Generating ASFV Antigen Adenoviruses

The recombinant adenoviruses expressing the selected ASFV antigens, as well as the negative control Ad-Luc (Ad-Luciferase), were generated, scaled up, and titrated as previously described (infectious focus units [IFU]) [17 (link),18 (link),19 (link)]. Thirty-five antigens were used to generate adenovirus expression constructs. Due to its large size, the pp220 polyprotein (Georgia 2007/1 isolate; GenBank Accession FR682468) sequence was split into two parts designated p220.1 and p220.2 (Table 1). Briefly, the polypeptide sequences of the ASFV antigens were modified to add in-frame, HA, and FLAG tags at their N- and C-termini, respectively. The modified protein sequences were used to generate synthetic genes (GenScript) which were codon-optimized for expression in swine. All the genes were then used to generate recombinant replication-incompetent adenoviruses encoding the ASFV genes (designated Ad5-ASFV 1 to 10) using the ViraPower™ Adenoviral Gateway™ Expression Kit (Thermo Fisher Scientific K493000). Antigen expression by the adenoviruses was confirmed as previously described [17 (link),18 (link),19 (link)] (Figure 1).