Abstract
Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.
Highlights
Key Contribution: Here we describe the generation, characterization, and efficacy of one Fab antibody fragment anti-Shiga toxin type 2 (Stx2) in protecting cells against Stx2 cytotoxic effects and its ability to neutralize Shiga toxins (Stx) produced by Shiga toxinproducing Escherichia coli (STEC) strains, which is demonstrated for the first time
The hemolytic uremic syndrome (HUS) in children is mostly caused by Shiga toxinproducing Escherichia coli (STEC) infection, which is responsible for outbreaks in the United States, Europe, South America, and Japan [1,2,3]
The FabF8:Stx2 was generated from the selection using purified Stx2a toxin and a human synthetic antibody phage display library developed by Persson et al [27]
Summary
The hemolytic uremic syndrome (HUS) in children is mostly caused by Shiga toxinproducing Escherichia coli (STEC) infection, which is responsible for outbreaks in the United States, Europe, South America, and Japan [1,2,3]. In addition to conventional antibodies, recombinant antibodies can be an attractive replacement to avoid animal immunization and other limitations of hybridoma technology, a successful, but cumbersome and costly approach to generate monoclonal antibodies [22,23] In this context, we may include a family of Stx2B-binding VHHs that neutralize Stx in vitro at a nanomolar to the subnanomolar range [24] and the FabC11:Stx generated by phage display technology and produced very efficiently using bacterial protein synthesis systems which were able to prevent Stx toxicity to human kidney cells and in mice [25,26]. Employing phage display antibody library F [27], a monovalent FabF8:Stx was generated, and efficiently produced in the bacterial system with neutralizing qualities against Stx. We introduce a novel and simple antitoxin agent as a new therapeutic option for STEC infections therapy
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