Development and characterization of a liposomal subunit vaccine against Neisseria Gonorrhoeae
Creators: name: Parmar, Manjeet M.
Publisher: name: University of British Columbia
Year: 2009

description: This thesis is concerned with the development and characterization of a subunit vaccine against Neisseria gonorrhoeae. The major gonococcal outer membrane protein, Protein I (Por), was selected as the subunit component because it is antigenically conserved between gonococcal strains. Isolated proteins, however, often do not elicit a protective immune response, either because they are not efficiently taken up by antigen presenting cells (APC) or because antibodies generated against the denatured protein do not recognize the native conformation. In the present research, therefore, Protein I was reconstituted into liposomes. These lipid bilayer structures should be capable of maintaining Por in its native conformation and may be efficiently accumulated by APC. The protein reconstitution process was characterized with regard to the efficiency of protein insertion and the ease of detergent removal. Subsequently, the Por proteoliposomes were characterized as to their size, lamellarity, Por orientation in the bilayer and antibody binding efficiency. The detergents octyl glucopyranoside (OGP) and sodium cholate were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. During OGP-mediated reconstitution, essentially complete protein incorporation was achieved at a protein-lipid ratio of 0.01:1. It was observed, however, that the degree of protein incorporation was dependent on the initial protein-lipid ratios. Increasing the concentration of Por protein relative to phospholipid in the reconstitution mixture resulted in inefficient protein incorporation at ratios of 0.02:1 or higher. Reconstitution studies using cholate indicated that protein insertion into liposomes was less efficient than during OGP-mediated reconstitution at the same initial protein-lipid ratios. Subsequent experiments examined Por reconstitution into liposomes consisting of a solely bilayer-forming lipid, 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) or mixtures of POPC with a non-bilayer-forming lipid, 1-palmitoyl, 2-oleoyl phosphatidylethanolamine (POPE). These studies showed no significant differences in incorporation as a function of POPC POPE ratio. Examination of Por orientation in these proteoliposomes suggested that over 80% of the protein was oriented facing outwards in the same "hairpin loop" fashion found in the native bacterial membrane. The conclusion from these studies is that OGP is a preferred detergent for protein reconstitution, providing efficient insertion into the liposomal bilayer in an orientation comparable to the native conformation. Por protein was also reconstituted into liposomes containing positively or negatively charged lipids. Protein reconstitution into systems composed of negatively charged lipids, 1- palmitoyl, 2-oleoyl phosphatidylserine (POPS) or 1-palmitoyl, 2-oleoyl phosphatidylglycerol (POPG), exhibited similar protein incorporation efficiencies as neutral POPC systems when the acidic lipid was present at 5% (by wt). However, increasing the amount of anionic lipid up to 25% resulted in a decrease in protein incorporation efficiency. Essentially complete protein incorporation was achieved when Por was reconstituted into positively charged, dioleyl dimethylammonium chloride- or DODAC-containing liposomes at 5, 10 or 25% DODAC. Interestingly, increasing the concentration of cationic lipid resulted in a shift of the protein/lipid peak towards the top of the isopycnic density gradient, indicating a decrease in density of the reconstituted systems. These results indicate that efficient protein incorporation can be achieved for liposomal systems of differing lipid composition. Reconstituted Por proteoliposomes were characterized by quasi-elastic light scattering size analysis (QELS) and cryo-electron microscopy (CTEM) to determine proteoliposome size and morphology. Such systems exhibited a mean vesicle diameter of greater than 0.3 iv microns and were observed as heterogeneous structures with regard to size and lamellarity using CTEM. A potential subunit vaccine would have to be sterilized before it could be safe for use in humans. Conventional techniques such as heat or steam sterilization would be inappropriate due to potential denaturation of the protein subunit. Terminal filtration through 0.2 micron filters would be adequate for vaccine sterilization; however, reconstituted systems with a mean diameter of 0.3 microns would be too large and unsuitable for direct sterile filtration. Therefore, these proteoliposomes would have to be size-reduced prior to the filtration/sterilization step. In this thesis, it is shown that reconstituted systems can be sizereduced to 100 nm unilamellar vesicles by extrusion, without significant loss of protein or lipid. These extruded systems were then suitable for sterilization by terminal filtration. In a comparative study, the reconstitution of meningococcal outer membrane protein (MOMP) was characterized with regard to degree of protein insertion and detergent removal. The rate of octyl glucoside removal during MOMP reconstitution followed the same kinetics as seen for gonococcal Por reconstitutions. However, the efficiency of protein incorporation was lower than that of Por incorporation at the same initial protein and lipid concentrations. In addition, MOMP reconstitution was examined in the presence of a zwitterionic detergent, Empigen BB, which has been shown to restore the antigenicity of purified meningococcal proteins. Isopycnic density gradient centrifugation studies showed that liposomes were not formed, and hence no protein incorporation occurred during dialysis from an Empigen BBcontaining reconstitution mixture. The results of this comparative study would suggest that protein reconstitution occurs more efficiently in the case of a single polypeptide, such as Por, than for mixtures of different membrane proteins, such as MOMP.