Structure of a High-affinity “Mimotope” Peptide Bound to HIV-1-neutralizing Antibody b12 Explains its Inability to Elicit gp120 Cross-reactive Antibodies

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Journal of Molecular Biology (Impact Factor: 3.96). 07/2007; 369(3):696-709. DOI: 10.1016/j.jmb.2007.01.060
Source: PubMed

ABSTRACT The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.

Download full-text


Available from: Ralph A Pantophlet, Jun 29, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Elucidating the network of interactions established by Interleukin-2 is a key step to understanding its role as a master regulator of the immune system. Binding of this cytokine by specific antibodies gives rise to different classes of immune complexes that boost or inhibit immune responses. The molecular bases of such functional dichotomy are likely related to the nature of the recognized epitopes, making it necessary to perform fine epitope mapping studies. The current work was aimed at developing a versatile platform to do so. This was accomplished by display of human and mouse Interleukin-2 on filamentous phages, together with extensive mutagenesis of both antigens and high throughput screening of binding properties of more than 200 variants. Detailed molecular pictures of the epitopes were thus delineated for four antibodies against either human or mouse Interleukin-2, which refined and, in some cases, modified the conclusions derived from previous mapping studies with peptide libraries. Overlapping surface patches on mouse Interleukin-2 that also coincide with the predicted interface between the cytokine and its receptor alpha chain were shown to be recognized by two monoclonal antibodies that promote enhancement of immune responses, shedding new light on the structural bases of their biological activity. Our strategy was powerful enough to reveal multiple binding details and could be used to map the epitopes recognized by other antibodies and to explore additional interactions involving Interleukin-2 and related cytokines, thus contributing to our understanding of the complex structure-function relationships within the immune system.
    Immunobiology 02/2012; DOI:10.1016/j.imbio.2012.02.009 · 3.18 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Peptide "mimics" (mimotopes) of linear protein epitopes and carbohydrate epitopes have been successfully used as immunogens to elicit cross-reactive antibodies against their cognate epitopes; however, immunogenic mimicry has been difficult to achieve for discontinuous protein epitopes. To explore this, we developed from phage-displayed peptide libraries optimized peptide mimics for three well-characterized discontinuous epitopes on hen egg lysozyme and horse cytochrome c. The peptides competed with their cognate antigens for antibody binding, displayed affinities in the nM range, and shared critical binding residues with their native epitopes. Yet, while immunogenic, none of the peptides elicited antibodies that cross-reacted with their cognate antigens. We analyzed the 3-D structure of the site within each discontinuous epitope that shared critical binding residues with its peptide mimic, and observed that in each case it formed a ridge-like patch on the epitope; in no case did it cover most or all of the epitope. Thus, the peptides' lack of immunogenic mimicry could be attributed to their inability to recapitulate the topological features of their cognate epitopes. Our results suggest that direct peptide immunizations are not a practical strategy for generating targeted antibody responses against discontinuous epitopes.
    Molecular Immunology 02/2010; 47(5):1137-48. DOI:10.1016/j.molimm.2009.10.015 · 3.00 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Several peptide mimics of a conserved H5N1 avian influenza virus neutralization site recognized by 8H5 mAb have been reported previously. In this study, the secondary and possibly higher structural orders of the peptide mimics 122 and 125 were investigated and found to be closely related to the specific binding with 8H5 mAb. These two peptide mimics were fused to three different carrier proteins, and the antibody binding activities were recovered in 4 of the 11 fusion proteins. HEV structural protein p239 and HBc were more suitable than the outer membrane protein T47 of the Treponema pallidum particle for the recovery of reactivity. The increase in the copy number of peptide mimics was important for the recovery of antibody-binding activity and the interaction between peptide and carrier protein may affect the spatial structure of both the peptide and the carrier protein. These results are likely to be of relevance for conformational peptide mimics in diagnostic tests, vaccine and inhibitors.
    Archives of Virology 01/2010; 155(1):19-26. DOI:10.1007/s00705-009-0542-2 · 2.28 Impact Factor