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

Abstract

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.

Immunoproteomic Approach of Extracellular Antigens From Paracoccidioides Species Reveals Exclusive B-Cell Epitopes

“…Similar analyses have already been performed in other works, where linear B-cell epitopes were employed for serological detection of several pathogenic organisms, such as Borrelia miyamotoiis (Tokarz et al, 2019), Toxoplasma gondii (Gatkowska et al, 2019), Treponema pallidum (Ogden et al, 2019), Leishmania spp. (Bremer Hinckel et al, 2019;Vale et al, 2019), and several human viruses (Amrun et al, 2019;Mutsvunguma et al, 2019;Yao et al, 2019). This reinforces the importance of these analyses, demonstrating the applications of linear B-cell epitopes in the diagnosis of human pathogenic organisms.…”

A systematic review of peptide-based serological tests for the diagnosis of leishmaniasis

“…[ 82 ], in which the cases were selected from a health service or hospital. To assess the performance of peptide-based tests, three different formats were reported: one study used an ICT format [ 7 ], two studies used phage-ELISA [ 15 , 62 ] and 19 studies used an ELISA technique [ 10 , 11 , 16 , 17 , 26 , 27 , 29 , 35 , 37 , 40 – 43 , 47 , 48 , 58 , 63 , 69 , 70 ]. In these studies using an ELISA method, a marked difference was observed in the amount of peptides used, which ranged from 0.25 μg to 20 μg per well.…”

“… Reference Peptide name given by authors Reference standard Index test Peptide concentration Clinical research phase Clinical form evaluated Case origin Causative Leishmania species Bremer Hinckel, B.C. et al ., 2019 [ 7 ] EpQ11 Parasitology, serology (rK39 or rK28) ICT (cassette) ND I VL Sudan ND ICT (dipstick) ND Carmelo, E. et al ., 2002 [ 10 ] 23061 Parasitology ELISA 20 μg/mL I CL Peru ND 23063 23065 23067 23069 23071 23073 Carvalho, A.M.R.S. et al ., 2018 [ 11 ] Peptide-1 Molecular technique ELISA 10 μg/well II VL Brazil …”

“… Reference Peptide sequence Peptide name given by authors Antigen name ID UniProt (protein or gene name) or NCBI accession number of antigen Bremer Hinckel, B.C. et al ., 2019 [ 7 ] NIRIHLGDTIRIAPCK EpQ11 Transitional endoplasmic reticulum ATPase, putative LDBPK_361420 Carmelo, E. et al ., 2002 [ 10 ] MFANSSAAAVTAASNSPQRS 23061 Histone H1 AF131892 SNSPQRSPRPSPKKAAVKKA 23063 KKAAAKKAAAKKAAPKKAAP 23065 KAAPKRAAPKRAAPKKAAPK 23067 APKKAAAKRAAKKSAPKKAV 23069 APKKAVKKAVKAAKKAVKKA 23071 AVKKAAKKATKRTAKKAAKK 23073 Carvalho, A.M.R.S. et al ., 2018 [ 11 ] SGAPRANNSGDASA Peptide-1 Stabilization of polarity axis, putative LINJ_30_2730 GLSGEGSPASPEPRLAGGGGGADTQSTT Peptide-2 DGKPKENQKTARES Peptide-3 Hypothetical protein, conserved LINJ_32_0280 VADSGSASSEDGGSAKP …”

“…Their use could improve the accuracy and robustness of diagnostic tests because they are devoid of uninformative or less informative epitopes responsible for background reactions [ 38 ]. Their chemical synthesis does not involve handling living organisms and can be standardized for a high level of purity and reproducibility, allowing the production of robust ELISA or ICT [ 7 , 42 ]. Peptide phage display technology, overlapping peptide libraries covering the entire selected protein sequence or in silico B-cell epitope prediction can be used to identify specific peptides [ 28 , 61 , 67 , 73 ].…”