Recombinant antibodies as biomolecules for the therapy of intoxications caused by Escherichia coli that produce Shiga toxins

• Grant number: 22/06099-8
• Support Opportunities: Regular Research Grants
• Start date: December 01, 2022
• End date: November 30, 2024
• Field of knowledge: Health Sciences - Collective Health - Preventive Medicine
• Principal Investigator: Roxane Maria Fontes Piazza
• Grantee: Roxane Maria Fontes Piazza
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated researchers: Flavia Sacerdoti ; María Marta Amaral

Abstract

Shiga toxin-producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli (EHEC), one of the six pathotypes of diarrheagenic E. coli, is a concern for human health, as it is associated with outbreaks of infections and food poisoning from ingesting contaminated food or water, particularly undercooked meats, raw vegetables and milk. These bacteria produce the most potent cytotoxins, Shiga toxins (Stx), which are responsible for the severe form of the disease, Hemolytic Uremic Syndrome (HUS), which turn in very serious sequelae that can lead to death. HUS is characterized by acute renal failure, thrombocytopenia and microangiopathic hemolytic anemia, being more common in children less than 5 years of age and the elderly. To date, there are no effective therapies against Stx-mediated HUS. Regarding the treatment of symptoms, antibiotics cannot be used, as the phages that encode the toxins are induced by cellular stress, therefore, antibiotics (especially those that target the genetic material) activate the SOS system of the bacteria, leading to increased of Stx production, and worsening patient intoxication. Treatment is then limited to hydration and symptom-supportive care. As an alternative, neutralization of the Stx toxin would be the best and most recommendable treatment of infection by these strains and prevention of HUS. The high specificity and affinity of antibodies are characteristics that have made these molecules tools used in a wide variety of applications in biotechnology. Alternatively, neutralization of the Stx toxin would be the best and most recommended treatment of infection by these strains and prevention of HUS. Thus, the present proposal intends to invest in these molecules with biotechnological potential that are the Fab antibodies, which is the variable portion of the antibody being composed of three complementarity determining regions also known as CDRs, these fragments are essential since they contain the binding site to the target antigen, in this way this fragment can be used due to the generated high specificity. The Fabs that will be tested were produced and characterized as to their specificity to their antigens and their dissociation constants were defined by surface plasmon resonance; they are: FabB6:Stx1 (3.99 X 10-8); FabC8:Stx1 (1.82 X 10-8 ); FabF8:Stx2 (1.64 X 10-8) and FabC11 that recognizes Stx1 (3.49 X 10-8) and Stx2 (7.54 X 10-9) and were able to neutralize the respective purified toxins in standard Vero cell neutralization assay. Thus, the aim of the present project is the validation of recombinant antibodies (Fab) as therapeutic biomolecules in the intoxication of infections caused by Escherichia coli that produce Shiga toxins. (AU)