Identification of conformational neutralizing epitopes an the capsid protein of canine calicivirus

Two neutralizing monoclonal antibodies (MAbs) against canine calicivirus (C aCV), which has a distinct antigenicity from feline calicivirus (FCV), were obtained. Both MAbs recognized conformational epitopes on the capsid prote in of CaCV and were used to identify these epitopes. Neutralization-resista nt variants of CaCV were selected in the presence of individual MAbs in a c ell culture. Cross-neutralization tests using the variants indicated that t he MAbs recognized functionally independent epitopes on the capsid protein. Recombinantly expressed ORF2 products (capsid precursors) of the variants showed no reactivity to the MAbs used for the selection, suggesting that th e resistance was induced by a failing in binding of the MAbs to the variant capsid proteins. Several nucleotide changes resulting in amino acid substi tutions in the capsid protein were found by sequence analysis, Reactivities of the MAbs to the revertant ORF2 products produced from each variant ORF2 by site-directed mutagenesis identified a single amino acid substitution i n each variant capsid protein responsible for the failure of MAb binding. T he amino acid residues related to forming the conformational neutralizing e pitopes were located in regions equivalent to the 5' and 3' hypervariable r egions of the FCV capsid protein, where antigenic sites were demonstrated i n previous studies. The recombinant ORF2 products expressed in bacteria fai led to induce neutralizing antibody, suggesting that neutralizing antibodie s were only generated when properly folded capsid protein was used as an an tigen. In CaCV, the conformational epitopes may play a more important role in neutralization than do linear epitopes.