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Shiga Toxin-producing Escherichia coli (STEC) intoxication and colonization in zebrafish model: an in vivo alternative for pathogenicity studies and therapeutic antibodies validation

Grant number: 17/17006-2
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): January 01, 2018
Effective date (End): December 31, 2019
Field of knowledge:Health Sciences - Collective Health
Principal Investigator:Roxane Maria Fontes Piazza
Grantee:Daniela Luz Hessel da Cunha
Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Shiga toxins (Stx1 and Stx2) produced by Shiga toxin-producing Escherichia coli (STEC), are potent AB5 type cytotoxins that cause a hemolytic uremic syndrome (HUS), generally leading intoxicated individuals to death, being an important public health problem. As existing therapies are not effective, a neutralization of Stx toxicity is the most indicated treatment. Antibodies high specificity and affinity have made these molecules tools used in the therapy of various diseases, furthermore, from the recombinant DNA technology, it is possible to develop recombinant antibody fragments as therapeutic tools. Using such technology, four Fab fragments of human recombinant antibodies (two against each Stx toxin) were obtained by Phage Display and expression in bacteria, LPS free. These different Fabs were characterized and proved to be efficient in their antigen recognition as well as in the cytotoxic effect neutralization in vitro, drawing attention as a promising measure for the Stx neutralization and HUS prevention. However, to validate this neutralizing capacity, in vivo tests are required. Existing animal models do not cover all factors relating to intoxication and infection. In this context, a vertebrate model is proposed, the zebrafish (Danio rerio), which presents as advantages: its small size, fast generation time, and genomic similarity with mammals; its use has produced relevant results in studies of pathogen-host relationships. Its well-developed innate cellular and adaptive immune systems make this model ideal for the study of infectious diseases In STEC however, little has been done in this vertebrate. Thus, we have consolidated a successful partnership with a Zebrafish platform of the Butantan Institute, where it was possible to take the first step in the development of a zebrafish embryo intoxication model, standardizing a toxicity test, use Stx, which results in a dose-dependent toxic effect. These results are essential for the present project, which aims to study Stx intoxication in this animal, including other development stages, as well as STEC isolate infection, with the purpose of launching a new experimental model, to facilitate the validation of therapeutic tools, in particular human recombinant antibodies. Once the model has been established, the study with outbreaks STEC strains will be optimized, as well as the characterization of other virulence factors associated with the infection.

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
LUZ, DANIELA; AMARAL, MARIA MARTA; SACERDOTI, FLAVIA; BERNAL, ALAN MAURO; QUINTILIO, WAGNER; MORO, ANA MARIA; PALERMO, MARINA SANDRA; IBARRA, CRISTINA; FONTES PIAZZA, ROXANE MARIA. Human Recombinant Fab Fragment Neutralizes Shiga Toxin Type 2 Cytotoxic Effects in vitro and in vivo. TOXINS, v. 10, n. 12 DEC 2018. Web of Science Citations: 1.
LUZ, DANIELA; SHIGA, EMERSON A.; CHEN, GANG; QUINTILIO, WAGNER; ANDRADE, FERNANDA B.; MARANHAO, ANDREA Q.; CAETANO, BRUNA A.; MITSUNARI, THAIS; SILVA, MIRIAM A.; ROCHA, LETICIA B.; MORO, ANA M.; SIDHU, SACHDEV S.; PIAZZA, ROXANE M. F. Structural Changes in Stx1 Engineering Monoclonal Antibody Improves Its Functionality as Diagnostic Tool for a Rapid Latex Agglutination Test. ANTIBODIES, v. 7, n. 1 MAR 2018. Web of Science Citations: 0.

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