Article

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.

0 Followers
 · 
103 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Design of an envelope-based immunogen capable of inducing a broadly neutralizing antibody response is thought to be key to the development of a protective HIV-1 vaccine. However, the broad diversity of viral variants and a limited ability to produce native envelope have hampered such design efforts. Here we describe adaptation of the yeast display system and use of a combinatorial protein engineering approach to permit directed evolution of HIV envelope variants. Because the intrinsic instability and complexity of this trimeric glycoprotein has greatly impeded the development of immunogens that properly represent the structure of native envelope, this platform addresses an essential need for methodologies with the capacity to rapidly engineer HIV spike proteins towards improved homogeneity, stability, and presentation of neutralizing epitopes. We report for the first time the display of a designed SOSIP gp140 on yeast, and the in vitro evolution of derivatives with greatly improved expression and binding to conformation-dependent antibodies. These efforts represent an initial and critical step toward the ability to rapidly engineer HIV-1 envelope immunogens via directed evolution.
    PLoS ONE 02/2015; 10(2):e0117227. DOI:10.1371/journal.pone.0117227 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.
    mAbs 11/2014; 6(6). DOI:10.4161/mabs.36144 · 4.73 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: HIV rapidly mutates to escape antibody detection, and B cells counter this mutation by continual evolution to restore recognition, serendipitously resulting in the evolution of neutralizing activity in a fraction of infected individuals. A recent Cell paper describes how antibody repertoires stochastically collaborated, shaping the viral swarm and utilizing viral immune evasion to their advantage.
    Trends in Immunology 08/2014; 35(9). DOI:10.1016/j.it.2014.07.009 · 12.03 Impact Factor

Full-text (2 Sources)

Download
27 Downloads
Available from
Jun 6, 2014