AttABseq: an attention-based deep learning prediction method for antigen-antibody binding affinity changes based on protein sequences.

The optimization of therapeutic antibodies through traditional techniques, such as candidate screening via hybridoma or phage display, is resource-intensive and time-consuming. In recent years, computational and artificial intelligence-based methods have been actively developed to accelerate and improve the development of therapeutic antibodies. In this study, we developed an end-to-end sequence-based deep learning model, termed AttABseq, for the predictions of the antigen-antibody binding affinity changes connected with antibody mutations. AttABseq is a highly efficient and generic attention-based model by utilizing diverse antigen-antibody complex sequences as the input to predict the binding affinity changes of residue mutations. The assessment on the three benchmark datasets illustrates that AttABseq is 120% more accurate than other sequence-based models in terms of the Pearson correlation coefficient between the predicted and experimental binding affinity changes. Moreover, AttABseq also either outperforms or competes favorably with the structure-based approaches. Furthermore, AttABseq consistently demonstrates robust predictive capabilities across a diverse array of conditions, underscoring its remarkable capacity for generalization across a wide spectrum of antigen-antibody complexes. It imposes no constraints on the quantity of altered residues, rendering it particularly applicable in scenarios where crystallographic structures remain unavailable. The attention-based interpretability analysis indicates that the causal effects of point mutations on antibody-antigen binding affinity changes can be visualized at the residue level, which might assist automated antibody sequence optimization. We believe that AttABseq provides a fiercely competitive answer to therapeutic antibody optimization.

Isolation of Rare Antigen-Specific Memory B Cells via Antigen Tetramers.

Immunological memory, which sets the foundation for the adaptive immune response, plays a key role in disease protection and prevention. Obtaining a deeper understanding of the mechanisms underlying this phenomenon can aide in research aimed to improve vaccines and therapies. Memory B cells (MBCs) are a fundamental component of immunological memory but can exist in rare populations that prove challenging to study. By combining fluorescent antigen tetramers with multiple enrichment processes, a highly streamlined method for identifying and sorting antigen-specific MBCs from human blood and lymphoid tissues can be achieved. With the output of this process being viable cells, there is a multitude of downstream operations that can be used in conjunction with the antigen-specific cell sorting outlined in this chapter. Single-cell RNA-sequencing paired with B cell repertoire sequencing, which can be linked to distinct antigens in a high-throughput fashion, is a downstream application widely used in disease and vaccination research. Incorporation of this protocol can lead to a variety of applications and a diversity of outcomes aiding in a deeper understanding of how immunological memory not only forms but is recalled and impacted by infection and vaccination.

ELISpots for Detecting Antigen-Specific Memory B Cells in Infection or Autoimmunity.

Enzyme-Linked Immunosorbent Spot assay (ELISpot) is an immunoassay used to quantify individual protein-specific secreting cells. Proteins secreted by cells cultured in ELISpot plates (96- or 8-well format) bind to a specific antigen bound to a PVDF membrane at the bottom of the well. A detection antibody followed by an enzymatic reaction is used to identify secreted protein bound to the membrane coated antigen. This reaction results in distinct "spots" on the membrane corresponding to individual protein secreting cells. While the design is similar to an ELISA, ELISpots quantify the number and relative amount of secreted protein on a single cell level, as opposed to an ELISA that reveals the concentration of secreted proteins from a population of cells. The sensitivity, robustness, and diversity of different antigens used by ELISpots have led to an array of research applications such as measuring cytokines from cytotoxic T cells in cancer and quantifying antibody specificity from B cells following vaccinations. Improvements have been made to assays measuring cytokines and antibodies on a single cell basis, such as intracellular flow cytometry. Yet the ability of an ELISpot to evaluate the quantity and quality of protein secretion on an individual cell basis remains unmatched. Here, we describe the use of a modified ELISpot assay to detect antigen-specific memory B cells in the setting of a viral infection and autoimmunity.

Expanded Antigen-Specific Elimination Assay to Measure Human CD8+ T Cell Cytolytic Potential.

Durable cellular immunity against pathogens is dependent upon a coordinated recall response to antigen by memory CD8+ T cells, involving their proliferation and the generation of secondary cytotoxic effector cells. Conventional assays measuring ex vivo cytotoxicity fail to capture this secondary cytolytic potential, especially in settings where cells have not been recently exposed to their cognate antigen in vivo. Here we describe the expanded antigen-specific elimination assay (EASEA), a flow cytometric endpoint assay to measure the capacity of human CD8+ T cells to expand in vitro upon antigen re-exposure and generate secondary effector cells capable of selectively eliminating autologous antigen-pulsed target cells across a range of effector-to-target ratios. Unlike alternative assays, EASEA avoids the hazards of radioactive labeling and viral infection and can be used to study responses to individual or pooled antigens of interest. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Expanded antigen-specific elimination assay.

An Analytical Protocol for Detecting Antibody Titer Levels in Serum/Saliva by Indirect Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) detects qualitatively and quantitatively the presence of antibodies or antigens in a sample. Due to its simplicity, high sensitivity, and user-friendliness, the test is widely used in laboratory research, clinical diagnoses, and food testing. This chapter describes the indirect semiquantitative ELISA protocol used to monitor antibody levels in animals and analyze the titer levels of specific antibodies against a target antigen in serum and saliva.

Assessment of the Antigen-Binding Capacity and Separation of Extracellular Vesicles Coated with Antigen-Specific Antibody Light Chains.

Many researchers are interested in the possibility of manipulating the targeting specificity of extracellular vesicles (EVs) for their use as physiological delivery vehicles for drugs and bioactive molecules. Our studies demonstrated the possibility of directing EVs toward the desired acceptor cell by coating them with antigen-specific antibody light chains. Here, we describe the methods for detection of the presence of antibody light chains on the EV surface, proving their ability to specifically bind the antigen and for separating the antigen-binding EV subpopulation.

Exploiting the Role of Features for Antigens-Antibodies Interaction Site Prediction.

Antibodies are a class of proteins that recognize and neutralize pathogens by binding to their antigens. They are the most significant category of biopharmaceuticals for both diagnostic and therapeutic applications. Understanding how antibodies interact with their antigens plays a fundamental role in drug and vaccine design and helps to comprise the complex antigen binding mechanisms. Computational methods for predicting interaction sites of antibody-antigen are of great value due to the overall cost of experimental methods. Machine learning methods and deep learning techniques obtained promising results.In this work, we predict antibody interaction interface sites by applying HSS-PPI, a hybrid method defined to predict the interface sites of general proteins. The approach abstracts the proteins in terms of hierarchical representation and uses a graph convolutional network to classify the amino acids between interface and non-interface. Moreover, we also equipped the amino acids with different sets of physicochemical features together with structural ones to describe the residues. Analyzing the results, we observe that the structural features play a fundamental role in the amino acid descriptions. We compare the obtained performances, evaluated using standard metrics, with the ones obtained with SVM with 3D Zernike descriptors, Parapred, Paratome, and Antibody i-Patch.

Temperature sensitivity of bat antibodies links metabolic state of bats with antigen-recognition diversity.

The bat immune system features multiple unique properties such as dampened inflammatory responses and increased tissue protection, explaining their long lifespan and tolerance to viral infections. Here, we demonstrated that body temperature fluctuations corresponding to different physiological states in bats exert a large impact on their antibody repertoires. At elevated temperatures typical for flight, IgG from the bat species Myotis myotis and Nyctalus noctula show elevated antigen binding strength and diversity, recognizing both pathogen-derived antigens and autoantigens. The opposite is observed at temperatures reflecting inactive physiological states. IgG antibodies of human and other mammals, or antibodies of birds do not appear to behave in a similar way. Importantly, diversification of bat antibody specificities results in preferential recognition of damaged endothelial and epithelial cells, indicating an anti-inflammatory function. The temperature-sensitivity of bat antibodies is mediated by the variable regions of immunoglobulin molecules. Additionally, we uncover specific molecular features of bat IgG, such as low thermodynamic stability and implication of hydrophobic interactions in antigen binding as well as high prevalence of polyreactivity. Overall, our results extend the understanding of bat tolerance to disease and inflammation and highlight the link between metabolism and immunity.

Ionic Liquid-Based Immunization Patch for the Transdermal Delivery of Antigens.

Herein, we report a transdermal patch prepared using an ionic liquid-based solid in oil (IL-S/O) nanodispersion and a pressure-sensitive adhesive (PSA) to deliver the macromolecular antigenic protein, ovalbumin (OVA). The IL-S/O nanodispersion and a PSA were first mixed at an equal weight ratio, then coated onto a release liner, and covered with a support film. To evaluate the effect of the PSA, three types of PSAs, DURO-TAK 87-4098, DURO-TAK 87-4287, and DURO-TAK 87-235A, were used to obtain the corresponding IL-S/O patches SP-4098, SP-4287, and SP-235A, respectively. The prepared IL-S/O patches were characterized for surface morphology, viscoelasticity, and moisture content. In vitro skin penetration and in vivo immunization studies of the IL-S/O patches were performed using Yucatan micropig skin and the C57BL/6NJc1 mice model, respectively. The SP-4098 and SP-4287 delivered 5.49-fold and 5.47-fold higher amounts of drug compared with the aqueous formulation. Although both patches delivered a similar amount of drug, SP-4287 was not detached fully from the release liner after 30 days, indicating low stability. Mice immunized with the OVA-containing SP-4098 produced a 10-fold increase in anti-OVA IgG compared with those treated with an aqueous formulation. These findings suggested that the IL-S/O patch may be a good platform for the transdermal delivery of antigen molecules.

New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis.

BACKGROUND: Bone marrow-derived mesenchymal stem cell (BMMSC)-based therapy has become a major focus for treating liver fibrosis/cirrhosis. However, although these cell therapies promote the treatment of this disease, the heterogeneity of BMMSCs, which causes insufficient efficacy during clinical trials, has not been addressed. In this study, we describe a novel Percoll-Plate-Wait procedure (PPWP) for the isolation of an active cell subset from BMMSC cultures that was characterized by the expression of neuroglial antigen 2 (NG2/BMMSCs). METHODS: By using the key method of PPWP and other classical biological techniques we compared NG2/BMMSCs with parental BMMSCs in biological and functional characteristics within a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis injury male C57BL/6 mouse model also in a culture system. Of note, the pathological alterations in the model is quite similar to humans'. RESULTS: The NG2/BMMSCs revealed more advantages compared to parentalBMMSCs. They exhibited greater proliferation potential than parental BMMSCs, as indicated by Ki-67 immunofluorescence (IF) staining. Moreover, higher expression of SSEA-3 (a marker specific for embryonic stem cells) was detected in NG2/BMMSCs than in parental BMMSCs, which suggested that the "stemness" of NG2/BMMSCs was greater than that of parental BMMSCs. In vivo studies revealed that an injection of NG2/BMMSCs into mice with ongoing DEN-induced liver fibrotic/cirrhotic injury enhanced repair and functional recovery to a greater extent than in mice treated with parental BMMSCs. These effects were associated with the ability of NG2/BMMSCs to differentiate into bile duct cells (BDCs). In particular, we discovered for the first time that NG2/BMMSCs exhibit unique characteristics that differ from those of parental BMMSCs in terms of producing liver sinusoidal endothelial cells (LSECs) to reconstruct injured blood vessels and sinusoidal structures in the diseased livers, which are important for initiating hepatocyte regeneration. This unique potential may also suggest that NG2/BMMSCs could be an novel off-liver progenitor of LSECs. Ex vivo studies revealed that the NG2/BMMSCs exhibited a similar trend to that of their in vivo in terms of functional differentiation responding to the DEN-diseased injured liver cues. Additionally, the obvious core role of NG2/BMMSCs in supporting the functions of BMMSCs in bile duct repair and BDC-mediated hepatocyte regeneration might also be a novel finding. CONCLUSIONS: Overall, the PPWP-isolated NG2/BMMSCs could be a novel effective cell subset with increased purity to serve as a new therapeutic tool for enhancing treatment efficacy of BMMSCs and special seed cell source (BDCs, LSECs) also for bioliver engineering.

Centrosome age breaks spindle size symmetry even in cells thought to divide symmetrically.

Centrosomes are the main microtubule-organizing centers in animal cells. Due to the semiconservative nature of centrosome duplication, the two centrosomes differ in age. In asymmetric stem cell divisions, centrosome age can induce an asymmetry in half-spindle lengths. However, whether centrosome age affects the symmetry of the two half-spindles in tissue culture cells thought to divide symmetrically is unknown. Here, we show that in human epithelial and fibroblastic cell lines centrosome age imposes a mild spindle asymmetry that leads to asymmetric cell daughter sizes. At the mechanistic level, we show that this asymmetry depends on a cenexin-bound pool of the mitotic kinase Plk1, which favors the preferential accumulation on old centrosomes of the microtubule nucleation-organizing proteins pericentrin, γ-tubulin, and Cdk5Rap2, and microtubule regulators TPX2 and ch-TOG. Consistently, we find that old centrosomes have a higher microtubule nucleation capacity. We postulate that centrosome age breaks spindle size symmetry via microtubule nucleation even in cells thought to divide symmetrically.

Ultrasensitive electrochemical immunosensor for the detection of C-reactive protein antigen.

Cardiovascular disease is one of the leading causes of premature death worldwide, and the determination of C-reactive protein (CRP) from human serum is of vital importance for the diagnosis of the disease. For this study, we have developed an electrochemical immunosensor based on onion-like carbon@polyacrylonitrile (OLC-PAN) for the detection of CRP antigens. This was accomplished by immobilizing CRP antibodies on a modified glassy carbon electrode (GCE). Several electrochemical techniques such as cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) were employed to evaluate the electrochemical detection of the CRP antigen. This ultrasensitive method for CRP antigen detection exhibited a very good logarithmic plot from -4.52 to -12.05 g mL-1 and a limit of detection (LOD) of 0.9 fg mL-1. The high selectivity, sensitivity, and stability of the developed electrochemical immunosensor would facilitate miniaturization for point-of-care applications and the efficient diagnosis of cardiovascular diseases.

Role of the afferent lymph as an immunological conduit to analyze tissue antigenic and inflammatory load.

The lymphatic fluid is the conduit by which part of the tissue "omics" is transported to the draining lymph node for immunosurveillance. Following cannulation of the pre-nodal cervical and mesenteric afferent lymphatics, herein we investigate the lymph proteomic composition, uncovering that its composition varies according to the tissue of origin. Tissue specificity is also reflected in the dendritic cell-major histocompatibility complex class II-eluted immunopeptidome harvested from the cervical and mesenteric nodes. Following inflammatory disruption of the gut barrier, the lymph antigenic and inflammatory loads are analyzed in both mice and subjects with inflammatory bowel diseases. Gastrointestinal tissue damage reflects the lymph inflammatory and damage-associated molecular pattern signatures, microbiome-derived by-products, and immunomodulatory molecules, including metabolites of the gut-brain axis, mapped in the afferent mesenteric lymph. Our data point to the relevance of the lymphatic fluid to probe the tissue-specific antigenic and inflammatory load transported to the draining lymph node for immunosurveillance.

Modulating Whey Proteins Antigenicity with Lactobacillus delbrueckii subsp. bulgaricus DLPU F-36 Metabolites: Insights from Spectroscopic and Molecular Docking Studies.

Numerous studies have highlighted the potential of Lactic acid bacteria (LAB) fermentation of whey proteins for alleviating allergies. Nonetheless, the impact of LAB-derived metabolites on whey proteins antigenicity during fermentation remains uncertain. Our objective was to elucidate the impact of small molecular metabolites on the antigenicity of α-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG). Through metabolomic analysis, we picked 13 bioactive small molecule metabolites from Lactobacillus delbrueckii subsp. bulgaricus DLPU F-36 for coincubation with α-LA and ß-LG, respectively. The outcomes revealed that valine, arginine, benzoic acid, 2-keto butyric acid, and glutaric acid significantly diminished the sensitization potential of α-LA and ß-LG, respectively. Moreover, chromatographic analyses unveiled the varying influence of small molecular metabolites on the structure of α-LA and ß-LG, respectively. Notably, molecular docking underscored that the primary active sites of α-LA and ß-LG involved in protein binding to IgE antibodies aligned with the interaction sites of small molecular metabolites. In essence, LAB-produced metabolites wield a substantial influence on the antigenic properties of whey proteins.

Administration sequence- and formation-dependent vaccination using acid-degradable polymeric nanoparticles with high antigen encapsulation capability.

Vaccines aim to efficiently and specifically activate the immune system via a cascade of antigen uptake, processing, and presentation by antigen-presenting cells (APCs) to CD4 and CD8 T cells, which in turn drive humoral and cellular immune responses. The specific formulation of vaccine carriers can not only shield the antigens from premature sequestering before reaching APCs but also favorably promote intracellular antigen presentation and processing. This study compares two different acid-degradable polymeric nanoparticles that are capable of encapsulating a moderately immunogenic antigen, GFP, at nearly full efficacy via electrostatic interactions or molecular affinity between His tag and Ni-NTA-conjugated monomners. This resulted in GFP-encapsulating NPs composed of ketal monomers and crosslinkers (KMX/GFP NPs) and NTA-conjugated ketal monomers and crosslinkers (NKMX/GFP NPs), respectively. Encapsulated GFP was found to be released more rapidly from NKMX/GFP NPs (electrostatic encapsulation) than from KMX/GFP NPs (affinity-driven encapsulation). In vivo vaccination studies demonstrated that while repeated injections of either NP formulation resulted in poorer generation of anti-GFP antibodies than injections of the GFP antigen itself, sequential injections of NPs and GFP as prime and booster vaccines, respectively, restored the humoral response. We proposed that NPs primarily assist APCs in antigen presentation by T cells, and B cells need to be further stimulated by free protein antigens to produce antibodies. The findings of this study suggest that the immune response can be modulated by varying the chemistry of vaccine carriers and the sequences of vaccination with free antigens and antigen-encapsulating NPs.

Toward enhancement of antibody thermostability and affinity by computational design in the absence of antigen.

Over the past two decades, therapeutic antibodies have emerged as a rapidly expanding domain within the field of biologics. In silico tools that can streamline the process of antibody discovery and optimization are critical to support a pipeline that is growing more numerous and complex every year. High-quality structural information remains critical for the antibody optimization process, but antibody-antigen complex structures are often unavailable and in silico antibody docking methods are still unreliable. In this study, DeepAb, a deep learning model for predicting antibody Fv structure directly from sequence, was used in conjunction with single-point experimental deep mutational scanning (DMS) enrichment data to design 200 potentially optimized variants of an anti-hen egg lysozyme (HEL) antibody. We sought to determine whether DeepAb-designed variants containing combinations of beneficial mutations from the DMS exhibit enhanced thermostability and whether this optimization affected their developability profile. The 200 variants were produced through a robust high-throughput method and tested for thermal and colloidal stability (Tonset, Tm, Tagg), affinity (KD) relative to the parental antibody, and for developability parameters (nonspecific binding, aggregation propensity, self-association). Of the designed clones, 91% and 94% exhibited increased thermal and colloidal stability and affinity, respectively. Of these, 10% showed a significantly increased affinity for HEL (5- to 21-fold increase) and thermostability (>2.5C increase in Tm1), with most clones retaining the favorable developability profile of the parental antibody. Additional in silico tests suggest that these methods would enrich for binding affinity even without first collecting experimental DMS measurements. These data open the possibility of in silico antibody optimization without the need to predict the antibody-antigen interface, which is notoriously difficult in the absence of crystal structures.

Extending Linker Sequences between Antigen-Recognition Modules Provides More Effective Production of Bispecific Nanoantibodies in the Periplasma of E. coli.

Technology of production of single-domain antibodies (NANOBODY® molecules, also referred to as nanoantibodies, nAb, or molecules based on other stable protein structures) and their derivatives to solve current problems in biomedicine is becoming increasingly popular. Indeed, the format of one small, highly soluble protein with a stable structure, fully functional in terms of specific recognition, is very convenient as a module for creating multivalent, bi-/oligo-specific genetically engineered targeting molecules and structures. Production of nAb in periplasm of E. coli bacterium is a very convenient and fairly universal way to obtain analytical quantities of nAb for the initial study of the properties of these molecules and selection of the most promising nAb variants. The situation is more complicated with production of bi- and multivalent derivatives of the initially selected nAbs under the same conditions. In this work, extended linker sequences (52 and 86 aa) between the antigen-recognition modules in the cloned expression constructs were developed and applied in order to increase efficiency of production of bispecific nanoantibodies (bsNB) in the periplasm of E. coli bacteria. Three variants of model bsNBs described in this study were produced in the periplasm of bacteria and isolated in soluble form with preservation of functionality of all the protein domains. If earlier our attempts to produce bsNB in the periplasm with traditional linkers no longer than 30 aa were unsuccessful, the extended linkers used here provided a significantly more efficient production of bsNB, comparable in efficiency to the traditional production of original monomeric nAbs. The use of sufficiently long linkers could presumably be useful for increasing efficiency of production of other bsNBs and similar molecules in the periplasm of E. coli bacteria.

Construction of multi-epitope vaccine against the Rhipicephalus microplus tick: an immunoinformatics approach.

Rhipicephalus microplus, known as the hard tick, is a vector for the parasites Babesia spp. and Anaplasma marginale, both of which can cause significant financial losses to the livestock industry. There is currently no effective vaccine for R. microplus tick infestations, despite the identification of numerous prospective tick vaccine candidates. As a result, the current research set out to develop an immunoinformatics-based strategy using existing methods for designing a multi-epitope based vaccination that is not only effective but also safe and capable of eliciting cellular and humoral immune responses. First, R. microplus proteins Bm86, Subolesin, and Bm95 were used to anticipate and link B and T-cell epitopes (HTL and CTL) to one another. Antigenicity testing, allergenicity assessment, and toxicity screening were just a few of the many immunoinformatics techniques used to identify potent epitopes. Multi-epitope vaccine design was chosen based on the antigenic score 0.935 that is promising vaccine candidate. Molecular docking was used to determine the nature of the interaction between TLR2 and the vaccine construct. Finally, molecular dynamic simulation was used to assess the stability and compactness of the resulting vaccination based on docking scores. The developed vaccine was shown to be stable, have immunogenic qualities, be soluble, and to have high expression by in silico cloning. These findings suggest that experimental investigation of the multi-epitope based vaccine designed in the current study will produce achievable vaccine candidates against R. microplus ticks, enabling more effective control of infestations.

Effect of Silencing subolesin and enolase impairs gene expression, engorgement and reproduction in Haemaphysalis longicornis (Acari: Ixodidae) ticks.

IMPORTANCE: Haemaphysalis longicornis is an obligate blood-sucking ectoparasite that has gained attention due its role of transmitting medically and veterinary significant pathogens and it is the most common tick species in Republic of Korea. The preferred strategy for controlling ticks is a multi-antigenic vaccination. Testing the efficiency of a combination antigen is a promising method for creating a tick vaccine. OBJECTIVE: The aim of the current research was to analyze the role of subolesin and enolase in feeding and reproduction of H. longicornis by gene silencing. METHODS: In this study, we used RNA interference to silence salivary enolase and subolesin in H. longicornis. Unfed female ticks injected with double-stranded RNA targeting subolesin and enolase were attached and fed normally on the rabbit's ear. Real-time polymerase chain reaction was used to confirm the extent of knockdown. RESULTS: Ticks in the subolesin or enolase dsRNA groups showed knockdown rates of 80% and 60% respectively. Ticks in the combination dsRNA (subolesin and enolase) group showed an 80% knockdown. Knockdown of subolesin and enolase resulted in significant depletion in feeding, blood engorgement weight, attachment rate, and egg laying. Silencing of both resulted in a significant (p < 0.05) reduction in tick engorgement, egg laying, egg hatching (15%), and reproduction. CONCLUSIONS AND RELEVANCE: Our results suggest that subolesin and enolase are an exciting target for future tick control strategies.