Computational structure-based approach to study chimeric antigens using a new protein scaffold displaying foreign epitopes.

The identification and recombinant production of functional antigens and/or epitopes of pathogens represent a crucial step for the development of an effective protein-based vaccine. Many vaccine targets are outer membrane proteins anchored into the lipidic bilayer through an extended hydrophobic portion making their recombinant production challenging. Moreover, only the extracellular loops, and not the hydrophobic regions, are naturally exposed to the immune system. In this work, the Domain 3 (D3) from Group B Streptococcus (GBS) pilus 2a backbone protein has been identified and engineered to be used as a scaffold for the display of extracellular loops of two Neisseria gonorrhoeae membrane proteins (PorB.1b and OpaB). A computational structure-based approach has been applied to the design of both the scaffold and the model antigens. Once identified the best D3 engineerable site, several different chimeric D3 displaying PorB.1b and OpaB extracellular loops were produced as soluble proteins. Each molecule has been characterized in terms of solubility, stability, and ability to correctly display the foreign epitope. This antigen dissection strategy allowed the identification of most immunogenic extracellular loops of both PorB.1b and OpaB gonococcal antigens. The crystal structure of chimeric D3 displaying PorB.1b immunodominant loop has been obtained confirming that the engineerization did not alter the predicted native structure of this epitope. Taken together, the reported data suggest that D3 is a novel protein scaffold for epitope insertion and display, and a valid alternative to the production of whole membrane protein antigens. Finally, this work describes a generalized computational structure-based approach for the identification, design, and dissection of epitopes in target antigens through chimeric proteins.

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.

Structural characterization of a cross-protective natural chimera of factor H binding protein from meningococcal serogroup B strain NL096.

Invasive meningococcal disease can cause fatal sepsis and meningitis and is a global health threat. Factor H binding protein (fHbp) is a protective antigen included in the two currently available vaccines against serogroup B meningococcus (MenB). FHbp is a remarkably variable surface-exposed meningococcal virulence factor with over 1300 different amino acid sequences identified so far. Based on this variability, fHbp has been classified into three variants, two subfamilies or nine modular groups, with low degrees of cross-protective activity. Here, we report the crystal structure of a natural fHbp cross-variant chimera, named variant1-2,3.x expressed by the MenB clinical isolate NL096, at 1.2 Å resolution, the highest resolution of any fHbp structure reported to date. We combined biochemical, site-directed mutagenesis and computational biophysics studies to deeply characterize this rare chimera. We determined the structure to be composed of two adjacent domains deriving from the three variants and determined the molecular basis of its stability, ability to bind Factor H and to adopt the canonical three-dimensional fHbp structure. These studies guided the design of loss-of-function mutations with potential for even greater immunogenicity. Moreover, this study represents a further step in the understanding of the fHbp biological and immunological evolution in nature. The chimeric variant1-2,3.x fHbp protein emerges as an intriguing cross-protective immunogen and suggests that identification of such naturally occurring hybrid proteins may result in stable and cross-protective immunogens when seeking to design and develop vaccines against highly variable pathogens.

Natural Human Immunity Against Staphylococcal Protein A Relies on Effector Functions Triggered by IgG3.

Staphylococcal protein A (SpA) is a multifunctional, highly conserved virulence factor of Staphylococcus aureus. By binding the Fc portion of all human IgG subclasses apart from IgG3, SpA interferes with antibody and complement deposition on the bacterial surface, impairing staphylococcal clearance by phagocytosis. Because of its anti-opsonic properties, SpA is not investigated as a surface antigen to mediate bacterial phagocytosis. Herein we investigate human sera for the presence of SpA-opsonizing antibodies. The screening revealed that sera containing IgG3 against SpA were able to correctly opsonize the target and drive Fcγ receptor-mediated interactions and phagocytosis. We demonstrated that IgG3 Fc is significantly more efficient in inducing phagocytosis of SpA-expressing S. aureus as compared to IgG1 Fc in an assay resembling physiological conditions. Furthermore, we show that the capacity of SpA antibodies to induce phagocytosis depends on the specific epitope recognized by the IgGs on SpA molecules. Overall, our results suggest that anti-SpA IgG3 antibodies could favor the anti-staphylococcal response in humans, paving the way towards the identification of a correlate of protection against staphylococcal infections.

Stability of Outer Membrane Vesicles-Based Vaccines, Identifying the Most Appropriate Methods to Detect Changes in Vaccine Potency.

Ensuring the stability of vaccines is crucial to successfully performing global immunization programs. Outer Membrane Vesicles (OMV) are receiving great attention as vaccine platforms. OMV are complex molecules and few data have been collected so far on their stability. OMV produced by bacteria, genetically modified to increase their spontaneous release, simplifying their production, are also known as Generalized Modules for Membrane Antigens (GMMA). We have performed accelerated stability studies on GMMA from different pathogens and verified the ability of physico-chemical and immunological methods to detect possible changes. High-temperature conditions (100 °C for 40 min) did not affect GMMA stability and immunogenicity in mice, in contrast to the effect of milder temperatures for a longer period of time (37 °C or 50 °C for 4 weeks). We identified critical quality attributes to monitor during stability assessment that could impact vaccine efficacy. In particular, specific recognition of antigens by monoclonal antibodies through competitive ELISA assays may replace in vivo tests for the potency assessment of GMMA-based vaccines.

Epitope and Paratope Mapping by HDX-MS Combined with SPR Elucidates the Difference in Bactericidal Activity of Two Anti-NadA Monoclonal Antibodies.

Characterization of antigen-antibody interactions is crucial for understanding antibody-mediated protection against pathogens, biopharmaceutical development, as well as evaluation of the immune response post vaccination. Bexsero is a multicomponent vaccine against Neisseria meningitidis serogroup B in which one of the key vaccine antigens is Neisserial adhesin A (NadA), a trimeric coiled-coil protein. Two NadA-specific monoclonal antibodies (mAbs) isolated from Bexsero-vaccinated individuals have been shown to have similar binding affinity and appear to recognize a similar antigen region, yet only one of the mAbs is bactericidal. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to perform an in-depth study of the interaction of the two mAbs with NadA antigen using a combined epitope and paratope mapping strategy. In addition, we use surface plasmon resonance (SPR) to investigate the stoichiometry of the binding of the two mAbs to NadA. While epitope mapping only identifies a clear binding impact of one of the mAbs on NadA, the paratope mapping analyses shows that both mAbs are binding to NadA through several complementarity determining regions spanning both heavy and light chains. Our results highlight the advantage of combined epitope and paratope mapping HDX-MS experiments and supporting biochemical experiments to characterize antigen-antibody interactions. Through this combined approach, we provide a rationale for how the binding stoichiometry of the two mAbs to the trimeric NadA antigen can explain the difference in bactericidal activity of the two mAbs.

4CMenB vaccine induces elite cross-protective human antibodies that compete with human factor H for binding to meningococcal fHbp.

Neisseria meningitidis serogroup B (MenB) is the leading cause of meningococcal meningitis and sepsis in industrialized countries, with the highest incidence in infants and adolescents. Two recombinant protein vaccines that protect against MenB are now available (i.e. 4CMenB and MenB-fHbp). Both vaccines contain the Factor H Binding Protein (fHbp) antigen, which can bind the Human Factor H (fH), the main negative regulator of the alternative complement pathway, thus enabling bacterial survival in the blood. fHbp is present in meningococcal strains as three main variants which are immunologically distinct. Here we sought to obtain detailed information about the epitopes targeted by anti-fHbp antibodies induced by immunization with the 4CMenB multicomponent vaccine. Thirteen anti-fHbp human monoclonal antibodies (mAbs) were identified in a library of over 100 antibody fragments (Fabs) obtained from three healthy adult volunteers immunized with 4CMenB. Herein, the key cross-reactive mAbs were further characterized for antigen binding affinity, complement-mediated serum bactericidal activity (SBA) and the ability to inhibit binding of fH to live bacteria. For the first time, we identified a subset of anti-fHbp mAbs able to elicit human SBA against strains with all three variants and able to compete with human fH for fHbp binding. We present the crystal structure of fHbp v1.1 complexed with human antibody 4B3. The structure, combined with mutagenesis and binding studies, revealed the critical cross-reactive epitope. The structure also provided the molecular basis of competition for fH binding. These data suggest that the fH binding site on fHbp v1.1 can be accessible to the human immune system upon immunization, enabling elicitation of human mAbs broadly protective against MenB. The novel structural, biochemical and functional data are of great significance because the human vaccine-elicited mAbs are the first reported to inhibit the binding of fH to fHbp, and are bactericidal with human complement. Our studies provide molecular insights into the human immune response to the 4CMenB meningococcal vaccine and fuel the rationale for combined structural, immunological and functional studies when seeking deeper understanding of the mechanisms of action of human vaccines.

Synergic complement-mediated bactericidal activity of monoclonal antibodies with distinct specificity.

The classical complement pathway is triggered when antigen-bound immunoglobulins bind to C1q through their Fc region. While C1q binds to a single Fc with low affinity, a higher avidity stable binding of two or more of C1q globular heads initiates the downstream reactions of the complement cascade ultimately resulting in bacteriolysis. Synergistic bactericidal activity has been demonstrated when monoclonal antibodies recognize nonoverlapping epitopes of the same antigen. The aim of the present work was to investigate the synergistic effect between antibodies directed toward different antigens. To this purpose, we investigated the bactericidal activity induced by combinations of monoclonal antibodies (mAbs) raised against factor H-binding protein (fHbp) and Neisserial Heparin-Binding Antigen (NHBA), two major antigens included in Bexsero, the vaccine against Meningococcus B, for prevention from this devastating disease in infants and adolescents. Collectively, our results show that mAbs recognizing different antigens can synergistically activate complement even when each single Mab is not bactericidal, reinforcing the evidence that cooperative immunity induced by antigen combinations can represent a remarkable added value of multicomponent vaccines. Our study also shows that the synergistic effect of antibodies is modulated by the nature of the respective epitopes, as well as by the antigen density on the bacterial cell surface.

A LONELY GUY protein of Bordetella pertussis with unique features is related to oxidative stress.

The Gram-negative bacterium B. pertussis is the causative agent of whooping cough. This infection is re-emerging and new features related to Bordetella pathogenesis and microbiology could be relevant to defeat it. Therefore, we focused our attention on BP1253, a predicted exported protein from B. pertussis erroneously classified as lysine decarboxylase. We showed that BP1253 shares the highly conserved motif PGGxGTxxE and the key catalytic amino-acid residues with newly structurally characterized "LONELY GUY" (LOG) proteins. Biochemical studies have confirmed that this protein functions as a cytokinin-activating enzyme since it cleaves the N-glycosidic linkage between the base and the ribose, leading to the formation of free bases, which are the active form of plant hormones called cytokinins. Remarkably, BP1253 selectively binds monophosphate nucleotides such as AMP, GMP and CMP, showing a wider variety in binding capacity compared to other LOGs. Cytokinin production studies performed with B. pertussis have revealed 6-O-methylguanine to be the physiological product of BP1253 in agreement with the higher activity of the enzyme towards GMP. 6-O-methylguanine is likely to be responsible for the increased sensitivity of B. pertussis to oxidative stress. Although BP1253 has a primary sequence resembling the hexameric type-II LOGs, the dimeric state and the presence of specific amino-acids suggests that BP1253 can be classified as a novel type-II LOG. The discovery of a LOG along with its product 6-O-methylguanine in the human pathogen B. pertussis may lead to the discovery of unexplored functions of LOGs, broadening their role beyond plants.

Structural basis for cooperativity of human monoclonal antibodies to meningococcal factor H-binding protein.

Monoclonal antibody (mAb) cooperativity is a phenomenon triggered when mAbs couples promote increased bactericidal killing compared to individual partners. Cooperativity has been deeply investigated among mAbs elicited by factor H-binding protein (fHbp), a Neisseria meningitidis surface-exposed lipoprotein and one of the key antigens included in both serogroup B meningococcus vaccine Bexsero and Trumenba. Here we report the structural and functional characterization of two cooperative mAbs pairs isolated from Bexsero vaccines. The 3D electron microscopy structures of the human mAb-fHbp-mAb cooperative complexes indicate that the angle formed between the antigen binding fragments (fAbs) assume regular angle and that fHbp is able to bind simultaneously and stably the cooperative mAbs pairs and human factor H (fH) in vitro. These findings shed light on molecular basis of the antibody-based mechanism of protection driven by simultaneous recognition of the different epitopes of the fHbp and underline that cooperativity is crucial in vaccine efficacy.

Pilus Assembly in Gram-Positive Bacteria.

Pili of Gram-positive bacteria are unique structures on the bacterial surface, assembled from covalently linked polypeptide subunits. Pilus assembly proceeds by transpeptidation reactions catalyzed by sortases, followed by covalent anchoring of the filament in the peptidoglycan layer. Another distinctive property is the presence of intramolecular isopeptide bonds, conferring extraordinary chemical and mechanical stability to these elongated structures. Besides their function in cell adhesion and biofilm formation, this section discusses possible application of pilus constituents as vaccine components against Gram-positive pathogens.

Exploring host-pathogen interactions through genome wide protein microarray analysis.

During bacterial pathogenesis extensive contacts between the human and the bacterial extracellular proteomes take place. The identification of novel host-pathogen interactions by standard methods using a case-by-case approach is laborious and time consuming. To overcome this limitation, we took advantage of large libraries of human and bacterial recombinant proteins. We applied a large-scale protein microarray-based screening on two important human pathogens using two different approaches: (I) 75 human extracellular proteins were tested on 159 spotted Staphylococcus aureus recombinant proteins and (II) Neisseria meningitidis adhesin (NadA), an important vaccine component against serogroup B meningococcus, was screened against ≈2300 spotted human recombinant proteins. The approach presented here allowed the identification of the interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting; and of the interaction between meningococcal NadA and human LOX-1 (low-density oxidized lipoprotein receptor), an endothelial receptor. The novel interactions between bacterial and human extracellular proteins here presented might provide a better understanding of the molecular events underlying S. aureus and N. meningitidis pathogenesis.

Identification of a Monoclonal Antibody Against Pneumococcal Pilus 1 Ancillary Protein Impairing Bacterial Adhesion to Human Epithelial Cells.

The adhesion of Streptococcus pneumoniae is a key step during colonization of human respiratory tract mucosae. Here we demonstrate that pneumococcal type I pilus significantly increases the adhesiveness of poorly adhering highly capsulated strains in vitro. Interestingly, preincubation of bacteria with antibodies against the major pilus backbone subunit (RrgB) or the adhesin component (RrgA) impaired pneumococcal association to human epithelial cells. Screening for anti-RrgA monoclonal antibodies specifically affecting the adhesive capacity of S. pneumoniae led to the identification of the monoclonal 11B9/61 antibody, which greatly reduced pilus-dependent cell contact. Proteomic-based epitope mapping of 11B9/61 monoclonal antibody revealed a well-exposed epitope on the D2 domain of RrgA as the target of this functional antibody. The data presented here confirm the importance of pilus I for S. pneumoniae pathogenesis and the potential use of antipilus antibodies to prevent bacterial colonization.

Defined conjugation of glycans to the lysines of CRM197 guided by their reactivity mapping.

Systematic characterisation of the reactivity of the lysine moieties in CRM197 towards N-hydroxysuccinimide linkers bearing alkynes or azides is described. This involves two-step conjugation of various glycans to CRM197 by click chemistry in a well-defined manner. By semiquantitative LC-MS/MS analysis of proteolytic digests of the conjugates formed, the reactivity of lysine residues in the protein was mapped and ranked. Computational analysis of the solvent accessibility of each lysine residue (based on the CRM197 crystal structure) established a correlation between reactivity and surface exposure. By this approach, conjugation involving lysine residues (normally a random process) can be controlled. It enables the preparation of lysine-mediated glycoconjugates with improved batch-to-batch reproducibility, thereby producing neo-glycoconjugates with more-consistent biological activity.

Two cross-reactive monoclonal antibodies recognize overlapping epitopes on Neisseria meningitidis factor H binding protein but have different functional properties.

Factor H binding protein (fHbp) is one of the main antigens of the 4-component meningococcus B (4CMenB) multicomponent vaccine against disease caused by serogroup B Neisseria meningitidis (MenB). fHbp binds the complement down-regulating protein human factor H (hfH), thus resulting in immune evasion. fHbp exists in 3 variant groups with limited cross-protective responses. Previous studies have described the generation of monoclonal antibodies (mAbs) targeting variant-specific regions of fHbp. Here we report for the first time the functional characterization of two mAbs that recognize a wide panel of fHbp variants and subvariants on the MenB surface and that are able to inhibit fHbp binding to hfH. The antigenic regions targeted by the two mAbs were accurately mapped by hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing partially overlapping epitopes on the N terminus of fHbp. Furthermore, while none of the mAbs had bactericidal activity on its own, a synergistic effect was observed for each of them when tested by the human complement serum bactericidal activity (hSBA) assay in combination with a second nonbactericidal mAb. The bases underlying fHbp variant cross-reactivity, as well as inhibition of hfH binding and cooperativity effect observed for the two mAbs, are discussed in light of the mapped epitopes.

Protective efficacy induced by recombinant Clostridium difficile toxin fragments.

Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.

Development of an influenza virus protein array using Sortagging technology.

Protein array technology is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work, we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as "Sortagging". LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular extracts were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly3 linker. Immobilized proteins were revealed by specific antibodies, and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purification steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technology has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes in order to develop protein arrays for high-throughput screening.

Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain.

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.

The two variants of the Streptococcus pneumoniae pilus 1 RrgA adhesin retain the same function and elicit cross-protection in vivo.

Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus composed of the backbone subunit RrgB and two ancillary proteins, RrgA and RrgC. RrgA is the major determinant of in vitro adhesion associated with pilus 1, is protective in vivo in mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the "head" of the protein, which contains the putative binding domains, whereas the elongated "stalk" was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the "head" domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

Adhesion determinants of the Streptococcus species.

Streptococci are clinically important Gram-positive bacteria that are capable to cause a wide variety of diseases in humans and animals. Phylogenetic analyses based on 16S rRNA sequences of the streptococcal species reveal a clustering pattern, reflecting, with a few exceptions, their pathogenic potential and ecological preferences. Microbial adhesion to host tissues is the initial critical event in the pathogenesis of most infections. Streptococci use multiple adhesins to attach to the epithelium, and their expression is regulated in response to environmental and growth conditions. Bacterial adhesins recognize and bind cell surface molecules and extracellular matrix components through specific domains that for certain adhesin families have been well defined and found conserved across the streptococcal species. In this review, we present the different streptococcal adhesin families categorized on the basis of their adhesive properties and structural characteristics, and, when available, we focus the attention on conserved functional domains.