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/tem/Australia/G70c/75 and inhibits its enzyme activity. To add ress the molecular mechanisms by which antibodies interact with neuram inidase and the requirements for successful escape from antibody inhib ition, we made amino acid substitutions in heavy chain CDRs of NC41. A ntibody proteins expressed as a single-chain Fv (scFv) fused with malt ose-binding protein were assayed for binding to NA by ELISA. Associati on constants (K-a) 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 K-a for intact NC41 antibody was 4 x 10(8) M-1 in the same a ssay, reflecting increased stability compared to that of the scFv. Mut ations in the scFv antibody had less of an effect on binding than muta tions in their partners on the NA, and modeling studies suggest that i nteractions involving the mutant antibody side chains occur, even with out 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 bin ding, most likely through the loss of interaction with NA residue Thr4 01. Substitution N31Q had no effect on K-a. NC41 residue Glu96 interac ts with NA critical contact Ser368, yet E96D showed only a 2-fold redu ction in binding to NA, apparently because the H bond can still form. Asp97 and Asn98 provide the most important interactions, but some bind ing is maintained when they are mutated, in contrast to their partners on the NA. The results are consistent with maturation of the immune r esponse, when the protein epitope is fixed while variation in the anti body paratope allows increasing affinity. Influenza viruses may exploi t this general mechanism since single amino acid changes in the epitop e allow the virus to escape from the antibody.