title: Influenza virus-like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice.
abstract: A strain-specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant-based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high-molecular-weight structures. H5-containing structures were purified and examination by transmission electron microscopy confirmed virus-like particle (VLP) assembly. High-performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP-producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 microg of purified influenza H5-VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 microg of H5-VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains.
authors.affiliation: Medicago Inc., 1020 Route de l'Eglise, Bureau 600, Québec, QC, Canada, G1V 3V9.
authors.forename: Marc-André
authors.initials: MA
authors.lastname: D'Aoust
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Lavoie
authors.forename: Manon M-J
authors.initials: MM
authors.lastname: Couture
authors.forename: Sonia
authors.initials: S
authors.lastname: Trépanier
authors.forename: Jean-Martin
authors.initials: JM
authors.lastname: Guay
authors.forename: Michèle
authors.initials: M
authors.lastname: Dargis
authors.forename: Sébastien
authors.initials: S
authors.lastname: Mongrand
authors.forename: Nathalie
authors.initials: N
authors.lastname: Landry
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Ward
authors.forename: Louis-P
authors.initials: LP
authors.lastname: Vézina
mesh_terms: D000818:Animals; D006385:Hemagglutination Inhibition Tests; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007252:Influenza Vaccines / Q000235:genetics / Q000276:immunology; D051379:Mice; D009975:Orthomyxoviridae / Q000276:immunology; D009976:Orthomyxoviridae Infections / Q000276:immunology*; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000821:virology*; D014026:Nicotiana / Q000235:genetics* / Q000821:virology*
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines