Structural characterization of the human immune response to the meningococcal vaccine antigen NHBA

Neisseria meningitidis is a human pathogen which can cause fatal sepsis and invasive meningococcal disease once it reaches the blood stream and the nervous system. Bexsero™ is the first broadly protective multicomponent vaccine against serogroup B N. meningitidis. Among itscomponents, the Neisserial Heparin Binding Antigen (NHBA) represents the less structurally and functionally characterized antigen of Bexsero and therefore has been the target of the current study.NHBA is a surface-exposed lipoprotein composed by two domains (C- and N-terminal), and while the structure of the C-terminal domain was solved by NMR, the structure of N-terminal region is still unknown and predicted to be largely unstructured. Knowledge of the NHBA full-length structure canprovide important insights towards the understanding of its biological function, as well as on its role as vaccine antigen.In this work we combined the information derived from the profiling the B-cell repertoire in response to vaccination with Bexsero with structural biology, in order to shed light on NHBA structure and on the molecular bases of its recognition by the human immune system. By using fragments antigen binding (Fabs) derived from human monoclonal antibodies (mAbs), we sought to provide highresolution epitope mapping of the NHBA immunogenic regions, which in turn could permit a deeper characterization of the molecular determinants of antibody binding and protective epitopes. Here we present a structural characterization of the first high-resolution crystal structures of three free human anti-NHBA Fabs of NHBA. These structures reveal features compatible with the binding of NHBA regions as previously determined by other low-resolution methods. Moreover, these Fabs provided important tools for structural studies through co-crystallization experiments with various NHBA fragments and constructs. While using Fabs that bind to the NHBA N-terminal region complexed with full length or sub-full length antigen did not result in crystal growth, a construct including solely the C-terminal domain yielded crystals that allowed solving the structure of the complex with a C-terminal Fab binder. Structural analysis highlighted the conservation of the epitope and lead to the identification of the key residues involved into NHBA-Fab recognition. Additionally, the comparison between bound and unbound Fab revealed an interesting fitting mechanism occurring during antigen recognition that provides further details into the elucidation of antibody binding. Combined together, these results enhance our structural and biophysical understanding of NHBA, and provide a platform for deeper analyses aimed at the elucidation of the molecular determinants of its immunogenicity

Moltemplate: A Tool for Coarse-Grained Modeling of Complex Biological Matter and Soft Condensed Matter Physics

Coarse-grained models have long been considered indispensable tools in the investigation of biomolecular dynamics and assembly. However, the process of simulating such models is arduous because unconventional force fields and particle attributes are often needed, and some systems are not in thermal equilibrium. Although modern molecular dynamics programs are highly adaptable, software designed for preparing all-atom simulations typically makes restrictive assumptions about the nature of the particles and the forces acting on them. Consequently, the use of coarse-grained models has remained challenging. Moltemplate is a file format for storing coarse-grained molecular models and the forces that act on them, as well as a program that converts moltemplate files into input files for LAMMPS, a popular molecular dynamics engine. Moltemplate has broad scope and an emphasis on generality. It accommodates new kinds of forces as they are developed for LAMMPS, making moltemplate a popular tool with thousands of users in computational chemistry, materials science, and structural biology. To demonstrate its wide functionality, we provide examples of using moltemplate to prepare simulations of fluids using many-body forces, coarse-grained organic semiconductors, and the motor-driven supercoiling and condensation of an entire bacterial chromosome

Structural characterization of the human immune response to the meningococcal vaccine antigen NHBA

Neisseria meningitidis is a human pathogen which can cause fatal sepsis and invasive meningococcal disease once it reaches the blood stream and the nervous system. Bexsero™ is the first broadly protective multicomponent vaccine against serogroup B N. meningitidis. Among itscomponents, the Neisserial Heparin Binding Antigen (NHBA) represents the less structurally and functionally characterized antigen of Bexsero and therefore has been the target of the current study.NHBA is a surface-exposed lipoprotein composed by two domains (C- and N-terminal), and while the structure of the C-terminal domain was solved by NMR, the structure of N-terminal region is still unknown and predicted to be largely unstructured. Knowledge of the NHBA full-length structure canprovide important insights towards the understanding of its biological function, as well as on its role as vaccine antigen.In this work we combined the information derived from the profiling the B-cell repertoire in response to vaccination with Bexsero with structural biology, in order to shed light on NHBA structure and on the molecular bases of its recognition by the human immune system. By using fragments antigen binding (Fabs) derived from human monoclonal antibodies (mAbs), we sought to provide highresolution epitope mapping of the NHBA immunogenic regions, which in turn could permit a deeper characterization of the molecular determinants of antibody binding and protective epitopes. Here we present a structural characterization of the first high-resolution crystal structures of three free human anti-NHBA Fabs of NHBA. These structures reveal features compatible with the binding of NHBA regions as previously determined by other low-resolution methods. Moreover, these Fabs provided important tools for structural studies through co-crystallization experiments with various NHBA fragments and constructs. While using Fabs that bind to the NHBA N-terminal region complexed with full length or sub-full length antigen did not result in crystal growth, a construct including solely the C-terminal domain yielded crystals that allowed solving the structure of the complex with a C-terminal Fab binder. Structural analysis highlighted the conservation of the epitope and lead to the identification of the key residues involved into NHBA-Fab recognition. Additionally, the comparison between bound and unbound Fab revealed an interesting fitting mechanism occurring during antigen recognition that provides further details into the elucidation of antibody binding. Combined together, these results enhance our structural and biophysical understanding of NHBA, and provide a platform for deeper analyses aimed at the elucidation of the molecular determinants of its immunogenicity.Neisseria meningitidis è un patogeno umano obbligato e un potenziale agente eziologico di sepsi e meningite, qualora entri in contatto con il flusso sanguigno o il sistema nervoso. Bexsero è il primo vaccino multicomponente in grado di fornire ampia protezione contro il sierogruppo B di N meningitidis. Tra i vari componenti di Bexsero, Neisserial Heparin Binding Antigen (NHBA) rappresenta l’antigene meno caratterizzato sia funzionalmente che strutturalmente e, per questa ragione, è stato selezionato come oggetto di studio nel presente lavoro. NHBA è una lipoproteina esposta sulla superficie del batterio e si compone di due domini (C- e N-terminale); mentre la struttura del dominio C-terminale è stata risolta tramite spettroscopia NMR, il dominio N-terminale è ancora strutturalmente non caratterizzato e predetto, per la maggior parte della sua sequenza, come non strutturato. La conoscenza delle caratteristiche strutturali della proteina intera, quindi, potrebbe essere utile per chiarire il ruolo che NHBA svolge sia dal punto di vista biologico sia come antigene vaccinale. In questo lavoro di tesi, informazioni derivanti dall’analisi del repertorio delle cellule B generate in risposta all’immunizzazione con Bexsero sono state combinante con tecniche di biologia strutturale (cristallografia a raggi X), in modo da delucidare la struttura completa di NHBA e le basi molecolari del suo riconoscimento da parte del sistema immunitario umano. Frammenti di anticorpi leganti l'antigene (Fabs), derivanti da anticorpi monoclonali umani (mAbs) sono stati utilizzati per la mappatura ad altarisoluzione degli epitopi immunogenici di NHBA, permettendo un’accurata caratterizzazione dal punto di vista molecolare delle regioni dell’antigene implicate nel legame con gli anticorpi. Dall’analisi delle strutture cristallografiche ad alta risoluzione di tre Fabs umani diretti controNHBA, è emerso che questi presentano a livello del paratopo caratteristiche che indicano la loro compatibilità con il legame a NHBA tramite regioni precedente mappate con tecniche a bassa risoluzione. Inoltre, questi Fabs si sono rivelati un importante strumento per studi strutturali attraverso esperimenti di co-cristallizzazione in complesso con NHBA. Purtroppo non è stato possibile ottenere cristalli idonei per esperimenti di diffrazione dalla co-cristallizzazione dei Fabs con l’intera NHBA o frammenti N-terminali di NHBA; al contrario, il dominio C-terminale dell’antigene in complesso con un Fab ha generato cristalli che hanno permesso di risolvere la prima struttura di un complesso tra Cterminale di NHBA e un Fab umano. L’analisi strutturale di questo complesso ha evidenziato l’estrema conservazione dell’epitopo e guidato l’identificazione dei residui chiave implicati nel riconoscimento tra NHBA e Fab. Inoltre, il confronto tra la forma complessata e non-complessata del Fab ha rivelato un 8 interessante meccanismo di legame che avviene nella fase di riconoscimento dell’antigene e che fornisce ulteriori dettagli sui meccanismi che regolano i legami tra anticorpi e antigeni. I risultati ottenuti in questo studio hanno contribuito a migliorare la conoscenza strutturale e biofisica di NHBA e forniscono una base per una futura analisi strutturale più accurata, volta a delucidare i dettagli molecolari della risposta immunitaria a NHBA

Wall painting pigments of Roman Empire age from Syria Palestina province (Israel)

This work is the first step of an extensive project aimed at chemical and mineralogical characterization of pigments of Roman Empire age throughout the provinces. Wall painting materials from two Roman archaeological sites in Israel (the ancient province of Iudaea, later known as Syria Palestina) were studied: the Roman villa of Lod, dated to the fourth century CE and the synagogue of Migdal, dated between the first century BCE and the second century CE. A total of 165 samples were investigated, producing multi-analytical information from optical microscopy, scanning electron microscopy supported by energy-dispersive X-ray spectroscopy, micro-Raman spectroscopy, X ray diffraction, infra-red spectroscopy, and portable energy-dispersive X-ray fluorescence spectroscopy. This study shows that materials and techniques in the Roman province of Syria Palestina follow Empire tendencies, although advantage was taken also of the local availability of raw materials. Seven different pigments were identified, including Egyptian blue and cinnabar, and the predominant wall painting technique used was fresco occasionally supported by secco retouching

Role of non-polyQ regions on the aggregation process by polyQ proteins into amyloid fibrils

Nine neurodegenerative disorders, referred to as polyglutamine diseases and including Huntington’s disease, are associated with the abnormal expansion of a polyglutamine tract inside nine unrelated proteins. This polyQ expansion is thought to be the major determinant in the development of neurotoxicity, triggering protein aggregation into amyloid fibrils. A large body of evidence however indicates that non-polyQ regions modulate the aggregation process triggered by polyQ expansions. The interplay between the polyQ tract and non-polyQ regions is complex and still not fully understood. In order to better understand it, we previously designed and characterized model polyQ proteins made of the beta-lactamase BlaP and a 23, 30, 55 or 79Q tract inserted in position 197 or 216. These chimeras recapitulate the aggregation properties of polyQ disease-associated proteins: there is a Q threshold for the formation of amyloid fibrils, and above the threshold, the longer the polyQ, the faster the aggregation. Moreover, the structure of BlaP (native or unfolded) and the position of insertion of the polyQ tract (197 versus 216) influence their aggregation properties. In this work, (i) we will discuss the role of the conformation of the host protein, BlaP, and of the location of the polyQ within BlaP on the different phases of amyloid fibril formation, the nucleation and elongation steps, using mainly quartz crystal microbalance (QCM), atomic force microscopy (AFM) and dynamic light scattering (DLS); and (ii) we will investigate the precise aggregation threshold and the modulating role of the N- and C-terminal polyQ flanking sequences in position 197 of BlaP by creating and characterizing new chimeras containing intermediate length polyQ tracts in position 197, or polyQ tracts inserted between two cleavage sites in position 197, respectively. Our results highlight a linear dependence of the polyQ length on the elongation rate whatever the insertion site and the conformation of BlaP. These two parameters however drastically influence the ability of a polyQ tract to trigger the nucleation and the elongation steps of amyloid fibril formation. Finally, we observed that the propensity to form amyloid fibrils and its rate seems to be largely dependent on the polyQ length and on the polyQ flanking sequences. Altogether our results contribute to identify the important species and elements (polyQ or non-polyQ regions, monomers, oligomers or fibrils) during the aggregation process into amyloid fibrils to interfere with the latter associated with neurotoxicity

Role of the polyQ length and non-polyQ regions during the aggregation process into amyloid fibrils of model polyQ proteins

Nine neurodegenerative disorders, referred to as polyglutamine diseases and including Huntington’s disease, are associated with the abnormal expansion of a polyglutamine tract inside nine unrelated proteins. This polyQ expansion is thought to be the major determinant in the development of neurotoxicity, triggering protein aggregation into amyloid fibrils. A large body of evidence however suggests that non-polyQ regions modulate the aggregation process triggered by polyQ expansions. The interplay between the polyQ tract and non-polyQ regions is complex and still not fully understood. In order to better understand it, we previously designed and characterized model polyQ proteins made of the beta-lactamase BlaP with 23, 30, 55 and 79Q inserted at position 197 or 216. Our first results had indicated that our model is relevant to study polyQ aggregation since it recapitulates the aggregation properties of polyQ disease-associated proteins: there is a Q-threshold for the spontaneous formation of amyloid fibrils in solution, and above the threshold, the longer the polyQ, the faster the aggregation. Moreover, the structure of BlaP and the position of insertion of the polyQ tract influence their aggregation properties in solution. This work aims to better understand, at the molecular level, (i) the precise role of the polyQ length (23, 30, 55, 61, 67, 73 and 79Q), (ii) the conformation of the host protein (native or unfolded BlaP), (iii) the location of the polyQ tract within BlaP (197 or 216), (iv) the flexibility of the polyQ flanking sequences, and (v) the origin of constraints applied by BlaP to the inserted polyQ tract (at its N- or C-terminal end) on the structural, thermodynamic and aggregation properties of BlaP-polyQ chimeras, using a wide range of biophysical techniques (e.g., spectroscopy methods, quartz crystal microbalance, atomic force microscopy and dynamic light scattering). The effect on the aggregation properties will be determined on the spontaneous aggregation into amyloid fibrils in solution, and on the nucleation and on the elongation steps of amyloid fibril formation. For this purpose, new chimeras containing 61, 67 and 73Q at position 197, or 55Q inserted at position 197 in between two different protease’s cleavage sites, that are relatively flexible, will be moreover created. Our results first demonstrate that the spontaneous aggregation into amyloid fibrils in solution is correlated to the polyQ length with an exponential growth function, and that the elongation rate is linearly correlated to the polyQ length, independently of the protein context (i.e., conformation of BlaP, and/or location of the polyQ tract, and/or polyQ peptides inserted or not within BlaP). However, the location of the polyQ tract inside BlaP, and/or its conformational state, and/or the flexibility of polyQ flanking sequences, and/or the origin of constraints applied to the polyQ tract drastically influence the ability of a polyQ tract to trigger the nucleation and/or the elongation step of amyloid fibrils (variation in the Q-threshold and in the absolute rate of both steps). Altogether, our results suggest that non-polyQ regions constitute an additional potential therapeutic target, more specific than drugs targeting the polyQ sequence, to interfere with the nucleation and/or the elongation of amyloid fibrils, associated to neurotoxicity. A possible drug could be constituted by a ligand specific to non-polyQ regions of disease-associated proteins, which further increases the constraints applied to the polyQ expansion to prevent the disease onset and/or progression

Structures of NHBA elucidate a broadly conserved epitope identified by a vaccine induced antibody

Neisserial heparin binding antigen (NHBA) is one of three main recombinant protein antigens in 4CMenB, a vaccine for the prevention of invasive meningococcal disease caused by Neisseria meningitidis serogroup B. NHBA is a surface-exposed lipoprotein composed of a predicted disordered N-terminal region, an arginine-rich region that binds heparin, and a C-terminal domain that folds as an anti-parallel β-barrel and that upon release after cleavage by human proteases alters endothelial permeability. NHBA induces bactericidal antibodies in humans, and NHBA-specific antibodies elicited by the 4CMenB vaccine contribute to serum bactericidal activity, the correlate of protection. To better understand the structural bases of the human antibody response to 4CMenB vaccination and to inform antigen design, we used X-ray crystallography to elucidate the structures of two C-terminal fragments of NHBA, either alone or in complex with the Fab derived from the vaccine-elicited human monoclonal antibody 5H2, and the structure of the unbound Fab 5H2. The structures reveal details on the interaction between an N-terminal β-hairpin fragment and the β-barrel, and explain how NHBA is capable of generating cross-reactive antibodies through an extensive conserved conformational epitope that covers the entire C-terminal face of the β-barrel. By providing new structural information on a vaccine antigen and on the human immune response to vaccination, these results deepen our molecular understanding of 4CMenB, and might also aid future vaccine design projects