Journal: iScience

Article Title: HIV-1 interaction with an O -glycan-specific bacterial lectin enhances virus infectivity and resistance to neutralizing antibodies

doi: 10.1016/j.isci.2024.110390

Figure Lengend Snippet: Capture of HIV-1 virions and recognition of gp120 by the SLBR-N lectin (A) Schematic of the experimental procedure. Virus was incubated with GST-tagged SLBR-N for 24 h at 37 o C and then glutathione beads were added. After the beads were pelleted, RT-qPCR or infectivity assays were performed on the beads while the supernatant was titrated on TZM.bl cells to measure residual infectivity. (B) The amount of CMU06 virus captured by SLBR-N-coated beads or control beads as measured by RT-qPCR. The experiment was repeated three times; data from two experiments, each performed in triplicates, are shown. A two-tailed t test was used to compare virus capture in the presence of SLBR-N versus no SLBR-N (control). (C) The residual virus infectivity in the supernatant after capture with SLRB-N-coated beads versus control beads was measured by titration on TZM.bl cells. The amount of residual virus was calculated relative to the control of each experiment (set to 100%). Mean and standard error from two repeat experiments, each in triplicate, are shown. (D) The infectivity of virus captured by SLBR-N coated beads or control beads upon incubation with TZM.bl cells as measured by luminescence (RLU). Experiment was performed in triplicate and repeated twice; mean and SD of triplicates from one experiment are shown. A two-way ANOVA with no multiple comparison test was used to compare SLBR-N treated vs. control.

Article Snippet: Recombinant gp120 A244 or JRFL (10 μg/mL) were coupled to Octet AR2G Biosensors (Sartorius) following the manufacturers protocol.

Techniques: Virus, Incubation, Quantitative RT-PCR, Infection, Control, Two Tailed Test, Titration, Comparison

Journal: iScience

Article Title: HIV-1 interaction with an O -glycan-specific bacterial lectin enhances virus infectivity and resistance to neutralizing antibodies

doi: 10.1016/j.isci.2024.110390

Figure Lengend Snippet: SLBR-N interaction with HIV-1 Env (A) SLBR-N binding to HIV-1 Env proteins of different virus strains was measured by ELISA. Recombinant soluble Env proteins were coated onto well surface and then treated with titrating amounts of GST-tagged SLBR-N. SLBR-N binding was detected by rabbit anti-GST antibody followed by HRP-conjugated anti-rabbit IgG. Background binding (SLBR-N binding to no Env) is indicated by dotted line. Mean and SD from duplicate wells are shown. (B) The binding affinity of SLBR-N for HIV-1 gp120 ZM109 was determined using Octet BLI. Recombinant gp120 was coupled to AR2G biosensors and then reacted with SLBR-N at the designated concentrations. (C) Recombinant ZM109 gp120 protein was subjected to SDS-PAGE, transferred to a nitrocellulose membrane and probed with SLBR-N lectin or an anti-gp120 mAb pool. The gp120 protein was treated with a mixture of neuraminidase and O -glycosidase to remove sialic acids and O -glycans. Loss of SLBR-N reactivity verified the O -glycan-dependent interaction between SLBR-N and HIV-1 Env. Experiments were repeated two or more times; data from one of the repeat experiments are shown.

Article Snippet: Recombinant gp120 A244 or JRFL (10 μg/mL) were coupled to Octet AR2G Biosensors (Sartorius) following the manufacturers protocol.

Techniques: Binding Assay, Virus, Enzyme-linked Immunosorbent Assay, Recombinant, SDS Page, Membrane

Journal: iScience

Article Title: HIV-1 interaction with an O -glycan-specific bacterial lectin enhances virus infectivity and resistance to neutralizing antibodies

doi: 10.1016/j.isci.2024.110390

Figure Lengend Snippet: O -glycan-specific lectins enhance cell-cell transfer of HIV-1 at the pre-fusion step Jurkat cells nucleofected with an HIV-1 clone bearing T/F clade B Env QH0692 and an mCherry reporter gene were used as donor cells, whereas primary CD4 T cells served as target cells. The two cell types were labeled with distinct dyes, mixed for 3 h, and mCherry+ HIV-1 transfer to target cells was monitored by flow cytometry. Donor cells were pre-treated with titrating amounts of lectin before mixing with target cells. O -glycan-specific lectins SLBR-N and MAL II were tested in comparison with high-mannose N -glycan-binding lectin GRFT. Anti-CD4 Leu3a mAb blocking gp120-CD4 engagement was tested as control. Viruses with WT Env or cleavage-defective RS Env (R508S/R511S) capable of binding CD4 but not virus fusion were tested in parallel. Four to six independent experiments were performed with primary CD4 T cells from three or five different individuals. Mean and standard error values were calculated from the total replicates of 9 for SLBR-N, 11 for MAL II, 7 for GRFT, and 7 for Leu3a. Mixed effects analysis with Dunnett’s multiple comparisons test was used to compare WT and RS with untreated control (set to 100%). ∗∗∗∗ p < 0.0001, ns: not significant ( p > 0.05).

Article Snippet: Recombinant gp120 A244 or JRFL (10 μg/mL) were coupled to Octet AR2G Biosensors (Sartorius) following the manufacturers protocol.

Techniques: Labeling, Flow Cytometry, Comparison, Binding Assay, Blocking Assay, Control, Virus

Journal: iScience

Article Title: HIV-1 interaction with an O -glycan-specific bacterial lectin enhances virus infectivity and resistance to neutralizing antibodies

doi: 10.1016/j.isci.2024.110390

Figure Lengend Snippet:

Article Snippet: Recombinant gp120 A244 or JRFL (10 μg/mL) were coupled to Octet AR2G Biosensors (Sartorius) following the manufacturers protocol.

Techniques: Virus, Recombinant, Plasmid Preparation, Cell Isolation, Clone Assay, DC Protein Assay, Software

Journal: Communications Medicine

Article Title: Convergence and divergence of B cell responses in two HIV-1 Env immunizations in Rhesus macaques

doi: 10.1038/s43856-025-00899-3

Figure Lengend Snippet: a NHP immunization scheme with multivalent HIV-1 gp120 Env cocktails delivered as DNA and monomeric proteins. i.d. intradermal, i.m. intramuscular. b Viremia time-course of the four NHPs included in this study post-seroconversion or after the fifteenth challenge for RVv15. Each NHP is color-coded as follows: red circles: RNs14; blue squares: RPt15; orange triangles: RSf15; green inverted triangles: RSf15. Dashed line at assay limit of sensitivity threshold (60 copies/ml). Measurements were performed in technical duplicates. Also see Supplementary Data . c Serum was collected four weeks prior to immunization and again at weeks 10 and 18 post-immunization. Peak serum neutralization data, defined as highest ID 50 value within the time course, are shown for the four NHPs in this study against two tier 1 viruses (C.MW965.26 and TH023.6) and four tier 2 viruses (A.92UG037.1, B.JR-FL, B.WITO4160.33, and C.Du172.17). Each NHP is color-coded as indicated in panel b. Lines indicate group mean and standard deviation. Measurements were performed in technical duplicates. Also see Supplementary Data . d Immunogen binding profiles of 734 B cells isolated from all four NHPs. Reactivity with individual and multiple gp120 Env immunogens is color coded as indicated: yellow: A.92UG037 gp120 only; orange: B.JR-FL gp120 only; red: C.93MW965 gp120 only; brown: AE.consensus gp120 only; light blue: 2 gp120 Envs; violet: 3 gp120 Envs; dark blue: all 4 gp120 Envs. Also see Supplementary Data . e Binding to individual immunogens of monoreactive B cells, expressed as percentage of the total number of monoreactive B cells. Data are shown as aggregate (“All NHPs”) and for each NHP. Reactivity with individual and multiple gp120 Env immunogens is color coded as indicated in panel d. Also see Supplementary Data . f Immunogen cross-reactivity of multireactive B cells, expressed as percentage of the total number of multireactive B cells, shown as aggregate (“All NHPs”) and for each NHP. Reactivity with individual and multiple gp120 Env immunogens is color coded as indicated in panel d. Also see Supplementary Data .

Article Snippet: Across all NHPs, immunogen-reactive B cells constituted 0.5–2% of the circulating memory B cells (RPt15: 1.31%, RNS14: 0.77%, RSf15: 2.04%, RVv15: 0.52%) (Supplementary Fig. ), which is in line with the frequency we previously reported for a human trial that used bivalent recombinant gp120 immunogens .

Techniques: Neutralization, Standard Deviation, Binding Assay, Isolation

Journal: Virologica Sinica

Article Title: Host factor Naf1 restricts HIV-1 infection of myeloid cells and compromises the capacity of dendritic cell to prime CD4 + T cell

doi: 10.1016/j.virs.2025.03.007

Figure Lengend Snippet: The induced expression of Naf1. A Naf1 expression in monocytes and MDDCs was detected by Western blotting. The gray density of protein strips was analyzed with ImageJ software and the relative value normalized with GAPDH was labeled below. B–E Induced expression of Naf1 in MDDCs and monocytes. MDDCs and CD14 + monocytes were treated with indicated dosages of pseudo-typed HIV-luc/JRFL, recombinant gp120 proteins, or replication competent HIV-1/AD8, for indicated time. The endogenous expression of Naf1 was detected with Western blotting. AZT (20 ​ng/mL) was added to inhibit the reverse transcriptase ( C ). In heat treated gp120 JRFL group, the recombinant gp120 proteins were prior-treated at 100 ​°C for 10 ​min ( D ). PDTC (50 ​nmol/L, NF-κB inhibitor) was added to block NF-κB signaling pathway ( E ). The concentration of gp120 proteins in ( D ) is 1 ​μg/mL, and 10 ​ng p24 gag amount of pseudo-typed HIV-1 were used in ( E ).

Article Snippet: Recombinant HIV-1 glycoprotein gp120 JRFL and gp120 HXB2 were purchased from eEnzyme (Gaithersburg, MD, USA).

Techniques: Expressing, Western Blot, Software, Labeling, Recombinant, Reverse Transcription, Blocking Assay, Concentration Assay

Journal: Virologica Sinica

Article Title: Host factor Naf1 restricts HIV-1 infection of myeloid cells and compromises the capacity of dendritic cell to prime CD4 + T cell

doi: 10.1016/j.virs.2025.03.007

Figure Lengend Snippet: Schematic illustration of Naf1 expression and its function. Naf1 has a higher expression in CD14 + monocytes than in MDDCs, and its expression in both types of cells can be induced by HIV-1 gp120 glycoproteins. By inhibiting NF–B signaling, Naf1 restricts HIV-1 infection in both types of cells, and reduces both the basal and LPS-stimulated cytokines in MDDCs. Moreover, Naf1 reduces ICAM-1 expression in MDDCs and thus hinders the capacity to prime resting CD4 + T cells.

Article Snippet: Recombinant HIV-1 glycoprotein gp120 JRFL and gp120 HXB2 were purchased from eEnzyme (Gaithersburg, MD, USA).

Techniques: Expressing, Infection

Journal: PLOS Pathogens

Article Title: Assessing bnAb potency in the context of HIV-1 envelope conformational plasticity

doi: 10.1371/journal.ppat.1012825

Figure Lengend Snippet: A. Schematic illustrating the experimental sCD4-induced opening of cell surface expressed Env leading to exposure of the co-receptor binding site. For detection by flow-cytometry mAb 17b was used as a co-receptor mimic. B. Top panel: Opening of cell surface expressed Envs by increasing concentrations of sCD4 was monitored by staining with mAb 17b. Env mutants were divided into two groups showing enhanced 17b binding in absence of sCD4 (right) or not (left) compared to JR-CSF wt Env. The N332A mutant served as additional control. All titrations were performed once. Color code of mutant sensitivity as in . Middle panel: magnification of top panel data indicating data points for 17b staining of individual Env mutants. Bottom panel: The bar graph depicts area under the curve values derived from the normalized MFI curves of the top panel graphs. C. Spearman correlation between 17b neutralization sensitivity of JR-CSF wildtype and mutant viruses and 17b binding according to B. Mutations directly affecting 17b binding were not included (see also ). D. Alanine-substitutions leading to moderate (blue) or high (red) generalized neutralization sensitivity of the JR-CSF Env were mapped onto the trimeric closed prefusion structure of the closely related JR-FL Env ectodomain (PDB: 5FYK; V1V2: yellow, V3: orange, β20-β21: brown, gp120: light grey, gp41: dark grey). Glycans on neutralization sensitive positions N156, N262 and N301 are depicted. The N197 glycan is not shown as JR-FL naturally lacks this PNGS.

Article Snippet: Gp120 (in-house produced affinity-purified JR-FLgp120 protein) and p24 (AG 6054, Aalto Bioreagents, Ireland) standards were prepared accordingly.

Techniques: Binding Assay, Flow Cytometry, Staining, Mutagenesis, Control, Derivative Assay, Neutralization, Glycoproteomics