Sars cov 2 spike s1 protein

Cryopreserved BAL samples (from the 1 week after second vaccination time point) were thawed and resuspended at a concentration of 3–4 million cells/mL in serum-free medium AIM V (Thermo Fisher Scientific). Poly I:C (2 μg/mL) was added to the cells in the presence or absence of 2 μg/mL of SARS-CoV-2 spike S1 protein (Sino Biological; endotoxin level: <0.001 U/μg). After 18 hours of culture at 37°C, 5% CO₂, supernatant was collected and frozen at –20°C for IFN-α ELISA and Chemokine/Cytokine Bioplex Assay using an LSRII cytometer. LEGENDplex NHP Chemokine/Cytokine Panel (13-plex, BioLegend) was used to measure the following 13 chemokines and cytokines: TNF-α, IL-1β, IL-6, IL-8, MIP1-α, MIP1-β, RANTES, MCP-1, IFN-γ, MIG, IP-10, ITAC, and Eotaxin. Pan–IFN-α (including subtypes α1, 2, 4, 5, 6, 7, 8, 10, 14, 16, and 17) ELISA kit (Mabtech) was used to measure the total concentration of IFN-α. Both assays were performed in accordance with the manufacturers’ instructions.

Western blot analysis was performed to detect RBD219-N1C1 as well as P. pastoris host cell protein (HCP). Five micrograms of purified protein was run on 14% Tris-Glycine gels under non-reducing and reducing conditions to detect RBD219-N1C1 and HCP, respectively. Proteins in gel were transferred to PVDF membranes and blocked with 5% dry milk in PBST (1× PBS with 0.05% Tween-20). RBD219-N1C1 was detected using a rabbit monoclonal antibody against the SARS-CoV-2 spike S1 protein (Sino Biological, Beijing, China; Cat#: 40150-R007) and goat anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (Invitrogen, Carlsbad, USA; Cat#: G21234). HCPs were detected using an anti-P. pastoris:HRP conjugate (2G) solution (Cygnus, Southport, USA; Cat#: F641-12). The blots were developed using ECL Prime Substrate System (Cytiva, Marlborough, USA).