Meningococcal disease in the United States--1986. Meningococcal Disease Study Group.

Active surveillance for invasive meningococcal disease was conducted during 1986 and 1987 in six areas of the United States with a total population of approximately 34 million persons. The incidence of meningococcal disease was 1.3:10(5). The highest incidence of disease among the surveillance areas was in Los Angeles County (1.65:10(5). Neisseria meningitidis serogroups B and C caused about equal amounts of disease, which reflects a recent increase in the incidence of group C disease. Group C caused more than half of the cases of meningococcal disease in Los Angeles and Tennessee but less than one-third of the cases in Missouri and Oklahoma. Multilocus enzyme electrophoresis demonstrated that a group of closely related isolates of N. meningitidis was prevalent in Los Angeles during the surveillance period and was associated with an increased incidence of meningococcal disease there.

Antibody response of adults to an aluminum hydroxide-adsorbed Neisseria meningitidis serotype 2b protein-group B polysaccharide vaccine.

A group B Neisseria meningitidis serotype protein vaccine was studied clinically in adults. The vaccine comprised lipopolysaccharide-depleted outer membrane vesicles from a serotype 2b strain, 3006-M2, noncovalently complexed with group B meningococcal polysaccharide. Volunteers received 25 micrograms each of protein and polysaccharide administered intramuscularly either in 0.9% NaCl or adsorbed onto aluminum hydroxide on weeks 0 and 6. Most individuals experienced mild local reactions, but there were no systemic reactions. Both vaccine formulations stimulated antibodies to the outer membrane proteins of serotypes 2a:P1.2 and 2b:P1.2, but higher levels were achieved with the aluminum hydroxide-adsorbed vaccine after two immunizations. Vaccine-induced antibodies were primarily IgG and were bactericidal for both a serotype 2a and a serotype 2b strain. Induction of bactericidal antibodies has been shown to be a major predictor of protection against meningococcal disease.

Dose-dependent changes in the antigenicity of bacterial endotoxin exposed to ionizing radiation.

The antigenic properties of the highly purified US reference standard endotoxin (RSE) exposed to varying doses of ionizing radiation were studied with double immunodiffusion, immunoelectrophoresis and immunoblotting. Rabbit RSE antisera identified 2 distinct major antigenic components for untreated RSE: one related to the O-polysaccharide side chain ("O-antigenic specificity"), the other to the R-core. Based on a serologic cross-reactivity of R-core of RSE (Escherichia coli 0113) with the R-core of the lipopolysaccharide from E. coli 0111, the core type of E. coli 0113 was identified as coli R3. Increasing exposure of RSE to ionizing radiation progressively destroyed all antigenic reactivities: at lower doses of radiation the rate of elimination differed for the 2 antigen classes. The O-polysaccharide was more sensitive to gamma-radiation than the R-core and the O-antigenicity was lost before that of the R-core. Endotoxin molecules containing incomplete R-core (radiation-induced or mutant) did not react with the RSE antiserum.

Immunotype epitopes of Neisseria meningitidis lipooligosaccharide types 1 through 8.

Lipooligosaccharides (LOS) of the eight immunotypes found in serogroup B Neisseria meningitidis were purified from their prototype strains grown in tryptic soy broth. Rabbit antisera to these LOS were prepared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining revealed that most of the LOS antigens contained two major components; the larger components had apparent molecular weights (Mrs) in the range of 4,800 +/- 300, and the smaller components had an apparent Mr of 4,300. Immunoblot analysis showed that the larger major component of an LOS, in general, was much more immunogenic because the rabbits produced antibodies exclusively or primarily to this component even though the LOS immunogen contained both large and small major components. Antibodies to the smaller 4,300-Mr components were infrequently observed but, when present, were cross-reactive with the same-size components of all heterologous LOS. Hence, the immunotype epitopes reside in the larger major components of all immunotypes except type 5, in which a smaller major component having an apparent Mr of 4,400 carries the epitope. Rabbit antisera to types 1, 5, and 6 were immunotype specific. Antisera to other types had cross-reactivities with some heterologous LOS, and the larger components, but not the 4,300-Mr components, of the LOS were primarily responsible for the cross-reactivities. This finding suggests that the larger components of cross-reactive LOS have a similar structure in addition to their type-specific sugar moieties. The LOS of N. meningitidis M986, a strain used for the production of a serotype 2a vaccine, was found to contain the immunotype 7 epitope.

Genetic structure of Neisseria meningitidis populations in relation to serogroup, serotype, and outer membrane protein pattern.

The genetic structure of populations of Neisseria meningitidis was examined by an analysis of electrophoretically demonstrable allelic variation at 15 genes encoding enzymes in 650 isolates of eight serogroups (A, B, C, W135, X, Y, Z, and 29E) and 38 nonserogroupable isolates. A total of 331 distinctive multilocus genotypes (electrophoretic types, ETs) was identified, among which mean genetic diversity per locus (H = 0.547) was greater than in Escherichia coli and other bacterial species thus far studied. The intercontinental distribution of some ETs and the recovery of organisms of identical genotype over periods of many years strongly suggest that the genetic structure of N. meningitidis is basically clonal as a consequence of low rates of recombination of chromosomal genes. Variation among strains in serogroup, serotype, and the electrophoretic pattern of the major outer membrane proteins has little relationship to the complex structure of populations revealed by enzyme electrophoresis, which involves 14 major lineages of clones diverging from one another at genetic distances greater than 0.50. Genetic diversity among ETs of isolates of the same serogroup was, on average, 84% of that in the total sample. Clones of serogroup A were unusual in being genotypically less heterogeneous than those of other serogroups and in forming a single phylogenetic group. Isolates of the same serotype or outer membrane protein pattern were also highly heterogeneous; on average, 87 and 97%, respectively, of the total species diversity was represented by ETs of the same serotype or outer membrane protein.

Genetic relationships and clonal population structure of serotype 2 strains of Neisseria meningitidis.

Two hundred and thirty-four strains of Neisseria meningitidis, including 94 serotype 2a, 111 serotype 2b, and 19 serotype 2c isolates, together with 10 isolates that were serotyped as 2 with polyvalent antiserum but did not react with monoclonal antibodies, were characterized by the electrophoretic mobilities of 15 metabolic enzymes. Of these enzymes, 14 were polymorphic, and 56 distinctive combinations of alleles at the enzyme loci (electrophoretic types) were identified, among which the mean genetic diversity per locus was 0.413, or about 75% of that recorded for the species N. meningitidis as a whole. Mean genetic diversity among electrophoretic types of the same serotype (2a, 2b, or 2c) was, however, on average, less than half the total species diversity, and no multilocus genotypes were shared between isolates of the different serotypes, which belong to distinctive clonal lineages. Recent temporal changes in the frequencies of recovery of pathogenic strains of serotypes 2a and 2b in South Africa and North America resulted from clone replacement in these populations rather than evolutionary modification of the serotype protein of the initially dominant clones.

Intercontinental spread of Neisseria meningitidis clones of the ET-5 complex.

The genetic structure of populations of Neisseria meningitidis was examined by an analysis of electrophoretically demonstrable allelic variation at 15 structural genes encoding enzymes in 688 isolates. Variation among strains in serogroup and serotype has little relationship to the complex structure of populations revealed by enzyme electrophoresis, which involves 14 major lineages of clones diverging from one another at more than half their genetic loci. Clones of one of these lineages, the ET-5 complex, have been identified as the causative agent of recent outbreaks and epidemics of meningococcal disease in Europe, South Africa, Latin America, and the United States. There is evidence that organisms of the ET-5 complex reached Florida via human immigrants from Cuba.

Appearance of new strains associated with group B meningococcal disease and their use for rapid vaccine development.

There has been a decrease in the prevalence of disease in the United States due to meningococcal serotypes 2a and 2b containing class 2 proteins with a concomitant increase in nonserotypable strains containing class 3 major outer membrane proteins. A new disease associated strain was identified using monoclonal antibodies as B:4:P1.15. Serotype 4 strains have been heretofore isolated almost only from carriers. This B:4:P1.15 strain predominated among group B disease isolates in Cuba from the late 1970s to the present and among Miami, Florida isolates recovered in 1981 and 1982. To determine whether protein vaccines for new strains or serotypes could be prepared using our present methods, a combined vaccine was prepared from a group B strain (B:8:P1.15) recovered during a recent outbreak in Virginia, and a serotype 2b strain, plus group C polysaccharide. The vaccine was prepared with aluminum hydroxide, or with trehalose dimycolate plus monophosphoryl lipid A, or without adjuvant. Four weeks after immunization antibody levels were much higher in mice that received vaccine containing adjuvant.

Intercontinental spread of a genetically distinctive complex of clones of Neisseria meningitidis causing epidemic disease.

Strains of Neisseria meningitidis responsible for an epidemic of meningococcal disease occurring in Norway since the mid-1970s and for recent increases in the incidence of disease in several other parts of Europe have been identified by multilocus enzyme electrophoresis as members of a distinctive group of 22 closely related clones (the ET-5 complex). Clones of this complex have also colonized South Africa, Chile, Cuba, and Florida, where they have been identified as the causative agents of recent outbreaks of meningococcal disease. There is strong circumstantial evidence that outbreaks of disease occurring in Miami in 1981 and 1982 were caused in large part by bacteria that reached Florida via human immigrants from Cuba.

Outer membrane proteins of Neisseria meningitidis: structure and importance in meningococcal disease.

Meningococcal meningitis is a serious disease with a high incidence in children. Since meningococcal surface structures play a role in invasion of the host and initiation of disease, host defense mechanisms are directed against these meningococcal structures. All virulent meningococci are encapsulated, but other factors are also involved. Only certain serotypes of group B and group C meningococci are strongly associated with disease. Our studies have been directed toward a better understanding of the structure of the meningococcal cell surface. There are five classes of major outer membrane proteins exposed on the cell surface, designated class 1 through 5, based in part upon the protein's molecular weight. Using monoclonal antibodies we have shown that antibodies to the class 1, 2 and 5 proteins of serotype 2 are bactericidal and therefore probably protective. Convalescent sera of meningococcal disease patients have elevated antibody levels to each of these classes of proteins. Outer membrane protein vaccines have been prepared, and clinically evaluated in adults and children as young as 6 mo of age. The vaccines induce serotype specific bactericidal antibodies in all age groups, but they induced lower levels in young children. We are now working to improve the immune response of children through the use of aluminium adjuvants.

Serotypes and polyacrylamide gel electrophoresis types among disease-associated isolates of group B Neisseria meningitidis in Spain, 1976-1979.

A high annual incidence of meningococcal meningitis and septicemia occurred in Spain from 1976 through 1980 with a peak of 19 cases per 100,000 population in 1979. Approximately 80% were caused by group B Neisseria meningitidis. Studies were undertaken to determine the distribution of groups, outer membrane protein serotypes and polyacrylamide gel electrophoresis (PAGE) types among 338 disease-associated group B isolates from six regions of Spain. The related serotypes 1, 8, and 15 accounted for 38% (129 of 338) of the isolates. Serotype 2, the major disease type in the United States, was responsible for 14% (48 of 338) of the disease in Spain and was prevalent in only one region. Forty-three percent (146 of 338) were nonserotypable. The predominant PAGE type among the nonserotypable strains was PAGE type IV (79%). These studies demonstrate the necessity of surveillance for selection of suitable serotypes to be included in protective group B meningococcal vaccines.

Sodium dodecyl sulfate-polyacrylamide gel typing system for characterization of Neisseria meningitidis isolates.

Thirty to fifty percent of group B and group C Neisseria meningitidis carrier isolates are not serotypable with existing outer membrane protein typing sera. A typing system based on differences in the outer membrane protein profiles after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was therefore developed as an adjunct to existing serotyping methods. Although most N. meningitidis strains contain several outer membrane proteins visible by SDS-PAGE, there are only one to three predominant proteins. The SDS-PAGE profiles of these major proteins were used to establish 10 different PAGE types. Greater than 95% of all meningococcal isolates, regardless of serogroup, fit into 1 of the 10 PAGE types. The outer membrane protein profile of individual strains after SDS-PAGE was constant when outer membrane fractions were prepared from the same strain on several different days. A comparison of gel profiles of meningococcal isolates obtained from different sites of the same patient revealed no significant differences among both major and minor proteins for isolate sets thus far examined. Characterization of strains by PAGE type can be a valuable epidemiological tool in addition to serotyping and in the absence of specific serotype antisera.

Strains of Neisseria meningitidis isolated from patients and their close contacts.

Neisseria meningitidis isolates from contacts, mostly family members, of 27 unrelated meningococcal disease patients were examined by serogrouping, serotyping, and a recently described sodium dodecyl sulfate-polyacrylamide gel electrophoresis typing procedure. Most of the isolates were serogroup B or C. Serotyping and sodium dodecyl sulfate-polyacrylamide gel electrophoresis typing now provide a more precise means than serogrouping for determining the epidemiological relationships among patient isolates and those of related carriers. In 70% of the families studied, all contact carriers had strains indistinguishable from that of the patient. In the other 30%, more than one meningococcal strain was recovered from the family. Sixty percent of the carrier isolates were recovered from adults. It was found that, among household contacts, the mother was most likely and the father was least likely to carry the disease isolate. Nonhousehold contacts were least likely to carry the disease isolate.

Five structural classes of major outer membrane proteins in Neisseria meningitidis.

Group B Neisseria meningitidis is thus far subdivided into 15 protein serotypes based on antigenically different major outer membrane proteins. Most serotypes have three or four major proteins in their outer membranes. Comparative structural analysis by chymotryptic 125I-peptide mapping was performed on these major proteins from the prototype strains as well as from six non-serotypable strains. The major outer membrane proteins from each of the serotypes were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the Laemmli system. Individual proteins within the gel slices were radioiodinated and digested with chymotrypsin, and then their 125I-peptides were separated by electrophoresis and chromatography on cellulose thin-layer plates. The peptide maps obtained by autoradiography were categorized into five different structural classes which correlated with the apparent molecular weights of proteins, i.e., 46 +/- 1K, 41 +/- 1K, 38 +/- 1K, 33 +/- 1K, and 28 +/- 1K. Each of the major outer membrane proteins within a strain had a distinctly different chymotryptic peptide map, indicating significant differences in the primary structure of these proteins. In contrast, outer membrane proteins of the same or very similar molecular weight from different serotype strains had similar, occasionally identical peptide maps, indicating a high degree of structural homology. The unique peptides from proteins of the same structural classes were often hydrophilic, whereas common peptides were often hydrophobic, suggesting that the serotype determinants reside within the variable hydrophilic regions of major outer membrane proteins.

Adherence of isolates of Neisseria meningitidis from patients and carriers to human buccal epithelial cells.

An in vitro assay was used to study the adherence of Neisseria meningitidis to human buccal epithelial cells. Both unencapsulated and encapsulated, piliated isolates obtained from throats of asymptomatic carriers demonstrated significantly higher levels of adherence to buccal cells than encapsulated, piliated isolates obtained from the blood and cerebrospinal fluid of patients (P < 0.001). Meningococcal adherence to buccal cells could not be correlated to a specific capsular polysaccharide serogroup, outer membrane protein serotype, or quantitative differences in pili. However, the data suggested that capsular polysaccharide impedes the adherence of meningococci to buccal cells, and the significantly smaller amount of capsular polysaccharide extracted from carrier isolates compared with case isolates (P < 0.001) could explain differences in meningococcal adherence to buccal cells. Increased adherence may facilitate host colonization, promote nasopharyngeal carriage, and possible reflect altered pathogenicity.

Rapid serogroup identification of Neisseria meningitidis by using antiserum agar: Prevalence of serotypes in a disease-free military population.

Nasopharyngeal cultures from 414 Marines were plated directly onto antiserum agar containing the antibiotics vancomycin, colistin, and nystatin for meningococcal isolation and serogroup identification. Meningococci were isolated from 267 Marines, giving a carrier prevalence of 64.5%. A total of 58% of the isolates could be placed into serogroups; of these 22.3% were group B, 4.7% were group C, 25.7% were group Y, 24.3% were group W135, and 23.0% were group 29E. No serogroup A organisms were recovered. Serotyping by agar gel double diffusion was performed on 148 strains. More than 70% of these strains were nontypable, and the disease-associated serotype 2 was present only in two group Y isolates. The same 148 isolates were also classified by major outer membrane protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis pattern IV was most common among all serogroups. The data demonstrate the effective use of antiserum agar for meningococcal surveillance and document the frequency of specific serotypes and polyacrylamide gel electrophoresis types among carrier isolates obtained from a nonrecruit military population.

Heat-modifiable outer membrane proteins of Neisseria meningitidis and their organization within the membrane.

Neisseria meningitidis group B serotype 2 strain M986 contains two predominant outer membrane proteins, with apparent molecular weights of 41,000 (protein b) and 28,000 (protein e). Heating of outer membrane vesicles at 56 degrees C for 20 min caused much of b** to disaggregate and denature into b (41,000 daltons). In contrast, protein e could be rapidly solubilized by SDS at room temperature into its monomeric state (e*), but it was not converted to its final higher apparent molecular weight of 28,000 (e) unless heated at 100 degrees C for 2 min. We propose that protein b exists in the membrane as trimers or tetramers in a transmembrane configuration and that protein e exists as subunits on the exterior surface of the outer membrane and has a highly ordered tertiary structure.