Inter-coat protein loading of active ingredients into Tobacco mild green mosaic virus through partial dissociation and reassembly of the virion.

Chemical pesticide delivery is a fundamental aspect of agriculture. However, the extensive use of pesticides severely endangers the ecosystem because they accumulate on crops, in soil, as well as in drinking and groundwater. New frontiers in nano-engineering have opened the door for precision agriculture. We introduced Tobacco mild green mosaic virus (TMGMV) as a viable delivery platform with a high aspect ratio and favorable soil mobility. In this work, we assess the use of TMGMV as a chemical nanocarrier for agriculturally relevant cargo. While plant viruses are usually portrayed as rigid/solid structures, these are "dynamic materials," and they "breathe" in solution in response to careful adjustment of pH or bathing media [e.g., addition of solvent such as dimethyl sulfoxide (DMSO)]. Through this process, coat proteins (CPs) partially dissociate leading to swelling of the nucleoprotein complexes-allowing for the infusion of active ingredients (AI), such as pesticides [e.g., fluopyram (FLP), clothianidin (CTD), rifampicin (RIF), and ivermectin (IVM)] into the macromolecular structure. We developed a "breathing" method that facilitates inter-coat protein cargo loading, resulting in up to ~ 1000 AIs per virion. This is of significance since in the agricultural setting, there is a need to develop nanoparticle delivery strategies where the AI is not chemically altered, consequently avoiding the need for regulatory and registration processes of new compounds. This work highlights the potential of TMGMV as a pesticide nanocarrier in precision farming applications; the developed methods likely would be applicable to other protein-based nanoparticle systems.

Systemic Administration of Cowpea Mosaic Virus Demonstrates Broad Protection Against Metastatic Cancers.

The key challenge in cancer treatment is prevention of metastatic disease which is therapeutically resistant and carries poor prognoses necessitating efficacious prophylactic approaches that prevent metastasis and recurrence. It is previously demonstrated that cowpea mosaic virus (CPMV) induces durable antitumor responses when used in situ, i.e., intratumoral injection. As a new direction, it is showed that CPMV demonstrates widespread effectiveness as an immunoprophylactic agent - potent efficacy is demonstrated in four metastatic models of colon, ovarian, melanoma, and breast cancer. Systemic administration of CPMV stimulates the innate immune system, enabling attack of cancer cells; processing of the cancer cells and associated antigens leads to systemic, durable, and adaptive antitumor immunity. Overall, CPMV demonstrated broad efficacy as an immunoprophylactic agent in the rejection of metastatic cancer.

Peptide-Driven Proton Sponge Nano-Assembly for Imaging and Triggering Lysosome-Regulated Immunogenic Cancer Cell Death.

Triggering lysosome-regulated immunogenic cell death (ICD, e.g., pyroptosis and necroptosis) with nanomedicines is an emerging approach for turning an "immune-cold" tumor "hot"-a key challenge faced by cancer immunotherapies. Proton sponge such as high-molecular-weight branched polyethylenimine (PEI) is excellent at rupturing lysosomes, but its therapeutic application is hindered by uncontrollable toxicity due to fixed charge density and poor understanding of resulted cell death mechanism. Here, a series of proton sponge nano-assemblies (PSNAs) with self-assembly controllable surface charge density and cell cytotoxicity are created. Such PSNAs are constructed via low-molecular-weight branched PEI covalently bound to self-assembling peptides carrying tetraphenylethene pyridinium (PyTPE, an aggregation-induced emission-based luminogen). Assembly of PEI assisted by the self-assembling peptide-PyTPE leads to enhanced surface positive charges and cell cytotoxicity of PSNA. The self-assembly tendency of PSNAs is further optimized by tuning hydrophilic and hydrophobic components within the peptide, thus resulting in the PSNA with the highest fluorescence, positive surface charge density, cell uptake, and cancer cell cytotoxicity. Systematic cell death mechanistic studies reveal that the lysosome rupturing-regulated pyroptosis and necroptosis are at least two causes of cell death. Tumor cells undergoing PSNA-triggered ICD activate immune cells, suggesting the great potential of PSNAs to trigger anticancer immunity.

Melanoma immunotherapy enabled by M2 macrophage targeted immunomodulatory cowpea mosaic virus.

We have developed nanoparticle formulations targeting M2 macrophages for cancer immunotherapy by conjugating high-affinity binding peptides to cowpea mosaic virus as an immunostimulatory adjuvant. We confirmed the targeting of and uptake by M2 macrophages in vitro and the therapeutic efficacy of the nanoparticles against murine melanoma in vivo.

Synergistic combination therapy using cowpea mosaic virus intratumoral immunotherapy and Lag-3 checkpoint blockade.

Immune checkpoint therapy (ICT) for cancer can yield dramatic clinical responses; however, these may only be observed in a minority of patients. These responses can be further limited by subsequent disease recurrence and resistance. Combination immunotherapy strategies are being developed to overcome these limitations. We have previously reported enhanced efficacy of combined intratumoral cowpea mosaic virus immunotherapy (CPMV IIT) and ICT approaches. Lymphocyte-activation gene-3 (LAG-3) is a next-generation inhibitory immune checkpoint with broad expression across multiple immune cell subsets. Its expression increases on activated T cells and contributes to T cell exhaustion. We observed heightened efficacy of a combined CPMV IIT and anti-LAG-3 treatment in a mouse model of melanoma. Further, LAG-3 expression was found to be increased within the TME following intratumoral CPMV administration. The integration of CPMV IIT with LAG-3 inhibition holds significant potential to improve treatment outcomes by concurrently inducing a comprehensive anti-tumor immune response, enhancing local immune activation, and mitigating T cell exhaustion.

Pharmacology of a Plant Virus Immunotherapy Candidate for Peritoneal Metastatic Ovarian Cancer.

Due to the increasing incidence of cancer, there is a need to develop new platforms that can combat this disease. Cancer immunotherapy is a platform that takes advantage of the immune system to recognize and eradicate tumors and metastases. Our lab has identified a plant virus nanoparticle, cowpea mosaic virus (CPMV) as a promising approach for cancer immunotherapy. When administered intratumorally, CPMV relieves the immune system of tumor-induced immunosuppression and reprograms the tumor microenvironment into an activated state to launch systemic antitumor immunity. The efficacy of CPMV has been tested in many tumor models and in canine cancer patients with promising results: tumor shrinkage, systemic efficacy (abscopal effect), and immune memory to prevent recurrence. To translate this drug candidate from the bench to the clinic, studies that investigate the safety, pharmacology, and toxicity are needed. In this work, we describe the efficacy of CPMV against a metastatic ovarian tumor model and investigate the biodistribution of CPMV after single or repeated intraperitoneal administration in tumor-bearing and healthy mice. CPMV shows good retention in the tumor nodules and broad bioavailability with no apparent organ toxicity based on histopathology. Data indicate persistence of the viral RNA, which remains detectable 2 weeks post final administration, a phenomenon also observed with some mammalian viral infections. Lastly, while protein was not detected in stool or urine, RNA was shed through excretion from mice; however, there was no evidence that RNA was infectious to plants. Taken together, the data indicate that systemic administration results in broad bioavailability with no apparent toxicity. While RNA is shed from the subjects, data suggest agronomical safety. This data is consistent with prior reports and provides support for translational efforts.

3D bioprinting cowpea mosaic virus as an immunotherapy depot for ovarian cancer prevention in a preclinical mouse model.

Implantable polymeric hydrogels loaded with immunostimulatory cowpea mosaic virus (CPMV) were fabricated using digital light processing (DLP) printing technology. The CPMV-laden hydrogels were surgically implanted into the peritoneal cavity to serve as depots for cancer slow-release immunotherapy. Sustained release of CPMV within the intraperitoneal space alleviates the need for repeated dosing and we demonstrated efficacy against ovarian cancer in a metastatic mouse model.

Study of uricase-polynorbornene conjugates derived from grafting-from ring-opening metathesis polymerization.

PEGylation has been the 'gold standard' in bioconjugation due to its ability to improve the pharmacokinetics and pharmacodynamics of native proteins. However, growing clinical evidence of hypersensitivity reactions to PEG due to pre-existing anti-PEG antibodies in healthy humans have raised concerns. Advancements in controlled polymerization techniques and conjugation chemistries have paved the way for the development of protein-polymer conjugates that can circumvent these adverse reactions while retaining the benefits of such modifications. Herein, we show the development of polynorbornene based bioconjugates of therapeutically relevant urate oxidase (UO) enzymes used in the treatment of gout synthesized by grafting-from ring-opening metathesis polymerization (ROMP). Notably, these conjugates exhibit comparable levels of bioactivity to PEGylated UO and demonstrate increased stability across varying temperatures and pH conditions. Immune recognition of conjugates by anti-UO antibodies reveal low protein immunogenicity following the conjugation process. Additionally, UO conjugates employing zwitterionic polynorbornene successfully avoid recognition by anti-PEG antibodies, further highlighting a potential replacement for PEG.

Pluronic F127 "nanoarmor" for stabilization of Cowpea mosaic virus immunotherapy.

Our lab demonstrated that intratumoral Cowpea mosaic virus (CPMV) is a potent antitumor immunotherapy when used as in situ vaccine. As we pave the way for human clinical translation, formulation chemistry needs to be optimized for long-term storage of the drug candidate. In this work, CPMV was nanoengineered with Pluronic F127 to realize liquid and gel formulations which mitigate structural changes and RNA release during long-term storage. We evaluated the CPMV-F127 formulations for their stability and biological activity through a combination of in vitro assays and efficacy in vivo using a B16F10 murine melanoma model. Results demonstrate that both F127 liquid and gel formulations preserve CPMV structure and function following extended periods of thermal incubation at 4°C, 25°C, and 37°C. Heat-incubated CPMV without formulation resulted in structural changes and inferior in vivo efficacy. In stark contrast, in vivo efficacy was preserved when CPMV was formulated and protected with the F127 "nanoarmor."

Virus-like Particles Armored by an Endoskeleton.

Many virus-like particles (VLPs) have good chemical, thermal, and mechanical stabilities compared to those of other biologics. However, their stability needs to be improved for the commercialization and use in translation of VLP-based materials. We developed an endoskeleton-armored strategy for enhancing VLP stability. Specifically, the VLPs of physalis mottle virus (PhMV) and Qß were used to demonstrate this concept. We built an internal polymer "backbone" using a maleimide-PEG15-maleimide cross-linker to covalently interlink viral coat proteins inside the capsid cavity, while the native VLPs are held together by only noncovalent bonding between subunits. Endoskeleton-armored VLPs exhibited significantly improved thermal stability (95 °C for 15 min), increased resistance to denaturants (i.e., surfactants, pHs, chemical denaturants, and organic solvents), and enhanced mechanical performance. Single-molecule force spectroscopy demonstrated a 6-fold increase in rupture distance and a 1.9-fold increase in rupture force of endoskeleton-armored PhMV. Overall, this endoskeleton-armored strategy provides more opportunities for the development and applications of materials.

Poly(Oxanorbornene)-Protein Conjugates Prepared by Grafting-to ROMP as Alternatives for PEG.

PEGylation is the gold standard in protein-polymer conjugation, improving circulation half-life of biologics while mitigating the immune response to a foreign substance. However, preexisting anti-PEG antibodies in healthy humans are becoming increasingly prevalent and elicitation of anti-PEG antibodies when patients are administered with PEGylated therapeutics challenges their safety profile. In the current study, two distinct amine-reactive poly(oxanorbornene) (PONB) imide-based water-soluble block co-polymers are synthesized using ring-opening metathesis polymerization (ROMP). The synthesized block-copolymers include PEG-based PONB-PEG and sulfobetaine-based PONB-Zwit. The polymers are then covalently conjugated to amine residues of lysozyme (Lyz) and urate oxidase (UO) using a grafting-to bioconjugation technique. Both Lyz-PONB and UO-PONB conjugates retained significant bioactivities after bioconjugation. Immune recognition studies of UO-PONB conjugates indicated a comparable lowering of protein immunogenicity when compared to PEGylated UO. PEG-specific immune recognition is negligible for UO-PONB-Zwit conjugates, as expected. These polymers provide a new alternative for PEG-based systems that retain high levels of activity for the biologic while showing improved immune recognition profiles.

Viral nanoparticle vaccines against S100A9 reduce lung tumor seeding and metastasis.

Metastatic cancer accounts for 90% of all cancer-related deaths and continues to be one of the toughest challenges in cancer treatment. A growing body of data indicates that S100A9, a major regulator of inflammation, plays a central role in cancer progression and metastasis, particularly in the lungs, where S100A9 forms a premetastatic niche. Thus, we developed a vaccine against S100A9 derived from plant viruses and virus-like particles. Using multiple tumor mouse models, we demonstrate the effectiveness of the S100A9 vaccine candidates in preventing tumor seeding within the lungs and outgrowth of metastatic disease. The elicited antibodies showed high specificity toward S100A9 without cross-reactivity toward S100A8, another member of the S100A family. When tested in metastatic mouse models of breast cancer and melanoma, the vaccines significantly reduced lung tumor nodules after intravenous challenge or postsurgical removal of the primary tumor. Mechanistically, the vaccines reduce the levels of S100A9 within the lungs and sera, thereby increasing the expression of immunostimulatory cytokines with antitumor function [(interleukin) IL-12 and interferonγ] while reducing levels of immunosuppressive cytokines (IL-10 and transforming growth factorß). This also correlated with decreased myeloid-derived suppressor cell populations within the lungs. This work has wide-ranging impact, as S100A9 is overexpressed in multiple cancers and linked with poor prognosis in cancer patients. The data presented lay the foundation for the development of therapies and vaccines targeting S100A9 to prevent metastasis.

Two decades of vaccine development against atherosclerosis.

Atherosclerosis is an immune-mediated chronic inflammatory disease that leads to the development of fatty plaques in the arterial walls, ultimately increasing the risk of thrombosis, stroke, and myocardial infarction. The immune response in this complex disease is both atheroprotective and pro-atherogenic, involving both innate and adaptive immunity. Current treatments include the adjustment of lifestyle factors, cholesterol-lowering drugs such as statins, and immunotherapy, whereas vaccine development has received comparatively little attention. In this review, we discuss the potential of antigen-specific vaccination as a preventative approach based on more than 20 years of research and innovation. Vaccination targets include proteins that are more abundant in atherosclerotic patients, such as oxidized low-density lipoprotein (LDL), apolipoprotein B-100, proprotein convertase subtilisin/kexin type-9 serine protease (PCSK9), cholesteryl ester transfer protein (CETP), and heat shock proteins HSP60 and HSP65. Immunization with such proteins or their peptide epitopes has been shown to induce T-cell activation, produce antigen-specific antibodies, reduce the size of atherosclerotic lesions, and/or reduce serum cholesterol levels. Vaccination against atherosclerosis therefore offers a new strategy to address the burden on healthcare systems caused by cardiovascular disease, the leading cause of death worldwide.

Neoadjuvant Intratumoral Immunotherapy with Cowpea Mosaic Virus Induces Local and Systemic Antitumor Efficacy in Canine Mammary Cancer Patients.

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients.

Transcriptomics of Canine Inflammatory Mammary Cancer Treated with Empty Cowpea Mosaic Virus Implicates Neutrophils in Anti-Tumor Immunity.

Canine inflammatory mammary cancer (IMC) is a highly aggressive and lethal cancer in dogs serving as a valuable animal model for its human counterpart, inflammatory breast cancer (IBC), both lacking effective therapies. Intratumoral immunotherapy (IT-IT) with empty cowpea mosaic virus (eCPMV) nanoparticles has shown promising results, demonstrating a reduction in tumor size, longer survival rates, and improved quality of life. This study compares the transcriptomic profiles of tumor samples from female dogs with IMC receiving eCPMV IT-IT and medical therapy (MT) versus MT alone. Transcriptomic analyses, gene expression profiles, signaling pathways, and cell type profiling of immune cell populations in samples from four eCPMV-treated dogs with IMC and four dogs with IMC treated with MT were evaluated using NanoString Technologies using a canine immune-oncology panel. Comparative analyses revealed 34 differentially expressed genes between treated and untreated samples. Five genes (CXCL8, S100A9, CCL20, IL6, and PTGS2) involved in neutrophil recruitment and activation were upregulated in the treated samples, linked to the IL17-signaling pathway. Cell type profiling showed a significant increase in neutrophil populations in the tumor microenvironment after eCPMV treatment. These findings highlight the role of neutrophils in the anti-tumor response mediated by eCPMV IT-IT and suggest eCPMV as a novel therapeutic approach for IBC/IMC.

TLR Agonists Delivered by Plant Virus and Bacteriophage Nanoparticles for Cancer Immunotherapy.

Toll-like receptors (TLRs) are promising targets in cancer immunotherapy due to their role in activating the immune system; therefore, various small-molecule TLR agonists have been tested in clinical applications. However, the clinical use of TLR agonists is hindered by their non-specific side effects and poor pharmacokinetics. To overcome these limitations, we used plant virus nanoparticles (VNPs) and bacteriophage virus-like particles (VLPs) as drug delivery systems. We conjugated TLR3 or TLR7 agonists to cowpea mosaic virus (CPMV) VNPs, cowpea chlorotic mottle virus (CCMV) VNPs, and bacteriophage Qß VLPs. The conjugation of TLR7 agonist, 2-methoxyethoxy-8-oxo-9-(4-carboxybenzyl)adenine (1V209), resulted in the potent activation of immune cells and promoted the production of pro-inflammatory cytokine interleukin 6. We found that 1V209 conjugated to CPMV, CCMV, and Qß reduced tumor growth in vivo and prolonged the survival of mice compared to those treated with free 1V209 or a simple admixture of 1V209 and viral particles. Nucleic acid-based TLR3 agonist, polyinosinic acid with polycytidylic acid (poly(I:C)), was also delivered by CPMV VNPs, resulting in enhanced mice survival. All our data suggest that coupling and co-delivery are required to enhance the anti-tumor efficacy of TLR agonists and simple mixing of the VLPs with the agonists does not confer a survival benefit. The delivery of 1V209 or poly(I:C) conjugated to VNPs/VLPs probably enhances their efficacy due to the multivalent presentation, prolongation of tumor residence time, and targeting of the innate immune cells mediated by the VNP/VLP carrier.

Physalis Mottle Virus-Like Nanocarriers with Expanded Internal Loading Capacity.

An ongoing challenge in precision medicine is the efficient delivery of therapeutics to tissues/organs of interest. Nanoparticle delivery systems have the potential to overcome traditional limitations of drug and gene delivery through improved pharmacokinetics, tissue targeting, and stability of encapsulated cargo. Physalis mottle virus (PhMV)-like nanoparticles are a promising nanocarrier platform which can be chemically targeted on the exterior and interior surfaces through reactive amino acids. Cargo-loading to the internal cavity is achieved with thiol-reactive small molecules. However, the internal loading capacity of these nanoparticles is limited by the presence of a single reactive cysteine (C75) per coat protein with low inherent reactivity. Here, we use structure-based design to engineer cysteine-added mutants of PhMV VLPs that display increased reactivity toward thiol-reactive small molecules. Specifically, the A31C and S137C mutants show a greater than 10-fold increased rate of reactivity towards thiol-reactive small molecules, and PhMV Cys1 (A31C), PhMV Cys2 (S137C), and PhMV Cys1+2 (double mutant) VLPs display up to three-fold increased internal loading of the small molecule chemotherapeutics aldoxorubicin and vcMMAE and up to four-fold increased internal loading of the MRI imaging reagent DOTA(Gd). These results further improve upon a promising plant virus-based nanocarrier system for use in targeted delivery of small-molecule drugs and imaging reagents in vivo.

In Vitro and Ex Planta Gold-Bonded and Gold-Mineralized Tobacco Mosaic Virus.

Biotemplated mineralization is a promising and ecofriendly approach to manufacture metal nanoparticles and composites with precise size control. Plant viruses are suitable templates for biomineralization because they are chemically robust and highly scalable through molecular farming. Here, we report a gold-nanoparticle-coated tobacco mosaic virus (TMV) synthesized in a test tube or in plant extracts making use of a TMV displaying a gold-binding peptide (GBP). The methods developed are a step toward engineered living materials, where gold nanowires could be formed in plant tissues for sensing or energy harvest applications.

Delivery of Nematicides Using TMGMV-Derived Spherical Nanoparticles.

Spherical nanoparticles (SNPs) from tobacco mild green mosaic virus (TMGMV) were developed and characterized, and their application for agrochemical delivery was demonstrated. Specifically, we set out to develop a platform for pesticide delivery targeting nematodes in the rhizosphere. SNPs were obtained by thermal shape-switching of the TMGMV. We demonstrated that cargo can be loaded into the SNPs during thermal shape-switching, enabling the one-pot synthesis of functionalized nanocarriers. Cyanine 5 and ivermectin were encapsulated into SNPs to achieve 10% mass loading. SNPs demonstrated good mobility and soil retention slightly higher than that of TMGMV rods. Ivermectin delivery to Caenorhabditis elegans using SNPs was determined after passing the formulations through soil. Using a gel burrowing assay, we demonstrate the potent efficacy of SNP-delivered ivermectin against nematodes. Like many pesticides, free ivermectin is adsorbed in the soil and did not show efficacy. The SNP nanotechnology offers good soil mobility and a platform technology for pesticide delivery to the rhizosphere.

Potato Virus X Inactivation and Characterization.

The nucleoprotein components of plant viruses self-assemble into monodisperse, nanoscale structures with a high degree of symmetry and polyvalency. Of particular interest are the filamentous plant viruses which provide uniform high aspect ratio nanostructures-such structures remain challenging to obtain using purely synthetic approaches. Potato virus X (PVX) has drawn interest by the materials science community because of its filamentous structure measuring 515 × 13 nm; and both genetic engineering and chemical conjugation methods have been reported to impart new functionalities and develop PVX-based nanomaterials for applications in the health and materials sector. Toward environmentally safe materials-i.e., materials that are not infectious toward crops, such as potato, we reported methods to inactivate PVX. In this chapter, we describe the three methods to inactivate PVX and render it non-infectious toward plants, while maintaining structure and function.