Mapping an epitope in EBNA-1 that is recognized by monoclonal antibodies to EBNA-1 that cross-react with dsDNA.
BACKGROUND
The Epstein Barr Virus (EBV) has been associated with the autoimmune disease, Systemic Lupus Erythematosus (SLE). EBV nuclear antigen-I (EBNA-1) is the major nuclear protein of EBV. We previously generated an IgG monoclonal antibody (MAb) to EBNA-1, 3D4, and demonstrated that it cross-reacts with double stranded DNA (dsDNA) and binds the 148 amino acid viral binding site (VBS) in the carboxyl region of EBNA-1. The aim of the present study was to characterize another antibody to EBNA-1 that cross-reacts with dsDNA, compare its immunoglobulin genes to 3D4, and finely map the epitope in EBNA-1 that is recognized by these cross-reactive antibodies.


METHODS
We generated an IgM MAb to EBNA-1, 16D2, from EBNA-1 injected mice and demonstrated by ELISA that it cross-reacts with dsDNA and binds the 148 amino acid VBS. We sequenced the variable heavy and light chain genes of 3D4 and 16D2 and compared V gene usage. To more finely map the epitope in EBNA-1 recognized by these MAbs, we examined their binding by ELISA to 15 overlapping peptides spanning the 148 amino acid domain.


RESULTS
Sequence analysis revealed that 3D4 and 16D2 utilize different VH and VL genes but identical JH and Jk regions with minimal junctional diversity. This accounts for similarities in their CDR3 regions and may explain their similar dual binding specificity. Epitope mapping revealed 3D4 and 16D2 bind the same peptide in the VBS. Based on the crystal structure of EBNA-1, we observed that this peptide resides at the base of an exposed proline rich loop in EBNA-1.


CONCLUSIONS
We have demonstrated that two MAbs that bind EBNA-1 and cross-react with dsDNA, recognize the same peptide in the VBS. This peptide may serve as a mimetope for dsDNA and may be of diagnostic and therapeutic value in SLE.
Department of ChemistryCity College of New York160 Convent AvenueNew YorkNew York10031; Graduate Program in BiochemistryGraduate Center of the City University of New York160 Convent AvenueNew YorkNew York10031.
Pragya
P
Yadav
Department of ChemistryCity College of New York160 Convent AvenueNew YorkNew York10031; Graduate Program in BiochemistryGraduate Center of the City University of New York160 Convent AvenueNew YorkNew York10031.
Matthew T
MT
Carr
Department of Biology City College of New York 160 Convent Avenue New York New York 10031.
Ruby
R
Yu
Department of Pathobiology, Sophie Davis School of Biomedical Education City College of New York 160 Convent Avenue New York New York 10031.
Alice
A
Mumbey-Wafula
Department of Pathobiology, Sophie Davis School of Biomedical Education City College of New York 160 Convent Avenue New York New York 10031.
Linda A
LA
Spatz
EBNA‐1; SLE; anti‐dsDNA antibodies; autoantibody; cross‐reactivity; molecular mimicry