Figure 5. Stem immunogens fail to selectively recall stem antibodies in preimmune mice and macaques.

Mice infected intranasally with PR8 and immunized on day 56 were analyzed for (A) serum endpoint total IgG titers measured by ELISA on day 56 (black box shows before immunization) and day 70 (white box shows 2 weeks after immunization) using HA-FL or HA stem proteins (n = 10 from 2 independent experiments with groups of 5 animals) and (B) frequency of GC B cells (B220⁺IgD^–CD38^loGL7⁺) stained with HA-FL and HA stem probes (PR8) measured by flow cytometry (n = 5). Box boundaries represent the 25^th and 75^th percentiles, the inner line represents the median, and whiskers represent minimum and maximum values. Data indicate the mean ± SEM. *P < 0.05, by Mann-Whitney U test. Macaques (n = 6) infected intranasally with A/Auckland/1/2009 and immunized on day 56 with seasonal IIV4 or HA stem immunogen were analyzed for (C) serum endpoint total IgG titers measured by ELISA using CA09 HA-FL (blue) or stabilized CA09 HA stem (red) proteins and (D) frequency of IgG⁺ memory B cells (CD19⁺IgD^–IgG⁺) binding CA09 HA-FL (blue) or stabilized CA09 HA stem (red) probes measured by flow cytometry within cryopreserved PBMC samples. Dotted lines denote the detection cutoff (dilution 1:100).