Antibodies to an Epstein Barr Virus protein that cross-react with dsDNA have pathogenic potential.

Pathogens such as the Epstein Barr virus (EBV) have long been implicated in the etiology of systemic lupus erythematosus (SLE). The Epstein Barr virus nuclear antigen I (EBNA-1) has been shown to play a role in the development of anti-nuclear antibodies characteristic of SLE. One mechanism by which EBV may play a role in SLE is molecular mimicry. We previously generated two monoclonal antibodies (mAbs) to EBNA-1 and demonstrated that they cross-react with double-stranded DNA (dsDNA). In the present study, we demonstrate that these mAbs have pathogenic potential. We show that they can bind to isolated rat glomeruli and that binding can be greatly diminished by pretreatment of glomeruli with DNase I, suggesting that these mAbs bind dsDNA in the kidney. We also demonstrate that these antibodies can deposit in the kidney when injected into mice and can induce proteinuria and elicit histopathological alterations consistent with glomerulonephritis. Finally, we show that these antibodies can cross-react with laminin and collagen IV in the extracellular matrix suggesting that direct binding to the glomerular basement membrane or mesangial matrix may also contribute to the antibody deposition in the kidney. In summary, our results indicate that EBNA-1 can elicit antibodies that cross-react with dsDNA, that can deposit in the kidney, and induce kidney damage. These results are significant because they support the role of a viral protein in SLE and lupus nephritis.

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

INTRODUCTION: 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. CONCLUSION: 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.

HIV therapy by a combination of broadly neutralizing antibodies in humanized mice.

Human antibodies to human immunodeficiency virus-1 (HIV-1) can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection. Previous work showed that antibodies exert selective pressure on the virus but escape variants emerge within a short period of time. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice. Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy, the longer half-life of antibodies led to control of viraemia for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in humanized mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals.

BAFF overexpression promotes anti-dsDNA B-cell maturation and antibody secretion.

Overexpression of BAFF is believed to play an important role in systemic lupus erythematosus and elevated levels of serum BAFF have been found in lupus patients. Excess BAFF also leads to overproduction of anti-dsDNA antibodies and a lupus-like syndrome in mice. In the present study, we use mice transgenic for the R4A-Cmu (IgM) heavy chain of an anti-dsDNA antibody, to study the effects of BAFF overexpression on anti-dsDNA B-cell regulation. We observe that overexpression of BAFF promotes anti-dsDNA B-cell maturation and secretion of antibody and enriches for transgenic anti-dsDNA B cells in the marginal zone and follicular splenic compartments. In addition, our data suggests that BAFF rescues a subset of anti-dsDNA B cells from a regulatory checkpoint in the transitional stage of development.

Loss of tolerance of anti-dsDNA B cells in mice overexpressing CD19.

Mice transgenic for the R4A-Cmu heavy chain of an anti-dsDNA antibody, maintain tolerance by anergy and deletion. In C57BL/6 mice overexpressing CD19, a molecule, which lowers the threshold for B cell activation, elevated levels of serum autoantibodies have been observed. In the present study, we wished to determine whether CD19 overexpression could alter the induction of tolerance in R4A-Cmu mice and lead to the secretion of transgenic anti-dsDNA antibodies. We, therefore, bred R4A-Cmu transgenic mice-to-mice transgenic for human CD19 (hCD19) and generated R4A-Cmu mice heterozygous and homozygous for hCD19. We, now report the spontaneous secretion of transgenic IgM anti-dsDNA antibody in the sera of R4A-Cmu mice overexpressing CD19, indicative of a loss of B cell tolerance. We observe that transgenic B cells secreting anti-dsDNA antibody in these mice are T independent and display a marginal zone like phenotype althought they do not reside in the MZ. In addition, they appear to be derived from the conventional B2 subset rather than the B1 subset. Interestingly, a subset of the anti-dsDNA B cells in these mice still display the phenotype and functional characteristics of anergic B cells. These B cells cannot be activated to secrete antibody following BCR crosslinking, however, they are hyper-responsive to activation by innate signaling mechanisms. This suggests that CD19 overexpression may promote anergic B cells to escape tolerance by converging with BCR independent pathways, thereby rendering these B cells hyper-responsive to innate signaling.

Expression of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) in the mouse can elicit the production of anti-dsDNA and anti-Sm antibodies.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by the production of anti-nuclear antibodies. The etiology of SLE is unknown, although several viruses including the Epstein-Barr virus (EBV) have been implicated. An increase in the frequency of EBV infection has been observed in SLE patients relative to normal individuals. Some patients with SLE develop antibodies that recognize a proline rich epitope in the ribonucleoprotein Sm B/B that is similar to an epitope in EBNA-1, a major nuclear antigen of EBV. In the present study we have cloned the EBNA-1 gene under the control of the CMV promoter in the vector pcDNA3. We now report for the first time that expression of the entire EBNA-1 protein in the mouse can elicit the production of IgG antibodies to Sm and to double-stranded DNA (dsDNA). Our data suggest that the anti-Sm response arises as a consequence of antigenic cross-reactivity by anti-EBNA-1 antibodies. These results support a possible association between EBV infection and SLE.