Low doses of αDCIR.Gagp24 expand a broad repertoire of HIV Gagp24-specific T cells.

<p><b>A.</b> Experimental procedure. ICS, intracellular staining. <b>B.</b> PBMCs from an HIV-infected patient (patient A15) were cultured for 10 days in medium alone (top panel), or with 0.3 nM hIgG4.Gagp24 (middle panel), or 0.3 nM αDCIR.Gagp24 (lower panel) and then rechallenged for 6 h with or without (-C) 5 clusters (CL) of 15-mer overlapping peptides covering HIV Gagp24 in the presence of brefeldin A prior to intracellular IFNγ staining. The flow cytometry profiles are gated on the overall CD3⁺ T cell population. Data are representative of 4 different patients. <b>C.</b> PBMCs from 4 HIV-infected patients (patient A1, A2. A12 and A15) were cultured for 10 days with 0.3 nM (filed symbols) or 30 pM (open symbols) of αDCIR.Gagp24 or hIgG4.Gagp24 and then restimulated for 48 hrs with 5 clusters (CL) of 15-mer overlapping peptides covering HIV Gagp24. The culture supernatants were then harvested and IFNγ produced by total T cells was analyzed by multiplex bead-based assay. Each patient is represented by a different symbol. Data are presented as mean of sum of HIV Gagp24 clusters. Differences between αDCIR.Gagp24 and hIgG4.Gagp24 were assessed by a matched paired t-test.</p

αDCIR.Gagp24 expands multifunctional HIV Gagp24-specific CD4⁺ T cells <i>in vitro</i>.

<p>PBMCs from HIV-infected patients (panel <b>A</b>, patient A15; panel <b>B</b>, patients A2, A1, A15) were cultured for 10 days with 0.3 nM of αDCIR.Gagp24 or hIgG4.Gag p24 or left unstimulated and then restimulated for 6 hrs with or without clusters of 15-mer overlapping peptides covering HIV Gagp24 in the presence of brefeldin A prior to intracellular cytokine staining. The profiles are gated on the overall CD4⁺ T cell populations, with the percentages of populations responding to the designated peptides shown in each plot. <b>A</b>. Coordinate analysis of TNFα versus MIP-1β, IFNγ or CD154 expression (patient A15). Data are representative of 4 different patients. <b>B</b>. Multifunctionality of the cytokine responses. Combined percentages of 5 CD4⁺ T cell populations producing different combinations of at least 3 cytokines (IFNγ, TNFα, MIP-1β or CD154). Data are presented as mean of sum of HIV Gagp24 clusters ± SEM for 3 patients. Differences between αDCIR.Gagp24 and hIgG4.Gagp24 were assessed by a matched paired t-test.</p

Immunization schedules used in this study.

<p>Immunization schedules used in this study.</p

Anti-HIV Gagp24 antibody avidity following vaccination.

<p>Animals were immunized i.d. three times at week 0, 6 and 15 with αDCIR.Gag24 or the molar equivalent of the Gagp24 protein with or without poly(I:C). Serum HIV Gagp24-specific IgG antibody avidity was measured by ELISA 2 weeks after the third injection (week 17) with αDCIR.Gagp24 or Gagp24 without adjuvant <b>(A)</b> or with poly(I:C) <b>(B)</b>. Relative avidity index (RAI) (%) of Gagp24-specific IgG for the individual monkeys is presented.</p

Immunization with αDCIR.Gagp24 without adjuvant generates high titers of HIV Gagp24 antibody responses in NHPs.

<p>Animals were immunized i.d. three times at week 0, 6 and 15 with αDCIR.Gag24 or control hIgG4.Gagp24 or the molar equivalent of the HIV Gagp24 protein with or without poly(I:C). HIV Gagp24-specific IgG antibodies in serum were assessed at indicated time points post-immunization with αDCIR.Gagp24 or hIgG4.Gagp24 or Gagp24 without adjuvant <b>(A)</b> or with poly(I:C) <b>(B)</b>. Dashed lines indicates times of immunization. The serial measurements of Gagp24-specific antibody responses collected on individual monkeys over time among the vaccine groups were summarized by calculating the area under the titration curves for each monkey at each time point, and longitudinal differences between the vaccine groups were assessed by linear mixed model analysis. Data are presented as mean ± SEM. *, p<0.01; **, p<0.001; ***, p<0.0001 compared with hIgG4.Gagp24 and Gagp24 without poly(I:C).</p

Characterization of the αDCIR.Gagp24 fusion rAbs.

<p><b>A.</b> Schematic representation and SDS-PAGE analysis under reducing conditions of the αDCIR.Gagp24 fusion protein. Expression constructs for chimeric mouse variable (V) region-human IgG4 constant (C) region αDCIR and isotype control recombinant antibodies (rAbs) were engineered with the HIV Gagp24 coding region fused in frame to the heavy (H) chain C-terminus (left panel). These constructs were co-transfected with a matching light (k) chain expression vector into stable CHO-S cell lines and the secreted fusion rAbs were purified from the culture supernatants by protein A-affinity chromatography and analyzed on reducing SDS-PAGE. Proteins were stained with Coomassie Blue (right panel). <b>B.</b> Specific binding of αDCIR.Gagp24 rAb. Monocytes (left panel), B cells (middle panel) and T cells (right panel) from an HIV-infected patient PBMCs were treated with 3 nM, 0.3 nM and 30 pM of αDCIR.Gagp24 and 3 nM hIgG4.Gagp24 fusion proteins, followed by incubation with an anti-HIV Gagp24 PE-conjugated antibody and then analyzed by flow cytometry. Blue traces are staining from αDCIR.Gagp24. The blue color gradient represents the different concentrations from light blue (3 nM) to dark blue (30 pM). Grey traces (staining by 3 nM hIgG4.Gagp24) and grey solid traces (untreated cells) are the background staining. Data are representative of 2 different patients.</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

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

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