Dielectric nanohole array metasurface for high-resolution near-field sensing and imaging.

Dielectric metasurfaces support resonances that are widely explored both for far-field wavefront shaping and for near-field sensing and imaging. Their design explores the interplay between localised and extended resonances, with a typical trade-off between Q-factor and light localisation; high Q-factors are desirable for refractive index sensing while localisation is desirable for imaging resolution. Here, we show that a dielectric metasurface consisting of a nanohole array in amorphous silicon provides a favourable trade-off between these requirements. We have designed and realised the metasurface to support two optical modes both with sharp Fano resonances that exhibit relatively high Q-factors and strong spatial confinement, thereby concurrently optimizing the device for both imaging and biochemical sensing. For the sensing application, we demonstrate a limit of detection (LOD) as low as 1 pg/ml for Immunoglobulin G (IgG); for resonant imaging, we demonstrate a spatial resolution below 1 µm and clearly resolve individual E. coli bacteria. The combined low LOD and high spatial resolution opens new opportunities for extending cellular studies into the realm of microbiology, e.g. for studying antimicrobial susceptibility.

Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies.

Neutralizing monoclonal antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent promising candidates for clinical intervention against coronavirus disease 2019 (COVID-19). We isolated a large number of nAbs from SARS-CoV-2-infected individuals capable of disrupting proper interaction between the receptor binding domain (RBD) of the viral spike (S) protein and the receptor angiotensin converting enzyme 2 (ACE2). However, the structural basis for their potent neutralizing activity remains unclear. Here, we report cryo-EM structures of the ten most potent nAbs in their native full-length IgG-form or in both IgG-form and Fab-form bound to the trimeric S protein of SARS-CoV-2. The bivalent binding of the full-length IgG is found to associate with more RBDs in the "up" conformation than the monovalent binding of Fab, perhaps contributing to the enhanced neutralizing activity of IgG and triggering more shedding of the S1 subunit from the S protein. Comparison of a large number of nAbs identified common and unique structural features associated with their potent neutralizing activities. This work provides a structural basis for further understanding the mechanism of nAbs, especially through revealing the bivalent binding and its correlation with more potent neutralization and the shedding of S1 subunit.

First case report of monoclonal IgG3-heavy-chain glomerulonephritis with microtubular structures.

Our patient was a 69-year-old woman admitted to our hospital for heavy proteinuria and hematuria. A renal biopsy showed findings similar to those of membranoproliferative glomerulonephritis associated with nodular lesions, and immunofluorescence showed marked deposits of IgG, C1q, and C3 on the peripheral capillary walls. IgG3 alone was observed on IgG subclass staining with no κ or λ light chains, and Congo red staining was negative. These findings suggested IgG3-heavy-chain deposition disease (HCDD). However, we did not find a deletion of the first heavy-chain constant domain, which is commonly observed in HCDD. Electron microscopy showed randomly arranged subendothelial microtubular structures with diameters of 70-90 nm. Altogether, the diagnosis of HCDD could not be made, although monoclonal IgG3 deposits in glomeruli were observed. This is the first case report of monoclonal IgG3-heavy-chain glomerulonephritis with subendothelial-based, randomly arranged microtubular structures with diameters of 70-90 nm.

Role of NMR in High Ordered Structure Characterization of Monoclonal Antibodies.

Obtaining high ordered structure (HOS) information is of importance to guarantee the efficacy and safety of monoclonal antibodies (mAbs) in clinical application. Assessment of HOS should ideally be performed in a non-invasive manner under their formulated storage conditions, as any perturbation can introduce unexpected detritions. However, most of the currently available techniques only indirectly report HOS of mAbs and/or require a certain condition to conduct the analyses. Besides, the flexible multidomain architecture of mAbs has hampered atomic-resolution structural analyses using X-ray crystallography and cryo-electron microscopy. In contrast, the ability of nuclear magnetic resonance (NMR) spectroscopy to structurally analyze biomolecules in various conditions in a non-invasive and quantitative manner is suitable to meet the needs. However, the application of NMR to mAbs is not straightforward due to the high molecular weight of the system. In this review, we will discuss how NMR techniques have been applied to HOS analysis of mAbs, along with the recent advances of the novel 15N direct detection NMR strategy that allows for obtaining the structural fingerprint of mAbs at lower temperatures under multiple formulation conditions. The potential application of these NMR strategies will benefit next-generation mAbs, such as antibody-drug conjugates and bispecific antibodies.

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies.

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

Capturing transient antibody conformations with DNA origami epitopes.

Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immunoglobulin Gs (IgGs) at room temperature. The DNA origami epitopes (DOEs) allows programmed spatial distribution of epitope spikes, which enables direct imaging of functional complexes with atomic force microscopy (AFM). We establish the critical dependence of the IgG avidity on the lateral distance of epitopes within 3-20 nm at the single-molecule level. High-speed AFM imaging of transient conformations further provides structural and dynamic evidence for the IgG avidity from monovalent to bivalent in a single event, which sheds light on various applications including virus neutralization, diagnostic detection and cancer immunotherapy.

Antibody-mediated complement activation in pathology and protection.

Antibody-dependent complement activity is associated not only with autoimmune morbidity, but also with antitumor efficacy. In infectious disease, both recombinant monoclonal antibodies and polyclonal antibodies generated in natural adaptive responses can mediate complement activity to protective, therapeutic or disease-enhancing effect. Recent advances have contributed to the structural resolution of molecular complexes involved in antibody-mediated complement activation, defining the avid nature of participating interactions and pointing to how antibody isotype, subclass, hinge flexibility, glycosylation state, amino acid sequence and the contextual nature of the cognate antigen/epitope are all factors that can determine complement activity through impact on antibody multimerization and subsequent recruitment of complement component 1q. Beyond the efficiency of activation, complement activation products interact with various cell types that mediate immune adherence, trafficking, immune education and innate functions. Similarly, depending on the anatomical location and extent of activation, complement can support homeostatic restoration or be leveraged by pathogens or neoplasms to enhance infection or promote tumorigenic microenvironments, respectively. Advances in means to suppress complement activation by intravenous immunoglobulin (IVIG), IVIG mimetics and complement-intervening antibodies represent proven and promising exploratory therapeutic strategies, while antibody engineering has likewise offered frameworks to enhance, eliminate or isolate complement activation to interrogate in vivo mechanisms of action. Such strategies promise to support the optimization of antibody-based drugs that are able to tackle emerging and difficult-to-treat diseases by improving our understanding of the synergistic and antagonistic relationships between antibody mechanisms mediated by Fc receptors, direct binding and the products of complement activation.

Molecular docking explores heightened immunogenicity and structural dynamics of acetaldehyde human immunoglobulin G adduct.

Acetaldehyde is a metabolite of ethanol, an important constituent of tobacco pyrolysis and the aldehydic product of lipid peroxidation. Acetaldehyde induced toxicity is mainly due to its binding to cellular macromolecules resulting in the formation of stable adducts accompanied by oxidative stress. The aim of this study was to characterize structural and immunological alterations in human immunoglobulin G (IgG) modified with acetaldehyde in the presence of sodium borohydride, a reducing agent. The IgG modifications were studied by various physicochemical techniques such as fluorescence and CD spectroscopy, free amino group estimation, 2,2-azobis 2-amidinopropane (AAPH) induced red blood cell hemolysis as well as transmission electron microscopy. Molecular docking was also employed to predict the preferential binding of acetaldehyde to IgG. The immunogenicity of native and acetaldehyde-modified IgG was investigated by immunizing female New Zealand white rabbits using native and modified IgG as antigens. Binding specificity and cross reactivity of rabbit antibodies was screened by competitive inhibition ELISA and band shift assays. The modification of human IgG with acetaldehyde results in quenching of the fluorescence of tyrosine residues, decrease in free amino group content, a change in the antioxidant property as well as formation of cross-linked structures in human IgG. Molecular docking reveals strong binding of IgG to acetaldehyde. Moreover, acetaldehyde modified IgG induced high titer antibodies (>1:12800) in the experimental animals. The antibodies exhibited high specificity in competitive binding assay toward acetaldehyde modified human IgG. The results indicate that acetaldehyde induces alterations in secondary and tertiary structure of IgG molecule that leads to formation of neo-epitopes on IgG that enhances its immunogenicity.

The solution structure of the human IgG2 subclass is distinct from those for human IgG1 and IgG4 providing an explanation for their discrete functions.

Human IgG2 antibody displays distinct therapeutically-useful properties compared with the IgG1, IgG3, and IgG4 antibody subclasses. IgG2 is the second most abundant IgG subclass, being able to bind human FcγRII/FcγRIII but not to FcγRI or complement C1q. Structural information on IgG2 is limited by the absence of a full-length crystal structure for this. To this end, we determined the solution structure of human myeloma IgG2 by atomistic X-ray and neutron-scattering modeling. Analytical ultracentrifugation disclosed that IgG2 is monomeric with a sedimentation coefficient (s20, w0) of 7.2 S. IgG2 dimer formation was ≤5% and independent of the buffer conditions. Small-angle X-ray scattering in a range of NaCl concentrations and in light and heavy water revealed that the X-ray radius of gyration (Rg ) is 5.2-5.4 nm, after allowing for radiation damage at higher concentrations, and that the neutron Rg value of 5.0 nm remained unchanged in all conditions. The X-ray and neutron distance distribution curves (P(r)) revealed two peaks, M1 and M2, that were unchanged in different buffers. The creation of >123,000 physically-realistic atomistic models by Monte Carlo simulations for joint X-ray and neutron-scattering curve fits, constrained by the requirement of correct disulfide bridges in the hinge, resulted in the determination of symmetric Y-shaped IgG2 structures. These molecular structures were distinct from those for asymmetric IgG1 and asymmetric and symmetric IgG4 and were attributable to the four hinge disulfides. Our IgG2 structures rationalize the existence of the human IgG1, IgG2, and IgG4 subclasses and explain the receptor-binding functions of IgG2.

Architecture of cell-cell adhesion mediated by sidekicks.

Cell-cell adhesion is important for cell growth, tissue development, and neural network formation. Structures of cell adhesion molecules have been widely studied by crystallography, revealing the molecular details of adhesion interfaces. However, due to technical limitations, the overall structure and organization of adhesion molecules at cell adhesion interfaces has not been fully investigated. Here, we combine electron microscopy and other biophysical methods to characterize the structure of cell-cell adhesion mediated by the cell adhesion molecule Sidekick (Sidekick-1 and Sidekick-2) and obtain 3D views of the Sidekick-mediated adhesion interfaces as well as the organization of Sidekick molecules between cell membranes by electron tomography. The results suggest that the Ig-like domains and the fibronectin III (FnIII) domains of Sidekicks play different roles in cell adhesion. The Ig-like domains mediate the homophilic transinteractions bridging adjacent cells, while the FnIII domains interact with membranes, resulting in a tight adhesion interface between cells that may contribute to the specificity and plasticity of cell-cell contacts during cell growth and neural development.

Structures of C1-IgG1 provide insights into how danger pattern recognition activates complement.

Danger patterns on microbes or damaged host cells bind and activate C1, inducing innate immune responses and clearance through the complement cascade. How these patterns trigger complement initiation remains elusive. Here, we present cryo-electron microscopy analyses of C1 bound to monoclonal antibodies in which we observed heterogeneous structures of single and clustered C1-immunoglobulin G1 (IgG1) hexamer complexes. Distinct C1q binding sites are observed on the two Fc-CH2 domains of each IgG molecule. These are consistent with known interactions and also reveal additional interactions, which are supported by functional IgG1-mutant analysis. Upon antibody binding, the C1q arms condense, inducing rearrangements of the C1r2s2 proteases and tilting C1q's cone-shaped stalk. The data suggest that C1r may activate C1s within single, strained C1 complexes or between neighboring C1 complexes on surfaces.

Characterization of methylglyoxal-modified human IgG by physicochemical methods.

Human IgG is a defence protein and quite reactive to dicarbonyls. In this study, methylglyoxal-induced modification of IgG was examined by various biochemical and biophysical methods. The methylglyoxal-induced changes in IgG were monitored by UV-visible and fluorescence spectroscopy, Fourier transform infrared spectroscopy, 1-anilinonaphthalene-8-sulfonic acid (ANS), and thermal denaturation studies. Aggregate formation was studied by Thioflavin T (ThT), Congo red (CR) and scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Spectroscopic studies suggested gross changes in MGO-modified IgG. Fluorogenic AGEs appeared during modification and the MGO-modified IgG gained thermostability. The reaction produced oxidative stress in the medium because carbonyl content increased manifold and sulfhydryl groups decreased. Enhanced binding of the MGO-modified IgG by Congo red and Thioflavin T suggests crosslinking and aggregation. This was supported by SEM and TEM results.

Glycation, oxidation and glycoxidation of IgG: a biophysical, biochemical, immunological and hematological study.

Glycation and oxidation induce structural alterations in the proteins in an interdependent manner with consequent pathological implications. The published literature presents wide range of modifications in conformational characteristics of proteins by glycation and oxidation; however, there is little data that could elaborate the cumulative effect of both the processes. This study has analysed the modifications in IgG by methylglyoxal (MG) (glycative stress), hydroxyl radical ([Formula: see text]) (oxidative stress) and by their combined action i.e. [Formula: see text] treatment of MG glycated IgG (glycoxidation). It further addresses the implications of the altered structural integrity of IgG on its immunological characteristics and impact on haematological parameters in rabbits. Using circular dichroism, FTIR, SDS-PAGE analysis, thioflavin-T fluorescence assay, congo red absorbance analysis, dynamic light scattering, transmission electron microscopy, ELISA, blood cell counts and rectal temperature studies, we report that the glycoxidative modification caused maximum alteration in the IgG as compared to the glycatively and oxidatively modified protein. Far-UV CD results confirmed the highest decline in the beta-pleated sheet content of the protein by glycoxidation. The damage led to the reduced flexibility and enhanced electronic interactions in IgG as observed by near-UV CD. Modifications caused cross-linking and adduct formation in the serum protein. The electron micrograph confirmed amorphous aggregation in modified IgG. The modifications increased the hydrodynamic radius of IgG by allowing the attachment of [Formula: see text] and MG residues. The glycoxidatively modified IgG induced the maximum antibody titres that showed high specificity towards the altered IgG. The glycoxidation of IgG leads to activation of inflammatory pathways.

Determining the Spatial Relationship of Membrane-Bound Aquaporin-4 Autoantibodies by STED Nanoscopy.

Determining the spatial relationship of individual proteins in dense assemblies remains a challenge for superresolution nanoscopy. The organization of aquaporin-4 (AQP4) into large plasma membrane assemblies provides an opportunity to image membrane-bound AQP4 antibodies (AQP4-IgG) and evaluate changes in their spatial distribution due to alterations in AQP4 isoform expression and AQP4-IgG epitope specificity. Using stimulated emission depletion nanoscopy, we imaged secondary antibody labeling of monoclonal AQP4-IgGs with differing epitope specificity bound to isolated tetramers (M1-AQP4) and large orthogonal arrays of AQP4 (M23-AQP4). Imaging secondary antibodies bound to M1-AQP4 allowed us to infer the size of individual AQP4-IgG binding events. This information was used to model the assembly of larger AQP4-IgG complexes on M23-AQP4 arrays. A scoring algorithm was generated from these models to characterize the spatial arrangement of bound AQP4-IgG antibodies, yielding multiple epitope-specific patterns of bound antibodies on M23-AQP4 arrays. Our results delineate an approach to infer spatial relationships within protein arrays using stimulated emission depletion nanoscopy, offering insight into how information on single antibody fluorescence events can be used to extract information from dense protein assemblies under a biologic context.

Antibodies: From novel repertoires to defining and refining the structure of biologically important targets.

Antibodies represent a highly successful class of molecules that bind a wide-range of targets in therapeutic-, diagnostic- and research-based applications. The antibody repertoire is composed of the building blocks required to develop an effective adaptive immune response against foreign insults. A number of species have developed novel genetic and structural mechanisms from which they derive these antibody repertoires, however, traditionally antibodies are isolated from human, and rodent sources. Due to their high-value therapeutic, diagnostic, biotechnological and research applications, much innovation has resulted in techniques and approaches to isolate novel antibodies. These approaches are bolstered by advances in our understanding of species immune repertoires, next generation sequencing capacity, combinatorial antibody discovery and high-throughput screening. Structural determination of antibodies and antibody-antigen complexes has proven to be pivotal to our current understanding of the immune repertoire for a range of species leading to advances in man-made libraries and fine tuning approaches to develop antibodies from immune-repertoires. Furthermore, the isolation of antibodies directed against antigens of importance in health, disease and developmental processes, has yielded a plethora of structural and functional insights. This review highlights the significant contribution of antibody-based crystallography to our understanding of adaptive immunity and its application to providing critical information on a range of human-health related indications.

Simulation and Theory of Antibody Binding to Crowded Antigen-Covered Surfaces.

In this paper we introduce a fully flexible coarse-grained model of immunoglobulin G (IgG) antibodies parametrized directly on cryo-EM data and simulate the binding dynamics of many IgGs to antigens adsorbed on a surface at increasing densities. Moreover, we work out a theoretical model that allows to explain all the features observed in the simulations. Our combined computational and theoretical framework is in excellent agreement with surface-plasmon resonance data and allows us to establish a number of important results. (i) Internal flexibility is key to maximize bivalent binding, flexible IgGs being able to explore the surface with their second arm in search for an available hapten. This is made clear by the strongly reduced ability to bind with both arms displayed by artificial IgGs designed to rigidly keep a prescribed shape. (ii) The large size of IgGs is instrumental to keep neighboring molecules at a certain distance (surface repulsion), which essentially makes antigens within reach of the second Fab always unoccupied on average. (iii) One needs to account independently for the thermodynamic and geometric factors that regulate the binding equilibrium. The key geometrical parameters, besides excluded-volume repulsion, describe the screening of free haptens by neighboring bound antibodies. We prove that the thermodynamic parameters govern the low-antigen-concentration regime, while the surface screening and repulsion only affect the binding at high hapten densities. Importantly, we prove that screening effects are concealed in relative measures, such as the fraction of bivalently bound antibodies. Overall, our model provides a valuable, accurate theoretical paradigm beyond existing frameworks to interpret experimental profiles of antibodies binding to multi-valent surfaces of different sorts in many contexts.

Immunoglobulin G Expression in Human Sperm and Possible Functional Significance.

Immunoglobulin G (IgG), the major molecule of the immune system, which was traditionally thought to be produced by differentiated B-lymphocytes, had recently been found in non-immune cells including spermatozoa of rabbit testis. To study if human sperms could produce IgG that might play a role in fertilization, we employed immunofluorescent staining, Western blot, in situ hybridization, RT-PCR (reverse transcription polymerase chain reaction) and immunoelectron microscope and found that human sperms were capable of synthesizing IgG. IgG protein and mRNA were detected in the cytoplasm, mainly the neck region of the sperm and IgG immunoreactivity was found to cover the entire sperm cell. The essential enzymes necessary for IgG synthesis and class switching, RAG1 (recombination activating gene 1), RAG2 (recombination activating gene 2) and AID (activation-induced cytidine deaminase), were also detected in the sperm cells. Furthermore, we found that anti-IgG antibody could inhibit sperm from penetrating Zona-free hamster egg with statistical significance. These discoveries suggested that immunoglobulin G could be produced by human sperms and it might play a role during fertilization.

Proteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects.

The glucose-regulated protein94 (Grp94) has been found in complexes with IgG in plasma of Type 1 (T1) diabetic subjects; however, the pathogenetic meaning of Grp94-IgG complexes has not yet been elucidated. To shed light on the nature and structure of these complexes in vivo, we conducted a proteomic analysis on plasma of both T1 diabetic subjects and healthy control subjects. IgG purified from plasma was submitted to 2D PAGE followed by Western blotting and mass analysis. Grp94 was detected in plasma of all diabetic but not control subjects and found linked with its N-terminus to the IgG heavy chain. Mass analysis of heavy chain of IgG that binds Grp94 also in vitro, forming stable complexes with characteristics similar to those of native ones, permitted identifying CH2 and CH3 regions as those involved in binding Grp94. At the electron microscopy, IgG from diabetic plasma appeared as fibrils of various lengthes and dimensions, suggestive of elevated aggregating tendency conferred to IgG by Grp94. The nonimmune nature of complexes turned out to be responsible for the particular stability and structure adopted by complexes in plasma of diabetic subjects. Results are of relevance to understanding the pathogenetic mechanisms underlying diabetes and its complications.

3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography.

Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1-3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.

Atomic force microscopy analysis of IgG films at hydrophobic surfaces: a promising method to probe IgG orientations and optimize ELISA tests performance.

IgG films are widely used in the field of immunoassays, especially in (double) antibody-sandwich ELISA tests where capture antibodies are coated on surfaces like polystyrene or hydrophobic self-assembled monolayers (h-SAMs). It is critical to analyze-at a molecular scale and under liquid conditions-the structure of the deposited IgG film in order to quantitatively address the efficiency of the ELISA test in terms of antigen detection. In this communication, we report an atomic force microscopy (AFM) analysis evidencing a strong relationship between immunological activities of mouse monoclonal anti-human interleukin-2 (IL-2) and 6 (IL-6) antibodies, thickness and roughness of the IgG monolayer adsorbed onto h-SAMs, and surface concentration of IgG molecules. Indirect information may be further obtained on antibody orientation. Collating the results obtained by AFM and those from ELISA tests leads us to conclude that antibodies like anti-IL-6 forming flat monolayers should be more efficient under ELISA detection conditions. In addition, the concentration of IgG in the coating suspension should be optimized to obtain a monolayer heavily populated by "end-on" adsorbed molecules, an orientation that is desirable for enhancing ELISA tests performance.