SDS-PAGE and native PAGE analysis of control and USP laser-treated plasma proteins.

<p>(A) On the left is shown the SDS-PAGE of control and laser-treated plasma; on the right, for comparison, is shown the SDS-PAGE of laser-treated MCMV virus adapted from Tsen <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111673#pone.0111673-Tsen3" target="_blank">[13]</a> (reprinted with permission from the Society of Photo-Optical Instrumentation Engineers). Control (untreated) or USP laser-treated plasma were boiled in reducing buffer and separated on a 10% gel. Proteins were visualized by Coomassie blue stain. The solid arrow indicates the location of low mobility detergent-resistant aggregates; the dotted arrow indicates missing band(s) corresponding to aggregated proteins. (B) Native PAGE of control and laser-treated plasma. Control (untreated) or USP laser-treated plasma were separated on a 10% gel. Proteins were visualized by Coomassie blue stain. Arrows indicate location of low mobility detergent-resistant aggregates.</p

Structural analysis of control and laser-treated fibrinogen protein.

<p>Control (untreated) or laser-treated fibrinogen protein was analyzed by circular dichroism. The red line indicates the CD spectrum of control fibrinogen, while the dotted black line indicates laser treated fibrinogen. The spectra (in Mean Residue Ellipticity, rescaled for concentration) show virtually complete overlap.</p

Retention of coagulation factor activity for USP laser-treated plasma.

<p>Retention of coagulation factor activity for USP laser-treated plasma.</p

Inactivation of viruses in plasma using a USP laser.

<p>Human plasma containing HIV (A), HAV (B), or MCMV (C) were treated with the USP laser. For the HIV-spiked plasma, viral titer was assessed by plaque assay in MAGI cells. For the HAV-spiked plasma, viral titer was assessed by plaque assay in fetal rhesus monkey kidney cells. For the MCMV-spiked plasma, viral titer was assessed by TCID₅₀ assay in murine embryonic fibroblast cells. Results are representative of triplicate experiments and are shown as means ± SEM.</p

Dynamic light scattering measurements on the aggregation state of fibrinogen.

<p>Dynamic light scattering measurements on the aggregation state of fibrinogen.</p

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 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-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 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