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Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis

BACKGROUND: The search for diagnostic biomarkers has been profiting from a growing number of high quality sequenced genomes and freely available bioinformatic tools. These can be combined with wet lab experiments for a rational search. Improved, point-of-care diagnostic tests for visceral leishmania...

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Autores principales: Bremer Hinckel, Bruno Cesar, Marlais, Tegwen, Airs, Stephanie, Bhattacharyya, Tapan, Imamura, Hideo, Dujardin, Jean-Claude, El-Safi, Sayda, Singh, Om Prakash, Sundar, Shyam, Falconar, Andrew Keith, Andersson, Bjorn, Litvinov, Sergey, Miles, Michael A., Mertens, Pascal
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522066/
https://www.ncbi.nlm.nih.gov/pubmed/31059497
http://dx.doi.org/10.1371/journal.pntd.0007353
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author Bremer Hinckel, Bruno Cesar
Marlais, Tegwen
Airs, Stephanie
Bhattacharyya, Tapan
Imamura, Hideo
Dujardin, Jean-Claude
El-Safi, Sayda
Singh, Om Prakash
Sundar, Shyam
Falconar, Andrew Keith
Andersson, Bjorn
Litvinov, Sergey
Miles, Michael A.
Mertens, Pascal
author_facet Bremer Hinckel, Bruno Cesar
Marlais, Tegwen
Airs, Stephanie
Bhattacharyya, Tapan
Imamura, Hideo
Dujardin, Jean-Claude
El-Safi, Sayda
Singh, Om Prakash
Sundar, Shyam
Falconar, Andrew Keith
Andersson, Bjorn
Litvinov, Sergey
Miles, Michael A.
Mertens, Pascal
author_sort Bremer Hinckel, Bruno Cesar
collection PubMed
description BACKGROUND: The search for diagnostic biomarkers has been profiting from a growing number of high quality sequenced genomes and freely available bioinformatic tools. These can be combined with wet lab experiments for a rational search. Improved, point-of-care diagnostic tests for visceral leishmaniasis (VL), early case detection and surveillance are required. Previous investigations demonstrated the potential of IgG1 as a biomarker for monitoring clinical status in rapid diagnostic tests (RDTs), although using a crude lysate antigen (CLA) as capturing antigen. Replacing the CLA by specific antigens would lead to more robust RDTs. METHODOLOGY: Immunoblots revealed L. donovani protein bands detected by IgG1 from VL patients. Upon confident identification of these antigens by mass spectrometry (MS), we searched for evidence of constitutive protein expression and presence of antigenic domains or high accessibility to B-cells. Selected candidates had their linear epitopes mapped with in silico algorithms. Multiple high-scoring predicted epitopes from the shortlisted proteins were screened in peptide arrays. The most promising candidate was tested in RDT prototypes using VL and nonendemic healthy control (NEHC) patient sera. RESULTS: Over 90% of the proteins identified from the immunoblots did not satisfy the selection criteria and were excluded from the downstream epitope mapping. Screening of predicted epitope peptides from the shortlisted proteins identified the most reactive, for which the sensitivity for IgG1 was 84% (95% CI 60—97%) with Sudanese VL sera on RDT prototypes. None of the sera from NEHCs were positive. CONCLUSION: We employed in silico searches to reduce drastically the output of wet lab experiments, focusing on promising candidates containing selected protein features. By predicting epitopes in silico we screened a large number of peptides using arrays, identifying the most promising one, for which IgG1 sensitivity and specificity, with limited sample size, supported this proof of concept strategy for diagnostics discovery, which can be applied to the development of more robust IgG1 RDTs for monitoring clinical status in VL.
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spelling pubmed-65220662019-05-31 Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis Bremer Hinckel, Bruno Cesar Marlais, Tegwen Airs, Stephanie Bhattacharyya, Tapan Imamura, Hideo Dujardin, Jean-Claude El-Safi, Sayda Singh, Om Prakash Sundar, Shyam Falconar, Andrew Keith Andersson, Bjorn Litvinov, Sergey Miles, Michael A. Mertens, Pascal PLoS Negl Trop Dis Research Article BACKGROUND: The search for diagnostic biomarkers has been profiting from a growing number of high quality sequenced genomes and freely available bioinformatic tools. These can be combined with wet lab experiments for a rational search. Improved, point-of-care diagnostic tests for visceral leishmaniasis (VL), early case detection and surveillance are required. Previous investigations demonstrated the potential of IgG1 as a biomarker for monitoring clinical status in rapid diagnostic tests (RDTs), although using a crude lysate antigen (CLA) as capturing antigen. Replacing the CLA by specific antigens would lead to more robust RDTs. METHODOLOGY: Immunoblots revealed L. donovani protein bands detected by IgG1 from VL patients. Upon confident identification of these antigens by mass spectrometry (MS), we searched for evidence of constitutive protein expression and presence of antigenic domains or high accessibility to B-cells. Selected candidates had their linear epitopes mapped with in silico algorithms. Multiple high-scoring predicted epitopes from the shortlisted proteins were screened in peptide arrays. The most promising candidate was tested in RDT prototypes using VL and nonendemic healthy control (NEHC) patient sera. RESULTS: Over 90% of the proteins identified from the immunoblots did not satisfy the selection criteria and were excluded from the downstream epitope mapping. Screening of predicted epitope peptides from the shortlisted proteins identified the most reactive, for which the sensitivity for IgG1 was 84% (95% CI 60—97%) with Sudanese VL sera on RDT prototypes. None of the sera from NEHCs were positive. CONCLUSION: We employed in silico searches to reduce drastically the output of wet lab experiments, focusing on promising candidates containing selected protein features. By predicting epitopes in silico we screened a large number of peptides using arrays, identifying the most promising one, for which IgG1 sensitivity and specificity, with limited sample size, supported this proof of concept strategy for diagnostics discovery, which can be applied to the development of more robust IgG1 RDTs for monitoring clinical status in VL. Public Library of Science 2019-05-06 /pmc/articles/PMC6522066/ /pubmed/31059497 http://dx.doi.org/10.1371/journal.pntd.0007353 Text en © 2019 Bremer Hinckel et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Bremer Hinckel, Bruno Cesar
Marlais, Tegwen
Airs, Stephanie
Bhattacharyya, Tapan
Imamura, Hideo
Dujardin, Jean-Claude
El-Safi, Sayda
Singh, Om Prakash
Sundar, Shyam
Falconar, Andrew Keith
Andersson, Bjorn
Litvinov, Sergey
Miles, Michael A.
Mertens, Pascal
Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis
title Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis
title_full Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis
title_fullStr Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis
title_full_unstemmed Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis
title_short Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis
title_sort refining wet lab experiments with in silico searches: a rational quest for diagnostic peptides in visceral leishmaniasis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522066/
https://www.ncbi.nlm.nih.gov/pubmed/31059497
http://dx.doi.org/10.1371/journal.pntd.0007353
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