Neutralizing antibodies to influenza viruses

Abstract

The present invention concerns methods and means for identifying, producing, and engineering neutralizing molecules against influenza A viruses, and to the neutralizing molecules produced. In particular, the invention concerns neutralizing molecules against various influenza A virus subtypes, including neutralizing antibodies against H5 and/or H3 and/or H1, such as, for example all of H1, H3, and H5 subtypes, and methods and means for making such molecules.

1 . A molecule, which is an antibody or antibody-like molecule, wherein said molecule
 (i) neutralizes more than one subtype and/or more than one isolate of an influenza A virus,   (ii) binds to a hemagglutinin (HA) antigen of the virus, and (iii) does not inhibit hemagglutination.   
     
     
         2 . The molecule of  claim 1 , which is a polypeptide comprising a VpreB sequence and/or a λ5 sequence. 
     
     
         3 . The molecule of  claim 1 , which is a polypeptide comprising a VpreB sequence fused to a λ5 sequence. 
     
     
         4 . The molecule of  claim 1 , which is a κ-like surrogate light chain (SLC) construct comprising a Vκ-like and/or a JCκ sequence. 
     
     
         5 . The molecule of  claim 1 , which is an antibody. 
     
     
         6 . The molecule of  claim 1  which is cross-reactive with at least two HA antigens selected from the group consisting of H1, H2, H3, H5, H6, H7, H8 and H9. 
     
     
         7 . The molecule of  claim 1  which is cross-reactive with at least two HA antigens selected from the group consisting of H1, H2, H3, H5, and H9. 
     
     
         8 . The molecule of  claim 1  which binds to an epitope of an H1 subtype of the HA antigen. 
     
     
         9 . The molecule of  claim 1  which binds to an epitope of an H5 subtype of the HA antigen. 
     
     
         10 . The molecule of  claim 1  which binds to an epitope of an H3 subtype of the HA antigen. 
     
     
         11 . The molecule of any one of  claims 8 ,  9 , or  10  wherein the epitope is displayed on the surface of an influenza A virus. 
     
     
         12 . The molecule of any one of  claims 8 ,  9 , or  10  which neutralizes at least one of the H5, H3, and H1 influenza A virus subtypes. 
     
     
         13 . The molecule of any one of  claims 8 ,  9 , or  10  which neutralizes more than one isolate of an H5 and/or H3 and/or H1 subtype of an influenza A virus. 
     
     
         14 . The molecule of  claim 1 , which does not prevent the globular head region of the influenza A virus from binding the surface of a cell. 
     
     
         15 . The molecule of  claim 1  wherein at least one of said viruses has the ability to infect humans. 
     
     
         16 . The molecule of  claim 1  wherein at least one of said isolates has been obtained from a human subject. 
     
     
         17 . The molecule of  claim 1  wherein at least one of said isolates has been obtained from a non-human animal. 
     
     
         18 . The molecule of  claim 17  wherein said non-human animal is a bird. 
     
     
         19 . The molecule of  claim 18  wherein said bird is a wild-fowl or a chicken. 
     
     
         20 . The molecule of  claim 1  which binds to an H1 HA antigen. 
     
     
         21 . The molecule of  claim 20  which binds to at least one additional HA antigen. 
     
     
         22 . The molecule of  claim 21  wherein said additional HA antigen is selected from the group consisting of H2, H3, H5, H6, H7, H8 and H9. 
     
     
         23 . The molecule of  claim 21  which additionally binds HA antigen H5. 
     
     
         24 . The molecule of  claim 21  which additionally binds HA antigens H3 and H9. 
     
     
         25 . The molecule of  claim 21  which additionally binds HA antigens H3, H5, and H9. 
     
     
         26 . An antibody or antibody-like molecule which binds essentially the same epitope as the epitope for an antibody or antibody-like molecule comprising a heavy chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:45, SEQ ID NO:9, and SEQ ID NO:61; or a consensus or variant sequence based upon said amino acid sequences. 
     
     
         27 . The antibody or antibody-like molecule of  claim 26  which binds essentially the same epitope as the epitope for an antibody or antibody-like molecule comprising a light chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID NO:160; or a consensus or variant sequence based upon said amino acid sequences. 
     
     
         28 . An antibody or antibody-like molecule which binds essentially the same epitope as the epitope for an antibody or antibody-like molecule comprising a heavy chain polypeptide comprising an amino acid sequence having the formula: X 1 -X 2 -Q-L-V-Q-S-G-X 3 -E-V-X 4 -K-P-G-X 5 -S-V-X 6 -X 7 -S-C-K-X 8 -S-G-G-X 9 -F-S-S-Y-A-X 10 -X 11 -W-V-R-Q-A-P-G-Q-G-L-E-W-M-G-X 12 -G-I-I-X 13 -X 14 -F-G-T-T-X 15 -N-Y-A-Q-K-F-Q-G-R-X 16 -T-X 17 -T-A-D-X 18 -X 19 -T-S-T-A-Y-M-E-L-S-S-L-R-S-X 20 -D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X 21 -X 22 -L-D-Y-W-G-X 23 -G-T-X 24 ; or a consensus or variant sequence based upon said amino acid sequences, or a fragment thereof;
 wherein X 1  is Q or E; X 2  is V or M; X 3  is A or T; X 4  is K or Q; X 5  is S or A; X 6  is K or R; X 7  is V or L; X 8  is A, T or V; X 9  is T, S or A; X 10  is I or V; X 11  is S or T; X 12  is G or A; X 13  is P or G; X 14  is I or M; X 15  is A or T; X 16  is V or L; X 17  is I, L, or M; X 18  is K or E; X 19  is S, L or M; X 20  is E or D; X 2  is S, T or N; X 22  is S or T; X 23  is Q, K, G or R; and X 24  is L, T or M.   
     
     
         29 . The antibody or antibody-like molecule of  claim 28  which binds essentially the same epitope as the epitope for an antibody or antibody-like molecule comprising a light chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID NO:160; or a consensus or variant sequence based upon said amino acid sequences. 
     
     
         30 . An antibody or antibody-like molecule comprising a heavy chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:45, SEQ ID NO:9, and SEQ ID NO:61; or a consensus or variant sequence based upon said amino acid sequences. 
     
     
         31 . The antibody or antibody-like molecule of  claim 30  further comprising a light chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID NO:160; or a consensus or variant sequence based upon said amino acid sequences. 
     
     
         32 . An antibody or antibody-like molecule comprising a heavy chain polypeptide comprising an amino acid sequence having the formula: X 1 -X 2 -Q-L-V-Q-S-G-X 3 -E-V-X 4 -K-P-G-X 5 -S-V-X 6 -X 7 -S-C-K-X 8 -S-G-G-X 9 -F-S-S-Y-A-X 10 -X 11 -W-V-R-Q-A-P-G-Q-G-L-E-W-M-G-X 12 -G-I-I-X 13 -X 14 -F-G-T-T-X 15 -N-Y-A-Q-K-F-Q-G-R-X 16 -T-X 17 -T-A-D-X 18 -X 19 -T-S-T-A-Y-M-E-L-S-S-L-R-S-X 20 -D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X 21 -X 22 -L-D-Y-W-G-X 23 -G-T-X 24 ; or a consensus or variant sequence based upon said amino acid sequences, or a fragment thereof;
 wherein X 1  is Q or E; X 2  is V or M; X 3  is A or T; X 4  is K or Q; X 5  is S or A; X 6  is K or R; X 7  is V or L; X 8  is A, T or V; X 9  is T, S or A; X 10  is I or V; X 11  is S or T; X 12  is G or A; X 13  is P or G; X 14  is I or M; X 15  is A or T; X 16  is V or L; X 17  is I, L, or M; X 18  is K or E; X 19  is S, L or M; X 20  is E or D; X 21  is S, T or N; X 22  is S or T; X 23  is Q, K, G or R; and X 24  is L, T or M.   
     
     
         33 . The antibody or antibody-like molecule of  claim 32  further comprising a light chain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID NO:160; or a consensus or variant sequence based upon said amino acid sequences. 
     
     
         34 . The antibody or antibody-like molecule of any one of  claims 26  to  33  which (i) neutralizes more than one subtype and/or more than one isolate of an influenza A virus, (ii) binds to a hemagglutinin (HA) antigen of the virus, and (iii) does not inhibit hem agglutination. 
     
     
         35 . A composition comprising a molecule according to any one of  claims 1  to  25 . 
     
     
         36 . A composition comprising an antibody or antibody-like molecule according to any one of  claims 26  to  34 . 
     
     
         37 . A molecule comprising an antibody heavy chain variable domain comprising at least one substitution in the surface exposed cluster determined by amino acid positions 52A, 53, 73, and 74, following Kabat amino acid numbering, wherein said molecule is capable of binding to and neutralizing a viral antigen. 
     
     
         38 . The molecule of  claim 37 , comprising a substitution at least one of amino acid positions 52A, 53, 73, and 74. 
     
     
         39 . The molecule of  claim 37 , comprising a substitution at all of amino acid positions 52A, 53, 73, and 74. 
     
     
         40 . The molecule of  claim 39 , further comprising a substitution at amino acid position 57. 
     
     
         41 . The molecule of  claim 39  comprising P52G, 153M, L73E, and S74L/M substitutions. 
     
     
         42 . The molecule of  claim 41  additionally comprising an A57T substitution. 
     
     
         43 . The molecule of  claim 42  additionally comprising a substitution at least one of amino acid positions 24, 34, 35 and 50. 
     
     
         44 . The molecule of  claim 43  comprising substitutions at all of amino acid positions 24, 34, 35 and 50. 
     
     
         45 . The molecule of  claim 44  comprising V24T, W34V, G35T and S50A substitutions. 
     
     
         46 . The molecule of any one of  claims 37  to  45  wherein the heavy chain variable domain sequence is from the V H  1e germ-line heavy chain. 
     
     
         47 . The molecule of  claim 46  wherein the rest of the heavy chain variable domain sequence retains the sequence of the V H  1e germ-line heavy chain. 
     
     
         48 . The molecule of  claim 46  wherein the V H  1e germ-line heavy chain variable domain comprises at least one additional conservative substitution. 
     
     
         49 . The molecule of any one of  claims 38  to  48 , further comprising a light chain sequence. 
     
     
         50 . The molecule of  claim 49  wherein the light chain sequence is an antibody λ or κ light chain sequence. 
     
     
         51 . The molecule of  claim 49  wherein the light chain sequence is a surrogate light chain sequence. 
     
     
         52 . The molecule of  claim 51  wherein the surrogate light chain sequence comprises a VpreB sequence and/or a λ5 sequence. 
     
     
         53 . The molecule of  claim 52 , wherein the surrogate light chain sequence comprises a VpreB sequence fused to a λ5 sequence. 
     
     
         54 . The molecule of  claim 51 , wherein the surrogate light chain sequence is a κ-like surrogate light chain (SLC) construct comprising a Vκ-like and/or a JCκ sequence. 
     
     
         55 . The molecule of any one of  claims 37  to  54 , wherein the viral antigen is selected from the group consisting of viral antigens from influenza viruses, HIV-1, HIV-2, HTLV-I and -II viruses, SARS coronavirus, herpes simplex virus, Epstein Barr virus, cytomegalovirus, HCV, HAV, HBV, HDV, HEV, toxoplasma gondii virus, treponema pallidium virus, human T-lymphotrophic virus, encephalitis virus, West Nile virus, Dengue virus, Varicella Zoster Virus, rubeola, mumps, and rubella. 
     
     
         56 . The molecule of  claim 55  wherein the viral antigen is from an influenza virus or an HIV-1 or HIV-2 virus. 
     
     
         57 . A vaccine effective against an influenza A virus, comprising a peptide or polypeptide functionally mimicking a neutralization epitope of a molecule according to any one of  claims 1  to  36 . 
     
     
         58 . A vaccine effective against a viral antigen, comprising a peptide or polypeptide functionally mimicking a neutralization epitope of a molecule according to any one of  claims 37  to  57 . 
     
     
         59 . A method for identifying an antibody capable of neutralizing an isolate of an H5 influenza A virus and/or an isolate of an H1 influenza A virus; or a subtype of an H5 influenza A virus and/or a subtype of an H1 influenza A virus, comprising identifying, in an antibody library, antibodies that react with both an H5 isolate and/or an H1 isolate; or an H5 subtype and/or an H1 subtype, and subjecting the antibodies identified to successive alternating rounds of selection, based on their ability to bind said H5 and/or H1 isolates or HA proteins; or said H5 and/or H1 subtypes or HA proteins, respectively. 
     
     
         60 . A collection of sequences shared by the neutralizing antibodies identified by the method of  claim 59 . 
     
     
         61 . A collection of sequences comprising one or more of the unique heavy and/or light chain sequences shown in Table 2 or a consensus or variant sequence based on said sequences. 
     
     
         62 . A neutralizing antibody identifiable by the method of  claim 59 , or a fragment thereof.