No abstract

Adult volunteers were injected with a combined meningococcal group A and group C polysaccharide vaccine. Immunoglobulin M (IgM) and IgG antibody levels against both polysaccharides were measured in serum samples taken 14 days as well as 3 years after vaccination. For both group A and group C polysaccharides, the IgM and IgG antibody levels at 14 days postvaccination were positively related. The IgM-to-IgG antibody ratio at 14 days postvaccination was an indicator for the persistence of both IgM and IgG antibodies during the next 3 years; a high ratio meant a short persistence, whereas a low ratio was associated with a long persistence.

A crude complex containing group C polysaccharide, outer membrane proteins, and lipopolysaccharide (LPS) was isolated from the cell-free culture liquid of Neisseria meningitidis serogroup C, serotype 2a. Group C polysaccharide and LPS were removed from this complex, resulting in an outer membrane complex and a purified complex, respectively. Analysis by electron microscopy showed the outer membrane origin of the crude complex and the outer membrane complex, whereas such a structure was absent in the purified complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of the three complexes were identical. Pyrolysis-mass spectrometry data correlated well with those obtained by the biochemical assays and suggested a low LPS content in the purified complex and a low polysaccharide content in the outer membrane complex. The purified complex was shown to be nonpyrogenic and could be prepared with the same yield as that of purified polysaccharide. The immunogenic activities of the complexes were studied in mice. The antibodies were measured by the enzyme-linked immunosorbent assay; and the bactericidal antibody assay. All complexes induced immunoglobulin G antibodies to group C polysaccharide as well as to the serotype antigen, although the removal of polysaccharide and LPS resulted in a reduction of the immunogenic activities of outer membrane complex and purified complex, respectively. A second dose of all complexes produced a clear booster effect of both antibody responses. The antibodies were bactericidal.

In a sandwich ELISA for tetanus antibodies, the influence of the tetanus toxoid concentration used for coating microtiter plates has been studied. The antibody levels by toxin neutralization bioassay and by ELISA were studied for a population with known immunization history. By decreasing the tetanus toxoid concentration in ELISA from 5 to 0.2 Lf/ml, a better correlation was found between the ELISA results and the bioassay titers, but sera from recently immunized people still showed high ELISA titers. This phenomenon cannot be ascribed to nonspecific reactions since sera from nonimmunized people are negative in both assays. All sera negative in ELISA had, however, a bioassay titer beneath 0.01 IU/ml.

Neisseria meningitidis group C polysaccharide-tetanus toxoid conjugate was prepared to obtain the polysaccharide component in a thymus-dependent form and to preserve the immunogenic properties of the tetanus toxoid component. Biochemical and immunochemical analyses of this conjugate revealed that (i) it was composed of equal amounts of polysaccharide and protein; (ii) the antigenic activity of the polysaccharide component was greatly reduced; (iii) it contained about 10o free polysaccharide; (iv) the composition was not homogeneous; and (v) only 5% of the tetanus toxoid component was present at the surface of the conjugate molecules. In this study, the influence of these characteristics on the antibody response to both components in mice was investigated. The doseresponse relationship, the persistence of antibodies, a possible antigenic competition, and the specificities of the antibodies induced were also studied. Our data suggest that the conjugate behaves as a pronounced thymus-dependent antigen, that the tetanus toxoid component is more immunogenic at lower dosages (0.8 and 20 ng) than equivalent doses of tetanus vaccine, that the presence of free polysaccharide does not influence the induction of antibodies to polysaccharide by the conjugate, and that no antibodies to new structures in the conjugate are induced. These characteristics favor the application of this conjugate as a vaccine for human use. Purified meningococcal groups A, C, W135, Y, and Z' (29E) induce protective bactericidal antibodies in humans and mice (9, 10, 17, 18, 39). These antibodies exert their protective effect by initiating complement-dependent bacteriolysis and opsonization (15, 16, 26). It is well known that no helper thymus-derived cells are involved in the immune response to purified polysaccharides from encapsulated bacteria in mice (5); most likely they are not involved in humans, either (6). As a consequence, they only induce an immunoglobulin M (IgM) antibody response in mice (3, 8, 9) and uncommitted individuals, e.g., very young children (22). Moreover, a second dose will not result in a booster reaction (4, 17). Such thymus independence is a drawback for their potential use as a vaccine. This drawback can be overcome by coupling the polysaccharides to thymus-dependent carrier proteins (1, 7-9, 13, 20, 29, 30, 33, 34). Polysaccharide-protein conjugates are serious candidates for vaccines for human use. Recently, we have described the preparation t Present address: Centraal Laboratorium van de Bloedtransfusiedienst van het Nederlandse Rode Kruis, 1006 AD Amsterdam, The Netherlands.