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The accepted correlate of protection for meningococcal disease is serum bactericidal activity. The importance of bactericidal activity was established by Goldschneider et al. (1969) who demonstrated an inverse relationship between disease incidence and bactericidal titres of >1 :4. The importance of antibody and complement mediated immune mechanisms is also emphasised by the increased susceptibility of complement deficient individuals to meningococcal disease. However, the absence of bactericidal activity does not necessarily indicate susceptibility and protection has been demonstrated in the absence of bactericidal activity. This has indicated the importance of other protection mechanisms such as opsonophagocytosis. In this study a high throughput antibody-mediated complement deposition assay has been developed to measure the deposition of both C3b/iC3b and C5b-9 onto Neisseria meningitidis. Antibody mediated C5b-9 deposition measured in this assay has been shown to correlate highly with bactericidal activity for several strains and C3b/iC3b deposition has been shown to correlate with opsonophagocytosis. This assay has also been shown to be reproducible. Measurement of antibody mediated C5b-9 and C3b/iC3b deposition will not be a replacement for opsonic killing assays or the accepted correlate SBA, but could be used as a tool to evaluate large panels of sera against multiple strains due to the very low serum volumes required. A respiratory burst assay has also been developed to measure both the uptake of fluorescently-stained bacteria and the induction of a respiratory burst response. This assay was optimised with respect to the reagents used to measure the respiratory burst response, bacterial stain and the assay buffers. Complement is an important reagent used in immunoassays to evaluate antibody mediated immunity to N. meningitidis. Due to the high levels of nasopharyngeal carriage in adults it is difficult to obtain a complement source without cross- reactive bactericidal activity, often resulting in the requirement for a source from a different individual for each strain. An IgG-depleted complement source has been developed for use in immunoassays. The method developed has been shown to be reproducible and effective at removing the IgG whilst retaining functional complement and will be a valuable tool for assessing immunity to N. meningitidis.

Neisseria meningitidis contains c-type and b-type cytochromes. Oxidation of c-type and b-type cytochromes by oxygen was observed in both cells grown under aerobic and denitrifying conditions, whereas oxidation of cytochromes by nitrite was only seen in cells grown under denitrifying conditions, and the predominant oxidizable cytochromes were b-type. These are likely to be associated with the oxidation of a b-haem containing nitric oxide reductase. Nitrite inhibits the oxidation of cytochromes by oxygen in a nitrite reductase-independent manner, indicating that nitrite may inhibit oxidase activity directly, as well as via the intermediate of denitrification, nitric oxide. Cytochromes c4 and c2 are major electron donors to the cbb3 oxidase. Both strains deficient in cytochrome c4 and c2 exhibits a growth defect under high level of oxygen. These growth defects are linked to their decreased oxygen consumption rate. The growth defect and the decreased oxygen consumption rate indicated that cytochrome c4 dominates electron transfer to cbb3 oxidase. Cytochrome c5 is an important electron donor to AniA nitrite reductase. A strain deficient in cytochrome c5 exhibits a growth defect under microaerobic conditions in the presence of nitrite. The mutant can not reduce nitrite to nitric oxide although AniA is expressed normally. A growth defect during high cell density culture under aerobic conditions suggests that cytochrome c5 is also an electron donor to ccb3 oxidase but to a lesser extents than c4 or c2. Lipid-modified azurin (Laz) was heterologously expressed and purified. The purified protein contains copper ion and can be oxidized or reduced. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. Laz can be oxidized by membrane extract in the presence of oxygen or nitrite. This is likely to be due to the activity of cbb3 oxidase and AniA nitrite reductase, respectively, in membrane extract. However the oxidation rate is very slow. A strain deficient in laz exhibits a growth defect during high cell density culture, similarly to that of c5 mutant. This suggests that Laz might be an electron donor to cbb3 oxidase.

Neisseria meningitidis (the meningococcus) is a Gram negative diplococcus which asymptomatically colonizes the human nasopharynx in about 10%-30% of the population. In rare cases it can invade the bloodstream and cross the blood brain barrier to cause sepsis and meningitis. Serogroup B strains are the major cause of disease in the UK. To gain insight into the process of colonization, I have studied the meningococcal transcriptome in a model of human nasopharyngeal colonization. As meningococcal colonization can last for weeks to months, investigating the meningococcal transcriptome over an extended period of time offers potentially important new insights into meningococcal biology unexplored in short time-course experiments as undertaken by others. Confluent layers of a human bronchial epithelial cell line (16HBE14) were infected with a serogroup B meningococcal strain (MC58) for different time periods (4h, 24h, 96h and 21d) prior to bacterial RNA extraction. Using microarray technology, the transcriptome of host cell-associated meningococci was investigated and expression of selected genes (some previously shown to be involved in host cell interaction/colonization, and others surface expressed vaccine candidates) was validated with quantitative real time PCR. The transcription profile at late time points (24 h, 96 h and 21 d) was compared to that at 4 h to identify genes potentially involved in prolonged bacteria host-cell association. A set of seven genes (NMB034230348) was identified which was up-regulated at later time points. By use of RT-PCR the operon structure of the locus was defined: one set of four and one pair of these genes are co-transcribed. MC58 kanR insertion mutants in five of these seven genes were prepared and studied in quantitative adherence and invasion assays. An impaired adherence and reduced invasion profile compared to the wild type suggests a potential role in sustained host cell association.

The development of vaccines against serogroup B Neisseria meningitidis to reduce the morbidity and mortality of meningococcal disease is a major public health priority. We developed a novel genetic screen for immunogens present on the bacterial surface using human immune sera with bactericidal activity. We found that two mutants lacking nmb1467 survived in high concentrations of sera from two patients, while the wild-type strain was killed. Biochemical assays using purified recombinant NMB1467 indicated that nmb1467 encodes an exopolyphosphatase (PPX) with the ability to hydrolyse inorganic polyphosphate (poly P). In addition, we demonstrated that the Δppx mutant has at least 2-fold more poly P than the wild-type strain. Therefore, we designated NMB1467 as PPX. We showed that N. meningitidis mutant lacking the ppx had an increased resistance against normal human complement system. Substitution of the glutamic acid at residue 147 of PPX with an alanine significantly reduced the enzymatic activity in vitro, and contributed to increased level of poly P in N. meningitidis and the resistance of bacteria against the complement-mediated killing. Levels of polysaccharide capsule and lipopolysaccharide (LPS) sialylation, two important virulence factors, were not affected by the loss of ppx in N. meningitidis. Using flow cytometry, we demonstrated that binding of factor H (fH), the negative regulator of the alternative pathway of complement activation, to the bacterial surface was increased in the strain lacking PPX. By Western blot analysis, we did not detect a significant change in the expression of the fH receptor, indicting another mechanism is involved in the fH binding to the bacterial surface and resistance of bacteria against complement-mediated killing.

Neisseria meningitidis is the most common cause of bacterial meningitis in the Western world and the second leading cause of mortality in 1-5 year-olds in the United Kingdom. A signature-tagged mutagenesis screen of approximately 3,000 insertional mutants of a serogroup B isolate of N. meningitidis, C311⁺, identified 73 genes required for pathogenesis in an infant rat model of meningococcal septicaemia. Homology-based searches indicate that two of the genes identified, NMB0342 and NMB0345, have homologues in other pathogenic bacteria and exist within a potential operon of seven genes (NMB0342-NMB0348). NBM0342 is a homologue of ispA (intracellular septation protein) of Shigella flexneri, required for effective intercellular spread and plaque formation in epithelial cells. NMB0345 is a homologue of cbf, a Campylobacter jejuni gene that encodes a 29 kDa protein which is a major antigenic peptide. PCR and Southern analyses of genes in the NMB0342-NMB0348 locus show that they are conserved across a wide range of pathogenic isolates and serogroups of N. meningitidis. However, these genes are present only in a subset of commensal strains. N. meningitidis mutants with transpoon insertions in NMB0342 and NMB0345 are highly attenuated when directly competed with the wild-type bacterium in the infant rat model of infection. Mutations have been introduced into other genes within the locus and the resulting mutants analyzed for their ability to cause systemic disease. Mutants with transpoon insertions in the NMB0343 and NMB0344 genes are significantly attenuated, while mutants with insertions in NMB0347 and NMB0348 are attenuated to a lesser degree. Complementation of the NMB0342 mutation by ectopic chromosomal integration of a wild-type copy of NMB0342 almost completely restores the virulence of the bacterium. In vitro cell-culture analyses and microscopic analysis of mutants in association with host cells demonstrate that a mutant with a transpoon insertion in NMB0345 is significantly reduced in its adherence to Chang epithelial cells compared to the wild-type bacterium. Whole blood and serum-sensitivity assays show that the NMB0342 and NMB0345 mutants are highly serum-sensitive compared to the wild-type bacterium. Preliminary experiments have demonstrated that immunization of mice with recombinant NMB0345 protein confers protection against challenge with the serogroup B isolate MC58. In vitro assays demonstrate that recombinant NMB0345 is an active peptidyl polyl cistrans isomerase (PPIase). Together, the results from these investigations support a role for genes of the NMB0342-NMB0348 locus in the pathogenesis of N. meningitidis infections. Further investigation is needed to assess the potential of NMB0345 as a vaccine candidate.

In the search for a vaccine providing comprehensive protection against meningococcal disease, one vaccine currently under development contains the immunogenic proteins PorA and FetA in meningococcal outer membrane vesicles (OMVs). To achieve high levels of coverage against disease-causing isolates, the antigenic variability of these proteins could be overcome using knowledge of meningococcal epidemiology and population structure. In this study, the possible implications of variable expression levels of PorA and FetA on vaccine efficacy were investigated. Producing OMVs containing consistent amounts of FetA is difficult due to iron-repressed expression; therefore, meningococcal strains were constructed which constitutively expressed FetA at increased levels for OMV vaccine production and analysis. In mice, OMVs from modified strains induced antibodies against both PorA and FetA. These antibodies acted synergistically in a serum bactericidal assay; however, antibodies against FetA were weakly bactericidal alone. The potential to increase levels of PorA- and FetA-specific bactericidal antibodies with a prime-boost strategy, using OMV and protein inoculums, was also tested. While successful for a weakly-immunogenic PorA variant, a similar strategy did not increase bactericidal activity against FetA. Although antibodies against FetA can be induced following OMV immunisation, sufficient antigen expression in target bacteria is also required for bactericidal killing; therefore, the variability and regulation of porA and fetA transcription was investigated in a range of isolates. Despite differences in regulation among clonal complexes, variable expression is unlikely to be an issue for vaccine coverage. In particular, regulation of fetA by iron is reduced in many isolates due to a deletion in the sequence bound by the regulatory protein, Fur. Therefore, a vaccine targeting PorA and FetA may provide high levels of protection against meningococcal disease; however, an alternative formulation or immunisation strategy is required to improve coverage against FetA.

Experimental meningitis was induced in pigmented guinea pigs by subarachnoid inoculation of \(1 \times 10^9\) Escherichia coli K-12 or \(3 \times 10^7\) CFU Streptococcus pneumoniae serotype 2 D39 (NCTC 7466) or PLN-A, \(\Delta\)NA1 or \(\Delta\)HY1, defined isogenic derivatives of D39 deficient in pneumolysin, neuraminidase or hyaluronidase respectively. All animals developed a meningeal inflammatory response and a labyrinthitis. Hearing loss in pneumococcal meningitis was measured by recording the evoked auditory nerve compound action potential from the round window membrane. Animals infected with PLN-A sustained significantly less hearing loss than those infected with wild-type D39 (12 dB vs. 50 dB 12 h post inoculation; P<0.0001), Neuraminidase deficiency did not alter the course of the meningeal inflammatory response nor affect hearing loss. The \(\Delta\)HY1 mutant survived poorly in the cerebrospinal fluid and blood but still caused hearing loss. Both pneumococcal and E. coli meningitis induced specific ultrastructural lesions in the organ of Corti as judged by high-resolution scanning and transmission electron microscopy, and these lesions were most severe with pneumolysin-sufficient pneumococcal infection. Microperfusion of \(5\times10^6\) CFU S.pneumoniae D39 directly into the scala tympani of guinea pigs also resulted in electrophysiological and ultrastructural damage to the organ of Corti that could be diminished by pretreatment with antibiotics. The data confirm the cochlea as the site of meningogenic deafness. They suggest that pneumolysin expression is chiefly responsible for meningogenic deafness and that if pneumococci invade the inner ear during bacterial meningitis, cochlear deafhess will rapidly ensue.

The primary approach to the control of meningococcal disease remains effective vaccination programmes in susceptible populations. Vaccines against serogroups A, C, W and Y offer broad protection against meningococci and both polysaccharide and conjugate quadrivalent vaccines are licensed for use in the UK. Previous studies have assessed the antibody response to meningococcal polysaccharide and conjugate vaccines, but there is limited information on the nature of the B cell response to these antigens. As part of a clinical trial using both polysaccharide (MenACWY-PS) and conjugate (MenACWY-CRM) vaccines in adult volunteers, this DPhil reports the analysis of subsets of antigen specific B-cells produced in response to either vaccine. Prior MenACWY-PS impaired the response to a subsequent dose of MenACWY-CRM. This may be due to MenACWY-PS driving terminal differentiation of antigen specific cells into plasma cells, without replenishment of the memory B cell pool. In addition, despite prior data indicating that it may act as a thymus dependent antigen, the serogroup A polysaccharide component of MenACWY-PS appears to behave in the same way as serogroup C, W & Y polysaccharide components. Antibody molecules recognise and bind to a multitude of conformational epitopes. This variability is enabled by the complexities of immunoglobulin variable domain gene recombination which can generate a vast potential repertoire of unique antibody molecules. However, the diversity of the antibody repertoire is more restricted against specific antigens and within defined B cell subsets. In this DPhil, ‘next generation’ sequencing technologies were used to investigate the diversity of the B cell variable domain before and after vaccination of adult volunteers. Individuals at baseline were found to have distinct antibody repertoires. Vaccination with a Haemophilus influenzae type b (Hib) conjugate vaccine resulted in an oligoclonal antibody response, with enrichment for Hib specific canonical antibody sequences.