Structure of antigenic sites on the haemagglutinin molecule of H5 avian influenza virus and phenotypic variation of escape mutants

To elucidate the structure of the antigenic sites of avian H5 influenza virus haemagglutinin (HA) we analysed escape mutants of a mouse-adapted variant of the H5N2 strain A/Mallard/Pennsylvania/10218/84. A panel of five anti-H5 monoclonal antibodies (mAbs) was used to select 16 escape mutants. The mutants were tested by ELISA and haemagglutination inhibition with this panel of anti-H5 mAbs and the HA genes of the mutants were sequenced. The sequencing demonstrated that the amino acid changes were grouped in two antigenic sites. One corresponded to site A in the H3 HA. The other contained areas that are separated in the amino acid sequence but are topographically close in the three-dimensional structure and partially overlap in the reactions with mAbs. This site corresponds in part to site B in the H3 structure; it also includes a region not involved in site B that partially overlaps site Sa in the H I HA and an antigenic area in H2 HA. Mutants with the amino acid change K152N, as well as those with the change D126N, showed reduced lethality in mice. The substitution D126N, creating a new glycosylation site, was accompanied by an increase in the sensitivity of the mutants to normal mouse serum inhibitors. Several amino acid changes in the H5 escape mutants occurred at the positions of reported changes in H2 drift variants. This coincidence suggests that the antigenic sites described and analysed here may be important for drift variation if H5 influenza virus ever appears as a pathogen circulating in humans.