Immune responses of a meningococcal A + W outer membrane vesicle (OMV) vaccine with and without aluminium hydroxide adjuvant in two different mouse strains.

Meningococci (Neisseria meningiditis) of serogroups A and W have caused large epidemics of meningitis in sub-Saharan Africa for decades, and affordable and multivalent vaccines, effective in all age groups, are needed. A bivalent serogroup A and W (A + W) meningococcal vaccine candidate consisting of deoxycholate-extracted outer membrane vesicles (OMV) from representative African disease isolates was previously found to be highly immunogenic in outbred mice when formulated with the adjuvant aluminium hydroxide (AH). OMV has been shown to have inherent adjuvant properties. In order to study the importance of AH and genetical differences between mice strains on immune responses, we compared the immunogenicity of the A + W OMV vaccine when formulated with or without AH in inbred C57BL/6J and BALB/cJ mice (Th1 and Th2 dominant strains, respectively). The immunogenicity of the vaccine was found to be comparable in the two mice strains despite their different immune profiles. Adsorption to AH increased anti-OMV IgG levels and serum bactericidal activity (SBA). The immune responses were increased by each dose for the adsorbed vaccine, but the third dose did not significantly improve the immunogenicity further. Thus, a vaccine formulation with the A and W OMV will likely benefit from including AH as adjuvant.

Characterization of the extent of a large outbreak of Legionnaires' disease by serological assays.

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.

Molecular characterization of clinical and environmental isolates of Legionella pneumophila in Norway, 2001-2008.

BACKGROUND: 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. METHODS: The isolates were characterized by 7-gene locus sequence-based typing and dot-blotting with monoclonal antibodies to various serogroups and subgroups. RESULTS: 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. CONCLUSIONS: 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.

Characterization of meningococcal serogroup B outer membrane vesicle vaccines from strain 44/76 after growth in different media.

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.

Protection by natural human immunoglobulin M antibody to meningococcal serogroup B capsular polysaccharide in the infant rat protection assay is independent of complement-mediated bacterial lysis.

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. 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.

Passive protection in the infant rat protection assay by sera taken before and after vaccination of teenagers with serogroup B meningococcal outer membrane vesicle vaccines.

From a previous published clinical trial among teenagers in Iceland [Perkins BA, Jonsdottir K, Briem H, Griffiths E, Plikaytis BD, Høiby EA, et al. J Infect Dis 1998;177:683--91], we evaluated a 25% stratified subset of sera, collected before vaccination and 6 weeks after the second vaccination with either the Norwegian (n=37) or the Cuban (n=35) serogroup B meningococcal outer membrane vesicle (OMV) vaccine or the control serogroup A/C capsular polysaccharide vaccine (n=20), for protective activity in an infant rat protection assay (IRPA). Protection was assessed with both the Norwegian (44/76-SL, B:15:P1.7,16:L3,7) and the Cuban (Cu 385, B:4:P1.19,15:L3,7) vaccine strain, and the results compared with serum bactericidal assay (SBA) titres and anti-OMV IgG antibody concentrations. An IRPA response was defined as a >or=10-fold rise in protective activity compared to pre-vaccination level. Forty-six percent (42/92) of the pre-vaccination sera showed protection with strain 44/76-SL compared to only 12% (11/92) with strain Cu 385. After the second dose, 22% (8/37) of those given the Norwegian vaccine showed IRPA responses with the homologous strain compared to 65% (24/37) in SBA. The corresponding numbers with the homologous strain for the Cuban vaccinees were 14% (5/35) and 29% (10/35), respectively. Among the controls, 15% (3/20) showed IRPA responses to 44/76-SL but none to Cu 385. Correlation between IRPA activity and SBA titres or anti-OMV IgG was low, especially for pre-vaccination sera against strain 44/76-SL. We conclude that the sensitivity of IRPA described herein may not be sufficient to evaluate serogroup B OMV vaccine responses from clinical samples.

Antibody specificities and effect of meningococcal carriage in icelandic teenagers receiving the Norwegian serogroup B outer membrane vesicle vaccine.

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 > or =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 > or =4-fold increases in SBA to this antigen.