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Complement activation is closely associated with plasma endotoxin levels in patients with meningococcal infections. This study assessed complement activation induced by purified Neisseria meningitidis lipopolysaccharide (Nm-LPS), native outer membrane vesicles (nOMVs), LPS-depleted outer membrane vesicles (dOMVs), wild-type meningococci, and an LPS-free mutant (lpxA(-)) from the same strain (44/76) in whole blood anticoagulated with the recombinant hirudin analogue. Complement activation products (C1rs-C1 inhibitor complexes, C4d, C3bBbP, and terminal SC5b-9 complex) were measured by double-antibody EIAs. Nm-LPS was a weak complement activator. Complement activation increased with preparations containing nOMVs, dOMVs, and wild-type bacteria at constant LPS concentrations. With the same protein concentration, complement activation induced by nOMVs, dOMVs, and the LPS-free mutant was equal. The massive complement activation observed in patients with fulminant meningococcal septicemia is, presumably, an indirect effect of the massive endotoxemia. Outer membrane proteins may be more potent complement activators than meningococcal LPSs.

Whole killed meningococci (Nm) and pertussis bacteria (Bp) were tested for mucosal immunogenicity and as mucosal adjuvants for an inactivated influenza virus vaccine given intranasally to unanaesthetized mice. Virus was given alone, or simply mixed with one of the bacterial preparations, in four doses at weekly intervals. The virus alone induced low but significant increases of influenza-specific IgG antibodies in serum measured by ELISA, whereas IgA responses in serum and saliva were insignificant compared to non-immunized controls. With Bp or Nm admixed, serum IgG and IgA and salivary IgA responses to the influenza virus were substantially augmented (P<0.005). However, this adjuvant effect of the bacterial preparations was not significant for responses in the intestine as measured by antibodies in faeces. Antibody responses to Bp itself, but not to Nm, were inhibited by the admixture of the virus vaccine. Moreover, the pertussis preparation induced salivary antibodies which cross-reacted with Nm. Whole-cell bacteria with inherent strong mucosal immunogenicity may also possess mucosal adjuvanticity for admixed particulate antigens which are weakly immunogenic by the nasal route.

Neisseria meningitidis, the cause of epidemic meningitis and acute lethal sepsis, synthesizes surplus lipopolysaccharides (LPSs) during growth, which are released as outer membrane vesicles (OMV) or "blebs". Meningococcal disease severity is related to plasma LPS levels. We have compared the biological activities of native outer membrane vesicles (nOMV) to those of purified Nm-LPS (Nm-LPS) and LPS-depleted OMV (dOMV) prepared from N. meningitidis. The LPS content of nOMV was determined spectrophotometrically by quantifying KDO and by silver-stained SDS-PAGE gels. The morphology of the preparations was studied by transmission electron microscopy. The Limulus amoebocyte lysate (LAL) assay was used to quantify LPS in the plasma solutions. The preparations were diluted in endotoxin-free heparin plasma to equal amounts of LPS (w/w) in the range 50-5000 pg/ml. The biological reactivity was tested by: (i) a monocyte target-assay (monocyte purity > or =96%); and (ii) a whole blood model, measuring the secretion of TNF-alpha and IL-6 induction of procoagulant activity in monocytes (PCA). In both models, nOMV induced dose-dependent cell responses (TNF-alpha, IL-6, PCA) similar to purified Nm-LPS, whereas dOMV induced minimal responses. However, LAL activity was significantly higher for nOMV than for purified Nm-LPS and dOMV. The cellular responses of purified Nm-LPS and nOMV were reduced (>95%) by a specific anti-CD14-antibody.

Antibodies against the class 4 outer membrane protein (OMP) from Neisseria meningitidis have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard beta-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane.

A Norwegian outer membrane vesicle (OMV) vaccine against group B meningococcal disease proved to be strongly immunogenic when administered intranasally in mice. The OMV preparation, made from Neisseria meningitidis and intended for parenteral use, was therefore given without adjuvant to human volunteers (n = 12) in the form of nose drops or nasal spray. Such immunizations, which were carried out at weekly intervals during a three-week period, were able to induce systemic antibodies with bactericidal activity in more than half of the individuals. In addition, all vaccinees developed marked increases in OMV-specific IgA antibodies in nasal secretions. The potential of the OMV particles as carriers for other less immunogenic antigens were elucidated in mice with use of whole inactivated influenza virus. Even though influenza virus alone did induce some systemic and salivary antibody responses after being administered intranasally, these responses were greatly augmented when the virus was presented together with OMVs. Thus, it is possible that a nasal OMV vaccine may induce protection against invasive meningococcal disease, and also that it might be used as a vehicle for nasal vaccines against other diseases.

We wanted to compare the potential protective capacity of antibodies to meningococcal lipopolysaccharides (LPS). The frequency of occurrence and degree of expression of the epitopes recognized by murine monoclonal antibodies (MAbs) to immunotypes L3,7,9 (9-2-L379) and L8 (2-1-L8) and to the LPS inner core (216-Lc and 217-Lc), were determined among 77 consecutive Norwegian meningococcal patient isolates from 1995. The immunotype L3,7,9 was strongly expressed by 95% of the isolates, whereas L8 was weakly to moderately expressed by 9%. The inner core epitopes, were widely distributed among the serogroup B organisms, but were proved weakly expressed. The bactericidal activity of the four MAbs to various selected strains, was found to correlate positively with the quantity of the LPS epitopes recognized by these four MAbs in the bacteria. When tested in the serum bactericidal assay (SBA), often a few percent of the colonies of the inocula survived high concentrations of the MAbs. The results indicate that escape from the bactericidal action could be achieved through: (i) selection of variants not expressing the LPS-epitope of the actual MAb, (ii) a relative reduction in the density of the LPS-epitope achieved by dilution with another LPS structure or (iii) other factors, not yet understood. In conclusion, antibodies to the L3,7,9 epitope seem to be of importance for protection, whereas antibodies to the epitopes of the LPS inner core or immunotype L8, are not likely to offer protection alone. However, in order to prevent escape through alteration of the LPS pattern of the microbes, various LPS structures should probably be present in the OMV vaccine.

We have compared gas chromatography and mass spectrometry (GC-MS) analysis with the Limulus amebocyte lysate (LAL) assay to quantify native meningococcal lipopolysaccharides (LPS) in five patient plasmas containing greater than 5 micrograms/liter by LAL. 3-Hydroxy lauric acid (3-OH-12:0) was used as a specific lipid A marker of neisserial LPS. The quantitative LAL results were confirmed by GC-MS (r = 0.98, P = 0.006). Seven patient plasmas were centrifuged at 103,000 g and the sedimentation behavior of native LPS compared with reference plasma proteins and with apo A1 and apo B100 representing high and low density lipoproteins. After 15 min of centrifugation, 84 +/- 2% (mean +/- SE) of the recovered LPS were found in the lower one-third of the centrifuged volume, whereas 6 +/- 1% remained in the upper one-third volume, indicating that meningococcal endotoxin circulates as complexes with high sedimentation coefficients. Bacterial outer membrane fragments were detected in the bottom fractions of three patient plasmas examined by means of electron microscopy. In three patient plasmas ultracentrifuged for 60 min at 103,000 g, the levels of apo A1 and apo B100 revealed minor changes, whereas only 1 +/- 1% of the recovered LPS remained in the upper one-third and 91 +/- 2% were found in the lower one-third volume. Few bioreactive LPS appear to be complexed with high and low density lipoproteins in meningococcal septic shock plasma.