An epitope‐imprinted piezoelectric diagnostic tool for Neisseria meningitidis detection

Neisseria meningitidis, a human‐specific bacterial pathogen causes bacterial meningitis by invading the meninges (outer lining) of central nervous system. It is the polysaccharide present on the bacterial capsid that distinguishes various serogroups of N. meningitidis and can be utilized as antigens to elicit immune response. A computational approach identified candidate T‐cell epitopes from outer membrane proteins Por B of N. meningitidis (MC58): (²⁷³KGLVDDADI²⁸² in loop VII and ¹⁷⁰GRHNSESYH¹⁷⁹ in loop IV) present on the exposed surface of immunogenic loops of class 3 outer membrane proteins allele of N. meningitidis. One of them, KGLVDDADI is used here for designing a diagnostic tool via molecularly imprinted piezoelectric sensor (molecularly imprinted polymer‐quartz crystal microbalance) for N. meningitidis strain MC58. Methacrylic acid, ethylene glycol dimethacrylate and azoisobutyronitrile were used as functional monomer, cross‐linker and initiator, respectively. The epitope can be simultaneously bound to methacrylic acid and fitted into the shape‐selective cavities. On extraction of epitope sequence from thus grafted polymeric film, shape‐selective and sensitive sites were generated on electrochemical quartz crystal microbalance crystal, ie, known as epitope imprinted polymers. Imprinting was characterized by atomic force microscopy images. The epitope‐imprinted sensor was able to selectively bind N. meningitidis proteins present in blood serum of patients suffering from brain fever. Thus, fabricated sensor can be used as a diagnostic tool for meningitis disease.     

Detection of oxytocin,atrial natriuretic peptide,and brain natriuretic peptide using novel imprinted polymers produced with amphiphilic monomers

The cystine‐bridged cyclic peptide hormones (CBCPHs) represent signature structural feature as well as unique biological activity. In this study, three CBCPHs have been identified and characterized, namely, oxytocin, atrial natriuretic peptides (ANPs), and brain natriuretic peptides (BNPs). Because research has shown that ANPs and BNPs are powerful diagnostic biomarkers for heart disease, a highly laudable endeavor would be to develop a novel sensor for detecting ANP or BNP levels. Therefore, an amphiphilic monomer Acr‐His‐NHNH‐Fmoc was synthesized to form molecularly imprinted polymers (MIPs) for targeted CBCPH detection. First, oxytocin, a cardiovascular hormone and a CBCPH, was used as a template to fabricate MIPs on quartz crystal microbalance (QCM) chips. On the other hand, fabricated selected ANP segment or BNP segment as an epitope is able to construct epitope‐mediated MIPs (EMIPs) for ANP or BNP. The developed oxytocin or ANP sensor reached a detection limitation of 0.1nM with the dissociation constants being 30pM for oxytocin and 20pM for ANP. Moreover, BNP sensor achieved a detection limitation of 2.89pM with an even lower K_d value as 2pM. Compared with the performance of EMIPs, the imprinted films showed high affinity and selectivity in special binding to CBCPHs. The developed MIPs‐QCM biosensors thus provide an improved sensing platform using an amphiphilic monomer and may be useful for applications toward cyclotides, cystine knot motifs, or insulin‐like peptides.     

Analysis of the human Ig isotype response to lactoferrin binding protein A from Neisseria meningitidis

An effective vaccine for serogroup B meningococci has yet to be developed and attention has turned to subcapsular antigens of the meningococcus as possible vaccine candidates. Iron binding proteins are being studied, with most interest focused on the transferrin binding proteins (TbpA and TbpB) and the ferric binding protein (FbpA). This study describes the purification of lactoferrin binding protein A (LbpA) from two meningococcal strains and assesses the human isotype-specific serum antibody response to these proteins in patients with proven meningococcal disease due to a range of phenotypes. Overall, fewer than 50% of sera contained IgG that recognised LbpA isolated from either strain and this antibody response was not uniform between the two proteins. There was some evidence that the antibody response varied between meningococcal phenotypes. This study demonstrates that LbpA does not induce a highly cross-reactive antibody response, indicating that it is unlikely to be an effective vaccine antigen.     

Purification of the Neisseria meningitidis transferrin binding protein-2 (TBP2) to homogeneity using column chromatography

Abstract A method for purifying TBP2 from N. meningitidis has been developed using affirnity chromatography on Tf-agarose followed by ion exchange chromatography; the Tf-binding activity of fractions was evaluated by a dot-blot technique. This method allowed the purification of the TBP2 in milder conditions than those used previously and to a high degree of homogeneity as was demonstrated by Coomassie brilliant blue or Silver training after SDS-PAGE electrophoresis. The SDS-PAGE profile of the material obtained in the Tf-agarose affinity chromatography step shows only two detectable proteins, identified as the TBP1 and the TBP2, with a small amount of contamination. Passage through a MonoQ HR anion exchange column, allowed the isolation of TBP2 in the absence of TBP1. Our results demonstrate the conservation of the antigenic reactivity of this protein, which produces monospecific serum with the antibodies elicited reacting exclusively with the TBP2 in whole outer membrane vesicles.     

Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology

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.     

Identification of a cation transport pathway in Neisseria meningitidis PorB

Neisseria meningitidis is the main causative agent of bacterial meningitis. In its outer membrane, the trimeric Neisserial porin PorB is responsible for the diffusive transport of essential hydrophilic solutes across the bilayer. Previous molecular dynamics simulations based on the recent crystal structure of PorB have suggested the presence of distinct solute translocation pathways through this channel. Although PorB has been electrophysiologically characterized as anion‐selective, cation translocation through nucleotide‐bound PorB during pathogenesis is thought to be instrumental for host cell death. As a result, we were particularly interested in further characterizing cation transport through the pore. We combined a structural approach with additional computational analysis. Here, we present two crystal structures of PorB at 2.1 and 2.65 Å resolution. The new structures display additional electron densities around the protruding loop 3 (L3) inside the pore. We show that these electron densities can be identified as monovalent cations, in our case Cs⁺, which are tightly bound to the inner channel. Molecular dynamics simulations reveal further ion interactions and the free energy landscape for ions inside PorB. Our results suggest that the crystallographically identified locations of Cs⁺ form a cation transport pathway inside the pore. This finding suggests how positively charged ions are translocated through PorB when the channel is inserted into mitochondrial membranes during Neisserial infection, a process which is considered to dissipate the mitochondrial transmembrane potential gradient and thereby induce apoptosis. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Identification and characterisation of a novel conserved outer membrane protein from Neisseria meningitidis

We have identified a homologue of the adhesin AIDA-I of Escherichia coli in Neisseria meningitidis. This gene was designated nhhA (Neisseria hia homologue), as analysis of the complete coding sequence revealed that it is more closely related to the adhesins Hia and Hsf of Haemophilus influenzae. The sequence of nhhA was determined from 10 strains, and found to be highly conserved. Studies of the localisation by Western immunoblot analysis of total cell proteins and outer membrane complex preparations and by immunogold electron microscopy revealed that NhhA is located in the outer membrane. A strain survey showed that nhhA is present in 85/85 strains of N. meningitidis representative of all the major disease-associated serogroups, based on Southern blot analysis. It is expressed in the majority of strains tested by Western immunoblot.     

A molecularly imprinted nanofilm‐based quartz crystal microbalance sensor for the real‐time detection of pirimicarb

This study aimed to prepare a novel quartz crystal microbalance (QCM) sensor for the detection of pirimicarb. Pirimicarb‐imprinted poly (ethylene glycol dimethacrylate‐N‐metacryloyl‐(l )‐tryptophan methyl ester) [p (EGDMA‐MATrp)] nanofilm (MIP) on the gold surface of a QCM chip was synthesized using the molecular imprinting technique. A nonimprinted p (EGDMA‐MATrp) nanofilm (NIP) was also synthesized using the same experimental technique. The MIP and NIP nanofilms were characterized via Fourier transform infrared spectroscopy attenuated total reflectance spectroscopy, contact angle, atomic force microscopy, and an ellipsometer. A competitive adsorption experiment on the sensor was performed to display the selectivity of the nanofilm. An analysis of the QCM sensor showed that the MIP nanofilm exhibited high sensitivity and selectivity for pirimicarb determination. A liquid chromatography‐tandem mass spectrometry method was prepared and validated to determine the accuracy and precision of the QCM sensor. The accuracy and precision of both methods were determined by a comparison of six replicates at three different concentrations to tomato samples extracted by using a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method. The limit of detection of the QCM sensor was found to be 0.028 nM. In conclusion, the QCM sensor showed good accuracy, with recovery percentages between 91 and 94%. Also, the pirimicarb‐imprinted QCM sensor exhibited a fast response time, reusability, high selectivity and sensitivity, and a low limit of detection. Therefore, it offers a serious alternative to the traditional analytical methods for pesticide detection in both natural sources and aqueous solutions.     

In vivo human immune response to transferrin-binding protein 2 and other iron-regulated proteins of Neisseria meningitidis

Abstract When grown under iron restriction, Neisseria meningitidis expresses new outer-membrane proteins, some of which are antigenic and potentially useful as vaccine components. This is particularly relevant to N. meningitidis serogroup B, against which neither polysaccharide nor conjugate vaccines are effective. We investigated recognition of N. meningitidis serogroup B outer-membrane antigens by three sera from patients recovered from meningitis. Recognition of antigens from the homologous strain provided information on in vivo expression during infection and immunogenicity, while cross-reactivity with outer membrane proteins from the other two strains and from another five strains in our collection allowed evaluation of antigenic heterogeneity. Our results demonstrate that transferrin-binding protein 2 (TBP2) is immunogenic in humans, to varying degrees depending on the strain, and that TBP2s (like the equivalent proteins of Haemophilus influenzae type b) are among the most important iron-regulated outer membrane antigens expressed during infection. Other immunogenic outer membrane proteins (some iron-regulated) are also expressed during infection; in a previous study in mouse, three of these proteins (with M _r of 50, 70 and 77 kDa) did not induce an immune response. Our cross-reactivity data provide some support for Robki et al.'s two-group classification of N. meningitidis strains, and provide evidence against the possibility that the antigenic domains shared by the TBP2s of all N . meningitidis strains induce immune responses in vivo.     

Evidence for the role of glycoprotein G of respiratory syncytial virus in binding of Neisseria meningitidis to HEp-2 cells

Abstract Viral glycoproteins G and F are expressed on the surface of cells infected with respiratory syncytial virus (RSV). We investigated the role of these proteins in the previously reported enhanced binding of Neisseria meningitidis to RSV-infected HEp-2 cells. Virus particles attached to bacteria were detected by immunofluorescence with flow cytometry. Binding of FITC-labelled bacteria to RSV-infected cells was significantly inhibited by monoclonal antibody against glycoprotein G. Unlabelled bacteria interfered with binding of the anti-G monoclonal antibody to these cells. These interactions were not found with a monoclonal antibody against glycoprotein F. We propose that glycoprotein G of RSV expressed on the surface of infected cells might act as an additional receptor for meningococci.     

Immunogenicity of in vitro folded outer membrane protein PorA of Neisseria meningitidis

In vitro folded and the denatured form of PorA P1.6 from Neisseria meningitidis strain M990 were used for immunization studies in mice. Previously, the antigen was isolated from cytoplasmic inclusion bodies, folded and purified. Its immunogenicity without adjuvant appeared to be low. The addition of the adjuvant QuilA, but not of galE lipooligosaccharide, considerably enhanced the immunogenicity. Moreover, when immunized with folded PorA P1.6 plus QuilA, a clear switch towards the IgG2a subclass of antibodies and concomitantly, the appearance of serum bactericidal activity, which is believed to be important for protective immunity, was observed. Hence, a tool for preparing vaccines against serogroup B meningococci devoid of endotoxin is available.     

The influence of mutation, recombination, population history, and selection on patterns of genetic diversity in Neisseria meningitidis

Patterns of genetic diversity within populations of human pathogens, shaped by the ecology of host-microbe interactions, contain important information about the epidemiological history of infectious disease. Exploiting this information, however, requires a systematic approach that distinguishes the genetic signal generated by epidemiological processes from the effects of other forces, such as recombination, mutation, and population history. Here, a variety of quantitative techniques were employed to investigate multilocus sequence information from isolate collections of Neisseria meningitidis, a major cause of meningitis and septicemia world wide. This allowed quantitative evaluation of alternative explanations for the observed population structure. A coalescent-based approach was employed to estimate the rate of mutation, the rate of recombination, and the size distribution of recombination fragments from samples from disease-associated and carried meningococci obtained in the Czech Republic in 1993 and a global collection of disease-associated isolates collected globally from 1937 to 1996. The parameter estimates were used to reject a model in which genetic structure arose by chance in small populations, and analysis of molecular variation showed that geographically restricted gene flow was unlikely to be the cause of the genetic structure. The genetic differentiation between disease and carriage isolate collections indicated that, whereas certain genotypes were overrepresented among the disease-isolate collections (the "hyperinvasive" lineages), disease-associated and carried meningococci exhibited remarkably little differentiation at the level of individual nucleotide polymorphisms. In combination, these results indicated the repeated action of natural selection on meningococcal populations, possibly arising from the coevolutionary dynamic of host-pathogen interactions.     

Immunoproteomic identification of the hypothetical protein NMB1468 as a novel lipoprotein ubiquitous in Neisseria meningitidis with vaccine potential

Many potential vaccine candidates for serogroup B Neisseria meningitidis (NMB) have been identified by reverse vaccinology, a genome-based approach. However, some candidates may be unseen owing to uncertain annotation or their peculiar properties. In this study, we describe the preparation and identification of a novel lipoprotein expressed in all meningococcal strains tested. mAb were first prepared from mice immunized with a meningococcal B strain isolated in Taiwan. Total proteins from the immunizing strain were separated by 2-DE in duplicate. Clone 4-7-3, which crossreacted to 174 tested meningococcal isolates, was used as the primary antibody for Western blotting. The immunoreactive spot was identified by LC-mass spectrometric analysis of the corresponding spot from the silver-stained gel and confirmed by molecular biology approach to be a novel lipoprotein encoded by the hypothetical NMB1468 gene. The potential use of this protein, designated Ag473/NMB1468, as a vaccine component was evaluated using the recombinant protein produced in Escherichia coli. Immunized mice were found to be protected from developing meningococcal disease after intraperitoneal inoculation with a lethal dose of meningococcal strain Nm22209, suggesting that Ag473/NMB1468 may be a promising vaccine candidate. This study also demonstrates the usefulness of the immunoproteomic approach in identification of novel vaccine candidates.     

Bactericidal antibody response to Neisseria meningitidis serogroup B in patients with bacterial meningitis: effect of immunization with an outer membrane protein vaccine

We evaluated the bactericidal antibody response to Neisseria meningitidis serogroup B in convalescent patients (n=65) from bacterial meningitis. Patients infected with B meningococci were stratified according to their vaccination status (Cuban BC vaccine) into group 1 (immunized) (n=12) and group 2 (non-immunized) (n=15). The results suggested that antibody titers > or =2 (log(2)) indicate a specific immune response to N. meningitidis. In group 1, 64% of patients had a significant antibody titer (> or =2) in their acute sera against a B:4:P1.15 strain, compared to only 21% of group 2 patients. All patients from group 1 without bactericidal antibodies in their acute sera had a significant increase (at least 2-fold increase in log(2) titers) in antibody titers in their convalescent sera, in contrast, to only 27% of patients from group 2 (P=0.06). Using mutant strains lacking OMP1 or OMP5, it was shown that OMP1 was an important antigen recognized by immunized patients but not by non-immunized patients.     

Cloning and partial sequence of transferrin-binding protein 2 of Neisseria meningitidis using a novel method: Twin N-terminal PCR

Abstract The genes encoding transferrin-binding proteins (TBPs) 1 and 2 of Neisseria meningitidis and N. gonorrhoeae were used as model loci in a novel method of cloning (twin N-terminal polymerase chain reaction; TNT-PCR) involving amplification between the 5' ends of two genes. Primers were based on N-terminal amino-acid sequences. A 2.1-kb product amplified from N. meningitidis strain SD (B15 P1.16) was cloned into a plasmid vector and partially sequenced. Translated sequence immediately downstream of the primer at both ends of this product correlated to the additional known N-terminal amino acids of TBP-1 and 2. The protein encoded by the cloned sequence reacted with TBP-2-specific antiserum. The size of products generated in TNT-PCR correlated exactly with the different sized TBP-2 produced by 10 strains of the Neisseria spp. examined, indicating successful cloning of the gene for TBP-2 and showing it to be adjacent to and preceding TBP-1 on the chromosome for both N. meningitidis and N. gonorrhoeae .     

A novel approach to study variable heavy chain gene usage in response to the capsular polysaccharide of Neisseria meningitidis serogroup C

The molecular mechanisms involved in the relatively poor immune response in the elderly are not clearly understood. Qualitative aspects of the immune response could be a possible explanation for the differential response to T-independent antigens in young adults and elderly. This study is directed towards elucidating the differential usage of variable heavy chain by young adult and elderly derived sequences in response to the capsular polysaccharide of Neisseria meningitidis serogroup C. We currently report findings of a preliminary study designed to test the feasibility of a novel approach to isolate antigen-specific B cells. Paramagnetic beads coated with an anti-idiotypic antibody, which mimics the capsular polysaccharide of N. meningitidis serogroup C, were used to select B cells. Analysis of the gene usage data indicates some unexpected differences in the use of variable chain heavy chain in the case of young adult versus elderly sequences. The elderly derived sequences use a more diverse array of V_H gene families in contrast to the young adult sequences, where the V_H gene family usage is restricted. Nearly half the young adult sequences utilize V_H3-15 germline sequence while only 25% of the elderly sequences use this germline sequence. There were interesting differences in the types of JH chain and the composition and length of CDR3 utilized by the two groups. Together, these significant differences may contribute towards the poor immune response to T-independent antigens in the elderly. These data validate the techniques used for these studies and suggest that it is pertinent to use this approach towards future investigations to elucidate gene usage in response to an antigen.     

The class 3 outer membrane protein (PorB) of Neisseria meningitidis: gene sequence and homology to the gonococcal porin PIA

The class 3 protein (PorB) is an important component of the meningococcal outer membrane. The structural gene (porB) encoding the class 3 protein has been cloned using primers suitable for the amplification of the corresponding chromosomal fragment by the polymerase chain reaction (PCR). The complete nucleotide sequence was determined and predicts a mature protein of 310 amino acids, preceded by a signal peptide of 19 residues. The predicted protein sequence of the class 3 protein exhibits essential structural homology to the gonococcal porin PIA. The class 3 protein encoding gene was expressed in Escherichia coli under the control of an inducible promoter.     

A computational approach to study functional monomer‐protein molecular interactions to optimize protein molecular imprinting

Molecular imprinting has become a promising approach for synthesis of polymeric materials having binding sites with a predetermined selectivity for a given analyte, the so‐called molecularly imprinted polymers (MIPs), which can be used as artificial receptors in various application fields. Realization of binding sites in a MIP involves the formation of prepolymerization complexes between a template molecule and monomers, their subsequent polymerization, and the removal of the template. It is believed that the strength of the monomer‐template interactions in the prepolymerization mixture influences directly on the quality of the binding sites in a MIP and consequently on its performance. In this study, a computational approach allowing the rational selection of an appropriate monomer for building a MIP capable of selectively rebinding macromolecular analytes has been developed. Molecular docking combined with quantum chemical calculations was used for modeling and comparing molecular interactions among a model macromolecular template, immunoglobulin G (IgG), and 1 of 3 electropolymerizable functional monomers: m‐phenylenediamine (mPD), dopamine, and 3,4‐ethylenedioxythiophene, as well as to predict the probable arrangement of multiple monomers around the protein. It was revealed that mPD was arranged more uniformly around IgG participating in multiple H‐bond interactions with its polar residues and, therefore, could be considered as more advantageous for synthesis of a MIP for IgG recognition (IgG‐MIP). These theoretical predictions were verified by the experimental results and found to be in good agreement showing higher binding affinity of the mPD‐based IgG‐MIP toward IgG as compared with the IgG‐MIPs generated from the other 2 monomers.