Rapid Transient Expression of Receptor-Binding Domain of SARS-CoV-2 and the Conserved M2e Peptide of Influenza A Virus Linked to Flagellin in Nicotiana benthamiana Plants Using Self-Replicating Viral Vector

The development of recombinant vaccines against SARS-CoV-2 and influenza A is an important task. The combination of the conserved influenza A antigen, the extracellular domain of the transmembrane protein M2 (M2e), and the receptor-binding domain of the SARS-CoV-2 spike glycoprotein (RBD) provides the opportunity to develop a bivalent vaccine against these infections. The fusion of antigens with bacterial flagellin, the ligand for Toll-like receptor 5 and potent mucosal adjuvant, may increase the immunogenicity of the candidate vaccines and enable intranasal immunization. In this study, we report the transient expression of RBD alone, RBD coupled with four copies of M2e, and fusions of RBD and RBD-4M2e with flagellin in Nicotiana benthamiana plants using the self-replicating potato virus X-based vector pEff. The yields of purified recombinant proteins per gram of fresh leaf tissue were about 20 µg for RBD, 50–60 µg for RBD-4M2e and the fusion of RBD with flagellin, and about 90 µg for RBD-4M2e fused to flagellin. Targeting to the endoplasmic reticulum enabled the production of glycosylated recombinant proteins comprising RBD. Our results show that plant-produced RBD and RBD-4M2e could be further used for the development of subunit vaccines against COVID-19 and a bivalent vaccine against COVID-19 and influenza A, while flagellin fusions could be used for the development of intranasal vaccines.