.
  • VHH heavy chain variable region
  • the CDRs are defined according to the IMGT, Kabat or Chothia numbering systems.
  • the Nanobody comprises:
  • VHH heavy chain variable region
  • CDRs complementarity determining regions
  • VHH heavy chain variable region
  • CDRs three complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs three complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • the Nanobody comprises a sequence as shown in any one of SEQ ID NOs: 10-29, 68, 69 or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.
  • the substitutions are conservative substitutions.
  • the fusion protein further comprises a linker.
  • the linker is a polypeptide, such as a flexible peptide or a rigid peptide.
  • the linker comprises one or more (e.g., 1, 2, or 3) sequences as shown in (GmS)n, wherein m is selected from an integer of 1 to 6, and n is selected from an integer of 1 to 6. In certain embodiments, m is 3, 4, or 5. In certain embodiments, n is 2, 3, or 4.
  • the linker has an amino acid sequence as shown in SEQ ID NO:39.
  • the immunogenic polypeptide and the Nanobody of the fusion protein are directly linked or linked via a linker.
  • the immunogenic polypeptide is located at the N-terminus or C-terminus of the fusion protein.
  • the fusion protein comprises, from N-terminus to C-terminus, in sequence: an immunogenic polypeptide and a nanobody; or, a nanobody and an immunogenic polypeptide; or, an immunogenic polypeptide, a linker and a nanobody; or, a nanobody, a linker and an immunogenic polypeptide.
  • the fusion protein further comprises a signal peptide and/or a tag.
  • the signal peptide is selected from tPA signal peptide, bee venom signal peptide. In certain embodiments, The signal peptide has an amino acid sequence as shown in SEQ ID NO: 31, 37 or 38.
  • the tag is a tag used for purification, for example, selected from a His tag or a GST tag.
  • the signal peptide is located at the N-terminus of the fusion protein.
  • the tag is located at the C-terminus of the fusion protein.
  • the fusion protein has an amino acid sequence as shown in any one of SEQ ID NO: 42-49, 50-56, 72-79, 80-87, 63, 64, 89.
  • the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein as described in the first aspect.
  • the nucleotide sequence is codon-optimized or non-optimized according to the codon preference of the host cell.
  • codons have degeneracy. That is, during the translation of proteins, each amino acid may correspond to one or more codons, for example, up to six codons. Different species (e.g., host cells) have great differences in the use of degenerate codons encoding a certain amino acid, and have different preferences. This preference phenomenon is referred to as "codon preference”. Therefore, as used herein, the term "codon preference" refers to the situation in which a species prefers to use certain specific codons to encode amino acids. Optimizing the sequence of nucleic acid molecules according to codon preference is particularly advantageous in some cases, for example, it may help to improve the expression level of the protein encoded by the nucleic acid molecule.
  • the present application provides a vector comprising the isolated nucleic acid molecule as described in the second aspect.
  • the vector is used to express (eg, express in vitro in a cell) a protein encoded by the isolated nucleic acid molecule.
  • the present application provides a host cell comprising the nucleic acid molecule as described in the second aspect or the vector as described in the third aspect.
  • the host cell is selected from a prokaryotic cell and a eukaryotic cell.
  • the prokaryotic cell is selected from the group consisting of an Escherichia coli cell and a Bacillus subtilis cell.
  • the eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, a plant cell, and an animal cell.
  • the animal cell is a mammalian cell (eg, a murine cell, a human cell).
  • the present application provides a method for expressing or producing the fusion protein as described in the first aspect, the method comprising culturing the host cell described in the fourth aspect under conditions allowing protein expression, and optionally, recovering or purifying the expressed fusion protein.
  • the present application provides a composition comprising at least one fusion protein as described in the first aspect.
  • the composition further comprises an assembling polypeptide.
  • the assembling polypeptides assemble into VLPs.
  • the fusion protein is attached to the VLP.
  • the present application provides a kit comprising: the fusion protein as described in the first aspect or a first nucleic acid molecule containing a nucleotide sequence encoding the fusion protein, and an assembly polypeptide or a second nucleic acid molecule containing a nucleotide sequence encoding the fusion protein.
  • the fusion protein or first nucleic acid molecule and the assembly polypeptide or second nucleic acid molecule are provided separately or in the form of a composition.
  • the kit further comprises a vector (eg, an expression vector).
  • a vector eg, an expression vector
  • the first nucleic acid molecule and the second nucleic acid molecule are contained on the same or different vectors.
  • the kit further comprises a buffer.
  • the buffer is selected from a phosphate buffer, a citrate buffer, a carbonate buffer, an acetate buffer, a barbituric acid buffer, a Tris buffer, or any combination thereof.
  • the buffer is PBS buffer.
  • the buffer further comprises a salt.
  • the salt is selected from NaCl, (NH 4 )SO 4 , NaSO 4 , NH 4 Cl, or any combination thereof.
  • the present application provides a particulate antigen, which comprises an assembly polypeptide in the form of VLP, and the fusion protein as described in the first aspect attached to the assembly polypeptide.
  • the fusion protein is attached to the VLP via the interaction of a Nanobody with the assembly polypeptide.
  • the VLP is attached to at least one fusion protein as described in the first aspect.
  • the VLP is further attached to an additional polypeptide or fusion protein (eg, a T cell epitope).
  • an additional polypeptide or fusion protein eg, a T cell epitope
  • the present application provides a method for preparing the particulate antigen as described in the eighth aspect, the method comprising: using the kit as described in the seventh aspect.
  • the method comprises contacting the assembly polypeptide with the fusion protein under conditions that allow for assembly of the VLP.
  • the conditions that allow VLP assembly are placing the assembled polypeptide in a solution.
  • the solution is a buffer comprising a salt.
  • the salt is selected from NaCl, (NH 4 )SO 4 , NaSO 4 , NH 4 Cl, or any combination thereof.
  • the method comprises: (i) adding the assembly polypeptide to a buffer and then contacting it with the fusion protein; or (ii) adding the assembly polypeptide and the fusion protein to a buffer together; or (iii) adding the fusion protein to a buffer and then adding the assembly polypeptide to the buffer;
  • the particulate antigen in the buffer is recovered or purified.
  • the present application provides a vaccine comprising the fusion protein as described in the first aspect, or the composition as described in the sixth aspect, or the particulate antigen as described in the eighth aspect, and an adjuvant.
  • the adjuvant is selected from aluminum salt adjuvants, zinc-aluminum mixed adjuvants (eg, FH002C), Freund's adjuvant, oil emulsion adjuvant, cytokine, TLR agonist, CpG adjuvant, liposome, AS01B adjuvant, or any combination thereof.
  • the present application provides a pharmaceutical composition comprising any one or more of (1) to (6):
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutically acceptable carrier and/or excipient is selected from a pH adjusting agent (e.g., phosphate Salt buffer), surfactant (e.g., cationic, anionic or non-ionic surfactant, such as Tween-80), adjuvant, ionic strength enhancer (e.g., sodium chloride), diluent, excipient, medium for containing or administering the therapeutic agent, and any combination.
  • a pH adjusting agent e.g., phosphate Salt buffer
  • surfactant e.g., cationic, anionic or non-ionic surfactant, such as Tween-80
  • adjuvant e.g., sodium chloride
  • ionic strength enhancer e.g., sodium chloride
  • the present application provides the use of the fusion protein as described in the first aspect, or the nucleic acid molecule as described in the second aspect, or the vector as described in the third aspect, or the host cell as described in the fourth aspect, or the composition as described in the sixth aspect, or the kit as described in the seventh aspect, or the particulate antigen as described in the eighth aspect in the preparation of a pharmaceutical composition or vaccine, wherein the pharmaceutical composition or vaccine is used to induce an immune response in a subject.
  • the immune response is a response to an immunogenic polypeptide and/or an assembled polypeptide.
  • the immune response is a T cell response (e.g., a CD4+ response or a CD8+ response).
  • the immune response is a B cell response.
  • the subject is a mammal, such as a human, monkey, or mouse.
  • the present application provides the use of the fusion protein as described in the first aspect, or the nucleic acid molecule as described in the second aspect, or the vector as described in the third aspect, or the host cell as described in the fourth aspect, or the composition as described in the sixth aspect, or the kit as described in the seventh aspect, or the particulate antigen as described in the eighth aspect in the preparation of a pharmaceutical composition or vaccine, wherein the pharmaceutical composition or vaccine is used to prevent and/or treat diseases and/or symptoms in a subject that benefit or are prevented by an immune response to an immunogenic polypeptide.
  • the disease and/or condition is caused by a tumor cell from which the immunogenic polypeptide is derived.
  • the disease and/or condition is caused by the pathogen (eg, virus, bacteria, fungus, parasite) from which the immunogenic polypeptide is derived.
  • pathogen eg, virus, bacteria, fungus, parasite
  • the disease and/or symptom is caused by a virus from which the immunogenic polypeptide is derived, e.g., chickenpox, novel coronavirus pneumonia, AIDS, genital warts, viral hepatitis (e.g., viral hepatitis B, viral hepatitis A, viral hepatitis C, viral hepatitis E), measles, mumps.
  • a virus from which the immunogenic polypeptide is derived e.g., chickenpox, novel coronavirus pneumonia, AIDS, genital warts, viral hepatitis (e.g., viral hepatitis B, viral hepatitis A, viral hepatitis C, viral hepatitis E), measles, mumps.
  • a virus from which the immunogenic polypeptide is derived e.g., chickenpox, novel coronavirus pneumonia, AIDS, genital warts, viral hepatitis (
  • the immunogenic polypeptide may be an RBD protein of a novel coronavirus or a fragment thereof.
  • the disease may be novel coronavirus pneumonia.
  • the immunogenic polypeptide may be the gE protein of VZV or a fragment thereof.
  • the disease may be varicella.
  • the immunogenic polypeptide may be a protein of HPV or a fragment thereof.
  • the disease may be condyloma acuminatum.
  • the immunogenic polypeptide may be a protein or fragment thereof of hepatitis B virus, hepatitis A virus, hepatitis C virus, hepatitis E virus.
  • the disease may be viral hepatitis (e.g., viral hepatitis B, viral hepatitis A, viral hepatitis C, viral hepatitis E).
  • the immunogenic polypeptide may be an envelope glycoprotein of the measles virus or a fragment thereof.
  • the disease may be measles.
  • the immunogenic polypeptide may be a protein of the mumps virus or a fragment thereof.
  • the disease may be mumps.
  • the subject is a mammal, such as a human, monkey, or mouse.
  • the present application provides a method for inducing an immune response in a subject, comprising administering to the subject an effective amount of the fusion protein as described in the first aspect, or the nucleic acid molecule as described in the second aspect, or the vector as described in the third aspect, or the host cell as described in the fourth aspect, or the composition as described in the sixth aspect, or the kit as described in the seventh aspect, or the granulated antigen as described in the eighth aspect, or the vaccine as described in the tenth aspect, or the pharmaceutical composition as described in the eleventh aspect.
  • the immune response is a response to an immunogenic polypeptide and/or an assembled polypeptide.
  • the immune response is a T cell response (e.g., a CD4+ response or a CD8+ response).
  • the immune response is a B cell response.
  • the subject is a mammal, such as a human, monkey, or mouse.
  • the present application provides a method for preventing and/or treating a disease and/or symptom in a subject that benefits or is prevented by an immune response to an immunogenic polypeptide, comprising administering to the subject an effective amount of the fusion protein as described in the first aspect, or the nucleic acid molecule as described in the second aspect, or the vector as described in the third aspect, or the host cell as described in the fourth aspect, or the composition as described in the sixth aspect, or the kit as described in the seventh aspect, or the granulated antigen as described in the eighth aspect, or the vaccine as described in the tenth aspect, or the pharmaceutical composition as described in the eleventh aspect.
  • the disease and/or condition is caused by a tumor cell from which the immunogenic polypeptide is derived.
  • the disease and/or condition is caused by the pathogen (eg, virus, bacteria, fungus, parasite) from which the immunogenic polypeptide is derived.
  • pathogen eg, virus, bacteria, fungus, parasite
  • the disease and/or symptom is caused by a virus from which the immunogenic polypeptide is derived, for example, chickenpox, COVID-19, AIDS, condyloma acuminatum, viral hepatitis (e.g., viral hepatitis B Inflammation, viral hepatitis A, viral hepatitis C, viral hepatitis E), measles, mumps.
  • a virus from which the immunogenic polypeptide is derived for example, chickenpox, COVID-19, AIDS, condyloma acuminatum, viral hepatitis (e.g., viral hepatitis B Inflammation, viral hepatitis A, viral hepatitis C, viral hepatitis E), measles, mumps.
  • a virus from which the immunogenic polypeptide is derived for example, chickenpox, COVID-19, AIDS, condyloma acuminatum, viral
  • the immunogenic polypeptide may be HIV Env protein or a fragment thereof (eg, gp160 protein, gp120 protein, gp41 protein).
  • the disease may be AIDS.
  • the immunogenic polypeptide may be an RBD protein of a novel coronavirus or a fragment thereof.
  • the disease may be novel coronavirus pneumonia.
  • the immunogenic polypeptide may be the gE protein of VSV or a fragment thereof.
  • the disease may be varicella.
  • the immunogenic polypeptide may be a protein of HPV or a fragment thereof.
  • the disease may be condyloma acuminatum.
  • the immunogenic polypeptide may be a protein or fragment thereof of hepatitis B virus, hepatitis A virus, hepatitis C virus, hepatitis E virus.
  • the disease may be viral hepatitis (e.g., viral hepatitis B, viral hepatitis A, viral hepatitis C, viral hepatitis E).
  • the immunogenic polypeptide may be an envelope glycoprotein of the measles virus or a fragment thereof.
  • the disease may be measles.
  • the immunogenic polypeptide may be a protein of the mumps virus or a fragment thereof.
  • the disease may be mumps.
  • the subject is a mammal, such as a human, monkey, or mouse.
  • the present application provides a system for preparing a particulate immunogenic polypeptide, comprising a first carrier and a second carrier, wherein the first carrier comprises a nucleotide sequence encoding a fusion protein, the fusion protein comprises an immunogenic polypeptide and an assembly polypeptide, and the second carrier comprises a nucleotide sequence encoding a nanobody; and the nanobody can specifically bind to the assembly polypeptide, and the assembly polypeptide can be assembled into a VLP;
  • the nucleotide sequence is codon-optimized or non-optimized according to the codon preference of the host cell.
  • the assembling polypeptide is selected from proteins of hepatitis E virus (HEV) or fragments thereof or variants thereof. In certain embodiments, the assembling polypeptide is as defined in the first aspect.
  • HEV hepatitis E virus
  • the fusion protein is as defined in the first aspect.
  • the present application provides a method for enhancing the immunogenicity of an immunogenic polypeptide, comprising preparing or obtaining a fusion protein containing the immunogenic polypeptide and a nanobody capable of specifically binding to an assembled polypeptide; and, The fusion protein is contacted with a VLP comprising the assembled polypeptide, thereby obtaining a particulate antigen comprising the immunogenic polypeptide attached to the VLP.
  • the method comprises: using the system of aspect 16. In some embodiments, the method comprises: (1) expressing or producing a fusion protein by a first vector, and expressing or producing an assembly polypeptide by a second vector; (2) contacting the fusion protein and the assembly polypeptide under conditions that allow VLP assembly.
  • the Nanobody is as defined in the first aspect.
  • the fusion protein is as defined in the first aspect.
  • the assembly polypeptide is assembled into a VLP.
  • the fusion protein is attached to the VLP via the interaction of a nanobody with the assembly polypeptide.
  • the term "immunogenic polypeptide” refers to a protein or polypeptide that can cause an immune response.
  • the immunogenic polypeptide is administered (directly or indirectly) to a subject, and can cause an immune response in the subject. It is understood by those skilled in the art that the immunogenic polypeptide may be naturally occurring, or may be a protein or polypeptide that is naturally produced or artificially introduced with mutations or variations (including but not limited to, substitutions, deletions and/or additions) but does not affect its biological activity (in this article, the biological activity is the ability to cause an immune response).
  • the immunogenic polypeptide may be a polypeptide or variant thereof derived from a non-pathogen (e.g., a tumor cell) or a pathogen (e.g., a virus, a bacterium, a fungus, a parasite or other pathogen).
  • a pathogen e.g., a virus, a bacterium, a fungus, a parasite or other pathogen.
  • the term "immune response” refers to the response of cells of the immune system (e.g., B cells, T cells, or monocytes) to stimulation.
  • the immune response is a response specific to a particular antigen (i.e., an antigen-specific response).
  • the immune response is a T cell response, such as a CD4+ response or a CD8+ response.
  • the immune response is a B cell response, which can result in the production of specific antibodies.
  • subviruses refers to a microorganism that does not have a complete viral structure, mainly including viroids, pseudovirions and prions.
  • SARS-Cov-2 RBD protein or “SARS-Cov-2 RBD” has the same meaning and is used interchangeably, which refers to the receptor binding site on the spike protein (S protein) of SARS-Cov-2.
  • S protein spike protein
  • the main function of the RBD protein is to recognize host cell surface receptors and mediate fusion with host cells. It is understood by those skilled in the art that different SARS-Cov-2 virus strains may contain RBD proteins of different sequences, but these RBD proteins have the same or similar biological properties. Therefore, in the present invention, the RBD protein not only includes the proteins shown in SEQ ID NO: 1-8, but also includes the RBD proteins of various SARS-Cov-2 virus strains.
  • the amino acid sequences of these RBD proteins can be obtained from public databases (such as the GenBank database), for example, the amino acid sequences shown in GenBank accession numbers OP077006.1, OP077005.1, and OP077003.1.
  • gE protein of VZV refers to a kind of envelope glycoprotein of VZV, which have the same meaning and can be used interchangeably. It is understood by those skilled in the art that different VZV strains may contain gE proteins with different sequences, but these gE proteins have the same or similar biological properties. Therefore, in the present invention, the gE protein not only includes the protein shown in SEQ ID NO:30, but also should include the gE proteins of various VZV strains. The amino acid sequences of these gE proteins can be obtained from public databases (such as the GenBank database).
  • Env of HIV-1 refers to the envelope protein on the surface of the HIV-1 virus, also referred to as "BGTSTIP” in this article.
  • BGTSTIP the envelope protein on the surface of the HIV-1 virus
  • Env proteins include not only the proteins shown in SEQ ID NO:34-35, but also Env proteins of various HIV-1 strains.
  • the amino acid sequences of these Env proteins can be obtained from public databases (such as the GenBank database).
  • the capsid protein encoded by the ORF2 gene in HEV also referred to as ORF2 protein
  • ORF2 protein also referred to as ORF2 protein
  • its fragments e.g., p239 protein, p495 protein
  • the sequence of the ORF2 protein is well known in the art, see, for example, DDBJ database accession number: D11092.
  • the sequence of the p239 protein is from amino acid 368 to amino acid 606 corresponding to the ORF2 protein.
  • the sequence of the p495 protein is from amino acid 112 to amino acid 606 corresponding to the ORF2 protein.
  • the term "assembly polypeptide” refers to a protein or polypeptide that can be assembled into a virus-like particle (VLP).
  • the assembly polypeptide is a coat protein of a natural virus or viroid.
  • the VLP assembled from the assembly polypeptide is similar in structure to the natural virus or viroid, the only difference being that it does not contain the genome of the natural virus or viroid.
  • the assembly polypeptide is an artificially prepared and/or screened polypeptide.
  • the VLP assembled from the assembly polypeptide is similar or dissimilar in structure to the natural virus or viroid.
  • virus-like particle is a multimeric particle whose structure is similar or dissimilar to that of a natural virus or subvirus. It has been demonstrated that proteins (e.g., capsid proteins, surface proteins, envelope proteins) of some viruses (e.g., HBV, HEV, HPV) can spontaneously form after recombinant expression in an appropriate expression system. VLP.
  • the protein to be tested can be placed in a buffer solution (e.g., PBS solution) at room temperature, and then the presence of VLPs can be detected.
  • a buffer solution e.g., PBS solution
  • Conventional techniques known in the art can be used to detect the presence of VLPs, such as electron microscopy, biophysical characterization, etc. For specific detection methods, see, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Virol. 68: 4503-4505.
  • capsid protein encoded by the ORF2 gene in HEV also called ORF2 protein
  • its fragments e.g., p239 protein, p495 protein
  • the assembled polypeptides include not only natural proteins or polypeptides that can be assembled into VLPs, but also proteins or polypeptides that are naturally produced or artificially introduced with mutations or variations (including but not limited to, substitutions, deletions and/or additions) based on the natural proteins but do not affect their biological functions (in this article, the biological function is the ability to assemble into VLPs). Therefore, in certain embodiments, the assembled polypeptides are selected from the ORF2 protein of hepatitis E virus (HEV) or its fragments or variants thereof. In certain embodiments, the assembled polypeptides are selected from p239 protein or its fragments or variants thereof, p495 protein or its fragments or variants thereof.
  • HEV hepatitis E virus
  • variant refers to a protein/polypeptide that has one or more (e.g., 1-10 or 1-5 or 1-3) amino acid differences (e.g., substitutions, deletions or additions) compared to the sequence of the protein/polypeptide from which it is derived, and which retains the biological activity of the protein/polypeptide from which it is derived.
  • amino acid differences e.g., substitutions, deletions or additions
  • the term "derived from” in "the immunogenic polypeptide is a polypeptide derived from an organism or a non-organism or an immunogenic variant thereof” means that the sequence of the polypeptide is derived from a sequence in an organism or a non-organism, but the method of obtaining the polypeptide is not limited to a specific production method.
  • the polypeptide may be naturally isolated, artificially synthesized, or obtained by genetic engineering recombination.
  • the term "particulated immunogenic polypeptide” refers to a collection of immunogenic polypeptides in the form of particles.
  • the particulate immunogenic polypeptide refers to an immunogenic polypeptide attached to a virus-like particle.
  • organism refers to a living individual or object. Except for a few species such as viruses, organisms are composed of cells. In certain embodiments, organisms include pathogens and non-pathogens.
  • pathogens refers to microorganisms (eg, bacteria, viruses, rickettsia, fungi), parasites, or other agents (eg, recombinant microorganisms) that can cause infection in humans, animals, or plants.
  • fusion protein refers to a protein composed of at least two separate protein or polypeptide amino acid sequences linked together.
  • the amino acid sequences of the two independent proteins or polypeptides can be directly connected or connected through a linker.
  • conservative substitution means an amino acid substitution that does not adversely affect or change the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • vector refers to a nucleic acid carrier into which a polynucleotide can be inserted.
  • a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell.
  • Vectors are well known to those skilled in the art, and include but are not limited to: plasmids (e.g., naked plasmids); phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phages or M13 phages, and viral vectors, etc.
  • plasmids e.g., naked plasmids
  • phagemids e.g., cosmids
  • artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)
  • bacteriophages such as lambda phages or M13 phages, and viral vectors, etc.
  • the term "host cell” refers to a cell that can be used to amplify or express exogenous genes, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, 293T cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts
  • CHO cells COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, 293T cells or human cells.
  • antibody refers to an immunoglobulin that is capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • Antibodies include antibodies of any type, such as IgG, IgA or IgM (or their subclasses), and antibodies do not need to belong to any particular class. Immunoglobulins can be assigned to different types depending on the amino acid sequence of the constant region of the antibody heavy chain.
  • immunoglobulins There are five major types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant regions corresponding to different types of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively.
  • Antibody light chains can be classified into ⁇ (kappa) and ⁇ (lambda) light chains.
  • the subunit structures and three-dimensional configurations of different types of immunoglobulins are well known.
  • the heavy chain constant region consists of 4 domains (CH1, hinge region, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain CL.
  • the constant domains are not directly involved in antibody binding to antigen, but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • VH and VL regions of antibodies can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antigen binding site, respectively.
  • the assignment of amino acids to regions or domains may follow the definitions of Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.
  • Chothia an immunoglobulin numbering system proposed by Chothia et al. that identifies CDRs based on the position of structural loop regions Classical rules for region boundaries (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • CDR complementarity determining region
  • the variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3.
  • CDR1, CDR2, and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al.
  • framework region or "FR” residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
  • single-domain antibody is also referred to as nanobody, and the two can be used interchangeably. It has the meaning commonly understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region, also referred to as VHH), which retains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibody. Nanobodies can be alpaca antibodies derived from camels, or shark antibodies derived from sharks.
  • Nanobodies can be screened for specificity in the same way as for intact antibodies using conventional techniques known to the person skilled in the art.
  • the expression "specific binding” refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed.
  • the strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction.
  • KD the equilibrium dissociation constant
  • the term "KD” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the term "adjuvant” refers to an immunopotentiator, which, when delivered into the body together with an antigen or in advance, can enhance the body's immune response to the antigen or change the type of immune response.
  • adjuvants including but not limited to aluminum salt adjuvants, zinc aluminum mixed adjuvants (e.g., FH002C), Freund's adjuvant, oil emulsion adjuvant, cytokine, TLR agonist, CpG adjuvant, liposome, AS01B adjuvant or a combination thereof.
  • the term “pharmaceutically acceptable” means that it is recognized in the pharmaceutical field that it can be used in animals, especially in humans.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed.
  • pH adjusters including but not limited to phosphate buffers
  • surfactants including but not limited to cationic, anionic or non-ionic surfactants, such as Tween-80
  • adjuvants including but not limited to chlorides
  • ionic strength enhancers including but not limited to chlorides
  • sodium diluents, excipients, media for containing or administering therapeutic agents, and any combination thereof.
  • the term "subject” refers to mammals, including but not limited to humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, etc.
  • the subject is a human.
  • the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • a disease prevention effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease
  • a disease treatment effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.
  • condition allowing VLP assembly refers to conditions under which the assembly polypeptide can be assembled into VLP.
  • a polypeptide or protein with assembly capability is placed in a liquid, which can self-assemble into VLP.
  • the condition allowing VLP assembly is to place the assembly polypeptide in a solution.
  • the solution will not contain a composition that is unfavorable for VLP assembly.
  • the solution contains a composition that is conducive to VLP assembly, for example, the solution is a buffer containing salt.
  • the present invention utilizes the ability of assembled polypeptides (e.g., ORF2 protein of HEV or its fragments or variants thereof, L1 protein of HPV or its fragments or variants thereof, surface protein of HBV or its fragments or variants thereof) to assemble into VLPs, fuses and expresses nano antibodies that specifically bind to the assembled polypeptides with immunogenic polypeptides, and utilizes the targeted binding properties of nano antibodies to the assembled polypeptides to display a variety of immunogenic polypeptides on the surface of VLPs formed by the assembled polypeptides, thereby obtaining granulated immunogenic polypeptides.
  • the present application provides a system and method for granulating immunogenic polypeptides.
  • the granulated immunogenic polypeptides have higher immunogenicity and can stimulate high-level immune responses, including B cell and T cell responses. Therefore, the granulated immunogenic polypeptides of the present invention are particularly suitable for vaccine production and vaccination, and have advantages in preventing and/or treating viral infections.
  • the system and method for granulating immunogenic polypeptides of the present invention use nanobodies.
  • the use of nanobodies has at least the following advantages: 1
  • the display of immunogenic polypeptides mediated by nanobodies is not affected by the N/C-terminal structure of the assembled polypeptides, and the construction strategy is more flexible.
  • the strategy of nanobody-mediated display will not affect the assembly and conformation of the assembled polypeptides, and has a wider range of applicability.
  • the fusion of nanobodies and immunogenic polypeptides is conducive to maintaining the immunogenic 4 Nanobodies of different epitopes can be combined and bound, which is beneficial to increase the proportion of immunogenic polypeptide display.
  • Nanobodies can be rationally designed based on the symmetry of the assembled polypeptides and the oligomeric state of the immunogenic polypeptides. 6
  • the nanoantibody-mediated display strategy can reduce the potential steric hindrance of immunogenic polypeptides.
  • Antibody-mediated display can directly target unmodified or modified VLPs. If the VLPs that have been marketed have good safety themselves, there is no need to consider their own drugability issues, and they have good convertibility.
  • Nanoantibodies as linkers can minimize their immunogenicity through humanization strategies well known in the art, reduce the body's unnecessary immune response, and achieve immunological focusing of immunogenic polypeptides.
  • Any immunogenic polypeptide can be granulated by the method of the present invention through fusion expression with nanoantibodies, which has universality and versatility. 10 Due to the small size of nanoantibodies, the exposure of the original major immune epitopes of the granulated carriers such as HEV ORF2, HPV L1 and HBV S can be retained by screening and identifying the binding positions of nanoantibodies. The granulated carriers of immunogenic polypeptides still have good immunogenicity and can be used as a combination vaccine. This strategy can also be used in the development of other combination vaccines, thus having great application prospects.
  • Figure 1 shows the screening process of the nanobodies of the present invention.
  • Figure 1A shows the results of agarose gel electrophoresis of bacterial library colony PCR.
  • Figure 1B shows the results of evaluating the diversity of the bacterial library after sequencing.
  • Figure 2 shows the SDS polyacrylamide gel electrophoresis results of various VHH-RBD proteins of the embodiments of the present invention.
  • Figure 2A is the electrophoresis results of various VHH-RBD fusion proteins produced and purified by insect cell expression system.
  • Figure 2B is the electrophoresis results of various VHH-RBD fusion proteins produced and purified by mammalian cell expression system.
  • M Molecular weight marker.
  • Figure 3 shows the SDS polyacrylamide gel electrophoresis results of various VHH-gE fusion proteins of the examples of the present invention.
  • M molecular weight marker
  • lanes 1-7 respectively show different VHH-gE fusion proteins.
  • Figure 4 shows the SDS-polyacrylamide gel electrophoresis results of various VHH-BGTSTIP proteins of the present invention. "+” indicates reducing conditions, and “-” indicates non-reducing conditions.
  • FIG. 5 shows the immunoblotting experiment of various VHH-RBD fusion proteins of the present invention.
  • Figure 5A shows a variety of VHH-RBD fusion proteins produced and purified by an insect cell expression system
  • M molecular weight marker
  • lanes 1-8 respectively show fusion proteins of different nanobodies and RBD.
  • Figure 5B shows a variety of VHH-RBD fusion proteins produced and purified by a mammalian cell expression system 293F cells.
  • FIG6 shows the results of Western Blot experiments of various candidate molecules VHH-gE according to the embodiments of the present invention.
  • M molecular weight marker
  • lanes 1-7 respectively show fusion proteins of different nanobodies and gE.
  • Figure 7 shows the results of enzyme-linked immunosorbent assay (ELISA) of various VHH-RBDs in the examples and various reported specific monoclonal antibodies.
  • Figure 7A shows the activity identification results of various VHH-RBD proteins produced and purified by insect cell expression system, and the antibodies used include broad-spectrum neutralizing antibodies JSR-105, JSR-551, JSR-209, m6D6, m7D6, and non-broad-spectrum neutralizing antibody 85F7.
  • Figure 7B shows the activity identification results of various VHH-RBD proteins produced and purified by mammalian cell expression system 293F cells, and the antibodies used include 3G11, 8H12, 13F10, 8B8, 9D3, and 3F9.
  • Figure 8 shows the results of enzyme-linked immunosorbent assay (ELISA) of various VHH-gE and various specific monoclonal antibodies of the embodiment.
  • the specific monoclonal antibodies used include 3H7, 4G4, 6B7, 11B11, 11B12, 13B6, 14G1, and 17B7.
  • FIG. 9 shows the results of enzyme-linked immunosorbent assay (ELISA) of various VHH-BGTSTIPs of the examples with various reported HIV-1 neutralizing antibodies or non-neutralizing antibodies.
  • ELISA enzyme-linked immunosorbent assay
  • Figure 10 shows the affinity analysis results of various VHH-RBDs and HEV-p239 proteins in the examples.
  • Figure 10A shows the affinity results of VHH-RBD and HEV-p239 proteins produced and purified by insect cell expression system
  • Figure 10B shows the affinity results of VHH-RBD and HEV-p239 proteins produced and purified by mammalian cell expression system 293F cells.
  • FIG. 11 shows the results of affinity analysis of various VHH-gEs of the Examples with HEV-p239 protein.
  • FIG. 12 shows the results of affinity analysis of various VHH-BGTSTIPs of the Examples with HEV-p239 protein.
  • Figure 13A shows the results of purification of P1-5B-RBD produced and purified by insect cell expression system through superdex 200 increase (high performance liquid chromatography molecular sieve) in an embodiment of the present invention.
  • Figure 13B shows the results of purification of P2-6D-RBD, P2-3E-RBD, and P2-10G-RBD produced and purified by mammalian cell expression system through superdex 200 increase in an embodiment of the present invention.
  • Figure 14A shows the purification profile and SDS-PAGE identification results of the complex formed by the P1-5B-RBD fusion protein purified by the insect cell expression system and HEV-p239.
  • Figure 14B shows the purification profile and SDS-PAGE identification results of the complex formed by the P2-3E-RBD, P2-10G-RBD and P2-6D-RBD fusion proteins purified by the mammalian cell expression system and HEV-p239, respectively.
  • Figure 15 shows the purification pattern and SDS-PAGE identification results of the complex of the candidate fusion protein P2-8C-gE and HEV-p239 of the embodiment of the present invention.
  • Figure 15A is the purification result of the complex.
  • Figure 15B is the SDS-PAGE identification result of the complex of P2-8C-gE and HEV-p239.
  • FIG. 16 shows the purification pattern and SDS-PAGE identification results of the complex of the candidate fusion protein P2-5C-BGTSTIP and HEV-p239 according to the embodiment of the present invention.
  • FIG. 17 shows the results of high performance size exclusion chromatography (HPSEC) of Example P2-8C-gE of the present invention.
  • Figure 18A shows the molecular size detection results of HEV-RBD complexes and HEV-p239 particles formed by VHH-RBD and HEV-p239 protein produced and purified by insect cell expression system in an embodiment of the present invention.
  • Figure 18B shows the molecular size detection results of three HEV-RBD complexes and HEV-p239 particles formed by VHH-RBD and HEV-p239 protein produced and purified by mammalian cell expression system.
  • Figure 19 shows the molecular particle size detection results of the HEV-gE complex sample of the present invention.
  • Figure 19A shows the molecular particle size of HEV-p239 particles
  • Figure 19B shows the molecular particle size of the HEV-gE complex.
  • FIG. 20 shows the molecular size detection results of the HEV-BGTSTIP complex and HEV-p239 particles according to an embodiment of the present invention.
  • Figure 21 shows the analytical ultracentrifugation results of HEV-p239 and RBD complexes constructed based on HEV-p239 particles in the present invention.
  • Figure 21A shows the analytical ultracentrifugation results of the complex particles prepared by the purified VHH-RBD produced by the insect cell expression system and HEV-p239 (right figure) and HEV-p239 (left figure).
  • Figure 21B shows the analytical ultracentrifugation results of three complex particles prepared by the purified VHH-RBD produced by the mammalian cell expression system 293F cells and HEV-p239.
  • Figure 22 shows the results of the analysis of HEV-p239 and the gE complex constructed based on HEV-p239 particles according to the present invention.
  • Figure 22A shows that HEV-p239 presents a single component with a sedimentation coefficient of 22S.
  • Figure 22B shows that the gE complex presents a single component with a sedimentation coefficient of 31S.
  • FIG. 23 shows the analytical ultracentrifugation results of the HEV-p239 and the Env(BGTSTIP) complex constructed based on the HEV-p239 particles according to the examples of the present invention.
  • Figure 24A shows the negative staining results of transmission electron microscopy of a complex sample prepared by HEV-p239 and purified VHH-RBD produced by an insect cell expression system in an embodiment of the present invention.
  • Figure 24B shows the negative staining results of transmission electron microscopy of a complex sample prepared by HEV-p239 and purified VHH-RBD produced by a mammalian expression system 293F cells in an embodiment of the present invention.
  • FIG. 25 shows the negative staining results of a transmission electron microscope of a complex sample prepared from HEV-p239 and VHH-gE fusion protein according to an embodiment of the present invention.
  • FIG. 26 shows the negative staining results of transmission electron microscopy of the HEV-p239 and the Env(BGTSTIP) complex constructed based on the HEV-p239 particles according to the example of the present invention.
  • Figure 27 shows the immunogenicity test results of the RBD particle antigen of the embodiment of the present invention.
  • Figure 27A is the serum binding and neutralization activity test results after mice were immunized with RBD particle antigen obtained by insect cell expression system.
  • Figures 27B and 27C are the serum binding activity and neutralization activity test results against wild-type SARS-CoV-2 pseudovirus after mice were immunized with RBD particle antigen obtained by mammalian cell expression system.
  • Figure 28 shows the neutralization test results of the antibodies induced by the RBD particle antigen combined with aluminum adjuvant in an embodiment of the present invention and the wild-type (WT) strain of the new coronavirus, the Gamma strain, and the BA.2 strain.
  • WT wild-type
  • FIG. 29 shows the immunogenicity test results of the gE particle antigen of the embodiment of the present invention.
  • FIG. 30 shows the results of live virus neutralization test of mouse immune serum against gE particle antigen of an example of the present invention.
  • FIG31 shows the flow cytometry immunoassay results of the monomeric gE antigen, gE particle antigen and control adjuvant according to the examples of the present invention.
  • FIG. 32 shows the ELISpot detection of cytokines in the immunization group of the monomeric gE antigen, gE particulate antigen and control adjuvant of the embodiment of the present invention.
  • Figure 33 shows the results of humanization transformation of Nanobody P1-5B of Example 1 of the present invention.
  • FIG34 shows the polyacrylamide electrophoresis results of the single-domain antibody fusion protein P1F8-BGTSTIP according to an embodiment of the present invention; wherein M is a molecular weight marker; “+” indicates reducing conditions, and “-” indicates non-reducing conditions.
  • Figure 35 shows the results of the enzyme-linked immunosorbent assay (ELISA) of the single-domain antibody fusion protein P1F8-BGTSTIP of the present invention and various reported HIV-1 broad-spectrum neutralizing antibodies and non-neutralizing antibodies.
  • ELISA enzyme-linked immunosorbent assay
  • 2G12, VRC01, PGT121, PGT121, SF12, and B12 are broad-spectrum neutralizing antibodies
  • 17b, F105, and F240 are non-neutralizing antibodies.
  • Figure 36 shows the affinity analysis results of the single-domain antibody fusion protein P1F8-BGTSTIP and HPV 58 VLP.
  • Figure 37 shows the high performance size exclusion chromatography (HPSEC) and SDS-PAGE identification results of the complex formed by P1F8-BGTSTIP and 58-VLP produced and purified by the mammalian expression system in the embodiment of the present invention.
  • the red curve represents the complex map formed by P1F8-BGTSTIP and 58VLP
  • the green curve represents the 58VLP map
  • the blue curve represents the fusion protein P1F8-BGTSTIP map.
  • FIG. 38 shows the complex formed by P1F8-BGTSTIP and 58VLP and the analytical ultracentrifugation results of 58VLP in the practice of the present invention.
  • FIG. 39 shows the negative staining results of a transmission electron microscope of a complex sample formed by P1F8-BGTSTIP and 58VLP in an example of the present invention.
  • FIG. 40 shows the results of the BGTSTIP-specific binding antibody titer test after mice were immunized with the 58-VLP-BGTSTIP particulate antigen in an example of the present invention.
  • Figure 41 shows the SDS-PAGE identification diagram of the particulate antigen formed after HBsAg binds to the nanobody fusion protein S2-gE in the present invention.
  • Figure 42 shows the molecular sieve identification diagram of the granulated antigen formed after HBsAg binds to the nanobody fusion protein S2-gE in the present invention.
  • Figure 43 shows the DLS identification diagram of the particulate antigen HBsAg-S2-gE (HBV-gE) formed after HBsAg binds to the nanobody fusion protein S2-gE in the present invention.
  • Figure 44 shows a negative staining electron micrograph of the particulate antigen HBsAg-S2-gE formed after HBsAg binds to the nanobody fusion protein S2-gE in the present invention.
  • Figure 45 shows the specific antibody titer results after HBsAg binds to the nanobody fusion protein S2-gE to form the particulate antigen HBsAg-S2-gE and S2-gE immunizes mice.
  • Example 1 Nanobody phage library construction and screening process
  • V is the volume of phage added (in ⁇ L)
  • T library is the phage titer
  • T is the phage titer (unit: pfu/mL)
  • D is the dilution multiple
  • N is the number of single colonies at the corresponding dilution multiple.
  • Figure 1 shows the screening process of the nanoantibody of the embodiment of the present invention.
  • Figure 1A shows the results of colony PCR agarose gel electrophoresis. 50 of the 51 randomly selected monoclones are positive clones, with a positive rate of 98%. The positive clone rate of the bacterial library meets the requirements.
  • Figure 1B is the protein sequence translated by software after sequencing. The sequence diversity comparison shows that all 50 sequences are independent sequences with good diversity. The diversity of the bacterial library meets the requirements.
  • V is the volume of helper phage added (in mL)
  • T helper-phage is the titer of helper phage used.
  • VHH nanoantibody heavy chain variable region
  • the VHH-RBD sequence in the insect cell expression system was constructed by connecting the RBD sequence (SEQ ID NO: 1-8) to the C-terminus of the VHH sequence (SEQ ID NO: 10-29), and then connecting the bee venom signal peptide (SEQ ID NO: 31) to the N-terminus of the above sequence to promote protein secretion expression.
  • the nucleotide sequence encoding the above amino acid sequence was synthesized by Bio-Industry, and the nucleotide sequence was constructed on the PIEX/bac-1 vector through the Nco I and BamH I restriction sites.
  • VHH-RBD proteins P1-5B-RBD, P2-2C-RBD, P1-1B-RBD, P1-1G-RBD, P2-4E-RBD, P2-3E-RBD, P2-3D-RBD, P2-3A-RBD
  • P1-5B-RBD, P2-2C-RBD, P1-1B-RBD, P1-1G-RBD, P2-4E-RBD, P2-3E-RBD, P2-3D-RBD, P2-3A-RBD were obtained through the insect cell expression system, and the sequences are shown in SEQ ID NO: 42-49 in Table 1.
  • VHH-gE fusion protein The same method was used to construct the sequence of the VHH-gE fusion protein, except that the RBD sequence connected to the C-terminus was replaced with the gE sequence (SEQ ID NO: 30).
  • VHH-gE proteins P2-10G-gE, P2-6D-gE, P2-5C-gE, P2-8C-gE, P2-1H-gE, P2-5G-gE, and P2-7D-gE
  • SEQ ID NO: 50-56 seven VHH-gE proteins (P2-10G-gE, P2-6D-gE, P2-5C-gE, P2-8C-gE, P2-1H-gE, P2-5G-gE, and P2-7D-gE) were obtained through the insect cell expression system, and their sequences are shown in SEQ ID NO: 50-56 in Table 1.
  • the VHH-RBD sequence in the mammalian cell expression system was constructed by sequentially connecting the (G4S)3 flexible protein linker (SEQ ID NO: 39), the RBD sequence (SEQ ID NO: 1-8), and the histidine tag (8-His tag) to the C-terminus of the VHH sequence (SEQ ID NO: 10-29), and introducing a signal peptide sequence (SEQ ID NO: 38) to promote protein secretion expression at the N-terminus.
  • the nucleotide sequence encoding the above amino acid sequence was synthesized by Universal Bio after codon optimization and cloned into the ECORI and Xbal restriction sites of the pcDNA3.1 vector.
  • VHH-RBD proteins (P1-1B-RBD, P1-5B-RBD, P2-2C-RBD, P2-5C-RBD, P2-3A-RBD, P2-3E-RBD, P2-4E-RBD, P2-5G-RBD) were obtained through the mammalian cell expression system, and their sequences are shown in SEQ ID No: 72 to 89 in Table 1.
  • VHH-BGTSTIP i.e., VHH-Env
  • the connected RBD sequence was replaced with the amino acid sequence of the full-length Env extracellular segment (SEQ ID NO: 34 or 35).
  • 8 VHH-BGTSTIP proteins P1-1B-BGTSTIP, P1-5B-BGTSTIP, P2-2C-BGTSTIP, P2-5C-BGTSTIP, P2-3A BGTSTIP, P2-3E-BGTSTIP, PA-4G-BGTSTIP, P2-7D-BGTSTIP
  • the sequences are shown in Table 1 as shown in SEO ID No: 80 to 87.
  • step (2) Collect the cell supernatant obtained in step (1), centrifuge at 500 g for 5 min, remove cell debris and debris, and store the supernatant at 4° C. in the dark. This is the P1 virus seed solution.
  • ESF921 culture medium to a 1L shake flask and culture H5 cells (purchased from Invitrogen, B855-02) with a density of 2 ⁇ 10 6 /mL and a survival rate of more than 90%.
  • Add the virus according to the corresponding MOI seal the bottle mouth with a sealing film, and culture it in a 27°C shaker at 120rpm. Take out the cells in the shake flask every day, observe and count them, and record the relevant data.
  • the appropriate MOI can ensure that more than 70% of the cells are diseased on the first day. On the second day, all cells are diseased, and the survival rate is about 80%. On the third day, the cells rupture and the survival rate drops to 30-50%. At this time, you can consider collecting the cells. Use The cells were collected by centrifugation at 10,000 rpm for 10 min, and the supernatant was separated and purified.
  • Purification medium Ni Sepharose 6 Fast Flow affinity medium
  • Buffer divided into A pump and B pump buffer, generally
  • a pump is 1 ⁇ PBS buffer (160g/L NaCL, 8.1mmol/LNa 2 HPO 4 , 1.5mmol/L KH 2 PO 4 , 2.7mmol/L KCL, pH7.4)
  • B pump is 1 ⁇ PBS+250mmol/L imidazole buffer;
  • PEI MW 25,000
  • VHH-RBD and VHH-BGTSTIP plasmids were placed in 50 ml of culture medium with PEI (MW 25,000) at a ratio of 1:2, mixed thoroughly, and then allowed to stand for 18 minutes. The mixture was then transferred to the above 450 ml of culture medium and suspended and cultured at 37°C, 5% CO 2 , 120 rpm for 6 days to express VHH-RBD and VHH-BGTSTIP proteins. During the transfection process, PEI was kept away from light.
  • PEI MW 25,000
  • VHH-RBD and VHH-BGTSTIP Purification of VHH-RBD and VHH-BGTSTIP in mammalian expression systems
  • the cell culture medium was collected and centrifuged at 7000g for 10 min in a JA-14 rotor to obtain the cell supernatant. After centrifugation at 20000g for 10 min, the supernatant was filtered twice with a 0.22 um pore size filter membrane, and the sample was used for the next step of Ni-excel column purification.
  • Purification medium Ni Sepharose excel affinity medium; Buffer: divided into A and B buffers, A liquid is 1 ⁇ PBS buffer, B pump is 1 ⁇ PBS+250mmol/L imidazole buffer;
  • Elution conditions Elute impurities with 20mM imidazole, and collect the 250mM imidazole elution product. Dialyze the eluate in 1 ⁇ PBS overnight, changing the dialysate twice. Harvest about 30ml of low-concentration target protein, Vivaspin 20ml, 100KD ultrafiltration concentration tube to 5ml for standby use. Prepare reduced and non-reduced samples from the collected elution samples, and perform SDS-PAGE gel electrophoresis according to the above method.
  • Figure 2 shows the results of SDS polyacrylamide gel electrophoresis of 8 kinds of VHH-RBD proteins prepared by the present invention.
  • Figure 2A is the electrophoresis results of various VHH-RBD fusion proteins produced and purified by an insect cell expression system.
  • M Molecular weight Marker, lanes 1-8 respectively show different VHH-RBD fusion proteins. The results show that after one-step purification of the VHH-RBD protein by affinity chromatography, the purity is about 90% and the molecular size is about 50KDa.
  • Figure 2B is the electrophoresis results of 10 kinds of VHH-RBD fusion proteins produced and purified by a mammalian cell expression system. The results show that the purity of the VHH-RBD protein is about 90%, and the molecular weight is about 50KDa.
  • FIG3 shows the SDS polyacrylamide gel electrophoresis results of 7 VHH-gE fusion proteins prepared by the present invention.
  • M molecular weight marker
  • lanes 1-7 respectively show different VHH-gE fusion proteins.
  • the results show that after one-step purification of the VHH-gE protein by affinity chromatography, the purity is about 80%, and the molecular size is about 85KDa.
  • FIG4 shows the SDS-polyacrylamide gel electrophoresis results of 8 VHH-BGTSTIP proteins prepared by the present invention. "+” indicates reducing conditions, and “-” indicates non-reducing conditions. The results show that after the VHH-BGTSTIP is purified by affinity chromatography, the purity is about 90% and the molecular weight is about 180KD.
  • Equal amounts of protein samples were mixed with loading buffer, boiled for 10 min, and loaded onto SDS-PAGE gels for western blotting (BioRad) according to laboratory standard protocols. Proteins were electrophoresed at 80 V for 70 min on a BioRad Miniprotean Tetra system, and the gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad) for 30 min at room temperature. The separated proteins were transferred to nitrocellulose membranes using a trans-blot turbo transfer system (Bio-Rad), blocked, and incubated with anti-His-HRP (1:5000 dilution) for 1 h. Unbound antibodies were removed by washing 5 times for 5 min each time, and then detected using a chemiluminescent substrate kit.
  • FIG5 shows the results of Western Blot experiments of various VHH-RBD fusion proteins of the embodiments of the present invention.
  • FIG5A shows 8 kinds of VHH-RBD fusion proteins produced and purified by an insect cell expression system, M: molecular weight marker, and lanes 1-8 respectively show fusion proteins of different nano antibodies and RBD. The results confirm that the 50KDa molecule is determined to be the target protein of the present invention.
  • FIG5B shows 10 kinds of VHH-RBD fusion proteins produced and purified by a mammalian cell expression system 293F cells. The results show that the purity of the VHH-RBD fusion protein is about 90%, and the molecular weight is about 50KDa. The results confirm that the 50KDa molecule is determined to be the target protein of the present invention.
  • FIG6 shows the results of Western Blot experiments of seven candidate molecules VHH-gE of the embodiments of the present invention.
  • M Molecular weight marker
  • lanes 1-7 respectively show fusion proteins of different nanobodies and gE. The results confirm that the 85KDa molecule is determined to be the target protein of the present invention.
  • FIG7 shows the results of enzyme-linked immunosorbent assay (ELISA) of various VHH-RBDs in the embodiment and various reported specific monoclonal antibodies.
  • FIG7A shows the activity identification results of 8 kinds of VHH-RBD proteins produced and purified by insect cell expression system.
  • the RBD antibodies used include broad-spectrum neutralizing antibodies VacW-105 (corresponding to JSR-105 in FIG7A ), JSR-551, VacW-209 (corresponding to JSR-209 in FIG7A ), 6D6 (corresponding to m6D6 in FIG7A ), 7D6 (corresponding to m7D6 in FIG7A ) (the specific information and sequences of these antibodies are obtained from the literature Ju B, Zheng Q, Guo H, Fan Q, Li T, Song S, Sun H, Shen S, Zhou X, Xue W, Cui L, Zhou B, Li S, Xia N, Zhang Z.I mmune escape by SARS-CoV-2 Omicron variant and structural basis of its effective neutralization by a broad neutralizing human antibody VacW-209. Cell Res. 2022 May; 32(5): 491-494. doi: 10.1038/s41422-022-00638-6. Epub 2022 Mar 8. PMID: 35260792; PMCID: PMC8902274.).
  • FIG. 7B shows the activity identification results of 10 VHH-RBD proteins produced and purified by the mammalian cell expression system 293F cells.
  • the antibodies used include 3G11, 8H12, 13F10, 8B8, 9D3, and 3F9 (these antibodies were prepared by conventional antibody preparation methods in the reference literature of this laboratory.
  • the SARS-CoV-2 surface spike protein S protein extracellular segment protein was expressed and purified in 293F cells. Then BALB/c mice were immunized, and blood was collected for serum detection at 0, 2, 3, and 5 weeks of immunization.
  • mice with higher mouse immune serum binding titers and neutralization titers were selected for spleen immunization.
  • 10 ⁇ g of mixed protein was injected into the mouse spleen, the mice were sutured and observed regularly, and a cell fusion experiment was performed three days later.
  • the cell supernatant was taken and the cell supernatant and SARS-CoV-2 were detected by indirect ELISA.
  • the S-2P trimer protein, RBD, S2 protein and SARS-CoV-1 S-2P protein were reacted, and the wells with strong binding ability to the corresponding proteins were selected for picking and cloning experiments. After more than three rounds of cloning, monoclonal antibodies were obtained.
  • FIG8 shows the results of enzyme-linked immunosorbent assay (ELISA) of VHH-gE of Example 7 and various specific monoclonal antibodies.
  • the gE-specific monoclonal antibodies used include 3H7, 4G4, 6B7, 11B11, 11B12, 13B6, 14G1, and 17B7 (the specific information and sequences of these antibodies are obtained from the literature Liu, J., Ye, X., Jia, J. et al. Serological Evaluation of Immunity to the Varicella-Zoster Virus Based on a Novel Competitive Enzyme-Linked Immunosorbent Assay. Sci Rep 6, 20577 (2016). https://doi.org/10.1038/srep20577).
  • the results show that the produced and purified VHH-gE can still maintain a complete and correct molecular conformation and has good binding activity with the gE-specific antibody.
  • FIG. 9 shows the results of enzyme-linked immunosorbent assay (ELISA) of various VHH-BGTSTIPs of the examples with various reported HIV-1 neutralizing antibodies or non-neutralizing antibodies. These antibodies were recombinantly expressed in 293F cells, and the antibody sequences were derived from NCBI (VRC01 (GeneBank: MK032237.1/GU980703.1), SF12 (GeneBank: MK722171.1/MK722164.1), 2G12 (GeneBank: OM484328.1/AF029237.1), PGT121 (GeneBank: JN201911.1/JN201894.1) or the article reported F105 (Wilkinson, RA, C. Piscitelli, M.
  • Biacore 8K was loaded with a CM5 chip and the pipeline was flushed with PBS-P buffer (PBS+0.5% P20, cytiva).
  • the ligand HEV-p239 (10 ug/ml) was centrifuged at high speed for 10 min.
  • HEV-p239 amino acid sequence as shown in SEQ ID NO: 40
  • was loaded for coupling time: 420s, flow rate 10ul/min.
  • use ethanolamine to perform the channel blocking program time: 420s, flow rate: 10ul/min).
  • Affinity detection Set the concentration gradient of 125nM, 62.5nM, 31.2nM, 15.6nM, 7.8nM, and 3.9nM fusion protein for sample loading detection.
  • the sample binding time is 120s
  • the dissociation time is 200s
  • the flow rate is 30ul/min.
  • Biacore 8K (GE) software was used to perform kinetic methods to fit the binding (Ka) and dissociation (Kd) curves, and to analyze and calculate the affinity (KD).
  • Figure 10 shows the affinity analysis results of various fusion proteins VHH-RBD and HEV-p239 protein in the embodiment.
  • Figure 10A shows the affinity results of VHH-RBD and HEV-p239 protein produced and purified by insect cell expression system, and the results show that various VHH-RBDs have nanomolar high affinity with HEV-p239 particles.
  • Figure 10B shows the affinity results of VHH-RBD and HEV-p239 protein produced and purified by mammalian cell expression system 293F cells, and the results show that VHH-RBD has nanomolar high affinity with HEV-p239 particles.
  • Figure 11 shows the results of affinity analysis of various fusion proteins VHH-gE and HEV-p239 protein in the examples. The results show that various VHH-gE have high affinity with HEV-p239 particles at the nanomolar level.
  • Figure 12 shows the results of affinity analysis of various fusion proteins VHH-BGTSTIP and HEV-p239 protein in the examples. The results show that various VHH-BGTSTIP and HEV-p239 particles have nanomolar high affinity.
  • Instrument system AKTA explorer 100 preparative liquid chromatography system produced by GE Healthcare (formerly Amershan Pharmacia).
  • Chromatography medium Superdex 200 increase (cytiva).
  • Buffer 20 mM phosphate buffer pH 7.4.
  • the samples are the samples in Example 1 and Example 5.
  • the elution procedure is: collect the penetration peak in segments.
  • Collect the product penetrated by Supedex 200 increase to obtain 5 mL of purified sample. Take 50 ⁇ L of each eluted product, add 10 ⁇ L of 6X Loading Buffer and mix well. After 10 minutes in 80°C water bath, take 10 ⁇ L and electrophoresed in 10% SDS-polyacrylamide gel at 120V voltage for 60 minutes. Then stain with Coomassie Brilliant Blue to display the electrophoresis bands.
  • FIG13A shows the results of purification of the fusion protein P1-5B-RBD produced and purified by an insect cell expression system in an embodiment of the present invention by superdex 200 increase (high performance liquid chromatography molecular sieve). The results show that the purity of the P1-5B-RBD protein reaches more than 95%.
  • FIG13B shows the results of purification of P2-6D-RBD, P2-3E-RBD, and P2-10G-RBD produced and purified by a mammalian cell expression system in an embodiment of the present invention by superdex 200 increase. The results show that all three fusion proteins present a single elution peak, indicating that the protein purity and homogeneity are high.
  • Figures 14-16 show the purification patterns and SDS-PAGE identification results of the complexes formed by various fusion proteins constructed by the present invention and HEV-p239.
  • Figure 14A shows the purification map and SDS-PAGE identification results of the complex formed by the P1-5B-RBD fusion protein purified by the insect cell expression system and HEV-p239.
  • the purple curve is significantly higher than the blue curve, proving that the fusion protein P1-5B-RBD specifically binds to the surface of HEV particles to form a complex.
  • Figure 14B shows the purification profiles and SDS-PAGE identification results of the complexes formed by P2-3E-RBD, P2-10G-RBD and P2-6D-RBD with HEV-p239, respectively, which were purified by the mammalian cell expression system of the candidate fusion protein of the embodiment of the present invention.
  • the results show that P2-3E-RBD, P2-10G-RBD and P2-6D-RBD formed complexes with HEV-p239 and could be purified by superdex 200 10/300 increase.
  • Figure 15 shows the purification pattern and SDS-PAGE identification results of the complex formed by the candidate fusion protein P2-8C-gE and HEV-p239 in the embodiment of the present invention.
  • Figure 15A shows the purification results of the complex. The results show that the retention volume of the complex in the superdex 200 increase (cytiva) chromatography column is 8 ml, showing a particle peak component, and a 14 ml peak component. The unbound P2-8C-gE indicated that the 8ml particle peak component had bound. Therefore, P2-8C-gE was saturatedly bound to the surface of HEV particles to form a complex.
  • Figure 16 shows the purification pattern and SDS-PAGE identification results of the complex of the candidate fusion protein P2-5C-BGTSTIP and HEV-p239 in the embodiment of the present invention.
  • the results show that the retention volume of the complex in the superose 6 chromatography column is about 9 ml, and the retention time of P2-5C-BGTSTIP is about 16 ml.
  • Different components are collected for SDS-PAGE identification.
  • the sample with an elution volume of 9 mL shows two bands in SDS-PAGE, confirming that after the complex is purified by superose 6, a complex sample is obtained at about 9 mL.
  • Instrument Waters. System flow rate: 0.5 mL/min for G3000PW XL . Wavelength 190-600 nm, column wavelengths 280 nm and 254 nm.
  • Operational procedures pre-equilibrate the column for 30 to 60 minutes until there is no significant change in the absorption value at 280 nm, and return the absorption value of the detector to zero.
  • Edit the chromatography operation method centrifuge the sample first, inject the sample to be analyzed into a 100 ⁇ L sample loop, set the automatic sample loading, run for 30 minutes, and observe that the retention time of the S trimer is about 14 minutes.
  • the results are shown in FIG. 17 .
  • the P2-8C-gE fusion protein of the present invention showed a single main peak without aggregate components, a retention time of about 14 min, and a purity of about 80%.
  • Example 8 Molecular size detection of RBD complex and gE complex (DLS)
  • DLS Dynamic Light Scattering
  • Operation process Turn on the instrument and preheat for 5 minutes. Prepare the sample to be tested (concentration 0.5 mg/ml, centrifuge at 12000 rpm for 5 minutes, take 50ul and add it to the sample cup). Set the test parameters: Test time: 300s each time, repeat 3 times for each sample.
  • FIG. 18 shows the purified VHH-RBD and HEV- The molecular particle size detection results of the HEV-RBD complex sample formed by the p239 protein.
  • Figure 18B shows the molecular particle size detection results of the HEV-RBD complex sample formed by the purified VHH-RBD and HEV-p239 protein produced by the mammalian cell expression system.
  • the results of Figures 18A and 18B show that the molecular particle size of HEV-RBD is significantly larger than that of HEV-p239 particles.
  • the particle size of the HEV-RBD complex is significantly larger than that of HEV-p239, which confirms that VHH-RBD can bind to the surface of HEV particles and maintain a particle state.
  • Figure 19 shows the molecular particle size detection results of the HEV-gE complex sample of the embodiment of the present invention.
  • the results show that the molecular particle size of HEV-p239 particles is 14.2nm, and the molecular particle size of HEV-gE is 38nm.
  • the results confirm that VHH-gE can bind to the surface of HEV particles and maintain the particle state.
  • Figure 20 shows the molecular particle size detection results of the HEV-BGTSTIP complex sample of the embodiment of the present invention.
  • the results show that the molecular particle size of the HEV-p239 particle is 15.9nm, and the particle size of the HEV-BGTSTIP complex is 25.2nm.
  • the results show that VHH-BGTSTIP can bind to the surface of HEV particles and maintain the particle state.
  • the instrument used was a Beckman XL-A analytical ultracentrifuge equipped with an optical detection system and an An-60Ti rotor.
  • sample pool installs the sample pool according to the operating instructions, add 400 ⁇ L of sample buffer (the same buffer as the sample) to the control pool, add 380 ⁇ L of sample (OD280 is about 0.8) to the sample pool, and balance the sample pool so that the weight difference is within 0.1 g.
  • Parameter settings temperature (20°C), Rmin (6.0cm), Rmax (7.2cm), wavelength (280nm), step speed (0.003cm), scan mode (continuous), data interval (30sec) and number of data (150scans). Set the centrifugal speed to 30000rpm.
  • the density and viscosity of the buffer solution and the partial microvolume of the known protein were calculated using SENDTERP software.
  • the RMSD value is required to be no greater than 0.01 and the residue map fluctuation is within 0.05.
  • Figure 21 shows the results of ultracentrifugation analysis of HEV-p239 and HEV-RBD complexes constructed based on HEV-p239 particles in the present invention.
  • Figure 21A shows the results of ultracentrifugation analysis of complex particles prepared by the purified VHH-RBD produced by the insect cell expression system and HEV-p239 (right figure) and HEV-p239 (left figure). The results show: HEV-p239 presents a single component with a sedimentation coefficient of 22S, and the HEV-RBD complex presents a single component with a sedimentation coefficient of 27S, which is significantly higher than the sedimentation coefficient (22S) of HEV-p239 particles.
  • FIG. 21B is the analytical ultracentrifugation results of the complex particles prepared by the purified VHH-RBD and HEV-p239 produced by the mammalian cell expression system 293F cells.
  • the results show that the HEV-RBD complex presents a single component, and the sedimentation coefficient is significantly higher than the sedimentation coefficient (22S) of HEV-p239 particles. It proves that it can maintain a stable particle morphology in an aqueous solution state, and RBD is firmly bound to the surface of HEV particles to form RBD particle proteins.
  • FIG22 shows the results of the analysis of the HEV-p239 (FIG. 22A) and the HEV-gE complex constructed based on the HEV-p239 particles (FIG. 22B) of the embodiment of the present invention.
  • the results show that HEV-p239 presents a single component with a sedimentation coefficient of 22S, and the HEV-gE complex presents a single component with a sedimentation coefficient of 31S, which is significantly larger than the sedimentation coefficient (22S) of the HEV-p239 particles.
  • the results confirm that the HEV-gE complex can maintain a stable particle morphology in the aqueous solution state, and gE is firmly bound to the surface of the HEV particles to form gE particle protein.
  • FIG. 23 shows the analytical ultracentrifugation results of the HEV-p239 and the Env(BGTSTIP) complex constructed based on the HEV-p239 particles according to the examples of the present invention.
  • the transmission electron microscope used was a Tecnai G2 Spirit 120kV (FEI). Phosphotungstic acid was used for negative staining.
  • Sample preparation First, a copper mesh (R2/2, 200 mesh, ThermoFisher Scientific) was treated with glow discharge hydrophilicity. Then, 5ul of a sample with a concentration of 0.5mg/ml was dripped onto the copper mesh. After standing at room temperature for 60s, the droplets were absorbed from the edge of the copper mesh with absorbent paper. After drying at room temperature, the sample was observed on the machine.
  • a copper mesh R2/2, 200 mesh, ThermoFisher Scientific
  • Figure 24A shows the transmission electron microscopy negative staining results of the complex sample prepared by HEV-p239 and the purified VHH-RBD produced by the insect cell expression system in the present invention.
  • Figure 24B shows the transmission electron microscopy negative staining results of the complex sample prepared by HEV-p239 and the purified VHH-RBD produced by the mammalian expression system 293F cells in the present invention. The results all show that the HEV-RBD complex presents a typical virus-like particle morphology. The results confirm that the HEV-RBD complex sample constructed in the present application is an RBD granulated antigen.
  • Figure 25 shows the negative staining results of the HEV-gE complex of the present invention.
  • the results show that the HEV-gE complex presents a typical virus-like particle morphology.
  • the results confirm that the HEV-gE complex sample constructed in the present application is a granulated antigen.
  • FIG. 26 shows negative staining of the transmission electron microscopy of the HEV-Env (BGTSTIP) complex of the present invention.
  • Results The results showed that the HEV-Env complex exhibited a typical virus-like particle morphology, confirming that the HEV-Env complex sample constructed in the present application was a particulate antigen.
  • mice Six-week-old Balb/C mice were selected and divided into four groups, with five mice in each group. Each group was immunized with HEV-RBD particles (immunization dose of 0.5 ⁇ g), HEV-RBD particles (immunization dose of 5 ⁇ g), P1-5B-RBD monomer (immunization dose of 0.5 ⁇ g), and P1-5B-RBD monomer (immunization dose of 5 ⁇ g) combined with aluminum adjuvant.
  • the left or right hind limb of the mice was injected intramuscularly (50 ⁇ L) at 0, 2, and 6 weeks. Ocular venous blood was collected at 0, 1, 2, 3, 4, 5, 6, 7, and 8 weeks, and blood was collected before injection at 0, 1, and 4 weeks.
  • mice Female, 6 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Twelve groups of immunized mice were set up, with 5 mice in each group. P2-3E-RBD, P2-10G-RBD, P2-6D-RBD and P239 were used to prepare particle complex antigens. Diluted with physiological saline to the required concentration, mixed with aluminum adjuvant in a volume ratio of 1:1 and incubated at 4 degrees overnight to allow the protein to adsorb on the adjuvant, and the mice were immunized intramuscularly at 0, 2, and 5 weeks (100uL, 50 ⁇ L each for the left and right hind legs). The mouse immunization scheme is set as shown in Table 4.
  • Eye blood was collected from mice every week, and mice were euthanized with carbon dioxide after nine weeks.
  • the blood samples were placed at 37°C for 30 minutes, centrifuged at 13,300 rpm for 10 minutes, and serum was collected and stored at -20 degrees for wild-type SARS-CoV-2 pseudovirus neutralization and binding antibody titer determination.
  • Figure 27 shows the immunogenicity test results of the HEV-RBD particle antigen of the embodiment of the present invention.
  • Figure 27A is the serum binding and neutralization activity test results after mice were immunized with HEV-RBD granular antigen obtained by the insect cell expression system.
  • Figures 27B and 27C are the serum binding activity and neutralization activity test results of wild-type SARS-CoV-2 pseudovirus after mice were immunized with HEV-RBD granular antigen obtained by the mammalian cell expression system
  • Figure 27D is the serum antibody binding titer after hamsters were immunized with HEV-RBD granular antigen obtained by the mammalian cell expression system.
  • Figure 28 shows the neutralization test results of the antibodies induced by the HEV-RBD particle antigen combined with aluminum adjuvant in an example of the present invention and the new coronavirus wild-type (WT) strain, Gamma strain, and BA.2 strain.
  • WT coronavirus wild-type
  • the HEV-RBD particle (shown as RBD CPX in the figure) 5ug immunization group can quickly establish an immune barrier after the first injection, producing about 3log binding antibody titers. After booster immunization, the binding antibody titer reached 5log, which is 500 times higher than the binding antibody titer produced by the P1-5B-RBD monomer, and did not decrease significantly in the eighth week. In the mouse immunization experiment, the binding antibody titer induced by HEV-RBD was significantly higher than that of the monomeric RBD protein.
  • P2-3E-RBD-p239, P2-10G-RBD-p239 and P2-6D-RBD-p239 complex particles can induce relatively higher binding antibody titers (compared with the corresponding VHH-RBD proteins), among which the binding antibody titers of the P2-3E-RBD-p239 and P2-10G-RBD-p239 complex immunization groups before the fifth injection were significantly higher than those of the corresponding VHH-RBD immunization groups (Figure 27B).
  • the pseudovirus neutralization results at week 6 showed that the complex particles formed by the three VHH-RBDs and p239 could induce more significant neutralizing antibody responses ( FIG. 27C ).
  • the neutralizing antibodies of HEV-RBD particles have broad-spectrum neutralizing ability and can effectively neutralize pseudoviruses (VSV-Spike) including wild strains of the new coronavirus, Gamma strains, and BA.2 strains.
  • VSV-Spike pseudoviruses
  • Example 12 Evaluation of immunogenicity of gE particles
  • mice Six-week-old Balb/C mice were selected and divided into 8 groups, with 5 mice in each group. The mice were immunized with 100 ⁇ g of HEV-gE particles (immunization dose of 0.1 ⁇ g), 100 ⁇ g of HEV-gE particles (immunization dose of 0.5 ⁇ g), 100 ⁇ g of HEV-gE particles (immunization dose of 5 ⁇ g), 100 ⁇ g of P2-8C-gE monomer (immunization dose of 0.1 ⁇ g), 100 ⁇ g of P2-8C-gE monomer (immunization dose of 0.5 ⁇ g), 100 ⁇ g of P2-8C-gE monomer (immunization dose of 5 ⁇ g), and 100 ⁇ g of P2-8C-gE monomer (immunization dose of 5 ⁇ g) in combination with aluminum adjuvant.
  • HEV-gE particles immunized with 100 ⁇ g of HEV-gE particles (immunization dose of 0.1
  • mice The left or right hind limbs of mice were injected intramuscularly (50 ⁇ L) at 0 and 2 weeks, respectively. Ocular venous blood was collected at 0, 1, 2, 3, 4, 5, and 6 weeks, respectively. The blood samples were centrifuged at 13000 g for 10 minutes, and the serum samples were stored at -20 °C.
  • the antigen-specific IgG and neutralizing antibody titers were determined by end-point enzyme-linked immunosorbent assay and attenuated virus (v-Oka strain) neutralization method, respectively.
  • Guinea pig serum purchased from Beijing Borsi Technology Co., Ltd., catalog number: BM361Y
  • v-Oka virus ATCC, catalog number VR-795
  • Figure 29 shows the immunogenicity test results of the gE particle antigen of the present invention.
  • the gE particle antigen combined with an adjuvant similar to AS01B can induce high titer binding antibodies (shown as XUA in the figure, up to about 6 log).
  • Figure 30 shows the results of the live virus neutralization test of mouse immune serum of HEV-gE particle antigen in the present invention.
  • the neutralizing antibodies induced by HEV-gE particle antigen are 2.8 times that of P2-8c-gE monomer.
  • the results confirm that expression on the surface of p239 particles can significantly improve the immunogenicity of gE antigen.
  • Example 13 Flow cytometry detection of cytokines in gE particle antigen immunization group
  • mice were immunized according to the method described in Example 6 (same adjuvant), and the subsequent experimental procedures were as follows:
  • Plating 96-well U-bottom plate, plate 200 ⁇ L (2-4 ⁇ 10 6 cells) per well, centrifuge at 400 g, 4°C for 5 min, and remove the supernatant;
  • Fixation/Permeabilization centrifuge at 2000 rpm, 4°C for 2 min, remove the supernatant, add 75 ⁇ L of Fixation/Permeabilization solution to each well to resuspend the cells, and treat at 4°C in the dark for 60 min;
  • Intracellular staining Use PE-conjugated anti-mouse IL-2 antibody (purchased from BD, Catalog No.: 554428) and APC-conjugated anti-mouse IFN- ⁇ antibody (purchased from BD, Catalog No.: 554413) for staining. Add 50 ⁇ L of fluorescent antibody (IL-2/IFN- ⁇ antibody, diluted with 1 ⁇ BD Perm/Wash solution) to each well, resuspend the cells, and treat at 4°C in the dark. 60 min, add 200 ⁇ L Perm Buffer and resuspend the cells;
  • the treated cells are filtered through a 200-mesh screen (the screen strips are spread on the surface of the hole, and the cell suspension is sucked vertically and slowly added with a discharge gun), and transferred to a flow cytometer tube;
  • Example 14 ELISpot detection of cytokines by gE particle antigen immunization group
  • the plasmid was prepared in large quantities using an endotoxin-free plasmid extraction kit (Tiangen Company). Then 293FT cells were transfected with PEI transient transfection for humanized nanobody expression. A total of two humanized nanobodies were obtained, named H1 and H2, respectively.
  • the full-length amino acid sequences of the humanized nanobodies are shown in SEQ ID NOs: 71 and 72, and the sequences of CDR1-3 are shown in SEQ ID NOs: 65-67.
  • PCDNA3.1-H1-RBD and PCDNA3.1-H2-RBD plasmids (the plasmids contain nucleotide sequences encoding fusion proteins H1-RBD and H1-RBD, respectively, wherein the amino acid sequences of H1-RBD and H1-RBD are shown in SEQ ID NOs: 63 and 64) were diluted with PEI in 5 ml of serum-free CD05 medium, and the two were thoroughly mixed and allowed to stand for 18 min to form a plasmid PEI complex.
  • the modified humanized nanobody has the same molecular activity as the parent.
  • H1-RBD and H2-RBD are RBD fusion proteins formed by the modified humanized nanobody
  • RBD-WT is the wild RBD protein.
  • Example 16 Evaluation of immunogenicity of HEV-Env particle antigen prepared by humanized nanobodies
  • mice female, 6 weeks old, purchased from Shanghai Slake Experimental Animal Co., Ltd. Six groups of immunized mice were set up, with 5 mice in each group.
  • This example uses P2-5C nanobody (the amino acid sequence of P2-5C nanobody is shown in SEQ ID NO: 21, and the sequence of CDR1-3 is shown in SEQ ID NO: 60-62).
  • the prepared complex particles and P2-5C-BGTSTIP antigen were diluted with physiological saline, mixed with aluminum adjuvant in a volume ratio of 1: 1, and the protein was adsorbed on the adjuvant, and the mice were immunized intramuscularly.
  • the mouse immunization scheme is set as shown in Table 5.
  • mice eyeball blood was collected before immunization.
  • the above immunization scheme was used for immunization.
  • the mouse eyeball blood was collected before each immunization.
  • Blood was collected from the eyeball after the sixth injection and the mice were dislocated.
  • the blood samples were placed at 37°C for 30 min, centrifuged at 13300 rpm for 10 min, and serum was collected for HIV-1 pseudovirus neutralization and antibody titer determination.
  • Nanobodies were screened for HPV L1 protein (SEQ ID NO: 71), and nanobody P1F8 (SEQ ID NO: 88) was obtained.
  • a (GGGGS) 3 flexible linker was added after the antibody sequence, and then coupled to the front of the BGTSTIP sequence.
  • the amino acid sequence of the constructed P1F8-BGTSTIP fusion protein is shown in SEQ ID NO: 89.
  • P1F8-BGTSTIP plasmid was extracted, and frozen 293F cells were taken from a -80°C refrigerator. After thawing at 37°C, centrifuged at 1300rpm for 4min, and the supernatant was discarded in a clean bench.
  • the cells were flicked and resuspended with 293freestyle medium incubated at 37°C in advance, and then transferred to a triangular flask containing 50mL of incubation medium, and suspended and cultured at 37°C, 5% CO 2, 120 rpm, and subcultured when the cell density reached 2.0*10 6 , and the culture system was gradually expanded.
  • PEI MW 25,000
  • the cells are collected in a sterile 50 mL tube and centrifuged at 1300 rpm for 4 min. After flicking the cells, they are resuspended in 37°C incubation medium and transferred to a conical flask containing 450 mL 37°C incubation medium and placed in a 37°C shaker for use.
  • the extracted P1F8-BGTSTIP plasmid and PEI (MW 25,000) were placed in 50 ml of culture medium at a ratio of 1:2, mixed thoroughly, and then allowed to stand for 18 minutes. The mixture was then transferred to the above 450 ml of culture medium and suspended and cultured at 37°C, 5% CO 2, 120 rpm for 6 days to express the P1F8-BGTSTIP protein. During the transfection process, the PEI was kept away from light.
  • the cell culture medium was collected and centrifuged at 7000g for 10 min in a JA-14 rotor to obtain the cell supernatant. After centrifugation at 20000g for 10 min, the supernatant was filtered twice with a 0.22 um pore size filter membrane, and the sample was used for the next step of Ni-excel column purification.
  • Purification medium Ni Sepharose excel affinity medium; Buffer: divided into A and B buffers, A liquid is 1 ⁇ PBS buffer, B pump is 1 ⁇ PBS+250mmol/L imidazole buffer;
  • Elution conditions Elute impurities with 20mM imidazole, collect 250mM imidazole elution products. Dialyze the eluate against 1 ⁇ PBS overnight, changing the dialysate twice. Harvest about 30ml of low-concentration target protein, Vivaspin 20ml, 100KD ultrafiltration concentrator tube is concentrated to 5ml for later use. The collected elution samples are prepared into reduced and non-reduced samples, and SDS-PAGE gel electrophoresis is performed according to the above method.
  • Figure 34 shows the SDS polyacrylamide gel electrophoresis results of P1F8-BGTSTIP protein; M: molecular weight marker; "+” indicates reduced SDS-PAGE; “-” indicates non-reduced SDS-PAGE.
  • M molecular weight marker
  • “+” indicates reduced SDS-PAGE
  • "-” indicates non-reduced SDS-PAGE.
  • the results show that the molecular weight of P1F8-BGTSTIP molecule is about 160KD under reducing conditions and is a multimer under non-reducing conditions, which is consistent with the theoretical molecular weight.
  • the fusion protein was diluted to 1 ⁇ g/mL and coated on a 96-well plate, 100 ⁇ L per well, and allowed to stand at room temperature for 2 h;
  • Figure 35 shows the results of the enzyme-linked immunosorbent assay of P1F8-BGTSTIP.
  • the results indicate that P1F8 has good binding activity with neutralizing antibodies such as VRC01, PGT121, PGT122 (JN201912.1/JN201895.1), 2G12, SF162, and B12, while the binding activity with 17b, F105, and F240 is very weak, indicating that P1F8-BGTSTIP well exposes the broad-spectrum neutralizing antibody epitopes without exposing non-neutralizing epitopes.
  • PBS PBS-P buffer
  • the ligand 58-VLP (10 ug/ml) was centrifuged at high speed for 10 min.
  • Affinity detection Set the concentration gradient of 800nM, 400nM, 200nM, 100nM, 50nM, and 25nM fusion protein for sample loading detection.
  • the sample binding time is 120s
  • the dissociation time is 300s
  • the flow rate is 30ul/min.
  • Biacore 8K (GE) software was used to perform kinetic methods to fit the binding (Ka) and dissociation (Kd) curves, and to analyze and calculate the affinity (KD).
  • Figure 36 shows the results of the affinity test between P1F8-BGTSTIP and 58VLP.
  • the results show that P1F8-
  • the affinity of BGTSTIP to the fusion protein is 4.66 ⁇ 10 -8 , indicating that the two can bind and the binding is relatively strong.
  • P1F8-BGTSTIP and 58-VLP were incubated in a water bath at 37°C for 30 min at a mass ratio of 5:1, centrifuged at 13300 rpm for 10 min, and then analyzed by high performance size exclusion chromatography (HPSEC).
  • Instrument Waters. System flow rate: 0.5 mL/min for G5000PW XL . Wavelength 190-600 nm, column wavelengths 280 nm and 254 nm.
  • Buffer Final buffer.
  • the P1F8-BGTSTIP-58 VLP complex collected through the above purification process was subjected to AUC analysis.
  • the instrument used was a Beckman XL-A analytical ultracentrifuge equipped with an optical detection system and an An-60Ti rotor.
  • sample pool installs the sample pool according to the operating instructions, add 400 ⁇ L of sample buffer (the same buffer as the sample) to the control pool, add 380 ⁇ L of sample (OD280 is about 0.8) to the sample pool, and balance the sample pool so that the weight difference is within 0.1 g.
  • Parameter settings temperature (20°C), Rmin (6.0cm), Rmax (7.2cm), wavelength (280nm), step speed (0.003cm), scanning mode (continuous), data interval (30sec) and number of data (150scans).
  • the density and viscosity of the buffer solution and the partial pressure of the known protein were calculated using SENDTERP software. Specific volume.
  • the sedimentation coefficient was analyzed using the Origin version of Nonlin and SEDFIT software.
  • the friction ratio of globulin f/f0 was preset to 1.2.
  • the analysis range was set according to the molecular weight and basic properties of the sample protein.
  • the calculation resolution was set to 100. Generally, the RMSD value was required to be no greater than 0.01 and the residue map fluctuation was within 0.05.
  • Figure 38 shows the results of the analysis of the P1F8-BGTSTIP and 58VLP complex of the present invention.
  • the results show that 58VLP presents a single component with a sedimentation coefficient of 119S ( Figure 38 right), and the P1F8-BGTSTIP-58VLP complex presents a single component with a sedimentation coefficient of 176.3S ( Figure 38 left), which is significantly higher than the sedimentation coefficient of 58VLP particles.
  • the results show that 1F8-BGTSTIP can form a particle complex with 58VLP and maintain a stable particle morphology in an aqueous solution state.
  • the transmission electron microscope used was a Tecnai G2 Spirit 120kV (FEI). Phosphotungstic acid was used for negative staining.
  • Sample preparation First, a copper mesh (R2/2, 200 mesh, ThermoFisher Scientific) was treated with glow discharge hydrophilicity. Then, 5ul of a sample with a concentration of 0.5mg/ml was dripped onto the copper mesh. After standing at room temperature for 60s, the droplets were absorbed from the edge of the copper mesh with absorbent paper. After drying at room temperature, the sample was observed on the machine.
  • a copper mesh R2/2, 200 mesh, ThermoFisher Scientific
  • Figure 39 shows the morphology of the complex particles under a transmission electron microscope. It can be clearly seen that the surface of the particles is covered with a layer of protein, indicating that P1F8-BGTSTIP is successfully displayed on the surface of HPV 58 VLP particles.
  • mice Six-week-old Balb/C mice were selected and divided into six groups, with five mice in each group, three high-dose groups and three low-dose groups.
  • P1F8-BGTSTIP-58VLP particles, P1F8-BGTSTIP, and BGTSTIP immunogens combined with aluminum adjuvant were used for immunization, and two immunization doses of 0.5ug and 5ug were set for each antigen.
  • the mice were immunized intramuscularly (100uL/mouse) at 0, 2, 4, and 6 weeks.
  • mice The eyeball blood of mice was collected before immunization, before each immunization, and after the sixth injection, the mice were dislocated.
  • the blood samples were placed at 37°C for 30 minutes, centrifuged at 13300 rpm for 10 minutes, and the serum was collected for specific binding antibody titer determination.
  • Figure 40 shows the immunogenicity test results of the P1F8-BGTSTIP-VLP particle antigen of the present invention.
  • Figure 40 shows the BGTSTIP-specific binding antibody titer test results (the left figure is the 0.5 ⁇ g group, and the right figure is the 5 ⁇ g group). The results show that in weeks 1-4, the binding titer of the complex particles is significantly higher than that of the single BGTSTIP and the single P1F8-BGTSTIP protein.
  • Example 19 Design and evaluation of nanoantibody fusion protein based on HBsAg VLP
  • VLP vectors were prepared based on the granulated antigen SHBs protein (SEQ ID NO: 73) of the hepatitis B vaccine that has been marketed, and gE fusion protein was prepared accordingly according to the method of the previous example, and HBV-gE granulated antigen was further prepared, and the immunogenicity of the granulated antigen was tested.
  • the specific experimental process is as follows:
  • HBsAg nanoantibody (1. Serruys, B., Houtte, FV, Verbrugghe, P., Leroux-Roels, G. & Vanlandschoot, P. Llama-derived single-domain intrabodies inhibit secretion of hepatitis B virions in mice. Hepatology 49, 39-49 (2009).) was selected for the experiment.
  • a (GGGGS)3 flexible linker was added after the antibody sequence and then coupled to the RBD and gE sequences to construct an RBD and gE fusion protein.
  • Extract plasmids take frozen 293F cells from -80°C refrigerator, thaw at 37°C, centrifuge at 1300rpm for 4min, discard supernatant in clean bench, flick cells and resuspend with 293freestyle medium incubated at 37°C in advance, transfer to a flask containing 50mL incubation medium, 37°C 5% CO 2 120 rpm suspension culture, subculture when cell density reaches 2.0*10 6 , and gradually expand the culture system.
  • PEI MW 25,000
  • the extracted plasmid and PEI (MW 25,000) were placed in 50 ml of culture medium at a ratio of 1:2, mixed thoroughly, and then allowed to stand for 18 minutes. The mixture was then transferred to the above 450 ml of culture medium and suspended and cultured at 37°C, 5% CO 2 , 120 rpm for 6 days to express the gE fusion protein. During the transfection process, PEI was kept away from light.
  • the cell culture medium was collected and centrifuged at 7000g for 10 min in a JA-14 rotor to obtain the cell supernatant. After centrifugation at 20000g for 10 min, the supernatant was filtered twice with a 0.22 um pore size filter membrane, and the sample was used for the next step of Ni-excel column purification.
  • Purification medium Ni Sepharose excel affinity medium; Buffer: divided into A and B buffers, A liquid is 1 ⁇ PBS buffer, B pump is 1 ⁇ PBS+250mmol/L imidazole buffer;
  • Elution conditions use 20mM imidazole to elute the impurities, and collect the 250mM imidazole elution product.
  • the dialysis solution was changed twice in 1 ⁇ PBS overnight.
  • About 30 ml of low-concentration target protein was harvested and concentrated to 5 ml using 20 ml of Vivaspin and 100KD ultrafiltration concentrator tube for later use.
  • the collected eluted samples were prepared and subjected to SDS-PAGE gel electrophoresis according to the above method.
  • Instrument system AKTA explorer 100 preparative liquid chromatography system produced by GE Healthcare (formerly Amershan Pharmacia).
  • Chromatography medium Superdex 200 increase (cytiva).
  • Buffer 20 mM phosphate buffer pH 7.4.
  • the samples are the samples in Example 1 and Example 5.
  • the elution procedure is: collect the penetration peak in segments.
  • Collect the product penetrated by Supedex 200 increase to obtain 5 mL of purified sample. Take 50 ⁇ L of each eluted product, add 10 ⁇ L of 6X Loading Buffer and mix well. After 10 minutes in 80°C water bath, take 10 ⁇ L and electrophoresed in 10% SDS-polyacrylamide gel at 120V voltage for 60 minutes. Then stain with Coomassie Brilliant Blue to display the electrophoresis bands.
  • DLS Dynamic Light Scattering
  • Operation process Turn on the instrument and preheat for 5 minutes. Prepare the sample to be tested (concentration 0.5 mg/ml, centrifuge at 12000 rpm for 5 minutes, take 50ul and add it to the sample cup). Set the test parameters: Test time: 300s each time, repeat 3 times for each sample.
  • the DLS results showed that the particle sizes of HBsAg alone and the HBsAg-S2-gE complex were different.
  • the particle size of HBsAg-S2-gE (HBV-gE) was significantly larger than that of HBsAg, indicating the formation of gE particulate antigen.
  • the transmission electron microscope used was a Tecnai G2 Spirit 120kV (FEI). Phosphotungstic acid was used for negative staining.
  • Sample preparation First, a copper mesh (R2/2, 200 mesh, ThermoFisher Scientific) was treated with glow discharge hydrophilicity. Then, 5ul of a sample with a concentration of 0.5mg/ml was dripped onto the copper mesh. After standing at room temperature for 60s, the droplets were absorbed from the edge of the copper mesh with absorbent paper. After drying at room temperature, the sample was observed on the machine.
  • a copper mesh R2/2, 200 mesh, ThermoFisher Scientific
  • the negative staining results showed that the size and morphology of the complex of HBsAg alone and HBsAg-S2-gE were different, indicating the formation of gE particulate antigen.
  • mice Six-week-old Balb/C mice were selected and divided into two groups, each with three mice. Each group was immunized with the HBsAg-gE complex prepared above and S2-gE alone, where the gE dose was the same, 5ug, and injected intramuscularly (50 ⁇ L) into the left or right hind limb of the mouse at 0 and 2 weeks, respectively. Ocular venous blood was collected at 0, 1, 2, 3, and 4 weeks, respectively. The blood samples were centrifuged at 13000g for 10 minutes, and the serum samples were stored at -20°C. The antigen-specific IgG titer was determined by end-point enzyme-linked immunosorbent assay.
  • the gE-specific titer results after two injections showed that the particulate antigen HBsAg-S2-gE could induce significantly higher antibody levels than S2-gE alone, showing an immune enhancement effect.

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Abstract

一种融合蛋白以及包含其的组合物、试剂盒、颗粒化的抗原、疫苗和药物组合物。还涉及融合蛋白以及包含其的组合物、试剂盒和颗粒化的抗原在制备药物组合物或疫苗中的用途。所述颗粒化的抗原尤其适合于疫苗生产和接种，在预防和/或治疗病毒感染方面具有优势。

Description

一种融合蛋白以及包含其的颗粒化的抗原 技术领域
本申请涉及生物医药领域，具体而言，本申请涉及一种融合蛋白以及包含其的组合物、试剂盒、颗粒化的抗原、疫苗和药物组合物。本申请还涉及融合蛋白以及包含其的组合物、试剂盒和颗粒化的抗原在制备药物组合物或疫苗中的用途。
背景技术
众所周知，疫苗是防控传染性疾病最为经济有效的方法，通过疫苗接种每年可挽救近600万人的生命。目前，基因工程疫苗多采用对病原体感染至关重要的蛋白/多糖作为抗原。虽然这类疫苗抗原的研发具有安全性高、工艺成熟等优点，能产生针对特定病原体的免疫保护，但经常面临体液免疫强度较差，很难实现长期保护，难以激活高效的细胞免疫等问题。因此研发安全、高效、广谱的新型免疫原是提升疫苗效力的关键。
颗粒化疫苗是抵御病原威胁的重要疫苗平台。目前主要有三种颗粒化疫苗获批上市，包括戊型肝炎(HEV)疫苗，人乳头瘤病毒疫苗(HPV)和乙型肝炎疫苗(HBV)。戊型肝炎疫苗是一种病毒样颗粒(Virus-like particles,VLP)疫苗，由大肠杆菌表达系统表达的戊型肝炎抗原片段—p239蛋白(aa368-606)经复性后能组装成直径25nm的颗粒形态，具有与天然病毒颗粒相似的结构构象，能够很好地模拟天然病毒抗原表位。目前，其已被成功地开发成为疫苗抗原，成为世界上第一支戊肝疫苗Hecolin，并于2012年在中国大陆上市，临床结果已证实其具有良好的预防性保护效果。目前上市的HPV疫苗包括二价疫苗，四价疫苗和九价疫苗。HPV疫苗由病毒的主要衣壳蛋白L1组装形成病毒样颗粒，可由昆虫细胞、酵母细胞和大肠杆菌等表达系统生产制备，在临床和上市后研究都表明HPV疫苗具有良好的安全性和保护效果。乙肝疫苗的研发历史悠久，目前国内外有多款乙肝疫苗上市。乙肝疫苗主要由表面抗原HBsAg的不同形式(SHBs，MHBs或LHBs)的S蛋白自发组装形成颗粒，目前主要由酵母系统、CHO系统生产。临床和真实世界研究表明乙肝疫苗能产生高滴度的保护性抗体，具有良好的有效性和安全性。综上所述，戊肝疫苗、HPV疫苗和乙肝疫苗中的颗粒化抗原生产具有经济高效、生产工艺稳定、抗原质量可控、可工业化批量生产、安全性良好、免疫原性强的优势，是进一步开发其他疫苗的良好基础和重要参考。
然而，很多的病毒抗原蛋白不具备组装成颗粒的能力，无法进行颗粒疫苗的研制， 单独的病毒抗原或者形成寡聚体抗原的免疫原性较差，难以开发成疫苗。如何将目标蛋白展示在颗粒表面是重组疫苗和药物递送载体设计的关键问题和主要目标。目前多价抗原的颗粒展示方法多采用和颗粒载体融合表达或基于SpyTag/SpyCatcher化学结合至颗粒载体的技术方法。但是，这些方法都具有很大的局限性。首先，融合表达方法的局限性在于，满足融合表达的颗粒载体必须具有暴露的N/C端以满足外源蛋白融合要求，对于没有暴露末端的颗粒载体，必须基于结构生物学、信息生物学探索其是否能够融合在其他位置。其次，融合表达策略还需考虑是否会影响目标分子的天然构象。并且，暴露的末端结构会限制目标蛋白展示位置，需要综合考虑空间位阻等诸多因素。基于spytag/spycatcher技术的局限性在于，这种连接方法受限于载体颗粒的结构特征。因此，探索出一种高效的将目标抗原展示于颗粒抗原表面的方法是本领域的重要目标，面临各种难题。
包膜病毒的膜蛋白是疫苗开发的主要免疫原靶标，但这些蛋白通常锚定在病毒表面的膜结构上，自身无法形成颗粒抗原，且通常具有高度疏水性，完整蛋白也难以直接通过表达而获得含膜的颗粒结构。新型冠状病毒是一种具有囊膜的RNA病毒，Spike蛋白是囊膜上的重要分子，主要参与受体识别与细胞膜融合。其中RBD是Spike蛋白上直接与宿主受体(ACE2)结合的蛋白结构域，也是中和抗体的主要靶标。因此以RBD蛋白作为新冠疫苗抗原具有很大价值。但由于RBD蛋白分子量较小，免疫原性不足，因此，如何提升RBD的免疫原性是关键问题。与RBD类似，gE蛋白是水痘带状疱疹病毒(Varicella-zoster virus,VZV)表面丰度最高的一种糖蛋白，也是疫苗研发的重要靶标。相较于新型冠状病毒和水痘带状疱症病毒而言，造成艾滋病的HIV-1病毒，其基因的变异率更高，使得艾滋病疫苗研发面临着更大的挑战。Env是HIV-1病毒表面主要的抗原物质，也是艾滋病疫苗研发的关键分子。全长的Env分子为跨膜蛋白，也称为gp160蛋白，通过删除胞内段可有效提高Env蛋白的可溶性表达，这种分子被称为gp140蛋白，形成类天然三聚体的gp140蛋白，在暴露中和表位及中和表位抗体应答方面，具有明显的优势。但即便如此，目前基于Env的艾滋病疫苗研发仍未取得成功，难点之一在于Env表面覆盖了超过50％的多糖分子，显著降低了抗原的免疫原性。
因此，在上述疫苗研发的种种难题下，提高抗原的免疫原性对于相应疫苗的研发与应用具有重要意义。
发明内容
本发明利用组装多肽(例如，HEV的ORF2蛋白或其片段或它们的变体，HPV的L1蛋白或其片段或它们的变体，HBV的表面抗原或其片段或它们的变体)能够组装成VLP的能力，将特异性结合所述组装多肽的纳米抗体与免疫原性多肽进行融合表达，并利用纳米抗体对所述组装多肽的靶向结合特性，将多种免疫原性多肽展示在由组装多肽形成的VLP的表面，获得了颗粒化的免疫原性多肽。由此，本申请提供了一种将免疫原性多肽颗粒化的系统和方法。本申请还证实了颗粒化的免疫原性多肽能够诱导机体产生高水平的体液免疫和细胞免疫应答。因此，本发明的颗粒化免疫原性多肽具有作为疫苗候选分子的潜能，在疫苗的设计和生产中具有通用性和可转化性的优势。
因此，在第一方面，本申请提供了一种融合蛋白，其包含免疫原性多肽和能够特异性结合病毒样颗粒(VLP)的纳米抗体；
在某些实施方案中，所述VLP是由组装多肽组装成的VLP。
在某些实施方案中，所述组装多肽是能够组装成VLP的多肽。
在某些实施方案中，所述组装多肽是天然病毒或类病毒的外壳蛋白。在此类实施方案中，所述组装多肽组装成的VLP与天然病毒或类病毒的结构相似，区别仅在于不含有天然病毒或类病毒的基因组。
在某些实施方案中，所述组装多肽是人工制备和/或筛选的能够组装成VLP的多肽。在此类实施方案中，所述组装多肽组装成的VLP与天然病毒或类病毒的结构相似或不相似。
在某些实施方案中，所述组装多肽选自戊型肝炎病毒(HEV)的蛋白或其片段或它们的变体，乙型肝炎病毒(HBV)的蛋白或其片段或它们的变体，人乳头瘤病毒(HPV)的蛋白或其片段或它们的变体，或其任意组合；其中，所述片段或变体保留了组装成VLP的能力。
在某些实施方案中，所述变体与其所源自的多肽的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个氨基酸的置换、缺失或添加)。在某些实施方案中，所述置换是保守置换。在某些实施方案中，所述多肽与其变体具有相同或相似的生物学活性。在某些优选的实施方案中，所述生物学活性为能够组装成VLP。
在某些实施方案中，所述组装多肽为HEV的ORF2蛋白或其片段或它们的变体。
在本发明中，术语“ORF2蛋白”是指，HEV病毒的基因组中第二个开放阅读框所编码的蛋白，ORF2蛋白或其片段具有自组装成VLP的能力。
在某些实施方案中，所述ORF2蛋白的片段选自p239蛋白，p495蛋白。
在某些实施方案中，所述组装多肽选自p239蛋白或其片段或它们的变体，p495蛋白或其片段或它们的变体。
在某些实施方案中，p239蛋白的序列为对应于ORF2蛋白的氨基酸序列的第368位氨基酸至第606位氨基酸。在某些实施方案中，p495蛋白的序列为对应于ORF2蛋白的氨基酸序列的第112位氨基酸至第606位氨基酸。
在某些实施方案中，所述ORF2蛋白具有如SEQ ID NO:9所示的氨基酸序列。在某些实施方案中，所述p239蛋白具有如SEQ ID NO:40所示的氨基酸序列。在某些实施方案中，所述p495蛋白具有如SEQ ID NO:41所示的氨基酸序列。
在某些实施方案中，所述组装多肽为HPV的衣壳蛋白L1或其片段或它们的变体。
HPV由蛋白外壳和核心DNA构成，外壳由主要衣壳蛋白(L1)和次要衣壳蛋白(L2)组成。在本发明中，术语“衣壳蛋白L1”或“HPV L1蛋白”或“L1蛋白”是指构成HPV外壳的L1蛋白。
在某些实施方案中，所述组装多肽具有如SEQ ID NO:74所示的氨基酸序列。
在某些实施方案中，所述组装多肽为HBV的表面蛋白(例如，乙型肝炎病毒表面抗原)或其片段或它们的变体。在某些实施方案中，所述组装多肽选自乙型肝炎病毒表面抗原(HBsAg)的LHBs蛋白或其片段或它们的变体，MHBs蛋白或其片段或它们的变体，SHBs蛋白或其片段或它们的变体。在某些实施方案中，所述组装多肽选自乙型肝炎病毒表面抗原(HBsAg)的SHBs蛋白或其片段或它们的变体。
乙型肝炎病毒表面抗原(HBsAg)由表面大蛋白(LHBs)、表面中蛋白(MHBs)和表面小蛋白(SHBs)组成。在本发明中，术语“SHBs蛋白”是指构成HBsAg的表面小蛋白。
在某些实施方案中，所述组装多肽具有如SEQ ID NO:73所示的氨基酸序列。
在某些实施方案中，所述免疫原性多肽是获自生物体或非生物体(例如，人工合成)的多肽或其免疫原性变体。
在某些实施方案中，所述变体与其所源自的多肽的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个的置换、缺失或添加)。在某些 实施方案中，所述置换是保守置换。在某些实施方案中，所述多肽与其变体具有相同或相似的生物学活性。在某些优选的实施方案中，所述生物学活性为能够引起免疫应答。
在某些实施方案中，所述生物体为病原体(例如，病毒，细菌，真菌，寄生虫)或非病原体。
在某些实施方案中，所述免疫原性多肽获自哺乳动物(例如，人)的非肿瘤细胞。在某些实施方案中，所述免疫原性多肽是前蛋白转化酶枯草杆菌蛋白酶/kexin9型(PCSK9)。
在某些实施方案中，所述免疫原性多肽获自哺乳动物(例如，人)的肿瘤细胞(例如，表达或过表达于肿瘤细胞表面)。在某些实施方案中，所述免疫原性多肽选自癌胚抗原(CEA)，甲胎蛋白(AFP)，癌抗原125(CA125)。
在某些实施方案中，所述免疫原性多肽获自病毒，细菌(例如，结核分枝杆菌(Mycobacterium tuberculosis))，真菌(例如，念珠菌(Nostoc))，寄生虫(例如，恶性疟原虫(Plasmodium falciparum))。
在某些实施方案中，所述病毒选自水痘带状疱疹病毒(VZV)，新型冠状病毒(SARS-CoV-2)，冠状病毒(SARS-COV-1)，人类免疫缺陷病毒I型(HIV-1)，人乳头瘤病毒，乙型肝炎病毒，甲型肝炎病毒，丙型肝炎病毒，戊型肝炎病毒，麻疹病毒，腮腺炎病毒，流感病毒，乙型脑炎病毒。
在某些实施方案中，所述组装多肽的序列源自病毒病原体，所述免疫原性多肽的序列源自非病毒病原体(例如，细菌，真菌，寄生虫)。
在某些实施方案中，所述组装多肽的序列源自病毒病原体，所述免疫原性多肽的序列源自另外的病毒病原体。
本发明的免疫原性多肽可以是来源于病毒表面或核芯的多肽。通常，免疫原性多肽可以是包含至少6个氨基酸残基的任何结构或功能的多肽。在一些实施方案中，免疫原性多肽的长度为6至10,000个氨基酸残基。在一些实施方案中，免疫原性多肽的长度25至2,000个氨基酸残基。在一些实施方案中，免疫原性多肽的长度为50至500个氨基酸残基。
在某些实施方案中，所述免疫原性多肽选自SARS-CoV-2的RBD蛋白或其免疫原性片段或变体，HIV-1的Env蛋白或其免疫原性片段(例如，gp140，gp160)或变体，VZV的gE蛋白或其免疫原性片段或变体。
在某些实施方案中，所述Env蛋白的片段选自gp160蛋白，gp120蛋白，gp41蛋白。
在某些实施方案中，所述Env蛋白具有如SEQ ID NO:34或35所示的氨基酸序列。在某些实施方案中，gp160蛋白的序列在GenBank以登录号AAB05604和AAD12142示出。在某些实施方案中，gp41的氨基酸序列在GenBank中以登录号CAD20975示出。
在某些实施方案中，所述RBD蛋白具有如SEQ ID NO:1-8任一项所示的氨基酸序列。
在某些实施方案中，所述gE蛋白具有如SEQ ID NO:30所示的氨基酸序列。
本领域技术人员有能力通过本领域已知的各种方法来制备能够特异性结合特定抗原的纳米抗体，例如，通过特定抗原免疫羊驼或鲨鱼，筛选出阳性克隆，通过测序获得抗体的重链的序列，然后，构建包含抗体的重链的序列的载体，在特定条件下转染宿主细胞，表达以获得纳米抗体。
因此，在确定了组装多肽的情况下，本领域技术人员能够制备并获得能够特异性结合组装多肽的纳米抗体。所以，本申请的纳米抗体并不局限于实施例所具体使用的具体形式(例如，纳米抗体)及具体序列。
在某些实施方案中，所述纳米抗体为驼源(例如，羊驼)抗体或鱼源(例如，鲨鱼)抗体。
在某些实施方案中，所述纳米抗体为嵌合抗体、人源化抗体或全人源抗体。
在某些实施方案中，所述融合蛋白包含2种，3种，或更多种免疫原性多肽。
在某些实施方案中，每一种免疫原性多肽各自独立地获自相同或不同的病原体(例如，病毒)。
在某些实施方案中，每一种免疫原性多肽为获自相同病原体(例如，病毒)的不同多肽。
在某些实例性的实施方案中，所述融合蛋白包含第一免疫原性多肽和第二免疫原性多肽，且第一免疫原性多肽为SARS-CoV-2的RBD蛋白，第二免疫原性多肽为VZV的gE蛋白。在某些实例性的实施方案中，所述纳米抗体特异性结合RBD蛋白和gE蛋白。
在某些实例性的实施方案中，所述融合蛋白包含第一免疫原性多肽，第二免疫原性多肽和第三免疫原性多肽，且第一免疫原性多肽为SEQ ID NO:1所示的RBD蛋白，第二免疫原性多肽为SEQ ID NO:2所示的RBD蛋白，第三免疫原性多肽为SEQ ID NO:3所示的RBD蛋白。在某些实施方案中，所述纳米抗体特异性结合SEQ ID NO:1-3所示的RBD蛋白。
在某些实施方案中，所述融合蛋白包含1种免疫原性多肽。在某些实施方案中，所述纳米抗体是纳米抗体，其特异性结合HEV，HBV和/或HPV的多肽。
在某些实施方案中，所述纳米抗体包含SEQ ID NO:10-29，68，69任一项所示的重链可变区(VHH)中含有的CDR-H1、CDR-H2以及CDR-H3。在某些实施方案中，所述CDR根据IMGT、Kabat或Chothia编号系统定义。
在某些实施方案中，所述纳米抗体，其包含：
(a)包含下述3个互补决定区(CDRs)的重链可变区(VHH)：序列为SEQ ID NO:90的VH CDR1、序列为SEQ ID NO:91的VH CDR2、序列为SEQ ID NO:92的VH CDR3；
(b)包含下述3个互补决定区(CDRs)的重链可变区(VHH)：序列为SEQ ID NO:93的VH CDR1、序列为SEQ ID NO:94的VH CDR2、序列为SEQ ID NO:95的VH CDR3；
(c)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:96的VH CDR1、序列为SEQ ID NO:97的VH CDR2、序列为SEQ ID NO:98的VH CDR3；
(d)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:99的VH CDR1、序列为SEQ ID NO:100的VH CDR2、序列为SEQ ID NO:101的VH CDR3；
(e)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:102的VH CDR1、序列为SEQ ID NO:103的VH CDR2、序列为SEQ ID NO:104的VH CDR3；
(f)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:105的VH CDR1、序列为SEQ ID NO:106的VH CDR2、序列为SEQ ID NO:107的VH CDR3；
(g)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:108的VH CDR1、序列为SEQ ID NO:109的VH CDR2、序列为SEQ ID NO:110的VH CDR3；
(h)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:111的VH CDR1、序列为SEQ ID NO:112的VH CDR2、序列为SEQ ID NO:113的 VH CDR3；
(i)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:114的VH CDR1、序列为SEQ ID NO:115的VH CDR2、序列为SEQ ID NO:116的VH CDR3；
(j)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:117的VH CDR1、序列为SEQ ID NO:118的VH CDR2、序列为SEQ ID NO:119的VH CDR3；
(k)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:120的VH CDR1、序列为SEQ ID NO:121的VH CDR2、序列为SEQ ID NO:122的VH CDR3；
(l)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:123的VH CDR1、序列为SEQ ID NO:124的VH CDR2、序列为SEQ ID NO:125的VH CDR3；
(m)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:126的VH CDR1、序列为SEQ ID NO:127的VH CDR2、序列为SEQ ID NO:128的VH CDR3；
(n)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:129的VH CDR1、序列为SEQ ID NO:130的VH CDR2、序列为SEQ ID NO:131的VH CDR3；
(o)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:132的VH CDR1、序列为SEQ ID NO:133的VH CDR2、序列为SEQ ID NO:134的VH CDR3；
(p)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:135的VH CDR1、序列为SEQ ID NO:136的VH CDR2、序列为SEQ ID NO:137的VH CDR3；
(q)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:138的VH CDR1、序列为SEQ ID NO:139的VH CDR2、序列为SEQ ID NO:140的VH CDR3；
(r)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:141的VH CDR1、序列为SEQ ID NO:142的VH CDR2、序列为SEQ ID NO:143的 VH CDR3；
(s)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:57的VH CDR1、序列为SEQ ID NO:58的VH CDR2、序列为SEQ ID NO:59的VH CDR3；
(t)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:60的VH CDR1、序列为SEQ ID NO:61的VH CDR2、序列为SEQ ID NO:62的VH CDR3；或
(u)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:65的VH CDR1、序列为SEQ ID NO:66的VH CDR2、序列为SEQ ID NO:67的VH CDR3。
在某些实施方案中，所述纳米抗体包含如SEQ ID NO:10-29，68，69任一项所示的序列或其变体；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)。在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述融合蛋白还包含连接体。
在某些实施方案中，所述连接体为多肽，例如柔性肽或刚性肽。
在某些实施方案中，所述连接体包含一个或几个(例如1个、2个或3个)如(GmS)n所示的序列，其中m选自1-6的整数，n选自1-6的整数。在某些实施方案中，m为3、4、或5。在某些实施方案中，n为2，3或4。
在某些实施方案中，所述连接体具有如SEQ ID NO:39所示的氨基酸序列。
在某些实施方案中，所述融合蛋白的免疫原性多肽和纳米抗体直接连接或通过连接体连接。
在某些实施方案中，所述免疫原性多肽位于融合蛋白的N端或C端。
在某些实施方案中，所述融合蛋白从N端至C端依次包含：免疫原性多肽和纳米抗体；或者，纳米抗体和免疫原性多肽；或者，免疫原性多肽，连接体和纳米抗体；或者，纳米抗体，连接体和免疫原性多肽。
在某些实施方案中，所述融合蛋白还包含信号肽和/或标签。
在某些实施方案中，所述信号肽选自tPA信号肽，蜂毒信号肽。在某些实施方案中， 所述信号肽具有如SEQ ID NO:31，37或38所示的氨基酸序列。
在某些实施方案中，所述标签为用于纯化的标签，例如选自His标签或GST标签。
在某些实施方案中，所述信号肽位于融合蛋白的N端。
在某些实施方案中，所述标签位于融合蛋白的C端。
在某些实施方案中，所述融合蛋白具有如SEQ ID NO:42-49，50-56，72-79，80-87，63，64，89任一项所示的氨基酸序列。
在第二方面，本申请提供了一种分离的核酸分子，其包含编码如第一方面所述的融合蛋白的核苷酸序列。
在某些实施方案中，所述核苷酸序列根据宿主细胞的密码子偏好性进行了密码子优化或未进行优化。
如本领域技术人员所知晓的，密码子存在简并性。即，在蛋白质的翻译过程中，每个氨基酸可对应1种或多种密码子，例如可对应多达6种密码子。不同的物种(例如，宿主细胞)在使用编码某一氨基酸的简并密码子时存在着很大的差异，有着不同的偏好。这种偏好现象即被称为“密码子偏好性”。因此，如本文中所使用的，术语“密码子偏好性”是指某一物种偏爱使用某些特定的密码子来编码氨基酸的情况。根据密码子偏好性来优化核酸分子的序列在某些情况下是特别有利的，例如，可能有助于提高核酸分子所编码的蛋白质的表达水平。
在第三方面，本申请提供了一种载体，其包含如第二方面所述的分离的核酸分子。在某些实施方案中，所述载体用于表达(例如，在细胞中体外表达)所述分离的核酸分子编码的蛋白。
在第四方面，本申请提供了一种宿主细胞，其包含如第二方面所述的核酸分子或如第三方面所述的载体。
在某些实施方案中，所述宿主细胞选自原核细胞和真核细胞。
在某些实施方案中，所述原核细胞选自大肠杆菌细胞和枯草芽孢杆菌细胞。
在某些实施方案中，所述真核细胞选自酵母细胞，昆虫细胞，植物细胞和动物细胞。
在某些实施方案中，所述动物细胞是哺乳动物细胞(例如，鼠细胞，人细胞)。
在第五方面，本申请提供了一种表达或产生如第一方面所述的融合蛋白的方法，所述方法包括，在允许蛋白质表达的条件下，培养第四方面所述的宿主细胞，以及任选地，回收或纯化所表达的融合蛋白。
在第六方面，本申请提供了一种组合物，其包含至少一种如第一方面所述的融合蛋白。
在某些实施方案中，述组合物还包含组装多肽。
在某些实施方案中，所述组装多肽组装成VLP。
在某些实施方案中，所述融合蛋白附接至所述VLP。
在第七方面，本申请提供了一种试剂盒，其包含：如第一方面所述的融合蛋白或含有编码其核苷酸序列的第一核酸分子，以及组装多肽或包含编码其核苷酸序列的第二核酸分子。
在某些实施方案中，所述融合蛋白或第一核酸分子和组装多肽或第二核酸分子是分开提供的，或者以组合物的形式提供。
在某些实施方案中，所述试剂盒还包含载体(例如，表达载体)。
在某些实施方案中，第一核酸分子与第二核酸分子包含于相同或不同的载体。
在某些实施方案中，所述试剂盒还包含缓冲液。
在某些实施方案中，所述缓冲液选自磷酸缓冲液，柠檬酸缓冲液，碳酸缓冲液，醋酸缓冲液，巴比妥酸缓冲液，Tris缓冲液，或其任意组合。
在某些实施方案中，所述缓冲液为PBS缓冲液。
在某些实施方案中，所述缓冲液还包含盐。
在某些实施方案中，所述盐选自NaCl，(NH4)SO4，NaSO4，NH4Cl，或其任何组合。
在第八方面，本申请提供了一种颗粒化的抗原，其包含以VLP形式存在的组装多肽，以及与组装多肽附接的如第一方面所述的融合蛋白。
在某些实施方案中，所述融合蛋白通过纳米抗体与所述组装多肽的相互作用附接至VLP。
在某些实施方案中，所述VLP附接至少一种如第一方面所述的融合蛋白。
在某些实施方案中，所述VLP还附接另外的多肽或融合蛋白(例如，T细胞表位)。
在第九方面，本申请提供了制备如第八方面所述的颗粒化的抗原的方法，所述方法包括：使用如第七方面所述的试剂盒。
在某些实施方案中，所述方法包括：在允许VLP组装的条件下，将组装多肽与融合蛋白接触。
在某些实施方案中，允许VLP组装的条件是将组装多肽置于溶液中。在某些实施方案中，所述溶液是包含盐的缓冲液。在某些实施方案中，所述盐选自NaCl，(NH4)SO4，NaSO4，NH4Cl，或其任何组合。
在某些实施方案中，所述方法包括：(i)将组装多肽加入缓冲液中，然后与融合蛋白接触；或者(ii)将组装多肽与融合蛋白一同加入缓冲液中；或者(iii)将融合蛋白加入缓冲液中，然后将组装多肽加入缓冲液中；
任选地，回收或纯化缓冲液中的颗粒化的抗原。
在第十方面，本申请提供了一种疫苗，其包含如第一方面所述的融合蛋白，或如第六方面所述的组合物，或如第八方面所述的颗粒化的抗原，以及佐剂。
在某些实施方案中，所述佐剂选自铝盐类佐剂，锌铝混合佐剂(例如FH002C)，弗氏佐剂，油乳佐剂，细胞因子，TLR激动剂，CpG佐剂，脂质体，AS01B佐剂，或其任意组合。
在第十一方面，本申请提供了一种药物组合物，其包含(1)至(6)的任意一项或多项：
(1)如第一方面所述的融合蛋白；
(2)如第二方面所述的核酸分子；
(3)如第三方面所述的载体；
(4)如第四方面所述的宿主细胞；
(5)如第六方面所述的组合物；
(6)如第八方面所述的颗粒化的抗原；
任选地，所述药物组合物还包含药学上可接受的载体和/或赋形剂。
在某些实施方案中，药学上可接受的载体和/或赋形剂选自pH调节剂(例如，磷酸 盐缓冲液)，表面活性剂(例如，阳离子，阴离子或者非离子型表面活性剂，例如Tween-80)，佐剂，离子强度增强剂(例如，氯化钠)，稀释剂，赋形剂，用于容纳或施用治疗剂的介质，以及任何组合。
在第十二方面，本申请提供了如第一方面所述的融合蛋白，或如第二方面所述的核酸分子，或如第三方面所述的载体，或如第四方面所述的宿主细胞，或如第六方面所述的组合物，或如第七方面所述的试剂盒，或如第八方面所述的颗粒化的抗原在制备药物组合物或疫苗中的用途，所述药物组合物或疫苗用于在受试者中诱导免疫应答。
在某些实施方案中，所述免疫应答是对免疫原性多肽和/或组装多肽的应答。在某些实施方案中，所述免疫应答是T细胞应答(例如CD4+应答或CD8+应答)。在某些实施方案中，所述免疫应答是B细胞应答。
在某些实施方案中，所述受试者为哺乳动物，例如人、猴或鼠。
在第十三方面，本申请提供了第一方面所述的融合蛋白，或如第二方面所述的核酸分子，或如第三方面所述的载体，或如第四方面所述的宿主细胞，或如第六方面所述的组合物，或如第七方面所述的试剂盒，或如第八方面所述的颗粒化的抗原在制备药物组合物或疫苗中的用途，所述药物组合物或疫苗用于在受试者中预防和/或治疗因对免疫原性多肽的免疫应答而受益或者被预防的疾病和/或症状。
在某些实施方案中，所述疾病和/或症状是由免疫原性多肽所源自的肿瘤细胞引起的。
在某些实施方案中，所述疾病和/或症状是由免疫原性多肽所源自的病原体(例如，病毒，细菌，真菌，寄生虫)引起的。
在某些实施方案中，所述疾病和/或症状是由免疫原性多肽所源自的病毒引起的，例如，水痘，新型冠状病毒肺炎，艾滋病，尖锐湿疣，病毒性肝炎(例如，病毒性乙型肝炎，病毒性甲型肝炎，病毒性丙型肝炎，病毒性戊型肝炎)，麻疹，腮腺炎。
在某些实施方案中，所述免疫原性多肽可以为新型冠状病毒的RBD蛋白或其片段。在此类实施方案中，疾病可以为新型冠状病毒肺炎。
在某些实施方案中，所述免疫原性多肽可以为VZV的gE蛋白或其片段。在此类实施方案中，疾病可以为水痘。
在某些实施方案中，所述免疫原性多肽可以为HPV的蛋白或其片段。在此类实施方案中，疾病可以为尖锐湿疣。
在某些实施方案中，所述免疫原性多肽可以为乙型肝炎病毒，甲型肝炎病毒，丙型肝炎病毒，戊型肝炎病毒的蛋白或其片段。在此类实施方案中，疾病可以为病毒性肝炎(例如，病毒性乙型肝炎，病毒性甲型肝炎，病毒性丙型肝炎，病毒性戊型肝炎)。
在某些实施方案中，所述免疫原性多肽可以为麻疹病毒的包膜糖蛋白或其片段。在此类实施方案中，疾病可以为麻疹。
在某些实施方案中，所述免疫原性多肽可以为腮腺炎病毒的蛋白或其片段。在此类实施方案中，疾病可以为腮腺炎。
在某些实施方案中，所述受试者为哺乳动物，例如人、猴或鼠。
在第十四方面，本申请提供了一种在受试者中诱导免疫应答的方法，包括给予受试者有效量的如第一方面所述的融合蛋白，或如第二方面所述的核酸分子，或如第三方面所述的载体，或如第四方面所述的宿主细胞，或如第六方面所述的组合物，或如第七方面所述的试剂盒，或如第八方面所述的颗粒化的抗原，或如第十方面所述的疫苗，或如第十一方面所述的药物组合物。
在某些实施方案中，所述免疫应答是对免疫原性多肽和/或组装多肽的应答。在某些实施方案中，所述免疫应答是T细胞应答(例如CD4+应答或CD8+应答)。在某些实施方案中，所述免疫应答是B细胞应答。
在某些实施方案中，所述受试者为哺乳动物，例如人、猴或鼠。
在第十五方面，本申请提供了一种在受试者中预防和/或治疗因对免疫原性多肽的免疫应答而受益或者被预防的疾病和/或症状，包括给予受试者有效量的如第一方面所述的融合蛋白，或如第二方面所述的核酸分子，或如第三方面所述的载体，或如第四方面所述的宿主细胞，或如第六方面所述的组合物，或如第七方面所述的试剂盒，或如第八方面所述的颗粒化的抗原，或如第十方面所述的疫苗，或如第十一方面所述的药物组合物。
在某些实施方案中，所述疾病和/或症状是由免疫原性多肽所源自的肿瘤细胞引起的。
在某些实施方案中，所述疾病和/或症状是由免疫原性多肽所源自的病原体(例如，病毒，细菌，真菌，寄生虫)引起的。
在某些实施方案中，所述疾病和/或症状是由免疫原性多肽所源自的病毒引起的，例如，水痘，新型冠状病毒肺炎，艾滋病，尖锐湿疣，病毒性肝炎(例如，病毒性乙型肝 炎，病毒性甲型肝炎，病毒性丙型肝炎，病毒性戊型肝炎)，麻疹，腮腺炎。
在某些实施方案中，所述免疫原性多肽可以为HIV的Env蛋白或其片段(例如，gp160蛋白，gp120蛋白，gp41蛋白)。在此类实施方案中，疾病可以为艾滋病。
在某些实施方案中，所述免疫原性多肽可以为新型冠状病毒的RBD蛋白或其片段。在此类实施方案中，疾病可以为新型冠状病毒肺炎。
在某些实施方案中，所述免疫原性多肽可以为VSV的gE蛋白或其片段。在此类实施方案中，疾病可以为水痘。
在某些实施方案中，所述免疫原性多肽可以为HPV的蛋白或其片段。在此类实施方案中，疾病可以为尖锐湿疣。
在某些实施方案中，所述免疫原性多肽可以为乙型肝炎病毒，甲型肝炎病毒，丙型肝炎病毒，戊型肝炎病毒的蛋白或其片段。在此类实施方案中，疾病可以为病毒性肝炎(例如，病毒性乙型肝炎，病毒性甲型肝炎，病毒性丙型肝炎，病毒性戊型肝炎)。
在某些实施方案中，所述免疫原性多肽可以为麻疹病毒的包膜糖蛋白或其片段。在此类实施方案中，疾病可以为麻疹。
在某些实施方案中，所述免疫原性多肽可以为腮腺炎病毒的蛋白或其片段。在此类实施方案中，疾病可以为腮腺炎。
在某些实施方案中，所述受试者为哺乳动物，例如人、猴或鼠。
在第十六方面，本申请提供了一种制备颗粒化免疫原性多肽的系统，其包括第一载体和第二载体，其中，第一载体包含编码融合蛋白的核苷酸序列，所述融合蛋白包含免疫原性多肽和组装多肽，第二载体包含编码纳米抗体的核苷酸序列；并且，所述纳米抗体能够特异性结合所述组装多肽，所述组装多肽能够组装成VLP；
在某些实施方案中，所述核苷酸序列根据宿主细胞的密码子偏好性进行了密码子优化或未进行优化。
在某些实施方案中，所述组装多肽选自戊型肝炎病毒(HEV)的蛋白或其片段或它们的变体。在某些实施方案中，所述组装多肽如第一方面所定义。
在某些实施方案中，所述融合蛋白如第一方面所定义。
在第十七方面，本申请提供了一种增强免疫原性多肽的免疫原性的方法，其包括，制备或获得含有所述免疫原性多肽和能够特异性结合组装多肽的纳米抗体的融合蛋白；和， 使所述融合蛋白与包含所述组装多肽的VLP接触，从而，获得包含附接至VLP的免疫原性多肽的颗粒化抗原。
在某些实施方案中，所述方法包括：使用第十六方面所述的系统。在某些实施方案中，所述方法包括：(1)通过第一载体表达或产生融合蛋白，通过第二载体表达或产生组装多肽；(2)在允许VLP组装的条件下，使融合蛋白和组装多肽接触。
在某些实施方案中，所述纳米抗体如第一方面所定义。
在某些实施方案中，所述融合蛋白如第一方面所定义。
在某些实施方案中，所述组装多肽组装为VLP。在某些实施方案中，所述融合蛋白通过纳米抗体与所述组装多肽的相互作用附接至VLP。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的病毒学、生物化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本文中，术语“免疫原性多肽”是指能够引起免疫应答的蛋白质或多肽。在某些实施方案中，将所述免疫原性多肽施用(直接或间接施用)于受试者，能够在受试者体内引起免疫应答。本领域技术人员理解，所述免疫原性多肽可以是天然存在的，也可以是天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加)但不影响其生物学活性的蛋白质或多肽(在本文中，该生物学活性为能够引起免疫应答)。因此，在本文中，免疫原性多肽可以是来源于非病原体(例如，肿瘤细胞)或病原体(例如，病毒、细菌、真菌、寄生虫或其他病原体)的多肽或其变体。
在本文中，术语“免疫应答”是指免疫系统的细胞(例如B细胞，T细胞或单核细胞)对刺激的应答。在一些实施方案中，免疫应答是对特定抗原特异性的应答(即抗原特异性应答)。在一些实施方案中，免疫应答是T细胞应答，例如CD4+应答或CD8+应答。在另一些实施方案中，免疫应答是B细胞应答，会导致特异性抗体的产生。
在本文中，术语“亚病毒(subviruses)”是一种不具有完整的病毒结构的微生物，主要包括类病毒、拟病毒和朊病毒。
在本文中，术语“SARS-Cov-2的RBD蛋白”或“SARS-Cov-2的RBD”具有相同的含义，可互换使用，其是指SARS-Cov-2的刺突蛋白(Spike protein,S蛋白)上的受体结 合结构域(RBD)。其主要功能是识别宿主细胞表面受体，介导与宿主细胞的融合。本领域技术人员理解，不同SARS-Cov-2病毒株可能含有不同序列的RBD蛋白，但这些RBD蛋白具有相同或相似的生物学特性。因此，在本发明中，RBD蛋白不仅包括SEQ ID NO:1-8所示的蛋白，而且应包括各种SARS-Cov-2病毒株的RBD蛋白。这些RBD蛋白的氨基酸序列可从公共数据库(例如GenBank数据库)获得，例如，GenBank登录号OP077006.1、OP077005.1、OP077003.1所示的氨基酸序列。
在本文中，术语“VZV的gE蛋白”、“VZV的gE”、“VZV的糖蛋白gE”是指VZV的一种囊膜糖蛋白，它们具有相同的含义，可互换使用。本领域技术人员理解，不同VZV病毒株可能含有不同序列的gE蛋白，但这些gE蛋白具有相同或相似的生物学特性。因此，在本发明中，gE蛋白不仅包括SEQ ID NO:30所示的蛋白，而且应包括各种VZV病毒株的gE蛋白。这些gE蛋白的氨基酸序列可从公共数据库(例如GenBank数据库)获得。
在本文中，术语“HIV-1的Env”是指HIV-1病毒表面的包膜蛋白(envelope)，在本文中也称为“BGTSTIP”。本领域技术人员理解，不同HIV-1病毒株可能含有不同序列的Env蛋白，但这些Env蛋白具有相同或相似的生物学特性。因此，在本发明中，Env蛋白不仅包括SEQ ID NO:34-35所示的蛋白，而且应包括各种HIV-1病毒株的Env蛋白。这些Env蛋白的氨基酸序列可从公共数据库(例如GenBank数据库)获得。
HEV中由ORF2基因编码的衣壳蛋白(也称为ORF2蛋白)及其片段(例如，p239蛋白，p495蛋白)已被证实具有组装成VLP的能力。ORF2蛋白的序列是本领域公知的，参见，例如，DDBJ数据库登录号：D11092。p239蛋白的序列为对应于ORF2蛋白的第368位氨基酸至第606位氨基酸。在某些实施方案中，p495蛋白的序列为对应于ORF2蛋白的第112位氨基酸至第606位氨基酸。
在本文中，术语“组装多肽”是指能够组装成病毒样颗粒(VLP)的蛋白或多肽。在某些实施方案中，所述组装多肽是天然病毒或类病毒的外壳蛋白。在此类实施方案中，所述组装多肽组装成的VLP与天然病毒或类病毒的结构相似，区别仅在于不含有天然病毒或类病毒的基因组。在某些实施方案中，所述组装多肽是人工制备和/或筛选的多肽。在此类实施方案中，所述组装多肽组装成的VLP与天然病毒或类病毒的结构相似或不相似。术语“病毒样颗粒(VLP)”是一种多聚体颗粒，其结构与天然的病毒或亚病毒相似或不相似。经证实，一些病毒(例如，HBV，HEV，HPV)的蛋白(例如，衣壳蛋白，表面蛋白，包膜蛋白)在适当的表达系统中重组表达之后，可以自发形成 VLP。
本领域技术人员可以通过已公开的方法来鉴定具有组装成VLP能力的蛋白或多肽，例如，可在室温下将待测蛋白置于缓冲液(例如，PBS溶液)中，然后检测VLP的存在。可以使用本领域中已知的常规技术来检测VLP的存在，例如，电子显微技术，生物物理表征等。具体检测方法可参见，例如，Baker et al.(1991)Biophys.J.60：1445-1456；以及Hagensee et al.(1994)J.Virol.68：4503-4505。例如，HEV中由ORF2基因编码的衣壳蛋白(也称为ORF2蛋白)及其片段(例如，p239蛋白，p495蛋白)已被证实具有组装成VLP的能力。
本领域技术人员可以理解，在本申请中，组装多肽不仅包含能够组装成VLP的天然蛋白或多肽，还包含在所述天然蛋白的基础上天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加)但不影响其生物学功能(在本文中，该生物学功能为能够组装成VLP)的蛋白或多肽。因此，在某些实施方案中，所述组装多肽选自戊型肝炎病毒(HEV)的ORF2蛋白或其片段或它们的变体。在某些实施方案中，所述组装多肽选自p239蛋白或其片段或它们的变体，p495蛋白或其片段或它们的变体。
根据本发明，当在蛋白/多肽的背景中使用时，术语“变体”是指这样的蛋白/多肽，与其所源自的蛋白/多肽的序列相比具有一个或多个(例如1-10个或1-5个或1-3个)氨基酸差异(例如，置换、缺失或添加)，并且其保留了其所源自的蛋白/多肽的生物学活性。
在本文中，“免疫原性多肽是来源于生物体或非生物体的多肽或其免疫原性变体”中的“来源于”是指该多肽的序列源自生物体或非生物体中的序列，但该多肽的获得方式不受限于具体的产生方式。在某些实施方案中，所述多肽可以是自然分离的，人工合成的，或通过基因工程重组等方式获得的。
在本文中，术语“颗粒化免疫原性多肽”是指以颗粒形式存在的具有免疫原性的多肽的集合体。在某些实施方案中，颗粒化免疫原性多肽是指附接于病毒样颗粒的免疫原性多肽。
在本文中，术语“生物体”是指有生命的个体或物体。除病毒等少数种类以外，生物体都是由细胞构成的。在某些实施方案中，生物体包含病原体和非病原体。
在本文中，术语“病原体(pathogens)”是指可造成人或动植物感染的微生物(例如，细菌、病毒、立克次氏体、真菌)、寄生虫或其他媒介(例如，微生物重组体)。
在本文中，术语“融合蛋白”是指由至少两个独立的蛋白或多肽的氨基酸序列连接 形成的重组蛋白。所述两个独立的蛋白或多肽的氨基酸序列可以直接连接，也可以通过连接体(linker)连接。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒(例如裸质粒)；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及病毒载体等。
如本文中所使用的，术语“宿主细胞”是指，可用于扩增或表达外源基因的细胞，其包括但不限于，如大肠杆菌或枯草芽孢杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞，293T细胞或人细胞等的动物细胞。
如本文中所使用的，术语“抗体”指能够通过位于免疫球蛋白分子可变区的至少一个抗原识别位点特异性结合靶(如碳水化合物、多核苷酸、脂质、多肽等)的免疫球蛋白 分子。抗体包括任何类型的抗体，例如IgG、IgA或IgM(或其亚类)，并且抗体不需要属于任何特定的类型。取决于抗体重链恒定区的氨基酸序列，免疫球蛋白可以分配到不同的类型。有五种主要类型的免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中几种可进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同类型的免疫球蛋白的重链恒定区分别被称为α、δ、ε、γ和μ。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。不同类型的免疫球蛋白的亚基结构和三维构型是众所周知的。重链恒定区由4个结构域(CH1、hinge region、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。
抗体的VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDRs和4个FRs组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat(Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义)，Chothia(由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883))和/或AbM(AbM CDR定义方式来源于Martin的相关研究(Martin ACR,Cheetham JC,Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268-9272))。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDRs，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat. Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。在本发明中，纳米抗体含有的CDR可根据本领域已知的各种编号系统确定。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
如本文中所使用的，术语“单域抗体(single-domain antibody,sdAb)”也称为纳米抗体(nanobody)，二者可以互换使用。其具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区，也称为VHH)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力(Holt,L.等人,生物技术趋势(Trends in Biotechnology),21(11):484-490，2003)。单域抗体。纳米抗体可以是源自驼类的羊驼抗体，也可以是源自鲨鱼的鲨鱼抗体。
可使用本领域技术人员已知的常规技术与用于完整抗体的方式相同的方式就特异性筛选纳米抗体。
如本文中所使用的，表述“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(KD)表示。在本发明中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
根据本发明，术语“佐剂”是指免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝盐类佐剂、锌铝混合佐剂(例如FH002C)、弗氏佐剂、油乳佐剂、细胞因子、TLR激动剂、CpG佐剂、脂质体、AS01B佐剂或其组合。
如本文中所使用的，术语“药学上可接受的”意指，制药领域公认的可用于动物，特别是可用于人的。如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂(包括但不限于磷酸盐缓冲液)，表面活性剂(包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80)，佐剂，离子强度增强剂(包括但不限于氯化 钠)，稀释剂，赋形剂，用于容纳或施用治疗剂的介质，以及其任何组合。
如本文中所使用的，术语“受试者”是指哺乳动物，包括但不限于，人，啮齿类动物(小鼠，大鼠，豚鼠)，狗，马，牛，猫，猪，猴，黑猩猩等。优选地，受试者是人。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病有效量是指，足以预防，阻止，或延迟疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“允许VLP组装的条件”是指组装多肽能够组装成VLP的条件。通常，将具有组装能力的多肽或蛋白置于液体中，其能够自组装成VLP。在某些实施方案中，允许VLP组装的条件是将组装多肽置于溶液中。在此类实施方案中，所述溶液不会含有不利于VLP组装的成分。在某些实施方案中，所述溶液含有利于VLP组装的成分，例如，所述溶液为含有盐的缓冲液。
发明的有益效果
本发明利用组装多肽(例如，HEV的ORF2蛋白或其片段或它们的变体，HPV的L1蛋白或其片段或它们的变体，HBV的表面蛋白或其片段或它们的变体)能够组装成VLP的能力，将特异性结合所述组装多肽的纳米抗体与免疫原性多肽进行融合表达，并利用纳米抗体对所述组装多肽的靶向结合特性，将多种免疫原性多肽展示在由组装多肽形成的VLP的表面，获得了颗粒化的免疫原性多肽。由此，本申请提供了一种将免疫原性多肽颗粒化的系统和方法。
并且，这种颗粒化的免疫原性多肽与普通的免疫原性多肽相比，具有较高的免疫原性，能够刺激产生高水平免疫反应，包括B细胞和T细胞反应。因此，本发明的颗粒化免疫原性多肽尤其适合于疫苗生产和接种，在预防和/或治疗病毒感染方面具有优势。
本发明的将免疫原性多肽颗粒化的系统和方法使用了纳米抗体，与现有技术相比，使用纳米抗体至少具有以下优势：①纳米抗体介导的免疫原性多肽的展示不受组装多肽N/C端结构的影响，构建策略更加灵活。②纳米抗体介导的展示的策略不会影响组装多肽的组装和构象，适用性更加广泛。③纳米抗体与免疫原性多肽融合有利于维持免疫原 性多肽构象。④不同表位的纳米抗体可以组合结合，有利于提高免疫原性多肽展示的比例。⑤纳米抗体可以基于组装多肽的对称性和免疫原性多肽的寡聚状态进行合理设计。⑥纳米抗体介导的展示策略可以降低免疫原性多肽潜在的空间位阻。⑦抗体介导的展示可以直接靶向未经修饰或改造的VLP，如果已上市的VLP自身具有良好的安全性，无需考虑其本身的成药性问题，具有良好的可转化性。⑧作为接头的纳米抗体可通过本领域熟知的人源化策略最大程度降低其免疫原性，减少机体产生没有必要的免疫应答，实现免疫原性多肽免疫聚焦。⑨任何免疫原性多肽通过与纳米抗体的融合表达，都可以通过本发明的方法实现免疫原性多肽的颗粒化，具备普适性和通用性。⑩纳米抗体由于体积较小，通过筛选和鉴定纳米抗体的结合位置，可以保留颗粒化载体如HEV ORF2、HPV L1和HBV S的原有主要免疫表位的暴露，免疫原性多肽的颗粒化后载体仍具有良好的免疫原性，可作为一种联合疫苗，这种策略也可用于其他联合疫苗的开发，从而，具有巨大的应用前景。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了本发明实施例纳米抗体的筛选过程。图1A显示了细菌文库菌落PCR琼脂糖凝胶电泳结果。图1B为测序后评价细菌文库多样性的结果。
图2显示了本发明实施例的多种VHH-RBD蛋白的SDS聚丙烯酰胺凝胶电泳结果。图2A为通过昆虫细胞表达系统生产纯化的多种VHH-RBD融合蛋白的电泳结果。图2B为通过哺乳动物细胞表达系统生产纯化的多种VHH-RBD融合蛋白的电泳结果。M:分子量Marker。
图3显示了本发明实施例的多种VHH-gE融合蛋白的SDS聚丙烯酰胺凝胶电泳结果。M:分子量Marker，1-7泳道分别显示为不同的VHH-gE的融合蛋白。
图4显示了本发明实施例的多种VHH-BGTSTIP蛋白的SDS-聚丙烯酰胺凝胶电泳结果。“+”表示还原条件，“-”表示非还原条件。
图5显示了本发明实施例的多种VHH-RBD的融合蛋白的免疫印迹实验(Western  Blot)的结果。图5A为通过昆虫细胞表达系统生产纯化的多种VHH-RBD融合蛋白，M:分子量Marker，1-8分别泳道显示为不同纳米抗体与RBD的融合蛋白。图5B为通过哺乳动物细胞表达系统293F细胞生产纯化的多种VHH-RBD融合蛋白。
图6显示了本发明实施例的多种候选分子VHH-gE的蛋白免疫印迹实验(Western Blot)的结果。M:分子量Marker，1-7分别泳道显示为不同纳米抗体与gE的融合蛋白。
图7显示了实施例多种VHH-RBD与多种已报道特异性单克隆抗体的酶联免疫吸附实验(ELISA)结果。图7A为通过昆虫细胞表达系统生产纯化的多种VHH-RBD蛋白的活性鉴定结果，所使用的抗体包括广谱中和抗体JSR-105、JSR-551、JSR-209、m6D6、m7D6，非广谱中和抗体85F7。图7B为通过哺乳动物细胞表达系统293F细胞生产纯化的多种VHH-RBD蛋白的活性鉴定结果，所使用的抗体包括3G11、8H12、13F10、8B8、9D3、3F9。
图8显示了实施例多种VHH-gE与多种特异性单克隆抗体的酶联免疫吸附实验(ELISA)结果。所使用的特异性单克隆抗体包括3H7、4G4、6B7、11B11、11B12、13B6、14G1、17B7。
图9显示了实施例多种VHH-BGTSTIP与多种报道的HIV-1中和抗体或非中和抗体的酶连免疫吸附实验(ELISA)结果。
图10显示为实施例多种VHH-RBD与HEV-p239蛋白的亲和力分析结果。图10A显示了通过昆虫细胞表达系统生产纯化获得的VHH-RBD与HEV-p239蛋白的亲和力结果，图10B为通过哺乳动物细胞表达系统293F细胞生产纯化的VHH-RBD与HEV-p239蛋白的亲和力结果。
图11显示了实施例多种VHH-gE与HEV-p239蛋白的亲和力分析结果。
图12显示了实施例多种VHH-BGTSTIP与HEV-p239蛋白的亲和力分析结果。
图13A显示了本发明实施例中通过昆虫细胞表达系统生产纯化的P1-5B-RBD经过superdex 200 increase(高效液相色谱分子筛)纯化的结果。图13B显示了本发明实施例中通过哺乳动物细胞表达系统生产纯化的P2-6D-RBD、P2-3E-RBD、P2-10G-RBD经过superdex 200 increase纯化的结果。
图14A显示了昆虫细胞表达系统纯化的P1-5B-RBD融合蛋白与HEV-p239形成的复合物的纯化图谱和SDS-PAGE鉴定结果。图14B显示为哺乳动物细胞表达系统纯化的P2-3E-RBD，P2-10G-RBD和P2-6D-RBD融合蛋白分别与HEV-p239形成的复合物的纯化图谱和SDS-PAGE鉴定结果。
图15显示了本发明实施例候选融合蛋白P2-8C-gE与HEV-p239的复合物纯化图谱和SDS-PAGE鉴定结果。图15A为复合物纯化结果。图15B为P2-8C-gE与HEV-p239的复合物的SDS-PAGE鉴定结果。
图16显示了本发明实施例候选融合蛋白P2-5C-BGTSTIP与HEV-p239的复合物纯化图谱和SDS-PAGE鉴定结果。
图17显示了本发明实施例P2-8C-gE的高效体积排阻色谱(HPSEC)的结果。
图18A显示了本发明实施例中通过昆虫细胞表达系统生产纯化的VHH-RBD与HEV-p239蛋白形成的HEV-RBD复合物与HEV-p239颗粒的分子粒径检测结果。图18B显示了通过哺乳动物细胞表达系统生产纯化的VHH-RBD与HEV-p239蛋白形成的三种HEV-RBD复合物与HEV-p239颗粒的分子粒径检测结果。
图19显示了本发明实施例HEV-gE复合物样品的分子粒径检测结果。图19A为HEV-p239颗粒的分子粒径，图19B为HEV-gE复合物的分子粒径。
图20显示了本发明实施例HEV-BGTSTIP复合物与HEV-p239颗粒的分子粒径检测结果。
图21显示了本发明实施例HEV-p239和基于HEV-p239颗粒构建的RBD复合物的分析超离结果。图21A为昆虫细胞表达系统生产纯化的VHH-RBD与HEV-p239制备的复合物颗粒(右图)以及HEV-p239(左图)的分析超离结果。图21B为通过哺乳动物细胞表达系统293F细胞生产纯化的VHH-RBD与HEV-p239制备的三种复合物颗粒和HEV-p239的分析超离结果。
图22显示了本发明实施例HEV-p239和基于HEV-p239颗粒构建的gE复合物的分析超离结果。图22A显示HEV-p239呈现单一组分，沉降系数为22S。图22B显示gE复合物呈现单一组分，沉降系数为31S。
图23显示了本发明实施例HEV-p239和基于HEV-p239颗粒构建的Env(BGTSTIP)复合物的分析超离结果。
图24A显示了本发明实施例HEV-p239和通过昆虫细胞表达系统生产纯化的VHH-RBD制备的复合物样品的透射电子显微镜负染结果。图24B显示了本发明实施例HEV-p239和通过哺乳动物表达系统293F细胞生产纯化的VHH-RBD制备的复合物样品的透射电子显微镜负染结果。
图25显示为本发明实施例HEV-p239和VHH-gE融合蛋白制备的复合物样品的透射电子显微镜负染结果。
图26显示了本发明实施例HEV-p239和基于HEV-p239颗粒构建的Env(BGTSTIP)复合物的透射电子显微镜负染结果。
图27显示了本发明实施例RBD颗粒抗原的免疫原性检测结果。图27A为通过昆虫细胞表达系统获得的RBD颗粒化抗原免疫小鼠后的血清结合及中和活性检测结果。图27B及图27C为通过哺乳动物细胞表达系统获得的RBD颗粒化抗原免疫小鼠后的血清结合活性及对野生型SARS-CoV-2假病毒的中和活性检测结果。
图28显示了本发明实施例RBD颗粒抗原配伍铝佐剂诱导产生的抗体与新冠野生型(WT)毒株、Gamma毒株、BA.2毒株的中和检测结果。
图29显示了本发明实施例gE颗粒抗原的免疫原性检测结果。
图30显示了本发明实施例gE颗粒抗原的小鼠免疫血清活病毒中和检测结果。
图31显示了本发明实施例单体gE抗原、gE颗粒抗原和对照配伍佐剂的流式细胞免疫检测结果。
图32显示了本发明实施例单体gE抗原、gE颗粒抗原和对照配伍佐剂的免疫组酶联免疫斑点检测细胞因子。
图33显示了本发明实施例P1-5B纳米抗体人源化改造结果。
图34显示了本发明实施例的单域抗体融合蛋白P1F8-BGTSTIP的聚丙烯酰胺电泳结果；其中，M为分子量Marker；“+”表示还原条件,“-”表示非还原条件。
图35显示了本发明实施例单域抗体融合蛋白P1F8-BGTSTIP与多种已报道的HIV-1广谱中和抗体及非中和抗体的酶联免疫吸附实验(ELISA)结果。其中2G12、VRC01、PGT121、PGT121、SF12、B12为广谱中和抗体；17b、F105和F240为非中和抗体。
图36显示了实施例单域抗体融合蛋白P1F8-BGTSTIP与HPV 58VLP的亲和力分析结果。
图37显示了本发明实施例中通过哺乳动物表达系统生产纯化的P1F8-BGTSTIP与58-VLP形成的复合物的高效体积排阻色谱(HPSEC)和SDS-PAGE鉴定结果。红色曲线代表P1F8-BGTSTIP与58VLP形成的复合物图谱，绿色曲线表示58VLP图谱，蓝色曲线表示为融合蛋白P1F8-BGTSTIP图谱。
图38显示了本发明实施中P1F8-BGTSTIP与58VLP形成的复合物和58VLP的分析超离结果。
图39显示了本发明实施例中P1F8-BGTSTIP和58VLP形成的复合物样品的透射电子显微镜负染结果。
图40显示了为本发明实施例中58-VLP-BGTSTIP颗粒化抗原免疫小鼠后BGTSTIP特异性的结合抗体滴度检测结果。
图41显示了本发明中HBsAg结合纳米抗体融合蛋白S2-gE后形成颗粒化抗原的SDS-PAGE鉴定图。
图42显示了本发明中HBsAg结合纳米抗体融合蛋白S2-gE后形成颗粒化抗原的分子筛鉴定图。
图43显示了本发明中HBsAg结合纳米抗体融合蛋白S2-gE后形成颗粒化抗原HBsAg-S2-gE(HBV-gE)的DLS鉴定图。
图44显示了本发明中HBsAg结合纳米抗体融合蛋白S2-gE后形成颗粒化抗原HBsAg-S2-gE的负染电镜图。
图45显示了本发明中HBsAg结合纳米抗体融合蛋白S2-gE后形成颗粒化抗原HBsAg-S2-gE与S2-gE免疫小鼠后的特异性抗体滴度结果。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。












具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1：纳米抗体噬菌体文库构建与筛选过程
1.RNA提取(Trizol法)
(1)将用Trizol保存的外周血淋巴细胞(获自羊驼)转移至1.5mL的离心管，加入1/5体积的氯仿混匀；
(2)室温静置5分钟后4℃12000g离心15分钟；
(3)小心将离心后的上清液转移到新的离心管；
(4)往新离心管中加入等体积的异丙醇；
(5)室温静置10分钟后4℃12000g离心10分钟；
(6)用1mL 75％乙醇清洗各管沉淀，7500g离心5分钟后，去除乙醇，干燥后，沉淀溶解于适量的无RNA酶的水中，合并所有样品，即为提取得到的总RNA。
2.反转录cDNA(Takara反转录试剂盒)
3.PCR扩增
表2.PCR扩增程序

4.酶切和连接
表3.酶切和连接程序
5.细菌文库和噬菌体文库构建
6.噬菌体筛选纯化、扩增
其中，V为加入的噬菌体体积(单位μL)，Tlibrary为噬菌体效价；
T(pfu/ml)＝N×D×400
其中，T为噬菌体效价(单位pfu/mL)，D为稀释倍数，N为相应稀释倍数上单菌落个数。
7.文库质量验证
其中，图1显示了本发明实施例纳米抗体的筛选过程。图1A显示了菌落PCR琼脂糖凝胶电泳结果，任意挑选的51个单克隆有50个为阳性克隆，阳性率为98％，细菌文库阳性克隆率符合要求。图1B为测序后使用软件翻译成蛋白质序列，序列多样性比对显示50个序列均为独立序列，多样性良好，细菌文库多样性符合要求。
8.单克隆ELISA检测
按照下面公式计算并每孔加入辅助噬菌体M13K07以使细菌个数：噬菌体数＝1：20：
其中，V为加入辅助噬菌体的体积(单位mL)，Thelper-phage为使用的辅助噬菌体效价。
9.将上述挑选出的阳性克隆进行ELISA二次验证
最终挑选出的阳性克隆送至生物公司进行测序和分析，共获得了20株纳米抗体的重链可变区(VHH)序列，这些纳米抗体的氨基酸序列如表1中SEQ ID NO:10-29所示，CDR1-3序列如表1中SEQ ID NO:57-62以及90-144所示。
实施例2：VHH-RBD，VHH-gE和VHH-BGTSTIP融合蛋白的制备和表达
1、融合蛋白的制备
昆虫细胞表达系统中VHH-RBD序列的构建方式为：在VHH序列(SEQ ID NO:10-29)的C端连接RBD序列(SEQ ID NO:1-8)，然后，在上述序列的N端连接蜂毒信号肽(SEQ ID NO:31)以促进蛋白分泌表达。由生工合成编码上述氨基酸序列的核苷酸序列，并通过Nco I和BamH I酶切位点将所述核苷酸序列构建在PIEX/bac-1载体上。最终，通过昆虫细胞表达系统获得了8种VHH-RBD蛋白(P1-5B-RBD，P2-2C-RBD，P1-1B-RBD，P1-1G-RBD，P2-4E-RBD，P2-3E-RBD，P2-3D-RBD，P2-3A-RBD)，序列分别如表1中的SEQ ID NO:42-49所示。
采用相同方法，构建了VHH-gE融合蛋白的序列，不同之处在于将C端连接的RBD序列替换为gE序列(SEQ ID NO:30)。最终，通过昆虫细胞表达系统获得了7种VHH-gE蛋白(P2-10G-gE，P2-6D-gE，P2-5C-gE，P2-8C-gE，P2-1H-gE，P2-5G-gE，P2-7D-gE)，序列分别如表1中的SEQ ID NO:50-56所示。
哺乳动物细胞表达系统中VHH-RBD序列的构建方式为：在VHH序列(SEQ ID NO:10-29)的C端依次连接(G4S)3柔性蛋白接头(SEQ ID NO:39)、RBD序列(SEQ ID NO:1-8)、组氨酸标签(8-His tag)，在N端引入促进蛋白分泌表达的信号肽序列(SEQ ID NO:38)。编码上述氨基酸序列的核苷酸序列经密码子优化后，由通用生物进行合成，并克隆到pcDNA3.1载体的ECORI和Xbal酶切位点之间。最终，通过哺乳动物细胞表达系统获得了8种VHH-RBD蛋白(P1-1B-RBD，P1-5B-RBD，P2-2C-RBD，P2-5C-RBD，P2-3A-RBD，P2-3E-RBD，P2-4E-RBD，P2-5G-RBD)，序列分别如表1中的SEQ ID No:72至89所示。
采用相同方法，构建了VHH-BGTSTIP(即，VHH-Env)融合蛋白的序列，不同之处在于将连接的RBD序列替换为全长Env胞外段的氨基酸序列(SEQ ID NO:34或35)。最终通过哺乳动物细胞表达系统获得了8种VHH-BGTSTIP蛋白(P1-1B-BGTSTIP，P1-5B-BGTSTIP，P2-2C-BGTSTIP，P2-5C-BGTSTIP，P2-3A BGTSTSIP，P2-3E-BGTSTIP，PA-4G-BGTSTIP，P2-7D-BGTSTIP)序列分别如表 1中的SEO ID No:80至87所示。
2、VHH-RBD，VHH-gE在昆虫表达系统中的表达
昆虫细胞的转染
(1)确定sf9细胞(购自Invitrogen，11496-015)或sf21细胞(购自Invitrogen，11497-013)处于对数增长期(1.5-2.5×106/mL)，生存率维持在90％以上。在24孔板中加入200μL培养基ESF 921(购自Expression systems,96-001-01)含2％FBS)，0.1μg Baculovirus DNA(购自Expression systems，91-002)和1ug pAc-S质粒，混合均匀。在50μL ESF921培养基(购自Expression systems,96-001-01)中加入1μL转染试剂(购自Expression systems，95-055-075)，混合均匀。将上述两者合成一管，混合均匀，在室温中静置30min。静置过程中清洗细胞(时间快到前进行)：待细胞完全贴壁后，用枪吸走培养基，再加入300μL ESF921培养基，动作迅速，以免使细胞失水，轻轻摇摆后，去掉培养基，再加入300μL ESF921培养基。时间到后，将约100μL的上述混合物滴加到每孔细胞中，均匀滴加。然后置于27℃孵育6h，弃去上清，补加500μL完全培养基(50％CCM3+50％TNM-FH(SIGMA-ALDRICH，T1032)+10％FBS)。
(2)收集步骤(1)中所得细胞上清，500g离心5min，去除细胞残骸和碎片，将上清于4℃避光存放，此为P1病毒种液。
(3)杆状病毒的扩增
确定sf9细胞或sf21细胞处于对数增长期(1.5-2.5×106/mL)，生存率维持在90％以上。在10cm板上铺8-10mL密度为6×105/mL个细胞。静置15min使细胞贴壁。加入P1病毒液约600μL，均匀滴入。置于27℃孵育3-4d。观察细胞病变现象。收集细胞上清，1000rpm离心5min，去除细胞残骸和碎片，并用0.22um滤膜过滤。将上清于4℃避光存放，此为P2病毒种液。P2病毒滴度约为106-107pfu/mL。P3可依照此方法在250mL摇瓶中按体积放大扩增病毒。
昆虫细胞蛋白表达操作
1L摇瓶中加入250mL ESF921培养基培养的密度为2×106/mL，生存率90％以上的H5细胞(购自Invitrogen，B855-02)。按相应MOI加入病毒量，用封口膜封住瓶口，置于27℃摇床中120rpm培养。每天取出摇瓶中细胞观察并计数，记录相关数据。合适的MOI能保证第一天细胞70％以上病变。第二天细胞全部病变，且生存率80％左右。第三天细胞出现破裂，生存率降低至30-50％。此时可考虑收集细胞。采用 10000rpm，离心10min来收集细胞，然后分离上清并进行纯化。
3、VHH-RBD，VHH-gE在昆虫表达系统中的纯化
利用AKTA系统进行Ni亲和层析纯化；
仪器系统：AKTA Pure型制备型液相色谱仪；
纯化介质：Ni Sepharose 6 Fast Flow亲和介质；缓冲液：分为A泵、B泵缓冲液，一般A泵为1×PBS缓冲液(160g/L NaCL，8.1mmol/LNa2HPO4，1.5mmol/L KH2PO4，2.7mmol/L KCL，pH7.4)，B泵为1×PBS+250mmol/L咪唑缓冲液；
系统流速：5mL/min；检测波长:UV@280nm
洗脱条件：用50mM咪唑缓冲液(250mmol/L咪唑缓冲液经1×PBS缓冲液稀释获得)洗脱杂蛋白，接着用1×PBS清洗，再用250mM咪唑缓冲液洗脱目的蛋白(S三聚体蛋白)；
收集250mM咪唑洗脱的产物，获得纯化的样品10mL。取洗脱产物各50μL，加入6X Loading Buffer 10μL混匀，于80℃水浴10min后取10μl于10％SDS-聚丙烯酰胺凝胶中以80V电压电泳120min。随后以考马斯亮兰染色显示电泳条带。
4、VHH-RBD和VHH-BGTSTIP在哺乳动物表达系统中的转染表达
-80℃冰箱取冻存的293F细胞，37℃化冻后1300rpm离心4min，于超净台内弃上清，轻弹细胞并用提前37℃温育的293freestyle培养基重悬后，转移至含50mL温育培养基的三角瓶中，37℃5％CO2 120转/min悬浮培养，待细胞密度达2.0*106时进行传代，逐渐扩大培养体系。待细胞足够量时，采用PEI(MW 25,000)瞬时转染293F细胞，无菌50mL管收取细胞，1300rpm离心4min，轻弹细胞后37℃温育培养基重悬，并转移至含有450mL 37℃温育培养基的三角瓶中，置于37℃摇床备用。
将提取的VHH-RBD、VHH-BGTSTIP质粒与PEI(MW 25,000)按1：2比例置于50ml培养基中充分混匀后静置18min，转移至上述450ml培养基中，37℃5％CO2 120转/min悬浮培养6天，进行VHH-RBD、VHH-BGTSTIP蛋白的表达。转染过程中注意PEI的避光操作。
5、VHH-RBD和VHH-BGTSTIP在哺乳动物表达系统中的纯化
瞬时转染6天后，收取细胞培养液，JA-14转头7000g离心10min后取细胞上清，20000g离心10min后取上清0.22um孔径滤膜过滤两次，以该样品进行下一步Ni-excel柱纯化。
利用AKTA系统进行Ni亲和层析纯化；
仪器系统：AKTA Pure型制备型液相色谱仪；
纯化介质：Ni Sepharose excel亲和介质；缓冲液：分为A、B缓冲液，A液为1×PBS缓冲液，B泵为1×PBS+250mmol/L咪唑缓冲液；
系统上样流速：8mL/min；检测波长:UV@280nm
系统洗脱流速：4ml/min；检测波长:UV@280nm
洗脱条件：用20mM咪唑洗脱杂蛋白，收集250mM咪唑洗脱产物。将洗脱液透析于1×PBS过夜，期间更换两次透析液。收获约30ml低浓度目的蛋白，Vivaspin20ml，100KD超滤浓缩管浓缩至5ml备用。收取的洗脱样品制备还原及非还原样品，按上述方法进行SDS-PAGE凝胶电泳。
6、实验结果
图2显示了本发明制备的8种VHH-RBD蛋白的SDS聚丙烯酰胺凝胶电泳结果。图2A为通过昆虫细胞表达系统生产纯化的多种VHH-RBD融合蛋白的电泳结果。M:分子量Marker，1-8泳道分别显示为不同的VHH-RBD的融合蛋白。结果显示:VHH-RBD蛋白经过亲和层析一步纯化后，纯度约为90％左右，分子大小约为50KDa。图2B为通过哺乳动物细胞表达系统生产纯化的10种VHH-RBD融合蛋白的电泳结果。结果显示，VHH-RBD蛋白纯度约为90％，分子量大小约为50KDa左右。
图3显示了本发明制备的7种VHH-gE融合蛋白的SDS聚丙烯酰胺凝胶电泳结果。M:分子量Marker，1-7泳道分别显示为不同的VHH-gE的融合蛋白。结果显示:VHH-gE蛋白经过亲和层析一步纯化后，纯度约为80％左右，分子大小约为85KDa。
图4显示了本发明制备的8种VHH-BGTSTIP蛋白的SDS-聚丙烯酰胺凝胶电泳结果。“+”表示还原条件，“-”表示非还原条件。结果表明，VHH-BGTSTIP经过亲和层析以步纯化后，纯度约为90％左右，分子量大小约为180KD。
实施例3：VHH-RBD和VHH-gE蛋白免疫印记实验
将等量的蛋白质样品与载入缓冲液混合，煮沸10分钟，按照实验室标准方案载入SDS-PAGE凝胶用于western blotting(BioRad)，蛋白在BioRad迷你protean Tetra系统80V下电泳70分钟，凝胶在室温下用考马斯亮蓝R-250(Bio-Rad)染色30分钟。分离的蛋白质使用反式印迹涡轮转移系统(Bio-Rad)转移到硝化纤维素膜上，封闭后，与anti-His-HRP(1:5000稀释)孵育1小时。清洗5次，每次清洗5分钟，以去除未结合的抗体，然后使用化学发光底物试剂盒检测。
图5显示了本发明实施例的多种VHH-RBD的融合蛋白的免疫印迹实验(Western Blot)的结果。图5A为通过昆虫细胞表达系统生产纯化的8种VHH-RBD融合蛋白，M:分子量Marker，1-8分别泳道显示为不同纳米抗体与RBD的融合蛋白。结果证实：50KDa的分子确定为本发明的目的蛋白。图5B为通过哺乳动物细胞表达系统293F细胞生产纯化的10种VHH-RBD融合蛋白，结果显示，VHH-RBD融合蛋白纯度约为90％，分子量大小约为50KDa左右。结果证实：50KDa左右的分子确定为本发明的目的蛋白。
图6显示了本发明实施例的7种候选分子VHH-gE的蛋白免疫印迹实验(Western Blot)的结果。M:分子量Marker，1-7分别泳道显示为不同纳米抗体与gE的融合蛋白。结果证实：85KDa的分子确定为本发明的目的蛋白。
实施例4：融合蛋白分子活性分析(ELISA)
(1)将融合蛋白稀释至1μg/mL包被96孔板，每孔100μL，室温静置2h；
(2)洗板1次，用牛血清蛋白稀释液(ED，200μL/孔)室温封闭2h；
(3)洗板1次，将对应的特异性单克隆抗体稀释至1ug/ml,首孔加入100ul，2倍系列稀释11个梯度，双孔重复，室温放置1h；
(4)洗板5次，将二抗GAH-HRP(1:5000)加入96孔板，100μL/孔，室温放置1h；
(5)洗板5次，室温显色10min，终止，酶标仪450nm波长处检测；使用GraphPad Prism 5(GraphPad,USA)软件进行数据分析。
图7显示为实施例多种VHH-RBD与多种已报道特异性单克隆抗体的酶联免疫吸附实验(ELISA)结果。图7A为通过昆虫细胞表达系统生产纯化的8种VHH-RBD蛋白的活性鉴定结果，所使用的RBD抗体包括广谱中和抗体VacW-105(对应图7A中JSR-105)、JSR-551、VacW-209(对应图7A中JSR-209)、6D6(对应图7A中m6D6)、7D6(对应图7A中m7D6)(这些抗体的具体信息以及序列获自文献Ju B,Zheng Q,Guo H,Fan Q,Li T,Song S,Sun H,Shen S,Zhou X,Xue W,Cui L,Zhou B,Li S,Xia N,Zhang Z.Immune escape by SARS-CoV-2 Omicron variant and structural basis of its effective neutralization by a broad neutralizing human antibody VacW-209.Cell Res.2022 May；32(5):491-494.doi:10.1038/s41422-022-00638-6.Epub 2022 Mar 8.PMID:35260792；PMCID:PMC8902274.)。
结果证明：昆虫细胞表达系统生产纯化的VHH-RBD仍然能够保持完整的、正确 的分子构象，与RBD特异性抗体具有良好的结合活性。图7B为通过哺乳动物细胞表达系统293F细胞生产纯化的10种VHH-RBD蛋白的活性鉴定结果，所使用的抗体包括3G11、8H12、13F10、8B8、9D3、3F9(这些抗体为本实验室参考文献中常规抗体制备方法而制备的。简而言之，在293F细胞中制备表达纯化了SARS-CoV-2表面刺突蛋白S蛋白胞外段蛋白。然后进行BALB/c小鼠免疫，在免疫第0、2、3、5周采血进行血清检测。免疫结束后选取小鼠免疫血清结合滴度中和滴度较高的两只小鼠进行脾免，将10μg混合蛋白注射到小鼠脾脏后，缝合小鼠并定时观察，三天后进行细胞融合实验。融合培养后取细胞上清利用间接ELISA方法检测细胞上清和SARS-CoV-2 S-2P三聚体蛋白、RBD、S2蛋白以及SARS-CoV-1S-2P蛋白反应，选取与对应蛋白结合能力较强的孔进行挑孔和克隆化实验，经过三轮以上克隆化后获得单克隆抗体。免疫腹水小鼠，抽取腹水，用protein A柱子纯化获得相应的单克隆抗体。)。结果证明：哺乳动物细胞表达系统生产纯化的VHH-RBD仍然能够保持完整的、正确的分子构象，与RBD特异性抗体具有良好的结合活性。并且，这些融合蛋白与抗体(例如，8H12、13F10、8B8)的结合活性优于RBD蛋白。
图8显示为实施例7种VHH-gE与多种特异性单克隆抗体的酶联免疫吸附实验(ELISA)结果。所使用的gE特异性单克隆抗体包括3H7、4G4、6B7、11B11、11B12、13B6、14G1、17B7(这些抗体的具体信息以及序列获自文献Liu,J.,Ye,X.,Jia,J.et al.Serological Evaluation of Immunity to the Varicella-Zoster Virus Based on a Novel Competitive Enzyme-Linked Immunosorbent Assay.Sci Rep 6,20577(2016).https://doi.org/10.1038/srep20577)。结果证明：生产纯化的VHH-gE仍然能够保持完整的、正确的分子构象，与gE特异性抗体具有良好的结合活性。
图9显示为实施例多种VHH-BGTSTIP与多种报道的HIV-1中和抗体或非中和抗体的酶连免疫吸附实验(ELISA)结果。这些抗体通过293F细胞重组表达，抗体序列来源于NCBI(VRC01(GeneBank:MK032237.1/GU980703.1)、SF12(GeneBank:MK722171.1/MK722164.1)、2G12(GeneBank:OM484328.1/AF029237.1)、PGT121(GeneBank:JN201911.1/JN201894.1)或文章报道F105(Wilkinson,R.A.,C.Piscitelli,M.Teintze,et al.Structure of the Fab fragment of F105,a broadly reactive anti-human immunodeficiency virus(HIV)antibody that recognizes the CD4binding site of HIV type 1 gp120.J Virol,2005.79(20):13060-13069.)、F240(Gohain,N.,W.D.Tolbert,C.Orlandi,et al.Molecular basis for epitope recognition by non-neutralizing anti-gp41antibody F240.Sci  Rep,2016.6:36685.)、17b(Carlo D.Rizzuto,Richard Wyatt,Nivia Herna ndez-Ramos,et al.A Conserved HIV gp120Glycoprotein Structure Involved in Chemokine Receptor Binding.Science,1998.280(19):1949-1953.)。结果显示本发明的多种VHH-BGTSTIP融合蛋白与多种中和抗体均具有良好的结合活性，而与非中和抗体结合活性较弱。说明293F细胞表达的VHH-BGTSTIP良好地呈现了Env上地一些中和抗体表位，而没有暴露一些非中和抗体表位。
实施例5：融合蛋白与HEV-p239的亲和力分析(HPR)
Biacore 8K装载CM5芯片，使用PBS-P缓冲液(PBS+0.5％P20，cytiva)冲洗管路。
将配体HEV-p239(10ug/ml)高速离心10min。
设置HEV-p239偶联程序：芯片通道活化(EDC：NHS＝1:1，流速10ul/min)，然后执行冲洗程序(PBS-P缓冲液，流速30ul/min)。HEV-p239(氨基酸序列如SEQ ID NO:40所示)上样偶联(时间:420s，流速10ul/min)。确定配体偶联量后用乙醇胺进行通道封闭程序(时间：420s，流速：10ul/min)。
亲和力检测：设置浓度梯度为125nM、62.5nM、31.2nM、15.6nM、7.8nM、3.9nM的融合蛋白进行上样检测。样品结合时间为120s，解离时间为200s，流速为30ul/min。
使用Biacore 8K(GE)软件采用动力学方法进行结合(Ka)解离(Kd)曲线拟合，并分析计算亲和力(KD)。
图10显示为实施例多种融合蛋白VHH-RBD与HEV-p239蛋白的亲和力分析结果。图10A为通过昆虫细胞表达系统生产纯化获得的VHH-RBD与HEV-p239蛋白的亲和力结果，结果显示：多种VHH-RBD均与HEV-p239颗粒有纳摩尔级的高亲和力。图10B为通过哺乳动物细胞表达系统293F细胞生产纯化的VHH-RBD与HEV-p239蛋白的亲和力结果，结果显示VHH-RBD与HEV-p239颗粒有纳摩尔级高亲和力。
图11显示为实施例多种融合蛋白VHH-gE与HEV-p239蛋白的亲和力分析结果。结果显示：多种VHH-gE均与HEV-p239颗粒有纳摩尔级的高亲和力。
图12显示为实施例多种融合蛋白VHH-BGTSTIP与HEV-p239蛋白的亲和力分析结果。结果显示：多种VHH-BGTSTIP与HEV-p239颗粒有纳摩尔级的高亲和力。
实施例6：融合蛋白及其复合物的分子筛色谱纯化
仪器系统：GE Healthcare(原Amershan Pharmacia)公司生产的AKTA explorer 100型制备型液相色谱系统。
层析介质：Superdex 200 increase(cytiva)。
柱体积：20cm×20mm。
缓冲液：20mM磷酸缓冲液pH7.4。
流速：0.7mL/min。
检测器波长：280nm。
样品为实施例1和实施例5中的样品。
洗脱程序为：分段收集收集穿透峰。
收集Supedex 200 increase穿透的产物，获得纯化的样品5mL。取洗脱产物各50μL，加入6X Loading Buffer 10μL混匀，于80℃水浴10min后取10μl于10％SDS-聚丙烯酰胺凝胶中以120V电压电泳60min。随后以考马斯亮兰染色显示电泳条带。
图13A显示了本发明实施例中通过昆虫细胞表达系统生产纯化的融合蛋白P1-5B-RBD经过superdex 200 increase(高效液相色谱分子筛)纯化的结果。结果显示P1-5B-RBD蛋白纯度达到95％以上。图13B显示了本发明实施例中通过哺乳动物细胞表达系统生产纯化的P2-6D-RBD、P2-3E-RBD、P2-10G-RBD经过superdex 200 increase纯化的结果。结果显示三种融合蛋白均呈现单一洗脱峰，说明蛋白纯度和均一性高。
图14-16显示了本发明构建的多种融合蛋白与HEV-p239形成的复合物的纯化图谱和SDS-PAGE鉴定结果。其中，
图14A显示了昆虫细胞表达系统纯化的P1-5B-RBD融合蛋白与HEV-p239形成的复合物的纯化图谱和SDS-PAGE鉴定结果，紫色曲线显著高于蓝色曲线，证明融合蛋白P1-5B-RBD特异性的结合在HEV颗粒表面，形成了复合物。
图14B显示了本发明实施例候选融合蛋白哺乳动物细胞表达系统纯化的P2-3E-RBD、P2-10G-RBD和P2-6D-RBD分别与HEV-p239形成的复合物的纯化图谱和SDS-PAGE鉴定结果。结果表明P2-3E-RBD、P2-10G-RBD和P2-6D-RBD与HEV-p239形成了复合物，且能经由superdex 200 10/300 increase得以纯化。
图15显示为本发明实施例候选融合蛋白P2-8C-gE与HEV-p239形成的复合物纯化图谱和SDS-PAGE鉴定结果。图15A为复合物纯化结果。结果显示：复合物在superdex 200 increase(cytiva)层析柱的保留体积为8ml，显示为颗粒峰组分，14ml峰 为未结合的P2-8C-gE，说明8ml的颗粒峰组分已经结合。因此P2-8C-gE饱和结合在HEV颗粒表面形成了复合物。SDS-PAGE结果证实：复合物泳道呈现出典型的双组分条带(HEV-p239＝20KDa，P2-8C-gE＝85KDa)，因此经过superdex 200 increase纯化所得的样品确定为gE复合物样品。
图16显示了本发明实施例候选融合蛋白P2-5C-BGTSTIP与HEV-p239的复合物纯化图谱和SDS-PAGE鉴定结果。结果表明，复合物在superose 6层析柱中的保留体积为9ml左右，P2-5C-BGTSTIP的保留时间约为16ml，收取不同组分进行SDS-PAGE鉴定，9mL洗脱体积的样品在SDS-PAGE中呈现出两条带，确认复合物经superose 6纯化后，在9mL左右获得复合物样品。
综上，上述实验结果证明，本申请成功纯化并获得了多种候选融合蛋白与HEV-p239形成的复合物颗粒。
实施例7：P2-8C-gE蛋白的高效体积排阻色谱(HPSEC)分析
仪器：Waters。系统流速：G3000PWXL为0.5mL/min。波长190～600nm，柱波长为280nm和254nm。
缓冲液：PBS。
操作流程：预先平衡层析柱30～60min至280nm吸收值无明显的变化，将检测器的吸收值归零。编辑层析运行方法，样品先离心，将待分析样品注入100μL样品环中，设置自动上样，运行30min，观察S三聚体的保留时间约为14min。
结果如图17所示，本发明实施例的P2-8C-gE融合蛋白呈现单独主峰，无聚集体组分，保留时间约14min，纯度约80％。
实施例8：RBD复合物与gE复合物分子粒径检测(DLS)
仪器：NanoBrook Series(Brookhaven instrument)。
功能模块：DLS(Dynamic Light Scattering)。
缓冲液：PBS。
操作流程：提前开启仪器预热5分钟。准备待检测样品(浓度0.5mg/ml，12000rpm离心5分钟，取50ul加入样品杯中)。设置检测参数：检测时间：300s每次，每个样品3次重复。
图18显示了本发明实施例中通过昆虫细胞表达系统生产纯化的VHH-RBD与HEV- p239蛋白形成的HEV-RBD复合物样品的分子粒径检测结果。图18B为通过哺乳动物细胞表达系统生产纯化的VHH-RBD与HEV-p239蛋白形成的HEV-RBD复合物样品的分子粒径检测结果。图18A和图18B的结果显示：HEV-RBD的分子粒径明显大于HEV-p239颗粒的分子粒径。HEV-RBD复合物的粒径比HEV-p239的粒径明显增大，证实VHH-RBD能够结合在HEV颗粒表面，并保持颗粒状态。
图19显示了本发明实施例HEV-gE复合物样品的分子粒径检测结果。结果显示：HEV-p239颗粒的分子粒径为14.2nm，HEV-gE的分子粒径为38nm。结果证实：VHH-gE能够结合在HEV颗粒表面，并保持颗粒状态。
图20显示了本发明实施例HEV-BGTSTIP复合物样品的分子粒径检测结果。结果显示：HEV-p239颗粒的分子粒径为15.9nm，HEV-BGTSTIP复合物粒径为25.2nm。结果表明，VHH-BGTSTIP能结合在HEV颗粒表面，并保持颗粒状态。
实施例9：分析超离法计算样品沉降系数
所使用的仪器为Beckman XL-A分析型超速离心机，其配有光学检测系统及An-60Ti转头。
按照操作说明安装样品池，对照池加入400μL的样品缓冲液(与样品统一缓冲液)，样品池加入380μL的样品(OD280为0.8左右)，配平样品池，重量差在0.1g以内。
将样品池放入An-60Ti转头中，在将转头置于Beckman XL-A型分析超速离心机腔内，装上光路检测器。
参数设定：温度(20℃)、Rmin(6.0cm)、Rmax(7.2cm)、波长(280nm)、步进速度(0.003cm)、扫描模式(continuous)、数据间隔(30sec)和数据的数目(150scans)。设置离心转速为30000rpm。
实验结束后使用SENDTERP软件计算缓冲液的密度和粘度，以及已知蛋白的偏微比容。使用Origin版的Nonlin和SEDFIT软件进行沉降系数的分析，预设球蛋白的摩擦比f/f0＝1.2，根据样品蛋白分子量和基本性质设定分析范围，计算分辨率设为100，一般要求RMSD值不大于0.01且残基图波动在0.05以内。
图21显示了本发明实施例HEV-p239和基于HEV-p239颗粒构建的HEV-RBD复合物的分析超离结果。图21A为昆虫细胞表达系统生产纯化的VHH-RBD与HEV-p239制备的复合物颗粒(右图)以及HEV-p239(左图)的分析超离结果。结果显示： HEV-p239呈现单一组分，沉降系数为22S，HEV-RBD复合物呈现单一组分，沉降系数为27S，比HEV-p239颗粒的沉降系数(22S)明显增大。结果证实：RBD复合物能在水溶液状态中保持稳定的颗粒形态，RBD稳固结合在HEV颗粒表面形成了RBD颗粒蛋白。图21B为通过哺乳动物细胞表达系统293F细胞生产纯化的VHH-RBD与HEV-p239制备的复合物颗粒的分析超离结果，结果表明：HEV-RBD复合物呈现单一组分，且沉降系数比HEV-p239颗粒的沉降系数(22S)明显增大。证明其能在水溶液状态中保持稳定的颗粒形态，RBD稳固结合在HEV颗粒表面形成了RBD颗粒蛋白。
图22显示了本发明实施例HEV-p239(图22A)和基于HEV-p239颗粒构建的HEV-gE复合物(图22B)的分析超离结果。结果显示：HEV-p239呈现单一组分，沉降系数为22S，HEV-gE复合物呈现单一组分，沉降系数为31S，比HEV-p239颗粒的沉降系数(22S)明显增大。结果证实：HEV-gE复合物能在水溶液状态中保持稳定的颗粒形态，gE稳固结合在HEV颗粒表面形成了gE颗粒蛋白。
图23显示了本发明实施例HEV-p239和基于HEV-p239颗粒构建的Env(BGTSTIP)复合物的分析超离结果。
实施例10：透射电子显微镜颗粒形态观察
所使用的透射电子显微镜为Tecnai G2 Spirit 120kV(FEI)。采用磷钨酸进行负染。
样品制备：先对铜网(R2/2，200目，ThermoFisher Scientific)进行辉光放电亲水处理。然后将5ul浓度为0.5mg/ml的样品滴加在铜网上，室温放置60s后用吸水纸从铜网边缘吸取液滴。置于室温晾干后上机观察。
图24A显示了本发明实施例HEV-p239和通过昆虫细胞表达系统生产纯化的VHH-RBD制备的复合物样品的透射电子显微镜负染结果。图24B显示了本发明实施例HEV-p239和通过哺乳动物表达系统293F细胞生产纯化的VHH-RBD制备的复合物样品的透射电子显微镜负染结果。结果均显示：HEV-RBD复合物呈现典型的病毒样颗粒形态。结果证实：本申请构建的HEV-RBD复合物样品为RBD颗粒化抗原。
图25显示为本发明实施例HEV-gE复合物的透射电子显微镜负染结果。结果显示：HEV-gE复合物呈现典型的病毒样颗粒形态。结果证实：本申请构建的HEV-gE复合物样品为颗粒化抗原。
图26显示了本发明实施例HEV-Env(BGTSTIP)复合物的透射电子显微镜负染 结果。结果显示，HEV-Env复合物呈现典型的病毒样颗粒形态，证实本申请构建的HEV-Env复合物样品为颗粒化抗原。
实施例11：HEV-RBD颗粒免疫原性评价
本实验方案经厦门大学实验动物管理伦理委员会批准。所有操作都严格按照动物伦理准则和批准的规程进行。
昆虫细胞表达系统的RBD颗粒免疫原性评价
选用6周龄的Balb/C小鼠，将其分为4组，每组5只，每组分别使用HEV-RBD颗粒(免疫剂量为0.5μg)、HEV-RBD颗粒(免疫剂量为5μg)、P1-5B-RBD单体(免疫剂量为0.5μg)以及P1-5B-RBD单体(免疫剂量为5μg)并结合铝佐剂进行免疫，分别于0周、2周和6周肌肉注射(50μL)小鼠的左或右后肢。分别于第0周、1周、2周、3周、4周、5周、6周、7周和8周采集眼球静脉血，其中第0周、1周和4周在注射前采血。血液样本13000g离心10分钟，得到血清样本在-20℃保存。用终点酶联免疫吸附法和野生型SARS-CoV-2假病毒(构建方法参考文献Xiong HL,Wu YT,Cao JL,Yang R,Liu YX,Ma J,Qiao XY,Yao XY,Zhang BH,Zhang YL,Hou WH,Shi Y,Xu JJ,Zhang L,Wang SJ,Fu BR,Yang T,Ge SX,Zhang J,Yuan Q,Huang BY,Li ZY,Zhang TY,Xia NS.Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus(VSV)pseudovirus and ACE2-overexpressing BHK21 cells.Emerg Microbes Infect.2020 Dec；9(1):2105-2113.doi:10.1080/22221751.2020.1815589.PMID:32893735；PMCID:PMC7534347.)中和法分别测定抗原特异性IgG和中和抗体滴度。
哺乳动物表达系统的RBD颗粒免疫原性评价
小白鼠：雌性，6周龄，购自北京维通利华实验动物技术有限公司。设置十二组免疫小鼠，每组5只小鼠。将P2-3E-RBD、P2-10G-RBD、P2-6D-RBD与P239制备颗粒复合物抗原。用生理盐水稀释至所需浓度，按体积比1：1的比例与铝佐剂混合并于4度过夜使蛋白吸附于佐剂上，于0、2、5周对小鼠进行肌肉免疫(100uL，左右后腿各50μL)。小鼠免疫方案设置如表4。
表4：小鼠免疫方案

每周采集小鼠眼球血，九周后对小鼠进行二氧化碳安乐死。血样于37℃放置30min后，13300rpm离心10min，收集血清保存于-20度用于野生型SARS-CoV-2假病毒中和及结合抗体滴度测定。
结果如图27和图28所示，图27显示为本发明实施例HEV-RBD颗粒抗原的免疫原性检测结果。图27A为通过昆虫细胞表达系统获得的HEV-RBD颗粒化抗原免疫小鼠后的血清结合及中和活性检测结果。图27B及图27C为通过哺乳动物细胞表达系统获得的HEV-RBD颗粒化抗原免疫小鼠后的血清结合活性及对野生型SARS-CoV-2假病毒的中和活性检测结果，图27D为哺乳动物细胞表达系统获得HEV-RBD颗粒化抗原免疫仓鼠后的血清抗体结合滴度。
图28显示为本发明实施例HEV-RBD颗粒抗原配伍铝佐剂诱导产生的抗体与新冠野生型(WT)毒株、Gamma毒株、BA.2毒株的中和检测结果。
结果显示，昆虫细胞表达的VHH-RBD或哺乳动物细胞表达的VHH-RBD展示在p239颗粒表面能显著提高RBD抗原的免疫原性。
具体来说，HEV-RBD颗粒(图中显示为RBD CPX)5ug免疫组在首针免疫后即可快速建立免疫屏障，产生约3log结合抗体滴度。加强免疫后结合抗体滴度达到5log，比P1-5B-RBD单体产生的结合抗体滴度高500倍，并在第八周没有明显降低。在小鼠免疫实验中，HEV-RBD诱导的结合抗体滴度显著高于单体RBD蛋白。在免疫剂量为0.5μg/只时，P2-3E-RBD-p239、P2-10G-RBD-p239和P2-6D-RBD-p239复合物颗粒能诱导出相对更高的结合抗体滴度(相较于对应的VHH-RBD蛋白)，其中P2-3E-RBD-p239、P2-10G-RBD-p239复合物免疫组在第五针前的结合抗体滴度明显高于对应的VHH-RBD免疫组的结合抗体滴度(图27B)。第六周的假病毒中和结果表明，三种VHH-RBD与p239形成的复合物颗粒均能诱导出更显著的中和抗体应答(图27C)。
并且，HEV-RBD颗粒的中和抗体具有广谱中和能力，能够有效中和包括新冠野生毒株、Gamma毒株、BA.2毒株在内的假病毒(VSV-Spike)。
实施例12：gE颗粒免疫原性评价
本实验方案经厦门大学实验动物管理伦理委员会批准。所有操作都严格按照动物伦理准则和批准的规程进行。
选用6周龄的Balb/C小鼠，将其分为8组，每组5只，每组分别使用HEV-gE颗 粒(免疫剂量为0.1μg)，HEV-gE颗粒(免疫剂量为0.5μg)，HEV-gE颗粒(免疫剂量为5μg)，P2-8C-gE单体(免疫剂量为0.1μg)，P2-8C-gE单体(免疫剂量为0.5μg)，P2-8C-gE单体(免疫剂量为5μg)，并结合铝佐剂进行免疫，分别于0周和2周肌肉注射(50μL)小鼠的左或右后肢。分别于第0周、1周、2周、3周、4周、5周、6周、采集眼球静脉血。血液样本13000g离心10分钟，得到血清样本在-20℃保存。用终点酶联免疫吸附法和减毒病毒(v-Oka毒株)中和法分别测定抗原特异性IgG和中和抗体滴度。
中和操作流程(ELISPOT法)：
1、豚鼠血清(购自北京博尔西科技有限公司，货号：BM361Y)和v-Oka病毒(ATCC，货号VR-795))干粉用病毒保护液复溶，其中补体用0.22μm小滤器过滤备用；
2、用病毒保护液将血清稀释50倍加入24孔板的首孔，2倍系列稀释4个梯度，与vOka病毒37℃共孵育1h；
3、将血清病毒混合液转至预先铺好ARPE-19细胞的24孔板，37℃共孵育1h，1h后弃掉液体补加F12培养基，37℃培养3天；
4、3天后，弃掉培养基，PBS清洗1次；固定液室温固定5min，弃掉固定液，通透液室温通透10min；
5、将一抗1B11-HRP(1:2000)加入24孔板，37℃放置1h；
6、洗板5次，室温显色5min，酶联斑点图像分析系统进行读点和数点；使用GraphPad Prism 5(GraphPad,USA)软件进行数据分析。
图29显示为本发明实施例gE颗粒抗原的免疫原性检测结果。gE颗粒抗原搭配AS01B类似佐剂可以诱导高滴度的结合抗体(图中显示为XUA，最高约6log)。
图30显示为本发明实施例HEV-gE颗粒抗原的小鼠免疫血清活病毒中和检测结果，HEV-gE颗粒抗原诱导的中和抗体是P2-8c-gE单体的2.8倍。结果证实在p239颗粒表面表达能显著提高gE抗原的免疫原性。
实施例13：gE颗粒抗原免疫组流式细胞术检测细胞因子
按照实施例6所述的方法免疫小鼠(相同佐剂)，后续实验操作如下：
a)取脾：颈椎脱臼处死小鼠，置于75％乙醇浸泡3-5min后，小鼠右躺，无菌取出脾脏(尽量去除脂肪)；
b)研磨：6孔细胞板放入筛网，加入含10％FBS的1640培养基，将脾脏置于筛网中(完全浸入培养基中)，用2mL注射器头研磨至看不见红色组织，将研磨后的细胞 转移至50mL管中，置于冰上；
c)400g，4℃离心5min，弃上清，止血钳敲击管底细胞沉淀松散；
d)细胞沉淀加入10mL预冷的RBC溶液，重悬，静置冰上裂解5min(期间颠倒混匀)；
e)400g，4℃离心5min，弃上清，止血钳敲击管底细胞沉淀松散；
f)细胞沉淀加入10mL预冷的培养基重悬，洗去红细胞沉淀或脂肪，吹匀后取50uL计数；
g)400g，4℃离心5min，弃上清，止血钳敲击管底细胞沉淀松散，加入一定量的培养基，稀释细胞数至2×107个/mL；
h)铺板：96孔U底板，每孔铺200μL(2～4×106个细胞)，400g，4℃离心5min，去上清；
i)加入100μL含有多肽((gE/gI)重叠多肽，用FACS溶液稀释，多肽终浓度为2μg/mL)的培养基重悬细胞，刺激18h；
j)加入20μL高尔基抑制剂(1：1000，用培养基稀释)，孵育6h；
k)400g，4℃离心2min，去上清，加入200μL FACS溶液(1×PBS+10％FBS)重悬细胞，离心，去上清；
(以下所有操作避光且保持在4℃的条件下进行)
l)细胞表面染色：用FITC偶联的抗小鼠CD4抗体(购自Biolegend，货号：100510)，PE-Cy7偶联的抗小鼠CD8α抗体(购自Biolegend，货号：100722)，以及LIVE/DEADTM可修复Aqua死细胞染色试剂(购自Invitrogen，货号：L34966)进行染色，每孔加入40μL surface(AQUA/CD4/CD8抗体，用FACS溶液稀释)，重悬细胞，避光4℃处理30～60min，加入200μL FACS溶液吹吸7～8次；
m)固定破膜：2000rpm，4℃离心2min，去上清，每孔加入75μL Fixation/Permeabilization溶液重悬细胞，避光4℃处理60min；
n)2000rpm，4℃离心2min，去上清；
o)每孔加入200μL 1×BD Perm/Wash溶液重悬细胞，2000rpm，4℃离心2min，去上清；
p)细胞内染色：用PE偶联的抗小鼠IL-2抗体(购自BD，货号：554428)和APC偶联的抗小鼠IFN-γ抗体(购自BD，货号：554413)进行染色，每孔加入50μL荧光抗体(IL-2/IFN-γ抗体，用1×BD Perm/Wash溶液稀释)重悬细胞，避光4℃处理 60min，加入200μL Perm Buffer，重悬细胞；
q)2000rpm，4℃离心2min，去上清，每孔加入200μL 1×BD Perm/Wash溶液重悬细胞；
r)处理好的细胞，用200目筛网过滤处理(将筛网条铺在孔的表面，排枪吸取细胞悬液垂直缓慢加入)，转移到流式细胞管中；
s)使用BD LSRFortessa X-20流式细胞仪测量样品，并通过FlowJo V10分析数据。
实验结果如图31所示，结果表明gE颗粒抗原激活的IFN-gamma阳性的CD4细胞是P2-8C-gE单体的1.65倍，激活的IFN-gamma阳性的CD8细胞是P2-8C-gE单体的8.42倍。结果证实：gE颗粒抗原在细胞免疫反应方面更有优势。
实施例14：gE颗粒抗原免疫组酶联免疫斑点检测细胞因子
在第8周取脾进行流式和Elispot检测T细胞反应，其中，流式检测方法如实施例6，ELISPOT检测细胞因子流程(所用试剂盒购自MABTECH，货号：3321-4HPW-10、3441-4HPW-10)如下：
(1)分离脾细胞，然后每孔铺50万个细胞，离心去上清，加入100μL gE多肽(0.15ug/100ul)的培养基重悬细胞，37℃刺激20h；
(2)弃掉培养基，无菌PBS清洗5次，将检测抗体R4-6A2-biotin(IFN-γ)和5H4-biotin(IL-2)稀释至1μg/mL，每孔加入100μL，室温孵育2h；
(3)无菌PBS清洗5次，每孔加入Streptavidin-ALP(1:1000)100μL，室温孵育1h；
(4)无菌PBS清洗5次，每孔加入底物溶液(BCIP/NBT-plus)100μL进行显色，直至斑点出现，洗板终止；
(5)将板扣干，酶联斑点图像分析系统进行读点和数点；使用GraphPad Prism 5(GraphPad,USA)软件进行数据分析。
结果如图32所示，结果证实：gE颗粒抗原激活的T细胞免疫反应显著高于P2-8C-gE单体蛋白。
实施例15：P1-5B纳米抗体人源化改造
使用在线软件对P1-5B纳米抗体(P1-5B纳米抗体全长氨基酸序列如SEQ ID NO:12所示，CDR1-3的序列如SEQ ID NO:57-59所示)进行CDR比对(IMGT/DomainGapAlign；http://www.bioinf.org.uk/abs/abnum/)。使用在线软件进 行同源序列比对检索(https://www.imgt.org/IMGT_vquest/input)，选择得分最高的人源序列进行CDR移植。选定的序列由生工合成，构建到PCDNA3.1载体。使用无内毒素质粒大提试剂盒大量制备质粒(天跟公司)。然后采用PEI瞬转方式转染293FT细胞进行人源化纳米抗体表达。共获得了两个人源化纳米抗体，分别命名为H1和H2，人源化纳米抗体全长氨基酸序列如SEQ ID NO:71和72所示，CDR1-3的序列如SEQ ID NO:65-67所示。
PEI瞬转HEK239FT细胞
PCDNA3.1-H1-RBD、PCDNA3.1-H2-RBD质粒(质粒分别含有编码融合蛋白H1-RBD和H1-RBD的核苷酸序列，其中，H1-RBD和H1-RBD的氨基酸序列如SEQ ID NO:63和64所示)PEI分别稀释于5ml的无血清CD05培养基中，并将两者充分混匀静置18min形成质粒PEI复合物，以PEI:质粒＝2：1的比例转染HEK293FT细胞，待转染6h后更换新鲜的CD05培养基，37℃，5％CO2恒温培养48h后添加100mlCD05补料培养基，96h后收获上清进行纯化，然后通过酶联免疫反应检测人源化纳米抗体的亲和力(ELISA参见实施例4)。
结果如图33所示：经改造后的人源化纳米抗体具有和亲本等同的分子活性。其中，H1-RBD和H2-RBD是经改造后的人源化纳米抗体所形成的RBD融合蛋白，RBD-WT是野生的RBD蛋白。
实施例16：人源化纳米抗体制备的HEV-Env颗粒抗原免疫原性评价
小白鼠：雌性，6周龄，购自上海斯莱克实验动物有限责任公司。设置六组免疫小鼠，每组5只小鼠。本实施例使用P2-5C纳米抗体(P2-5C纳米抗体氨基酸序列如SEQ ID NO:21所示，CDR1-3的序列如SEQ ID NO:60-62所示)。将制备的复合物颗粒及P2-5C-BGTSTIP抗原用生理盐水稀释，按体积比1：1的比例与铝佐剂混合，使蛋白吸附于佐剂上，对小鼠进行肌肉免疫。小鼠免疫方案设置如表5。
表5：小鼠免疫方案
在免疫前采集小鼠眼球血，前述的免疫方案进行免疫，每次免疫前采集小鼠眼球 血，免疫至第六针后采集眼球血后对小鼠进行断颈处理。血样于37℃放置30min后，13300rpm离心10min，收集血清用于HIV-1假病毒中和和抗体滴度测定。
我们采集0-6针次的免疫血清进行了病毒中和实验。
实施例17:P1F8-BGTSTIP融合蛋白的表达及鉴定
P1F8-BGTSTIP融合蛋白设计及表达
针对HPV L1蛋白(SEQ ID NO:71)筛选纳米抗体，获得了纳米抗体P1F8(SEQ ID NO:88)，在该抗体序列后添加(GGGGS)3柔性接头，随后偶联到BGTSTIP序列前，构建的P1F8-BGTSTIP融合蛋白的氨基酸序列如SEQ ID NO:89所示。提取P1F8-BGTSTIP质粒，-80℃冰箱取冻存的293F细胞，37℃化冻后1300rpm离心4min，于超净台内弃上清，轻弹细胞并用提前37℃温育的293freestyle培养基重悬后，转移至含50mL温育培养基的三角瓶中，37℃5％CO2 120转/min悬浮培养，待细胞密度达2.0*106时进行传代，逐渐扩大培养体系。待细胞足够量时，采用PEI(MW 25,000)瞬时转染293F细胞，无菌50mL管收取细胞，1300rpm离心4min，轻弹细胞后37℃温育培养基重悬，并转移至含有450mL 37℃温育培养基的三角瓶中，置于37℃摇床备用。
将提取的P1F8-BGTSTIP质粒与PEI(MW 25,000)按1：2比例置于50ml培养基中充分混匀后静置18min，转移至上述450ml培养基中，37℃5％CO2 120转/min悬浮培养6天，进行P1F8-BGTSTIP蛋白的表达。转染过程中注意PEI的避光操作。
P1F8-BGTSTIP融合蛋白的纯化
瞬时转染6天后，收取细胞培养液，JA-14转头7000g离心10min后取细胞上清，20000g离心10min后取上清0.22um孔径滤膜过滤两次，以该样品进行下一步Ni-excel柱纯化。
利用AKTA系统进行Ni亲和层析纯化；
仪器系统：AKTA Pure型制备型液相色谱仪；
纯化介质：Ni Sepharose excel亲和介质；缓冲液：分为A、B缓冲液，A液为1×PBS缓冲液，B泵为1×PBS+250mmol/L咪唑缓冲液；
系统上样流速：8mL/min；检测波长:UV@280nm
系统洗脱流速：4ml/min；检测波长:UV@280nm
洗脱条件：用20mM咪唑洗脱杂蛋白，收集250mM咪唑洗脱产物。将洗脱液透析于1×PBS过夜，期间更换两次透析液。收获约30ml低浓度目的蛋白，Vivaspin20ml， 100KD超滤浓缩管浓缩至5ml备用。收取的洗脱样品制备还原及非还原样品，按上述方法进行SDS-PAGE凝胶电泳。图34显示了P1F8-BGTSTIP蛋白的SDS聚丙烯酰胺凝胶电泳结果；M:分子量Marker；“+”表示还原SDS-PAGE；“-”表示非还原SDS-PAGE。结果显示，P1F8-BGTSTIP分子在还原条件下分子量约为160KD，在非还原条件下为多聚体，与理论分子量相符。
P1F8-BGTSTIP蛋白分子活性分析(ELISA)
将融合蛋白稀释至1μg/mL包被96孔板，每孔100μL，室温静置2h；
(2)洗板1次，用牛血清蛋白稀释液(ED，200μL/孔)室温封闭2h；
(3)洗板1次，将对应的特异性单克隆抗体稀释至1ug/ml,首孔加入100ul，3倍系列稀释，双孔重复，室温放置1h；
(4)洗板5次，将二抗GAH-HRP(1:5000)加入96孔板，100μL/孔，室温放置1h；
(5)洗板5次，室温显色10min，终止，酶标仪450nm波长处检测；使用GraphPad Prism 5(GraphPad,USA)软件进行数据分析。
图35显示了P1F8-BGTSTIP的酶联免疫吸附实验结果，结果表明，P1F8与VRC01、PGT121、PGT122(JN201912.1/JN201895.1)、2G12、SF162、B12这些中和抗体具有良好的结合活性，而与17b、F105及F240的结合活性很弱，说明P1F8-BGTSTIP很好地暴露了广谱中和抗体表位，而没有暴露非中和表位。
P1F8-BGTSTIP与HPV 58VLP的亲和力分析(SPR)
装载CM5芯片，使用PBS-P缓冲液PBS冲洗管路。
将配体58-VLP(10ug/ml)高速离心10min。
设置58-VLP偶联程序：芯片通道活化(EDC：NHS＝1:1，流速10ul/min)，然后执行冲洗程序(PBS缓冲液，流速30ul/min)。58VLP上样偶联(时间:420s，流速10ul/min)。确定配体偶联量后用乙醇胺进行通道封闭程序(时间：420s，流速：10ul/min)。
亲和力检测：设置浓度梯度为800nM、400nM、200nM、100nM、50nM、25nM的融合蛋白进行上样检测。样品结合时间为120s，解离时间为300s，流速为30ul/min。
使用Biacore 8K(GE)软件采用动力学方法进行结合(Ka)解离(Kd)曲线拟合，并分析计算亲和力(KD)。
图36显示了P1F8-BGTSTIP与58VLP的亲和力测定结果，结果显示，P1F8- BGTSTIP与融合蛋白的亲和力为4.66×10-8，表明两者能结合，且结合较牢固。
实施例18：HPV-Env融合蛋白(P1F8-BGTSTIP)与HPV 58-VLP颗粒复合物的制备及鉴定
P1F8-BGTSTIP复合物制备
P1F8-BGTSTIP和58-VLP按质量比5:1的比例在37℃水浴锅中孵育30min，13300rpm离心10min后，进行高效体积排阻色谱(HPSEC)分析。
仪器：Waters。系统流速：G5000PWXL为0.5mL/min。波长190～600nm，柱波长为280nm和254nm。
缓冲液：终缓。
操作流程：预先平衡层析柱60min至280nm吸收值无明显的变化，将检测器的吸收值归零。编辑层析运行方法，将待分析样品注入100μL样品环中，设置自动上样，运行30min。图37(左)显示了单独的HPV 58VLP、P1F8-BGTSTIP、BGTSTIP以及P1F8-BGTSTIP-58VLP复合物的HPSEC图谱。结果显示复合物的出峰时间为12min，相较于58VLP提前。我们收取复合物组分，进行了SDS-PAGE凝胶电泳。结果显示，收取的12min的样品显示了P1F8-BGTSTIP和58-VLP两个分子量大小的条带，说明P1F8-BGTSTIP与58VLP成功形成复合物，出峰时间约为12min左右。
P1F8-BGTSTIP-58VLP颗粒化抗原的分析超离(AUC)
将通过上述纯化过程收取的P1F8-BGTSTIP-58VLP复合物进行AUC分析。
所使用的仪器为Beckman XL-A分析型超速离心机，其配有光学检测系统及An-60Ti转头。
按照操作说明安装样品池，对照池加入400μL的样品缓冲液(与样品统一缓冲液)，样品池加入380μL的样品(OD280为0.8左右)，配平样品池，重量差在0.1g以内。
将样品池放入An-60Ti转头中，在将转头置于Beckman XL-A型分析超速离心机腔内，装上光路检测器。
参数设定：温度(20℃)、Rmin(6.0cm)、Rmax(7.2cm)、波长(280nm)、步进速度(0.003cm)、扫描模式(continuous)、数据间隔(30sec)和数据的数目(150scans)。设置复合物离心转速为7000rpm，融合蛋白离心转速为30000rpm。
实验结束后使用SENDTERP软件计算缓冲液的密度和粘度，以及已知蛋白的偏微 比容。使用Origin版的Nonlin和SEDFIT软件进行沉降系数的分析，预设球蛋白的摩擦比f/f0＝1.2，根据样品蛋白分子量和基本性质设定分析范围，计算分辨率设为100，一般要求RMSD值不大于0.01且残基图波动在0.05以内。
图38显示了本发明P1F8-BGTSTIP与58VLP复合物的分析超离结果。结果显示：58VLP呈现单一组分，沉降系数为119S(图38右)，P1F8-BGTSTIP-58VLP复合物呈现单一组分，沉降系数为176.3S(图38左)，比58VLP颗粒的沉降系数明显增大。结果表明：1F8-BGTSTIP能与58VLP形成颗粒复合物，并在水溶液状态中保持稳定的颗粒形态。
P1F8-BGTSTIP-58VLP颗粒化抗原的透射电子显微镜形态观察
所使用的透射电子显微镜为Tecnai G2 Spirit 120kV(FEI)。采用磷钨酸进行负染。
样品制备：先对铜网(R2/2，200目，ThermoFisher Scientific)进行辉光放电亲水处理。然后将5ul浓度为0.5mg/ml的样品滴加在铜网上，室温放置60s后用吸水纸从铜网边缘吸取液滴。置于室温晾干后上机观察。
图39显示了复合物颗粒在透色电子显微镜下的形态，可明显看到颗粒表面覆盖着一层蛋白，说明P1F8-BGTSTIP成功展示在了HPV 58VLP颗粒表面。
P1F8-BGTSTIP-58VLP颗粒抗原免疫原性评估
本实验方案经厦门大学实验动物管理伦理委员会批准。所有操作都严格按照动物伦理准则和批准的规程进行。
选用6周龄的Balb/C小鼠，将其分为6组，每组5只，高低剂量组各3组，分别使用P1F8-BGTSTIP-58VLP颗粒、P1F8-BGTSTIP、BGTSTIP免疫原结合铝佐剂进行免疫，每种抗原设置0.5ug和5ug两种免疫剂量。于0、2、4、6周对小鼠进行肌肉免疫(100uL/只)。
在免疫前采集小鼠眼球血，每次免疫前采集小鼠眼球血，免疫至第六针后采集眼球血后对小鼠进行断颈处理。血样于37℃放置30min后，13300rpm离心10min，收集血清用于特异性结合抗体滴度测定。
图40显示为本发明实施例P1F8-BGTSTIP-VLP颗粒抗原的免疫原性检测结果。图40显示为BGTSTIP特异性的结合抗体滴度检测结果(左图为0.5μg组，右图为5μg组)。结果表明，在第1-4周，复合物颗粒的结合滴度显著高于单独的BGTSTIP和单独的P1F8-BGTSTIP蛋白。
实施例19：基于乙肝颗粒化抗原HBsAg VLP的纳米抗体融合蛋白设计和评估
本实施例根据已上市的乙肝疫苗的颗粒化抗原SHBs蛋白(SEQ ID NO:73)制备VLP载体，并按照前述实施例的方法相应制备gE的融合蛋白，进一步制备HBV-gE颗粒化抗原，并测试颗粒化抗原的免疫原性。具体实验过程如下：
gE融合蛋白设计及表达
选取已报道的HBsAg的纳米抗体(1.Serruys,B.,Houtte,F.V.,Verbrugghe,P.,Leroux-Roels,G.&Vanlandschoot,P.Llama-derived single-domain intrabodies inhibit secretion of hepatitis B virions in mice.Hepatology 49,39-49(2009).)进行实验，在该抗体序列后添加(GGGGS)3柔性接头，随后偶联到RBD和gE的序列前，构建了RBD和gE融合蛋白。提取质粒，-80℃冰箱取冻存的293F细胞，37℃化冻后1300rpm离心4min，于超净台内弃上清，轻弹细胞并用提前37℃温育的293freestyle培养基重悬后，转移至含50mL温育培养基的三角瓶中，37℃5％CO2 120转/min悬浮培养，待细胞密度达2.0*106时进行传代，逐渐扩大培养体系。待细胞足够量时，采用PEI(MW 25,000)瞬时转染293F细胞，无菌50mL管收取细胞，1300rpm离心4min，轻弹细胞后37℃温育培养基重悬，并转移至含有450mL 37℃温育培养基的三角瓶中，置于37℃摇床备用。
将提取的质粒与PEI(MW 25,000)按1：2比例置于50ml培养基中充分混匀后静置18min，转移至上述450ml培养基中，37℃5％CO2 120转/min悬浮培养6天，进行gE融合蛋白的表达。转染过程中注意PEI的避光操作。
gE融合蛋白的纯化
瞬时转染6天后，收取细胞培养液，JA-14转头7000g离心10min后取细胞上清，20000g离心10min后取上清0.22um孔径滤膜过滤两次，以该样品进行下一步Ni-excel柱纯化。
利用AKTA系统进行Ni亲和层析纯化；
仪器系统：AKTA Pure型制备型液相色谱仪；
纯化介质：Ni Sepharose excel亲和介质；缓冲液：分为A、B缓冲液，A液为1×PBS缓冲液，B泵为1×PBS+250mmol/L咪唑缓冲液；
系统上样流速：8mL/min；检测波长:UV@280nm
系统洗脱流速：4ml/min；检测波长:UV@280nm
洗脱条件：用20mM咪唑洗脱杂蛋白，收集250mM咪唑洗脱产物。将洗脱液透析 于1×PBS过夜，期间更换两次透析液。收获约30ml低浓度目的蛋白，Vivaspin20ml，100KD超滤浓缩管浓缩至5ml备用。收取的洗脱样品制备样品，按上述方法进行SDS-PAGE凝胶电泳。
实验结果显示，获得了表达量较高的纳米抗体的融合蛋白，将其命名为S2-gE。如图41所示，SDS聚丙烯酰胺凝胶电泳结果显示S2-gE与理论分子量相符。
融合蛋白及其复合物的分子筛色谱纯化
仪器系统：GE Healthcare(原Amershan Pharmacia)公司生产的AKTA explorer100型制备型液相色谱系统。
层析介质：Superdex 200 increase(cytiva)。
柱体积：20cm×20mm。
缓冲液：20mM磷酸缓冲液pH7.4。
流速：0.7mL/min。
检测器波长：280nm。
样品为实施例1和实施例5中的样品。
洗脱程序为：分段收集收集穿透峰。
收集Supedex 200 increase穿透的产物，获得纯化的样品5mL。取洗脱产物各50μL，加入6X Loading Buffer 10μL混匀，于80℃水浴10min后取10μl于10％SDS-聚丙烯酰胺凝胶中以120V电压电泳60min。随后以考马斯亮兰染色显示电泳条带。
如图42所示，SDS聚丙烯酰胺凝胶电泳结果显示HBsAg与HBsAg-S2-gE(HBsAg和S2-gE的复合物峰)吸收不同的峰，HBsAg-S2-gE的颗粒峰变大且保留时间提前。表明S2-gE可与HBsAg形成颗粒化抗原。
HBV-gE复合物分子粒径检测(DLS)
仪器：NanoBrook Series(Brookhaven instrument)。
功能模块：DLS(Dynamic Light Scattering)。
缓冲液：PBS。
操作流程：提前开启仪器预热5分钟。准备待检测样品(浓度0.5mg/ml，12000rpm离心5分钟，取50ul加入样品杯中)。设置检测参数：检测时间：300s每次，每个样品3次重复。
如图43所示，DLS结果显示单独的HBsAg与HBsAg-S2-gE的复合物粒径不同， HBsAg-S2-gE(HBV-gE)的粒径明显大于HBsAg，表明形成gE颗粒化抗原。
透射电子显微镜颗粒形态观察
所使用的透射电子显微镜为Tecnai G2 Spirit 120kV(FEI)。采用磷钨酸进行负染。
样品制备：先对铜网(R2/2，200目，ThermoFisher Scientific)进行辉光放电亲水处理。然后将5ul浓度为0.5mg/ml的样品滴加在铜网上，室温放置60s后用吸水纸从铜网边缘吸取液滴。置于室温晾干后上机观察。
如图44所示，负染结果显示单独的HBsAg与HBsAg-S2-gE的复合物大小形态不同，表明形成gE颗粒化抗原。
gE颗粒免疫原性评价
本实验方案经厦门大学实验动物管理伦理委员会批准。所有操作都严格按照动物伦理准则和批准的规程进行。
选用6周龄的Balb/C小鼠，将其分为2组，每组3只，每组分别使用前述制备的HBsAg-gE复合物和单独的S2-gE进行免疫，其中gE剂量同为5ug，分别于0周和2周肌肉注射(50μL)小鼠的左或右后肢。分别于第0周、1周、2周、3周、4周采集眼球静脉血。血液样本13000g离心10分钟，得到血清样本在-20℃保存。用终点酶联免疫吸附法测定抗原特异性IgG滴度。
如图45所示，针对免疫两针后的gE特异性滴度结果显示，颗粒化抗原HBsAg-S2-gE可以诱导比单独S2-gE显著更高的抗体水平。显示出免疫增强效果。

Claims (22)

  1. 一种融合蛋白，其包含免疫原性多肽和能够特异性结合病毒样颗粒(VLP)的纳米抗体；
    优选地，所述VLP是由组装多肽组装成的VLP。
  2. 如权利要求1所述的融合蛋白，其中，所述组装多肽是能够组装成VLP的多肽；
    优选地，所述组装多肽是天然病毒或类病毒的外壳蛋白，或者，是人工制备和/或筛选的多肽。
    优选地，所述组装多肽选自戊型肝炎病毒(HEV)的蛋白或其片段或它们的变体，乙型肝炎病毒(HBV)的蛋白或其片段或它们的变体，人乳头瘤病毒(HPV)的蛋白或其片段或它们的变体，或其任意组合；其中，所述片段或变体保留了组装成VLP的能力；
    优选地，所述组装多肽为HEV的ORF2蛋白或其片段或它们的变体；优选地，所述ORF2蛋白的片段选自p239蛋白，p495蛋白；
    优选地，所述组装多肽选自p239蛋白或其片段或它们的变体，p495蛋白或其片段或它们的变体；
    优选地，所述变体与其所源自的蛋白的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述置换是保守置换；
    优选地，所述ORF2蛋白具有如SEQ ID NO:9所示的氨基酸序列；优选地，所述p239蛋白具有如SEQ ID NO:40所示的氨基酸序列；优选地，所述p495蛋白具有如SEQ ID NO:41所示的氨基酸序列；
    优选地，所述组装多肽为HPV的衣壳蛋白L1或其片段或它们的变体；
    优选地，所述组装多肽具有如SEQ ID NO:74所示的氨基酸序列；
    优选地，所述组装多肽为HBV的表面蛋白(例如，乙型肝炎病毒表面抗原)或其片段或它们的变体；
    优选地，所述组装多肽选自乙型肝炎病毒表面抗原(HBsAg)的LHBs蛋白或其片段或它们的变体，MHBs蛋白或其片段或它们的变体，SHBs蛋白或其片段或它们的变体；
    优选地，所述组装多肽为乙型肝炎病毒表面抗原(HBsAg)的SHBs蛋白或其片段或它们的变体；
    优选地，所述组装多肽具有如SEQ ID NO:73所示的氨基酸序列。
  3. 如权利要求1或2所述的融合蛋白，其中，所述免疫原性多肽是来源于生物体或非生物体(例如，人工合成)的多肽或其免疫原性变体；
    优选地，所述生物体为病原体(例如，病毒，细菌，真菌，寄生虫)或非病原体；
    优选地，所述免疫原性多肽获自哺乳动物(例如，人)的肿瘤细胞；优选地，所述免疫原性多肽选自癌胚抗原(CEA)，甲胎蛋白(AFP)，癌抗原125(CA125)；
    优选地，所述免疫原性多肽获自病毒，细菌(例如，结核分枝杆菌(Mycobacterium tuberculosis))，真菌(例如，念珠菌(Nostoc))，寄生虫(例如，恶性疟原虫(Plasmodium falciparum))；
    优选地，所述病毒选自水痘带状疱疹病毒(VZV)，新型冠状病毒(SARS-CoV-2)，冠状病毒(SARS-COV-1)，人类免疫缺陷病毒I型(HIV-1)，人乳头瘤病毒，乙型肝炎病毒，甲型肝炎病毒，丙型肝炎病毒，戊型肝炎病毒，麻疹病毒，腮腺炎病毒，流感病毒，乙型脑炎病毒；
    优选地，所述免疫原性多肽选自SARS-CoV-2的RBD蛋白或其免疫原性片段或变体，HIV-1的Env蛋白或其免疫原性片段(例如，gp140，gp160)或变体，VZV的gE蛋白或其免疫原性片段或变体；
    优选地，所述变体与其所源自的蛋白的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述置换是保守置换；
    优选地，所述RBD蛋白具有如SEQ ID NO:1-8任一项所示的氨基酸序列；
    优选地，所述Env蛋白具有如SEQ ID NO:34或35所示的氨基酸序列；
    优选地，所述gE蛋白具有如SEQ ID NO:30所示的氨基酸序列。
  4. 如权利要求1-3任一项所述的融合蛋白，其中，所述纳米抗体为驼源(例如，羊驼)抗体或鱼源(例如，鲨鱼)抗体；
    优选地，所述纳米抗体为嵌合抗体、人源化抗体或全人源抗体；
    优选地，所述融合蛋白包含2种，3种，或更多种免疫原性多肽；
    优选地，每一种免疫原性多肽各自独立地获自相同或不同的病原体(例如，病毒)；
    优选地，每一种免疫原性多肽为获自相同病原体(例如，病毒)的不同多肽；
    优选地，所述融合蛋白包含1种免疫原性多肽；
    优选地，所述纳米抗体特异性结合HEV，HBV和/或HPV的多肽；
    更优选地，所述纳米抗体包含SEQ ID NO:10-29，68，69任一项所示的重链可变区(VHH)中含有的CDR-H1、CDR-H2以及CDR-H3；优选地，所述CDR根据IMGT、Kabat或Chothia编号系统定义；
    优选地，所述纳米抗体，其包含：
    (a)包含下述3个互补决定区(CDRs)的重链可变区(VHH)：序列为SEQ ID NO:90的VH CDR1、序列为SEQ ID NO:91的VH CDR2、序列为SEQ ID NO:92的VH CDR3；
    (b)包含下述3个互补决定区(CDRs)的重链可变区(VHH)：序列为SEQ ID  NO:93的VH CDR1、序列为SEQ ID NO:94的VH CDR2、序列为SEQ ID NO:95的VH CDR3；
    (c)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:96的VH CDR1、序列为SEQ ID NO:97的VH CDR2、序列为SEQ ID NO:98的VH CDR3；
    (d)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:99的VH CDR1、序列为SEQ ID NO:100的VH CDR2、序列为SEQ ID NO:101的VH CDR3；
    (e)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:102的VH CDR1、序列为SEQ ID NO:103的VH CDR2、序列为SEQ ID NO:104的VH CDR3；
    (f)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:105的VH CDR1、序列为SEQ ID NO:106的VH CDR2、序列为SEQ ID NO:107的VH CDR3；
    (g)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:108的VH CDR1、序列为SEQ ID NO:109的VH CDR2、序列为SEQ ID NO:110的VH CDR3；
    (h)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:111的VH CDR1、序列为SEQ ID NO:112的VH CDR2、序列为SEQ ID NO:113的VH CDR3；
    (i)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:114的VH CDR1、序列为SEQ ID NO:115的VH CDR2、序列为SEQ ID NO:116的VH CDR3；
    (j)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:117的VH CDR1、序列为SEQ ID NO:118的VH CDR2、序列为SEQ ID NO:119的VH CDR3；
    (k)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:120的VH CDR1、序列为SEQ ID NO:121的VH CDR2、序列为SEQ ID NO:122的VH CDR3；
    (l)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:123的VH CDR1、序列为SEQ ID NO:124的VH CDR2、序列为SEQ ID NO:125的VH CDR3；
    (m)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:126的VH CDR1、序列为SEQ ID NO:127的VH CDR2、序列为SEQ ID NO:128的VH CDR3；
    (n)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO: 129的VH CDR1、序列为SEQ ID NO:130的VH CDR2、序列为SEQ ID NO:131的VH CDR3；
    (o)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:132的VH CDR1、序列为SEQ ID NO:133的VH CDR2、序列为SEQ ID NO:134的VH CDR3；
    (p)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:135的VH CDR1、序列为SEQ ID NO:136的VH CDR2、序列为SEQ ID NO:137的VH CDR3；
    (q)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:138的VH CDR1、序列为SEQ ID NO:139的VH CDR2、序列为SEQ ID NO:140的VH CDR3；
    (r)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:141的VH CDR1、序列为SEQ ID NO:142的VH CDR2、序列为SEQ ID NO:143的VH CDR3；
    (s)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:57的VH CDR1、序列为SEQ ID NO:58的VH CDR2、序列为SEQ ID NO:59的VH CDR3；
    (t)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:60的VH CDR1、序列为SEQ ID NO:61的VH CDR2、序列为SEQ ID NO:62的VH CDR3；或
    (u)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:65的VH CDR1、序列为SEQ ID NO:66的VH CDR2、序列为SEQ ID NO:67的VH CDR3；
    更优选地，所述纳米抗体包含如SEQ ID NO:10-29，68，69任一项所示的序列或它们的变体；其中，所述变体能够特异性结合组装多肽，并且与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)；优选地，所述的置换是保守置换。
  5. 如权利要求1-4任一项所述的融合蛋白，所述融合蛋白还包含连接体；
    优选地，所述连接体为多肽，例如柔性肽或刚性肽；
    优选地，所述连接体包含一个或几个(例如1个、2个或3个)如(GmS)n所示的序列，其中m选自1-6的整数，n选自1-6的整数；优选地，m为3、4、或5；优选地，n为2，3或4；
    更优选地，所述连接体具有如SEQ ID NO:39所示的氨基酸序列；
    优选地，所述融合蛋白的免疫原性多肽和纳米抗体直接连接或通过连接体连接；
    优选地，所述免疫原性多肽位于融合蛋白的N端或C端；
    优选地，所述融合蛋白从N端至C端依次包含：免疫原性多肽和纳米抗体；或者，纳米抗体和免疫原性多肽；或者，免疫原性多肽，连接体和纳米抗体；或者，纳米抗体，连接体和免疫原性多肽。
  6. 如权利要求1-5任一项所述的融合蛋白，所述融合蛋白还包含信号肽和/或标签；
    优选地，所述信号肽具有如SEQ ID NO:31，37或38所示的氨基酸序列；
    优选地，所述标签为用于纯化的标签，例如选自His标签或GST标签；
    优选地，所述信号肽位于融合蛋白的N端；
    优选地，所述标签位于融合蛋白的C端；
    优选地，所述融合蛋白具有如SEQ ID NO:42-49，50-56，72-79，80-87，63，64，89任一项所示的氨基酸序列。
  7. 一种分离的核酸分子，其包含编码权利要求1-6任一项所述的融合蛋白的核苷酸序列；
    优选地，所述核苷酸序列根据宿主细胞的密码子偏好性进行了密码子优化或未进行优化。
  8. 一种载体，其包含权利要求7所述的分离的核酸分子；优选地，所述载体用于表达(例如，在细胞中体外表达)所述分离的核酸分子编码的蛋白。
  9. 一种宿主细胞，其包含权利要求7所述的核酸分子或权利要求8所述的载体；
    优选地，所述宿主细胞选自原核细胞和真核细胞；
    优选地，所述原核细胞选自大肠杆菌细胞和枯草芽孢杆菌细胞；
    优选地，所述真核细胞选自酵母细胞，昆虫细胞，植物细胞和动物细胞；
    优选地，所述动物细胞是哺乳动物细胞(例如，鼠细胞，人细胞)。
  10. 一种表达或产生权利要求1-6任一项所述的融合蛋白的方法，所述方法包括，在允许蛋白质表达的条件下，培养权利要求9的宿主细胞，以及任选地，回收或纯化所表达的融合蛋白。
  11. 一种组合物，其包含至少一种权利要求1-6任一项所述的融合蛋白；
    优选地，所述组合物还包含组装多肽；
    优选地，所述组装多肽以VLP形式存在；
    优选地，所述融合蛋白附接至所述VLP。
  12. 一种试剂盒，其包含：权利要求1-6任一项所述的融合蛋白或含有编码其核苷酸 序列的第一核酸分子，以及组装多肽或包含编码其核苷酸序列的第二核酸分子；
    优选地，所述核苷酸序列根据宿主细胞的密码子偏好性进行了密码子优化或未进行优化；
    优选地，所述融合蛋白或第一核酸分子和组装多肽或第二核酸分子是分开提供的，或者以组合物的形式提供；
    优选地，所述试剂盒还包含载体(例如，表达载体)；优选地，第一核酸分子与第二核酸分子包含于相同或不同的载体；
    任选地，所述试剂盒还包含缓冲液；
    优选地，所述缓冲液选自磷酸缓冲液，柠檬酸缓冲液，碳酸缓冲液，醋酸缓冲液，巴比妥酸缓冲液，Tris缓冲液，或其任意组合；
    优选地，所述缓冲液为PBS缓冲液；
    优选地，所述缓冲液还包含盐；
    优选地，所述盐选自NaCl，(NH4)SO4，NaSO4，NH4Cl，或其任何组合。
  13. 一种颗粒化的抗原，其包含以VLP形式存在的组装多肽，以及与组装多肽附接的权利要求1-6任一项所述的融合蛋白；
    优选地，所述融合蛋白通过纳米抗体与所述组装多肽的相互作用附接至VLP；
    优选地，所述VLP附接至少一种如权利要求1-6任一项所述的融合蛋白；
    优选地，所述VLP还附接另外的多肽或融合蛋白(例如，T细胞表位)。
  14. 制备权利要求13所述的颗粒化的抗原的方法，所述方法包括：使用权利要求12所述的试剂盒；
    优选地，所述方法包括：在允许VLP组装的条件下，将组装多肽与融合蛋白接触；任选地，回收或纯化缓冲液中的颗粒化的抗原。
  15. 一种疫苗，其包含权利要求1-6任一项所述的融合蛋白，或权利要求11所述的组合物，或权利要求13所述的颗粒化的抗原，以及佐剂；
    优选地，所述佐剂选自铝盐类佐剂，锌铝混合佐剂(例如FH002C)，弗氏佐剂，油乳佐剂，细胞因子，TLR激动剂，CpG佐剂，脂质体，AS01B佐剂，或其任意组合。
  16. 一种药物组合物，其包含(1)至(6)的任意一项或多项：
    (1)权利要求1-6任一项所述的融合蛋白；
    (2)权利要求7所述的核酸分子；
    (3)权利要求8所述的载体；
    (4)权利要求9所述的宿主细胞；
    (5)权利要求11所述的组合物；
    (6)权利要求13所述的颗粒化的抗原；
    任选地，所述药物组合物还包含药学上可接受的载体和/或赋形剂。
  17. 权利要求1-6任一项所述的融合蛋白，或权利要求7所述的核酸分子，或权利要求8所述的载体，或权利要求9所述的宿主细胞，或权利要求11所述的组合物，或权利要求12所述的试剂盒，或权利要求13所述的颗粒化的抗原在制备药物组合物或疫苗中的用途，所述药物组合物或疫苗用于在受试者中诱导免疫应答；
    优选地，所述免疫应答是对免疫原性多肽的应答；优选地，所述免疫应答是T细胞应答(例如CD4+应答或CD8+应答)；优选地，所述免疫应答是B细胞应答；
    优选地，所述受试者为哺乳动物，例如人、猴或鼠。
  18. 权利要求1-6任一项所述的融合蛋白，或权利要求7所述的核酸分子，或权利要求8所述的载体，或权利要求9所述的宿主细胞，或权利要求11所述的组合物，或权利要求12所述的试剂盒，或权利要求13所述的颗粒化的抗原在制备药物组合物或疫苗中的用途，所述药物组合物或疫苗用于在受试者中预防和/或治疗因对免疫原性多肽的免疫应答而受益或者被预防的疾病和/或症状；
    优选地，所述疾病和/或症状是由免疫原性多肽所源自的肿瘤细胞或病原体(例如，病毒，细菌，真菌，寄生虫)引起的；
    优选地，所述疾病和/或症状是由免疫原性多肽所源自的病毒引起的，例如，水痘，新型冠状病毒肺炎，艾滋病，尖锐湿疣，病毒性肝炎(例如，病毒性乙型肝炎，病毒性甲型肝炎，病毒性丙型肝炎，病毒性戊型肝炎)，麻疹，腮腺炎；
    优选地，所述受试者为哺乳动物，例如人、猴或鼠。
  19. 一种在受试者中诱导免疫应答的方法，包括给予受试者有效量的权利要求1-6任一项所述的融合蛋白，或权利要求7所述的核酸分子，或权利要求8所述的载体，或权利要求9所述的宿主细胞，或权利要求11所述的组合物，或权利要求12所述的试剂盒，或权利要求13所述的颗粒化的抗原，或权利要求所述的15的疫苗，或权利要求16所述的药物组合物；
    优选地，所述免疫应答是对免疫原性多肽；优选地，所述免疫应答是T细胞应答(例如CD4+应答或CD8+应答)；优选地，所述免疫应答是B细胞应答；
    优选地，所述受试者为哺乳动物，例如人、猴或鼠。
  20. 一种在受试者中预防和/或治疗因对免疫原性多肽的免疫应答而受益或者被预防的疾病和/或症状，包括给予受试者有效量的权利要求1-6任一项所述的融合蛋白，或权利要求7所述的核酸分子，或权利要求8所述的载体，或权利要求9所述的宿主细胞，或权利要求11所述的组合物，或权利要求12所述的试剂盒，或权利要求13所述 的颗粒化的抗原，或权利要求所述的15的疫苗，或权利要求16所述的药物组合物；
    优选地，所述疾病和/或症状是由免疫原性多肽所源自的肿瘤细胞或病原体(例如，病毒，细菌，真菌，寄生虫)引起的；
    优选地，所述疾病和/或症状是由免疫原性多肽所源自的病毒引起的，例如，水痘，新型冠状病毒肺炎，艾滋病，尖锐湿疣，病毒性肝炎(例如，病毒性乙型肝炎，病毒性甲型肝炎，病毒性丙型肝炎，病毒性戊型肝炎)，麻疹，腮腺炎；
    优选地，所述受试者为哺乳动物，例如人、猴或鼠。
  21. 一种制备颗粒化免疫原性多肽的系统，其包括第一载体和第二载体，其中，第一载体包含编码融合蛋白的核苷酸序列，所述融合蛋白包含免疫原性多肽和组装多肽，第二载体包含编码纳米抗体的核苷酸序列；并且，所述纳米抗体能够特异性结合所述组装多肽，所述组装多肽能够组装成VLP；
    优选地，所述核苷酸序列根据宿主细胞的密码子偏好性进行了密码子优化或未进行优化；
    优选地，所述组装多肽选自戊型肝炎病毒(HEV)的蛋白或其片段或它们的变体；优选地，所述组装多肽如权利要求2所定义；
    优选地，所述融合蛋白如权利要求1-6任一项所定义。
  22. 一种增强免疫原性多肽的免疫原性的方法，其包括，制备或获得含有所述免疫原性多肽和能够特异性结合组装多肽的纳米抗体的融合蛋白；和，使所述融合蛋白与包含所述组装多肽的VLP接触，从而，获得包含附接至VLP的免疫原性多肽的颗粒化抗原；
    优选地，所述方法包括：使用权利要求21所述的系统；优选地，所述方法包括：(1)通过第一载体表达或产生融合蛋白，通过第二载体表达或产生组装多肽；(2)在允许VLP组装的条件下，使融合蛋白和组装多肽接触；
    优选地，所述纳米抗体如权利要求4所定义；
    优选地，所述融合蛋白如权利要求1-6任一项所定义；
    优选地，所述组装多肽组装为VLP；优选地，所述融合蛋白通过纳米抗体与所述组装多肽的相互作用附接至VLP。
PCT/CN2023/129973 2022-11-07 2023-11-06 一种融合蛋白以及包含其的颗粒化的抗原 WO2024099273A1 (zh)

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