Kinesin motor domain of Leishmania donovani as a future vaccine candidate

Abstract

Visceral leishmaniasis (VL) is one of the important parasitic diseases, with approximately 350 million people at risk. Due to the nonavailability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. The present study was carried out to examine the immunological potential of kinesin protein from the microtubule locus of Leishmania donovani as a suitable vaccine candidate. In silico analysis of this region revealed clusters of major histocompatibility complex class I and II binding epitopes in its motor domain region. A recombinant protein was expressed from this region and named rLvacc. The antigenicity and immunogenicity studies of this protein by Western blot analysis revealed that rLvacc is strongly recognized by sera from acute VL patients. To evaluate its immunogenicity, peripheral blood mononuclear cells from cured VL patients were separated, and a lymphocyte proliferation assay was carried out in the presence of rLvacc. After lymphocyte proliferation, the pooled culture supernatant was assayed for anti-rLvacc antibody titers using an enzyme-linked immunosorbent assay. The results showed that immunoglobulin G2 (IgG2) subtype antibodies were predominant, while IgG1 subtype antibodies were produced in very low titers. On the basis of these ex vivo preliminary findings, its immunogenicity was studied in BALB/c mice. Vaccination with the DNA construct generated a good cellular immune response with significant increases in gamma interferon and interleukin-2 (IL-2) cytokine levels (Th1), but no increase in IL-4 levels (Th2). Taken together, our findings suggest the kinesin motor domain region of L. donovani as a potential vaccine candidate against visceral leishmaniasis.

FIG. 1.

Western blot analysis of rLvacc (35-kDa kinesin motor domain) in kala-azar patients. Lane M, prestained molecular mass marker; lane 1, rLacc probed with VL-positive patient serum; lane 2, rLacc probed with VL-positive patient serum; lane 3, rLvacc probed with VL-positive patient serum; lane M1, prestained molecular mass marker; lanes 2 and 3, rLacc probed with VL-negative patient serum.

FIG. 2.

Lymphoproliferative responses to rLvacc in cured and healthy kala-azar patients. The proliferative response in cured VL patients (mean OD, 0.4628 ± 0.014) was compared to nonendemic healthy controls (mean OD, 0.1046 ± 0.003).

FIG. 3.

IgG2 and IgG1 production in response to rLvacc stimulation in cured kala-azar patients. rLvacc-stimulated IgG2 production (mean OD, 1.5 ± 0.5) was compared to production in unstimulated PBMCs of the same kala-azar patients (mean OD, 0.26 ± 0.06). The IgG1 production in the induced (mean OD, 0.43 ± 0.03) and the uninduced PBMCs (mean OD, 0.39 ± 0.04) is also shown (P < 0.3).

FIG. 4.

In vitro expression of the kinesin motor domain from a DNA vaccine construct. Lane 1, purified rLacc probed with VL-positive patient serum; lane 2, in vitro-expressed kinesin motor domain probed with VL-positive patient serum; lane 3, in vitro-expressed kinesin motor domain probed with VL-positive patient serum; lane 4, prestained molecular mass marker.

FIG. 5.

Splenocyte proliferative responses (mean OD) to rLvacc and rKE16 in kinesin motor domain-vaccinated and kinesin immunodominant repeat domain-vaccinated mice. Splenocytes from mice injected with kinesin motor domain-containing DNA showed proliferative responses against the rLvacc with a mean OD of 0.4060 ± 0.013. The mean proliferative response in the empty plasmid group was 0.1275 ± 0.006. The mice injected with the kinesin immunodominant vaccine construct had proliferative responses against the rKE16 with a mean OD of 0.2442 ± 0.021.

FIG. 6.

Cytokine response to rLvacc and rKE16 stimulation in splenocytes from kinesin motor domain-vaccinated and kinesin immunodominant repeat domain-vaccinated mice. The rLvacc-stimulated splenocytes showed low IFN-γ, IL-2, and IL-4 levels (200.8 ± 12.14, 118.0 ± 3.95, and 63.83 ± 7.12 pg/ml, respectively) in the control empty plasmid group, whereas the kinesin motor domain-vaccinated mice had higher levels of IFN-γ and IL-2 (1,695 ± 52.52 and 413.3 ± 12.89 pg/ml, respectively) but lower levels of IL-4 (57.67 ± 3.69 pg/ml). The splenocytes from the kinesin immunodominant-vaccinated group showed a mixed profile with IFN-γ, IL-2, and IL-4 levels of 419.2 ± 41.48, 179.2 ± 12.94, and 285.5 ± 12.44 pg/ml, respectively.

FIG. 7.

Cytokine response to whole-cell lysate of L. donovani (KE-16) in splenocytes from the kinesin motor domain-vaccinated group. The mice had higher levels of IFN-γ and IL-2 (1,134 ± 92.40 and 406.7 ± 30.29 pg/ml) but lower levels of IL-4 (60.50 ± 2.51 pg/ml).