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

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.

A candidate vaccine for human visceral leishmaniasis based on a specific T cell epitope-containing chimeric protein protects mice against Leishmania infantum infection

“…ChimeraT induced lymphoproliferation in PBMC cultures from VL patients after treatment and from healthy subjects, in addition to stimulating IFN-γ production. In this context, ChimeraT could be considered as a promising candidate for future translational studies in humans, due to the induction of markers of immunogenicity from cells isolated from non-exposed healthy individuals, as observed in previous studies with other candidate vaccine antigens [58][59][60][61] .…”

Liposomal Formulation of ChimeraT, a Multiple T-Cell Epitope-Containing Recombinant Protein, Is a Candidate Vaccine for Human Visceral Leishmaniasis

“…Initially, spleens were collected from BALB/c mice that had been vaccinated with ChimeraT/liposome, ChimeraT/saponin, ChimeraT/saline, saline, saponin, or empty liposome but were not infected with L. infantum . Spleen cells were cultured and stimulated with ChimeraT and SLA, in order to examine the specificity of the cytokine response to the vaccinating antigen and to the parasite protein extract, respectively [ 58 , 59 , 60 , 61 , 62 , 63 ]. Spleen cell supernatants were collected, and the production of selected Th1 and Th2-type cytokines was evaluated as markers of the cellular immune response and as an indicator of the potential efficacy of the immunogen against infection by the parasites [ 64 , 65 , 66 ].…”

An immunoproteomics approach to identifyLeishmania infantumproteins to be applied for the diagnosis of visceral leishmaniasis and human immunodeficiency virus co-infection

“…On the basis of the identified proteins, some of them were already showed to be antigenic in the VL, such as tubulins and heat shock proteins; which are parasite housekeeping proteins. These antigens are known to induce cellular and/or humoral response in the course of Leishmania infection in mammalian hosts, and they have shown a diagnostic value for VL (Menezes-Souza et al ., 2014; Costa et al ., 2019; Humbert et al ., 2019). In this context, to partially validate our immunoproteomics findings, we selected the β -tubulin protein to perform ELISA experiments against our serological panel composed by VL, HIV, VL/HIV, Chagas disease, leprosy, tuberculosis and malaria patients sera; as well as healthy subjects samples.…”