Journal: The Journal of Experimental Medicine

Article Title: Design and characterization of HIV-1 vaccine candidates to elicit antibodies targeting multiple epitopes

doi: 10.1084/jem.20250693

Figure Lengend Snippet: Design and biochemical characterization of 3nv.2 SOSIP. (A) Design of 426c-based triple immunogen to present CD4bs (blue), V3 (green), and V2 (purple) epitopes. (B) Schematics of single (IGT2), double (IGT2–RC1), and triple (3nv.2) immunogen constructs used in this study. (C and D) 3nv.2 and 3nv.1 triple immunogen characterization by (C) SEC and (D) SDS-PAGE. (E) Top: 2D class averages demonstrating 3nv.2 is predominantly trimeric. Bottom: 6.6-Å single-particle 3nv.2 cryo-EM density map. (F) Schematic for the generation of SOSIP–mi3 nanoparticles using the SpyCatcher–SpyTag system. (G) Characterization of purified SOSIP–mi3 nanoparticles by SDS-PAGE. R, reduced; NR, non-reduced; SC, SpyCatcher. (H) Negative-stain EM of SOSIP–mi3 nanoparticles. Scale bar = 100 nm.

Article Snippet: Biotinylated SOSIP timers were immobilized on streptavidin-coated 96-well plates (Thermo Fisher Scientific) at a concentration of 2–5 μg/ml in TBS-T (20 mM Tris [pH 8.0], 150 mM NaCl, and 0.1% Tween 20) supplemented with 1% BSA for 1 h at room temperature.

Techniques: Construct, SDS Page, Single Particle, Cryo-EM Sample Prep, Purification, Staining

Journal: The Journal of Experimental Medicine

Article Title: Design and characterization of HIV-1 vaccine candidates to elicit antibodies targeting multiple epitopes

doi: 10.1084/jem.20250693

Figure Lengend Snippet: 3nv.2 binds to iGLs targeting three bNAb epitopes. (A) SPR sensorgrams of SOSIP–mi3 nanoparticles injected over bNAb iGLs at a concentration of ∼0.5 mg/ml. First row: The CD4bs-specific single immunogen IGT2 only binds to CD4bs bNAb precursors (IOMA iGL, left), and not V3 (10-1074/PGT121 iGL, middle) or V2 (PG9 or PG16 iGL, right) bNAb precursors. Second row: Incorporating V3-targeting mutations into IGT2 creates an immunogen that binds CD4bs and V3 bNAb precursors, but not V2 bNAb precursors. Third row: Incorporating V3- and V2-targeting residues into the CD4bs-targeting IGT2 SOSIP creates an immunogen (3nv.2) that binds to the CD4bs (IOMA iGL, left), V3 (10-1074/PGT121 iGL, middle), and V2 (PG9 or PG16 iGL, right) bNAb precursors. Fourth row: The parental 426c Env does not bind to any of the bNAb precursors. (B) 3nv.2 SOSIP injected in a dilution series over bNAb iGLs starting at top concentrations of 10 µM or 5 µM as indicated. 3nv.2 SOSIP binds to multiple CD4bs precursors, including IOMA iGL (left), BG24 iGL (middle), and VRC01 iGL (right). (C) 3nv.2 SOSIP injected in a dilution series over bNAb UCAs and an iGL starting at a top concentration of 10 µM. 3nv.2 SOSIP binds to multiple V3 precursors, including BG18 iGL (left), DH270 UCA (middle), and BF520 UCA (right). Representative sensorgrams are from at least two independent experiments. UCA, unmutated common ancestor; RU, resonance unit.

Article Snippet: Biotinylated SOSIP timers were immobilized on streptavidin-coated 96-well plates (Thermo Fisher Scientific) at a concentration of 2–5 μg/ml in TBS-T (20 mM Tris [pH 8.0], 150 mM NaCl, and 0.1% Tween 20) supplemented with 1% BSA for 1 h at room temperature.

Techniques: Injection, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Design and characterization of HIV-1 vaccine candidates to elicit antibodies targeting multiple epitopes

doi: 10.1084/jem.20250693

Figure Lengend Snippet: Data processing of the 3nv.2 SOSIP dataset. * denotes the dataset that is deposited in the EMDB.

Article Snippet: Biotinylated SOSIP timers were immobilized on streptavidin-coated 96-well plates (Thermo Fisher Scientific) at a concentration of 2–5 μg/ml in TBS-T (20 mM Tris [pH 8.0], 150 mM NaCl, and 0.1% Tween 20) supplemented with 1% BSA for 1 h at room temperature.

Techniques: