rMELEISH: A Novel Recombinant Multiepitope-Based Protein Applied to the Serodiagnosis of Both Canine and Human Visceral Leishmaniasis

Abstract

Background: visceral leishmaniasis (VL) is a critical public health problem in over ninety countries. The control measures adopted in Brazil have been insufficient when it comes to preventing the spread of this overlooked disease. In this context, a precise diagnosis of VL in dogs and humans could help to reduce the number of cases of this disease. Distinct studies for the diagnosis of VL have used single recombinant proteins in serological assays; however, the results have been variable, mainly in relation to the sensitivity of the antigens. In this context, the development of multiepitope-based proteins could be relevant to solving such problem.

Methods: a chimeric protein (rMELEISH) was constructed based on amino acid sequences from kinesin 39 (k39), alpha-tubulin, and heat-shock proteins HSP70 and HSP 83.1, and tested in enzyme-linked immunosorbent (ELISA) for the detection of L. infantum infection using canine (n = 140) and human (n = 145) sera samples.

Results: in the trials, rMELEISH was able to discriminate between VL cases and cross-reactive diseases and healthy samples, with sensitivity and specificity values of 100%, as compared to the use of a soluble Leishmania antigenic extract (SLA).

Conclusions: the preliminary data suggest that rMELEISH has the potential to be tested in future studies against a larger serological panel and in field conditions for the diagnosis of canine and human VL.

Keywords: dogs; humans; leishmaniasis; recombinant chimeric protein; serodiagnosis; visceral leishmaniasis.

Figure 1

SDS–PAGE analysis of the rMELEISH. (A) 12% SDS–PAGE of the accumulation of the rMELEISH post induction with 1 mM IPTG. Lane M: Unstained Protein Weight Marker Standards (Thermo Scientific, Waltham, MA, USA) Lanes 1–4; induction times of 0, 0.5, 1.5, and 2.5 (h), respectively. (B) Western blotting analysis of the crude extract after 2.5 h of induction. (C) The protein was eluted in 5 fractions using 0.5 mL elution buffer containing different concentrations of imidazole. M: Unstained Protein Weight Marker Standards (Thermo Scientific, Waltham, MA, USA), Lane 1: flow through (FT); lanes 2–4: wash; lanes 5–6: elutions with 50 mM imidazole; lanes 7–8: elutions with 100 mM imidazole, which is considered the best purification condition, and lanes 9–10: elutions with 200 mM imidazole.

Figure 2

Structural analysis of rMELEISH by fluorescence and circular dichroism. (A) Fluorescence spectra of the rMELEISH as a function of pH 4.0, 7.0, and 9.0 at 25 °C. The fluorescence emission bands were red shifted by 2 nm from 340 nm (pH 7.0) to 342 nm (pH 4.0 and 9.0). (B) Far-UV CD spectra of the rMELEISH at pH 4.0, 7.0, and 9.0 and 25 °C. The typical CD bands of the secondary structure were not observed at pH values of 4.0 and 9.0 and a very low signal appears at pH 7.0.

Figure 3

ELISA reactivity for the diagnosis of human visceral leishmaniasis. ELISA assays were performed using sera samples from visceral leishmaniasis patients (HVL; n = 35), sera from healthy subjects living in regions where the disease is endemic (HCea; n = 30); and sera from patients with Chagas Disease (CD, n = 25), leprosy (HD, n = 10), tuberculosis (TB, n = 10), malaria (MAL, n = 10), and HIV (HIV; n = 25). ROC curves were constructed with the individual OD values for each serum sample against rMELEISH (panel A) or L. infantum SLA (panel B), and the data are shown. The dotted lines represent the cut-off value obtained by the ROC curves, which were used to obtain the sensitivity, specificity, and AUC of the antigens. The mean of each group is also shown.

Figure 4

ELISA assays using the antigens for the diagnosis of canine visceral leishmaniasis. ELISA experiments were performed using sera from asymptomatic (CVLa; n = 20) and symptomatic (CVLs; n = 25) visceral leishmaniasis dogs, sera from healthy dogs living in endemic (HCea; n = 30) or non-endemic (HCnea; n = 20) areas, or those immunized with the Leish-Tec^® vaccine (n = 20); samples were additionally taken from animals infected with Ehrlichia canis (Ec) n = 15) or Babesia canis (Bc) n = 10). ROC curves were constructed with the individual OD values for each serum sample against rMELEISH (panel A) or L. infantum SLA (panel B), and the data are shown. The dotted lines represent the cut-off value obtained by the ROC curves, which were used to obtain the sensitivity, specificity, and AUC of the antigens. The mean of each group is also shown.