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The vaccine potential of meningococcal Omp85 was studied by comparing the immune responses of genetically modified deoxycholate-extracted outer membrane vesicles, expressing five-fold higher levels of Omp85, with wild-type vesicles. Groups (n = 6-12) of inbred and outbred mouse strains (Balb/c, C57BL/6, OFI and NMRI) were immunized with the two vaccines, and the induced antibody levels and bactericidal and opsonic activities measured. Except for Balb/c mice, which were low responders, the genetically modified vaccine raised high Omp85 antibody levels in all mouse strains. In comparison, the wild-type vaccine gave lower antibody levels, but NMRI mice responded to this vaccine with the same high levels as the modified vaccine in the other strains. Although the vaccines induced strain-dependent Omp85 antibody responses, the mouse strains showed high and similar serum bactericidal titres. Titres were negligible with heterologous or PorA-negative meningococcal target strains, demonstrating the presence of the dominant bactericidal PorA antibodies. The two vaccines induced the same opsonic titres. Thus, the genetically modified vaccine with high Omp85 antibody levels and the wild-type vaccine induced the same levels of functional activities related to protection against meningococcal disease, suggesting that meningococcal Omp85 is a less attractive vaccine antigen.

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.

The aims of the study were to determine the molecular characteristics of a collection of Legionella pneumophila isolates from 45 cases with Legionnaires' disease and from 96 environmental samples, received by the national reference laboratory in Norway between 2001 and 2008, to use these characteristics to identify links between cases and suspected sources of infection, and to compare the isolate characteristics with those in other European countries.The isolates were characterized by 7-gene locus sequence-based typing and dot-blotting with monoclonal antibodies to various serogroups and subgroups.The clinical isolates represented 12.6% of the 357 cases notified in Norway between 2001 and 2008, during which 3 outbreaks of L. pneumophila serogroup 1 occurred. Outbreak cases constituted 62.2% of the cases, followed by travel-associated (24.4%) and sporadic cases (11.1%). Forty-two (93.3%) of the clinical and 69 (71.9%) of the environmental isolates were serogroup 1, and 39 (86.7%) and 50 (52.1%) isolates, respectively, carried the monoclonal antibody (Mab) 3/1 virulence-associated epitope. The clinical isolates belonged to 17 sequence types and the environmental isolates to 19 sequence types. neuA was not detected in 23 environmental isolates.Matching characteristics of sequence types and monoclonal subgroups for case and environmental isolates were obtained for all 3 outbreaks and for 2 of 5 cases of sporadic disease. Sampling during the outbreaks accounted for the higher proportion of serogroup 1 and Mab 3/1-positive environmental isolates in comparison with other European strain collections.

This study presents detailed analyses of total and specific serum antibody levels among 26 and 24 adult volunteers before vaccination and after the third dose of the meningococcal serogroup B outer membrane vesicle (OMV) vaccines MeNZB and MenBvac, respectively, in a clinical trial in New Zealand (V. Thornton, D. Lennon, K. Rasanathan, J. O’Hallahan, P. Oster, J. Stewart, S. Tilman, I. Aaberge, B. Feiring, H. Nokleby, E. Rosenqvist, K. White, S. Reid, K. Mulholland, M. J. Wakefield, and D. Martin, Vaccine 24:1395‐1400, 2006). With the homologous vaccine strains as targets, both vaccines induced significant increases in serum bactericidal and opsonophagocytic activities and in the levels of immunoglobulin G (IgG) to OMV antigens in an enzyme-linked immunosorbent assay (ELISA) and to live meningococci by flow cytometry. They also induced high levels of activity against the heterologous strains, particularly in terms of opsonophagocytic activity and IgG binding to live bacteria. The antibody levels with the homologous and heterologous strains in the four assays showed high and significant positive correlations. Specific IgG binding to 10 major OMV antigens in each vaccine was measured by scanning of immunoblots; ELISAs for two antigens, lipopolysaccharide and Neisseria surface protein A (NspA), were also performed. Both vaccines elicited significant increases in IgG binding to all homologous and heterologous OMV antigens except NspA. The total IgG band intensity on the blots correlated significantly with the IgG levels determined by the OMV ELISA and flow cytometry. In conclusion, the results of the various immunological assays showed that both OMV vaccines gave rise to high levels of specific and cross-reacting antibodies. Since 1991, an epidemic of meningococcal disease in New Zealand has caused over 200 deaths and nearly 6,000 cases of disease in a population of 4 million people (www.moh.govt.nz). Most of the cases are caused by serogroup B strains, and from 1991 through 2004, 86% of these expressed the P1.7-2,4 (P1.7b,4) PorA and belonged to the sequence type 41/44 complex (lineage III) (13, 15). The majority of these strains also expressed the serotype 4 PorB protein (14). In contrast to the other capsular meningococcal polysaccharides, group B polysaccharide is poorly immunogenic in humans (65); and vaccines based on subcapsular antigens, such as outer membrane proteins or outer membrane vesicles (OMVs) from various group B strains, have been developed and used in clinical trials (6, 9, 12, 18, 48). The experience of the Norwegian Institute of Public Health (NIPH) with the development and production of the OMV vaccine (MenBvac) for the protection trial in Norway (6, 19) led to a partnership with Chiron Vaccines (now Novartis Vaccines & Diagnostics) and the New Zealand Ministry of Health, in which NIPH developed and produced a tailor-made OMV vaccine (MeNZB) from a representative strain of the New Zealand epidemic (NZ98/254)

Background In May 2005, a long-distance outbreak of Legionnaires’ disease (LD) caused by Legionella pneumophila serogroup 1 occurred in south-east Norway. The initial outbreak investigation without serology identified 56 laboratory-confirmed LD cases of whom 10 died. However, 116 patients with community-acquired pneumonia might belong to the outbreak based on epidemiological investigations, but acute laboratory tests other than serology were negative or not performed. To assess the true extent of the outbreak, we evaluated two serological assays in order to reclassify the 116 patients with indeterminate case status. Methods Two polyvalent antibody tests, a serogroup 1–6 immunofluorescence assay (IFA) and a serogroup 1–7 enzyme-linked immunosorbent assay (ELISA) were used. They were evaluated with cases defined as culture- or urinary antigen positive LD patients (n = 40) and non-cases defined as confirmed non-LD patients (n = 39) and healthy control subjects (n = 62). The 116 patients, who were negative in culture, polymerase chain reaction and/or urinary antigen tests, were analysed by the same serological assays. Antibodies to the outbreak strain were determined by immunoblotting. Results In the evaluation study, the sensitivity and specificity of a ≥4-fold IFA titre change was 38% and 100%, respectively, with corresponding values of 30% and 99% for seroconversion in ELISA. A single high positive IFA titre yielded sensitivity and specificity of 73% and 97%, respectively, with corresponding values of 68% and 96% for a single high immunoglobulin (Ig) G and/or IgM in ELISA. Based on this evaluation, the following serological testing identified 47 more LD cases, and the outbreak thus comprised 103 cases with a case fatality rate of 10%. About the same proportion (70%) of the urinary antigen positive and negative LD cases had antibodies to the serogroup-specific lipopolysaccharide of the outbreak strain. In addition to the 103 LD cases, Legionella infection could not be verified or excluded in 32 patients based on epidemiology and/or lack of microbiological sampling. Conclusions The acute-phase tests (culture, polymerase chain reaction, and urinary antigen) identified less than 55% of the 103 patients in this outbreak. Serological testing thus remains an important supplement for diagnosis of LD and for determination of outbreak cases.

Neisseria meningitidis, an important cause of bacterial meningitis and septicemia worldwide, is associated with high mortality and serious sequelae. Natural immunity against meningococcal disease develops with age, but the specificity and functional activity of natural antibodies associated with protection are poorly understood. We addressed this question by using a selected subset of prevaccination sera (n= 26) with convergent or discrepant serum bactericidal activity (SBA) and infant rat protective activity (IRPA) against the serogroup B meningococcal strain 44/76-SL (B:15:P1.7,16) from Icelandic teenagers (B. A. Perkins et al., J. Infect. Dis. 177:683-691, 1998). The sera were analyzed by opsonophagocytic activity (OPA) assay, immunoblotting, immunoglobulin G (IgG) quantitation against live meningococcal cells by flow cytometry, and enzyme immunosorbent assay (EIA). High levels of SBA and OPA were reflected in distinct IgG binding to major outer membrane proteins and/or lipopolysaccharide in immunoblots. However, we could not detect any specific antibody patterns on blots that could explain IRPA. Only IgM antibody to group B capsular polysaccharide (B-PS), measured by EIA, correlated positively (r= 0.76,P< 0.001) with IRPA. Normal human sera (NHS;n= 20) from healthy Finnish children of different ages (7, 14, and 24 months and 10 years) supported this finding and showed an age-related increase in IRPA that coincided with the acquisition of B-PS specific IgM antibody. The protection was independent of complement-mediated bacterial lysis, as detected by the inability of NHS to augment SBA in the presence of human or infant rat complement and the equal protective activity of NHS in rat strains with fully functional or C6-deficient complement.

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.

IgG antibody levels to the meningococcal PorA and PorB proteins in 56 acute and convalescent phase sera from 25 patients with meningococcal disease were compared by immunoblotting and ELISA. Heat-treated outer membrane vesicles from strain 44/76 (B:15:P1.7, 16) served as antigens for immunoblotting, whereas purified P1.7,16 PorA and P15 PorB from the same strain were used as antigens in the ELISA. In the blotting method, IgG binding to the porins was determined by digital scanning of the immunoreactive bands and calculated relative to the PorA binding of a reference serum on each blot. The coefficient of variation for the reference serum was 21.6% (a total of 144 strips) with smaller variations for each day's experiments. Blotting of all 56 sera at the standard 1/200 dilution measured anti-PorA and anti-PorB levels that correlated with those obtained by ELISA (Spearman rank-order correlation coefficient r(s)=0.48; P

The ET-15 clone within the electrophoretic type (ET)-37 complex of Neisseria meningitidis was first detected in Canada in 1986 and has since been associated with outbreaks of meningococcal disease in many parts of the world. While the majority of the strains of the ET-37 complex are serosubtype P1.5,2, serosubtype determination of ET-15 strains may often be incomplete, with either only one or none of the two variable regions (VRs) of the serosubtype PorA outer membrane protein reacting with monoclonal antibodies. DNA sequence analysis of the porA gene from ET-15 strains with one or both unidentified serosubtype determinants was undertaken to identify the genetic basis of the lack of reaction with the monoclonal antibodies. Fourteen different porA alleles were identified among 38 ET-15 strains from various geographic origins. The sequences corresponding to subtypes P1.5a,10d, P1.5,2, P1.5,10d, P1.5a,10k, and P1.5a,10a were identified in 18, 11, 2, 2, and 1 isolate, respectively. Of the remaining four strains, which all were nonserosubtypeable, two had a stop codon within the VR1 and the VR2, respectively, while in the other two the porA gene was interrupted by the insertion element, IS 1301 . Of the strains with P1.5,2 sequence, one had a stop codon between the VR1 and VR2, one had a four-amino-acid deletion outside the VR2, and another showed no expression of PorA on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results reveal that numerous genetic events have occurred in the porA gene of the ET-15 clone in the short time of its epidemic spread. The magnitude of microevolutionary mechanisms available in meningococci and the remarkable genetic flexibility of these bacteria need to be considered in relation to PorA vaccine development.

Two reference monoclonal antibodies against the meningococcal P1.15 subtype PorA, MN3C5C and 2-1-P1.15, showed only partial concordant recognition of meningococcal isolates. Cyanogen bromide cleavage of P1.19,15 PorA, peptide mapping, and sequencing of porA regions demonstrated that 2-1-P1.15 was specific for subtype P1.19, and henceforth it is to be redesignated as 2-1-P1.19.

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 105 and 106 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 μg/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.

Outer membrane vesicles (OMVs) from group B meningococci induced both serum and mucosal antibodies when given as a nasal and rectal vaccine to mice. Cholera toxin (CT) enhanced the antibody responses in serum both after nasal and rectal immunizations, and the mucosal responses after rectal immunizations only. Nasal immunizations, however, were most effective, with mucosal responses which were not dependent on the use of CT. The serum bactericidal activity was similarly not enhanced by CT, indicating that the positive effect of CT on the serum IgG level was not including bactericidal activity. A small nasal booster dose induced antibody responses in serum as far as eight months after intranasal and subcutaneous immunizations, and in saliva after intranasal immunizations. Nasal vaccines may thus be favorably combined with parenteral vaccines.

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 β-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.

Sera from vaccinees and controls who contracted serogroup B meningococcal disease during the blinded and open parts of a two-dose protection trial in Norway were compared for antigen-specific and bactericidal antibodies against vaccine strain 44/76 (B:15:P1.7,16). From 16 of 20 (80%) vaccinees and 26 of 35 (74%) controls, one or more serum samples ( n = 104) were collected during the acute phase (1 to 4 days), early convalescent phase (5 to 79 days), and late convalescent phase (8 to 31 months) after onset of disease. Binding of immunoglobulin G (IgG) to the major outer membrane antigens (80- and 70-kDa proteins, class 1, 3, and 5 proteins, and lipopolysaccharide [LPS]) on immunoblots was measured by digital image analysis. Specific IgG levels in vaccinees increased from acute to early convalescent phases, followed by a decline, while controls showed a small increase over time. Vaccinees had significantly higher levels than controls against class 1 and 3 porins and LPS in acute sera, against all antigens during early convalescence, and against class 1 and 3 porins in the later sera. Vaccinees who were infected with strains expressing subtype P1.7,16 proteins demonstrated a level of IgG binding to protein P1.7,16 with early-convalescent-phase sera that was fourfold higher than that of those infected with other strains. Bactericidal titers in serum against the vaccine strain were 192-fold higher for vaccinees than those for controls during early convalescence, but similarly low levels were found during late convalescence. A vaccine-induced anamnestic response of specific and functional antibody activities was thus shown, but the decrease in protection over time after vaccination indicated that two vaccine doses did not induce sufficient levels of long-term protective antibodies.

Immunoassays employ a range of methods to detect and quantify antigens or antibodies and to study the composition of antigens. This chapter describes four useful immunoassays for serological characterization of antigens of Neisseria meningitidis: whole-cell enzyme-linked immunosorbent assay (ELISA); dot blot, colony blot, and immunoblot. Serological characterization of N. meningitidis antigens is valuable for epidemiological studies as well as for identifying immunologically important antigens in vaccine development (1,2). Typing of N. meningitidis is based on the immunological detection of specific epitopes on the outer-membrane proteins (OMP) and lipopolysaccharides (LPS) (3), and panels of monoclonal antibodies (MAbs) have been developed by several laboratories to refine the serological classification system (4-8). Differences in capsular polysaccharides determine the meningococcal serogroup, whereas the serotypes and serosubtypes are based on antigenic differences of the PorB OMP and PorA OMP, respectively (3). The PorA protein contains two variable loops (VR1 and VR2), each of which determine a dis- tinct set of serosubtypes. Thus the serosubtypes of an isolate can include two independent designations (9-11). Variation in the oligosaccharide moiety of the LPS determines the immunotype, and more than one epitope can be present in the same population of a single isolate (6,12). In the current typing scheme the classification is given as [serogroup]: [serotype]: [serosubtype]: [immunotype], e.g., B: 15:P1.7,16:L3,7,9.

This chapter will describe the use of the immunoblotting method for analysing antibody specificities towards meningococcal antigens in sera from vaccinees, receiving group B meningococcal outer-membrane vesicle (OMV) vaccines, or from patients, falling ill with meningococcal disease. The power of the blotting technique lies in the simultaneous identification of many immunogenic components in a sample. Within the meningococcal field, this method has been employed in analyzes of antibody specificities of sera from vaccinees (1-6), patients (7-10), and carriers (11,12) as well as for characterization of OMV vaccines (13,14). Some of these studies present various ways of quantifying the blotting results (5,7,9,11).

Following a long-distance outbreak of Legionnaires' disease from an industrial air scrubber in Norway in 2005, a seroepidemiological study measuring levels of immunoglobulin G (IgG) and IgM antibodies to Legionella pneumophila was performed with a polyvalent enzyme-linked immunosorbent assay. One year after the outbreak, IgG levels in employees ( n = 213) at the industrial plant harboring the scrubber and in blood donors ( n = 398) from the outbreak county were low but significantly higher ( P ≤ 0.002) than those in blood donors ( n = 406) from a nonexposed county. No differences in IgM levels among the three groups were found after adjustment for gender and age. Home addresses of the seroresponders in the exposed county clustered to the city of the outbreak, in contrast to the scattering of addresses of the seroresponding donors in the nonexposed county. Factory employees who operated at an open biological treatment plant had significantly higher IgG and IgM levels ( P ≤ 0.034) than those working >200 m away. Most of the healthy seroresponders among the factory employees worked near this exposure source. Immunoblotting showed that IgG and IgM antibodies in 82.1% of all seroresponders were directed to the lipopolysaccharide of the L. pneumophila serogroup 1 outbreak strain. In conclusion, 1 year after the long-distance industrial outbreak a small increase in IgG levels of the exposed population was observed. The open biological treatment plant within the industrial premises, however, constituted a short-distance exposure source of L. pneumophila for factory employees working nearby.

Typing of meningococci with a panel of serotype and subtype specific monoclonal antibodies (MAbs) was compared in co-agglutination, dot-blotting and ELISA tests. Twenty reference strains, 50 case isolates and 133 throat isolates from healthy carriers were studied. The typing results with dot-blotting and ELISA were identical, whereas co-agglutination gave different results for three case and 24 carrier strains. The distribution of serotypes and subtypes among the strains is reported. The combination of the subtypes P1.1 and P1.15 in a serotype 15 patient strain was observed. With one case strain and 15 carrier strains, neither serotype nor subtype could be determined. Non-typable and non-subtypable isolates were further characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Co-agglutination is useful for typing small numbers of strains with a few MAbs, but less suitable for large-scale typing than the other two methods. Dot-blotting needs less expensive equipment, smaller volumes of antibodies and fewer manipulations than ELISA.

Two new methods for serogrouping of meningococci, whole-cell ELISA and dot-blotting, with monoclonal antibodies against serogroups A, B, C, Y and W135 were compared with slide-agglutination applying polyclonal sera. In addition to a panel of strains with previously determined serogroups by slide-agglutination, two strain collections of meningococci were studied: 1) 50 strains isolated from patients with systemic meningococcal disease in Norway during the winter 1987-1988; 2) 133 throat strains isolated from asymptomatic carriers over the same period. For the disease strains all three methods gave identical results, whereas some carrier strains which were non-agglutinable or polyagglutinable by slide-agglutination were serogroupable by the two other methods. All the systemic strains and about half of the carrier strains were serogroupable. We find that whole-cell ELISA and dot-blotting are specific, easy to read and more sensitive compared to slide-agglutination, but the former methods are at present limited by the availability of monoclonal antibodies against only serogroups A, B, C, Y and W135.

The aim of this study was to measure the seroprevalence to mumps in Norwegian conscripts belonging to the first children vaccination cohorts that had been offered two doses of MMR vaccine. The seroprevalence to mumps was 76% with the Microimmune assay and 85% with the Enzygnost assay. We also compared the performance of the Microimmune assay for detection of mumps- and measles-specific IgG antibodies in 340 paired serum and oral fluid samples from the conscripts and evaluated the effect of revaccination. Mumps-specific IgG antibodies were detected in only 61% of the oral fluids. In contrast, high levels of measles-specific IgG antibodies were detected in both the serum and oral fluid samples. Based on these results, we are only able to recommend the use of oral fluid for surveillance of measles in Norway. Our results may also indicate that the seroprevalence necessary to interrupt transmission of mumps has not been reached in vaccinated young adult Norwegians. Seroconversion was observed in all initially measles seronegative conscripts after revaccination, whereas 23 of 27 initially mumps seronegative conscripts failed to seroconvert.

Fulminant meningococcal sepsis has been termed the prototypical lipopolysaccharide (LPS)-mediated gram-negative septic shock. Systemic inflammation by activated complement and cytokines is important in the pathogenesis of this disease. We investigated the involvement of meningococcal LPS in complement activation, complement-dependent inflammatory effects, and cytokine or chemokine production. Whole blood anticoagulated with lepirudin was stimulated with wild-typeNeisseria meningitidisH44/76 (LPS+), LPS-deficientN. meningitidisH44/76lpxA(LPS−), or purified meningococcal LPS (NmLPS) at concentrations that were relevant to meningococcal sepsis. Complement activation products, chemokines, and cytokines were measured by enzyme-linked immunosorbent assays, and granulocyte CR3 (CD11b/CD18) upregulation and oxidative burst were measured by flow cytometry. The LPS+and LPS−N. meningitidisstrains both activated complement effectively and to comparable extents. Purified NmLPS, used at a concentration matched to the amount present in whole bacteria, did not induce any complement activation. Both CR3 upregulation and oxidative burst were also induced, independent of LPS. Interleukin-1β (IL-1β), tumor necrosis factor alpha, and macrophage inflammatory protein 1α production was predominantly dependent on LPS, in contrast to IL-8 production, which was also markedly induced by the LPS−meningococci. In this whole blood model of meningococcal sepsis, complement activation and the immediate complement-dependent inflammatory effects of CR3 upregulation and oxidative burst occurred independent of LPS.

Antibody specificities of pre- and postvaccination serum samples from 40 (53%) teenagers who received three doses of the Norwegian Neisseria meningitidis serogroup B vaccine (B:15:P1.7,16) during a previous trial in Iceland (Perkins et al., J. Infect. Dis. 177: 683-691, 1998) were analyzed with serum bactericidal activity (SBA) and immunoblotting assays with reference and isogenic meningococcal H44/76 vaccine strains. The H44/76 variants demonstrated significant vaccine-induced SBA to P1.7,16 PorA and Opc but not to PorB, Opa5.5, and a heterologous PorA protein. On blots, immunoglobulin G levels to all these proteins increased significantly after vaccination. Measurement of SBA to the two main variable regions (P1.7 and P1.16) on the P1.7,16 PorA with PorA deletion mutants revealed significantly higher activity to the P1.7,− and P1.−,16 mutants compared to the P1.7,16 strain, indicating exposure of new accessible epitopes. Only 12 (30%) serum samples showed distinct decreases with these or the P1.−,− mutant, with most samples containing SBA to the P1.7 and P1.16 combination. In contrast, P1.16-specific antibodies were mainly found on blots. Thirteen of the vaccinees (32.5%) were carriers of meningococci at the time of the third dose, of whom four (30.8%) harbored strains of the ET-5 complex. Carriage of P1.15 strains was generally reflected in ≥4-fold increases in SBA and distinct immunoglobulin G binding to the P1.19,15 PorA on blots. Although vaccination did not elicit bactericidal activity to the serotype 15 PorB, most carriers of serotype 15 strains showed ≥4-fold increases in SBA to this antigen.

Meningitis caused by Neisseria meningitidis serogroup W-135 was diagnosed in a 14-year-old girl with a history of neonatal septicemia and meningitis caused by group B streptococci type III. C2 deficiency type I was found in the patient and her healthy sister. Both sisters were vaccinated with tetravalent meningococcal vaccine and a conjugate Haemophilus influenzae type b vaccine. Three main points emerged from the analysis. First, vaccination resulted in serum bactericidal responses demonstrating anticapsular antibody-mediated recruitment of the alternative pathway. Second, addition of C2 to prevaccination sera produced bactericidal activity in the absence of anticapsular antibodies, which suggested that the bactericidal action of antibodies to subcapsular antigens detected in the sera might strictly depend on the classical pathway. A third point concerned a previously unrecognized type of blocking activity. Thus, postvaccination sera of the healthy sister contained IgG that inhibited killing of serogroup W-135 in C2-deficient serum, and the deposition of C3 on enzyme-linked immunosorbent assay plates coated with purified W-135 polysaccharide. Our findings suggested blocking to be serogroup-specific and dependent on early classical pathway components. Retained opsonic activity probably supported post-vaccination immunity despite blocking of the bactericidal activity. The demonstration of functional vaccination responses with recruitment of alternative pathway-mediated defense should encourage further trial of capsular vaccines in classical pathway deficiency states.

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

A whole-cell pertussis vaccine, each dose consisting of 250 microg of protein, was given intranasally four times at weekly intervals to six adult volunteers. All vaccinees responded with increases in nasal fluid IgA antibodies to Bordetella pertussis whole-cell antigen. Three vaccinees with high nasal antibody responses also developed increased serum IgA and IgG antibodies to this antigen. Salivary antibody responses to the whole-cell antigen, as well as antibodies in serum and secretions to pertussis toxin (PT) and filamentous haemagglutinin (FHA) were negligible, except for a moderate increase in nasal fluid antibodies to FHA. Unexpectedly, the same vaccinees developed significant rises in nasal and salivary IgA antibodies to meningococcal outer-membrane antigens, whereas corresponding serum IgA and IgG antibodies were unchanged. Thus it appears that mucosal immunisation may induce secretory antibodies with broader specificities than can be found in serum.

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.

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.

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 μg 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.

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.

Summary 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 Bacillussubtilis, 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.

Summary: The class 3 outer-membrane protein (OMP) of Neisseria meningitidis is a potential target for bactericidal and opsonic antibodies in humans. Synthetic peptides spanning the class 3 OMP from the vaccine strain 44/76 (B: 15:P1.7,16:L3,7) were synthesized on pins and screened with serum obtained from Norwegian adolescents immunized with a meningococcal serogroup B outer-membrane vesicle (OMV) vaccine. A strong IgG response to a single peptide (19FHQNGQVTEVTT30) located within loop 1 (VR1) was stimulated after three doses of OMV vaccine in three vaccinees selected on the basis of their antibody response to class 3 OMP. No clear linear B-cell epitopes were recognized by four different murine serotype 15-specific mAbs. A 23mer peptide (D63b2) containing loop 1 of the class 3 OMP was synthesized, and the IgG responses were measured in pre- and post-vaccination serum from 27 vaccinees. Specific IgG rose significantly in 37% of vaccinees 6 weeks after the second dose and in 74% of the vaccinees 6 weeks after the third dose of the OMV vaccine. Most immune sera reacted distinctly on immunoblots with denatured class 3 OMP, and the immunoblotting reactivity correlated strongly with concentration of the IgG antibodies specific for peptide D63b2. When added to a post-vaccination serum from one vaccinee, peptide D63b2 competed efficiently with the class 3 OMP for specific antibody binding on immunoblots and in pin ELISA. The results show that the significant part of the humoral response to the meningococcal class 3 OMP elicited by vaccination with the Norwegian OMV vaccine was directed against a single continuous epitope.

An increase in B:15:P1.12 meningococci among isolates from patients with Neisseria meningitidis infection in Norway in recent years led to further characterization of such strains. Between 1987 and 1992, B:15:P1.12 strains constituted 9.8% (24 strains) of B:15 isolates. The B:15:P1.12 strains belonged to the electrophoretic type 5 (ET-5) complex, but 17 (71%) strains were a new clone (ET-5c) not found elsewhere in the world. All but one strain of ET-5c were responsible for a localized outbreak of systemic meningococcal disease in western Norway. A novel monoclonal antibody (202,G-12), developed against the unknown variable region 2 on the class 1 protein of one of these strains, bound to 19 of the 15:P1.12 strains, 4 strains bound the subtype P1.13 reference monoclonal antibody MN24H10.75, and the remaining strain showed no reaction. Sequencing of porA genes demonstrated a series of nine threonine residues in the deduced variable region 2 of the latter strain, while four and five threonine residues were found in the corresponding regions of strains reacting with the monoclonal antibodies 202,G-12 and MN24H10.75, respectively. Epitope mapping with synthetic peptides showed that 202,G-12 bound to a sequence of 11 amino acids which included the four threonine residues specific for subtype P1.13a. Immunoglobulin G antibodies against the P1.7,16 subtype protein, induced in volunteers after vaccination with the Norwegian meningococcal vaccine, did not cross-react on immunoblots with the subtype protein of clone ET-5c. Thus, postvaccination class 1 protein antibodies, assumed to be protective, may not be effective against infection with the new clone.

To estimate the extent of meningococcal carriage in the Norwegian population and to investigate the relationship of several characteristics of the population to the carrier state, 1,500 individuals living in rural and small-town areas near Oslo were selected at random from the Norwegian National Population Registry. These persons were asked to complete a questionnaire and to volunteer for a bacteriological tonsillopharyngeal swab sampling. Sixty-three percent of the selected persons participated in the survey. Ninety-one (9.6%) of the volunteers harbored Neisseria meningitidis. The isolates were serogrouped, serotyped, tested for antibiotic resistance, and analyzed by multilocus enzyme electrophoresis. Eight (8.8%) of the 91 isolates represented clones of the two clone complexes that have been responsible for most of the systemic meningococal disease in Norway in the 1980s. Age between 15 and 24, male sex, and active and passive smoking were found to be independently associated with meningococcal carriage in logistic regression analyses. Working outside the home and having an occupation in transportation or industry also increased the risk for meningococcal carriage in individuals older than 17, when corrections for gender and smoking were made. Assuming that our sample is representative of the Norwegian population, we estimated that about 40,000 individuals in Norway are asymptomatic carriers of isolates with epidemic potential. Thus, carriage eradication among close contacts of persons with systemic disease is unlikely to have a significant impact on the overall epidemiological situation.

Based on differences in reaction pattern with monoclonal antibodies against the P1.16 epitope, a new variant of the class 1 protein in Neisseria meningitidis serogroup B was identified in Norway. A single amino acid deletion was revealed when the part of the gene region encoding the second variable region of the protein was sequenced. This new variant was designated P1.16c. About 5% of the B:15:P1.7,16 strains in Norway from the time period 1987-1991 were P1.16c. In a localized area in Southern Norway, 5/8 (62%) of the P1.7,16 strains were P1.16c. The P1.16b mutant, recently described in England, was not found among the Norwegian meningococcal isolates. Strains carrying the P1.16c mutation showed increased resistance to bactericidal killing, not only by P1.16-specific monoclonal antibodies, but also by the sera from individuals immunized with a vaccine based on outer membranes from a B:15:P1.7,16 strain.

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.

Summary Dot-blot analysis of whole-cell suspensions of meningococci showed that 81% of B:15:P1.16 strains from patients reacted with a monoclonal antibody (MAb) against subtype P1.7. The remaining strains, which did not react on dot-blots or in ELISA, demonstrated the P1.7 subtype epitope on immunoblots after denaturation of the cells with sodium dodecyl sulphate. The monomeric class 1 proteins of the two P1.16 subtype variants had slightly different mol. wts, but bound the P1.7 antibody equally well. These results were explained by a deletion of three codons in the gene encoding the first variable region of the P1.16 class 1 protein. The deletion accounted for the non-exposure of the P1.7 epitope on native cells. Other patient strains, with subtypes P1.3, P1.9 or without any known subtype, also showed a binding site for the P1.7 MAb, which became available only after denaturation. Demonstration of inaccessible epitopes may have consequences for subtype designations and vaccine development.

A survey is given of the efforts made to inform the general public, the potential vaccinees and their parents, and the health care personnel about meningococcal disease in general and the vaccination trial in particular, as a preparation for the meningococcal outer membrane vesicle serogroup B vaccine (MenB-vaccine "Folkehelsa") trials in secondary school students and military conscripts in Norway. Our case reporting system, supplementing the official notification, concerning even vaguely suspected cases in the age cohorts involved, is described. The efforts made to collect clinical material as well as laboratory and clinical data from 221 registered suspected cases are delineated. We also briefly summarize our cerebrospinal fluid antigen detection methods and diagnostic meningococcal serology work on these suspected cases. The compiled information on findings done at the admitting hospital of the possible cases and the additional diagnostic data provided at the National Institute of Public Health were put at the disposal of the independent Diagnosis Review Committee (DRC) as a basis for their diagnostic decisions before code opening for the meningococcal serogroup B outer membrane vesicle vaccine protection trial 3 June 1991.

The reactions of serogroup A strains of Neisseria meningitidis with one monoclonal antibody specific for serotype 21 and three different monoclonal antibodies specific for serotype 4 were compared with those of serogroup B strains previously assigned to serotype 4. Antibody binding was studied by enzyme-linked immunosorbent assay (ELISA), dot blotting, and immunoblotting. Characterization of the isolates by the electrophoretic mobilities of 14 metabolic enzymes showed 50 multilocus enzyme genotypes. All except two genotypes fell into three distinct clusters: I, IIa and IIb. The enzyme genotypes of serogroup B strains were mainly in cluster I, and 88% of the serogroup A strains had genotypes in clusters IIa and IIb. Serogroup B strains generally reacted with all three serotype 4 monoclonal antibodies in ELISA and dot blotting but with only two in immunoblots. Serogroup A strains showed two different reactions in the blotting methods: either binding of the serotype 21 antibody only or binding of this and two of the three serotype 4 monoclonal antibodies. Strains of the first pattern were in clusters I and IIa, whereas all but two strains in cluster IIb were of the second pattern. In ELISA, an additional reaction of two of the serotype 4 monoclonal antibodies with serogroup A isolates was observed. The different binding of these two monoclonal antibodies in ELISA and the blotting methods appeared to result from heat inactivation of the meningococcal cells and use of detergent-containing reagents in ELISA. The results show that the serotype of serogroup A strains is distinct from serotype 4 of serogroup B strains.

IgG and IgG subclass antibodies to the outer membrane antigens from Neisseria meningitidis (serogroup B., serotype 15:P1.16) were quantitated by an enzyme-linked immunosorbent assay (ELISA) in sera from 40 patients with group B:15:P1.16 meningococcal disease and 24 volunteers immunized with a serotype 15:P1.16 outer membrane vesicle vaccine. A second injection was given 6 weeks after the first immunization. Patient sera obtained two and six weeks after onset of the disease had significantly higher levels of total IgG, IgG1, IgG2, and IgG3 antibodies to the outer membrane antigens than acute sera, convalescent sera from patients with systemic non-meningococcal bacterial infections and sera from healthy controls. The levels of total IgG and IgG 1 remained high one and three years later. Sera from the vaccinees showed high levels of total IgG and IgG 1 6, 12 and 26 weeks after the first immunization and high levels of IgG3 6 weeks after the second immunization. No increase of IgG2 or IgG4 levels was observed in the postimmunization sera. Immunoblotting of three convalescent sera demonstrated individual patterns of IgG subclass binding to various outer membrane antigens with most distinct binding of IgG 1 and IgG3 antibodies to the class 1 protein, the H.8 lipoprotein and the lipopolysaccharide. Since IgGl and IgG3 are the most effective antibodies for complement activation and phagocytosis, group B meningococcal disease and immunization with the serotype 15:P1.16 outer membrane vesicle vaccine stimulate production of those IgG subclasses which have the strongest opsonic and bactericidal activity.

Two new methods for serogrouping of meningococci, whole-cell ELISA and dot-blotting, with monoclonal antibodies against serogroups A, B, C, Y and W135 were compared with slide-agglutination applying polyclonal sera. In addition to a panel of strains with previously determined serogroups by slide-agglutination, two strain collections of meningococci were studied: 1) 50 strains isolated from patients with systemic meningococcal disease in Norway during the winter 1987-1988; 2) 133 throat strains isolated from asymptomatic carriers over the same period. For the disease strains all three methods gave identical results, whereas some carrier strains which were non-agglutinable or polyagglutinable by slide-agglutination were serogroupable by the two other methods. All the systemic strains and about half of the carrier strains were serogroupable. We find that whole-cell ELISA and dot-blotting are specific, easy to read and more sensitive compared to slide-agglutination, but the former methods are at present limited by the availability of monoclonal antibodies against only serogroups A, B, C, Y and W135.

1. 1. The concentrations of perchloric acid (PCA) soluble proteins, expressed per weight of the acid-insoluble proteins and of DNA, varied 10 and 30 fold, respectively, in the 11 rat tissues studied. 2. 2. The gel electrophoretic patterns of the PCA-soluble tissue extracts could be divided into two groups, the first one resembling that of the chromosomal high-mobility group and H1 proteins, the second one being more dominated by lower mol. wt proteins. 3. 3. The results indicated that tissues with PCA-soluble proteins belonging to the first group also had lower average cellular concentrations of the PCA-soluble and insoluble proteins.

Three murine IgG1 monoclonal antibodies (MoAb) were produced against the glucose/mannose-specific two-chain mitogen from seeds of Lathyrus odoratus (sweet pea) belonging to the Vicieae tribe of the Leguminosae family. Their antigenic specificities were tested against subunits of two-chain and one-chain legume lectins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose filters. Different binding to native and detergent-treated lectins in dot-blots indicated that two of the antibodies bound to conformational epitopes which by immunoblotting were shown to be expressed on the heavy subunits from all the five tested two-chain lectins and on the six one-chain lectins, including PHA (Phaseolus vulgaris) and Con A (Canavalia ensiformis). However, ELISA cross-inhibition studies showed that these two antibodies bound to different epitopes on the lectin molecules. The third MoAb reacted with a continuous epitope not revealed by classical taxonomy since only three of five heavy subunits of the two-chain lectins stained with the antibody. Comparison of the known amino-acid sequences of the chains indicates eight positions as possible binding sites for this antibody.

The antibody response of 30 volunteers vaccinated with a complex of group B polysaccharide and outer membrane vesicles (OMV) from serotype 2a Neisseria meningitidis and of 3 individuals who received a placebo vaccine was determined by immunoblotting. OMV were separated by sodium dodecyl sulfate-gel electrophoresis and electrotransferred to nitrocellulose filters. Binding of immunoglobulin G (IgG), IgA, and IgM antibodies in the human sera to OMV components was detected with class-specific peroxidase-conjugated antibodies. The immunoblotting results were also related to the bactericidal activity of the sera and the meningococcal carrier status of the volunteers. Before vaccination weakly reactive bands in the molecular weight range of 140,000 to 10,000 were observed on the blots. Sera from carriers showed more marked bands. Individual patterns of increased reactivity were seen 6 weeks after vaccination. The main immunoreactive components of OMV corresponded to a molecular weight of 43,000 (class 1 protein), 30,000 (class 5 proteins), and 22,000. IgG antibodies in postvaccination sera of high bactericidal titers showed distinct binding to the 43,000-molecular-weight antigen. Meningococcal carriers had antibodies against an antigen of 22,000 molecular weight; in polyacrylamide gels this component did not stain with Coomassie brilliant blue or silver. The marked binding of IgG antibodies to the class 5 proteins decreased considerably between weeks 6 and 25 after vaccination. Periodate oxidation of OMV abolished the binding of IgG antibodies to the class 5 proteins, whereas the antigenicity of the 43,000-molecular-weight (class 1 protein) and 22,000-molecular-weight antigens was unaffected.

Restoration of IgG antibody binding to heat-denatured meningococcal outer membrane proteins has been studied on immunoblots with a series of 14 detergents. Nitrocellulose strips with the blotted proteins were incubated with the detergents and sera from human volunteers vaccinated with meningococcal membrane proteins. Zwitterionic and ionic detergents, containing substituted quarternary ammonium or amino groups with a minimum of 10 C atoms in the alkyl chain, restored the antigenicity of the serotype-specific class 2 porin protein. The concentrations of the Zwittergent detergents necessary for activation decreased with increasing alkyl chain length of the homologues. Only zwitterionic detergents renatured the class 1 protein. Both proteins were weakly antigenic in the presence of the nonionic detergents Triton X-100 and Tween 20. Meningococcal lipopolysaccharide restored antibody binding to the porin, but not to the class 1 protein. Similar concentrations of lipopolysaccharides from two other gram-negative bacteria had no effect.

The immunoglobulin G (IgG) subclass distribution of antibodies against the major outer membrane proteins from serotype 2a Neisseria meningitidis in human vaccinees was studied by immunoblotting. The volunteers received two doses of a noncovalent complex of group B polysaccharide and outer membrane material from the same meningococcal strain. Six weeks after the first vaccination the antibodies mounted against the class 1 and 5 proteins belonged mainly to the IgG1 and IgG3 subclasses. However, the binding of IgG3 to the class 5 proteins decreased markedly in serum samples taken after 25 weeks. Antibody binding to the serotype-specific class 2 protein was dependent on renaturation of the antigen by a dipolar ionic detergent (R. E. Mandrell and W. D. Zollinger, J. Immunol. Methods 67:1-11, 1984). The immune response against this protein showed more individual variation and consisted of IgG1 or IgG3 or both, often combined with IgG4.

The gluten lectin was isolated by affinity chromatography, separated by sodium dodecyl sulphate-gel electrophoresis together with purified wheat germ agglutinin (WGA) and electrotransferred to nitrocellulose filters. The binding pattern of anti-WGA to the blotted filters confirmed the presence of WGA in gluten. A lectin from rice bran and white rice flour, respectively, was isolated by affinity chromatography. Both lectins reacted with anti-WA in immunoblotting. As patients with coeliac disease are known to tolerate rice flour, the finding of a WGA-like lectin questioned the suggestion that WGA in gluten is involved in the pathogenesis of coeliac disease. A second lectin was also isolated from rice flour which reacted only with antibodies against soybean lectin on immunoblots. This may indicate a contamination of soybean proteins in rice flour.