Display of peptides or proteins in an ordered, repetitive array, such as on the surface of a
virus-like particle, is known to induce an enhanced immune response relative to
vaccination with the “free”
protein antigen. The 2100
coat proteins comprising the rod-shaped
capsid of
Tobacco mosaic virus (TMV) can accommodate short
peptide insertions into the
primary sequence, but the display of larger
protein moieties on the virion surface by genetic fusions to the
capsid protein has not been possible. Since TMV lacks surface exposed residues compatible with commonly available
linker chemistries, we employed a randomized
library approach to introduce a reactive
lysine at the externally located at the amino-terminus of the
coat protein. We found that we could easily control the extent of virion conjugation and demonstrated stoichiometric
biotinylation of the introduced
lysine. To characterize this modular platform for the display of
heterologous proteins, we bound a model
antigen (
streptavidin (SA)-
green fluorescent protein (GFP), expressed and purified from plants) to the surface of TMV, creating a GFP-SA decorated
virus particle. Rapid and
quantitative determination of the level of TMV
capsid decoration was accomplished by subjecting the complex to
amino acid analysis and solving the family of linear equations relating the pmoles of each residue to the known
amino acid composition of the complex components. We obtained a GFP-SA
tetramer loading of 26%, which corresponds to display of approximately 2200 GFP moieties per intact virion. We evaluated the
immunogenicity of GFP decorated virions in both mice and guinea pigs, and found augmented humoral IgG titers in both species, relative to unbound GFP-SA
tetramer. In mice, we observed a detectable humoral immune response after only a single immunization with the TMV-protein complex. By demonstrating the presentation of whole proteins, this study expands the utility of TMV as a vaccine
scaffold beyond that which is possible by genetic manipulation.