Engineering plant-virus like particles to control the aspect ratio for nanovaccine design

• Conrad Gravuer, Chemical Engineering, University of Delaware

• Kevin Solomon, Department of Chemical and Biomolecular Engineering, University of Delaware

The efficacy of nanoparticle-based vaccines depends on targeted antigen delivery to immune cells to elicit a strong immune response. Precise control over particle aspect ratio is essential for cellular uptake, intracellular trafficking, biodistribution, tissue penetration, and immunogenicity. Rod-shaped plant viruses, such as barley stripe mosaic virus (BSMV), are promising candidates for vaccine delivery because they self-assemble into protein nanorods on an RNA template, incorporating hundreds to thousands of copies of capsid protein (CP) subunits. We leverage a bacterial expression system to produce non-infectious BSMV virus-like particles (VLPs) in which the viral genome is replaced with a user-defined RNA template for control over aspect ratio. However, traditional single-promoter expression systems for CP and RNA template have demonstrated that increasing RNA template lengths does not result in proportional increases in rod length, limiting precise control over aspect ratio. To address this, we developed a bacterial expression system that enables orthogonal control of RNA and CP expression via separate inducible promoters. Protein gel electrophoresis confirmed successful protein overexpression in soluble lysate fractions, as well as rod formation in ultracentrifuged virus-like particles (VLPs). Future work will include characterizing encapsidated RNA and assessing how varying RNA template length influences rod length. By enabling better control over stoichiometry, structural uniformity, and aspect ratio, this research supports the development of BSMV VLPs as a versatile platform for vaccine delivery.