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Title: Investigating the therapeutic potential of genetically modified Neisseria lactamica
Author: Pounce, Zoe Caroline
ISNI:       0000 0004 7656 4726
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2018
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Neisseria meningitidis colonises the human nasopharynx and can invade through the epithelium to cause rapidly progressing, potentially fatal disease. Carriage of the commensal Neisseria lactamica correlates with decreased colonisation with N. meningitidis and reduced invasive meningococcal disease (IMD), making N. lactamica a candidate for an anti-meningococcal vaccine or probiotic. Intranasal inoculation of N. lactamica showed a good safety profile, but only 34% of those challenged with this species became colonised. Intramuscular injection of N. lactamicaderived outer membrane vesicles (OMVs) demonstrated their potential as a mucosal adjuvant but with limited anti-meningococcal immunogenicity. This project investigated the use of genetically modified (GM) N. lactamica to enhance the potential of this organism to protect against IMD. N. lactamica previously proved resistant to genetic modification. Here we showed that a significant barrier to transformation is the activity of restriction enzymes, which was partially overcome by the incorporation of exclusively hypermethylated cytosine residues into donor DNA, by bespoke PCR amplification. Although it was observed that N. lactamica Y92-1009 is highly resistant to heterologous genetic recombination in vivo, in vitro and in biofilms, this technique allowed us to accurately target genetic modifications to various chromosomal loci in this strain. Multiple factors affecting the efficiency of transformation were identified, including the extent of homology between donor and chromosomal DNA and the number of DNA uptake sequences. Transformation was found to be dependent on the presence of type 4 pili, which were also required for efficient interaction of N. lactamica with epithelial cells. Meningococcal immunogen PorA was selected for the proof of principle that heterologous antigens could be expressed in N. lactamica. With the view to progress this strain to human challenge, the protein was introduced in an antibiotic-resistance-free manner, using the deletion and re-introduction of β-galactosidase as a marker of transformation on X-Gal-containing agar. PorA P1.7,16 was expressed at modest levels but the expression of other PorA types was unsuccessful. PorA P1.7,16 was presented on the surface of bacteria and on OMVs. Intraperitoneal injection of mice with PorA-containing OMVs induced production of serum bactericidal antibodies (SBA) that were active against both N. meningitidis H44/76 and MC58. A genetic enhancer sequence was identified upstream of PorA in N. meningitidis and was used to modulate PorA expression in N. lactamica. In order for N. lactamica to tolerate phase variable expression of PorA, the enhancer sequence was truncated and expression reduced. At this reduced level, a panel of three PorA-expressing strains of N. lactamica were assessed for progression to human challenge. Whilst strains were comparable, genetically stable and antibiotic susceptible, the low-level PorA expression meant that phase variation could not be detected and OMVs derived from these strains did not induce SBA activity in mice. This thesis presents the first steps in developing GM strains of N. lactamica with the potential to reduce nasopharyngeal colonisation with N. meningitidis and to vaccinate against invasive meningococcal disease.
Supervisor: Read, Robert ; Laver, Jay Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available