Search

Ovarian cancer ranks fifth in cancer deaths amongst women, and most patients are diagnosed with late-stage and disseminated diseases. Surgical debulking and chemotherapy remove most of the tumor burden and provide a short period of remission; however, most patients experience cancer relapse and eventually succumb to the disease. Therefore, there is an urgent need for the development of vaccines to prime anti-tumor immunity and prevent its recurrence. Here we developed vaccine formulations composed of a mixture of irradiated cancer cells (ICCs, providing the antigen) and cowpea mosaic virus (CPMV) adjuvants. More specifically we compared the efficacy of co-formulated vs. mixtures of ICCs and CPMV. Specifically, we compared co-formulations where the ICCs and CPMV are bonded through natural CPMV-cell interactions or chemical coupling vs. mixtures of PEGylated CPMV and ICCs, where PEGylation of CPMV prevents ICC interactions. Flow cytometry and confocal imaging provided insights into the composition of the vaccines and their efficacy was tested using a mouse model of disseminated ovarian cancer. 67% of the mice receiving the co-formulated CPMV-ICCs survived the initial tumor challenge, and 60% of the surviving mice rejected tumors in a re-challenge experiment. In stark contrast, simple mixtures of the ICCs and (PEGylated) CPMV adjuvants were ineffective. Overall, this study highlights the importance of the co-delivery of cancer antigens and adjuvants in ovarian cancer vaccine development.

Brownian motion of Cowpea mosaic virus (CPMV) in water was measured using small-angle X-ray photon correlation spectroscopy (SA-XPCS) at 19.2 µs time resolution. It was found that the decorrelation time τ(Q) = 1/DQ 2 up to Q = 0.091 nm -1 . The hydrodynamic radius R H determined from XPCS using Stokes-Einstein diffusion D = kT/(6πηR H ) is 43% larger than the geometric radius R 0 determined from SAXS in the 0.007 M K 3 PO 4 buffer solution, whereas it is 80% larger for CPMV in 0.5 M NaCl and 104% larger in 0.5 M (NH 4 ) 2 SO 4 , a possible effect of aggregation as well as slight variation of the structures of the capsid resulting from the salt-protein interactions., (open access.)

Despite advances in laparoscopic surgery combined with neoadjuvant and adjuvant therapy, colon cancer management remains challenging in oncology. Recurrence of cancerous tissue locally or in distant organs (metastasis) is the major problem in colon cancer management. Vaccines and immunotherapies hold promise in preventing cancer recurrence through stimulation of the immune system. We and others have shown that nanoparticles from plant viruses, such as cowpea mosaic virus (CPMV) nanoparticles, are potent immune adjuvants for cancer vaccines and serve as immunostimulatory agents in the treatment or prevention of tumors. While being noninfectious toward mammals, CPMV activates the innate immune system through recognition by pattern recognition receptors (PRRs). While the particulate structure of CPMV is essential for prominent immune activation, the proteinaceous architecture makes CPMV subject to degradation in vivo ; thus, CPMV immunotherapy requires repeated injections for optimal outcome. Frequent intraperitoneal (IP) injections however are not optimal from a clinical point of view and can worsen the patient's quality of life due to the hospitalization required for IP administration. To overcome the need for repeated IP injections, we loaded CPMV nanoparticles in injectable chitosan/glycerophosphate (GP) hydrogel formulations, characterized their slow-release potential, and assessed the antitumor preventative efficacy of CPMV-in-hydrogel single dose versus soluble CPMV (single and prime-boost administration). Using fluorescently labeled CPMV-in-hydrogel formulations, in vivo release data indicated that single IP injection of the hydrogel formulation yielded a gel depot that supplied intact CPMV over the study period of 3 weeks, while soluble CPMV lasted only for one week. IP administration of the CPMV-in-hydrogel formulation boosted with soluble CPMV for combined immediate and sustained immune activation significantly inhibited colon cancer growth after CT26 IP challenge in BALB/c mice. The observed antitumor efficacy suggests that CPMV can be formulated in a chitosan/GP hydrogel to achieve prolonged immunostimulatory effects as single-dose immunotherapy against colon cancer recurrence. The present findings illustrate the potential of injectable hydrogel technology to accommodate plant virus nanoparticles to boost the translational development of effective antitumor immunotherapies.

Prognosis and treatment of metastatic cancer continues to be one of the most difficult and challenging areas of oncology. Treatment usually consists of chemotherapeutics, which may be ineffective due to drug resistance, adverse effects, and dose-limiting toxicity. Therefore, novel approaches such as immunotherapy have been investigated to improve patient outcomes and minimize side effects. S100A9 is a calcium-binding protein implicated in tumor metastasis, progression, and aggressiveness that modulates the tumor microenvironment into an immunosuppressive state. S100A9 is expressed in and secreted by immune cells in the pre-metastatic niche, as well as, post-tumor development, therefore making it a suitable targeted for prophylaxis and therapy. In previous work, it is demonstrated that cowpea mosaic virus (CPMV) acts as an adjuvant when administered intratumorally. Here, it is demonstrated that systemically administered, S100A9-targeted CPMV homes to the lungs leading to recruitment of innate immune cells. This approach is efficacious both prophylactically and therapeutically against lung metastasis from melanoma and breast cancer. The current research will facilitate and accelerate the development of next-generation targeted immunotherapies administered as prophylaxis, that is, after surgery of a primary breast tumor to prevent outgrowth of metastasis, as well as, therapy to treat established metastatic disease., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Background: Virus nanoparticles have been extensively studied over the past decades for theranostics applications. Viruses are well-characterized, naturally occurring nanoparticles that can be produced in high quantity with a high degree of similarity in both structure and composition., Objectives: The plant virus Cowpea Mosaic Virus (CPMV) has been innovatively used as a nanoscaffold. Utilization of the internal cavity of empty Virus-Like Particles (VLPs) for the inclusion of therapeutics within the capsid has opened many opportunities in drug delivery and imaging applications., Methods: The encapsidation of magnetic materials and anticancer drugs was achieved. Superscript CPMV denotes molecules attached to the external surface of CPMV and CPMV Subscript denotes molecules within the interior of the capsid., Results: Here, the generation of novel VLPs incorporating iron-platinum nanoparticles T CPMV FePt and cisplatin (Cis) ( T CPMV Cis ) is reported. T CPMV Cis exhibited a cytotoxic IC 50 of T CPMV Cis on both A549 and MDA-MB-231 cell lines of 1.8 μM and 3.9 μM, respectively after 72 hours of incubation. The T CPMV FePt were prepared as potential MRI contrast agents., Conclusion: Cisplatin loaded VLP ( T CPMV Cis ) is shown to enhance cisplatin cytotoxicity in cancer cell lines with its potency increased by 2.3-folds., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cowpea mosaic virus (CPMV) is a plant virus that has been developed for multiple biomedical and nanotechnology applications, including immunotherapy. Two key platforms are available: virus nanoparticles (VNPs) based on the complete CMPV virion, including the genomic RNA, and virus-like nanoparticles (VLPs) based on the empty CPMV (eCPMV) virion. It is unclear whether these platforms differ in terms of immunotherapeutic potential. We therefore compared their physicochemical properties and immunomodulatory activities following in situ vaccination of an aggressive ovarian tumor mouse model (ID8-Defb29/Vegf-A). In physicochemical terms, CPMV and eCPMV were very similar, and both significantly increased the survival of tumor-bearing mice and showed promising antitumor efficacy. However, they demonstrated distinct yet overlapping immunostimulatory effects due to the presence of virus RNA in wild-type particles, indicating their suitability for different immunotherapeutic strategies. Specifically, we found that the formulations had similar effects on most secreted cytokines and immune cells, but the RNA-containing CPMV particles were uniquely able to boost populations of potent antigen-presenting cells, such as tumor-infiltrating neutrophils and activated dendritic cells. Our results will facilitate the development of CPMV and eCPMV as immunotherapeutic vaccine platforms with tailored responses. IMPORTANCE The engagement of antiviral effector responses caused by viral infection is essential when using viruses or virus-like particles (VLPs) as an immunotherapeutic agent. Here, we compare the chemophysical and immunostimulatory properties of wild-type cowpea mosaic virus (CPMV) (RNA containing) and eCPMV (RNA-free VLPs) produced from two expression systems (agrobacterium-based plant expression system and baculovirus-insect cell expression). CPMV and eCPMV could each be developed as novel adjuvants to overcome immunosuppression and thus promote tumor regression in ovarian cancer (and other tumor types). To our knowledge, this is the first study to define the immunotherapeutic differences between CPMV and eCPMV, which is essential for the further development of biomedical applications for plant viruses and the selection of rational combinations of immunomodulatory reagents., (Copyright © 2019 American Society for Microbiology.)

The icosahedral capsid of cowpea mosaic virus is formed by 60 copies of the large (L) and small (S) coat protein subunits. The 24-amino-acid C-terminal peptide of the S coat protein can undergo proteolytic cleavage without affecting particle stability or infectivity. Mutagenic studies have shown that this sequence is involved in particle assembly, virus movement, RNA encapsidation and suppression of gene silencing. However, it is unclear how these processes are related, and which part(s) of the sequence are involved in each process. Here, we have analysed the effect of mutations in the C-terminal region of the S protein on the assembly of empty virus-like particles and on the systemic movement of infectious virus. The results confirmed the importance of positively charged amino acids adjacent to the cleavage site for particle assembly and revealed that the C-terminal 11 amino acids are important for efficient systemic movement of the virus.

Contemporary immunotherapies, e.g., those that target the CTLA-4 and PD-1/PD-L1 axis, act on T cells to reinstate their antitumor activity. An alternative, and possibly more powerful approach is to target and reprogram the innate immune system within the tumor microenvironment. To this end, blockade of CD47 has been demonstrated as an attractive approach. Blockade of CD47 inhibits antiphagocytic signals therefore inducing macrophage phagocytosis of cancer cells. CD47 blockade also primes antitumor T-cell responses by either activating antigen-presenting cells or inhibiting interactions between CD47 on cancer cells and the matricellular protein thrombospondin-1 on T cells. Here, a combination immunotherapy is identified using cowpea mosaic virus (CPMV) in situ vaccination and CD47-blocking antibodies. The CPMV in situ vaccine synergizes with CD47 blockade, because CPMV in situ vaccination activates the innate immune system, leading to recruitment and activation of phagocytes. Therefore, the combination therapy targets monocytes and boosts their ability of cancer cell phagocytosis, in turn priming the adaptive immune system leading to a potent antitumor immune response. This work presents a novel strategy to promote macrophage activity to kill tumor cells, and hold promise to enhance T cells targeted immunotherapies by inducing both innate and adaptive arms of immune system., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In situ cancer vaccination that uses immune stimulating agents is revolutionizing the way that cancer is treated. In this realm, viruses and noninfectious virus-like particles have gained significant traction in reprogramming the immune system to recognize and eliminate malignancies. Recently, cowpea mosaic virus-like particles (VLPs) have shown exceptional promise in their ability to fight a variety of cancers. However, the current methods used to produce CPMV VLPs rely on agroinfiltration in plants. These protocols remain complicated and labor intensive and have the potential to introduce unwanted immunostimulatory agents, like lipopolysaccharides. This Letter describes a simple "post-processing" method to remove RNA from wild-type CPMV, while retaining the structure and function of the capsid. Lyophilization was able to eject encapsulated RNA to form lyo-eCPMV and, when purified, eliminated nearly all traces of encapsulated RNA. Lyo-eCPMV was characterized by cryo-electron microscopy single particle reconstruction to confirm the structural integrity of the viral capsid. Finally, lyo-eCPMV showed  equivalent anticancer efficacy as eCPMV, produced by agroinfiltration, when using an invasive melanoma model. These results describe a straightforward method to prepare CPMV VLPs from infectious virions.

Protein-corona formation in body fluids and/or entrapment of nanoparticles in protein matrices (e.g. food and mucus) can hinder the delivery of nanoparticles, irrespective of the route of administration. Here we demonstrate that certain viral nanoparticles (VNPs) can evade the adhesion of a broad panel of macromolecules from several biological milieus. We also show that the permeability of VNPs through mucin gels is far superior to that of synthetic nanoparticles. The non-sticky nature of VNPs implies that they will be able to readily cross most non-specific protein and glycoprotein barriers encountered, ubiquitously, upon administration through mucosal, and non-mucosal routes.

Stem cells can interact and respond to the extracellular nanoscale environment. Viral nanoparticles have been utilized as building blocks to control cell growth and differentiation. By integrating stem cell research and virus nanoparticle chemistry together, a systematic analysis of the effects of nanotopography on stem cell differentiation can be accomplished. The fabrication of thin films of the viral nanoparticles is particularly valuable for such studies. Here, we describe two methods to fabricate plant virus-based thin films and procedures to study the osteogenic differentiation of mesenchymal stem cells on plant virus-based substrates. The method makes use of wild-type tobacco mosaic virus (wt-TMV), RGD-modified TMV (TMV-RGD), turnip yellow mosaic virus (TYMV), cowpea mosaic virus (CPMV), turnip vein clearing virus (TVCV), and potato virus X (PVX) for development of bone tissue engineering biomaterials.

We present a facile method for preparing hierarchical assemblies of cowpea mosaic virus (CPMV) nanoparticles adsorbed onto patterned polypyrrole copolymer arrays, which can be released as a freely standing and microporous polymer-protein membrane with a Janus-type structure. The patterning protocol is based on colloidal sphere lithography wherein a sacrificial honeycomb pattern composed of colloidal polystyrene (PS) microspheres is assembled on an electrode. A thin layer of polypyrrole film is electropolymerized within the interstices of the template and monitored using an electrochemical quartz crystal microbalance with dissipation (EC-QCM-D) and microscopy. Dissolving the PS template reveals an inverse opaline pattern capable of electrostatically capturing the CPMV particles. Through an electrochemical trigger, the polypyrrole-CPMV delaminates from the surface producing a self-sustaining polymer-protein membrane that can potentially be used for sensing and nanocargo applications.

Metallic nanoscale 3D architectures concentrate electromagnetic energy at precise spatial locations to enable sensing and photocatalysis applications. We have developed solution-based methods to reproducibly fabricate 3D gold nanostructures useful as efficient surface-enhanced Raman spectroscopy (SERS) biosensors. Virus capsids were recruited as templates to assemble gold nanoparticles on their surfaces at well-defined locations to prepare the nanoscale 3D structures. Cowpea mosaic virus (CPMV) and its variants were selected as specific templates due to their high symmetry, scalability, and stability, which have proven useful in materials science applications. While the methods described herein were optimized for the CPMV capsids, they also provide a useful starting point for researchers who are working toward the nanoassembly of metal nanoparticles on other protein scaffolds.

Plant virus capsids are attractive entities for nanotechnological applications because of their variation in shape and natural assembly ability. This chapter describes the production and modification of three differently shaped plant virus capsids for silica mineralization purposes. The chosen plant viruses exhibit either an icosahedral (cowpea mosaic virus, CPMV), or a flexuous rod-like structure (potato virus X, PVX), or a rigid rod-like shape (tobacco mosaic virus, TMV), and are well-known and frequently used plant viruses for biotechnological applications. We describe the production (including genetic or chemical modification) and purification of the plant viruses or of empty virus-like particles in the case of CPMV, as well as the characterization of these harvested templates. The mineralization procedures and differences in the protocols specific to the distinct viruses are described, and the analyses of the mineralization results are explained.

Photodynamic therapy (PDT) is a promising avenue for greater treatment efficacy of highly resistant and aggressive melanoma. Through photosensitizer attachment to nanoparticles, specificity of delivery can be conferred to further reduce potential side effects. While the main focus of PDT is the destruction of cancer cells, additional targeting of tumor-associated macrophages also present in the tumor microenvironment could further enhance treatment by eliminating their role in processes such as invasion, metastasis, and immunosuppression. In this study, we investigated PDT of macrophages and tumor cells through delivery using the natural noninfectious nanoparticle cowpea mosaic virus (CPMV), which has been shown to have specificity for the immunosuppressive subpopulation of macrophages and also targets cancer cells. We further explored conjugation of CPMV/dendron hybrids in order to improve the drug loading capacity of the nanocarrier. Overall, we demonstrated effective elimination of both macrophage and tumor cells at low micromolar concentrations of the photosensitizer when delivered with the CPMV bioconjugate, thereby potentially improving melanoma treatment., Competing Interests: The authors declare no competing financial interest.

Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C terminus of the small (S) subunit. The intact C terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Nanotechnology has tremendous potential to contribute to cancer immunotherapy. The 'in situ vaccination' immunotherapy strategy directly manipulates identified tumours to overcome local tumour-mediated immunosuppression and subsequently stimulates systemic antitumour immunity to treat metastases. We show that inhalation of self-assembling virus-like nanoparticles from cowpea mosaic virus (CPMV) reduces established B16F10 lung melanoma and simultaneously generates potent systemic antitumour immunity against poorly immunogenic B16F10 in the skin. Full efficacy required Il-12, Ifn-γ, adaptive immunity and neutrophils. Inhaled CPMV nanoparticles were rapidly taken up by and activated neutrophils in the tumour microenvironment as an important part of the antitumour immune response. CPMV also exhibited clear treatment efficacy and systemic antitumour immunity in ovarian, colon, and breast tumour models in multiple anatomic locations. CPMV nanoparticles are stable, nontoxic, modifiable with drugs and antigens, and their nanomanufacture is highly scalable. These properties, combined with their inherent immunogenicity and demonstrated efficacy against a poorly immunogenic tumour, make CPMV an attractive and novel immunotherapy against metastatic cancer.

Virus-based nanoparticles (VNPs) have been used for a wide range of applications, spanning basic materials science and translational medicine. Their propensity to self-assemble into precise structures that offer a three-dimensional scaffold for functionalization has led to their use as optical contrast agents and related biophotonics applications. A number of fluorescently labeled platforms have been developed and their utility in optical imaging demonstrated, yet their optical properties have not been investigated in detail. In this study, two VNPs of varying architectures were compared side-by-side to determine the impact of dye density, dye localization, conjugation chemistry, and microenvironment on the optical properties of the probes. Dyes were attached to icosahedral cowpea mosaic virus (CPMV) and rod-shaped tobacco mosaic virus (TMV) through a range of chemistries to target particular side chains displayed at specific locations around the virus. The fluorescence intensity and lifetime of the particles were determined, first using photochemical experiments on the benchtop, and second in imaging experiments using tissue culture experiments. The virus-based optical probes were found to be extraordinarily robust under ultrashort, pulsed laser light conditions with a significant amount of excitation energy, maintaining structural and chemical stability. The most effective fluorescence output was achieved through dye placement at optimized densities coupled to the exterior surface avoiding conjugated ring systems. Lifetime measurements indicate that fluorescence output depends not only on spacing the fluorophores, but also on dimer stacking and configurational changes leading to radiationless relaxation-and these processes are related to the conjugation chemistry and nanoparticle shape. For biological applications, the particles were also examined in tissue culture, from which it was found that the optical properties differed from those found on the benchtop due to effects from cellular processes and uptake kinetics. Data indicate that fluorescent cargos are released in the endolysosomal compartment of the cell targeted by the virus-based optical probes. These studies provide insight into the optical properties and fates of fluorescent proteinaceous imaging probes. The cellular release of cargo has implications not only for virus-based optical probes, but also for drug delivery and release systems.

In this contribution, optically active and paramagnetic micelles of the ligand 1,4,7,10-tetraaza-1-(1-carboxymethylundecane)-4,7,10-triacetic acid cyclododecane (DOTAC10) have been incorporated inside capsids of the cowpea chlorotic mottle virus (CCMV) protein through a hierarchical process of self-assembly triggered by self-assembly. The DOTAC10 ligand was used to complex Gd(III), in order to form paramagnetic micelles, as well as to encapsulate an amphiphilic Zn(II) phthalocyanine (ZnPc) dye that optically confirmed the encapsulation of the micelles. The incorporation of ZnPc molecules in the paramagnetic micelles led to high capsid loading of both Gd(III) and ZnPc, as the micelles were stabilized by the amphiphilic dye encapsulation. The resulting protein cage nanoparticles (PCNs) show an improved r1 relaxivity, suggesting the possible use of these nanostructures as contrast agents (CAs) for magnetic resonance imaging (MRI). Since the encapsulated ZnPc dye also has a potential therapeutic value, the present results represent a first step towards the consecution of fully self-assembled PCNs for multimodal imaging and therapy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Unlabelled: Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58-kDa protein (P58) encoded by RNA2 has not been resolved. P58 and the movement protein (MP) of BPMV are two largely identical proteins differing only at their N termini, with P58 extending MP upstream by 102 amino acid residues. In this report, we unveil a unique role for P58. We show that BPMV RNA2 accumulation in infected cells was abolished when the start codon of P58 was eliminated. The role of P58 does not require the region shared by MP, as RNA2 accumulation in individual cells remained robust even when most of the MP coding sequence was removed. Importantly, the function of P58 required the P58 protein, rather than its coding RNA, as compensatory mutants could be isolated that restored RNA2 accumulation by acquiring new start codons upstream of the original one. Most strikingly, loss of P58 function could not be complemented by P58 provided in trans, suggesting that P58 functions in cis to selectively promote the accumulation of RNA2 copies that encode a functional P58 protein. Finally, we found that all RNA1-encoded proteins are cis-acting relative to RNA1. Together, our results suggest that P58 probably functions by recruiting the RNA1-encoded polyprotein to RNA2 to enable RNA2 reproduction., Importance: Bean pod mottle virus (BPMV) is one of the most important pathogens of the crop plant soybean, yet its replication mechanism is not well understood, hindering the development of knowledge-based control measures. The current study examined the replication strategy of BPMV RNA2, one of the two genomic RNA segments of this virus, and established an essential role for P58, one of the RNA2-encoded proteins, in the process of RNA2 replication. Our study demonstrates for the first time that P58 functions preferentially with the very RNA from which it is translated, thus greatly advancing our understanding of the replication mechanisms of this and related viruses. Furthermore, this study is important because it provides a potential target for BPMV-specific control, and hence could help to mitigate soybean production losses caused by this virus.

Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA virus of Secoviridae. We recently reported that a 137 nucleotide (nt) stretch (#263-399) of the 466 nt 5' untranslated region (5' UTR) of BPMV RNA2 can be deleted without compromising BPMV propagation in host plants [Lin et al., J. Gen. Virol. 94 (2013) 1415-1420]. Here we demonstrate that nonviral insertions of up to 625 nt is tolerated by the same region. Furthermore, one insertion mutant underwent recombination in infected plants, leading to the truncation of nt #250-361, thus extending the dispensable sequence to 150 nt (nt #250-399). We are unaware of any other (+) RNA virus that tolerates insertion/deletion of these sizes (625 nt/150 nt) within its 5' UTR. Importantly, tolerance of large insertions within the RNA2 5' UTR offers a novel, more convenient site for incorporating host gene fragments, making BPMV a more versatile vector of virus-induced gene silencing., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Due to the nanoscale size and the strictly controlled and consistent morphologies of viruses, there has been a recent interest in utilizing them in nanotechnology. The structure, surface chemistries and physical properties of many viruses have been well elucidated, which have allowed identification of regions of their capsids which can be modified either chemically or genetically for nanotechnological uses. In this review we focus on the use of such modifications for the functionalization and production of viruses and empty viral capsids that can be readily decorated with metals in a highly tuned manner. In particular, we discuss the use of two plant viruses (Cowpea mosaic virus and Tobacco mosaic virus) which have been extensively used for production of novel metal nanoparticles (<100nm), composites and building blocks for 2D and 3D materials, and illustrate their applications., (© 2013 Elsevier Inc. All rights reserved.)

The development of methods for the production of empty Cowpea mosaic virus (CPMV) virus-like particles (VLPs) that are devoid of RNA, eVLPs, has renewed promise in CPMV capsid technologies. The recombinant nature of CPMV eVLP production means that the extent and variety of genetic modifications that may be incorporated into the particles is theoretically much greater than those that can be made to infectious CPMV virions due to restrictions on viral propagation of the latter. Free of the infectious agent, the genomic RNA, these particles are now finding potential uses in vaccine development, in vivo imaging, drug delivery, and other nanotechnology applications that make use of internal loading of the empty particles. Here we describe methods for the genetic modification and production of CPMV eVLPs and describe techniques useful for their characterization.

Polyelectrolyte surface-modified cowpea mosaic virus (CPMV) can be used for the templated synthesis of narrowly dispersed gold nanoparticles. Cationic polyelectrolyte, poly(allylamine) hydrochloride, is electrostatically bound to the external surface of the virus capsid. The polyelectrolyte-coated CPMV promotes adsorption of aqueous gold hydroxide anionic species, prepared from gold(III) chloride and potassium carbonate, that are easily reduced to form CPMV-templated gold nanoparticles. The process is simple and environmentally benign using only water as solvent at ambient temperature.

Templated mineralization of virus particles provides routes to narrowly dispersed nanoparticles that are not readily prepared by other means. The templated mineralization of metal or metal oxide on the external surface of wild-type cowpea mosaic virus (CPMV), a plant virus, is facilitated by increasing the external surface negative charge. This is achieved by the chemical modification of surface lysine groups by succinic anhydride. Hence, for example, treatment of charge-modified CPMV succinamate with a 1:2 mixture of iron(II) and iron(III) salts, followed by raising the pH to 10.2, led to the formation of narrowly dispersed, CPMV-templated, magnetite (Fe3O4) nanoparticles.

Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus of the family Secoviridae. Its RNA1 encodes all proteins needed for genome replication and is capable of autonomous replication. By contrast, BPMV RNA2 must utilize RNA1-encoded proteins for replication. Here, we sought to identify RNA elements in RNA2 required for its replication. The exchange of 5' untranslated regions (UTRs) between genome segments revealed an RNA2-specific element in its 5' UTR. Further mapping localized a 66 nucleotide region that was predicted to fold into an RNA stem-loop structure, designated SLC. Additional functional analysis indicated the importance of the middle portion of the stem and an adjacent two-base mismatch. This is the first report of a cis-acting RNA element in RNA2 of a bipartite secovirus.

Chemical addressability of viral particles has played a pivotal role in adapting these biogenic macromolecules for various applications ranging from medicine to inorganic catalysis. Cowpea mosaic virus possesses multiple features that are advantageous for the next generation of virus-based nanotechnology: consistent multimeric assemblies dictated by its genetic code, facile large scale production, and lack of observable toxicity in humans. Herein, the chemistry of the viral particles is extended with the use of Cu-free strain-promoted azide-alkyne cycloaddition reaction, or SPAAC reaction. The elimination of Cu, its cocatalyst and reducing agent, simplifies the reaction scheme to a more straightforward approach, which can be directly applied to living systems. As a proof of concept, the viral particles modified with the azadibenzylcyclooctyne functional groups are utilized to trigger and amplify a weak fluorescent signal (azidocoumarin) in live cell cultures to visualize the non-natural sugars. Future adaptations of this platform may be developed to enhance biosensing applications.

The plant virus, Cowpea mosaic virus (CPMV), is developed as a carrier of the chemotherapeutic drug doxorubicin (DOX). CPMV-DOX conjugate, in which eighty DOX molecules are covalently bound to external surface carboxylates of the viral nanoparticle (VNP), shows greater cytotoxicity than free DOX toward HeLa cells when administered at low dosage. At higher concentrations, CPMV-DOX cytotoxicity is time-delayed. The CPMV conjugate is targeted to the endolysosomal compartment of the cells, in which the proteinaceous drug carrier is degraded and the drug released. This study is the first demonstrating the utility of CPMV as a drug delivery vehicle.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. (1) and (2) for further information on VNPs). From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies. In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

There remains a tremendous need to develop targeted therapeutics that can both image and localize the toxic effects of chemotherapeutics and antagonists on diseased tissue while reducing adverse systemic effects. These needs have fostered the development of a nanotechnology-based approach that can combine targeting and toxicity potential. In this study, CPMV nanoparticles were chemically modified with the dye Alexa Flour 488 and were also tandemly modified with PEG1000 followed by AF488; and the derivatized nanoparticles were subsequently added to macrophages stimulated with either LPS (M1) or IL-4 (M2). Previously published studies have shown that M1/M2 macrophages are both present in an inflammatory microenvironment (such as a tumor microenvironment and atherosclerosis) and play opposing yet balancing roles; M2 macrophages have a delayed and progressive onset in the tumor microenvironment (concomitant with an immunosuppression of M1 macrophages). In this study, we show higher uptake of CPMV-AF488 and CPMV-PEG-AF488 by M2 macrophages compared to M1 macrophages. M1 macrophages showed no uptake of CPMV-PEG-AF488. More specifically, M2 macrophages are known to be up-regulated in early atherosclerosis plaque. Indeed, previous work showed that M2 macrophages in plaque also correlate with CPMV internalization. These studies emphasize the potential effectiveness of CPMV as a tailored vehicle for targeting tumor macrophages involved in cancer metastasis or vascular inflammation and further highlight the potential of CPMV in targeted therapeutics against other diseases.

Surface engineering of plant virus capsids via cationization (1) and stoichiometric coupling of a polymer surfactant coronal layer (2) produces a highly concentrated, solvent-free liquid virus at 28 °C. These ionic bionanoconstructs are viscoelastic, retain plant infectivity and can be dispersed in a range of organic solvents for aerosol delivery., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Viral nanoparticles (VNPs) based on plant viruses such as Cowpea mosaic virus (CPMV) can be used for a broad range of biomedical applications because they present a robust scaffold that allows functionalization by chemical conjugation and genetic modification, thereby offering an efficient drug delivery platform that can target specific cells and tissues. VNPs such as CPMV show natural affinity to cells; however, cellular uptake is inefficient. Here we show that chemical modification of the CPMV surface with a highly reactive, specific and UV-traceable hydrazone linker allows bioconjugation of polyarginine (R5) cell penetrating peptides (CPPs), which can overcome these limitations. The resulting CPMV-R5 particles were taken up into a human cervical cancer cell line (HeLa) more efficiently than native particles. Uptake efficiency was dependent on the density of R5 peptides on the surface of the VNP; particles displaying 40 R5 peptides per CPMV (denoted as CPMV-R5H) interact strongly with the plasma membrane and are taken up into the cells via an energy-dependent mechanism whereas particles displaying 10 R5 peptides per CPMV (CPMV-R5L) are only slowly taken up. The fate of CPMV-R5 versus native CPMV particles within cells was evaluated in a co-localization time course study. It was indicated that the intracellular localization of CPMV-R5 and CPMV differs; CPMV remains trapped in Lamp-1 positive endolysosomes over long time frames; in contrast, 30-50% of the CPMV-R5 particles transitioned from the endosome into other cellular vesicles or compartments. Our data provide the groundwork for the development of efficient drug delivery formulations based on CPMV-R5.

Aims: Detection of atherosclerosis has generally been limited to the late stages of development, after cardiovascular symptoms present or a clinical event occurs. One possibility for early detection is the use of functionalized nanoparticles. The aim of this study was the early imaging of atherosclerosis using nanoparticles with a natural affinity for inflammatory cells in the lesion., Materials & Methods: We investigated uptake of cowpea mosaic virus by macrophages and foam cells in vitro and correlated this with vimentin expression. We also examined the ability of cowpea mosaic virus to interact with atherosclerotic lesions in a murine model of atherosclerosis., Results & Conclusion: We found that uptake of cowpea mosaic virus is increased in areas of atherosclerotic lesion. This correlated with increased surface vimentin in the lesion compared with nonlesion vasculature. In conclusion, cowpea mosaic virus and its vimentin-binding region holds potential for use as a targeting ligand for early atherosclerotic lesions, and as a probe for detecting upregulation of surface vimentin during inflammation.

The use of polyelectrolyte surface-modified Cowpea mosaic virus (CPMV) for the templated synthesis of narrowly dispersed gold nanoparticles is described. The cationic polyelectrolyte, poly(allylamine) hydrochloride (PAH), is electrostatically bound to the external surface of the virus capsid; the polyelectrolyte promotes the adsorption of anionic gold complexes, which are then easily reduced, under mild conditions, to form a metallic gold coating. As expected, the templated gold nanoparticles can be further modified with thiol reagents. In contrast, reaction of polyelectrolyte-modified CPMV (CPMV-PA) with preformed gold nanoparticles results in the self-assembly of large, hexagonally packed, tessellated-spheres.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level(1,2,3,) highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM)( 6). The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) (7, 8, 9,10,11). Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor(7,12). PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect (7, 10,11). The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, and uptake, making them particularly well suited to molecular imaging applications. Herein is reported the stepwise synthesis and characterization of NIR viral nanoparticles targeted to gastrin-releasing peptide receptors that are over-expressed in human prostate cancers. The pan-bombesin analogue, [β-Ala11, Phe13, Nle14]bombesin-(7-14), is conjugated to cowpea mosaic virus particles functionalized with an NIR dye (Alexa Fluor 647) and polyethylene glycol (PEG) using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. Targeting and uptake in human PC-3 prostate cells is demonstrated in vitro. Tumor homing is observed using human prostate tumor xenografts on the chicken chorioallantoic membrane model using intravital imaging. Further development of this viral nanoparticle platform may open the door to potential clinical noninvasive molecular imaging strategies., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Assembling and interconnecting the building blocks of nanoscale devices and being able to electronically address or measure responses at the molecular level remains an important challenge for nanotechnology. Here we show the usefulness of bottom-up self-assembly for building electronic nanosensors from multiple components that have been designed to interact in a controlled manner. Cowpea mosaic virus was used as a scaffold to control the positions of gold nanoparticles. The nanoparticles were then interconnected using thiol-terminated conjugated organic molecules, resulting in a three-dimensional conductive network. Biotin molecules were attached to the virus scaffold using linkers to act as molecular receptors. We demonstrated that binding avidin to the biotin receptors on the self-assembled nanosensors causes a significant change in the network conductance that is dependent on the charge of the avidin protein., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The external surface of the plant virus Cowpea mosaic virus (CPMV) can be chemically modified with peptides that direct specific mineralization processes. Subsequent mineralization of the peptide-CPMV conjugates produces monodisperse nanoparticles of ca. 32 nm diameter coated with, for example, cobalt-platinum, iron-platinum or zinc sulfide, which cannot be readily prepared by other methods. This route is particularly attractive as it avoids the need to genetically engineer the protein surface of the virus to provide chimaeras for templated-mineralization. The synthetic procedure is environmentally friendly, as it proceeds at ambient temperature and pressure, in aqueous solvent. Further, the methodology is demonstrated to be generally applicable by the mineralization of a peptide-modified multiwalled carbon nanotube.

Plant viruses are considered as nanobuilding blocks that can be used as synthons or templates for novel materials. Cowpea mosaic virus (CPMV) particles have been shown to template the fabrication of metallic nanoparticles by an electroless deposition metallization process. Palladium ions were electrostatically bound to the virus capsid and, when reduced, acted as nucleation sites for the subsequent metal deposition from solution. The method, although simple, produced highly monodisperse metallic nanoparticles with a diameter of ca. ≤35 nm. CPMV-templated particles were prepared with cobalt, nickel, iron, platinum, cobalt-platinum and nickel-iron.

A naturally occurring nanoparticle, the plant virus Cowpea mosaic virus, can be decorated with ferrocene derivatives, of various linker lengths with amine and carboxylate groups, on the external surface using a range of conjugation strategies. The multiple, organometallic, redox-active ferrocene moieties on the outer surface of the virus are electrochemically independent with reduction potentials that span a potential window of 0.16 V that are dependent on the site of modification and the nature of the ferrocene derivative. The number of ferrocenes coupled to each virus ranges from about 100 to 240 depending upon the conjugation site and the linker length and these redox active units can provide multielectron reservoirs.

The broadly neutralizing antibody 2G12 recognizes a conserved cluster of high-mannose glycans on the surface envelope spike of HIV, suggesting that the "glycan shield" defense of the virus can be breached and may, under the right circumstances, serve as a vaccine target. In an attempt to recreate features of the glycan shield semisynthetically, oligomannosides were coupled to surface lysines on the icosahedral capsids of bacteriophage Q beta and cowpea mosaic virus (CPMV). The Q beta glycoconjugates, but not CPMV, presented oligomannose clusters that bind the antibody 2G12 with high affinity. However, antibodies against these 2G12 epitopes were not detected in immunized rabbits. Rather, alternative oligomannose epitopes on the conjugates were immunodominant and elicited high titers of anti-mannose antibodies that do not crossreact with the HIV envelope. The results presented reveal important design considerations for a carbohydrate-based vaccine component for HIV., ((c) 2010 Elsevier Ltd. All rights reserved.)

A highly ordered assembly of biological molecules provides a powerful means to study the organizational principles of objects at the nanoscale. Two-dimensional cowpea mosaic virus arrays were assembled in an ordered manner on mica using osmotic depletion effects and a drop-and-dry method. The packing of the virus array was controlled systematically from rhombic packing to hexagonal packing by modulating the concentrations of poly(ethylene glycol) surfactant in the virus solutions. The orientation and packing symmetry of the virus arrays were found to be tuned by the concentrations of surfactants in the sample solutions. A phenomenological model for the present system is proposed to explain the assembly array morphology under the influence of the surfactant. Steric and electrostatic complementarity of neighboring virus capsids is found to be the key factors in controlling the symmetry of packing.

A molecular-level model is used to study the mechanical response of empty cowpea chlorotic mottle virus (CCMV) and cowpea mosaic virus (CPMV) capsids. The model is based on the native structure of the proteins that constitute the capsids and is described in terms of the C(alpha) atoms. Nanoindentation by a large tip is modeled as compression between parallel plates. Plots of the compressive force versus plate separation for CCMV are qualitatively consistent with continuum models and experiments, showing an elastic region followed by an irreversible drop in force. The mechanical response of CPMV has not been studied, but the molecular model predicts an order of magnitude higher stiffness and a much shorter elastic region than for CCMV. These large changes result from small structural changes that increase the number of bonds by only 30% and would be difficult to capture in continuum models. Direct comparison of local deformations in continuum and molecular models of CCMV shows that the molecular model undergoes a gradual symmetry breaking rotation and accommodates more strain near the walls than the continuum model. The irreversible drop in force at small separations is associated with rupturing nearly all of the bonds between capsid proteins in the molecular model, while a buckling transition is observed in continuum models.

N-(2-Hydroxypropyl)methacrylamide (HPMA) co-polymers containing disulfide and carbonyl thiazolidine-2-thione (TT) reactive groups in their side-chains (pHPMA-TT) were used as reductively removable chemical modification of the surface of cowpea mosaic viruses (CPMV). CPMV was used in this study as a model particle for viral gene delivery. The co-polymer reaction with CPMV surfaces carried out in aqueous solution was evaluated by monitoring the changes in the weight-average molecular weight and hydrodynamic size of the viruses using light scattering methods. The reaction conditions were optimized. The surface modification of CPMV with pHPMA-TT under selected conditions (concentrations of a coating polymer (c(p)) and NaCl) has not influenced the size distribution of the viral particles. The uncharged polymers bound to the viral surface via biodegradable S-S bonds can be fully removed by the exchange reaction with reductive dithiothreitol. To achieve optimal covering of viral surfaces, the positively charged reactive polymers (with or without biodegradable S-S bonds) should be applied at low concentrations (c(p)=0.1-0.5 mg/ml) and in presence of NaCl (0.15 M).

The use of an engineered variant of a plant virus, Cowpea mosaic virus (CPMV), as a template for directed mineralization provides an environmentally benign route to monodisperse iron-platinum nanoparticles of approximately 30 nm diameter.

Previous studies have shown that a functionalized viral nanoparticle can be used as a fluorescent signal-generating element and enhance detection sensitivity for immunoassays and low density microarrays. In this study, we further tested this ability in commercial DNA microarrays, including Affymetrix high density resequencing microarray. Optimum conditions for NeutrAvidin and dye coupling to a double-cysteine mutant of cowpea mosaic virus (CPMV) were found to be comparable to the commonly used streptavidin-phycoerythrin (SAPE) for high density resequencing microarray. A 3-fold signal enhancement in comparison to Cy5-dCTP controls was obtained when using nanoparticles on control scorecard expression microarrays. Hybridization results from commercially available 8000 rat expression arrays indicate an increment of 14% on the detected features when the virus complex was used as the staining reagent in comparison to Cy5-dCTP controls. The current work shows the utility of the CPMV-dye nanoparticles as a detection reagent in well-established detection platforms.

Cowpea mosaic virus (CPMV) was analyzed by CZE and the two separated subcomponents recorded at approximately 2.2 and approximately 2.7 min were respectively designated to be the slow electrophoretic form of CPMV (CPMV(s)) and the fast electrophoretic form of CPMV (CPMV(f)), the two electrophoretic forms of CPMV, by the following methods: first, CZE analysis of the biospecific complexes of CPMV and anti-CPMV polyclonal antibody proved the properties of CPMV; second, isolation and CZE analysis of CPMV(f) and CPMV(s). Because CPMV(s) was precipitated and CPMV(f) was left in the supernatant at pH 5.6, the only one peak at approximately 2.7 min from the supernatant was assigned for CPMV(f), while the peak at approximately 2.2 min dominating for the precipite was deemed to be CPMV(s). Third, viral characterization by directly analyzing the capsid proteins using MALDI-TOF-MS. Based on the theoretical molecular weights calculated from the sequence of the capsid proteins, the three peaks at m/z 21 516.75, 23 745.8 and 42 509.22 for natural CPMV were destined for the small (S) protein of CPMV(f), the S protein of CPMV(s) and the large (L) protein of both of them, respectively. As expected, the non-appearance of the peak at m/z 23 745.8 for the isolated CPMV(f) sample indicated the absence of CPMV(s), and the peak at m/z 23 745.8 was predominant in the spectrum for the precipitated CPMV(s) sample. After the confirmation of CPMV(s) and CPMV(f), the CZE separation of them was optimized. The developed analysis method has proven useful in investigating the stability of CPMV and the effect of 2,4-dinitrophenyl-decoration on the modification of the electrophoretical behavior of CPMV.

There is a trend toward viral-based hybrid systems to furnish viral nanoparticles with enhanced features, for function beyond a delivery vehicle. Such hybrids have included Nanogold for microwave release, quantum dots and fluorescent moieties, to provide simultaneous imaging capabilities, and iron oxide particles for image enhancement in MRI. Other systems are the subject of ongoing and vigorous research. Nanogold surface decoration of cow pea mosaic virus (CPMV) to form NG-CPMV hybrids were explored to release fluorescent carriers using microwave energy as a model system in this presentation. Thus, emergent viral-based systems will have increasingly sophisticated architectures to provide versatile functions. Zeta potential (ZP) is a powerful tool to probe the electrostatic surface potential of biological materials and remains an untapped method for studying the interaction of nanoparticles with cells. An enormous effort is being made to study nanoparticle-cell interaction, but current throughput solutions (e.g., flow cytometry) cannot differentiate between surface-attached or endocytosed particles, while standard fluorescence microscopy is tedious and costly. CPMV-WT and other mutants (CPMV-T184C, CPMV-L189C) were studied using ZP methods and rationalized based on variations in their surface exposed residue character. Understanding such subtle changes can discretely alter the cell surface interactions due to charge affinity. Applying sensitive ZP measurements on viral nanoparticles is useful to elucidating the characteristics of the surface charge and the potential interaction modes with cell surfaces they may encounter. Thus, ZP can be a unique and efficient tool for studying cell-virus interactions and aid in development of future therapeutic strategies.