Cowpea chlorotic mottle virus (CCMV) is a positive-sense RNA virus that can be repurposed for gene delivery applications. Understanding the self-assembly process of the virus enabled to remove its genome and replace it with desired nucleic acids, and we and others have previously reported using CCMV virus-like particle (VLP) to encapsulate siRNA, mRNA, as well as CpG oligodeoxynucleotides. In this study, the CCMV VLP was applied to encapsulate two different formats of anti-miR-181a oligonucleotides: naked RNA and chemically stabilized RNA to knockdown highly regulated miR-181a in ovarian cancer cells. miR-181a expression in ovarian tumors is associated with high aggressiveness, invasiveness, resistance to chemotherapy, and overall poor prognosis. Therefore, miR-181a is an important target for ovarian cancer therapy. qPCR data and cancer cell migration assays demonstrated higher knockdown efficacy when anti-miR-181a oligonucleotides were encapsulated and delivered using the VLPs resulting in reduced cancer cell invasiveness. Importantly, delivery of anti-miR-181a oligonucleotide into cells could be achieved without the aid of a transfection agent or surface modification. These results highlight the opportunity of plant-derived VLPs as nucleic acid carriers.

Cowpea mosaic virus (CPMV) is a nucleoprotein nanoparticle that functions as a highly potent immunomodulator when administered intratumorally and is used as an in situ vaccine. CPMV in situ vaccination remodels the tumor microenvironment and primes a highly potent, systemic, and durable antitumor immune response against the treated and untreated, distant metastatic sites (abscopal effect). Potent efficacy was demonstrated in multiple tumor mouse models and, most importantly, in canine cancer patients with spontaneous tumors. Data indicate that presence of anti-CPMV antibodies are not neutralizing and that in fact opsonization leads to enhanced efficacy. Plant viruses are part of the food chain, but to date, there is no information on human exposure to CPMV. Therefore, patient sera were tested for the presence of immunoglobulins against CPMV, and indeed, >50% of deidentified patient samples tested positive for CPMV antibodies. To get a broader sense of plant virus exposure and immunogenicity in humans, we also tested sera for antibodies against tobacco mosaic virus (>90% patients tested positive), potato virus X (<20% patients tested positive), and cowpea chlorotic mottle virus (no antibodies were detected). Further, patient sera were analyzed for the presence of antibodies against the coliphage Qβ, a platform technology currently undergoing clinical trials for in situ vaccination; we found that 60% of patients present with anti-Qβ antibodies. Thus, data indicate human exposure to CPMV and other plant viruses and phages. Next, we thought to address agronomical safety; i.e., we examined the fate of CPMV after intratumoral treatment and oral gavage (to mimic consumption by food). Because live CPMV is used, an important question is whether there is any evidence of shedding of infectious particles from mice or patients. CPMV is noninfectious toward mammals; however, it is infectious toward plants including black-eyed peas and other legumes. Biodistribution data in tumor-bearing and healthy mice indicate little leaching from tumors and clearance via the reticuloendothelial system followed by biliary excretion. While there was evidence of shedding of RNA in stool, there was no evidence of infectious particles when plants were challenged with stool extracts, thus indicating agronomical safety. Together these data aid the translational development of CPMV as a drug candidate for cancer immunotherapy.

Many plant virus-like particles (VLPs) utilized in nanotechnology are 30-nm icosahedrons. To expand the VLP platforms, we produced VLPs of Cytoplasmic type citrus leprosis virus (CiLV-C) in Nicotiana benthamiana. We were interested in CiLV-C because of its unique bacilliform shape (60-70 nm × 110-120 nm). The CiLV-C capsid protein (p29) gene was transferred to the pTRBO expression vector transiently expressed in leaves. Stable VLPs were formed, as confirmed by agarose gel electrophoresis, transmission electron microscopy and size exclusion chromatography. Interestingly, the morphology of the VLPs (15.8 ± 1.3 nm icosahedral particles) differed from that of the native bacilliform particles indicating that the assembly of native virions is influenced by other viral proteins and/or the packaged viral genome. The smaller CiLV-C VLPs will also be useful for structure-function studies to compare with the 30-nm icosahedrons of other VLPs.

Coronavirus disease 2019 (COVID-19) is a highly transmissible disease that has affected more than 90% of the countries worldwide. At least 17 million individuals have been infected, and some countries are still battling first or second waves of the pandemic. Nucleic acid tests, especially reverse transcription polymerase chain reaction (RT-PCR), have become the workhorse for early detection of COVID-19 infection. Positive controls for the molecular assays have been developed to validate each test and to provide high accuracy. However, most available positive controls require cold-chain distribution and cannot serve as full-process control. To overcome these shortcomings, we report the production of biomimetic virus-like particles (VLPs) as SARS-CoV-2 positive controls. A SARS-CoV-2 detection module for RT-PCR was encapsidated into VLPs from a bacteriophage and a plant virus. The chimeric VLPs were obtained either by in vivo reconstitution and coexpression of the target detection module and coat proteins or by in vitro assembly of purified detection module RNA sequences and coat proteins. These VLP-based positive controls mimic SARS-CoV-2 packaged ribonucleic acid (RNA) while being noninfectious. Most importantly, we demonstrated that the positive controls are scalable, stable, and can serve broadly as controls, from RNA extraction to PCR in clinical settings.

Prostate-specific membrane antigen (PSMA) is a membrane-bound protein that is preferentially expressed in the prostate gland and induced in many prostate cancers, making it an important target for new diagnostics and therapeutics. To improve the efficacy of nanoparticle formulations for the imaging and/or eradication of prostate cancer, we synthesized the PSMA-binding glutamic acid derivative DUPA and conjugated it to the external surface of tobacco mosaic virus (TMV) particles. DUPA-targeted TMV was subsequently loaded with the antineoplastic agent mitoxantrone (MTO) or conjugated internally with the fluorescent dye cyanine 5 (Cy5). We found that TMV particles could be efficiently decorated with DUPA and loaded with MTO or Cy5 while maintaining structural integrity. DUPA-targeted TMV particles were able to bind more efficiently to the surface of PSMA⁺ LNCaP cells compared to non-targeted TMV; but there was little difference in binding efficiency between targeted and untargeted TMV when we tested PSMA^- PC3 cells (both cell lines are prostate cancer cell lines). DUPA-targeted TMV particles were internalized by LNCaP cells enabling drug delivery. Finally, we loaded the DUPA-targeted TMV particles and untargeted control particles with MTO to test their cytotoxicity against LNCaP cells in vitro. The cytotoxicity of the TMV-MTO particles (IC₅₀ = 10.2 nM) did not differ significantly from that of soluble MTO at an equivalent dose (IC₅₀ = 12.5 nM) but the targeted particles (TMV-DUPA-MTO) were much more potent (IC₅₀ = 2.80 nM). The threefold increase in cytotoxicity conferred by the DUPA ligand suggests that MTO-loaded, DUPA-coated TMV particles are promising as a therapeutic strategy for PSMA⁺ prostate cancer and should be advanced to preclinical testing in mouse models of prostate cancer.

Antimicrobial resistance is a global health threat that is exacerbated by the overuse and misuse of antibiotics in medicine and agriculture. As an alternative to conventional antimicrobial drugs, phage therapy involves the treatment of infected patients with a bacteriophage that naturally destroys bacterial pathogens. With the re-emergence of phage therapy, novel tools are needed to study phages. In this work we set out to screen and isolate peptide candidates that bind to phages and act as affinity tags. Such peptides functionalized with an imaging agent could serves as versatile tools for tracking and imaging of phages. Specifically, we screened a phage display library for peptides that bind to the Good Vibes phage (GV), which lyses the bacterial pathogen Pseudomonas aeruginosa. Isolated monoclonal library phages featured a highly conserved consensus motif, LPPIXRX. The corresponding peptide WDLPPIGRLSGN was synthesized with a GGGSK linker and conjugated to cyanine 5 or biotin. The specific binding of the LPPIXRX motif to GV in vitro was confirmed using an enzyme-linked immunosorbent assay. We demonstrated imaging and tracking of GV in bacterial populations using the fluorescent targeting peptide and flow cytometry. In conclusion, we developed fluorescent labeled peptides that can bind to bacteriophage GV specifically, which may enable real-time analysis of phage in vivo and monitor the efficacy of phage therapy.