Immunochemistry and structural variation of the class 1 outer membrane protein of Neisseria meningitidis

Meningococcal meningitis is a severe life threatening disease for which no adequate vaccine exists. A number of studies have suggested that the class 1 outer membrane protein, which is responsible for subtype specificity, represents the most promising target for a protective immune response. Comparison of the predicted amino acid sequence of the class 1 protein from three meningococcal strains of different subtypes revealed a very high degree of homology, with major differences restricted to two variable regions (VR1 and VR2). Using overlapping synthetic oligopeptides corresponding to each of the three amino acid sequences, the epitopes recognised by protective subtype specific mAbs were mapped to either VR1 or VR2. Comparison of the predicted amino acid sequence of the class 1 protein from an epidemic disease isolate (MC58) with a reference strain (H4476) of the same subtype revealed a single amino acid substitution (D to N) in MC58 caused by a point mutation, located in VR2 within the P1.16 epitope. The normal bactericidal activity of P1.16 specific mAbs for P1.16 strains was abolished for MC58. Investigation of a number of strains demonstrated that isolates carrying the same point mutation were responsible for cases of meningococcal meningitis around England and Wales. These epitope mapping results were used to generate a model predicting the arrangement of the class 1 protein in the outer membrane (van der Ley et al., 1991). This model suggests that the P1.16 epitope is at the apex of a large surface exposed loop. Computer modelling of the P1.16 and P1.16b epitopes demonstrated significant changes in conformation by the D to N change. In order to define and examine more subtype specificities, PCR technology was used to selectively amplify VR1 and VR2 sequences from a number of strains of varying subtype specificity. Comparison of the predicted amino acid sequences of each variable region, in conjunction with subtype specificities and detailed epitope mapping, permitted the identification of sequences defining a number of new subtypes. Examination of all of the variable regions sequenced, suggested the occurrence of recombinational events creating new combinations of subtype specificities. The PCR technology developed for the sequencing work was also used succesfully to amplify meningococcal specific sequences from clinical samples of CSF. This technology therefore permits detailed molecular epidemiological studies of disease isolates. To examine the possibility of eliciting an immune response to the class 1 protein, rabbits and mice were immunised with synthetic peptides corresponding to the P1.16 and P1.16b epitopes. Cross reactive immune responses were elicited to peptide, isolated protein and native protein in outer membranes. However, significant bactericidal activity against strains MC58 and H44/76 could not be demonstrated in the anti-peptide sera. Epitope mapping revealed that the immune responses were not directed precisely at the bactericidal P1.16 epitope. This suggests that precise targeting of the immune response is required to produce a protective immune response. The results presented in this thesis have therefore produced detailed knowledge on the immunochemistry of an important meningococcal surface antigen and will be of particular value in studies designed to induce protective immunity against meningococcal infection.