mRNA Vaccine Manufacturing Process

Antigens encoded by the mRNA vaccines were derived from the ancestral SARS-CoV-2 Wuhan-Hu-1 strain (GenBank MN908947.3). Nucleoside-modified mRNAs expressing SARS-CoV-2 full-length N (mRNA-N) or prefusion-stabilized S protein with two proline mutations (mRNA-S-2P) were synthesized by in vitro transcription using T7 RNA polymerase (MegaScript, Thermo Fisher Scientific) on linearized plasmid templates, as previously reported (26 (link)). Uridine triphosphate was replaced with one-methylpseudouridine (m1Ψ)-5′-triphosphate (TriLink, catalog no. N-1081) for producing nucleoside-modified mRNAs. Polyadenylated tails were added to the end of modified mRNAs for optimized protein expression. In vitro transcribed mRNAs were capped using ScriptCap m7G capping system and ScriptCap 2′-O-methyltransferase kit (ScriptCap, CellScript) (26 (link)), followed by purification using the cellulose purification method as previously described (27 (link)). Purified mRNAs were analyzed by agarose gel electrophoresis and were kept frozen at −20°C. The mRNAs were formulated into LNPs using an ethanolic lipid mixture of ionizable cationic lipid and an aqueous buffer system, as previously reported (28 (link), 57 (link)). Formulated mRNA-LNPs were prepared according to RNA concentrations (1 μg/μl) and were stored at −80°C for animal immunizations.