Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

Abstract

BackgroundStx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes.Methods and FindingsIn this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA.ConclusionIn this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.

Diarrheagenic Escherichia coli

“…Stx2-scFv was obtained from a bacteria-induced culture and showed diagnostic ability; the scFv fragment was able to recognize the majority of Stx2-producing strains, with 79.3% sensitivity (confidence interval of 60.3 to 92%), and no reactivity was observed with the non-producing strains, indicating as high as 100% specificity (confidence interval of 86.8–100%) 210 . It is worth mentioning that none of the commercially available immunoenzymatic tests for Stx1/2 toxin detection employ recombinant antibodies produced in bacteria, which indeed will reduce the costs of the diagnostic assays 198 …”

Shiga-Toxin E. coli Hemolytic Uremic Syndrome: Review of Management and Long-term Outcome

“…As yet, clinical efficacy has not been convincingly demonstrated. Research efforts to develop agents for neutralization of Shiga-toxin are ongoing, such as the development of humanized recombinant antibody fragments directed against Stx2 which effectively neutralize cytotoxicity in vitro [86].…”

Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

“…EspA and intimin proteins of EHEC O157:H7 were targets for the development of scFvs that recognize both antigens in O157 and O111 EHEC tested [ 36 ]. For STEC, some anti-Stx2 scFv were able to recognize and neutralize the Stx2 toxin [ 37 , 38 , 39 , 40 ].…”