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Variation in nine enzymes in 152 isolates of Neisseria meningitidis from Norway (118 from blood or cerebrospinal fluid of patients with systemic disease and 34 from the pharynx of healthy carriers) was analysed by starch-gel electrophoresis. All nine enzymes were polymorphic and the number of allozymes (electromorphs) identified per locus ranged from 3 to 12, with a mean of 6.1. Among the 152 isolates, 55 unique combinations of electromorphs (electrophoretic types, ETs) were distinguished. Twenty ETs were represented among the carrier isolates and 37 among the systemic isolates; hence, only two ETs were found in both groups of isolates. ET-5 was identified 67 times among the 118 systemic isolates (58%), indicating an association of this ET with invasiveness; ET-5 was also the most common type among the carrier isolates (18%). Genetic similarity between ETs was analysed by pairwise comparison of all 55 ETs with respect to the number of electromorphs by which they differed. No evidence of a general genetic difference between carrier and case isolates was found. Two well-defined clusters of ETs were observed, each including one of the two most common ETs identified among the systemic isolates (ET-5 and ET-37), together with isolates differing from them only at one or two loci. All isolates of ET-5 and ET-37, as well as their closely related variants defined by the similarity matrix, were resistant to sulphonamide, independent of their antigenic characteristics and isolation site. The extensive allozyme variation among isolates of the same serogroup demonstrated the limited value of serogrouping as an epidemiological tool. All but one isolate of serotype 15:P1.16 were electrophoretically similar, as were all the 2a:P1.2 isolates. The 15:P1.15 isolates, however, were genetically heterogeneous. The distribution of alleles in genotypes identified among the systemic isolates indicated that genetic recombination may occur in natural populations of N. meningitidis.

To improve the diagnosis of systemic meningococcal disease we used a nested polymerase chain reaction (nPCR) test to detect meningococcal DNA in cerebrospinal fluid (CSF) samples from patients. The nPCR test was based on the gene coding for the PorA outer membrane protein, a major antigen of Neisseria meningitidis, which is the basis for determining the subtype of the strains. The method was tested on CSF samples from 87 patients with various disease aetiology, including 37 patients with systemic meningococcal disease. It was also applied to all 67 CSF samples from culture-negative patients with suspected meningococcal disease which were collected in the course of the Norwegian serogroup B vaccination trials, between 1987 and 1993. Of the 67 culture-negative CSF samples, 10 were nPCR positive. Two of the 67 CSF were positive with the antigen test, and both of these were nPCR positive. Serum pairs from 46 of the 67 culture-negative patients were analysed for serological serogroup response in an enzyme-linked immunosorbent assay (ELISA) against meningococcal capsular polysaccharides. Nine of the 10 nPCR-positive CSF samples belonged to patients with serological response judged as either typical of, or compatible with systemic meningococcal disease. Sequence analysis of the nPCR products was performed to determine the subtype of the infecting meningococcal strains. Four of the 10 positive CSF samples were infected with a strain of subtype P1.7,16, which is similar to the epidemic strain used to produce the vaccine. Inclusion of nPCR for diagnosis of meningococcal meningitis did not significantly alter the results of the vaccination trial, but added important information about the strain causing disease in 5 of 13 serogroup B cases without preserved isolates.

Aase A, Høiby EA, Michaelsen TE. Opsonophagocytic and Bactericidal Activity Mediated by Purified IgG Subclass Antibodies After Vaccination with the Norwegian Group B Meningococcal Vaccine. Scand J Immunol 1998;47:388-396 To study how the different immunoglobulin (Ig)G subclass antibodies may confer protection against systemic meningococcal disease, we isolated IgG1, IgG2 and IgG3 antibodies from plasma from vaccinees immunized with the Norwegian meningococcal outer membrane vesicle vaccine. Four IgG1, one IgG2 and four IgG3 preparations were purified. The IgG2 and IgG3 subclass preparations were free from contaminating subclasses, whereas the IgG1 preparations contained from 0 to 14% of IgG2 and/or IgG3. Immunoblotting against whole-cell meningococcal antigens showed broad specificities of the various preparations, both within and between subclasses. These subclass preparations were tested for opsonophagocytic and bactericidal activity. As targets we used two different variants of the meningococcal vaccine strain, with low (44/76-SL) and high (44/76-1) expression of the outer membrane protein Opc. Using polymorphonuclear leucocytes as effector cells in the presence of human complement, all three IgG subclass preparations revealed high, and similar, opsonophagocytic activities against 44/76-SL, whereas against 44/76-1 the IgG2 preparation showed a reduced activity and most IgG3 preparations were slightly more active than the IgG1 preparations. Regarding bactericidal activity, all the three subclasses were highly active against 44/76-SL. Against 44/ 76-1 the bactericidal activities were somewhat more varied: all IgG1 and three IgG3 preparations exhibited higher activities than against 44/76-SL. Due to the low concentration in the IgG2 preparations, only a weak activity was seen against 44/76-1. One IgG3 preparation that was highly opsonophagocytic revealed no bactericidal activity against either of the two bacterial variants examined. In conclusion, we have shown that the IgG subclass effector functions differ from person to person, but that antibodies of IgG1, IgG2 and IgG3 subclasses, judged by their behaviour in the functional tests, may all contribute to protection against meningococcal disease.

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