Immunoreactivity of LAg and SLA with Leishmania infected human serum samples.
Leishmania promastigote membrane antigen (LAg) and soluble leishmanial antigen (SLA) were isolated from L. donovani promastigote culture and subjected to 10% polyacrylamide gel electrophoresis. LAg and SLA were resolved in their components of different molecular masses through SDS PAGE and share many immunogenic proteins. LAg contains approximately 25-30 polypeptides whereas SLA is a mixture of about ten dominant polypeptides (Fig. 1).
One of the most challenging objectives for Leishmania-specific antibody-based serodiagnosis is to differentiate an active VL patient from the past infection. In this regard reactivity of LAg and SLA with the active VL, one month cured and six months follow-up VL cases were evaluated through ELISA. We observed that there was comparatively higher antibody titer in the active VL sera against LAg in comparison to other study groups. However, cured VL patients just after the treatment show a significant decrease in the antibody titer but 84.16% positivity in ELISA (Fig. 2A). Follow-up patient sera after six months of treatment showed a remarkable decrease in the antibody titer with no positive reactivity against LAg and the mean was below the cut off line. No cross reaction of LAg was found with other diseases and healthy controls. Similar study was also performed with SLA. Both active and one month cured VL patients showed 100% positivity with SLA (Fig. 2B). However, 7.69% of the follow-up patients were still positive with SLA. Healthy controls and other diseases, however, did not show cross reactivity with SLA. These results reveal that LAg and SLA have strong serodiagnostic potential for VL. Both LAg and SLA could not satisfactorily differentiate active VL from past infections following one month post therapy. However, after six months of infection, antibody titers significantly decreased below the cut off and become 100% negative for LAg. Therefore in the next step, we isolated different components of LAg for immunoreactive studies of individual antigens.
Electroelution of LAg antigens and its reactivity in ELISA.
Several studies have been undertaken for the serodiagnosis of VL using whole cell lysate, total membrane antigens and other recombinant proteins from Leishmania. However, very few attempts have been made to distinguish between active diseases from past infections of VL. Therefore we took the initiative to use the immunoreactive potential of different membrane fractions in a simpler and effective approach through ELISA. In this course, nine polypeptides of LAg, 31, 34, 36, 45, 51, 63, 72, 91, and 97 kDa were eluted out electrophoretically from Coomassie stained gels and subjected to SDS-PAGE (Fig. 3). To differentiate active disease from cured VL and follow-up patients ELISA were carried out with all the nine electroeluted antigens.
Diagnostic and prognostic value of electroeluted proteins.
Nine electroeluted antigens were evaluated for their ability to distinguish active disease from other diseases and healthy controls as well as from the cured VL and follow-up samples. Cut off values were set for each eluted antigen where antibody titer above the cut off line was considered as positive and below as negative in ELISA. Cut offs for VL positivity were obtained from ROC curve where maximum sensitivity and specificity were achieved in comparison with healthy controls. Out of the nine antigens, 100% sensitivity with active VL sera was observed with all electroeluted antigens. The specificity of the antigens was calculated based on the cross reactivity of the antigens with healthy control and other diseases. With healthy control samples tested, the mean optical density values for all the electroeluted antigens were below the cut off line. 31, 34, 36, 51, and 97 kDa proteins found to be 100% specific whereas antigens 63 and 91 kDa showed more than 90% specificity. The reactivity of eluted antigens with other diseases showed 100% specificity with 34 and 51 kDa antigens followed by 91 and 97 kDa with 95.75% specificity. Therefore, for the purpose of diagnosis antigens 34 and 51 kDa proteins we found to be best to distinguish active VL from healthy controls and other diseases. The percent sensitivity and specificity of all the electroeluted antigens are listed in Table 1.
The study to differentiate active VL from cured and past infections using electroeluted antigens demonstrated considerable differences among the patients’ groups (Fig. 4). There was a statistically considerable decline in the serum IgG levels against all the antigens in the one month cured patient sera compared to their respective active disease sera. However, except 63, 72 and 91kDa none of the antigens showed mean optical density values below its cut off. Recognition of electroeluted antigens with sera after one month cure ranged from 8.69-100% which is still positive and not ideal for the differentiation of the diseased state from cure. Follow-up patients who recovered from VL six months post treatment showed a significant decline in the IgG levels against 31, 34, 51, 63, 72, and 91kDa antigens and their levels were comparable to the healthy control sera the mean lying below the cut off. Amongst all the antigens, 72 and 91 kDa showed the most promising results with 100% negative reactivity in follow-up patients.
Evaluation of electroeluted proteins for in vitro cytokine analysis with PBMCs of cured VL patients.
Immunity to leishmaniasis is known to depend on protective cellular responses against the parasites. In this regard antigen-induced cytokine production from the culture supernatants from PBMCs of cured VL individuals were evaluated who are known to present positive CMI. In order to assess the immunogenicity of eluted proteins, 31, 34, 51, 63, 72 and 91 kDa were used to stimulate the PBMCs of cured VL patients for the production of protective cytokines IFN-g and IL-12, and anti-inflammatory cytokines, IL-10 and TGF-β, which promote disease progression. IFN-g is the most dominant Th1 cytokine required to control Leishmania infection. Analysis of cytokines revealed variable levels of IFN-g production when PBMCs were stimulated with electroeluted antigens whereas unstimulated cultures produced negligible IFN-g (Fig. 5A). Prominent levels of IFN-g were produced by the LAg as a whole. Significantly high levels of IFN-g were produced by 51 and 63 kDa antigens followed by 31 and 72 kDa antigens when compared with unstimulated cultures. In comparison 34 and 91 kDa antigen produced low levels of IFN-g. IL-12 is another protective cytokine produced by the macrophages and in turn, activates the T cells to produce IFN-g and accelerates the leishmanicidal function. Similar to the LAg stimulated cultures, significantly elevated levels of IL-12 were produced by 51and 63 kDa antigens followed by 31 and 72 kDa (Fig. 5B). 34 and 91 kDa antigens produced comparatively lower levels of IL-12.
IL-10 and TGF-β are two immunosuppressive cytokines that promote parasite survival and help in disease progression in VL infection. Our study demonstrated that LAg stimulated PBMCs produced moderate levels of IL-10. 34 and 91 kDa antigens showed maximum levels of IL-10. In contrast, comparatively low levels of IL-10 were produced by 31, 51, 63, and 72 kDa electroeluted antigens (Fig. 5C). Low levels of TGF-β were produced by the unstimulated cultures, whereas, 31, 51, 63 and 72 kDa antigens produced median levels of TGF-β (Fig. 5D). Again, 34 and 91 kDa antigens produced highest levels of TGF-β similar to that produced by LAg stimulated cultures (Fig. 5D).
Identification of electroeluted proteins
Out of nine fractions of LAg evaluated in this study, except 34 and 45 kDa all proteins were identified in our earlier reports (Table 2). Therefore, we identified the remaining two proteins through MALDI-TOF mass spectrometry and matched with Leishmania sequence. The 34 kDa protein was identified as Leishmania analogue of the receptors of activated C kinase (LACK) whereas 45 kDa protein was β tubulin (Supplementary Fig. 1).