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.

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.

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.

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.

Production of two SARS-CoV-2 neutralizing antibodies with different potencies in .

Monoclonal antibodies are considered to be highly effective therapeutic tools for the treatment of mild to moderate COVID-19 patients. In the present work, we describe the production of two SARS-CoV-2 human IgG1 monoclonal antibodies recognizing the spike protein receptor-binding domain (RBD) and endowed with neutralizing activity (nAbs) in plants. The first one, mAbJ08-MUT, was previously isolated from a COVID-19 convalescent patient and Fc-engineered to prolong the half-life and reduce the risk of antibody-dependent enhancement.

Development of a Novel Assay Based on Plant-Produced Infectious Bursal Disease Virus VP3 for the Differentiation of Infected From Vaccinated Animals.

Infectious bursal disease virus is the causative agent of Gumboro disease, a severe infection that affects young chickens and is associated with lymphoid depletion in the bursa of Fabricius. Traditional containment strategies are based either on inactivated or live-attenuated vaccines. These approaches have several limitations such as residual virulence or low efficacy in the presence of maternally derived antibodies (MDA) but, most importantly, the impossibility to detect the occurrence of natural infections in vaccinated flocks.

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.

The expression in plants of an engineered VP2 protein of Infectious Bursal Disease Virus induces formation of structurally heterogeneous particles that protect from a very virulent viral strain.

Infectious Bursal Disease Virus (IBDV), the etiological agent of Gumboro disease, causes mortality and immunosuppression in chickens and major losses to poultry industry worldwide. The IBDV major capsid protein VP2 is considered the best candidate for the production of novel subunit vaccines. This structural protein contains the major conformational epitopes responsible for the induction of IBDV neutralizing antibodies in chickens and has been demonstrated able to form supramolecular structures in yeast and insect cells.

Plant Molecular Farming as a Strategy Against COVID-19 - The Italian Perspective.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 37,000 people in Italy and has caused widespread socioeconomic disruption. Urgent measures are needed to contain and control the virus, particularly diagnostic kits for detection and surveillance, therapeutics to reduce mortality among the severely affected, and vaccines to protect the remaining population. Here we discuss the potential role of plant molecular farming in the rapid and scalable supply of protein antigens as reagents and vaccine candidates, antibodies for virus detection and passive immunotherapy, other therapeutic proteins, and virus-like particles as novel vaccine platforms.

Production and Functional Characterization of a Recombinant Predicted Pore-Forming Protein (TVSAPLIP12) of in Plants.

Pore-forming proteins (PFPs) are a group of functionally versatile molecules distributed in all domains of life, and several microbial pathogens notably use members of this class of proteins as cytotoxic effectors. Among pathogenic protists, , and display a range of pore-forming toxins belonging to the Saposin-Like Proteins (Saplip) family: Amoebapores and Naegleriapores. Following the genome sequencing of , we identified a gene family of 12 predicted saposin-like proteins (TvSaplips): this work focuses on investigating the potential role of TvSaplips as cytopathogenetic effectors.

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.

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.

Characterization Of Blood-Brain Barrier Crossing And Tumor Homing Peptides By Molecular Dynamics Simulations.

Introduction: The new frontier of tumor diagnosis and treatment relies on the development of delivery strategies capable of allowing the specific targeting of the diagnostic agents/chemotherapeutics, avoiding side effects. In the case of brain tumors, achieving this goal is made more difficult by the presence of the blood-brain barrier (BBB). Peptides have been revealed as excellent candidates for both BBB crossing and specific cancer homing.

Complete genome analysis and time scale evolution of very virulent infectious bursal disease viruses isolated from recent outbreaks in Morocco.

Emerging of very virulent infectious bursal disease virus (vvIBDV) genotype in poultry flocks in Morocco were characterized. VP2 sequence analysis showed that the strains of Moroccan vvIBDV genotypes clustered separately from classic and vaccine strains reference of IBDV. The full-length genome of four Moroccan vvIBDV strains was determined, in order to get a more exhaustive molecular characterization allowing to conduct the evolution time scale and speculations on their origin.

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.

Molecular characterization and phylogenetic analysis of very virulent infectious bursal disease virus circulating in Morocco during 2016-2017.

Very virulent infectious bursal disease virus (vvIBDV), the cause of significant economic losses in many poultry-producing areas, has been present in Morocco since 1991. In spite of the introduction of vaccination, disease outbreaks are frequently observed. To ascertain if vaccines failure may be due to the emergence of new strains, the aim of this study was to perform for the first time the molecular characterization of vvIBDV strains circulating in Morocco by focusing on the hypervariable region (HVR) of the VP2 protein, which is frequently used for molecular epidemiology and phylogenetic studies.

A biodistribution study of two differently shaped plant virus nanoparticles reveals new peculiar traits.

Self-assembling plant virus nanoparticles (pVNPs) have started to be explored as nanometre-sized objects for biomedical applications, such as vaccine or drug delivery and imaging. Plant VNPs may be ideal tools in terms of biocompatibility and biodegradability endowed with a wide diversity of symmetries and dimensions, easy chemical/biological engineering, and rapid production in plants. Recently, we defined that icosahedral Tomato bushy stunt virus (TBSV) and filamentous Potato virus X (PVX) are neither toxic nor teratogenic.

The Two-Faced Potato Virus X: From Plant Pathogen to Smart Nanoparticle.

Potato virus X (PVX) is a single-stranded RNA plant virus, historically investigated in light of the detrimental effects on potato, the world's fourth most important food commodity. The study of the interactions with cells, and more generally with the plant, both locally and systemically, significantly contributed to unveil the mechanisms underlying gene silencing, fundamental not only in plant virology but also in the study of gene expression regulation. Unraveling the molecular events of PVX infection paved the way for the development of different viral expression vectors and consequential applications in functional genomics and in the biosynthesis of heterologous proteins in plants.

In vitro and in vivo toxicity evaluation of plant virus nanocarriers.

The use of biological self-assembling materials, plant virus nanoparticles in particular, appears very intriguing as it allows a great choice of symmetries and dimensions, easy chemical and biological engineering of both surface and/or internal cavity as well as safe and rapid production in plants. In this perspective, we present an initial evaluation of the safety profile of two structurally different plant viruses produced in Nicotiana benthamiana L. plants: the filamentous Potato virus X and the icosahedral Tomato bushy stunt virus.

Heat-shock proteins as dendritic cell-targeting vaccines--getting warmer.

Heat-shock proteins (hsp) provide a natural link between innate and adaptive immune responses by combining the ideal properties of antigen carriage (chaperoning), targeting and activation of antigen-presenting cells (APC), including dendritic cells (DC). Targeting is achieved through binding of hsp to distinct cell surface receptors and is followed by antigen internalization, processing and presentation. An improved understanding of the interaction of hsp with DC has driven the development of numerous hsp-containing vaccines, designed to deliver antigens directly to DC.

Heat-shock protein 70 from plant biofactories of recombinant antigens activate multiepitope-targeted immune responses.

Although a physiological role of heat-shock proteins (HSP) in antigen presentation and immune response activation has not been directly demonstrated, their use as vaccine components is under clinical trial. We have previously demonstrated that the structure of plant-derived HSP70 (pHSP70) can be superimposed to the mammalian homologue and similarly to the mammalian counterpart, pHSP70-polypeptide complexes can activate the immune system. It is here shown that pHSP70 purified from plant tissues transiently expressing the influenza virus nucleoprotein are able to induce both the activation of major histocompatibility complex class I-restricted polyclonal T-cell responses and antibody production in mice of different haplotypes without the need of adjuvant co-delivery.

LC-MS/MS methods for absolute quantification and identification of proteins associated with chimeric plant oil bodies.

Oil bodies (OBs) are plant cell organelles that consist of a lipid core surrounded by a phospholipid monolayer embedded with specialized proteins such as oleosins. Recombinant proteins expressed in plants can be targeted to OBs as fusions with oleosin. This expression strategy is attractive because OBs are easily enriched and purified from other cellular components, based on their unique physicochemical properties.

Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants.

Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques.

Transient expression systems for plant-derived biopharmaceuticals.

In the molecular farming area, transient expression approaches for pharmaceutical proteins production, mainly recombinant monoclonal antibodies and vaccines, were developed almost two decades ago and, to date, these systems basically depend on Agrobacterium-mediated delivery and virus expression machinery. We survey here the current state-of-the-art of this research field. Several vectors have been designed on the basis of DNA- and RNA-based plant virus genomes and viral vectors are used both as single- and multicomponent expression systems in different combinations depending on the protein of interest.