Polyclonal B Cell Differentiation and Loss of Gastrointestinal Tract Germinal Centers in the Earliest Stages of HIV-1 Infection

The antibody response to HIV-1 does not appear in the plasma until approximately 2–5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1–specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4+ T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses.

The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses

Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses

Plasmablast Response to Primary Rhesus Cytomegalovirus (CMV) Infection in a Monkey Model of Congenital CMV Transmission.

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide and the leading infectious cause of neurologic deficits and hearing loss in newborns. Development of a maternal HCMV vaccine to prevent vertical virus transmission is a high priority, yet protective maternal immune responses following acute infection are poorly understood. To characterize the maternal humoral immune response to primary CMV infection, we investigated the plasmablast and early antibody repertoire using a nonhuman primate model with two acutely rhesus CMV (RhCMV)-infected animals-a CD4+ T cell-depleted dam that experienced fetal loss shortly after vertical RhCMV transmission and an immunocompetent dam that did not transmit RhCMV to her infant. Compared to the CD4+ T cell-depleted dam that experienced fetal loss, the immunocompetent, nontransmitting dam had a more rapid and robust plasmablast response that produced a high proportion of RhCMV-reactive antibodies, including the first identified monoclonal antibody specific for soluble and membrane-associated RhCMV envelope glycoprotein B (gB). Additionally, we noted that plasmablast RhCMV-specific antibodies had variable gene usage and maturation similar to those observed in a monkey chronically coinfected with simian immunodeficiency virus (SIV) and RhCMV. This study reveals characteristics of the early maternal RhCMV-specific humoral immune responses to primary RhCMV infection in rhesus monkeys and may contribute to a future understanding of what antibody responses should be targeted by a vaccine to eliminate congenital HCMV transmission. Furthermore, the identification of an RhCMV gB-specific monoclonal antibody underscores the possibility of modeling future HCMV vaccine strategies in this nonhuman primate model

Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses

<div><p>Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an <i>in vitro</i> flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (<i>i</i>.<i>e</i>. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (<i>i</i>.<i>e</i>. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (<i>i</i>.<i>e</i>. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine-induced antibodies and therapeutic antibodies will enable a better understanding of their capacity to prevent and/or control HIV-1 infection <i>in vivo</i>.</p></div

IgG3 has enhanced phagocytosis potency across multiple HIV-1 epitopes.

<p><b>A.</b> Epitope-matched IgG3 and IgG1 mAbs were tested for HIV-1_92TH023-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>B.</b> Data from antibody paratopes positive for phagocytosis (CH27, CH28, HG107, 7B2, CH31) were aggregated by subclass. Box-and-whisker plots indicate 25^th and 75^th percentiles by box and minimum and maximum scores by whisker. <b>C.</b> The differences in phagocytosis score were compared between IgG1_SEK and IgG3 using a linear mixed effects model.</p

IgG3 shows greater HIV-1 virion internalization than IgG1, independent of Env protein binding.

<p><b>A-D.</b> 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-1_BaL-Tomato virions (N = 19 independent experiments representing 11 different donors) (B) and HIV-1_92TH023-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 25^th and 75^th 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). <b>E-H.</b> 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-1_92Th023 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 (K_D), association rate (k_on), and dissociation rate (k_off) are shown for 3 independent experiments (G), and their respective median values are also shown (H).</p

ImageStream imaging of IgG and IgA-mediated virion internalization shows distinct internalized virus puncta.

<p><b>A.</b> Fluorescent infectious HIV-1_BaL-Tomato virions were spinoculated and incubated with freshly isolated monocytes and antibodies for antibody-mediated virion internalization to occur. Virion internalization was visualized with ImageStream^X 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>B.</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). <b>C.</b> 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). <b>D.</b> 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). <b>E.</b> The percentage of fluorescence intensity comparing the 5-pixel-eroded image to the original image is shown for each CH31 antibody form. <b>F.</b> 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). <b>G.</b> The distribution of spot counts is shown for the cells in each CH31 antibody condition. <b>H.</b> The mean number of viral foci is shown for each condition.</p

HIV-1 IgA mediates phagocytosis of beads and virions in primary monocytes through FcαRI (CD89).

<p><b>A.</b> To investigate the ability of IgA to mediate phagocytosis in THP-1 cells, immune complexes were prepared <i>in vitro</i> 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>B.</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). <b>C-D.</b> 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005817#sec002" target="_blank">Methods</a>) (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. <b>E-F.</b> 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.</p