Related Experiment Video
Updated: Nov 11, 2025
Engineering Antiviral Agents via Surface Plasmon Resonance
Published on: June 14, 2022
Characterization of a novel mCH3 conjugated anti-PcrV scFv molecule
Samira Komijani1,2, Elham Bayat3,2, Elham Rismani4
1Department of Biotechnology School of Biology, Alzahra University, Tehran, Iran.
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.
Insights
Researchers developed novel recombinant antibody fragments targeting Pseudomonas aeruginosa PcrV. These fragments showed biological activity against bacterial hemolysis and cytotoxicity, with one variant demonstrating superior serum stability for potential cystic fibrosis treatments.
Area of Science:
- Biotechnology and Pharmaceutical Sciences
- Infectious Diseases and Microbiology
- Immunology and Molecular Biology
Background:
- Pseudomonas aeruginosa (PA) is a significant pathogen causing severe nosocomial infections, particularly in cystic fibrosis patients.
- The PcrV protein is a critical virulence factor of PA, making it a promising target for therapeutic interventions.
- Recombinant antibody fragments offer potential for targeted therapies with improved characteristics.
Purpose of the Study:
- To evaluate the physicochemical structure, biological activity, and serum stability of a recombinant anti-PcrV single-chain variable fragment (scFv) fused to an mCH3cc domain (YFL001) and a standalone scFv (YFL002).
- To assess the molecular interactions of YFL001 and YFL002 with PcrV.
- To determine the therapeutic potential of these antibody fragments against PA infections.
Main Methods:
- Computational analysis (in silico) of stereochemical properties and molecular interactions.
- Expression and purification of recombinant proteins (YFL001, YFL002) in E. coli using Ni-NTA affinity chromatography.
- Assessment of biological activity through inhibition of PA-induced hemolysis and cytotoxicity assays on lung epithelial cells.
- Structural analysis using circular dichroism spectroscopy and NMR.
- Evaluation of serum stability using ELISA.
Main Results:
- In silico analysis confirmed satisfactory stereochemical quality and identified common interaction residues between scFv-mCH3/scFv and PcrV.
- Both YFL001 and YFL002 demonstrated significant inhibition of hemolysis by ExoU-positive PA strains and exhibited cytotoxicity effects on lung epithelial cells.
- ELISA revealed that the YFL001 molecule possessed longer serum stability compared to YFL002.
Conclusions:
- The recombinant anti-PcrV antibody fragments (YFL001 and YFL002) exhibit promising biological activity against Pseudomonas aeruginosa.
- YFL001 demonstrates enhanced serum stability, suggesting its potential as a more effective therapeutic candidate.
- These findings warrant further investigation of YFL001 and YFL002 in preclinical animal models for treating PA infections.
