SpyCatcher-AP205 VLPs were expressed in bacteria and purified as described (48 (link), 88 (link)). SpyTagged SOSIP immunogens were incubated at a 2 to 3–fold molar excess with SpyCatcher-VLPs for 12 to 24 hours at room temperature in phosphate-buffered saline (PBS). Conjugated VLPs were separated from free Env trimers by SEC on a Superdex 200 (fig. S7D) or Superose 6 (fig. S7E) column. Conjugation of Env trimers was verified by SDS-PAGE (fig. S7C). Immunogen concentrations for immunizations were estimated by comparing to known amounts of the analogous unconjugated Env trimer on a SDS-PAGE gel.
Conjugated VLPs were examined by nsEM (fig. S7A and D). Purified VLPs were diluted to about 10 μg/mL immediately before adding 3 μL to a glow-discharged ultrathin C film on a holey carbon support film, 400 mesh, Cu grid (Ted Pella). After blotting, the grids were stained by uranyl acetate and then imaged using a FEI Tecnai T12 transmission electron microscope operating at 120 keV with a Gatan Ultrascan 2k × 2k CCD camera. Each image was collected using a 1 second exposure at about 2 μm defocus and 42,000× magnification, resulting in 2.5 Å per pixel.