Citation
- Authors: Powell AE. et al.
- Year: 2021
- Journal: ACS Cent Sci 7 183-199
- Applications: in vitro / DNA / FectoPRO
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
Expression and Purification of SARS-CoV-2 Antigens, mAbs, and Soluble ACE2:
- All antigens for immunization, mAbs, and soluble human ACE2-Fc were expressed and purified from Expi293F cells. Expi293F cells were cultured using 66% FreeStyle 293 Expression/33% Expi293 Expression medium (ThermoFisher) and grown in polycarbonate baffled shaking flasks at 37 °C and 8% CO2 while shaking at 120 rpm.
- Cells were transfected at a density of approximately (3–4) × 106 cells/mL. Transfection mixtures were made by adding 568 μg of maxi-prepped DNA to 113 mL of culture medium (per liter of transfected cells) followed by addition of 1.48 mL of FectoPro (Polyplus). For mAbs, cells were transfected with a 1:1 ratio of HC:LC plasmid DNA. Mixtures were incubated at room temperature for 10 min and then added to cells. Cells were immediately boosted with d-glucose (4 g/L final concentration) and 2-propylpentanoic (valproic) acid (3 mM final concentration).
- Cells were harvested 3–5 days post-transfection via centrifugation at 7000g for 15 min. Culture supernatants were filtered with a 0.22 μm filter.
Abstract
The development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (SΔC-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines, and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of SΔC-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with SΔC-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.
