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Journal of Virology, May 2009, p. 4861-4870, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02537-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Robert J. Luallen,1 Hu Fu,1 Caroline Agrawal-Gamse,2 Innocent Mboudjeka,1 Wei Huang,3 Fang-Hua Lee,2 Lai-Xi Wang,3 Robert W. Doms,2 and Yu Geng1*

ProSci Incorporated, 12170 Flint Place, Poway, California 92064,1 Department of Microbiology, University of Pennsylvania, 225 Johnson Pavilion, Philadelphia, Pennsylvania 19104,2 Institute of Human Virology, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, Maryland 212013

Received 9 December 2008/ Accepted 19 February 2009

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 {Delta}pmr1 strain deficient in hyperglycosylation. 2G12 binding to this protein, identified as Pst1, was enhanced by adding the {Delta}mnn1 deletion to the {Delta}pmr1 background, ensuring the exposure of terminal {alpha}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 Man8GlcNAc2 structures in {Delta}och1 {Delta}mnn1 {Delta}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.

* Corresponding author. Mailing address: ProSci Inc., 12170 Flint Place, Poway, CA 92064. Phone: (858) 513-2638. Fax: (858) 513-2769. E-mail: yugeng{at}prosci-inc.com

{triangledown} Published ahead of print on 4 March 2009.

Journal of Virology, May 2009, p. 4861-4870, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02537-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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