Peptide-Functionalized and Drug-Loaded Tomato Bushy Stunt Virus Nanoparticles Counteract Tumor Growth in a Mouse Model of Shh-Dependent Medulloblastoma.

Sonic hedgehog medulloblastoma (SHH-MB) accounts for 25-30% of all MBs, and conventional therapy results in severe long-term side effects. New targeted therapeutic approaches are urgently needed, drawing also on the fields of nanoparticles (NPs). Among these, plant viruses are very promising, and we previously demonstrated that tomato bushy stunt virus (TBSV), functionalized on the surface with CooP peptide, specifically targets MB cells. Here, we tested the hypothesis that TBSV-CooP can specifically deliver a conventional chemotherapeutic drug (i.e., doxorubicin, DOX) to MB in vivo. To this aim, a preclinical study was designed to verify, by histological and molecular methods, if multiple doses of DOX-TBSV-CooP were able to inhibit tumor progression of MB pre-neoplastic lesions, and if a single dose was able to modulate pro-apoptotic/anti-proliferative molecular signaling in full-blown MBs. Our results demonstrate that when DOX is encapsulated in TBSV-CooP, its effects on cell proliferation and cell death are similar to those obtained with a five-fold higher dose of non-encapsulated DOX, both in early and late MB stages. In conclusion, these results confirm that CooP-functionalized TBSV NPs are efficient carriers for the targeted delivery of therapeutics to brain tumors.

Atomic structure of potato virus X, the prototype of the Alphaflexiviridae family.

Potato virus X (PVX) is a positive-sense single-stranded RNA (ssRNA) filamentous plant virus belonging to the Alphaflexiviridae family, considered in recent years as a tool for nanotechnology applications. We present the cryo-electron microscopy structure of the PVX particle at a resolution of 2.2 Å. The well-defined density of the coat proteins and of the genomic RNA allowed a detailed analysis of protein-RNA interactions, including those mediated by solvent molecules. The particle is formed by repeated segments made of 8.8 coat proteins, forming a left-handed helical structure. The RNA runs in an internal crevice along the virion, packaged in 5-nucleotide repeats in which the first four bases are stacked in the classical way, while the fifth is rotated and nearly perpendicular. The resolution of the structure described here suggests a mechanism for the virion assembly and potentially provides a platform for the rational design of antiviral compounds and for the use of PVX in nanotechnology.

Tomato Bushy Stunt Virus Nanoparticles as a Platform for Drug Delivery to Shh-Dependent Medulloblastoma.

Medulloblastoma (MB) is a primary central nervous system tumor affecting mainly young children. New strategies of drug delivery are urgent to treat MB and, in particular, the SHH-dependent subtype-the most common in infants-in whom radiotherapy is precluded due to the severe neurological side effects. Plant virus nanoparticles (NPs) represent an innovative solution for this challenge. Tomato bushy stunt virus (TBSV) was functionally characterized as a carrier for drug targeted delivery to a murine model of Shh-MB. The TBSV NPs surface was genetically engineered with peptides for brain cancer cell targeting, and the modified particles were produced on a large scale using Nicotiana benthamiana plants. Tests on primary cultures of Shh-MB cells allowed us to define the most efficient peptides able to induce specific uptake of TBSV. Immunofluorescence and molecular dynamics simulations supported the hypothesis that the specific targeting of the NPs was mediated by the interaction of the peptides with their natural partners and reinforced by the presentation in association with the virus. In vitro experiments demonstrated that the delivery of Doxorubicin through the chimeric TBSV allowed reducing the dose of the chemotherapeutic agent necessary to induce a significant decrease in tumor cells viability. Moreover, the systemic administration of TBSV NPs in MB symptomatic mice, independently of sex, confirmed the ability of the virus to reach the tumor in a specific manner. A significant advantage in the recognition of the target appeared when TBSV NPs were functionalized with the CooP peptide. Overall, these results open new perspectives for the use of TBSV as a vehicle for the targeted delivery of chemotherapeutics to MB in order to reduce early and late toxicity.

Optimised production of an anti-fungal antibody in Solanaceae hairy roots to develop new formulations against Candida albicans.

BACKGROUND: Infections caused by fungi are often refractory to conventional therapies and urgently require the development of novel options, such as immunotherapy. To produce therapeutic antibodies, a plant-based expression platform is an attractive biotechnological strategy compared to mammalian cell cultures. In addition to whole plants, hairy roots (HR) cultures can be used, representing an expression system easy to build up, with indefinite growth while handled under containment conditions. RESULTS: In this study the production in HR of a recombinant antibody, proved to be a good candidate for human immunotherapy against fungal infections, is reported. Expression and secretion of this antibody, in an engineered single chain (scFvFc) format, by HR from Nicotiana benthamiana and Solanum lycopersicum have been evaluated with the aim of directly using the deriving extract or culture medium against pathogenic fungi. Although both Solanaceae HR showed good expression levels (up to 68 mg/kg), an optimization of rhizosecretion was only obtained for N. benthamiana HR. A preliminary assessment to explain this result highlighted the fact that not only the presence of proteases, but also the chemical characteristics of the growth medium, can influence antibody yield, with implications on recombinant protein production in HR. Finally, the antifungal activity of scFvFc 2G8 antibody produced in N. benthamiana HR was evaluated in Candida albicans growth inhibition assays, evidencing encouraging results. CONCLUSIONS: Production of this anti-fungal antibody in HR of N. benthamiana and S. lycopersicum elucidated factors affecting pharming in this system and allowed to obtain promising ready-to-use immunotherapeutics against C. albicans.

Current state-of-the-art in the use of plants for the production of recombinant vaccines against infectious bursal disease virus.

Infectious bursal disease is a widely spread threatening contagious viral infection of chickens that induces major damages to the Bursa of Fabricius and leads to severe immunosuppression in young birds causing significant economic losses for poultry farming. The etiological agent is the infectious bursal disease virus (IBDV), a non-enveloped virus belonging the family of Birnaviridae. At present, the treatment against the spread of this virus is represented by vaccination schedules mainly based on inactivated or live-attenuated viruses. However, these conventional vaccines present several drawbacks such as insufficient protection against very virulent strains and the impossibility to differentiate vaccinated animals from infected ones. To overcome these limitations, in the last years, several studies have explored the potentiality of recombinant subunit vaccines to provide an effective protection against IBDV infection. In this review, we will give an overview of these novel types of vaccines with special emphasis on current state-of-the-art in the use of plants as "biofactories" (plant molecular farming). In fact, plants have been thoroughly and successfully characterized as heterologous expression systems for the production of recombinant proteins for different applications showing several advantages compared with traditional expression systems (Escherichia coli, yeasts and insect cells) such as absence of animal pathogens in the production process, improved product quality and safety, reduction of manufacturing costs, and simplified scale-up.

Functional characterization of a plant-produced infectious bursal disease virus antigen fused to the constant region of avian IgY immunoglobulins.

Infectious bursal disease virus (IBDV) is the cause of an economically important highly contagious disease of poultry, and vaccines are regarded as the most beneficial interventions for its prevention. In this study, plants were used to produce a recombinant chimeric IBDV antigen for the formulation of an innovative subunit vaccine. The fusion protein (PD-FcY) was designed to combine the immunodominant projection domain (PD) of the viral structural protein VP2 with the constant region of avian IgY (FcY), which was selected to enhance antigen uptake by avian immune cells. The gene construct encoding the fusion protein was transiently expressed in Nicotiana benthamiana plants and an extraction/purification protocol was set up, allowing to reduce the contamination by undesired plant compounds/proteins. Mass spectrometry analysis of the purified protein revealed that the glycosylation pattern of the FcY portion was similar to that observed in native IgY, while in vitro assays demonstrated the ability of PD-FcY to bind to the avian immunoglobulin receptor CHIR-AB1. Preliminary immunization studies proved that PD-FcY was able to induce the production of protective anti-IBDV-VP2 antibodies in chickens. In conclusion, the proposed fusion strategy holds promises for the development of innovative low-cost subunit vaccines for the prevention of avian viral diseases.

Plant-Produced Viral Nanoparticles as a Functionalized Catalytic Support for Metabolic Engineering.

Substrate channeling could be very useful for plant metabolic engineering; hence, we propose that functionalized supramolecular self-assembly scaffolds can act as enzymatic hubs able to perform reactions in close contiguity. Virus nanoparticles (VNPs) offer an opportunity in this context, and we present a functionalization strategy to display different enzymes on the outer surface of three different VNPs produced in plants. Tomato bushy stunt virus (TBSV) and Potato virus X (PVX) plant viruses were functionalized by the genetic fusion of the E-coil peptide coding sequence to their respective coat proteins genes, while the enzyme lichenase was tagged with the K-coil peptide. Immobilized E-coil VNPs were able to interact in vitro with the plant-produced functionalized lichenase, and catalysis was demonstrated by employing a lichenase assay. To prove this concept in planta, the Hepatitis B core (HBc) virus-like particles (VLPs) were similarly functionalized by genetic fusion with the E-coil sequence, while acyl-activating enzyme 1, olivetolic acid synthase, and olivetolic acid cyclase enzymes were tagged with the K-coil. The transient co-expression of the K-coil-enzymes together with E-coil-VLPs allowed the establishment of the heterologous cannabinoid precursor biosynthetic pathway. Noteworthy, a significantly higher yield of olivetolic acid glucoside was achieved when the scaffold E-coil-VLPs were employed.

A plant derived multifunctional tool for nanobiotechnology based on Tomato bushy stunt virus.

Structure, size, physicochemical properties and production strategies make many plant viruses ideal protein based nanoscaffolds, nanocontainers and nano-building blocks expected to deliver a multitude of applications in different fields such as biomedicine, pharmaceutical chemistry, separation science, catalytic chemistry, crop pest control and biomaterials science. Functionalization of viral nanoparticles through modification by design of their external and internal surfaces is essential to fully exploit the potentiality of these objects. In the present paper we describe the development of a plant derived multifunctional tool for nanobiotechnology based on Tomato bushy stunt virus. We demonstrate the ability of this system to remarkably sustain genetic modifications and in vitro chemical derivatizations of its outer surface, which resulted in the successful display of large chimeric peptides fusions and small chemical molecules, respectively. Moreover, we have defined physicochemical conditions for viral swelling and reversible viral pore gating that we have successfully employed for foreign molecules loading and retention in the inner cavity of this plant virus nanoparticles system. Finally, a production and purification strategy from Nicotiana benthamiana plants has been addressed and optimized.

Targeting of Tomato Bushy Stunt Virus with a Genetically Fused C-End Rule Peptide.

Homing peptides are widely used to improve the delivery of drugs, imaging agents, and nanoparticles (NPs) to their target sites. Plant virus-based particles represent an emerging class of structurally diverse nanocarriers that are biocompatible, biodegradable, safe, and cost-effective. Similar to synthetic NPs, these particles can be loaded with imaging agents and/or drugs and functionalized with affinity ligands for targeted delivery. Here we report the development of a peptide-guided Tomato Bushy Stunt Virus (TBSV)-based nanocarrier platform for affinity targeting with the C-terminal C-end rule (CendR) peptide, RPARPAR (RPAR). Flow cytometry and confocal microscopy demonstrated that the TBSV-RPAR NPs bind specifically to and internalize in cells positive for the peptide receptor neuropilin-1 (NRP-1). TBSV-RPAR particles loaded with a widely used anticancer anthracycline, doxorubicin, showed selective cytotoxicity on NRP-1-expressing cells. Following systemic administration in mice, RPAR functionalization conferred TBSV particles the ability to accumulate in the lung tissue. Collectively, these studies show the feasibility of the CendR-targeted TBSV platform for the precision delivery of payloads.

Consumer Awareness and Acceptance of Biotechnological Solutions for Gluten-Free Products.

Celiac disease is an immune-mediated disorder caused by the ingestion of gluten proteins. The gluten-free diet is currently the only therapy to achieve the symptoms' remission. Biotechnological approaches are currently being explored to obtain safer and healthier food for celiacs. This article analyzes consumer awareness and acceptance of advanced biotechnologies to develop gluten-free products. An online snowball sampling questionnaire was proposed to 511 Italian participants, selected among celiac and non-celiac people, from December 2020 to January 2021, during the second wave of the COVID-19 pandemic. Overall, 64% of respondents favor food biotechnology, as long as it has benefits for health or the environment. Moreover, biotechnology perception differs according to education level and type. A total of 65% of the survey participants would taste gluten-free products obtained through a biotechnological approach, and 57% would buy them at a higher price than the current market price. Our results show a change in public opinion about the usefulness of food biotechnology and its moral acceptability compared to 20 years ago. However, the study of public opinion is very complex, dealing with individuals with social, economic, and cultural differences. Undoubtedly, the scientific dissemination of genetic biotechnologies must be more effective and usable to increase the level of citizens' awareness.