Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Yadav P

PROVIDER: S-EPMC5004290 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Yadav Pragya P Carr Matthew T MT Yu Ruby R Mumbey-Wafula Alice A Spatz Linda A LA

Immunity, inflammation and disease 20160802 3

<h4>Introduction</h4>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 ...[more]

PMID: 27621818

Similar Datasets

Project description:Dengue virus (DV) infection causes a spectrum of clinical diseases ranging from dengue fever to a life-threatening dengue hemorrhagic fever. Four distinct serotypes (DV1-4), which have similar genome sequences and envelope protein (E protein) antigenic properties, were divided. Among these 4 serotypes, DV1 usually causes predominant infections and fast diagnosis and effective treatments are urgently required to prevent further hospitalization and casualties.To develop antibodies specifically targeting and neutralizing DV1, we immunized mice with UV-inactivated DV1 viral particles and recombinant DV1 E protein from residue 1 to 395 (E395), and then generated 12 anti-E monoclonal antibodies (mAbs) as the candidates for a series of characterized assays such as ELISA, dot blot, immunofluorescence assay, western blot, and foci forming analyses.Among the mAbs, 10 out of 12 showed cross-reactivity to four DV serotypes as well as Japanese encephalitis virus (JEV) in different cross-reactivity patterns. Two particular mAbs, DV1-E1 and DV1-E2, exhibited strong binding specificity and neutralizing activity against DV1 and showed no cross-reactivity to DV2, DV3, DV4 or JEV-infected cells as characterized by ELISA, dot blot, immunofluorescence assay, western blot, and foci forming analyses. Using peptide coated indirect ELISA, we localized the neutralizing determinants of the strongly inhibitory mAbs to a sequence-unique epitope on the later-ridge of domain III of the DV1 E protein, centered near residues T346 and D360 (346TQNGRLITANPIVTD360). Interestingly, the amino acid sequence of the epitope region is highly conserved among different genotypes of DV1 but diverse from DV2, DV3, DV4 serotypes and other flaviviruses.Our results showed two selected mAbs DV1-E1 and DV1-E2 can specifically target and significantly neutralize DV1. With further research these two mAbs might be applied in the development of DV1 specific serologic diagnosis and used as a feasible treatment option for DV1 infection. The identification of DV1 mAbs epitope with key residues can also provide vital information for vaccine design.

Project description:Enterotoxigenic Escherichia coli (ETEC) is a leading diarrheagenic bacterial pathogen among travelers and children in resource-limited regions. Adherence to host intestinal cells mediated by ETEC fimbriae is believed to be a critical first step in ETEC pathogenesis. These fimbriae are categorized into related classes based on sequence similarity, with members of the class 5 fimbrial family being the best characterized. The eight related members of the ETEC class 5 fimbrial family are subdivided into three subclasses (5a, 5b, and 5c) that share similar structural arrangements, including a fimbrial tip adhesin. However, sequence variability among the class 5 adhesins may hinder the generation of cross-protective antibodies. To better understand functional epitopes of the class 5 adhesins and their ability to induce intraclass antibody responses, we produced 28 antiadhesin monoclonal antibodies (MAbs) to representative adhesins CfaE, CsbD, and CotD, respectively. We determined the MAb cross-reactivities, localized the epitopes, and measured functional activities as potency in inhibition of hemagglutination induced by class 5 fimbria-bearing ETEC. The MAbs' reactivities to a panel of class 5 adhesins in enzyme-linked immunosorbent assays (ELISAs) revealed several reactivity patterns, including individual adhesin specificity, intrasubclass specificity, intersubclass specificity, and class-wide cross-reactivity, suggesting that some conserved epitopes, including two conserved arginines, are shared by the class 5 adhesins. However, the cross-reactive MAbs had functional activities limited to strains expressing colonization factor antigen I (CFA/I), coli surface antigen 17 (CS17), or CS1, suggesting that the breadth of functional activities of the MAbs was more restricted than the repertoire of cross-reactivities measured by ELISA. The results imply that multivalent adhesin-based ETEC vaccines or prophylactics need more than one active component to reach broad protection.

Dataset Information

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

Introduction

Methods

Results

Conclusion

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure