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J. Virol. doi:10.1128/JVI.00412-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

An engineered Saccharomyces cerevisiae strain binds the broadly neutralizing HIV-1 antibody 2G12 and elicits mannose-specific, gp120 binding antibodies

ProSci Incorporated, 12170 Flint Place, Poway, CA 92064; Department of Microbiology, University of Pennsylvania, 225 Johnson Pavilion, Philadelphia, PA 19104; Department of Surgery, Duke University Medical Center, Box 2926, DUMC, Durham, NC 27710; Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd NE, Room 4035, Atlanta, GA 30322

^* To whom correspondence should be addressed. Email: yugeng{at}prosci-inc.com.

	Abstract

The glycan shield of the 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 were identified that were efficiently recognized by 2G12. 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 och1mnn1mnn4 yeast produced sera that recognized a broad range of HIV-1 and 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. The fact that yeast are genetically pliable, and can be grown easily and inexpensively, will make it 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.