A Yeast Glycoprotein Shows High-Affinity Binding to the Broadly Neutralizing Human Immunodeficiency Virus Antibody 2G12 and Inhibits gp120 Interactions with 2G12 and DC-SIGN

Abstract

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein contains numerous N-linked carbohydrates that shield conserved peptide epitopes and promote trans infection by dendritic cells via binding to cell surface lectins. The potent and broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose-type oligosaccharides on the gp120 subunit of Env, revealing a conserved and highly exposed epitope on the glycan shield. To find an effective antigen for eliciting 2G12-like antibodies, we searched for endogenous yeast proteins that could bind to 2G12 in a panel of Saccharomyces cerevisiae glycosylation knockouts and discovered one protein that bound weakly in a ⌬pmr1 strain deficient in hyperglycosylation. 2G12 binding to this protein, identified as Pst1, was enhanced by adding the ⌬mnn1 deletion to the ⌬pmr1 background, ensuring the exposure of terminal ␣1,2-linked mannose residues on the D1 and D3 arms of high-mannose glycans. However, optimum 2G12 antigenicity was found when Pst1, a heavily N-glycosylated protein, was expressed with homogenous Man 8 GlcNAc 2 structures in ⌬och1 ⌬mnn1 ⌬mnn4 yeast. Surface plasmon resonance analysis of this form of Pst1 showed high affinity for 2G12, which translated into Pst1 efficiently inhibiting gp120 interactions with 2G12 and DC-SIGN and blocking 2G12-mediated neutralization of HIV-1 pseudoviruses. The high affinity of the yeast glycoprotein Pst1 for 2G12 highlights its potential as a novel antigen to induce 2G12-like antibodies.The human immunodeficiency virus (HIV) has evolved numerous means to evade the humoral immune response, including a two-receptor mechanism for entry that recesses and protects highly conserved binding sites in the gp120 subunit of the viral envelope (Env) protein, trimerization of Env to further protect neutralizing epitopes readily exposed on the monomer, and rapid and continual mutation in the face of immune selective pressure (8, 9). Another highly effective defense mechanism is found in the extensive array of oligosaccharides covering gp120, with approximately 25

Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens

“…The identification of its epitope as a cluster of Manα1→2Man termini within the intrinsic mannose patch of gp120 (17) has encouraged the development of vaccines based around biological (19,27,(31)(32)(33) and synthetic Manα1→2Man clusters (34)(35)(36)(37)(38). A major limitation of any vaccine based on 2G12 recognition is the absence of its cognate epitope on the epidemiologically important clade C. The specific Asn residues required for 2G12 neutralization are absent in clade C isolates, explaining their resistance to 2G12.…”

In-Solution Virus Capture Assay Helps Deconstruct Heterogeneous Antibody Recognition of Human Immunodeficiency Virus Type 1

“…Most likely, MPER MAbs capture Env(Ϫ) virions through weak membrane interactions. 2G12 specifically targets a cluster of high mannose glycans on gp120, but it has also been shown to bind to clusters of synthetic oligomannose (4,40,77), a yeast glycoprotein (46,47), and 293T cells treated with oligomannose-modifying drugs (75). Thus, Env-independent virus capture by 2G12 is most likely mediated through weak interactions with virion-incorporated host glycoproteins.…”

Rapid development of glycan-specific, broad, and potent anti–HIV-1 gp120 neutralizing antibodies in an R5 SIV/HIV chimeric virus infected macaque

“…3C), demonstrating that the plasma bNAb specificities bind to epitopes distinct from that recognized by 2G12. As a second approach, we adsorbed the plasma with TM-Pst1, a yeast glycoprotein displaying homogenous Man 8 GlcNAc 2 glycans that has high affinity for 2G12 and can inhibit 2G12 neutralization of pseudoviruses (20). Interestingly, a large fraction of the broad and potent plasma-neutralizing activity could be adsorbed with TM-Pst1, suggesting that the broadly neutralizing plasma antibodies bind directly to Man 8 GlcNAc 2 glycans (Fig.…”