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...
