Study design for testing immunogenicity of αLOX-1.Env gp140 in NHP.

<p>Study design for testing immunogenicity of αLOX-1.Env gp140 in NHP.</p

Analysis of HIV-specific T cell responses.

<p>The upper pie charts show the percentage of epitope specificities for (A) CD4⁺ T cell responses and (B) CD8⁺ T cell responses from PBMCs sampled at week 6 (2 weeks post NYVAC-KC administration, NHP groups G1 and G2 only) and week 22 (2 weeks post αLOX-1.Env gp140 administration) as defined by IFNγ⁺ T cells specific to each peptide pool. The lower pie charts show analysis of multifunctional HIV-specific T cell responses. ICS analysis of HIV-specific (C) CD4⁺ (left 6 pies) and (D) CD8⁺ (right 6 pies) T cells in PBMC samples taken at week 6 (2 weeks post NYVAC-KC administration, G1 Nkc2Lp3Nkc and G2 Nkc2Lg3Nkc only) and week 22 (2 weeks post αLOX-1.Env gp140 administration). The data show the breakdown of IFNγ⁺ (labeled as G), IL-2⁺ (labeled as 2) and TNFα⁺ (labeled as T) T cells specific to the combined HIV peptide pools (n = 6 NHP for G1, G2; n = 4 NHP for G3, G4). The lower linear presentation in (C) and (D) shows the same data as the pie charts, but includes data points for individual NHPs.</p

Analysis of Env-specific IgA binding levels.

<p>Plasma samples taken at week -2 (baseline), week 22 (2 weeks post αLOX-1.Env gp140 administrations), and week 32 (2 weeks post NYVAC-KC boost) were measured by binding antibody multiplex assay against the Env proteins indicated above each panel. Each panel shows anti-Env IgA binding units (MFI) of individual NHPs NHP (n = 6 for G1, G2; n = 4 for G3, G4). Plotting details are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153484#pone.0153484.g003" target="_blank">Fig 3</a>, except IgA levels were log₁₀-transformed. Non-responders (open circles) did not meet the pre-specified criteria for vaccine-induced positivity as outlined in the Methods.</p

Analysis of HIV Env-specific cytokine-producing T cells.

<p>(A) Env peptide-specific CD4⁺ T cell responses and (B) Env peptide-specific CD8⁺ T cell responses from PBMC samples taken at week 6 (2 weeks post NYVAC-KC administration for G1 and G2 only) (blue circles) and week 22 (2 weeks post the final αLOX-1.Env gp140 administration) (red circles). The data are based on total CD4⁺ and CD8⁺ T cells and show the sum of IFNγ⁺, IL-2⁺, and TNFα⁺ T cells specific to the Env peptide pools for individual NHP as filled circles. The grey horizontal bars are the median value for the group NHP (n = 6 for G1, G2; n = 4 for G3, G4) and the vertical grey bars are the IQR.</p

Design and physical properties of αLOX-1.Env gp140 fusion protein.

<p>(A) Schematic representation showing antibody (grey) fused at the H chain C-terminus to HIV Env gp140 (black). (B) Analysis of 3 μg purified αLOX-1.Env gp140 fusion protein (lane 2) or 3 μg purified αLOX-1 protein (lane 3) by reducing SDS-PAGE stained with Coomassie Blue and location of protein molecular weight markers (lane 1) is indicated in kDa. Unglycosylated mass of αLOX-1 L chain is ca. 23,800 Da and of αLOX-1.Env gp140 H chain is ca. 127,570 Da. (C) Size exclusion gel chromatography analysis of αLOX-1.Env gp140. The proteins were run separately on an TSK G4000SW column in PBS at 0.5 ml/min. αLOX-1.Env gp140 is shown in the solid line, trimeric gp140 is shown in the dashed line, and monomeric gp140 is shown in the dotted line. For molecular weight calibration the NativeMark Protein Standard (Life Technologies, LC0725) was used and their peak positions are indicated. (D) Equilibrium competition binding analysis of αLOX-1.HIV Env gp140 interaction with human LOX-1. Beads coated with human LOX-1 ectodomain were incubated overnight with 10 ng/ml of the parental mouse αLOX-1 mAb and varying concentrations of αLOX-1.Env gp140 or humanized αLOX-1 IgG4 without fused antigen (X axis units are nM competing human αLOX-1 proteins), then probed with PE-labeled anti-mouse IgG, and analyzed by flow cytometry (Y axis units are mean fluorescence intensity). Black circles are an irrelevant control human IgG4 mAb, grey squares are human αLOX-1.Env gp140, and vertical strokes are humanized αLOX-1 without fused antigen.</p

Serum Env gp140-specific IgG responses to αLOX-1.Env gp140 administration.

<p>(A) Responses in NHP (n = 6 per group) primed with NYVAC-KC virus at week 0 and week 4 (X axis values), followed by αLOX-1.Env gp140 administrations at weeks 12, 16, and 20, with an additional NYVAC-KC boost at week 30 are shown. (B) Responses in NHP primed with αLOX-1.Env gp140 administrations at weeks 12, 16, and 20 (n = 4 per group), with a NYVAC-KC virus boost at week 30 are shown. Open symbols are with poly ICLC co-administration and closed symbols are with GLA co-administration. Reciprocal of the half maximal titration by solid-phase ELISA versus Env gp140 protein is plotted for individual NHPs. Bars are the median. Response rates, based on 1/EC₅₀ >2, are shown above the X-axis and are 100% unless otherwise indicated.</p

Analysis of neutralizing antibody titers.

<p>Plasma samples taken at peak response week 22 (2 weeks post αLOX-1.Env gp140 administrations) and week 32 (2 weeks post NYVAC-KC boost) for groups 1–4 (G1-G4) were tested for neutralizing titers versus each indicated virus (MW965.26 or TH023.6). Log to base 10 of titers inhibiting replication by 50% for individual NHP (n = 6 for G1, G2; n = 4 for G3, G4) are shown. Boxes represent the middle 95^th percentile. Horizontal lines are the median. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153484#pone.0153484.s004" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153484#pone.0153484.s005" target="_blank">S3</a> Tables show similar tests against other viruses.</p

Analysis of Env binding serum IgG antibody titers.

<p>Plasma samples taken at week -2 (baseline), week 22 (2 weeks post αLOX-1.Env gp140 vaccinations), and week 32 (2 weeks post NYVAC-KC boost) were measured by binding antibody multiplex assay against the indicated Env proteins: (A) 96ZM651 gp140, (B) C.1086C_V1_V2 Tags, (C) Con S gp140 CFI, (D) 00MSA 4076, and (E) gp70_B.CaseA-V1_V2. Each panel shows anti-Env IgG titers represented as an area under the titration curve (AUC) of individual NHP (n = 6 for G1, G2; n = 4 for G3, G4). Boxplots show the 25th, 50th, and 75th percentiles of the AUC distribution for each group. Filled and open circles denote positive and negative responders, respectively, and the percent of positive responders is noted above each boxplot. AUC comparisons between groups where the Wilcoxon rank sum test p-value is < 0.05 are denoted with a *. Note that part of the data (weeks -2, 22, 32) in (A) is replicated independently in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153484#pone.0153484.g002" target="_blank">Fig 2</a>.</p