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

Abstract

Background: Visceral leishmaniasis (VL), is a parasitic disease that causes serious medical consequences if treatment is delayed. Despite a decline in the number of VL cases in the Indian subcontinent, the commencement of the disease in newer areas continues to be a major concern. Although serological diagnosis mainly by immunochromatographic tests has been found to be effective, a test of cure in different phases of treatment is still desired. Even though a good prophylactic response has been obtained in murine models by a number of vaccine candidates, few have been proposed for human use.

Methods: In this study, nine antigenic components (31, 34, 36, 45, 51, 63, 72, 91 and 97 kDa) of Leishmania promastigote membrane antigens (LAg), were electroeluted and evaluated through ELISA to diagnose and distinguish active VL from one month cured and six months post-treatment patients. Further, to investigate the immunogenicity of electroeluted proteins, human PBMCs of cured VL patients were stimulated with 31, 34, 51, 63, 72 and 91 kDa proteins.

Results: We found that 34 and 51 kDa proteins show 100% sensitivity and specificity with healthy controls and other diseases. After six months post-treatment, antibodies to 72 and 91 kDa antigens show a significant decline to almost normal levels. This suggests that 34 and 51 kDa proteins are efficient in diagnosis, whereas 72 and 91 kDa proteins may be used to monitor treatment outcome. In another assay, 51 and 63 kDa proteins demonstrated maximum ability to upregulate IFN-γ and IL-12 with minimum induction of IL-10 and TGF-β. The results indicating that 51 and 63 kDa proteins could be strong candidates for human immunization against VL. In contrast, 34 and 91 kDa proteins demonstrated a reverse profile and may not be a good vaccine candidate.

Conclusions: The preliminary data obtained in this study proposes the potential of some of the antigens in Leishmania diagnosis and for test of cure. Additionally, some antigens demonstrated good immunoprophylactic cytokine production through T cell-mediated immune response, suggesting future vaccine candidates for VL. However, further studies are necessary to explore these antigens in diagnosis and to access the long-term immune response.

Keywords: Biochemistry; Cytokines; Diagnosis; Immunology; Leishmaniasis; Parasitology; Th1/Th2; Vaccination.

Fig. 1

SDS-PAGE of purified proteins. Antigens, SLA and LAg were separated using SDS-PAGE and stained with Coomassie Blue to observe the individual proteins of each antigen. Lane M1: protein molecular weight markers; Lane L1: SLA; Lane L2: LAg

Fig. 2

Indirect ELISA using purified antigens LAg and SLA. Optical density values were obtained by ELISA for the detection of serum antibodies against antigens LAg (a) and SLA (b). Paired serum samples (n = 13) from confirmed active VL cases before the treatment (AVL), cured VL one month after treatment (CVL), and six-month follow-ups (FU) were investigated against purified leishmanial antigens, LAg and SLA. Serum samples from 13 healthy individuals (HC) and 13 from other diseases were also tested. The cut-off values were selected from the ROC curve where 100% sensitivity and specificity were achieved. Each point represents an average of triplicate values obtained from a single sample

Fig. 3

SDS-PAGE of electroeluted proteins of LAg. Electroeluted antigens 31, 34, 36, 45, 51, 63, 72, 91 and 97 kDa were run on 10% SDS-PAGE. Lane L1: marker; Lane L2: 31 kDa; Lane L3: 34 kDa; Lane L4: 36 kDa; Lane L5: 45 kDa; Lane L6: 51 kDa; Lane L7: 63 kDa; Lane L8: 72 kDa; Lane L9: 91 kDa; Lane L10: 97 kDa. The figure depicted here was obtained from two separate gels

Fig. 4

Indirect ELISA using electroeluted antigens. IgG reactivity of eluted antigens 31 (a), 34 (b), 36 (c), 45 (d), 51 (e), 63 (f), 72 (g), 91 (h) and 97 kDa (i) with serum samples from 23 VL patients at three different time points that is active VL before the treatment (AVL), after one month cured VL (CVL), and after six months follow-up cases (FU). The study also included sera from symptomatically similar other diseases (OD; n = 23) and healthy controls (HC; n = 23). The horizontal lines denote the mean value for each group. The dotted lines denote the cut-off values selected from the ROC curve where maximum sensitivities and specificities were achieved. Each point represents an average of triplicate values obtained from a single sample

Fig. 5

Sandwich ELISA for the cytokine assay. In vitro cytokine production is shown by the PBMCs of VL-treated and healthy individuals in ELISA upon stimulation with LAg and by the PBMCs of treated VL patients with electroeluted 31, 34, 51, 63, 72 and 91 kDa proteins for 96 h, (n = 6). Unstimulated PBMCs were included here as control. Cytokines were measured by ELISA for IFN-γ production (a), IL-12 production (b), IL-10 production (c), TGF-β production (d), IFN-γ/IL-10 ratio (e) and IFN-γ/ TGF-β ratio (f). Unpaired Studentʼs t-tests were used to analyse the data