title: Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana.
abstract: Filoviruses (Ebola and Marburg viruses) cause severe and often fatal haemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identifies Ebola and Marburg viruses as 'category A' pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost-effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C-terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co-expression of the GP1-heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of N. benthamiana produced assembled immunoglobulin, which was purified by ammonium sulphate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size-exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti-Ebola virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential for a plant-expressed EIC as a human vaccine.
authors.affiliation: Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, Arizona, USA.
authors.forename: Waranyoo
authors.initials: W
authors.lastname: Phoolcharoen
authors.forename: Seong H
authors.initials: SH
authors.lastname: Bhoo
authors.forename: Huafang
authors.initials: H
authors.lastname: Lai
authors.forename: Julian
authors.initials: J
authors.lastname: Ma
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Arntzen
authors.forename: Qiang
authors.initials: Q
authors.lastname: Chen
authors.forename: Hugh S
authors.initials: HS
authors.lastname: Mason
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D000914:Antibodies, Viral / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D015922:Complement C1q / Q000235:genetics / Q000378:metabolism; D029043:Ebolavirus / Q000235:genetics / Q000276:immunology*; D005260:Female; D017791:Geminiviridae / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D019142:Hemorrhagic Fever, Ebola / Q000517:prevention & control*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D010641:Phenotype; D018515:Plant Leaves; D011993:Recombinant Fusion Proteins; D012093:Replicon; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D014611:Vaccination; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C113189:envelope glycoprotein, Ebola virus; D015922:Complement C1q