Journal: bioRxiv

Article Title: Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies

doi: 10.1101/2023.09.13.557082

Figure Lengend Snippet: a , SDS-PAGE of purified 1059 gp120 b , Binding of CD4bs and V1V2 bnAbs to soluble 1059 gp120. c , Comparison of CD4bs and V1V2 bnAbs binding (1 µg/ml) to soluble gp120 of AD8 and 1059. d , Comparison of AD8 and 1059 pseudovirus neutralization by bnAbs from ( c ). Color code and bnAb order in panels c and d is identical. *, IC 50 > maximal value tested. Data are average + SEM of at least 2 independent experiments, each performed at least in duplicate. e , We incubated recombinant pseudoviruses carrying the different Envs with CD4-negative, CCR5-expressing Cf2Th cells in the presence of indicated concentrations of sCD4. Between 48-72 hours post infection we measured the activity of firefly luciferase reporter protein (relative light units) to assess viral entry. Data shown are representative results from one of 2 independent experiments performed in triplicate + standard deviation (SD).

Article Snippet: The membrane was blocked with 5% blotting-grade blocker (catalog number 1706404, Bio-Rad) in PBS (5%MPBS), washed with PBS, and incubated for 1-hour on a shaker with serum from a person living with HIV-1 (1:30,000 dilution) and sheep anti-gp120 IgG (1:30,000 dilution; catalog number 288, NIH AIDS reagent program) both diluted in 5%MPBS.

Techniques: SDS Page, Purification, Binding Assay, Comparison, Neutralization, Incubation, Recombinant, Expressing, Infection, Activity Assay, Luciferase, Standard Deviation

Journal: bioRxiv

Article Title: Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies

doi: 10.1101/2023.09.13.557082

Figure Lengend Snippet: a , We designed different 1059 SOSIP variants based on published studies and current knowledge. Env changes associated with each variant are shown and highlighted in green relative to the WT (red). b , Western blot of different 1059 SOSIP variants expressed in 293F cells for 4 days and analyzed directly from cell supernatants. We detected the SOSIP Env proteins using 1:10,000 dilution of serum of PLWH + 0.5μg/ml of JR52 antibody, which recognizes the D7324 epitope, followed by anti-human (1:10,000 dilution) + anti-mouse (1:20,000 dilution), both conjugated to horseradish peroxidase (HRP). Lanes: 1, ladder; 2, HIV-1 AD8 gp120 control; 3, TPA WT; 4, TPA P22A (contains amino acid Alanine at position 22); 5, DS; 6, V5.2.8a; 7, v4.1. TPA, signal peptide of the Tissue Plasminogen Activator. TPA-1059 SOSIP exhibited the highest expression levels and was selected for further characterization in all studies described in the main text. c , Comparison of size exclusion chromatography (SEC) profile of 1059 and BG505 SOSIP v6 trimers (both contained the signal peptide derived from the TPA). d , ELISA of 17b antibody binding to 1059 and BG505 SOSIPs in the presence and absence of sCD4. e , SEC profiles of 1059-SOSIP and BG505-SOSIP preparations used for determining structures of the unliganded Envs. The dashed vertical lines indicate the fractions that were pooled for downstream studies.

Article Snippet: The membrane was blocked with 5% blotting-grade blocker (catalog number 1706404, Bio-Rad) in PBS (5%MPBS), washed with PBS, and incubated for 1-hour on a shaker with serum from a person living with HIV-1 (1:30,000 dilution) and sheep anti-gp120 IgG (1:30,000 dilution; catalog number 288, NIH AIDS reagent program) both diluted in 5%MPBS.

Techniques: Variant Assay, Western Blot, Control, Expressing, Comparison, Size-exclusion Chromatography, Derivative Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: bioRxiv

Article Title: Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies

doi: 10.1101/2023.09.13.557082

Figure Lengend Snippet: a , Side and top views of unliganded incompletely closed 1059-SOSIP structure at 3.6 Å. b-d, Structural determinants of incompletely closed SOSIP. b, Conformational flexibility. Overlay of 10 rigid fitted sub-class models of 1059 or BG505 SOSIPs purified and analyzed under identical conditions. Color bar indicates the C-alpha root mean square deviation between those structures. We used one protomer (noted with an asterisk) for superposition and calculated the C-alpha root the mean deviation between the structures. Number of particles used, and resolution of each sub-class model are provided in . c, Asymmetry. We measured the inter-protomer distances between residues 343 of gp120 α2 helix of different protomers in each structure to assess asymmetry. Left, interprotomer distances of different soluble Envs, including 2 asymmetric structures (PDB: 5FUU & 5U1F) that were obtained with cryo-EM and refined with C1 symmetry and four symmetric structures (PDB: 4ZMJ, 5ACO, 5TZ3 and 5V8L) that were obtained either by X-ray crystallography or by cryo-EM and refined with C3 symmetry except the 5V8L. *, SOSIP structures solved in this study by cryo-EM and refined with C1 symmetry. Right, a statistical analysis of the difference in asymmetry level between the 1059 and BG505 SOSIPs calculated from the 10 respective subclasses for each SOSIP. Asymmetry was calculated based on deviation from inter-protomer distances as described in Methods. P, two-tailed P value of Mann-Whitney U-test. d, Mobility. CryoSPARC 3D variability analysis revealed disruption of the trimer apex and outward movement of the V1/V2 loop (also see Supplementary Movies, principal component 3 motion). Two snapshots are shown where the V1/V2 loop (yellow) is shifting outward from the trimer (pink circles). e, A structure of open 1059-SOSIP when in complex with 17b. f, DEER spectroscopy data for interprotomer distance distributions for V1 spin labels in V1V2 (residue 173), bridging sheet (residue 202), and inner domain (residue 106) for unliganded and sCD4-bound 1059 SOSIPs. Vertical solid and dashed lines represent interprotomer Cα distance measurements at target sites for closed (PDB: 5T3Z) and sCD4-liganded (PDB: 5VN3) structures, respectively. Residue 173 was disordered for sCD4-Env structure (distance not shown). Grey dotted line indicates the upper limit for reliably measuring distances using DEER technology. SOSIP trimers were purified by GNL/SEC ( a-d ) or 2G12 Ab/SEC ( e-f ).

Article Snippet: The membrane was blocked with 5% blotting-grade blocker (catalog number 1706404, Bio-Rad) in PBS (5%MPBS), washed with PBS, and incubated for 1-hour on a shaker with serum from a person living with HIV-1 (1:30,000 dilution) and sheep anti-gp120 IgG (1:30,000 dilution; catalog number 288, NIH AIDS reagent program) both diluted in 5%MPBS.

Techniques: Purification, Cryo-EM Sample Prep, Two Tailed Test, MANN-WHITNEY, Disruption, Spectroscopy, Residue

Journal: bioRxiv

Article Title: Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies

doi: 10.1101/2023.09.13.557082

Figure Lengend Snippet: Effects of bnAbs targeting gp120 V1/V2 loop (PG9, PG16, and PGT145) and bnAbs targeting gp120 V3 glycan (PGT 121, PGT126, and PGT128) on the entry of pseudoviruses displaying the K160N ( a ) and E332N ( b ) CH040 Env variants. Data shown (mean + SD) are representative results from one of at least 2 independent experiments performed in duplicate.

Article Snippet: The membrane was blocked with 5% blotting-grade blocker (catalog number 1706404, Bio-Rad) in PBS (5%MPBS), washed with PBS, and incubated for 1-hour on a shaker with serum from a person living with HIV-1 (1:30,000 dilution) and sheep anti-gp120 IgG (1:30,000 dilution; catalog number 288, NIH AIDS reagent program) both diluted in 5%MPBS.

Techniques: Glycoproteomics

Journal: bioRxiv

Article Title: Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies

doi: 10.1101/2023.09.13.557082

Figure Lengend Snippet: a , Representative micrograph and b , cryo-EM 2D class averages for 1059SOSIP-N6-10-1074 cryo-EM data collection. c , Gold-standard Fourier shell correlation (FSC) plot was used to estimate structure global resolution of 1059SOSIP-N6-10-1074 complex. Estimate is reciprocal of spatial frequency at Fourier shell correlation value of 0.143 (horizontal dashed line). d , Local resolution estimation for the 1059SOSIP-N6-10-1074 complex. Rigid body fits of a protomer for DU172.17 gp120-N6 (PDB: 5TE7) (e) and BG505-N6 FR3-03 scFV-3H109 Fab-35022 scFV (PDB: 6NM6, 3H109 Fab and 35022 scFv not shown) (f) shown in cartoon in 1059SOSIP-N6-10-1074 EM density. g-i , Rigid body fits of trimers (top) and protomers (bottom) for structure models of BG505-3BNC117 (PDB: 5V8M) ( g ), BG505-1-18-10-1074 (PDB: 6UDJ) ( h ), and BG505-IOMA-10-1074 (PDB:5T3Z) ( i ). Fits are shown in cartoon in 1059SOSIP-N6-10-1074 EM density map.

Article Snippet: The membrane was blocked with 5% blotting-grade blocker (catalog number 1706404, Bio-Rad) in PBS (5%MPBS), washed with PBS, and incubated for 1-hour on a shaker with serum from a person living with HIV-1 (1:30,000 dilution) and sheep anti-gp120 IgG (1:30,000 dilution; catalog number 288, NIH AIDS reagent program) both diluted in 5%MPBS.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies

doi: 10.1101/2023.09.13.557082

Figure Lengend Snippet: a , Sensitivity to cold exposure of VRC01-resistant Envs, isolated from the VRC01 or placebo arms, and of VRC01-sensitive Envs, isolated from the placebo arm. b, Statistical analysis of difference between cold sensitivity of VRC01-resistant and VRC01-sensitive Envs. Half life on ice was calculated by fitting zero-order decay curves to the residual infection data (panel a ). A dot line represents the maximal experimental time (96 hours) tested. c, Sensitivity of Envs from a single VRC01-resistant strain (H704_1835_150_RE_p002s_2484A) to VRC01 and to internal-epitope antibodies that target different gp120 domains. d, Left - principal component analysis (PCA) of AMP Envs (6 VRC01-resistant & 7 VRC01-sensitive) clustered VRC01 sensitivity opposite to E51, 17b, 246-D and cold sensitivities. All Envs were resistant to 19b and 697-30D and thus these antibodies were not included. Right - statistical analysis of the difference between the sensitivity of VRC01-resistant and VRC01-sensitive Envs to 3 Abs (E51, 17b, and 246-D) identified by PCA. IC50s were calculated from dose-response curves . No differences were identified for all other Env ligands or for comparison of VRC01-resistant and VRC01-sensitive for each antibody separately . e, Sensitivity of VRC01-resistant (left) and VRC01-sensitive (right) Envs to CD4bs bnAbs. f, Binding of VRC01 to soluble AMP gp120s by ELISA. g, 2G12-nornalized VRC01 binding to AMP gp120s. t1/2 values or IC50s higher than the maximal concentration/time tested (96 hours ( b ), 100 µg/ml ( d ) and 50 µg/ml ( e )) were set to an arbitrary 100 hours ( b ), 200 µg/ml ( d ) and 100 µg/ml concentrations ( e ), which are labeled with red letters and an asterisk on the Y-axis. P, two-tailed P value of Mann-Whitney U-test. ns, not significant. Results are the average of 2-5 independent experiments, each performed in duplicate.

Article Snippet: The membrane was blocked with 5% blotting-grade blocker (catalog number 1706404, Bio-Rad) in PBS (5%MPBS), washed with PBS, and incubated for 1-hour on a shaker with serum from a person living with HIV-1 (1:30,000 dilution) and sheep anti-gp120 IgG (1:30,000 dilution; catalog number 288, NIH AIDS reagent program) both diluted in 5%MPBS.

Techniques: Isolation, Infection, Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Labeling, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Conformational flexibility of HIV-1 envelope glycoproteins modulates transmitted / founder sensitivity to broadly neutralizing antibodies

doi: 10.1101/2023.09.13.557082

Figure Lengend Snippet: a , Sensitivity of VRC01-resistant (grey) and VRC01-sensitive Envs (green) to internal-epitope antibodies and soluble CD4. Results are the average of 2-5 independent experiment, each performed in 2-4 replicates. b , SDS-PAGE (left) and western blot (right) of gp120 of VRC01-resistant HIV-1 strains. gp120 IDs are the same as those used in .

Article Snippet: The membrane was blocked with 5% blotting-grade blocker (catalog number 1706404, Bio-Rad) in PBS (5%MPBS), washed with PBS, and incubated for 1-hour on a shaker with serum from a person living with HIV-1 (1:30,000 dilution) and sheep anti-gp120 IgG (1:30,000 dilution; catalog number 288, NIH AIDS reagent program) both diluted in 5%MPBS.

Techniques: SDS Page, Western Blot