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Reduced epitope counts (1) for peptidic sequences of uniform length varying only at a single-residue position. Each count corresponds to a set of 20 peptides representing every standard proteinogenic amino-acid residue at the variable-residue position. Functional similarity is equated with either fractional aligned-sequence identity (“□”; (3) and (4)) or the Shannon information entropy for differential epitope binding ((5) through (9)). In the latter case, counts were based on steric incompatibility only (“▽”; (7) and Figure 2), both steric incompatibility and cavity formation (“⋄”; c Δ V i j k in (8)), or steric incompatibility with both cavity formation and hydrogen bonding (“△”; (8) and (9) and Table 1).
Source publication
Epitope-based design of vaccines, immunotherapeutics, and immunodiagnostics is complicated by structural changes that radically alter immunological outcomes. This is obscured by expressing redundancy among linear-epitope data as fractional sequence-alignment identity, which fails to account for potentially drastic loss of binding affinity due to si...
Citations
... Nonetheless, no research has been done to determine the sequence similarity percentage that may prevent cross-reactivity between the recognition of selfpeptides and epitopes (74). When it comes to lengthy epitopes like CD4+ T cell and B-cell epitopes, a 70% cut-off has been defined as conservative by a strategy intended to reduce epitope redundancy (75). In terms of nonameric CD8+ T cell epitopes, it has been noted that residue positions 2 and 9 are crucial for determining epitope binding to MHC-I. ...
The current global outbreak of monkeypox (MPX) disease, caused by Monkeypox virus (MPXV), has resulted in 16 thousand infection cases, five deaths, and has been declared a global health emergency of international concern by the World Health Organization. Given current challenges in the safety of existing vaccines, a vaccine to prevent MPX infection and/or onset of symptoms would significantly advance disease management. In this context, a multi-epitope-based vaccine could be a well-suited approach. Herein, we searched a publicly accessible database (Virus Pathogen Database and Analysis Resource) for MPXV immune epitopes from various antigens. We prioritized a group of epitopes (10 CD8+ T cells and four B-cell epitopes) using a computer-aided technique based on desirable immunological and physicochemical properties, sequence conservation criteria, and non-human homology. Three multi-epitope vaccines were constructed (MPXV-1–3) by fusing finalized epitopes with the aid of appropriate linkers and adjuvant (beta-defensin 3, 50S ribosomal protein L7/L12, and Heparin-binding hemagglutinin). Codon optimization and in silico cloning in the pET28a (+) expression vector ensure the optimal expression of each construct in the Escherichia Coli system. Two and three-dimensional structures of the constructed vaccines were predicted and refined. The optimal binding mode of the construct with immune receptors [Toll-like receptors (TLR2, TLR3, and TLR4)] was explored by molecular docking, which revealed high docking energies of MPXV-1–TLR3 (–99.09 kcal/mol), MPXV-2–TLR3 (–98.68 kcal/mol), and MPXV-3–TLR2 (–85.22 kcal/mol). Conformational stability and energetically favourable binding of the vaccine-TLR2/3 complexes were assessed by performing molecular dynamics simulations and free energy calculations (Molecular Mechanics/Generalized Born Surface Area method). In silico immune simulation suggested that innate, adaptive, and humoral responses will be elicited upon administration of such potent multi-epitope vaccine constructs. The vaccine constructs are antigenic, non-allergen, non-toxic, soluble, topographically exposed, and possess favourable physicochemical characteristics. These results may help experimental vaccinologists design a potent MPX vaccine.
... First, the computational prediction of HLAbinding can be inaccurate especially for certain HLA alleles (18,19). Second, previous works have suggested that the overrepresentation of highly similar sequences due to collection bias in the IEDB could influence the analysis results (20,21). Consequently, we kept allele-peptide pairs if the binding of the peptide to the reported HLA allele was also verified empirically and excluded similar sequences using an iterative method (Methods and SI Appendix, Fig. S1). ...
Significance
It is well established that peptides that are dissimilar to human proteins are more immunogenic. However, the immune system is still unable to recognize a large fraction of highly dissimilar peptides found in a wide variety of pathogens. We propose that this phenomenon could be explained by the mechanism of T cell positive selection. During this process, only those cells survive that recognize human peptides on the surface of thymic epithelial cells. As self-peptides mediate positive selection, the immune system is unable to recognize many nonself peptides, most of which are highly dissimilar to human peptides.
... There, a 70% threshold value is used for protein sequence analysis (77). Such cutoff has been described as conservative by an approach aimed at reducing epitope redundancy, particularly in relation to long epitopes such as CD4 + T cell and B cell epitopes (78). Regarding nonameric CD8 + T cell epitopes, it has been described that residue positions 2 and 9 are key to determine epitope binding to MHC-I, whereas residues 3-6 and 8 are engaged in T cell receptor (TCR) motif recognition (79). ...
Trypanosoma cruzi infection causes Chagas disease, which affects 7 million people worldwide. Two drugs are available to treat it: benznidazole and nifurtimox. Although both are efficacious against the acute stage of the disease, this is usually asymptomatic and goes undiagnosed and untreated. Diagnosis is achieved at the chronic stage, when life-threatening heart and/or gut tissue disruptions occur in ~30% of those chronically infected. By then, the drugs' efficacy is reduced, but not their associated high toxicity. Given current deficiencies in diagnosis and treatment, a vaccine to prevent infection and/or the development of symptoms would be a breakthrough in the management of the disease. Current vaccine candidates are mostly based on the delivery of single antigens or a few different antigens. Nevertheless, due to the high biological complexity of the parasite, targeting as many antigens as possible would be desirable. In this regard, an epitope-based vaccine design could be a well-suited approach. With this aim, we have gone through publicly available databases to identify T. cruzi epitopes from several antigens. By means of a computer-aided strategy, we have prioritized a set of epitopes based on sequence conservation criteria, projected population coverage of Latin American population, and biological features of their antigens of origin. Fruit of this analysis, we provide a selection of CD8⁺ T cell, CD4⁺ T cell, and B cell epitopes that have <70% identity to human or human microbiome protein sequences and represent the basis toward the development of an epitope-based vaccine against T. cruzi.
... Recent advances in computational and bioinformatic analyses using extensively accumulated data of epitope-paratope interfaces make it possible to group T cell receptors of common specificity using grouping algorithms of lymphocyte interactions by paratope hotspots 29 . Furthermore, these advances have also revealed the functional redundancy of epitopes based on physicochemical similarities at a level of amino acid residues involved in the antigenic cross-reaction 30 . In addition to these accumulated data, further comprehensive understanding of the physicochemical environment of epitope-paratope interfaces including both "on-targets" and "off-targets" may make it possible to evaluate and predict more accurately antigenicity and immunogenicity, and these advances will bring about therapeutic Abs and vaccines with high specificity and without adverse effects. ...
It is critical for development of high-quality antibodies in research and diagnostics to predict accurately their cross-reactivities with “off-target” molecules, which potentially induce false results. Herein, we report a good example of such a cross-reactivity for an off-target due to a stereochemical environment of epitopes, which does not simply depend on amino acid sequences. We found that significant subpopulation of a polyclonal peptide antibody against Bcnt (Bucentaur) (anti-BCNT-C antibody) cross-reacted with a completely different protein, glutamine synthetase (GS), and identified four amino acids, GYFE, in its C-terminal region as the core amino acids for the cross-reaction. Consistent with this finding, the anti-BCNT-C antibody strongly recognized endogenously and exogenously expressed GS in tissues and cultured cells by Western blotting and immunohistochemistry. Furthermore, we elucidated that the cross-reaction is caused by a spatial similarity between the stereochemical environments formed by amino acid residues, including the GYFE of GS and the GYIE of Bcnt, rather than by their primary sequences. These results suggest it is critical to comprehensively analyze antibody interactions with target molecules including off-targets with special attention to the physicochemical environments of epitope-paratope interfaces to decrease the risk of false interpretations of results using antibodies in science and clinical applications.
Background
Background: B-cell epitope prediction for antipeptide antibody responses enables peptide-based vaccine design and related translational applications. This entails estimating epitope-paratope 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
Objective: This work aimed to develop a sequence-based physicochemical approach to variable-length B-cell epitope prediction for antipeptide paratopes recognizing flexibly disordered targets.
Method
Method: 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 non-polyproline 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
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
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
The Bucentaur (BCNT) protein family is characterized by a conserved amino acid sequence at the C-terminus (BCNT-C domain) and plays an essential role in gene expression and chromosomal maintenance in yeast and Drosophila. The mammalian Bucentaur/Craniofacial developmental protein 1 (Bcnt/Cfdp1) is also a tentative component of the Srcap (SNF2-Related CBP Activator Protein) chromatin remodeling complex, but little is known about its properties, partly because few antibodies are available to examine the endogenous protein. In this paper, we assigned the western blot signal against the mouse Bcnt/ Cfdp1 as a doublet of approximately 45 kDa using anti-Bcnt/Cfdp1 antibodies, which were generated against either of two unrelated immunogens, BCNT-C domain or mouse N-terminal peptide, and in addition, the Cfdp1 knockdown mouse ES cell line and bovine tissue were used as potential negative controls. Moreover, LC-MS/MS analysis of the corresponding doublet to the Flag-tagged mouse Bcnt/Cfdp1 that was constitutively expressed in a HEK293 cell exhibited that the upper band was much more phosphorylated than the lower band with preferential Ser phosphorylation in the WESF motif of BCNT-C domain. Western blot analysis with these evaluated antibodies indicated a preferential expression of Bcnt/Cfdp1 in the early stages of brain development of mouse and rat, which is consistent with a data file of the expression of Bcnt/Cfdp1 mRNA.
The design of synthetic vaccine peptides and other constructs (e.g., for developing immunodiagnostics) is informed by B-cell epitope prediction for antipeptide paratopes, which crucially depends on physicochemically and biologically meaningful interpretation of pertinent experimental data as regards paratope-epitope binding, with negative data being particularly problematic as they may be due to artefacts of immunization and immunoassays. Yet, the problem posed by negative data remains to be comprehensively addressed in a manner that clearly defines their role in the further development of B-cell epitope prediction. Hence, published negative data were surveyed and analyzed herein to identify key issues impacting on B-cell epitope prediction. Data were retrieved via searches using the Immune Epitope Database (IEDB) and review of underlying primary sources in literature to identify said issues, which include (1) inherent tendency toward false-negative data with use of solid-phase immunoassays and/or monoclonal paratopes, (2) equivocal data (i.e., both positive and negative data obtained from similar experiments), and (3) failure of antipeptide paratopes to cross-react with antigens of covalent structure and/or conformation different from that of the peptide immunogens despite apparent identity between curated epitope sequences. Analysis of experimental details thus focused on negative data from fluid-phase (e.g., immunoprecipitation) assays for detection of polyclonal paratope-epitope binding. Underlying literature references were reviewed to confirm the identification of negative data included for analysis. Furthermore, data from assays to detect cross-reaction of antipeptide antibody with protein antigen were included only if supported by positive data on either the corresponding reaction of the same antibody with peptide antigen or cross-reaction of said antibody with denatured protein antigen, to exclude the possibility that negative data on cross-reaction were due to absence of antipeptide paratopes in the first place (e.g., because of failed immunization due to insufficient immunogenicity and/or immune tolerance). Among currently available negative binding data on antipeptide antibodies, very few are on polyclonal responses yet also clearly attributable to conformational differences between peptide immunogens and native cognate proteins thereof. This dearth of negative data suitable for benchmarking B-cell epitope prediction conceivably could be addressed by generating positive data on binding of polyclonal antipeptide antibodies to cognate-protein sequences (e.g., in solid-phase immunoassays using unfolded protein antigen) to complement negative data on failure of the same antibodies to cross-react with native protein (e.g., in fluid-phase immunoassays, without artefactual covalent modification of antigens that tends to produce false-negative results). As regards cross-reactive binding of native cognate proteins by antipeptide antibodies (e.g., as mechanistic basis for novel vaccines and immunotherapeutics), negative data are most informative where attributable to conformational differences between peptide immunogens and target proteins. This is favored by careful peptide-immunogen design (e.g., avoiding covalent backbone and sidechain differences vis-a-vis target protein sequence) and positive data on antibody binding of the target protein sequence (e.g., in unfolded protein) paired with negative data on the same antibody using native protein antigen (e.g., from fluid- rather than solid-phase assays).
