Development of complement-activating immunotherapeutic complexes (CoMiX) targeting multidrug-resistant Pseudomonas aeruginosa
Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative opportunistic pathogen capable of causing a variety of life-threatening acute and chronic infections. P. aeruginosa evades complement-mediated cytotoxicity and phagocytosis by binding Factor H (FH), the main inhibitor of the alternative pathway. Factor H-related protein 4 (FHR4) was shown to compete with FH and act as a positive regulator of complement attack. We have developed CoMiX molecules that exploit the activating properties of FHR4 (CoMiX-FHR4) or the Fc-region (CoMiX-Fc) and target the O11 or Psl polysaccharides on the surface of bacterial cells.
The CoMiX molecules and controls lacking the oligomerization scaffold have been generated in HEK293T cells. The binding of molecules to 28 clinical isolates and one reference strain of P. aeruginosa was analyzed by whole cell ELISA. Subsequently, the effect of the anti-O11 CoMiX-Fc on C3b and C5b9 deposition was measured by ELISA, while the direct killing activity was assessed using the complement-dependent killing assay.
The molecules targeting the Psl exopolysaccharide bound to ~80% of clinical isolates, whereas the molecules specific to O11-polysaccharide recognized only ~20% of the analyzed strains. Anti-O11 CoMiX-Fc dose-dependently promoted C3b and C5b9 deposition, therefore upregulated terminal pathway activation. Anti-O11 CoMiX-Fc (1 μg) significantly increased C3b deposition (p<0.001) and C5b9 deposition (p<0.001) on clinical isolates and the reference strain. Supplementing human serum with 1 μg of anti-O11 CoMiX-Fc resulted in a ~50% (p<0.01) reduction for the clinical isolates in the number of Colony Forming Units/plate compared to serum alone.
In conclusion, we have developed CoMiX molecules as a potential treatment against multi-drug resistant P. aeruginosa. When bound to bacteria anti-O11 CoMiX-Fc molecules not only increased C3b and C5b9 deposition on the bacterial cell surface, but also induced the direct lysis of bacteria.

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