title: Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV.
abstract: Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb.
authors.affiliation: a The Hotung Molecular Immunology Group; Institute for Infection & Immunity; St George's ; University of London ; London , UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Paul
authors.forename: Rajko
authors.initials: R
authors.lastname: Reljic
authors.forename: Katja
authors.initials: K
authors.lastname: Klein
authors.forename: Pascal M W
authors.initials: PM
authors.lastname: Drake
authors.forename: Craig
authors.initials: C
authors.lastname: van Dolleweerd
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.forename: Markus
authors.initials: M
authors.lastname: Windwarder
authors.forename: Elsa
authors.initials: E
authors.lastname: Arcalis
authors.forename: Eva
authors.initials: E
authors.lastname: Stoger
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.forename: Catherine
authors.initials: C
authors.lastname: Cosgrove
authors.forename: Angela
authors.initials: A
authors.lastname: Bartolf
authors.forename: Susan
authors.initials: S
authors.lastname: Baden
authors.forename: Julian K-C
authors.initials: JK
authors.lastname: Ma
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000276:immunology; D001826:Body Fluids / Q000276:immunology / Q000378:metabolism; D005260:Female; D006031:Glycosylation; D006678:HIV / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007071:Immunoglobulin A, Secretory / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000032:analysis / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014621:Vagina / Q000276:immunology / Q000378:metabolism; D014771:Virion / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D054299:env Gene Products, Human Immunodeficiency Virus / Q000276:immunology / Q000378:metabolism
chemical_list: D057134:Antibodies, Neutralizing; D007071:Immunoglobulin A, Secretory; D011134:Polysaccharides; D011994:Recombinant Proteins; D054299:env Gene Products, Human Immunodeficiency Virus
keywords: DC-SIGN, dendritic cell – specific intercellular adhesion molecule 3 grabbing non-integrin; HIV microbicide; LFM, leaf fresh mass; SC, secretory component; SEM, standard error of the mean; SIgA, secretory IgA; antibody stability; glycosylation; molecular farming; secretory IgA