Breadth of binding and HAI activity of serum Ab from i. n. + i. p Breadth of binding and HAI activity of serum Ab from i.n. + i.p. vaccinated groups (COBRA: P1 [n = 15], X3 [n = 15], X6 [n = 15]; seasonal: Chile/83 [n = 10], Sing/86 [n = 5], NC/99 [n = 10], and Brisb/07 [n = 10], and pandemic H1N1: CA/09 [n = 6]), using COBRA, seasonal, or pandemic H1N1 viral strains. Breadth of binding and HAI activity of serum Ab from i.n. + i.p. vaccinated groups (COBRA: P1 [n = 15], X3 [n = 15], X6 [n = 15]; seasonal: Chile/83 [n = 10], Sing/86 [n = 5], NC/99 [n = 10], and Brisb/07 [n = 10], and pandemic H1N1: CA/09 [n = 6]), using COBRA, seasonal, or pandemic H1N1 viral strains. White dots indicate the IgG binding titer against COBRA (P1, X3, and X6) and multiple H1N1 rHA expressed as area under curve (AUC) of the obtained OD414 values from 3-fold serial diluted sera on the different rHA proteins. The y-axes on the right of each graph are expressed as antilog of Log10. The continuous black lines indicate the AUC values obtained on cH6/1 rHA protein and approximately representing the contribution of stem-binding Abs. Black bars indicate the HAI titers (left y-axis) against COBRA (P1, X3, and X6) and multiple H1N1 viruses (x-axis). Titers below the limit of detection were assigned a value of 5 for the purpose of calculating the geometric mean titer and are not visible on this scale. The y-axes along the left of each graph are expressed as Log2. Values are reported as absolute mean values plus SEM obtained in a single experiment and using sera from different mouse groups (A–H). Data are representative of three independent experiments evaluating the breadth of binding and HAI activity of sera from the same mouse groups. For each independent experiment, mouse sera were assayed in duplicate. Black dotted lines delimiting the gray area indicate the 1:40–1:80 HAI titer range. Giuseppe A. Sautto et al. ImmunoHorizons 2018;2:226-237 Copyright © 2018 The Authors