Production of Plant Virus-Derived Hybrid Nanoparticles Decorated with Different Nanobodies

Viral nanoparticles (VNPs) are self-assembled nanometric complexes whose size and shape are similar to those of the virus from which they are derived. VNPs are arousing great attention due to potential biotechnological applications in fields like nanomedicine and nanotechnology because they allow the presentation of polypeptides of choice linked to the virus structural proteins. Starting from tobacco etch virus (TEV), a plant plus-strand RNA virus that belongs to the genus Potyvirus (family Potyviridae), here we describe the development of recombinant hybrid VNPs in Nicotiana benthamiana plants able of exposing simultaneously different proteins on their surface. This system is based on the synergic coinfection of TEV and potato virus X (PVX; Potexvirus), in which PVX provides a second TEV CP in trans allowing a mixed assembly. We first generated genetically modified hybrid VNPs simultaneously displaying green and red fluorescent proteins on their surface. A population of decorated and nondecorated CPs resulting from the insertion of the picornavirus F2A ribosomal escape peptide was required for viral particle assembly. Correct assembly of the recombinant mosaic VNPs presenting the exogenous peptides was successfully observed by immunoelectron microscopy. We next achieved the production of hybrid VNPs expressing a nanobody against SARS-CoV-2 and a fluorescent reporter protein, whose functionality was demonstrated by ELISA and dot-blot assay. Finally, we engineered the production of hybrid multivalent VNPs carrying two different nanobodies against distinct epitopes of the same SARS-CoV-2 antigenic protein, emulating a nanobody cocktail. These plant-produced recombinant mosaic VNPs, which are filamentous and flexuous in shape, presenting two different fused proteins on the surface, represent a molecular tool with several potential applications in biotechnology.