Viral Nanoparticles Research Articles

Enzymatic activity of individual bioelectrocatalytic viral nanoparticles: dependence of catalysis on the viral scaffold and its length.

The enzymatic activity of tobacco mosaic virus (TMV) nanorod particles decorated with an integrated electro-catalytic system, comprising the quinoprotein glucose-dehydrogenase (PQQ-GDH) enzyme and ferrocenylated PEG chains as redox mediators, is probed at the individual virion scale by atomic force microscopy-scanning electrochemical atomic force microscopy (AFM-SECM). A marked dependence of the catalytic activity on the particle length is observed. This finding can be explained by electron propagation along the viral backbone, resulting from electron exchange between ferrocene moieties, coupled with enzymatic catalysis. Thus, the use of a simple 1D diffusion/reaction model allows the determination of the kinetic parameters of the virus-supported enzyme. Comparative analysis of the catalytic behavior of the Fc-PEG/PQQ-GDH system assembled on two differing viral scaffolds, TMV (this work) and bacteriophage-fd (previous work), reveals two distinct kinetic effects of scaffolding: An enhancement of catalysis that does not depend on the virus type and a modulation of substrate inhibition that depends on the virus type. AFM-SECM detection of the enzymatic activity of a few tens of PQQ-GDH molecules, decorating a 40 nm-long viral domain, is also demonstrated, a record in terms of the lowest number of enzyme molecules interrogated by an electrochemical imaging technique.

Emerging Potential of Plant Virus Nanoparticles (PVNPs) in Anticancer Immunotherapies.

Cancer immunotherapies using plant virus nanoparticles (PVNPs) have achieved considerable success in preclinical studies. PVNP based nanoplatforms can be endogenous immune adjuvants and act as nanocarriers that stabilize and deliver cancer antigens and exogenous immune adjuvants. Although they do not infect mammalian cells, PVNPs are viruses and they are variably recognized by pathogen pattern recognition receptors (PRR), activate innate immune cells including antigen-presenting cells (APCs), and increase the expression of costimulatory molecules. Novel immunotherapy strategies use them as in situ vaccines (ISV) that can effectively inhibit tumor growth after intratumoral administration and generate expanded systemic antitumor immunity. PVNPs combined with other tumor immunotherapeutic options and other modalities of oncotherapy can improve both local and systemic anti-tumor immune responses. While not yet in clinical trials in humans, there is accelerating interest and research of the potential of PVNPs for ISV immune therapy for cancer. Thus, antitumor efficacy of PVNPs by themselves, or loaded with soluble toll-like receptor (TLR) agonists and/or cancer antigens, will likely enter human trials over the next few years and potentially contribute to next-generation antitumor immune-based therapies.

Purification of Tomato Bushy Stunt Virus Particles by One-Step Hydroxyapatite Column Chromatography

The main aim of this work was to develop a time-saving and cost-effective purification method of infectious plant viral nanoparticles. Virions of Tomato bushy stunt virus (TBSV), which is a member of Tombusvirus genus, were purified by one-step Bio-gel HT Hydroxyapatite (HA) column chromatography. Extracts from Nicotiana benthamiana plants infected with TBSV were directly loaded onto the HA column and eluted by 10 mM sodium phosphate buffer (pH 6.8). A specificity of virions has been confirmed by immunoblotting and electron microscopy. Homogeneity of virions was tested by SDS-PAGE, where only 41 kDa polypeptide bands referring to the capsid protein of TBSV were detected by Coomassie staining. The biological infectious activity of a purified material was demonstrated by observing TBSV-specific symptoms observed in N. benthamiana plants at 7‒10 days of post-inoculation (dpi). Moreover, purified virions were used for immunization of the BALb/c mouse to raise primary antibodies against the TBSV virus. Our results show that in low concentrations of sodium phosphate buffer total proteins extracted from infected plants adsorb to HA sorbent, while viral particles do not adsorb to the HA matrix and flow throw column due to Ca2+ ions implicated in TBSV virions’ structure. This highly effective and simple virus purification protocol can also be used for the isolation of other plant virions.

An Up-to-Date Review of Natural Nanoparticles for Cancer Management.

Cancer represents one of the leading causes of morbidity and mortality worldwide, imposing an urgent need to develop more efficient treatment alternatives. In this respect, much attention has been drawn from conventional cancer treatments to more modern approaches, such as the use of nanotechnology. Extensive research has been done for designing innovative nanoparticles able to specifically target tumor cells and ensure the controlled release of anticancer agents. To avoid the potential toxicity of synthetic materials, natural nanoparticles started to attract increasing scientific interest. In this context, this paper aims to review the most important natural nanoparticles used as active ingredients (e.g., polyphenols, polysaccharides, proteins, and sterol-like compounds) or as carriers (e.g., proteins, polysaccharides, viral nanoparticles, and exosomes) of various anticancer moieties, focusing on their recent applications in treating diverse malignancies.

Plant Virus Nanoparticles for Anti-cancer Therapy.

Plant virus nanoparticles (VNPs) are inexpensive to produce, safe, biodegradable and efficacious as treatments. The applications of r plant virus nanoparticles range from epitope carriers for vaccines to agents in cancer immunotherapy. Both VNPs and virus-like particles (VLPs) are highly immunogenic and are readily phagocytosed by antigen presenting cells (APCs), which in turn elicit antigen processing and display of pathogenic epitopes on their surfaces. Since the VLPs are composed of multiple copies of their respective capsid proteins, they present repetitive multivalent scaffolds which aid in antigen presentation. Therefore, the VLPs prove to be highly suitable platforms for delivery and presentation of antigenic epitopes, resulting in induction of more robust immune response compared to those of their soluble counterparts. Since the tumor microenvironment poses the challenge of self-antigen tolerance, VLPs are preferrable platforms for delivery and display of self-antigens as well as otherwise weakly immunogenic antigens. These properties, in addition to their diminutive size, enable the VLPs to deliver vaccines to the draining lymph nodes in addition to promoting APC interactions. Furthermore, many plant viral VLPs possess inherent adjuvant properties dispensing with the requirement of additional adjuvants to stimulate immune activity. Some of the highly immunogenic VLPs elicit innate immune activity, which in turn instigate adaptive immunity in tumor micro-environments. Plant viral VLPs are nontoxic, inherently stable, and capable of being mass-produced as well as being modified with antigens and drugs, therefore providing an attractive option for eliciting anti-tumor immunity. The following review explores the use of plant viruses as epitope carrying nanoparticles and as a novel tools in cancer immunotherapy.

Bioconjugation of Active Ingredients to Plant Viral Nanoparticles Is Enhanced by Preincubation with a Pluronic F127 Polymer Scaffold.

Proteinaceous nanoparticles can be used to deliver large payloads of active ingredients, which is advantageous in medicine and agriculture. However, the conjugation of hydrophobic ligands to hydrophilic nanocarriers such as plant viral nanoparticles (plant VNPs) can result in aggregation by reducing overall solubility. Given the benefits of hydrophilic nanocarrier platforms for targeted delivery and multivalent ligand display, coupled with the versatility of hydrophobic drugs, contrast agents, and peptides, this is an issue that must be addressed to realize their full potential. Here, we report two preincubation strategies that use a Pluronic F127 polymer scaffold to prevent the aggregation of conjugated plant VNPs: a plant VNP-polymer precoat (COAT) and an active ingredient formulation combined with a plant VNP-polymer precoat (FORMCOAT). The broad applications of these modified conjugation strategies were highlighted by testing their compatibility with three types of bioconjugation chemistry: N-hydroxysuccinimide ester-amine coupling, maleimide-thiol coupling, and copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry). The COAT and FORMCOAT strategies promoted efficient bioconjugation and prevented the aggregation that accompanies conventional bioconjugation methods, thus improving the stability, homogeneity, and translational potential of plant VNP conjugates in medicine and agriculture.

Development of a SARS-CoV-2 Vaccine Candidate Using Plant-Based Manufacturing and a Tobacco Mosaic Virus-like Nano-Particle.

Stable, effective, easy-to-manufacture vaccines are critical to stopping the COVID-19 pandemic resulting from the coronavirus SARS-CoV-2. We constructed a vaccine candidate CoV-RBD121-NP, which is comprised of the SARS-CoV-2 receptor-binding domain (RBD) of the spike glycoprotein (S) fused to a human IgG1 Fc domain (CoV-RBD121) and conjugated to a modified tobacco mosaic virus (TMV) nanoparticle. In vitro, CoV-RBD121 bound to the host virus receptor ACE2 and to the monoclonal antibody CR3022, a neutralizing antibody that blocks S binding to ACE2. The CoV-RBD121-NP vaccine candidate retained key SARS-CoV-2 spike protein epitopes, had consistent manufacturing release properties of safety, identity, and strength, and displayed stable potency when stored for 12 months at 2–8 °C or 22–28 °C. Immunogenicity studies revealed strong antibody responses in C57BL/6 mice with non-adjuvanted or adjuvanted (7909 CpG) formulations. The non-adjuvanted vaccine induced a balanced Th1/Th2 response and antibodies that recognized both the S1 domain and full S protein from SARS2-CoV-2, whereas the adjuvanted vaccine induced a Th1-biased response. Both adjuvanted and non-adjuvanted vaccines induced virus neutralizing titers as measured by three different assays. Collectively, these data showed the production of a stable candidate vaccine for COVID-19 through the association of the SARS-CoV-2 RBD with the TMV-like nanoparticle.

Three Alternative Treatment Protocols for the Efficient Inactivation of Potato Virus X.

Filamentous nanomaterials are flexible with a high aspect ratio, conferring unique mechanical, electromagnetic, and optical properties; promoting tissue penetration; and allowing the formation of hierarchical superstructures. The fabrication of synthetic nanofilaments with uniform properties is challenging, but this can be addressed by the use of filamentous plant viruses such as potato virus X (PVX), which are produced as monodisperse structures from a genetic template. To take advantage of PVX without risks to agriculture and the environment, it is necessary to inactivate the virus efficiently without disrupting its chemical and material properties. Herein, we report experiments showing that PVX can be completely inactivated by exposure to UV irradiation (0.5 J cm-2) or chemical treatment (1 mM β-propiolactone or 10 mM formalin) without interfering with the chemical addressability of lysine or cysteine residues, which are typically used as conjugation handles for virus nanoparticle functionalization.

Interaction of virus-like particles and nanoparticles with inorganic materials for biosensing: An exciting approach

In recent years, the implementation of virus-like particles (VLPs) and viral nanoparticles (VNPs) into biosensing devices have been extensively studied due to their ease of surface modification into specific bio-recognizing elements by both chemical and genetic approaches. Some authors have already reported hybrid VLPs/VNPs as promising alternatives due to their biocompatibility and tunable physicochemical properties. Furthermore, incorporating inorganic compounds into VLPs/VNPs systems for improving the optical and electrochemical biosensing performance is somehow a current approach. VNPs of bacteriophages have served as nanoscale models that offer unique structural properties such as pathogen detection. In this sense, non-infectious VLPs/VNPs and inorganic materials represent an opportunity in research, drug innovation, and sensing for further comprehension and early identification of diseases.

Tumor Microenvironment Responsive Pepper Mild Mottle Virus-Based Nanotubes for Targeted Delivery and Controlled Release of Paclitaxel.

Plant virus nanoparticles (PVNPs) have been widely used for drug delivery, antibody development and medical imaging because of their good biodegradation and biocompatibility. Particles of pepper mild mottle virus (PMMoV) are elongated and may be useful as drug carriers because their shape favours long circulation, preferential distribution and increased cellular uptake. Moreover, its effective degradation in an acidic microenvironment enables a pH-responsive release of the encapsulated drug. In this study, genetic engineering techniques were used to form rod-shaped structures of nanoparticles (PMMoV) and folated-modified PMMoV nanotubes were prepared by polyethylene glycol (PEG) to provide targeted delivery of paclitaxel (PTX). FA@PMMoV@PTX nanotubes were designed to selectively target tumor cells and to release the encapsulated PTX in response to pH. Efficient cell uptake of FA@PMMoV@PTX nanotubes was observed when incubated with tumor cells, and FA@PMMoV@PTX nanotubes had superior cytotoxicity to free PTX, as reflected by cell survival and apoptosis. This system is a strong candidate for use in developing improved strategies for targeted treatment of tumors.

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.

Gene Therapy to Modulate Alpha-Synuclein in Synucleinopathies.

The protein alpha-Synuclein (α-Syn) is a key contributor to the etiology of Parkinson’s disease (PD) with aggregation, trans-neuronal spread, and/or depletion of α-Syn being viewed as crucial events in the molecular processes that result in neurodegeneration. The exact succession of pathological occurrences that lead to neuronal death are still largely unknown and are likely to be multifactorial in nature. Despite this unknown, α-Syn dose and stability, autophagy-lysosomal dysfunction, and inflammation, amongst other cellular impairments, have all been described as participatory events in the neurodegenerative process. To that end, in this review we discuss the logical points for gene therapy to intervene in α-Syn-mediated disease and review the preclinical body of work where gene therapy has been used, or could conceptually be used, to ameliorate α-Syn induced neurotoxicity. We discuss gene therapy in the traditional sense of modulating gene expression, as well as the use of viral vectors and nanoparticles as methods to deliver other therapeutic modalities.

Plant Viral Nanoparticle Conjugated with Anti-PD-1 Peptide for Ovarian Cancer Immunotherapy.

Immunotherapy holds tremendous potential in cancer therapy, in particular, when treatment regimens are combined to achieve synergy between pathways along the cancer immunity cycle. In previous works, we demonstrated that in situ vaccination with the plant virus cowpea mosaic virus (CPMV) activates and recruits innate immune cells, therefore reprogramming the immunosuppressive tumor microenvironment toward an immune-activated state, leading to potent anti-tumor immunity in tumor mouse models and canine patients. CPMV therapy also increases the expression of checkpoint regulators on effector T cells in the tumor microenvironment, such as PD-1/PD-L1, and we demonstrated that combination with immune checkpoint therapy improves therapeutic outcomes further. In the present work, we tested the hypothesis that CPMV could be combined with anti-PD-1 peptides to replace expensive antibody therapies. Specifically, we set out to test whether a multivalent display of anti-PD-1 peptides (SNTSESF) would enhance efficacy over a combination of CPMV and soluble peptide. Efficacy of the approaches were tested using a syngeneic mouse model of intraperitoneal ovarian cancer. CPMV combination with anti-PD-1 peptides (SNTSESF) resulted in increased efficacy; however, increased potency against metastatic ovarian cancer was only observed when SNTSESF was conjugated to CPMV, and not added as a free peptide. This can be explained by the differences in the in vivo fates of the nanoparticle formulation vs. the free peptide; the larger nanoparticles are expected to exhibit prolonged tumor residence and favorable intratumoral distribution. Our study provides new design principles for plant virus-based in situ vaccination strategies.

Trivalent Subunit Vaccine Candidates for COVID-19 and Their Delivery Devices.

The COVID-19 pandemic highlights the need for platform technologies enabling rapid development of vaccines for emerging viral diseases. The current vaccines target the SARS-CoV-2 spike (S) protein and thus far have shown tremendous efficacy. However, the need for cold-chain distribution, a prime-boost administration schedule, and the emergence of variants of concern (VOCs) call for diligence in novel SARS-CoV-2 vaccine approaches. We studied 13 peptide epitopes from SARS-CoV-2 and identified three neutralizing epitopes that are highly conserved among the VOCs. Monovalent and trivalent COVID-19 vaccine candidates were formulated by chemical conjugation of the peptide epitopes to cowpea mosaic virus (CPMV) nanoparticles and virus-like particles (VLPs) derived from bacteriophage Qβ. Efficacy of this approach was validated first using soluble vaccine candidates as solo or trivalent mixtures and subcutaneous prime-boost injection. The high thermal stability of our vaccine candidates allowed for formulation into single-dose injectable slow-release polymer implants, manufactured by melt extrusion, as well as microneedle (MN) patches, obtained through casting into micromolds, for prime-boost self-administration. Immunization of mice yielded high titers of antibodies against the target epitope and S protein, and data confirms that antibodies block receptor binding and neutralize SARS-CoV and SARS-CoV-2 against infection of human cells. We present a nanotechnology vaccine platform that is stable outside the cold-chain and can be formulated into delivery devices enabling single administration or self-administration. CPMV or Qβ VLPs could be stockpiled, and epitopes exchanged to target new mutants or emergent diseases as the need arises.

Gene interfered-ferroptosis therapy for cancers

Although some effective therapies have been available for cancer, it still poses a great threat to human health and life due to its drug resistance and low response in patients. Here, we develop a ferroptosis-based therapy by combining iron nanoparticles and cancer-specific gene interference. The expression of two iron metabolic genes (FPN and LCN2) was selectively knocked down in cancer cells by Cas13a or microRNA controlled by a NF-κB-specific promoter. Cells were simultaneously treated by iron nanoparticles. As a result, a significant ferroptosis was induced in a wide variety of cancer cells. However, the same treatment had little effect on normal cells. By transferring genes with adeno-associated virus and iron nanoparticles, the significant tumor growth inhibition and durable cure were obtained in mice with the therapy. In this work, we thus show a cancer therapy based on gene interference-enhanced ferroptosis.

Application of Plant Viruses in Biotechnology, Medicine, and Human Health.

Plant-based nanotechnology programs using virus-like particles (VLPs) and virus nanoparticles (VNPs) are emerging platforms that are increasingly used for a variety of applications in biotechnology and medicine. Tobacco mosaic virus (TMV) and potato virus X (PVX), by virtue of having high aspect ratios, make ideal platforms for drug delivery. TMV and PVX both possess rod-shaped structures and single-stranded RNA genomes encapsidated by their respective capsid proteins and have shown great promise as drug delivery systems. Cowpea mosaic virus (CPMV) has an icosahedral structure, and thus brings unique benefits as a nanoparticle. The uses of these three plant viruses as either nanostructures or expression vectors for high value pharmaceutical proteins such as vaccines and antibodies are discussed extensively in the following review. In addition, the potential uses of geminiviruses in medical biotechnology are explored. The uses of these expression vectors in plant biotechnology applications are also discussed. Finally, in this review, we project future prospects for plant viruses in the fields of medicine, human health, prophylaxis, and therapy of human diseases.

Analysis of Engineered Tobacco Mosaic Virus and Potato Virus X Nanoparticles as Carriers for Biocatalysts.

Plant virus nanoparticles are promising candidates for the development of novel materials, including nanocomposites and scaffolds/carriers for functional molecules such as enzymes. Their advantages for enzyme immobilization include a modular organization, a robust and programmable structure, and a simple, cost-effective production. However, the activity of many enzymes relies on posttranslational modification and most plant viruses replicate in the cytoplasm, so functional enzymes cannot be displayed on the virus surface by direct coat protein fusions. An alternative display system to present the Trichoderma reesei endoglucanase Cel12A on potato virus X (PVX) using SpyTag/SpyCatcher (ST/SC) technology was recently developed by the authors, which allows the carrier and enzyme to be produced separately before isopeptide conjugation. Although kinetic analysis clearly indicated efficient biocatalyst activity, the PVX carrier interfered with substrate binding. To overcome this, the suitability of tobacco mosaic virus (TMV) was tested, which can also accommodate a larger number of ST peptides. We produced TMV particles displaying ST as a new platform for the immobilization of enzymes such as Cel12A, and compared its performance to the established PVX-ST platform in terms of catalytic efficiency. Although more enzyme molecules were immobilized on the TMV-ST particles, we found that the rigid scaffold and helical spacing significantly affected enzyme activity.

Reasons for success and lessons learnt from nanoscale vaccines against COVID-19.

Almost all currently used vaccines against COVID-19 consist of either non-viral or viral nanoparticles. Here we attempt to understand the reasons behind the success of such advanced nanoscale vaccine technologies compared with clinically established conventional vaccines, and the lessons to be learnt from this potentially transformative development in the adoption and acceptance of nanotechnology for medicine.

Producing Plant Virus Patterns with Defined 2D Structure

In nanobiotechnology, viral nanoparticles have come into focus as interesting nano building blocks. In this context, the formation of 2D and 3D structures is of particular interest. Herein, the creation of defined 2D patterns of an icosahedral plant virus, the tomato bushy stunt virus (TBSV), by means of different techniques is reported on: the top‐down lithography ebeam and focused ion beam (FIB) as well as the bottom‐up fluidic force microscope (FluidFM) approach. The obtained layer structures are imaged by scanning force and scanning electron microscopy. The data show that a defined 2D structure can successfully be created either top down by FIB or bottom up by FluidFM. Electron beam lithography is not able to remove viruses from the substrate under the chosen conditions. FIB has an advantage if larger areas covered with viruses combined with smaller areas without being desired. FluidFM is advantageous if only small areas with viruses are required. A further benefit is that the uncovered areas are not affected. The pattern formation in FluidFM is influenced not only by the spotting parameters, but in particular by the drying process. Deegan and Marangoni effects are shown to play a role if the spotted droplets are not very small.

Frontiers in Bioengineering and Biotechnology: Plant Nanoparticles for Anti-Cancer Therapy.

Naturally occurring viral nanomaterials have gained popularity owing to their biocompatible and biodegradable nature. Plant virus nanoparticles (VNPs) can be used as nanocarriers for a number of biomedical applications. Plant VNPs are inexpensive to produce, safe to administer and efficacious as treatments. The following review describes how plant virus architecture facilitates the use of VNPs for imaging and a variety of therapeutic applications, with particular emphasis on cancer. Examples of plant viruses which have been engineered to carry drugs and diagnostic agents for specific types of cancer are provided. The drug delivery system in response to the internal conditions is known as stimuli response, recently becoming more applicable using plant viruses based VNPs. The review concludes with a perspective of the future of plant VNPs and plant virus-like particles (VLPs) in cancer research and therapy.