| Summary: | 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.
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