Generation and characterization of a trackable plant‐made influenza H5 virus‐like particle (VLP) containing enhanced green fluorescent protein (eGFP)

ABSTRACT Medicago, Inc. has developed an efficient virus‐like particle (VLP) vaccine production platform using the Nicotiana benthamiana expression system, and currently has influenza‐based products targeting seasonal/pandemic hemagglutinin (HA) proteins in advanced clinical trials. We wished to generate a trackable HA‐based VLP that would allow us to study both particle assembly in plants and VLP interactions within the mammalian immune system. To this end, a fusion protein was designed, composed of H5 (from influenza A/Indonesia/05/2005 [H5N1]) with enhanced green fluorescent protein (eGFP). Expression of H5‐eGFP in N. benthamiana produced brightly fluorescent ~160 nm particles resembling H5‐VLPS. H5‐eGFP‐VLPs elicited anti‐H5 serologic responses in mice comparable to those elicited by H5‐VLPs in almost all assays tested (hemagglutination inhibition/IgG(total)/IgG1/IgG2b/IgG2a:IgG1 ratio), as well as a superior anti‐GFP IgG response (mean optical density = 2.52 ±0.16 sem) to that elicited by soluble GFP (mean optical density = 0.12 ± 0.06 sem). Confocal imaging of N. benthamiana cells expressing H5‐eGFP displayed large fluorescent accumulations at the cell periphery, and draining lymph nodes from mice given H5‐eGFP‐VLPs via footpad injection demonstrated bright fluorescence shortly after administration (10 min), providing proof of concept that the H5‐eGFP‐protein/VLPs could be used to monitor both VLP assembly and immune trafficking. Given these findings, this novel fluorescent reagent will be a powerful tool to gain further fundamental insight into the biology of influenza VLP vaccines.—Young, K. R., Arthus‐Cartier, G., Yam, K. K., Lavoie, P.‐O., Landry, N., D'Aoust, M.‐A., Vézina, L.‐P., Couture, M. M.‐J., Ward, B. J. Generation and characterization of a trackable plant‐made influenza H5 virus‐like particle (VLP) containing enhanced green fluorescent protein (eGFP). FASEB J. 29, 3817‐3827 (2015). www.fasebj.org