Protein Fraction with Immunogenic Potential and Low Toxicity Isolated from the Cell Wall of Neisseria meningitidis Group B

Abstract

Several fractions were extracted from the cell envelope (CE) of Neisseria meningitidis group B and characterized with regard to their morphology, antigenicity, protein composition, and toxicity. Whole bacterial cells were suspended in a medium of low ionic strength and disrupted in a French pressure cell. The crude CE thus obtained were separated into cell membrane (CM) enriched and cell wall (CW) enriched fractions on sucrose density gradients. In addition, CM and CW fractions were separated from CE on the basis of differential solubility in the nonionic detergent, Triton X-100. The Triton-insoluble fraction, containing primarily CW components, was further treated with a mixture of Triton and ethylenediaminetetraacetic acid, which was shown to remove additional protein and most of the lipopolysaccharide. Electron microscope examination of the various fractions revealed typical unit membrane structures in the case of CM, or large, open segments in the case of CW. The Triton-insoluble and especially the Triton-ethylenediaminetetraacetic acid-insoluble fractions consisted of small vesicular structures. All fractions, except the Triton-soluble fraction, when assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were shown to contain one major protein component accounting for more than 50% of the total. Sera from rabbits immunized with the various fractions formed precipitin lines in immunodiffusion tests against the homologous and some of the heterologous fractions. High-titer bactericidal antibodies were also demonstrated in these sera when tested against the homologous strains. Toxicity studies in rats sensitized with lead acetate indicate that the level of contamination of Triton-insoluble/Triton-ethylenediaminetetraacetic acid-insoluble fractions with lipopolysaccharide was significantly smaller than that of the other fractions.