Antigenic mimicking with cysteine-based cyclized peptides reveals a previously unknown antigenic determinant on E2 glycoprotein of classical swine fever virus

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, 753RYLASLHKKALPTSV767, on the double-looped peptides. This epitope region has not been revealed previously in the literature. Both substitution-scanning of residues 753RYLASLHKKALPTSV767 on a double-looped peptide and site-directed mutagenesis of expressed E2 demonstrated that residues 761K, 763L and 764P were critical for the reactivity with mAbs. In addition, the up- and downstream residues 753R, 754Y, 755L and 765T were also crucial. Alignment showed that this stretch of amino acids was relatively conserved among various CSFVs. Thus, we identified a motif 753RYLASLHKKALPT765, which may be part of group-specific antigen and important for the structural integrity of conformational epitope recognition.