EPITOPE MAPPING OF THE PSEUDOMONAS-AERUGINOSA MAJOR OUTER-MEMBRANE PORIN PROTEIN OPRF

The Pseudomonas aeruginosa major outer membrane protein OprF has been proposed for use as a vaccine and as a target for immunotherapeutic and diagnostic monoclonal antibodies, The well-conserved epitopes for 10 surface-reactive, OprF-specific monoclonal antibodies were localized by both overlapping peptide analysis and immunodetection of OprF peptides generated by cyanogen bromide and the protease papain. Three of the monoclonal antibodies bound to specific overlapping octapeptides, which had been synthesized on 160 pins to cover the entire 326 amino acids of OprF. The highest reactivities were as follows: MA7-1 to the pin with attached peptide GTYETGNK (amino acids 55 to 62), MA7-2 to NLADFMKQ (amino acids 237 to 244), and MA5-8 to TAEGRAIN (amino acids 307 to 314). The other monoclonal antibodies showed no reactivity, indicating that they do not recognize linear epitopes, Two polyclonal sera were also tested and demonstrated weak reactivity with discrete regions of OprF, suggesting that the majority of antibodies produced might recognize conformational epitopes, Utilizing defined peptides generated with cyanogen bromide and papain, the conformational epitopes recognized by the seven monoclonal antibodies were localized to regions that were 42 to 90 amino acids long, These regions were located on two adjacent loops in the middle of an amended structural model of OprF.