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Agrawal-Gamse C.,University of Pennsylvania | Luallen R.J.,ProSci Incorporated | Liu B.,ProSci Incorporated | Fu H.,ProSci Incorporated | And 3 more authors.
Journal of Virology | Year: 2011

The HIV envelope (Env) protein uses a dense coat of glycans to mask conserved domains and evade host humoral immune responses. The broadly neutralizing antibody 2G12, which binds a specific cluster of high-mannose glycans on HIV Env, shows that the glycan shield can also serve as a target for neutralizing antibodies. We have described a triple mutant Saccharomyces cerevisiae strain that expresses high-mannose glycoproteins that bind to 2G12. When used to immunize rabbits, this yeast elicits antibodies that bind to gp120-associated glycans but fail to neutralize virus. Here we sought to determine the reason for these discordant results. Affinity purification of sera over columns conjugated with three 2G12-reactive yeast glycoproteins showed that these proteins could adsorb 80% of the antibodies that bind to gp120 glycans. Despite binding to monomeric gp120, these mannose-specific antibodies failed to bind cell surface-expressed trimeric Env. However, when Env was expressed in the presence of the mannosidase inhibitor kifunensine to force retention of high-mannose glycans at all sites, the purified antibodies gained the abilities to bind trimeric Env and to strongly and broadly neutralize viruses produced under these conditions. Combined, these data show that the triple mutant yeast strain elicits antibodies that bind to high-mannose glycans presented on the HIV envelope, but only when they are displayed in a manner not found on native Env trimers. This implies that the underlying structure of the protein scaffold used to present the high-mannose glycans may be critical to allow elicitation of antibodies that recognize trimeric Env and neutralize virus. Copyright © 2011, American Society for Microbiology. All Rights Reserved.


Zhang H.,ProSci Incorporated | Fu H.,ProSci Incorporated | Luallen R.J.,ProSci Incorporated | Luallen R.J.,University of California at San Diego | And 4 more authors.
Vaccine | Year: 2015

The glycan shield on the human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein has drawn attention as a target for HIV-1 vaccine design given that an increasing number of potent and broadly neutralizing antibodies (bNAbs) recognize epitopes entirely or partially comprised of high mannose type N-linked glycans. In an attempt to generate immunogens that target the glycan shield of HIV-1, we previously engineered a triple mutant (TM) strain of Saccharomyces cerevisiae that results in exclusive presentation of high mannose type N-glycans, and identified five TM yeast glycoproteins that support strong binding of 2G12, a bNAb that targets a cluster of high mannose glycans on the gp120 subunit of Env. Here, we further analyzed the antigenicity and immunogenicity of these proteins in inducing anti-HIV responses. Our study demonstrated that the 2G12-reactive TM yeast glycoproteins efficiently bound to recently identified bNAbs including PGT125-130 and PGT135 that recognize high mannose glycan-dependent epitopes. Immunization of rabbits with a single TM yeast glycoprotein (Gp38 or Pst1), when conjugated to a promiscuous T-cell epitope peptide and coadministered with a Toll-like receptor 2 agonist, induced glycan-specific HIV-1 Env cross-reactive antibodies. The immune sera bound to both synthetic mannose oligosaccharides and gp120 proteins from a broad range of HIV-1 strains. The purified antibodies recognized and captured virions that contain both complex- and high mannose-type of N-glycans, and potently neutralized virions from different HIV-1 clades but only when the virions were enforced to retain high mannose N-glycans. This study provides insights into the elicitation of anti-carbohydrate, HIV-1 Env-cross reactive antibodies with a heterologous glycoprotein and may have applications in the design and administration of immunogens that target the viral glycan shield for development of an effective HIV-1 vaccine. © 2015 Elsevier Ltd.


PubMed | ProSci Incorporated and University of Pennsylvania
Type: Journal Article | Journal: Vaccine | Year: 2015

The glycan shield on the human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein has drawn attention as a target for HIV-1 vaccine design given that an increasing number of potent and broadly neutralizing antibodies (bNAbs) recognize epitopes entirely or partially comprised of high mannose type N-linked glycans. In an attempt to generate immunogens that target the glycan shield of HIV-1, we previously engineered a triple mutant (TM) strain of Saccharomyces cerevisiae that results in exclusive presentation of high mannose type N-glycans, and identified five TM yeast glycoproteins that support strong binding of 2G12, a bNAb that targets a cluster of high mannose glycans on the gp120 subunit of Env. Here, we further analyzed the antigenicity and immunogenicity of these proteins in inducing anti-HIV responses. Our study demonstrated that the 2G12-reactive TM yeast glycoproteins efficiently bound to recently identified bNAbs including PGT125-130 and PGT135 that recognize high mannose glycan-dependent epitopes. Immunization of rabbits with a single TM yeast glycoprotein (Gp38 or Pst1), when conjugated to a promiscuous T-cell epitope peptide and coadministered with a Toll-like receptor 2 agonist, induced glycan-specific HIV-1 Env cross-reactive antibodies. The immune sera bound to both synthetic mannose oligosaccharides and gp120 proteins from a broad range of HIV-1 strains. The purified antibodies recognized and captured virions that contain both complex- and high mannose-type of N-glycans, and potently neutralized virions from different HIV-1 clades but only when the virions were enforced to retain high mannose N-glycans. This study provides insights into the elicitation of anti-carbohydrate, HIV-1 Env-cross reactive antibodies with a heterologous glycoprotein and may have applications in the design and administration of immunogens that target the viral glycan shield for development of an effective HIV-1 vaccine.


PubMed | ProSci Incorporated
Type: Journal Article | Journal: Glycobiology | Year: 2010

Design of an envelope glycoprotein (Env)-based vaccine against human immunodeficiency virus type-1 (HIV-1) is complicated by the large number of N-linked glycans that coat the protein and serve as a barrier to antibody-mediated neutralization. Compared to normal mammalian glycoproteins, high-mannose-type glycans are disproportionately represented on the gp120 subunit of Env. These N-glycans serve as a target for a number of anti-HIV molecules that bind terminal alpha1,2-linked mannose residues, including lectins and the monoclonal antibody 2G12. We created a Saccharomyces cerevisiae glycosylation mutant, Deltamnn1Deltamnn4, to expose numerous terminal Manalpha1,2-Man residues on endogenous hypermannosylated glycoproteins in the yeast cell wall. Immunization of rabbits with whole cells from this mutant induced antibodies that bound to a broad range of Env proteins, including clade A, B, and C of HIV and simian immunodeficiency virus (SIV). The gp120 binding activity of these immune sera was due to mannose-specific immunoglobulin, as removal of high-mannose glycans and alpha1,2-linked mannoses from gp120 abrogated serum binding. Glycan array analysis with purified IgG demonstrated binding mainly to glycans with Manalpha1,2-Manalpha1,2-Man trisaccharides. Altogether, these data demonstrate the immunogenicity of exposed polyvalent Manalpha1,2-Manalpha1,2-Man structures on the yeast cell wall mannan and their ability to induce antibodies that bind to the HIV Env protein. The yeast strain and sera from this study will be useful tools for determining the type of mannose-specific response that is needed to develop neutralizing antibodies to the glycan shield of HIV.

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