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Int. J. Mol. Sci. 2019, 20(8), 1812; https://doi.org/10.3390/ijms20081812

Leishmania infantum β-Tubulin Identified by Reverse Engineering Technology through Phage Display Applied as Theranostic Marker for Human Visceral Leishmaniasis

1
Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte 30130-100, Minas Gerais, Brazil
2
Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Uberlândia 38405-320, Minas Gerais, Brazil
3
Departamento de Patologia Clínica, COLTEC, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Minas Gerais, Brazil
4
Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria
5
Department of Medical Microbiology and Immunology, University of California-Davis, Davis, CA 95616, USA
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Received: 24 February 2019 / Revised: 30 March 2019 / Accepted: 4 April 2019 / Published: 12 April 2019
(This article belongs to the Section Molecular Biology)
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Abstract

Two Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the β-tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN-γ and lower IL-10 production was found. View Full-Text
Keywords: phage display; β-tubulin protein; Leishmania infantum; scFv antibody; diagnosis; vaccine phage display; β-tubulin protein; Leishmania infantum; scFv antibody; diagnosis; vaccine
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Costa, L.E.; Alves, P.T.; Carneiro, A.P.; Dias, A.C.S.; Fujimura, P.T.; Araujo, G.R.; Tavares, G.S.V.; Ramos, F.F.; Duarte, M.C.; Menezes-Souza, D.; Briza, P.; Briza, F.F.; Coelho, E.A.F.; Goulart, L.R. Leishmania infantum β-Tubulin Identified by Reverse Engineering Technology through Phage Display Applied as Theranostic Marker for Human Visceral Leishmaniasis. Int. J. Mol. Sci. 2019, 20, 1812.

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