Immunoproteomic Identification and Characterization of Leishmania Membrane Proteins as Non-Invasive Diagnostic Candidates for Clinical Visceral Leishmaniasis

Abstract

Visceral leishmaniasis (VL), a potentially fatal disease is an outcome of infection caused by the parasite Leishmania donovani. The clinical diagnostic tests for this disease are still related to invasive tissue aspiration or serological immunochromatography. Advancements in immunoproteomics such as two-dimensional gel electrophoresis, mass spectrometry, B cell epitope prediction, and peptide synthesis have enabled researchers to discover newer biomarkers for disease diagnosis. In this study, we have screened several urine-reactive leishmanial membrane proteins as potential biomarker candidates. In the immunoblot assay, three proteins 51, 55 and 63 kDa showed 100% reactivity to the urine of 47 VL patients and nonreactive to 18 healthy and other diseases. Mass spectrometry revealed the identity of 51, 55 and 63 kDa proteins as elongation factor 1α (EF1-α), α-tubulin, and glycoprotein 63, respectively. B cell reactive epitopes of these proteins were mapped through bioinformatic tools and one epitope from each protein that had the highest score were synthesized. All the three native electroeluted proteins and their corresponding synthetic peptides were tested through ELISA for reactivity with VL and control urine samples. While all three demonstrated good reactivity, the diagnostic performance of EF1-α was the best. Our findings illustrate the use of urine-based proteomic approach for biomarker discovery in non-invasive clinical diagnosis of VL.

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

“…In addition, the epitope topology in the protein structure is important. To check which epitopes were hydrophilic and can be present in the protein surface, we used ProtScale software, as performed by Ejazi et al (2018). Thus, we selected only the hydrophilic, non-homologous to other related organisms and exclusive epitopes of the Paracoccidioides complex ( Table 2).…”

Investigation of the antigenicity and protective efficacy of Leishmania promastigote membrane antigens in search of potential diagnostic and vaccine candidates against visceral leishmaniasis

“…In the present study, two purified leishmanial antigens, LAg and SLA, together with nine electroeluted proteins of LAg were evaluated for serodiagnosis of VL as well as to differentiate active VL from past infection. LAg has been earlier reported to demonstrate strong potential for the diagnosis of VL by detecting antigen-specific antibodies in serum and urine samples [10,24]. SLA has also been reported in vaccine-mediated protection in murine VL [22,25].…”

Proteomics and Leishmaniasis: Potential Clinical Applications

“…Urine of healthy subjects and of patients with malaria, tuberculosis, viral infections and typhoid fever were employed as controls. Samples analyzed 6 months after treatment did not recognize the three antigens . These results suggest that ELISA may be employed as a specific and sensitive tool for detection of urine antibodies against Leishmania in patients with VL …”

“…Apart from serum, urine may also be used as a source for antibodies for diagnostic tests. Antibodies present in the urine of 47 patients infected with L. donovani selectively recognized Leishmania proteins such as GP63, α‐tubulin and EF‐1α . Urine of healthy subjects and of patients with malaria, tuberculosis, viral infections and typhoid fever were employed as controls.…”