Plant viral nanoparticles (VNPs) are attractive to researchers in nanomedicine due to their safety, easy production, resistance, and straightforward functionalization. In this paper, we developed and successfully purified a VNP derived from turnip mosaic virus (TuMV), a well-known plant pathogen, that exhibits high affinity for immunoglobulins G (IgG) thanks to its functionalization with the Z domain from staphylococcal protein A via gene fusion. We selected cetuximab as a model IgG to prove the versatility of this novel TuMV VNP by developing a fluorescent nanoplatform to mark tumoral cells of the line Cal33, from tongue squamous cell carcinoma. We observed that the fluorescent VNP-cetuximab bound selectively to Cal33 and were internalized by confocal microscopy, revealing the potential of this nanotool in cancer research.

Since its introduction, the Triangulation number has been the most successful and ubiquitous scheme for classifying spherical viruses. However, despite its many successes, it fails to describe the relative angular orientations of proteins, as well as their radial mass distribution within the capsid. It also fails to provide any insight into critical sites of stability, modifications or possible mutations. We show how classifying spherical viruses using icosahedral point arrays, introduced by Keef and Twarock, unveils new geometric rules and constraints for understanding virus stability and key locations for exterior and interior modifications. We present a modified fitness measure which classifies viruses in an unambiguous and rigorous manner, irrespective of local surface chemistry, steric hinderance, solvent accessibility or triangulation number. We then utilize these point arrays to explain the immutable surface loops of bacteriophage MS2, the relative reactivity of surface lysines in CPMV and the non-quasiequivalent flexibility of the HBV dimers. We explain how using sister and double point arrays can function as predictive tools for site directed modifications in other systems. This success builds on our previous work showing that viruses place their protruding features along the great circles of the asymmetric unit, demonstrating that viruses indeed adhere to these geometric constraints.

A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by western blot, ELISA and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy-, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and 44 samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5 % and a specificity of 100 % for the certified IVD. There were 7 samples with an uncertain outcome. Our VLP-ELISA showed superior performance with a sensitivity of 97.5 %, a specificity of 100 %, and only 3 uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen.

Cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses is a key step. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by an affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. It was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications.