Defining critical residues in the epitope for a HIV-neutralizing monoclonal antibody using phage display and peptide array technologies.
A peptide display library [Scott and Smith, Science 249 (1990) 386-390] was constructed that expressed 1.5 x 10(8) unique 20-amino-acid (aa) peptides fused to the N-terminus of the pIII coat protein of filamentous phage fd. This phage display library (PDL-20) was prepared using a degenerate oligodeoxyribonucleotide designed to minimize bias towards most aa. Characterization of the PDL-20 showed that all aa were present at the expected frequency and that there was no positional bias. Screening of this library with a HIV-1 isotype MN envelope reactive monoclonal antibody (mAb 58.2) using two different panning procedures showed that the biopanning technique was sensitive to one phage in 10(8). Analysis of peptide sequences from panning the mAb identified a core antibody recognition sequence of four aa residues (GPGR) and two preferred flanking residues on either side. This epitope occurred at various locations within the random aa segment demonstrating an absence of positional or nearest neighbor effects. Parallel panning experiments using an array of 266 synthetic peptides identified an epitope similar to that defined by the phage display library.
Jellis
C L
CL
Repligen Corporation, Cambridge, MA 02139.
Cradick
T J
TJ
Rennert
P
P
Salinas
P
P
Boyd
J
J
Amirault
T
T
Gray
G S
GS
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D001483:Base Sequence; D000939:Epitopes; D015497:HIV-1 / Q000276:immunology*; D017912:Inovirus; D008969:Molecular Sequence Data; D010455:Peptides; D011993:Recombinant Fusion Proteins
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes; D010455:Peptides; D011993:Recombinant Fusion Proteins