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.

Reprogramming the antigen specificity of B cells using genome-editing technologies.

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.

Optimal Sequential Immunization Can Focus Antibody Responses against Diversity Loss and Distraction.

Affinity maturation is a Darwinian process in which B lymphocytes evolve potent antibodies to encountered antigens and generate immune memory. Highly mutable complex pathogens present an immense antigenic diversity that continues to challenge natural immunity and vaccine design. Induction of broadly neutralizing antibodies (bnAbs) against this diversity by vaccination likely requires multiple exposures to distinct but related antigen variants, and yet how affinity maturation advances under such complex stimulation remains poorly understood. To fill the gap, we present an in silico model of affinity maturation to examine two realistic new aspects pertinent to vaccine development: loss in B cell diversity across successive immunization periods against different variants, and the presence of distracting epitopes that entropically disfavor the evolution of bnAbs. We find these new factors, which introduce additional selection pressures and constraints, significantly influence antibody breadth development, in a way that depends crucially on the temporal pattern of immunization (or selection forces). Curiously, a less diverse B cell seed may even favor the expansion and dominance of cross-reactive clones, but only when conflicting selection forces are presented in series rather than in a mixture. Moreover, the level of frustration due to evolutionary conflict dictates the degree of distraction. We further describe how antigenic histories select evolutionary paths of B cell lineages and determine the predominant mode of antibody responses. Sequential immunization with mutationally distant variants is shown to robustly induce bnAbs that focus on conserved elements of the target epitope, by thwarting strain-specific and distracted lineages. An optimal range of antigen dose underlies a fine balance between efficient adaptation and persistent reaction. These findings provide mechanistic guides to aid in design of vaccine strategies against fast mutating pathogens.

Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity.

The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10-6), outperforming other sequence- and structure-based models.

Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity.

A structurally distinct human mycoplasma protein that generically blocks antigen-antibody union.

We report the discovery of a broadly reactive antibody-binding protein (Protein M) from human mycoplasma. The crystal structure of the ectodomain of transmembrane Protein M differs from other known protein structures, as does its mechanism of antibody binding. Protein M binds with high affinity to all types of human and nonhuman immunoglobulin G, predominantly through attachment to the conserved portions of the variable region of the κ and λ light chains. Protein M blocks antibody-antigen union, likely because of its large C-terminal domain extending over the antibody-combining site, blocking entry to large antigens. Similar to the other immunoglobulin-binding proteins such as Protein A, Protein M as well as its orthologs in other Mycoplasma species could become invaluable reagents in the antibody field.

The antigenic evolution of influenza: drift or thrift?

It is commonly assumed that antibody responses against the influenza virus are polarized in the following manner: strong antibody responses are directed at highly variable antigenic epitopes, which consequently undergo 'antigenic drift', while weak antibody responses develop against conserved epitopes. As the highly variable epitopes are in a constant state of flux, current antibody-based vaccine strategies are focused on the conserved epitopes in the expectation that they will provide some level of clinical protection after appropriate boosting. Here, we use a theoretical model to suggest the existence of epitopes of low variability, which elicit a high degree of both clinical and transmission-blocking immunity. We show that several epidemiological features of influenza and its serological and molecular profiles are consistent with this model of 'antigenic thrift', and that identifying the protective epitopes of low variability predicted by this model could offer a more viable alternative to regularly update the influenza vaccine than exploiting responses to weakly immunogenic conserved regions.

Deciphering the molecular bases of the biological effects of antibodies against Interleukin-2: a versatile platform for fine epitope mapping.

Elucidating the network of interactions established by Interleukin-2 is a key step to understanding its role as a master regulator of the immune system. Binding of this cytokine by specific antibodies gives rise to different classes of immune complexes that boost or inhibit immune responses. The molecular bases of such functional dichotomy are likely related to the nature of the recognized epitopes, making it necessary to perform fine epitope mapping studies. The current work was aimed at developing a versatile platform to do so. This was accomplished by display of human and mouse Interleukin-2 on filamentous phages, together with extensive mutagenesis of both antigens and high throughput screening of binding properties of more than 200 variants. Detailed molecular pictures of the epitopes were thus delineated for four antibodies against either human or mouse Interleukin-2, which refined and, in some cases, modified the conclusions derived from previous mapping studies with peptide libraries. Overlapping surface patches on mouse Interleukin-2 that also coincide with the predicted interface between the cytokine and its receptor alpha chain were shown to be recognized by two monoclonal antibodies that promote enhancement of immune responses, shedding new light on the structural bases of their biological activity. Our strategy was powerful enough to reveal multiple binding details and could be used to map the epitopes recognized by other antibodies and to explore additional interactions involving Interleukin-2 and related cytokines, thus contributing to our understanding of the complex structure-function relationships within the immune system.

Dynamic antibody-binding properties in the pathogenesis of HIT.

Rapid laboratory assessment of heparin-induced thrombocytopenia (HIT) is important for disease recognition and management. The utility of contemporary immunoassays to detect antiplatelet factor 4 (PF4)/heparin antibodies is hindered by detection of antibodies unassociated with disease. To begin to distinguish properties of pathogenic anti-PF4/heparin antibodies, we compared isotype-matched monoclonal antibodies that bind to different epitopes: KKO causes thrombocytopenia in an in vivo model of HIT, whereas RTO does not. KKO binding to PF4 and heparin is specifically inhibited by human HIT antibodies that activate platelets, whereas inhibition of RTO binding is not differentially affected. Heparin increased the avidity of KKO binding to PF4 without affecting RTO, but it did not increase total binding or binding to nontetrameric PF4(K50E). Single-molecule forced unbinding demonstrated KKO was 8-fold more reactive toward PF4 tetramers and formed stronger complexes than RTO, but not to PF4(K50E) dimers. KKO, but not RTO, promoted oligomerization of PF4 but not PF4(K50E). This study reveals differences in the properties of anti-PF4 antibodies that cause thrombocytopenia not revealed by ELISA that correlate with oligomerization of PF4 and sustained high-avidity interactions that may simulate transient antibody-antigen interactions in vivo. These differences suggest the potential importance of epitope specificity in the pathogenesis of HIT.

The immune inhibitory complex CD200/CD200R is developmentally regulated in the mouse brain.

The CD200/CD200R inhibitory immune ligand-receptor system regulates microglial activation/quiescence in adult brain. Here, we investigated CD200/CD200R at different stages of postnatal development, when microglial maturation takes place. We characterized the spatiotemporal, cellular, and quantitative expression pattern of CD200 and CD200R in the developing and adult C57/BL6 mice brain by immunofluorescent labeling and Western blotting. CD200 expression increased from postnatal day 1 (P1) to P5-P7, when maximum levels were found, and decreased to adulthood. CD200 was located surrounding neuronal bodies, and very prominently in cortical layer I, where CD200(+) structures included glial fibrillary acidic protein (GFAP)(+) astrocytes until P7. In the hippocampus, CD200 was mainly observed in the hippocampal fissure, where GFAP(+) /CD200(+) astrocytes were also found until P7. CD200(+) endothelium was seen in the hippocampal fissure and cortical blood vessels, notably from P14, showing maximum vascular CD200 in adults. CD200R(+) cells were a population of ameboid/pseudopodic Iba1(+) microglia/macrophages observed at all ages, but significantly decreasing with increasing age. CD200R(+) /Iba1(+) macrophages were prominent in the pial meninges and ventricle lining, mainly at P1-P5. CD200R(+) /Iba1(+) perivascular macrophages were observed in cortical and hippocampal fissure blood vessels, showing maximum density at P7, but being prominent until adulthood. CD200R(+) /Iba1(+) ameboid microglia in the cingulum at P1-P5 were the only CD200R(+) cells in the nervous tissue. In conclusion, the main sites of CD200/CD200R interaction seem to include the molecular layer and pial surface in neonates and blood vessels from P7 until adulthood, highlighting the possible role of the CD200/CD200R system in microglial development and renewal.

Distribution of alarin immunoreactivity in the mouse brain.

Alarin is a 25 amino acid peptide that belongs to the galanin peptide family. It is derived from the galanin-like peptide gene by a splice variant, which excludes exon 3. Alarin was first identified in gangliocytes of neuroblastic tumors and later shown to have a vasoactive function in the skin. Recently, alarin was demonstrated to stimulate food intake as well as the hypothalamic-pituitary-gonadal axis in rodents, suggesting that it might be a neuromodulatory peptide in the brain. However, the individual neurons in the central nervous system that express alarin have not been identified. Here, we determined the distribution of alarin-like immunoreactivity (alarin-LI) in the adult murine brain. The specificity of the antibody against alarin was demonstrated by the absence of labeling after pre-absorption of the antiserum with synthetic alarin peptide and in transgenic mouse brains lacking neurons expressing the GALP gene. Alarin-LI was observed in different areas of the murine brain. A high intensity of alarin-LI was detected in the accessory olfactory bulb, the medial preoptic area, the amygdala, different nuclei of the hypothalamus such as the arcuate nucleus and the ventromedial hypothalamic nucleus, the trigeminal complex, the locus coeruleus, the ventral chochlear nucleus, the facial nucleus, and the epithelial layer of the plexus choroideus. The distinct expression pattern of alarin in the adult mouse brain suggests potential functions in reproduction and metabolism.

A novel laboratory technique demonstrating the influences of RHD zygosity and the RhCcEe phenotype on erythrocyte D antigen expression.

D antigen is the most immunogenic and clinically relevant antigen within the complex Rh blood group system. Variability of D antigen expression was first described decades ago but has rarely been investigated quantitatively, particularly in the context of RHD zygosity along with RhCcEe serological phenotype. With IRB approval, 107 deidentified blood samples were analyzed. Rh phenotypes were determined serologically by saline technique using monoclonal antibodies against D, C, c, E, and e antigens. RHD zygosity was determined using both PCR-restriction fragment length polymorphisms and quantitative real-time PCR techniques. A novel and robust method was developed for quantitation of erythrocyte D antigen sites using calibrated microspheres and flow cytometry, allowing correlation of D antigen density with RHD zygosity and expression of Rh CcEe antigens. Subjects homozygous for RHD expressed nearly twice the number of D antigen sites compared with RHD hemizygotes (33,560 ± 8,222 for DD versus 17,720 ± 4,471 for Dd, P < 0.0001). Expression of c or E antigens was associated with significantly increased erythrocyte D antigen expression, whereas presence of C or e antigens reduced expression. These data and this novel quantitation method will be important for future studies investigating the clinical relevance of D antigen variability.

Antigen-binding abilities of anti-nephrin antibody are prescribed by signal sequence of expression vector in genetic immunization.

BACKGROUND: Nephrin is an essential protein for maintaining the normal structure of podocyte foot processes and the glomerular filtration barrier. To analyze the mechanism of proteinuria and nephrotic syndrome, we previously reported on a new method of producing polyclonal anti-nephrin antibody by genetic immunization. In the present paper, we investigate the effect of signal peptide sequence cDNA on the characteristics of polyclonal anti-nephrin antibodies induced by genetic immunization. METHODS: Five fragments of nephrin cDNA with or without signal peptide sequence were inserted into the pTARGET™ vector. Rats were immunized with these vectors by the gene gun method. Sera obtained from rats at 14 weeks following immunization were analyzed by Western blotting, immunoprecipitation, flow cytometry and immunohistochemistry. RESULTS: Four different antibodies induced by cDNA encoding nephrin protein fragments without signal peptide showed antigen site-specific binding to fragmented glycosylation-disturbed nephrin proteins. These antibodies also reacted to a glycosylation-disturbed full-length nephrin protein (inhibited by tunicamycin), but did not react to either a native nephrin protein or a fully glycosylated conformational nephrin protein. Four different antibodies induced by cDNA encoding nephrin fragments with signal peptide showed an antigen site-specific binding to glycosylation-disturbed nephrin protein fragments. In addition, these antibodies reacted to both a native nephrin protein and a full-length glycosylated conformational nephrin protein. CONCLUSIONS: The absence of signal peptide sequence cDNA in the expression vector produced antibodies specific for glycosylation-disturbed proteins, while its presence produced antibodies that bound to native or fully glycosylated conformational protein.

Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation.

During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.

Combined genotypic and phenotypic killer cell Ig-like receptor analyses reveal KIR2DL3 alleles displaying unexpected monoclonal antibody reactivity: identification of the amino acid residues critical for staining.

In humans, recent clinical and experimental data from hematopoietic stem cell transplantation revealed that donor-derived alloreactive NK cells exert a beneficial graft versus leukemia effect. The existence of donor-derived alloreactive NK cells can be predicted on the basis of donor killer cell Ig-like receptor (KIR) gene profile and HLA class I typing of both donor and recipient. Moreover, the size of the alloreactive NK cell population can be directly assessed by the combined use of anti-KIR-specific mAb. In this study, in an attempt to improve the definition of alloreactive NK cell subsets, we assessed the KIR genotype and phenotype in a cohort of 44 donors. This approach allowed the identification of two different KIR2DL3 alleles (KIR2DL3*005 and the novel allele KIR2DL3*015) that did not react with the anti-KIR2DL3-specific ECM41 mAb. In contrast, both alleles were recognized at the cell surface by several mAb reacting with KIR2DL2/L3/S2. Notably, KIR2DL3*005 was also stained by the anti-KIR2DL1/S1-specific EB6B and 11PB6 mAb. Functional analysis revealed that, despite its particular mAb reactivity, the specificity of KIR2DL3*005 for HLA-C molecules did not differ from that of other KIR2DL2/L3 alleles. Finally, site-directed mutagenesis demonstrated that glutamine at position 35 is required for ECM41 staining, whereas glutamic acid 35 and arginine 50 are relevant for staining with EB6B or 11PB6 mAb. Our present data represent a substantial progress in the characterization of the NK cell repertoire and an improved phenotypic/functional definition of given KIR(+) subsets.

Substitution of certain amino acids in a short peptide causes a significant difference in their immunoreactivities with antibodies against different epitopes: evidence for possible folding of the peptide on a nitrocellulose or PVDF membrane.

Substitution of amino acids in a peptide caused remarkable differences in its immunoreactivities with antibodies against 3 epitopes in the immobilized peptide. The observed differences in immunoreactivities among the peptides were not due to the differences in efficiencies of their transfer onto nitrocellulose or PVDF membranes. Rather, possible folding of the peptide on the membrane was considered to be the reason for their distinct immunoreactivities with the antibodies.

Reverse transfection using antibodies against a cell surface antigen in mammalian adherent cell lines.

Reverse transfection from a solid surface has the potential to deliver genes to various cells more efficiently than conventional methods. However, the effective gene delivery from a solid surface requires an optimized extracellular matrix (ECM) for the coating of glass slides, dependent on the nature of the cells. In a search for an appropriate substrate for the universal application to multiple types of cell, we focused on cell surface antigens and examined the effects of antibodies raised against them on gene transfer from an antibody-coated surface. We found that a coating of CD29-specific antibody allowed the most effective delivery of genes by reverse transfection in every type of cell that we examined. Our results suggest that reverse transfection with antibodies against CD29 might provide a universal tool for gene delivery and cell array-based analyses.

Mass spectrometric approaches for elucidation of antigenantibody recognition structures in molecular immunology.

Mass spectrometric approaches have recently gained increasing access to molecular immunology and several methods have been developed that enable detailed chemical structure identification of antigen-antibody interactions. Selective proteolytic digestion and MS-peptide mapping (epitope excision) has been successfully employed for epitope identification of protein antigens. In addition, "affinity proteomics" using partial epitope excision has been developed as an approach with unprecedented selectivity for direct protein identification from biological material. The potential of these methods is illustrated by the elucidation of a beta-amyloid plaque-specific epitope recognized by therapeutic antibodies from transgenic mouse models of Alzheimer's disease. Using an immobilized antigen and antibody-proteolytic digestion and analysis by high resolution Fourier transform ion cyclotron resonance mass spectrometry has lead to a new approach for the identification of antibody paratope structures (paratope-excision; "parex-prot"). In this method, high resolution MS-peptide data at the low ppm level are required for direct identification of paratopes using protein databases. Mass spectrometric epitope mapping and determination of "molecular antibody-recognition signatures" offer high potential, especially for the development of new molecular diagnostics and the evaluation of new vaccine lead structures.