Adenovirus-based Epigraph HA Vaccine

The three Epigraph HA genes (immunogens) were codon-optimized for human gene expression and synthesized by GenScript. These Epigraph genes were then cloned into a replication-defective E1/E3 deleted Adenovirus type 5 vector using the Ad-Easy Adenoviral Vector System (Agilent). The Epigraph HA genes were individually cloned into the pShuttle-CMV plasmid and cotransformed with pAd-Easy-1 (HAdV-5 genome) into BJ5183 cells for homologous recombination into the E1 region of the HAdV-5 genome^{17 (link),39 (link)}. The linearized recombinant pAd-Epigraph plasmid DNA was transfected into 293 cells using the PolyFect Transfection Reagent (Qiagen). Virus was amplified by sequential passages in 293 cells until a final amplification using a Corning 10-cell stack (~6300 cm²). The virus was purified by 2 sequential CsCl ultracentrifuge gradients, desalted using Econo-Pac 10DG Desalting Columns (Bio-Rad), and stored at −80 °C in 20 mM Tris, 100 mM NaCl, 1 mM MgCl₂, 10% glycerol (pH 8.0). Virus particles (vp) were quantitated by OD260. The infectious units per mL were determined using the AdenoX Rapid Titer kit according to the manufacturer’s instructions (Clontech Laboratories).