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. 2021 Jan 26;11:602254. doi: 10.3389/fimmu.2020.602254

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Copyright © 2021 Fisher, Padula, Podack, O’Neill, Seavey, Jayaraman, Jasuja and Strbo

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Schematic of gp96-Ig and SARS-CoV-2 protein S constructs used to generate vaccine cells HEK-293-gp96-Ig-S and AD-100-gp96-Ig-S. (A) Each panel presents the protein expressed by the DNA (black outline) for the gp96-Ig and SARS-CoV-2 protein S vaccine antigen. Gp96-Ig and SARS-CoV-2-S DNA were cloned into the mammalian expression vectors B45 and pcDNA 3.1, which are transfected into HEK-293 and AD100. Stably transfected vaccine cell clones (1A, 1A6, 1D6) were generated after selection with L-Histidinol and Neomycin; (B) One million 293-gp96-Ig-S and AD-100-gp96-Ig-S (1D6) cells were plated in 1 ml for 24 h and gp96-Ig production in the supernatant was determined by ELISA using anti-human IgG antibody for detection with human IgG1 (0.5 ug/ml) as a standard; (C) Cell lysates were analyzed under reduced conditions by SDS-PAGE and Western blotting using anti protein S antibody and recombinant protein S1 as a positive control; (D) IF for protein S (in green) expressed in AD100-gp96-Ig-S cells using rabbit anti-SARS-CoV-2 S antibody and anti-rabbit Ig-AF488 as secondary antibody. AD100 was used as a negative control and β-actin for protein quantification. Original magnification 40× with DAPI nuclear staining shown in blue. DNA, deoxyribonucleic acid; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; N, amino terminus; C, carboxy terminus; IF, immunofluorescence; TM, transmembrane domain; KDEL, retention signal; CH2 CH3 gamma 1, heavy chain of IgG1. See text for explanation.