9G4+ antibodies from HIV-1 infected patients bind apoptotic lymphocytes.

<p>Unfractionated serum from normal donors (n=16), SLE (n=21) and HIV- infected (n=57) were used in apoptotic binding assays. (<b>A</b>-<b>C</b>) The Jurkat Human T cell line was treated with camptothecin to induce apoptosis then incubated with patient serum. Serum binding to cells was detected using FITC- labeled 9G4 rat anti-human monoclonal antibody. Apoptotic Cell Gate was drawn around cells stained positive for viability dye, representing the dying cell population. (<b>C</b>) The shaded histogram represents cells incubated with PBS in the absence of patient serum. The thin-line represents the antibody binding from a normal serum donor and the Bold-line is representative of 9G4+ antibody binding from an HIV infected patient. (<b>D</b>) Data from all samples are plotted. The dashed-line represents the normal donor mean plus 2 standard deviations. Significance determined by Mann-Whitney test.</p

Auto-antigen microarray profiles of 9G4+ IgG isolated from HIV-infected patients.

<p>Plasma, 9G4+ and 9G4- antibodies were screened for IgG binding to 85 auto-antigens. Columns represent HIV samples, rows represent antigens and color maps to log₁₀ signal (gray indicates lower limit of detection, LLOD). Columns are organized by sample type (unfractionated plasma, 9G4+, 9G4-) and subject order within groups is the same for each type. Rows are clustered based on Euclidean distance to facilitate visualization. Antigens that resulted in no signal are listed to the right of the heat map. Antigens in red indicate higher 9G4+ binding relative to 9G4- based on a Wilcoxon signed-rank test at p<0.05. Average signal data where either the signal intensity was greater than 100 or the signal-to-noise ratio was greater than 2 were considered to be above the LLOD; these data were background-subtracted (based on PBS control per antigen) and then log₁₀ transformed.</p

9G4+ antibodies isolated from HIV-1 infected patients exhibit less Cardiolipin and ANA autoreactivity than 9G4+ isolated from SLE patients.

<p>Purified 9G4+ antibodies (from SLE [n=8] and HIV-1 infected patients [n=8]) were added to Cardiolipin (<b>A</b>) and HEp2 lysate (<b>B</b>) coated ELISA plates. Anti-IgG was used to detect antibody binding. (<b>B</b>) Cardiolipin IgG (GPLU/ml) and (<b>C</b>) ANA IgG (arbitrary units) were calculated based on manufacturer guidelines, and data were plotted. Each dot represents a sample. Significant differences between HIV and SLE patients were observed in 9G4+ antibody binding to cardiolipin and ANA as determined by Mann-Whitney test. (<b>C</b>) Slides coated with HEp-2 cells were incubated with patient antibodies, detected with FITC conjugated anti-IgG, and visualized with a fluorescence microscope. Dilutions of patient plasma were selected based on manufacturer guidelines while fractionated samples were used at 100 µg/ml. Representative images for unfractionated plasma, 9G4+ and 9G4- antibody fractions from an SLE patient and HIV patient are presented. </p

9G4+ antibodies purified from HIV- infected patients bind to HIV gp140 and neutralize HIV-1.

<p>9G4+ antibodies isolated from SLE patients (n=6) and PLWH (n=8) normalized to equal concentrations, were serially diluted in triplicate and added to ELISA plates coated with oligomeric YU2 gp140 (<b>A</b>) Influenza vaccine (<b>C</b>) or CMV lysate (<b>D</b>) and binding detected by anti-IgG. (<b>B</b>) 9G4+ antibodies were tested for inhibition of pseudotyped HIV-1 SF-162 infection of TZM-bl cells. Infectivity was measured by luciferase assay, and relative inhibitions were calculated after normalizing to internal controls. Each symbol represents the mean value of a patient. Significance determined by Mann-Whitney test.</p

9G4+ antibodies from SLE and HIV-infected patients bind B cells.

<p>Tonsil cells were incubated with either patient plasma, purified 9G4+ antibodies from patients’ plasma or a positive control 9G4+ monoclonal antibody then stained with fluorescently -labeled antibody cocktails. (<b>A</b>) Gating strategy: lymphocytes were gated based on size and granularity followed by an exclusion of doublets. Dead cells were excluded using a viability stain then live cells were then gated by expression of CD3 and CD19. CD3-CD19+ B cells were then further divided by IgD and CD27 markers. The open histogram represents the no antibody source control (PBS alone). The solid histogram is representative of 9G4+ antibody binding B cells. (<b>B</b>) B cell binding of 9G4+ antibodies purified from SLE (n=6) and HIV- infected (n=8) patients was assessed. Significance was determined by Mann-Whitney test.</p