MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2011 / Despite being a fulminant pathogen, Neisseria meningitidis (meningococcus) is part of the commensal flora of the human nasopharynx. Globally, five meningococcal serogroups (A, B, C, Y and W135) cause the majority of invasive disease. Most serogroup B cases occur sporadically but may be endemic or epidemic within a geographic region. In South Africa, there are limited data on invasive serogroup B clones and the antigenic diversity of certain meningococcal outer membrane proteins. This study examined the molecular epidemiology of serogroup B meningococci in South Africa from 2002 through 2006. Invasive meningococcal isolates were submitted to a national laboratory-based surveillance system. For this study, serogroup B isolates were characterised by pulsed-field gel electrophoresis (PFGE), PorA, FetA and multilocus sequence (MLST) typing. PorA, FetA and multilocus sequence (MLST) typing were performed on all 2005 isolates (n=58) and randomly selected isolates from other years (n=25). A total of 2144 invasive cases were reported over the study period. Of these, 76% (1627/2144) had viable isolates available for serogrouping and 307 (19%) were serogroup B. Serogroup B cases were reported from across the country however the majority were from the Western Cape province. The highest incidence of serogroup B was in children less than 5 years of age. Isolates displayed a high level of diversity by PFGE. Despite this diversity the majority of serogroup B meningococci collected over the 5-year period could be grouped into several clonal clusters representative of global invasive MLST clonal complexes. Overall, the most predominant MLST clones in South Africa were ST-32/ET-5 and ST-41/44/lineage 3. In addition, at least 19 PorA types and 16 FetA types were determined among selected isolates. Globally invasive serogroup B disease is caused by heterogeneous strains however, prolonged outbreaks in several countries have been due to strains of the ST-32/ET-5 and ST-41/44/lineage 3 clonal complexes. At present, serogroup B disease in South Africa is not dominated by an epidemic clone, however, global clonal complexes ST-32/ET-5 and ST-41/44/lineage 3 are circulating in Western Cape and Gauteng, respectively.

Development of a broadly cross-protective capsular group B meningococcal (MenB) vaccine has been hampered by poor capsular immunogenicity and often diverse and poorly cross-protective subcapsular antigens. The MenB MC58 strain genome has facilitated the discovery of novel, relatively conserved vaccine candidates. The four-component MenB (4CMenB) investigational vaccine contains factor H-binding protein (fHbp; variant 1), neisserial heparin-binding antigen (NHBA), Neisserial adhesin A (NadA) and PorA P1.4-containing outer membrane vesicles. The latter are known to elicit protection against homologous strains. Clinical trials have demonstrated protective responses in infants and adults against isolates expressing homologous PorA or fHbp (subvariant 1.P1), or heterologous NadA (variant 2). Cross-protective responses have also been demonstrated in adults and, to a lesser extent, infants, against isolates expressing heterologous fHbp variant 1 subvariants. The contribution of NHBA is still poorly understood. MenB currently accounts for 87% of invasive meningococcal disease in England and Wales. The proportion of disease due to the ST-269 clonal complex (cc269) peaked at 45.6% in 2006 and is currently approximately 24.2%. The aims of this study were (i) to genotypically assess potential 4CMenB coverage against recent English and Welsh invasive disease isolates and, specifically, cc269 isolates from England and Wales and other countries, (ii) to compare phenotypic expression levels of the 4CMenB antigens (excluding PorA) among typical cc269 isolates, and (iii) to assess 4CMenB responses against typical cc269 isolates among healthy adults administered 4CMenB.Full length alleles for fHbp variant 1, NHBA and NadA variants 1, 2 and 3 were present in 64.6%, >99% and 7.1%, respectively, of English and Welsh invasive disease isolates from 2007/8. Between 67.5% and >99% (adults) or 25.7% and 43.5% (infants) of the isolates were predicted to be covered by 4CMenB. cc269 comprised two antigenically distinct lineages (clusters) centred around ST-269 and ST-275, respectively. These accounted for 57% and 40% of cc269 in 2007/8. Both clusters effectively lacked nadA and PorA P1.4. The predominant fHbp;NHBA profiles represented by the respective clusters were 1.P15;P0021 and (1.P13 or 2.P19);P0017. Between 77.4% and 100% (adults) or 2.2% and 27.1% (infants) of cc269 isolates from 2007/8 were predicted to be covered by 4CMenB. Estimates for infants were conservative due to e.g. the exclusion of NHBA. Serum bactericidal antibody (SBA) analyses targeting typical fHbp variant 1-expressing cc269 strains, indicated high levels of coverage among adults administered 4CMenB. Notable differences among genotypically matched isolates e.g. in terms of SBA geometric mean titres, were not reflected in the relative fHbp and NHBA expression levels. Such differences may lead to conflicting estimates of coverage in infant populations. Whilst these are investigated further it seems prudent to use typical isolates giving mid-range responses when assessing SBA, and therefore protection, among infants. Potential 4CMenB coverage of cc269 and the broader meningococcal population in England and Wales was high among adults and encouraging among infants when compared to that of existing MenB vaccines.

Neisseria meningitidis causes 500,000 cases of meningitis and septicaemia annually worldwide, with a mortality rate of approximately 10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opa proteins are major meningococcal outer membrane proteins, and a limited number of Opa variants have been associated with hyperinvasive serogroup B meningococci, suggesting their use as a potential novel vaccine. Immunisation of mice with recombinant Opa elicited high levels of meningococcal-specific serum bactericidal antibody (SBA), demonstrating proof in principle of this approach. Opa proteins mediate bacterial adherence to host cells and modulate human cellular immunity, and there are conflicting data regarding their effects on CD4⁺ T cells. opa genes from N. meningitidis strain H44/76 were cloned into the plasmid vector pBluescript, disrupted using antibiotic resistance cassettes and transformed into H44/76 to sequentially disrupt the four opa genes. This produced a unique panel of 15 isogenic Opa-deficient strains, including an Opa-negative strain, which enabled investigation of the immunomodulatory role of surface-expressed Opa proteins. There was no consistent effect of Opa expressed on the surface of OMVs and inactivated bacteria on CD4⁺ T cells, with significant heterogeneity of responses between individuals. The rate of Opa phase variation was between 10^-3 and 10^-4, and increased 180-fold following transformation of bacteria with unrelated DNA. These data support further investigation of Opa as a potential meningococcal vaccine component, and highlight the importance of host and bacterial factors in the development of OMV vaccines.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

N. meningitidis is a common commensal of the human upper respiratory tract and a leading cause of bacterial meningitis and septicemia worldwide. The classical pathway of complement (C) is essential for both naturally acquired and vaccine induced immunity against N. meningitidis. Qualitative and/or quantitative differences in anti-meningococcal antibodies (Abs) in serum is one reason for variations in C-dependent bactericidal Ab activity among individuals. I showed that IgG isolated from select individuals could block killing of group B meningococci by Abs that were otherwise bactericidal. Ligand overlay immunoblots revealed that these blocking IgG Abs were directed against a meningococcal antigen called H.8, Killing of meningococci in reactions containing bactericidal mAbs and human blocking Abs was restored when blocking Ab binding to meningococci was inhibited (or competed for) using either synthetic peptides corresponding to H.8 or a non-blocking mAb against H.8. Further, genetic deletion of H.8 from target organisms abrogated blocking. The Fc region of the blocking IgG was required for blocking because F(ab)2 fragments alone generated by pepsin treatment were ineffective. Blocking required IgG glycosylation; deglycosylation of blocking IgG with peptide:N-glycanase (PNGase) eliminated blocking. C4 deposition mediated by a bactericidal mAb directed against a meningococcal vaccine candidate, called factor H-binding protein (fHbp), was reduced by blocking Ab. Anti-fHbp-mediated C4 deposition was unaffected, however, by deglycosylated blocking IgG. Although preliminary, our data suggests blocking of serum bactericidal activity by human anti-H.8 blocking antibody may require mannan-binding lectin (MBL), which itself is a complement activator. Also, whether MBL recruits a complement inhibitor(s) that facilitates blocking remains to be determined. In conclusion, we have identified H.8 as a meningococcal target for novel blocking antibodies that are commonly found in human serum. Blocking Ab may reduce the efficacy of meningococcal vaccines. We propose that outer membrane vesicle-containing meningococcal vaccines may be more efficacious if purged of subversive immunogens such as H.8.