Isolation of a neutralizing human RSV antibody from a dominant, non-neutralizing immune repertoire by epitope-blocked panning.
We isolated a large panel of human Abs directed against the respiratory syncytial virus (RSV) Ag from combinatorial phage display libraries. Following initial differentiation of the Fabs by BstNI restriction patterns, DNA sequence analysis revealed 10 different classes of VH paired with more than 35 different VL genes. All the Fabs bound with high affinity to the F Ag. However, most Fabs competed with the binding of a representative member of this group, suggesting that the Fabs recognized a common epitope on the F Ag, and none of them neutralized virus in vitro. To suppress repetitive isolation of these non-neutralizing Abs, a representative Fab was included during panning to block this common epitope on the F Ag. By this "epitope-blocked panning" approach, two novel Fabs, encoded by unique VH and VL genes, were isolated from a previously screened library. Competition binding analysis confirmed that the Fabs recognized epitopes distinct from that of the previously isolated Fabs. One of these Fabs, 516, neutralized RSV in cell culture. These activities of Fab-516 were retained upon its genetic conversion to a mAb (IgG1) and expression in mammalian cells. Our results suggest that the RSV F glycoprotein presents a dominant, non-neutralizing epitope to the human immune system, which may serve in evasion of host defenses. However, less prevalent, fusion-inhibiting Abs were revealed by blockade of this epitope during the panning process.
Tsui
P
P
Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
Tornetta
M A
MA
Ames
R S
RS
Bankosky
B C
BC
Griego
S
S
Silverman
C
C
Porter
T
T
Moore
G
G
Sweet
R W
RW
D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D017104:Bacteriophage M13 / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D005822:Genetic Vectors / Q000276:immunology; D006801:Humans; D016056:Immunodominant Epitopes / Q000235:genetics / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000276:immunology*; D017421:Sequence Analysis
D000914:Antibodies, Viral; D000956:Antigens, Viral; D016056:Immunodominant Epitopes; D007140:Immunoglobulin Fab Fragments