FACS analysis showed that the five antibodies bound to all 13 H5 variants tested at levels quite similar to F10, for which a crystal structure had been generated to define this epitope. Thus, half of the neutralizing and a surprising 10% of all antibodies induced by pandemic H1N1 infection bound to a conserved, critical epitope on the HA stalk. By comparison, none of 50 H1N1 strain-specific antibodies that we had previously isolated after annual vaccination before the 2009 pandemic had this reactivity (unpublished data). The frequency of pandemic-induced, stem-reactive antibodies (5/46) versus those from annual vaccine (0/50) is significantly greater (Chi-square test, P = 0.02). Further, this specificity is only rarely seen in human memory B cells (Corti et al., 2010) or from phagedisplay libraries (Sui et al., 2009 ). These observations support avian influenza strain (Fig. 3 B and Fig. S2 A) . In addition, these five antibodies cross-compete for a similar epitope that was not over-lapping with the HAI + antibodies (Fig. 4 A, epitope-1). These antibodies are competitively inhibited by a commercial antibody referred to as C179 that binds this HA stalk region (Okuno et al., 1993) , and four of five of these antibodies are encoded by the hallmark VH1-69 gene (Ekiert et al., 2009; Sui et al., 2009) . To verify HA stalk reactivity, these five antibodies were tested for binding to H5 variants predicted to affect the stalk epitope by the crystal structure, and their binding patterns were compared with that of the prototypical stalk antibody (mAb F10; Sui et al., 2009; Fig. 4 B) . Each H5 variant has a single residue mutation in the stalk region and was transiently expressed on 293T cells. (A) Competition ELISA assays were used to determine the similarity in specificity between the various neutralizing antibodies. Shown is the percentage of competition of each antibody in an ELISA binding assay against all other neutralizing antibodies. A 10-fold molar excess of unlabeled antibody was used to inhibit a biotinylated antibody. Percent competition is calculated as the reduction in absorbance relative to the level of inhibition of any particular antibody against itself. Colors indicate degree of inhibition of antibody binding, as indicated. Antibody C179 is a commercial antibody that binds to the stalk region of the HA molecule identifying epitope-1. Epitope-2 and -3 are each on the HA-head active site. 1000-2G06 and the nonneutralizing, but HAbinding, antibodies had no competition with any of the other HA-reactive antibodies and are therefore not shown. VH gene usage of the individual antibodies is listed on the right. All assays were performed in duplicate. (B) Plasmids encoding full-length WT H5-TH04 (A/Thailand/2-SP-33/2004 [H5N1]) and its mutants were transiently transfected into 293T cells. 24 h after transfection, cells were harvested for FACS analysis, and binding of indicated antibodies was tested at 10 µg/ml. The cell surface HA expression of each of the mutants were verified with a ferret anti-H5N1 serum (not depicted). Antibody F10 was one of the antibodies used to characterize the HA stalk epitope by x-ray crystallography (Sui et al., 2009 ) and served as a positive control for the binding pattern expected of HA stalk-reactive antibodies to these HA mutants.