Fusion protein and granulated antigen containing same

Abstract

A fusion protein, and compositions, kits, granulated antigens, vaccines and pharmaceutical compositions comprising the same. The invention also relates to a fusion protein and a composition comprising the fusion protein, a kit and application of the granulated antigen in preparation of a pharmaceutical composition or a vaccine. The granulated antigens are particularly suitable for vaccine production and vaccination and have advantages in the prevention and / or treatment of viral infections.

Description

A fusion protein and a particle-bound antigen containing the same Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, to a fusion protein, and compositions, kits, granulated antigens, vaccines, and pharmaceutical compositions comprising the fusion protein. This application also relates to the use of the fusion protein, and compositions, kits, and granulated antigens comprising the fusion protein in the preparation of pharmaceutical compositions or vaccines. Background Art

[0002] It is well known that vaccines are the most cost-effective method for preventing and controlling infectious diseases, saving nearly 6 million lives annually. Currently, genetically engineered vaccines often use proteins / polysaccharides that are essential for pathogen infection as antigens. While these vaccine antigens offer advantages such as high safety, mature technology, and the ability to generate immune protection against specific pathogens, they often face challenges such as poor humoral immunity, difficulty achieving long-term protection, and difficulty activating effective cellular immunity. Therefore, developing safe, efficient, and broad-spectrum novel immunogens is key to improving vaccine efficacy.

[0003] Particle vaccines are an important vaccine platform for combating pathogenic threats. Currently, three major particle vaccines have been approved for marketing, including hepatitis E (HEV), human papillomavirus (HPV), and hepatitis B (HBV). The hepatitis E vaccine is a virus-like particle (VLP) vaccine. The hepatitis E antigen fragment, p239 protein (aa368-606), expressed in an Escherichia coli expression system, is renatured and assembled into particles 25 nm in diameter. These particles have a structural conformation similar to that of natural virus particles and are able to effectively mimic natural viral antigen epitopes. Currently, they have been successfully developed into vaccine antigens, becoming the world's first hepatitis E vaccine, Hecolin. It was launched in mainland China in 2012, and clinical results have confirmed its excellent preventive protective effect. Currently available HPV vaccines include bivalent, quadrivalent, and nine-valent vaccines. HPV vaccines are assembled from the virus's major capsid protein L1 to form virus-like particles, which can be produced and prepared by expression systems such as insect cells, yeast cells, and Escherichia coli. Clinical and post-marketing studies have shown that HPV vaccines have good safety and protective effects. Hepatitis B vaccine research and development has a long history, and there are currently several hepatitis B vaccines on the market both domestically and internationally. Hepatitis B vaccines are mainly composed of S proteins of different forms of surface antigen HBsAg (SHBs, MHBs, or LHBs) that spontaneously assemble to form particles. They are currently mainly produced by yeast systems and CHO systems. Clinical and real-world studies have shown that hepatitis B vaccines can produce high titers of protective antibodies and have good efficacy and safety. In summary, the production of granulated antigens in hepatitis E vaccines, HPV vaccines, and hepatitis B vaccines has the advantages of being economical and efficient, with stable production processes, controllable antigen quality, industrial mass production, good safety, and strong immunogenicity. They are a good foundation and important reference for the further development of other vaccines.

[0004] However, many viral antigen proteins lack the ability to assemble into particles, making particle vaccines impractical. Isolated viral antigens or oligomeric antigens have poor immunogenicity, making vaccine development difficult. Displaying target proteins on particle surfaces is a key issue and primary goal in the design of recombinant vaccines and drug delivery vehicles. Currently, methods for displaying multivalent antigens on particles often rely on fusion expression with particle vectors or chemical conjugation to particle vectors using SpyTag / SpyCatcher technology. However, these approaches have significant limitations. First, fusion expression methods require that particle vectors suitable for fusion expression possess exposed N / C termini to accommodate exogenous protein fusion. For particle vectors without exposed termini, structural biology and information biology must be used to explore whether fusion at other locations is feasible. Second, fusion expression strategies must consider whether they will affect the native conformation of the target molecule. Furthermore, exposed termini can restrict target protein display locations, necessitating comprehensive consideration of factors such as steric hindrance. A limitation of SpyTag / SpyCatcher technology is that this conjugation method is constrained by the structural characteristics of the vector particles. Therefore, exploring an efficient method to display target antigens on the surface of particle antigens is an important goal in this field, but it faces various difficulties.

[0005] Membrane proteins of enveloped viruses are primary immunogenic targets for vaccine development. However, these proteins are typically anchored to the membrane structure on the viral surface and cannot form particle antigens on their own. They are also highly hydrophobic, making it difficult to directly express intact proteins into membrane-containing particle structures. The novel coronavirus is an enveloped RNA virus. The spike protein is a key molecule on the envelope, primarily involved in receptor recognition and cell membrane fusion. The RBD, the protein domain on the spike protein that directly binds to the host receptor (ACE2), is also the primary target for neutralizing antibodies. Therefore, using the RBD protein as an antigen in a COVID-19 vaccine holds great promise. However, due to its small molecular weight and limited immunogenicity, enhancing its immunogenicity is a key issue. Similar to the RBD, the gE protein is the most abundant glycoprotein on the surface of the varicella-zoster virus (VZV) and is also a key target for vaccine development. Compared to the novel coronavirus and VZV, HIV-1, the virus that causes AIDS, has a higher genetic mutation rate, making HIV vaccine development more challenging. Env is the main antigenic substance on the surface of the HIV-1 virus and is also a key molecule in the development of AIDS vaccines. The full-length Env molecule is a transmembrane protein, also known as the gp160 protein. By deleting the intracellular segment, the soluble expression of the Env protein can be effectively improved. This molecule is called the gp140 protein, and the formation of a natural trimer-like gp140 protein has obvious advantages in exposing neutralizing epitopes and neutralizing epitope antibody responses. However, despite this, the development of AIDS vaccines based on Env has not yet been successful. One of the difficulties is that the surface of Env is covered with more than 50% polysaccharide molecules, which significantly reduces the immunogenicity of the antigen.

[0006] Therefore, in the face of the various difficulties in vaccine development mentioned above, improving the immunogenicity of antigens is of great significance for the development and application of corresponding vaccines.

[0007] Summary of the Invention

[0008] The present invention utilizes the ability of assembled polypeptides (for example, ORF2 protein of HEV or its fragments or variants thereof, L1 protein of HPV or its fragments or variants thereof, surface antigen of HBV or its fragments or variants thereof) to assemble into VLPs, fuses nano antibodies that specifically bind to the assembled polypeptides with immunogenic polypeptides for expression, and utilizes the targeted binding properties of nano antibodies to the assembled polypeptides to display a variety of immunogenic polypeptides on the surface of VLPs formed by the assembled polypeptides, thereby obtaining granulated immunogenic polypeptides. Thus, the present application provides a system and method for granulating immunogenic polypeptides. The present application also confirms that granulated immunogenic polypeptides can induce the body to produce high levels of humoral immunity and cellular immune responses. Therefore, the granulated immunogenic polypeptides of the present invention have the potential to be vaccine candidate molecules, and have the advantages of versatility and convertibility in the design and production of vaccines.

[0009] Therefore, 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);

[0010] In certain embodiments, the VLP is a VLP assembled from an assembly polypeptide.

[0011] In certain embodiments, the assembly polypeptide is a polypeptide capable of assembling into a VLP.

[0012] In certain embodiments, the assembly polypeptide is a coat protein of a natural virus or viroid. In such embodiments, the VLP assembled from the assembly polypeptide is similar in structure to the natural virus or viroid, except that it does not contain the genome of the natural virus or viroid.

[0013] In certain embodiments, the assembling polypeptide is an artificially prepared and / or screened polypeptide capable of being assembled into a VLP. In such embodiments, the VLP that the assembling polypeptide is assembled into is similar or dissimilar to the structure of a natural virus or viroid.

[0014] In certain embodiments, the assembly polypeptide is selected from a protein of hepatitis E virus (HEV) or a fragment thereof or a variant thereof, a protein of hepatitis B virus (HBV) or a fragment thereof or a variant thereof, a protein of human papillomavirus (HPV) or a fragment thereof or a variant thereof, or any combination thereof; wherein the fragment or variant retains the ability to assemble into VLP.

[0015] In certain embodiments, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5 amino acid substitutions, deletions or additions) compared to the sequence of the polypeptide from which it is derived. In certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the polypeptide and its variant have the same or similar biological activity. In certain preferred embodiments, the biological activity is the ability to be assembled into VLPs.

[0016] In certain embodiments, the assembly polypeptide is the ORF2 protein of HEV or a fragment or variant thereof.

[0017] 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. The ORF2 protein or its fragment has the ability to self-assemble into VLPs.

[0018] In certain embodiments, the fragment of the ORF2 protein is selected from the group consisting of p239 protein and p495 protein.

[0019] In certain embodiments, the assembling polypeptide is selected from p239 protein or a fragment thereof or a variant thereof, p495 protein or a fragment thereof or a variant thereof.

[0020] In some embodiments, the sequence of the p239 protein is from amino acid position 368 to amino acid position 606 of the amino acid sequence of the ORF2 protein. In some embodiments, the sequence of the p495 protein is from amino acid position 112 to amino acid position 606 of the amino acid sequence of the ORF2 protein.

[0021] In certain embodiments, the ORF2 protein has the amino acid sequence shown in SEQ ID NO: 9. In certain embodiments, the p239 protein has the amino acid sequence shown in SEQ ID NO: 40. In certain embodiments, the p495 protein has the amino acid sequence shown in SEQ ID NO: 41.

[0022] In certain embodiments, the assembly polypeptide is the HPV capsid protein L1 or a fragment or variant thereof.

[0023] HPV is composed of a protein coat and core DNA. The coat is composed of the major capsid protein (L1) and the 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 coat.

[0024] In certain embodiments, the assembling polypeptide has the amino acid sequence shown in SEQ ID NO:74.

[0025] In certain embodiments, the assembled polypeptide is a surface protein of HBV (e.g., hepatitis B virus surface antigen) or a fragment thereof or a variant thereof. In certain embodiments, the assembled polypeptide is selected from the LHBs protein of hepatitis B virus surface antigen (HBsAg) or a fragment thereof or a variant thereof, the MHBs protein or a fragment thereof or a variant thereof, the SHBs protein or a fragment thereof or a variant thereof. In certain embodiments, the assembled polypeptide is selected from the SHBs protein of hepatitis B virus surface antigen (HBsAg) or a fragment thereof or a variant thereof.

[0026] Hepatitis B virus surface antigen (HBsAg) is composed of large surface protein (LHBs), middle surface protein (MHBs) and small surface protein (SHBs). In the present invention, the term "SHBs protein" refers to the small surface protein that constitutes HBsAg.

[0027] In certain embodiments, the assembling polypeptide has the amino acid sequence shown in SEQ ID NO:73.

[0028] In certain embodiments, the immunogenic polypeptide is a polypeptide obtained from an organism or a non-organism (eg, artificially synthesized) or an immunogenic variant thereof.

[0029] In certain embodiments, the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5 substitutions, deletions, or additions) compared to the sequence of the polypeptide from which it is derived. In certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the polypeptide and its variant have the same or similar biological activity. In certain preferred embodiments, the biological activity is the ability to elicit an immune response.

[0030] In certain embodiments, the organism is a pathogen (eg, virus, bacteria, fungus, parasite) or a non-pathogen.

[0031] In certain embodiments, the immunogenic polypeptide is obtained from a non-tumor cell of a mammal (eg, a human). In certain embodiments, the immunogenic polypeptide is proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0032] In certain embodiments, the immunogenic polypeptide is obtained from a tumor cell of a mammal (e.g., a human) (e.g., expressed or overexpressed on the surface of a tumor cell). In certain embodiments, the immunogenic polypeptide is selected from carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and cancer antigen 125 (CA125).

[0033] In certain embodiments, the immunogenic polypeptide is obtained from a virus, a bacterium (eg, Mycobacterium tuberculosis), a fungus (eg, Candida species), or a parasite (eg, Plasmodium falciparum).

[0034] In certain embodiments, the virus is selected from varicella-zoster virus (VZV), novel coronavirus (SARS-CoV-2), coronavirus (SARS-COV-1), human immunodeficiency virus type 1 (HIV-1), human papillomavirus, hepatitis B virus, hepatitis A virus, hepatitis C virus, hepatitis E virus, measles virus, mumps virus, influenza virus, and Japanese encephalitis virus.

[0035] In certain embodiments, the sequence of the assembled polypeptide is derived from a viral pathogen and the sequence of the immunogenic polypeptide is derived from a non-viral pathogen (eg, bacteria, fungi, parasites).

[0036] In certain embodiments, the sequence of the assembling polypeptide is derived from a viral pathogen and the sequence of the immunogenic polypeptide is derived from another viral pathogen.

[0037] The immunogenic polypeptides of the present invention can be polypeptides derived from the surface or core of a virus. Generally, the immunogenic polypeptide can be any structural or functional polypeptide comprising at least 6 amino acid residues. In some embodiments, the immunogenic polypeptide is 6 to 10,000 amino acid residues in length. In some embodiments, the immunogenic polypeptide is 25 to 2,000 amino acid residues in length. In some embodiments, the immunogenic polypeptide is 50 to 500 amino acid residues in length.

[0038] In certain embodiments, the immunogenic polypeptide is selected from the RBD protein of SARS-CoV-2 or an immunogenic fragment or variant thereof, the Env protein of HIV-1 or an immunogenic fragment (e.g., gp140, gp160) or variant thereof, the gE protein of VZV or an immunogenic fragment or variant thereof.

[0039] In certain embodiments, the fragment of the Env protein is selected from the group consisting of gp160 protein, gp120 protein, and gp41 protein.

[0040] In certain embodiments, the Env protein has an amino acid sequence as shown in SEQ ID NO: 34 or 35. In certain embodiments, the sequence of the gp160 protein is shown in GenBank as Accession Nos. AAB05604 and AAD12142. In certain embodiments, the amino acid sequence of gp41 is shown in GenBank as Accession No. CAD20975.

[0041] In certain embodiments, the RBD protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1-8.

[0042] In certain embodiments, the gE protein has the amino acid sequence shown in SEQ ID NO:30.

[0043] Those skilled in the art are capable of preparing Nanobodies that can specifically bind to specific antigens by various methods known in the art, for example, by immunizing alpacas or sharks with specific antigens, screening out positive clones, obtaining the sequence of the heavy chain of the antibody by sequencing, and then constructing a vector comprising the sequence of the heavy chain of the antibody, transfecting host cells under specific conditions, and expressing them to obtain Nanobodies.

[0044] Therefore, when the assembly polypeptide is determined, those skilled in the art can prepare and obtain nanobodies that can specifically bind to the assembly polypeptide. Therefore, the nanobodies of the present application are not limited to the specific forms (e.g., nanobodies) and specific sequences specifically used in the examples.

[0045] In certain embodiments, the Nanobody is a camelid (eg, alpaca) antibody or a fish (eg, shark) antibody.

[0046] In certain embodiments, the Nanobody is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0047] In certain embodiments, the fusion protein comprises two, three, or more immunogenic polypeptides.

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

[0049] In certain embodiments, each immunogenic polypeptide is a different polypeptide obtained from the same pathogen (eg, virus).

[0050] 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.

[0051] In certain exemplary embodiments, the fusion protein comprises a first immunogenic polypeptide, a second immunogenic polypeptide, and a third immunogenic polypeptide, and the first immunogenic polypeptide is the RBD protein shown in SEQ ID NO: 1, the second immunogenic polypeptide is the RBD protein shown in SEQ ID NO: 2, and the third immunogenic polypeptide is the RBD protein shown in SEQ ID NO: 3. In certain embodiments, the Nanobody specifically binds to the RBD proteins shown in SEQ ID NOs: 1-3.

[0052] In certain embodiments, the fusion protein comprises 1 immunogenic polypeptide.In certain embodiments, the Nanobody is a Nanobody that specifically binds to a polypeptide of HEV, HBV and / or HPV.

[0053] In certain embodiments, the Nanobody comprises CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VHH) shown in any one of SEQ ID NO: 10-29, 68, 69. In certain embodiments, the CDRs are defined according to the IMGT, Kabat or Chothia numbering systems.

[0054] In certain embodiments, the Nanobody comprises:

[0055] (a) a heavy chain variable region (VHH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 90, VH CDR2 of SEQ ID NO: 91, and VH CDR3 of SEQ ID NO: 92;

[0056] (b) a heavy chain variable region (VHH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 93, VH CDR2 of SEQ ID NO: 94, and VH CDR3 of SEQ ID NO: 95;

[0057] (c) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 96, VH CDR2 of SEQ ID NO: 97, and VH CDR3 of SEQ ID NO: 98;

[0058] (d) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 99, VH CDR2 of SEQ ID NO: 100, and VH CDR3 of SEQ ID NO: 101;

[0059] (e) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 102, VH CDR2 of SEQ ID NO: 103, and VH CDR3 of SEQ ID NO: 104;

[0060] (f) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 105, VH CDR2 of SEQ ID NO: 106, and VH CDR3 of SEQ ID NO: 107;

[0061] (g) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 108, VH CDR2 of SEQ ID NO: 109, and VH CDR3 of SEQ ID NO: 110;

[0062] (h) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 111, VH CDR2 of SEQ ID NO: 112, and VH CDR3 of SEQ ID NO: 113;

[0063] (i) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 114, VH CDR2 of SEQ ID NO: 115, and VH CDR3 of SEQ ID NO: 116;

[0064] (j) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 117, VH CDR2 of SEQ ID NO: 118, and VH CDR3 of SEQ ID NO: 119;

[0065] (k) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 120, VH CDR2 of SEQ ID NO: 121, and VH CDR3 of SEQ ID NO: 122;

[0066] (1) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 123, VH CDR2 of SEQ ID NO: 124, and VH CDR3 of SEQ ID NO: 125;

[0067] (m) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 126, VH CDR2 of SEQ ID NO: 127, and VH CDR3 of SEQ ID NO: 128;

[0068] (n) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 129, VH CDR2 of SEQ ID NO: 130, and VH CDR3 of SEQ ID NO: 131;

[0069] (o) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 132, VH CDR2 of SEQ ID NO: 133, and VH CDR3 of SEQ ID NO: 134;

[0070] (p) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 135, VH CDR2 of SEQ ID NO: 136, and VH CDR3 of SEQ ID NO: 137;

[0071] (q) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 138, VH CDR2 of SEQ ID NO: 139, and VH CDR3 of SEQ ID NO: 140;

[0072] (r) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 141, VH CDR2 of SEQ ID NO: 142, and VH CDR3 of SEQ ID NO: 143;

[0073] (s) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 57, VH CDR2 of SEQ ID NO: 58, and VH CDR3 of SEQ ID NO: 59;

[0074] (t) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 60, VH CDR2 of SEQ ID NO: 61, and VH CDR3 of SEQ ID NO: 62; or

[0075] (u) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs): VH CDR1 of SEQ ID NO: 65, VH CDR2 of SEQ ID NO: 66, and VH CDR3 of SEQ ID NO: 67.

[0076] In certain embodiments, the Nanobody comprises a sequence as shown in any one of SEQ ID NOs: 10-29, 68, 69, or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived. In certain embodiments, the substitutions are conservative substitutions.

[0077] In certain embodiments, the fusion protein further comprises a linker.

[0078] In certain embodiments, the linker is a polypeptide, such as a flexible peptide or a rigid peptide.

[0079] In certain embodiments, the linker comprises one or more (e.g., 1, 2, or 3) sequences as shown in (GmS)n, wherein m is selected from an integer from 1 to 6, and n is selected from an integer from 1 to 6. In certain embodiments, m is 3, 4, or 5. In certain embodiments, n is 2, 3, or 4.

[0080] In certain embodiments, the linker has the amino acid sequence shown in SEQ ID NO:39.

[0081] In certain embodiments, the immunogenic polypeptide and the Nanobody of the fusion protein are directly linked or linked via a linker.

[0082] In certain embodiments, the immunogenic polypeptide is located at the N-terminus or C-terminus of the fusion protein.

[0083] In certain 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.

[0084] In certain embodiments, the fusion protein further comprises a signal peptide and / or a tag.

[0085] In certain embodiments, the signal peptide is selected from tPA signal peptide, bee venom signal peptide. In certain embodiments, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 31, 37 or 38.

[0086] In certain embodiments, the tag is a tag for purification, for example, selected from a His tag or a GST tag.

[0087] In certain embodiments, the signal peptide is located at the N-terminus of the fusion protein.

[0088] In certain embodiments, the tag is located at the C-terminus of the fusion protein.

[0089] In certain embodiments, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 42-49, 50-56, 72-79, 80-87, 63, 64, and 89.

[0090] In a second aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein as described in the first aspect.

[0091] In certain embodiments, the nucleotide sequence is codon-optimized or non-optimized according to the codon preference of the host cell.

[0092] As known to those skilled in the art, codons have degeneracy. That is, during the translation of a protein, each amino acid may correspond to one or more codons, for example, up to six codons. Different species (e.g., host cells) have very different preferences when using degenerate codons to encode a certain amino acid. 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 certain specific codons to encode an amino acid. Optimizing the sequence of a nucleic acid molecule according to codon preference is particularly advantageous in some cases, for example, it may help to improve the expression level of the protein encoded by the nucleic acid molecule.

[0093] In a third aspect, the present application provides a vector comprising the isolated nucleic acid molecule as described in the second aspect. In certain embodiments, the vector is used to express (eg, express in vitro in a cell) a protein encoded by the isolated nucleic acid molecule.

[0094] In a fourth aspect, the present application provides a host cell comprising the nucleic acid molecule as described in the second aspect or the vector as described in the third aspect.

[0095] In certain embodiments, the host cell is selected from a prokaryotic cell and a eukaryotic cell.

[0096] In certain embodiments, the prokaryotic cell is selected from the group consisting of an Escherichia coli cell and a Bacillus subtilis cell.

[0097] 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.

[0098] In certain embodiments, the animal cell is a mammalian cell (eg, a murine cell, a human cell).

[0099] In a fifth aspect, the present application provides a method for expressing or producing the fusion protein as described in the first aspect, the method comprising culturing the host cell described in the fourth aspect under conditions allowing protein expression, and optionally, recovering or purifying the expressed fusion protein.

[0100] In a sixth aspect, the present application provides a composition comprising at least one fusion protein as described in the first aspect.

[0101] In certain embodiments, the composition further comprises an assembling polypeptide.

[0102] In certain embodiments, the assembling polypeptides assemble into VLPs.

[0103] In certain embodiments, the fusion protein is attached to the VLP.

[0104] In a seventh aspect, the present application provides a kit comprising: the fusion protein as described in the first aspect or a first nucleic acid molecule containing a nucleotide sequence encoding the fusion protein, and an assembly polypeptide or a second nucleic acid molecule containing a nucleotide sequence encoding the fusion protein.

[0105] In certain 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.

[0106] In certain embodiments, the kit further comprises a vector (eg, an expression vector).

[0107] In certain embodiments, the first nucleic acid molecule and the second nucleic acid molecule are contained on the same or different vectors.

[0108] In certain embodiments, the kit further comprises a buffer.

[0109] In certain embodiments, the buffer is selected from phosphate buffer, citrate buffer, carbonate buffer, acetate buffer, barbituric acid buffer, Tris buffer, or any combination thereof.

[0110] In certain embodiments, the buffer is PBS buffer.

[0111] In certain embodiments, the buffer further comprises a salt.

[0112] In certain embodiments, the salt is selected from NaCl, (NH4)SO4, NaSO4, NH4Cl, or any combination thereof.

[0113] In an eighth aspect, the present application provides a particulate antigen, which comprises an assembly polypeptide in the form of VLP, and the fusion protein as described in the first aspect attached to the assembly polypeptide.

[0114] In certain embodiments, the fusion protein is attached to the VLP via the interaction of a Nanobody with the assembly polypeptide.

[0115] In certain embodiments, the VLP is attached to at least one fusion protein as described in the first aspect.

[0116] In certain embodiments, the VLP is further attached to an additional polypeptide or fusion protein (eg, a T cell epitope).

[0117] In a ninth aspect, the present application provides a method for preparing the particulate antigen as described in the eighth aspect, the method comprising: using the kit as described in the seventh aspect.

[0118] In certain embodiments, the method comprises contacting the assembly polypeptide with the fusion protein under conditions that allow VLP assembly.

[0119] In certain embodiments, the condition allowing VLP to assemble is to place the assembling polypeptide in a solution. In certain embodiments, the solution is a buffer solution comprising a salt. In certain embodiments, the salt is selected from NaCl, (NH 4 ) SO 4 , NaSO 4 , NH 4 Cl, or any combination thereof.

[0120] In certain 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 the buffer;

[0121] Optionally, the particulate antigen in the buffer is recovered or purified.

[0122] In the tenth aspect, the present application provides a vaccine comprising the fusion protein as described in the first aspect, or the composition as described in the sixth aspect, or the granulated antigen as described in the eighth aspect, and an adjuvant.

[0123] In certain embodiments, the adjuvant is selected from aluminum salt adjuvants, zinc-aluminum mixed adjuvants (eg, FH002C), Freund's adjuvant, oil emulsion adjuvant, cytokine, TLR agonist, CpG adjuvant, liposome, AS01B adjuvant, or any combination thereof.

[0124] In the eleventh aspect, the present application provides a pharmaceutical composition comprising any one or more of (1) to (6):

[0125] (1) The fusion protein according to the first aspect;

[0126] (2) the nucleic acid molecule according to the second aspect;

[0127] (3) The carrier according to the third aspect;

[0128] (4) the host cell according to the fourth aspect;

[0129] (5) The composition according to the sixth aspect;

[0130] (6) The granulated antigen as described in the eighth aspect;

[0131] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0132] In certain embodiments, the pharmaceutically acceptable carrier and / or excipient is selected from a pH adjuster (e.g., phosphate buffer), a surfactant (e.g., a cationic, anionic, or nonionic surfactant, such as Tween-80), an adjuvant, an ionic strength enhancer (e.g., sodium chloride), a diluent, an excipient, a medium for containing or administering the therapeutic agent, and any combination thereof.

[0133] In the twelfth aspect, the present application provides the use of the fusion protein as described in the first aspect, or the nucleic acid molecule as described in the second aspect, or the vector as described in the third aspect, or the host cell as described in the fourth aspect, or the composition as described in the sixth aspect, or the kit as described in the seventh aspect, or the particulate antigen as described in the eighth aspect in the preparation of a pharmaceutical composition or vaccine, wherein the pharmaceutical composition or vaccine is used to induce an immune response in a subject.

[0134] In certain embodiments, the immune response is a response to an immunogenic polypeptide and / or an assembled 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.

[0135] In certain embodiments, the subject is a mammal, such as a human, monkey, or mouse.

[0136] In the thirteenth aspect, the present application provides the use of the fusion protein as described in the first aspect, or the nucleic acid molecule as described in the second aspect, or the vector as described in the third aspect, or the host cell as described in the fourth aspect, or the composition as described in the sixth aspect, or the kit as described in the seventh aspect, or the particulate antigen as described in the eighth aspect in the preparation of a pharmaceutical composition or vaccine, wherein the pharmaceutical composition or vaccine is used to prevent and / or treat diseases and / or symptoms in a subject that benefit or are prevented by an immune response to the immunogenic polypeptide.

[0137] In certain embodiments, the disease and / or condition is caused by a tumor cell from which the immunogenic polypeptide is derived.

[0138] In certain embodiments, the disease and / or condition is caused by the pathogen (eg, virus, bacteria, fungus, parasite) from which the immunogenic polypeptide is derived.

[0139] In certain embodiments, the disease and / or symptom is caused by a virus from which the immunogenic polypeptide is derived, e.g., chickenpox, novel coronavirus pneumonia, AIDS, genital warts, viral hepatitis (e.g., viral hepatitis B, viral hepatitis A, viral hepatitis C, viral hepatitis E), measles, mumps.

[0140] In certain embodiments, the immunogenic polypeptide can be the RBD protein of the novel coronavirus or a fragment thereof. In such embodiments, the disease can be novel coronavirus pneumonia.

[0141] In certain embodiments, the immunogenic polypeptide may be the gE protein of VZV or a fragment thereof. In such embodiments, the disease may be varicella.

[0142] In certain embodiments, the immunogenic polypeptide may be a protein of HPV or a fragment thereof. In such embodiments, the disease may be condyloma acuminata.

[0143] In certain embodiments, the immunogenic polypeptide can be a protein or fragment thereof of hepatitis B virus, hepatitis A virus, hepatitis C virus, hepatitis E virus. In such embodiments, the disease can be viral hepatitis (e.g., viral hepatitis B, viral hepatitis A, viral hepatitis C, viral hepatitis E).

[0144] In certain embodiments, the immunogenic polypeptide may be an envelope glycoprotein of the measles virus or a fragment thereof. In such embodiments, the disease may be measles.

[0145] In certain embodiments, the immunogenic polypeptide may be a protein of the mumps virus or a fragment thereof. In such embodiments, the disease may be mumps.

[0146] In certain embodiments, the subject is a mammal, such as a human, monkey, or mouse.

[0147] In the fourteenth aspect, the present application provides a method for inducing an immune response in a subject, comprising administering to the subject an effective amount of the fusion protein as described in the first aspect, or the nucleic acid molecule as described in the second aspect, or the vector as described in the third aspect, or the host cell as described in the fourth aspect, or the composition as described in the sixth aspect, or the kit as described in the seventh aspect, or the particulate antigen as described in the eighth aspect, or the vaccine as described in the tenth aspect, or the pharmaceutical composition as described in the eleventh aspect.

[0148] In certain embodiments, the immune response is a response to an immunogenic polypeptide and / or an assembled 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.

[0149] In certain embodiments, the subject is a mammal, such as a human, monkey, or mouse.

[0150] In the fifteenth aspect, the present application provides a method for preventing and / or treating a disease and / or symptom in a subject that benefits or is prevented by an immune response to an immunogenic polypeptide, comprising administering to the subject an effective amount of the fusion protein as described in the first aspect, or the nucleic acid molecule as described in the second aspect, or the vector as described in the third aspect, or the host cell as described in the fourth aspect, or the composition as described in the sixth aspect, or the kit as described in the seventh aspect, or the granulated antigen as described in the eighth aspect, or the vaccine as described in the tenth aspect, or the pharmaceutical composition as described in the eleventh aspect.

[0151] In certain embodiments, the disease and / or condition is caused by a tumor cell from which the immunogenic polypeptide is derived.

[0152] In certain embodiments, the disease and / or condition is caused by the pathogen (eg, virus, bacteria, fungus, parasite) from which the immunogenic polypeptide is derived.

[0153] In certain embodiments, the disease and / or symptom is caused by a virus from which the immunogenic polypeptide is derived, e.g., chickenpox, novel coronavirus pneumonia, AIDS, genital warts, viral hepatitis (e.g., viral hepatitis B, viral hepatitis A, viral hepatitis C, viral hepatitis E), measles, mumps.

[0154] In certain embodiments, the immunogenic polypeptide may be HIV Env protein or a fragment thereof (eg, gp160 protein, gp120 protein, gp41 protein). In such embodiments, the disease may be AIDS.

[0155] In certain embodiments, the immunogenic polypeptide can be the RBD protein of the novel coronavirus or a fragment thereof. In such embodiments, the disease can be novel coronavirus pneumonia.

[0156] In certain embodiments, the immunogenic polypeptide may be the gE protein of VSV or a fragment thereof. In such embodiments, the disease may be varicella.

[0157] In certain embodiments, the immunogenic polypeptide may be a protein of HPV or a fragment thereof. In such embodiments, the disease may be condyloma acuminata.

[0158] In certain embodiments, the immunogenic polypeptide can be a protein or fragment thereof of hepatitis B virus, hepatitis A virus, hepatitis C virus, hepatitis E virus. In such embodiments, the disease can be viral hepatitis (e.g., viral hepatitis B, viral hepatitis A, viral hepatitis C, viral hepatitis E).

[0159] In certain embodiments, the immunogenic polypeptide may be an envelope glycoprotein of the measles virus or a fragment thereof. In such embodiments, the disease may be measles.

[0160] In certain embodiments, the immunogenic polypeptide may be a protein of the mumps virus or a fragment thereof. In such embodiments, the disease may be mumps.

[0161] In certain embodiments, the subject is a mammal, such as a human, monkey, or mouse.

[0162] In a sixteenth aspect, the present application provides a system for preparing a particulate immunogenic polypeptide, comprising a first carrier and a second carrier, wherein the first carrier comprises a nucleotide sequence encoding a fusion protein, the fusion protein comprising an immunogenic polypeptide and an assembly polypeptide, and the second carrier comprises a nucleotide sequence encoding a nanobody; and the nanobody is capable of specifically binding to the assembly polypeptide, and the assembly polypeptide is capable of assembling into a VLP;

[0163] In certain embodiments, the nucleotide sequence is codon-optimized or non-optimized according to the codon preference of the host cell.

[0164] In certain embodiments, the assembling polypeptide is selected from a protein of hepatitis E virus (HEV) or a fragment thereof or a variant thereof. In certain embodiments, the assembling polypeptide is as defined in the first aspect.

[0165] In certain embodiments, the fusion protein is as defined in the first aspect.

[0166] In the seventeenth aspect, the present application provides a method for enhancing the immunogenicity of an immunogenic polypeptide, comprising preparing or obtaining a fusion protein containing the immunogenic polypeptide and a nanobody capable of specifically binding to an assembled polypeptide; and contacting the fusion protein with a VLP containing the assembled polypeptide, thereby obtaining a particulate antigen containing the immunogenic polypeptide attached to the VLP.

[0167] In certain embodiments, the method comprises: using the system of aspect 16. In certain embodiments, the method comprises: (1) expressing or producing a fusion protein via a first vector and expressing or producing an assembly polypeptide via a second vector; and (2) contacting the fusion protein and the assembly polypeptide under conditions that allow VLP assembly.

[0168] In certain embodiments, the Nanobody is as defined in the first aspect.

[0169] In certain embodiments, the fusion protein is as defined in the first aspect.

[0170] In certain embodiments, the assembly polypeptide is assembled into a VLP.In certain embodiments, the fusion protein is attached to the VLP via the interaction of a nanobody with the assembly polypeptide.

[0171] Definition of terms

[0172] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0173] As used herein, the term "immunogenic polypeptide" refers to a protein or polypeptide capable of eliciting an immune response. In certain embodiments, the immunogenic polypeptide is administered (directly or indirectly) to a subject to induce an immune response in the subject. It will be understood by those skilled in the art that the immunogenic polypeptide may be naturally occurring, or may be a protein or polypeptide that is naturally produced or artificially introduced with mutations or variations (including but not limited to, substitutions, deletions and / or additions) that do not affect its biological activity (in this article, the biological activity is the ability to induce an immune response). Therefore, in this article, the immunogenic polypeptide may be a polypeptide or variant thereof derived from a non-pathogen (e.g., a tumor cell) or a pathogen (e.g., a virus, bacteria, fungus, parasite or other pathogen).

[0174] As used herein, the term "immune response" refers to the response of cells of the immune system (e.g., B cells, T cells, or monocytes) to stimulation. 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, which can result in the production of specific antibodies.

[0175] In this article, the term "subviruses" refers to microorganisms that do not have a complete viral structure, mainly including viroids, pseudovirions and prions.

[0176] Herein, the terms "RBD protein of SARS-Cov-2" or "RBD of SARS-Cov-2" have the same meaning and are used interchangeably, and refer to the receptor binding domain (RBD) on the spike protein (S protein) of SARS-Cov-2. Its main function is to recognize host cell surface receptors and mediate fusion with host cells. It is understood by those skilled in the art that different SARS-Cov-2 virus strains may contain RBD proteins of different sequences, but these RBD proteins have the same or similar biological properties. Therefore, in the present invention, RBD proteins include not only the proteins shown in SEQ ID NO: 1-8, 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 (such as the GenBank database), for example, the amino acid sequences shown in GenBank accession numbers OP077006.1, OP077005.1, and OP077003.1.

[0177] Herein, the terms "VZV gE protein," "VZV gE," and "VZV glycoprotein gE" refer to a VZV envelope glycoprotein. These terms have the same meaning and are used interchangeably. Those skilled in the art will appreciate that different VZV strains may contain gE proteins with different sequences, but these gE proteins possess the same or similar biological properties. Therefore, in the present invention, gE protein includes not only the protein set forth in SEQ ID NO:30, but also gE proteins from various VZV strains. The amino acid sequences of these gE proteins can be obtained from public databases (e.g., GenBank).

[0178] 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." Those skilled in the art will appreciate 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 those shown in SEQ ID NOs: 34-35, but also Env proteins from various HIV-1 strains. The amino acid sequences of these Env proteins can be obtained from public databases (e.g., GenBank database).

[0179] The capsid protein encoded by the ORF2 gene in HEV (also referred to as ORF2 protein) and its fragments (e.g., p239 protein, p495 protein) have been shown to be capable of assembling into VLPs. The sequence of the ORF2 protein is well known in the art, see, for example, DDBJ database accession number: D11092. The sequence of the p239 protein corresponds to amino acids 368 to 606 of the ORF2 protein. In certain embodiments, the sequence of the p495 protein corresponds to amino acids 112 to 606 of the ORF2 protein.

[0180] Herein, the term "assembly polypeptide" refers to a protein or polypeptide that can be assembled into a virus-like particle (VLP). In certain embodiments, the assembly polypeptide is a coat protein of a natural virus or viroid. In such embodiments, the VLP assembled from the assembly polypeptide is similar in structure to the natural virus or viroid, the only difference being that it does not contain the genome of the natural virus or viroid. In certain embodiments, the assembly polypeptide is an artificially prepared and / or screened polypeptide. In such embodiments, the VLP assembled from the assembly polypeptide is similar or dissimilar in structure to the natural virus or viroid. The term "virus-like particle (VLP)" is a multimeric particle whose structure is similar or dissimilar to that of a natural virus or subvirus. It has been demonstrated that proteins (e.g., capsid proteins, surface proteins, envelope proteins) of some viruses (e.g., HBV, HEV, HPV) can spontaneously form VLPs after recombinant expression in an appropriate expression system.

[0181] Those skilled in the art can identify proteins or polypeptides capable of assembling into VLPs by published methods. For example, the protein to be tested can be placed in a buffer solution (e.g., PBS solution) at room temperature and then the presence of VLPs can be detected. Conventional techniques known in the art can be used to detect the presence of VLPs, such as electron microscopy, biophysical characterization, and the like. Specific detection methods can be found in, for example, 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 in HEV (also known as ORF2 protein) and its fragments (e.g., p239 protein, p495 protein) have been shown to have the ability to assemble into VLPs.

[0182] Those skilled in the art will understand that, in the present application, the assembled polypeptides include not only natural proteins or polypeptides that can be assembled into VLPs, but also proteins or polypeptides that are naturally produced or artificially introduced with mutations or variations (including but not limited to, substitutions, deletions and / or additions) on the basis of the natural proteins but do not affect their biological functions (in this article, the biological function is the ability to assemble into VLPs). Therefore, in certain embodiments, the assembled polypeptides are selected from the ORF2 protein of hepatitis E virus (HEV) or its fragments or variants thereof. In certain embodiments, the assembled polypeptides are selected from p239 protein or its fragments or variants thereof, p495 protein or its fragments or variants thereof.

[0183] According to the present invention, when used in the context of a protein / polypeptide, the term "variant" refers to a protein / polypeptide that has one or more (e.g., 1-10 or 1-5 or 1-3) amino acid differences (e.g., substitutions, deletions or additions) compared to the sequence of the protein / polypeptide from which it is derived, and which retains the biological activity of the protein / polypeptide from which it is derived.

[0184] As used herein, the phrase "an immunogenic polypeptide is a polypeptide derived from an organism or a non-organism, or an immunogenic variant thereof" means that the sequence of the polypeptide is derived from a sequence in an organism or a non-organism, but the method of obtaining the polypeptide is not limited to a specific production method. In certain embodiments, the polypeptide may be isolated from nature, artificially synthesized, or obtained through genetic engineering recombination.

[0185] As used herein, the term "particulated immunogenic polypeptide" refers to an aggregate of immunogenic polypeptides in the form of particles. In certain embodiments, the particulate immunogenic polypeptide refers to an immunogenic polypeptide attached to a virus-like particle.

[0186] As used herein, the term "organism" refers to a living individual or object. Except for a few species such as viruses, organisms are composed of cells. In certain embodiments, organisms include pathogens and non-pathogens.

[0187] As used herein, the term "pathogens" refers to microorganisms (eg, bacteria, viruses, rickettsiae, fungi), parasites, or other agents (eg, recombinant microorganisms) that can cause infection in humans, animals, or plants.

[0188] 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. The amino acid sequences of the two independent proteins or polypeptides can be linked directly or through a linker.

[0189] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. 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 substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the 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).

[0190] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids (e.g., naked plasmids); phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and viral vectors.

[0191] As used herein, the term "host cell" refers to a cell that can be used to amplify or express exogenous genes, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, 293T cells or human cells.

[0192] As used herein, the term "antibody" refers to an immunoglobulin molecule that is capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Antibodies include any type of antibody, such as IgG, IgA or IgM (or its subclass), and antibodies do not need to belong to any specific class. Depending on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulins can be assigned to different types. There are five main types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to different types of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. Antibody light chains can be classified as κ (kappa) 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 is composed of four domains (CH1, hinge region, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a single domain, CL. The constant domain does not directly participate in antibody-antigen binding but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0193] The VH and VL regions of an antibody can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL region consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site. The assignment of amino acids to regions or domains may follow the definitions of Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), Chothia (an immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position 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 AbM (the AbM CDR definition system is derived from the relevant studies of Martin ACR, Cheetham JC, Rees et al. AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272)).

[0194] 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. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In the present invention, the CDRs contained in the Nanobodies can be determined according to various numbering systems known in the art.

[0195] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0196] As used herein, the term "single-domain antibody (sdAb)" is also referred to as nanobody, and the two can be used interchangeably. It has the meaning commonly understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region, also referred to as VHH), which retains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibody. Nanobodies can be alpaca antibodies derived from camels or shark antibodies derived from sharks.

[0197] Nanobodies can be screened for specificity in the same way as for intact antibodies using conventional techniques known to the person skilled in the art.

[0198] As used herein, the expression "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the equilibrium constant for the dissociation of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0199] According to the present invention, the term "adjuvant" refers to an immunopotentiator that, when delivered to the body along with an antigen or in advance, can enhance the body's immune response to the antigen or alter the type of immune response. There are many types of adjuvants, including but not limited to aluminum salt adjuvants, zinc-aluminum mixed adjuvants (e.g., FH002C), Freund's adjuvant, oil-emulsion adjuvants, cytokines, TLR agonists, CpG adjuvants, liposomes, AS01B adjuvants, or combinations thereof.

[0200] As used herein, the term "pharmaceutically acceptable" means that it is generally recognized in the pharmaceutical field that it can be used in animals, particularly in humans. As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH regulators (including but not limited to phosphate buffers), surfactants (including but not limited to cationic, anionic or nonionic surfactants, such as Tween-80), adjuvants, ionic strength enhancers (including but not limited to sodium chloride), diluents, excipients, media for containing or administering therapeutic agents, and any combination thereof.

[0201] As used herein, the term "subject" refers to mammals, including but not limited to humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, etc. Preferably, the subject is a human.

[0202] 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 prophylactic effective amount is an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutic effective amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and any other concurrently administered treatments.

[0203] As used herein, the term "conditions allowing VLP assembly" refers to conditions under which the assembly polypeptide can be assembled into VLPs. Typically, a polypeptide or protein with assembly capability is placed in a liquid, which can self-assemble into VLPs. In certain embodiments, the condition allowing VLP assembly is to place the assembly polypeptide in a solution. In such embodiments, the solution will not contain components that are detrimental to VLP assembly. In certain embodiments, the solution contains components that are detrimental to VLP assembly, for example, the solution is a buffer solution containing salts.

[0204] Advantageous Effects of the Invention

[0205] The present invention utilizes the ability of assembled polypeptides (e.g., HEV ORF2 protein or fragments or variants thereof, HPV L1 protein or fragments or variants thereof, HBV surface protein or fragments or variants thereof) to assemble into VLPs, fuses nanobodies that specifically bind to the assembled polypeptides with immunogenic polypeptides for expression, and utilizes the targeted binding properties of nanobodies to the assembled polypeptides to display multiple immunogenic polypeptides on the surface of VLPs formed by the assembled polypeptides, thereby obtaining granulated immunogenic polypeptides. Thus, the present application provides a system and method for granulating immunogenic polypeptides.

[0206] Furthermore, compared to conventional immunogenic polypeptides, these granulated immunogenic polypeptides have higher immunogenicity and can stimulate high-level immune responses, including B and T cell responses. Therefore, the granulated immunogenic polypeptides of the present invention are particularly suitable for vaccine production and vaccination, and offer advantages in preventing and / or treating viral infections.

[0207] The system and method for granulating immunogenic polypeptides of the present invention use nanoantibodies. Compared with the existing technology, the use of nanoantibodies has at least the following advantages: ① The display of immunogenic polypeptides mediated by nanoantibodies is not affected by the N / C-terminal structure of the assembled polypeptide, and the construction strategy is more flexible. ② The nanoantibody-mediated display strategy will not affect the assembly and conformation of the assembled polypeptide, and has a wider applicability. ③ The fusion of nanoantibodies with immunogenic polypeptides is conducive to maintaining the conformation of immunogenic polypeptides. ④ Nanoantibodies with different epitopes can be combined and bound, which is conducive to increasing the proportion of immunogenic polypeptide display. ⑤ Nanoantibodies can be rationally designed based on the symmetry of the assembled polypeptide and the oligomeric state of the immunogenic polypeptide. ⑥ The nanoantibody-mediated display strategy can reduce the potential steric hindrance of immunogenic polypeptides. ⑦ Antibody-mediated display can directly target unmodified or modified VLPs. If the VLPs already on the market have good safety themselves, there is no need to consider their own drugability issues, and they have good convertibility. ⑧Nano antibodies used as linkers can minimize their immunogenicity through humanization strategies well known in the art, reduce the body's production of unnecessary immune responses, and achieve immune focusing of immunogenic polypeptides. ⑨Any immunogenic polypeptide can be granulated by the method of the present invention through fusion expression with nano antibodies, which has universality and versatility. ⑩Due to the small size of nano antibodies, the exposure of the original major immune epitopes of granulated carriers such as HEV ORF2, HPV L1 and HBV S can be retained by screening and identifying the binding sites of nano antibodies. The carrier of the immunogenic polypeptide still has good immunogenicity after granulation and can be used as a combination vaccine. This strategy can also be used in the development of other combination vaccines, thus having huge application prospects.

[0208] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0209] Figure 1 shows the screening process of the nanobodies of the present invention. Figure 1A shows the results of agarose gel electrophoresis of bacterial library colony PCR. Figure 1B shows the results of evaluating the diversity of the bacterial library after sequencing.

[0210] Figure 2 shows the SDS polyacrylamide gel electrophoresis results of various VHH-RBD proteins from the examples of the present invention. Figure 2A shows the electrophoresis results of various VHH-RBD fusion proteins produced and purified using an insect cell expression system. Figure 2B shows the electrophoresis results of various VHH-RBD fusion proteins produced and purified using a mammalian cell expression system. M: Molecular weight marker.

[0211] Figure 3 shows the results of SDS polyacrylamide gel electrophoresis of various VHH-gE fusion proteins according to the examples of the present invention. M: molecular weight marker, lanes 1-7 respectively show different VHH-gE fusion proteins.

[0212] Figure 4 shows the SDS-polyacrylamide gel electrophoresis results of various VHH-BGTSTIP proteins according to the present invention. "+" indicates reducing conditions, and "-" indicates non-reducing conditions.

[0213] Figure 5 shows the results of Western blot experiments of various VHH-RBD fusion proteins of the embodiments of the present invention. Figure 5A shows various VHH-RBD fusion proteins produced and purified by an insect cell expression system. M: molecular weight marker, lanes 1-8 respectively show fusion proteins of different nanobodies and RBD. Figure 5B shows various VHH-RBD fusion proteins produced and purified by a mammalian cell expression system 293F cells.

[0214] Figure 6 shows the results of Western Blot analysis of various candidate VHH-gE molecules according to the present invention. M: molecular weight marker, lanes 1-7 show fusion proteins of different nanobodies and gE, respectively.

[0215] Figure 7 shows the results of enzyme-linked immunosorbent assays (ELISAs) of various VHH-RBD proteins from the examples and various reported specific monoclonal antibodies. Figure 7A shows the activity assay results of various VHH-RBD proteins produced and purified using an insect cell expression system. The antibodies used include the broadly neutralizing antibodies JSR-105, JSR-551, JSR-209, m6D6, and m7D6, and the non-broadly neutralizing antibody 85F7. Figure 7B shows the activity assay results of various VHH-RBD proteins produced and purified using the mammalian cell expression system 293F cells. The antibodies used include 3G11, 8H12, 13F10, 8B8, 9D3, and 3F9.

[0216] Figure 8 shows the results of enzyme-linked immunosorbent assay (ELISA) of various VHH-gE and various specific monoclonal antibodies of the embodiment. The specific monoclonal antibodies used include 3H7, 4G4, 6B7, 11B11, 11B12, 13B6, 14G1, and 17B7.

[0217] FIG9 shows the results of enzyme-linked immunosorbent assay (ELISA) of various VHH-BGTSTIPs of the examples with various reported HIV-1 neutralizing antibodies or non-neutralizing antibodies.

[0218] Figure 10 shows the affinity analysis results of various VHH-RBDs and HEV-p239 proteins from the Examples. Figure 10A shows the affinity analysis results for VHH-RBDs produced and purified using an insect cell expression system, and Figure 10B shows the affinity analysis results for VHH-RBDs produced and purified using a mammalian cell expression system, 293F cells.

[0219] FIG11 shows the affinity analysis results of various VHH-gEs of the Examples to HEV-p239 protein.

[0220] FIG12 shows the affinity analysis results of various VHH-BGTSTIPs of the Examples and HEV-p239 protein.

[0221] Figure 13A shows the results of superdex 200 increase (HPLC molecular sieve) purification of P1-5B-RBD produced and purified by an insect cell expression system in an embodiment of the present invention. Figure 13B shows the results of superdex 200 increase purification of P2-6D-RBD, P2-3E-RBD, and P2-10G-RBD produced and purified by a mammalian cell expression system in an embodiment of the present invention.

[0222] Figure 14A shows the purification profile and SDS-PAGE identification results of the complex formed between the P1-5B-RBD fusion protein purified from an insect cell expression system and HEV-p239. Figure 14B shows the purification profile and SDS-PAGE identification results of the complex formed between the P2-3E-RBD, P2-10G-RBD, and P2-6D-RBD fusion proteins purified from a mammalian cell expression system and HEV-p239.

[0223] Figure 15 shows the purification profile and SDS-PAGE analysis of the complex of candidate fusion protein P2-8C-gE and HEV-p239 from an example of the present invention. Figure 15A shows the purification results of the complex. Figure 15B shows the SDS-PAGE analysis of the complex of P2-8C-gE and HEV-p239.

[0224] FIG16 shows the purification pattern and SDS-PAGE identification results of the complex of the candidate fusion protein P2-5C-BGTSTIP and HEV-p239 according to the embodiment of the present invention.

[0225] FIG17 shows the results of high performance size exclusion chromatography (HPSEC) of Example P2-8C-gE of the present invention.

[0226] Figure 18A shows the molecular size measurement results of HEV-RBD complexes and HEV-p239 particles formed by VHH-RBD and HEV-p239 protein produced and purified using an insect cell expression system in an embodiment of the present invention. Figure 18B shows the molecular size measurement results of three HEV-RBD complexes and HEV-p239 particles formed by VHH-RBD and HEV-p239 protein produced and purified using a mammalian cell expression system.

[0227] Figure 19 shows the molecular size detection results of HEV-gE complex samples according to an embodiment of the present invention. Figure 19A shows the molecular size of HEV-p239 particles, and Figure 19B shows the molecular size of HEV-gE complexes.

[0228] FIG. 20 shows the molecular size detection results of the HEV-BGTSTIP complex and HEV-p239 particles according to an embodiment of the present invention.

[0229] Figure 21 shows the analytical ultracentrifugation results of HEV-p239 and RBD complexes constructed based on HEV-p239 particles in accordance with the present invention. Figure 21A shows the analytical ultracentrifugation results of complex particles prepared from purified VHH-RBD produced in an insect cell expression system and HEV-p239 (right panel) and HEV-p239 (left panel). Figure 21B shows the analytical ultracentrifugation results of three complex particles prepared from purified VHH-RBD produced in a mammalian cell expression system, 293F cells, and HEV-p239.

[0230] Figure 22 shows the results of ultracentrifugation analysis of HEV-p239 and gE complexes constructed based on HEV-p239 particles according to an embodiment of the present invention. Figure 22A shows that HEV-p239 is a single component with a sedimentation coefficient of 22S. Figure 22B shows that the gE complex is a single component with a sedimentation coefficient of 31S.

[0231] FIG. 23 shows the analytical ultracentrifugation results of the HEV-p239 and Env(BGTSTIP) complex constructed based on HEV-p239 particles according to an embodiment of the present invention.

[0232] Figure 24A shows the negative staining results of a transmission electron microscopy (TEM) of a complex sample prepared from HEV-p239 and a VHH-RBD produced and purified using an insect cell expression system. Figure 24B shows the negative staining results of a complex sample prepared from HEV-p239 and a VHH-RBD produced and purified using a mammalian 293F cell expression system.

[0233] FIG25 shows the negative staining results of a transmission electron microscope of a complex sample prepared from HEV-p239 and VHH-gE fusion protein according to an embodiment of the present invention.

[0234] FIG26 shows the negative staining results of transmission electron microscopy of the HEV-p239 and Env (BGTSTIP) complex constructed based on HEV-p239 particles according to an embodiment of the present invention.

[0235] Figure 27 shows the immunogenicity test results of RBD particle antigens according to the present invention. Figure 27A shows the results of serum binding and neutralization activity tests after mice were immunized with RBD particle antigens obtained via an insect cell expression system. Figures 27B and 27C show the results of serum binding activity and neutralization activity against wild-type SARS-CoV-2 pseudovirus after mice were immunized with RBD particle antigens obtained via a mammalian cell expression system.

[0236] Figure 28 shows the neutralization test results of the antibodies induced by the RBD particle antigen combined with aluminum adjuvant in an embodiment of the present invention and the new coronavirus wild-type (WT) strain, Gamma strain, and BA.2 strain.

[0237] FIG29 shows the immunogenicity test results of the gE particle antigen according to the embodiment of the present invention.

[0238] FIG30 shows the live virus neutralization test results of mouse immune serum against gE particle antigen according to an embodiment of the present invention.

[0239] FIG31 shows the flow cytometry immunoassay results of monomeric gE antigen, gE particulate antigen and control adjuvant according to the examples of the present invention.

[0240] FIG32 shows the ELISpot detection of cytokines in the immunization group of monomeric gE antigen, gE particulate antigen and control adjuvant according to the embodiment of the present invention.

[0241] Figure 33 shows the results of humanization transformation of Nanobody P1-5B according to Example 1 of the present invention.

[0242] FIG34 shows the polyacrylamide electrophoresis results of the single-domain antibody fusion protein P1F8-BGTSTIP according to an embodiment of the present invention; wherein M is a molecular weight marker; “+” indicates reducing conditions, and “-” indicates non-reducing conditions.

[0243] Figure 35 shows the results of an enzyme-linked immunosorbent assay (ELISA) comparing the single-domain antibody fusion protein P1F8-BGTSTIP of an embodiment of the present invention with various reported broadly neutralizing and non-neutralizing antibodies to HIV-1. Among them, 2G12, VRC01, PGT121, PGT121, SF12, and B12 are broadly neutralizing antibodies; 17b, F105, and F240 are non-neutralizing antibodies.

[0244] FIG36 shows the affinity analysis results of the single-domain antibody fusion protein P1F8-BGTSTIP and HPV 58 VLP.

[0245] Figure 37 shows the high performance size exclusion chromatography (HPSEC) and SDS-PAGE identification results of the complex formed by P1F8-BGTSTIP and 58-VLP produced and purified by a mammalian expression system in an embodiment of the present invention. The red curve represents the complex formed by P1F8-BGTSTIP and 58VLP, the green curve represents the 58VLP spectrum, and the blue curve represents the fusion protein P1F8-BGTSTIP spectrum.

[0246] FIG38 shows the complex formed by P1F8-BGTSTIP and 58VLP and the analytical ultracentrifugation results of 58VLP in accordance with the present invention.

[0247] FIG39 shows the negative staining results of a transmission electron microscope of a complex sample formed by P1F8-BGTSTIP and 58VLP according to an embodiment of the present invention.

[0248] FIG40 shows the results of BGTSTIP-specific binding antibody titer detection after mice were immunized with the 58-VLP-BGTSTIP particulate antigen according to an embodiment of the present invention.

[0249] Figure 41 shows the SDS-PAGE identification of the particulate antigen formed after HBsAg binds to the nanobody fusion protein S2-gE in the present invention.

[0250] Figure 42 shows a molecular sieve identification diagram of the granulated antigen formed after HBsAg binds to the nanobody fusion protein S2-gE in the present invention.

[0251] Figure 43 shows the DLS identification diagram of the particulate antigen HBsAg-S2-gE (HBV-gE) formed after HBsAg binds to the nanobody fusion protein S2-gE in the present invention.

[0252] Figure 44 shows a negative-stained electron micrograph of the particulate antigen HBsAg-S2-gE formed after HBsAg binds to the nanobody fusion protein S2-gE in the present invention.

[0253] Figure 45 shows the specific antibody titer results after HBsAg binds to the nanobody fusion protein S2-gE to form the particulate antigen HBsAg-S2-gE and S2-gE immunization mice.

[0254] Sequence information

[0255] Information on the partial sequences involved in the present invention is provided in Table 1 below. DETAILED DESCRIPTION

[0256] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0257] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on 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., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0258] In addition, if specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. It is understood that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention. All publications and other references mentioned herein are incorporated herein by reference in their entirety.

[0259] Example 1: Nanobody phage library construction and screening process

[0260] 1. RNA Extraction (Trizol Method)

[0261] (1) Transfer peripheral blood lymphocytes (obtained from alpacas) preserved with Trizol to a 1.5 mL centrifuge tube and add 1 / 5 volume of chloroform to mix;

[0262] (2) After standing at room temperature for 5 minutes, centrifuge at 12,000 g for 15 minutes at 4°C;

[0263] (3) Carefully transfer the supernatant after centrifugation to a new centrifuge tube;

[0264] (4) Add an equal volume of isopropanol to a new centrifuge tube;

[0265] (5) After standing at room temperature for 10 minutes, centrifuge at 12,000 g for 10 minutes at 4°C;

[0266] (6) Wash the pellet in each tube with 1 mL of 75% ethanol, centrifuge at 7500 g for 5 minutes, remove the ethanol, dry the pellet, and dissolve it in an appropriate amount of RNase-free water. Combine all samples to obtain the extracted total RNA.

[0267] 2. Reverse transcription of cDNA (Takara reverse transcription kit)

[0268] 3. PCR Amplification

[0269] Table 2. PCR amplification program

[0270] 4. Enzyme Digestion and Ligation

[0271] Table 3. Enzyme digestion and ligation procedures

[0272] 5. Construction of bacterial and phage libraries

[0273] 6. Phage screening, purification and amplification

[0274] Where, V is the volume of phage added (unit: μL), T library is the phage titer;

[0275] T (pfu / ml) = N × D × 400

[0276] Wherein, 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.

[0277] 7. Library Quality Verification

[0278] Among them, Figure 1 shows the screening process of the nanoantibodies of the embodiment of the present invention. Figure 1A shows the results of colony PCR agarose gel electrophoresis. 50 of the 51 randomly selected monoclonal clones were positive clones, with a positive rate of 98%. The positive clone rate of the bacterial library meets the requirements. Figure 1B is the protein sequence translated into protein sequence using software after sequencing. Sequence diversity comparison shows that all 50 sequences are independent sequences with good diversity. The diversity of the bacterial library meets the requirements.

[0279] 8. Monoclonal ELISA detection

[0280] Calculate the number of bacteria and phages according to the following formula and add helper phage M13K07 to each well so that the number of bacteria: number of phages = 1:20:

[0281] Where V is the volume of helper phage added (unit: mL), T helper-phage is the helper phage titer used.

[0282] 9. Perform ELISA secondary verification on the positive clones selected above

[0283] The positive clones finally selected were sent to a biological company for sequencing and analysis, and a total of 20 heavy chain variable region (VHH) sequences of nanobodies were obtained. The amino acid sequences of these nanobodies are shown in SEQ ID NOs: 10-29 in Table 1, and the CDR1-3 sequences are shown in SEQ ID NOs: 57-62 and 90-144 in Table 1.

[0284] Example 2: Preparation and expression of VHH-RBD, VHH-gE and VHH-BGTSTIP fusion proteins

[0285] 1. Preparation of fusion protein

[0286] The VHH-RBD sequence was constructed in the insect cell expression system by connecting the RBD sequence (SEQ ID NO: 1-8) to the C-terminus of the VHH sequence (SEQ ID NO: 10-29), and then connecting the bee venom signal peptide (SEQ ID NO: 31) to the N-terminus of the above sequence to promote protein secretion expression. The nucleotide sequence encoding the above amino acid sequence was synthesized by Bio-Engineering, and the nucleotide sequence was constructed on the PIEX / bac-1 vector through the Nco I and BamH I restriction sites. Finally, 8 VHH-RBD proteins (P1-5B-RBD, P2-2C-RBD, P1-1B-RBD, P1-1G-RBD, P2-4E-RBD, P2-3E-RBD, P2-3D-RBD, P2-3A-RBD) were obtained through the insect cell expression system. The sequences are shown in SEQ ID NO: 42-49 in Table 1.

[0287] The same method was used to construct the sequence of the VHH-gE fusion protein, except that the C-terminal RBD sequence was replaced with the gE sequence (SEQ ID NO: 30). Ultimately, 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. The sequences are shown in SEQ ID NOs: 50-56 in Table 1, respectively.

[0288] The VHH-RBD sequence was constructed in a mammalian cell expression system by sequentially attaching a (G4S)3 flexible protein linker (SEQ ID NO:39), an RBD sequence (SEQ ID NO:1-8), and an 8-His tag to the C-terminus of the VHH sequence (SEQ ID NO:10-29). A signal peptide sequence (SEQ ID NO:38) was introduced at the N-terminus to promote protein secretion. The nucleotide sequence encoding the amino acid sequence was codon-optimized, synthesized by Universal Biotechnology, and cloned into the pcDNA3.1 vector between the ECORI and Xbal restriction sites. 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 through a mammalian cell expression system, and their sequences are shown in SEQ ID Nos: 72 to 89 in Table 1, respectively.

[0289] The same method was used to construct the sequence of the VHH-BGTSTIP (i.e., VHH-Env) fusion protein, except that the connected RBD sequence was replaced with the amino acid sequence of the full-length Env extracellular segment (SEQ ID NO: 34 or 35). Finally, 8 VHH-BGTSTIP proteins (P1-1B-BGTSTIP, P1-5B-BGTSTIP, P2-2C-BGTSTIP, P2-5C-BGTSTIP, P2-3A BGTSTSIP, P2-3E-BGTSTIP, PA-4G-BGTSTIP, P2-7D-BGTSTIP) were obtained through a mammalian cell expression system. The sequences are shown in SEQ ID Nos: 80 to 87 in Table 1.

[0290] 2. Expression of VHH-RBD and VHH-gE in insect expression systems

[0291] Transfection of insect cells

[0292] (1) Determine whether sf9 cells (purchased from Invitrogen, 11496-015) or sf21 cells (purchased from Invitrogen, 11497-013) are in the logarithmic growth phase (1.5-2.5×10 6 / mL), maintaining a viability above 90%. Add 200 μL of ESF 921 culture medium (purchased from Expression systems, 96-001-01) containing 2% FBS, 0.1 μg of Baculovirus DNA (purchased from Expression systems, 91-002), and 1 μg of pAc-S plasmid to a 24-well plate and mix thoroughly. Add 1 μL of transfection reagent (purchased from Expression systems, 95-055-075) to 50 μL of ESF921 culture medium (purchased from Expression systems, 96-001-01) and mix thoroughly. Combine the two in a single tube, mix thoroughly, and incubate at room temperature for 30 minutes. Wash the cells during this incubation period (do this shortly before the timer expires): Once the cells have fully adhered, remove the culture medium with a pipette and add 300 μL of ESF921 culture medium. Move quickly to avoid dehydrating the cells. After gently rocking, remove the culture medium and add another 300 μL of ESF921 culture medium. After the time is up, add approximately 100 μL of the above mixture evenly to each well of cells. Then, incubate at 27°C for 6 hours, discard the supernatant, and add 500 μL of complete culture medium (50% CCM3 + 50% TNM-FH (SIGMA-ALDRICH, T1032) + 10% FBS).

[0293] (2) Collect the cell supernatant obtained in step (1), centrifuge at 500g for 5 minutes, remove cell debris and debris, and store the supernatant at 4°C in the dark. This is the P1 virus seed solution.

[0294] (3) Amplification of baculovirus

[0295] Determine whether sf9 cells or sf21 cells are in the logarithmic growth phase (1.5-2.5×10 6 / mL), the survival rate was maintained above 90%. 8-10mL of the solution was spread on a 10cm plate at a density of 6×10 5 / mL cells. Let it stand for 15 minutes to allow the cells to adhere. Add about 600μL of P1 virus solution and drip evenly. Incubate at 27℃ for 3-4 days. Observe the cytopathic effect. Collect the cell supernatant, centrifuge at 1000rpm for 5 minutes, remove cell debris and debris, and filter with a 0.22um filter membrane. Store the supernatant at 4℃ in the dark. This is the P2 virus seed solution. The P2 virus titer is about 10 6 -10 7 P3 can be amplified by volume in a 250 mL shake flask according to this method.

[0296] Insect cell protein expression

[0297] 250 mL of ESF921 medium was added to a 1 L shake flask and the culture density was 2 × 10 6 / mL, H5 cells with a survival rate of more than 90% (purchased from Invitrogen, B855-02). Add the virus according to the corresponding MOI, seal the bottle mouth with sealing film, and culture it in a 27°C shaker at 120rpm. Take out the cells in the shaker every day, observe and count them, and record the relevant data. The appropriate MOI can ensure that more than 70% of the cells are pathological on the first day. On the second day, all the cells are pathological, and the survival rate is about 80%. On the third day, the cells rupture and the survival rate drops to 30-50%. At this time, consider collecting the cells. Use 10000rpm and centrifuge for 10 minutes to collect the cells, then separate the supernatant and purify them.

[0298] 3. Purification of VHH-RBD and VHH-gE in insect expression systems

[0299] Ni affinity chromatography purification was performed using the AKTA system;

[0300] Instrument system: AKTA Pure preparative liquid chromatograph;

[0301] Purification medium: Ni Sepharose 6 Fast Flow affinity medium; Buffer: divided into A pump and B pump buffer, generally A pump is 1× PBS buffer (160g / L NaCl, 8.1mmol / LNa2HPO4, 1.5mmol / L KH2PO4, 2.7mmol / L KCl, pH 7.4), B pump is 1× PBS + 250mmol / L imidazole buffer;

[0302] System flow rate: 5mL / min; detection wavelength: UV@280nm

[0303] Elution conditions: elution of contaminants with 50 mM imidazole buffer (250 mmol / L imidazole buffer diluted with 1× PBS buffer), followed by washing with 1× PBS, and then elution of the target protein (S trimer protein) with 250 mM imidazole buffer;

[0304] Collect the product eluted with 250 mM imidazole to obtain 10 mL of purified sample. Take 50 μL of each eluted product, add 10 μL of 6X Loading Buffer, mix thoroughly, incubate in an 80°C water bath for 10 minutes, then apply 10 μL of the sample to a 10% SDS-polyacrylamide gel for electrophoresis at 80 V for 120 minutes. Then, stain with Coomassie Brilliant Blue to visualize the electrophoretic bands.

[0305] 4. Transfection and expression of VHH-RBD and VHH-BGTSTIP in mammalian expression systems

[0306] Take the frozen 293F cells from -80℃ refrigerator, thaw at 37℃, centrifuge at 1300rpm for 4min, discard the supernatant in a clean bench, flick the cells and resuspend them in 293freestyle medium incubated at 37℃ in advance, transfer them to a triangular flask containing 50mL incubation medium, and culture them in suspension at 37℃, 5% CO2, 120 rpm, until the cell density reaches 2.0*10 6 Subculture the cells regularly and gradually expand the culture system. When sufficient cells are present, transiently transfect 293F cells with PEI (MW 25,000). Harvest the cells in a sterile 50 mL tube and centrifuge at 1300 rpm for 4 minutes. Gently flick the cells and resuspend them in 37°C incubation medium. Transfer the cells to a flask containing 450 mL of 37°C incubation medium and place them in a shaker at 37°C until ready to use.

[0307] The extracted VHH-RBD and VHH-BGTSTIP plasmids were added to PEI (MW 25,000) at a ratio of 1:2 in 50 ml of culture medium, mixed thoroughly, and allowed to stand for 18 minutes. The cells were then transferred to the aforementioned 450 ml of culture medium and cultured in suspension at 37°C, 5% CO2, and 120 rpm for 6 days to express the VHH-RBD and VHH-BGTSTIP proteins. During transfection, the PEI was kept away from light.

[0308] 5. Purification of VHH-RBD and VHH-BGTSTIP in mammalian expression systems

[0309] Six days after transient transfection, the cell culture medium was collected and centrifuged at 7000 g for 10 min in a JA-14 rotor. The cell supernatant was collected and then centrifuged at 20000 g for 10 min. The supernatant was filtered twice with a 0.22 μm pore size filter membrane. The sample was then used for the next step of Ni-excel column purification.

[0310] Ni affinity chromatography purification was performed using the AKTA system;

[0311] Instrument system: AKTA Pure preparative liquid chromatograph;

[0312] Purification medium: Ni Sepharose Excel affinity medium; Buffer: divided into A and B buffers, A buffer is 1× PBS buffer, B pump is 1× PBS + 250mmol / L imidazole buffer;

[0313] System loading flow rate: 8mL / min; detection wavelength: UV@280nm

[0314] System elution flow rate: 4ml / min; detection wavelength: UV@280nm

[0315] Elution conditions: Elute contaminants with 20 mM imidazole, then collect the 250 mM imidazole elution product. Dialyze the eluate against 1× PBS overnight, changing the dialysate twice. Harvest approximately 30 ml of low-concentration target protein, concentrate to 5 ml using 20 ml of Vivaspin and a 100 kDa ultrafiltration concentrator for later use. Prepare reduced and non-reduced samples from the collected elution sample and perform SDS-PAGE gel electrophoresis as described above.

[0316] 6. Experimental Results

[0317] Figure 2 shows the SDS polyacrylamide gel electrophoresis results of 8 VHH-RBD proteins prepared by the present invention. Figure 2A is the electrophoresis results of various VHH-RBD fusion proteins produced and purified by an insect cell expression system. M: molecular weight marker, lanes 1-8 respectively show different VHH-RBD fusion proteins. The results show that after one-step purification by affinity chromatography, the purity of the VHH-RBD protein is about 90%, and the molecular size is about 50KDa. Figure 2B is the electrophoresis results of 10 VHH-RBD fusion proteins produced and purified by a mammalian cell expression system. The results show that the purity of the VHH-RBD protein is about 90%, and the molecular weight is about 50KDa.

[0318] Figure 3 shows the results of SDS-polyacrylamide gel electrophoresis of seven VHH-gE fusion proteins prepared according to the present invention. M: Molecular weight marker. Lanes 1-7 represent different VHH-gE fusion proteins. The results show that after one-step purification by affinity chromatography, the VHH-gE proteins have a purity of approximately 80% and a molecular weight of approximately 85 kDa.

[0319] Figure 4 shows the results of SDS-polyacrylamide gel electrophoresis of eight VHH-BGTSTIP proteins prepared according to the present invention. "+" indicates reducing conditions, and "-" indicates non-reducing conditions. The results show that after affinity chromatography and purification, the VHH-BGTSTIP proteins have a purity of approximately 90% and a molecular weight of approximately 180 kDa.

[0320] Example 3: VHH-RBD and VHH-gE protein immunoblotting experiments

[0321] 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 on a BioRad MiniProtean Tetra system, and the gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad) for 30 minutes at room temperature. The separated proteins were transferred to nitrocellulose membranes using a trans-blot turbo transfer system (Bio-Rad). After blocking, the membranes were incubated with anti-His-HRP (1:5000 dilution) for 1 hour. Unbound antibodies were removed by washing five times for 5 minutes each, and then detected using a chemiluminescent substrate kit.

[0322] Figure 5 shows the results of immunoblotting experiments (Western Blot) of various VHH-RBD fusion proteins of the embodiments of the present invention. Figure 5A shows 8 kinds of VHH-RBD fusion proteins produced and purified by an insect cell expression system. M: molecular weight marker, and lanes 1-8 respectively show fusion proteins of different nano antibodies and RBD. The results confirmed that the 50KDa molecule was determined to be the target protein of the present invention. Figure 5B shows 10 kinds of VHH-RBD fusion proteins produced and purified by the mammalian cell expression system 293F cells. The results show that the purity of the VHH-RBD fusion protein is about 90%, and the molecular weight is about 50KDa. The results confirmed that the molecule of about 50KDa is determined to be the target protein of the present invention.

[0323] Figure 6 shows the results of a Western blot analysis of seven candidate VHH-gE molecules from the examples of the present invention. M: Molecular weight marker. Lanes 1-7 represent fusion proteins of different Nanobodies with gE. The results confirm that the 85 kDa molecule is the target protein of the present invention.

[0324] Example 4: Fusion protein molecule activity analysis (ELISA)

[0325] (1) Dilute the fusion protein to 1 μg / mL and coat a 96-well plate, 100 μL per well, and incubate at room temperature for 2 h.

[0326] (2) Wash the plate once and block with bovine serum albumin diluent (ED, 200 μL / well) at room temperature for 2 h;

[0327] (3) Wash the plate once, dilute the corresponding specific monoclonal antibody to 1 μg / ml, add 100 μl to the first well, perform 11 2-fold serial dilutions, repeat in duplicate wells, and incubate at room temperature for 1 hour.

[0328] (4) Wash the plate five times, add the secondary antibody GAH-HRP (1:5000) to the 96-well plate, 100 μL / well, and incubate at room temperature for 1 h;

[0329] (5) Wash the plate five times, develop the color at room temperature for 10 min, stop the reaction, and detect at a wavelength of 450 nm using a microplate reader. Data analysis was performed using GraphPad Prism 5 (GraphPad, USA) software.

[0330] FIG7 shows the results of enzyme-linked immunosorbent assay (ELISA) of various VHH-RBDs in the examples and various reported specific monoclonal antibodies. Figure 7A shows the activity identification results of 8 VHH-RBD proteins produced and purified by an insect cell expression system. The RBD antibodies used include the broadly neutralizing antibody 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) (the specific information and sequences of these antibodies are obtained from the literature Ju B, Zheng Q, Guo H, Fan Q, Li T, Song S, Sun H, Shen S, Zhou X, Xue W, Cui L, Zhou B, Li S, Xia N, Zhang Z. Immune escape by SARS-CoV-2 Omicron variant and structural basis of its effective neutralization by a broad neutralizing human antibody VacW-209. Cell Res. 2022). May;32(5):491-494.doi:10.1038 / s41422-022-00638-6.Epub 2022 Mar 8.PMID:35260792;PMCID:PMC8902274.).

[0331] The results demonstrated that the VHH-RBD purified by the insect cell expression system maintained an intact and correct molecular conformation and exhibited excellent binding activity with RBD-specific antibodies. Figure 7B shows the activity assay results of 10 VHH-RBD proteins produced and purified using the mammalian cell expression system 293F cells. The antibodies used included 3G11, 8H12, 13F10, 8B8, 9D3, and 3F9 (these antibodies were prepared using conventional antibody preparation methods in our laboratory's reference literature). Briefly, the extracellular domain of the SARS-CoV-2 surface spike protein (S) was expressed and purified in 293F cells. BALB / c mice were then immunized, and blood was collected for serum testing at 0, 2, 3, and 5 weeks after immunization. After immunization, two mice with high immune serum binding and neutralization titers were selected for spleen immunization. 10 μg of the mixed protein was injected into the spleen of the mice. The mice were then sutured and observed regularly. Three days later, a cell fusion assay was performed. After fusion culture, the cell supernatant was collected and analyzed for the presence of SARS-CoV-2 using an indirect ELISA. The S-2P trimer, RBD, S2 protein, and SARS-CoV-1 S-2P protein were reacted. Wells with strong binding to the corresponding proteins were selected for well picking and cloning experiments. After more than three rounds of cloning, monoclonal antibodies were obtained. Mice with ascites were immunized, ascites was extracted, and the corresponding monoclonal antibodies were purified using a protein A column. The results demonstrated that the purified VHH-RBD produced in a mammalian cell expression system retained an intact and correct molecular conformation and exhibited strong binding activity with RBD-specific antibodies. Furthermore, these fusion proteins exhibited superior binding activity to antibodies (e.g., 8H12, 13F10, and 8B8) compared to the RBD proteins.

[0332] Figure 8 shows the results of enzyme-linked immunosorbent assays (ELISAs) of the VHH-gE from Example 7 with various specific monoclonal antibodies. The gE-specific monoclonal antibodies used included 3H7, 4G4, 6B7, 11B11, 11B12, 13B6, 14G1, and 17B7 (detailed information and sequences of these antibodies were obtained from 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 demonstrate that the purified VHH-gE maintains an intact, correct molecular conformation and exhibits good binding activity with the gE-specific antibodies.

[0333] FIG9 shows the results of enzyme-linked immunosorbent assay (ELISA) of various VHH-BGTSTIPs of the examples with various reported HIV-1 neutralizing antibodies or non-neutralizing antibodies. These antibodies were recombinantly expressed in 293F cells. The antibody sequences were obtained from NCBI (VRC01 (GeneBank: MK032237.1 / GU980703.1), SF12 (GeneBank: MK722171.1 / MK722164.1), 2G12 (GeneBank: OM484328.1 / AF029237.1), PGT121 (GeneBank: JN201911.1 / JN201894.1) or the article reporting F105 (Wilkinson, RA, 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., WD Tolbert, C. Orlandi, et al. Molecular basis for epitope recognition by non-neutralizing anti-gp41antibody F240. Sci Rep, 2016.6: 36685.), 17b (Carlo D. Rizzuto, Richard Wyatt,Nivia Herna ndez-Ramos,et al.A Conserved HIV gp120Glycoprotein Structure Involved in Chemokine Receptor Binding. Science, 1998. 280(19): 1949-1953.). The results showed that the various VHH-BGTSTIP fusion proteins of the present invention had good binding activity with various neutralizing antibodies, but weak binding activity with non-neutralizing antibodies. This suggests that the VHH-BGTSTIP expressed in 293F cells effectively presents some neutralizing antibody epitopes on Env while not exposing some non-neutralizing antibody epitopes.

[0334] Example 5: Affinity Analysis of Fusion Protein and HEV-p239 (HPR)

[0335] The Biacore 8K was loaded with a CM5 chip, and the pipeline was flushed with PBS-P buffer (PBS+0.5% P20, cytiva).

[0336] The ligand HEV-p239 (10 ug / ml) was centrifuged at high speed for 10 min.

[0337] The HEV-p239 coupling protocol was as follows: chip channel activation (EDC:NHS = 1:1, flow rate 10 μl / min), followed by a wash (PBS-P buffer, flow rate 30 μl / min). HEV-p239 (amino acid sequence shown in SEQ ID NO:40) was loaded for coupling (time: 420 s, flow rate 10 μl / min). After determining the amount of ligand coupled, the channel was blocked with ethanolamine (time: 420 s, flow rate 10 μl / min).

[0338] Affinity assay: A concentration gradient of 125 nM, 62.5 nM, 31.2 nM, 15.6 nM, 7.8 nM, and 3.9 nM fusion protein was loaded for assay. The sample association time was 120 s, the dissociation time was 200 s, and the flow rate was 30 μl / min.

[0339] Biacore 8K (GE) software was used to perform kinetic analysis to fit the binding (Ka) and dissociation (Kd) curves and calculate the affinity (KD).

[0340] Figure 10 shows the affinity analysis results of various VHH-RBD fusion proteins from the Examples for HEV-p239. Figure 10A shows the affinity analysis of VHH-RBD produced and purified via an insect cell expression system for HEV-p239, demonstrating that various VHH-RBD proteins exhibit high nanomolar affinity for HEV-p239 particles. Figure 10B shows the affinity analysis of VHH-RBD produced and purified via a mammalian cell expression system (293F) for HEV-p239, demonstrating high nanomolar affinity for HEV-p239 particles.

[0341] Figure 11 shows the affinity analysis results of various fusion proteins VHH-gE and HEV-p239 protein in the examples. The results show that various VHH-gE proteins have high nanomolar affinity for HEV-p239 particles.

[0342] Figure 12 shows the affinity analysis results of various fusion proteins VHH-BGTSTIP and HEV-p239 protein in the examples. The results show that various VHH-BGTSTIP have high nanomolar affinity for HEV-p239 particles.

[0343] Example 6: Molecular sieve chromatography purification of fusion protein and its complex

[0344] Instrument system: AKTA explorer 100 preparative liquid chromatography system produced by GE Healthcare (formerly Amershan Pharmacia).

[0345] Chromatographic medium: Superdex 200 increase (cytiva).

[0346] Column volume: 20 cm × 20 mm.

[0347] Buffer: 20 mM phosphate buffer pH 7.4.

[0348] Flow rate: 0.7 mL / min.

[0349] Detector wavelength: 280nm.

[0350] The samples are the samples in Example 1 and Example 5.

[0351] The elution procedure is: segmented collection to collect the penetration peak.

[0352] Collect the product that penetrated through the Supedex 200 increase to obtain 5 mL of purified sample. Take 50 μL of each eluted product, add 10 μL of 6X Loading Buffer, mix thoroughly, incubate in an 80°C water bath for 10 minutes, then apply 10 μL of the sample to a 10% SDS-polyacrylamide gel for electrophoresis at 120 V for 60 minutes. Then, stain with Coomassie Brilliant Blue to visualize the electrophoretic bands.

[0353] Figure 13A shows the results of superdex 200 increase (high-performance liquid chromatography molecular sieve) purification of the fusion protein P1-5B-RBD produced and purified by an insect cell expression system in an embodiment of the present invention. The results show that the purity of the P1-5B-RBD protein reaches more than 95%. Figure 13B shows the results of superdex 200 increase purification of P2-6D-RBD, P2-3E-RBD, and P2-10G-RBD produced and purified by a mammalian cell expression system in an embodiment of the present invention. The results show that all three fusion proteins present a single elution peak, indicating high protein purity and homogeneity.

[0354] Figures 14-16 show the purification patterns and SDS-PAGE identification results of the complexes formed by various fusion proteins constructed by the present invention and HEV-p239.

[0355] Figure 14A shows the purification profile and SDS-PAGE identification results of the complex formed by the P1-5B-RBD fusion protein purified from the insect cell expression system and HEV-p239. The purple curve is significantly higher than the blue curve, demonstrating that the fusion protein P1-5B-RBD specifically binds to the surface of HEV particles to form a complex.

[0356] Figure 14B shows the purification profiles and SDS-PAGE analysis of complexes formed between P2-3E-RBD, P2-10G-RBD, and P2-6D-RBD, respectively, and HEV-p239, purified from a mammalian cell expression system using candidate fusion proteins from the present invention. The results demonstrate that P2-3E-RBD, P2-10G-RBD, and P2-6D-RBD form complexes with HEV-p239 and can be purified using Superdex 200 at a 10 / 300 increase.

[0357] Figure 15 shows the purification profile and SDS-PAGE identification results of the complex formed by the candidate fusion protein P2-8C-gE and HEV-p239 according to an example of the present invention. Figure 15A shows the purification results of the complex. The results show that the retention volume of the complex on a Superdex 200 Increase (Cytiva) column is 8 ml, representing the particle peak fraction. The 14 ml peak represents unbound P2-8C-gE, indicating that the 8 ml particle peak fraction is bound. Therefore, P2-8C-gE is saturatedly bound to the surface of HEV particles, forming a complex. SDS-PAGE results confirm that the complex lane exhibits a typical two-component band (HEV-p239 = 20 kDa, P2-8C-gE = 85 kDa). Therefore, the sample purified by Superdex 200 Increase is confirmed to be a gE complex sample.

[0358] Figure 16 shows the purification profile and SDS-PAGE analysis of the complex of the candidate fusion protein P2-5C-BGTSTIP and HEV-p239 from an example of the present invention. The results show that the retention volume of the complex in the Superose 6 column is approximately 9 ml, and the retention time of P2-5C-BGTSTIP is approximately 16 ml. SDS-PAGE analysis of the different fractions shows two bands in the 9 ml elution volume, confirming that the complex sample was obtained in approximately 9 ml after Superose 6 purification.

[0359] In summary, the above experimental results prove that the present application successfully purified and obtained complex particles formed by multiple candidate fusion proteins and HEV-p239.

[0360] Example 7: High Performance Size Exclusion Chromatography (HPSEC) Analysis of P2-8C-gE Protein

[0361] Instrument: Waters. System flow rate: G3000PW XL The flow rate is 0.5 mL / min. The wavelength is 190-600 nm, and the column wavelengths are 280 nm and 254 nm.

[0362] Buffer: PBS.

[0363] Procedure: Pre-equilibrate the column for 30-60 minutes until there is no significant change in the absorbance at 280 nm. Return the detector to zero. Create a chromatography run method, centrifuge the sample, inject the sample to be analyzed into a 100 μL sample loop, set the autosampler, and run for 30 minutes. Observe that the retention time of the S trimer is approximately 14 minutes.

[0364] The results are shown in FIG17 . The P2-8C-gE fusion protein of the present invention showed a single main peak without aggregate components, a retention time of about 14 minutes, and a purity of about 80%.

[0365] Example 8: Molecular size detection of RBD complex and gE complex (DLS)

[0366] Instrument: NanoBrook Series (Brookhaven instrument).

[0367] Functional module: DLS (Dynamic Light Scattering).

[0368] Buffer: PBS.

[0369] Procedure: Preheat the instrument for 5 minutes. Prepare the sample to be tested (concentration 0.5 mg / ml, centrifuge at 12,000 rpm for 5 minutes, and add 50 μl to the sample cup). Set the test parameters: Test time: 300 seconds per test, repeat three times for each sample.

[0370] Figure 18 shows the molecular size measurement results of a HEV-RBD complex sample formed by VHH-RBD and HEV-p239 protein, produced and purified using an insect cell expression system, according to an embodiment of the present invention. Figure 18B shows the molecular size measurement results of a HEV-RBD complex sample formed by VHH-RBD and HEV-p239 protein, produced and purified using a mammalian cell expression system. The results in Figures 18A and 18B show that the molecular size of HEV-RBD is significantly larger than that of HEV-p239 particles. The significantly larger size of the HEV-RBD complex than that of HEV-p239 confirms that VHH-RBD can bind to the surface of HEV particles and maintain a granular state.

[0371] Figure 19 shows the molecular size measurement results of HEV-gE complex samples from examples of the present invention. The results show that the molecular size of HEV-p239 particles is 14.2 nm, and the molecular size of HEV-gE is 38 nm. These results confirm that VHH-gE can bind to the surface of HEV particles and maintain their particle state.

[0372] Figure 20 shows the molecular size measurement results of the HEV-BGTSTIP complex sample from an example of the present invention. The results show that the molecular size of the HEV-p239 particle is 15.9 nm, and the HEV-BGTSTIP complex is 25.2 nm. These results demonstrate that VHH-BGTSTIP can bind to the surface of HEV particles and maintain their particle state.

[0373] Example 9: Calculation of sample sedimentation coefficient by analytical ultracentrifugation method

[0374] The instrument used was a Beckman XL-A analytical ultracentrifuge equipped with an optical detection system and an An-60Ti rotor.

[0375] Install the sample pool according to the operating instructions, add 400 μL of sample buffer (the same buffer as the sample) to the control pool, add 380 μL of sample (OD280 is about 0.8) to the sample pool, and balance the sample pool so that the weight difference is within 0.1 g.

[0376] The sample pool was placed in the An-60Ti rotor, and the rotor was placed in the cavity of the Beckman XL-A analytical ultracentrifuge, and an optical path detector was installed.

[0377] Parameter settings: 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 sec), and number of data (150 scans). Set the centrifugal speed to 30,000 rpm.

[0378] After the experiment, SENDTERP software was used to calculate the density and viscosity of the buffer, as well as the partial microvolume of the known proteins. Sedimentation coefficients were analyzed using the Origin versions of Nonlin and SEDFIT. The friction ratio f / f0 was set to 1.2 for globular proteins. The analysis range was set based on the molecular weight and basic properties of the sample protein, and the calculation resolution was set to 100. The RMSD value was generally required to be no greater than 0.01, and the residue map fluctuation was within 0.05.

[0379] Figure 21 shows the analytical ultracentrifugation results of HEV-p239 and the HEV-RBD complex constructed based on HEV-p239 particles in an embodiment of the present invention. Figure 21A shows the analytical ultracentrifugation results of the complex particles prepared by the purified VHH-RBD produced by the insect cell expression system and HEV-p239 (right figure) and HEV-p239 (left figure). The results show that: HEV-p239 presents a single component with a sedimentation coefficient of 22S, and the HEV-RBD complex presents a single component with a sedimentation coefficient of 27S, which is significantly higher than the sedimentation coefficient of HEV-p239 particles (22S). The results confirm that the RBD complex can maintain a stable particle morphology in an aqueous solution state, and the RBD is firmly bound to the surface of the HEV particles to form RBD particle protein. Figure 21B shows the analytical ultracentrifugation results of complex particles prepared from purified VHH-RBD produced in the mammalian 293F cell expression system and HEV-p239. The results demonstrate that the HEV-RBD complex is a single component with a significantly increased sedimentation coefficient compared to the sedimentation coefficient (22S) of HEV-p239 particles. This demonstrates that the complex maintains a stable particle morphology in aqueous solution, with the RBD firmly bound to the surface of HEV particles, forming RBD particle proteins.

[0380] Figure 22 shows the analytical ultracentrifugation results for HEV-p239 (Figure 22A) and the HEV-gE complex constructed based on HEV-p239 particles (Figure 22B), according to an embodiment of the present invention. The results show that HEV-p239 exhibits a single component with a sedimentation coefficient of 22S, while the HEV-gE complex exhibits a single component with a sedimentation coefficient of 31S, significantly higher than the sedimentation coefficient of HEV-p239 particles (22S). The results confirm that the HEV-gE complex maintains a stable granular form in aqueous solution, with gE firmly bound to the surface of HEV particles, forming gE granule protein.

[0381] FIG. 23 shows the analytical ultracentrifugation results of the HEV-p239 and Env(BGTSTIP) complex constructed based on HEV-p239 particles according to an embodiment of the present invention.

[0382] Example 10: Transmission electron microscopy particle morphology observation

[0383] The transmission electron microscope used was a Tecnai G2 Spirit 120 kV (FEI). Negative staining was performed using phosphotungstic acid.

[0384] Sample preparation: First, a copper grid (R2 / 2, 200 mesh, ThermoFisher Scientific) was treated with glow discharge hydrophilization. Then, 5 μL of a 0.5 mg / mL sample was dripped onto the grid. After standing at room temperature for 60 seconds, the droplet was removed from the edge of the grid with absorbent paper. The grid was allowed to air dry at room temperature before being inspected.

[0385] Figure 24A shows transmission electron microscopy (TEM) negative staining of a complex sample prepared from HEV-p239 and purified VHH-RBD produced using an insect cell expression system. Figure 24B shows transmission electron microscopy negative staining of a complex sample prepared from HEV-p239 and purified VHH-RBD produced using a mammalian 293F cell expression system. Both results show that the HEV-RBD complex exhibits a typical virus-like particle morphology. These results confirm that the HEV-RBD complex sample constructed in this application is a granulated RBD antigen.

[0386] Figure 25 shows negative-staining transmission electron microscopy results of the HEV-gE complex from an example of the present invention. The results show that the HEV-gE complex exhibits typical virus-like particle morphology. The results confirm that the HEV-gE complex sample constructed in this application is a particulate antigen.

[0387] Figure 26 shows the negative-staining transmission electron microscopy results of the HEV-Env (BGTSTIP) complex of the present invention. The results show that the HEV-Env complex exhibits a typical virus-like particle morphology, confirming that the HEV-Env complex sample constructed in this application is a particulate antigen.

[0388] Example 11: Evaluation of immunogenicity of HEV-RBD particles

[0389] This experimental protocol was approved by the Experimental Animal Care Ethics Committee of Xiamen University. All operations were performed in strict accordance with animal ethics guidelines and approved protocols.

[0390] Evaluation of the immunogenicity of RBD particles expressed in insect cells

[0391] Six-week-old Balb / C mice were divided into four groups of five. Each group was immunized with HEV-RBD particles (immunization dose of 0.5 μg), HEV-RBD particles (immunization dose of 5 μg), P1-5B-RBD monomers (immunization dose of 0.5 μg), or P1-5B-RBD monomers (immunization dose of 5 μg) in combination with aluminum adjuvant. The mice were injected intramuscularly (50 μL) into the left or right hind limb at 0, 2, and 6 weeks. Ocular venous blood was collected at 0, 1, 2, 3, 4, 5, 6, 7, and 8 weeks, with blood drawn before injection at 0, 1, and 4 weeks. Blood samples were centrifuged at 13,000 g for 10 minutes, and serum samples were stored at -20°C. End-point enzyme-linked immunosorbent assay and wild-type SARS-CoV-2 pseudovirus (constructed according to 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.) Antigen-specific IgG and neutralizing antibody titers were determined by neutralization method.

[0392] Evaluation of the immunogenicity of RBD particles in mammalian expression systems

[0393] Female mice, 6 weeks old, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Twelve groups of 5 mice were immunized. P2-3E-RBD, P2-10G-RBD, P2-6D-RBD, and P239 were used to prepare particle complex antigens. These were diluted with saline to the desired concentration, mixed with aluminum adjuvant at a 1:1 volume ratio, and allowed to adsorb to the adjuvant overnight at 4°C. Mice were immunized intramuscularly (100 μL, 50 μL each in the left and right hind legs) at 0, 2, and 5 weeks. The mouse immunization protocol is shown in Table 4.

[0394] Table 4: Mouse immunization schedule

[0395] Eye blood was collected weekly from mice, and mice were euthanized by carbon dioxide after nine weeks. Blood samples were incubated at 37°C for 30 minutes, centrifuged at 13,300 rpm for 10 minutes, and serum was collected and stored at -20°C for determination of wild-type SARS-CoV-2 pseudovirus neutralization and binding antibody titers.

[0396] The results are shown in Figures 27 and 28. Figure 27 shows the immunogenicity test results of the HEV-RBD particle antigen of the present invention. Figure 27A shows the serum binding and neutralization activity test results after mice were immunized with HEV-RBD particle antigen obtained through an insect cell expression system. Figures 27B and 27C show the serum binding activity and neutralization activity test results against wild-type SARS-CoV-2 pseudovirus after mice were immunized with HEV-RBD particle antigen obtained through a mammalian cell expression system. Figure 27D shows the serum antibody binding titer after hamsters were immunized with HEV-RBD particle antigen obtained through a mammalian cell expression system.

[0397] Figure 28 shows the neutralization test results of the antibodies induced by the HEV-RBD particle antigen combined with aluminum adjuvant in an embodiment of the present invention and the new coronavirus wild-type (WT) strain, Gamma strain, and BA.2 strain.

[0398] The results showed that displaying VHH-RBD expressed in insect cells or mammalian cells on the surface of p239 particles could significantly enhance the immunogenicity of RBD antigen.

[0399] Specifically, the group immunized with 5 μg of HEV-RBD particles (shown as RBD CPX in the figure) quickly established an immune barrier after the first dose, producing a binding antibody titer of approximately 3 log. After booster immunization, the binding antibody titer reached 5 log, which is 500 times higher than the binding antibody titer produced by the P1-5B-RBD monomer, and did not decrease significantly in the eighth week. In mouse immunization experiments, the binding antibody titer induced by HEV-RBD was significantly higher than that induced by monomeric RBD protein. At an immunization dose of 0.5 μg / mouse, P2-3E-RBD-p239, P2-10G-RBD-p239, and P2-6D-RBD-p239 complex particles induced relatively higher binding antibody titers (compared to the corresponding VHH-RBD proteins). The binding antibody titers of the P2-3E-RBD-p239 and P2-10G-RBD-p239 complex immunization groups were significantly higher than those of the corresponding VHH-RBD immunization groups before the fifth injection (Figure 27B). Pseudovirus neutralization results at week 6 showed that all three VHH-RBD-p239 complex particles induced a more significant neutralizing antibody response (Figure 27C).

[0400] In addition, the neutralizing antibodies of HEV-RBD particles have broad-spectrum neutralizing ability and can effectively neutralize pseudoviruses (VSV-Spike) including wild-type coronavirus strains, Gamma strains, and BA.2 strains.

[0401] Example 12: Evaluation of immunogenicity of gE particles

[0402] This experimental protocol was approved by the Experimental Animal Care Ethics Committee of Xiamen University. All operations were performed in strict accordance with animal ethics guidelines and approved protocols.

[0403] Six-week-old Balb / C mice were divided into eight groups of five mice each. 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), respectively, in combination with aluminum adjuvant. The mice were injected intramuscularly (50 μL) into the left or right hind limb at 0 and 2 weeks. Ocular venous blood was collected at 0, 1, 2, 3, 4, 5, and 6 weeks. Blood samples were centrifuged at 13,000 g for 10 minutes, and serum samples were stored at −20°C. Antigen-specific IgG and neutralizing antibody titers were determined by endpoint enzyme-linked immunosorbent assay and attenuated virus (v-Oka strain) neutralization assay, respectively.

[0404] Neutralization operation process (ELISPOT method):

[0405] 1. Guinea pig serum (purchased from Beijing Borsi Technology Co., Ltd., catalog number: BM361Y) and v-Oka virus (ATCC, catalog number VR-795) powder were reconstituted with virus protection solution, and complement was filtered through a 0.22 μm filter for later use;

[0406] 2. Dilute the serum 50-fold with virus protection solution and add it to the first well of a 24-well plate. Make 4 2-fold serial dilutions and incubate with vOka virus at 37°C for 1 hour.

[0407] 3. Transfer the serum-virus mixture to a 24-well plate pre-plated with ARPE-19 cells and incubate at 37°C for 1 hour. After 1 hour, discard the liquid and add F12 medium. Incubate at 37°C for 3 days.

[0408] 4. After 3 days, discard the culture medium and wash once with PBS; fix with fixative at room temperature for 5 minutes, discard the fixative, and permeabilize with permeabilization solution at room temperature for 10 minutes;

[0409] 5. Add the primary antibody 1B11-HRP (1:2000) to a 24-well plate and incubate at 37°C for 1 hour.

[0410] 6. Wash the plate five times, develop the color at room temperature for 5 min, read and count the spots using an enzyme-linked dot-spot image analysis system, and analyze the data using GraphPad Prism 5 (GraphPad, USA) software.

[0411] Figure 29 shows the immunogenicity test results of the gE particle antigen of the present invention. The gE particle antigen combined with an adjuvant similar to AS01B can induce high titer binding antibodies (shown as XUA in the figure, up to about 6 log).

[0412] Figure 30 shows the results of a live virus neutralization assay using mouse immune sera against HEV-gE particle antigens from an example of the present invention. The neutralizing antibodies induced by HEV-gE particle antigens were 2.8 times higher than those induced by P2-8c-gE monomers. This result confirms that surface expression of p239 particles significantly enhances the immunogenicity of gE antigens.

[0413] Example 13: Flow cytometry detection of cytokines in gE particle antigen immunization group

[0414] Mice were immunized according to the method described in Example 6 (same adjuvant), and the subsequent experimental procedures were as follows:

[0415] a) Spleen removal: Mice were sacrificed by cervical dislocation and soaked in 75% ethanol for 3-5 minutes. The mice were then placed in right lateral recumbency, and the spleen was removed aseptically (fat removed as much as possible).

[0416] b) Grinding: Place a 6-well plate on a mesh and add 1640 medium containing 10% FBS. Place the spleen on the mesh (completely immersed in the medium). Grind with a 2 mL syringe until no red tissue is visible. Transfer the ground cells to a 50 mL tube and place on ice.

[0417] c) Centrifuge at 400g for 5 min at 4°C, discard the supernatant, and tap the bottom of the tube with hemostats to loosen the cell pellet.

[0418] d) Add 10 mL of pre-chilled RBC solution to the cell pellet, resuspend, and place on ice for 5 minutes (invert and mix thoroughly).

[0419] e) Centrifuge at 400 g for 5 min at 4°C, discard the supernatant, and tap the bottom of the tube with hemostats to loosen the cell pellet.

[0420] f) Resuspend the cell pellet in 10 mL of pre-chilled culture medium, wash away any red blood cell pellet or fat, and then aspirate to homogenize the cell pellet and take 50 μL for counting.

[0421] g) Centrifuge at 400g for 5 min at 4°C, discard the supernatant, tap the bottom of the tube with hemostats to loosen the cell pellet, add a certain amount of culture medium, and dilute the cell count to 2 × 10 7 / mL;

[0422] h) Plating: 96-well U-bottom plate, 200 μL (2-4×10 6 cells), centrifuged at 400 g for 5 min at 4 °C, and the supernatant was discarded;

[0423] i) Add 100 μL of culture medium containing peptides ((gE / gI) overlapping peptides, diluted with FACS solution, final peptide concentration of 2 μg / mL) to resuspend the cells and stimulate for 18 hours;

[0424] j) Add 20 μL of Golgi inhibitor (1:1000, diluted in culture medium) and incubate for 6 h;

[0425] k) Centrifuge at 400g for 2 min at 4°C, remove the supernatant, add 200 μL FACS solution (1× PBS + 10% FBS) to resuspend the cells, centrifuge again, and remove the supernatant;

[0426] (All the following operations were carried out in the dark and kept at 4°C)

[0427] l) Cell surface staining: FITC-conjugated anti-mouse CD4 antibody (purchased from Biolegend, catalog number: 100510), PE-Cy7-conjugated anti-mouse CD8α antibody (purchased from Biolegend, catalog number: 100722), and LIVE / DEAD TMFixable Aqua Dead Cell Stain Reagent (purchased from Invitrogen, Cat. No. L34966) was used for staining. 40 μL of surface (AQUA / CD4 / CD8 antibody, diluted in FACS solution) was added to each well, and the cells were resuspended in the dark at 4°C for 30-60 min. 200 μL of FACS solution was added and pipetted 7-8 times.

[0428] m) Fixation / Permeabilization: Centrifuge at 2000 rpm for 2 min at 4°C, remove the supernatant, add 75 μL of Fixation / Permeabilization solution to each well to resuspend the cells, and incubate at 4°C in the dark for 60 min.

[0429] n) Centrifuge at 2000 rpm, 4°C for 2 min, and discard the supernatant;

[0430] o) Add 200 μL of 1× BD Perm / Wash solution to each well to resuspend the cells. Centrifuge at 2000 rpm at 4°C for 2 min and discard the supernatant.

[0431] p) Intracellular staining: PE-conjugated anti-mouse IL-2 antibody (BD, Catalog No. 554428) and APC-conjugated anti-mouse IFN-γ antibody (BD, Catalog No. 554413) were used for staining. 50 μL of fluorescent antibody (IL-2 / IFN-γ antibody, diluted in 1× BD Perm / Wash solution) was added to each well, and the cells were resuspended. The cells were incubated at 4°C in the dark for 60 min. 200 μL of Perm Buffer was added and the cells were resuspended.

[0432] q) Centrifuge at 2000 rpm, 4°C for 2 min, remove the supernatant, and add 200 μL of 1× BD Perm / Wash solution to each well to resuspend the cells;

[0433] r) Filter the treated cells through a 200-mesh sieve (place the sieve strips on the surface of the well and slowly add the cell suspension vertically using a pipette), and transfer them to a flow cytometer tube;

[0434] s) Samples were measured using a BD LSRFortessa X-20 flow cytometer, and the data were analyzed by FlowJo V10.

[0435] The experimental results are shown in Figure 31. The results show that gE particle antigen activated 1.65 times more IFN-gamma-positive CD4 cells and 8.42 times more IFN-gamma-positive CD8 cells than P2-8C-gE alone. These results confirm that gE particle antigen has a greater advantage in cellular immune responses.

[0436] Example 14: ELISpot detection of cytokines using gE particle antigen immunization group

[0437] At week 8, spleens were harvested for flow cytometry and Elispot assays of T cell responses. The flow cytometry assay was as described in Example 6, and the ELISPOT assay protocol for cytokines (using kits purchased from MABTECH, catalog numbers: 3321-4HPW-10, 3441-4HPW-10) was as follows:

[0438] (1) Isolate splenocytes, then plate 500,000 cells per well, centrifuge and remove the supernatant, add 100 μL of culture medium containing gE polypeptide (0.15 μg / 100 μL) to resuspend the cells, and stimulate at 37°C for 20 h;

[0439] (2) Discard the culture medium, wash with sterile PBS five times, dilute the detection antibodies R4-6A2-biotin (IFN-γ) and 5H4-biotin (IL-2) to 1 μg / mL, add 100 μL to each well, and incubate at room temperature for 2 h;

[0440] (3) Wash with sterile PBS five times, add 100 μL of Streptavidin-ALP (1:1000) to each well, and incubate at room temperature for 1 h;

[0441] (4) Wash with sterile PBS five times, add 100 μL of substrate solution (BCIP / NBT-plus) to each well for color development, and continue washing until spots appear;

[0442] (5) The plate was dried and the spots were read and counted using an enzyme-linked dot-spot image analysis system; data were analyzed using GraphPad Prism 5 (GraphPad, USA) software.

[0443] The results are shown in FIG32 , which confirmed that the T cell immune response activated by gE particulate antigen was significantly higher than that of P2-8C-gE monomer protein.

[0444] Example 15: Humanized transformation of P1-5B nanobody

[0445] 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 CDRs 1-3 are shown in SEQ ID NOs:57-59) was aligned using online software (IMGT / DomainGapAlign; http: / / www.bioinf.org.uk / abs / abnum / ). Homologous sequence alignment was performed using online software (https: / / www.imgt.org / IMGT_vquest / input), and the humanized sequence with the highest score was selected for CDR transplantation. The selected sequence was synthesized by Sangon and constructed into the pcDNA3.1 vector. Plasmids were prepared in large quantities using an endotoxin-free plasmid extraction kit (Tiangen). 293FT cells were then transiently transfected with PEI for expression of the humanized nanobody. 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 sequences of CDRs 1-3 are shown in SEQ ID NOs:65-67.

[0446] PEI transiently transfected HEK239FT cells

[0447] PCDNA3.1-H1-RBD and PCDNA3.1-H2-RBD plasmids (the plasmids contain nucleotide sequences encoding fusion proteins H1-RBD and H1-RBD, respectively, wherein the amino acid sequences of H1-RBD and H1-RBD are shown in SEQ ID NO: 63 and 64) were diluted with PEI in 5 ml of serum-free CD05 medium, and the two were thoroughly mixed and allowed to stand for 18 minutes to form a plasmid PEI complex. HEK293FT cells were transfected at a ratio of PEI: plasmid = 2: 1. Fresh CD05 medium was replaced 6 hours after transfection, and 100 ml of CD05 feed medium was added after constant temperature culture at 37 ° C, 5% CO2 for 48 hours. After 96 hours, the supernatant was harvested for purification, and the affinity of the humanized nanoantibody was detected by enzyme-linked immunosorbent assay (ELISA, see Example 4).

[0448] The results are shown in Figure 33: The modified humanized Nanobodies have the same molecular activity as the parent. Among them, H1-RBD and H2-RBD are RBD fusion proteins formed by the modified humanized Nanobodies, and RBD-WT is the wild-type RBD protein.

[0449] Example 16: Evaluation of the immunogenicity of HEV-Env particle antigens prepared by humanized nanobodies

[0450] Mice: Female, 6 weeks old, purchased from Shanghai Slake Laboratory Animal Co., Ltd. Six groups of immunized mice were set up, with 5 mice in each group. This example uses P2-5C nanobody (the amino acid sequence of P2-5C nanobody is shown in SEQ ID NO: 21, and the sequence of CDR1-3 is shown in SEQ ID NO: 60-62). The prepared complex particles and P2-5C-BGTSTIP antigen were diluted with physiological saline, mixed with aluminum adjuvant in a volume ratio of 1: 1, and the protein was adsorbed on the adjuvant, and the mice were immunized intramuscularly. The mouse immunization scheme is set as shown in Table 5.

[0451] Table 5: Mouse immunization schedule

[0452] Immunizations were performed according to the aforementioned protocol. Eye blood was collected before each immunization and after the sixth immunization. The mice were then dislocated by cervical dislocation. Blood samples were incubated at 37°C for 30 minutes and centrifuged at 13,300 rpm for 10 minutes. Serum was collected for HIV-1 pseudovirus neutralization and antibody titer determination.

[0453] We collected immune sera from injections 0-6 and conducted virus neutralization experiments.

[0454] Example 17: Expression and identification of P1F8-BGTSTIP fusion protein

[0455] Design and expression of P1F8-BGTSTIP fusion protein

[0456] Nanobodies were screened against the HPV L1 protein (SEQ ID NO: 71) to obtain the nanobody P1F8 (SEQ ID NO: 88). A (GGGGS)3 flexible linker was added after the antibody sequence and then coupled to the front of the BGTSTIP sequence. The amino acid sequence of the constructed P1F8-BGTSTIP fusion protein is shown in SEQ ID NO: 89. The P1F8-BGTSTIP plasmid was extracted, and frozen 293F cells were taken from a -80°C refrigerator. After thawing at 37°C, they were centrifuged at 1300 rpm for 4 minutes. The supernatant was discarded in a clean bench, the cells were flicked and resuspended in 293freestyle medium incubated at 37°C in advance, and then transferred to a triangular flask containing 50 mL of incubation medium. The suspension culture was carried out at 37°C, 5% CO2, and 120 rpm. When the cell density reached 2.0*10 6 Subculture the cells regularly and gradually expand the culture system. When sufficient cells are present, transiently transfect 293F cells with PEI (MW 25,000). Harvest the cells in a sterile 50 mL tube and centrifuge at 1300 rpm for 4 minutes. Gently flick the cells and resuspend them in 37°C incubation medium. Transfer the cells to a flask containing 450 mL of 37°C incubation medium and place them in a shaker at 37°C until ready to use.

[0457] The extracted P1F8-BGTSTIP plasmid and PEI (MW 25,000) were added to 50 ml of culture medium at a ratio of 1:2, mixed thoroughly, and allowed to stand for 18 minutes. The culture was then transferred to the aforementioned 450 ml of culture medium and cultured in suspension at 37°C, 5% CO2, and 120 rpm for 6 days to express the P1F8-BGTSTIP protein. During transfection, the PEI was kept away from light.

[0458] Purification of P1F8-BGTSTIP fusion protein

[0459] Six days after transient transfection, the cell culture medium was collected and centrifuged at 7000 g for 10 min in a JA-14 rotor. The cell supernatant was collected and then centrifuged at 20000 g for 10 min. The supernatant was filtered twice with a 0.22 μm pore size filter membrane. The sample was then used for the next step of Ni-excel column purification.

[0460] Ni affinity chromatography purification was performed using the AKTA system;

[0461] Instrument system: AKTA Pure preparative liquid chromatograph;

[0462] Purification medium: Ni Sepharose Excel affinity medium; Buffer: divided into A and B buffers, A buffer is 1× PBS buffer, B pump is 1× PBS + 250mmol / L imidazole buffer;

[0463] System loading flow rate: 8mL / min; detection wavelength: UV@280nm

[0464] System elution flow rate: 4ml / min; detection wavelength: UV@280nm

[0465] Elution conditions: Elute impurities with 20 mM imidazole, and collect the 250 mM imidazole elution product. Dialyze the eluate against 1× PBS overnight, changing the dialysate twice. Harvest approximately 30 ml of low-concentration target protein, concentrate to 5 ml using 20 ml of Vivaspin and a 100 kD ultrafiltration concentrator for later use. Prepare reduced and non-reduced samples from the collected elution sample and perform SDS-PAGE gel electrophoresis as described above. Figure 34 shows the SDS-polyacrylamide gel electrophoresis results for the P1F8-BGTSTIP protein; M: molecular weight marker; "+" indicates reduced SDS-PAGE; "-" indicates non-reduced SDS-PAGE. The results show that the P1F8-BGTSTIP molecule has a molecular weight of approximately 160 kD under reducing conditions and is a multimer under non-reducing conditions, consistent with the theoretical molecular weight.

[0466] P1F8-BGTSTIP protein molecular activity analysis (ELISA)

[0467] The fusion protein was diluted to 1 μg / mL and coated on a 96-well plate, with 100 μL per well, and allowed to stand at room temperature for 2 h;

[0468] (2) Wash the plate once and block with bovine serum albumin diluent (ED, 200 μL / well) at room temperature for 2 h;

[0469] (3) Wash the plate once, dilute the corresponding specific monoclonal antibody to 1 μg / ml, add 100 μl to the first well, make 3-fold serial dilutions, repeat in duplicate, and incubate at room temperature for 1 hour;

[0470] (4) Wash the plate five times, add the secondary antibody GAH-HRP (1:5000) to the 96-well plate, 100 μL / well, and incubate at room temperature for 1 h;

[0471] (5) Wash the plate five times, develop the color at room temperature for 10 min, stop the reaction, and detect at a wavelength of 450 nm using a microplate reader. Data analysis was performed using GraphPad Prism 5 (GraphPad, USA) software.

[0472] Figure 35 shows the results of the enzyme-linked immunosorbent assay of P1F8-BGTSTIP. The results indicate that P1F8 has good binding activity with neutralizing antibodies such as VRC01, PGT121, PGT122 (JN201912.1 / JN201895.1), 2G12, SF162, and B12, while the binding activity with 17b, F105, and F240 is very weak, indicating that P1F8-BGTSTIP well exposes the broad-spectrum neutralizing antibody epitopes without exposing non-neutralizing epitopes.

[0473] Affinity analysis of P1F8-BGTSTIP and HPV 58 VLP (SPR)

[0474] Load the CM5 chip and rinse the pipeline with PBS-P buffer PBS.

[0475] The ligand 58-VLP (10 ug / ml) was centrifuged at high speed for 10 min.

[0476] The 58-VLP coupling protocol was as follows: chip channel activation (EDC:NHS = 1:1, flow rate 10 μl / min), followed by a wash cycle (PBS buffer, flow rate 30 μl / min). 58 VLP sample coupling was performed (time: 420 s, flow rate 10 μl / min). After determining the ligand coupling amount, the channel was blocked with ethanolamine (time: 420 s, flow rate 10 μl / min).

[0477] Affinity assay: A concentration gradient of 800 nM, 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM fusion protein was loaded for assay. The sample association time was 120 s, the dissociation time was 300 s, and the flow rate was 30 μl / min.

[0478] Biacore 8K (GE) software was used to perform kinetic analysis to fit the binding (Ka) and dissociation (Kd) curves and calculate the affinity (KD).

[0479] FIG36 shows the affinity determination results of P1F8-BGTSTIP and 58VLP. The results show that the affinity of P1F8-BGTSTIP and fusion protein is 4.66×10 -8 , indicating that the two can be combined and the combination is relatively strong.

[0480] Example 18: Preparation and Identification of HPV-Env Fusion Protein (P1F8-BGTSTIP) and HPV 58-VLP Particle Complexes

[0481] Preparation of P1F8-BGTSTIP complex

[0482] P1F8-BGTSTIP and 58-VLP were incubated in a water bath at 37°C for 30 min at a mass ratio of 5:1, centrifuged at 13,300 rpm for 10 min, and then analyzed by high-performance size exclusion chromatography (HPSEC).

[0483] Instrument: Waters. System flow rate: G5000PW XL The flow rate is 0.5 mL / min. The wavelength is 190-600 nm, and the column wavelengths are 280 nm and 254 nm.

[0484] Buffer: Final buffer.

[0485] Operational procedures: Pre-equilibrate the chromatography column for 60 minutes until there is no significant change in the absorption value at 280 nm, and return the absorption value of the detector to zero. Edit the chromatography operation method, inject the sample to be analyzed into a 100 μL sample loop, set automatic loading, and run for 30 minutes. Figure 37 (left) shows the HPSEC spectra of individual HPV 58VLP, P1F8-BGTSTIP, BGTSTIP, and P1F8-BGTSTIP-58VLP complexes. The results showed that the peak time of the complex was 12 minutes, which was earlier than that of 58VLP. We collected the complex components and performed SDS-PAGE gel electrophoresis. The results showed that the sample collected for 12 minutes showed two molecular weight bands of P1F8-BGTSTIP and 58-VLP, indicating that P1F8-BGTSTIP and 58VLP successfully formed a complex, and the peak time was about 12 minutes.

[0486] Analytical ultracentrifugation (AUC) of P1F8-BGTSTIP-58 VLP particle antigen

[0487] The P1F8-BGTSTIP-58 VLP complex recovered through the above purification process was subjected to AUC analysis.

[0488] The instrument used was a Beckman XL-A analytical ultracentrifuge equipped with an optical detection system and an An-60Ti rotor.

[0489] Install the sample pool according to the operating instructions, add 400 μL of sample buffer (the same buffer as the sample) to the control pool, add 380 μL of sample (OD280 is about 0.8) to the sample pool, and balance the sample pool so that the weight difference is within 0.1 g.

[0490] The sample pool was placed in the An-60Ti rotor, and the rotor was placed in the cavity of the Beckman XL-A analytical ultracentrifuge, and an optical path detector was installed.

[0491] Parameter settings: 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 sec), and number of data (150 scans). The centrifugation speed for the complex was set to 7000 rpm, and the centrifugation speed for the fusion protein was set to 30,000 rpm.

[0492] After the experiment, SENDTERP software was used to calculate the density and viscosity of the buffer, as well as the partial microvolume of the known proteins. Sedimentation coefficients were analyzed using the Origin versions of Nonlin and SEDFIT. The friction ratio f / f0 was set to 1.2 for globular proteins. The analysis range was set based on the molecular weight and basic properties of the sample protein, and the calculation resolution was set to 100. The RMSD value was generally required to be no greater than 0.01, and the residue map fluctuation was within 0.05.

[0493] Figure 38 shows the analytical ultracentrifugation results of the complex of the present invention, P1F8-BGTSTIP, and 58VLP. The results show that 58VLP exhibits a single component with a sedimentation coefficient of 119S (Figure 38, right), while the P1F8-BGTSTIP-58VLP complex exhibits a single component with a sedimentation coefficient of 176.3S (Figure 38, left), significantly higher than that of 58VLP particles. These results demonstrate that 1F8-BGTSTIP can form a particle complex with 58VLP and maintain a stable particle morphology in aqueous solution.

[0494] Transmission electron microscopy morphology observation of P1F8-BGTSTIP-58 VLP granulated antigen

[0495] The transmission electron microscope used was a Tecnai G2 Spirit 120 kV (FEI). Negative staining was performed using phosphotungstic acid.

[0496] Sample preparation: First, a copper grid (R2 / 2, 200 mesh, ThermoFisher Scientific) was treated with glow discharge hydrophilization. Then, 5 μL of a 0.5 mg / mL sample was dripped onto the grid. After standing at room temperature for 60 seconds, the droplet was removed from the edge of the grid with absorbent paper. The grid was allowed to air dry at room temperature before being inspected.

[0497] Figure 39 shows the morphology of the complex particles under a transmission electron microscope. It can be clearly seen that the surface of the particles is covered with a layer of protein, indicating that P1F8-BGTSTIP is successfully displayed on the surface of HPV 58 VLP particles.

[0498] Evaluation of the immunogenicity of P1F8-BGTSTIP-58 VLP particle antigen

[0499] This experimental protocol was approved by the Experimental Animal Care Ethics Committee of Xiamen University. All operations were performed in strict accordance with animal ethics guidelines and approved protocols.

[0500] Six-week-old Balb / C mice were divided into six groups of five mice each, with three high-dose and three low-dose groups. They were immunized with P1F8-BGTSTIP-58 VLP particles, P1F8-BGTSTIP, and BGTSTIP immunogens in an aluminum adjuvant, respectively. Each antigen was administered at two doses, 0.5 μg and 5 μg. Mice were immunized intramuscularly (100 μL / mouse) at 0, 2, 4, and 6 weeks.

[0501] Eye blood was collected from mice before immunization, before each immunization, and after the sixth immunization. The mice were then dislocated by cervical dislocation. Blood samples were incubated at 37°C for 30 minutes and centrifuged at 13,300 rpm for 10 minutes. Serum was collected for specific binding antibody titer determination.

[0502] Figure 40 shows the immunogenicity test results of the P1F8-BGTSTIP-VLP particle antigen of the present invention. Figure 40 shows the BGTSTIP-specific binding antibody titer test results (left figure is the 0.5 μg group, right figure is the 5 μg group). The results show that in weeks 1-4, the binding titer of the complex particles was significantly higher than that of BGTSTIP alone and P1F8-BGTSTIP protein alone.

[0503] Example 19: Design and evaluation of nanobody fusion proteins based on HBsAg VLPs

[0504] In this example, a VLP vector was prepared based on the granulated antigen SHBs protein (SEQ ID NO: 73) from the commercially available hepatitis B vaccine. A gE fusion protein was prepared according to the methods of the previous examples. HBV-gE granulated antigen was further prepared and the immunogenicity of the granulated antigen was tested. The specific experimental procedures are as follows:

[0505] Design and expression of gE fusion protein

[0506] A reported HBsAg nanoantibody (1. Serruys, B., Houtte, FV, Verbrugghe, P., Leroux-Roels, G. & Vanlandschoot, P. Llama-derived single-domain intrabodies inhibit secretion of hepatitis B virions in mice. Hepatology 49, 39-49 (2009).) was selected for the experiment. A (GGGGS)3 flexible linker was added after the antibody sequence and then coupled to the RBD and gE sequences to construct an RBD and gE fusion protein. To extract the plasmid, take frozen 293F cells from a -80°C refrigerator, thaw at 37°C, and centrifuge at 1300 rpm for 4 min. Discard the supernatant in a clean bench, flick the cells, and resuspend them in 293freestyle medium incubated at 37°C in advance. Transfer the cells to a flask containing 50 mL of incubation medium and culture them in suspension at 37°C, 5% CO2, 120 rpm, until the cell density reaches 2.0*10 6 Subculture the cells regularly and gradually expand the culture system. When sufficient cells are present, transiently transfect 293F cells with PEI (MW 25,000). Harvest the cells in a sterile 50 mL tube and centrifuge at 1300 rpm for 4 minutes. Gently flick the cells and resuspend them in 37°C incubation medium. Transfer the cells to a flask containing 450 mL of 37°C incubation medium and place them in a shaker at 37°C until ready to use.

[0507] The extracted plasmid and PEI (MW 25,000) were placed in 50 ml of culture medium at a ratio of 1:2, mixed thoroughly, and allowed to stand for 18 minutes. The cells were then transferred to the aforementioned 450 ml of culture medium and cultured in suspension at 37°C, 5% CO2, and 120 rpm for 6 days to express the gE fusion protein. During transfection, the PEI was kept away from light.

[0508] Purification of gE fusion protein

[0509] Six days after transient transfection, the cell culture medium was collected and centrifuged at 7000 g for 10 min in a JA-14 rotor. The cell supernatant was collected and then centrifuged at 20000 g for 10 min. The supernatant was filtered twice with a 0.22 μm pore size filter membrane. The sample was then used for the next step of Ni-excel column purification.

[0510] Ni affinity chromatography purification was performed using the AKTA system;

[0511] Instrument system: AKTA Pure preparative liquid chromatograph;

[0512] Purification medium: Ni Sepharose Excel affinity medium; Buffer: divided into A and B buffers, A buffer is 1× PBS buffer, B pump is 1× PBS + 250mmol / L imidazole buffer;

[0513] System loading flow rate: 8mL / min; detection wavelength: UV@280nm

[0514] System elution flow rate: 4ml / min; detection wavelength: UV@280nm

[0515] Elution conditions: Elute contaminants with 20 mM imidazole, then collect the 250 mM imidazole elution product. Dialyze the eluate against 1× PBS overnight, changing the dialysate twice. Harvest approximately 30 ml of low-concentration target protein and concentrate to 5 ml using 20 ml of Vivaspin and a 100 kDa ultrafiltration concentrator for later use. Prepare the collected eluted sample and perform SDS-PAGE gel electrophoresis as described above.

[0516] The experimental results showed that a high-expression nanobody fusion protein was obtained, which was named S2-gE. As shown in Figure 41, SDS polyacrylamide gel electrophoresis results showed that S2-gE was consistent with the theoretical molecular weight.

[0517] Purification of fusion proteins and their complexes by molecular sieve chromatography

[0518] Instrument system: AKTA explorer 100 preparative liquid chromatography system produced by GE Healthcare (formerly Amershan Pharmacia).

[0519] Chromatographic medium: Superdex 200 increase (cytiva).

[0520] Column volume: 20 cm × 20 mm.

[0521] Buffer: 20 mM phosphate buffer pH 7.4.

[0522] Flow rate: 0.7 mL / min.

[0523] Detector wavelength: 280nm.

[0524] The samples are the samples in Example 1 and Example 5.

[0525] The elution procedure is: collect the penetration peak in segments.

[0526] Collect the product that penetrated through the Supedex 200 increase to obtain 5 mL of purified sample. Take 50 μL of each eluted product, add 10 μL of 6X Loading Buffer, mix thoroughly, incubate in an 80°C water bath for 10 minutes, then apply 10 μL of the sample to a 10% SDS-polyacrylamide gel for electrophoresis at 120 V for 60 minutes. Then, stain with Coomassie Brilliant Blue to visualize the electrophoretic bands.

[0527] As shown in Figure 42, SDS polyacrylamide gel electrophoresis results showed that HBsAg and HBsAg-S2-gE (the complex peak of HBsAg and S2-gE) absorbed different peaks. The HBsAg-S2-gE particle peak became larger and had an earlier retention time, indicating that S2-gE can form particulate antigens with HBsAg.

[0528] HBV-gE complex molecular size detection (DLS)

[0529] Instrument: NanoBrook Series (Brookhaven instrument).

[0530] Functional module: DLS (Dynamic Light Scattering).

[0531] Buffer: PBS.

[0532] Procedure: Preheat the instrument for 5 minutes. Prepare the sample to be tested (concentration 0.5 mg / ml, centrifuge at 12,000 rpm for 5 minutes, and add 50 μl to the sample cup). Set the test parameters: Test time: 300 seconds per test, repeat three times for each sample.

[0533] As shown in FIG43 , DLS results showed that the particle sizes of HBsAg alone and the complex of HBsAg-S2-gE were different. The particle size of HBsAg-S2-gE (HBV-gE) was significantly larger than that of HBsAg, indicating the formation of gE particulate antigen.

[0534] Transmission electron microscopy particle morphology observation

[0535] The transmission electron microscope used was a Tecnai G2 Spirit 120 kV (FEI). Negative staining was performed using phosphotungstic acid.

[0536] Sample preparation: First, a copper grid (R2 / 2, 200 mesh, ThermoFisher Scientific) was treated with glow discharge hydrophilization. Then, 5 μL of a 0.5 mg / mL sample was dripped onto the grid. After standing at room temperature for 60 seconds, the droplet was removed from the edge of the grid with absorbent paper. The grid was allowed to air dry at room temperature before being inspected.

[0537] As shown in FIG44 , negative staining results showed that the size and morphology of the complex of HBsAg alone and HBsAg-S2-gE were different, indicating the formation of gE particulate antigen.

[0538] Evaluation of immunogenicity of gE particles

[0539] This experimental protocol was approved by the Experimental Animal Care Ethics Committee of Xiamen University. All operations were performed in strict accordance with animal ethics guidelines and approved protocols.

[0540] Six-week-old Balb / C mice were selected and divided into two groups of three. Each group was immunized with the previously prepared HBsAg-gE complex and S2-gE alone. The gE dose was the same, 5 μg, injected intramuscularly (50 μL) into the left or right hind limb at week 0 and week 2, respectively. Ocular venous blood was collected at week 0, week 1, week 2, week 3, and week 4. 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.

[0541] As shown in Figure 45, the gE-specific titer results after two immunizations showed that the particulate antigen HBsAg-S2-gE induced significantly higher antibody levels than S2-gE alone, demonstrating an immune-enhancing effect.

Claims

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

2. The fusion protein according to claim 1, wherein The assembly polypeptide is a polypeptide capable of being assembled into a VLP; Preferably, the assembled polypeptide is a coat protein of a natural virus or viroid, or is an artificially prepared and / or screened polypeptide. Preferably, the assembly polypeptide is selected from a protein of hepatitis E virus (HEV) or a fragment thereof or a variant thereof, a protein of hepatitis B virus (HBV) or a fragment thereof or a variant thereof, a protein of human papillomavirus (HPV) or a fragment thereof or a variant thereof, or any combination thereof; wherein the fragment or variant retains the ability to assemble into VLP; Preferably, the assembly polypeptide is the ORF2 protein of HEV or a fragment thereof or a variant thereof; preferably, the fragment of the ORF2 protein is selected from the group consisting of p239 protein and p495 protein; Preferably, the assembly polypeptide is selected from p239 protein or its fragment or variant thereof, p495 protein or its fragment or variant thereof; Preferably, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence of the protein from which it is derived; preferably, the substitutions are conservative substitutions; Preferably, the ORF2 protein has the amino acid sequence shown in SEQ ID NO: 9; preferably, the p239 protein has the amino acid sequence shown in SEQ ID NO: 40; preferably, the p495 protein has the amino acid sequence shown in SEQ ID NO: 41; Preferably, the assembly polypeptide is HPV capsid protein L1 or a fragment or variant thereof; Preferably, the assembly polypeptide has the amino acid sequence shown in SEQ ID NO: 74; Preferably, the assembled polypeptide is a surface protein of HBV (e.g., hepatitis B virus surface antigen) or a fragment or variant thereof; Preferably, the assembled polypeptide is selected from the group consisting of LHBs protein of hepatitis B virus surface antigen (HBsAg) or a fragment thereof or a variant thereof, MHBs protein or a fragment thereof or a variant thereof, SHBs protein or a fragment thereof or a variant thereof; Preferably, the assembled polypeptide is the SHBs protein of hepatitis B virus surface antigen (HBsAg) or a fragment thereof or a variant thereof; Preferably, the assembly polypeptide has the amino acid sequence shown in SEQ ID NO:

73.

3. The fusion protein according to claim 1 or 2, wherein The immunogenic polypeptide is a polypeptide derived from an organism or a non-organism (e.g., artificially synthesized) or an immunogenic variant thereof; Preferably, the organism is a pathogen (e.g., virus, bacteria, fungus, parasite) or a non-pathogen; Preferably, the immunogenic polypeptide is obtained from a tumor cell of a mammal (eg, a human); preferably, the immunogenic polypeptide is selected from carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), cancer antigen 125 (CA125); Preferably, the immunogenic polypeptide is obtained from a virus, a bacterium (e.g., Mycobacterium tuberculosis), a fungus (e.g., Candida species), or a parasite (e.g., Plasmodium falciparum); Preferably, the virus is selected from varicella-zoster virus (VZV), novel coronavirus (SARS-CoV-2), coronavirus (SARS-COV-1), human immunodeficiency virus type 1 (HIV-1), human papillomavirus, hepatitis B virus, hepatitis A virus, hepatitis C virus, hepatitis E virus, measles virus, mumps virus, influenza virus, Japanese encephalitis virus; Preferably, the immunogenic polypeptide is selected from the RBD protein of SARS-CoV-2 or an immunogenic fragment or variant thereof, the Env protein of HIV-1 or an immunogenic fragment (e.g., gp140, gp160) or variant thereof, the gE protein of VZV or an immunogenic fragment or variant thereof; Preferably, the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence of the protein from which it is derived; preferably, the substitutions are conservative substitutions; Preferably, the RBD protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1-8; Preferably, the Env protein has the amino acid sequence shown in SEQ ID NO: 34 or 35; Preferably, the gE protein has the amino acid sequence shown in SEQ ID NO:

30.

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

5. The fusion protein according to any one of claims 1 to 4, further comprising a linker; Preferably, the linker is a polypeptide, such as a flexible peptide or a rigid peptide; Preferably, the linker comprises one or several (e.g., 1, 2, or 3) sequences as shown in (GmS)n, wherein m is selected from an integer of 1-6, and n is selected from an integer of 1-6; preferably, m is 3, 4, or 5; preferably, n is 2, 3, or 4; More preferably, the linker has the amino acid sequence shown in SEQ ID NO: 39; Preferably, the immunogenic polypeptide and the Nanobody of the fusion protein are directly linked or connected through a linker; Preferably, the immunogenic polypeptide is located at the N-terminus or C-terminus of the fusion protein; Preferably, the fusion protein comprises, from N-terminus to C-terminus, in sequence: an immunogenic polypeptide and a nanobody; or, a nanobody and an immunogenic polypeptide; or, an immunogenic polypeptide, a linker and a nanobody; or, a nanobody, a linker and an immunogenic polypeptide.

6. The fusion protein according to any one of claims 1 to 5, further comprising a signal peptide and / or a tag; Preferably, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 31, 37 or 38; Preferably, the tag is a tag for purification, for example, selected from a His tag or a GST tag; Preferably, the signal peptide is located at the N-terminus of the fusion protein; Preferably, the tag is located at the C-terminus of the fusion protein; Preferably, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 42-49, 50-56, 72-79, 80-87, 63, 64, 89.

7. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1 to 6; Preferably, the nucleotide sequence is codon-optimized or non-optimized according to the codon preference of the host cell.

8. A vector comprising the isolated nucleic acid molecule of claim 7; preferably, the vector is used to express (eg, express in vitro in a cell) a protein encoded by the isolated nucleic acid molecule.

9. A host cell comprising the nucleic acid molecule of claim 7 or the vector of claim 8; Preferably, the host cell is selected from prokaryotic cells and eukaryotic cells; Preferably, the prokaryotic cells are selected from Escherichia coli cells and Bacillus subtilis cells; Preferably, the eukaryotic cell is selected from yeast cells, insect cells, plant cells and animal cells; Preferably, the animal cell is a mammalian cell (eg, a mouse cell, a human cell).

10. A method for expressing or producing the fusion protein of any one of claims 1 to 6, comprising culturing the host cell of claim 9 under conditions allowing protein expression, and optionally recovering or purifying the expressed fusion protein.

11. A composition comprising at least one fusion protein according to any one of claims 1 to 6; Preferably, the composition further comprises an assembly polypeptide; Preferably, the assembled polypeptide is in the form of VLP; Preferably, the fusion protein is attached to the VLP.

12. A kit comprising: the fusion protein according to any one of claims 1 to 6 or a first nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein, and an assembly polypeptide or a second nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein; Preferably, the nucleotide sequence is codon-optimized or not codon-optimized according to the codon preference of the host cell; Preferably, 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; Preferably, the kit further comprises a vector (eg, an expression vector); preferably, the first nucleic acid molecule and the second nucleic acid molecule are contained in the same or different vectors; Optionally, the kit further comprises a buffer; Preferably, the buffer is selected from phosphate buffer, citrate buffer, carbonate buffer, acetate buffer, barbituric acid buffer, Tris buffer, or any combination thereof; Preferably, the buffer is PBS buffer; Preferably, the buffer further comprises salt; Preferably, the salt is selected from NaCl, (NH4)SO4, NaSO4, NH4Cl, or any combination thereof.

13. A particulate antigen comprising an assembly polypeptide in the form of VLP and the fusion protein of any one of claims 1 to 6 attached to the assembly polypeptide; Preferably, the fusion protein is attached to the VLP via the interaction of the Nanobody with the assembly polypeptide; Preferably, the VLP is attached to at least one fusion protein according to any one of claims 1 to 6; Preferably, the VLP is also attached to an additional polypeptide or fusion protein (eg, a T cell epitope).

14. A method for preparing the particulate antigen according to claim 13, comprising: Using the kit according to claim 12; Preferably, the method comprises: contacting the assembly polypeptide with the fusion protein under conditions that allow VLP assembly; Optionally, the particulate antigen in the buffer is recovered or purified.

15. A vaccine comprising the fusion protein according to any one of claims 1 to 6, or the composition according to claim 11, or the particulate antigen according to claim 13, and an adjuvant; Preferably, the adjuvant is selected from aluminum salt adjuvants, zinc-aluminum mixed adjuvants (eg, FH002C), Freund's adjuvant, oil emulsion adjuvant, cytokine, TLR agonist, CpG adjuvant, liposome, AS01B adjuvant, or any combination thereof.

16. A pharmaceutical composition comprising any one or more of (1) to (6): (1) The fusion protein according to any one of claims 1 to 6; (2) The nucleic acid molecule according to claim 7; (3) The vector according to claim 8; (4) The host cell according to claim 9; (5) The composition according to claim 11; (6) The granulated antigen according to claim 13; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

17. Use of the fusion protein of any one of claims 1 to 6, or the nucleic acid molecule of claim 7, or the vector of claim 8, or the host cell of claim 9, or the composition of claim 11, or the kit of claim 12, or the particulate antigen of claim 13 in the preparation of a pharmaceutical composition or vaccine for inducing an immune response in a subject; Preferably, the immune response is a response to an immunogenic polypeptide; preferably, the immune response is a T cell response (e.g., a CD4+ response or a CD8+ response); preferably, the immune response is a B cell response; Preferably, the subject is a mammal, such as a human, monkey or mouse.

18. Use of the fusion protein of any one of claims 1 to 6, or the nucleic acid molecule of claim 7, or the vector of claim 8, or the host cell of claim 9, or the composition of claim 11, or the kit of claim 12, or the particulate antigen of claim 13 in the preparation of a pharmaceutical composition or vaccine for preventing and / or treating a disease and / or symptom that would benefit from or be prevented by an immune response to the immunogenic polypeptide in a subject; Preferably, the disease and / or condition is caused by a tumor cell or a pathogen (e.g., virus, bacteria, fungus, parasite) from which the immunogenic polypeptide is derived; Preferably, the disease and / or symptom is caused by a virus from which the immunogenic polypeptide is derived, for example, chickenpox, novel coronavirus pneumonia, AIDS, genital warts, viral hepatitis (e.g., viral hepatitis B, viral hepatitis A, viral hepatitis C, viral hepatitis E), measles, mumps; Preferably, the subject is a mammal, such as a human, monkey or mouse.

19. A method of inducing an immune response in a subject, comprising administering to the subject an effective amount of the fusion protein of any one of claims 1 to 6, or the nucleic acid molecule of claim 7, or the vector of claim 8, or the host cell of claim 9, or the composition of claim 11, or the kit of claim 12, or the particulate antigen of claim 13, or the vaccine of claim 15, or the pharmaceutical composition of claim 16; Preferably, the immune response is to an immunogenic polypeptide; preferably, the immune response is a T cell response (e.g., a CD4+ response or a CD8+ response); preferably, the immune response is a B cell response; Preferably, the subject is a mammal, such as a human, monkey or mouse.

20. A method for preventing and / or treating a disease and / or symptom in a subject that benefits from or is prevented by an immune response to an immunogenic polypeptide, comprising administering to the subject an effective amount of the fusion protein of any one of claims 1 to 6, or the nucleic acid molecule of claim 7, or the vector of claim 8, or the host cell of claim 9, or the composition of claim 11, or the kit of claim 12, or the particulate antigen of claim 13, or the vaccine of claim 15, or the pharmaceutical composition of claim 16; Preferably, the disease and / or condition is caused by a tumor cell or a pathogen (e.g., virus, bacteria, fungus, parasite) from which the immunogenic polypeptide is derived; Preferably, the disease and / or symptom is caused by a virus from which the immunogenic polypeptide is derived, for example, chickenpox, novel coronavirus pneumonia, AIDS, genital warts, viral hepatitis (e.g., viral hepatitis B, viral hepatitis A, viral hepatitis C, viral hepatitis E), measles, mumps; Preferably, the subject is a mammal, such as a human, monkey or mouse.

21. A system for preparing a granulated immunogenic polypeptide, comprising a first carrier and a second carrier, 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; and 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 not codon-optimized according to the codon preference of the host cell; Preferably, the assembly polypeptide is selected from the group consisting of proteins of hepatitis E virus (HEV) or fragments thereof or variants thereof; preferably, the assembly polypeptide is as defined in claim 2; Preferably, the fusion protein is as defined in any one of claims 1-6.

22. 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 assembled polypeptide; and, contacting the fusion protein with a VLP comprising the assembled polypeptide, thereby obtaining a particulate antigen comprising the immunogenic polypeptide attached to the VLP; Preferably, the method comprises: using the system of claim 21; preferably, the method comprises: (1) expressing or producing the fusion protein via a first vector and expressing or producing the assembly polypeptide via a second vector; (2) contacting the fusion protein and the assembly polypeptide under conditions that allow VLP assembly; Preferably, the Nanobody is as defined in claim 4; Preferably, the fusion protein is as defined in any one of claims 1 to 6; Preferably, the assembling polypeptide is assembled into a VLP; preferably, the fusion protein is attached to the VLP via the interaction of the Nanobody with the assembling polypeptide.

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