High-resolution epitope mapping for monoclonal antibodies to the structural protein Erns of classical swine fever virus using peptide array and random peptide phage display approaches.
The structural glycoprotein E(rns) (an envelope protein with RNase activity) of classical swine fever virus (CSFV) is not well characterized with respect to its antigenic structure and organization. Here, we investigated the antigenic sites on E(rns) by raising mAbs against the Escherichia coli expressed E(rns) of CSFV strain Alfort/187 and defined the B-cell epitopes recognized by these antibodies. Eighteen mAbs to E(rns) were identified and they were classified as either immunoglobulin subclass G1 or G2b. Using an array of overlapping 12-mer peptides, spanning aa 27-227 of E(rns), the epitopes for 12 mAbs were mapped to a high resolution of six to eight residues, which cluster in five discrete locations, ¹³GIWPEKIC³⁸ (group I), ⁶⁵NYTCCKLQ⁷² (group II), ¹²⁷QARNRPTT¹³⁴ (group III), ¹⁴⁵SFAGTVIE¹⁵² (group IV) and ¹⁶¹VEDILY¹⁶⁶ (group V). Two mAbs recognize two or more antigenic determinants, including the group II epitope. The epitopes for four other mAbs could not be mapped using the overlapping 12-mer peptides. Random peptide phage display with one mAb from each of all the groups except group V further identified some conserved residues that may be critical for binding antibodies, i.e. Trp³³ in the epitope of group I, Leu⁷¹ in the epitope of group II, Gln¹²⁷ and Apn¹³⁰ in the epitope of group III, and Ser¹⁴⁵ and Gly¹⁴⁸ in the epitope of group IV. This study has provided new insights into the structure and organization of epitopes on the CSFV E(rns) and valuable epitope information for the rational design of vaccines, drugs and diagnostic immunoassays for CSFV.
Lin
Min
M
Canadian Food Inspection Agency, Ottawa Laboratory (Fallowfield), Ottawa, ON, Canada. linm@inspection.gc.ca
McRae
Heather
H
Dan
Hanhong
H
Tangorra
Erin
E
Laverdiere
Amanda
A
Pasick
John
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000276:immunology* / Q000302:isolation & purification; D006692:Classical Swine Fever Virus / Q000276:immunology*; D017124:Conserved Sequence; D018604:Epitope Mapping / Q000379:methods*; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D005260:Female; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D040081:Protein Array Analysis; D012260:Ribonucleases / Q000276:immunology*; D015678:Viral Structural Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D018985:Epitopes, B-Lymphocyte; D007074:Immunoglobulin G; D019151:Peptide Library; D015678:Viral Structural Proteins; D012260:Ribonucleases