Comparison of pre-infection NAb responses among vaccine and placebo recipients as measured with tier 1 and tier 2 reference strains. NAbs in plasma samples from 90 randomly selected vaccine recipients and 30 randomly selected placebo recipients, all of whom who were uninfected at the time of blood draw (2 weeks post fourth inoculation), were assessed against HIV-1_MN, SF162.LS and a panel of 12 subtype B tier 2 reference strains. Positive response rates (frequency of positive results at ≥1:10 plasma dilution), titers of NAbs and M-B curves were derived from results obtained in the TZM-bl (A) and U87.CD4.CCR5.CXCR4 (B) assays. For the box plots of NAb titers (middle panel), 25% of values lie below the box, 25% lie above the box, and 50% lie below the horizontal line (the median) inside the box. Vertical lines above the box extend to a distance 50% greater than the height of the box; points beyond this are unusually high values (outliers). Subject-specific and group averages in M-B plots are shown as light and heavy lines, respectively, and are for the tier 2 viruses only.

Comparison of pre-infection NAb responses among vaccine and placebo recipients as measured with viruses from trial participants. Plasma samples in were assessed for neutralizing activity against viruses from 27 trial participants obtained at the earliest available post-infection time point. A. Neutralization response rates and the titers of NAbs. The first 13 viruses from the left are from vaccine recipients and the second 14 viruses are from placebo recipients. B. M-B curves to the vaccine recipient isolate panel and to the placebo recipient isolate panel (top) and differences in AUC of M-B curves for the placebo and vaccine isolate panels (bottom). Subject-specific and group averages in M-B plots are shown as light and heavy lines, respectively. All results in A and B were obtained in the U87.CD4.CCR5.CXCR4 assay. Parallel assessments in the TZM-bl assay were not performed.

Breadth of pre-infection NAbs against tier 1 and tier 2 viruses among vaccine and placebo recipients. A. Plasma samples from 24 randomly selected vaccine recipients and 5 placebo recipients (2 weeks post fourth immunization, prior to infection) among the same 120 trial participants in were assayed against HIV-1_MN, SF162.LS, six additional tier 1 viruses and one prototypic tier 2 virus (JR-FL) in the TZM-bl assay. Plasma samples were assayed at eight dilutions starting at 1:20. NAb titers <20 were assigned a value of 10. Results are shown for vaccine recipients only. Results with placebo recipient plasmas were low (SS1196.1, four samples with NAb titers of 29–59; MW965.26, one sample with a NAb titer of 31) or negative (all remaining tests). Positive response rate (% of values ≥50 neutralization) is shown above each scatter plot. B. Serum samples from additional vaccine and placebo recipients (n=20 each) were tested for neutralizing activity at a 1:10 dilution in the TZM-bl assay against the 12 subtype B tier 2 reference strains (same as , excluding tier 1 viruses MN and SF162.LS). Many of these same samples (16 vaccine and 17 placebo recipients) were also assayed against 8 tier 2 transmitted/founder clade B strains (WEAU-d15.410.787, BB1006-11.C3.1601, BB1054-07.TC4.1499, BB1056-10.TA11.1826, BB1012-11.TC21, 6240.08.TA.4622, 6244.13.B5.4576, 62357.14.D3.4589); sufficient quantities were not available for all samples to be assayed against this latter panel of viruses. Serum samples prior to the first inoculation (pre-immune) and 2 weeks post fourth inoculation (prior to infection) were assayed in triplicate on the same assay plate. Percent neutralization was calculated by dividing the average RLU of pre-immune serum by the average RLU of post-immune serum, subtracting this result from 1 and multiplying by 100. For each subject and each tier 2 panel (12 reference viruses and 8 transmitted/founder viruses), the average of the percent neutralization values across the isolates in the panel was computed. These averages were compared between the vaccine and placebo groups for each panel with Mann Whitney tests, and were compared between the two panels with a paired data Wilcoxon signed-rank test. Solid symbols, vaccine recipients; open symbols, placebo recipients.

Comparison of post-infection NAb responses among vaccine and placebo recipients. NAbs were assessed in plasma samples from 14 vaccine recipients and 14 placebo recipients 12–24 months after diagnosis of infection. All subjects were antiretroviral therapy naïve at the time of plasma collection. A. Assays with MN, SF162.LS and the subtype B reference panel of tier 2 viruses. B. Assays with viruses from trial participants. In the top two diagrams, the first 13 viruses from the left are from vaccine recipients and the second 14 viruses are from placebo recipients. Autologous virus/plasma combinations in the middle diagram (Neutralization Response Levels) are indicated by an asterisk. All results in A and B were obtained in the U87.CD4.CCR5.CXCR4 assay. Subject-specific and group averages in M-B plots are shown as light and heavy lines, respectively, and are for the tier 2 viruses only.

Comparison of pre-infection NAb responses in vaccine recipients to post-infection NAb responses in placebo recipients. Plasma from 90 vaccine recipients (2 weeks post fourth inoculation) and 14 placebo recipients (1–2 years post diagnosis) were assayed against MN, SF162.LS and the subtype B reference panel of tier 2 viruses. A. TZM-bl assay. B. U87.CD4.CCR5.CXCR4 assay. Subject-specific and group averages in M-B plots are shown as light and heavy lines, respectively, and are for the tier 2 viruses only.

Comparison of pre-infection NAb responses among men and women vaccine recipients (n=90 evaluated in ) as measured with the tier 1 and tier 2 reference strains evaluated in . Positive response rates (frequency of positive results at ≥1:10 plasma dilution), titers of NAbs and M-B curves were derived from results obtained in the TZM-bl (A) and U87.CD4.CCR5.CXCR4 (B) assays. Subject-specific and group averages in M-B plots are shown as light and heavy lines, respectively, and are for the tier 2 viruses only.