Preparation and physicochemical and immunological characterization of polysaccharide-outer membrane protein complexes of Neisseria meningitidis

Abstract

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.