Fusion protein and particulate antigen containing the same

Abstract

The present invention provides fusion proteins, as well as compositions, kits, particulate antigens, vaccines, and pharmaceutical compositions comprising the same. The present invention also relates to the use of fusion proteins, as well as compositions, kits, and particulate antigens comprising the same, in the preparation of pharmaceutical compositions or vaccines. Particulate antigens are particularly suitable for the production and administration of vaccines and have advantages in terms of the prevention and / or treatment of viral infections.

Description

[Technical Field]

[0001] The present application relates to the field of biopharmaceuticals, and in particular to a fusion protein and a composition, kit, particulate antigen, vaccine, and pharmaceutical composition containing the same. The present application also relates to the use of the fusion protein and a composition, kit, and particulate antigen containing the same in the manufacture of a pharmaceutical composition or vaccine. [Background technology]

[0002] Vaccines are the most cost-effective and effective method for preventing and controlling infectious diseases, and it is well known that vaccination can save nearly 6 million lives each year. Currently, genetically engineered vaccines mainly use proteins / polysaccharides that are crucial for pathogen infection as antigens. The development of such vaccine antigens has the advantages of high safety and mature technology, and can provide immune protection against specific pathogens. However, they often face problems such as insufficient humoral immunity, difficulty in achieving long-term protection, and difficulty in activating efficient cellular immunity. Therefore, the development of new immunogens that are safe, efficient, and have a broad spectrum is key to improving vaccine efficacy.

[0003] Vaccines based on particulate antigens are an important vaccine platform for combating pathogen threats. Currently, three major particulate vaccines have been approved for marketing, including hepatitis E virus (HEV) vaccines, human papillomavirus (HPV) vaccines, and hepatitis B virus (HBV) vaccines. The HEV vaccine is a virus-like particle (VLP) vaccine. The HEV antigen fragment, p239 protein (amino acids 368–606), is expressed in Escherichia coli (Escherichia coli) and can assemble into particles with a diameter of 25 nm after renaturation. It has a structural conformation similar to that of natural virus particles and can fully mimic natural viral antigen epitopes. Currently, the p239 protein has been successfully developed as a vaccine antigen, resulting in the world's first HEV vaccine, Hecolin. Hecolin was launched in mainland China in 2012, and clinical results have confirmed its excellent preventive and protective effects. Currently available HPV vaccines include bivalent, quadrivalent, and nonavalent vaccines. HPV vaccines are produced by assembling the virus's major capsid protein, L1, to form virus-like particles. They can be produced and prepared using expression systems such as insect cells, yeast cells, and Escherichia coli. Clinical trials and post-marketing surveillance have confirmed that HPV vaccines have good safety and protective effects. Research and development of HBV vaccines has a long history. Many HBV vaccines are currently available on the international market. HBV vaccines are primarily composed of different forms of the S protein of the surface antigen HBsAg (SHB, MHB, or LHB), which naturally assemble to form particles. Currently, they are primarily produced using yeast and CHO systems. Clinical trials and real-world clinical studies have shown that HBV vaccines can produce high titers of protective antibodies and have good efficacy and safety.In summary, the production of particulate antigens for HEV vaccine, HPV vaccine and HBV vaccine has the advantages of economic efficiency, stable production process, controllable antigen quality, industrial mass production, good safety and strong immunogenicity, which provides a good basis and important reference for the further development of other vaccines.

[0004] However, many viral proteins lack the ability to assemble into particles, making it impossible to develop particle-based vaccines. Furthermore, isolated viral antigens or oligomeric antigens often exhibit poor immunogenicity, posing significant challenges to vaccine development. How to display target proteins on the particle surface is an important challenge and a primary goal in the design of recombinant vaccines and drug delivery vehicles. Currently, multivalent antigen display on particle carriers is achieved by fusion expression techniques or by using chemical conjugation strategies based on SpyTag / SpyCatcher. However, these methods have significant limitations. First, a major limitation of the fusion expression method is the requirement that the particle carrier must have an exposed N- or C-terminus. For particle carriers lacking exposed termini, other suitable insertion sites need to be explored based on structural biology and bioinformatics analyses. Second, fusion expression requires consideration of various factors, such as the native conformation of the target molecule and steric hindrance, all of which require comprehensive consideration. A limitation of SpyTag / SpyCatcher-based technology is that the coupling method is limited by the structural characteristics of the particle carrier. Therefore, finding an efficient method for displaying target antigens on the surface of particle carriers is an important goal in the field, but faces various difficult challenges.

[0005] Membrane proteins of enveloped viruses are major immunogenic targets for vaccine development. However, these proteins are typically anchored to membrane structures on the viral surface and cannot form particulate antigens by themselves. Furthermore, their high hydrophobicity makes it difficult to directly express full-length proteins in the form of membrane-bound particles. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped RNA virus, and the spike protein is a key molecule on the envelope that is primarily involved in receptor recognition and cell membrane fusion. Among the domains of the spike protein, the receptor-binding domain (RBD) directly binds to the host receptor angiotensin-converting enzyme 2 (ACE2) and serves as the primary target for neutralizing antibodies. Therefore, the RBD is considered an important immunogen. However, due to its small molecular weight and poor immunogenicity, how to improve the immunogenicity of the RBD remains a key challenge. Like the RBD, the gE protein is the most abundant glycoprotein on the surface of varicella-zoster virus (VZV) and is also an important target for vaccine development. Compared to SARS-CoV-2 and varicella-zoster virus, human immunodeficiency virus type 1 (HIV-1), which causes acquired immunodeficiency syndrome (AIDS), faces a greater challenge in HIV vaccine development due to its higher rate of genetic mutation. Env is the major antigen on the surface of the HIV-1 virus and is a key molecule in HIV vaccine development. The full-length Env molecule is a transmembrane protein known as gp160. Deletion of the cytoplasmic tail significantly improves the solubility of the expressed Env, resulting in a molecule called gp140. This protein retains a native-like trimeric structure, demonstrating clear advantages in exposing neutralizing epitopes and eliciting antibody responses. Nevertheless, development of an Env-based HIV vaccine has remained unsuccessful, in part due to the fact that more than 50% of the Env surface is covered with glycan molecules, which significantly attenuates the immunogenicity of Env. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, enhancing the immunogenicity of antigens is of great significance for the development and application of vaccines. [Means for solving the problem]

[0007] The present invention utilizes the ability of assembly polypeptides, such as the HEV ORF2 protein or fragments or variants thereof, the HPV L1 protein or fragments or variants thereof, and the HBV surface antigen or fragments or variants thereof, to form virus-like particles (VLPs). By fusing nanobodies that specifically bind to these assembly polypeptides with immunogenic polypeptides and utilizing the targeting binding characteristics of the nanobodies to the assembly polypeptides, multiple immunogenic polypeptides are displayed on the surface of the VLPs formed by the assembly polypeptides, thereby resulting in particulate display of the immunogenic polypeptides. Thus, the present application provides a system and method for particulate display of immunogenic polypeptides. Furthermore, particulate immunogenic polypeptides have been demonstrated to be capable of inducing robust humoral and cellular immune responses. Thus, the particulate immunogenic polypeptides of the present invention hold potential as vaccine candidate molecules and offer the advantages of universality and translatability for vaccine design and production.

[0008] Thus, in a first aspect, the present application provides a fusion protein comprising an immunogenic polypeptide and a nanobody capable of specifically binding to a virus-like particle (VLP).

[0009] In certain embodiments, the VLP is a VLP assembled from assembly polypeptides.

[0010] In certain embodiments, the assembly polypeptide is a polypeptide capable of assembling into a VLP.

[0011] In certain embodiments, the assembly polypeptides are capsid proteins of naturally occurring viruses or virus-like bodies, and in such embodiments, the VLPs assembled by the assembly polypeptides are similar in structure to naturally occurring viruses or virus-like bodies, except that they do not contain the genome of the naturally occurring viruses or virus-like bodies.

[0012] In certain embodiments, the assembly polypeptides are artificially prepared and / or screened polypeptides capable of assembling into VLPs, and in such embodiments, the VLPs assembled by the assembly polypeptides may or may not be structurally similar to naturally occurring viruses or virus-like entities.

[0013] In certain embodiments, the assembly polypeptide is selected from the group consisting of a Hepatitis E virus (HEV) protein or a fragment thereof or a variant thereof, a Hepatitis B virus (HBV) protein or a fragment thereof or a variant thereof, a Human Papillomavirus (HPV) protein or a fragment thereof or a variant thereof, and any combination thereof, wherein the fragment or variant retains the ability to assemble into a VLP.

[0014] In some embodiments, the variants have one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence of the polypeptide from which they are derived. In some embodiments, the substitutions are conservative substitutions. In some embodiments, the polypeptide and its variants have the same or similar biological activity. In some preferred embodiments, the biological activity is the ability to assemble into a VLP.

[0015] In some embodiments, the assembly polypeptide is the ORF2 protein of HEV, or a fragment or variant thereof.

[0016] In the present invention, the term "ORF2 protein" refers to the protein encoded by the second open reading frame in the genome of the HEV virus, and the ORF2 protein or a fragment thereof has the ability to self-assemble into a VLP.

[0017] In some embodiments, the fragment of the ORF2 protein is selected from the group consisting of a p239 protein and a p495 protein.

[0018] In some embodiments, the assembly polypeptide is selected from the group consisting of a p239 protein, or a fragment or variant thereof, and a p495 protein, or a fragment or variant thereof.

[0019] In some embodiments, the p239 protein has a sequence corresponding to amino acids 368 to 606 of the amino acid sequence of the ORF2 protein. In some embodiments, the p495 protein has a sequence corresponding to amino acids 112 to 606 of the amino acid sequence of the ORF2 protein.

[0020] In some embodiments, the ORF2 protein has the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the p239 protein has the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the p495 protein has the amino acid sequence set forth in SEQ ID NO: 41.

[0021] In some embodiments, the assembly polypeptide is HPV capsid protein L1, or a fragment or variant thereof.

[0022] HPV consists of a protein capsid and core DNA, and the capsid is composed of a major capsid protein (L1) and a minor capsid protein (L2). In the present invention, the term "capsid protein L1" or "HPV L1 protein" or "L1 protein" refers to the L1 protein that constitutes the HPV capsid.

[0023] In certain embodiments, the assembly polypeptide has the amino acid sequence set forth in SEQ ID NO:74.

[0024] In certain embodiments, the assembly polypeptide is a surface protein of HBV (e.g., hepatitis B virus surface antigen) or a fragment or variant thereof. In certain embodiments, the assembly polypeptide is selected from an LHB protein or a fragment or variant thereof of the hepatitis B virus surface antigen (HBsAg), an MHB protein or a fragment or variant thereof, or an SHB protein or a fragment or variant thereof. In certain embodiments, the assembly polypeptide is selected from an SHB protein or a fragment or variant thereof of the hepatitis B virus surface antigen (HBsAg).

[0025] Hepatitis B virus surface antigen (HBsAg) is composed of a large surface protein (L protein) (LHB), a middle surface protein (M protein) (MHB), and a small surface protein (S protein) (SHB). In the present invention, the term "SHB protein" refers to the small surface protein that constitutes HBsAg.

[0026] In certain embodiments, the assembly polypeptide has the amino acid sequence set forth in SEQ ID NO:73.

[0027] In certain embodiments, the immunogenic polypeptide is a biologically derived or non-biologically derived (eg, artificially synthesized) polypeptide or an immunogenic variant thereof.

[0028] In certain embodiments, variants have one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence of the polypeptide from which they are derived. In certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the polypeptide and its variants have the same or similar biological activity. In certain preferred embodiments, the biological activity is the ability to induce an immune response.

[0029] In certain embodiments, the organism is a pathogen (eg, a virus, bacterium, fungus, parasite) or a non-pathogen.

[0030] In certain embodiments, the immunogenic polypeptide is derived from a non-tumor cell of a mammal (e.g., a human). In certain embodiments, the immunogenic polypeptide is proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0031] In certain embodiments, the immunogenic polypeptide is derived from (e.g., expressed or overexpressed on the surface of) a tumor cell in a mammal (e.g., a human). In certain embodiments, the immunogenic polypeptide is selected from the group consisting of carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and cancer antigen 125 (CA125).

[0032] In certain embodiments, the immunogenic polypeptide is derived from a virus, a bacterium (e.g., Mycobacterium tuberculosis), a fungus (e.g., Nostoc), or a parasite (e.g., Plasmodium falciparum).

[0033] In certain embodiments, the virus is selected from the group consisting of varicella-zoster virus (VZV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), 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 encephalitis B virus (Japanese encephalitis virus).

[0034] In certain embodiments, the assembly polypeptide has a sequence derived from a viral pathogen and the immunogenic polypeptide has a sequence derived from a non-viral pathogen (eg, a bacterium, fungus, parasite).

[0035] In certain embodiments, the assembly polypeptide has a sequence derived from a viral pathogen and the immunogenic polypeptide has a sequence derived from another viral pathogen.

[0036] Immunogenic polypeptides of the present invention can be polypeptides derived from the surface or core of a virus. Generally, immunogenic polypeptides can be polypeptides of any structure or function comprising at least six amino acid residues. In some embodiments, the length of an immunogenic polypeptide is between 6 and 10,000 amino acid residues. In some embodiments, the length of an immunogenic polypeptide is between 25 and 2,000 amino acid residues. In some embodiments, the length of an immunogenic polypeptide is between 50 and 500 amino acid residues.

[0037] In certain embodiments, the immunogenic polypeptide is selected from the group consisting of a SARS-CoV-2 RBD protein or an immunogenic fragment thereof or a variant thereof, an HIV-1 Env protein or an immunogenic fragment thereof (e.g., gp140, gp160) or a variant thereof, and a VZV gE protein or an immunogenic fragment thereof or a variant thereof.

[0038] In certain embodiments, the fragment of an Env protein is selected from the group consisting of a gp160 protein, a gp120 protein, and a gp41 protein.

[0039] In certain embodiments, the Env protein has the amino acid sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 35. In certain embodiments, the gp160 protein has the sequence set forth in GenBank under accession numbers AAB05604 and AAD12142. In certain embodiments, the gp41 protein has the amino acid sequence set forth in GenBank under accession number CAD20975.

[0040] In certain embodiments, the RBD protein has the amino acid sequence set forth in any one of SEQ ID NO:1 to SEQ ID NO:8.

[0041] In certain embodiments, the gE protein has the amino acid sequence set forth in SEQ ID NO:30.

[0042] Those skilled in the art will appreciate that Nanobodies capable of specifically binding to a particular antigen can be prepared by various methods known in the art, for example, by immunizing alpacas or sharks with a particular antigen, screening positive clones, and obtaining the sequence of the antibody heavy chain by sequencing. A vector containing the heavy chain sequence is then constructed and transfected into host cells under defined conditions for expression and production of the Nanobody.

[0043] Thus, once an assembly polypeptide is determined, one skilled in the art can prepare and obtain a Nanobody capable of specifically binding to the assembly polypeptide. Thus, the Nanobodies of the present application are not limited to the particular forms (e.g., Nanobodies) and particular sequences used in the examples.

[0044] In certain embodiments, the nanobody is a camelid-derived (e.g., alpaca-derived) antibody or a fish-derived (e.g., shark-derived) antibody.

[0045] In certain embodiments, the Nanobody is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0046] In certain embodiments, the fusion protein comprises two, three, or more different immunogenic polypeptides.

[0047] In certain embodiments, each type of immunogenic polypeptide is independently derived from the same or a different pathogen (eg, virus).

[0048] In certain embodiments, each type of immunogenic polypeptide is a different polypeptide from the same pathogen (eg, virus).

[0049] In certain exemplary embodiments, the fusion protein comprises a first immunogenic polypeptide and a second immunogenic polypeptide, wherein the first immunogenic polypeptide is the RBD protein of SARS-CoV-2 and the second immunogenic polypeptide is the gE protein of VZV. In certain exemplary embodiments, the nanobody specifically binds to the RBD protein and the gE protein.

[0050] In certain exemplary embodiments, the fusion protein comprises a first immunogenic polypeptide, a second immunogenic polypeptide, and a third immunogenic polypeptide, wherein the first immunogenic polypeptide is the RBD protein set forth in SEQ ID NO: 1, the second immunogenic polypeptide is the RBD protein set forth in SEQ ID NO: 2, and the third immunogenic polypeptide is the RBD protein set forth in SEQ ID NO: 3. In certain embodiments, the Nanobody specifically binds to the RBD proteins set forth in SEQ ID NOs: 1 to 3.

[0051] In certain embodiments, the fusion protein comprises one immunogenic polypeptide, hi certain embodiments, the Nanobody is a Nanobody that specifically binds to a polypeptide of HEV, HBV and / or HPV.

[0052] In certain embodiments, the Nanobody comprises CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VHH) set forth in any one of SEQ ID NOs: 10 to 29, 68, and 69. In certain embodiments, the CDRs are defined according to the IMGT, Kabat, or Chothia numbering system.

[0053] In certain embodiments, the Nanobody is (a) a heavy chain variable region (VHH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 90, VH CDR2 set forth in SEQ ID NO: 91, and VH CDR3 set forth in SEQ ID NO: 92; (b) a heavy chain variable region (VHH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 93, a VH CDR2 set forth in SEQ ID NO: 94, and a VH CDR3 set forth in SEQ ID NO: 95; (c) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 96, VH CDR2 set forth in SEQ ID NO: 97, and VH CDR3 set forth in SEQ ID NO: 98; (d) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 99, VH CDR2 set forth in SEQ ID NO: 100, and VH CDR3 set forth in SEQ ID NO: 101; (e) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 102, VH CDR2 set forth in SEQ ID NO: 103, and VH CDR3 set forth in SEQ ID NO: 104; (f) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 105, VH CDR2 set forth in SEQ ID NO: 106, and VH CDR3 set forth in SEQ ID NO: 107; (g) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 108, VH CDR2 set forth in SEQ ID NO: 109, and VH CDR3 set forth in SEQ ID NO: 110; (h) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 111, VH CDR2 set forth in SEQ ID NO: 112, and VH CDR3 set forth in SEQ ID NO: 113; (i) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 114, VH CDR2 set forth in SEQ ID NO: 115, and VH CDR3 set forth in SEQ ID NO: 116; (j) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 117, VH CDR2 set forth in SEQ ID NO: 118, and VH CDR3 set forth in SEQ ID NO: 119; (k) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 120, VH CDR2 set forth in SEQ ID NO: 121, and VH CDR3 set forth in SEQ ID NO: 122; (l) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 123, VH CDR2 set forth in SEQ ID NO: 124, and VH CDR3 set forth in SEQ ID NO: 125; (m) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 126, VH CDR2 set forth in SEQ ID NO: 127, and VH CDR3 set forth in SEQ ID NO: 128; (n) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 129, VH CDR2 set forth in SEQ ID NO: 130, and VH CDR3 set forth in SEQ ID NO: 131; (o) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 132, VH CDR2 set forth in SEQ ID NO: 133, and VH CDR3 set forth in SEQ ID NO: 134; (p) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 135, VH CDR2 set forth in SEQ ID NO: 136, and VH CDR3 set forth in SEQ ID NO: 137; (q) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 138, a VH CDR2 set forth in SEQ ID NO: 139, and a VH CDR3 set forth in SEQ ID NO: 140; (r) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 141, VH CDR2 set forth in SEQ ID NO: 142, and VH CDR3 set forth in SEQ ID NO: 143; (s) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 57, VH CDR2 set forth in SEQ ID NO: 58, and VH CDR3 set forth in SEQ ID NO: 59; (t) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 60, VH CDR2 set forth in SEQ ID NO: 61, and VH CDR3 set forth in SEQ ID NO: 62; or (u) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 65, VH CDR2 set forth in SEQ ID NO: 66, and VH CDR3 set forth in SEQ ID NO: 67; Includes:

[0054] In certain embodiments, the Nanobody comprises a sequence as set forth in any one of SEQ ID NOs: 10 to 29, 68, 69, or a variant thereof, wherein the variant comprises one or several amino acid substitutions, deletions or additions (e.g. one, two or three amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0055] In certain embodiments, the fusion protein further comprises a linker.

[0056] In certain embodiments, the linker is a polypeptide, for example, a flexible or rigid peptide.

[0057] In certain embodiments, the linker is m S) n where m is an integer selected from 1 to 6 and n is an integer selected from 1 to 6. In certain embodiments, m is 3, 4, or 5. In certain embodiments, n is 2, 3, or 4.

[0058] In certain embodiments, the linker has the amino acid sequence set forth in SEQ ID NO:39.

[0059] In certain embodiments, the immunogenic polypeptide and the Nanobody of the fusion protein are directly linked or linked via a linker.

[0060] In some embodiments, the immunogenic polypeptide is located at the N-terminus or C-terminus of the fusion protein.

[0061] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, 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.

[0062] In some embodiments, the fusion protein further comprises a signal peptide and / or a tag.

[0063] In some embodiments, the signal peptide is selected from the group consisting of a tPA signal peptide and a bee venom signal peptide, hi some embodiments, the signal peptide has the amino acid sequence set forth in SEQ ID NO:31, SEQ ID NO:37, or SEQ ID NO:38.

[0064] In some embodiments, the tag is a purification tag, for example, selected from a His tag or a GST tag.

[0065] In some embodiments, the signal peptide is located at the N-terminus of the fusion protein.

[0066] In some embodiments, the tag is located at the C-terminus of the fusion protein.

[0067] In certain embodiments, the fusion protein has an amino acid sequence set forth in any one of SEQ ID NOs: 42 to 49, 50 to 56, 72 to 79, 80 to 87, 63, 64, and 89.

[0068] In a second aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein according to the first aspect.

[0069] In certain embodiments, the nucleotide sequence is codon-optimized or non-optimized depending on the codon preferences of the host cell.

[0070] As known to those skilled in the art, codons have degeneracy. That is, during translation into protein, each amino acid can correspond to one or more codons, for example, up to six codons. Different species (e.g., host cells) have significant differences and different preferences in the use of degenerate codons to encode certain amino acids. This preference phenomenon is referred to as "codon preference." Therefore, as used herein, the term "codon preference" refers to a situation in which a species prefers to use a certain codon to encode an amino acid. Optimizing the sequence of a nucleic acid molecule according to codon preference can be particularly advantageous in some cases, and can be useful, for example, to increase the expression level of a protein encoded by the nucleic acid molecule.

[0071] In a third aspect, the present application provides a vector comprising the isolated nucleic acid molecule of the second aspect. In certain embodiments, the vector is used for expression (e.g., in vitro expression in a cell) of a protein encoded by the isolated nucleic acid molecule.

[0072] In a fourth aspect, the present application provides a host cell comprising a nucleic acid molecule according to the second aspect or a vector according to the third aspect.

[0073] In certain embodiments, the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell.

[0074] In certain embodiments, the prokaryotic cell is selected from the group consisting of an E. coli cell and a Bacillus subtilis cell.

[0075] In certain embodiments, the eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, a plant cell, and an animal cell.

[0076] In certain embodiments, the animal cell is a mammalian cell (eg, a mouse cell, a human cell).

[0077] In a fifth aspect, the present application provides a method of expressing or producing a fusion protein according to the first aspect, the method comprising culturing a host cell according to the fourth aspect under conditions that allow expression of the protein, and optionally recovering or purifying the expressed fusion protein.

[0078] In a sixth aspect, the present application provides a composition comprising at least one fusion protein according to the first aspect.

[0079] In certain embodiments, the composition further comprises an assembly polypeptide.

[0080] In certain embodiments, the assembly polypeptide is in the form of a VLP.

[0081] In some embodiments, the fusion protein is attached to a VLP.

[0082] In a seventh aspect, the present application provides a kit comprising a fusion protein according to 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 assembly polypeptide.

[0083] In some embodiments, 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.

[0084] In some embodiments, the kit further comprises a vector (eg, an expression vector).

[0085] In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule are contained in the same or different vectors.

[0086] In some embodiments, the kit further comprises a buffer.

[0087] In some embodiments, the buffer is selected from the group consisting of phosphate buffer, citrate buffer, carbonate buffer, acetate buffer, barbiturate buffer, Tris buffer, and any combination thereof.

[0088] In some embodiments, the buffer is a PBS buffer.

[0089] In some embodiments, the buffer further comprises a salt.

[0090] In some embodiments, the salt is selected from the group consisting of NaCl, (NH4)SO4, NaSO4, NH4Cl, and any combination thereof.

[0091] In an eighth aspect, the present application provides a particulate antigen comprising an assembly polypeptide in the form of a VLP and a fusion protein according to the first aspect attached to the assembly polypeptide.

[0092] In certain embodiments, the fusion protein is attached to the VLP through the interaction of the Nanobody with the assembly polypeptide.

[0093] In certain embodiments, the VLP is attached to at least one fusion protein according to the first aspect.

[0094] In certain embodiments, the VLP is also attached to an additional polypeptide or fusion protein (eg, a T cell epitope).

[0095] In a ninth aspect, the present application provides a method for preparing a particulate antigen according to the eighth aspect, the method comprising using a kit according to the seventh aspect.

[0096] In certain embodiments, the method comprises contacting the assembly polypeptide with the fusion protein under conditions that allow for VLP assembly.

[0097] In certain embodiments, the conditions that allow VLP assembly include placing the assembly polypeptides in a solution. In certain embodiments, the solution is a buffer containing a salt. In certain embodiments, the salt is selected from the group consisting of NaCl, (NH4)SO4, NaSO4, NH4Cl, and any combination thereof.

[0098] In some embodiments, 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 together to a buffer; or (iii) adding the fusion protein to a buffer and then adding the assembly polypeptide to a buffer; Optionally, recovering or purifying the particulate antigen in the buffer; Includes:

[0099] In a tenth aspect, the present application provides a vaccine comprising a fusion protein according to the first aspect, or a composition according to the sixth aspect, or a particulate antigen according to the eighth aspect, and an adjuvant.

[0100] In some embodiments, the adjuvant is selected from the group consisting of an aluminum salt adjuvant, a mixed zinc / aluminum adjuvant (e.g., FH002C), Freund's adjuvant, an oil emulsion adjuvant, a cytokine, a TLR agonist, a CpG adjuvant, a liposome, an AS01B adjuvant, and any combination thereof.

[0101] In an eleventh aspect, the present application provides a method for manufacturing a semiconductor device comprising: (1) the fusion protein according to the first aspect; (2) a nucleic acid molecule according to the second aspect; (3) the vector according to the third aspect; (4) a host cell according to the fourth aspect; (5) the composition according to the sixth aspect; (6) The particulate antigen according to the eighth aspect; A pharmaceutical composition comprising any one or more of: Optionally, further comprising a pharmaceutically acceptable carrier and / or excipient; Pharmaceutical compositions are provided.

[0102] In certain embodiments, the pharmaceutically acceptable carrier and / or additive is selected from the group consisting of a pH adjusting agent (e.g., phosphate buffer), a surfactant (e.g., a cationic, anionic, or non-ionic surfactant, e.g., Tween-80), an adjuvant, an ionic strength enhancer (e.g., sodium chloride), a diluent, an excipient, a vehicle for containing or administering a therapeutic agent, and any combination thereof.

[0103] In a twelfth aspect, the present application provides use of a fusion protein according to the first aspect, or a nucleic acid molecule according to the second aspect, or a vector according to the third aspect, or a host cell according to the fourth aspect, or a composition according to the sixth aspect, or a kit according to the seventh aspect, or a particulate antigen according to the eighth aspect, in the manufacture of a pharmaceutical composition or vaccine, wherein the pharmaceutical composition or vaccine is used to induce an immune response in a subject.

[0104] In certain embodiments, the immune response is a response to the immunogenic polypeptide and / or assembly polypeptide. In certain embodiments, the immune response is a T cell response (e.g., a CD4+ response or a CD8+ response). In certain embodiments, the immune response is a B cell response.

[0105] In certain embodiments, the subject is a mammal, such as a human, monkey, or mouse.

[0106] In a thirteenth aspect, the present application provides the use of a fusion protein according to the first aspect, or a nucleic acid molecule according to the second aspect, or a vector according to the third aspect, or a host cell according to the fourth aspect, or a composition according to the sixth aspect, or a kit according to the seventh aspect, or a particulate antigen according to the eighth aspect, in the manufacture of a pharmaceutical composition or vaccine, wherein the pharmaceutical composition or vaccine is used for the prevention and / or treatment of a disease and / or condition in a subject in which an immune response to the immunogenic polypeptide is beneficial or can be prevented thereby.

[0107] In certain embodiments, the disease and / or condition is caused by the tumor cells from which the immunogenic polypeptide is derived.

[0108] In certain embodiments, the disease and / or condition is caused by a pathogen (eg, a virus, bacterium, fungus, parasite) from which the immunogenic polypeptide is derived.

[0109] In certain embodiments, the disease and / or condition is caused by the virus from which the immunogenic polypeptide is derived, e.g., chickenpox, COVID-19, AIDS, genital warts, viral hepatitis (e.g., hepatitis B, hepatitis A, hepatitis C, hepatitis E), measles, or mumps.

[0110] In certain embodiments, the immunogenic polypeptide may be the RBD protein of SARS-CoV-2 or a fragment thereof, and in such embodiments, the disease may be COVID-19.

[0111] In certain embodiments, the immunogenic polypeptide may be the gE protein of VZV or a fragment thereof, hi such embodiments, the disease may be varicella.

[0112] In certain embodiments, the immunogenic polypeptide may be a protein or fragment thereof of HPV, hi such embodiments, the disease may be genital warts.

[0113] In certain embodiments, the immunogenic polypeptide may be a protein or fragment thereof derived from hepatitis B virus, hepatitis A virus, hepatitis C virus, or hepatitis E virus. In such embodiments, the disease may be viral hepatitis (e.g., hepatitis B virus, hepatitis A virus, hepatitis C virus, or hepatitis E virus).

[0114] In certain embodiments, the immunogenic polypeptide may be an envelope glycoprotein or fragment thereof derived from the measles virus, hi such embodiments, the disease may be measles.

[0115] In certain embodiments, the immunogenic polypeptide may be a protein or fragment thereof derived from the mumps virus, hi such embodiments, the disease may be mumps.

[0116] In certain embodiments, the subject is a mammal, such as a human, monkey, or mouse.

[0117] In a fourteenth aspect, the present application provides a method of inducing an immune response in a subject, the method comprising administering to the subject an effective amount of a fusion protein according to the first aspect, or a nucleic acid molecule according to the second aspect, or a vector according to the third aspect, or a host cell according to the fourth aspect, or a composition according to the sixth aspect, or a kit according to the seventh aspect, or a particulate antigen according to the eighth aspect, or a vaccine according to the tenth aspect, or a pharmaceutical composition according to the eleventh aspect.

[0118] In certain embodiments, the immune response is a response to the immunogenic polypeptide and / or assembly polypeptide. In certain embodiments, the immune response is a T cell response (e.g., a CD4+ response or a CD8+ response). In certain embodiments, the immune response is a B cell response.

[0119] In certain embodiments, the subject is a mammal, such as a human, monkey, or mouse.

[0120] In a fifteenth aspect, the present application provides a method for preventing and / or treating a disease and / or condition in a subject in which an immune response to an immunogenic polypeptide is beneficial or prevented thereby, the method comprising administering to the subject an effective amount of a fusion protein according to the first aspect, or a nucleic acid molecule according to the second aspect, or a vector according to the third aspect, or a host cell according to the fourth aspect, or a composition according to the sixth aspect, or a kit according to the seventh aspect, or a particulate antigen according to the eighth aspect, or a vaccine according to the tenth aspect, or a pharmaceutical composition according to the eleventh aspect.

[0121] In certain embodiments, the disease and / or condition is caused by the tumor cells from which the immunogenic polypeptide is derived.

[0122] In certain embodiments, the disease and / or condition is caused by a pathogen (eg, a virus, bacterium, fungus, parasite) from which the immunogenic polypeptide is derived.

[0123] In certain embodiments, the disease and / or condition is caused by the virus from which the immunogenic polypeptide is derived, e.g., chickenpox, COVID-19, AIDS, genital warts, viral hepatitis (e.g., hepatitis B, hepatitis A, hepatitis C, hepatitis E), measles, or mumps.

[0124] In certain embodiments, the immunogenic polypeptide may be an HIV Env protein or a fragment thereof (e.g., gp160 protein, gp120 protein, gp41 protein). In such embodiments, the disease may be AIDS.

[0125] In certain embodiments, the immunogenic polypeptide may be an RBD protein or fragment thereof from SARS-CoV-2, and in such embodiments, the disease may be COVID-19.

[0126] In certain embodiments, the immunogenic polypeptide may be the gE protein of VZV or a fragment thereof, hi such embodiments, the disease may be chickenpox.

[0127] In certain embodiments, the immunogenic polypeptide may be a protein or fragment thereof of HPV, hi such embodiments, the disease may be genital warts.

[0128] In certain embodiments, the immunogenic polypeptide may be a protein or fragment thereof derived from hepatitis B virus, hepatitis A virus, hepatitis C virus, or hepatitis E virus. In such embodiments, the disease may be viral hepatitis (e.g., hepatitis B virus, hepatitis A virus, hepatitis C virus, or hepatitis E virus).

[0129] In certain embodiments, the immunogenic polypeptide may be an envelope glycoprotein or fragment thereof derived from the measles virus, hi such embodiments, the disease may be measles.

[0130] In certain embodiments, the immunogenic polypeptide may be a protein or fragment thereof derived from the mumps virus, hi such embodiments, the disease may be mumps.

[0131] In certain embodiments, the subject is a mammal, such as a human, monkey, or mouse.

[0132] In a sixteenth aspect, the present application provides a system for preparing a particulate immunogenic polypeptide, the system comprising a first vector and a second vector, wherein the first vector comprises a nucleotide sequence encoding a fusion protein, the fusion protein comprising an immunogenic polypeptide and an assembly polypeptide, and the second vector comprises a nucleotide sequence encoding a nanobody, wherein the nanobody can specifically bind to the assembly polypeptide, and the assembly polypeptide can assemble into a VLP.

[0133] In certain embodiments, the nucleotide sequence is codon-optimized or non-optimized depending on the codon preferences of the host cell.

[0134] In certain embodiments, the assembly polypeptide is selected from a protein of Hepatitis E Virus (HEV) or a fragment or variant thereof. In certain embodiments, the assembly polypeptide is as defined in the first aspect.

[0135] In certain embodiments, the fusion protein is as defined in the first aspect.

[0136] In a seventeenth aspect, the present application provides a method for enhancing the immunogenicity of an immunogenic polypeptide, the method comprising preparing or obtaining a fusion protein comprising the immunogenic polypeptide and a Nanobody capable of specifically binding to an assembly polypeptide, and contacting the fusion protein with a VLP comprising the assembly polypeptide, thereby obtaining a particulate antigen comprising the immunogenic polypeptide attached to the VLP.

[0137] In some embodiments, the method comprises using the system described in 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, and (2) contacting the fusion protein and the assembly polypeptide under conditions that allow VLP assembly.

[0138] In some embodiments, the Nanobody is as defined in the first aspect.

[0139] In some embodiments, the fusion protein is as defined in the first aspect.

[0140] In some embodiments, the assembly polypeptide assembles into a VLP, hi some embodiments, the fusion protein is attached to the VLP through the interaction of the Nanobody with the assembly polypeptide.

[0141] Definition of Terms In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the experimental procedures of virology, biochemistry, and immunology used herein are all routine procedures widely used in the corresponding fields. In addition, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0142] As used herein, the term "immunogenic polypeptide" refers to a protein or polypeptide that can induce an immune response. In certain embodiments, an immunogenic polypeptide can be administered (directly or indirectly) to a subject, thereby inducing an immune response in the subject. It will be understood by those skilled in the art that an immunogenic polypeptide can be a naturally occurring or a protein or polypeptide that has naturally occurring or artificially introduced mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) that do not affect its biological activity (biological activity, as used herein, is the ability to induce an immune response). Thus, as used herein, an immunogenic polypeptide can be a polypeptide or variant thereof derived from a non-pathogen (e.g., tumor cell) or a pathogen (e.g., virus, bacteria, fungus, parasite, or other pathogen).

[0143] As used herein, the term "immune response" refers to the response of a cell of the immune system (e.g., a B cell, a T cell, or a monocyte) to a stimulus. In some embodiments, the immune response is a response specific to a particular antigen (i.e., an antigen-specific response). In some embodiments, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In other embodiments, the immune response is a B cell response that results in the production of specific antibodies.

[0144] As used herein, the term "subvirus" refers to a microorganism that does not have a complete virus structure, and primarily includes virus-like bodies, mimics, and prions.

[0145] As used herein, the terms "SARS-CoV-2 RBD protein" and "SARS-CoV-2 RBD" have the same meaning and can be used interchangeably. This term refers to the receptor-binding domain (RBD) on the spike protein (S protein) of SARS-CoV-2. The primary function of the RBD is to recognize host cell surface receptors and mediate fusion with the host cell. Different SARS-CoV-2 virus strains may contain RBD proteins with different sequences, but it is understood by those skilled in the art that these RBD proteins have the same or similar biological properties. Therefore, in the present invention, the RBD protein includes not only the proteins set forth in SEQ ID NOs: 1 to 8, but also the RBD proteins of various SARS-CoV-2 virus strains. The amino acid sequences of these RBD proteins can be obtained from public databases (e.g., GenBank database), for example, the amino acid sequences set forth in GenBank under accession numbers OP077006.1, OP077005.1, and OP077003.1.

[0146] As used herein, the terms "VZV gE protein," "VZV gE," and "VZV glycoprotein gE" refer to a type of VZV envelope glycoprotein, have the same meaning, and can be used interchangeably. Different VZV virus strains may contain gE proteins with different sequences, but it is understood by those skilled in the art that these gE proteins have the same or similar biological properties. Therefore, in the present invention, the gE protein includes not only the protein set forth in SEQ ID NO: 30, but also gE proteins of various VZV strains. The amino acid sequences of these gE proteins can be obtained from public databases (e.g., the GenBank database).

[0147] As used herein, the term "HIV-1 Env" refers to the envelope protein on the surface of the HIV-1 virus, also referred to herein as "BGTSTIP." It is understood by those skilled in the art that different HIV-1 strains may contain Env proteins with different sequences, but these Env proteins have the same or similar biological properties. Therefore, in the present invention, Env proteins include not only the proteins set forth in SEQ ID NOs: 34 and 35, but also Env proteins of various HIV-1 strains. The amino acid sequences of these Env proteins can be obtained from public databases (e.g., the GenBank database).

[0148] The capsid protein encoded by the ORF2 gene of HEV (also referred to as ORF2 protein) and fragments thereof (e.g., p239 protein, p495 protein) have been confirmed to have the ability to assemble into VLPs. The sequence of the ORF2 protein is well known in the art. See, for example, DDBJ database accession number: D11092. The p239 protein has a sequence corresponding to amino acids 368 to 606 of the ORF2 protein. In certain embodiments, the p495 protein has a sequence corresponding to amino acids 112 to 606 of the ORF2 protein.

[0149] As used herein, the term "assembly polypeptide" refers to a protein or polypeptide that can assemble into a virus-like particle (VLP). In certain embodiments, the assembly polypeptide is a capsid protein of a naturally occurring virus or virus-like body. In such embodiments, the VLP assembled by the assembly polypeptide is structurally similar to a naturally occurring virus or virus-like body, except that it does not contain the genome of the naturally occurring virus or virus-like body. In certain embodiments, the assembly polypeptide is an artificially prepared and / or screened polypeptide. In such embodiments, the VLP assembled by the assembly polypeptide may or may not be structurally similar to a naturally occurring virus or virus-like body. The term "virus-like particle (VLP)" refers to a multimeric particle that may or may not be structurally similar to a naturally occurring virus or subvirus. It has been determined that proteins (e.g., capsid proteins, surface proteins, envelope proteins) of several viruses (e.g., HBV, HEV, HPV) can spontaneously form VLPs after recombinant expression in an appropriate expression system.

[0150] Those skilled in the art can identify proteins or polypeptides capable of assembling into VLPs by published methods, for example, by placing the protein to be tested in a buffer solution (e.g., PBS solution) at room temperature and then detecting the presence of VLPs. The presence of VLPs can be detected using conventional techniques known in the art, such as electron microscopy, biophysical characterization, etc. Specific detection methods can be found, for example, in Baker et al. (1991) Biophys. J. 60: 1445-1456 and Hagensee et al. (1994) J. Virol. 68: 4503-4505. For example, the capsid protein encoded by the ORF2 gene of HEV (also referred to as ORF2 protein) and fragments thereof (e.g., p239 protein, p495 protein) have been confirmed to have the ability to assemble into VLPs.

[0151] Those skilled in the art will understand that, in the present application, assembly polypeptides include not only naturally occurring proteins or polypeptides that can assemble into VLPs, but also proteins or polypeptides that have mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) that are naturally occurring or artificially introduced based on naturally occurring proteins but that do not affect their biological function (as used herein, biological function is the ability to assemble into VLPs). Thus, in some embodiments, the assembly polypeptide is selected from the ORF2 protein of hepatitis E virus (HEV), or a fragment or variant thereof. In some embodiments, the assembly polypeptide is selected from the p239 protein, or a fragment or variant thereof, or the p495 protein, or a fragment or variant thereof.

[0152] In accordance with the present invention, the term "variant" when used in the context of a protein / polypeptide 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 variant retains the biological activity of the protein / polypeptide from which it is derived.

[0153] As used herein, the phrase "an immunogenic polypeptide is a polypeptide of biological or non-biological origin or an immunogenic variant thereof" means that the sequence of the polypeptide is derived from a sequence in a living or non-living organism, but the method for obtaining the polypeptide is not limited to a particular production method. In certain embodiments, the polypeptide may be naturally isolated, artificially synthesized, or obtained by genetic recombination.

[0154] As used herein, the term "particulate immunogenic polypeptide" refers to a collection of immunogenic polypeptides in the form of a particle. In certain embodiments, particulate immunogenic polypeptide refers to an immunogenic polypeptide attached to a virus-like particle.

[0155] As used herein, the term "organism" refers to a living individual or object. Except for a few species, such as viruses, organisms are made up of cells. In certain embodiments, organisms include pathogens and non-pathogens.

[0156] As used herein, the term "pathogen" refers to a microorganism (e.g., bacteria, virus, rickettsia, fungus), parasite or other vector (e.g., recombinant microorganism) that can cause infectious diseases in humans, animals and plants.

[0157] As used herein, the term "fusion protein" refers to a recombinant protein formed by linking the amino acid sequences of at least two independent proteins or polypeptides, which may be linked directly or via a linker.

[0158] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the expected properties of a protein / polypeptide containing the amino acid sequence. For example, 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 replacement of an amino acid residue with an amino acid residue having a similar side chain, for example, replacement with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., a residue having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993), Kobayashi et al. Protein Eng. 12(10):879-884 (1999), and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0159] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements carried by the vector are expressed in the host cell. Vectors are well known to those skilled in the art, including, but 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 λ phage or M13 phage, and viral vectors.

[0160] As used herein, the term "host cell" refers to cells that can be used to amplify or express exogenous genes, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, 293T cells or human cells.

[0161] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target (e.g., a carbohydrate, a polynucleotide, a lipid, a polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Antibodies include antibodies of any type, e.g., IgG, IgA, or IgM (or subtypes thereof), and antibodies need not belong to any particular type. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the antibody heavy chain constant region. There are five major types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subtypes (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different types of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Antibody light chains can be classified into κ (kappa) light chains and λ (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 four 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 binding the antibody to an antigen but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0162] The VH and VL regions of an antibody can also be subdivided into alternating highly variable regions (called complementarity-determining regions (CDRs)) and more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form an antigen-binding site, respectively. The assignment of amino acids to various regions or domains is based on the Kabat definition (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883), the Chothia definition (the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the location of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917, Chothia et al. (1989) Nature 342:878-883)) and / or the AbM definition (AbM The CDR definition method can be followed from the related work by Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272).

[0163] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3. The exact 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. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one skilled in the art can readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In the present invention, the CDRs contained in a Nanobody can be determined according to various numbering systems known in the art.

[0164] As used herein, the term "framework region" or "FR" residues refers to amino acid residues in antibody variable regions other than the CDR residues as defined above.

[0165] As used herein, the term "single domain antibody (sdAb)" is also referred to as nanobody, and the two terms may be used interchangeably. This term has the meaning commonly understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric antibody variable domain (e.g., a single heavy chain variable region, also referred to as VHH) that retains the ability to specifically bind to the same antigen as a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). A single domain antibody or nanobody may be an alpaca antibody derived from a camel or a shark antibody derived from a shark.

[0166] The specificity of Nanobodies can be screened in the same manner as for intact antibodies, using conventional techniques known to those skilled in the art.

[0167] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and 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.

[0168] In accordance with the present invention, the term "adjuvant" refers to an immunopotentiating agent that, when delivered to the body together with or before an antigen, can enhance or modify the type of immune response of the body to the antigen. There are many types of adjuvants, including, but not limited to, aluminum salt adjuvants, mixed zinc and aluminum adjuvants (e.g., FH002C), Freund's adjuvant, oil emulsion adjuvants, cytokines, TLR agonists, CpG adjuvants, liposomes, AS01B adjuvants, or combinations thereof.

[0169] As used herein, the term "pharmaceutically acceptable" means that it is recognized in the pharmaceutical field that it can be used in animals, particularly humans. As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredient, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences (ed. by Gennaro AR), 19th ed., PA: Mack Publishing Company, 1995), including, but not limited to, pH adjusting agents (including, but not limited to, phosphate buffers), surfactants (including, but not limited to, cationic, anionic, or non-ionic surfactants, e.g., Tween-80), adjuvants, ionic strength enhancers (including, but not limited to, sodium chloride), diluents, excipients, vehicles for containing or administering therapeutic agents, and any combination thereof.

[0170] As used herein, the term "subject" refers to a mammal, including, but not limited to, humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, etc. Preferably, the subject is a human.

[0171] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A disease-therapeutic effective amount refers to an amount sufficient to cure or at least partially prevent a disease and its complications in a patient already suffering from the disease. Determining such effective amounts is well within the capabilities of one skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's overall condition, e.g., age, weight, and sex, the form of drug administration, and other concurrently administered treatments, etc.

[0172] As used herein, the term "conditions permitting VLP assembly" refers to conditions under which assembly polypeptides can assemble into VLPs. Typically, when an assembly-competent polypeptide or protein is placed in a liquid, the polypeptide or protein can self-assemble into a VLP. In certain embodiments, conditions permitting VLP assembly are when the assembly polypeptide is placed in a solution. In such embodiments, the solution does not contain components that do not contribute to VLP assembly. In certain embodiments, the solution contains components that contribute to VLP assembly, for example, the solution is a buffer containing salt. [Effects of the Invention]

[0173] In the present invention, the ability of assembly polypeptides (e.g., HEV ORF2 protein or a fragment or variant thereof, HPV L1 protein or a fragment or variant thereof, HBV surface antigen or a fragment or variant thereof) to assemble into VLPs is utilized to fusion-express an immunogenic polypeptide with a Nanobody that specifically binds to the assembly polypeptide, and the targeting binding properties of the Nanobody to the assembly polypeptide are utilized to display various immunogenic polypeptides on the surface of the VLP formed from the assembly polypeptide, thereby obtaining particulate immunogenic polypeptides. Thus, the present application provides a system and method for particulating immunogenic polypeptides.

[0174] Furthermore, compared with conventional immunogenic polypeptides, such particulate immunogenic polypeptides have higher immunogenicity and can stimulate high-level immune responses, including B cell and T cell responses. Therefore, the particulate immunogenic polypeptides of the present invention are particularly suitable for vaccine production and vaccination, and are advantageous for the prevention and / or treatment of viral infections.

[0175] The system and method for particle-forming immunogenic polypeptides of the present invention use nanobodies. The use of nanobodies offers at least the following advantages over conventional techniques: 1. Nanobody-mediated display of immunogenic polypeptides is not affected by the N- / C-terminal structure of the assembly polypeptide, thereby enabling more flexible assembly strategies. 2. Nanobody-mediated display of immunogenic polypeptides does not affect the assembly and conformation of the assembly polypeptide, thereby providing broader applicability. 3. Fusion of nanobodies to immunogenic polypeptides contributes to maintaining the conformation of the immunogenic polypeptide. 4. Nanobodies with different epitopes can be combined, which contributes to increasing the presentation rate of the immunogenic polypeptide. 5. Nanobodies can be rationally designed based on the symmetry of the oligomeric state of the assembly polypeptide and the immunogenic polypeptide. 6. Nanobody-mediated display of immunogenic polypeptides can reduce potential steric hindrance of the immunogenic polypeptide. 7. In this system and method, unmodified or modified VLPs can be used directly. If a commercially available VLP has good safety characteristics, there is no need to consider the druggability of the VLP itself, and it has good conversion potential. 8. The immunogenicity of nanobodies as linkers can be minimized by humanization strategies well known in the art, thereby reducing unnecessary immune responses in the body and focusing immunity on the immunogenic polypeptide. 9. The method of the present invention allows any immunogenic polypeptide to be made into a particle through fusion expression with a nanobody, making it universal and versatile. 10. Due to the small size of nanobodies, screening and identifying the binding site of nanobodies can preserve the exposure of the original major immune epitopes of particle carriers such as HEV ORF2, HPV L1, and HBV S. Therefore, the carrier after particleization of immunogenic polypeptides still has good immunogenicity and can be used as a combination vaccine. This strategy can also be used to develop other combination vaccines, and therefore has great potential for application.

[0176] Although the embodiments of the present invention will be described in detail in conjunction with the accompanying drawings and examples, those skilled in the art will understand that the following drawings and examples are used only to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following drawings and detailed description of the preferred embodiments. [Brief explanation of the drawings]

[0177] [Figure 1A] FIG. 1 shows the nanobody screening process in an embodiment of the present invention, showing the results of colony PCR agarose gel electrophoresis for a bacterial library. [Figure 1B] The results of evaluating the diversity of the bacterial library after sequencing are shown. [Figure 2A] 1 is a photograph showing the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of various VHH-RBD proteins produced and purified using an insect cell expression system in an example of the present invention. M represents a molecular weight marker. [Figure 2B] 1 is a photograph showing the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of various VHH-RBD proteins produced and purified using a mammalian cell expression system in an example of the present invention. M represents a molecular weight marker. [Figure 3] 1 is a photograph showing the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of various VHH-gE fusion proteins in an example of the present invention, where M represents a molecular weight marker and lanes 1 to 7 each represent a different VHH-gE fusion protein. [Figure 4] 1 is a photograph showing the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of various VHH-BGTSTIP proteins in an example of the present invention, where "+" indicates reducing conditions and "-" indicates non-reducing conditions. [Figure 5A]FIG. 1 is a photograph showing the results of Western blot of various VHH-RBD fusion proteins in an example of the present invention, which were produced using an insect cell expression system and purified. M represents a molecular weight marker, and lanes 1 to 8 each represent a different nanobody-RBD fusion protein. [Figure 5B] FIG. 1 is a photograph showing the results of Western blot of various VHH-RBD fusion proteins in an example of the present invention, which were produced using the mammalian cell expression system 293F cells and purified. M represents a molecular weight marker, and lanes 1 to 8 each represent a different nanobody-RBD fusion protein. [Figure 6] 1 is a photograph showing the results of Western blot of various candidate VHH-gE molecules in an example of the present invention, where M represents a molecular weight marker and lanes 1 to 7 each show a different fusion protein of Nanobody and gE. [Figure 7A] FIG. 1 is a graph showing the results of ELISA for various VHH-RBDs and various reported specific monoclonal antibodies in the Examples, showing the activity identification results of various VHH-RBD proteins produced and purified using an insect cell expression system, including broad-spectrum neutralizing antibodies JSR-105, JSR-551, JSR-209, m6D6, m7D6, and the non-broad-spectrum neutralizing antibody 85F7. [Figure 7B] FIG. 1 is a graph showing the results of ELISA for various VHH-RBDs and various reported specific monoclonal antibodies in the Examples. The graph shows the activity identification results for various VHH-RBD proteins produced and purified using the mammalian cell expression system 293F cells, and the antibodies include 3G11, 8H12, 13F10, 8B8, 9D3, and 3F9. [Figure 8] 1 is a graph showing the results of ELISA for various VHH-gEs and various specific monoclonal antibodies in the examples, including 3H7, 4G4, 6B7, 11B11, 11B12, 13B6, 14G1, and 17B7. [Figure 9]1 is a graph showing the results of ELISA of various VHH-BGTSTIPs and various reported HIV-1 neutralizing or non-neutralizing antibodies in an example. [Figure 10A] 1 is a graph showing the results of affinity analysis of various VHH-RBDs for HEV-p239 protein in an example, showing the affinity results for HEV-p239 protein of VHH-RBDs produced and purified using an insect cell expression system. [Figure 10B] 1 is a graph showing the results of affinity analysis of various VHH-RBDs for HEV-p239 protein in an example, showing the affinity results for HEV-p239 protein of VHH-RBDs produced and purified using the mammalian cell expression system 293F cells. [Figure 11] 1 is a graph showing the results of affinity analysis of various VHH-gEs for HEV-p239 protein in an example. [Figure 12] 1 is a graph showing the results of affinity analysis of various VHH-BGTSTIPs for HEV-p239 protein in an example. [Figure 13A] 1 is a graph showing the results of purification by Superdex 200 increase (high performance liquid chromatography molecular sieve) of P1-5B-RBD produced and purified using an insect cell expression system in an example of the present invention. [Figure 13B] 1 is a graph showing the results of purification using Superdex 200 increase of P2-6D-RBD, P2-3E-RBD, and P2-10G-RBD, which were produced and purified using a mammalian cell expression system in an example of the present invention. [Figure 14A] FIG. 1 shows the purification profile of the complex formed by the P1-5B-RBD fusion protein purified from an insect cell expression system and HEV-p239, and the results of its identification by SDS-PAGE. [Figure 14B]Figure 1 shows the purification profiles and SDS-PAGE identification of complexes formed by P2-3E-RBD fusion protein, P2-10G-RBD fusion protein, and P2-6D-RBD fusion protein and HEV-p239, each purified from a mammalian cell expression system. [Figure 15A] FIG. 1 shows the purification profile and SDS-PAGE identification results of the complex formed by the candidate fusion protein P2-8C-gE and HEV-p239 in an example of the present invention, and shows the results of complex purification. [Figure 15B] FIG. 1 shows the purification profile and SDS-PAGE identification results of the complex formed by the candidate fusion protein P2-8C-gE and HEV-p239 in an example of the present invention. FIG. 2 shows the SDS-PAGE identification results of the complex formed by P2-8C-gE and HEV-p239. [Figure 16] FIG. 1 shows the purification profile of the complex formed by the candidate fusion protein P2-5C-BGTSTIP and HEV-p239 in an example of the present invention, and the results of identification by SDS-PAGE. [Figure 17] 1 is a graph showing the results of HPSEC of P2-8C-gE in an example of the present invention. [Figure 18A] 1 is a graph showing the molecular size detection results of the HEV-RBD complex formed by VHH-RBD and HEV-p239 protein produced and purified using an insect cell expression system in an example of the present invention, and the HEV-p239 particle. [Figure 18B] 1 is a graph showing the molecular size detection results of three HEV-RBD complexes formed by VHH-RBD and HEV-p239 protein produced and purified using a mammalian cell expression system in an example of the present invention, and HEV-p239 particles. [Figure 19] 19A and 19B are graphs showing the molecular size detection results of HEV-gE complex samples of the present invention, where Fig. 19A shows the molecular size of HEV-p239 particles, and Fig. 19B shows the molecular size of HEV-gE complexes. [Figure 20]1 is a graph showing the results of detecting the molecular sizes of HEV-BGTSTIP complexes and HEV-p239 particles in an example of the present invention. [Figure 21A] This figure shows the results of analytical ultracentrifugation of HEV-p239 and RBD complexes constructed based on HEV-p239 particles in an example of the present invention. The figure also shows the results of analytical ultracentrifugation of complex particles prepared from purified VHH-RBD and HEV-p239 produced using an insect cell expression system (right panel), and HEV-p239 (left panel). [Figure 21B] 1 shows the results of analytical ultracentrifugation of HEV-p239 and RBD complexes constructed based on HEV-p239 particles in an example of the invention, and also shows the results of analytical ultracentrifugation of three types of complex particles prepared from purified VHH-RBD and HEV-p239 produced using the mammalian cell expression system 293F cells. [Figure 22] 22A and 22B are graphs showing analytical ultracentrifugation results for HEV-p239 and gE complexes constructed based on HEV-p239 particles in an example of the present invention. Figure 22A shows that HEV-p239 exhibits a single component with a sedimentation coefficient of 22S. Figure 22B shows that the gE complex exhibits a single component with a sedimentation coefficient of 31S. [Figure 23] 1 is a graph showing the results of analytical ultracentrifugation of HEV-p239 and Env(BGTSTIP) complexes constructed based on HEV-p239 particles in an example of the present invention. [Figure 24A] 1 is a photograph showing the results of negative staining of a transmission electron microscope of a complex sample prepared from HEV-p239 and VHH-RBD produced and purified using an insect cell expression system in an example of the present invention. [Figure 24B] 1 is a photograph showing the results of negative staining of a transmission electron microscope of a complex sample prepared from HEV-p239 and VHH-RBD produced and purified using mammalian expression system 293F cells in an example of the present invention. [Figure 25] 1 is a photograph showing the results of transmission electron microscopy negative staining of a complex sample prepared from HEV-p239 and a VHH-gE fusion protein of the present invention. [Figure 26] 1 is a photograph showing the results of negative staining of transmission electron microscopy of HEV-p239 and Env(BGTSTIP) complexes constructed based on HEV-p239 particles in an example of the present invention. [Figure 27A] 1 is a graph showing the immunogenicity detection results of RBD particulate antigens in an example of the present invention, showing the detection results of serum binding and neutralizing activity after immunizing mice with RBD particulate antigens obtained using an insect cell expression system. [Figure 27B1] This shows the results of detecting serum binding activity and wild-type SARS-CoV-2 pseudovirus neutralizing activity after immunizing mice with RBD particulate antigen obtained by a mammalian cell expression system. [Figure 27B2] This shows the results of detecting serum binding activity and wild-type SARS-CoV-2 pseudovirus neutralizing activity after immunizing mice with RBD particulate antigen obtained by a mammalian cell expression system. [Figure 27C] This shows the results of detecting serum binding activity and wild-type SARS-CoV-2 pseudovirus neutralizing activity after immunizing mice with RBD particulate antigen obtained by a mammalian cell expression system. [Figure 27D] Not specified [Figure 28] This is a graph showing the results of neutralization detection of antibodies induced by RBD particulate antigen and aluminum adjuvant in an example of the present invention against the wild-type (WT), gamma, and BA.2 strains of SARS-CoV-2. [Figure 29] 1 is a graph showing the immunogenicity detection results of gE particulate antigen in an example of the present invention. [Figure 30] 1 is a graph showing the results of live virus neutralization detection in mouse immune serum against gE particulate antigen in an example of the present invention. [Figure 31] 1 is a graph showing the results of flow cytometry immunoassay of monomeric gE antigen, gE particulate antigen, and control adjuvant in an example of the present invention. [Figure 32]FIG. 1 shows the results of enzyme-linked immunospot assays detecting cytokines in groups immunized with monomeric gE antigen, gE particulate antigen, and a control adjuvant in an example of the present invention. [Figure 33] Graph showing the results of humanization of Nanobody P1-5B in an example of the present invention. [Figure 34] 1 is a photograph showing the results of polyacrylamide electrophoresis of the single domain antibody fusion protein P1F8-BGTSTIP in an example of the present invention, where M represents a molecular weight marker, "+" indicates reducing conditions, and "-" indicates non-reducing conditions. [Figure 35] FIG. 1 shows the results of ELISA of the single domain antibody fusion protein P1F8-BGTSTIP in an example of the present invention and various reported broad-spectrum neutralizing and non-neutralizing antibodies against HIV-1 (2G12, VRC01, PGT121, PGT121, SF12, and B12 are broad-spectrum neutralizing antibodies, and 17b, F105, and F240 are non-neutralizing antibodies). [Figure 36] 1 is a graph showing the results of affinity analysis of the single domain antibody fusion protein P1F8-BGTSTIP for HPV 58 VLP in an example. [Figure 37] 1 shows the results of high-performance size-exclusion chromatography (HPSEC) and SDS-PAGE analysis of the complex formed by P1F8-BGTSTIP and 58VLP, which was produced and purified using a mammalian expression system in an example of the present invention. The red curve represents the spectrum of the complex formed by P1F8-BGTSTIP and 58VLP, the green curve represents the spectrum of 58VLP, and the blue curve represents the spectrum of the fusion protein P1F8-BGTSTIP. [Figure 38] 1 is a graph showing the results of analytical ultracentrifugation of a complex formed by P1F8-BGTSTIP and 58VLP and 58VLP in an example of the present invention. [Figure 39] 1 is a photograph showing the results of negative staining of a transmission electron microscope of a complex sample formed by P1F8-BGTSTIP and 58VLP in an example of the present invention. [Figure 40] 1 is a graph showing the titer detection results of BGTSTIP-specific binding antibodies after mice were immunized with the particulate antigen 58VLP-BGTSTIP in an example of the present invention. [Figure 41] FIG. 1 shows the SDS-PAGE identification of particulate antigens formed after binding of HBsAg with the nanobody fusion protein S2-gE of the invention. [Figure 42] FIG. 1 shows the molecular sieve identification of particulate antigens formed after binding of HBsAg with the nanobody fusion protein S2-gE of the invention. [Figure 43] FIG. 1 shows the DLS identification of the particulate antigen HBsAg-S2-gE (HBV-gE) formed after binding of HBsAg with the nanobody fusion protein S2-gE of the invention. [Figure 44] 1 is a negative staining transmission electron microscope image of the particulate antigen HBsAg-S2-gE formed after binding of HBsAg with the nanobody fusion protein S2-gE of the invention. [Figure 45] 1 is a graph showing the specific antibody titer results of mice immunized with HBsAg-S2-gE, a particulate antigen formed after binding of HBsAg to the nanobody fusion protein S2-gE of the invention, and mice immunized with S2-gE. DETAILED DESCRIPTION OF THE INVENTION

[0178] Sequence information Some sequence information relevant to the present invention is shown in Table 1 below.

[0179] [Table 1-1]

[0180] [Table 1-2]

[0181] [Table 1-3]

[0182] Table 1-4

[0183] Table 1-5

[0184] Table 1-6

[0185] Table 1-7

[0186] Table 1-8

[0187] Table 1-9

[0188] Table 1-10

[0189] Table 1-11

[0190] Table 1-12

[0191] Table 1-13

[0192] [Table 1-14]

[0193] [Table 1-15]

[0194] Specific Models for Implementing the Invention The invention will now be described with reference to the following examples, which are intended to illustrate (but not limit) the invention.

[0195] Unless otherwise specified, the molecular biological experimental methods and immunoassays used in the present invention are essentially performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. The use of restriction endonucleases follows the conditions recommended by the product manufacturers. Those skilled in the art will understand that the examples are intended to illustrate the present invention and do not limit the scope of protection claimed by the present invention.

[0196] Furthermore, for examples where no specific conditions are indicated, the examples were carried out under conventional conditions or those recommended by the manufacturer. All drugs or equipment used for which no manufacturer is indicated are conventional products that can be purchased commercially. Those skilled in the art will appreciate that the examples are provided to illustrate the invention and do not limit the scope of protection sought by the invention. This disclosure and all other references mentioned herein are incorporated by reference in their entirety. [Example]

[0197] Example 1: Nanobody phage library construction and screening process 1. RNA extraction (Trizol method) (1) Peripheral blood lymphocytes (obtained from alpacas) preserved in Trizol were transferred to a 1.5 mL centrifuge tube, and 1 / 5 volume of chloroform was added and mixed; (2) After standing at room temperature for 5 minutes, centrifugation was carried out at 12,000 g for 15 minutes at 4°C; (3) After centrifugation, the supernatant was carefully transferred to a new centrifuge tube; (4) An equal volume of isopropanol was added to the new centrifuge tube; (5) After standing at room temperature for 10 minutes, centrifugation was carried out at 4°C and 12000 g for 10 minutes; (6) The precipitate in each tube was washed with 1 mL of 75% ethanol, centrifuged at 7500 g for 5 minutes to remove the ethanol, and dried. The precipitate was then dissolved in an appropriate amount of ribonuclease-free water, and all samples were combined to obtain extracted total RNA.

[0198] 2. Reverse transcription of cDNA (Takara Bio Inc. reverse transcription kit)

[0199] 3. PCR Amplification

[0200] [Table 2]

[0201] 4. Enzymatic Digestion and Ligation

[0202] [Table 3]

[0203] 5. Construction of Bacterial and Phage Libraries

[0204] 6. Phage Screening, Purification, and Amplification V(μl)=10 12 / T ライブラリー ×1000 (where V is the volume of the added phage (unit: μL), and T ライブラリー was the phage titer) T(pfu / ml) = N × D × 400 where T is the phage titer (unit: pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0205] 7. Library Quality Verification Figures 1A and 1B show the nanobody screening process of an embodiment of the present invention. Figure 1A shows the results of colony PCR agarose gel electrophoresis. Of 51 randomly selected single clones, 50 were positive clones, resulting in a positive rate of 98%, which met the requirements for the positive clone rate of the bacterial library. Figure 1B shows the protein sequences obtained by translation using software after sequencing. Sequence diversity comparison showed that all 50 sequences were independent and had good diversity, meeting the requirements for the diversity of the bacterial library.

[0206] 8. Monoclonal ELISA Detection After calculation according to the following formula, the auxiliary phage M13K07 was added to each well so that the ratio of bacteria number to phage number was 1:20: V (ml) = OD600 × 1.6 × 10 11 / T ヘルパー-ファージ (where V is the volume of the supplementary phage added (unit: mL), and Tヘルパー-ファージ was the titer of the auxiliary phage used).

[0207] 9. Performing Secondary ELISA Validation on Selected Positive Clones The final positive clones were sent to a biotech company for sequencing and analysis, yielding a total of 20 Nanobody heavy chain variable region (VHH) sequences. The amino acid sequences of these Nanobodies are shown in SEQ ID NOs: 10 to 29 in Table 1, and the CDR1 to CDR3 sequences are shown in SEQ ID NOs: 57 to 62 and 90 to 144 in Table 1.

[0208] Example 2: Preparation and expression of VHH-RBD fusion proteins, VHH-gE fusion proteins, and VHH-BGTSTIP fusion proteins 1. Fusion Protein Preparation The construction of VHH-RBD sequences in an insect cell expression system involved the following: RBD sequences (SEQ ID NO: 1 to SEQ ID NO: 8) were linked to the C-terminus of VHH sequences (SEQ ID NO: 10 to SEQ ID NO: 29), and then a bee venom signal peptide (SEQ ID NO: 31) was linked to the N-terminus of the above sequences to facilitate protein secretion and expression. Nucleotide sequences encoding the above amino acid sequences were synthesized by Sangon Biotech, and the nucleotide sequences were constructed on the PIEX / bac-1 vector via the NcoI and BamHI restriction sites. Finally, eight VHH-RBD proteins (P1-5B-RBD, P2-2C-RBD, P1-1B-RBD, P1-1G-RBD, P2-4E-RBD, P2-3E-RBD, P2-3D-RBD, and P2-3A-RBD) were obtained using the insect cell expression system. Their sequences are shown in SEQ ID NO: 42 to SEQ ID NO: 49 in Table 1.

[0209] VHH-gE fusion proteins were constructed using the same method, except that the RBD sequence linked to the C-terminus of the VHH sequence was replaced with the gE sequence (SEQ ID NO: 30). Finally, 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 using an insect cell expression system. Their sequences are shown in SEQ ID NOs: 50 to 56 in Table 1.

[0210] The VHH-RBD sequence was constructed in a mammalian cell expression system using the following procedure: a (G4S)3 flexible protein linker (SEQ ID NO: 39), RBD sequences (SEQ ID NOs: 1 to 8), and a histidine tag (8-His tag) were sequentially linked to the C-terminus of the VHH sequence (SEQ ID NOs: 10 to 29). A signal peptide sequence (SEQ ID NO: 38) was introduced at the N-terminus to facilitate protein secretion and expression. The nucleotide sequences encoding the above amino acid sequences were codon-optimized and then synthesized by Tongyong Shengwu Co., Ltd. and cloned between the ECORI and XbaI restriction sites of the pcDNA3.1 vector. Finally, eight VHH-RBD proteins (P1-1B-RBD, P1-5B-RBD, P2-2C-RBD, P2-5C-RBD, P2-3A-RBD, P2-3E-RBD, P2-4E-RBD, and P2-5G-RBD) were obtained using a mammalian cell expression system. The sequences are shown in Table 1 as SEQ ID NO: 72 to SEQ ID NO: 89.

[0211] VHH-BGTSTIP (i.e., VHH-Env) fusion proteins were constructed using the same method, except that the linked RBD sequence was replaced with the full-length Env extracellular segment amino acid sequence (SEQ ID NO: 34 or SEQ ID NO: 35). Finally, eight VHH-BGTSTIP proteins (P1-1B-BGTSTIP, P1-5B-BGTSTIP, P2-2C-BGTSTIP, P2-5C-BGTSTIP, P2-3A-BGTSTIP, P2-3E-BGTSTIP, PA-4G-BGTSTIP, and P2-7D-BGTSTIP) were obtained using a mammalian cell expression system. Their sequences are shown in SEQ ID NOs: 80 to 87 in Table 1.

[0212] 2. Expression of VHH-RBD and VHH-gE in an insect expression system Transfection into insect cells (1) sf9 cells (purchased from Invitrogen, 11496-015) or sf21 cells (purchased from Invitrogen, 11497-013) were in the logarithmic growth phase (1.5 × 10 6 / mL ~ 2.5 × 10 6The viability was maintained at >90% (per mL). 200 μL of ESF921 medium (purchased from Expression Systems, Inc., 96-001-01) containing 2% FBS, 0.1 μg of baculovirus DNA (purchased from Expression Systems, Inc., 91-002), and 1 μg of pAc-S plasmid was added to a 24-well plate and mixed thoroughly. 1 μL of transfection reagent (purchased from Expression Systems, Inc., 95-055-075) was added to 50 μL of ESF921 medium (purchased from Expression Systems, Inc., 96-001-01) and mixed thoroughly. These two reagents were combined into a single tube, mixed thoroughly, and allowed to stand at room temperature for 30 minutes. During the incubation period, the cells were washed with the following solution (performed near the end of incubation): After the cells had completely attached to the wall, the medium was removed with a pipette, and then 300 μL of ESF921 medium was added in a quick motion to avoid cell dehydration. After gentle shaking, the medium was removed and 300 μL of ESF921 medium was added again. After the specified time had elapsed, approximately 100 μL of the above mixture was added dropwise evenly to each well containing cells. The incubation was then carried out at 27°C for 6 hours, after which the supernatant was discarded and 500 μL of complete culture medium (50% CCM3 + 50% TNM-FH (Sigma-Aldrich, T1032) + 10% FBS) was added.

[0213] (2) The cell supernatant obtained in step (1) was collected and centrifuged at 500 g for 5 minutes, the cell residue and debris were discarded, and the supernatant was stored at 4°C in the dark and used as the P1 virus seed solution.

[0214] (3) Baculovirus amplification sf9 or sf21 cells were in the logarithmic growth phase (1.5 × 10 6 / mL ~ 2.5 × 10 6 / mL) and maintained a survival rate of over 90%. 58-10 mL of cells at a density of 1000 μg / mL were coated onto a 10 cm plate and left to stand for 15 minutes to allow the cells to adhere to the wall. Approximately 600 μL of P1 virus solution was added dropwise evenly and incubated at 27°C for 3-4 days. Cytopathic effects were observed. The cell supernatant was collected, centrifuged at 1000 rpm for 5 minutes to remove cell debris and then filtered through a 0.22 μm filter. The supernatant was stored at 4°C in the dark and used as the P2 virus seed solution. The P2 virus titer was approximately 10 6 pfu / mL ~10 7 pfu / mL. P3 virus can be amplified according to this method in a volume-scaled manner in 250 mL shake flasks.

[0215] Manipulating protein expression in insect cells Density is 2×10 6 H5 cells (purchased from Invitrogen, B855-02) with a viability of over 90% and a viral load of 1 / mL were cultured in 250 mL of ESF921 culture medium in a 1 L shake flask. Virus was added in an amount corresponding to the MOI, the flask's neck was sealed with sealing film, and the cells were cultured at 27°C in a shaker at 120 rpm. Cells in the shake flask were removed daily for observation and counting, and relevant data were recorded. An appropriate MOI ensured that over 70% of the cells were infected on day 1. On day 2, all cells were infected, with a viability of approximately 80%. On day 3, the cells burst, and viability dropped to 30%-50%. At this point, the cells were considered suitable for harvesting. The cells were collected by centrifugation at 10,000 rpm for 10 minutes, and the supernatant was then separated and purified.

[0216] 3. Purification of VHH-RBD and VHH-gE in an insect expression system The AKTA system was used for Ni affinity chromatography purification; Instrument system: AKTA Pure type preparative liquid chromatograph; Purification medium: Ni Sepharose 6 Fast Flow affinity medium; Buffer: Pump A buffer and Pump B buffer, generally, Pump A buffer was 1x PBS buffer (160 g / L NaCl, 8.1 mmol / L Na2HPO4, 1.5 mmol / L KH2PO4, 2.7 mmol / L KCl, pH 7.4), and Pump B buffer was 1x PBS + 250 mmol / L imidazole buffer; System flow rate: 5 mL per minute; Detection wavelength: UV@280 nm Elution conditions: impurities were eluted using 50 mM imidazole buffer (obtained by diluting 250 mmol / L imidazole buffer with 1× PBS buffer), followed by washing with 1× PBS, and then the target protein (S trimer protein) was eluted with 250 mM imidazole buffer.

[0217] The elution products with 250 mM imidazole were collected to obtain 10 mL of purified sample. 50 μL of each elution product was taken, 10 μL of 6× loading buffer was added, mixed thoroughly, and placed in an 80°C water bath for 10 minutes. 10 μL was then taken and electrophoresed on a 10% SDS-polyacrylamide gel at 80 V for 120 minutes. Subsequently, Coomassie Brilliant Blue staining was performed to visualize the electrophoretic bands.

[0218] 4. Transfection and Expression of VHH-RBD and VHH-BGTSTIP in Mammalian Expression Systems Frozen 293F cells were removed from a -80°C refrigerator, thawed at 37°C, centrifuged at 1300 rpm for 4 minutes, the supernatant was discarded in a clean bench, the cells were gently flicked, resuspended in 293 freestyle medium pre-incubated at 37°C, transferred to a flask containing 50 mL of incubation medium, and cultured at 37°C, 5% CO2, and 120 rpm until a cell density of 2.0 × 10 6The culture was gradually expanded by subculturing when the number of cells reached 100. When the number of cells reached 100, 293F cells were transiently transfected with PEI (MW 25000). The cells were collected in a sterile 50 mL tube and centrifuged at 1300 rpm for 4 minutes. The cells were gently flicked and resuspended in incubation medium at 37°C. The flask was then transferred to an Erlenmeyer flask containing 450 mL of incubation medium at 37°C and placed in a shaker at 37°C for later use.

[0219] The extracted VHH-RBD and VHH-BGTSTIP plasmids and PEI (MW 25000) were added to 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 450 ml of the above culture medium, suspended, and cultured at 37°C, 5% CO2, and 120 rpm for 6 days to express the VHH-RBD and VHH-BGTSTIP proteins. Care was taken to handle the PEI in the dark during the transfection process.

[0220] 5. Purification of VHH-RBD and VHH-BGTSTIP in a mammalian expression system Six days after transient transfection, the cell culture medium was collected and centrifuged at 7,000 g for 10 minutes in a JA-14 rotor to remove the cell supernatant. After centrifugation at 20,000 g for 10 minutes, the supernatant was removed and filtered twice using a 0.22 μm pore size membrane. The sample was used for the next step of Ni-excel column purification.

[0221] Ni affinity chromatography purification was performed using the AKTA system; Instrument system: AKTA Pure preparative liquid chromatograph; Purification medium: Ni Sepharose excel affinity medium; Buffer: Pump A buffer and Pump B buffer, Pump A buffer was 1x PBS buffer, Pump B buffer was 1x PBS + 250mmol / L imidazole buffer; System loading flow rate: 8 mL per minute; detection wavelength: UV@280 nm System elution flow rate: 4 ml per minute; Detection wavelength: UV@280 nm Elution conditions: Protein impurities were eluted using 20 mM imidazole, and the eluted product with 250 mM imidazole was collected. The eluate was dialyzed overnight against 1x PBS, with the dialysate replaced twice during the dialysis period. Approximately 30 ml of low-concentration target protein was collected and concentrated to 5 ml using a Vivaspin 20 ml, 100 KDa ultrafiltration tube for later use. The collected elution samples were divided into reduced and non-reduced samples and subjected to SDS-PAGE according to the method described above.

[0222] 6. Experimental Results Figures 2A and 2B show the results of SDS-PAGE of eight types of VHH-RBD proteins prepared according to the present invention. Figure 2A shows the results of electrophoresis of various VHH-RBD fusion proteins produced and purified using an insect cell expression system. M represents a molecular weight marker, and lanes 1 to 8 each represent a different VHH-RBD fusion protein. The results showed that after one-step purification by affinity chromatography, the VHH-RBD proteins had a purity of approximately 90% and a molecular size of approximately 50 kDa. Figure 2B shows the results of electrophoresis of 10 types of VHH-RBD fusion proteins produced and purified using a mammalian cell expression system. The results showed that the VHH-RBD proteins had a purity of approximately 90% and a molecular weight of approximately 50 kDa.

[0223] Figure 3 shows the results of SDS-PAGE of seven types of VHH-gE fusion proteins prepared according to the present invention. M represents a molecular weight marker, and lanes 1 to 7 each represent a different VHH-gE fusion protein. The results showed that after one step of affinity chromatography purification, the VHH-gE protein had a purity of approximately 80% and a molecular weight of approximately 85 kDa.

[0224] Figure 4 shows the results of SDS-PAGE of the eight VHH-BGTSTIP proteins prepared according to the present invention. "+" indicates reducing conditions, and "-" indicates non-reducing conditions. The results showed that after one-step affinity chromatography purification, the VHH-BGTSTIP proteins had a purity of approximately 90% and a molecular weight of approximately 180 KD.

[0225] Example 3: Immunoblotting experiments of VHH-RBD and VHH-gE proteins Equal amounts of protein samples were mixed with loading buffer, boiled for 10 minutes, and loaded onto SDS-PAGE gels for Western blotting (BioRad) according to standard laboratory protocols. Proteins were electrophoresed at 80 V for 70 minutes in a BioRad mini protean Tetra system, and the gels were stained with Coomassie Brilliant Blue R-250 (BioRad) for 30 minutes at room temperature. Separated proteins were transferred to nitrocellulose membranes using a Trans-Blot turbo transfer system (BioRad), blocked, and incubated with anti-His-HRP (1:5000 dilution) for 1 hour. Unbound antibody was removed by five 5-minute washes, followed by detection using a chemiluminescent substrate kit.

[0226] Figures 5A and 5B show the results of immunoblotting experiments (Western blots) of various VHH-RBD fusion proteins in examples of the present invention. Figure 5A shows the results for eight types of VHH-RBD fusion proteins produced and purified using an insect cell expression system. M represents a molecular weight marker, and lanes 1 to 8 each show a different nanobody-RBD fusion protein. The results confirm that the 50 KDa molecule was determined to be the target protein of the present invention. Figure 5B shows the results for ten types of VHH-RBD fusion proteins produced and purified using a mammalian cell expression system, 293F cells. The results showed that the VHH-RBD fusion proteins had a purity of approximately 90% and a molecular weight of approximately 50 KDa. The results confirm that the 50 KDa molecule was determined to be the target protein of the present invention.

[0227] Figure 6 shows the results of Western blot of seven candidate molecules, VHH-gE, from an example of the present invention. M represents a molecular weight marker, and lanes 1 to 7 show fusion proteins of different Nanobodies and gE, respectively. The results confirm that the 85 kDa molecule was determined to be the target protein of the present invention.

[0228] Example 4: Molecular activity analysis of fusion proteins (ELISA) (1) The fusion protein to be tested was diluted to 1 μg / mL, coated onto a 96-well plate at 100 μL per well, and allowed to stand at room temperature for 2 hours; (2) The plate was washed once and blocked with a diluent containing bovine serum albumin (ED, 200 μL / well) at room temperature for 2 hours; (3) The plate was washed once, and the corresponding specific monoclonal antibody was diluted to 1 μg / mL and added to the first well in a volume of 100 μl, and two-fold serial dilutions were performed with 11 gradients, with two replicate wells for each gradient, and the plate was left to stand at room temperature for 1 hour; (4) The plate was washed five times, and the secondary antibody GAH-HRP (1:5000) was added to the 96-well plate at 100 μL / well, and the plate was left to stand at room temperature for 1 hour; (5) The plate was washed five times, and color development was allowed to proceed at room temperature for 10 minutes, after which it was stopped. Detection was carried out at a wavelength of 450 nm using a microplate reader, and GraphPad Prism 5 (GraphPad, USA) software was used for data analysis.

[0229] The ELISA results for various VHH-RBDs and various reported specific monoclonal antibodies are shown in Figures 7A and 7B. Figure 7A shows the activity identification results for eight VHH-RBD proteins produced and purified using an insect cell expression system. The RBD antibodies included the broad-spectrum neutralizing antibodies VacW-105 (corresponding to JSR-105 in Figure 7A), JSR-551, VacW-209 (corresponding to JSR-209 in Figure 7A), 6D6 (corresponding to m6D6 in Figure 7A), and 7D6 (corresponding to m7D6 in Figure 7A) (specific information and sequences of these antibodies can be found in reference: 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 the 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. Retrieved from

[0230] The results confirmed that the VHH-RBD proteins produced and purified using the insect cell expression system still maintained their intact and accurate molecular conformation and had good binding activity to RBD-specific antibodies. Figure 7B shows the activity identification results of 10 VHH-RBD proteins produced and purified using the mammalian cell expression system, 293F cells. The antibodies included 3G11, 8H12, 13F10, 8B8, 9D3, and 3F9 (these antibodies were prepared in our laboratory by conventional antibody preparation methods described in references). Briefly, the extracellular segment of the SARS-CoV-2 surface spike protein S protein was expressed in 293F cells and purified. BALB / c mice were then immunized, and blood was collected for serum detection at 0, 2, 3, and 5 weeks after immunization. After immunization, two mice showed higher mouse immune serum binding titers and neutralization titers, and these mice were selected for spleen immunization. After injecting 10 μg of the mixed protein into the mouse spleen, the incision of the mouse was sutured and observed regularly. Three days later, a cell fusion experiment was performed. After fusion and culture, the cell supernatant was removed and analyzed for SARS-CoV-2 S-2P trimer protein, RBD, S2 protein, and SARS-CoV-1 The cell supernatants were detected for binding to the S-2P protein by indirect ELISA. Wells showing strong binding to the corresponding proteins were selected for picking and cloning experiments, and monoclonal antibodies were obtained after more than three rounds of cloning. Ascites mice were immunized, and the ascites fluid was extracted and purified using a protein A column to obtain the corresponding monoclonal antibodies. The results showed that the VHH-RBDs produced and purified using a mammalian cell expression system still maintained their complete and accurate molecular conformation and exhibited good binding activity to RBD-specific antibodies. Furthermore, the binding activity of these fusion proteins to antibodies (e.g., 8H12, 13F10, and 8B8) was better than that of the RBD protein itself.

[0231] Figure 8 shows the ELISA results using various specific monoclonal antibodies against seven types of VHH-gE in the examples. The gE-specific monoclonal antibodies included 3H7, 4G4, 6B7, 11B11, 11B12, 13B6, 14G1, and 17B7 (specific information and sequences of these antibodies were obtained from the following reference: 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 showed that the produced and purified VHH-gE still maintained a complete and accurate molecular conformation and had good binding activity to the gE-specific antibodies.

[0232] FIG. 9 shows the results of ELISA using various reported HIV-1 neutralizing or non-neutralizing antibodies against various VHH-BGTSTIPs in the examples. These antibodies were recombinantly expressed in 293F cells, and their sequences were identified in 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 reported F105 antibody (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 CD4 binding 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-gp41 antibody F240. Sci Rep, 2016. 6:36685), and 17b (Carlo D. Rizzuto, Richard Wyatt, Nivia Herna ndez-Ramos, et al. A Conserved HIV gp120 Glycoprotein Structure Involved in Chemokine Receptor Binding. Science, 1998. 280(19):1949-1953). The results showed that various VHH-BGTSTIP fusion proteins of the present invention had good binding activity to various neutralizing antibodies, but lower binding activity to non-neutralizing antibodies.This indicates that VHH-BGTSTIP expressed in 293F cells sufficiently presented several neutralizing antibody epitopes on Env, while not exposing several non-neutralizing antibody epitopes.

[0233] Example 5: Affinity analysis of fusion proteins for HEV-p239 (SPR) A CM5 chip was loaded onto a Biacore 8K, and the pipeline was flushed with PBS-P buffer (PBS + 0.5% P20, Cytiva).

[0234] Ligand HEV-p239 (10 μg / mL) was centrifuged at high speed for 10 min.

[0235] The HEV-p239 coupling program was set up: the chip channel was activated (EDC:NHS = 1:1, flow rate: 10 μl per minute), followed by a flushing program (PBS-P buffer, flow rate: 30 μl per minute). HEV-p239 (amino acid sequence shown in SEQ ID NO: 40) was loaded for coupling (time: 420 s, flow rate: 10 μl per minute). After determining the amount of ligand for coupling, ethanolamine was used for the channel blocking procedure (time: 420 s, flow rate: 10 μl per minute).

[0236] Affinity detection: A concentration gradient of fusion protein was set up at 125 nM, 62.5 nM, 31.2 nM, 15.6 nM, 7.8 nM, and 3.9 nM and loaded for detection. The sample association time was set to 120 s, the dissociation time to 200 s, and the flow rate to 30 μl per minute.

[0237] Using Biacore 8K (Cytiva) software, association (Ka) / dissociation (Kd) curve fitting was performed by the kinetic method, and affinity (KD) was analyzed and calculated.

[0238] Figures 10A and 10B show the affinity analysis results for HEV-p239 protein of various VHH-RBD fusion proteins of the present example. Figure 10A shows the affinity results for HEV-p239 protein of VHH-RBDs produced and purified using an insect cell expression system. The results showed that all of the various VHH-RBDs had high affinity for HEV-p239 particles at the nanomolar level. Figure 10B shows the affinity results for HEV-p239 protein of VHH-RBDs produced and purified using a mammalian cell expression system, 293F cells. The results showed that VHH-RBDs had high affinity for HEV-p239 particles at the nanomolar level.

[0239] The affinity analysis results of various VHH-gE fusion proteins for HEV-p239 protein are shown in Figure 11. The results showed that all of the various VHH-gE fusion proteins have high affinity for HEV-p239 particles at the nanomolar level.

[0240] The affinity analysis results of various VHH-BGTSTIP fusion proteins for HEV-p239 protein in the examples are shown in Figure 12. The results showed that all of the various VHH-BGTSTIPs had high affinity for HEV-p239 particles at the nanomolar level.

[0241] Example 6: Purification of fusion proteins and their complexes by molecular sieve chromatography Instrument system: AKTA explorer 100 preparative liquid chromatography system manufactured by GE Healthcare (formerly Amershan Pharmacia). Chromatography medium: Superdex 200 increase (Cytiva). Column volume: 20 cm x 20 mm. Buffer: 20 mM phosphate buffer, pH 7.4. Flow rate: 0.7mL per minute. Detector wavelength: 280 nm. The samples were those in Example 1 and Example 5. The elution procedure included: collecting the permeate peak in fractions.

[0242] The products that permeated through Supedex 200 increase were collected to obtain 5 mL of purified samples. 50 μL of each elution product was taken, 10 μL of 6x loading buffer was added, and mixed thoroughly. After placing in an 80°C water bath for 10 minutes, 10 μL of the mixture was taken and electrophoresed on a 10% SDS-polyacrylamide gel at 120 V for 60 minutes. The electrophoretic bands were then visualized using Coomassie Brilliant Blue staining.

[0243] Figure 13A shows the results of purification of the fusion protein P1-5B-RBD, produced and purified using an insect cell expression system in an example of the present invention, using Superdex 200 increase (high-performance liquid chromatography molecular sieve). The results indicated that the purity of the P1-5B-RBD protein reached more than 95%. Figure 13B shows the results of purification of P2-6D-RBD, P2-3E-RBD, and P2-10G-RBD, produced and purified using a mammalian cell expression system in an example of the present invention, using Superdex 200 increase. The results indicated that all three fusion proteins showed a single elution peak, indicating high purity and homogeneity of these proteins.

[0244] 14 to 16 show the purification chromatograms and the results of identification by SDS-PAGE of the complexes formed by various fusion proteins constructed in the present invention and HEV-p239.

[0245] Figure 14A shows the chromatogram of the complex formed between the P1-5B-RBD fusion protein purified from insect cell expression system and HEV-p239, and its SDS-PAGE analysis. The purple curve was significantly higher than the blue curve, demonstrating that the fusion protein P1-5B-RBD specifically bound to the surface of HEV particles and formed a complex.

[0246] Figure 14B shows the purification chromatograms and SDS-PAGE analysis of the complexes formed between HEV-p239 and P2-3E-RBD, P2-10G-RBD, and P2-6D-RBD, which were purified from mammalian cell expression systems as candidate fusion proteins in this example. The results showed that P2-3E-RBD, P2-10G-RBD, and P2-6D-RBD formed complexes with HEV-p239 and could be purified using Superdex 200 10 / 300 increase.

[0247] Figure 15 shows the purification chromatogram and SDS-PAGE results of the complex formed by the candidate fusion protein P2-8C-gE and HEV-p239 in this example. Figure 15A shows the results of the complex purification. The results show that the complex retention volume on a Superdex 200 Increase (Cytiva) chromatography column was 8 ml, which is expressed as the particle peak component. The 14 ml peak indicates unbound P2-8C-gE, which therefore indicates that the 8 ml particle peak component is bound. Therefore, P2-8C-gE bound to the surface of HEV particles to form a complex at saturation. The SDS-PAGE results confirmed that the complex lane showed typical two-component bands (HEV-p239 = 20 kDa, P2-8C-gE = 85 kDa). Therefore, the sample purified by Superdex 200 Increase was determined to be a gE complex sample.

[0248] Figure 16 shows the purification chromatogram and SDS-PAGE results of the complex of the candidate fusion protein P2-5C-BGTSTIP and HEV-p239 in this example. The results show that the retention volume of the complex on the Superose 6 column was approximately 9 ml, while the retention volume of P2-5C-BGTSTIP was approximately 16 ml. SDS-PAGE analysis revealed that distinct components were collected. For the sample with an elution volume of 9 ml, two bands were observed on SDS-PAGE, confirming that the complex sample was obtained in approximately 9 ml after purification with Superose 6.

[0249] In summary, the above experimental results demonstrated that complex particles formed by various candidate fusion proteins and HEV-p239 in this application could be successfully purified and obtained.

[0250] Example 7: Analysis of P2-8C-gE protein by high performance size exclusion chromatography (HPSEC) Equipment: Waters. System flow rate: G3000PW XL Flow rate: 0.5 mL per minute. Wavelength: 190 nm to 600 nm. Column wavelength: 280 nm and 254 nm. Buffer: PBS. Operation process: The column was pre-equilibrated for 30 to 60 minutes until no significant change in absorbance at 280 nm was observed. The detector absorbance was reset to zero. The chromatography operation method was edited to first centrifuge the sample, then inject the sample to be analyzed into a 100 μL sample loop, set to automatic loading, and run the instrument for 30 minutes. The retention time of the S trimer was observed to be approximately 14 minutes.

[0251] The results are shown in Figure 17. The P2-8C-gE fusion protein of the present invention showed a single major peak without any aggregated components, had a retention time of about 14 minutes, and a purity of about 80%.

[0252] Example 8: Molecular size detection (DLS) of RBD complex and gE complex Equipment: NanoBrook Series (Brookhaven Instruments). Functional module: DLS (Dynamic Light Scattering). Buffer: PBS. Operation process: The instrument was activated and preheated for 5 minutes. The test sample was prepared (concentration: 0.5 mg / ml, centrifugation: 12000 rpm for 5 minutes, 50 μl was taken and added to the sample cup). Detection parameters were set: detection time: 300 seconds each time, 3 replicates for each sample.

[0253] Figures 18A and 18B show the molecular size detection results for HEV-RBD complex samples formed by VHH-RBD and HEV-p239 protein, which were produced and purified using an insect cell expression system in an example of the present invention. Figure 18B shows the molecular size detection results for HEV-RBD complex samples formed by VHH-RBD and HEV-p239 protein, which were produced and purified using a mammalian cell expression system. The results in Figures 18A and 18B indicate that the molecular size of HEV-RBD is significantly larger than that of HEV-p239 particles. The size of the HEV-RBD complex was significantly larger than that of HEV-p239, confirming that VHH-RBD bound to the surface of HEV particles and maintained the particle state.

[0254] Figure 19 shows the molecular size detection results for the HEV-gE complex sample in this example. The results showed that the molecular size of the HEV-p239 particle was 14.2 nm, and that of the HEV-gE particle was 38 nm. The results confirmed that VHH-gE bound to the surface of the HEV particle and maintained its particle state.

[0255] Figure 20 shows the molecular size detection results for the HEV-BGTSTIP complex sample of the present invention. The results showed that the molecular size of the HEV-p239 particle was 15.9 nm, and the size of the HEV-BGTSTIP complex was 25.2 nm. The results indicated that VHH-BGTSTIP bound to the surface of HEV particles and was able to maintain the particle state.

[0256] Example 9: Calculation of sample sedimentation coefficient by analytical ultracentrifugation The instrument used was a Beckman XL-A analytical ultracentrifuge equipped with an optical detection system and an An-60Ti rotor.

[0257] The sample pools were loaded according to the manufacturer's instructions. 400 μL of sample buffer was added to the control pool (the same buffer used for the samples), and 380 μL of sample (OD280 approximately 0.8) was added to the sample pool. The sample pools were allowed to equilibrate until their weights were within 0.1 g.

[0258] The sample pool was placed in an An-60Ti rotor, and the rotor was placed in the cavity of a Beckman XL-A analytical ultracentrifuge, and an optical path detector was attached.

[0259] The parameters were set as follows: temperature (20°C), Rmin (6.0 cm), Rmax (7.2 cm), wavelength (280 nm), step speed (0.003 cm), scan mode (continuous), data interval (30 s), and data number (150 scans). The centrifugation speed was set to 30,000 rpm.

[0260] After the experiment was completed, the density and viscosity of the buffer solution and the partial specific volume of the known protein were calculated using SENDTERP software. The sedimentation coefficient was analyzed using Nonlin software and the Origin version of SEDFIT software. In this analysis, the globulin friction ratio 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, and the RMSD value was generally required to be 0.01 or less, and the variation of the residual map was within 0.05.

[0261] Figures 21A and 21B show analytical ultracentrifugation results for HEV-p239 and HEV-RBD complexes constructed based on the HEV-p239 particles in the examples of the present invention. Figure 21A shows the analytical ultracentrifugation results for complex particles prepared from purified VHH-RBD and HEV-p239 produced using an insect cell expression system (right panel), and HEV-p239 (left panel). The results showed that HEV-p239 was a single component with a sedimentation coefficient of 22S, while the HEV-RBD complex was a single component with a sedimentation coefficient of 27S, which was significantly higher than the sedimentation coefficient of HEV-p239 particles (22S). The results confirmed that the RBD complexes maintained a stable particle morphology in aqueous solution and that the RBD tightly bound to the surface of HEV particles to form RBD particle proteins. Figure 21B shows the analytical ultracentrifugation results of the complex particles prepared from purified VHH-RBD and HEV-p239 produced in the mammalian expression system 293F cells. The results show that the HEV-RBD complex is a single component, and its sedimentation coefficient is significantly higher than that of HEV-p239 particles (22S). This confirms that the HEV-RBD complex can maintain a stable particle morphology in aqueous solution and that RBD is tightly bound to the surface of HEV particles to form the RBD particle protein.

[0262] Figure 22 shows the analytical ultracentrifugation results of HEV-p239 (Figure 22A) and HEV-gE complexes constructed based on HEV-p239 particles (Figure 22B). The results showed that HEV-p239 was a single component with a sedimentation coefficient of 22S, while the HEV-gE complexes were a single component with a sedimentation coefficient of 31S, which was significantly higher than the sedimentation coefficient of HEV-p239 particles (22S). The results confirmed that the HEV-gE complexes could maintain a stable particle morphology in aqueous solution and that gE tightly bound to the surface of HEV particles to form gE particle proteins.

[0263] FIG. 23 shows the results of analytical ultracentrifugation of the HEV-p239 and Env(BGTSTIP) complex constructed based on the HEV-p239 particle in an example of the present invention.

[0264] Example 10: Observation of particle morphology by transmission electron microscope The transmission electron microscope used was a Tecnai G2 Spirit 120 kV (FEI). Phosphotungstic acid was used for negative staining.

[0265] Sample preparation: First, a copper mesh (R2 / 2, 200 mesh, ThermoFisher Scientific) was subjected to glow discharge hydrophilization treatment. Next, 5 μl of a sample with a concentration of 0.5 mg / ml was dropped onto the copper mesh. After leaving it to stand at room temperature for 60 seconds, the droplet was absorbed from the edge of the copper mesh using absorbent paper. After drying at room temperature, the copper mesh was observed under the microscope described above.

[0266] Figure 24A shows the negative staining transmission electron microscopy results of a complex sample prepared with HEV-p239 and VHH-RBD produced and purified using an insect cell expression system in an example of the present invention. Figure 24B shows the negative staining transmission electron microscopy results of a complex sample prepared with HEV-p239 and VHH-RBD produced and purified using a mammalian expression system, 293F cells, in an example of the present invention. All results indicated that the HEV-RBD complex exhibited a typical virus-like particle morphology. These results confirmed that the HEV-RBD complex sample constructed in this application is an RBD particulate antigen.

[0267] Figure 25 shows the negative staining results of transmission electron microscopy of the HEV-gE complex in the example of the present invention. The results showed that the HEV-gE complex exhibited a typical virus-like particle morphology. The results confirmed that the HEV-gE complex sample constructed in this application was a particulate antigen.

[0268] Figure 26 shows the negative staining results of transmission electron microscopy of the HEV-Env(BGTSTIP) complex in the example of the present invention. The results showed that the HEV-Env complex exhibited a typical virus-like particle morphology, confirming that the HEV-Env complex sample constructed in this application was a particulate antigen.

[0269] Example 11: Evaluation of immunogenicity of HEV-RBD particles This experimental protocol was approved by the Experimental Animal Management Ethics Committee of Xiamen University, and all procedures were performed in strict accordance with animal ethics guidelines and approved procedures.

[0270] Evaluation of immunogenicity of RBD particles expressed in insect cells Six-week-old Balb / C mice were selected and divided into four groups, each with five mice. Each group was immunized with HEV-RBD particles (immunization dose 0.5 μg), HEV-RBD particles (immunization dose 5 μg), P1-5B-RBD monomer (immunization dose 0.5 μg), or a combination of P1-5B-RBD monomer (immunization dose 5 μg) and aluminum adjuvant via intramuscular injection (50 μL) into the left or right hind limb at 0, 2, and 6 weeks. Ophthalmic vein blood samples were collected at 0, 1, 2, 3, 4, 5, 6, 7, and 8 weeks. Blood samples were collected before injection at 0, 1, and 4 weeks. Blood samples were centrifuged at 13,000 g for 10 minutes, and the resulting serum samples were stored at -20°C. Antigen-specific IgG and neutralizing antibody titers were determined by end-point enzyme-linked immunosorbent assay and a neutralization method based on wild-type SARS-CoV-2 pseudovirus, respectively (construction method is described in reference; 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.

[0271] Evaluation of immunogenicity of RBD particles in mammalian expression systems Mice: Female, 6 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Twelve groups of immunized mice were prepared, with five mice in each group. P2-3E-RBD, P2-10G-RBD, P2-6D-RBD, and p239 were used to prepare particulate complex antigens. The particulate complex antigens were diluted to the required concentration with saline, mixed with aluminum adjuvant at a 1:1 volume ratio, and adsorbed onto the adjuvant overnight at 4°C. Mice were immunized intramuscularly at 0, 2, and 5 weeks (100 μL total; 50 μL each for the left and right hind legs). The immunization scheme for mice is shown in Table 4.

[0272] [Table 4]

[0273] Blood was collected from the eyes of the mice once a week, and after 9 weeks, the mice were euthanized by carbon dioxide. Blood samples were placed at 37°C for 30 minutes and then centrifuged at 13,300 rpm for 10 minutes. Serum was collected and stored at -20°C for wild-type SARS-CoV-2 pseudovirus neutralization and binding antibody titer determination.

[0274] The results are shown in Figures 27 and 28. Figures 27A-D show the immunogenicity detection results of the particulate antigen HEV-RBD in an example of the present invention. Figure 27A shows the results of serum binding and neutralizing activity detection after immunizing mice with the particulate antigen HEV-RBD obtained using an insect cell expression system. Figures 27B1-2 and 27C show the results of serum binding activity and wild-type SARS-CoV-2 pseudovirus neutralizing activity detection after immunizing mice with the particulate antigen HEV-RBD obtained using a mammalian cell expression system, and Figure 27D shows the serum antibody binding titer after immunizing hamsters with the particulate antigen HEV-RBD obtained using a mammalian cell expression system.

[0275] Figure 28 shows the results of neutralization detection of antibodies against the wild-type (WT), gamma, and BA.2 strains of SARS-CoV-2 induced by the combination of the particulate antigen HEV-RBD of an example of the present invention and aluminum adjuvant.

[0276] The results showed that the immunogenicity of RBD antigens could be significantly improved by displaying VHH-RBD expressed in insect cells or VHH-RBD expressed in mammalian cells on the surface of p239 particles.

[0277] Specifically, in the group immunized with 5 μg of HEV-RBD particles (denoted RBD CPX in the figure), the immune barrier was rapidly established after the first immunization, with binding antibody titers reaching approximately 3 logs. After the booster immunization, the binding antibody titer reached 5 logs, which was 500-fold higher than the binding antibody titer elicited by P1-5B-RBD monomer and did not significantly decrease over 8 weeks. In mouse immunization experiments, the binding antibody titer induced by HEV-RBD was significantly higher than that induced by the monomeric RBD protein. At an immunization dose of 0.5 μg per mouse, the P2-3E-RBD-p239, P2-10G-RBD-p239, and P2-6D-RBD-p239 conjugate particles induced relatively high binding antibody titers (compared to the corresponding VHH-RBD proteins). The binding antibody titers of the P2-3E-RBD-p239 conjugate and P2-10G-RBD-p239 conjugate immunized groups before the fifth injection were significantly higher than those of the corresponding VHH-RBD immunized groups (Figure 27B). The pseudovirus neutralization results at week 6 demonstrated that the conjugate particles formed by the three VHH-RBD and p239 conjugates were able to induce significant neutralizing antibody responses (Figure 27C).

[0278] Furthermore, neutralizing antibodies against HEV-RBD particles had broad-spectrum neutralizing activity and were able to effectively neutralize pseudoviruses (VSV-Spike) including wild-type, gamma, and BA.2 strains of SARS-CoV-2.

[0279] Example 12: Evaluation of immunogenicity of gE particles This experimental protocol was approved by the Xiamen University Laboratory Animal Care and Ethics Committee, and all procedures were performed in strict accordance with animal ethics guidelines and approved procedures.

[0280] Six-week-old Balb / C mice were selected and divided into eight groups, each with five mice. Each group was immunized with HEV-gE particles (immunization dose: 0.1 μg), HEV-gE particles (immunization dose: 0.5 μg), HEV-gE particles (immunization dose: 5 μg), P2-8C-gE monomer (immunization dose: 0.1 μg), P2-8C-gE monomer (immunization dose: 0.5 μg), or P2-8C-gE monomer (immunization dose: 5 μg) in combination with aluminum adjuvant via intramuscular injection (50 μL) into the left or right hind leg at 0 and 2 weeks, respectively. Ophthalmic vein blood samples were collected at 0, 1, 2, 3, 4, 5, and 6 weeks, respectively. Blood samples were centrifuged at 13,000 g for 10 min, and the resulting serum samples were stored at −20° C. 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.

[0281] Neutralization procedure (ELISPOT method): 1. Guinea pig serum (purchased from Beijing Boerxi Technology Co., Ltd., Cat. No.: BM361Y) and v-Oka virus (ATCC, Cat. No.: VR-795) dry powder were reconstituted with virus stock solution, and the complement was filtered through a 0.22 μm miniature filter for later use; 2. The serum was diluted 50-fold with virus stock solution, added to the first well of a 24-well plate, subjected to two-fold serial dilutions in a four-step gradient, and incubated with vOka virus at 37°C for 1 hour; 3. The serum-virus mixture was transferred to a 24-well plate pre-coated with ARPE-19 cells and incubated at 37°C for 1 hour, after which the liquid was discarded, replenished with F12 medium, and cultured at 37°C for 3 days; 4. After 3 days, the medium was discarded, the plate was washed once with PBS, fixed with fixation solution at room temperature for 5 minutes, the fixation solution was discarded, and permeabilization was performed with permeabilization solution at room temperature for 10 minutes; 5. Primary antibody 1B11-HRP (1:2000) was added to the 24-well plate, and the plate was left at 37°C for 1 hour; 6. The plate was washed five times and allowed to develop for 5 minutes at room temperature. The spots were read and counted using an enzyme-linked spot image analysis system, and GraphPad Prism 5 (GraphPad, USA) software was used for data analysis.

[0282] Figure 29 shows the immunogenicity detection results of gE particulate antigen in an example of the present invention. It was shown that the combination of gE particulate antigen and AS01B-like adjuvant can induce binding antibodies at high titer levels (shown as XUA in Figure 29, up to approximately 6 logs).

[0283] Figure 30 shows the results of live virus neutralization detection of mouse immune sera against HEV-gE particulate antigen in an example of the present invention. The neutralizing antibodies induced by HEV-gE particulate antigen were 2.8-fold higher than those induced by P2-8c-gE monomer. These results confirmed that expression on the surface of p239 particles significantly improved the immunogenicity of gE antigen.

[0284] Example 13: Flow cytometric detection of cytokines in groups immunized with gE particulate antigen Mice were immunized according to the method described in Example 6 (using the same adjuvant), and the subsequent experimental procedures were as follows: a) Spleen removal: Mice were sacrificed by cervical dislocation and immersed in 75% ethanol for 3-5 minutes. The mice were placed on their right side, and the spleens were aseptically removed (fat was removed as much as possible); b) Mashing: A 6-well cell plate was placed in a mesh, 1640 culture medium containing 10% FBS was added, the spleen was placed in the mesh (completely immersed in the culture medium), and the red tissue was mashed with the head of a 2 mL syringe until the red tissue was no longer visible. The mashed cells were transferred to a 50 mL tube and placed on ice; c) Centrifugation was carried out at 400 g for 5 minutes at 4°C, the supernatant was discarded, and the cell pellet at the bottom of the tube was loosened by tapping with a hemostat; d) 10 mL of pre-chilled RBC solution was added to the cell pellet, resuspended, and placed on ice for 5 minutes (mixed by inversion during this period); e) Centrifugation was carried out at 400 g for 5 minutes at 4°C, the supernatant was discarded, and the cell pellet at the bottom of the tube was loosened by tapping with a hemostat; f) 10 mL of pre-chilled culture medium was added to the cell pellet, resuspended, washed to remove any red blood cell pellet or fat, pipetted to homogenize, and then 50 μL of cells were removed and counted; g) Centrifuge at 400 g for 5 minutes at 4°C, discard the supernatant, loosen the cell pellet at the bottom of the tube by tapping with a hemostat, add a certain amount of culture medium, and the cell count is 2 x 10 7 / mL; h) 200 µL (2 x 10 cells) per well of a 96-well U-bottom plate 6 pieces~4×10 6 pcs) were coated and centrifuged at 400 g and 4°C for 5 minutes, and the supernatant was discarded; i) Cells were resuspended by adding 100 μL of culture medium containing polypeptide ((gE / gI) overlapping polypeptide diluted with FACS solution to a final polypeptide concentration of 2 μg / mL) and stimulated for 18 hours; j) 20 μL of Golgi inhibitor (1:1000, diluted with culture medium) was added and incubated for 6 hours; k) Centrifugation was carried out at 400 g for 2 minutes at 4°C, the supernatant was discarded, 200 μL of FACS solution (1×PBS+10% FBS) was added to the cells, they were resuspended, centrifuged, and the supernatant was discarded; (All of the following procedures were carried out in the dark at 4°C.) l) Cell surface staining: Cells were stained using FITC-conjugated anti-mouse CD4 antibody (purchased from Biolegend, Cat. No.: 100510), PE-Cy7-conjugated anti-mouse CD8α antibody (purchased from Biolegend, Cat. No.: 100722), and LIVE / DEAD™ fixed Aqua dead cell staining reagent (purchased from Invitrogen, Cat. No.: L34966), resuspended by adding 40 μL of surface antibody (AQUA / CD4 / CD8 antibody, diluted with FACS solution) per well, treated at 4°C in the dark for 30 to 60 minutes, added 200 μL of FACS solution, and pipetted 7 or 8 times; m) Fixation / Permeabilization: Centrifugation was performed at 2000 rpm at 4°C for 2 minutes, the supernatant was discarded, and the cells were resuspended by adding 75 μL of Fixation / Permeabilization solution per well, and treated at 4°C in the dark for 60 minutes; n) Centrifugation was carried out at 2000 rpm for 2 minutes at 4°C, and the supernatant was discarded; o) Cells were resuspended by adding 200 μL of 1× BD Perm / Wash solution per well, centrifuged at 2000 rpm at 4°C for 2 minutes, and the supernatant was discarded; p) Intracellular staining: Cells were stained using PE-conjugated anti-mouse IL-2 antibody (purchased from BD, Cat. No.: 554428) and APC-conjugated anti-mouse IFN-γ antibody (purchased from BD, Cat. No.: 554413), resuspended by adding 50 μL of fluorescent antibody (IL-2 / IFN-γ antibody, diluted with 1× BD Perm / Wash solution) per well, incubated at 4°C in the dark for 60 minutes, and resuspended by adding 200 μL of Perm Buffer; q) Centrifugation was carried out at 2000 rpm for 2 minutes at 4°C, the supernatant was discarded, and 200 μL of 1× BD Perm / Wash solution was added to each well to resuspend the cells; r) The treated cells were filtered through a 200-mesh screen (a piece of screen was placed on the surface of the well, and the cell suspension was slowly added by pipetting with the pipette held vertically), and then transferred to a flow cytometer; s) Samples were measured using a BD LSRFortessa X-20 flow cytometer and data were analyzed using FlowJo V10.

[0285] The experimental results are shown in Figure 31. The number of IFN-gamma positive CD4 cells activated by gE particulate antigen was 1.65 times that of IFN-gamma positive CD4 cells activated by P2-8C-gE monomer, and the number of IFN-gamma positive CD8 cells activated by gE particulate antigen was 8.42 times that of IFN-gamma positive CD8 cells activated by P2-8C-gE monomer. The results confirmed that gE particulate antigen has more advantages in inducing cellular immune responses.

[0286] Example 14: Enzyme-linked immunospot assay of cytokines in groups immunized with gE particulate antigen At week 8, the spleens were collected and subjected to flow cytometry and Elispot detection of T cell responses. The flow cytometry method was the same as in Example 6, and the process of ELISPOT detection of cytokines (kits used were purchased from MABTECH, Cat. Nos. 3321-4HPW-10 and 3441-4HPW-10) was as follows: (1) Splenocytes were isolated and then plated at 500,000 cells per well, centrifuged to remove the supernatant, and 100 μL of gE polypeptide (0.15 μg / 100 μl) culture medium was added to resuspend the cells, followed by stimulation at 37°C for 20 hours; (2) The culture medium was discarded, the cells were washed five times with sterile PBS, and the detection antibodies R4-6A2-biotin (IFN-γ) and 5H4-biotin (IL-2) were diluted to 1 μg / mL and added at 100 μL per well and incubated at room temperature for 2 hours; (3) The cells were washed five times with sterile PBS, and streptavidin-ALP (1:1000) was added at 100 μL per well and incubated at room temperature for 1 h; (4) The cells were washed five times with sterile PBS, and 100 μL of substrate solution (BCIP / NBT-plus) was added per well for color development until spots appeared, and then the plate was washed; (5) The plates were inverted and dried, and the spots were read and counted using an enzyme-linked immunospot image analysis system. GraphPad Prism 5 (GraphPad, USA) software was used for data analysis.

[0287] The results are shown in Figure 32. It was confirmed that the T cell immune response activated by the gE particulate antigen was significantly higher than that activated by the P2-8C-gE monomer protein.

[0288] Example 15: Humanization of P1-5B Nanobody The P1-5B nanobody (the full-length amino acid sequence of the P1-5B nanobody is shown in SEQ ID NO: 12, and the sequences of CDR1 to CDR3 are shown in SEQ ID NO: 57 to SEQ ID NO: 59) was subjected to CDR alignment using online software (IMGT / DomainGapAlign; http: / / www.bioinf.org.uk / abs / abnum / ). A homologous sequence alignment search was performed using online software (https: / / www.imgt.org / IMGT_vquest / input), and the humanized sequence with the highest score was selected for CDR-grafting. The selected sequence was synthesized at Sangon Biotech and constructed in the PCDNA3.1 vector. This plasmid was prepared in large quantities using an endotoxin-free plasmid large-scale extraction kit (Tiangen Company). 293FT cells were then transfected by PEI transient transfection to express the humanized nanobody. A total of two humanized Nanobodies were obtained, designated H1 and H2. The full-length amino acid sequences of the humanized Nanobodies are shown in SEQ ID NOs: 71 and 72, and the CDR1 to CDR3 sequences are shown in SEQ ID NOs: 65 to 67.

[0289] PEI transient transfection of HEK239FT cells The PCDNA3.1-H1-RBD and PCDNA3.1-H2-RBD plasmids (these plasmids contained the nucleotide sequences H1-RBD and H1-RBD, respectively, encoding the fusion proteins. The amino acid sequences of H1-RBD and H1-RBD are shown in SEQ ID NOs:63 and 64) and PEI were diluted in 5 ml of serum-free CD05 medium, mixed thoroughly, and allowed to stand for 18 minutes to form a plasmid-PEI complex. The complex was then used at a PEI:plasmid ratio of 2:1 to transfect HEK293FT cells. Fresh CD05 medium was replaced 6 hours after transfection. After 48 hours of culture at a constant temperature of 37°C and 5% CO2, 100 ml of CD05 medium was replenished. After 96 hours, the supernatant was collected for purification, and the affinity of the humanized nanobody was then detected by enzyme-linked immunosorbent assay (ELISA, see Example 4).

[0290] The results are shown in Figure 33: the engineered humanized Nanobodies had the same molecular activity as their parent, where H1-RBD and H2-RBD are RBD fusion proteins formed by the engineered humanized Nanobodies, and RBD-WT is the wild-type RBD protein.

[0291] Example 16: Evaluation of immunogenicity of particulate antigen HEV-Env prepared by humanized nanobody White mice: female, 6 weeks old, purchased from Shanghai Silaike Experimental Animal Co., Ltd. Six groups of immunized mice were prepared, with each group consisting of five mice. In this example, the P2-5C nanobody (the amino acid sequence of the P2-5C nanobody is shown in SEQ ID NO: 21, and the CDR1 to CDR3 sequences are shown in SEQ ID NO: 60 to SEQ ID NO: 62) was used. The prepared complex particles and P2-5C-BGTSTIP antigen were diluted with physiological saline and mixed with aluminum adjuvant at a volume ratio of 1:1 so that the protein was adsorbed to the adjuvant. Mice were immunized intramuscularly. The mouse immunization scheme is shown in Table 5.

[0292] [Table 5]

[0293] Before immunization, blood was collected from the eyes of the mice, and immunizations were performed according to the above immunization scheme. Blood was collected from the eyes of the mice before each immunization. After the sixth injection, blood was collected from the eyes, and the mice were then sacrificed by cervical dislocation. Blood samples were placed at 37°C for 30 minutes, centrifuged at 13,300 rpm for 10 minutes, and serum was collected for HIV-1 pseudovirus neutralization and antibody titer determination.

[0294] Immune sera from injections 0 to 6 were collected for virus neutralization experiments.

[0295] Example 17: Expression and identification of P1F8-BGTSTIP fusion protein Design and expression of P1F8-BGTSTIP fusion protein Nanobody P1F8 (SEQ ID NO: 88) was obtained by screening the nanobody against the HPV L1 protein (SEQ ID NO: 71). A (GGGGS)3 flexible linker was added after the antibody sequence and then coupled before the BGTSTIP sequence. The amino acid sequence of the constructed P1F8-BGTSTIP fusion protein is shown in SEQ ID NO: 89. The P1F8-BGTSTIP plasmid was extracted. Frozen 293F cells were removed from a -80°C refrigerator, thawed at 37°C, centrifuged at 1300 rpm for 4 minutes, the supernatant was discarded in a clean bench, the cells were flicked, resuspended in 293 freestyle medium pre-incubated at 37°C, transferred to a flask containing 50 mL of incubation medium, and cultured in suspension at 37°C, 5% CO2, and 120 rpm. When the cell density reached 2.0 x 10 6The culture system was gradually expanded by subculturing the cells when they reached a density of 1000 kJ / ml. When the cells were sufficient, PEI (MW 25000) was used to transiently transfect 293F cells, and the cells were collected in a sterile 50 mL tube and centrifuged at 1300 rpm for 4 minutes. The cells were then gently flicked and resuspended in incubation medium at 37°C. The cells were transferred to a flask containing 450 mL of culture medium incubated at 37°C and placed on a shaker at 37°C for later use.

[0296] The extracted P1F8-BGTSTIP plasmid and PEI (MW 25000) were added to 50 ml of culture medium at a 1:2 ratio, thoroughly mixed, and then allowed to stand for 18 minutes. The mixture was then transferred to 450 ml of the above culture medium, suspended, and cultured at 37°C, 5% CO2, and 120 rpm for 6 days to express the P1F8-BGTSTIP protein. Care was taken to handle the PEI in the dark during the transfection process.

[0297] Purification of P1F8-BGTSTIP fusion protein Six days after transient transfection, the cell culture medium was collected and centrifuged at 7,000 g for 10 minutes in a JA-14 rotor to obtain the cell supernatant. After centrifugation at 20,000 g for 10 minutes, the supernatant was removed and filtered twice using a 0.22 μm pore size membrane filter. The sample was used for the next step of Ni-excel column purification.

[0298] Ni affinity chromatography purification was performed using the AKTA system; Instrument system: AKTA Pure type preparative liquid chromatograph; Purification medium: Ni Sepharose excel affinity medium; Buffer: Pump A buffer and Pump B buffer, Pump A buffer was 1x PBS buffer, Pump B buffer was 1x PBS + 250mmol / L imidazole buffer; System loading flow rate: 8 mL per minute; detection wavelength: UV@280 nm System elution flow rate: 4 ml per minute; Detection wavelength: UV@280 nm Elution conditions: Protein impurities were eluted with 20 mM imidazole, and the 250 mM imidazole elution product was collected. The eluate was dialyzed overnight against 1x PBS, with the dialysate replaced twice during the dialysis period. Approximately 30 ml of low-concentration target protein was collected and concentrated to 5 ml using a Vivaspin 20 ml, 100 kDa ultrafiltration concentration tube for later use. The collected elution samples were divided into reduced and non-reduced samples, and SDS-PAGE gel electrophoresis was performed as described above. Figure 34 shows the SDS-PAGE results of the P1F8-BGTSTIP protein; M represents the molecular weight marker, "+" indicates reduced SDS-PAGE, and "-" indicates non-reduced SDS-PAGE. The results indicated that the P1F8-BGTSTIP molecule had a molecular weight of approximately 160 kDa under reduced conditions and was polymeric under non-reduced conditions, consistent with the theoretical molecular weight.

[0299] Activity analysis of P1F8-BGTSTIP protein molecule (ELISA) The fusion protein was diluted to 1 μg / mL and coated onto a 96-well plate at 100 μL per well and left at room temperature for 2 h; (2) The plate was washed once and blocked with a diluent containing bovine serum albumin (ED, 200 μL / well) at room temperature for 2 hours; (3) The plate was washed once, the corresponding specific monoclonal antibody was diluted to 1 μg / mL, 100 μl was added to the first well, a 3-fold serial dilution was performed, duplicate well replicates were prepared, and the plate was left at room temperature for 1 hour; (4) The plate was washed five times, and the secondary antibody GAH-HRP (1:5000) was added to the 96-well plate at 100 μL / well, and the plate was left to stand at room temperature for 1 hour; (5) The plate was washed five times, and color development was carried out at room temperature for 10 minutes, followed by detection at a wavelength of 450 nm using a microplate reader. Data analysis was performed using GraphPad Prism 5 (GraphPad, USA) software.

[0300] The results of enzyme-linked immunosorbent assay of P1F8-BGTSTIP are shown in Figure 35. The results showed that P1F8 had good binding activity to the neutralizing antibodies VRC01, PGT121, PGT122 (JN201912.1 / JN201895.1), 2G12, SF162, and B12, but very weak binding activity to 17b, F105, and F240, indicating that P1F8-BGTSTIP sufficiently exposes broad-spectrum neutralizing antibody epitopes but not non-neutralizing target epitopes.

[0301] Affinity analysis (SPR) of P1F8-BGTSTIP and HPV 58 VLP A CM5 tip was attached and the pipeline was rinsed with PBS-P buffer PBS.

[0302] Ligand 58 VLP (10 μg / mL) was centrifuged at high speed for 10 minutes.

[0303] The 58 VLP coupling program was set: the chip channel was activated (EDC:NHS = 1:1, flow rate: 10 μl per minute), followed by a flushing program (PBS buffer, flow rate: 30 μl per minute). 58 VLPs were loaded for coupling (time: 420 s, flow rate: 10 μl per minute). Once the amount of ligand coupled was determined, a channel blocking program was performed using ethanolamine (time: 420 s, flow rate: 10 μl per minute).

[0304] Affinity detection: A concentration gradient of fusion proteins was prepared and loaded for detection at 800 nM, 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM. Samples were prepared with an association time of 120 s, a dissociation time of 300 s, and a flow rate of 30 μl per minute.

[0305] Using Biacore 8K (Cytiva) software, association (Ka) / dissociation (Kd) curve fitting was performed by the kinetic method, and affinity (KD) was analyzed and calculated.

[0306] Figure 36 shows the results of determining the affinity of P1F8-BGTSTIP for 58 VLP. The results showed that the affinity of P1F8-BGTSTIP for the fusion protein was 4.66 x 10 -8 It was therefore shown that these two can bind and that the binding is relatively strong.

[0307] Example 18: Preparation and identification of HPV-Env fusion protein (P1F8-BGTSTIP) and HPV 58 VLP particle complexes Preparation of P1F8-BGTSTIP complex P1F8-BGTSTIP and 58VLP were incubated at a mass ratio of 5:1 in a water bath at 37°C for 30 minutes, centrifuged at 13,300 rpm for 10 minutes, and then analyzed by high-performance size-exclusion chromatography (HPSEC).

[0308] Equipment: Waters. G5000PW XL The system flow rate for the column was 0.5 mL per minute. The wavelengths were 190 nm to 600 nm, and the column wavelengths were 280 nm and 254 nm. Buffer: Stop buffer. Operation process: The column was pre-equilibrated for 60 minutes until there was no significant change in the absorbance at 280 nm, and then the detector absorbance was reset to zero. The chromatography operation method was edited, and the sample to be analyzed was injected into a 100 μL sample loop. Automatic loading was set, and the instrument was run for 30 minutes. Figure 37 (left side) shows the HPSEC chromatograms of individual HPV 58 VLP, P1F8-BGTSTIP, BGTSTIP, and the P1F8-BGTSTIP-58 VLP complex. The results showed that the peak time of the complex was 12 minutes, which was earlier than that of 58 VLP. The complex components were collected and subjected to SDS-PAGE gel electrophoresis. The results showed that the sample collected at 12 minutes showed a band corresponding to the sum of the molecular weights of P1F8-BGTSTIP and 58VLP, thus indicating that a complex was successfully formed between P1F8-BGTSTIP and 58VLP, with a peak time of approximately 12 minutes.

[0309] Analytical ultracentrifugation (AUC) of particulate antigen P1F8-BGTSTIP-58 VLP The P1F8-BGTSTIP-58 VLP complex collected by the above purification process was subjected to AUC analysis.

[0310] The instrument used was a Beckman XL-A analytical ultracentrifuge equipped with an optical detection system and an An-60Ti rotor.

[0311] The sample pools were loaded according to the operating instructions, 400 μL of sample buffer (same buffer as the sample) was added to the control pool, and 380 μL of sample (OD280 approximately 0.8) was added to the sample pool, and the sample pools were equilibrated to within 0.1 g of weight difference.

[0312] The sample pool was placed in an An-60Ti rotor, and the rotor was placed in the cavity of a Beckman XL-A analytical ultracentrifuge, and an optical path detector was attached.

[0313] The parameters were set as follows: temperature (20°C), Rmin (6.0 cm), Rmax (7.2 cm), wavelength (280 nm), step speed (0.003 cm), scan mode (continuous), data interval (30 s), and data number (150 scans). The centrifugation speed for the complex was 7000 rpm, and for the fusion protein it was 30,000 rpm.

[0314] After the experiment was completed, the density and viscosity of the buffer solution and the partial specific volume of the known protein were calculated using SENDTERP software. The sedimentation coefficient was analyzed using Nonlin software and the Origin version of SEDFIT software, and the friction ratio of globulin f / f 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 0.01 or less, and the variation of the residual map was within 0.05.

[0315] Figure 38 shows the analytical ultracentrifugation results of the complex of P1F8-BGTSTIP of the present invention and 58VLP. The results showed that 58VLP was a single component with a sedimentation coefficient of 119S (Figure 38, right panel), and the P1F8-BGTSTIP-58VLP complex was a single component with a sedimentation coefficient of 176.3S (Figure 38, left panel), which was significantly higher than the sedimentation coefficient of 58VLP particles. The results indicated that 1F8-BGTSTIP formed a particle complex with 58VLP and was able to maintain a stable particle morphology in aqueous solution.

[0316] Transmission electron microscopic observation of the morphology of particulate antigen P1F8-BGTSTIP-58 VLP The transmission electron microscope used was a Tecnai G2 Spirit 120 kV (FEI). Negative staining was performed using phosphotungstic acid.

[0317] Sample preparation: First, a copper mesh (R2 / 2, 200 mesh, ThermoFisher Scientific) was subjected to glow discharge hydrophilization treatment. Next, 5 μl of a sample with a concentration of 0.5 mg / ml was dropped onto the copper mesh and left at room temperature for 60 seconds. After that, the droplet was absorbed from the edge of the copper mesh using absorbent paper. After drying at room temperature, the copper mesh was observed under the microscope described above.

[0318] The morphology of the complex particles under transmission electron microscopy is shown in Figure 39. It was clearly observed that the surface of the particles was covered with a layer of protein, thus indicating that P1F8-BGTSTIP was successfully displayed on the surface of HPV 58 VLP particles.

[0319] Evaluation of immunogenicity of particulate antigen P1F8-BGTSTIP-58 VLP This experimental protocol was approved by the Xiamen University Laboratory Animal Care and Ethics Committee, and all procedures were performed in strict accordance with animal ethics guidelines and approved procedures.

[0320] Six-week-old Balb / C mice were selected and divided into six groups, three high-dose groups and three low-dose groups, with five mice per group. P1F8-BGTSTIP-58VLP particles, P1F8-BGTSTIP, and BGTSTIP immunogens in combination with aluminum adjuvant were used for immunization, with two immunization doses of 0.5 μg and 5 μg for each antigen. Mice were immunized intramuscularly (100 μl / mouse) at 0, 2, 4, and 6 weeks.

[0321] Mice were ocularly bled before each immunization. After the sixth injection, mice were sacrificed by cervical dislocation. Blood samples were placed at 37°C for 30 minutes, centrifuged at 13,300 rpm for 10 minutes, and serum was collected for specific binding antibody titer determination.

[0322] Figure 40 shows the immunogenicity detection results of the particulate antigen P1F8-BGTSTIP-VLP in an example of the present invention. Figure 40 also shows the results of detecting BGTSTIP-specific binding antibody titers (the left graph shows the 0.5 μg group, and the right graph shows the 5 μg group). The results showed that from weeks 1 to 4, the binding titer of the complex particles was significantly higher than that of single BGTSTIP and single P1F8-BGTSTIP proteins.

[0323] Example 19: Design and evaluation of nanobody fusion proteins based on Hepatitis B particulate antigen HBsAg VLP In this example, the VLP carrier was prepared based on the commercial HBV vaccine particulate antigen SHB protein (SEQ ID NO: 73), and the gE fusion protein was prepared according to the method of the previous example, and the particulate antigen HBV-gE was further prepared, and the immunogenicity of the particulate antigen was detected. The specific experimental process was as follows:

[0324] Design and expression of gE fusion proteins The previously reported HBsAg nanobody (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)) was selected for this study. A (GGGGS)3 flexible linker was added to the end of the antibody sequence, which was then coupled to the RBD and gE sequences to construct RBD and gE fusion proteins. To extract the plasmid, frozen 293F cells were removed from a -80°C refrigerator, thawed at 37°C, centrifuged at 1300 rpm for 4 minutes, the supernatant was discarded in a clean bench, the cells were gently flicked, resuspended in 293freestyle culture medium pre-incubated at 37°C, transferred to a flask containing 50 mL of the incubated culture medium, suspended, and cultured at 37°C, 5% CO2, and 120 rpm until the cell density reached 2.0 × 10 6 The culture system was gradually expanded by subculturing when the number of cells reached 100. When the number of cells reached 100, 293F cells were transiently transfected using PEI (MW 25000). The cells were collected in a sterile 50 mL tube and centrifuged at 1300 rpm for 4 minutes. The cells were gently flicked and resuspended in culture medium incubated at 37°C. The flasks were then placed in 450 mL of culture medium incubated at 37°C on a shaker at 37°C for later use.

[0325] The plasmid and PEI (MW 25000) were added to 50 ml of culture medium at a ratio of 1:2, mixed thoroughly, and then allowed to stand for 18 minutes. The mixture was transferred to 450 ml of the above culture medium, suspended, and cultured at 37°C, 5% CO2, and 120 rpm for 6 days to express the gE fusion protein. Care was taken to handle the PEI in the dark during the transfection process.

[0326] Purification of gE fusion proteins Six days after transient transfection, the cell culture medium was collected and centrifuged at 7,000 g for 10 minutes in a JA-14 rotor to obtain the cell supernatant. After centrifugation at 20,000 g for 10 minutes, the supernatant was removed and filtered twice through a 0.22 μm pore size membrane filter. This sample was used for the next step of Ni-excel column purification.

[0327] Ni affinity chromatography purification was performed using the AKTA system; Instrument system: AKTA Pure type preparative liquid chromatograph; Purification medium: Ni Sepharose excel affinity medium; Buffer: Pump A buffer and Pump B buffer, Pump A buffer was 1x PBS buffer, Pump B buffer was 1x PBS + 250mmol / L imidazole buffer; System loading flow rate: 8 mL per minute; detection wavelength: UV@280 nm System elution flow rate: 4 ml per minute; Detection wavelength: UV@280 nm Elution conditions: Protein impurities were eluted with 20 mM imidazole, and the 250 mM imidazole elution product was collected. The eluate was dialyzed overnight against 1x PBS, with the dialysate replaced twice during the dialysis period. Approximately 30 ml of low-concentration target protein was collected and concentrated to 5 ml using a Vivaspin 20 ml, 100 KDa ultrafiltration concentration tube for later use. The collected elution sample was prepared according to the method described above and subjected to SDS-PAGE gel electrophoresis.

[0328] The experimental results showed that a high expression level of the nanobody fusion protein was obtained, and the fusion protein was named S2-gE. As shown in Figure 41, the SDS-PAGE results indicated that S2-gE was consistent with the theoretical molecular weight.

[0329] Purification of fusion proteins and their complexes by molecular sieve chromatography Instrument system: AKTA explorer 100 preparative liquid chromatography system manufactured by GE Healthcare (formerly Amershan Pharmacia). Chromatography medium: Superdex 200 increase (Cytiva). Column volume: 20 cm x 20 mm. Buffer: 20 mM phosphate buffer, pH 7.4. Flow rate: 0.7mL per minute. Detector wavelength: 280 nm. The samples were those in Example 1 and Example 5. Elution procedure: The permeate peak is collected in fractions.

[0330] The products that permeated through Supedex 200 increase were collected to obtain 5 mL of purified samples. 50 μL of each elution product was taken, 10 μL of 6x loading buffer was added, and mixed thoroughly. After placing in an 80°C water bath for 10 minutes, 10 μL was taken and electrophoresed on a 10% SDS-polyacrylamide gel at 120 V for 60 minutes. Coomassie Brilliant Blue staining was then performed to reveal the electrophoretic bands.

[0331] As shown in Figure 42, the SDS-PAGE results showed that the absorption peaks of HBsAg and HBsAg-S2-gE (the peak of the complex of HBsAg and S2-gE) were different, and the particle peak of HBsAg-S2-gE was larger and had a shorter retention time, indicating that S2-gE could form a particulate antigen with HBsAg.

[0332] DLS (Digital Light Spectroscopy) of HBV-gE complex molecules Equipment: NanoBrook Series (Brookhaven Instruments). Functional module: DLS (Dynamic Light Scattering). Buffer: PBS. Operation process: The instrument was activated and preheated for 5 minutes. The test sample was prepared (concentration: 0.5 mg / ml, centrifugation: 12000 rpm for 5 minutes, 50 μl was taken and added to the sample cup). Detection parameters were set: detection time: 300 seconds each time, 3 replicates for each sample.

[0333] As shown in Figure 43, the DLS results indicated that the particle size of single HBsAg was different from that of the HBsAg-S2-gE complex. The particle size of HBsAg-S2-gE (HBV-gE) was significantly larger than that of HBsAg, thus indicating that gE particulate antigen was formed.

[0334] Observation of particle morphology by transmission electron microscopy The transmission electron microscope used was a Tecnai G2 Spirit 120 kV (FEI). Negative staining was performed using phosphotungstic acid.

[0335] Sample preparation: First, a copper mesh (R2 / 2, 200 mesh, ThermoFisher Scientific) was subjected to glow discharge hydrophilization treatment. Next, 5 μl of a sample with a concentration of 0.5 mg / ml was dropped onto the copper mesh. After leaving it at room temperature for 60 seconds, the droplet was absorbed from the edge of the copper mesh using absorbent paper. After drying at room temperature, the copper mesh was observed under the microscope described above.

[0336] As shown in Figure 44, the negative staining results showed that the size and morphology of single HBsAg were different from those of the HBsAg-S2-gE complex, thus indicating that gE particulate antigen was formed.

[0337] Evaluation of the immunogenicity of gE particles This experimental protocol was approved by the Xiamen University Laboratory Animal Care and Ethics Committee, and all procedures were performed in strict accordance with animal ethics guidelines and approved procedures.

[0338] 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 as described above and single S2-gE. The gE dose was 5 μg for both groups, and the mice were intramuscularly injected (50 μL) into the left or right hind limb at 0 and 2 weeks, respectively. Ophthalmic vein blood was collected at 0, 1, 2, 3, and 4 weeks, respectively. Blood samples were centrifuged at 13,000 g for 10 minutes, and serum samples were stored at -20°C. Antigen-specific IgG titers were determined by end-point enzyme-linked immunosorbent assay.

[0339] As shown in Figure 45, the gE-specific titer results after two injections for immunization indicated that the particulate antigen HBsAg-S2-gE could induce significantly higher antibody levels than single S2-gE, and had an immune-enhancing effect.

Claims

1. A fusion protein comprising an immunogenic polypeptide and a nanobody capable of specifically binding to a virus-like particle (VLP), Preferably, the VLP is a VLP assembled from assembly polypeptides, in the fusion protein.

2. One or more of the following: (1) The assembly polypeptide is a polypeptide capable of assembling into a VLP; (2) The assembly polypeptide is a capsid protein of a natural virus or virus-like body, or is an artificially prepared and / or screened polypeptide; (3) The assembly polypeptide is selected from the group consisting of a hepatitis E virus (HEV) protein or a fragment thereof or a variant thereof, a hepatitis B virus (HBV) protein or a fragment thereof or a variant thereof, a human papillomavirus (HPV) protein or a fragment thereof or a variant thereof, and any combination thereof, wherein the fragment or variant retains the ability to assemble into a VLP. (4) The assembly polypeptide is an ORF2 protein of HEV, a fragment thereof, or a mutant thereof. (5) The assembly polypeptide is selected from the group consisting of a p239 protein, a fragment thereof, or a mutant thereof, and a p495 protein, a fragment thereof, or a mutant thereof. (6) The assembly polypeptide is a surface protein of HBV, or a fragment or mutant thereof. (7) The assembly polypeptide is selected from the group consisting of an LHB protein or a fragment or a variant thereof, an MHB protein or a fragment or a variant thereof, and an SHB protein or a fragment or a variant thereof of the hepatitis B virus surface antigen (HBsAg). The fusion protein of claim 1 ,

3. One or more of the following: (1) The variant has one, two, or three amino acid substitutions, deletions, or additions compared to the sequence of the protein from which it is derived; (2) The ORF2 protein has the amino acid sequence shown in SEQ ID NO:

9. (3) The p239 protein has the amino acid sequence shown in SEQ ID NO:

40. (4) The p495 protein has the amino acid sequence shown in SEQ ID NO:

41. (5) The assembly polypeptide is HPV capsid protein L1 or a fragment or mutant thereof. (6) The assembly polypeptide is the SHB protein of hepatitis B virus surface antigen (HBsAg), or a fragment or mutant thereof. (7) The assembly polypeptide has the amino acid sequence set forth in SEQ ID NO: 73 or 74. The fusion protein of claim 2 , characterized in that

4. One or more of the following: (1) The immunogenic polypeptide is a polypeptide of biological or non-biological origin or an immunogenic variant thereof; (2) The immunogenic polypeptide is a polypeptide derived from a pathogen or a non-pathogen, or an immunogenic variant thereof; (3) The immunogenic polypeptide is obtained from a mammalian tumor cell. (4) The immunogenic polypeptide is selected from the group consisting of carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and cancer antigen 125 (CA125). (5) The immunogenic polypeptide is derived from a virus, bacterium, fungus, or parasite. The fusion protein of claim 1 ,

5. One or more of the following: (1) The virus is selected from the group consisting of varicella-zoster virus (VZV), severe acute respiratory syndrome coronavirus 2 (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 encephalitis B virus; (2) The immunogenic polypeptide is selected from the group consisting of an RBD protein of SARS-CoV-2 or an immunogenic fragment thereof or a mutant thereof, an Env protein of HIV-1 or an immunogenic fragment thereof or a mutant thereof, and a gE protein of VZV or an immunogenic fragment thereof or a mutant thereof; (3) The variant has one, two, or three amino acid substitutions, deletions, or additions compared to the sequence of the protein from which it is derived. (4) The RBD protein has an amino acid sequence shown in any one of SEQ ID NOs: 1 to 8. (5) The Env protein has the amino acid sequence shown in SEQ ID NO: 34 or SEQ ID NO:

35. (6) The gE protein has the amino acid sequence shown in SEQ ID NO:

30. The fusion protein of claim 1 ,

6. One or more of the following: (1) The nanobody is a camel-derived antibody or a fish-derived antibody. (2) The nanobody is an alpaca-derived antibody or a shark-derived antibody. (3) The nanobody is a chimeric antibody, a humanized antibody, or a fully human antibody. (4) The fusion protein comprises two, three, or more types of immunogenic polypeptides. (5) Each type of immunogenic polypeptide is independently obtained from the same or different pathogens; (6) Each type of immunogenic polypeptide is a different polypeptide obtained from the same pathogen; (7) The fusion protein contains one type of immunogenic polypeptide. (8) The nanobody is a nanobody that specifically binds to a polypeptide of HEV, HBV, and / or HPV. (9) The Nanobody comprises CDR-H1, CDR-H2, and CDR-H3 contained in a heavy chain variable region (VHH) set forth in any one of SEQ ID NOs: 10 to 29, 68, and 69, wherein the CDRs are defined according to the IMGT, Kabat, or Chothia numbering system. The fusion protein of claim 1 , 7. The nanobody of claim 1, (a) a heavy chain variable region (VHH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 having the sequence set forth in SEQ ID NO:90, a VH CDR2 having the sequence set forth in SEQ ID NO:91, and a VH CDR3 having the sequence set forth in SEQ ID NO:92; (b) a heavy chain variable region (VHH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 having the sequence set forth in SEQ ID NO:93, a VH CDR2 having the sequence set forth in SEQ ID NO:94, and a VH CDR3 having the sequence set forth in SEQ ID NO:95; (c) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO:96, a VH CDR2 set forth in SEQ ID NO:97, and a VH CDR3 set forth in SEQ ID NO:98; (d) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO:99, a VH CDR2 set forth in SEQ ID NO:100, and a VH CDR3 set forth in SEQ ID NO:101; (e) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 102, a VH CDR2 set forth in SEQ ID NO: 103, and a VH CDR3 set forth in SEQ ID NO: 104; (f) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 105, a VH CDR2 set forth in SEQ ID NO: 106, and a VH CDR3 set forth in SEQ ID NO: 107; (g) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 108, a VH CDR2 set forth in SEQ ID NO: 109, and a VH CDR3 set forth in SEQ ID NO: 110; (h) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 111, a VH CDR2 set forth in SEQ ID NO: 112, and a VH CDR3 set forth in SEQ ID NO: 113; (i) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 114, a VH CDR2 set forth in SEQ ID NO: 115, and a VH CDR3 set forth in SEQ ID NO: 116; (j) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 117, VH CDR2 set forth in SEQ ID NO: 118, and VH CDR3 set forth in SEQ ID NO: 119; (k) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 120, VH CDR2 set forth in SEQ ID NO: 121, and VH CDR3 set forth in SEQ ID NO: 122; (l) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 123, VH CDR2 set forth in SEQ ID NO: 124, and VH CDR3 set forth in SEQ ID NO: 125; (m) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 set forth in SEQ ID NO: 126, VH CDR2 set forth in SEQ ID NO: 127, and VH CDR3 set forth in SEQ ID NO: 128; (n) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 129, a VH CDR2 set forth in SEQ ID NO: 130, and a VH CDR3 set forth in SEQ ID NO: 131; (o) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 132, a VH CDR2 set forth in SEQ ID NO: 133, and a VH CDR3 set forth in SEQ ID NO: 134; (p) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 135, a VH CDR2 set forth in SEQ ID NO: 136, and a VH CDR3 set forth in SEQ ID NO: 137; (q) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 138, a VH CDR2 set forth in SEQ ID NO: 139, and a VH CDR3 set forth in SEQ ID NO: 140; (r) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 141, a VH CDR2 set forth in SEQ ID NO: 142, and a VH CDR3 set forth in SEQ ID NO: 143; (s) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO:57, a VH CDR2 set forth in SEQ ID NO:58, and a VH CDR3 set forth in SEQ ID NO:59; (t) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 60, a VH CDR2 set forth in SEQ ID NO: 61, and a VH CDR3 set forth in SEQ ID NO: 62; or (u) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO:65, a VH CDR2 set forth in SEQ ID NO:66, and a VH CDR3 set forth in SEQ ID NO:67; Including, More preferably, the Nanobody comprises a sequence as set forth in any one of SEQ ID NOs: 10 to 29, SEQ ID NO: 68, SEQ ID NO: 69 or a variant thereof, wherein the variant is capable of specifically binding to the assembly polypeptide and comprises one, two or three amino acid substitutions, deletions or additions compared to the sequence from which it is derived. The fusion protein of claim 6.

8. The fusion protein of claim 1 , further comprising a linker, a signal peptide and / or a tag.

9. One or more of the following: (1) The linker is a polypeptide. (2) The linker comprises one, two or three sequences represented by (G m S) n , where m is an integer selected from 1 to 6, and n is an integer selected from 1 to 6; (3) The linker has the amino acid sequence shown in SEQ ID NO:

39. (4) The immunogenic polypeptide and the Nanobody of the fusion protein are directly linked or linked through the linker. (5) The immunogenic polypeptide is located at the N-terminus or C-terminus of the fusion protein. (6) The fusion protein comprises, from N-terminus to C-terminus, the immunogenic polypeptide and the Nanobody; Or, said Nanobody and said immunogenic polypeptide; Or, the immunogenic polypeptide, the linker, and the Nanobody; or comprising said Nanobody, said linker, and said immunogenic polypeptide; The fusion protein according to claim 8, characterized in that

10. One or more of the following: (1) The signal peptide has an amino acid sequence shown in SEQ ID NO: 31, SEQ ID NO: 37, or SEQ ID NO:

38. (2) The tag is a purification tag. (3) The signal peptide is located at the N-terminus of the fusion protein. (4) The tag is located at the C-terminus of the fusion protein. (5) The fusion protein has an amino acid sequence shown in any one of SEQ ID NOs: 42 to 49, 50 to 56, 72 to 79, 80 to 87, 63, 64, and 89. The fusion protein according to claim 8, characterized in that

11. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of claim 1.

12. A vector comprising the isolated nucleic acid molecule of claim 11.

13. 12. A host cell comprising the nucleic acid molecule of claim 11 or a vector comprising said isolated nucleic acid molecule.

14. 10. A method for expressing or producing the fusion protein of claim 1, comprising culturing a host cell containing an isolated nucleic acid molecule encoding the fusion protein under conditions that allow expression of the protein, and optionally recovering or purifying the expressed fusion protein.

15. A composition comprising at least one fusion protein according to claim 1, Optionally, the composition further comprises an assembly polypeptide.

16. The assembly polypeptide is present in the form of a VLP; or The composition of claim 15 , wherein the fusion protein is attached to a VLP.

17. 10. A kit comprising a fusion protein of claim 1 or a first nucleic acid molecule containing a nucleotide sequence encoding said fusion protein, and an assembly polypeptide or a second nucleic acid molecule containing a nucleotide sequence encoding said assembly polypeptide, Optionally, the kit further comprises a vector and / or a buffer.

18. One or more of the following: (1) the nucleotide sequence is codon-optimized or non-optimized according to the codon preferences of the host cell; (2) The fusion protein or the first nucleic acid molecule and the assembly polypeptide or the second nucleic acid molecule are provided separately or in the form of a composition; (3) The first nucleic acid molecule and the second nucleic acid molecule are contained in the same or different vectors. (4) The buffer solution is selected from the group consisting of a phosphate buffer solution, a citrate buffer solution, a carbonate buffer solution, an acetate buffer solution, a barbiturate buffer solution, a Tris buffer solution, and any combination thereof. 、 18. The kit of claim 17,

19. A particulate antigen comprising an assembly polypeptide in the form of a VLP and the fusion protein of claim 1 attached to said assembly polypeptide.

20. The particulate antigen, wherein the particulate antigen is one or more of the following: (1) the fusion protein is attached to a VLP through the interaction of the Nanobody with the assembly polypeptide; (2) the VLP is attached to at least one fusion protein; (3) The VLP also has an additional polypeptide or fusion protein attached to it. The particulate antigen according to claim 19, characterized in that:

21. 20. A method for preparing the particulate antigen of claim 19, comprising: A method comprising contacting the assembly polypeptide with the fusion protein under conditions that allow VLP assembly, and optionally recovering or purifying the particulate antigen in a buffer solution.

22. An adjuvant and one of the following: A fusion protein according to claim 1, or a composition comprising at least one of the fusion proteins; or 1. A vaccine comprising an assembly polypeptide in the form of a VLP and a particulate antigen comprising the fusion protein attached to the assembly polypeptide, Preferably, the vaccine wherein the adjuvant is selected from the group consisting of an aluminum salt adjuvant, a mixed zinc and aluminum adjuvant, Freund's adjuvant, an oil emulsion adjuvant, a cytokine, a TLR agonist, a CpG adjuvant, a liposome, an AS01B adjuvant, and any combination thereof.

23. （１）～（６）： (1) The fusion protein according to claim 1; (2) a nucleic acid molecule encoding the fusion protein; (3) a vector comprising an isolated nucleic acid molecule encoding the fusion protein; (4) a host cell containing an isolated nucleic acid molecule encoding the fusion protein; (5) a composition comprising at least one of the fusion proteins; (6) a particulate antigen comprising an assembly polypeptide in the form of a VLP and the fusion protein attached to the assembly polypeptide; and A pharmaceutical composition optionally further comprising a pharmaceutically acceptable carrier and / or excipient.

24. 1. A method of inducing an immune response in a subject, comprising administering to the subject: A fusion protein according to claim 1, or a nucleic acid molecule encoding the fusion protein; or a vector comprising the isolated nucleic acid molecule encoding the fusion protein; or a host cell comprising an isolated nucleic acid molecule encoding the fusion protein; or a composition comprising at least one of the fusion proteins; or a kit comprising the fusion protein and an assembly polypeptide; or a particulate antigen comprising an assembly polypeptide in the form of a VLP and said fusion protein attached to said assembly polypeptide; or a vaccine comprising said fusion protein together with an adjuvant; or administering an effective amount of a pharmaceutical composition comprising the fusion protein; Preferably, said immune response is a response to an immunogenic polypeptide, preferably said immune response is a T cell or B cell response; Preferably, the subject is a human.

25. 1. A method of preventing and / or treating a disease and / or condition in a subject in which an immune response to an immunogenic polypeptide is beneficial or prevented, comprising administering to said subject: A fusion protein according to claim 1, or a nucleic acid molecule encoding the fusion protein; or a vector comprising the isolated nucleic acid molecule encoding the fusion protein; or a host cell comprising an isolated nucleic acid molecule encoding the fusion protein; or a composition comprising at least one of the fusion proteins; or a kit comprising the fusion protein and an assembly polypeptide; or a particulate antigen comprising an assembly polypeptide in the form of a VLP and said fusion protein attached to said assembly polypeptide; or a vaccine comprising said fusion protein together with an adjuvant; or administering an effective amount of a pharmaceutical composition comprising the fusion protein; Preferably, the disease and / or condition is caused by a tumor cell or a pathogen (e.g., a virus, bacterium, fungus, parasite) from which the immunogenic polypeptide is derived; Preferably, the disease and / or condition is caused by the virus from which the immunogenic polypeptide is derived, such as chickenpox, COVID-19, AIDS, condyloma acuminata, viral hepatitis (e.g., hepatitis B, hepatitis A, hepatitis C, hepatitis E), measles, or mumps; Preferably, the subject is a human.

26. A system for preparing a particulate immunogenic polypeptide, comprising: a first vector and a second vector, wherein the first vector comprises a nucleotide sequence encoding a fusion protein, the fusion protein comprising the immunogenic polypeptide and an assembly polypeptide; and the second vector comprises a nucleotide sequence encoding a nanobody, wherein the nanobody is capable of specifically binding to the assembly polypeptide, and the assembly polypeptide is capable of assembling into a VLP. Preferably, the nucleotide sequence is codon-optimized or non-optimized according to the codon preferences of the host cell, Preferably, the assembly polypeptide is selected from a protein of Hepatitis E Virus (HEV) or a fragment or variant thereof; Preferably, the fusion protein is as defined in claim 1.

27. A method for enhancing the immunogenicity of an immunogenic polypeptide, comprising: preparing or obtaining a fusion protein comprising the immunogenic polypeptide and a Nanobody capable of specifically binding to an assembly polypeptide; and contacting the fusion protein with a VLP comprising the assembly polypeptide, thereby obtaining a particulate antigen comprising the immunogenic polypeptide attached to a VLP; Preferably, the fusion protein is as defined in claim 1, Preferably, said assembly polypeptide assembles into said VLP, and preferably said fusion protein is attached to said VLP through the interaction of said Nanobody with said assembly polypeptide.

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