A co-formulated vaccine of irradiated cancer cells and cowpea mosaic virus improves ovarian cancer rejection.

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.

Direct measurement of Stokes-Einstein diffusion of Cowpea mosaic virus with 19 µs-resolved XPCS.

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/DQ2 up to Q = 0.091 nm-1. The hydrodynamic radius RH determined from XPCS using Stokes-Einstein diffusion D = kT/(6πηRH) is 43% larger than the geometric radius R0 determined from SAXS in the 0.007 M K3PO4 buffer solution, whereas it is 80% larger for CPMV in 0.5 M NaCl and 104% larger in 0.5 M (NH4)2SO4, a possible effect of aggregation as well as slight variation of the structures of the capsid resulting from the salt-protein interactions.

Injectable Hydrogel Containing Cowpea Mosaic Virus Nanoparticles Prevents Colon Cancer Growth.

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.

S100A9-Targeted Cowpea Mosaic Virus as a Prophylactic and Therapeutic Immunotherapy against Metastatic Breast Cancer and Melanoma.

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.

A Potential MRI Agent and an Anticancer Drug Encapsulated within CPMV Virus-Like Particles.

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. SuperscriptCPMV denotes molecules attached to the external surface of CPMV and CPMVSubscript denotes molecules within the interior of the capsid. RESULTS: Here, the generation of novel VLPs incorporating iron-platinum nanoparticles TCPMVFePt and cisplatin (Cis) (TCPMVCis) is reported. TCPMVCis exhibited a cytotoxic IC50 of TCPMVCis on both A549 and MDA-MB-231 cell lines of 1.8 µM and 3.9 µM, respectively after 72 hours of incubation. The TCPMVFePt were prepared as potential MRI contrast agents. CONCLUSION: Cisplatin loaded VLP (TCPMVCis) is shown to enhance cisplatin cytotoxicity in cancer cell lines with its potency increased by 2.3-folds.

Cowpea Mosaic Virus Nanoparticles and Empty Virus-Like Particles Show Distinct but Overlapping Immunostimulatory Properties.

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.

Amino acids at the exposed C-terminus of the S coat protein of cowpea mosaic virus play different roles in particle formation and viral systemic movement.

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.

CD47 Blockade and Cowpea Mosaic Virus Nanoparticle In Situ Vaccination Triggers Phagocytosis and Tumor Killing.

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.

Freeze-Drying To Produce Efficacious CPMV Virus-like Particles.

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.

Viral nanoparticles can elude protein barriers: exploiting rather than imitating nature.

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.

Nanomanufacture of Free-Standing, Porous, Janus-Type Films of Polymer-Plant Virus Nanoparticle Arrays.

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.

Bioinspired Silica Mineralization on Viral Templates.

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.

Dual-Functionalized Virus-Gold Nanoparticle Clusters for Biosensing.

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.

Fabrication of Plant Virus-Based Thin Films to Modulate the Osteogenic Differentiation of Mesenchymal Stem Cells.

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.

Utilizing Viral Nanoparticle/Dendron Hybrid Conjugates in Photodynamic Therapy for Dual Delivery to Macrophages and Cancer Cells.

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.

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus.

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.

In situ vaccination with cowpea mosaic virus nanoparticles suppresses metastatic cancer.

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.

Interface of physics and biology: engineering virus-based nanoparticles for biophotonics.

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.

Self-assembly triggered by self-assembly: optically active, paramagnetic micelles encapsulated in protein cage nanoparticles.

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.