Mapping an epitope in EBNA-1 that is recognized by monoclonal antibodies to EBNA-1 that cross-react with dsDNA

Abstract

IntroductionThe 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.MethodsWe 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.ResultsSequence 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.ConclusionWe 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.

Engineered resistance to Nosema bombycis by in vitro expression of a single-chain antibody in Sf9-III cells

“…The cDNA of G4 hybridoma cell line was PCR-amplified with the RT-PCR Kit (Promega, Madison, Wisconsin, USA). Heavy-chain and light-chain variable regions were cloned using the following two degenerate primer pairs: VH-F: ′, VH-R: [ 23 ] and VL-F: [ 24 ], VL-R: [ 25 ]. The two variable regions were then ligated to pMT19-T for sequence analysis.…”

Epstein Barr Virus and Autoimmune Responses in Systemic Lupus Erythematosus

“…Antibodies towards different regions of EBNA-1 protein cross-react with SLE autoantigens SmB, SmD, as well as Ro ( 67 ). Monoclonal antibodies generated from mice immunized with EBNA-1 cross-react with dsDNA ( 68 , 69 ). Cross-reactivity between the anti-EBNA-1 response and anti- complement component C1q response has also been shown.…”

Molecular mimicry, genetic homology, and gene sharing proteomic “molecular fingerprints” using an EBV (Epstein-Barr virus)-derived microarray as a potential diagnostic method in autoimmune disease

“…A correlation between antigen-specific IgG levels to specific EBV proteins has been reported in SLE, MS (multiple sclerosis), and other autoimmune syndromes [4][5][6][7][8][9]. Since the vast majority of the adult population is positive for EBV, it would be useful to have additional laboratory analysis for clinical evaluation and monitoring of autoimmune disease in patients based on response to past EBV infection [10][11][12][13].…”