Monoclonal antibodies against Henipavirus glycoprotein G and encoding nucleic acids thereof

Abstract

Provided is an anti-Henipavirus monoclonal antibody having broad spectrum neutralization activity, wherein the antibody comprises a macaque variable region and a human constant region. The antibody of the present invention has good binding activity to both Nipah virus glycoprotein G and Hendra virus glycoprotein G, can effectively neutralize Nipahpseudovirus and Hendra pseudovirus, and can be used for preparing drugs for treating Henipavirus diseases.

Claims

1. A monoclonal antibody against Henipavirus glycoprotein G, wherein the amino acid sequence of the CDR1, CDR2, and CDR3, of the heavy chain variable region of the said antibody and the amino acid sequence of the CDR1, CDR2, and CDR3 of the light chain variable region of the said antibody are respectively shown as the following sequence combinations: 26-33, 51-58, 97-116 of SEQ ID NO:1 and 27-36, 54-56, 93-100 of SEQ ID NO:3, or 26-33, 51-58, 97-117 of SEQ ID NO:5 and 27-32, 50-52, 89-97 of SEQ ID NO:7, or 26-33, 51-58, 97-115 of SEQ ID NO:9 and 27-32, 50-52, 89-97 of SEQ ID NO: 11, or 26-33, 51-58, 97-109 of SEQ ID NO:13 and 27-37, 51-53, 90-100 of SEQ ID NO:15.

2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the said antibody and the amino acid sequence of the light chain variable region of the said antibody are respectively shown as the following sequence combinations: SEQ ID NO:1 and SEQ ID NO:3, or SEQ ID NO:5 and SEQ ID NO:7, or SEQ ID NO:9 and SEQ ID NO:11, or SEQ ID NO:13 and SEQ ID NO:15.

3. The monoclonal antibody of claim 2, wherein the antibody comprises a heavy chain constant region and a light chain constant region, and wherein the amino acid sequence of the heavy chain constant region of the said antibody is shown as SEQ ID NO:17, and the amino acid sequence of the light chain constant region of the said antibody is shown as SEQ ID NO: 19 or SEQ ID NO:21.

4. An isolated nucleic acid encoding the variable region of the heavy chain and light chain of the monoclonal antibody of claim 1, wherein the sequence of the isolated nucleic acid encoding the variable region of the heavy chain and the sequence of the isolated nucleic acid encoding the variable region of the light chain of the monoclonal antibody are respectively shown as the following sequence combinations: SEQ ID NO:2 and SEQ ID NO:4, or SEQ ID NO:6 and SEQ ID NO:8, or SEQ ID NO:10 and SEQ ID NO:12, or SEQ ID NO: 14 and SEQ ID NO: 16.

5. The isolated nucleic acid of claim 4, wherein the sequence of the isolated nucleic acid encoding a heavy chain constant region is shown as SEQ ID NO:18, and the sequence of the isolated nucleic acid encoding a light chain constant region is shown as SEQ ID NO:20 or SEQ ID NO:22.

6. A functional element expressing the isolated nucleic acid of claim 5, wherein the functional element is a linear expression cassette.

7. A host cell comprising the functional element of claim 6.

8. The host cell of claim 7, wherein the cell is an Expi 293F cell or a CHO-S cell.

9. A method of preparing the monoclonal antibody of claim 1 as a therapeutic drug for treating Henipavirus disease.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Sorting of rhesus monkey memory B cells;

(2) FIG. 2. Capillary electrophoresis of nested PCR products;

(3) FIG. 3. Distribution of ELISA OD.sub.450-630 nm values for screening of binding antibodies;

(4) FIG. 4. The curves of antibody binding with antigen in ELISA detection;

(5) FIG. 5. Affinity determination of 1E5 to Henipavirus G protein;

(6) FIG. 6. The neutralization curves of mAbs against HeV pseudovirus;

(7) FIG. 7. The neutralization curves of mAbs against NiV-MY pseudovirus;

(8) FIG. 8. The neutralization activity of mAbs against NiV-BD pseudovirus;

(9) FIG. 9. Competitive inhibition on the binding of Henipavirus G protein with receptor ephrin-B3 by mAbs;

(10) FIG. 10. Competitive inhibition on the binding of Henipavirus G protein with receptor ephrin-B2 by mAbs;

(11) FIG. 11. The neutralization activity of mAbs against HeV-G-D582N variant pseudovirus.

EXAMPLES

(12) The invention is further described below with reference to specific embodiments, and the advantages and characteristics of the invention will become clearer with the description. However, these embodiments are only exemplary, and do not constitute any limitation on the protection scope defined by the claims of the invention.

Example 1. Screening and Preparation of Antibodies

(13) 1. Collection of Blood Samples

(14) Female rhesus monkeys were immunized with adenovirus vector Nipah virus candidate vaccine, recombinant NiV G protein and recombinant HeV G protein three times by intramuscular injection on day 0, 28, and 49, respectively. Finally, blood samples of the rhesus monkey were collected on day 77.

(15) 2. Labeling of NiV-BD G with FITC

(16) The NiV-BD G was labeled with fluorescein isothiocyanate (FITC) to sort antigen-specific memory B cells. Method is described as below: 1) FITC (SIGMA, F4274) is dissolved in dimethyl sulfoxide at a final concentration of 20 mg/mL. Take 100 L of NiV-BD G (about 3.3 mg/mL) and add carbonate buffer (pH=9.6) to 400 L. 2) Add 8 UL FITC to the NiV-BD G solution and incubate for 3 h at 4 C. in the dark. 3) Buffer-exchange the protein into PBS using a 30 kDa centrifugal concentrator tube until the filtrate is transparent and colorless. Wrap the labeled protein in tin foil paper and store it at 4 C. until use.
3. Flow Sorting of Memory B Cells

(17) PBMCs are isolated from blood samples using a Ficoll density gradient centrifugation method, details are described as follows: 1) Take fresh EDTA anticoagulant whole blood and dilute the whole blood with the same volume of PBS. 2) Add the separation solution to the centrifuge tube, and slowly spread the diluted blood sample above the surface of the separation solution to keep the interface between the two liquid surfaces clear. Separation solution, anticoagulated blood, PBS (or normal saline) volume is 1:1:1. 3) After Balancing, the tube is centrifuged at 800g, 3rd gear acceleration and deceleration, room temperature, for 30 min. After centrifugation, the bottom of the tube is red blood cells, the middle layer is the separation solution, the top layer is the plasma/tissue homogenate layer. The thin and dense white film between the plasma layer and the separation solution layer is mononuclear cells (including lymphocytes and monocytes). Carefully pipette the mononuclear cells into another centrifuge tube. 4) Dilute the cells with PBS and gently invert and mix well. The tube is centrifuged at 300g, room temperature, for 10 min. Discard supernatant and repeat twice. Finally, the lymphocytes are resuspended in PBS for later use. 5) Count 510.sup.5 cells in a volume of 50 L PBS, add the five fluorescent dyes recommended in the following table, and incubate for 1 h at 4 C. in the dark.

(18) TABLE-US-00001 TABLE 1 Fluorescent dyes used for cell sorting Marker Fluorescence Company/Cat. No. Volume Antigen FITC SIGMA, F4274 4 g IgG PE BD, 555787 15 L CD19 APC-AF 700 Beckman, IM2470 5 L CD3 PerCP BD, 552851 10 L CD27 PC7 Beckman, A54823 10 L 6) Wash cells 2-3 times with PBS containing 2% FBS and resuspend in 400 L FPBS. Remove cell clusters with a 40 m cell filter, and store at 4 C. in the dark for sorting. 7) NiV-BD G-specific single memory B cells are sorted by a cell sorter (Beckman, MoFlo XDP) using a strategy of IgG.sup.+/CD3.sup./CD19.sup.+/CD27.sup.+/NiV-BD G.sup.+. Each single cell is directly sorted into 96-well plates contains 20U RNase inhibitor and 20 L RNase free water in each well. Store plates at 80 C. Cell sorting result is shown in FIG. 1. Cells circled by R7 box in the figure are characterized by IgG.sup.+/CD3.sup./CD19.sup.+/CD27.sup.+/NiV-BD G.sup.+, which are NiV-BD G-specific memory B cells.
4. Amplification of Antibody Genes by Single Cell PCR
1) Reverse Transcription PCR

(19) A total of 1124 NiV-BDG-specific memory B cells were obtained by flow sorting. The SuperScript III reverse transcription kit was used to perform reverse transcription polymerase chain reaction (PCR). The mixed system was prepared according to the instructions and directly added to 96-well plates containing single cells for PCR reaction. Reaction conditions: 42 C., 10 min; 25 C., 10 min; 50 C., 60 min; 94 C., 5 min. The reaction system and conditions are described as follows.

(20) TABLE-US-00002 TABLE 2 Reaction system for reverse transcription PCR Component Volume Template (sorted single cells) 20 L Random primer 3 L dNTP 1 L 10 buffer 3 L 0.1M DTT 1 L MgCl.sub.2 2 L RNaseOUT 1 L SuperScript III 0.5 L
2) Nested PCR

(21) Reverse transcription products were used as the template, and two rounds of nested PCR reactions were performed to amplify H, K, and A genes. The detail process is described as follows.

(22) The first-round nested PCR reaction system is listed in Table 3.

(23) TABLE-US-00003 TABLE 3 The reaction system for the first-round of nested PCR Component Volume Template (Reverse transcription products) 1 L Mixed primers (H//) 1.5/1/1 L dNTP 2 L 10 buffer 2.5 L TransStart Taq DNA polymerase 0.5 L RNase-free water to 25 L

(24) The first-round of nested PCR reaction conditions: firstly pre-denaturation 5 min at 95 C.; then 40 cycles of denaturation 30 s at 95 C., annealing 30 s at 57 C., elongation 45 s at 72 C.; finally elongation 10 min at 72 C.

(25) The first-round nested PCR primers are listed in Table 4.

(26) TABLE-US-00004 TABLE4 Primersforfirst-roundnestedPCR Primer Sequence 5VH1.L1 ATGGACTKGACCTGGAGG 5VH2.L1 ATGGACACGCTTTGCTCC 5VH3A.L1 ATGGAGTTKGGGCTGAGCTG 5VH3B.L1 ATGGAGTTTGKRCTGAGCTGG 5VH3C.L1 ATGGAGTCRTGGCTGAGCTGG 5VH3D.L1 ATGGAGTTTGTGCTGAGTTTGG 5VH4.L1 ATGAAGCACCTGTGGTTC 5VH5A.L1 ATGGGGTCAACTGCCATC 5VH5B.L1 ATGGGGTCCACCGTCACC 5VH6.L1 ATGTCTGTCTCCTTCCTCA 5VH7.L1 ATGGACCTCACCTGGAGC 3IgG(Outer) GGAAGGTGTGCACGCCGCTGGTC 5VK1A.L1 ATGGACATGAGGGTCCCCGC 5VK1B.L1 GGCTCCTKCTGCTCTGGCTC 5VK2.L1 ATGARGYTCCCTGCTCAG 5VK3.L1 ATGGAARCCCCAGCWCAGC 5VK4.L1 ATGGTGTCACAGACCCAAGTC 5VK5.L1 ATGGCATCCCAGGTTCASC 5VK6A.L1 ATGTTGTCTCCATCACAACTC 5VK6B.L1 ATGGTGTCCCCATTGCAACTC 5VK7.L1 ATGGGGTCCTGGGCTCC 3Kappa(Outer) GTCCTGCTCTGTGACACTCTC 5VL1.L1 ATGGCCTGGTYYCCTCTC 5VL2/7/10.L1 ATGGCCTGGRCTCTGCTCC 5VL3A.L1 ATGGCCTGGATTCCTCTC 5VL3B.L1 ATGGCCTGGACCTTTCTC 5VL3C.L1 ATGGCCTGGACCCCTCCC 5VL4A.L1 ATGGCCTGGGTCTCCTTC 5VL4B.L1 ATGGCCTGGACCCCACTC 5VL5/11.L1 ATGGCCTGGACTCCTCTC 5VL6.L1 ATGGCCTGGGCTCCACTCC 5VL8.L1 ATGGCCTGGATGATGCTTC 5VL9.L1 ATGGCCTGGGCTCCTCTG 3Lamda(Outer) TGTTGCTCTGTTTGGAGGG

(27) The second-round nested PCR reaction system is listed in Table 5.

(28) TABLE-US-00005 TABLE 5 The reaction system for the second-round of nested PCR Component Volume Template (Products of the first-round nested PCR) 1.6 L Mixed primers (H//) 3.2/1.6/1.6 L dNTP 3.2 L 10 buffer 4 L TransStart Taq DNA polymerase 0.8 L RNase-free water to 40 L

(29) The second-round nested PCR primers are listed in Table 6.

(30) TABLE-US-00006 TABLE6 Primersforsecond-roundnestedPCR Primer Sequence H 5VH1A.SE TGGCAGCAGCTACAGGTGC 5VH1B.SE TGACAGCAGCTACAGGCGC 5VH1C.SE TGGCAGCAGCAACAGGCAC 5VH2.SE GTCCCGTCCTGGGTCTTGTC 5VH3A.SE GCTGTTTGGAGAGGTGTCCAGTGTG 5VH3B.SE GCCATATTAGAAGGTGTCCAGTGTG 5VH3C.SE GCTCTTTTGAAAGGTGTCCAGTGTG H5VH3D.SE GCTATTTTAAGAGGTGTCCAGTGTG 5VH3E.SE GCTATTTTAAAAGGTGTCCAGTGTG 5VH4.SE AGCTCCCAGATGGGTCYTGTCC 5VH5.SE GCTGTTCTCCARGGAGTCTGTG 5VH6.SE GGCCTCCCATGGGGTGTC 5VH7A.SE GCAGCAACAGGTGCCCACTC 5VH7B.SE GCAGCAACAGGCACCCACTC 3IgG(Inner) GTTCAGGGAAGTAGTCCTTGAC 5VK1/2.SE CTCCCAGGTGCCAGATGTGA 5VK1B.SE GGTCCCTGGRTCCAGTGGG 5VK3A.SE TGGCTCCCAGGTACCACYGGA 5VK3B.SE TGGATCCCGGATGCCGCCG 5VK3C.SE TGGCTTCCGGATACCACTGGA 5VK4.SE CTGGATCTCTGGTGTCTGTGG 5VK5.SE CCTTTGGATCTCTGMTGCCAGG 5VK6.SE TGGGTTCCAGTCTCCAAGGG 5VK7.SE TGTGCTCCAGGCTGCAATGG 3Kappa(Inner) ATTCAGCAGGCACACAACAGAG 5VL1A.SE CTGTGCAGGGTCCTGGGCC 5VL1B.SE CTGCACAGGGTCCYGGGCC 5VL2.SE TCACTCAGGGCACAGGATCC 5VL3A.SE CGCCCTCTGCACAGTCTCTGTGG 5VL3B.SE CACTCTCTGCACAGGTTCCGTGG 5VL4A.SE TTCATTTTCTCCACAGGTCTCTGTG 5VL4B.SE CTTCACTGCAGAGGTGTCTCTC 5VL5.SE CACTGCACAGGTTCCCTCTC 5VL6.SE CTGCACAGGGTCTTGGGCTG 5VL8.SE GCTTATGGCTCAGGAGTGGA 3Lamda(Inner) AGACACACTAGTGTGGCCTTG

(31) The reaction conditions for the second-round of nested PCR are the same as those of the first-round of nested PCR.

(32) 3) Capillary Electrophoresis

(33) After the nested PCR, the amplified products were analyzed by capillary electrophoresis using the QIAxcel DNA Fast Analysis Cartridge. Positive clones with paired light and heavy chains were selected for sequencing, and the variable region sequences of the antibody were analyzed by Vector NTI software and IMGT website. The results of nested PCR capillary electrophoresis are shown in FIG. 2.

(34) 5. Expression of Antibodies Using Linear Expression Cassettes

(35) Through the above single-cell PCR reaction, 254 paired antibody sequences were obtained, and the antibody was rapidly expressed by constructing linear expression cassettes.

(36) Firstly, promoter-leader sequence fragments, constant region fragments (synthetized by Sangon Biotech, the heavy chain constant region sequence is shown as SEQ ID NO:17, the DNA coding sequence is shown as SEQ ID NO:18; the constant region sequence of the kappa light chain is shown as SEQ ID NO:19, the DNA coding sequence is shown as SEQ ID NO:20; the constant region sequence of the lambda light chain is shown as SEQ ID NO:21, and the DNA coding sequence is shown as SEQ ID NO:22), and poly A-tail fragments (Genbank accession number: X03896.1) were obtained by PCR. Then amplify the antibody variable region fragments, and the PCR reaction system is listed in Table 7.

(37) TABLE-US-00007 TABLE 7 Reaction system for amplifying variable region fragments Component Volume Template (Products of the second-round nested PCR) 0.5 L Mixed primers 0.3 L dNTP 2 L 10 buffer 2 L TransStart Taq DNA polymerase 0.25 L Deionized water to 20 L

(38) PCR reaction conditions: firstly pre-denaturation at 95 C. for 5 min; then 30 cycles of 95 C., 30 s; 60 C., 30 s; 72 C., 30 s; finally elongation at 72 C. for 10 min.

(39) Take the amplified promoter-leader sequence fragment, constant region-poly A tail fragment and variable region fragment as templates, and use CMV-UP and TK-PolyA as primers, to perform overlapped extension PCR to amplify linear expression cassettes of H, and chains. PCR products were identified by nucleic acid electrophoresis. The reaction system for amplifying the full-length linear expression cassettes is listed in Table 8.

(40) TABLE-US-00008 TABLE 8 Reaction system for amplifying full-length linear expression cassettes Component Volume Template 1 (promoter-leader sequence fragment) 10 ng Template 2 (constant region-poly A tail fragment) 10 ng Template 3 (variable region fragment) 0.5 L Upstream primer (CMV-UP) 2.5 L (10 M) Downstream primer (TK-Poly A) 2.5 L (10 M) dNTP 4 L 10 buffer 5 L TransStart Taq DNA polymerase 1 L Deionized water to 50 L

(41) PCR reaction conditions: firstly pre-denaturation at 95 C. for 5 min; then 30 cycles of 95 C., 30 s; 60 C., 30 s; 72 C., 3 min; finally elongation at 72 C. for 10 min.

(42) PCR reaction products are directly recovered with the OMEGA kit and quantified with Nano (GE Healthcare). One day before transfection, 210.sup.4 cells in 150 L medium were seeded into each well of 96-well plates. On the day of transfection, took 0.2 g of each light and heavy chain, added 0.8 L of Turbofect transfection reagent, diluted to 40 L with DMEM medium, and incubated at room temperature for 15 min after mixing. The mixtures were slowly added dropwise to 96-well plates and then cultured in a 37 C. incubator for 48 h.

(43) 6. Screening of Antibodies with Binding Capacity by ELISA

(44) 1) One day before the experiment, microplates are coated with 100 L of NiV-BD G at 1 g/mL and incubated overnight at 4 C. in a humid box. 2) On the following day, plates are washed 5 times with a plate washer (BIO-TEK, 405_LS). After adding 100 L of blocking buffer to each well, plates are incubated at 37 C. for 1 h. 3) After washed by plater-washer, plates are added 100 L of the transfected cell culture supernatant and then incubated at 37 C. for 1 h. 4) After washed, plates are added 100 L of HRP-labelled goat anti-human IgG (Abcam, Ab97225) at a dilution of 1:10,000, and then incubated at 37 C. for 1 h. 5) After washed, plates are added 100 L of TMB substrate for 6 min in the dark at room temperature, followed by addition of 50 L stop solution. Optical density at a 450-630 nm is read on a microplate reader.

(45) Results: Taking 0.1 as the cutoff value of optical density. Fifty-nine antibodies that can specifically bind to NiV-BDG were screened from 254 amplified positive clones. These antibodies were further expressed, purified and verified. Distribution of OD values for ELISA screening of antibodies with binding capacity is shown in FIG. 3.

(46) 7. Construction of the Expression Plasmid Construction and Preparation of the Antibody

(47) The expression plasmids are constructed and then the antibodies are preparation by expression. The method is described as follows: 1) Full-length genes of heavy chain and light chain linear expression cassettes are digested with EcoR I (NEB, R3101) and Not I (NEB, R3189), then ligated into pcDNA3.4 expression plasmids. 2) 15 g of pcDNA3.4-H and 15 g of pcDNA3.4-L are co-transfected into 30 mL Expi293 system (Life, A14524), and cells are cultured at 125 rpm, 5% CO.sub.2 for 72 h. 3) The expression supernatant is collected by centrifugation at 3000g for 10 min. Antibodies are purified using a rProtein A column. Antibodies are buffer-exchanged into PBS and then quantified by BCA protein quantification kit (Thermo Scientific, 23225).

Example 2. Detection of the Binding Capacity of the Antibody by ELISA

(48) 1) One day before the assay, microplates are coated with 100 L of NiV-BD/MY G or HeV G (NiV-BD G, Genbank: AY988601.1; NiV-MY G, Genbank: FN869553.1; HeV G, Genbank: NC_001906.3) at 1 g/mL and incubated overnight at 4 C. 2) On the following day, after washing 5 times by a plate washer, adding 100 L of blocking buffer to each well, plates are incubated at room temperature for 1 h. 3) Wash plates. Add 150 L of antibodies at a concentration of 20 g/mL to the first well, and add 100 L of dilution solution to the remaining wells. Transfer 50 L from the first well to the second well, and so on, dilute at a gradient of 1:3, with a final volume of 100 L per well. Incubate plates for 1 h at room temperature. 4) Wash plates. Add 100 L of HRP-labelled goat anti-human IgG (Abcam, Ab97225) at a dilution of 1:10,000 into each well, and then incubated at room temperature for 1 h. 5) Wash plates. Plates are added 100 L of TMB substrate for 6 min in the dark at room temperature, followed by addition of 50 L stop solution. 6) Read optical density at a 450-630 nm on a microplate reader.

(49) As shown in FIG. 4, antibodies have good binding capacity to the protein G of NiV-BD, NiV-MY and HeV. Among them, the half effective concentration (EC.sub.50) values of antibody 1B6 are 28.43 ng/mL, 63.92 ng/mL and 52.93 ng/mL, respectively; the EC.sub.50 values of antibody 1E5 are 2.6 ng/ml, 0.85 ng/ml and 0.34 ng/ml, respectively; the EC.sub.50 values of antibody 2A4 are 4.50 ng/ml, 11.23 ng/ml and 7.37 ng/mL, respectively; the EC.sub.50 values of antibody 2E7 are 15.4 ng/ml, 26.69 ng/ml and 70.05 ng/ml, respectively.

(50) The sequences of the above-mentioned four antibodies are sequenced. The nucleotide sequences of the heavy chain variable region of 1B6 is shown as SEQ ID NO:2; the nucleotide sequences of the light chain variable region of 1B6 is shown as SEQ ID NO:4; the amino acid sequences of the heavy chain variable regions of 1B6 is shown as SEQ ID NO:1; the amino acid sequences of the light chain variable regions of 1B6 is shown as SEQ ID NO:3; further analysis on the amino acid sequences of the heavy chain and the light chain variable region shows, the amino acid sequences of the CDR1, CDR2 and CDR3 region of the heavy chain variable region are respectively shown as 26-33, 51-58, and 97-116 of SEQ ID NO:1, the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown as 27-36, 54-56, and 93-100 of SEQ ID NO:3.

(51) The nucleotide sequences of the heavy chain variable region of 1E5 is shown as SEQ ID NO:6; the nucleotide sequences of the light chain variable region of 1E5 is shown as SEQ ID NO:8; the amino acid sequences of the heavy chain variable regions of 1E5 is shown as SEQ ID NO:5; the amino acid sequences of the light chain variable regions of 1E5 is shown as SEQ ID NO:7; further analysis on the amino acid sequences of the heavy chain and the light chain variable region shows, the amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26-33, 51-58, and 97-117 of SEQ ID NO:5, the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown as 27-32, 50-52, and 89-97 of SEQ ID NO:7.

(52) The nucleotide sequences of the heavy chain variable region of 2A4 is shown as SEQ ID NO:10; the nucleotide sequences of the light chain variable region of 2A4 is shown as SEQ ID NO: 12; the amino acid sequences of the heavy chain variable regions of 2A4 is shown as SEQ ID NO:9; the amino acid sequences of the light chain variable regions of 2A4 is shown as SEQ ID NO:11; further analysis on the amino acid sequences of the heavy chain and the light chain variable region shows, the amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown as 26-33, 51-58, and 97-115 of SEQ ID NO:9, the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown as 27-32, 50-52, and 89-97 of SEQ ID NO:11.

(53) The nucleotide sequences of the heavy chain variable region of 2E7 is shown as SEQ ID NO:14; the nucleotide sequences of the light chain variable region of 2E7 is shown as SEQ ID NO: 16; the amino acid sequences of the heavy chain variable regions of 2E7 is shown as SEQ ID NO: 13; the amino acid sequences of the light chain variable regions of 2E7 is shown as SEQ ID NO:15; further analysis on the amino acid sequences of the heavy chain and the light chain variable region shows, the amino acid sequences of CDR1, CDR2 and CDR3 region of the heavy chain variable region are respectively shown as 26-33, 51-58, and 97-109 of SEQ ID NO:13, the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown as 27-37, 51-53, and 90-100 of SEQ ID NO:15.

(54) Four monoclonal antibodies have the same human heavy chain and light chain constant regions. The sequence of the polynucleotide encoding the heavy chain constant region is shown as SEQ ID NO:18, and the sequence of the polynucleotide encoding the light chain constant region is shown as SEQ ID NO:20 or SEQ ID NO:22, the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO: 17, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO: 19 or SEQ ID NO:21.

Example 3. Affinity Determination of Antibody 1E5

(55) 1) Dilute 1E5 to concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM, respectively. 2) Prepare the Octet RED instrument (fortBIO, Pall Corp, USA) and set the kinetic detection method in the companion software Data Analysis Software v9.0. The method includes 5 steps: baseline, loading, baseline, association and dissociation, and the duration of each step is set to 100 s, 180 s, 60 s, 300 s and 600 s respectively. 3) Place the antibody, antigen, and PBS buffer, and then start the assay. After the experiment, the software DataAnalysis was used for data processing, and the equilibrium dissociation constant KD value and the binding dissociation curve of 1E5 with Henipavirus G proteins are fitted and calculated. As shown in FIG. 5, A, B and C are the curves of 1E5 binding and dissociation with HeV G, NiV-BD G, and NiV-MY G, respectively. The calculation results of the KD value are shown in the table below.

(56) TABLE-US-00009 TABLE 9 Affinity of 1E5 to Henipavirus G protein Antigen NiV-BD G NiV-MY G HeV G KD 0.171 nM <0.001 nM 0.785 nM

(57) 1E5 has high affinity to all three G proteins, and the affinity constant KD is less than 1 nM. The highest affinity is to NiV-MY G and the lowest is to HeV G.

Example 4. Pseudovirus Neutralization Assay to Evaluate the Neutralizing Capacity of the Antibodies

(58) Package of human immunodeficiency virus (HIV)-backboneNiV-BD, NiV-MY and HeVpseudoviruses to evaluate the neutralizing capacity of monoclonal antibodies in vitro (Dimple Khetawat, C.C.B., A functional Henipavirus envelope glycoprotein pseudotyped lentivirus assay system. Virology Journal 2010. 7(312)). Method is as below: 1) Dilute antibodies with DMEM medium, add 75 L of antibody at 5 g/mL to the first well of 96-well culture plates, and add 50 L of DMEM medium to the remaining wells. 2) Transfer 25 L of liquid from the first well into the second well, mix well, and so on, dilute at a ratio of 1:3, and the final volume of each well is 50 L. Add 50 L pseudovirus to each well and incubate at 37 C. for 1 h. 3) Count 293T cells and seed 100 L cells at a density of 210.sup.5 cells/mL to each well. Place the culture plate in a 37 C. incubator for 36-48 h. 4) Take out plates. Carefully remove the medium. Add 100 L of cell lysate to each well and shake at 400 rpm for 15 min on a shaker. Centrifuge at 3000 rpm for 10 min at room temperature. After mixing the lyophilized detection substrate and buffer of the luciferase detection system (Promega, E1501), then fill them in the GLOMAX 96 Microplate Luminometer (Promega) detection loops. Transfer 20 L of the lysis supernatant and read the fluorescence value. Calculate the protection rate of the antibody on the cells.

(59) The results are shown in FIG. 6, FIG. 7 and FIG. 8, 1B6, 1E5, 2A4 and 2E7 can effectively neutralize three pseudoviruses of HIV-NiV-BD, -NiV-MY, and -HeV in vitro. Among them, the neutralizing capacity of 1B6, 1E5 and 2A4 increased with the increase of its concentration, and nearly 100% neutralization against the three pseudotyped Henipaviruses could be achieved at a concentration of 1 g/mL. As for HeVpseudovirus, the half inhibiting concentration (IC.sub.50) values of 1B6, 1E5 and 2A4 are 16.31 ng/mL, 5.74 ng/mL and 28.96 ng/mL, respectively, and 2E7 could be nearly 100% neutralized at 5 g/mL. As for NiV-BD and NiV-MY pseudovirus, all four antibodies have good neutralizing activity, and the IC.sub.50 values against NiV-MY pseudovirus are 27.15 ng/mL, 19.03 ng/mL, 48.60 ng/mL and 80.79 ng/ml, respectively. These results indicate that the four monoclonal antibodies 1B6, 1E5, 2A4 and 2E7 have broad-spectrum neutralizing capacity, and can simultaneously neutralize Nipah virus and Hendra virus of the Henipavirus genus.

Example 5. Competition Experiment

(60) The capacity of monoclonal antibody inhibiting the binding of Henipavirus G protein with the receptor was evaluated through Luminex microsphere competitive inhibition assay. The method is described as follows: 1) Add 10 L of 10 g monoclonal antibody to the first well, and then dilute it by two times successively. 2) Add 1.25 ng of receptor ephrin-B2 or ephrin-B3 to each well in a volume of 10 L. Add 10 L of prepared microspheres (containing 1500 NiV-BD/MY G-coupled microspheres respectively) to each well, and incubate on a shaker for 60 min. 3) Add 10 L of SAPE (concentration of 12 g/mL) to each well and incubate on a shaker for 30 min. 4) Wash 3 times with 100 L assay buffer and read on Luminex MAGPIX instrument.

(61) The curves of antibodies competitively inhibiting of the binding of Henipavirus G protein with receptor ephrin-B2/B3 are shown in FIGS. 9 and 10. The results show that 1E5 and 2A4 can effectively inhibit the binding of Henipavirus G protein with receptor ephrin-B2/B3 binding; 1B6 and 2E7 can effectively inhibit the binding of Nipah virus G protein with the receptor ephrin-B2/B3, but fail to inhibit Hendra virus G protein binding with the receptor. It is suggested that antibodies 1E5 and 2A4 are likely to play a neutralizing role by inhibiting the binding of Henipavirus G protein with receptor ephrin-B2/B3, while 1B6 and 2E7 may have other neutralizing mechanisms against Hendra virus.

Example 6. Escape Variant Neutralization Experiments

(62) The reported antibody m102.4-escape variant HeV G-D582N (synthesized by Sangon Biotech, Genbank:NC_001906.3. The exceptionally large genome of Hendra virus: support for creation of a new genus within the family Paramyxoviridae. J. Virol. 74 (21), 9972-9979 (2000)) pseudovirus was used to perform neutralization experiments. The amino acid D at position 582 was mutated to N during synthesis, and the pseudovirus was packaged according to Example 4. The results are shown in FIG. 11. When the antibody concentration was 1 g/mL, mAbs 1B6, 1E5 and 2A4 had over 60% inhibition activity against D582N pseudovirus, 2E7 had 50% neutralization activity, and the remaining antibodies were all below 20%. This indicates that the antibodies disclosed in the invention can be used to neutralize the Henipavirus mutant strain escaping from 102.4, which have different binding epitopes from that of m102.4.

INDUSTRIAL APPLICABILITY

(63) The invention provides a series of anti-Henipavirus monoclonal antibodies with broad-spectrum neutralizing capacity and their application in the preparation of medicines. The monoclonal antibodies are easy to be industrially produced and have industrial practicability.