Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis
| Autor: | Tegwen Marlais, Shyam Sundar, Bruno C Bremer Hinckel, Pascal Mertens, Andrew K. I. Falconar, Sayda El-Safi, Tapan Bhattacharyya, Jean-Claude Dujardin, Hideo Imamura, Sergey V. Litvinov, Stephanie Airs, Michael A. Miles, Björn Andersson, Om Prakash Singh |
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| Přispěvatelé: | Clinical sciences, Medical Genetics, Department of Bio-engineering Sciences |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Leishmania Donovani Life Cycles B Cells Physiology RC955-962 Antibodies Protozoan Protozoology Biochemistry Epitope White Blood Cells 0302 clinical medicine Antibodies Protozoan/analysis Leishmania donovani/genetics Animal Cells Arctic medicine. Tropical medicine Zoonoses Immune Physiology Medicine and Health Sciences Antigens Protozoan/analysis Leishmaniasis Protozoans Leishmania Immune System Proteins biology Leishmaniasis Visceral/diagnosis Eukaryota Genomics Peptides/analysis 3. Good health Infectious Diseases Biomarker (medicine) Leishmaniasis Visceral Engineering and Technology Protozoan Life Cycles Diagnostic Tests Routine/methods Public aspects of medicine RA1-1270 Cellular Types Research Article Neglected Tropical Diseases In silico Immune Cells 030231 tropical medicine Immunology Leishmania donovani Antigens Protozoan Computational biology Microbiology Sensitivity and Specificity 03 medical and health sciences Immunoglobulin G/analysis Antigen medicine Parasitic Diseases Genetics Prototypes Humans Computer Simulation Antigens Antibody-Producing Cells Comparative genomics Protozoan Infections Blood Cells Diagnostic Tests Routine Promastigotes Public Health Environmental and Occupational Health Organisms Biology and Life Sciences Proteins Computational Biology Cell Biology Comparative Genomics biology.organism_classification medicine.disease Tropical Diseases Parasitic Protozoans 030104 developmental biology Epitope mapping Visceral leishmaniasis Technology Development Immunoglobulin G Human medicine Peptides Biomarkers Developmental Biology |
| Zdroj: | PLoS neglected tropical diseases PLoS Neglected Tropical Diseases PLoS Neglected Tropical Diseases, Vol 13, Iss 5, p e0007353 (2019) |
| ISSN: | 1935-2727 |
| Popis: | 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. Author summary Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan parasites of the Leishmania donovani complex. Without treatment, VL is fatal. Although diagnostic techniques, mainly based on the detection of anti-Leishmania antibodies are available, invasive procedures such as microscopy from spleen or bone marrow aspirates are still required for the diagnosis of seronegative VL suspects, for the detection of recurrent cases and to confirm cure after successful treatment. Previous investigations showed the potential of IgG1 as a biomarker of post-chemotherapeutic relapse for VL in rapid diagnostic tests (RDTs) sensitised with crude lysate antigen (CLA). Here we employed in silico tools to search for desired protein features in a large number of L. donovani antigens detected by human IgG1 in western blots. We then employed prediction algorithms to profile epitopes from the shortlisted proteins. We screened a panel of high-scoring peptides in a high-throughput manner using arrays, with low reagent consumption. The most reactive peptide was adapted to RDTs, showing promising results of both sensitivity and specificity. This peptide has the potential of replacing the CLAs in IgG1 RDTs. Thus we believe that in silico tools can be used to optimise wet lab experiments for a rational search of biomarkers. |
| Databáze: | OpenAIRE |
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