NEUTRALIZING ANTIBODIES IN THE INTESTINAL MUCOSA ARE ESSENTIAL TO CONTROL SHIGA TOXIN- PRODUCING Escherichia coli (STEC) GASTROINTESTINAL INFECTION IN MICE

ALAN MAURO BERNAL; FERNANDO NICOLÁS SOSA; YINA MARÍA CARPINTERO POLANCO; ROMINA JIMENA FERNÁNDEZ BRANDO; MARÍA VICTORIA RAMOS; MARTÍN RUMBO; MARINA SANDRA PALERMO

Congreso; LXXI Reunión Anual de la Sociedad Argentina de Inmunología; 2023

The humoral response in the intestine against pathogens such as STEC is fundamental to define the evolution to self-limited forms or systemic complications. We previously demonstrated that B cell stimulation and the early antibody production are fundamental in protection against STEC infections in BALB/c mice (BALB). The objective of this work is to characterize the mechanism by which local and/or systemic specific anti-STEC antibodies (aSTECAb) mediate protection against systemic complications and death after STEC infection in BALB.To achieve this, BALB were gastrointestinally infected with a non-lethal dose of STEC (1x1010 CFU/mouse) at weaning; to increase the antibody titer, they were challenged two more times with STEC (5x1010 CFU/mouse) at 10-day intervals. Mice were bled 7 days after the 1st and 2nd infection to determine plasma aSTECAb titer. Seven days after the 3rd infection, mice were euthanized, and plasma and feces collected to determine the aSTECAb titer reached by ELISA. To characterize the functionality of these antibodies, in vitro opsonization assays were carried out by incubating feces (1/2 dilution) or plasma (1/10 dilution) with STEC (1x107 UFC) for 2 h and subsequent analysis by flow cytometry using an anti-mouse IgA or IgG antibody, respectively, coupled to FITC. Also, the neutralizing capacity of these antibodies was analyzed by bacterial growth inhibition in tryptic soy broth determining the optical density at 600 nm (OD600) every 20 min for 18 h, and motility in soft agar by measuring diffusion halos at 48 h (103 UFC with 1/10 diluted plasma for growth and motility inhibition. Only plasma was selected for these assays as it is a sterile sample). The statistical analyzes used were ANOVA, Student’s parametric t test or non-parametric t test, as appropriate.IgG aSTECAb titer in plasma was 2048 and IgA aSTECAb titer in feces was 1024 after the 3rd infection (n=4 mice) (levels of aSTECAb measured as OD492 in plasma at 1/2048 dilution: 0.427±0.17/ 0.07±0.11 infected vs control mice, p