Remission-Stage Ovarian Cancer Cell Vaccine with Cowpea Mosaic Virus Adjuvant Prevents Tumor Growth.

Ovarian cancer is the deadliest gynecological malignancy. Though most patients enter remission following initial interventions, relapse is common and often fatal. Accordingly, there is a substantial need for ovarian cancer therapies that prevent relapse. Following remission generated by surgical debulking and chemotherapy, but prior to relapse, resected and inactivated tumor tissue could be used as a personalized vaccine antigen source. The patient's own tumor contains relevant antigens and, when combined with the appropriate adjuvant, could generate systemic antitumor immunity to prevent relapse. Here, we model this process in mice to investigate the optimal tumor preparation and vaccine adjuvant. Cowpea mosaic virus (CPMV) has shown remarkable efficacy as an immunostimulatory cancer therapy in ovarian cancer mouse models, so we use CPMV as an adjuvant in a prophylactic vaccine against a murine ovarian cancer model. Compared to its codelivery with tumor antigens prepared in three other ways, we show that CPMV co-delivered with irradiated ovarian cancer cells constitutes an effective prophylactic vaccine against a syngeneic model of ovarian cancer in C57BL/6J mice. Following two vaccinations, 72% of vaccinated mice reject tumor challenges, and all those mice survived subsequent rechallenges, demonstrating immunologic memory formation. This study supports remission-stage vaccines using irradiated patient tumor tissue as a promising option for treating ovarian cancer, and validates CPMV as an antitumor vaccine adjuvant for that purpose.

Affinity of plant viral nanoparticle potato virus X (PVX) towards malignant B cells enables cancer drug delivery.

Non-Hodgkin's B cell lymphomas (NHL) include a diverse set of neoplasms that constitute ∼90% of all lymphomas and the largest subset of blood cancers. While chemotherapy is the first line of treatment, the efficacy of contemporary chemotherapies is hampered by dose-limiting toxicities. Partly due to suboptimal dosing, ∼40% of patients exhibit relapsed or refractory disease. Therefore more efficacious drug delivery systems are urgently needed to improve survival of NHL patients. In this study we demonstrate a new drug delivery platform for NHL based on the plant virus Potato virus X (PVX). We observed a binding affinity of PVX towards malignant B cells. In a metastatic mouse model of NHL, we show that systemically administered PVX home to tissues harboring malignant B cells. When loaded with the chemotherapy monomethyl auristatin (MMAE), the PVX nanocarrier enables effective delivery of MMAE to human B lymphoma cells in a NHL mouse model leading to inhibition of lymphoma growth in vivo and improved survival. Thus, PVX nanoparticle is a promising drug delivery platform for B cell malignancies.

Endosomal toll-like receptors play a key role in activation of primary human monocytes by cowpea mosaic virus.

The plant virus, cowpea mosaic virus (CPMV), has demonstrated a remarkable capacity to induce anti-tumour immune responses following direct administration into solid tumours. The molecular pathways that account for these effects and the capacity of CPMV to activate human cells are not well defined. Here, we examine the ability of CPMV particles to activate human monocytes, dendritic cells (DCs) and macrophages. Monocytes in peripheral blood mononuclear cell cultures and purified CD14+ monocytes were readily activated by CPMV in vitro, leading to induction of HLA-DR, CD86, PD-L1, IL-15R and CXCL10 expression. Monocytes released chemokines, CXCL10, MIP-1α and MIP-1ß into cell culture supernatants after incubation with CPMV. DC subsets (pDC and mDC) and monocyte-derived macrophages also demonstrated evidence of activation after incubation with CPMV. Inhibitors of spleen tyrosine kinase (SYK), endocytosis or endocytic acidification impaired the capacity of CPMV to activate monocytes. Furthermore, CPMV activation of monocytes was partially blocked by a TLR7/8 antagonist. These data demonstrate that CPMV activates human monocytes in a manner dependent on SYK signalling, endosomal acidification and with an important contribution from TLR7/8 recognition.

In Situ Vaccination of Tumors Using Plant Viral Nanoparticles.

Viral nanoparticles are self-assembling units that are being developed and applied for a variety of applications. While most clinical uses involve animal viruses, a plant-derived virus, cowpea mosaic virus (CPMV) has been shown to have antitumor properties in mice when applied as in situ vaccine. Here we describe the production and characterization of CPMV and its use as in situ vaccines in the context of cancer. Subsequent analyses to obtain efficacy or mechanistic data are also detailed.

Slow-Release Formulation of Cowpea Mosaic Virus for In Situ Vaccine Delivery to Treat Ovarian Cancer.

The plant viral nanoparticle cowpea mosaic virus (CPMV) is shown to be an effective immunotherapy for ovarian cancer when administered as in situ vaccine weekly, directly into the intraperitoneal (IP) space in mice with disseminated tumors. While the antitumor efficacy is promising, the required frequency of administration may pose challenges for clinical implementation. To overcome this, a slow release formulation is developed. CPMV and polyamidoamine generation 4 dendrimer form aggregates (CPMV-G4) based on electrostatic interactions and as a function of salt concentration, allowing for tailoring of aggregate size and release of CPMV. The antitumor efficacy of a single administration of CPMV-G4 is compared to weekly administration of soluble CPMV in a mouse model of peritoneal ovarian cancer and found to be as effective at reducing disease burden as more frequent administrations of soluble CPMV; a single injection of soluble CPMV, does not significantly slow cancer development. The ability of CPMV-G4 to control tumor growth following a single injection is likely due to the continued presence of CPMV in the IP space leading to prolonged immune stimulation. This enhanced retention of CPMV and its antitumor efficacy demonstrates the potential for viral-dendrimer hybrids to be used for delayed release applications.

Interactions Between Plant Viral Nanoparticles (VNPs) and Blood Plasma Proteins, and Their Impact on the VNP In Vivo Fates.

Plant viral nanoparticles (VNPs) are currently being developed as novel vessels for delivery of diagnostic and therapeutic cargos to sites of disease. With a rapid increase in the number of VNP variants and their potential applications in nanomedicine, the properties they acquire in the bloodstream need to be investigated. Biomolecules present in plasma are known to adsorb onto the surface of nanomaterials (including VNPs), forming a biointerface called the protein corona, which is capable of reprogramming the properties of VNPs. Here we describe a few general methods to isolate and study the VNP-protein corona complexes, in order to evaluate the impact of protein corona on molecular recognition of VNPs by target cells, and clearance by phagocytes. We outline procedures for in vivo screening of VNP fates in a mouse model, which may be useful for evaluation of efficacy and biocompatibility of different VNP based formulations.

Treatment of Canine Oral Melanoma with Nanotechnology-Based Immunotherapy and Radiation.

The presence and benefit of a radiation therapy-associated immune reaction is of great interest as the overall interest in cancer immunotherapy expands. The pathological assessment of irradiated tumors rarely demonstrates consistent immune or inflammatory response. More recent information, primarily associated with the "abscopal effect", suggests a subtle radiation-based systemic immune response may be more common and have more therapeutic potential than previously believed. However, to be of consistent value, the immune stimulatory potential of radiation therapy (RT) will clearly need to be supported by combination with other immunotherapy efforts. In this study, using a spontaneous canine oral melanoma model, we have assessed the efficacy and tumor immunopathology of two nanotechnology-based immune adjuvants combined with RT. The immune adjuvants were administered intratumorally, in an approach termed "in situ vaccination", that puts immunostimulatory reagents into a recognized tumor and utilizes the endogenous antigens in the tumor as the antigens in the antigen/adjuvant combination that constitutes a vaccine. The radiation treatment consisted of a local 6 × 6 Gy tumor regimen given over a 12 day period. The immune adjuvants were a plant-based virus-like nanoparticle (VLP) and a 110 nm diameter magnetic iron oxide nanoparticle (mNPH) that was activated with an alternating magnetic field (AMF) to produce moderate heat (43 °C/60 min). The RT was used alone or combined with one or both adjuvants. The VLP (4 × 200 µg) and mNPH (2 × 7.5 mg/gram tumor) were delivered intratumorally respectively during the RT regimen. All patients received a diagnostic biopsy and CT-based 3-D radiation treatment plan prior to initiating therapy. Patients were assessed clinically 14-21 days post-treatment, monthly for 3 months following treatment, and bimonthly, thereafter. Immunohistopathologic assessment of the tumors was performed before and 14-21 days following treatment. Results suggest that addition of VLPs and/or mNPH to a hypofractionated radiation regimen increases the immune cell infiltration in the tumor, extends the tumor control interval, and has important systemic therapeutic potential.

Speciation of Phenanthriplatin and Its Analogs in the Core of Tobacco Mosaic Virus.

Efficient loading of drugs in novel delivery agents has the potential to substantially improve therapy by targeting the diseased tissue while avoiding unwanted side effects. Here we report the first systematic study of the loading mechanism of phenanthriplatin and its analogs into tobacco mosaic virus (TMV), previously used by our group as an efficient carrier for anticancer drug delivery. A detailed investigation of the preferential uptake of phenanthriplatin in its aquated form (∼2000 molecules per TMV particle versus ∼1000 for the chlorido form) is provided. Whereas the net charge of phenanthriplatin analogs and their ionic mobilities have no effect on loading, the reactivity of aqua phenanthriplatin with the glutamates, lining the interior walls of the channel of TMV, has a pronounced effect on its loading. MALDI-MS analysis along with NMR spectroscopic studies of a model reaction of hydroxy-phenanthriplatin with acetate establish the formation of stable covalent adducts. The increased number of heteroaromatic rings on the platinum ligand appears to enhance loading, possibly by stabilizing hydrophobic stacking interactions with TMV core components, specifically Pro102 and Thr103 residues neighboring Glu97 and Glu106 in the channel. Electron transfer dissociation MS/MS fragmentation, a technique that can prevent mass-condition-vulnerable modification of proteins, reveals that Glu97 preferentially participates over Glu106 in covalent bond formation to the platinum center.

Encapsulation of Plant Viral Particles in Calcite Crystals.

The concept of biomineralization and encapsulation of organic molecules into inorganic matrices to alter and enhance their physical properties has been evolved and perfected in natural systems. Being inspired by the natural biomineralization of foreign components into calcite, here the inclusion of a plant virus, cowpea mosaic virus (CPMV) of 5.4% by mass into crystals of calcite is reported. The viral particles are labeled with a fluorescent tag (Alexa Fluor 532), and are observed within the calcite matrix using confocal fluorescence microscopy. Upon encapsulation, the calcite crystals exhibit an irregular and aggregated morphology, as visualized with atomic force and electron microscopy. The viral particles protected inside the calcite crystals are able to resist harsh chemical agents. While spherical viral particles such as CPMV can be easily included in calcite, viruses such as the tobacco mosaic virus are not compatible with the host, presumably due to their high aspect ratio. The results provide a simple and scalable method to incorporate viral particles into inorganic matrix, and could prove useful in thermal stabilization of sensitive viral biological agents such as vaccines in the future.

Effect of intra-tumoral magnetic nanoparticle hyperthermia and viral nanoparticle immunogenicity on primary and metastatic cancer.

Although there is long association of medical hyperthermia and immune stimulation, the relative lack of a quantifiable and reproducible effect has limited the utility and advancement of this relationship in preclinical/clinical cancer and non-cancer settings. Recent cancer-based immune findings (immune checkpoint modulators etc.) including improved mechanistic understanding and biological tools now make it possible to modify and exploit the immune system to benefit conventional cancer treatments such as radiation and hyperthermia. Based on the prior experience of our research group including; cancer-based heat therapy, magnetic nanoparticle (mNP) hyperthermia, radiation biology, cancer immunology and Cowpea Mosaic Virus that has been engineered to over express antigenic proteins without RNA or DNA (eCPMV/VLP). This research was designed to determine if and how the intra-tumoral delivery of mNP hyperthermia and VLP can work together to improve local and systemic tumor treatment efficacy. Using the C3H mouse/MTG-B mammary adenocarcinoma cell model and the C57-B6 mouse/B-16-F10 melanoma cancer cell model, our data suggests the appropriate combination of intra-tumoral mNP heat (e.g. 43°C/30-60 minutes) and VLP (100 µg/200 mm3 tumor) not only result in significant primary tumor regression but the creation a systemic immune reaction that has the potential to retard secondary tumor growth (abscopal effect) and resist tumor rechallenge. Molecular data from these experiments suggest treatment based cell damage and immune signals such as Heat Shock Protein (HSP) 70/90, calreticulin, MTA1 and CD47 are potential targets that can be exploited to enhance the local and systemic (abscopal effect) immune potential of hyperthermia cancer treatment.

Hypo-fractionated Radiation, Magnetic Nanoparticle Hyperthermia and a Viral Immunotherapy Treatment of Spontaneous Canine Cancer.

It has recently been shown that cancer treatments such as radiation and hyperthermia, which have conventionally been viewed to have modest immune based anti-cancer effects, may, if used appropriately stimulate a significant and potentially effective local and systemic anti-cancer immune effect (abscopal effect) and improved prognosis. Using eight spontaneous canine cancers (2 oral melanoma, 3 oral amelioblastomas and 1 carcinomas), we have shown that hypofractionated radiation (6 x 6 Gy) and/or magnetic nanoparticle hyperthermia (2 X 43°C / 45 minutes) and/or an immunogenic virus-like nanoparticle (VLP, 2 x 200 µg) are capable of delivering a highly effective cancer treatment that includes an immunogenic component. Two tumors received all three therapeutic modalities, one tumor received radiation and hyperthermia, two tumors received radiation and VLP, and three tumors received only mNP hyperthermia. The treatment regimen is conducted over a 14-day period. All patients tolerated the treatments without complication and have had local and distant tumor responses that significantly exceed responses observed following conventional therapy (surgery and/or radiation). The results suggest that both hypofractionated radiation and hyperthermia have effective immune responses that are enhanced by the intratumoral VLP treatment. Molecular data from these tumors suggest Heat Shock Protein (HSP) 70/90, calreticulin and CD47 are targets that can be exploited to enhance the local and systemic (abscopal effect) immune potential of radiation and hyperthermia cancer treatment.

Photon Management through Virus-Programmed Supramolecular Arrays.

Photon extraction and capture efficiency is a complex function of the material's composition, its molecular structure at the nanoscale, and the overall organization spanning multiple length scales. The architecture of the material defines the performance; nanostructured features within the materials enhance the energy efficiency. Photon capturing materials are largely produced through lithographic, top-down, manufacturing schemes; however, there are limits to the smallest dimension achievable using this technology. To overcome these technological barriers, a bottom-up nanomanufacturing is pursued. Inspired by the self-programmed assembly of virus arrays in host cells resulting in iridescence of infected organisms, virus-programmed, nanostructured arrays are studied to pave the way for new design principles in photon management and biology-inspired materials science. Using the nanoparticles formed by plant viruses in combination with charged polymers (dendrimers), a bottom-up approach is illustrated to prepare a family of broadband, low-angular dependent antireflection mesoscale layered materials for potential application as photon management coatings. Measurement and theory demonstrate antireflectance and phototrapping properties of the virus-programmed assembly. This opens up new bioengineering principles for the nanomanufacture of coatings and films for use in LED lighting and photovoltaics.

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.

Serum albumin 'camouflage' of plant virus based nanoparticles prevents their antibody recognition and enhances pharmacokinetics.

Plant virus-based nanoparticles (VNPs) are a novel class of nanocarriers with unique potential for biomedical applications. VNPs have many advantageous properties such as ease of manufacture and high degree of quality control. Their biocompatibility and biodegradability make them an attractive alternative to synthetic nanoparticles (NPs). Nevertheless, as with synthetic NPs, to be successful in drug delivery or imaging, the carriers need to overcome several biological barriers including innate immune recognition. Plasma opsonization can tag (V)NPs for clearance by the mononuclear phagocyte system (MPS), resulting in shortened circulation half lives and non-specific sequestration in non-targeted organs. PEG coatings have been traditionally used to 'shield' nanocarriers from immune surveillance. However, due to broad use of PEG in cosmetics and other industries, the prevalence of anti-PEG antibodies has been reported, which may limit the utility of PEGylation in nanomedicine. Alternative strategies are needed to tailor the in vivo properties of (plant virus-based) nanocarriers. We demonstrate the use of serum albumin (SA) as a viable alternative. SA conjugation to tobacco mosaic virus (TMV)-based nanocarriers results in a 'camouflage' effect more effective than PEG coatings. SA-'camouflaged' TMV particles exhibit decreased antibody recognition, as well as enhanced pharmacokinetics in a Balb/C mouse model. Therefore, SA-coatings may provide an alternative and improved coating technique to yield (plant virus-based) NPs with improved in vivo properties enhancing drug delivery and molecular imaging.