Postgenomics of Neisseria meningitidis for vaccines development

Meningococcal disease is a global problem. Multivalent (A, C, Y, W135) conjugate vaccines have been developed and licensed; however, an effective vaccine against serogroup B has not yet become available. Outer membrane vesicle (OMV) vaccines have been used to disrupt serogroup B epidemics and outbreaks. Postgenomic technologies have been useful in aiding the discovery of new protein vaccine candidates. Moreover, proteomic technologies enable large-scale identification of membrane and surface-associated proteins, and provide suitable methods to characterize and standardize the antigen composition of OMV-based vaccines.     

Postgenomics of Neisseria meningitidis for vaccines development

Meningococcal disease is a global problem. Multivalent (A, C, Y, W135) conjugate vaccines have been developed and licensed; however, an effective vaccine against serogroup B has not yet become available. Outer membrane vesicle (OMV) vaccines have been used to disrupt serogroup B epidemics and outbreaks. Postgenomic technologies have been useful in aiding the discovery of new protein vaccine candidates. Moreover, proteomic technologies enable large-scale identification of membrane and surface-associated proteins, and provide suitable methods to characterize and standardize the antigen composition of OMV-based vaccines.     

Immunity against Neisseria meningitidis Serogroup C in the Dutch Population before and after Introduction of the Meningococcal C Conjugate Vaccine

Background

In 2002 a Meningococcal serogroup C (MenC) conjugate vaccine, with tetanus toxoid as carrier protein, was introduced in the Netherlands as a single-dose at 14 months of age. A catch-up campaign was performed targeting all individuals aged 14 months to 18 years. We determined the MenC-specific immunity before and after introduction of the MenC conjugate (MenCC) vaccine.

Methods and Findings

Two cross-sectional population-based serum banks, collected in 1995/1996 (n = 8539) and in 2006/2007 (n = 6386), were used for this study. The main outcome measurements were the levels of MenC polysaccharide(PS)-specific IgG and serum bactericidal antibodies (SBA) after routine immunization, 4–5 years after catch-up immunization or by natural immunity. There was an increasing persistence of PS-specific IgG and SBA with age in the catch-up immunized cohorts 4–5 years after their MenCC immunization (MenC PS-specific IgG, 0.25 µg/ml (95%CI: 0.19–0.31 µg/ml) at age 6 years, gradually increasing to 2.34 µg/ml,(95%CI: 1.70–3.32 µg/ml) at age 21–22 years). A comparable pattern was found for antibodies against the carrier protein in children immunized above 9 years of age. In case of vaccination before the age of 5 years, PS-specific IgG was rapidly lost. For all age-cohorts together, SBA seroprevalence (≥8) increased from 19.7% to 43.0% in the pre- and post-MenC introduction eras, respectively. In non-immunized adults the SBA seroprevalence was not significantly different between the pre- and post-MenC introduction periods, whereas PS-specific IgG was significantly lower in the post-MenC vaccination (GMT, age ≥25 years, 0.10 µg/ml) era compared to the pre-vaccination (GMT, age ≥25 years, 0.43 µg/ml) era.

Conclusion

MenCC vaccination administered above 5 years of age induced high IgG levels compared to natural exposure, increasing with age. In children below 14 months of age and non-immunized cohorts lower IgG levels were observed compared to the pre-vaccination era, whereas functional levels remained similar in adults. Whether the lower IgG poses individuals at increased risk for MenC disease should be carefully monitored. Large-scale introduction of a MenCC vaccine has led to improved protection in adolescents, but in infants a single-dose schedule may not provide sufficient protection on the long-term and therefore a booster-dose early in adolescence should be considered.     

Hemagglutination by Neisseria meningitidis

The direct agglutination of erythrocytes by Neisseria meningitidis was studied as a marker for adherence. Hemagglutination (HA) was studied by slide test (5-min incubation) and by dilutions in microtitre plates (20-h incubation). Meningococci that were freshly isolated from subjects agglutinated only human cells by slide test but human, dog, rabbit, guinea pig, and rat cells were agglutinated in the microtitre system. Newly isolated strains were piliated and HA positive but pili were lost after 10 passages on agar, and bacteria became HA negative. HA could be maintained by "affinity culturing," which selected markedly adhesive bacteria: erythrocytes with adherent meningococci were isolated and cultured on agar. This procedure was repeated daily. HA titres were unaffected by mannose but were reduced by sonic disruption, trypsinization, ultraviolet irradiation, heating (65 degrees C), and formaldehyde. Encapsulated (serogroupable) bacteria had low HA titres compared with nongroupable strains, and purified capsular polysaccharides A and C inhibited HA. Meningococcal HA is probably mediated by pili and modified by other factors such as encapsulation. Colonial variation was not a reliable indicator of piliation, and HA is best used for this purpose.     

In vitro resistance mechanisms of Neisseria meningitidis against neutrophil extracellular traps

Neisseria meningitidis (Nm) is a leading cause of septicemia in childhood. Nm septicemia is unique with respect to very quick disease progression, high in vivo bacterial replication rate and its considerable mortality. Nm circumvents major mechanisms of innate immunity such as complement system and phagocytosis. Neutrophil extracellular traps (NETs) are formed from neutrophils during systemic infection and are suggested to contain invading microorganisms. Here, we investigated the interaction of Nm with NETs. Both, meningococci and spontaneously released outer membrane vesicles (SOMVs) were potent NET inducers. NETs were unable to kill NET bound meningococci, but slowed down their proliferation rate. Using Nm as model organism we identified three novel mechanisms how bacteria can evade NET‐mediated killing: (i) modification of lipid A of meningococcal LPS with phosphoethanolamine protected Nm from NET‐bound cathepsin G; (ii) expression of the high‐affinity zinc uptake receptor ZnuD allowed Nm to escape NET‐mediated nutritional immunity; (iii) binding of SOMVs to NETs saved Nm from NET binding and the consequent bacteriostatic effect. Escape from NETs may contribute to the most rapid progression of meningococcal disease. The induction of NET formation by Nm in vivo might aggravate thrombosis in vessels ultimately directing to disseminated intravascular coagulation (DIC).     

An Outer Membrane Receptor of Neisseria meningitidis Involved in Zinc Acquisition with Vaccine Potential

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract.     

Construction of Opa-Positive and Opa-Negative Strains of Neisseria meningitidis to Evaluate a Novel Meningococcal Vaccine

Neisseria meningitidis is a major global pathogen causing invasive disease with a mortality of 5–10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opacity-associated adhesin (Opa) proteins are major meningococcal outer membrane proteins, which have shown recent promise as a potential novel vaccine. Immunisation of mice with different Opa variants elicited high levels of meningococcal-specific bactericidal antibodies, demonstrating proof in principle for this approach. Opa proteins are critical in meningococcal pathogenesis, mediating bacterial adherence to host cells, and modulating human cellular immunity via interactions with T cells and neutrophils, although there are conflicting data regarding their effects on CD4⁺ T cells. We constructed Opa-positive and Opa-negative meningococcal strains to allow further evaluation of Opa as a vaccine component. All four opa genes from N. meningitidis strain H44/76 were sequentially disrupted to construct all possible combinations of N. meningitidis strains deficient in one, two, three, or all four opa genes. The transformations demonstrated that homologous recombination of exogenous DNA into the meningococcal chromosome can occur with as little as 80 bp, and that minor sequence differences are permissible. Anti-Opa bactericidal antibody responses following immunisation of mice with recombinant Opa were specific to the Opa variant used in immunisation. No immunomodulatory effects were observed when Opa was contained within meningococcal outer membrane vesicles (OMVs), compared to Opa-negative OMVs. These observations support the incorporation of Opa in meningococcal vaccines.     

Vascular colonization by Neisseria meningitidis

Bacterial infection of human vasculature can lead to unregulated systemic activation of coagulation and innate immunity and rapidly becomes life threatening. Neisseria meningitidis is a vascular pathogen that causes fatal sepsis and meningitis. Post-mortem histological analysis of tissues from individuals infected with N. meningitidis show large bacterial aggregates in close association with the vascular wall of small vessels. The ability of this bacterium to colonize blood vessel endothelium is likely to impact its capacity to both multiply in the blood stream and reach the brain. This process will be referred to as vascular colonization. Recent work has described a number of early steps in N. meningitidis vascular colonization, from attachment to proliferation and dissemination, focusing on the bacterial-host interaction.     

Nitric oxide participates in the immune response against Neisseria meningitidis serogroup B

The present report explores the role of nitric oxide into the immune response against Neisseria meningitidis serogroup B. Here we show that NO mediates the alphaTNF increase induced by N. meningitidis derived lipopolysaccharides (LPS), at the same time that participates in the bactericidal activity of resting or gammaIFN activated macrophages and plays a role in the specific DTH and IgG response induced by a commercial anti-meningococcal vaccine. Our findings suggest a positive role for NO at the final effector mechanisms and in the early events driving the immunity against N. meningitidis, suggesting also an insight into its role in endotoxic shock.     

Sulphur acquisition by Neisseria meningitidis

Group B Neisseria meningitidis (SD1C) was grown on defined medium supplemented with each of a variety of sulphur compounds as the sole source of sulphur. The organism grew on sulphate, sulphite, bisulphite, thiosulphate, dithionite, hydrosulphide, thiocyanate, L-cysteine, L-cystine, reduced glutathione, methionine, mercaptosuccinate, and lanthionine, but not on dithionate unless previously sulphur starved. Good growth was seen on concentrations of sulphate or thiosulphate as low as 10 microM. When pregrown on and subsequently starved for sulphate, the meningococcus showed enhanced transport capacity for this ion. Optimal conditions for assessing sulphur transport by active sulphur-limited cells were determined. The maximal sulphate uptake velocity was 9.3 nmol sulphate X mg protein-1 X min-1, and the apparent Km was 1.4 microM, far below human nasopharyngeal or serum sulphate levels.     

Candidate Neisseria meningitidis NspA vaccine

The highly conserved NspA protein has been found in the outer membrane of every Neisseria meningitidis strain tested so far. Two monoclonal antibodies (MAbs) directed against this protein were used to demonstrate that biologically important epitopes of the NspA protein are exposed at the surface of serologically distinct meningococcal strains. Analysis of sera collected from mice that survived a deadly meningococcal challenge following immunization with recombinant NspA protein (rNspA) revealed the presence of cross-reactive antibodies which efficiently attached to and killed the four serogroup B strains tested. These data are additional proof that the NspA protein is exposed at the surface of intact meningococcal cells, which is an important characteristic for a vaccine candidate.     

Role of Factor H Binding Protein in Neisseria meningitidis Virulence and Its Potential as a Vaccine Candidate To Broadly Protect against Meningococcal Disease

SUMMARY

Neisseria meningitidis is a Gram-negative microorganism that exists exclusively in humans and can cause devastating invasive disease. Although capsular polysaccharide-based vaccines against serogroups A, C, Y, and W135 are widely available, the pathway to a broadly protective vaccine against serogroup B has been more complex. The last 11 years has seen the discovery and development of the N. meningitidis serogroup B (MnB) outer membrane protein factor H binding protein (fHBP) as a vaccine component. Since the initial discovery of fHBP, a tremendous amount of work has accumulated on the diversity, structure, and regulation of this important protein. fHBP has proved to be a virulence factor for N. meningitidis and a target for functional bactericidal antibodies. fHBP is critical for survival of meningococci in the human host, as it is responsible for the primary interaction with human factor H (fH). Binding of hfH by the meningococcus serves to downregulate the host alternative complement pathway and helps the organism evade host innate immunity. Preclinical studies have shown that an fHBP-based vaccine can elicit serum bactericidal antibodies capable of killing MnB, and the vaccine has shown very encouraging results in human clinical trials. This report reviews our current knowledge of fHBP. In particular, we discuss the recent advances in our understanding of fHBP, its importance to N. meningitidis, and its potential role as a vaccine for preventing MnB disease.     

The composition of immune sera against Neisseria meningitidis studied by high-performance liquid chromatography

The comparative study of the composition of immune rabbit sera to N. meningitidis, as well as nonimmune sera, has been made by the methods of HPLC and radial immunodiffusion. The quantitative evaluation of the main serum proteins by the two methods has shown the coincidence of the results yielded by these methods. To study the total level of IgM and IgG in the sera under study, a simple and rapid HPLC technique is proposed. The study of the stability of sera during storage (at 4-6 degrees C) has revealed that immune sera show greater stability during storage under such conditions in comparison with sera obtained from nonimmune animals.     

The R-type lipopolysaccharides of Neisseria meningitidis

The lipopolysaccharides of all the different serogroups of Neisseria meningitidis are of the "R" type despite the morphologically smooth appearance and the demonstrated virulence of the organisms from which they were derived. This was confirmed when each of the lipopolysaccharides was found to be devoid of detectable O-antigen side chains, giving only a low "molecular" weight core oligosaccharide when subjected to mild acid hydrolysis. The cores were modified by dephosphorylation and subjected to sugar and methylation analysis by gas-liquid chromatography. Although all the different cores contained identical components (glucose, galactose, glucosamine, heptose, and 2-keto-3-deoxyoctonate) they could be separated into three distinct categories according to their galactose:glucose ratios. These categories are typified by the cores obtained from groups A, C, and 29-e which have galactose:glucose ratios of 1:2, 2:2, and 2:1, respectively. The modified cores were methylated and analyzed by gas chromatography--mass spectrometry and on the basis of differences in the derived methylated sugars the cores could again be divided into the same three categories as above. This structural diversity also results in some serological specificity as demonstrated by the complete serogroup specificity of the group A lipopolysaccharide.     

Functional genomics of Neisseria meningitidis pathogenesis

The pathogenic bacterium Neisseria meningitidis is an important cause of septicemia and meningitis, especially in childhood. The establishment and maintenance of bacteremic infection is a pre-requisite for all the pathological sequelae of meningococcal infection. To further understand the genetic basis of this essential step in pathogenesis, we analyzed a library of 2,850 insertional mutants of N. meningitidis for their capacity to cause systemic infection in an infant rat model. The library was constructed by in vitro modification of Neisseria genomic DNA with the purified components of Tn10 transposition. We identified 73 genes in the N. meningitidis genome that are essential for bacteremic disease. Eight insertions were in genes encoding known pathogenicity factors. Involvement of the remaining 65 genes in meningocoocal pathogenesis has not been demonstrated previously, and the identification of these genes provides insights into the pathogenic mechanisms that underlie meningococcal infection. Our results provide a genome-wide analysis of the attributes of N. meningitidis required for disseminated infection, and may lead to new interventions to prevent and treat meningococcal infection.     

Carriage of Neisseria meningitidis and Neisseria lactamica in northern Greece

In response to an increase in the number of cases of invasive meningococcal disease (IMD) in northern regions of Greece, a survey was carried out to determine if there was an increase in carriage of Neisseria meningitidis, particularly in areas where there have been increases in immigrant populations from neighbouring countries. The second objective was to determine if there was an increase in the serogroup C:2a:P1.5,2 a phenotype associated with recent outbreaks or changes in antibiotic sensitivities. As carriage of Neisseria lactamica is associated with development of natural immunity to IMD, the third objective was to determine the carriage rate of N. lactamica in this population. Among 3167 individuals tested, meningococci were isolated from 334 (10.5%). Compared with our previous studies, the proportion of meningococcal carriers was significantly increased among children in secondary education (11.3%) (chi2=9.67, P<0.005) and military recruits (37.4%) (chi2=21.11, P<0.000). Only 5/334 (1.5%) isolates expressed the phenotype associated with the increase in IMD in Greece. N. lactamica was isolated from 146/3167 (4.6%) participants. It was isolated from 71/987 (7.2%) children attending primary or nursery schools; however, the highest proportion of carriers (11.3%) was found in the boarding school for young Albanian men. In the 21-59-year age range, the majority of N. lactamica isolates (22/25, 88%) were from women, probably due to closer or more prolonged contact with children in the primary school age range. Smoking was significantly associated with isolation of meningococci from men but not from women. Penicillin-insensitive strains (25/334, 7.5%) were identified in all four regions examined; the majority (14/25, 56%) were obtained from military personnel. We conclude that there was a higher proportion of carriers in the population of northern Greece; however, the increase in carriage rate was not associated with the influx of immigrants from neighbouring countries, and there was not a higher incidence of the C:2a:P1.5,2 strain responsible for increased disease activity in Greece in either the immigrant or local populations.     

Serological studies of ungroupable Neisseria meningitidis

Verification that Slaterus' Neisseria meningitidis serotypes X, Y, and Z are groups distinct from each other and from groups A, B, C, and D was made by use of the tube agglutination test on absorbed and unabsorbed antisera. A significant number of meningococcal strains in this country, which could not be classified serologically with standard antisera prepared to Branham's neotype A, B, C, and D strains, were grouped specifically with antisera prepared to the Slaterus types. The strains grouped as X, Y, and Z were from various geographical areas of the United States and were isolated from both carriers and cases. Over a 2-year period, the cultures tested ranged in predominance in descending order as follows: group B, C, Y, X, Z, A, and D. It is recommended that Slaterus' types should be considered as standard groups and follow in alphabetical order with the standard A, B, C, and D groups; i.e., X would be designated as group E, Y as group F, and Z as group G. It was observed that false-grouping cross-reactions could be greatly reduced by reconstituting the lyophilized grouping antisera in 50% glycerol-water. Of 99 cultures which could not be specifically grouped with antisera reconstituted in distilled water, 19 were specifically grouped with antisera reconstituted in 50% glycerol-water.