J. Okada and others Fig. 4. Summary of FCM analyses of 98 mAbs that bound to HA and showed neutralizing activity. Plus (+) indicates that the Ab reacted with HA expressed on the cell surface. Minus (”) indicates that the Ab did not react with the HA expressed on the cell surface at all. Blanks indicate that the analysis was not performed. Recognized site: the location of the epitope recognized by respective clones. (a) The first set of clones, (b) the second set and (c) the third set. that some memory B cells persist in the body for a long time; in the most extreme case, memory B cells could persist for a lifetime. The donor had the chance to be infected almost every year by novel influenza viruses that had drifted away from previous viruses. Therefore, some pre-existing memory B cells produced Abs that could neutralize the novel viruses, but others produced Abs that could not neutralize the novel viruses. It might be possible that even if B cells become memory cells, their numbers gradually decrease, and eventually they disappear from the body without further Ag stimulation. In Okada et al. (2010), we compared 50 aa of the five antigenic sites of 12 strains whose amino acids were variable during the period 1968–2004. While no mutations were introduced into site C between 1977 and 1993, many mutations were introduced into sites A and B. According to this hypothesis the stability of the epitopes would greatly affect the fate of memory B cells. Another situation should also be considered to gain insight into memory B cells. When the donor was infected with novel viruses, the generation of new B cells and the activation of pre-existing memory B cells occurred simultaneously. New amino acids were always found at several residues in a newly emergent strain with the exception of the 1970 strain (see Fig. 5 in Okada et al., 2010). When the amino acid sequences were compared between the 1970 and 1973 strains, different amino acids were found at seven residues in the five antigenic sites. However, there were no clones that neutralized the 1973 strain but did not neutralize the 1968 and 1970 strains. It is possible that such clones were generated by infection with the 1973 virus but they did not become long-lived memory B cells. Thus, we speculate that the fate of newly generated B cells is determined by whether Abs produced by pre- existing memory B cells are able to neutralize the newly emergent viruses. As long as the pre-existing memory B cells produce neutralizing Abs effective against novel viruses, it might be difficult for newly generated B cells against the novel viruses to become long-lived memory cells. The majority of the clones were classified into three groups with distinct strain specificity, 1968–1973, 1977– 1993 and 1997–2003; we suspect that this is because the largest change in antigenic epitopes was observed between Fig. 5. Comparison of the sites assigned by the EMAC method with those determined by the isolation of escape mutants. (a) Amino acid sequences of Wyoming/03 HA, those replaced in the chimaeric HA and those in the escape mutants are indicated. Bars indicate the same amino acid as those of Wyo/03. (b) Locations of the epitopes identified by the EMAC method and by the isolation of escape mutants are indicated in the 3D structure of HA. The illustrations were constructed by using the structure of H3 HA [Protein Data Bank (PDB) accession code 1HA0] according to molecular graphics viewer RasMol 2.7.5. Amino acids indicated in various colours correspond to those used in (a). 332 Journal of General Virology 92