Broad-spectrum monoclonal anti-flu B antibody and uses thereof

Abstract

Provided are a broad-spectrum monoclonal anti-Flu B antibody, cell strains generating the antibody, and a composition comprising the antibody; also provided are uses of the antibody for diagnosing, preventing and/or treating an infection of the Flu B and/or diseases caused by the infection.

Claims

1. A hybridoma cell line, selected from the group consisting of: 1) Hybridoma cell line 12G6, deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO: C201527; 2) Hybridoma cell line 7G6, deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO: C201435; and 3) Hybridoma cell line 11B10, deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO: C201432.

2. A monoclonal antibody or an antigen binding fragment thereof, comprising complementary determining regions (CDR) of a heavy chain variable region (VH) and complementary determining regions (CDR) of a light chain variable region (VL), wherein the monoclonal antibody or antigen binding fragment thereof comprises: (1) VH CDR1-3 with amino acid sequences set forth in SEQ ID NO: 1-3, respectively, and VL CDR1-3 with amino acid sequences set forth in SEQ ID NO: 4-6, respectively; (2) VH CDR1-3 with amino acid sequences set forth in SEQ ID NO: 7-9, respectively, and VL CDR1-3 with amino acid sequences set forth in SEQ ID NO: 10-12, respectively; or (3) VH CDR1-3 with amino acid sequences set forth in SEQ ID NO: 13-15, respectively, and VL CDR1-3 with amino acid sequences set forth in SEQ ID NO: 16-18, respectively.

3. The monoclonal antibody or antigen binding fragment thereof according to claim 2, wherein the monoclonal antibody or antigen binding fragment thereof comprises: (1) VH set forth in SEQ ID NO: 19 and VL set forth in SEQ ID NO: 20; (2) VH set forth in SEQ ID NO: 21 and VL set forth in SEQ ID NO: 22; or (3) VH set forth in SEQ ID NO: 23 and VL set forth in SEQ ID NO: 24.

4. The monoclonal antibody or antigen binding fragment thereof according to claim 2, wherein the monoclonal antibody or antigen binding fragment thereof has one or more of the following features: (1) the monoclonal antibody or antigen binding fragment thereof is selected from Fab, Fab, F(ab).sub.2, Fd, Fv, dAb, complementary determining region fragment, single chain antibody, mouse antibody, humanized antibody, chimeric antibody, or bispecific or poly-specific antibody; (2) the monoclonal antibody comprises non-CDR region, and the non-CDR region is from a species other than murine species; (3) the monoclonal antibody is a monoclonal antibody produced by Hybridoma cell line 12G6, 7G6 or 11B10, wherein the Hybridoma cell lines 12G6, 7G6 and 11B10 have been deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO C201527, CCTCC NO: C201435 and CCTCC NO: C201432, respectively; (4) the monoclonal antibody or antigen binding fragment thereof can specifically bind to HA1 domain of HA protein of Yamagata lineage and/or Victoria lineage of influenza B viruses; (5) the monoclonal antibody or antigen binding fragment thereof has hemagglutination-inhibiting activity against Yamagata lineage of influenza B virus and/or Victoria lineage of influenza B virus; (6) the monoclonal antibody or antigen binding fragment thereof has neutralizing activity, and can neutralize Yamagata lineage of influenza B virus and/or Victoria lineage of influenza B virus; (7) the monoclonal antibody or antigen binding fragment thereof has an activity of inhibiting entry of Yamagata lineage and/or Victoria lineage of influenza B viruses into a host cell; (8) the monoclonal antibody or antigen binding fragment thereof has an activity of inhibiting release of Yamagata lineage and/or Victoria lineage of influenza B viruses from a host cell; (9) the monoclonal antibody or antigen binding fragment thereof has an activity of inhibiting membrane fusion of Yamagata lineage and/or Victoria lineage of influenza B viruses with a host cell; (10) the monoclonal antibody or antigen binding fragment thereof has an activity of triggering ADCC against Yamagata lineage and/or Victoria lineage of influenza B viruses; and (11) the monoclonal antibody or antigen binding fragment thereof has an activity of triggering CDC against Yamagata lineage and/or Victoria lineage of influenza B viruses.

5. A pharmaceutical composition, comprising the monoclonal antibody or antigen binding fragment thereof according to claim 2 or an anti-idiotype antibody which is specifically directed to the idiotype of said monoclonal antibody, and a pharmaceutically acceptable carrier and/or excipient.

6. A composition, comprising anyone of the following: (1) the monoclonal antibody or antigen binding fragment thereof according to claim 2; (2) an isolated nucleic acid molecule encoding the monoclonal antibody or antigen binding fragment thereof of (1); (3) a vector comprising the isolated nucleic acid molecule of (2); (4) an isolated host cell comprising the isolated nucleic acid molecule of (2); and (5) an anti-idiotype antibody, which is specifically directed to the idiotype of the monoclonal antibody of (1).

7. A kit, comprising the monoclonal antibody or antigen binding fragment thereof according to claim 2 with or without a detectable marker; and optionally, a second antibody, which specifically recognizes the monoclonal antibody or antigen binding fragment thereof and is optionally labeled by a detectable marker.

8. An isolated nucleic acid molecule, encoding the monoclonal antibody or antigen binding fragment thereof according to claim 2.

9. A vector, comprising the isolated nucleic acid molecule according to claim 8.

10. An isolated host cell, comprising the isolated nucleic acid molecule according to claim 8 or a vector comprising said isolated nucleic acid molecule.

11. An anti-idiotype antibody, which is specifically directed to the idiotype of the monoclonal antibody according to claim 2.

12. A method for producing a monoclonal antibody or antigen binding fragment thereof, comprising culturing the host cell according to claim 10 under suitable conditions to express the monoclonal antibody or antigen binding fragment thereof, and recovering the monoclonal antibody or antigen binding fragment thereof from the cell culture.

13. A method for detecting the presence or level of influenza B virus or HA protein thereof in a sample, comprising (1) contacting the monoclonal antibody or antigen binding fragment thereof according to claim 2 with a sample having or suspected to have influenza B virus or HA protein thereof, and (2) detecting any binding between the monoclonal antibody or antigen binding fragment thereof and HA protein of influenza B virus.

14. A method for neutralizing virulence of influenza B virus in a sample, comprising contacting a sample comprising influenza B virus with the monoclonal antibody or antigen binding fragment thereof according to claim 2.

15. A method for diagnosing whether a subject is infected by influenza B virus, comprising (1) contacting the monoclonal antibody or antigen binding fragment thereof according to claim 2 with a sample from a subject that is suspected to be infected by influenza B virus, and (2) detecting the presence of influenza B virus in the sample by detecting any binding between the monoclonal antibody or antigen binding fragment thereof and HA protein of influenza B virus.

16. A method for inhibiting or treating an infection by influenza B virus or a disease associated with the infection in a subject, comprising administering a prophylactically or therapeutically effective amount of the monoclonal antibody or antigen binding fragment thereof according to claim 2 to a subject in need thereof.

17. The method of claim 16, characterized by one or more of the following features: (1) the subject is a human; (2) the influenza B virus is selected from Yamagata lineage and Victoria lineage of influenza B viruses; (3) the monoclonal antibody or antigen binding fragment thereof is administered alone, or in combination with an additional anti-influenza agent; and (4) the disease associated with the infection is influenza.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the three-dimensional structures of the key amino acid sites in the epitopes recognized by the monoclonal antibodies 12G6 (FIG. 1A), 7G6 (FIG. 1B) and 11B10 (FIG. 1C). In FIG. 1A, receptor binding sites of HA are marked in green, and the amino acid sites recognized by the monoclonal antibody 12G6 are marked in red. The results show that the key epitope amino acids recognized by the monoclonal antibody 12G6 are at position 156, position 176 and position 183 of HA. In FIG. 1B, receptor binding sites of HA are marked in green, and the amino acid sites recognized by the monoclonal antibody 7G6 are marked in red. The results show that the key epitope amino acids recognized by the monoclonal antibody 7G6 are at position 156, position 165 and position 180 of HA. In FIG. 1C, receptor binding sites of HA are marked in green, and the amino acid sites recognized by the monoclonal antibody 11B10 are marked in red. The results show that the key epitope amino acid recognized by the monoclonal antibody 11B10 is at position 180 of HA.

(2) FIG. 2 shows results of ELISA assays using the monoclonal antibodies 7G6 and 11B10 to detect viruses B/Florida/04/2006 (Yamagata) (FIG. 2A), B/Malaysia/2506/2004 (Victoria) (FIG. 2B), A/Brisbane/20/2007 (H3N2) (FIG. 2C) and A/NewCalidonia/20/1999 (H1N1) (FIG. 2D), wherein the horizontal ordinate represents the dilution fold of the monoclonal antibody, and the longitudinal coordinate represents the ELISA result (OD.sub.450). The results show that both of the monoclonal antibodies 7G6 and 11B10 have a strong binding reactivity with the two lineages of influenza B viruses, B/Florida/04/2006 (Yamagata) and B/Malaysia/2506/2004 (Victoria), but have no specific reactivity with influenza A viruses, A/Brisbane/20/2007 (H3N2) and A/NewCalidonia/20/1999 (H1N1).

(3) FIG. 3 shows the protective effect of the monoclonal antibody 12G6 in mice infected by influenza B virus B/Florida/04/2006 (FL04-MA) and B/Brisbane/60/2008 (BR60-MA).

(4) FIG. 3A and FIG. 3B show changes in survival rate and body weight of mice in negative control group (PBS-NC), B/Florida/04/2006 virus-infected control group (Flu B Viral cont) and therapeutic group (12G6-10 mg/kg), respectively. FIG. 3C and FIG. 3D show changes in survival rate and body weight of mice in negative control group (PBS-NC), B/Brisbane/60/2008 virus-infected control group (Flu B Viral cont) and therapeutic group (12G6-10 mg/kg), respectively.

(5) The results show that no significant weight fluctuation was observed in the mice in the negative control group during the whole experiment, while significant weight loss was observed in the mice in the two virus-infected control groups. All the mice in the B/Florida/04/2006 virus control group died 8 days after infection, and all the mice in the B/Brisbane/60/2008 virus control group died 10 days after infection. For the two influenza B viruses, the intervention by injection of the antibody 12G6 at a dose of 10 mg/kg can enable the infected mice to regain the body weight, and enable the infected mice to survive normally for 14 days, with a treatment effectiveness of 100%.

(6) FIG. 4 shows the protective effect of the monoclonal antibody 7G6 in mice infected by influenza B virus B/Florida/04/2006 (FL04-MA) and B/Brisbane/60/2008 (BR60-MA).

(7) FIG. 4A and FIG. 4B show changes in survival rate and body weight of mice in negative control group (PBS-NC), B/Florida/04/2006 virus-infected control group (Flu B Viral cont) and therapeutic group (7G6-10 mg/kg), respectively. FIG. 4C and FIG. 4D show changes in survival rate and body weight of mice in negative control group (PBS-NC), B/Brisbane/60/2008 virus-infected control group (Flu B Viral cont) and therapeutic group (7G6-10 mg/kg), respectively.

(8) The results show that no significant weight fluctuation was observed in the mice in the negative control group during the whole experiment, while significant weight loss was observed in the mice in the two virus-infected control groups. All the mice in the B/Florida/04/2006 virus control group died 8 days after infection, and all the mice in the B/Brisbane/60/2008 virus control group died 5 days after infection. For the two influenza B viruses, the intervention by injection of the antibody 7G6 at a dose of 10 mg/kg can enable the infected mice to regain the body weight, and enable the infected mice to survive normally for 14 days, with a treatment effectiveness of 100%.

(9) FIG. 5 shows the protective effect of the monoclonal antibody 11B10 in mice infected by influenza B virus B/Florida/04/2006 (FL04-MA) and B/Brisbane/60/2008 (BR60-MA).

(10) FIG. 5A and FIG. 5B show changes in survival rate and body weight of mice in negative control group (PBS-NC), B/Florida/04/2006 virus-infected control group (Flu B Viral cont) and therapeutic group (11B10-10 mg/kg), respectively. FIG. 5C and FIG. 5D show changes in survival rate and body weight of mice in negative control group (PBS-NC), B/Brisbane/60/2008 virus-infected control group (Flu B Viral cont) and therapeutic group (11B10-10 mg/kg), respectively.

(11) The results show that no significant weight fluctuation was observed in the mice in the negative control group during the whole experiment, while significant weight loss was observed in the mice in the two virus-infected control groups. All the mice in the B/Florida/04/2006 virus control group died 8 days after infection, and all the mice in the B/Brisbane/60/2008 virus control group died 8 days after infection. For the two influenza B viruses, the intervention by injection of the antibody 11B10 at a dose of 10 mg/kg can enable the infected mice to regain the body weight, and enable the infected mice to survive normally for 14 days, with a treatment effectiveness of 100%.

(12) FIG. 6 shows the immunofluorescence analysis results of MDCK cells infected by influenza B virus B/Florida/04/2006 (Yamagata) or B/Brisbane/60/2008 (Victoria), wherein, prior to being used to infect MDCK cells, the influenza B virus was incubated with the monoclonal antibody 12G6, a polyclonal antiserum against B/Florida/4/2006 virus (B/FL. antiserum), a polyclonal antiserum against B/Malaysia/2506/2004 virus (B/Mal. antiserum) or PBS (an antibody-free control), respectively.

(13) FIG. 7 shows the results on the staining of MDCK cells infected by influenza B virus B/Florida/4/2006 (Yamagata) or B/Brisbane/60/2008 (Victoria) with Giemsa Stain, wherein after infection with the virus, the MDCK cells were incubated with the antibody 12G6 at 0 g/ml, 5 g/ml, 20 g/ml or 100 g/ml.

(14) FIG. 8 shows the results of immunoblotting assay for detecting NP protein in the culture supernatant and cell lysate of MDCK cells, wherein the MDCK cells were infected with influenza B virus B/Florida/4/2006 (Yamagata) or B/Brisbane/60/2008 (Victoria), and after the infection, were incubated in a culture medium (a control free of antibody) or with a given concentration of the monoclonal antibody 12G6 (2 g/ml, 0.2n/ml or 0.02 g/ml; diluted in the culture medium) or a given concentration of a negative control antibody (20 g/ml or 2 g/ml; diluted in the culture medium).

(15) FIG. 9 shows the analytic results of ADCC (FIG. 9A) and CDC (FIG. 9B) against influenza virus Massachusetts/02/2012-like (Yamagata) and B/Brisbane/60/2008 (Victoria) triggered by the monoclonal antibody 12G6 and the negative control antibody; wherein Ctr: negative control antibody (anti-HIV mAb 5G6).

(16) Sequence Information

(17) Information of the sequences involved in the invention is provided in the following Table 1.

(18) TABLE-US-00001 TABLE1 SEQID NO: Sequencedepiction 1 theaminoacidsequenceofheavychainCDR1of12G6 GYTFTDYY 2 theaminoacidsequenceofheavychainCDR2of12G6 VNPYSGGT 3 theaminoacidsequenceofheavychainCDR3of12G6 ARWDYGVYEGYIDY 4 theaminoacidsequenceoflightchainCDR1of12G6 EKIYSN 5 theaminoacidsequenceoflightchainCDR2of12G6 AAI 6 theaminoacidsequenceoflightchainCDR3of12G6 QHFWGTPLT 7 theaminoacidsequenceofheavychainCDR1of7G6 GYTFTDYN 8 theaminoacidsequenceofheavychainCDR2of7G6 IYPNNGGT 9 theaminoacidsequenceofheavychainCDR3of7G6 VRSGAYYFNYLVPYFDY 10 theaminoacidsequenceoflightchainCDR1of7G6 ESVDIYGNSF 11 theaminoacidsequenceoflightchainCDR2of7G6 LAS 12 theaminoacidsequenceoflightchainCDR3of7G6 QQNYEDPWT 13 theaminoacidsequenceofheavychainCDRIof11B10 GYTFTGYN 14 theaminoacidsequenceofheavychainCDR2of11B10 IYPNNGVT 15 theaminoacidsequenceofheavychainCDR3of11B10 VRSGAYYVNYLVPYFDY 16 theaminoacidsequenceoflightchainCDR1of11B10 ESIDIYGNSF 17 theaminoacidsequenceoflightchainCDR2of11B10 RAS 18 theaminoacidsequenceoflightchainCDR3of11B10 QQNYEDPWT 19 theaminoacidsequenceofheavychainvariableregion(VH)of12G6 EVHLQQSGPELVKPGASVKMSCEASGYTFTDYYVAWVKQSPGESFEWIGRVNPYS GGTSYNQKFKGKATLIVDKSSSTAYMELSSLTSEDSAVYYCARWDYGVYEGYIDY WGQGSALTV 20 theaminoacidsequenceoflightchainvariableregion(VL)of12G6 DIQMTQSPASLSVSVGETVTITCRASEKIYSNLAWYQQKEGKSPQLLVYAAIRLAD GVPSRFSGSGSGTQFSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELK 21 theaminoacidsequenceofheavychainvariableregion(VH)of7G6 EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMHWVKQSLGKSLEWIGYIYPNN GGTGYNQKFESKATLTVDNSSSTAYMELRTLTSEDSAVYYCVRSGAYYFNYLVPY FDYWGQGTTLTVSS 22 theaminoacidsequenceoflightchainvariableregion(VL)of7G6 NIVLTQSPASLAVSLGQRATISCRASESVDIYGNSFMHWYQQKPGQPPKWYLASK LECGVCARFNGSGCRTDFTLAIDPVEGDDGATYYCQQNYEDPWTFGGGTKLEIK 23 theaminoacidsequenceofheavychainvariableregion(VH)of11B10 EVQLQQSGPELVKPGASVKISCKASGYTFTGYNMHWVKQSHGKSLEWIGKIYPNN GVTGYNQEFRSKATLTVDNSSSTAYMELRSLTSEDSAIYFCVRSGAYYVNYLVPYF DYWGQGTTLTVSS 24 theaminoacidsequenceoflightchainvariableregion(VL)of11B10 NIVLTQSPASLAVSPGQRATISCRASESIDIYGNSFM+56IWYQKKPGQPPKLLIYRASNL ESGVPARFNGSGSRTDFTLTIDPVEGDDGATYYCQQNYEDPWTFGGGTKLEIK 25 thenucleotidesequenceencodingtheheavychainvariableregion(VH)of12G6 GAGGTCCACCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTG AAGATGTCCTGTGAGGCTTCTGGATACACATTCACTGACTACTACGTGGCCTGG GTGAAGCAGAGCCCTGGAGAAAGCTTTGAGTGGATTGGACGTGTTAATCCTTAC AGTGGTGGTACTAGTTACAACCAGAAGTTCAAGGGCAAGGCCACATTGATTGTT GACAAGTCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACATCTGAGGA CTCTGCGGTCTATTACTGTGCTAGATGGGACTATGGTGTCTACGAGGGGTACAT TGACTACTGGGGCCAAGGCTCCGCTCTCACAGTC 26 thenucleotidesequenceencodingthelightchainvariableregion(VL)of12G6 GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACT GTCACCATCACATGTCGAGCAAGTGAGAAAATTTACAGTAATTTAGCATGGTAT CAGCAGAAAGAGGGAAAATCTCCTCAGCTCCTGGTCTATGCTGCAATAAGATTA GCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCC CTCAAGATCAACAGCCTGCAGTCTGAAGATTTTGGGACTTATTACTGTCAACAT TTTTGGGGTACTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA 27 thenucleotidesequenceencodingtheheavychainvariableregion(VH)of7G6 GAGGTCCAGCTTCAGCAGTCAGGACCTGAGCTGGTGAAACCTGGGGCCTCAGT GAAGATATCCTGCAAGGCTTCTGGATACACATTCACTGACTACAACATGCACTG GGTGAAGCAGAGCCTTGGAAAGAGCCTTGAGTGGATTGGATATATTTATCCTAA CAATGGTGGTACTGGCTACAATCAGAAGTTCGAGAGTAAGGCCACATTGACTGT AGACAATTCCTCCAGCACAGCCTACATGGAGCTCCGCACCCTGACATCTGAGGA CTCTGCAGTCTATTACTGTGTAAGATCAGGAGCCTACTATTTTAACTACCTAGTC CCCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 28 thenucleotidesequenceencodingthelightchainvariableregion(VL)of7G6 AACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGG GCCACCATATCTTGCAGAGCCAGTGAAAGTGTTGATATTTATGGCAATAGTTTT ATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATTTATCTT GCATCCAAACTAGAATGTGGGGTGTGTGCCAGGTTCAATGGCAGTGGGTGTAG GACAGACTTCACCCTCGCTATTGATCCTGTGGAGGGTGATGATGGTGCAACCTA TTACTGTCAGCAAAATTATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCT GGAAATCAAA 29 thenucleotidesequenceencodingtheheavychainvariableregion(VH)of11B10 GAGGTCCAGCTTCAGCAGTCAGGACCTGAGCTGGTGAAACCTGGGGCCTCAGT GAAGATATCCTGCAAGGCTTCTGGATACACATTCACTGGCTACAACATGCACTG GGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAAAAATTTATCCTA ACAATGGTGTTACTGGCTACAACCAGGAGTTCAGGAGCAAGGCCACATTGACT GTAGACAATTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAG GACTCTGCAATCTATTTCTGTGTAAGATCAGGAGCCTACTATGTTAACTACCTA GTCCCCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTTTCCTCA 30 thenucleotidesequenceencodingthelightchainvariableregion(VL)of11B10 AACATTGTGCTGACCCAATCTCCAGCCTCTTTGGCTGTGTCTCCAGGGCAGAGG GCCACCATATCCTGCAGAGCCAGTGAAAGTATTGATATTTATGGCAATAGTTTT ATGCACTGGTACCAGAAGAAACCAGGACAGCCACCCAAACTCCTCATTTATCGT GCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAATGGCAGTGGGTCTAGG ACAGACTTCACCCTCACCATTGATCCTGTGGAGGGTGATGATGGTGCAACCTAT TACTGTCAACAAAATTATGAGGATCCGTGGACGTTCGGTGGAGGTACCAAGCTG GAAATCAAA 31 primerMVJkR 5-CCGTTTGKATYTCCAGCTTGGTSCC-3 32 primerMVDJhR 5-CGGTGACCGWGGTBCCTTGRCCCCA-3 33 primer12G6VhMuIgVh5-B2 5-ATGGACTCCAGGCTCAATTTAGTTTTCCT-3 34 primer12G6VkMuIgkV15-F4 5-ATGAAGTTGCCTGTTAGGCTGTTGGTGCT-3 35 primer7G6VhMuIgVh5-B2 5-ATGGACTCCAGGCTCAATTTAGTTTTCCT-3 36 primer7G6VkMuIgkV15-B 5-ATGGAGACAGACACACTCCTGCTAT-3 37 primer11B10VhMuIgVh5B1 5-ATGRAATGSASCTGGGTYWTYCTCTT-3 38 primer11B10VkMuIgkV15-G2 5-ATGGTYCTYATVTCCTTGCTGTTCTGG-3 39 theaminoacidsequenceofHAproteinofB/Xiamen/1346/2008(Victorialineage) MVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETR GKLCPKCLNCTDLDVALGRPKCTGNIPSARVSILHEVRPVTSGCFPIMHDRTKIRQL PNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNVTNGNGFFATMAWAVPKN DNNKTATNSLTIEVPYICTEGEDQITVWGFHSDNETQMAKLYGDSKPQKFTSSANG VTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWC ASGKSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKL ANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADL KSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQI ELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIA AGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFVVY MVSRDNVSCFHS 40 theaminoacidsequenceofHAproteinofB/Singapore/3/1964 MVVTSNADRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTQTR GKLCPNCLNCTDLDVALGRPKCMGTIPSAKASILHEVKPVTSGCFPIMHDRTKIRQL PNLLRGYENIRLSARNVINAETAPGGPYIVGTSGSCPNVTNGKGFFATMAWAVPKN KNKTATNPLTVEVPYICTKGEDQITVWGFHSDNEAQMVTLYGDSKPQKFTSSANG VTTHYVSQIGGFPNQTEDEGLQQSGRIVVDYMVQKPGKTGTIVYQRGVLLPQKVW CASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLK LANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAAD LKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISS QIELAVLLSNEGIINSEDEHILLALERKLKKMLGPSAVDIGNGCFETKIIKCNQTCLDR IAAGTFNAGEFSLPTEDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFIV YMVSRDNVFLLHLSIRK

(19) Description of Deposition of Biological Materials

(20) The invention relates to the following biological materials deposited in China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China):

(21) Hybridoma cell line 12G6, with a deposition number of CCTCC NO: C201527, deposited on Apr. 10, 2015;

(22) Hybridoma cell line 7G6, with a deposition number of CCTCC NO: C201435, deposited on Mar. 26, 2014; and

(23) Hybridoma cell line 11B10, with a deposition number of CCTCC NO: C201432, deposited on Mar. 26, 2014.

SPECIFIC MODES FOR CARRYING OUT THE INVENTION

(24) The present invention is illustrated by reference to the following examples (which are not intended to limit the scope of the present invention).

(25) Unless indicated otherwise, the molecular biological experimental methods and immunological assays used in the present invention are carried out substantially in accordance with the methods as described in Sambrook J et al., Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; restriction enzymes are used under the conditions recommended by manufacturers of the products. In the case where the concrete conditions are not indicated in the examples, the examples are carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatuses, the manufacturers of which are not indicated, are conventional products that are commercially available. Those skilled in the art understand that the examples are used for illustrating the present invention, but not intended to limit the scope of the present invention.

Example 1: Preparation of Monoclonal Antibodies Against HA Protein of Influenza B Virus

(26) 1. Preparation of Virus Antigen

(27) MDCK cells were inoculated with four strains of influenza B virus, i.e., B/Xiamen/891/2006 (Yamagata), B/Xiamen/1346/2008 (Victoria), B/Xiamen/N697/2012 (Yamagata), and B/Xiamen/3043/2006 (Victoria), respectively. After incubating the cells at 37 C. for 2 days, the supernatant was collected to get the amplified viruses. Live viruses were collected, and were inactivated with 0.03% formalin at 4 C. The inactivated viruses were subjected to HA titration to determine the titer of the inactivated viruses (Note: please refer to WHO Operation Guideline for the particular steps of HA titration). B/Xiamen/891/2006 (Yamagata), B/Xiamen/1346/2008 (Victoria), B/Xiamen/N697/2012 (Yamagata), and B/Xiamen/3043/2006 (Victoria) were strains of influenza B virus isolated in the inventor's laboratory.

(28) 2. Experimental Mice:

(29) 6-Week old, SPF-grade female Balb/C mice were provided by Experimental Animal Center, Xiamen University.

(30) 3. Preparation of Hybridoma:

(31) Hybridoma cells secreting monoclonal antibodies were obtained by standard in vivo immunization method and PEG fusion method; please refer to Ed Harlow et al., Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory 1988 for detail. The brief process was as followed.

(32) 3.1 Immunization of Mice:

(33) The titers of the inactivated viruses were adjusted to 128HA, and then the mice were immunized by means of sequential immunization. In brief, firstly, the virus B/Xiamen/891/2006 (Yamagata) was mixed and emulsified with an equal volume of Freund's complete adjuvant (CFA), and then was administered to limbs of mice by multi-point intramuscular injection for primary immunization, at an amount of 400 ul for each mouse. The viruses B/Xiamen/1346/2008 (Victoria), B/Xiamen/N697/2012 (Yamagata), and B/Xiamen/3043/2006 (Victoria) were separately mixed and emulsified with incomplete Freund's adjuvant (IFA), and then administered to the mice for booster immunization at 14 d, 28 d and 42 d after the primary immunization, respectively. Finally, the booster immunization was performed to the spleens of mice at 56 d after the primary immunization, wherein the immunogen was a mixture of the above virus solutions mixed at an equal volume 100 ul for each mouse. 3 d after the immunization, spleens of the mice were taken for fusion experiment.

(34) 3.2 Cell Fusion:

(35) The spleen was ground to obtain a suspension of spleen cells, and then was mixed with mouse myeloma cells SP2/0 in exponential growth phase. Cell fusion was carried out in the presence of PEG1500. The fused cells were re-suspended in 400 ml fusion culture medium, and then were seeded to 20 96-well plates for culture. The fusion culture medium was a RPMI1640 complete screening culture medium containing HAT and 20% FBS.

(36) 3.3 Screening of Hybridoma:

(37) After culturing the fused cells in the 96-well plates for 10 days, the cell supernatant was taken for Hemagglutination-Inhibition (HI) assay and ELISA. The viruses for detection were B/Xiamen/891/2006 (Yamagata) and B/Xiamen/1346/2008 (Victoria). For HI assay, the antibodies secreted in the positive wells should be able to inhibit the agglutination of influenza B virus and red blood cell; for ELISA, the antibodies secreted in the positive wells should be able to specifically react with the influenza B virus coated onto the polystyrene plate. The positive clones as screened were subjected to cloning for three times, so as to obtain hybridoma cell lines capable of stably secreting antibodies. Finally, 41 hybridoma cells lines against HA protein of influenza B virus, including 12G6, 7G6, and 11B10, were obtained.

(38) 3.4 Culture of Hybridoma:

(39) 41 Stable hybridoma cell lines were subjected to amplification culture in a CO.sub.2 incubator, and then were transferred from a 96-well plate to a 24-well plate, and later to a 50 ml cell bottle for further culture. Then, the cells collected from the cell bottle were injected to peritoneal cavities of mice. 7-10 d later, ascites containing monoclonal antibodies were drawn from the peritoneal cavities of mice.

(40) 4. Purification of Monoclonal Antibodies

(41) The ascites containing monoclonal antibodies were precipitated with a 50% ammonium sulfate solution. The precipitate obtained was then dissolved in PBS, and then purified through Protein A column in AKTA system to get the purified monoclonal antibodies. The purity of the purified monoclonal antibodies was identified by SDS-PAGE.

Example 2: Identification of Broad-Spectrum Monoclonal Antibodies Recognizing HA Protein of Different HA Lineages of Influenza B Virus

(42) Representative stains of influenza B virus, which were isolated at different times from different regions and represented different variant types, and representative stains of influenza A virus as control, were selected. HI assay was used to determine the cross-reactivity of said 41 monoclonal antibodies with different lineages of influenza B virus variant strains. Please refer to WHO Operation Guideline for the HI assay. According to the reactivity of the monoclonal antibodies with the influenza virus strains, three broad-spectrum monoclonal antibodies 12G6, 7G6 and 11B10, which could recognize Yamagata and Victoria lineages of influenza B virus simultaneously (i.e., recognize HA protein of different HA lineages of influenza B virus), were identified (Table 2).

(43) The monoclonal antibodies 12G6, 7G6 and 11B10 could specifically react with Yamagata lineage of influenza B virus and Victoria lineage of influenza B virus, and showed a good broad-spectrum reactivity with different HA lineages. The monoclonal antibody 7G6 could react with unlineaged influenza B viruses discovered at an early stage, Yamagata lineage of influenza B virus and Victoria lineage of influenza B virus. Among all the influenza B viruses used in the assay, the monoclonal antibody 7G6 could react with all of them except for the two virus strains, i.e., B/Harbin/7/1994 (Yamagata lineage) and B/Great Lakes/1739/1954, showing a very broad reactive spectrum.

(44) The monoclonal antibodies 12G6 and 11B10 could specifically react with a part of unlineaged influenza B viruses discovered at an early stage, a part of Yamagata lineage of influenza B viruses and a part of Victoria lineage of influenza B viruses. The monoclonal antibody 11B10 had a broader reactivity spectrum than the monoclonal antibody 12G6 in the case of Yamagata lineage and Victoria lineage of viruses; while the monoclonal antibody 12G6 had a broader reactivity spectrum than the monoclonal antibody 11B10 in the case of the unlineaged influenza B viruses discovered at an early stage.

(45) The results showed that although the monoclonal antibodies 12G6, 7G6 and 11B10 were slightly different in terms of the reactivity with some virus strains, all of them are broad-spectrum monoclonal antibodies having specificity for recognizing different HA lineages of influenza B viruses.

(46) TABLE-US-00002 TABLE 2 Activity of monoclonal antibodies 12G6, 7G6 and 11B10 in HI assay (HI titer) HI titer of mAbs HA Lineage Virus 12G6 7G6 11B10 Unlineaged B/Lee/1940 6400 1600 100 influenza B B/Great Lakes/1739/1954 1600 100 100 viruses discov- B/Maryland/1/1959 100 6400 100 ered at an B/Taiwan/2/1962 100 12800 800 early stage B/Singapore/3/1964 400 3200 100 Yamagata B/Harbin/7/1994 200 100 100 lineage B/Florida/4/2006 12800 25600 400 B/Xiamen/891/206 100 25600 800 B/Xiamen/756/2007 100 12800 400 B/Xiamen/1147/2008 100 25600 800 B/Xiamen/N697/2012 100 25600 800 Victoria B/Hong Kong/330/2001 3200 25600 800 lineage B/Malaysia/2506/2004 3200 6400 3200 B/Xiamen/3043/2006 100 12800 6400 B/Xiamen/165/2007 100 12800 3200 B/Brisbane/60/2008 3200 3200 100 B/Brisbane/33/2008 3200 3200 100 B/Xiamen/1346/2008 100 6400 3200 B/Xiamen/N639/2010 100 3200 400 B/Xiamen/N678/2012 100 6400 400 Seasonal H3N2 A/Brisbane/10/2007 100 100 100 Note: HI titer refers to the maximum dilution fold at which the monoclonal antibody can completely inhibit HA activity of virus; wherein 100 means no reactivity.

Example 3. Analysis on Neutralizing Activity of Monoclonal Antibodies 12G6, 7G6 and 11B10

(47) Neutralization activity/titer is an important index for assessing whether a monoclonal antibody has a potential of preventing and treating a disease. In the Example, microwell neutralization assay was used to determine the neutralization activity of the monoclonal antibodies 12G6, 7G6 and 11B10 against the representative strains of different lineages of influenza B viruses (please refer to Hulse-Post et al., PNAS. 2005, 102: 10682-7 for the method). The experimental results were shown in Table 3. Three monoclonal antibodies (12G6, 7G6 and 11B10) had broad-spectrum cross-neutralizing activity for unlineaged influenza B virus discovered at an early stage, Yamagata lineage of influenza B virus and Victoria lineage of influenza B virus. The neutralizing activity of the monoclonal antibody 7G6 was substantively consistent with the HI activity. The monoclonal antibody 7G6 had neutralizing activity for all the tested influenza B viruses except for the two viruses, i.e., B/Harbin/7/1994 (Yamagata lineage) and B/Great Lakes/1739/1954, and showed a relatively broad neutralizing activity spectrum and a very strong reactivity. The neutralizing activity spectrum of the monoclonal antibody 11B10 was slightly broader than the HI activity spectrum. In particular, the monoclonal antibody 11B10 could neutralize almost all the influenza B viruses (except for B/Harbin/7/1994) during 1962-2012. The neutralizing activity spectrum of the monoclonal antibody 12G6 was quite different from the HI activity spectrum, and the monoclonal antibody 12G6 could neutralize all the influenza B viruses during 1940-2012, and showed a very strong and very broad-spectrum neutralizing activity.

(48) TABLE-US-00003 TABLE 3 Activity of monoclonal antibodies 12G6, 7G6 and 11B10 in neutralization assay Neutralization titer of mAbs Lineage Virus 12G6 7G6 11B10 Unline- B/Lee/1940 6400 1600 100 aged influ- B/Great Lakes/1739/1954 25600 100 100 enza B B/Maryland/1/1959 3200 800 100 virus B/Taiwan/2/1962 1600 12800 800 discovered B/Singapore/3/1964 6400 25600 800 at an early stage Yamagata B/Harbin/7/1994 3200 100 100 lineage B/Florida/4/2006 25600 25600 6400 B/Xiamen/891/206 1600 25600 800 B/Xiamen/756/2007 1600 12800 400 B/Xiamen/1147/2008 1600 25600 800 B/Xiamen/N697/2012 800 25600 800 Victoria B/Hong Kong/330/2001 25600 25600 25600 lineage B/Malaysia/2506/2004 12800 25600 25600 B/Xiamen/3043/2006 3200 12800 6400 B/Xiamen/165/2007 6400 12800 3200 B/Brisbane/60/2008 25600 12800 3200 B/Brisbane/33/2008 25600 25600 800 B/Xiamen/1346/2008 3200 6400 3200 B/Xiamen/N639/2010 3200 3200 400 B/Xiamen/N678/2012 1600 6400 400 Seasonal A/Brisbane/10/2007 100 100 100 H3N2 Note: 100, means no reactivity.

Example 4. Identification of Key Epitope Amino Acids of Monoclonal Antibodies 12G6, 7G6 and 11B10

(49) In the Example, monoclonal antibodies 12G6, 7G6 and 11B10 were used to induce and screen influenza B virus strains having escape mutations. The screened escape viruses were subjected to plaque-purification, amplification culture, gene retrieve, sequencing, and structural localization of escape mutation sites, so as to determine the region in which the epitope recognized by monoclonal antibodies 12G6, 7G6 and 11B10 was present, and the key epitope amino acids recognized.

(50) 1. Materials and Methods:

(51) (1) Parent virus: influenza B viruses, B/Singapore/3/1964 and B/Xiamen/1346/2008 (Victoria lineage), were selected as parent viruses for escape mutation screening.

(52) (2) Monoclonal antibody: purified 12G6, 7G6 and 11B10

(53) (3) Escape-screening method: 10.sup.5 TCID.sub.50 parent virus was homogeneously mixed with a single monoclonal antibody, wherein the final concentration of the monoclonal antibody was 1 mg/ml, and the total volume was 1 ml. The virus-monoclonal antibody complex was incubated at room temperature for 4 h, and then was used for infecting MDCK cells pre-coated on a 96-well plate. After incubating the cells with the virus-monoclonal antibody complex for 2 h, the complex was removed, a maintenance medium containing 50 ug/ml monoclonal antibody was added, and the cells were further cultured at 37 C. for 48 h. The culture supernatant was subjected to hemagglutination assay. The viruses in the wells determined to be positive were escape viruses. After three cycles of plaque cloning, stable escape virus strains were obtained.

(54) (4) Localization of escape mutation sites in the escape virus strains: the obtained escape virus strain was subjected to RT-PCR to obtain the structural gene of the virus, and the structural gene thus obtained was sequenced and was aligned with the structural gene of the parent virus so as to identify the escape mutations and the mutation sites.

(55) (5) Localization of key epitope amino acids in 3D structure of HA: the 3D structure file of A/Florida/4/2006 (Yamagata) virus HA protein (PDB No.: 4FQJ) was downloaded from PDB database. The 3D plotting software Pymol was used to localize the receptor binding sites of HA and the key epitope amino acid sites recognized by 12G6, 7G6, 11B10 in 3D structure of HA.

(56) 2. Results and Analysis

(57) The escape mutation results showed that all the escape mutation sites were present in the gene encoding HA1 domain of influenza B virus HA protein.

(58) In particular, the escape mutation results for the monoclonal antibody 12G6 (Table 4) showed that the escape mutation sites involved the amino acid residues at positions 156, 176 and 183 (numbering from the signal peptide, the same below) of HA protein (SEQ ID NO: 39) of B/Xiamen/1346/2008 (Victoria lineage). 15 escape virus strains were obtained by B/Xiamen/1346/2008 (Victoria lineage)-based virus escape screening, among which 4 virus strains had mutation at position 156 of HA (G156W), 4 virus strains had mutation at position 176 of HA (P176Q), and 7 virus strains had mutation at position 183 of HA (T183K). Said three amino acid sites were identified as key epitope amino acids recognized by monoclonal antibody 12G6, and the localization thereof in the 3D structure of HA was shown in FIG. 1A.

(59) TABLE-US-00004 TABLE 4 The amino acid mutation sites of HA from the monoclonal antibody 12G6-induced escape strain Parent virus escape mutation site in HA B/Xiamen/1346/2008 G156W (4/15) P176Q (4/15) T183K (7/15)

(60) The escape mutation results for the monoclonal antibody 7G6 (Table 5) showed that the escape mutation sites involved the amino acid residues at positions 156, 165 and 180 of HA protein (SEQ ID NO: 40) of B/Singapore/3/1964. 16 escape virus strains were obtained by B/Singapore/3/1964-based virus escape screening, among which 6 virus strains had mutation at position 156 of HA (G156W), 4 virus strains had mutation at position 165 of HA (K165E), and 6 virus strains had mutation at position 180 of HA (N180T). Said three amino acid sites were identified as key epitope amino acids recognized by monoclonal antibody 7G6, and the localization thereof in the 3D structure of HA was shown in FIG. 1B.

(61) TABLE-US-00005 TABLE 5 The amino acid mutation sites of HA from the monoclonal antibody 7G6-induced escape strain Parent virus escape mutation site in HA B/Singapore/3/1964 G156W (6/16) K165E (4/16) N180T (6/16)

(62) The escape mutation results for the monoclonal antibody 11B10 (Table 6) showed that the escape mutation sites involved the amino acid residue at position 180 of HA protein (SEQ ID NO: 39) of B/Xiamen/1346/2008 (Victoria lineage). 4 escape virus strains were obtained by B/Xiamen/1346/2008 (Victoria lineage)-based virus escape screening, and all had mutation at position 180 of HA (N180K). This showed that the amino acid at position 180 of HA protein was the key epitope amino acid recognized by monoclonal antibody 11B10, and the localization thereof in the 3D structure of HA was shown in FIG. 1C.

(63) TABLE-US-00006 TABLE 6 The amino acid mutation sites of HA from the monoclonal antibody 11B10-induced escape strain Parent virus escape mutation site in HA B/Xiamen/1346/2008 N180K (4/4)

(64) The experimental results showed that the monoclonal antibodies 12G6, 7G6 and 11B10 recognized similar epitopes, and the epitopes they recognized were all present in HA1 domain of influenza virus HA protein, and were close to receptor binding site (RBS) in the spatial structure.

Example 5. Separation and Sequence Analysis of Light Chain Gene and Heavy Chain Gene of Monoclonal Antibodies 12G6, 7G6 and 11B10

(65) About 10.sup.7 hybridoma cells were cultured by means of semi-adherence. The adhered cells were suspended by blowing, and transferred to a new 4 ml centrifuge tube. After centrifugation at 1500 rpm for 3 min, the cell precipitate was collected and re-suspended in 100 l sterile PBS (pH=7.45), and then transferred to a new 1.5 ml centrifuge tube. 800 l Trizol (Roche, Germany) was added, mixed gently by reverse mixing, and then standing for 10 min. 200 l chloroform was added, shaken vigorously for 15 s, standing for 10 min, and then centrifuged at 4 C., 12000 rpm for 15 min. The supernatant liquid was transferred to a new 1.5 ml centrifuge tube, and an equal volume of isopropanol was added, mixed, standing for 10 min, and then centrifuged at 4 C., 12000 rpm for 10 min. The supernatant was removed, 600 l of 75% ethanol was added for washing, and then centrifuged at 4 C., 12000 rpm for 5 min. The supernatant was removed, and the precipitate was dried under vacuum at 60 C. for 5 min. The transparent precipitate was dissolved in 70 ul DEPC H.sub.2O, and was collected into two tubes. To each tube, 1 l reverse transcription primer was added, wherein the reverse transcription primer added to one tube was MVJkR (5-CCGTTTGKATYTCCAGCT TGGTSCC-3) (SEQ ID NO: 31), for amplification of the gene encoding the light chain variable region, and the reverse transcription primer added to the other tube was MVDJhR (5-CGGTGACCGWGGTBCCTTGRCCCCA-3) (SEQ ID NO: 32), for amplification of the gene encoding the heavy chain variable region. To each tube, 1 l dNTP (Sangon Biotech (Shanghai) Co., Ltd.) was added, and the tubes were placed in 72 C. water bath for 10 min and then immediately in an ice bath for 5 min. 10 l 5 reverse transcription buffer, 1 l AMV (10 u/ul, Promega), and 1 l Rnasin (40 u/l, Promega) were added. After mixing the mixture well, RNA was reverse-transcripted into cDNA at 42 C.

(66) The gene of the variable region of the antibody was separated by polymerase chain reaction (PCR) method. The primer set (as shown in Table 7) and another two designed and synthesized downstream primers MVJkR (SEQ ID NO:31) and MVDJhR (SEQ ID NO:32) (synthesized by ShangHai Boya Company) were used, wherein MVJkR was a downstream primer for amplification of the gene encoding the light variable region, and MVDJhR was a downstream primer for amplification of the gene encoding the heavy variable region. The templates were the two cDNA as synthesized in the previous step. PCR conditions were: 94 C. 5 min; (94 C. 40 s, 53 C. 1 min, 72 C. 50 s)35 cycles; 72 C. 15 min. The fragments of interest were recovered and were cloned to pMD 18-T vector, and then were sent to ShangHai Boya Company for sequencing. By blast alignment, the gene sequences encoding the antibody variable regions were determined, and the corresponding amino acid sequences were determined.

(67) By the above method, the genes encoding the antibody variable regions were cloned from the hybridoma cell lines 12G6, 7G6, 11B10, and the amino acid sequences of complementary determinant regions (CDRs) of the monoclonal antibodies were determined by reference to Kabat method (Kabat E A, Wu T T, Perry H M, Gottesman K S, Coeller K. Sequences of proteins of immunological interest, U.S Department of Health and Human Services, PHS, NIH, Bethesda, 1991). The results were shown in Tables 8a-8b.

(68) TABLE-US-00007 TABLE7 Sequencesofupstreamprimersforamplificationofvariableregion genesofmonoclonalantibodies12G6,7G6,11B10 Variable Nameofupstream regiongene primer Sequenceofupstreamprimer 12G6Vh MuIgVh5-B2 5-ATGGACTCCAGGCTCAATTTAGTTTTCCT-3 (SEQIDNO:33) 12G6Vk MuIgkVI5-F4 5-ATGAAGTTGCCTGTTAGGCTGTTGGTGCT-3 (SEQIDNO:34) 7G6Vh MuIgVh5-B2 5-ATGGACTCCAGGCTCAATTTAGTTTTCCT-3 (SEQIDNO:35) 7G6Vk MuIgkV15-B 5-ATGGAGACAGACACACTCCTGCTAT-3 (SEQIDNO:36) 11B10Vh MuIgVh5-B1 5-ATGRAATGSASCTGGGTYWTYCTCTT-3 (SEQIDNO:37) 11B10Vk MuIgkVI5-G2 5-ATGGTYCTYATVTCCTTGCTGTTCTGG-3 (SEQIDNO:38)

(69) TABLE-US-00008 TABLE8a AminoacidsequencesofCDRsofmonoclonalantibodies12G6,7G6,11B10 CDR Monoclonalantibody No. 12G6 7G6 11B10 heavy CDR1 GYTFTDYY GYTFTDYN GYTFTGYN chain (SEQIDNO:1) (SEQIDNO:7) (SEQIDNO:13) CDR2 VNPYSGGT IYPNNGGT IYPNNGVT (SEQIDNO:2) (SEQIDNO:8) (SEQIDNO:14) CDR3 ARWDYGVYEGYIDY VRSGAYYFNYLVPYFDY VRSGAYYVNYLVPYFDY (SEQIDNO:3) (SEQIDNO:9) (SEQIDNO:15) light CDR1 EKIYSN ESVDIYGNSF ESIDIYGNSF chain (SEQIDNO:4) (SEQIDNO:10) (SEQIDNO:16) CDR2 AAI LAS RAS (SEQIDNO:5) (SEQIDNO:11) (SEQIDNO:17) CDR3 QHFWGTPLT QQNYEDPWT QQNYEDPWT (SEQIDNO:6) (SEQIDNO:12) (SEQIDNO:18)

(70) TABLE-US-00009 TABLE8b Aminoacidsequencesandnucleotidesequencesofvariableregionsof monoclonalantibodies12G6,7G6,11B10 aminoacidsequenceof EVHLQQSGPELVKPGASVKMSCEASGYTFTDYYVAWVKQSPGESFEWIG VHof12G6(SEQID RVNPYSGGTSYNQKFKGKATLIVDKSSSTAYMELSSLTSEDSAVYYCAR NO:19) WDYGVYEGYIDYWGQGSALTV aminoacidsequenceof DIQMTQSPASLSVSVGETVTITCRASEMYSNLAWYQQKEGKSPQLLVYA VLof12G6(SEQID AIRLADGVPSRFSGSGSGTQFSLKINSLQSEDFGTYYCQHFWGTPLTFGAG NO:20) TKLELK aminoacidsequenceof EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMHWVKQSLGKSLEWIG VHof7G6(SEQID YIYPNNGGTGYNQKFESKATLTVDNSSSTAYMELRTLTSEDSAVYYCVRS NO:21) GAYYFNYLVPYFDYWGQGTTLTVSS aminoacidsequenceof NIVLTQSPASLAVSLGQRATISCRASESVDIYGNSFMHWYQQKPGQPPKL VLof7G6(SEQID LIYLASKLECGVCARFNGSGCRTDFTLAFDPVEGDDGATYYCQQNYEDP NO:22) WTFGGGTKLEIK aminoacidsequenceof EVQLQQSGPELVKPGASVKISCKASGYTFTGYNMHWVKQSHGKSLEWIG VHof11B10(SEQID KIYPNNGVTGYNQEFRSKATLTVDNSSSTAYMELRSLTSEDSAIYFCVRS NO:23) GAYYVNYLVPYFDYWGQGTTLTVSS aminoacidsequenceof NIVLIQSPASLAVSPGQRATISCRASESIDIYGNSFMHWYQKKPGQPPKLLI VLof11B10(SEQID YRASNLESGVPARFNGSGSRTDFILTIDPVEGDDGATYYCQQNYEDPWT NO:24) FGGGTKLEIK nucleotidesequenceof GAGGTCCACCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGC VHgeneof12G6 TTCAGTGAAGATGTCCTGTGAGGCTTCTGGATACACATTCACTGACTA (SEQIDNO:25) CTACGTGGCCTGGGTGAAGCAGAGCCCTGGAGAAAGCTTTGAGTGGA TTGGACGTGTTAATCCTTACAGTGGTGGTACTAGTTACAACCAGAAGT TCAAGGGCAAGGCCACATTGATTGTTGACAAGTCCTCCAGCACAGCCT ACATGGAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACT GTGCTAGATGGGACTATGGTGTCTACGAGGGGTACATTGACTACTGGG GCCAAGGCTCCGCTCTCACAGTCTC nucleotidesequenceof GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGA VLgeneof12G6 GAAACTGTCACCATCACATGTCGAGCAAGTGAGAAAATTTACAGTAA (SEQIDNO:26) TTTAGCATGGTATCAGCAGAAAGAGGGAAAATCTCCTCAGCTCCTGGT CTATGCTGCAATAAGATTAGCAGATGGTGTGCCATCAAGGTTCAGTGG CAGTGGATCAGGCACACAGTTTTCCCTCAAGATCAACAGCCTGCAGTC TGAAGATTTIGGGACTTATTACTGTCAACATTTTTGGGGTACTCCICTC ACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC nucleotidesequenceof GAGGTCCAGCTTCAGCAGTCAGGACCTGAGCTGGTGAAACCTGGGGC VHgeneof7G6(SEQ CTCAGTGAAGATATCCTGCAAGGCTTCTGGATACACATTCACTGACTA IDNO:27) CAACATGCACTGGGTGAAGCAGAGCCTTGGAAAGAGCCTTGAGTGGA TTGGATATATTTATCCTAACAATGGTGGTACTGGCTACAATCAGAAGT TCGAGAGTAAGGCCACATTGACTGTAGACAATTCCTCCAGCACAGCCT ACATGGAGCTCCGCACCCTGACATCTGAGGACTCTGCAGTCTATTACT GTGTAAGATCAGGAGCCTACTATTTTAACTACCTAGTCCCCTACTTTG ACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA nucleotidesequenceof AACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGC VLgeneof7G6(SEQ AGAGGGCCACCATATCTTGCAGAGCCAGTGAAAGTGTTGATATTTATG IDNO:28) GCAATAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCC AAACTCCTCATTTATCTTGCATCCAAACTAGAATGTGGGGTGTGTGCC AGGTTCAATGGCAGTGGGTGTAGGACAGACTTCACCCTCGCTATTGAT CCTGTGGAGGGTGATGATGGTGCAACCTATTACTGTCAGCAAAATTAT GAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC nucleotidesequenceof GAGGTCCAGCTTCAGCAGTCAGGACCTGAGCTGGTGAAACCTGGGGC VHgeneof11B10 CTCAGTGAAGATATCCTGCAAGGCTTCTGGATACACATTCACTGGCTA (SEQIDNO:29) CAACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGA TTGGAAAAATTTATCCTAACAATGGTGTTACTGGCTACAACCAGGAGT TCAGGAGCAAGGCCACATTGACTGTAGACAATTCCTCCAGCACAGCCT ACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAATCTATTTCT GTGTAAGATCAGGAGCCTACTATGTTAACTACCTAGTCCCCTACTTTG ACTACTGGGGCCAAGGCACCACTCTCACAGTTTCCTCA nucleotidesequenceof AACATTGTGCTGACCCAATCTCCAGCCTCTTTGGCTGTGTCTCCAGGG VLgeneof11B10 CAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTATTGATATTTAT (SEQIDNO:30) GGCAATAGTTTTATGCACTGGTACCAGAAGAAACCAGGACAGCCACC CAAACTCCTCATTTATCGTGCATCCAACCTAGAATCTGGGGTCCCTGC CAGGTTCAATGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGA TCCTGTGGAGGGTGATGATGGTGCAACCTATTACTGTCAACAAAATTA TGAGGATCCGTGGACGTTCGGTGGAGGTACCAAGCTGGAAATCAAAC

Example 6. Application of Monoclonal Antibodies 7G6 and 11B10 for Detection of Influenza B Virus

(71) 1. Materials and Methods

(72) (1) Preparation of influenza virus: a given amount of the following influenza virus strains: B/Florida/04/2006 (Yamagata), B/Malaysia/2506/2004(Victoria), A/Brisbane/20/2007 (H3N2), A/NewCalidonia/20/1999(H1N1), were inactivated with 0.03% formalin at 4 C. The inactivated virus was subjected to sucrose density gradient centrifugation in a super-centrifuge, and centrifuged at 4 C., 25200 rpm for 3 h. The virus precipitate was dissolved in 1PBS at 4 C. overnight. The super-centrifuged virus was determined by HA titration, to determine the titer of the virus solution.

(73) (2) Monoclonal antibody: the monoclonal antibodies 7G6 and 11B10 as prepared above; the concentration of the monoclonal antibody was 1 mg/ml.

(74) (3) ELISA Experiment:

(75) The titer of the super-centrifuged virus was adjusted to 128HA, and pre-coated onto a 96-well polystyrene ELISA plate, at 200 ul per well. The 96-well plate was then blocked with a blocking liquid. The monoclonal antibody to be tested was diluted to 0.1 mg/ml as the initial concentration, and then subjected to two-fold gradient dilution for 15 times. The diluted monoclonal antibody was added to the ELISA plate, at a volume of 100 ul per well, and incubated at 37 C. for 30 min. The ELISA plate was washed with ELISA washing solution (PBST) for 5 times, and 100 ul diluted HRP-labeled secondary antibody was then added, and the resultant mixture was incubated at 37 C. for 30 min. After washing the ELISA plate with PBST for 5 times, a chromogenic agent was added, and the color was developed for 20 min. The absorption value A450 was then read on an ELISA Reader.

(76) 2. Results and Analysis

(77) ELISA results were shown in FIG. 2. As shown in FIG. 2, monoclonal antibodies 7G6 and 11B10 had a strong binding reactivity with two HA lineages (i.e., Yamagata and Victoria) of influenza B viruses (B/Florida/04/2006 (Yamagata) and B/Malaysia/2506/2004(Victoria)), but had no specific reactivity with the representative strains of influenza A virus (e.g., A/Brisbane/20/2007(H3N2) and A/NewCalidonia/20/1999(H1N1)).

(78) The results showed that the broad-spectrum monoclonal antibodies 7G6 and 11B10 can be used to specifically detect at least two HA lineages of influenza B viruses.

Example 7. Application of Monoclonal Antibodies 12G6, 7G6 and 11B10 for Treatment of Influenza Virus Infection

(79) Passive immunotherapy of infectious diseases with antibodies is a potentially effective route for anti-viral treatment. It was demonstrated by in vitro neutralization assay in microwells in the previous Examples that the monoclonal antibodies according to the invention had high neutralizing activity against at least two HA lineages (e.g., Yamagata and Victoria) of influenza B viruses isolated from different regions at different times. The monoclonal antibodies according to the invention were characterized by a broad neutralizing reactivity spectrum against at least two HA lineages (e.g., Yamagata and Victoria) of influenza B viruses, and a high neutralizing titer. To further demonstrate the anti-viral effect of the monoclonal antibodies according to the invention in vivo, the monoclonal antibodies according to the invention was verified for the anti-viral therapeutic effect on the infection by Yamagata lineage and Victoria lineage of influenza B virus in an Animal Biosafety laboratory, based on Yamagata lineage and Victoria lineage of influenza B virus-infected Balb/C mice. The experiment was as followed.

(80) (1) Materials and Methods

(81) Animal: Balb/C mice, SPF, 6-8-week-old, female, weighed about 20 g.

(82) Monoclonal antibody: 12G6, 7G6, 11B10

(83) Mouse adaptive strain of influenza B virus:

(84) a mouse adaptive strain of Yamagata lineage of influenza B virus: B/Florida/04/2006, called FL04-MA for short;

(85) a mouse adaptive strain of Victoria lineage of influenza B virus: B/Brisbane/60/2008, called BR60-MA for short.

(86) Anesthetic: Isoflorane

(87) Animal grouping: mice were sent to the Animal Biosafety laboratory one day ahead, and were divided into groups, with 5 mice per cage, designated as G1, G2, etc.; the weight of each mouse was recorded, the regimens were shown in Table 9.

(88) Virus infection: Yamagata lineage virus B/Florida/04/2006 was pre-diluted to 10.sup.5 TCID.sub.50/ul, Victoria lineage virus B/Brisbane/60/2008 was pre-diluted to 10.sup.6 TCID.sub.50/ul, and the mice were inoculated with the virus at a dose of 50 l/mouse. Before inoculation, mice were anesthetized with isoflorane, and then infected by inoculation of virus via nasal cavity.

(89) MAb intervention: 24 h (dpi.1) after virus infection, the mice in antibody-treatment group were injected with antibody at a given dose, i.e. 10 mg/kg, wherein the injection volume was MOW/mouse.

(90) Observation: 1-14 days after virus infection, weight, survival, and the corresponding behavioral symptoms of mice were recorded every day.

(91) TABLE-US-00010 TABLE 9 Experimental regimen Group Note Infectious virus MAb intervention Number of mice 12G6 experimental regimen G1 Blank control group PBS PBS 5 G2 Y lineage therapeutic group FL04-MA 12G6 10 mg/kg 5 G3 Y lineage virus control group FL04-MA 5 G4 Blank control group PBS PBS 5 G5 V lineage therapeutic group BR60-MA 12G6 10 mg/kg 5 G6 V lineage virus control group BR60-MA 5 7G6 experimental regimen G7 Blank control group PBS PBS 5 G8 Y lineage therapeutic group FL04-MA 7G6 10 mg/kg 5 G9 Y lineage virus control group FL04-MA 5 G10 Blank control group PBS PBS 5 G11 V lineage therapeutic group BR60-MA 7G6 10 mg/kg 5 G12 V lineage virus control group BR60-MA 5 11B10 experimental regimen G13 Blank control group PBS PBS 5 G14 Y lineage therapeutic group FL04-MA 11B10 10 mg/kg 5 G15 Y lineage virus control group FL04-MA 5 G16 Blank control group PBS PBS 5 G17 V lineage therapeutic group BR60-MA 11B10 10 mg/kg 5 G18 V lineage virus control group BR60-MA 5

(92) (2) Results and Analysis

(93) Mice were separately infected by Yamagata lineage of influenza B virus FL04-MA and Victoria lineage of influenza B virus BR60-MA at a one-week lethal dose. 1 day after infection, the antibody was injected to the caudal vein of mice in the therapeutic group. The therapeutic effect of the monoclonal antibody was determined by measuring the body weight and calculating the survival rate of mice in each group. The experimental results were shown in FIGS. 3-5.

(94) The experimental results in FIG. 3-5 showed that no significant weight fluctuation was observed in mice in the negative control group during the whole experiment; significant weight loss was observed in two virus-infected control groups. All the mice in FL04-MA virus control group died 8 days after infection; and all the mice in BR60-MA virus control group died 5-10 days after infection. In the case of said two influenza B viruses, 12G6, 7G6, 11B10 antibody-injection intervention at a dose of 10 mg/kg could lead to weight recovery in the infected mice (FIGS. 3B, 3D, 4B, 4D, 5B, and 5D). In addition, the survival of mice was observed 1-14 days after virus infection. The results showed that monoclonal antibody 12G6, 7G6, 11B10 at a dose of 10 mg/kg could enable the mice infected by virus survive normally for 14 days, and the treatment effectiveness reached 100% (FIGS. 3A, 3C, 4A, 4C, 5A, and 5C). The results showed that the broad-spectrum monoclonal antibodies according to the invention can effectively prevent and treat the infection by the two HA lineages of influenza B viruses and the diseases caused thereby.

Example 8. Function of Monoclonal Antibody 12G6

(95) 1. Monoclonal Antibody 12G6 can Inhibit the Entry of Influenza B Virus into a Host Cell.

(96) (1) Materials and Methods

(97) Virus: B/Florida/4/2006(Yamagata), and B/Brisbane/60/2008 (Victoria);

(98) HA-specific antibody: monoclonal antibody 12G6, polyclonal antiserum against B/Florida/4/2006 virus, and polyclonal antiserum against B/Malaysia/2506/2004 virus;

(99) rabbit polyclonal antiserum specifically against influenza B virus NP protein;

(100) GAM-FITC (FITC-labeled goat anti-mouse antibody, green fluorescence);

(101) Cell: MDCK cell;

(102) The monoclonal antibody 12G6 (10 g/ml), polyclonal antiserum against B/Florida/4/2006 virus, polyclonal antiserum against B/Malaysia/2506/2004 virus, and PBS (an antibody-free control) were separately incubated with two lineages of influenza B viruses at 37 C. for 1 h. The incubated mixture was then added to the monolayer MDCK cells cultured in a 96-well plate, and the cells were further cultured for 16-18 h. After the culture, the cells were washed with the culture medium for three times. The cells were then subjected to DAPI staining (blue fluorescence) with a commercialized kit; and the cells were subjected to immunofluorescence analysis using rabbit polyclonal antiserum against influenza B virus NP protein (as a first antibody) and GAM-FITC (as a second antibody, green fluorescence).

(103) (2) Results and Analysis

(104) The experimental results were shown in FIG. 6. The results showed that when PBS was used for incubation, both of the two lineages of influenza B viruses tested could enter MDCK cells (cells showed green fluorescence). When polyclonal antiserum against B/Florida/4/2006 virus was used for incubation, B/Florida/4/2006 (Yamagata) could not enter MDCK cells (cells showed blue fluorescence), while B/Brisbane/60/2008 (Victoria) could enter MDCK cells (cells showed green fluorescence). When polyclonal antiserum against B/Malaysia/2506/2004 virus was used for incubation, B/Brisbane/60/2008(Victoria) could not enter MDCK cells (cells showed blue fluorescence), while B/Florida/4/2006(Yamagata) could enter MDCK cells (cells showed green fluorescence). When monoclonal antibody 12G6 was used for incubation, neither of the two lineages of influenza B viruses could enter MDCK cells (cells showed blue fluorescence). The results showed that monoclonal antibody 12G6 could inhibit the entry of the two lineages of influenza B viruses into a host cell.

(105) 2. Monoclonal Antibody 12G6 can Inhibit the Membrane Fusion of Influenza B Virus to a Cell.

(106) (1) Materials and Methods

(107) Virus: B/Florida/4/2006 (Yamagata), and B/Brisbane/60/2008 (Victoria);

(108) HA-specific antibody: monoclonal antibody 12G6

(109) Experimental reagents: Giemsa Stain, 10 mM MES (Sigma, Catalog number: M3671-250G) and 10 mM HEPES (Sigma, Catalog number: H3375, pH 5.5), and TPCK trypsin (Sigma, Catalog number: T1426);

(110) Cell: MDCK cells;

(111) At multiplicity of infection MOI=0.3, MDCK cells were infected by B/Florida/4/2006 (Yamagata) and B/Brisbane/60/2008(Victoria), respectively. 24 hours later, the cells were washed with the culture medium for three times, to remove the residuary virus. 2.5 mg/ml TPCK trypsin was then added to the cells. After incubation at 37 C. for 15 min, the cells were washed with the culture medium for three times, to remove the residuary trypsin. Later, a given concentration of antibody 12G6 (0 g/ml, 5 g/ml, 20 g/ml or 100 g/ml) was added to the cells, and incubated at 37 C. for 30 min. The antibody solution was then removed, and the cells were incubated in 10 mM MES and 10 mM HEPES (pH 5.5) at 37 C. for 2 min (allosterism easily occurs for HA protein under acidic conditions, thereby promoting the fusion of a viral envelope to a cell membrane). After washing with the culture medium for three times (after washing, the virus/cell culture environment turned from acidic to neutral), and the cells infected by virus were further cultured at 37 C. for 3 h. Later, the cells were fixed, and stained with Giemsa Stain, and membrane fusion occurred or not in the cells was observed. If an antibody can inhibit the fusion of a viral envelop to a cell membrane, no syncytium resulted from membrane fusion can be observed after staining. On the contrary, if an antibody cannot inhibit the fusion of a viral envelop to a cell membrane, syncytium resulted from membrane fusion can be observed after staining.

(112) (2) Results and Analysis

(113) The experimental results were shown in FIG. 7. The results showed that when no antibody 12G6 was used for incubating cells (i.e., 0 g/ml antibody), both of the two lineages of influenza B viruses tested could result in membrane fusion of the MDCK cells (i.e., a lot of syncytia appeared). When 5 g/ml or 20 g/ml antibody 12G6 was used for incubation, the membrane fusion of MDCK cells was significantly inhibited (i.e., the number of syncytia decreased significantly), and 20 g/ml antibody had a stronger inhibition than 5 g/ml antibody. Moreover, when 100 g/ml antibody 12G6 was used for incubation, membrane fusion of MDCK cells were completely inhibited (i.e., no syncytia appeared). The results showed that monoclonal antibody 12G6 could inhibit the membrane fusion of the two lineages of influenza B viruses to a cell; and the inhibitory activity of monoclonal antibody 12G6 was dose-dependent.

(114) 3. Monoclonal Antibody 12G6 can Inhibit the Release of Influenza B Virus from a Host Cell.

(115) (1) Materials and Methods

(116) Virus: B/Florida/4/2006 (Yamagata), and B/Brisbane/60/2008 (Victoria)

(117) HA-specific antibody: monoclonal antibody 12G6, and negative control antibody (anti-HIV mAb 5G6);

(118) rabbit polyclonal antiserum specifically against NP protein of influenza B virus;

(119) GAM-HRP;

(120) Cell: MDCK cells;

(121) MDCK cells were inoculated onto a 96-well plate at a density of 40000 cells/well. 4 hours later, and an excessive amount of influenza B virus was added to the cells, to infect the cells. 3 h after the infection, the virus solution was removed, and the cells were washed with PBS for 3 times, to remove the free viruses. To the cell culture plate, a culture medium (a control free of antibody), or a given concentration of monoclonal antibody 12G6 (2 g/ml, 0.2 g/ml or 0.02 m/ml; diluted in the culture medium), or a given concentration of negative control antibody (20 g/ml or 2 g/ml; diluted in the culture medium), was added. After further incubation at 37 C. for 16-18 h, the cell supernatant and cell lysate were collected, respectively, and immunoblotting assay was carried out by using rabbit polyclonal antiserum (as a first antibody) against NP protein of influenza B virus and GAM-HRP (as a second antibody).

(122) (2) Results and Analysis

(123) The experimental results were shown in FIG. 8. The results showed that when no antibody or a negative control antibody was used for incubating the MDCK cells infected by influenza B virus, a significant amount of NP protein could be detected in the cultured supernatant and cell lysate. This showed that the two lineages of influenza B viruses could be proliferated in MDCK cells, and had been released from the cells; and the negative control antibody could not inhibit the release of influenza B virus from the host cells. In contrast, when monoclonal antibody 12G6 was used for incubating the MDCK cells infected by influenza B virus, NP protein could be detected in the cell lysate, but the amount of NP protein in the cultured supernatant decreased with the increase of the concentration of the monoclonal antibody 12G6. When the concentration of monoclonal antibody 12G6 reached 2 m/ml, no NY protein was detected in the cultured supernatant, indicating that the release of influenza B virus was completely inhibited. These results showed that monoclonal antibody 12G6 could inhibit the release of the lineages of influenza B viruses from the host cells, and the inhibitory activity of monoclonal antibody 12G6 was dose-dependent.

(124) 4. Monoclonal Antibody 12G6 has ADCC and CDC Activity.

(125) By the method as described by Srivastava V. et al., J Virol, 2013 May, 87(10):5831-40, ADCC and CDC activity of monoclonal antibody 12G6 were determined.

(126) (1) Materials and Methods

(127) Virus: B/Massachusetts/02/2012-like (Yamagata lineage), and B/Brisbane/60/2008 (Victoria lineage);

(128) HA-specific antibody: 12G6, and negative control antibody (anti-HIV mAb 5G6);

(129) Cell: MDCK cells, and mouse NK cells;

(130) Cell staining reagent: PKH-67(SIGMA-ALDRICH, Catalog number: PKH67GL; as conventional cell membrane dye), and 7-AAD (eBioscience, Catalog number: 00-6993-50; as nucleic acid dye, for identifying dead cells);

(131) Mouse NK Cell Isolation Kit (NK Cell Isolation Kit II mouse, manufacturer: MACS, Catalog number: 130-096-892) was used to isolate NK cells from mouse spleen (i.e., effector cell), for further use. At multiplicity of infection MOI=10, MDCK cells (i.e., target cells) were infected by influenza B virus. 3 h later, at a cell concentration of 110.sup.5 cells/mL, 100 l cells were seeded in a 96-well plate. After culturing for 1 h, the membrane of the MDCK cells was stained with PKH-67 dye. After staining, the antibody to be tested was diluted to a given concentration (20 g/ml, 2 g/ml, or 0.5 g/ml), and added to the cells in the culture plate at a volume of 50 l/well, and the cells were then incubated at 37 C. for 15 min. Later, for ADCC assay, the effector cells (100 l) were added to the cells in the culture plate, at a ratio of effector cells to target cells being 50:1; for CDC assay, 100-fold diluted guinea pig serum (1000) as a complement, was added to the cells in the culture plate. The culture plate was incubated at 37 C. for 2 h, and then the dye 7-AAD was added at a volume of 1 l/well, and the resultant mixture was incubated for 5 min. After the incubation, the cells were analyzed by flow cytometer, and the percentage of the dead target cells was calculated. In addition, the experiment was repeated under the conditions where no antibody was used, as background control, which represented the spontaneous lysis rate of the infected target cells incubated with the effector cells. In addition, 1% Triton X-100 (in place of the antibody to be tested) was used to repeat the experiment, as positive control, which represented the maximum lysis rate of the infected target cells incubated with the effector cells.

(132) The fluorescence staining states for these cells were as followed: living effector cells, no fluorescence; dead effector cells, 7-AAD staining (far-red light); living target cells, PKH-67 staining (green light); dead target cells, PKH-67 and 7-AAD double staining (far-red light and green light). ADCC and CDC activity were calculated as followed:
ADCC activity or CDC activity=(percentage of dead target cells in a test grouppercentage of dead target cells in a background control group)/(percentage of dead target cells in a positive control grouppercentage of dead target cells in a background control group)*100%

(133) (2) Results and Analysis

(134) The experimental results were shown in FIG. 9. The results showed that when a negative control antibody was used for incubation, no obvious lysis occurred in the virus-infected MDCK cells in the presence of NK cells or guinea pig serum. This showed that the negative control antibody could not trigger the ADCC and CDC against the tested two lineages of influenza B viruses. In contrast, when the antibody 12G6 was used for incubation, obvious lysis occurred in the virus-infected MDCK cells in the presence of NK cells or guinea pig serum; and, with the increase of the antibody concentration used, the lysis of MDCK cells was enhanced. These results showed that monoclonal antibody 12G6 could trigger the ADCC and CDC against the tested two lineages of influenza B viruses; and the activity of monoclonal antibody 12G6 was dose-dependent.

(135) The results showed that the antibody 12G6 had multiple functional activities: capable of inhibiting the entry of a virus into a host cell, capable of inhibiting the membrane fusion of a virus to a cell, capable of inhibiting the release of a virus from a host cell, and capable of triggering antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against a virus. The antibody 12G6 can neutralize influenza B virus by the five activities, and thereby prevent and treat infection by influenza B virus.

(136) Although the specific embodiments of the invention have been described in detail, those skilled in the art would understand that, according to all the disclosed teachings, various modifications and changes can be made, and that such modifications and changes are within the scope of the present invention. The scope of the present invention is given by the appended claims and any equivalents thereof.