Constructs of nanobody s43 and uses thereof

Abstract

The application provides a construct based on a nanobody S43 specifically binding to SARS-CoV-2 RBD (including a multivalent nanobody and a nanobody fusion protein), a related product thereof and application thereof; the construct based on the nanobody S43 specifically binding to SARS-CoV-2 RBD (including the multivalent nanobody and the nanobody fusion protein) can effectively inhibit SARS-CoV-2 infection and infection of a variant strain thereof, can be administered in a nebulization mode, can directly reach the lung, has a relatively fast effect and has a long half-life, and provides a more effective treatment strategy for preventing or treating a new coronavirus and a variant strain thereof.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a construct of nanobody S43 and application thereof, more particularly to a multivalent nanobody, a nanobody fusion protein based on nanobody S43 specifically binding to SARS-CoV-2 RBD, a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a transformed cell comprising the polynucleotide, the nucleic acid construct or the expression vector, and a pharmaceutical composition comprising any of the above products, and the use of the above products in the preparation of a medicament for preventing or treating novel coronavirus, and in the preparation of a reagent or kit for detecting novel coronavirus or diagnosing novel coronavirus infection. BACKGROUND

[0002] The epidemic caused by the novel coronavirus (SARS-CoV-2) of the family Coronaviridae is still spreading worldwide. In addition, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and other viruses belonging to the family Coronaviridae are also major pathogens of the human respiratory system, mainly transmitted through droplets, aerosols and contact, and have strong infectivity.

[0003] The current outbreak of COVID-19 has promoted the development of various vaccines and antiviral drugs. Vaccination can effectively prevent the occurrence of severe infectious diseases, but the vaccine is suitable for uninfected people, and the development cycle is long and the clinical research process is complex. For confirmed patients, only antiviral drugs can be used for treatment, one of which is therapeutic antibody drugs, mainly neutralizing antibodies; neutralizing antibody drugs mainly bind to antigens on the surface of pathogenic microorganisms, prevent specific molecules expressed by pathogenic microorganisms from binding to cell surface receptors, and achieve the effect of "neutralization". The SARS-CoV-2 virus surface has a glycosylated spike protein (S), which can interact with the host cell receptor protein ACE2 and trigger membrane fusion, so blocking the binding of S protein to ACE2 is an effective way to treat COVID-19 infection.

[0004] Conventional monoclonal antibodies are generally administered by intravenous injection, however, the drug concentration of monoclonal antibodies administered by intravenous injection from the systemic circulation into the lungs is very low, which greatly reduces the antiviral effect of the neutralizing antibodies themselves, resulting in the inability to effectively reduce the viral load in the lungs. The novel coronavirus initially infects the upper respiratory tract, and the first interaction with the immune system mainly occurs on the respiratory mucosal surface. In view of this, for the novel coronavirus infected through the respiratory tract, it is necessary to consider, design and develop suitable antibody drugs from the perspective of mucosal immunity in addition to focusing on serum antibodies. For example, aerosol administration can achieve higher local concentration of antibody drugs in the respiratory tract, which can more effectively block viral infection when the virus invades.

[0005] Nanobodies have been paid much attention as therapeutic drugs, for example, Caplacizumab (Cabivi TM ) developed by Ablynx company is the first nanobody drug approved for the treatment of acquired thrombotic thrombocytopenic purpura, and ALX-0171 is a trivalent form of nanobody for the treatment of pediatric respiratory syncytial virus (RSV) infection, which is administered by aerosol and has entered the clinical phase II (https: / / clinicaltrials.gov), which indicates that nanobody drugs have safety and feasibility.

[0006] Therefore, it is of potential clinical application value and prospect to develop nanobody drugs for the novel coronavirus suitable for respiratory mucosal immunity. SUMMARY

[0007] Invention objectives

[0008] The present application aims to provide a construct based on the nanobody S43 specifically binding to SARS-CoV-2 RBD (including multivalent nanobody and nanobody fusion protein), a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a transformed cell comprising the polynucleotide, the nucleic acid construct or the expression vector, and a pharmaceutical composition comprising any of the above products, and the use thereof in the preparation of a drug for preventing or treating the novel coronavirus, and the use thereof in the preparation of a reagent or kit for detecting the novel coronavirus or diagnosing the infection of the novel coronavirus.

[0009] The construct based on the nanobody S43 specifically binding to the RBD of SARS-CoV-2 of the present application (including the multivalent nanobody and the nanobody fusion protein) can effectively inhibit the infection of SARS-CoV-2 and its mutant strains, can be administered by nebulization, can directly reach the lungs, has a faster effect and a longer half-life, and provides a more effective treatment strategy for the infection of the new coronavirus and its mutant strains.

[0010] Solution

[0011] To achieve the above-mentioned object, the present application provides the following technical solutions.

[0012] In a first aspect, the present application provides a multivalent nanobody comprising two or more VHH chains of a nanobody specifically binding to the RBD of SARS-CoV-2, wherein the VHH chain of the nanobody specifically binding to the RBD of SARS-CoV-2 comprises the following CDRs:

[0013] a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 1 (i.e., GFTLDYYAIG),

[0014] a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 2 (i.e., CISSNNSTYYADSVKG), and

[0015] a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 3 (i.e., EPDYSGVYYYTCGWTDFGS).

[0016] Preferably, the amino acid sequences of the FR1-4 are as set forth in SEQ ID NO: 4 (i.e., QVQLQESGGGLVQPGGSLRLTCAPS), SEQ ID NO: 5 (i.e., WFRQAPGKEREGVS), SEQ ID NO: 6 (i.e., RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA), and SEQ ID NO: 7 (i.e., WGQGTQVTVSS), respectively.

[0017] In a preferred embodiment, the VHH chain of the Nanobody specifically binding to SARS-CoV-2 RBD has an amino acid sequence as set forth in SEQ ID NO: 8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 8; preferably, the amino acid sequence of the VHH chain is as set forth in SEQ ID NO: 8 below: wherein the underlined parts are the framework regions FR1-4, and the blacked parts are CDR1, CDR2 and CDR3 of the heavy chain variable region, respectively.

[0018] In a preferred embodiment I, the multivalent Nanobody is composed of two or more, preferably three, VHH chains of the Nanobody specifically binding to SARS-CoV-2 RBD connected by a Linker;

[0019] wherein the Linker is (GGGGS)n, wherein n = 1, 2, 3, or 4, preferably n = 2 or 3.

[0020] As a further preferred embodiment of embodiment I, the multivalent Nanobody is a trivalent Nanobody having an amino acid sequence as set forth in SEQ ID NO: 9:

[0021] QVQLQESGGGLVQPGGSLRLTCAPSGFTLDYYAIGWFRQAPGKEREGVSCISSNNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEPDYSGVYYYTCGWTDFGSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLTCAPSGFTLDYYAIGWFRQAPGKEREGVSCISSNNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEPDYSGVYYYTCGWTDFGSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVQPGGSLRLTCAPSGFTLDYYAIGWFRQAPGKEREGVSCISSNNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEPDYSGVYYYTCGWTDFGSWGQGTQVTVSS (SEQ ID NO: 9).

[0022] In a preferred embodiment II, the multivalent Nanobody is an IgM pentamer formed by a fusion protein having the structure from N- to C-terminus as shown in formula (I):

[0023] A-L-B (I)

[0024] wherein,

[0025] A is a single VHH chain of the Nanobody specifically binding to the SARS-CoV-2 RBD, or a multivalent Nanobody as described in the preferred embodiment I above;

[0026] B is an Fc fragment of human IgM; preferably, the Fc fragment of human IgM has the amino acid sequence as shown in SEQ ID NO: 10 (i.e., VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTD QVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQD TAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNA TFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSI LTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY) or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as shown in SEQ ID NO: 10;

[0027] L is (GGGGS)m, wherein m = 0, 1, 2, 3, or 4.

[0028] As a preferred embodiment of the fusion protein described above, it has the amino acid sequence as shown in SEQ ID NO: 11:

[0029] QVQLQESGGGLVQPGGSLRLTCAPSGFTLDYYAIGWFRQAPGKEREGVSCISSNN STYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEPDYSGVYYYTCGW TDFGSWGQGTQVTVSSVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVS WLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRG LTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQN GEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPK GVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTS APMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTL YNVSLVMSDTAGTCY (SEQ ID NO: 11).

[0030] In a second aspect, the present application provides a Nanobody fusion protein, the structure of which from N-terminal to C-terminal is shown as formula (I):

[0031] A-L-B (I)

[0032] wherein,

[0033] A is a VHH chain of a Nanobody specifically binding to SARS-CoV-2 RBD, which is defined as above in the first aspect; or, A is a multivalent Nanobody according to the preferred specific embodiment I of the first aspect, i.e., a multivalent Nanobody composed of two or more, preferably three, VHH chains of said Nanobody specifically binding to SARS-CoV-2 RBD, which are connected by a Linker, wherein said Linker is (GGGGS)n, wherein n = 1, 2, 3, or 4, preferably n = 2 or 3;

[0034] B is an Fc fragment of human IgM; preferably, the Fc fragment of human IgM has an amino acid sequence as set forth in SEQ ID NO: 10, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 10;

[0035] L is (GGGGS)m, wherein m = 0, 1, 2, 3, or 4.

[0036] As a preferred embodiment of the fusion protein described above, it has an amino acid sequence as set forth in SEQ ID NO: 11.

[0037] In a third aspect, the present application provides a polynucleotide encoding the multivalent nanobody according to the first aspect described above, or encoding the nanobody fusion protein according to the second aspect described above.

[0038] In specific embodiments, the polynucleotide is DNA or mRNA;

[0039] Preferably, the polynucleotide encodes a multivalent Nanobody according to the preferred embodiment I of the first aspect described above, further preferably the polynucleotide comprises SEQ ID NO: 12 (i.e., CAGGTCCAACTCCAAGAGAGCGGCGGCGGCCTCGTCCAACCCGGAGGATCACTC AGACTCACATGCGCCCCAAGCGGCTTCACACTCGACTACTACGCCATCGGCTGGTT CAGACAAGCCCCCGGCAAAGAGAGAGAAGGAGTGTCTTGCATTAGCAGCAACAA CAGCACCTACTACGCCGACAGTGTCAAAGGAAGATTCACCATCAGCAGGGACAAC GCTAAGAACACCGTGTATCTCCAGATGAACTCACTGAAGCCCGAGGACACCGCCG TGTACTACTGCGCCGCCGAGCCCGACTACAGCGGCGTTTACTACTACACCTGCGGA TGGACCGACTTCGGCAGCTGGGGCCAAGGAACCCAAGTCACCGTGAGCAGCGGA GGCGGAGGAAGCGGCGGTGGAGGAAGTGGCGGAGGCGGATCTGGGGGGGGAGG ATCAGGCGGAGGAGGAAGCCAGGTGCAGCTGCAGGAGAGCGGAGGAGGACTGG TGCAGCCAGGAGGAAGCCTGAGACTGACATGCGCACCAAGCGGATTCACACTGGACTATTATGCTATCGGATGGTTCAGACAGGCCCCTGGAAAAGAGAGAGAGGGGGT GAGCTGCATCAGCAGCAATAACTCCACATACTACGCCGATAGCGTCAAGGGGAGG TTCACTATTAGCAGGGACAATGCAAAGAACACAGTGTACCTGCAGATGAACAGCC TGAAGCCCGAAGACACCGCCGTCTACTACTGCGCAGCCGAGCCCGATTACAGCGG CGTGTACTACTACACATGCGGATGGACAGACTTCGGCTCCTGGGGCCAAGGCACC CAAGTGACCGTGTCAAGCGGAGGCGGGGGGAGCGGAGGAGGTGGAAGTGGAGGGGGGGGATCTGGCGGGGGAGGAAGTGGAGGAGGAGGATCACAGGTGCAGCTCCAGGAGAGCGGGGGAGGACTGGTCCAGCCAGGAGGGAGCCTGAGACTCACATGTGCACCCAGCGGATTTACACTGGATTATTACGCCATCGGATGGTTTAGGCAGGCACCCGGGAAAGAGAGAGAGGGCGTGAGCTGCATTAGCAGTAATAACAGCACCTATTACGCCGACTCAGTGAAGGGGCGGTTCACCATAAGCAGGGATAACGCCAAGAACACCGTCTACCTGCAGATGAATAGCCTGAAACCCGAAGACACAGCCGTGTACTATTGCGCCGCCGAACCCGACTACTCTGGAGTGTACTACTATACCTGCGGCTGGACCGACTTTGGCAGCTGGGGGCAAGGCACCCAGGTGACCGTGAGCAGT) or a nucleotide sequence represented by (GGGGGGATCTGGCGGGGGAGGAAGTGGAGGAGGAGGATCACAGGTGCAGCTCCAGGAGAGCGGGGGAGGACTGGTCCAGCCAGGAGGGAGCCTGAGACTCACATGTGCACCCAGCGGATTTACACTGGATTATTACGCCATCGGATGGTTTAGGCAGGCACCCGGGAAAGAGAGAGAGGGCGTGAGCTGCATTAGCAGTAATAACAGCACCTATTACGCCGACTCAGTGAAGGGGCGGTTCACCATAAGCAGGGATAACGCCAAGAACACCGTCTACCTGCAGATGAATAGCCTGAAACCCGAAGACACAGCCGTGTACTATTGCGCCGCCGAACCCGACTACTCTGGAGTGTACTACTATACCTGCGGCTGGACCGACTTTGGCAGCTGGGGGCAAGGCACCCAGGTGACCGTGAGCAGT) ;

[0040] Preferably, the polynucleotide encodes a Nanobody fusion protein as described in the second aspect above, further preferably the polynucleotide comprises SEQ ID NO: 13 (i.e. CAGGTGCAGCTGCAGGAGAGCGGAGGAGGGCTGGTGCAGCCCGGAGGAAGCCTG AGACTGACCTGCGCCCCCAGCGGATTCACCCTGGATTATTATGCTATTGGCTGGTTTAGGCAGGCTCCCGGCAAAGAGAGAGAGGGGGTGTCATGCATTAGCAGCAATAACT CAACCTACTACGCCGACAGCGTCAAGGGACGCTTCACCATTTCCAGGGACAACGC TAAGAACACCGTGTATCTCCAGATGAATAGCCTGAAGCCCGAGGACACCGCAGTG TACTACTGCGCCGCCGAGCCCGACTACAGCGGTGTGTATTACTACACCTGCGGATG GACCGACTTCGGCAGCTGGGGCCAGGGAACCCAGGTGACAGTGAGCAGCGTGAT CGCCGAGCTGCCCCCCAAGGTGAGCGTGTTCGTGCCCCCTAGAGACGGCTTCTTC GGCAACCCTAGAAAGAGCAAGCTGATCTGCCAAGCCACCGGCTTCTCCCCTAGAC AGATCCAAGTGAGCTGGCTGAGAGAGGGCAAGCAAGTGGGCAGCGGCGTCACAA CAGACCAAGTGCAAGCCGAGGCCAAGGAGAGCGGCCCCACCACCTACAAGGTGA CAAGCACCCTGACCATCAAGGAGAGCGACTGGCTGGGGCAGAGCATGTTCACCTG CAGAGTGGACCACAGAGGCCTGACCTTTCAGCAGAACGCTAGCAGCATGTGCGTG CCCGACCAAGACACCGCCATCAGAGTGTTCGCCATCCCCCCTAGCTTCGCTAGCAT CTTCCTGACCAAGAGCACCAAGCTGACCTGCCTCGTGACCGATCTGACCACCTAC GACAGCGTGACCATCAGCTGGACAAGACAGAACGGCGAGGCCGTGAAGACCCACACCAACATCAGCGAGAGCCACCCCAACGCCACCTTCAGCGCCGTGGGCGAGGCTAGCATCTGCGAGGACGACTGGAACAGCGGCGAGAGATTCACCTGCACCGTGACCCA CACCGACCTGCCTAGCCCCCTGAAGCAGACCATCAGCAGACCCAAGGGCGTGGCC CTGCACAGACCCGACGTGTACCTGCTGCCCCCCGCTAGAGAGCAGCTGAACCTGA GAGAGAGCGCCACCATCACCTGCCTGGTGACCGGCTTTAGCCCCGCTGACGTGTT CGTGCAGTGGATGCAGAGAGGGCAGCCCCTGAGCCCCGAGAAGTACGTGACAAG CGCCCCCATGCCCGAGCCCCAAGCCCCCGGCAGATACTTCGCCCACAGCATCCTG ACCGTGAGCGAGGAAGAGTGGAACACCGGCGAGACCTACACCTGCGTGGTGGCC CACGAGGCCCTGCCCAACAGAGTGACCGAGAGAACCGTGGACAAGAGCACCGGC AAGCCCACCCTGTACAACGTGAGCCTGGTGATGAGCGACACCGCCGGCACCTGCT AC).

[0041] In a fourth aspect, the present application provides a nucleic acid construct comprising the polynucleotide of the third aspect described above, and optionally, at least one expression regulatory element operably linked to the polynucleotide. For example, a histidine tag, a stop codon, and the like.

[0042] In a fifth aspect, the present application provides an expression vector comprising the nucleic acid construct of the fourth aspect described above.

[0043] In a sixth aspect, the present application provides a transformed cell comprising the polynucleotide of the third aspect described above, the nucleic acid construct of the fourth aspect described above, or the expression vector of the fifth aspect described above.

[0044] In a seventh aspect, the present application provides a pharmaceutical composition comprising the multivalent nanobody according to the first aspect described above, the nanobody fusion protein according to the second aspect described above, the polynucleotide according to the third aspect described above, the nucleic acid construct according to the fourth aspect described above, the expression vector according to the fifth aspect described above, or the transformed cell according to the sixth aspect described above, and a pharmaceutically acceptable carrier and / or excipient.

[0045] In a specific embodiment, the pharmaceutical composition can be in the form of a nasal spray, an oral preparation, a suppository, or a parenteral preparation.

[0046] Preferably, the nasal spray is selected from the group consisting of an aerosol, a spray, and a powder spray.

[0047] Preferably, the oral preparation is selected from the group consisting of a tablet, a powder, a pill, a granule, a fine granule, a soft / hard capsule, a film-coated agent, a pellet, a sublingual tablet, and a paste.

[0048] Preferably, the parenteral preparation is a transdermal agent, an ointment, a plaster, a topical liquid, an injectable or a bolus preparation.

[0049] The amount of the effective component of the pharmaceutical composition of the present application to be administered varies depending on the subject, the organ of the subject, the symptoms, the method of administration, and the like, and can be determined according to the judgment of a doctor, taking into account the type of the dosage form, the method of administration, the age and weight of the patient, the symptoms of the patient, and the like.

[0050] In an eighth aspect, the present application provides use of the multivalent nanobody according to the first aspect described above, the nanobody fusion protein according to the second aspect described above, the polynucleotide according to the third aspect described above, the nucleic acid construct according to the fourth aspect described above, the expression vector according to the fifth aspect described above, the transformed cell according to the sixth aspect described above, or the pharmaceutical composition according to the seventh aspect described above, in the preparation of a medicament for preventing and / or treating a novel coronavirus infection.

[0051] In a specific embodiment, the novel coronavirus can be a SARS-CoV-2 original strain and / or a SARS-CoV-2 variant strain.

[0052] Preferably, the SARS-CoV-2 variant strain is Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2) strain, Omicron (B.1.1.529) sub-lineage BA.1 strain or Omicron (B.1.1.529) sub-lineage BA.2 strain; further preferably, the SARS-CoV-2 variant strain is Delta (B.1.617.2) strain, Omicron (B.1.1.529) sub-lineage BA.1 strain or Omicron (B.1.1.529) sub-lineage BA.2 strain.

[0053] In a ninth aspect, the present application provides use of the multivalent nanobody according to the first aspect described above, the nanobody fusion protein according to the second aspect described above, the polynucleotide according to the third aspect described above, the nucleic acid construct according to the fourth aspect described above, the expression vector according to the fifth aspect described above or the transformed cell according to the sixth aspect described above in the preparation of a reagent or kit for detecting a novel coronavirus or for diagnosing a novel coronavirus infection.

[0054] In specific embodiments, the novel coronavirus can be SARS-CoV-2 original strain and / or SARS-CoV-2 variant strain;

[0055] Preferably, the SARS-CoV-2 variant strain is Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2) strain, Omicron (B.1.1.529) sub-lineage BA.1 strain or Omicron (B.1.1.529) sub-lineage BA.2 strain; further preferably, the SARS-CoV-2 variant strain is Delta (B.1.617.2) strain, Omicron (B.1.1.529) sub-lineage BA.1 strain or Omicron (B.1.1.529) sub-lineage BA.2 strain.

[0056] In a tenth aspect, the present application provides a novel coronavirus detection kit comprising the multivalent nanobody according to the first aspect described above, the nanobody fusion protein according to the second aspect described above, the polynucleotide according to the third aspect described above, the nucleic acid construct according to the fourth aspect described above, the expression vector according to the fifth aspect described above or the transformed cell according to the sixth aspect described above.

[0057] Beneficial effects

[0058] The present application is directed to the development of nanobody constructs for the new coronavirus, and the constructs based on nanobody S43 of the present application can all bind to SARS-CoV-2 RBD with high affinity, and can neutralize SARS-CoV-2 prototype strains and a series of mutant strains of pseudovirus and live virus with high neutralization activity, which all indicate that the constructs based on nanobody S43 are novel coronavirus (SARS-CoV-2) nanobodies that can bind to SARS-CoV-2 RBD with high affinity and have high neutralization activity.

[0059] In particular, the inventors have demonstrated through a series of experiments that the trivalent nanobody (TS43) and IgM pentamer form (MS43) based on nanobody S43 of the present application have significantly improved neutralization activity and significantly prolonged half-life compared to their monomer (i.e., nanobody S43), which achieves mucosal immunity, can limit the reproduction and further crossing of the mucosal barrier of the virus, controls the mucosal transmission of the virus, provides a potential nebulizable antibody new drug for the clinical prevention and treatment of the new coronavirus, and can achieve sensitive and reliable detection of the new coronavirus. BRIEF DESCRIPTION OF DRAWINGS

[0060] One or more embodiments are illustrated by way of example in the figures that form a part of this patent document. These example embodiments do not necessarily represent the full scope of the embodiments. The word “example” as used herein means “serving as an example, instance, or illustration.” Any implementation described herein as “example” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0061] Figure 1 is a structural schematic diagram of the nanobody constructs TS43 and MS43 constructed in embodiment 1 of the present application;

[0062] Figure 2 is a chromatogram of S43 protein molecular sieve chromatography and SDS-PAGE identification results described in embodiment 1 of the present application;

[0063] Figure 3 is a chromatogram of TS43 protein molecular sieve chromatography and SDS-PAGE identification results described in embodiment 1 of the present application;

[0064] Figure 4 is a chromatogram of MS43 protein molecular sieve chromatography and SDS-PAGE identification results described in embodiment 1 of the present application;

[0065] Figure 5Figure 1 is a graph showing the SDS-PAGE identification results of SARS-CoV-2 RBD-his protein (A), RBD-his protein of variant strain Omicron (B.1.1.529) subtype BA.1 (B), and RBD-his protein of Omicron (B.1.1.529) subtype BA.2 (C) as described in Example 2 of the present application.

[0066] Figure 6 Figure 2 is a graph showing the neutralization effect of the three antibodies on SARS-CoV-2 prototype strain pseudovirus infection as determined in Example 5 of the present application.

[0067] Figure 7 Figure 3 is a graph showing the neutralization effect of the three antibodies on SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus infection as determined in Example 5 of the present application.

[0068] Figure 8 Figure 4 is a graph showing the neutralization effect of the three antibodies on SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.1 pseudovirus infection as determined in Example 5 of the present application.

[0069] Figure 9 Figure 5 is a graph showing the neutralization effect of the three antibodies on SARS-CoV-2 variant strain Omicron (B.1.1.529) subtype BA.2 pseudovirus infection as determined in Example 5 of the present application.

[0070] Figure 10 Figure 6 is a graph showing the neutralization activity of the three antibodies on pseudovirus before and after nebulization as determined in Example 7 of the present application; wherein A is the neutralization activity of nanobody S43 on SARS-CoV-2 prototype strain pseudovirus before and after nebulization, B is the neutralization activity of nanobody construct TS43 on SARS-CoV-2 prototype strain pseudovirus, and C is the neutralization activity of nanobody construct MS43 on SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus. DETAILED DESCRIPTION

[0071] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application. Unless otherwise clearly indicated, in the entire specification and claims, the term "comprise" or its variants such as "contain" or "include" are understood to include the stated element or component without excluding other elements or components.

[0072] In addition, in order to better illustrate the present application, a large number of specific details are given in the specific embodiments below. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some embodiments, the raw materials, elements, methods, means and the like which are well known to those skilled in the art are not described in detail, so as to highlight the main idea of the present application.

[0073] The present application will be described in detail below.

[0074] Definitions

[0075] "Nanobody", i.e. "heavy-chain single-domain antibody", this kind of antibody only contains one heavy chain variable region (VHH, variable domain of heavy chain of heavy-chain antibody), compared with other antibodies, the light chain is naturally missing.

[0076] Due to the biophysical advantages of nanobodies themselves, they can be easily aerosolized and directly delivered to the lungs through inhalers to treat respiratory viral infections, and are considered to be very potential antibody drugs.

[0077] When referring to ligand / receptor, antibody / antigen or other binding pairs, "specific" binding refers to the determination of the presence or absence of a binding reaction between a protein, for example, a nanobody of the present application and a SARS-CoV-2 RBD protein in a heterogeneous population of proteins and / or other biological reagents. Therefore, under the specified conditions, a specific ligand / antigen binds to a specific receptor / antibody, and does not bind to other proteins present in the sample in a significant amount.

[0078] The chemical materials used in the following examples of the present application, such as reagents, enzymes, culture media, antibiotics and milk, are commercially available products, for example, TRIzol is purchased from Invitrogen, and Superscript II First-Strand Synthesis System for RT-PCR kit is purchased from Invitrogen.

[0079] Some commonly used biological materials, such as competent cells, vectors, helper phages, cells to be transformed, etc., are also commercially available products, for example, pCAGGS vector is purchased from MiaoLing Plasmid; 293F cells, HEK293T cells, etc. are purchased from ATCC; Series Sensor Chip SA chip is purchased from GE Healthcare; Vero cells are purchased from ATCC CCL81.

[0080] Some synthetic biological materials, such as primers, sequences, etc., which are materials that need to be artificially synthesized, are all entrusted to synthesis companies to complete, for example, the coding sequence of TS43 in the present application is synthesized by Beijing Qikexing Biological Technology Co., Ltd.

[0081] Example 1: Construction, expression and purification of trivalent form (TS43) and IgM pentamer form (MS43) antibodies based on nanobody S43

[0082] The structural schematic diagram of the single-chain nanobody and its trivalent form and IgM pentamer form in this example is shown as Figure 1 .

[0083] The basic nanobody S43 used is obtained by immunizing a llama with SARS-CoV-2 S protein, constructing an antibody library, and screening using phage display technology in the laboratory; the amino acid sequence of the VHH chain of the single-chain nanobody S43 is shown as SEQ ID NO: 8, which can specifically bind to SARS-CoV-2 RBD with high affinity (binding constant is 1.2E-10±1.4E-11M), and can neutralize SARS-CoV-2 pseudovirus with high neutralization activity in the pseudovirus neutralization experiment, which all indicate that S43 nanobody is a new coronavirus (SARS-CoV-2) llama-derived nanobody that can bind to SARS-CoV-2 RBD with high affinity and has high neutralization activity.

[0084] The coding sequence of the single-chain nanobody S43VHH chain (as shown in SEQ ID NO: 14) was connected with a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15) at the 5' end and the coding sequence of a hexa-His-tag and a translation termination codon TGA at the 3' end, which was constructed into a pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI, and then the obtained recombinant vector was transfected into 293F cells (purchased from Invitrogen) to express the S43-his protein. The cell culture liquid containing the target protein was purified by nickel ion affinity chromatography (HisTrap TM excel (GE Healthcare) and gel filtration chromatography (Superdex TM 200 Increase 10 / 300 GL column (GE Healthcare), and the relatively pure target protein can be obtained. The SDS-PAGE identification size of the S43-his protein is about 15 KD, and the result is shown in Figure 2

[0085] The coding sequence of three nanobody S43VHH chains as shown in SEQ ID NO: 14 was connected in a head-to-tail form in series through two (GGGGS)3 sequences (directly synthesized by Beijing Chengke Biological Technology Co., Ltd.), which was connected with a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15) at the 5' end and the coding sequence of a hexa-His-tag and a translation termination codon TGA at the 3' end, which was constructed into a pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI, and then the obtained recombinant vector was transfected into 293F cells (purchased from Invitrogen) to express the TS43-his protein. The cell culture liquid containing the target protein was purified by nickel ion affinity chromatography (HisTrap TM excel (GE Healthcare) and gel filtration chromatography (Superdex TM 200 Increase 10 / 300 GL column (GE Healthcare), and the relatively pure target protein can be obtained. The SDS-PAGE identification size of the S43-his protein is about 15 KD, and the result is shown in Figure 3

[0086] ​​The coding sequence of the S43 VHH chain (as shown in SEQ ID NO: 14) was connected to the coding sequence of the Fc of human IgM antibody (as shown in SEQ ID NO: 16) by homologous recombination, a signal peptide (ATMHSSALLCCLVLLTGVRA, SEQ ID NO: 15) was connected at the 5' end, and a translation termination codon TGA was connected at the 3' end. The sequence was constructed into a pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI, to obtain a pCAGGS-S43-IgM Fc recombinant expression vector. The coding sequence of the J chain (as shown in SEQ ID NO: 17) was connected to a translation termination codon TGA at the 3' end, and the sequence was constructed into a pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI, to obtain a pCAGGS-J chain recombinant expression vector. The two recombinant expression vectors pCAGGS-S43-IgM Fc and pCAGGS-J chain were co-transfected into 293F cells (purchased from Invitrogen) to express S43-IgM Fc fusion protein and J chain, and the two were self-assembled to form MS43 protein in the form of IgM. The obtained MS43 protein was purified by HiTrap TM IgM Purification HP (GE Healthcare) and Superose TM 6 increase 10 / 300GL (GE Healthcare) and identified by SDS-PAGE. The SDS-PAGE identification size of the MS43 protein was about 70KD, and the results are shown in Figure 4

[0087] Example 2: Expression and purification of SARS-CoV-2 and RBD of its mutant strains

[0088] A 6-histidine tag (hexa-His-tag) coding sequence and a translation termination codon TGA were connected at the 3' end of the coding sequence of the RBD protein of the original strain of SARS-CoV-2 (the amino acid sequence is shown in SEQ ID NO: 18), and the sequence was constructed into a pCAGGS vector (purchased from Invitrogen) through restriction enzyme sites EcoRI and XhoI. Then, the obtained recombinant vector was transfected into 293F cells (purchased from Invitrogen) to express SARS-CoV-2 RBD-his protein.

[0089] ​The coding sequence of the 6 histidine tag (hexa-His-tag) and the translation termination codon TGA were connected to the 3' end of the coding sequence of the RBD protein of SARS-CoV-2 mutant strain Omicron (B.1.1.529) subtype BA.1 (the amino acid sequence of which is shown as SEQ ID NO: 19) and the RBD protein of SARS-CoV-2 mutant strain Omicron (B.1.1.529) subtype BA.2 (the amino acid sequence of which is shown as SEQ ID NO: 20), respectively, and expressed by the bac-to-bac baculovirus expression system (Invitrogen). The pFastbac1 plasmid containing the target gene was transformed into DH10Bac competent cells to generate recombinant bacmids. The recombinant bacmids were transfected into Sf9 cells to amplify the virus, and protein expression was performed in Hi5 cells. After 48 hours of expression, the supernatant of the Hi5 cells was collected, and the soluble protein was purified by nickel affinity chromatography using HisTrap TM excel (GE Healthcare) through nickel affinity chromatography.

[0090] The cell culture fluid containing the target protein was passed through a nickel ion affinity chromatography column HisTrap TM excel (GE Healthcare) and gel filtration chromatography Superdex TM 200Increase 10 / 300 GL column (GE Healthcare). After purification, the target protein can be obtained with relatively high purity. The SDS-PAGE identification of SARS-CoV-2 RBD-his protein, RBD-his protein of mutant strain Omicron (B.1.1.529) subtype BA.1, and RBD-his protein of mutant strain Omicron (B.1.1.529) subtype BA.2 showed a size of about 30KD, as shown in Figure 5 A-C.

[0091] Example 3: Surface plasmon resonance technology for detecting the binding ability of each antibody to the RBD protein of SARS-CoV-2 original strain and its mutant strain

[0092] Surface plasmon resonance analysis was performed using Biacore 8K (Biacore Inc.). The specific steps are as follows:

[0093] The single-chain nanobodies S43 and its constructs TS43 and MS43 prepared in the above examples were biotinylated and then immobilized on a Series Sensor Chip SA chip (Cytiva Life Sciences); the RBD proteins of the SARS-CoV-2 original strain and its variants prepared in the above examples were diluted by 2 times with PBST buffer (2.7 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 0.05% Tween) from low concentration to high concentration and loaded onto the chip one by one. The calculation of the binding kinetic constant was performed by using BIAevaluation software 8K (Biacore, Inc.) software. The equilibrium dissociation constant (Kd) between each antibody and each RBD was calculated by using the following equation: Kd = (Rmax - Ymin) / 2Ymax D As shown in Table 1, the results of Table 1 show that the single-chain nanobody S43 and its constructs TS43 and MS43 can bind to the RBD proteins of the SARS-CoV-2 original strain and the Omicron subtypes BA.1 and BA.2 of the variant strain with high affinity. Table 1, the affinity results between the single-chain nanobody S43 and its constructs TS43 and MS43 and the RBD of the SARS-CoV-2 original strain and the variant strain

[0094]

[0095] Example 4: Packaging of SARS-CoV-2 original strain and variant strain pseudovirus

[0096] 1) The genes encoding the 18th amino acid of the S protein of the SARS-CoV-2 original strain (WT) and the variant strains Delta (B.1.617.2), Omicron (B.1.1.529) subtype BA.1 and Omicron (B.1.1.529) subtype BA.2 were removed, and the remaining sequence of the S protein was synthesized (synthesis service was provided by Suzhou Jinyu Zhi), obtaining the nucleotide sequences of SARS-CoV-2-WT-S-del18, B.1.617.2-S-del18, B.1.1.529-BA.1-S-del18 and B.1.1.529-BA.2-S-del18 genes, the sequences are shown in SEQ ID NO: 21-24, respectively.

[0097] 2) The protein gene obtained in 1) was respectively cloned into pCAGGS vector to obtain expression plasmids pCAGGS-SARS-CoV-2-WT-S-del18, pCAGGS-B.1.617.2-S-del18, pCAGGS-B.1.1.529-BA.1-S-del18 and pCAGGS-B.1.1.529-BA.2-S-del18.

[0098] The packaging steps of SARS-CoV-2 original strain and variant strain pseudovirus are as follows:

[0099] a. Cell preparation: HEK293T cells (purchased from ATCC CRL-3216) were plated in a 10 cm cell culture dish, and the second day cell confluence density was about 80%. The culture solution was DMEM medium containing 10% FBS.

[0100] b. Transfection: 30 μg of the expression plasmid of each S protein in step 2) above was transfected with PEI per 10 cm cell culture dish. The target plasmid was mixed with PEI at a ratio of 1:3 before transfection. The culture solution (DMEM medium containing 10% FBS) was changed 4-6 h after transfection, and the cells were cultured at 37°C for 24 h.

[0101] c. Virus addition: The pseudovirus packaging backbone virus G*VSV-delG (purchased from Wuhan Privy Brain Science and Technology Co., Ltd.) was added to the transfected HEK293T cells above, incubated at 37°C for 2 h, the culture solution (DMEM medium containing 10% FBS) was changed, and VSV-G antibody (hybridoma cells expressing the antibody were purchased from ATCC cell bank) was added, and the culture was continued in the incubator for 30 h.

[0102] d. Virus collection: The supernatant was centrifuged at 3000 rpm for 10 min, filtered through a 0.45 μm sterile filter in a clean bench, the cell debris was removed, aliquoted, and stored in a -80°C refrigerator.

[0103] Pseudovirus of SARS-CoV-2 original strain (SARS-CoV-2 WT) and variant strains Delta (B.1.617.2), Omicron (B.1.1.529) subtype BA.1, and Omicron (B.1.1.529) subtype BA.2 were obtained, respectively.

[0104] Example 5: Detection of antibody neutralization of pseudovirus infection

[0105] The purified single-chain nanobody S43 and its constructs TS43 and MS43 (prepared in Example 1) were diluted 5 times by gradient to the 9th gradient (2.56 pg / mL), and the diluent was mixed with 1.6 x 10 4 TCID50 The SARS-CoV-2 prototype strain and variant strains obtained in Example 4 were mixed respectively, and incubated at 37°C for 1 h, and then added to a 96-well plate pre-seeded with Vero cells (purchased from ATCC CCL81). After 18-20 hours of incubation, detection was performed by CQ1 Confocal Quantitative Image Cytometer (Yokogawa). According to the number of cells with GFP fluorescence, the neutralization ability of the antibodies to the pseudovirus of the above-mentioned SARS-CoV-2 prototype strain and variant strains Delta, BA.1 and BA.2 was calculated, and the results were as shown in FIG. 2, and the statistical results were as shown in Table 2; the results showed that the neutralization effect of the constructs TS43 and MS43 was improved compared with the single-chain nanobody S43. Figures 6-9 The results showed that the neutralization effect of the constructs TS43 and MS43 was improved compared with the single-chain nanobody S43.

[0106] Table 2, Neutralization ability of single-chain nanobody S43 and its constructs TS43 and MS43 to the pseudovirus of SARS-CoV-2 prototype strain and variant strains

[0107]

[0108] Note: IC 50 (μg / mL) is the half-inhibitory concentration of the antibody. * indicates that the determination at the highest concentration of 10 mg / mL still did not reach 100% inhibition rate.

[0109] Example 6: Detection of antibody neutralization of live virus infection

[0110] In this example, the neutralization effect of each antibody to the live virus of the new coronavirus was determined by a live virus neutralization test based on cytopathic effect (CPE). The specific steps are as follows:

[0111] The purified single-chain nanobody S43 and its TS43 and MS43 (prepared in Example 1) were diluted by 2 times to the 11th gradient, 4 repeated wells for each gradient, and 50 μL for each well. The dilution was incubated with an equal volume of 100 TCID 50 of SARS-CoV-2 prototype strain or its variant strains Delta, Omicron subtype BA.1 at 37°C. After 1 hour, the mixture was added to the suspended Vero cells, and incubated at 37°C for 3 days. The cytopathic effect was observed and recorded. The IC 50 of the nanobody and its constructs was calculated using GraphPad Prism 7.0. The experiments were all carried out in a biosafety level 3 laboratory (BSL3) of the China Disease Prevention and Control Center.

[0112] The neutralizing effects of single-chain nanobody S43 and its TS43 and MS43 on the live virus of the original strain and its variant strain of SARS-CoV-2 are shown in Table 3. The results in Table 3 show that TS43 and MS43 have good inhibitory effects on the live virus of both the original strain and its variant strain of SARS-CoV-2.

[0113] Table 3. Neutralizing ability of single-chain nanobody S43 and its constructs TS43 and MS43 against live SARS-CoV-2 original strains and variant strains.

[0114]

[0115] Note: IC 50 (μg / mL) is the half-inhibitory concentration of the antibody.

[0116] Example 7: Detection of antibody stability before and after nebulization

[0117] Using an Aerogen Solo (Aerogen Inc., Chicago, USA) nebulizer, single-chain nanobody S43 and its constructs TS43 and MS43 were nebulized, respectively. The nebulized antibodies were then collected using an all-glass SKC (Eighty Four, PA, USA) containing 20 mL of PBS, and a pseudovirus neutralization test was performed as described in Example 5. The results are as follows... Figure 10 As shown, A represents the neutralizing activity of nanobody S43 against SARS-CoV-2 prototype strain pseudovirus before and after nebulization; B represents the neutralizing activity of nanobody construct TS43 against SARS-CoV-2 prototype strain pseudovirus; and C represents the neutralizing activity of nanobody construct MS43 against SARS-CoV-2 variant strain Delta (B.1.617.2) pseudovirus. Figure 10 The results showed that the nanobody constructs TS43 and MS43 of the present invention maintained stable neutralizing activity against pseudoviruses of the SARS-CoV-2 prototype strain or its variant strains before and after nebulization, suggesting that they are both suitable for administration via nebulization.

[0118] The above results indicate that the S43-based nanobody constructs TS43 and MS43 of the present invention have the potential to be developed into high- and medium-activity antibody drugs, particularly nebulized drugs, for the treatment of novel coronavirus and its variant strains.

[0119] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application. SEQUENCE LISTING <110> Institute of Microbiology, Chinese Academy of Sciences <120> Constructs of Nanobody S43 and Applications Thereof <130> 1087-220048F <160> 24 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> CDR1 sequence of VHH chain of Nanobody S43 <400> 1 Gly Phe Thr Leu Asp Tyr Tyr Ala Ile Gly 1 5 10 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> CDR2 sequence of VHH chain of Nanobody S43 <400> 2 Cys lie Ser Ser Asn Asn Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 3 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> CDR3 sequence of VHH chain of Nanobody S43 <400> 3 Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr Asp 1 5 10 15 Phe Gly Ser <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR1 sequence of VHH chain of Nanobody S43 <400> 4 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Pro Ser 20 25 <210> 5 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR2 sequence of VHH chain of Nanobody S43 <400> 5 Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Gly Val Ser 1 5 10 <210> 6 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR3 sequence of VHH chain of Nanobody S43 <400> 6 Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gin 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> FR4 sequence of VHH chain of Nanobody S43 <400> 7 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 8 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of VHH chain of Nanobody S43 <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Pro Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Asn Asn Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr 100 105 110 Asp Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 9 <211> 431 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of a trivalent nanobody <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Pro Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Asn Asn Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr 100 105 110 Asp Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly 145 150 155 160 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Thr Cys Ala Pro 165 170 175 Ser Gly Phe Thr Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala 180 185 190 Pro Gly Lys Glu Arg Glu Gly Val Ser Cys Ile Ser Ser Asn Asn Ser 195 200 205 Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 210 215 220 Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu 225 230 235 240 Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Pro Asp Tyr Ser Gly Val 245 250 255 Tyr Tyr Tyr Thr Cys Gly Trp Thr Asp Phe Gly Ser Trp Gly Gln Gly 260 265 270 Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 275 280 285 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 290 295 300 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 305 310 315 320 Ser Leu Arg Leu Thr Cys Ala Pro Ser Gly Phe Thr Leu Asp Tyr Tyr 325 330 335 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 340 345 350 Ser Cys Ile Ser Ser Asn Asn Ser Thr Tyr Tyr Ala Asp Ser Val Lys 355 360 365 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 370 375 380 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 385 390 395 400 Ala Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr 405 410 415 Asp Phe Gly Ser Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser 420 425 430 <210> 10 <211> 349 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Fc fragment of human IgM <400> 10 Val lie Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg 1 5 10 15 Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu lie Cys Gin Ala 20 25 30 Thr Gly Phe Ser Pro Arg Gin lie Gin Val Ser Trp Leu Arg Glu Gly 35 40 45 Lys Gin Val Gly Ser Gly Val Thr Thr Asp Gin Val Gin Ala Glu Ala 50 55 60 Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr lie 65 70 75 80 Lys Glu Ser Asp Trp Leu Gly Gin Ser Met Phe Thr Cys Arg Val Asp 85 90 95 His Arg Gly Leu Thr Phe Gin Gin Asn Ala Ser Ser Met Cys Val Pro 100 105 110 Asp Gin Asp Thr Ala lie Arg Val Phe Ala lie Pro Pro Ser Phe Ala 115 120 125 Ser lie Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp 130 135 140 Leu Thr Thr Tyr Asp Ser Val Thr lie Ser Trp Thr Arg Gin Asn Gly 145 150 155 160 Glu Ala Val Lys Thr His Thr Asn lie Ser Glu Ser His Pro Asn Ala 165 170 175 Thr Phe Ser Ala Val Gly Glu Ala Ser lie Cys Glu Asp Asp Trp Asn 180 185 190 Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser 195 200 205 Pro Leu Lys Gin Thr lie Ser Arg Pro Lys Gly Val Ala Leu His Arg 210 215 220 Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gin Leu Asn Leu Arg 225 230 235 240 Glu Ser Ala Thr lie Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp 245 250 255 Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys 260 265 270 Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro Gly Arg Tyr 275 280 285 Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu Trp Asn Thr Gly 290 295 300 Glu Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro Asn Arg Val 305 310 315 320 Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu Tyr Asn 325 330 335 Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr 340 345 <210> 11 <211> 476 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of a nanobody fusion protein <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Pro Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gin Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Asn Asn Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Glu Pro Asp Tyr Ser Gly Val Tyr Tyr Tyr Thr Cys Gly Trp Thr 100 105 110 Asp Phe Gly Ser Trp Gly Gin Gly Thr Gin Val Thr Val Ser Ser Val 115 120 125 Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg Asp 130 135 140 Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gin Ala Thr 145 150 155 160 Gly Phe Ser Pro Arg Gin Ile Gin Val Ser Trp Leu Arg Glu Gly Lys 165 170 175 Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala Lys 180 185 190 Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile Lys 195 200 205 Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr Cys Arg Val Asp His 210 215 220 Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro Asp 225 230 235 240 Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser Phe Ala Ser 245 250 255 Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp Leu 260 265 270 Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln Asn Gly Glu 275 280 285 Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala Thr 290 295 300 Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn Ser 305 310 315 320 Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser Pro 325 330 335 Leu Lys Gin Thr lie Ser Arg Pro Lys Gly Val Ala Leu His Arg Pro 340 345 350 Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gin Leu Asn Leu Arg Glu 355 360 365 Ser Ala Thr lie Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp Val 370 375 380 Phe Val Gin Trp Met Gin Arg Gly Gin Pro Leu Ser Pro Glu Lys Tyr 385 390 395 400 Val Thr Ser Ala Pro Met Pro Glu Pro Gin Ala Pro Gly Arg Tyr Phe 405 410 415 Ala His Ser lie Leu Thr Val Ser Glu Glu Glu Trp Asn Thr Gly Glu 420 425 430 Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro Asn Arg Val Thr 435 440 445 Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu Tyr Asn Val 450 455 460 Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr 465 470 475 <210> 12 <211> 1293 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Coding sequence of a trivalent nanobody <400> 12 caggtccaac tccaagagag cggcggcggc ctcgtccaac ccggaggatc actcagactc 60 acatgcgccc caagcggctt cacactcgac tactacgcca tcggctggtt cagacaagcc 120 cccggcaaag agagagaagg agtgtcttgc attagcagca acaacagcac ctactacgcc 180 gacagtgtca aaggaagatt caccatcagc agggacaacg ctaagaacac cgtgtatctc 240 cagatgaact cactgaagcc cgaggacacc gccgtgtact actgcgccgc cgagcccgac 300 tacagcggcg tttactacta cacctgcgga tggaccgact tcggcagctg gggccaagga 360 acccaagtca ccgtgagcag cggaggcgga ggaagcggcg gtggaggaag tggcggaggc 420 ggatctgggg ggggaggatc aggcggagga ggaagccagg tgcagctgca ggagagcgga 480 ggaggactgg tgcagccagg aggaagcctg agactgacat gcgcaccaag cggattcaca 540 ctggactatt atgctatcgg atggttcaga caggcccctg gaaaagagag agagggggtg 600 agctgcatca gcagcaataa ctccacatac tacgccgata gcgtcaaggg gaggttcact 660 attagcaggg acaatgcaaa gaacacagtg tacctgcaga tgaacagcct gaagcccgaa 720 gacaccgccg tctactactg cgcagccgag cccgattaca gcggcgtgta ctactacaca 780 tgcggatgga cagacttcgg ctcctggggc caaggcaccc aagtgaccgt gtcaagcgga 840 ggcgggggga gcggaggagg tggaagtgga ggggggggat ctggcggggg aggaagtgga 900 ggaggaggat cacaggtgca gctccaggag agcgggggag gactggtcca gccaggaggg 960 agcctgagac tcacatgtgc acccagcgga tttacactgg attattacgc catcggatgg 1020 tttaggcagg cacccgggaa agagagagag ggcgtgagct gcattagcag taataacagc 1080 acctattacg ccgactcagt gaaggggcgg ttcaccataa gcagggataa cgccaagaac 1140 accgtctacc tgcagatgaa tagcctgaaa cccgaagaca cagccgtgta ctattgcgcc 1200 gccgaacccg actactctgg agtgtactac tatacctgcg gctggaccga ctttggcagc 1260 tgggggcaag gcacccaggt gaccgtgagc agt 1293 <210> 13 <211> 1428 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Coding sequence of a Nanobody fusion protein <400> 13 caggtgcagc tgcaggagag cggaggaggg ctggtgcagc ccggaggaag cctgagactg 60 acctgcgccc ccagcggatt caccctggat tattatgcta ttggctggtt taggcaggct 120 cccggcaaag agagagaggg ggtgtcatgc attagcagca ataactcaac ctactacgcc 180 gacagcgtca agggacgctt caccatttcc agggacaacg ctaagaacac cgtgtatctc 240 cagatgaata gcctgaagcc cgaggacacc gcagtgtact actgcgccgc cgagcccgac 300 tacagcggtg tgtattacta cacctgcgga tggaccgact tcggcagctg gggccaggga 360 acccaggtga cagtgagcag cgtgatcgcc gagctgcccc ccaaggtgag cgtgttcgtg 420 ccccctagag acggcttctt cggcaaccct agaaagagca agctgatctg ccaagccacc 480 ggcttctccc ctagacagat ccaagtgagc tggctgagag agggcaagca agtgggcagc 540 ggcgtcacaa cagaccaagt gcaagccgag gccaaggaga gcggccccac cacctacaag 600 gtgacaagca ccctgaccat caaggagagc gactggctgg ggcagagcat gttcacctgc 660 agagtggacc acagaggcct gacctttcag cagaacgcta gcagcatgtg cgtgcccgac 720 caagacaccg ccatcagagt gttcgccatc ccccctagct tcgctagcat cttcctgacc 780 aagagcacca agctgacctg cctcgtgacc gatctgacca cctacgacag cgtgaccatc 840 agctggacaa gacagaacgg cgaggccgtg aagacccaca ccaacatcag cgagagccac 900 cccaacgcca ccttcagcgc cgtgggcgag gctagcatct gcgaggacga ctggaacagc 960 ggcgagagat tcacctgcac cgtgacccac accgacctgc ctagccccct gaagcagacc 1020 atcagcagac ccaagggcgt ggccctgcac agacccgacg tgtacctgct gccccccgct 1080 agagagcagc tgaacctgag agagagcgcc accatcacct gcctggtgac cggctttagc 1140 cccgctgacg tgttcgtgca gtggatgcag agagggcagc ccctgagccc cgagaagtac 1200 gtgacaagcg cccccatgcc cgagccccaa gcccccggca gatacttcgc ccacagcatc 1260 ctgaccgtga gcgaggaaga gtggaacacc ggcgagacct acacctgcgt ggtggcccac 1320 gaggccctgc ccaacagagt gaccgagaga accgtggaca agagcaccgg caagcccacc 1380 ctgtacaacg tgagcctggt gatgagcgac accgccggca cctgctac 1428 <210> 14 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Coding sequence for Nanobody S43 VHH chain <400> 14 caggtgcagc tgcaggagag cggaggaggg ctggtgcagc ccggaggaag cctgagactg 60 acctgcgccc ccagcggatt caccctggat tattatgcta ttggctggtt taggcaggct 120 cccggcaaag agagagaggg ggtgtcatgc attagcagca ataactcaac ctactacgcc 180 gacagcgtca agggacgctt caccatttcc agggacaacg ctaagaacac cgtgtatctc 240 cagatgaata gcctgaagcc cgaggacacc gcagtgtact actgcgccgc cgagcccgac 300 tacagcggtg tgtattacta cacctgcgga tggaccgact tcggcagctg gggccaggga 360 cccggcaaag agagagaggg ggtgtcatgc attagcagca ataactcaac ctactacgcc 180GAGGTGCAGCTGTGGAGCAGCTGGAGTCTGGGGGAGGCTCTAATGACTTTAGCCTGGTGGTAAC <210> 15 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Signal peptide sequence <400> 15 Ala Thr Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr 1 5 10 15 Gly Val Arg Ala 20 <210> 16 <211> 1047 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Coding sequence for Fc of human IgM antibody <400> 16 gtgatcgccg agctgccccc caaggtgagc gtgttcgtgc cccctagaga cggcttcttc 60 ggcaacccta gaaagagcaa gctgatctgc caagccaccg gcttctcccc tagacagatc 120 caagtgagct ggctgagaga gggcaagcaa gtgggcagcg gcgtcacaac agaccaagtg 180 caagccgagg ccaaggagag cggccccacc acctacaagg tgacaagcac cctgaccatc 240 aaggagagcg actggctggg gcagagcatg ttcacctgca gagtggacca cagaggcctg 300 acctttcagc agaacgctag cagcatgtgc gtgcccgacc aagacaccgc catcagagtg 360 ttcgccatcc cccctagctt cgctagcatc ttcctgacca agagcaccaa gctgacctgc 420 ctcgtgaccg atctgaccac ctacgacagc gtgaccatca gctggacaag acagaacggc 480 gaggccgtga agacccacac caacatcagc gagagccacc ccaacgccac cttcagcgcc 540 gtgggcgagg ctagcatctg cgaggacgac tggaacagcg gcgagagatt cacctgcacc 600 gtgacccaca ccgacctgcc tagccccctg aagcagacca tcagcagacc caagggcgtg 660 gccctgcaca gacccgacgt gtacctgctg ccccccgcta gagagcagct gaacctgaga 720 gagagcgcca ccatcacctg cctggtgacc ggctttagcc ccgctgacgt gttcgtgcag 780 tggatgcaga gagggcagcc cctgagcccc gagaagtacg tgacaagcgc ccccatgccc 840 gagccccaag cccccggcag atacttcgcc cacagcatcc tgaccgtgag cgaggaagag 900 TGGAACACCG GC GAGACCTAC ACCTGC GTG GTGGCCCACG AGGCCCTGCC CAACAGAGTG 960 ACC GAGAGAA CC GTGGACAA GAGC ACCGGC AAGCCC ACCCT GTACAACGT GAGCCTGGTG 1020 ATGAGCGACA CCGCCGGCAC CTGCTAC 1047 <210> 17 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Coding sequence for J chain <400> 17 ATGAAGAACC ACC TGCTGTT CTGGGGCGTG CTGGCCGTGT T CATCAAGGC CGTGCACGTG 60 AAGGCCCAAG AGGACGAGAG AATCGTGCTG GTGGACAACA AGTGCAAGTG CGCTAGAATC 120 ACAAGCAGAA T CATCAGAAG CAGCGAGGAC CCAACGAGGA CATCGTGGAG AGAAACATC 180 AGAATCATCG TGCCCCTGAA CAACAGAGAG AACATCAGCG ACCCCACAAG CCCCCTGAGA 240 ACAAGATTCG TG TACCACTG AGCGACCTGTG CAAGAGTGCGACCCCACCGAGGTGGAG 300 CTGGACAATC AGATCGTGAC CGCCACACAG AGCAACATCT GCGACGAGGA CAGCGCCACC 360 gagacctgct acacctacga cagaaacaag tgctacaccg ccgtggtgcc cctggtgtac 420 ggcggcgaga ccaagatggt ggagaccgcc ctgacccccg acgcctgcta ccccgac 477 <210> 18 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of RBD protein of SARS-CoV-2 original strain <400> 18 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Arg 1 5 10 15 Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu 20 25 30 Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 35 40 45 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 50 55 60 Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 65 70 75 80 Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 85 90 95 Phe Val Ile Arg Gly Asp Glu Val Arg Gin He Ala Pro Gly Gin Thr 100 105 110 Gly Lys He Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 115 120 125 Cys Val He Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly 130 135 140 Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro 145 150 155 160 Phe Glu Arg Asp He Ser Thr Glu He Tyr Gin Ala Gly Ser Thr Pro 165 170 175 Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gin Ser Tyr 180 185 190 Gly Phe Gin Pro Thr Asn Gly Val Gly Tyr Gin Pro Tyr Arg Val Val 195 200 205 Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 210 215 220 Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 225 230 235 <210> 19 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of RBD protein of variant strain Omicron (B.1.1.529) sublineage BA.1 <400> 19 Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr 1 5 10 15 Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala 20 25 30 Ala His Ser Ala Phe Ala Arg Val Gln Pro Thr Glu Ser Ile Val Arg 35 40 45 Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala 50 55 60 Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn 65 70 75 80 Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu Ala Pro Phe Phe Thr 85 90 95 Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe 100 105 110 Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg 115 120 125 Gln Ile Ala Pro Gly Gin Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys 130 135 140 Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys 145 150 155 160 Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe 165 170 175 Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile 180 185 190 Tyr Gin Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys 195 200 205 Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro Thr Tyr Gly Val Gly 210 215 220 His Gin Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala 225 230 235 240 Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn 245 250 255 Lys Cys Val Asn Phe 260 <210> 20 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> MISC_FEATURE <223> Amino acid sequence of RBD protein of variant strain Omicron (B.1.1.529) sublineage BA.2 <400> 20 Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr 1 5 10 15 Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala 20 25 30 Ala His Ser Ala Phe Ala Arg Val Gln Pro Thr Glu Ser Ile Val Arg 35 40 45 Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp Glu Val Phe Asn Ala 50 55 60 Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn 65 70 75 80 Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Phe Ala Pro Phe Phe Ala 85 90 95 Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe 100 105 110 Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asn Glu Val Ser 115 120 125 Gln Ile Ala Pro Gly Gin Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys 130 135 140 Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Lys 145 150 155 160 Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe 165 170 175 Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile 180 185 190 Tyr Gin Ala Gly Asn Lys Pro Cys Asn Gly Val Ala Gly Phe Asn Cys 195 200 205 Tyr Phe Pro Leu Arg Ser Tyr Gly Phe Arg Pro Thr Tyr Gly Val Gly 210 215 220 His Gin Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala 225 230 235 240 Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn 245 250 255 Lys Cys Val Asn Phe 260 <210> 21 <211> 3789 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Nucleotide sequence of SARS-CoV-2-WT-S-del18 gene <400> 21 atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc aatgcgtgaa cctgaccaca 60 agaacacagc tgccccccgc ctacaccaac agcttcacaa gaggcgtgta ctaccccgac 120 aaggtgttca gaagcagcgt cctccacagc acccaagacc tgttcctgcc cttcttcagc 180 aacgtgacct ggttccacgc catcagcggc accaacggca ccaagagatt cgacaacccc 240 gtgctgccct tcaacgacgg cgtgtacttc gctagcaccg agaagagcaa catcatcaga 300 ggctggatct tcggcaccac cctggacagc aaaacacaga gcctgctgat cgtgaacaac 360 gccacaaacg tggtgatcaa ggtgtgcgag tttcagttct gcaacgaccc cttcctgggc 420 gtgtaccaca agaacaacaa gagctggatg gagagcgagt tccgggtgta cagcagcgcc 480 aacaactgca ccttcgagta cgtgagccaa cccttcctga tggacctgga gggcaagcaa 540 ggcaatttta agaacctgag agagttcgtg ttcaagaaca tcgacggcta cttcaagatc 600 tacagcaagc acacccccat caacctggtg agagacctgc cccaaggctt cagcgccctg 660 gagcccctgg tggacctgcc catcggcatc aacatcacaa gatttcagac cctgctggcc 720 ctgcacagaa gctatctgac ccccggcgac agcagcagcg gctggaccgc cggcgccgcc 780 gcttactacg tgggctacct gcagcctaga accttcctgc tgaagtacaa cgagaacggc 840 acaatcaccg acgccgtcga ctgcgccctg gaccccctga gcgagaccaa gtgcaccctg 900 aagagcttca ccgtggagaa gggcatctat cagacaagca acttcagagt gcagcccacc 960 gagagcatcg tgagattccc caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 1020 gccacaagat tcgctagcgt gtacgcctgg aacagaaaga gaatcagcaa ctgcgtggcc 1080 gactacagcg tgctgtacaa cagcgctagc ttcagcacct tcaagtgcta cggcgtcagc 1140 cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcaga 1200 ggcgacgagg tgagacagat cgcccccggg cagaccggca agatcgccga ctacaactac 1260 aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaacaa cctggactcc 1320 aaggtgggcg gcaactacaa ctacctgtac agactgttca gaaagagcaa cctgaagccc 1380 ttcgagagag acatcagcac cgagatctac caagccggca gcaccccctg caacggcgtg 1440 gagggcttca actgctactt ccccctgcag agctacggct ttcagcccac ctacggcgtg 1500 ggctatcagc cctacagagt ggtcgtgctg agcttcgagc tgctgcacgc ccccgccacc 1560 gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttcaacttc 1620 aacggcctca ccgggaccgg cgtgctgacc gagagcaaca agaagttcct gcctttccaa 1680 cagttcggca gagacatcga cgacaccacc gacgccgtca gagaccctca gaccctggag 1740 atcctggaca tcacaccctg cagcttcggc ggcgtgagcg tgatcacccc cggcaccaac 1800 acaagcaacc aagtggccgt gctgtaccaa ggcgtgaact gcaccgaggt gcccgtggcc 1860 atccacgccg atcagctgac ccccacctgg agagtgtaca gcaccggcag caacgtgttt 1920 cagacaagag ccggctgcct gatcggcgcc gagcacgtga acaacagcta cgagtgcgac 1980 atccccatcg gcgccggcat ctgcgctagc tatcagacac agaccaacag ccacagaaga 2040 GCTAGAAGCG TGGCTAGCCA AAGCATCATC GCCTACACCA TGAGCCTGGG CGCCGAGAAC 2100 AGCGTGGCCT ACAGCAACAA CAGCATCGCC ATCCCCACCA ACTTCACCAT CAGCGTGACC 2160 ACCgAAATCC TGCCTGTGAG CATGACCAAG ACAAGCGTGG ACTGCACCAT GTACATCTGC 2220 GGCGACAGCA CCGAGTGCAG CAACCTGCTC CTGCAGTACG GCAGCTTCTG CATTCACTTG 2280 AACAGAGCCC TGACCggCAT CGCCGTGGAG CAAGACAAGA ACACCCAAGA GGTGTTCGCC 2340 CAAGTGAAGC AGATCTACAA GACCCCCCCC ATCAAGGACT TCggCGGCTT CAACITCAGC 2400 CAATCCTGCC TGACCCTAGC AAGCCTAGCA AGAGAAGCTT CATCGAGGAC CTGCTGTTc 2460 AACAAGGTGA CCCTGGCCGA CGCCGGCTTC ATCAAGCAGT ACGGCGACTG CCTGGGCGAC 2520 ATCGCCGTCA GAGACCTGAT CTGCGCTCAG AAGTTCAACG GCCTGACCCT GCTGCCCCCC 2580 CTGCTGACCG ACGAGATGAT CGCTCAGTAC ACAAGCGCCC TGCTCGCTGG CACCATCACA 2640 AGCGGGTGGA CCTTCGGCGC CggGGCCGCC CTGCAGATCC CCTTCGCCAT GCAGATGGCC 2700 TACAGATTC A ACggCATCgg CGTGACACAG AACGTGCTGT ACGAGAATCA GAAGCTGATC 2760 gccaatcagt tcaacagcgc catcggcaag atccaagaca gcctgagcag caccgctagc 2820 gccctgggca agctgcaaga cgtggtgaat cagaacgccc aagccctgaa caccctggtg 2880 aagcagctga gcagcaactt cggcgccatc agcagcgtgc tgaacgacat cctggctaga 2940 ctggacaagg tggaggccga ggtgcagatc gatagactga tcaccggcag actgcagagc 3000 ctgcagacct acgtgacaca gcagctgatc agagccgccg agatcagagc tagcgccaac 3060 ctggccgcca ccaagatgag cgagtgcgtg ctggggcaga gcaagagat ggacttctgc 3120 ggcaagggct accacctgat gagcttccct cagagcgcc cccacggcgt ggtgttcctg 3180 cacgtgacct acgtgcccgc ccaagagaag aacttcacca ccgcccccgc catctgccac 3240 gacggcaagg cccacttccc tagagagggc gtgttcgtga gcaacggcac ccactggttc 3300 gtgacacaga gaaacttcta cgagcctcag atcatcacca cccacaacac cttcgtgagc 3360 ggcaactgcg acgtggtgat cggcatcgtg aacaacaccg tgtacgaccc tctgcagccc 3420 gagctggaca gcttcaagga ggagctggac aagtacttca agaaccacac aagccccgac 3480 gtggacctgg gcgacatcag cgggatcaac gctagcgtgg tgaacattca gaaggaaatc 3540 gacagactga atgaggtggc caagaacctg aacgagagcc tgatcgacct gcaagagctg 3600 ggcaagtacg agcagtacat caagtggccc tggtacatct ggctgggctt catcgccggc 3660 ctgatcgcca tcgtgatggt gaccatcatg ctgtgctgca tgacaagctg ctgctcctgt 3720 ctgaaggggt gctgcagctg cggcagctgc tgcaaggact acaaggacga tgacgacaag 3780 ggcccctga 3789 <210> 22 <211> 3792 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Nucleotide sequence of the B.1.617.2-S-del18 gene <400> 22 atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgagaaca 60 cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120 aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180 aacgtgacat ggttccacgc catccacgtg tctggcacaa acggcacaaa gcggttcgac 240 aaccccgtgc tccctttcaa cgacggcgtg tacttcgcca gcaccgagaa gtctaacatt 300 atccggggct ggattttcgg caccacactc gactctaaga cacagtccct cctgattgtg 360 aacaacgcca caaacgtggt gattaaggtg tgcgagttcc agttctgcaa cgaccctttc 420 ctggacgtgt actaccacaa gaacaacaag tcttggatgg agtctggcgt gtactctagc 480 gccaacaact gcaccttcga gtacgtgtcc cagcctttcc tcatggacct ggagggcaag 540 cagggcaact tcaagaacct gagagagttc gtgttcaaga acattgacgg ctacttcaag 600 atttactcta agcacacccc aattaacctc gtgagggacc tccctcaggg cttctccgtg 660 ttagaaccac tggtggacct ccctattggc attaacatca cacgcttcca gacactgctc 720 gccctccacc ggtcttacct gaccccaggc gactctagct ctggctggac agccggcgcc 780 gccgcctact acgtgggcta cctgcagcct aggaccttcc tcctgaagta caacgagaac 840 ggcacaatta ccgacgccgt ggactgcgcc ctggacccac tgtccgagac aaagtgcaca 900 ctgaagtcct tcacagtgga gaagggcatt taccagacat ctaacttccg ggtgcagcct 960 acagagtcta ttgtgcggtt cccaaacatc acaaacctgt gccctttcgg cgaggtgttc 1020 aacgccaccc ggttcgcctc tgtgtacgcc tggaaccgga agcggatctc taactgcgtg 1080 gccgactact ccgtgctgta caactccgcc tctttctcta cattcaagtg ctacggcgtg 1140 tcccctacaa agctgaacga cctgtgcttc accaacgtgt acgccgactc tttcgtgatt 1200 agaggcgacg aggtgaggca gattgccccc ggccagacag gcaagatcgc cgactacaac 1260 tacaagctgc ccgacgactt cacaggctgc gtgatcgcct ggaactctaa caacctggac 1320 tctaaggtgg gcggcaacta caactacaga tacagactgt tccggaagtc taacctgaag 1380 ccattcgaga gggacattag caccgagatt taccaggccg gctctaagcc atgcaacggc 1440 gtggagggct tcaactgcta cttcccactg cagtcctacg gcttccagcc tacaaacggc 1500 gtgggctacc agccttaccg ggtggtggtg ctgtctttcg agctgctcca cgcccccgcc 1560 acagtgtgcg gcccaaagaa gagcacaaac ctcgtgaaga acaagtgcgt gaacttcaac 1620 ttcaacggcc tcacaggcac aggcgtgctc accgagtcta acaagaagtt cctccctttc 1680 cagcagttcg gccgcgacat tgccgacacc accgacgccg tgcgggaccc tcagacactg 1740 gaaattctcg acatcacccc ttgcagcttc ggcggcgtgt ccgtgatcac cccaggcaca 1800 aacacatcta accaggtggc cgtgctgtac cagggcgtga actgcaccga ggtgccagtg 1860 gccatccacg ccgaccagct caccccaaca tggagggtgt acagcacagg ctctaacgtg 1920 ttccagaccc gggccggctg cctcattggc gccgagcacg tgaacaactc ttacgagtgc 1980 gacatcccta ttggcgccgg catttgcgcc tcttaccaga cccagacaaa ctctagacgg 2040 agagcccggt ctgtggcctc tcagagcatt attgcctaca ccatgtctct gggcgccgag 2100 aactctgtgg cctactctaa caactctatt gccatcccta caaacttcac aatttctgtg 2160 accaccgaga ttctcccagt gtctatgacc aagacatctg tggactgcac catgtacatt 2220 tgcggcgact ccaccgagtg ctctaacctc ctgctccagt acggctcttt ctgcacccag 2280 ctcaaccgcg ccctgacagg catcgccgtg gagcaggaca agaacaccca ggaggtgttc 2340 GCCCAGGTGA AGCAGATTTA CAAGACCCCC CCAATTAAGG ACTTCGGCGG CTTCAACTTC 2400 TCTCAGATTC TCCCCGACCC ATCCAAGCCT AGCAAGCGGT CCTTCATCGA GGACCTCCTG 2460 TTCAACAAGG TGACACTGGC CGACGCCGGC TTCATTAAGC AGTACGGCGA CTGCCTGGGC 2520 GACATCGCCG CCCGGGACCT GATTTGCGCC CAGAAGTTCA ACGGCCTCAC AGTGCTCCCC 2580 CCACTGCTCA CCGACGAGAT GATTGCCCAG TACACATCTG CCCTCCTGGC CGGCACAATT 2640 ACATCTGGCT GGACCTTCGG CGCCGGCGCC GCCCTGCAGA TCCCTTTCGC CATGCAGATG 2700 GCCTACCGCT TCAACGGCAT CGGCgtGAC ACAGAACGTG CTGTACGAAG ACCAGAAGCT G 2760 ATCGCCAACC AGTTCAACAG CGCCATTGGC AAGATTCAgg ACTCTCTGAG CAGCACAGCC 2820 AGCGCCCTGG GCAAGCTGCA GAACGTGGTG AACCAGAACG CCCAGGCCCT GAACACACTG 2880 GTGAAGCAGC GTGTCTTCTA ACTTCGGCGC CATTTC TAGC GTGCTGAACG ACATTCTGTC G 2940 CggCTGGACA AGGTGGAGGC CGAGGTGCAG ATTGACAGG CTATACACAG GCAGACTGCAG 3000 TCTCTGCAGA CATACGTGAC CCAGCAGCTG ATTAGAGCCG CCGAGATTAG AGCCTCCGCC 3060 aacctggccg ccaccaagat gagcgagtgc gtgctcggcc agtctaagcg ggtggacttc 3120 tgcggcaagg gctaccacct catgtctttc cctcagtccg cccctcacgg cgtggtgttc 3180 ctccacgtga catacgtgcc cgcccaggag aagaacttca ccacagcccc cgccatttgc 3240 cacgacggca aggcccactt ccctagggag ggcgtgttcg tgtctaacgg cacccactgg 3300 ttcgtgaccc agcggaactt ctacgagcct cagattatta ccacagacaa cacattcgtg 3360 agcggcaact gcgacgtggt gattggcatt gtgaacaaca cagtgtacga cccactgcag 3420 cctgagttgg actctttcaa ggaggaactc gacaagtact tcaagaacca cacatctcct 3480 gacgtggacc tgggcgacat tagcggcatt aacgcctctg tggtgaacat tcagaaggag 3540 attgacagac tgaacgaggt ggccaagaac ctgaacgagt ctctcattga cctgcaggag 3600 ctgggcaagt acgagcagta cattaagtgg ccttggtaca tttggctggg cttcattgcc 3660 ggcctgatcg ccattgtgat ggtgaccatc atgctgtgct gcatgacatc ttgctgcagc 3720 tgcctgaagg gctgctgctc ttgcggctct tgctgcaagg actacaagga cgacgatgac 3780 aagggacctt aa 3792 <210> 23 <211> 3795 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <220> <221> misc_feature <223> Nucleotide sequence of B.1.1.529-BA.1-S-del18 gene <400> 23 atgttcgtgt tcctcgtgct cctgcctctg gtgtctagcc agtgcgtgaa cctgaccaca 60 cggacccagc tccctcccgc ctacacaaac tctttcaccc ggggcgtgta ctaccccgac 120 aaggtgttcc ggtctagcgt gctccactct acacaggacc tgttcctccc tttcttcagc 180 aacgtgacat ggttccacgt gatctctggc acaaacggca caaagcggtt cgacaacccc 240 gtgctccctt tcaacgacgg cgtgtacttc gccagcattg agaagtctaa cattatccgg 300 ggctggattt tcggcaccac actcgactct aagacacagt ccctcctgat tgtgaacaac 360 gccacaaacg tggtgattaa ggtgtgcgag ttccagttct gcaacgaccc tttcctggac 420 cacaagaaca acaagtcttg gatggagtct gagttcagag tgtactctag cgccaacaac 480 tgcaccttcg agtacgtgtc ccagcctttc ctcatggacc tggagggcaa gcagggcaac 540 ttcaagaacc tgagagagtt cgtgttcaag aacattgacg gctacttcaa gatttactct 600 aagcacaccc caattattgt gagggaacca gaagacctcc ctcagggctt ctccgcctta 660 gaaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc 720 ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780 gcctactacg tgggctacct gcagcctagg accttcctcc tgaagtacaa cgagaacggc 840 acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900 aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960 gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020 gccacccggt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080 gactactccg tgctgtacaa cctggcccct ttcttcacat tcaagtgcta cggcgtgtcc 1140 cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200 GGCGACGAGG TGAGGCA GAT TGCC CCCGGC CAGACAGGCA ACATCGCCG A CTACAAC TAC 1260 AAGCTGCCCG AC GACTTCAC AGGCTGC GTG ATCGCCTGG A CTCTTCAA C AAGCTGGAC TCT 1320 AAGGTGTCTG GCAACTACAA CTACCTGTAC AGACTGTTCC GGAAGTCTAA CCTGAAGCCA 1380 TTCGAGAGGG ACATTAGCAC CGAGATTTAC CAGGCCGGCA ACAAGCCATG CAACGGCGTG 1440 GCCGGCTTCA ACTGCTACTT CCCACTGCGC TCCTACTCCT TCCGGCCTAC ATACGGCGTG 1500 GGCCACCAGC CTTACC GGGTG GTGGTGCTG TCTTTCGAGC TGCTCCACGC CCCCGCCACA 1560 GTGTGCGGCC CAAAGAAGAG CACAAACCTC GTGAAGAACA AGTGC GTGA CTTCAACTTC 1620 AACGGCCTCA AGGGCACAGG CGTGCTCACC GAGTCTAAC AAGAAGTTCCT CCCTTTCCAG 1680 CAGTTCGGCC GCGACATTGC CGACACCACC GACGCCGTGC GGGACCCTCA GACACTGGAA 1740 ATTCTCGACA TCACCCCTTG CAGCTTCGGC GGCGTGTCCG TGATCACCCC AGGCACAAAC 1800 ACATCTAACCA GGTGGCCGTG CTGTACCAGG GC GTGAAC TGC ACCGAGGTG CAGTGGCC 1860 ATCCACGCCG ACCAGCTCAC CCAACATGGA GG GTGTACAGC ACAGGCTCTAAC GTGTT C 1920 caaacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980 atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040 gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100 tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160 accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220 ggcgactcca ccgagtgctc taacctcctg ctccagtacg gctctttctg cacccagctc 2280 aagcgcgccc tgacaggcat cgccgtggag caggacaaga acacccagga ggtgttcgcc 2340 caggtgaagc agatttacaa gaccccccca attaagtact tcggcggctt caacttctct 2400 cagattctcc ccgacccatc caagcctagc aagcggtcct tcattgagga cctcctgttc 2460 aacaaggtga cactggccga cgccggcttc attaagcagt acggcgactg cctgggcgac 2520 attgccgccc gggacctgat ttgcgcccag aagttcaagg gcctcacagt gctcccccca 2580 ctgctcaccg acgagatgat tgcccagtac acatctgccc tcctggccgg cacaattaca 2640 TCTGGCTGGACCTTCGGCGCCGGCGCCGCCCTGCAGATCCCTTTCGCCAT GCAGATGGCC 2700 TACCgCTTCAACGGCATCGGCgtGAcaCAGAACGTGCTGTACGAGAACCA GAAGCTGATC 2760 GCCAACCAGTTCAACAGCGCCATTGGCAAGATTCAGGACTCTCTGA GCAGCACAGCCAGC 2820 GCCCTGGGCAAGCTGCAGGACGTGgtGAACCACAACGCCCAggCCCTGAAC ACACCTGgtG 2880 AAGCAGCTGTTCTTAAGTTcGGCGCCATTTCTAGCGTGCTGAACGA CATTTTCTCGG 2940 CTGGACAAGGTGGAGGCCGAGGTGCAGATtgACAGGCTCATCACAGGcAGACTGCAGTCT 3000 CTGCAGACATACGTGACCAGCAGCTGATTAGAGCCGCCGAGATTAGAG CCTCCGCCAAC 3060 CTGGCCGCCACCAAGATGAGCGAGTGCgtGCtcGGCCAGTCTAAGCGGGT GGACTTCTGC 3120 GGCAAGGGCTACCACCCATGTCTTTCCCTCAGTCCGCCCTCacGGCgtGgtgtTCCTC 3180 CACGTGACATACGTGCCCgCCCAGGAGAAGAaCTTCACCAcAGCCCCCGCC ATTTGCCAC 3240 GACGGCAAGGCCCACTTCCCTAGGGAGGGCgtGTTCGTGTCtAACGGCACCCACTGgtTC 3300 GTGACCCAGCGGAACCTTCTACGAGCCTCAGATTATTACCAcAGACAAcACATTcGTGAGC 3360 ggcaactgcg acgtggtgat tggcattgtg aacaacacag tgtacgaccc actgcagcct 3420 gagttggact ctttcaagga ggaactcgac aagtacttca agaaccacac atctcctgac 3480 gtggacctgg gcgacattag cggcattaac gcctctgtgg tgaacattca gaaggagatt 3540 gacagactga acgaggtggc caagaacctg aacgagtctc tcattgacct gcaggagctg 3600<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​cggacccaga gctacacaaa ctctttcacc cggggcgtgt actaccccga caaggtgttc 120 cggtctagcg tgctccactc tacacaggac ctgttcctcc ctttcttcag caacgtgaca 180 tggttccacg ccatccacgt gtctggcaca aacggcacaa agcggttcga caaccccgtg 240 ctccctttca acgacggcgt gtacttcgcc agcaccgaga agtctaacat tatccggggc 300 tggattttcg gcaccacact cgactctaag acacagtccc tcctgattgt gaacaacgcc 360 acaaacgtgg tgattaaggt gtgcgagttc cagttctgca acgacccttt cctggacgtg 420 tactaccaca agaacaacaa gtcttggatg gagtctgagt tcagagtgta ctctagcgcc 480 aacaactgca ccttcgagta cgtgtcccag cctttcctca tggacctgga gggcaagcag 540 ggcaacttca agaacctgag agagttcgtg ttcaagaaca ttgacggcta cttcaagatt 600 tactctaagc acaccccaat taacctcggc agggacctcc ctcagggctt ctccgcctta 660 gaaccactgg tggacctccc tattggcatt aacatcacac gcttccagac actgctcgcc 720 ctccaccggt cttacctgac cccaggcgac tctagctctg gctggacagc cggcgccgcc 780 gcctactacg tgggctacct gcagcctagg accttctcc tgaagtacaa cgagaacggc 840 acaattaccg acgccgtgga ctgcgccctg gacccactgt ccgagacaaa gtgcacactg 900 aagtccttca cagtggagaa gggcatttac cagacatcta acttccgggt gcagcctaca 960 gagtctattg tgcggttccc aaacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020 gccacccggt tcgcctctgt gtacgcctgg aaccggaagc ggatctctaa ctgcgtggcc 1080 gactactccg tgctctacaa cttcgcccc ttcttcgcct tcaagtgcta cggcgtgtcc 1140 cctacaaagc tgaacgacct gtgcttcacc aacgtgtacg ccgactcttt cgtgattaga 1200 ggcaacgagg tgagccagat tgccccggc cagacaggca acatcgccga ctacaactac 1260 aagctgcccg aggacttcac aggctgcgtg atcgcctgga actctaacaa gctggactct 1320 aaggtgggcg gcaactacaa ctacctgtac agactgttcc ggaagtctaa cctgaagcca 1380 ttcgagaggg acattagcac cgagatttac caggccggca acaagccatg caacggcgtg 1440 gccggcttca actgctactt cccactgcgg tcctacggct tccggcctac atacggcgtg 1500 ggccaccagc cttaccgggt ggtggtgctg tctttcgagc tgctccacgc ccccgccaca 1560 gtgtgcggcc caaagaagag cacaaacctc gtgaagaaca agtgcgtgaa cttcaacttc 1620 aacggcctca caggcacagg cgtgctcacc gagtctaaca agaagttcct ccctttccag 1680 cagttcggcc gcgacattgc cgacaccacc gacgccgtgc gggaccctca gacactggaa 1740 attctcgaca tcaccccttg cagcttcggc ggcgtgtccg tgatcacccc aggcacaaac 1800 acatctaacc aggtggccgt gctgtaccag ggcgtgaact gcaccgaggt gccagtggcc 1860 atccacgccg accagctcac cccaacatgg agggtgtaca gcacaggctc taacgtgttc 1920 cagacccggg ccggctgcct cattggcgcc gagtacgtga acaactctta cgagtgcgac 1980 atccctattg gcgccggcat ttgcgcctct taccagaccc agacaaagtc tcaccggaga 2040 gcccggtctg tggcctctca gagcattatt gcctacacca tgtctctggg cgccgagaac 2100 tctgtggcct actctaacaa ctctattgcc atccctacaa acttcacaat ttctgtgacc 2160 accgagattc tcccagtgtc tatgaccaag acatctgtgg actgcaccat gtacatttgc 2220 ggcgactccaccgagtgctctaacctcctgctccagtacggctctttctgcacccagctc 2280 aagcgcgccctgacaggcatcgccgtggagcaggacaagaacacccaggaggtgttcgcc 2340 caggtgaagcagatttacaagacccccccaattaagtacttcggcggcttcaacttctct 2400 cagattctcccccgacccatccaagcctagcaagcggtccttcattgaggacctcctgttc 2460 aacaaggtgacactggccgacgccggcttcattaagcagtacggcgactgcctgggcgac 2520 attgccgcccgggacctgatttgcgcccagaagttcaacggcctcacagtgctcccccca 2580 ctgctcaccgacgagatgattgcccagtacacatctgccctcctggccggcacaattaca 2640 tctggctggaccttcggcgccggcgccgccctgcagatccctttcgccatgcagatggcc 2700 taccgcttcaacggcatcggcgtgacacagaacgtgctgtacgagaaccagaagctgatc 2760 gccaaccagttcaacagcgccattggcaagattcaggactctctgagcagcacagccagc 2820 gccctgggcaagctgcaggacgtggtgaaccacaacgcccaggccctgaacacactggtg 2880 aagcagctgtcttctaagttcggcgccattagc 2913

Claims

1. A multivalent nanobody comprising two or more VHH chains of a nanobody that specifically binds to SARS-CoV-2 RBD, wherein, The VHH chain of the nanobody specifically binding to SARS-CoV-2 RBD comprises the following CDRs: CDR1 with an amino acid sequence as set forth in SEQ ID NO: 1, CDR2 with an amino acid sequence as set forth in SEQ ID NO: 2, and CDR3 with an amino acid sequence as set forth in SEQ ID NO:

3.

2. The multivalent nanobody of claim 1, wherein The VHH chain of the nanobody specifically binding to SARS-CoV-2 RBD further comprises four framework regions FR1-4, which are sequentially interspersed with the CDR1, CDR2 and CDR3.

3. The multivalent nanobody of claim 2, wherein The amino acid sequences of the FR1-4 are set forth in SEQ ID NOs: 4, 5, 6, and 7, respectively.

4. The multivalent nanobody of claim 1, wherein, The VHH chain of the nanobody specifically binding to SARS-CoV-2 RBD has an amino acid sequence as set forth in SEQ ID NO: 8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

8.

5. The multivalent nanobody according to any one of claims 1-4, which is composed of two or more VHH chains of the nanobody specifically binding to SARS-CoV-2 RBD connected by a Linker. wherein The Linker is (GGGGS)n, wherein n = 1, 2, 3, or 4.

6. The multivalent nanobody of claim 5, wherein, The multivalent nanobody is a trivalent nanobody, which is composed of three VHH chains of the nanobody specifically binding to SARS-CoV-2 RBD connected by a Linker.

7. The multivalent nanobody according to claim 6, characterized in that, The trivalent nanobody has an amino acid sequence as set forth in SEQ ID NO:

9.

8. The multivalent nanobody of any one of claims 1-4, wherein, The multivalent nanobody is an IgM pentamer formed by a fusion protein having a structure from N-terminus to C-terminus as set forth in formula (I): A-L-B (I); wherein, A is the multivalent nanobody according to any one of claims 5-7; B is an Fc fragment of human IgM; L is (GGGGS)m, wherein m = 0, 1, 2, 3, or 4. The Fc fragment of human IgM has an amino acid sequence as set forth in SEQ ID NO:

10.

9. The multivalent nanobody of claim 8, wherein, The multivalent nanobody is an IgM pentamer formed by a fusion protein having a structure from N-terminus to C-terminus as set forth in formula (I):

10. The multivalent nanobody of any one of claims 1-4, wherein, A-L-B (I); wherein, A is a single VHH chain of the nanobody specifically binding to SARS-CoV-2 RBD; B is an Fc fragment of human IgM; L is (GGGGS)m, wherein m = 0, 1, 2, 3, or 4. The Fc fragment of human IgM has an amino acid sequence as set forth in SEQ ID NO:

10. The fusion protein has an amino acid sequence as set forth in SEQ ID NO:

11.

11. The multivalent nanobody of claim 10, wherein, The nanobody fusion protein has a structure from N-terminus to C-terminus as set forth in formula (I):

12. The multivalent nanobody of claim 10, wherein, A-L-B (I); wherein, 13. A nanobody fusion protein, characterized in that, ​ ​ ​ A is a VHH chain of a nanobody specifically binding to SARS-CoV-2 RBD, the VHH chain of a nanobody specifically binding to SARS-CoV-2 RBD being as defined in any one of claims 1-4, or A is a multivalent nanobody according to any one of claims 5-7; B is an Fc fragment of human IgM; L is (GGGGS)m, wherein m = 0, 1, 2, 3, or 4.

14. The Nanobody fusion protein of claim 13, wherein, The Fc fragment of human IgM has an amino acid sequence as set forth in SEQ ID NO:

10.

15. The nanobody fusion protein of claim 13, wherein, The nanobody fusion protein has an amino acid sequence as set forth in SEQ ID NO:

11.

16. A polynucleotide encoding the multivalent nanobody of any one of claims 1 to 12, or encoding the nanobody fusion protein of any one of claims 13 to 15.

17. The polynucleotide of claim 16, wherein The polynucleotide is DNA or mRNA.

18. The polynucleotide of claim 16, wherein The polynucleotide encodes the multivalent nanobody of any one of claims 5 to 7.

19. The polynucleotide of claim 18, wherein, The polynucleotide comprises a nucleotide sequence as set forth in SEQ ID NO:

12.

20. The polynucleotide of claim 16, wherein The polynucleotide encodes the nanobody fusion protein of claim 6.

21. The polynucleotide of claim 20, wherein, The polynucleotide comprises a nucleotide sequence as set forth in SEQ ID NO:

13.

22. A nucleic acid construct comprising the polynucleotide of any one of claims 16 to 21.

23. The nucleic acid construct of claim 22, wherein, The nucleic acid construct further comprises at least one expression regulatory element operably linked to the polynucleotide.

24. An expression vector comprising the nucleic acid construct of claim 22 or 23.

25. A transformed cell comprising the polynucleotide of any one of claims 16 to 21, the nucleic acid construct of claim 22 or 23, or the expression vector of claim 24.

26. A pharmaceutical composition comprising the multivalent nanobody of any one of claims 1 to 12, the nanobody fusion protein of any one of claims 13 to 15, the polynucleotide of any one of claims 16 to 21, the nucleic acid construct of claim 22 or 23, the expression vector of claim 24, or the transformed cell of claim 25, and a pharmaceutically acceptable carrier and / or excipient.

27. The pharmaceutical composition of claim 26, wherein, The pharmaceutical composition is in the form of a nasal spray, an oral preparation, a suppository, a transdermal preparation, an ointment, a plaster, a liquid for external use, or an injectable preparation.

28. The pharmaceutical composition of claim 27, wherein, The nasal spray is selected from the group consisting of an aerosol, a spray, and a powder spray; The oral preparation is selected from the group consisting of a tablet, a powder, a pill, a granule, a soft / hard capsule, and a film-coated agent; The injectable preparation is a bolus preparation.

29. The pharmaceutical composition of claim 28, wherein, The tablet is a sublingual tablet; The powder is a powder for dispersing; The pill is a small pill; The granule is a fine granule.

30. Use of the multivalent Nanobody of any one of claims 1 to 5, 8-9, the Nanobody fusion protein of any one of claims 13 to 15, the polynucleotide of any one of claims 16 to 21, the nucleic acid construct of claim 22 or 23, the expression vector of claim 24, the transformed cell of claim 25, or the pharmaceutical composition of any one of claims 26 to 29 for the manufacture of a medicament for the prevention and / or treatment of a SARS-CoV-2 infection.

31. Use of the multivalent Nanobody of any one of claims 1 to 12, the Nanobody fusion protein of any one of claims 13 to 15, the polynucleotide of any one of claims 16 to 21, the nucleic acid construct of claim 22 or 23, the expression vector of claim 24, or the transformed cell of claim 25 for the manufacture of a reagent or kit for the detection of SARS-CoV-2 or for the diagnosis of a SARS-CoV-2 infection.

32. The use according to claim 30 or 31, characterized in that The SARS-CoV-2 is a SARS-CoV-2 original strain and / or a SARS-CoV-2 variant strain.

33. The use according to claim 32, wherein The SARS-CoV-2 variant strain is an Alpha, Beta, Gamma, Kappa, Delta strain, an Omicron sublineage BA.1 strain, or an Omicron sublineage BA.2 strain.

34. The use of claim 32, wherein, The SARS-CoV-2 variant strain is a Delta strain, an Omicron sublineage BA.1 strain, or an Omicron sublineage BA.2 strain.

35. A SARS-CoV-2 detection kit comprising the multivalent Nanobody of any one of claims 1 to 12, the Nanobody fusion protein of any one of claims 13 to 15, the polynucleotide of any one of claims 16 to 21, the nucleic acid construct of claim 22 or 23, the expression vector of claim 24, or the transformed cell of claim 25.

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