Several recent studies support our findings regarding antigenic regions R1-R4 in the stem region of HA. In efforts aimed at characterizing antibodies with cross-neutralizing activity for various influenza virus subtypes [10, 13, 31] , it was found that most of these antibodies bind to the HA stem region and interfere with conformational changes of the protein critical for membrane fusion. For example, in the case of antibody CR6261 (binding to HA of the SC1919 virus, H1 type), two antigenic regions on HA stem were revealed: helix A in the HA2 region, and the adjacent HA1 region [32] . The critical sites in helix A (corresponding to residues 390-391, 393-395, 398, 401-402, 405 in H1N1 HA) fall within the R2 region identified in this study, whereas the hydrophobic interaction sites in the adjacent HA1 region (corresponding to residues 38-42, 306-307 in H1N1 HA) fall into regions R3 and R4 of this study, respectively. More recently, it was reported that for a different cross-neutralizing antibody that targets a broad range of H3 subtype virus strains, 12D1, the dominant contacts on the antigen (corresponding to residues 425-455 in H1N1 HA) [37] correlate well with the R1 region found in this study. Furthermore, vaccination in mice using a HA2-based synthetic peptide mimicking the 12D1 epitope was shown to provide protection against influenza viruses of H3N2, H5N1, and H1N1 subtypes [38] . Taken together, our approach should be useful for dissecting antigenic regions on the HA stem, and providing clues for designing more potent peptide vaccines in inducing cross-neutralizing antibodies than the entire HA protein [32] .