Epitope mapping of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) and canine leishmaniasis diagnosis using a derived synthetic bi-epitope

Abstract

BackgroundLeishmaniasis is one of the most important zoonotic diseases spread in Latin America. Since many species are involved in dog infection with different clinical manifestations, the development of specific diagnostic tests is mandatory for more accurate disease control and vaccine strategies.Methodology/Principal findingsSeventy-five 15-mer peptides covering the sequence of recombinant Leishmania donovani virulence factor A2 (recLdVFA2) protein were prepared by Spot synthesis. Membrane-bound peptides immunoreactivity with sera from dogs immunized with recLdVFA2 and with a specific anti-recLdVFA2 monoclonal antibody allowed mapping of continuous B-cell epitopes. Five epitopes corresponding to the N-terminal region of recLdVFA2 (MKIRSVRPLVVLLVC, RSVRPLVVLLVCVAA, RPLVVLLVCVAAVLA, VVLLVCVAAVLALSA and LVCVAAVLALSASAE, region 1–28) and one located within the repetitive units (PLSVGPQAVGLSVG, regions 67–81 and 122–135) were identified. A 34-mer recLdVFA2-derived bi-epitope containing the sequence MKIRSVRPLVVLLVC linked to PLSVGPQAVGLSVG by a Gly-Gly spacer was chemically synthesized in its soluble form. The synthetic bi-epitope was used as antigen to coat ELISA plates and assayed with dog sera for in vitro diagnosis of canine visceral leishmaniasis (CVL). The assay proved to be highly sensitive (98%) and specific (99%).Conclusions/SignificanceOur work suggests that synthetic peptide-based ELISA strategy may be useful for the development of a sensitive and highly specific serodiagnosis for CVL or other parasitic diseases.

Leishmania infantum Virulence Factor A2 Protein: Linear B-Cell Epitope Mapping and Identification of Three Main Linear B-Cell Epitopes in Vaccinated and Naturally Infected Dogs

“…Ninety percent (90%) of the animals in CVL group presented positive serology for VQ34 compared to 30% of the asymptomatic group. In a previous work of epitope mapping from A2 protein using the Spot-synthesis technique, Mendes et al ( 65 ) described a high-reactivity B cell epitope in the serum of infected CVL animals, located within the repeating units PLSVGPQAVGLSVG (regions 67–81 and 122–135). Interestingly, the linear B-cell epitope VQ34 predicted in silico present these same repetitive units (region 67–95).…”

Recombinant Protein Containing B-Cell Epitopes of Different Loxosceles Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits

“…The epitopes from metalloproteinase LALP-1 and HYAL LiHYAL from L. intermedia spider venom were mapped in the first part of this work, by the SPOT method. This methodological approach was found extremely useful to rapidly map continuous epitopes ( 40 – 42 ). We limited the number of epitopes used in the rMEPLox to avoid unknown folding which could hide epitopes of interest in the recombinant protein structure.…”

New antigens for the serological diagnosis of human visceral leishmaniasis identified by immunogenomic screening

“…Part of the specificity problems of the target proteins could be solved by the identification of B-cell epitopes, with a high score, and analysis of the divergence of these regions with targets present in the host and in microorganisms that have cross-reactivity with VL [27, 47]. The use of synthetic peptides as antigens in diagnosis of VL may limit cross reactivity, as described to, B-cell epitopes mapped by immunoinformatic approaches [20, 24, 51]. Here, we selected divergent epitopes in relation to proteins enconded by T .…”

“…Bioinformatic and immunoproteomic approaches has been used to discovery more sensitive and specific antigens [19, 20]. The strategy is based on in silico analysis of protein sequences to predict B-cell epitopes.…”