(A and B) Histograms showing binding of macaque serum FH to live meningococci of group B strain H44/76 when incubated in 1:150 dilutions of predose 1 (dashed line) or postdose 3 macaque sera (solid line). Gray-filled histogram shows bacteria that were not incubated with macaque serum. (A) Representative macaque immunized with the control (WT) FHbp antigen showing higher FH binding in postimmunization than preimmunization serum (i.e., no inhibition and slight enhancement of FH binding). (B) Representative macaque immunized with the mutant FHbp vaccine showing lower FH binding in postimmunization than preimmunization serum (i.e., inhibition). (C) Representative sera from an adult human immunized with 3 doses of a meningococcal serogroup B vaccine (MenB-FHbp) containing FHbp that binds FH. The postdose 3 (solid line) shows approximately 10-fold more binding of FH to meningococci than the preimmunization serum (dashed line). The human sera were tested at a 1:600 dilution. (D and E) Binding of macaque serum FH to strain H44/76 before dose 1 and 3 weeks after 3 doses of WT (D) or mutant FHbp antigen (E). All sera were tested at a 1:150 dilution. After immunization, there was a significant increase in serum FH binding in the WT FHbp group (MFI before vaccination of 487 increasing to 766 one month after dose 3, P = 0.003). In contrast, in the mutant FHbp group, there was a significant decrease in FH binding (MFI of 493 before immunization, which decreased to 294 after immunization, P = 0.005). The number of animals per groups is the same as in Figure 2. (F) Ratios of MFI of serum FH binding (postdose 3/predose 1) for each individual animal. Each symbol represents median ratio determined in 2–3 independent assays. Ratios ≥ 1 signify no inhibition or enhanced FH binding. Ratios < 1 indicate inhibition of FH binding. **P < 0.01, comparing median ratio of mutant or control FHbp vaccine group to a theoretical ratio of 1.0 by a one sample Wilcoxon signed rank test (2-tailed).