Abstract
Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni–NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.
Highlights
Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients
In order to identify an anti-PcrV MAb possessing enhanced inhibition of the T3SS, Warrener et al developed a panel of novel antibodies amongst which 29D2 and V2L2MD provided the strongest protection against P. aeruginosa infection in an animal model[13]
A monomeric CH3 domain (mCH3) encoding gene fragment was inserted at 5′ end of anti-PcrV scFv and the YFL001 expression cassette was designed (Fig. 1A,B)
Summary
Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. In 2009, Baer et al engineered a human Fab antibody fragment against PcrV protein which competed with MAb166 in binding to the same e pitopes[11]. This Fab molecule showed significant potency in in vitro and in vivo TTSS-neutralizing activity very similar to that of the Mab[166] against lethal doses of PA. KB001 is a PEGylated recombinant human Fab antibody fragment designed against PcrV12 This antibody represented potential activity in a model of mouse pulmonary infection in which reduced mortality and effective clearance of bacteria was observed within the infected lungs. The aim of the present study was to evaluate the physicochemical and biological properties of the recombinantly mCH3 conjugated anti-PcrV scFv molecule in in vitro conditions
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