Tecnai spirit t12

Fab/IgG-SOSIP complexes were made by incubating Fabs or IgGs with AMC009 SOSIP trimers with a 4-fold and 2-fold molar excess of antibody, respectively, for 30 minutes at RT. The Fab/IgG-SOSIP complexes were loaded onto glow-discharged, carbon-coated Cu400 EM grids at a concentration of 30 ng/μl in TBS for 10 seconds. The grids were blotted to remove excess sample and the Fab/IgG-SOSIP complexes were then stained with 2% (w/v) uranyl formate. Grids were immediately blotted and a second stain with 2% (w/v) uranyl formate was applied for 30 seconds, followed by a final blot to remove excess stain. A Tecnai Spirit T12 (FEI) (120kV, 52,000x magnification) equipped with an Eagle 4K CCD (FEI/Thermo Fisher) or Tecnai T20 (FEI) (200kV, 62,000x magnification) equipped with a TemCam F416 CMOS (TVIPS)was used to image the grids. Image collection was performed using Leginon and data processing was carried out as previously described [60 (link),61 (link)]. 2D classification and 3D sorting was performed with Relion v3.0 [62 (link)], and UCSF Chimera [63 (link)] and Segger [64 (link)] were used to visualize and segment the EM maps, respectively.

Purified complexes were diluted to 0.03 mg/ml using 1X TBS pH 7.4, deposited on glow-discharged carbon coated copper mesh grids, and stained for 90 sec with 2% uranyl formate (w/v). Samples were imaged either on a FEI Tecnai Spirit T12 transmission electron microscope (120 keV, 52,000x mag, 2.06 Å per pixel, -1.5 μm defocus) equipped with an FEI Eagle 4k x 4k CCD camera or a FEI TF20 transmission electron microscope (200 keV, 62,000x mag, 1.78 Å per pixel, -1.5 μm defocus) equipped with a TVIPS TemCam F416 CMOS 4k x 4k camera. Collection of raw micrographs was automated with the Leginon (Suloway et al., 2005 (link)) software and stored in the Appion (Lander et al., 2009 (link)) database. For each mouse group complex, enough micrographs were collected to have ≥ 100k particles for data processing. Particles were picked using DogPicker (Voss et al., 2009 (link)), and further data processing was performed in RELION 3.0 (Scheres, 2012 (link)). For EMPEM with SARS-CoV-2 spike, an initial model was generated from a published SARS-CoV-2 S protein structure (PDB: 6VYB (Walls et al., 2020 (link))) and used during data processing. Map interpretation and segmentation was performed in UCSF Chimera (Pettersen et al., 2004 (link)).

The details of serum and sample preparation to obtain polyclonal fabs for electron microscopy were previously described²¹ . Briefly, IgG was isolated using Protein A (Cytiva) from 1 mL NHP sera (drawn at week 12 post first immunization). Papain (Sigma Aldrich) was used to digest IgG to antigen-binding fragments (Fab). Trimer-fab complexes were prepared and incubated for an overnight by mixing 15 µg of BG505 MD39 SOSIP with 1 mg of fab mixture (containing Fc and residual Papain). On the next day, the complexes were purified using a Superdex 200 Increase 10/300 GL gel filtration column (Cytiva). Purified complexes were concentrated and diluted to a final concentration of 0.03 mg/mL, which were adsorbed on glow-discharged carbon coated copper mesh grids and stained with 2% (w/v) uranyl formate. Electron microscopy images were collected on an FEI Tecnai Spirit T12 equipped with an FEI Eagle 4k x 4k CCD camera (120 keV, 2.06 Å/pixel) and processed using Relion 3.0^{46 (link)} following standard 2D and 3D classification procedures. Leginon was used to automate EM data collection. UCSF Chimera v1.13^{47 (link)} was used to generate the composite maps, and the representative maps with identified epitopes have been deposited to the Electron Microscopy Data Bank under accession codes listed in Supplementary Fig. 7b.

Formvar/carbon-coated EM grids (Gilder Nickel Grid, Electron microscopy Sciences, PA, USA) were placed Formvar/carbon side down on top of the exosome sample drops for 10 minutes at room temperature. The grids were removed, blotted with filter paper and placed onto drops of freshly prepared 2.0% uranylacetate aqueous solution for one minute. The excess uranylacetate solution was removed, and air-dried. The images of the exosomes were captured using the FEI Tecnai™ Spirit (T12) transmission electron microscopes (Hillsboro, Oregon, USA) with a Gatan US4000 4kx4k charge coupled device (CCD) camera.

Fabs or IgGs were complexed with SOSIP trimers for 30 min at RT with a 4-fold and 2-fold molar excess of Fab and IgG, respectively. Fab/IgG-SOSIP complexes were diluted to 30 ng/µl in TBS and loaded onto glow-discharged, carbon-coated Cu400 EM grids for 10 s followed by blotting to remove excess sample. The Fab/IgG-SOSIP complexes were then stained with 2% (w/v) uranyl formate and immediately blotted, followed by a second stain with 2% (w/v) uranyl formate for 30 s before blotting to remove excess stain. Image collection was performed on a Tecnai Spirit T12 (FEI) (120 kV, ×52,000 magnification) equipped with an Eagle 4 K CCD (FEI/Thermo Fisher) camera or Tecnai T20 (FEI) (200 kV, ×62,000 magnification) equipped with a TemCam F416 CMOS (TVIPS) camera using Leginon. Data processing was performed as follows^{57 (link),58 (link)}. 2D classification and 3D sorting was performed using Relion v3.0⁵⁹ and EM maps were visualized and segmented using UCSF Chimera^{60 (link)} and Segger^{61 (link)}, respectively.