RBD and Monoclonal Antibody Expression RBD, monoclonal antibodies, and soluble human ACE2-Fc were expressed and purified from Expi293F cells. Expi293F cells were cultured in 66% Freestyle/33% Expi medium and grown in TriForest polycarbonate shaking flasks at 37 °C in 8% CO2. One day prior to transfection, cells were spun down at 300g and resuspended to a density of 3 × 106 cells/mL in fresh medium. The next day, cells were diluted and transfected at a density of approximately 3–4 × 106 cells/mL. Transfection mixtures were made by adding maxi-prepped DNA, culture medium, and FectoPRO (Polyplus) to cells to a ratio of 0.5–0.8 μg:100 μL:1.3 μL:900 μL. For example, for a 100 mL transfection, 50–80 μg of DNA was added to 10 mL of culture medium, and then, 130 μL of FectoPRO was added to that mixture. Following mixing and a 10 min incubation, the resultant transfection cocktail was added to 90 mL of cells. The cells were harvested 3–5 days after transfection by spinning the cultures at >7000g for 15 min. Supernatants were filtered using a 0.22 μm filter.

Vaccine scaffolds and carrier proteins increase the immunogenicity of subunit vaccines. Here, we developed, characterized, and demonstrated the efficacy of a novel microparticle vaccine scaffold comprised of bacterial peptidoglycan (PGN), isolated as an entire sacculi. The PGN microparticles contain bio-orthogonal chemical handles allowing for site-specific attachment of immunogens. We first evaluated the purification, integrity, and immunogenicity of PGN microparticles derived from a variety of bacterial species. We then optimized PGN microparticle modification conditions; Staphylococcus aureus PGN microparticles containing azido-d-alanine yielded robust conjugation to immunogens. We then demonstrated that this vaccine scaffold elicits comparable immunostimulation to the conventional carrier protein, keyhole limpet hemocyanin (KLH). We further modified the S. aureus PGN microparticle to contain the SARS-CoV-2 receptor-binding domain (RBD)─this conjugate vaccine elicited neutralizing antibody titers comparable to those elicited by the KLH-conjugated RBD. Collectively, these findings suggest that chemically modified bacterial PGN microparticles are a conjugatable and biodegradable microparticle scaffold capable of eliciting a robust immune response toward an antigen of interest.