Production of Potyvirus-Derived Nanoparticles Decorated with a Nanobody in Biofactory Plants

Abstract

Viral nanoparticles (VNPs) have recently attracted attention for their use as building blocks for novel materials to support a range of functions of potential interest in nanotechnology and medicine. Viral capsids are ideal for presenting small epitopes by inserting them at an appropriate site on the selected coat protein (CP). VNPs presenting antibodies on their surfaces are considered highly promising tools for therapeutic and diagnostic purposes. Due to their size, nanobodies are an interesting alternative to classic antibodies for surface presentation. Nanobodies are the variable domains of heavy-chain (VHH) antibodies from animals belonging to the family Camelidae, which have several properties that make them attractive therapeutic molecules, such as their small size, simple structure, and high affinity and specificity. In this work, we have produced genetically encoded VNPs derived from two different potyviruses—the largest group of RNA viruses that infect plants—decorated with nanobodies. We have created a VNP derived from zucchini yellow mosaic virus (ZYMV) decorated with a nanobody against the green fluorescent protein (GFP) in zucchini (Cucurbita pepo) plants. As reported for other viruses, the expression of ZYMV-derived VNPs decorated with this nanobody was only made possible by including a picornavirus 2A splicing peptide between the fused proteins, which resulted in a mixed population of unmodified and decorated CPs. We have also produced tobacco etch virus (TEV)-derived VNPs in Nicotiana benthamiana plants decorated with the same nanobody against GFP. Strikingly, in this case, VNPs could be assembled by direct fusion of the nanobody to the viral CP with no 2A splicing involved, likely resulting in fully decorated VNPs. For both expression systems, correct assembly and purification of the recombinant VNPs was confirmed by transmission electron microscope; the functionality of the CP-fused nanobody was assessed by western blot and binding assays. In sum, here we report the production of genetically encoded plant-derived VNPs decorated with a nanobody. This system may be an attractive alternative for the sustainable production in plants of nanobody-containing nanomaterials for diagnostic and therapeutic purposes.

A Multifunctionalized Potyvirus-Derived Nanoparticle That Targets and Internalizes into Cancer Cells

“…Apart from cancer research, a nanoplatform based on VNPs with high affinity for IgGs has other multiple applications in the field of antigen sensing. Two other potyviruses (zucchini yellow mosaic virus and tobacco etch virus) functionalized with nanobodies have been recently developed through genome engineering, revealing the attractiveness of this group of plant viruses for antibody presentation [33]. Also, our VLPs-Z domain go a step further as they can readily bind other IgGs of interest and they can be double-functionlalized with other compounds through chemical conjugation, giving them a huge versatility as a novel nanoplatform.…”

A Multifunctionalized Potyvirus-Derived Nanoparticle That Targets and Internalizes into Cancer Cells

“…Apart from cancer research, a nanoplatform based on VNPs with a high affinity for IgGs has multiple applications in the field of antigen sensing. Two other potyviruses (zucchini yellow mosaic virus and tobacco etch virus) functionalized with nanobodies have recently been developed through genome engineering, revealing the attractiveness of this group of plant viruses for antibody presentation [48]. In addition, our VLPs-Z domain constructs go a step further as they can readily bind other IgGs of interest and can be double-functionalized with other compounds through chemical conjugation, giving them great versatility as a novel nanoplatform.…”

Plant virus‐derived nanoparticles decorated with genetically encoded SARS‐CoV‐2 nanobodies display enhanced neutralizing activity

“…Regarding genetically encoding VNPs, zucchini yellow mosaic virus (ZYMV; Potyvirus ), plum pox virus (PPV; Potyvirus ) and TuMV were developed to express short peptides at the N‐terminal end of CP (Martínez‐Turiño and García, 2020 ). TEV VNPs, with a genetically encoded nanobody against GFP, have also been recently described (Martí et al ., 2022 ).…”