SINGLE-DOMAIN ANTIBODY AGAINST CPV AND USES THEREOF

Abstract

A single-domain antibody or an antigen-binding fragment thereof for neutralizing canine parvovirus (CPV) including a heavy chain encompassing a variable region. The variable region includes three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3. The three complementarity-determining regions CDR1, CDR2, and CDR3 have a combination of different amino acid sequences. The antibodies effectively prevent CPV from infecting F81 cells, exhibiting high binding affinity and specificity, and significant neutralizing activity against CPV. Consequently, the antibody sequences and expression vectors of the disclosure are valuable tools for various applications in CPV epitope identification, prevention, treatment, and diagnosis.

Claims

1. A single-domain antibody or an antigen-binding fragment thereof for neutralizing canine parvovirus (CPV), the single-domain antibody or antigen-binding fragment thereof comprising a heavy chain encompassing a variable region, and the variable region comprising three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3; the three complementarity-determining regions CDR1, CDR2, and CDR3 having a combination of amino acid sequences as follows: (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 49, and CDR3 shown in SEQ ID NO: 97; (2) CDR1 shown in SEQ ID NO: 2, CDR2 shown in SEQ ID NO: 50, and CDR3 shown in SEQ ID NO: 98; (3) CDR1 shown in SEQ ID NO: 3, CDR2 shown in SEQ ID NO: 51, and CDR3 shown in SEQ ID NO: 99; (4) CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 52, and CDR3 shown in SEQ ID NO: 100; (5) CDR1 shown in SEQ ID NO: 5, CDR2 shown in SEQ ID NO: 53, and CDR3 shown in SEQ ID NO: 101; (6) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 54, and CDR3 shown in SEQ ID NO: 102; (7) CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 55, and CDR3 shown in SEQ ID NO: 103; (9) CDR1 shown in SEQ ID NO: 8, CDR2 shown in SEQ ID NO: 56, and CDR3 shown in SEQ ID NO: 104; (10) CDR1 shown in SEQ ID NO: 9, CDR2 shown in SEQ ID NO: 57, and CDR3 shown in SEQ ID NO: 105; (12) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 58, and CDR3 shown in SEQ ID NO: 106; (13) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 59, and CDR3 shown in SEQ ID NO: 107; (14) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 60, and CDR3 shown in SEQ ID NO: 108; (15) CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 61, and CDR3 shown in SEQ ID NO: 109; (17) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 62, and CDR3 shown in SEQ ID NO: 110; (18) CDR1 shown in SEQ ID NO: 15, CDR2 shown in SEQ ID NO: 63, and CDR3 shown in SEQ ID NO: 111; (19) CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 64, and CDR3 shown in SEQ ID NO: 112; (20) CDR1 shown in SEQ ID NO: 17, CDR2 shown in SEQ ID NO: 65, and CDR3 shown in SEQ ID NO: 113; (21) CDR1 shown in SEQ ID NO: 18, CDR2 shown in SEQ ID NO: 66, and CDR3 shown in SEQ ID NO: 114; (22) CDR1 shown in SEQ ID NO: 19, CDR2 shown in SEQ ID NO: 67, and CDR3 shown in SEQ ID NO: 115; (24) CDR1 shown in SEQ ID NO: 20, CDR2 shown in SEQ ID NO: 68, and CDR3 shown in SEQ ID NO: 116; (25) CDR1 shown in SEQ ID NO: 21, CDR2 shown in SEQ ID NO: 69, and CDR3 shown in SEQ ID NO: 117; (26) CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 70, and CDR3 shown in SEQ ID NO: 118; (27) CDR1 shown in SEQ ID NO: 23, CDR2 shown in SEQ ID NO: 71, and CDR3 shown in SEQ ID NO: 119; (29) CDR1 shown in SEQ ID NO: 24, CDR2 shown in SEQ ID NO: 72, and CDR3 shown in SEQ ID NO: 120; (30) CDR1 shown in SEQ ID NO: 25, CDR2 shown in SEQ ID NO: 73, and CDR3 shown in SEQ ID NO: 121; (31) CDR1 shown in SEQ ID NO: 26, CDR2 shown in SEQ ID NO: 74, and CDR3 shown in SEQ ID NO: 122; (32) CDR1 shown in SEQ ID NO: 27, CDR2 shown in SEQ ID NO: 75, and CDR3 shown in SEQ ID NO: 123; (33) CDR1 shown in SEQ ID NO: 28, CDR2 shown in SEQ ID NO: 76, and CDR3 shown in SEQ ID NO: 124; (34) CDR1 shown in SEQ ID NO: 29, CDR2 shown in SEQ ID NO: 77, and CDR3 shown in SEQ ID NO: 125; (35) CDR1 shown in SEQ ID NO: 30, CDR2 shown in SEQ ID NO: 78, and CDR3 shown in SEQ ID NO: 126; (36) CDR1 shown in SEQ ID NO: 31, CDR2 shown in SEQ ID NO: 79, and CDR3 shown in SEQ ID NO: 127; (39) CDR1 shown in SEQ ID NO: 32, CDR2 shown in SEQ ID NO: 80, and CDR3 shown in SEQ ID NO: 128; (40) CDR1 shown in SEQ ID NO: 33, CDR2 shown in SEQ ID NO: 81, and CDR3 shown in SEQ ID NO: 129; (42) CDR1 shown in SEQ ID NO: 34, CDR2 shown in SEQ ID NO: 82, and CDR3 shown in SEQ ID NO: 130; (43) CDR1 shown in SEQ ID NO: 35, CDR2 shown in SEQ ID NO: 83, and CDR3 shown in SEQ ID NO: 131; (44) CDR1 shown in SEQ ID NO: 36, CDR2 shown in SEQ ID NO: 84, and CDR3 shown in SEQ ID NO: 132; (45) CDR1 shown in SEQ ID NO: 37, CDR2 shown in SEQ ID NO: 85, and CDR3 shown in SEQ ID NO: 133; (46) CDR1 shown in SEQ ID NO: 38, CDR2 shown in SEQ ID NO: 86, and CDR3 shown in SEQ ID NO: 134; (47) CDR1 shown in SEQ ID NO: 39, CDR2 shown in SEQ ID NO: 87, and CDR3 shown in SEQ ID NO: 135; (48) CDR1 shown in SEQ ID NO: 40, CDR2 shown in SEQ ID NO: 88, and CDR3 shown in SEQ ID NO: 136; (49) CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 89, and CDR3 shown in SEQ ID NO: 137; (50) CDR1 shown in SEQ ID NO: 42, CDR2 shown in SEQ ID NO: 90, and CDR3 shown in SEQ ID NO: 138; (51) CDR1 shown in SEQ ID NO: 43, CDR2 shown in SEQ ID NO: 91, and CDR3 shown in SEQ ID NO: 139; (52) CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 92, and CDR3 shown in SEQ ID NO: 140; (53) CDR1 shown in SEQ ID NO: 45, CDR2 shown in SEQ ID NO: 93, and CDR3 shown in SEQ ID NO: 141; (54) CDR1 shown in SEQ ID NO: 46, CDR2 shown in SEQ ID NO: 94, and CDR3 shown in SEQ ID NO: 142; (55) CDR1 shown in SEQ ID NO: 47, CDR2 shown in SEQ ID NO: 95, and CDR3 shown in SEQ ID NO: 143; or (56) CDR1 shown in SEQ ID NO: 48, CDR2 shown in SEQ ID NO: 96, and CDR3 shown in SEQ ID NO: 144; and CDR1 has a sequence represented by one of SEQ ID NO: 1 to SEQ ID NO: 48, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to one of SEQ ID NO: 1 to SEQ ID NO: 48, or a sequence variant having no more than 8 amino acid substitutions from one of SEQ ID NO: 1 to SEQ ID NO: 48; CDR2 has a sequence represented by one of SEQ ID NO: 49 to SEQ ID NO: 96, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to one of SEQ ID NO: 49 to SEQ ID NO: 96, or a sequence variant having no more than 8 amino acid substitutions from one of SEQ ID NO: 49 to SEQ ID NO: 96; and CDR3 has a sequence represented by one of SEQ ID NO: 97 to SEQ ID NO: 144, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to one of SEQ ID NO: 97 to SEQ ID NO: 144, or a sequence variant having no more than 8 amino acid substitutions from one of SEQ ID NO: 97 to SEQ ID NO: 144.

2. The single-domain antibody or an antigen-binding fragment thereof of claim 1, being selected from Fab, Fab, F(ab)2, Fv, single-chain Fv (scFv), single-chain Fab, diabody, single-domain antibody or sdAb (nanobody), camelid Ig, IgNAR, F(ab)3 fragment, bispecific scFv, (scFv)2, mini antibody, bispecific antibody, trispecific antibody, tetraspecific antibody, and disulfide-stabilized Fv protein (dsFv).

3. The single-domain antibody or an antigen-binding fragment thereof of claim 1, further comprising a constant region selected from porcine IgG, human IgG, and chicken IgY.

4. The single-domain antibody or an antigen-binding fragment thereof of claim 3, wherein the constant region IgG is IgG1, IgG2a, IgG2b, and/or IgG4.

5. The single-domain antibody or an antigen-binding fragment thereof of claim 1, wherein the CPV is isolated from CPV-2a strain.

6. A nucleic acid molecule encoding the single-domain antibody or an antigen-binding fragment thereof of claim 1.

7. A recombinant vector or host cell comprising the nucleic acid molecule of claim 6.

8. The recombinant vector or host cell of claim 7, wherein the recombinant vector is selected from DNA, RNA, viral vectors, transposons, or a combination thereof.

9. The recombinant vector or host cell of claim 8, wherein the viral vectors comprise lentivirus, adenovirus, adeno-associated virus (AAV), retrovirus, or a combination thereof.

10. A chimeric antibody comprising the single-domain antibody or antigen-binding fragment thereof of claim 1.

11. A fusion or conjugate comprising the single-domain antibody or antigen-binding fragment thereof of claim 1 and a heterologous molecule to which the fusion or conjugate is attached.

12. The fusion or conjugate of claim 11, wherein the heterologous molecule is proteins, peptides, markers, drugs, or cytotoxic agents.

13. A composition comprising the single-domain antibody or antigen-binding fragment thereof of claim 1, a nucleic acid molecule encoding the single-domain antibody or antigen-binding fragment thereof, a recombinant vector or host cell comprising the single-domain antibody or antigen-binding fragment thereof, a chimeric antibody comprising the single-domain antibody or antigen-binding fragment thereof, and/or a fusion or conjugate comprising the single-domain antibody or antigen-binding fragment thereof.

14. A method for diagnosis, prevention, and/or treatment of viral infections comprising administering a patient in need thereof the single-domain antibody or antigen-binding fragment thereof of claim 1, a nucleic acid molecule encoding the single-domain antibody or antigen-binding fragment thereof, a recombinant vector or host cell comprising the single-domain antibody or antigen-binding fragment thereof, a chimeric antibody comprising the single-domain antibody or antigen-binding fragment thereof, and/or a fusion or conjugate comprising the single-domain antibody or antigen-binding fragment thereof.

15. A kit for detecting CPV comprising an instruction manual and a reagent for detecting the single-domain antibody or antigen-binding fragment thereof of claim 1, a nucleic acid molecule encoding the single-domain antibody or antigen-binding fragment thereof, a recombinant vector or host cell comprising the single-domain antibody or antigen-binding fragment thereof, a chimeric antibody comprising the single-domain antibody or antigen-binding fragment thereof, and/or a fusion or conjugate comprising the single-domain antibody or antigen-binding fragment thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0097] FIG. 1 shows the expression and purification of CPV antigen according to one example of the disclosure;

[0098] FIG. 2 shows the detection results of specific antibodies and neutralizing antibodies 4 weeks after immunizing a camel with antigen using Freund's adjuvant, according to one example of the disclosure;

[0099] FIG. 3 is an electrophoresis image showing the amplification of VHH sequences through nested PCR (first round of PCR), according to one example of the disclosure;

[0100] FIG. 4 is an electrophoresis image showing the amplification of VHH sequences through nested PCR (second round of PCR), according to one example of the disclosure;

[0101] FIG. 5 shows the sequences of 56 VHH sequences according to one example of the disclosure;

[0102] FIG. 6 shows a vector map of pNFCG1-EB containing Fc-VHH according to one example of the disclosure;

[0103] FIG. 7 shows the results of an ELISA assay for evaluating the binding affinity of VHH antibodies against CPV, according to one example of the disclosure; and

[0104] FIG. 8 shows the neutralizing activity of VHH antibodies according to one example of the disclosure.

DETAILED DESCRIPTION

[0105] To further illustrate the disclosure, embodiments detailing a single-domain antibody against CPV and uses thereof are described below. It should be noted that the following embodiments are intended to describe and not to limit the disclosure.

[0106] The term antibody, as used herein, refers to a polypeptide that include at least the variable region of the light chain or the heavy chain of an immunoglobulin. The variable region specifically recognizes and binds to an antigen. The term encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, single-chain antibodies, multi-chain antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, chimeric antibodies, humanized antibodies, full-length antibodies, and antibody fragments, as long as the antibody exhibits the desired antigen-binding activity.

[0107] The term antigen-binding fragment, as described herein, refers to a portion or segment of a whole antibody that has fewer amino acid residues than the whole antibody but can still bind to an antigen or compete with the whole antibody (i.e., the antibody from which the antigen-binding fragment is derived) for binding. The antigen-binding fragment can be prepared through recombinant DNA technology or by enzymatic or chemical cleavage of the complete antibody. The antigen-binding fragment includes, but is not limited to, Fab, Fab, F(ab)2, Fv, single-chain Fv (scFv), single-chain Fab, diabody, single-domain antibody or nanobody (sdAb), camelid Ig, IgNAR, F(ab)3 fragment, double scFv, (scFv)2, mini antibody, bispecific antibodies, trispecific antibody, tetraspecific antibody, and disulfide-stabilized Fv proteins (dsFv). The term also includes genetically engineered forms, such as chimeric antibody (e.g., humanized mouse antibody), hybrid antibody, and antigen-binding fragments thereof.

[0108] The term variant, as described herein, refers to antibodies that have been modified by 1-20 amino acid substitutions, deletions, and/or insertions in the target antibody region (such as the variable region of the heavy chain or the light chain, or the CDR regions of the heavy chain or the light chain), while substantially retaining the biological properties of the unmodified antibody. In one aspect, the disclosure provides variants of the antibodies described herein. In one example, the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (such as antigen-binding capacity or neutralizing capacity) of the unmodified antibody. The modifications can be applied individually or in combination to the variable region of the heavy chain, the variable region of the light chain, or each CDR region.

[0109] The term chimeric antibody, as described herein, refers to a recombinant protein that incorporates variable regions, including CDRs, derived from an antibody from one species, while the constant regions are sourced from a human antibody. Alternatively, the constant regions of the chimeric antibody may be obtained from other species, such as cats or dogs.

[0110] The term isolated, as described herein, refers to a substance or component that has been obtained through artificial means, separated from its natural state. The term does not imply absolute purity. Instead, the term acknowledges that there may be other impurities present in the isolated substance, provided these impurities do not affect the activity or function of the primary substance.

[0111] The term sequence identity, as used herein, refers to the degree of similarity between two or more biological sequences. The similarity is assessed within a sliding window that compares individual nucleotides in nucleic acids or amino acids in proteins. The percent sequence identity can be calculated as follows: aligning the two sequences within the sliding window to achieve the optimal alignment; identifying positions where nucleotide bases (e.g., A, T, C, G, I) or amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are identical in both sequences; counting a total number of matching positions; dividing the total number of matching positions by a total number of positions in the sliding window (i.e., the size of the sliding window); and multiplying the result by 100 to obtain the percent sequence identity. The optimal alignment for determining the percent sequence identity can be achieved in various methods and software tools, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in related art can adjust parameters and select suitable algorithms to achieve the optimal alignment over the full-length sequence being compared or within specific sequence regions of interest.

[0112] The term vector, as used herein, refers to a construct capable of introducing one or more genes or sequences of interest into a host cell and, preferably, to facilitate the expression of the genes or sequences within the host cell. The vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or bacteriophage vectors, DNA or RNA expression vectors combined with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0113] The terms host cell, as used herein, are used interchangeably to refer to cells that have been introduced with exogenous nucleic acids, including progeny of the cells. Host cells include transformants and transformed cells, which encompass primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell and may contain mutations. Cells that exhibit the same function or biological activity as those screened or selected from initially transformed cells are included.

[0114] The terms neutralizing antibodies, as used herein, refer to antibodies that bind to or interact with a target antigen and prevent the target antigen from binding to a binding partner, such as a receptor, thereby inhibiting or blocking the biological response that would result from the interaction of the target antigen with the binding partner.

[0115] The experimental methods and materials mentioned in the following examples are conventional and can be obtained through conventional commercial sources unless specified otherwise.

Example 1 Preparation of VHH Antibody Against CPV

1.1 Construction of a Phage Display Library

[0116] Two healthy, appropriately aged camels, designated P-1 #and P-2 #, were selected for the immunization process. The two camels were immunized four times with an inactivated CPV (canine parvovirus) antigen. After the fourth immunization, 200 mL of peripheral blood was collected from each camel. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood. RNA was extracted from the PBMCs and used to construct a camel VHH antibody library. The camel VHH antibody library had a capacity of 1.310.sup.9 unique VHH antibodies and a cloning positivity rate of 90%, with no contamination from aggressive bacteriophages.

1.1.1 Proliferation, Inactivation, and Purification of CPV Antigen

[0117] CPV-2a strain was cultured in F81 cells and inactivated using BEI (binary ethyleneimine) for 28 hours at 30 C. After incubation, the cell culture was centrifuged at 4000g for 30 minutes to remove cell debris. The supernatant was then concentrated 1000-fold using a 100 kDa membrane, and a concentrated CPV-2a particles were obtained. A 10-50% sucrose density gradient was prepared. The concentrated CPV-2a particles were ultra-centrifugated at 250,000g for 2 hours to form a CPV-2a band. The CPV-2a band was collected and dialyzed into PBS. The purified CPV-2a particles were observed using transmission electron microscopy (the results were shown in FIG. 1).

1.1.2 Camel Immunization and PBMC Separation

[0118] 400 g of SVA antigen was mixed with an equal volume of Freund's adjuvant and thoroughly emulsified; the resulting emulsion was administered subcutaneously at multiple sites along the neck of each camel; the initial immunization employed Freund's complete adjuvant; for subsequent booster immunizations, Freund's incomplete adjuvant was used; and the immunizations were administered at 14-day intervals.

[0119] After the 2nd, 3rd, and 4th immunizations, a small amount of serum was collected and tested for antibody titers using ELISA array and neutralization assay. The results of antibody titers are depicted in FIG. 2, showing that the serum from camel P-1 #achieved a titer of 1:640,000, while the serum from camel P-2 #reached a titer of 1:2,560,000. The two antibody titer values indicate a strong immune response and satisfactory antibody production in both camels. After confirming satisfactory antibody titers, 200 mL of peripheral blood was collected from each camel. PBMCs were isolated using the Ficoll density gradient centrifugation method. The isolated PBMCs from the two camels were combined to construct the camel VHH antibody library.

[0120] As shown in FIG. 2, the specificity and neutralizing antibody levels were stable by the 3rd and 4th weeks.

1.1.3 RNA Extraction

[0121] An appropriate amount of Trizol was added to the isolated PBMCs to ensure thorough cell lysis. The lysate was centrifuged at 13000 rpm. The precipitate was discarded, and 200 L of chloroform was added to each 1 mL of supernatant from the lysate. The resulting mixture was vigorously shaken for 30 seconds, incubated on ice for 5 minutes, and centrifuged at 13000 rpm for 10 minutes. After centrifugation, the solution was separated into three layers. 350-500 L of the upper aqueous phase was carefully pipetted out, transferred into a centrifugal tube, mixed with an equal volume of isopropanol, and incubated at 20 C. for 30 minutes. After centrifugation at 13000 rpm for 10 minutes, the supernatant was discarded. The RNA pellet was washed twice with 1 mL of 75% cold ethanol and air-dried. DEPC water was treated with RRI (RNase Inhibitor) according to the amount of the RNA pellet. The dried RNA pellet was dissolved in the pre-treated DEPC water.

1.1.4 Insertion of Exogenous Genes into Phage Vectors

[0122] The isolated RNA was reverse transcribed into cDNA via RT-PCR using oligo-dT primers. In the first round of PCR, the cDNA was used as the template along with two primers: an upstream primer (SEQ ID NO: 145: GTC.CTG.GCT.GCT.CTT.CTA.CAA.GG) that binds to the variable region of the heavy chain of the VHH antibody and a downstream primer (SEQ ID NO: 146: GGT.ACG.TGC.TGT.TGA.ACT.GTT.CC) that binds to the constant region (CH2) of heavy chain of the VHH antibody. As shown in FIG. 3, the first round of PCR reaction yielded a 600 bp DNA band. The DNA band was used as a template in the second round of PCR.

[0123] Restriction sites were added to two VHH-specific primers used in the second round of PCR to ensure that the VHH gene can be easily inserted into the vector: the second round of PCR employed the two VHH-specific primers:

TABLE-US-00001 aforwardprimerwitharestrictionsite: SEQIDNO:147: AACATGCCATGACTCGCGGCTCAACCGGCCATGGCTGAKGTBCAGCTGCA GGCGTCTGGRGGAGG; and areverseprimerwitharestrictionsite: SEQIDNO:148: GTTATTATTATTCAGATTATTAGTGCGGCCGCTGGAGACGGTGACCWGGG TCC

[0124] As shown in FIG. 4, the second round of PCR reaction yielded a VHH antibody gene.

[0125] The second round of PCR reaction yielded a VHH gene; a vector used for phage display was selected and digested with the same restriction enzymes that correspond to the restriction sites on the two specific primers; the VHH gene were also digested with the same restriction enzymes and ligated into the pre-digested vector using T4 DNA ligase; the ligation product was precipitated with ethanol to remove salts; and the purified ligation product was introduced into TG1 phage component cells via electroporation to construct a phage-displayed VHH antibody library with a capacity of 1.310.sup.9 different clones as shown in Table 1.

TABLE-US-00002 TABLE 1 A phage-displayed VHH antibody library Detection Plate Number 1 Total Number of Competent 5 5 Cells Number of Clones 1320 1320 Library Capacity 5 200 10.sup.3 (1320/1) = 1.3 10.sup.9 Calculation

1.2 Screening of VHH Antibodies Against CPV

1.2.1 Selecting of Phages with Binding Affinity to CPV

[0126] 1. Library preparation: 1.0010.sup.13 pfu (plaque-forming units) of the phage-displayed VHH antibody library were used. A target protein was coated on a solid support at a concentration of 10 g/mL. Before screening, the background from a blank plate and a control plate coated with 10 g/mL of S protein was subtracted. The unbound phages were washed using 0.1% PBST, and then the bound phages were eluted from the solid support using Trypsin. The target enrichment rate was 1.0410.sup.5.

[0127] 2. First round of selecting: 6.0010.sup.12 pfu of the phage-displayed VHH antibody library were used. The target protein was coated on a solid support at a concentration of 5 g/mL. Before screening, the background from a normal plate and a control plate coated with 5 g/mL of S protein was subtracted. The unbound phages were washed using 0.2% PBST and then eluted using from the solid support using Trypsin. The target enrichment rate was 1.0410.sup.5, the control enrichment rate was 9.2610.sup.5, and the blank enrichment rate was 2.0410.sup.5.

[0128] 2. Second round of selecting: 5.0010.sup.12 pfu of the phage-displayed VHH antibody library were used. The target protein was coated on a solid support at a concentration of 5 g/mL. Before screening, the background from a normal plate and a control plate coated with 5 g/mL of S protein was subtracted. The unbound phages were washed using 0.3% PBST and then eluted from the solid support using Trypsin. The target enrichment rate was 8.3310.sup.3, the control enrichment rate was 2.5010.sup.7, and the blank enrichment rate was 3.1310.sup.7.

1.2.2 Screening of Monoclonal Antibodies Using Phage ELISA

[0129] After the second round of selecting, the eluted phages were used to infect TG1 phage component cells. Individual bacterial clones were randomly selected, and each bacterial clone produced monoclonal phages. Four 96-well were coated with the monoclonal phages. A target protein and a control protein were then added to test the binding specificity of the monoclonal phage to the target protein compared to the control protein. The four 96-well plates were then blocked with skim milk. HRP-conjugated anti-M13 phage antibody was added as a secondary antibody, and then the binding affinity of the monoclonal phages to the target protein were detected and quantified.

[0130] Specifically: [0131] 1. Coating: A target protein was diluted in a coating buffer with a pH of 9.6; 50 L of the diluted target protein solution was added to each well of the ELISA plate; and the ELISA plate was then placed in a humidified chamber and incubated at 37 C. for 1 hour, or alternatively, at 4 C. overnight. [0132] 2. Blocking: the unbound diluted coating buffer was discarded from the wells; the ELISA plate was inverted onto a clean paper towel and gently tapped to remove any residual liquid; the ELISA plate was washed with PBS; 300 L of a blocking buffer was added into each well of the ELISA plate; and the ELISA plate was incubated at 37 C. for 1 hour. [0133] 3. Primary antibody incubation: the unbound phage solution was discarded from the wells; the ELISA plate was inverted onto a clean paper towel and gently tapped to remove any residual liquid; and the ELISA plate was washed with PBS; HRP-conjugated anti-M13 phage antibody was diluted 1:1000 in 1% Milk-PBS and added (50 L/well); and the ELISA plate was then incubated at 37 C. for 1 hour. [0134] 4. Color development: after incubation, the unbound HRP-conjugated anti-M13 phage antibody was discarded; the ELISA plate was inverted onto a clean paper towel and gently tapped to remove any residual liquid; the ELISA plate was washed four times with PBS; TMB (3,3,5,5-tetramethylbenzidine) substrate solution was added (50 L/well); 2M H.sub.2SO.sub.4 was added to each well to stop the reaction; and absorbance of the stopped reaction was measured at 450 nm using a microplate reader.

1.2.3 Sequencing of Monoclonal Clones:

[0135] A total of 218 monoclonal phages were selected and sent to a sequencing company for sequence analysis. The sequences obtained were categorized based on the CDR3 region. From the analysis, 56 unique VHH sequences were identified (as shown in FIG. 5). The 56 unique VHH sequences were selected used for subsequent experiments.

1.3 In Vitro Expression of Recombinant VHH Antibodies

1.3.1 Construction of Fc Fusion Vector and Insertion of VHH Sequences

[0136] The VHH sequences were inserted into the pNFCG1-EB vector (as shown in FIG. 6) to form a recombinant plasmid.

1.3.2 Transfection of Recombinant Vectors into 293T Cells

[0137] Human embryonic kidney cells (293T cells) were collected, resuspended in culture medium to achieve a concentration of 110.sup.6 cells/mL, and incubated at 37 C. with 5% CO.sub.2 to form a cell suspension. 1 g of the recombinant vector was diluted in 40 L of KPM medium and mixed; 5 L of T1 transfection regent (T1:recombinant plasmid=1:5) was diluted in 40 L of KPM medium, mixed, and incubated at room temperature for 5 minutes; after incubation, the diluted T1 transfection regent was added to the diluted recombinant plasmid solution, mixed, and incubated at room temperature for 30 minutes to form a transfection complex. The transfection complex was added to the cell suspension, mixed, and incubated at 37 C. with 5% CO.sub.2 to form transfected cells. After 72 hours of incubation, the supernatant was collected and analyzed to detect and measure the activity and expression levels of the recombinant VHH antibodies produced by the transfected cells.

Example 2 Detection of Specificity and Binding Activity of Recombinant VHH Antibodies Against CPV

[0138] 1. Coating: Antigen P was diluted to a concentration of 5 g/mL in a carbonate buffer with a pH of 9.6; 100 L of the diluted antigen P was added to each well of a 96-well ELISA plate; and the 96-well ELISA plate was then incubated overnight at 4 C.

[0139] 2. Blocking: The unbound coating buffer was discarded from the wells; the 96-well ELISA plate was washed three times with PBST; each well was blocked with 300 L of a 4% skim milk in PBS; and the 96-well ELISA plate was incubated at 37 C. for 2 hours.

[0140] 3. Sample addition: The unbound blocking solution was discarded; the 96-well ELISA plate was washed three times with PBST; samples, along with PBS and medium for the control group, were added to the wells (100 L/well); and the 96-well ELISA plate was then incubated at 37 C. for 1 hour.

[0141] 4. Secondary antibody incubation: After incubation, the unbound samples were discarded, and the 96-well ELISA plate was washed three times with PBST; the goat anti-human IgG-HRP (Fc) antibody, diluted 1:5000, was added (100 L/well); and the 96-well ELISA plate was then incubated at 37 C. for 1 hour.

[0142] 5. Color development: The unbound goat anti-human IgG-RP (Fc) antibody was discarded; the 96-well ELISA plate was washed five times with PBST; TMB substrate solution was added (100 l/well) and developed in the dark; after color development, 50 l of 2M hydrochloric acid (HCl) was added to each well to stop the reaction; and the absorbance of the stopped reaction was measured at 450 nm (A450) using a microplate reader

[0143] As shown in FIG. 7 and Table 2, the 56 selected VHH antibodies exhibit both binding affinity and specificity compared to the Control group and the control group.

TABLE-US-00003 TABLE 2 Binding affinity of 56 VHH antibodies to CPV Antibody number A450 value 1 1.88 2 1.72 3 1.26 4 1.56 5 1.68 6 1.67 7 1.52 8 1.62 9 1.84 10 1.88 11 1.68 12 1.87 13 1.66 14 1.55 15 1.67 16 1.50 17 1.67 18 1.75 19 1.55 20 1.62 21 1.62 22 1.94 23 0.26 24 1.57 25 1.54 26 1.74 27 1.48 28 1.50 29 1.43 30 1.67 31 1.72 32 1.61 33 1.59 34 1.73 35 1.44 36 1.73 37 1.58 38 1.86 39 1.65 40 1.71 41 1.50 42 1.55 43 1.41 44 1.50 45 1.53 46 1.75 47 1.54 48 1.48 49 1.55 50 1.61 51 1.53 52 1.53 53 1.70 54 1.53 55 1.67 56 1.59 Fc 0.12 Control 0.13

Example 3 Identification of Neutralizing Activity of VHH Antibodies Against CPV

[0144] A neutralization assay was performed as follows: the VHHT antibodies, derived from the final supernatant in Example 1, were prepared at a concentration of 1 g/mL, with serial dilutions starting from 1:2; each dilution was repeated across four sample wells; the diluted VHHT antibodies were incubated with 100 Tissue Culture Infectious Dose (TCID50) of the CH-HuB-2017 virus at 37 C. for 1 hour. 50 L of IBRS-2 cells was added to each well of a 96-well plate; the 96-well plate was then incubated at 37 C. with 500 CO.sub.2 for 72 hours; after incubation, the wells were examined using an inverted microscope to observe cytopathic effects; and the neutralizing antibody titers were calculated using the Reed-Muench method.

[0145] As shown in FIG. 8, out of the 56 VHH antibodies tested for neutralizing activity against CPV, 8 VHH antibodies, specifically antibodies 8, 11, 16, 23, 28, 37, 38, and 41, did not exhibit any neutralizing activity. The remaining 48 VHH antibodies demonstrated varying levels of neutralizing activity. Among the 48 VHHT antibodies, antibodies 2, 10, 45, 48, and 55 showed a neutralizing activity with a titer of 16 Furthermore, antibodies 12, 42, 43, and 47 exhibited even higher neutralizing activity, with a titer of: 32:1.

TABLE-US-00004 TABLE 3 Neutralizing activity of 56 VHH antibodies against CPV Antibody Neutralizing number activity 1 8:1 2 16:1 3 8:1 4 8:1 5 8:1 6 8:1 7 8:1 8 0 9 4:1 10 16:1 11 0 12 32:1 13 8:1 14 2:1 15 4:1 16 0 17 8:1 18 4:1 19 4:1 20 8:1 21 16:1 22 8:1 23 0 24 4:1 25 4:1 26 16:1 27 2:1 28 0 29 4:1 30 2:1 31 4:1 32 4:1 33 4:1 34 2:1 35 8:1 36 8:1 37 0 38 0 39 2:1 40 2:1 41 0 42 32:1 43 32:1 44 4:1 45 16:1 46 4:1 47 32:1 48 16:1 49 8:1 50 8:1 51 4:1 52 4:1 53 8:1 54 2:1 55 16:1 56 2:1 Fc 0 Control 0

TABLE-US-00005 TABLE4 Sequencesof48VHHantibodieswith neutralizingactivityagainstCPV Antibody SEQIDNO CDR-1 SEQIDNO CDR-2 SEQIDNO CDR-3 1 SEQID GFAFSRYPM SEQID STIFPGGDT SEQID DGIGVSSP NO:1 NO:49 PY NO:97 2 SEQID GYTYRWCN SEQID SRVISDGTT SEQID GLRTGDTQ NO:2 M NO:50 A NO:98 GCTNY 3 SEQID GYTYSSYC SEQID ATIDSYGY SEQID SGRLQASGG NO:3 M NO:51 TS NO:99 YCYIHSHAY NY 4 SEQID VYTISNPKK SEQID AGIHRGVG SEQID RFCLTDYGL NO:4 CM NO:52 TTY NO:100 GDFNRLYD Y 5 SEQID GYTYSAGS SEQID AVDSGVDG SEQID FRGWGLTL NO:5 M NO:53 ITA NO:101 DRRRFNY 6 SEQID GFTFSNYGM SEQID SGIETGGGS SEQID GVVGISNP NO:6 NO:54 TY NO:102 7 SEQID RFTYNNGW SEQID AAIYTTAG SEQID EKHSGATW NO:7 L NO:55 STF NO:103 AAGRYTY 9 SEQID TYTYKIYSM SEQID AAIDSDGS SEQID ETYGWEVA NO:8 NO:56 TI NO:104 GPSALSSYE YNY 10 SEQID GFTFSNYGM SEQID SGIETGGGS SEQID GTIGISSP NO:9 NO:57 TY NO:105 12 SEQID GYISSSDYM SEQID ATLFTGSTT SEQID TTGALWWP NO:10 NO:58 TY NO:106 AREALRNT NFAY 13 SEQID GVTHTSNC SEQID ARIFSGAG SEQID TYVLYARSC NO:11 M NO:59 GITNTY NO:107 RITNEYNY 14 SEQID GVTHTSNC SEQID AFMHTADS SEQID DSRYRPYYS NO:12 M NO:60 TTR NO:108 SCPRSTDDLY 15 SEQID GVTGRICTM SEQID SRITRAGDT SEQID RYGSFCTSN NO:13 NO:61 T NO:109 R 17 SEQID GFTFSHYDM SEQID SGINSDGG SEQID GTIGISEP NO:14 NO:62 DTY NO:110 18 SEQID GDTYSRYIM SEQID ATISADGY SEQID SRSNWYGL NO:15 NO:63 TG NO:111 SLRRSDFAY 19 SEQID KYSYSTDC SEQID ASIDSERGT SEQID HSWSTVCG NO:16 M NO:64 S NO:112 GVDWSESA GNV 20 SEQID GSSDLYNW SEQID AAIYMRIE SEQID AFLPRGIQA NO:17 M NO:65 RTH NO:113 SWATTLDPT MYPS 21 SEQID MYGIRNLY SEQID AAISPSAGG SEQID DDWLHTGY NO:18 M NO:66 TY NO:114 LSPKEYHL 22 SEQID GFRFSSYGM SEQID SGISNDGSE SEQID GLVGVTTP NO:19 NO:67 AY NO:115 24 SEQID EATYNDFC SEQID AAITTEGSY SEQID KRFCYSRVR NO:20 M NO:68 TY NO:116 SLTSSEYSQ 25 SEQID KLTYCTYD SEQID SGIDSTGST SEQID VLGGSACR NO:21 M NO:69 S NO:117 GGPY 26 SEQID RDTSSRNCM SEQID ASIRSGIGA SEQID GFCPYGPSL NO:22 NO:70 TN NO:118 HLASYAY 27 SEQID SMSNINCM SEQID ALLYTRAS SEQID DSYNCHLGS NO:23 NO:71 GTI NO:119 WYHFTRY 29 SEQID GYIDSDYCM SEQID ATIANFKPI SEQID VSRCGVVPP NO:24 NO:72 NO:120 NYH 30 SEQID GFTYVSYC SEQID GTIDSTLST SEQID TDIRSSCWH NO:25 M NO:73 K NO:121 ASIRGPDFG Y 31 SEQID GLTDVSYIM SEQID ACITDGGP SEQID TRGYGFTLR NO:26 NO:74 TA NO:122 SADYKY 32 SEQID GFTFRNYY SEQID SGIDIWGV SEQID ETDPFSGGS NO:27 M NO:75 TTD NO:123 WGGETGDY 33 SEQID GFTFSNYW SEQID SSISTGGTT SEQID PRHVEGSST NO:28 M NO:76 Y NO:124 GTSNFDERG K 34 SEQID GITYRVCSR SEQID SSISNDGVP SEQID SCRRVEWTI NO:29 NO:77 A NO:125 RYDDY 35 SEQID GNTYTSRL SEQID AAIGDYGT SEQID KRWGGIWS NO:30 M NO:78 SIY NO:126 TVTDYNY 36 SEQID GYNYKTYR SEQID AVIDSDGS SEQID DTAFVGEV NO:31 M NO:79 TK NO:127 VTLDQYEY NY 39 SEQID GDTHRTYFI SEQID ATMAESGV SEQID AYGYGLSL NO:32 NO:80 TG NO:128 RPHDYRY 40 SEQID GYAYRPYC SEQID ATINIDGST SEQID ETMGWDRC NO:33 M NO:81 S NO:129 SLLGYEYNY 42 SEQID SGITYSRYC SEQID ASIDTVGST SEQID STVTVGYEY NO:34 M NO:82 N NO:130 EPGPYCSGG 43 SEQID GFTISRNAM SEQID SGIERGGG SEQID GIGVSEP NO:35 NO:83 GPTY NO:131 44 SEQID GYAFSTYSM SEQID AVINKYGT SEQID HLNLWIGR NO:36 NO:84 TK NO:132 HVPPAEIFD Y 45 SEQID GYTFKDYC SEQID AIISPYGGW SEQID GPSYCSRAY NO:37 M NO:85 D NO:133 IQTAPY 46 SEQID RYTYRTYC SEQID AAMNSDG SEQID ESYGFEIAT NO:38 M NO:86 NTV NO:134 LTPDEYNY 47 SEQID GFFVGSHLM SEQID ASIGDTSTI SEQID AREGSWGW NO:39 NO:87 G NO:135 SRRERDFAY 48 SEQID GYRYSTYC SEQID AAIDADGF SEQID GCNFDGGS NO:40 M NO:88 TS NO:136 DIRYGLDY 49 SEQID GYRYSTYC SEQID ALINTRGD SEQID PLLPGGACS NO:41 M NO:89 STS NO:137 RTALVPDFR Y 50 SEQID GYTFSSYCL SEQID VTICGSGTP SEQID SLRYYCSPR NO:42 NO:90 T NO:138 SDFTY 51 SEQID EYTYSNYY SEQID ACLNNGGI SEQID STYLRGACR NO:43 M NO:91 STY NO:139 DYNYAY 52 SEQID EYAYSNAY SEQID ATLYISIGR SEQID HRGSCWDE NO:44 M NO:92 TK NO:140 TIFPANFES 53 SEQID GYYGYTYN SEQID ARISLSSGT SEQID DLTQYARN NO:45 NNMCM NO:93 SI NO:141 CGRLLNFYE SPS 54 SEQID EYSYINCM SEQID ACMYYPG SEQID GVRIWGISC NO:46 NO:94 GTTT NO:142 LHNEYRY 55 SEQID GYTYRSYC SEQID AAIGSDGS SEQID DLAGIGDGD NO:47 M NO:95 TR NO:143 YCYPNVDE YNY 56 SEQID GYSYTSNFM SEQID AAIDTRGV SEQID GVGFGYSLS NO:48 NO:96 RTF NO:144 GKYYKY

[0146] It will be obvious to those skilled in the art that changes and modifications may be made, and therefore, the aim in the appended claims is to cover all such changes and modifications.