Investigating the Role of Antigen Orientation on the Immune Response Elicited by Neisseria meningitidis Factor H Binding Protein on GMMA.

GMMA are outer membrane vesicles (OMVs) released from Gram-negative bacteria genetically modified to enhance OMVs formation that have been shown to be optimal systems to enhance immunogenicity of protein antigens. Here, we selected Neisseria meningitidis factor H binding protein (fHbp) and used the conjugation chemistry as a tool to alter antigen orientation on GMMA. Indeed, fHbp was randomly linked to GMMA or selectively attached via the N-terminus to mimic native presentation of the protein on the bacterial surface. Interestingly, protein and peptide array analyses confirmed that antibodies induced by the selective and the random conjugates showed a pattern very similar to fHbp natively expressed on bacterial surfaces or to the recombinant protein mixed with GMMA, respectively. However, the two conjugates elicited antibodies with similar serum bactericidal activity against meningococcal strains, superior to the protein alone or physically mixed with GMMA. Presentation of fHbp on GMMA strongly enhances the functional immune response elicited by the protein but its orientation on the bacterial surface does not have an impact. This study demonstrates the flexibility of the GMMA platform as a display and delivery system for enhancing antigen immunogenicity and further supports the use of such promising technology for the development of effective vaccines.

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.

Computational modeling of microfluidic data provides high-throughput affinity estimates for monoclonal antibodies.

Affinity measurement is a fundamental step in the discovery of monoclonal antibodies (mAbs) and of antigens suitable for vaccine development. Innovative affinity assays are needed due to the low throughput and/or limited dynamic range of available technologies. We combined microfluidic technology with quantum-mechanical scattering theory, in order to develop a high-throughput, broad-range methodology to measure affinity. Fluorescence intensity profiles were generated for out-of-equilibrium solutions of labelled mAbs and their antigen-binding fragments migrating along micro-columns with immobilized cognate antigen. Affinity quantification was performed by computational data analysis based on the Landau probability distribution. Experiments using a wide array of human or murine antibodies against bacterial or viral, protein or polysaccharide antigens, showed that all the antibody-antigen capture profiles (n = 841) generated at different concentrations were accurately described by the Landau distribution. A scale parameter W, proportional to the full-width-at-half-maximum of the capture profile, was shown to be independent of the antibody concentration. The W parameter correlated significantly (Pearson's r [p-value]: 0.89 [3 × 10-8]) with the equilibrium dissociation constant KD, a gold-standard affinity measure. Our method showed good intermediate precision (median coefficient of variation: 5%) and a dynamic range corresponding to KD values spanning from ~10-7 to ~10-11 Molar. Relative to assays relying on antibody-antigen equilibrium in solution, even when they are microfluidic-based, the method's turnaround times were decreased from 2 days to 2 h. The described computational modelling of antibody capture profiles represents a fast, reproducible, high-throughput methodology to accurately measure a broad range of antibody affinities in very low volumes of solution.

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.

Integration of high-throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi-subunit vaccine antigen.

Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. Individual clone expansion and phenotype-based ranking is performed initially for hundreds or thousands of mini-scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200-1,200 cell colony stage, within 14 days of the single cell cloning in static 96-well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single-step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top-ranked clones identified using an established iterative clone screening approach. Using a complex, multi-subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed-batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.

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.

NadA3 Structures Reveal Undecad Coiled Coils and LOX1 Binding Regions Competed by Meningococcus B Vaccine-Elicited Human Antibodies.

Neisseria meningitidis serogroup B (MenB) is a major cause of sepsis and invasive meningococcal disease. A multicomponent vaccine, 4CMenB, is approved for protection against MenB. Neisserial adhesin A (NadA) is one of the main vaccine antigens, acts in host cell adhesion, and may influence colonization and invasion. Six major genetic variants of NadA exist and can be classified into immunologically distinct groups I and II. Knowledge of the crystal structure of the 4CMenB vaccine component NadA3 (group I) would improve understanding of its immunogenicity, folding, and functional properties and might aid antigen design. Here, X-ray crystallography, biochemical, and cellular studies were used to deeply characterize NadA3. The NadA3 crystal structure is reported; it revealed two unexpected regions of undecad coiled-coil motifs and other conformational differences from NadA5 (group II) not predicted by previous analyses. Structure-guided engineering was performed to increase NadA3 thermostability, and a second crystal structure confirmed the improved packing. Functional NadA3 residues mediating interactions with human receptor LOX-1 were identified. Also, for two protective vaccine-elicited human monoclonal antibodies (5D11, 12H11), we mapped key NadA3 epitopes. These vaccine-elicited human MAbs competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. The data presented provide a significant advance in the understanding of the structure, immunogenicity and function of NadA, one of the main antigens of the multicomponent meningococcus B vaccine.IMPORTANCE The bacterial microbe Neisseria meningitidis serogroup B (MenB) is a major cause of devastating meningococcal disease. An approved multicomponent vaccine, 4CMenB, protects against MenB. Neisserial adhesin A (NadA) is a key vaccine antigen and acts in host cell-pathogen interactions. We investigated the 4CMenB vaccine component NadA3 in order to improve the understanding of its immunogenicity, structure, and function and to aid antigen design. We report crystal structures of NadA3, revealing unexpected structural motifs, and other conformational differences from the NadA5 orthologue studied previously. We performed structure-based antigen design to engineer increased NadA3 thermostability. Functional NadA3 residues mediating interactions with the human receptor LOX-1 and vaccine-elicited human antibodies were identified. These antibodies competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. Our data provide a significant advance in the overall understanding of the 4CMenB vaccine antigen NadA.

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 ß-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 ß-hairpin fragment and the ß-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 ß-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.

Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein.

Stability is one of the critical attributes of a protein-based therapeutic or vaccine product, which is directly linked to product quality and efficacy. Elucidating protein degradation pathways is required to obtain thorough understanding of the product and ensure degradation products are properly monitored. We observed a unique protein degradation involving nonenzyme catalyzed loss of a complete N-linked glycan under stress condition from an engineered respiratory syncytial virus (RSV) prefusion F protein (RSVPreF3). Investigations involving mass spectrometry, molecular modeling, and mutagenesis revealed that the glycan shedding was site-specific, dependent on structural elements, and required a glycine residue immediately following the site of glycosylation. The glycan loss did not negatively affect the binding between the main immunogenic epitope Site Ø and the neutralizing antibody D25. Further study indicated that the glycan shedding followed a similar but different mechanism than that of conventional deamidation. Since glycosylation is an important attribute for many recombinant therapeutic proteins or vaccine antigens, the finding from this study suggests the need to monitor this new type of degradation, especially when glycosylation has an impact on efficacy or safety.

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.

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.

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.

Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA.

Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis.

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.

Exploiting chimeric human antibodies to characterize a protective epitope of Neisseria adhesin A, one of the Bexsero vaccine components.

Neisseria adhesin A (NadA) is one of the antigens of Bexsero, the recently licensed multicomponent vaccine against serogroup B Neisseria meningitidis (MenB). NadA belongs to the class of oligomeric coiled-coil adhesins and is able to mediate adhesion and invasion of human epithelial cells. As a vaccine antigen, NadA has been shown to induce high levels of bactericidal antibodies; however, the domains important for protective response are still unknown. In order to further investigate its immunogenic properties, we have characterized the murine IgG1 mAb (6E3) that was able to recognize the 2 main antigenic variants of NadA on the surface of MenB strains. The epitope targeted by mAb 6E3 was mapped by hydrogen-deuterium exchange mass spectrometry and shown to be located on the coiled-coil stalk region of NadA (aa 206-249). Although no serum bactericidal activity was observed for murine IgG1 mAb 6E3, functional activity was restored when using chimeric antibodies in which the variable regions of the murine mAb 6E3 were fused to human IgG3 constant regions, thus confirming the protective nature of the mAb 6E3 epitope. The use of chimeric antibody molecules will enable future investigations of complement-mediated antibody functionality independently of the Fc-mediated differences in complement activation.

Molecular Engineering of Ghfp, the Gonococcal Orthologue of Neisseria meningitidis Factor H Binding Protein.

Knowledge of the sequences and structures of proteins produced by microbial pathogens is continuously increasing. Besides offering the possibility of unraveling the mechanisms of pathogenesis at the molecular level, structural information provides new tools for vaccine development, such as the opportunity to improve viral and bacterial vaccine candidates by rational design. Structure-based rational design of antigens can optimize the epitope repertoire in terms of accessibility, stability, and variability. In the present study, we used epitope mapping information on the well-characterized antigen of Neisseria meningitidis factor H binding protein (fHbp) to engineer its gonococcal homologue, Ghfp. Meningococcal fHbp is typically classified in three distinct antigenic variants. We introduced epitopes of fHbp variant 1 onto the surface of Ghfp, which is naturally able to protect against meningococcal strains expressing fHbp of variants 2 and 3. Heterologous epitopes were successfully transplanted, as engineered Ghfp induced functional antibodies against all three fHbp variants. These results confirm that structural vaccinology represents a successful strategy for modulating immune responses, and it is a powerful tool for investigating the extension and localization of immunodominant epitopes.

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.

Apo, Zn2+-bound and Mn2+-bound structures reveal ligand-binding properties of SitA from the pathogen Staphylococcus pseudintermedius.

The Gram-positive bacterium Staphylococcus pseudintermedius is a leading cause of canine bacterial pyoderma, resulting in worldwide morbidity in dogs. S. pseudintermedius also causes life-threatening human infections. Furthermore, methicillin-resistant S. pseudintermedius is emerging, resembling the human health threat of methicillin-resistant Staphylococcus aureus. Therefore it is increasingly important to characterize targets for intervention strategies to counteract S. pseudintermedius infections. Here we used biophysical methods, mutagenesis, and X-ray crystallography, to define the ligand-binding properties and structure of SitA, an S. pseudintermedius surface lipoprotein. SitA was strongly and specifically stabilized by Mn2+ and Zn2+ ions. Crystal structures of SitA complexed with Mn2+ and Zn2+ revealed a canonical class III solute-binding protein with the metal cation bound in a cavity between N- and C-terminal lobes. Unexpectedly, one crystal contained both apo- and holo-forms of SitA, revealing a large side-chain reorientation of His64, and associated structural differences accompanying ligand binding. Such conformational changes may regulate fruitful engagement of the cognate ABC (ATP-binding cassette) transporter system (SitBC) required for metal uptake. These results provide the first detailed characterization and mechanistic insights for a potential therapeutic target of the major canine pathogen S. pseudintermedius, and also shed light on homologous structures in related staphylococcal pathogens afflicting humans.

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.

EsiB, a novel pathogenic Escherichia coli secretory immunoglobulin A-binding protein impairing neutrophil activation.

UNLABELLED: In this study, we have characterized the functional properties of a novel Escherichia coli antigen named EsiB (E. coli secretory immunoglobulin A-binding protein), recently reported to protect mice from sepsis. Gene distribution analysis of a panel of 267 strains representative of different E. coli pathotypes revealed that esiB is preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains. These findings were supported by the presence of anti-EsiB antibodies in the sera of patients affected by urinary tract infections (UTIs). By solving its crystal structure, we observed that EsiB adopts a superhelical fold composed of Sel1-like repeats (SLRs), a feature often associated with bacterial proteins possessing immunomodulatory functions. Indeed, we found that EsiB interacts with secretory immunoglobulin A (SIgA) through a specific motif identified by an immunocapturing approach. Functional assays showed that EsiB binding to SIgA is likely to interfere with productive FcαRI signaling, by inhibiting both SIgA-induced neutrophil chemotaxis and respiratory burst. Indeed, EsiB hampers SIgA-mediated signaling events by reducing the phosphorylation status of key signal-transducer cytosolic proteins, including mitogen-activated kinases. We propose that the interference with such immune events could contribute to the capacity of the bacterium to avoid clearance by neutrophils, as well as reducing the recruitment of immune cells to the infection site. IMPORTANCE: Pathogenic Escherichia coli infections have recently been exacerbated by increasing antibiotic resistance and the number of recurrent contagions. Attempts to develop preventive strategies against E. coli have not been successful, mainly due to the large antigenic and genetic variability of virulence factors, but also due to the complexity of the mechanisms used by the pathogen to evade the immune system. In this work, we elucidated the function of a recently discovered protective antigen, named EsiB, and described its capacity to interact with secretory immunoglobulin A (SIgA) and impair effector functions. This work unravels a novel strategy used by E. coli to subvert the host immune response and avoid neutrophil-dependent clearance.