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.     

Bordetella pertussis fimbriae are effective carrier proteins in Neisseria meningitidis serogroup C conjugate vaccines

Serogroup C meningococcal conjugate vaccines generally use diphtheria or tetanus toxoids as the protein carriers. The use of alternative carrier proteins may allow multivalent conjugate vaccines to be formulated into a single injection and circumvent potential problems of immune suppression in primed individuals. Bordetella pertussis fimbriae were assessed as carrier proteins for Neisseria meningitidis serogroup C polysaccharide. Fimbriae were conjugated to the polysaccharide using modifications of published methods and characterised by size exclusion chromatography; co-elution of protein and polysaccharide moieties confirmed conjugation. The conjugates elicited boostable IgG responses to fimbriae and serogroup C polysaccharide in mice, and IgG:IgM ratios indicated that the responses were thymus-dependent. High bactericidal antibody titres against a serogroup C strain of N. meningitidis were also observed. In a mouse infection model, the conjugate vaccine protected against lethal infection with N. meningitidis. Therefore, B. pertussis fimbriae are effective carrier proteins for meningococcal serogroup C polysaccharide and could produce a vaccine to protect against meningococcal disease and to augment protection against pertussis.     

Neisseria meningitidis serogroup B: laboratory correlates of protection

Meningococcal disease in the Western countries is frequently caused by Neisseria meningitidis serogroup B. Major efforts have been made to develop a safe and efficacious vaccine against this serogroup which is suitable for use in infants and young children. To assess the quality of the immune response after vaccination with candidate vaccines, laboratory correlates of protection are needed. For serogroups A and C, serum bactericidal activity (SBA) is a well established predictor for protection, but for serogroup B other mechanisms besides SBA may also be involved in conferring protection from disease. Several laboratory methods for identification and evaluation of the immunogenicity of possible vaccine antigens are described in this review.     

Epitopes of serogroup B Neisseria meningitidis analysed in vitro and directly from cerebrospinal fluid

Two type B15 P1.16 strains of Neisseria meningitidis were examined by immunogold electron microscopy for accessibility of two outer membrane protein (OMPs) to monoclonal antibodies. Both strains exhibited cell-to-cell variation of one epitope of the Class 3 OMP (P3.15) and one of the Class 1 OMPs (P1.16) when grown in vitro. One strain, a nasopharyngeal isolate revealed this variation to be growth-phase independent and double labelling of both epitopes showed independent variation. CSF containing N. meningitidis was stored in liquid nitrogen without laboratory processing at the time of isolation of the second strain. Direct analysis of the organisms showed no cell-to-cell variation and immunoglobulin G on the surface. However, while there were similar labelling densities of the Class 1 epitope in vivo compared with either strain grown in vitro, there was a lower labelling density of the Class 3 epitope in vivo that was not caused by freeze-thawing. This reduction may be due to decreased expression of this epitope in vivo which casts doubts on the use of the Class 2/3 OMP as a vaccine candidate.     

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.     

Iron and outer membrane proteins in the susceptibility of Neisseria meningitidis to human serum

The proportion of carrier-isolated Neisseria meningitidis strains sensitive to human serum (37.2%) was found to be significantly higher than that of case-isolated ones (4.1%), although the difference is too low to consider serum-resistance responsible for invasion in this microorganism. Serum-susceptibility was not related to the existence of specific outer membrane proteins, as is the case of N. gonorrhoeae. Iron restriction induced iron-regulated outer membrane proteins in each strain (but not the same proteins in all strains) but without any detectable effect on serum-susceptibility. Iron excess was also unable to induce changes in the susceptibility of N. meningitidis to human serum.     

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.     

Identification and characterization of auxotrophs of Neisseria meningitidis produced by Tn916 mutagenesis

Abstract Fourteen Tn 916 mutants of Neisseria meningitidis strain NMB were identified as auxotrophs. Among these were eight amino acid auxotrophs, with five different phenotypes, and three isolates restricted in carbon source utilization, growing in the presence of glucose but not on L-lactate, D-lactate, pyruvate, or casamino acids as principal carbon sources.     

Epidemiological evaluation of Neisseria meningitidis serogroup B by pulsed-field gel electrophoresis

Abstract Genomic DNA from 25 strains of serogroup B Neisseria meningitidis was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Spe I. N. meningitidis genomic DNA displayed considerable diversity. The diversity we observed among these strains was stable and included isolates from an outbreak that were phenotypically identical. This confirms the value of macrorestriction profiling and PFGE in providing epidemiologically stable strain markers for typing meningococci.     

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.     

Analysis of Neisseria lactamica antigens putatively implicated in acquisition of natural immunity to Neisseria meningitidis

Sera from healthy human volunteers, patients convalescent from meningococcal meningitis, and mice immunized with outer membrane proteins from Neisseria meningitidis and Neisseria lactamica strains were used to analyze and identify antigens cross-reactive to both neisserial species. All classes of meningococcal proteins except class 1 (PorA) and class 5 cross-reacted with N. lactamica proteins and two other proteins of 65 and 55 kDa (an iron-regulated protein). Results obtained with the mouse sera demonstrate that cross-reactive antibodies can be elicited by either N. meningitidis or N. lactamica. These results support the suggestion that N. lactamica contributes to the development of natural immunity against N. meningitidis during the first years of life. The use of vaccines containing proteins other than PorA could interfere in colonization of mucosal surfaces by N. lactamica, hampering the natural mechanisms of immunity acquisition in humans. Only convalescent sera reacted with the 55 and 65 kDa proteins, which suggests that they might be relevant for pathogenicity.     

A mouse model utilising human transferrin to study protection against Neisseria meningitidis serogroup B induced by outer membrane vesicle vaccination

We have previously developed a mouse model based on transient bacteraemia in normal B10.M mice to evaluate the protective efficacy of outer membrane vesicle vaccines against serogroup B meningococci. To obtain a course of infection similar to that observed in man, we have in this work modified the mouse model by administration of human holo-transferrin upon bacterial challenge. Co-challenge with holo-transferrin induced increasing bacteraemia and subsequent death in normal non-immune mice, but not in vaccinated animals. The model system is dependent on challenge with meningococci expressing the transferrin receptor which is obtained by culturing the bacteria under iron restriction. The modified model system for protection against meningococcal infection presented here makes it possible to measure outer membrane vesicle vaccine induced protection by using bacteraemia as well as survival as parameters.     

Association of iron-regulated outer membrane proteins of Neisseria meningitidis with the RmpM (class 4) protein

The RmpM (class 4) protein of Neisseria meningitidis has previously been shown to be associated with the outer membrane porins. In the present study, we demonstrate that this protein forms complexes with the lactoferrin receptor LbpA, the transferrin receptor TbpA and the siderophore receptor FrpB as well. This complexation apparently resulted in a stabilization of oligomeric forms of these iron-regulated proteins. In vitro experiments further revealed a reduced ability to acquire iron from human lactoferrin in the rmpM mutant. Furthermore, all TonB-dependent receptors investigated here appeared to exist as oligomers (probably dimers), suggesting that this is a general feature of this class of proteins.     

Rapid molecular identification of Neisseria meningitidis isolates using the polymerase chain reaction followed by single-stranded conformation polymorphism analysis

Typing of Neisseria meningitidis strains is currently performed with conventional and molecular methods. Our objectives were: first, to develop a polymerase chain reaction (PCR) followed by single-stranded conformation polymorphism (SSCP) analysis of the PorA gene (VR1 region) to distinguish N. meningitidis subtypes and second, to evaluate the method for the identification and characterization of N. meningitidis in patient specimens. SSCP analysis of the VR1 region of the PorA1/2 gene from 126 N. meningitidis strains and 29 clinical samples identified seven SSCP types (SP-1 to SP-7); four strains were not typeable by the method. Classification according to the SSCP methods and serosubtype agreed for 122 of the 126 typeable strains (96.8%). For the 24-culture positive clinical samples, serosubtype and SSCP agreed in all cases. Five samples, which were culture-negative but obtained from children during an apparent outbreak of meningococcal disease in a primary school, presented identical SSCP classification for each sample (SP-2). PCR-SSCP is a rapid and cost-effective method for typing N. meningitidis strains that could provide important early information in the surveillance of suspected meningococcal outbreaks, particularly when culture-negative specimens constitutes the main source of material to analyze.     

Immunologic and genetic characterization of lipooligosaccharide variants in a Neisseria meningitidis serogroup C strain

Neisseria meningitidis shows great variation in expression of structurally different lipooligosaccharides (LOS) on its cell surface. To better understand the LOS diversity that may occur within an individual strain, a group C wild-type strain, BB305-Tr4, and two stable isogenic LOS variants, Tr5 and Tr7, were selected for this study. SDS-PAGE analysis showed a size reduction of Tr5 and Tr7 LOS compared to that of Tr4. Immunoblotting showed that parental Tr4 LOS reacted with L1, L2 and L3,7 antibodies, variant Tr5 LOS with L1 and L6 antibodies, while Tr7 LOS was non-typeable. Genetic analysis showed that the gene organization at the lgt-1 locus in the three strains was lgtZ,C,A,B,H4 in Tr4, lgtZ,C,A,H4 in Tr5 and lgtZ,C,A,H9 in Tr7. The genetic differences in the three strains were consistent with their phenotypic changes. Sequence comparison revealed two independent recombination events. The first was the recombination of repeated DNA fragments in the flanking regions to delete lgtB in Tr5. The second was the recombination of a fragment of two genes, lgtB and lgtH4, to create an inactive lgtH9 allele with a mosaic structure in Tr7. These findings suggest that besides phase variation, homologous recombination can contribute to the genetic diversity of the lgt locus and to the generation of LOS variation in N. meningitidis.     

Identification of two major families of transferrin receptors among Neisseria meningitidis strains based on antigenic and genomic features

Abstract The transferrin receptor or transferrin-binding proteins (Tbps) of 50 strains of Neisseria meningitidis belonging to different serogroups were examined by Western blotting using two rabbit antisera raised against Tbp purified from N. meningitidis strains B16B6 and M982. On the basis of the reactivity of Tbp2 with the antisera two patterns were observed and allowed the classification of 74% of the strains in group I (M982-like strains) and 26% in group II (B16B6-like strains). Southern blot analysis was performed on the genomic DNA of 16 meningococcal strains and showed that under stringent conditions, the tbp2 probes were specific for each group identified. Both immunological and genomic analyses have led to the identification within N. meningitidis strains of two major families distinguished on the basis of the characteristics of Tbp2 molecules, independently of serogroup, type or subtype.     

Effect of a booster dose of serogroup B meningococcal vaccine on antibody response to Neisseria meningitidis in mice vaccinated with different immunization schedules

The generation and maintenance of memory antibody response by different primary immunization schedules with the Cuban-produced outer membrane protein based vaccine was investigated in a murine model. We analyzed the duration of the antibody response (IgG-ELISA and bactericidal titer) and the effect of a booster dose on the antibody response. The IgG avidity index was determined in an attempt to find a marker for memory development. This study also included an analysis of IgG subclasses induced by primary and booster immunization. The specificity of bactericidal antibodies was investigated using local strains of the same serotype/serosubtype (4,7:P1.19,15) as the vaccine strain and mutant strains lacking major outer membrane proteins. A significant recall response was induced by a booster dose given 7 months after a primary series of 2, 3 or 4 doses of vaccine. The primary antibody response showed a positive dose-effect. In contrast, a negative dose-effect was found on the booster bactericidal antibody response. There was a significant increase in IgG1 levels after the fourth and booster doses. Three doses of vaccine were required to induce a significant increase in IgG avidity. Two injections of vaccine induced a significant antibody response to PorA protein, while 4 injections induced a larger range of specificities.     

Analysis of Moraxella catarrhalis outer membrane antigens cross-reactive with Neisseria meningitidis and Neisseria lactamica

Mouse sera against outer membrane proteins from Moraxella catarrhalis, Neisseria meningitidis and Neisseria lactamica, and human sera from both healthy individuals and patients convalescing from meningococcal meningitis were used to identify cross-reactive antigens. Mouse anti-N. meningitidis and anti-N. lactamica sera recognized 77, 62 and 32 kDa outer membrane antigens in M. catarrhalis strains; on the contrary, the meningococcal porin PorB (38-42 kDa) was recognized by one of the two anti-M. catarrhalis sera. Human sera from both healthy individuals and patients convalescing from meningococcal meningitis also showed cross-reactive antibodies against these proteins. The existence of cross-reactive antigens in M. catarrhalis and N. meningitidis (as well as in N. lactamica) could favor the development of natural immunization against both pathogens.