Search Results (527)

Sepsis is a fetal disease that requires a clear diagnostic biomarker for timely antibiotic treatment. Recent research has identified a pyroptosis-inducing epitope known as P5-5 in tetranectin (TN), a plasma protein produced by monocytes. Previously, we produced a 12F1 monoclonal antibody against the P5-5 and discovered that it could not only diagnose the presence but also monitor the progress of sepsis in the clinic. In the current study, we further investigated the structure site of the P5-5 and the recognition mechanism between the 12F1 mAb and the P5-5 epitope. To this end, 10 amino acids (NDALYEYLRQ) in the P5-5 were individually mutated to alanine, and their binding to the mAb was tested to confirm the most significant antigenic recognition sites. In the meanwhile, the spatial conformation of 12F1 mAb variable regions was modeled, and the molecular recognition mechanisms in detail of the mAb to the P5-5 epitope were further studied by molecular docking. Following epitope prediction and experimental verification, we demonstrated that the motif “DALYEYL” in the epitope sequence position 2−8 of TN-P5-5 is the major binding region for mAb recognition, in which two residues (4L and 8L) were essential for the interaction between the P5-5 epitope and the 12F1 mAb. Therefore, our study greatly narrowed down the previously reported motif from ten to seven amino acids and identified two Leu as critical contact residues. Finally, a single-chain variable fragment (scFv) from the 12F1 hybridoma was constructed, and it was confirmed that the identified motif and residues are prerequisites for the strong binding between P5-5 and 12F1. Altogether, the data of the present work could serve as a theoretic guide for the clinical design of biosynthetic drugs by artificial intelligence to treat sepsis. Full article

Porcine Rotavirus (PoRV), a predominant causative agent of neonatal diarrhea in piglets, shares substantial genetic homology with human rotavirus and represents a considerable threat to both public health and the global swine industry in the absence of specific antiviral interventions. The VP6 protein, an internal capsid component, is characterized by exceptional sequence conservation and robust immunogenicity, rendering it an ideal candidate for viral genotyping and vaccine development. In the present study, the recombinant plasmid pET28a(+)-VP6 was engineered to facilitate the high-yield expression and purification of the VP6 antigen. BALB/c mice were immunized to generate monoclonal antibodies (mAbs) through hybridoma technology, and the antigenic specificity of the resulting mAbs was stringently validated. Subsequently, a panel of truncated protein constructs was designed to precisely map linear B-cell epitopes, followed by comparative conservation analysis across diverse PoRV strains. Functional validation demonstrated that all three mAbs exhibited high-affinity binding to VP6, with a peak detection titer of 1:3,000,000 and exclusive specificity toward PoRVA. These antibodies effectively recognized representative genotypes such as G3 and X1, while exhibiting no cross-reactivity with unrelated viral pathogens; however, their reactivity against other PoRV serogroups (e.g., types B and C) remains to be further elucidated. Epitope mapping identified two novel linear B-cell epitopes, ¹²⁸YIKNWNLQNR¹³⁷ and ¹³⁸RQRTGFVFHK¹⁴⁷, both displaying strong sequence conservation among circulating PoRV strains. Collectively, these findings provide a rigorous experimental framework for the functional dissection of VP6 and reinforce its potential as a valuable diagnostic and immunoprophylactic target in PoRV control strategies. Full article

The rapid evolution of SARS-CoV-2 has underscored the need for a detailed understanding of antibody binding mechanisms to combat immune evasion by emerging variants. In this study, we investigated the interactions between Class I neutralizing antibodies—BD55-1205, BD-604, OMI-42, P5S-1H1, and P5S-2B10—and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein using multiscale modeling, which combined molecular simulations with the ensemble-based mutational scanning of the binding interfaces and binding free energy computations. A central theme emerging from this work is that the unique binding strength and resilience to immune escape of the BD55-1205 antibody are determined by leveraging a broad epitope footprint and distributed hotspot architecture, additionally supported by backbone-mediated specific interactions, which are less sensitive to amino acid substitutions and together enable exceptional tolerance to mutational escape. In contrast, BD-604 and OMI-42 exhibit localized binding modes with strong dependence on side-chain interactions, rendering them particularly vulnerable to escape mutations at K417N, L455M, F456L and A475V. Similarly, P5S-1H1 and P5S-2B10 display intermediate behavior—effective in some contexts but increasingly susceptible to antigenic drift due to narrower epitope coverage and concentrated hotspots. Our computational predictions show strong agreement with experimental deep mutational scanning data, validating the accuracy of the models and reinforcing the value of binding hotspot mapping in predicting antibody vulnerability. This work highlights that neutralization breadth and durability are not solely dictated by epitope location, but also by how binding energy is distributed across the interface. The results provide atomistic insight into mechanisms driving resilience to immune escape for broadly neutralizing antibodies targeting the ACE2 binding interface—which stems from cumulative effects of structural diversity in binding contacts, redundancy in interaction patterns and reduced vulnerability to mutation-prone positions. Full article

Synthetic biology has fundamentally advanced cell engineering and helped to develop effective therapeutics such as chimeric antigen receptor (CAR)-T cells. For these applications, the detection, localization, and quantification of heterologous fusion proteins assembled from interchangeable building blocks is of high importance. The V5 tag, a 14-residue epitope tag, offers promising characteristics for these applications but has only rarely been used in this context. Thus, we have systematically evaluated the murine anti-V5 tag antibody mu_SV5-Pk1 as well as its humanized version, hu_SV5-Pk1, to analyze cells expressing V5-tagged receptors in samples from various in vitro and in vivo experiments. We found that the V5 tag signal on cells is affected by certain fixation and detachment reagents. Immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) mouse tissue samples was performed to sensitively detect cells in tissue. We improved IHC by applying the hu_SV5-Pk1 monoclonal antibody (mAb) to avoid cross-reactivity within and unspecific background signals arising on fixed mouse tissue. Conversely, the absence of unspecific binding by the mu_SV5-Pk1 mAb was evaluated on 46 human normal or cancer tissues. Our findings present a robust toolbox for utilizing the V5 tag and cognate antibodies in synthetic biology applications. Full article

Background/Objectives: For viral entry into host cells, the spike (S) protein of coronavirus (CoV) uses its S1 domain to bind to the host receptor and S2 domain to mediate the fusion between virion and cellular membranes. The S1 domain acquired multiple mutations as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolved to give rise to Variant of Concerns (VOCs) but the S2 domain has limited changes. In particular, the stem helix in S2 did not change significantly and it is fairly well-conserved across multiple beta-CoVs. In this study, we generated a murine mAb 7B2 binding to the stem helix of SARS-CoV-2. Methods: MAb 7B2 was isolated from immunized mouse and its neutralization activity was evaluated using microneutralization, plaque reduction and cell–cell fusion assays. Bio-layer interferometry was used to measure binding affinity and AlphaFold3 was used to model the antibody–antigen interface. Results: MAb 7B2 has lower virus neutralizing and membrane block activities when compared to a previously reported stem helix-binding human mAb S2P6. Alanine scanning and AlphaFold3 modeling reveals that residues K1149 and D1153 in S form a network of polar interactions with the heavy chain of 7B2. Conversely, S2P6 binding to S is not affected by alanine substitution at K1149 and D1153 as indicated by the high ipTM scores in the predicted S2P6-stem helix structure. Conclusions: Our detailed characterization of the mechanism of inhibition of 7B2 reveals its distinctive binding model from S2P6 and yields insights on multiple neutralizing and highly conserved epitopes in the S2 domain which could be key components for pan-CoV vaccine development. Full article

Glycans on the surface of all immune cells are the product of diverse post-translational modifications (glycosylation) that affect almost all proteins and possess enormous structural heterogeneity. Their bioinformational content is decoded by glycan-binding proteins (lectins, GBPs), such as C-type lectins, including selectins, galectins, and Siglecs. Glycans located on the surface of immune cells are involved in many immunological processes through interactions with GBPs. Lectins recognize changes in the glycan epitopes; distinguish among host (self), microbial (non-self), and tumor (modified self) antigens; and consequently regulate immune responses. Understanding GBP–glycan interactions accelerates the development of glycan-targeted therapeutics in severe diseases, including inflammatory and autoimmune diseases and cancer. This review will discuss N- and O-glycosylations and glycosyltransferases involved in the biosynthesis of carbohydrate epitopes and address how interactions between glycan epitopes and GBPs are crucial in immune responses. The pivotal role of the glycan antigen tetrasaccharide sialyl Lewis x in mediating immune and tumor cell trafficking into the extravascular site will be discussed. Next, the role of glycans in modulating bacterial, fungal, viral, and parasitic infections and cancer will be surveyed. Finally, the role of glycosylation in antibodies and carbohydrate vaccines will be analyzed. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major viral threat to swine, causing significant economic loss in the global pig farming industry. This virus includes two major genotypes, PRRSV1 and PRRSV2, both characterized by high mutation rates and genetic variability, complicating the development of a universally effective vaccine and disease control. To address this challenge, this study utilizes immunoinformatics tools to identify conserved epitopes and design a multi-epitope vaccine candidate against PRRSV based on reverse vaccinology. The complete sequences of PRRSV-encoded proteins were retrieved worldwide, and the conserved fragments were identified through the alignment of polypeptide sequences. Subsequent screening was conducted to screen epitopes for their potential to be safe and to activate B cells, HTLs (helper T cells), and CTLs (cytotoxic T cells). By conjugating the selected epitopes with distinct adjuvant proteins, three vaccine candidates were designed and termed PRRSV-vaccine (PRRSV-V-1, PRRSV-V-2, and PRRSV-V-3, respectively). Furthermore, systematic evaluations of their physicochemical properties, structural stability, binding with pattern recognition receptors, and induction of the host immune system were performed. PRRSV-V-2 had the most promising physicochemical and structural characteristics, strong binding with toll-like receptors (TLR3 and TLR8), and the most vigorous reactions to host immune responses. As the most promising candidate, the recombinant PRRSV plasmid was in silico designed for expression in Escherichia coli. Our study proposed a novel approach to PRRSV vaccine development against PRRSV, offering a promising strategy for controlling the infection across diverse PRRSV strains in swine. Despite providing significant insights into vaccine design through computational methods, the results of this study remain predictive. So, it is open for the experimental validations of the scientific community to ensure its actual immunological properties, especially the safety and efficacy. Full article

African swine fever virus (ASFV) is a highly virulent pathogen that causes nearly 100% mortality in acute infections and poses persistent risks. Effective containment of ASFV outbreaks requires rapid and reliable diagnostic tools. The p54 protein, a key structural component of ASFV, has emerged as an important target for serological detection. Herein, the recombinant p54 protein (amino acids 53–184) was expressed in Escherichia coli, and three mouse monoclonal antibodies (mAbs) (IgG1/kappa subtype) were developed. Among these mAbs, the mAb 1F9 specifically recognized the B-cell epitope ⁶⁶IQFINPYQDQQ⁷⁶, which is conserved across different genotypes of ASFV, suggesting that the epitope may serve as a valuable target for serological detection of ASFV. Structural modeling analysis revealed that this epitope is surface-exposed on the p54 protein, with ⁶⁷Gln and ⁶⁸Phe identified as critical residues for 1F9 binding. Moreover, a blocking ELISA based on the mAb 1F9 was established for detecting ASFV-specific antibodies in clinical serum samples, achieving a coincidence rate exceeding 95%. These findings demonstrate that mAb 1F9, targeting a conserved and accessible region of p54, represents a valuable tool for ASFV serodiagnosis, surveillance, and outbreak management. Full article

Eggs are a great source of protein in the human diet. They are consumed in tens of millions of tons globally per year. In addition, egg proteins, which are known food allergens, are included in many food products due to their excellent techno-functional properties. Hen’s eggs are the most consumed, but other edible avian eggs are occasionally used as gourmet ingredients or delicacies. With a high presence in the food market, the risk of accidental exposure to egg allergens is high. Hen egg allergy ranks among the top three food allergens in infants and young children. The complex structure and similar physicochemical properties of egg proteins limit their separation and purification, making further research challenging. Egg composition is influenced by age, disease, medicine, and environmental stress, and the target protein is often present in negligible amounts or polymorphic forms. To investigate the immunoreactivity of proteins from eggs of different bird species, it is necessary to consistently and quantitatively extract and purify proteins while avoiding harsh conditions. The conformational shape of allergens is impacted by denaturation, which can remove or expose IgE-binding epitopes and change the allergenic potential of proteins. This review presents findings from a literature survey on the isolation and purification strategies utilized for egg allergens from culinary-relevant bird eggs. Full article

B Cell Maturation Antigen (BCMA) has gained considerable attention as a target in directed therapies for multiple myeloma (MM) treatment, via immunoglobulin-based bispecific T cell engagers or CAR T cell strategies. We describe the development of alternative, non-immunoglobulin BCMA-recognising affinity proteins, based on the small (58 aa) three-helix bundle affibody scaffold. A first selection campaign using a naïve affibody phage library resulted in the isolation of several BCMA-binding clones with different kinetic profiles. One clone showing the slowest dissociation kinetics was chosen as the template for the construction of two second-generation libraries. Characterization of output clones from selections using these libraries led to the identification of clone 1-E6, which demonstrated low nM affinity to BCMA and high thermal stability. Biosensor experiments showed that 1-E6 interfered with the binding of BCMA to both its natural ligand APRIL and to the clinically evaluated anti-BCMA monoclonal antibody belantamab, suggesting overlapping epitopes. A fluorescently labelled head-to-tail homodimer construct of 1-E6 showed specific binding to the BCMA⁺ MM.1s cell line in both flow cytometry and fluorescence microscopy. Taken together, the results suggest that the small anti-BCMA affibody 1-E6 could be an interesting alternative to antibody-based affinity units in the development of BCMA-targeted therapies and diagnostics. Full article

Background: Brucellosis poses a significant public health challenge, necessitating effective vaccine development. Current vaccines have limitations such as safety concerns and inadequate mucosal immunity. This study aims to develop an FcRn-targeted mucosal Brucella vaccine by fusing the human Fc domain with Brucella’s multi-epitope protein (MEV), proposing a novel approach for human brucellosis prevention. Methods: The study developed a recombinant antigen (h-tFc-MEV) through computational analyses to validate antigenicity, structural stability, solubility, and allergenic potential. Molecular simulations confirmed FcRn binding. The vaccine was delivered orally via chitosan nanoparticles in murine models. Immunization was compared to MEV-only immunization. Post-challenge assessments were conducted to evaluate protection against Brucella colonization. Mechanistic studies investigated dendritic cell activation and antigen presentation. Results: Computational analyses showed that the antigen had favorable properties without allergenic potential. Molecular simulations demonstrated robust FcRn binding. In murine models, oral delivery elicited enhanced systemic immunity with elevated serum IgG titers and amplified CD4+/CD8+ T-cell ratios compared to MEV-only immunization. Mucosal immunity was evidenced by significant IgA upregulation across multiple tracts. Long-term immune memory persisted for six months. Post-challenge assessments revealed markedly reduced Brucella colonization in visceral organs. Mechanistic studies identified FcRn-mediated dendritic cell activation through enhanced MHC-II expression and antigen presentation efficiency. Conclusions: The FcRn-targeted strategy establishes concurrent mucosal and systemic protective immunity against Brucella infection. This novel vaccine candidate shows potential for effective human brucellosis prevention, offering a promising approach to address the limitations of current vaccines. Full article

Antibody lead discovery, crucial for immunotherapy development, requires identifying candidates with potent binding affinities to target antigens. Recent advances in protein language models have opened promising avenues to tackle this challenge by predicting antibody–antigen interactions (AAIs). Despite their appeals, precisely detecting binding sites (i.e., paratopes and epitopes) within the complex landscape of long-sequence biomolecules remains challenging. Herein, we propose MambaAAI, a bio-inspired model built upon the Mamba architecture, designed to predict AAIs and identify binding sites through selective attention mechanisms. Technically, we employ ESM-2, a pre-trained protein language model to extract evolutionarily enriched representations from input antigen and antibody sequences, which are modeled as residue-level interaction matrixes. Subsequently, a dual-view Mamba encoder is devised to capture important binding patterns, by dynamically learning embeddings of interaction matrixes from both antibody and antigen perspectives. Finally, the learned embeddings are decoded using a multilayer perceptron to output interaction probabilities. MambaAAI provides a unique advantage, relative to prior techniques, in dynamically selecting bio-enhancing residue sites that contribute to AAI prediction. We evaluate MambaAAI on two large-scale antibody–antigen neutralization datasets, and in silico results demonstrate that our method marginally outperforms the state-of-the-art baselines in terms of prediction accuracy, while maintaining robust generalization to unseen antibodies and antigens. In further analysis of the selective attention mechanism, we found that MambaAAI successfully uncovers critical epitope and paratope regions in the SARS-CoV-2 antibody examples. It is believed that MambaAAI holds great potential to discover lead candidates targeting specific antigens at a lower burden. Full article

Nanoclays have gained attention in biological applications due to their biocompatibility, low toxicity, and cost-effectiveness. Layered double hydroxides (LDHs) are synthetic nanoclays that have been used as adjuvants and antigen carriers in nanovaccines developed through passive bioconjugation. However, performing active bioconjugation to bind antigens covalently and generate subunit nanovaccines remains unexplored. In this study, we investigated the synthesis, functionalization, and active conjugation of LDH nanoparticles to produce subunit nanovaccines with peptides from SARS-CoV-2. The synthesis of Mg-Al LDHs via a coprecipitation and hydrothermal treatment rendered monodisperse particles averaging 100 nm. Their functionalization with (3-aminopropyl)triethoxysilane was better than it was with other organosilanes. Glutaraldehyde was used as a linker to bind lysine as a model biomolecule to establish the best conditions for reductive amination. Finally, two peptides, P₂ and P₅ (epitopes of the SARS-CoV-2 spike protein), were bound on the surface of the LDH to produce two subunit vaccine candidates, reaching peptide concentrations of 125 and 270 µg/mL, respectively. The particles were characterized using DLS, TEM, XRD, TGA, DSC, and FTIR. The cytotoxicity studies revealed that the conjugate with P₂ was non-toxic up to 250 µg/mL, while the immunogenicity studies showed that this conjugate induced similar IgG titers to those reached when aluminum hydroxide was used as an adjuvant. Full article

In this study, we conducted a comprehensive analysis of the interactions between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and four neutralizing antibodies—S309, S304, CYFN1006, and VIR-7229. Using integrative computational modeling that combined all-atom molecular dynamics (MD) simulations, mutational scanning, and MM-GBSA binding free energy calculations, we elucidated the structural, energetic, and dynamic determinants of antibody binding. Our findings reveal distinct dynamic binding mechanisms and evolutionary adaptation driving the broad neutralization effect of these antibodies. We show that S309 targets conserved residues near the ACE2 interface, leveraging synergistic van der Waals and electrostatic interactions, while S304 focuses on fewer but sensitive residues, making it more susceptible to escape mutations. The analysis of CYFN-1006.1 and CYFN-1006.2 antibody binding highlights broad epitope coverage with critical anchors at T345, K440, and T346, enhancing its efficacy against variants carrying the K356T mutation, which caused escape from S309 binding. Our analysis of broadly potent VIR-7229 antibody binding to XBB.1.5 and EG.5 Omicron variants emphasized a large and structurally complex epitope, demonstrating certain adaptability and compensatory effects to F456L and L455S mutations. Mutational profiling identified key residues crucial for antibody binding, including T345, P337, and R346 for S309 as well as T385 and K386 for S304, underscoring their roles as evolutionary “weak spots” that balance viral fitness and immune evasion. The results of the energetic analysis demonstrate a good agreement between the predicted binding hotspots, reveal distinct energetic mechanisms of binding, and highlight the importance of targeting conserved residues and diverse epitopes to counteract viral resistance. Full article

Immunoassays for cardiac troponin, such as the Elecsys^® hs-TnT, have become the gold standard for myocardial infarction diagnostics. While various protein/chemical factors affecting the troponin complex and thus its diagnostic accuracy have been investigated, the role of coding single-nucleotide polymorphisms remains underexplored. To evaluate potential cSNP-induced interference with antibody binding in the Elecsys^® hs-TnT immunoassay, we applied ITEM-FOUR, a mass spectrometry-based method that quantifies changes in antibody binding upon amino acid substitutions in epitope peptides. Candidate cSNPs were selected from the dbSNP database and were mapped to human cardiac troponin T by molecular modeling. Consuming micromolar antibody concentrations and microliter sample volumes, two wild-type and 17 cSNP-derived variant epitope peptides—six for monoclonal antibody M7 and eleven for monoclonal antibody M11.7—were investigated to reveal the binding motifs “V₁₃₁-K₁₃₄-E₁₃₈-A₁₄₂” for M7 and “E₁₄₆-I₁₅₀-R₁₅₄-E₁₅₇” for M11.7. Loss of binding to M11.7 was observed for substitutions Q148R (rs730880232), R154W (rs483352832), and R154Q (rs745632066), whereas the E138K (rs730881100) exchange disrupted binding of M7. Except for cSNP Q148R, they are associated with cardiomyopathies, placing affected individuals at risk of both underlying heart disease and false-negative hs-TnT assay results in cases of myocardial infarction. Our results highlight the need to account for cSNP-related interferences in antibody-based diagnostics. ITEM-FOUR offers a powerful approach for tackling this challenge, fostering next-generation assay development. Full article