Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

Autor(es)

Luz, Daniela ^[1] ; Chen, Gang ; Maranhao, Andrea Q. ^{[2, 3]} ; Rocha, Leticia B. ^[1] ; Sidhu, Sachdev ; Piazza, Roxane M. F. ^[1]

Afiliação do(s) autor(es)

^[1] Inst Butantan, Lab Bacteriol, Sao Paulo - Brazil

^[2] Univ Toronto, Banting & Best Dept Med Res, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON - Canada

^[3] Univ Brasilia, Lab Imunol, Brasilia, DF - Brazil

• Tipo de documento: Artigo Científico
• Fonte: PLoS One; v. 10, n. 3 MAR 19 2015.
• Citações Web of Science: 11

Resumo

Background Stx toxin is a member of the AB(5) family of bacterial toxins: the active A subunit has N-glyco-sidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra) osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes. Methods and Findings In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA. Conclusion In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro. (AU)

• Processo FAPESP: 11/12928-2 - Novo desafio para o diagnóstico de Escherichia coli diarreiogênica: obtenção de anticorpos recombinantes contra diferentes fatores de virulência
• Beneficiário: Roxane Maria Fontes Piazza
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 13/03160-9 - Novas estratégias para obtenção de anticorpos recombinantes contra as toxinas Stx1 e Stx2 de Escherichia coli produtora da toxina de Shiga
• Beneficiário: Daniela Luz Hessel da Cunha
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Doutorado
• Processo FAPESP: 10/20148-4 - Anticorpos recombinantes (scFv) contra a proteína EspB e as toxinas Stx1 e Stx2: obtenção e caracterização dessas ferramentas para o Imunodiagnóstico de Escherichia coli enteropatogênica (EPEC) e Escherichia coli produtora da Toxina de Shiga (STEC)
• Beneficiário: Daniela Luz Hessel da Cunha
• Modalidade de apoio: Bolsas no Brasil - Doutorado