Epitope Mapping of Monoclonal Antibodies using Synthetic Oligosaccharides Uncovers Novel Aspects of Immune Recognition of the Psl Exopolysaccharide of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic Gram‐negative bacterium that can cause life‐threatening infections in critically ill and cystic fibrosis patients. The Psl exopolysaccharide of P. aeruginosa offers an attractive serotype‐independent antigen for the development of immunotherapies. Here, the first chemical synthesis of a panel of oligosaccharides derived from the exopolysaccharide of P. aeruginosa by a synthetic strategy that efficiently deals with the stereoselective installation of several β‐mannosides and the formation of a mannoside that is extended by saccharide moieties at C‐1, C‐2, and C‐3 in a crowded 1,2,3‐cis configuration is described. The approach was employed to prepare tetra‐, penta‐, and hexa‐ and decasaccharide part structures. The compounds were employed to define the epitope requirements of several functionally active monoclonal antibodies (mAbs) that can bind three distinct epitopes of Psl (class I, II, and III). The class II mAb reacted potently with each oligosaccharide indicating its epitope resides within the tetrasaccharide and does not require the branched mannoside of Psl. The class III antibody did not bind the tetra‐ or pentasaccharide; however, it did react potently with the hexasaccharide and weakly with the decasaccharide, suggesting a terminal glucoside is required for optimal binding. Unexpectedly, the class I mAb did not bind any of the oligosaccharides indicating that Psl contains a yet to be elucidated sub‐stoichiometric isoform. This study demonstrates that functional activity of a mAb does not only depend on the avidity of binding but also on the location of an epitope within a bacterial polysaccharide. The results also provide a strong impetus to analyze further the structure of Psl to identify the class I epitope, that is expected to provide an attractive target for the development of a synthetic vaccine for P. aeruginosa.

Anti-Psl Targeting of Pseudomonas aeruginosa Biofilms for Neutrophil-Mediated Disruption

“…Since the anti-Psl mAbs recognize unique epitopes of Psl on planktonic P . aeruginosa (class I, class II, and class III; 29 ), we sought to evaluate whether these epitopes were expressed within mature biofilms. To do this, we grew mature PAO1 biofilms under either flow (18 hour biofilms) or static conditions (24 hour biofilms) followed by staining and analysis via confocal laser scanning microscopy (CLSM) using fluorescently labeled versions of each anti-Psl mAb.…”

“…To identify anti-Psl mAb epitope binding requirements, we constructed a panel of synthetic Psl oligosaccharides followed by evaluation of anti-Psl mAb binding to each (Supplementary Fig. S1B–E ) 29 . The class II epitope binding mAb, WapR001, recognized all oligosaccharide derivatives, while the class III epitope binding mAb, WapR016, recognized the hexasaccharide containing a terminal glucose residue and weakly bound to a decasaccharide (comprised of two Psl pentasaccharide units) (Supplementary Fig.…”

“…The class II epitope binding mAb, WapR001, recognized all oligosaccharide derivatives, while the class III epitope binding mAb, WapR016, recognized the hexasaccharide containing a terminal glucose residue and weakly bound to a decasaccharide (comprised of two Psl pentasaccharide units) (Supplementary Fig. S1B ) 29 . Unexpectedly, mAbs that bound the class I epitope (Cam003 or its affinity optimized derivative, Psl0096), which were also the most active in promoting opsonophagocytic killing (OPK) or in preventing P .…”

“…aeruginosa binding to epithelial cells 28 , did not bind any of the synthetized oligosaccharides. Interestingly, this epitope was associated with an acyl chain modification that is sensitive to mild alkaline exposure 29 .…”

Vaccines forPseudomonas aeruginosa: a long and winding road

“…In both cases, Cam-003 was effective at diminishing the infection [145]. Recently the epitopes of this mAb and other reacting with Psl have been mapped [146]. …”