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Journal of Virology, July 2008, p. 6447-6457, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00412-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

An Engineered Saccharomyces cerevisiae Strain Binds the Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody 2G12 and Elicits Mannose-Specific gp120-Binding Antibodies

ProSci Incorporated, 12170 Flint Place, Poway, California 92064,¹ Department of Microbiology, University of Pennsylvania, 225 Johnson Pavilion, Philadelphia, Pennsylvania 19104,² Department of Surgery, Duke University Medical Center, Box 2926, DUMC, Durham, North Carolina 27710,³ Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd. NE, Room 4035, Atlanta, Georgia 30322⁴

Received 25 February 2008/ Accepted 16 April 2008

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 Man₈GlcNAc₂, 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 och1 mnn1 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 Man1,2Man residues, and binding to gp120 was abrogated by glycosidase removal of high-mannose glycans and terminal Man1,2Man 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.

Published ahead of print on 23 April 2008.