Epitope profiling using computational structural modelling demonstrated on coronavirus-binding antibodies.

Identifying the epitope of an antibody is a key step in understanding its function and its potential as a therapeutic. Sequence-based clonal clustering can identify antibodies with similar epitope complementarity, however, antibodies from markedly different lineages but with similar structures can engage the same epitope. We describe a novel computational method for epitope profiling based on structural modelling and clustering. Using the method, we demonstrate that sequence dissimilar but functionally similar antibodies can be found across the Coronavirus Antibody Database, with high accuracy (92% of antibodies in multiple-occupancy structural clusters bind to consistent domains). Our approach functionally links antibodies with distinct genetic lineages, species origins, and coronavirus specificities. This indicates greater convergence exists in the immune responses to coronaviruses than is suggested by sequence-based approaches. Our results show that applying structural analytics to large class-specific antibody databases will enable high confidence structure-function relationships to be drawn, yielding new opportunities to identify functional convergence hitherto missed by sequence-only analysis.

Why current quantitative serology is not quantitative and how systems immunology could provide solutions.

Determination of the presence of antibodies against infectious agents, self-antigens, allogeneic antigens and environmental antigens is the goal of medical serology. Along with the standardization of these tests the community also started to use the expression "quantitative serology," referring to the fact that arbitrary units are used for the expression of results. In this review I will argue against the use of the term quantitative serology for current tests. Because each test and each antibody isotype determination uses its own references, the term semiquantitative better describes these methods. The introduction of really quantitative serology could both benefit from and drive forward systems immunological approach to immunity.

Fixing and Binding Antibodies to Suspension Cells in Preparation for Staining.

Staining of suspension cells after fixation, described here, is normally used only to detect cell-surface antigens. Once cells are fixed and permeabilized, the antibodies are added. The antibodies can be labeled directly or they can be detected by using a labeled secondary reagent that will bind specifically to the primary antibody. Detection reagents for cell staining can be labeled with fluorochromes, enzymes, gold, or iodine. Because the cells are in suspension, the washing steps are tedious, and care should be taken not to centrifuge for long durations or at high speeds.

A compact vocabulary of paratope-epitope interactions enables predictability of antibody-antigen binding.

Antibody-antigen binding relies on the specific interaction of amino acids at the paratope-epitope interface. The predictability of antibody-antigen binding is a prerequisite for de novo antibody and (neo-)epitope design. A fundamental premise for the predictability of antibody-antigen binding is the existence of paratope-epitope interaction motifs that are universally shared among antibody-antigen structures. In a dataset of non-redundant antibody-antigen structures, we identify structural interaction motifs, which together compose a commonly shared structure-based vocabulary of paratope-epitope interactions. We show that this vocabulary enables the machine learnability of antibody-antigen binding on the paratope-epitope level using generative machine learning. The vocabulary (1) is compact, less than 104 motifs; (2) distinct from non-immune protein-protein interactions; and (3) mediates specific oligo- and polyreactive interactions between paratope-epitope pairs. Our work leverages combined structure- and sequence-based learning to demonstrate that machine-learning-driven predictive paratope and epitope engineering is feasible.

Changes in epigenetic profiles throughout early childhood and their relationship to the response to pneumococcal vaccination.

BACKGROUND: Pneumococcal infections are a major cause of morbidity and mortality in young children and immaturity of the immune system partly underlies poor vaccine responses seen in the young. Emerging evidence suggests a key role for epigenetics in the maturation and regulation of the immune system in health and disease. The study aimed to investigate epigenetic changes in early life and to understand the relationship between the epigenome and antigen-specific antibody responses to pneumococcal vaccination. METHODS: The epigenetic profiles from 24 healthy children were analyzed at 12 months prior to a booster dose of the 13-valent pneumococcal conjugate vaccine (PCV-13), and at 24 months of age, using the Illumina Methylation 450 K assay and assessed for differences over time and between high and low vaccine responders. RESULTS: Our analysis revealed 721 significantly differentially methylated positions between 12 and 24 months (FDR < 0.01), with significant enrichment in pathways involved in the regulation of cell-cell adhesion and T cell activation. Comparing high and low vaccine responders, we identified differentially methylated CpG sites (P value < 0.01) associated with HLA-DPB1 and IL6. CONCLUSION: These data imply that epigenetic changes that occur during early childhood may be associated with antigen-specific antibody responses to pneumococcal vaccines.

Hiding in plain sight: structure and sequence analysis reveals the importance of the antibody DE loop for antibody-antigen binding.

Antibody variable domains contain "complementarity-determining regions" (CDRs), the loops that form the antigen binding site. CDRs1-3 are recognized as the canonical CDRs. However, a fourth loop sits adjacent to CDR1 and CDR2 and joins the D and E strands on the antibody v-type fold. This "DE loop" is usually treated as a framework region, even though mutations in the loop affect the conformation of the CDRs and residues in the DE loop occasionally contact antigen. We analyzed the length, structure, and sequence features of all DE loops in the Protein Data Bank (PDB), as well as millions of sequences from HIV-1 infected and naïve patients. We refer to the DE loop as H4 and L4 in the heavy and light chains, respectively. Clustering the backbone conformations of the most common length of L4 (6 residues) reveals four conformations: two κ-only clusters, one λ-only cluster, and one mixed κ/λ cluster. Most H4 loops are length-8 and exist primarily in one conformation; a secondary conformation represents a small fraction of H4-8 structures. H4 sequence variability exceeds that of the antibody framework in naïve human high-throughput sequences, and both L4 and H4 sequence variability from λ and heavy germline sequences exceed that of germline framework regions. Finally, we identified dozens of structures in the PDB with insertions in the DE loop, all related to broadly neutralizing HIV-1 antibodies (bNabs), as well as antibody sequences from high-throughput sequencing studies of HIV-infected individuals, illuminating a possible role in humoral immunity to HIV-1.

In vivo Induction of Functional Inhibitory IgG Antibodies by a Hypoallergenic Bet v 1 Variant.

Allergic sensitization to the major allergen Bet v 1 represents the dominating factor inducing a vast variety of allergic symptoms in birch pollen allergic patients worldwide, including the pollen food allergy syndrome. In order to overcome the huge socio-economic burden associated with allergic diseases, allergen-specific immunotherapy (AIT) as a curative strategy to manage the disease was introduced. Still, many hurdles related to this treatment exist making AIT not the patients' first choice. To improve the current situation, the development of hypoallergen-based drug products has raised attention in the last decade. Herein, we investigated the efficacy of the novel AIT candidate BM4, a hypoallergenic variant of Bet v 1, to induce treatment-relevant cross-reactive Bet v 1-specific IgG antibodies in two different mammals, Wistar rats and New Zealand White rabbits. We further analyzed the cross-reactivity of BM4-induced Wistar rat antibodies with the birch pollen-associated food allergens Mal d 1 and Cor a 1, and the functional capability of the induced antibodies to act as IgE-blocking IgG antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of rat IgG1, IgG2a, IgG2b, and IgE, as well as rabbit IgG and IgE antibodies. To address the functional relevance of the induced IgG antibodies, the capacity of rat sera to suppress binding of human IgE to Bet v 1 was investigated by using an inhibition ELISA and an IgE-facilitated allergen-binding inhibition assay. We found that the treatment with BM4 induced elevated Bet v 1-specific IgG antibody titers in both mammalian species. In Wistar rats, high BM4-specific IgG1, IgG2a, and IgG2b titers (104 to 106) were induced, which cross-reacted with wild-type Bet v 1, and the homologous allergens Mal d 1 and Cor a 1. Rat allergen-specific IgG antibodies sustained upon treatment discontinuation. Sera of rats immunized with BM4 were able to significantly suppress binding of human IgE to the wild-type allergens and CD23-mediated human IgE-facilitated Bet v 1 binding on B cells. By contrast, treatment-induced IgE antibody levels were low or undetectable. In summary, BM4 induced a robust IgG immune response that efficiently blocked human IgE-binding to wild-type allergens, underscoring its potential therapeutic value in AIT.

Structural Aspects of the Allergen-Antibody Interaction.

The development of allergic disease involves the production of IgE antibodies upon allergen exposure in a process called sensitization. IgE binds to receptors on the surface of mast cells and basophils, and subsequent allergen exposure leads to cross-linking of IgE antibodies and release of cell mediators that cause allergy symptoms. Although this process is quite well-understood, very little is known about the epitopes on the allergen recognized by IgE, despite the importance of the allergen-antibody interaction for the allergic response to occur. This review discusses efforts to analyze allergen-antibody interactions, from the original epitope mapping studies using linear peptides or recombinant allergen fragments, to more sophisticated technologies, such as X-ray crystallography and nuclear magnetic resonance. These state-of-the-art approaches, combined with site-directed mutagenesis, have led to the identification of conformational IgE epitopes. The first structures of an allergen (egg lysozyme) in complex with Fab fragments from IgG antibodies were determined in the 1980s. Since then, IgG has been used as surrogate for IgE, due to the difficulty of obtaining monoclonal IgE antibodies. Technical developments including phage display libraries have contributed to progress in epitope mapping thanks to the isolation of IgE antibody constructs from combinatorial libraries made from peripheral blood mononuclear cells of allergic donors. Most recently, single B cell antibody sequencing and human hybridomas are new breakthrough technologies for finally obtaining human IgE monoclonal antibodies, ideal for epitope mapping. The information on antigenic determinants will facilitate the design of hypoallergens for immunotherapy and the investigation of the fundamental mechanisms of the IgE response.

Challenge to ABO blood type barrier in living donor liver transplantation.

ABO incompatible living donor liver transplantation has the potential to expand the donor pool for patients with end stage liver diseases on the expense of challenges to overcome immunological barriers across blood type. There is a profound impact of age on incidence and severity of antibody mediated rejection (AMR). Even children older than 1 year have chances of AMR; children aged 8 years or older have risks of hepatic necrosis similar to adult liver recipients. The mechanism of AMR is based on circulatory disturbances secondary to inflammation and injury of the vascular endothelium caused by an antibody-antigen-complement reaction. The strategy to overcome ABO blood type barrier is based on both pre-transplant desensitization and adequate treatment of this phenomenon. Nowadays, rituximab is the standard means of desensitization but unfortunately an insufficient aid to treat AMR. Because of low incidence (less than 5% in the rituximab era), in practice of AMR only some case reports about the treatment of clinical AMR are available in the literature. Initial experiences revealed that the proteasome inhibitor, bortezomib might be a promising treatment based on its capacity to deplete plasma cell agents. Although ABO blood type barrier has been counteracted in 95% of patients by applying "rituximab-desensitization", many issues, such as prediction of high-risk patients of infection and AMR and secure treatment strategies for evoked AMR, remain to be resolved.

Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2.

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.

Complement activation by human IgG antibodies to galactose-α-1,3-galactose.

Some human antibodies may paradoxically inhibit complement activation on bacteria and enhance pathogen survival in humans. This property was also claimed for IgG antibodies reacting with terminal galactose-α-1,3-galactose (Galα3Gal; IgG anti-αGal), a naturally occurring and abundant antibody in human plasma that targets numerous different pathogens. To reinvestigate these effects, we used IgG anti-αGal affinity isolated from a pool of normal human IgG and human hypogammaglobulinaemia serum as a complement source. Flow cytometry was performed to examine antibody binding and complement deposition on pig erythrocytes, Escherichia coli O86 and Streptococcus pneumoniae serotype 9V. Specific nanobodies were used to block the effect of single complement factors and to delineate the complement pathways involved. IgG anti-αGal was capable of activating the classical complement pathway on all the tested target cells. The degree of activation was exponentially related to the density of bound antibody on E. coli O86 and pig erythrocytes, but more linearly on S. pneumoniae 9V. The alternative pathway of complement amplified complement deposition. Deposited C3 fragments covered the activating IgG anti-αGal, obstructing its detection and highlighting this as a likely general caveat in studies of antibody density and complement deposition. The inherent capacity for complement activation by the purified carbohydrate reactive IgG anti-αGal was similar to that of normal human IgG. We propose that the previously reported complement inhibition by IgG anti-αGal relates to suboptimal assay configurations, in contrast to the complement activating property of the antibodies demonstrated in this paper.

Investigation of anomalous charge variant profile reveals discrete pH-dependent conformations and conformation-dependent charge states within the CDR3 loop of a therapeutic mAb.

During the development of a therapeutic monoclonal antibody (mAb-1), the charge variant profile obtained by pH-gradient cation exchange chromatography (CEX) contained two main peaks, each of which exhibited a unique intrinsic fluorescence profile and demonstrated inter-convertibility upon reinjection of isolated peak fractions. Domain analysis of mAb-1 by CEX and liquid chromatography-mass spectrometry indicated that the antigen-binding fragment chromatographed as two separate peaks that had identical mass. Surface plasmon resonance binding analysis to antigen demonstrated comparable kinetics/affinity between these fractionated peaks and unfractionated starting material. Subsequent molecular modeling studies revealed that the relatively long and flexible complementarity-determining region 3 (CDR3) loop on the heavy chain could adopt two discrete pH-dependent conformations: an "open" conformation at neutral pH where the HC-CDR3 is largely solvent exposed, and a "closed" conformation at lower pH where the solvent exposure of a neighboring tryptophan in the light chain is reduced and two aspartic acid residues near the ends of the HC-CDR3 loop have atypical pKa values. The pH-dependent equilibrium between "open" and "closed" conformations of the HC-CDR3, and its proposed role in the anomalous charge variant profile of mAb-1, were supported by further CEX and hydrophobic interaction chromatography studies. This work is an example of how pH-dependent conformational changes and conformation-dependent changes to net charge can unexpectedly contribute to perceived instability and require thorough analytical, biophysical, and functional characterization during biopharmaceutical drug product development.

B Cell Activation and Response Regulation During Viral Infections.

Acute viral infections are characterized by rapid increases in viral load, leading to cellular damage and the resulting induction of complex innate and adaptive antiviral immune responses that cause local and systemic inflammation. Successful antiviral immunity requires the activation of many immune cells, including T cells, natural killer cells, and macrophages. B cells play a unique part through their production of antibodies that can both neutralize and clear viral particles before virus entry into a cell. Protective antibodies are produced even before the first exposure of a pathogen, through the regulated secretion of so-called natural antibodies that are generated even in the complete absence of prior microbial exposure. An early wave of rapidly secreted antibodies from extrafollicular (EF) responses draws on the preexisting naive or memory repertoire of B cells to induce a strong protective response that in kinetics tightly follows the clearance of acute infections, such as with influenza virus. Finally, the generation of germinal centers (GCs) provides long-term protection through production of long-lived plasma cells and memory B cells, which shape and broaden the B cell repertoire for more effective responses following repeat exposures. In this study, we review B cell responses to acute viral infections, primarily influenza virus, from the earliest nonspecific B-1 cell to early, antigen-specific EF responses and finally to GC responses. Throughout, we address known factors that lead to distinct B cell response outcomes and discuss how their functions effect viral clearance, highlighting the critical contributions of each response type to the induction of highly protective antiviral humoral immunity.

Specificity of human natural antibodies referred to as anti-Tn.

To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα-Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g., 6-O-Su-GalNAcα and GalNAcα1-3Galß1-3(4)GlcNAcß. The binding with several bacterial polysaccharides that have no structural resemblance to the affinity ligand GalNAcα was quite unexpected. Given that GalNAcα is considered the key fragment of the Tn antigen, it is surprising that these antibodies bind weakly GalNAcα-OSer and do not bind a wide variety of GalNAcα-OSer/Thr-containing mucin glycopeptides. At the same time, we have observed specific binding to cells having Tn-positive glycoproteins containing similar glycopeptide motifs in a conformationally rigid macromolecule. Thus, specific recognition of the Tn antigen apparently requires that the naturally occurring "anti-Tn" IgM recognize a complex epitope comprising the GalNAcα as an essential component and a fairly long amino acid sequence where the amino acids adjacent to GalNAcα do not contact the antibody paratope; i.e., the antibodies recognize a spatial epitope or a molecular pattern rather than a classical continuous sequence. In addition, we have not found any increase in the binding of natural antibodies when GalNAcα residues were clustered. These results may help in further development of anticancer vaccines based on synthetic Tn constructs.

Ras isoforms selectively regulate antigen-specific immune response.

H-/K-Ras and N-Ras isoforms were proposed to lack functional specificities due to similarity in 1-165 amino acids. As recent studies implied Ras isoform-specific developmental effects, we examined their functional specificity using Leishmania major infection, anti-hapten antibody response and carrier-specific T cell response. While N-Ras overexpression increased L. major infection in resistant C57BL/6 mice, H-Ras or K-Ras overexpression reduced the infection in susceptible BALB/c mice. These Ras isoforms differentially regulated anti-TNP antibody response in TNP-Ova-primed, but not in TNP-Ficoll- or TNP-LPS-primed, BALB/c mice. Ras isoform-specific silencing selectively modulated Ova-specific T cell response. The data indicate Ras isoform-specific regulation of antigen-specific immune response.

Fast and Efficient Fc-Specific Photoaffinity Labeling To Produce Antibody-DNA Conjugates.

Antibody-DNA conjugates are powerful tools for DNA-assisted protein analysis. Growing usage of these methods demands efficient production of high-quality conjugates. We developed an easy and fast synthesis route yielding covalent antibody-DNA conjugates with a defined conjugation site and low batch-to-batch variability. We utilize the Z domain from protein A, containing the unnatural amino acid 4-benzoylphenylalanine (BPA) for photoaffinity labeling of the antibodies' Fc region. Z(xBPA) domains are C-terminally modified with triple-glycine (G3)-modified DNA-oligonucleotides via enzymatic Sortase A coupling. We show reliable modification of the most commonly used IgG's. To prove our conjugates' functionality, we detected antibody-antigen binding events in an assay called Droplet Barcode Sequencing for Protein analysis (DBS-Pro). It confirms not only retained functionality for both conjugate parts but also the potential of using DBS-Pro for quantifying protein abundances. As intermediates are easily storable and our approach is modular, it offers a convenient strategy for screening various antibody-DNA conjugates using the same starting material.

Clinical Application Evaluation of a Fourth-Generation HIV Antigen Antibody Combination Screening Assay.

BACKGROUND: Human immunodeficiency virus infection and acquired immunodeficiency syndrome (HIV/AIDS) are infectious diseases with high mortality. Early diagnosis is crucial. Combining HIV antibody and p24 antigen, the Elecsys HIV combi PT assay is a fourth generation HIV screening assay. The sensitivity and specificity of this assay was examined. METHODS: A total of 111,556 samples was conducted from January 1, 2016, to June 30, 2018 in Zhongshan Hospital of Yat-Sen University. We conducted a fourth-generation HIV test of retrospective HIV screening samples and assessed the reliability of using signal-to-cutoff (S/CO) ratios to distinguish false positive HIV antibody reactions and analyzed false positives. RESULTS: A total of 122 specimens were confirmed as HIV-1 infected by western blot (WB) and HIV nucleic acid assays. The median S/CO ratio for HIV false positive specimens was 3.27, while for the HIV-infected specimen it was 391.7. Receiver operating characteristic (ROC) analysis showed that the best diagnostic point for HIV was 22.85 S/CO. The sensitivity, specificity, and Youden index were 100%, 97.8%, and 0.978, respectively. The highest false positive rate of 26.4% was found in patients with malignant tumors and blood diseases. CONCLUSIONS: The results of this study show that the fourth-generation Elecsys HIV combination PT test can identify early HIV infected and can be a useful adjunct to help clinicians to manage the disease by viral load testing and starting an appropriate therapy. Our research data provides a reference for subsequent research and HIV testing in the region.

The autoantibody response to cyclic citrullinated collagen type II peptides in rheumatoid arthritis.

OBJECTIVES: The detection of anti-citrullinated peptide antibodies (ACPAs) is a serological hallmark of RA. Autoantibodies reactive with collagen type II (CII) are present in RA sera and synovial fluid and are potentially pathogenic. Here, we investigate the prevalence and specificity of the autoantibody responses to defined citrullinated cyclic peptides derived from CII in a China RA cohort. METHODS: Using bead-based multiplex assay, we examined the presence of autoantibodies binding to 54 cyclic 17-mer citrullinated CII peptides, encompassing all citrullinate epitopes in CII, and the corresponding unmodified peptides in 415 RA patients, in addition to 304 patients with OA. Furthermore, the autoantibody responses to a selected set of 10 cyclic citrullinated peptides were also examined in 203 healthy individuals. RESULTS: Autoantibody responses to cyclic citrullinated CII peptides were higher in RA patients as compared with OA patients or healthy individuals, whereas little or negligible antibody responses to cyclic unmodified CII peptides were observed. Interestingly, several novel citrullinated CII epitopes were identified. Antibodies to these novel citrullinated CII epitopes showed not only substantial overlapping reactivities but also had unique specificities. CONCLUSION: We found a high prevalence of autoantibodies against cyclic citrullinated CII in the sera of patients in a China RA cohort. The present study revealed heterogeneous binding patterns against novel citrullinated CII epitopes, which may help to stratify RA patients into different subgroups.