Prime-boost using heterologous env sequences does not augment antibody responses. Binding antibody responses were measured by an human immunodeficiency virus type 1 (HIV-1) envelope binding antibody multiplex assay using a consensus clade A envelope (A), a consensus clade B envelope (B), and the V1V2 variable region (C). Antibodies specific for the CD4 binding site were assessed by differential binding to the clade B isolate YU2 gp120 old core and the D368R mutant (D). Positive responders are shown in red and negative responders in blue (A, B, and C), with background responses (to medium alone) subtracted. Boxplots show the distribution of responses among the positive responders only. The box indicates the median and interquartile range (IQR); the whiskers extend to 1.5 times the IQR from the upper or lower quartile. The response rates are indicated along the x-axis. Abbreviation: MFI, mean fluorescence intensity.