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.

Neisseria meningitidis, also known as the meningococcus, is a globally spread obligate human bacterium causing meningitis and/or septicaemia. It is responsible for epidemics in both developed and developing countries. Untreated invasive meningococcal disease is often fatal, and despite modern intensive care units, the mortality is still remarkably high (approximately 10%). The continuously increasing antibiotic resistance in many bacterial pathogens is a serious public health threat worldwide and there have been numerous reports of emerging resistance in meningococci during the past decades. In paper I, the gene linked to reduced susceptibility to penicillins, the penA gene, was examined. The totally reported variation in all published penA genes was described. The penA gene was highly variable (in total 130 variants were identified). By examination of clinical meningococcal isolates, the association between penA gene sequences and penicillin susceptibility could be determined. Isolates with reduced susceptibility displayed mosaic structures in the penA gene. Two closely positioned nucleotide polymorphisms were identified in all isolates with reduced penicillin susceptibility and mosaic structured penA genes. These alterations were absent in all susceptible isolates and were successfully used to detect reduced penicillin susceptibility by real-time PCR and pyrosequencing in paper II. In papers III and IV, antibiotic susceptibility and characteristics of Swedish and African meningitis belt meningococcal isolates were comprehensively described. Although both populations were mainly susceptible to the antibiotics used for treatment and prophylaxis, the proportion of meningococci with reduced penicillin susceptibility was slightly higher in Sweden. A large proportion of the African isolates was resistant to tetracycline and erythromycin. In paper V, the gene linked to rifampicin resistance, the rpoB gene, was examined in meningococci from 12 mainly European countries. Alterations of three amino acids in the RpoB protein were found to always and directly lead to rifampicin resistance. A new breakpoint for rifampicin resistance in meningococci was suggested. The biological cost of the RpoB alterations was investigated in mice. The pathogenicity/virulence was significantly lower in rifampicin resistant mutants as compared with susceptible wild-type bacteria.

Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target. Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation. Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections. This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).