| abstract |
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. |