Epitope Mapping of Human Polyclonal Antibodies to the fHbp Antigen of a Neisseria Meningitidis Vaccine by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Antigen-antibody interactions play a key role in the immune response post vaccination and the mechanism of action of antibody-based biopharmaceuticals. 4CMenB is a multicomponent vaccine against Neisseria meningitidis serogroup B in which factor H binding protein (fHbp) is one of the key antigens. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to identify epitopes in fHbp recognized by polyclonal antibodies (pAb) from two human donors (HDs) vaccinated with 4CMenB. Our HDX-MS data reveal several epitopes recognized by the complex mixture of human pAb. Furthermore, we show that the pAb from the two HDs recognize the same epitope regions. Epitope mapping of total pAb and purified fHbp-specific pAb from the same HD reveals that the two antibody samples recognize the same main epitopes, showing that HDX-MS based epitope mapping can, in this case at least, be performed directly using total IgG pAb samples that have not undergone Ab-selective purification. Two monoclonal antibodies (mAb) were previously produced from B-cell repertoire sequences from one of the HDs and used for epitope mapping of fHbp with HDX-MS. The epitopes identified for the pAb from the same HD in this study, overlap with the epitopes recognized by the two individual mAbs. Overall, HDX-MS epitope mapping appears highly suitable for simultaneous identification of epitopes recognized by pAb from human donors and to thus both guide vaccine development and study basic human immunity to pathogens, including viruses.

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.

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.

Cocrystal structure of meningococcal factor H binding protein variant 3 reveals a new crossprotective epitope recognized by human mAb 1E6.

The 4 component meningococcus B vaccine (4CMenB) vaccine is the first vaccine containing recombinant proteins licensed for the prevention of invasive meningococcal disease caused by meningococcal serogroup B strains. 4CMenB contains 3 main recombinant proteins, including the Neisseria meningitidis factor H binding protein (fHbp), a lipoprotein able to bind the human factor H. To date, over 1000 aa sequences of fHbp have been identified, and they can be divided into variant groups 1, 2, and 3, which are usually not crossprotective. Nevertheless, previous characterizations of a small set (n = 10) of mAbs generated in humans after 4CMenB immunization revealed 2 human Fabs (huFabs) (1A12, 1G3) with some crossreactivity for variants 1, 2, and 3. This unexpected result prompted us to examine a much larger set of human mAbs (n = 110), with the aim of better understanding the extent and nature of crossreactive anti-fHbp antibodies. In this study, we report an analysis of the human antibody response to fHbp, by the characterization of 110 huFabs collected from 3 adult vaccinees during a 6-mo study. Although the 4CMenB vaccine contains fHbp variant 1, 13 huFabs were also found to be crossreactive with variants 2 and 3. The crystal structure of the crossreactive huFab 1E6 in complex with fHbp variant 3 was determined, revealing a novel, highly conserved epitope distinct from the epitopes recognized by 1A12 or 1G3. Further, functional characterization shows that human mAb 1E6 is able to elicit rabbit, but not human, complement-mediated bactericidal activity against meningococci displaying fHbp from any of the 3 different variant groups. This functional and structural information about the human antibody response upon 4CMenB immunization contributes to further unraveling the immunogenic properties of fHbp. Knowledge gained about the epitope profile recognized by the human antibody repertoire could guide future vaccine design.-Bianchi, F., Veggi, D., Santini, L., Buricchi, F., Bartolini, E., Lo Surdo, P., Martinelli, M., Finco, O., Masignani, V., Bottomley, M. J., Maione, D., Cozzi, R. Cocrystal structure of meningococcal factor H binding protein variant 3 reveals a new crossprotective epitope recognized by human mAb 1E6.

Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide.

Despite substantial progress in the prevention of group B Streptococcus (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen remains a leading cause of neonatal infection. Capsular polysaccharide conjugate vaccines have been tested in phase I/II clinical studies, showing promise for further development. Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. In this study, we report the structure of the epitope recognized by a monoclonal antibody with opsonophagocytic activity and representative of the protective response against type III GBS polysaccharide. The structure and the atomic-level interactions were determined by saturation transfer difference (STD)-NMR and X-ray crystallography using oligosaccharides obtained by synthetic and depolymerization procedures. The GBS PSIII epitope is made by six sugars. Four of them derive from two adjacent repeating units of the PSIII backbone and two of them from the branched galactose-sialic acid disaccharide contained in this sequence. The sialic acid residue establishes direct binding interactions with the functional antibody. The crystal structure provides insight into the molecular basis of antibody-carbohydrate interactions and confirms that the conformational epitope is not required for antigen recognition. Understanding the structural basis of immune recognition of capsular polysaccharide epitopes can aid in the design of novel glycoconjugate vaccines.

Native State Organization of Outer Membrane Porins Unraveled by HDx-MS.

Hydrogen-deuterium exchange (HDx) associated with mass spectrometry (MS) is emerging as a powerful tool to provide conformational information about membrane proteins. Unfortunately, as for X-ray diffraction and NMR, HDx performed on reconstituted in vitro systems might not always reflect the in vivo environment. Outer-membrane vesicles naturally released by Escherichia coli were used to carry out analysis of native OmpF through HDx-MS. A new protocol compatible with HDx analysis that avoids hindrance from the lipid contents was setup. The extent of deuterium incorporation was in good agreement with the X-ray diffraction data of OmpF as the buried ß-barrels incorporated a low amount of deuterium, whereas the internal loop L3 and the external loops incorporated a higher amount of deuterium. Moreover, the kinetics of incorporation clearly highlights that peptides segregate well in two distinct groups based exclusively on a trimeric organization of OmpF in the membrane: peptides presenting fast kinetics of labeling are facing the complex surrounding environment, whereas those presenting slow kinetics are located in the buried core of the trimer. The data show that HDx-MS applied to a complex biological system is able to reveal solvent accessibility and spatial arrangement of an integral outer-membrane protein complex.

Neisseria meningitidis factor H-binding protein bound to monoclonal antibody JAR5: implications for antibody synergy.

Factor H-binding protein (fHbp) is an important antigen of Neisseria meningitidis that is capable of eliciting a robust protective immune response in humans. Previous studies on the interactions of fHbp with antibodies revealed that some anti-fHbp monoclonal antibodies that are unable to trigger complement-mediated bacterial killing in vitro are highly co-operative and become bactericidal if used in combination. Several factors have been shown to influence such co-operativity, including IgG subclass and antigen density. To investigate the structural basis of the anti-fHbp antibody synergy, we determined the crystal structure of the complex between fHbp and the Fab (fragment antigen-binding) fragment of JAR5, a specific anti-fHbp murine monoclonal antibody known to be highly co-operative with other monoclonal antibodies. We show that JAR5 is highly synergic with monoclonal antibody (mAb) 12C1, whose structure in complex with fHbp has been previously solved. Structural analyses of the epitopes recognized by JAR5 and 12C1, and computational modeling of full-length IgG mAbs of JAR5 and 12C1 bound to the same fHbp molecule, provide insights into the spatial orientation of Fc (fragment crystallizable) regions and into the possible implications for the susceptibility of meningococci to complement-mediated killing.

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.

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).

Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen.

Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens.

Auto ADP-ribosylation of NarE, a Neisseria meningitidis ADP-ribosyltransferase, regulates its catalytic activities.

NarE is an arginine-specific mono-ADP-ribosyltransferase identified in Neisseria meningitidis that requires the presence of iron in a structured cluster for its enzymatic activities. In this study, we show that NarE can perform auto-ADP-ribosylation. This automodification occurred in a time- and NAD-concentration-dependent manner; was inhibited by novobiocin, an ADP-ribosyltransferase inhibitor; and did not occur when NarE was heat inactivated. No reduction in incorporation was evidenced in the presence of high concentrations of ATP, GTP, ADP-ribose, or nicotinamide, which inhibits NAD-glycohydrolase, impeding the formation of free ADP-ribose. Based on the electrophoretic profile of NarE on auto-ADP-ribosylation and on the results of mutagenesis and mass spectrometry analysis, the auto-ADP-ribosylation appeared to be restricted to the addition of a single ADP-ribose. Chemical stability experiments showed that the ADP-ribosyl linkage was sensitive to hydroxylamine, which breaks ADP-ribose-arginine bonds. Site-directed mutagenesis suggested that the auto-ADP-ribosylation site occurred preferentially on the R(7) residue, which is located in the region I of the ADP-ribosyltransferase family. After auto-ADP-ribosylation, NarE showed a reduction in ADP-ribosyltransferase activity, while NAD-glycohydrolase activity was increased. Overall, our findings provide evidence for a novel intramolecular mechanism used by NarE to regulate its enzymatic activities.

Bactericidal human monoclonal antibody 1B1 shows specificity for meningococcal factor H binding protein variant 2 and displaces human factor H.

Thousands of disease cases and hundreds of deaths occur globally each year due to invasive meningococcal disease. Neisseria meningitidis serogroup B (MenB) is the leading cause of such disease in developed countries. Two vaccines, 4CMenB and MenB-fHbp, that protect against MenB are available and include one or two forms respectively of factor H binding protein (fHbp), a key protective antigen. Studies of circulating meningococci have identified over 1380 different fHbp amino acid sequences, which form three immunologically distinct clusters, termed variants 1, 2, and 3. Neither of the current vaccines contains a variant 2 antigen, which is less well characterized than fHbp variants 1 and 3. We characterized the interaction of fHbp variant 2 with humAb 1B1 using biochemical methods and live meningococcal assays. Further, we determined the crystal structure of the complex at 2.4 Å resolution, clearly revealing the epitope and providing the first detailed report of an antibody with distinct specificity for fHbp variant 2. Extensive mutagenesis and binding studies elucidated key hotspots in the interface. This combination of structural and functional studies provides a molecular explanation for the bactericidal potency and specificity of humAb 1B1 for fHbp variant 2. Our studies, focused on fHbp variant 2, expand the understanding of this previously under characterized group of the vast family of variants of fHbp, a virulence factor present on all meningococci. Moreover, the definition of a protective conformational epitope on fHbp variant 2 may support the design and development of novel variant 2-containing MenB vaccines affording greater breadth of protection.

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.

The factor H binding protein of Neisseria meningitidis interacts with xenosiderophores in vitro.

The factor H binding protein (fHbp) is a key virulence factor of Neisseria meningitidis that confers to the bacterium the ability to resist killing by human serum. The determination of its three-dimensional structure revealed that the carboxyl terminus of the protein folds into an eight-stranded ß barrel. The structural similarity of this part of the protein to lipocalins provided the rationale for exploring the ability of fHbp to bind siderophores. We found that fHbp was able to bind in vitro siderophores belonging to the cathecolate family and mapped the interaction site by nuclear magnetic resonance. Our results indicated that the enterobactin binding site was distinct from the site involved in binding to human factor H and stimulates new hypotheses about possible multiple activities of fHbp.

Structure of the C-terminal domain of Neisseria heparin binding antigen (NHBA), one of the main antigens of a novel vaccine against Neisseria meningitidis.

Neisseria heparin binding antigen (NHBA), also known as GNA2132 (genome-derived Neisseria antigen 2132), is a surface-exposed lipoprotein from Neisseria meningitidis that was originally identified by reverse vaccinology. It is one the three main antigens of a multicomponent vaccine against serogroup B meningitis (4CMenB), which has just completed phase III clinical trials in infants. In contrast to the other two main vaccine components, little is known about the origin of the immunogenicity of this antigen, and about its ability to induce a strong cross-bactericidal response in animals and humans. To characterize NHBA in terms of its structural/immunogenic properties, we have analyzed its sequence and identified a C-terminal region that is highly conserved in all strains. We demonstrate experimentally that this region is independently folded, and solved its three-dimensional structure by nuclear magnetic resonance. Notably, we need detergents to observe a single species in solution. The NHBA domain fold consists of an 8-strand ß-barrel that closely resembles the C-terminal domains of N. meningitidis factor H-binding protein and transferrin-binding protein B. This common fold together with more subtle structural similarities suggest a common ancestor for these important antigens and a role of the ß-barrel fold in inducing immunogenicity against N. meningitidis. Our data represent the first step toward understanding the relationship between structural, functional, and immunological properties of this important vaccine component.

Structural and biochemical characterization of NarE, an iron-containing ADP-ribosyltransferase from Neisseria meningitidis.

NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the family of ADP-ribosyltransferases (ADPRT) and catalyzes the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE is further the first ADPRT which could be shown to bind iron through a Fe-S center, which is crucial for the catalytic activity. Here we present the NMR solution structure of NarE, which shows structural homology to other ADPRTs. Using NMR titration experiments we could identify from Chemical Shift Perturbation data both the NAD binding site, which is in perfect agreement with a consensus sequence analysis between different ADPRTs, as well as the iron coordination site, which consists of 2 cysteines and 2 histidines. This atypical iron coordination is also capable to bind zinc. These results could be fortified by site-directed mutagenesis of the catalytic region, which identified two functionally crucial residues. We could further identify a main interaction region of NarE with antibodies using two complementary methods based on antibody immobilization, proteolytic digestion, and mass spectrometry. This study combines structural and functional features of NarE providing for the first time a characterization of an iron-dependent ADPRT.

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.

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.