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

Abstract

The chemokine receptor CCR5 contains seven transmembrane‐spanning domains. It binds chemokines and acts as co‐receptor for macrophage (m)‐tropic (or R5) 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 structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme‐linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C‐HASIYDFGS‐C (3A9 / 1), and 5C7 most frequently identified C‐PHWLRDLRV‐C (5C7 / 1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences 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 the 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 third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4‐dependent reactivity with gp120 of a primary, m‐tropic HIV‐1 isolate. Thus reactivity of antibodies raised to CCR5 against phage‐displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.

Induction of Autoantibodies to CCR5 in Macaques and Subsequent Effects upon Challenge with an R5-Tropic Simian/Human Immunodeficiency Virus

“…3A9 has been demonstrated to bind to a conformational epitope of CCR5 that includes the EC1 domain (29). Whole blood from a naïve pig-tailed macaque was incubated with a MAb against CD95 along with IgG from either a vaccinated (97P026) or a control (96P080) macaque and then reacted with a limiting amount of PE-labeled 3A9.…”

Circulating human CD4 and CD8 T cells do not have large intracellular pools of CCR5

“…In initial flow cytometric experiments, human PBMCs were gated for expression of both CD3 and CD4 or CD8 and were stained with 3 monoclonal anti-CCR5 antibodies recognizing different domains. [14][15][16][17] In agreement with the findings of Achour et al, 11 we found that, although cells from each donor indicated variable but universally low levels of CCR5 staining on nonpermeabilized cells, uniformly strong signals were obtained on fixed and permeabilized cells ( Figure 1A). Control GHOST (3) parental cell lines that do not express CCR5, however, also gave positive results when the same anti-CCR5 antibodies were used to stain fixed and permeabilized cells ( Figure 1B).…”

Peptide Ligands Selected with CD4-Induced Epitopes on Native Dualtropic HIV-1 Envelope Proteins Mimic Extracellular Coreceptor Domains and Bind to HIV-1 gp120 Independently of Coreceptor Usage

“…1C) (3). Interestingly, some peptide sequences contained additional amino acid motifs present in extracellular loops 1 and 3 of CCR5, which were previously described to be present in the mimotopes that we selected with two HIV-neutralizing MAbs against CCR5 (MAbs 3A9 and 5C7) and to be involved in HIV-1 entry (33,34,42). Due to its high reactivity, frequent selection, and strong sequence similarity to different extracellular regions of coreceptors, phage a was further analyzed.…”