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Dissecting the specificities of human antibody responses following disease caused by serogroup A meningococci may be important for the development of improved vaccines. We performed a study of Ethiopian patients during outbreaks in 2002 and 2003. Sera were obtained from 71 patients with meningitis caused by bacteria of sequence type 7, as confirmed by PCR or culture, and from 113 Ethiopian controls. Antibody specificities were analyzed by immunoblotting (IB) against outer membrane antigen extracts of a reference strain and of the patients' own isolates and by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) levels against lipooligosaccharide (LOS) L11 and the proteins NadA and NspA. IB revealed that the main antigens targeted were the proteins PorA, PorB, RmpM, and Opa/OpcA, as well as LOS. MenA disease induced significant increases in IgG against LOS L11 and NadA. The IgG levels against LOS remained elevated following disease, whereas the IgG anti-NadA levels returned to acute-phase levels in the late convalescent phase. Among adults, the anti-LOS IgG levels were similar in acute-phase patient sera as in control sera, whereas anti-NadA IgG levels were significantly higher in acute-phase sera than in controls. The IgG antibody levels against LOS and NadA correlated moderately but significantly with serum bactericidal activity against MenA strains. Future studies on immune response during MenA disease should take into account the high levels of anti-MenA polysaccharide IgG commonly found in the population and seek to clarify the role of antibodies against subcapsular antigens in protection against MenA disease.

To elucidate critical components of protective immune responses induced during the natural course of serogroup A meningococcal disease, we studied acute-, early-convalescent-, and late-convalescent-phase sera from Ethiopian patients during outbreaks in 2002 to 2003. Sera were obtained from laboratory-confirmed patients positive for serogroup A sequence type 7 (ST-7) meningococci (A:4/21:P1.20,9) (n = 71) and from Ethiopian controls (n = 113). The sera were analyzed using an enzyme-linked immunosorbent assay to measure levels of immunoglobulin G (IgG) against serogroup A polysaccharide (APS) and outer membrane vesicles (OMVs) and for serum bactericidal activity (SBA) using both rabbit and human complement sources. Despite relatively high SBA titers and high levels of IgG against APS and OMVs in acute-phase patient sera, significant increases were seen in the early convalescent phase. Antibody concentrations returned to acute-phase levels in the late convalescent phase. Considering all patients' sera, a significant but low correlation (r = 0.46) was observed between SBA with rabbit complement (rSBA) using an ST-5 reference strain and SBA with human complement (hSBA) using an ST-7 strain from Ethiopia. While rSBA demonstrated a significant linear relation with IgG against APS, hSBA demonstrated significant linear relationships with IgG against both APS and OMV. This study indicates that antibodies against both outer membrane proteins and APS may be important in providing the protection induced during disease, as measured by hSBA. Therefore, outer membrane proteins could also have a role as components of future meningococcal vaccines for the African meningitis belt.

There is currently no standardized serum bactericidal antibody (SBA) assay for evaluating immune responses to meningococcal outer membrane vesicle or protein vaccines. Four laboratories, Manchester Health Protection Agency (MC HPA), New Zealand Institute of Environmental Science and Research Limited (NZ ESR), Norwegian Institute of Public Health (NIPH), and Chiron Vaccines (Chiron), measured SBA titers in the same panel of human sera (n=76) from laboratory staff (n=21) vaccinated with MenBvac. Blood samples were collected prevaccination, prior to each of the three doses of MenBvac given at 6-week intervals, and 6 weeks following the third dose. Initial results showed a number of discrepancies in results between the four participating laboratories. The greatest effect on titers appeared to be due to differences among laboratories in the maintenance of the meningococcal serogroup B test strain, 44/76-SL. A repeat study was conducted using the same frozen isolate (meningococcal serogroup B test strain 44/76-SL), freshly distributed to all four laboratories. Using SBA titers from the tilt method for all samples, and using MC HPA as the comparator, the results were as follows for NZ ESR, NIPH, and Chiron, respectively, using log(10) titers: correlation coefficients (r) were 0.966, 0.967, and 0.936; intercepts were 0.08, 0.15, and 0.17; and slopes were 0.930, 0.851, and 0.891. In both prevaccination and postvaccination samples from 15 subjects assayed by all four laboratories, similar increases in SBA (fourfold or greater) were observed (for 11, 11, 9, and 9 subjects for MC HPA, NZ ESR, NIPH, and Chiron, respectively), and similar percentages of subjects with SBA titers of>or=4 p revaccination and 6 weeks following each dose were found. The SBA assay has been harmonized between the four different laboratories with good agreement on seroconversion rates, n-fold changes in titers, and percentages of subjects with SBA titers of >or=4.

Mice immunised intranasally with multiple doses of outer membrane vesicles (OMVs) from group B meningococci developed antibody responses that depended on the interval between doses. High levels of antibodies in saliva and extracts of faeces were induced within 4 weeks after an OMV vaccine had been given at weekly intervals, whereas the antibody responses in these samples were negligible when given four times at 1-day or 1-h intervals, or as one large dose. Only modest responses were obtained in serum after 4 weeks, however, whether the vaccine had been given repeatedly at any schedule, including the 1-week interval, or as one dose. On the other hand, two large doses given 8 weeks apart induced booster antibody responses in both serum and secretions that matched the responses from a second series of the four smaller doses. Intranasal immunisations may thus stimulate immunological memory more rapidly in secretions than in serum. In order to secure adequate systemic responses by a minimum of doses, nasal vaccines should therefore be given at intervals longer than 4 weeks, in harmony with the intervals recommended for injectable vaccines.

The mouse humoral immune response toward native or detergent-extracted outer membrane vesicles (NOMVs and DOMVs, respectively) from Neisseria meningitidis was determined after intranasal immunization. Both preparations elicited high frequencies of NOMV-specific antibody-forming cells (AFCs) locally in the nasal associated lymphoid tissue (NALT) after three or four weekly doses. The diffuse NALT (D-NALT) contained ca. 10-fold more NOMV-specific AFCs than those observed in the mediastinal lymph node, spleen, and bone marrow. AFCs observed in the D-NALT were primarily immunoglobulin A positive (IgA(+)) and were maintained for at least 1 month. In contrast, the organized NALT (O-NALT) contained low numbers of AFCs, and the response was relatively short-lived. In other lymphoid tissues, AFCs producing various IgG subclasses and IgM were present with IgG2b-producing AFCs being dominant or codominant with IgA or IgG2a. In serum and in all of the tissues examined, with the exception of the NALT, NOMVs clearly induced a stronger antibody response and a broader range of antibody isotypes than DOMVs. The development of NOMV-specific AFCs in spleen and bone marrow after intranasal immunization was slow compared to intravenous immunization but, once established, the intranasally elicited responses increased steadily for at least 75 days. NOMV-specific antibodies induced via several routes of immunization had high bactericidal activities in serum. Our results indicated that intranasally administered OMVs induced strong local and systemic antibody responses in mice that were relatively long-lived.

We studied the in vitro protective activities of human immunoglobulin G1 (IgG1), IgG3, and IgM antibodies against group B meningococci by constructing sets of chimeric mouse-human antibodies (chIgG1, chIgG3, and chIgM, respectively) with identical binding regions against the P1.7 and P1.16 epitopes on PorA. This was done by cloning the V genes of three mouse hybridoma antibodies and subsequently transfecting vectors containing the homologous heavy- and light-chain genes into NSO cells. Cell clones secreting intact human chIgG1, chIgG3, or chIgM antibodies originating from three parent mouse antibodies were isolated. The functional affinities appeared to be similar for all human isotypes and surprisingly also for the pentameric chIgM antibody. chIgG1 exhibited greater serum bactericidal activity (SBA) than chIgG3, while chIgG3 was more efficient in inducing a respiratory burst (RB) associated with opsonophagocytosis than chIgG1 was. On the other hand, chIgM exhibited SBA similar to that of chIgG1, but it exhibited much higher RB activity than chIgG3 and chIgG1 exhibited. The antibodies against the P1.16 epitope were more efficient in terms of SBA than the antibodies against the P1.7 epitope were; thus, 10- to 40-fold-lower concentrations of antibodies against P1.16 than of antibodies against P1.7 were needed to induce SBA. On the other hand, antibodies against these epitopes were equally effective in inducing RB. Our results revealed differences in the functional activities of human chIgG1, chIgG3, and chIgM antibodies against meningococci, which might influence their protective effects against meningococcal disease.

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.

The infant rat infection model previously developed to evaluate protective ability of passively administered murine antibodies to group B meningococcal (MenB) surface antigens was adapted for human sera. Several challenge doses were tested, aiming at sensitive detection of protection with little interassay variability. Doses of 10(5) and 10(6) colony forming units of strain IH5341 (MenB:15:P1.7,16) injected intraperitoneally gave consistently high levels of bacteremia and meningitis developed in 6 h in 50-100% of the pups. A monoclonal antibody mAb735 to the MenB capsule, injected 1-2 h before bacterial challenge, gave full protection at a dose of 2 microg/pup. Sera from adult volunteers immunized with a MenB outer membrane vesicle vaccine reproducibly reduced bacterial counts in the blood and cerebrospinal fluid, whereas a normal human serum, lacking bactericidal and opsonophagocidal activity, was unprotective.

Context: Meningococcal disease occurs worldwide, and serogroup B disease accounts for a large proportion of cases. Although persons younger than 4 years are at greatest risk for serogroup B meningococcal disease, vaccine efficacy has not been demonstrated in this age group., Objective: To evaluate serum bactericidal activity (SBA) against homologous vaccine type strains and a heterologous Chilean epidemic strain of Neisseria meningitidis as a potential correlate for vaccine efficacy., Design: Double-blind, randomized controlled trial conducted between March 14 and July 20, 1994. All blood samples were taken by December 1994., Setting: Santiago, Chile, where a clonal serogroup B meningococcal disease epidemic began in 1993., Participants: Infants younger than 1 year (n = 187), children aged 2 to 4 years (n = 183), and adults aged 17 to 30 years (n = 173)., Intervention: Participants received 3 doses of outer-membrane protein (OMP) meningococcal vaccine developed in either Cuba or Norway or a control vaccine, with each dose given 2 months apart. Blood samples were obtained at baseline, prior to dose 3, and at 4 to 6 weeks after dose 3., Main Outcome Measure: Immune response, defined as a 4-fold or greater rise in SBA titer 4 to 6 weeks after dose 3 compared with prevaccination titer., Results: Children and adult recipients of either meningococcal vaccine were more likely than controls to develop an immune response to the heterologous epidemic strain. After 3 doses of vaccine, 31% to 35% of children responded to the vaccine vs 5% to placebo; 37% to 60% of adults responded to vaccine vs 4% to placebo (P<.05 vs control for all). Infants, however, did not respond. In contrast, against homologous vaccine type strains, the response rate was 67% or higher among children and adults and 90% or higher among infants (P<.001 vs control for all). Subsequent SBA against 7 isogenic homologous target strains identified class 1 OMP as the immunodominant antigen., Conclusions: These data suggest that neither serogroup B OMP meningococcal vaccine would confer protection during a heterologous epidemic. However, epidemic strain-specific vaccines homologous for class 1 OMP are promising candidates for the control of epidemic serogroup B meningococcal disease.

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 nasal vaccine, consisting of outer membrane vesicles (OMVs) from group B Neisseria meningitidis, was given to 12 volunteers in the form of nose drops or nasal spray four times at weekly intervals, with a fifth dose 5 months later. Each nasal dose consisted of 250 microg of protein, equivalent to 10 times the intramuscular dose that was administered twice with a 6-week interval to 11 other volunteers. All individuals given the nasal vaccine developed immunoglobulin A (IgA) antibody responses to OMVs in nasal secretions, and eight developed salivary IgA antibodies which persisted for at least 5 months. Intramuscular immunizations did not lead to antibody responses in the secretions. Modest increases in serum IgG antibodies were obtained in 5 volunteers who had been immunized intranasally, while 10 individuals responded strongly to the intramuscular vaccine. Both the serum and secretory antibody responses reached a maximum after two to three doses of the nasal vaccine, with no significant booster effect of the fifth dose. The pattern of serum antibody specificities against the different OMV components after intranasal immunizations was largely similar to that obtained with the intramuscular vaccine. Five and eight vaccinees in the nasal group developed persistent increases in serum bactericidal titers to the homologous meningococcal vaccine strain expressing low and high levels, respectively, of the outer membrane protein Opc. Our results indicate that meningococcal OMVs possess the structures necessary to initiate systemic as well as local mucosal immune responses when presented as a nasal vaccine. Although the serum antibody levels were less conspicuous than those after intramuscular vaccinations, the demonstration of substantial bactericidal activity indicates that a nonproliferating nasal vaccine might induce antibodies of high functional quality.

Outer membrane vesicle (OMV) vaccines were made from Neisseria meningitidis strain 44/76 and its two short-chain lipopolysaccharide (LPS) mutants, Mu-1 and Mu-4. Only the 44/76 vaccine contained LPS with the host antigen lacto-N-neotetraose. The protein composition of the vaccines was similar. The LPS carbohydrate chain length proved to influence drastic changes in the LPS immunogenicity as well as the outer membrane proteins (OMPs) ability to elicit functional antibodies in mice. Only LPS in the Mu-1 and Mu-4 vaccines were immunogenic, and the 44/76 vaccine differed also by not inducing antibodies to the class 4 OMP. The Mu-1 vaccine, with a LPS carbohydrate chain comprising only two residues of 2-keto-3-deoxy-octonic acid, induced lower bactericidal activity and less antibodies to the class 1 OMP, compared to the two other vaccines. This indicates that LPS of a certain carbohydrate chain length is required for adequate exposure of the class 1 OMP epitopes essential for inducing bactericidal antibodies.

Two monoclonal antibodies (mAbs) designated 144,H-3 (IgG2a) and 218,C-5 (IgM) were produced after immunization of mice with two different heat-treated and sonicated pneumococcal strains. Western blotting, with solubilized proteins from different bacterial genera and from mammalian lymphocytes, showed that both mAbs reacted with a protein of approximately 12 kDa in all 66 strains of eubacteria examined, representing 27 different species. The 12 kDa protein was isolated by immunoaffinity chromatography. Subsequent preparative Western blotting enabled N-terminal amino acid sequence analysis by microsequencing. A high degree of amino acid sequence similarity with eubacterial ribosomal proteins L7/L12 was demonstrated. One of the mAbs (144,H-3) also cross-reacted in Western blotting with a 43 kDa protein, but only from streptococci. The 43 kDa protein carrying the common streptococcal epitope was isolated and sequenced in the N-terminal region. A high degree of amino acid sequence identity was found to elongation factor Ts from Escherichia coli.

An ELISA method was developed to quantitate gravimetrically (microgram/ml) the IgG subclass response against a Norwegian vaccine composed of outer membrane vesicles (OMV) isolated from a Neisseria meningitidis B:15:P1.7,16 epidemic strain. Chimeric mouse-human anti-hapten NIP (5-iodo-4-hydroxy-3-nitrophenacetyl) antibodies of each subclass were used for calibration purposes. Before vaccination, low amounts of IgG1 and IgG2 antibodies against OMV were detectable in all vaccinees, whereas IgG3 was only detectable in one of the 21 vaccinees. After vaccination, IgG1 antibodies dominated the response followed by IgG3 and low to moderate levels of IgG2 antibodies. IgG4 was only detectable at very low levels in a few vaccinees. All sera showed close to parallel dose-response curves to each other for IgG1 and IgG3, whereas the IgG2 curves were not parallel to chimeric IgG2 and could thus not be quantitated gravimetrically. For IgG3, 1/3 of the vaccinee sera showed non-parallel dose-response curves to the rest of the vaccinee sera and to chimeric IgG3 and could not be gravimetrically quantitated. The rest of the sera showed parallel dose-response curves with the chimeric IgG3 and gravimetric quantitation was possible. This study illustrates that chimeric antibodies can be used as calibrators to quantitate IgG subclass antibody responses against OMV in gravimetric units and that the vaccine mainly induces IgG1 and IgG3 antibodies in humans.

The antibody kinetics in sera from 27 adults after three doses of the Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine was studied. The vaccinees received the third dose 4 to 5 years after the first two. Antibody responses against outer membrane proteins (OMPs) and lipopolysaccharides were studied by enzyme-linked immunosorbent assay and immunoblotting and with serum bactericidal assays (SBA) with three variants of the vaccine strain, 44/76. Six weeks after the second injection, the geometric mean (GM) of the levels of immunoglobulin G (IgG) against OMVs was about sevenfold higher than that of prevaccination levels, and 74% of the vaccinees developed a greater-than-twofold rise in SBA titer. After 6 months, the GM of IgG levels declined to about threefold higher, and after 4 to 5 years it declined to about twofold higher, than that before vaccination. The third dose induced a rapid increase in SBA titers in 96% of the vaccinees, and the GM of levels of IgG against OMVs rose to about 14-fold the prevaccination level. One year later, the IgG antibody levels had dropped to 4.6-fold the prevaccination level, but 88% of the vaccinees still showed bactericidal activity. The response after the two first doses was higher in individuals with prevaccination antibodies, but no such effect was found after three doses. The use of defined mutants in SBA and linear multiple regression analyses indicated that among the major OMPs, antibodies to the Opc and class 1 proteins made the most important single contributions to the bactericidal activity against the vaccine strain, but it also demonstrated the importance of antibodies against other antigens. After three doses, 68% of the vaccinees showed a significant SBA response against a strain lacking both the Opc and the class 1 proteins. Three doses converted almost all subjects to SBA responders and gave higher antibody levels and relatively less serosubtype-specific bactericidal activity than did two doses, probably indicating a broader cross-protection against heterologous strains.

Antibodies to the meningococcal serosubtype-specific P1.7,16 protein and its variable regions (VR) were analysed in 28 convalescent sera drawn 8-36 months after systemic meningococcal disease by immunoblotting and enzyme immunoassay (EIA) methods. EIA antigens were the meningococcal P1.7,16 protein, produced in Bacillus subtilis, and peptides covering its VR1 (P1.7 region) and VR2 (P1.16 region) inserted into a bacterial penicillinase protein. In the immunoblotting method, three meningococcal reference strains were used; they expressed either the P1.7,16 protein, or only its VR1 or VR2 epitopes in their class 1 proteins. Both methods showed a strong IgG response in four sera to P1.7,16 and VR2, but not to VR1; 18 sera had no or weak anti-class 1 protein activity. The six remaining sera were positive only on blots. The VR2-specific sera had 30-fold higher bactericidal activity than those with negligible P1.7,16 responses. Previous vaccination of the patients with a B:15:P1.7,16 meningococcal vaccine was associated with a strong anti-P1.7,16 and anti-VR2 booster response that declined with time. The subtype-specific antibody activity in some sera indicated colonisation after disease by meningococci with class 1 proteins different from the strain that had caused disease.

Opsonic activity in sera from 27 military recruits vaccinated with the Norwegian meningococcal serogroup B outer membrane vesicle vaccine was measured as respiratory burst with polymorphonuclear leukocytes as the effector cells and meningococci of the epidemic strain as the target. The results were compared with antimeningococcal IgG antibodies against an outer membrane vesicle coat in an enzyme-linked immunosorbent assay and with serum bactericidal activity. The vaccinees were immunized twice, with a 6-week interval between the two. The serum samples studied were collected at day zero, after 6 weeks, and after 12 weeks. Both serum bactericidal activity and respiratory burst were measured by adding external serum as the complement source. The results revealed a significant increase in specific IgG response, serum bactericidal activity, and respiratory burst after vaccination. We found a highly significant correlation between the responses in all three assays (P < 0.0001). The highest correlation was found between respiratory burst and antimeningococcal IgG response (r = 0.93). This result strongly indicates that respiratory burst is mediated almost exclusively by IgG antibodies. The correlation between antimeningococcal IgG response and serum bactericidal activity was slightly lower (r = 0.83). The correlation between respiratory burst and serum bactericidal activity was further reduced (r = 0.78), and some of the sera revealed a marked preference for only one of the activities. This result means that respiratory burst and serum bactericidal activity in part are induced by different mediators, and to obtain a more complete picture of the potential protective activity, both assays should be applied to survey a vaccine trial.

Monoclonal human IgG SS269 reacts with Neisseria meningitidis expressing the P1.7 PorA protein and with linear peptides containing NGGAS, which accounts for the P1.7 specificity. Murine monoclonal antibody to P1.7 reacts with peptides containing the overlapping epitope, ASGQ. The human and murine antibodies have similar affinities. The low avidity human antibody is very inefficient at stimulating complement-mediated bactericidal killing while the high avidity murine antibody efficiently kills bacteria. However, efficient opsonophagocytosis was mediated even at low concentrations of the human antibody and in the absence of complement, suggesting that low avidity antibodies might be protective against disease.

The opsonic activity of sera from healthy volunteers immunized with an outer membrane vesicle vaccine prepared from a Neisseria meningitidis (class 3, 44/76, B:15:P1.7,16), characteristic of the present Norwegian epidemic, has been examined. A marked increase in the phagocytosis of class 3 and nontypeable strains of different serogroups, serotypes, and serosubtypes was demonstrated in the presence of postvaccination sera. Sera from vaccinees also caused a significant increase in leukocyte oxidative metabolism as measured by luminol-enhanced chemoluminescence during phagocytosis of class 3 and nontypeable meningococci. An increase in serum opsonins cross-reacting with class 2 (type 2a) meningococci of different serogroups was not observed, suggesting that future meningococcal outer membrane vesicle vaccine preparations should contain both class 2 and class 3 porins in geographic areas where both class 2 and class 3 strains cause disease.

The 5C protein is expressed by the strain of Neisseria meningitidis (44/76) used for production of the Norwegian meningococcal group B outer membrane vesicle vaccine and is included in the final formulation of this vaccine. The immunoglobulin G antibody response to 5C stimulated by vaccination, systemic meningococcal disease, and carriage was measured using ELISAs with synthetic liposomes as antigen and by immunoblotting. Increased levels of IgG were found in paired sera from all three groups. The antibodies were bactericidal to meningococci of serogroups A and B that expressed large amounts of 5C but not to meningococci expressing smaller amounts. There was a linear correlation between bactericidal titer and units of IgG to 5C.

Antibody responses after vaccination with three different formulations of a new meningococcal group B outer membrane vesicle (OMV) vaccine have been studied with the ELISA technique using four different antigens. Sera from about 1200 vaccinees participating in steps 1, 2, 3 and 6 of the phase II clinical trials in Norway were analysed. The effects of non-covalently complexing the OMV antigen to group C polysaccharide (C-PS) and of adsorbing OMV (with and without C-PS) to aluminium hydroxide (AH) were studied. All three vaccine formulations were highly immunogenic in humans. Adsorption of the vaccine to AH had a relatively small effect on the immune response, but the results indicated that the booster response was stronger with the adsorbed than with the unadsorbed vaccines. Some increase in the immune response against OMV was also observed by non-covalent complexing OMV with C-PS, particularly after the second dose. In most of the vaccinees the antibody levels were significantly reduced 6 to 12 months after vaccination. Adsorption of the vaccine to AH had no effect on the antibody response against C-PS. Comparison with bactericidal activity of the same sera was done. A highly significant correlation was observed between the bactericidal titres and the levels of IgG antibodies against OMV and class 5C protein, whereas the correlation between antibody levels against lipopolysaccharide and the bactericidal activity was poor.

Results from the serum bactericidal assay (BA) after immunization of human volunteers with the Norwegian serogroup B meningococcal outer membrane vesicle vaccine are surveyed. In the phase II trials with adults we found very high seroconversion rates (greater than 98%) against the vaccine strain in the BA. Details in the antigenic composition of the inoculum used in the BA seem very important as shown here by finding lower bactericidal titres with teenager sera when tested with a variant of the standard inoculum. The present preliminary report corresponds to the presentation given at the Report Meeting on the Norwegian Meningococcal Vaccine Trial, Oslo, 12 September, 1991.

To elucidate pathogenic aspects and serodiagnostic possibilities for meningococcal disease, we investigated levels of specific antimeningococcal immunoglobulin G (IgG), IgA, and IgM in serum by using an enzyme-linked immunosorbent assay with outer membrane antigen prepared from a Neisseria meningitidis B:15:P1.16 strain. Serum samples were drawn on hospital admission as well as during convalescence from patients suspected of purulent meningitis or meningococcal septicemia, and single samples were drawn from population controls. A total of 637 samples were examined blindly. On admission, the average antimeningococcal immunoglobulin levels were about the same in the meningococcal disease patients as in the population controls. Septicemic patients, however, had significantly lower values. During one week the mean specific immunoglobulin levels in meningococcal-disease patients increased 6 times for IgG, 14 times for IgA, and 5 times for IgM. Children younger than 1 year showed a modest and more slowly developing antibody response. There were no statistically significant differences in average antibody responses among patients infected with meningococci of different serotypes. At 100% specificity, the increase in IgG, IgA, and IgM yielded diagnostic sensitivities for meningococcal disease of 84, 52, and 66%, respectively. One of seven serum pairs from the patient control group with unknown etiology was positive for meningococcal disease in this assay. The patients with meningococcal disease originally diagnosed only by clinical signs and symptoms showed a slightly lower rate of seroconversion than the patients in whom the diagnosis was supported by test results showing a systemic Neisseria meningitidis isolate.

Antibody responses of adult volunteers given a vaccine containing meningococcal capsular polysaccharides (serogroups A, C, Y, and W-135) noncovalently complexed with serotype 2b:P1.2 and 15:P1.16 outer membrane proteins have been studied. Sera were analyzed by enzyme-linked immunosorbent assay methods for immunoglobulin G (IgG), IgM, and IgA antibodies and for bactericidal activities against the homologous strains. The vaccination was performed as a double-blind experiment with 47 volunteers, of whom 23 received the protein-polysaccharide vaccine and 24 received the control preparation containing the polysaccharides only. Ten additional persons volunteered for the protein-polysaccharide vaccine. Before vaccination, carriers of meningococci had significantly higher levels of specific IgG and IgA and also higher bactericidal activities than noncarriers. At 2 weeks postvaccination we found significant IgG and bactericidal antibody responses against both the 2b:P1.2 and 15:P1.16 strains in about 70% of the protein-polysaccharide vaccinees. The immune response induced by disease was compared with that induced by vaccination by analyzing paired sera from 13 survivors of serogroup B serotype 15 meningococcal disease. We found that the mean specific IgG level in acute-phase sera was lower than average in prevaccination sera from the vaccinees but similar to that of healthy noncarriers before vaccination. The convalescent-phase sera showed IgG responses similar to those of the vaccinees, but the IgM response to disease was significantly higher than after vaccination. The immune response to disease caused by serogroup B serotype 15 meningococci was found by enzyme-linked immunosorbent assay analysis to be about the same with outer-membrane antigens from a serotype 2b strain as it was with antigens from a serotype 15 strain.