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.

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.

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

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. This nAb produced in mammalian cells, delivered in a single intramuscular administration during a Phase I clinical study, was shown to (i) be safe and effectively protect against major variants of concern, and (ii) have some neutralizing activity against the recently emerged omicron variant in a cytopathic-effect-based microneutralization assay (100% inhibitory concentration, IC100 of 15 µg/mL). The second antibody, mAb675, previously isolated from a vaccinated individual, showed an intermediate neutralization activity against SARS-CoV-2 variants. Different accumulation levels of mAbJ08-MUT and mAb675 were observed after transient agroinfiltration in Nicotiana benthamiana plants knocked-out for xylosil and fucosil transferases, leading to yields of ~35 and 150 mg/kg of fresh leaf mass, respectively. After purification, as a result of the proteolytic events affecting the hinge-CH2 region, a higher degradation of mAb675 was observed, compared to mAbJ08-MUT (~18% vs. ~1%, respectively). Both nAbs showed a human-like glycosylation profile, and were able to specifically bind to RBD and compete with angiotensin-converting enzyme 2 binding in vitro. SARS-CoV-2 neutralization assay against the original virus isolated in Wuhan demonstrated the high neutralization potency of the plant-produced mAbJ08-MUT, with levels (IC100 < 17 ng/mL) comparable to those of the cognate antibody produced in a Chinese hamster ovary cell line; conversely, mAb675 exhibited a medium neutralization potency (IC100 ~ 200 ng/mL). All these data confirm that plant expression platforms may represent a convenient and rapid production system of potent nAbs to be used both in therapy and diagnostics in pandemic emergencies.

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. Therefore, the development of novel vaccination strategies allowing the differentiation of infected from vaccinated animals (DIVA) is a priority. Recently, commercial vectored and experimental subunit vaccines based on VP2 have been proved effective in protecting from clinical disease and posed the basis for the development of novel DIVA strategies. In this study, an engineered version of the VP3 protein of IBDV (His-VP3) was produced in plants, successfully purified from Nicotiana benthamiana leaves, and used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-VP3 antibodies. The His-VP3 ELISA was validated with a panel of 180 reference sera and demonstrated to have 100% sensitivity (95% CI: 94.7-100.0) and 94.17% specificity (95% CI: 88.4-97.6). To evaluate the application of His-VP3 ELISA as a DIVA test, the novel assay was used to monitor, in combination with a commercial kit, detecting anti-VP2 antibodies, the immune response of chickens previously immunized with an inactivated IBDV vaccine, a recombinant Turkey herpes virus carrying the VP2 of IBDV (HVT-ND-IBD) or with plant-produced VP2 particles. The combined tests correctly identified the immune status of the vaccinated specific pathogen free white-leghorn chickens. Moreover, the His-VP3 ELISA correctly detected MDA against VP3 in commercial broiler chicks and showed that antibody titers fade with time, consistent with the natural decrease of maternally derived immunity. Finally, the novel assay, in combination with a VP2-specific ELISA, demonstrated its potential application as a DIVA test in chickens inoculated with VP2-based vaccines, being able to detect the seroconversion after challenge with a very virulent IBDV strain.

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.

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. The aim of this study was to express an engineered version of the VP2 protein (His-pVP2) to verify its ability to self-assemble into virus-like particles in plants. The recombinant VP2 was transiently expressed by agroinfiltration in Nicotiana benthamiana and transmission electron microscopy of sucrose density gradient fractions revealed the presence of a mixed population of differently shaped particles ranging from spherical capsids, with a diameter between ~25 and ~70 nm, to tubular structures, with variable length (from 100 to 400 nm). The recombinant VP2-based particles when used for the intramuscular immunization of specific-pathogen-free chicks resulted able to induce the production of anti-IBDV specific antibodies at titers comparable to those induced by a commercial vaccine. Moreover, all the immunized birds survived to the challenge with a Moroccan very virulent IBDV strain with no major histomorphological alterations of the Bursa of Fabricius, similarly to what obtained with the commercial inactivated vaccine.

Production and Functional Characterization of a Recombinant Predicted Pore-Forming Protein (TVSAPLIP12) of Trichomonas vaginalis in Nicotiana benthamiana 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, Entamoeba histolytica, and Naegleria fowleri display a range of pore-forming toxins belonging to the Saposin-Like Proteins (Saplip) family: Amoebapores and Naegleriapores. Following the genome sequencing of Trichomonas vaginalis, 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. We provide evidence that TvSaplip12 gene expression is potently upregulated upon T. vaginalis contact with target cells. We cloned and expressed recombinant TvSaplip12 in planta and we demonstrate haemolytic, cytotoxic, and bactericidal activities of rTvSaplip12 in vitro. Also, evidence for TvSaplip subcellular discrete distribution in cytoplasmic granules is presented. Altogether, our results highlight the importance of TvSaplip in T. vaginalis pathogenesis, depicting its involvement in the cytolytic and bactericidal activities during the infection process, leading to predation on host cells and resident vaginal microbiota for essential nutrients acquisition. This hence suggests a potential key role for TvSaplip12 in T. vaginalis pathogenesis as a candidate Trichopore.

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.

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.

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. Nanoparticles (NPs), functionalized with BBB crossing and tumor homing (TH) peptides, are emerging as smart theranostic systems. However, there is still poor knowledge concerning the molecular structure and dynamical properties of these peptides, essential requirements for a suitable functionalization of the delivery systems themselves. METHODS: In this work, by means of molecular dynamics (MD) simulations, we have extensively characterized the structural and dynamical behavior of several peptides, known to be endowed of BBB crossing and TH properties. RESULTS: The simulations point out that, on the basis of their conformational dynamics, the peptides can be classified in two main groups: 1) peptides assuming a specific structural conformation, a feature that could be important for interacting with the molecular target but that may limit their use as functionalizing molecules and 2) highly flexible peptides whose interaction with the target may be independent of a particular structural conformation and that may represent good candidates for the functionalization of theranostic NP-based platforms. DISCUSSION: Such findings may be useful for the de novo designing of NP-based delivery systems.

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. We report here the results of an interdisciplinary study aimed to define for the first time the biodistribution of unlabelled, unpegylated, underivatized TBSV and PVX by proved detecting antibodies. These data add new insights on the in vivo behaviour of these nano-objects and demonstrate that the pVNPs under scrutiny are each intrinsically endowed with peculiar properties foreshadowing different applications in molecular medicine.

Plant-Derived Chimeric Virus Particles for the Diagnosis of Primary Sjögren Syndrome.

Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX), chimeric virus particles, and Cowpea mosaic virus, empty virus-like particles to display a linear peptide (lipo) derived from human lipocalin, which is immunodominant in Sjögren's syndrome (SjS) and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.

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. In vitro haemolysis assay was used to test the cytotoxic effects, which could arise by pVNPs interaction with cellular membranes, while early embryo assay was used to evaluate toxicity and teratogenicity in vivo. Data indicates that these structurally robust particles, still able to infect plants after incubation in serum up to 24h, have neither toxic nor teratogenic effects in vitro and in vivo. This work represents the first safety-focused characterization of pVNPs in view of their possible use as drug delivery carriers.

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.

The use of plants for the production of therapeutic human peptides.

Peptides have unique properties that make them useful drug candidates for diverse indications, including allergy, infectious disease and cancer. Some peptides are intrinsically bioactive, while others can be used to induce precise immune responses by defining a minimal immunogenic region. The limitations of peptides, such as metabolic instability, short half-life and low immunogenicity, can be addressed by strategies such as multimerization or fusion to carriers, to improve their pharmacological properties. The remaining major drawback is the cost of production using conventional chemical synthesis, which is also difficult to scale-up. Over the last 15 years, plants have been shown to produce bioactive and immunogenic peptides economically and with the potential for large-scale synthesis. The production of peptides in plants is usually achieved by the genetic fusion of the corresponding nucleotide sequence to that of a carrier protein, followed by stable nuclear or plastid transformation or transient expression using bacterial or viral vectors. Chimeric plant viruses or virus-like particles can also be used to display peptide antigens, allowing the production of polyvalent vaccine candidates. Here we review progress in the field of plant-derived peptides over the last 5 years, addressing new challenges for diverse pathologies.

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. These experiments showed that coat protein-peptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time.

Plant-produced potato virus X chimeric particles displaying an influenza virus-derived peptide activate specific CD8+ T cells in mice.

Plant viruses can be genetically modified to produce chimeric virus particles (CVPs) carrying heterologous peptides. The efficacy of plant-produced CVPs in inducing antibody responses specific to the displayed peptide has been extensively demonstrated. To determine if plants can be used to produce CVPs able to activate peptide-specific major histocompatibility complex (MHC) class I-restricted CD8+ T cells, potato virus X (PVX) has been engineered to display the H-2D(b)-restricted epitope ASNENMETM of influenza A virus nucleoprotein (NP). Engineering criteria were devised to comply not only with plant virus genetic stability and infectivity but also with antigen processing rules. The immunological properties of different doses of endotoxin-free preparations of CVPs or unmodified PVX have been evaluated by s.c. immunizing C57BL/6J mice and testing at different time intervals splenocyte responses by interferon gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay. These experiments demonstrated that CVPs activate ASNENMTEM-specific CD8+ T cells. Remarkably, the best response was achieved without adjuvant co-delivery. These results represent the proof of concept that well-designed plant virus carriers of epitopes produced in plant can reasonably be used into peptide vaccine formulations aimed to activate cell-mediated immune responses.

Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells.

BACKGROUND: Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa) and a truncated form (p25, 25 kDa). Here we report the expression and purification of HIV Nef from transgenic tobacco. RESULTS: We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure. CONCLUSION: We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

Peptide display on Potato virus X: molecular features of the coat protein-fused peptide affecting cell-to-cell and phloem movement of chimeric virus particles.

The potexvirus Potato virus X (PVX) can be modified genetically to generate chimeric virus particles (CVPs) carrying heterologous peptides fused to coat protein (CP) subunits. A spontaneous PVX mutant expressing a truncated, but functional, form of the CP has been isolated. With the aim of exploiting this virus to display peptides useful for vaccine formulations, two novel viral expression vectors based on pPVX201 (bearing the wild-type PVX genome) were constructed encoding the truncated CP. Both vectors were able to produce infectious virus particles in planta and were used to insert a panel of sequences encoding peptides of biopharmaceutical interest as N-terminal fusions to the truncated cp gene. The analysis of infection progression induced by the different constructs enabled identification of two important structural features of the fused peptide, namely tryptophan content and isoelectric point, critically affecting the formation of PVX CVPs and virus movement through the plant. These results are discussed in view of the rising interest in engineered plant viruses for development of peptide-based epitope vaccines.

In planta production of two peptides of the Classical Swine Fever Virus (CSFV) E2 glycoprotein fused to the coat protein of potato virus X.

BACKGROUND: Classical Swine Fever (CSFV) is one of the most important viral infectious diseases affecting wild boars and domestic pigs. The etiological agent of the disease is the CSF virus, a single stranded RNA virus belonging to the family Flaviviridae. All preventive measures in domestic pigs have been focused in interrupting the chain of infection and in avoiding the spread of CSFV within wild boars as well as interrupting transmission from wild boars to domestic pigs. The use of plant based vaccine against CSFV would be advantageous as plant organs can be distributed without the need of particular treatments such as refrigeration and therefore large areas, populated by wild animals, could be easily covered. RESULTS: We report the in planta production of peptides of the classical swine fever (CSF) E2 glycoprotein fused to the coat protein of potato virus X. RT-PCR studies demonstrated that the peptide encoding sequences are correctly retained in the PVX construct after three sequential passage in Nicotiana benthamiana plants. Sequence analysis of RT-PCR products confirmed that the epitope coding sequences are replicated with high fidelity during PVX infection. Partially purified virions were able to induce an immune response in rabbits. CONCLUSION: Previous reports have demonstrated that E2 synthetic peptides can efficiently induce an immunoprotective response in immunogenized animals. In this work we have showed that E2 peptides can be expressed in planta by using a modified PVX vector. These results are particularly promising for designing strategies for disease containment in areas inhabited by wild boars.