title: Expression-system-dependent modulation of HIV-1 envelope glycoprotein antigenicity and immunogenicity.
abstract: Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly related Env surface (gp120) antigens produced in different systems: (a) mammalian (293 FreeStyle cells; 293F) cells in the presence of kifunensine, which impart only high-mannose glycans; (b) insect cells (Spodoptera frugiperda, Sf9), which confer mainly paucimannosidic glycans; (c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic), which impart high-mannose, hybrid and complex glycans without sialic acid; and (d) 293F cells, which impart high-mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9-derived and wild-type mammalian-cell-derived material that is predicted to affect ligand binding sites proximal to glycans. Modeling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4 binding and CD4-induced sites compared to those expressed in the system that do, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic-acid-containing gp120 was significantly more immunogenic than the sialylated version when administered in two different adjuvants, and induced higher titers of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic-acid-imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations that should be considered when selecting expression systems for glycosylated antigens to be used for structure-function studies and for vaccine use.
authors.affiliation: The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK; Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Leopold
authors.initials: L
authors.lastname: Kong
authors.affiliation: The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
authors.forename: Neil C
authors.initials: NC
authors.lastname: Sheppard
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7BN, UK.
authors.forename: Guillaume B E
authors.initials: GBE
authors.lastname: Stewart-Jones
authors.affiliation: The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
authors.forename: Cynthia L
authors.initials: CL
authors.lastname: Robson
authors.affiliation: School of Biological Sciences, University of Reading, Reading RG6 6UR, UK.
authors.forename: Hongying
authors.initials: H
authors.lastname: Chen
authors.affiliation: School of Biological Sciences, University of Reading, Reading RG6 6UR, UK.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Xu
authors.affiliation: The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
authors.forename: George
authors.initials: G
authors.lastname: Krashias
authors.affiliation: Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Camille
authors.initials: C
authors.lastname: Bonomelli
authors.affiliation: Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Scanlan
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Kwong
authors.affiliation: Wright-Fleming Institute, Division of Medicine, Imperial College London, Norfolk Place, London, W2 1PG, UK.
authors.forename: Simon A
authors.initials: SA
authors.lastname: Jeffs
authors.affiliation: School of Biological Sciences, University of Reading, Reading RG6 6UR, UK.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Jones
authors.affiliation: The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK. Electronic address: quentin.sattentau@path.ox.ac.uk.
authors.forename: Quentin J
authors.initials: QJ
authors.lastname: Sattentau
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015704:CD4 Antigens / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D005822:Genetic Vectors; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D006031:Glycosylation; D015483:HIV Antibodies / Q000097:blood; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017422:Sequence Analysis, DNA; D018411:Spodoptera; D055672:Static Electricity
chemical_list: D000956:Antigens, Viral; D015704:CD4 Antigens; D006023:Glycoproteins; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007074:Immunoglobulin G; D011134:Polysaccharides; D011994:Recombinant Proteins; C514793:gp120 protein, Human immunodeficiency virus 1
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