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Optimization and characterization of recombinant antibodies and their application for diagnosis and therapy of Shiga, heat-labile and heat-stable enterotoxins

Grant number: 15/17178-2
Support type:Regular Research Grants
Duration: November 01, 2015 - October 31, 2017
Field of knowledge:Biological Sciences - Microbiology
Principal Investigator:Roxane Maria Fontes Piazza
Grantee:Roxane Maria Fontes Piazza
Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
Assoc. researchers:Monica Valdyrce dos Anjos Lopes Ferreira

Abstract

Diarrheal disease is a worldwide public health major problem responsible for about 1.5 million children under five years old deaths per year, and about 30-40% of acute diarrhea episodes in the world are caused by diarrheagenic Escherichia coli, especially the pathotypes responsible for acute diarrhea/ traveler's diarrhea and hemorrhagic colitis/hemolytic uremic syndrome, enterotoxigenic E. coli (ETEC) and Shiga toxin- producing E. coli (STEC), respectively. ETEC produces both heat-stable toxin (ST) and heat-labile (LT) toxins and STEC produces the potent Stx cytotoxins. These toxins are the main virulence factors therefore excellent diagnosis targets for diarrhea caused by these pathogens and intoxication therapy. Antibodies are among the best tools for the development of both immunodiagnostic tests as well as toxin neutralization therapy. The recombinant DNA technology allowed the development of recombinant antibodies, such as scFv and Fab fragments in a faster and lower cost way, been produced even in bacteria hosts, one advantage compared to the monoclonal produced by hybridomas, which are time expending and laborious to obtain it. Over the years our research group at the Bacteriology Laboratory of the Butantan Institute have been successfully obtained antibodies, including recombinant as well as developing and standardizing diagnostic methods for diarrheagenic Escherichia coli epidemiologically important pathotypes. In this context, this project aims to obtain optimum molecules (stable, easy production and low cost) for the diagnosis and treatment of intoxication by enterotoxin LT, ST and Stx. For LT / ST toxins, we aim to improve the previously obtained recombinant antibodies anti-LT/ST - in order to optimize their production, stability and half-life, thus having a more efficient tool for ETEC immunodiagnosis - and selection of Fab fragments able to neutralize the toxins, thus allowing the intoxication treatment by ETEC. On the other hand, for Stx toxin, we already have promising recombinants, but they still need to be validated as tools for diagnosis of STEC (scFv) and tested in animal models in vivo to assess their therapeutic potential in preventing Stx intoxication and hemolytic uremic syndrome (Fab/IgG4). (AU)