Development and characterization of a liposomal subunit vaccine against Neisseria gonorrhoeae

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. (Abstract shortened by UMI.)...