The effects of intramuscular immunization against influenza on FVIII immunogenicity. (A) Mice were immunized intramuscularly with a scaled standard human dose (4.8 μg/kg) within 24 hours of the first of 7 rhFVIII (6 IU, ∼240 IU/kg) infusions. Blood was collected by cardiac puncture 4 weeks later and plasma was isolated by centrifugation. (B) Incidence of FVIII-specific IgG assessed by indirect ELISA. Total cohort sizes are indicated above each bar. (C) Incidence of FVIII inhibitors. (D) Titers of influenza- and FVIII-specific IgG among FVIII responders. (E) Comparison of inhibitory activity among FVIII responders. (F) FVIII-specific IgG subclasses IgG1, IgG2a, and IgG2b among FVIII responders. The horizontal lines and error bars represent the mean and SEM. Fisher’s exact and Mann-Whitney U tests were used where appropriate. ND, not detectable; n.s., not significant.