Cr3022

RBD and spike ELISAs were modified from Amanat et al. and conducted as previously described [6 (link),8 (link)]. ELISA plates were coated either with RBD (Sino Biological, Wayne, PA, USA), nucleocapsid (Gift from B. Geiss Lab) or spike protein (Sino Biological, Wayne, PA, USA). Samples and controls were added after 1 h of blocking (1X PBS, 3% milk powder, 0.1% tween). Positive controls included convalescent patient serum (gift of R. Goodrich) and monoclonal antibody CR3022 (Absolute Antibody, Boston, MA, USA). Negative control serum was charcoal inactivated pooled human serum collected in 2015 (Jackson Immuno Research, West Grove, PA, USA). Secondary antibody was added followed by development and reading via spectrophotometer (Thermo Fisher, Dallas, TX, USA).

2.5 μg/ml of monoclonal antibody CR3022 (Absolute Antibody) or 3 μg/ml of HEK293-produced human ACE2-Fc (Klausberger et al., 2021 (link)) were coated (50 μl/well) in bicarbonate buffer or PBS, respectively, onto NUNC MaxiSorp 96 well plates (Thermo Fisher Scientific) overnight at 4°C. Plates were washed three times with PBS supplemented with 0.1% (v/v) Tween 20 (PBST) and subsequently blocked for 1 h with 1% (w/v) BSA in PBST. Purified RBD was diluted in PBST supplemented with 1% BSA (twofold dilution series: 1–0.0625 μg/ml) and incubated for 2 h. The plates were washed 3 times with PBST and incubated for 1.5 h with biotin-conjugated anti-His antibody (Thermo Fisher Scientific) diluted 1:1000 in PBST + 1% (w/v) BSA. After washing, plates were incubated for 30 min with streptavidin-horseradish peroxidase (HRP) conjugate (Roche) diluted 1:5000 in PBST + 1% (w/v) BSA. After 3 washes, substrate solution [10 mM sodium acetate, pH 5 + 1:60 diluted TMB-stock solution (0.4% (w/v) tetramethylbenzidine (Fluka) in DMSO) + 1:300 diluted H₂O₂ (0.6% in H₂O)] was applied (150 μl/well) and plates were incubated for 5–10 min with shaking. Reactions were stopped by the addition of 1 M sulfuric acid (25 μl/well) and absorbance was measured at 450 nm on a Tecan Sunrise Microplate reader using a reference wavelength of 620 nm.

Spodoptera frugiperda (Sf9) cells were used for generation of recombinant baculoviruses and for subsequent virus amplification. For production of VLPs Trichoplusia ni (Tnao38) cells [28 (link)] were used. Virus was grown at an MOI of 0.01 and infected cells were incubated for up to 6 days before harvesting the supernatant. Virus infection for protein expression and VLP assembly employed an MOI of 5 and incubation for 4 h, after which the cells were pelleted and re-suspended in fresh culture medium to maximize expression levels [29 (link),30 (link)]. Infected cells were then incubated for a further 72 h by which time cell viability measured by trypan blue exclusion was ~50%. The resultant supernatant was harvested, clarified by centrifugation twice at 4500× g, 4 °C for 20 min, and the VLPs then pelleted by centrifugation through a 25% sucrose cushion, at 100,000× g, 4 °C for 100 min. VLPs were re-suspended in 5 mL of PBS and purified by centrifugation on a 20–60% sucrose gradient at 100,000× g, 10 °C for 18 h. The gradients were fractionated and the presence of S protein assessed by dot blot with CR3022 (Absolute Antibody, Oxford, UK). The pixel density in each dot was assessed using Image J [31 (link)].