Antibody Neutralization of HIV-1 Crossing the Blood-Brain Barrier

Abstract

HIV-1 can cross the blood-brain barrier (BBB) to penetrate the brain and infect target cells, causing neurocognitive disorders as a result of neuroinflammation and brain damage. Here, we examined whether antibodies targeting the HIV-1 envelope glycoproteins interfere with the transcytosis of virions across the human BBB endothelium. We found that although the viral envelope spike gp160 is required for optimal endothelial cell endocytosis, no anti-gp160 antibodies blocked the BBB transcytosis of HIV-1 in vitro Instead, both free viruses and those in complex with antibodies transited across endothelial cells in the BBB model, as observed by confocal microscopy. HIV-1 infectious capacity was considerably altered by the transcytosis process but still detectable, even in the presence of nonneutralizing antibodies. Only virions bound by neutralizing antibodies lacked posttranscytosis infectivity. Overall, our data support the role of neutralizing antibodies in protecting susceptible brain cells from HIV-1 infection despite their inability to inhibit viral BBB endocytic transport.IMPORTANCE HIV-1 can cross the blood-brain barrier (BBB) to penetrate the brain and infect target cells, causing neurocognitive disorders as a result of neuroinflammation and brain damage. The HIV-1 envelope spike gp160 is partially required for viral transcytosis across the BBB endothelium. But do antibodies developing in infected individuals and targeting the HIV-1 gp160 glycoproteins block HIV-1 transcytosis through the BBB? We addressed this issue and discovered that anti-gp160 antibodies do not block HIV-1 transport; instead, free viruses and those in complex with antibodies can transit across BBB endothelial cells. Importantly, we found that only neutralizing antibodies could inhibit posttranscytosis viral infectivity, highlighting their ability to protect susceptible brain cells from HIV-1 infection.

Keywords: HIV-1; antibodies; blood brain barrier; neutralization; transcytosis.

FIG 1

HIV-1 transcytosis across the BBB endothelium. (A) In vitro BBB endothelial cell system. The phase-contrast image shows an hCMEC/D3 endothelial cell monolayer separating apical (a) and basal (b) compartments when grown at confluence on a Transwell membrane (bar, 25 μm). HIV-1 virions alone or in the presence of antibodies applied to the apical compartment transcytose and are quantified in the basal medium by p24 ELISA. (B) Transendothelial electrical resistance (TEER) values over time of hCMEC/D3 endothelial cell monolayers. Means of quadruplicate values ± standard errors of the means (SEM) from three independent experiments are shown. Transcytosis experiments were performed at day 8 (black arrow). (C) Dot plots comparing the diffusion of FITC-labeled DEAE-dextran molecules from the apical to the basal compartment when passing through the Transwell membrane without cells (black) and with hCMEC/D3 cells (blue) at day 5 (D5; n = 12) and day 8 (D8; n = 8). Means of quadruplicate values ± SEM from two or three independent experiments are shown. (D) Immunofluorescence staining of hCMEC/D3 monolayers with DAPI (blue) and anti-VE-cadherin, anti-JAM-A, and anti-ZO-1 antibodies (green) at day 7 of culture. Bar, 20 μm. (E) Dot plots show the percent transcytosis of NLAD8 viruses incubated with or without non-HIV-1 isotype control (Iso) and of NL-ΔEnv alone (8 Transwells each). Means of quadruplicate values from two independent experiments are shown. ***, P < 0.001 (Mann-Whitney test); ns, not significant. (F) 3D confocal microscopy image (left) showing nuclei (blue) and NL4.3 HIV-1-GFP viruses (green). Bar, 5 μm. Box (top right) and violin plot (bottom right) show the distribution of fluorescently labeled NL4.3 virions across the z-stack depth of hCMEC/D3 cells using a representative 7.7-μm z-stack confocal microscopy acquisition.

FIG 2

HIV-1 virions in the presence of anti-gp160 antibodies transcytose across the BBB. (A) Dot plots comparing the in vitro transcytosis of HIV-1 virions (cell-free NLAD8 [CCR5-tropic]; cell-free and MOLT cell-produced NL4.3 [CXCR4-tropic]) across the hCMEC/D3 cell endothelium (as a percentage of normalized viral input) alone (No Ab) and in the presence of selected bNAbs, NnAbs and non-HIV-1 mGO53 control IgG antibodies. Means of quadruplicate values from one or two experiments are shown. (B) Same as for panel A but with CH058 T/F virions. Means of triplicate values from two experiments are shown. (C) Dot plots comparing the percentage of in vitro transcytosis of NLAD8 alone (No Ab) and in the presence of non-HIV-1 isotype control mGO53 and combinations of anti-HIV-1 gp160 antibodies (pool 1, 10-1074, 3BNC117, 10E8 and PGDM1400; pool 2, 10-1074, 3BNC117, 10E8, PGDM1400, 10-188, and 5-25). Means of quadruplicate values from two independent experiments are shown. (D) Graphs showing the ELISA binding of gp140-immunoabsorbed serum IgG antibodies from selected elite neutralizers (pt1, pt2, and pt10) against YU2 gp140 and p24 proteins. Means of duplicate values ± standard deviations (SD) are shown. Vertical dotted lines indicate the IgG concentration (1 μg/ml) used in the experiments presented in panel E. (E) Dot plots comparing the percentage of in vitro transcytosis of NLAD8 and NL-ΔEnv virions alone (No Ab) and in the presence of the non-HIV-1 isotype control mGO53, purified serum IgG antibodies from a healthy donor (hd2) (54), and immunopurified polyclonal anti-gp140 antibodies, as shown in panel D. Means of quadruplicate (NLAD8) or triplicate (NL-ΔEnv) values are shown. Groups in panels A, B, C, and E were compared to the no-Ab controls using Dunn's multiple-comparison test. *, 0.035 < P < 0.05; ns, not significant.

FIG 3

bNAbs neutralize HIV-1 transcytosed through the BBB endothelium. (A) Representative images from the 3D reconstruction of the confocal microscopy experiments on hCMEC/D3 cell monolayers with GFP-labeled HIV-1 (green) in the presence of mGO53, 5-25 and 3BNC117 antibodies (red). Yellow objects represent HIV-1 virions collocating with antibodies. Nuclei and intercellular junctions were stained with DAPI (blue) and anti-VE cadherin antibody (gray), respectively. Side views of the 3D confocal microscopy pictures across the depth of endothelial cells are shown below each image. (B) Graphs comparing colocalization using Pearson coefficient (r) and percentage of overlap between fluorescent HIV-1 viruses and mGO53, 5-25, and 3BNC117 antibodies, per cell and per field. Values of the Pearson coefficient and frequency of overlapping virus-IgG objects were compared between groups using Dunn's multiple-comparison and Mann-Whitney tests, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001; **** P ≤ 0.0001. (C) Dot plots show the number of fluorescent objects corresponding to HIV-1 NL4.3 virus alone (green) or antibodies IgG alone (red) or virus-antibody complexes (HIV_NL4.3-IgG; yellow) according to the distance across the z-stack depth. Quantification was made using three representative 4- to 5-μm z-stack acquisitions. (D) Dot plots comparing the normalized infectivity (NI; calculated as RLU/p24 concentration [nanograms per milliliter) of HIV-1 virions transcytosed across the BBB in the absence (No Ab) or presence of a non-HIV-1 isotype control (mGO53), HIV-1 gp160 NnAbs, and bNAbs. Ctrl, HIV-1 applied to Transwell membranes without endothelial cells. Each replicate per condition (n = 4) was tested in triplicate for infectivity. Dotted lines indicate basal levels of luminescence given by TZM-bl cells alone. (E) Same as for panel D but with various concentrations of 3BNC117 IgG antibodies. Each replicate (n = 3 or 4) per independent experiment (n = 2) was tested in triplicate for infectivity. Groups in panels D and E were compared to no-Ab controls using Dunn's multiple-comparison test. *, P < 0.05; ** P < 0.01; ns, non significant.