Expression of class 5 antigens by meningococcal strains obtained from patients in Brazil and evaluation of two new monoclonal antibodies.

Determining the profile of antigen expression among meningococci is important for epidemiologic surveillance and vaccine development. To this end, two new mouse monoclonal antibodies (MAbs) have been derived against Neisseria meningitidis proteins (class 5). The MAbs were reactive against outer membrane antigens and were bactericidal. Selected anti-class 5 MAbs [(5.1)-3E6-2; (5.3)-3BH4-C7; (5.4)-1BG11-C7; (5.5)-3DH-F5G9 also 5F1F4-T3(5.c)], and the two new monoclonal antibodies C14F10Br2 (5.8) and 7F11B5Br3 (5.9), were then tested against different meningococcal strains, (63 strains of serogroup A, 60 strains of serogroup C (from 1972 to 1974); and 136 strains of serogroup B (from 1992) meningococci). Our results demonstrated that the expression of class 5 proteins in the N. meningitidis B Brazilian strains studied is highly heterogeneous. The serotypes and subtypes of B:4:P1.15, B:4:P1.9, B:4:P1.7, B:4:P1.3, B:4:P1.14, B:4:P1.16, B:4:NT, and B:NT:NT were detected in N. meningitidis B serogroups. The strains C:2a:P1.2 and A:4.21:P1.9 were dominant in the C and A serogroups, respectively. Serogroup B organisms expressed the class 5 epitopes 5.4 (18%), 5.5 (22%), 5.8 (3.6%), 5.9 (8.0%) and 5c (38%). Serogroup C expressed class 5 epitopes 5.1 (81%), 5.4 (35%), 5.5 (33%) and 5.9 (5.0%); and serogroup A showed reactivity directed at the class 5 protein 5c (47%); and reactivity was present with the new monoclonal antibody, 5.9 (5.5%). We conclude that the two new MAbs are useful in detecting important group B, class 5 antigens, and that a broad selection of serogroup B, class 5 proteins would be required for an effective vaccine based on the class 5 proteins.

Efficacy, safety, and immunogenicity of a meningococcal group B (15:P1.3) outer membrane protein vaccine in Iquique, Chile. Chilean National Committee for Meningococcal Disease.

A meningococcal group B (15:P1.3) outer membrane protein vaccine was tested for efficacy in a randomized, double-blind controlled study in Iquique, Chile. A total of 40 811 volunteers, ages 1-21 years, enrolled in the study. Volunteers received two doses of vaccine six weeks apart by jet injector. Both the experimental vaccine and the control vaccine (Menomune, A, C, Y and W135 meningococcal polysaccharide vaccine) were well tolerated with minor side-effects. Active surveillance for suspected cases of meningococcal disease was conducted for 20 months in Iquique. Eighteen cases of group B meningococcal disease were confirmed during the 20 months. Efficacy was estimated to be 51% (p = 0.11) for all ages combined. In children aged 1-4 no protection was evident, but in volunteers aged 5-21 vaccine efficacy was 70% (p = 0.045). The IgG antibody response by ELISA was characterized by a large booster effect after the second dose, followed by a substantial drop in antibody levels by 6 months. The youngest children had the highest responses. The bactericidal antibody response, on the other hand, was characterized by the lack of a significant booster response, higher responses in the older children, and an increase in the geometric mean titer in the later months of the study in the older children.

The use of filter paper monoclonal antibodies in a Dot-blot test for typing Neisseria meningitidis B.

A simple method for the collection, preservation, shipment, and testing of minute amounts of dried monoclonal antibodies for typing Neisseria meningitidis B is described. The monoclonal antibodies collected on filter paper were extracted in PBS and evaluated by Dot-blot employing whole cells of N. meningitidis B as antigen. The dried filter paper with monoclonal antibodies could be stored at room temperature for as long as 30 days without detectable changes in antibody response when used for typing outer membrane antigens of N. meningitidis B.

The use of filter paper monoclonal antibodies in a Dot-blot test for typing Neisseria meningitidis B

A simple method for the collection, preservation, shipment, and testing of minute amounts of dried monoclonal antibodies for typing Neisseria meningitidis B is described. The monoclonal antibodies collected on filter paper were extracted in PBS and evaluated by Dot-blot employing whole cells of N. meningitidis B as antigen. The dried filter paper with monoclonal antibodies could be stored at room temperature for as long as 30 days without detectable changes in antibody response when used for typing outer membrane antigens of N. meningitidis B

A rapid and sensitive PCR strategy employed for amplification and sequencing of porA from a single colony-forming unit of Neisseria meningitidis.

The predicted amino acid sequence was determined for the class-1 outer membrane protein, PorA, from a B:15:P1.7,3 strain of Neisseria meningitidis that is currently causing an epidemic of meningitis in Northern Chile. The P1.7,3 PorA showed a unique sequence in the exposed loop 4 of the putative porin structure that is different from all the reported PorA sequences. Based on the nucleotide (nt) sequence of the P1.7,3 porA, we designed two sets of PCR (polymerase chain reaction) primers that specifically amplified porA from any N. meningitidis strain, and a third set of primers that amplified porA only from the P1.7,3 strain. Using these primers, we developed a sensitive double hot-start nested PCR (HNPCR) strategy that could amplify porA and generate nt sequence from as low as a single colony-forming unit. This strategy consisted of three phases of PCR. The first two phases were designed to generate amplified target DNA that could be directly visualized by ethidium bromide staining starting from one to two molecules of Neisseria genome. The third phase was designed to generate a sequence of several hundred nt directly from the amplified DNA. A number of culture-negative cerebrospinal fluid samples from individuals suspected of meningitis during a vaccine trial were analyzed by this strategy to obtain more accurate information on the actual number of cases that occurred in the study and the non-study populations. The basic HNPCR strategy described here could be applied to amplify and sequence target DNAs from any low-copy-number biological sample.

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.