Monoclonal antibody development advances immunological research in horses.

Host immune analyses require specific reagents to identify cellular and soluble components of the immune system. These immune reagents are often species-specific. For horses, various immunological tools have been developed and tested by different initiatives during the past decades. This article summarizes the development of well characterized monoclonal antibodies (mAbs) for equine immune cells, immunoglobulin isotypes, cytokines, and chemokines.

Machine learning evaluation for identification of M-proteins in human serum.

Serum electrophoresis (SPEP) is a method used to analyze the distribution of the most important proteins in the blood. The major clinical question is the presence of monoclonal fraction(s) of antibodies (M-protein/paraprotein), which is essential for the diagnosis and follow-up of hematological diseases, such as multiple myeloma. Recent studies have shown that machine learning can be used to assess protein electrophoresis by, for example, examining protein glycan patterns to follow up tumor surgery. In this study we compared 26 different decision tree algorithms to identify the presence of M-proteins in human serum by using numerical data from serum protein capillary electrophoresis. For the automated detection and clustering of data, we used an anonymized data set consisting of 67,073 samples. We found five methods with superior ability to detect M-proteins: Extra Trees (ET), Random Forest (RF), Histogram Grading Boosting Regressor (HGBR), Light Gradient Boosting Method (LGBM), and Extreme Gradient Boosting (XGB). Additionally, we implemented a game theoretic approach to disclose which features in the data set that were indicative of the resulting M-protein diagnosis. The results verified the gamma globulin fraction and part of the beta globulin fraction as the most important features of the electrophoresis analysis, thereby further strengthening the reliability of our approach. Finally, we tested the algorithms for classifying the M-protein isotypes, where ET and XGB showed the best performance out of the five algorithms tested. Our results show that serum capillary electrophoresis combined with decision tree algorithms have great potential in the application of rapid and accurate identification of M-proteins. Moreover, these methods would be applicable for a variety of blood analyses, such as hemoglobinopathies, indicating a wide-range diagnostic use. However, for M-protein isotype classification, combining machine learning solutions for numerical data from capillary electrophoresis with gel electrophoresis image data would be most advantageous.

Bi-isotype immunoglobulins enhance antibody-mediated neutrophil activity against Plasmodium falciparum parasites.

Background: Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay. Methods: We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG-IgA Fc region chimera would enhance the level of ADRB activity. Results: We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG-IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31. Conclusions: We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites.

The aryl hydrocarbon receptor differentially modulates the expression profile of antibody isotypes in a human B-cell line.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant and high affinity ligand for the aryl hydrocarbon receptor (AhR). In animal models, AhR activation by TCDD generally inhibits antibody secretion. However, it is less clear if this translates to human antibody production. Using a human Burkitt lymphoma B-cell line (CL-01) that can be stimulated to secrete Ig and undergo class switch recombination to other Ig isotypes, the current study evaluated the effects of AhR activation or antagonism on the human Ig isotypic expression profile with CD40L+IL-4 stimulation. Our results suggest that AhR agonists (TCDD and indirubin) have little to no effect on IgM or IgA secretion, which were also not induced with stimulation. However, AhR activation significantly inhibited stimulation-induced IgG secretion, an effect reversed by the AhR antagonist CH223191. Evaluation of Ig heavy chain (IgH) constant region gene expression (ie Cµ, Cγ1-4, Cα1-2, and Cε that encode for IgM, IgG1-4, IgA1-2, and IgE, respectively) demonstrated differential effects. While Cµ and Cα2 transcripts were unaffected by stimulation or AhR agonists, AhR activation significantly inhibited stimulation-induced Cγ2-4 and Cε mRNA transcripts, which was reversed by AhR antagonism. Notably, AhR antagonism in the absence of exogenous AhR ligands significantly increased IgG and IgA secretion as well as the expression of Cγ2-4 and Cε. These results suggest that modulation of AhR activity differentially alters the IgH isotypic expression profile and antibody secretion that may be partly dependent on cellular stimulation. Since a variety of chemicals from anthropogenic, industrial, pharmaceutical, dietary, and bacterial sources bind the AhR, the ability of environmental exposures to alter AhR activity (i.e. activate or inhibit) may have a direct influence on immune function and antibody-relevant disease conditions.

The immunoglobulin heavy chain super enhancer controls class switch recombination in developing B cells.

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to replace the initial IgM by another antibody class (IgG, IgE or IgA). CSR is preceded by transcription of the IgH constant genes and is controlled by the super-enhancer 3' regulatory region (3'RR) in an activation-specific manner. The 3'RR is composed of four enhancers (hs3a, hs1-2, hs3b and hs4). In mature B cells, 3'RR activity correlates with transcription of its enhancers. CSR can also occur in primary developing B cells though at low frequency, but in contrast to mature B cells, the transcriptional elements that regulate the process in developing B cells are ill-known. In particular, the role of the 3'RR in the control of constant genes' transcription and CSR has not been addressed. Here, by using a mouse line devoid of the 3'RR and a culture system that highly enriches in pro-B cells, we show that the 3'RR activity is indeed required for switch transcription and CSR, though its effect varies in an isotype-specific manner and correlates with transcription of hs4 enhancer only.

Aberrant B cell receptor signaling in circulating naïve and IgA+ memory B cells from newly-diagnosed autoantibody-positive rheumatoid arthritis patients.

OBJECTIVE: Altered B cell receptor (BCR) signaling has been implicated in the pathogenesis of rheumatoid arthritis (RA). Here we aimed to identify signaling aberrations in autoantibody-positive and autoantibody-negative RA patients by performing a comprehensive analysis of the BCR signaling cascade in different B cell subsets. METHODS: We first optimized phosphoflow cytometry for an in-depth analysis of BCR signaling across immunoglobulin isotypes in healthy donors. Subsequently, we compared BCR signaling in circulating B cell subsets from treatment-naïve, newly-diagnosed autoantibody-positive RA and autoantibody-negative RA patients and healthy controls (HCs). RESULTS: We observed subset-specific phosphorylation patterns of the BCR signalosome in circulating B cells from healthy donors. Compared with HCs, autoantibody-positive RA patients displayed enhanced responses to BCR stimulation for multiple signaling proteins, specifically in naïve and IgA+ memory B cells. Whereas in unstimulated healthy donor B cells, the phosphorylation status of individual signaling proteins showed only limited correlation, BCR stimulation enhanced the interconnectivity in phosphorylation within the BCR signalosome. However, this strong interconnectivity within the BCR signalosome in stimulated B cells from HCs was lost in RA, especially in autoantibody-positive RA patients. Finally, we observed strong correlations between SYK and BTK protein expression, and IgA and IgG anti-citrullinated protein antibody concentrations in serum from autoantibody-positive RA patients. CONCLUSION: Collectively, the isotype-specific analysis of multiple key components of the BCR signalosome identified aberrant BCR signaling responses in treatment-naïve autoantibody-positive RA patients, particularly in naïve B cells and IgA+ memory B cells. Our findings support differential involvement of dysregulated BCR signaling in the pathogenesis of autoantibody-positive and autoantibody-negative RA.

Changes in RDW according to prognostic predictors in newly diagnosed multiple myeloma.

RDW is an erythrocyte index that increase in multiple myeloma, in which it appears to have an important role in predicting outcome. For this reason, we performed a retrospective analysis to evaluate the relationships of RDW with some important prognostic predictors. Specifically, in a cohort of 190 newly diagnosed multiple myeloma patients, we have examined the behaviour of RDW and its trend in relation to the ISS stage and other prognostic factors, such as albumin, beta-2 microglobulin, LDH and bone marrow plasma cell infiltration. We performed the analysis in the entire cohort of patients and in the three different disease isotypes (Light chain, IgA, and IgG multiple myeloma). The evaluation of RDW in the different isotypes was made with the Kruskal-Wallis test, integrated by the Dunn test. The comparison between the subgroups allocated above and below the median value of each prognostic factor, was made with the Mann-Whitney test. From our analysis, we observed that RDW is higher in the IgA multiple myeloma, and it increases significantly from ISS I to III. Moreover, RDW increases in the presence of lower albumin values, higher levels of beta2-microglobulin and LDH and in the presence of a greater bone marrow plasma cell infiltrate.

Waldenström Disease in a Renal Transplant: a Case Report and Review of the Literature.

BACKGROUND: We report through this case, the exceptional occurrence of Waldenström's macroglobulinemia in a renal transplant. METHODS: A 65-year-old diabetic man, who had a kidney transplant in 2008, presented to the hospital in 2020 for ketoacid decompensation. The blood ionogram showed hyperproteinemia at 102 g/L. Electrophoresis and immunofixation of serum proteins revealed a monoclonal immunoglobulin of IgM Kappa isotype numbered at 46 g/L. Confirmation of Waldenström's disease was made by myelogram and immunophenotyping of tumor cells. RESULTS: The diagnosis adopted for our case is Waldenström's disease which occurred 12 years after the kidney transplantation. CONCLUSIONS: Post-transplant lymphoproliferative syndromes are secondary to immunosuppressive therapy, the main concern in this case is the involvement of the graft with the risk of losing its function, hence the interest of monitoring and identifying any hyperproteinemia.

Interaction of Asn297-Linked Glycan Ligands with the Fc Fragment of the Immunoglobulin Class G1: A Molecular Dynamics Simulation Study.

The allosteric modulation of the homodimeric H10-03-6 protein to glycan ligands L1 and L2, and the STAB19 protein to glycan ligands L3 and L4, respectively, has been studied by molecular dynamics simulations and free energy calculations. The results revealed that the STAB19 protein has a significantly higher affinity for L3 (-11.38 ± 2.32 kcal/mol) than that for L4 (-5.51 ± 1.92 kcal/mol). However, the combination of the H10-03-6 protein with glycan L2 (1.23 ± 6.19 kcal/mol) is energetically unfavorable compared with that of L1 (-13.96 ± 0.35 kcal/mol). Further, the binding of glycan ligands L3 and L4 to STAB19 would result in the significant closure of the two CH2 domains of the STAB19 conformation with the decrease of the centroid distances between the two CH2 domains compared with the H10-03-6/L1/L2 complex. The CH2 domain closure of STAB19 relates directly to the formation of new hydrogen bonds and hydrophobic interactions between the residues Ser239, Val240, Asp265, Glu293, Asn297, Thr299, Ser337, Asp376, Thr393, Pro395, and Pro396 in STAB19 and glycan ligands L3 and L4, which suggests that these key residues would contribute to the specific regulation of STAB19 to L3 and L4. In addition, the distance analysis revealed that the EF loop in the H10-03-6/L1/L2 model presents a high flexibility and partial disorder compared with the stabilized STAB19/L3/L4 complex. These results will be helpful in understanding the specific regulation through the asymmetric structural characteristics in the CH2 and CH3 domains of the H10-03-6 and STAB19 proteins.

The multilevel extensive diversity across the cynomolgus macaque captured by ultra-deep adaptive immune receptor repertoire sequencing.

The extent to which AIRRs differ among and within individuals remains elusive. Via ultra-deep repertoire sequencing of 22 and 25 tissues in three cynomolgus macaques, respectively, we identified 84 and 114 novel IGHV and TRBV alleles, confirming 72 (85.71%) and 100 (87.72%) of them. The heterogeneous V gene usage patterns were influenced, in turn, by genetics, isotype (for BCRs only), tissue group, and tissue. A higher proportion of intragroup shared clones in the intestinal tissues than those in other tissues suggests a close intra-intestinal adaptive immunity network. Significantly higher mutation burdens in the public clones and the inter-tissue shared IgM and IgD clones indicate that they might target the shared antigens. This study reveals the extensive heterogeneity of the AIRRs at various levels and has broad fundamental and clinical implications. The data generated here will serve as an invaluable resource for future studies on adaptive immunity in health and diseases.

MCT1-governed pyruvate metabolism is essential for antibody class-switch recombination through H3K27 acetylation.

Monocarboxylate transporter 1 (MCT1) exhibits essential roles in cellular metabolism and energy supply. Although MCT1 is highly expressed in activated B cells, it is not clear how MCT1-governed monocarboxylates transportation is functionally coupled to antibody production during the glucose metabolism. Here, we report that B cell-lineage deficiency of MCT1 significantly influences the class-switch recombination (CSR), rendering impaired IgG antibody responses in Mct1f/fMb1Cre mice after immunization. Metabolic flux reveals that glucose metabolism is significantly reprogrammed from glycolysis to oxidative phosphorylation in Mct1-deficient B cells upon activation. Consistently, activation-induced cytidine deaminase (AID), is severely suppressed in Mct1-deficient B cells due to the decreased level of pyruvate metabolite. Mechanistically, MCT1 is required to maintain the optimal concentration of pyruvate to secure the sufficient acetylation of H3K27 for the elevated transcription of AID in activated B cells. Clinically, we found that MCT1 expression levels are significantly upregulated in systemic lupus erythematosus patients, and Mct1 deficiency can alleviate the symptoms of bm12-induced murine lupus model. Collectively, these results demonstrate that MCT1-mediated pyruvate metabolism is required for IgG antibody CSR through an epigenetic dependent AID transcription, revealing MCT1 as a potential target for vaccine development and SLE disease treatment.

The distribution and function of teleost IgT.

Given the uniquely close relationship between fish and aquatic environments, fish mucosal tissues are constantly exposed to a wide array of pathogenic microorganisms in the surrounding water. To maintain mucosal homeostasis, fish have evolved a distinct mucosal immune system known as mucosal-associated lymphoid tissues (MALTs). These MALTs consist of key effector cells and molecules from the adaptive immune system, such as B cells and immunoglobulins (Igs), which play crucial roles in maintaining mucosal homeostasis and defending against external pathogen infections. Until recently, three primary Ig isotypes, IgM, IgD, and IgT, have been identified in varying proportions within the mucosal secretions of teleost fish. Similar to the role of mucosal IgA in mammals and birds, teleost IgT plays a predominant role in mucosal immunity. Following the identification of the IgT gene in 2005, significant advances have been made in researching the origin, evolution, structure, and function of teleost IgT. Multiple IgT variants have been identified in various species of teleost fish, underscoring the remarkable complexity of IgT in fish. Therefore, this study provides a comprehensive review of the recent advances in various aspects of teleost IgT, including its genomic and structural features, the diverse distribution patterns within various fish mucosal tissues (the skin, gills, gut, nasal, buccal, pharyngeal, and swim bladder mucosa), its interaction with mucosal symbiotic microorganisms, and its immune responses towards diverse pathogens, including bacteria, viruses, and parasites. We also highlight the existing research gaps in the study of teleost IgT, suggesting the need for further investigation into the functional aspects of IgT and IgT+ B cells. This research is aimed at providing valuable insights into the immune functions of IgT and the mechanisms underlying the immune responses of fish against infections.

RPA guides UNG to uracil in ssDNA to facilitate antibody class switching and repair of mutagenic uracil at the replication fork.

Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sµ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.

Understanding Fc function for rational vaccine design against pathogens.

Antibodies represent the primary correlate of immunity following most clinically approved vaccines. However, their mechanisms of action vary from pathogen to pathogen, ranging from neutralization, to opsonophagocytosis, to cytotoxicity. Antibody functions are regulated both by antigen specificity (Fab domain) and by the interaction of their Fc domain with distinct types of Fc receptors (FcRs) present in immune cells. Increasing evidence highlights the critical nature of Fc:FcR interactions in controlling pathogen spread and limiting the disease state. Moreover, variation in Fc-receptor engagement during the course of infection has been demonstrated across a range of pathogens, and this can be further influenced by prior exposure(s)/immunizations, age, pregnancy, and underlying health conditions. Fc:FcR functional variation occurs at the level of antibody isotype and subclass selection as well as post-translational modification of antibodies that shape Fc:FcR-interactions. These factors collectively support a model whereby the immune system actively harnesses and directs Fc:FcR interactions to fight disease. By defining the precise humoral mechanisms that control infections, as well as understanding how these functions can be actively tuned, it may be possible to open new paths for improving existing or novel vaccines.

Monitoring of antibody levels in healthcare workers after inactivated coronavirus disease 19 vaccination.

OBJECTIVE: Because of the coronavirus disease 19 pandemic, studies on vaccination are being conducted in our country as well as across the world. In this study, the antibody levels in healthcare workers vaccinated with two doses of inactivated vaccine and the factors affecting these levels were investigated. METHODS: Randomly selected volunteers from healthcare workers, who had been vaccinated with two doses of inactivated vaccine in January to February 2021, were included in the study. Blood samples were drawn twice, 1 month and 6 months after the second dose vaccine (CoronaVac:Sinovac Life Science Co, Ltd, Beijing, China). The antibody levels were determined by the chemiluminescence microparticle immunoassay method using kits for quantitative detection of immunoglobulin class G antibodies to severe acute respiratory syndrome coronavirus 2. RESULTS: The mean antibody levels of 129 volunteers were 1232.5 (min: 103 to max: 7151) AU/mL in the first month and 403.5 (min: 23 to max: 4963) AU/mL in the sixth month. According to the survey results, 91 (71%) volunteers had not been diagnosed with coronavirus disease 19 before vaccination. The antibody levels 1 month and 6 months after the second dose of vaccination were significantly higher in those who had been diagnosed with coronavirus disease 19 before vaccination than in those who had not. It was found that age, gender, fast food, or healthy nutrition had no effect on antibody levels. CONCLUSION: Vaccines are very important both to protect against coronavirus disease 19 and to experience only a mild form of the disease. Immunoglobulin class G levels formed after vaccination may be affected by many factors and may decrease over time.

Spatial transcriptomics deconvolution at single-cell resolution using Redeconve.

Computational deconvolution with single-cell RNA sequencing data as reference is pivotal to interpreting spatial transcriptomics data, but the current methods are limited to cell-type resolution. Here we present Redeconve, an algorithm to deconvolute spatial transcriptomics data at single-cell resolution, enabling interpretation of spatial transcriptomics data with thousands of nuanced cell states. We benchmark Redeconve with the state-of-the-art algorithms on diverse spatial transcriptomics platforms and datasets and demonstrate the superiority of Redeconve in terms of accuracy, resolution, robustness, and speed. Application to a human pancreatic cancer dataset reveals cancer-clone-specific T cell infiltration, and application to lymph node samples identifies differential cytotoxic T cells between IgA+ and IgG+ spots, providing novel insights into tumor immunology and the regulatory mechanisms underlying antibody class switch.

Vaccine-induced SARS-CoV-2 antibody response: the comparability of S1-specific binding assays depends on epitope and isotype discrimination.

Background: Quantification of the SARS-CoV-2-specific immune response by serological immunoassays is critical for the management of the COVID-19 pandemic. In particular, neutralizing antibody titers to the viral spike (S) protein have been proposed as a correlate of protection (CoP). The WHO established the First International Standard (WHO IS) for anti-SARS-CoV-2 immunoglobulin (Ig) (NIBSC 20/136) to harmonize binding assays with the same antigen specificity by assigning the same unitage in binding antibody units (BAU)/ml. Method: In this study, we analyzed the S1-specific antibody response in a cohort of healthcare workers in Germany (n = 76) during a three-dose vaccination course over 8.5 months. Subjects received either heterologous or homologous prime-boost vaccination with ChAdOx1 nCoV-19 (AstraZeneca) and BNT162b2 (Pfizer-BioNTech) or three doses of BNT162b2. Antibodies were quantified using three anti-S1 binding assays (ELISA, ECLIA, and PETIA) harmonized to the WHO IS. Serum levels of neutralizing antibodies were determined using a surrogate virus neutralization test (sVNT). Binding assays were compared using Spearman's rank correlation and Passing-Bablok regression. Findings: All assays showed good correlation and similar antibody kinetics correlating with neutralizing potential. However, the assays show large proportional differences in BAU/ml. ECLIA and PETIA, which detect total antibodies against the receptor- binding domain (RBD) within the S1 subunit, interact similarly with the convalescent plasma-derived WHO IS but differently with vaccine serum, indicating a high sensitivity to the IgG/IgM/IgA ratio. Conclusion: All three binding assays allow monitoring of the antibody response in COVID-19-vaccinated individuals. However, the assay-specific differences hinder the definition of a common protective threshold in BAU/ml. Our results highlight the need for the thoughtful use of conversion factors and consideration of method-specific differences. To improve the management of future pandemics and harmonize total antibody assays, we should strive for reference material with a well-characterized Ig isotype composition.

Construction of Semisynthetic Shark vNAR Yeast Surface Display Antibody Libraries.

The adaptive immune system of sharks comprises a unique heavy chain-only antibody isotype, termed immunoglobulin new antigen receptor (IgNAR), in which antigen binding is mediated by a single variable domain, referred to as vNAR. In recent years, efforts were made to harness these domains for biomedical and biotechnological applications particularly due to their high affinity and specificity combined with a small size and high stability. Herein, we describe protocols for the construction of semisynthetic, CDR3-randomized vNAR libraries for the isolation of target-specific paratopes by yeast surface display. Additionally, we provide guidance for affinity maturation of a panel of antigen-enriched vNAR domains through CDR1 diversification of the FACS-selected, antigen-enriched population and sublibrary establishment.

Organically Templated Uranyl Sulfates and Selenates: Structural Complexity and Crystal Chemical Restrictions for Isotypic Compounds Formation.

This paper reviews the state of the art in the structural chemistry of organically templated uranyl sulfates and selenates, which are considered as the most representative groups of U-bearing synthetic compounds. In total, there are 194 compounds known for both groups, the crystal structures of which include 84 various organic molecules. Structural studies and topological analysis clearly indicate complex crystal chemical limitations in terms of the isomorphic substitution implementation, since the existence of isotypic phases has to date been confirmed only for 24 compounds out of 194, which is slightly above 12%. The structural architecture of the entire compound depends on the combination of the organic and oxyanion parts, changes in which are sometimes realized even while maintaining the topology of the U-bearing complex. An increase in the size of the hydrocarbon part and number of charge functional groups of the organic cation leads to the formation of rare and more complex topologies. In addition, the crystal structures of two novel uranyl sulfates and one uranyl selenate, templated by isopropylammonium cations, are reported.