N-propionylated group B meningococcal polysaccharide mimics a unique epitope on group B Neisseria meningitidis.

Antibodies induced in mice by the N-propionyl (N-Pr)-group B meningococcal polysaccharide (GBMP)-tetanus toxoid (TT) conjugate were bactericidal for GBM organisms independent of protein serotype. The antisera contained two populations of N-Pr-GBMP-specific antibodies, only one of which crossreacted with the GBMP. Particularly significant was the fact that the bactericidal activity was mainly associated with the population of antibodies that did not crossreact with the GBMP. Therefore it can be inferred from the above evidence that the N-Pr-GBMP mimics a unique epitope on the surface of GBM organisms that is not present on the exogenous GBMP.

Age-related immunogenicity of meningococcal polysaccharide vaccine in aboriginal children and adolescents living in a Northern Manitoba reserve community.

OBJECTIVE: To determine the total and functional serogroup C antibody response to a quadrivalent meningococcal polysaccharide vaccine in a group of aboriginal infants, children and adolescents. A secondary objective was to determine their prevalence of meningococcal carriage. DESIGN: Open prospective, before and after intervention study. SUBJECTS: Aboriginal children ages 0.5 to 19.9 years, living in a single Northern community and eligible for a public health immunization campaign conducted in all Manitoba native reserve communities to control a meningococcal serogroup C, electrophoretic type (ET) 15 outbreak. No outbreak cases had occurred in the community at the time of the study. METHODS: Total serogroup C capsular polysaccharide antibody (CPA) and functional bactericidal antibody (BA) responses were measured by enzyme-linked immunosorbent assay and bactericidal assay, respectively. RESULTS: Neisseria meningitidis was recovered from the oropharynx of 13 (5.2%) of 249 aboriginal children including 4 (1.6%) serogroup C isolates, all with the designation C:2a:P1.2,5 ET15. Paired sera from 152 children were available for assay. For CPA the geometric mean concentrations and proportions with > or =2 microg/ml before and after immunization were 0.69, 18% and 12.3, 96%, respectively. A significant increase in serum CPA was achieved by children of all ages, with the greatest response occurring after age 11 years. Among infants < lyear old 89% achieved concentrations of > or =2 microg/ml. For BA the pre- and post-vaccine geometric mean titers were 1.02 and 45.9. The response was significantly associated with age. BA titers > or =1:8 were present, before and after immunization, respectively, in 0 and 0% of infants <1 year old, 0 and 20% of 1- to 1.4-year-olds, 0 and 50% of 1.5- to 1.9-year-olds and 1 and 100% of > or =2-year-olds. CONCLUSION: The age-related total and functional group C meningococcal antibody response after quadrivalent polysaccharide vaccine among aboriginals is similar to that reported for Caucasian children. After age 2 all children made excellent CPA and BA responses. In the younger age groups the BA response was blunted but 82 to 95% achieved CPA titers of > or =2 microg/ml.

Enzyme-linked immunosorbent assay with polyvalent gonococcal antigen.

An indirect enzyme-linked immunosorbent assay (ELISA) with a mixture of eight different gonococcal outer-membrane proteins (OMP) as coating antigen was evaluated for detection of gonococcal antibody in 507 sera obtained from patients selected from high-risk and low-risk populations. The indirect ELISA method was more specific and sensitive when the polyvalent antigen was used than when OMP from only one serotype was used. Past episodes of the gonorrhoea had a significant influence on the seropositivity of the test. In a selected low-risk population the specificity of the assay was 94% and in a selected high-risk population the sensitivity was 78%. When sera from 24 asymptomatic individuals were tested the sensitivity was 83%. The ELISA polyvalent-antigen test should be useful as an aid for the detection of gonorrhoea in populations with a low prevalence.

Immunogenic and protective properties of meningococcal serotype 2a protein in the hen-embryo model.

The ability of serotype 2 outer membrane protein (SP-2) of Neisseria meningitidis strain M986 serogroup B, serotype 2a (B, 2a) to stimulate antibody formation in hens and to confer protection against meningococcal challenge to embryos from immunised hens was investigated. Hens, housed with roosters, were immunised with 100 micrograms of SP-2 once a week for 5 weeks to maintain consistent levels of serum antibody during the study. Antibodies in sera of hens, yolks and plasma of 13-day-old embryos reacted in enzyme-linked immunosorbent assays and immunodiffusion with outer membrane vesicles (OMV) from serotypes 2a, 2b and 2c and most of the other prototype group B strains of N. meningitidis. Cross-reactivity of hen sera with most OMV appeared after only one injection of SP-2. Embryos from immunised hens were protected against challenge with up to 10,000 LD50 doses of either the homologous strain M986 (B, 2a) or strain M1011 (B, 2a). Protection was also evident against strains 614 (W135, 2a), S5896 (Y, 2c) and 2241 (C, 2a), but not against strain 78704 (C, 2a) despite strong cross-reactivity of antibody with OMV of this strain. Embryos were only partially protected against strain 78069 (B, 2b) and were fully susceptible to strain 2996 (B, 2b). Some protection was also obtained against meningococcal strains M1080 (B, 1), M982 (B,9), S3032 (B, 12), 79001 (B, 12), 79694 (B, 15-related) but not strain 77252 (B, nontypable). These results suggest that proteins extracted from both serotype 2a and 2b meningococci would provide the broadest protection against infection with group B, serotype 2 meningococci and that antibody, presumably directed against common peptides in the major outer membrane proteins, can prevent infection by some other disease-associated serotypes of N. meningitidis.

Multilocus enzyme electrophoresis for characterization of Listeria monocytogenes isolates: results of an international comparative study.

Multilocus enzyme electrophoresis (MEE) is a standard technique that is used to elucidate the epidemiology of a variety of bacterial species. Recently, the method has been employed by several laboratories for investigations of clinical and foodborne isolates of Listeria monocytogenes. To assess the sensitivity and reproducibility of MEE in characterising L. monocytogenes isolates for epidemiological purposes and, ultimately, to agree on a standard protocol, seven laboratories participated in a blinded study of 80 strains. The strain collection included both epidemiologically related and unrelated isolates. Each laboratory used its own protocol for MEE. The number of enzymes that were assayed by the laboratories ranged from 8 to 23, and the total number of identified electrophoretic types (ETs) varied between 14 and 25. Of the II pairs of duplicate strains, the number of pairs recognised as identical by the seven laboratories ranged from 3 to 10 (median = 8). From 10 to 18 (median = 15) of the 22 groups of epidemiological related strains were recognised as homogeneous by the different laboratories. The discriminatory power of the method, calculated using Simpson's index of diversity for 69 strains (80 strains minus the 11 duplicates), ranged from 0.827 to 0.925. This relatively low discriminatory power is a consequence of a somewhat low genetic diversity of L. monocytogenes compared to other bacterial species. Efforts should be pursued to standardise the method in order to improve the intra- and inter-laboratory reproducibility.

The structure of the capsular polysaccharide obtained from a new serogroup (L) of Neisseria meningitidis.

A newly isolated serogroup of Neisseria meningitidis (serogroup L), obtained from contacts of a patient with meningococcal meningitis, elaborates a structurally unique, capsular polysaccharide. The polysaccharide contains only 2-acetamido-2-deoxy-D-glucosyl and phosphate constituents in the molar ratio of 3:1, and is composed of the following repeating unit.

The panmictic nature of Neisseria meningitidis serogroup B during a period of endemic disease in Canada.

Three hundred and one (301) strains of Neisseria meningitidis serogroup B, isolated from patients with meningococcal disease during the years 1994-1996, were subjected to multilocus enzyme electrophoresis, serotyping, and serosubtyping. Based on the analyses of 14 enzyme loci, 177 electrophoretic types (ETs) were identified. Of these, 136 were represented by single isolates and 41 were represented by multiple isolates (range 2-31). The mean genetic diversity for isolates was 0.444 and for ETs was 0.440. The index of association (I(A)) between loci was 0.530 +/- 0.08 for isolates and 0.256 +/- 0.10 for ETs. Cluster analysis revealed the presence of 39 lineages each represented by a single ET or clusters of ETs. The most common serotypes were 4, 15, and 14 and accounted for 84 (28.0%), 53 (17.6%), and 32 (10.6%) of the isolates, respectively, and were dispersed amongst 46 ETs (1-122), 35 ETs (3-165), and 26 ETs (18-76), respectively. The 109 (36.6%) nontypable (NT) isolates were amongst 74 ETs (6-177). The mean genetic diversity for serotypes 4, 15, and 14 and NT isolates was 0.368, 0.371, 0.343, and 0.442, respectively, and for ETs was 0.363, 0.354, 0.397, and 0.440, respectively. Combinations of serotypes and serosubtypes (number of isolates) that occurred most frequently were 4:P1.14 (17), 14:P1.16 (16), NT:P1.16 (16), 15:P1.16 (13), and NT:P1.13 (13). The majority of group B disease in Canada during 1994-1996 was caused by meningococci of considerable genetic diversity, and reflects a situation of endemic disease. However, the results also indicate that organisms belonging to the ET-5 complex, which has been responsible for outbreaks of group B disease globally for several decades, have been introduced into the country.

Improved antiserum agar method for the serogroup differentiation of Neisseria meningitidis Y and W135.

Rabbit antisera prepared to meningococcal serogroups Y and W135 strains were compared with horse antisera using the antiserum agar method (ASA) for the serogroup identification of Neisseria meningitidis. Thirty-seven group Y strains formed immunoprecipitates with the Y rabbit serum only, whereas the same Y strains formed immunoprecipitates with both the Y and W135 horse antisera. Forty-seven W135 strains formed specific immunoprecipitates with both the rabbit and horse W135 antisera by ASA. None of the 166 meningococcal isolates, representative of other meningococcal serogroups, formed immunoprecipitates with the groups Y and W135 rabbit or horse antisera. Use of specific Y and W135 rabbit antisera in ASA provides an improved technique for the serogroup differentiation of groups Y and W135 meningococci.

Type and strain variation in induction of L forms of Neisseria gonorrhoeae.

Strains of Neisseria gonorrhoeae were inoculated onto brain heart infusion (Difco) agar supplemented with penicillin and polyvinylpyrrolidone (PVP) as a stabilizer and cultivated in a candle extinction jar. L-form colonies became visible after a few days. They continued to grow and were viable for up to 38 days. The extent of inducibility of L forms of N. gonorrhoeae was examined semiquantitatively. It was found to be constant for each type and strain and to depend only slightly on the amount of penicillin added to the medium. None of the types of one strain produced L-form colonies. In another strain, avirulent types (T3, T4) showed more ability to produce L forms than virulent types (T1, T2) and no L forms were produced by the subtypes of T1 and T2-T1a and T2a. In a third strain, only T4 produced L forms. Electron microscopic studies showed that the L forms consisted of a number of membranous vesicles and a variety of cell types such as those completely lacking cell walls and those with only remnants of cell walls. The results indicate the existence of subtypes of T1 and T2 of gonococci and the intrinsic inducibility of gonococcal types and strains to produce L forms.

Enzyme-linked immunosorbent assays for the detection of Neisseria gonorrhoeae specific antibodies.

An indirect enzyme-linked immunosorbent assay (ELISA) using rigid polystyrene microtiter plates was adapted to detect specific gonococcal antibodies against outer membrane-complex antigens extracted from Neisseria gonorrhoeae. The concentration of antigen to obtain maximum coating of the well was 10 micrograms protein per millilitre. The optimal binding of the primary antibody and enzyme-conjugated antimmunoglobulin was achieved after 1 h at 37 degrees C. Under these conditions using gonococcal antisera, no cross-reactivity was observed with outer membrane antigens extracted from Neisseria meningitidis serogroups B, C, X, Y, and W135. Neisseria meningitidis serogroup A demonstrated low levels of cross-reactivity. All the non-pathogenic Neisseria spp. tested were negative (absorbance value at 400 nm/30 min less than 0.15). The reaction of immune serum against outer membrane complex absorbed to the microwells was completely inhibited with soluble-specific antigen but not with purified N. gonorrhoeae lipopolysaccharide. Quantitative inhibition permitted the measurement of low levels of antigen (0.5 microgram/ml). The detection of N. gonorrhoeae antibody with ELISA is specific and highly sensitive.

Adjuvant and immunogenic properties of gonococcal R-type lipopolysaccharide in reaginic antibody formation in mice.

The ability of gonococcal R-type lipopolysaccharide (LPS) to function as an adjuvant and as an antigen in the formation of IgE and IgG1 antibody responses in mice was investigated. LPS failed to induce LPS-specific IgE or IgG1 under a variety of experimental conditions. LPS was capable of enhancing IgE and IgG1 antibody responses to gonococcal protein (ZAB), but its adjuvant effect was weaker than that of A1(OH)3. The LPS-induced anti-ZAB IgE antibody titers showed a cycling phenomenon with time, but the IgG1 response was delayed and peaked only once.

Conjugation of meningococcal lipopolysaccharide R-type oligosaccharides to tetanus toxoid as route to a potential vaccine against group B Neisseria meningitidis.

Oligosaccharides were obtained by the mild acid hydrolysis of the lipopolysaccharides from a number of different strains of Neisseria meningitidis, serotypes L2, L3, L4, L5, and L10. The dephosphorylated oligosaccharides were conjugated to tetanus toxoid as their 2-(4-isothiocyanatophenyl)-ethylamine derivatives, which resulted in the incorporation of from 18 to 38 oligosaccharides per molecule of tetanus toxoid. When injected in rabbits, the conjugates produced oligosaccharide-specific antibodies which were predominantly serologically specific but which also exhibited some cross-reactivity. These serological results can be attributed to regions of structural dissimilarity and similarity within the oligosaccharides. The oligosaccharide-specific antibodies were also lipopolysaccharide serotype specific, thus indicating that the oligosaccharides are the determinants associated with this serotype specificity. Consistent with the serological results, the conjugate antisera were bactericidal for the homologous serotype meningococcal organisms and in some cases for heterologous serotype organisms.

Identification of Neisseria gonorrhoeae from primary cultures by a slide agglutination test.

Hen antigonococcal lipopolysaccharide hen serum was used in a simple slide agglutination test for the identification of Neisseria gonorrhoeae from primary isolates.

Serotypes among Neisseria meningitidis serogroups B and C strains isolated in Canada.

Antisera made to prototype serogroup B strains of Neisseria meningitidis were used to serotype, by agar gel double diffusion, 262 meningococcal serogroups B and C strains isolated in Canada. The strains included 93 from patients and 169 from carriers. Serotype 2 was associated with 39 of 75 (52%) of group B strains and 14 of 18 (77.8%) of group C strains isolated from patients. The group B strains were mainly (87.2%) serotype 2b, while the majority (92.2%) of group C strains was serotype 2a. Other serotypes (including a new provisional serotype) represented 25.3 and 5.5% of groups B and C strains, respectively. The new serotype accounted for 13% of the group B strains. Approximately 23% of the strains isolated from patients were nontypable. The distribution of serotype 2, nontype 2 (other serotypes), and nontypable strains isolated from carriers was 2.1, 36.6, and 61.3%, respectively, for group B meningococci and 22.2, 29.6, and 48.25, respectively, for group C meningococci. Serotype 11 was the most prominent of the strains isolated from carriers. Approximately 7% of all the strains were multiple serotypes. Serotype 2 is an important virulence marker associated with meningococcal groups B and C disease in Canada, with serotypes 2a and 2b being markedly associated with groups C and B meningococcal disease, respectively.

Structural determination of the capsular polysaccharide of Neisseria meningitidis group I: a two-dimensional NMR analysis.

The capsular polysaccharide antigen of Neisseria meningitidis group I was isolated by Cetavlon precipitation and purified by ion-exchange chromatography. The structure of the I polysaccharide was determined largely by comprehensive proton and carbon-13 nuclear magnetic resonance studies in which both one-dimensional and two-dimensional experiments were carried out directly on the I polysaccharide. The I polysaccharide is composed of the repeating unit----4)alpha-L-GulpNAcA(1----3)[4-OAc]beta-D-ManpNA-cA(-- --in which the former residue adopts the 4C1 (L) conformation and the latter residue adopts the 4C1 (D) conformation. The one-bond coupling between the anomeric carbon and proton (1J13C,H) of the 2-acetamido-2-deoxy-beta-D-mannuronopyranosyl residue is not consistent with its beta-D configuration. This anomalous value of 1J13C,H for this residue is due to through-space anisotropy effects on its anomeric proton, generated by the proximity of the carboxyl group of the neighboring 2-acetamido-2-deoxy-alpha-L-guluronopyranosyl residue. The O-acetyl substituents of the I polysaccharide are essential for its antigenicity to group I polysaccharide-specific antibodies.

Role of typhoid antigens in protection and pathogenicity for mice.

Swiss white mice immunized with acetone-killed vaccines prepared from strains of Salmonella typhosa, S. typhimurium, and mouse-virulent S. typhimurium hybrids which had acquired, by conjugal genetic transfer, the S. typhosa antigens 9, Vi, and d were challenged with the S. typhimurium hybrids and with the S. typhimurium parent strain. The results of these experiments suggested that the Salmonella somatic antigens were important in conferring protection against death in this system. The S. typhosa Vi antigen did not appear to play any significant role in this protection. The S. typhimurium hybrids employed in these studies did not show any loss of mouse virulence as the consequence of acquisition of various combinations of the S. typhosa somatic, flagella, or Vi antigens, nor did S. typhosa hybrids which had acquired the somatic antigen of S. typhimurium show any increase in mouse virulence.

Organism resembling Neisseria gonorrhoeae and Neisseria meningitidis.

A problem isolate resembling Neisseria gonorrhoeae and Neisseria meningitidis is reported. Growth and biochemical characteristics indicated the organism to be N. meningitidis, whereas serological characteristics indicated it to be N. gonorrhoeae. This vaginal isolate may be a genetically transformed gonococcus with the ability to utilize maltose. Conversely, it may be a meningococcus which has acquired antigenic determinants of N. gonorrhoeae.

Protective efficacy of mouse serum to the N-propionyl derivative of meningococcal group B polysaccharide.

The protective properties of antibodies induced by immunization of mice with a conjugate of tetanus toxoid and the N-propionyl derivative of group B meningococcal polysaccharide (N-Pr-GBMP-TT) have been investigated. Mice immunized with the conjugate produced antibodies which were bactericidal for Neisseria meningitidis strains B:2b:P1.Ham and B:15:P1.16. Passive protection studies indicated that the conjugate serum completely eliminated or reduced considerably levels of bacteremia by the same strains in mice. There was no bactericidal activity or passive protection against a strain of N. meningitidis C:2b:P1.2. Following absorption of the conjugate serum with GBMP the non-absorbed antibody, directed to N-Pr-GBMP, was bactericidal and protected mice against bacteremia with group B meningococci. Thus N-Pr-GBMP antibodies which do not bind to the GBMP are protective in vitro and in vivo.

Unique intermolecular bactericidal epitope involving the homosialopolysaccharide capsule on the cell surface of group B Neisseria meningitidis and Escherichia coli K1.

The N-propionylated group B meningococcal polysaccharide mimics a unique bactericidal epitope on the surface of group B meningococci and Escherichia coli K1. This was confirmed when both the above organisms were able to absorb the bactericidal antibodies from a mouse-anti-N-propionylated group B meningococcal polysaccharide-tetanus toxoid conjugate serum. By using affinity columns it was possible to divide the conjugate antiserum into three distinct populations of both group B polysaccharide cross-reactive and non-cross-reactive antibodies, one of which contained most of the bactericidal activity. The cross-reactive (IgG1) antibodies were absorbed by an affinity column in which the group B polysaccharide was linked to the solid support by a long spacer arm, thereby isolating a population of non-cross-reactive (IgG1) antibodies. Surprisingly the above column also retained another population of non-cross-reactive (IgG2a) and (IgG2b) antibodies which contained most of the bactericidal activity. These latter antibodies were not absorbed by a similar group B polysaccharide-affinity column in which a short spacer arm was employed. Thus the above experiments not only effected a separation of highly bactericidal antibodies but also provided evidence that the long spacer arm is functional in the binding of the bactericidal antibodies to the affinity column. This indicates that the bactericidal epitope is mimicked by the group B polysaccharide in the presence of the long spacer arm, which supports the hypothesis that the epitope is polysaccharide-associated and is probably intermolecular in nature.