Universal affinity epitope polypeptides, antibodies and uses thereof for human rhinoviruses

Abstract

The present invention belongs to the field of immunobiology and relates to an epitope polypeptide, such as a universal affinity epitope polypeptide of human rhinovirus and uses thereof, and to an antibody that can bind to the above-mentioned epitope polypeptide and uses thereof. The present invention further relates to the use of the affinity epitope polypeptide or the antibody in the manufacture of a medicament or method for the treatment and / or prophylaxis and / or diagnosis of human rhinovirus and / or the identification of human rhinovirus titer and / or the identification of human rhinovirus neutralizing antibody potency. The affinity epitope polypeptide and the antibody of the present invention can be used in a medicament or method for the treatment and / or prophylaxis and / or diagnosis of human rhinovirus and / or the identification of human rhinovirus titer and / or the identification of human rhinovirus neutralizing antibody potency.

Description

Technical Field

[0001] This invention relates to the fields of immunology and molecular virology, particularly to the prevention and treatment of human rhinovirus. Specifically, this invention relates to polypeptides that can be used to prevent or treat human rhinovirus infection, recombinant proteins comprising such polypeptides and carrier proteins, and the uses of such polypeptides and recombinant proteins. This invention also relates to antibodies against such polypeptides, and their use for detecting, preventing, and / or treating human rhinovirus infection and / or diseases caused by said infection. Background Technology

[0002] Human rhinovirus (HRV) belongs to the genus Enterovirus of the family Picornaviridae. HRV has the most diverse serotypes among viruses that infect humans. Based on their genetic characteristics, HRV can be divided into three main groups: A, B, and C. According to the latest virus classification report (2019 edition) published by the International Committee on Taxonomy of Viruses (ICTV), HRV-A includes 80 serotypes such as HRV-A1, 1B, 2, and 7–13; HRV-B includes 30 serotypes such as HRV-B3–6, 14, 17, and 26–27; and HRV-C includes 55 serotypes such as HRV-C1–51 and 54–57.

[0003] HRV infection typically presents with self-limiting common cold symptoms, with an average course of about one week. However, recent studies have shown that HRV infection can also cause lower respiratory tract infections such as bronchiolitis and pneumonia, and is closely associated with severe respiratory diseases such as acute wheezing, asthma exacerbations, persistent cough, dyspnea in children, and chronic obstructive pulmonary disease in adults. In addition, HRV can co-infect with other respiratory viruses such as coronaviruses, increasing the disease burden and risk of severe illness (Li et al., 2018. J Infect. 76(3): 311-13; David et al., 2020. JAMA. 323(20): 2085-86). Besides mixed viral infections, HRV can also co-infect with bacteria, leading to a series of diseases such as otitis media, sinusitis, and pharyngitis (Chistopher et al., 2017. Front Microbiol. 8: 2412). HRV can infect people of all ages, but infants, the elderly, and immunocompromised individuals are more susceptible and experience more severe illness. HRV infection can provide some immunity, but it is short-lived and there is almost no cross-protection between different types of the virus.

[0004] HRV virus particles are non-enveloped, nearly spherical in shape, and approximately 27–30 nm in diameter. Their structure mainly consists of an outer capsid and internal nucleic acid. The HRV capsid exhibits icosahedral symmetry and is composed of four structural proteins, VP1, VP2, VP3, and VP4, arranged in a specific pattern. VP1–3 are exposed on the surface of the capsid, while VP4 is embedded inside and tightly connected to the internal nucleic acid. Different HRV genotypes share similar genome structures, all being single-stranded positive-sense RNA, approximately 7200 bp in length. The entire HRV genome contains only one open reading frame (ORF), with untranslated regions (UTRs) at the 5' and 3' ends. This unique ORF encodes a polyprotein approximately 2200 amino acids long, which is subsequently hydrolyzed by a viral protease into three precursor proteins, P1, P2, and P3. P1 can be further cleaved into 4 capsid proteins (VP4, VP2, VP1, VP3), P2 can be cleaved into 3 non-structural proteins (2A, 2B, 2C), and P3 can be cleaved into 4 non-structural proteins (3A, 3B, 3C, 3D). Protein 2C is one of the most conserved non-structural proteins in small RNA viruses. It has ATPase activity and participates in the rearrangement of host cell membrane proteins and the formation of the viral replication complex, as well as in the process of viral evasion of the host's innate immunity (Oberste et al., 2004. J Gen Virol. 85: 1597; Du et al., 2015. Sci Rep. 5: 14302; Kevin et al., 2020. PNAS. 117(44): 27598-607).

[0005] The numerous HRV serotypes and the scarcity of cross-immune reactions between different serotypes pose significant challenges to the prevention and control of HRV infection. Therefore, the discovery of broad-spectrum affinity epitopes, the acquisition of antibodies with broad-spectrum binding capabilities, and the development of universal detection methods for HRV, as well as effective preventative and therapeutic drugs (such as antibodies and vaccines), are of great significance. Summary of the Invention

[0006] The inventors of this application, through extensive research, identified universal affinity epitope peptides in human rhinoviruses, which can induce antibodies with specific binding activity against at least two (e.g., 2, 5, 10, 15, 20, 24) subtypes of human rhinoviruses. Based on this, this application also provides recombinant proteins comprising such epitope peptides and carrier proteins, antibodies against said epitope peptides or recombinant proteins, cell lines that produce said antibodies, and their uses.

[0007] Isolated polypeptides or their variants

[0008] Therefore, in one aspect, this application provides an isolated polypeptide or a variant thereof, the polypeptide consisting of at least 13 consecutive amino acid residues of the human rhinovirus (HRV) 2C protein, including amino acid residues 151-163 of the protein, and the variant differs from the polypeptide from which it is derived only in the substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) amino acid residues, and retains the biological function of the polypeptide from which it is derived (e.g., inducing antibodies with specific binding activity against at least two (e.g., 2, 5, 10, 15, 20, 24) HRV subtypes).

[0009] In some embodiments, the polypeptide consists of no more than 100 (e.g., no more than 95, no more than 90, no more than 85, no more than 80, no more than 75, no more than 70, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, or no more than 30) consecutive amino acid residues of the HRV 2C protein.

[0010] In some embodiments, the polypeptide consists of 13 to 30 (e.g., 13 to 25, 13 to 21, 13 to 17, 13 to 15) consecutive amino acid residues of the HRV 2C protein.

[0011] In some embodiments, the variants contain substitutions (e.g., conserved substitutions) at amino acid positions corresponding to the 156th and / or 159th amino acid position of the HRV 2C protein.

[0012] Conservative substitutions are well known in the art and include, but are not limited to, the following variations: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0013] In some embodiments, the polypeptide or a variant thereof comprises a structure as shown in YSLPPX1PKX2FDGY (SEQ ID NO:52);

[0014] Wherein, X1 is selected from: (i) amino acid residues D, A, S, N and (ii) amino acid residues that are conserved substitutions relative to (i);

[0015] X2 is selected from: (i) amino acid residues Y and H and (ii) amino acid residues that are conserved substitutions relative to (i).

[0016] In some embodiments, the HRV 2C protein has a sequence as shown in SEQ ID NO:9 or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it.

[0017] In some embodiments, amino acid residues 151-163 of the HRV 2C protein are as shown in any one of SEQ ID NOs:1, 30-34.

[0018] In some embodiments, the polypeptide comprises, or is composed of, amino acid residues 151-163, 149-165, 147-167, or 145-169 of the HRV 2C protein.

[0019] In some embodiments, the polypeptide comprises or consists of the sequence shown in any one of SEQ ID NOs:1, 30-37.

[0020] In particular, the polypeptides or variants thereof of the present invention can be fused with a carrier protein to enhance the immunogenicity of the polypeptides or variants thereof, so that the polypeptides or variants thereof can be recognized by the body's immune system and induce an immune response.

[0021] Therefore, in another aspect, this application provides a recombinant protein comprising the isolated polypeptide or a variant thereof as described above, and a carrier protein, wherein the recombinant protein is not a naturally occurring protein or a fragment thereof.

[0022] In some embodiments, the polypeptide or variant thereof is optionally linked to a carrier protein via a linker (e.g., a rigid or flexible linker, such as a peptide linker containing one or more glycines and / or one or more serines).

[0023] In some embodiments, the carrier protein is selected from CRM197 protein or a fragment thereof, HBcAg or a fragment thereof, WHcAg or a fragment thereof, keyhole hemocyanin, human serum albumin, bovine serum albumin, bovine thyroglobulin, and ovalbumin.

[0024] In some embodiments, the carrier protein is HBcAg or a fragment thereof, and amino acids 79-80 of HBcAg are replaced by the polypeptide or a variant thereof. In some embodiments, the polypeptide or a variant thereof is linked to HBcAg or a fragment thereof via a linker. In some embodiments, the HBcAg fragment comprises or consists of aa1-149 of HBcAg, for example, comprising the sequence shown in SEQ ID NO:4. In some embodiments, the recombinant protein has an amino acid sequence as shown in any one of SEQ ID NO:5, 44-51.

[0025] On the other hand, this application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide or a variant or recombinant protein as described above.

[0026] On the other hand, this application provides a carrier comprising the isolated nucleic acid molecules as described above.

[0027] On the other hand, this application provides a host cell comprising the isolated nucleic acid molecules as described above or the vector as described above.

[0028] Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., Escherichia coli cells), and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0029] On the other hand, this application provides a method for preparing the polypeptide or variant thereof as described above or the recombinant protein as described above, comprising culturing the host cell as described above under suitable conditions and recovering the polypeptide or variant thereof or recombinant protein from the cell culture.

[0030] It is understood that the polypeptide or its variants or the recombinant protein described above can be prepared by the above method, or by artificial chemical synthesis, or by other biochemical or molecular biological methods known to those skilled in the art.

[0031] On the other hand, this application provides virus-like particles (VLPs) displaying isolated polypeptides or variants thereof as described above on their surface.

[0032] In some embodiments, the virus-like particle comprises or is composed of a fusion protein, the fusion protein comprising a polypeptide or a variant thereof as described above, and a carrier protein. In some embodiments, the polypeptide or a variant thereof is linked to the carrier protein via a linker.

[0033] In some embodiments, the carrier protein is HBcAg protein or a fragment thereof (e.g., HBcAg aa 1-149). In some embodiments, amino acids 79-80 of HBcAg are replaced with the polypeptide or a variant thereof. In some embodiments, the fusion protein has an amino acid sequence as shown in any one of SEQ ID NO: 5, 44-51.

[0034] On the other hand, this application provides compositions comprising the polypeptide or variant thereof as described above, recombinant protein, isolated nucleic acid molecule, vector, host cell or VLP.

[0035] In some embodiments, the composition is a pharmaceutical composition or an immunogenic composition (e.g., a vaccine).

[0036] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier and / or excipient (e.g., an adjuvant). In some embodiments, the pharmaceutically acceptable carrier and / or excipient comprises an adjuvant. The adjuvant used for co-administration or included in the immunogenic composition according to the invention should preferably be a potentially safe, well-tolerated, and effective adjuvant in humans. Such adjuvants are well known to those skilled in the art.

[0037] In some embodiments, the composition comprises one or more of the peptides or variants thereof, and these peptides or variants thereof may be single or in tandem, modified or unmodified, coupled to other proteins or not coupled to other proteins.

[0038] In some embodiments, the composition comprises one or more of the recombinant proteins.

[0039] In some embodiments, the composition comprises one or more of the VLPs.

[0040] The pharmaceutical or immunogenic compositions described above can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The immunogenic compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. Furthermore, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0041] The pharmaceutical or immunogenic compositions described above may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose.

[0042] The pharmaceutical or immunogenic compositions described above should be administered in an amount sufficient to induce an immune response against HRV. The appropriate amount of immunogen may be determined based on the specific disease or condition to be treated or prevented, its severity, the age of the subject, and other individual attributes of the subject, such as the subject's general health status and the robustness of their immune system. The determination of effective dosage is also guided by animal model studies, followed by human clinical trials, and by administration regimens that significantly reduce the occurrence or severity of the target disease symptoms or condition in the subjects.

[0043] On the other hand, this application provides the use of the polypeptides or variants thereof, recombinant proteins, isolated nucleic acid molecules, vectors, host cells or VLPs as described above in the preparation of formulations for inducing an immune response against HRV in a subject and / or for preventing and / or treating HRV infection or HRV infection-related diseases (e.g., respiratory infections, such as pneumonia, asthma) in a subject.

[0044] In some implementations, the preparation is a vaccine.

[0045] On the other hand, this application provides a method for inducing an immune response against HRV in a subject and / or for preventing and / or treating HRV infection or HRV infection-related diseases (e.g., respiratory infections such as pneumonia, asthma) in a subject, comprising: administering an effective amount of the polypeptide or a variant thereof, recombinant protein, VLP, or composition as described above to a subject in need. In some embodiments, the subject is a human.

[0046] For prophylactic application, the peptide or variant thereof, recombinant protein, isolated nucleic acid molecule, vector, host cell, or VLP or composition as described above are provided prior to any symptoms, such as prior to infection. Prophylactic administration is intended to prevent or improve any subsequent infection to reduce the expected severity, duration, or extent of symptoms of infection and / or related disease after exposure to or suspected exposure to the virus or after actual onset of infection. Thus, in some embodiments, the subject to be treated is someone who has HRV infection or is at risk of developing HRV infection, for example, due to exposure to or potential exposure to HRV.

[0047] For therapeutic applications, the polypeptide or variant thereof, recombinant protein, isolated nucleic acid molecule, vector, host cell or VLP or composition described above are provided at or after the onset of symptoms of disease or infection, such as after the onset of symptoms of HRV infection or after a diagnosis of HRV infection.

[0048] Antibodies and their binding fragments

[0049] In one aspect, this application also provides monoclonal antibodies and antigen-binding fragments thereof, wherein the monoclonal antibody is capable of specifically binding to the polypeptide or its variants, recombinant proteins or VLPs as described above.

[0050] In some embodiments, the monoclonal antibody is capable of specifically binding to amino acid residues 151-163 of the HRV 2C protein.

[0051] In some embodiments, the monoclonal antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody (e.g., scFv), murine antibody, humanized antibody, fully human antibody, chimeric antibody (e.g., human-mouse chimeric antibody), or bispecific or multispecific antibody.

[0052] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0053] (a) Heavy chain variable regions (VH) of the following three complementarity-determining regions (CDRs):

[0054] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:27, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0055] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:28, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0056] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:29, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids);

[0057] And / or,

[0058] (b) The variable regions (VL) of the light chain in the following three complementary determinant regions (CDRs):

[0059] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:24, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0060] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:25, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0061] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:26, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids).

[0062] In some implementations, the CDR described in any one of (i)-(vi) is defined according to the IMGT numbering system.

[0063] In some implementations, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0064] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0065] (a) The following three heavy chain CDRs: VH CDR1 with sequence SEQ ID NO: 27, VHCDR2 with sequence SEQ ID NO: 28, and VH CDR3 with sequence SEQ ID NO: 29; and / or the following three light chain CDRs: VLCDR1 with sequence SEQ ID NO: 24, VL CDR2 with sequence SEQ ID NO: 25, and VL CDR3 with sequence SEQ ID NO: 26;

[0066] or,

[0067] (b) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:23; and / or three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:19.

[0068] In some implementations, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT, or Chothia numbering system.

[0069] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0070] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0071] (i) The sequence shown in SEQ ID NO: 23;

[0072] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 23; or

[0073] (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 23;

[0074] and

[0075] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0076] (iv) The sequence shown in SEQ ID NO: 19;

[0077] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 19; or

[0078] (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 19.

[0079] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0080] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH comprising the sequence shown in SEQ ID NO:23 and a VL comprising the sequence shown in SEQ ID NO:19.

[0081] In some implementations, the antibody or its antigen-binding fragment is humanized.

[0082] In some embodiments, the antibody or its antigen-binding fragment comprises a framework region sequence derived from human immunoglobulin. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain framework region sequence derived from a human heavy chain germline sequence and a light chain framework region sequence derived from a human light chain germline sequence.

[0083] In some embodiments, the antibody or its antigen-binding fragment further comprises a constant region derived from mouse or human immunoglobulins. In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises a heavy chain constant region derived from mouse or human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antibody or its antigen-binding fragment comprises a light chain constant region derived from mouse or human immunoglobulins (e.g., κ or λ).

[0084] On the other hand, this application provides a monoclonal antibody and its antigen-binding fragment, which is produced by or derived from hybridoma cell line 9A5, wherein hybridoma cell line 9A5 is deposited at the China Center for Type Culture Collection (CCTCC) and has accession number CCTCC NO. C2021141.

[0085] In some embodiments, the antibody or its antigen-binding fragment as described above specifically recognizes at least two (e.g., 2, 5, 10, 15, 20, 24) HRV subtypes. In some embodiments, the antibody or its antigen-binding fragment specifically recognizes one or more of the following: HRV-A1B, HRV-A2, HRV-A9, HRV-A16, HRV-A21, HRV-A23, HRV-A29, HRV-A34, HRV-A36, HRV-A40, HRV-A43, HRV-A49, HRV-A54, HRV-A59, HRV-A77, HRV-A78, HRV-A89, HRV-B5, HRV-B6, HRV-B14, HRV-B17, HRV-C11, HRV-C15, and HRV-C23.

[0086] In some embodiments, the representative strain of HRV-A1B is B632 (GenBank accession number D00239). In some embodiments, the representative strain of HRV-A2 is HGP (GenBank accession number AB079139). In some embodiments, the representative strain of HRV-A9 is 211 (GenBank accession number AB079146). In some embodiments, the representative strain of HRV-A16 is 11757 (GenBank accession number AB079152). In some embodiments, the representative strain of HRV-A21 is 47 (GenBank accession number AB079157). In some embodiments, the representative strain of HRV-A23 is 5124 (GenBank accession number AB079159). In some embodiments, the representative strain of HRV-A29 is 5582 (GenBank accession number AB079165). In some embodiments, the representative strain of HRV-A34 is 137-3 (GenBank accession number AB079170). In some embodiments, the representative strain of HRV-A36 is 324H (GenBank accession number AB079172). In some embodiments, the representative strain of HRV-A40 is 1794 (GenBank accession number FJ445129). In some embodiments, the representative strain of HRV-A43 is 58750 (GenBank accession number FJ445131). In some embodiments, the representative strain of HRV-A49 is 8213 (GenBank accession number AB079185). In some embodiments, the representative strain of HRV-A54 is F01-3774 (GenBank accession number AB079190). In some embodiments, the representative strain of HRV-A59 is 611-CV35 (GenBank accession number AB079195). In some embodiments, the representative strain of HRV-A77 is 130-63 (GenBank accession number FJ445154). In some embodiments, the representative strain of HRV-A78 is 2030-65 (GenBank accession number FJ445183). In some embodiments, the representative strain of HRV-A89 is 41467 (GenBank accession number M16248). In some embodiments, the representative strain of HRV-B5 is Norman (GenBank accession number AB079142). In some embodiments, the representative strain of HRV-B6 is Thompson (GenBank accession number AB079143).In some embodiments, the representative strain of HRV-B14 is 1059 (GenBank accession number EU870450). In some embodiments, the representative strain of HRV-B17 is 33342 (GenBank accession number EF173420). In some embodiments, the representative strain of HRV-C11 is SC1065 (GenBank accession number KY369877). In some embodiments, the representative strain of HRV-C15 is W10 (GenBank accession number GU219984). In some embodiments, the representative strain of HRV-C23 is 8713-MY-10 (GenBank accession number KJ675506).

[0087] On the other hand, this application provides isolated nucleic acid molecules that encode antibodies or antigen-binding fragments thereof as described above, or their heavy chain variable regions and / or light chain variable regions.

[0088] On the other hand, this application provides a vector comprising the isolated nucleic acid molecules as described above. In some embodiments, the vector is a cloning vector or an expression vector.

[0089] On the other hand, this application provides a host cell containing isolated nucleic acid molecules as described above or a vector as described above.

[0090] On the other hand, this application provides hybridoma cell line 9A5, which is deposited at the China Center for Type Culture Collection (CCTCC) and has accession number CCTCC NO.C2021141.

[0091] On the other hand, this application provides a method for preparing the antibody or antigen-binding fragment thereof as described above, comprising culturing host cells or hybridoma cells as described above under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cells or hybridoma cell culture.

[0092] On the other hand, this application provides conjugates comprising an antibody or an antigen-binding fragment thereof as described above, and a detectable label linked to the antibody or the antigen-binding fragment thereof.

[0093] In some embodiments, the detectable label is selected from enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent protein), radionuclides, or biotin.

[0094] On the other hand, this application provides a kit comprising an antibody or its antigen-binding fragment or conjugate as described above.

[0095] In some embodiments, the kit comprises the conjugates described above.

[0096] In some embodiments, the kit comprises an antibody or antigen-binding fragment thereof as described above, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof. In some embodiments, the second antibody is specific to an antibody of the species (e.g., mouse or human) from which the constant region contained in the antibody or antigen-binding fragment of the present invention originates. In some embodiments, the second antibody is an anti-immunoglobulin (e.g., mouse or human immunoglobulin) antibody, such as an anti-IgG antibody. In some embodiments, the second antibody is an anti-mouse IgG antibody or an anti-human IgG antibody. In some embodiments, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0097] On the other hand, this application also provides a method for detecting the presence or level of HRV in a sample, which includes using an antibody or antigen-binding fragment thereof as described above or a conjugate as described above.

[0098] In some embodiments, the method is used for therapeutic, diagnostic, or non-therapeutic and non-diagnostic purposes. For example, the method of the present invention can be applied to a subject to diagnose, prevent, or treat a disease caused by human rhinovirus in the subject. Furthermore, the method of the present invention can be applied to in vitro samples (e.g., cell cultures, viral cultures, etc.) to determine the presence or level of rhinovirus in the sample, or the presence or titer of rhinovirus neutralizing antibodies.

[0099] In some embodiments, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0100] In some embodiments, the HRV comprises at least two (e.g., two, five, ten, fifteen, twenty, or twenty-four) subtypes. In some embodiments, the HRV comprises one or more of the following: HRV-A1B, HRV-A2, HRV-A9, HRV-A16, HRV-A21, HRV-A23, HRV-A29, HRV-A34, HRV-A36, HRV-A40, HRV-A43, HRV-A49, HRV-A54, HRV-A59, HRV-A77, HRV-A78, HRV-A89, HRV-B5, HRV-B6, HRV-B14, HRV-B17, HRV-C11, HRV-C15, and HRV-C23.

[0101] In some embodiments, the method includes using the conjugates described above.

[0102] In some embodiments, the method includes using an antibody or antigen-binding fragment thereof as described above, and the method further includes using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin) to detect the antibody or antigen-binding fragment thereof.

[0103] In some embodiments, the method includes: (1) contacting the sample with an antibody or an antigen-binding fragment of the present invention; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of HRV or cells infected with HRV.

[0104] On the other hand, this application provides the use of the antibody or its antigen-binding fragment as described above in the preparation of a diagnostic reagent for detecting the presence or level of HRV in a sample, and / or for diagnosing whether a subject is infected with HRV.

[0105] In some embodiments, the detection reagent detects the presence or level of HRV in a sample by means of the method described above.

[0106] In some embodiments, the sample is a bodily fluid sample (e.g., respiratory secretions, whole blood, plasma, serum, saliva excretions, or urine) from a subject (e.g., a mammal, preferably a human).

[0107] On the other hand, this application provides a method for detecting the neutralizing activity of HRV neutralizing antibodies or screening for HRV neutralizing antibodies, which includes using an antibody as described above or an antigen-binding fragment thereof or a conjugate as described above.

[0108] In some embodiments, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0109] In some embodiments, the method includes: (i) incubating a test sample containing the antibody to be tested with HRV; (ii) incubating the product of step (i) with cells; (iii) incubating the antibody or its antigen-binding fragment or the conjugate with the cell product of step (ii) (e.g., after membrane permeation treatment); and (iv) determining the number of cells labeled by the antibody or its antigen-binding fragment or the conjugate, thereby determining the neutralizing activity of the antibody to be tested against HRV.

[0110] On the other hand, this application provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0111] On the other hand, this application provides the use of the antibody or its antigen-binding fragment, isolated nucleic acid molecule, vector, host cell or hybridoma cell as described above for the preparation of a drug for the prevention and / or treatment of HRV infection or HRV infection-related diseases (e.g., respiratory infections, such as pneumonia, asthma) in a subject.

[0112] In some implementations, the subject is a mammal, such as a human.

[0113] In some embodiments, the antibody or its antigen-binding fragment may be used alone or in combination with other pharmaceutically active agents.

[0114] On the other hand, this application provides a method for preventing and / or treating HRV infection or HRV infection-related disease in a subject (e.g., a human), comprising: administering to a subject in need an effective amount of the antibody or antigen-binding fragment thereof as described above, or a pharmaceutical composition as described above.

[0115] The antibodies or antigen-binding fragments or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The antibodies or antigen-binding fragments or pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody or antigen-binding fragment of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0116] The antibodies or antigen-binding fragments thereof of the present invention, or the pharmaceutical compositions thereof, may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the antibodies or antigen-binding fragments thereof or the pharmaceutical compositions thereof of the present invention are administered by intravenous injection or bolus.

[0117] Beneficial effects

[0118] This invention provides broad-spectrum affinity epitopes for different HRV types, and antibodies against such epitopes. The epitope peptides of this invention can induce specific immune responses against at least two (e.g., 2, 5, 10, 15, 20, 24) subtypes of human rhinovirus; the antibodies of this invention have broad-spectrum binding ability against different HRV types and can be used for the detection, prevention, and / or treatment of human rhinovirus infection and / or diseases caused by said infection (e.g., respiratory infections).

[0119] Terminology Definition

[0120] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0121] As used herein, when referring to the amino acid position of HRV nonstructural protein 2C, reference is made to the amino acid sequence of nonstructural protein 2C of HRV-C15 strain (W10) (the sequence shown in SEQ ID NO: 9). For example, the expression "amino acid residues 151-163 of HRV nonstructural protein 2C (aa 151-163)" refers to amino acid residues 151-163 of the polypeptide shown in SEQ ID NO: 9. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions, such as different HRV types) can be naturally generated or artificially introduced into the amino acid sequence of HRV nonstructural protein 2C without affecting its biological function. For example, in different rhinovirus strains (e.g., HRV-A1B, HRV-A2, HRV-A9, HRV-A16, HRV-A21, HRV-A23, HRV-A29, HRV-A34, HRV-A36, HRV-A40, HRV-A43, HRV-A49, HRV-A54, HRV-A59, HRV-A77, HRV-A78, HRV-A89, HRV-B5, HRV-B6, HRV-B14, HRV-B17, HRV-C11, HRV-C15, or HRV-C23), the amino acid sequence of non-structural protein 2C may naturally vary (mutations or variations) without affecting its biological function. Therefore, in this invention, the term "HRV non-structural protein 2C" should include all such sequences, including, for example, the sequence shown in SEQ ID NO: 9, as well as its natural or artificial variants. Furthermore, when describing a sequence fragment of rhinovirus nonstructural protein 2C, it includes not only the sequence fragment of SEQ ID NO: 9, but also the corresponding sequence fragment in its natural or artificial variants. For example, the expression "aa 151-163 of rhinovirus nonstructural protein 2C" includes amino acid residues 151-163 of SEQ ID NO: 9, and the corresponding fragment in its variants (natural or artificial). According to the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to the fragment located at the equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity.

[0122] Furthermore, according to the complete genome of the HRV-C15 virus strain (GU219984), the aa151-163 segments of the HRV non-structural protein 2C (e.g., the sequence shown in SEQ ID NO:1) correspond to the aa1238-1250 amino acid sequence translated from the rhinovirus genome. Therefore, in this invention, the expression "aa 151-163 segments of the HRV non-structural protein 2C" and the expression "aa 1238-1250 amino acid sequence translated from the HRV genome" have equivalent meanings and can be used interchangeably.

[0123] As used herein, the term "HBcAg" refers to the core antigen protein of hepatitis B virus (HBV), which is well known to those skilled in the art (see, for example, NCBI GENBANK database accession number: AAO63517.1). When referring to the amino acid positions of HBcAg, reference is made to the sequence shown in SEQ ID NO:4. For example, the expression "amino acid residues 1-149 of HBcAg (aa 1-149)" refers to amino acid residues 1-149 of the polypeptide shown in SEQ ID NO:4. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions, such as different genotypes or subtypes of HBcAg) can be naturally generated or artificially introduced into the amino acid sequence of HBcAg without affecting its biological function. Therefore, in this invention, the term "HBcAg" should include all such sequences, including, for example, the sequence shown in SEQ ID NO:4 and its natural or artificial variants. Furthermore, when describing a sequence fragment of HBcAg, it includes not only the sequence fragment of SEQ ID NO: 4, but also the corresponding sequence fragment in its natural or artificial variants. For example, the expression "amino acid residues 1-149 of HBcAg" includes amino acid residues 1-149 of SEQ ID NO: 4, and the corresponding fragment in its variants (natural or artificial). According to the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to the fragment located at the equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity.

[0124] As used herein, the term “WHcAg” refers to the core protein of marmot hepatitis virus, which is well known to those skilled in the art (see, for example, NCBI GENBANK database accession number: ADE19018.1).

[0125] As used herein, the term "CRM197 (Cross-Reacting Materials 197)" refers to a non-toxic mutant of diphtheria toxin (DT) that differs from wild-type diphtheria toxin in that the amino acid residue at position 52 is changed from Gly to Glu (G. Gianninini, R. Rappuoli, G. Ratti et al., Nucleic Acids Research. 1984.12: 4063-4070). Diphtheria toxin is well known to those skilled in the art (see, for example, Choe S, Bennett M, Fujii G, et al., Nature. 1992.357: 216-222), and its amino acid sequence can be found, for example, in GenBank accession number AAV70486.1.

[0126] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid sequence or nucleic acid sequence to best align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between two sequences is a function of the number of identity positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In some embodiments, the two sequences are of the same length.

[0127] The determination of percentage identity between two sequences can also be achieved using mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as an improvement upon that in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are integrated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0128] As used herein, the term "variant," in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide that contains an amino acid sequence altered by the introduction of amino acid residue substitutions, deletions, or additions. As used herein, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limited to, polypeptides can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, variants have functions similar to, identical to, or improved upon those of the polypeptide or peptide from which they are derived.

[0129] As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 3 consecutive amino acid residues, at least 4 consecutive amino acid residues, at least 5 consecutive amino acid residues, at least 6 consecutive amino acid residues, at least 7 consecutive amino acid residues, at least 8 consecutive amino acid residues, at least 9 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 11 consecutive amino acid residues, at least 12 consecutive amino acid residues, at least 13 consecutive amino acid residues, at least 14 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 16 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 30 consecutive amino acid residues, or more. In a specific embodiment, the fragment of the polypeptide retains at least one function of the polypeptide.

[0130] As used herein, the term "epitope" refers to a portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein, referred to as linear epitopes or conformational epitopes, respectively. Epitopes formed from consecutive amino acids are typically retained upon protein denaturation, while epitopes formed from ternary folding are typically lost upon protein denaturation. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation.

[0131] As used herein, the term "epitaphide" refers to a peptide on an antigen that can serve as an epitope. In some cases, an epitope peptide alone can be specifically recognized / bound by an antibody targeting that epitope. In other cases, it may be necessary to fuse the epitope peptide with a carrier protein so that the epitope peptide can be recognized by a specific antibody. As used herein, the term "carrier protein" refers to a protein that can act as a carrier for an epitope peptide, i.e., it can insert the epitope peptide at a specific location (e.g., inside the protein, at the N-terminus, or at the C-terminus) so that the epitope peptide can be presented and thus recognized by antibodies or the immune system. Such carrier proteins are well known to those skilled in the art, including, for example, HPV L1 protein (where an epitope peptide can be inserted between amino acids 130-131 or 426-427 of the protein, see Slupetzky, K. et al., J Gen Virol, 2001, 82:2799-2804; Varsani, A. et al., J Virol, 2003, 77:8386-8393), HBV core antigen (where an epitope peptide can replace amino acids 79-81 of the protein, see Koletzki, D. et al. Biol Chem, 1999, 380:325-333), and marmot hepatitis virus core protein (where an epitope peptide can replace amino acids 79-81 of the protein, see Sabine). J.Virol. 1998, 72(6): 4997), CRM197 protein (which can link epitope peptides to the N-terminus or C-terminus of the protein or fragments thereof). Optionally, a linker can be used between the epitope peptide and the carrier protein to facilitate the folding of both.

[0132] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding, but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The allocation of amino acids in each region or domain can follow the definitions in 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.

[0133] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residue in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains 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, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0134] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat, Chothia, or IMGT numbering system. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the IMGT numbering system.

[0135] As used herein, the term “framework region” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0136] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0137] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety”. See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabody, single-domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), probody, and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0138] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain is also included when the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0139] As used herein, the term “Fd” refers to an antibody fragment consisting of VH and CH1 domains; the term “dAb fragment” refers to an antibody fragment consisting of VH domains (Ward et al., Nature 341:544 546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; the term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term “Fab' fragment” refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0140] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0141] As used herein, the term "Fc" refers to an antibody fragment formed by the disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0142] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some embodiments of the present invention, scFv can form di-scFv, which refers to two or more individual scFvs linked together to form an antibody. In some embodiments of the present invention, scFv can form (scFv)2, which refers to two or more individual scFvs linked together in parallel to form an antibody.

[0143] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0144] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0145] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0146] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)). In some embodiments, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a mouse antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0147] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase sequence homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Typically, at least one or two, but usually all three (heavy and / or light immunoglobulin chains) of the receptor CDR of the humanized antibody are replaced by donor CDRs. The immunoglobulin providing the CDR is referred to as the "donor," and the immunoglobulin providing the framework is referred to as the "receptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., rabbit) antibody, and the receptor framework can be a naturally occurring human framework, or a sequence having approximately 85%, 90%, 95%, 99%, or higher sequence identity compared to it. Humanized antibodies typically retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, etc. Donor antibodies can be antibodies from mice, rats, rabbits, or non-human primates that have the desired properties (e.g., antigen specificity, affinity, reactivity, etc.).

[0148] The chimeric or humanized antibodies of this invention can be prepared based on the sequence of monoclonal antibodies produced by immunized animals (e.g., mice). The DNA encoding the heavy and light chains can be obtained from targeted hybridomas or specific B cells from immunized animals and engineered using standard molecular biology techniques to contain human immunoglobulin sequences.

[0149] To prepare chimeric antibodies, methods known in the art can be used to ligate the variable region of an immunoglobulin from an immunized animal (e.g., a mouse) to the constant region of a human immunoglobulin (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). For example, DNA encoding VH can be operatively ligated to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferred to be an IgG1 or IgG4 constant region. For example, DNA encoding VL can be operatively ligated to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant regions are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, USDA Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. Light chain constant regions can be κ or λ constant regions, but κ constant regions are generally preferred.

[0150] To prepare humanized antibodies, the CDR region of an immunized animal (e.g., a mouse) can be transplanted into a human frame sequence using methods known in the art (see Winter's U.S. Patent No. 5,225,539; Queen et al.'s U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). The human frame sequence can be derived, for example, from a germline antibody gene. The term "germline antibody gene" refers to a sequence in the genome of an organism that encodes an immunoglobulin and has not undergone a maturation process that would result in genetic rearrangements and mutations leading to the expression of a specific immunoglobulin. "Heavy chain germline genes" refer to germline antibody genes or gene fragments encoding the heavy chain of immunoglobulins, including the V gene (variable), D gene (diversity), J gene (joining), and C gene (constant). Similarly, "light chain germline genes" refer to germline antibody genes or gene fragments encoding the light chain of immunoglobulins, including the V gene (variable), J gene (joining), and C gene (constant). In this invention, the amino acid sequence encoded by the germline antibody gene or germline antibody gene fragment is also called a "germline sequence," with the amino acid sequence encoded by the heavy chain germline gene being called the heavy chain germline sequence, and the amino acid sequence encoded by the light chain germline gene being called the light chain germline sequence. Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from professional databases (e.g., IMGT, UNSWIg, NCBI, or VBASE2).

[0151] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K0) of that interaction. D () indicates. In this invention, the term "K" is used. D"" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. The specific binding properties between the two molecules can be determined using methods known in the art, such as surface plasmon resonance (SPR) in a BIACORE instrument.

[0152] As used herein, the detectable markers described in this invention can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridine esters, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), thermal markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers.

[0153] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0154] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, 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, or human cells.

[0155] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). 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 and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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).

[0156] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0157] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means 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 adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art as being capable of stabilizing the desired activity of the active ingredient in the pharmaceutical product, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable fluids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0158] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when administered to the body along with or before an antigen, 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 adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal studies. Aluminum hydroxide adjuvant is more frequently used in clinical trials.

[0159] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., HRV infection) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0160] As used herein, the term "subject" refers to a mammal, such as a human. In some embodiments, the subject (e.g., a human) has HRV infection or a disease associated with HRV infection, or is at risk of having such a disease.

[0161] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., HRV infection) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., HRV infection); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0162] As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment to bind to antigenic proteins on a virus, thereby preventing the virus from infecting cells and / or maturing and / or releasing viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent viral amplification, thereby inhibiting or eliminating viral infection. Attached Figure Description

[0163] Figure 1The peptide 2C01 was shown. Figure 1 A) 2CO2 ( Figure 1 B) and 2CO3 ( Figure 1 C) Results of sequence conservation analysis in 117 virus strains.

[0164] Figure 2 The SDS-PAGE electrophoresis results of the fusion proteins C149-2C(aa 12-20), C149-2C(aa 151-163), C149-2C(aa 186-193), C149-2C(aa 149-165), C149-2C(aa 147-167), and C149-2C(aa 145-169) are shown.

[0165] Figure 3 Electron micrographs of virus-like particles assembled from recombinant proteins C149-2C(aa 12-20), C149-2C(aa 151-163), C149-2C(aa 186-193), C149-2C(aa 149-165), C149-2C(aa 147-167), and C149-2C(aa 145-169).

[0166] Figure 4 The image shows an OD (450 / 620) intensity rectangle plot of the binding affinity between the monoclonal antibody 9A5 screened by ELISA and peptides 2C01, 2C02 and 2C03.

[0167] Figure 5 The image shows an intensity rectangle plot of OD (450 / 620) of the binding affinity between the monoclonal antibody 9A5-HRP screened by ELISA and the proteins C149-2C(aa 151-163), C149-2C(aa 12-20), and C149-2C(aa 186-193).

[0168] Figure 6 This is an immunofluorescence image of H1-HeLa cells infected with rhinovirus using monoclonal antibody 9A5.

[0169] Figure 7 The number of spots on virus-infected H1-Hela cells was shown by ELISPOT assay using monoclonal antibody 9A5.

[0170] Sequence information

[0171] The descriptions of the sequences involved in this application are provided in the table below.

[0172] Table 1: Sequence Information

[0173]

[0174]

[0175]

[0176]

[0177] Note: R = A or G; K = G or T

[0178] Instructions on the Preservation of Biological Materials

[0179] This invention relates to the following biological materials that have been deposited at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China):

[0180] Hybridoma cell line 9A5, with accession number CCTCC No. C2021141, deposited on May 28, 2021, at Luojia Mountain, Wuchang District, Wuhan (postcode 430072). Detailed Implementation Plan

[0181] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0182] Example 1: Design of highly conserved peptides for human rhinovirus

[0183] In this embodiment, the amino acid sequences of human rhinovirus protein 2C from different serotypes were analyzed for homology using Clustal software. The virus strains used included 117 strains such as HRV-A1B, HRV-A2, HRV-B14, and HRV-C15, as detailed in Table 2. Homology analysis identified three highly conserved peptides located in human rhinovirus protein 2C: peptide 2C01 (aa 151-163): YSLPPDPKYFDGY (SEQ ID NO:1); peptide 2C02 (aa 12-20): CNAAKGLEW (SEQ ID NO:2); and peptide 2C03 (aa 186-193): FCQMVSTT (SEQ ID NO:3). The amino acid sequences of these three peptides (with lengths of 13, 9, and 8 amino acids, respectively) showed high conservation and homology in all 117 virus strains. The sequence conservation analysis results for these three peptides are shown below. Figure 1 As shown.

[0184] Table 2. Information on virus strains selected for amino acid sequence alignment

[0185]

[0186]

[0187] The aa 151-163 of the non-structural protein 2C of the above 117 viral strains are provided in Table 3 (SEQ ID NOs: 1, 30-34).

[0188] Table 3. Amino acid sequences of aa 151-163 of non-structural protein 2C of strain 117 viruses.

[0189]

[0190]

[0191] Example 2: Preparation of highly conserved human rhinovirus polypeptide antiserum

[0192] Based on the amino acid sequences of the three peptides screened in Example 1, three polypeptides (synthesized by Genscript Biotech Inc.) were designed and synthesized, designated as: 2C01 (hereinafter also referred to as 2C(aa 151-163), SEQ ID NO:1), 2C02 (hereinafter also referred to as 2C(aa 12-20), SEQ ID NO:2), and 2C03 (hereinafter also referred to as 2C(aa 186-193), SEQ ID NO:3). The synthesized polypeptides were used to immunize 6-week-old BALB / c female mice. Each polypeptide was used to immunize five mice in parallel (numbered: mice 1 to mice 5), with an immunization dose of 100 μg per mouse. The initial immunization was mixed with Freund's complete adjuvant (purchased from Sigma, catalog number: F5881), followed by booster immunizations every two weeks, each time mixed with Freund's incomplete adjuvant (purchased from Sigma, catalog number: F5881). A total of three booster immunizations were administered, with blood collected on day 14 after the third booster immunization.

[0193] Example 3: Preparation of antiserum against highly conserved human rhinovirus polypeptide variants

[0194] This example analyzes the amino acid sequences (aa) 151-163 of the non-structural protein 2C of 117 human rhinovirus strains, and designs and synthesizes five site-mutated and three sequence-extended peptide variants (synthesized by Genscript Biotech Inc.): peptide variant 2C04 (aa 151-163, Y159H): YSLPPPDPKHFDGY (SEQ ID NO: 30); peptide variant 2C05 (aa151-163, D156A): YSLPPAPKYFDGY (SEQ ID NO: 31); peptide variant 2C06 (aa151-163, D156S): YSLPPSPKYFDGY (SEQ ID NO: 32); peptide variant 2C07 (aa151-163, D156N & Y159H): YSLPPNPKHFDGY (SEQ ID NO: 30). NO: 33); peptide variant 2C08 (aa151-163, D156N): YSLPPNPKYFDGY (SEQ ID NO: 34); peptide variant 2C09 (aa149-165): QVYSLPPDPKYFDGYDQ (SEQ ID NO: 35); peptide variant 2C10 (aa147-167): EAQVYSLPPDPKYFDGYDQQQ (SEQ ID NO: 36); peptide variant 2C11 (aa145-169): TQEAQVYSLPPDPKYFDGYDQQQVV (SEQ ID NO: 37). Eight synthetic peptide variants were used to immunize 6-week-old BALB / c female mice. Each peptide was used to immunize five mice in parallel (numbered 1 to 5). The immunization dose was 100 μg per mouse. The initial immunization was mixed with Freund's complete adjuvant (purchased from Sigma, catalog number: F5881). Boosters were administered every two weeks thereafter, with Freund's incomplete adjuvant (purchased from Sigma, catalog number: F5881) mixed with each booster. A total of three booster doses were administered. Blood samples were collected on day 14 after the third booster dose.

[0195] Example 4: Identification of the binding activity of highly conserved human rhinovirus peptide antiserum to human rhinovirus.

[0196] The polypeptide antiserum obtained in Examples 2 and 3 was used to detect different representative strains of human rhinovirus groups A, B, and C using indirect immunofluorescence assay. The representative human rhinovirus strains were derived from: group A1B (HRV-A1B), group A2 (HRV-A2), group A9 (HRV-A9), group A16 (HRV-A16), group A21 (HRV-A21), group A23 (HRV-A23), group A29 (HRV-A29), group A34 (HRV-A34), group A36 (HRV-A36), group A40 (HRV-A40), group A43 (HRV-A43), and group A49 (HRV-A43). The GenBank accession numbers for these 24 representative human rhinoviruses are shown in Table 4. Among them, HRV-A49, HRV-A54, HRV-A59, HRV-A77, HRV-A78, HRV-A89, HRV-B5, HRV-B6, HRV-B14, HRV-B17, HRV-C11, HRV-C15, and HRV-C23 were obtained in our laboratory through the construction of infectious clones and virus rescue; the rest were purchased from ATCC.

[0197] Table 4 Virus GenBank Login Numbers

[0198]

[0199] The steps of the immunofluorescence method are as follows:

[0200] H1-HeLa cells (purchased from ATCC) cultured in DMEM (purchased from GIBCO) medium containing 10% FBS (purchased from ABW) were sputtered at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 24-well cell culture plates; after cell adhesion, 10 cells were infected per well. 3 TCID 50Human rhinovirus; after 20 hours, the cell culture supernatant was aspirated, and 500 μL of fixative (PBS solution containing 4% paraformaldehyde) was added to each well for fixation. The cells were incubated at room temperature in the dark for 30 minutes; after 30 minutes, the fixative was aspirated, and 500 μL of 0.3% Triton X-ray solution was added to each well. Cells were permeated with X-100 solution and incubated at room temperature for 10 min. After 10 min, the permeabilization solution was aspirated, and cells were washed three times with PBS solution, which was then aspirated. A suitable amount of blocking solution (PBS solution containing 2% BSA) was added for blocking, and the cells were incubated at 37°C for 1 h. After 1 h, the blocking solution was aspirated, and 1:100 diluted polypeptide immunosera was added, and the cells were incubated at 37°C for 1 h. After 1 h, the serum was aspirated, and cells were washed three times with PBS solution, which was then aspirated. FITC-conjugated goat anti-mouse antibody was added at a 1:500 dilution, and the cells were incubated at 37°C for 30 min. After 30 min, the antibody was aspirated, and cells were washed three times with PBS solution, which was then aspirated. DAPI staining solution was added at a 1:2000 dilution and incubated at room temperature in the dark for 5 min. After 5 min, the DAPI staining solution was aspirated, and cells were washed three times with PBS solution, which was then aspirated. The cells were mounted and observed under a fluorescence microscope. The results are shown in Table 5.

[0201] Table 5. Immunofluorescence binding activity of peptide antiserum to H1-HeLa cells infected with rhinovirus.

[0202]

[0203] Note: + indicates positive; - indicates negative.

[0204] Table 5 shows that antiserum against peptide 2C01 and its variants 2C04–2C11 can cross-bind to H1-HeLa cells infected with 24 representative human rhinoviruses, showing positive green fluorescence; while antiserum against peptides 2C02 or 2C03 cannot cross-bind to H1-HeLa cells infected with 24 representative human rhinoviruses, and does not show green fluorescence. This result indicates that: (1) anti-peptide 2C01 (i.e., peptide 2C(aa151-163)) and its variants (i.e., 2C04(aa151-163)) are effective against peptides 2C01 and 2C03, respectively. Immune sera against 151-163, Y159H), 2C05 (aa151-163, D156A), peptide variants 2C06 (aa151-163, D156S), 2C07 (aa151-163, D156N&Y159H), 2C08 (aa151-163, D156N), 2C09 (aa149-165), 2C10 (aa147-167), and 2C11 (aa145-169) can specifically cross-bind to different subtypes of human rhinovirus; peptide 2C01 and its peptide variants may contain universal affinity epitopes; (2) Immune sera against peptide 2C02 or 2C03 cannot specifically cross-bind to different subtypes of human rhinovirus; peptide 2C02 and 2C03 do not contain universal affinity epitopes. These results also indicate that not all conserved peptides contain universal affinity epitopes. On the contrary, in many cases, conserved peptides (such as 2CO2 and 2CO3) cannot be used as universal affinity epitopes and cannot induce the production of universal affinity antibodies.

[0205] Example 5: Antiserum of peptide 2C01 was used to detect human rhinovirus infection and titer.

[0206] When the dilution is sufficient, rhinovirus infection of cells can be followed by enzyme-linked immunospot assay (ELISA) to produce a signal detectable by an Elispot instrument. The number of positive cells with the signal, i.e., the number of infected cells, can be considered as the number of infectious units of rhinovirus used in the infection experiment, thus allowing the detection of rhinovirus titer using a serial dilution spot counting method. In this example, three representative rhinoviruses (HRV-A2, HRV-B14, and HRV-C15) from Example 4 were selected for titer determination. The specific steps are as follows:

[0207] H1-HeLa cells were seeded in 96-well cell culture plates at a density of 1×10⁶ cells / well. 4Cells / well, cultured in DMEM medium containing 2% FBS. Incubate at 33℃ for 10 h. Take one portion of the original virus culture solution and serially dilute it 5-fold with serum-free DMEM medium to obtain 6 concentration gradients of virus dilution per 100 μL of medium: 100 μL, 20 μL, 4 μL, 0.8 μL, 0.16 μL, and 0.032 μL. Add 100 μL of each gradient of virus dilution to H1-HeLa cells pre-coated in 96-well cell culture plates, with 3 replicates per well for each gradient. Incubate the 96-well plates at 33℃. After 14–24 hours of incubation, detect the virus using an enzyme-linked immunospot assay.

[0208] (1) Remove the culture medium from each well, add 100 μL of fixative (PBS solution containing 0.5% formaldehyde) to each well, and let stand at room temperature in the dark for 1 h;

[0209] (2) After 1 hour, remove the fixative, add 100 μL of 1% Triton X-100 solution to each well to permeate the cells, and let stand at room temperature for 0.5 hours;

[0210] (3) After 0.5 h, remove the permeation solution, add PBST solution to each well to wash the cells three times, and then remove the PBST solution.

[0211] (4) Add 100 μL of the antiserum of polypeptide 2C01 prepared in Example 2 to each well. The polypeptide antiserum is diluted 3000 times with enzyme diluent (buffer solution of 20 mM PBS + 0.5% casein + 2% gelatin + 0.1% preservative, the same below) and placed in 37°C for 1 h to react.

[0212] (5) After 1 hour, the serum was removed, and PBST solution was added to each well to wash the cells three times. Then the PBST solution was removed.

[0213] (6) Add 100 μL of HRP-conjugated goat anti-mouse antibody diluted 1:5000 to each well and incubate at 37°C for 30 min.

[0214] (7) After 30 min, remove the reaction solution, add PBST solution to each well to wash the cells three times, and then remove the PBST solution; add 100 μL TMB color development solution to each well and place it at 37℃ for 15 min for color development.

[0215] (8) After 15 minutes, remove the developing solution from the plate to stop the color development.

[0216] (9) Start the ELISPOT (Series 3B) spot detection instrument. Place the cell culture plate after the colorimetric reaction on the instrument's sampling plate and use the instrument to detect the number of blue cells in each well, which is the number of infected cells. For detailed operating procedures of ELISPOT, please refer to the instrument's instruction manual.

[0217] H1-Hela cells that had not undergone infection experiments were used as a control group.

[0218] Rhinovirus titer calculation method: Infectious titer (IU / mL) = Number of infected cells in the test well / Volume of virus stock solution in the test well (mL). Where, Number of infected cells in the test well = Average number of chromogenic cells in the test wells - Average number of chromogenic cells in the negative wells. In the above formula, "Number of infected cells in the test wells" and "Volume of virus stock solution in the test wells" are selected from the test wells with an infected cell count closest to 50 and not less than 50, along with their corresponding rhinovirus stock solution volume. The results are shown in Table 6. The volume of virus stock solution corresponds to the number of infected cells (an average of 3 wells), which can be substituted into the above formula to calculate the viral titer.

[0219] Table 6. Relationship between the dosage of rhinovirus and the number of cells infected in the corresponding well.

[0220]

[0221] Based on the above results, the viral titers of the three representative rhinoviruses, HRV-A2, HRV-B14, and HRV-C15, are 9.375 × 10⁻⁶. 4 (IU / mL), 8.625×10 4 (IU / mL) and 7.875×10 4 (IU / mL). The results of this example show that the antiserum of the human rhinovirus universal affinity peptide 2C01 can determine the infection titers of different representative rhinoviruses.

[0222] Example 6: Preparation of antiserum against human rhinovirus

[0223] Immunogen preparation: Prepare 3 10cm cell culture dishes and seed them with H1-HeLa cells. When the H1-HeLa cells in the culture dishes reach 80% confluence, replace the culture medium with serum-free DMEM medium and seed with HRV-A2, HRV-B14, or HRV-C15 viruses, respectively, at a viral dose of 10. 5 TCID 50 Gently shake to ensure even distribution and incubate at 33°C. When complete cytopathic effects occur, collect the cells and supernatant, and repeatedly freeze-thaw three times in a -80°C ultra-low temperature freezer. Centrifuge at 25000g for 10 min at 4°C to remove cell debris; store the centrifuged viral supernatant at -80°C for later use.

[0224] Primary immunization of mice: 6-8 week old female BALB / c mice were immunized at multiple sites using the above-mentioned immunogens, including subcutaneous injections in the back, groin, footpads, and limbs, with a dose of 500 μL per mouse per injection. Five mice (numbered 1 to 5) were immunized in parallel with each virus. The primary immunization was mixed with Freund's complete adjuvant, followed by booster immunizations every two weeks, each time mixed with Freund's incomplete adjuvant, for a total of two booster injections. On day 14 after the third immunization, 200 μL of periorbital venous blood was collected for titer determination.

[0225] Example 7: Antiserum of peptide 2C01 used to detect the titer of neutralizing antibodies against human rhinovirus.

[0226] In this embodiment, antiserum containing the affinity peptide 2C01 was used in an enzyme-linked immunospot assay to detect the neutralizing antibody titer of human rhinovirus. This method has the advantages of high efficiency and accuracy, and is more conducive to improving work efficiency, making it more suitable for high-throughput detection of different rhinovirus subtypes. The specific method is described below:

[0227] 1. Seed H1-HeLa cells into 96-well cell culture plates (1×10⁻⁶ cells / well). 4 / hole).

[0228] 2. After 10 hours, the samples to be tested (monoclonal antibody samples, serum samples, ascites samples, cell culture supernatant samples, cell lysis buffer samples, etc.) were serially diluted with serum-free DMEM (starting from the stock solution, perform 2-fold serial dilutions, dilute 8 times, and perform 3 replicates for each dilution).

[0229] 3. Take 50 μL of the sample to be tested from each well and dilute it with 50 μL of rhinovirus diluted in serum-free DMEM (10). 4 TCID 50 Mix. Incubate at 37°C for 2 hours.

[0230] 4. Take the culture medium from the 96-well cell culture plate pre-coated with H1-HeLa cells, add the incubated mixture, incubate at 33°C for 14–24 h, and then perform an enzyme-linked immunospot reaction.

[0231] 5. Remove the culture medium from each well, add 100 μL of fixative (PBS solution containing 0.5% formaldehyde) to each well, and let stand at room temperature in the dark for 1 hour.

[0232] 6. Remove the fixative, add 100 μL of 1% Triton X-100 solution to each well to permeate the cells, and incubate at room temperature for 30 min.

[0233] 7. Remove the permeabilizing solution, add PBST solution to each well to wash the cells three times, and then discard the PBST solution.

[0234] 8. Add 100 μL of the antiserum of polypeptide 2C01 prepared in Example 2 to each well. The polypeptide antiserum is diluted 3000 times with enzyme dilution and then placed in 37°C for 1 h.

[0235] 9. After 1 hour, aspirate the serum, add PBST solution to each well to wash the cells three times, and then aspirate the PBST solution.

[0236] 10. Add 100 μL of HRP-conjugated goat anti-mouse antibody diluted 1:5000 to each well and incubate at 37°C for 30 min.

[0237] 11. After 30 min, remove the reaction solution, add PBST solution to each well to wash the cells three times, and then discard the PBST solution; add 100 μL TMB chromogenic solution to each well and incubate at 37℃ for 15 min.

[0238] 10. After 15 minutes, remove the developing solution from the plate to stop the color development process.

[0239] 11. Start the ELISPOT speckle detection instrument and count the number of blue cells in each well, which represents the number of infected cells. For detailed operating procedures, please refer to the ELISPOT instrument's instruction manual.

[0240] Meanwhile, H1-HeLa cells that had not undergone infection experiments were set up as negative wells, and H1-HeLa cells that had only undergone viral infection were set up as positive wells, with 5 wells in each 96-well plate.

[0241] The highest dilution factor achieving an infection inhibition rate of 50% or higher was used as the neutralizing titer of the sample. Infection inhibition rate for each sample = (1 - number of infected cells in sample wells / (total number of stained cells in positive wells / 5 - total number of stained cells in negative wells / 5)) × 100%. Number of infected cells in sample wells = average number of stained cells in sample wells - total number of stained cells in negative wells / 3.

[0242] In this embodiment, six mouse polyclonal antibody sera prepared in Example 6 were randomly selected for the detection of neutralizing antibodies against human rhinovirus. The results of the neutralizing antibody detection (as shown in Table 7) show that the antiserum of peptide 2C01 can be used to detect the titer of neutralizing antibodies against human rhinovirus; the mouse polyclonal antibody sera can neutralize HRV-A2, HRV-B14, or HRV-C15 viruses.

[0243] Table 7. Results of detection of neutralizing antibody titers in mouse polyclonal antibody serum

[0244]

[0245] Example 8: Fusion expression of human rhinovirus universal affinity epitope peptide with HBcAg protein

[0246] 1. Construction of the pTO-T7-C149 expression vector

[0247] Those skilled in the art know that HBcAg has strong T-cell immunogenicity. The fusion of exogenous peptides into the MIR (mayor immunodominant region, aa 78-83) within HBcAg does not alter its particle polymerization characteristics, antigenic activity, or immunogenicity, and can expose exogenous epitopes on the particle surface (see Fu et al., (2009). Comparative immunogenicity evaluations of influenza A virus M2 peptide as recombinant virus like particle or conjugate vaccines in mice and monkeys. Vaccine 27(9): 1440-1447; Jin et al., (2007). Protective immune responses against foot-and-mouth disease virus by vaccination with a DNA vaccine expressing virus-like particles. Viral Immunol 20(3): 429-440; and Yin et al., (2010). Hepatitis B viruscore protein as an epitope vaccine carrier: a review. Sheng Wu Gong Cheng XueBao 26(4): 431-438.).

[0248] Taking advantage of the property that HBcAg 1-149aa can be expressed in Escherichia coli in the form of virus-like particles, the inventors inserted these 149 aaa into the E. coli expression vector pTO-T7 (the preparation steps of this vector can be found in the reference Luo Wenxin, Zhang Jun, Yang Haijie et al., Construction and preliminary application of a prokaryotic high-efficiency expression vector with enhancer [J]. Chinese Journal of Biotechnology, 2000, 16(5): 578-581) (other commonly used E. coli expression vectors in this field can also be used). The 79th and 80th amino acids (corresponding to the amino acid PA) of the HBcAg B cell dominant epitope segment were replaced by a linker (SEQ ID NO: 8). GGATCC was introduced at 265nt-270nt and GAATTC was introduced at 274nt-279nt (relative to pTO-T7-C149) to introduce two restriction endonuclease recognition sites (BamHI and EcoRI) to construct the mutant HBc expression plasmid pTO-T7-C149. The foreign protein is inserted at the 93rd amino acid of C149.

[0249] Primers were designed for the non-structural proteins 2C(aa 151-163) (2C01, SEQ ID NO:1), 2C(aa 12-20) (2C02, SEQ ID NO:2), and 2C(aa 186-193) (2C03, SEQ ID NO:3) of human rhinovirus (W10(HRV-C15)) (primer sequences are shown in Table 8). The resulting fusion proteins were inserted into the HBcAg protein and named C149-2C(aa151-163) or C149-2C01 (SEQ ID NO:5), C149-2C(aa 12-20) or C149-2C02 (SEQ ID NO:6), and C149-2C(aa 186-193) or C149-2C03 (SEQ ID NO:7), respectively. The amino acid sequence of the non-structural protein 2C of human rhinovirus (W10(HRV-C15)) is shown in SEQ ID NO:9.

[0250] Table 8: Cloning primer sequences used for fusion proteins

[0251]

[0252] The upstream and downstream primers for synthesizing the 2C region were cross-linked to obtain a fragment with sticky BamHI and EcoRI ends (for specific cloning procedures, refer to the reference, Xu L, et al. 2014. Protection against Lethal Enterovirus 71 Challenge in Mice by a Recombinant Vaccine Candidate Containing a Broadly Cross-Neutralizing Epitope within the VP2 EF Loop. Theranostics 4:498-513.). 1 μL of each primer was added to 48 μL of ddH2O, mixed, and incubated at 94 °C for 4 min, then at 70 °C for 10 min, and slowly cooled to room temperature. Simultaneously, the vector pTO-T7-C149 was double-digested with BamHI and EcoRI, and the recovered vector was ligated to the cross-linked fragment. The ligand was transformed into E. coli ER2566 (purchased from NEB) for expression identification and plasmid restriction enzyme digestion identification (with pTO-T7-C149 as a blank control). The plasmids that were correctly identified were inserted into the desired target fragment and named C149-2C (aa 151-163) or C149-2C01 (SEQ ID NO:5), C149-2C (aa 12-20) or C149-2C02 (SEQ ID NO:6), C149-2C (aa 186-193) or C149-2C03 (SEQ ID NO:7), respectively.

[0253] ER2566 bacteria expressing the above plasmids and ER2566 bacteria containing the pTO-T7-C149 empty plasmid were cultured at 37°C with shaking until OD600 reached approximately 0.5. Then, they were transferred at a 1:1000 ratio to 500 mL LB (containing kanamycin sulfate) for expansion. When OD600 reached approximately 0.8, 500 μL of IPTG was added, and the cells were induced at 30°C for 6 h. Cells were collected at 4°C and centrifuged at 8000 rpm for 10 min. The supernatant was discarded, and the cell pellet was resuspended in 10 mL / bottle of TB8.0 buffer. The cells were then sonicated in an ice-water bath. Sonication conditions: operating time: 3 min / bottle; pulse: 2 sec, 4 sec pause; output power: 55%. The cells were centrifuged at 12000 rpm for 10 min, and the supernatant was retained. SDS-PAGE analysis confirmed that all fusion proteins were expressed in the supernatant (SDS-PAGE results are shown in the figure). Figure 2 (As shown).

[0254] Because the expression supernatant contained many contaminating proteins, the inventors used the following method to purify the protein and promote its spontaneous assembly into particles: The supernatant obtained after sonication was placed in a 65°C water bath for heat denaturation for 30 min, followed by centrifugation at 12000 rpm for 10 min, and the supernatant was collected. An equal volume of the supernatant was mixed with a saturated ammonium sulfate solution, incubated in an ice-water bath for 30 min, and then centrifuged at 12000 rpm for 10 min, retaining the precipitate. The purified protein was then subjected to PBS buffer to promote spontaneous assembly into particles, which were then observed under an electron microscope. The successfully assembled particles were uniform hollow spheres, as shown in the electron microscopy results. Figure 3 As shown.

[0255] The purified and assembled fusion protein was used to immunize 6-week-old BALB / c female mice. Five mice (numbered 1 to 5) were immunized in parallel for each displayed peptide. The immunization dose was 100 μg / mouse. The initial immunization was mixed with Freund's complete adjuvant, and booster immunizations were performed every two weeks thereafter, with Freund's incomplete adjuvant mixed in each booster immunization. A total of three booster immunizations were performed. Blood was collected on day 14 after the third booster immunization.

[0256] Example 9: Binding activity of immune serum containing fusion proteins C149-2C01, C149-2C02, and C149-2C03 to human rhinovirus

[0257] The antisera containing the C149-2C01, C149-2C02, and C149-2C03 fusion proteins obtained in Example 8 were used to detect different representative rhinoviruses using immunofluorescence. The immunofluorescence method was performed according to Example 4, and the experimental results are shown in Table 9.

[0258] Table 9. Immunofluorescence binding activity of C149-2C01, C149-2C02, and C149-2C03 immune sera to H1-HeLa cells infected with rhinovirus.

[0259]

[0260] Note: + indicates positive; - indicates negative.

[0261] The results showed that the C149-2C01 fusion protein peptide antiserum could cross-bind with HRV-A1B, HRV-A2, HRV-A9, HRV-A16, HRV-A21, HRV-A23, HRV-A29, HRV-A34, HRV-A36, HRV-A40, HRV-A43, HRV-A49, HRV-A54, HRV-A59, HRV-A77, HRV-A78, and HRV-A 24 representative rhinoviruses, including HRV-B5, HRV-B6, HRV-B14, HRV-B17, HRV-C11, HRV-C15, and HRV-C23, were tested. Infected H1-HeLa cells showed green fluorescence, while uninfected H1-HeLa cells (controls) showed no reaction. Furthermore, the antiserum containing the C149-2C02 and C149-2C03 fusion proteins failed to specifically cross-bind to different subtypes of human rhinovirus. This result demonstrates that the carrier protein can present the epitope peptide of this invention in the form of virus-like particles. Based on these experimental results, the 2C01 peptide and its fusion protein C149-2C01 were selected for further experiments.

[0262] Example 10: Fusion expression of a human rhinovirus universal affinity epitope peptide elongation variant with HBcAg protein

[0263] Following the method described in Example 8, primers were designed for the extended variants of the human rhinovirus (W10(HRV-C15)) nonstructural protein 2C01: 2C(aa149-165)(2C09, SEQ ID NO: 35), 2C(2C10, aa147-167)(SEQ ID NO: 36), and 2C(2C11, aa145-169)(SEQ ID NO: 37) (primer sequences are shown in Table 10). The resulting fusion proteins, inserted into the HBcAg protein, were named C149-2C(aa149-165) or C149-2C09 (SEQ ID NO: 49); C149-2C(aa147-167) or C149-2C10 (SEQ ID NO: 50); and C149-2C(aa145-169) or C149-2C11 (SEQ ID NO: 37), respectively. NO:51).

[0264] In addition, fusion proteins C149-2C04(SEQ ID NO:44), C149-2C05(SEQ ID NO:45), C149-2C06(SEQ ID NO:46), C149-2C07(SEQ ID NO:47), and C149-2C08(SEQ ID NO:48) containing the inserted peptides 2C04(SEQ ID NO:30), 2C05(SEQ ID NO:31), 2C06(SEQ ID NO:32), 2C07(SEQ ID NO:33), or 2C08(SEQ ID NO:34) were obtained by the same method.

[0265] Table 10: Cloning primer sequences used for extending variant fusion proteins

[0266]

[0267]

[0268] The upstream and downstream primers of the synthesized 2C extended variant region were interleaved, and the C149-2C extended variant fusion protein was expressed and identified according to the fusion protein expression method in Example 8. The resulting fusion protein expression supernatant was then analyzed by SDS-PAGE, and all extended variant fusion proteins were expressed in the supernatant (SDS-PAGE results are shown below). Figure 2 (As shown).

[0269] The obtained fusion protein was purified using the method described in Example 8. The purified protein was then placed in PBS buffer to promote spontaneous assembly into particles. Electron microscopy revealed that the successfully assembled particles were uniform hollow spheres. The electron microscopy results are shown below. Figure 3 As shown.

[0270] The purified and assembled fusion protein was used to immunize 6-week-old BALB / c female mice. Five mice (numbered 1 to 5) were immunized in parallel for each displayed peptide. The immunization dose was 100 μg / mouse. The initial immunization was mixed with Freund's complete adjuvant, and booster immunizations were performed every two weeks thereafter, with Freund's incomplete adjuvant mixed in each booster immunization. A total of three booster immunizations were performed. Blood was collected on day 14 after the third booster immunization.

[0271] Example 11: Binding activity of immune serum containing fusion proteins C149-2C (aa 149-165), C149-2C (aa 147-167), and C149-2C (aa145-169) to human rhinovirus.

[0272] The obtained extended variant fusion proteins C149-2C (aa 149-165), C149-2C (aa 147-167), and C149-2C (aa 145-169) antisera were used to detect different representative rhinoviruses using immunofluorescence. The immunofluorescence method was performed according to Example 4, and the experimental results are shown in Table 11.

[0273] Table 11. Immunofluorescence binding activity of C149-2C (aa 149-165), C149-2C (aa 147-167), and C149-2C (aa 145-169) immune sera to H1-HeLa cells infected with rhinovirus.

[0274]

[0275]

[0276] Note: + indicates positive; - indicates negative.

[0277] The results prove that C149-2C(aa 149-165), C149-2C(aa 147-167), and C149-2C(aa The fusion protein peptide antiserums (145-169) were able to cross-bind to 24 representative rhinoviruses, including HRV-A1B, HRV-A2, HRV-A9, HRV-A16, HRV-A21, HRV-A23, HRV-A29, HRV-A34, HRV-A36, HRV-A40, HRV-A43, HRV-A49, HRV-A54, HRV-A59, HRV-A77, HRV-A78, HRV-A89, HRV-B5, HRV-B6, HRV-B14, HRV-B17, HRV-C11, HRV-C15, and HRV-C23. Infected H1-HeLa cells showed green fluorescence, while uninfected H1-HeLa cells (control) showed no reaction. This result indicates that not only can the C149-2C01 carrier protein present the epitope polypeptide of the present invention in the form of virus-like particles, but also the extended regions C149-2C(aa 149-165), C149-2C(aa 147-167), and C149-2C(aa 145-169) along its original region can present the variant extended epitope polypeptide of the present invention in the form of virus-like particles.

[0278] Example 12: Immunosal of fusion protein C149-2C01 was used to detect human rhinovirus infection and titer.

[0279] The C149-2C01 fusion protein antiserum obtained in Example 8 was used to detect the titers of rhinoviruses (HRV-A2, HRV-B14, and HRV-C15) using a serial dilution spot counting method. Specific procedures are detailed in Example 5, and the experimental results are shown in Table 12 below.

[0280] Table 12. Relationship between the amount of rhinovirus used for infection and the number of cells infected in the corresponding well.

[0281]

[0282] Based on the above results, the viral titers of the three representative rhinoviruses, HRV-A2, HRV-B14, and HRV-C15, are 7.375 × 10⁻⁶. 4 (IU / mL), 8.875×10 4 (IU / mL) and 1.538×10 5 (IU / mL). The results of this example show that antiserum against the fusion protein C149-2C01 can determine the infection titers of different representative rhinoviruses.

[0283] Example 13: Immunosal of fusion protein C149-2C01 was used to detect the titer of neutralizing antibodies against human rhinovirus.

[0284] In this example, using enzyme-linked immunospot assay, we used the C149-2C01 fusion protein antiserum obtained in Example 8 to detect the titer of human rhinovirus neutralizing antibodies. The specific method is described in Example 7.

[0285] In this embodiment, six mouse polyclonal antibody sera prepared in Example 6 were randomly selected for the detection of neutralizing antibodies against human rhinovirus. The results of the neutralizing antibody detection are shown in Table 13. The results show that the C149-2C01 immune serum can be used to detect the titer of neutralizing antibodies against human rhinovirus; the mouse polyclonal antibody sera can neutralize HRV-A2, HRV-B14, or HRV-C15 viruses.

[0286] Table 13. Results of detection of neutralizing antibody titers in mouse polyclonal antibody serum

[0287]

[0288] Example 14: Preparation of monoclonal antibodies against universal affinity epitope peptides against human rhinovirus

[0289] (1) Mouse immunization

[0290] 1. Preparation of immunogen: The fusion protein C149-2C01 prepared in Example 8 was used as the immunogen. Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations. No adjuvant was added for the last booster immunization 72 hours before fusion.

[0291] 2. Basic immunization of mice: Six- to eight-week-old female BALB / c mice were injected at multiple sites using the immunogen prepared in step 1 above, including subcutaneous injections in the back, groin, paw pads, and intramuscular injections in the limbs, with a dosage of 500 μL per mouse per injection. Booster immunizations were administered every two weeks. Before each immunization, 100–200 μL of periorbital venous blood was collected for titer determination. Immunization was stopped once the mouse serum titer reached a plateau, and cell fusion experiments were performed after a two-month rest period.

[0292] 3. A booster immunization 72 hours before cell fusion is crucial for stimulating an antibody response; therefore, mice should be immunized 72 hours prior to fusion. Mice were immunized by directly administering 100 μL of 0.5 mg / mL recombinant protein (C149-2C01) to the spleen. Before spleen immunization, mice were anesthetized with ether, the abdominal skin was opened to remove the spleen, and 100 μL of antigen was injected longitudinally along the spleen. The abdominal incision was then quickly sutured.

[0293] (2) Preparation and screening of fusion hybridoma cells

[0294] 1. Preparation of mouse macrophages: (i) Sacrifice a 6-week-old BALB / c mouse, rinse with tap water, and soak in 75% ethanol solution for 5 minutes. Place the mouse in a clean bench with its abdomen facing upwards. Lift the skin of the mouse's abdomen with forceps, make a small incision, and use large forceps to tear the skin upwards and downwards to fully expose the abdomen. (ii) Lift the peritoneum with sterile forceps, use a pair of scissors to make a small incision in the center of the peritoneum, and inject an appropriate amount of culture medium into the peritoneal cavity through the small incision using a 1 mL pipette. Carefully stir the culture medium in the peritoneal cavity with the pipette, and finally aspirate the culture medium into a centrifuge tube. (iii) Dissolve the peritoneal cell fluid in HAT culture medium or HT culture medium to a final concentration of 2 × 10⁻⁶. 5 / mL. (iv) Add the cells to a 96-well cell culture plate and incubate in an incubator. Alternatively, the cells can be directly mixed with fused cells and then added to a 96-well cell culture plate.

[0295] 2. Preparation of mouse thymocytes: (i) A BALB / c mouse approximately 13 days old was euthanized by cervical indwelling, rinsed with tap water, and immersed in 75% ethanol solution for 5 minutes. The mouse was placed in a clean bench with its abdomen facing upwards. (ii) The skin of the mouse's abdomen was lifted with forceps, and the outer skin of the abdomen and chest was cut open. (iii) The thoracic cavity was cut open with another pair of clean scissors, and the milky white thymus was removed with forceps. The thymus was ground and passed through a 200-mesh cell sieve to obtain thymus feeder cell solution.

[0296] 3. Preparation of mouse myeloma cells: (i) The mouse myeloma cell line Sp2 / 0-Ag14 (Sp2 / 0) is easy to culture and has a high fusion rate, making it the most ideal fusion cell line currently available. However, care should be taken to avoid over-dilution of this cell line (density below 3 × 10⁻⁶).5 Myeloma cells grow poorly under conditions of low concentration ( / mL) and alkaline pH (pH>7.3). (ii) Culture myeloma cells in 20% FBS RPMI-1640 medium (purchased from ATCC). When the cell number is 10... 4 ~10 6 At a density of 1 × 10⁶ / mL, cells exhibit logarithmic growth, appearing round, translucent, uniform in size, with clear edges, neatly arranged, and semi-densely distributed. Generally, this occurs during mid-logarithmic growth (approximately 1–5 × 10⁶ / mL). 5 / mL), which can be diluted and passaged at a ratio of 1:5 to 1:10. Select cells in the logarithmic growth phase with vigorous growth and good morphology for fusion. The viable cell count of the fused myeloma cells should be above 95%. (iii) Before fusion, transfer the myeloma cells from the culture flask to a centrifuge tube and wash 3 times with RPMI-1640 medium (1000 rpm, 5 min). Resuspend the cells in RPMI-1640 medium and count them. (iv) Generally, mouse myeloma cells are resuscitated starting 5 days before fusion. Each fusion requires approximately 6 flasks of 35cm³ cells. 2 Sp2 / 0 cells.

[0297] 4. Preparation of immune spleen cells: (i) Take the BALB / c mice to be fused prepared in step (1) above, remove the eyeballs to bleed them to death, collect the blood to make antiserum, which can be used as a positive control for antibody detection. Rinse the mice with tap water and soak them in 75% ethanol solution for 5 min, then place them on a dissecting plate in a laminar flow hood in a right lateral decubitus position. (ii) Open the abdominal cavity and remove the spleen, cut it into small pieces with scissors and place them on a 200-mesh cell sieve, then grind them with a grinding rod (syringe core). Add RPMI-1640 culture medium dropwise using a blowpipe. (iii) Add an appropriate amount of RPM-1640 culture medium, let stand for 3-5 min, and transfer 2 / 3 of the suspension into a 50 mL plastic centrifuge tube. Repeat the above process 2-3 times. (iv) Wash the cells 3 times with RPMI-1640 culture medium (centrifuge at 1000 rpm for 10 min). (v) Resuspend the cells in RPMI-1640 culture medium and count them.

[0298] 5. Preparation of hybridoma cells using PEG fusion accelerator: (i) Before fusion, equilibrate 1 mL of PEG-1500, 10 mL of RPMI-1640 serum-free medium, and 200 mL of complete medium to 37°C. (ii) Mix the prepared myeloma cells with spleen cells in a 50 mL centrifuge tube (1×10⁻⁶ cells / mL). 8 Spleen cells and 1×10 7Myeloma cells (approximately 10:1) were centrifuged at 1500 rpm for 8 min, and the bottom of the tube was gently tapped to loosen the cells into a paste. (iii) 0.8 mL of PEG was added to the centrifuge tube over 60 s while gently stirring. Immediately afterward, 10 mL of pre-warmed 37°C RPM-1640 complete culture medium was added and gently stirred. Finally, RPMI-1640 culture medium was added to a final volume of 40 mL, and the tube was centrifuged at 1000 rpm for 5 min. (iv) The supernatant was discarded, and a small amount of HT culture medium was used to disperse the cells. Then, the prepared HT culture medium was added. The cells were transferred to a 96-well cell culture plate, 100 μL per well, and incubated in a CO2 incubator. (v) After 12 h, 100 μL of HAT complete culture medium was prepared and added to each well. After 5 days, the cell supernatant in the wells was replaced with HT complete culture medium at a rate of 50%–100%. After 9–14 days, the supernatant was collected for analysis.

[0299] 6. Hybridoma screening: Indirect ELISA was used for screening. 100 ng / well of recombinant antigen was coated, 50 μL of cell supernatant was added for detection, and positive clones were selected.

[0300] 7. Cloning of Hybridoma Cells: The culture wells containing positive supernatant typically contain two or more hybridoma colonies. Some colonies may not secrete antibodies or may secrete antibodies not required for the experiment; only one colony will secrete the specific antibody. Therefore, clonal culture methods must be used to separate them and select the desired hybridoma cells. The most commonly used clonal culture method is the limiting dilution method. Cells are first serially diluted to a specific concentration and then seeded into 96-well cell culture plates, ensuring that only one cell grows in each well. Generally, a single-clone positive hybridoma cell line needs to be cloned 2-3 times until 100% positive, at which point it is considered a stable clone. A stable hybridoma cell line, 9A5, was obtained, with accession number CCTCC No. C2021141, deposited on May 28, 2021, at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Wuchang, Wuhan (Postcode 430072).

[0301] (3) Induction of monoclonal antibody ascites

[0302] 1. Take 2-3 BALB / c mice and inject 0.5 mL of liquid paraffin oil into their peritoneal cavity.

[0303] 2.1 Weeks later, hybridoma cells in the logarithmic growth phase were treated by centrifugation at 1000 rpm for 5 min, and the supernatant was discarded. The hybridoma cells were resuspended in serum-free culture medium, and the cell number was adjusted to (1–2) × 10⁻⁶. 6 / mL, 0.5mL was injected intraperitoneally into each mouse.

[0304] 3.7–10 days later, after the mice's abdomens became significantly distended, they were euthanized by cervical insemination. The mice were rinsed with tap water and immersed in 75% ethanol for 5 minutes. With the mice facing upwards, their limbs were fixed to the dissection table using an injection needle. The abdominal skin was lifted with forceps, a small incision was made, and then cut open from both sides towards the back of the mouse. The skin was then torn open with large forceps to fully expose the abdomen. The peritoneum was lifted with sterile ophthalmic forceps, a small incision was made in the center of the peritoneum, and all ascites fluid was aspirated from the abdominal cavity through a 1 mL pipette. All ascites fluid was mixed and placed in a centrifuge tube, centrifuged at 3000 rpm for 20 minutes, and then the supernatant was collected.

[0305] (4) Purification of monoclonal antibody ascites: The ascites was purified by octanoic acid-saturated ammonium sulfate precipitation and Protein A affinity chromatography (purchased from GE Healthcare, USA) to obtain purified monoclonal antibody 9A5.

[0306] (5) Identification of antibody subtypes: The fusion protein antigen was diluted 100-fold with 20mM PB 7.4 and coated onto a 96-well ELISA plate at 100 μL / well. Coating was carried out at 37°C for 2 h. The plate was washed once with PBST, and non-specific binding sites were blocked with the relevant blocking buffer (20mM PB 7.4 containing 150mM NaCl, 0.5% casein, and 0.002% gelatin) at 200 μL / well, and incubated overnight at 4°C. 100 μL of a 500-fold diluted monoclonal antibody was added to the ELISA plate and incubated at 37°C for 1 h. The plate was washed 5 times with PBST, and goat anti-mouse secondary antibodies labeled with HRP (anti-IgG1, IgG2a, IgG2b, IgG3, and IgM) were added respectively. Incubation was carried out at 37°C for 30 min. The plate was washed 5 times with PBST, and TMB chromogenic buffer was added for 15 min of development. The reaction was stopped with stop buffer, and the readings were recorded using a TECAN sunrise microplate reader. The results showed that monoclonal antibody 9A5 was the IgG2a subtype.

[0307] (6) HRP labeling of monoclonal antibodies: All operations were performed under light-protected conditions: (i) Weigh 2 mg of HRP powder and dissolve it in 0.1 mL of ddH2O. (ii) Weigh 2 mg of 9A5 antibody and dissolve it in 0.1 mL of ddH2O. Slowly mix 0.1 mL of NaIO4 solution with 0.1 mL of HRP and let it stand at 4°C for 30 min. (iii) Add 2 μL of ethylene glycol and let it stand at room temperature in the dark for 30 min. (iv) Add an equal volume (0.2 + 0.2 + 0.05) mL of CB buffer (pH 9.6, 50 mM) to make the pH of the solution alkaline. (v) Transfer the reaction solution from the centrifuge tube to a dialysis bag containing the antibody and dialyze against CB buffer at 4°C for 4 h, changing the buffer once. (vi) Dissolve 0.4 mg NaBH4 in 20 μL of ddH2O and add to a dialysis bag. Incubate at 4°C for 2 h, shaking once every 30 min. (vii) Add an equal volume of 50% saturated ammonium sulfate (SAS) solution, precipitate at 4°C for 1 h, centrifuge at 12000 rpm for 10 min, and discard the supernatant (be careful to invert the SAS completely to avoid residual SAS affecting the antibody). Finally, resuspend the precipitate in 500 μL of buffer (50% glycerol and 10% NBS) and store at -20°C.

[0308] (7) Properties analysis of Anti-C149-2C01 mouse monoclonal antibody (9A5)

[0309] 1. The synthetic peptides (2CO1, 2CO2, 2CO3) from Example 2 were coated into 96-well ELISA plates at a concentration of 1 μg / well. After washing the plates once, the non-specific binding sites were blocked with blocking buffer (20 mM PB7.4 containing 150 mM NaCl, 0.5% casein, and 0.002% gelatin). The plates were blocked at 37°C for 2 h and then dried for later use.

[0310] 2. Dilute antibody 9A5 to a concentration of 2 μg / mL (the dilution buffer is formulated the same as the blocking buffer), add 100 μL to each well, and incubate at 37°C for 30 min. Wash the plate 5 times with PBST, add HRP-conjugated goat anti-mouse antibody diluted 1:5000, and incubate at 37°C for 30 min. Wash the plate 5 times with PBST, add chromogenic buffer for 15 min, add stop solution to stop the reaction, and read the values ​​on a microplate reader.

[0311] 3. The fusion proteins (C149-2C01, C149-2C02, C149-2C03) prepared in Example 8 were coated into 96-well ELISA plates at a concentration of 200 ng / well. After washing the plates once, the non-specific binding sites were blocked with blocking buffer, and the plates were blocked at 37°C for 2 h before being dried for later use.

[0312] 4. Dilute the antibody-enzyme-labeled antibody obtained in step (6) above at a ratio of 1:1000 (the dilution solution formula is the same as the blocking solution), add 100 μL to each well, and incubate at 37°C for 30 min. Wash the plate 5 times with PBST, add the colorimetric solution for 15 min, add the stop solution to stop the reaction, and read the value on a microplate reader.

[0313] The results are shown in Figure 4 and Figure 5 . Figure 4 The results showed that the 9A5 antibody could bind to the 2C01 peptide and had good activity. Figure 5 The results showed that the 9A5-HRP enzyme-labeled antibody could bind to the C149-2C01 recombinant protein and exhibited good activity. The following examples were performed using either 9A5 or 9A5-HRP.

[0314] Example 15: Binding activity of monoclonal antibody 9A5 to human rhinovirus

[0315] The monoclonal antibody 9A5 prepared in Example 14 was used to detect different representative rhinoviruses using immunofluorescence. The immunofluorescence method procedure was the same as in Example 4, and the experimental results are as follows. Figure 6 As shown in the figure. The results indicate that 9A5 can cross-bind with 24 rhinoviruses, including HRV-A1B, HRV-A2, HRV-A9, HRV-A16, HRV-A21, HRV-A23, HRV-A29, HRV-A34, HRV-A36, HRV-A40, HRV-A43, HRV-A49, HRV-A54, HRV-A59, HRV-A77, HRV-A78, HRV-A89, HRV-B5, HRV-B6, HRV-B14, HRV-B17, HRV-C11, HRV-C15, and HRV-C23, in H1-HeLa cells, exhibiting green fluorescence. In contrast, uninfected H1-HeLa cells (control) showed no reaction, indicating that the monoclonal antibody 9A5 can specifically cross-bind with different subtypes of rhinovirus. These results indicate that 9A5 is a universal affinity antibody and that 2C01 contains a universal affinity epitope.

[0316] Example 16: Monoclonal antibody 9A5 used to detect human rhinovirus infection and titer.

[0317] The monoclonal antibody 9A5 prepared in Example 14 was used to detect the rhinovirus titer using a serial dilution spot count method. In this example, 10 representative rhinoviruses from Example 4 were selected for titer determination. Specific steps are detailed in Example 5, and the experimental results are shown in Table 14. Figure 7 middle.

[0318] Table 14. Relationship between the dosage of rhinovirus and the number of cells infected in the corresponding well.

[0319]

[0320] According to Table 14 and Figure 7 The results showed that the viral titers of 10 representative rhinoviruses, namely HRV-A1B, HRV-A2, HRV-A16, HRV-A36, HRV-A40, HRV-A78, HRV-A89, HRV-B5, HRV-B14, and HRV-C15, were 8.75 × 10⁻⁶. 4 (IU / mL), 1.513×10 5 (IU / mL), 7.75×10 4 (IU / mL), 6.75×10 4 (IU / mL), 8.25×10 4 (IU / mL), 9.5×10 4 (IU / mL), 2.875×10 4 (IU / mL), 6.375×10 4 (IU / mL), 2.5×10 4 (IU / mL) and 8.25×10 4 (IU / mL). The results of this example demonstrate that monoclonal antibody 9A5 can determine the infection titers of different representative rhinoviruses.

[0321] Example 17: Monoclonal antibody 9A5 used to detect the titer of neutralizing antibodies against human rhinovirus.

[0322] In this example, using enzyme-linked immunospot assay, we used the monoclonal antibody 9A5 prepared in Example 14 to detect the neutralizing antibody titer of human rhinovirus. The specific method is described in Example 7.

[0323] In this embodiment, six mouse polyclonal antibody sera prepared in Example 5 were randomly selected for the detection of neutralizing antibodies against human rhinovirus. The results are shown in Table 15. The results indicate that monoclonal antibody 9A5 can be used to detect the neutralizing antibody titer against human rhinovirus; mouse polyclonal antibody sera can neutralize HRV-A2, HRV-B14, or HRV-C15 viruses.

[0324] Table 15. Results of detection of neutralizing antibody titers in mouse polyclonal antibody serum

[0325]

[0326] Example 18: Sequencing of the light and heavy chain variable regions of monoclonal antibody 9A5

[0327] Semi-adherent culture 10 79A5 mouse hybridoma cells were suspended by blowing onto the adherent cells with a pipette and transferred to a new 4 mL centrifuge tube. The cells were centrifuged at 1500 rpm for 3 min. The cell pellet was collected, resuspended in 100 μL of sterile PBS buffer, and transferred to a new 1.5 mL centrifuge tube. 800 μL of Trizol (Roche, Germany) was added, and the mixture was gently inverted to mix. The mixture was allowed to stand for 10 min. 200 μL of chloroform was added, and the mixture was vigorously vortexed for 15 s. The mixture was allowed to stand for 10 min, and then centrifuged at 12000 rpm for 15 min at 4 °C. The supernatant was transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added. The mixture was allowed to stand for 10 min. The mixture was centrifuged at 12000 rpm for 5 min at 4 °C, the supernatant was discarded, and the pellet was dried under vacuum at 60 °C for 5 min. The clear pellet was dissolved in 70 μL of DEPC H2O, and 1 μL of reverse transcription primer Oligo(dT) was added. (12-18) Add 1 μL of dNTP (purchased from Promega) and place in a 72°C water bath for 10 min, then immediately place in an ice bath for 5 min. Add 20 μL of 5× reverse transcription buffer, 2 μL of AMV (10 U / μL, purchased from Promega), and 1 μL of Rnasin (40 U / μL, purchased from Promega). Mix well and reverse transcribe RNA into cDNA at 42°C.

[0328] The light chain gene was isolated using the aforementioned cDNA as a template. PCR amplification was performed with MVkF-G1 (SEQ ID NO:16) as the upstream primer and MVkR-M13 (SEQ ID NO:17) as the downstream primer, yielding a DNA fragment of approximately 500 bp. The PCR conditions were: 95℃ for 3 min, 23 cycles (95℃ for 15 s, 56℃ for 30 s, 72℃ for 30 s), and 72℃ for 5 min. The fragment was then recovered and sequenced. BLAST alignment confirmed the sequence as the 9A5 light chain, with the variable region encoding the gene shown in SEQ ID NO:18, and its encoded amino acid sequence shown in SEQ ID NO:19.

[0329] The heavy chain gene was isolated using the cDNA described above as a template. PCR amplification was performed with MVhF-C1 (SEQ ID NO:20) as the upstream primer and MVhR-M13 (SEQ ID NO:21) as the downstream primer, yielding a DNA fragment of approximately 500 bp. The PCR conditions were: 95℃ for 3 min, 23 cycles (95℃ for 15 s, 56℃ for 30 s, 72℃ for 30 s), and 72℃ for 5 min. The fragment was then recovered and sequenced. The sequence was confirmed as the 9A5 heavy chain sequence after BLAST alignment. The gene encoding the variable region is shown in SEQ ID NO:22, and its encoded amino acid sequence is shown in SEQ ID NO:23.

[0330] In addition, the CDR sequences (SEQ ID NOs: 24-29) of the light and heavy chains of monoclonal antibody 9A5 were determined based on the IMGT database (http: / / www.imgt.org / IMGT_vquest / analysis).

[0331] Although certain specific embodiments of the invention have been described in detail above, those skilled in the art will understand that various modifications and substitutions can be made to the details without departing from the spirit and intent of the invention, based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The scope of the invention is defined only by the appended claims and any equivalents thereof.

Claims

1. An isolated polypeptide or a variant thereof, said polypeptide comprising at least 13 consecutive amino acid residues of human rhinovirus (HRV) 2C protein, and including amino acid residues 151-163 of said protein; The amino acid residues at positions 151-163 of the HRV 2C protein are shown in SEQ ID NO: 1; Furthermore, the polypeptide consists of no more than 25 consecutive amino acid residues of the HRV 2C protein; Furthermore, (i) the variant has an amino acid residue at position 156 of the HRV 2C protein replaced with A, S, or N, or (ii) the variant has an amino acid residue at position 159 of the HRV 2C protein replaced with H, or (iii) the variant has an amino acid residue at position 156 of the HRV 2C protein replaced with N and an amino acid residue at position 159 of the HRV 2C protein replaced with H.

2. The isolated polypeptide or a variant thereof of claim 1, wherein, The polypeptide consists of 13 to 21 consecutive amino acid residues of the HRV 2C protein.

3. The isolated polypeptide or a variant thereof of claim 1, wherein, The HRV 2C protein has the sequence shown in SEQ ID NO:

9.

4. The isolated polypeptide of claim 1 or a variant thereof, comprising or consisting of the sequence shown in any one of SEQ ID NOs: 1, 30-37.

5. A recombinant protein comprising an isolated polypeptide or a variant thereof of any one of claims 1-4, and a carrier protein, wherein the recombinant protein is not a naturally occurring protein or a fragment thereof.

6. The recombinant protein of claim 5, wherein, The polypeptide or its variants are optionally linked to a carrier protein via a linker.

7. The recombinant protein of claim 6, wherein, The connector can be rigid or flexible.

8. The recombinant protein of claim 6, wherein, The linker is selected from peptide linkers containing one or more glycines and / or one or more serines.

9. The recombinant protein of claim 5, wherein, The carrier protein is selected from CRM197 protein or a fragment thereof, HBcAg or a fragment thereof, WHcAg or a fragment thereof, keyhole hemocyanin, human serum albumin, bovine serum albumin, bovine thyroglobulin, and ovalbumin.

10. The recombinant protein of claim 5, wherein, The carrier protein is HBcAg or a fragment thereof, and the 79th-80th amino acids of HBcAg are replaced by the polypeptide or a variant thereof.

11. The recombinant protein of claim 10, wherein, The polypeptide or its variants are linked to HBcAg or fragments thereof via linkers.

12. The recombinant protein of claim 10, wherein, The HBcAg fragment contains or is composed of HBcAg aa 1-149.

13. The recombinant protein of claim 5, having an amino acid sequence as shown in any one of SEQ ID NO: 5, 44-51.

14. An isolated nucleic acid molecule encoding a polypeptide or a variant thereof as claimed in any one of claims 1-4, or a recombinant protein as claimed in any one of claims 5-13.

15. A vector comprising the isolated nucleic acid molecule of claim 14.

16. A host cell comprising the isolated nucleic acid molecule of claim 14 or the vector of claim 15.

17. A method for preparing the polypeptide or a variant thereof of any one of claims 1-4 or the recombinant protein of any one of claims 5-13, comprising culturing the host cell of claim 16 under suitable conditions and recovering the polypeptide or a variant thereof or the recombinant protein from the cell culture.

18. A virus-like particle (VLP) having on its surface the isolated polypeptide or a variant thereof as described in any one of claims 1-4.

19. The virus-like particle of claim 18, wherein, The virus-like particles comprise or are composed of a fusion protein, the fusion protein being composed of a polypeptide or a variant thereof as described in any one of claims 1-4, and a carrier protein.

20. The virus-like particle of claim 19, wherein, The polypeptide or its variants are linked to the carrier protein via linkers.

21. The virus-like particle of claim 19, wherein, The carrier protein is HBcAg protein or a fragment thereof.

22. The virus-like particle of claim 19, wherein, The carrier protein is HBcAg aa 1-149.

23. The virus-like particle of claim 21, wherein, Replace amino acids 79-80 of HBcAg with the polypeptide or a variant thereof.

24. The virus-like particle of claim 19, wherein, The fusion protein has an amino acid sequence as shown in any one of SEQ ID NO: 5, 44-51.

25. A composition comprising a polypeptide or a variant thereof of any one of claims 1-4, a recombinant protein of any one of claims 5-13, an isolated nucleic acid molecule of claim 14, a vector of claim 15, a host cell of claim 16, or a VLP of any one of claims 18-24.

26. The composition of claim 25, wherein, The composition is a pharmaceutical composition or an immunogenic composition.

27. The composition of claim 25, wherein, The composition also contains a pharmaceutically acceptable carrier and / or excipient.

28. The composition of claim 25, wherein, The composition also contains an adjuvant.

29. The composition of claim 25, wherein, The composition is a vaccine.

30. The composition of claim 25, wherein, The composition comprises one or more of the peptides or variants thereof, and these peptides or variants thereof may be single or in tandem, modified or unmodified, coupled to other proteins or not coupled to other proteins.

31. The composition of claim 25, wherein, The composition comprises one or more of the recombinant proteins.

32. The composition of claim 25, wherein, The composition comprises one or more of the VLPs.

33. Use of the polypeptide of any one of claims 1-4 or a variant thereof, the recombinant protein of any one of claims 5-13, the isolated nucleic acid molecule of claim 14, the vector of claim 15, the host cell of claim 16, or the VLP of any one of claims 18-24 in the preparation of formulations for inducing an immune response against HRV in a subject and / or for preventing HRV infection or HRV-related diseases in a subject.

34. The use of claim 33, wherein, The diseases associated with HRV infection include respiratory infections.

35. The use of claim 33, wherein, The preparation is a vaccine.

36. An antibody or its antigen-binding fragment, comprising: (a) The following three heavy chain CDRs: VH CDR1 with sequence SEQ ID NO: 27, VHCDR2 with sequence SEQ ID NO: 28, and VH CDR3 with sequence SEQ ID NO: 29; and the following three light chain CDRs: VLCDR1 with sequence SEQ ID NO: 24, VL CDR2 with sequence LVS, and VL CDR3 with sequence SEQ ID NO: 26; or, (b) The three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 23; and the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:

19.

37. The monoclonal antibody of claim 36 or its antigen-binding fragment, wherein, The three CDRs contained in the VH and the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system.

38. The antibody or antigen-binding fragment thereof of claim 36, comprising: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO: 23; or (ii) A sequence that has at least 80% sequence identity with the sequence shown in SEQ ID NO: 23; and (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iii) The sequence shown in SEQ ID NO: 19; or (iv) A sequence that has at least 80% sequence identity with the sequence shown in SEQ ID NO:

19.

39. The antibody of claim 36 or an antigen-binding fragment thereof, comprising: a VH comprising the sequence shown in SEQ ID NO: 23 and a VL comprising the sequence shown in SEQ ID NO:

19.

40. The antibody or antigen-binding fragment thereof of claim 36, wherein it is humanized.

41. The antibody of claim 36 or its antigen-binding fragment, wherein, The antibody or its antigen-binding fragment contains a framework region sequence derived from human immunoglobulin.

42. The antibody of claim 36 or its antigen-binding fragment, wherein, The antibody or its antigen-binding fragment comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence, and a light chain framework region sequence derived from a human light chain germline sequence.

43. The antibody or antigen-binding fragment thereof of claim 36, further comprising a constant region derived from mouse or human immunoglobulin.

44. The antibody of claim 36 or its antigen-binding fragment, wherein, The heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from mouse or human immunoglobulin, and the light chain of the antibody or its antigen-binding fragment includes a light chain constant region derived from mouse or human immunoglobulin.

45. The antibody of claim 36 or its antigen-binding fragment, wherein, The antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, scFv, and / or the antibody is selected from: murine antibody, humanized antibody, fully human antibody, chimeric antibody, bispecific or multispecific antibody.

46. ​​A monoclonal antibody and its antigen-binding fragment, produced by hybridoma cell line 9A5, wherein, Hybridoma cell line 9A5 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. C2021141.

47. The antibody or antigen-binding fragment thereof according to any one of claims 36-46, wherein, The antibody or its antigen-binding fragment specifically recognizes at least two HRV subtypes.

48. The antibody or antigen-binding fragment thereof of any one of claims 36-46, wherein, The antibody or its antigen-binding fragment specifically recognizes 2, 5, 10, 15, 20 or 24 HRV subtypes.

49. The antibody or antigen-binding fragment thereof according to any one of claims 36-46, wherein, The antibody or its antigen-binding fragment specifically recognizes one or more of HRV-A1B, HRV-A2, HRV-A9, HRV-A16, HRV-A21, HRV-A23, HRV-A29, HRV-A34, HRV-A36, HRV-A40, HRV-A43, HRV-A49, HRV-A54, HRV-A59, HRV-A77, HRV-A78, HRV-A89, HRV-B5, HRV-B6, HRV-B14, HRV-B17, HRV-C11, HRV-C15, and HRV-C23.

50. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described in any one of claims 36-49, or a variable region of the heavy chain and a variable region of the light chain thereof.

51. A vector comprising the isolated nucleic acid molecule of claim 50.

52. A host cell comprising the isolated nucleic acid molecule of claim 50 or the vector of claim 51.

53. Hybridoma cell line 9A5, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. C2021141.

54. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 36-49, comprising culturing a host cell according to claim 52 or a hybridoma cell according to claim 53 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell or hybridoma cell culture.

55. A conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 36-49, and a detectable label linked to said antibody or antigen-binding fragment thereof.

56. The conjugate of claim 55, wherein, The detectable markers are selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, and biotin.

57. A kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 36-49 or the conjugate as described in claim 55 or 56.

58. The kit of claim 57, wherein, The kit comprises the conjugate as described in claim 55 or 56.

59. The kit of claim 57, comprising the antibody or antigen-binding fragment thereof as described in any one of claims 36-49, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof.

60. The kit of claim 59, wherein, The second antibody also includes a detectable marker.

61. The kit of claim 60, wherein, The detectable markers are selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, and biotin.

62. A method for detecting the presence or level of HRV in a sample for non-diagnostic purposes, comprising using an antibody or antigen-binding fragment thereof as described in any one of claims 36-49 or a conjugate as described in claim 55 or 56.

63. The method of claim 62, wherein, The method described is an immunological detection.

64. The method of claim 62, wherein, The HRV contains at least two subtypes.

65. The method of claim 62, wherein, The HRV includes 2, 5, 10, 15, 20, or 24 subtypes.

66. The method of claim 62, wherein, The HRV includes one or more of the following: HRV-A1B, HRV-A2, HRV-A9, HRV-A16, HRV-A21, HRV-A23, HRV-A29, HRV-A34, HRV-A36, HRV-A40, HRV-A43, HRV-A49, HRV-A54, HRV-A59, HRV-A77, HRV-A78, HRV-A89, HRV-B5, HRV-B6, HRV-B14, HRV-B17, HRV-C11, HRV-C15, and HRV-C23.

67. The method of claim 62, comprising using the conjugate as described in claim 55 or 56.

68. The method of claim 62, comprising using an antibody or antigen-binding fragment thereof as described in any one of claims 36-49, and the method further comprising using a second antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof.

69. The method of claim 68, wherein, The detectable markers are selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, and biotin.

70. Use of the antibody or antigen-binding fragment thereof according to any one of claims 36-49 in the preparation of a diagnostic reagent for detecting the presence or level of HRV in a sample, and / or for diagnosing whether a subject is infected with HRV.

71. The use of claim 70, wherein, The sample was a bodily fluid sample from the subject.

72. A method for detecting the neutralizing activity of HRV neutralizing antibodies or screening for HRV neutralizing antibodies, comprising using an antibody or antigen-binding fragment thereof as described in any one of claims 36-49 or a conjugate as described in claims 55 or 56; and the method is for non-diagnostic purposes.

73. The method of claim 72, wherein, The method described is an immunological detection.

74. The method of claim 72, wherein, The method includes: (i) incubating a test sample containing the antibody to be tested with HRV; (ii) incubating the product of step (i) with cells; (iii) incubating the antibody or its antigen-binding fragment or the conjugate with the cell product of step (ii); and (iv) determining the number of cells labeled by the antibody or its antigen-binding fragment or the conjugate, thereby determining the neutralizing activity of the antibody to be tested against HRV.

75. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 36-49, and a pharmaceutically acceptable carrier and / or excipient.

76. Use of the antibody or antigen-binding fragment thereof according to any one of claims 36-49, the isolated nucleic acid molecule according to claim 50, the vector according to claim 51, the host cell according to claim 52, or the hybridoma cell according to claim 53 for the preparation of a medicament for the prevention and / or treatment of HRV infection or HRV infection-related disease in a subject.

77. The use of claim 76, wherein, The diseases associated with HRV infection include respiratory infections.

78. The use of claim 76, wherein, The antibody or its antigen-binding fragment may be used alone or in combination with other pharmaceutically active agents.

Images