Fig 1.
HIV-1 IgA mediates phagocytosis of beads and virions in primary monocytes through FcαRI (CD89).
A. To investigate the ability of IgA to mediate phagocytosis in THP-1 cells, immune complexes were prepared in vitro by mixing IgA with ConSgp140-conjugated 1 μm fluorescent beads. Immune complexes were then added to THP-1 cells, and the uptake of IgA-ConSgp140 immune complexes was analysed by flow cytometry. Representative histograms of bead uptake by THP-1 cells for CD4bs bNAb CH31 mIgA2 and negative control anti-influenza mAb CH65 mIgA2 phagocytosis activity are shown. Red traces represent antibody-mediated internalization of beads, while the black trace represents background internalization of beads in the absence of antibody, and the grey solid area is the negative control without inclusion of beads. B. THP-1 cells and primary monocytes were phenotyped for expression of FcαRI, FcγRI, FcγRII, and FcγRIII by fluorescent antibody staining and flow cytometry. Compensated MFI values are reported (N = 2 independent experiments for THP-1 cells, N = 5 independent experiments for primary monocytes representing 5 different primary monocyte donors). C-D. Phagocytosis of IgA/ConSgp140 1μm bead immune complexes by primary monocytes is shown by a representative histogram of bead uptake for CD4bs bNAb CH31 mIgA2 and anti-influenza mAb CH65 mIgA2 phagocytosis activity (C). Bead phagocytosis was quantified using a phagocytosis score (see Methods) (D). The dashed line indicates background phagocytosis levels, measured by the mean + 3 SD of relevant negative controls. Results from 2 independent experiments are shown. E-F. To identify whether blocking of FcαRI reduces IgA-mediated phagocytosis, primary monocytes were incubated with various concentrations of anti-FcαRI (CD89) antibody for at least 1.5 hours at 4°C before addition to immune complexes made from 1 μm or 0.2 μm ConSgp140-conjugated fluorescent beads. Representative histograms of 1 μm bead uptake by monocytes in the presence of CH31 dIgA2 at 0, 1, 5, and 25 μg/ml anti-CD89 are shown (E). Anti-CD89-mediated blocking of antibody-mediated phagocytosis was quantified by taking the difference in phagocytosis score between experiments conducted in the presence of 5 μg/ml and 0 μg/ml anti-CD89 (F). Results from 3 independent experiments are shown.
Fig 2.
IgG1 mediates greater phagocytosis than mIgA1 and mIgA2 for virions but not for beads.
A-B. To compare phagocytosis efficiencies of different immunoglobulin isotypes, mIgA1, mIgA2 and IgG1 was incubated with ConSgp140-conjugated 1 μm fluorescent beads (N = 4 independent experiments representing 2 different donors) (A) or with HIV-1BaL-Tomato virus (N = 10 independent experiments representing 8 different donors) (B), and the uptake of immune complexes by primary monocytes was analysed by flow cytometry. Box plots represent the range of phagocytosis scores, while box-and-whisker plots indicate 25th and 75th percentiles by box and minimum and maximum scores by whisker. Horizontal black dashed lines indicate limit of detection, as calculated using the mean + 3 SD of negative controls in the corresponding assays. C. The differences in phagocytosis score among immunoglobulin isotypes mIgA1, mIgA2, and IgG1 were compared pairwise using a Sign test.
Fig 3.
IgG3 shows greater HIV-1 virion internalization than IgG1, independent of Env protein binding.
A-D. Wild type CH31 IgG3 and IgG1 were tested for internalization of ConSgp140-conjugated 1 μm fluorescent beads (N = 8 independent experiments representing 6 different donors) (A), HIV-1BaL-Tomato virions (N = 19 independent experiments representing 11 different donors) (B) and HIV-192TH023-Tomato virions (N = 12 independent experiments representing 6 different donors) (C) in human primary monocytes. Anti-influenza mAb CH65 in each subclass backbone were also tested as negative controls. Box-and-whisker plots indicate 25th and 75th percentiles by box, and minimum and maximum scores by whisker. Horizontal black dashed line indicates limit of detection, as calculated using the mean + 3 SD of negative controls in the corresponding assays. The differences in phagocytosis score were compared between IgG1 and IgG3 using a Sign test (D). E-H. To examine if differences in phagocytosis were due to different binding to HIV-1 Env, antibody binding to HIV-1 Env protein was tested using biolayer interferometry. Antibodies (CH31 and CH65 IgG1 and IgG3) were loaded on a Human IgG Capture sensor, and binding to HIV-192Th023 gDneg gp120 monomer protein in solution was tested (E). Specific binding curves of gp120 binding to CH31 IgG1 and IgG3 (light blue and dark blue lines respectively) are shown along with 1:1 Langmuir model fitted curves (red lines) (F). Dissociation constant (KD), association rate (kon), and dissociation rate (koff) are shown for 3 independent experiments (G), and their respective median values are also shown (H).
Fig 4.
ImageStream imaging of IgG and IgA-mediated virion internalization shows distinct internalized virus puncta.
A. Fluorescent infectious HIV-1BaL-Tomato virions were spinoculated and incubated with freshly isolated monocytes and antibodies for antibody-mediated virion internalization to occur. Virion internalization was visualized with ImageStreamX Mark II (EMD Millipore), collecting more than 10,000 images per setup. Representative images are shown for the CH31 CD4bs bNAb antibody engineered in IgG3, IgG1, and mIgA1 backbones, control anti-influenza CH65 antibodies, and two control conditions without antibody and without virus/antibody respectively. B. Total virus fluorescence was quantified for each antibody. Virus fluorescence was quantified using the mean fluorescence intensity of all single, focused cell images for each antibody (~5,000 images each). C. To exclude surface-bound virions, a mask was applied to demarcate the internal portion of the cell, as defined by the erosion of 5 pixels into the bright-field perimeter of the cell. Two representative cells are shown, the upper row showing a cell with mostly excluded surface-localized virus, and the lower row showing a cell with both surface and deep internalized virus (left, bright-field; middle, virus fluorescence; right, virus fluorescence with blue mask demarcating internal portion of the cell). D. Internal virus fluorescence was quantified for each antibody condition. Virus fluorescence was quantified using the mean fluorescence intensity of all single, focused cell images for each antibody (~5,000 images each). E. The percentage of fluorescence intensity comparing the 5-pixel-eroded image to the original image is shown for each CH31 antibody form. F. To count viral foci, a mask determined by the ImageStream IDEAS Spot Wizard algorithm was applied, representing the areas with peak brightness defined by a spot-to-background ratio of 2.0. Spots within this mask were counted. Two representative cells are shown, the upper row showing a cell with 1 virus foci, and the bottom row showing a cell with 14 virus foci (left, bright-field; middle, virus fluorescence; right, virus fluorescence with blue mask demarcating the applied mask for peak brightness). G. The distribution of spot counts is shown for the cells in each CH31 antibody condition. H. The mean number of viral foci is shown for each condition.
Fig 5.
IgG3 has enhanced phagocytosis potency across multiple HIV-1 epitopes.
A. Epitope-matched IgG3 and IgG1 mAbs were tested for HIV-192TH023-Tomato virion phagocytosis in human primary monocytes. Phagocytosis-positive antibodies are shown (N = 4 independent experiments). Box plots represent the range of phagocytosis scores. Horizontal black dashed line indicates limit of detection, as calculated using the mean + 3 SD of negative controls in the corresponding assays. B. Data from antibody paratopes positive for phagocytosis (CH27, CH28, HG107, 7B2, CH31) were aggregated by subclass. Box-and-whisker plots indicate 25th and 75th percentiles by box and minimum and maximum scores by whisker. C. The differences in phagocytosis score were compared between IgG1_SEK and IgG3 using a linear mixed effects model.
Fig 6.
Mucosal HIV-1 specific polyclonal IgG from vaginal wecks from HIV-1+ women can capture virions and mediate internalization of infectious HIV-1BaL.
A. HIV-1 envelope binding profile is shown for purified IgG of 4 HIV-1+ women (PTID16, PTID32, PTID34, PTID01) positive for infectious and non-infectious virus capture. Binding responses to gp41 and ConSgp140 reached saturation. Specific activity for BaL gp120, gp70_B.CaseA_V1_V2, and RSC3 is shown, and samples with FI-background<100 were classified negative. Classification of VRC01 like CD4bs binding antibodies is indicated by the ratio of binding MFI of the CD4bs-exposed RSC3 to CD4bs mutants RSC3Δ371, RSC3G367R, and RSC3Δ371P363N, and classification of CD4i antibodies is indicated by CD4i differential, the ratio of binding MFI of HxB2 core to HxB2 core I420R. B. The ability of the mucosal HIV-1+ purified IgG samples to mediate uptake of HIV-1BaL-Tomato by THP-1 cells was analysed by flow cytometry. A representative flow cytometry diagram of virion internalization mediated by mucosal IgG isolated from a vaginal weck from a HIV-1+ woman (PTID16) is shown alongside a representative diagram for a negative control (RSV-specific Palivizumab). C. The ability of mucosal IgG in chronically infected women to mediate virus capture and virion internalization was quantified. Blue circles represent phagocytosis scores for HIV-1BaL-Tomato virions in THP-1 cells (N = 2 independent experiments), while green squares represent virus capture percentages as measured by RT-qPCR (N = 3 independent experiments).