Plant virus nanoparticles (VNPs) have multiple advantages over their synthetic counterparts including the cost-effective large-scale manufacturing of uniform particles that are easy to functionalize. Tobacco mosaic virus (TMV) is one of the most promising VNP scaffolds, reflecting its high aspect ratio and ability to carry and/or display multivalent therapeutic ligands and contrast agents. Here we investigated the circulation, protein corona, immunogenicity, and organ distribution/clearance of TMV particles internally co-labeled with cyanine 5 (Cy5) and chelated gadolinium (Gd) for dual tracking by fluorescence imaging and optical emission spectrometry, with or without an external coating of polydopamine (PDA) to confer photothermal and photoacoustic capabilities. The PDA-coated particles (Gd-Cy5-TMV-PDA) showed a shorter plasma circulation time and broader distribution to organs of the reticuloendothelial system (liver, lungs, and spleen) than uncoated Gd-Cy5-TMV particles (liver and spleen only). The Gd-Cy5-TMV-PDA particles were surrounded by 2-10-fold greater protein corona (containing mainly immunoglobulins) compared to Gd-Cy5-TMV particles. However, the enzyme-linked immunosorbent assay (ELISA) revealed that PDA-coated particles bind 2-fold lesser to anti-TMV antibodies elicited by particle injection than uncoated particles, suggesting that the PDA coat enables evasion from systemic antibody surveillance. Gd-Cy5-TMV-PDA particles were cleared from organs after 8 days compared to 5 days for the uncoated particles. The slower tissue clearance of the coated particles makes them ideal for theranostic applications by facilitating sustained local delivery in addition to multimodal imaging and photothermal capabilities. We have demonstrated the potential of PDA-coated proteinaceous nanoparticles for multiple biomedical applications.

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.

Despite aggressive multi-modality treatment with surgery, radiation and chemotherapies, malignant glioma inevitably recurs and has dismal survival rates. Recent progress in immunotherapy has led to a resurgence of interest, and immunotherapies are being investigated for treatment of glioma. However, the unique brain anatomy and a highly immunosuppressive glioma microenvironment pose significant challenges to achieving efficacy. Thus, there is a critical need for assessment of next-generation immunotherapies for glioma. In this study, we have investigated the efficacy of the nanoparticle platform technology based on plant-derived Cowpea mosaic virus like particles (empty CPMV or eCPMV) to instigate a potent immune response against intracranial glioma. CPMV immunotherapy has been shown to efficiently reverse the immunosuppressive tumor microenvironments in pre-clinical murine models of dermal melanoma and metastatic melanoma, metastatic breast cancer, intraperitoneal ovarian cancer and in canine patients with oral melanoma. In the present study, we demonstrate that in situ administration of CPMV immunotherapy in the setting of glioma can effectively recruit unique subset of effector innate and adaptive immune cells to the brain parenchyma while reducing immune suppressive cellular population, leading to regression of intracranial glioma. The in situ CPMV nanoparticle vaccine offers a potent yet safe and localized immunotherapy for intracranial glioma.

The immunosuppressive tumor microenvironment enables cancer to resist immunotherapies. We have established that intratumoral administration of plant-derived Cowpea mosaic virus (CPMV) nanoparticles as an in situ vaccine overcomes the local immunosuppression and stimulates a potent anti-tumor response in several mouse cancer models and canine patients. CPMV does not infect mammalian cells but acts as a danger signal that leads to the recruitment and activation of innate and subsequently, adaptive immune cells. In the present study we addressed whether other icosahedral viruses or virus-like particles (VLPs) of plant, bacteriophage and mammalian origin can be similarly employed as intratumoral immunotherapy. Our results indicate that CPMV in situ vaccine outperforms Cowpea chlorotic mottle virus (CCMV), Physalis mosaic virus (PhMV), Sesbania mosaic virus (SeMV), bacteriophage Qβ VLPs, or Hepatitis B virus capsids (HBVc). Furthermore, ex vivo and in vitro assays reveal unique features of CPMV that makes it an inherently stronger immune stimulant.

Biologics - medications derived from a biological source - are increasingly used as pharmaceuticals, for example, as vaccines. Biologics are usually produced in bacterial, mammalian or insect cells. Alternatively, plant molecular farming, that is, the manufacture of biologics in plant cells, transgenic plants and algae, offers a cheaper and easily adaptable strategy for the production of biologics, in particular, in low-resource settings. In this Review, we discuss current vaccination challenges, such as cold chain requirements, and highlight how plant molecular farming in combination with advanced materials can be applied to address these challenges. The production of plant viruses and virus-based nanotechnologies in plants enables low-cost and regional fabrication of thermostable vaccines. We also highlight key new vaccine delivery technologies, including microneedle patches and material platforms for intranasal and oral delivery. Finally, we provide an outlook of future possibilities for plant molecular farming of next-generation vaccines and biologics.

The COVID-19 pandemic caused by SARS-CoV-2 sparked intensive research into the development of effective vaccines, 50 of which have been approved thus far, including the novel mRNA-based vaccines developed by Pfizer and Moderna. Although limiting the severity of the disease, the mRNA-based vaccines presented drawbacks, such as the cold chain requirement. Moreover, antibody levels generated by these vaccines decline significantly after 6 months. These vaccines deliver mRNA encoding the full-length spike (S) glycoprotein of SARS-CoV-2, but must be updated as new strains and variants of concern emerge, creating a demand for adjusted formulations and booster campaigns. To overcome these challenges, we have developed COVID-19 vaccine candidates based on the highly conserved SARS CoV-2, 809-826 B-cell peptide epitope (denoted 826) conjugated to cowpea mosaic virus (CPMV) nanoparticles and bacteriophage Qβ virus-like particles, both platforms have exceptional thermal stability and facilitate epitope delivery with inbuilt adjuvant activity. We evaluated two administration methods: subcutaneous injection and an implantable polymeric scaffold. Mice received a prime-boost regimen of 100 μg per dose (2 weeks apart) or a single dose of 200 μg administered as a liquid formulation, or a polymer implant. Antibody titers were evaluated longitudinally over 50 weeks. The vaccine candidates generally elicited an early Th2-biased immune response, which stimulates the production of SARS-CoV-2 neutralizing antibodies, followed by a switch to a Th1-biased response for most formulations. Exceptionally, vaccine candidate 826-CPMV (administered as prime-boost, soluble injection) elicited a balanced Th1/Th2 immune response, which is necessary to prevent pulmonary immunopathology associated with Th2 bias extremes. While the Qβ-based vaccine elicited overall higher antibody titers, the CPMV-induced antibodies had higher avidity. Regardless of the administration route and formulation, our vaccine candidates maintained high antibody titers for more than 50 weeks, confirming a potent and durable immune response against SARS-CoV-2 even after a single dose.

The use of microneedles has facilitated the painless localized delivery of drugs across the skin. However, their efficacy has been limited by slow diffusion of molecules and often requires external triggers. Herein, an autonomous and degradable, active microneedle delivery platform is introduced, employing magnesium microparticles loaded within the microneedle patch, as the built-in engine for deeper and faster intradermal payload delivery. The magnesium particles react with the interstitial fluid, leading to an explosive-like rapid production of H₂ bubbles, providing the necessary force to breach dermal barriers and enhance payload delivery. The release kinetics of active microneedles is evaluated in vitro by measuring the amount of IgG antibody (as a model drug) that passed through phantom tissue and a pigskin barrier. In vivo experiments using a B16F10 mouse melanoma model demonstrate that the active delivery of anti-CTLA-4 (a checkpoint inhibitor drug) results in greatly enhanced immune response and significantly longer survival. Moreover, spatially resolved zones of active and passive microneedles allow a combinatorial rapid burst response along with slow, sustained release, respectively. Such versatile and effective autonomous dynamic microneedle delivery technology offers considerable promise for a wide range of therapeutic applications, toward a greatly enhanced outcome, convenience, and cost.

The development of vaccines against coronaviruses has focused on the spike (S) protein, which is required for the recognition of host-cell receptors and thus elicits neutralizing antibodies. Targeting conserved epitopes on the S protein offers the potential for pan-beta-coronavirus vaccines that could prevent future pandemics. We displayed five B-cell epitopes, originally identified in the convalescent sera from recovered severe acute respiratory syndrome (SARS) patients, on the surface of the cowpea mosaic virus (CPMV) and evaluated these formulations as vaccines. Prime-boost immunization of mice with three of these candidate vaccines, CPMV-988, CPMV-1173, and CPMV-1209, elicited high antibody titers that neutralized the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro and showed an early Th1-biased profile (2-4 weeks) transitioning to a slightly Th2-biased profile just after the second boost (6 weeks). A pentavalent slow-release implant comprising all five peptides displayed on the CPMV elicited anti-S protein and epitope-specific antibody titers, albeit at a lower magnitude compared to the soluble formulations. While the CPMV remained intact when released from the PLGA implants, processing results in loss of RNA, which acts as an adjuvant. Loss of RNA may be a reason for the lower efficacy of the implants. Finally, although the three epitopes (988, 1173, and 1209) that were found to be neutralizing the SARS-CoV were 100% identical to the SARS-CoV-2, none of the vaccine candidates neutralized the SARS-CoV-2 in vitro suggesting differences in the natural epitope perhaps caused by conformational changes or the presence of N-linked glycans. While a cross-protective vaccine candidate was not developed, a multivalent SARS vaccine was developed. The technology discussed here is a versatile vaccination platform that can be pivoted toward other diseases and applications that are not limited to infectious diseases.