Determination Of Epitopes Research Articles

Computational prediction of multiple antigen epitopes.

Identifying antigen epitopes is essential in medical applications, such as immunodiagnostic reagent discovery, vaccine design, and drug development. Computational approaches can complement low-throughput, time-consuming, and costly experimental determination of epitopes. Currently available prediction methods, however, have moderate success predicting epitopes, which limits their applicability. Epitope prediction is further complicated by the fact that multiple epitopes may be located on the same antigen and complete experimental data is often unavailable. Here, we introduce the antigen epitope prediction program ISPIPab that combines information from two feature-based methods and a docking-based method. We demonstrate that ISPIPab outperforms each of its individual classifiers as well as other state-of-the-art methods, including those designed specifically for epitope prediction. By combining the prediction algorithm with hierarchical clustering, we show that we can effectively capture epitopes that align with available experimental data while also revealing additional novel targets for future experimental investigations.

Integrating Dynamic Network Analysis with AI for Enhanced Epitope Prediction in PD-L1:Affibody Interactions.

Understanding binding epitopes involved in protein-protein interactions and accurately determining their structure are long-standing goals with broad applicability in industry and biomedicine. Although various experimental methods for binding epitope determination exist, these approaches are typically low throughput and cost-intensive. Computational methods have potential to accelerate epitope predictions; however, recently developed artificial intelligence (AI)-based methods frequently fail to predict epitopes of synthetic binding domains with few natural homologues. Here we have developed an integrated method employing generalized-correlation-based dynamic network analysis on multiple molecular dynamics (MD) trajectories, initiated from AlphaFold2Multimer structures, to unravel the structure and binding epitope of the therapeutic PD-L1:Affibody complex. Both AlphaFold2 and conventional molecular dynamics trajectory analysis were ineffective in distinguishing between two proposed binding models, parallel and perpendicular. However, our integrated approach, utilizing dynamic network analysis, demonstrated that the perpendicular mode was significantly more stable. These predictions were validated using a suite of experimental epitope mapping protocols, including cross-linking mass spectrometry and next-generation sequencing-based deep mutational scanning. Conversely, AlphaFold3 failed to predict a structure bound in the perpendicular pose, highlighting the necessity for exploratory research in the search for binding epitopes and challenging the notion that AI-generated protein structures can be accepted without scrutiny. Our research underscores the potential of employing dynamic network analysis to enhance AI-based structure predictions for more accurate identification of protein-protein interaction interfaces.

Computational Prediction of Multiple Antigen Epitopes.

Identifying antigen epitopes is essential in medical applications, such as immunodiagnostic reagent discovery, vaccine design, and drug development. Computational approaches can complement low-throughput, time-consuming, and costly experimental determination of epitopes. Currently available prediction methods, however, have moderate success predicting epitopes, which limits their applicability. Epitope prediction is further complicated by the fact that multiple epitopes may be located on the same antigen and complete experimental data is often unavailable. Here, we introduce the antigen epitope prediction program ISPIPab that combines information from two feature-based methods and a docking-based method. We demonstrate that ISPIPab outperforms each of its individual classifiers as well as other state-of-the-art methods, including those designed specifically for epitope prediction. By combining the prediction algorithm with hierarchical clustering, we show that we can effectively capture epitopes that align with available experimental data while also revealing additional novel targets for future experimental investigations. raji@yu.edu. Supplementary data are available at Bioinformatics online.

Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping

Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.

#2086 Understanding B cell memory autoreactivity in idiopathic membranous nephropathy for tailored therapy

Abstract Background and Aims Idiopathic membranous nephropathy (iMN) is an autoantibody-mediated glomerular disease, and one of the leading causes of nephrotic syndrome in adults. iMN is caused by the formation of immune complexes composed by IgG autoantibodies and target antigens expressed on podocytes. The main autoantigen is the phospholipase A2 receptor1 (PLA2R1) and the antibodies are mainly belonging to the IgG4 isotype. Those immune complexes are responsible for profound changes in the structure and function of the podocytes and disruption of the glomerular barrier. Nevertheless, despite complement components at the site of glomerular injury have been found in biopsies from patients with MN, many factors involved in the pathogenesis are still unclear. We overall aim at characterizing both the serological and the memory cellular component in iMN patients, with identification of dominant B cell epitopes recognized by circulating autoantibodies and autoreactive memory B cells, which could be potentially used to deplete PLA2R1 specific memory B cells and plasma blasts. Method We analyzed the serum from a cohort of 159 iMN patients collected at diagnosis without any previous or concomitant treatment as well as a similar number of healthy controls’ samples. An ELISA-based quantification method has been used to estimate the concentration of circulating anti-PLA2R1 antibodies. Furthermore, we processed 27 additional blood samples from independent iMN patients as well as age and gender matched healthy controls to extract and analyze peripheral blood mononuclear cells by flow cytometry (BD FACS Fortessa) and identify the major B and T cell populations via multi-color staining. In order to identify and clone human anti-PLA2R1 antibodies from primary memory B cells we reproduced the experimental workflow exactly as described in Lindner J.M. et al. Immunity 2019. The isolated antibodies were then characterized with analytical methods (affinity, binding, epitope determination and complement activation ability) and used in competition assays. Results A deep analysis of the serum reactivity has been performed and iMN patients showed a high prevalence of autoantibodies against PLA2R1, mostly of high affine IgG4 subclass with a broad concentration range (1-20ug/ml). A concomitant IgG1 response versus PLA2R1 has been also detected in the serum in different proportion with IgG4. An extended immunophenotyping of circulating peripheral blood mononuclear cells revealed an altered B and T cell compartment in iMN patients. In particular, we described an expansion of circulating CD27neg IgG4 memory B cells, IgG4 plasma blasts, CD4+ T follicular helper cells and CX3CR1+ T peripheral helper cells. Moreover, we isolated and cloned five very specific, highly affine, and somatically hypermutated patient's derived anti-PLA2R1 antibodies. Competition experiments between patient sera and patients’ derived monoclonal antibodies showed a strong inhibitory effect, indicating the presence of a shared epitope recognized on the surface of the CysR domain of PLA2R1. The identified epitope has also shown an important role in complement activation, despite a component patient-specific seems to be involved as well. Conclusion Overall, we performed a comprehensive characterization of the autoreactive B cell memory compartment in iMN, shedding lights on the complexity of the autoantibody repertoire. Indeed, the investigation of the granularity of the anti-PLA2R1 response in term of IgG1 versus IgG4 isotypes provided a potential new way to stratify the patients’ population and potentially tailored the therapy or follow the disease progression. Moreover, the isolation of patient's derived anti-PLA2R1 antibodies opened the possibility of using them the as tools to establish new models directly from the in vivo-selected specificities. Finally, the identification of an immunodominant epitope within PLA2R1 shared among the patients is crucial to develop molecules aimed at depletion of PLA2R1 specific memory B cells and plasma blasts in the patients.

Glycosylated SARs Cov 2 interaction with plant lectins.

Lectins are non-immune carbohydrate-binding proteins/glycoproteins that are found everywhere in nature, from bacteria to human cells. They have also been a valuable biological tool for the purification and subsequent characterisation of glycoproteins due to their carbohydrate binding recognition capacity. Antinociceptive, antiulcer, anti-inflammatory activities and immune modulatory properties have been discovered in several plant lectins, with these qualities varying depending on the lectin carbohydrate-binding site. The Coronavirus of 2019 (COVID-19) is a respiratory disease that has swept the globe, killing millions and infecting millions more. Despite the availability of COVID-19 vaccinations and the vaccination of a huge portion of the world's population, viral infection rates continue to rise, causing major concern. Part of the reason for the vaccine's ineffectiveness has been attributed to repeated mutations in the virus's epitope determinant elements. The surface of the Coronavirus envelope is heavily glycosylated, with approximately sixty N-linked oligomannose, composite, and hybrid glycans covering the core of Man3GlcNAc2Asn. Some O-linked glycans have also been discovered. Many of these glyco-chains have also been subjected to multiple mutations, with only a few remaining conserved. As a result, numerous plant lectins with specificity for these viral envelope sugars have been discovered to interact preferentially with them and are being investigated as a potential future tool to combat coronaviruses such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by preventing viral attachment to the host. The review will discuss the possible applications of plant lectins as anti-coronaviruses including SARS-CoV-2, antinociceptive, anti-inflammation and its immune modulating effect.

Early acquisition of S-specific Tfh clonotypes after SARS-CoV-2 vaccination is associated with the longevity of anti-S antibodies.

SARS-CoV-2 vaccines have been used worldwide to combat COVID-19 pandemic. To elucidate the factors that determine the longevity of spike (S)-specific antibodies, we traced the characteristics of S-specific T cell clonotypes together with their epitopes and anti-S antibody titers before and after BNT162b2 vaccination over time. T cell receptor (TCR) αβ sequences and mRNA expression of the S-responded T cells were investigated using single-cell TCR- and RNA-sequencing. Highly expanded 199 TCR clonotypes upon stimulation with S peptide pools were reconstituted into a reporter T cell line for the determination of epitopes and restricting HLAs. Among them, we could determine 78 S epitopes, most of which were conserved in variants of concern (VOCs). After the 2nd vaccination, T cell clonotypes highly responsive to recall S stimulation were polarized to follicular helper T (Tfh)-like cells in donors exhibiting sustained anti-S antibody titers (designated as 'sustainers'), but not in 'decliners'. Even before vaccination, S-reactive CD4+ T cell clonotypes did exist, most of which cross-reacted with environmental or symbiotic microbes. However, these clonotypes contracted after vaccination. Conversely, S-reactive clonotypes dominated after vaccination were undetectable in pre-vaccinated T cell pool, suggesting that highly responding S-reactive T cells were established by vaccination from rare clonotypes. These results suggest that de novo acquisition of memory Tfh-like cells upon vaccination may contribute to the longevity of anti-S antibody titers.

Early acquisition of S-specific Tfh clonotypes after SARS-CoV-2 vaccination is associated with the longevity of anti-S antibodies

SARS-CoV-2 vaccines have been used worldwide to combat COVID-19 pandemic. To elucidate the factors that determine the longevity of spike (S)-specific antibodies, we traced the characteristics of S-specific T cell clonotypes together with their epitopes and anti-S antibody titers before and after BNT162b2 vaccination over time. T cell receptor (TCR) αβ sequences and mRNA expression of the S-responded T cells were investigated using single-cell TCR- and RNA-sequencing. Highly expanded 199 TCR clonotypes upon stimulation with S peptide pools were reconstituted into a reporter T cell line for the determination of epitopes and restricting HLAs. Among them, we could determine 78 S epitopes, most of which were conserved in variants of concern (VOCs). After the 2nd vaccination, T cell clonotypes highly responsive to recall S stimulation were polarized to follicular helper T (Tfh)-like cells in donors exhibiting sustained anti-S antibody titers (designated as ‘sustainers’), but not in ‘decliners’. Even before vaccination, S-reactive CD4+ T cell clonotypes did exist, most of which cross-reacted with environmental or symbiotic microbes. However, these clonotypes contracted after vaccination. Conversely, S-reactive clonotypes dominated after vaccination were undetectable in pre-vaccinated T cell pool, suggesting that highly responding S-reactive T cells were established by vaccination from rare clonotypes. These results suggest that de novo acquisition of memory Tfh-like cells upon vaccination may contribute to the longevity of anti-S antibody titers.

New potent vaccine against brucellosis based on multi-epitope prediction method of inf C protein. In silico study

PurposeBrucellosis infection could be eradicated by using an effective vaccine. Inf C gene expression generates one of the main Brucella pathogenesis proteins. The purpose of this study was to design a new vaccine against Brucella disease by in silico determination of epitopes of inf C protein. MethodsIn the first, Inf C amino acid sequences were extracted from the UniProt database and subjected to in silico analysis, including multiple sequence alignment, conserved region determination, allergenicity, antigenicity, and toxicity of the selected epitopes for TCL, HTL, and BCl. Vaccine-target (MHC alleles and TLRs) interactions, binding affinities, and dynamical stabilities were inspected through molecular docking and molecular dynamic simulation (MD) using Cluspro 2 server and GROMACS packages respectively. Further, the codon adaptation of the designed vaccine was determined by the JCat server and the obtained sequence was cloned in pET19b(+)vector by pDRAW32 software. Finally, the ability of the newer vaccine to stimulate the immune response was assessed using a computational immune simulation. ResultsResults allowed us to select a peptide vaccine on basis of its good binding affinities with TLR-8 allele. The multi-peptide vaccine showed also to be highly antigenic, non-allergenic, non-toxic, and potential expression in E coli. Results showed also good stability of the vaccine–TLR8 complex and strong cellular and humoral immune response after three in silico injections of the vaccine construct. ConclusionAll these theoretical results reveal that the conserved region of inf C protein could be used for designing of a new potent vaccine against Brucella.

New potent vaccine against brucellosis based on multi-epitope prediction method of inf C protein. In silico study

PurposeBrucellosis infection could be eradicated by using an effective vaccine. Inf C gene expression generates one of the main Brucella pathogenesis proteins. The purpose of this study was to design a new vaccine against Brucella disease by in silico determination of epitopes of inf C protein. MethodsIn the first, Inf C amino acid sequences were extracted from the UniProt database and subjected to in silico analysis, including multiple sequence alignment, conserved region determination, allergenicity, antigenicity, and toxicity of the selected epitopes for TCL, HTL, and BCl. Vaccine-target (MHC alleles and TLRs) interactions, binding affinities, and dynamical stabilities were inspected through molecular docking and molecular dynamic simulation (MD) using Cluspro 2 server and GROMACS packages respectively. Further, the codon adaptation of the designed vaccine was determined by the JCat server and the obtained sequence was cloned in pET19b(+)vector by pDRAW32 software. Finally, the ability of the newer vaccine to stimulate the immune response was assessed using a computational immune simulation. ResultsResults allowed us to select a peptide vaccine on basis of its good binding affinities with TLR-8 allele. The multi-peptide vaccine showed also to be highly antigenic, non-allergenic, non-toxic, and potential expression in E coli. Results showed also good stability of the vaccine–TLR8 complex and strong cellular and humoral immune response after three in silico injections of the vaccine construct. ConclusionAll these theoretical results reveal that the conserved region of inf C protein could be used for designing of a new potent vaccine against Brucella.

Determination of Protein Monoclonal-Antibody Epitopes by a Combination of Structural Proteomics Methods.

Structural proteomics techniques are useful for the determination of protein interaction interfaces. Each technique provides orthogonal structural information on the structure and the location of protein interaction sites. Here, we have characterized a monoclonal antibody epitope for a protein antigen by a combination of differential photoreactive surface modification (SM), cross-linking (CL), differential hydrogen-deuterium exchange (HDX), and epitope extraction/excision. We found that experimental data from different approaches agree with each other in determining the epitope of the monoclonal antibody on the protein antigens using the HIV-1 p24-mAb E complex as an illustrative example. A combination of these multiple structural proteomics approaches results in a detailed picture of the interaction of the proteins and increases confidence in the determination of the final structure of the protein interaction interface. Data are available via ProteomeXchange with identifier PXD040902.

Unmasking Arterial-resident Autoreactive B cells involved in Atherosclerosis and Peripheral Artery Disease Progression

Abstract Atherosclerosis activates the immune system, including B cells which secrete antibodies that bind antigens released by necrotic cells within plaques and may promote inflammation. To identify these antibodies, we isolated activated B cells from atherosclerotic ApoE-deficient mice aortas and human tibial arteries with peripheral artery disease post-amputation. Single-cell sequencing of the B cell receptors identified 35 mouse and 48 human overrepresented disease-relevant heavy and light chain pairings, which were sequentially expressed as antibody. Epitope determination was performed by human proteome array, and 14 mouse and 21 human protein targets were found with significant affinity and specificity. Mouse antigens were scrutinized by immunohistochemistry to confirm presence in inflamed plaques, and expression of these proteins were used to interrogate autoreactive antibody titers in diseased sera by ELISA. ApoE-deficient mice were immunized with the 6 most promising antigens. Serum antibody measurement showed strong, rapid, and early onset of antigen-specific antibody responses in the immunized mice compared to an adjuvant only control group. Strikingly, we saw decreased plaque accumulation in the immunized animals, suggesting antigen responses may clear pathogenic antigens before disease develops. Likewise, human antigen ELISAs comparing antigen-specific serum antibody titers to patient Framingham score for cardiovascular risk supported a role for two antigens in disease. To confirm these antigens as potential biomarkers, we will characterize the emergence of their antibody counterparts using a historical library of serum samples from individuals enrolled in the Baltimore Longitudinal Study on Aging. This work is directly funded by the National Institutes of Health – National Institute on Aging’s Intramural Research Program in the Lab of Molecular Biology and Immunology

Combining Phage Display Technology with In Silico-Designed Epitope Vaccine to Elicit Robust Antibody Responses against Emerging Pathogen Tilapia Lake Virus.

Antigen epitope identification is a critical step in the vaccine development process and is a momentous cornerstone for the development of safe and efficient epitope vaccines. In particular, vaccine design is difficult when the function of the protein encoded by the pathogen is unknown. The genome of Tilapia lake virus (TiLV), an emerging virus from fish, encodes protein functions that have not been elucidated, resulting in a lag and uncertainty in vaccine development. Here, we propose a feasible strategy for emerging viral disease epitope vaccine development using TiLV. We determined the targets of specific antibodies in serum from a TiLV survivor by panning a Ph.D.-12 phage library, and we identified a mimotope, TYTTRMHITLPI, referred to as Pep3, which provided protection against TiLV after prime-boost vaccination; its immune protection rate was 57.6%. Based on amino acid sequence alignment and structure analysis of the target protein from TiLV, we further identified a protective antigenic site (399TYTTRNEDFLPT410) which is located on TiLV segment 1 (S1). The epitope vaccine with keyhole limpet hemocyanin (KLH-S1399-410) corresponding to the mimotope induced the tilapia to produce a durable and effective antibody response after immunization, and the antibody depletion test confirmed that the specific antibody against S1399-410 was necessary to neutralize TiLV. Surprisingly, the challenge studies in tilapia demonstrated that the epitope vaccine elicited a robust protective response against TiLV challenge, and the survival rate reached 81.8%. In conclusion, this study revealed a concept for screening antigen epitopes of emerging viral diseases, providing promising approaches for development and evaluation of protective epitope vaccines against viral diseases. IMPORTANCE Antigen epitope determination is an important cornerstone for developing efficient vaccines. In this study, we attempted to explore a novel approach for epitope discovery of TiLV, which is a new virus in fish. We investigated the immunogenicity and protective efficacy of all antigenic sites (mimotopes) identified in serum of primary TiLV survivors by using a Ph.D.-12 phage library. We also recognized and identified the natural epitope of TiLV by bioinformatics, evaluated the immunogenicity and protective effect of this antigenic site by immunization, and revealed 2 amino acid residues that play important roles in this epitope. Both Pep3 and S1399-410 (a natural epitope identified by Pep3) elicited antibody titers in tilapia, but S1399-410 was more prominent. Antibody depletion studies showed that anti-S1399-410-specific antibodies were essential for neutralizing TiLV. Our study demonstrated a model for combining experimental and computational screens to identify antigen epitopes, which is attractive for epitope-based vaccine development.

Rational identification of potent and broad sarbecovirus-neutralizing antibody cocktails from SARS convalescents.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages have escaped most receptor-binding domain (RBD)-targeting therapeutic neutralizing antibodies (NAbs), which proves that previous NAb drug screening strategies are deficient against the fast-evolving SARS-CoV-2. Better broad NAb drug candidate selection methods are needed. Here, we describe a rational approach for identifying RBD-targeting broad SARS-CoV-2 NAb cocktails. Based on high-throughput epitope determination, we propose that broad NAb drugs should target non-immunodominant RBD epitopes to avoid herd-immunity-directed escape mutations. Also, their interacting antigen residues should focus on sarbecovirus conserved sites and associate with critical viral functions, making the antibody-escaping mutations less likely to appear. Following these criteria, a featured non-competing antibody cocktail, SA55+SA58, is identified from a large collection of broad sarbecovirus NAbs isolated from SARS-CoV-2-vaccinated SARS convalescents. SA55+SA58 potently neutralizes ACE2-utilizing sarbecoviruses, including circulating Omicron variants, and could serve as broad SARS-CoV-2 prophylactics to offer long-term protection, especially for individuals who are immunocompromised or with high-risk comorbidities.

Epitope Mapping Using the Cell-Based 2 × Alanine Substitution Method About the Anti-mouse CXCR6 Monoclonal Antibody, Cx6Mab-1.

An anti-mouse CXC chemokine receptor 6 (mCXCR6) monoclonal antibody (mAb), Cx6Mab-1, was developed recently. Cx6Mab-1 is applicable for flow cytometry, Western blotting, and enzyme-linked immunosorbent assay. The purpose of this study is to determine the binding epitope of Cx6Mab-1 using 2 × alanine mutated mCXCR6. Analysis of flow cytometry revealed that Cx6Mab-1 did not recognize S8A-A9G, L10A-Y11A, D12A-G13A, and H14A-Y15A mutants of mCXCR6. The results clearly indicate that the binding epitope of Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Asp12, Gly13, His14, and Tyr15 of mCXCR6. The successful determination of the Cx6Mab-1 epitope might contribute to the pathophysiological investigation of mCXCR6.

Determination of crucial epitopes in the sperm protein calsperin employing synthetic peptides and monoclonal antibodies.

The chaperone protein calsperin is exclusively expressed in the testes and is essential for sperm migration from the uterus into the oviduct. During spermatogenesis, calsperin interacts with ADAM3, a spermatozoon membrane protein required for fertilization. In this study, we characterized a calsperin epitope by using two monoclonal antibodies and resin-bound calsperin peptides, which were tested for reactivity using a modified enzyme-linked immunosorbent assay. An epitope located at the C-terminal end of calsperin corresponding to amino acids 228 WEKHFLDAS237 was identified. Three hot spot amino acids were essential for antibody binding whereas the remaining amino acids in the identified epitope appeared to be essential for bringing the critical contact residues into an α-helix structure. No notable sequence similarity was determined between the identified calsperin epitope and calreticulin, a chaperone homologue with sequence similarity, indicating that the identified epitope was specific for calsperin. Characterization of the calsperin epitope and of the two antibodies tested may be used in assays for further characterization of calsperin, where knowledge about the binding sites is necessary, for example, in sandwich assays. Moreover, studies like these may be used to study the function of calsperin during spermatogenesis and fertilization in detail and to develop new male contraception methods by targeting calsperin and mediating neutralization of its function.

Determination of a distinguished interferon gamma epitope recognized by monoclonal antibody relating to autoantibody associated immunodeficiency

Anti-interferon gamma autoantibodies (anti-IFN-γ autoAbs) neutralize the IFN-γ-mediated functions, contributing to immunodeficiency. A particular autoAb in patient serum had been previously demonstrated to recognize the same determinant on IFN-γ as the neutralizing anti-IFN-γ monoclonal antibody clone B27 (B27 mAb). This study explored the epitope recognized by B27 mAb. The specific peptide sequence recognized by B27 mAb, TDFLRMMLQEER, was retrieved from a phage display random peptide library. Sequence alignment and homology modeling demonstrated that the queried phage peptide sequence and structure were similar to amino acids at position 27–40 (TLFLGILKNWKEES) of the human IFN-γ. This determinant resides in the contact surface of IFN-γ and interferon gamma receptor 1. To elucidate the crucial amino acids, mutations were introduced by substituting T27 and T27F29L30 with alanine or deleting the amino acid residues T27–L33. The binding of B27 mAb to IFN-γ T27A using western blotting was lesser than that to wild-type. The interaction with triple mutant and T27–L33 deletion mutant using western blotting and sandwich ELISA was abolished. The finding demonstrated that T27, F29, and L30 are critical residues in the B27 antigenic determinant. Identification of the functional domain of IFN-γ decrypted the relevance of neutralizing autoAb in adult-onset immunodeficiency.

AbSE Workflow: Rapid Identification of the Coding Sequence and Linear Epitope of the Monoclonal Antibody at the Single-cell Level.

Monoclonal antibody (mAb) has been widely used in immunity research and disease diagnosis and therapy. Antibody sequence and epitope are the prerequisites and basis of mAb applications, which determine the properties of antibodies and make the preparation of antibody-based molecules controllable and reliable. Here, we present the antibody sequence and epitope identification (AbSE) workflow, a time-saving and cost-effective route for rapid determination of antibody sequence and linear epitope of mAb even at the single-cell level. The feasibility and accuracy of the AbSE workflow were demonstrated through the identification and validation of the coding sequence and epitope of antihuman serum albumin (antiHSA) mAb. It can be inferred that the AbSE workflow is a powerful and universal approach for paired antibody-epitope sequence identification. It may characterize antibodies not only on a single hybridoma cell but also on any other antibody-secreting cells.

Arterial antibodies promote atherosclerotic plaque development in mice

Abstract Atherosclerosis is characterized by the progressive buildup of cholesterol-rich plaques within the arterial wall. Plaques activate the immune system, of which B cells play a major role. Antibodies bind autoantigens released from necrotic cells and form immune complexes to increase inflammation. We isolated aorta-associated B cells from atherosclerotic ApoE-deficient mice on a high-fat diet and performed single cell sequencing of the B cell receptors to obtain antibody pairs. 32 over-represented antibodies were identified and then expressed in Expi293F cells. Epitope determination of these antibodies was performed against a human proteome array, and 15 potential autoantigens bound the monoclonal antibodies with significant affinity and specificity. The 12 most-promising antigens were scrutinized for their location in plaque tissue by immunohistochemistry using the cognate antibodies. Nine of these relevant antigens were then successfully synthesized using the pET bacterial expression system, and purified proteins were used to interrogate antibodies in human and mouse sera by ELISA. Mice were then immunized with the 5 best antigens to confirm their immunogenicity. The relevant antigens and antibodies are now being tested to see if they will protect or promote atherosclerosis development, and if they can be used as biomarkers in human disease. This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.

In Silico Prediction of Cross-Reactive Epitopes of Tropomyosin from Shrimp and Other Arthropods Involved in Allergy.

Tropomyosin in shellfish is considered a major cross-reactive allergen in house dust mites and cockroaches; however, the specific epitopes have not been elucidated. Therefore, this study aimed to identify the consensus antigenic determinant among shrimp, house dust mites, and cockroaches using in silico methods. The protein sequences of tropomyosin, including Der f 10, Mac r 1, Pen a 1, Pen m 1, Per a 7, and Bla g 7, were retrieved from the UniProt database. The 3D structures were derived from the AlphaFold or modeled using the Robetta. The determination of linear epitopes was performed by AlgPRED and BepiPRED for B cell epitope, and NetMHCIIpan and NetMHCII for T cell epitope, while Ellipro was used to evaluate conformational epitopes. Fourteen peptides were discovered as the consensus linear B cell epitopes, while seventeen peptides were identified as linear T cell epitopes specific to high-frequency HLA-DR and HLA-DQ alleles. The conformational determination of B cell epitopes provided nine peptides, in which residues 209, 212, 255–256, and 258–259 were found in both linear B cell and linear T cell epitope analysis. This data could be utilized for further in vitro study and may contribute to immunotherapy for allergic diseases associated with tropomyosin.