The development of the Pseudomonas aeruginosa outer membrane protein OprF as a presentation vector for foreign antigenic determinants

• Main Author: Wong, Rebecca Suk Yi
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/7463

A variety of systems have been developed to improve the presentation of foreign antigenic determinants (‘epitopes’) by inserting them in the context of carrier proteins. The goals of this study were to develop the Pseudomonas aeruginosa outer membrane protein OprF as a carrier for foreign epitopes and to study the effect of the mode of presentation on the antigenicity of the presented epitope. The model epitope used in this study was the 4-amino acid repeating epitope (NANP) of the circumsporozoite protein of the malaria parasite, Plasmodium falciparum. Linker-insertion mutagenesis was carried out to create 11 “permissive” sites which allowed the insertion of 4 extra amino acids. Two series of OprF::malarial epitope hybrid proteins, the positional hybrids and the multiplerepeat hybrids, were constructed by inserting oligonucleotides encoding the epitope into the linker-insertion sites of oprF. The effects of the insertion position and the length of the epitope on its antigenicity were studied by ELISA using outer membranes and by whole cell dot blot analysis. It was shown that the antigenicity of the epitope varied when inserted at different positions of OprF, while it increased with the length of the epitope at two of the three insertion positions studied. These data were employed to revise the membrane topology model of OprF and have improved our understanding of the epitopes recognized by the OprF-specific monoclonal antibodies. Generalizations about the influence of surrounding amino acids on the antigenicity of the inserted epitope are proposed. A targeted study of immunogenicity showed that a 19-amino acid malarial epitope was significantly more immunogenic than a 7-amino acid epitope when inserted at an N-terminal insertion site of OprF. A parallel immunogenicity study of two versions of glutathione S-transferase (GST) : :malarial epitope fusion proteins demonstrated that neither an 11- nor a 19- amino acid epitope fused to the C-terminus of GST was immunogenic. This study demonstrated for the first time that OprF can be used as a carrier to generate and detect anti-epitope antibodies in immunized animals and in immunoassays respectively.