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ABSTRACT: The highly conserved cluster of high-mannose glycans on the HIV-1 envelope glycoprotein, gp120, has been highlighted as a target for neutralizing antibodies. 2G12, the first HIV-1 anti-glycan neutralizing antibody described, binds with an unusual domain-exchanged structure that creates a high-affinity multivalent binding surface. It is an interesting challenge for rational vaccine design to generate immunogens capable of eliciting domain-exchanged 2G12-like responses. We recently showed that di-mannose recognition by the variable domains of 2G12 is independent of domain exchange, but that exchange is critical for virus neutralization. Carbohydrate-based immunogens aimed at inducing 2G12-like antibodies may need to drive both di-mannose recognition and domain exchange through interaction with B cell receptors. Here we assessed the ability of such immunogens to activate mouse B cell lines displaying domain-exchanged 2G12 wild-type (2G12-WT), a non-domain-exchanged Y-shaped variant (2G12-I19R) and 2G12 germ line (2G12-gl). We show that several immunogens, including heat-killed yeast and bacteria, can activate both 2G12-WT and 2G12-I19R B cells. However, only discrete clusters of high-mannose glycans, as on recombinant forms of the HIV-1 envelope trimer and oligodendrons, activate 2G12-WT B cells. Furthermore, no immunogen tested activated 2G12-gl cells. Our results support the hypothesis that in order to drive domain exchange of an anti-mannose antibody response, a boost with an immunogen displaying discrete clusters of high-mannose glycans not recognized by conventional Y-shaped antibodies will be required. Additionally, a molecule capable of activating 2G12-gl cells might also be required. The results highlight 2G12-expressing mouse B cells as potentially useful tools for carbohydrate immunogen screening.
Journal of Virology 12/2012; · 5.40 Impact Factor
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Robert Pejchal,
Katie J Doores,
Laura M Walker,
Reza Khayat,
Po-Ssu Huang, Sheng-Kai Wang,
Robyn L Stanfield,
Jean-Philippe Julien,
Alejandra Ramos,
Max Crispin, [......],
Ten Feizi,
Christopher N Scanlan,
Chi-Huey Wong,
John P Moore,
William C Olson,
Andrew B Ward,
Pascal Poignard,
William R Schief,
Dennis R Burton,
Ian A Wilson
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ABSTRACT: The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man(9) at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificity. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.
Science 11/2011; 334(6059):1097-103. · 31.20 Impact Factor
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Laura M Walker,
Michael Huber,
Katie J Doores,
Emilia Falkowska,
Robert Pejchal,
Jean-Philippe Julien, Sheng-Kai Wang,
Alejandra Ramos,
Po-Ying Chan-Hui,
Matthew Moyle, [......],
Pham Phung,
Steven Fling,
Chi-Huey Wong,
Sanjay Phogat,
Terri Wrin,
Melissa D Simek,
Wayne C Koff,
Ian A Wilson,
Dennis R Burton,
Pascal Poignard
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ABSTRACT: Broadly neutralizing antibodies against highly variable viral pathogens are much sought after to treat or protect against global circulating viruses. Here we probed the neutralizing antibody repertoires of four human immunodeficiency virus (HIV)-infected donors with remarkably broad and potent neutralizing responses and rescued 17 new monoclonal antibodies that neutralize broadly across clades. Many of the new monoclonal antibodies are almost tenfold more potent than the recently described PG9, PG16 and VRC01 broadly neutralizing monoclonal antibodies and 100-fold more potent than the original prototype HIV broadly neutralizing monoclonal antibodies. The monoclonal antibodies largely recapitulate the neutralization breadth found in the corresponding donor serum and many recognize novel epitopes on envelope (Env) glycoprotein gp120, illuminating new targets for vaccine design. Analysis of neutralization by the full complement of anti-HIV broadly neutralizing monoclonal antibodies now available reveals that certain combinations of antibodies should offer markedly more favourable coverage of the enormous diversity of global circulating viruses than others and these combinations might be sought in active or passive immunization regimes. Overall, the isolation of multiple HIV broadly neutralizing monoclonal antibodies from several donors that, in aggregate, provide broad coverage at low concentrations is a highly positive indicator for the eventual design of an effective antibody-based HIV vaccine.
Nature 08/2011; 477(7365):466-70. · 36.28 Impact Factor
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Angewandte Chemie International Edition 02/2011; 50(7):1608-12. · 13.45 Impact Factor
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Rena D Astronomo,
Eiton Kaltgrad,
Andrew K Udit, Sheng-Kai Wang,
Katie J Doores,
Cheng-Yuan Huang,
Ralph Pantophlet,
James C Paulson,
Chi-Huey Wong,
M G Finn,
Dennis R Burton
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ABSTRACT: The broadly neutralizing antibody 2G12 recognizes a conserved cluster of high-mannose glycans on the surface envelope spike of HIV, suggesting that the "glycan shield" defense of the virus can be breached and may, under the right circumstances, serve as a vaccine target. In an attempt to recreate features of the glycan shield semisynthetically, oligomannosides were coupled to surface lysines on the icosahedral capsids of bacteriophage Q beta and cowpea mosaic virus (CPMV). The Q beta glycoconjugates, but not CPMV, presented oligomannose clusters that bind the antibody 2G12 with high affinity. However, antibodies against these 2G12 epitopes were not detected in immunized rabbits. Rather, alternative oligomannose epitopes on the conjugates were immunodominant and elicited high titers of anti-mannose antibodies that do not crossreact with the HIV envelope. The results presented reveal important design considerations for a carbohydrate-based vaccine component for HIV.
Chemistry & biology 04/2010; 17(4):357-70. · 6.52 Impact Factor
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ABSTRACT: It is widely accepted that the heavily glycosylated glycoprotein gp120 on the surface of HIV-1 shields peptide epitopes from recognition by the immune system and may promote infection in vivo by interaction with dendritic cells and transport to tissue rich in CD4(+) T cells such as lymph nodes. A conserved cluster of oligomannose glycans on gp120 has been identified as the epitope recognized by the broadly HIV-1-neutralizing monoclonal antibody 2G12. Oligomannose glycans are also the ligands for DC-SIGN, a C-type lectin found on the surface of dendritic cells. Multivalency is fundamental for carbohydrate-protein interactions, and mimicking of the high glycan density on the virus surface has become essential for designing carbohydrate-based HIV vaccines and antiviral agents. We report an efficient synthesis of oligomannose dendrons, which display multivalent oligomannoses in high density, and characterize their interaction with 2G12 and DC-SIGN by a glycan microarray binding assay. The solution and the surface binding analysis of 2G12 to a prototype oligomannose dendron clearly demonstrated the efficacy of dendrimeric display. We further showed that these glycodendrons inhibit the binding of gp120 to 2G12 and recombinant dimeric DC-SIGN with IC(50) in the nanomolar range. A second-generation Man(9) dendron was identified as a potential immunogen for HIV vaccine development and as a potential antiviral agent.
Proceedings of the National Academy of Sciences 04/2008; 105(10):3690-5. · 9.68 Impact Factor
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ABSTRACT: Carbohydrate-protein interactions on surface and in solution were quantitatively measured by a glycan microarray. Assessing carbohydrate affinities is typically difficult due to weak affinities and limited sources of structurally complex glycans. We described here a sensitive, high-throughput, and convenient glycan microarray technology for the simultaneous determination of a wide variety of parameters in a single experiment using small amounts of materials. Assay systems based on this technology were developed to analyze multivalent interactions and determine the surface dissociation constant (KD,surf) for surface-coated mannose derivatives with mannose binding lectins and antibodies. Competition experiments that employed monovalent ligands in solution yielded KD and Ki values in solution similar to equilibrium binding constants obtained in titration microcalorimetry and surface plasmon resonance experiments.
Journal of the American Chemical Society 10/2007; 129(36):11177-84. · 9.91 Impact Factor
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ABSTRACT: Developing tools for investigating the cellular activity of glycans will help to delineate the molecular basis for aberrant glycosylation in pathological processes such as cancer. Metabolic oligosaccharide engineering, which inserts sugar-reporting groups into cellular glycoconjugates, represents a powerful method for imaging the localization, trafficking, and dynamics of glycans and isolating them for glyco-proteomic analysis. Herein, we show that the alkyne-reporting group can be incorporated into cellular glycans. The alkyne group is a small, inert, bio-orthogonal handle that can be chemoselectively labeled by using the Cu(I) catalyzed [3 + 2] azide-alkyne cycloaddition, or click chemistry. Alkynyl sugar monomers, based on fucose (Fuc) and N-acetylmannosamine (ManNAc), were incorporated into fucosylated and sialylated glycans in several cancer cell lines, allowing for cell surface and intracellular visualization of glycoconjugates, as well as, observation of alkyne-bearing glycoproteins. Similarly to our previous results with an azido Fuc/alkynyl probe system, we demonstrated that click-activated fluorogenic probes are practical tools for efficiently and selectively labeling alkynyl-modified glycans. Because Fuc and sialic acid are terminal glycan residues with a notably increased presence in many tumors, we hope that our method will provide useful information about their roles in cancer and ultimately can be used for diagnostic and therapeutic purposes.
Proceedings of the National Academy of Sciences 03/2007; 104(8):2614-9. · 9.68 Impact Factor
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M Germana Sanna, Sheng-Kai Wang,
Pedro J Gonzalez-Cabrera,
Anthony Don,
David Marsolais,
Melanie P Matheu,
Sindy H Wei,
Ian Parker,
Euijung Jo,
Wei-Chieh Cheng,
Michael D Cahalan,
Chi-Huey Wong,
Hugh Rosen
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ABSTRACT: Sphingosine 1-phosphate (S1P, 1) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S1P1 receptors. We used reversible chemical probes (i) to gain mechanistic insights into S1P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo-active chiral S1P1 receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S1P1 tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S1P-S1P1 'set points' among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention.
Nature Chemical Biology 09/2006; 2(8):434-41. · 14.69 Impact Factor
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ChemBioChem 01/2006; 6(12):2176-80. · 3.94 Impact Factor
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ABSTRACT: Sphingosine 1-phosphate type 1 (S1P(1)) receptor agonists cause sequestration of lymphocytes in secondary lymphoid organs by a mechanism that is not well understood. One hypothesis proposes that agonists act as 'functional antagonists' by binding and internalizing S1P(1) receptors on lymphocytes; a second hypothesis proposes instead that S1P(1) agonists act on endothelial cells to prevent lymphocyte egress from lymph nodes. Here, two-photon imaging of living T cells in explanted lymph nodes after treatment with S1P(1) agonists or antagonists has provided insight into the mechanism by which S1P(1) agonists function. The selective S1P(1) agonist SEW2871 caused reversible slowing and 'log-jamming' of T cells between filled medullary cords and empty sinuses, whereas motility was unaltered in diffuse cortex. Removal or antagonist competition of SEW2871 permitted recovery of T cell motility in the parenchyma of the medulla and resumption of migration across the stromal endothelial barrier, leading to refilling of sinuses. Our results provide visualization of transendothelial migration of T cells into lymphatic sinuses and suggest that S1P(1) agonists act mainly on endothelial cell S1P(1) receptors to inhibit lymphocyte migration.
Nature Immunology 01/2006; 6(12):1228-35. · 26.01 Impact Factor
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Angewandte Chemie International Edition 01/2005; 43(47):6496-500. · 13.45 Impact Factor
