title: An engineered Saccharomyces cerevisiae strain binds the broadly neutralizing human immunodeficiency virus type 1 antibody 2G12 and elicits mannose-specific gp120-binding antibodies.
abstract: The glycan shield of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein serves as a barrier to antibody-mediated neutralization and plays a critical role in transmission and infection. One of the few broadly neutralizing HIV-1 antibodies, 2G12, binds to a carbohydrate epitope consisting of an array of high-mannose glycans exposed on the surface of the gp120 subunit of the Env protein. To produce proteins with exclusively high-mannose carbohydrates, we generated a mutant strain of Saccharomyces cerevisiae by deleting three genes in the N-glycosylation pathway, Och1, Mnn1, and Mnn4. Glycan profiling revealed that N-glycans produced by this mutant were almost exclusively Man(8)GlcNAc(2), and four endogenous glycoproteins that were efficiently recognized by the 2G12 antibody were identified. These yeast proteins, like HIV-1 gp120, contain a large number and high density of N-linked glycans, with glycosidase digestion abrogating 2G12 cross-reactivity. Immunization of rabbits with whole Delta och1 Delta mnn1 Delta mnn4 yeast cells produced sera that recognized a broad range of HIV-1 and simian immunodeficiency virus (SIV) Env glycoproteins, despite no HIV/SIV-related proteins being used in the immunization procedure. Analyses of one of these sera on a glycan array showed strong binding to glycans with terminal Man alpha1,2Man residues, and binding to gp120 was abrogated by glycosidase removal of high-mannose glycans and terminal Man alpha1,2Man residues, similar to 2G12. Since S. cerevisiae is genetically pliable and can be grown easily and inexpensively, it will be possible to produce new immunogens that recapitulate the 2G12 epitope and may make the glycan shield of HIV Env a practical target for vaccine development.
authors.affiliation: ProSci Incorporated, 12170 Flint Place, Poway, CA 92064, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Luallen
authors.forename: Jianqiao
authors.initials: J
authors.lastname: Lin
authors.forename: Hu
authors.initials: H
authors.lastname: Fu
authors.forename: Karen K
authors.initials: KK
authors.lastname: Cai
authors.forename: Caroline
authors.initials: C
authors.lastname: Agrawal
authors.forename: Innocent
authors.initials: I
authors.lastname: Mboudjeka
authors.forename: Fang-Hua
authors.initials: FH
authors.lastname: Lee
authors.forename: David
authors.initials: D
authors.lastname: Montefiori
authors.forename: David F
authors.initials: DF
authors.lastname: Smith
authors.forename: Robert W
authors.initials: RW
authors.lastname: Doms
authors.forename: Yu
authors.initials: Y
authors.lastname: Geng
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000914:Antibodies, Viral / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D000080908:Broadly Neutralizing Antibodies; D004797:Enzyme-Linked Immunosorbent Assay; D005818:Genetic Engineering / Q000379:methods; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120 / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D007106:Immune Sera / Q000276:immunology; D047468:Immunoprecipitation; D008358:Mannose / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000276:immunology* / Q000821:virology
chemical_list: C529157:2G12 monoclonal antibody; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000080908:Broadly Neutralizing Antibodies; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007106:Immune Sera; D008358:Mannose