Critical interactions in binding antibody NC41 to influenza N9 neuraminidase: amino acid contacts on the antibody heavy chain.

Antibody NC41 binds to the subtype N9 neuraminidase (NA) of influenza virus A/tern/Australia/ G70c/75 and inhibits its enzyme activity. To address the molecular mechanisms by which antibodies interact with neuraminidase and the requirements for successful escape from antibody inhibition, we made amino acid substitutions in heavy chain CDRs of NC41. Antibody proteins expressed as a single-chain Fv (scFv) fused with maltose-binding protein were assayed for binding to NA by ELISA. Association constants (Ka) for wild-type and mutant scFvs are as follows: wild type, 2 x 10(7) M-1; Asn31-->Gln, 2 x 10(7) M-1; Glu96-->Asp, 1 x 10(7) M-1; Asp97-->Lys, 6 x 10(6) M-1; and Asn98-->Gln, 8 x 10(6) M-1. The Ka for intact NC41 antibody was 4 x 10(8) M-1 in the same assay, reflecting increased stability compared to that of the scFv. Mutations in the scFv antibody had less of an effect on binding than mutations in their partners on the NA, and modeling studies suggest that interactions involving the mutant antibody side chains occur, even without taking increased flexibility into account. Asp97 forms a salt link with NA critical contact Lys434; of the four mutants, D97K shows the largest reduction in binding to NA. Mutant N98Q also shows reduced binding, most likely through the loss of interaction with NA residue Thr401. Substitution N31Q had no effect on Ka. NC41 residue Glu96 interacts with NA critical contact Ser368, yet E96D showed only a 2-fold reduction in binding to NA, apparently because the H bond can still form. Asp97 and Asn98 provide the most important interactions, but some binding is maintained when they are mutated, in contrast to their partners on the NA. The results are consistent with maturation of the immune response, when the protein epitope is fixed while variation in the antibody paratope allows increasing affinity. Influenza viruses may exploit this general mechanism since single amino acid changes in the epitope allow the virus to escape from the antibody.