ABSTRACT
Neisserial adhesin A (NadA) is a meningococcal surface protein included as recombinant antigen in 4CMenB, a protein-based vaccine able to induce protective immune responses against Neisseria meningitidis serogroup B (MenB). Although NadA is involved in the adhesion/invasion of epithelial cells and human myeloid cells, its function in meningococcal physiology is still poorly understood. To clarify the role played by NadA in the host-pathogen interaction, we sought to identify its cellular receptors. We screened a protein microarray encompassing 2,846 human and 297 mouse surface/secreted recombinant proteins using recombinant NadA as probe. Efficient NadA binding was revealed on the paired sialic acid-binding immunoglobulin-type lectins receptors 5 and 14 (Siglec-5 and Siglec-14), but not on Siglec-9 therein used as control. The interaction was confirmed by biochemical tools with the determination of the KD value in the order of nanomolar and the identification of the NadA binding site by hydrogen-deuterium exchange coupled to mass spectrometry. The N-terminal domain of the Siglec-5 that recognizes the sialic acid was identified as the NadA binding domain. Intriguingly, exogenously added recombinant soluble Siglecs, including Siglec-9, were found to decorate N. meningitidis surface in a NadA-dependent manner. However, Siglec-5 and Siglec-14 transiently expressed in CHO-K1 cells endorsed NadA binding and increased N. meningitidis adhesion/invasion while Siglec-9 did not. Taken together, Siglec-5 and Siglec-14 satisfy all features of NadA receptors suggesting a possible role of NadA in the acute meningococcal infection.IMPORTANCEBacteria have developed several strategies for cell colonization and immune evasion. Knowledge of the host and pathogen factors involved in these mechanisms is crucial to build efficacious countermoves. Neisserial adhesin A (NadA) is a meningococcal surface protein included in the anti-meningococcus B vaccine 4CMenB, which mediates adhesion to and invasion of epithelial cells. Although NadA has been shown to bind to other cell types, like myeloid and endothelial cells, it still remains orphan of a defined host receptor. We have identified two strong NadA interactors, Siglec-5 and Siglec-14, which are mainly expressed on myeloid cells. This showcases that NadA is an additional and key player among the Neisseria meningitidis factors targeting immune cells. We thus provide novel insights on the strategies exploited by N. meningitidis during the infection process, which can progress to a severe illness and death.
Subject(s)
Adhesins, Bacterial , Antigens, CD , Antigens, Differentiation, Myelomonocytic , Bacterial Adhesion , Host-Pathogen Interactions , Lectins , Humans , Adhesins, Bacterial/metabolism , Adhesins, Bacterial/genetics , Antigens, CD/metabolism , Antigens, CD/genetics , Lectins/metabolism , Lectins/genetics , Lectins/immunology , Animals , Antigens, Differentiation, Myelomonocytic/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Protein Binding , Mice , CHO Cells , Cricetulus , Neisseria meningitidis/genetics , Neisseria meningitidis/metabolism , Neisseria meningitidis/immunology , Recombinant Proteins/metabolism , Recombinant Proteins/genetics , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/genetics , Epithelial Cells/microbiology , Epithelial Cells/metabolism , Epithelial Cells/immunology , Meningococcal Infections/microbiology , Meningococcal Infections/immunology , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/genetics , Neisseria meningitidis, Serogroup B/genetics , Neisseria meningitidis, Serogroup B/immunology , Neisseria meningitidis, Serogroup B/metabolismABSTRACT
Predictions of vaccine efficacy against Neisseria meningitidis serogroup B (NmB) disease are hindered by antigenic variability, limiting the representativeness of individual NmB isolates. A qualitative human serum bactericidal assay using endogenous complements of individual subjects (enc-hSBA) enables large panels of NmB isolates to be tested. A 110-isolate panel was randomly selected from 442 invasive NmB isolates from United States cases reported to the Centers for Disease Control (CDC) from 2000 to 2008. Typing analyses confirmed the 110-isolate panel is representative of the 442 isolates. The genetic features of the 110-isolate panel were compared against over 4,200 invasive NmB isolates collected from 2000 to 2018 in the United States, Australia, Canada, and nine European countries. Clonal complexes in the 110-isolate panel are also present in each geographical region; cumulative percentages show that these account for around 81% of the clonal complexes found in NmB isolates in other panels. For the antigens (fHbp, NHBA, PorA1.4, NadA) included in the currently licensed meningococcal serogroup B (MenB) vaccines, specifically considering the presence of at least one antigen with a matched genotype, the 110-isolate panel represents approximately 89% of the NmB isolates circulating worldwide, ranging from 87% for the European isolates to 95% and 97% for NmB isolates in the United States and Australia, respectively. The 110-isolate panel includes the most prevalent clonal complexes and genetic variants of MenB vaccine antigens found in a multinational collection of invasive NmB isolates. This panel is useful for assessing the efficacy of MenB vaccines in clinical trials worldwide. IMPORTANCE Neisseria meningitidis serogroup B (NmB) is a major cause of invasive meningococcal disease (IMD). Predicting the effectiveness of vaccines against NmB is difficult because NmB is an uncommon disease and because antigens targeted by meningococcal serogroup B (MenB) vaccines have highly variable genetic features and expression levels. Therefore, a large number of NmB isolates from different regions would need to be tested to comprehensively assess vaccine effectiveness. We examined a panel of 110 isolates obtained from NmB IMD cases in the United States and compared the genetic features of this panel with those of panels from different countries around the world. We found the 110-isolate panel included the most common clonal complexes and genetic variants of MenB vaccine antigens that exist in the global collections of invasive NmB isolates. This confirms the value of the NmB 110-isolate panel in understanding the effectiveness of MenB vaccines in clinical trials worldwide.
Subject(s)
Meningococcal Infections , Meningococcal Vaccines , Neisseria meningitidis, Serogroup B , Humans , United States , Antigens, Bacterial/genetics , Meningococcal Infections/prevention & control , GenotypeABSTRACT
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.
ABSTRACT
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.
Subject(s)
Antibodies/immunology , Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Meningitis, Meningococcal/immunology , Meningococcal Vaccines/administration & dosage , Neisseria meningitidis/immunology , Adult , Antibodies/blood , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Complement Factor H/immunology , Complement Factor H/metabolism , Humans , Meningitis, Meningococcal/metabolism , Meningitis, Meningococcal/microbiology , Meningitis, Meningococcal/prevention & controlABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Complement Factor H/immunology , Epitopes/immunology , Meningococcal Vaccines/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Complement Factor H/genetics , Complement Factor H/metabolism , Crystallography, X-Ray , Epitopes/genetics , Epitopes/metabolism , Genetic Variation , Humans , Meningococcal Infections/microbiology , Meningococcal Infections/prevention & control , Meningococcal Vaccines/administration & dosage , Models, Molecular , Neisseria meningitidis/drug effects , Neisseria meningitidis/immunology , Neisseria meningitidis/physiology , Protein Binding , Protein ConformationABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/chemistry , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Antibodies, Monoclonal/immunology , Blood Bactericidal Activity , Complement Factor H/metabolism , Epitope Mapping , Humans , Meningococcal Vaccines/immunology , Microscopy, Electron, Transmission , Surface Plasmon ResonanceABSTRACT
The discovery of vaccine antigens through whole genome sequencing (WGS) contrasts with the classical hypothesis-driven laboratory-based analysis of microbes to identify components to elicit protective immunity. This radical change in scientific direction and action in vaccine research is captured in the term reverse vaccinology. The complete genome sequence of an isolate of Neisseria meningitidis serogroup B (MenB) was systematically analyzed to identify proteins predicted to be secreted or exported to the outer membrane. This identified hundreds of genes coding for potential surface-exposed antigens. These were amplified, cloned in expression vectors and used to immunize mice. Antisera against 350 recombinant antigens were obtained and analyzed in a panel of immunological assays from which 28 were selected as potentially protective based on the -antibody dependent, complement mediated- serum bactericidal activity assay. Testing of these candidate vaccine antigens, using a large globally representative strain collection of Neisseria species isolated from cases of disease and carriage, indicated that no single component would be sufficient to induce broad coverage and that a "universal" vaccine should contain multiple antigens. The final choice of antigens to be included was based on cross-protective ability, assayed by serum bactericidal activity and maximum coverage of the extensive antigenic variability of MenB strains. The resulting multivalent vaccine formulation selected consisted of three recombinant antigens (Neisserial Heparin Binding Antigen or NHBA, Factor H binding protein or fHbp and Neisseria Adhesin A or NadA). To improve immunogenicity and potential strain coverage, an outer membrane vesicle component obtained from the epidemic New Zealand strain (OMVNz) was added to the formulation to create a four component vaccine, called 4CMenB. A series of phase 2 and 3 clinical trials were conducted to evaluate safety and tolerability and to estimate the vaccine effectiveness of human immune responses at different ages and how these were affected by various factors including concomitant vaccine use and lot-to-lot consistency. 4CMenB was approved in Europe in 2013 and introduced in the National Immunization Program in the UK starting from September 2015 when the vaccine was offered to all newborns using a 2, 4, and 12 months schedule., The effectiveness against invasive MenB disease measured at 11 months after the study start and 5 months after the second vaccination was 83% and there have been no safety concerns.
Subject(s)
Meningitis, Meningococcal/immunology , Meningitis, Meningococcal/prevention & control , Meningococcal Vaccines/immunology , Neisseria meningitidis, Serogroup B/immunology , Vaccinology/methods , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Computational Biology , Genes, Bacterial , Humans , Meningococcal Vaccines/genetics , Mice , Neisseria meningitidis, Serogroup B/genetics , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Whole Genome SequencingABSTRACT
Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections most commonly in immunocompromised, cystic fibrosis (CF) and burns patients. The pilin and Pseudomonas lectins 1 (PA-IL) and 2 (PA-IIL) are known glycan-binding proteins of P. aeruginosa that are involved in adherence to host cells, particularly CF host airways. Recently, new P. aeruginosa surface proteins were identified by reverse vaccinology and tested in vivo as potential vaccine antigens. Three of these, namely PSE17-1, PSE41-5 and PSE54, were screened for glycan binding using glycan arrays displaying glycan structures representative of those found on human cells. Surface plasmon resonance was used to confirm the lectin activity of these proteins, and determined affinities with several host glycans to be in the nanomolar range. PSE17-1 binds hyaluronic acid and sialyl Lewis A and X. PSE41-5 binds terminal ß-linked galactose structures, Lewis and ABO blood group antigens. PSE54 binds to ABO blood group antigens and some terminal ß-linked galactose. All three proteins are novel lectins of P. aeruginosa with potential roles in infection of host cells.
Subject(s)
Bacterial Proteins/metabolism , Lectins/metabolism , Polysaccharides/metabolism , Pseudomonas Infections/metabolism , Pseudomonas aeruginosa/physiology , Bacterial Adhesion , Humans , Pseudomonas Infections/prevention & control , Pseudomonas Vaccines/metabolism , Virulence Factors/metabolismABSTRACT
INTRODUCTION: Neisseria meningitidis serogroup B (MenB) is the most common cause of bacterial meningitis in many industrialized countries and occurs at any age. The highest incidence is in infants aged <1 year, followed by children and adolescents. Four-component MenB vaccine (4CMenB, Bexsero) is the only MenB vaccine authorized for use in all age-groups. Experience with 4CMenB is growing as it is implemented in different countries/age-groups encompassing university students, children, adolescents, and infant mass vaccination programs. AREAS COVERED: An update of recently available data describing the mechanism of immunogenicity of 4CMenB and real-world evidence of vaccine effectiveness and disease impact. We discuss the appropriate age for vaccination to maximize population impacts. EXPERT COMMENTARY: Invasive meningococcal disease is uncommon and sufficiently powered efficacy studies were not feasible during 4CMenB development. Additionally, several thousand genetically diverse invasive MenB strains circulate globally, varying widely in surface protein expression. This posed significant challenges in predicting clinical protection with MenB vaccines. Five years of 4CMenB use post-licensure confirm the clinical benefit of vaccination as predicted during development. Preliminary evidence suggests an extended impact on other meningococcal serogroups and Neisseria gonorrhoeae.
Subject(s)
Meningitis, Meningococcal/prevention & control , Meningococcal Vaccines/administration & dosage , Neisseria meningitidis, Serogroup B/immunology , Adolescent , Age Factors , Child , Humans , Infant , Meningitis, Meningococcal/epidemiology , Meningitis, Meningococcal/immunology , Meningococcal Vaccines/immunology , Vaccination/methodsABSTRACT
High incidence, severity and increasing antibiotic resistance characterize Pseudomonas aeruginosa infections, highlighting the need for new therapeutic options. Vaccination strategies to prevent or limit P. aeruginosa infections represent a rational approach to positively impact the clinical outcome of risk patients; nevertheless this bacterium remains a challenging vaccine target. To identify novel vaccine candidates, we started from the genome sequence analysis of the P. aeruginosa reference strain PAO1 exploring the reverse vaccinology approach integrated with additional bioinformatic tools. The bioinformatic approaches resulted in the selection of 52 potential antigens. These vaccine candidates were conserved in P. aeruginosa genomes from different origin and among strains isolated longitudinally from cystic fibrosis patients. To assess the immune-protection of single or antigens combination against P. aeruginosa infection, a vaccination protocol was established in murine model of acute respiratory infection. Combinations of selected candidates, rather than single antigens, effectively controlled P. aeruginosa infection in the in vivo model of murine pneumonia. Five combinations were capable of significantly increase survival rate among challenged mice and all included PA5340, a hypothetical protein exclusively present in P. aeruginosa. PA5340 combined with PA3526-MotY gave the maximum protection. Both proteins were surface exposed by immunofluorescence and triggered a specific immune response. Combination of these two protein antigens could represent a potential vaccine to prevent P. aeruginosa infection.
Subject(s)
Genome, Bacterial , Genomics , Pseudomonas Infections/immunology , Pseudomonas Vaccines/genetics , Pseudomonas Vaccines/immunology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/immunology , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Cystic Fibrosis/complications , Disease Models, Animal , Genome-Wide Association Study , Genomics/methods , Humans , Mice , Open Reading Frames , Pseudomonas Infections/etiology , Pseudomonas Infections/mortality , Pseudomonas Infections/prevention & control , Pseudomonas Vaccines/administration & dosage , Pseudomonas aeruginosa/isolation & purificationABSTRACT
Streptococcus pneumoniae is a leading cause of morbidity and mortality globally. The Pilus-1 proteins, RrgA, RrgB and RrgC of S. pneumoniae have been previously assessed for their role in infection, invasive disease and as possible vaccine candidates. In this study we have investigated the glycan binding repertoire of all three Pilus-1 proteins, identifying that the tip adhesin RrgA has the broadest glycan recognition of the three proteins, binding to maltose/cellobiose, α/ß linked galactose and blood group A and H antigens. RrgB only bound mannose, while RrgC bound a subset of glycans also recognized by RrgA. Adherence of S. pneumoniae TIGR4 to epithelial cells was tested using four of the oligosaccharides identified through the glycan array analysis as competitive inhibitors. The blood group H trisaccharide provided the best blocking of S. pneumoniae TIGR4 adherence. Adherence is the first step in disease, and host glycoconjugates are a common target for many adhesins. This study has identified Pilus-1 proteins as new lectins involved in the targeting of host glycosylation by S. pneumoniae.
Subject(s)
Fimbriae, Bacterial/metabolism , Lectins/metabolism , Streptococcus pneumoniae/metabolism , A549 Cells , Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Cell Line, Tumor , Cellobiose/metabolism , Epithelial Cells/metabolism , Fimbriae Proteins/metabolism , Galactose/metabolism , Humans , Maltose/metabolism , Protein Binding/physiology , Virulence Factors/metabolismABSTRACT
INTRODUCTION: The successful development of two broadly protective vaccines targeting Neisseria meningitidis serogroup B (MenB); 4CMenB and rLP2086, is the most significant recent advance in meningococcal disease prevention. Areas covered: Here we review the principles underlying the development of each vaccine and the novel methods used to estimate vaccine coverage. We update clinical and post-licensure experience with 4CMenB and rLP2086. Expert commentary: The immunogenicity and acceptable safety profile of 4CMenB and rLP2086 has been demonstrated in clinical trials. Continuing uncertainties exist around the appropriate age groups to be immunized, the degree and duration of efficacy, and the impact on nasopharyngeal carriage which has implications for strategies to interrupt transmission and maximize herd protection effects. Universal vaccination programs such as those undertaken in Quebec and the United Kingdom are providing important information on these issues. The potential for MenB vaccines to prevent infection by other serogroups appears promising, and the impact of MenB vaccines on other pathogenic neisserial species with similar surface proteins warrants further investigation.
Subject(s)
Meningococcal Infections/epidemiology , Meningococcal Infections/prevention & control , Meningococcal Vaccines/administration & dosage , Meningococcal Vaccines/immunology , Neisseria meningitidis, Serogroup B/immunology , Carrier State/epidemiology , Carrier State/prevention & control , Clinical Trials as Topic , Global Health , Humans , Meningococcal Vaccines/adverse effects , Product Surveillance, PostmarketingABSTRACT
Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by Neisseria meningitidis strains and an antigen of the Bexsero® vaccine. NHBA binds heparin through a conserved Arg-rich region that is the target of two proteases, the meningococcal NalP and human lactoferrin (hLf). In this work, in vitro studies showed that recombinant NHBA protein was able to bind epithelial cells and mutations of the Arg-rich tract abrogated this binding. All N-terminal and C-terminal fragments generated by NalP or hLf cleavage, regardless of the presence or absence of the Arg-rich region, did not bind to cells, indicating that a correct positioning of the Arg-rich region within the full length protein is crucial. Moreover, binding was abolished when cells were treated with heparinase III, suggesting that this interaction is mediated by heparan sulfate proteoglycans (HSPGs). N. meningitidis nhba knockout strains showed a significant reduction in adhesion to epithelial cells with respect to isogenic wild-type strains and adhesion of the wild-type strain was inhibited by anti-NHBA antibodies in a dose-dependent manner. Overall, the results demonstrate that NHBA contributes to meningococcal adhesion to epithelial cells through binding to HSPGs and suggest a possible role of anti-Bexsero® antibodies in the prevention of colonization.
Subject(s)
Antibodies, Bacterial/genetics , Antibodies, Bacterial/metabolism , Bacterial Adhesion , Bacterial Outer Membrane Proteins/metabolism , Carrier Proteins/metabolism , Epithelial Cells/microbiology , Meningococcal Infections/drug therapy , Neisseria meningitidis/physiology , Antibodies, Bacterial/chemistry , Binding Sites , Cell Line , Gene Knockout Techniques , Heparan Sulfate Proteoglycans/metabolism , Heparin/metabolism , Humans , Lactoferrin/metabolism , Membrane Transport Proteins/metabolism , Meningococcal Infections/microbiology , Meningococcal Infections/prevention & control , Mutation , Neisseria meningitidis/immunology , Recombinant Proteins/metabolism , Serine Endopeptidases/metabolismABSTRACT
We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete "hot spots" with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/chemistry , Bacteriophage lambda/genetics , Epitopes/genetics , High-Throughput Nucleotide Sequencing , Peptide Library , Animals , MiceABSTRACT
We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a component of the anti-group B meningococcus Bexsero® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.
Subject(s)
Antibodies, Monoclonal/immunology , Bacterial Outer Membrane Proteins/immunology , Carrier Proteins/immunology , Epitope Mapping/methods , Peptide Library , Animals , Bacterial Outer Membrane Proteins/genetics , Carrier Proteins/genetics , Cross Reactions , High-Throughput Nucleotide Sequencing , Mass Spectrometry/methods , Mice , Neisseria meningitidis, Serogroup B/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
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.
Subject(s)
Host-Pathogen Interactions , Neisseria meningitidis/physiology , Protein Array Analysis/methods , Staphylococcus aureus/physiology , Adhesins, Bacterial/chemistry , Adhesins, Bacterial/metabolism , Animals , Bacterial Proteins/metabolism , Binding Sites , CHO Cells , Complement C1q/metabolism , Cricetulus , Humans , Protein Binding , Recombinant Proteins/metabolism , Scavenger Receptors, Class E/metabolismABSTRACT
There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero(®) anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes.
Subject(s)
Adhesins, Bacterial/immunology , Antibodies, Bacterial/immunology , Antibodies, Monoclonal/immunology , Cell Surface Display Techniques/methods , Epitope Mapping/methods , Neisseria meningitidis/immunology , Animals , Bacterial Vaccines/immunology , Meningococcal Vaccines , Peptide Fragments/immunologyABSTRACT
Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Neisseria meningitidis, Serogroup B/metabolism , Amino Acid Sequence , Antigens, Bacterial/genetics , Antigens, Bacterial/immunology , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Genetic Variation , Humans , Mass Spectrometry/methods , Meningitis, Meningococcal/immunology , Meningitis, Meningococcal/microbiology , Meningococcal Vaccines/immunology , Neisseria meningitidis, Serogroup B/classification , Neisseria meningitidis, Serogroup B/genetics , Phylogeny , Species SpecificityABSTRACT
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.
Subject(s)
Adhesins, Bacterial/immunology , Antibodies, Bacterial/immunology , Epitopes/immunology , Meningococcal Infections/immunology , Meningococcal Vaccines/immunology , Neisseria/immunology , Animals , Deuterium Exchange Measurement/methods , Epitope Mapping/methods , Humans , MiceABSTRACT
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.