Development of Nanobodies as Theranostic Agents against CMY-2-Like Class C ß-Lactamases.

Three soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against the CMY-2 ß-lactamase behaved as inhibitors. The structure of the complex VHH cAbCMY-2(254)/CMY-2 showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes into the catalytic site. The ß-lactamase inhibition pattern followed a mixed profile with a predominant noncompetitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binders. Our study identified a binding site that can be targeted by a new class of ß-lactamase inhibitors designed on the sequence of the paratope. Furthermore, the use of mono- or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enables the development of the first generation of enzyme-linked immunosorbent assay (ELISA) for the detection of CMY-2 produced by CMY-2-expressing bacteria, irrespective of resistotype.

Ara h 2-specific IgE epitope-like peptides inhibit the binding of IgE to Ara h 2 and suppress lgE-dependent effector cell activation.

BACKGROUND: Clinical and experimental analyses indicate a pathognomonic role for allergen IgE crosslinking through epitope-paratope interactions as a major initial step in the cascade leading to effector cell activation and clinical manifestations of lgE-mediated food allergies. We aimed to undertake the initial development and assessment of Ara h 2-specific IgE epitope-like peptides that can bind to allergen-specific IgE paratopes and suppress effector cell activation. METHODS: We performed biopanning, screening, IgE binding, selection and mapping of peptides. We generated synthetic peptides for use in all functional experiments. ImmunoCAP inhibition, basophil and mast cell activation tests, with LAD2 cells, a human mast cell line were performed. Twenty-six children or young adults who had peanut allergy were studied. RESULTS: We identified and selected three linear peptides (DHPRFNRDNDVA, DHPRYGP and DHPRFST), and immunoblot analyses revealed binding to lgE from peanut-allergic individuals. The peptide sequences were aligned to the disordered region corresponding to the loop between helices 2 and 3 of Ara h 2, and conformational mapping showed that the peptides match the surface of Ara h 2 and h 6 but not other peanut allergens. In ImmunoCAP inhibition experiments, the peptides significantly inhibit the binding of IgE to Ara h 2 (p < .001). In basophil and mast cell activation tests, the peptides significantly suppressed Ara h 2-induced effector cell activation (p < .05) and increased the half-maximal Ara h 2 effective concentration (p < .05). Binding of the peptides to specific IgEs did not induce activation of basophils or mast cells. CONCLUSIONS: These studies show that the indicated peptides reduce the allergenic activity of Ara h 2 and suppress lgE-dependent basophil and mast cell activation. These observations may suggest a novel therapeutic strategy for food allergy based on epitope-paratop blocking.

Epitope-Analyzer: A structure-based webtool to analyze broadly neutralizing epitopes.

The antigenic epitope regions of pathogens (e.g., viruses) are recognized by antibodies (Abs) and subsequently cleared by the host immune system, thereby protecting us from disease. Some of these epitopes are conserved among different variants or subgroups of pathogens (e.g., Influenza (FLU) viruses, Coronaviruses), hence can be targeted for potential broad-neutralization. Here we report a web-based tool, Epitope Analyzer (EA), that rapidly identifies conformational epitope and paratope residues in an antigen-antibody complex structure. Furthermore, the tool provides the ways and means to analyze broadly neutralizing epitopes by comparing the equivalent epitope residues in similar antigen structures. The similarity in the epitope residues between (multiple) pairs of similar antigen molecules suggest the presence of conserved epitopes that can be targeted by broadly neutralizing antibodies. These details can be used as a guide in developing effective treatments, such as the design of novel vaccines and formulation of cocktail of broadly neutralizing antibodies, against multiple variants or subgroups of viruses. The web application can be freely accessed from the URL, http://viperdb.scripps.edu/ea.php.

Peptide Antibodies: Current Status.

Peptide antibodies have become one of the most important classes of reagents in molecular biology and clinical diagnostics. For this reason, methods for their production and characterization continue to be developed, including basic peptide synthesis protocols, peptide-conjugate production and characterization, conformationally restricted peptides, immunization procedures, etc. Detailed mapping of peptide antibody epitopes has yielded important information on antibody-antigen interaction in general and specifically in relation to antibody cross-reactivity and theories of molecular mimicry. This information is essential for detailed understanding of paratope-epitope dynamics, design of antibodies for research, design of peptide-based vaccines, development of therapeutic peptide antibodies, and de novo design of antibodies with predetermined specificity.

Structural insights into the unique recognition module between α-synuclein peptide and nanobody.

Nanobodies are single-domain fragments of antibodies with comparable specificity and affinity to antibodies. They are emerging as versatile tools in biology due to their relatively small size. Here, we report the crystal structure of a specific nanobody Nbα-syn01, bound to a 14 amino acid long peptide of α-synuclein (αSyn), a 140-residue protein whose aggregation is associated with Parkinson's disease. The complex structure exhibits a unique binding pattern where the αSyn peptide replaces the N-terminal region of nanobody. Recognition is mediated principally by extended main chain interaction of the αSyn peptide and specificity of the interaction lies in the central 48-52 region of αSyn peptide. Structure-guided truncation of Nbα-syn01 shows tighter binding to αSyn peptide and improved inhibition of α-synuclein aggregation. The structure of the truncated complex was subsequently determined and was indistinguishable to full length complex as the full-length form had no visible electron density for the N-terminal end. These findings reveal the molecular basis for a previously unobserved binding mode for nanobody recognition of α-synuclein, providing an explanation for the enhanced binding, and potential for an alternate framework for structure-based protein engineering of nanobodies to develop better diagnostic and therapeutic tools.

Prediction of Variable-Length B-Cell Epitopes for Antipeptide Paratopes Using the Program HAPTIC.

BACKGROUND: B-cell epitope prediction for antipeptide antibody responses enables peptide-based vaccine design and related translational applications. This entails estimating epitopeparatope binding free-energy changes from antigen sequence; but attempts to do so assuming uniform epitope length (e.g., of hexapeptide sequences, each spanning a typical paratope diameter when fully extended) have neglected empirically established variation in epitope length. OBJECTIVE: This work aimed to develop a sequence-based physicochemical approach to variablelength B-cell epitope prediction for antipeptide paratopes recognizing flexibly disordered targets. METHODS: Said approach was developed by analogy between epitope-paratope binding and protein folding modeled as polymer collapse, treating paratope structure implicitly. Epitope-paratope binding was thus conceptually resolved into processes of epitope compaction, collapse and contact, with epitope collapse presenting the main entropic barrier limiting epitope length among nonpolyproline sequences. The resulting algorithm was implemented as a computer program, namely the Heuristic Affinity Prediction Tool for Immune Complexes (HAPTIC), which is freely accessible via an online interface (http://badong.freeshell.org/haptic.htm). This was used in conjunction with published data on representative known peptide immunogens. RESULTS: HAPTIC predicted immunodominant epitope sequences with lengths limited by penalties for both compaction and collapse, consistent with known paratope-bound structures of flexibly disordered epitopes. In most cases, the predicted association constant was greater than its experimentally determined counterpart but below the predicted upper bound for affinity maturation in vivo. CONCLUSION: HAPTIC provides a physicochemically plausible means for estimating the affinity of antipeptide paratopes for sterically accessible and flexibly disordered peptidic antigen sequences by explicitly considering candidate B-cell epitopes of variable length.

Beyond B-Cell Epitopes: Curating Positive Data on Antipeptide Paratope Binding to Support Peptide-Based Vaccine Design.

BACKGROUND: B-cell epitope prediction is a computational approach originally developed to support the design of peptide-based vaccines for inducing protective antibody-mediated immunity, as exemplified by neutralization of biological activity (e.g., pathogen infectivity). Said approach is benchmarked against experimentally obtained data on paratope-epitope binding; but such data are curated primarily on the basis of immune-complex structure, obscuring the role of antigen conformational disorder in the underlying immune recognition process. OBJECTIVE: This work aimed to critically analyze the curation of epitope-paratope binding data that are relevant to B-cell epitope prediction for peptide-based vaccine design. METHODS: Database records on neutralizing monoclonal antipeptide antibody immune-complex structure were retrieved from the Immune Epitope Database (IEDB) and analyzed in relation to other data from both IEDB and external sources including the Protein Data Bank (PDB) and published literature, with special attention to data on conformational disorder among paratope-bound and unbound peptidic antigens. RESULTS: Data analysis revealed key examples of antipeptide antibodies that recognize conformationally disordered B-cell epitopes and thereby neutralize the biological activity of cognate targets (e.g., proteins and pathogens), with inconsistency noted in the mapping of some epitopes due to reliance on immune-complex structural details, which vary even among experiments utilizing the same paratope-epitope combination (e.g., with the epitope forming part of a peptide or a protein). CONCLUSION: The results suggest an alternative approach to curating paratope-epitope binding data based on neutralization of biological activity by polyclonal antipeptide antibodies, with reference to immunogenic peptide sequences and their conformational disorder in unbound antigen structures.

Identification of Unique Peptides for SARS-CoV-2 Diagnostics and Vaccine Development by an In Silico Proteomics Approach.

Ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus strains is posing new COVID-19 diagnosis and treatment challenges. To help efforts to meet these challenges we examined data acquired from proteomic analyses of human SARS-CoV-2-infected cell lines and samples from COVID-19 patients. Initially, 129 unique peptides were identified, which were rigorously evaluated for repeats, disorders, polymorphisms, antigenicity, immunogenicity, toxicity, allergens, sequence similarity to human proteins, and contributions from other potential cross-reacting pathogenic species or the human saliva microbiome. We also screened SARS-CoV-2-infected NBHE and A549 cell lines for presence of antigenic peptides, and identified paratope peptides from crystal structures of SARS-CoV-2 antigen-antibody complexes. We then selected four antigen peptides for docking with known viral unbound T-cell receptor (TCR), class I and II peptide major histocompatibility complex (pMHC), and identified paratope sequences. We also tested the paratope binding affinity of SARS-CoV T- and B-cell peptides that had been previously experimentally validated. The resultant antigenic peptides have high potential for generating SARS-CoV-2-specific antibodies, and the paratope peptides can be directly used to develop a COVID-19 diagnostics assay. The presented genomics and proteomics-based in-silico approaches have apparent utility for identifying new diagnostic peptides that could be used to fight SARS-CoV-2.

Epitope and Paratope Mapping Reveals Temperature-Dependent Alterations in the Dengue-Antibody Interface.

Uncovering mechanisms of antibody-mediated neutralization for viral infections requires epitope and paratope mapping in the context of whole viral particle interactions with the antibody in solution. In this study, we use amide hydrogen/deuterium exchange mass spectrometry to describe the interface of a dengue virus-neutralizing antibody, 2D22, with its target epitope. 2D22 binds specifically to DENV2, a serotype showing strain-specific structural expansion at human host physiological temperatures of 37°C. Our results identify the heavy chain of 2D22 to be the primary determinant for binding DENV2. Temperature-mediated expansion alters the mode of interaction of 2D22 binding. Importantly, 2D22 interferes with the viral expansion process and offers a basis for its neutralization mechanism. The relative magnitude of deuterium exchange protection upon antibody binding across the various epitope loci allows a deconstruction of the antibody-viral interface in host-specific environments and offers a robust approach for targeted antibody engineering.