Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: implications for the tertiarystructure of the receptor and for an N-terminal binding site for HIV-1 gp120

The chemokine receptor CCR5 contains seven transmembrane-spanning domains. it binds chemokines and acts as co-receptor for macrophage (m)-tropic (or R 5) strains of HIV-1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)-constrained phage-displayed random peptide library to ascertain contact residues and define tertiary s tructures of possible epitopes on CCR5. Reactivity of antibodies with phago topes was established by enzyme-linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C-HASIYDFGS-C (3A9/1), and 5C7 most frequently iden tified C-PHWLRDLRV-C (5C7/1). Corresponding peptides were synthesized. Phag otopes and synthetic peptides reacted in ELISA with corresponding antibodie s and synthetic peptides inhibited antibody binding to the phagotopes. Reac tivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagoto pes and the corresponding peptide selected by the alternative mAb. The sequ ences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9/1, the motif SIYD aligned to residues at t he N terminus and FG to residues on the first extracellular loop; for 5C7/1 , residues at the N terminus, first extracellular loop, and possibly the th ird extracellular loop could be aligned and so would contribute to the mimo tope. The synthetic peptides corresponding to the isolated phagotopes showe d a CD4-dependent reactivity with gp120 of a primary, m-tropic HIV-1 isolat e. Thus reactivity of antibodies raised to CCR5 against phage-displayed pep tides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.