Generation and epitope mapping of novel neutralizing monoclonal antibodies against glycoprotein E2 of CSFV

Classical swine fever virus (CSFV) is a highly contagious and economically important pathogen threatening pig industry worldwide, the envelope glycoprotein E2 of CSFV is the dominant antigen inducing strong antiviral neutralizing immunity. In this study, 7 monoclonal antibodies (mAbs) with neutralizing potency were generated using E2 protein of CSFV Shimen strain (SM) expressed by eukaryotic cells. Their reactivity with 116 CSFV strains in cell cultures and E2 proteins of 10 subgenotypes in western blots showed different CSFV spectrums they recognized. Of them, three (HCL-001, HCL-005 and HCL-010) reacted with all CSFV subgenotypes, while HCL-014 and HCL-002 reacted with most CSFV strains, except for some variants in genotype 2.3. In contrast, mAb HCL-009 reacted only with a few subgenotype 1.1 strains including SM, field strains and some vaccine strains. Interestingly, mAb HCL-018 reacted only with SM and field subgenotype 1.1 strains, not with any vaccine strains. Further epitope mapping using chimeric and site-directed mutated E2 proteins showed that HCL-001, HCL-005 and HCL-010 recognized a conservative epitope motif ¹⁴³SPT¹⁴⁵,L¹⁴⁷, and HCL-002 recognized a conformational epitope with key aa motifs of ⁹⁵GDD⁹⁷,¹⁵⁷RX(D/E)K(R)XFXXR¹⁶⁴. HCL-014 recognized a new conservative epitope with key aa motifs of ⁴¹D,⁵⁸XNVVXRR⁶⁴. HCL-009 and HCL-018 recognized the epitope with key aa motifs of ³⁶D,⁴⁰ND⁴¹,⁴⁵KXI⁴⁷ and ⁶⁹LHXGXLLT⁷⁶, respectively. Taken together, present study has provided not only new insights into the antigenic structure of E2 protein, but also key reagents for antigenic characterization of CSFV strains and development of antibody assay for evaluation of the vaccination efficacy.