BackgroundSeveral genetic and environmental factors have been linked to Systemic Lupus Erythematosus (SLE). One environmental trigger that has a strong association with SLE is the Epstein Barr Virus (EBV). Our laboratory previously demonstrated that BALB/c mice expressing the complete EBNA-1 protein can develop antibodies to double stranded DNA (dsDNA). The present study was undertaken to understand why anti-dsDNA antibodies arise during the immune response to EBNA-1.Methodology/Principal FindingsIn this study, we demonstrated that mouse antibodies elicited in response to EBNA-1 cross-react with dsDNA. First, we showed that adsorption of sera reactive with EBNA-1 and dsDNA, on dsDNA cellulose columns, diminished reactivity with EBNA-1. Next, we generated mononclonal antibodies (MAbs) to EBNA-1 and showed, by several methods, that they also reacted with dsDNA. Examination of two cross-reactive MAbs—3D4, generated in this laboratory, and 0211, a commercial MAb—revealed that 3D4 recognizes the carboxyl region of EBNA-1, while 0211 recognizes both the amino and carboxyl regions. In addition, 0211 binds moderately well to the ribonucleoprotein, Sm, which has been reported by others to elicit a cross-reactive response with EBNA-1, while 3D4 binds only weakly to Sm. This suggests that the epitope in the carboxyl region may be more important for cross-reactivity with dsDNA while the epitope in the amino region may be more important for cross-reactivity with Sm.Conclusions/SignificanceIn conclusion, our results demonstrate that antibodies to the EBNA-1 protein cross-react with dsDNA. This study is significant because it demonstrates a direct link between the viral antigen and the development of anti-dsDNA antibodies, which are the hallmark of SLE. Furthermore, it illustrates the crucial need to identify the epitopes in EBNA-1 responsible for this cross-reactivity so that therapeutic strategies can be designed to mask these regions from the immune system following EBV exposure.

Several studies have shown an association between Systemic Lupus Erythematosus (SLE) and the Epstein Barr Virus (EBV). Our laboratory recently demonstrated that BALB/c mice injected with the major EBV nuclear antigen, EBNA-1, developed antibodies to EBNA-1 that cross-react with double stranded DNA (dsDNA). We generated monoclonal antibodies (MAbs) to EBNA-1 from EBNA-1 injected mice and demonstrated by anti-dsDNA ELISA, dsDNA affinity columns, and Crithidia luciliae assays, that these antibodies cross-react with dsDNA. Studies are underway to determine the pathogenicity of these MAbs. We have been mapping the epitope in EBNA-1 that serves as a peptide mimotope for dsDNA. We now demonstrate that the mimotope is contained within the carboxyl region of the EBNA-1 protein, between amino acids 459 and 607. This region contains well-defined secondary structure, which suggests that the basis for the cross-reactivity with dsDNA could be due to a conformational epitope in EBNA-1. Studies are in progress to further map this cross-reactive epitope. Identification of this epitope may help in designing diagnostic and therapeutic strategies that can mask the epitope from the immune system following EBV infection and thereby prevent the development of antibodies that cross-react with dsDNA.

Several genetic and environmental factors have been linked to Systemic Lupus Erythematosus (SLE). One environmental trigger that has a strong association with SLE is the Epstein Barr Virus (EBV). Our laboratory previously demonstrated that BALB/c mice expressing the EBNA-1 protein, developed antibodies to double stranded DNA (dsDNA). The present study was undertaken to determine the basis for this anti-dsDNA response. We now demonstrate that BALB/c mice immunized with recombinant EBNA-1 protein develop antibodies to EBNA-1 that cross-react with dsDNA. Close examination of two monoclonal antibodies to EBNA-1 has demonstrated their reactivity with dsDNA but not unrelated proteins and absorption on dsDNA cellulose columns removes their reactivity to both dsDNA and EBNA-1. In addition, these monoclonal antibodies recognize different regions of the EBNA-1 protein suggesting that more than one epitope in EBNA-1 may be the target of antibodies that cross-react with dsDNA. These results support the association of SLE with EBV and emphasize the need to prevent EBV infection or to mask EBNA-1 protein from the immune system following EBV exposure, in order to minimize the risk of autoimmunity.