Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12 - PubMed (original) (raw)
Comparative Study
Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12
M B Zwick et al. J Virol. 2001 Jul.
Abstract
Human monoclonal antibody (MAb) b12 recognizes a conformational epitope that overlaps the CD-4-binding site of the human immunodeficiency virus type 1 (HIV-1) envelope. MAb b12 neutralizes a broad range of HIV-1 primary isolates and protects against primary virus challenge in animal models. We report here the discovery and characterization of B2.1, a peptide that binds specifically to MAb b12. B2.1 was selected from a phage-displayed peptide library by using immunoglobulin G1 b12 as the selecting agent. The peptide is a homodimer whose activity depends on an intact disulfide bridge joining its polypeptide chains. Competition studies with gp120 indicate that B2.1 occupies the b12 antigen-binding site. The affinity of b12 for B2.1 depends on the form in which the peptide is presented; b12 binds best to the homodimer as a recombinant polypeptide fused to the phage coat. Originally, b12 was isolated from a phage-displayed Fab library constructed from the bone marrow of an HIV-1-infected donor. The B2.1 peptide is highly specific for b12 since it selected only phage bearing b12 Fab from this large and diverse antibody library.
Figures
FIG. 1
Analysis of binding of biotinylated IgG1 b12 to B2.1 phage (B2.1φ) and B2.1 synthetic peptide (B2.1pep) by ELISA. Competition for IgG1 b12 binding to plate-adsorbed B2.1 phage by the following in-solution competitors is shown: 2 × 1010 B2.1 phage, 300 μM B2.1 synthetic peptide, 100 nM gp120Ba-L (gp120), f88-4 phage (f88 φ), and unrelated peptide G45B, whose sequence is VERSKAFSNCYPYDVPDYASLRS. BSA is bovine serum albumin, and n. c. indicates no in-solution competitor. O.D.405–490, optical density at 405 minus 490 nm.
FIG. 2
SDS-PAGE analysis of the f88-4 wild-type phage (f88) and recombinant B2.1 phage (all others). Phage were left untreated or treated with DTT, NEM, or NEM followed by DTT and then analyzed by SDS-PAGE. Monomeric (M) and dimeric (D) recombinant pVIII proteins are shown. Proteins in similar gels were either silver stained or transferred to a membrane and subjected to Western blotting with anti-phage Ab or IgG1 b12. Panels: A, silver-stained gel; B, Western blot using IgG1 b12 to show the reactive dimer; C, Western blot using rabbit anti-phage Ab to show the wild-type and recombinant pVIII proteins.
FIG. 3
Titration of Fab b12 (A), IgG1 b12 (B), and murine anti-B2.1 peptide serum (C) on different immobilized antigens. Twofold dilutions of Fab and IgG1 b12 and fourfold mouse serum dilutions were reacted with biotinylated B2.1 directly adsorbed to ELISA wells (bio-B2.1), biotinylated B2.1 bound to immobilized streptavidin (SA+bio-B2.1), gp120Ba-L, B2.1 recombinant phage, f88-4 phage, bovine serum albumin (BSA), ovalbumin, and streptavidin (SA). O.D., optical density.
FIG. 4
Kinetics of binding of IgG1 b12 to B2.1 peptide in-solution. (A) Percent free Ab versus molar concentration of peptide; data (diamonds) and the best-fit theoretical curve are shown. (B) Percent error from fit of the data in Fig. 3A to the best-fit curves calculated for a range of K _d_s. The 95% confidence interval calculated for this experiment is 1.3 to 3.7 μM.
FIG. 5
Structure of the D loop of gp120, residues 273 to 285, taken from the HXB2 HIV-1 isolate. The sequence of this region, whose alpha-carbon backbone is shown in red, is RSVNFTDNAKTII. Residues shared with the B2.1 peptide, HERSYMFSDLENRCI, are in bold type.
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