U bottom 96 well tissue culture plates

5x10⁵ PBMCs per well were seeded in U-bottom 96-well tissue culture plates (Greiner Bio-One, Austria) and stimulated with 10 ng/ml lipopolysaccharides (LPS) derived from Escherichia coli O55:B5 (Sigma-Aldrich, MO, USA), 10⁶/ml heat-killed Staphylococcus aureus, 10⁶/ml heat-killed Candida albicans, R848 (InvivoGen), or heat-inactivated(30 minutes, 60°C) SARS-CoV-2 Wuhan-Hu-1 strain (NRW-42 isolate, GISAID accession number: EPI_ISL_425126) (35 (link)) with 1 µg/ml soluble purified mouse anti-human CD28 (BD Biosciences, New Jersey, USA), for 24 hours and 7 days in the presence of 10% pooled human serum. A control condition without any stimulant was included. Cells were incubated at 37°C with 5% CO2. Stimulations with R848 and SARS-CoV-2 were performed later with cryopreserved cells, while the rest of the stimulations were done with freshly isolated PBMCs. Concentrations of TNF-α, IL-6, IL-1β, IL-1Ra, and IFN-γ in culture supernatants were determined using DuoSet ELISA kits (R&D Systems, MN, USA). IFNα concentrations were determined using VeriKine Human Interferon Alpha ELISA Kit (PBL Assay Science, NJ, USA) according to the manufacturer’s instructions. Supernatants from 7 days stimulations were used for IFN-γ, and the remaining cytokines were measured in 24-hour supernatants.

Proteins were isolated from Lysinibacillus sphaericus CCM 2177 (SbpA) and subsequently purified as reported elsewhere^{37 (link)}. To reconstitute the S-layer protein solution, 5 mg of lyophilized protein was dissolved in guanidine hydrochloride (5 M in 50 mM Tris-(hydroxymethyl)aminomethane x HCl buffer, pH 7.2) and dialyzed against Milli-Q-water (Millipore, Austria) for 1 h at 4 °C. Afterward, the protein solution was centrifuged at 1300 rpm for 15 min at 4 °C to remove self- assembly products. The concentration was adjusted to 100 μg/mL in a recrystallization buffer (0.5 mM Tris-(hydroxymethyl)aminomethane, 10 mM CaCl₂, pH 9), 250 μL of the solution were added to U-bottom 96-well tissue culture plates (Greiner-Bio-One, Austria) and incubated over night at room temperature.