Structural and computational design of a SARS-CoV-2 spike antigen with improved expression and immunogenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern challenge the efficacy of approved vaccines, emphasizing the need for updated spike antigens. Here, we use an evolutionary-based design aimed at boosting protein expression levels of S-2P and improving immunogenic outcomes in mice. Thirty-six prototype antigens were generated in silico and 15 were produced for biochemical analysis. S2D14, which contains 20 computationally designed mutations within the S2 domain and a rationally engineered D614G mutation in the SD2 domain, has an ~11-fold increase in protein yield and retains RBD antigenicity. Cryo-electron microscopy structures reveal a mixture of populations in various RBD conformational states. Vaccination of mice with adjuvanted S2D14 elicited higher cross-neutralizing antibody titers than adjuvanted S-2P against the SARS-CoV-2 Wuhan strain and four variants of concern. S2D14 may be a useful scaffold or tool for the design of future coronavirus vaccines, and the approaches used for the design of S2D14 may be broadly applicable to streamline vaccine discovery.

Effective Multivalent Oriented Presentation of Meningococcal NadA Antigen Trimers by Self-Assembling Ferritin Nanoparticles.

The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuable technology for vaccines. On the nanoparticle surface, highly ordered, repetitive arrays of antigens can mimic their geometric arrangement on virion surfaces and elicit stronger humoral responses than soluble viral antigens. More recently, bacterial antigens have been presented on self-assembling protein nanoparticles and have elicited protective antibody and effective T-helper responses, further supporting the nanoparticle platform as a universal approach for stimulating potent immunogenicity. Here, we present the rational design, structural analysis, and immunogenicity of self-assembling ferritin nanoparticles displaying eight copies of the Neisseria meningitidis trimeric adhesin NadA. We engineered constructs consisting of two different NadA fragments, head only and head with stalk, that we fused to ferritin and expressed in Escherichia coli. Both fusion constructs self-assembled into the expected nanoparticles as determined by Cryo electron microscopy. In mice, the two nanoparticles elicited comparable NadA antibody levels that were 10- to 100-fold higher than those elicited by the corresponding NadA trimer subunits. Further, the NadAferritin nanoparticles potently induced complement-mediated serum bactericidal activity. These findings confirm the value of self-assembling nanoparticles for optimizing the immunogenicity of bacterial antigens and support the broad applicability of the approach to vaccine programs, especially for the presentation of trimeric antigens.

Self-assembling protein nanoparticles and virus like particles correctly display ß-barrel from meningococcal factor H-binding protein through genetic fusion.

Recombinant protein-based vaccines are a valid and safer alternative to traditional vaccines based on live-attenuated or killed pathogens. However, the immune response of subunit vaccines is generally lower compared to that elicited by traditional vaccines and usually requires the use of adjuvants. The use of self-assembling protein nanoparticles, as a platform for vaccine antigen presentation, is emerging as a promising approach to enhance the production of protective and functional antibodies. In this work we demonstrated the successful repetitive antigen display of the C-terminal ß-barrel domain of factor H binding protein, derived from serogroup B Meningococcus on the surface of different self-assembling nanoparticles using genetic fusion. Six nanoparticle scaffolds were tested, including virus-like particles with different sizes, geometries, and physicochemical properties. Combining computational and structure-based rational design we were able generate antigen-fused scaffolds that closely aligned with three-dimensional structure predictions. The chimeric nanoparticles were produced as recombinant proteins in Escherichia coli and evaluated for solubility, stability, self-assembly, and antigen accessibility using a variety of biophysical methods. Several scaffolds were identified as being suitable for genetic fusion with the ß-barrel from fHbp, including ferritin, a de novo designed aldolase from Thermotoga maritima, encapsulin, CP3 phage coat protein, and the Hepatitis B core antigen. In conclusion, a systematic screening of self-assembling nanoparticles has been applied for the repetitive surface display of a vaccine antigen. This work demonstrates the capacity of rational structure-based design to develop new chimeric nanoparticles and describes a strategy that can be utilized to discover new nanoparticle-based approaches in the search for vaccines against bacterial pathogens.

Transplantation Without Overimmunosuppression (TWO) study protocol: a phase 2b randomised controlled single-centre trial of regulatory T cell therapy to facilitate immunosuppression reduction in living donor kidney transplant recipients.

INTRODUCTION: Regulatory T cell (Treg) therapy has been demonstrated to facilitate long-term allograft survival in preclinical models of transplantation and may permit reduction of immunosuppression and its associated complications in the clinical setting. Phase 1 clinical trials have shown Treg therapy to be safe and feasible in clinical practice. Here we describe a protocol for the TWO study, a phase 2b randomised control trial of Treg therapy in living donor kidney transplant recipients that will confirm safety and explore efficacy of this novel treatment strategy. METHODS AND ANALYSIS: 60 patients will be randomised on a 1:1 basis to Treg therapy (TR001) or standard clinical care (control). Patients in the TR001 arm will receive an infusion of autologous polyclonal ex vivo expanded Tregs 5 days after transplantation instead of standard monoclonal antibody induction. Maintenance immunosuppression will be reduced over the course of the post-transplant period to low-dose tacrolimus monotherapy. Control participants will receive a standard basiliximab-based immunosuppression regimen with long-term tacrolimus and mycophenolate mofetil immunosuppression. The primary endpoint is biopsy proven acute rejection over 18 months; secondary endpoints include immunosuppression burden, chronic graft dysfunction and drug-related complications. ETHICS AND DISSEMINATION: Ethical approval has been provided by the National Health Service Health Research Authority South Central-Oxford A Research Ethics Committee (reference 18/SC/0054). The study also received authorisation from the UK Medicines and Healthcare products Regulatory Agency and is being run in accordance with the principles of Good Clinical Practice, in collaboration with the registered trials unit Oxford Clinical Trials Research Unit. Results from the TWO study will be published in peer-reviewed scientific/medical journals and presented at scientific/clinical symposia and congresses. TRIAL REGISTRATION NUMBER: ISRCTN: 11038572; Pre-results.

Improved epitope resolution of the prefusion trimer-specific antibody AM14 bound to the RSV F glycoprotein.

Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens.

Convergent structural features of respiratory syncytial virus neutralizing antibodies and plasticity of the site V epitope on prefusion F.

Respiratory syncytial virus (RSV) is a global public health burden for which no licensed vaccine exists. To aid vaccine development via increased understanding of the protective antibody response to RSV prefusion glycoprotein F (PreF), we performed structural and functional studies using the human neutralizing antibody (nAb) RSB1. The crystal structure of PreF complexed with RSB1 reveals a conformational, pre-fusion specific site V epitope with a unique cross-protomer binding mechanism. We identify shared structural features between nAbs RSB1 and CR9501, elucidating for the first time how diverse germlines obtained from different subjects can develop convergent molecular mechanisms for recognition of the same PreF site of vulnerability. Importantly, RSB1-like nAbs were induced upon immunization with PreF in naturally-primed cattle. Together, this work reveals new details underlying the immunogenicity of site V and further supports PreF-based vaccine development efforts.

Crystal structures of human Fabs targeting the Bexsero meningococcal vaccine antigen NHBA.

Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA.

Crystal structure of FhuD at 1.6†Šresolution: a ferrichrome-binding protein from the animal and human pathogen Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is a leading cause of disease in dogs, and zoonosis causes human infections. Methicillin-resistant S. pseudintermedius strains are emerging, resembling the global health threat of S. aureus. Therefore, it is increasingly important to characterize potential targets for intervention against S. pseudintermedius. Here, FhuD, an S. pseudintermedius surface lipoprotein implicated in iron uptake, was characterized. It was found that FhuD bound ferrichrome in an iron-dependent manner, which increased the thermostability of FhuD by >15 °C. The crystal structure of ferrichrome-free FhuD was determined via molecular replacement at 1.6 Å resolution. FhuD exhibits the class III solute-binding protein (SBP) fold, with a ligand-binding cavity between the N- and C-terminal lobes, which is here occupied by a PEG molecule. The two lobes of FhuD were oriented in a closed conformation. These results provide the first detailed structural characterization of FhuD, a potential therapeutic target of S. pseudintermedius.

Structural and Computational Biology in the Design of Immunogenic Vaccine Antigens.

Vaccination is historically one of the most important medical interventions for the prevention of infectious disease. Previously, vaccines were typically made of rather crude mixtures of inactivated or attenuated causative agents. However, over the last 10-20 years, several important technological and computational advances have enabled major progress in the discovery and design of potently immunogenic recombinant protein vaccine antigens. Here we discuss three key breakthrough approaches that have potentiated structural and computational vaccine design. Firstly, genomic sciences gave birth to the field of reverse vaccinology, which has enabled the rapid computational identification of potential vaccine antigens. Secondly, major advances in structural biology, experimental epitope mapping, and computational epitope prediction have yielded molecular insights into the immunogenic determinants defining protective antigens, enabling their rational optimization. Thirdly, and most recently, computational approaches have been used to convert this wealth of structural and immunological information into the design of improved vaccine antigens. This review aims to illustrate the growing power of combining sequencing, structural and computational approaches, and we discuss how this may drive the design of novel immunogens suitable for future vaccines urgently needed to increase the global prevention of infectious disease.

Neisseria meningitis GNA1030 is a ubiquinone-8 binding protein.

Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is composed of 3 main recombinant proteins and an outer membrane vesicle component. One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA), is present as a fusion protein with the accessory protein genome-derived neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene encoding for GNA1030 is present and highly conserved in all Neisseria strains, and although orthologs are present in numerous species, its biologic function is unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly involved in the electron transport chain and with antioxidant properties. Disc diffusion assays on the wild-type and knockout mutant of GNA1030, in the presence of various compounds, suggested that GNA1030 is not involved in oxidative stress or electron chain transport per se, although it contributes to constitutive refilling of the inner membrane with Ub8. These studies shed light on an accessory protein present in Bexsero and reveal functional insights into the family of related proteins. On the basis of our findings, we propose to name the protein neisseria ubiquinone binding protein (NUbp).

Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody.

Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5-15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SpyCEP, a candidate antigen for a vaccine against Streptococcus pyogenes.

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen against which an effective vaccine does not yet exist. The S. pyogenes protein SpyCEP (S. pyogenes cell-envelope proteinase) is a surface-exposed subtilisin-like serine protease of 1647 amino acids. In addition to its auto-protease activity, SpyCEP is capable of cleaving interleukin 8 and related chemokines, contributing to GAS immune-evasion strategies. SpyCEP is immunogenic and confers protection in animal models of GAS infections. In order to structurally characterize this promising vaccine candidate, several SpyCEP protein-expression constructs were designed, cloned, produced in Escherichia coli, purified by affinity chromatography and subjected to crystallization trials. Crystals of a selenomethionyl form of a near-full-length SpyCEP ectodomain were obtained. The crystals diffracted X-rays to 3.3 Šresolution and belonged to space group C2, with unit-cell parameters a=139.2, b=120.4, c=104.3 Å, ß=111°.

Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197.

CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). Being naturally nontoxic, CRM197 is an ideal carrier protein for conjugate vaccines against encapsulated bacteria and is currently used to vaccinate children globally against Haemophilus influenzae, pneumococcus, and meningococcus. To understand the molecular basis for lack of toxicity in CRM197, we determined the crystal structures of the full-length nucleotide-free CRM197 and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA), both at 2.0-Å resolution. The structures show for the first time that the overall fold of CRM197 and DT are nearly identical and that the striking functional difference between the two proteins can be explained by a flexible active-site loop that covers the NAD binding pocket. We present the molecular basis for the increased flexibility of the active-site loop in CRM197 as unveiled by molecular dynamics simulations. These structural insights, combined with surface plasmon resonance, NAD hydrolysis, and differential scanning fluorimetry data, contribute to a comprehensive characterization of the vaccine carrier protein, CRM197.

Rational design of a meningococcal antigen inducing broad protective immunity.

The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.

Structural characterization of the Hepatitis C Virus NS3 protease from genotype 3a: the basis of the genotype 1b vs. 3a inhibitor potency shift.

The first structural characterization of the genotype 3a Hepatitis C Virus NS3 protease is reported, providing insight into the differential susceptibility of 1b and 3a proteases to certain inhibitors. Interaction of the 3a NS3 protease with a P2-P4 macrocyclic and a linear phenethylamide inhibitor was investigated. In addition, the effect of the NS4A cofactor binding on the conformation of the protease was analyzed. Complexation of NS3 with the phenethylamide inhibitor significantly stabilizes the protease but binding does not involve residues 168 and 123, two key amino acids underlying the different inhibition of genotype 1b vs. 3a proteases by P2-P4 macrocycles. Therefore, we studied the dynamic behavior of these two residues in the phenethylamide complex, serving as a model of the situation in the apo 3a protein, in order to explore the structural basis of the inhibition potency shift between the proteases of the genotypes 1b and 3a.

Binding of a noncovalent inhibitor exploiting the S' region stabilizes the hepatitis C virus NS3 protease conformation in the absence of cofactor.

We present the first structure of a noncovalent inhibitor bound to the protease domain of hepatitis C virus NS3 protein (NS3p), solved by NMR. The inhibitor exploits interactions with the S' region of NS3p to form a long-lived complex, although the absence of negative charges strongly reduces the association rate. The inhibitor stabilizes the N-terminal domain of NS3p and the substrate-binding site, and correctly aligns catalytic His-Asp residues. These actions were previously attributed exclusively to the cofactor NS4A, which interacts with the N-terminal domain of the NS3p and functions as an activator in vivo. The structure of the inhibitor/NS3p complex is very similar to that of the NS3p-NS4A complex, showing that binding of the NS4A cofactor is not the only event leading to a stable active-site conformation.

DNA-PK contributes to the phosphorylation of AIRE: importance in transcriptional activity.

The autoimmune regulator (AIRE) protein is a key mediator of the central tolerance for tissue specific antigens and is involved in transcriptional control of many antigens in thymic medullary epithelial cells (mTEC). Mutations in the AIRE gene cause a rare disease named autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Here we report using GST pull-down assay, mass-spectrometry and co-immunoprecipitation that a heterotrimeric complex of DNA-Dependent Protein Kinase (DNA-PK), consisting of Ku70, Ku80 and DNA-PK catalytic subunit (DNA-PKcs), is a novel interaction partner for AIRE. In vitro phosphorylation assays show that the residues Thr68 and Ser156 are DNA-PK phosphorylation sites in AIRE. In addition, we demonstrate that DNA-PKcs is expressed in AIRE positive mTEC cell population and that introduction of mutations into the AIRE phosphorylation sites decrease the capacity of AIRE to activate transcription from reporter promoters. In conclusion, our results suggest that phosphorylation of the AIRE protein at Thr68 and Ser156 by DNA-PK influences AIRE transactivation ability and might have impact on other aspects of the functional regulation of the AIRE protein.

(1)H, (13)C and (15)N backbone assignments for the ligand binding domain of the Pseudomonas aeruginosa virulence regulator LasR.

LasR regulates toxin production in Pseudomonas aeruginosa and its inhibition can attenuate the virulence of this opportunistic human pathogen. To aid studies of interactions with inhibitors, we report the NMR backbone assignments for the dimeric LasR ligand-binding domain.

NMR structure of the first PHD finger of autoimmune regulator protein (AIRE1). Insights into autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) disease.

Mutations in the autoimmune regulator protein AIRE1 cause a monogenic autosomal recessively inherited disease: autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE1 is a multidomain protein that harbors two plant homeodomain (PHD)-type zinc fingers. The first PHD finger of AIRE1 is a mutational hot spot, to which several pathological point mutations have been mapped. Using heteronuclear NMR spectroscopy, we determined the solution structure of the first PHD finger of AIRE1 (AIRE1-PHD1), and characterized the peptide backbone mobility of the domain. We performed a conformational analysis of pathological AIRE1-PHD1 mutants that allowed us to rationalize the structural impact of APECED-causing mutations and to identify an interaction site with putative protein ligands of the AIRE1-PHD1 domain. The structure unequivocally exhibits the canonical PHD finger fold, with a highly conserved tryptophan buried inside the structure. The PHD finger is stabilized by two zinc ions coordinated in an interleaved (cross-brace) scheme. This zinc coordination resembles RING finger domains, which can function as E3 ligases in the ubiquitination pathway. Based on this fold similarity, it has been suggested that PHD fingers might also function as E3 ligases, although this hypothesis is controversial. At variance to a previous report, we could not find any evidence that AIRE1-PHD1 has an intrinsic E3 ubiquitin ligase activity, nor detect any direct interaction between AIRE1-PHD1 and its putative cognate E2. Consistently, we show that the AIRE1-PHD1 structure is clearly distinct from the RING finger fold. Our results point to a function of the AIRE1-PHD1 domain in protein-protein interactions, which is impaired in some APECED mutations.