A peptide inhibitor of HIV‐1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120

Abstract

MAb 2G12 neutralizes HIV-1 by binding with high affinity to a cluster of high-mannose oligosaccharides on the envelope glycoprotein, gp120. Screening of phage-displayed peptide libraries with 2G12 identified peptides that bind specifically, with K d s ranging from 0.4 to 200 μM. The crystal structure of a 21-mer peptide ligand in complex with 2G12 Fab was determined at 2.8 Å resolution. Comparison of this structure with previous structures of 2G12-carbohydrate complexes revealed striking differences in the mechanism of 2G12 binding to peptide vs. carbohydrate. The peptide occupies a site different from, but adjacent to, the primary carbohydrate-binding site on 2G12, and makes only slightly fewer contacts to the Fab than Man 9 GlcNAc 2 (51 vs. 56, respectively). However, only two antibody contacts with the peptide are hydrogen bonds in contrast to six with Man 9 GlcNAc 2 , and only three of the antibody residues that interact with Man 9 GlcNAc 2 also contact the peptide. Thus, this mechanism of peptide binding to 2G12 does not support structural mimicry of the native carbohydrate epitope on gp120, since it neither replicates the oligosaccharide footprint on the antibody nor most of the contact residues. Moreover, 2G12.1 peptide is not an immunogenic mimic of the 2G12 epitope, since antisera produced against it did not bind gp120.-Menendez, A., Calarese, D. A., Stanfield, R. L., Chow, K. C., Scanlan, C. N., Kunert, R., Katinger, H., Burton, D. R., Wilson, I. A., Scott, J. K. A peptide inhibitor of HIV-1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptHuman monoclonal antibody (MAb) 2G12 efficiently neutralizes a broad range of HIV-1 primary isolates in vitro (1-3) and protects from in vivo viral challenge in macaques in combination with other antibodies (4-6). MAb 2G12 binds with high affinity to a unique, conserved epitope on the HIV-1 envelope that is formed by a cluster of N-linked, high-mannose glycan groups on the "silent" face of gp120 (7-10). Crystal structures of 2G12 Fab alone, and in complex with oligosaccharides Man 9 GlcNAc 2 and Manα1-2Man, revealed that two Fab monomers assemble into an interlocked, domain-swapped dimer, that forms an extensive, multivalent binding surface (11). Four Man 9 GlcNAc 2 moieties were observed bound to the Fab dimer in the crystal structure, occupying the two canonical antigen-binding sites, and two novel ones located at the assembled interface of the two interlocked V H domains. Since the identification of its epitope, MAb 2G12 has received significant attention as a template for HIV-1 vaccine development. Considerable effort has been directed at characterizing its oligosaccharide-binding profile (12)(13)(14)(15), and the generation of novel antigens in the form of synthetic oligomannoses (13,16,17) or short, synthetic glycopeptides (18-20). In an attempt to generate immunogens that elicit 2G12-like antibodies, we chose to isolate peptides t...

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“…For example, Menendez et al isolated peptides specific for MAb 2G12. The crystal structure of MAb 2G12 bound to a "mimotope" peptide shows that the peptide occupies a different site on the Ab than the cognate carbohydrate (150). Unsurprisingly, immunization of rabbits with recombinant phage-bearing 2G12 peptides did not elicit Abs that showed cross-reactivity with gp120, although antipeptide Ab reactivity reached half-maximal titers of ϳ800 to 1,600 after four immunizations.…”

Antibody WN1 222-5 mimics Toll-like receptor 4 binding in the recognition of LPS

“…Thus, in contrast to their great immunological significance during infectious disease, still relatively little is known about carbohydrate recognition by antibodies at the structural level. Whereas cavity-or groove-shaped antibody-combining sites have been observed in most cases, a unique mechanism of binding has been observed for the HIV-1 neutralizing antibody 2G12, binding clusters of carbohydrates from the silent face of gp120 by using "domain swapping" (19,23,24).…”

Developing strategies to enhance and focus humoral immune responses using filamentous phage as a model antigen

“…multiple factors, including the nature of the displayed molecule and/or the method of its display ( Table 1). Specifically, we speculate that, as reported for other peptides, [26][27][28] the native antigenic structure of MD10 may be stabilized by the peptide bond with pVIII (since it was originally selected from a phage-displayed peptide library as a recombinant fusion to the N-terminus of pVIII); this may be particularly important for short peptides (MD10 is 6 residues long) that are less likely to adopt stable folded conformations as free peptides in solution. 29 In support of this, others have found synthetic MD10 peptide conjugated to other carrier proteins to be very poorly immunogenic.…”