Proscan: a structure-based proline design web server.

The ability to control protein conformations and dynamics through structure-based design has been useful in various scenarios, including engineering of viral antigens for vaccines. One effective design strategy is the substitution of residues to proline amino acids, which due to its unique cyclic side chain can favor and rigidify key backbone conformations. To provide the community with a means to readily identify and explore proline designs for target proteins of interest, we developed the Proscan web server. Proscan provides assessment of backbone angles, energetic and deep learning-based favorability scores, and other parameters for proline substitutions at each position of an input structure, along with interactive visualization of backbone angles and candidate substitution sites on structures. It identifies known favorable proline substitutions for viral antigens, and was benchmarked against datasets of proline substitution stability effects from deep mutational scanning and thermodynamic measurements. This tool can enable researchers to identify and prioritize designs for prospective vaccine antigen targets, or other designs to favor stability of key protein conformations. Proscan is available at: https://proscan.ibbr.umd.edu.

TCRmodel2: high-resolution modeling of T cell receptor recognition using deep learning.

The cellular immune system, which is a critical component of human immunity, uses T cell receptors (TCRs) to recognize antigenic proteins in the form of peptides presented by major histocompatibility complex (MHC) proteins. Accurate definition of the structural basis of TCRs and their engagement of peptide-MHCs can provide major insights into normal and aberrant immunity, and can help guide the design of vaccines and immunotherapeutics. Given the limited amount of experimentally determined TCR-peptide-MHC structures and the vast amount of TCRs within each individual as well as antigenic targets, accurate computational modeling approaches are needed. Here, we report a major update to our web server, TCRmodel, which was originally developed to model unbound TCRs from sequence, to now model TCR-peptide-MHC complexes from sequence, utilizing several adaptations of AlphaFold. This method, named TCRmodel2, allows users to submit sequences through an easy-to-use interface and shows similar or greater accuracy than AlphaFold and other methods to model TCR-peptide-MHC complexes based on benchmarking. It can generate models of complexes in 15 minutes, and output models are provided with confidence scores and an integrated molecular viewer. TCRmodel2 is available at https://tcrmodel.ibbr.umd.edu.

Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate.

SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.

Structural insights into protection against a SARS-CoV-2 spike variant by T cell receptor diversity.

T cells play a crucial role in combatting SARS-CoV-2 and forming long-term memory responses to this coronavirus. The emergence of SARS-CoV-2 variants that can evade T cell immunity has raised concerns about vaccine efficacy and the risk of reinfection. Some SARS-CoV-2 T cell epitopes elicit clonally restricted CD8+ T cell responses characterized by T cell receptors (TCRs) that lack structural diversity. Mutations in such epitopes can lead to loss of recognition by most T cells specific for that epitope, facilitating viral escape. Here, we studied an HLA-A2-restricted spike protein epitope (RLQ) that elicits CD8+ T cell responses in COVID-19 convalescent patients characterized by highly diverse TCRs. We previously reported the structure of an RLQ-specific TCR (RLQ3) with greatly reduced recognition of the most common natural variant of the RLQ epitope (T1006I). Opposite to RLQ3, TCR RLQ7 recognizes T1006I with even higher functional avidity than the WT epitope. To explain the ability of RLQ7, but not RLQ3, to tolerate the T1006I mutation, we determined structures of RLQ7 bound to RLQ-HLA-A2 and T1006I-HLA-A2. These complexes show that there are multiple structural solutions to recognizing RLQ and thereby generating a clonally diverse T cell response to this epitope that assures protection against viral escape and T cell clonal loss.

Design of a native-like secreted form of the hepatitis C virus E1E2 heterodimer.

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

T cell receptors employ diverse strategies to target a p53 cancer neoantigen.

Adoptive cell therapy with tumor-specific T cells can mediate durable cancer regression. The prime target of tumor-specific T cells are neoantigens arising from mutations in self-proteins during malignant transformation. To understand T cell recognition of cancer neoantigens at the atomic level, we studied oligoclonal T cell receptors (TCRs) that recognize a neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by the major histocompatibility complex class I molecule HLA-A2. We previously reported the structures of three p53R175H-specific TCRs (38-10, 12-6, and 1a2) bound to p53R175H and HLA-A2. The structures showed that these TCRs discriminate between WT and mutant p53 by forming extensive interactions with the R175H mutation. Here, we report the structure of a fourth p53R175H-specific TCR (6-11) in complex with p53R175H and HLA-A2. In contrast to 38-10, 12-6, and 1a2, TCR 6-11 makes no direct contacts with the R175H mutation, yet is still able to distinguish mutant from WT p53. Structure-based in silico mutagenesis revealed that the 60-fold loss in 6-11 binding affinity for WT p53 compared to p53R175H is mainly due to the higher energetic cost of desolvating R175 in the WT p53 peptide during complex formation than H175 in the mutant. This indirect strategy for preferential neoantigen recognition by 6-11 is fundamentally different from the direct strategies employed by other TCRs and highlights the multiplicity of solutions to recognizing p53R175H with sufficient selectivity to mediate T cell killing of tumor but not normal cells.

An Antigenically Diverse, Representative Panel of Envelope Glycoproteins for Hepatitis C Virus Vaccine Development.

BACKGROUND & AIMS: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays. METHODS: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps. RESULTS: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones. CONCLUSIONS: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.

CoV3D: a database of high resolution coronavirus protein structures.

SARS-CoV-2, the etiologic agent of COVID-19, exemplifies the general threat to global health posed by coronaviruses. The urgent need for effective vaccines and therapies is leading to a rapid rise in the number of high resolution structures of SARS-CoV-2 proteins that collectively reveal a map of virus vulnerabilities. To assist structure-based design of vaccines and therapeutics against SARS-CoV-2 and other coronaviruses, we have developed CoV3D, a database and resource for coronavirus protein structures, which is updated on a weekly basis. CoV3D provides users with comprehensive sets of structures of coronavirus proteins and their complexes with antibodies, receptors, and small molecules. Integrated molecular viewers allow users to visualize structures of the spike glycoprotein, which is the major target of neutralizing antibodies and vaccine design efforts, as well as sets of spike-antibody complexes, spike sequence variability, and known polymorphisms. In order to aid structure-based design and analysis of the spike glycoprotein, CoV3D permits visualization and download of spike structures with modeled N-glycosylation at known glycan sites, and contains structure-based classification of spike conformations, generated by unsupervised clustering. CoV3D can serve the research community as a centralized reference and resource for spike and other coronavirus protein structures, and is available at: https://cov3d.ibbr.umd.edu.

High-throughput modeling and scoring of TCR-pMHC complexes to predict cross-reactive peptides.

MOTIVATION: The binding of T-cell receptors (TCRs) to their target peptide MHC (pMHC) ligands initializes the cell-mediated immune response. In autoimmune diseases such as multiple sclerosis, the TCR erroneously recognizes self-peptides as foreign and activates an immune response against healthy cells. Such responses can be triggered by cross-recognition of the autoreactive TCR with foreign peptides. Hence, it would be desirable to identify such foreign-antigen triggers to provide a mechanistic understanding of autoimmune diseases. However, the large sequence space of foreign antigens presents an obstacle in the identification of cross-reactive peptides. RESULTS: Here, we present an in silico modeling and scoring method which exploits the structural properties of TCR-pMHC complexes to predict the binding of cross-reactive peptides. We analyzed three mouse TCRs and one human TCR isolated from a patient with multiple sclerosis. Cross-reactive peptides for these TCRs were previously identified via yeast display coupled with deep sequencing, providing a robust dataset for evaluating our method. Modeling query peptides in their associated TCR-pMHC crystal structures, our method accurately selected the top binding peptides from sets containing more than a hundred thousand unique peptides. AVAILABILITY AND IMPLEMENTATION: Analyses were performed using custom Python and R scripts available at https://github.com/weng-lab/antigen-predict. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Structural and energetic profiling of SARS-CoV-2 receptor binding domain antibody recognition and the impact of circulating variants.

The SARS-CoV-2 pandemic highlights the need for a detailed molecular understanding of protective antibody responses. This is underscored by the emergence and spread of SARS-CoV-2 variants, including Alpha (B.1.1.7) and Delta (B.1.617.2), some of which appear to be less effectively targeted by current monoclonal antibodies and vaccines. Here we report a high resolution and comprehensive map of antibody recognition of the SARS-CoV-2 spike receptor binding domain (RBD), which is the target of most neutralizing antibodies, using computational structural analysis. With a dataset of nonredundant experimentally determined antibody-RBD structures, we classified antibodies by RBD residue binding determinants using unsupervised clustering. We also identified the energetic and conservation features of epitope residues and assessed the capacity of viral variant mutations to disrupt antibody recognition, revealing sets of antibodies predicted to effectively target recently described viral variants. This detailed structure-based reference of antibody RBD recognition signatures can inform therapeutic and vaccine design strategies.

Structure-Based Design of Hepatitis C Virus E2 Glycoprotein Improves Serum Binding and Cross-Neutralization.

An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of broadly neutralizing antibodies (bNAbs) in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.IMPORTANCE Hepatitis C virus infects approximately 1% of the world's population, and no vaccine is currently available. Due to the high variability of HCV and its ability to actively escape the immune response, a goal of HCV vaccine design is to induce neutralizing antibodies that target conserved epitopes. Here, we performed structure-based design of several epitopes of the HCV E2 envelope glycoprotein to engineer its antigenic properties. Designs were tested in vitro and in vivo, demonstrating alteration of the E2 antigenic profile in several cases, and one design led to improvement of cross-neutralization of heterologous viruses. This represents a proof of concept that rational engineering of HCV envelope glycoproteins can be used to modulate E2 antigenicity and optimize a vaccine for this challenging viral target.

Broadly neutralizing antibodies from an individual that naturally cleared multiple hepatitis C virus infections uncover molecular determinants for E2 targeting and vaccine design.

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1-6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1-6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.

In Vivo and In Vitro Potency of Polyphosphazene Immunoadjuvants with Hepatitis C Virus Antigen and the Role of Their Supramolecular Assembly.

Two well-defined synthetic polyphosphazene immunoadjuvants, PCPP and PCEP, were studied for their ability to potentiate the immune response to the hepatitis C virus (HCV) E2 glycoprotein antigen in vivo. We report that PCEP induced significantly higher serum neutralization and HCV-specific IgG titers in mice compared to other adjuvants used in the study: PCPP, Alum, and Addavax. PCEP also shifted the response toward the desirable balanced Th1/Th2 immunity, as evaluated by the antibody isotype ratio (IgG2a/IgG1). The in vivo results were analyzed in the context of antigen-adjuvant molecular interactions in the system and in vitro immunostimulatory activity of formulations. Asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) analysis showed that both PCPP and PCEP spontaneously self-assemble with the E2 glycoprotein with the formation of multimeric water-soluble complexes, which demonstrates the role of polyphosphazene macromolecules as vaccine delivery vehicles. Intrinsic in vitro immunostimulatory activity of polyphosphazene adjuvants, which was assessed using a mouse macrophage cell line, revealed comparable activities of both polymers and did not provide an explanation of their in vivo performance. However, PCEP complexes with E2 displayed greater stability against agglomeration and improved in vitro immunostimulatory activity compared to those of PCPP, which is in line with superior in vivo performance of PCEP. The results emphasize the importance of often neglected antigen-polyphosphazene self-assembly mechanisms in formulations, which can provide important insights on their in vivo behavior and facilitate the establishment of a structure-activity relationship for this important class of immunoadjuvants.

Geometrical characterization of T cell receptor binding modes reveals class-specific binding to maximize access to antigen.

Recognition of antigenic peptides bound to major histocompatibility complex (MHC) proteins by αß T cell receptors (TCRs) is a hallmark of T cell mediated immunity. Recent data suggest that variations in TCR binding geometry may influence T cell signaling, which could help explain outliers in relationships between physical parameters such as TCR-pMHC binding affinity and T cell function. Traditionally, TCR binding geometry has been described with simple descriptors such as the crossing angle, which quantifies what has become known as the TCR's diagonal binding mode. However, these descriptors often fail to reveal distinctions in binding geometry that are apparent through visual inspection. To provide a better framework for relating TCR structure to T cell function, we developed a comprehensive system for quantifying the geometries of how TCRs bind peptide/MHC complexes. We show that our system can discern differences not clearly revealed by more common methods. As an example of its potential to impact biology, we used it to reveal differences in how TCRs bind class I and class II peptide/MHC complexes, which we show allow the TCR to maximize access to and "read out" the peptide antigen. We anticipate our system will be of use in not only exploring these and other details of TCR-peptide/MHC binding interactions, but also addressing questions about how TCR binding geometry relates to T cell function, as well as modeling structural properties of class I and class II TCR-peptide/MHC complexes from sequence information. The system is available at https://tcr3d.ibbr.umd.edu/tcr_com or for download as a script.

Antigenicity and Immunogenicity of Differentially Glycosylated Hepatitis C Virus E2 Envelope Proteins Expressed in Mammalian and Insect Cells.

The development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic, and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex- and high-mannose-type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose-type glycans. Mass spectrometry demonstrated that all 11 predicted N-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but that N-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains A to E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than did HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specific N-glycans proximal to these epitopes.IMPORTANCE The development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein.

TCR3d: The T cell receptor structural repertoire database.

SUMMARY: T cell receptors (TCRs) are critical molecules of the adaptive immune system, capable of recognizing diverse antigens, including peptides, lipids and small molecules, and represent a rapidly growing class of therapeutics. Determining the structural and mechanistic basis of TCR targeting of antigens is a major challenge, as each individual has a vast and diverse repertoire of TCRs. Despite shared general recognition modes, diversity in TCR sequence and recognition represents a challenge to predictive modeling and computational techniques being developed to predict antigen specificity and mechanistic basis of TCR targeting. To this end, we have developed the TCR3d database, a resource containing all known TCR structures, with a particular focus on antigen recognition. TCR3d provides key information on antigen binding mode, interface features, loop sequences and germline gene usage. Users can interactively view TCR complex structures, search sequences of interest against known structures and sequences, and download curated datasets of structurally characterized TCR complexes. This database is updated on a weekly basis, and can serve the community as a centralized resource for those studying T cell receptors and their recognition. AVAILABILITY AND IMPLEMENTATION: The TCR3d database is available at https://tcr3d.ibbr.umd.edu/.

TCRmodel: high resolution modeling of T cell receptors from sequence.

T cell receptors (TCRs), along with antibodies, are responsible for specific antigen recognition in the adaptive immune response, and millions of unique TCRs are estimated to be present in each individual. Understanding the structural basis of TCR targeting has implications in vaccine design, autoimmunity, as well as T cell therapies for cancer. Given advances in deep sequencing leading to immune repertoire-level TCR sequence data, fast and accurate modeling methods are needed to elucidate shared and unique 3D structural features of these molecules which lead to their antigen targeting and cross-reactivity. We developed a new algorithm in the program Rosetta to model TCRs from sequence, and implemented this functionality in a web server, TCRmodel. This web server provides an easy to use interface, and models are generated quickly that users can investigate in the browser and download. Benchmarking of this method using a set of nonredundant recently released TCR crystal structures shows that models are accurate and compare favorably to models from another available modeling method. This server enables the community to obtain insights into TCRs of interest, and can be combined with methods to model and design TCR recognition of antigens. The TCRmodel server is available at: http://tcrmodel.ibbr.umd.edu/.

Peptide-MHC (pMHC) binding to a human antiviral T cell receptor induces long-range allosteric communication between pMHC- and CD3-binding sites.

T cells generate adaptive immune responses mediated by the T cell receptor (TCR)-CD3 complex comprising an αß TCR heterodimer noncovalently associated with three CD3 dimers. In early T cell activation, αß TCR engagement by peptide-major histocompatibility complex (pMHC) is first communicated to the CD3 signaling apparatus of the TCR-CD3 complex, but the underlying mechanism is incompletely understood. It is possible that pMHC binding induces allosteric changes in TCR conformation or dynamics that are then relayed to CD3. Here, we carried out NMR analysis and molecular dynamics (MD) simulations of both the α and ß chains of a human antiviral TCR (A6) that recognizes the Tax antigen from human T cell lymphotropic virus-1 bound to the MHC class I molecule HLA-A2. We observed pMHC-induced NMR signal perturbations in the TCR variable (V) domains that propagated to three distinct sites in the constant (C) domains: 1) the Cß FG loop projecting from the Vß/Cß interface; 2) a cluster of Cß residues near the Cß αA helix, a region involved in interactions with CD3; and 3) the Cα AB loop at the membrane-proximal base of the TCR. A biological role for each of these allosteric sites is supported by previous mutational and functional studies of TCR signaling. Moreover, the pattern of long-range, ligand-induced changes in TCR A6 revealed by NMR was broadly similar to that predicted by the MD simulations. We propose that the unique structure of the TCR ß chain enables allosteric communication between the TCR-binding sites for pMHC and CD3.

Global mapping of antibody recognition of the hepatitis C virus E2 glycoprotein: Implications for vaccine design.

The E2 envelope glycoprotein is the primary target of human neutralizing antibody response against hepatitis C virus (HCV), and is thus a major focus of vaccine and immunotherapeutics efforts. There is emerging evidence that E2 is a highly complex, dynamic protein with residues across the protein that are modulating antibody recognition, local and global E2 stability, and viral escape. To comprehensively map these determinants, we performed global E2 alanine scanning with a panel of 16 human monoclonal antibodies (hmAbs), resulting in an unprecedented dataset of the effects of individual alanine substitutions across the E2 protein (355 positions) on antibody recognition. Analysis of shared energetic effects across the antibody panel identified networks of E2 residues involved in antibody recognition and local and global E2 stability, as well as predicted contacts between residues across the entire E2 protein. Further analysis of antibody binding hotspot residues defined groups of residues essential for E2 conformation and recognition for all 14 conformationally dependent E2 antibodies and subsets thereof, as well as residues that enhance antibody recognition when mutated to alanine, providing a potential route to engineer E2 vaccine immunogens. By incorporating E2 sequence variability, we found a number of E2 polymorphic sites that are responsible for loss of neutralizing antibody binding. These data and analyses provide fundamental insights into antibody recognition of E2, highlighting the dynamic and complex nature of this viral envelope glycoprotein, and can serve as a reference for development and rational design of E2-targeting vaccines and immunotherapeutics.

How structural adaptability exists alongside HLA-A2 bias in the human αß TCR repertoire.

How T-cell receptors (TCRs) can be intrinsically biased toward MHC proteins while simultaneously display the structural adaptability required to engage diverse ligands remains a controversial puzzle. We addressed this by examining αß TCR sequences and structures for evidence of physicochemical compatibility with MHC proteins. We found that human TCRs are enriched in the capacity to engage a polymorphic, positively charged "hot-spot" region that is almost exclusive to the α1-helix of the common human class I MHC protein, HLA-A*0201 (HLA-A2). TCR binding necessitates hot-spot burial, yielding high energetic penalties that must be offset via complementary electrostatic interactions. Enrichment of negative charges in TCR binding loops, particularly the germ-line loops encoded by the TCR Vα and Vß genes, provides this capacity and is correlated with restricted positioning of TCRs over HLA-A2. Notably, this enrichment is absent from antibody genes. The data suggest a built-in TCR compatibility with HLA-A2 that biases receptors toward, but does not compel, particular binding modes. Our findings provide an instructional example for how structurally pliant MHC biases can be encoded within TCRs.