Tetanus toxoid

As previously described^{25 (link)}, a custom multiplex bead array was designed and coupled with SARS-CoV-2 spike 1 (Sino Biological), spike 2 (ACRO Biosystems), RBD (BEI Resources) and nucleoprotein (ACRO Biosystems), as well as SARS and hCoV (229E, NL63, HKU1, OC43) spikes and nucleoproteins (Sino Biological) (Supplementary Table 5). In addition, SARS-CoV-2 and HKU-1 spike trimers (kind gifts from Adam K. Wheatley), as well as SARS-CoV and NL63 spike trimers (BPS Bioscience) were also coupled. Tetanus toxoid (Sigma-Aldrich), influenza hemagglutinin (H1Cal2009; Sino Biological) and SIV gp120 (Sino Biological) were included in the assay as positive and negative controls respectively. Antigens were covalently coupled to magnetic carboxylated beads (Bio Rad) using a two-step carbodiimide reaction and blocked with 0.1% BSA, before being resuspended and stored in PBS 0.05% sodium azide.

For allogeneic Mixed Lymphocyte Reactions (MLRs), human peripheral blood mononuclear cells were obtained from healthy volunteers via leukapheresis after provision of informed written consent, and in accordance with the principals of the Declaration of Helsinki and National Institutes of Health (NIH) guidelines for human subjects, through protocols approved by the Institutional Review Boards of the Cleveland Clinic (08–957) and Kent State University (18–421), and collected cells were separated via countercurrent centrifugal elutriation into monocyte-rich and lymphocyte-rich fractions [24 (link)]. Co-cultures of allogeneic activated dendritic cells and lymphocytes derived from elutriated cells were set up as described previously [23 (link)] in the presence or absence of 1–10 µM simvastatin, and IFN-γ assessed in co-culture supernatants 72 h later as described previously [23 (link)]. For EliSpot assays, human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from CTL Corporation (Shaker Heights, OH, USA) and stimulated in vitro with viral peptide ‘plus’ peptide pool (10 µL/well; CTL corp) or Tetanus Toxoid (2 µg/mL; Sigma) in the presence or absence of 1–10 µM simvastatin in 96-well IFN-γ EliSpot assay plates (CTL corp). After 24 h, incubation plates were developed as per manufacturer’s protocol and analyzed as described previously [23 (link)].

Analytical grades of n-hexane, chloroform, ethyl acetate, methanol, hydrogen peroxide, hydrochloric acid, ethanol, DPPH (1,1-diphenyl-2-picrylhydrazyl), epinephrine, trichloroacetic acid, thiobarbituric acid, ovalbumin, and tetanus toxoid were all obtained from Sigma Chemicals CO, USA. All laboratory reagents including distilled water were freshly prepared when required.

A custom multiplex bead array was designed (Supplemental Table 3) and coupled with SARS-CoV-1 and SARS-CoV-2 spike-1 (stem, Sino Biological), spike-2 (head, ACRO Biosystems), RBD (BEI Resources), and N (ACRO Biosystems), as described previously (45 (link)). In addition, SARS-CoV-2 spike trimers (provided in-house) and SARS-CoV-2 spike trimers (BPS Bioscience) were included. Tetanus toxoid (Sigma-Aldrich), influenza hemagglutinin (H1Cal2009, Sino Biological), and SIV-gp120 (Sino Biological) were included as positive and negative control antigens, respectively. Antigens were covalently coupled to magnetic carboxylated beads (Bio-Rad) using a 2-step carbodiimide reaction and blocked with 0.1% BSA, before being resuspended and stored in PBS with 0.05% sodium azide until use.

MaxiSorp^® ELISA plates (Thermo Scientific) were coated with 0.5 μg of nucleases or control proteins diluted in 1 × PBS and incubated overnight at room temperature. Plates were then washed and incubated with a 1% (w/v) BSA (Sigma-Aldrich)/1 × PBS solution (1% BSA-PBS) for an hour at room temperature. After another washing step, wells were incubated in duplicate for 1 h at room temperature with 48 separate serum samples (BioChemed) taken from randomly selected donors (1:50 dilution in 1% BSA-PBS). Plates were then washed and incubated for an hour at room temperature with a peroxidase-labeled goat anti-human (Fcγ fragment-specific) secondary antibody (Jackson Immuno Research), diluted 1:50,000 in 1% BSA-PBS. The assay was developed using a 3,3′,5,5′-tetramethylbenzidine Liquid Substrate System kit (Sigma-Aldrich), according to the manufacturer's specifications. Antibody titers were reported as average absorbance values measured at 450 nm. Tetanus toxoid was purchased from Sigma-Aldrich.