ENABLING SURFACE FUNCTIONALIZATION OF BARLEY STRIPE MOSAIC VIRUS-LIKE PARTICLES FOR VACCINE DEVELOPMENT
Date
2025-05
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Virus-like particles (VLPs) have tremendous potential for medical applications,
including theranostics, vaccines, and drug delivery. Of particular interest are rod-shaped
plant viruses, such as barley stripe mosaic virus (BSMV), due to their precise helical
structure, tunable size, biocompatibility, and sustainable bioproduction. However,
classical manufacturing in plants is slow and inflexible due to the requirement of host
infectivity. Our lab was the first to generate bacterially derived BSMV VLPs, which
offer an opportunity for flexible, high-density surface functionalization with antigens
and other ligands. We hypothesized that cysteine residues and peptidic ligands could be
inserted at the surface-exposed C-terminus of BSMV VLPs for modular surface
functionalization, enabling the use of BSMV VLPs as vectors for antigen delivery and
subsequent presentation on macrophages. Cysteine, which is absent in the native coat
protein, contains a thiol group that can undergo selective click chemistries such as the
thiol-ene and thiol-Michael reactions. I was able to create a BSMV VLP rod with
cysteine on its surface. TEM imaging confirmed that the VLPs maintained structural
integrity after cysteine incorporation. We validated the functionality of our cysteine displaying VLPs via conjugation to maleimide-linked fluorescent dyes, which we then
used to demonstrate uptake of BSMV VLPs into murine macrophages. Model
ovalbumin antigen fusions onto the cysteine-functional VLPs were also compatible with
protein expression and particle assembly, as confirmed by protein gel electrophoresis
and TEM, respectively. The cysteine motifs in these ovalbumin antigen fusion VLPs
were still accessible and chemically reactive, enabling the creation of dual-functional
VLPs that present both antigen and a fluorescent marker. Furthermore, the model
antigens displayed by the VLPs were effectively processed and presented on MHC-I
complexes by murine macrophages in a dose-dependent manner. Antigen-presenting
macrophages activated the B3Z CD8+ T cell hybridoma, which selectively recognizes
MHC-I loaded with ovalbumin antigen. Our experiments are the first demonstration of
BSMV VLP’s capacity for self-adjuvanted antigen delivery. Collectively, these results
help to establish BSMV VLPs as a modular platform for nanovaccine development.