Fig. 6. The antigenic site of HA recognized by 98 mAbs that showed binding and neutralizing activity against H3 influenza viruses. Illustrations of the 3D model were constructed in the same way as described in Fig. 5(b). The amino acids that formed a receptor-binding site are marked in grey. When loss of Ab binding was observed in some chimaeric HA, the amino acids replaced in the chimaera were marked in the same way as in Fig. 1(b). http://vir.sgmjournals.org Epitope mapping of neutralizing Abs against H3N2 virus 1973 and 1977 as well as between 1993 and 1997. The Abs produced by pre-existing B cells could not effectively neutralize the viruses that were newly emergent between 1973 and 1977 as well as between 1993 and 1997. Although there could be other possible explanations, an analysis of another Ab library from a donor who was born in 1944 gave essentially the same results as those described in this paper (unpublished results). According to these hypotheses, the reason why memory B cells producing Abs that recognized site C became dominant during 1977 and 1993 could be explained as follows. When amino acid sequences were compared between the 1973 and 1977 strains, different amino acids were found at 15 residues in the seven antigenic sites (in this case, B1 and B2 as well as C1 and C2 were counted as different sites). Therefore, it was possible that most of the Abs produced by pre-existing B cells were not effective against the newly emergent viruses. Then, many kinds of B cells that produced neutralizing Abs against the emergent viruses were generated by infection and played roles in protection against the viruses. Since various sites should have been immunogenic, the epitopes recognized by such Abs were located not only at site C but also at sites A and B. Initially, all of these B cells had equal chances of becoming long-lived memory B cells. However, while mutations were introduced into various residues in sites A, B1 and B2 during 1977–1993, no difference was generated in sites C1 and C2. Therefore, the memory B cells that produced Abs against sites C1 and C2 were stimulated every year by infection. On the other hand, the growth of B cells producing Abs that bound to site A or B was not enhanced after the introduction of mutations into the relevant residues, and the numbers of these B cells gradually decreased. In the present analysis, a set of Abs recognizing site C played major roles in protection of a donor against the influenza virus for around 20 years. Since we have analysed B cells from only two donors to date, it is premature to generalize our observations to humans. Recently, however, several papers reported the presence of a neutralizing epitope that was commonly found among the H1 and H5 strains (Ekiert et al., 2009; Kashyap et al., 2008; Sui et al., 2009; Throsby et al., 2008). If the epitope whose sequence has been highly conserved is generally immunogenic in humans, it might be possible and desirable to develop a new type of vaccine that can specifically stimulate the growth of B cells that produce Abs against such a neutralizing epitope. We also found that Abs recognizing site C do not show HI activity. Traditionally, the global influenza surveillance network has adopted the HI assay for characterization of the antigenic properties of influenza viruses. Since we could not isolate escape mutants under the presence of any of the mAbs that recognize site C (unpublished results), it is possible that the alteration of sequences in site C is a much rarer event than that of sites A and B. Thus, we suggest that when the antigenic and genetic evolution of influenza viruses is examined, the 333