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Inspired by the specificity of α-(2,9)-sialyl epitopes in bacterial capsular polysaccharides (CPS), a doubly fluorinated disaccharide has been validated as a vaccine lead against Neisseria meningitidis serogroups C and/or B. Emulating the importance of fluorine in drug discovery, this molecular editing approach serves a multitude of purposes, which range from controlling α-selective chemical sialylation to mitigating competing elimination. Conjugation of the disialoside with two carrier proteins (CRM197 and PorA) enabled a semisynthetic vaccine to be generated; this was then investigated in six groups of six mice. The individual levels of antibodies formed were compared and classified as highly glycan-specific and protective. All glycoconjugates induced a stable and long-term IgG response and binding to the native CPS epitope was achieved. The generated antibodies were protective against MenC and/or MenB; this was validated in vitro by SBA and OPKA assays. By merging the fluorinated glycan epitope of MenC with an outer cell membrane protein of MenB, a bivalent vaccine against both serogroups was created. It is envisaged that validation of this synthetic, fluorinated disialoside bioisostere as a potent antigen will open new therapeutic avenues.

An extensive morphological analysis of the Neisseria meningitidis cell envelope, including serogroup B capsule and outer membrane, based on atomic force microscopy (AFM) together with mechanical characterization by force spectroscopic measurements, has been carried out. Three meningococcal strains were used: the encapsulated serogroup B strain B1940, and the isogenic mutants B1940 siaD(+C) (lacking capsule), and B1940 cps (lacking both capsule and lipooligosaccharide outer core). AFM experiments with the encapsulated strain B1940 provided unprecedented images of the meningococcal capsule, which seems to be characterized by protrusions ("bumps") with the lateral dimensions of about 30 nm. Measurement of the Young's modulus provided quantitative assessment of the property of the capsule to confer resistance to mechanical stress. Moreover, Raman spectroscopy gave a fingerprint by which it was possible to identify the specific molecular species of the three strains analyzed, and to highlight major differences between them., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Factor H binding protein (fHbp) is a key virulence factor of Neisseria meningitidis and a main component of the two licensed vaccines against serogroup B meningococcus (Bexsero and Trumenba). fHbp is a surface-exposed lipoprotein that enables the bacterium to survive in human blood by binding the human complement regulator factor H (fH). When used as vaccine, the protein induces antibodies with potent bactericidal activity. While the fHbp gene is present in the majority of N. meningitidis serogroup B isolates, the expression level varies up to 15 times between different strains and more than 700 different sequence variants have been described. Antigenically, the protein has been divided into three variants or two subfamilies. The 3D structure of fHbp alone, in combination with fH or in complex with bactericidal antibodies, has been key to understanding the molecular details of the protein. In this article, we will review the biochemical and immunological properties of fHbp, and its key role in meningococcal pathogenesis, complement regulation, and immune evasion., (© 2020 Federation of European Biochemical Societies.)

Lytic transglycosylases (LTs) are a class of enzymes important for the recycling and metabolism of peptidoglycan (PG). LTs cleave the β-1,4-glycosidic bond between N -acetylmuramic acid (MurNAc) and GlcNAc in the PG glycan strand, resulting in the concomitant formation of 1,6-anhydro- N -acetylmuramic acid and GlcNAc. No LTs reported to date have utilized chitins as substrates, despite the fact that chitins are GlcNAc polymers linked via β-1,4-glycosidic bonds, which are the known site of chemical activity for LTs. Here, we demonstrate enzymatically that LtgA, a non-canonical, substrate-permissive LT from Neisseria meningitidis utilizes chitopentaose ((GlcNAc) 5 ) as a substrate to produce three newly identified sugars: 1,6-anhydro-chitobiose, 1,6-anhydro-chitotriose, and 1,6-anhydro-chitotetraose. Although LTs have been widely studied, their complex reactions have not previously been visualized in the crystalline state because macromolecular PG is insoluble. Here, we visualized the cleavage of the glycosidic bond and the liberation of GlcNAc-derived residues by LtgA, followed by the synthesis of atypical 1,6-anhydro-GlcNAc derivatives. In addition to the newly identified anhydro-chitin products, we identified trapped intermediates, unpredicted substrate rearrangements, sugar distortions, and a conserved crystallographic water molecule bound to the catalytic glutamate of a high-resolution native LT. This study enabled us to propose a revised alternative mechanism for LtgA that could also be applicable to other LTs. Our work contributes to the understanding of the mechanisms of LTs in bacterial cell wall biology., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Bivalent rLP2086 (Trumenba), a vaccine for prevention of Neisseria meningitidis serogroup B (NmB) disease, was licensed for use in adolescents and young adults after it was demonstrated that it elicits antibodies that initiate complement-mediated killing of invasive NmB isolates in a serum bactericidal assay with human complement (hSBA). The vaccine consists of two factor H binding proteins (fHBPs) representing divergent subfamilies to ensure broad coverage. Although it is the surrogate of efficacy, an hSBA is not suitable for testing large numbers of strains in local laboratories. Previously, an association between the in vitro fHBP surface expression level and the susceptibility of NmB isolates to killing was observed. Therefore, a flow cytometric meningococcal antigen surface expression (MEASURE) assay was developed and validated by using an antibody that binds to all fHBP variants from both fHBP subfamilies and accurately quantitates the level of fHBP expressed on the cell surface of NmB isolates with mean fluorescence intensity as the readout. Two collections of invasive NmB isolates ( n = 1,814, n = 109) were evaluated in the assay, with the smaller set also tested in hSBAs using individual and pooled human serum samples from young adults vaccinated with bivalent rLP2086. From these data, an analysis based on fHBP variant prevalence in the larger 1,814-isolate set showed that >91% of all meningococcal serogroup B isolates expressed sufficient levels of fHBP to be susceptible to bactericidal killing by vaccine-induced antibodies. IMPORTANCE Bivalent rLP2086 (Trumenba) vaccine, composed of two factor H binding proteins (fHBPs), was recently licensed for the prevention of N. meningitidis serogroup B (NmB) disease in individuals 10 to 25 years old in the United States. This study evaluated a large collection of NmB isolates from the United States and Europe by using a flow cytometric MEASURE assay to quantitate the surface expression of the vaccine antigen fHBP. We find that expression levels and the proportion of strains above the level associated with susceptibility in an hSBA are generally consistent across these geographic regions. Thus, the assay can be used to predict which NmB isolates are susceptible to killing in the hSBA and therefore is able to demonstrate an fHBP vaccine-induced bactericidal response. This work significantly advances our understanding of the potential for bivalent rLP2086 to provide broad coverage against diverse invasive-disease-causing NmB isolates., (Copyright © 2018 McNeil et al.)

Neisseria meningitidis express numerous virulence factors that enable it to interact with diverse microenvironments within the host, during both asymptomatic nasopharyngeal colonization and invasive disease. Many of these interactions involve bacterial or host glycans. In order to characterise the meningococcal glycointeractome, glycan arrays representative of structures found on human cells, were used as a screening tool to investigate host glycans bound by N. meningitidis. Arrays probed with fluorescently labelled wild-type MC58 revealed binding to 223 glycans, including blood group antigens, mucins, gangliosides and glycosaminoglycans. Mutant strains lacking surface components, including capsule, lipooligosaccharide (LOS), Opc and pili, were investigated to identify the factors responsible for glycan binding. Surface plasmon resonance and isothermal calorimetry were used to confirm binding and determine affinities between surface components and host glycans. We observed that the L3 LOS immunotype (whole cells and purified LOS) bound 26 structures, while L8 only bound 5 structures. We further demonstrated a direct glycan-glycan interaction between purified L3 LOS and Thomsen-Friedenreich (TF) antigen, with a K D of 13 nM. This is the highest affinity glycan-glycan interaction reported to date. These findings highlight the diverse glycointeractions that may occur during different stages of meningococcal disease, which could be exploited for development of novel preventative and therapeutic strategies.

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.

Recently approved in the EU, US, Australia, and Canada, 4CMenB (Bexsero(®), GSK Vaccines) is a multi-component meningococcal B (MenB) vaccine containing 3 surface exposed recombinant proteins (fHbp, NadA, and NHBA) and New Zealand strain outer membrane vesicles (NZ OMV) containing PorA 1.4. The accepted correlate of protection to assess response to MenB vaccines, the serum bactericidal assay with human complement, is impractical for large panels of strains with diverse antigenic profile and expression. Therefore, the Meningococcal Antigen Typing System (MATS) was developed to identify MenB strains with a high likelihood of being covered by 4CMenB. MATS is used to assess MenB strain coverage without requiring sera, an advantage for testing large panels of bacterial isolates. MATS provides an accurate, conservative estimate of 4CMenB coverage. In a public-private partnership, 10 reference laboratories around the world were established and standardized to facilitate the timely collection and analysis of regional data. MATS has global public health implications for informing local policy makers of the predicted effect of the implementation of the 4CMenB vaccine. Coverage estimates are similar to or better than other recently approved vaccines, ranging from 66% to 91%. The use of MATS in post-vaccine implementation surveillance could provide data regarding vaccine effectiveness in the field and duration of protection on a global scale that will aid in the development of vaccine booster schedules, if necessary. This MATS approach could potentially be applied rapidly to assess epidemiology of other bacterial pathogens and coverage by other protein-based vaccines., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Meningococcal surface proteins capable of evoking a protective immune response are candidates for inclusion in protein-based vaccines against serogroup B Neisseria meningitidis (NmB). In this study, a 2-dimensional (2-D) gel-based platform integrating surface and immune-proteomics was developed to characterize NmB surface protein antigens. The surface proteome was analyzed by differential 2-D gel electrophoresis following treatment of live bacteria with proteinase K. Alongside, proteins recognized by immune sera from mice challenged with live meningococci were detected using 2-D immunoblots. In combination, seventeen proteins were identified including the well documented antigens PorA, OpcA and factor H-binding protein, previously reported potential antigens and novel potential immunogens. Results were validated for the macrophage infectivity potentiator (MIP), a recently proposed NmB vaccine candidate. MIP-specific antisera bound to meningococci in whole-cell ELISA and facilitated opsonophagocytosis and deposition of complement factors on the surface of meningococcal isolates of different serosubtypes. Cleavage by proteinase K was confirmed in western blots and shown to occur in a fraction of the MIP expressed by meningococci suggesting transient or limited surface exposure. These observations add knowledge for the development of a protein NmB vaccine. The proteomic workflow presented here may be used for the discovery of vaccine candidates against other pathogens., Biological Significance: This study presents an integrated proteomic strategy to identify proteins from N. meningitidis with desirable properties (i.e. surface exposure and immunogenicity) for inclusion in subunit vaccines against bacterial meningitis. The effectiveness of the method was demonstrated by the identification of some of the major meningococcal vaccine antigens. Information was also obtained about novel potential immunogens as well as the recently described potential antigen macrophage infectivity potentiator which can be useful for its consideration as a vaccine candidate. Additionally, the proteomic strategy presented in this study provides a generic 2-D gel-based platform for the discovery of vaccine candidates against other bacterial infections., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (±1500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteine-dependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis.

Neisseria meningitidis may be classified according to the lipooligosaccharide immunotype. We show that this classification can be achieved by PCR genotyping of the genes involved in the lipooligosaccharide inner-core biosynthesis, lpt3, lpt6, lgtG and lot3. Genotyping data correlated well (90-100%) with mass spectrometry data and was, therefore, applied to screen a random subset of recent N. meningitidis serogroup B isolates from Europe. Analysis of the proportion of the different lipooligosaccharide types highlighted the predominance of L3 strains. Surprisingly, high rates of L2 type strains were found in Spain (17%, versus 2.5% in Germany and 1.9% in the United Kingdom). Therefore, we also investigated further these Spanish L2 strains in an attempt to explain such prevalence despite the known sensitivity of L2 immunotype to complement. We explored the hypothesis that these strains express high amounts of factor H-binding protein (fHbp), but we found, on the contrary, that L2 strains express low or undetectable amounts of fHbp. Our findings suggest that, in addition to a genetic analysis, a multivalent approach may be necessary to estimate the effectiveness of a N. meningitidis serogroup B vaccine., (Copyright © 2012. Published by Elsevier Masson SAS.)

The investigational multicomponent meningococcus serogroup B vaccine (4CMenB) targets the antigenetically variable population of serogroup B meningococci. Forty-one strains of capsule null locus (cnl) meningococci, which are frequent among healthy carriers, were selected from nine sequence types (ST), which belong to four clonal complexes (cc), and three countries. They were antigen sequence typed and analyzed for antigen expression to predict whether these strains harbor the genes and express the four vaccine antigens of 4CMenB as measured by the meningococcal antigen typing system (MATS). The PorA variant used in the vaccine was not found. The nadA gene was absent in all but one strain, which did not express the antigen in vitro. Only strains of clonal complex ST-198 harbored a factor H binding protein (FHBP) allele of the cross-reactive variant 1 family which is included in the vaccine. All these strains expressed the antigen. Five variants of the Neisserial heparin binding antigen (NHBA) gene were identified. Expression of NHBA was observed in all strains with highest levels in ST-198 cc and ST-845. The data suggest a potential impact of 4CMenB immunization at least on cnl meningococci of the ST-198 cc and ST-845., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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

Background: In the absence of an efficacious broadly protective vaccine, serogroup B Neisseria meningitidis (MenB) is the leading cause of bacterial meningitis and septicemia in many industrialized countries. An investigational recombinant vaccine that contains 3 central proteins; Neisserial adhesin A (NadA), factor H binding protein (fHBP) and Neisserial heparin binding antigen (NHBA) has been developed. These antigens have been formulated with and without outer membrane vesicles (rMenB+OMV and rMenB, respectively) from the New Zealand epidemic strain (B:4:P1.7-2,4). In this trial, we assessed the immunogenicity of these formulations in infants, who are at greatest risk of contracting MenB disease., Methods: A total of 147 infants from the United Kingdom were enrolled and randomly assigned to receive rMenB or rMenB+OMV at 2, 4, 6, and 12 months of age or a single dose at 12 months of age. Serum samples taken before and after vaccination were assayed in a standardized serum bactericidal antibody assay against 7 MenB strains. Local and systemic reactogenicity were recorded for 7 days after each vaccination. Analysis was according to protocol., Results: After 3 doses, both vaccines were immunogenic against strains expressing homologous or related NadA and fHBP. rMenB+OMV demonstrated greater immunogenicity than did rMenB and was immunogenic against strains expressing homologous PorA. Both vaccines elicited anamnestic responses after the fourth dose. For both vaccines, responses were lower against strains expressing heterologous fHBP variants and after a single dose at 12 months., Conclusions: The rMenB+OMV vaccine has the potential to protect infants from MenB disease, although the breadth of protection afforded to heterologous antigens requires additional investigation.

In this study, we evaluated the effect of the growth medium on the composition and immunogenicity of meningococcal outer membrane vesicle (OMV) vaccines after cultivation of the Norwegian serogroup B 44/76 vaccine strain in either Frantz' or modified Catlin-6 media (MC.6M). Differential proteomic analysis revealed that 97% of the OMV proteins maintained the same levels in the two preparations. However, a number of differentially expressed proteins, including TdfH, OpcA, OMP NMB0088, hypothetical NMB2134, lipoprotein NMB1126/1164 and NspA, increased significantly in OMVs produced from bacteria grown in the MC.6M. Together with increased lipopolysaccharide levels, the increased expression of these proteins was associated with significantly higher serum bactericidal titres in mice immunized with the MC.6M OMV vaccine. The high resolution two-dimensional separation of the OMVs on a large-format gel across a pH range of 3-11 resolved around 2000 protein spots from which 75 proteins were identified by mass spectrometry., (Copyright 2010 Elsevier Ltd. All rights reserved.)

GNA2132 (Genome-derived Neisseria Antigen 2132) is a surface-exposed lipoprotein discovered by reverse vaccinology and expressed by genetically diverse Neisseria meningitidis strains (Pizza et al. 2000). The protein induces bactericidal antibodies against most strains of Meningococccus and has been included in a multivalent recombinant vaccine against N. meningitidis serogroup B. Structure determination of GNA2132 is important for understanding the antigenic properties of the protein in view of increased efficiency vaccine development. We report practically complete (1)H, (13)C and (15)N assignment of the detectable spectrum of a highly conserved C-terminal region of GNA2132 (residues 245-427) in micellar solution, a medium used to improve the spectral quality. The first 32 residues of our construct up to residue 277 were not visible in the spectrum, presumably because of line broadening due to solvent and/or conformational exchange. Secondary structure predictions based on chemical shift information indicate the presence of an all beta-protein with eight beta strands.

Bacterial endotoxin interacts with the human immune system via complex immunological pathways. The evaluation of endotoxicity is important in the development of safe vaccines and immunomodulatory therapeutics. The Limulus amebocyte lysate (LAL) assay is generally accepted by the FDA for use for the quantification of lipopolysaccharide (LPS), while the rabbit pyrogen test (RPT) is used to estimate pyrogenicity during early development and production. Other in vitro assays, such as cytokine release assays with human whole blood (WB) or peripheral blood mononuclear cells (PBMCs), have also been used and may better estimate the human immunological response to products containing novel LPS molecules. In this study, WB and PBMC interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) release assays were used to estimate the endotoxic activities of purified LPS and native outer membrane vesicle (NOMV) vaccines derived from wild-type (hexa-acylated lipid A) and genetically detoxified (penta- and tetra-acylated lipid A) group B Neisseria meningitidis. A method for quantification of the differences in endotoxicity observed in the WB and PBMC assays is elucidated. The LAL assay was shown to be relatively insensitive to lipid A variations, and the RPT was less sensitive than the cytokine release assay with WB. The IL-6 and TNF-alpha assays with WB but not the assays with PBMCs distinguished between vaccines containing LPS from penta- and tetra-acylated strains. The high degree of sensitivity of the WB system to LPS variations and the presumed relevance of the use of human tissues to predict toxicity in humans suggest that this assay may be particularly well suited for the safety evaluation of vaccines and therapeutics containing acylation variants of LPS.

This work presents the results from a study of the protein composition of outer membrane vesicles from VA-MENGOC-BC (Finlay Institute, Cuba), an available vaccine against serogroup B Neisseria meningitidis. Proteins were identified by means of SCAPE, a 2DE-free method for proteome studies. More than one hundred proteins were detected by tandem liquid chromatographymass spectrometry analysis of fractions enriched in peptides devoid of histidine or arginine residues, providing a detailed description of the vaccine. A bioinformatic analysis of the identified components resulted in the identification of 31 outer membrane proteins and three conserved hypothetical proteins, allowing the cloning, expression, purification and immunological study of two of them (NMB0088 and NMB1796) as new antigens.

Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOS-enriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.

The physico-chemical characterization of NadADelta(351-405), a recombinant protein discovered by reverse vaccinology, component of a candidate vaccine against Neisseria meningitidis serotype B is presented. Analytical methods like mass spectrometry, electrophoresis, optical spectroscopy and SEC-MALLS have been applied to unveil the structure of NadADelta(351-405), and to evaluate Product-Related Substances. Moreover, analysis of the protein after intentional denaturation has been applied in order to challenge the chosen methods and to determine their appropriateness and specificity. All the obtained results were inserted in a model allowing in-depth understanding of the antigen NadADelta(351-405): it is present in solution as a homo-trimer, retaining a high percentage of alpha-helix secondary structure, and able to reassemble from monomeric subunits after thermal denaturation; this structural organization is consistent with that foreseen for MenB NadA (Neisseria Adhesin A). The analytical data sets produced during process development for clinical phases I-III material confirm product quality and manufacturing consistency.

Endotoxins represent one of the most potent classes of microbial immunoactive components that can cause pulmonary inflammation. The aim of this study was to compare the inflammatory potency of two types of Neisseria meningitidis endotoxins (lipooligosaccharides) in lungs: wild type (hexaacylated, LOS(wt)) and mutant type (pentaacylated, LOS(msbB)), and to determine the importance of MD-2 in endotoxin responses in lungs in vivo. Endotoxin-normoresponsive mice (BALB/c) were exposed to selected doses of penta- and hexaacylated lipooligosaccharides (LOS) by nasal aspiration. Cellular and cytokine/chemokine inflammatory responses in bronchoalveolar lavage were measured at 1-, 4-, 8-, 16-, 24-, and 48-hour time points. MD-2-null mice were exposed to one dose of hexaacylated LOS and inflammatory responses were measured after 4 and 24 hours. Inhalation of hexaacylated LOS resulted in strong inflammatory responses, while pentaacylated LOS was much less potent in inducing increases of neutrophils, TNF-alpha, macrophage inflammatory protein-1 alpha, IL-6, granulocyte colony-stimulating factor, and IL-1 beta concentration in bronchoalveolar lavage. Similar kinetics of inflammatory responses in lungs were found in both types of endotoxin exposures. Inhalation of hexaacylated LOS in MD-2-null mice resulted in significantly lower numbers of neutrophils in bronchoalveolar lavage than in normoresponsive mice. Markedly lower inflammatory potency of pentaacylated LOS was observed compared with hexaacylated LOS. Hyporesponsiveness in MD-2-null mice after nasal aspiration of wild-type LOS indicate its essential role in airway responsiveness to endotoxin.

The objective of this work was to conduct an in vivo comparison of nanoparticles and microparticles as vaccine delivery systems. Poly (lactide-co-glycolide) (PLG) polymers were used to create nanoparticles size 110 nm and microparticles of size 800-900 nm. Protein antigens were then adsorbed to these particles. The efficacy of these delivery systems was tested with two protein antigens. A recombinant antigen from Neisseria meningitides type B (MenB) was administered intramuscularly (i.m.) or intraperitonealy (i.p.). An antigen from HIV-1, env glycoprotein gp140 was administered intranasally (i.n.) followed by an i.m. boost. From three studies, there were no differences between the nanoparticles and micro-particles formulations. Both particles led to comparable immune responses in mice. The immune responses for MenB (serum bactericidal activity and antibody titers) were equivalent to the control of aluminum hydroxide. For the gp140, the LTK63 was necessary for high titers. Both nanoparticles and microparticles are promising delivery systems.

Current experimental vaccines against serogroup B Neisseria meningitidis are based on meningococcal outer membrane (OM) proteins present in outer membrane vesicles (OMV) in which toxic lipopolysaccharide is depleted by detergent extraction. Knowledge of the composition of OM and OMV is essential for developing new meningococcal vaccines based on defined antigens. In the current study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nanocapillary liquid chromatography-tandem mass spectrometry were used to investigate the proteomes of OM and OMV from meningococcal strain MC58 and OM from a lipopolysaccharide-deficient mutant. The analysis of OM revealed a composition that was much more complex than the composition that has been reported previously; a total of 236 proteins were identified, only 6.4% of which were predicted to be located in the outer membrane. The most abundant proteins included not only the well-established major OM proteins (PorA, PorB, Opc, Rmp, and Opa) but also other proteins, such as pilus-associated protein Q (PilQ) and a putative macrophage infectivity protein. All of these proteins were also present in OMV obtained by extraction of the OM with deoxycholate. There were markedly increased levels of some additional proteins in OM from the lipopolysaccharide-deficient mutant, including enzymes that contribute to the tricarboxylic acid cycle. In all the preparations, the proteins not predicted to have an OM location were predominantly periplasmic or cytoplasmic or had an unknown location, and relatively few cytoplasmic membrane proteins were detected. However, several proteins that have previously been identified as potential vaccine candidates were not detected in either OM preparations or in OMV. These results have important implications for the development and use of vaccines based on outer membrane proteins.

The most popular approach for proteomic analysis is based on the combination of two-dimensional electrophoresis (2DE) and mass spectrometry. Although very effective, the method suffers from a number of limitations, the most serious one being the necessity of expensive and sophisticated instrumentation to be operated by skilled personnel. Here, we propose an alternative approach, which is particularly useful when one is interested to establish if a subset of proteins is present in a complex protein mixture derived from a sequenced organism. The method is based on amplification of the genes whose products are under investigation. The amplified genes are used in transcription and translation reactions and the derived radio-labeled proteins are separated by 2DE. The gel is autoradiographed and the autoradiograph is superimposed on the 2D gel (sample gel) from which the protein mixture from the organism has been separated. The matching between the autoradiographic spots and the protein spots of the sample gel allows immediate protein identification.

Cochleate are highly stable structures with promising immunological features. Cochleate structures are usually obtaining from commercial lipids. Proteoliposome derived Cochleate are derived from an outer membrane vesicles of Neisseria meningitidis B. Previously, we obtained Cochleates using dialysis procedures. In order to increase the production process, we used a crossflow system (CFS) that allows easy scale up to obtain large batches in an aseptic environment. The raw material and solutions used in the production process are already approved for human application. This work demonstrates that CFS is very efficient process to obtain Cochleate structures with a yield of more than 80% and the immunogenicity comparable to that obtained by dialysis membrane.

Cochleate structures obtained from the outer membrane of Neisseria meningitidis serotype B have demonstrated to be high immunogenicity when administrated by intramuscular, oral or intranasal routes, and could be used as adjuvant and meningococcal nasal vaccine candidate. Due to the microparticulate nature of Cochleate it is necessary to control the particle size since it capture by cells of the immune system could be affected by this aspect. We combined optic microscopy and immunisation experiments to select the optimum particle size. Six different processes of producing Cochleate obtaining were evaluated and different mechanical stress conditions were carried out to homogenize and modulate the particles size. The more immunogenic particles were selected on the basis of the levels of specific IgA and IgG antibodies induced after intranasal immunisation in mice. The best treatment parameter for mechanical stress of the Cochleate was prolonged treatment with untrasonic low frequency waves.

Peptide fragments of conservative sites of PorA, OpaB, and NspA proteins of the outer membrane of serogroup B meningococci were synthesized. These peptides caused a pronounced protective effect in immunized mice infected with virulent homologous and heterologous strains of serogroups B and A meningococci. The protective effect appreciably increased, if the studied peptides were associated in a polycomponent preparation, which can be used in the construction of meningococcal bivalent B+A vaccine.

GNA33 is a membrane-bound lipoprotein with murein hydrolase activity that is present in all Neisseria species and well conserved in different meningococcal isolates. The protein shows 33% identity to a lytic transglycolase (MltA) from Escherichia coli and has been shown to be involved in the degradation of both insoluble murein sacculi and unsubstituted glycan strands. To study the function of the gene and its role in pathogenesis and virulence, a knockout mutant of a Neisseria meningitidis serogroup B strain was generated. The mutant exhibited retarded growth in vitro. Transmission electron microscopy revealed that the mutant grows in clusters which are connected by a continuous outer membrane, suggesting a failure in the separation of daughter cells. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatant revealed that the mutant releases several proteins in the medium. The five most abundant proteins, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, belong to the outer membrane protein family. Finally, the mutant showed an attenuated phenotype, since it was not able to cause bacteremia in the infant rat model. We conclude that GNA33 is a highly conserved lipoprotein which plays an important role in peptidoglycan metabolism, cell separation, membrane architecture, and virulence.

The detection of antibodies specific to meningococcal lipo-oligosaccharides (LOSs; outer-core-->inner-core-->lipid A) in sera of patients convalescent from meningococcal infection suggests the potential use of LOS as a vaccine to combat pathogenic Neisseria spp. Removal of the outer-core region, which expresses glycans homologous to human blood-group antigens, is a required first-step in order to avoid undesirable immunological reactions following vaccination. To this end, we describe here the structural makeup of the LOS produced by serogroup B N. meningitidis NMB isogenic phosphoglucomutase (Pgm) mutant (NMB-R6). The dominant LOS types produced by NMB-R6 expressed a deep-truncated inner-core region, GlcNAc-(1-->2)-LDHepII-(1-->3)-LDHepI-(1-->5)-[Kdo-2-->4]-Kdo-->lipid A, with one PEA unit attached at either O-6 or O-7 of LDHepII, or with two simultaneously PEA moieties attached at O-3 and O-6 or O-3 and O-7 of the same unit. Unexpectedly, this mutation did not completely deactivate the production of Glc, as some LOS molecules were observed to carry Glc at O-4 of LDHepI and at O-3 of LDHepII. A glycoconjugate vaccine comprised of NMB-R6 LOSs is currently being evaluated in our laboratory.