Peptide immunoarrays for rationale development of vaccines with enhanced cross-reactivity

We determined epitope-specific IgG antibody responses in convalescent plasma of ferrets challenged with ancestral SARS-CoV-2 and compared that to plasma collected pre-infection. Each plasma sample was reacted with a high-density peptide microarray representing the complete proteome of SARS-CoV-2 as 15 mer peptides with 11 amino acid overlap and homologs of spike glycoprotein, nucleoprotein, membrane protein, and envelope small membrane protein from related human coronaviruses. Binding signatures were compared between plasma collected pre- and post-infection using the web service tool EPIphany.Blood was collected into lithium-heparin blood collection tubes from ferrets prior to virus exposure and 4 weeks post-infection. After centrifugation, plasma was transferred into a new tube and stored at -80C. Plasma samples were thawed once before use in immunoarray assays. Peptide microarrays were blocked in Tris-buffered saline (TBS), pH 7.2 supplemented with 0.05% v/v Tween-20 (TBS-T) and 3% w/v bovine serum albumin fraction V (BSA; diluent) for 30 min. Plasma was diluted 1:100 in diluent and incubated for 2 h. Each array was washed with 5 exchanges of TBS-T, and once with sterile deionized distilled water. Plasma IgG antibodies were detected using Alexa Fluor 647 conjugated goat anti-ferret IgG, Fc(gamma) fragment specific antibody (Jackson ImmunoResearch, 109-605-098) diluted to 1 μg/mL in diluent and incubated for 45 min in the dark. Washes were carried out as previously described, and slides dried by centrifugation for 5 min at 800×g.Peptide microarrays were imaged using a GenePix Professional 4200A microarray scanner (MDS Analytical Technologies, Toronto ON, Canada) equipped with a 635 nm laser and fluorescence captured using a 655 to 695 nm filter. Images were scanned at 10 µm resolution and data acquired using GenePix software (version 6.0). Data pre-processing and analysis was performed using the web-based service EPIphany <br>