Antibody epitopes on the neuraminidase of a recent H3N2 influenza virus (A/Memphis/31/98)

Abstract

We have characterized monoclonal antibodies raised against the neuraminidase (NA) of a Sydney-like influenza virus (A/Memphis/31/98, H3N2) in a reassortant virus A/NWS/33(HA)-A/Mem/31/98(NA) (H1N2) and nine escape mutants selected by these monoclonal antibodies. Five of the antibodies use the same heavy chain VDJ genes and may not be independent. Another antibody, Mem5, uses the same V(H) and J genes with a different D gene and different isotype. Sequence changes in escape mutants selected by these antibodies occur in two loops of the NA, at amino acid 198, 199, 220, or 221. These amino acids are located on the opposite side of the NA monomer to the major epitopes found in N9 and early N2 NAs. Escape mutants with a change at 198 have reduced NA activity compared to the wild-type virus. Asp198 points toward the substrate binding pocket, and we had previously found that a site-directed mutation of this amino acid resulted in a loss of enzyme activity (M. R. Lentz, R. G. Webster, and G. M. Air, Biochemistry 26:5351-5358, 1987). Mutations at residue 199, 220, or 221 did not alter the NA activity significantly compared to that of wild-type NA. A 3.5-A structure of Mem5 Fab complexed with the Mem/98 NA shows that the Mem5 antibody binds at the sites of escape mutation selected by the other antibodies.

FIG. 1.

Antibody inhibition curves of NWS-Mem/98 and the escape mutants. Purified viruses were titrated with antibodies Mem4 (a) and Mem5 (b).

FIG. 2.

Sequences of the heavy chains of monoclonal antibodies (with Kabat numbering). The heavy chain (VDJ) amino acid sequences are assembled from N-terminal protein sequence and cDNA sequences. The N-terminal sequences of the light chains were obtained by Edman degradation and subtraction of the heavy chain. X, not determined.

FIG. 3.

Location of escape mutations in recent and early N2 NAs. The amino acid positions where changes have been selected by monoclonal antibodies (mAb) are indicated by the colored bars: green, X-7(F1) (RI/5⁺/57) escape mutants; blue, Tokyo/67 escape mutants, including escape mutants of Tokyo/67 selected by antibodies made against A/Jap/305/57 (H2N2) and A/Texas/77 (H3N2); red, Mem/31/98 (Sydney-like) escape mutants. Data for X-7(F1) and Tokyo/67 escape mutants are from references , , , , and 43).

FIG. 4.

(A) Stereo view of the location of escape mutations on the three-dimensional structure of N2 (A/Tokyo/67) NA (39). The color coding is the same as in Fig. 3. Sialic acid in the active site is shown as a space-filling model. (B) Comparison of crystal structures of NC41 Fab-N9 NA (left) and Mem5 Fab complexed with Mem/98 NA as solved by molecular replacement at 3.5 Å by using Tokyo/67 NA and the NC41 Fv domain (37) as starting models (right). The NA (green) is in the same orientation in both complexes. The antibody heavy chains are red, and light chains are blue.