Antigenic mimicking with cysteine-based cyclized peptides reveals a previously unknown antigenic determinant on E2...

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies in infected pigs. The conformational epitope(s) on B/C domains were mapped to the N-terminal 90 residues of E2 between amino acids 690 and 779 (Chang et al., 2010a). To mimic the conformational epitopes, a set of synthetic cyclized peptides spanning the B/C domains of E2 were used to react with monoclonal antibodies (mAbs) against E2 and with swine anti-CSFV polyclonal sera. All antibodies recognized a highest common element, (753)RYLASLHKKALPTSV(767), on the double-looped peptides. This epitope region has not been revealed previously in the literature. Both substitution-scanning of residues (753)RYLASLHKKALPTSV(767) on a double-looped peptide and site-directed mutagenesis of expressed E2 demonstrated that residues (761)K, (763)L and (764)P were critical for the reactivity with mAbs. In addition, the up- and downstream residues (753)R, (754)Y, (755)L and (765)T were also crucial. Alignment showed that this stretch of amino acids was relatively conserved among various CSFVs. Thus, we identified a motif (753)RYLASLHKKALPT(765), which may be part of group-specific antigen and important for the structural integrity of conformational epitope recognition.