Suitability of potyviral recombinant virus-like particles bearing a complete food allergen for immunotherapy vaccines

Abstract

Virus-like particles (VLPs) have been gaining attention as potential platforms for delivery of cargos in nanomedicine. Although animal viruses are largely selected due to their immunostimulatory capacities, VLPs from plant viruses constitute a promising alternative to be considered. VLPs derived from Turnip mosaic virus (TuMV) have proven to present a tridimensional structure suited to display molecules of interest on their surface, making them interesting tools to be studied in theragnostic strategies. Here, we study their potential in the treatment of food allergy by genetically coupling TuMV-derived VLPs to Pru p 3, one of the most dominant allergens in Mediterranean climates. VLPs-Pru p 3 were generated by cloning a synthetic gene encoding the TuMV coat protein and Pru p 3, separated by a linker, into a transient high-expression vector, followed by agroinfiltration in Nicotiana benthamiana plants. The generated fusion protein self-assembled in planta to form the VLPs, which were purified by exclusion chromatography. Their elongated morphology was confirmed by electron microscopy and their size (~400 nm), and monodispersity was confirmed by dynamic light scattering. Initial in vitro characterization confirmed that they were able to induce proliferation of human immune cells. This proliferative capability was enhanced when coupled with the natural lipid ligand of Pru p 3. The resultant formulation, called VLP-Complex, was also able to be transported by intestinal epithelial cells, without affecting the monolayer integrity. In light of all these results, VLP-Complex was furtherly tested in a mouse model of food allergy. Sublingual administration of VLP-Complex could effectively reduce some serological markers associated with allergic responses in mice, such as anti-Pru p 3 sIgE and sIgG2a. Noteworthy, no associated macroscopic, nephritic, or hepatic toxicity was detected, as assessed by weight, blood urea nitrogen (BUN) and galectin-3 analyses, respectively. Our results highlight the standardized production of allergen-coated TuMV-VLPs in N. benthamiana plants. The resulting formula exerts notable immunomodulatory properties without the need for potentially hazardous adjuvants. Accordingly, no detectable toxicity associated to their administration was detected. As a result, we propose them as good candidates to be furtherly studied in the treatment of immune-based pathologies.

Isopeptide Bonding In Planta Allows Functionalization of Elongated Flexuous Proteinaceous Viral Nanoparticles, including Non-Viable Constructs by Other Means

“…Immunogold electron microscopy showed that only ST-CP + SC-VIP co-agroinfiltration was marked with colloidal gold ( Figure 7 ). Unlike constructs with GFP, TuMV VLPs-VIP were shorter than free virions, something already observed in a construct functionalized with the allergen Pru p 3 [ 11 ].…”

“…Surface display of GFP is not the only example of a successful functionalization of TuMV eVLPs through genetic fusion. Recently, our group developed another eVLP functionalized with the complete allergen Pru p 3 to test a potential novel food allergy treatment in allergic mice [ 11 ]. In this case, Pru p 3 was physically separated from the CP through a flexible linker to avoid undesired interactions as well.…”

“…Among these, tobacco mosaic virus (TMV) and potato virus X (PVX) have been widely used [ 4 ]. In our own work, we have a long track record in the development of VNPs from turnip mosaic virus (TuMV) for multiple nanobiotechnological applications [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ]. TuMV is a potyvirus, flexuous elongated viruses, whose long virions (750 nm long and 13.5 nm wide) are made of over 2000 CP molecules, and of which eVLPs can be formed in planta [ 16 ].…”

“…This feature makes it possible to potentially bind more molecules of interest per VNP. So far, TuMV eVLPs were functionalized via genetic fusion, where the protein of interest was expressed in planta together with the CP, with or without a flexible linker in between [ 11 ]. Genetic fusion allows the functionalization of all CP available in the eVLPs, which, in the case of TuMV, is approximately 2000 copies of the protein of interest per eVLP.…”

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

“…Most of these strategies relied on the production of VLPs by assembling chimeric CPs, generally presenting short antigenic peptides (Yuste‐Calvo et al ., 2019 ). In a recent work, the correct expression of turnip mosaic virus (TuMV; genus Potyvirus ) VLPs presenting a food allergen peptide of 90 residues was achieved by the addition of a linker between the fused proteins (Pazos‐Castro et al ., 2022 ). 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 ).…”