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.

High-Yield Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope and Receptor Binding Domain of SARS-CoV-2 in Plants Using Viral Vectors.

Capsid protein of Hepatitis E virus (HEV) is capable of self-assembly into virus-like particles (VLPs) when expressed in Nicotiana benthamiana plants. Such VLPs could be used as carriers of antigens for vaccine development. In this study, we obtained VLPs based on truncated coat protein of HEV bearing the M2e peptide of Influenza A virus or receptor-binding domain of SARS-CoV-2 spike glycoprotein (RBD). We optimized the immunogenic epitopes' presentation by inserting them into the protruding domain of HEV ORF2 at position Tyr485. The fusion proteins were expressed in Nicotiana benthamiana plants using self-replicating potato virus X (PVX)-based vector. The fusion protein HEV/M2, targeted to the cytosol, was expressed at the level of about 300-400 µg per gram of fresh leaf tissue and appeared to be soluble. The fusion protein was purified using metal affinity chromatography under native conditions with the final yield about 200 µg per gram of fresh leaf tissue. The fusion protein HEV/RBD, targeted to the endoplasmic reticulum, was expressed at about 80-100 µg per gram of fresh leaf tissue; the yield after purification was up to 20 µg per gram of fresh leaf tissue. The recombinant proteins HEV/M2 and HEV/RBD formed nanosized virus-like particles that could be recognized by antibodies against inserted epitopes. The ELISA assay showed that antibodies of COVID-19 patients can bind plant-produced HEV/RBD virus-like particles. This study shows that HEV capsid protein is a promising carrier for presentation of foreign antigen.