Determination of the epitope 264 on the hemagglutinin molecule of influenza H1N1 virus by site-specific mutagenesis.

Full-length cDNA of HA gene derived from B-1-23 virus, a variant of A/USSR/90/77 (A/USSR/77) (H1N1) which had been selected with monoclonal antibody (mAb) 264 and contained an amino acid change at position 190 of the HA1 region, was cloned into the SV40 expression vector. Hemabsorbing or fusion activities of the HA protein expressed on CV-1 cells infected with the recombinant virus were indistinguishable from those of the authentic HA protein on B-1-23 virus infected cells. The antigenicity of the expressed HA protein was examined with five monoclonal antibodies to the HA of A/USSR/77 virus. The HA protein reacted with all mAbs except for mAb 264. To define the area of epitope 264, we prepared seven HA cDNAs modified by site-specific mutagenesis and cloned them into the SV40 expression vector (SVHA). The single base change from G to A at position 642 of B-1-23 HA cDNA replaced Asn at position 190 of the HA protein with Asp. The resulting HA protein, the amino acid sequence of which was the same as that in A/USSR/77, regained the binding activity with mAb 264. F. L. Raymond et al. (1984, In "Segmented Negative Strand Viruses" (R. W. Compans and D. H. L. Bishop, Eds.), pp. 253-258, Academic Press, Orlando; 1986, Virology 148, 275-287) and A. P. Kendal et al. (1984, In "Modern Approaches to Vaccines," pp. 151-157, Harvard Univ. Press, Cambridge) showed that changed amino acid residues 219(Lys) and 227(Glu) in A/Brazil/11/78 and 189(Lys) and 225(Asp) in A/Lackland/3/78 were presumably included in the area of epitope 264 by the nucleotide sequence analysis. Of those amino acids, our results show it is the residues 190 and 219 and possibly residue 189 that are involved in the epitope 264, but neither residue 225 nor 227.