title: 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.
authors.affiliation: *Research Institute of McGill University Health Centre and Department of Experimental Medicine, McGill University, Montréal, Québec, Canada; and Medicago, Incorporated, Québec, Québec, Canada.
authors.forename: Katie R
authors.initials: KR
authors.lastname: Young
authors.affiliation: *Research Institute of McGill University Health Centre and Department of Experimental Medicine, McGill University, Montréal, Québec, Canada; and Medicago, Incorporated, Québec, Québec, Canada.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Arthus-Cartier
authors.affiliation: *Research Institute of McGill University Health Centre and Department of Experimental Medicine, McGill University, Montréal, Québec, Canada; and Medicago, Incorporated, Québec, Québec, Canada.
authors.forename: Karen K
authors.initials: KK
authors.lastname: Yam
authors.affiliation: *Research Institute of McGill University Health Centre and Department of Experimental Medicine, McGill University, Montréal, Québec, Canada; and Medicago, Incorporated, Québec, Québec, Canada.
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Lavoie
authors.affiliation: *Research Institute of McGill University Health Centre and Department of Experimental Medicine, McGill University, Montréal, Québec, Canada; and Medicago, Incorporated, Québec, Québec, Canada.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Landry
authors.affiliation: *Research Institute of McGill University Health Centre and Department of Experimental Medicine, McGill University, Montréal, Québec, Canada; and Medicago, Incorporated, Québec, Québec, Canada.
authors.forename: Marc-André
authors.initials: MA
authors.lastname: D'Aoust
authors.affiliation: *Research Institute of McGill University Health Centre and Department of Experimental Medicine, McGill University, Montréal, Québec, Canada; and Medicago, Incorporated, Québec, Québec, Canada.
authors.forename: Louis-Philippe
authors.initials: LP
authors.lastname: Vézina
authors.affiliation: *Research Institute of McGill University Health Centre and Department of Experimental Medicine, McGill University, Montréal, Québec, Canada; and Medicago, Incorporated, Québec, Québec, Canada.
authors.forename: Manon M-J
authors.initials: MM
authors.lastname: Couture
authors.affiliation: *Research Institute of McGill University Health Centre and Department of Experimental Medicine, McGill University, Montréal, Québec, Canada; and Medicago, Incorporated, Québec, Québec, Canada brian.ward@mcgill.ca.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Ward
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D005260:Female; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000276:immunology; D007252:Influenza Vaccines / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D000914:Antibodies, Viral; D007252:Influenza Vaccines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: H5 hemagglutinin; fluorescent particle; immune response; particle assembly