Human antibody 2G12 neutralizes a broad range of HIV-1 isolates. Hence, molecular characterization of its epitope, which corresponds to a conserved cluster of oligomannoses on the viral envelope glycoprotein gp120, is a high priority in HIV vaccine design. A prior crystal structure of 2G12 in complex with Man 9GlcNAc2 highlighted the central importance of the D1 arm in antibody binding. To characterize the specificity of 2G12 more precisely, we performed solution-phase ELISA, carbohydrate microarray analysis, and cocrystallized Fab 2G12 with four different oligomannose derivatives (Man 4, Man5, Man7, and Man8) that compete with gp120 for binding to 2G12. Our combined studies reveal that 2G12 is capable of binding both the D1 and D3 arms of the Man 9GlcNAc2 moiety, which would provide more flexibility to make the required multivalent interactions between the antibody and the gp120 oligomannose cluster than thought previously. These results have important consequences for the design of immunogens to elicit 2G12-like neutralizing antibodies as a component of an HIV vaccine. 2G12 antibody ͉ HIV vaccine ͉ oligomannoses There is widespread agreement that the most promising approach to contain the ongoing HIV pandemic is through the development of an effective vaccine (1, 2). However, HIV vaccine design has faced many difficulties including, prominently, the lack of an immunogen able to elicit broadly neutralizing antibodies (Abs). The feasibility of developing such an immunogen is suggested by a small panel of broadly neutralizing human monoclonal antibodies (mAbs) that have been isolated from seropositive donors (3). One of these Abs, 2G12, recognizes a conserved and unusually dense cluster of oligomannose residues on the ''silent face'' of gp120, the major envelope protein of HIV-1 (4, 5). Many lectins have been identified that can bind to envelope and act antivirally, but 2G12 remains the only known anti-carbohydrate protein that has been specifically elicited to HIV-1 in an adaptive immune response (6). 2G12 has an unusual architecture in which the arms of the IgG swap variable heavy domains, creating a domainswapped dimer of Fabs (7). The crystal structure of Fab 2G12 complexed with Man 9 GlcNAc 2 indicated that the conventional Ab-binding sites are occupied by the D1 arms of the Man 9 GlcNAc 2 moieties (1; Fig. 1) (7). The terminal Man␣1-2Man residues of the D1 branch account for 85% of the Fab contacts to Man 9 GlcNAc 2 , although the disaccharide by itself is 50-fold less potent in binding to 2G12 than Man 9 GlcNAc 2 (7).Based on these structural results, several initiatives have been launched to design novel immunogens that will elicit 2G12-like Abs (8-12). Recently, we described the design and synthesis of novel antigens, oligomannoses 2-6 (Fig. 1), that bind to mAb 2G12 (13). The Man␣1-2Man-containing oligomannoses 4, 5, and 6 were identified as new epitope mimics that inhibit the binding of gp120 to mAb 2G12 as well as, or better than, Man 9 GlcNAc 2 (13). Encouraged by this result, we now report the desig...

A covalent array for the display of complex oligosaccharides in microtiter plates has been developed. This strategy is conducive to the display of carbohydrates to proteins of interest such as lectins and antibodies, including the broadly neutralizing antibody 2G12 against HIV envelope oligomannose and can be cleaved from the surface for further characterization by mass spectrometry. The system was used to probe the multivalent interaction of 2G12 with an optimal epitope (Kd 0.1 muM).

The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man 4 ) that corresponds to the D1 arm of Man 9 GlcNAc 2 inhibits 2G12 binding to gp120 as efficiently as Man 9 GlcNAc 2 itself, indicating the potential use of Man 4 as a building block for creating immunogens. Here, we describe the development of neoglycoconjugates displaying variable copy numbers of Man 4 on bovine serum albumin (BSA) molecules by conjugation to Lys residues. The increased valency enhances the apparent affinity of 2G12 for Man 4 up to a limit which is achieved at ϳ10 copies per BSA molecule, beyond which no further enhancement is observed. Immunization of rabbits with BSA-(Man 4 ) 14 elicits significant serum Ab titers to Man 4 . However, these Abs are unable to bind gp120. Further analysis reveals that the elicited Abs bind a variety of unbranched and, to a lesser extent, branched Man 9 derivatives but not natural N-linked oligomannose containing the chitobiose core. These results suggest that Abs can be readily elicited against the D1 arm; however, potential differences in the presentation of Man 4 on neoglycoconjugates, compared to glycoproteins, poses challenges for eliciting anti-mannose Abs capable of crossreacting with gp120 and HIV-1.

An HIV‐1 vaccine may eventually be developed from oligomannoses synthesized by a reactivity‐based one‐pot self‐condensation strategy. The compounds were tested for their ability to inhibit the binding of gp120 to the broadly neutralizing HIV‐1 antibody 2G12. New epitope mimics (see structure for example) were identified that inhibit this binding as well as, or better than the natural epitope Man9GlcNAc2 (Man=mannose, Glc=glucose).

The total synthesis of the sialic acid-containing antigenic epitope fucose GM1 (Fuc-GM1) by an improved reactivity-based one-pot synthetic strategy is reported. Based on a thioglycoside reactivity database, three saccharide building blocks, 3, 4, and 5, were designed and prepared to incorporate a descending order of reactivity toward thiophilic activation. Using the reactivity-based one-pot synthetic method, the fully protected Fuc-GM1 glycoside 2 was furnished in a facile manner, which was globally deprotected to give the Fuc-GM1 glycoside 1. In addition, using the promoter system 1-(benzensulfinyl)piperidine͞trifluoromethanesulfonic anhydride, the product yield was improved and the reaction time was reduced in comparison with the N-iodosuccinimide͞trifluoro-methanesulfonic acid-and dimethyl (thiomethyl) sulfonium trifluoromethanesulfonate-promoted systems.synthetic vaccine F ucose GM 1 (Fuc-GM 1 ) ganglioside was first isolated from bovine thyroid tissue in 1979 (1). It is comprised of a hexasaccharide carbohydrate moiety and a ceramide-reducing end component. Within this carbohydrate framework is a tetrasaccharide (sugars a, b, d, and e) bearing a branched sialic acid residue (c) and a terminal fucose (f) (Fig. 1). It is found specifically in the tumor tissue of small-cell lung cancer (SCLC). SCLC accounts for 20% of lung cancer, which remains one of the leading causes of death in the United States (2). Unlike other cancer antigens, Fuc-GM 1 has a more restricted distribution in normal tissue, suggesting that this carbohydrate antigen may be a good target for active immunization. Development of an anti-(Fuc-GM 1 ) vaccine and mAb could potentially be of importance for diagnosis and immunotherapy of these tumors (3, 4). However, one of the barriers preventing effective production of an anticancer vaccine is the limited supply of chemically pure Fuc-GM 1 oligosaccharide. Despite the development of various oligosaccharide synthetic methods (5-8), the first synthesis of Fuc-GM 1 glycoside was reported by Allen and Danishefsky (9) two decades after its initial discovery. This elegant strategy incorporated the sulfonamide glycosidation method (10) in conjunction with a [3 ϩ 3] convergent glycosylation, ultimately leading to the target glycoside. It required protecting group and anomeric leaving group manipulations and encountered a problem with stereoselective formation of the ␤(1,4) glycosidic bond between a bulky trisaccharide donor and the poor nucleophilic trisaccharide acceptor.We envisioned that the incorporation of our programmable reactivity-based one-pot strategy (8) in the synthesis of the Fuc-GM 1 could simplify this complicated synthetic operation. In brief, the reactivity-based one-pot strategy is based on a developed competitive HPLC assay to assess quantitatively the reactivity of different thioglycosides, the so-called relative reactivity value (RRV). Such information is then used to guide the reactivity-based one-pot synthesis of an oligosaccharide without protecting group manipulation and intermed...