Isolation and characterization of human neutralizing antibodies to rabies virus derived from a recombinant immune antibody library.
Résumé
A human immune Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from rabies virus hyperimmune volunteers on phagemid vector. The size of the constructed Fab library was 2 x 10(7) Escherichia coli transformants. After four rounds of panning on whole inactivated rabies virus (PV-11), phage clones displaying rabies virus-specific human Fab were selected. The specificity of soluble Fab antibody fragments, derived from positive phage clones was verified by ELISA. Among 20 specific Fab clones, the genetic sequence of 6 of them (FabRV01, FabRV02, FabRV03, FabRV04, FabRV05, and FabRV06) was analyzed. The variable heavy (VH) and variable light (VL) domains were found to share 90% and 93% homology with sequences encoded by the corresponding human germline genes, respectively. The soluble Fab fragments, expressed in Escherichia coli were purified by a single step Nickel-NTA affinity chromatography via a hexa-histidine tag and their binding specificities to rabies virus were confirmed. Three of the Fab antibodies, FabRV01, FabRV02 and FabRV03, showed binding characteristics to rabies virus glycoprotein antigenic site III with affinities in the K(D) range 7 x 10(-9) to 5 x 10(-8)M. The Fab fragments showed dose-dependent neutralization properties for the challenge virus standard (CVS-11).