Antigenic variation in the outer membrane proteins of Neisseria meningitidis and the suitability of the H.8 antigen for use in immunisation against meningococcal disease

Tinsley, Colin Richard (1990) Antigenic variation in the outer membrane proteins of Neisseria meningitidis and the suitability of the H.8 antigen for use in immunisation against meningococcal disease. University of Southampton, Doctoral Thesis.

Abstract

Inter- and intra-strain variation in the surface-exposed proteins of N. meningitidis was investigated in order to compare the variability with that which had been reported for the closely-related N. gonorrhoeae and to identify antigens possessing conserved epitopes which were stably expressed by the bacteria. Radioimmune precipitation, SDS-PAGE of surface radioiodinated disease isolates and isolated outer membrane vesicles, and western blotting techniques demonstrated variability in the major outer membrane proteins and pilin both between strains and during the course of infection. Several monoclonal antibodies recognised an antigen which was common to all pathogenic Neisseriae, and absent from most commensal organisms. The stable expression of this antigen suggested that it might have an important role in pathogenesis and made it an attractive choice for further study. The pathogenic Neisseria antigen was shown to have unusual properties which, together with its peculiarity to pathogenic species and apparent molecular mass range, it shared with the H.8 antigen found by Cannon and coworkers (Infection and Immunity, (1984), 43 ,994-999). Further similarities have confirmed that these are the same protein. Two published methods for purification of the antigen were investigated: Gel filtration followed by ion exchange chromatography, or extraction into phenol, precipitation of lipopolysaccharide, and lipophilic gel filtration followed by reverse-phase HPLC. However, better results were obtained after development of an affinity chromatography system with an anti-H.8 monoclonal antibody immobilised on a cyanogen bromide-sepharose column. Due to difficulties in staining the antigen after SDS-PAGE a quantitative spot blot assay was developed for its detection, based on the ELISA principle. Amino acid analysis showed the antigen to be rich in alanine, glutamine, and proline and lacking in sulphur-containing amino acids. Immunisation of mice with purified antigen from one strain of meningococcus produced antisera reactive with the H.8 antigens of the homologous and heterologous strains. However, this serum and the H.8-directed monoclonal antibodies were ineffective in an in vitro complement-mediated bactericidal assay. Initial experiments showed poor opsonic activity for a monoclonal antibody. Synthetic oligopeptides corresponding to regions of the primary sequence of the H.8 antigen were used to define the epitopes recognised by the serum and monoclonal antibodies. Since neither polyclonal sera nor monoclonal antibodies directed against a variety of epitopes showed biological activity it appears that immunisation with the H.8 antigen would not elicit protection against meningococcal disease.

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