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Human anti-A? IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

ABSTRACT: Soluble non-fibrillar assemblies of amyloid-beta (A?) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-A? immunotherapy is a promising and advanced therapeutic strategy, but the precise A? species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse A? conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic A? dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized A? extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on A?'s conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic A? monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-A? NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-A? NAbs are warranted.

SUBMITTER: Welzel AT

PROVIDER: S-EPMC3507685 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Human anti-Aβ IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

Welzel Alfred T AT Williams Angela D AD McWilliams-Koeppen Helen P HP Acero Luis L Weber Alfred A Blinder Veronika V Mably Alex A Bunk Sebastian S Hermann Corinna C Farrell Michael A MA Ehrlich Hartmut J HJ Schwarz Hans P HP Walsh Dominic M DM Solomon Alan A O'Nuallain Brian B

PloS one 20121127 11

Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational e ...[more]

PMID: 23209707

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Project description:BackgroundPrecise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs.ResultsWe present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu), the antibody mAb Bo2C11 targeting the C(2) domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples the conformational epitopes as determined by the X-ray crystal structures of the antibody-antigen complexes, were found within the highest scoring patches of EpiSearch, covering in most cases more than 50% residues of experimental observed conformational epitopes. Input options of the program include mapping of a single peptide or a set of peptides on the antigen structure, and the results of the calculation can be visualized on our interactive web server.AvailabilityUsers can access the EpiSearch from our web server http://curie.utmb.edu/episearch.html.

Project description:A number of machine learning-based predictors have been developed for identifying immunogenic T-cell epitopes based on major histocompatibility complex (MHC) class I and II binding affinities. Rationally selecting the most appropriate tool has been complicated by the evolving training data and machine learning methods. Despite the recent advances made in generating high-quality MHC-eluted, naturally processed ligandome, the reliability of new predictors on these epitopes has yet to be evaluated. This study reports the latest benchmarking on an extensive set of MHC-binding predictors by using newly available, untested data of both synthetic and naturally processed epitopes. 32 human leukocyte antigen (HLA) class I and 24 HLA class II alleles are included in the blind test set. Artificial neural network (ANN)-based approaches demonstrated better performance than regression-based machine learning and structural modeling. Among the 18 predictors benchmarked, ANN-based mhcflurry and nn_align perform the best for MHC class I 9-mer and class II 15-mer predictions, respectively, on binding/non-binding classification (Area Under Curves = 0.911). NetMHCpan4 also demonstrated comparable predictive power. Our customization of mhcflurry to a pan-HLA predictor has achieved similar accuracy to NetMHCpan. The overall accuracy of these methods are comparable between 9-mer and 10-mer testing data. However, the top methods deliver low correlations between the predicted versus the experimental affinities for strong MHC binders. When used on naturally processed MHC-ligands, tools that have been trained on elution data (NetMHCpan4 and MixMHCpred) shows better accuracy than pure binding affinity predictor. The variability of false prediction rate is considerable among HLA types and datasets. Finally, structure-based predictor of Rosetta FlexPepDock is less optimal compared to the machine learning approaches. With our benchmarking of MHC-binding and MHC-elution predictors using a comprehensive metrics, a unbiased view for establishing best practice of T-cell epitope predictions is presented, facilitating future development of methods in immunogenomics.

Project description:BackgroundThe identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides.MethodsTwenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay.ResultsThe peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%.ConclusionsThe epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the development of diagnostic reagents that could improve upon the sensitivity and specificity of currently available diagnostic tests. Overall, the results provide further evidence of the usefulness of identifying specific linear B-cell epitopes for improving diagnostic tools.

Dataset Information

Human anti-A? IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

Publications

Human anti-Aβ IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

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