Kinesin Motor Domain of Leishmania donovani as a Future Vaccine Candidate

In silico analysis of the kinesin peptide sequence.To determine the immunogenic domains in the kinesin protein of L. donovani, we carried out in silico analysis of the kinesin peptide sequence using the two promiscuous major histocompatibility complex (MHC) class I and II binding peptide prediction servers (available at http://www.imtech.res.in/raghava and http://www.jenner.ac.uk/MHCpred ) (27). These servers predict the specific epitopes by using a neural network and physiochemical properties of the epitopes. Our analysis revealed clusters of MHC-I and -II binding epitopes with high affinity for cytotoxic and helper T cells in the kinesin motor domain region. Further amino acid sequence analysis revealed the kinesin motor domain region in the Leishmania major, Leishmania infantum, and Leishmania braziliensis proteome also. The kinesin motor domain of Leishmania donovani KE-16 (AAT40474) shares 94% homology with L. major strain Friedlin (CAJO3170), 83% homology with L. infantum JPCM5 (CAM66680), and 77% homology with L. braziliensis MHOM/BR/75/M2904 (CAM37580). Moreover, we were able to identify MHC binding epitopic regions in kinesin motor domain of these strains also. There is homology in the epitopic regions identified in the L. major strain Friedlin amino acid sequence. However, there is much heterogeneity in the epitopic regions identified in L. infantum JPCM5 and L. braziliensis MHOM/BR/75/M2904. This analysis reveals that the kinesin motor domain may be a promising leishmania vaccine candidate.

On the basis of these in silico analyses, primers capable of amplifying the open reading frame of the kinesin motor domain (bp 361 to 1350) of Leishmania donovani were designed with sequences for the sense primer 5′-CCA ATG CAT TGG ATG CAC CCT TCC ACT GTG CGG-3′ and antisense primer 5′-GGG CCC GTC GAT CAC GCG CCC GAG CGT CGT-3′. Using these primers, the kinesin motor domain region was amplified from the KE-16 strain of Leishmania donovani (GenBank accession no. AY615886). The amplified product was isolated, purified, and cloned in the pGEM-T vector, and later it was sequenced (Microsynth, Switzerland) to verify the insertion.

Expression and Western blot analysis of rLvacc using VL patient serum.rLvacc was expressed as an N-terminal histidine-tagged (six-His) protein in the pRSET-C vector (Invitrogen, San Diego, CA) and purified twice to homogeneity by affinity chromatography on a Ni-nitrilotriacetic acid (NTA) resin column (Qiagen, Chatsworth, CA), following a standard protocol as described earlier (28). Briefly, Escherichia coli BL21(DE3) logarithmic-phase cells were transformed with the pRSET-C plasmid and induced for 4 h with 1 mM isopropyl-β-d-thiogalactoside. Following induction, the bacterial cells were lysed and the recombinant proteins were purified using a Ni-NTA agarose column (Qiagen) under native conditions following the manufacturer's protocol. The lysate was centrifuged at 10,000 × g for 30 min at 4°C to sediment the cellular debris. The supernatant was aspirated, and an aliquot of 50 μl was stored at −20°C for records and controls. One milliliter of 50% Ni-NTA slurry was added to 4 ml of cleared lysate and mixed gently by shaking (200 rpm on a rotary shaker) at 4°C for 60 min. The lysate-Ni-NTA mixture was then loaded onto a column with the bottom outlet capped. The flowthrough was collected after removing the bottom cap, the column was washed twice with 4 ml of wash buffer, and the fractions were collected for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The protein was eluted four times with 0.5 ml of imidazole buffer, and the eluate was collected in four tubes. The optimal imidazole concentration for protein elution was determined by eluting initially with increasing concentration of imidazole viz., 40, 80, 160, and 240 mM, and analyzing the fractions by 12% SDS-PAGE. The recombinant proteins were optimally eluted at a 160 mM imidazole concentration. The eluted protein was then finally concentrated, and small protein contaminants (<10 kDa) were removed by size exclusion centrifugation (Amicon, Beverly, MA). rLvacc was subsequently purified, and possible lipopolysaccharide contamination was also removed with a polymyxin B-agarose column (Qiagen, Chatsworth, CA). The molecular mass of the 361-amino-acid-long rLvacc peptide was estimated to be 35 kDa by molecular mass prediction software (Protein calculator v3.2; http://www.scripps.edu/∼cdputnam/protcalc.html ).

In order to check the presence of antibodies against rLvacc in VL patient serum, Western blot analysis was carried out. The purified rLvacc protein was resolved on 12% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, MA) using a semidry blotting apparatus (Bio-Rad) following the manufacturer's protocol. The membrane was blocked with 5% skimmed milk, washed thrice with phosphate-buffered saline (PBS) with 0.1% Tween 20, pH 7.0 (PBS-T) for 10 min each, and incubated thereafter with patient serum samples (1:50 dilution in PBS, pH 7.0, 1% bovine serum albumin [BSA]) for 1 h at room temperature. The membrane was washed thrice with PBS-T and incubated for 1 h at room temperature with a 1:1,000 dilution of biotin-conjugated anti-human immunoglobulin G (IgG; in PBS, pH 7.0, 1% BSA). The membrane was incubated with secondary antibody, washed, and incubated with primary substrate avidine-conjugated to horseradish peroxidase. After washing thrice for 10 min each with PBS, pH 7.0, 0.1% Tween 20, the blots were developed using diaminobenzidine (DAB; Amresco, OH) and 0.1% H₂O₂ as substrate, and bands were visualized by the naked eye.

Induction study and cell proliferation assay.After institutional ethical committee approval, 10 serologically and parasitologically confirmed and patients successfully treated for kala-azar (all male, with ages ranging from 19 to 48 years [29 ± 8 years, mean ± standard deviation]) were included in the study. Five healthy controls (24 to 45 years old [31 ± 8]) from nonendemic regions were also included. All kala-azar patients hailed from endemic rural areas of Bihar, Eastern India. From these patients, 10 ml heparinized blood was collected in sterile tubes and peripheral blood mononuclear cells (PBMCs) were separated using density gradient centrifugation over Histopaque 1077 (Sigma-Aldrich, St. Louis, MO). PBMCs from five nonendemic healthy controls were also separated using the same protocol. Mononuclear cells were resuspended in RPMI 1640 (Hi-Media, India) supplemented with 25 mM HEPES, 0.2% sodium bicarbonate, and 10% fetal calf serum. To the medium, 100 units each of penicillin and streptomycin dissolved in 100 μl of sterile PBS were added. These cells (2 × 10⁵) were distributed in triplicate in 96-well round-bottom microtiter plates (TPP, Switzerland) in a final volume of 200 μl. Cultures were stimulated either with rLvacc (5 μg/well) or concanavalin A (1 μg/well) for 72 h, and cell viability was assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Aldrich, St. Louis, MO) as described elsewhere, with minor modifications (22). In brief, MTT solution (10 μl, 5 mg/ml) was added to 200 μl of medium and incubated for 3 h at 37°C. After incubation the culture medium was removed and dark blue formazan crystals were dissolved in acidic isopropanol (100 μl of 0.04 N HCl in isopropanol). Color formation was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (Biotek Instruments). Wells with concanavalin A served as positive controls, while wells without any stimulation served as negative controls.

Immune response against the expressed protein.The immunogenicity of rLvacc was measured by ELISA as described earlier (12). In brief, a 96-well ELISA plate (Dynatech) was coated with rLvacc at a concentration of 100 ng/well overnight in bicarbonate buffer (pH 9.2) at 4°C, and the next morning it was washed three times with the washing buffer PBS-T (PBS with Tween 20, pH 7.2) in an automated ELISA plate washer (Atlantis, Austria). The wells were blocked with 1% BSA in PBS (pH 7.2) and incubated for 1 h at room temperature followed by washing the plate again three times with PBS-T. The PBMC culture supernatants were aspirated and diluted 1:50 in PBS, and 200 μl of diluted supernatant was added to each antigen-coated well and incubated at 37°C for 1 hour. After 1 h, the plate was again washed in the automated ELISA plate washer, followed by addition of 100 μl of anti-human IgG1- and IgG2-biotin conjugate (Sigma-Aldrich, St. Louis, MO) in each well (including control wells), and incubated for 60 min, followed by washing; 100 μl of avidin-horseradish peroxidase (HRP) conjugate was added to each well, and the plate was incubated for 30 min at room temperature. Finally, the plate was again washed three times under the same conditions as mentioned above, followed by addition of 50 μl of chromogenic substrate to each well. The reaction was stopped after 0.5 hour by addition of 1 N H₂SO₄ solution, and absorbance was measured at 450 nm using an ELISA plate reader (Biotek Instruments). Each test was performed with appropriate negative and positive controls. The mean optical density (OD) values of nonendemic healthy controls + 2 standard deviations were taken as the cutoff.

Generation of the kinesin motor domain DNA vaccine construct.The DNA vaccine expressing the kinesin motor domain of Leishmania donovani was prepared by restriction digestion of the kinesin motor domain gene (993 bp) from a pGEM-T vector using BstX1 and Apa1 restriction enzymes and cloned into the mammalian expression vector pVAX-1 (Invitrogen). This vector is a shuttle vector that can be screened easily using Escherichia coli, and it also has a cytomegalovirus promoter for high expression levels in a wide range of mammalian cells, with a kanamycin resistance gene as a selection marker.

For generating the DNA vaccine construct, the fragment was digested, purified, and ligated cohesively in the vector by using T4 DNA ligase. After ligation, the recombination-deficient and endonuclease-deficient E. coli DH5α strain was transformed using a pVAX1-kinesin motor domain construct. All the recombinant molecules generated were screened using antibiotic pressure selection. Finally, the plasmid DNA was purified from the recombinant clones. Apart from the kinesin motor domain construct, a DNA vaccine expressing a 468-bp kinesin immunodominant repeat construct was also generated and included as control.

In vitro expression analysis of clones.The ability of the recombinant construct to express the kinesin motor domain was studied in vitro using a transcription and translation (TNT)-coupled system (Promega) according to the manufacturer's instruction. TNT uses a coupled transcription/translation reaction for in vitro protein synthesis. Transcription and translation take place simultaneously in the reaction; while the RNA polymerase transcribes the template gene, the ribosomes provided by the TNT start to translate the 5′ end of nascent mRNA. To confirm the expression, Western blot analysis was carried out with kala-azar patient sera using a standard methodology, as described above.

Immunogenicity study of the DNA vaccine construct.Six BALB/c mice, 6 to 8 weeks of age, were used in each group for all immunization studies. For intramuscular vaccination, 100 μg of plasmid DNA (in 50 μl of PBS) was injected into the midpoint of the left thigh muscle. The mice were immunized twice at a fortnightly interval. Another set of six mice injected with empty plasmid served as controls. Another set of six mice was also included as a control and was vaccinated with the kinesin immunodominant repeat domain (nonmotor) construct, which is predominantly a B-cell epitopic region. The injected mice were bled and sacrificed after 2 weeks of final injections. Spleens were removed from mice under aseptic conditions on a sterile dish containing RPMI 1640 medium. Single-cell suspensions were prepared by grinding the spleen with the disk bottom of the plunger of a 10-ml syringe. To the suspension, 10 ml of RPMI 1640 medium was added and mixed to homogeneity. The dish containing cell suspension was kept undisturbed for 2 min, and the clear supernatant was pipetted out slowly. Cells were pelleted by centrifuging at 4°C at 250 × g (Sigma 2K centrifuge) for 10 min. The pellet containing erythrocytes and splenocytes was collected and washed once with 0.9% ammonium chloride to lyse erythrocytes. The remaining cells (splenocytes) were resuspended to a density of 2.5 × 10⁶ cells/ml in RPMI 1640 containing 10% fetal calf serum and 0.05 μM 2-mercaptoethanol. The cell suspension was then distributed as 200-μl aliquots (5 × 10⁵ cells) in 96-well plates. After addition of the mitogen concanavalin A (1 μg/well), rLvacc (1 μg/well), or rKE16 (1 μg/well), the cells were incubated for 3 days at 37°C in an atmosphere containing 5% CO₂ and 95% humidity (30). Proliferation was measured in an MTT assay (22).

IFN-γ, IL-2, and IL-4 detection in splenocyte cultures of mice in response to vaccination.The splenocyte cultures (isolated from mouse spleens) were set up in 96-well culture plates at a concentration of 5 × 10⁵ cells/well, followed by addition of antigens/mitogens, with each group set up in triplicate. After 48 h, the supernatants were harvested carefully from each well for cytokine measurements. IFN-γ, IL-2, and IL-4 in the supernatants were determined by using an Endogen mouse IFN-γ, IL-2, and IL-4 enzyme-linked immunosorbent assay kit (Pierce Biotechnology, Inc.) following the protocol recommended by the manufacturer.

Immunoproliferative response to whole parasite.The present study was carried out to check immune reactivation to whole parasite following vaccination. For this we isolated splenocytes from kinesin motor domain construct-vaccinated mice and carried out a splenocyte proliferation assay in the presence of whole-cell lysate (1 μg/well) and further estimated the cytokine profile using the protocol described above.

Statistical analysis.Statistical analysis of the results and graphic creations were done with the Graph Pad Prism program (version 3.0) and Microsoft Excel (version 7.0) for general statistical calculations, such as arithmetic means and standard deviations. P values of <0.05 were considered significant.

rLvacc showed strong immunoreactivity with sera from cured VL patients.The cloning and expression of the kinesin motor domain in pRSET-C resulted in high-level expression of the rLvacc protein with a molecular mass of 35 kDa (after removal of the His tag). We obtained an expression level of 2.6 mg of purified recombinant protein from a 1-liter culture and a purity of >96%. The DNA content of the purified protein sample was approximately ≤9 ng/ml, which is well within the WHO recommendations on biological standardization for recombinant proteins. While assessing the antibody response against the recombinant rLvacc, the sera from VL patients reacted with rLvacc in Western blotting (Fig. 1). Sera from healthy controls did not react with rLvacc at all.

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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.

rLvacc stimulation induced a strong proliferative response in PBMCs from cured VL patients.The results of the proliferative response of PBMCs against rLvacc at a 5-μg/well concentration showed significantly higher stimulation in cured kala-azar patients (mean OD, 0.4628 ± 0.014) (Fig. 2) than nonendemic healthy controls (mean OD, 0.1046 ± 0.003) (Fig. 2). The difference was statistically significant (P < 0.0001). Concavalin A controls showed a good proliferative response in both cured kala-azar patients and healthy controls, indicating the procedural sensitivity as shown in Fig. 2.

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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).

rLvacc stimulation induced strong IgG2 production in PBMCs from VL patients.Supernatants from the rLvacc-stimulated PBMCs of cured kala-azar patients had a higher IgG2 response (mean OD, 1.5 ± 0.5) than uninduced PBMCs of the same kala-azar patients (mean OD, 0.26 ± 0.06). The difference was statistically significant (P < 0.0001). However, the IgG1 response was not significantly different between the induced and the uninduced PBMCs (mean OD, 0.43 ± 0.03 versus 0.39 ± 0.04; P = 0.3) (Fig. 3).

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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).

Evaluation of the DNA vaccine construct in BALB/c mice.The DNA vaccine candidate, expressing the L. donovani kinesin motor protein, was prepared by cloning in a pVAX-1 vector. The in vitro translation system confirmed the expression of the kinesin motor domain from the DNA vaccine construct (Fig. 4). Following generation of the vaccine candidate, BALB/c mice were immunized and were finally bled 2 weeks after the final injections. Splenocytes were isolated from both control and test groups of mice, and proliferation assays were carried out, followed by cytokine profile analysis using an ELISA. Statistically insignificant (P = 0.09) proliferative responses were observed against expressed protein rLvacc in the splenocytes from mice injected with empty plasmid (0.1275 ± 0.006). However, mice in the group injected with the kinesin motor domain DNA vaccine construct generated a strong proliferative response against rLvacc (0.4060 ± 0.013). The difference was statistically significant (P < 0.0001). The mouse group injected with kinesin immunodominant repeat domain constructs also showed a good proliferative response against rKE16. Concanavalin A controls showed a good proliferative response in all vaccinated groups and the empty plasmid group (Fig. 5).

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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.

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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.

Levels of IFN-γ, IL-2, and IL-4 in response to rLvacc.The supernatants of rLvacc-stimulated splenocytes from control and vaccinated mice were assayed for IFN-γ, IL-2, and IL-4 concentrations. 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 controls injected with empty plasmid, whereas the vaccinated mice showed significantly (P < 0.0001) 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 difference in IL-4 levels between the vaccinated and the empty plasmid group was statistically insignificant (P = 0.4587) (Fig. 6). The mice injected with the kinesin immunodominant construct showed a mixed cytokines profile. The levels of IFN-γ, IL-2, and IL-4 were 419.2 ± 41.48, 179.2 ± 12.94, and 285.5 ± 12.44 pg/ml, respectively (Fig. 6).

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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.

Immune response to whole parasite.Stimulation with whole-cell lysate resulted in a Th1-specific cytokine profile. This mouse group showed significantly (P < 0.0001) 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) (Fig. 7).

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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).