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Introduction: VHHs (VH of heavy-chain-only antibodies) represent a unique alternative to Q7 conventional antibodies because of their smaller size, comparable binding affinity and biophysical properties. Method: In this study, we systematically analyzed VHH NGS sequences from 22 Alpacas and structure data from public database. Results: VHHs in Alpaca can be grouped into five main types with multiple distinct sequence and structure features. Based on the existence of hallmark residues in FR2 region, VHHs can be classified into two groups: nonclassical VHHs (without hallmark residues) and classical VHHs (with hallmark residues). Based on VHH hallmark residues at 42 position (IMGT numbering, FR2 region) and number of cysteines, we found that Alpaca classical VHHs can be further separated into three main types: F_C2 VHHs with F (phenylalanine) at position 42 and having 2 cysteines within sequences, Y_C2 VHHs with Y (tyrosine) at position 42 and having 2 cysteines, and F_C4 with F at position 42 and having 4 cysteines. Non-classical VHHs can be further separated into 2 types based on germlines mapped: N_V3 for VHHs mapped to V3 germlines and N_V4 for V4 germlines. Based on whether FR2 residues are involved in binding, two kinds of paratopes can be identified. Different types of VHHs showed distinct associations with these two paratopes and displayed significant differences in paratope size, residue usage and other structure features. Discussion: Such results will have significant implications in VHH discovery, engine e ring, and design for innovative therapeutics. [ABSTRACT FROM AUTHOR]

We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody–protein interactions by using hydrogen–deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody. [ABSTRACT FROM AUTHOR]

Antibodies, through their ability to target virtually any epitope, play a key role in driving the adaptive immune response in jawed vertebrates. The binding domains of standard antibodies are their variable light (VL) and heavy (VH) domains, both of which present analogous complementarity-determining region (CDR) loops. It has long been known that the VH CDRs contribute more heavily to the antigenbinding surface (paratope), with the CDR-H3 loop providing a major modality for the generation of diverse paratopes. Here, we provide evidence for an additional role of the VL domain as a modulator of CDR-H3 structure, using a diverse set of antibody crystal structures and a large set of molecular dynamics simulations. We show that specific attributes of the VL domain such as subtypes, CDR canonical forms and genes can influence the structural diversity of the CDR-H3 loop, and provide a physical model for how this effect occurs through inter-loop contacts and packing of CDRs against each other. Our results indicate that the rigid minor loops fine-tune the structure of CDR-H3, thereby contributing to the generation of surfaces complementary to the vast number of possible epitope topologies, and provide insights into the interdependent nature of CDR conformations, an understanding of which is important for the rational antibody design process. [ABSTRACT FROM AUTHOR]

Antibodies are the largest class of biotherapeutics. However, in recent years, single-domain antibodies have gained traction due to their smaller size and comparable binding affinity. Antibodies (Abs) and single-domain antibodies (sdAbs) differ in the structures of their binding sites: most significantly, singledomain antibodies lack a light chain and so have just three CDR loops. Given this inherent structural difference, it is important to understand whether Abs and sdAbs are distinguishable in how they engage a binding partner and thus, whether they are suited to different types of epitopes. In this study, we use non-redundant sequence and structural datasets to compare the paratopes, epitopes and antigen interactions of Abs and sdAbs. We demonstrate that even though sdAbs have smaller paratopes, they target epitopes of equal size to those targeted by Abs. To achieve this, the paratopes of sdAbs contribute more interactions per residue than the paratopes of Abs. Additionally, we find that conserved framework residues are of increased importance in the paratopes of sdAbs, suggesting that they include non-specific interactions to achieve comparable affinity. Furthermore, the epitopes of sdAbs are only marginally less accessible than those of Abs: we posit that this may be explained by differences in the orientation and compaction of sdAb and Ab CDR-H3 loops. Overall, our results have important implications for the engineering and humanization of sdAbs, as well as the selection of the best modality for targeting a particular epitope. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a +sense single-strand RNA virus. The virus has four major surface proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), respectively. The constitutive proteins present a high grade of symmetry. Identifying a binding site is difficult. The virion is approximately 50–200 nm in diameter. Angiotensin-converting enzyme 2 (ACE2) acts as the cell receptor for the virus. SARS-CoV-2 has an increased affinity to human ACE2 compared with the original SAR strain. Topological space, and its symmetry, is a critical component in molecular interactions. By exploring this space, a suitable ligand space can be characterized accordingly. A spike protein (S) computational model in a complex with ACE 2 was generated using silica methods. Topological spaces were probed using high computational throughput screening techniques to identify and characterize the topological space of both SARS and SARS-CoV-2 spike protein and its ligand space. In order to identify the symmetry clusters, computational analysis techniques, together with statistical analysis, were utilized. The computations are based on crystallographic protein data bank PDB-based models of constitutive proteins. Cartesian coordinates of component atoms and some cluster maps were generated and analyzed. Dihedral angles were used in order to compute a topological receptor space. This computational study uses a multimodal representation of spike protein interactions with some fragment proteins. The chemical space of the receptors (a dimensional volume) suggests the relevance of the receptor as a drug target. The spike protein S of SARS and SARS-CoV-2 is analyzed and compared. The results suggest a mirror symmetry of SARS and SARS-CoV-2 spike proteins. The results show thatSARS-CoV-2 space is variable and has a distinct topology. In conclusion, surface proteins grant virion variability and symmetry in interactions with a potential complementary target (protein, antibody, ligand). The mirror symmetry of dihedral angle clusters determines a high specificity of the receptor space. [ABSTRACT FROM AUTHOR]

CD59 is an abundant immuno-regulatory human protein that protects cells from damage by inhibiting the complement system. CD59 inhibits the assembly of the Membrane Attack Complex (MAC), the bactericidal pore-forming toxin of the innate immune system. In addition, several pathogenic viruses, including HIV-1, escape complement-mediated virolysis by incorporating this complement inhibitor in their own viral envelope. This makes human pathogenic viruses, such as HIV-1, not neutralised by the complement in human fluids. CD59 is also overexpressed in several cancer cells to resist the complement attack. Consistent with its importance as a therapeutical target, CD59-targeting antibodies have been proven to be successful in hindering HIV-1 growth and counteracting the effect of complement inhibition by specific cancer cells. In this work, we make use of bioinformatics and computational tools to identify CD59 interactions with blocking antibodies and to describe molecular details of the paratope–epitope interface. Based on this information, we design and produce paratope-mimicking bicyclic peptides able to target CD59. Our results set the basis for the development of antibody-mimicking small molecules targeting CD59 with potential therapeutic interest as complement activators. [ABSTRACT FROM AUTHOR]

Several human monoclonal Abs for treating Influenza have been evaluated in clinical trials with limited success despite demonstrating superiority in preclinical animal models including mice. To conduct efficacy studies in mice, human monoclonal Abs are genetically engineered to contain mouse heavy chain constant domain to facilitate the engagement of Fc-receptors on mouse immune effector cells. Although studies have consistently reported discrepancies in Ab effectiveness following genetic engineering, the structural and mechanistic basis for these inconsistencies remain uncharacterized. Here, we use homology modeling to predict variable region (VR) analogous monoclonal Abs possessing human IgG1, mouse IgG1, and mouse IgG2a heavy chain constant domains. We then examine predicted 3D structures for variations in the spatial location and orientation of corresponding paratope amino acid residues. By structurally aligning crystal structures of Fabs in complex with hemagglutinin (HA), we show that corresponding paratope amino acid residues for VR-analogous human IgG1, mouse IgG1, and mouse IgG2a monoclonal Abs interact differentially with HA suggesting that their epitopes might not be identical. To demonstrate that variations in the paratope 3D fine architecture have implications for Ab specificity and effectiveness, we genetically engineered VR-analogous human IgG1, human IgG4, mouse IgG1, and mouse IgG2a monoclonal Abs and explored their specificity and effectiveness in protecting MDCK cells from infection by pandemic H1N1 and H3N2 Influenza viruses. We found that VR-analogous monoclonal Abs placed on mouse heavy chain constant domains were more efficacious at protecting MDCK cells from Influenza virus infection relative to those on human heavy chain constant domains. Interestingly, mouse but not human heavy chain constant domains increased target breadth in some monoclonal Abs. These data suggest that heavy chain constant domain sequences play a role in shaping Ab repertoires that go beyond class or sub-class differences in immune effector recruitment. This represents a facet of Ab biology that can potentially be exploited to improve the scope and utilization of current therapeutic or prophylactic candidates for influenza. [ABSTRACT FROM AUTHOR]

Combinatorial biology methods such as phage and yeast display, suitable for the generation and screening of huge numbers of protein fragments and mutated variants, have been useful when dissecting the molecular details of the interactions between antibodies and their target antigens (mainly those of protein nature). The relevance of these studies goes far beyond the mere description of binding interfaces, as the information obtained has implications for the understanding of the chemistry of antibody–antigen binding reactions and the biological effects of antibodies. Further modification of the interactions through combinatorial methods to manipulate the key properties of antibodies (affinity and fine specificity) can result in the emergence of novel research tools and optimized therapeutics. [ABSTRACT FROM AUTHOR]

The dynamic interplay between virus and host plays out across many interacting surfaces as virus and host evolve continually in response to one another. In particular, epitope-paratope interactions (EPIs) between viral antigen and host antibodies drive much of this evolutionary race. In this review, we describe a series of recent studies examining aspects of epitope complexity that go beyond two interacting protein surfaces as EPIs are typically understood. To structure our discussion, we present a framework for understanding epitope complexity as a spectrum along a series of axes, focusing primarily on 1) epitope biochemical complexity (e.g., epitopes involving N-glycans) and 2) antigen conformational/dynamic complexity (e.g., epitopes with differential properties depending on antigen state or fold-axis). We highlight additional epitope complexity factors including epitope tertiary/quaternary structure, which contribute to epistatic relationships between epitope residues within- or adjacent-to a given epitope, as well as epitope overlap resulting from polyclonal antibody responses, which is relevant when assessing antigenic pressure against a given epitope. Finally, we discuss how these different forms of epitope complexity can limit EPI analyses and therapeutic antibody development, as well as recent efforts to overcome these limitations. [ABSTRACT FROM AUTHOR]

Antibody‐mediated enzyme formation is a phenomenon first described in 1968 and further studied by molecular Immunologists and Biochemists over the following five decades. The present review is made mainly by analyzing the 27 articles concerned with AMEF that appeared over the course of 47 years, commenting 16 original figures selected to be re‐printed in AMEF's Legacy. We, the reviewers, started by revisiting our own "insider's" experience of discovery, and followed by considering all results, our own and of members of other AMEF Labs. We had planned to conclude the review by correlating the various AMEF mutants to a detailed knowledge of the consensus betaGal structure. However, we became aware of several "robust" papers, published between 1989 and 2014, by authors outside of AMEF Labs. We familiarly called this surge: "The Second Wave" and adorned it with a doodle in Hokusai style. We were thrilled and happy to take them on board and properly examined their data. A team of this second wave had imagined unique uses for AMEF, and new doors to modern biotechnology. Another one had used AMEF as Tool and Marker to attain high levels of crystallography, solving puzzles of conformation, and ultimate structure. Together, they doubled our motivation to review AMEF. Serendipity gives us back the pleasure of finding, a treat at any age. [ABSTRACT FROM AUTHOR]

Background: A conformational epitope (CE) is composed of neighboring amino acid residues located on an antigenic protein surface structure. CEs bind their complementary paratopes in B-cell receptors and/or antibodies. An effective and efficient prediction tool for CE analysis is critical for the development of immunology-related applications, such as vaccine design and disease diagnosis. Results: We propose a novel method consisting of two sequential modules: matching and prediction. The matching module includes two main approaches. The first approach is a complete sequence search (CSS) that applies BLAST to align the sequence with all known antigen sequences. Fragments with high epitope sequence identities are identified and the predicted residues are annotated on the query structure. The second approach is a spiral vector search (SVS) that adopts a novel surface spiral feature vector for large-scale surface patch detection when queried against a comprehensive epitope database. The prediction module also contains two proposed subsystems. The first system is based on knowledge-based energy and geometrical neighboring residue contents, and the second system adopts combinatorial features, including amino acid contents and physicochemical characteristics, to formulate corresponding geometric spiral vectors and compare them with all spiral vectors from known CEs. An integrated testing dataset was generated for method evaluation, and our two searching methods effectively identified all epitope regions. The prediction results show that our proposed method outperforms previously published systems in terms of sensitivity, specificity, positive predictive value, and accuracy. Conclusions: The proposed method significantly improves the performance of traditional epitope prediction. Matching followed by prediction is an efficient and effective approach compared to predicting directly on specific surfaces containing antigenic characteristics. [ABSTRACT FROM AUTHOR]

B-cells can neutralize pathogenic molecules by targeting them with extreme specificity using receptors secreted or expressed on their surface (antibodies). This is achieved via molecular interactions between the paratope (i.e., the antibody residues involved in the binding) and the interacting region (epitope) of its target molecule (antigen). Discerning the rules that define this specificity would have profound implications for our understanding of humoral immunogenicity and its applications. The aim of this work is to produce improved, antibody-specific epitope predictions by exploiting features derived from the antigens and their cognate antibodies structures, and combining them using statistical and machine learning algorithms. We have identified several geometric and physicochemical features that are correlated in interacting paratopes and epitopes, used them to develop a Monte Carlo algorithm to generate putative epitopes-paratope pairs, and train a machine-learning model to score them. We show that, by including the structural and physicochemical properties of the paratope, we improve the prediction of the target of a given B-cell receptor. Moreover, we demonstrate a gain in predictive power both in terms of identifying the cognate antigen target for a given antibody and the antibody target for a given antigen, exceeding the results of other available tools. [ABSTRACT FROM AUTHOR]

The ability of the human immune system to generate antibodies to any given antigen can be strongly influenced by immunoglobulin V-gene allelic polymorphisms. However, previous studies have provided only limited examples. Therefore, the prevalence of this phenomenon has been unclear. By analyzing >1,000 publicly available antibody-antigen structures, we show that many V-gene allelic polymorphisms in antibody paratopes are determinants for antibody binding activity. Biolayer interferometry experiments further demonstrate that paratope allelic polymorphisms on both heavy and light chains often abolish antibody binding. We also illustrate the importance of minor V-gene allelic polymorphisms with low frequency in several broadly neutralizing antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Overall, this study not only highlights the pervasive impact of V-gene allelic polymorphisms on antibody binding but also provides mechanistic insights into the variability of antibody repertoires across individuals, which in turn have important implications for vaccine development and antibody discovery. [Display omitted] • Analysis of V-gene allelic polymorphisms in 1,048 antibody-antigen complex structures • Many antibodies contain allelic polymorphisms predicted to disrupt binding • These include antibodies with different V genes and to different pathogens • Some broadly neutralizing antibodies depend on minor V-gene allelic polymorphisms By analyzing >1,000 publicly available antibody-antigen complex structures, Yuan et al. demonstrate that antibody binding activity is often influenced by V-gene allelic polymorphisms. This result provides mechanistic insights into the variability of antibody repertoires across individuals, which in turn have important implications for vaccine development and antibody discovery. [ABSTRACT FROM AUTHOR]

Microcontact printing (µCP) is a practical and versatile approach to create nanostructured patterns of biomolecular probes, but it involves conformational changes on the patterned bioreceptors that often lead to a loss on the biological activity of the resulting structures. Herein we introduce indirect µCP to create functional patterns of bioreceptors on solid substrates. This is a simple strategy that relies on physisorbing biomolecular probes of interest in the nanostructured gaps that result after patterning backfilling agents by standard µCP. This study presents the approach, assesses bovine serum albumin as backfilling agent for indirect µCP on different materials, reports the limitations of standard µCP on the functionality of patterned antibodies, and demonstrates the capabilities of indirect µCP to solve this issue. Bioreceptors were herein structured as diffractive gratings and used to measure biorecognition events in label-free conditions. Besides, as a preliminary approach towards sensing biomarkers, this work also reports the implementation of indirect µCP in an immunoassay to detect human immunoglobulin E. [ABSTRACT FROM AUTHOR]

Nanobodies (Nbs) are a class of antigen-binding protein derived from camelid immune systems, which achieve equivalent binding affinities and specificities to classical antibodies (Abs) despite being comprised of only a single variable domain. Here, we use a data set of 156 unique Nb:antigen complex structures to characterize Nb–antigen binding and draw comparison to a set of 156 unique Ab:antigen structures. We analyse residue composition and interactions at the antigen interface, together with structural features of the paratopes of both data sets. Our analysis finds that the set of Nb structures displays much greater paratope diversity, in terms of the structural segments involved in the paratope, the residues used at these positions to contact the antigen and furthermore the type of contacts made with the antigen. Our findings suggest a different relationship between contact propensity and sequence variability from that observed for Ab VH domains. The distinction between sequence positions that control interaction specificity and those that form the domain scaffold is much less clear-cut for Nbs, and furthermore H3 loop positions play a much more dominant role in determining interaction specificity. [ABSTRACT FROM AUTHOR]

Since the death of Jean Genet, his name and oeuvre have been the subject of heated debate. Influential critics have argued that Genet was an aristocratic anti-Semite, rather than a revolutionary poet who took sides with the outcasts. In this article, I analyze the positions, patterns, and strategies of this multifaceted debate, suggesting that provocation and marginalization constitute an integral part of Genet's aesthetics. In the act of judging Genet from historical, political, and ethical perspectives, the critics operate as executors of his literary project, confirming the paratopic position the writer presumably desired. [ABSTRACT FROM AUTHOR]

Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process. [ABSTRACT FROM AUTHOR]

Affinity maturation is associated with reduced malleability of the paratope that optimizes an antibody to bind to the bonafide antigen with high specificity and affinity. However, it has been illustrated that mature antibodies tend to exhibit promiscuity despite acquisition of a relatively rigid binding pocket. Such an attribute is contrary to the established paradigm of specificity in antigen recognition. In this review, an explicit dissection of the underlying mechanisms fostering such versatility in mature antibodies has been done. Polyspecificity is essentially achieved by undergoing minimal structural rearrangement at the paratope complemented with plasticity in interaction with antigen. Besides, the structural invariance of the antigen across species could modulate mature antibody specificity. Polyreactivity has been well documented for germline antibodies as broad spectrum antibody repertoire amplification is primarily governed by recombination event of the genetic machinery, which is further expanded at the structural and functional level of interaction. Degenerate specificity in antigen recognition obviates the need to produce distinct antibody for every incoming epitope © 2015 IUBMB Life, 67(7):498-505, 2015 [ABSTRACT FROM AUTHOR]

The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories. However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens. Here, we review the important contributions that protein crystallography has made so far to vaccine research and development. We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors. We cover the critical role of high-resolution epitope mapping by reviewing structures of complexes between antigens and their cognate neutralizing, or protective, antibody fragments. Most importantly, we provide recent examples where structural insights obtained via protein crystallography have been used to design novel optimized vaccine antigens. This review aims to illustrate the value of protein crystallography in the emerging discipline of structural vaccinology and its impact on the rational design of vaccines. [ABSTRACT FROM AUTHOR]

ABSTRACT The identification of immunogenic regions on the surface of antigens, which are able to stimulate an immune response, is a major challenge for the design of new vaccines. Computational immunology aims at predicting such regions-in particular B-cell epitopes-but is far from being reliably applicable on a large scale. To gain understanding into the factors that contribute to the antigen-antibody affinity and specificity, we perform a detailed analysis of the amino acid composition and secondary structure of antigen and antibody surfaces, and of the interactions that stabilize the complexes, in comparison with the composition and interactions observed in other heterodimeric protein interfaces. We make a distinction between linear and conformational B-cell epitopes, according to whether they consist of successive residues along the polypeptide chain or not. The antigen-antibody interfaces were shown to differ from other protein-protein interfaces by their smaller size, their secondary structure with less helices and more loops, and the interactions that stabilize them: more H-bond, cation-π, amino-π, and π-π interactions, and less hydrophobic packing; linear and conformational epitopes can clearly be distinguished. Often, chains of successive interactions, called cation/amino-π and π-π chains, are formed. The amino acid composition differs significantly between the interfaces: antigen-antibody interfaces are less aliphatic and more charged, polar and aromatic than other heterodimeric protein interfaces. Moreover, paratopes and epitopes-albeit to a lesser extent-have amino acid compositions that are distinct from general protein surfaces. This specificity holds promise for improving B-cell epitope prediction. Proteins 2014; 82:1734-1746. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Characterization of interactions between antigens and antibodies is of utmost importance both for fundamental understanding of the binding and for development of advanced clinical diagnostics. Here, fluorescence line-narrowing (FLN) spectroscopy was used to study physicochemical interactions between 3-hydroxybenzo[ a]pyrene (3OH-BaP, as antigen) and a variety of solvent matrices (as model systems) or anti-polycyclic aromatic hydrocarbon antibodies (anti-PAH). We focused the studies on the specific physicochemical interactions between 3OH-BaP and different, previously obtained, monoclonal and recombinant anti-PAH antibodies. Control experiments performed with non-binding monoclonal antibodies and bovine serum albumin (BSA) indicated that nonspecific interactions did not affect the FLN spectrum of 3OH-BaP. The spectral positions and relative intensities of the bands in the FLN spectra are highly dependent on the molecular environment of the 3OH-BaP. The FLN bands correlate with different vibrational modes of 3OH-BaP which are affected by interactions with the molecular environment (π-π interactions, H-bonding, or van-der-Waals forces). Although the analyte (3OH-BaP) was the same for all the antibodies investigated, different binding interactions could be identified from the FLN spectra on the basis of structural flexibility and conformational multiplicity of the antibodies' paratopes. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The function of antibodies (Abs) involves specific binding to antigens (Ags) and activation of other components of the immune system to fight pathogens.The six hypervariable loops within the variable domains of Abs, commonly termed complementarity determining regions (CDRs), are widely assumed to be responsible for Ag recognition, while the constant domains are believed to mediate effector activation. Recent studies and analyses of the growing number of available Ab structures, indicate that this clear functional separation between the two regions may be an oversimplification. Some positions within the CDRs have been shown to never participate in Ag binding and some off-CDRs residues often contribute critically to the interaction with the Ag. Moreover, there is now growing evidence for non-local and even allosteric effects in Ab-Ag interaction in which Ag binding affects the constant region and vice versa. This review summarizes and discusses the structural basis of Ag recognition, elaborating on the contribution of different structural determinants of the Ab to Ag binding and recognition. We discuss the CDRs, the different approaches for their identification and their relationship to the Ag interface. We also review what is currently known about the contribution of non-CDRs regions to Ag recognition, namely the framework regions (FRs) and the constant domains.The suggested mechanisms by which these regions contribute to Ag binding are discussed. On the Ag side of the interaction, we discuss attempts to predict B-cell epitopes and the suggested idea to incorporate Ab information into B-cell epitope prediction schemes. Beyond improving the understanding of immunity, characterization of the functional role of different parts of the Ab molecule may help in Ab engineering, design of CDR-derived peptides, and epitope prediction. [ABSTRACT FROM AUTHOR]

The primary immunogenic component of the currently approved anthrax vaccine is the protective antigen (PA) unit of the binary toxin system. PA-specific antibodies neutralize anthrax toxins and protect against infection. Recent research has determined that in humans, only antibodies specific for particular determinants are capable of effecting toxin neutralization, and that the neutralizing epitopes recognized by these antibodies are distributed throughout the PA monomer. The mechanisms by which the majority of these epitopes effect neutralization remain unknown. In this report we investigate the process by which a human monoclonal antibody specific for the amino-terminal domain of PA neutralizes lethal toxin in an in vitro assay of cytotoxicity, and find that it neutralizes LT by blocking the requisite cleavage of the amino-terminal 20 kD portion of the molecule (PA20) from the remainder of the PA monomer. We also demonstrate that the epitope recognized by this human monoclonal does not encompass the 166RKKR169 furin recognition sequence in domain 1 of PA. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus (SARS-CoV) caused a worldwide epidemic in late 2002/early 2003 and a second outbreak in the winter of 2003/2004 by an independent animal-to-human transmission. The GD03 strain, which was isolated from an index patient of the second outbreak, was reported to resist neutralization by the human monoclonal antibodies (hmAbs) 80R and 53.1. which can potently neutralize isolates from the first outbreak. Here we report that two hmAbs, m396 and 5230.15, potently neutralized GD03 and representative isolates from the first SARS outbreak (Urbani, Tor2) and from palm civets (SZ3, SZ16). These antibodies also protected mice challenged with the Urbani or recombinant viruses bearing the GD03 and SZ16 spike (S) glycoproteins. Both antibodies competed with the SARS-CoV receptor, ACE2. for binding to the receptor-binding domain (RBD), suggesting a mechanism of neutralization that involves interference with the SARS-CoV-ACE2 interaction. Two putative hot-spot residues in the RBD (Ile-489 and Tyr-491) were identified within the SARS-CoV spike that likely contribute to most of the m396-binding energy. Residues Ile-489 and Tyr-491 are highly conserved within the SARS-CoV spike, indicating a possible mechanism of the m396 cross-reactivity. Sequence analysis and mutagenesis data show that m396 might neutralize all zoonotic and epidemic SARS-CoV isolates with known sequences, except strains derived from bats. These antibodies exhibit cross-reactivity against isolates from the two SARS outbreaks and palm civets and could have potential applications for diagnosis, prophylaxis, and treatment of SARS-CoV infections. [ABSTRACT FROM AUTHOR]

The new antigen receptor (IgNAR) antibodies from sharks are disulphide bonded dimers of two protein chains, each containing one variable and five constant domains. Three types of IgNAR variable domains have been discovered, with Type 3 appearing early in shark development and being overtaken by the antigen-driven affinity-matured Type 1 and 2 response. Here, we have determined the first structure of a naturally occurring Type 2 IgNAR variable domain, and identified the disulphide bond that links and stabilizes the CDR1 and CDR3 loops. This disulphide bridge locks the CDR3 loop in an 'upright' conformation in contrast to other shark antibody structures, where a more lateral configuration is observed. Further, we sought to model the Type 3 isotype based on the crystallographic structure reported here. This modeling indicates (1) that internal Type 3-specific residues combine to pack into a compact immunoglobulin core that supports the CDR loop regions, and (2) that despite apparent low-sequence variability, there is sufficient plasticity in the CDR3 loop to form a conformationally diverse antigen-binding surface. [ABSTRACT FROM AUTHOR]

The evolution of mutations in SARS-CoV-2 at antigenic sites that impact neutralizing antibody responses in humans poses a risk to immunity developed through vaccination and natural infection. The highly successful RNA-based vaccines have enabled rapid vaccine updates that incorporate mutations from current variants of concern (VOCs). It is therefore important to anticipate future antigenic mutations as the virus navigates the heterogeneous global landscape of host immunity. Toward this goal, we survey epitope-paratope interfaces of anti-SARS-CoV-2 antibodies to map an antigenic space that captures the role of each spike protein residue within the polyclonal antibody response directed against the ACE2-receptor binding domain (RBD) or the N-terminal domain (NTD). In particular, the antigenic space map builds on recently published epitope definitions by annotating epitope overlap and orthogonality at the residue level. We employ the antigenic space map as a framework to understand how mutations on nine major variants contribute to each variant's evasion of neutralizing antibodies. Further, we identify constellations of mutations that span the orthogonal epitope regions of the RBD and NTD on the variants with the greatest antibody escape. Finally, we apply the antigenic space map to predict which regions of antigenic space—should they mutate—may be most likely to complementarily augment antibody evasion for the most evasive and transmissible VOCs. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) is a global health burden that causes acute and chronic hepatitis. To develop an HBV-neutralizing antibody that effectively prevents HBV infection, we previously generated a human anti-preS1 monoclonal antibody (1A8) that binds to genotypes A–D and validated its HBV-neutralizing activity in vitro. In the present study, we aimed to determine the fine epitope and paratope of 1A8 to understand the mechanism of HBV neutralization. We performed alanine-scanning mutagenesis on the preS1 (aa 19–34, genotype C) and the heavy (HCDR) and light (LCDR) chain complementarity-determining regions. The 1A8 recognized the three residues (Leu22, Gly23, and Phe25) within the highly conserved receptor-binding motif (NPLGFFP) of the preS1, while four CDR residues of 1A8 were critical in antigen binding. Structural analysis of the epitope–paratope interaction by molecular modeling revealed that Leu100 in the HCDR3, Ala50 in the HCDR2, and Tyr96 in the LCDR3 closely interacted with Leu22, Gly23, and Phe25 of the preS1. Additionally, we found that 1A8 also binds to the receptor-binding motif (NPLGFLP) of infrequently occurring HBV. The results suggest that 1A8 may broadly and effectively block HBV entry and thus have potential as a promising candidate for the prevention and treatment of HBV infection. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Prediction of antibody-antigen binding is a central question in immunology • A motif vocabulary of paratope-epitope interactions governs antibody specificity • Proof of principle that antibody-antigen binding is predictable • Implications for de novo antibody and (neo-)epitope design Prediction of antibody-antigen binding is a central question in immunology and of high relevance for predictive antibody and vaccine design. Akbar et al. prove the predictability of antibody-antigen binding by discovering a universal, compact, and immunity-specific motif vocabulary of paratope-epitope interactions. [ABSTRACT FROM AUTHOR]

At the 10th Human Genome Mapping (HGM10) Workshop, in New Haven, for the first time, immunoglobulin (IG) or antibody and T cell receptor (TR) variable (V), diversity (D), joining (J), and constant (C) genes were officially recognized as 'genes', as were the conventional genes. Under these HGM auspices, IMGT®, the international ImMunoGeneTics information system®, was created in June 1989 at Montpellier (University of Montpellier and CNRS). The creation of IMGT® marked the birth of immunoinformatics, a new science, at the interface between immunogenetics and bioinformatics. The accuracy and the consistency between genes and alleles, sequences, and three-dimensional (3D) structures are based on the IMGT Scientific chart rules generated from the IMGT-ONTOLOGY axioms and concepts: IMGT standardized keywords (IDENTIFICATION), IMGT gene and allele nomenclature (CLASSIFICATION), IMGT standardized labels (DESCRIPTION), IMGT unique numbering and IMGT Collier de Perles (NUMEROTATION). These concepts provide IMGT® immunoinformatics insights for antibody V and C domain structure and function, used for the standardized description in IMGT® web resources, databases and tools, immune repertoires analysis, single cell and/or high-throughput sequencing (HTS, NGS), antibody humanization, and antibody engineering in relation with effector properties. [ABSTRACT FROM AUTHOR]

Background: Antibody, the primary effector molecule of the immune system, evolves after initial encounter with the antigen from a precursor form to a mature one to effectively deal with the antigen. Antibodies of a lineage diverge through antigen-directed isolated pathways of maturation to exhibit distinct recognition potential. In the context of evolution in immune recognition, diversity of antigen cannot be ignored. While there are reports on antibody lineage, structural perspective with respect to diverse recognition potential in a lineage has never been studied. Hence, it is crucial to evaluate how maturation leads to topological tailoring within a lineage enabling them to interact with significantly distinct antigens. Results: A data-driven approach was undertaken for the study. Global experimental mouse and human antibody-antigen complex structures from PDB were compiled into a coherent database of germline-linked antibodies bound with distinct antigens. Structural analysis of all lineages showed variations in CDRs of both H and L chains. Observations of conformational adaptation made from analysis of static structures were further evaluated by characterizing dynamics of interaction in two lineages, mouse VH1–84 and human VH5–51. Sequence and structure analysis of the lineages explained that somatic mutations altered the geometries of individual antibodies with common structural constraints in some CDRs. Additionally, conformational landscape obtained from molecular dynamics simulations revealed that incoming pathogen led to further conformational divergence in the paratope (as observed across datasets) even while maintaining similar overall backbone topology. MM-GB/SA analysis showed binding energies to be in physiological range. Results of the study are coherent with experimental observations. Conclusions: The findings of this study highlight basic structural principles shaping the molecular evolution of a lineage for significantly diverse antigens. Antibodies of a lineage follow different developmental pathways while preserving the imprint of the germline. From the study, it can be generalized that structural diversification of the paratope is an outcome of natural selection of a conformation from an available ensemble, which is further optimized for antigen interaction. The study establishes that starting from a common lineage, antibodies can mature to recognize a wide range of antigens. This hypothesis can be further tested and validated experimentally. [ABSTRACT FROM AUTHOR]