Comparison of two single-chain antibodies that neutralize canine parvovirus: analysis of an antibody-combining site and mechanisms of neutralization.
We cloned the heavy- and light-chain variable domains of two monoclonal antibodies that recognize each of the two major neutralizing antigenic sites of the canine parvovirus (CPV) capsid. After expression in Escherichia coli as single-chain variable domains (scFv) with glycine-serine linker sequences, both scFv bound CPV capsids with the same specificity as the intact IgG, but with 10- to 20-fold lower avidity. Both scFvs neutralized CPV infectivity with efficiency similar to that of the IgG. Although both IgGs inhibited hemagglutination by CPV, only one scFv was inhibiting. The binding of one of the antibodies has previously been analyzed by cryoelectron microscopic reconstruction and the epitope-binding residues predicted. Mutagenesis of predicted contact residues in three heavy-chain complementarity-determining regions (CDR) showed that mutants of CDR1 or CDR3 reduced the binding of the scFv by about 10-fold compared with the wild-type scFv, while no effect was seen for one mutant of CDR2. The levels of neutralization of CPV and of hemagglutination inhibition by the scFv mutants were proportional to their reduction in binding affinity compared with the wild type. Neither scFv blocked virus binding to host cells, but they both caused aggregation of the capsids and appeared to affect the process of infection after virus uptake into the cells.
Yuan
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James A. Baker Institute for Animal Health, Cornell University, Ithaca, New York, 14853, USA.
Parrish
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D002213:Capsid / Q000276:immunology; D002460:Cell Line; D003001:Cloning, Molecular; D004285:Dogs; D007135:Immunoglobulin Variable Region / Q000276:immunology; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D017993:Parvovirus, Canine / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007135:Immunoglobulin Variable Region