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

Abstract

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.

Plant-Produced Nanoparticles Based on Artificial Self-Assembling Peptide Bearing the Influenza M2e Epitope

“…However, the yield of 19S_SAP_Sp_4M2eh was about ten times lower than the previously reported maximum level for the pEff expression system [42]. Some M2e fusions were also expressed in plants using the pEff system at higher levels, including up to 1 mg/g for flagellin-M2e [57], and 300-400 µg/g for M2e fused to the Hepatitis E capsid protein [58]. Nevertheless, the expression level of 19S_SAP_Sp_4M2eh was several times higher than those achieved using a similar expression system for the production of M2e-bearing VLPs based on the core antigen of the hepatitis B virus in plants [59].…”

The Re-Emergence of Hepatitis E Virus in Europe and Vaccine Development

“…Chimeric vaccines are composed of the immunogenic protein from two or more viruses, and they induce an immunogenic response against more than one disease. In recent years, HEV has been a part of dual-vaccination efforts against foot-and-mouth disease (FMDV) [ 261 ], hepatitis B virus (HBV) [ 262 ], porcine circovirus 2 (PCV2) [ 263 ], influenza virus [ 215 , 217 , 264 ], hepatitis A virus (HAV) [ 265 , 266 ], norovirus (NoV) and astrovirus (AstV) [ 267 ], and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [ 268 ]. The formation of chimeric proteins can vary based on the chosen host cell expression system, which is also the case in the formation of non-chimeric proteins.…”

Challenges and Opportunities in the Process Development of Chimeric Vaccines

“…Recently, the truncated coat protein of the hepatitis E virus (HEV) was used as a carrier to generate M2e peptide-based influenza A and receptor binding domain (RBD)-based SARS-CoV-2 chimeric VLPs in Nicotiana benthamiana . The fusion constructs HEV/M2e and HEV/RBD were successfully expressed and purified with final yields of 200 μg/g and 20 μg/g fresh leaf tissue, respectively [ 141 ].…”