Maturation of neutralizing Ab responses with repeated immunizations. Neutralizing activity in plasma collected 2 wk after the second and fifth inoculations of Env-immunized macaques (red; n = 6) and controls (blue; n = 6) using the TZM-bl pseudovirus assay. The analysis was performed once, at multiple dilutions of each plasma sample. Data from individual animals are shown as the reciprocal dilution giving 50% neutralization (ID₅₀) with group means ± SEM indicated. Clade B HIV-1 isolates that are sensitive (tier 1) or resistant (tier 2) to neutralization were examined. The viruses tested were: tier 1 clade B viruses MN, HxBc2, SF162, and BaL.01 (top left); and Tier 2 clade B viruses YU2, 89.6, 6535, and ADA (top right). The clade A virus DJ263 (bottom left) and the clade C virus MW965 (bottom middle) were also examined. Plasma was also tested for the presence of coreceptor binding site-directed Abs (CoRbs Abs) that neutralize virus pseudotyped with HIV-2 Env in the presence of subinhibitory concentration of soluble CD4 (sCD4; bottom right). ID₅₀ values between inoculations two and five were compared with repeated measures ANOVA. ***, P < 0.001.