Development of anti-Env binding Abs in Env trimer-inoculated macaques. (A) Schematic representation of the study design. Inoculations (black arrows) were given monthly for 5 mo with Env trimers in Abisco-100 + CpG to six monkeys (F123-F128). Six other monkeys were inoculated with Abisco-100 + CpG alone. Blood (red arrowheads) was collected 1 and 2 wk after each inoculation. Bone marrow (blue arrowheads) was sampled 3 wk after the second and fourth protein inoculation. The animals were challenged (green arrow) with SHIV-SF162P4 virus 5 wk after the last vaccination. (B) Env-binding IgG and IgA Abs were measured 2 wk after each inoculation (Pre, preimmunization; Imm 1, first immunization). Binding titers are presented as Log₁₀ OD50 titer, and the black line corresponds to the mean of six monkeys. (C) Avidity index was measured 2 wk after each inoculation with the individual monkeys (n = 6) shown. The experiment was repeated at two independent occasions with similar results for the IgG measurements and once for the IgA measurements. (D) Avidity index against gp140-F, gp120-F, and gp120ΔV1/2/3-F in samples collected 2 wk after immunizations two and five (n = 6). The experiment was performed once. (E) Comparison of avidity index against the different Env antigens in the same samples as in D. The experiment was performed once. The box plots illustrate the following: horizontal line, median; plus, mean; box, interquartile range; whiskers, min/max and error bars, +SEM. Significance was evaluated using ANOVA. ns, nonsignificant. *, P ≤ 0.05; **, for P ≤ 0.01; ***, for P ≤ 0.001.