Nr 52308

r-Ad-S is a rAd5 vector containing the gene of the full-length SARS-CoV-2 stabilized S protein, under control of the cytomegalovirus (CMV) promoter. rAd5 vaccine constructs were created based on the published DNA sequence of SARS-CoV-2 publicly available as GenBank Accession No. MN908947.3. The published amino acid sequences of the SARS-CoV-2 S were used to create recombinant plasmids containing transgenes cloned into the E1 region of Adenovirus Type 5 (rAd5) (37), using the same vector backbone used in prior clinical trials for oral rAd tablets (11, 38). All vaccines were grown in the Expi293F suspension cell-line (Thermo Fisher Scientific) and purified by CsCl density centrifugation. The spike protein vaccine was provided by BEI Resources, NR-52308, Spike Glycoprotein (Stabilized) from SARS-Related Coronavirus 2, Recombinant from Baculovirus, NR-52308.

Purified VLP samples were mixed with lithium dodecyl sulfate (LDS) sample buffer (1× final concentration) and heated at 100 °C for 5 min. Samples were loaded onto 4–12% Bis-Tris gels (Invitrogen, Waltham, MA, USA) and transferred to a nitrocellulose membrane. Membranes were blocked with 5% non-fat dry milk in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20). Primary antibodies were added to membranes and incubated overnight: anti-N (1:1000 dilution, Sino Biological, Beijing China, 40143-T62), anti-M (antibody cocktail: i. (1:250 dilution, ProSci, Fort Collins, CO, USA, 3529) and ii. (1:250 dilution, Invitrogen, PA1-41160)), or anti-S (1:1000 dilution, generated in house via immunization of mice with purified S protein (BEI Resources, NIAID, Manassas, VA, USA, NR-52308)). Membranes were washed 3 times with TBS-T and subsequently incubated for 1 h with HRP-conjugated secondary antibody: anti-rabbit (1:6000 dilution, Invitrogen, 65-6120) or anti-mouse (1:6000 dilution, Invitrogen, 62-6520). Blots were developed with SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific) and imaged using an Azure Biosystems C600 Imaging System. Densitometry analysis was performed using AzureSpot software (Azure Biosystems, Dublin, CA, USA). Purified recombinant S protein was used as a standard (BEI Resources, NR-52308).