J. Okada and others but designed to be the 1997 type. Thus, we concluded that group 5 clones bind to site E (Fig. 4b). All 14 clones in group 6 bound to site C1/C2. F020-334 in group 7 was judged to bind to site C2 since the interaction between F020-334 and F85/276C2 was negative. On the other hand, the interaction between F020-334 and Y77/ 276C2 was judged to be positive. However, the interaction between F020-334 and site C1 and C2-chimaeric HA of Yamanashi/77 and Fukuoka/85 strains was largely decreased according to the raw data. Therefore, we concluded that F020-334 bound to site C1/C2. F037-115 of group 12 bound to site B1, and six clones of group 19 recognized site E. The remaining clones, which were six clones in groups 8–11 and 26 clones in groups 13–18, were judged to bind to site C1/C2 (Fig. 4b). The clones in the third set recognized a rather diverse set of epitopes (Fig. 4c). The majority of clones in groups 20 and 25–27 were judged to bind site to B1/B2. F010-073 of group 21 bound to site A/B1, and clones of group 23 bound to site E/C2. F004-136 of group 24 did not bind to W03/142A or W03/189B2 but did bind to all five other chimaeras. Therefore, we concluded that it recognized site A/B2, although the results from Sydney/97 chimaeras were rather marginal. The epitope recognized by the clones belonging to 29 did not appear to be strictly fixed, but we speculate that they might recognize site B2 and the surrounding region. Further validation of the EMAC method Traditionally, an analysis of escape mutants is used to identify epitopes. We compared the results obtained by the EMAC method with the results from an analysis of escape mutants, as indicated in Fig. 5. The epitope recognized by F004-104 of group 20 and F004-136 of group 24 had been revealed by the analysis of escape mutants (Okada et al., 2010). Escape mutants were isolated by incubating A/ Wyoming/3/2003 viruses with IgG1 Ab. In the case of F004- 104, two types of variation, Y159N and D190E, were identified, and the 159th and 190th residues were localized in sites B1 and B2, respectively (Fig. 5a). According to the EMAC method F004-104 did not react with either W03/ 156B1 or W03/189B2 (Fig. 4c); thus, the epitope recognized by the clone was mapped to site B1/B2. In the escape mutants isolated in the presence of F004-136, the mutation was identified to be K145Q. According to the EMAC method, F004-136 did not react with either W03/142A or W03/189B2 (Fig. 4c); thus, the epitope recognized by the clone was mapped to site A/B2 (Fig. 5a). As indicated in Fig. 5b, the amino acid residues that were mutated in the escape mutants were located in the sites that were changed in the chimaeras. Haemagglutination inhibition (HI) activity Since the HI activity has been routinely measured by many researchers (Smith et al., 2004; Wang et al., 1986), we 330 examined the HI activity of the 98 mAbs whose epitopes were analysed by the EMAC method (data not shown). The results can be summarized as follows: (i) the majority of the clones recognizing A and/or B site had strong HI activity; (ii) the clones recognizing site E generally showed HI activity but not strong activity; and (iii) the clones recognizing site C did not show any HI activity. Since HI activity is the ability of Ab to block the interaction between HA and sialic acid, the regularity observed is reasonable. DISCUSSION In a previous paper (Okada et al., 2010), we reported the strain specificity of clones that could represent the in vivo repertoire of neutralizing mAbs against H3N2 influenza viruses from a donor born in 1960. These clones could be divided into three major groups showing distinct strain specificity: 1968–1973, 1977–1993 and 1997–2003. In the present study, we determined the location of epitopes recognized by these mAbs. We developed a new method, EMAC, that allowed us to comprehensively identify the location of epitopes recognized by numerous clones. While the EMAC method does not provide direct evidence showing the location of an epitope, it is highly plausible that the epitopes identified by this method are indeed correct. All the locations identified were superimposed on the 3D structure of the membrane-distal half of HA, which includes five antigenic sites. Fig. 6 illustrates the sites recognized by mAbs with neutralizing activity that were isolated from the donor in June 2004. As indicated in this figure, all five antigenic sites were immunogenic in the donor. A set of B cells producing Abs that can react with viruses are generated by immunization through infection and/or vaccination. Afterwards they will take various courses under further stimulation with the Ags. Many B cells disappear while others become memory cells. Since all of the clones that were analysed in the present study were highly mutated (see Table 2 in Okada et al., 2010), it is likely that they represent the repertoire of memory B cells. In our experiments, we analysed the Ab repertoire formed by B cells isolated from the peripheral blood of a donor in June 2004. Therefore, the clones classified into groups 20– 29 should reflect the Ab repertoire generated by infection and/or vaccination during the period 1997–2003. The epitopes mapped by using Sydney/97 and Wyoming/2003 HAs were distributed in all five antigenic sites. On the other hand, the majority of the clones that were classified into groups 4–19 recognized the antigenic site C. The presence of two kinds of memory B cells, relatively short- lived cells and long-lived cells, may explain the mechanism for generating such differences. For example, the clones that neutralized the 1968–1973 strains would not have been stimulated by Ags after viruses with which they were unable to react became dominant in the circulation. Despite the lack of stimulation, these memory B cells remained in the donor’s body for more than 30 years. Therefore, it appears Journal of General Virology 92