Neutralizing anti-influenza a virus antibodies and uses thereof

Abstract

The invention relates to antibodies, and antigen binding fragments thereof, that specifically bind to an epitope in the stem region of an influenza A hemagglutinin trimer and neutralize a group 1 subtype and a group 2 subtype of influenza A virus. The invention also relates to nucleic acids that encode, immortalized B cells and cultured single plasma cells that produce, and to epitopes that bind to such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies, antibody fragments, and epitopes in screening methods as well as in the diagnosis, treatment and prevention of influenza A virus infection.

1 . An isolated antibody, or an antigen-binding fragment thereof comprising a heavy chain variable region having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 59 or 55; and a light chain variable region having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 57 or 61, that neutralizes infection of a group 1 subtype and a group 2 subtype of influenza A virus and specifically binds to an epitope in the stem region of an influenza A hemagglutinin (HA) trimer, and wherein said antibody, or antigen-binding fragment thereof, is produced in transfected cells at titers of at least 3 fold higher than the titer at which FI6 variant 2 is produced. 
     
     
         2 . An isolated antibody, or an antigen-binding fragment thereof of  claim 1 , comprising a heavy chain variable region having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 55; and a light chain variable region having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 57, that neutralizes infection of a group 1 subtype and a group 2 subtype of influenza A virus and specifically binds to an epitope in the stem region of an influenza A hemagglutinin (HA) trimer, and wherein said antibody, or antigen-binding fragment thereof, is produced in transfected cells at titers of at least 3 fold higher than the titer at which FI6 variant 2 is produced. 
     
     
         3 . An isolated antibody, or an antigen-binding fragment thereof of  claim 1 , comprising a heavy chain variable region having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 59; and a light chain variable region having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 57, that neutralizes infection of a group 1 subtype and a group 2 subtype of influenza A virus and specifically binds to an epitope in the stem region of an influenza A hemagglutinin (HA) trimer, and wherein said antibody, or antigen-binding fragment thereof, is produced in transfected cells at titers of at least 3 fold higher than the titer at which FI6 variant 2 is produced. 
     
     
         4 . An isolated antibody, or an antigen-binding fragment thereof of  claim 1 , comprising a heavy chain variable region having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 59; and a light chain variable region having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 61, that neutralizes infection of a group 1 subtype and a group 2 subtype of influenza A virus and specifically binds to an epitope in the stem region of an influenza A hemagglutinin (HA) trimer, and wherein said antibody, or antigen-binding fragment thereof, is produced in transfected cells at titers of at least 3 fold higher than the titer at which FI6 variant 2 is produced. 
     
     
         5 . The isolated antibody, or an antigen-binding fragment thereof of  claim 1 , that neutralizes infection of a group 1 subtype and a group 2 subtype of influenza A virus and comprises: (i) the heavy chain CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NOs: 1, 41 and 43, respectively, or as set forth in SEQ ID NOs: 1, 41 and 42, respectively; and (ii) the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NOs: 4, 5 and 6, respectively, or as set forth in SEQ ID NOs: 44, 5 and 6, respectively. 
     
     
         6 . The isolated antibody, or antigen binding fragment thereof of  claim 1 , wherein the antibody is a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, Fab, Fab′, F(ab′)2, Fv or scFv. 
     
     
         7 . The isolated antibody, or antigen-binding fragment thereof of  claim 1 , wherein said antibody, or antigen-binding fragment thereof, specifically binds to an influenza A HA of subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16. 
     
     
         8 . A nucleic acid molecule comprising a polynucleotide encoding the antibody, or an antigen binding fragment thereof, of  claim 1 . 
     
     
         9 . A vector comprising the nucleic acid molecule of  claim 8 . 
     
     
         10 . A cell expressing the antibody or an antigen binding fragment thereof of  claim 1 . 
     
     
         11 . A pharmaceutical composition comprising the antibody or antigen-binding fragment of  claim 1 , and a pharmaceutically acceptable diluent or carrier. 
     
     
         12 . A method for diagnosing influenza A virus infection in a subject, the method comprising contacting the antibody or antigen-binding fragment thereof of  claim 1  with a sample from the subject. 
     
     
         13 . A method for monitoring the quality of anti-influenza A virus vaccines, comprising contacting the vaccine with the antibody or antigen-binding fragment thereof of  claim 1  and determining whether the vaccine contains a specific epitope in correct conformation. 
     
     
         14 . A method of reducing influenza A virus infection, or lowering the risk of influenza A virus infection, comprising: administering to a subject in need thereof, a prophylactically or therapeutically effective amount of the antibody or antigen binding fragment thereof of  claim 1 . 
     
     
         15 . An isolated antibody, or an antigen-binding fragment thereof comprising a heavy chain variable region having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 59 or 55; and a light chain variable region having at least 90% sequence identity to the amino acid sequence as set forth in SEQ ID NOs: 57 or 61, that neutralizes infection of a group 1 subtype and a group 2 subtype of influenza A virus and specifically binds to an epitope in the stem region of an influenza A hemagglutinin (HA) trimer, wherein the heavy and light chain of said antibody, or antigen binding fragment thereof, contact amino acids in a first, proximal monomer and a second, distal, right monomer of said HA trimer, and wherein said antibody, or antigen-binding fragment thereof, is produced in transfected cells at titers of at least 3 fold higher than the titer at which FI6 variant 2 is produced, in which:
 a. the heavy chain of said antibody, or antigen-binding fragment thereof, contacts the amino acid at position 318 in HA1 and amino acid residues at positions 18, 19, 20, 21, 38, 41, 42, 45, 49, 53, and 57 in HA2 of said first or second monomer, and wherein said monomer is uncleaved or cleaved;   b. the light chain of said antibody, or antigen-binding fragment thereof, contacts amino acid residues at positions 38, 39, and 43 in HA2 of said proximal monomer, and amino acid residues at positions 327, 328, and 329 in HA1 and 1, 2, 3, and 4 in HA2 of said distal right monomer, and wherein said proximal and said distal right monomers are uncleaved;   c. the light chain of said antibody, or antigen-binding fragment thereof, contacts amino acid residues at positions 38, 39, 42, and 46 in HA2 of said proximal monomer and amino acid residues at positions 321 and 323 in HA1 and 7 and 11 in HA2 of said distal right monomer, and wherein said proximal and said distal right monomers are cleaved;   d. the antibody, or antigen-binding fragment thereof specifically binds to an epitope that comprises the amino acid at position 318 of HA1 and amino acid residues at positions 18, 19, 20, 21, 38, 39, 41, 42, 43, 45, 48, 49, 53, 56, and 57 of HA2 of said proximal monomer, and the amino acid residues at positions 327, 328, 329 of HA1 and amino acid residues at positions 1, 2, 3, and 4 of HA2 polypeptide of said distal right monomer, wherein said proximal and said distal right monomers are uncleaved;   e. the antibody, or antigen-binding fragment thereof specifically binds to an epitope that comprises the amino acid at position 318 of HA1 and the amino acid residues at positions 18, 19, 20, 21, 38, 39, 41, 42, 45, 46, 49, 52, 53, and 57 of HA2 of said proximal monomer, and the amino acid residues at positions 321 and 323 of HA1 and amino acid residues at positions 7 and 11 of HA2 of said distal right monomer, wherein said proximal and said distal right monomers are cleaved; or the antibody, or   f. the antigen-binding fragment thereof specifically binds to an epitope that comprises the amino acid at position 329 of HA1 and the amino acid residues at positions 1, 2, 3, and 4 of HA2, wherein said HA1 and HA2 are present in an uncleaved monomer of said HA trimer.   
     
     
         16 . The isolated antibody, or an antigen-binding fragment thereof of  claim 15 , that neutralizes infection of a group 1 subtype and a group 2 subtype of influenza A virus and comprises: (i) the heavy chain CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NOs: 1, 41 and 43, respectively, or as set forth in SEQ ID NOs: 1, 41 and 42, respectively; and (ii) the light chain CDR1, CDR2, and CDR3 sequences as set forth in SEQ ID NOs: 4, 5 and 6, respectively, or as set forth in SEQ ID NOs: 44, 5 and 6, respectively. 
     
     
         17 . The antibody, or antigen binding fragment thereof of  claim 15 , wherein the antibody is a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, Fab, Fab′, F(ab′)2, Fv or scFv. 
     
     
         18 . The antibody, or antigen-binding fragment thereof of  claim 15 , wherein said antibody, or antigen-binding fragment thereof, specifically binds to an influenza A HA of subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16.