FABF8:STX2 RECOMBINANT MONOCLONAL ANTI- BODIES AVOID THE DELETERIOUS EFFECTS OF SHIGA TOXIN TYPE 2 ON HUMAN MICROVASCULAR ENDOTHELIAL CELLS

GOMEZ FERNANDO D; LUZ DANIELA; PIAZZA ROXANE MP; PRESTA AGOSTINA; IBARRA CRISTINA; SACERDOTI FLAVIA; AMARAL MARÍA MARTA

Congreso; Reunión de Sociedades de Biociencias.; 2021

Sociedad Argentina de Investigación Clínica

Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenicEscherichia coli (STEC) infections is the principal cause of acuterenal injury in pediatric age groups in Argentina. Neither a licensedvaccine nor effective therapy for HUS is available for humans. Pre-viously, we demonstrated the in vitro cytotoxic effects of Shiga toxintype 2 (Stx2) on human glomerular endothelial cells (HGEC). Re-cently, recombinant antibodies against Stx2, produced in bacteria,were developed, and characterized. In this work, we studied the abil-ity of anti-Stx2 FabF8:Stx2 antibody to neutralize the Stx2 activity onprimary cultures of HGEC. Cells were plated in 96-well plates andgrown to con uence. Then, cells were treated in growth-arrestedconditions for 72 h with different pre-incubations (1 h at 37oC) orco-incubations of FabF8 with Stx2. Antibodies were used from 10μg/mL to 0.001 μg/mL and Stx2 at the dilution required to kill 50%of cells (0.5 ng/mL). Finally, cell viability was assessed by neutralred uptake. In addition, cells were seeded on gelatine coated glasscoverslips and then treated, as it was previously mentioned, with1 μg/mL FabF8 and 0.5 ng/mL Stx2, during 72 h. Percentage ofnecrotic and apoptotic cells were established by uorescence mi-croscopy after staining with acridine orange/ethidium bromide. Un-der both conditions evaluated, FabF8:Stx2 signi cantly neutralized,in a dose-dependent manner, the cytotoxic effects caused by 0.5ng/mL Stx2 in HGEC (p <0.05, n = 3). HGEC viability was protect-ed by 10 μg/mL FabF8 in about 67.5% at the co-incubation con-dition, and about 83% at the pre-incubation condition. Additionally,FabF8:Stx2 signi cantly prevented HGEC necrosis (pre: 60%; co:92.5%) and apoptosis (pre: 93% and co:75%) (p <0.05, n = 3). Theresults demonstrate the high ef ciency of FabF8:Stx2 to avoid thecytotoxic effects of Stx2 on HGEC, therefore, they could be usedas a therapeutic strategy to prevent the renal damage described inpatients with HUS.