Journal: PLoS ONE

Article Title: Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

doi: 10.1371/journal.pone.0076139

Figure Lengend Snippet: Overlay of binding of varying concentrations of wild-type SF162 gp120 to (A) sCD4, (C) b12 mAb, (E) VRC01 mAb, (G) 17b mAb and (I) 17b mAb (+sCD4). Overlay of binding of varying concentrations of disulfide-stabilized SF162 gp120 (gp120 L1-SS-L2) to (B) sCD4, (D) b12 mAb, (F) VRC01 mAb, (H) 17b mAb and (J) 17b mAb (+sCD4). ∼250–500 RUs of sCD4 or mAbs, b12, VRC01 or 17b, were immobilized directly onto a CM5 sensor chip via amine coupling. Varying concentrations of gp120s, either wild-type of disulfide-stabilized, were then injected at 80 µl/min, at 25°C with HBS-EP buffer as running buffer. The experimental curves were then fitted to a 1∶1 Langmuir binding model using BIAevaluation software 3.2 (BIAcore Inc). The red lines indicate the experimentally derived curves while the black lines indicate curves generated after fitting the experimental data to the 1∶1 Langmuir binding model (BIAevaluation software 3.0). Kon (association rate, in 1/Ms), Koff (dissociation rate, in 1/s) and Kd (dissociation constant, in M) are indicated for each paired (ligand-analyte) interaction, derived by fitting the experimental data to the 1∶1 Langmuir binding model. Chi2 (of ≤1) refers to goodness of fit of the statistical model to observed experimental data.

Article Snippet: Soluble CD4 (sCD4) was purchased from Progenics Pharmaceuticals (Tarrytown, NY).

Techniques: Binding Assay, Injection, Software, Derivative Assay, Generated

Journal: PLoS ONE

Article Title: Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

doi: 10.1371/journal.pone.0076139

Figure Lengend Snippet: Individual rabbit sera were tested for neutralizing activity against HIV-2 (7312A/V434M) in the absence (open bars) or presence (filled bars) of sCD4. The 50% neutralizing titers are shown for the pre-immune (black) and 2wp3 (red) serum samples. The antigens used for immunization are indicated above each graph (A – gp120, B – gp120 L1-SS-L2, C – gp140, D- gp140 L1-SS-L2); the numbers on the x-axis indicate the rabbit numbers. The dotted lines indicate the lowest dilution tested, 1∶20, that serves as the background.

Article Snippet: Soluble CD4 (sCD4) was purchased from Progenics Pharmaceuticals (Tarrytown, NY).

Techniques: Activity Assay

Journal: The Journal of Infectious Diseases

Article Title: Pathological Role of Anti-CD4 Antibodies in HIV-Infected Immunologic Nonresponders Receiving Virus-Suppressive Antiretroviral Therapy

doi: 10.1093/infdis/jix223

Figure Lengend Snippet: Clinical Characteristics of Study Participants

Article Snippet: We thank the AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, for providing human soluble CD4 recombinant protein (Progenics); and Dr Paul Parren (Genmab), for providing human monoclonal anti-CD4 antibody (zanolimumab).

Techniques:

Journal: The Journal of Infectious Diseases

Article Title: Pathological Role of Anti-CD4 Antibodies in HIV-Infected Immunologic Nonresponders Receiving Virus-Suppressive Antiretroviral Therapy

doi: 10.1093/infdis/jix223

Figure Lengend Snippet: Increased plasma anti-CD4 immunoglobulin G (IgG) levels in aviremic antiretroviral therapy (ART)–recipient immunologic nonresponders. A, Median absolute numbers of peripheral CD4+ and CD8+ T cells, assessed by flow cytometry, in healthy controls, responders, and nonresponders. B, Median plasma levels of anti-CD4 IgG in healthy controls, responders, nonresponders, and long-term nonprogressors (LTNPs). C and D, Correlations between plasma levels of anti-CD4 IgG and peripheral CD4+ T-cell counts in all ART-recipient aviremic human immunodeficiency virus (HIV)–infected subjects (C) and healthy controls (D). E, Median plasma levels of soluble CD4 (sCD4) antigen in healthy controls, responders, and nonresponders. Statistical analyses were performed using the Mann-Whitney U test (unpaired).

Article Snippet: We thank the AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, for providing human soluble CD4 recombinant protein (Progenics); and Dr Paul Parren (Genmab), for providing human monoclonal anti-CD4 antibody (zanolimumab).

Techniques: Flow Cytometry, Infection, MANN-WHITNEY

Journal: The Journal of Infectious Diseases

Article Title: Pathological Role of Anti-CD4 Antibodies in HIV-Infected Immunologic Nonresponders Receiving Virus-Suppressive Antiretroviral Therapy

doi: 10.1093/infdis/jix223

Figure Lengend Snippet: Anti-CD4 antibody-dependent natural killer (NK) cell activation. CD4+ T cells were cultured with purified anti-CD4 immunoglobulin G (IgG) from nonresponders or control antibodies and cocultured with NK cells at a ratio of 1:1. Intracellular CD107a and interferon γ (IFN-γ) expression in NK cells was analyzed by flow cytometry. A, Dot plots from a representative donor show CD107a and IFN-γ expression in NK cells in response to different concentrations of anti-CD4 antibodies from nonresponders. B, Percentages of NK cells expressing IFN-γ and CD107a in response to different concentrations of anti-CD4 IgG from 5 different nonresponders in vitro. Analyses were performed by the Friedman paired nonparametric test. C, Median percentages of NK cells expressing IFN-γ and CD107a in a mixed culture of CD4+ T cells and NK cells in the presence of anti-CD4 IgG from nonresponders, anti-CD4 IgG from nonresponders pretreated with sCD4 (control 1), anti-CD4 IgG–depleted total IgG from nonresponders (control 2), and zanolimumab (positive control) at 5 μg/mL in vitro. Analyses were performed using the Mann-Whitney U test (unpaired). D, Median percentages of NK cells expressing CD107a in healthy controls, responders, and nonresponders ex vivo. Analyses were performed using the Mann-Whitney U test (unpaired). FSC, forward scatter; SSC, side scatter.

Article Snippet: We thank the AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, for providing human soluble CD4 recombinant protein (Progenics); and Dr Paul Parren (Genmab), for providing human monoclonal anti-CD4 antibody (zanolimumab).

Techniques: Activation Assay, Cell Culture, Purification, Expressing, Flow Cytometry, In Vitro, Positive Control, MANN-WHITNEY, Ex Vivo

Journal: The Journal of Infectious Diseases

Article Title: Pathological Role of Anti-CD4 Antibodies in HIV-Infected Immunologic Nonresponders Receiving Virus-Suppressive Antiretroviral Therapy

doi: 10.1093/infdis/jix223

Figure Lengend Snippet: Anti-CD4 antibody dependent natural killer (NK) cell–mediated cytolysis to primary CD4+ T cells. CD4+ T cells were cultured with purified anti-CD4 immunoglobulin G (IgG) and control antibodies and cocultured with NK cells at a ratio of 1:3 in vitro, and the CD4+ T-cell cytolysis percentage was analyzed by flow cytometry. A, Dot plots from 1 representative donor were showing CD4+ T-cell frequencies and annexin V binding in response to different concentrations of anti-CD4 IgG from nonresponders. B, Percentages of CD4+ T cells undergoing cytolysis in response to different concentrations of anti-CD4 IgG from 5 different nonresponders through antibody-dependent cell-mediated cytotoxicity (ADCC). Analyses were performed by the Friedman paired nonparametric test. C, Median percentages of CD4+ T cells undergoing cytolysis in response to anti-CD4 IgG from nonresponders and controls at 5 μg/mL in vitro. Analyses were performed using the Mann-Whitney U test (unpaired). D, Percentages of CD4+ T cells undergoing apoptosis in response to different concentrations of anti-CD4 IgG from 5 different nonresponders through ADCC. Analyses were performed by the Friedman paired nonparametric test. E, Percentages of CD4+ T cells undergoing apoptosis in response to anti-CD4 IgG from nonresponders and controls at 5 μg/mL in vitro. Analyses were performed using the Mann-Whitney U test (unpaired). SSC, side scatter.

Article Snippet: We thank the AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, for providing human soluble CD4 recombinant protein (Progenics); and Dr Paul Parren (Genmab), for providing human monoclonal anti-CD4 antibody (zanolimumab).

Techniques: Cell Culture, Purification, In Vitro, Flow Cytometry, Binding Assay, MANN-WHITNEY

Journal: The Journal of Infectious Diseases

Article Title: Pathological Role of Anti-CD4 Antibodies in HIV-Infected Immunologic Nonresponders Receiving Virus-Suppressive Antiretroviral Therapy

doi: 10.1093/infdis/jix223

Figure Lengend Snippet: CD4+ T-cell activation in response to purified anti-CD4 immunoglobulin G (IgG) from immunologic nonresponders. A, Overlapped dot plots (1 representative donor) and the median CD4 mean fluorescence intensity (MFI) expression on CD4+ T cells treated with anti-CD4 IgG (red) and anti-CD4 IgG–depleted total IgG (blue) in vitro. B and C, Median CD4 MFI expression on naive CD4+ T cells (B) and memory CD4+ T cells (C) in healthy controls, responders, and nonresponders ex vivo. D, Overlapped dot plots (1 representative donor) and median percentages of CD38 and HLA-DR coexpression on memory CD4+ T cells treated with anti-CD4 IgG (red) and anti-CD4 IgG–depleted total IgG in vitro. E, Median percentages of CD38 and HLA-DR coexpression on memory CD4+ T cells in healthy controls, responders, and nonresponders ex vivo. Analyses were performed using the Mann-Whitney U test (unpaired). SSC, side scatter.

Article Snippet: We thank the AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, for providing human soluble CD4 recombinant protein (Progenics); and Dr Paul Parren (Genmab), for providing human monoclonal anti-CD4 antibody (zanolimumab).

Techniques: Activation Assay, Purification, Fluorescence, Expressing, In Vitro, Ex Vivo, MANN-WHITNEY

Journal: The Journal of Infectious Diseases

Article Title: Pathological Role of Anti-CD4 Antibodies in HIV-Infected Immunologic Nonresponders Receiving Virus-Suppressive Antiretroviral Therapy

doi: 10.1093/infdis/jix223

Figure Lengend Snippet: Memory and naive CD4+ T cells. A, Comparison of apoptotic induction between naive CD4+ and memory CD4+ T cells by purified anti-CD4 immunoglobulin G (IgG) from plasma of 5 different nonresponders in antibody-dependent cell-mediated cytotoxicity assays in vitro. Data are median fold change (interquartile range) from anti-CD4 IgG to anti-CD4 IgG–depleted total IgG. B, Median ratio of memory CD4+ T cells to naive CD4+ T cells in healthy controls, responders, and nonresponders ex vivo. C, Median absolute counts of memory CD4+ and naive CD4+ T cells in healthy controls, responders, and nonresponders ex vivo. Analyses were performed using the Mann-Whitney U test (unpaired).

Article Snippet: We thank the AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, for providing human soluble CD4 recombinant protein (Progenics); and Dr Paul Parren (Genmab), for providing human monoclonal anti-CD4 antibody (zanolimumab).

Techniques: Purification, In Vitro, Ex Vivo, MANN-WHITNEY